Daurichromenic Acid from the Chinese Traditional Medicinal Plant Rhododendron dauricum Inhibits Sphingomyelin Synthase and Aβ Aggregation

Species of the genus Rhododendron have been used in traditional Chinese medicine, with the medicinal herb “Manshanfong” used as an expectorant and for the treatment of acute bronchitis. Daurichromenic acid (DCA), a constituent of Rhododendron dauricum, is a meroterpenoid with antibacterial, anti-HIV, and anti-inflammatory activities. However, the mechanisms underlying these pharmacologic activities are poorly understood. To develop new drugs based on DCA, more information is required regarding its interactions with biomolecules. The present study showed that DCA inhibits the activity of the enzyme sphingomyelin synthase, with an IC50 of 4 µM. The structure–activity relationships between DCA and sphingomyelin synthase were evaluated using derivatives and cyclized hongoquercin A. In addition, DCA was found to inhibit amyloid β aggregation. These results may help in the design of effective drugs based on DCA.


Introduction
Traditional medicinal plants are promising sources of naturally occurring drugs, especially of natural products targeting membrane proteins, including membrane receptors and enzymes. Daurichromenic acid (DCA), a meroterpenoid consisting of orsellinic acid and sesquiterpene moieties, was isolated from the leaves of Rhododendron dauricum, a plant growing in the wild in northern China, eastern Siberia, and the Japanese island of Hokkaido [1]. This species of Rhododendron has been used in traditional Chinese medicine, with the medicinal herb "Manshanfong" used as an expectorant and to treat various diseases, such as acute and chronic bronchitis [2]. DCA has also been reported to have antibacterial activity against Gram-positive bacteria [3], as well as having anti-HIV [4], and anti-inflammatory [5] activity, suggesting that DCA may be a medicinal resource in the development of derivatives that can act as novel drugs to treat these conditions. Although DCA exhibits various pharmacological activities, including the induction of cell death in cultured cells [6], its molecular mechanisms of activity remain unclear. Thus, a deeper understanding of its mechanisms

Preparation of Daurichromenic Acid (1) and Its Derivatives (2-7)
The identity of DCA (1) isolated from Rhododendron dauricum was confirmed by 1 H-and 13 C-nuclear magnetic resonance (NMR) and by electrospray ionization-mass spectrometry (ESI-MS) and compared with previously reported values [22]. To understand the structure-activity relationship (SAR) between DCA and its target molecules, DCA derivatives were synthesized. Compound (2) was prepared from DCA by selective methyl esterification of its carboxylic acid moiety by TMS-CH 2 N 2 . Compound (3) was prepared by protecting the carboxylic acid and hydroxy groups with iodomethane, and compound (4) was prepared by hydrolysis of the methyl ester group of compound (3). Subsequently, hongoquercin A (7), which has been isolated from an unidentified terrestrial fungus [23] and exhibits antibacterial properties toward methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium [24,25], was prepared from compound (2). In the first step, compound (2) was treated with FeCl 3 to obtain the cyclized derivative compound 5 (5). The methyl ester group of (5) was hydrolyzed to yield compound (6), and the double bond at the benzyl position of compound (6) was reduced by Pd/C and H 2 to obtain hongoquercin A (7) in moderate yield (Scheme 1). Molecules 2020, 25, x 3 of 11 Scheme 1. Preparation and synthesis of DCA (1) and its derivatives.

