marine drugs Article
5-Hydroxycyclopenicillone, a New β-Amyloid Fibrillization Inhibitor from a Sponge-Derived Fungus Trichoderma sp. HPQJ-34 Fang Fang 1 , Jiaying Zhao 1 , Lijian Ding 1 , Chunhui Huang 1 , C. Benjamin Naman 2 ID , Shan He 1,3, * ID , Bin Wu 4 , Peng Zhu 1 , Qijun Luo 3 , William H. Gerwick 2 ID , Xiaojun Yan 3 , Qinwen Wang 5 , Zaijun Zhang 6 and Wei Cui 1,5, * 1
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Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, Ningbo University, Ningbo 315211, China; [email protected] (F.F.); [email protected] (J.Z.); [email protected] (L.D.); [email protected] (C.H.); [email protected] (P.Z.) Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA; [email protected] (C.B.N.); [email protected] (W.H.G.) Key Laboratory of Marine Biotechnology of Zhejiang Province, Ningbo University, Ningbo 315211, China; [email protected] (Q.L.); [email protected] (X.Y.) Ocean College, Zhejiang University, Hangzhou 310058, China; [email protected] School of Medicine, Ningbo University, Ningbo 315211, China; [email protected] Institute of New Drug Research and Guangzhou Key Laboratory of Innovative Chemical Drug Research in Cardio-cerebrovascular Diseases, College of Pharmacy, Jinan University, Guangzhou 510632, China; [email protected] Correspondence: [email protected] (S.H.); [email protected] (W.C.); Tel.: +86-574-8760-0458 (S.H.); +86-574-8760-9589 (W.C.)
Received: 17 May 2017; Accepted: 16 August 2017; Published: 19 August 2017
Abstract: A new cyclopentenone, 5-hydroxycyclopeni cillone (1), was isolated together with three known compounds, ar-turmerone (2), citreoisocoumarin (3), and 6-O-methyl-citreoisocoumarin (4), from a culture of the sponge-derived fungus Trichoderma sp. HPQJ-34. The structures of 1–4 were characterized using comprehensive spectroscopic analyses. The absolute configuration of 1 was determined by comparison of electronic circular dichroism (ECD) spectra with literature values used for the reported analogue, cyclopenicillone (5), which was not isolated in this research. Compound 1 was shown to scavenge 2,2-diphenyl-1-picrylhydrazyl free radicals, and decrease β-amyloid (Aβ) fibrillization in vitro. Moreover, 1 significantly reduced H2 O2 -induced neurotoxicity in SH-SY5Y cells. These findings suggested that compound 1, a newly discovered cyclopentenone, has moderate anti-oxidative, anti-Aβ fibrillization properties and neuroprotective effects, and might be a good free radical scavenger. Keywords: sponge-derived fungus; cyclopentenone; Trichoderma sp. HPQJ-34; Alzheimer’s disease
1. Introduction Marine fungi continue to be an important source for the discovery of structurally diverse secondary metabolites with a wide variety of biological activities [1–10]. Fungi isolated from sponges are well-known as prolific producers of new natural products, and a considerable number of these have displayed promising biological and pharmacological properties such as antiviral, antibacterial, antitumor, antifouling, anti-inflammatory, as well as immunomodulatory activity [11–14]. In the search for new pharmaceutical leads in sponge-associated fungi, the organic extract of Trichoderma sp. HPQJ-34, isolated from the marine sponge Hymeniacidon perleve, was found to have potent Mar. Drugs 2017, 15, 260; doi:10.3390/md15080260
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Bioactivity-guided chromatographic separation of the EtOAc extract obtained from the fermentation anti-oxidative, anti-Aβ fibrillization properties and neuroprotective effects during preliminary testing. Bioactivity-guided chromatographic separation thecyclopentenone, EtOAc extract obtained from the fermentation(1), broth of this strain resulted in the isolation of a of new 5-hydroxycyclopenicillone Bioactivity-guided chromatographic separation of the EtOAc extract obtained from the fermentation brothwith of this strain resulted in the isolation of a new cyclopentenone, 5-hydroxycyclopenicillone along three known compounds: ar-turmerone (2) [15], citreoisocoumarin (3) [16] and(1), 6-Obroth of this strain resulted in the isolation of a new cyclopentenone, 5-hydroxycyclopenicillone along with three known compounds: ar-turmerone (2) [15], citreoisocoumarin (3) [16] and 6-Omethyl-citreoisocoumarin (4) [17] (Figure 1).The details of the isolation, the structure identification (1), along with three known compounds: ar-turmerone (2) [15], citreoisocoumarin (3) [16] and (4) [17] (Figure 1).The details of thethese isolation, the metabolites structure identification ormethyl-citreoisocoumarin elucidation, and the biological activities associated with fungal are reported 6-O-methyl-citreoisocoumarin (4) [17] (Figure 1).The details of the isolation, the structure identification or elucidation, and the biological activities associated with these fungal metabolites are reported below. orbelow. elucidation, and the biological activities associated with these fungal metabolites are reported below.
Figure 1. The chemical structures of 1–4, isolated from the sponge-derived fungus Trichoderma Figure isolated from fromthe thesponge-derived sponge-derivedfungus fungus Trichoderma Figure1.1.The Thechemical chemicalstructures structures of of 1–4, isolated Trichoderma sp. sp. sp. HPQJ-34. HPQJ-34. HPQJ-34.
2. 2. Results and Discussion Resultsand andDiscussion Discussion 2. Results 2.1. Isolation and Taxonomy the 2.1. Isolationand andTaxonomy Taxonomyof theProducing Producing Microorganism Microorganism 2.1. Isolation ofofthe Producing Microorganism The fungal strain Trichoderma sp. HPQJ-34 was isolated isolated fromthe the spongeHymeniacidon Hymeniacidon perleve Thefungal fungalstrain strainTrichoderma Trichoderma sp. sp. HPQJ-34 HPQJ-34 was perleve The was isolatedfrom from thesponge sponge Hymeniacidon perleve collected from Zhejiang, China. ItItwas was identifiedasasTrichoderma Trichoderma HPQJ-34 according collected fromDongji DongjiIsland, Island,Zhejiang, Zhejiang, China. sp.sp. HPQJ-34 according collected from Dongji Island, It wasidentified identified as Trichoderma sp. HPQJ-34 according to tomorphological (ITSrDNA rDNAsequence) sequence) analyses. A BLASTN employing morphological and and molecular molecular (ITS analyses. A BLASTN searchsearch employing the to morphological and molecular (ITS rDNA sequence) analyses. A BLASTN search employing the Chain Reaction (PCR)-amplified Internal Transcribed SpacerSpacer (ITS) rDNA sequence (640 thePolymerase Polymerase Chain Reaction (PCR)-amplified Internal Transcribed (ITS) rDNA sequence Polymerase Chain Reaction (PCR)-amplified Internal Transcribed Spacer (ITS) rDNA sequence (640 bp)bp) indicated that that the strain was closely relatedrelated to Trichoderma harzianum strain MGQ2 (99% (T) (640 indicated the strain was closely to Trichoderma harzianum strain (T) MGQ2 bp) indicated that the strain was closely related to Trichoderma harzianum strain MGQ2 (T) (99% similarity). A phylogenetic tree was using using the neighbor-joining methodmethod corrected with (99% similarity). A phylogenetic tree constructed was constructed the neighbor-joining corrected similarity). A phylogenetic tree was constructed using the neighbor-joining method corrected with the Jukes–Cantor algorithm (Figure 2), also showing that the HPQJ-34 strain is a member of the genus with the Jukes–Cantor algorithm (Figure 2), also showing that the HPQJ-34 strain is a member of the theTrichoderma Jukes–Cantor algorithm (Figure 2), also showing that the HPQJ-34 strain is a member of the genus [18]. [18]. genus Trichoderma Trichoderma [18]. 95
HPQJ34 HPQJ34
97 95 KC342029.1_Trichoderma_harzianum_strain_MGQ2 77 60 63 46
63
60
77
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KC342029.1_Trichoderma_harzianum_strain_MGQ2 EU280124.1_Trichoderma_hamatum_strain_DAOM_167057 EU280124.1_Trichoderma_hamatum_strain_DAOM_167057 KY102298.1_Candida_parapsilosis_culture-collection_CBS_11892
KT310169.1_Trichophyton_tonsurans_strain_CBS_127.97 KY102298.1_Candida_parapsilosis_culture-collection_CBS_11892 XM_002851228.1_Arthroderma_otae_CBS_113480 KT310169.1_Trichophyton_tonsurans_strain_CBS_127.97
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XM_003010622.1_Arthroderma_benhamiae_CBS_112371 XM_002851228.1_Arthroderma_otae_CBS_113480 XM_001402349.2_Aspergillus_niger_CBS_513.88 XM_003010622.1_Arthroderma_benhamiae_CBS_112371 63 61
63
XM_001210850.1_Aspergillus_terreus_NIH2624 XM_001402349.2_Aspergillus_niger_CBS_513.88 KP171180.1_Grosmannia_clavigera_strain_KW_1407 XM_001210850.1_Aspergillus_terreus_NIH2624
EU747605.1_Trichiurus_haumela_clone_Trha68 61
KP171180.1_Grosmannia_clavigera_strain_KW_1407
EU747605.1_Trichiurus_haumela_clone_Trha68 0.2
0.2 Figure 2. Phylogenetic tree built with Molecular Evolutionary Genetics Analysis (MEGA) 5.05 based onFigure nearly2.complete ITS rDNA gene sequences of Evolutionary HPQJ-34. Genetics Analysis (MEGA) 5.05 based Phylogenetic tree built with Molecular on nearly complete ITS rDNA gene sequences of HPQJ-34. Figure 2. Phylogenetic tree built with Molecular Evolutionary Genetics Analysis (MEGA) 5.05 based on nearly complete ITS rDNA gene sequences of HPQJ-34.
