880 Chem. Pharm. Bull. 64, 880–885 (2016) Vol. 64, No. 7 Special Collection of Papers This article is dedicated to Professor Satoshi Ōmura in celebration of his 2015 Nobel Prize. Regular Article Aromatase Inhibitory Activity of Geranylated Coumarins, Mammeasins C and D, Isolated from the Flowers of Mammea siamensis Kiyofumi Ninomiya,a Kanae Shibatani,a Mayumi Sueyoshi,a Saowanee Chaipech,a,b Yutana Pongpiriyadacha,c Takao Hayakawa,a Osamu Muraoka,a and Toshio Morikawa*,a a Pharmaceutical Research and Technology Institute, Kindai University; 3–4–1 Kowakae, Higashi-osaka, Osaka 577–8502, Japan: b Faculty of Agro-Industry, Rajamangala University of Technology Srivijaya; Thungsong, Nakhonsithammarat 80110, Thailand: and c Faculty of Science and Technology, Rajamangala University of Technology Srivijaya; Thungsong, Nakhonsithammarat 80110, Thailand. Received March 2, 2016; accepted March 17, 2016 A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit enzymatic activity against aromatase (IC5016.5 µg/mL). From the extract, two new geranylated coumarins, mam- measins C (1) and D (2), were isolated together with seven coumarins: 8-hydroxy-5-methyl-7-(3,7-dimethyl- octa-2,6-dienyl)-9-(2-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (9), 8-hydroxy-5-meth- yl-7-(3,7-dimethyl-octa-2,6-dienyl)-9-(3-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2-de]chromen-2-one (10), mammeas A/AA (14), A/AB (15), A/AA cyclo D (18), E/BA (23), and E/BC cyclo D (25). The structures of 1 and 2 were elucidated on the basis of spectroscopic evidence. Among the isolates including 17 previously reported coumarins, 1 (IC502.7 µM), 2 (3.6 µM), and mammea B/AB cyclo D (21, 3.1 µM) showed relatively strong inhibitory activities comparable to the activity of the synthetic nonsteroidal aromatase inhibitor ami- noglutethimide (2.0 µM). Key words Mammea siamensis; mammeasin; aromatase inhibitor; geranylated coumarin; Calophyllaceae Mammea siamensis (MIQ.) T. ANDERS. is a species of flower- estrogens and estrogen receptors are widely studied molecular ing plant in the Calophyllaceae family and is widely distrib- targets.28–30) The presence of high concentrations of estrogen uted in Thailand, Laos, Cambodia, Vietnam, and Myanmar. in breast tissue increases the risk of developing breast cancer The flowers of this plant have been used for preparing a heart and the ability of immature breast tissue cells to strongly bind tonic in Thai traditional medicine (“Sarapi” in Thai).1–10) to carcinogens, decreasing their DNA repair capacity.31,32) Several coumarins,1–7) xanthones,8,9) triterpenoids,10) and Aromatase, a CYP19 enzyme, is the rate-limiting enzyme steroids10) have been isolated from the flowers,1,2,6,7,10) seeds,3,9) in the conversion of testosterone and androstenedione to the twigs,4,8) and bark5) of this plant. In the course of our char- estrogens, estrone and estradiol.26–30,32–34) It is involved in the acterization studies on bioactive constituents in Thai natural final step of the estrogen biosynthetic pathway and its selec- medicine,1,11–25) we reported that the methanol extract of the tive inhibition will not affect the production of other steroids flowers of M. siamensis and its coumarin constituents showed in the pathway.32,35–37) The source of estrogen production in inhibitory effects on nitric oxide production in lipopolysac- breast cancer tissues is intratumoral aromatase, and thus, inhi- charide-activated RAW264.7 cells.1) Further studies revealed bition of aromatase may inhibit the growth stimulation effect that the methanol extract inhibited enzymatic activity against of estrogens in breast cancer tissues. Therefore, aromatase is aromatase. Separation of the active constituents in the extract considered a useful therapeutic target in the treatment and allowed us to isolate two new geranylated coumarins, mam- prevention of estrogen-dependent breast cancer.32) measins C (1) and D (2). This paper describes the isolation The dried flowers of M. siamensis (collected from Na- and structure elucidation of these new coumarins (1, 2) and khonsithammarat Province, Thailand) were extracted with the inhibitory effects of the coumarin constituents (1–26) on methanol under reflux (25.66% from the dried flowers). The 26) aromatase. methanol extract was partitioned into an EtOAc–H2O (1 : 1, v/v) mixture to furnish an EtOAc-soluble fraction (6.84%) Results and Discussion and an aqueous phase. The aqueous phase was subjected to Effects of the Methanol