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Abstract

A series of new thiazacridine agents were synthesized and evaluated as antitumor agents, in terms of not only their cytotoxicity but also their selectivity. The cytotoxicity assay confirmed that all compounds showed cytotoxic activity and selectivity. The new compound, 3-acridin-9-ylmethyl-5-(5-bromo-1H-indol-3-ylmethylene)-thiazolidine-2,4-dione (LPSF/AA29 – 7a), proved to be the most promising compound as it presents lower half-maximal inhibitory concentration (IC₅₀) values (ranging from 0.25 to 68.03 µM) depending on cell lineage. In HepG2 cells, the lowest IC₅₀ value was exhibited by 3-acridin-9-ylmethyl-5-(4-piperidin-1-yl-benzylidene)-thiazolidine-2,4-dione (LPSF/AA36 – 7b; 46.95 µM). None of the synthesized compounds showed cytotoxic activity against normal cells (IC₅₀ > 100 µM). The mechanism of death induction and cell cycle effects was also evaluated. Flow cytometric analysis revealed that the compounds LPSF/AA29 – 7a and LPSF/AA36 – 7b significantly increased the percentage of apoptotic cells and induced G2/M arrest in the cell cycle progression. Therefore, these new thiazacridine derivatives constitute promising antitumor agents whose cytotoxicity and selectivity properties indicate they have potential to contribute to or serve as a basis for the development of new cancer drugs in the future.

Keywords

Acridine anticancer activity cell cycle death induction mechanisms thiazolidine

Introduction

Acridines and their structural analogues constitute a class of important molecules for the development of promising chemotherapeutic agents.¹ The planar structure of their aromatic rings provides them with the ability to intercalate with the base pairs of DNA.² A series of biological activities have been reported in the literature for these molecules such as analgesic, antiviral, antimicrobial, antiprion, anti-inflammatory, and anticancer activities.^3

–8

In cancer chemotherapy, acridines can lead to cell cycle arrest and apoptosis,^7
–9 as their major biological targets are enzymes related to DNA synthesis and cell proliferation like topoisomerases (Topo I and Topo II) and telomerase.^10
–12 Amsacrine is one of the most well-known acridine derivatives and was among the first to be used clinically as an anticancer agent. It is considered to be an inhibitor of topoisomerase II.^13,14

Amsacrine has a cytotoxic potential that is highly effective in the treatment of acute leukemia and lymphomas; however, it is less effective on solid tumors.¹⁵ On the other hand, thiazolidinediones (TZDs), also known as glitazones, belong to a class of molecules that increase insulin sensitivity and were used in the treatment of type II diabetes mellitus for a long time.¹⁶ These molecules have high affinity for the peroxisome proliferator–activated receptor γ (PPARγ), a nuclear receptor with a well-established role in lipid homeostasis.^17,18 Additionally, studies have shown that PPARγ is expressed in many cancer lineages modulating cell proliferation and apoptosis.¹⁹

The TZD compounds, PPARγ agonists, constitute emergent antitumor agents against multiple cancers,^20,21 but some studies have also indicated antiproliferative activity of TZDs in cancer cell lines independent of PPARγ agonism.^22
–24 In summary, the key mechanisms involved in anticancer activity following TZD treatment are inhibition of cell growth, induction of cell death by apoptosis, and inhibition of cell invasion.^20,24,25

Therefore, the strategy of our group was to develop new alternatives for the treatment of cancer using the molecular hybridization technique to unite the nuclei of acridine and TZD molecules to obtain a new class of compounds, the thiazacridines. Here, we describe the synthesis of eight new thiazacridines and results of in vitro cytotoxicity evaluation against different cancer cell lineages, including their selective potential and effects on the cell cycle and the induction of programmed death in tumor lines.

Materials and methods

Chemical

The synthesis of all intermediates was performed in parallel to obtain the thiazolidine-2,4-dione nucleus (2) and esters of Cope (6a–g), which allowed for the synthesis of the new thiazacridine derivatives (7a–g).⁸ All melting points (mp) were determined using a Quimis-340.27 apparatus (Quimis, Brazil). Infrared spectroscopy (IR) was performed on an IRPrestige-21 spectrometer (Shimadzu, Japan). Proton nuclear magnetic resonance (¹H NMR) spectra were recorded at 27°C on a Varian Plus 300 MHz instrument (Varian, Palo Alto, CA, USA) with tetramethylsilane (TMS) as an internal standard and dimethyl sulfoxide (DMSO)-d ₆ as a solvent. Chemical shifts (δ ppm) were determined using TMS as the internal standard and the coupling constants (J) are given in Hertz. Reaction progress was determined by thin layer chromatography on 0.25-mm silica gel plates (Merck, Germany). Mass spectra were obtained using liquid chromatography/mass spectrometry with a HCT Ultra from Bruker Daltonics (Billerica, MA, USA) and were performed by electrospray ionization in positive or negative mode. The base peak of the MS spectrum was set to 100 (in percentage), and the height of the other peaks was measured relative to the base peak. All reagents used in the present work were of analytical grade.

