1-Deoxynojirimycin | Hif signal

Dual-Targeting Antiproliferation Hybrids Derived from 1‑Deoxynojirimycin and Kaempferol Induce MCF‑7 Cell Apoptosis through the Mitochondria-Mediated Pathway

Ran Zhang,# Yueyue Zhang,# Xiangdong Xin, Gaiqun Huang, Ning Zhang, Qinglei Zeng, Liumei Tang, Thomas Attaribo, Kwang Sik Lee, Byung Rae Jin, and Zhongzheng Gui

ancer and diabetes, two highly complex and prevalent diseases, both have a tremendous eﬀect on human health.1 Tumor incidence and tumor-related mortality in patients with diabetes are signiﬁcantly higher than in patients without diabetes. In addition, approXimately 17% of patients with malignant tumors have diabetes.2 These two prevalent diseases are diagnosed in the same individual more frequently than would be expected by chance.3 For patients who have cancer and diabetes as comorbidities, medicinal therapy is a potential treatment option. Metformin, the ﬁrst-line agent for type 2 diabetes mellitus, also exerts anticancer eﬀects in patients with or without metabolic disorders by inhibiting oXidative phosphorylation and improving the tumor micro-

stimulated glucose uptake, and prevention of oXidative damage in pancreatic β-cells.8 Evidence suggests that the intake of kaempferol may reduce the risk of developing certain types of cancer. The cancer preventive eﬀect of kaempferol is mostly associated with the inhibition of cancer cell proliferation by apoptosis induction.9,10 Indeed, kaempferol inhibits various cancer cells by triggering apoptosis, activating cysteine proteases, preventing the accumulation of reactive oXygen species (ROS), and downregulating epithelial-mesenchymal transition-related proteins and the inﬂammation-related proteins cyclooXygenase and phosphoinositide 3-kinase/ protein kinase B signaling pathways.6,8,11,12 In addition, the
impressive antitumor activity of kaempferol may result from its

environment.4 However, it still causes common adverse eﬀects such as diarrhea, nausea, vomiting, bloating, fatigue, and indigestion and is susceptible to drug resistance.5 Therefore, great interest has been focused on the development of natural compounds that not only promote the progression of diabetes but also improve the cure rate of cancer.
Kaempferol, a ﬂavonoid present in several medicinal herbs and foods, has been reported to exert diverse pharmacological activities including anti-inﬂammatory, antidiabetic, antioXidant, and antimicrobial activities in preclinical studies.6,7 The antidiabetic eﬀect of kaempferol may be associated with the stimulation of glycogen synthesis, improvement of insulin-

suitable lipophilicity and high transfer eﬃciency.13 Therefore, kaempferol is used commonly as a chemosensitizer in combination with other chemotherapy drugs, such as 5- ﬂuorouracil and cisplatin, to enhance antitumor eﬃcacy and© 2021 American Chemical Societ educe side eﬀects.14,15 Although kaempferol shows numerous potential health beneﬁts, its low bioavailability has been
manner. This eﬀect lowers the blood glucose level, thereby reducing the bioavailability of glucose and exerting a caloric

associated with challenges to its application.16 Kaempferol is usually modiﬁed by methylation, alkylation, and sulfation to

restriction eﬀect in tumor cells.28,29 Furthermore, 1-deoXy- nojirimycin inhibits cancer cell proliferation by oXidative

ameliorate its solubility and bioavailability.17−19 For example, kaempferol-SO3-Ga, a sulfonate kaempferol−gallium complex, showed greater water solubility and antioXidant activity than free kaempferol.20 Kaempferol−piperidine, a primary amino- methyl derivative, exhibited more potential cytotoXic activity than kaempferol in HeLa cells.21
1-DeoXynojirimycin, a representative natural compound, was ﬁrst isolated from the bark of the mulberry tree.22 It is known for its antidiabetic eﬀects that are mediated by inhibiting the activity of α-glucosidase and regulating the expression of certain mRNAs and proteins.23 Recently, 1-deoXynojirimycin has been the focus of attention because of its therapeutic eﬀects against diabetes24 and hyperlipidemia25 and its

stress30 and caloric restriction29 and curbs tumor cell metastasis by interfering with cell surface-binding molecules and the expression of matriX metalloproteinase.31 In addition, 1-deoXynojirimycin restricts the development of gastric cancer from its antioXidant and anti-inﬂammatory eﬀects32 and by inducing apoptosis through the Bcl-2/Bax signaling pathway under caloric restriction.29 However, the poor lipophilicity and rapid degradation of 1-deoXynojirimycin prevents the main- tenance of an eﬃcient and prolonged eﬀect.23 Therefore, various derivatives of 1-deoXynojirimycin have been synthe- sized to prolong its half-life and exert multitarget eﬀects by introducing active groups to its N atom, providing a useful
strategy for developing novel candidates.33−35 Recently,

cardioprotective26 and anticancer eﬀects.27 Cancer cells are
quinazoline-1-deoXynojirimycin derivatives, a series of com-

well known to use glycolysis as a source of energy and require more glucose (energy) than normal cells. Since its structure is similar to that of glucose, 1-deoXynojirimycin can penetrate cells rapidly to potently inhibit α-glucosidase in a competitive

pounds with hybrid quinazoline backbones, have been reported as promising antitumor and hypoglycemic agents, since they are highly potent dual inhibitors of epidermal growth factor receptor and α-glucosidase.36

In addition, it has been reported that alkyl chains can tune Table 1. Calculated and Experimental Log P Values and the
the cytotoXicity of 1-deoXynojirimycin derivatives.33 For α-Glucosidase Inhibitory IC50 of 1-Deoxynojirimycin

example, N-(8-(3-ethynylphenoXy)octyl-1-deoXynojirimycin, linking 1-deoXynojirimycin with 3-ethynylphenol via a decane chain, exhibited a 20-fold higher antitumor potency than 1- deoXynojirimycin.33 Therefore, a series of 1-deoXynojirimycin− kaempferol compounds were designed and synthesized, in which the amino group of 1-deoXynojirimycin was linked to the 3-hydroXy of kaempferol via an alkyl chain linker, aiming to improve the cytotoXicity of 1-deoXynojirimycin. This strategy was used to develop agents that exert intracellular dual functions of 1-deoXynojirimycin and kaempferol by inhibiting the activity of α-glucosidase and the expression of cyclo-

