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Asperidines A–C, pyrrolidine and piperidine derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178
Bioorganic & Medicinal Chemistry 26 (2018) 4502–4508
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Asperidines A–C, pyrrolidine and piperidine derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178
T
⁎
Patima Phainuphonga, Vatcharin Rukachaisirikula, , Saowanit Saithonga, Souwalak Phongpaichitb, Jariya Sakayarojc, Chutima Srimaroengd, Atcharaporn Ontawongd,e, Acharaporn Duangjaie, Paradorn Muangnilf, Chatchai Muanprasatf a
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c School of Science, Walailak University, Tha Sala, Nakhon Si Thammarat 80161, Thailand d Department of Physiology, Faculty of Medicine, Chiang Mai University, Mueang District, Chiang Mai 50200, Thailand e Division of Physiology, School of Medical Sciences, University of Phayao, Mueang District, Phayao 56000, Thailand f Excellent Center for Drug Discovery and Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Aspergillus sclerotiorum Pyrrolidine derivative Piperidine derivative CFTR inhibitor Antioxidant Cytotoxicity
One new pyrrolidine derivative, asperidine A (1), and two new piperidine derivatives, asperidines B (2) and C (3), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 together with two known alkaloids. Compound 3 possessed an unprecedented 7-oxa-1-azabicyclo[3.2.1]octane skeleton with four chiral centers. Their structures were determined by spectroscopic evidence. The absolute configurations of compounds 2 and 3 were established using Mosher’s method and further confirmed for compound 3 by X-ray crystallographic data. Compound 2 dose-dependently inhibited the CFTR-mediated chloride secretion in T84 cells with an IC50 value of 0.96 μM whereas 3 displayed the same activity with the IC50 value of 58.62 μM. Compounds 2 and 3 also significantly reduced intracellular ROS under both normal and H2O2-treated conditions compared with their respective controls in a dose-dependent manner without cytotoxic effect on Caco-2 cells. In addition, compound 3 was inactive against noncancerous Vero cells whereas compound 2 was considered to be inactive with the IC50 value of > 10 μM.
1. Introduction Fungi in the genus Aspergillus have been recognized as prolific sources of new bioactive secondary metabolites which were documented in recent years. Over the past few years, many new metabolites were isolated from the genus Aspergillus. Some of them exhibited interesting biological activities such as cytotoxic activity against HepG2 cell lines,1 HMG-CoA reductase inhibitory activity,2 α-glucosidase inhibitory activity3 and antiproliferative activity against the H460 cell lines.4 In our ongoing search for new bioactive metabolites from soilderived fungi, we have investigated secondary metabolites produced by the soil fungus Aspergillus sclerotiorum PSU-RSPG178 isolated from a soil sample collected from the Plant Genetic Conservation Project under the Royal Initiation of Her Royal Highness Princess Maha Chakri Sirindhorn at Ratchaprapa Dam in Suratthani Province, Thailand. Recently, we have reported the isolation of three new and four known lovastatin analogues2 as well as seven new γ-butenolide and furanone derivatives
⁎
together with six known compounds5 from the crude extracts of A. sclerotiorum PSU-RSPG178. Further chemical investigation of the broth ethyl acetate extract led to the isolation of one new pyrrolidine derivative, asperidine A (1), and 3-[[5-(1,1-dimethyl-2-propen-1-yl)-1Himidazol-4-yl]methylene]-6-methyl-(6S)-2,5-piperazinedione (4) which was isolated as a natural product for the first time.6 Moreover, two new piperidine derivatives, asperidines B (2) and C (3), were obtained from the mycelial hexane extract whereas nicotinic acid (5)7 was isolated from the mycelial ethyl acetate extract. Compounds 2 and 3 were evaluated for HMG-CoA reductase (HMGR) and cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory activities, free radical-scavenging activity and cytotoxicity against human colon cancer (Caco-2) cell lines and African green monkey kidney fibroblast (Vero) cells.
Corresponding author. E-mail address: [email protected] (V. Rukachaisirikul).
https://doi.org/10.1016/j.bmc.2018.07.036 Received 2 June 2018; Received in revised form 20 July 2018; Accepted 22 July 2018 Available online 23 July 2018 0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
Bioorganic & Medicinal Chemistry 26 (2018) 4502–4508
P. Phainuphong et al.
OH
3 9
11
7
5
3'
N H
1'
O
7'
1 5 13
9
11
1
3
N
7
OH
5'
3'
HN
N
HN
15
13
11
9
1
N
7
O
7'
1'
O OH
OH 5
NH
4
15
2
7.33 (t, J = 7.6 Hz, 2H) and 7.30 (m, 1H)], three methine protons [δH 4.23 (brt, J = 3.1 Hz, 1H), 3.58 (dt, J = 7.4 and 3.1 Hz, 1H) and 3.56 (m, 1H)], two sets of nonequivalent methylene protons [δH 3.22 (dd, J = 13.9 and 7.2 Hz, 1H)/3.00 (dd, J = 13.9 and 7.6 Hz, 1H) and 2.45 (ddd, J = 14.1, 10.1 and 5.0 Hz, 1H)/1.74 (td, J = 14.1 and 5.0 Hz, 1H)] and a n-heptyl side chain [δH (1.80, m, 1H/1.75, m, 1H), 1.33 (m, 10H) and 0.90 (t, J = 6.9 Hz, 3H)]. The 13C NMR spectrum (Table 1) consisted of signals for one quaternary carbon (δC 136.7), three resonances for five aromatic methine carbons [δC 128.8 (×2), 128.5 (×2) and 126.8], three methine carbons (δC 69.3, 66.4 and 58.7), eight methylene carbons (δC 38.5, 34.2, 31.9, 31.5, 28.9, 28.8, 26.4 and 22.3) and one methyl carbon (δC 13.0). In the HMBC spectrum, Hab-1′ (δH 3.22 and 3.00) displayed the correlations to C-2′ (δC 136.7), C-3′ (δC 128.8) and C-7′ (δC 128.8) of the monosubstituted benzene ring (Table 1), indicating the presence of a benzyl unit having four degrees of unsaturation. Based on the 1H–1H COSY correlations of H-2 (δH 3.58)/Hab-1′ and H-3 (δH 4.23), and Hab-4 (δH 2.45 and 1.74)/H-3 and H-5 (δH 3.56), the chemical shifts of C-2 (δC 66.4) and C-5 (δC 58.7) and the remaining one degree of unsaturation, a 5-substituted 2-benzylpyrrolidine moiety was established. A substituent at C-3 was a hydroxy group due to the chemical shift of C-3 (δC 69.3). The remaining signals in the 1H and 13C NMR spectra together with the molecular formula established the n-heptyl side chain which was attached at C-5 according to a HMBC correlation of H-5 with C-6 (δC 34.2) of the n-heptyl unit and 1 H–1H COSY correlations from Hab-6 (δH 1.80 and 1.75) to H-5. In the NOEDIFF experiments, irradiation of Ha-4 enhanced the signal intensity of H-3, Hb-4 and H-5 whereas the signal intensity of H-2 was affected after irradiation of H-3 (Fig. 2). These results indicated a cis-relationship of H-2, H-3 and H-5. Comparison of the specific rotation of 1, ([α ]26 D = −13.8, c 1.0, MeOH), with that of (−)-(2S,3S,5R)-2-benzyl-310 hydroxy-5-nonylpyrrolidine ([α ]20 indicated D = −15.6, c 1.0, MeOH) that they possessed the same absolute configurations. Therefore, 1 was assigned as a 5-n-heptyl derivative of the above pyrrolidine derivative. Asperidine B (2) was obtained as a colorless gum with the molecular formula C21H35NO determined by the HRESIMS peak at m/z 318.2797 [M+H]+. The UV and IR spectra were similar to those of 1, suggesting the presence of a benzene chromophore and a hydroxy group. The 1H NMR spectroscopic data (Table 2) contained signals for five aromatic protons of a monosubstituted benzene [δH 7.30 (d, J = 8.1 Hz, 2H), 7.28 (t, J = 8.1 Hz, 2H) and 7.18 (m, 1H)], three methine protons [δH 3.80 (brs, 1H), 2.25 (ddd, J = 9.3, 6.3 and 5.0 Hz, 1H) and 2.10 (m, 1H)], three sets of nonequivalent methylene protons [δH 2.88 (dd, J = 12.9 and 9.3 Hz, 1H)/2.84 (dd, J = 12.9 and 5.1 Hz, 1H), 2.20 (m, 1H)/1.40 (ddd, J = 13.8, 6.3 and 1.2 Hz, 1H) and 1.71 (m, 1H)/1.26 (m, 1H)] and a n-octyl side chain [δH 1.27 (m, 14H) and 0.88 (t, J = 6.6 Hz, 3H)] and one methyl group (δH 2.33, s, 3H). The 13C NMR spectroscopic data (Table 2) displayed signals for one quaternary (δC 139.5), three resonances for five aromatic methine [δC 129.4 (×2), 128.4 (×2) and 126.0], three methine (δC 73.6, 70.5 and 65.8), ten methylene (δC 39.4, 35.0, 33.7, 31.9, 29.9, 29.7, 29.6, 29.3, 26.3 and 22.7) and two methyl (δC 38.7 and 14.1) carbons. A benzyl unit was established by HMBC correlations (Table 2) from Hab-1′ (δH 2.88 and 2.84) to C-2′ (δC 139.5), C-3′ (δC 129.4) and C-7′ (δC 129.4) of the monosubstituted benzene ring. A 6-substituted 2-benzyl-N-methylpiperidine moiety was constructed according to the following 1H–1H COSY correlations: H-2 (δH 2.25)/Hab-1′ and H-3 (δH 3.80), Hab-4 (δH 2.20 and 1.40)/H-3 and Hab-5 (δH 1.71 and 1.26), and Hab-5/H-6 (δH 2.10) together with HMBC correlations of H3-15 (δH 2.33) with C-2 (δC 73.6) and C-6 (δC 65.8). A substituent at C-3 (δC 70.5) was assigned to be a hydroxy group due to its chemical shift. Based on the remaining signals in the 1H and 13C NMR spectra along with the molecular formula, the n-octyl side chain was constructed. HMBC correlations of Hab5 with C-7 (δC 26.3) attached the n-octyl side chain at C-6. Irradiation of H-2 enhanced signal intensity of H-3 and H-6 in the NOEDIFF experiment (Fig. 2), indicating a cis-relationship of H-2, H-3 and H-6. The absolute configuration at C-3 was assigned as S on the basis of Mosher’s
5'
1
O
N
3'
3
5
5'
1' 7'
