Synthesis and cytotoxic effects on HeLa cervical cancer cells of ( E )-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidine

Abstract

Seven (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidines were synthesized via a tandem Ugi 4CC/S_N cyclization sequence starting from a Baylis-Hillman-derived acid, primary amines, arylglyoxals, and isocyanides in one pot. In addition, the cytotoxicity of these compounds on the cervical cancer cell line HeLa was studied by MTT assay. The results showed that most of the compounds could inhibit the proliferation of HeLa cells significantly.

Introduction

Heterocyclic compounds are widely used in the fields of pesticides, medicinal chemistry, and industry because of their useful properties. Fluorine-containing compounds have been widely used as pharmaceuticals due to their excellent lipophilicity, hydrophobicity, and biological activities. Therefore, the synthesis of heterocyclic compounds containing a fluorine atom is of interest to drug researchers.

Heterocycles containing a pyrrolidinone are of importance because they have many applications in pharmaceuticals, chemicals, and other fields. ¹ They have significant antimuscarinic, ² antiviral, ³ antiepileptic, ⁴ and anti-HIV activities. ⁵ Atigadda et al. ⁶ reported a substituted 2-pyrrolidinone that was a low nanomolar inhibitor of neuraminidase. Wu and Feldkamp ⁷ utilized a pyrrolidinone as a scaffold in a novel selective inhibitor of matrix metalloproteinases (MMPs).

The classic synthetic methodology for the construction of pyrrolidinones is focused primarily on the cyclization of a dialkyl itaconate with different primary amines. ⁸ In addition, pyrrolidinones have been prepared by other methods, such as rearrangements of lactams, ⁹ radical addition cyclization reactions, cycloaddition reactions, and transition-metal-catalyzed intramolecular allylic alkylation reaction. ¹⁰

Studies showed that the alkaloids Rhopaladins A–D (see Figure 1), which exist in marine cysts, have significant cytotoxicity to human tumor cells. ¹¹ Rhopaladin B inhibited a cyclin-dependent kinase (CDK4) and tyrosine kinase C-erbB2 (IC₅₀ was 12.5 and 7.4 μg mL⁻¹, respectively),^12,13 in particular. The data show that C-erbB2 expression is a late event in cervical carcinogenesis. ¹⁴ Cancer incidence and mortality are rapidly growing worldwide. There were about 570,000 cases and 310,000 deaths worldwide in 2018. The incidence of cervical cancer ranked fourth and was the fourth leading cause of cancer in women. ¹⁵

Figure 1.

The strategy for the choice of 2-aroyl-4-arylidene-5-oxopyrrolidines as targets.

Results and discussion

We have previously synthesized 2-aroyl-4-arylidene-5-oxopyrrolidines via tandem Ugi and cyclization reactions (see Figure 1) ¹⁶ and found they are biological isochores of Rhopaladins because of the five-membered nitrogen heterocycles with arylidene and aroyl groups. In order to study the anti-cervical cancer activity of these compounds, we decided to synthesize fluorine-containing (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidines. Herein, we report the synthesis of seven (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidines via a tandem Ugi 4CC/S_N cyclization sequence starting from a Baylis-Hillman-derived acid, primary amines, arylglyoxals, and isocyanides in one pot. In addition, the cytotoxicity of these compounds to HeLa cells was studied by the MTT assay.

The optimized conditions ¹⁶ for the fluorine-containing Baylis-Hillman-derived acid 1, arylglyoxals 2, amines 3, and isocyanides 4 used Cs₂CO₃ as a base to adjust the pH of the solution to 6 during the reaction process (Scheme 1). All reactions proceeded efficiently and seven (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidines were obtained (see Table 1) in 79%–91% yields.

Scheme 1.

Synthesis of (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidines 6.

Table 1.

Synthesis of compound 6.

Product	Ar2	R1	R2	Yield (%) a
6a	4-ClC6H4	PhCH2	c-C6H11	89
6b	4-BrC6H4	PhCH2	c-C6H11	91
6c	4-NO2C6H4	PhCH2	c-C6H11	85
6d	4-ClC6H4	PhCH2	t-Bu	81
6e	4-BrC6H4	PhCH2	t-Bu	83
6f	4-NO2C6H4	PhCH2	t-Bu	83
6g	4-ClC6H4	i-Pr	c-C6H11	79

a

Isolated yields.