Compounds

compound calcd log P α
exptl log P -glucosidase inhibitory IC50 (μM)
1 −2.40 −0.68 ± 0.09 8.03 ± 0.59
8 2.37 0.49 ± 0.04 40.56 ± 4.08
9 3.89 1.65 ± 0.02 1.78 ± 0.08
10 5.40 2.92 ± 0.12 0.24 ± 0.01
In addition, compound 1 is well known to exhibit

oXygenase-2 (COX-2). Furthermore, because of the poor

antidiabetic eﬀects by competitively inhibiting α-glucosi-

lipophilicity and low bioavailability of these compounds, it was investigated whether the conjugation of kaempferol with 1- deoXynojirimycin via an alkyl chain linker would simulta-

dases.42 Therefore, the inhibitory activity of 8−10 against α- glucosidase was analyzed using 1 as a reference compound.43 As shown in Table 1, all tested compounds potently inhibited

neously improve the lipophilicity of 1-deoXynojirimycin and
increase the cytotoXicity of kaempferol. Finally, the apoptotic pathway of tumor cells activated by 1-deoXynojirimycin−
kaempferol was studied.

α-glucosidase, and compound 8 (IC50 of 40.56 ± 4.08 μM) was the least active among the compounds. Compounds 9 and 10 were more potent against α-glucosidase than compound 1, with IC50 values of 1.78 ± 0.08 and 0.24 ± 0.01 μM, which

■ RESULTS AND DISCUSSION
Synthesis and Characterization of Compounds. The
chemical structures of compounds 8−10 (Figure 1) and their synthetic routes are outlined in Scheme 1. First, compound 4 was prepared according to a previously reported proce- dure.37,38 Kaempferol (2) was allowed to react with dimethyl sulfate in the presence of K2CO3 in acetone to protect the hydroXy groups of 2 and obtain the intermediate 3. This intermediate was then treated with AlBr3 to remove the
protecting group at C-3 and yield compound 4. Subsequently, intermediates 5−7 and the target compounds 8−10 were synthesized in accordance with previously published meth- ods.34 Brieﬂy, the hydroXy group at C-3 of intermediate 4 was treated with linear dibromoalkane to obtain compounds 5−7. Then, the target compounds 8−10 were obtained by treating
intermediates 5−7 with 1-deoXynojirimycin (1) in the
presence of K2CO3 in dry dimethylformamide (DMF). The resulting compounds were puriﬁed using column chromatog- raphy, and their structures were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS). The purity of compounds 8−10 was over 95% by analysis using high-performance liquid chromatography (HPLC) (Figures S1−S26, Supporting Information).
Lipophilicity of Compound 10 Contributed to
Inhibiting α-Glucosidase Activity. Lipophilicity is a critical factor in the rational design of certain drugs.35 Generally, log P values in the range of 0.5−3.0 are regarded as optimal for drug absorption and distribution.39 To explore the eﬀect of introducing compound 2 and alkyl chains on the lipophilicity of 1, log P values of 1 and its derivatives 8−10 were calculated using Molinspiration Cheminformatics (miLogP) and also were measured by the traditional shake-ﬂask method.40,41 As shown in Table 1, compound 8 exhibited the poorest lipophilicity among compounds 8−10 with an experimental log P of 0.49 ± 0.04, which was close to that of 1 (log P =
−0.68 ± 0.09). However, the experimental log P values of 9 and 10 were 1.65 ± 0.02 and 2.92 ± 0.12, respectively, which was in the range of 0.5−3.0. This observation indicated that the presence of 2 and an alkyl chain aﬀected the lipophilicity of 1, while increasing the alkyl chain length increased the lipophilicity of the compound.

were approXimately 4.5- and 33.5- fold lower than that of compound 1 (8.03 ± 0.59 μM), respectively. Moreover, the α- glucosidase inhibitory activity of compound 10 was 7.4-fold higher than that of compound 9, indicating that the former exerted an excellent α-glucosidase inhibitory activity. These results suggested that the lipophilicity of compound 10 contributed to enhancing its absorption and subsequent binding to the catalytic site of α-glucosidase, thereby exerting excellent α-glucosidase inhibitory activity.
Compound 10 Exhibited Toxicity in Tumor Cells. The cytotoXicity of compound 8−10 was tested at 48 h using a 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against a panel of human cancer cell lines including breast (MCF-7 and HCC-1937), colon (HCT-116), gastric (BGC-823), liver (HepG-2), and lung cancer lines (A549), using compounds 1 and 2 and physical miXtures of 1 and 2 (1:1 molar ratio) as references. As shown in Table 2, the IC50 values of all the reference compounds and compound 8 were >10 μM. However, as expected, compound 10 exhibited the highest cytotoXicity among compounds 8−10 against all the cancer cell lines tested, with IC50 values ranging from 3.6 to
7.8 μM. Additionally, a monoclonal formation assay also conﬁrmed that 10 had a major eﬀect on the proliferation of MCF-7 cells (Figure 2a). These results suggested the superior antiproliferative eﬀects of compound 10 and prompted a further investigation of its cytotoXic mechanism in MCF-7 cells.
Compound 10 Inhibited MCF-7 Cell Migration. Metastasis is an important event in the later stages of cancer progression,44 and the inhibition of migration is a key step in neoplasm metastasis45 and is an essential target for eﬀective cancer treatment. Therefore, a wound-healing assay was conducted to investigate whether compound 10 represses the migration of MCF-7 cells. The results obtained showed that the wounds on untreated cells re-formed 82.9% healing after 24 h. However, the wounds of cells treated with 1, 2, and their combination at 10 μM showed 78.4%, 79.9%, and 67.6% healing, respectively. Notably, the wounds of MCF-7 cells displayed 56.5% and 46.0% healing after incubation with 10 at
5 and 10 μM, respectively, suggesting that 10 eﬀectively
suppressed the migration of MCF-7 cells (Figure 2b and Figure S27, Supporting Information).

