Structures of Mammeasins P and Q, Coumarin-Related Polysubstituted Benzofurans, from the Thai Medicinal Plant Mammea siamensis (Miq.) T. Anders.: Anti-Proliferative Activity of Coumarin Constituents against Human Prostate Carcinoma Cell Line LNCaP

A methanol extract of the flowers of Mammea siamensis (Miq.) T. Anders. (Calophyllaceae) showed anti-proliferative activity against human prostate carcinoma LNCaP cells (IC50 = 2.0 µg/mL). Two new coumarin-related polysubstituted benzofurans, mammeasins P (1) and Q (2), and a known polysubstituted coumarin mammea B/AC cyclo F (39) were isolated from the extract along with 44 previously reported polysubstituted coumarin constituents (3–38 and 40–47). The structures of two new compounds (1 and 2) were determined based on their spectroscopic properties derived from the physicochemical evidence including NMR and MS analyses and taking the plausible generative pathway into account. Among the coumarin constituents, mammeasins A (3, IC50 = 1.2 µM) and B (4, 0.63 µM), sugangin B (18, 1.5 µM), kayeassamins E (24, 3.0 µM) and G (26, 3.5 µM), and mammeas E/BA (40, 0.88 µM), E/BB (41, 0.52 µM), and E/BC (42, 0.12 µM) showed relatively potent anti-proliferative activity.


Introduction
Prostate cancer, a hormonally driven cancer, is the second most frequent malignancy in men worldwide [1][2][3][4]. It may be asymptomatic at an early stage, often has an indolent course, and may require minimal or even no treatment. However, the most frequent complaints are difficulty with urination, increased urination frequency, and nocturia, all of which may also arise from prostatic hypertrophy. A more advanced stage of the disease may present with urinary retention and back pain, as the axial skeleton is the most common site of bone metastasis [4]. The main therapeutic option for advanced prostate cancer is androgen deprivation therapy, which has limited clinical outcomes. However, its therapeutic benefit does not last long, and most patients develop metastatic castration-resistant prostate cancer. After progression into castration-resistant prostate cancer, several chemotherapeutic drugs are used. Chemotherapy is the standard first-line treatment for recurrent metastatic castration-resistant prostate cancer, and relapse eventually occurs due to drug resistance [5]. Therefore, there is a strong demand for the development of new therapeutic molecules against prostate cancer.

Anti-proliferative Effects of the Coumarin Constituents against Human Prostate Carcin LNCaP Cells
Chemical studies on the flowers of M. siamensis allowed the isolation of the above-mentioned new coumarin-related polysubstituted benzofurans, mammeasins

Cell Culture Assay
Experiments were performed in accordance with previously reported methods [40,41], with slight modifications. LNCaP clone FGC (89110211) was purchased from KAC (Kyoto, Japan). Cells were cultured in RPMI 1640 medium (FUJIFILM Wako) supplemented with 10% FBS, 1 mM sodium pyruvate, 100 U/mL penicillin G, and 100 µg/mL streptomycin at 37 • C in a 5% CO 2 environment. LNCaP cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well in 100 µL/well medium, and 100 µL/well of medium containing a test sample was added after an initial incubation of 24 h. Cell viability was detected after 96 h of incubation using the Cell Counting Kit-8 (CCK-8). CCK-8 was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). The O.D. of the yellow-colored formazan Pharmaceuticals 2023, 16, 231 9 of 12 solution was measured using a microplate reader at 450 nm (reference: 650 nm) ( Table S1). The IC 50 value was determined graphically, and the inhibition (%) was calculated using the following formula: Each test compound was dissolved in DMSO and added to the medium (final concentration in 0.1% DMSO).

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

Conclusions
The methanol extract of the flowers of M. siamensis (Miq.) showed anti-proliferative activity against human prostate carcinoma LNCaP cells (IC 50 = 2.0 µg/mL). Two new coumarin-related polysubstituted benzofurans, mammeasins P (1) and Q (2), were isolated, and their structures were elucidated based on their spectroscopic properties derived from the physicochemical evidence including NMR and MS analyses as well as considering the plausible generative pathway. We have already achieved the total syntheses of mammeasins C (5) and D (6), which were isolated as new compounds from the same plant material [42], and we would like to conduct the similar synthetic studies for 1 and 2 to further confirm the stated properties in the future.