Experimental

3-Acridin-9-ylmethyl-5-(5-bromo-1H-indol-3-ylmethylene)-thiazolidine-2,4-dione (LPSF/AA29 – 7a)

C₂₆H₁₆BrN₃O₂S. mp: 298°C. Yield: 53.25%. CCD (n-hexane:ethyl acetate, 7:3) R_f 0.4. IR (potassium bromide (KBr), cm⁻¹): 3412 (R₂N-H), 1732 (C=O), 1681 (C=O), 1595 (C=C), 1369 (C–N), 750 (C–H), 731 (C=C), 599 (C–Br). ¹H NMR (400 MHz, DMSO-d ₆): δ 5.93 (s, 2H, N-CH₂), 7.35 (d, 1H, J = 8.4 Hz, Ar-H, i pos), 7.44 (d, 1H, J = 8.8 Hz, Ar-H, h pos), 7.68 (t, 2H, J = 15.6 Hz, j = 8.0 Hz, c pos), 7.8 (s, 1H, =CH), 7.86 (t, 2H, J = 15, 2 Hz, j = 8.4 Hz, d pos), 8.18 (m, 4H, a, g, j pos), 8.50 (d, 2H, J = 8.8 Hz, Acr-H, b pos). MS m/z [%]: 514 [(M + 1)⁺, 43.4], 516 [(M + 3)⁺, 47.1], 251 [100]. HRMS (ESI): calculated for C₂₆H₁₆BrN₃O₂S [M−H]⁻ 513.0147. Found 512.0026.

3-Acridin-9-ylmethyl-5-(3,4-bis-benzyloxy-benzylidene)-thiazolidine-2,4-dione (LPSF/AA35 – 7b)

C₃₈H₂₈N₂O₄S. mp: 232°C. Yield: 54.44%. CCD (n-hexane:ethyl acetate, 7:3) R_f 0.38. IR (KBr, cm⁻¹): 1737 (C=O), 1667 (C=O), 1585 (C=C), 1381 (C–N), 1265 (C–O), 751 (C–H), 697 (C=C). ¹H NMR (400 MHz, DMSO-d ₆): 5.17 (s, 2H, O-CH₂), 5.21 (s, 2H, O-CH₂), 5.91 (s, 2H, N-CH₂), 7.15–7.22 (m, 3H, Ar-H), 7.27–7.45 (m, 10H, Ar-H), 7.67 (t, 2H, J = 12 Hz, j = 8, c pos), 7.84 (s, 1H, =CH), 7.86 (d, 1H, J = 7.2 Hz, Acr-H, d pos), 8.18 (d, 1H, J = 8.79 Hz, Acr-H, a pos), 8.44 (d, 1H, J = 8.4 Hz, Acr-H, b pos). HRMS (ESI): calculated for C₃₈H₂₈N₂O₄S [M + H]⁺ 608.1770. Found 609.1856.

3-Acridin-9-ylmethyl-5-(4-piperidin-1-yl-benzylidene)-thiazolidine-2,4-dione (LPSF/AA36 – 7c)

C₂₉H₂₅N₃O₂S. mp: 232–233°C. Yield: 39.93%. CCD (n-hexane:ethyl acetate, 7:3) R_f 0.33. IR (KBr, cm⁻¹): 1677 (C=O), 1576 (C=C), 1377 (C–N). ¹H NMR 400 MHz (δ ppm, DMSO-d ₆) 1.57 (s, 6H, –CH, j, k pos), 3.36–3.37 (m, 4H, N-CH₂, i pos), 5.91 (s, 2H, N-CH₂, e pos), 6.99 (d, 2H, J = 9.2 Hz, h pos), 7.39 (d, 2H, J = 9.2 Hz, g pos), 7.63 (t, 2H, J = 14.4 Hz, j = 6.8 Hz, Acr-H, c pos), 7.79 (s, 1H, =CH, f pos), 7.85 (t, 2H, J = 14 Hz, j = 6.8 Hz, Acr-H, d pos), 8.18 (d, 2H, J = 8.8 Hz, Acr-H, a pos), 8.47 (d, 2H, J = 8.8 Hz, Acr-H, b pos). HRMS (ESI): calculated for C₂₉H₂₅N₃O₂S [M+H]⁺ 479.1667. Found 480.1710.

