s2fieldsofstudy: Chemistry Biology

text:
Development of Semisynthetic Apoptosis-Inducing Agents Based on Natural Phenolic Acids Scaffold: Design, Synthesis and In-Vitro Biological Evaluation

A crucial target in drug research is magnifying efficacy and decreasing toxicity. Therefore, using natural active constituents as precursors will enhance both safety and biological activities. Despite having many pharmacological activities, caffeic and ferulic acids showed limited clinical usage due to their poor bioavailability and fast elimination. Therefore, semisynthetic compounds from these two acids were prepared and screened as anticancer agents. In this study, CA and FA showed very potent anticancer activity against Caco-2 cells. Consequently, eighteen derivatives were tested against the same cell line. Four potent candidates were selected for determination of the selectivity index, where compound 10 revealed a high safety margin. Compound 10 represented a new scaffold and showed significant cytotoxic activity against Caco-2. Cell-cycle analysis and evaluation of apoptosis showed that derivatives 10, 7, 11, 15 and 14 showed the highest proportion of cells in a late apoptotic stage.

Introduction
Cancer has the highest social and economic burden among all human diseases, and coupled with that, in the coming four decades it is anticipated to leap from the second to the first place to become the leading cause of death worldwide [1].
Over the years, several natural anticancer molecules were used as scaffolds for the synthesis of anticancer drugs, such as vinblastine and vincristine from Catharanthus alkaloids [2], paclitaxel from Yew trees [3], and podophyllotoxin from Podophyllum resins [4].
Ferulic acid has been used in healthy food and nutrition restoratives due to its wellknown antioxidant properties, and due to this biological effect, it can be used in Alzheimer's disease [25,26]. It is highly recommended for diabetic patients [27]. Moreover, FA has a reported neuroprotective effect [28][29][30] and relieves ischemia-reperfusion-induced cell apoptosis [31,32]. FA also has a chemo-preventive activity against carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats [33,34]. Equally important is its antiproliferative action on osteosarcoma cells where it induces apoptosis by blocking the PI3K/Akt pathway [35].
It is highly recommended for diabetic patients as it can alleviate oxidative stress and also has an antihyperglycemic effect. [27] Recently, several phenolic compounds were tested against a spectrum of severe pathological conditions and both CA and FA showed potential results against coronavirus-based infections [36].
The aforementioned biological activities are achieved through a variety of mechanisms; the anticancer activity of both CA and FA is exerted by two mechanisms, acting as primary antioxidants that eliminate reactive oxygen species (ROS) and as secondary antioxidants (pro-oxidants) preventing the splitting of peroxides by metal ion chelation (iron and copper) [37,38]. Thus, inhibiting oxidative degradation of essential biological molecules (lipids, amino acids, polyunsaturated fatty acids and DNA bases) and preventing the formation of cellular lesions [25,[39][40][41][42].
As antioxidants, the study of the structure activity relationship of both acids and their derivatives showed that the hydroxy groups in caffeic and ferulic acids proved to be a pre-requisite for anti-oxidant activity, through their free radical scavenging and metal chelation properties. The 4-hydroxy moiety supports the resonance around the whole molecule resulting in higher stability due to the formation of the phenoxy radical. It is also accepted that a second 3-hydroxy substitution in caffeic acid promotes the antioxidant quality due to the formation of a stable ortho-quinone structure. Equally, in ferulic acid, the methoxy group enhances the resonance of the aromatic ring due to its electron-donating attributes. The acids have an additional aspect where the COOH tail provides lipid support and resonates with its neighboring unsaturation, offering alternative sites to prevent membrane deterioration by free radicals while the inclusion of H-donating groups in the side chain such as amide moiety accelerates the activity [43][44][45] (Figure 2). Caffeic acid and its different derivatives were reported to possess many biological activities such as immunostimulants [9][10][11][12], antibacterial [13,14], anti-inflammatory [15], cardio protective [15,16], anti-atherosclerotic [9,17], antidiabetic [15,18], antiproliferative [19,20], hepatoprotective [21], anticancer [9,[13][14][15][22][23][24], and Alzheimer's disease [25].
Ferulic acid has been used in healthy food and nutrition restoratives due to its wellknown antioxidant properties, and due to this biological effect, it can be used in Alzheimer's disease [25,26]. It is highly recommended for diabetic patients [27]. Moreover, FA has a reported neuroprotective effect [28][29][30] and relieves ischemia-reperfusion-induced cell apoptosis [31,32]. FA also has a chemo-preventive activity against carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats [33,34]. Equally important is its antiproliferative action on osteosarcoma cells where it induces apoptosis by blocking the PI3K/Akt pathway [35].
It is highly recommended for diabetic patients as it can alleviate oxidative stress and also has an antihyperglycemic effect. [27] Recently, several phenolic compounds were tested against a spectrum of severe pathological conditions and both CA and FA showed potential results against coronavirus-based infections [36].
The aforementioned biological activities are achieved through a variety of mechanisms; the anticancer activity of both CA and FA is exerted by two mechanisms, acting as primary antioxidants that eliminate reactive oxygen species (ROS) and as secondary antioxidants (pro-oxidants) preventing the splitting of peroxides by metal ion chelation (iron and copper) [37,38]. Thus, inhibiting oxidative degradation of essential biological molecules (lipids, amino acids, polyunsaturated fatty acids and DNA bases) and preventing the formation of cellular lesions [25,[39][40][41][42].
As antioxidants, the study of the structure activity relationship of both acids and their derivatives showed that the hydroxy groups in caffeic and ferulic acids proved to be a prerequisite for anti-oxidant activity, through their free radical scavenging and metal chelation properties. The 4-hydroxy moiety supports the resonance around the whole molecule resulting in higher stability due to the formation of the phenoxy radical. It is also accepted that a second 3-hydroxy substitution in caffeic acid promotes the antioxidant quality due to the formation of a stable ortho-quinone structure. Equally, in ferulic acid, the methoxy group enhances the resonance of the aromatic ring due to its electron-donating attributes. The acids have an additional aspect where the COOH tail provides lipid support and resonates with its neighboring unsaturation, offering alternative sites to prevent membrane deterioration by free radicals while the inclusion of H-donating groups in the side chain such as amide moiety accelerates the activity [43][44][45] (Figure 2).
As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49]. Molecules 2022, 27, x FOR PEER REVIEW 3 of 20 Figure 2. SAR of caffeic acid and ferulic acid as antioxidants.
As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1)  Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:   It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1)

H
As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1) were synthesized ( Figure 3) via reacting the respective phenolic acids with a panel of amines, adopting N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent. Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:  As cell apoptosis promoters, it was established that CA and FA play a distinctive role in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelial growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1) were synthesized ( Figure 3) via reacting the respective phenolic acids with a panel of amines, adopting N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent.
Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:

