- Email: [email protected]
Lodopyridones B and C from a marine sediment-derived bacterium Saccharomonospora sp.
Bioorganic & Medicinal Chemistry Letters 27 (2017) 3123–3126
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Lodopyridones B and C from a marine sediment-derived bacterium Saccharomonospora sp. Tu Cam Le a,d, Chae-Yoon Yim a,d, Songhee Park b, Nikita Katila b, Inho Yang a, Myoung Chong Song a, Yeo Joon Yoon a, Dong-Young Choi b, Hyukjae Choi b,⇑, Sang-Jip Nam a,⇑, William Fenical c,⇑ a b c
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California-San Diego, CA 92093-0204, USA
a r t i c l e
i n f o
Article history: Received 12 April 2017 Revised 9 May 2017 Accepted 11 May 2017 Available online 12 May 2017 Keywords: Lodopyridone Saccharomonospora BACE1 inhibitor Marine natural products
a b s t r a c t HPLC-UV guided isolation of the culture broth of a marine bacterium Saccharomonospora sp. CNQ-490 has led to the isolation of two new natural products, lodopyridones B and C (1 and 2) along with the previously reported lodopyridone A (3). Their chemical structures were established from the interpretation of 2D NMR spectroscopic data and the comparison of NMR data with the lodopyridone A (3). Lodopyridones B and C (1 and 2) possess the thiazole, and chloroquinoline groups which are characteristic features of these molecules. Lodopyridones A–C show weak inhibitory activities on the b-site amyloid precursor protein cleaving enzyme 1 (BACE1). Ó 2017 Elsevier Ltd. All rights reserved.
Actinomycetes are well known sources for the discovery of bioactive secondary metabolites and are recognized to produce 45% of known microbial secondary metabolites.1–4 Recent studies also reveal that marine actinomycetes are becoming a unique source for discovering structurally unique bioactive natural products.5–10 The marine actinomycete strain CNQ-490, was isolated from a sediment sample collected from the La Jolla Submarine Canyon in La Jolla California. This strain, which was identified as distantly related to the genus Saccharomonospora, requires seawater for growth indicating a high degree of adaptation to marine habitats. Previous chemical studies of this strain involved the identification of an unprecedented alkaloid, lodopyridone A (3), bearing an ethanolamine, a thiomethyl-substituted 4-pyridone, thiazole, and chloroquinoline moieties.10 A facile total synthesis of lodopyridone A has subsequently been reported in relatively high yield.11 Strain CNQ-490 has also recently been the subject of genome mining resulting in the heterologous expression of a daptomycin class lipopeptide, taromycin A, by TAR gene cluster cloning.12 Given the uniqueness of this unprecedented alkaloid class, we examined the culture extracts of this strain by HPLC-UV guided
⇑ Corresponding authors. E-mail addresses: [email protected] (H. Choi), [email protected] (S.-J. Nam), [email protected] (W. Fenical). d These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2017.05.035 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
fractionation, and now report the isolation of two new lodopyridone congeners, lodopyridones B and C (1 and 2). The details of the isolation and structure elucidation of these new metabolites and their biological activities against the important enzyme Beta-secretase (BACE1) are reported.
Lodopyridone B (1) was identified to have the molecular formula C25H35 23ClN4O5S2 from HRESIMS data of a protonated molecule [M+H]+ at m/z 559.0878 (calcd for C25H35 24ClN4O5S2, 559.0877) and by comparison with its NMR data along with the previous reported data on lodopyridone A (3).10 Compound 1 showed a similar UV pattern with strong absorptions at 219, 257, 315, 331 and 346 nm analogous to those of 3. IR absorptions at 1720, 1654 and 1589 cm 1 indicated the presence of carbonyl and amide functional groups. The 1H NMR spectrum of 1 exhibited six aromatic
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protons (dH 8.55, 8.33, 8.29, 8.25, 8.14 and 7.86 ppm), four methyl singlets [an acetyl (dH 1.99), a thiomethyl (dH 2.30), an aminomethyl (dH 3.36) and a methoxy (dH 3.67)] along with two methylene proton groups (dH 4.14 and 3.51) and one exchangeable proton (NH, dH 9.14). Overall 13C and 2D NMR data also indicated the presence of four methyls, two methylenes, six methines and thirteen unprotonated carbons. The planar structure of compound 1 was established from further interpretation of 2D NMR spectroscopic data. O S
O O
H N
O N
O
Cl N S
N COSY HMBC
The chloroquinoline moiety was constructed based on COSY and HMBC correlations (Fig. 1). The COSY spectrum revealed two pairs of ortho-coupled aromatic protons [H-11/ H-12 (dH 8.33/8.55, J = 8.4 Hz), and H-16/H-17 (dH 7.86/8.14, J = 9.0 Hz)]. A weak meta-coupling was also observed between H-14 (dH 8.25)
Fig. 1. COSY and key HMBC correlations of lodopyridone B (1).