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The new molecule, 5-hydroxycyclopenicillone (1), was obtained as a yellow oil. Compound 1 has the molecular formula C13H20O4, which was determined from a sodiated molecular ion adduct 2.2. Elucidation peakStructure in the High-resolution electrospray ionisation mass spectrometry (HRESIMS) at m/z 263.1247 + [M +The Na]new (calcd. for C 13H20O4Na+, 263.1254). The 1H NMR spectrum of 1 in CD3OD showed two molecule, 5-hydroxycyclopenicillone (1), was obtained as a yellow oil. Compound 1 has methyl doublets at δH C 1.65H(3H, d, J = 4.8 Hz, CH3-6′) and 1.75 (3H, d, J = 1.0 Hz, CH3-6), one methyl the molecular formula 13 20 O4 , which was determined from a sodiated molecular ion adduct peak in singlet at δH 1.23 (3H, s, CH3-7), two olefinicmass methines at δH 5.49 (1H, m, CH-4′) and 5.48 (1H, CH-+ the High-resolution electrospray ionisation spectrometry (HRESIMS) at m/z 263.1247 [Mm, + Na] 5′), two oxygenated methines at δ H 4.49 (1H, d, J = 1.0 Hz, CH-4) and 4.72 (1H, dd, J = 8.6, 4.8 Hz, CH+ 1 (calcd. for C13 H20 O4 Na , 263.1254). The H NMR spectrum of 1 in CD3 OD showed two methyl 1′), as well as signals with complex coupling patterns attributed to two methylenes between δ H 1.76 0 doublets at δH 1.65 (3H, d, J = 4.8 Hz, CH3 -6 ) and 1.75 (3H, d, J = 1.0 Hz, CH3 -6), one methyl singlet and 1).CH The-7), four degrees of methines unsaturation in the molecular formula of 1, together 0 0 at δH2.12 1.23(Table (3H, s, two olefinic at δinherent 3 H 5.49 (1H, m, CH-4 ) and 5.48 (1H, m, CH-5 ), 13C NMR and with data showing the presence of one carbonyl and four olefinic carbons in the two oxygenated methines at δH 4.49 (1H, d, J = 1.0 Hz, CH-4) and 4.72 (1H, dd, J = 8.6, 4.8 Hz, CH-10 ), Distortionless Enhancement by coupling Polarization Transfer (DEPT)tospectra, indicatedbetween that the structure of as well as signals with complex patterns attributed two methylenes δH 1.76 and 1 possesses one ring [19]. Selected Heteronuclear Multiple Bond Correlation (HMBC) correlations, 2.12 (Table 1). The four degrees of unsaturation inherent in the molecular formula of 1, together with shown in Figure including from CH3-7 and to C-1, and C-5, from -6CtoNMR C-1, and C-2, Distortionless and C-3, and data showing the 3, presence of one carbonyl fourC-4 olefinic carbons inCH the313 from CH-4 to by C-3Polarization indicated the presence of a 3-substituted 4,5-dihydroxy-2,5-dimethylcyclopent-2Enhancement Transfer (DEPT) spectra, indicated that the structure of 1 possesses one enone ring system in 1. This assignment was further supported by the homoallylic coupling (J = 1.0 ring [19]. Selected Heteronuclear Multiple Bond Correlation (HMBC) correlations, shown in Figure 3, Hz) observed between CH-4 and CH 3-6. Consecutive 1H–1H COrrelated SpectroscopY (COSY) including from CH3 -7 to C-1, C-4 and C-5, from CH3 -6 to C-1, C-2, and C-3, and from CH-4 to C-3 correlations CH3of -6′a to CH-5′, then CH-4′, CH-3′, CH-2′ and finally CH-1′ suggested a in 1indicated the from presence 3-substituted 4,5-dihydroxy-2,5-dimethylcyclopent-2-enone ring system disubstituted 1-hydroxyhex-4-ene moiety as an additional substructure of 1. This was reinforced by 1. This assignment was further supported by the homoallylic coupling (J = 1.0 Hz) observed between HMBCand correlations observed from 3-6′ to C-4′ and C-5′, as well as from CH-2′ to C-1′, C-3′, and0 C1 H–1CH CH-4 CH3 -6. Consecutive H COrrelated SpectroscopY (COSY) correlations from CH3 -6 to 4′. Finally, the connection between the two structural fragments was established as a C-3 to C-1′ 0 0 0 0 0 CH-5 , then CH-4 , CH-3 , CH-2 and finally CH-1 suggested a 1-disubstituted 1-hydroxyhex-4-ene linkage by the HMBC correlation observed from CH-2′ to C-3, thus completing the planar structure moiety as an additional substructure of 1. This was reinforced by HMBC correlations observed from of 1. 0 0 0 0 0 0 0 CH -6 to C-4 and C-5 , as well as from CH-2 to C-1 , C-3 , and C-4 . Finally, the connection between 3
the two structural fragments was established as a C-3 to C-10 linkage by the HMBC correlation observed Table 1. The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data of compound 1 (in CD3OD). from CH-20 to C-3, thus completing the planar structure of 1. 1H–1H COSY Position δC, Type δH, Mult. (J in Hz) HMBC NOESY 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) data of compound 1 (in CD OD). Table 1. The 3 1 210.4, C 2 135.8, C 1 H–1 H COSY Position δC , Type δH , Mult. (J in Hz) HMBC NOESY 3 171.7, C 1 4 75.3,210.4, CH C 4.49, d (1.0) 3 7, 2′ 2 135.8, 5 74.5, C C 3 171.7, C 6 8.4,75.3, CH3CH 1.75, d (1.0) 1, 2, 3 7, 20 1′ 4 4.49, d (1.0) 3 7 23.1, CH 3 1.23, s 1, 4, 5 4 5 74.5, C 6 8.4, CH3 1.75, (1.0) 4.8) 1, 2,2,33, 4, 2′, 3′ 10 1′ 69.4, CH 4.72, ddd(8.6, 2′ 6, 2′, 3′ 7 23.1, CH3 s 8.6, 5.5), 1, 4, 5 4 1.86, dtd1.23, (14.2, 2′ 36.6,69.4, CHCH 2 4, 1′ 10 4.72, dd (8.6, 4.8) 201′, 3′ 2, 3, 4, 3, 20 ,1′, 30 3′, 4′6, 20 , 30 1.76, m 1.86, dtd (14.2, 8.6, 5.5), 0 0 0 0 0 0 0 2 36.6, 1 , 32′, 4′ 3, 1 , 3 , 41′, 5′ 4, 1 3′ 29.8, CHCH 2 2 2.12,mm 1.76, 0 5′ 4′ 131.6, CH 5.49,m m 3′ 30 29.8, CH2 2.12, 20 , 43′, 10 , 50 0 40 131.6, 5.49, 30 , 54′, 30 5′ 126.7, CHCH 5.48,m m 6′ 3′ 6′ 50 126.7, CH 5.48, m 40 , 60 5′ 30 4′, 5′ 60 6′ 18.1, CH 3 1.64, d (4.8) 5′ 0 0 0 0 0
6
18.1, CH3
1.64, d (4.8)
5
4,5
5
Figure 3. Selected HMBC and 11H–11 H COSY correlations observed for 1. Figure 3. Selected HMBC and H– H COSY correlations observed for 1.
