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. 2025 Apr 14. Online ahead of print. doi: 10.1039/d5md00179j

Design, synthesis and anti-proliferative activity of 3-aryl-evodiamine derivatives†

Ming-Li Zhou a,b, Nan-Ling Liu a, Lian Sun a, Xiaohuan Li a, Jin-Bu Xu a,c,✉, Feng Gao a,c,✉
PMCID: PMC12224201  PMID: 40621061

Abstract

Evodiamine and its analogues have received significant attention owing to their antitumor activity. In this work, Pd-catalyzed Suzuki–Miyaura coupling was employed as a key reaction to diversify the natural product evodiamine at the C3 position. A library of new 3-aryl-evodiamine derivatives (6a–6ae) was synthesized. The in vitro antitumor activity of these compounds were evaluated against various cancer cell lines (HCT116, 4T1, and HepG2). Most of them showed better cytotoxicity than evodiamine. Herein, the primary structure–activity relationship of 3-aryl-evodiamine derivatives was briefly discussed. In particular, the methylsulfonylbenzene derivative 6y exhibited excellent anti-proliferative activity against HCT116 (IC50 = 0.58 ± 0.04 μM) and 4T1 cells (IC50 = 0.99 ± 0.07 μM), providing a promising evodiamine analogue for antitumor drug development. This work would offer new insights for the development of evodiamine-based compounds.

Evodiamine and its analogues have received significant attention owing to their antitumor activity.graphic file with name d5md00179j-ga.jpg

1. Introduction

Natural products serve as important inspirations in the field of new drug development. Evodiamine (Evo, 1) is a naturally occurring quinazolinocarboline alkaloid isolated from the Chinese herbal medicine Evodia rutaecarpa (rutaceae).1 It has a wide spectrum of pharmacological properties, such as cytotoxicity, anti-neuroinflammation, and analgesic effects.2,3 Anticancer activity of evodiamine has been extensively studied over the past few decades. As a potential multi-target anti-cancer compound,4 evodiamine exhibits moderate antiproliferative activity only at high concentrations (for example, IC50(A549) = 100 μM, IC50(MDA-MB-435) = 20 μM, and IC50(HCT116) = 100 μM), limiting its clinical development.5 Natural product diversification is essential for developing new drugs with enhanced specificity.6 Therefore, many efforts involving the structural modification of evodiamine have been made, hoping to develop highly active evodiamine derivatives.5,7–13 In recent years, Sheng's group have continuously conducted many ingenious works on the structural optimization of evodiamine,5,7–10 developing a series of novel anti-cancer compounds such as 3-fluoro-10-hydroxy-evodiamine, 3-amino-10-hydroxy-evodiamine, and 3-chloro-10-hydroxy-thio-evodiamine (Fig. 1). Prior structure–activity relationship studies of these evodiamine derivatives indicate that the C-3 position of the E ring is a key active site for structural optimization of evodiamine. Furthermore, 3-methyl-14-phenyl-evodiamine showed strong anticancer potency,13 indicating that introducing aromatic rings into evodiamine could enhance its activity.

Fig. 1. Structures of evodiamine and its analogues.

Fig. 1

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The biphenyl unit is a common pharmacophore in small-molecule drugs.14 Tazemetostat (Fig. 2), a methyltransferase inhibitor, is approved by FDA in 2020 for the treatment of epithelioid sarcoma.15 Brequinar is a potent dihydroorotate dehydrogenase inhibitor, which has been evaluated in multiple clinical trials as a potential anticancer drug.16 Natural products possessing a biphenyl moiety, clusiparalicoline A17 and garmultine A,18 have also exhibited remarkable cytotoxic activities. In this work, we envisioned the introduction of aromatic groups into the C-3 position of evodiamine to design novel evodiamine derivatives containing a biphenyl structural unit. The palladium-catalyzed Suzuki–Miyaura coupling is an important reaction for forming C–C bonds,19 providing a highly selective and efficient chemical method to synthesize diversified 3-aryl-evodiamine derivatives. Therefore, as part of our effort to find novel antitumor bioactive molecules,20 we performed the functional modification of the natural drug evodiamine to generate a C-3-arylated diverse molecular library employing the Suzuki–Miyaura cross-coupling reaction as the key synthetic method. The cytotoxicity of the obtained novel evodiamine analogues (6a–6ae) was also evaluated. Furthermore, the preliminary structure–activity relationship was discussed in this work.

Fig. 2. Structural modification strategy reported in this work.

Fig. 2

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2. Results and discussion

2.1. Chemistry

The synthetic route of 3-aryl evodiamine derivatives (6a–6ae) is outlined in Scheme 1. At the initial stage of the investigation, commercially available evodiamine (1) was used as the starting material. Di-tert-butyldicarbonate (Boc2O) was employed to protect the nitrogen at the N-13 position, providing N-Boc-evodiamine (2) (Scheme 1). Compound 2 was halogenated with NBS to produce bromide 3. The single-crystal X-ray diffraction analysis (Fig. 3) elucidated that the Br atom is located at the C-3 position in compound 3. Subsequently, deprotection in the presence of NaOMe (30 wt% in methanol) in a THF solution provided the key intermediate 3-bromo-evodiamine (4),21 which was used in the Suzuki–Miyaura cross-coupling reactions. After that, 3-bromo-evodiamine (4) and phenylboronic acid (5a) as the model substrates were employed to optimize the Suzuki–Miyaura cross-coupling reaction condition. After screening different Pd catalysts (Pd(OAc)2, Pd(dppf)Cl2, Pd[P(C6H5)3]4), bases (KF, K3PO4, and K2CO3), and solvents (THF/H2O, THF, 1,4-dioxane, and toluene) (Table 1), Pd[P(C6H5)3]4 (10 mol%) catalyzing the reaction with K2CO3 at 80 °C for 12 h in THF/H2O (10 : 1) was found to be the optimized reaction condition, providing the biphenyl evodiamine analogue 6a in 85% yield (entry 7).

Scheme 1. Synthetic route for evodiamine derivatives 6a–6ae.

Scheme 1

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Fig. 3. Single-crystal X-ray structure of compound 3.

Fig. 3

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Table 1. Optimization of reaction conditionsa.

graphic file with name d5md00179j-u1.jpg
Entry Catalyst Ligand Base Solvent Yieldb/time
1 Pd(OAc)2 (10% mol) Dicyclohexylphenylphosphine KF THF/H2O (10 : 1) 80%/24 h
2 Pd(OAc)2 (10% mol) 2-(Di-tert-butylphosphino)-biphenyl KF THF/H2O (10 : 1) 42%/24 h
3 Pd(OAc)2 (10% mol) BINAP KF THF/H2O (10 : 1) 53%/5 h
4 Pd(OAc)2 (10% mol) X-PHOS KF THF/H2O (10 : 1) 50%/24 h
5 Pd(dppf)Cl2 (10% mol) — KF THF/H2O (10 : 1) 80%/24 h
6 Pd[P(C6H5)3]4 (10% mol) — K3PO4 THF/H2O (10 : 1) 40%/12 h
7 Pd[P(C6H5)3]4 (10% mol) — K2CO3 THF/H2O (10 : 1) 85%/12 h
8 Pd[P(C6H5)3]4 (10% mol) — K2CO3 THF 55%/12 h
9 Pd[P(C6H5)3]4 (10% mol) — K2CO3 1,4-Dioxane 52%/12 h
10 Pd[P(C6H5)3]4 (10% mol) — K2CO3 Toluene 0%/12 h
11 Pd[P(C6H5)3]4 (5% mol) — K2CO3 THF/H2O (10 : 1) 40%/12 h
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a

Reaction conditions: 4 (0.2 mmol, 1.0 equiv.), 5a (0.3 mmol, 1.5 equiv.), in solvent (1 mL).

b

Isolated yield.

