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Article

Synthesis and In Vitro Anticancer Activity of Pyrrolidone Derivatives Bearing 3,4,5-Trimethoxyphenyl Moiety as a Promising Anticancer Scaffold

by
Povilas Kavaliauskas
1,2,3,4,
Birutė Sapijanskaitė-Banevič
1,
Birutė Grybaitė
1,
Eglė Mickevičiūtė
5,
Kazimieras Anusevičius
1,
Andrew Garcia
2,
Ethan Naing
2,
Rūta Petraitienė
2,3,
Vidmantas Petraitis
2,3,
Ramunė Grigalevičiūtė
4 and
Vytautas Mickevičius
1,*
1
Department of Organic Chemistry, Kaunas University of Technology, 50254 Kaunas, Lithuania
2
Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine of Cornell University, New York, NY 10065, USA
3
Institute of Infectious Diseases and Pathogenic Microbiology, 59116 Prienai, Lithuania
4
Biological Research Center, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania
5
Department of Information Systems, Kaunas University of Technology, 51368 Kaunas, Lithuania
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(24), 11784; https://doi.org/10.3390/app142411784
Submission received: 18 November 2024 / Revised: 10 December 2024 / Accepted: 13 December 2024 / Published: 17 December 2024
(This article belongs to the Special Issue Research on Organic and Medicinal Chemistry)
Browse Figures
Versions Notes

Abstract

:
A series of 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives–hydrazones, N-ethylhydrazones, pyrrole, pyrazole, oxadiazole, and triazole were synthesized and evaluated for their anticancer activity using human A549 pulmonary epithelial cells (ATCC CCl-185). The in vitro viability inhibitory effects of the compounds were assessed using the MTT assay. The characterization of the anticancer activity of the synthesized compounds showed that the incorporation of 1,3,4-oxadiazolethione and 4-aminotriazolethione rings into the molecular structures obviously enhances the anticancer activity against human A549 lung epithelial cells, reducing their viability to 28.0% and 29.6%, respectively. The anticancer activity of these azole derivatives was significantly higher than that of cytarabine. Further studies are needed to better optimize 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives and enhance their in vitro anticancer activity.

1. Introduction

Cancer is the second leading cause of death worldwide after cardiac diseases and is the leading cause of death in more than 50 countries [1]. Among all cancer cases, breast, lung, and colon cancers continue to be the most common types.
According to the World Health Organization, cancer, being a major public health, societal, and economic issue and the world’s biggest killer in many centuries, is responsible for almost one in six deaths globally [2]. It accounts for nearly 10 million fatal outcomes every year. The incidence of new cases of cancer is increasing most rapidly. There were 14.1 million new cancer cases in 2012 worldwide [3], and data taken from the Global Cancer Observatory show the increase to 20 million in 2022. It is predicted that by 2040 there will be 29.9 million cases of cancer [4]. According to the latest data, in 2024 in the United States alone, 2 million new cases of cancer will be diagnosed, and over 600,000 people will die from the disease [5]. The data are truly ominous.
The increasing burden of cancer continues to have an increasing negative impact on individuals, communities, and health care systems worldwide, and this impact is likely to grow further as morbidity and mortality continue to increase. The high death rate from cancer is a reminder of the need for more effective treatments. Currently, there are several methods of suppressing and treating cancer, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, biological therapy, and photodynamic therapy [6]. At the same time, cancer treatment is becoming more and more individualized, and cancer as a whole is increasingly turning into a collection of increasingly rare tumors, the differentiation of which is no longer based on histological criteria alone. This phenomenon challenges the methods of oncological disease treatment and drug development [7].
Medical advances through artificial intelligence, precision oncology, DNA sequencing, and other technologies and techniques designed to innovate cancer treatment and diagnostics are extending the lives of cancer patients [8,9]. One of the important ways to treat the uncontrolled division and growth of cancer cells, which causes various cancer types [10], is the use of drugs that interfere with the synthesis of nucleic acids, DNA/RNA, and suppress their normal function [11,12]. Although novel therapeutics targeting more selective pathways are crucially needed to overcome challenging anticancer-resistance mechanisms or target advanced and metastatic processes.
Although there are many methods and a large supply of anticancer drugs, only increasing the selectivity of existing anticancer drugs or the discovery of new classes of more selective drugs would significantly improve patient survival. In order to continue to fight with this insidious, difficult, and often fatal disease, the discovery and development of completely new cancer therapeutics is essential [13,14,15].
Among the abundance of biologically effective organic compounds, the 3,4,5-trimethoxyphenyl (TMP) scaffold is the structural pharmacophore of many biologically active substances with diverse bioactivities (Figure 1). This moiety is widely studied and clearly visible in the molecular structures of various scientific works and exhibits remarkable multifunctionality or specific targeting, surpassing the effectiveness of other derivatives at similar concentrations [16,17,18,19,20,21]. For example, combretastatin inhibits cell growth at nanomolar concentrations, exhibiting inhibitory effects even on multidrug-resistant cancer cell lines [22]. Trimethoprim is typically utilized to hinder the activities of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes, respectively [23]. Oxiracetam constitutes a separate group of therapeutics due to its unique selectivity for brain areas involved in the procedures of acquiring knowledge and memory processes and its safe profile (Figure 1) [24]. These features make them suitable for chronic therapy in aged patients [25].
Compounds with the above-mentioned fragment in the molecular structures display remarkable anti-inflammatory effects by inhibiting cyclooxygenase-1 and -2 and cytokines TNF-α and IL-6. Also, they have shown antiviral activity by inhibiting reverse transcriptase, as well as against influenza or hepatitis C viruses. In addition, 3,4,5-trimethoxyphenyl derivatives were also found to be potent agents against malaria, leishmaniasis, and trypanosomiasis, thus confirming their potential as antiparasitic agents. Furthermore, these compounds have been associated with anti-Alzheimer, anti-depressant, and anti-migraine properties, thereby expanding their therapeutic scope and demonstrating a significant versatility in biological activity that is crucial for the further medicinal chemistry development [26,27,28,29,30].
Another medical application of TMP compounds is anticancer research. Extensive studies of trimethoxyphenyl-containing derivatives in the field have shown that they exhibit anti-cancer potency by effectively inhibiting tubulin, P-glycoprotein, and platelet-derived growth factor receptor β and a series of enzymes like Hsp90, TrxR, HLSD1, and ALK2 [25]. Investigations revealed their promising GI values against NCSLC lung cancer, HCT-116 colorectal carcinoma, and SK-BR-3 breast cancer cell lines [31]. The TMP moiety is critical in maintaining the proper molecular conformations required for optimal interaction with tubulin and maximal antiproliferative activity [32,33]. Also, derivatives bearing the 3,4,5-trimethoxyphenyl scaffold were found to be potent and highly selective phosphodiesterase 5 inhibitors [34].
In this study, we have synthesized a new series of 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide derivatives, and their molecular structures were confirmed by NMR and IR spectroscopy techniques and microanalysis data. These new compounds were evaluated for their anticancer activity using the A549 human lung adenocarcinoma in vitro model.

