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

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.


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,

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.

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 sub-stitutions 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 CH 2 Cl 2 /H 2 O=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 SeO 2 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. of salvinal and benzofuranlignan derivatives. The synthesis steps of salv are shown in Scheme 1. Isoeugenol as a starting material was reacted w diacetateuse (IDA) to obtain salvinal (4) in a four-step reaction (Sche substitutions in the C-3 and C-5 positions of the benzofuran skeleton w and evaluated for their impact on anticancer activity. In order to examine phenyl substituent at the C-2 position of compound 1, we used com template for further alkylation (that resulted in compounds 5-9) and acy (that resulted in compounds 10-12). Compound 1 was treated with DDQ mixture solvent of CH2Cl2/H2O=4/1 to initiate a reaction, which could sel arylpropene to arylpropenal, forming compound 13 under the room Compound 2 was further oxidized with SeO2 in refluxing EtOH to yield The structural difference between compound 14 and salvinal (4) is the sid 5 position, and the anticancer activities of the two are only slightly differ the impact of phenyl substituent at the C-2 position, alkylation reaction with compound 14 in order to obtain compound 15, as shown in Scheme Oxidative coupling of methyl ferulate with IDA generated benzofura (Scheme 2). Compound 16 was further acylated to obtain com Dehydrogenation of compound 17 using DDQ in 1,4-dioxane under compound 21. Compound 21 was dissolved in the mixture solvent of CH with 10% Pd/C as catalysis under a hydrogen atmosphere to yield compo both hydrogenation and hydrolysis reactions. Benylation of compound bromide and K2CO3 in acetone under reflux afforded compound 23 in a hi By using LAH in dry THF, two methyl carboxylates were reduced to hy form compound 24. We also used Ag2O for the oxidative coupling of m form compound 25, which was then acetylated to form compound 26. 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 CH 3 OH 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 K 2 CO 3 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 Ag 2 O for the oxidative coupling of methyl caffeate to form compound 25, which was then acetylated to form compound 26. Int. J. Mol. Sci. 2023, 24,

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. 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.

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 IC 50 of compound 1 and salvinal against KB cells was 5.6 µM and 5.0 µM, respectively. The IC 50 of salvinal derivatives against KB and HONE-1 cells is shown in Table 1. 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 IC 50 less than 0.4 µM, four showed IC 50 in the range of 0.4-5.0 µM, three showed IC 50 in the range of 5-10 µM, twelve showed IC 50 in the range of 10-38 µM, and two showed IC 50 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 IC 50 values of 0.137 µM compared to salvinal, with IC 50 of 5.0 µM. The test compounds exhibited similar IC 50 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 IC 50 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.

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28 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. 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.

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

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 IC 50 of compounds 4, 19, 20, 25, and 26 against KB, KB-Vin10, and KB-7D cells is shown in Table 2. 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.

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  4, 19, 20, 25, and 26. MAP-rich tubulins were incubated at 37 • C in the absence or presence of test compounds. Absorbance at 350 nm was measured every 30 s for 30 min and is presented as the increased polymerized microtubule.
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.

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 ( 1 H-NMR: 300 MHz, 13 C-NMR: 75 MHz)(Bruker, Fällanden, Switzerland) with CDCl 3 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.

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 CH 2 Cl 2 ) was added dropwise to the solution of IDA (10.0 g, 30.2 mmol) in 100 mL of CH 2 Cl 2 (dry with CaH 2 ) at room temperature for 4 h. After 48 h, NaHCO 3 (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; 1 (11), 91 (15).

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 MgCl 2 , 1 mM GTP, and 1% (v/v) DMSO). A 350 was monitored every 30 s for 30 min, using the PowerWave X Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). The increase in A 350 indicated the increase in tubulin polymerization; 100% polymerization was defined as the AUC of the untreated control.

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 IC 50 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.