Synthesis of Novel Pyrazole-Oxindole Conjugates with Cytotoxicity in Human Cancer Cells via Apoptosis

Abstract

A novel series of pyrazole-oxindole conjugates were prepared and characterized as potential cytotoxic agents by FTIR, NMR and HRMS. The cytotoxic activity of these compounds was tested in the Jurkat acute T cell leukemia, CEM acute lymphoblastic leukemia, MCF10A mammary epithelial and MDA-MB 231 triple negative breast cancer cell line. Among the tested conjugates, 5-methyl-3-((3-(1-phenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6h emerged as the most cytotoxic with a CC₅₀of 4.36 +/− 0.2 μM against Jurkat cells. The mechanism of cell death induced by 6h was investigated through the Annexin V-FITC assay via flow cytometry. Reactive oxygen species (ROS) accumulation, mitochondrial health and the cell cycle progression were also evaluated in cells exposed to 6h. Results demonstrated that 6h induces apoptosis in a dose-response manner, without generating ROS and/or altering mitochondrial health. In addition, 6h disrupted the cell cycle distribution causing an increase in DNA fragmentation (Sub G0-G1), and an arrest in the G0-G1 phase. Taken together, the 6h compound revealed a strong potential as an antineoplastic agent evidenced by its cytotoxicity in leukemia cells, the activation of apoptosis and restriction of the cell cycle progression.

Graphical Abstract

Introduction

Cancer is the second leading cause of death worldwide. In 2020 approximately 10 million deaths were attributed to cancer.^[1] However, many cancers are curable if are promptly detected and treated adequately.^[1]

Nitrogen-based heterocyclic compounds are present in most biologically active natural products and are commonly found in traditional medications.^[2,3] Nearly 75% of FDA approved drugs are nitrogen- and sulfur-based heterocyclic compounds.^[4] Therefore, they have become attractive targets to medicinal chemists for drug design and discovery. For instance, indolin-2,3-dione based compounds are a promising class of heterocyclic compounds with diverse pharmacological activities.^[5] The C-3 carbon of indolin-2,3-dione is a highly reactive group[6]. Nucleophilic addition, condensation and spiroannulation reactions at C-3 of indolin-2,3-dione lead to 2-oxindole derivatives.^[6]

Moreover, the pyrazole and indolin-2-one based compounds have presented extensive biological properties, such as antitumoral,^[7,8,9,10] anti-inflammatory,^[11] and antimicrobial activities.^[11,12,13] Similarly the 2-oxindole derivatives have been previously described as cytotoxic,^[14,15] antimicrobial,^[16] antiviral,^[17] Ligase-1^[18] and BCL₂ inhibitors,^[19,20] and as anticancer agents^[21,22] (Figure 1). The objective of the present investigation was to synthesize and characterize pyrazoles with 2-oxindole conjugates (6a-6k) as anti-cancer agents.

Figure 1.

Structures of anticancer pyrazole and 2-oxindole derivatives

Results and discussion

Chemistry

1-aryl-2-(1-arylethylidene)hydrazines (3) were prepared by reacting various acetophenones (1) with aryl hydrazine (2) in alcohol. 1,3-Diaryl-1H-pyrazole-4-carbaldehydes (4) were prepared by Vilsmeier–Haack reaction. The title compounds (6) 3-((1,3-diaryl-1H-pyrazole-4-yl)methylene)indolin-2-ones were reported in Scheme 1 were prepared by refluxing various 2-oxindoles (5) with 1,3-diaryl-1H-pyrazole-4-carbaldehydes (4) in methyl alcohol with catalytic amount of piperidine. The compounds were characterized by Fourier Transform Infra Red (FTIR), ¹H and ¹³C Nuclear Magnetic Resonance (NMR) & High Resolution Mass Spectrometry (HRMS) confirms the authenticity of the compounds.

Scheme 1.

Synthesis of 3-((1,3-diaryl-1H-pyrazol-4-yl)methylene)indolin-2-one.

Reagents and conditions: a) Ethyl alcohol, reflux b) Dimethylformamide and Phosphorous oxychloride c) Methyl alcohol and piperidine.

