Daryamide Analogues from a Marine-Derived Streptomyces species

Feb 22, 2017 - Peng Fu, Scott La, and John B. MacMillan. Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas...
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Daryamide Analogues from a Marine-Derived Streptomyces species Peng Fu, Scott La, and John B. MacMillan* Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, United States S Supporting Information *

ABSTRACT: Three new cyclohexene amine derivatives, daryamides D−F (1−3), a new arylamine derivative, carpatamide D (4), and a new ornithine lactamization derivative, ornilactam A (5), were isolated from the marinederived Streptomyces strain SNE-011. Their structures, including absolute configurations, were elucidated on the basis of spectroscopic analysis and chemical methods. The carpatamide skeleton could be considered as the biosynthetic precursor of the daryamides.

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ur laboratory has been invested in the discovery of biologically active natural products from marine-derived bacteria, which have been a tremendous resource of novel molecules.1 In addition to molecules with potent biological activity, these organisms have provided novel pathways in natural product biosynthesis.2 As part of our studies to search for new molecules with selective activity against nonsmall-cell lung cancer (NSCLC), we investigated the secondary metabolites of a cytotoxic fraction from a Streptomyces sp. (SNE-011). As part of these studies, we earlier identified three new arylamine derivatives, carpatamides A−C, that possessed moderate cytotoxicity against NSCLC cell lines.3 Investigation of a new culture from Streptomyces sp. strain SNE-011 to identify additional active molecules led to the isolation of a series of carpatamide analogues, as identified by a unique UV signature at approximately 280 nm. Chemical investigation on those fractions resulted in the isolation of three new cyclohexene amine derivatives, daryamides D−F (1−3), a new arylamine derivative, carpatamide D (4), and a new cyclic ornithine derivative, ornilactam A (5). Structurally, compounds 1−3 are manumycin-type metabolites4 and are closely related to daryamides A−C, which were discovered from the marine Streptomyces sp. strain CNQ-085.5 The daryamides (1−3) provide a stereochemical challenge that we solved via a combination of NMR and ECD approaches. Although computational approaches to ECD have been extensively reported in the literature,6 we feel that the use of orthogonal approaches to stereochemical assignment is critical when possible. Although these molecules do not have any activity (cytotoxicity, antibacterial), the structures could provide a starting point for studying unique biotransformations.

RESULTS AND DISCUSSION Daryamide D (1) was isolated as a yellow oil. Its molecular formula was C18H26N2O5 based on the HRESIMS data. Analysis of its NMR spectra and comparison with spectra of carpatamide B (6)3 indicated that compound 1 also contains a 7-methylocta-2,4-dienoic acid residue, which was confirmed by the COSY correlations extending from H-2′ through H3-8′/ H3-9′ (Figure 1). The NOE correlations between H-2′ and H-4′ and between H-3′ and H-5′ (Figure 1), and the typical coupling constants (∼15 Hz) confirmed the configurations of the double bonds as 2′E and 4′E. The COSY correlation of H2-7 and H2-8, and the HMBC correlations of H2-7/H2-8 to carbonyl C-9 (δC 178.8) and two exchangeable proton signals (δH 7.28/6.71, 9-NH2, measured in DMSO-d6) to C-9 (Figure 1) indicated the presence of a propionic amide residue. The 13C NMR signals at δC 58.1 (C-2) and 55.6 (C-3) were attributable to an epoxide, whose protons were observed at δH 3.25 (H-2, dd, J = 3.8, 2.1 Hz) and 3.41 (H-3, dd, J = 3.8, 1.8 Hz). H-2 and H-3 were coupled to an olefinic proton at δH 5.81 (H-6, d, J = 2.1 Hz) and an oxygenated methine proton at δH 4.50 (H-4, d, J = 1.8 Hz), respectively. The COSY correlations of H-2/H-3/H-4 and the HMBC correlations of H-2 to C-6, H-3 to C-1/C-5, H-4 to C-5, and H-6 to C-4/C-5 (Figure 1) suggested a 2,3-epoxy5-cyclohexene moiety. One exchangeable proton signal was observed at δH 9.41 (5-NH, s) in the 1H NMR spectrum (measured in DMSO-d6). This NH proton signal showed key HMBC correlations to C-4/C-6/C-1′ (Figure 1), which suggested that the 7-methylocta2,4-dienoic acid residue was connected with 2,3-epoxy-5cyclohexene moiety through 5-NH. The propionic amide residue was connected with C-1 (δC 71.7), which was confirmed by the HMBC correlations of H2-7 to C-1/C-2/C-6 (Figure 1). A methylation reaction was carried out using iodomethane and sodium hydride to generate 1a, whose structure was determined by HRESIMS and 2D NMR data (Figure 1). In the spectra of 1a, five new methyl groups (two −OCH3 and three −NCH3) were observed, which combined with the HMBC Received: January 4, 2017 Published: February 22, 2017