Table 2. Cytotoxicity Proﬁles of Compounds in Diﬀerent Human Cell Lines

compound IC50 (μM)
MCF-7 HCC-1937 HepG-2 HCT-116 BGC-823 A549
1a 2a
1+2b >10 >10 >10 >10 >10 >10
8 >10 >10 >10 >10 >10 >10
9 >10 >10 >10 8.8 ± 0.03 >10 7.6 ± 1.04
10 3.6 ± 0.02 7.1 ± 0.46 7.7 ± 0.98 7.8 ± 0.06 4.5 ± 0.07 5.1 ± 0.13
a1 and 2 were used as positive control. bAn equimolar miXture of compounds 1 and 2.

Figure 2. (a) Clonogenic capacity of MCF-7 cells measured by a colony-forming assay. The cells were cultured with the tested compounds at various concentrations for 48 h and incubated for 2 weeks until the formation of large colonies. (b) The cell migration eﬀects of the compounds on MCF-7 cell migration were examined using a wound-healing assay. Wound images were captured at 0 and 24 h using an inverted ﬂuorescence microscope analyzer. The term MiXture indicates a combination of 1 (10 μM) and 2 (10 μM). 1 and 2 were used as positive controls.

Compound 10 Arrested the Cell Cycle at the S Phase. To investigate the inﬂuence of compound 10 on cell cycle progression, ﬂow cytometry was used to evaluate the cell cycle distribution of MCF-7 cells after 24 h of incubation with 10. As shown in Figure 3a and d, the population of cells in the S phase changed from 25.64% (control group) to 31.41%, 29.49%, and
28.89% after treatment with 1, 2, and their combination (1:1 molar ratio) at 20 μM, respectively. In contrast, the population of cells in the S phase increased to 56.48% after incubation with 10 under the same conditions, suggesting that the introduction of 2 enhanced the ability of the 1-deoXynojir- imycin derivatives evaluated to arrest the cell cycle at the S phase. In addition, the S phase is associated with DNA synthesis and plays a pivotal role in cell cycle progression. These results suggest that compound 10 interferes with DNA synthesis and regulates cell cycle progression.
Compound 10 Elicited MCF-7 Cell Apoptosis. To determine whether the superior antiproliferative eﬀects of compound 10 over the other agents were associated with cell apoptosis, the Hoechst 33342 staining method was used to study the apoptosis of MCF-7 cells qualitatively. As shown in Figure 3b, cell shrinkage and nuclear changes were observed to be more palpable after treatment with compound 10 at 20 μM than after treatment with the other compounds at equimolar concentrations. To conﬁrm the accuracy of these experiments, ﬂow cytometry was used to analyze quantitatively percentage of apoptosis. As shown in Figure 3c and e, MCF-7 cells treated with compound 10 showed a concentration-dependent increase in apoptosis (6.48% and 67.80%) at 10 and 20 μM,

respectively, which was consistent with the apoptosis trend exhibited with Hoechst 33342 staining. In contrast, the percentage of apoptosis was 5.17% for the combination (1:1 molar ratio) at 20 μM, which was much lower than that for compound 10 at the same concentration. These results indicate that compound 10 elicited MCF-7 cell apoptosis and exerted preferential toXicity against MCF-7 cells in a dose- dependent manner.
Compound 10 Induced Mitochondrial Membrane Potential Collapse. The mitochondria play an important role in the regulation of apoptotic cell death,46 and the loss of mitochondrial membrane potential (Δψm) has been implicated as an early event in cell apoptosis.47 To determine whether apoptosis induced by compound 10 was related to mitochondrial dysfunction, MCF-7 cells were treated with 10 at the indicated concentrations for 24 h and Δψm was examined using ﬂuorescence microscopy with JC-1 staining. Untreated cells were used as a negative control, and cells treated with 1, 2, and their combination (1:1 molar ratio) were positive controls. As shown in Figure 4a, the mitochondria in control cells showed an intense red ﬂuorescence. However, the red ﬂuorescence decreased whereas the green ﬂuorescence increased after treatment of MCF-7 cells with 10 at 5 and 10 μM for 24 h, and the eﬀect was especially intense at 10 μM. These results indicated that compound 10 eﬀectively induced Δψm collapse, leading to mitochondrial dysfunction and ﬁnally triggered apoptotic cell death.
Compound 10 Augmented ROS Accumulation.
Mitochondrial dysfunction is also associated with mitochon-
Figure 3. (a) Cell cycle distribution was tested using ﬂow cytometric analysis. MCF-7 cells were incubated with various compounds at the indicated concentrations for 24 h. (b) MCF-7 cells were stained with Hoechst 33342 staining and visualized using a ﬂuorescence microscope. (c) The image of MCF-7 cell apoptosis was induced by the tested compounds at various concentrations for 24 h using annexin V-FITC/PI staining. (d) DNA content in MCF-7 cell cycle distribution. (e) Comparison of MCF-7 cell apoptosis in various compounds. The term MiXture indicates a combination of 1 (20 μM) and 2 (20 μM).