Chemistry
The synthesis of CA and FA derivatives 3-20 is displayed in Figure 3. Preparation of these candidates was carried out by reacting the naturally obtained caffeic or ferulic acids with different amines using N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent for the preparation of the amide linkage starting from the acids. Separated amides 3-20 were purified by column chromatography. The main idea is screening the effect of changing the alkyl/cycloalkyl/aralkyl/aryl/heterocyclic moiety of the amide functionality on the biological activity. Compounds 1 and 2 exhibited a sky-blue color in UV, which turns to a greenish yellow color on exposure to ammonia vapor and spraying with AlCl3, indicating that they are phenolic acid in nature. 1 H NMR spectrum of 1 showed the characteristic signals for caffeic acid [50], which was characterized by two trans-olefinic protons at δ 6.16 and δ 7.41 ppm with a large coupling constant δ 15.88 Hz, assigned to H-8 and H-7, respectively. An ABX system was displayed at δ 6.76, 6.95 and δ 7.04 ppm assigned to H-5, H-6 and H-2, respectively. The 13 C NMR showed the nine characteristic carbon signals for 3,4-dihydroxy trans cinnamic acid with two oxy carbons at δ 146.0 and δ 148.5 (C-3 and 4), one carbonyl carbon at δ 168.3 (C-9), two trans olefinic carbons at δ 145.07 and δ 115.5 ppm, in addition to C-1 at δ 126.1, C-2 at δ 115.1, C-5 at δ 116.2 and C-6 at δ 21.6. 1 H NMR in encouraging cell apoptosis in human cervical cancer cells through inhibition of the Bcl-2 activity [46,47] and in non-small lung cancer cells (NSCLC) through the MAPK pathway [48] In another pathway, it was observed that the acids are responsible for suppressing hepatocellular carcinoma cells (HCC) via inhibiting the expression of vascular endothelia growth factor (VEGF), which in turn impedes angiogenesis of the tumor cells [49].
It is worth mentioning that both CA and FA are highly selective, imparting cytotoxicity to tumor cells but not to normal cells; on the other hand, they are highly metabolized in the body, therefore jeopardizing their stability. By evaluating the two major derivatives-amides and esters-the amides possessed potent cytotoxic activity in addition to high in-vivo stability when put in comparison with the ester derivatives. Following in the same vein, 18 amide compounds (Table 1) were synthesized ( Figure 3) via reacting the respective phenolic acids with a panel of amines, adopting N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent.
Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:   Therefore, the current study was conducted to verify the anti-tumor activity of CA and FA versus their semisynthetic derivatives on different cell lines and explore the underlying mechanisms using the following scheme:

Chemistry
The synthesis of CA and FA derivatives 3-20 is displayed in Figure 3. Preparation of these candidates was carried out by reacting the naturally obtained caffeic or ferulic acids with different amines using N,N'-dicyclohexylcarbodiimide (DCC) as a coupling agent for the preparation of the amide linkage starting from the acids. Separated amides 3-20 were purified by column chromatography. The main idea is screening the effect of changing the alkyl/cycloalkyl/aralkyl/aryl/heterocyclic moiety of the amide functionality on the biological activity. Compounds 1 and 2 exhibited a sky-blue color in UV, which turns to a greenish yellow color on exposure to ammonia vapor and spraying with AlCl 3, indicating that they are phenolic acid in nature. 1 H NMR spectrum of 1 showed the characteristic signals for caffeic acid [50], which was characterized by two trans-olefinic protons at δ 6.16 and δ 7.41 ppm with a large coupling constant δ 15.88 Hz, assigned to H-8 and H-7, respectively. An ABX system was displayed at δ 6.76, 6.95 and δ 7.04 ppm assigned to H-5, H-6 and H-2, respectively. The 13 C NMR showed the nine characteristic carbon signals for 3,4-dihydroxy trans cinnamic acid with two oxy carbons at δ 146.0 and δ 148.5 (C-3 and 4), one carbonyl carbon at δ 168.3 (C-9), two trans olefinic carbons at δ 145.07 and δ 115.5 ppm, in addition to C-1 at δ 126.1, C-2 at δ 115.1, C-5 at δ 116.2 and C-6 at δ 21.6. 1 H NMR spectrum of 2 showed the characteristic signals for ferulic acid [51], which was characterized by two trans-olefinic protons at δ 6.35 and 7.48 ppm with large coupling constant 15.88 Hz, assigned to H-8 and H-7, respectively. An ABX system was affirmed by these signals at δ 6.79, 7.07 and 7.28 ppm, corresponding to H-5, H-6 and H-2, respectively. The methoxy group at C-3 was manifested by the singlet signal at δ 3.82 ppm and integrated as three protons (Supporting Information).
IR spectra of all the candidates showed the characteristic carbonyl band of amide between 1626-1662 cm −1 while the NH band appeared in the range of 3510-3240 cm −1 for all compounds except 3 and 12, which are tertiary amides. 1 H NMR spectra of compounds 3-20 exhibited the appearance of the doublet signal of HC=CH in the range of δ 5.70-7.73 ppm with large coupling constant 15.00-15.90 Hz. Compounds 3 and 12 revealed the multiplet signals in their 1 H NMR spectra at δ 1.04-1.85 ppm assigned to CH 2 of piperazine ring. 1 H NMR spectra of compounds 5, 14 and 17 showed a series of singlet signals in the range of δ 2.26-2.29 ppm due to CH 3 group while the 11 protons corresponding to the cyclohexyl group in compounds 6 and 15 were confirmed by the appearance of multiplet signals in the range of δ 1-3.35 ppm. Compound 8 was the only CA derivative that showed a singlet signal corresponding to OCH 3 group at δ 3.63 ppm while 16 showed an additional OCH 3 group at δ 3.81 ppm. The appearance of singlet signals at δ 5.59 and 3.19 ppm in 1 H NMR spectra of 9 and 18 respectively confirmed the presence of N-CH 2 (Supporting Information). Compounds 11 and 20 revealed the presence of NH 2 group at δ 9.17 and 4.94 ppm, respectively. 13 C NMR spectrum of all compounds confirmed the presence of the characteristic C=O group, showing signals in the range of δ 160.7-170.9 ppm.

Biological Activity 2.2.1. Cell Viability Determination by MTT Assay
The cytotoxicity of the parent compounds 1 and 2 was tested against breast (MCF-7), liver (HepG2), lung epithelial (A459), colorectal (Caco-2) and pancreatic (PANC-1) cancer cell lines. Their cytotoxicity was compared to doxorubicin (DOX) and their selectivity was tested on Vero cells ( Table 2). The cytotoxicity of both 1 and 2 was lower than that of DOX but the selectivity index (SI) was significantly higher than DOX (Supporting Information). Caco-2 showed the highest sensitivity to both acids 1 and 2 (IC 50 = 0.41 µM for both caffeic and ferulic acids) and thus it was selected for testing the cytotoxicity of the 18 semisynthetic candidates. All the synthesized amides were far more potent than the parent acids by 10-fold or more with a slight reduction in the SI of these derivatives (Table 3). It is worth mentioning that caffeic acid derivatives were more potent than ferulic acid derivatives. Among the CA amide derivatives 3-11, compound 10 was the most potent against the Caco-2 cancer cell line with an IC 50 of 0.009 ± 0.09 µM with a SI 14-fold greater than its IC 50 followed by compounds 11, 7 and 9 with an IC 50 of 0.01 ± 0.11, 0.02 ± 0.11 and 0.03 ± 0.29 µM, respectively. The potency of the rest of the CA amide derivatives ranged from IC 50 = 0.05 µM to 0.08 µM, which was still much more potent than the parent acid. Regarding the FA amide derivatives 12-20, compound 20 revealed the highest potency with an IC 50 of 0.01 ± 0.63 µM followed by compounds 15, 17 and 19 with IC 50 of 0.02 ± 0.17, 0.02 ± 0.21 and 0.02 ± 0.65 µM sequentially. Moreover, compounds 12, 13, 14, 16 and 18 showed very good cytotoxicity against Caco-2 with an IC 50 ranging from 0.04 µM to 0.08 µM. The derivatives were much more potent than the parent acid with an excellent SI (Table 3). To further confirm this, cell-cycle analysis and apoptosis assays were performed and the gene expression of p53, caspase 3, Bax, MMP-2 and MMP-9 was quantified.