3 Fig. 1. Structures of compounds 1–5 isolated from Aspergillus sclerotiorum PSURSPG178.
2. Results and discussion All alkaloids (1–5) (Fig. 1) were purified using chromatographic techniques and their structures were elucidated by using various spectroscopic techniques. The relative configuration was assigned according to the NOEDIFF data. The absolute configuration of 1 was established by comparison of the specific rotation with that of a known and structurally related compound. In addition, the absolute configurations of the secondary alcohol at C-3 in 2 and C-4 in 3 were established by Mosher’s method.8,9 Moreover, the absolute configuration of 3 was confirmed by the X-ray data. Asperidine A (1) was obtained as a colorless solid, melting at 97–98 °C, with the molecular formula C18H29NO determined by the HRESIMS peak at m/z 276.2327 [M+H]+, indicating the presence of five degrees of unsaturation. It exhibited UV absorption bands at 207 and 256 nm for an aromatic chromophore while an IR absorption band was observed at 3325 cm−1 for hydroxy and amino groups. The 1H NMR spectroscopic data (Table 1) displayed signals for five aromatic protons of a monosubstituted benzene [δH 7.35 (d, J = 7.6 Hz, 2H),
Table 1 1 H and 13C NMR data of asperidine A (1) in CD3OD. Position
1 δC,a type
δH,b mult. (J, Hz)
HMBC
2 3 4
66.4, CH 69.3, CH 38.5, CH2
5 6
58.7, CH 34.2, CH2
1′, 2′ 2, 5 2, 3, 5, 6 2, 3, 5, 6 4, 6 7, 8 7, 8
7 8 9 10 11 12 1′
28.9, 26.4, 28.8, 31.5, 22.3, 13.0, 31.9,
3.58, dt (7.4, 3.1) 4.23, brt (3.1) a: 2.45, ddd (14.1, 10.1, 5.0) b: 1.74, td (14.1, 5.0) 3.56, m a: 1.80, m b: 1.75, m 1.33, m 1.33, m 1.33, m 1.33, m 1.33, m 0.90, t (6.9) a: 3.22, dd (13.9, 7.2) b: 3.00, dd (13.9, 7.6)
2′ 3′ 4′ 5′ 6′ 7′
136.7, 128.8, 128.5, 126.8, 128.5, 128.8,
7.35, 7.33, 7.30, 7.33, 7.35,
1′, 5′, 7′ 2′ 3′, 4′, 6′, 7′ 2′ 1′, 3′, 5′
a b
CH2 CH2 CH2 CH2 CH2 CH3 CH2 C CH CH CH CH CH
d (7.6) t (7.6) m t (7.6) d (7.6)
10, 11 2, 3, 2′, 3′, 7′ 2, 3, 2′, 3′, 7′
Recorded at 75 MHz. Recorded at 300 MHz. 4503
Bioorganic & Medicinal Chemistry 26 (2018) 4502–4508
P. Phainuphong et al.
Fig. 2. Key NOEDIFF data of compounds 1 and 2.
method using the (S)- and (R)-MTPA esters (Fig. 3). Consequently, the remaining absolute configurations at C-2 and C-6 were identified to be S and R, respectively. Therefore, asperidine B had the structure 2. Asperidine C (3) was obtained as colorless crystals, melting at 95–96 °C, with the molecular formula C21H33NO2 determined by the HRESIMS peak at m/z 332.2590 [M+H]+, indicating the presence of six degrees of unsaturation. The UV spectrum was similar to that of 1, suggesting the presence of a benzene chromophore. The IR spectrum showed an absorption band for a hydroxy group at 3393 cm−1. The 1H NMR spectroscopic data (Table 2) contained signals for five aromatic protons of a monosubstituted benzene [δH 7.36 (dd, J = 7.2 and 1.7 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H) and 7.23 (m, 1H)], four methine protons [δH 5.26 (s, 1H), 3.99 (brt, J = 6.9 Hz, 1H), 2.63 (m, 1H) and 2.60 (dd, J = 6.9 and 4.5 Hz, 1H)], two sets of nonequivalent methylene protons [δH 3.32 (dd, J = 12.0 and 4.5 Hz, 1H)/2.76 (d, J = 12.0 Hz, 1H) and 2.05 (m, 1H)/1.44 (m, 1H)] and a n-nonyl side chain [δH (1.76, m, 1H/ 1.74, m, 1H), 1.39 (m, 2H), 1.26 (m, 12H) and 0.88 (t, J = 6.9 Hz, 3H)]. The 13C NMR spectroscopic data (Table 2) consisted of signals for one quaternary carbon (δC 142.3), three resonances for five aromatic methine carbons [δC 128.2 (×2), 127.1 and 125.6 (×2)], four methine carbons (δC 80.4, 68.1, 65.2 and 53.4), nine resonances for ten methylene carbons (δC 55.5, 35.4, 34.6, 31.9, 29.7, 29.6 (×2), 29.3, 26.3 and 22.7) and one methyl carbon (δC 14.1). The following correlations were observed in the 1H–1H COSY spectrum: H-3 (δH 2.60)/Hab-2 (δH 3.32 and 2.76), H-4 (δH 3.99) and H-1′ (δH 5.26), and Hab-5 (δH 2.05
0.00
- 0.01
- 0.09 - 0.10
- 0.02 - 0.08
OMTPA + 0.11 + 0.10
- 0.01 - 0.01 - 0.03
N - 0.01
- 0.01
- 0.01
'
+ 0.03 + 0.07
- 0.01
'
' '
+ 0.14 + 0.11
+ 0.10
- 0.02
Fig. 3. Δδ (=δS − δR) values for (S)- and (R)-MTPA-esters of compound 2.
and 1.44)/H-4 and H-6 (δH 2.63). These results together with HMBC correlations (Table 2) from Hab-2 and Hab-5 to C-3 (δC 53.4), C-4 (δC 68.1) and C-6 (δC 65.2) and those from H-1′ to C-2′ (δC 142.3), C-3′ (δC 125.6) and C-7′ (δC 125.6) constructed a 1,4,6-trisubstituted-3-(1′substituted benzyl)piperidine having five degrees of unsaturation. The molecular formula and 1H and 13C NMR spectroscopic data established the n-nonyl side chain which was attached at C-6 on the basis of HMBC correlations of H-6 with C-7 (δC 34.6) and C-8 (δC 26.3) and 1H–1H COSY correlations of H-6 and Hab-7 (δH 1.76 and 1.74). Substituents at C-4 and C-1′ (δC 80.4) were assigned to be oxy groups according to their chemical shifts. On the basis of degree of unsaturation, a bond between the N atom and either the oxygen atom at C-4 or C-1′ should be formed. The X-ray data of 3 with Cu Kα monochromated radiation displayed the formation of the bond between the nitrogen atom and C-1′ oxygen atom
Table 2 1 H and 13C NMR data of asperidines B (2) and C (3) in CDCl3. Position
2
3
δC,a type
δH,b mult. (J, Hz)
HMBC
δC,a type
δH,b mult. (J, Hz)
HMBC
2
73.6, CH
2.25, ddd (9.3, 6.3, 5.0)
3, 6, 15, 1′, 2′
55.5, CH2
3 4
70.5, CH 39.4, CH2 35.0, CH2
6 7
65.8, CH 26.3, CH2
2, 2, 4, 7 2,
3, 4, 6, 1′ 3, 4, 6, 1′ 4, 5, 1′, 2′ 1′
5
3.80, brs a: 2.20, m b: 1.40, ddd (13.8, 6.3, 1.2) a: 1.71, m b: 1.26, m 2.10, m 1.27, m
a: 3.32, dd (12.0, 4.5) b: 2.76, d (12.0) 2.60, dd (6.9, 4.5) 3.99, brt (6.9)
8 9 10 11 12 13 14 15 1′
31.9, 29.9, 29.7, 29.6, 29.3, 22.7, 14.1, 38.7, 33.7,
a: 2.05, m b: 1.44, m 2.63, m a: 1.76, m b: 1.74, m 1.39, m 1.26, m 1.26, m 1.26, m 1.26, m 1.26, m 1.26, m 0.88, t (6.9) 5.26, s
3, 3, 5, 8, 8, 6,
2′ 3′ 4′ 5′ 6′ 7′
139.5, 129.4, 128.4, 126.0, 128.4, 129.4,
7.36, 7.31, 7.23, 7.31, 7.36,
5′ 2′, 5′ 3′, 7′ 5′, 7′ 5′
a b
CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH3 CH2 C CH CH CH CH CH
3, 5, 6 3, 5, 6 6, 7 8
35.4, CH2 65.2, CH 34.6, CH2
1.27, m 1.27, m 1.27, m 1.27, m 1.27, m 1.27, m 0.88, t (6.6) 2.33, s a: 2.88, dd (12.9, 9.3) b: 2.84, dd (12.9, 5.1)
12, 13 2, 6 2, 3, 2′, 3′, 7′ 2, 3, 2′, 3′, 7′
7.30, 7.28, 7.18, 7.28, 7.30,
1′, 2′, 3′, 5′, 1′,
d (8.1) t (8.1) m t (8.1) d (8.1)
53.4, CH 68.1, CH
2′, 4′, 5′ 5′ 7′ 7′ 2′, 5′, 6′
Recorded at 75 MHz. Recorded at 300 MHz. 4504
26.3, 29.7, 29.6, 29.6, 29.3, 31.9, 22.7, 14.1, 80.4,
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH
142.3, 125.6, 128.2, 127.1, 128.2, 125.6,
C CH CH CH CH CH
dd (7.2, 1.7) t (7.2) m t (7.2) dd (7.2, 1.7)
4, 4, 7, 9 9 7,
6 6, 7 8
9
12, 13, 15 13, 14 2, 3, 4, 2′, 3′, 7′
Bioorganic & Medicinal Chemistry 26 (2018) 4502–4508
P. Phainuphong et al.
Fig. 4. ORTEP drawing of compound 3.
