Citation

Na Gao, Zhi-Chun Shang, Pei Yu, Jie Luo, Kai-Li Jian, Ling-Yi Kong, Ming-Hua Yang. Alkaloids from the endophytic fungus Penicillium brefeldianum and their cytotoxic activities[J]. Chin. Chem. Lett., 2017, 28(6): 1194-1199  

Na Gao, Zhi-Chun Shang, Pei Yu, Jie Luo, Kai-Li Jian, Ling-Yi Kong, Ming-Hua Yang    

Chemical investigations have revealed that Penicillium spp. are proficient in using different synthases, like polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), to creat a vast variety of secondary metabolites [1-5]. Various metabolites with wide ranges of bioactivties, such as anticancer, antimicrobial, anti-inflammatory and insecticidal were isolated from the culture of Penicillium spp. [6-9]. It makes the Penicillium genus a rich and hot source of natural products. P. brefeldianum is one of the well kown Penicillium sp. Its famous product is the 16-membered macrolide antibiotic brefeldin A, an intracellular protein transport inhibitor that also can lead to differentiation and apoptosis of a wide variety of human cancer cells [10, 11]. Recently, during the investigation of an endophytic P. brefeldianum XMK-2 from the rhizome of Pinellia ternata, we isolated one new indoloditerpene, 6, 7-dehydropaxilline (1), one new indole-diketopiperazine, spirotryprostatin F (2), and one new 13-membered macrolide, N-demethylmelearoride A (3), together with 8 known indoloditerpene and indole-diketopiperazine alkaloids (Fig. 1). To the best of our knowledge, the indoloditerpenes and the 13-membered macrolide were reported from this species for the fist time. Meanwhile, when evaluating their bioactivities, these metabolites showed different cytotoxic activities against three human tumor cell lines. The new compounds 2 and 3 showed moderate cytotoxicities against HepG2 and MDA-MB-231 cells, and the known 11 was the most effective one that comparable to the positive control cisplatin. Herein, the details of isolation and structural elucidation of 1-3 are presented, in addition with the cytotoxic evaluation of all the isolates.

Figure 1

	Download: JPG larger image
Fig. 1. Structures of compounds 1-11.

Compound 1 was obstained as a yellow powder. Its molecular formula C₂₇H₃₁NO₄ was determined by the positive HRESIMS ion at m/z 434.2325 [M+H]⁺ (calcd.for C₂₇H₃₂NO₄, 434.2326), revealing 13 degrees of unsaturation. The ESIMS fragment ion at m/z 130 [(C₉H₈N)⁺], as well as the characteristic UV absorptions at 229 and 277 nm, indicated the presence of an indole moiety in 1 [12, 13]. Its ¹H NMR data (Table 1) also displayed the diagnostic indole singals of the NH at δ_H 7.74 (br s) and the four aromatic protons at δ_H 7.45 (dd, 1H, J = 6.3, 1.9 Hz, H-20), 7.31 (dd, 1H, J = 6.7, 1.9 Hz, H-23), and 7.10 (m, 2H, H-21, 22). The remaining protons were two olefinic signals at δ_H 6.04 (br s, 1H, H-11) and 5.76 (m, 1H, H-6), four methyl singlets at δ_H 1.37 (s, 3H, H₃-25), 1.33 (s, 3H, H₃-28), 1.28 (s, 3H, H₃-29), and 1.07 (s, 3H, H₃-26), four methylenes at δ_H 3.27 (d, 1H, J = 17.7 Hz, H-5a)/2.13 (dd, 1H, J = 17.7, 6.4 Hz, H-5b), 2.75 (dd, 1H, J = 12.0, 6.3 Hz, H-17b)/2.47 (dd, 1H, J = 12.0, 10.1 Hz, H-17a), 2.00 (m, 1H, H-15a)/1.84 (m, 1H, H-15b), and 1.98 (m, 1H, H-14a)/1.96 (m, 1H, H-14b), and two methines at δ_H 4.23 (br s, 1H, H-9) and 2.81 (m, 1H, H-16). Based on the HSQC spectrum, all the above corresponding carbons could be found in its ¹³C NMR spectrum (Table 1), which also showed additional resonances of one carbonyl at δ_C 196.9 and two oxygenated quaternary carbons at δ_C 76.0 and 73.9. These functional groups accounted for 9 indices of hydrogen deficiency, and the remaining four ones implied other tetracyclic rings in 1. Further NMR data comparison with those of 13-desoxypaxilline (5) revealed the same carbon skeleton of them, which was confimred by the HMBC correlations (Fig. 2). Differently, the HMBC correlations between H-11/H₃-26/H-5b and C-13 (δ_C 76.0) revealed the hydroxylation of C-13. The HMBC correlations from H-6 to C-4/C-7/C-12, from H₂-5 to C-6/C-7, and from H-11 to C-7 also established the double bond at Δ⁶⁽⁷⁾. Therefore, its planar structure was identified as 6, 7-dehydropaxilline.