Under the optimized reaction conditions in hand, 3-aryl evodiamine derivatives (6b–6ae) were prepared by reacting intermediate 4 with arylboronic acids 5b–5ae in moderate to good yields. As shown in Scheme 1, various substrates were compatible with the reaction. Benzene ring bearing electron-donating substituents (methyl, isopropyl, methoxyl, thiomethyl, and hydroxy groups) participated in the coupling reaction to afford the desired 6b–6j in yields of 66–86%. The substrates 5k–5s with stronger electron-withdrawing groups (acylamino, fluoro, chloro, trifluoromethyl, and cyanide) on the benzene ring provided the corresponding products 6k–6s in good yields of 67–88%. Substrates 5t–5x containing unsaturated groups were also feasible under mild reaction conditions to produce compounds 6t–6x with yields ranging from 63% to 75%. The coupling substrates possessing methylsulfonyl (5y) or isopropylsulfonyl (5z) groups at the para-position in the aryl moiety smoothly underwent the coupling process, respectively, giving products 6y (85%) and 6z (83%). However, pyridine-4-boronic acid (5aa) only provided the coupling product 6aa in 30% low yield. This could be due to the low electron cloud density of nitrogen atom's para-position, and the reactivity of the substrate 4-pyridineboronic acid (5aa) was reduced. Furthermore, 6-methoxyl-3-pyridineboronic acid (5ab), 6-chloro-3-pyridineboronic acid (5ac), 6-fluoro-3-pyridineboronic acid (5ad), and 3-furanboronic acid (5ae) were well tolerated, leading to coupling compounds 6aa–6ae in 72–87% yields. However, 2,6-difluorophenylboronic acid did not generate the product under the conditions, indicating that aryl boric acid with large steric hindrance was not applicable to the C3 cross-coupling process. In addition, 4-carboxyphenylboronic acid did not participate in the reaction, which might be influenced by the acidic carboxyl group.

2.2. Biology

2.2.1. Cytotoxic activity

According to previous reports, evodiamine and its derivatives effectively inhibited the proliferation and migration of HCT116 (human colorectal adenocarcinoma cells)22,23 and HepG-2 (human liver cancer cells).24 In addition, some of these compounds had anti-proliferative effects on triple-negative breast cancer (TNBC) cells.25 The tumor growth and metastatic spread of 4T1 murine TNBC cells closely mimic human breast cancer, which is a commonly used model for antitumor activity evaluation in vitro. Therefore, HCT116, HepG-2, and 4T1 cell lines were used to evaluate the antiproliferative activity of the synthesized evodiamine derivatives (6a–6ae) through the MTT method. Camptothecin was employed as a positive control. The 50% inhibitory concentration (IC50) values of the tested compounds were tested after treatment for 48 h (Table 2). The results showed differential potency for these derivatives on different cancer cells. Compared with evodiamine, some of the derivatives had enhanced cytotoxic activity against HCT116 and 4T1, showing that introducing an aromatic ring into C-3 position to form a planar conjugated biphenyl structure was advantageous for enhancing the activity of evodiamine. However, 3-aryl evodiamine derivatives were not sensitive HepG2 cell line, only few derivatives (6n, 6r, 6s, 6v, 6w, 6ac, and 6ad) presented moderate potency (IC50 range 2.11–11.27 μM).

Table 2. In vitro antitumor activity of evodiamine derivatives 6a–6ae.
graphic file with name d5md00179j-u2.jpg
Compound R IC50a (μM)
HCT116 4T1 HepG2
6a H >12.5 15.20 ± 0.99 >12.5
6b 4′-Me >12.5 10.31 ± 0.90 >12.5
6c 3′-Me 11.07 ± 0.33 >25 >12.5
6d 4′-iPr >12.5 >25 >12.5
6e 4′-OMe >12.5 >25 >12.5
6f 3′,5′-OMe >12.5 13.49 ± 0.64 >12.5
6g 4′-S–Me 6.61 ± 0.29 11.37 ± 0.27 >12.5
6h 3′-S–Me 8.34 ± 0.05 13.03 ± 0.26 >12.5
6i 2′-S–Me >12.5 >25 >12.5
6j 4′-OH >12.5 >25 >12.5
6k 4′-CO–NH2 0.84 ± 0.29 >25 >12.5
6l 4′-Cl >12.5 >25 >12.5
6m 3′-Cl >12.5 >25 >12.5
6n 2′-Cl 3.30 ± 0.15 >25 7.38 ± 0.03
6o 4′-F >12.5 >25 >12.5
6p 3′,5′-F >12.5 >25 >12.5
6q 4′-CF3 >12.5 >25 >12.5
6r 3′,5′-CF3 5.29 ± 0.53 5.87 ± 0.10 11.27 ± 0.98
6s 4′-CN 3.94 ± 0.19 12.52 ± 0.42 7.64 ± 0.26
6t 4′-CH–CH2 12.43 ± 0.60 11.56 ± 0.21 >12.5
6u 4′-CHO 7.88 ± 0.27 10.59 ± 0.57 >12.5
6v 4′-CO–Me 1.71 ± 0.17 >25 2.80 ± 0.27
6w 4′-CO–OMe 2.17 ± 0.61 4.40 ± 0.17 2.11 ± 0.08
6x 4’-CO–Ph >12.5 >25 >12.5
6y 4′-SO2–Me 0.58 ± 0.04 0.99 ± 0.07 >12.5
6z 4′-SO2–iPr 2.50 ± 0.24 4.44 ± 0.44 >12.5
6aa — >12.5 >25 >12.5
6ab 6′-OMe >12.5 >25 >12.5
6ac 6′-Cl 3.62 ± 0.50 2.46 ± 0.12 2.80 ± 0.38
6ad 6′-F 6.09 ± 0.82 5.27 ± 0.20 4.23 ± 0.14
6ae — 3.69 ± 0.05 >25 >12.5
EVO — >12.5 >25 >25
Camptothecin — <0.1 >25 <0.1
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a

MTT method; the cells were incubated with the indicated compounds for 48 h (mean ± SD, n = 3).

By introducing various aromatic ring structures with electron-donating or electron-withdrawing groups, the electron cloud distribution of the aromatic ring moiety had impact on the biological activity (Fig. 4). In general, the phenyl ring possessing electron-withdrawing groups (compounds 6k–6z) generally had higher activity than that having electron-donating groups (compounds 6b–6j). The introduction of methyl at para-position on the terminal aromatic group (6c, IC50 = 11.07 μM) provided antiproliferative effects on the HCT116 cell. However, shifting the methyl to meta-position (6b, IC50 = 10.31 μM) was beneficial for cytotoxicity on the 4T1 cell. However, introducing isopropyl at the para-position (6d) was noneffective regarding antiproliferative activity (IC50 > 12.5 μM against the three cell lines). An improvement of the cytotoxicity was observed for the derivatives having thiomethyl at the para- or meta-position on the benzene ring. For example, analogues 6g and 6h showed good activity for HCT116 and 4T1 cells (IC50 range from 6.61 to 13.03 μM). However, compound 6i which had a thiomethyl group at the ortho-position was inactive. Benzamide analogue 6k showed anti-proliferative activity particularly for the HCT116 cell line with an IC50 of 0.84 μM. For the derivatives with electron-withdrawing groups, halogen monosubstituted derivatives were unfavourable for the activity with the exception of compound 6n (IC50 = 3.30 μM against HCT116). When trifluoromethyl were introduced, the number of groups and substituting position had obvious effects on the cytotoxicity activity of target compounds, such as 6q and 6r. The IC50 values of compound 6r against HCT116, 4T1, and HepG2 cells were lower than 12 μM. In contrast, 6q was noneffective against three cell lines. It could be due to a greater electron-withdrawing effect of 3,5-bis(trifluoromethyl)phenyl (6r) than that of 4-fluoromethyl phenyl (6q). Sulfonyl derivatives 6y and 6z exhibited a higher inhibitory activity against HCT-116 (IC50 = 0.58 and 2.50 μM, respectively) and 4T1 (IC50 = 0.99 and 4.44 μM, respectively) cells, which were more potent than that of the parent compound evodiamine. In particular, the methylsulfonyl derivative 6y (IC50 = 0.58 against HCT-116 and 0.99 against 4T1) had the best activity, highlighting it was a potential novel antitumor candidate. However, when evodiamine was modified by introducing a pyridine ring on C-3 to produce derivative 6aa, the cytotoxic activity of 6aa was unsatisfactory. Nevertheless, introducing F and Cl on the pyridine ring improved the activity against the three cancerous cell lines, such as compounds 6ac (IC50 range 2.46–3.62 μM) and 6ad (IC50 range 4.23–6.09 μM). The enhanced activity could be attributed to the introduced halogen atoms that decreased the basicity of the pyridine unit and enhanced lipophilicity. Besides, the halogen atoms might form halogen bonds, exerting potentially beneficial effects in protein–ligand interaction.