OMTPA
- 0.01
- 0.01 - 0.01
- 0.03 - 0.01 - 0.08 - 0.01 - 0.01
0.00 - 0.01
- 0.01
- 0.01
- 0.01 - 0.03
+ 0.11 + 0.05 + 0.03
N
O
'
+ 0.09
+ 0.26
'
known that human HMGR, the rate-limiting enzyme in cholesterol biosynthesis, has active catalytic site at the cytoplasmic domain as the target for binding and inhibiting by statins.11 Previous study demonstrated that the synthetic medium ring lactams affected with the broad range of HMGR inhibition from 32 to 76% by occupying with the pyridinium moiety on NADP+ which were different mechanisms from pravastatin/HMGR interaction and inhibition.12 Thus, 2 may additively apply with statins for cholesterol lowering effect. The mechanism of action is being investigated. Further investigation on the cytotoxicity of 2 and 3 on Caco-2 cells was performed using MTT assays. The results showed that the viability of Caco-2 cells did not change after 24 h exposure to piperidine derivatives at the concentration ranging from 12.5 to 100 μM (Fig. 7). Based on this data, effects of 2 and 3 on reactive oxygen species (ROS) in Caco-2 cells were carried out using the same concentration range. As shown in Fig. 8, both 2 and 3 significantly reduced intracellular ROS under both normal and H2O2-treated conditions compared with their respective controls in a dose-dependent manner, indicating that both 2 and 3 exerted anti-oxidative effect in intestinal epithelial cells and 2 was more potent than 3. Based on these results together with the above cytotoxic data, it is concluded that both 2 and 3 prevented the accumulation of intracellular ROS without cytotoxic effect. As ROS production could induce oxidative stress, which has been reported to be the major mediator in various pathological diseases, including cancer, neurological disorders, atherosclerosis, hypertension, cardiovascular diseases, diabetes, and metabolic syndromes,13 2 and 3 could have pleiotropic effect as an effective antioxidant for prevention of several diseases. The effect of 2 and 3 on CFTR-mediated chloride secretion in human intestinal epithelial (T84) cells was also evaluated using short-circuit current analysis. As shown in Fig. 9, 2 dose-dependently inhibited the CFTR-mediated chloride secretion induced by forskolin with the IC50 0.96 μM and with near complete inhibition at 10 μM. In addition, 3 was found to inhibit the CFTR-mediated chloride secretion with the IC50 value of 58.62 μM. These results indicated that 2 and 3 inhibited CFTRmediated chloride secretion in human intestinal epithelia, a process known to be involved in the pathogenesis of secretory diarrheas.
+ 0.01
' '
+ 0.26
+ 0.01 + 0.01
Fig. 5. Δδ (=δS − δR) values for (S)- and (R)-MTPA-esters of compound 3.
and also provide absolute configurations at C-3, C-4, C-6 and C-1′ which were R, S, R and S, respectively (Fig. 4). Moreover, the S configuration at C-4 was confirmed by Mosher’s method using the (S)- and (R)-MTPA esters (Fig. 5). Accordingly, compound 3 possessed an unprecedented 7oxa-1-azabicyclo[3.2.1]octane skeleton. Compounds 2 and 3 which were obtained in sufficient amount were tested for HMGR and CFTR inhibitory activities, free radical-scavenging activity and cytotoxicity against noncancerous Vero cells. Compound 3 was inactive against Vero cells whereas 2 was considered to be inactive with the IC50 value of 51.50 μM. Compound 2 at the concentration of 200 μM inhibited HMGR activity by 29% whereas 3 showed no inhibitory effect against HMGR (Fig. 6). In addition, 1 µM of pravastatin, a positive control, was able to inhibit HMGR activity by 86%. These results revealed that 2 exhibited novel effect as a HMGR inhibitor similar to pravastatin. Although the low affinity for binding to catalytic subunit of 2 was observed, the structurally dissimilar to statins should take into account. It has been
3. Conclusion Five alkaloids including one new pyrrolidine derivative, asperidine A (1), and two new piperidine derivatives, asperidines B (2) and C (3), together with two known compounds (4 and 5) were produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Compound 3 possessed a new 7-oxa-1-azabicyclo[3.2.1]octane skeleton with four chiral centers in the core unit whereas compound 4 was isolated as a natural product for the first time. Compounds 2 and 3 were assayed for
Fig. 6. Inhibitory effect of 2 and 3 on HMG-CoA reductase activity. HMGR activity was determined in the absence (control) or presence of pravastatin (1 μM) or piperidine derivatives; 2 and 3 (200 μM). Data are expressed as percent of inhibition. *p < 0.05 compared with control (n = 3). 4505
Bioorganic & Medicinal Chemistry 26 (2018) 4502–4508
P. Phainuphong et al.
Fig. 7. Effect of 2 and 3 on the viability of Caco-2 cells. Cells were incubated with 2 and 3 at the doses of 12.5, 25, 50, 75 and 100 µM for 24 h. Subsequently, the cells were washed and treated with 5 mg/mL of MTT for the next 4 h. The absorbance was measured at 570 nm and the data are expressed as means of percent control ± S.E.M (n = 5).
4.2. Fungal material
HMGR and CFTR inhibitory activities, free radical-scavenging activity and cytotoxicity against noncancerous Vero cells. Compound 2 displayed mild HMGR inhibitory activity, interesting anti-oxidative effect in Caco-2 cells and promising CFTR inhibitory activity. In addition, it was noncytotoxic to noncancerous Vero cells.
The soil fungus PSU-RSPG178 was isolated from a soil sample from Suratthani province, Thailand and deposited as BCC56851 at BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, Genbank accession number KC478521. The fungus PSU-RSPG178 was identified as Aspergillus sclerotiorum by morphological characteristics and the analysis of an internal transcribed spacer (ITS1-5.8S-ITS2) rDNA using universal fungal primers,14 which was described in our previous report.2
4. Experimental 4.1. General experimental procedures The melting points were determined on an Electrothermal 9100 melting point apparatus and reported without correction. Optical rotations were recorded on a JASCO P-1020 polarimeter. The ultraviolet (UV) absorption spectra were measured in MeOH on a Perkin-Elmer Lambda 45 spectrophotometer. The infrared (IR) spectra were recorded neat using a Perkin-Elmer 783 FTS165 FT-IR spectrometer. Mass spectra were obtained from a liquid chromatograph-mass spectrometer (2090, LCT, Waters, Micromass). 1H and 13C NMR spectra were recorded on a 300 MHz Bruker FTNMR Ultra Shield spectrometer. Chemical shifts are expressed in δ (parts per million, ppm) referring to the tetramethylsilane peak. Thin-layer chromatography (TLC) and preparative TLC (PTLC) were performed on silica gel 60 GF254 (Merck). Column chromatography (CC) was carried out on Sephadex® LH-20 with MeOH, silica gel (Merck) type 60 (230–400 mesh ASTM) or type 100 (70–230 mesh ASTM), or on reversed phase C18 silica gel.
4.3. Fermentation, extraction and isolation Fermentation and extraction procedures were previously reported.2 The broth extract (9.43 g) was separated by CC over Sephadex® LH-20 using MeOH to give three fractions (A-C). Fraction A (1.11 g) was fractionated by CC over Sephadex® LH-20 using MeOH/CH2Cl2 (1:1) to afford four fractions (A1-A4). Fraction A3 (368.7 mg) was purified by CC over silica gel using a gradient of MeOH/CH2Cl2 (2:98 → 100:0) to give five fractions (A3A-A3E). Compound 1 (3.1 mg) was obtained as a colorless solid from fraction A3E. Fraction B (8.26 g) was separated by CC over silica gel using a gradient of EtOAc/petroleum ether (15:85 → 100:0) to afford nine fractions (B1-B9). Fraction B7 (283.1 mg) was further separated by CC over silica gel using a gradient of MeOH/ CH2Cl2 (4:96 → 100:0) to give seven fractions (B7A-B7G). Fraction B7D (33.7 mg) was rechromatographed on CC over silica gel using MeOH/ CH2Cl2 (4:96) followed by PTLC using MeOH/CH2Cl2 (8:92) as a mobile
Fig. 8. Anti-oxidative effect of 2 and 3 in Caco-2 cells. Cells were treated with 2 and 3 at indicated concentrations (12.5–100 µM) for 24 h and incubated with 10 µM DCFH-DA for 1 h at 37 °C in the dark followed by a-30 min incubation in the absence (A) or the presence (B) of H2O2. Values represents means ± S.E.M. (n = 3), * p < 0.05; **p < 0.01 compared to control cells in the absence of H2O2; #p < 0.05; ##p < 0.01 compared to control cells exposed with H2O2. 4506
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Fig. 9. Effect of compounds 2 and 3 on CFTR-mediated chloride secretion in human intestinal epithelial (T84) cells.
phase (3 runs) and subsequent PTLC using EtOAc/CH2Cl2 (2:3) as a mobile phase (2 runs) to provide 4 (1.8 mg). The mycelial hexane extract (1.40 g) was separated by CC over Sephadex® LH-20 using CHCl3/ MeOH (1:1) to give three fractions (CH1–CH3). Fraction CH2 (1.06 g) was fractionated by CC over silica gel using a gradient of MeOH/CH2Cl2 (0:100 → 100:0) to give five fractions (CH2A–CH2E). Fraction CH2C (298.3 mg) was further separated by CC over silica gel using a gradient of EtOAc/petroleum ether (20:80 → 70:30) to give six fractions (CH2C1–CH2C6). Fraction CH2C3 (100.9 mg) was separated by CC over reversed phase C18 silica gel using a gradient of MeOH/H2O (85:15 → 100:0) followed by CC over silica gel using a gradient of MeOH/CH2Cl2 (0.1:99.9 → 5:95) and rechromatographed on CC over silica gel using CH2Cl2/MeOH/petroleum ether (70:2:28) to afford 3 (20.7 mg). Fraction CH2D (369.4 mg) was further purified by CC over silica gel using a gradient of EtOAc/petroleum ether (10:90 → 100:0) to give 2 (20.7 mg). The mycelial ethyl acetate extract (4.34 g) was separated using the same procedure as the broth extract to afford four fractions (CE1-CE4). Fraction CE3 (2.47 g) was rechromatographed on CC over silica gel using a gradient of MeOH/CH2Cl2 (0:100 → 100:0) to give six fractions (CE3A-CE3F). Fraction CE3F (412.4 mg) was separated using the same procedure as the broth extract to afford five fractions (CE3F1-CE3F5). Fraction CE3F4 (62.9 mg) was purified by CC over silica gel using MeOH/CH2Cl2 (4:96) followed by PTLC using MeOH/CH2Cl2 (4:96) as a mobile phase (3 runs) to provide 5 (2.1 mg).