Table 1

Table 1 1H NMR and 13C NMR spectroscopic data of compounds 1-3.

Table 1 1H NMR and 13C NMR spectroscopic data of compounds 1-3.

Figure 2

	Download: JPG larger image
Fig. 2. The key HMBC and ROESY correlations of 1.

The relative configuration of 1 was established by ROESY experiment (Fig. 2). The correlations of H₃-26/H-14b/H-16 indicated that they were on the same face of the chair form of ring D. The ROESY correlations between H₃-25 and H-15a/H-14a/ H-5a placed them in the opposite facial orientation. Therefore, the D/E rings adopted the trans positional relationship [13, 14], and consequently 13-OH was in the α-orientation of ring D. Furthermore, the ROESY correlations between H₃-26 and H-5b, between H₃-26 and gem-dimethyl H₃-28/H₃-29, implied the a-orientation of H-9.

The absolute configuration of compound 1 was determined by the circular dichroism (CD) exciton chirality method [15-17]. In the CD spectrum of compound 1 (Fig. 3), a negative cotton effect of the α, β, γ, δ-unsaturated ketone (C-6/7/12/11/10) at 239 nm (Δε -21.2) and a positive cotton effect of the indole moiety at 224 nm (Δε +7.0) were observed. The negative chirality arose from the anticlockwise manner of above two chromophores in space, which was only possible under the 3S, 4R, 13S, and 16S configurations. Thus, compound 1 was determined as description.

Figure 3

	Download: JPG larger image
Fig. 3. The CD spectra of 1 (A) and 2 (B) in MeOH.

Compound 2 was isolated as a pale yellow powder. On the basis of positive HRESIMS ion at m/z 462.2385 [M+H]⁺ (calcd. for C₂₇H₃₂N₃O₄, 462.2387), its molecular formula was determined as C₂₇H₃₁N₃O₄. Analysis of its 1D NMR data established the similar indole-diketopiperazine to 10 and 11, especially when observing the 1, 2, 4-trisubstituted benzene ring, the two prenyl, the proline residue, and three amide/carbonyl alike carbons (Table 1). But since the additional olefinic proton at δ_H 5.72 (br s, 1H, H-10) in 2 gave HMBC correlations (Fig. 4) to C-11 and C-19, the double bond at Δ¹⁰⁽¹¹⁾ was established. More importantly, H-10 showed HMBC correlations to C-4, C-20 and the distinctive quaternary carbon C-3 at δ_C 61.4, and both H-20 and H₂-25 had HMBC correlations with the carbonyl C-2 at δ_C 176.7. These key evidence indicated that the sprio[pyrrolidin-3, 3'-oxindole] ring system in 2 was similar to spirotryprostatin B [18, 19], which also explained the HMBC correlations from H-20 to C-3, C-4 and C-10. Further analyzing the HMBC correlations confirmed its planar structure.