SMS Inhibition by DCA and Its Derivatives
The ability of DCA and its derivatives to inhibit the SMS isozymes SMS1 and SMS2 was evaluated using a cell lysate assay and the fluorescent substrate C6-NBD (4-nitrobenzo-2-oxa-1,3diazole)-Cer. DCA (1) and compounds 4, 6, and 7 showed relatively moderate inhibitory activities, with IC50 values of 7, 17, 9, and 4 µM, respectively, for SMS1, and 4, 10, 7, and 5 µM, respectively, for SMS2 ( Figure 1), similar to other natural compounds [13,14]. These findings indicated that the methyl esters of DCA derivatives, compounds 2, 3, and 5, were inactive in these assays. Thus, the carboxylic acid group of DCAs are essential moieties for their inhibition of SMSs. However, the SMS has not been succeeded in its crystallization. Therefore, a direct comparison with the previously isolated SMS inhibitor ginkgolic acid [13] has been examined. The chemical similarity of ginkogolic acid to DCA, as it is a long alkyl chain and a carboxylic acid moiety in an aromatic ring, shows that these two inhibitors also have the same functional group participation to inhibit SMS. Further, a previous report [26] concerning the mode of action for Aβ aggregation inhibition for small molecules gave the insight that the crucial role of a hydrophobic-side hydrocarbon chain and an aromatic ring as well as its two hydrophilic functions causes significant interaction with the amyloid. Scheme 1. Preparation and synthesis of DCA (1) and its derivatives.

SMS Inhibition by DCA and Its Derivatives
The ability of DCA and its derivatives to inhibit the SMS isozymes SMS1 and SMS2 was evaluated using a cell lysate assay and the fluorescent substrate C6-NBD (4-nitrobenzo-2-oxa-1,3-diazole)-Cer. DCA (1) and compounds 4, 6, and 7 showed relatively moderate inhibitory activities, with IC 50 values of 7, 17, 9, and 4 µM, respectively, for SMS1, and 4, 10, 7, and 5 µM, respectively, for SMS2 ( Figure 1), similar to other natural compounds [13,14]. These findings indicated that the methyl esters of DCA derivatives, compounds 2, 3, and 5, were inactive in these assays. Thus, the carboxylic acid group of DCAs are essential moieties for their inhibition of SMSs. However, the SMS has not been succeeded in its crystallization. Therefore, a direct comparison with the previously isolated SMS inhibitor ginkgolic acid [13] has been examined. The chemical similarity of ginkogolic acid to DCA, as it is a long alkyl chain and a carboxylic acid moiety in an aromatic ring, shows that these two inhibitors also have the same functional group participation to inhibit SMS. Further, a previous report [26] concerning the mode of action for Aβ aggregation inhibition for small molecules gave the insight that the crucial role of a hydrophobic-side hydrocarbon chain and an aromatic ring as well as its two hydrophilic functions causes significant interaction with the amyloid.  (6) and (d) derivative (7). IC50 values were measured using a cell lysate assay. SMS-expressing cell lysates and compounds were incubated for 3 h at 37 °C, and the extracted fluorescent lipids were directly analyzed by HPLC. Each point represents the mean ± SD of triplicate assays.

Inhibition of Aβ Aggregation by DCA and Its Derivatives
Alzheimer's disease (AD) is a multifactorial disease, which is believed to be caused by complex interactions among several contributing patho-mechanisms. The ability of DCA and its derivatives, along with other natural compounds, ginkgolic acids (8, 9) ( Figure 2) and malabaicone A-C (10, 11 and 12), to inhibit Aβ42 aggregation was evaluated by microtiter-scale high-throughput screening (MSHTS) assays [27,28]. EC50 values were measured from inhibition curves, in which the percent SDs of fluorescence intensities were plotted against the concentration of each compound ( Figure 3). Here, 30 nM quantum dot amyloid beta (QDAβ) and 30 µM of Aβ were incubated with different concentrations to evaluate the EC50 of all derivatives. DCA and its derivative compound 4 showed relatively potent inhibition activities, with EC50 values of 57 and 74 µM, respectively, similar to the EC50 of rosmarinic acid (60 µM) [27]. The inhibitory activities of hongoquercin A (7) and ginkgolic acids (8,9) for Aβ aggregation were lower, with EC50 values of 150-300 µM. The inhibitory activities of the other cyclized DCAs and malabaricones were lower ( Figure S2). These results indicate that DCA, a terpenoid containing a benzopyran moiety, is a candidate inhibitor of Aβ aggregation, and that the carboxylic acid group on aromatics and hydrophobic long alkyl chains may be required for this activity.   (6) and (d) derivative (7). IC 50 values were measured using a cell lysate assay. SMS-expressing cell lysates and compounds were incubated for 3 h at 37 • C, and the extracted fluorescent lipids were directly analyzed by HPLC. Each point represents the mean ± SD of triplicate assays.