The structures of compounds 6a–g were confirmed by their spectroscopic data. For example, the ¹H NMR spectrum of 6a shows two doublets at δ 3.84 and δ 3.38 ppm due to the CH₂ of the pyrrolidine ring. The CONH signals are found at δ 6.46–5.93 ppm as singlets. The signals of the CH₂ appear at δ 5.16–4.92 and δ 4.56–4.48 ppm as two doublets. Signals attributable to the ArHs and vinyl-H are found at δ 8.21–7.06 ppm as multiplets and a singlet. The ¹³C NMR spectrum for 6a showed C=O carbons at δ 194.8, 170.4, and 166.8 ppm, and the C–F carbon at 162.8 ppm as a doublet. The quaternary carbon of the pyrrolidinone ring resonates at δ 74.7–73.7 ppm. The MS spectrum of 6a shows a molecular ion peak and M⁺ - 4-ClC₆H₄CO at m/z 544 and 405 with 2% and 44% abundance, respectively. The configuration of the double bond is known to be retained after the tandem Ugi and cyclization reactions. ¹⁶

The MTT assay was used to test the cytotoxic activity of the target compounds 6 in vitro. The inhibition of the compounds 6 on HeLa cell proliferation was tested under gradient concentration. In this experiment, cisplatin was used as the positive control. The results showed that there were four compounds, 6a, 6c, 6d, and 6f, which showed good inhibitory activity on the proliferation of cervical cancer HeLa cells. Their IC₅₀ values were 6.2, 5.4, 3.7, and 4.9 μmol L⁻¹, respectively, while the corresponding IC₅₀ value of cisplatin was 21.3 μmol L⁻¹ (see Table 2). Analysis of the structure–activity relationship shows that the aryl group Ar² has an influence on the antitumor activity of the compound. When the benzene ring of Ar² contains bromine, the antitumor activity was relatively poor, but the activity was better when the benzene ring contains a nitro group; the influence of groups of R¹ and R² are relatively small.

Table 2.

Toxicity of compound 6 on HeLa cells in vitro.

Compound	IC50 (μmol L−1)
6a	6.2
6b	40.7
6c	5.4
6d	3.7
6e	51.5
6f	4.9
6g	34.2
Cisplatin	21.3

Conclusion

Seven (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidines have been synthesized via a tandem Ugi 4CC/S_N cyclization starting from a Baylis-Hillman-derived acid, primary amines, arylglyoxals, and isocyanides in one pot. All are new compounds which have not been reported before. In the experimental procedure, the Ugi reaction intermediates do not need to be separated and purified, but directly used in the following reaction. The cytotoxicities of compounds 6 on HeLa cells in vitro were studied. It was found that 6a, 6c, 6d, and 6f had a good inhibitory activity on HeLa cell proliferation.

Experimental

Melting points were determined using a X-4 model apparatus and were uncorrected. MS were measured on a Finnigan Trace MS spectrometer. IR were recorded on a PE-983 infrared spectrometer as KBr pellets with absorption in cm⁻¹. NMR were recorded in CDCl₃ or [D₆] DMSO on a Varian Mercury 600 or 400 spectrometer and resonances relative to TMS. Elementary analyses were taken on a Vario EL III elementary analysis instrument.

General procedure for the synthesis of 6a–g

A mixture of 2-(bromomethyl)-3-(4-fluorobenzyl)-2-propenoic acid 1 (1 mmol) and primary amine 2 (1 mmol) was stirred in methanol (5 mL) at room temperature for 10 min and then isocyanide 4 (1 mmol) and the arylglyoxal 3 (1 mmol) were added consecutively to the solution. The mixture was stirred at room temperature and the pH value of the solution was adjusted to 6 using Cs₂CO₃ during the reaction process. After stirring for 12 h, the mixture was chilled and the resulted solid were then filtered, recrystallized from ether to give product compounds 6 as white solid.