2. Materials and Methods

2.1. Synthesis

Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The reaction course and purity of the synthesized compounds were monitored by TLC using aluminum plates precoated with silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts were reported in (δ) ppm relative to tetramethylsilane (TMS), with the residual solvent as an internal reference (DMSO-d6, δ = 2.50 ppm for 1H and δ = 39.52 ppm for 13C). Data are reported as follows: chemical shift, multiplicity, coupling constant [Hz], integration, and assignment. Melting points were determined with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and were uncorrected. Elemental analyses (C, H, and N) were conducted using the Elemental Analyzer CE-440 (Exeter Analytical, Inc., North Chelmsford, MA, USA), and their results were found to be in good agreement (±0.3%) with the calculated values. IR spectra (ν, cm−1) were recorded on a Perkin–Elmer Spectrum BX FT–IR spectrometer (Perkin–Elmer Inc., Waltham, MA, USA) using KBr pellets.
  • 5-Oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid (2)
A mixture of 3,4,5-trimethoxyaniline (1) (20 g, 109 mmol), itaconic acid (21.3 g, 164 mmol), and water (200 mL) was heated at reflux for 7 h. After completion of the reaction, the mixture was cooled, the formed crystalline solid was filtered off and washed with water, then diethyl ether. The crude product was purified by dissolving it in 5% sodium hydroxide solution, filtering, and acidifying the filtrate with hydrochloric acid to pH 1 (the procedure was performed twice).
Grayish powder, yield 24.6 g (76.3%), m. p. 140–142 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.64–2.82 (m, 2H, CH2CO), 3.27–3.39 (m, 1H, CH), 3.63, 3.76 (2s, 9H, 3OCH3), 3.92–4.10 (m, 2H, NCH2), 6.97 (s, 2H, HAr), 12.82 (br s, 1H, OH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 34.08, 35.36, 50.43, 55.94, 60.15, 97.91, 134.27, 135.24, 152.71, 171.76, 174.27 ppm.
IR (KBr): νmax: 3497 (OH), 1736, 1667 (2C=O), 1273, 1235 (3OCH3) cm−1.
Anal. Calcd. for C14H17NO6, %: C 56.95; H 5.80; N 4.74. Found: C 56.83; H 5.71; N 4.65.
  • Methyl 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylate (3)
To a solution of carboxylic acid 2 (22.13 g, 75 mmol) in methanol (200 mL), conc. sulfuric acid (3 mL) was added dropwise, and the mixture was heated at reflux for 8 h. Afterwards, the solvent was evaporated under reduced pressure, and then the residue was neutralized with 10% sodium carbonate solution to pH 6. The obtained solid was filtered off, washed with plenty of water, and recrystallized from methanol.
Dark grayish solid, yield 19.48 g (84%), m. p. 94–95 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.67–2.84 (m, 2H, CH2CO), 3.40–3.59 (m, 1H, CH), 3.63 (s, 3H, OCH3), 3.69 (s, 3H, COOCH3), 3.76 (s, 6H, 2OCH3), 3.93–4.13 (m, 2H, NCH2), 6.96 (s, 2H, HAr) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 34.83, 35.13, 51.17, 52.18, 55.91, 60.10, 97.95, 134.30, 135.09, 152.67, 171.41, 173.11 ppm.
IR (KBr): νmax: 1745, 1695 (2C=O), 1257, 1236 (3OCH3) cm−1.
Anal. Calcd. for C15H19NO6, %: C 58.25; H 6.19; N 4.53. Found: C 58.17; H 6.11; N 4.47
  • 5-Oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (4)
To a solution of methyl ester 3 (7.73 g, 24 mmol) in propan-2-ol (50 mL), hydrazine monohydrate (4.20 g, 75 mmol) was added dropwise, and the mixture was heated at reflux for 3 h, then cooled, and the formed crystalline precipitate was filtered off, washed with propan-2-ol, and recrystallized from propan-2-ol.
Light violet solid, yield 5.26 g (68%), m. p. 161–162 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.57–2.74 (m, 2H, CH2CO), 3.09–3.19 (m, 1H, CH), 3.63 (s, 3H, OCH3), 3.76 (s, 6H, 2OCH3), 3.82–4.05 (m, 2H, NCH2), 4.32 (s, 2H, NH2), 6.97 (s, 2H, HAr), 9.28 (s, 1H, NH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 34.02, 35.85, 51.13, 55.89, 60.11, 97.79, 134.17, 135.24, 152.65, 171.44, 171.92 ppm.
IR (KBr): νmax: 3326, 3188, (NH2, NH), 1673 (2C=O), 1275 1239 (3OCH3) cm−1.
Anal. Calcd. for C14H19N3O5, %: C 54.36; H 6.19; N 13.58. Found: C 54.29; H 6.11; N 13.50.
  • 5-Oxo-N′-(2-oxoindolin-3-ylidene)-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (5)
A mixture of carbohydrazide 4 (0.5 g, 1.6 mmol), isatin (0.36 g, 2.4 mmol), and propan-2-ol (20 mL) was heated at reflux for 7 h, then cooled. The formed crystalline precipitate was filtered off, washed with propan-2-ol and ethyl ether, and recrystallized from propan-2-ol.
Yellow solid, yield 0.4 g (68%), m. p. 193–195 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.65–2.91 (m, 2H, CH2CO), 3.64, 3.77 (2s, 9H, 3OCH3), 3.98–4.19 (m, 3H, NCH2, CH), 6.90 (d, 1H, J = 7.7 Hz, HAr), 6.99 (s, 2H, HAr), 7.01–7.15 (m, 1H, HAr), 7.39 (t, 1H, J = 6.8 H, HAr), 8.14 (s, 1H, HAr), 10.82 (s, 1H, NHisatin), 11.36 (br s, 1H, NHCO) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 33.50, 35.23, 50.51, 56.03, 60.12, 98.09, 110.80, 121.81, 134.31, 134.96, 143.61 152.63, 164.74, 171.84 ppm.
IR (KBr): νmax: 3308, (2NH), 1739, 1697 (3C=O), 1276, 1236 (3OCH3) cm−1.
Anal. Calcd. for C22H22N4O6, %: C 60.27; H 5.06; N 12.78. Found: C 60.19; H 4.58; N 12.69.
  • N′-(7-bromo-2-oxoindolin-3-ylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (6)
A mixture of carbohydrazide 4 (0.5 g, 1.6 mmol), 7-bromoisatin (0.55 g, 2.4 mmol), and propan-2-ol (20 mL) was heated at reflux for 6.5 h, then cooled. The formed crystalline precipitate was filtered off, washed with propan-2-ol and ethyl ether, and recrystallized from propan-2-ol.
Light yellow powder, yield 0.53g (63%), m. p. 217–219 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.68–2.97 (m, 2H, CH2CO), 3.63, 3.76 (2s, 9H, 3OCH3), 3.97–4.26 (m, 3H, NCH2, CH), 6.98 (s, 2H, HAr), 7.04 (t, 1H, J = 7.8 Hz, HAr), 7.58 (d, 1H, J = 8.0 Hz, HAr), 7.60–8.24 (m, 1H, HAr), 11.12 (s, 0.15H, NHisatin), 11.58 (s, 0.85H, NHisatin), 12.55 (s, 0.70H, NHCO), 13.02 (s, 0.30H, NHCO) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 31.90, 34.76, 49.72, 55.72, 59.86, 97.94, 103.13, 119.65, 121.60, 123.00, 123.91, 133.92, 134.15, 134.90, 134.96, 141.32 152.47, 162.34, 171.32, 171.41, 174.27 ppm.
IR (KBr): νmax: 3136, (2NH), 1742, 1683, 1656 (3C=O), 1282, 1238 (3OCH3) cm−1.
Anal. Calcd. for C22H21BrN4O6, %: C 51.08; H 4.09; N 10.83. Found: C 50.97; H 3.96; N 10.74.
  • General procedure for the preparation of hydrazones 7, 8
A solution of hydrazide 4 (0.5 g, 1.6 mmol) and the corresponding aldehyde (1.8 mmol) in propan-2-ol (10 mL) was refluxed for 7–11 h. After completion of the reaction, the mixture was cooled, the formed precipitate was filtered off, washed with propan-2-ol and diethyl ether, and dried. The obtained compounds were purified by recrystallization from propan-2-ol.
  • N′-((5-nitrofuran-2-yl)methylene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (7)
Light yellow solid, yield 0.7g (86%), m. p. 213–215 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.66–2.88 (m, 2H, CH2CO), 3.63, 3.76 (2s, 9H, 3OCH3), 3.94–4.15 (m, 3H, NCH2, CH), 6.98 (s, 2H, HAr), 7.21–7.32 (m, 1H, HHet), 7.74–7.82 (m, 1H, HHet), 7.98 (s, 0.6H, CH=N), 8.19 (s, 0.40H, CH=N), 11.98 (s, 0.60H, CONH), 12.02 (s, 0.4H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.57, 34.88, 35.11, 35.66, 50.31, 50.77, 55.90, 60.11, 97.88, 98.10, 114.59, 114.75, 115.51, 131.87, 134.27, 134.32, 135.01, 135.17, 135.20, 151.56. 151.61, 151.77, 151.96, 152.68, 169.26, 171.59, 171.76, 174.09 ppm.
IR (KBr): νmax: 3190, (NH), 1687, 1668 (2C=O), 1251, 1204 (3OCH3) cm−1.
Anal. Calcd. for C19H20N4O8, %: C 52.78; H 4.66; N 12.96. Found: C 52.68; H 4.57; N 12.89.
  • N′-((5-nitrothiophen-2-yl)methylene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (8)
Yellow solid, yield 0.46 g (64%), m. p. 219–221 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.69–2.88 (m, 2H, CH2CO), 3.63, 3.76 (2s, 9H, 3OCH3), 3.92–4.19 (m, 3H, NCH2, CH), 6.99 (s, 2H, HAr), 7.50–7.61 (m, 1H, HHet), 8.06–8.14 (m, 1H, HHet), 8.20 (s, 0.65H, CH=N), 8.48 (s, 0.35H, CH=N), 12.00 (s, 1H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.82, 34.82, 35.12, 35.59, 50.29, 50.81, 55.90, 60.11, 97.88, 98.05, 129.27, 129.81, 130.64, 134.29, 135.16, 135.22, 136.93, 140.63, 146.47, 146.57, 150.56. 152.67, 169.18, 171.62, 171.71, 173.82 ppm.
IR (KBr): νmax: 3115, (NH), 1692, 1665 (2C=O), 1266, 1219 (3OCH3) cm−1.
Anal. Calcd. for C19H20N4O7S, %: C 50.89; H 4.50; N 12.49. Found: C 51.80; H 4.43; N 12.39.
  • General procedure for the preparation of hydrazones 9–18