Cell Cycle Progression
The potential candidates with anti-tumor effect on the Caco-2 cell line along with a high SI were chosen for cell-cycle analysis. The cell-cycle distribution and cell death were analyzed using Annexin V-FITC/PI staining and flow cytometry (Table 4, Figure 4). The potential candidates that showed potent anticancer activity against the Caco-2 cell line and high SI were chosen to investigate how they will change the cell-cycle distribution. 16.51% respectively) which showed a more significant effect than the parent compound 2 itself (13.22%), whereas the derivatives 17, 18 and 20 showed non-significant decrease in the apoptotic population compared to parent compound 2. (Table 4)

Evaluation of Apoptosis by Annexin V-FITC Staining
Quantification of the different types of apoptotic cells was of our interest due to the accumulation of cells at the G2/M phase during cell-cycle analysis following the treatment with the parent compounds 1 and 2 and their potential candidates.
After Caco-2 cells were treated with parent compounds 1 and 2 and their derivatives,

Flow Cytometry Analysis Showing Induction of Apoptosis
Cell-cycle analysis showed that the treatment of Caco-2 cells with the parent compounds and their derivatives resulted in an increase in the cell population in the G2/M phase, indicating apoptosis. In the case of caffeic acid derivatives, the highest induction of apoptosis was seen in compounds 10, 7 and 11 (23.42%, 18.26% and 14.04%), respectively, which showed a more significant increase than the caffeic acid itself (11.31%). On the contrary, compounds 5 and 9 showed a non-significant increase in the apoptotic population compared to the parent compound 1 (Table 4).
Concerning Ferulic acid derivatives, the accumulation in the Pre-G1 phase and thus the highest induction of apoptosis was observed with candidates 15 and 14 (25.17% and 16.51% respectively) which showed a more significant effect than the parent compound 2 itself (13.22%), whereas the derivatives 17, 18 and 20 showed non-significant decrease in the apoptotic population compared to parent compound 2. (Table 4)

Evaluation of Apoptosis by Annexin V-FITC Staining
Quantification of the different types of apoptotic cells was of our interest due to the accumulation of cells at the G2/M phase during cell-cycle analysis following the treatment with the parent compounds 1 and 2 and their potential candidates.
After Caco-2 cells were treated with parent compounds 1 and 2 and their derivatives, they were stained with annexin V-FITC/PI, and then flow cytometry was used to detect the cell-cycle distribution as shown in Figure 5 and Table 5.    The results of the evaluation of apoptosis revealed that potential candidates 7, 10, 11, 14 and 15 showed the highest proportion of cells in the late apoptotic stage (stained by Annexin V-FITC and PI).

Gene Expression Caffeic Acid and Ferulic Acid Derivatives Enhanced Pro-Apoptotic Genes
The effect of caffeic acid 1 and its amide derivatives 5, 7, 9, 10 and 11 as well as ferulic acid 2 and its derivatives 14, 15, 17, 18, and 20 on the expression of pro-apoptotic genes were tested. β-actin gene was used as a control gene. Gene expression was chosen over Western blotting for more accurate quantification and thus comparison. The gene expression of the p53, caspase 3 and Bax genes was measured. Most of the derivatives showed an increased expression of the three genes, which was evidenced by increasing apoptotic effects. In the case of caffeic acid derivatives, compound 10 showed the highest overexpression with an increase of 14-to 16-fold in the three genes, followed by 7 and 11 ( Figure 6). Regarding the ferulic acid derivatives, 15 showed the highest overexpression of 10-fold in the p53 and 15-fold in the caspase 3 and Bax genes compared to the parent acid ( Figure 7).

Caffeic Acid and Ferulic Acid Derivatives Inhibit Metastasis Enhancing Genes
Matrix metalloproteinases (MMPs) appear to play an important role in the formation and progression of human cancers. MMPs are involved in the breakdown of the extracellular matrix, which is a crucial stage in tumor invasion and spread [52]. A literature review revealed that both 1 and 2 decrease the expression of both MMP-2 and 9, which may lead to decrease in enzymatic activity [53].
The derivatives of both 1 and 2 showed a significant decrease in the expression of both MMP2 and MMP9. Out of the most potential candidates, 17 and 7 showed the greatest inhibition in both groups (Figures 8 and 9).

Structure-Activity Relationship Correlation
The parent phenolic acids 1 and 2 were tested against five cancer cell lines: Caco-2, A549, PANC-1, MCF7, and Hep G2, as well as one normal Vero cell line using DOX as positive reference. The parent compounds showed significant antitumor activity against Caco-2 cell line and with excellent selectivity index (SI). Consequently, all the newly synthesized amide derivatives 3-20 were evaluated for their cytotoxic activity against Caco-2 cancer cell line. The SI of amide derivatives was determined using the Vero normal cell line. They showed excellent cytotoxic activity, much more potent than the parent acids 1 and 2. Based on the phenolic acids scaffold, the amide derivatives were designed to have all the features of both acids as well as the stable amide linkage. All the amide derivatives 3-20 showed very promising anticancer activity against Caco-2 cell line and very good SI. So, changing the carboxylic acid moiety into an amide functionality increased the biological activity and did not destroy the selectivity index of the parent compounds. Examining the structure of the six most active compounds (7, 10, 11, 14, 15 and 17) (Figure 10), starting from the anticancer activity against Caco-2 cell line and excellent SI, followed by cell-cycle analysis and apoptosis assays, then the effect of these candidates on gene expression of p53, caspase 3, Bax, MMP-2 and MMP-9, it was found that besides all the features of both acids and the amide linkage, five compounds out of six have aromatic amide moieties. The presence of p-chloro on the phenyl ring of the amide linkage resulted in the most cytotoxic compound of all the six candidates. Replacing the chloro atom in compound 10 with bromo in compound 7 decreases the antitumor activity. Moreover, it was found that the presence of the cycloalkyl group of the amide linkage in the case of compound 15 increased the induction of apoptosis and showed the highest proportion of cells in late apoptotic stage. In case of enhancing the pro apoptotic genes (p53, caspase 3 and Bax), compound 10 (with p-chlorophenyl) revealed the highest activity. Furthermore, in case of inhibiting the metastasis-enhancing genes, compound 17 with m-tolyl group was the most active one. were tested. β-actin gene was used as a control gene. Gene expression was chosen over Western blotting for more accurate quantification and thus comparison. The gene expression of the p53, caspase 3 and Bax genes was measured. Most of the derivatives showed an increased expression of the three genes, which was evidenced by increasing apoptotic effects. In the case of caffeic acid derivatives, compound 10 showed the highest overexpression with an increase of 14-to 16-fold in the three genes, followed by 7 and 11 ( Figure  6). Regarding the ferulic acid derivatives, 15 showed the highest overexpression of 10-fold in the p53 and 15-fold in the caspase 3 and Bax genes compared to the parent acid ( Figure  7). Figure 6. The effect of CA and its derivatives on the expression of P53, Caspase 3 and Bax genes by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. * Significant from CA at p < 0.05, ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001.

Caffeic Acid and Ferulic Acid Derivatives Inhibit Metastasis Enhancing Genes
Matrix metalloproteinases (MMPs) appear to play an important role in the formation and progression of human cancers. MMPs are involved in the breakdown of the extracellular matrix, which is a crucial stage in tumor invasion and spread [52]. A literature review revealed that both 1 and 2 decrease the expression of both MMP-2 and 9, which may lead to decrease in enzymatic activity [53].
The derivatives of both 1 and 2 showed a significant decrease in the expression of both MMP2 and MMP9. Out of the most potential candidates, 17 and 7 showed the greatest inhibition in both groups (Figures 8 and 9). The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001. Figure 8. The effect of CA and its derivatives on the expression of MMP-2 and MMP-9 by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. ** Significant from CA at p < 0.01, *** Significant from CA at p < 0.001. Figure 9. The effect of FA and its derivatives on the expression of MMP-2 and MMP-9 genes by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. *** Significant from FA at p < 0.001. Figure 9. The effect of FA and its derivatives on the expression of MMP-2 and MMP-9 genes by RT-qPCR. The results were represented as mean of fold changes ± SD. β-actin gene was used as a control gene. *** Significant from FA at p < 0.001.