(E)-1-Benzyl-2-(4-chlorobenzoyl)-N-cyclohexyl-4-(4-fluorobenzylidene)-5-oxopyrrolidine-2-carboxamide (6a): white solid (0.48 g, yield: 89%). m.p.: 193–194 °C. ¹H NMR (CDCl₃, 600 MHz): δ 7.70 (d, J = 7.2 Hz, 2H, ArH), 7.47 (s, 1H, =CH), 7.41–7.07 (m, 11H, ArH), 6.29 (s, 1H, NH), 5.16 (d, J = 16.2 Hz, 1H, NCH₂^a), 4.51 (d, J = 15.6 Hz, 1H, NCH₂^b), 3.84 (d, J = 17.4 Hz, 1H, CH₂^a), 3.40–3.37 (m, 2H, NCH and CH₂^b), 1.52–0.79 (m, 10H, 5CH₂). ¹³C NMR (CDCl₃, 100 MHz): δ 194.8, 170.4, 166.8, 162.8 (d, J = 250 Hz), 140.2, 136.3, 131.8, 131.5, 131.0, 130.5, 128.9, 128.3, 127.5, 127.2, 125.5, 116.0, 115.8, 73.9, 49.4,46.9, 35.3, 31.7, 25.1, 24.5. MS m/z (%): 544 (M⁺, 2), 405 (44), 280 (24), 139 (21), 91 (100). Anal. Calcd for C₃₂H₃₀ClFN₂O₃: C, 70.52; H, 5.55; N, 5.14; found: C, 70.47; H, 5.61; N, 5.07.

(E)-1-Benzyl-2-(4-bromobenzoyl)-N-cyclohexyl-4-(4-fluorobenzylidene)-5-oxopyrrolidine-2-carboxamide (6b): white solid (0.53 g, yield: 91%). m.p.: 197–198 °C. ¹H NMR (CDCl₃, 600 MHz): δ 7.60 (d, J = 9.0 Hz, 2H, ArH), 7.54 (s, 1H, =CH), 7.52–7.07 (m, 11H, ArH), 6.18 (s, 1H, NH), 5.16 (d, J = 16.2 Hz, 1H, NCH₂^a), 4.50 (d, J = 15.6 Hz, 1H, NCH₂^b), 3.80 (d, J = 17.4 Hz, 1H, CH₂^a), 3.40–3.37 (m, 2H, NCH and CH₂^b), 1.52–0.77 (m, 10H, 5CH₂). ¹³C NMR (CDCl₃, 100 MHz): δ 194.8, 170.3, 166.7, 162.8 (d, J = 250 Hz), 136.3, 132.1, 131.8, 131.5, 130.9, 130.5, 128.9, 128.3, 127.4, 127.1, 125.5, 116.0, 115.8, 74.0, 49.4, 46.8, 35.2, 31.7, 25.1, 24.5. MS m/z (%): 589 (M⁺+1, 2), 463 (10), 405 (24), 280 (21), 185 (16), 91 (100). Anal. Calcd for C₃₂H₃₀ BrFN₂O₃: C, 65.20; H, 5.13; N, 4.75; found: C, 65.24; H, 5.19; N, 4.68.

(E)-1-Benzyl-N-cyclohexyl-4-(4-fluorobenzylidene)-2-(4-nitrobenzoyl)-5-oxopyr-rolidine-2-carboxamide (6c): white solid (0.47 g, yield: 85%). m.p.: 201–202 °C. ¹H NMR (CDCl₃, 600 MHz): δ 8.19 (d, J = 6.6 Hz, 2H, ArH), 7.84 (d, J = 9.0 Hz, 2H, ArH), 7.52 (s, 1H, =CH), 7.41–7.08 (m, 9H, ArH), 6.46 (s, 1H, NH), 4.92 (d, J = 15.0 Hz, 1H, NCH₂^a), 4.56 (d, J = 15.6 Hz, 1H, NCH₂^b), 3.70 (d, J = 18.6 Hz, 1H, CH₂^a), 3.47–3.44 (m, 2H, NCH and CH₂^b), 1.68–0.82 (m, 10H, 5CH₂). ¹³C NMR (CDCl₃, 100 MHz): δ 195.1, 170.4, 167.2, 162.9 (d, J = 250 Hz), 149.9, 138.8, 136.2, 132.3, 131.7, 131.7, 129.9, 128.5, 128.1, 124.7, 123.5, 116.2, 116.0, 73.7, 49.4, 46.9, 35.5, 32.2, 25.1, 24.6. MS m/z (%): 555 (M⁺, 2), 430 (11), 405 (42), 280 (14), 150 (16), 91 (100). Anal. Calcd for C₃₂H₃₀FN₃O₅: C, 69.18; H, 5.44; N, 7.56; found: C, 69.13; H, 5.49; N, 7.51.