Fig. 4. Relationship between the C-3 substituent group and cytotoxicity activity.

Fig. 4

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Topoisomerases I (Top I) are the key biomacromolecules which take part in the progression of cellular processes such as replication, recombination, and transcription.26 It has been confirmed that Top I is a common molecular target of evodiamine and its analogues.5 These compounds bind to the Top I-DNA complex, causing DNA double strand breaks and ultimately tumour cell apoptosis. In this work, some synthesized 3-aryl-evodiamine derivatives had enhanced antiproliferative activity than that of evodiamine. Introducing aromatic groups might generate additional interactions with aromatic residues of Top I, enhancing the molecular activity. The inhibitory effect of these 3-aryl-evodiamine derivatives towards Top I need to be explored in further research.

2.2.2. Effect on cell apoptosis of compound 6y

The preliminary MTT cell viability assay showed that compound 6y could inhibit HCT-116 and 4T1 cell proliferation with an IC50 of 0.58 and 0.99, respectively. Therefore, in order to further evaluate the antiproliferative potency of 6y, the cell colony formation assay was performed. Fig. 5 presents the notable cell colony reduction and superior activity of 6y to that of parent compound evodiamine at the same concentration (5 μM), with the antiproliferation activities being dose-dependent.

Fig. 5. Compound 6y inhibited the colony formation abilities in cancer cells. (A) Colony formation of HCT-116 cells treated with 6y (5.0 and 0.5 μM) and Evo (5.0 μM), and the colony numbers of each group calculated. (B) Colony formation of 4T1 cells treated with 6y (5.0 and 0.5 μM) and Evo (5.0 μM), and the colony numbers of each group calculated. All statistical data are presented as mean ± SD of three experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control group.

Fig. 5

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2.2.3. Migration inhibition effect of compound 6y

Metastasis of tumor cells is a pivotal factor in cancer death. The effect of the compound on the migration of cancer cells is also an indicator to evaluate its anti-tumor activity. The scratch assay was conducted to estimate the migration inhibition ability of derivative 6y towards HCT-116 and 4T1 cell lines. As shown in Fig. 6, the scratched area of the blank control decreased distinctly. Compared with evodiamine, the healing ratio of cell scratches was slower after treatment with compound 6y for 24 h.

Fig. 6. Compound 6y inhibited migration towards HCT-116 and 4T1 cells. Cells were treated with 6y and Evo for 24 h. The scratch assay was used to measure migration capabilities of the cells. The wound closure ratio represents the level of cell migration ability (scale bar, 200 μm). All statistical data are presented as mean ± SD of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control group.

Fig. 6

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3. Conclusion

In conclusion, a series of 3-arylevodiamine derivatives (6a–6ae) were designed and synthesized. The in vitro antiproliferative activity of these synthesized compounds against HCT116, 4T1, and HepG2 cancer cell lines was evaluated. The result showed that most of evodiamine analogues had enhanced the cytotoxic activity when compared with the parent compound. Among them, compound 6y with a methylsulfonylbenzene unit exhibited remarkable cytotoxicity against HCT116 (IC50 = 0.58 ± 0.04 μM) and 4T1 (IC50 = 0.99 ± 0.07 μM), which could be developed as a potential antitumor drug.

4. Experimental

4.1. Chemistry

Evodiamine was purchased from Shanghai McLean Biochemical Technology Co., Ltd., with a purity of >98%. Unless otherwise specified, all reagents of analytical or chemical grade used in this investigation were commercially available and used directly without further purification. Analytical TLC was performed on silica gel plates (GF245) and visualized by UV irradiation (254 nm), by spraying with Dragendorff's reagent or staining with iodine. Column chromatography was carried out using silica gel (200–300 mesh, Qingdao Sea Chemical Factory, Qingdao, People's Republic of China) under pressure. 1H and 13C NMR spectra were recorded in DMSO-d6 at ambient temperature using Bruker AV 400 or 600 nuclear magnetic resonance instruments. 1H NMR and 13C NMR: chemical shifts were recorded in ppm relative to tetramethylsilane (TMS) and internally referenced to the residual solvent signal (for 1H NMR: DMSO-d6 = 2.50 ppm; for 13C NMR: DMSO-d6 = 39.52 ppm). (+)-HRESIMS spectra were recorded using a Waters Acquity UPLC/Xevo G2-S Q-Tof mass spectrometer in the electro-spray ionization (ESI) mode at a flow rate of 0.3 mL min−1 and a gradient starting at MeOH/H2O + 0.1% FA (3/7) and ramping to 100% MeOH in 5 min. The mass spectrometer was calibrated with sodium formate. Leucine enkephalin (556.2771 Da, positive ion mode) was used as the reference compound. The detailed ion source parameters are as follows: capillary, 2 kV; sampling cone, 40; source offset, 80; source temperatures, 150 °C; desolvation temperatures, 500 °C; cone gas, 50 L h−1; desolvation gas, 800 L h−1; mass range, 50–1200 m/z.

4.1.1. Synthesis of compound 2

Evodiamine (1, 1.0 g, 3.30 mmol) was dissolved in dry THF (30 mL). Subsequently, di-tert-butyldicarbonate (Boc2O, 1.08 g, 4.95 mmol, 1.5 equiv.) and 4-dimethylaminopyridine (DMAP, 40 mg, 0.33 mmol, 0.1 equiv.) were added. The reaction was stirred at room temperature for 2 h until completion. Then, the mixture was evaporated to afford crude products. For removing a small amount of unconsumed raw material and DMAP, the Boc-protected evodiamine (2) (1.30 g, 98% yield) was purified using flash column chromatography over silica gel eluting with PE/EtOAc.

Compound 2, light yellow amorphous powder, 98% yield; column chromatography on silica gel (eluent: PE/EtOAc = 10/1); Rf = 0.60 (PE/EtOAc = 3/1); 1H NMR (600 MHz, DMSO-d6) δ 8.21 (d, J = 7.8 Hz, 1H), 7.90 (dd, J = 7.8, 1.8 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.52 (t, J = 6.6 Hz, 1H), 7.43 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 6.20 (s, 1H), 4.65 (dd, J = 20.4, 5.4 Hz, 1H), 3.15–3.10 (m, 1H), 3.04–3.01 (m, 1H), 2.79–2.74 (m, 1H), 2.42 (s, 3H), 1.45 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 163.4, 150.0, 149.0, 136.4, 133.0, 128.3, 127.7, 127.1, 125.6, 123.1, 121.7, 121.5, 121.5, 119.7, 119.3, 115.1, 84.2, 67.8, 37.9, 34.8, 27.3 × 3, 20.1; HRESIMS (m/z) 404.1968 [M + H]+ (calcd for C24H26N3O3, 404.1974).

4.1.2. Synthesis of compound 3

A solution of compound 2 (1.30 g, 3.22 mmol) in dry DCM (25 mL) was stirred to dissolve under an argon atmosphere. After that, N-bromosuccinimide (NBS, 0.63 g, 3.54 mmol, 1.1 equiv.) was added. The reaction was stirred at room temperature for 3 h and then quenched by H2O (20 mL). The solution was extracted with CH2Cl2 (20 mL × 3). The organic layer was washed with brine, dried over anhydrous Na2SO4, and evaporated to afford crude products. The compound 3 (1.32 g, 85% yield) was purified by column chromatography over silica gel eluting with PE/EtOAc.

