Synthesis and Structure–Activity Relationship of Salvinal Derivatives as Potent Microtubule Inhibitors

Abstract

Salvinal is a natural lignan isolated from the roots of Salvia mitorrhiza Bunge (Danshen). Previous studies have demonstrated its anti-proliferative activity in both drug-sensitive and -resistant cancer cell lines, with IC₅₀ values ranging from 4–17 µM. In this study, a series of salvinal derivatives was synthesized and evaluated for the structure–activity relationship. Among the twenty-four salvinal derivatives, six compounds showed better anticancer activity than salvinal. Compound 25 displayed excellent anticancer activity, with IC₅₀ values of 0.13–0.14 µM against KB, KB-Vin10 (overexpress MDR/Pgp), and KB-7D (overexpress MRP) human carcinoma cell lines. Based on our in vitro microtubule depolymerization assay, compound 25 showed depolymerization activity in a dose-dependent manner. Our findings indicate that compound 25 is a promising anticancer agent with depolymerization activity that has potential for the management of malignance.

1. Introduction

The dynamic equilibrium between tubulin and microtubules refers to the balance between the assembly and disassembly of microtubules, which is critical for various cellular processes, such as cell shape maintenance, intracellular transport, and cell division [1,2]. Microtubules are long, tubular structures made up of αβ-tubulin dimers that polymerize to form a rigid yet dynamic network within the cell; this becomes a significant component of the cytoskeleton [3,4,5]. Microtubules play a crucial role in the cell cycle, where they form the spindle apparatus that separates the chromosomes during mitosis [3,6]. As a result, microtubules have become a popular target for cancer therapies, as their disruption can prevent cancer cell proliferation [1,3,6]. Tubulin-binding agents that interfere with microtubule systems are commonly used in the treatment of hematological malignancies and solid tumors [4]. These agents can be classified into two categories based on their mechanism of action and the effects they have on microtubule polymerization [1,4,7]. The first category comprises drugs that inhibit polymerization, such as the vinca alkaloids [4]. These drugs bind to tubulin and prevent it from polymerizing into microtubules [1,4,7]. This disrupts the formation of the spindle fibers required for proper cell division, leading to cell death [1,3,4]. The second category comprises drugs that stabilize microtubules, such as taxanes and epothilones [4]. These drugs bind to microtubules and enhance their stability, which results in prolonged mitotic arrest and eventual cell death [1,3,4]. Taxanes and epothilones act by binding to the microtubule plus-end, inhibiting depolymerization and resulting in the accumulation of microtubules [4,8]. Tubulin-binding agents are a class of compounds primarily derived from natural sources; they encompasses a vast array of agents exhibiting a diverse range of chemical structures, such as paclitaxel, epothilone A, vinblastine, combretastatin A-4, colchicine, dolas-tatin 10, and chamaecypanone C [4,7,9,10,11]. Despite the differences in their structures, these agents all share a common characteristic—the ability to interfere with the dynamics of microtubules [1,7,10]. This interference can lead to a number of cellular effects, including mitotic arrest and cell death [1,3,10].

Salvinal, 5-(3-hydroxypropyl)-7-methyoxy-2-(3′-methoxy-4′-hydroxyphenyl)-3-benzo[b] furancarbaldehyde, which is originally extracted from the roots of Salvia miltiorrhizae Bunge, has been shown to possess strong cytotoxic properties against the progression of tumors [12]. Salvinal’s anticancer mechanism is associated with its ability to inhibit microtubules in both drug-sensitive and drug-resistant cell lines by binding to the colchicine binding domain of tubulin [12]. The promising anticancer properties of salvinal have generated considerable interest in creating and enhancing its therapeutic potential through the development of various derivatives or analogs using chemical synthesis methods. This study aims to design and synthesize several salvinal derivatives, assess their structure–activity relationships (SARs), and identify the most promising lead compounds for further investigation.

2. Results and Discussion

In dividing cells, microtubules are organized into the mitotic spindle, which is crucial for proper chromosome segregation during cell division [1]. Microtubule-binding agents are a class of drugs that target microtubules and have been proven to be highly effective in treating certain types of cancer [4]. These drugs function by either stabilizing or destabilizing microtubules, resulting in disrupted mitotic spindle formation, cell cycle arrest and, ultimately, cell death [4]. In our previous research, we discovered promising antitubulin compounds derived from various sources, including natural and synthetic products. One of these leading compounds, salvinal, was extracted from the roots of S. miltiorrhizae using chloroform. However, due to the low concentrations of active compounds found in plants, it is necessary to synthesize larger quantities of these compounds for further evaluation of their biological activity.

In this current study, we synthesized a series of derivatives of salvinal to evaluate their structure-activity relationships (SARs). Our findings indicate that compound 25 exhibited the most potent anticancer activity and is therefore a promising candidate for further investigation.

2.1. Synthesis of Salvinal Derivatives

Previously, we reported a synthetic route of salvinal using isoeugenol as a starting material, as reacted with iodobenzene diacetateuse (IDA) in a four-step reaction with a yield of 23% [13]. The convenient synthesis method shortens the preparation procedure of salvinal and benzofuranlignan derivatives. The synthesis steps of salvinal derivatives are shown in Scheme 1. Isoeugenol as a starting material was reacted with iodobenzene diacetateuse (IDA) to obtain salvinal (4) in a four-step reaction (Scheme 1). Various substitutions in the C-3 and C-5 positions of the benzofuran skeleton were synthesized and evaluated for their impact on anticancer activity. In order to examine the influence of phenyl substituent at the C-2 position of compound 1, we used compound 1 as the template for further alkylation (that resulted in compounds 5–9) and acylation reactions (that resulted in compounds 10–12). Compound 1 was treated with DDQ (1.1 eqa) in the mixture solvent of CH₂Cl₂/H₂O=4/1 to initiate a reaction, which could selectively oxidize arylpropene to arylpropenal, forming compound 13 under the room temperature. Compound 2 was further oxidized with SeO₂ in refluxing EtOH to yield compound 14. The structural difference between compound 14 and salvinal (4) is the side chain at the C-5 position, and the anticancer activities of the two are only slightly different. To evaluate the impact of phenyl substituent at the C-2 position, alkylation reaction was conducted with compound 14 in order to obtain compound 15, as shown in Scheme 1.

Oxidative coupling of methyl ferulate with IDA generated benzofuran compound 16 (Scheme 2). Compound 16 was further acylated to obtain compounds 17–20. Dehydrogenation of compound 17 using DDQ in 1,4-dioxane under reflux afforded compound 21. Compound 21 was dissolved in the mixture solvent of CH₃OH and AcOH with 10% Pd/C as catalysis under a hydrogen atmosphere to yield compound 22 through both hydrogenation and hydrolysis reactions. Benylation of compound 22 using benzyl bromide and K₂CO₃ in acetone under reflux afforded compound 23 in a high yield of 85%. By using LAH in dry THF, two methyl carboxylates were reduced to hydroxymethyl to form compound 24. We also used Ag₂O for the oxidative coupling of methyl caffeate to form compound 25, which was then acetylated to form compound 26.

2.2. Structure–Activity Relationship

Salvinal consists of a benzofuran skeleton with a phenyl moiety at the C-2 position. Here, 24 salvinal derivatives with various substitutions in the C-2, 3, 5, and 7 positions were prepared for their structure–activity relationship study. The antiproliferative effect of salvinal derivatives was evaluated by methylene blue assay in two epithelial tumor cell lines (KB and HONE-1). The anticancer activity of salvinal derivatives was compared with compounds 1 and 4 (salvinal). The IC₅₀ of compound 1 and salvinal against KB cells was 5.6 μM and 5.0 μM, respectively. The IC₅₀ of salvinal derivatives against KB and HONE-1 cells is shown in

Table 1. Growth inhibition of salvinal derivatives against human cancer cells.

Compound	IC50 Values a
	KB (μM) b	HONE-1 (μM)
1	5.6 ± 0.1	5.7 ± 0.1
2	6.9 ± 0.2	n.d.
4	5.0 ± 0.1	4.5 ± 0.1
5	21.6 ± 2.7	28.6 ± 5.0
6	40.0 ± 7.3	n.d.
7	42.9 ±3.4	n.d.
8	33.0 ± 1.4	n.d.
9	13.1± 0	n.d.
10	16.1 ± 0.07	n.d.
11	28.1 ± 3.3	33.8 ± 0.3
12	25.4 ± 3.46	n.d.
13	n.d.	6.8 ± 0.5
14	4.8 ± 1.1	4.5 ± 0.1
15	14.9 ± 1.4	28.6 ± 5.0
16	13.8 ± 0.1	12.8 ± 2.1
17	6.7 ± 0.4	3.7 ± 0.1
18	2.0 ± 0.1	2.1 ± 0.1
19	1.2 ± 0.2	1.6 ± 0.1
20	4.9 ± 0.3	5.0 ± 0.1
21	30.3 ± 0.4	n.d.
22	37.8 ± 0.5	45.6 ± 2.4
23	30.8 ± 4.8	39.6 ± 0.5
24	22.0 ± 6.2	25.0 ± 2.8
25	0.137 ± 0.008	0.316 ± 0.025
26	0.326 ± 0.037	0.643 ± 0.053
VP-16	1.1 ± 0.2 c	n.d.