The formation pyrazole-4-carbaldehydes (4) by the reaction were confirmed by its FTIR spectra, which displayed the presence of carbaldehyde (-CHO) and absence of carbonyl (C=O) and amine (-NH₂) stretching. In ¹H NMR spectra, peak for –CHO exhibited between ⸹ 9.98–10.02 ppm. Structures of 3-((1,3-diaryl-1H-pyrazole-4-yl)methylene)indolin-2-ones (6) were established by the absence of carbaldehyde (-CHO) in their respective FTIR spectrums. The presence of proton for –NH in the compounds 3-((1,3-diaryl-1H-pyrazole-4-yl)methylene)indolin-2-ones (6) were exhibited between ⸹ 10.67–10.53 ppm and aromatic protons between ⸹ 8.43–6.74 ppm and for –OCH₃ around ⸹ 3.87–3.85 ppm and for –CH₃ between ⸹ 2.42–2.26 ppm. In the ¹³C spectra of 3-((1,3-diaryl-1H-pyrazole-4-yl)methylene)indolin-2-ones (6) showed peaks around ⸹ 168 ppm for C=O of indolin-2-one, and peaks between ⸹ 159.75–109.46 ppm for aromatic carbons, peak at ⸹ 55.27 ppm for –OCH₃ and for peaks between ⸹ 20.92–20.67 ppm for –CH₃. The high-resolution mass spectrometry (HRMS) of these compounds further confirmed the assigned structures.

Biology

Cytotoxicity assays revealed 6h as the most potent of the pyrazole-oxindole compounds.

The DNS assay was performed to evaluate the cytotoxic activity of the synthesized compounds in Jurkat cells. 6h and 6j emerged as the most cytotoxic compounds after 48 h of exposure to the cells with 6h being the most active with a CC₅₀ of 4.36 +/− 0.2 μM and 6j with a CC₅₀ of 7.77μM (Table 1). The other synthesized compounds (6c, 6e, 6f, 6g, 6j and 6k) displayed minor cytotoxicity with CC₅₀’s estimated above 20 μM. Moreover, three of the compounds (6a, 6b and 6d) were unable to be examined at concentrations higher than 1 μM due to poor solubility (Table 1). The most cytotoxically active compound 6h was selected for further characterization and examination of mode of action.

Table 1.

Cytotoxicity of the synthesized compounds determined in cancer cells after 48 h and 72 h of exposure.

CC50 (μM)**
Compound	MCF-10A	MDA-MB-231	CEM	Jurkat
	72 h		48 h
6a	>1*	>1*	>1*	>1*
6b	>1*	>1*	>1*	>1*
6c	>20	>20	>20	>20
6d	>1*	>1*	>1*	>1
6e	>20	>20	>20	>20
6f	>20	>20	>20	>20
6g	13.02±0.49	>20	>20	>20
6h	14.75±1.48	>20	7.14±0.92	4.36±0.2
6i	9.37±0.42	>20	>20	>20
6j	8.59±0.31	>20	7.66±0.19	7.77±0.21
6k	>20	>20	>20	>20

^*Concentrations higher than 1μM formed crystals and were excluded from the experiment

^**The CC₅₀ indicates the cytotoxic concentration at which 50% of the population dies.

6h induces apoptosis in Jurkat cells.

To elucidate the mechanism of cell death induced by 6h, phosphatidylserine (PS) externalization was evaluated on Jurkat cells after 24 h of exposure to the compound, by means of the annexin V-FITC PI assay and flow cytometry. PS externalization is a common feature of apoptosis, where PS is transferred from the cytoplasmic space to the cell surface on cells undergoing apoptosis.^[23] Results demonstrated that 6h induces a significant amount of PS externalization in a dose-response trend when compared to vehicle control (**p< 0.01; Figure 1A). These data suggest that 6h triggers apoptosis as the mechanism to induce cell death.

6h does not promote Reactive Oxygen Species (ROS) production in Jurkat cells.

To investigate if apoptosis induced by 6h involves ROS overproduction and accumulation, ROS was quantified in cells treated with 6h for 18 h using the Carboxy-H₂DCFDA oxidative stress indicator and measured via flow cytometry. When ROS is overproduced in cells oxidation occurs which can cause cellular damage of Deoxyribonucleic acid (DNA), proteins and other macromolecules leading to the activation of apoptosis.^[24] Our results showed that 6h does not activate ROS overproduction and/or accumulation in Jurkat cells (Figure 1B) when compared to solvent control dimethyl sulfoxide (DMSO). As expected, the H₂O₂ positive control displayed the highest amount of accumulated ROS (***p < 0.001). These results suggest that 6h activates pathways that are ROS-independent.

