Perezone and its phenyl glycine derivative induce cytotoxicity via caspases on human glial cancer cells

Abstract The new phenyl glycine derivative of perezone was obtained in a single reaction step in ca. 80% yield which showed remarkable cytotoxic activity against the astrocytoma U-251 cell line. After 24 h of exposure, both perezone (IC50 = 6.83 ± 1.64 µM) and its phenyl glycine derivative (2.60 ± 1.69 µM) showed cytotoxic effect on U-251 cells but were five times less cytotoxic on the non-tumoral SVGp12 cell line (IC50 = 28.54 ± 1.59 and 31.87 ± 1.54 µM respectively). Both compounds induced cellular morphological changes (pyknosis or cytoplasmic vacuolization) and increased the expression of caspases 3, 8, and 9 genes related to apoptosis. In the acute toxicity study, phenyl glycine perezone (DL50 = 2000 mg/Kg) demonstrated to be less toxic than perezone (DL50 = 500 mg/Kg). Phenylglycine-perezone can envisage a beneficial therapeutic potential. Graphical Abstract

It is striking that since its discovery in 1852, the correct chemical structure of perezone was proposed in 1965 (Wagner et al. 1965;Walls et al. 1965).After that, perezone has received attention for its broad purported medicinal properties for many decades, including our group, which previously reported the synthesis of several amino perezone derivatives with cytotoxic activity against different cancer cell lines (Enríquez et al. 1995).In addition, several products of quinones with amino acids exhibit properties such as bactericide (Iyer and Szybalski 1963), antibiotic-like activity (Cooper and Haines 1929), or apoptotic agents, as reported with indolyl quinone derivatives (Escobedo-González et al. 2017), antioxidant (Lozada et al. 2012), and cytotoxic (Enríquez et al. 1995;Lozada et al. 2012).
Phenylglycine is a bioactive chiral building block (Wang et al. 2001) and is used in treating diseases such as cystic fibrosis (Pedemonte et al. 2005).It is reasonable to expect that such molecular fragments contribute to the observed cytotoxic activity.Since perezone and its derivatives exhibit a high cytotoxic effect versus several cancer cell lines previously reported, additional studies became necessary to elucidate the type of cellular death induced by perezone and its phenyl glycine derivative using morphological studies, bright-field microscopy, and caspase 3, 8 and 9 gene expression.
In the present work, we analyzed the activity of perezone and its phenyl glycine derivative against astrocytoma (U-251) and non-tumoral astrocyte (SVGp12) cell lines; the phenyl glycine perezone derivative stands out in high potency and low toxicity, thus providing ground to consider it a potential therapeutic agent against astrocytoma.

Results and discussion
In this work, perezone was modified by a Michael addition reaction with phenyl glycine methyl ester (Scheme 1), rendered in good yield (>80%); spectroscopic techniques and X-ray diffraction characterized this new compound.
Scheme 1.General synthesis pathway of phenyl glycine perezone derivative.

Structural characterization of phenyl glycine perezone derivative
The 1D and 2D 1 H, and 13 C NMR spectra (see S2-S10) agree with the expected signals for phenylglycine perezone derivative and agree with related compounds (Enríquez et al. 1995).Assignments are shown in Figure S1 and Table S1 (supplementary material).The reaction of perezone with amines occurs through the nucleophilic attack of nitrogen to C6 on the quinone ring.Using zinc acetate as a catalyzer increases the yield of the chemical reaction (Enríquez et al. 1995).
The 1 H NMR spectrum of phenyl glycine perezone derivative (Figure S2) shows multiplets at 8.37 ppm and 7.48 ppm corresponding to the AÁBB'C system of phenyl ring; phenol protons are at 6.17 ppm (br, OH-7) and 5.45 ppm (br, OH-6); the multiple at 5.11 ppm (H-21) shows vicinal couplings to (CH 2 -20) and allylic couplings (to CH 3 -23 and CH 3 -24); proton (H-18) is observed at 3.48 ppm as a complex multiplet coupled to two gem protons (CH 2 -19) and one vicinal methyl (CH 3 -25); the singlet at 2.59 ppm corresponds to CH 3 -17.Methylene group CH 2 -19 is an AB system (2.05 and 1.79 ppm) with complex vicinal couplings, and CH 2 -20 is an AB system at 1.94 and 1.87 ppm.The allylic interaction between the methyl group at 1.65 ppm and proton H-21 is observed through 1D nOe after selective inversion of a signal at 5.11 ppm.The singlet at 1.46 ppm corresponds to CH 3 -24.Finally, CH 3 -25, at 1.41 ppm, is a doublet from coupling to the chiral proton H-18.
The IR spectrum shows characteristic bands for aliphatic CH, CH 2, and CH 3 at 2964.50 and 2927.99 cm −1 , carbonyl (C = O, ester) in 1703.60 cm −1 , a broad band at 3400.48 cm −1 , characteristic of hydroxyl phenolic groups, and a band at 1615.30 cm −1 corresponding to the C = N bond.The MS shows the molecular ion at m/z 379 + , which is consistent with the molecular formula C 23 H 26 O 4 N and the structure of phenyl glycine perezone derivative (see Figures S11-S13).