© 2017 American Chemical Society and American Society of Pharmacognosy

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Figure 2. Comparative analysis of coupling constants for the epoxide moiety and C-4 in 1. Figure 1. Key 2D NMR correlations for the structural assignment of 1−5 and 1a.

correlations of 1-OCH3 to C-1, 4-OCH3 to C-4, 5-NCH3 to C-5/C-1′, and 9-NCH3 to C-9 (Figure 1), further confirmed the structure of 1. The trans configuration between the epoxy ring and the hydroxy group at C-4 was determined by the accurate comparison of the coupling constants (JH2−H3 = 3.8 Hz, JH3−H4 = 1.8 Hz) of 2 with those of the examples with a similar ring system (Figures 2 and S1).7 The cis compounds, gymnastatin D (8)8 and 9,9 showed larger coupling constants between H-2 and H-3 and between H-3 and H-4 (8: JH2−H3 = 4.1 Hz, JH3−H4 = 2.9 Hz; 9: JH2−H3 = 4.2 Hz, JH3−H4 = 2.4 Hz) than those of trans compound 7 (JH2−H3 = 3.8 Hz, JH3−H4 = 2.0 Hz).9 In the 1D NOE spectrum of 1a, the NOEs between H2-7 and 4-OCH3 and between 9-NCH3 and 4-OCH3 could be observed (Figure 1), which indicated that 1-OH and 4-OH of 1 were located on opposing sides of the cyclohexene ring system. Meanwhile, the NOE correlations of H-2/H2-7 and H-3/4-OCH3 (Figure 1) also suggested the relative configurations of C-2/C-1 and C-3/ C-4, respectively. Thus, the relative configurations of 1 were fully determined as trans between the epoxy ring and 4-OH and cis between the epoxy ring and 1-OH. The absolute configuration of C-4 was determined using the modified Mosher’s method.10 When reacted with (R)- and (S)-MTPA chloride, compound 1 gave the corresponding (S)- and (R)- MTPA esters 1b and 1c, respectively. During preparation of the Mosher’s esters, the carboxyamide was transformed into a nitrile. The structures of 1b and 1c were confirmed by 13C chemical shifts and 2D NMR correlations (Figure 3). The observed chemical shift differences of the H-2 and H-3 protons (ΔδS − R) measured in CDCl3 were −0.04 and −0.07, respectively (Figure 3). These results defined the R configuration of C-4. There are some anomalies on the amide side-chain and H-6 in the Mosher’s results. We thought that the MTPA plane was not fixed properly in CDCl3. So, we measured the 1H NMR spectra of 1b and 1c in DMSO-d6, and the results further confirmed the suggested absolute configuration (Figure 3). Moreover, the Dess−Martin oxidation of 1

Figure 3. Δδ (= δS − δR) values for (S)- and (R)-MTPA esters of 1. 13 C chemical shifts and key 2D NMR correlations of 1b in CDCl3.

was carried out to generate 1d.11 The empirical inverse octant rule for cyclohexene oxides was used to determine the absolute configuration of 1d.12 The conformation of (2S, 3R)-1d, as shown in Figure 4, was placed in accordance with the inverse octant rule model. The functional group, oxirane oxygen atom, lying in the lower left quadrant was responsible for the positive Cotton effect at λmax (Δε) 353 (+0.9) nm (Figure 4). Although the unsaturated side chain might be expected to affect the ECD spectrum, the long distance to the carbonyl and its flexible conformation led us to ignore this effect. We attempted to use the quantum chemical calculation method to confirm the absolute configuration of 1. The predicted ECD spectrum was obtained by the TDDFT [B3LYP/ 6-31G(d)] method.13 It showed a strong negative Cotton effect at λmax 315 nm, which was not observed in the measured ECD spectrum (Figure 5). It was considered that the conformational unstability of the 7-methylocta-2,4-dienoic amide chain caused 1097