and H-16 (dH 7.86) with a coupling constant of 2.2 Hz. HMBC NMR correlations from H-11 (dH 8.33) to C-13 (dC 129.1), from H12 (dH 8.55) to C-10 (dC 150.5) / C-18 (dC 145.5), from H-14 (dH 8.25) to C-16 (dC 130.8)/C-18 (dC 145.5), from H-16 (dH 7.86) to C-14 (dC 126.9)/C-18 (dC 145.5) and from H-17 (dH 8.14) to C-13 (dC 129.1)/C-15 (dC 132.0) confirmed the presence of the chloroquinoline moiety. COSY NMR correlations of H2-19 (dH 3.51) with H2-20 (dH 4.14)/ NH (dH 9.14) and HMBC correlations from NH to C-19 (dC 37.8) and carbonyl C-1 (dC 161.1) allowed the ethanolamine moiety to be assigned. The presence of a singlet methyl group (dH 1.99, dC 20.6) and a carbonyl carbon C-21 (dC 170.2), as well as the observation of the downfield-shifted H2-20 methylene protons (dH 4.14) compared to those of 3 suggested that an acetyl group was attached to the end of the ethanolamine moiety. HMBC correlations from the NCH3 to C-2 and C-6 implied the presence of a nitrogen bearing ring structure. HMBC correlations for the three non-protonated carbons at dC 119.8 (C-3), dC 170.8 (C-4), dC 147.0 (C-5) were not observed due to the lack of neighboring protons. Furthermore, the downfield-shifted aromatic proton (H-8) and the HMBC correlations from the singlet aromatic proton H-8 to C-6/C-9 only established the presence of the thiazole ring. This difficulty was also observed for lodopyridone A (3), whose chemical structure was solved from the interpretation of X-ray crystallographic data.12 However, considering almost identical UV spectra of 1 and 3 and the comparison of carbon chemical shifts of 1 to those of 3 indicated that compound 1 also contained the 4-pyridone and thiazole components. Therefore, the structure of lodopyridone B (1) was defined as 20-acetyl-lodopyridone A (Fig. 1). The molecular formula of lodopyridone C (2) was determined as C23H35 21ClN4O5S2 on the basis of a protonated molecular ion peak at 1 m/z 533.0729 [M+H]+ (calcd for C23H35 22ClN4O5S2, 533.0720). The H
Fig. 2. BACE1 inhibitory activities of lodopyridones A–C (1–3).
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T.C. Le et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3123–3126 Table 1 NMR spectroscopic data for Lodopyridones B (1) and C (2) in DMSO-d6, d in ppm. No.
