Synthesis, In Vitro, and In Vivo Investigations of Pterostilbene-Tethered Analogues as Anti-Breast Cancer Candidates

Pterostilbene has been found to be an active scaffold with anti-breast cancer (BC) action. In this study, fourteen pterostilbene-tethered analogues (2A–2N) were prepared and screened in vitro against MDA-MB-231 and MCF-7 cells. Meanwhile, their structures were characterized using 1H-NMR, 13C-NMR, and HRMS (ESI) spectroscopy techniques. Among them, analogue 2L displayed the most potent anti-proliferation effect on MDA-MB-231 (IC50 = 10.39 μM) and MCF-7 cells (IC50 = 11.73 μM). Furthermore, the meaningful structure–activity relationships suggested that the introduction of a saturated six-membered nitrogen heterocyclic ring into the side chain favored anti-BC capacity. Biological observations indicated that 2L could cause the typical morphological changes in apoptosis, namely an increase in reactive oxygen species level and a loss of mitochondrial membrane potential in BC cells. Importantly, 2L could induce mitochondrial-mediated apoptosis by regulating the expression of caspase-related proteins. Consistent with the results of our in vitro study, 2L apparently inhibited tumor growth in MDA-MB-231 xenograft mice without obvious toxicity. These findings revealed that 2L is expected to be a promising anti-BC lead compound that merits further investigations.


Introduction
Breast cancer (BC) is one of the most prevalent malignancies and the second leading cause of cancer death among women worldwide [1,2]. Triple-negative breast cancer (TNBC), the most aggressive subtype of BC, features the absence of three classical membrane receptors (estrogen receptor, ER; progesterone receptor, PR; human epidermal growth factor receptor 2 positive, HER2) [3]. The limited therapeutic options for TNBC result in an undesirable prognosis and a high recurrence rate [4]. At present, conventional chemotherapy still occupies an important role in the combination therapy model for BC [5,6]. Unfortunately, its efficacy is hampered by several factors including drug resistance, severe side effects, and low selectivity [7,8]. To overcome these obstacles, some biological molecules, such as tubulin protein, cyclin-dependent kinase (CDK), epidermal growth factor (EGF), and Ras have been considered as appealing targets for antitumor drugs in the recent decades [9]. Therefore, the development of new chemotherapeutic agents for the treatment of BC is necessary.
Pterostilbene (3,5-dimethoxy-4 -hydroxy-trans-stilbene, PTE), a natural polyphenolic product, abundantly exists in Pterocarpus marsupium, blueberries, nuts, and Vitis vinifera The discovery of anti-BC drug candidates has always been a part of our endeavors [36]. Meanwhile, encouraged by the above research background, we synthesized fourteen novel PTE-tethered derivatives (PTEs, 2A-2N) in this study. Subsequently, all the target products were evaluated for their in vitro anti-proliferative activity against MDA-MB-231 and MCF-7 cells. At the same time, the structure-activity relationships (SARs) were preliminarily discussed. The most outstanding anti-BC candidate (2L) was screened to further investigate its effects on proliferation, reactive oxygen species, mitochondrial membrane potential, and apoptosis in vitro. Of note, the in vivo study suggested that 2L exerted a significant anti-tumor potency in an MDA-MB-231 xenograft tumor model. These results indicated that 2L has the potential to develop into a novel anti-BC lead compound. The discovery of anti-BC drug candidates has always been a part of our endeavors [36]. Meanwhile, encouraged by the above research background, we synthesized fourteen novel PTE-tethered derivatives (PTEs, 2A-2N) in this study. Subsequently, all the target products were evaluated for their in vitro anti-proliferative activity against MDA-MB-231 and MCF-7 cells. At the same time, the structure-activity relationships (SARs) were preliminarily discussed. The most outstanding anti-BC candidate (2L) was screened to further investigate its effects on proliferation, reactive oxygen species, mitochondrial membrane potential, and apoptosis in vitro. Of note, the in vivo study suggested that 2L exerted a significant anti-tumor potency in an MDA-MB-231 xenograft tumor model. These results indicated that 2L has the potential to develop into a novel anti-BC lead compound.