Figure 4

	Download: JPG larger image
Fig. 4. The key HMBC and ROESY correlations of 2.

The relative configuration of 2 was elucidated as the same as that of spirotryprostatin B. The ROESY correlations (Fig. 4) between H-5/H-21/H-20 and H-10, indicated that the two planes of the sprioring system were arranged in an approximately vertical manner and that H-5 and H-21 are in close spatial proximity. The ROESY correlation between H-14 and H-20 showed that they were in the same facial orientation.

The CD exciton chirality method was also used to determine the absolute configuration of 2. In its CD spectrum (Fig. 3), a positive cotton effect was observed, which originated from the exciton coupling between chromophores of the α, β-unsaturated amide (C-10/11/19) at 242 nm (Δε +62.0) and the oxindole moiety at 221 nm (Δε -46.7). The clockwise manner of two chromophores in space established the absolute configuration of C-3 (3S). Combined with the relative configuration, C-14 and C-20 were therefore determined as 14S and 20S, respectively. Thus, 2 was identified as the derivative of spirotryprostatin B, and was named spirotryprostatin F.

Compound 3 was obstained as colorless amorphous solid. The molecular formula of 3, C₂₉H₄₅NO₄, was determined through the positive ion at m/z 494.3237 [M+Na]⁺, which indicated 8° of unsaturation. Although the molecular formula indicated that 3 was another alkaloid, its NMR data was quite different from those of other isolates. Its 1D NMR data showed signals of a 1, 4-disubstituted benzene ring, a prenyl, and two carbonyls. These above functional groups account for 7 indices of hydrogen deficiency, which meant that the remaining sixteen aliphatic carbons only occupied one degree of unsaturation. Thus 3 might be a macrolide. According to detial 2D NMR analysis, 3 was established to have the same skeleton as melearoride A, PF1163A, and PF1163B [20-22]. The HMBC correlations (Fig. 5) from H-9 to C-7, C-10 and C-16 indicated the ester bond between C-9 and C-16. The NH signal at δ_H 5.68 (d, J = 9.5 Hz) was confirmed by the HMBC correlations from δ_H 5.68 to C-1 (δ_C 173.0) and C-17 (δ_C 53.8), which also approved the amido linkage of C-1/C-17. Meanwhile, the HMBC correlations from H₂-25 to C-22, C-26 and C-27, indicated an oxygenated prenyl to C-22, and the HMBC correlations from H₃-15 to C-5, C-6 and C-7 indicated a methyl to C-6.

Figure 5

	Download: JPG larger image
Fig. 5. The key HMBC and 1H-1H COSY correlations of 3.

Same as those for the known analogues the key ROESY correlations of H-6/H-9/H-10a and of H-17/H-10b indicated that H-6 and H-9 were on the same face of the macrolide ring system, while H-17 was in the opposite direction. The same relative configurations along with the similar negative rotation ( -55.9) also indicated 3 had the same stereochemical structure as those of melearoride A and PF1163B, which was elucidated by total synthesis and single crystal X-ray diffraction [20-24]. Therefore, 3 was named as N-demethylmelearoride A.

All compounds (1-11) were evaluated for their cytotoxic activities against three human tumor cell lines (Table 2). The new compounds 2 and 3 showed moderate activities to the MDAMB-231 and HepG2 cell lines. The structure-activity relationships of 4 and its analogues (1, 5, 6, and 7) indicated that the presence of α, β-unsaturated carbonyl obviously decreased the cytotoxicities to all three cell lines. Meanwhile, indole-diketopiperazines with 11b-hydroxyl in 11 showed most potent cytotoxicities, followed by 11ahydroxyl in 8, 9, and 10 successively, which indicated that the presence of the 11β-hydroxyl in 11 showed most potent cytotoxicities, followed by 11α-hydroxyl in 8, 9, and 10 successively, which indicated that the presence of the 11β-hydroxyl was important for their cytotoxicities.