A mixture of carbohydrazide 4 (0.5 g, 1.6 mmol), the corresponding aldehyde (1.7 mmol), and propan-2-ol (10 mL) was refluxed for 3–8 h. Afterwards, the reaction mixture was cooled, the obtained product was filtered off, washed with propan-2-ol and diethyl ether, and recrystallized from the indicated solvent.
  • N′-benzylidene-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (9)
White solid, yield 0.46 g (72%), m. p. 142–144 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.68–2.88 (m, 2H, CH2CO), 3.63, 3.76. 3.77 (3s, 9H, 3OCH3), 3.92–4.18 (m, 3H, NCH2, CH), 6.99 (s, 2H, HAr), 7.34–7.76 (m, 5H, HAr), 8.04 (s, 0.65H, CH=N), 8.24 (s, 0.35H, CH=N), 11.59 (s, 0.65H, CONH), 11.65 (s, 0.35H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.72, 34.76, 35.21, 35.74, 50.45, 51.00, 55.91, 60.11, 97.85, 98.07, 126.90, 127.10, 128.85, 129.93, 130.14, 134.13, 134.23, 134.28, 135.22, 135.28, 143.69, 147.08, 152.67, 168.64, 171.78, 171.92, 173.60 ppm.
IR (KBr): νmax: 3133, (NH), 1680, 1655 (2C=O), 1296, 1280, 1233 (3OCH3) cm−1.
Anal. Calcd. for C21H23N3O5, %: C 63.47; H 5.83; N 10.57. Found: C 63.39; H 5.75; N 10.48.
  • N′-(4-chlorobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (10)
White solid, yield 0.59 g (85%), m. p. 184–186 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.68–2.87 (m, 2H, CH2CO), 3.63, 3.76. 3.77 (3s, 9H, 3OCH3), 3.95–4.17 (m, 3H, NCH2, CH), 6.99 (s, 2H, HAr), 7.42–7.78 (m, 4H, HAr), 8.03 (s, 0.65H, CH=N), 8.22 (s, 0.35H, CH=N), 11.65 (s, 0.65H, CONH), 11.71 (s, 0.35H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.67, 34.75, 35.20, 35.72, 50.41, 51.96, 55.91, 60.11, 97.84, 98.06, 128.56, 128.73, 128.91, 128.94, 133.09, 134.23, 134.28, 134.35, 134.59, 135.21, 135.26, 142.43, 145.76, 152.67, 168.74, 171.76, 171.89, 173.67 ppm.
IR (KBr): νmax: 3129, (NH), 1681, 1651 (2C=O), 1292, 1279, 1232 (3OCH3) cm−1.
Anal. Calcd. for C21H22ClN3O5, %: C 58.40; H 5.13; N 9.73. Found: C 58.29; H 5.05; N 9.64.
  • N′-(4-bromobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (11)
White solid, yield 0.7 g (91%), m. p. 210–211 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.69–2.89 (m, 2H, CH2CO), 3.63, 3.76. 3.77 (3s, 9H, 3OCH3), 3.92–4.18 (m, 3H, NCH2, CH), 6.99 (s, 2H, HAr), 7.52–7.73 (m, 4H, HAr), 8.01 (s, 0.64H, CH=N), 8.20 (s, 0.36H, CH=N), 11.65 (s, 0.64H, CONH), 11.71 (s, 0.36H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.67, 34.74, 35.19, 35.71, 50.40, 50.95, 55.91, 60.11, 97.84, 98.06, 123.12, 123.38, 128.79, 128.96, 131.82, 131.85, 133.42, 134.22, 134.27, 135.21, 135.25, 142.53, 145.84, 152.67, 168.75, 171.75, 171.88, 173.67 ppm.
IR (KBr): νmax: 3145, (NH), 1689, 1667 (2C=O), 1275, 1221 (3OCH3) cm−1.
Anal. Calcd. for C21H22BrN3O5, %: C 52.95; H 4.66; N 8.82. Found: C 52.84; H 4.54; N 8.75.
  • N′-(4-fluorobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (12)
White solid, yield 0.58 g (87%), m. p. 177–179 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.71–2.89 (m, 2H, CH2CO), 3.63, 3.76. 3.77 (3s, 9H, 3OCH3), 3.91–4.24 (m, 3H, NCH2, CH), 6.99 (s, 2H, HAr), 7.17–7.85 (m, 4H, HAr), 8.03 (s, 0.65H, CH=N), 8.23 (s, 0.35H, CH=N), 11.60 (s, 0.65H, CONH), 11.65 (s, 0.35H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.67, 34.74, 35.20, 35.73, 50.43, 50.99, 55.91, 60.11, 97.85, 98.07, 115.77, 115.99, 129,05, 129.13, 129.24, 129.32, 130.75, 134.28, 135.27, 142.56, 145.96, 152.67, 161.78, 164.24, 168.66, 171.77, 171.91, 173.60 ppm.
IR (KBr): νmax: 3126, (NH), 1678, 1651 (2C=O), 1282, 1232 (3OCH3) cm−1.
Anal. Calcd. for C21H22FN3O5, %: C 60.72; H 5.34; N 10.12. Found: C 60.64; H 5.23; N 10.04.
  • N′-(4-methoxybenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (13)
White solid, yield 0.43 g (62%), m. p. 226–228 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.67–2.88 (m, 2H, CH2CO), 3.63, 3.76. 3.79 (3s, 12H, 4OCH3), 3.92–4.20 (m, 3H, NCH2, CH), 6.89–7.10 (m, 4H, HAr), 7.64 (d, J = 8.0 Hz, 2H, HAr), 7.98 (s, 0.63H, CH=N), 8.16 (s, 0.37H, CH=N), 11.46 (s, 0.63H, CONH), 11.51 (s, 0.37H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.69, 34.72, 35.21, 35.76, 50.49, 51.05, 55.30, 55.91, 60.11, 97.84, 98.06, 114.32, 126.66, 126,73, 128.48, 128.71, 134.21, 134.26, 135.23, 135.29, 143.96, 146.96, 152.67, 160.69, 160.89, 168.36, 171.82, 171.96, 173.33 ppm.
IR (KBr): νmax: 3219, (NH), 1691, 1666 (2C=O), 1294, 1251 (4OCH3) cm−1.
Anal. Calcd. for C22H25N3O6, %: C 61.82; H 5.90; N 9.83. Found: C 61.71; H 5.78; N 9.70.
  • N′-(2,4-difluorobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (14)
White solid, yield 0.6 g (86%), m. p. 179–181 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.71–2.88 (m, 2H, CH2CO), 3.63, 3.76. (2s, 9H, 3OCH3), 3.92–4.17 (m, 3H, NCH2, CH), 6.98 (s, 2H, HAr), 7.09–8.10 (m, 3H, HAr), 8.19 (s, 0.67H, CH=N), 8.40 (s, 0.33H, CH=N), 11.69 (s, 0.67H, CONH), 11.78 (s, 0.33H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.69, 34.82, 35.14, 35.67, 50.35, 50.90, 55.91, 60.11, 97.85, 98.06, 104.50, 112.51, 112.72, 118.49, 128.20, 134.24, 134.28, 135.20, 135.25, 135.86, 152.67, 168.71, 171.71, 171.86, 173.67 ppm.
IR (KBr): νmax: 3121, (NH), 1680, 1651 (2C=O), 1296, 1234 (3OCH3) cm−1.
Anal. Calcd. for C21H21F2N3O6, %: C 58.20; H 4.88; N 9.70. Found: C 58.08; H 4.70; N 9.58.
  • 5-Oxo-N′-(3,4,5-trimethoxybenzylidene)-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (15)
White solid, yield 0.68 g (86%), m. p. 185–187 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.67–2.91 (m, 2H, CH2CO), 3.63, 3.69. 3.75, 3.77, 3.81, 38.2 (6s, 18H, 6OCH3), 3.92–4.20 (m, 3H, NCH2, CH), 7.00 (s, 4H, HAr), 7.95 (s, 0.60H, CH=N), 8.15 (s, 0.40H, CH=N), 11.61 (s, 1H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.54, 34.71, 35.29, 35.77, 50.51, 51.07, 55.90, 55.96, 60.12, 97.88, 98.05, 104.20, 104.32, 129.64, 134.24, 134.29, 135.22, 135.29, 139.10, 139.25, 143.58, 147.07, 152.68, 153.20, 168.63, 171.80, 172.00, 173.69 ppm.
IR (KBr): νmax: 3179, (NH), 1695, 1673 (2C=O), 1290, 1234 (6OCH3) cm−1.
Anal. Calcd. for C24H29N3O8, %: C 59.13; H 6.00; N 8.62. Found: C 58.98; H 5.91; N 8.54.
  • N′-(2-chlorobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (16)
Light violet solid, yield 0.5 g (72%), m. p. 136–138 °C (from propan-2-ol). 1H NMR (400 MHz, DMSO-d6) δ: 2.71–2.89 (m, 2H, CH2CO), 3.63, 3.76, 3.77. (3s, 9H, 3OCH3), 3.94–4.17 (m, 3H, NCH2, CH), 6.87–8.07 (m, 6H, HAr), 8.43 (s, 0.65H, CH=N), 8.62 (s, 0.35H, CH=N), 11.77 (s, 0.65H, CONH), 11.88 (s, 0.35H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.68, 34.85, 35.17, 35.66, 50.39, 50.91, 55.91, 60.11, 97.84, 98.06, 126.87, 126.92, 127.64, 127.68, 129,92, 131.60, 132.99, 133.17, 134.23, 135.21, 135.25, 139.76, 142.96, 152.67, 168.79, 171.73, 171.87, 173.76 ppm.
IR (KBr): νmax: 3133, (NH), 1673 (2C=O), 1296, 1276, 1235 (3OCH3) cm−1.
Anal. Calcd. for C21H22ClN3O5, %: C 58.40; H 5.13; N 9.73. Found: C 58.43; H 5.08; N 9.78.
  • N′-(3-chlorobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (17)
Grayish powder, yield 0.53 g (76%), m. p. 170–172 °C (from acetone).
1H NMR (400 MHz, DMSO-d6) δ: 2.70–2.88 (m, 2H, CH2CO), 3.63, 3.76, 3.77. (3s, 9H, 3OCH3), 3.92–4.18 (m, 3H, NCH2, CH), 7.00 (s, 2H, HAr), 7.38–7.82 (m, 4H, HAr), 8.02 (s, 0.65H, CH=N), 8.21 (s, 0.35H, CH=N), 11.70 (s, 0.65H, CONH), 11.78 (s, 0.35H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.58, 34.76, 35.24, 35.70, 50.43, 50.94, 55.90, 60.10, 97.85, 98.03, 125.67, 125.76, 126.10, 126.45, 129,57, 130.70, 130.74, 133.64, 133.71, 134.26, 135.20, 135.27, 136.36, 136.41, 142.15, 145.39, 152.67, 168.84, 171.73, 171.90, 173.84 ppm.
IR (KBr): νmax: 3134, (NH), 1682 (2C=O), 1269, 1235 (3OCH3) cm−1.
Anal. Calcd. for C21H22ClN3O5, %: C 58.40; H 5.13; N 9.73. Found: C 58.30; H 5.02; N 9.63.
  • N′-(2-chloro-5-nitrobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (18)
Light brown powder, yield 0.66 g (86%), m. p. 199–201 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 2.71–2.92 (m, 2H, CH2CO), 3.63, 3.76, 3.77. (3s, 9H, 3OCH3), 3.95–4.21 (m, 3H, NCH2, CH), 6.99 (s, 2H, HAr), 7.78–8.66 (m, 4H, HAr, CH=N), 11.95 (s, 0.60H, CONH), 12.09 (s, 0.40H, CONH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 32.59, 34.92, 35.16, 35.61, 50.36, 50.79, 55.88, 60.10, 97.85, 98.02, 121.12, 125.08, 125.29, 131.58, 132.84, 134.28, 135.17, 135.22, 138.08, 138.92, 139.06, 146.68, 146.79, 152.67, 169.13, 171.62, 171.80, 173.99 ppm.
IR (KBr): νmax: 3101, (NH), 1673 (2C=O), 1281, 1235 (3OCH3) cm−1.
Anal. Calcd. for C21H21ClN4O7, %: C 52.89; H 4.44; N 11.75. Found: C 52.79; H 4.35; N 11.62.
  • General procedure for the synthesis of N-alkylated hydrazones 19–22