1a dC, mult.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 SCH3 OCH3 NCH3 OH NH a b c
2b c
161.1, C 148.8, C 119.8, C 170.8, C 147.0, C 136.8, C 145.0, C 126.3, CH 168.4, C 150.5, C 118.4, CH 137.4, CH 129.1, C 126.9, CH 132.0, C 130.8, CH 130.7, CH 145.5, C 37.8, CH2 62.1, CH2 170.2, C 20.6, CH3 16.1, CH3 59.4, CH3 39.1, CH3
dH (J in Hz)
dC, mult.c
HMBC
7.86, dd (9.0, 2.2) 8.14, d (9.0)
17 16
14, 18 13, 15
3.51, br m 4.14, br m
NH, 20 19
21
157.7, C 146.2, C 157.4, C 169.0, C 147.8, C 137.4, C 144.7, C 126.7, CH 168.6, C 150.5, C 118.4, CH 137.4, CH 129.1, C 126.9, CH 132.0, C 131.1, CH 130.8, CH 145.5, C 41.8, CH2 59.1, CH2
21 3 5 2, 6
37.7, CH3 59.5, CH3 38.6, CH3
8.29, s
6, 9
8.33, d (8.4) 8.55, d (8.4)
12 11
8.25, d (2.2)
1.99, 2.30, 3.67, 3.36,
9, 13 10, 14, 18 12, 16, 18
s s s s
9.14, t (5.6) 1
COSY
19
1, 19
dH (J in Hz)
COSY
8.31, s
8.32, d (8.5) 8.55, d (8.5)
6, 9
12 11
8.25, d (2.2) 17 16
3.31, m 3.52, m
NH, 20 19
br s s s s
9, 13 10, 14, 18 12, 16, 18
7.86, dd (9.0, 2.5) 8.13, d (9.0)
3.04, 3.72, 3.37, 4.70, 9.08,
HMBC
14, 18 13, 15
5 2, 6 19
13
300 MHz for H NMR. 75 MHz for C NMR, 500 MHz for 2D NMR. 500 MHz for 1H NMR, 125 MHz for 13C NMR. Numbers of attached protons were determined by analysis of 2D NMR spectroscopic data.
Fig. 3. Inhibition of Ab40 and Ab42 release by lodopyridone A (1).
and 13C NMR spectroscopic data for 2 were almost identical to those of 3, which indicated that they share the same carbon backbone. Interpretation of 2D NMR spectroscopic data and the comparison of the carbon chemical shifts of 2 to those of 3 permitted the ethanolamine, 4-pyridone, thiazole, and chloroquinoline components to be established. The observation that the chemical shift of C-3 (dC 157.4) was significantly deshielded, coupled with the deshielded chemical shift of the sulfur-attached methyl (dH 3.04, dC 37.7) group suggested that compound 2 had a methyl sulfoxide group at C-3 rather than the thiomethyl group. Actinomycetes related to the genus Saccharomonospora, (family Pseudonocardiaceae), was first identified by Nonomura and Ohara in 1971.13 To date, eleven new species with validly reported names are described and a number of biological and chemical investigations on these microbes have been shown it to be one of the promising sources for discovering novel bioactive secondary metabolites.14,15 Lodopyridone A was reported in 2009 as a structurally unprecedented and rare alkaloid.10 Since the first report of the lodopyri-
done A, related metabolites possessing a similar origin and structural components has now been expanded. Consequently, lodopyridones B and C are the only new entries to this unique class of natural products. The biological activities for lodopyridones A–C were investigated in several bioassays. Lodopyridones A–C displayed neither agonistic activity on peroxisome proliferator-activated receptors (PPARs) nor antibacterial activities against several pathogens. In the past, thiazole-containing natural products have been reported as acetylcholinesterase (AChE) inhibitors,16,17 and one thiazole containing AChE inhibitor, acotiamide, was launched in Japan.18 On this basis, lodopyridones A–C were examined for inhibitory activity against acetylcholinesterase. Lodopyridones did not exhibit any inhibitory activity on this enzyme up to 100 mM. Lodopyridones A–C (1–3) were found to possess weak inhibitory activity on b-site amyloid precursor protein cleaving enzyme 1 (BACE1). The metabolites did not show BACE1 inhibition below 100 nM, but illustrated dose-dependent inhibition when tested at 1 mM and above (Fig. 2) (Table 1).