Table 2

Table 2 Cytotoxicities of compounds 1–11 in three human tumor cell lines with MTT method.

Table 2 Cytotoxicities of compounds 1–11 in three human tumor cell lines with MTT method.

Three new alkaloids, 6, 7-dehydropaxilline (1), spirotryprostatin F (2), and N-demethylmelearoride A (3), along with 8 known alkaloids were isolated from the endophytic P. brefeldianum. These metablites are biosynthetically through different pathways that combined polyketide, peptide and terpenoid biosynthesis. The indoloditerpenes and the 13-membered macrolide were reported from this species for the fist time, which enriched the metabolic diversity of Penicillium sp. In addition, some of these compounds showed moderate cytotoxicities against human tumor cell lines, which should be further investigated.

Optical rotations were measured with a Jasco P-1020 polarimeter. UV spectra were acquired with a Shimadzu UV-2450 spectrophotometer. CD spectra were recorded on a Jasco J-810 spectrometer. IR spectra (KBr disks, in cm^-1) were obtained on a Bruker Tensor 27 spectrometer. The NMR spectra (¹H: 500 MHz, ¹³C: 125 MHz) were measured at 300 K with a Bruker Avance Ⅲ NMR instrument with TMS as internal standard. CDCl₃ (δ_H = 7.26 and δ_C = 77.16 for CDCl₃) was used as solvent. Mass spectra were obtained on an Agilent 6520 B UPLC-Q-TOF spectrometer. Column chromatography (CC) was carried out using silica gel (Qingdao Marine Chemical Co. Ltd., Qingdao, China), Sephadex LH-20 (Pharmacia, Stockholm, Sweden), RPC18 silica (40-63 mm, FuJi, Japan). The fractions obtained from column chromatography were detected by TLC with precoated silica gel GF254 (Qingdao Haiyang Chemical Co., Ltd., China) plates. Analytical HPLC was performed with an Agilent 1200 Series instrument with a DAD detector by using a shim-pack VPODS column (4.6 mm × 250 mm; Agilent Technologies, Palo Alto, CA, USA). Preparative HPLC was carried out using a Shimadzu LC-8A equipped with a Shim-pack RP-C₁₈ column (20 mm × 200 mm, i.d., Shimadzu, Tokyo, Japan) with a flow rate of 10.0 mL/min, detected by a binary channel UV detector.

The fungal strain was isolated from the rhizome of Pinellia ternata that collected from suburb of Nanjing, Jiangsu Province, People's Republic of China, in October 2014. It was identified as Penicillium brefeldianum by the morphological method and sequence analysis of internal transcribed spacer (ITS) rDNA (GenBank accession No. KM243921.1). The strain was cultured on potato dextrose agar (PDA) at 28 ℃. After 7 days, agar plugs were cut into small pieces (about 0.3 cm × 0.3 cm × 0.3 cm) under aseptic condition, 10-15 pieces of which were inoculated to a Erlenmeyer flask (500 mL), containing 200 mL of potato dextrose liquid medium. Then the flasks were incubated on a rotary shaker at 28 ℃ and 170 rpm for 6 days to prepare the seed culture. Fermentation was carried out in 10 Erlenmeyer flasks (500 mL), each containing 80 g of rice and 120 mL distilled H₂O, and cultivated at 28 ℃ for 35 days. The solid culture medium was soaked overnight and sterillizing at 121 ℃ for 20 min before using.