Compound 3, yellow amorphous powder, 85% yield; column chromatography on silica gel (eluent: PE/EtOAc = 10/1); Rf = 0.65 (PE/EtOAc = 3/1); 1H NMR (600 MHz, DMSO-d6) δ 8.20 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 3.0 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.66 (dd, J = 9.0, 3.0 Hz, 1H), 7.42 (t, J = 6.6 Hz, 1H), 7.34 (t, J = 8.4 Hz, 1H), 7.16 (d, J = 9.0 Hz, 1H), 6.21 (s, 1H), 4.61 (dd, J = 10.8, 3.0 Hz, 1H), 3.16–3.11 (m, 1H), 3.06 (d, J = 16.2 Hz, 1H), 2.80–2.74 (m, 1H), 2.48 (s, 3H), 1.47 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 162.4, 148.9, 148.9, 136.3, 135.5, 130.5, 127.2, 127.1, 125.8, 123.2, 122.7, 121.6, 121.3, 119.4, 115.2, 112.7, 84.4, 67.7, 38.3, 34.4, 27.4 × 3, 20.0; HRESIMS (m/z) 483.3869 [M + H]+ (calcd for C24H25BrN3O3, 483.3860).

4.1.3. Synthesis of compound 4 (ref. 27)

To facilitate the complete dissolution of compound 3 (1.32 g, 2.74 mmol), a volume of 15 mL of ultra-dry tetrahydrofuran (THF) was utilized. The resulting solution was cooled in an ice bath to maintain a low-temperature environment. Sodium methoxide (NaOMe, 30 wt% in methanol) (840 μL, 15.07 mmol, 5.5 equiv.) was then introduced dropwise with careful monitoring to ensure gradual and controlled addition. Once the reaction is complete, quench it by adding H2O (15 mL) and then extract the mixture with ethyl acetate (20 mL × 4). The organic layers were combined, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 4 (1.02 g, 98%).

Compound 4, white amorphous powder, 98% yield; Rf = 0.65 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.14 (s, 1H), 7.82 (d, J = 2.4 Hz, 1H), 7.61 (dd, J = 9.0, 2.4 Hz, 1H),7.46 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.11 (t, J = 6.6 Hz, 1H), 7.02–6.99 (m, 2H), 6.17 (s, 1H), 4.62 (dd, J = 10.8, 3.0 Hz, 1H), 3.24–3.20 (m, 1H), 2.96 (s, 3H), 2.94–2.89 (m, 1H), 2.79 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 163.1, 147.5, 136.5, 135.9, 130.4, 130.0, 125.9, 121.9, 120.1, 119.0, 118.9, 118.2, 111.7, 111.5, 111.1, 69.9, 41.3, 36.3, 19.4; HRESIMS (m/z) 382.0561 [M + H]+ (calcd for C19H17BrN3O, 382.0555). The NMR data of 4 was consistent with that of the compound reported in the literature.23

4.1.4. General procedure for the preparation of 6a–6ae

Compound 4 (1.0 equiv.), various arylboronic acid (5a–5ae, 1.2 equiv.), Pd[P(C6H5)3]4 (10 mol%), and K2CO3 (2 equiv.) were dissolved in THF/H2O (10 : 1, v : v). The resulting solution was refluxed at 80 °C under an argon atmosphere for a duration of 12 h. Following the completion of the reaction and subsequent cooling to room temperature, H2O (2 mL) was added to the mixture. Extraction was then performed using ethyl acetate (5 mL × 4). The combined organic layers were washed thoroughly with brine and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure (in vacuo). The residue was purified by silica gel column chromatography eluting with CH2Cl2/EtOAc or CH2Cl2/MeOH to obtain the desired products 6a–6ae in 30–87% yields.

Compound 6a, white amorphous powder, 85% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.55 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 8.4, 2.4 Hz, 1H), 7.59 (d, J = 7.2 Hz, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 7.2 Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.13–7.10 (m, 2H), 7.02 (t, J = 7.2 Hz, 1H), 6.20 (s, 1H), 4.67 (dd, J = 12.6, 5.4 Hz, 1H), 3.27–3.23 (m, 1H), 3.02 (s, 3H), 2.99–2.93 (m, 1H), 2.80–2.77 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 164.4, 147.8, 139.4, 136.5, 131.9, 131.7, 131.0, 129.0 × 2, 127.0, 126.0, 126.1 × 2, 125.8, 121.9, 119.0, 118.8, 118.3, 117.3, 111.7, 111.5, 70.1, 41.4, 36.5, 19.5; HRESIMS (m/z) 380.1755 [M + H]+ (calcd for C25H22N3O, 380.1763).

Compound 6b, yellow amorphous powder, 86% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.60 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.06 (s, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.77 (dd, J = 8.4, 2.4 Hz, 1H), 7.50–7.47 (m, 3H), 7.36 (d, J = 8.4 Hz 1H), 7.24 (d, J = 7.8 Hz, 2H), 7.12–7.10 (m, 2H), 7.01 (t, J = 7.8 Hz, 1H), 6.18 (s, 1H), 4.66 (dd, J = 12.6, 5.4 Hz, 1H), 3.26–3.21 (m, 1H), 2.98 (s, 3H), 2.96–2.92 (m, 1H), 2.80–2.77 (dd, J = 15.6, 4.8 Hz, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 164.4, 147.7, 136.5, 136.5, 136.2, 131.9, 131.6, 130.8, 129.6 × 2, 126.0, 125.9 × 2, 125.5, 121.9, 119.0, 119.0, 118.2, 117.5, 111.7, 111.5, 70.0, 41.3, 36.5, 20.7, 19.5; HRESIMS (m/z) 394.1924 [M + H]+ (calcd for C26H24N3O, 394.1919).

Compound 6c, white amorphous powder, 68% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.65 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.02 (s, 1H), 7.77 (dd, J = 8.4, 2.4 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.42 (s, 1H), 7.39–7.36 (m, 2H), 7.31 (t, J = 7.6 Hz, 1H), 7.13–7.10 (m, 3H), 7.01 (t, J = 7.4 Hz, 1H), 6.20 (s, 1H), 4.67 (dd, J = 12.6, 5.4 Hz, 1H), 3.27–3.22 (m, 1H), 3.01 (s, 3H), 2.99–2.95 (m, 1H), 2.81–2.77 (m, 1H), 2.36 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 164.3, 147.7, 139.4, 138.1, 136.4, 131.8, 131.8, 130.9, 128.9, 127.6, 126.7, 126.0, 125.7, 123.1, 121.9, 118.9, 118.8, 118.2, 117.3, 111.7, 111.5, 70.1, 41.3, 36.5, 21.1, 19.5; HRESIMS (m/z) 394.1920 [M + H]+ (calcd for C26H24N3O, 394.1919).

Compound 6d, yellow amorphous powder, 78% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.70 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.00 (br s, 1H), 7.75 (br d, J = 8.4 Hz, 1H), 7.52 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 7.8 Hz, 2H), 7.12–7.10 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.19 (s, 1H), 4.67 (dd, J = 12.6, 5.4 Hz, 1H), 3.27–3.22 (m, 1H), 2.99 (s, 3H), 2.94–2.89 (m, 1H), 2.81–2.77 (m, 1H), 1.21 (d, J = 7.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 164.4, 147.7, 147.2, 137.0, 136.5, 131.9, 131.7, 130.9, 130.0, 126.9 × 2, 126.1, 126.0 × 2, 125.5, 121.9, 119.0, 118.2, 117.5, 111.7, 111.5, 70.1, 41.3, 36.5, 33.1, 23.9 × 2, 19.5; HRESIMS (m/z) 422.2241 [M + H]+ (calcd for C28H28N3O, 422.2232).

Compound 6e, yellow amorphous powder, 85% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.55 (CH2Cl2/EtOAc = 20/1); 1H NMR (600 MHz, DMSO-d6) δ 11.07 (s, 1H), 7.98 (br s, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 6.6 Hz, 2H), 7.48 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.12–7.15 (m, 2H), 7.02–6.99 (m, 3H), 6.17 (s, 1H), 4.67–4.64 (dd, J = 13.8, 4.8 Hz, 1H), 3.78 (s, 3H), 3.25–3.21 (m, 1H), 2.95–2.94 (m, 4H), 2.81–2.78 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 164.3, 158.6, 147.4, 136.5, 131.9, 131.8, 131.3, 130.7, 127.2 × 2, 126.0, 125.2, 121.9, 119.3, 118.9, 118.2, 117.8, 114.4 × 2, 111.7, 111.5, 69.9, 55.1, 41.1, 36.5, 19.5; HRESIMS (m/z) 410.1862 [M + H]+ (calcd for C26H24N3O2, 410.1869).