Chemistry
Coinciding with the previous literature [18][19][20][21][22], our tentative bioassay screening found that PTE possessed anti-proliferation properties against MDA-MB-231 (TNBC, IC 50 = 63.59 µM) and MCF-7 (ER + BC, IC 50 = 44.26 µM) cells. In this research, we aimed to obtain lead chemical entities with improved anti-BC activity through the structural optimization of PTE. As outlined in Scheme 1, a series of PTE analogues (2A-2N) were readily synthesized. PTE was reacted with 1,3-dibromopropane in the presence of potassium carbonate and acetone under reflux conditions to afford intermediate 1. The title compounds (2A-2N) were prepared from 1 by nucleophilic substitution with appropriate aromatic amines, alicyclic amines, and anilines. All derivatives were purified through recrystallization or column chromatography with good yields, and their structures were confirmed using 1 H-NMR, 13 C-NMR, and HRMS (ESI) spectra.

Chemistry
Coinciding with the previous literature [18][19][20][21][22], our tentative bioassay screening found that PTE possessed anti-proliferation properties against MDA-MB-231 (TNBC, IC50 = 63.59 μM) and MCF-7 (ER + BC, IC50 = 44.26 μM) cells. In this research, we aimed to obtain lead chemical entities with improved anti-BC activity through the structural optimization of PTE. As outlined in Scheme 1, a series of PTE analogues (2A-2N) were readily synthesized. PTE was reacted with 1,3-dibromopropane in the presence of potassium carbonate and acetone under reflux conditions to afford intermediate 1. The title compounds (2A-2N) were prepared from 1 by nucleophilic substitution with appropriate aromatic amines, alicyclic amines, and anilines. All derivatives were purified through recrystallization or column chromatography with good yields, and their structures were confirmed using 1 H-NMR, 13 C-NMR, and HRMS (ESI) spectra.

Evaluation of Anti-BC Activity
The in vitro anti-proliferative activity of all synthesized analogues against MDA-MB-231 and MCF-7 cells was tested using the 3-(4,5-dimethylthiazol-2-yl)-3,5-phenytetrazoliumromide (MTT) method. Dox, a valuable anticancer drug, is highly effective in the treatment of various hematopoietic malignancies and solid tumors. Usually, Dox has been used as a positive control in many basic studies, especially drug screening assay [37,38]. As listed in Table 1, the IC50 values of most analogues (1, 2A-2J) against BC cells were approximately greater than 100 μM, suggesting that they were almost inactive against BC cells at a 100 μM concentration. Only analogues 2K-2N exerted the potential to moderate inhibition activity against BC cells with IC50 values ranging from 10.39 to 49.34 μM, which were obviously weaker than those of the positive control (Dox). Compared with the prototype drug (PTE), the derivatives 2K, 2L, and 2N possessed excellent anti-BC activity, most impressively analogue 2L. By contrast, analogue 2M exhibited an inhibitory capacity against BC cells comparable to that of PTE. In the meantime, the cytotoxic activity of 2K-2N, PTE, and Dox against MCF-10A (human breast epithelial cell line) was examined. Notably, the most active analogue 2L showed a weak cytotoxicity against MCF-10A (IC50 = 68.87 μM) compared with analogues 2K and 2N. The preliminary SARs revealed that the implantation of aromatic moieties into the terminal alkyl fragment did not improve the anti-BC property (2A-2I vs. 1, PTE). By contrast, the substitution of a saturated six-membered nitrogen heterocyclic ring on the side chain is extremely favorable for enhancing