To a solution of the corresponding hydrazone 9–15 (1.0 mmol), potassium hydroxide (3.0 mmol), potassium carbonate (3.0 mmol) in DMF (5 mL), and ethyl iodide (3.5 mmol) was added dropwise. The obtained mixture was stirred at room temperature for 24 h. Then, the reaction mixture was diluted with 20–25 mL of water. The formed precipitate was filtered off, washed with water and diethyl ether, then dried and recrystallized from propan-2-ol (19–21) or a water and 1,4-dioxane mixture (22).
  • N′-benzylidene-N-ethyl-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (19)
White solid, yield 0.26 g (62%), m. p. 155–156 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 1.11 (t, J = 6.9 Hz, 3H, CH3), 2.72–2.89 (m, 2H, CH2CO), 3.63, 3.75 (2s, 9H, 3OCH3), 3.96–4.17 (m, 4H, NCH2, CH2), 4.26–4.38 (m, 1H, CH), 6.98 (s, 2H, HAr), 7.38–7.87 (m, 5H, HAr), 8.11 (s, 1H, CH=N) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 10.95, 33.34, 35.17, 35.76, 50.81, 55.90, 60.10, 98.09, 127.12, 128.81, 129.73, 134.28, 134.83, 135.26, 140.46, 152.66, 171.88, 172.72 ppm.
IR (KBr): νmax: 1689, 1667 (2C=O), 1271, 1254, 1232 (3OCH3) cm−1.
Anal. Calcd. for C23H27N3O5, %: C 64.93; H 6.40; N 9.88. Found: C 64.85; H 6.32; N 9.74.
  • N′-(4-chlorobenzylidene)-N-ethyl-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (20)
White solid, yield 0.35 g (76%), m. p. 128–129 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 1.11 (t, J = 6.9 Hz, 3H, CH3), 2.73–2.88 (m, 2H, CH2CO), 3.63, 3.75 (2s, 9H, 3OCH3), 3.95–4.16 (m, 4H, NCH2, CH2), 4.26–4.37 (m, 1H, CH), 6.98 (s, 2H, HAr), 7.51 (d, J = 7.9 Hz, 2H, HAr), 7.83 (d, J = 7.9 Hz, 2H, HAr), 8.11 (s, 1H, CH=N) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 10.91, 33.25, 35.32, 50.77, 55.91, 60.11, 98.10, 128.76, 128.90, 133.79, 134.17, 134.30, 135.25, 139.31, 152.67, 171.86, 172.79 ppm.
IR (KBr): νmax: 1681 (2C=O), 1271, 1261, 1239 (3OCH3) cm−1.
Anal. Calcd. for C23H26ClN3O5, %: C 60.06; H 5.70; N 9.14. Found: C 59.95; H 5.64; N 9.00.
  • N′-(4-bromobenzylidene)-N-ethyl-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (21)
White solid, yield 0.4 g (79%), m. p. 127–128 °C (from propan-2-ol).
1H NMR (400 MHz, DMSO-d6) δ: 1.10 (t, J = 6.9 Hz, 3H, CH3), 2.72–2.88 (m, 2H, CH2CO), 3.63, 3.75 (2s, 9H, 3OCH3), 3.96–4.15 (m, 4H, NCH2, CH2), 4.25–4.37 (m, 1H, CH), 6.98 (s, 2H, HAr), 7.65 (d, J = 8.0 Hz, 2H, HAr), 7.76 (d, J = 8.1 Hz, 2H, HAr), 8.10 (s, 1H, CH=N) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 10.91, 33.25, 35.33, 35.74, 50.77, 55.91, 60.11, 98.10, 122.94, 128.99, 131.81, 134.13, 134.30, 135.24, 139.39, 152.66, 171.85, 172.79 ppm.
IR (KBr): νmax: 1681 (2C=O), 1270, 1260, 1238 (3OCH3) cm–1.
Anal. Calcd. for C23H26BrN3O5, %: C 54.77; H 5.20; N 8.33. Found: C 54.68; H 5.11; N 8.24.
  • N-ethyl-N′-(4-fluorobenzylidene)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (22)
White solid, yield 0.37 g (84%), m. p. 71–72 °C (from water–1,4-dioxane mixture).
1H NMR (400 MHz, DMSO-d6) δ: 1.11 (t, J = 6.9 Hz, 3H, CH3), 2.72–2.88 (m, 2H, CH2CO), 3.63, 3.75 (2s, 9H, 3OCH3), 3.97–4.16 (m, 4H, NCH2, CH2), 4.26–4.38 (m, 1H, CH), 6.98 (s, 2H, HAr), 7.23 (t, J = 8.5 Hz, 2H, HAr), 7.82–7.92 (m, 2H, HAr), 8.12 (s, 1H, CH=N) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 10.94, 33.27, 35.23, 35.75, 50.79, 55.90, 60.09, 98.08, 115.73, 115.94, 129.21, 129.29, 131.44, 131.47, 134.29, 135.26, 139.42, 152.66, 161.69, 164.15, 171.87, 172.70 ppm.
IR (KBr): νmax: 1681 (2C=O), 1270, 1260, 1238 (3OCH3) cm−1.
Anal. Calcd. for C23H26FN3O5, %: C 62.29; H 5.91; N 9.48. Found: C 62.19; H 5.82; N 9.39.
  • 4-(3,5-Dimethyl-1H-pyrazole-1-carbonyl)-1-(3,4,5-trimethoxyphenyl)pyrrolidin-2-one (23)
A mixture of hydrazide 4 (0.98 g, 3.2 mmol), 2,4-pentanedione (0.5 g, 5.2 mmol), and propan-2-ol (10 mL) was refluxed for 3 h. The solvent was evaporated under reduced pressure until dryness was achieved, and the oily product was triturated with hexane. The obtained solid was filtered off and washed with hexane and ethyl ether. The crude product was purified by recrystallization from propan-2-ol.
Light brown powder, yield 0.82 g (70%), m. p. 120–121 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.22, 2.51 (2s, 6H, 2CH3), 2.78–2.94 (m, 2H, CH2CO), 4.02–4.23 (m, 2H, NCH2), 3.64, 3.77 (2s, 9H, 3OCH3), 4.44–4.55 (m, 1H, CH), 6.25 (s, 1H, C=CH), 6.98 (s, 2H, HAr) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 13.58, 14.07, 35.18, 35.29, 50.57, 55.92, 60.10, 98.16, 111.69, 134.37, 135.08, 143.90, 152.19, 152.68, 171.46, 172.71 ppm.
IR (KBr): νmax: 1720, 1710 (2C=O), 1272, 1255, 1238 (3OCH3) cm−1.
Anal. Calcd. for C19H23N3O5, %: C 61.12; H 6.21; N 11.25. Found: C 60.97; H 6.14; N 11.15.
  • N-(2,5-dimethyl-1H-pyrrol-1-yl)-5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxamide (24)
To a solution of hydrazide 4 (1.0 g, 3.2 mmol) in propan-2-ol (10 mL), 2,5-hexanedione (0.74 g, 6.5 mmol) and glacial acetic acid (0.5 mL) were added. The reaction mixture was stirred and refluxed for 3 h. Then, it was cooled to room temperature, the precipitated product was filtered off, washed with water and ethyl ether, and recrystallized from a 1,4-dioxane–water mixture or toluene.
Light brown powder, yield 0.95 g (76%), m. p. 149–150 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.00 (s, 6H, 2CH3), 2.67–2.94 (m, 2H, CH2CO), 3.39–3.51 (m, 1H, CH), 3.64, 3.78 (2s, 9H, 3OCH3), 3.97–4.16 (m, 2H, NCH2), 5.65 (s, 2H, CH=CH), 6.99 (s, 2H, HAr), 10.90 (s, 1H, NH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 10.94, 10,99, 33.99, 36.00, 50.65, 55.93, 60.12, 98.11, 103.10, 126,75, 134.37, 135.13, 152.70, 171.39, 172.89 ppm.
IR (KBr): νmax: 3260 (CONH), 1709 (2C=O), 1271, 1239 (3OCH3) cm−1.
Anal. Calcd. for C20H25N3O5, %: C 62.00; H 6.50; N 10.85. Found: C 61.92; H 6.39; N 10.74.
  • 4-(5-Thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-1-(3,4,5-trimethoxyphenyl)pyrrolidin-2-one (25)
To a solution of potassium hydroxide (1.8 g, 32 mmol) in methanol (40 mL), carbon disulfide (2.4 g, 32 mmol) was added dropwise. The obtained mixture was stirred at room temperature for 15–20 min, and the methanolic solution of acid hydrazide 4 (1 g, 3.2 mmol/15 mL) was poured over. The reaction mixture was refluxed for 21 h. Then, the methanol was evaporated under reduced pressure, the residue was poured with water (30 mL), and the mixture was acidified with diluted hydrochloric acid (1:1) to pH 4. The crystalline solid was filtered off, washed with plenty of water, and recrystallized from propan-2-ol.
Light brown powder, yield 0.8 g (70%), m. p. 163–164 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.00 (s, 6H, 2CH3), 2.78–3.03 (m, 2H, CH2CO), 3.63, 3.77 (2s, 9H, 3OCH3), 3.88–4.27 (m, 3H, NCH2, CH), 6.97 (s, 2H, HAr), 14.39 (br s, 1H, NH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 27.82, 35.24, 50.37, 55.93, 60.12, 98.09, 134.42, 134.93, 152.69, 163.85, 170.88, 178.07 ppm.
IR (KBr): νmax: 3138 (NH), 1689 (2C=O), 1274, 1230 (3OCH3) cm−1.
Anal. Calcd. for C15H17N3O5S, %: C 51.27; H 4.88; N 11.96. Found: C 51.17; H 4.72; N 11.82.
  • 4-(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)-1-(3,4,5-trimethoxyphenyl)pyrrolidin-2-one (26)
A mixture of hydrazide 4 (1.5 g, 4.8 mmol), potassium hydroxide (2.72 g, 48 mmol), carbon disulfide (3.69 g, 48 mmol), and methanol (50 mL) was refluxed for 18 h. Then, the solvent was removed under reduced pressure, and the residue was poured with diethyl ether (25 mL). The crystalline solid was filtered off, washed with ethyl ether, and dried. The obtained product was dissolved in the mixture of solvents (propan-2-ol (40 mL), 1,4-dioxane (10 mL), and water (10 mL)), and hydrazine monohydrate (0.73 g, 14.4 mmol) was added, and the mixture was refluxed for 24 h. After completion of the reaction, volatile solvents were removed on a rotary evaporator. The crude product was poured with water (15 mL), and the mixture was acidified with diluted hydrochloric acid (1:1) to pH 6. The crystalline solid was filtered off, washed with plenty of water, and recrystallized from methanol.
Light brown powder, yield 1.15 g (65%), m. p. 101–102 °C.
1H NMR (400 MHz, DMSO-d6) δ: 2.00 (s, 6H, 2CH3), 2.82–3.03 (m, 2H, CH2CO), 3.63, 3.76 (2s, 9H, 3OCH3), 3.82–4.26 (m, 3H, NCH2, CH), 5.58 (s, 2H, NH2), 6.97 (s, 2H, HAr), 13.63 (s, 1H, NH) ppm.
13C NMR (101 MHz, DMSO-d6) δ: 27.13, 35.40, 50.92, 55.92, 60.14, 97.93, 134.31, 135.17, 152.63, 152.68, 167.28, 171.62, ppm.
IR (KBr): νmax: 3261, 3164 (NH2, NH), 1692, 680 (2C=O), 1274, 1236 (3OCH3) cm−1.
Anal. Calcd. for C15H19N5O4S, %: C 49.31; H 5.24; N 19.17. Found: C 49.20; H 5.15; N 19.05.