3-Acridin-9-ylmethyl-5-(4-morpholin-4-yl-benzylidene)-thiazolidine-2,4-dione (LPSF/AA-39 – 7d)

C₂₈H₂₃N₃O₃S. mp: 243°C. Yield: 41.83%. CCD (n-hexane:ethyl acetate, 7:3) R_f 0.43. IR (KBr, cm⁻¹): 1731 (C=O), 1678 (C=O), 1585 (C=C), 1373 (C–N), 1122 (C–O), 753 (C–H). ¹H NMR 400 MHz (δ ppm, DMSO-d ₆) 3.26 (t, 4H, J = 10 Hz, j = 4.8 Hz, morpholin, i pos), 3.70 (t, 4H, J = 9.6 Hz, j = 4.8 Hz, morpholin, j pos), 5.90 (s, 2H, N-CH₂, e pos), 7.01 (d, 2H, J = 9.2 Hz, Ar-H, h pos), 7.42 (d, 2H, J = 9.2 Hz, Ar-H, g pos), 7.67 (t, 2H, J = 15.6 Hz, j = 8.8 Hz, Acr-H, c pos), 7.81 (s, 14, C=CH, f pos), 7.85 (t, 2H, J = 15.2 Hz, j = 8.8 Hz, Acr-H, d pos), 8.18 (d, 2H, J = 8.8 Hz, Acr-H, a pos), 8.47 (d, 2H, J = 8.8 Hz, Acr-H, b pos). HRMS (ESI): calculated for C₂₈H₂₃N₃O₃S [M + H]⁺ 481.1460. Found 482.1512.

3-Acridin-9-ylmethyl-5-(4-pyridin-2-yl-benzylidene)-thiazolidine-2,4-dione (LPSF/AA-40 – 7e)

C₂₉H₁₉N₃O₂S. mp: 269°C. Yield: 55.63%. CCD (n-hexane:ethyl acetate, 7:3) R_f 0.26. IR (KBr, cm⁻¹) 1745 (C=O), 1685 (C=O), 1597 (C=C), 1373 (C–N), 752 (C–H). ¹H NMR 400 MHz (δ ppm, DMSO-d ₆) 5.92 (s, 2H, N-CH₂, e pos), 6.14 (s, 2H, O-CH₂, benzodioxole, j pos), 6.92–7.05 (m, 3H, Ar-H), 7.69 (t, 2H, J = 15.2 Hz, j = 80 Hz, Acr-H, c pos), 7.78 (s, 1H, C=CH, f pos), 8.36 (t, 2H, J = 15.2 Hz, j = 8.0 Hz, Acr-H, d pos), 8.18 (d, 2H, J = 8.8 Hz, Acr-H, a pos), 8.44 (d, 2H, J = 9.2 Hz, Acr-H, b pos). HRMS (ESI): calculated for C₂₉H₁₉N₃O₂S [M + H]⁺ 473.1198. Found 474.0589.

3-Acridin-9-ylmethyl-5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione (LPSF/AA-41 – 7f)

C₂₅H₁₆N₂O₄S. mp: 252–253°C. Yield: 70.29%. CCD (n-hexane:ethyl acetate, 7:3) R_f 0.46. IR (KBr, cm⁻ ¹) 1737 (C=O), 1689 (C=O), 1614 (C=C), 1381 (C–N), 1244 (C–O), 1062 (C–O), 750 (C–H), 719 (C=C). ¹H NMR 400 MHz (δ ppm, DMSO-d ₆) 5.94 (s, 2H, N-CH₂), 7.39 (m, 1H, J = 12 Hz, Ar-H, k pos), 7.6 (t, 4H, J = 12 Hz, Ar-H, c, g pos), 7.88 (m, 3H, J = 36 Hz, Ar-H, d, j pos), 7.99 (s, 1H, =CH), 8.04 (d, 1H, 8 Hz, Ar-H, i pos), 8.19 (d, 1H, 8 Hz, Ar-H, a pos), 8.23 (d, 1H, 8 Hz, Ar-H, h pos), 8.47 (d, 2H, J = 8 Hz, Ar-H, b pos), 8.69 (d, 2H, J = 8 Hz, Ar-H, l pos). HRMS (ESI): calculated for C₂₅H₁₆N₂O₄S [M + H]⁺ 440.0831. Found 441.0281.