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Lodopyridones A–C (1–3) exhibited BACE1 inhibition at 60%, 51% and 42% at the concentration of 100 mM, respectively (a drug control, isoliquiritigenin, IC50 = 51.5 lM).19 We further analyzed the inhibitory effects of lodopyridone A on b-amyloid (1–40) (Ab40) or b-amyloid (1–42) (Ab42) production in SH-SY5Y cells. Ab40 and Ab42 productions were reduced by lodopyridone A up to 41% and 49% of the control at 1 mM, respectively, while isoliquiritigenin decreased the level to 48% and 50% of the control at 51.5 lM. Thus, lodopyridone A exhibited a mild, but dose dependent inhibition on release of Ab40 and Ab42 (Fig. 3). BACE1 is essential enzyme for the production of b-amyloid which is considered to induce neurotoxicity in Alzheimer’s brain.20,21 BACE1 inhibitor MK-8931, is currently in the human clinical trials.20,21 However, the treatment of neurodegenerative diseases in the future will require a much larger diversity of chemical scaffolds for the development of novel BACE1 inhibitors. Acknowledgments The present study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning under grant NRF-2014R1A1A2057302 (to HC) and NRF2014R1A1A1003492 (to SJN), and by the US National Cancer Institute (NIH) under grant R37 CA044848 (to WF). A. Supplementary data Supplementary data (1D and 2D NMR spectroscopic data of 1 and 2, General experimental procedures, strain isolation and
fermentation, extraction and isolation, BACE1 inhibitory activity assay, b-amyloid assay in SH-SY5Y cell) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. bmcl.2017.05.035. References 1. Bérdy J. J Antibiot. 2005;58:1–26. 2. Genilloud O, González I, Salazar O, Martín J, Tormo JR, Vicenle F. J Ind Microbiol Biotechnol. 2011;38:375–389. 3. Sharma M. Int J Curr Microbiol App Sci. 2014;3:801–832. 4. Baltz RH. Microbe. 2007;2:125–131. 5. a) Fenical W, Jensen PR. Nat Chem Biol. 2006;2:666–673; b) Hu Y, Chen J, Hu G, et al. Mar Drugs. 2015;13:202–221. 6. Zotchev SB. J Biotechnol. 2012;158:168–175. 7. Das S, Lyla PS, Khan A. Curr Sci. 2006;90:1325–1335. 8. Subramani R, Albersberg W. Microbiol Res. 2012;167:571–580. 9. Manivasaga P, Venkatesan J, Sivakumar K, Kim SK. Microbiol Res. 2013;168:311–332. 10. Maloney KN, MacMillan JB, Kauffman CA, et al. Org Lett. 2009;23:5422–5424. 11. Burckhardt T, Harms K, Koert U. Org Lett. 2012;14:4674–4677. 12. Yamanaka K, Reynolds KA, Kersten RD, et al. Proc Natl Acad Sci. 2014;111:1957–1962. 13. Nonomura H, Ohara Y. J Ferment Technol. 1971;49:895–903. 14. Veyisoglu A, Sazak A, Cetin D, Guven K, Sahin N. Int J Syst Evol Microbiol. 2013;63:3782–3786. 15. a) Singh B, Parshad R, Khajuria RK, et al. Tetrahedron Lett. 2013;54:6695–6699; b) Tamura A, Takeda I. J Antibiot. 1975;28:395–397. 16. Nagal AA, Liston DR, Jung S, et al. J Med Chem. 1995;38:1084–1089. 17. Ronco C, Sorin G, Nachon F, et al. Bioorg Med Chem. 2009;17:4523–4536. 18. Nolan ML, Scott LJ. Drugs. 2013;73:1377–1383. 19. Yang EJ, Kim GS, Noh H, Shin YS, Song KS. J Funct Foods. 2015;18:445–454. 20. Yan R, Vassar R. Lancet Neurol. 2014;13:319–329. 21. Choi DY, Choi H. Arch Pharm Res. 2015;38:139–170.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Lodopyridones B and C from a marine sediment-derived bacterium Saccharomonospora sp. Tu Cam Le a,d, Chae-Yoon Yim a,d, Songhee Park b, Nikita Katila b, Inho Yang a, Myoung Chong Song a, Yeo Joon Yoon a, Dong-Young Choi b, Hyukjae Choi b,⇑, Sang-Jip Nam a,⇑, William Fenical c,⇑ a b c
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California-San Diego, CA 92093-0204, USA
a r t i c l e
i n f o
Article history: Received 12 April 2017 Revised 9 May 2017 Accepted 11 May 2017 Available online 12 May 2017 Keywords: Lodopyridone Saccharomonospora BACE1 inhibitor Marine natural products
a b s t r a c t HPLC-UV guided isolation of the culture broth of a marine bacterium Saccharomonospora sp. CNQ-490 has led to the isolation of two new natural products, lodopyridones B and C (1 and 2) along with the previously reported lodopyridone A (3). Their chemical structures were established from the interpretation of 2D NMR spectroscopic data and the comparison of NMR data with the lodopyridone A (3). Lodopyridones B and C (1 and 2) possess the thiazole, and chloroquinoline groups which are characteristic features of these molecules. Lodopyridones A–C show weak inhibitory activities on the b-site amyloid precursor protein cleaving enzyme 1 (BACE1). Ó 2017 Elsevier Ltd. All rights reserved.