Structure-Activity Relationship Correlation
The parent phenolic acids 1 and 2 were tested against five cancer cell lines: Caco-2, A549, PANC-1, MCF7, and Hep G2, as well as one normal Vero cell line using DOX as positive reference. The parent compounds showed significant antitumor activity against Caco-2 cell line and with excellent selectivity index (SI). Consequently, all the newly synthesized amide derivatives 3-20 were evaluated for their cytotoxic activity against Caco-2 cancer cell line. The SI of amide derivatives was determined using the Vero normal cell line. They showed excellent cytotoxic activity, much more potent than the parent acids 1 and 2. Based on the phenolic acids scaffold, the amide derivatives were designed to have all the features of both acids as well as the stable amide linkage. All the amide derivatives 3-20 showed very promising anticancer activity against Caco-2 cell line and very good SI. So, changing the carboxylic acid moiety into an amide functionality increased the biological activity and did not destroy the selectivity index of the parent compounds. Examining the structure of the six most active compounds (7, 10, 11, 14, 15 and 17) (Figure 10), starting from the anticancer activity against Caco-2 cell line and excellent SI, followed by cell-cycle analysis and apoptosis assays, then the effect of these candidates on gene expression of p53, caspase 3, Bax, MMP-2 and MMP-9, it was found that besides all the features of both acids and the amide linkage, five compounds out of six have aromatic amide moieties. The presence of p-chloro on the phenyl ring of the amide linkage resulted in the most cytotoxic compound of all the six candidates. Replacing the chloro atom in compound 10 with bromo in compound 7 decreases the antitumor activity. Moreover, it was found that the presence of the cycloalkyl group of the amide linkage in the case of compound 15 increased the induction of apoptosis and showed the highest proportion of cells in late apoptotic stage. In case of enhancing the pro apoptotic genes (p53, caspase 3 and Bax), compound 10 (with p-chlorophenyl) revealed the highest activity. Furthermore, in case of inhibiting the metastasis-enhancing genes, compound 17 with m-tolyl group was the most active one.

General
Silica gel 60 (70-230 mesh ASTM; Fluka, Steinheim, Germany), Diaion HP-20 AG, Sephadex LH-20 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and silica gel GF254 precoated thin layer plates (Fluka, Steinheim, Germany) were used. Chromatograms detections were performed under UV light (at 254 and 366 nm) and sprayed by p-anisaldehyde sulphuric acid spray reagent. Melting points (°C, uncorrected) were determined on Stuart apparatus and the given values were uncorrected. Progress of the reaction was monitored by thin layer chromatography (TLC) using aluminum sheets precoated with UV fluorescent silica gel (MERCK, Rahway, NJ, USA, 60 F 254), and spots were visualized by UV lamp. The solvent system used was dichloromethane: methanol (in different

General
Silica gel 60 (70-230 mesh ASTM; Fluka, Steinheim, Germany), Diaion HP-20 AG, Sephadex LH-20 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and silica gel GF 254 precoated thin layer plates (Fluka, Steinheim, Germany) were used. Chromatograms detections were performed under UV light (at 254 and 366 nm) and sprayed by p-anisaldehyde sulphuric acid spray reagent. Melting points ( • C, uncorrected) were determined on Stuart apparatus and the given values were uncorrected. Progress of the reaction was monitored by thin layer chromatography (TLC) using aluminum sheets precoated with UV fluorescent silica gel (MERCK, Rahway, NJ, USA, 60 F 254), and spots were visualized by UV lamp. The solvent system used was dichloromethane: methanol (in different ratios). The IR spectra (cm −1 ) were determined using KBr discs on a Shimadzu IR8400s Spectrophotometer (Microanalytical Unit, Faculty of Pharmacy, Cairo University, Cairo, Egypt). 1 H NMR and 13 C NMR spectra were performed on Bruker 400-BB 400 MHz Spectrophotometer, microanalytical unit, Faculty of Pharmacy, Cairo University, Cairo, Egypt, using tetramethylsilane (TMS) as internal standard, and chemical shift values were recorded in ppm on δ scales. Peak multiplicities were designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Elemental analyses were performed at the Regional Center for Mycology and Biotechnology, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. The data were statistically evaluated using the Graph Pad Prism 6 program. Data were reported as mean ± SD of the triplicates of each experiment. One-way ANOVA with multiple comparisons post-hoc tests were used for analysis. Results were considered significant at p < 0.05.

Plant Material
The grains of oats (Avena sativa L. Family Poaceae) were obtained from the Agricultural Research Center, Giza, Egypt. The plants were authenticated at Botany Department, Faculty of Science, Cairo University, Giza, Egypt. Voucher samples no (3-10-2018) of the plants were deposited at the Museum of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

Extraction and Isolation of Caffeic and Ferulic Acids
The powdered oats (2 kg) were extracted with 80% ethanol at 55 • C for 165 min (14 L × three times), the ethanolic extract was evaporated under vacuum at 40 • C. The ethanolic residue (350 gm) was suspended in distilled water (1 L) and defatted by methylene chloride (1 L × 3 times), then the defatted aqueous solution was fractionated over a Dianion column using 100% water, methanol-water (1:1 v/v) and 100% methanol. The solvent in each case was evaporated under vacuum to obtain three fractions 100% water fraction (75 gm), MeOH-water (1:1) fraction (55 gm) and 100% methanol fraction 67 gm.

Annexin V/Propidium Iodide (PI) Apoptosis Assay by Flow Cytometry
In order to determine the percentage of viable, apoptotic and necrotic cells after treatment with our compounds, double-staining with fluorescein isothiocyanate (FITC) and PI was performed. Annexin V-FITC Apoptosis Detection Kit (BioVision, CA, USA) was used. Briefly, cultured Caco-2 were treated with the tested compounds or DMSO for 24h. Cells were then collected by centrifugation at 2000 rpm for 5 min, and the pellet was washed with PBS then centrifuged again at 2000 rpm for 5 min then resuspended in 500µL of the kit's binding buffer before adding the Annexin V-FITC and PI. The cells were then incubated at room temperature in the dark for 5 min. Annexin V-FITC binding was measured using FACS Calibur flow cytometer (BD biosciences, USA) (Ex = 488 nm, Em = 533 nm, FL1 filter for annexin and FL2 filter for PI).

Quantitative Polymerase Chain Reaction (qPCR) and RNA Extraction
The treated cells were harvested and the total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer's instruction. The primers of the required genes (Table 6) were used for SYBR Green qRT-PCR using a one-step RT-PCR kit with SYBR Green (Bio-Rad). qRT-PCR was performed on Rotor Gene Q (Qiagen). The mRNA levels of p53, Bcl-2, Caspase-3, Bax, MMP-2, and MMP-9 were calculated using the 2 −∆∆CT method with the endogenous β-actin mRNA as control to normalize the values. Rotor-Gene 6000 Series Software was used for data analysis. and their HGNC ID was added ( Table 6) Table 6. Primers used in qRT-PCR.