4.3.3. Asperidine C (3) Colorless crystals; mp 95–96 °C; [α ]25 D −158.3 (c 0.1, CHCl3); UV (MeOH) λmax (log ε): 209 (3.72), 259 (2.37) nm; IR (neat)νmax 3393 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z: [M +H]+ 332.2590 (calcd for C21H34NO2, 332.2590).
4.3.1. Asperidine A (1) Colorless solid; mp 97–98 °C; [α ]26 D −13.8 (c 1.0, MeOH); UV (MeOH) λmax (log ε): 207 (4.02), 256 (2.48) nm; IR (neat)νmax 3325 cm−1; 1H and 13C NMR data, see Table 1, HRESIMS m/z: [M +H]+ 276.2327 (calcd for C18H30NO, 276.2327).
4.6. X-ray crystallography
4.4. Preparation of the (R)- and (S)-MTPA ester derivative of 28,9 Pyridine (100 μL) and (+)-(R)-MTPACl (40 μL) were added to a CH2Cl2 solution (300 μL) of 2 (2.5 mg). The reaction mixture was stirred at room temperature overnight. After removal of the solvent, the mixture was purified by CC over silica gel using acetone/hexane (3:17) to afford the (S)-MTPA ester (3.7 mg). 2 (2.6 mg) was treated in a similar way with (−)-(S)-MTPACl and, after purification by CC over silica gel, (R)-MTPA ester (3.0 mg) was obtained. 4.5. Preparation of the (R)- and (S)-MTPA ester derivative of 38,9 Pyridine (100 μL) and (+)-(R)-MTPACl (40 μL) were added to a CH2Cl2 solution (300 μL) of 3 (2.1 mg). The reaction mixture was stirred at room temperature overnight. After removal of the solvent, the mixture was purified by CC over silica gel using MeOH/CH2Cl2 (1:199) to afford the (S)-MTPA ester (3.8 mg). 3 (2.1 mg) was treated in a similar way with (−)-(S)-MTPACl and, after purification by CC over silica gel, (R)-MTPA ester (2.7 mg) was obtained.
The crystal data of 3 (Fig. 4) was selected for data collection which was performed on a on a XtaLAB Synergy-S Hypix-600HE diffractometer equipped with Cu Kα monochromated radiation (λ = 1.5408 Å) source at low temperature of 100 K. The automatic crystal screening, data collection and data reduction process were performed using CrysAlis PRO version 1.171.39.30t. This structure was solved using direct methods with SHELXT and refined with the fullmatrix least-squares methods based on F2 with the SHELXL program.15 All non hydrogen atoms were found from the electron density maps and refined anisotropically. All hydrogen atoms of this compound were
4.3.2. Asperidine B (2) Colorless gum; [α ]25 D −39.1 (c 0.2, CHCl3); UV (MeOH) λmax (log ε): 210 (4.22), 260 (2.86) nm; IR (neat)νmax 3382 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z: [M+H]+ 318.2797 (calcd for C21H36NO, 318.2797). 4507
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was measured by Synergy™ HT microplate reader (Biotek, VT) at excitation and emission wavelengths of 495 nm and 527 nm, respectively.
determined from the residual electron density and refined isotropically. The WinGXv2014.116 and Mercury programs17 were used to prepare the materials and molecular graphic for publication. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit No. CCDC 1839357, 3). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax: +44-(0)223-336033 or e-mail: [email protected]). Crystal Data of 3 (CCDC 1839357): C21H33NO2, M = 331.48, T = 100(1) K, monoclinic space group P21, a = 6.68920(10) Å, b = 7.16530(10) Å, c = 19.7476(2) Å, V = 943.94(2) Å3, α = γ = 90°, β = 94.2200(10)°, Z = 2, Dcalc = 1.166 Mg/m3, crystal dimensions 0.095 × 0.089 × 0.043 mm3, μ = 0.570 mm−1, F(0 0 0) = 364, 17,536 reflections measured, 3447 unique (Rint = 0.0311). The final refinement gave R1 = 0.0247 and wR2 = 0.0655 [I > 2σ(I)]. Absolute structure parameter = −0.02(8).
4.7.6. Evaluation of the effect of test compounds on CFTR-mediated intestinal chloride secretion T84 cells were seeded and cultured on porous membrane of Snapwell inserts (1 cm2 surface area; Corning-Costar Corp., Corning, NY, USA) at a density of 500,000 cells/insert. After transepithelial electrical resistance of > 1000 Ω.cm2 was reached, insert was mounted in Ussing Chambers. Apical and basolateral hemichambers were filled with symmetrical Kreb’s solution containing 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2 (pH 7.3), and 10 mM glucose, and were continuously bubbled with 95% O2/5% CO2 mixture and maintained at 37 °C. The cAMP-induced chloride secretion was induced using forskolin (20 μM).20 Subsequently, tested substances were added into apical solutions at varied concentrations. The half maximal inhibitory concentration (IC50) was calculated using Hill’s equation.
4.7. Bioassays 4.7.1. Cytotoxicity assay The activity assay against Vero cells was performed in triplicate employing the method described by Hunt and co-workers.18 Ellipticine, the standard drug, displayed the IC50 value of 4.06 μM.
Acknowledgments V. Rukachaisirikul thanks the Royal Property Bureau Foundation and National Science and Technology Development Agency for the NSTDA Chair Professor grant (The Fourth Grant). P. Phainuphong is grateful to the TRF through the Royal Golden Jubilee Ph.D. program (Grant number PHD/0031/2555) and Prince of Songkla University (PSU-Ph.D. scholarship) for a joint funding scholarship. The Center of Excellence for Innovation in Chemistry (PERCH-CIC) and Prince of Songkla University Graduate School are acknowledged for partial support. Finally, the National Center for Genetic Engineering and Biotechnology (BIOTEC) is acknowledged for cytotoxicity assay.
4.7.2. HMGR activity assay Inhibitory effect on HMGR activity was determined using a cell-free HMG-CoA reductase assay kit according to the manufacturer’s protocol (Sigma-Aldrich, MO). Briefly, the reaction mixture containing 1× buffer, NADPH, HMG CoA, and piperidine derivatives (200 µM) or pravastatin (1 μM) (positive control) were added into the 96-well plates (Corning Inc., NY, USA). Subsequently, catalytic domains of human HMGR were added to initiate the reaction at 37 °C. The oxidation rate of HMGR was analyzed by measuring absorbance at 340 nm for 10 min at 20-s interval using Synergy™ HT microplate reader (Biotek, VT, USA). The data were expressed as percent inhibition of control.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmc.2018.07.036.
4.7.3. Cell culture preparation Caco-2 and T84 cells were purchased from the American Type Culture Collection (ATCC) (VA, USA). Cells (passage 2nd–22nd) were cultured in DMEM containing DMEM/F-12 (Sigma-Aldrich, MO) containing 1.2 g/L NaHCO3 supplemented with 10% FBS and 100 unit/mL penicillin–streptomycin solution (Life technology, NY). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C.
References [1]. Liang Z, Zhang T, Zhang X, Zhang J, Zhao C. Molecules. 2015;20:1424–1433. [2]. Phainuphong P, Rukachaisirikul V, Saithong S, et al. J Nat Prod. 2016;79:1500–1507. [3]. Zhang L-H, Feng B-M, Zhao Y-Q, et al. Bioorg Med Chem Lett. 2016;26:346–350. [4]. Liu L, Wang L, Bao L, et al. Org Lett. 2017;19:942–945. [5]. Phainuphong P, Rukachaisirikul V, Tadpetch K, et al. Phytochemistry. 2017;137:165–173. [6]. Couladouros E, Magos A, Strongilos A. WO2005/003102A1. [7]. Trisuwan K, Rukachaisirikul V, Sukpondma Y, Phongpaichit S, Preedanon S, Sakayaroj J. Tetrahedron. 2010;66:4484–4489. [8]. Ohtani I, Kusumi T, Kashman Y, Kakisawa H. J Am Chem Soc. 1991;113:4092–4096. [9]. Arunpanichlert J, Rukachaisirikul V, Sukpondma Y, Phongpaichit S, Supaphon O, Sakayaroj J. Arch Pharm Res. 2011;34:1633–1637. [10]. Kanazawa A, Gillet S, Delair P, Greene AE. J Org Chem. 1998;63:4660–4663. [11]. Friesen JA, Rodwell VW. Genome Biol. 2004;5:248. [12]. Perchellet J-PH, Perchellet EM, Crow KR, et al. Int J Mol Med. 2009;24:633–643. [13]. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. WAO J. 2012;5:9–19. [14]. White TJ, Bruns T, Lee S, Taylor JW. PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press, Inc.; 1990:315–322. [15]. Sheldrick GM. Acta Crystallogr C Sect Struct Chem. 2015;71:3–8. [16]. Farrugia LJ. J Appl Crystallogr. 2012;45:849–854. [17]. Macrae CF, Bruno IJ, Chisholm JA, et al. J Appl Crystallogr. 2008;41:466–470. [18]. Hunt L, Jordan M, De Jesus M, Wurm FM. Biotechnol Bioeng. 1999;65:201–205. [19]. Lluis JM, Buricchi F, Chiarugi P, Morales A, Fernandez-Checa JC. Cancer Res. 2007;67:7368–7377. [20]. Pongkorpsakol P, Pathomthongtaweechai N, Srimanote P, Soodvilai S, Chatsudthipong V, Muanprasat C. PLOS Negl Trop Dis. 2014;8:e3119.
