Design,Synthesis,and Anticancer Activity of Natural Product Hybrids With Paclitaxel Side Chain Inducing Apoptosis in Human Colon Cancer Cells

Abstract

Based on the strong activity dependence of paclitaxel (PTX; Taxol) or docetaxel (Taxotere) on the C-13 side chain, a small library of dehydroepiandrosterone, cholesterol, vitamin D2, and alkaloids talatisamine and songorine-PTX hybrids have been synthesized and evaluated for in vitro anticancer activity by MTT assay against human breast (MCF-7), colon (HCT116), lung carcinoma (A549), and renal adenocarcinoma (786-0) cancer cell lines. Most hybrids (11b, 12b, 13b, 15b, and 18b) reduced the growth of MCF-7 and 786-0 cells with low PTX sensitivity in vitro. Among the synthesized compounds, hybrid 11b was better in inhibiting the growth of the 4 cells than PTX. A relatively low IC50 value of compound 11b (8.16 ± 0.04 μM) was also examined after exposure for 48 hours. Hybrid 11b showed a proapoptotic effect in HCT116 cells evaluated by Annexin V/propidium iodide binding assay. The level of hybrid 11b leading to protective cell death in HCT116 cells was detected using western blot and not easily observed in our basic examinations.

Taxol (1) and its semisynthetic derivatives docetaxel (Taxotere, 2) and cabazitaxel (Jevana, 3) (Figure 1) are the most widely used drugs in chemotherapy and anticancer. ¹ Their mechanism of action is closely related to their ability to promote polymerization of tubulin into microtubules by binding microtubules and stabilizing the cell division cycle of the G2/M phase. ^2,3 Based on the important role of taxoids, structure-activity relationships (SARs) have been studied in order to determine the minimal structural requirements necessary for biological activity, and the results highlighted the indispensable role of the unique C-13 side chains. ^{4 -9} In addition, this has been justified by some successful studies of paclitaxel (PTX) mimics in which baccatin core was replaced by simplified structures, but retaining part of the 3-dimensional configuration and phenylisoserine side chains of PTX . ^{5,10 -15}

Figure 1

Structures of paclitaxel, docetaxel, and cabazitaxel.

Most of rings and side chains belonging to PTX has been modified in numerous attempts of modern chemists. ¹⁶ In contrast, less attention has been paid to the hybridization of PTX with biologically active natural products. Some PTX-thiocolchicine hybrids were synthesized by Danieli et al, ¹⁷ who found that, compared with PTX, 7-acetylpaclitaxel-thiocolchicine and N-debenzoyl-N-boc-paclitaxel- thiocolchicine have lower anticancer effect. 2′-Acetylpaclitaxel-thiocolchicine hybrid, however, has certain effects because acylation of the 2-OH group is adverse to tubulin polymerization.

Wittman et al ¹⁸ reported 3 PTX-chlorambucil hybrids and evaluated their cytotoxicity against the HCT116 human colon tumor cell lines which were not as excellent as PTX, but can provide extra antitumor effects when they were treated with PTX-resistant M109/taxlR cell lines. Six PTX-daunorubicin hybrids were synthesized by Wandless et al, ¹⁹ but none of them had more cytotoxicity than the parent monomers. Lack of analysis of the deep SAR of PTX has consistently limited the development of a synergistic combination of PTX with other drugs until Smith et al ²⁰ designed 5 hybrids by utilizing a fully flexible (+)-discodermolide and a portion of the PTX side chain according to the inoccupation of the aromatic pocket occupied by the PTX side chain in the potential binding modes. Compared to the parent molecules, a 2- to 8-fold increase in antiproliferative activity was observed using the A549 and MCF-7 cancer cell lines. These studies indicate that active PTX-based hybrids can extend the medical utility of taxol.

Because of its complex structure, especially its baccatin component, obtaining PTX became prohibitively expensive, and it also prevents the large-scale production of synthetic PTX. Therefore, a baccatin-free compound with a taxane-like conformation which retains appreciable antitumor activity can be widely used in the field of anticancer for its simple synthetic technology and accessible materials. In our previous study, the assays of in vitro stabilizing microtubule and its antiproliferative activity indicated that PTX which mimics oxetane D-rings and side chains shows moderate antitumor activities. ⁶ The structures and configurations of some natural compounds like dehydroepiandrosterone (DHEA), cholesterol, vitamin D2, talatisamine, and songorine are similar to the taxane. In addition, these natural products are economical and easy to obtain. These benefits encourage us to synthesize taxane-like natural skeleton-C13 side chain hybrids to study simulated taxane skeletons and we expect to extend the therapeutic use of PTX to PTX-resistant or insensitive tumors. Herein we report the synthesis and in vitro anticancer activity of 8 PTX-natural product hybrids, in which the expensive baccatin core is structurally flexible to DHEA, cholesterol, vitamin D2, talatisamine, and songorine. Preliminary antitumor mechanism of PTX-DHEA hybrid (11b) against HCT116 cells was also studied by western blot experiment.

Results and Discussion

We designed and synthesized a series of new hybrids which consisted of DHEA (6), cholesterol (7), vitamin D2 (8), and diterpenoid alkaloid talatisamine (9) and songorine (10) subunits. These compounds were used to replace the natural baccatin scaffold, respectively.(Figure 2)

Figure 2

Structures of hybrids 11b to 18b.

For replacement of the baccatin core of toxoids (6-10) in different candidates, esterification was carried out with the protected PTX or docetaxel side chain, 1[3-benzoyl-2-(4-methoxyphenyl)-4-phenyloxazolidine-5-carboxylicacid] or 2[3-(tert-butoxycarbonyl)-2-(4-methoxyphenyl)-4-phenyloxazoli-dine-5-carboxylic acid], under standard conditions of N′-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diaminemonohydrochloride (EDC) and 4-dimethylaminopryidine (DMAP) in dichloromethane or methanol at room temperature. The corresponding esters (11a-18a) were obtained in yields ranging from 48.8% to 86.8%. According to the report of protected side chain, ²¹ acid-mediated oxaolidine cleavages of the crude esters 11a to 18a were then carried out with p-toluenesulfonic acid (PTSA) in methanol at room temperature to afford the desired hybrids 11b to 18b in 59.3% to 78.5% yields (Scheme 1).