Compound 6f, white amorphous powder, 66% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.50 (CH2Cl2/EtOAc = 20 : 1); 1H NMR (600 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.00 (d, J = 1.8 Hz, 1H), 7.78 (dd, J = 8.4, 2.4 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.01 (t, J = 7.8 Hz, 1H), 6.70 (br s, 2H), 6.45 (s, 1H), 6.21 (s, 1H), 4.68 (dd, J = 13.2, 5.4 Hz, 1H), 3.79 (s, 6H), 3.28–3.23 (m, 1H), 3.04 (s, 3H), 3.00–2.94 (m, 1H), 2.79 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 164.3, 160.9 × 2, 47.9, 141.7, 136.4, 132.1, 131.6, 131.0, 126.1, 125.9, 121.9, 118.9, 118.5, 118.2, 116.9, 111.7, 111.5, 104.2 × 2, 99.0, 70.2, 55.2 × 2, 41.4, 36.5, 19.4; HRESIMS (m/z) 440.1970 [M + H]+ (calcd for C27H26N3O3, 440.1974).

Compound 6g, yellow amorphous powder, 81% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.60 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.01 (br s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.4, 2H), 7.48 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 7.8 Hz, 2H), 7.12–7.10 (m, 2H), 7.01 (t, J = 7.8 Hz, 1H), 6.19 (s, 1H), 4.68 (dd, J = 13.2, 6.0 Hz, 1H), 3.27–3.22 (m, 1H), 3.01 (s, 3H), 2.98–2.93 (m, 1H), 2.80 (dd, J = 15.0, 4.8 Hz, 1H), 2.49 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 164.3, 147.7, 136.8, 136.5, 136.0, 131.5, 131.1, 130.9, 126.6 × 2, 126.5 × 2, 126.1, 125.4, 121.9, 119.0, 118.8, 118.2, 117.3, 111.7, 111.5, 70.1, 41.4, 36.5, 19.5, 14.8; HRESIMS (m/z) 426.1643 [M + H]+ (calcd for C26H24N3SO3, 426.1640).

Compound 6h, white amorphous powder, 66% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.65 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.80 (dd, J = 8.4, 2.4 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.42 (s, 1H), 7.37–7.36 (m, 3H), 7.21–7.19 (m, 1H), 7.12–7.10 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.21 (s, 1H), 4.68 (dd, J = 12.6, 5.4 Hz, 1H), 3.28–3.23 (m, 1H), 3.05 (s, 3H), 3.00–2.94 (m, 1H), 2.80 (dd, J = 15.6, 4.8 Hz, 1H), 2.51 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 164.3, 147.8, 140.2, 138.9, 136.4, 132.0, 131.0, 131.0, 129.5, 126.1, 125.9, 124.4, 123.3, 122.7, 121.9, 118.9, 118.5, 118.2, 117.0, 111.7, 111.5, 70.2, 41.4, 36.5, 19.4, 14.6; HRESIMS (m/z) 426.1635 [M + H]+ (calcd for C26H24N3SO3, 426.1640).

Compound 6i, yellow amorphous powder, 83% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.55 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.04 (s, 1H), 7.78 (d, J = 2.4 Hz, 1H), 7.48 (dd, J = 8.4, 2.4 Hz, 2H), 7.39 (d, J = 8.4, 1H), 7.36–7.33 (m, 1H), 7.31 (d, J = 6.6 Hz, 1H), 7.20–7.16 (m, 2H), 7.12 (t, J = 7.2 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 6.22 (s, 1H), 4.67 (dd, J = 12.6, 6.0 Hz, 1H), 3.26–3.21 (m, 1H), 3.03 (s, 3H), 2.98–2.93 (m, 1H), 2.80 (dd, J = 15.6, 4.8 Hz, 1H), 2.35 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 164.1, 147.6, 139.0, 136.8, 136.4, 134.3, 131.3, 131.0, 129.6, 128.4, 128.0, 126.1, 124.9, 124.6, 121.9, 118.9, 118.2, 117.9, 116.2, 111.7, 111.5, 70.1, 41.3, 36.5, 19.4, 15.0; HRESIMS (m/z) 426.1632 [M + H]+ (calcd for C26H24N3SO3, 426.1640).

Compound 6j, yellow amorphous powder, 78% yield; column chromatography on silica gel (eluent: CH2Cl2/MeOH = 20/1); Rf = 0.40 (CH2Cl2/MeOH = 20/1); 1H NMR (600 MHz, DMSO-d6) δ 11.16 (s, 1H), 9.62 (s, 1H), 7.95 (br s, 1H), 7.69 (dd, J = 8.4, 2.4 Hz, 1H), 7.47 (d, J = 7.4 Hz, 1H), 7.41–7.40 (m, 2H), 7.37 (d, J = 7.8 Hz, 1H), 7.10–7.08 (m, 2H), 7.01–7.00 (m, 1H), 6.85 (dd, J = 9.0, 2.4 Hz, 2H), 6.15 (s, 1H), 4.67–4.65 (m, 1H), 3.24–3.20 (m, 1H), 2.93–2.92 (m, 4H), 2.81–2.79 (m, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.3, 156.8, 147.3, 136.5, 132.5, 131.1, 130.6, 130.2, 127.1 × 2, 126.0, 124.9, 121.8, 119.4, 118.9, 118.2, 117.9, 115.8 × 2, 111.7, 111.5, 69.8, 41.0, 36.5, 19.5; HRESIMS (m/z) 396.1703 [M + H]+ (calcd for C25H22N3O2, 396.1712).

Compound 6k, white amorphous powder, 72% yield; column chromatography on silica gel (eluent: CH2Cl2/MeOH = 20/1); Rf = 0.40 (CH2Cl2/MeOH = 20/1); 1H NMR (600 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.08 (d, J = 2.4 Hz, 1H), 7.99 (br s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.86 (dd, J = 8.4, 2.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.33 (br s, 1H), 7.10 (t, J = 8.4 Hz, 2H), 7.01 (t, J = 7.4 Hz, 1H), 6.23 (s, 1H), 4.68 (dd, J = 13.2, 6.0 Hz, 1H), 3.28–3.23 (m, 1H), 3.09 (s, 3H), 3.00–2.95 (m, 1H), 2.79 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 167.6, 164.3, 148.0, 142.0, 136.4, 132.5, 132.0, 131.1, 130.2, 128.2 × 2, 126.1, 126.0, 125.6 × 2, 121.9, 119.0, 118.3, 118.2, 116.7, 111.7, 111.5, 70.3, 41.6, 36.5, 19.4; HRESIMS (m/z) 423.1816 [M + H]+ (calcd for C26H23N4O2, 423.1821).

Compound 6l, white amorphous powder, 71% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.60 (CH2Cl2/EtOAc = 40 : 1); 1H NMR (600 MHz, DMSO-d6) δ 11.02 (s, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 8.4, 2.4 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 3H), 7.36 (d, J = 6.0 Hz, 1H), 7.12–7.09 (m, 2H), 7.00 (t, J = 7.8 Hz, 1H), 6.22 (s, 1H), 4.68 (dd, J = 13.2, 6.0 Hz, 1H), 3.28–3.23 (m, 1H), 3.06 (s, 3H), 2.99–2.94 (m, 1H), 2.79–2.76 (dd, J = 15.6, 4.8 Hz, 1H);13C NMR (100 MHz, DMSO-d6) δ 164.2, 147.9, 138.2, 136.4, 131.7, 131.7, 131.0, 130.0, 128.9 × 2, 127.7 × 2, 126.0, 125.7, 121.9, 118.9, 118.5, 118.2, 116.9, 111.7, 111.5, 70.2, 41.5, 36.5, 19.4; HRESIMS (m/z) 414.1370 [M + H]+ (calcd for C25H21ClN3O, 414.1373).

