Phytochemicals with Chemopreventive Activity Obtained from the Thai Medicinal Plant Mammea siamensis (Miq.) T. Anders.: Isolation and Structure Determination of New Prenylcoumarins with Inhibitory Activity against Aromatase

With the aim of searching for phytochemicals with aromatase inhibitory activity, five new prenylcoumarins, mammeasins K (1), L (2), M (3), N (4), and O (5), were isolated from the methanolic extract of Mammea siamensis (Miq.) T. Anders. flowers (fam. Calophyllaceae), originating in Thailand. The stereostructures of 1–5 were elucidated based on their spectroscopic properties. Among the new compounds, 1 (IC50 = 7.6 µM) and 5 (9.1 µM) possessed relatively strong inhibitory activity against aromatase, which is a target of drugs already used in clinical practice for the treatment and prevention of estrogen-dependent breast cancer. The analysis through Lineweaver–Burk plots showed that they competitively inhibit aromatase (1, Ki = 3.4 µM and 5, 2.3 µM). Additionally, the most potent coumarin constituent, mammea B/AB cyclo D (31, Ki = 0.84 µM), had a competitive inhibitory activity equivalent to that of aminoglutethimide (0.84 µM), an aromatase inhibitor used in therapeutics.


Inhibitory Activity against Aromatase
In a recent exploratory study on the bioactive constituents of M. siamansis, several coumarins exhibited antiproliferative and apoptotic effects in several human cancer cell lines. Furthermore, their mechanisms of action have also been characterized [29][30][31][32][33]. We have also reported that these coumarin constituents exhibit antiproliferative and apoptotic effects against human digestive tract carcinoma cell lines and human breast cancer MCF-7 [22]. Breast cancer is one of the malignant carcinomas associated with the highest morbidity and mortality in women [34,35]. The presence of high estrogen concentrations in breast tissue increases the risk of developing breast cancer. Estrogen and estrogen receptors play an important role in the development and progression of hormone-dependent breast cancer [36]. Aromatase is a key enzyme in estrogen biosynthesis, as it catalyzes the conversion of androgens (testosterone and androstenediol) to estrogens (estradiol and estrone). Since intra-tumoral aromatase is the source of estrogen production in breast cancer tissues, aromatase inhibitors have been widely used in clinical practice as chemotherapeutic agents against hormone-dependent breast cancer [37]. Based on their chemical structures, aromatase inhibitors are classified into two categories: steroidal and non-ste-

Inhibitory Activity against Aromatase
In a recent exploratory study on the bioactive constituents of M. siamansis, several coumarins exhibited antiproliferative and apoptotic effects in several human cancer cell lines. Furthermore, their mechanisms of action have also been characterized [29][30][31][32][33]. We have also reported that these coumarin constituents exhibit antiproliferative and apoptotic effects against human digestive tract carcinoma cell lines and human breast cancer MCF-7 [22]. Breast cancer is one of the malignant carcinomas associated with the highest morbidity and mortality in women [34,35]. The presence of high estrogen concentrations in breast tissue increases the risk of developing breast cancer. Estrogen and estrogen receptors play an important role in the development and progression of hormone-dependent breast cancer [36]. Aromatase is a key enzyme in estrogen biosynthesis, as it catalyzes the conversion of androgens (testosterone and androstenediol) to estrogens (estradiol and estrone). Since intra-tumoral aromatase is the source of estrogen production in breast cancer tissues, aromatase inhibitors have been widely used in clinical practice as chemotherapeutic agents against hormone-dependent breast cancer [37]. Based on their chemical structures, aromatase inhibitors are classified into two categories: steroidal and non-steroidal [38]. The structures of steroidal aromatase inhibitors closely resemble those of the substrates of aromatase enzymes, such as testosterone and androstenediol. Exemestane, a clinically used steroidal aromatase inhibitor, is metabolized to an intermediate, which attaches irreversibly to the active site of the enzyme, thereby blocking its activity. These inhibitors are known as "suicide inhibitors" [37]. On the other hand, non-steroidal aromatase inhibitors (e.g., aminoglutethimide, anastrozole, and letrozole, etc.) are generally reversible, and the inhibition of estrogen synthesis is dependent on the continuous presence of the drug [39]. Owing to the development of resistance to aromatase inhibitors and their side effects, the need for improved aromatase inhibitors remains [40,41]. Therefore, new non-steroidal natural products with aromatase inhibitory activity are being investigated [36,[42][43][44][45]. During our studies of characterization of the Thai medicinal plant M. siamensis, we found that the methanolic extract and several isolated coumarin constituents exhibited inhibitory activity against aromatase [19]. Continuing the chemical study on M. siamemsis, we have so far isolated 42 coumarin constituents, as summarized in Figure 5.
We analyzed inhibition kinetics using Lineweaver-Burk plots to determine the mode of inhibition of coumarins that showed strong inhibitory activities against human aromatase. In the assay system, we fixed the enzyme concentration, changed the substrate concentration, and obtained the kinetic parameters of the enzyme-catalyzed reaction using Lineweaver-Burk double reciprocal plot 1/[V] vs. 1/[S]. The inhibition constant K i indicates the potency of an inhibitor and equals the concentration required to produce halfmaximal inhibition [46]. The K i value was obtained from the intersection of the secondary plot with the x-axis (apparent K m /V max vs. inhibitor). Thus, the first-generation aromatase inhibitor aminoglutethimide showed a competitive inhibition of aromatase characterized by a K i value of 0.84 µM, as shown in Table 6 and Figure 6, which is consistent with the results of a previous report [47]. Among the active coumarin constituents from M.

