Malignancies are a kind of the most serious diseases that damage human health in the modern world. Although the efficacy of chemotherapy for the majority of cancer types has improved over the last three decades,the high toxic effects of chemotherapeutic drugs,causing a severe reduction in quality of life,are still formidable problems in clinical medicine^[1]. Therefore,many researchers have begun to investigate natural products,due to their antitumor activities and low side-effects^[2].

Citrinin derivatives are naturally occurring polyketone compounds from microbes,which have been reported to possess many kinds of bioactivities,such as enzyme inhibitory^[3],antifouling^[3],antioxidant^[4],antitumor^[8] and antimicrobial^[9] properties. Among these bioactivities,the antitumor property is getting the most attention. Penicillium citrinum is a rich source of various citrinin derivatives^[10]. Our previous chemical investigation of P. citrinum resulted in the isolation of a new citrinin derivative,namely,citrinacetal^[4]. Our continuing search for bioactive compounds from this organism has further resulted in the isolation of another new derivative,namely,penicitrinol L (1),along with two known analogues penidicitrinin B (2)^[11] and pennicitrinone A (3)^[3] (Figure 1). In this paper,we report the isolation,structural elucidation and bioactivity of these metabolites.

Figure 1 Structures of compounds 1-3

Compound 1,which is trivially named as penicitrinol L,was obtained as yellow gum and was analyzed to have the molecular formula C₁₈H₂₀O₄ through negative high-resolution electrospray ionization mass spectroscopy (HR-ESI-MS) (m/z: 299.126 4 [M-H]^-,Calcd. for C₁₈H₁₉O₄: 299.128 3). Its NMR data (Table 1) revealed eighteen carbon signals,including three methyls,eight methines,and seven quaternary carbons.

Table

Table 1 NMR data for 1 (500 MHz for 1H,125 MHz for 13C,DMSO-d6) Position δC δH (J in Hz) COSY HMBC (H→C) 1 71.4 5.54 (1H,s) 3,8,9,10,1',2',6' 3 71.2 3.48 (1H,m) 4,11 1,10,11,12 4 35.9 2.56 (1H,m) 3,12 3,9,10,11,12 5 111.7 6 154.6 7 99.8 6.19 (1H,s) 5,6,8,9 8 151.4 9 113.7 10 138.5 11 19.5 1.10 (3H,d,6.4) 3 3,4 12 19.4 1.05 (3H,d,6.8) 4 3,4,10 13 10.8 1.97 (3H,s) 5,6,10 1' 133.0 2'/6' 129.3 6.93 (2H,d,8.5) 3'/5' 1,4' 3'/5' 114.3 6.63 (2H,d,8.5) 2'/6' 1',4' 4' 156.2 4'-OH 9.21 (1H,s) 3',4',5' 6-OH 8.93 (1H,s) 5,6,7 8-OH 8.74 (1H,s) 7,8,9

Table 1 NMR data for 1 (500 MHz for 1H,125 MHz for 13C,DMSO-d6)

The plane structure of 1 was revealed through COSY and HMBC spectrum analyses (Table 1). The COSY correlations of H-2'/6' with H-3'/5',as well as the HMBC correlations from H-2'/6' to C-4',H-3'/5' to C-1' and C-4',and 4'-OH to C-3',C-4' and C-5' demonstrated the existence of a 1,4-substituted benzene ring. The COSY correlations of H-3 with H-4 and H-11,and H-4 with H-3 and H-12,as well as the HMBC correlations from H-1 to C-3,C-9 and C-10,H-3 to C-1 and C-10,and H-4 to C-9 and C-10 demonstrated the existence of a multisubstituted dihydropyrane. The HMBC correlations of H-13 with C-5,C-6 and C-10,H-7 with C-5,C-6,C-8 and C-9,6-OH with C-5,C-6 and C-7,as well as 8-OH with C-7,C-8 and C-9 linked the second benzene ring to the dihydropyrane via C-9 and C-10. Furthermore,the HMBC correlations of H-1 with C-1',C-2' and C-6',as well as H-2'/6' with C-1 linked first benzene ring to the dihydropyrane via C-1 and C-1'.