Inhibition of Aβ Aggregation by DCA and Its Derivatives
Alzheimer's disease (AD) is a multifactorial disease, which is believed to be caused by complex interactions among several contributing patho-mechanisms. The ability of DCA and its derivatives, along with other natural compounds, ginkgolic acids (8, 9) ( Figure 2) and malabaicone A-C (10, 11 and 12), to inhibit Aβ 42 aggregation was evaluated by microtiter-scale high-throughput screening (MSHTS) assays [27,28]. EC 50 values were measured from inhibition curves, in which the percent SDs of fluorescence intensities were plotted against the concentration of each compound ( Figure 3). Here, 30 nM quantum dot amyloid beta (QDAβ) and 30 µM of Aβ were incubated with different concentrations to evaluate the EC 50 of all derivatives. DCA and its derivative compound 4 showed relatively potent inhibition activities, with EC 50 values of 57 and 74 µM, respectively, similar to the EC 50 of rosmarinic acid (60 µM) [27]. The inhibitory activities of hongoquercin A (7) and ginkgolic acids (8,9) for Aβ aggregation were lower, with EC 50 values of 150-300 µM. The inhibitory activities of the other cyclized DCAs and malabaricones were lower ( Figure S2). These results indicate that DCA, a terpenoid containing a benzopyran moiety, is a candidate inhibitor of Aβ aggregation, and that the carboxylic acid group on aromatics and hydrophobic long alkyl chains may be required for this activity.
relatively potent inhibition activities, with EC50 values of 57 and 74 µM, respectively, similar to the EC50 of rosmarinic acid (60 µM) [27]. The inhibitory activities of hongoquercin A (7) and ginkgolic acids (8,9) for Aβ aggregation were lower, with EC50 values of 150-300 µM. The inhibitory activities of the other cyclized DCAs and malabaricones were lower ( Figure S2). These results indicate that DCA, a terpenoid containing a benzopyran moiety, is a candidate inhibitor of Aβ aggregation, and that the carboxylic acid group on aromatics and hydrophobic long alkyl chains may be required for this activity.  (8,9). Figure 2. ginkgolic acids (8,9).

DCA and Compound (4) Inhibit Both SMS and Aβ Aggregation
These studies' findings indicated that DCA and its derivative compound 4 could be new dual inhibitors of SMS activity and Aβ42 aggregation. DCA was recently reported to inhibit the aggregation of Aβ40 [29], similar to our findings that DCA inhibits the aggregation of Aβ42. Furthermore, SAR studies of the ability of a terpenoid containing a benzopyran moiety to inhibit both SMS activity and Aβ42 aggregation suggested that benzoic acid and hydrophobic long alkyl chains are essential for both activities (Table 1).