(E)-1-Benzyl-N-tert-butyl-2-(4-chlorobenzoyl)-4-(4-fluorobenzylidene)-5-oxopyrrolidine-2-carboxamide (6d): white solid (0.42 g, yield: 81%). m.p.: 212–213 °C. ¹H NMR (CDCl₃, 600 MHz): δ 7.74 (d, J = 6.6 Hz, 2H, ArH), 7.52 (s, 1H, =CH), 7.44–7.09 (m, 11H, ArH), 5.93 (s, 1H, NH), 5.12 (d, J = 15.6 Hz, 1H, NCH₂^a), 4.48 (d, J = 16.8 Hz, 1H, NCH₂^b), 3.92 (d, J = 17.4 Hz, 1H, CH₂^a), 3.37 (d, J = 17.4 Hz, 1H, CH₂^b), 0.99 (s, 9H, 3CH 3). ¹³C NMR (CDCl₃, 100 MHz): δ 195.0, 170.4, 166.2, 162.8 (d, J = 250 Hz), 140.3,136.5, 131.7, 131.5, 131.0, 130.5, 128.9, 128.5, 127.2, 125.5, 116.0, 115.8, 109.8, 74.7, 52.4, 46.9, 35.1, 27.6. MS m/z (%): 518 (M⁺, 2), 419 (18), 379 (29), 280 (24), 139 (26), 91 (100). Anal. Calcd for C₃₀H₂₈ClFN₂O₃: C, 69.43; H, 5.44; N, 5.40; found: C, 69.37; H, 5.49; N, 5.34.

(E)-1-Benzyl-2-(4-bromobenzoyl)-N-tert-butyl-4-(4-fluorobenzylidene)-5-oxopyrrolidine-2-Carboxamide (6e): white solid (0.46 g, yield: 83%). m.p.: 209–210 °C. ¹H NMR (CDCl₃, 600 MHz): δ 7.65 (d, J = 8.4 Hz, 2H, ArH), 7.55 (d, J = 8.4 Hz, 2H, ArH), 7.51 (s, 1H, =CH), 7.44 (d, J = 6.6 Hz, 2H, ArH), 7.22–7.07 (m, 7H, ArH), 5.94 (s, 1H, NH), 5.12 (d, J = 16.2 Hz, 1H, NCH₂^a), 4.48 (d, J = 16.2 Hz, 1H, NCH₂^b), 3.92 (d, J = 17.4 Hz, 1H, CH₂^a), 3.37 (d, J = 17.4 Hz, 1H, CH₂^b), 0.99 (s, 9H, 3CH₃). ¹³C NMR (CDCl₃, 100 MHz): δ 195.2, 170.4, 166.1, 162.8 (d, J = 250 Hz), 136.5, 132.1, 131.9, 131.4, 131.6, 131.0, 130.6, 129.1, 128.5, 127.1, 125.5, 116.0, 115.8, 74.7, 52.4, 46.9, 35.2, 35.1, 27.5, 27.2. MS m/z (%): 563(M⁺+1, 4), 465 (18), 379 (33), 280 (21), 183 (23), 91 (100). Anal. Calcd for C₃₀H₂₈BrFN₂O₃: C, 63.95; H, 5.01; N, 4.97; found: C, 63.89; H, 5.06; N, 4.92.