^a Cells were treated with various concentrations of test compounds for 72 h. Cell survival was determined by methylene blue assay. Each IC₅₀ value was calculated as described in Experimental Method. Each value represents the mean ± S.D. of three independent experiments. ^b The KB cell line was originally derived from an epidermal carcinoma of the mouth but has now been shown to have HeLa characteristics. ^c The IC₅₀ value of VP-16 for KB cell lines is derived from our previous publication [12]. n.d. —not done.

. The results of the anticancer activity against KB cells revealed that of the 24 salvinal derivatives in this series, except for compound 13, two (compounds 25 and 26) showed IC₅₀ less than 0.4 μM, four showed IC₅₀ in the range of 0.4–5.0 μM, three showed IC₅₀ in the range of 5–10 μM, twelve showed IC₅₀ in the range of 10–38 μM, and two showed IC₅₀ more than 38 μM. Six compounds (14, 18, 19, 20, 25, and 26) (Figures S1–S16) showed more potent anticancer activity than salvinal. Compound 25 showed the best anticancer activity against KB cells, with IC₅₀ values of 0.137 μM compared to salvinal, with IC₅₀ of 5.0 μM. The test compounds exhibited similar IC₅₀ values against both KB and HONE-1 cells, which indicated that they have similar anticancer activity against these two cells.

The newly synthesized benzofuran compounds can be divided into two subclasses, benzofuran and dihydrobenzofuran, as shown in Figure 1. In the dihydrobenzofuran series compounds, by using compound 1 as the template for further modification, we found that the change of C-4′ position of 2-phenyl portion, either to the ether (compounds 5–9) or the ester (compounds 10–12) substituents, decreased the inhibitory activity; the trend almost paralleled the increase in carbon number of substituents in compounds 5–8 and 10–12. This observation suggests that hydroxyl group of the C-4′ position on the phenyl ring is an important functional group for the anticancer activity against KB cells. Modification of the C-5 position with a propenal group (compound 13) resulted in a comparable anticancer activity to the parental compound 1 containing a propenyl group. Interestingly, when the C-3 and C-5 positions were substituted by acrylic methyl and carboxyl methyl esters (compound 16), respectively, the IC₅₀ value was larger compared to compound 1. The C-4′ position of compound 16 was esterified to the corresponding ester compounds 17–20, most of which showed better anticancer activity than salvinal (4). We found that the change of the C-2 substituent with a 3,4-dihydroxyphenyl moiety (compound 25) showed the best anticancer activity in this series of compounds, indicating that the catechol moiety is an important functionality for anticancer potency. Compound 25 acetylated to form compound 26 also showed good anticancer activities. Phenolic esters with triacyl groups exhibited lower polarity than the original phenols, and they will express more penetrable potency through cell membrane. The essential functionality was proposed as the catechol moiety, because triacetyl groups can be hydrolyzed to the original trihydroxyl groups by esterase in the internals of cancer cells.

In the benzofuran series compounds, modification of the C-2 position with a methyl group (2) or the C-5 position with a propenal group (14) showed comparable anticancer activity to salvinal (4). The C-3 and C-5 positions were substituted by the acrylic methyl and acetyl groups, respectively, as well as the acetylation of the C-4′ position (21), showing obviously weaker anticancer activity than compound 4. The modification of the C-4′ position with a benzyloxyl group, together with the C-5 position with a propenal (15), an acrylic methyl (23), or a propanol (24) group, decreased their cytotoxic activity.

Based on our SAR analysis, we can conclude that the change of the benzyloxyl group on the benzofuran or dihydrobenzofuran backbone (compound 9) showed better anticancer activity in KB cells than the alkyloxy analogs (as compounds 5, 6, 7, and 8) but weaker anticancer activity than original hydroxyl compound (as compound 1). Further formation of acyl substituents on the C-4′ position of the benzene ring from 16 could improve the anticancer activity (compared compound 16 with compounds 17, 18, 19, and 20). Additionally, substitution of the methyl group at C-3 and the 1-propenyl group at C-5 (compound 1) with carboxylic methyl ester and acrylic methyl ester (compound 16), respectively, would attenuate the cytotoxic activity. The hydroxyl group of the C-4′ position in compound 16 was acylated as ethanoate (17), benzoate (18), butanoate (19), and isobutanoate (20), increasing the potential for cytotoxic activity. Furthermore, comparing the anticancer activity between compounds 21 and 17 indicated that dihydrobenzefuran derivatives had more cytotoxic activity than benzofuran derivatives.

2.3. Drug Resistance Analysis

Drug resistance is a serious problem that restricts the use of microtubule-interfering drugs for clinical therapy [14]. We selected compounds 4, 19, 20, 25, and 26 of salvinal derivatives to further examine the efficacy against KB and KB drug-resistant cell lines. The IC₅₀ of compounds 4, 19, 20, 25, and 26 against KB, KB-Vin10, and KB-7D cells is shown in

Table 2. Growth inhibition of salvinal derivatives against KB-derived resistance cancer cell lines.

Compound	KB (μM) a	KB-VIN10 (μM)	R.I. b	KB-7D (μM)	R.I.
	(Parental)	(MDR+)		(MRP+)
Vincristine	0.4 ± 0.1 (nM)	90.1 ± 7.4 (nM)	225.2	1.2 ± 0.4 (nM)	3
VP-16 c	1.1 ± 0.2	23 ± 3	20.9	54 ± 3.5	49.1
4d	5.0 ± 0.1	3.7 ± 0.1	0.7	3.4 ± 0.2	0.7
19	1.2 ± 0.2	0.7 ± 0	0.6	0.5 ± 0.2	0.4
20	4.9 ± 0.3	2.8 ± 0.2	0.6	2.4 ± 0.1	0.5
25	0.137± 0.008	0.141 ± 0.003	1.0	0.138 ± 0.004	1.0
26	0.326 ± 0.037	0.269 ± 0.032	0.8	0.251 ± 0.007	0.8

^a Cells were treated with various concentrations of test compounds for 72 h. Cell survival was determined by methylene blue assay. Each IC₅₀ value was calculated as described in Experimental Method. ^b Resistant index. ^c The IC₅₀ values of vincristine and VP-16 for all three cell lines are derived from our previous publication [12]. ^d The IC₅₀ values of Compound 4 and its derivatives (Compound 19, 20, 25, and 26) for KB parental cell lines are reported in Table 1.

. The data we obtained indicate that compounds 4, 19, 20, 25, and 26 possess a certain level of inhibitory activity against the proliferation of cancer cell lines KB and HONE1. Table 2 reveals that the results obtained for the HONE-1 cell line were highly consistent with the findings we observed in KB cells [15]. Despite overexpression of drug -resistant efflux protein (MDR/Pgp or MRP) in KB-Vin10 and KB-7D cells, the compounds 4, 19, 20, 25, and 26 showed comparative cytotoxic activity for both the parental cell line and MRP- or MDR-overexpressing counterparts. Compounds 4, 19, 20, 25, and 26 manifest similar potency, regardless of the cell’s MDR or MRP status, suggesting that they are not substrates for these efflux pumps.

2.4. Inhibition of Tubulin Polymerization

In this study, the depolymerization activity of compounds 4, 19, 20, 25, and 26 on pure MAP-rich tubulins were assessed in vitro. As shown in Figure 2, compounds 4, 19, 20, and 25 demonstrated a concentration-dependent inhibition of tubulin polymerization, while compound 26 did not affect the microtubule assembly.

The findings of our study are intriguing. The compounds 4, 19, 20, and 25 and colchicine have been found to depolymerize microtubules in vitro in a dose-dependent manner. It is hypothesized that compounds 4, 19, 20, and 25 directly bind to tubulin/microtubules, leading to their depolymerization. However, it is noteworthy that compound 26 did not show any effect on the microtubule assembly. This suggests that the mechanism of action for its anticancer properties may be different from the other compounds. Therefore, further studies are needed to investigate the specific pathways through which these compounds exert their anticancer effects.

3. Experimental Method

3.1. General

The Infrared (IR) spectra were measured on a Nicolet MAG NA-IR 550 Spectrometer Series Ⅱ (Nicolet, Madison, WI, USA). The NMR Spectra were collected using a Brucker M-300 WT FT-300 (¹H-NMR: 300 MHz, ¹³C-NMR: 75 MHz)(Bruker, Fällanden, Switzerland) with CDCl₃ as the solvent. The EI-MS data were collected using a JEOL JMS-HX 300 Mass spectrometer (JEOL, Tokyo, Japan). Silica gel (Merck 70-230 mesh ASTM) was used for the column chromatography, and pre-coated silica gel (Merck 60 F-254) plates were used for the TLC analyses.