General
The following instruments were used to obtain spectroscopic data: specific rotation, JASCO P-2200 polarimeter (JASCO Corporation, Tokyo, Japan, l = 5 cm); UV spectra, Shi-

Inhibitory Effects against Human Recombinant Aromatase
The experiments were performed according to a previously described method [18]. Briefly, a test sample was dissolved in dimethyl sulfoxide (DMSO), and the solution was diluted with potassium phosphate buffer (50 mM, pH 7.4) containing MgCl 2 (0.5 mM) to obtain the test sample solution (concentration of DMSO: 2%). An enzyme/substrate solution in the buffer (20 µL, 1.6 µM DBF, 8 nM human recombinant aromatase) and the test sample solution (20 µL) were mixed in a 96-well half-area black microplate (Greiner Bio-One, Frickenhausen, Germany) at 37 • C for 10 min. The enzymatic reaction was initiated by adding NADPH solution (40 µL, 500 µM) at 37 • C for 30 min. After 30 min of incubation, NaOH (30 µL, 2 mM) was added, and the reaction mixture was incubated at 37 • C for 2 h to induce fluorescent signals (final DMSO concentration, 0.5%; aromatase, 2 nM; and NADPH, 250 µM). Fluorescence was measured using a fluorescence microplate reader (SH-9000, CORONA ELECTRIC Co., Ltd., Ibaraki, Japan) at an excitation wavelength of 435 nm and emission wavelength of 535 nm. Experiments were performed in triplicate, and the IC 50 values were determined graphically. The aromatase inhibitor, aminoglutethimide, was used as the reference compound.

Kinetic Analysis of Inhibitory Activity against Human Recombinant Aromatase Using Lineweaver-Burk Plots
Experiments were performed using a previously described protocol [18], modified by using various concentrations of testosterone (0.4-4 µM) as substrates instead of DBF and the plate was heated at 37 • C for 15 min. After the reaction, the enzyme was inactivated by heating in a boiling water bath for 2 min. An estradiol EIA kit (Oxford Biomedical Research, Inc., Oxford, MI, USA) was used to develop an estradiol standard curve to determine the concentration of estradiol produced and to correlate the concentration of estradiol with the reaction velocity. The mode of inhibition was analyzed using the Lineweaver-Burk plot of the inverse of the reaction velocity of estradiol plotted on the vertical axis and the inverse of the final concentration of the substrate on the horizontal axis, with and without the test substance ( Figure S27).

Statistics
Values are expressed as mean ± standard mean error (S.E.M.). One-way analysis of variance (ANOVA), followed by Dunnett's test, was used for statistical analysis. Probability (p) values of less than 0.05 were considered significant.

Conclusions
Five new prenylcoumarins, mammeasins K-O (1-5), were isolated from the methanolic extract of the flowers of M. siamensis, a plant originating from Thailand. The stereostructures of 1-5 were elucidated based on their spectroscopic properties. Fifteen coumarin constituents, including 1 (IC 50 = 7.6 µM) and 5 (9.1 µM), kayaessamin I (23, 9.3 µM), and mammea A/AA cyclo F (39, 9. Data Availability Statement: The data supporting the findings of this study are available from the corresponding author upon reasonable request.