Actinomycetes are well known sources for the discovery of bioactive secondary metabolites and are recognized to produce 45% of known microbial secondary metabolites.1–4 Recent studies also reveal that marine actinomycetes are becoming a unique source for discovering structurally unique bioactive natural products.5–10 The marine actinomycete strain CNQ-490, was isolated from a sediment sample collected from the La Jolla Submarine Canyon in La Jolla California. This strain, which was identified as distantly related to the genus Saccharomonospora, requires seawater for growth indicating a high degree of adaptation to marine habitats. Previous chemical studies of this strain involved the identification of an unprecedented alkaloid, lodopyridone A (3), bearing an ethanolamine, a thiomethyl-substituted 4-pyridone, thiazole, and chloroquinoline moieties.10 A facile total synthesis of lodopyridone A has subsequently been reported in relatively high yield.11 Strain CNQ-490 has also recently been the subject of genome mining resulting in the heterologous expression of a daptomycin class lipopeptide, taromycin A, by TAR gene cluster cloning.12 Given the uniqueness of this unprecedented alkaloid class, we examined the culture extracts of this strain by HPLC-UV guided
⇑ Corresponding authors. E-mail addresses: [email protected] (H. Choi), [email protected] (S.-J. Nam), [email protected] (W. Fenical). d These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2017.05.035 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
fractionation, and now report the isolation of two new lodopyridone congeners, lodopyridones B and C (1 and 2). The details of the isolation and structure elucidation of these new metabolites and their biological activities against the important enzyme Beta-secretase (BACE1) are reported.
Lodopyridone B (1) was identified to have the molecular formula C25H35 23ClN4O5S2 from HRESIMS data of a protonated molecule [M+H]+ at m/z 559.0878 (calcd for C25H35 24ClN4O5S2, 559.0877) and by comparison with its NMR data along with the previous reported data on lodopyridone A (3).10 Compound 1 showed a similar UV pattern with strong absorptions at 219, 257, 315, 331 and 346 nm analogous to those of 3. IR absorptions at 1720, 1654 and 1589 cm 1 indicated the presence of carbonyl and amide functional groups. The 1H NMR spectrum of 1 exhibited six aromatic
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protons (dH 8.55, 8.33, 8.29, 8.25, 8.14 and 7.86 ppm), four methyl singlets [an acetyl (dH 1.99), a thiomethyl (dH 2.30), an aminomethyl (dH 3.36) and a methoxy (dH 3.67)] along with two methylene proton groups (dH 4.14 and 3.51) and one exchangeable proton (NH, dH 9.14). Overall 13C and 2D NMR data also indicated the presence of four methyls, two methylenes, six methines and thirteen unprotonated carbons. The planar structure of compound 1 was established from further interpretation of 2D NMR spectroscopic data. O S
O O
H N
O N
O
Cl N S
N COSY HMBC
The chloroquinoline moiety was constructed based on COSY and HMBC correlations (Fig. 1). The COSY spectrum revealed two pairs of ortho-coupled aromatic protons [H-11/ H-12 (dH 8.33/8.55, J = 8.4 Hz), and H-16/H-17 (dH 7.86/8.14, J = 9.0 Hz)]. A weak meta-coupling was also observed between H-14 (dH 8.25)
Fig. 1. COSY and key HMBC correlations of lodopyridone B (1).