Scheme 1

Synthesis of paclitaxel hybrids. (a) 4 or 5, N′-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diaminemonohydrochloride, 4-dimethylaminopryidine, CH₂Cl₂, or CH₃OH, 48.8% to 86.8%; (b) p-toluenesulfonic acid, MeOH, 59.3% to 78.5%.

At the beginning of our assessment, the inhibition of the 8 hybrids on human breast cancer (MCF-7), colorectal cancer (HCT116), lung cancer (A549), and renal adenocarcinoma (786-0) cancer cell lines was evaluated by MTT analysis. ²² The results showed that all compounds inhibited the growth of HCT116 and A549 cells at a concentration of 50 µM (Figure 3). Surprisingly, compared with compound 11b, which was replaced by DHEA and PTX control, baccatin scaffold showed a better inhibition effect of cell growth of all the above cells. For PTX-resistant human mammary gland (MCF-7), most compounds including 11b, 12b, 13b, 15b, and 18b were superior to PTX in inhibiting cell line growth. Four hybrids (11b, 13b, 17b, and 18b) also showed therapeutic potential to inhibit PTX-resistant 786-0 cell growth.

Figure 3

The inhibitory rates of the hybrids.

According to the desirable inhibitory effect of compound 11b on the growth of the 4 cancer cell lines, we examined its antiproliferative effect in HCT116 human colon cancer cells and drew the dose-response (D/R) curves. With the exposure time increased, the amount of IC₅₀ marginally decreased, and there was nearly no difference when the exposure time was over 48 hours (Table 1). Specifically, IC₅₀ of hybrid 11b was 10.2 ± 0.02 µM after exposure to drug for 24 hours, followed by 8.16 ± 0.04 and 8.34 ± 0.04 µM for 48 and 72 hours, respectively. The cells showed a slightly greater sensitivity to the change of concentration when exposed to 24 hours, with a tiny steep slope, compared to a long exposure time (Figure 4). According to existing reports, ^23,24 PTX caused less than 50% survival of HCT116 cell saturation doses around 2.5 nanomoles after 1 or 3 days. Therefore, hybrid 11b does not possess antiproliferative activity at similar dose.

Table 1

Cytotoxicity of Compound 11b Against HCT116 Human Colon Cancer Cells After Different Exposure Times.

HCT116	IC50 (μM)
24 hours	10.2 ± 0.02
48 hours	8.16 ± 0.04
72 hours	8.34 ± 0.22

Figure 4

Dose-response curve of 11b.

The work direction of hybrid 11b will be continuously improved under the guidance of PTX. In order to observe the nature of hybrid 11b-induced cell death, we performed flow cytometry analysis with Annexin V-FITC/propidium iodide (PI) in 11b-treated HCT116 cells. As shown in Figure 5, on enhancing the compound concentration, the increased population of the 2 kinds of apoptotic cells and a consequent decrease of live cells indicated the induction of apoptosis by compound 11b. To be more specific, compared with corresponding cells in the control, it has been observed that the proportion of necrotic and late apoptotic cells in 20 nM hybrid control increased 10-fold. From 23.97% to 52.75% of cells, necrotic and apoptotic cells increased due to the rise in the contribution of the compound.

Figure 5

Compound 11b inducing apoptosis by the Annexin V-FITC method.

As hybrid 11b showed positive anticancer activity in MTT assay and flow cytometry analysis with Annexin V-FITC/PI, we detected the expression of apoptotic markers affected by hybrid 11b in HCT116 cells by western blot analysis (Figure 6). Using actin (66009-I-Ig, Proteintech, IL, USA) as control, treatment with compound 11b in a dose-dependent manner (5, 10, 15 µM) resulted in a slight increase of Bax (2772, CST) and the obvious cleavage of PARP1 (9532, CST). The decrease in PARP1 indicated that compound 11b could promote apoptosis of HCT116 cells in a dose-dependent manner.

Figure 6

Levels of apoptosis-related proteins Bax and PARP1.

A computer modeling study was then performed to determine whether compound 11b acted on the same target of PTX (Figures 7 and 8). The 3-dimensional structure file of beta-tubulin (PDB ID: 6I2I) was downloaded from the Protein Crystal Database (PDB). Molecular docking with compound 11b and beta-tubulin by Discovery Studio software platform showed that -Cdocker Interaction Energy was 48.6621 and -Cdocker Energy was -16.6521. The results demonstrated a general binding affinity of compound 11b and beta-tubulin. This may due to the difference between modes and the real internal environment.

Figure 7

Molecular docking result of compound 11b and beta-tubulin.

Figure 8

Molecular docking pattern of compound 11b.

Conclusions

In summary, 8 PTX-based hybrids were prepared and their inhibitory levels in the growth of 4 cancer cell lines were evaluated in vitro. Most compounds (11b, 13b, 14b, 15b, and 18b) could be beneficial to extend the therapeutic utility of PTX to tumors with low chemosensitivity or relatively high PTX resistance (A549 and 786-0 cell lines). All hybrids reduced the growth of HCT116 and A549 cell lines in vitro, among which compound 11b having the flexible baccatin counterpart was the most active. Besides the C13-side chain which is from the pharmacophore of PTX, this may be due to DHEA, a steroidal hormone, which can prevent the cancer of rodents, and studies show that low circulating levels of DHEA have been associated with a higher incidence of breast cancer in women. ^25,26 Replacing the baccatin scaffold with alkaloidstalatisamine and songorine showed a weaker ability to inhibit growth in allfour cell lines than with paclitaxel, which indicates baccatin and isoformswith relatively rigid skeleton are useful in attempt to the design taxane-likehybrid.

Moreover, hybrid 11b, which had significant inhibitory effects on the 4 cancer cell lines, showed an IC₅₀ value of 8.16 ± 0.04 µM only after exposure for 48 hours. After comparison, the research for active PTX hybrids that are similar to PTX in terms of anticancer activity is challenging through our simple replacement of baccatin scaffold. Hybrid 11b induced the apoptosis in HCT116 human colon cancer cells according to flow cytometry analysis and immunoblot analysis of the apoptosis-related proteins, which might assist in the development of better anticancer agents based on compound 11b in the future.