Figure 4. (a) Mitochondrial membrane potential Δψm and (b) the ROS and (c) Ca2+ level after treatment with the tested compounds at the indicated concentrations for 24 h using ﬂuorescence microscopy. The term MiXture indicates a combination of 1 (10 μM) and 2 (10 μM).

drial production of ROS, which has been implicated in the induction or enhancement of apoptosis.48 Therefore, increased accumulation of ROS in MCF-7 cells was detected using the

ﬂuorescent probe 2′,7′-dichlorodihydroﬂuorescein diacetate (DCHF-DA) after treatment with the test compounds for 24
h.49 As shown in Figure 4b, green ﬂuorescence intensity in cells
Figure 5. (a) Western blotting analysis of COX-2, Bcl-2, and Bax protein expression in MCF-7 cells cultured with compound 10 (10 μM), 1 (10 μM), and 2 (10 μM) and a miXture for 24 h, respectively. (b) Statistical analysis of protein expression relative to β-actin. The term MiXture indicates a combination of 1 (10 μM) and 2 (10 μM).

treated with 10 at 10 μM was much higher than that in cells in the other groups, suggesting that compound 10 signiﬁcantly increased the intracellular ROS level, which coincided with the decrease in Δψm. Taken together, the results showing Δψm reduction and ROS generation indicated that 10 induced MCF-7 cell apoptosis through the mitochondrial pathway.50
Compound 10 Increased Mitochondrial Ca2+ Inﬂux in MCF-7 Cells. The accumulation of ROS in cells can induce
oXidative stress in the endoplasmic reticulum (ER) and promote the release of Ca2+ from internal stores into the cytoplasm.51 EXcessive Ca2+ released from the ER leads to

undecane chain, was further studied to elucidate the possible antitumor mechanism, related to its enhanced lipophilicity and antiproliferation eﬀects against MCF-7 cells. Compared with 1, 2, and their combination, compound 10 inhibited signiﬁcantly the migration of MCF-7 cells, caused cell cycle arrest in the S phase, and reduced the levels of COX-2. Moreover, compound 10 induced MCF-7 cell apoptosis through the mitochondrial dysfunction pathway by mediating Δψm disruption, increasing intracellular levels of ROS and Ca2+, downregulating Bcl-2 expression, and upregulating Bax expression. In addition, an anti-α-glucosidase analysis indicated that compound 10

cancer cell death and dysregulation. Therefore, the change in Ca2+ levels after treatment with compound 10 was examined using the Ca2+ indicator Fluo-4 AM via ﬂuorescence microscopy. As shown in Figure 4c, the Ca2+ levels were increased signiﬁcantly in a dose-dependent manner after treatment with 10 and was much higher than that following

exerted a more potent α-glucosidase inhibitory eﬀect than compound 1, which may have been mediated by reducing blood glucose levels and reducing the energy supply of MCF-7 cells. Taken together, these results indicate that the conjugation of two natural products, 1-deoXynojirimycin and kaempferol, to form one drug molecule might provide a useful

treatment with other compounds, which was consistent with the level of ROS production. The intracellular accumulation of ROS induced by compound 10 probably altered the permeability of the mitochondrial membrane, leading to the

strategy for the development of antitumor and antidiabetic agents.
■ EXPERIMENTAL SECTION

1-deoXynojirimycin-based

release of excessive Ca2+ from the ER, which was ﬂuXed into the mitochondria and may have eventually induced apoptosis. Compound 10 Regulated COX-2 and Apoptosis- Related Protein Expression. COX-2 protein is the primary target of anti-inﬂammatory drugs.52 Kaempferol has been reported to downregulate COX-2 expression and exert anti- inﬂammatory eﬀects.6,26 Therefore, Western blotting was used to investigate whether compound 10 would reduce the levels of COX-2 protein. As shown in Figure 5a and b, the expression of COX-2 was decreased signiﬁcantly after the treatment of MCF-7 cells with 10, which was similar to the eﬀects of 2 and a miXture of 1 and 2. The abnormal expression of COX-2 also aﬀected the sensitivity of cancer cells to chemotherapy drugs by interfering with the Bcl-2 family.53 The Bcl-2 family plays vital roles in the regulation of the mitochondrial apoptotic pathway, and the Bax/Bcl-2 ratio has been proposed as a key factor in determining cell apoptosis.54,55 Compared with the control group, treatment of MCF-7 cells with compound 10 resulted in lower levels of Bcl-2 proteins, higher level of Bax proteins, and higher Bax/Bcl-2 ratio. These ﬁndings further indicated that compound 10 induced apoptosis of MCF-7 cell
via the mitochondrial pathway.

CONCLUSIONS
In conclusion, three novel 1-deoXynojirimycin derivatives were designed and synthesized, compounds 8−10, with the aim of improving the lipophilicity and bioavailability of compound 1. Among them, compound 10, a fusion of 1 and 2 with an

General Experimental Procedures. All chemicals were acquired from commercial sources and used without further puriﬁcation unless noted otherwise. 1-DeoXynojirimycin, dimethyl sulfate, 1,5-dibromo- pentane, 1,8-dibromooctane, and 1,11-dibromoundecane were purchased from Energy-Chemical Co., Ltd. (Shanghai, People’s Republic of China). Kaempferol was obtained from Tianjin Heowns Biochem LLC. MTT, propidium iodide (PI), annexin V-FITC/PI assay kit, crystal violet, medium, and fetal bovine serum (FBS) were from Solarbio. Bcl-2 monoclonal antibody, Bax monoclonal antibody, COX-2 monoclonal antibody, and β-tubulin monoclonal antibody were from Proteintech. Cells were counted with the Countstar cell automatic counter from Advanced Lab Instrument and Technology Co., Ltd. 1H NMR and 13C NMR spectra were measured with a Bruker spectrometer. The NMR spectra were processed and analyzed using the MestReNova software package. HRMS was determined by a Bruker solanX 70 FT-MS.
Purity of Target Compounds. The purity of target compounds 8−10 was determined by HPLC. HPLC analysis was carried out on a Shimadzu Prominence HPLC system equipped with a Venusil XBP- C18 column (5 μm, 150 Å, 250 mm × 4.6 mm). HPLC proﬁles were recorded by a UV detector at 367 nm at room temperature. A linear
gradient of 5−95% solvent B (methanol) in solvent A (0.1% formic acid in water) in 10 min and then constant 95% solvent B in 10−25 min was used to determine purity at a ﬂow rate of 1 mL min−1. The purity of the compounds 8−10 was conﬁrmed to be >95% (Table S1, Supporting Information), which was based on three parallel
experiments. The mobile phase used for HPLC is shown in Table 3. Synthesis of 3,5,7-Trimethoxy-2-(4-methoxyphenyl)-4H-chro- men-4-one (3). To a solution of compound 2 (6.00 g, 20.96 mmol) and K2CO3 (23.20 g, 160.00 mmol) in 5 mL of acetone was slowly added dimethyl sulfate (9.94 mL, 100.00 mmol); then the