3.2. Chemistry

The salvinal derivatives were synthesized at the laboratory of Professor Yueh-Hsiung Kuo of the Tsuzuki Institute for Traditional Medicine, China Medical University (Taichung, Taiwan).

Compound 1 was prepared from isoeugenol by using IDA (iodobenzene diacetate). The solution of isoeugenol (10.0 g in 100 mL of CH₂Cl₂) was added dropwise to the solution of IDA (10.0 g, 30.2 mmol) in 100 mL of CH₂Cl₂ (dry with CaH₂) at room temperature for 4 h. After 48 h, NaHCO₃ (3 g) was added to the solution and stirred for 1 h. The mixture was filtrated, and the filtrate was evaporated under reduced pressure to give a yellow oil. Then, the residue was purified by Si gel column chromatography to give 1 as a colorless solid (3.9 g, 40% yield; with solvent system EtOAc: hexane = 1:9); mp 123–124 °C; ¹H-NMR (CDCl₃) δ_H 6.96 (s, 1H, H-4), 6.88 (d, J = 8.1 Hz, 1H, H-6′), 6.86 (d, J = 8.1 Hz, 1H, H-5′), 6.77 (s, 1H, H-6), 6.75 (s, 1H, H-2′), 6.35 (dd, J = 15.6, 1.0 Hz, 1H, H-1″), 6.09 (dq, J = 15.6, 6.6 Hz, 1H, H-2″), 5.64 (s, 1H, 4′-OH), 5.08 (d, J = 9.5 Hz, 1H, H-2), 3.87 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.43 (m, 1H, H-3), 1.85 (dd, J = 6.6, 1.0 Hz, 3H, H-3″), 1.36 (d, J = 6.8 Hz, 3H, Me-C-3); ¹³C-NMR (CDCl₃) δ_C 146.5 (C-7), 146.4 (C-3′), 145.6 (C-7a), 143.9 (C-4′), 137.2 (C-1′), 132.0 (C-3a), 131.9 (C-5), 130.8 (C-1″), 123.2 (C-6′), 119.7 (C-2″), 114.0 (C-4), 113.2 (C-2′), 109.1 (C-5′), 108.8 (C-6), 93.6 (C-2), 55.7 (C-7-OMe), 51.7 (C-3′-OMe), 45.4 (C-3), 18.2 (C-3″), 17.4 (C-3-Me); IR (KBr film) ν_max 3446, 3023, 2964, 1608, 1515, 1460, 1337, 1275, 1141, 1033, 962 cm⁻¹; EI-MS m/z (%) (70 eV) 326 (M⁺, 45; C₂₀H₂₂O₄), 202 (30), 178 (45), 151 (100), 137 (8), 119 (11), 91 (15).

Compound 2 was prepared from 1 by using DDQ (dichlorodicyanobenzoquinone). Compound 1 (2.12 g) and DDQ (3.24 g) were resolved in 50 mL of 1,4-dioxane. The solution was then refluxed. After 48 h, the solution was filtrated, and the filtrate was evaporated under reduced pressure. Then, the residue was purified by Si gel column chromatography to give 2 as a colorless solid (1.81 g, 83% yield; with solvent system EtOAc: hexane = 1:8); mp 221–222 °C; ¹H-NMR (CDCl₃) δ_H 9.68 (d, J = 7.6 Hz, 1H, H-3″), 7.55 (d, J = 15.9 Hz, 1H, H-1″), 7.26–7.30 (m, 3H, H-4, H-6, H-6′), 6.99 (d, J = 8.0 Hz, 1H, H-5′), 6.97 (s, 1H, H-2′), 6.70 (dd, J = 15.9, 7.6 Hz, 1H, H-2″), 5.85 (br s, 1H, Ph-OH), 4.03 (s, 3H, MeO-C-7), 3.95 (s, 3H, MeO-C-3′), 2.40 (s, 3H, Me-C-3); ¹³C-NMR (CDCl₃) δ_C 193.6, 153.9, 146.7, 146.2, 145.2, 144.6, 133.5, 129.6, 127.4, 123.0, 120.8, 114.6, 113.8, 110.1, 109.4, 105.6, 56.1, 9.5; IR (KBr film) ν_max 3540, 2949, 2811, 2727, 1674, 1616, 1513, 1214, 1128, 972 cm⁻¹; EI-MS m/z (%) (70 eV) 338 (M⁺, 100; C₂₀H₁₈O₅), 326 (21), 310 (100), 295 (14), 267 (15), 151 (13), 137 (14), 69 (17), 57 (19).

Compound 3 was prepared from 2 using Adam’s catalyst reduction reaction. The solution of 2 (1.33 g in 20 mL of CH₃OH) with 10% PtO₂/H₂O (96.3 mg) was stirred under H₂ at room temperature. After 6 h, the mixture was filtrated, and the filtrate was evaporated under reduced pressure. Then, the residue was purified by Si gel column chromatography to give 3 as a colorless solid (1.28 g, 95% yield; with solvent system EtOAc: hexane = 2:5); mp 165–166 °C; ¹H-NMR (CDCl₃) δ_H 7.30 (d, J = 2.0 Hz, 1H, H-4), 7.26 (dd, J = 8.2, 1.6 Hz, 1H, H-6′), 6.98 (d, J = 8.2 Hz, 1H, H-5′), 6.91 (d, J = 1.0 Hz, 1H, H-2′), 6.63 (d, J = 2.0 Hz, 1H, H-6), 5.73 (s, 1H, Ph-OH), 4.01 (s, 3H, MeO-C-7), 3.96 (s, 3H, MeO-C-3′), 3.71 (t, J = 6.5 Hz, 2H, H-3″), 2.79 (t, J = 7.9 Hz, 2H, H-1″), 2.38 (s, 3H, Me-C-3), 1.95 (m, 2H, H-2″); ¹³C-NMR (CDCl₃) δ_C 151.4, 146.6, 145.7, 144.7, 136.9, 133.0, 123.8, 120.6, 114.5, 110.8, 109.5, 107.4, 62.4, 56.1, 34.8, 32.6, 9.60; IR (KBr film) ν_max 3431, 2940, 2851, 1602, 1516, 1455, 1386, 1222, 1052, 793 cm⁻¹; EI-MS m/z (%) (70 eV) 342 (M⁺, 5; C₂₀H₂₂O₅), 340 (100), 324 (37), 312 (26), 297 (19), 284 (20), 148 (13), 97 (16), 91 (18), 69 (23), 57 (28).

Compound 4 was prepared from 3 using SeO₂ oxidative reaction. The solution of 3 (0.64 g in 20 mL of EtOH) with SeO₂ (0.42 g) was refluxed. After 12 h, the mixture was evaporated under reduced pressure. Then, 30 mL of EtOAc was added, the mixture was filtrated by celite, and the filtrate was evaporated under reduced pressure. Then, the residue was purified by Si gel column chromatography to give 4 as a colorless solid (0.48 g, 72% yield; with solvent system EtOAc: hexane = 1:5); mp 173–174 °C; ¹H-NMR (CDCl₃) δ_H 10.25 (s, 1H, CHO), 7.64 (s, 1H, H-2′), 7.37 (d, J = 8.0 Hz, 1H, H-6′), 7.35 (s, 1H, H-4), 7.04 (d, J = 8.0 Hz, 1H, H-5′), 6.73 (s, 1H, H-6), 6.11 (br s, 1H, Ph-OH), 4.00 (s, 3H, OMe), 3.97 (s, 3H, OMe), 3.69 (t, J = 6.5 Hz, 2H, H-3″), 2.80 (t, J = 7.3 Hz, 2H, H-1″), 1.94 (m, 2H, H-2″); ¹³C-NMR (CDCl₃) δ_C 186.8, 165.9, 148.7, 146.9, 144.6, 141.6, 139.9, 127.3, 123.7, 120.6, 116.7, 115.0, 113.5, 111.0, 108.8, 62.2, 56.3, 56.1, 34.7, 32.5; IR (KBr film) ν_max 3513, 3435, 2940, 2864, 1637, 1601, 1522, 1490, 1409, 1273, 1139, 1061, 818 cm⁻¹; EI-MS m/z (%) (70 eV) 356 (M⁺, 60; C₂₀H₂₀O₆), 312 (100), 269 (7), 197 (6), 152 (6), 137 (6), 126 (4), 105 (4), 91 (4), 55 (4) (Figures S1–S4).