2.2. Biology

2.2.1. Cell Lines and Culture Conditions

Human A549 pulmonary epithelial cells (ATCC CCl-185). Cells were cultured in DMEM/F12 medium enriched with GlutaMAX (ThermoFisher Scientific, Waltham, MA, USA), penicillin–streptomycin (PenStrep) (ThermoFisher Scientific, Waltham, MA, USA), and 10% heat-inactivated fetal bovine serum (FBS) (ThermoFisher Scientific, Waltham, MA, USA). Both cell lines were cultivated at 37 °C under 5% CO2 atmospheric conditions.

2.2.2. Cytotoxicity Assay

The in vitro inhibitory effects of the compounds were assessed using the MTT assay, as described in previous studies [35,36,37,38,39,40,41]. The A549 was seeded into 96-well plates at a concentration of 1 × 104 cells per well. Following an overnight incubation at 37 °C with 5% CO2, the cells were treated with compounds at a concentration of 100 μM, and this treatment was carried out in triplicate. After a 20-h exposure period, the MTT reagent was introduced, and the cells were subsequently incubated for an additional 4 h. To serve as a cytotoxicity control, a 1% solution of sodium dodecyl sulfate (SDS) was added as a control agent before the addition of MTT reagent. The formazan product resulting from the MTT assay was extracted using anhydrous dimethyl sulfoxide (DMSO). The optical density of the samples was determined using a microplate reader (Multiscan, ThermoFisher Scientific), specifically at a wavelength of 570 nm. To ascertain the percentage of A549 cell viability, the following formula was applied: ([AE − AB]/[AC − AB]) × 100%, with AE representing the absorbance of the experimental samples, AC representing the absorbance of untreated samples, and AB representing the absorbance of blank controls [42].

2.2.3. Statistical Analysis

The results are expressed as mean ± standard deviation (SD). Statistical analyses were performed with Prism (GraphPad Software version 9) San Diego, CA, USA), using an unpaired t-test. p < 0.05 was considered significant.