DCA and Compound (4) Inhibit Both SMS and Aβ Aggregation
These studies' findings indicated that DCA and its derivative compound 4 could be new dual inhibitors of SMS activity and Aβ 42 aggregation. DCA was recently reported to inhibit the aggregation of Aβ 40 [29], similar to our findings that DCA inhibits the aggregation of Aβ 42 . Furthermore, SAR studies of the ability of a terpenoid containing a benzopyran moiety to inhibit both SMS activity and Aβ 42 aggregation suggested that benzoic acid and hydrophobic long alkyl chains are essential for both activities (Table 1). Natural products are a source of potential pharmacological agents with an abundance of novel chemical entities with various biological activities. The goal of our study is to identify potent SMS inhibitors from a natural source. In this study, we found that DCA (1), which is isolated from a Rhododendron dauricum, is a potent SMS inhibitor. Leaves of Rhododendron dauricum are reported as anti-HIV, used for the treatment of many diseases, and are also involved in cell proliferation, as well as being one of the well-known, practically used Chinese traditional medicines. DCA (1) itself possesses several biological activities, popularly anti-HIV and antibacterial, but its molecular mechanisms have not yet been examined. To completely understand the pharmalogical behavior of DCA (1) with sphingomyelin synthase enzyme, a series of DCA derivatives 2-6 and (−) hongoquercin A (7) were synthesized to evaluate functional group participation in the enzyme inhibiton. Structures of isolated and all synthesized compounds were analyzed using various spectroscopic methods such as UV, IR, ESI-MS, and optical rotation for synthesized compounds. Compounds with SMS inhibition properties against cell-based assays and the fluorescent substrate C6-NBD (4-nitrobenzo-2-oxa-1,3-diazole)-Cer share a carboxylic acid moiety which is also related to anti-aggregative properties against amyloid beta protein. MSHTS assays were used to evaluate amyloid beta aggregation activity using DCA and their derivatives. DCA (1) and compound 4 with the presence of a carboxylic acid group tend to inhibit both SMS and Aβ aggregation inhibition. Our work, to the best of our knowledge, is the first report on daurichromenic acid and derivatives of DCA and compound 4 as a dual inhibitor for sphingomyelin synthase and amyloid beta aggregation.

Extraction and Isolation of Active Compounds from Rhododendron dauricum
Rhododendron dauricum leaves were collected in Hokkaido, Japan, dried, and ground into powder. The dried powder, weighing 81 g, was extracted three times with 400 mL methanol for 24 h each at room temperature. The methanol extracts were combined and concentrated under reduced pressure, yielding a black residue weighing 24.2 g. The residue was dissolved in 20% MeOH in water (500 mL) Molecules 2020, 25, 4077 7 of 11 and partitioned three times with 200 mL hexane, three times with 200 mL Et 2 O, and three times with 200 mL EtOAc. The solvents were evaporated, and SMS activities were assessed in the hexane, Et 2 O, EtOAc, and water residues. The hexane fraction was more active than the Et 2 O fraction, whereas the EtOAc and water fractions were inactive. The active hexane fraction, weighing 3.8 g, was further fractionated by column chromatography on a spherical silica gel of diameter 40-50 µm (Kanto Silica Gel 60). The active component was identified as DCA, a finding confirmed by 1 H-NMR and ESI-MS [20]. Yield: 1.53% (1.24 g). Yellow oil; 1

Methyl Ester of DCA (2)
A solution of TMS-CH 2 N 2 in hexane was added to a solution of DCA (compound 1) (100 mg, 0.270 mmol) in methanol (5 mL) and diethyl ether (5 mL) at 0 • C until the color of the solution became yellow. The reaction mixture was stirred at 0 • C for 0.5 h and at room temperature for 1 h. The reaction was quenched with acetic acid and concentrated under vacuum to yield a residue, which was dissolved in hexane and purified by silica gel column chromatography using hexane/EtOAc (9.5:0.5) as an eluent, resulting in ester 2 [21] at a yield of 97%. Colorless oil; 1  A solution of 6 M NaOH (5 mL) was added to a solution of compound 3 (50.0 mg, 0.130 mmol) in methanol (2 mL) and THF (2 mL) at room temperature, and the reaction mixture was stirred at 100 • C for 2 h. The reaction mixture was brought to room temperature, the solvent was evaporated, and the residue was neutralized with 2 N HCl and extracted with EtOAc. The organic layer was concentrated, and the residue was dissolved in hexane and purified by silica gel column chromatography using hexane/EtOAc (9. Ferric chloride (81 mg, 499 µmol) was added to a solution of 2 (194 mg, 505 µmol) in toluene (10 mL) at room temperature and the solution stirred for 8 h until the disappearance of the starting material. The toluene was evaporated, and the residue dissolved in 10 mL of water, followed by extraction with ethyl acetate. The combined organic layer was dried over Na 2 SO 4 , and the residue was dissolved in hexane and purified by silica gel column chromatography using hexane/EtOAc (9.5:0.5) as an eluent, resulting in compound 5 as a white solid (yield, 46%). 1  in methanol (1 mL) and THF (2 mL). The mixture was refluxed for 2 h, acidified with 2% HCl, and extracted with CH 2 Cl 2 . The combined organic layer was washed with saline solution, dried over Na 2 SO 4 , and concentrated. The crude residue was purified by silica gel column chromatography using hexane/EtOAc (4:1) as an eluent, resulting in compound 6 [20] as a white solid (yield 84%). 1