X-ray diffraction
A suitable single crystal of phenyl glycine perezone derivative was obtained by controlled nucleation and convection using slow evaporation of an EtAcO/MeOH solution (see Figures S14 and S15).
The crystal of phenyl glycine perezone derivative has a monoclinic system in space group P2 1 (Table S2).

Cytotoxic activity of perezone and phenyl glycine perezone
A comparative study of perezone and phenylglycine perezone showed cytotoxic activity on U-251 cells (IC 50 = 6.83 ± 1.64 and 2.60 ± 1.69 µM, respectively), and five times less cytotoxic on the non-tumoral SVGp12 cell line (IC 50 = 28.54 ± 1.59 and 31.87 ± 1.54 µM, respectively).As can be appreciated in Figure S16, phenyl glycine perezone preserves cytotoxic activity with a value less than perezone against the U-251 cancer cell line.Individual curves of IC 50 are found in the supplementary material (Figure S17).
Previous syntheses of amino derivatives of perezone have demonstrated cytotoxic activity, and some have revealed potential as anticancer agents (Alducín-Suárez 1998; Wang et al. 2001;Lozada et al. 2012;Mehndiratta et al. 2016).In the present work, the phenyl glycine perezone derivative demonstrated cytotoxicity against the U-251 cancer cell line related to caspase-dependent pathways.
Phenyl glycine perezone turns out more cytotoxic than perezone against U-251 cancer cells and less toxic against non-tumoral astrocytes SVGp12 cell line.The latter fact is advantageous since in the therapeutics of brain cancer, drugs that are selective against malignant cells but not healthy ones, become a priority.Out of the high number of analog derivatives of perezone prepared in previous studies (Alducín-Suárez 1998;Lozada et al. 2012), this new derivative deserves attention due to its promising features.

Cellular morphological changes at IC 50 value
Figure S18 illustrates cellular changes induced by perezone and phenylglycine perezone in the glioblastoma cell line U-251.Perezone induced pyknosis, while the phenylglycine perezone induced cytoplasmic vacuolization, both changes associated with early apoptosis (Mooren and Krüger 2015;Xu et al. 2019).However, the morphological changes mentioned before are not specific to any cell death process (Aki et al. 2012;Shubin et al. 2016).Conversely, the non-tumoral astrocytes (SVGp12) showed no significant morphological changes when exposed to both compounds.
The changes in cell morphology carried out in this study provide insight into the mechanism of cell death induced by these two compounds.Thus, the signs of apoptosis could be detected through the presence of vesicles, pyknosis, and nuclear withdrawal (Xu et al. 2019).Furthermore, the current design focuses on the induction of apoptosis since programmed cell death is not associated with inflammatory events or negative prognosis signs (Naik et al. 1996;Lee et al. 2018;Xu et al. 2019).It should be noted that the increment of caspases is observed in the U-251 cancer cell line within the 24 h period when exposed to both compounds.

Gene expression of genes involved in apoptosis caspases 8, 9, and 3 by RT-qPCR
The expression of genes of caspases 3, 8, and 9 was analyzed because these proteins are involved in the extrinsic and intrinsic pathways of apoptosis (McIlwain et al. 2013;Mooren and Krüger 2015).As a result, there were no changes in the expression of genes in SVGp12, at IC 50 value, by perezone and its derivative.However, in cell line U-251, there were significant changes in the expression of caspases by compounds.Thus, perezone induced a ca.6× increase with caspases 8, 9, and 3 (6.57,6.21, and 5.93, respectively) versus control (see Figure S19).Furthermore, phenylglycine perezone also induces the corresponding increase to a lesser degree (ca.2-fold).These data support that cell death occurs mainly via caspase-dependent, which can be associated with apoptosis and necrosis death type (Lehman-McKeeman 2013).Consistent with our findings, several indolylquinone analogs of perezone have been reported to induce apoptosis against the breast cancer cell line MDA-MB-231 (Escobedo-González et al. 2017), isoperezone-induced cell death through cell-dependent and -independent caspase mechanisms (Sánchez- Torres et al. 2010) and perezone and its angelate induced cytotoxicity by apoptosis in glioblastoma U373 cell line (Hernández-Rodríguez et al. 2022).Although the apoptosis mechanism is preferred in antineoplastic agents because it is unrelated to inflammatory response, other types of cell death, such as necrosis, are also induced by compounds (Naik et al. 1996;Lee et al. 2018;Xu et al. 2019).
This represents a new potential alternative for cancer treatment since the chemical addition of the amino acid phenylglycine to the perezone molecule guarantees a compound with high potency (IC 50 = 2.60 ± 1.69 µM) compared to perezone (IC 50 = 6.83 ± 1.64) with a death cellular caspase-dependent pathways, in contrast with most existing anticancer drugs, which induce necrosis (Richards et al. 2011;Lekshmi et al. 2017).