Mitochondrial health is maintained after 6h exposure in Jurkat cells.

Under physiological and pathological circumstances, the induction of apoptosis is generally associated with ROS and mitochondria.^[24] The role of mitochondria in apoptosis has been well documented and revealed that B-cell lymphoma-2 (BCL-2) family proteins can form open pores in the outer mitochondrial membrane leading to the exit of cytochrome c, causing the mitochondrial membrane potential (ΔΨm) to collapse, an event that will irremediably bring the cell to apoptosis.^[25] To examine if 6h was able to provoke mitochondrial damage/depolarization, the ΔΨm was evaluated in cells exposed to 6h for 5 h by means of the MitoProbe JC-1 assay kit and flow cytometry. Our data revealed that 6h does not cause mitochondrial depolarization in Jurkat cells, displaying low percentages (18.3% and 18.7% for CC₅₀ and 2x CC₅₀, respectively) similar to untreated (13.2%) and solvent (20.9%) controls (Figure 1C). These data suggest that 6h apoptosis induction may be mediated by death receptors at the cell surface through the extrinsic apoptotic pathway.

6h impairs cell cycle progression in Jurkat cells.

Analysis of the potential effects of 6h on the cell cycle progression was evaluated in cells treated with 6h for 72 h using a Nuclear Isolation Medium containing 4’,6’-diamidino-2-phenylindole DAPI (NIM-DAPI) and flow cytometry. Treatments of CC₂₅ (2.18 μM) and CC₅₀ (4.36 μM) concentrations of 6h were included in this assay with the purpose of avoiding excessive DNA fragmentation that could interfere with the distribution of the cell cycle phases. A moderate but significant increase in DNA fragmentation (sub G0/G1) was observed in 6h (p< 0.01) treated cells as well as in the H₂O₂ (*p< 0.05) and etoposide (***p < 0.001) controls (Figure 2A). Additionally, a significant cell cycle arrest was seen in the G0/G1 subpopulation of cells (**p < 0.01, ***p< 0.001; Figure 2B). A small but also significant reduction in the G2/M (tetraploid) phase was observed (***p < 0.001; Figure 2D), likely due to the increases seen in the sub-G0/G1 and G0/G1 subpopulations. And as expected, etoposide displayed a significant arrest in the S hyperdiploid phase. Hence, our data demonstrated that 6h alters the normal cell cycle distribution by an increase in the sub-G0/G1 apoptotic subpopulation and an arrest in the G0/G1 diploid phase.

Figure 2.

6h triggers apoptosis in a dose-response fashion, without originating ROS and preserving mitochondrial membrane potential in acute T cell leukemia cells. A) 6h induced significant PS externalization after 24 h of exposure. Jurkat cells were treated with CC₅₀ (4.36 μM) and 2x CC₅₀ (8.72 μM) concentrations, stained with annexin V-FITC and PI and read via flow cytometry. Cells that resulted positive for FITC were considered as early apoptotic, and those positive for FITC and PI were annotated as late apoptotic. Bar graph display percentages of total apoptosis values (early and late apoptosis; red bars). Additionally, PI positive cells were accounted as necrotic (grey bars). B) 6h does not cause ROS accumulation in Jurkat cells after 18 h of exposure. Treated cells were stained with a Carboxy H₂-DCFDA oxidative stress indicator and analyzed by flow cytometry. No differences were observed in the 6h treated cells when compared to the DMSO control. C) Mitochondrial health was maintained after 6h treatment in Jurkat cells. Mitochondrial membrane potential was evaluated after 5 h of exposure to 6h using the Mito-Probe JC-1 assay and flow cytometry. No significant increase in cells with depolarized mitochondria was observed for the 6h treatment. H₂O₂, untreated and DMSO were included as controls in A, B and C. Two-tailed student’s paired t tests were accomplished to obtain statistical analyses in these series of experiments. P-values are annotated with asterisks, (*p< 0.05, **p < 0.01 and ***p < 0.001) and represent treatments compared to the solvent control (DMSO).