Evaluation of Anti-BC Activity
The in vitro anti-proliferative activity of all synthesized analogues against MDA-MB-231 and MCF-7 cells was tested using the 3-(4,5-dimethylthiazol-2-yl)-3,5-phenytetrazoliumromide (MTT) method. Dox, a valuable anticancer drug, is highly effective in the treatment of various hematopoietic malignancies and solid tumors. Usually, Dox has been used as a positive control in many basic studies, especially drug screening assay [37,38]. As listed in Table 1, the IC 50 values of most analogues (1, 2A-2J) against BC cells were approximately greater than 100 µM, suggesting that they were almost inactive against BC cells at a 100 µM concentration. Only analogues 2K-2N exerted the potential to moderate inhibition activity against BC cells with IC 50 values ranging from 10.39 to 49.34 µM, which were obviously weaker than those of the positive control (Dox). Compared with the prototype drug (PTE), the derivatives 2K, 2L, and 2N possessed excellent anti-BC activity, most impressively analogue 2L. By contrast, analogue 2M exhibited an inhibitory capacity against BC cells comparable to that of PTE. In the meantime, the cytotoxic activity of 2K-2N, PTE, and Dox against MCF-10A (human breast epithelial cell line) was examined. Notably, the most active analogue 2L showed a weak cytotoxicity against MCF-10A (IC 50 = 68.87 µM) compared with analogues 2K and 2N. The preliminary SARs revealed that the implantation of aromatic moieties into the terminal alkyl fragment did not improve the anti-BC property (2A-2I vs. 1, PTE). By contrast, the substitution of a saturated six-membered nitrogen heterocyclic ring on the side chain is extremely favorable for enhancing anti-BC efficacy (2L-2N vs. 1, PTE, 2A-2J). Based on the preceding analysis, analogue 2L was selected as the most promising anti-BC agent and used in the follow-up study.

Effect of 2L on BC Cells Proliferation
To investigate the underlying mechanism of action of 2L, we firstly inspected the effect of 2L on the growth inhibition of MDA-MB-231 and MCF-7 cells by MTT assay. As depicted in Figure 2, the viability of MDA-MB-231 and MCF-7 cells decreased accordingly with the increase in 2L concentration. These data suggested that 2L significantly restrained the proliferation of BC cells in a concentration-and time-dependent manner. Compared to the control group, the viability of BC cells was not evidently influenced by 2L within 24 h at the dosages of 1.1, 3.3, and 10 µM. Hence, these concentrations below the IC 50 values of 2L were chosen for the following experiments.
anti-BC efficacy (2L-2N vs. 1, PTE, 2A-2J). Based on the preceding analysis, analogue 2L was selected as the most promising anti-BC agent and used in the follow-up study.

Effect of 2L on BC Cells Proliferation
To investigate the underlying mechanism of action of 2L, we firstly inspected the effect of 2L on the growth inhibition of MDA-MB-231 and MCF-7 cells by MTT assay. As depicted in Figure 2, the viability of MDA-MB-231 and MCF-7 cells decreased accordingly with the increase in 2L concentration. These data suggested that 2L significantly restrained the proliferation of BC cells in a concentration-and time-dependent manner. Compared to the control group, the viability of BC cells was not evidently influenced by 2L within 24 h at the dosages of 1.1, 3.3, and 10 μM. Hence, these concentrations below the IC50 values of 2L were chosen for the following experiments.

Effect of 2L on Apoptosis of BC Cells
To observe whether 2L could cause the apoptosis of BC cells, Hoechst 33,342 staining was carried out. As shown in Figure 3A,D, the nuclei of BC cells in the control groups presented well-distributed fluorescence with a uniform circular and elliptical shape. However, the 2L-treated groups exhibited the typical morphological changes in programmed apoptosis, including membrane blebbing, nuclear condensation, cell shrinkage, and apoptotic body formation. Subsequently, flow cytometric analysis of annexin V/PI was conducted. The apoptotic percentages in the control groups were about 3.4% (MDA-MB-231 cell) and 3.3% (MCF-7 cell), while the apoptosis proportion in the 2L-treated groups increased to 12.7% and 14.0%, respectively ( Figure 3B,E). At the same time, the results of the quantitative analysis showed that the total population of apoptotic BC cells in 2L-treated