Compound 5 was prepared from 1 using alkylation reaction. MeI (66.2 mg) and K₂CO₃ (100.3 mg) were added to the solution of 1 (100.4 mg, in 10 mL of acetone), and then the solution was refluxed. After 6 h, the mixture was filtrated, and the filtrate was evaporated under reduced pressure. Then, the residue was purified by Si gel column chromatography to give 5 as a colorless solid (92.8 mg, 88% yield; with solvent system EtOAc: hexane = 1:9); mp 118–119 °C; ¹H-NMR (CDCl₃) δ_H 6.96 (s, 1H, H-4), 6.94 (dd, J = 8.1, 1.7 Hz, 1H, H-6′), 6.82 (d, J = 8.1 Hz, 1H, H-5′), 6.77 (s, 1H, H-6), 6.75 (s, 1H, H-2′), 6.34 (br d, J = 15.8 Hz, 1H, H-1″), 6.09 (dq, J = 15.8, 6.6 Hz, 1H, H-2″), 5.09 (d, J = 9.5 Hz, 1H, H-2), 3.87 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.85 (s, 3H, OMe), 3.44 (m, 1H, H-3), 1.85 (dd, J = 6.6, 1.3 Hz, 3H, H-3″), 1.36 (d, J = 6.9 Hz, 3H, Me-C-3); ¹³C-NMR (CDCl₃) δ_C 149.1, 146.5, 144.1, 137.4, 133.2, 132.6, 132.2, 130.9, 130.2, 127.4, 123.4, 119.2, 113.3, 110.8, 109.5, 109.2, 93.6, 55.9, 45.5, 18.3, 17.6; IR (KBr film) ν_max 3014, 2963, 2882, 1604, 1517, 1464, 1270, 1151, 1023, 957, 855, 817 cm⁻¹; EI-MS m/z (%) (70 eV) 340 (M⁺, 28; C₂₁H₂₄O₄), 204 (25), 192 (40), 168 (28), 165 (100), 153 (27), 125 (13), 81 (14), 77 (23), 69 (29).

Compound 6 was prepared from 1 using alkylation reaction; the reaction was similar to the preparation of 5. Compound 1 (100.4 mg) gave 6 as a colorless solid (93.4 mg, 85% yield; with solvent system EtOAc: hexane = 1:9); mp 106–107 °C; ¹H-NMR (CDCl₃) δ_H 6.96 (s, 1H, H-4), 6.91 (dd, J = 8.1, 1.9 Hz, 1H, H-6′), 6.82 (d, J = 8.1 Hz, 1H, H-5′), 6.77 (s, 1H, H-6), 6.75 (d, J = 1.9 Hz, 1H, H-2′), 6.35 (dq, J = 15.6, 1.4 Hz, 1H, H-1″), 6.09 (dq, J = 15.6, 6.2 Hz, 1H, H-2″), 5.09 (d, J = 9.3 Hz, 1H, H-2), 4.08 (q, J = 6.9 Hz, 2H, OCH₂CH₃), 3.87 (s, 3H, OMe), 3.84 (s, 3H, OMe), 3.45 (m, 1H, H-3), 1.85 (dd, J = 6.2, 1.4 Hz, 3H, H-3″), 1.44 (t, J = 6.9 Hz, 3H, OCH₂CH₃), 1.37 (d, J = 6.7 Hz, 3H, Me-C-3); ¹³C-NMR (CDCl₃) δ_C 149.4, 148.4, 146.5, 144.1, 137.4, 133.2, 132.5, 132.1, 130.9, 130.2, 127.4, 123.4, 119.2, 113.3, 112.3, 109.8, 109.2, 93.6, 64.3, 55.9, 55.8, 45.5, 18.3, 17.6, 14.7; IR (KBr film) ν_max 3011, 2965, 2884, 1600, 1517, 1461, 1339, 1227, 1031, 957, 856 cm⁻¹; EI-MS m/z (%) (70 eV) 354 (M⁺, 14; C₂₂H₂₆O₄), 266 (14), 206 (80), 179 (100), 151 (89), 119 (10), 91 (17), 77 (13).

Compound 7 was prepared from 1 using alkylation reaction; the reaction was similar to the preparation of 5. Compound 1 (100.4 mg) afforded 7 as a colorless solid (94.8 mg, 83% yield; with solvent system EtOAc: hexane = 1:9); mp 109–110 °C; ¹H-NMR (CDCl₃) δ_H 6.96 (s, 1H, H-4), 6.91 (dd, J = 8.1, 1.1 Hz, 1H, H-6′), 6.81 (d, J = 8.1 Hz, 1H, H-5′), 6.76 (s, 1H, H-6), 6.75 (s, 1H, H-2′), 6.35 (dq, J = 15.6, 1.0 Hz, 1H, H-1″), 6.09 (dq, J = 15.6, 6.6 Hz, 1H, H-2″), 5.09 (d, J = 9.5 Hz, 1H, H-2), 3.95 (t, J = 6.4 Hz, 2H, OCH₂CH₂CH₃), 3.87 (s, 3H, OMe), 3.84 (s, 3H, OMe), 3.44 (m, 1H, H-3), 1.78–1.88 (m, 5H, H-3″, OCH₂CH₂CH₃), 1.33 (d, J = 6.4 Hz, 3H, Me-C-3), 1.01 (t, J = 7.4 Hz, 3H, OCH₂CH₂CH₃); ¹³C-NMR (CDCl₃) δ_C 149.4, 148.6, 146.5, 144.1, 133.2, 132.5, 132.1, 130.9, 130.2, 123.3, 119.1, 113.2, 112.5, 109.9, 109.2, 93.6, 70.4, 55.9, 55.8, 45.5, 29.6, 22.4, 18.3, 17.5, 10.3; IR (KBr film) ν_max 2965, 2884, 1600, 1518, 1462, 1267, 1145, 1031, 957, 856, 807 cm⁻¹; EI-MS m/z (%) (70 eV) 368 (M⁺, 27; C₂₃H₂₈O₄), 280 (12), 220 (40), 193 (44), 178 (23), 151 (100), 140 (33), 97 (32), 77 (44), 57 (63).

Compound 8 was prepared from 1 using alkylation reaction; the reaction was similar to the preparation of 5. Compound 1 (100.4 mg) obtained 8 as a colorless solid purified on SiO₂ column chromatography (114.2 mg, 82% yield; with solvent system EtOAc: hexane = 1:9); mp 107–108 °C; ¹H-NMR (CDCl₃) δ_H 6.96 (s, 1H, H-4), 6.91 (dd, J = 8.1, 1.1 Hz, 1H, H-6′), 6.84 (d, J = 8.1 Hz, 1H, H-5′), 6.77 (s, 1H, H-6), 6.75 (d, J = 1.1 Hz, 1H, H-2′), 6.35 (d, J = 15.6 Hz, 1H, H-1″), 6.09 (dq, J = 15.6, 6.5 Hz, 1H, H-2″), 5.09 (d, J = 9.4 Hz, 1H, H-2), 4.50 (m, 1H, OCH(CH₃)₂), 3.87 (s, 3H, OMe), 3.84 (s, 3H, OMe), 3.46 (m, 1H, H-3), 1.85 (d, J = 6.5 Hz, 3H, H-3″), 1.36 (d, J = 7.7 Hz, 3H, Me-C-3), 1.34 (d, J = 6.3 Hz, 6H, OCH(CH₃)₂); ¹³C-NMR (CDCl₃) δ_C 150.4, 147.3, 144.9, 133.2, 132.9, 132.1, 130.9, 123.3, 119.1, 115.4, 113.2, 110.3, 109.2, 93.6, 71.4, 56.0, 55.9, 55.8, 45.4, 21.9, 18.3, 17.6; IR (KBr film) ν_max 2975, 2935, 1603, 1508, 1461, 1333, 1269, 1138, 1035, 958, 856, 820 cm⁻¹; EI-MS m/z (%) (70 eV) 368 (M⁺, 5; C₂₃H₂₈O₄), 326 (8), 280 (10), 220 (12), 178 (59), 151 (100), 140 (28), 91 (9), 71 (8), 57 (13).

Compound 9 was prepared from 1 using alkylation reaction; the reaction was similar to the preparation of 5. Compound 1 (100.4 mg) yielded 9 as a colorless solid on SiO₂ column chromatography (103.3 mg, 80% yield; with solvent system EtOAc: hexane = 1:9); mp 165–166 °C; ¹H-NMR (CDCl₃) δ_H 7.27–7.42 (m, 5H, OCH₂Ph), 6.98 (s, 1H, H-4), 6.86 (dd, J = 8.2, 1.5 Hz, 1H, H-6′), 6.82 (d, J = 8.2 Hz, 1H, H-5′), 6.76 (s, 1H, H-6), 6.74 (d, J = 1.5 Hz, 1H, H-2′), 6.34 (dq, J = 16.0, 1.2 Hz, 1H, H-1″), 6.05 (dq, J = 16.0, 6.7 Hz, 1H, H-2″), 5.14 (s, 2H, OCH₂Ph), 5.08 (d, J = 9.6 Hz, 1H, H-2), 3.87 (s, 3H, OMe), 3.84 (s, 3H, OMe), 3.44 (m, 1H, H-3), 1.85 (dd, J = 6.7, 1.2 Hz, 3H, H-3″), 1.35 (d, J = 6.8 Hz, 3H, Me-C-3); ¹³C-NMR (CDCl₃) δ_C 149.7, 148.1, 146.5, 144.0, 137.0, 133.1, 132.1, 130.8, 128.4, 127.7, 127.1, 123.3, 119.0, 113.6, 113.2, 110.0, 109.2, 93.5, 70.9, 55.9, 55.8, 45.4, 18.3, 17.6; IR (KBr film) ν_max 3069, 3002, 2961, 2871, 1602, 1516, 1460, 1268, 1220, 1141, 1031, 947, 742 cm⁻¹; EI-MS m/z (%) (70 eV) 416 (M⁺, 3; C₂₇H₂₈O₄), 328 (8), 268 (25), 241 (10), 177 (42), 151 (32), 139 (14), 91 (100), 65 (8).