In general, this work reveals a successful hybridization by analyzing the SAR. The use of baccatin-free hybrid component could be an effective strategy to establish PTX-based hybrid library. However, it was turned out that the promising compound 8b with potential anticancer activity should be improved in terms of reducing dose-dependent manner or increasing the therapeutic potential for inducing apoptosis.

Experimental

General Experimental Procedures

Unless otherwise stated, reagents were commercially available and used without further purification. The side chains 4 and 5 were purchased from Yunnan Hongdou Pharmaceuticals Ltd with more than 99.5% enantiomeric purity. DHEA (6), cholesterol (7), and vitamin D2 (8) were purchased from Aladdin Reagents Company. The diterpenoid alkaloids talatisamine (9) and songorine (10) were extracted from Delphinium delavayi Franch. HRESIMS data were measured using a Q-TOF micro mass spectrometer (Waters). NMR spectra were recorded on Bruker AV 600 or 400 spectrometers. Chemical shifts (δ) are reported in units of parts per million downfield from tetramethylsilane.

General Procedure for the Esterification Reaction of Compounds 6 to 10 With Protected Side Chain

A suspension of one of the starting compounds 6 to 10 (0.1 mmol), protected side chain 4 or 5 (0.12 mmol), EDC (0.12 mmol), and DMAP (0.1 mmol) in dry dichloromethane (3 mL) was stirred at room temperature for 24 hours until TLC showed complete consumption of the starting compounds. Then the reaction mixture was diluted with water (3 mL) and extracted by DCM (5 mL) 3 times. The combined organic layer was washed with H₂O (5 mL × 2) and brine (5 mL × 2), dried over MgSO₄, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford the corresponding ester. The yield and spectral data of each compound are given below.

General Procedure for the Deprotection of the Phenylisoserine Side Chain

Ester compounds 11a to 18a (0.05 mmol) were added to MeOH solution and treated with PTSA (0.2 mmol). The reaction mixture was stirred at room temperature for 4 hours and then diluted with an excess of ethyl acetate. The organic layer was washed with saturated aqueous solution of NaHCO₃ and brine, dried over MgSO₄, filtered, and concentrated. The crude mixture was purified by column chromatography on silica gel to afford the desired compound.

Compound 11a (Figure S1)

The reaction was carried out with 57.6 mg (0.2 mmol) of compound 6, 80.6 mg (0.2 mmol) of protected side chain 4, 45.84 mg (0.12 mmol) EDC, and 24.4 mg (0.1 mmol) DMAP in a mixture of toluene (3 mL) to give crude product. After purification by column chromatography on silica gel (petroleum ether/EtOAc = 5:1), compound 11a (101 mg, 75.8%) was obtained as white powder. ¹H NMR (600 MHz, CDCl₃) δ 7.50 to 7.27 (m, 9 H), 7.22 (t, J = 7.7 Hz, 3 H), 6.85 (s, 2 H), 5.44 (d, J = 4.8 Hz, 1 H), 5.38 (d, J = 5.1 Hz, 1 H), 4.83 (s, 1 H), 4.78 to 4.70 (m, 1 H), 3.81 (s, 3 H), 2.46 (ddd, J = 19.3, 8.7, 5.2 Hz, 1 H), 2.39 (d, J = 7.9 Hz, 2 H), 2.17 to 2.04 (m, 3 H), 1.99 to 1.81 (m, 6 H), 1.67 (dd, J = 16.7, 7.4 Hz, 4 H), 1.50 (ddd, J = 26.0, 13.2, 5.4 Hz, 3 H), 1.31 (dd, J = 14.5, 5.3 Hz, 2 H), 1.06 (s, 3 H), 0.89 (s, 2 H). MS (ESI) m/z: [(M + H)⁺, 674.34].

Compound 12a

The reaction was carried out with 126.3 mg (0.3 mmol) of compound 7, 120.9 mg (0.3 mmol) of protected side chain 4, 68.76 mg EDC (0.36 mmol), and 36.6 mg (0.4 mmol) DMAP in a mixture of CH₂Cl₂ (3 mL) to give a crude product. After purification by column chromatography on silica gel (petroleum ether/EtOAc = 1:2), compound 12a was obtained as white powder (118 mg, 48.8%). ¹H NMR (400 MHz, CDCl₃) δ 7.36 to 7.27 (m, 7 H), 7.25 to 7.18 (m, 4 H), 6.87 (d, J = 8.3 Hz, 3 H), 4.96 (t, J = 5.0 Hz, 1 H), 4.75 (s, 1 H), 3.82 (s, 3 H), 3.28 (d, J = 9.3 Hz, 6 H), 3.18 to 3.08 (m, 3 H), 3.02 to 2.96 (m, 4 H), 2.63 to 2.58 (m, 1 H), 2.56 to 2.34 (m, 6 H), 2.18 to 2.14 (m, 1 H), 2.06 (d, J = 7.8 Hz, 1 H), 1.97 (dd, J = 11.6, 5.1 Hz, 2 H), 1.90 (d, J = 8.1 Hz, 1 H), 1.84 (dd, J = 11.0, 6.3 Hz, 2 H), 1.81 to 1.76 (m, 1 H), 1.65 (d, J = 6.9 Hz, 3 H), 1.52 (dd, J = 14.3, 7.6 Hz, 1 H), 1.42 (t, J = 12.5 Hz, 1 H), 1.25 (s, 3 H), 1.05 (t, J = 7.1 Hz, 3 H). MS (ESI) m/z: [(M + H)⁺, 822.44].