Compound 6m, white amorphous powder, 82% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.65 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.02 (s, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.64 (s, 1H), 7.57 (d, J = 6.0, 1H), 7.47–7.43 (m, 2H), 7.37–7.35 (m, 2H), 7.12–7.09 (m, 2H), 7.01 (t, J = 7.4 Hz, 1H), 6.23 (s, 1H), 4.68 (dd, J = 12.6, 5.4 Hz, 1H), 3.28–3.23 (m, 1H), 3.09 (s, 3H), 3.01–2.95 (m, 1H), 2.79 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 148.0, 141.6, 136.4, 133.7, 132.0, 131.1, 130.8, 129.6, 126.6, 126.1, 126.0, 125.7, 124.7, 121.9, 119.0, 118.2, 118.2, 116.6, 111.7, 111.5, 70.3, 41.6, 36.5, 19.4; HRESIMS (m/z) 414.1370 [M + H]+ (calcd for C25H21ClN3O, 414.1373).

Compound 6n, white amorphous powder, 68% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.55 (CH2Cl2/EtOAc = 40/1); 1H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H), 7.81 (d, J = 3.6 Hz, 1H), 7.56–7.52 (m, 2H), 7.48 (d, J = 7.8 Hz, 1H), 7.40–7.34 (m, 4H), 7.13–7.08 (m, 2H), 7.01 (t, J = 10.2 Hz, 1H), 6.24 (s, 1H), 4.67 (dd, J = 19.2, 6.6 Hz, 1H), 3.28–3.21 (m, 1H), 3.07 (s, 3H), 3.01–2.92 (m, 1H), 2.78 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 147.7, 139.0, 136.4, 134.5, 131.4, 131.3, 131.1, 129.9, 129.7, 129.0, 128.6, 127.6, 126.1, 121.9, 119.0, 118.2, 117.7, 116.0, 111.8, 111.5, 70.3, 41.5, 36.5, 19.4; HRESIMS (m/z) 414.1370 [M + H]+ (calcd for C25H21ClN3O, 414.1373).

Compound 6o, light yellow amorphous powder, 67% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.55 (CH2Cl2/EtOAc = 40 : 1); 1H NMR (600 MHz, DMSO-d6) δ 11.04 (s, 1H), 7.99 (d, J = 2.4 Hz, 1H), 7.77 (dd, J = 8.4, 2.4 Hz, 1H), 7.64–7.62 (m, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.25 (t, J = 8.4 Hz, 2H), 7.12–7.10 (m, 2H), 7.02 (t, J = 6.6 Hz, 1H), 6.20 (s, 1H), 4.68 (dd, J = 11.4, 4.2 Hz, 1H), 3.27–3.22 (m, 1H), 3.02 (s, 3H), 2.99–2.93 (m, 1H), 2.80 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 162.3 (J = 240 Hz), 147.7, 136.4, 135.9 (J = 3 Hz), 131.8, 130.9, 130.7, 128.0 × 2 (d, J = 9 Hz), 126.0, 125.7, 121.9, 118.9, 118.7, 118.2, 117.2, 115.8 × 2 (d, J = 21 Hz), 111.7, 111.5, 70.1, 41.3, 36.5, 19.4; HRESIMS (m/z) 398.1675 [M + H]+ (calcd for C25H21FN3O, 398.1669).

Compound 6p, white amorphous powder, 88% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.55 (CH2Cl2/EtOAc = 40/1); 1H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 2.4 Hz, 1H), 7.84 (dd, J = 8.4, 2.4 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.37–7.32 (m, 3H), 7.16–7.07 (m, 3H), 7.02–7.00 (m, 1H), 6.25 (s, 1H), 4.68 (dd, J = 12.8, 4.8 Hz, 1H), 3.27–3.23 (m, 1H), 3.14 (s, 3H), 3.03–2.95 (m, 1H), 2.78 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 163.8 (J = 15 Hz), 162.2 (J = 15 Hz), 148.2, 143.2, 136.4, 132.2, 131.3, 128.4, 126.2, 126.1, 121.9, 119.0, 118.2, 117.7, 116.1, 111.7, 111.5, 109.1 (J = 6 Hz), 108.9 (J = 4.5 Hz), 102.0 (J = 30 Hz), 70.5, 41.8, 36.5, 19.4; HRESIMS (m/z) 416.1579 [M + H]+ (calcd for C25H20F2N3O, 416.1574).

Compound 6q, white amorphous powder, 67% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.70 (CH2Cl2/EtOAc = 40/1); 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.88–7.81 (m, 3H), 7.76 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.14–7.09 (m, 2H), 7.01 (t, J = 8.0 Hz, 1H), 6.25 (s, 1H), 4.69 (dd, J = 13.2, 4.8 Hz, 1H), 3.30–3.23 (m, 1H), 3.13 (s, 3H), 3.04–2.93 (m, 1H), 2.78–2.73 (m, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 148.1, 143.4, 136.4, 132.1, 131.2, 129.2, 127.2 (J = 30 Hz), 126.6, 126.2 × 2, 126.1, 125.8 (J = 32 Hz), 125.3 (J = 270 Hz), 121.9, 119.0, 118.2, 118.1, 116.5, 111.7, 111.5, 70.4, 41.7, 36.5, 19.4; HRESIMS (m/z) 448.1631 [M + H]+ (calcd for C26H21F3N3O, 448.1637).

Compound 6r, yellow amorphous powder, 84% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.70 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.23 (s, 2H), 8.10 (s, 1H), 7.98–7.97 (m, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.10–7.09 (m, 2H), 7.00 (t, J = 7.2 Hz, 1H), 6.29 (s, 1H), 4.69 (dd, J = 13.2, 6.0 Hz, 1H), 3.30–3.26 (m, 1H), 3.21 (s, 3H), 3.04–2.98 (m, 1H), 2.76–2.73 (br d, J = 15.0 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 148.1, 142.1, 136.3, 132.6, 132.6 (J = 6.6 Hz), 131.5, 131.0, 130.8, 127.3, 126.7, 126.5, 126.5 (J = 4.0 Hz), 126.1, 124.3 (J = 268.0 Hz), 121.9, 120.0 (J = 5.2 Hz), 119.0, 118.1, 117.5 (J = 263.8 Hz), 115.7, 111.7, 111.4, 70.6, 42.0, 36.6, 19.3; HRESIMS (m/z) 516.1520 [M + H]+ (calcd for C27H20F6N3O, 516.1511).

Compound 6s, yellow amorphous powder, 75% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.45 (CH2Cl2/EtOAc = 20/1); 1H NMR (600 MHz, DMSO-d6) δ 10.98 (s, 1H), 8.07 (s, 1H), 7.87–7.79 (m, 5H), 7.46 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.11–7.09 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.26 (s, 1H), 4.68 (dd, J = 12.6, 5.4 Hz, 1H), 3.29–3.24 (m, 1H), 3.16 (s, 3H), 3.02–2.96 (m, 1H), 2.77 (dd, J = 15.6, 5.4 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 164.2, 148.2, 143.9, 136.4, 132.9 × 2, 132.8, 132.2, 131.3, 128.6, 126.6 × 2, 126.3, 126.1, 122.0, 119.0, 118.2, 117.7, 116.1, 111.7, 111.5, 109.1, 70.5, 41.9, 36.5, 19.4; HRESIMS (m/z) 405.1720 [M + H]+ (calcd for C26H21N4O, 405.1715).

Compound 6t, yellow amorphous powder, 75% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 100/1); Rf = 0.60 (CH2Cl2/EtOAc = 40/1); 1H NMR (600 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.05 (s, 1H), 7.81 (d, J = 8.4 Hz 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.53–7.51 (m, 2H), 7.48 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.12–7.10 (m, 2H), 7.01 (t, J = 7.8 Hz, 1H), 6.77 (dd, J = 18.0, 10.8 Hz, 1H), 6.21 (s, 1H), 5.86 (d, J = 17.4 Hz 1H), 5.27 (d, J = 10.8 Hz 1H), 4.68–4.65 (m, 1H), 3.27–3.22 (m, 1H), 3.03 (s, 3H), 2.99–2.94 (m, 1H), 2.80–2.77 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 164.8, 148.2, 139.2, 136.9, 136.7, 136.2, 132.1, 131.5, 131.4, 127.2 × 2, 126.6 × 2, 126.5, 126.0, 122.4, 119.4, 119.2, 118.7, 117.6, 114.6, 112.2, 112.0, 70.6, 41.8, 36.9, 19.9; HRESIMS (m/z) 406.1913 [M + H]+ (calcd for C27H24N3O, 406.1919).