Effect of 2L on Apoptosis of BC Cells
To observe whether 2L could cause the apoptosis of BC cells, Hoechst 33,342 staining was carried out. As shown in Figure 3A,D, the nuclei of BC cells in the control groups presented well-distributed fluorescence with a uniform circular and elliptical shape. However, the 2L-treated groups exhibited the typical morphological changes in programmed apoptosis, including membrane blebbing, nuclear condensation, cell shrinkage, and apoptotic body formation. Subsequently, flow cytometric analysis of annexin V/PI was conducted. The apoptotic percentages in the control groups were about 3.4% (MDA-MB-231 cell) and 3.3% (MCF-7 cell), while the apoptosis proportion in the 2L-treated groups increased to 12.7% and 14.0%, respectively ( Figure 3B,E). At the same time, the results of the quantitative analysis showed that the total population of apoptotic BC cells in 2L-treated groups was obviously elevated compared with the control groups ( Figure 3C,F). These data indicated that analogue 2L could induce apoptosis in BC cells.
groups was obviously elevated compared with the control groups ( Figure 3C,F). These data indicated that analogue 2L could induce apoptosis in BC cells.

Effect of 2L on DNA Content of BC Cells
It is commonly acknowledged that intranucleosomal DNA fragmentation is a crucial hallmark of cell apoptosis [39]. Next, we assessed the influence of 2L on the DNA content of PI-stained BC cells using flow cytometry. As shown in Figure 4A,C, the 2L-treated groups exhibited a significant increase in the number of BC cells in the G0/G1 phase compared with the control groups. The relative proportion of MDA-MB-231 cells in the G0/G1 phase increased from 49.5% (control group) to 61.7% (2L-treated group). In turn,

Effect of 2L on DNA Content of BC Cells
It is commonly acknowledged that intranucleosomal DNA fragmentation is a crucial hallmark of cell apoptosis [39]. Next, we assessed the influence of 2L on the DNA content of PI-stained BC cells using flow cytometry. As shown in Figure 4A,C, the 2L-treated groups exhibited a significant increase in the number of BC cells in the G0/G1 phase compared with the control groups. The relative proportion of MDA-MB-231 cells in the G0/G1 phase increased from 49.5% (control group) to 61.7% (2L-treated group). In turn, treatment with 2L led to an apparent enhancement of the number of MCF-7 cells, from 51.7% to 63.0%. In particular, we found that 2L could result in an increased percentage of subG1 apoptotic cells (up from 1.19% to 7.99% in MDA-MB-231 cells; from 0.93% to 7.83% in MCF-7 cells). An augmentation of DNA distribution in the SubG1 peak was representative of the occurrence of apoptosis in 2L-exposed groups, which further confirmed that 2L could induce the apoptosis of BC cells. In addition, the quantitative analysis of DNA distribution for the subG1, G0/G1, S, and G2/M phases appeared to show similar alterations ( Figure 4B,D). These data indicated that 2L could restrict the proliferation of BC cells through G0/G1 phase cell cycle arrest. treatment with 2L led to an apparent enhancement of the number of MCF-7 cells, from 51.7% to 63.0%. In particular, we found that 2L could result in an increased percentage of subG1 apoptotic cells (up from 1.19% to 7.99% in MDA-MB-231 cells; from 0.93% to 7.83% in MCF-7 cells). An augmentation of DNA distribution in the SubG1 peak was representative of the occurrence of apoptosis in 2L-exposed groups, which further confirmed that 2L could induce the apoptosis of BC cells. In addition, the quantitative analysis of DNA distribution for the subG1, G0/G1, S, and G2/M phases appeared to show similar alterations ( Figure 4B,D). These data indicated that 2L could restrict the proliferation of BC cells through G0/G1 phase cell cycle arrest.

Effect of 2L on Mitochondrial Membrane Potential
It is widely known that the pathways of cell apoptosis are mediated by mitochondria, endoplasmic reticulum stress, and death receptors [40]. Among them, intrinsic mitochondrial apoptosis has been considered as the canonical pathway of cell apoptosis [41]. In general, mitochondrial membrane potential (MMP) is a critical parameter for reflecting mitochondrial configuration and function [42]. To explore whether 2L-induced apoptosis was involved with the disruption of mitochondrial membrane integrity, the variation of MMP (ΔΨm) was detected using fluorescent probe JC-1. Compared with the control group, the green fluorescence intensity in 2L-treated BC cells was obviously enhanced, whereas the red fluorescence intensity was correspondingly attenuated ( Figure 5A,B). Moreover, the results of flow cytometry manifested that the ΔΨ m level of MDA-MB-231 and MCF-7 cells sharply fell to 83.9% and 85.0%, respectively, after incubation with 2L for 24 h (Figure 5C,E). In line with the above result, the quantitative analysis results showed that the proportion of red/green fluorescence in 2L-administered groups drastically declined compared to control groups ( Figure 5D,F). These observations indicated that 2L could lead to a reduction in MMP, thus, triggering the early apoptosis of BC cells.