Because the 1 H NMR signals for CH-40 and CH-50 in 1 significantly overlapped, J coupling analysis was precluded. Therefore, the double bond connecting these carbons was assigned as trans
of a J-coupling partner for CH-4 (a sharp singlet in 1 and a broad singlet in 5), accompanied by proximal deshielding effects on C-5 (shifted to δC 74.5 in 1 from 50.9 in 5) and C-7 (shifted to δC 23.1 in 1 from 13.8 in 5) [19]. These observations further supported the structure assignment of 1, and strongly suggested a shared relative, or possibly absolute, configuration between the two structural analogues (Figure 4, panel A), because the NMR parameter of chemical shift (δ), especially for the 13C Mar. Drugs 2017, 15, 260 4 of 12 nucleus, is particularly sensitive to perturbations in conformation and differences in configuration. It was also observed that, for 1, as previously reported for 5 [19], the NOE between CH3-6 and 13 CCH-2ʹ CH-1ʹ CH-4 and a restricted free rotation about toIfC-1′ basedalong on thewith deshielded NMRindicated shift of the terminal methyl carbon, C-60 the (δC C-3 18.1). thebond. C-40 toThis C-50 isdouble likely bond due to a combination ofthis steric hindrance resulting inflexibility of the was cis-configured, terminal methyl carbon from wouldthe be relative significantly more shielded, cyclopentenone ring, along with an intramolecular hydrogen bond. The latter was indicated bythe a in the range of approximately 10–13 ppm [19]. The NMR data for this terminus of 1, and in fact −1 in the spectrum of 1, as was previously described for 5 [19]. Both OH-1′ broad IR stretch at 3369 cm entire 1-hydroxyhex-4-ene moiety, closely matched the reported values for the structural analogue and OH-4 are expected to exhibit hydrogen in 1 and 5, however, OH-5 may also cyclopenicillone (5), which was not isolatedbonding in this study [19]. In fact, the NMR data forinteract 1 and 5 with OH-4 in the case of 1. To examine this further, an energy minimized computational molecular were remarkably similar (Supplementary Materials Table S11), with obvious differences including the model generatedpartner for both and 5,(ausing Hartree–Fock to visualize their preferred loss ofwas a J-coupling for1 CH-4 sharpthe singlet in 1 and amethod, broad singlet in 5), accompanied by conformations (Figure 4, panel B). Because both the molecular modeling and NMR data collected for proximal deshielding effects on C-5 (shifted to δC 74.5 in 1 from 50.9 in 5) and C-7 (shifted to δC 23.1 in 11and strongly suggest a shared conformation for these two molecules, and because from5 13.8 in 5) [19]. These observations further supported the structure assignment of 1,the andabsolute strongly configuration of 5 was previously characterized, including the between use of theoretically predicted ECD suggested a shared relative, or possibly absolute, configuration the two structural analogues 13 spectra for its low energy molecular modeling conformers, it was reasoned that an electronic circular (Figure 4, panel A), because the NMR parameter of chemical shift (δ), especially for the C nucleus, is dichroism (ECD) spectrum could be used to resolve the and absolute configuration of 1. particularly sensitive to perturbations in conformation differences in configuration.
Figure 4. Structural comparison of 1 with reported analogue 5. Panel A: Chemical structures Figure 4. Structural comparison of 1 with reported analogue 5. Panel A: Chemical structures of 1 and of 1 and 5. Panel B: Energy-minimized computational molecular models of 1 and 5, showing a 5. Panel B: Energy-minimized computational molecular models of 1 and 5, showing a similar similar conformation. conformation.
It was 1, asestablished previouslyas reported for 5 [19], between The usealso of observed ECD has that, beenfor well a powerful tool the forNOE assigning theCH absolute 3 -6 and 0 along with CH-4 and CH-20 indicated a restricted free rotation about the C-3 to C-10 bond. CH-1 configuration of natural products [20–22]. In the experimental (ECD) spectrum of 1, the local maxima likely due to aeffect combination steric hindrance resulting from the relative inflexibility of the ofThis the is positive Cotton (CE) wasofobserved at 246 nm, and negative CEs were observed around cyclopentenone ring, along with an intramolecular hydrogen bond. The latter was indicated by a 321 and 214 nm. By comparison, the experimental ECD spectrum of 5 was similar to that of 1, with − 1 broad IR stretch CE at 3369 cm atin the spectrum of 1, asby was previously described 5 [19]. Both OH-10 obvious positive maxima 240 nm accompanied negative CEs around 326for and 209 nm (Figure arecases, expected to exhibitpositive hydrogen bonding in 1 and however, OH-5 may second also interact 5)and [19].OH-4 In both the observed Davydov splitting for5,positive first, negative CEs with OH-4 in the case of 1. To examine this further, an energy minimized computational molecular can be attributed to a positive angle of rotation present between the enone and alkene chromophores, generated 1 and 5,models using the Hartree–Fock method, to visualize preferred asmodel also was suggested by for the both molecular presented in Figure 4. As a result, their the absolute conformationsof(Figure panel B). Because both molecular modeling and data collected for configuration 1 was 4, determined to match thethe reported configuration of NMR 5, or 4(S), 5(S),1′ (R). 1 and 5 strongly suggest a shared conformation for these two molecules, and because the absolute Consequently, the structure of 1 was unambiguously established and named as 5configuration of 5 was previously characterized, including the use of theoretically predicted ECD hydroxycyclopenicillone (1) {IUPAC: (4S,5S)-4,5-dihydroxy-3-[(R,E)-1-hydroxyhex-4-en-1-yl]-2,5spectra for its low energy molecular modeling conformers, it was reasoned that an electronic circular dimethylcyclopent-2-en-1-one}. dichroism (ECD) spectrum could be used to resolve the absolute configuration of 1. The use of ECD has been well established as a powerful tool for assigning the absolute configuration of natural products [20–22]. In the experimental (ECD) spectrum of 1, the local maxima of the positive Cotton effect (CE) was observed at 246 nm, and negative CEs were observed around 321 and 214 nm. By comparison, the experimental ECD spectrum of 5 was similar to that of 1, with obvious positive CE maxima at 240 nm accompanied by negative CEs around 326 and 209 nm (Figure 5) [19]. In both cases, the observed positive Davydov splitting for positive first, negative second CEs can be attributed to a positive angle of rotation present between the enone and alkene chromophores, as also suggested by the molecular models presented in Figure 4. As a result, the absolute configuration of 1 was determined to match the reported configuration of 5, or 4(S), 5(S),10 (R). Consequently, the structure of 1 was unambiguously established and named as 5-hydroxycyclopenicillone (1) {IUPAC: (4S,5S)-4,5-dihydroxy-3-[(R,E)-1-hydroxyhex-4-en-1-yl]-2,5-dimethylcyclopent-2-en-1-one}.
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Figure 5. The electronic circular dichroism (ECD) spectra of 5-hydroxycyclopenicillone (1) and Figure 5. The electronic circular dichroism (ECD) spectra of 5-hydroxycyclopenicillone (1) and cyclopenicillone (5); from reference [19]. Figure 5. The electronic circular[19]. dichroism (ECD) spectra of 5-hydroxycyclopenicillone (1) and cyclopenicillone (5); from reference cyclopenicillone (5); from reference [19].
In addition to 1, three known metabolites were isolated during the course of this research. These wereInidentified, by their metabolites spectrometric and isolated spectroscopic data literature values, as In addition 1, threeknown known were isolated during during the course ofwith this research. These addition to comparing 1,tothree metabolites were the course of this research. were identified, by comparing their spectrometric and spectroscopic data with literature values, as ar-turmerone (2) [15], citreoisocoumarin [16], and 6-O-methyl-citreoisocoumarin (4) [17]. values, These were identified, by comparing their(3) spectrometric and spectroscopic data with literature ar-turmerone [15], citreoisocoumarin (3) andand 6-O-methyl-citreoisocoumarin (4) [17]. (4) [17]. as ar-turmerone (2)(2) [15], citreoisocoumarin (3)[16], [16], 6-O-methyl-citreoisocoumarin 2.3. Biological Activities 2.3. Biological Activities 2.3. Biological Activities The effects of 5-hydroxycyclopenicillone wereevaluated evaluated the scavenging free radicals. The effects of 5-hydroxycyclopenicillone (1) (1) were for for the scavenging of free of radicals. The effects of 5-hydroxycyclopenicillone (1) were evaluated for the scavenging of radicals. As shown in Figure 6, compound 1 concentration–dependency reduced the content of DPPH free As shown in Figure 6, compound 1 concentration–dependency reduced the content of DPPHfree free As shown in Figure 6, compound 1 concentration–dependency reduced the content of DPPH free radicals, suggesting it might moderate free scavenger. These anti-oxidative effects effects radicals, suggesting that that it might actact asasa amoderate freeradical radical scavenger. These anti-oxidative ofsuggesting 1 might beto due toitpresence the presence of multiple hydroxy Many anti-oxidants, assuch vitamin radicals, that might act a moderate freegroups. radical scavenger. Thesesuch anti-oxidative effects of 1 might be due the ofas multiple hydroxy groups. Many anti-oxidants, as vitamin C and arepresence useful for the delay of onset and relief of symptoms Alzheimer’s disease (AD) of 1 might bevitamin due toE,the multiple hydroxy groups. Manyofanti-oxidants, such as vitamin C and vitamin E, are useful for theof delay of onset and relief of symptoms of Alzheimer’s disease (AD) [23]. Therefore, it was hypothesized that 1, a new cyclopentenone with moderate free radical C and vitamin E, are useful for the delay of onset and relief of symptoms of Alzheimer’s disease [23]. Therefore, it was hypothesized that 1, a new cyclopentenone with moderate free radical scavenging properties, might be effective in AD models. (AD) [23]. Therefore, was hypothesized a new cyclopentenone with moderate free radical scavenging properties,it might be effective inthat AD1,models. scavenging properties, might be effective in AD models.