Conclusions

This study revealed a novel pyrazole-oxindole conjugate 6h as an anti-cancer agent that demonstrated cytotoxicity against acute T cell leukemia through the apoptotic pathway. 6h induced apoptosis in a dose-response manner without inducing oxidative stress and/or altering the mitochondrial membrane potential, therefore, involving mitochondria-independent pathways of apoptosis. Also, this compound altered the cell cycle distribution by arresting cells in the G0/G1 phase and by a significant increment in DNA fragmentation (Sub G0/G1). These pyrazole-oxindole conjugates represent a potential novel series of anti-cancer drugs with the possibility to treat acute T cell leukemia.

Materials and Methods

Chemicals and reagents

Silica gel plates were used for TLC by using CHCl₃/MeOH in various proportions. The FTIR spectra were recorded in KBr on a Jasco 430+; the ¹H and ¹³CNMR spectra were recorded in CDCl₃/ DMSO-d₆ on a Bruker (400 / 100 MHz), and J values were reported in hertz (Hz). The intermediate 1-phenyl-2-(1-arylethylidene)hydrazine (3), 3-aryl-1-phenyl-1H-pyrazole-4-carbaldehyde (4) and various 2-oxindoles (5) were prepared as per literature.^[26]

Synthesis of compounds

General procedure for the preparation of 3-((1,3-diaryl-1H-pyrazol-4-yl)methylene) indolin-2-one (6)

1,3-diaryl-1H-pyrazole-4-carbaldehyde (0.001 M) (4) and indolin-2-one (0.001 M) (5) were refluxed in methyl alcohol (15 ml) in presence of catalytic amount of piperidine. After completion of reaction, filtered the product and purified from suitable solvent.

3-((1,3-Diphenyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6a

Yield 61 %; m.p.262–265 °C; IR_KBr(cm⁻¹) ν_max: 3160, 3055, 3021, 2940, 2889, 2824, 1685, 1608, 1526, 1505, 1470, 1385, 1356, 1201. 1H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.67 (s, 1H, NH), 9.90 (s, 1H), 7.92 (d, 2H, J=9.6), 7.74 (d, 2H, J=9.6), 7.60 (tm, 6H, J=16), 7.46–7.42 (m, 2H), 7.20 (t, 1H, J=16), 6.96 (t, 1H, J=16), 6.88 (d, 1H, J=7.6). ¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.56, 154.66, 140.25, 138.98, 131.81, 131.64, 129.87, 129.05, 128.93, 128.84, 128.49, 127.36, 124.46, 124.36, 123.99, 121.08, 119.15, 119.10, 115.43, 109.44. HR-MS [C₂₄H₁₇N₃O]+: 364.1446; Calc. 363.4200.

3-((1-Phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6b

Yield 63%; m.p.285–287 °C; IR_KBr (cm⁻¹) ν_max: 3162, 3068, 3021, 2937, 2889, 2825, 1685, 1616, 1600, 1525, 1509, 1469, 1380, 1349, 1202.1H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.65 (s, 1H, -NH), 9.87 (s, 1H), 7.89 (d, 2H, J=8.4), 7.61–7.56 (m, 4H, ar), 7.49 (s, 1H), 7.44–7.38 (m, 4H), 7.19 (t, 1H, J=16.4), 6.95 (t, 1H, J=16), 6.86 (d, 1H, J=7.6), 2.41 (s, 3H, -CH₃). ¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.58, 154.72, 140.22, 139.00, 138.33, 131.73, 129.85, 129.51, 128.95, 128.79, 128.43, 127.29, 124.59, 124.40, 123.80, 121.07, 119.06, 115.39, 109.43, 20.92. HR-MS [C₂₅H₁₉N₃O]+: 378.1604; Calc. 377.4470.

3-((3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)indolin-2-one 6c

Yield 60%; IR_KBr (cm⁻¹) ν_max: 3158, 3079, 3032, 2964, 2898, 2834, 1701, 1600, 1524, 1465, 1348, 1205. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm):10.67 (s, 1H, NH), 9.88 (s, 1H), 8.43 (d, 2H, J=8.8), 8.03 (d, 2H, J=8.8), 7.92 (d, 2H, J=8.8), 7.62–7.55 (m, 4H), 7.45 (t, 1H, J=14.8), 7.21 (t, 1H, J=15.6), 6.96 (t, 1H, J=15.6), 6.86 (d, 1H, J=7.6).¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.48, 152.20, 147.39, 140.43, 138.82, 132.27, 130.08, 129.94, 129.73, 128.80, 127.72, 125.06, 124.59, 124.15, 123.74, 121.09, 119.80, 119.29, 119.22, 115.93, 109.46. HR-MS [C₂₄H₁₆N₄O₃]^-: 407.1146; Calc. 408.4170.