Effect of 2L on Mitochondrial Membrane Potential
It is widely known that the pathways of cell apoptosis are mediated by mitochondria, endoplasmic reticulum stress, and death receptors [40]. Among them, intrinsic mitochondrial apoptosis has been considered as the canonical pathway of cell apoptosis [41]. In general, mitochondrial membrane potential (MMP) is a critical parameter for reflecting mitochondrial configuration and function [42]. To explore whether 2L-induced apoptosis was involved with the disruption of mitochondrial membrane integrity, the variation of MMP (∆Ψm) was detected using fluorescent probe JC-1. Compared with the control group, the green fluorescence intensity in 2L-treated BC cells was obviously enhanced, whereas the red fluorescence intensity was correspondingly attenuated (Figure 5A,B). Moreover, the results of flow cytometry manifested that the ∆Ψ m level of MDA-MB-231 and MCF-7 cells sharply fell to 83.9% and 85.0%, respectively, after incubation with 2L for 24 h (Figure 5C,E). In line with the above result, the quantitative analysis results showed that the proportion of red/green fluorescence in 2L-administered groups drastically declined compared to control groups ( Figure 5D,F). These observations indicated that 2L could lead to a reduction in MMP, thus, triggering the early apoptosis of BC cells.

Effect of 2L on Reactive Oxygen Species
An increasing number of studies have emphasized the pivotal role of reactive oxygen species (ROS) homeostasis in cellular processes (e.g., proliferation, migration, and differentiation) and signaling cascades. Nevertheless, the abnormal elevation of ROS levels will initiate cell apoptosis and damage [43,44]. For this reason, we next determined if 2L could stimulate the massive production of ROS using 2,7-dichloro-dihydrofluorescein diacetate (DCFH-DA) fluorescence staining. As shown in Figure 6A,F, the number of BC cells

Effect of 2L on Reactive Oxygen Species
An increasing number of studies have emphasized the pivotal role of reactive oxygen species (ROS) homeostasis in cellular processes (e.g., proliferation, migration, and differentiation) and signaling cascades. Nevertheless, the abnormal elevation of ROS levels will initiate cell apoptosis and damage [43,44]. For this reason, we next determined if 2L could stimulate the massive production of ROS using 2,7-dichloro-dihydrofluorescein diacetate (DCFH-DA) fluorescence staining. As shown in Figure 6A,F, the number of BC cells emitting green fluorescence was substantially increased in 2L-administered groups, and the fluorescence intensity was also strengthened. Immediately after, flow cytometry was em-ployed to quantitatively analyze the ROS production of 2L-treated BC cells ( Figure 6B,G). Compared with the control groups, the accumulation of ROS was significantly augmented in 2L-treated groups. Likewise, the mean ROS-associated fluorescence intensity of 2Ltreated MDA-MB-231 and MCF-7 cells was greatly increased ( Figure 6C,H). To further demonstrate the role of ROS in the 2L-induced apoptosis, the cells were exposed to 5 mM NAC (a ROS scavenger) [45]. As depicted in Figure 6D,I, the apoptosis ratios (early and late stages) plummeted from 16.1% to 8.8% in MDA-MB-231 cells and from 14.5% to 6.7% in MCF-7 cells following 2L + NAC treatment. Again, the quantitative analysis showed a similar tendency, indicating that NAC significantly attenuated 2L-induced apoptosis ( Figure 6E,J). Consequently, 2L could trigger the apoptosis of BC cells via initiating the excessive generation of ROS.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of emitting green fluorescence was substantially increased in 2L-administered groups, a the fluorescence intensity was also strengthened. Immediately after, flow cytometry w employed to quantitatively analyze the ROS production of 2L-treated BC cells (Figu 6B,G). Compared with the control groups, the accumulation of ROS was significantly au mented in 2L-treated groups. Likewise, the mean ROS-associated fluorescence intens of 2L-treated MDA-MB-231 and MCF-7 cells was greatly increased ( Figure 6C,H). To f ther demonstrate the role of ROS in the 2L-induced apoptosis, the cells were exposed t mM NAC (a ROS scavenger) [45]. As depicted in Figure 6D,I, the apoptosis ratios (ea and late stages) plummeted from 16.1% to 8.8% in MDA-MB-231 cells and from 14.5% 6.7% in MCF-7 cells following 2L + NAC treatment. Again, the quantitative analy showed a similar tendency, indicating that NAC significantly attenuated 2L-induc apoptosis ( Figure 6E,J). Consequently, 2L could trigger the apoptosis of BC cells via in ating the excessive generation of ROS.