Compound 13a

The reaction was carried out with 126.3 mg (0.3 mmol) of compound 7, 119.7 mg (0.3 mmol) of protected side chain 5, 68.76 mg (0.36 mmol) EDC, and 36.6 mg (0.4 mmol) DMAP in a mixture of CH₂Cl₂ (3 mL) to give a crude product. After purification by column chromatography on silica gel (petroleum ether/EtOAc = 1:1), compound 13a was obtained as white powder (131 mg, 54.4%). ¹H NMR (400 MHz, CDCl₃) δ 7.44 to 7.35 (m, 4 H), 7.33 to 7.27 (m, 3 H), 6.91 (d, J = 8.6 Hz, 2 H), 4.49 (d, J = 1.7 Hz, 2 H), 3.82 (s, 3 H), 3.30 (s, 3 H), 3.24 (s, 3 H), 3.11 to 3.04 (m, 6 H), 2.97 (d, J = 9.0 Hz, 1 H), 2.89 (s, 1 H), 2.52 to 2.41 (m, 4 H), 2.36 to 2.21 (m, 3 H), 2.04 (dd, J = 15.4, 6.7 Hz, 1 H), 1.99 to 1.90 (m, 3 H), 1.87 to 1.79 (m, 1 H), 1.72 (d, J = 19.9 Hz, 3 H), 1.65 to 1.58 (m, 2 H), 1.55 (d, J = 7.2 Hz, 2 H), 1.44 (dd, J = 14.6, 8.8 Hz, 2 H), 1.24 (d, J = 5.6 Hz, 1 H), 1.10 (s, 9 H), 1.01 (t, J = 7.1 Hz, 3 H). MS (ESI) m/z: [(M + H)⁺, 802.47].

Compound 14a

The reaction was carried out with 107.1 mg (0.3 mmol) of compound 8, 120.9 mg (0.3 mmol) of protected side chain 4, 68.76 mg (0.36 mmol) EDC, and 36.6 mg (0.4 mmol) DMAP in a mixture of CH₂Cl₂ (3 mL) to give a crude product. After purification by column chromatography on silica gel (petroleum ether/EtOAc = 1:2), compound 14a was obtained as white powder (98.7 mg, 44.3%). ¹H NMR (400 MHz, CDCl₃) δ 7.75 (d, J = 7.3 Hz, 2 H), 7.48 (dd, J = 15.0, 7.6 Hz, 3 H), 7.41 (t, J = 7.4 Hz, 2 H), 7.35 (t, J = 7.5 Hz, 2 H), 7.07 (d, J = 8.8 Hz, 1 H), 5.66 (d, J = 8.9 Hz, 1 H), 5.34 to 5.10 (m, 3 H), 4.58 (t, J = 10.5 Hz, 1 H), 4.33 (s, 1 H), 3.54 (s, 1 H), 3.06 (d, J = 3.3 Hz, 1 H), 2.57 (ddd, J = 31.7, 19.1, 10.1 Hz, 5 H), 2.30 (dd, J = 21.9, 8.4 Hz, 2 H), 2.14 to 2.00 (m, 2 H), 1.83 to 1.73 (m, 2 H), 1.59 to 1.33 (m, 5 H), 1.25 (s, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 0.91 to 0.84 (m, 1 H), 0.73 (s, 3 H). MS (ESI) m/z: [(M + H)⁺, 789.44].

Compound 15a

The reaction was carried out with 107.1 mg (0.3 mmol) of compound 8, 119.7 mg (0.3 mmol) of protected side chain 5, 68.76 mg (0.36 mmol) EDC, and 36.6 mg (0.4 mmol) DMAP in a mixture of CH₂Cl₂ (3 mL) to give a crude product. After purification by column chromatography on silica gel (petroleum ether/EtOAc = 1:2), compound 15a was obtained as white powder (124 mg, 64.3%). ¹H NMR (400 MHz, CDCl₃) δ 7.44 to 7.28 (m, 7 H), 6.89 (d, J = 8.6 Hz, 2 H), 6.25 (s, 1 H), 5.24 (s, 1 H), 5.17 to 5.07 (m, 2 H), 4.58 (d, J = 6.6 Hz, 1 H), 4.25 (d, J = 6.4 Hz, 1 H), 3.81 (s, 3 H), 3.28 (s, 1 H), 2.87 (d, J = 3.0 Hz, 1 H), 2.68 to 2.53 (m, 2 H), 2.50 to 2.41 (m, 3 H), 2.27 (d, J = 11.3 Hz, 1 H), 2.18 (d, J = 5.1 Hz, 1 H), 2.04 to 1.90 (m, 2 H), 1.73 to 1.55 (m, 5 H), 1.42 to 1.31 (m, 3 H), 1.24 (dd, J = 12.4, 3.8 Hz, 2 H), 1.15 to 1.05 (m, 3 H), 0.99 (s, 9 H), 0.75 (s, 3 H). MS (ESI) m/z: [(M + H)⁺, 747.41].

Compound 16a

The reaction was carried out with 39.6 mg (0.1 mmol) of compound 9, 40.3 mg (0.1 mmol) of protected side chain 4, 22.9 mg (0.12 mmol) EDC, and 12.2 mg (0.1 mmol) DMAP in a mixture of CH₂Cl₂ (3 mL) to give a crude product, After purification by column chromatography on silica gel (petroleum ether/EtOAc = 5:1), compound 16a was obtained as white powder (67.8 mg, 86.8%). ¹H NMR (400 MHz, CDCl₃) δ 7.32 (dd, J = 25.5, 7.6 Hz, 8 H), 7.25 to 7.19 (m, 3 H), 6.83 (d, J = 7.7 Hz, 3 H), 6.27 (d, J = 11.2 Hz, 1 H), 6.02 (d, J = 11.2 Hz, 1 H), 5.20 (t, J = 5.9 Hz, 2 H), 5.13 to 5.03 (m, 2 H), 4.85 (d, J = 2.0 Hz, 2 H), 3.81 (s, 3 H), 2.89 to 2.78 (m, 1 H), 2.61 (dd, J = 13.4, 3.2 Hz, 1 H), 2.44 (dd, J = 13.4, 7.7 Hz, 1 H), 2.35 to 2.26 (m, 1 H), 2.23 to 2.12 (m, 1 H), 2.08 to 1.94 (m, 4 H), 1.86 (dd, J = 12.8, 6.5 Hz, 1 H), 1.73 (ddd, J = 22.6, 16.3, 8.2 Hz, 5 H), 1.58 to 1.42 (m, 4 H), 1.31 (ddd, J = 18.8, 15.0, 9.1 Hz, 4 H), 1.03 (d, J = 6.6 Hz, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 0.84 (t, J = 6.4 Hz, 6 H), 0.56 (s, 3 H). MS (ESI) m/z: [(M + H)⁺, 782.47].