Compound 6u, yellow amorphous powder, 67% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.40 (CH2Cl2/EtOAc = 20/1); 1H NMR (600 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.01 (s, 1H), 8.12 (d, J = 1.8 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.91 (dd, J = 8.4, 2.4 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.13–7.10 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.27 (s, 1H), 4.69 (dd, J = 13.2, 6.0 Hz, 1H), 3.30–3.25 (m, 1H), 3.16 (s, 3H), 3.03–2.98 (m, 1H), 2.79 (dd, J = 15.6, 5.4 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 192.5, 164.2, 148.2, 145.1, 136.3, 134.5, 132.3, 131.3, 130.2 × 2, 129.2, 126.4 × 2, 126.3, 126.1, 121.9, 119.0, 118.2, 117.9, 116.2, 111.7, 111.4, 70.4, 41.8, 36.5, 19.3; HRESIMS (m/z) 408.1718 [M + H]+ (calcd for C26H22N3O2, 408.1712).

Compound 6v, yellow amorphous powder, 63% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.40 (CH2Cl2/EtOAc = 20/1); 1H NMR (600 MHz, DMSO-d6) δ 11.00 (s, 1H), 8.10 (s, 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.87 (d, J = 8.4 Hz 1H), 7.76 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.10 (t, J = 9.6 Hz, 2H), 7.00 (t, J = 7.2 Hz, 1H), 6.25 (s, 1H), 4.68–4.65 (m, 1H), 3.28–3.24 (m, 1H), 3.12 (s, 3H), 3.01–2.95 (m, 1H), 2.78–2.75 (m, 1H), 2.58 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 197.3, 164.2, 148.1, 143.7, 136.4, 135.0, 132.1, 131.2, 129.5, 129.0 × 2, 126.1, 126.1, 125.9 × 2, 121.9, 119.0, 118.2, 118.0, 116.4, 111.7, 111.4, 70.4, 41.7, 36.5, 26.7, 19.4; HRESIMS (m/z) 422.1875 [M + H]+ (calcd for C27H24N3O2, 422.1869).

Compound 6w, white amorphous powder, 65% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.40 (CH2Cl2/EtOAc = 40/1); 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.10 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.88 (dd, J = 8.4, 2.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.13–7.09 (m, 2H), 7.00 (t, J = 6.8 Hz, 1H), 6.25 (s, 1H), 4.69 (dd, J = 13.2, 6.0 Hz, 1H), 4.32 (q, J = 7.2 Hz, 2H), 3.30–3.22 (m, 1H), 3.12 (s, 3H), 3.02–2.95 (m, 1H), 2.79 (dd, J = 15.6, 4.8 Hz, 1H), 1.32 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, DMSO-d6) δ 165.6, 164.3, 148.1, 143.8, 136.4, 132.1, 131.2, 129.9 × 2, 129.5, 128.0, 126.1, 126.1, 126.0 × 2, 121.9, 119.0, 118.2, 118.0, 116.4, 111.7, 111.5, 70.4, 60.7, 41.7, 36.5, 19.4, 14.2; HRESIMS (m/z) 438.1815 [M + H]+ (calcd for C28H26N3O3, 452.1974).

Compound 6x, white amorphous powder, 71% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.40 (CH2Cl2/EtOAc = 40 : 1); 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.13 (d, J = 2.4 Hz, 1H), 7.90 (dd, J = 8.4, 2.4 Hz, 1H), 7.80 (br s, 4H), 7.76–7.74 (m, 2H), 7.70–7.66 (m, 1H), 7.59 (t, J = 8.0 Hz, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.6, 1H), 7.14–7.09 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.26 (s, 1H), 4.69 (dd, J = 13.2, 6.0 Hz, 1H), 3.30–3.23 (m, 1H), 3.13 (s, 3H), 3.04–2.95 (m, 1H), 2.79 (dd, J = 16.0, 5.2 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 195.3, 164.2, 148.1, 143.5, 137.3, 136.4, 135.0, 132.5, 132.1, 131.2, 130.6 × 2, 129.6, 129.5 × 2, 128.6 × 2, 126.2, 126.1, 125.9 × 2, 121.9, 119.0, 118.2, 118.0, 116.4, 111.7, 111.5, 70.4, 41.7, 36.5, 19.4; HRESIMS (m/z) 484.2033 [M + H]+ (calcd for C32H26N3O2, 484.2025).

Compound 6y, white amorphous powder, 85% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 20/1); Rf = 0.40 (CH2Cl2/EtOAc = 10/1); 1H NMR (600 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.10 (d, J = 2.4 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.88–7.87 (m, 3H), 7.46 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.12–7.09 (m, 2H), 7.00 (t, J = 7.4 Hz, 1H), 6.27 (s, 1H), 4.68 (dd, J = 8.4, 3.6 Hz, 1H), 3.29–3.25 (m, 1H), 3.27 (s, 3H), 3.16 (s, 3H), 3.02–2.97 (m, 1H), 2.78–2.74 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 164.2, 148.2, 144.3, 138.8, 136.2, 132.3, 131.3, 128.7, 127.7 × 2, 126.6 × 2, 126.3, 126.1, 121.9, 119.0, 118.1, 117.8, 116.2, 111.7, 111.4, 70.5, 43.6, 41.8, 36.5, 19.3; HRESIMS (m/z) 458.1533 [M + H]+ (calcd for C26H24N3O3S, 458.1538).

Compound 6z, white amorphous powder, 83% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 20/1); Rf = 0.50 (CH2Cl2/EtOAc = 10/1); 1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 2.4 Hz, 1H), 7.90–7.84 (m, 5H), 7.47 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.13–7.08 (m, 2H), 7.02 (t, J = 8.0 Hz, 1H), 6.27 (s, 1H), 4.69 (m, J = 13.2, 4.8 Hz, 1H), 3.28–3.23 (m, 2H), 3.16 (s, 3H), 3.04–2.96 (m, 1H), 2.79 (dd, J = 15.6, 4.8 Hz, 1H), 1.17 (d, J = 6.8 Hz, 6H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 148.2, 144.4, 136.2, 134.7, 132.3, 131.3, 129.3 × 2, 128.7, 126.5 × 2, 126.4, 126.1, 121.9, 119.0, 118.2, 117.8, 116.2, 111.7, 111.5, 70.5, 54.2, 41.8, 36.5, 19.4, 15.2 × 2; HRESIMS (m/z) 486.1861 [M + H]+ (calcd for C28H28N3O3S, 486.1851).

Compound 6aa, yellow amorphous powder, 30% yield; Column chromatography on silica gel (Eluent: CH2Cl2/MeOH = 20/1); Rf = 0.40 (CH2Cl2/MeOH = 20/1); 1H NMR (600 MHz, DMSO-d6) δ 10.98 (s, 1H), 8.56 (d, J = 5.4 Hz, 2H), 8.13 (d, J = 2.4 Hz, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 5.4 Hz, 2H), 7.45 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.12–7.09 (m, 2H), 7.01 (t, J = 7.8 Hz, 1H), 6.27 (s, 1H), 4.68 (dd, J = 13.2, 6.0 Hz, 1H), 3.30–3.25 (m, 1H), 3.17 (s, 3H), 3.03–2.97 (m, 1H), 2.77 (dd, J = 15.6, 4.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 150.2 × 2, 148.5, 146.2, 136.4, 132.0, 132.0, 131.4, 126.1, 1261, 122.0, 120.2, 120.3, 119.0, 118.2, 117.6, 116.0, 111.7, 111.5, 70.5, 41.9, 36.5, 19.4; HRESIMS (m/z) 486.1861 [M + H]+ (calcd for C24H21N4O, 381.1715).

Compound 6ab, yellow amorphous powder, 80% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 10/1); Rf = 0.30 (CH2Cl2/EtOAc = 10/1); 1H NMR (600 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.40 (s, 1H), 7.98 (s, 1H), 7.95 (d, J = 8.4 Hz 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.13–7.10 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.88 (d, J = 9.0 Hz, 1H), 6.20 (s, 1H), 4.68 (dd, J = 12.6, 5.4 Hz, 1H), 3.88 (s, 3H), 3.27 (t, J = 12.0 Hz, 1H), 3.02 (s, 3H), 2.99–2.94 (m, 1H), 2.79–2.77 (d, J = 13.8 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 162.7, 147.7, 144.0, 137.1, 136.4, 131.5, 130.9, 128.8, 128.6, 126.0, 125.5, 121.9, 118.9, 118.2, 117.4, 111.7, 111.5, 110.5, 110.6, 70.1, 53.2, 41.3, 36.5, 19.4; HRESIMS (m/z) 411.1827 [M + H]+ (calcd for C25H23N4O2, 411.1821).