3. Results and Discussion

3.1. Chemistry

In our previous studies, we have reported the synthesis and in vitro anticancer and antimicrobial activity of 5-oxo-1-arylpyrrolidine-3-carbohydrazide derivatives [35,36]. The results were promising for further exploration of this scaffold to enhance the anticancer activity and establish the structure–activity-dependent (SAR) properties of 5-oxo-1-arylpyrrolidine-3-carbohydrazide derivatives. In this study, we used commercially available 3,4,5-trimethoxyphenylamine (1) that was used for further transformations to obtain various derivatives.
We decided to maintain the pyrrolidone scaffold in the molecular design due to its versatile role in medicinal chemistry, particularly due to its conformational rigidity and ability to participate in hydrogen bonding interactions. These properties could potentially enhance the binding affinity and specificity of small molecules. The additional moieties in the designed compounds were selected to complement the physicochemical properties imparted by the pyrrolidone core. Specifically, the 3,4,5-trimethoxyphenyl group was incorporated for its electron-donating characteristics, which enhance the compound’s overall stability and bioactivity by modulating electronic effects in the molecule.
The key intermediate, 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide (4), was achieved by a synthetic route illustrated in Scheme 1. Firstly, 3,4,5-trimethoxyphenylamine 1 was reacted with itaconic acid to obtain carboxylic acid 2, which was then subjected to acid-catalyzed esterification. In the next step, the obtained methyl ester 3 was converted to the target hydrazide 4 in the reaction with hydrazine monohydrate in propan-2-ol.
Compound 1 was used as a commercially available starting reagent for further chemical transformations. The diversity of the biological properties of hydrazones [37] and their suitability for the development of new drugs led to the incorporation of the synthesis of this type of compound, hoping that their combination with the trimethoxyphenyl moiety in the molecules will affect their biological properties. To incorporate 2-oxoindolin-3-ylidene fragment, hydrazide 4 was involved in the reaction with the corresponding isatin. The interaction in propan-2-ol at reflux continued for 7 (5) or 6.5 (6) h, and products were isolated in 68 or 63% yield. The 1H NMR spectrum of compound 6 in DMSO showed a proton signal of the C(O)-NH group at 12.55 and 13.02 ppm, while the N-H signal of the isatin moiety was visible at 11.12 and 11.25 ppm [38]. In the 13C NMR spectrum, the isatin C=O carbon spectral line was observed at 174.27 ppm, and the amide and 5-oxopyrrolidine C=O carbon resonances were observed at 171.32 and 171.41 ppm, respectively (Supplementary Files, Figures S9 and S10).
A synthetic route to hydrazones having heterocyclic 7, 8, and aromatic 9–18 rings in the structures shown in Scheme 1 consists of the reactions of hydrazide 4 with a slight excess of the corresponding aldehyde in propan-2-ol at reflux for 3–11 h. The 1H NMR spectra of compounds 7 and 8 showed singlets in the ranges of 7.98 and 8.48 and between 11.98 and 12.02 ppm, which belong to the protons of the CH=N and CONH, respectively. Multiplets integrated for one proton in the intervals of 7.21–7.61 and 7.74–8.14 ppm were attributed to the protons of the 2CH groups of the 5-nitrothien-2-yl fragment (Supplementary Files, Figures S11 and S13).
Regarding compounds 9–18, for example, we can examine the NMR spectra of compound 13, which has identical spectra to the remaining hydrazones. The 1H NMR spectrum of 13 showed two singlets of protons of the CH=N fragment at 7.98 and 8.16 ppm, and two singlets at 11.46 and 11.51 ppm for CONH [33]. The remaining signals of the methoxy groups, 5-oxopyrrolidine fragment, and aromatic rings were observed in the region as it was expected. The integral intensities of these signals correspond to the molecular structure of hydrazone 13. In the 13C NMR spectrum of compound 13, the carbon spectral lines were in good agreement with the target structure (Supplementary Materials).
In the next step, to improve the biological properties of hydrazones, some of them, viz. 9–12, were alkylated with ethyl iodide in DMF in the presence of potassium hydroxide and potassium carbonate in the reaction mixture. The reactions were carried out at room temperature for 24 h. Products 19–22 were isolated by adding water to the reaction mixtures. Comparison of the NMR spectra of parent compounds 9–12 and products 19–22 indicated an obvious difference between them and proved that N-ethylation had taken place. The 1H NMR spectra of these derivatives showed triplets at around 1.11 ppm with J of 6.9 Hz for the protons of the CH3CH2 group, and two protons of the CH2 of the same group were expressed as multiplets approx. in the range of 3.96 to 4.16 ppm (Supplementary Files, Figures S35, S37, S39 and S41).
As an effort toward the synthesis of small molecules as biologically active compounds, the 3,5-dimethylpyrazole-23 and 2,5-dimethylpyrrole-based 24 structures were prepared. Such molecules were easily obtained by using the condensation of acid hydrazide with diketones approach as depicted in Scheme 2. The targeted compounds were characterized using spectral methods, which data corroborated the assumed structures.
To generate 3,5-dimethylpyrazole 23, condensation was carried out using 1.6 equiv. of pentane-2,4-dione in propan-2-ol at reflux for 3 h. To generate compound 24, having a 2,5-dimethylpyrrole cycle, hydrazide 4 was reacted with two equiv. of hexane-2,5-dione and the reaction was catalyzed by glacial acetic acid.
Next, the intramolecular cyclization of acid hydrazide 4 with carbon disulfide was carried out and resulted in 1,3,4-oxadiazole-2(3H)-thione 25 in 70% yield. The analysis of the NMR spectra of the initial compound 4 and its cyclized derivative 25 showed strong differences indicating the formation of product 25. In the 1H NMR spectrum of 4, the NH proton was observed at 9.28 ppm, whereas in the same spectrum of 25, the NH singlet was shifted to 14.35 ppm and demonstrated the thiol–thione tautomerism. The examination of the 13C NMR spectrum of oxadiazole thione 25 revealed the correspondence of the peaks of carbon atoms with the structure of the compound, and the resonance line at 178.07 ppm confirmed the presence of the C=S group in the formed structure (Supplementary Files, Figures S47 and S48).
4-Amino-1,2,4-triazole 26 was prepared in a two-step procedure from hydrazide 4 by reaction with carbon disulfide and subsequent interaction of the resulting N-potassium thiocarbamate salt with hydrazine monohydrate. Compound 26 was isolated in 65% yield. The spectral data of 5.58 and 13.63 (1H NMR, NH2NH) ppm, as well as 152.68 and 167.28 (13C NMR, N-C=N, C=S) ppm, were in accord with the structure of the 4-amino-1.2.4-triazole cycle (Supplementary Files, Figures S49 and S50).
It should be noted that the structures of all target compounds 2–26 were characterized by IR, 1H and 13C NMR spectroscopic methods, and microanalysis data, and they fully corroborated the assumed structures.