Acute toxicity
All mice treated with perezone at the dosage of 2000 mg/Kg showed the following signs of toxicity: (i) isolation behavior, (ii) abdominal contortions, (iii) diarrhea, (iv) body weight loss (see Figure S20), and (v) death of 66% animals; however, the calculated DL 50 was 500 mg/Kg.In contrast, 50% of the animals treated with phenyl glycine perezone at 2000 mg/Kg presented diarrhea and only 33% dead.Therefore, the calculated median lethal dose of phenyl glycine perezone was 2000 mg/Kg.
Although these compounds are cytotoxic against several cancer cell lines in vitro, there are not enough reports of the biological activity in vivo of perezone or its derivatives.The present data supported that perezone is more toxic than its phenyl glycine derivative in mice.These results complement the preliminary report by Jiménez-Cardoso et al. (1986) about the hepatic damage induced by perezone in rodents related to mitochondrial impairments.
It is important to emphasize that natural plant products represent a valuable source of anti-neoplastic compounds.Of all the small compounds employed as anti-neoplastic drugs in the last decade, only 49% were phytocompounds or derivatives (Hernández-Rodríguez et al. 2022).Although, this research has analyzed the cytotoxicity and toxicity activity of the natural product perezone and its synthetic derivative, new complementary studies (in vitro and in vivo) are needed to determine the efficacy and safety of perezone and phenyl glycine perezone.

Chemicals
All chemicals were commercially available, and solvents were purified with conventional methods before use (Armarego and Chai 2009).The natural product perezone was extracted from the roots of Acourtia in hot hexane, as previously reported (Lozada et al. 2012).

Physical measurements
Melting points were determined on an electrothermal engineering IA9100X1 melting point apparatus and were uncorrected.

Spectroscopic determination
1 H and 13 C NMR spectra were recorded in CDCl 3 on a Bruker Avance 700 MHz with TMS as an internal reference.IR absorption spectra were recorded in the range of 4000-230 cm −1 as a thin film on a BRUKER Tensor 27 spectrophotometer.Mass spectrometry was recorded in a JEOL SX102A equipment in electronic impact (EI) mode with an ionization potential of 70 eV.Single-crystal X-ray diffraction (SCXRD) was determined in a Smart Apex, Bruker diffractometer, with Mo radiation (λ= 0.71073 Å), CCD two-dimensional detector, and a low-temperature device.Data collection and processing were performed with APEX and SAINT-Plus software suits (Bruker APEX3 2014 andBruker SAINT 2014).All structures were solved using the SHELX-2013 program and refined by a Full-matrix least-squares procedure on F 2 using SHELX-2008 (Sheldrick 2008).

Cell culture and growth conditions
U-251 and SVGp12 cells were cultured in EMEM and DMEM-F12 medium, respectively.Both were supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% non-essential amino acids, 100 μg/mL of streptomycin sulphate, and 100 μg/mL of penicillin/streptomycin under the following conditions: 37 °C, 5% CO 2 and high relative humidity.At confluence, cells were detached from dishes using 0.025% trypsin for 10 min and were collected by centrifugation at 1500 rpm for 5 min.

Cytotoxicity analysis by crystal violet staining
The cytotoxicity of perezone and phenylglycine perezone derivative was tested against U-251 and SVGp12 cell lines.The viability of the cells used in the experiments exceeded 95%, as determined with crystal violet assay, according to Feoktistova et al. (2016).Results were expressed as a reduction of viability considering the control cell (untreated) viability of each cell line as 100%.Later, the inhibitory concentration 50 (IC 50 ) was calculated according to the protocol of Monks et al. (1991).Briefly, a dose-response plot was fitted to a curve by non-linear regression; the concentration corresponding to the growth inhibition of 50% was considered the IC 50 .All data were represented as mean ± standard error (SEM).