Compound 17a

The reaction was carried out with 39.6 mg (0.1 mmol) of compound 9, 39.9 mg (0.1 mmol) of protected side chain 5, 22.9 mg (0.12 mmol) EDC, and 12.2 mg (0.1 mmol) DMAP in a mixture of CH₂Cl₂ (3 mL) to give a crude product. After purification by column chromatography on silica gel (petroleum ether/EtOAc = 5:1), compound 17a was obtained as white powder (66.6 mg, 85.7%). ¹H NMR (400 MHz, CDCl₃) δ 7.40 to 7.35 (m, 6 H), 7.32 to 7.28 (m, 1 H), 6.90 (t, J = 8.8 Hz, 2 H), 6.13 (d, J = 11.2 Hz, 1 H), 6.01 (d, J = 11.2 Hz, 1 H), 5.18 (dd, J = 17.2, 10.5 Hz, 2 H), 5.03 (s, 1 H), 4.82 (s, 2 H), 4.53 (d, J = 4.3 Hz, 1 H), 3.82 (s, 3 H), 2.88 to 2.78 (m, 1 H), 2.36 to 2.28 (m, 1 H), 2.16 (dd, J = 11.4, 7.0 Hz, 3 H), 2.00 (tt, J = 18.1, 9.0 Hz, 4 H), 1.86 (dd, J = 12.9, 6.5 Hz, 1 H), 1.69 (t, J = 10.0 Hz, 4 H), 1.62 (s, 1 H), 1.48 (dd, J = 13.2, 6.8 Hz, 4 H), 1.36 to 1.22 (m, 4 H), 1.06 (d, J = 5.9 Hz, 9 H), 1.02 (d, J = 6.6 Hz, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 0.83 (t, J = 6.5 Hz, 6 H), 0.56 (s, 3 H). MS (ESI) m/z: [(M + H)⁺, 762.50].

Compound 18a

The reaction was carried out with 38.6 mg (0.1 mmol) of compound 10, 40.3 mg (0.1 mmol) of protected side chain 4, 22.9 mg (0.12 mmol) EDC, and 12.2 mg (0.1 mmol) DMAP in a mixture of toluene (3 mL) to give a crude product. After purification by column chromatography on silica gel (petroleum ether/EtOAc = 5:1), compound 18a was obtained as white powder (63.5 mg, 82.4%). ¹H NMR (400 MHz, CDCl₃) δ 7.40 to 7.27 (m, 8 H), 7.22 (t, J = 7.6 Hz, 3 H), 6.89 (t, J = 32.2 Hz, 3 H), 5.38 (dd, J = 20.5, 4.6 Hz, 2 H), 4.83 (s, 1 H), 4.78 to 4.68 (m, 1 H), 3.82 (s, 3 H), 2.36 (d, J = 7.3 Hz, 2 H), 2.06 to 1.93 (m, 3 H), 1.91 to 1.81 (m, 3 H), 1.63 to 1.54 (m, 4 H), 1.53 to 1.47 (m, 3 H), 1.43 (dd, J = 10.6, 4.3 Hz, 1 H), 1.34 (d, J = 7.5 Hz, 3 H), 1.27 to 1.22 (m, 3 H), 1.19 to 1.05 (m, 7 H), 1.03 (s, 4 H), 0.92 (d, J = 6.5 Hz, 3 H), 0.87 (dd, J = 6.6, 1.7 Hz, 6 H), 0.68 (s, 3 H). MS (ESI) m/z: [(M + H)⁺, 772.49].

Compound 11b

The reaction was carried out with 0.05 mmol (33.6 mg) of compound 11a in MeOH (1.5 mL) and treated with PTSA (0.2 mmol, 38.2 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 11b (21 mg, 75.7%) was obtained as white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.81 to 7.71 (m, 2 H), 7.54 to 7.29 (m, 7 H), 6.98 (d, J = 9.1 Hz, 1 H), 5.77 (dd, J = 9.3, 1.8 Hz, 1 H), 5.40 (dd, J = 12.8, 5.0 Hz, 1 H), 4.78 to 4.68 (m, 1 H), 4.61 (s, 1 H), 2.50 to 2.23 (m, 4 H), 2.07 (dd, J = 18.3, 8.4 Hz, 2 H), 1.98 to 1.80 (m, 5 H), 1.67 to 1.61 (m, 3 H), 1.42 (s, 1 H), 1.28 to 1.23 (m, 5 H), 1.04 (d, J = 5.6 Hz, 4 H), 0.88 (d, J = 2.5 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 166.9, 141.1, 139.7, 138.9, 134.4, 131.8, 128.8, 128.8, 128.0, 127.1, 127.0, 122.4, 121.0, 73.5, 71.7, 54.8, 51.9, 50.4, 47.6, 42.3, 38.0, 37.3, 36.8, 35.9, 31.5, 30.9, 27.5, 22.0, 20.5, 19.4, 13.6. MS (ESI) m/z: [(M + H)⁺, 556.30].