Effect of 2L on Apoptosis-Associated Proteins
Given that 2L led to the production of intracellular ROS and loss of MMP, we speculated that 2L-induced cell apoptosis was closely related to the mitochondria-dependent signaling pathway. Additionally, the mitochondrial pathway is modulated by the Bcl-2 family, caspase family, and oncogenes (e.g., C-myc, p53) [46]. In view of this, we continued to examine the expression of apoptosis-related proteins by Western blotting assay. As shown in Figure 7A,B,E,F, the expression of anti-apoptosis protein Bcl-2 was markedly downregulated in 2L-treated cells, while the level of pro-apoptosis protein Bax was upregulated. Meanwhile, 2L could increase the protein expression of cleaved caspase-9 and cleaved caspase-3. As we know, cleaved caspase-3 can activate its proteolytic activity, which further leads to the cleavage of poly(ADP-ribose) polymerase (PARP) [47]. Also, it was found that 2L could effectively elevate the protein level of cleaved PARP. Generally, caspase-dependent apoptosis can be hindered by a classic caspase inhibitor Z-VAD-FMK [48]. To further determine whether 2L induced caspase-dependent apoptosis, we carried out flow cytometry to analyze the apoptosis ratio of BC cells pretreated with Z-VAD-FMK (20 µM). As described in Figure 7C,G, the percentages of apoptotic cells in 2L-alone groups were 13.1% (MDA-MB-231 cells) and 12.2% (MCF-7 cells), but the apoptosis ratios in 2L + Z-VAD-FMK groups sharply dropped to 6.2% and 6.5%, respectively. At the same time, the results of quantitative analysis manifested that the apoptosis proportion of BC cells was significantly declined in the 2L + Z-VAD-FMK groups compared with the 2L-only groups ( Figure 7D,H). These results indicated that the pretreatment of BC cells with Z-VAD-FMK could partly reverse the apoptosis of BC cells induced by 2L. Overall, 2L could activate apoptosis-related proteins, thereby inducing the apoptosis of BC cells through the mitochondrial pathway.

Evaluation of the Anti-Tumor Efficacy of 2L In Vivo
Considering the anti-tumor potency of 2L in vitro, we next constructed the MDA-MB-231-derived xenograft mouse model to perform the in vivo anti-BC study. As illustrated in Figure 8A,B, 2L effectively suppressed tumor size and growth in a dose-response curve. After the dissection of tumor tissues, the tumor volumes, weights, and tumor growth inhibition (TGI) ratios were calculated. The 2L-treated groups showed an evident reduction in average tumor volume and weight. The TGI values of 2L reached 42.0% and 69.9% at 25 and 50 mg/kg doses, respectively ( Figure 8C,D). Again, the fluctuations in body weight in 2L-administered groups were approximately the same as those in the control group, indicating that 2L had no obvious toxicity in nude mice throughout the whole study ( Figure 8E). Similar to the in vitro cell-based study, the apoptosis induced by 2L was apparent in tumor tissues. Compared with the control group, the protein expression of Bax and cleaved PARP were noticeably increased in 2L-treated groups, while the level of Bcl-2 protein was decreased ( Figure 8F). These data suggested that 2L remarkably inhibited the tumor proliferation of an MDA-MB-231 xenograft through the induction of cell apoptosis.