(E)-1-Benzyl-N-tert-butyl-4-(4-fluorobenzylidene)-2-(4-nitrobenzoyl)-5-oxopyrrolidine-2-carboxamide (6f): white solid (0.44 g, yield: 83%). m.p.: 225–226 °C. ¹H NMR (CDCl₃, 600 MHz): δ 8.21 (d, J = 8.4 Hz, 2H, ArH), 7.87 (d, J = 8.4 Hz, 2H, ArH), 7.57 (s, 1H, =CH), 7.45 (d, J = 6.6 Hz, 2H, ArH), 7.20–7.09 (m, 7H, ArH), 6.20 (s, 1H, NH), 4.96 (d, J = 16.2 Hz, 1H, NCH₂^a), 4.55 (d, J = 15.6 Hz, 1H, NCH₂^b), 3.73 (d, J = 17.4 Hz, 1H, CH₂^a), 3.46 (d, J = 18.6 Hz, 1H, CH₂^b), 0.80 (s, 9H, 3CH₃). ¹³C NMR (CDCl₃, 100 MHz): δ 195.5, 170.6, 166.7, 162.9 (d, J = 250 Hz), 150.0, 138.7, 136.3, 132.2, 131.7, 130.8, 130.0, 128.6, 127.7, 124.7, 123.5, 116.1, 115.9, 74.4, 52.5, 47.0, 35.5, 27.7. MS m/z (%): 529 (M⁺, 9), 412 (13), 379 (12), 280 (21), 183 (16), 150 (23), 91 (100). Anal. Calcd for C₃₀H₂₈BrFN₂O₃: C, 68.04; H, 5.33; N, 7.93; found: C, 68.09; H, 5.39; N, 7.85.

(E)-2-(4-Chlorobenzoyl)-N-cyclohexyl-4-(4-fluorobenzylidene)-1-isopropyl-5-oxopyrrolidine-2-carboxamide (6g): white solid (0.39 g, yield: 79%). m.p.: 225–226 °C. ¹H NMR (CDCl₃, 600 MHz): δ 7.80 (d, J = 7.8 Hz, 2H, ArH), 7.46–7.06 (m, 7H, =CH and ArH), 6.27 (s, 1H, NH), 3.82–7.06 (m, 4H, NCH NCH₂ and CH₂^a), 3.22 (d, J = 18.0 Hz, 1H, CH₂^b), 1.94–1.11 (m, 15H, 6CH₂ and CH₃). ¹³C NMR (CDCl₃, 100 MHz): δ 195.3, 169.0, 167.6, 162.6 (d, J = 250 Hz), 140.2, 131.8, 131.4, 130.5, 129.7, 129.0, 127.2, 115.9, 115.7,74.7, 58.1, 49.1, 35.5, 32.1, 25.1, 24.6, 19.7, 18.2. MS m/z (%): 496 (M⁺, 4), 370 (40), 357 (95), 315(61), 216 (47), 190 (76), 139 (100), 55 (16). Anal. Calcd for C₂₈H₃₀ClFN₂O₃: C, 67.67; H, 6.08; N, 5.64; found: C, 67.61; H, 6.14; N, 5.59.

MTT assay

Cell proliferation was assessed using the MTT method. ¹⁷ The evaluation of cell population growth is based on the capability of living cells to reduce the yellow product MTT to a blue product, formazan, by a reduction reaction occurring in the mitochondria. Cervical carcinoma cell line HeLa was cultured in Dulbecco’s Modified Eagle Medium (DMEM) which contained 10% fetal bovine serum (FBS, Gibco, USA). The cells were grown in an incubator with a humidified atmosphere of 5% CO₂ at 37 °C. HeLa cells in logarithmic growth phase were digested with 0.25% Trypsin EDTA solution. The cells were incubated for 24 h in 96-well plates at a density of 5 × 10³ cells/well. The cells were treated with varying concentrations of compound 6 (see Table 2) for 48 h, cisplatin was used as positive control, and cultured in the same medium containing 0.5 g mL⁻¹ MTT for 4 h. Then, the generating formazan crystals were dissolved in dimethyl sulfoxide (150 µL). The absorbance at 490 nm (A490 nm) was measured using an enzyme-labeled meter (BioTEK INC, Biotek MQX200). The effect of compound 6 treatment on cell viability was evaluated relative to the readings of control cells. The A490 nm represented the number of surviving cells.

Supplemental Material

Supporting_Information – Supplemental material for Synthesis and cytotoxic effects on HeLa cervical cancer cells of (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidine

Supplemental material, Supporting_Information for Synthesis and cytotoxic effects on HeLa cervical cancer cells of (E)-2-aroyl-4-(4-fluorobenzylidene)-5-oxopyrrolidine by Hongmei Wang, XiuLian Zhu, Shouheng Deng, Qinhua Chen and Xiaohua Zeng in Journal of Chemical Research