Table 3. Mobile Phase of HPLC Analyses for the Purities of All Target Compound25.00 5 95

miXture was stirred at 50 °C. When the reactant was consumed completely, an appropriate amount of dilute NaOH solution was added to quench the reaction and adjust the pH to 7. The reaction miXture was evaporated, and the residue was dissolved in water, extracted with ethyl acetate three times, and then dried over anhydrous MgSO4. The solution was concentrated under a vacuum, and the crude product obtained was puriﬁed by column chromatography to obtain compound 3 as a pale yellow solid: yield 90%; 1H NMR (400 MHz, CDCl3) δ 8.07 (2H, d, J = 6 Hz), 7.00 (2H, d, J = 6 Hz), 6.50 (1H, d, J = 1.2 Hz), 6.33 (1H, d, J = 1.6 Hz), 3.95 (3H, s), 3.89−3.87 (9H, m); 13C NMR (101 MHz, CDCl3) δ
174.06, 163.81, 161.10, 160.92, 158.76, 152.61, 141.02, 129.79,
123.18, 113.87, 109.40, 95.69, 92.36, 59.87, 56.38, 55.76, 55.39.
Synthesis of 3-Hydroxy-5,7-dimethoxy-2-(4-methoxyphenyl)-4H- chromen-4-one (4). To a solution of AlBr3 (22.60 g, 38.00 mmol) in acetonitrile (400 mL) in an ice bath was added compound 3 (13.00 g,
38.00 mmol). The miXture was stirred at room temperature for 2 h.
Then, the reaction solution was diluted with 2% hydrochloric acid and stirred at 75 °C for 25 min, and distilled water was added to dilute the

164.05, 161.24, 160.76, 158.63, 152.35, 139.77, 130.11, 123.15,
114.34, 108.96, 96.26, 93.35, 71.80, 56.51, 56.40, 55.80, 35.59, 32.72,
29.91, 29.43, 29.18, 28.58, 28.00, 25.91; HRMS m/z 583.16620 [M + Na]+ (calcd for C29H37BrO6, 583.16712); 601.13824 [M + K]+ (calcd for C29H37BrO6, 601.13901).
General Procedure for the Synthesis of 8−10. The intermediate5, 6, or 7 (0.20 mmol)was added to anhydrous DMF (2 mL), followed by addition of K2CO3 (0.40 mmol) and
compound 1 (0.18 mmol) to the miXture. The reaction was then stirred at 80 °C for 12 h, ﬁltered, and evaporated under vacuum to remove the solvent. The residue was puriﬁed using column chromatography to obtain the desired compounds 8−10 as a pale yellow solid.
5,7-Dimethoxy-2-(4-methoxyphenyl)-3-((5-((2R,3R,4R,5S)-3,4,5- trihydroxy-2-(hydroxymethyl)piperidin-1-yl)pentyl)oxy)-4H-chro- men-4-one (8): yield 30%; 1H NMR (400 MHz, DMSO-d6) δ 8.04 (2H, d, J = 8.8 Hz), 7.11 (2H, d, J = 8.8 Hz), 6.80 (1H, d, J = 2 Hz),
6.48 (1H, d, J = 2 Hz), 4.72 (2H, s), 4.14−4.12 (1H, m), 3.88−3.84
(9H, m), 3.72 (1H, d, J = 10.8 Hz), 1.97−1.91 (2H, m), 1.63 (1H, s), 1.38−1.22 (8H, m), 0.94−0.85 (6H, m); 13C NMR (101 MHz, DMSO-d6) δ 172.21, 166.95, 160.78, 160.24, 158.14, 151.89, 139.26,
131.65, 131.58, 129.61, 128.63, 122.57, 113.96, 108.41, 95.82, 92.88,
71.10, 67.34, 56.04, 55.95, 55.35, 51.95, 28.32, 27.17, 23.18, 22.37,
18.84, 13.88, 10.77; HRMS m/z 560.24940 [M + H]+ (calcd for
C29H37NO8, 560.24957); purity (HPLC) 99%.
5,7-Dimethoxy-2-(4-methoxyphenyl)-3-((8-((2R,3R,4R,5S)-3,4,5- trihydroxy-2-(hydroxymethyl)piperidin-1-yl)octyl)oxy)-4H-chro-