4.7.4. Cell viability assay Cell viability was measured by MTT assays. Briefly, the cells were plated at a density of 2.5 × 104 cells/well in a 96-well plate for 24 h. Cells were then incubated with different concentrations of piperidine derivatives (12.5, 25, 50, 75 and 100 μM) and MTT solution (5 mg/mL) for 4 h. At the end of experiment, dimethylsulfoxide (DMSO) was added to dissolve the blue crystals and absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed relatively to the absorbance of control. 4.7.5. Determination of ROS The intracellular ROS levels in Caco-2 cells were quantitated using fluorescent dye 20,70-dichlorfluoresceindiacetate (DCFH-DA) (SigmaAldrich, MO) as described previously.19 Caco-2 cells were treated with piperidine derivatives at various concentrations (12.5–100 μM) for 24 h. Cells were washed and incubated with 10 μM DCFH-DA for 1 h at 37 °C in the dark. Subsequently, 0.5 mM of H2O2 will be added in each well for 30 min and cells were washed twice. The fluorescence intensity
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Asperidines A–C, pyrrolidine and piperidine derivatives from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178
T
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Patima Phainuphonga, Vatcharin Rukachaisirikula, , Saowanit Saithonga, Souwalak Phongpaichitb, Jariya Sakayarojc, Chutima Srimaroengd, Atcharaporn Ontawongd,e, Acharaporn Duangjaie, Paradorn Muangnilf, Chatchai Muanprasatf a
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c School of Science, Walailak University, Tha Sala, Nakhon Si Thammarat 80161, Thailand d Department of Physiology, Faculty of Medicine, Chiang Mai University, Mueang District, Chiang Mai 50200, Thailand e Division of Physiology, School of Medical Sciences, University of Phayao, Mueang District, Phayao 56000, Thailand f Excellent Center for Drug Discovery and Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Aspergillus sclerotiorum Pyrrolidine derivative Piperidine derivative CFTR inhibitor Antioxidant Cytotoxicity
One new pyrrolidine derivative, asperidine A (1), and two new piperidine derivatives, asperidines B (2) and C (3), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178 together with two known alkaloids. Compound 3 possessed an unprecedented 7-oxa-1-azabicyclo[3.2.1]octane skeleton with four chiral centers. Their structures were determined by spectroscopic evidence. The absolute configurations of compounds 2 and 3 were established using Mosher’s method and further confirmed for compound 3 by X-ray crystallographic data. Compound 2 dose-dependently inhibited the CFTR-mediated chloride secretion in T84 cells with an IC50 value of 0.96 μM whereas 3 displayed the same activity with the IC50 value of 58.62 μM. Compounds 2 and 3 also significantly reduced intracellular ROS under both normal and H2O2-treated conditions compared with their respective controls in a dose-dependent manner without cytotoxic effect on Caco-2 cells. In addition, compound 3 was inactive against noncancerous Vero cells whereas compound 2 was considered to be inactive with the IC50 value of > 10 μM.
1. Introduction Fungi in the genus Aspergillus have been recognized as prolific sources of new bioactive secondary metabolites which were documented in recent years. Over the past few years, many new metabolites were isolated from the genus Aspergillus. Some of them exhibited interesting biological activities such as cytotoxic activity against HepG2 cell lines,1 HMG-CoA reductase inhibitory activity,2 α-glucosidase inhibitory activity3 and antiproliferative activity against the H460 cell lines.4 In our ongoing search for new bioactive metabolites from soilderived fungi, we have investigated secondary metabolites produced by the soil fungus Aspergillus sclerotiorum PSU-RSPG178 isolated from a soil sample collected from the Plant Genetic Conservation Project under the Royal Initiation of Her Royal Highness Princess Maha Chakri Sirindhorn at Ratchaprapa Dam in Suratthani Province, Thailand. Recently, we have reported the isolation of three new and four known lovastatin analogues2 as well as seven new γ-butenolide and furanone derivatives
⁎
together with six known compounds5 from the crude extracts of A. sclerotiorum PSU-RSPG178. Further chemical investigation of the broth ethyl acetate extract led to the isolation of one new pyrrolidine derivative, asperidine A (1), and 3-[[5-(1,1-dimethyl-2-propen-1-yl)-1Himidazol-4-yl]methylene]-6-methyl-(6S)-2,5-piperazinedione (4) which was isolated as a natural product for the first time.6 Moreover, two new piperidine derivatives, asperidines B (2) and C (3), were obtained from the mycelial hexane extract whereas nicotinic acid (5)7 was isolated from the mycelial ethyl acetate extract. Compounds 2 and 3 were evaluated for HMG-CoA reductase (HMGR) and cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory activities, free radical-scavenging activity and cytotoxicity against human colon cancer (Caco-2) cell lines and African green monkey kidney fibroblast (Vero) cells.
Corresponding author. E-mail address: [email protected] (V. Rukachaisirikul).
https://doi.org/10.1016/j.bmc.2018.07.036 Received 2 June 2018; Received in revised form 20 July 2018; Accepted 22 July 2018 Available online 23 July 2018 0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
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OH
3 9
11
7
5
3'
N H
1'
O
7'
1 5 13
9
11
1
3
N
7
OH
5'
3'
HN
N
HN
15
13
11
9
1
N
7
O
7'
1'
O OH
OH 5
NH
4
15
2
7.33 (t, J = 7.6 Hz, 2H) and 7.30 (m, 1H)], three methine protons [δH 4.23 (brt, J = 3.1 Hz, 1H), 3.58 (dt, J = 7.4 and 3.1 Hz, 1H) and 3.56 (m, 1H)], two sets of nonequivalent methylene protons [δH 3.22 (dd, J = 13.9 and 7.2 Hz, 1H)/3.00 (dd, J = 13.9 and 7.6 Hz, 1H) and 2.45 (ddd, J = 14.1, 10.1 and 5.0 Hz, 1H)/1.74 (td, J = 14.1 and 5.0 Hz, 1H)] and a n-heptyl side chain [δH (1.80, m, 1H/1.75, m, 1H), 1.33 (m, 10H) and 0.90 (t, J = 6.9 Hz, 3H)]. The 13C NMR spectrum (Table 1) consisted of signals for one quaternary carbon (δC 136.7), three resonances for five aromatic methine carbons [δC 128.8 (×2), 128.5 (×2) and 126.8], three methine carbons (δC 69.3, 66.4 and 58.7), eight methylene carbons (δC 38.5, 34.2, 31.9, 31.5, 28.9, 28.8, 26.4 and 22.3) and one methyl carbon (δC 13.0). In the HMBC spectrum, Hab-1′ (δH 3.22 and 3.00) displayed the correlations to C-2′ (δC 136.7), C-3′ (δC 128.8) and C-7′ (δC 128.8) of the monosubstituted benzene ring (Table 1), indicating the presence of a benzyl unit having four degrees of unsaturation. Based on the 1H–1H COSY correlations of H-2 (δH 3.58)/Hab-1′ and H-3 (δH 4.23), and Hab-4 (δH 2.45 and 1.74)/H-3 and H-5 (δH 3.56), the chemical shifts of C-2 (δC 66.4) and C-5 (δC 58.7) and the remaining one degree of unsaturation, a 5-substituted 2-benzylpyrrolidine moiety was established. A substituent at C-3 was a hydroxy group due to the chemical shift of C-3 (δC 69.3). The remaining signals in the 1H and 13C NMR spectra together with the molecular formula established the n-heptyl side chain which was attached at C-5 according to a HMBC correlation of H-5 with C-6 (δC 34.2) of the n-heptyl unit and 1 H–1H COSY correlations from Hab-6 (δH 1.80 and 1.75) to H-5. In the NOEDIFF experiments, irradiation of Ha-4 enhanced the signal intensity of H-3, Hb-4 and H-5 whereas the signal intensity of H-2 was affected after irradiation of H-3 (Fig. 2). These results indicated a cis-relationship of H-2, H-3 and H-5. Comparison of the specific rotation of 1, ([α ]26 D = −13.8, c 1.0, MeOH), with that of (−)-(2S,3S,5R)-2-benzyl-310 hydroxy-5-nonylpyrrolidine ([α ]20 indicated D = −15.6, c 1.0, MeOH) that they possessed the same absolute configurations. Therefore, 1 was assigned as a 5-n-heptyl derivative of the above pyrrolidine derivative. Asperidine B (2) was obtained as a colorless gum with the molecular formula C21H35NO determined by the HRESIMS peak at m/z 318.2797 [M+H]+. The UV and IR spectra were similar to those of 1, suggesting the presence of a benzene chromophore and a hydroxy group. The 1H NMR spectroscopic data (Table 2) contained signals for five aromatic protons of a monosubstituted benzene [δH 7.30 (d, J = 8.1 Hz, 2H), 7.28 (t, J = 8.1 Hz, 2H) and 7.18 (m, 1H)], three methine protons [δH 3.80 (brs, 1H), 2.25 (ddd, J = 9.3, 6.3 and 5.0 Hz, 1H) and 2.10 (m, 1H)], three sets of nonequivalent methylene protons [δH 2.88 (dd, J = 12.9 and 9.3 Hz, 1H)/2.84 (dd, J = 12.9 and 5.1 Hz, 1H), 2.20 (m, 1H)/1.40 (ddd, J = 13.8, 6.3 and 1.2 Hz, 1H) and 1.71 (m, 1H)/1.26 (m, 1H)] and a n-octyl side chain [δH 1.27 (m, 14H) and 0.88 (t, J = 6.6 Hz, 3H)] and one methyl group (δH 2.33, s, 3H). The 13C NMR spectroscopic data (Table 2) displayed signals for one quaternary (δC 139.5), three resonances for five aromatic methine [δC 129.4 (×2), 128.4 (×2) and 126.0], three methine (δC 73.6, 70.5 and 65.8), ten methylene (δC 39.4, 35.0, 33.7, 31.9, 29.9, 29.7, 29.6, 29.3, 26.3 and 22.7) and two methyl (δC 38.7 and 14.1) carbons. A benzyl unit was established by HMBC correlations (Table 2) from Hab-1′ (δH 2.88 and 2.84) to C-2′ (δC 139.5), C-3′ (δC 129.4) and C-7′ (δC 129.4) of the monosubstituted benzene ring. A 6-substituted 2-benzyl-N-methylpiperidine moiety was constructed according to the following 1H–1H COSY correlations: H-2 (δH 2.25)/Hab-1′ and H-3 (δH 3.80), Hab-4 (δH 2.20 and 1.40)/H-3 and Hab-5 (δH 1.71 and 1.26), and Hab-5/H-6 (δH 2.10) together with HMBC correlations of H3-15 (δH 2.33) with C-2 (δC 73.6) and C-6 (δC 65.8). A substituent at C-3 (δC 70.5) was assigned to be a hydroxy group due to its chemical shift. Based on the remaining signals in the 1H and 13C NMR spectra along with the molecular formula, the n-octyl side chain was constructed. HMBC correlations of Hab5 with C-7 (δC 26.3) attached the n-octyl side chain at C-6. Irradiation of H-2 enhanced signal intensity of H-3 and H-6 in the NOEDIFF experiment (Fig. 2), indicating a cis-relationship of H-2, H-3 and H-6. The absolute configuration at C-3 was assigned as S on the basis of Mosher’s
5'
1
O
N
3'
3
5
5'
1' 7'