3-Acridin-9-ylmethyl-5-(1-phenyl-1H-pyrazol-4-ylmethylene)-thiazolidine-2,4-dione (LPSF/AA-48 – 7g)

C₂₇H₁₈N₄O₂S. mp: 239–240°C. Yield: 69.05%. CCD (n-hexane:ethyl acetate, 7:3) R_f 0.5. IR (KBr, cm⁻ ¹) 1732 (C=O), 1678 (C=O), 1612 (C=C), 1373 (C–N), 1328 (C–N aromatic), 752 (C–H), 686 (C=C). Hz (δ ppm, DMSO-d ₆) 5.92 (s, 2H, –CH₂), 7.38 (t, 1H, J = 16 Hz, j = 7.6 Hz, Ar-H, k pos), 7.53 (t, 2N, J = 16 Hz, j = 8 Hz, Ar-H, j pos), 7.6 (t, 2H, J = 16 Hz, j = 7.2 Hz, Ar-H, i pos), 7.84–7.89 (m, SH, Ar-H, =CH, cdf pos), 8.09 (s, 1H, HC=N), 8.1 (d, 2H, J = 8.4 Hz, Acr-H, a pos), 8.46 (d, 2H, J = 8.8 Hz, Acr-H, b pos), 8.82 (s, 1H, =CH). HRMS (ESI): calculated for C₂₇H₁₈N₄O₂S [M + H]⁺ 462.1150. Found 463.0565.

Biological

Cell culture conditions

The cytotoxicity of the compounds was tested against five human tumor cell lines: NG97 (glioblastoma), HepG2 (human hepatocarcinoma), T47D (human ductal carcinoma), Raji (Burkitt’s lymphoma), and Jurkat (T-cell leukemia). All cell lineages were obtained from the Rio de Janeiro Tissue Cell Bank except for the NG97 cells that were kindly provide by Professor Roger Chammas (University of São Paulo). Cells were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum, 2 mM glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin at 37°C in a humidified atmosphere of 5% CO₂.

Cytotoxicity assay

The new derivatives were dissolved in DMSO and then diluted in culture medium to obtain final concentrations ranging from 1 µM to 100 µM. For all experiments, the cells were plated in 96-well plates (10⁴ cells/well). After 24 h, the compounds were added to each well and incubated for 72 h. Amsacrine (Sigma-Aldrich Co., St. Louis, MO, USA) was used as a positive control, absence of compound as a negative, and DMSO (0.1%) as a vehicle control.

Neoplastic viability was quantitated and assayed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich Co., St. Louis, MO, USA) method. After all treatments, 20 µL of MTT solution (0.5 mg/mL) was added to each well and incubated for 3 h at 37°C. Formazan, the product of MTT reduction, was dissolved in 20% sodium dodecyl sulfate, and absorbance was measured using a multiplate reader (ELX 800; Biotek, Winooski, VT, USA). The compound effect was quantified as a percentage of the control absorbance of the reduced dye in the 0.1% DMSO-treated group at 570 nm. Then it was calculated half-maximal inhibitory concentration (IC₅₀) from the percentage of viable cells.

Cytotoxicity against human peripheral blood mononuclear cells

Briefly, heparinized blood was diluted in an equal volume of phosphate-buffered saline (PBS). Blood samples came from healthy, nonsmoking volunteers, who had not taken any drugs for at least 15 days prior to sample collection (n = 15). Peripheral blood mononuclear cells (PBMCs) were isolated by a standard method of density-gradient centrifugation over Ficoll Paque Plus (1.077 g/mL; GE, Sweden) and then cell counting was performed using 0.4% (w/v) trypan blue (Sigma-Aldrich Co., St. Louis, MO, USA) exclusion.^25,26 Cells were only used when viability was >98%. All donors signed an informed consent form for the study, which was approved by the Human Research Ethics Committee of the UFPE Health Sciences Center (CEP/CCS/UFPE-11006). Cells were plated in 96-well plates (10⁶ cells/well). After 24 h, the compounds at 1–250 µM were added and then incubated for 2 days (48 h); after this procedure, the steps outlined in the previous section were performed. The selectivity index (SI) was calculated as the ratio of IC_{50 normal cells}/IC_{50 cancer cells} according to Islam et al.²⁷ Compounds with high selectivity have SI value >3.