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with those of 2 revealed that the signals of the isopropyl group (C-6′, C-7′, and C-8′) were replaced by propyl signals. This structure was confirmed by the COSY correlations of H-5′/ H-6′/ H2-7′/H3-8′. Compound 3 has the same absolute configuration as 1 and 2, which was confirmed by their identical ECD spectra (Figure 5). Carpatamide D (4) was obtained as a yellow oil. Its molecular formula was determined as C20H27NO6 according to its HRESIMS peak at m/z 378.1913 [M + H] +. The 13C NMR spectrum displayed 20 signals that were classified by HSQC as six nonprotonated carbons (two carbonyls and four olefinic carbons), seven methine groups (six olefinic carbons and one sp3 methine carbon), five sp3 methylene carbons, and two methyl carbons (Table 2). The presence of a 7-methylocta-2,4dienoic acid residue could be indicated by the comparison of NMR data with those of carpatamide B (6)3 and daryamide D (1) (Tables 1 and 2). Two aromatic proton signals at δH 6.41 (H-3, s) and 6.84 (H-6, s), along with the corresponding HMBC correlations of H-3 to C-1/C-5 and H-6 to C-2/C-4 (Figure 1), indicated a 1,2,4,5-tetrasubstituted benzene unit. Two coupled methylene signals at δC 26.2 (C-7) and 36.5 (C-8), together with the key HMBC correlations of H2-7 to C-2/C-6/C-9 and H2-8 to C-1/C-9, confirmed that a propionic acid residue was connected at C-1 (Figure 1). Another two coupled methylene signals at δC 52.3 (C-1″) and 60.0 (C-2″), and the HMBC correlations of H2-2″ to C-1″ and H2-1″ to C-5/C-1′, indicated that a hydroxyethyl unit was connected with 5-N. These analyses revealed that compound 4 was a 5-Nhydroxyethyl analogue of carpatamide B. The trans conformation of the amide was determined by the NOE correlation between H-2′ and H-6 (Figure 1). The molecular formula of ornilactam A (5) was determined as C14H22N2O2 on the basis of its HRESIMS spectrum. Analysis of its 1H and 13C NMR spectra showed that it had the same (2E,4E)-7-methylocta-2,4-dienoic acid unit as 1 (Tables 1 and 2). The rest of the 13C signals were classified by HSQC as three methylene carbons, one sp3 methine carbon, and one carbonyl. The COSY correlations of H-2/H2-3/H2-4/H2-5, and the key HMBC correlations of H-2 to C-1/C-4, H-3 to C-1, and H-5 to C-1 (Figure 1) suggested the presence of an ornithine lactamization moiety. The 7-methylocta-2,4-dienoic acid unit was connected with the 2-NH through an amide bond, which was confirmed by the HMBC correlation of H-2 to C-1′ (Figure 1). The absolute configuration of the ornithine moiety was determined as L- by Marfey’s method.14 The 1-fluoro-2,4-dinitrophenyl-5-L-valine amide (L-FDVA) derivatives of the acid hydrolysates of 5 and authentic L-ornithine, and the D-FDVA derivative of authentic L-ornithine were obtained for HPLC analysis. The results showed that the L-FDVA derivative of the acid hydrolysates of 5 gave the same retention time as the L-FDVA derivative of authentic L-ornithine (Figure S3). Although the biosynthetic pathway of the manumycin type of metabolites has been studied, some of the biosynthetic steps are still not clear; however evidence has suggested that the cyclohexene moiety is most likely derived from the 3-amino-4hydroxybenzoic acid (3,4-AHBA).15 Thus, the carpatamide skeleton may be the precursor to the daryamides. Compounds 1−5 did not show cytotoxicity to nonsmall cell lung cancer cell lines HCC366, A549, HCC44, and H2122. We also tested their antibacterial activities against Pseudomonas aeruginosa and Bacillus subtilis. However, they did not show significant antibacterial activity.