Compound 10 was prepared from 1 using esteration reaction. CH₃COCl (0.3 mL) and Et₃N (0.4 mL) were added to the solution of 1 (107.6 mg in 10mL of CHCl₃), and then the solution was refluxed. After 3 h, the mixture was added to ice water (10 mL) and extracted by EtOAc (2 × 10 mL). The organic layers were combined and then washed with 1N HCl and aqueous NaHCO₃ and subsequently evaporated under reduced pressure. Then, the residue was purified by Si gel column chromatography to give 10 as a colorless solid (117.4 mg, 92% yield; with solvent system EtOAc: hexane = 1:9); mp 154–155 °C; ¹H-NMR (CDCl₃) δ_H 7.04 (s, 1H, H-4), 7.02 (d, J = 8.2 Hz, 1H, H-5′), 6.94 (dd, J = 8.2, 1.7 Hz, 1H, H-6′), 6.77 (s, 1H, H-6), 6.74 (s, 1H, H-2′), 6.34 (dq, J = 15.4, 1.1 Hz, 1H, H-1″), 6.09 (dq, J = 15.4, 6.6 Hz, 1H, H-2″), 5.14 (d, J = 9.1 Hz, 1H, H-2), 3.88 (s, 3H, OMe), 3.80 (s, 3H, OMe), 3.46 (m, 1H, H-3), 2.29 (s, 3H, OAc), 1.85 (dd, J = 6.6, 1.1 Hz, 3H, H-3″), 1.38 (d, J = 6.7 Hz, 3H, Me-C-3); ¹³C-NMR (CDCl₃) δ_C 168.9, 151.2, 146.4, 144.1, 139.6, 139.2, 133.0, 132.3, 130.8, 123.6, 122.6, 118.6, 113.3, 110.2, 109.3, 93.0, 55.9, 55.8, 45.7, 20.6, 18.3, 17.9; IR (KBr film) ν_max 2968, 2938, 2881, 1769, 1608, 1507, 1461, 1202, 1152, 1035, 966, 860 cm⁻¹; EI-MS m/z (%) (70 eV) 368 (M⁺, 37; C₂₂H₂₄O₅), 326 (100), 182 (19), 172 (38), 140 (60), 127 (42), 98 (23), 85 (46), 71 (24), 57 (29).

Compound 11 was prepared from 1 using esteration reaction; the reaction was similar to the preparation of 10 with propanoyl chloride and triethylamine. Compound 1 (121.1 mg) afforded 11 as a colorless solid (133.6 mg, 95% yield; with solvent system EtOAc: hexane = 1:9); mp 175–176 °C; ¹H-NMR (CDCl₃) δ_H 7.03 (s, 1H, H-4), 6.98 (d, J = 8.0 Hz, 1H, H-5′), 6.94 (dd, J = 8.0, 1.4 Hz, 1H, H-6′), 6.77 (s, 1H, H-6), 6.74 (d, J = 1.4 Hz, 1H, H-2′), 6.34 (dq, J = 15.6, 1.1 Hz, 1H, H-1″), 6.11 (dq, J = 15.6, 6.8 Hz, 1H, H-2″), 5.13 (d, J = 9.2 Hz, 1H, H-2), 3.88 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.45 (m, 1H, H-3), 2.59 (q, J = 7.4 Hz, 2H, CH₂CH₃), 1.86 (dd, J = 6.8, 1.1 Hz, 3H, H-3″), 1.38 (d, J = 6.8 Hz, 3H, Me-C-3), 1.25 (t, J = 7.4 Hz, 3H, CH₂CH₃); ¹³C-NMR (CDCl₃) δ_C 172.5, 151.2, 146.5, 144.1, 139.7, 139.1, 133.0, 132.4, 130.9, 123.6, 122.6, 118.7, 113.3, 110.3, 109.3, 93.1, 56.0, 45.8, 29.7, 27.3, 18.3, 17.9, 9.1; IR (KBr film) ν_max 2928, 2858, 1768, 1605, 1499, 1465, 1274, 1125, 1032, 954, 822, 759 cm⁻¹; EI-MS m/z (%) (70 eV) 382 (M⁺, 25; C₂₃H₂₆O₅), 326 (100), 314 (7), 199 (6), 149 (11), 97 (16), 85 (15), 71 (22), 57 (34).

Compound 12 was prepared from 1 using esteration reaction; the reaction was similar to the preparation of 10 with butanoyl chloride and triethylamine. Compound 1 (120.4 mg) gave 12 as a colorless solid (131.2 mg, 92% yield; with solvent system EtOAc: hexane = 1:9); mp 174–175 °C; ¹H-NMR (CDCl₃) δ_H 7.03 (s, 1H, H-4), 6.95–6.97 (m, 2H, H-5′, H-6′), 6.77 (s, 1H, H-6), 6.75 (s, 1H, H-2′), 6.35 (dq, J = 15.6, 1.0 Hz, 1H, H-1″), 6.10 (dq, J = 15.6, 6.6 Hz, 1H, H-2″), 5.14 (d, J = 9.2 Hz, 1H, H-2), 3.88 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.43 (m, 1H, H-3), 2.54 (t, J = 7.3 Hz, 2H, CH₂CH₂CH₃), 1.85 (dd, J = 6.6, 1.0 Hz, 3H, H-3″), 1.78 (sex, J = 7.3 Hz, 2H, CH₂CH₂CH₃), 1.39 (d, J = 6.8 Hz, 3H, Me-C-3), 0.99 (t, J = 7.3 Hz, 3H, CH₂CH₂CH₃); ¹³C-NMR (CDCl₃) δ_C 171.5, 151.1, 146.4, 144.0, 139.6, 139.0, 132.9, 132.3, 130.8, 123.4, 122.5, 118.5, 113.2, 110.2, 109.3, 93.0, 55.8, 55.7, 45.7, 35.7, 18.4, 18.2, 17.7, 14.0; IR (KBr film) ν_max 3018, 2968, 2879, 1772, 1607, 1512, 1123, 1031, 954, 826, 596, 537 cm⁻¹; EI-MS m/z (%) (70 eV) 396 (M⁺, 27; C₂₄H₂₈O₅), 326 (100), 238 (8), 178 (27), 151 (24), 140 (25), 126 (23), 85 (21), 71 (54).

Compound 13 was prepared from 1 using DDQ (dichlorodicyanobenzoquinone). Compound 1 (1.15 g, 3.50 mmol) and DDQ (0.870 g, 3.80 mmol) were dissolved in the mixture of CH₂Cl: H₂O = 4:1 (10 mL) and stirred for 48 h. After filtration, the product in the filtrate was purified using Si gel column chromatography. It gave compound 13 (1.05 g, 88% yield); mp 177–178°C; ¹H-NMR (CDCl₃) δ_H 9.64 (d, J = 7.6 Hz, 1H, H-3″), 7.41 (d, J = 15.8 Hz, 1H, H-1″), 7.02 (s, 1H, H-4), 6.99 (s, 1H, H-6), 6.97 (d, J = 8.1 Hz, 1H, H-6′), 6.89 (s, 1H, H-2′), 6.87 (1d, J = 8.1Hz, 1H, H-5′), 6.60 (dd, J = 15.8, 7.6 Hz, H-2″), 5.66 (s, 1H, ph-OH), 5.18 (d, J = 9.2 Hz, 1H, H-2), 3.50 (m, 1H, H-3), 1.40 (d, J = 6.8 Hz, 3H, C-3-Me); ¹³C-NMR (CDCl₃) δ_C 193.6, 153.2, 150.6, 146.7, 146.0, 144.6, 134.0, 131.2, 128.1, 126.3, 119.9, 117.3, 114.3, 111.8, 108.9, 94.5, 56.0, 55.9, 45.1, 17.7; IR (KBr film) ν_max 3486, 2985, 2852, 2851, 2734, 1684, 1620, 1478, 1133, 821 cm⁻¹; EI-MS m/z (%) (70 eV) 340 (M⁺, 100; C₂₀H₂₀O₅), 325 (7), 137 (15), 97 (15), 71 (18), 57 (30).