Conclusions
Despite the high efficacy of CA and FA, they are limited by their high metabolism and consequent low bioavailability. Therefore, we were challenged to synthesize highly stable and biologically active compounds. Henceforth, we designed and investigated 18 congeners of amide nature; they were all superior to their parent acids in terms of cytotoxic activity against Caco-2. The selected candidates for cell-cycle analysis and gene expression assays were those displaying superior cytotoxicity and high SI using Vero normal cells.
Subsequently, the assays offered six remarkably active compounds, 7, 10, 11, 14, 15 and 17, and five of them comprised an aromatic ring adjacent to the amide bridge. In accordance with the SAR, the highest cytotoxicity was evident amongst the aromatic derivative with p-chloro phenyl 10, displaying excellent cytotoxic activity, this was deduced when the cytotoxicity decreased after the chloro residue was replaced with a bromo substitution. It could also be concluded that overall, the caffeic acid amides surpassed the ferulic analogues.
We are planning to further study other amide derivatives and assess their activity in a suitable animal model.


First synthetic report and antioxidant potential of natural product (±)-5,7-dihydroxy-8-methyl-3-(2',4'-dihydroxybenzyl)chroman-4-one from Chinese medicine Gan Luo Xin pill

Abstract The first synthetic route of naturally occurring (±)-5,7-dihydroxy-8-methyl-3-(2’,4’-dihydroxybenzyl)chroman-4-one (1) from Gan Luo Xin pill was successfully accomplished. The synthetic route has been developed retro-synthetically in 9 simple steps with a high yield of ∼80%. The synthetic protocol was developed using readily available starting material phloroglucinol. The key intermediate 2,4,6-trihydroxy-3-methyl acetophenone (4) was synthesized via Vilsmeier–Haack reaction, followed by reduction using sodium cyanoborohydride and acylation reaction. LC-MS, IR, 1H NMR, 13C NMR of 1 have been analyzed to confirm the structure of (±)-5,7-dihydroxy-8-methyl-3-(2’,4’-dihydroxybenzyl)chroman-4-one (1) and found in agreement with the natural molecule. The target compound showed 97% and 87% antioxidant activity in DPPH and ABTS assay at 1 mg/ml concentration, respectively. The compound (1) also showed ferric ion reducing activity with the absorbance of 0.18 at 700 nm. The present study could be useful in developing synthetic routes of other potential naturally occurring homoisoflavonoid. Graphical abstract

Introduction
Natural products are untapped sources of enormous valuable substances. Researchers periodically explore them, just like digging the mines for valuable minerals and getting recognition (Ahmad et al. 2016(Ahmad et al. , 2017. To date, innumerable natural products are isolated and evaluated for bioprospecting potential (Atanasov et al. 2021). Still, the luster of natural products never declined with time. Secondary metabolites produced by plants and fungi exhibit various pharmaceutical properties (Hussein and El-Anssary 2018). They remain an integral part of traditional medicine in many countries. The Gan Luo Xin pill is a traditional Chinese medical formula composed of 20 types of herbs (such as Panax ginseng, Astragalus membranaceus, Polygonatum sibiricum, and Crataegus pinnatifida, etc.) that has been shown effective for the treatment of hepatitis B (Li et al. 2014). It exhibits immunomodulatory and anti-inflammatory activities in therapeutic research and has been proven curative in a clinical trial. In 2014, Li et al. reported the isolation and structural determination of two new homoisoflavonoids, (±)-5,7-dihydroxy-8-methyl-3-(2 0 ,4 0 -dihydroxybenzyl)chroman-4-one (1) and (±)-5,7-dihydroxy-6,8-dimethyl-3-(2 0 ,4 0 -dihydroxybenzyl)chroman-4-one (2), along with two known homoisoflavonoids, 5,7-dihydroxy-6-methyl-3-(2 0 ,4 0 -dihydroxybenzyl)chroman-4-one (3) and disporopsin (4). The structures of compounds 1-4 reported from the Gan Luo Xin pill by Li et al. (2014) have been displayed in Figure 1. Dimethyl analogs of homoisoflavonoid (2) reported from Genus Polygonatum have anti-inflammatory activity (Zhao et al. 2019) and insulin-stimulated glucose uptake  activity. Compound 3 is also reported from Liriope platyphylla roots with anti-platelet activity (Tsai et al. 2013). Disporopsin (4) possesses cytotoxicity against MCF7 and A549 cell lines (Nguyen et al. 2006). Natural product isolation is reasonably necessary but suffers from limitations such as tedious isolation, environmental stress, low yield, seasonal and species variation. The synthesis of natural products can be helpful to overcome these limitations so that they can be available throughout the year. Our research group has previously developed the synthetic route for other natural products such as rugosaflavonoid A and podocarflavone A and evaluated them for biological activities (Puranik and Srivastava 2017;Puranik et al. 2021). Therefore, we focused on developing the synthetic route via retrosynthesis followed by characterization of recently isolated (±)-5,7-dihydroxy-8-methyl-3-(2 0 ,4 0 -dihydroxybenzyl)chroman-4-one (1) by the easy, economical and convenient method and evaluated it for antioxidant activity by DPPH, ABTS and ferric ion reducing assays. All the synthesized intermediates and target molecule (1) were spectroscopically evaluated by FT-IR, 1 H NMR, 13 C NMR, followed by LCMS analysis.

Antioxidant activity
The pure isomer (3 R)-5,7-dihydroxy-8-methyl-3-(2 0 ,4 0 -dihydroxybenzyl)-chroman-4-one of compound (1) reported from Polygonatum odoratum possess antioxidant potential ; hence the antioxidant activity of the compound (1) was also evaluated by DPPH, ABTS and Ferric ion reducing assays. The target homoisoflavonoid (1) showed DPPH radical scavenging activity of 97% at 1 mg/ml concentration. It also showed ABTS radical scavenging activity of 87% at the same concentration. Ferric ion reducing ability can be observed by the increase in absorbance at 700 nm in the ferric ion reducing assay. In this regard, the synthesized homoisoflavonoid (1) showed increased absorbance at 700 nm with an increasing concentration from 0.06-1mg/ml. The results are compared with BHA, BHT, and ascorbic acid. The % inhibition graphs of DPPH and ABTS radicals have been given in supplementary file 1 ( Figure S5).

Experimental section
All the intermediates 2-9 are synthesized and characterized by 1H NMR. Their methodologies are given in supplementary file 1 (Supplementary file 1). It also contains the LCMS, 1 H NMR, and 13 C NMR spectra of compound 1 and graphs of DPPH, ABTS, and ferric ion reducing assays.

Antioxidant activity of homoisoflavonoid (1)
The compound was tested for free radical scavenging assays using DPPH, ABTS as per the reported protocol (Srivastava et al. 2012). The ferric ion reducing assay was performed to identify its reducing ability at 700 nm. The detail protocols are mentioned in Supplementary file1

Conclusion
Natural products represent a wide variety of molecules with enormous bioprospecting potential. The development of synthetic routes can enhance their availability throughout the year to reduce environmental stress by cutting the plants repeatedly. The developed synthetic route of (±)-5,7-dihydroxy-8-methyl-3-(2 0 ,4 0 -dihydroxybenzyl) chroman-4-one (1) in 9 steps with economically available reagents will work as an insight for the chemist to do biologically active natural product synthesis. The synthesized natural product (1) showed antioxidant activity, enhancing its application as a ROS scavenger.