(−) Hongoquercin A (7)
A solution of 10% Pd/C (0.028 mmol) was added to a solution of 6 (0.140 mmol) in EtOAc (10 mL) while stirring, and the reaction mixture was further stirred overnight at 40 • C under a H 2 atmosphere. The solid was filtered off and the filtrate was concentrated under a vacuum. The residue was redissolved in hexane and purified by silica gel column chromatography using hexane/EtOAc (4:1) as an eluent, resulting in compound 7 (yield 74%) as a white solid. Its structure was confirmed by 1 H-and 13 C-NMR and ESI-MS and compared with previous results [18]. 1

SMS Assay
ZS/SMS1 and ZS/SMS2 cells (protein concentration 0.1 µg/µL) were diluted in 20 mM Tris-buffer (pH 7.5) and sonicated. A 1 µL aliquot of each compound at the desired concentration was added to 100 µL aliquots of the cell lysates and the solutions were incubated at 37 • C for 30 min. A 1 µL aliquot of C6-NBD-ceramide was added to each solution and the solutions were incubated for 3 h at 37 • C. The reactions were stopped by addition of 400 µL of MeOH/CHCl 3 [1/2 (v/v)], and the mixtures were shaken and centrifuged at 1500 rpm for 5 min. The formation of C6-NBD-sphingomyelin was quantified by HPLC determination of its peak area. Inhibitory activity was quantified by a reverse-phase HPLC assay using a JACSO HPLC system, equipped with a PU-2089 Plus and FP-2020 Plus set at λex = 470 nm and λem = 530 nm. A 50 × 4.6 YMC-Pack Diol-120-NP column (5-µm particle size) was used with the mobile phase (IPA/hexane/water) at a flow rate of 1.0 mL/min

Aβ Inhibition Aggregation Assay
The aggregation inhibitory activities of plant extracts, fractions, DCA, DCA derivatives, and naturally occurring SMS inhibitors (compounds 8, 9, and 10) were measured using a modified MSHTS system [1,25]. Various concentrations of each were incubated with 30 nM QD-labeled Aβ 40 (QDAβ), and 30 µM Aβ 42 in PBS containing 5% EtOH and 3% DMSO at 37 • C for 24 h in 1536-well plates (782096, Greiner) (Kremsmünster, Austria). The center of each well as viewed with an inverted fluorescence microscope (TE2000, Nikon)) (Tokyo, Japan) equipped with a color CCD camera (DP72, Olympus) (Tokyo, Japan), and the central region of each well were measured with ImageJ software Ver 1.53b (NIH) to determine the standard deviations (SD) of fluorescence intensities of 40,000 pixels (200 × 200 pixels: 320 × 320 µm) [29,30]. The SD values, which were approximately proportional to the amount of aggregates, were plotted against the concentrations of the putative inhibitors to generate inhibition curves, and the EC 50 values were determined.

Conclusions
The present study showed that DCA, a terpenoid from the leaves of Rhododendron dauricum, is a natural inhibitor of sphingomyelin synthase. Several of its derivatives, including compounds 4, 6, and 7, also showed good inhibitory activity against SMS. In addition, DCA and compound 4 were found to inhibit Aβ 42 aggregation. These properties suggest that DCA is a suitable candidate for further development as a new drug or medicinal supplement to treat diseases of abnormal lipid metabolism and to prevent Alzheimer's disease.