Norstaminane- and isopimarane-type diterpenes of Orthosiphon stamineus from Okinawa Suresh Awale, a Yasuhiro Tezuka, a Arjun H. Banskota, a Seikichi Shimoji, b Kazuhiko Taira c and Shigetoshi Kadota a,p a Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630-Sugitani, Toyama 930-0194, Japan b Okinawa Health Food Development Cooperative, 1075 Tsuhako aza Sashiki, Okinawa 901-1414, Japan c Faculty of Education, University of the Ryukyus, 1-Senbaru, Nishihara-cho, Okinawa 903-0129, Japan Received 10 April 2002; accepted 13 May 2002 Abstract ÐNine novel highly oxygenated and structurally diverse diterpenes, named norstaminolactone A (1), norstaminols B and C (2 and 3), secoorthosiphols A±C (4 ± 6) and orthosiphols R±T (7 ± 9) have been isolated from the aerial part of Orthosiphon stamineus cultivated in Okinawa Prefecture, Japan. Norstaminolactone A (1) is the ®rst representative of a biogenetically unusual norstaminane-type diterpene bearing a nitrogen atom. Norstaminol C (3) possessed a framework presumed to be biosynthesized from the staminane-type diterpene. Secoorthosiphols A±C (4 ± 6) possessed an unprecedented structural feature of the opened ring A system, encountered for the ®rst time in isopimarane-type diterpenes. Secoorothosiphol C (6) also represents the ®rst example of biogenetically unique and unconventional secoisopimarane-type diterpene bearing a cyano group. Norstaminolactone A (1) showed a potent antiproliferative activity with an IC 50 value of 2.16 mg/mL against highly liver metastatic colon 26-L5 carcinoma cell line. q 2002 Elsevier Science Ltd. All rights reserved. 1. Introduction Traditional medicines are widely used alongside with modern medicine in many countries of Southeast Asia and play an important role in promoting a health care system. 1 Orthosiphon (O.) stamineus Benth. [syn.: O. aristatus (Bl.) Miq., O. grandi¯orus Bold., O. spicatus (Thumb) Bak.; Lamiaceae] is one of the popular traditional folk medicine extensively used in Southeast Asia for the treatment of wide range of diseases: in Indonesia for rheumatism, diabetes, hypertension, tonsillitis, epilepsy, menstrual disorder, gonorrhea, syphilus, renal calculus, gallstone etc.; 2 in Vietnam for urinary lithiasis, edema, eruptive fever, in¯uenza, hepatitis, jaundice and biliary lithiasis; 3 and in Myanmar to alleviate diabetes, urinary tract and renal diseases. 4 Owing to its bene®cial pharmaceutical utility, it is under systematic cultivation in Okinawa Prefecture, Japan and is locally known as `Neko no hige' or, `Kumis kucing' both meaning `Cats whisker', and consumed as a healthy Java tea to facilitate body detoxi®cation. In our search for cancer antiproliferative agents from natural sources, we isolated a series of highly oxygenated isopimarane-type diterpenes and the diterpenes with a novel carbon-frame- work named `staminane' from Orthosiphon stamineus of Vietnam and Myanmar. 5±8 Likewise, from Indonesian O. stamineus, neoorthosiphols A and B with the `staminane' carbon-framework were reported by Shibuya et al. 9 However, there are no reports on the staminane-type diterpenes in O. stamineus from Okinawa, though they have been reported to contain isopimarane-type diterpenes, orthosiphols A, B, D and E. 10,11 Thus, we have investigated the constituents of O. stamineus cultivated in Okinawa Prefecture and isolated nine novel highly oxygenated diterpenes having biogenetically diverse structures based on the staminane and isopimarane carbon-frameworks. We herein report the isolation and structure elucidation of the new diterpenes by spectroscopic techniques as well as chemical methods, together with their antiproliferative activities (Chart 1). 2. Results and discussion Air-dried aerial parts of O. stamineus from Okinawa were extracted with re¯uxing MeOH and the MeOH extract was successively partitioned into hexane, CHCl 3 , EtOAc, BuOH and H 2 O fractions. The CHCl 3 fraction was subjected to a series of chromatographic separation and preparative TLC to afford nine new highly oxygenated diterpenes together with nine known diterpenes. The new diterpenes were one 16-norstaminane-type diterpene named norstaminolactone A(1), two 14-norstaminane-type diterpenes named nor- staminolactone A (1) and norstaminols B (2) and C (3), three secoisopimarane-type dieterpenes named seco- orthosiphols A±C (4 ± 6) and three isopimarane-type Tetrahedron 58 (2002) 5503±5512 Pergamon TETRAHEDRON 0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0040-4020(02)00508-2 Keywords: Orthosiphon stamineus; staminane; norstaminolactone A; isopimarane; secoorthosiphol; antiproliferative activity. p Corresponding author. Tel.: 181-76-434-2281x2825; fax: 181-76-434- 5059; e-mail: kadota@ms.toyama-mpu.ac.jp