3 Fig. 1. Structures of compounds 1–5 isolated from Aspergillus sclerotiorum PSURSPG178.
2. Results and discussion All alkaloids (1–5) (Fig. 1) were purified using chromatographic techniques and their structures were elucidated by using various spectroscopic techniques. The relative configuration was assigned according to the NOEDIFF data. The absolute configuration of 1 was established by comparison of the specific rotation with that of a known and structurally related compound. In addition, the absolute configurations of the secondary alcohol at C-3 in 2 and C-4 in 3 were established by Mosher’s method.8,9 Moreover, the absolute configuration of 3 was confirmed by the X-ray data. Asperidine A (1) was obtained as a colorless solid, melting at 97–98 °C, with the molecular formula C18H29NO determined by the HRESIMS peak at m/z 276.2327 [M+H]+, indicating the presence of five degrees of unsaturation. It exhibited UV absorption bands at 207 and 256 nm for an aromatic chromophore while an IR absorption band was observed at 3325 cm−1 for hydroxy and amino groups. The 1H NMR spectroscopic data (Table 1) displayed signals for five aromatic protons of a monosubstituted benzene [δH 7.35 (d, J = 7.6 Hz, 2H),
Table 1 1 H and 13C NMR data of asperidine A (1) in CD3OD. Position
1 δC,a type
δH,b mult. (J, Hz)
HMBC
2 3 4
66.4, CH 69.3, CH 38.5, CH2
5 6
58.7, CH 34.2, CH2
1′, 2′ 2, 5 2, 3, 5, 6 2, 3, 5, 6 4, 6 7, 8 7, 8
7 8 9 10 11 12 1′
28.9, 26.4, 28.8, 31.5, 22.3, 13.0, 31.9,
3.58, dt (7.4, 3.1) 4.23, brt (3.1) a: 2.45, ddd (14.1, 10.1, 5.0) b: 1.74, td (14.1, 5.0) 3.56, m a: 1.80, m b: 1.75, m 1.33, m 1.33, m 1.33, m 1.33, m 1.33, m 0.90, t (6.9) a: 3.22, dd (13.9, 7.2) b: 3.00, dd (13.9, 7.6)
2′ 3′ 4′ 5′ 6′ 7′
136.7, 128.8, 128.5, 126.8, 128.5, 128.8,
7.35, 7.33, 7.30, 7.33, 7.35,
1′, 5′, 7′ 2′ 3′, 4′, 6′, 7′ 2′ 1′, 3′, 5′
a b
CH2 CH2 CH2 CH2 CH2 CH3 CH2 C CH CH CH CH CH
d (7.6) t (7.6) m t (7.6) d (7.6)
10, 11 2, 3, 2′, 3′, 7′ 2, 3, 2′, 3′, 7′
Recorded at 75 MHz. Recorded at 300 MHz. 4503
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Fig. 2. Key NOEDIFF data of compounds 1 and 2.
method using the (S)- and (R)-MTPA esters (Fig. 3). Consequently, the remaining absolute configurations at C-2 and C-6 were identified to be S and R, respectively. Therefore, asperidine B had the structure 2. Asperidine C (3) was obtained as colorless crystals, melting at 95–96 °C, with the molecular formula C21H33NO2 determined by the HRESIMS peak at m/z 332.2590 [M+H]+, indicating the presence of six degrees of unsaturation. The UV spectrum was similar to that of 1, suggesting the presence of a benzene chromophore. The IR spectrum showed an absorption band for a hydroxy group at 3393 cm−1. The 1H NMR spectroscopic data (Table 2) contained signals for five aromatic protons of a monosubstituted benzene [δH 7.36 (dd, J = 7.2 and 1.7 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H) and 7.23 (m, 1H)], four methine protons [δH 5.26 (s, 1H), 3.99 (brt, J = 6.9 Hz, 1H), 2.63 (m, 1H) and 2.60 (dd, J = 6.9 and 4.5 Hz, 1H)], two sets of nonequivalent methylene protons [δH 3.32 (dd, J = 12.0 and 4.5 Hz, 1H)/2.76 (d, J = 12.0 Hz, 1H) and 2.05 (m, 1H)/1.44 (m, 1H)] and a n-nonyl side chain [δH (1.76, m, 1H/ 1.74, m, 1H), 1.39 (m, 2H), 1.26 (m, 12H) and 0.88 (t, J = 6.9 Hz, 3H)]. The 13C NMR spectroscopic data (Table 2) consisted of signals for one quaternary carbon (δC 142.3), three resonances for five aromatic methine carbons [δC 128.2 (×2), 127.1 and 125.6 (×2)], four methine carbons (δC 80.4, 68.1, 65.2 and 53.4), nine resonances for ten methylene carbons (δC 55.5, 35.4, 34.6, 31.9, 29.7, 29.6 (×2), 29.3, 26.3 and 22.7) and one methyl carbon (δC 14.1). The following correlations were observed in the 1H–1H COSY spectrum: H-3 (δH 2.60)/Hab-2 (δH 3.32 and 2.76), H-4 (δH 3.99) and H-1′ (δH 5.26), and Hab-5 (δH 2.05
0.00
- 0.01
- 0.09 - 0.10
- 0.02 - 0.08
OMTPA + 0.11 + 0.10
- 0.01 - 0.01 - 0.03
N - 0.01
- 0.01
- 0.01
'
+ 0.03 + 0.07
- 0.01
'
' '
+ 0.14 + 0.11
+ 0.10
- 0.02
Fig. 3. Δδ (=δS − δR) values for (S)- and (R)-MTPA-esters of compound 2.
and 1.44)/H-4 and H-6 (δH 2.63). These results together with HMBC correlations (Table 2) from Hab-2 and Hab-5 to C-3 (δC 53.4), C-4 (δC 68.1) and C-6 (δC 65.2) and those from H-1′ to C-2′ (δC 142.3), C-3′ (δC 125.6) and C-7′ (δC 125.6) constructed a 1,4,6-trisubstituted-3-(1′substituted benzyl)piperidine having five degrees of unsaturation. The molecular formula and 1H and 13C NMR spectroscopic data established the n-nonyl side chain which was attached at C-6 on the basis of HMBC correlations of H-6 with C-7 (δC 34.6) and C-8 (δC 26.3) and 1H–1H COSY correlations of H-6 and Hab-7 (δH 1.76 and 1.74). Substituents at C-4 and C-1′ (δC 80.4) were assigned to be oxy groups according to their chemical shifts. On the basis of degree of unsaturation, a bond between the N atom and either the oxygen atom at C-4 or C-1′ should be formed. The X-ray data of 3 with Cu Kα monochromated radiation displayed the formation of the bond between the nitrogen atom and C-1′ oxygen atom
Table 2 1 H and 13C NMR data of asperidines B (2) and C (3) in CDCl3. Position
2
3
δC,a type
δH,b mult. (J, Hz)
HMBC
δC,a type
δH,b mult. (J, Hz)
HMBC
2
73.6, CH
2.25, ddd (9.3, 6.3, 5.0)
3, 6, 15, 1′, 2′
55.5, CH2
3 4
70.5, CH 39.4, CH2 35.0, CH2
6 7
65.8, CH 26.3, CH2
2, 2, 4, 7 2,
3, 4, 6, 1′ 3, 4, 6, 1′ 4, 5, 1′, 2′ 1′
5
3.80, brs a: 2.20, m b: 1.40, ddd (13.8, 6.3, 1.2) a: 1.71, m b: 1.26, m 2.10, m 1.27, m
a: 3.32, dd (12.0, 4.5) b: 2.76, d (12.0) 2.60, dd (6.9, 4.5) 3.99, brt (6.9)
8 9 10 11 12 13 14 15 1′
31.9, 29.9, 29.7, 29.6, 29.3, 22.7, 14.1, 38.7, 33.7,
a: 2.05, m b: 1.44, m 2.63, m a: 1.76, m b: 1.74, m 1.39, m 1.26, m 1.26, m 1.26, m 1.26, m 1.26, m 1.26, m 0.88, t (6.9) 5.26, s
3, 3, 5, 8, 8, 6,
2′ 3′ 4′ 5′ 6′ 7′
139.5, 129.4, 128.4, 126.0, 128.4, 129.4,
7.36, 7.31, 7.23, 7.31, 7.36,
5′ 2′, 5′ 3′, 7′ 5′, 7′ 5′
a b
CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH3 CH2 C CH CH CH CH CH
3, 5, 6 3, 5, 6 6, 7 8
35.4, CH2 65.2, CH 34.6, CH2
1.27, m 1.27, m 1.27, m 1.27, m 1.27, m 1.27, m 0.88, t (6.6) 2.33, s a: 2.88, dd (12.9, 9.3) b: 2.84, dd (12.9, 5.1)
12, 13 2, 6 2, 3, 2′, 3′, 7′ 2, 3, 2′, 3′, 7′
7.30, 7.28, 7.18, 7.28, 7.30,
1′, 2′, 3′, 5′, 1′,
d (8.1) t (8.1) m t (8.1) d (8.1)
53.4, CH 68.1, CH
2′, 4′, 5′ 5′ 7′ 7′ 2′, 5′, 6′
Recorded at 75 MHz. Recorded at 300 MHz. 4504
26.3, 29.7, 29.6, 29.6, 29.3, 31.9, 22.7, 14.1, 80.4,
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH
142.3, 125.6, 128.2, 127.1, 128.2, 125.6,
C CH CH CH CH CH
dd (7.2, 1.7) t (7.2) m t (7.2) dd (7.2, 1.7)
4, 4, 7, 9 9 7,
6 6, 7 8
9
12, 13, 15 13, 14 2, 3, 4, 2′, 3′, 7′
Bioorganic & Medicinal Chemistry 26 (2018) 4502–4508
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Fig. 4. ORTEP drawing of compound 3.