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Tetrahydroquinolinone derivatives exert antiproliferative effect on lung cancer cells through apoptosis induction

The anticancer properties of quinolones is a topic of interest among researchers in the scientific world. Because these compounds do not cause side effects, unlike the commonly used cytostatics, they are considered a promising source of new anticancer drugs. In this work, we designed a brief synthetic pathway and obtained a series of novel 8-phenyltetrahydroquinolinone derivatives functionalized with benzyl-type moieties at position 3. The compounds were synthesized via classical reactions such as nucleophilic substitution, solvent lysis, and condensation. Biological evaluation revealed that 3-(1-naphthylmethyl)-4-phenyl-5,6,7,8-tetrahydro-1H-quinolin-2-one (4a) exhibited potent cytotoxicity toward colon (HTC-116) and lung (A549) cancer cell lines. Analysis of the mechanism of action of compounds showed that compound 4a induced cell cycle arrest at the G2/M phase, leading to apoptotic cell death via intrinsic and extrinsic pathways. Taken together, the findings of the study suggest that tetrahydroquinolinone derivatives bearing a carbonyl group at position 2 could be potential lead compounds to develop anticancer agents for the treatment of lung cancers.

Apoptosis or programmed cell death is a basic physiological process that plays a key role in the maintenance of tissue homeostasis. It is genetically regulated as a normal physiological response to many stimuli and is associated with other processes such as aging and embryogenesis. Disorders in apoptosis mechanisms can lead to various diseases, such as cancers. The disability of cancerous cells to keep up the balance between proliferation and death results in the development of abnormal tissue and formation of solid tumors 1,2 . Apoptosis is a double-track mechanism that occurs via extrinsic and intrinsic pathways. The extrinsic pathway involves transmembrane death receptor-mediated interactions, whereas the intrinsic pathway is mediated by mitochondria and starts with the binding of BAX/BAK protein to the mitochondrial membrane leading to the release of cytochrome c 3 . Both pathways converge at the same point-caspases-regulated execution. The majority of cytostatic drugs used in anticancer therapy cause burdensome side effects in patients; therefore, there is a constant search for novel chemical compounds that are safe. Quinolones are a family of compounds characterized by antibacterial properties. However, research indicates that some of them exhibit potential anticancer properties, especially apoptosis activation. It has been proven that ciprofloxacin, which is the most active fluoroquinolone, can activate apoptosis in breast, bladder, and prostate cancers, colorectal carcinoma, and melanoma. At the molecular level, this chemotherapeutic causes an adverse increase in the concentration of BAX protein which results in differences in the BAX:BCL-2 ratio. It also enhances the expression of p53 protein and activation of caspases [4][5][6][7][8] . Another fluoroquinolone, enoxacin, also works by activating apoptosis. It has been shown that programmed cell death is induced in prostate cancer by a significant increase in CASP3 mRNA and cleaved PARP expression as well as mitochondrial depolarization 9 . Levofloxacin, which is also a fluoroquinolone, induces apoptosis in breast and lung cancer through a caspase-dependent pathway and mitochondrial disfunction 10 . Obviously, several wellknown chemotherapeutic agents from the quinolone family display anticancer properties 11 . One of the newer quinolones, voreloxin, which is currently in clinical trials for the treatment of acute myeloid leukemia, shows a high affinity to eukaryotic type II topoisomerase and induces apoptosis through double-strand DNA breaks. Because the stable quinolone core is characterized by a favorable toxicity profile, voreloxin does not generate www.nature.com/scientificreports/ reactive oxygen species and free radicals unlike the widely used anthracyclines. Hence, this compound is much less harmful to the off-target tissues compared to the commonly used chemotherapeutic drugs 12,13 .
Over the past few years, researchers have proposed several interesting short synthesis pathways for the formation of various quinolinone cores. A.M.Y. Moustafa and S.B. Bakare described the synthesis of new 7-hydroxy quinolinone derivatives based on parent 7-hydroxy-4-methyl-2-oxo-1-amino quinolone. A series of new chemical compounds containing different substituents at positions 3-8 were assessed for their biological activity against breast cancer. It was observed that the most active compound A (Fig. 1) increased the level of active caspase-3, leading to the induction of apoptosis via mitochondrial pathway 14 . An interesting study demonstrated the synthesis of hybrid molecules containing cinnamic acid and 1-amino-4-methyl-2-quinolinone derivatives. The final products were synthesized via pyrogallol and ethyl acetoacetate condensation, resulting in the formation of 7,8-dihydroxy-4-methyl-coumarin which was then treated with hydrazine in pyridine to obtain a desirable 2-quinolinone core. Biological studies showed that compound B, which was qualified for further assessment, caused an increase in apoptotic cell fraction in the HCT-116 cell line 15 . Zhou P. et al. also proposed a few-step synthetic pathway for novel 4(1H)-quinoline derivatives based on the lead compound 7-chloro-4(1H)-quinolone characterized by poor solubility. In this study, five novel, functionalized chemical compounds were obtained via a previously described procedure to improve the solubility of final products. All these compounds exhibited antitumor activity; however, structure C also displayed proapoptotic properties and increased the level of Bak protein, inducing cell cycle arrest at the G2/M phase via a p53-dependent pathway in the HepG2 cell line 16 . Another study described the brief synthesis of ring-fused quinolinones. P. Arsenyan et al. proposed simple methods for the synthesis of substituted selenopheno [2,3-c] and selenopheno [3,2-c] quinolinones D 17 . Biological evaluation of compounds showed that these derivatives induced apoptosis via an intrinsic pathway in human breast adenocarcinoma, estrogen-positive (MCF-7) tumor cell line 18 . A. Chilin et al. reported a novel, more convenient pathway for synthesizing 1,6,8-trimethylfuro[2,3-h]quinolin-2-one derivatives E, which induced programmed cell death through mitochondrial depolarization. The synthesized compounds exerted a cytotoxic effect in Jurkat cells in the dark, which was dependent on ADH-catalyzed oxidation 19 . Finally, new tetrahydroquinoline derivatives synthesized in our previous work demonstrated potent anticancer activity on human colon cancer cells (HTC-116) and nonsmall cell lung cancer cells (A549). The literature review and experience derived from this project inspired us to propose and evaluate a short synthetic route for the synthesis of new tetrahydroquinoline derivatives modified with benzyl-type substituents at position 3 20 .
Chemistry. Synthesis of tetrahydroquinoline derivatives modified at position 3. We synthesized the new tetrahydroquinoline derivatives through the following three-step pathway (Fig. 2a). The starting compound was ethyl benzoylacetate (1). In the first step, ethyl benzoylacetate and a variety of benzyl-type halides were subjected to the S N 2 reaction. The reaction was carried out in dimethylformamide (DMF) in the presence of K 2 CO 3 . Then, the resulting benzyl-type derivatives of ethyl benzoylacetate (2a-e) were subjected to ammonolysis in 24% ammonia water solution. The corresponding benzoylopropanamides (3a-e) were condensed with cyclohexanone in the presence of TsOH and anhydrous MgSO 4 to obtain desirable tetrahydroquinolinones (4a-e) modified with benzyl-type substituents at position 3 21 . After biological evaluation, the most active 3-(1-naphthylmethyl)-4-phenyl-5,6,7,8-tetrahydro-1H-quinolin-2-one (4a) was converted into chlor 22 and methoxy 23 derivatives (5, 6) (Fig. 2b). It is worth mentioning that in some cases tautomers and rotamers were observed, which is described in the supplementary information.  www.nature.com/scientificreports/  www.nature.com/scientificreports/ of treatment and presented in Table 1. The results of the biological evaluation showed that compounds 4b, 4c, 4d and 4e did not have any effect on the tested cell lines in the range of 0-50 µM, whereas compounds 5, 4a, and 6 significantly decreased the viability of colon cancer cells (HCT-116) with an IC 50 value of approximately 13 µM. Additionally, 4a and 6 (A549) had an IC 50 value of 11.33 ± 0.67 and 40.18 ± 0.94 µM, respectively, and exhibited almost no suppressive effect on human normal HEK293 kidney cells in tested concentrations compared to the effect of compounds on A549 and HCT-116 cells (Fig. 3). In this assay, the IC 50 value of 5-FU and ETP was recorded as a positive control. Based on the observations, 4a was recognized as the most potent, and was therefore chosen for further analysis of the mechanisms underlying the antiproliferative action. Next, the effect of 4a on the number of cell-forming clones was examined by the colony formation assay. As shown in Fig. 3, the number of observed colonies was found to be significantly decreased after treatment in comparison to the DMSO-treated control, and this effect was dose-dependent.
Compound 4a induces sub-G 1 -phase cell cycle arrest in A549 cells. The antiproliferative effect of 4a was further assessed by examining the progression of the cell cycle (Fig. 4). The tested compound induced cell cycle arrest at the G 2 phase in A549 cells. The fraction of cells in this phase increased significantly from 3.2% (DMSO-treated cells) to 15.5% and 33.8% (cells treated with the compound for 24 and 48 h, respectively). The observed changes corresponded with a time-dependent decrease in the fraction of cells at the G 0 /G 1 phase and a concomitant increase in the sub-G 0 phase.