Figure 4. ECD spectrum and the representation of the empirical inverse octant rule for cyclohexene oxide of 1d.

the abnormal results. Therefore, we must be cautious about using calculation methods to determine absolute configurations of the molecules with flexible side groups. Daryamide E (2) was thought to be an analogue of daryamide D due to its similar UV absorption. The molecular formula was determined as C17H24N2O5 by HRESIMS, with one CH2 less than daryamide D (1). The 1D NMR spectra were similar to 1 except for the absence of signals for one methylene group in the 7-methylocta-2,4-dienoic acid moiety. So, we deduced that the 7-methylocta-2,4-dienoic acid unit in 1 was replaced by 6-methylhepta-2,4-dienoic acid in 2. The COSY correlations extending from H-2′ through H3-7′ and H3-8′, along with the HMBC data (Figure 1) confirmed this change. The (2′E,4′E)- configuration of the double bonds was confirmed by the NOE correlations between H-2′ and H-4′ and between H-3′ and H-5′ (Figure 1), and the large coupling constants (∼15 Hz). The nearly identical NMR chemical shifts of the 2,3-epoxy-5-cyclohexene core between 1 and 2 (Table 1) suggested they showed the same relative configuration. The ECD Cotton effects of 2 were nearly identical to those observed in 1 (Figure 5), indicating the same absolute configuration. The molecular formula of daryamide E (3) was also determined to be C17H24N2O5 by HRESIMS, which was an isomer of 2. Comparison of its 1H and 13C NMR spectra (Table 1)

Figure 5. ECD spectra of compounds 1−3. 1098

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Table 1. 1H (600 MHz) and 13C (100 MHz) NMR Data for Compounds 1−3 1 (in CD3OD) no.

δC

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 9-NH2 5-NH

71.7, C 58.1, CH 55.6, CH 64.6, CH 134.7, C 116.3, CH 37.1, CH2 31.2, CH2 178.8, C 168.0, C 122.9, CH 143.6, CH 130.8, CH 143.9, CH 43.4, CH2 29.5, CH 22.7, CH3 22.7, CH3

δH, mult. (J in Hz) 3.25, dd (3.8, 2.1) 3.41, dd (3.8, 1.8) 4.50, d (1.8) 5.81, d (2.1) 1.99, m 2.34, m

6.01, d (15.0) 7.20, dd (15.0, 10.7) 6.22, dd (15.0, 10.7) 6.15, dt (15.1, 7.2) 2.08, dd (7.2, 6.8) 1.72, m 0.93, d (6.7) 0.93, d (6.7)

1 (in DMSO-d6) δC 69.5, C 56.9, CH 53.9, CH 62.8, CH 132.7, C 113.9, CH 36.4, CH2 29.7, CH2 174.5, C 164.8, C 123.4, CH 140.2, CH 129.6, CH 141.4, CH 41.6, CH2 27.8, CH 22.2, CH3 22.2, CH3

2 (in CD3OD)

δH, mult. (J in Hz) 3.11, dd (3.9, 2.1) 3.25, dd (3.9, 1.9) 4.30, d (1.9) 5.99, d (2.1) 1.76, m 2.17, m; 2.09, m

6.09, d (15.1) 7.06, dd (15.1, 10.8) 6.21, dd (15.1, 10.9) 6.12, dt (15.1, 7.2) 2.03, dd (7.2, 7.0) 1.67, m 0.87, d (6.7) 0.87, d (6.7) 7.28, s; 6.71, s 9.41, s

δC 71.7, C 58.1, CH 55.6, CH 64.6, CH 134.7, C 116.3, CH 37.1, CH2 31.2, CH2 178.8, C 168.0, C 123.0, CH 143.9, CH 126.9, CH 151.6, CH 32.8, CH 22.3, CH3 22.3, CH3

3 (in CD3OD)

δH, mult. (J in Hz) 3.25, dd (3.9, 2.1) 3.41, dd (3.8, 1.8) 4.50, d (1.8) 5.81, d (2.1) 1.99, m 2.34, m

6.03, d (15.1) 7.19, dd (15.1, 10.7) 6.20, dd (15.2, 10.7) 6.12, dd (15.2, 6.8) 2.43, m 1.05, d (6.8) 1.05, d (6.8)

δC

δH, mult. (J in Hz)