Evaluation of the Anti-Tumor Efficacy of 2L In Vivo
Considering the anti-tumor potency of 2L in vitro, we next constructed the MDA-MB-231-derived xenograft mouse model to perform the in vivo anti-BC study. As illustrated in Figure 8A,B, 2L effectively suppressed tumor size and growth in a dose-response curve. After the dissection of tumor tissues, the tumor volumes, weights, and tumor growth inhibition (TGI) ratios were calculated. The 2L-treated groups showed an evident trol group, indicating that 2L had no obvious toxicity in nude mice throughout the whole study ( Figure 8E). Similar to the in vitro cell-based study, the apoptosis induced by 2L was apparent in tumor tissues. Compared with the control group, the protein expression of Bax and cleaved PARP were noticeably increased in 2L-treated groups, while the level of Bcl-2 protein was decreased ( Figure 8F). These data suggested that 2L remarkably inhibited the tumor proliferation of an MDA-MB-231 xenograft through the induction of cell apoptosis.

General Procedures
All chemical reagents and solvents were purchased from commercial suppliers and used without further purification. Fresh THF and DCM were distilled from sodium wire and calcium hydride, respectively. The reactions were monitored by analytical thin layer chromatography (TLC) on Merck Silica Gel 60 F254 plates, which were visualized using UV radiation (254 nm) or further exposed to an ethanolic solution of concentrated sulfuric acid and anisaldehyde. The chromatographic purification was performed on silica gel

General Procedures
All chemical reagents and solvents were purchased from commercial suppliers and used without further purification. Fresh THF and DCM were distilled from sodium wire and calcium hydride, respectively. The reactions were monitored by analytical thin layer chromatography (TLC) on Merck Silica Gel 60 F254 plates, which were visualized using UV radiation (254 nm) or further exposed to an ethanolic solution of concentrated sulfuric acid and anisaldehyde. The chromatographic purification was performed on silica gel (Qingdao Ocean Chemical Silicone Factory, Qingdao, China, 100-200 mesh, 200-300 mesh). The 1 H and 13 C NMR spectra were recorded on a Bruker AV-600 spectrometer using CDCl 3 or DMSO-d 6 as the solvent and tetramethylsilane as the internal standard. Signal multiplicity was designated as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, dt = doublet of triplets, br = broad, and dd = doublet of doublets. The HR-ESI/MS measurements were taken with an Agilent G6230 TOF mass spectrometer.

General Procedure for the Synthesis of the Title Compounds 2A-2N
Here, 1,3-dibromopropane (1.5 equiv.) was slowly added to a mixed solution of PTE (1.0 equiv.) and anhydrous K 2 CO 3 (2.5 equiv.) in acetone. After the almost complete consumption of PTE, the mixture was filtered. The filtrate was evaporated in vacuo and extracted three times with ethyl acetate (EtOAc). The combined organic phases were dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The resulting product was purified by flash chromatography column (petroleum ether/EtOAc = 50:1) to give intermediate 1.
The appropriate aromatic amines, alicyclic amines, and anilines (1.8 equiv.) were gradually added to a stirred solution of 1 (1 equiv.) and KOH (3.0 equiv.) in MeCN for 6 h at 80 • C. After the completion of the reaction, the mixture was cooled to room temperature, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography to furnish the corresponding analogues 2A-2N. times with phosphate-buffered saline (PBS), followed by staining with Hoechst 33,342 in the dark for 5 min. The nuclear images were observed with a DMI3000B fluorescence microscope (Leica, Solms, Germany), and the changes in nuclear morphology were captured.

Cell Apoptosis Analysis
MDA-MB-231 and MCF-7 cells (3 × 10 5 cells/well) were seeded into 6-well plates. After that, the cells were treated with 2L (0, 10 µM) for 24 h. Cells were harvested, washed repeatedly with cold phosphate-buffered saline (PBS), and resuspended in 195 µL of binding buffer. Then, 4 µL annexin V-FITC reagent (Beyotime) and 8 µL propidium iodide (PI, Beyotime) were added to the samples and gently mixed. After incubation for 15 min in the dark at room temperature, the stained cells were immediately detected by a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with the annexin V-FITC/PI apoptosis method.