Figure 6. 5-hydroxycyclopenicillone (1) scavenges DPPH free radicals in a concentration-dependent manner. Compound 1 or vitamin C (VitC) at indicated concentrations were added to 0.2 mM DPPH
Figure 6. 6. 5-hydroxycyclopenicillone 5-hydroxycyclopenicillone (1) (1) scavenges scavenges DPPH DPPH free free radicals radicals in in aa concentration-dependent concentration-dependent Figure manner. Compound 1 or vitamin C (VitC) at indicated concentrations were added to to 0.2 0.2 mM mM DPPH DPPH manner. Compound 1 or vitamin C (VitC) at indicated concentrations were added solution for 20 min. The concentration of DPPH free radicals was measured by evaluating the absorbance at 517 nm. Data are presented as the mean ± SEM of three separate experiments; ** p < 0.01 vs. the control group (ANOVA and Dunnett’s test).
solution for 20 min. The concentration of DPPH free radicals was measured by evaluating the absorbance at 517 nm. Data are presented as the mean ± SEM of three separate experiments; ** p < 0.01 vs. the control group (ANOVA and Dunnett’s test).
Previous studies have shown that Aβ peptides accumulate in the brain of AD patients [24]. Aβ Mar. Drugs 2017, 15, 260 6 of 12 peptides can form highly toxic oligomers and fibrils, which are considered as the key pathogenic factors of AD [25]. Aβ oligomers and fibrils can further aggregate into plaques, the hallmark of AD. studies have inhibit shown that peptidesof accumulate in the brain of AD patientsdisease[24]. Therefore,Previous drugs that could the Aβ formation Aβ fibrillization might produce Aβ peptides can form highly toxic oligomers and fibrils, which are considered as the key pathogenic modifying effects for AD treatment [26]. Thus, the effect of 1 on the formation of Aβ fibrils was factors of AD [25]. Aβ oligomers and fibrils can further aggregate into plaques, the hallmark of AD. measured. As shown in Figure 7, co-incubation of 1 and Aβ monomers led to a decreased formation Therefore, drugs that could inhibit the formation of Aβ fibrillization might produce disease-modifying of Aβ fibrils when compared to the incubation of Aβ monomers alone. Moreover, curcumin, a effects for AD treatment [26]. Thus, the effect of 1 on the formation of Aβ fibrils was measured. positive control, also inhibited the formation of Aβ fibrils led at about the same level asofcompound As shown in Figure 7, co-incubation of 1 and Aβ monomers to a decreased formation Aβ fibrils 1 (Figure 7). These results suggested that 5-hydroxycyclopenicillone (1) could effectively inhibit when compared to the incubation of Aβ monomers alone. Moreover, curcumin, a positive control, also the formation of Aβ inhibited thefibrils. formation of Aβ fibrils at about the same level as compound 1 (Figure 7). These results suggested that 5-hydroxycyclopenicillone (1) could effectively inhibit the formation of Aβ fibrils.
Figure 7. 5-Hydroxycyclopenicillone (1) significantly decreases the formation of Aβ1-42 fibrils. Aβ1-42
Figuremonomers 7. 5-Hydroxycyclopenicillone significantly the formation Aβ1-42 fibrils. for Aβ1-42 (10 µM) were incubated(1) with or without decreases 1 or curcumin at indicatedofconcentrations monomers μM) were incubated orthe without 1 orTcurcumin at indicated for 3 3 days.(10 Aβ fibrils were measuredwith using thioflavin (ThT) assay. ** p < 0.01 concentrations vs. Aβ1-42 group Dunnett’s test).using the thioflavin T (ThT) assay. ** p < 0.01 vs. Aβ1-42 group (ANOVA days. (ANOVA Aβ fibrilsand were measured and Dunnett’s test). Besides Aβ fibrils, oxidative stress also plays an important role in AD pathogenesis [27]. Hydrogen peroxide (H2 O widely used to establish stress-induced models,[27]. Besides Aβ fibrils, oxidative stress also plays oxidative an important role in neurotoxicity AD pathogenesis 2 ), an agent can increase intracellular reactive and promoteoxidative neurotoxicity via its acting neurotoxicity on various Hydrogen peroxide (H2O2), an agentoxygen widelyspecies used to establish stress-induced macromolecules [28]. Therefore, the neuroprotective effects of 5-hydroxycyclopenicillone on on models, can increase intracellular reactive oxygen species and promote neurotoxicity via its (1) acting H2 O in SH-SY5Y cells was investigated. cells were pre-treated (1) 2 –induced neuronal various macromolecules [28].loss Therefore, the neuroprotective effectsSH-SY5Y of 5-hydroxycyclopenicillone with 1 or vitamin C for 2 h, followed by treatment with 200 µM of 30% H2 O2 (in H2 O) for 24 h. on H2O2–induced neuronal loss in SH-SY5Y cells was investigated. SH-SY5Y cells were pre-treated Cell viability was determined by the MTT assay. In Figure 8, panel A shows that 1 significantly with 1 or vitamin C for 2 h, followed by treatment with 200 μM of 30% H2O2 (in H2O) for 24 h. Cell reduced H2 O2– induced neuronal death in a concentration-dependent manner, demonstrating that viability was determined by the MTT assay.neuroprotectant. In Figure 8, panel A shows that 1 with significantly reduced 5-hydroxycyclopenicillone is moderately Moreover, treatment 50 µM 1 alone H2O2–for induced death in suggesting a concentration-dependent that 526 h wasneuronal not cytotoxic to cells, that this agent may bemanner, quite safe.demonstrating Fluorescein diacetate hydroxycyclopenicillone is moderately neuroprotectant. Moreover, treatment with 50 μM 1 alone (FDA)/ propidium iodide (PI) double staining for both live cells and dead cells further confirmed for 26 h was cytotoxic to dramatically cells, suggesting that this agent may be neuronal quite safe. Fluorescein that 1not at 30 and 50 µM protected against H2 O2 –induced death in SH-SY5Ydiacetate cells (Figure 8, paneliodide B). (FDA)/ propidium (PI) double staining for both live cells and dead cells further confirmed that
1 at 30 and 50 μM dramatically protected against H2O2–induced neuronal death in SH-SY5Y cells (Figure 8, panel B).