The relative configuration of 1 was assigned on the basis of the NOESY correlations of H-1 and H-4 with H-11 as well as H-3 with H-12 as shown in Figure 2. The absolute configuration of 1 was determined by comparison of its specific optical rotation value ([α]_D +60.5) with those of penicitrinol C ([α]_D +33.2)^[1] and penicitrinol F ([α]_D +24.8)^[2],which suggested that the absolute configuration of 1 was also 1R,3R and 4S. So,the structure of 1 was elucidated as shown in Figure 1 and named as penicitrinol L.

Figure 2 Key NOESY correlations of compound 1

The cytotoxic effects of compounds 1-3 were evaluated using the MTT method on the HL-60 and A375 cancer cell lines^[3]. Compound 1 showed modest cytotoxic activity against HL-60 cell line with the IC₅₀ value of 22.7 μg·mL^-1,while compound 3 showed weak cytotoxic activity against A375 cell line with the IC₅₀ value of 65.4 μg·mL^-1.

Optical rotations were obtained from a Shenguang SGW-1 digital polarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer. ¹H NMR,¹³C NMR,DEPT spectra and 2D NMR were recorded on a BRUKER BIOSPIN AVANCE III spectrometer using TMS as the internal standard. ESI-MS were obtained by an AGILENT 1200/Q-TOF 6510 LC mass spectrometer. Semipreparative HPLC was performed using an ODS column (ODS-A,10 mm × 250 mm,5 µm) at 5 mL·min^-1.

The fungus P. citrinumwas isolated from marine sediments collected from Langqi Island,Fujian,China. It was identified according to its morphological characteristics and ITS by Beijing Sunbiotech Co.,Ltd,and preserved in our laboratory at -80 ℃. The producing strain was prepared on Martin medium and stored at 4 ℃.

The fungus was cultured under static conditions at 28 ℃ for 30 days in 1 000 mL conical flasks containing the liquid medium (400 mL/flask) composed of soytone (0.1%),soluble starch (1.0%),and seawater (60%). The fermented whole broth (80 L) was filtered through cheesecloth to separate the supernatant from the mycelia. The former was concentrated under reduced pressure to about a quarter of the original volume and then extracted three times with EtOAc to give an EtOAc solution,while the latter was extracted two times with acetone. The acetone solution was concentrated under reduced pressure to afford an aqueous solution. The aqueous solution was extracted two times with EtOAc to give another EtOAc solution. Both EtOAc solutions were combined and concentrated under reduced pressure to give a crude extract (18.9 g).

The crude extract (18.9 g) was separated into 8 fractions on a silica gel column using a step gradient elution of petroleum ether,CH₂Cl₂,and MeOH. Fraction 3 (3.1 g) eluted with petroleum ether/CH₂Cl₂ (1∶3) was further purified on a silica gel column using a step gradient elution of petroleum ether,CH₂Cl₂,and MeOH. Subfraction 3-2 (0.9 g) eluted with CH₂Cl₂ was further purified on a Sephadex LH-20 (CH₂Cl₂/MeOH,1∶1),a reversed-phase column (MeOH/H₂O,3∶2) and semipreparative HPLC (45% MeCN),yielding compounds 1 (5.3 mg),2 (4.8 mg),and 3(8.1 mg).

Penicitrinol L (1): yellow gum; [α]_D²⁵ +60.5 (c 0.1,MeOH); ¹H and ¹³C NMR see Table 1; HR-ESI-MS m/z 299.126 4 [M-H]^- (Calcd for C₁₈H₁₉O₄: 299.128 3); UV λ_max (CH₃CN) nm(log ε): 284 (3.44).

Cytotoxic activity was evaluated by the MTT method^[11,12]. The IC₅₀ values were obtained using the Bliss method.