Flow cytometry – Bromodeoxyuridine incorporation assay

Cells were plated (0.5 × 10⁶ cells/well) in 6-well plates and incubated at 37°C for 24 h. Following this period, the cells were treated with complete medium (blank), LPSF/AA29 (Raji, Jurkat, NG97, and T47D), LPSF/AA36 (HepG2), and amsacrine (all lineages) in concentrations previously determined by IC₅₀ and then incubated for 48 h at 37°C. After treatment, analysis of the potential induction of cell death was performed using the Apo-BrdU kit (eBiosciences, San Diego, CA, USA), following the manufacturer’s recommendations. At this stage, the cells were labeled with bromodeoxyuridine (BrdU) triphosphate nucleotides and incubated for 1 h at 37°C, followed by detection by staining with Fluorescein isothiocyanate (FITC)-anti-BrdU monoclonal antibody and then a solution of propidium iodide (PI) and RNAse A. The percentage of BrdU-labeled cells was then analyzed by flow cytometry using an Accuri C6 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) flow cytometer and data analysis was by means of the Accuri C6 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Flow cytometry – PI staining for cell cycle analysis

Cells were plated (0.5 × 10⁶ cells/well) in 6-well plates and treated as in previous section. After 24 h, cells were harvested and washed with PBS and fixed with ice-cold 70% EtOH overnight at −20°C.²⁸ Prior to analysis, cells were incubated with PI (5 μg/mL)/RNase A (100 μg/mL) in PBS for 30 min on ice. Cellular DNA content was analyzed using an Accuri C6 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) flow cytometer. The cell cycle profile was subsequently analyzed using the Accuri C6 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Statistical analysis

All experiments were performed in triplicate in three independent experiments. IC₅₀ values and 95% confidence intervals were obtained by nonlinear regression with the OriginPro program (version 8.0; OriginLab, Northampton, MA, USA). Statistical significance was tested with a two-tailed, unpaired Student’s t-test in relation to the untreated or positive controls.

Results and discussion

Synthesis

Thiazacridine derivatives were prepared as shown in Figures 1 and 2. The compound 3-acridin-9-ylmethyl-thiazolidine-2,4-dione (LPSF/AA1A, 3) was synthesized by the N-alkylation reaction of thiazolidine-2,4-dione (2) with 9-bromomethyl-acridine (1) in the presence of sodium hydroxide using ethanol as a solvent. The mixture was stirred at 60°C for 7 h. In parallel, the synthesis of cyanocinnamic esters (LPSF/IP, 6a–g) was performed using Knoevenagel condensation with aromatic aldehydes (5a–g) and ethyl cyanoacetate (4) in toluene and in the presence of morpholine at 110°C. The Michael reaction of 3-acridin-9-ylmethyl-thiazolidine-2,4-dione, compound 3, with different cyanoacrylates, compounds 6a–g, was carried out in the presence of morpholine, with ethanol as a solvent and heated at 60°C. The formation of the exocyclic double bond at position 5 of acridin-9-ylmethyl-thiazolidine-2,4-dione (LPSF/AA1A, 3) was the result of the action of the morpholine, which leads to the formation of a carbanion, leading to the new thiazacridine derivatives (LPSF/AAs, 7a–g). The presence of an arylidene proton peak in the ¹H NMR spectra of the compounds confirmed that the Michael reaction was successful (Figure 3). The identity of the compounds was confirmed by mass spectrometry in positive or negative mode, and the IR spectra of the compounds showed peaks characteristic of acridine and thiazolidine moiety.⁸

Figure 1.

Synthesis diagram of the thiazacridine 7b–g compounds. (I) Sodium hydroxide and ethanol, 60°C; (II) toluene and morpholine, 110°C; (III) ethanol and morpholine, 60°C.

Figure 2.

Synthesis diagram of thiazacridine derivative LPSF/AA29 – 7a. (I) Ethanol and morpholine, 60°C.

Figure 3.

Hydrogen identification for ¹H NMR interpretation: thiazacridine nucleus (a–e), radicals attached to the position 5 of the thiazolidine nucleus (f–k). ¹H NMR: proton nuclear magnetic resonance.

In vitro cytotoxic activity in tumor cells

The new thiazacridines synthesized, LPSF/AA29 – 7a, LPSF/AA35 – 7b, LPSF/AA36 – 7c, LPSF/AA39 – 7d, LPSF/AA40 – 7e, LPSF/AA41 – 7f, and LPSF/AA48 – 7g, exhibit cytotoxicity against all tested lineages, with IC₅₀ values ranging from 0.25 µM to 97.11 µM. The compound LPSF/AA29 – 7a showed the best results in this screening with the lowest IC₅₀ values for all tested tumor cell lines, except HepG2. For this lineage, LPSF/AA36 – 7c showed the most cytotoxic activity. The potential effects of the new thiazacridine compounds on the viability of tumor cells are summarized in Table 1.

Table 1.