Compound 6ac, yellow amorphous powder, 37% yield; column chromatography on silica gel (eluent: CH2Cl2/MeOH = 20/1); Rf = 0.40 (CH2Cl2/MeOH = 20 : 1); 1H NMR (600 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.09 (dd, J = 8.4, 2.4 Hz, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.85 (dd, J = 8.4, 2.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.12–7.09 (m, 2H), 7.00 (t, J = 8.4 Hz, 1H), 6.25 (s, 1H), 4.67 (dd, J = 13.2, 5.4 Hz, 1H), 3.29–3.24 (m, 1H), 3.13 (s, 3H), 3.01–2.96 (m, 1H), 2.78 (dd, J = 15.6, 5.4 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 148.5, 148.1, 147.1, 137.0, 136.3, 134.4, 132.0, 131.2, 126.3, 126.1, 126.1, 124.3, 121.9, 119.0, 118.1, 118.1, 116.4, 111.7, 111.4, 70.4, 41.7, 36.5, 19.3; HRESIMS (m/z) 415.1320 [M + H]+ (calcd for C24H20ClN4O, 415.1326).

Compound 6ad, yellow amorphous powder, 84% yield; column chromatography on silica gel (eluent: CH2Cl2/MeOH = 50/1); Rf = 0.65 (CH2Cl2/MeOH = 20/1); 1H NMR (400 MHz, DMSO-d6) δ 11.01 (s, 1H), 8.46 (d, J = 2.4 Hz, 1H), 8.20 (td, J = 8.4, 2.8 Hz, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 8.4, 2.4 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.21 (dd, J = 8.4, 2.8 Hz, 1H), 7.13–7.09 (m, 2H), 7.00 (t, J = 7.2 Hz, 1H), 6.23 (s, 1H), 4.68 (dd, J = 12.8, 6.0 Hz, 1H), 3.30–3.232 (m, 1H), 3.10 (s, 3H), 3.02–2.94 (m, 1H), 2.79 (dd, J = 15.6, 5.4 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 163.0 (J = 232 Hz), 148.0, 144.7 (J = 15 Hz), 139.7 (J = 8 Hz), 136.4, 133.7 (J = 4.5 Hz), 132.1, 131.2, 126.9, 126.1 (J = 1.5 Hz), 121.9, 118.9, 118.3, 118.2, 116.7, 111.7, 111.5, 109.7, 109.5, 70.3, 41.6, 36.5, 19.4; HRESIMS (m/z) 399.1628 [M + H]+ (calcd for C24H20FN4O, 399.1621).

Compound 6ae, yellow amorphous powder, 87% yield; column chromatography on silica gel (eluent: CH2Cl2/EtOAc = 50/1); Rf = 0.40 (CH2Cl2/EtOAc = 40/1); 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.12 (br s, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 2.4 Hz, 1H), 7.71–7.70 (m, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 8.4, 1H), 7.13–7.07 (m, 2H), 7.03–6.99 (m, 1H), 6.90 (dd, J = 2.0, 0.8 Hz, 1H), 6.15 (s, 1H), 4.68 (dd, J = 12.8, 4.0 Hz, 1H), 3.26–3.19 (m, 1H), 2.97–2.90 (m, 1H), 2.92 (s, 3H), 2.82 (dd, J = 15.6, 5.4 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 164.2, 147.6, 144.2, 138.6, 136.5, 130.9, 130.6, 126.0, 125.2, 124.7, 124.2, 121.9119.4, 118.9, 118.2, 117.8, 111.7, 111.5, 108.6, 69.8, 41.1, 36.4, 19.5; HRESIMS (m/z) 370.1561 [M + H]+ (calcd for C23H20N3O2, 370.1556).

4.2. X-ray crystal structure analyses

The measurement of compound 3 was performed using a Bruker APEX-II CCD diffractometer with Mo Kα radiation (λ = 0.71073 Å). The crystal was kept at 273.15 K during data collection.

Crystallographic data of 3. C24H24BrN3O3, mass (M) = 482.37 g mol−1, monoclinic, P212121, a = 10.6793(5) Å, b = 17.3490(7) Å, c = 12.0900(4) Å, α = 90°, β = 92.5510(10)°, γ = 90°, V = 2237.76(16) Å3, Z = 4, F(000) = 992.0, ρcalc = 1.432 g cm−3. A total of 55 076 reflections were measured (4.482° ≤ 2θ ≤ 54.956°), containing 5137 unique reflections (Rint = 0.0627, Rsigma = 0.0309), which were used in all calculations. The final R1 and wR2 values were 0.0348 (I > 2σ(I)) and 0.0800, respectively. The final R1 and wR2 values were 0.0604 (all data) and 0.0921 (all data), respectively. The goodness of fit on F2 was 1.009.

4.3. In vitro cytotoxicity assay

HCT-116 (human colon cancer cells), 4T1 (murine breast cancer cells), and HepG2 (human liver cancer cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The antiproliferative activity of compounds against cancer cell lines was measured by an MTT assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from SigmaAldrich (St. Louis, MO, USA). The cells were incubated in a humidified atmosphere at 37 °C with 5% CO2. Cells were cultured at a density of 5 × 103 cells per well in 96-well plates. After overnight incubation, the cells were treated with tested compounds and then incubated for 48 h. Then, 20 μL of MTT solution (5 mg mL−1) was added to each well, and the plate was incubated for 4 h. Subsequently, the medium was removed and 150 μL DMSO was added into each well to dissolve the formazan. The absorbance was recorded at 490 nm using a microplate reader (SpectraMax CMax Plus, Molecular Devices, Sunnyvale, CA, USA). All experiments were conducted three times. The software GraphPad Prism 8 was used for linear regression analysis to obtain the IC50 value and SD value.

4.4. Colony formation assay

HCT116 (1 × 103 cells per well) and 4T1 (1 × 103 cells per well) cells were seeded into 6-well plates and then cultured for 24 h. The cells were treated with the indicated concentration of 6y, evodiamine or vehicle control. After 14 days, the cells were fixed with methanol and stained with 1% crystal purple. The number of colonies was counted.

4.5. Scratch assay

HCT116 and 4T1 cells were cultured into 6-well plates and then scratch wounded by sterilized pipettes. The cells were washed with PBS and cultured with the indicated concentration of 6y and evodiamine. After 24 h, images were acquired using a phase contrast microscope.

4.6. Statistical analysis

All the data were confirmed by at least three independent experiments, and all the results were expressed as mean ± standard deviation (SD). The data were conducted using the GraphPad Prism version 8.0.1 software with one- or two-way analysis of variance. A P value < 0.05 was considered statistically significant.

Author contributions

All authors contributed equally to this article.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-OLF-D5MD00179J-s001
MD-OLF-D5MD00179J-s002

Acknowledgments

This research was financially supported by grants from the National Natural Science Foundation of China (22277101 and 82404470), the Fundamental Research Funds for the Central Universities of China (2682024KJ015 and 2682023ZTPY078), the Sichuan Science and Technology Program (2025ZNSFSC0689 and 2025ZNSFSC1731), and the Postdoctoral Research Funds for Yibin Research Institute of Southwest Jiaotong University (BSHBZ20240070102). The Analysis and Testing Centre of Southwest Jiaotong University is acknowledged for obtaining NMR data.

†

Electronic supplementary information (ESI) available. CCDC 2426211. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5md00179j

Data availability

The data supporting this article have been included as part of the ESI.†

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-OLF-D5MD00179J-s001
MD-OLF-D5MD00179J-s001.pdf (4.6MB, pdf)
MD-OLF-D5MD00179J-s002
MD-OLF-D5MD00179J-s002.cif (29.1KB, cif)

Data Availability Statement

The data supporting this article have been included as part of the ESI.†

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