3.2. In Vitro Biological Activity Characterization

The Anticancer Activity Characterization of Pyrrolidone Derivatives 2–26
After synthesizing and characterizing the pyrrolidone derivatives (compounds 2–26), we subsequently aimed to evaluate their in vitro anticancer activity utilizing the A549 non-small cell lung cancer cell line as a model. To identify the most promising pyrrolidone derivatives exhibiting anticancer properties, we initially exposed the A549 cells to a fixed concentration of 100 µM for each compound. The post-treatment cell viability was subsequently measured and compared to the cytotoxic effects induced by standard chemotherapy agents, including doxorubicin, cisplatin, and cytarabine (Figure 2). The following chemotherapy agents were chosen due to their use in clinical oncology alone or in combination to treat and manage various lung-derived cancers.
The initial testing revealed that both starting materials, carboxylic acid (compound 2) and methyl ester (compound 3), exhibited no significant anticancer activity against A549 cells, maintaining viabilities of 92.7% and 95.4%, respectively, after a 24-hour exposure. However, the transformation of the methyl ester to hydrazide (compound 4) resulted in a marked increase in anticancer activity, as evidenced by a reduced cell viability of 69.7%. The further incorporation of an unsubstituted or substituted isatin moiety into the hydrazide framework yielded compounds 5 and 6 (Scheme 1). Notably, compound 5 (R=H) demonstrated significant anticancer activity, decreasing A549 cell viability to 43.8%. In contrast, the substitution of the hydrogen radical with a bromine atom in compound 6 markedly enhanced the anticancer efficacy, reducing A549 viability to 27.5%. Furthermore, the anticancer activity of compound 6 was found to be significantly greater than that induced by cytarabine (AraC), which resulted in a cell viability of 53.4% (p < 0.05) (Figure 2).
The reaction of hydrazide 4 with various heterocyclic aldehydes yielded compounds that contained nitrofuran (compound 7) and nitrothiophene (compound 8) substitutions. Among these derivatives, the nitrofuran derivative (compound 7) exhibited a reduction in A549 viability to 67.8%. Conversely, the nitrothiophene derivative (compound 8) displayed particularly promising anticancer activity, with a notable decrease in A549 cell viability to 34.7% (Figure 2).
The further reaction of hydrazide 4 with various aromatic aldehydes yielded a series of compounds featuring aromatic substitutions (compounds 9–18). The incorporation of a phenyl group in compound 9 resulted in a reduction in A549 cell viability to 74.8%. Subsequent substitutions of halogen radicals at the para position of the phenyl ring led to the synthesis of compounds 10–12, which exhibited moderate anticancer activity. Specifically, compound 10, bearing a chlorine substituent at the para position, decreased A549 viability to 56.0%. Additionally, substitution of chlorine with bromine or fluorine in compounds 11 and 12 further reduced A549 viability to 52.5% and 38.4%, respectively. However, the introduction of a methoxy group at the para position in compound 13 diminished the anticancer activity, resulting in 74.1% viability. Furthermore, ortho- and para-halogen substitutions (R = 2,4-diFC₆H₃) did not enhance anticancer activity, as demonstrated by compound 14, which maintained a viability of 66.5% (Figure 2).
In contrast, the incorporation of a 2,4,6-trimethoxyphenyl substituent in compound 15 yielded favorable anticancer activity, reducing A549 viability to 37.8%. Subsequent substitutions of chlorines at the ortho and meta positions of the phenyl ring resulted in compounds 16 and 17, both exhibiting similar anticancer activity with viabilities of 66.1% and 66.3%, respectively. Conversely, the introduction of a 2-chloro-4-nitrophenyl substituent in compound 18 led to a reduction in A549 viability to 44.1% (Figure 2).
Following the exploration of the anticancer activity of pyrrolidone derivatives with aromatic substitutions, we aimed to investigate whether alkylation at the nitrogen position would enhance anticancer efficacy. To this end, ethyl iodide was reacted with pyrrolidone derivatives, yielding compounds 19–22 with N-ethyl substitutions. The alkylated compound 19, which contained an aromatic substituent (R = phenyl), resulted in a reduction in A549 viability to 72.5%, comparable to that of the non-alkylated derivative (compound 9). The alkylation of the para-chlorine substituted derivative produced compound 20, which reduced A549 viability to 67.9%. Interestingly, the alkylation of the bromine-containing derivative yielded compound 21, exhibiting moderate anticancer activity with a viability of 51.3%. Lastly, the N-alkylated derivative containing a 4-fluorophenyl substituent resulted in a reduced anticancer activity, maintaining A549 viability at 69.8% (Figure 2).
After investigating the effects of heterocyclic and aromatic substitutions, we further aimed to characterize the impact of incorporating nitrogen-containing heterocycles on anticancer activity. Compound 23, featuring a 3,5-dimethylpyrazole substituent, reduced A549 viability to 44.1%, while the 2,5-dimethylpyrrole derivative (compound 24) demonstrated weaker activity, resulting in a viability of 51.0%. Notably, derivatives containing oxadiazole thione (compound 25) and triazole thione (compound 26) cores exhibited the strongest anticancer activity, reducing A549 viability to 28.0% and 29.6%, respectively. The anticancer activity of compounds 25 and 26 was significantly higher than that of cytarabine (p < 0.05) (Figure 2).
After characterizing the structure-dependent anticancer activity of synthesized compounds, we then moved forward and selected the most promising candidates with significantly (p < 0.05) stronger anticancer activity than AraC and evaluated their dose–response kinetics in A549 cells. A549 cells were treated with increasing concentrations of the selected compounds (6, 25, and 26) or AraC, and cell viability was assessed post-treatment (Figure 3). All tested compounds demonstrated significant cytotoxic activity (p < 0.05) relative to the untreated control. Notably, the halogenated derivative (compound 6) exhibited cytotoxicity comparable to that of AraC (Figure 3). Additionally, the oxadiazole thione (compound 25) and triazole thione (compound 26) showed more potent cytotoxic effects than those induced by AraC.
Collectively, these data demonstrate that the pyrrolidone derivatives demonstrate structure-dependent anticancer activity against A549 cells in vitro. The most promising compounds demonstrated comparable or stronger anticancer activity than AraC, a standard FDA-approved chemotherapeutic agent. These pyrrolidone derivatives could serve as a starting scaffold for the future hit-to-lead optimization to enhance the in vitro anticancer activity.

4. Conclusions

New 5-oxo-1-(3,4,5-trimethoxyphenyl)-pyrrolidine-3-carboxylic acid derivatives were synthesized. The results of this study revealed that among the synthesized compounds, 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carbohydrazide derivatives having 7-bromo-2-oxoindolin-3-ylidene, 5-nitrothiophene, 1,3,4-oxadiazolethione, and 4-aminotriazolethione substituents demonstrated the highest anticancer activity against human A549 pulmonary epithelial cells. The research data show that the synthesized derivatives with the 3,4,5-trimethoxyphenyl)pyrrolidine framework exhibit structure-dependent anticancer activity against A549 cells in vitro and may be applied in future studies in the search for efficient anticancer agents. Further studies utilizing a broader range of cancer cell lines, coupled with a hit-to-lead optimization strategy, are necessary to develop more potent anticancer candidates suitable for early preclinical research. Additionally, in-depth investigations are essential to elucidate the mechanistic activity of these compounds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app142411784/s1. Figure S1: 1H NMR of compound 2, Figure S2: 13C NMR of compound 2, Figure S3: 1H NMR of compound 3, Figure S4: 13C NMR of compound 3, Figure S5: 1H NMR of compound 4, Figure S6: 13C NMR of compound 4, Figure S7: 1H NMR of compound 5, Figure S8: 13C NMR of compound 5, Figure S9: 1H NMR of compound 6, Figure S10: 13C NMR of compound 6, Figure S11: 1H NMR of compound 7, Figure S12: 13C NMR of compound 7, Figure S13: 1H NMR of compound 8, Figure S14: 13C NMR of compound 8, Figure S15: 1H NMR of compound 9, Figure S16: 13C NMR of compound 9, Figure S17: 1H NMR of compound 10, Figure S18: 13C NMR of compound 10, Figure S19: 1H NMR of compound 11, Figure S20: 13C NMR of compound 11, Figure S21: 1H NMR of compound 12, Figure S22: 13C NMR of compound 12, Figure S23: 1H NMR of compound 13, Figure S24: 13C NMR of compound 13, Figure S25: 1H NMR of compound 14, Figure S26: 13C NMR of compound 14, Figure S27: 1H NMR of compound 15, Figure S28: 13C NMR of compound 15, Figure S29: 1H NMR of compound 16, Figure S30: 13C NMR of compound 16, Figure S31: 1H NMR of compound 17, Figure S32: 13C NMR of compound 17, Figure S33: 1H NMR of compound 18, Figure S34: 13C NMR of compound 18, Figure S35: 1H NMR of compound 19, Figure S36: 13C NMR of compound 19, Figure S37: 1H NMR of compound 20, Figure S38: 13C NMR of compound 20, Figure S39: 1H NMR of compound 21, Figure S40: 13C NMR of compound 21, Figure S41: 1H NMR of compound 22, Figure S42: 13C NMR of compound 22, Figure S43: 1H NMR of compound 23, Figure S44: 13C NMR of compound 23, Figure S45: 1H NMR of compound 24, Figure S46: 13C NMR of compound 24, Figure S47: 1H NMR of compound 25, Figure S48: 13C NMR of compound 25, Figure S49: 1H NMR of compound 26, Figure S50: 13C NMR of compound 26, Figure S51: IR spectrum of compound 2, Figure S52: IR spectrum of compound 3, Figure S53: IR spectrum of compound 4, Figure S54: IR spectrum of compound 5, Figure S55: IR spectrum of compound 6, Figure S56: IR spectrum of compound 7, Figure S57: IR spectrum of compound 8, Figure S58: IR spectrum of compound 9, Figure S59: IR spectrum of compound 10, Figure S60: IR spectrum of compound 11, Figure S61: IR spectrum of compound 12, Figure S62: IR spectrum of compound 13, Figure S63: IR spectrum of compound 14, Figure S64: IR spectrum of compound 15, Figure S65: IR spectrum of compound 16, Figure S66: IR spectrum of compound 17, Figure S67: IR spectrum of compound 18, Figure S68: IR spectrum of compound 19, Figure S69: IR spectrum of compound 20, Figure S70: IR spectrum of compound 21, Figure S71: IR spectrum of compound 22, Figure S72: IR spectrum of compound 23, Figure S73: IR spectrum of compound 24, Figure S74: IR spectrum of compound 25, Figure S75: IR spectrum of compound 26.