Cytotoxicity of new derivatives on various human tumor cell lines and nontumor cells (PBMCs) – IC₅₀ (µM).^a

Compounds	Cell line
Compounds	HepG2	Jurkat	NG97	Raji	T47D	PBMC
(LPSF/AA29 – 7a)	68.03	0.25	39.59	34.20	31.3	181.47
(LPSF/AA35 – 7b)	48.63	76.66	79.31	80.33	92.31	>250
(LPSF/AA36 – 7c)	46.95	86.79	81.34	68.31	73.14	>250
(LPSF/AA39 – 7d)	55.04	78.18	74.02	71.06	87.49	>250
(LPSF/AA40 – 7e)	87.67	82.24	80.13	86.40	86.42	>250
(LPSF/AA41 – 7f)	97.11	71.40	83.19	82.31	51.13	>250
(LPSF/AA48 – 7g)	65.20	68.26	69.34	71.41	67.11	>250
Amsacrine	1.51	1.1	0.245	4.15	22.87	3.7

PBMC: peripheral blood mononuclear cell.

^aResults are of three independent experiments (performed in triplicate) and expressed as mean values, showing a maximum variation of 10%.

Based on the results, it is suggested that the addition of substituted 5-bromo-1H-indole to the thiazacridine core potentially contributed to the in vitro cytotoxic activity against the majority of lineages tested, compared with the other derivatives synthesized. A large number of compounds containing the indole nucleus have been evaluated in the last decade and have been shown to be active against several tumor cell lines. Examples of such cell lines include RH30 (rhabdomyosarcoma), MDA-MB 231 (breast cancer), PaCa-2 (pancreatic cancer cells), A549 (non-small-cell lung cancer), H460 (human lung cancer cells), HT-29 (human colorectal cancer cells), SMMC-7721 (human liver cancer cells), MCF-7 (breast cancer cells), IGR-OV-1 (ovary cancer cells), T47D (breast cancer cells), and CoLo-205 (colon cancer cells), among others.^29

–33 Additionally, the substituent 5-bromo-1H-indole, when added to phthalazine molecules, also showed good antitumor activity.³⁴ Su et al.³⁵ highlighted the presence of a bromide substituent in the indole ring that led to the increased anticancer activity against PLC5 cells of compounds derived from obatoclax. Thus, we can infer that bromine also contributes to the toxic effect of the molecule. Although the compounds LPSF/AA36 – 7c, LPSF/AA39 – 7d, and LPSF/AA40 – 7e are the most structurally similar compared to the other compounds synthesized, only 7c was shown to be a promising cytotoxic agent against the HepG2 line. The good activity of 7c can be explained by some structural factors, such as absence of oxygen in position 4 of the benzylidene ring and absence of resonance in the second ring (piperidine), when compared to 7d and 7e, respectively. Moreover, the piperidine nucleus is more basic than the pyridine nucleus, thus the nitrogen of piperidine is able to transfer its pair of electrons more easily, which therefore allows increased interaction with the biological receptor. Recently, it has been reported that N-containing heterocycles are considered especially “privileged” structures for the synthesis and development of new drugs.^36,37

Compounds 7b (bis-benzyloxy substituent), 7f (benzodioxole substituent), and 7g (phenylpyrazole substituent) were the second most active in HepG2, T47D, and Jurkat cell lines, respectively.

In vitro cytotoxic activity in normal cells

In this study, the selective potential of these thiazacridine derivatives was also evaluated. For this reason, nontumor cells were also exposed to treatment with the new compounds and with the positive control, amsacrine. It was found that the newly synthesized compounds showed IC₅₀ values >250 µM, against PBMCs, except LPSF/AA29 – 7a which exhibited IC₅₀ = 181.47 μM. The IC₅₀ value of the positive control amsacrine was 3.73 µM (Table 1).

As shown in Table 2, all the synthesized compounds showed higher SI for HepG2, Raji, and T47D, when compared with amsacrine. The agents LPSF/AA35 – 7b, LPSF/AA39 – 7d, LPSF/40 – 7e, LPSF/AA41 – 7f, and LPSF/AA48 – 7g were selective for Jurkat; in addition, LPSF/AA29 – 7a showed this lineage the highest SI in this study. While all the compounds have shown selectivity for NG97, with SI >3, the amsacrine was more selective.

Table 2.

Selectivity index of new thiazacridine agents and amsacrine.