Compound 14 was prepared from 2 using SeO₂ oxidative reaction. The solution of 2 (212.4 mg in 20 mL of EtOH) with SeO₂ (0.14 g, 1.24 mmol) was refluxed. After 12 h, the mixture was evaporated under reduced pressure. Then, 30 mL of EtOAc was added, the mixture was filtrated by celite, and the filtrate was evaporated under reduced pressure. Then, the residue was purified by Si gel column chromatography to give 14 as a colorless solid (170.3 mg, 77% yield; with solvent system EtOAc: hexane = 1:5); mp 234–235 °C; ¹H-NMR (CDCl₃) δ_H 10.3 (s, 1H, OHC-C-3), 9.72 (d, J = 7.7 Hz, 1H, H-3″), 8.05 (d, J = 1.9 Hz, 1H, H-4), 7.58 (d, J = 15.9 Hz, 1H, H-1″), 7.41 (dd, J = 8.0, 1.8 Hz, 1H, H-6′), 7.37 (d, J = 1.9 Hz, 1H, H-6), 7.09 (d, J = 1.8 Hz, 1H, H-2′), 7.07 (d, J = 8.0 Hz, 1H, H-5′), 6.75 (dd, J = 15.9, 7.7 Hz, 1H, H-2″), 6.02 (br s, 1H, Ph-OH), 4.06 (s, 3H, OMe), 4.01 (s, 3H, OMe); ¹³C-NMR (CDCl₃) δ_C 194.3, 186.6, 165.7, 153.8, 150.4, 148.2, 145.0, 143.7, 132.3, 128.4, 127.4, 123.1, 118.5, 116.2, 116.0, 115.3, 112.4, 107.0, 56.2, 55.9; IR (KBr film) ν_max 3488, 2930, 2854, 2734, 1680, 1619, 1513, 1478, 1437, 1279, 1133, 1027, 822 cm⁻¹; EI-MS m/z (%) (70 eV) 352 (M⁺, 100; C₂₀H₁₆O₆), 323 (40), 296 (15), 281 (20), 253 (33), 181 (18), 165 (20), 152 (23), 105 (13), 69 (14).

Compound 15 was prepared from 14 using alkylation reaction; the reaction was similar to the preparation of 5. Compound 14 (100.5 mg) obtained 15 as a colorless solid (126.2 mg, 87% yield; with solvent system EtOAc: hexane = 1:9); mp 265–266 °C; ¹H-NMR (CDCl₃) δ_H 10.27 (s, 1H, OHC-C-3), 9.68 (d, J = 7.6 Hz, 1H, H-3″), 8.01 (s, 1H, H-4), 7.53 (d, J = 15.6 Hz, 1H, H-1″), 7.35–7.45 (m, 7H, CH₂Ph, H-6, H-6′), 7.05 (s, 1H, H-2′), 7.00 (d, J = 8.0 Hz, 1H, H-5′), 6.72 (dd, J = 15.6, 7.6 Hz, 1H, H-2″), 5.22 (s, 2H, CH₂Ph), 4.04 (s, 3H, OMe), 3.97 (s, 3H, OMe); ¹³C-NMR (CDCl₃) δ_C 193.4, 186.3, 166.2, 152.9, 151.2, 149.9, 145.3, 144.6, 145.3, 144.6, 136.1, 132.0, 128.7, 128.3, 128.1, 127.8, 127.2, 122.9, 120.8, 116.6, 113.5, 111.8, 106.6, 70.9, 56.3, 56.1; IR (KBr film) ν_max 3060, 2947, 2841, 2739, 1680, 1607, 1517, 1477, 1271, 1126, 1028, 970, 730 cm⁻¹; EI-MS m/z (%) (70 eV) 442 (M⁺, 53; C₂₇H₂₂O₆), 351 (54), 105 (11), 91 (100), 65 (5).

Compound 16 was prepared from methyl ferulate using IDA; the procedure was similar to the preparation of 1 from isoeugenol. Methyl ferulate (10.0 g) yielded 16 as a colorless solid (7.3 g, 73% yield; with solvent system EtOAc: hexane = 1:9 on open column); mp 96–97 °C; ¹H-NMR (CDCl₃) δ_H 7.64 (d, J = 15.9 Hz, 1H, H-1″), 7.17 (s, 1H, H-4), 7.00 (s, 1H, H-6), 6.88 (s, 3H, H-2′, H-5′, H-6′), 6.30 (d, J = 15.9 Hz, 1H, H-2″), 6.09 (d, J = 8.1 Hz, 1H, H-2), 5.63 (s, 1H, Ph-OH), 4.33 (d, J = 8.1 Hz, 1H, H-3), 3.89 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.81 (s, 3H, COOMe), 3.79 (s, 3H, COOMe); ¹³C-NMR (CDCl₃) δ_C 170.7, 167.6, 146.0, 144.7, 144.6, 131.3, 128.5, 125.6, 119.4, 117.8, 115.5, 114.5, 112.0, 108.7, 87.4, 56.0, 55.9, 55.4, 52.8, 51.6; IR (KBr film) ν_max 3396, 3011, 2956, 2849, 1741, 1637, 1606, 1496, 1440, 1287, 837, 612 cm⁻¹; EI-MS m/z (%) (70 eV) 414 (M⁺, 95; C₂₂H₂₂O₈), 382 (100), 350 (73), 280 (15), 266 (12), 167 (8), 151 (7), 137 (6), 58 (18).

Compound 17 was prepared from 16 using ethanoyl chloride in triethylamine; the reaction was similar to the preparation of 10. Compound 16 (1.36 g) could give 17 as a colorless solid (1.40 g, 91% yield; with solvent system EtOAc: hexane = 1:9); mp 98–99 °C; ¹H-NMR (CDCl₃) δ_H 7.63 (d, J = 15.8 Hz, 1H, H-1″), 7.17 (s, 1H, H-4), 6.97–7.00 (m, 3H, H-6, H-5′, H-6′), 6.30 (d, J = 15.8 Hz, 1H, H-2″), 6.16 (d, J = 8.0 Hz, 1H, H-2), 4.32 (d, J = 8.0 Hz, 1H, H-3), 3.91 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.80 (s, 3H, CO₂Me), 3.79 (s, 3H, CO₂Me), 2.29 (s, 3H, COMe); ¹³C-NMR (CDCl₃) δ_C 170.5, 168.8, 167.5, 151.2, 149.7, 144.6, 144.5, 139.7, 138.4, 128.7, 125.3, 122.9, 118.1, 117.8, 115.6, 112.1, 109.9, 86.6, 56.0, 55.8, 55.4, 52.8, 51.5, 20.5; IR (KBr film) ν_max 3073, 2955, 2848, 1769, 1742, 1710, 1638, 1604, 1499, 1443, 1275, 1196, 1150, 835 cm⁻¹; EI-MS m/z (%) (70 eV) 456 (M⁺, 2; C₂₄H₂₄O₉), 412 (4), 400 (5), 310 (7), 250 (6), 204 (10), 148 (100), 131 (51), 130 (17), 97 (17), 69 (23), 57 (31).

Compound 18 was prepared from 16 using benzoyl chloride and triethylamine; the reaction was similar to the preparation of 10. Compound 16 (74.1 mg) afforded 18 as a colorless solid (87.1 mg, 94% yield; with solvent system EtOAc: hexane = 1:9); mp 143–144 °C; ¹H-NMR (CDCl₃) δ_H 8.19 (d, J = 7.5 Hz, 2H, -COPh (o)), 7.64 (d, J = 15.6 Hz, 1H, H-1″), 7.59 (t, J = 7.5 Hz, 1H, -COPh (p)), 7.48 (t, J = 7.5 Hz, 2H, -COPh (m)), 7.19 (s, 1H, H-4), 7.13 (d, J = 8.0 Hz, 1H, H-5′), 7.00–7.05 (m, 4H, H-6, H-2′, H-6′), 6.67 (d, J = 15.6 Hz, 1H, H-2″), 6.20 (d, J = 8.0 Hz, 1H, H-2), 4.36 (d, J = 8.0 Hz, 1H, H-3), 3.92 (s, 3H, OMe), 3.84 (s, 3H, OMe), 3.79 (s, 6H, CO₂Me); ¹³C-NMR (CDCl₃) δ_C 170.6, 167.6, 164.6, 151.6, 149.8, 144.6, 140.1, 138.5, 133.5, 130.3, 129.2, 128.8, 128.5, 125.5, 123.2, 118.2, 117.9, 115.7, 112.2, 110.2, 86.8, 56.2, 56.0, 55.6, 52.9, 51.6; IR (KBr film) ν_max 3069, 2950, 2852, 1734, 1641, 1607, 1506, 1446, 1274, 1209, 1028, 846, 704 cm⁻¹; EI-MS m/z (%) (70 eV) 518 (M⁺, 35; C₂₉H₂₆O₉), 486 (2), 455 (3), 382 (2), 264 (3), 220 (3), 160 (6), 105 (100), 77 (21), 57 (7).