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Figure (1) protects protects against against HH2O neuronaldeath death 2O 2 -inducedneuronal Figure8. 8. 5-hydroxycyclopenicillone 5-hydroxycyclopenicillone (1) 2-induced in in a a concentration-dependent manner in SH-SY5Y cells. (A): SH-SY5Y cells were treated with 1 or vitamin concentration-dependent manner in SH-SY5Y cells. (A): SH-SY5Y cells were treated with 1 or vitamin C (VitC) at at indicated exposedto to200 200μM µMofof30% 30%HH 2O 2 (in 2 O). C (VitC) indicatedconcentrations. concentrations.After After 22 h, h, cells cells were were exposed 2O 2 (in H2H O). MTT assay was used O22exposure. exposure.Data Dataare are expressed MTT assay was usedtotomeasure measurecell cellviability viability after after 24 24 h of H22O expressed as as thethe percentage ofof control SEMof ofthree threeseparate separateexperiments; experiments; < 0.05 percentage controland andare arepresented presentedas asthe themean mean ± ± SEM * p*
5-Hydroxycyclopenicillone, a New β-Amyloid Fibrillization Inhibitor from a Sponge-Derived Fungus Trichoderma sp. HPQJ-34 Fang Fang 1 , Jiaying Zhao 1 , Lijian Ding 1 , Chunhui Huang 1 , C. Benjamin Naman 2 ID , Shan He 1,3, * ID , Bin Wu 4 , Peng Zhu 1 , Qijun Luo 3 , William H. Gerwick 2 ID , Xiaojun Yan 3 , Qinwen Wang 5 , Zaijun Zhang 6 and Wei Cui 1,5, * 1
2
3 4 5 6
*
Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, Ningbo University, Ningbo 315211, China; [email protected] (F.F.); [email protected] (J.Z.); [email protected] (L.D.); [email protected] (C.H.); [email protected] (P.Z.) Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA; [email protected] (C.B.N.); [email protected] (W.H.G.) Key Laboratory of Marine Biotechnology of Zhejiang Province, Ningbo University, Ningbo 315211, China; [email protected] (Q.L.); [email protected] (X.Y.) Ocean College, Zhejiang University, Hangzhou 310058, China; [email protected] School of Medicine, Ningbo University, Ningbo 315211, China; [email protected] Institute of New Drug Research and Guangzhou Key Laboratory of Innovative Chemical Drug Research in Cardio-cerebrovascular Diseases, College of Pharmacy, Jinan University, Guangzhou 510632, China; [email protected] Correspondence: [email protected] (S.H.); [email protected] (W.C.); Tel.: +86-574-8760-0458 (S.H.); +86-574-8760-9589 (W.C.)
Received: 17 May 2017; Accepted: 16 August 2017; Published: 19 August 2017
Abstract: A new cyclopentenone, 5-hydroxycyclopeni cillone (1), was isolated together with three known compounds, ar-turmerone (2), citreoisocoumarin (3), and 6-O-methyl-citreoisocoumarin (4), from a culture of the sponge-derived fungus Trichoderma sp. HPQJ-34. The structures of 1–4 were characterized using comprehensive spectroscopic analyses. The absolute configuration of 1 was determined by comparison of electronic circular dichroism (ECD) spectra with literature values used for the reported analogue, cyclopenicillone (5), which was not isolated in this research. Compound 1 was shown to scavenge 2,2-diphenyl-1-picrylhydrazyl free radicals, and decrease β-amyloid (Aβ) fibrillization in vitro. Moreover, 1 significantly reduced H2 O2 -induced neurotoxicity in SH-SY5Y cells. These findings suggested that compound 1, a newly discovered cyclopentenone, has moderate anti-oxidative, anti-Aβ fibrillization properties and neuroprotective effects, and might be a good free radical scavenger. Keywords: sponge-derived fungus; cyclopentenone; Trichoderma sp. HPQJ-34; Alzheimer’s disease
1. Introduction Marine fungi continue to be an important source for the discovery of structurally diverse secondary metabolites with a wide variety of biological activities [1–10]. Fungi isolated from sponges are well-known as prolific producers of new natural products, and a considerable number of these have displayed promising biological and pharmacological properties such as antiviral, antibacterial, antitumor, antifouling, anti-inflammatory, as well as immunomodulatory activity [11–14]. In the search for new pharmaceutical leads in sponge-associated fungi, the organic extract of Trichoderma sp. HPQJ-34, isolated from the marine sponge Hymeniacidon perleve, was found to have potent Mar. Drugs 2017, 15, 260; doi:10.3390/md15080260
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Bioactivity-guided chromatographic separation of the EtOAc extract obtained from the fermentation anti-oxidative, anti-Aβ fibrillization properties and neuroprotective effects during preliminary testing. Bioactivity-guided chromatographic separation thecyclopentenone, EtOAc extract obtained from the fermentation(1), broth of this strain resulted in the isolation of a of new 5-hydroxycyclopenicillone Bioactivity-guided chromatographic separation of the EtOAc extract obtained from the fermentation brothwith of this strain resulted in the isolation of a new cyclopentenone, 5-hydroxycyclopenicillone along three known compounds: ar-turmerone (2) [15], citreoisocoumarin (3) [16] and(1), 6-Obroth of this strain resulted in the isolation of a new cyclopentenone, 5-hydroxycyclopenicillone along with three known compounds: ar-turmerone (2) [15], citreoisocoumarin (3) [16] and 6-Omethyl-citreoisocoumarin (4) [17] (Figure 1).The details of the isolation, the structure identification (1), along with three known compounds: ar-turmerone (2) [15], citreoisocoumarin (3) [16] and (4) [17] (Figure 1).The details of thethese isolation, the metabolites structure identification ormethyl-citreoisocoumarin elucidation, and the biological activities associated with fungal are reported 6-O-methyl-citreoisocoumarin (4) [17] (Figure 1).The details of the isolation, the structure identification or elucidation, and the biological activities associated with these fungal metabolites are reported below. orbelow. elucidation, and the biological activities associated with these fungal metabolites are reported below.
Figure 1. The chemical structures of 1–4, isolated from the sponge-derived fungus Trichoderma Figure isolated from fromthe thesponge-derived sponge-derivedfungus fungus Trichoderma Figure1.1.The Thechemical chemicalstructures structures of of 1–4, isolated Trichoderma sp. sp. sp. HPQJ-34. HPQJ-34. HPQJ-34.
2. 2. Results and Discussion Resultsand andDiscussion Discussion 2. Results 2.1. Isolation and Taxonomy the 2.1. Isolationand andTaxonomy Taxonomyof theProducing Producing Microorganism Microorganism 2.1. Isolation ofofthe Producing Microorganism The fungal strain Trichoderma sp. HPQJ-34 was isolated isolated fromthe the spongeHymeniacidon Hymeniacidon perleve Thefungal fungalstrain strainTrichoderma Trichoderma sp. sp. HPQJ-34 HPQJ-34 was perleve The was isolatedfrom from thesponge sponge Hymeniacidon perleve collected from Zhejiang, China. ItItwas was identifiedasasTrichoderma Trichoderma HPQJ-34 according collected fromDongji DongjiIsland, Island,Zhejiang, Zhejiang, China. sp.sp. HPQJ-34 according collected from Dongji Island, It wasidentified identified as Trichoderma sp. HPQJ-34 according to tomorphological (ITSrDNA rDNAsequence) sequence) analyses. A BLASTN employing morphological and and molecular molecular (ITS analyses. A BLASTN searchsearch employing the to morphological and molecular (ITS rDNA sequence) analyses. A BLASTN search employing the Chain Reaction (PCR)-amplified Internal Transcribed SpacerSpacer (ITS) rDNA sequence (640 thePolymerase Polymerase Chain Reaction (PCR)-amplified Internal Transcribed (ITS) rDNA sequence Polymerase Chain Reaction (PCR)-amplified Internal Transcribed Spacer (ITS) rDNA sequence (640 bp)bp) indicated that that the strain was closely relatedrelated to Trichoderma harzianum strain MGQ2 (99% (T) (640 indicated the strain was closely to Trichoderma harzianum strain (T) MGQ2 bp) indicated that the strain was closely related to Trichoderma harzianum strain MGQ2 (T) (99% similarity). A phylogenetic tree was using using the neighbor-joining methodmethod corrected with (99% similarity). A phylogenetic tree constructed was constructed the neighbor-joining corrected similarity). A phylogenetic tree was constructed using the neighbor-joining method corrected with the Jukes–Cantor algorithm (Figure 2), also showing that the HPQJ-34 strain is a member of the genus with the Jukes–Cantor algorithm (Figure 2), also showing that the HPQJ-34 strain is a member of the theTrichoderma Jukes–Cantor algorithm (Figure 2), also showing that the HPQJ-34 strain is a member of the genus [18]. [18]. genus Trichoderma Trichoderma [18]. 95
HPQJ34 HPQJ34
97 95 KC342029.1_Trichoderma_harzianum_strain_MGQ2 77 60 63 46
63
60
77
97
KC342029.1_Trichoderma_harzianum_strain_MGQ2 EU280124.1_Trichoderma_hamatum_strain_DAOM_167057 EU280124.1_Trichoderma_hamatum_strain_DAOM_167057 KY102298.1_Candida_parapsilosis_culture-collection_CBS_11892
KT310169.1_Trichophyton_tonsurans_strain_CBS_127.97 KY102298.1_Candida_parapsilosis_culture-collection_CBS_11892 XM_002851228.1_Arthroderma_otae_CBS_113480 KT310169.1_Trichophyton_tonsurans_strain_CBS_127.97
46
XM_003010622.1_Arthroderma_benhamiae_CBS_112371 XM_002851228.1_Arthroderma_otae_CBS_113480 XM_001402349.2_Aspergillus_niger_CBS_513.88 XM_003010622.1_Arthroderma_benhamiae_CBS_112371 63 61
63
XM_001210850.1_Aspergillus_terreus_NIH2624 XM_001402349.2_Aspergillus_niger_CBS_513.88 KP171180.1_Grosmannia_clavigera_strain_KW_1407 XM_001210850.1_Aspergillus_terreus_NIH2624
EU747605.1_Trichiurus_haumela_clone_Trha68 61
KP171180.1_Grosmannia_clavigera_strain_KW_1407
EU747605.1_Trichiurus_haumela_clone_Trha68 0.2
0.2 Figure 2. Phylogenetic tree built with Molecular Evolutionary Genetics Analysis (MEGA) 5.05 based onFigure nearly2.complete ITS rDNA gene sequences of Evolutionary HPQJ-34. Genetics Analysis (MEGA) 5.05 based Phylogenetic tree built with Molecular on nearly complete ITS rDNA gene sequences of HPQJ-34. Figure 2. Phylogenetic tree built with Molecular Evolutionary Genetics Analysis (MEGA) 5.05 based on nearly complete ITS rDNA gene sequences of HPQJ-34.