Cellular morphological changes at IC 50 value
1 × 10 4 cells were seeded on sterile circular 12 mm coverslips previously treated with poly-L-lysine (1:10; 5 min) on a 24 wells plate, adding 300 μL of medium to each well.Cells were left to attach to the coverslips for 24 h.Then, the medium was aspirated, and cells were exposed to the perezone (IC 50 = 6.83 ± 1.64 µM) or phenyl glycine perezone derivative (IC 50 = 2.60 ± 1.69 µM) in 300 μL of the medium.After 24 h of exposure, the cells were washed with 500 μL of saline phosphates buffer (PBS, pH = 7.4) for 5 min, and after the PBS was aspired, the cells were fixed with absolute methanol at −20 °C for 5 min.The fixed cells were rewashed with 500 μL of PBS and allowed to dry.The dry cells were stained with 500 μL of 10% crystal violet for 30 s, the excess was removed by washing with distilled water, and the stained preparation was allowed to dry again and mounted on coverslips.The cells were observed under an optical microscope Leica DMi8 at 40× magnifications using bright clear microscopy, and microphotographs were taken to analyze cell morphology.

Gene expression of caspases 8, 9, and 3 by RT-qPCR
30,000 cells were plated in 24 well plates and were exposed to perezone or phenyl glycine perezone at IC 50 values (6.83 µM and 2.6 µM, respectively), following the experimental protocol previously described.After 24 h of treatment, total RNA was isolated with TRIzol reagent (acquired from Invitrogen, Catalogue:15596-026) in accordance with the manufacturer's instructions, and cDNA was reverse transcribed using RevertAid First Strand cDNA Synthesis (acquired from Thermo Scientific, Catalogue: K1622) in accordance with the manufacturer's instructions.Real-time quantification was performed using a Maxima SYBR Green qPCR Master Mix (2X) kit (acquired from Thermo Scientific, Catalogue: K0252) using specific primers listed in Table S9.Reactions were performed on an AriaMx Real-time PCR System from Agilent®.The thermal profile was the following: initial denaturation 95 °C for 10 min, 40 cycles of denaturation 95 °C for 15 s, and amplification annealing/extension 58 °C for 60 s ending with a melting curve.The relative expression levels were analyzed with the 2 −ΔΔCt algorithm (Livak and Schmittgen 2001;Pfaffl 2001) using GAPDH as the housekeeping gene.

In vivo acute toxicity study
The studies were authorized by the bioethics committee of the ENCB-IPN under the license number CEI-ENCB-004/2015.

Animals
Female nulliparous NIH mice (35 ± 10 g body weight) were obtained from the Bioterio of Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional.Animals were acclimated for seven days under standard housing conditions: 12 × 12 h light/ dark cycle, 50-55% relative humidity, and 25 °C of room temperature.Rodent standard diet and water were made available ad libitum.The use of animals follows international and national guidelines for laboratory animal welfare.

Acute toxicity test
Acute toxicity of perezone and phenyl glycine perezone was investigated according to the OECD guideline 423, a single oral dose for 14 days (OECD 2001).Mice fasted four hours before the administration of compounds.Perezone and phenyl perezone (300 and 2000 mg/kg) were dissolved in peanut oil (vehicle) and administered to mice (n = 3 first step and n = 3 s step).An additional group of six animals was administered with the vehicle as control.All animals were observed during the next three hours post-administration and the next fourteen days to identify any behavioral and physical signs related to the toxicity of compounds.After that, the number of alive and dead animals was recorded, and the median lethal dose (LD 50 ) was determined according to the algorithm described in the OECD guideline 423.

Statistical analysis
Data from IC 50 determination and caspases 3, 8, and 9 gene expression were analyzed using a One-way ANOVA followed by Student Newman Keuls test for comparison between groups.Data from gene expression of caspases were analyzed using a two-way ANOVA and a post-hoc Sidak's test to confirm where the differences occurred between groups.* p ≤ 0.05, *** p ≤ 0.001 denote significant differences.Data of the body weight of mice was analyzed by Two-way ANOVA with a Tukey post-hoc test.Values of p < 0.05 were considered statistically significant.GraphPad Prism version 9.0.0 for windows GraphPad software San Diego California USA, www.graphpad.com.

Conclusion
The present phenyl glycine perezone stands out among many other amino acid derivatives of the naturally occurring quinone perezone.Moreover, it has high cytotoxic activity on the U-251 astrocytoma cell line at 2.6 ± 1.69 µM and significantly increases the expression of caspase 8, 9, and 3 genes.These findings are promising and suggest that perezone and phenyl glycine perezone could induce apoptosis signaling in glioblastoma cells U-251.In vivo, the median lethal doses of perezone and phenyl glycine perezone were 500 and 2000 mg/Kg, respectively.The present results can be exploited and are helpful in the search for new therapeutic agents for treating brain cancer and the safety profile.