Compound 19 was prepared from 16 using butanoyl chloride in triethylamine; the reaction was similar to the preparation of 10. Compound 16 (91.6 mg) gave 19 as a colorless solid (96.4 mg, 90% yield; with solvent system EtOAc: hexane = 1:9); mp 155–156 °C; ¹H-NMR (CDCl₃) δ_H 7.62 (d, J = 15.8 Hz, 1H, H-1″), 7.17 (s, 1H, H-4), 6.97–7.01 (m, 3H, H-6, H-5′, H-6′), 6.30 (d, J = 15.8 Hz, 1H, H-2″), 6.16 (d, J = 8.0 Hz, 1H, H-2), 4.32 (d, J = 8.0 Hz, 1H, H-3), 3.91 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.78 (s, 6H, CO₂Me), 2.53 (t, J = 7.3 Hz, 2H, CH₂CH₂CH₃), 1.75 (sex, J = 7.3 Hz, 2H, CH₂CH₂CH₃), 1.02 (t, J = 7.3 Hz, 3H, CH₂CH₂CH₃); ¹³C-NMR (CDCl₃) δ_C 171.6, 170.6, 167.6, 151.4, 149.8, 144.7, 144.6, 140.0, 138.3, 128.8, 125.5, 123.1, 118.2, 117.9, 115.7, 112.2, 110.1, 86.8, 56.2, 55.9, 55.5, 52.9, 51.6, 35.8, 18.5, 13.5; IR (KBr film) ν_max 3003, 2960, 2850, 1766, 1742, 1711, 1608, 1507, 1434, 1275, 1141, 1030, 843 cm⁻¹; EI-MS m/z (%) (70 eV) 484 (M⁺, 41; C₂₆H₂₈O₉), 414 (38), 382 (100), 350 (33), 323 (10), 290 (7), 166 (7), 71 (17) (Figures S5–S8).

Compound 20 was prepared from 16 using isobutanoyl chloride in triethylamine; the reaction was similar to the preparation of 10. Compound 16 (91.6 mg) obtained 20 as a colorless solid (85.6 mg, 88% yield; with solvent system EtOAc: hexane = 1:9); mp 132–133 °C; ¹H-NMR (CDCl₃) δ_H 7.63 (d, J = 15.8 Hz, 1H, H-1″), 7.17 (s, 1H, H-4), 6.96–6.98 (m, 4H, H-2′, H-5′, H-6′, H-6), 6.30 (d, J = 15.8 Hz, 1H, H-2″), 6.16 (d, J = 8.0 Hz, 1H, H-2), 4.32 (d, J = 8.0 Hz, 1H, H-3), 3.95 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.76 (s, 3H, CO₂Me), 3.74 (s, 3H, CO₂Me), 2.81 (m, 1H, CH(CH₃)₂), 1.27 (d, J = 7.1 Hz, 6H, CH(CH₃)₂); ¹³C-NMR (CDCl₃) δ_C 175.1, 170.6, 167.5, 151.4, 149.8, 144.7, 144.6, 140.1, 138.2, 128.8, 125.5, 123.0, 118.2, 117.9, 115.7, 112.2, 110.1, 86.8, 56.1, 56.0, 55.5, 52.9, 51.6, 33.9, 18.9; IR (KBr film) ν_max 2952, 2849, 1762, 1605, 1508, 1464, 1281, 848, 757 cm⁻¹; EI-MS m/z (%) (70 eV) 484 (M⁺, 45; C₂₆H₂₈O₉), 415 (27), 382 (100), 350 (28), 290 (5), 235 (7), 167 (4), 71 (13) (Figures S9–S13).

Compound 21 was prepared from 17 using DDQ (dichlorodicyanobenzoquinone) dehydrogenative and oxidative coupling reaction; the reaction was similar to the preparation of 2. Compound 17 (1.10 g) afforded 21 as a colorless solid (0.93 g, 85% yield; with solvent system EtOAc: hexane = 1:5); mp 176–177 °C; ¹H-NMR (CDCl₃) δ_H 7.81 (s, 1H, H-2′), 7.79 (d, J = 16.0 Hz, 1H, H-1″), 7.78 (d, J = 1.3 Hz, 1H, H-4), 7.65 (dd, J = 8.3, 2.0 Hz, 1H, H-6′), 7.13 (d, J = 8.3 Hz, 1H, H-5′), 7.01 (d, J = 1.3 Hz, 1H, H-6), 6.44 (d, J = 16.0 Hz, 1H, H-2″), 4.03 (s, 3H, OMe), 3.95 (s, 3H, OMe), 3.91 (s, 3H, CO₂Me), 3.81 (s, 3H, CO₂Me), 2.33 (s, 3H, OAc); ¹³C-NMR (CDCl₃) δ_C 168.6, 167.4, 163.9, 160.5, 150.7, 145.3, 145.2, 141.5, 131.5, 129.0, 127.6, 122.6, 122.3, 117.1, 116.1, 113.8, 109.2, 106.0, 56.1, 56.0, 51.8, 51.7, 20.6; IR (KBr film) ν_max 3096, 2951, 2846, 1769, 1721, 1633, 1605, 1468, 1258, 1045, 885, 855, 597 cm⁻¹; EI-MS m/z (%) (70 eV) 454 (M⁺, 10; C₂₄H₂₂O₉), 412 (100), 382 (15), 349 (8), 323 (2), 228 (2), 151 (2), 91 (2), 69 (3).

Compound 22 was prepared from 21 using hydrogenation reductive reaction. The solution of 21 (50.6 mg in 10 mL of CH₃OH and 1 mL of CH₃COOH) with 10% Pd/C (10 mg) was stirred under H₂ at room temperature. After 6 h, the mixture was filtrated. After removing acid with aqueous NaHCO₃, the product residue was purified by Si gel column chromatography to give 22 as a colorless solid (43.8 mg, 95% yield; with solvent system EtOAc: hexane = 1:5); mp 134–135 °C; ¹H-NMR (CDCl₃) δ_H 7.67 (d, J = 1.7 Hz, 1H, H-2′), 7.59 (dd, J = 8.6, 1.7 Hz, 1H, H-6′), 7.41 (d, J = 1.1 Hz, 1H, H-4), 6.98 (d, J = 8.6 Hz, 1H, H-5′), 6.68 (d, J = 1.1 Hz, 1H, H-6), 5.88 (s, 1H, Ph-OH), 3.99 (s, 3H, OMe), 3.96 (s, 3H, OMe), 3.92 (s, 3H, CO₂Me), 3.68 (s, 3H, CO₂Me), 3.04 (t, J = 7.5 Hz, 2H, H-1″), 2.69 (t, J = 7.5 Hz, 1H, H-2″); ¹³C-NMR (CDCl₃) δ_C 173.4, 164.6, 147.7, 145.9, 144.7, 141.6, 137.3, 129.0, 123.7, 121.5, 114.1, 113.7, 112.2, 107.8, 56.1, 56.0, 51.6, 51.5, 34.6, 31.5; IR (KBr film) ν_max 3424, 2954, 2855, 1714, 1602, 1514, 1449, 1276, 1209, 1046, 829, 787 cm⁻¹; EI-MS m/z (%) (70 eV) 414 (M⁺, 100; C₂₂H₂₂O₈), 383 (9), 354 (10), 341 (25), 323 (47), 170 (5), 161 (3), 151 (2).

Compound 23 was prepared from 22 using alkylation reaction; the reaction was similar to the preparation of 9. Compound 22 (150.4 mg) afforded 23 as a colorless solid (146.4 mg, 80% yield; with solvent system EtOAc: hexane = 1:9); mp 78–79 °C; ¹H-NMR (CDCl₃) δ_H 7.67 (d, J = 2.1 Hz, 1H, H-2′), 7.57 (dd, J = 8.7, 2.1 Hz, 1H, H-6′), 7.28–7.44 (m, 6H, OCH₂Ph, H-4), 6.93 (d, J = 8.7 Hz, 1H, H-5′), 6.67 (d, J = 1.2 Hz, 1H, H-6), 5.21 (s, 2H, OCH₂Ph), 3.98 (s, 3H, OMe), 3.95 (s, 3H, OMe), 3.91 (s, 3H, CO₂Me), 3.67 (s, 3H, OMe-C-3), 3.04 (t, J = 8.1 Hz, 2H, H-1″), 2.68 (t, J = 8.1 Hz, 1H, H-2″); ¹³C-NMR (CDCl₃) δ_C 173.3, 164.6, 161.1, 150.0, 148.9, 144.7, 141.7, 137.4, 136.7, 129.0, 128.6, 127.9, 127.2, 122.9, 122.4, 113.7, 113.1, 112.9, 108.0, 107.9, 70.8, 56.2, 56.1, 51.6, 51.5, 36.4, 31.6; IR (KBr film) ν_max 2933, 2857, 1737, 1717, 1603, 1511, 1265, 1236, 1147, 1096, 1048, 742 cm⁻¹; EI-MS m/z (%) (70 eV) 504 (M⁺, 28; C₂₉H₂₈O₈), 413 (100), 382 (7), 353 (5), 341 (7), 323 (11), 91 (45).