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The new molecule, 5-hydroxycyclopenicillone (1), was obtained as a yellow oil. Compound 1 has the molecular formula C13H20O4, which was determined from a sodiated molecular ion adduct 2.2. Elucidation peakStructure in the High-resolution electrospray ionisation mass spectrometry (HRESIMS) at m/z 263.1247 + [M +The Na]new (calcd. for C 13H20O4Na+, 263.1254). The 1H NMR spectrum of 1 in CD3OD showed two molecule, 5-hydroxycyclopenicillone (1), was obtained as a yellow oil. Compound 1 has methyl doublets at δH C 1.65H(3H, d, J = 4.8 Hz, CH3-6′) and 1.75 (3H, d, J = 1.0 Hz, CH3-6), one methyl the molecular formula 13 20 O4 , which was determined from a sodiated molecular ion adduct peak in singlet at δH 1.23 (3H, s, CH3-7), two olefinicmass methines at δH 5.49 (1H, m, CH-4′) and 5.48 (1H, CH-+ the High-resolution electrospray ionisation spectrometry (HRESIMS) at m/z 263.1247 [Mm, + Na] 5′), two oxygenated methines at δ H 4.49 (1H, d, J = 1.0 Hz, CH-4) and 4.72 (1H, dd, J = 8.6, 4.8 Hz, CH+ 1 (calcd. for C13 H20 O4 Na , 263.1254). The H NMR spectrum of 1 in CD3 OD showed two methyl 1′), as well as signals with complex coupling patterns attributed to two methylenes between δ H 1.76 0 doublets at δH 1.65 (3H, d, J = 4.8 Hz, CH3 -6 ) and 1.75 (3H, d, J = 1.0 Hz, CH3 -6), one methyl singlet and 1).CH The-7), four degrees of methines unsaturation in the molecular formula of 1, together 0 0 at δH2.12 1.23(Table (3H, s, two olefinic at δinherent 3 H 5.49 (1H, m, CH-4 ) and 5.48 (1H, m, CH-5 ), 13C NMR and with data showing the presence of one carbonyl and four olefinic carbons in the two oxygenated methines at δH 4.49 (1H, d, J = 1.0 Hz, CH-4) and 4.72 (1H, dd, J = 8.6, 4.8 Hz, CH-10 ), Distortionless Enhancement by coupling Polarization Transfer (DEPT)tospectra, indicatedbetween that the structure of as well as signals with complex patterns attributed two methylenes δH 1.76 and 1 possesses one ring [19]. Selected Heteronuclear Multiple Bond Correlation (HMBC) correlations, 2.12 (Table 1). The four degrees of unsaturation inherent in the molecular formula of 1, together with shown in Figure including from CH3-7 and to C-1, and C-5, from -6CtoNMR C-1, and C-2, Distortionless and C-3, and data showing the 3, presence of one carbonyl fourC-4 olefinic carbons inCH the313 from CH-4 to by C-3Polarization indicated the presence of a 3-substituted 4,5-dihydroxy-2,5-dimethylcyclopent-2Enhancement Transfer (DEPT) spectra, indicated that the structure of 1 possesses one enone ring system in 1. This assignment was further supported by the homoallylic coupling (J = 1.0 ring [19]. Selected Heteronuclear Multiple Bond Correlation (HMBC) correlations, shown in Figure 3, Hz) observed between CH-4 and CH 3-6. Consecutive 1H–1H COrrelated SpectroscopY (COSY) including from CH3 -7 to C-1, C-4 and C-5, from CH3 -6 to C-1, C-2, and C-3, and from CH-4 to C-3 correlations CH3of -6′a to CH-5′, then CH-4′, CH-3′, CH-2′ and finally CH-1′ suggested a in 1indicated the from presence 3-substituted 4,5-dihydroxy-2,5-dimethylcyclopent-2-enone ring system disubstituted 1-hydroxyhex-4-ene moiety as an additional substructure of 1. This was reinforced by 1. This assignment was further supported by the homoallylic coupling (J = 1.0 Hz) observed between HMBCand correlations observed from 3-6′ to C-4′ and C-5′, as well as from CH-2′ to C-1′, C-3′, and0 C1 H–1CH CH-4 CH3 -6. Consecutive H COrrelated SpectroscopY (COSY) correlations from CH3 -6 to 4′. Finally, the connection between the two structural fragments was established as a C-3 to C-1′ 0 0 0 0 0 CH-5 , then CH-4 , CH-3 , CH-2 and finally CH-1 suggested a 1-disubstituted 1-hydroxyhex-4-ene linkage by the HMBC correlation observed from CH-2′ to C-3, thus completing the planar structure moiety as an additional substructure of 1. This was reinforced by HMBC correlations observed from of 1. 0 0 0 0 0 0 0 CH -6 to C-4 and C-5 , as well as from CH-2 to C-1 , C-3 , and C-4 . Finally, the connection between 3
the two structural fragments was established as a C-3 to C-10 linkage by the HMBC correlation observed Table 1. The 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data of compound 1 (in CD3OD). from CH-20 to C-3, thus completing the planar structure of 1. 1H–1H COSY Position δC, Type δH, Mult. (J in Hz) HMBC NOESY 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) data of compound 1 (in CD OD). Table 1. The 3 1 210.4, C 2 135.8, C 1 H–1 H COSY Position δC , Type δH , Mult. (J in Hz) HMBC NOESY 3 171.7, C 1 4 75.3,210.4, CH C 4.49, d (1.0) 3 7, 2′ 2 135.8, 5 74.5, C C 3 171.7, C 6 8.4,75.3, CH3CH 1.75, d (1.0) 1, 2, 3 7, 20 1′ 4 4.49, d (1.0) 3 7 23.1, CH 3 1.23, s 1, 4, 5 4 5 74.5, C 6 8.4, CH3 1.75, (1.0) 4.8) 1, 2,2,33, 4, 2′, 3′ 10 1′ 69.4, CH 4.72, ddd(8.6, 2′ 6, 2′, 3′ 7 23.1, CH3 s 8.6, 5.5), 1, 4, 5 4 1.86, dtd1.23, (14.2, 2′ 36.6,69.4, CHCH 2 4, 1′ 10 4.72, dd (8.6, 4.8) 201′, 3′ 2, 3, 4, 3, 20 ,1′, 30 3′, 4′6, 20 , 30 1.76, m 1.86, dtd (14.2, 8.6, 5.5), 0 0 0 0 0 0 0 2 36.6, 1 , 32′, 4′ 3, 1 , 3 , 41′, 5′ 4, 1 3′ 29.8, CHCH 2 2 2.12,mm 1.76, 0 5′ 4′ 131.6, CH 5.49,m m 3′ 30 29.8, CH2 2.12, 20 , 43′, 10 , 50 0 40 131.6, 5.49, 30 , 54′, 30 5′ 126.7, CHCH 5.48,m m 6′ 3′ 6′ 50 126.7, CH 5.48, m 40 , 60 5′ 30 4′, 5′ 60 6′ 18.1, CH 3 1.64, d (4.8) 5′ 0 0 0 0 0
6
18.1, CH3
1.64, d (4.8)
5
4,5
5
Figure 3. Selected HMBC and 11H–11 H COSY correlations observed for 1. Figure 3. Selected HMBC and H– H COSY correlations observed for 1.