OMTPA
- 0.01
- 0.01 - 0.01
- 0.03 - 0.01 - 0.08 - 0.01 - 0.01
0.00 - 0.01
- 0.01
- 0.01
- 0.01 - 0.03
+ 0.11 + 0.05 + 0.03
N
O
'
+ 0.09
+ 0.26
'
known that human HMGR, the rate-limiting enzyme in cholesterol biosynthesis, has active catalytic site at the cytoplasmic domain as the target for binding and inhibiting by statins.11 Previous study demonstrated that the synthetic medium ring lactams affected with the broad range of HMGR inhibition from 32 to 76% by occupying with the pyridinium moiety on NADP+ which were different mechanisms from pravastatin/HMGR interaction and inhibition.12 Thus, 2 may additively apply with statins for cholesterol lowering effect. The mechanism of action is being investigated. Further investigation on the cytotoxicity of 2 and 3 on Caco-2 cells was performed using MTT assays. The results showed that the viability of Caco-2 cells did not change after 24 h exposure to piperidine derivatives at the concentration ranging from 12.5 to 100 μM (Fig. 7). Based on this data, effects of 2 and 3 on reactive oxygen species (ROS) in Caco-2 cells were carried out using the same concentration range. As shown in Fig. 8, both 2 and 3 significantly reduced intracellular ROS under both normal and H2O2-treated conditions compared with their respective controls in a dose-dependent manner, indicating that both 2 and 3 exerted anti-oxidative effect in intestinal epithelial cells and 2 was more potent than 3. Based on these results together with the above cytotoxic data, it is concluded that both 2 and 3 prevented the accumulation of intracellular ROS without cytotoxic effect. As ROS production could induce oxidative stress, which has been reported to be the major mediator in various pathological diseases, including cancer, neurological disorders, atherosclerosis, hypertension, cardiovascular diseases, diabetes, and metabolic syndromes,13 2 and 3 could have pleiotropic effect as an effective antioxidant for prevention of several diseases. The effect of 2 and 3 on CFTR-mediated chloride secretion in human intestinal epithelial (T84) cells was also evaluated using short-circuit current analysis. As shown in Fig. 9, 2 dose-dependently inhibited the CFTR-mediated chloride secretion induced by forskolin with the IC50 0.96 μM and with near complete inhibition at 10 μM. In addition, 3 was found to inhibit the CFTR-mediated chloride secretion with the IC50 value of 58.62 μM. These results indicated that 2 and 3 inhibited CFTRmediated chloride secretion in human intestinal epithelia, a process known to be involved in the pathogenesis of secretory diarrheas.
+ 0.01
' '
+ 0.26
+ 0.01 + 0.01
Fig. 5. Δδ (=δS − δR) values for (S)- and (R)-MTPA-esters of compound 3.
and also provide absolute configurations at C-3, C-4, C-6 and C-1′ which were R, S, R and S, respectively (Fig. 4). Moreover, the S configuration at C-4 was confirmed by Mosher’s method using the (S)- and (R)-MTPA esters (Fig. 5). Accordingly, compound 3 possessed an unprecedented 7oxa-1-azabicyclo[3.2.1]octane skeleton. Compounds 2 and 3 which were obtained in sufficient amount were tested for HMGR and CFTR inhibitory activities, free radical-scavenging activity and cytotoxicity against noncancerous Vero cells. Compound 3 was inactive against Vero cells whereas 2 was considered to be inactive with the IC50 value of 51.50 μM. Compound 2 at the concentration of 200 μM inhibited HMGR activity by 29% whereas 3 showed no inhibitory effect against HMGR (Fig. 6). In addition, 1 µM of pravastatin, a positive control, was able to inhibit HMGR activity by 86%. These results revealed that 2 exhibited novel effect as a HMGR inhibitor similar to pravastatin. Although the low affinity for binding to catalytic subunit of 2 was observed, the structurally dissimilar to statins should take into account. It has been
3. Conclusion Five alkaloids including one new pyrrolidine derivative, asperidine A (1), and two new piperidine derivatives, asperidines B (2) and C (3), together with two known compounds (4 and 5) were produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Compound 3 possessed a new 7-oxa-1-azabicyclo[3.2.1]octane skeleton with four chiral centers in the core unit whereas compound 4 was isolated as a natural product for the first time. Compounds 2 and 3 were assayed for
Fig. 6. Inhibitory effect of 2 and 3 on HMG-CoA reductase activity. HMGR activity was determined in the absence (control) or presence of pravastatin (1 μM) or piperidine derivatives; 2 and 3 (200 μM). Data are expressed as percent of inhibition. *p < 0.05 compared with control (n = 3). 4505
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Fig. 7. Effect of 2 and 3 on the viability of Caco-2 cells. Cells were incubated with 2 and 3 at the doses of 12.5, 25, 50, 75 and 100 µM for 24 h. Subsequently, the cells were washed and treated with 5 mg/mL of MTT for the next 4 h. The absorbance was measured at 570 nm and the data are expressed as means of percent control ± S.E.M (n = 5).
4.2. Fungal material
HMGR and CFTR inhibitory activities, free radical-scavenging activity and cytotoxicity against noncancerous Vero cells. Compound 2 displayed mild HMGR inhibitory activity, interesting anti-oxidative effect in Caco-2 cells and promising CFTR inhibitory activity. In addition, it was noncytotoxic to noncancerous Vero cells.
The soil fungus PSU-RSPG178 was isolated from a soil sample from Suratthani province, Thailand and deposited as BCC56851 at BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, Genbank accession number KC478521. The fungus PSU-RSPG178 was identified as Aspergillus sclerotiorum by morphological characteristics and the analysis of an internal transcribed spacer (ITS1-5.8S-ITS2) rDNA using universal fungal primers,14 which was described in our previous report.2
4. Experimental 4.1. General experimental procedures The melting points were determined on an Electrothermal 9100 melting point apparatus and reported without correction. Optical rotations were recorded on a JASCO P-1020 polarimeter. The ultraviolet (UV) absorption spectra were measured in MeOH on a Perkin-Elmer Lambda 45 spectrophotometer. The infrared (IR) spectra were recorded neat using a Perkin-Elmer 783 FTS165 FT-IR spectrometer. Mass spectra were obtained from a liquid chromatograph-mass spectrometer (2090, LCT, Waters, Micromass). 1H and 13C NMR spectra were recorded on a 300 MHz Bruker FTNMR Ultra Shield spectrometer. Chemical shifts are expressed in δ (parts per million, ppm) referring to the tetramethylsilane peak. Thin-layer chromatography (TLC) and preparative TLC (PTLC) were performed on silica gel 60 GF254 (Merck). Column chromatography (CC) was carried out on Sephadex® LH-20 with MeOH, silica gel (Merck) type 60 (230–400 mesh ASTM) or type 100 (70–230 mesh ASTM), or on reversed phase C18 silica gel.
4.3. Fermentation, extraction and isolation Fermentation and extraction procedures were previously reported.2 The broth extract (9.43 g) was separated by CC over Sephadex® LH-20 using MeOH to give three fractions (A-C). Fraction A (1.11 g) was fractionated by CC over Sephadex® LH-20 using MeOH/CH2Cl2 (1:1) to afford four fractions (A1-A4). Fraction A3 (368.7 mg) was purified by CC over silica gel using a gradient of MeOH/CH2Cl2 (2:98 → 100:0) to give five fractions (A3A-A3E). Compound 1 (3.1 mg) was obtained as a colorless solid from fraction A3E. Fraction B (8.26 g) was separated by CC over silica gel using a gradient of EtOAc/petroleum ether (15:85 → 100:0) to afford nine fractions (B1-B9). Fraction B7 (283.1 mg) was further separated by CC over silica gel using a gradient of MeOH/ CH2Cl2 (4:96 → 100:0) to give seven fractions (B7A-B7G). Fraction B7D (33.7 mg) was rechromatographed on CC over silica gel using MeOH/ CH2Cl2 (4:96) followed by PTLC using MeOH/CH2Cl2 (8:92) as a mobile
Fig. 8. Anti-oxidative effect of 2 and 3 in Caco-2 cells. Cells were treated with 2 and 3 at indicated concentrations (12.5–100 µM) for 24 h and incubated with 10 µM DCFH-DA for 1 h at 37 °C in the dark followed by a-30 min incubation in the absence (A) or the presence (B) of H2O2. Values represents means ± S.E.M. (n = 3), * p < 0.05; **p < 0.01 compared to control cells in the absence of H2O2; #p < 0.05; ##p < 0.01 compared to control cells exposed with H2O2. 4506
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Fig. 9. Effect of compounds 2 and 3 on CFTR-mediated chloride secretion in human intestinal epithelial (T84) cells.