Compound 24 was prepared from 23 using lithium aluminum hydride (LAH) reduction reaction. The solution of 23 (122.3 mg, in 20 mL of dry THF) was cooled to –10 °C before LAH (150.3 mg) was added. The solution was stirred under –10 °C. After 8 h, wet THF (10 mL) was added to the solution dropwise to quench the reaction. The solution was adjusted to pH = 4 using HCl (3N), and then the solution was evaporated under reduced pressure. The solution was extracted by EtOAc (3 × 100 mL). The combined organic layer was washed with saturated NaHCO₃ solution and brine and dry (Na₂SO₄), and the solvent was removed under reduced pressure to give a residue. Then, the residue was purified using Si gel column chromatography to give 24 as a colorless solid (103.2 mg, 95% yield; with solvent system EtOAc: hexane = 1:4); mp 187–188 °C; ¹H-NMR (CDCl₃) δ_H 7.29–7.45 (m, 7H, OCH₂Ph, H-2′, H-6′), 7.07 (s, 1H, H-4), 6.95 (d, J = 8.3 Hz, 1H, H-5′), 6.65 (s, 1H, H-6), 5.20 (s, 2H, OCH₂Ph), 4.88 (s, 2H, HOCH₂-C-3), 3.98 (s, 3H, OMe), 3.96 (s, 3H, OMe), 3.70 (t, J = 7.2 Hz, 2H, H-3″), 2.79 (t, J = 7.2 Hz, 2H, H-1″), 1.94 (quin, J = 7.2 Hz, 2H, H-2″); ¹³C-NMR (CDCl₃) δ_C 154.2, 149.7, 149.6, 148.9, 144.8, 141.6, 137.7, 137.6, 136.8, 136.7, 136.6, 131.1, 128.6, 127.9, 127.3, 127.2, 123.4, 120.4, 113.9, 113.8, 113.6, 111.0, 110.8, 107.7, 70.9, 62.2, 60.4, 56.2, 56.1, 34.6, 32.4; IR (KBr film) ν_max 2933, 2857, 1737, 1717, 1603, 1511, 1265, 1236, 1147, 1096, 1048, 742 cm⁻¹; HR-ESI-MS (M+H)⁺ m/z 449.1954; (C₂₇H₂₉O₆).

Preparation of 25: Methyl caffeate (762.2 mg) was dissolved in a mixture of benzene (20 mL) and acetone (30 mL), and then Ag₂O (1.82 g) was added. The reaction mixture was stirred at room temperature for 60 h. The precipitation was removed with filtration, and the filtrate gave 25 (306.9 mg, 45% yield). Compound 25: mp 187–188 °C; ¹H-NMR (CDCl₃) δ_H 7.54 (d, J = 15.9 Hz, 1H, H-1″), 7.05 (s, 1H, H-4), 6.99 (s, 1H, H-6), 6.85 (d, J = 1.9 Hz, 1H, H-2′), 6.82 (d, J = 8.1 Hz, H-5′), 6.76 (dd, J = 8.1, 1.9 Hz, 1H, H-6′), 6.24 (d, J = 15.9 Hz, 1H, H-2″), 6.02 (d, 2H, J = 7.4 Hz, 1H, H-2), 4.26 (d, J = 7.4 Hz, 1H, H-3), 3.79 (s, 3H, -OCH₃), 3.77 (s, 3H, OCH₃); ¹³C-NMR (CDCl₃) δ_C 171.0, 168.3, 148.5, 145.0, 144.7, 144.4, 144.0, 140.4, 132.0, 128.7, 125.4, 118.7, 117.6, 115.5, 115.4, 113.0, 87.2, 55.6, 53.0, 51.8; IR (KBr film) ν_max 3397, 2958, 1739, 1697, 1609, 1506, 1444, 1281, 1198, 980, 854, 814 cm⁻¹; EI-MS m/z (%) (70 eV) 386 (M⁺, 37; C₂₀H₁₈O₈), 354 (35), 322 (100), 294 (27), 267 (13), 194 (55), 163 (52), 134 (14) (Figures S13–S16).

Compound 26 was prepared from 25 (510.7 mg) under usual acetylation conditions using Ac₂O and pyridine. Compound 26 (609.6 mg, 90% yield), mp 137–139 °C; ¹H-NMR (CDCl₃) δ_H 7.59 (d, J = 15.8 Hz, 1H, H-1″), 7.42 (s, 1H, H-4), 7.28 (dd, J = 8.4, 1.9 Hz, 1H, H-6′), 7.21 (d, J = 1.9 Hz, 1H, H-2′), 7.19 (s, 1H, H-6), 7.17 (d, J = 8.4 Hz, 1H, H-5′), 6.29 (d, J = 15.8 Hz, 1H, H-2″), 6.19 (d, J = 7.4 Hz, 1H, H-2), 4.28 (d, J=7.4 Hz, 1H, H-3), 3.83 and 3.78 (s each, 3H-OCH₃), 2.30 (s, 3H, OAc), and 2.27 (s, 6H, OAc); IR (KBr film) ν_max 3074, 3016, 1776, 1739, 1716, 1643, 1612, 1591, 1273, 1203, 1176 cm⁻¹; HR-ESI-MS ((M+H)⁺ m/z 513.4708; C₂₆H₂₅O₁₁).

3.3. Chemicals

Colchicine, paclitaxel (Taxol), and vincristine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Microtubule-associated protein (MAP)-rich tubulin was from Cytoskeleton, Inc. (Denver, CO, USA). Other chemicals not specified were from Sigma or Merck (Darmstadt, Germany) with standard analytical or higher grade.

3.4. Cell Cultures

Human cancer cell lines (KB, HONE1) used in this study were procured from American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in RPMI 1640 medium.

To obtain KB-resistant cell lines, we followed the protocol described below. The protocol involved exposing exponentially growing cells to increasing concentrations of etoposide (VP-16) and vincristine over a period of six months. Initially, cells were exposed to the IC₅₀ concentration obtained from a methylene blue assay. Subsequently, cells were subcultured and maintained in RPMI 1640 medium containing vincristine and supplemented with 10% FBS and 1% penicillin/streptomycin. The concentration of the drugs was incrementally increased approximately 1.5-fold in the initial steps and 1.25-fold in the final steps. This process was repeated every four weeks until the final resistant sublines were obtained. Cryopreserved aliquots of cell sublines were taken at each incremental concentration. We collected three resistant sublines, named KB-Vin10 and KB-7D cells.

All cell cultures were supplemented with 10% fetal bovine serum, 2 μM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated in a humidified atmosphere (95% air and 5% CO₂) at 37 °C. KB-Vin10 was a cell line resistant to vincristine and overexpressing the MDR drug efflux protein. KB-7D cells were VP16-resistant cells and overexpressed MRP. All resistant cell lines were incubated in drug-free medium for 3 days before harvesting for the growth inhibition assay.

3.5. Growth Inhibition Assay

In vitro growth inhibition was assessed with the methylene blue assay [16]. Briefly, exponentially growing cells were seeded into 24-well culture plates at a density of 10,000 cells/mL/well and allowed to adhere overnight. Cells were incubated with various concentrations of drugs for 72 h. Then, we measured A₅₉₅ of the resulting solution from 1% N-lauroylsarcosine exaction. The 50% growth inhibition (IC₅₀) was calculated based on the A₅₉₅ of untreated cells (taken as 100%). The values shown are the means and standard errors of at least three independent experiments performed in duplicate.

3.6. In Vitro Microtubule Polymerization Assay

This assay was conducted in a 96-well UV microplate, as described previously [17]. A total of 0.24 mg MAP-rich tubulin was mixed with various concentrations of drugs and incubated at 37 °C in 120 μL reaction buffer (100 mM PIPES, pH 6.9, 1.5 mM MgCl₂, 1 mM GTP, and 1% (v/v) DMSO). A₃₅₀ was monitored every 30 s for 30 min, using the PowerWave X Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). The increase in A₃₅₀ indicated the increase in tubulin polymerization; 100% polymerization was defined as the AUC of the untreated control.

4. Conclusions

In this study, we synthesized a series of salvinal derivatives and evaluated their structure–activity relationship (SAR) in terms of antiproliferation in KB and HONE1 cancer cell lines. Compound 25 exhibited exceptional anticancer activity, with an IC₅₀ of 0.137 μM. Its anticancer activity was attributed to the depolymerization of microtubules, which may lead to cell death in tumor cells. Moreover, the effectiveness of compound 25 was not significantly affected by drug resistance caused by MDR or MRP overexpression. The anticancer potential of compound 25 warrants further investigation and development as a promising agent for cancer treatment.