Because the 1 H NMR signals for CH-40 and CH-50 in 1 significantly overlapped, J coupling analysis was precluded. Therefore, the double bond connecting these carbons was assigned as trans
of a J-coupling partner for CH-4 (a sharp singlet in 1 and a broad singlet in 5), accompanied by proximal deshielding effects on C-5 (shifted to δC 74.5 in 1 from 50.9 in 5) and C-7 (shifted to δC 23.1 in 1 from 13.8 in 5) [19]. These observations further supported the structure assignment of 1, and strongly suggested a shared relative, or possibly absolute, configuration between the two structural analogues (Figure 4, panel A), because the NMR parameter of chemical shift (δ), especially for the 13C Mar. Drugs 2017, 15, 260 4 of 12 nucleus, is particularly sensitive to perturbations in conformation and differences in configuration. It was also observed that, for 1, as previously reported for 5 [19], the NOE between CH3-6 and 13 CCH-2ʹ CH-1ʹ CH-4 and a restricted free rotation about toIfC-1′ basedalong on thewith deshielded NMRindicated shift of the terminal methyl carbon, C-60 the (δC C-3 18.1). thebond. C-40 toThis C-50 isdouble likely bond due to a combination ofthis steric hindrance resulting inflexibility of the was cis-configured, terminal methyl carbon from wouldthe be relative significantly more shielded, cyclopentenone ring, along with an intramolecular hydrogen bond. The latter was indicated bythe a in the range of approximately 10–13 ppm [19]. The NMR data for this terminus of 1, and in fact −1 in the spectrum of 1, as was previously described for 5 [19]. Both OH-1′ broad IR stretch at 3369 cm entire 1-hydroxyhex-4-ene moiety, closely matched the reported values for the structural analogue and OH-4 are expected to exhibit hydrogen in 1 and 5, however, OH-5 may also cyclopenicillone (5), which was not isolatedbonding in this study [19]. In fact, the NMR data forinteract 1 and 5 with OH-4 in the case of 1. To examine this further, an energy minimized computational molecular were remarkably similar (Supplementary Materials Table S11), with obvious differences including the model generatedpartner for both and 5,(ausing Hartree–Fock to visualize their preferred loss ofwas a J-coupling for1 CH-4 sharpthe singlet in 1 and amethod, broad singlet in 5), accompanied by conformations (Figure 4, panel B). Because both the molecular modeling and NMR data collected for proximal deshielding effects on C-5 (shifted to δC 74.5 in 1 from 50.9 in 5) and C-7 (shifted to δC 23.1 in 11and strongly suggest a shared conformation for these two molecules, and because from5 13.8 in 5) [19]. These observations further supported the structure assignment of 1,the andabsolute strongly configuration of 5 was previously characterized, including the between use of theoretically predicted ECD suggested a shared relative, or possibly absolute, configuration the two structural analogues 13 spectra for its low energy molecular modeling conformers, it was reasoned that an electronic circular (Figure 4, panel A), because the NMR parameter of chemical shift (δ), especially for the C nucleus, is dichroism (ECD) spectrum could be used to resolve the and absolute configuration of 1. particularly sensitive to perturbations in conformation differences in configuration.
Figure 4. Structural comparison of 1 with reported analogue 5. Panel A: Chemical structures Figure 4. Structural comparison of 1 with reported analogue 5. Panel A: Chemical structures of 1 and of 1 and 5. Panel B: Energy-minimized computational molecular models of 1 and 5, showing a 5. Panel B: Energy-minimized computational molecular models of 1 and 5, showing a similar similar conformation. conformation.
It was 1, asestablished previouslyas reported for 5 [19], between The usealso of observed ECD has that, beenfor well a powerful tool the forNOE assigning theCH absolute 3 -6 and 0 along with CH-4 and CH-20 indicated a restricted free rotation about the C-3 to C-10 bond. CH-1 configuration of natural products [20–22]. In the experimental (ECD) spectrum of 1, the local maxima likely due to aeffect combination steric hindrance resulting from the relative inflexibility of the ofThis the is positive Cotton (CE) wasofobserved at 246 nm, and negative CEs were observed around cyclopentenone ring, along with an intramolecular hydrogen bond. The latter was indicated by a 321 and 214 nm. By comparison, the experimental ECD spectrum of 5 was similar to that of 1, with − 1 broad IR stretch CE at 3369 cm atin the spectrum of 1, asby was previously described 5 [19]. Both OH-10 obvious positive maxima 240 nm accompanied negative CEs around 326for and 209 nm (Figure arecases, expected to exhibitpositive hydrogen bonding in 1 and however, OH-5 may second also interact 5)and [19].OH-4 In both the observed Davydov splitting for5,positive first, negative CEs with OH-4 in the case of 1. To examine this further, an energy minimized computational molecular can be attributed to a positive angle of rotation present between the enone and alkene chromophores, generated 1 and 5,models using the Hartree–Fock method, to visualize preferred asmodel also was suggested by for the both molecular presented in Figure 4. As a result, their the absolute conformationsof(Figure panel B). Because both molecular modeling and data collected for configuration 1 was 4, determined to match thethe reported configuration of NMR 5, or 4(S), 5(S),1′ (R). 1 and 5 strongly suggest a shared conformation for these two molecules, and because the absolute Consequently, the structure of 1 was unambiguously established and named as 5configuration of 5 was previously characterized, including the use of theoretically predicted ECD hydroxycyclopenicillone (1) {IUPAC: (4S,5S)-4,5-dihydroxy-3-[(R,E)-1-hydroxyhex-4-en-1-yl]-2,5spectra for its low energy molecular modeling conformers, it was reasoned that an electronic circular dimethylcyclopent-2-en-1-one}. dichroism (ECD) spectrum could be used to resolve the absolute configuration of 1. The use of ECD has been well established as a powerful tool for assigning the absolute configuration of natural products [20–22]. In the experimental (ECD) spectrum of 1, the local maxima of the positive Cotton effect (CE) was observed at 246 nm, and negative CEs were observed around 321 and 214 nm. By comparison, the experimental ECD spectrum of 5 was similar to that of 1, with obvious positive CE maxima at 240 nm accompanied by negative CEs around 326 and 209 nm (Figure 5) [19]. In both cases, the observed positive Davydov splitting for positive first, negative second CEs can be attributed to a positive angle of rotation present between the enone and alkene chromophores, as also suggested by the molecular models presented in Figure 4. As a result, the absolute configuration of 1 was determined to match the reported configuration of 5, or 4(S), 5(S),10 (R). Consequently, the structure of 1 was unambiguously established and named as 5-hydroxycyclopenicillone (1) {IUPAC: (4S,5S)-4,5-dihydroxy-3-[(R,E)-1-hydroxyhex-4-en-1-yl]-2,5-dimethylcyclopent-2-en-1-one}.
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Figure 5. The electronic circular dichroism (ECD) spectra of 5-hydroxycyclopenicillone (1) and Figure 5. The electronic circular dichroism (ECD) spectra of 5-hydroxycyclopenicillone (1) and cyclopenicillone (5); from reference [19]. Figure 5. The electronic circular[19]. dichroism (ECD) spectra of 5-hydroxycyclopenicillone (1) and cyclopenicillone (5); from reference cyclopenicillone (5); from reference [19].
