A fresh chloro-trinoreremophilane sesquiterpene 1 three fresh chlorinated eremophilane sesquiterpenes 2-4 together with a known compound eremofortine C (5) were isolated from an Antarctic deep-sea derived fungus sp. Ocean is inherently regarded as a harsh habitat for native microorganisms due to perpetual low temps and lack of nutrients among additional factors [1 2 However in the case of fungi inhabiting the Antarctic deep sea the aforementioned intense conditions arranged the manifestation of unusual biosynthetic mechanisms that may lead to unique secondary metabolites [3]. Undeniably the exploitation of these peculiar metabolic pathways represents a new chance for the finding of bioactive secondary metabolites [4]. Therefore the research community has been urged to explore the untapped metabolic reservoir originating from deep-sea fungi in order to combat human diseases [5]. In our efforts to search for novel active compounds from your secondary metabolites of deep-sea derived microorganisms [6 7 8 a fungus identified as sp. PR19N-1 was from a deep-sea sediment collected in Prydz Bay (?1000 m). Its draw out exhibited brine shrimp lethality activity. Studies on the active constituents of this fungus led to the isolation of four fresh chlorinated eremophilane sesquiterpenes 1-4 along with a known compound eremofortine C (5) [9 10 (Number 1). Herein we describe their isolation structure elucidation and in vitro cytotoxicity evaluation. Number GRS 1 Constructions of compounds 1-5. 2 Results and Conversation Compound 1 was acquired as an optically active colorless oil. The molecular method CX-4945 of C14H15ClO4 was founded through HRESIMS data ([M + Na]+ 305.0543 calcd. 305.0557) indicating seven two times relationship equivalents. The IR spectrum showed absorption bands characteristic for hydroxyl carbonyl and double relationship moieties at 3292 1731 1634 cm?1 respectively. One-dimensional NMR data (Table 1 Table 2) unveiled 8 sp2 deshielded carbons (1 × OC=O 3 × CH=C 1 × C=O) and 6 sp3 shielded carbons (3 × CH3 2 × CH 1 × C) indicating the presence of two rings in the molecule. The two fused six-membered rings were defined by considerable analysis of HMBC mix peaks from your diagnostic methyls H3-11 to C-4 C-5 C-6 and C-10 H3-12 to C-3 C-4 C-5 as well as from your olefinic protons H-2 to C-1 C-3 C-4 and C-10 H-6 to C-4 C-5 C-8 C-10 and C-11 and H-9 to C-1 C-5 C-7 and C-10. Considerable analysis of MS and NMR data led us to a trinor-eremophilene core CX-4945 with an 8-oxo-1(2) 9 unit [11 12 The hydroxyl group attached to C-7 was situated using HMBC correlations (Number 2) between the exchangeable proton (OH-7) and C-6 C-7 and C-8. In addition an acetoxy group was assigned to C-3 via HMBC correlations between H-14 and C-13 and between H-3 and C-13. Furthermore the COSY-defined spin system H-2/H-3/H-4/H3-12 along with the lack of an olefinic proton transmission at C-1 in the HMQC spectrum indicated the location of a chlorine atom at C-1. Table 1 13 NMR data for compounds 1-4 (150 MHz δ ppm). Table 2 1 NMR data for compounds 1-4 (600 MHz δ ppm in Hz). Number 2 The key 2D NMR correlations for compounds 1-4. The relative configuration of the trinor-eremophilane core was deduced on the basis of NOE-difference experiments (Number 2). The resonances of H-12 and H-14 were notably enhanced as a result of irradiating CH3-11 indicating that the axial-methyl at C-5 the equatorial-methyl at C-4 and the acetyl group at CX-4945 C-3 were co-facial. The coupling constants (3299.1060 (calcd. 299.1050). Important 1H and 13C NMR resonances (Table 1 Table 2) especially for the shielded methyl organizations at δH 1.04 (= 7.0 Hz) and δH 1.20 led us to consider an eremophilane-type sesquiterpene skeleton for 2. The presence of an epoxide moiety with 13C peaks at C-7 (δC 61.8) and C-11 (δC 67.3) was suggested by comparison of the 13C NMR spectrum with those of 5a [9 10 and was confirmed by HMBC CX-4945 correlations (Number 2) from H-13 to C-7 C-11 and C-12 from H2-6 to C-7 C-10 C-11 and C-14 and from H-12 to CX-4945 C-7 C-11 and C-13. According to the 1H-1H COSY correlation between 12-OH and H2-12 the sole primary alcohol was also located at C-12. Therefore the above evidence suggested 2 and 5a experienced the same substructure b (Number 1) [9 10 Careful analysis of the NMR data of 2 indicated the ring A was related to that in compound 1. The main differences of CX-4945 them were the 3-acetoxy group replaced by 3-OH which was confirmed by.