3-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)indolin-2-one 6d

Yield 63%; IR_KBr (cm⁻¹) ν_max: 3164, 3072, 3025, 2936, 2890, 2835, 1685, 1604, 1530, 1508, 1489, 1382, 1352, 1207. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.66 (s, 1H, NH), 9.87 (s, 1H), 7.89 (d, 2H, J=9.2), 7.65 (d, 2H, J=8.4), 7.58 (t, 2H, J=16), 7.49 (s, 1H), 7.46–7.39 (m, 2H, ar), 7.21–7.13 (td, 3H, J=16, 8.8), 6.95 (t, 1H, J=16), 6.86 (d, 1H, J=7.6), 3.85 (s, 3H, -OCH₃).¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.59, 159.79, 154.57, 140.19, 139.00, 131.66, 130.36, 129.84, 128.40, 127.24, 124.71, 124.42, 123.94, 123.68, 121.04, 119.09, 119.01, 115.31, 114.39, 109.41, 55.27. HR-MS [C₂₅H₁₉N₃O₂]⁺: 394.1559; Calc. 393.4460.

3-((3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)indolin-2-one 6e

Yield 65%; IR_KBr (cm⁻¹) ν_max: 3160, 3072, 3034, 2944, 2901, 2842, 1700, 1685, 1619, 1605, 1521, 1504, 1469, 1384, 1350, 1208. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.65 (s, 1H, NH), 9.87 (s, 1H0, 7.89 (d, 2H, J=8.4), 7.74 (d, 2H, J=8.4), 7.65 (dt, 4H, J=8.8, 16), 7.49 (s, 1H), 7.43 (t, 1H, J=13.4), 7.19 (t, 1H, J=16), 6.95 (t, 1H, J=16), 6.86 (d, 1H, J=7.6). HR-MS [C₂₄H₁₆ClN₃O]⁺: 398.1508; Calc. 397.8620.

3-((3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)indolin-2-one 6f

Yield 58%; m.p.>300 °C; IR_KBr (cm⁻¹) ν_max: 3162, 3076, 3027, 2944, 2895, 2833, 1727, 1696, 1615, 1531, 1504, 1468, 1367, 1343, 1210. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.63 (s, 1H, NH), 9.94 (s, 1H), 8.41 (s, 1H, Coumarin-H), 7.89–7.86 (m, 3H, ar), 7.73–7.69 (m, 1H, ar), 7.65–7.58 (m, 4H, ar), 7.53 (d, 1H, J=8), 7.45–7.41(m, 2H, ar), 7.17 (t, 1H, J=16.4), 6.92 (t, 1H, J=16), 6.85 (d, 1H, J=7.6). HR-MS [C₂₇H₁₇N₃O₃]⁺: 432.1346; Calc. 431.4510.

3-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-5-methylindolin-2-one 6g

Yield 61%; m.p.>300 °C; IR_KBr (cm⁻¹) ν_max: 3242, 3159, 3070, 3025, 2940, 2912, 2852, 1691, 1661, 1614, 1530, 1503, 1488, 1385, 1350, 1195. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm):10.59 (s, 1H, NH), 9.90 (s, 1H), 7.91 (d, 2H, J=7.2), 7.73–7.43 (m, 9H, ar), 7.26 (s, 1H), 7.02 (d, 1H, J=8), 6.76 (d, 1H, J=7.2), 2.26 (s, 3H, -CH₃).¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.63, 154.60, 138.96, 138.02, 131.74, 131.66, 129.80, 129.01, 128.94, 128.89, 128.78, 127.28, 124.35, 124.22, 124.01, 119.51, 119.04, 115.41, 109.18, 20.67. HR-MS [C₂₅H₁₉N₃O]⁺: 378.1608; Calc. 377.4470.