miXture, followed by the removal of acetonitrile using a rotary evaporator. After storing in a refrigerator for 24 h, the solution was ﬁltered and the precipitate was collected. Then, compound 4 was obtained by chromatography using a silica gel column as a pale yellow solid: yield 88%; 1H NMR (400 MHz, DMSO-d6) δ 8.94 (1H, s), 8.15 (2H, d, J = 5.6 Hz), 7.10 (2H, d, J = 5.6 Hz), 6.81 (1H, s), 6.46
(1H, s), 3.89 (3H, s), 3.86 (3H, s), 3.84 (3H, s); 13C NMR (101 MHz, DMSO-d6) δ 171.02, 163.61, 160.04, 159.95, 158.01, 141.66,
137.76, 128.62, 123.45, 113.93, 106.23, 95.57, 92.69, 56.11, 55.90,
55.26; HRMS m/z 351.08476 [M + Na]+ (calcd for C18H16O,
351.08446).
General Procedure for the Synthesis of 5−7. To a solution of compound 4 (0.92 mmol) and K2CO3 (4.57 mmol) in acetone (5 mL) was added 1,5-dibromopentane, 1,8-dibromooctane, or 1,11- dibromoundecane (4.57 mmol), and the miXture was allowed to reﬂuX overnight. After completion of the reaction, the solvent was removed under reduced pressure. The resulting residue was puriﬁed via silica
gel column chromatography to obtain the desired product compounds 5−7, as pale yellow solids.
3-((5-Bromopentyl)oxy)-5,7-dimethoxy-2-(4-methoxyphenyl)- 4H-chromen-4-one (5): yield 75%; 1H NMR (400 MHz, DMSO-d6)
δ 8.02 (2H, d, J = 8.8 Hz), 7.11 (2H, d, J = 8.8 Hz), 6.78 (1H, s), 6.47
(1H, s), 3.88−3.83 (11H, m), 3.50 (2H, t, J = 6.8 Hz), 1.82−1.75

men-4-one (9): yield 23%; 1H NMR (400 MHz, DMSO-d6) δ 8.02
(2H, d, J = 8.8 Hz), 7.09 (2H, d, J = 9.2 Hz), 6.77 (1H, d, J = 1.6 Hz),
6.47 (1H, d, J = 2 Hz), 4.76 (2H, s), 3.90−3.83 (11H, m), 3.72 (1H,
d, J = 11.2 Hz), 3.58 (1H, s), 3.37 (2H, s), 3.23 (1H, s), 3.09−3.04
(1H, m), 2.95−2.91 (1H, m), 2.51 (2H, s), 1.98 (2H, s), 1.59 (2H, t, J = 6.8 Hz), 1.36−1.20 (11H, m); 13C NMR (101 MHz, DMSO-d6) δ 172.19, 163.56, 160.74, 160.23, 158.13, 151.92, 139.24, 129.63,
122.60, 113.87, 108.42, 95.78, 92.84, 77.02, 72.67, 71.30, 68.68, 66.50,
56.96, 56.01, 55.92, 55.33, 54.89, 52.03, 44.67, 29.41, 28.96, 28.75,
26.88, 25.41; HRMS m/z 624.27614 [M + Na]+ (calcd for C32H43NO10, 624.27847); purity (HPLC) 95%.
5,7-Dimethoxy-2-(4-methoxyphenyl)-3-((11-((2R,3R,4R,5S)-3,4,5- trihydroxy-2-(hydroxymethyl)piperidin-1-yl)undecyl)oxy)-4H-chro- men-4-one (10): yield 22%; 1H NMR (600 MHz, DMSO-d6) δ 8.02 (2H, d, J = 9.0 Hz), 7.09 (2H, d, J = 9 Hz), 6.78 (1H, d, J = 2.4 Hz),
6.47 (1H, d, J = 2.4 Hz), 4.69 (2H, s), 4.10 (1H, s), 3.90−3.88 (5H,
m), 3.84 (6H, d, J = 4.2 Hz), 3.71 (1H, d, J = 10.2 Hz), 3.56 (1H, s),
3.22 (1H, s), 3.05 (1H, s), 2.92 (1H, s), 2,73 (2H, s), 2,39 (1H, s),
1.93 (2H, s), 1.60−1.56 (2H, m), 1.37 (2H, s), 1.28−1.19 (15H, m);
13C NMR (150 MHz, DMSO-d6) δ 172.70, 164.09, 161.27, 160.77,
158.65, 152.44, 139.78, 130.16, 123.14, 114.39, 108.96, 96.32, 93.40,
71.84, 56.55, 56.44, 55.83, 55.38, 52.55, 29.90, 29.56, 29.49, 29.44,
29.20, 27.45, 25.92; HRMS m/z 644.34251 [M + H]+ (calcd for

(2H, m), 1.67−1.60 (2H, m), 1.49−1.42 (2H, m); 13C NMR (101
MHz, DMSO-d6) δ 172.16, 163.57, 160.76, 158.14, 151.95, 139.21,

129.63, 122.56, 113.93, 108.41, 95.79, 92.86, 71.00, 56.02, 55.93,
55.33, 35.05, 31.85, 28.53, 24.19; HRMS m/z 499.07221 [M + Na]+

Calculation and Measurement of log P. The log Po/w values were calculated using the online program Molinspiration (http://

(calcd for C23

www.molinspiration.com/). The actual log P values of compound 1
and its derivatives 8−10 were obtained using the traditional octanol−

3-((8-Bromooctyl)oxy)-5,7-dimethoxy-2-(4-methoxyphenyl)-4H- chromen-4-one (6): yield 71%; 1H NMR (400 MHz, DMSO-d6) δ 8.02 (2H, d, J = 9.2 Hz), 7.10 (2H, d, J = 8.8 Hz), 6.78 (1H, s), 6.47
(1H, s), 3.90−3.83 (11H, m), 3.51 (2H, t, J = 6.8 Hz), 1.80−1.73
(2H, m), 1.62−1.55 (2H, m), 1.34−1.15 (8H, m); 13C NMR (101 MHz, DMSO-d6) δ 172.18, 163.57, 160.75, 160.25, 158.14, 151.91,
139.25, 129.64, 122.62, 113.88, 108.43, 95.80, 92.87, 71.28, 56.02,
55.93, 55.33, 35.13, 32.19, 29.35, 28.51, 28.03, 27.41, 25.32; HRMS
m/z 541.11905 [M + Na]+ (calcd for C26H31BrO6, 541.12017).
3-((11-Bromoundecyl)oxy)-5,7-dimethoxy-2-(4-methoxyphenyl)- 4H-chromen-4-one (7): yield 69%; 1H NMR (600 MHz, DMSO-d6) δ 8.00 (2H, d, J = 9.2 Hz), 7.08 (2H, d, J = 8.4 Hz), 6.75 (1H, d, J =
1.8 Hz), 6.45 (1H, d, J = 2.4 Hz), 3.89−3.83 (11H, m), 3.49 (2H, t, J
= 7.2 Hz), 1.78−1.74 (2H, m), 1.60−1.55 (2H, m), 1.36−1.31 (2H, m), 1.18 (12H, s); 13C NMR (150 MHz, DMSO-d6) δ 172.66,