Mitochondrial Membrane Potential (MMP) Assay
The MMP was assayed using a JC-1 assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. In brief, MDA-MB-231 and MCF-7 cells were plated in 24-well plates at a density of 6 × 10 4 cells per well. After intervention with 2L (0, 10 µM) for 24 h, the cells were washed once with PBS. Cell culture solution (0.5 mL) and JC-1 working solution (0.5 mL) were successively added, mixed well, and incubated at 37 • C for 20 min in the dark. After washing twice with 1 × JC-1 staining buffer, the cells were resuspended in fresh culture medium. The graphical results were observed under a fluorescence microscope.
MDA-MB-231 and MCF-7 cells were incubated in 6-well plates at a density of 3 × 10 5 cells per dish and exposed to 2L for 24 h. Cell culture media (1 mL) and JC-1 staining working solution (1 mL) were added, thoroughly mixed, and incubated at 37 • C for 20 min. After washing twice with 1 × JC-1 staining buffer, the cells were collected and resuspended in 500 µL JC-1 staining buffer and then instantly analyzed using a flow cytometry.

Measurement of Intracellular ROS Levels
The Reactive Oxygen Species Assay Kit was used to measure intracellular ROS levels. MDA-MB-231 and MCF-7 cells (6 × 10 4 cells/well) were seeded onto 24-well plates and treated with 2L (0, 10 µM) for 24 h. Subsequently, the cells were washed with PBS and stained with dichlorodihydrofluorescein diacetate (DCFH-DA, 10 µM) at 37 • C for 20 min in the dark. Following three washes with serum-free DMEM, the fluorescence alteration was photographed using a fluorescence microscope.
MDA-MB-231 and MCF-7 cells were plated in 6-well plates with a density of 3 × 10 5 cells/well. After exposure to 2L for 24 h, the cells were incubated with 10 µM DCFH-DA at 37 • C for 20 min in the dark. Later, the cells were slightly washed three times with serumfree DMEM and centrifuged (1000 rpm, 5 min). The collected cells were resuspended in PBS and loaded into a flow cytometer.

In Vivo Models
BALB/c nude mice (female, 6-7 weeks) were purchased from Changsheng Biotechnology Co., Ltd. (Liaoning, China). MDA-MB-231 cells (5 × 10 6 cells/mouse) were injected subcutaneously into the right back of the nude mice. When the tumor volume reached approximately 55 mm 3 , the female mice were randomly divided into two treatment groups (25, 50 mg/kg) and a vehicle group (equal volume of normal saline) with five mice in each group. Throughout the experiment, 2L and saline were intraperitoneally injected every other day for 10 treatments. At the same time, the changes in tumor volume and body weight of the nude mice were tracked and recorded. The tumor volumes were assessed using the formula: V (mm 3 ) = (length × width 2 )/2. At the end of the experimental period, tumor tissues were harvested, weighed, and collected. The TGI rate was calculated by the average tumor weight of the 2L-treated and control groups.

Statistical Analysis
All data were presented as mean ± SEM from three independent experiments and statistically analyzed using Student's t test. A one-way analysis of variance (ANOVA) and least significant difference (LSD) post hoc test were used to calculate group differences (SPSS version 17 software). * p < 0.05, ** p < 0.01, *** p < 0.001, and ns (no significant) were considered as the statistical significance.

Conclusions
In conclusion, fourteen pterostilbene-tethered analogues were smoothly achieved and evaluated for their anti-BC efficacy. The primary structure-activity relationship demonstrated that the existence of a saturated nitrogen heterocycle on the side chain was beneficial for anti-BC behavior. Among all of the analogues, 2L exhibited the best inhibitory potency on MDA-MB-231 and MCF-7 cells. Biological studies revealed that 2L could cause the morphological alteration, aberrant ROS generation, MMP depolarization, and the induction of apoptosis-related proteins, thus, triggering the mitochondrial-dependent apoptotic pathway. More importantly, 2L could evidently restrain the growth of an MDA-MB-231 tumor xenograft via inducing apoptosis. Collectively, these results implied the potential of 2L as an attractive anti-BC drug candidate.