Compound 12b

The reaction was carried out with 0.1 mmol (82.2 mg) of compound 12a in MeOH (2 mL) and treated with PTSA (0.4 mmol, 76.3 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 12b (54.2 mg, 78.4%) was obtained as white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.88 to 7.81 (m, 2 H), 7.53 to 7.46 (m, 2 H), 7.42 (t, J = 7.4 Hz, 4 H), 7.32 (t, J = 7.3 Hz, 2 H), 5.66 (dd, J = 8.8, 2.4 Hz, 1 H), 4.94 (dd, J = 10.7, 5.7 Hz, 1 H), 4.59 (d, J = 2.6 Hz, 1 H), 3.29 (s, 3 H), 3.24 (s, 3 H), 3.16 (s, 3 H), 3.07 (dd, J = 9.7, 4.3 Hz, 2 H), 2.99 to 2.93 (m, 2 H), 2.53 to 2.34 (m, 6 H), 2.19 to 2.07 (m, 3 H), 1.98 (dd, J = 17.3, 9.5 Hz, 4 H), 1.88 to 1.85 (m, 1 H), 1.84 to 1.81 (m, 1 H), 1.76 (dd, J = 11.7, 5.6 Hz, 2 H), 1.65 (dd, J = 14.7, 7.4 Hz, 1 H), 1.56 (d, J = 7.2 Hz, 1 H), 1.48 to 1.36 (m, 2 H), 1.26 (d, J = 7.1 Hz, 1 H), 1.22 (dd, J = 9.9, 4.0 Hz, 1 H), 1.04 (dd, J = 9.2, 5.1 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 171.9, 167.2, 138.8, 134.4, 131.6, 128.6, 127.8, 127.5, 127.1, 85.8, 82.5, 79.6, 74.3, 73.4, 62.5, 59.6, 56.5, 56.3, 55.1, 53.1, 49.5, 48.7, 47.1, 46.0, 45.0, 44.9, 40.4, 38.6, 35.9, 32.7, 29.8, 28.3, 26.2, 24.8, 21.1, 14.3, 13.7. MS (ESI) m/z: [(M + H)⁺, 721.44].

Compound 13b

The reaction was carried out with 0.1 mmol (80.2 mg) of compound 13a in MeOH (2 mL) and treated with PTSA (0.4 mmol, 76.3 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 13b (49 mg, 71.6%) was obtained as white powder. ¹H NMR (600 MHz, CDCl₃) δ 7.38 to 7.27 (m, 5 H), 5.05 (d, J = 17.5 Hz, 1 H), 4.44 (s, 1 H), 3.34 (d, J = 8.2 Hz, 3 H), 3.30 (s, 3 H), 3.27 (s, 3 H), 3.13 to 3.08 (m, 2 H), 3.00 (d, J = 9.1 Hz, 2 H), 2.54 to 2.43 (m, 4 H), 2.30 (s, 1 H), 2.21 (dd, J = 17.7, 9.9 Hz, 1 H), 2.06 (t, J = 9.5 Hz, 1 H), 2.01 (d, J = 7.3 Hz, 3 H), 1.85 to 1.75 (m, 3 H), 1.64 (d, J = 6.5 Hz, 4 H), 1.53 (dd, J = 14.8, 8.1 Hz, 1 H), 1.45 (s, 7 H), 1.25 (s, 7 H), 1.07 (t, J = 6.8 Hz, 3 H), 0.88 (t, J = 6.9 Hz, 1 H). ¹³C NMR (100 MHz, CDCl₃) δ 155.5, 128.4, 127.7, 127.1, 85.8, 82.3, 79.6, 75.7, 74.5, 73.4, 72.9, 68.3, 63.1, 62.6, 59.6, 56.9, 56.3, 53.1, 49.5, 48.8, 47.2, 46.1, 45.2, 45.0, 40.5, 38.6, 37.2, 32.9, 32.8, 32.0, 28.5, 28.4, 27.3, 26.3, 25.0, 24.9, 14.2, 13.7. MS (ESI) m/z: [(M + H)⁺, 685.40].

Compound 14b

The reaction was carried out with 0.1 mmol (74.2 mg) of compound 14a in MeOH (2 mL) and treated with PTSA (0.4 mmol, 76.3 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 14b (37 mg, 59.3%) was obtained as white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (d, J = 7.3 Hz, 2 H), 7.48 (dd, J = 15.0, 7.6 Hz, 3 H), 7.41 (t, J = 7.4 Hz, 2 H), 7.35 (t, J = 7.5 Hz, 2 H), 7.07 (d, J = 8.8 Hz, 1 H), 5.66 (d, J = 8.9 Hz, 1 H), 5.34 to 5.10 (m, 3 H), 4.58 (t, J = 10.5 Hz, 1 H), 4.33 (s, 1 H), 3.54 (s, 1 H), 3.06 (d, J = 3.3 Hz, 1 H), 2.57 (ddd, J = 31.7, 19.1, 10.1 Hz, 5 H), 2.30 (dd, J = 21.9, 8.4 Hz, 2 H), 2.14 to 2.00 (m, 2 H), 1.83 to 1.73 (m, 2 H), 1.59 to 1.33 (m, 5 H), 1.25 (s, 3 H), 1.17 (d, J = 7.1 Hz, 3 H), 0.91 to 0.84 (m, 1 H), 0.73 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 209.4, 172.6, 166.9, 150.7, 139.3, 134.6, 131.7, 128.8, 128.7, 127.9, 127.2, 127.1, 112.0, 77.3, 73.7, 65.5, 57.1, 54.9, 54.0, 50.6, 50.5, 50.1, 49.8, 44.3, 37.7, 37.6, 35.3, 34.2, 31.9, 29.8, 26.5, 25.7, 22.9, 13.6. MS (ESI) m/z: [(M + H)⁺, 671.40].

Compound 15b

The reaction was carried out with 0.1 mmol (73.8 mg) of compound 15a in MeOH (2 mL) and treated with PTSA (0.4 mmol, 76.3 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 15b (48.7 mg, 78.5%) was obtained as white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.40 to 7.27 (m, 5 H), 5.54 (d, J = 9.4 Hz, 1 H), 5.29 (d, J = 1.2 Hz, 1 H), 5.18 (d, J = 9.5 Hz, 3 H), 4.37 (d, J = 17.5 Hz, 2 H), 3.55 (d, J = 13.7 Hz, 1 H), 3.09 (d, J = 2.7 Hz, 1 H), 2.70 (dd, J = 12.2, 7.2 Hz, 1 H), 2.58 to 2.51 (m, 3 H), 2.44 to 2.33 (m, 2 H), 2.30 (d, J = 4.5 Hz, 1 H), 2.11 (dd, J = 9.7, 6.1 Hz, 2 H), 2.07 (s, 1 H), 1.82 (dt, J = 16.5, 8.3 Hz, 3 H), 1.51 to 1.43 (m, 3 H), 1.41 (d, J = 7.4 Hz, 9 H), 1.26 (d, J = 7.3 Hz, 3 H), 1.17 (t, J = 7.1 Hz, 4 H), 1.11 (t, J = 7.1 Hz, 1 H), 0.77 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 209.0, 172.5, 154.8, 150.7, 139.0, 128.5, 127.7, 126.9, 112.0, 79.8, 77.7, 73.9, 66.3, 57.0, 55.7, 54.0, 50.8, 50.4, 50.2, 49.9, 44.4, 42.9, 40.1, 37.7, 37.6, 35.3, 34.2, 32.0, 29.8, 28.4, 25.8, 23.0, 21.5, 14.2, 13.6, 13.2. MS (ESI) m/z: [(M + H)⁺, 635.36].