5-methyl-3-((3-(1-phenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6h

Yield 60%; m.p.>300 °C; IR_KBr (cm⁻¹) ν_max: 3160, 3050, 3025, 2914, 2855, 1684, 1600, 1532, 1506, 1488, 1382, 1345, 1205. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.58 (s, 1H, NH), 9.88 (s, 1H), 7.89–7.87 (m, 2H, ar), 7.59–7.26 (m, 9H, ar), 7.01–6.99 (m, 1H, ar), 6.76–6.74 (m, 1H, ar), 2.42 (s, 3H, -CH₃), 2.26 (s, 3H, -CH₃).¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.65, 154.64, 138.99, 138.26, 137.99, 131.69, 129.79, 129.47, 128.89, 128.81, 127.22, 124.39, 124.15, 124.06, 119.48, 119.02, 115.35, 109.16, 20.87, 20.68. HR-MS [C₂₆H₂₁N₃O]⁺: 392.1760; Calc. 391.4740.

3-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-5-methylindolin-2-one 6i

Yield 55%; m.p.>300 °C; IR_KBr (cm⁻¹) ν_max: 3167, 3069, 3024, 2966, 2817, 2835, 1682, 1626, 1601, 1530, 1508, 1455, 1377, 1338, 1208. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm):10.54 (s, 1H, NH), 9.88 (s, 1H), 7.89 (d, 2H, J=7.6), 7.66 (d, 2H,J=8.4), 7.59 (t, 2H, J=16), 7.45–7.40 (m, 2H, ar), 7.28 (s, 1H, ar), 7.17 (d, 2H, J=8.8), 7.01 (d, 1H, J=7.6), 6.76 (d, 1H, J=7.6), 3.87 (s, 3H, -OCH₃), 2.27 (s, 3H, -CH₃).¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.66, 159.75, 154.51, 138.99, 137.96, 131.61, 130.31, 129.79, 128.86, 127.17, 124.41, 124.27, 123.97, 119.46, 118.97, 115.28, 114.38, 109.16, 20.69. HR-MS [C₂₆H₂₁N₃O₂]⁺: 408.1716; Calc. 407.4730.

3-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-5-methylindolin-2-one 6j

Yield 58%; m.p.>300 °C; IR_KBr (cm⁻¹) ν_max: 3164, 3070, 3018, 2947, 2914, 2832, 1683, 1600, 1524, 1505, 1488, 1379, 1342, 1204. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.55 (s, 1H, NH), 9.88 (s, 1H), 7.90 (d, 2H, J=8.4), 7.76 (d, 2H, J=8.4), 7.67 (d, 2H, J=8.4), 7.60 (t, 2H, J=16), 7.45–7.43 (m, 2H, ar), 7.35 (s, 1H), 7.02 (d, 1H, J=7.6), 6.76 (d, 1H, J=8), 2.27 (s, 3H, -CH₃). ¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.60, 153.30, 138.90, 138.05, 133.62, 131.87, 130.69, 130.56, 129.82, 128.98, 127.37, 124.58, 124.30, 123.71, 119.81, 119.09, 115.45, 109.15, 20.67. HR-MS [C₂₅H₁₈ClN₃O]⁺: 412.1214; Calc. 411.8890.

5-methyl-3-((3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)indolin-2-one 6k

Yield 59%; m.p.>300 °C; IR_KBr (cm⁻¹) ν_max: 3166, 3072, 3021, 2923, 2862, 2818, 1728, 1692, 1610, 1531, 1507, 1489, 1341, 1212. ¹H NMR (400 MHz, DMSO-d₆, ⸹/ppm): 10.53 (s, 1H, NH), 9.95 (s, 1H), 8.42 (s, 1H, Coum-H), 7.90 (d, 3H, J=8.4), 7.72 (t, 1H, J=15.6), 7.63–7.59 (m, 3H, ar), 7.55 (d, 1H, J=8.4), 7.46–7.42 (m, 3H, ar), 7.00 (d, 1H, J=7.6), 6.74 (d, 1H, J=7.6), 2.26 (s, 3H, -CH₃).¹³C NMR (100 MHz, DMSO-d₆, ⸹/ppm): 167.82, 159.62, 153.83, 150.00, 144.13, 138.86, 137.90, 132.41, 131.61, 129.85, 129.64, 128.93, 128.79, 127.42, 125.26, 124.68, 123.64, 120.30, 120.08, 119.14, 119.06, 117.57, 116.24, 109.02, 20.72. HR-MS [C₂₈H₁₉N₃O₃]⁺: 446.1507; Calc. 445.4780.