Compounds	Cell line
Compounds	HepG2	Jurkat	NG97	Raji	T47D
(LPSF/AA29 – 7a)	2.67	725.88	4.58	5.31	5.80
(LPSF/AA35 – 7b)	5.14	3.26	3.15	3.11	2.71
(LPSF/AA36 – 7c)	5.32	2.88	3.07	3.66	3.42
(LPSF/AA39 – 7d)	4.54	3.20	3.38	3.52	2.86
(LPSF/AA40 – 7e)	2.85	3.04	3.12	2.89	2.89
(LPSF/AA41 – 7f)	2.57	3.50	3.01	3.04	4.89
(LPSF/AA48 – 7g)	3.83	3.66	3.61	3.50	3.73
Amsacrine	2.45	3.36	15.10	0.89	0.16

These results demonstrate that this new series of compounds exhibit low cytotoxicity to nontumor cells, thus demonstrating their selective property. Poor selectivity of traditional chemotherapy is associated with considerable damages to normal cells, limiting their therapeutic effectiveness. Consequently, drugs with tumor-selective cytotoxicity without impairment of normal cell population are potent candidates for cancer treatment.^38,39

Effects of LPSF/AA29 and LPSF/AA36 on the induction of apoptosis

Once it was observed that the new thiazacridine derivatives induced cell death, LPSF/AA29 – 7a and LPSF/AA36 – 7c compounds were submitted to the assay of potential induction of death by flow cytometry. The cell lines Jurkat, Raji, NG97, and T47D were used as they are more sensitive to the derivative LPSF/AA29 – 7a, while HepG2 cells were used to evaluate LPSF/AA36 – 7c for the same reason. Amsacrine was used as a positive control for all lineages.

The results of the evaluation of the potential for inducing apoptosis showed that LPSF/AA29 – 7a and LPSF/AA36 – 7c derivatives significantly increased the percentage of apoptotic cells in all tested lineages, compared to untreated cells and cells treated with amsacrine (Figures 4 and 5(a)). The increase in induction of apoptosis in HepG2 cells by LPSF/AA36 – 7c was the most significant in this study, both compared to untreated cells (p < 0.0001) and those treated with amsacrine (p < 0.0001). The LPSF/AA29 – 7a showed the greatest potential to induce apoptosis (p < 0.0001) in T47D when compared with untreated cells.

Figure 4.

Analysis of the potential induction of apoptosis. (a) Jurkat cells – LPSF/AA29 versus NTC (p = 0.0376) and versus amsacrine (p = 0.0268). (b) Raji cells – LPSF/AA29 versus NTC (p = 0.0025) and versus amsacrine (p = 0.0322). (c) NG97 cells – LPSF/AA29 versus NTC (p = 0.0002) and versus amsacrine (p = 0.0010). (d) T47D cells – LPSF/AA29 versus NTC (p < 0.0001) and versus amsacrine (p = 0.0014). Values were expressed as percentage of apoptotic cells. *p < 0.05; **p < 0.01; ***p < 0.001. NTC: nontreated cells.

Figure 5.

(a) Analysis of the potential induction of apoptosis: LPSF/AA36 versus NTC (p < 0.0001) and versus amsacrine (p < 0.0001). (b) The effect of LPSF/AA36 and amsacrine on the cell cycle: LPSF/AA36 – sub-G1 (p < 0.0001) and G2/M (p = 0.0008); amsacrine – sub-G1 (p < 0.0001). Values were expressed as percentage of apoptotic cells and cell distribution in each phase of the cycle. ***p < 0.001. NTC: nontreated cells.

Resistance to cell death is one of the hallmarks of cancer and is a principal biological capability acquired during the multistep development of human tumors.⁴⁰ Consequently, understanding the mechanisms for induction of cell death could be useful in cancer treatment.⁴¹ Therefore, the synthesis and identification of potential agents that induce cell death, in particular apoptosis, have aroused the interest of the scientific community worldwide.^25,42,43 However, a very common phenomenon of anticancer therapies is the induction of resistance to cell death, thus new molecular studies need to be conducted to better understand the action of LPSF/AA29 – 7a and LPSF/AA36 – 7c in the pathways that regulate apoptosis.