Extract from the Flowers of M. Diaion HP-20 column chromatography (H2O→MeOH) to give siamensis against Human Recombinant Aromatase Breast H2O- and MeOH-eluted fractions (13.50, 4.22%, respectively), cancer is one of the most common reasons for mortality in as described previously.1) As shown in Table 1, the methanol 26,27) women. Estrogens and estrogen receptors are well known extract had an inhibitory effect on aromatase (IC50=16.5 µg/ to play an important role in the development and progres- mL). A bioassay-guided fractionation revealed that the EtOAc- sion of hormone-dependent breast cancer; for this reason, soluble and MeOH-eluted fractions also showed aromatase in- * To whom correspondence should be addressed. e-mail: [email protected] © 2016 The Pharmaceutical Society of Japan Vol. 64, No. 7 (2016) Chem. Pharm. Bull. 881 Table 1. Inhibitory Effects of the Methanol Extract from the Flowers of M. siamensis and Its Fractions against Human Recombinant Aromatase Inhibition (%) IC50 (µg/mL) 3 µg/mL 10 µg/mL 30 µg/mL 100 µg/mL MeOH Extract 13.2±3.2 41.8±1.6** 80.0±3.1** 97.5±0.6** 16.5 EtOAc-Soluble fraction 46.3±4.4** 89.0±1.2** 100.1±0.6** 99.9±0.5** 2.9 MeOH-Eluted fraction 2.0±3.9 70.7±1.2** 93.0±1.0** 96.9±0.9** 8.5 H2O-Eluted fraction −4.7±4.8 1.2±2.2 −1.7±2.1 −0.1±4.0 >100 Each value represents the mean±S.E.M. (N=3). Significantly different from the control, ** p<0.01. Fig. 1. Coumarin Constituents (1–26) from Flowers of M. siamensis hibitory activities (IC50=2.9, 8.5 µg/mL, respectively), whereas dienyl)-9-(2-methyl-1-oxobutyl)-4,5-dihydropyrano[4,3,2- 4) the H2O-eluted fraction showed no noticeable activity. de] chromen-2-one (9, 0.0015%), 8-hydroxy-5-methyl-7- Isolation of Coumarin Constituents from the Methanol (3,7-dimethyl-octa-2,6-dienyl)-9-(3-methyl-1-oxobutyl)-4,5- Extract In our previous report we described the isola- dihydropyrano[4,3,2-de] chromen-2-one4) (10, 0.0012%), mam- tion of 17 coumarins: mammeasins A (3, 0.0293%) and meas A/AA38) (14, 0.0494%), A/AB38) (15, 0.0048%), A/AA B (4, 0.0115%), surangins B (5, 0.0271%), C (6, 0.0571%), cyclo D38) (18, 0.0035%), E/BA39) (23, 0.0045%), and E/BC and D (7, 0.0632%), kayeassamins A (8, 0.0578%), E (11, cyclo D2) (25, 0.0058%), using normal-phase silica gel and 0.0113%), F (12, 0.0390%), and G (13, 0.0171%), mam- reversed-phase octadecylsilane (ODS) column chromatogra- measins A/AC (16, 0.1056%), A/AD (17, 0.0022%), A/AB phy, and finally, HPLC (Fig. 1). cyclo D (19, 0.0097%), A/AC cyclo D (20, 0.0109%), B/AB Structures of Mammeasins C (1) and D (2) Mammea- cyclo D (21, 0.0016%), B/AC cyclo D (22, 0.0062%), and E/ sin C (1) was obtained as pale yellow oil. Its IR spectrum BB (24, 0.0194%), and deacetylmammea E/BC cyclo D (26, showed absorption bands at 1748 and 1634 cm−1 assignable to 0.0073%), β-amyrin (0.0072%), and benzoic acid (0.0043%).1) an α,β-unsaturated γ-lactone moiety and a chelated aryl keto In the present study we additionally isolated two new gera- group.1,2,4) The UV spectrum exhibited absorption maxima nylated coumarins, mammeasins C (1, 0.0008%) and D (2, at 221, 292, and 328 nm, similar to those of 5,7-dioxygenated 0.0047%), from the active EtOAc-soluble fraction together with coumarins.1,2,4) The electron ionization (EI)-MS spectrum of 1 seven coumarins: 8-hydroxy-5-methyl-7-(3,7-dimethyl-octa-2,6- showed a molecular ion peak at m/z 424 (M+), and the molecu- 882 Chem. Pharm. Bull. Vol. 64, No. 7 (2016) lar formula was determined as C26H32O5 by high-resolution of 1 were quite similar to those of 9 and 10, except for the sig- (HR)-EI-MS measurement. The 1H- and 13C-NMR spectra of 1 nals due to the 1-oxo-alkyl moiety.4) The 1H–1H COSY experi- (Tables 2, 3, CDCl3) were assigned with the aid of distortion- ment on 1 indicated the presence of partial structures, as in- less enhancement by polarization transfer (DEPT), 1H–1H cor- dicated by the bold lines in Fig. 2. In the HMBC experiment, relation spectroscopy (COSY), 1H-detected heteronuclear mul- long-range correlations were observed between the following tiple quantum coherence (HMQC), and heteronuclear multiple proton and carbon pairs: 3-H and 2,4,6b-C; 4-H and 3,6b-C; bond connectivity (HMBC) experiments (Fig. 2). The spectra 1″-H2 and 6a,7,8-C; 2″-H and 7,3″,5″-C; 4″-H2 and 3″-C; 5″-H3 showed signals assignable to three secondary and three vinyl and 2″–4″-C; 7″-H2 and 9″,10″-C; 9″-H3 and 7″,8″,10″-C; 10″-H3 methyls [δ 1.26, 1.27 (3H each, both d, J=6.6 Hz, 3‴-H3, and 7″–9″-C; 2‴-H and 1‴-C.