Compound 16b

The reaction was carried out with 0.05 mmol (39.2 mg) of compound 16a in MeOH (1.5 mL) and treated with PTSA (0.2 mmol, 38.2 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 16b (24.5 mg, 74.3%) was obtained as white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.78 to 7.73 (m, 2 H), 7.53 to 7.48 (m, 1 H), 7.43 (dt, J = 8.7, 4.3 Hz, 4 H), 7.36 (dd, J = 10.0, 4.8 Hz, 2 H), 7.32 to 7.26 (m, 1 H), 7.00 (d, J = 9.2 Hz, 1 H), 6.20 (d, J = 11.2 Hz, 1 H), 6.02 (d, J = 11.2 Hz, 1 H), 5.74 (dt, J = 9.5, 4.8 Hz, 1 H), 5.19 (t, J = 6.0 Hz, 2 H), 5.09 to 5.02 (m, 2 H), 4.84 (d, J = 2.1 Hz, 1 H), 4.63 (s, 1 H), 3.36 (d, J = 14.5 Hz, 1 H), 2.80 (dd, J = 11.9, 3.5 Hz, 1 H), 2.60 (dd, J = 13.4, 3.8 Hz, 1 H), 2.48 to 2.35 (m, 2 H), 2.22 to 2.14 (m, 1 H), 2.08 to 1.93 (m, 4 H), 1.85 (dd, J = 12.7, 6.7 Hz, 1 H), 1.74 to 1.64 (m, 4 H), 1.49 to 1.44 (m, 2 H), 1.37 to 1.24 (m, 5 H), 1.02 (t, J = 5.7 Hz, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 0.83 (t, J = 6.5 Hz, 6 H), 0.55 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.5, 166.9, 144.2, 142.8, 138.8, 135.7, 134.3, 133.7, 132.0, 131.8, 128.8, 128.8, 128.0, 127.1, 127.0, 122.9, 117.5, 113.2, 74.8, 73.3, 56.5, 54.8, 45.9, 42.9, 42.0, 40.5, 33.2, 32.1, 31.7, 29.8, 29.2, 27.9, 23.7, 22.3, 21.2, 21.1, 20.0, 19.7, 17.7, 12.3. MS (ESI) m/z: [(M + H)⁺, 664.43].

Compound 17b

The reaction was carried out with 0.1 mmol (74.2 mg) of compound 17a in MeOH (1.5 mL) and treated with PTSA (0.2 mmol, 38.2 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 17b (23.4 mg, 71.1%) was obtained as white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.37 to 7.27 (m, 5 H), 6.22 (d, J = 11.2 Hz, 1 H), 6.04 (d, J = 11.2 Hz, 1 H), 5.40 (d, J = 9.4 Hz, 1 H), 5.20 (t, J = 5.9 Hz, 2 H), 5.07 (s, 2 H), 4.86 (s, 1 H), 3.19 (d, J = 3.7 Hz, 1 H), 2.85 to 2.77 (m, 1 H), 2.61 (dd, J = 13.4, 3.8 Hz, 1 H), 2.49 to 2.37 (m, 2 H), 2.21 (ddd, J = 13.4, 8.9, 4.9 Hz, 1 H), 2.05 to 1.94 (m, 4 H), 1.84 (dt, J = 9.1, 5.4 Hz, 2 H), 1.68 (dd, J = 24.2, 15.8 Hz, 5 H), 1.51 to 1.46 (m, 2 H), 1.41 (s, 8 H), 1.33 (d, J = 9.5 Hz, 2 H), 1.26 (s, 1 H), 1.02 (d, J = 6.6 Hz, 3 H), 0.92 (d, J = 6.8 Hz, 3 H), 0.83 (t, J = 6.5 Hz, 7 H), 0.56 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.5, 155.1, 144.3, 142.7, 139.4, 135.7, 133.8, 132.1, 128.6, 127.7, 126.9, 122.9, 117.6, 113.1, 79.8, 77.3, 74.5, 73.6, 56.5, 56.0, 45.9, 42.9, 42.0, 40.5, 40.5, 33.2, 32.1, 31.7, 29.8, 29.2, 28.3, 27.9, 23.7, 22.3, 21.2, 21.1, 20.0, 19.9, 19.7, 17.7, 12.4, 11.5. MS (ESI) m/z: [(M + H)⁺, 644.46].