Effects of LPSF/AA29 – 7a and LPSFAA/36 – 7c on the cell cycle

The cell cycle is an event of great importance and its disorganization is related to the development of diseases such as cancer.⁴⁴ At this stage, the effects of LPSF/AA29 – 7a and LPSF/AA36 – 7c on the cell cycle were investigated. The choice for testing these compounds was the same for evaluating the potential induction of apoptosis, namely these compounds presented the best cytotoxicity results. The results showed that both compounds induced a significant increase in the percentage of cells in phases sub-G1 and G2/M compared to untreated cells (Figures 5(b) and 6). Amsacrine was shown to increase the percentage of cells in phase sub-G1 when compared to untreated cells (p < 0.05). The increase in sub-G1 peak after treatment with LPSF/AA29 – 7a, LPSF/AA36 – 7c, and amsacrine, measured by the subdiploid population, is considered to be an indicator of cell death,⁴⁵ which is consistent with the results of viability assay and induction of cell death assays. In relation to G2/M arrest, this result can be considered as a further delay in the cell cycle, so that the reorganization of various processes may occur at this stage, including the regulation of chromosome segregation and the attempt to resolve nonrepairable DNA damage.⁴⁶ Molecular studies have revealed that synthetic and natural compounds are capable of inducing G2/M cell cycle arrest and promote cell death by apoptosis, demonstrating that the stop cycle at this stage is also associated with cell death mechanisms.^47,48 The importance of G2/M cell cycle arrest in the mediation of cell death can be exemplified by paclitaxel, one of the most commonly used drugs to treat breast and ovarian cancer, which acts specifically on this phase of the cycle, thereby inducing apoptosis.^49,50

Figure 6.

The effect of LPSF/AA29 and amsacrine on the cell cycle. (a) Jurkat cells – LPSF/AA29 – sub-G1 (p = 0.0006) and G2/M (p = 0.0015); amsacrine – sub-G1 (p = 0.0105). (b) Raji cells – LPSF/AA29 – sub-G1 (p = 0.0012) and G2/M (p = 0.0078); amsacrine – sub-G1 (p = 0.0001). (c) NG97 cells – LPSF/AA29 – sub-G1 (p = 0.0023) and G2/M (p = 0.0126); amsacrine – sub-G1 (p < 0.0001). (d) T47D cells – LPSF/AA29 – sub-G1 (p = 0.0023) and G2/M (p < 0.0001; amsacrine – sub-G1 (p < 0.0001). Values were expressed in percentage (%) of cell distribution in each phase of the cycle. *p < 0.05; **p < 0.01; ***p < 0.001. NTC: nontreated cells.

Conclusion

In conclusion, a new series of thiazacridine derivatives was synthesized and had its physical and chemical characteristics defined. Moreover, in vitro studies demonstrated that all compounds showed antiproliferative activity against various cancer cell lineages and exhibited more selectivity than the positive control, amsacrine. The compound LPSF/AA29 – 7a, which has the 5-bromo-1H-indole substituent, showed the greatest antiproliferative activity against Jurkat, Raji, T47D, and NG97 among all compounds. Against HepG2, the highest activity was shown by LPSF/AA36 – 7c, which has a piperidine nucleus. These results suggest a close structure–activity relationship between the compounds and lineages tested. The test results of induction of cell death and cell cycle progression have shown that LPSF/AA29 – 7a and LPSF/AA36 – 7c are potential inducers of apoptosis and significant inhibitors of cell cycle progression at the G2/M phase. In this way, LPSF/AA29 – 7a and LPSF/AA36 – 7c proved able to exert cytotoxic effects against the tested lineages. However, further studies are needed to define their effects in terms of molecular pathways. Therefore, new thiazacridine derivatives constitute promising antitumor agents because they are effective and less toxic and may contribute to the development of new cancer drugs in the future.

Footnotes

Acknowledgement

The authors would like to thank the Brazilian National Research Council (CNPq), the Research Foundation of Pernambuco State (FACEPE), National Institute for Science and Technology in Pharmaceutical Innovation (INCT_if), and Coordination for the Improvement of Higher Education Personnel (CAPES) for student fellowships.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Brazilian National Research Council (CNPq), the Research Foundation of Pernambuco State (FACEPE), National Institute for Science and Technology in Pharmaceutical Innovation (INCT_if), and Coordination for the Improvement of Higher Education Personnel (CAPES).

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New thiazacridine agents: Synthesis,physical and chemical characterization,and in vitro anticancer evaluation

Abstract

Keywords

Introduction

Materials and methods

Chemical

Experimental

Biological

Cell culture conditions

Cytotoxicity assay

Cytotoxicity against human peripheral blood mononuclear cells

Flow cytometry – Bromodeoxyuridine incorporation assay

Flow cytometry – PI staining for cell cycle analysis

Statistical analysis

Results and discussion

Synthesis

In vitro cytotoxic activity in tumor cells

In vitro cytotoxic activity in normal cells

Effects of LPSF/AA29 and LPSF/AA36 on the induction of apoptosis

Effects of LPSF/AA29 – 7a and LPSFAA/36 – 7c on the cell cycle

Conclusion

Footnotes

Acknowledgement

Declaration of Conflicting Interests

Funding

References