Bio-evaluations

Cell culture conditions

The Jurkat acute T cell leukemia and CEM acute lymphoblastic leukemia cell lines were grown in RPMI-1640 culture medium (Hyclone, Logan, UT, USA), whereas the MBA-MB-231 triple-negative breast cancer cell line was grown in DMEM (CORNING, Corning, NY, USA), both mediums were supplemented with 10% heat-inactivated fetal bovine serum (Peak Serum, CO, USA) 100 u/mL of penicillin, and 100 μg/mL of streptomycin (Lonza, Walkersville, MD, USA). In addition, the MCF-10A mammary epithelial cell line was cultured in DMEM F/12 media supplemented with 10 μg/mL of insulin, 20 ng/mL of epidermal growth factor (EGF), and 0.5 μg/mL of hydrocortisone, as well as, 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. All cell lines were incubated at 37°C in a humidifier with 5% CO₂ atmosphere.

Differential Nuclear Staining Assay

In order to analyze the potential cytotoxicity of the compounds that were synthesized, the Differential Nuclear Staining Assay (DNS) was used which has been validated for high throughput compound screening.^[27] The DNS assay involves the use of two dyes, propidium iodide (PI) and Hoechst. While Hoechst stains the nuclei of healthy and dead cells, PI can only penetrate and stain nuclei of cells with compromised membranes. Cells with positive fluorescence for both dyes; Hoechst (blue) and PI (red) are categorized as the dead cell population.^[27] For the initial cytotoxicity screening, 10,000 cells per well were seeded in 100μL of culture media in a 96-well microplate and incubated overnight. A concentration gradient of the compounds from 0.1to 10 μM was tested. Untreated cells, 1% v/v DMSO solvent, and 1mM of H₂O₂ were included as controls. At least three technical replicates were evaluated for each experimental point, as well as for the controls. After adding the treatments, cells were incubated for 48 hours at optimal conditions. Two hours before the end of the incubation period, a combination of Hoescht and PI (1μg/mL final concentration) was added to each well and cells were then incubated for the remaining 2 hours. Afterward, the Image Xpress Pico (Molecular Devices, San Jose, CA) system was used to acquire four contiguous images per well creating 2×2 montages with a4X objective, for the Hoechst and PI individual fluorescent channels. Images were analyzed to obtain percentages of living and dead cells using the Cell Reporter Xpress software.

CC₅₀ calculations

The CC₅₀ is defined as the compound concentration needed to kill 50% of the cell population. After obtaining percentages of living and dead cells, the CC₅₀ values were calculated for each experimental compound by using a linear interpolation equation (https://www.johndcook.com/interpolator.html).^[28,29] The compound with the lowest CC₅₀ value was selected for further characterization.

Apoptosis assay

The mechanism of cell dead induced by 6h was evaluated in Jurkat cells using the Annexin V-FITC/PI assay kit (Beckman coulter; IM3546) and flow cytometry. On day one, 100,000 cells per well in 1 ml of culture media were seeded in a transparent flat-bottom 24-well plate and incubated overnight at optimal conditions (37 °C, 5% CO₂). The next day, cells were treated with the CC₅₀ (4.36 μM) and 2x CC₅₀ (8.72 μM) concentrations of the 6h compound for 24 hours. 1% v/v DMSO (vehicle), untreated and 1mM H₂O₂ (positive control for death) were included as controls. Cells were then collected in flow cytometry tubes and stained with a mixture of PI and annexin V-FITC according to the manufacturer’s instructions. Samples were immediately analyzed using the GALLIOS flow cytometer (Beckman Coulter, Brea, CA, USA). Approximately 10,000 events (cells) were acquired per sample and analyzed using the Kaluza 1.3 software tool (Beckman Coulter).

