Three new Lycopodium alkaloids from Huperzia carinata and Huperzia squarrosa Sakornrat Thorroad a , Prateep Worawittayanont b , Nisachon Khunnawutmanotham c , Nitirat Chimnoi d , Apiwan Jumruksa b , Somsak Ruchirawat a, b, e , Nopporn Thasana a, b, e, * a Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand b Program on Chemical Biology, Chulabhorn Graduate Institute, Laksi, Bangkok 10210, Thailand c Laboratory of Organic Synthesis, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand d Laboratory of Natural Products, Chulabhorn Research Institute, Laksi, Bangkok 10210, Thailand e Center of Excellence on Environmental Health and Toxicology, Commission on Higher Educaton (CHE), Ministry of Education, Bangkok, Thailand article info Article history: Received 18 June 2014 Received in revised form 5 August 2014 Accepted 18 August 2014 Available online 23 August 2014 Keywords: Huperziaceae Huperzia carinata Huperzia squarrosa Lycopodium alkaloid Acetylcholine esterase abstract Eleven Lycopodium alkaloids with lycodine-type, lycopodine-type, and fawcettimine-related skeletons were isolated from the whole plants of Huperzia carinata (Desv. Ex. Poir.) Trevis and Huperzia squarrosa (G. Forst) Trevis (Huperziaceae). Among them, 8,15-dihydrohuperzine A (2), lycocarinatine A (3), and lycoposerramine U N-oxide (11) are new compounds. The structures of these new alkaloids were elu- cidated on the basis of 2D NMR correlations. Some of these isolated alkaloids were assayed for acetyl- cholinesterase (AChE) inhibitory activity. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Huperziaceae plants have been reported to contain Lycopodium alkaloids having complex skeleton characteristics 1 with a well known biological activity for acetylcholinesterase (AChE) in- hibition. 2 Since huperzine A (1) was first isolated from Huperzia serrata (Thunb. ex Murray) Trevis, a traditional Chinese folk med- icine, 3 it has inspired many research groups to study the phyto- chemical constituents in the Lycopodium plants. 4 The Lycopodium plants are abundantly found in the Indo-Pacific region, however, the investigation of the alkaloid constituents of these club mosses in Thailand has been lacking. We have previously reported the isolation and structure elucidation of triterpenoids from Lycopo- dium phlegmaria. 5 As part of our ongoing research of Lycopodium plants, we re- ported here the investigation of the constituents of Huperzia car- inata (Desv. Ex. Poir.) Trevis and Huperzia squarrosa (G. Forst.) Trevis (Huperziaceae), two of the club mosses found in Thailand and Malaysian peninsula. 6 Our study revealed the diversity of various structural types of the Lycopodium alkaloids in these club mosses. In this paper, we described the isolation and structure elucidation of three new alkaloids (2, 3, and 11). The AChE inhibitory activity of some of these isolated alkaloids is also investigated. 2. Results and discussion H. carinata was purchased from a plant market in Bangkok, Thailand and was macerated and extracted with MeOH. The alka- loid extract was prepared by acidebase extraction, and purified by repeated column chromatography using Sephadex-LH20 and HPLC to afford two new alkaloids named 8,15-dihydrohuperzine A (2, 0.000028%), and lycocarinatine A (3, 0.000005%) together with nine known alkaloids including huperzine A (1 , 0.0008%), 3 12- epilycodine N-oxide (4, 0.00013%), 7 lycodoline (5, 0.000055%), 8,9 gnidioidine (6, 0.00021%), 9 lycoposerramine K (7 , 0.000037%), 10 fawcettimine (8, 0.000031%), 11 and phlegmariurine B (9, 0.000039%). 12 H. squarrosa was also purchased from the same plant market in Bangkok, Thailand. The alkaloid extract was prepared by the pro- cedure similar to that used for H. carinata. The alkaloid fractions were purified bycolumn chromatography using Sephadex-LH20 and HPLC to afford a new fawcettimine-related alkaloid named lycoposerr- amine U N-oxide (13, 0.000028%), together with four known * Corresponding author. Tel.: þ66 2553 8555; fax: þ66 2553 8527; e-mail address: nopporn@cri.or.th (N. Thasana). Contents lists available at ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet http://dx.doi.org/10.1016/j.tet.2014.08.042 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved. Tetrahedron 70 (2014) 8017e8022