Author Contributions

Conceptualization: V.P. and V.M.; methodology, P.K., B.S.-B., B.G., K.A., V.P. and V.M.; validation, B.S.-B., B.G., K.A., R.P. and V.P.; formal analysis, P.K., B.S.-B., B.G., K.A., E.M., A.G., E.N., R.P., V.P., R.G. and V.M.; investigation, P.K., B.S.-B., B.G., K.A., A.G., E.N., R.P. and R.G.; data curation, V.P. and V.M.; writing—original draft, P.K. and B.S.-B.; writing—review and editing, P.K., B.S.-B., E.M. and V.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article. Compounds are available upon request from the corresponding author.

Acknowledgments

We thank the supportive staff of Kaunas University of Technology and Weill Cornell Medicine of Cornell University, who assisted with this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 11784 g001
Figure 1. Structures of approved drugs.
Figure 1. Structures of approved drugs.
Applsci 14 11784 g001
Applsci 14 11784 sch001
Scheme 1. Synthesis and some chemical transformations of acid hydrazide 4. Reagents and conditions: (i) itaconic acid, H2O, Δ, 7 h; (ii) MeOH, H2SO4, Δ, 8 h; (iii) N2H4·H2O, i-PrOH, Δ, 3 h; (iv) the corresponding isatin, i-PrOH, Δ, 6.5–7 h; (v) the corresponding heterocyclic aldehyde, i-PrOH, Δ, 11 (7) or 7 (8) h; (vi) the corresponding aromatic aldehyde, i-PrOH, Δ, 3–8 h; (vii) the corresponding hydrazone 9–15, KOH, K2CO3, DMF, EtI (dropwise), r.t., stirring for 24 h, H2O. 5 R = H; 6 R = Br; 7 X = O; 8 X = S; 9, 19 Ar = C6H5; 10, 20 Ar = 4-ClC6H4; 11, 21 Ar = 4-BrC6H4; 12, 22 Ar = 4-FC6H4; 13 Ar = 4-CH3OC6H4; 14 Ar = 2,4-diFC6H3; 15 Ar = 3,4,5-tri(CH3O)C6H2; 16 Ar = 2-ClC6H4; 17 Ar = 3-ClC6H4; 18 Ar = 2-Cl-4-NO2C6H3.
Scheme 1. Synthesis and some chemical transformations of acid hydrazide 4. Reagents and conditions: (i) itaconic acid, H2O, Δ, 7 h; (ii) MeOH, H2SO4, Δ, 8 h; (iii) N2H4·H2O, i-PrOH, Δ, 3 h; (iv) the corresponding isatin, i-PrOH, Δ, 6.5–7 h; (v) the corresponding heterocyclic aldehyde, i-PrOH, Δ, 11 (7) or 7 (8) h; (vi) the corresponding aromatic aldehyde, i-PrOH, Δ, 3–8 h; (vii) the corresponding hydrazone 9–15, KOH, K2CO3, DMF, EtI (dropwise), r.t., stirring for 24 h, H2O. 5 R = H; 6 R = Br; 7 X = O; 8 X = S; 9, 19 Ar = C6H5; 10, 20 Ar = 4-ClC6H4; 11, 21 Ar = 4-BrC6H4; 12, 22 Ar = 4-FC6H4; 13 Ar = 4-CH3OC6H4; 14 Ar = 2,4-diFC6H3; 15 Ar = 3,4,5-tri(CH3O)C6H2; 16 Ar = 2-ClC6H4; 17 Ar = 3-ClC6H4; 18 Ar = 2-Cl-4-NO2C6H3.
Applsci 14 11784 sch001
Applsci 14 11784 sch002
Scheme 2. Synthesis of azole derivatives 23–26. Reagents and conditions: (i) pentane-2,4-dione, i-PrOH, Δ, 3 h; (ii) hexane-2,5-dione, i-PrOH, Δ, 3 h; (iii) KOH + MeOH, CS2, rt, stirring 15–20 min, 4 + MeOH, Δ, 21 h, H2O, aq. HCl (1:1) to pH 4; (iv) KOH, CS2, MeOH, Δ, 18 h, reduced pressure, Et2O, i-PrOH+1,4-dioxane + H2O, N2H4·H2O, Δ, 24 h, reduced pressure, H2O, aq. HCl (1:1) to pH 6.
Scheme 2. Synthesis of azole derivatives 23–26. Reagents and conditions: (i) pentane-2,4-dione, i-PrOH, Δ, 3 h; (ii) hexane-2,5-dione, i-PrOH, Δ, 3 h; (iii) KOH + MeOH, CS2, rt, stirring 15–20 min, 4 + MeOH, Δ, 21 h, H2O, aq. HCl (1:1) to pH 4; (iv) KOH, CS2, MeOH, Δ, 18 h, reduced pressure, Et2O, i-PrOH+1,4-dioxane + H2O, N2H4·H2O, Δ, 24 h, reduced pressure, H2O, aq. HCl (1:1) to pH 6.
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Figure 2. The in vitro anticancer activity of pyrrolidone derivatives 2–26 against A549 human non-small cell lung cancer cells. The cells were exposed to a fixed 100 µM concentration of compounds, or doxorubicin (DOX), cisplatin (CP), and citarabin (AraC), for 24 h, and then post-treatment viability was measured using an MTT assay. The line on the graph indicates a 50% inhibition threshold. The data are shown as mean ± SD of three experimental replicas. *—p < 0.05.
Figure 2. The in vitro anticancer activity of pyrrolidone derivatives 2–26 against A549 human non-small cell lung cancer cells. The cells were exposed to a fixed 100 µM concentration of compounds, or doxorubicin (DOX), cisplatin (CP), and citarabin (AraC), for 24 h, and then post-treatment viability was measured using an MTT assay. The line on the graph indicates a 50% inhibition threshold. The data are shown as mean ± SD of three experimental replicas. *—p < 0.05.
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Figure 3. The selected compounds 6, 25, and 26 demonstrate promising dose-dependent anticancer activity in the A549 human lung carcinoma cell line. The cells were exposed to increasing concentrations of compounds or cytarabine (AraC) for 24 h, and post-treatment viability was measured using an MTT assay. The data are shown as mean ± SD of three experimental replicas.
Figure 3. The selected compounds 6, 25, and 26 demonstrate promising dose-dependent anticancer activity in the A549 human lung carcinoma cell line. The cells were exposed to increasing concentrations of compounds or cytarabine (AraC) for 24 h, and post-treatment viability was measured using an MTT assay. The data are shown as mean ± SD of three experimental replicas.
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MDPI and ACS Style

Kavaliauskas, P.; Sapijanskaitė-Banevič, B.; Grybaitė, B.; Mickevičiūtė, E.; Anusevičius, K.; Garcia, A.; Naing, E.; Petraitienė, R.; Petraitis, V.; Grigalevičiūtė, R.; et al. Synthesis and In Vitro Anticancer Activity of Pyrrolidone Derivatives Bearing 3,4,5-Trimethoxyphenyl Moiety as a Promising Anticancer Scaffold. Appl. Sci. 2024, 14, 11784. https://doi.org/10.3390/app142411784

AMA Style

Kavaliauskas P, Sapijanskaitė-Banevič B, Grybaitė B, Mickevičiūtė E, Anusevičius K, Garcia A, Naing E, Petraitienė R, Petraitis V, Grigalevičiūtė R, et al. Synthesis and In Vitro Anticancer Activity of Pyrrolidone Derivatives Bearing 3,4,5-Trimethoxyphenyl Moiety as a Promising Anticancer Scaffold. Applied Sciences. 2024; 14(24):11784. https://doi.org/10.3390/app142411784

Chicago/Turabian Style

Kavaliauskas, Povilas, Birutė Sapijanskaitė-Banevič, Birutė Grybaitė, Eglė Mickevičiūtė, Kazimieras Anusevičius, Andrew Garcia, Ethan Naing, Rūta Petraitienė, Vidmantas Petraitis, Ramunė Grigalevičiūtė, and et al. 2024. "Synthesis and In Vitro Anticancer Activity of Pyrrolidone Derivatives Bearing 3,4,5-Trimethoxyphenyl Moiety as a Promising Anticancer Scaffold" Applied Sciences 14, no. 24: 11784. https://doi.org/10.3390/app142411784

APA Style

Kavaliauskas, P., Sapijanskaitė-Banevič, B., Grybaitė, B., Mickevičiūtė, E., Anusevičius, K., Garcia, A., Naing, E., Petraitienė, R., Petraitis, V., Grigalevičiūtė, R., & Mickevičius, V. (2024). Synthesis and In Vitro Anticancer Activity of Pyrrolidone Derivatives Bearing 3,4,5-Trimethoxyphenyl Moiety as a Promising Anticancer Scaffold. Applied Sciences, 14(24), 11784. https://doi.org/10.3390/app142411784

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