Compound 18b

The reaction was carried out with 0.1 mmol (39.9 mg) of compound 18a in MeOH (2 mL) and treated with PTSA (0.4 mmol, 76.3 mg) for 4 hours at room temperature. After purification (petroleum ether/EtOAc = 1:1), the expected compound 18b (24.5 mg, 75.2%) was obtained as white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.76 (dd, J = 5.2, 3.3 Hz, 2 H), 7.50 (d, J = 7.4 Hz, 1 H), 7.48 to 7.40 (m, 4 H), 7.36 (dd, J = 8.1, 6.7 Hz, 2 H), 7.30 (d, J = 7.2 Hz, 1 H), 7.03 (d, J = 9.0 Hz, 1 H), 5.76 (dd, J = 9.2, 2.0 Hz, 1 H), 5.38 (d, J = 5.2 Hz, 1 H), 4.80 to 4.67 (m, 1 H), 4.60 (s, 1 H), 3.35 (s, 1 H), 2.47 to 2.37 (m, 1 H), 2.36 to 2.29 (m, 1 H), 2.04 to 1.93 (m, 2 H), 1.87 to 1.78 (m, 3 H), 1.64 (s, 2 H), 1.52 (d, J = 6.7 Hz, 1 H), 1.50 to 1.47 (m, 1 H), 1.47 to 1.42 (m, 2 H), 1.39 to 1.29 (m, 4 H), 1.26 (s, 4 H), 1.09 (ddd, J = 18.4, 14.2, 6.1 Hz, 7 H), 1.02 (s, 3 H), 0.91 (d, J = 6.5 Hz, 3 H), 0.87 (d, J = 1.8 Hz, 3 H), 0.85 (d, J = 1.7 Hz, 3 H), 0.70 to 0.64 (m, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.5, 166.8, 139.4, 138.9, 134.4, 131.8, 128.8, 128.8, 127.9, 127.1, 127.0, 123.2, 76.9, 73.5, 56.8, 56.2, 54.8, 50.1, 42.4, 39.8, 39.6, 38.0, 36.9, 36.7, 36.3, 35.9, 32.0, 31.9, 29.8, 28.3, 28.1, 27.6, 24.4, 23.9, 22.9, 22.7, 21.1, 19.4, 18.8, 11.9. MS (ESI) m/z: [(M + H)⁺, 654.45].

Bioassays

MCF-7, HCT116, A549, and 786-0 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MTT (M2128) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used in this study were as follows: Bax (2772, CST), PARP (9532, CST), β-actin (66009-I-Ig, Proteintech).

Culture of Cell Lines

Cells were cultured in 175 cm² flasks that were filled with L-glutamine with 10% heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin, and 50 mg/L streptomyocin in a humidified, 5% CO₂, 37°C incubator. The medium was changed every second day and cells were subcultured at ~80% confluency every 4 to 5 days using 0.1% trypsin.

Cytotoxicity Assay by MTT Assay

MTT assay was employed for this primary determination as we did in previous report. ¹ The in vitro inhibitory activities of 8 hybrids on 4 cells were evaluated. Cells were cultured in 96-well plates with a density of 5 × 10³ for 1 day and then, respectively, pre-treated at 37℃ for 24 hours with each compound 11b to 18b (50 μM, n = 3). After that, 10 μL/well of 5 mg/mL MTT (Sigma) was added to all wells. The plates were incubated for an additional 4 hours, the supernatants were removed, and then 150 μL of dimethyl sulfoxide (DMSO) was added to all the wells to dissolve the dark blue crystals. The plates were then read on a microplate reader (Wellscan MK3, Labsystems Dragon) at a wavelength of 570 nm. The inhibition rate was calculated as follows:

Inhibition rate = (mean OD control - mean OD drug treated)/(mean OD control - mean OD vacuity) × 100%.

Experimental Details for Obtaining IC₅₀ Values

HCT116 human colon cancer cells were cultured in medium with 10% FBS. HCT116 human colon cancer cells were added to each well of a 96-well plate. Increasing concentrations of hybrid 11b were added 18 hours after plating. IC₅₀ values, the concentration of agent that inhibits cell growth by 50%, were determined after 24, 48, or 72 hours at 37℃, respectively, using the MTT method.

Determination of Apoptosis by Annexin V and Propidium Iodide Staining

Fluorescence intensity was analyzed using a flow cytometer (exposure of apoptotic cells was measured by adding Annexin V-FITC; Immunotech Coulter). The addition of PI can distinguish apoptoticcells (Annexin positive and PI-negative) from necrotic cells (Annexin-negativeand PI-positive). The HCT116 human colon cancer cells (2 × 10⁵ cells) were seeded on 6-well plates and incubated for 12 hours when cells are at 70% to 80% confluency. Then, cells were treated with hybrid 11b (0, 5, 10, 20 μM) in a dose-dependent manner for 24 hours at 37℃ in a CO₂ incubator, and DMSO was used in the control group. Supernatants were removed from culture dishes and adherent cells detached with trypsin-EDTA. The cells were collected by centrifugation, washed with PBS, and resuspended using 1 × binding buffer at 1 × 10⁶ cells/mL. Then, 100 mL of the cell suspension (1 × 10⁵ cells) was transferred to a 5 mL culture tube and incubated with 5 mL Annexin V-FITC and 5 mL PI for 15 minutes at room temperature in the dark. Then, 400 mL of 1 × binding buffer was added in a 5 mL culture tube.

Western Blot Analysis

After chemical treatment, cells were washed 3 times with PBS and lysed for 30 minutes at 4°C, followed by centrifugation at 15 000 r/min for 10 minutes at 4°C. Protein concentrations of the supernatants were assayed using the BCA protein assay kit.

According to our method in previous report, ² accoraliquots of supernatants were added to SDS denaturing buffer and boiled for 10 minutes and transferred electrophoretically onto PVDF membranes. Membranes were blocked with 5% nonfat dry milk in TTBS (Tris-buffered saline containing 0.1% Tween 20) for 1 hour at room temperature with shaking and washed 4 times with TTBS before overnight incubation with the primary antibody at 4°C. Membranes were washed 4 to 5 times, 20 minutes each, with TTBS and then incubated with the secondary antibody conjugated to horseradish peroxidase for 1 hour at room temperature. Proteins were detected with an enhanced chemiluminescence reagent and visualized by ChemiDoc MP imaging system and quantified by densitometry using Quantity One 4.52 software.

Supplemental Material

Supplementary material - Supplemental material for Design, Synthesis, and Anticancer Activity of Natural Product Hybrids With Paclitaxel Side Chain Inducing Apoptosis in Human Colon Cancer Cells

Supplemental material, Supplementary material, for Design, Synthesis, and Anticancer Activity of Natural Product Hybrids With Paclitaxel Side Chain Inducing Apoptosis in Human Colon Cancer Cells by Ling-Li Zheng, Guan Wen, Yun-Xin Yao, Xiao-Huan Li and Feng Gao in Natural Product Communications