and H-16 (dH 7.86) with a coupling constant of 2.2 Hz. HMBC NMR correlations from H-11 (dH 8.33) to C-13 (dC 129.1), from H12 (dH 8.55) to C-10 (dC 150.5) / C-18 (dC 145.5), from H-14 (dH 8.25) to C-16 (dC 130.8)/C-18 (dC 145.5), from H-16 (dH 7.86) to C-14 (dC 126.9)/C-18 (dC 145.5) and from H-17 (dH 8.14) to C-13 (dC 129.1)/C-15 (dC 132.0) confirmed the presence of the chloroquinoline moiety. COSY NMR correlations of H2-19 (dH 3.51) with H2-20 (dH 4.14)/ NH (dH 9.14) and HMBC correlations from NH to C-19 (dC 37.8) and carbonyl C-1 (dC 161.1) allowed the ethanolamine moiety to be assigned. The presence of a singlet methyl group (dH 1.99, dC 20.6) and a carbonyl carbon C-21 (dC 170.2), as well as the observation of the downfield-shifted H2-20 methylene protons (dH 4.14) compared to those of 3 suggested that an acetyl group was attached to the end of the ethanolamine moiety. HMBC correlations from the NCH3 to C-2 and C-6 implied the presence of a nitrogen bearing ring structure. HMBC correlations for the three non-protonated carbons at dC 119.8 (C-3), dC 170.8 (C-4), dC 147.0 (C-5) were not observed due to the lack of neighboring protons. Furthermore, the downfield-shifted aromatic proton (H-8) and the HMBC correlations from the singlet aromatic proton H-8 to C-6/C-9 only established the presence of the thiazole ring. This difficulty was also observed for lodopyridone A (3), whose chemical structure was solved from the interpretation of X-ray crystallographic data.12 However, considering almost identical UV spectra of 1 and 3 and the comparison of carbon chemical shifts of 1 to those of 3 indicated that compound 1 also contained the 4-pyridone and thiazole components. Therefore, the structure of lodopyridone B (1) was defined as 20-acetyl-lodopyridone A (Fig. 1). The molecular formula of lodopyridone C (2) was determined as C23H35 21ClN4O5S2 on the basis of a protonated molecular ion peak at 1 m/z 533.0729 [M+H]+ (calcd for C23H35 22ClN4O5S2, 533.0720). The H
Fig. 2. BACE1 inhibitory activities of lodopyridones A–C (1–3).
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T.C. Le et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 3123–3126 Table 1 NMR spectroscopic data for Lodopyridones B (1) and C (2) in DMSO-d6, d in ppm. No.
1a dC, mult.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 SCH3 OCH3 NCH3 OH NH a b c
2b c
161.1, C 148.8, C 119.8, C 170.8, C 147.0, C 136.8, C 145.0, C 126.3, CH 168.4, C 150.5, C 118.4, CH 137.4, CH 129.1, C 126.9, CH 132.0, C 130.8, CH 130.7, CH 145.5, C 37.8, CH2 62.1, CH2 170.2, C 20.6, CH3 16.1, CH3 59.4, CH3 39.1, CH3
dH (J in Hz)
dC, mult.c
HMBC
7.86, dd (9.0, 2.2) 8.14, d (9.0)
17 16
14, 18 13, 15
3.51, br m 4.14, br m
NH, 20 19
21
157.7, C 146.2, C 157.4, C 169.0, C 147.8, C 137.4, C 144.7, C 126.7, CH 168.6, C 150.5, C 118.4, CH 137.4, CH 129.1, C 126.9, CH 132.0, C 131.1, CH 130.8, CH 145.5, C 41.8, CH2 59.1, CH2
21 3 5 2, 6
37.7, CH3 59.5, CH3 38.6, CH3
8.29, s
6, 9
8.33, d (8.4) 8.55, d (8.4)
12 11
8.25, d (2.2)
1.99, 2.30, 3.67, 3.36,
9, 13 10, 14, 18 12, 16, 18
s s s s
9.14, t (5.6) 1
COSY
19
1, 19
dH (J in Hz)
COSY
8.31, s
8.32, d (8.5) 8.55, d (8.5)
6, 9
12 11
8.25, d (2.2) 17 16
3.31, m 3.52, m
NH, 20 19
br s s s s
9, 13 10, 14, 18 12, 16, 18
7.86, dd (9.0, 2.5) 8.13, d (9.0)
3.04, 3.72, 3.37, 4.70, 9.08,
HMBC
14, 18 13, 15
5 2, 6 19
13
300 MHz for H NMR. 75 MHz for C NMR, 500 MHz for 2D NMR. 500 MHz for 1H NMR, 125 MHz for 13C NMR. Numbers of attached protons were determined by analysis of 2D NMR spectroscopic data.