Reactive Oxygen Species (ROS) accumulation assay

Reactive oxygen species formation was examined in Jurkat cells treated with the 6h compound using the oxidative stress indicator; 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-H₂ DCFDA; Invitrogen C400). Cells were plated at a density of 100,000 cells in 1 ml of culture media per well in 24-well plates, and incubated overnight at optimal conditions. Cells were then treated with 6h CC₅₀ (4.36 μM) and 2x CC₅₀ (8.72 μM) concentrations and left for 18 hours at 37 °C, 5% CO₂. Triplicates for each treatment were included, and 100 mM of H₂O₂, and untreated cells were incorporated as positive and negative controls of death, respectively. A vehicle control (1% v/v DMSO) was also included. Cells were harvested in flow cytometry tubes and centrifuged at 262g for 5 min. Supernatants were decanted and cell pellets resuspended in 1 ml of PBS containing the carboxy-H₂ DCFDA reagent. Cells were incubated at 37 °C for 1 hour, and then were centrifuged for 5 min at 262g. Supernatants were decanted and 500 μl of pre-warmed PBS were added to each sample. A 20 min recovery time was allowed by incubating cells at 37 °C. Intracellular esterases in cells with accumulated ROS will transform the nonfluorescent molecule (Carboxy-H2DCFDA) into its green-fluorescent form by removing the acetate groups. Cells were immediately examined via the Gallios flow cytometer using the FL2 detector for green fluorescent signal, and at least 10,000 events per sample were collected. Data was analyzed using the Kaluza 1.3 software.

Mitochondrial depolarization assay

The mitochondrial membrane potential of Jurkat cells treated with the 6h compound was evaluated by using the MitoProbe JC-1 assay kit (Life Technologies; M34152) by flow cytometry. Cells were seeded at a density of 100,000 cells in 1 ml of culture media per well in a clear 24-well plate and incubated overnight at optimal conditions. The following day, cells were treated with the CC₅₀ and 2x CC₅₀ concentrations of 6h as previously mentioned. After treatment cells were incubated for 5 hours at 37 °C, 5% CO₂ with the cationic polychromatic JC-1(5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) reagent following manufacturer’s instructions. Cells were analyzed via the GALLIOS flow cytometer and 10,000 events were collected per sample and data was analyzed using the Kaluza 1.3 software. If mitochondria are depolarized (lost membrane potential), the JC-1 dye organize in monomers emitting a green fluorescent signal, whereas if the mitochondria is unaffected, the membrane electric potential will arrange JC-1 as aggregates emitting a red fluorescence^[30].

Cell cycle distribution analysis

The cell cycle distribution analysis was accomplished in cells exposed to 6h by using a Nuclear Isolation Media containing DAPI (NIM-DAPI; Kerafast ECT004) which permeabilizes the cell and stains DNA to allow its quantification by flow cytometry. On day one, Jurkat cells were seeded at a concentration of 100,000 cells in 1 mL of culture media per well in 24-well plates. The following day the cells were treated in triplicate for a 72-hr period with 6h compound at CC₂₅ (2.18 μM) and CC₅₀ (4.36 μM) concentrations and the controls included were 1% v/v DMSO, 1 mM H₂O₂, and 100 mM of etoposide as well as untreated cells. Cells were harvested and centrifuged for 5 min at 262g and resuspended in 100 μl of pre-warmed PBS and 200 μl of the NIM-DAPI solution were added to each sample and were lightly vortexed. Cell suspensions were immediately examined by flow cytometry and 100,000 events were acquired for a well-defined cell cycle distribution profile. Data was analyzed via the Kaluza 1.3 Flow Cytometry Software^[31,32].

Statistical analyses

For the biological assays the p-values were calculated using a two-tailed paired Student’s t-test to determine statistical significance between two groups, and denoted with asterisks (*p < 0.05, **p < 0.01 and ***p < 0.001).

Figure 3.

6h disrupts the normal cell cycle progression by arresting cells in G0/G1 phase. Jurkat cells were treated with CC₅₀ (4.36 μM) and CC₂₅ (2.18 μM) concentrations of 6h for 72 h. Cells were stained with NIM-DAPI and quantified via flow cytometry. A significant increase in DNA fragmentation (sub G0/G1; A) and cell cycle arrest in the G0/G1 diploid phase (B) were detected. A significant decrease in G2/M was also noticed (D). 1% DMSO, 1 mM H₂O₂ and 100 mM of etoposide were used as controls. Statistical significance of the treatments was calculated against the solvent control (DMSO) using a two-tailed student’s paired t tests (*p < 0.05, **p < 0.01 and ***p < 0.001).