Compound 4a induces caspase-dependent apoptosis in A549 cells.
To further explain the mechanism by which 4a induced cell death in cancer cells, a flow cytometric analysis was performed by dual-staining the cells with 7-aminoactinomycin D (7-AAD) and Annexin V-fluorescein isothiocyanate (FITC). Annexin-V is a Ca 2+ -dependent protein with potent affinity to phosphatidylserine 24 . During early apoptosis, cells lose plasma membrane asymmetry through, for example, the externalization of phosphatidylserine. Once exposed to the outside of the cell surface, phosphatidylserine can be recognized by Annexin-V, and quantified 25 . On the other hand, 7-AAD is a dye that cannot penetrate the intact cell membrane, and allows differentiating between necrotic and late apoptotic cells from living and early apoptotic cells. As shown in Fig. 5, exposure of A549 cells to 4a at its IC 50 concentration for 6 h caused a significant decrease in the percentage of surviving cells (20.76 ± 1.24%, p = 0.0004), with a simultaneous increase in the fraction of early-apoptotic cells [7-AAD (-), Annexin V-FITC (+); 14.03 ± 1.74%, p = 0.0078] in comparison to DMSO-treated control cells (Fig. 5). Treatment with the tested compound for 24 h caused 3.9-fold augmentation in apoptosis [early: 7-AAD (-), Annexin V-FITC (+); late: 7-AAD (+), Annexin V-FITC (+)] in comparison to the vehicle. Further incubation of cells with 4a for 48 h led to a significant increase in the percentage of apoptotic cells (p < 0.0001), while the level of the necrotic fraction was similar to that in control.
Compound 4a induces apoptosis via intrinsic and extrinsic pathways. Apoptosis induction via both intrinsic and extrinsic pathways is mediated by caspase cascade events 26 . Therefore, in this study, the ability of 4a to activate www.nature.com/scientificreports/ two effector caspases (caspase-3 and caspase-7) was evaluated by flow cytometry. As shown in Fig. 5, after 6 h of exposure to this compound, a slight increase (2.32 ± 0.05-fold) in the activity of caspase-3/7 was observed in A549-treated cells. After 24 and 48 h of incubation, remarkable activation of caspase-3/7 (3.9 ± 0.33-fold, p = 0.04) was observed in comparison to the vehicle. Furthermore, the expression of several apoptosis-related proteins was measured by Western blotting. As shown in Fig. 6, treatment of A549 cells with 4a resulted in time-dependent modulation of expression of all investigated proteins. The expression of BCL-2 protein was significantly decreased, while the expression of BID was slightly activated and that of BAD and BAX protein was higher than that in the control group. Treatment with 4a also led to the disappearance of the band corresponding to intact PARP-1 resulting in its proteolytic cleavage and the formation of an 85-kDa fragment. In addition, all the examined caspases (caspase-3, caspase-8, and caspase-9) were cleaved into their corresponding catalytically active forms. Expression of AIF remained unchanged in comparison to DMSO-treated control, which confirmed that treatment with 4a induced caspasedependent cell death 27 .
Structure-activity relationship (SAR). During our work, we noticed that the IC 50 values of presented compounds determined for HCT-116 and A549 strongly suggested that the (1-naphthyl)methyl moiety present at position 3 was crucial for the anticancer activity of the synthesized tetrahydroquinolinone derivatives (Fig. 7). Among the synthesized compounds, the best result was achieved for compound 4a with the amide group. Modifications of compounds at position 2 with a chlorine atom or methoxy group increased their IC 50 values. Therefore, alternatively, any other functionalization should be performed in the cyclohexyl ring or at position 4. The presented conclusions should be considered while designing subsequent derivatives from this subgroup.