In addition to 1, three known metabolites were isolated during the course of this research. These wereInidentified, by their metabolites spectrometric and isolated spectroscopic data literature values, as In addition 1, threeknown known were isolated during during the course ofwith this research. These addition to comparing 1,tothree metabolites were the course of this research. were identified, by comparing their spectrometric and spectroscopic data with literature values, as ar-turmerone (2) [15], citreoisocoumarin [16], and 6-O-methyl-citreoisocoumarin (4) [17]. values, These were identified, by comparing their(3) spectrometric and spectroscopic data with literature ar-turmerone [15], citreoisocoumarin (3) andand 6-O-methyl-citreoisocoumarin (4) [17]. (4) [17]. as ar-turmerone (2)(2) [15], citreoisocoumarin (3)[16], [16], 6-O-methyl-citreoisocoumarin 2.3. Biological Activities 2.3. Biological Activities 2.3. Biological Activities The effects of 5-hydroxycyclopenicillone wereevaluated evaluated the scavenging free radicals. The effects of 5-hydroxycyclopenicillone (1) (1) were for for the scavenging of free of radicals. The effects of 5-hydroxycyclopenicillone (1) were evaluated for the scavenging of radicals. As shown in Figure 6, compound 1 concentration–dependency reduced the content of DPPH free As shown in Figure 6, compound 1 concentration–dependency reduced the content of DPPHfree free As shown in Figure 6, compound 1 concentration–dependency reduced the content of DPPH free radicals, suggesting it might moderate free scavenger. These anti-oxidative effects effects radicals, suggesting that that it might actact asasa amoderate freeradical radical scavenger. These anti-oxidative ofsuggesting 1 might beto due toitpresence the presence of multiple hydroxy Many anti-oxidants, assuch vitamin radicals, that might act a moderate freegroups. radical scavenger. Thesesuch anti-oxidative effects of 1 might be due the ofas multiple hydroxy groups. Many anti-oxidants, as vitamin C and arepresence useful for the delay of onset and relief of symptoms Alzheimer’s disease (AD) of 1 might bevitamin due toE,the multiple hydroxy groups. Manyofanti-oxidants, such as vitamin C and vitamin E, are useful for theof delay of onset and relief of symptoms of Alzheimer’s disease (AD) [23]. Therefore, it was hypothesized that 1, a new cyclopentenone with moderate free radical C and vitamin E, are useful for the delay of onset and relief of symptoms of Alzheimer’s disease [23]. Therefore, it was hypothesized that 1, a new cyclopentenone with moderate free radical scavenging properties, might be effective in AD models. (AD) [23]. Therefore, was hypothesized a new cyclopentenone with moderate free radical scavenging properties,it might be effective inthat AD1,models. scavenging properties, might be effective in AD models.
Figure 6. 5-hydroxycyclopenicillone (1) scavenges DPPH free radicals in a concentration-dependent manner. Compound 1 or vitamin C (VitC) at indicated concentrations were added to 0.2 mM DPPH
Figure 6. 6. 5-hydroxycyclopenicillone 5-hydroxycyclopenicillone (1) (1) scavenges scavenges DPPH DPPH free free radicals radicals in in aa concentration-dependent concentration-dependent Figure manner. Compound 1 or vitamin C (VitC) at indicated concentrations were added to to 0.2 0.2 mM mM DPPH DPPH manner. Compound 1 or vitamin C (VitC) at indicated concentrations were added solution for 20 min. The concentration of DPPH free radicals was measured by evaluating the absorbance at 517 nm. Data are presented as the mean ± SEM of three separate experiments; ** p < 0.01 vs. the control group (ANOVA and Dunnett’s test).
solution for 20 min. The concentration of DPPH free radicals was measured by evaluating the absorbance at 517 nm. Data are presented as the mean ± SEM of three separate experiments; ** p < 0.01 vs. the control group (ANOVA and Dunnett’s test).
Previous studies have shown that Aβ peptides accumulate in the brain of AD patients [24]. Aβ Mar. Drugs 2017, 15, 260 6 of 12 peptides can form highly toxic oligomers and fibrils, which are considered as the key pathogenic factors of AD [25]. Aβ oligomers and fibrils can further aggregate into plaques, the hallmark of AD. studies have inhibit shown that peptidesof accumulate in the brain of AD patientsdisease[24]. Therefore,Previous drugs that could the Aβ formation Aβ fibrillization might produce Aβ peptides can form highly toxic oligomers and fibrils, which are considered as the key pathogenic modifying effects for AD treatment [26]. Thus, the effect of 1 on the formation of Aβ fibrils was factors of AD [25]. Aβ oligomers and fibrils can further aggregate into plaques, the hallmark of AD. measured. As shown in Figure 7, co-incubation of 1 and Aβ monomers led to a decreased formation Therefore, drugs that could inhibit the formation of Aβ fibrillization might produce disease-modifying of Aβ fibrils when compared to the incubation of Aβ monomers alone. Moreover, curcumin, a effects for AD treatment [26]. Thus, the effect of 1 on the formation of Aβ fibrils was measured. positive control, also inhibited the formation of Aβ fibrils led at about the same level asofcompound As shown in Figure 7, co-incubation of 1 and Aβ monomers to a decreased formation Aβ fibrils 1 (Figure 7). These results suggested that 5-hydroxycyclopenicillone (1) could effectively inhibit when compared to the incubation of Aβ monomers alone. Moreover, curcumin, a positive control, also the formation of Aβ inhibited thefibrils. formation of Aβ fibrils at about the same level as compound 1 (Figure 7). These results suggested that 5-hydroxycyclopenicillone (1) could effectively inhibit the formation of Aβ fibrils.
Figure 7. 5-Hydroxycyclopenicillone (1) significantly decreases the formation of Aβ1-42 fibrils. Aβ1-42
Figuremonomers 7. 5-Hydroxycyclopenicillone significantly the formation Aβ1-42 fibrils. for Aβ1-42 (10 µM) were incubated(1) with or without decreases 1 or curcumin at indicatedofconcentrations monomers μM) were incubated orthe without 1 orTcurcumin at indicated for 3 3 days.(10 Aβ fibrils were measuredwith using thioflavin (ThT) assay. ** p < 0.01 concentrations vs. Aβ1-42 group Dunnett’s test).using the thioflavin T (ThT) assay. ** p < 0.01 vs. Aβ1-42 group (ANOVA days. (ANOVA Aβ fibrilsand were measured and Dunnett’s test). Besides Aβ fibrils, oxidative stress also plays an important role in AD pathogenesis [27]. Hydrogen peroxide (H2 O widely used to establish stress-induced models,[27]. Besides Aβ fibrils, oxidative stress also plays oxidative an important role in neurotoxicity AD pathogenesis 2 ), an agent can increase intracellular reactive and promoteoxidative neurotoxicity via its acting neurotoxicity on various Hydrogen peroxide (H2O2), an agentoxygen widelyspecies used to establish stress-induced macromolecules [28]. Therefore, the neuroprotective effects of 5-hydroxycyclopenicillone on on models, can increase intracellular reactive oxygen species and promote neurotoxicity via its (1) acting H2 O in SH-SY5Y cells was investigated. cells were pre-treated (1) 2 –induced neuronal various macromolecules [28].loss Therefore, the neuroprotective effectsSH-SY5Y of 5-hydroxycyclopenicillone with 1 or vitamin C for 2 h, followed by treatment with 200 µM of 30% H2 O2 (in H2 O) for 24 h. on H2O2–induced neuronal loss in SH-SY5Y cells was investigated. SH-SY5Y cells were pre-treated Cell viability was determined by the MTT assay. In Figure 8, panel A shows that 1 significantly with 1 or vitamin C for 2 h, followed by treatment with 200 μM of 30% H2O2 (in H2O) for 24 h. Cell reduced H2 O2– induced neuronal death in a concentration-dependent manner, demonstrating that viability was determined by the MTT assay.neuroprotectant. In Figure 8, panel A shows that 1 with significantly reduced 5-hydroxycyclopenicillone is moderately Moreover, treatment 50 µM 1 alone H2O2–for induced death in suggesting a concentration-dependent that 526 h wasneuronal not cytotoxic to cells, that this agent may bemanner, quite safe.demonstrating Fluorescein diacetate hydroxycyclopenicillone is moderately neuroprotectant. Moreover, treatment with 50 μM 1 alone (FDA)/ propidium iodide (PI) double staining for both live cells and dead cells further confirmed for 26 h was cytotoxic to dramatically cells, suggesting that this agent may be neuronal quite safe. Fluorescein that 1not at 30 and 50 µM protected against H2 O2 –induced death in SH-SY5Ydiacetate cells (Figure 8, paneliodide B). (FDA)/ propidium (PI) double staining for both live cells and dead cells further confirmed that
1 at 30 and 50 μM dramatically protected against H2O2–induced neuronal death in SH-SY5Y cells (Figure 8, panel B).
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Figure (1) protects protects against against HH2O neuronaldeath death 2O 2 -inducedneuronal Figure8. 8. 5-hydroxycyclopenicillone 5-hydroxycyclopenicillone (1) 2-induced in in a a concentration-dependent manner in SH-SY5Y cells. (A): SH-SY5Y cells were treated with 1 or vitamin concentration-dependent manner in SH-SY5Y cells. (A): SH-SY5Y cells were treated with 1 or vitamin C (VitC) at at indicated exposedto to200 200μM µMofof30% 30%HH 2O 2 (in 2 O). C (VitC) indicatedconcentrations. concentrations.After After 22 h, h, cells cells were were exposed 2O 2 (in H2H O). MTT assay was used O22exposure. exposure.Data Dataare are expressed MTT assay was usedtotomeasure measurecell cellviability viability after after 24 24 h of H22O expressed as as thethe percentage ofof control SEMof ofthree threeseparate separateexperiments; experiments; < 0.05 percentage controland andare arepresented presentedas asthe themean mean ± ± SEM * p*