Fig. 3. Inhibition of Ab40 and Ab42 release by lodopyridone A (1).
and 13C NMR spectroscopic data for 2 were almost identical to those of 3, which indicated that they share the same carbon backbone. Interpretation of 2D NMR spectroscopic data and the comparison of the carbon chemical shifts of 2 to those of 3 permitted the ethanolamine, 4-pyridone, thiazole, and chloroquinoline components to be established. The observation that the chemical shift of C-3 (dC 157.4) was significantly deshielded, coupled with the deshielded chemical shift of the sulfur-attached methyl (dH 3.04, dC 37.7) group suggested that compound 2 had a methyl sulfoxide group at C-3 rather than the thiomethyl group. Actinomycetes related to the genus Saccharomonospora, (family Pseudonocardiaceae), was first identified by Nonomura and Ohara in 1971.13 To date, eleven new species with validly reported names are described and a number of biological and chemical investigations on these microbes have been shown it to be one of the promising sources for discovering novel bioactive secondary metabolites.14,15 Lodopyridone A was reported in 2009 as a structurally unprecedented and rare alkaloid.10 Since the first report of the lodopyri-
done A, related metabolites possessing a similar origin and structural components has now been expanded. Consequently, lodopyridones B and C are the only new entries to this unique class of natural products. The biological activities for lodopyridones A–C were investigated in several bioassays. Lodopyridones A–C displayed neither agonistic activity on peroxisome proliferator-activated receptors (PPARs) nor antibacterial activities against several pathogens. In the past, thiazole-containing natural products have been reported as acetylcholinesterase (AChE) inhibitors,16,17 and one thiazole containing AChE inhibitor, acotiamide, was launched in Japan.18 On this basis, lodopyridones A–C were examined for inhibitory activity against acetylcholinesterase. Lodopyridones did not exhibit any inhibitory activity on this enzyme up to 100 mM. Lodopyridones A–C (1–3) were found to possess weak inhibitory activity on b-site amyloid precursor protein cleaving enzyme 1 (BACE1). The metabolites did not show BACE1 inhibition below 100 nM, but illustrated dose-dependent inhibition when tested at 1 mM and above (Fig. 2) (Table 1).
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Lodopyridones A–C (1–3) exhibited BACE1 inhibition at 60%, 51% and 42% at the concentration of 100 mM, respectively (a drug control, isoliquiritigenin, IC50 = 51.5 lM).19 We further analyzed the inhibitory effects of lodopyridone A on b-amyloid (1–40) (Ab40) or b-amyloid (1–42) (Ab42) production in SH-SY5Y cells. Ab40 and Ab42 productions were reduced by lodopyridone A up to 41% and 49% of the control at 1 mM, respectively, while isoliquiritigenin decreased the level to 48% and 50% of the control at 51.5 lM. Thus, lodopyridone A exhibited a mild, but dose dependent inhibition on release of Ab40 and Ab42 (Fig. 3). BACE1 is essential enzyme for the production of b-amyloid which is considered to induce neurotoxicity in Alzheimer’s brain.20,21 BACE1 inhibitor MK-8931, is currently in the human clinical trials.20,21 However, the treatment of neurodegenerative diseases in the future will require a much larger diversity of chemical scaffolds for the development of novel BACE1 inhibitors. Acknowledgments The present study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning under grant NRF-2014R1A1A2057302 (to HC) and NRF2014R1A1A1003492 (to SJN), and by the US National Cancer Institute (NIH) under grant R37 CA044848 (to WF). A. Supplementary data Supplementary data (1D and 2D NMR spectroscopic data of 1 and 2, General experimental procedures, strain isolation and
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