Discussion
From the perspective of the latest published data, it is worth noticing that new small-molecule compounds from the 2-and 4-quinolones group, which are often structurally based on antibacterial fluoroquinolones manifest an attractive activity in many cancer cell lines. It's likely that compounds containing similar quinolone scaffold will exhibit activity towards the same cancer cell lines.   www.nature.com/scientificreports/ and enoliztion of used compounds. Due to the fact that we're interested in this type of condensations, it is possible that in the near future we will propose some synthetic paths which will solve this problem. Over the last several years, there is an increasing need for anticancer drugs that can perturb the proliferation of tumor cells by inhibiting various stages of the cell cycle 28 . In the present study, we examined the effect of compound 4a on the progression of the cell cycle and observed that it arrested the cell cycle at the G 2 /M phase in A549 cells, leading to cell apoptotic cell death.. Antimitotic properties of quinoline-based compounds were also observed by others: e.g. Metwally et al. showed that pyrimido [4,5-c]quinoline-1(2H)-one ring skeleton exhibited high cytotoxicity towards fibrosarcoma HT-1080 cells inducing G 2 /M arrest and apoptosis, whereas a Yang`s group presented quinoline derivative MPT0B392 as a strong antimitotic agent against acute leukemia 29,30 . Many studies reported that the antimitotic activity of quinolone results from their interaction with tubulin at the colchicine-binding site, disrupting microtubule organization; however, in the case of the 4a, the exact mechanism of blocking the G 2 /M cell cycle remains unresolved and will be subject of the future studies [31][32][33][34] . Caspase-3 and caspase-7 are members of the cysteine-aspartic acid protease family, which play an important role in executing apoptotic death 35 . Once activated, both these enzymes cleave multiple key structural and repair proteins. Compound 4a stimulated the activation of both caspase-3 and caspase-7, which could have triggered the antiproliferative effect on A549 cells. Caspase activation in response to anticancer chemotherapy can occur via two major pathways: intrinsic pathway mediated by mitochondria, or extrinsic pathway mediated by receptors. The mitochondria-mediated pathway is initiated by the activation of proapoptotic BCL-2 effector molecules, such as BAX and BAK, which leads to the permeabilization of mitochondrial outer membrane and subsequently the release of cytochrome c from the mitochondrial intermembrane space to the cytosol. Once released, it binds to its partner, Apaf-1 protein, forming an apoptosome complex, which induces the activation of pro-caspase-9 36 . The extrinsic pathway is activated by the stimulation of death receptors (e.g. Fas (CD95/APO-1)), TNF receptors (e.g. TNF/R1/p55/ CD120), or TNF-related apoptosis-inducing ligand 37 . Ligation of TNF-receptor family proteins results in the assembly of DISC, and the subsequent recruitment of caspase-8 which occurs through the interaction between the death effector domain of caspase 8 and the Fas-associated death domain. Then, caspase 8 is oligomerized and activated by self-cleavage, after which it initiates downstream apoptotic events, including the activation of effector caspases such as caspase-3 38 .
The results of Western blotting indicated that exposure of A549 cells to 4a diminished the expression of the antiapoptotic protein BCL-2, while enhancing the expression of the proapoptotic protein BAX, in comparison to the control group. Moreover, the BAX:BCL-2 ratio was significantly increased, which suggests the permeabilization of the mitochondrial outer membrane 39 . The level of the proapoptotic molecule BAD was also elevated  www.nature.com/scientificreports/ upon treatment with 4a. Huang et al. reported that the loss of BAD expression may indicate a poor prognosis in patients with nonsmall cell lung cancer, due to increased resistance to the treatment 40 . Therefore, high BAD expression may be considered to suppress tumorigenesis. In this study, together, with the modulating members of the BCL-2 family, treatment with 4a led to the cleavage of procaspase-9, promoting mitochondria-mediated apoptosis. However, the levels of cleaved caspase-8, which is the initiator caspase of the extrinsic apoptotic pathway, were also increased in the treated cells in comparison to the control cells 41 . Additionally, exposure to 4a induced proteolytic cleavage of PARP-1, which catalyzes the transfer of ADP-ribose polymers onto itself and the proteins activated in response to double-strand DNA breaks. It should be mentioned that cleavage of PARP-1, which is initiated by caspase-3 and caspase-7, is known as a hallmark of apoptosis 42 . Activation of both intrinsic and extrinsic apoptotic pathways by quinolinone was also reported by Janecka`s group, which showed that 2-ethyl-3-methyliden-1-tosyl-2,3-dihydroquinolin-4-(1H)-one exhibited cytotoxic activity by down-regulation MAPK pathway in myeloid leukemia HL-60 cell line 43 . On the other hand, Beretta et al. revealed that cytotoxic properties of quinoline-pyrrolidine gamma-lactam alkaloid towards prostate cancer cells result from activation of transmembrane receptor-mediated interactions 44 ; whereas Chiu et al. showed that quinoline derivative BPIQ induced mitochondrial apoptosis in lung cancer models 45 . Potent anticancer activities of quinolones have been found against various cancer cell lines and targets, including topoisomerase, protein tyrosine kinases, histone deacetylase, and many others 46 . Interestingly, inhibition of all mentioned targets may trigger G 2 /M cell cycle arrest and apoptotic cell death, similar to 4a. In conclusion, the newly synthesized tetrahydroquinolinone derivatives containing (1-naphthyl)methyl moiety at position 3 reduced the activity of non-small cell lung cancer cells in a time-dependent manner. Among them, compound 4a inhibited colony formation and proliferation of A549 cells. Further analysis of this compound revealed that it induced apoptosis through both intrinsic and extrinsic apoptotic pathways. These findings may be valuable for the future development of anticancer agents based on tetrahydroquinolinone derivatives. All culture media were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (penicillin 62.6 µg/ml and streptomycin 40 µg/ml; Sigma-Aldrich). Both cancer and normal cells were cultured at 37 °C under a humidified atmosphere with 5% or 10% CO 2 and routinely screened for Mycoplasma contamination. All materials and reagents were obtained from Corning unless stated otherwise.

Methods
Cell viability assay. The cytotoxic effect of the synthesized compounds was assessed by the MTT (4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide; Sigma-Aldrich) assay. Briefly, the cells were seeded into 96-well transparent flat-bottom plates and allowed to attach overnight. On the next day, the cells were treated with compounds in the range of 0-50 µM for 72 h. Dimethyl sulfoxide (DMSO; Merck) and 5-fluorouracil (5-FU; Sigma-Aldrich) were used as a reference. After exposure to the compounds, 20 μl of the MTT solution at a concentration of 4 mg/ml was added to each well, and the plates were incubated again for 2-3 h at 37 °C. Next, the media in the wells was replaced with 100 μl DMSO to solubilize the formazan crystals. The absorbance was measured at 540 nm using an ASYS UVM340 microplate reader (Biochrom Ltd.). The IC 50 value of the compounds was calculated using GraphPad Prism 9 software, based on the curves plotted with survival as a function of dose, averaged from three independent experiments.
Colony formation assay. HCT-116 and A549 cells were seeded into six-well plates at a density of 500 cells per well and allowed to attach overnight. On the next day, the cells were pretreated with compound 4a at different concentrations or with 1% v/v DMSO for 24 h. After incubation, the cells were washed and cultured for 9 days. Next, the cells were fixed with methanol for 20 min and stained with 0.5% crystal violet (Sigma-Aldrich). Finally, the plates were dried overnight and imaged using a UVITEC Cambridge imaging system. Quantification was performed with ImageJ software. The surviving fraction was calculated as previously described 47 . Each experiment was repeated three times.
Cell cycle progression. A549 and HCT-116 cells were incubated with the tested compounds at their IC 50 concentrations for 24, 48, and 72 h. DMSO (1% v/v) was used as a reference (Sigma-Aldrich). After incubation, the cells were collected in trypsin, fixed with 75% ice-cold ethanol, and stored overnight at − 20 °C. Then, the cells were stained with 20 μg/ml propidium iodide (Sigma-Aldrich) and 50 μg/ml RNaseA (Thermo Fisher Scientific) in phosphate-buffered saline (PBS) for 30 min at room temperature (RT). The content of DNA in cells was determined by flow cytometry (Guava EasyCyte 8 cell sorter; Merck Millipore) using FlowJo software v10. Each experiment was repeated three times.
Apoptosis and caspase-3/7 assay. Cells were seeded in a 5-cm 2 dish at a density of 15 × 10 5 cells per dish and allowed to attach overnight at 37 °C. Then, compounds at IC 50 concentrations were added to the medium. After 6, 24, and 48 h, the cells were harvested and washed with PBS. Subsequently, the cells were resuspended in the binding buffer and stained with Annexin V Alexa Fluor™ 488 conjugate (#A13201; Thermo Fisher Scientific) for the apoptosis assay and with CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (#C10427; Thermo www.nature.com/scientificreports/ Fisher Scientific) for the activation of caspase-3/7, as per the manufacturer's instructions. After incubation, the samples were immediately analyzed using a Guava EasyCyte 8 cell sorter (Merck Millipore) and FlowJo software v10. Each experiment was repeated three times.
Western blotting. The expression of apoptosis-related proteins was determined by Western blotting.
Briefly, A549 cells were treated with compound 4a at its IC 50 concentration for 6, 24, and 48 h. The cells treated with 1% v/v DMSO served as the negative control. The total protein content was extracted from the cells using the NP-40 cell lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 0.5% Nonidet P-40, cOmplete Mini EDTA-free Protease Inhibitor Cocktail). Then, 30 µg of protein extract was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were transferred to microporous polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% bovine serum albumin in tris-buffered saline-Tween 20 (TBST) buffer (0.2 M Tris-base, 0.137 M NaCl, 0.1% Tween 20) for 1 h at RT, and incubated overnight with primary antibodies at 4 °C. The blots were washed three times with TBST and incubated with appropriate peroxidase-conjugated secondary antibodies for 1 h at RT. Then, the blots were washed in TBST for 30 min and developed using an enhanced chemiluminescence detection reagent kit (Thermo Fisher Scientific) and a ChemiDoc XRS + Imaging System (Bio-Rad). All materials and reagents were obtained from Sigma-Aldrich unless stated otherwise. The antibodies used in Western blotting are listed in Table S1.
Statistical analyses. Statistical analyses were performed using GraphPad Prism 9 software. Data were obtained from at least three independent experiments and are presented as mean ± SEM. Statistical significance was calculated in comparison to the DMSO-treated control (1% v/v) using one-way ANOVA (post hoc Dunnet's test) unless stated otherwise.

Data availability
The datasets presented in the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/