phase (3 runs) and subsequent PTLC using EtOAc/CH2Cl2 (2:3) as a mobile phase (2 runs) to provide 4 (1.8 mg). The mycelial hexane extract (1.40 g) was separated by CC over Sephadex® LH-20 using CHCl3/ MeOH (1:1) to give three fractions (CH1–CH3). Fraction CH2 (1.06 g) was fractionated by CC over silica gel using a gradient of MeOH/CH2Cl2 (0:100 → 100:0) to give five fractions (CH2A–CH2E). Fraction CH2C (298.3 mg) was further separated by CC over silica gel using a gradient of EtOAc/petroleum ether (20:80 → 70:30) to give six fractions (CH2C1–CH2C6). Fraction CH2C3 (100.9 mg) was separated by CC over reversed phase C18 silica gel using a gradient of MeOH/H2O (85:15 → 100:0) followed by CC over silica gel using a gradient of MeOH/CH2Cl2 (0.1:99.9 → 5:95) and rechromatographed on CC over silica gel using CH2Cl2/MeOH/petroleum ether (70:2:28) to afford 3 (20.7 mg). Fraction CH2D (369.4 mg) was further purified by CC over silica gel using a gradient of EtOAc/petroleum ether (10:90 → 100:0) to give 2 (20.7 mg). The mycelial ethyl acetate extract (4.34 g) was separated using the same procedure as the broth extract to afford four fractions (CE1-CE4). Fraction CE3 (2.47 g) was rechromatographed on CC over silica gel using a gradient of MeOH/CH2Cl2 (0:100 → 100:0) to give six fractions (CE3A-CE3F). Fraction CE3F (412.4 mg) was separated using the same procedure as the broth extract to afford five fractions (CE3F1-CE3F5). Fraction CE3F4 (62.9 mg) was purified by CC over silica gel using MeOH/CH2Cl2 (4:96) followed by PTLC using MeOH/CH2Cl2 (4:96) as a mobile phase (3 runs) to provide 5 (2.1 mg).
4.3.3. Asperidine C (3) Colorless crystals; mp 95–96 °C; [α ]25 D −158.3 (c 0.1, CHCl3); UV (MeOH) λmax (log ε): 209 (3.72), 259 (2.37) nm; IR (neat)νmax 3393 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z: [M +H]+ 332.2590 (calcd for C21H34NO2, 332.2590).
4.3.1. Asperidine A (1) Colorless solid; mp 97–98 °C; [α ]26 D −13.8 (c 1.0, MeOH); UV (MeOH) λmax (log ε): 207 (4.02), 256 (2.48) nm; IR (neat)νmax 3325 cm−1; 1H and 13C NMR data, see Table 1, HRESIMS m/z: [M +H]+ 276.2327 (calcd for C18H30NO, 276.2327).
4.6. X-ray crystallography
4.4. Preparation of the (R)- and (S)-MTPA ester derivative of 28,9 Pyridine (100 μL) and (+)-(R)-MTPACl (40 μL) were added to a CH2Cl2 solution (300 μL) of 2 (2.5 mg). The reaction mixture was stirred at room temperature overnight. After removal of the solvent, the mixture was purified by CC over silica gel using acetone/hexane (3:17) to afford the (S)-MTPA ester (3.7 mg). 2 (2.6 mg) was treated in a similar way with (−)-(S)-MTPACl and, after purification by CC over silica gel, (R)-MTPA ester (3.0 mg) was obtained. 4.5. Preparation of the (R)- and (S)-MTPA ester derivative of 38,9 Pyridine (100 μL) and (+)-(R)-MTPACl (40 μL) were added to a CH2Cl2 solution (300 μL) of 3 (2.1 mg). The reaction mixture was stirred at room temperature overnight. After removal of the solvent, the mixture was purified by CC over silica gel using MeOH/CH2Cl2 (1:199) to afford the (S)-MTPA ester (3.8 mg). 3 (2.1 mg) was treated in a similar way with (−)-(S)-MTPACl and, after purification by CC over silica gel, (R)-MTPA ester (2.7 mg) was obtained.
The crystal data of 3 (Fig. 4) was selected for data collection which was performed on a on a XtaLAB Synergy-S Hypix-600HE diffractometer equipped with Cu Kα monochromated radiation (λ = 1.5408 Å) source at low temperature of 100 K. The automatic crystal screening, data collection and data reduction process were performed using CrysAlis PRO version 1.171.39.30t. This structure was solved using direct methods with SHELXT and refined with the fullmatrix least-squares methods based on F2 with the SHELXL program.15 All non hydrogen atoms were found from the electron density maps and refined anisotropically. All hydrogen atoms of this compound were
4.3.2. Asperidine B (2) Colorless gum; [α ]25 D −39.1 (c 0.2, CHCl3); UV (MeOH) λmax (log ε): 210 (4.22), 260 (2.86) nm; IR (neat)νmax 3382 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z: [M+H]+ 318.2797 (calcd for C21H36NO, 318.2797). 4507
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was measured by Synergy™ HT microplate reader (Biotek, VT) at excitation and emission wavelengths of 495 nm and 527 nm, respectively.
determined from the residual electron density and refined isotropically. The WinGXv2014.116 and Mercury programs17 were used to prepare the materials and molecular graphic for publication. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit No. CCDC 1839357, 3). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax: +44-(0)223-336033 or e-mail: [email protected]). Crystal Data of 3 (CCDC 1839357): C21H33NO2, M = 331.48, T = 100(1) K, monoclinic space group P21, a = 6.68920(10) Å, b = 7.16530(10) Å, c = 19.7476(2) Å, V = 943.94(2) Å3, α = γ = 90°, β = 94.2200(10)°, Z = 2, Dcalc = 1.166 Mg/m3, crystal dimensions 0.095 × 0.089 × 0.043 mm3, μ = 0.570 mm−1, F(0 0 0) = 364, 17,536 reflections measured, 3447 unique (Rint = 0.0311). The final refinement gave R1 = 0.0247 and wR2 = 0.0655 [I > 2σ(I)]. Absolute structure parameter = −0.02(8).
4.7.6. Evaluation of the effect of test compounds on CFTR-mediated intestinal chloride secretion T84 cells were seeded and cultured on porous membrane of Snapwell inserts (1 cm2 surface area; Corning-Costar Corp., Corning, NY, USA) at a density of 500,000 cells/insert. After transepithelial electrical resistance of > 1000 Ω.cm2 was reached, insert was mounted in Ussing Chambers. Apical and basolateral hemichambers were filled with symmetrical Kreb’s solution containing 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2 (pH 7.3), and 10 mM glucose, and were continuously bubbled with 95% O2/5% CO2 mixture and maintained at 37 °C. The cAMP-induced chloride secretion was induced using forskolin (20 μM).20 Subsequently, tested substances were added into apical solutions at varied concentrations. The half maximal inhibitory concentration (IC50) was calculated using Hill’s equation.
4.7. Bioassays 4.7.1. Cytotoxicity assay The activity assay against Vero cells was performed in triplicate employing the method described by Hunt and co-workers.18 Ellipticine, the standard drug, displayed the IC50 value of 4.06 μM.
Acknowledgments V. Rukachaisirikul thanks the Royal Property Bureau Foundation and National Science and Technology Development Agency for the NSTDA Chair Professor grant (The Fourth Grant). P. Phainuphong is grateful to the TRF through the Royal Golden Jubilee Ph.D. program (Grant number PHD/0031/2555) and Prince of Songkla University (PSU-Ph.D. scholarship) for a joint funding scholarship. The Center of Excellence for Innovation in Chemistry (PERCH-CIC) and Prince of Songkla University Graduate School are acknowledged for partial support. Finally, the National Center for Genetic Engineering and Biotechnology (BIOTEC) is acknowledged for cytotoxicity assay.
4.7.2. HMGR activity assay Inhibitory effect on HMGR activity was determined using a cell-free HMG-CoA reductase assay kit according to the manufacturer’s protocol (Sigma-Aldrich, MO). Briefly, the reaction mixture containing 1× buffer, NADPH, HMG CoA, and piperidine derivatives (200 µM) or pravastatin (1 μM) (positive control) were added into the 96-well plates (Corning Inc., NY, USA). Subsequently, catalytic domains of human HMGR were added to initiate the reaction at 37 °C. The oxidation rate of HMGR was analyzed by measuring absorbance at 340 nm for 10 min at 20-s interval using Synergy™ HT microplate reader (Biotek, VT, USA). The data were expressed as percent inhibition of control.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmc.2018.07.036.
4.7.3. Cell culture preparation Caco-2 and T84 cells were purchased from the American Type Culture Collection (ATCC) (VA, USA). Cells (passage 2nd–22nd) were cultured in DMEM containing DMEM/F-12 (Sigma-Aldrich, MO) containing 1.2 g/L NaHCO3 supplemented with 10% FBS and 100 unit/mL penicillin–streptomycin solution (Life technology, NY). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C.
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4.7.4. Cell viability assay Cell viability was measured by MTT assays. Briefly, the cells were plated at a density of 2.5 × 104 cells/well in a 96-well plate for 24 h. Cells were then incubated with different concentrations of piperidine derivatives (12.5, 25, 50, 75 and 100 μM) and MTT solution (5 mg/mL) for 4 h. At the end of experiment, dimethylsulfoxide (DMSO) was added to dissolve the blue crystals and absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed relatively to the absorbance of control. 4.7.5. Determination of ROS The intracellular ROS levels in Caco-2 cells were quantitated using fluorescent dye 20,70-dichlorfluoresceindiacetate (DCFH-DA) (SigmaAldrich, MO) as described previously.19 Caco-2 cells were treated with piperidine derivatives at various concentrations (12.5–100 μM) for 24 h. Cells were washed and incubated with 10 μM DCFH-DA for 1 h at 37 °C in the dark. Subsequently, 0.5 mM of H2O2 will be added in each well for 30 min and cells were washed twice. The fluorescence intensity
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