water shake-ﬂask method, in accordance with the previously published reports.56 First, the standard concentration absorbance curves of compound 1 and its derivatives in n-octanol and phosphate-buﬀered saline (PBS) were constructed using data obtained from an ultraviolet (UV) spectrometer. Then, the miXture of n-octanol and PBS was shaken to saturation, left overnight, and then separated before use. The compounds were dissolved in PBS-saturated n-octanol, incubated with an equal volume of n-octanol-saturated PBS, and stirred. After 3 h, the two phases were separated. The absorbance of these compounds in each phase was measured using a UV spectrometer, and the concentrations in PBS (Cw) and n-octanol (Co) were obtained from the standard curves. The log P values were calculated using the following formula: log P = log Co/Cw. All experiments were repeated independently three times.

Stability of Compounds 8−10 in PBS/DMF Buffer. Com- pounds 8−10 were incubated in a PBS/DMF (99:1, v/v) at 37 °C at diﬀerent times and then checked by HPLC. The conditions of the stability analysis were the same as that of the purity analysis (Figure S28−S30, Supporting Information).
Screening of α-Glucosidase Inhibitory Activity. p-Nitro-
phenyl-α-D-glucopyranoside (pNPG) was used as a substrate to evaluate the α-glucosidase inhibitory activity of the test samples. α- Glucosidase (0.02 mg/mL) was premiXed with 50 μL of each compound at varying concentrations made up in phosphate buﬀer at pH 6.8 and incubated for 5 min at 37 °C. pNPG (1 mM) was added to initiate the reaction, and the miXture was further incubated at 37
°C for 30 min; then it was terminated by the addition of 60 μL of 0.1 M Na2CO3 to a ﬁnal volume of 250 μL. α-Glucosidase activity was determined spectrophotometrically at a wavelength of 405 nm using a microplate reader (Inﬁnite F50, Tecan) to measure the quantity of p- nitrophenol released from pNPG. The assay was performed in triplicate, and the concentration of the extract required to inhibit 50% of α-glucosidase activity under the assay conditions was deﬁned as IC50.
Cell Cultures. The human gastric cancer (BGC-823), breast carcinoma (MCF-7 and HCC-1937), colon cancer (HCT-116), non- small-cell lung cancer (A549), and liver cancer (HepG-2) cell lines were purchased from the American Type Culture Collection. Cells were cultured in DMEM (for MCF-7 and A549) or RPMI1640 (for HCT-116, HCC-1937, HepG-2, and BGC-823) medium containing 10% FBS and maintained in a humidiﬁed atmosphere of 5% CO2 at 37 °C.
Cell Viability and Proliferation Analysis. The cytotoXicity of 1, 2, compounds 8−10, and their combination against diﬀerent cell lines (MCF-7, HCC-1937, HepG-2, HCT-116, BGC-823, and A549) was
evaluated using the MTT assay. Brieﬂy, the cells (3 × 103/well) were seeded in 96-well plates and incubated in an incubator at 37 °C and 5% CO2 for 24 h. Then, the cells were treated with diﬀerent concentrations of the test compounds and incubated for 48 h, followed by the addition of 10 μL of MTT (5 mg/mL in PBS) to each well and incubation for 4 h at 37 °C. The purple formazan crystals that formed subsequently were dissolved by adding 100 μL of dimethyl sulfoXide (DMSO) to each well after the culture medium was removed. The absorbance was measured at a wavelength of 570 nm using a microplate reader, and the results were recorded. The mean and standard deviation of triplicate values of each concentration were calculated, and IC50 was determined using SPSS 16.0 software. Colony Formation Assay. The aim of this assay was to test the inhibitory activity of compound 10 against the cancer cell colonization. Cellular proliferation, which is the ability of cancer cells to maintain a clonogenic capacity, was assessed using a colony formation assay. MCF-7 cells were seeded in a siX-well plate at a density of 500 cells/well and incubated for 3 days until the cells attached to wells. Compounds 1 and 2, alone or in combination, and 10 were added to the cells at various concentrations for 48 h. Then, the cell culture was replenished with fresh medium every 3−4 days
and incubated for 2 weeks until large colonies were formed. Next, the cells were washed twice with PBS, ﬁXed at 25 °C with 4%