The solid rice material was extracted with equivoluminal EtOAc three times at room temperature, and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (13.0 g). The EtOAc extract was fractionated by silica gel CC using petroleum ether-EtOAc (v/v, 1:0, 60:1, 40:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1) gradient elution to give nine fractions (Fr.1-Fr.9). Fr.6 (497.7 mg) was submitted to Sephadex LH-20 CC with CH₂Cl₂-MeOH (1:1) as eluent, giving seven subfractions (a-g) after combination according to TLC. The subfraction Fr.6-f (104.0 mg) was further purified by preparative HPLC with MeOH-H₂O (87:13), to give 4 (51.0 mg, t_R = 15.6 min), 5 (5.5 mg, t_R = 6.9 min), 7 (3.9 mg, t_R = 9.1 min), respectively. The subfraction Fr.6-g (18.9 mg) was further purified by preparative HPLC with MeOH-H₂O (78:22), to give 6 (10.9 mg, t_R = 13.8 min). The subfraction Fr.6-d (199.8 mg) was further fractionated by medium pressure preparative HPLC and purified subsequently by preparative HPLC with MeOH-H₂O (87:13), to give 3 (5.1 mg, t_R = 18.5 min). Fr.7 (577.7 mg) was also submitted to Sephadex LH-20 CC with CH₂Cl₂-MeOH (1:1) as eluent, giving six subfractions (a-f) after combination according to TLC. The subfraction Fr.7-e (38.7 mg) was further purified by preparative HPLC with MeOH-H₂O (67:33), to give 1 (5.7 mg, t_R = 26.4 min). The subfraction Fr.7-c (281.1 mg) was further purified by medium pressure preparative HPLC with MeOH-H₂O (66:34), to give 8 (10.9 mg, t_R = 24.3 min) and 10 (8.6 mg, t_R = 45.1 min). The subfraction Fr.7-b (105.1 mg) was further purified by preparative HPLC with MeOH-H₂O (82:18), to give 9 (5.1 mg, t_R = 15.4 min). Fr.8 and Fr.9 were combined to over a reversedphase C₁₈ column with MeOH-H₂O (v/v, 30:70-100:0) to obtain six subfractions (C_a-C_f). The subfraction C_c (65.0 mg) was further purified by preparative HPLC with MeOH-H₂O (65:35), to give 2 (2.6 mg, t_R = 17.6 min) and 11 (2.6 mg, t_R = 31.5 min).

Compound 1: Yellow powder; [α]_D ²⁵ +20.0 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 204 (4.06), 229 (4.18), 277 (3.53), 321(3.38) nm; IR (KBr) v_max 3435, 2922, 1638, 1384, 1262, 1171, 1109, 622 cm^-1; CD (MeOH) λ_max (Δε) 208 (-17.6), 224 (+7.0), 239 (-21.2), 263 (+10.9) 327 (+29.2) and 372 (-17.3) nm; ¹H and ¹³C NMR data, see Table 1; HR-ESIMS m/z 434.2325 [M+H]⁺ (calcd. for C₂₇H₃₂NO₄, 434.2326).

Compound 2: Pale yellow powder; [α]_D ²⁵ -27.8 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 205 (4.14), 216(4.15), 271 (3.68) nm; IR (KBr) v_max 3447, 2925, 2853, 1638, 1383, 1098 cm^-1; CD (MeOH) λ_max (Δε) 202 (-13.1), 221(-46.7), 242 (+62.0), 271 (-18.4) and 310 (+1.4) nm; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 462.2385 [M+H]⁺ (calcd. for C₂₇H₃₂N₃O₄, 462.2387).

Compound 3: Colorless amorphous solid; [α]_D ²⁵ -55.9 (c 0.13, MeOH); UV (MeOH) λ_max (log ε) 204 (4.18), 226 (4.07), 277(3.16) nm; IR (KBr) v_max 3422, 1729, 1640, 1513, 682 cm^-1; ¹H and ¹³C NMR data, see Table 1; HR-ESIMS m/z 494.3237 [M+Na]⁺ (calcd. for C₂₉H₄₅NNaO₄, 494.3241).

Cytotoxic activities were evaluated against three human tumor cell lines by the MTT method [25]. Cisplatin and doxorubicin were used as the positive control medicine.

This research work was funded by the National Natural Science Foundation of China (No. 81503218), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_15R63), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Fundamental Research Funds for the Central Universities (No. 2016ZZD010).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.02.022.