test compounds at the indicated concentrations for 24 h. Next, the cells were washed twice with cold PBS, collected using trypsin, resuspended in ice-cold 70% ethanol, and stored at −20 °C for 48 h. The residual ethanol in the tube was washed oﬀ, and the samples were resuspended in 250 μL of PBS containing 2.5 μL of RNase A and incubated at 37 °C for 30 min before adding PI dye solution (12.5 μL of 1 mg/mL solution). After 30 min of staining, the results were detected using a ﬂow cytometer (Beckman CytoFLEX) and processed by ModFit LT.
Hoechst 33342 Staining. Nuclear chromatin condensation is a critical characteristic of apoptotic cells. MCF-7 cells at a density of 1
× 105 cells/well were cultured in siX-well plates under 5% CO2 at 37
°C for 24 h. The cells were then treated with various compounds at the indicated concentrations for 24 h. Then the solution was removed, and the cells were washed with ice-cold PBS three times and stained with 1 μg/mL Hoechst 33342 for 15 min at 37 °C in the dark. This was followed by washing twice with PBS, and the stained cells were visualized using a ﬂuorescence microscope.
Apoptosis Analysis Using Annexin V-FITC/PI Staining. MCF- 7 cells were seeded in siX-well plates at a density of 2 × 105 cells/well and cultured at 37 °C for 24 h. After attachment, the cells were treated with the compounds at the indicated concentrations for 24 h. Then, the cells were washed with ice-cold PBS, digested, and collected into tubes. The next staining step was conducted following the instructions of the annexin V-FITC/PI apoptosis detection kit, and apoptosis was detected using ﬂow cytometry (BD FACS Verse). Data were analyzed by FlowJo7.6.
Mitochondrial Membrane Potential Assay. JC-1 is an ideal ﬂuorescent probe widely used to detect mitochondrial membrane potential. MCF-7 cells were added to siX-well plates at a density of 1 × 105 cells/well and incubated at 37 °C for 24 h. The cells were then treated with fresh medium containing diﬀerent compounds at the indicated concentrations for 24 h. Then, the cells were washed with ice-cold PBS three times and stained with JC-1 for 20 min at 37 °C in the dark. Finally, after further washing with JC-1 staining buﬀer, images of the cells were captured using an inverted ﬂuorescence microscope.
Assessment of Intracellular ROS Levels. DCHF-DA is usually utilized to induce ROS formation. MCF-7 cells were seeded in siX-well plates at a density of 1 × 105 cells/well and cultured at 37 °C overnight. Then, the medium was replaced with a fresh one containing diﬀerent test compounds at the indicated concentrations followed by incubation for 24 h. Following this, the cells were washed with PBS and treated with 10 μM DCFH-DA for 30 min at 37 °C. Finally, the stained cells were washed with DMEM three times and visualized using a ﬂuorescence microscope.
Evaluation of Intracellular Ca2+ Content. Fluo-4 AM crosses the cell membrane and is cleaved into Fluo-4 by endogenous esterases and trapped inside the cells. Fluo-4 speciﬁcally combines with Ca2+ to produce a strong ﬂuorescence. The concentration of intracellular free Ca2+ was determined using a Fluo-4 AM dye. MCF-7 cells were seeded in siX-well plates at a density of 1 × 105 cells/well, grown in DMEM with 10% FBS, and incubated at 37 °C for 24 h. Then, the medium was replaced with a fresh one containing the test compounds

paraformaldehyde for 15 min, stained with crystal purple (0.1%) for 15 min, washed with water, and photographed using a digital camera. Wound-Healing Assay. The eﬀects of compound 10 on breast tumor cell migration were examined using a wound-healing assay. Brieﬂy, MCF-7 cells were inoculated in siX-well plates at a density of 2
× 105 cells/well and cultured to a nearly conﬂuent monolayer in complete DMEM containing 10% FBS. Then, a sterilized 200 μL pipet tip was used to create a wound across the cells, and PBS was slowly added to the wells to eliminate cell debris. Then, the cells were treated with various compounds at the indicated concentrations. Wound images were captured at 0 and 24 h using an inverted ﬂuorescence microscope analyzer.
Cell Cycle Arrest Assay. MCF-7 cells were seeded in siX-well plates at a density of 2 × 105 cells/well, grown in DMEM supplemented with 10% FBS, and incubated at 37 °C for 24 h. Then, the medium was replaced with fresh medium containing the

at diﬀerent concentrations. After being treated for 24 h, cells were washed three times with PBS and stained with 2 μM Fluo-4 AM for 30 min at 37 °C in the dark, followed by washing three times with PBS. The ﬂuorescence images were captured using an inverted ﬂuorescence microscope.
Western Blot Analysis. The active preferred MCF-7 cells were seeded, cultured until they reached a density of 80%, treated with 10 μM of the test compounds, and then further cultured at 37 °C for 24
h. Proteins were extracted using a lysis buﬀer, and the lysate was stored at −20 °C. The protein concentrations were quantiﬁed using a BCA protein concentration detection kit (Solarbio). Then, the proteins were separated using 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred onto polyvinyli- dene diﬂuoride membranes. The membranes were then blocked with 5% nonfat milk in Tris-buﬀered saline with 0.1% Tween-20 (TBST) for 4 h and incubated with primary antibodies at 4 °C overnight,

shaking gently. Then, the membranes were washed with TBST and further incubated with the secondary antibodies for 1.5 h. All membranes were washed three times with TBST for 30 min, and protein blots were detected using a chemiluminescence reagent (Biosharp) and a Tanon automated chemiluminescence imaging analysis system.

ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.1c00014.
1H NMR and 13C NMR spectra for compounds 3−10, HRMS spectra for compounds 4−10, HPLC assessment of purity and stability for compounds 8−10, statistical analysis of migration in MCF-7 cells after treatment with
compound 10 (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Zhongzheng Gui − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212100, People’s Republic of China; Email: [email protected]
Authors
Ran Zhang − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212100, People’s Republic of China; orcid.org/0000-0003-3661- 7785
Yueyue Zhang − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s
Republic of China
Xiangdong Xin − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China
Gaiqun Huang − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China; Sericultural Research Institute,
Sichuan Academy of Agricultural Sciences, Nanchong, Sichuan 637000, People’s Republic of China
Ning Zhang − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China
Qinglei Zeng − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China
Liumei Tang − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China
Thomas Attaribo − School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, People’s Republic of China
Kwang Sik Lee − College of Natural Resources and Life Science, Dong-A University, Busan 49315, Republic of Korea
Byung Rae Jin − College of Natural Resources and Life Science, Dong-A University, Busan 49315, Republic of Korea
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jnatprod.1c00014

Author Contributions
#R.Z. and Y.Z. contributed equally and are joint ﬁrst authors.
Notes
The authors declare no competing ﬁnancial interest.
■ ACKNOWLEDGMENTS
Financial support for this research was provided by the Jiangsu
Province Policy Guidance Project (BX2019072) and the Jiangsu Project of Science and Technology (XZ-SZ201925).
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