71.7, C 58.1, CH 55.6, CH 64.6, CH 134.7, C 116.3, CH 37.1, CH2 31.2, CH2 178.8, C 168.0, C 122.8, CH 143.7, CH 129.9, CH 144.9, CH 36.1, CH2 23.1, CH2 14.0, CH3

3.25, dd (3.8, 2.1) 3.41, dd (3.8, 1.8) 4.50, d (1.8) 5.80, d (2.1) 1.99, m 2.34, m

6.01, d (15.0) 7.19, dd (15.0, 10.7) 6.24, dd (15.2, 10.8) 6.15, dt (15.2, 6.8) 2.17, td (7.0, 7.0) 1,48, qt (7.4, 7.4) 0.94, t (7.4)

Table 2. 1H (600 MHz) and 13C (100 MHz) NMR Data for Compounds 4 and 5 in CD3OD 4

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no.

δC

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″

121.4, C 157.6, C 104.7, CH 153.7, C 121.8, C 131.8, CH 26.2, CH2 36.5, CH2 178.7, C 170.4, C 121.0, CH 143.5, CH 131.3, CH 143.1, CH 43.2, CH2 29.5, CH 22.7, CH3 22.7, CH3 52.3, CH2 60.0, CH2

5 δH, mult. (J in Hz)

δC

δH, mult. (J in Hz)

6.41, s

172.9, C 50.9, CH 28.9, CH2 22.3, CH2 42.8, CH2

4.38, dd (10.8, 6.1) 2.14, m; 1.79, m 1.94, m; 1.88, m 3.30, overlapped

168.8, C 122.9, CH 142.5, CH 130.9, CH 143.1, CH 43.3, CH2 29.5, CH 22.7, CH3 22.7, CH3

5.93, d (15.2) 7.15, dd (15.1, 10.7) 6.20, dd (15.1, 10.8) 6.10, dt (15.1, 7.3) 2.07, t (7.1) 1.71, m 0.92, d (6.7) 0.92, d (6.7)

6.84, s 2.78, m 2.53, t (7.4)

5.72, d (15.1) 7.13, dd (15.1, 9.9) 6.01, dd (15.1, 9.7) 6.06, dt (15.1, 6.8) 2.00, t (6.5) 1.66, m 0.88, d (6.7) 0.88, d (6.7) 3.80, m; 3.74, m 3.74, m; 3.65, m

provided by The Scripps Research Institute, La Jolla, CA. Lowresolution LC/ESI-MS data were measured using an Agilent 1200 series LC/MS system with a reversed-phase C18 column (Phenomenex Luna, 150 mm × 4.6 mm, 5 μm) at a flow rate of 0.7 mL/min. Preparative HPLC was performed on an Agilent 1200 series instrument with a DAD detector, using a reversed-phase C18 column (Phenomenex Luna, 250 × 10.0 mm, 5 μm), a phenyl-hexyl column (Phenomenex Luna, 250 × 10.0 mm, 5 μm), or an EVO C18 column (Phenomenex Kinetex, 100 × 4.6 mm, 2.6 μm). Sephadex LH-20 (GE Healthcare) and ODS (50 mm, Merck) were used for column chromatography. Artificial seawater was used in microbial fermentations as described in a previous reference.16 Collection and Phylogenetic Analysis of Strain SNE-011. The actinomycete Streptomyces sp. SNE-011 was isolated as previously described.3

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded with an AUTOPOL AP IV-6W polarimeter equipped with a halogen lamp (589 nm). UV spectra were recorded on a Shimadzu UV-1601 UV−VIS spectrophotometer. ECD spectra were measured on JASCO J-815 spectrometer. IR spectra were obtained on a PerkinElmer Spectrum 1000 FT-IR Spectrometer. 1H and 2D NMR spectroscopic data were recorded at 600 or 400 MHz in CD3OD, DMSO-d6, CDCl3, or pyridine-d5 solution on Varian System spectrometer, and chemical shifts were referenced to the corresponding residual solvent signal (δH/C 3.31/49.00 for CD3OD, δH/C 2.50/39.52 for DMSO-d6, δH/C 7.26/77.16 for CDCl3, and δH/C 8.74/150.35 for pyridine-d5). 13C NMR spectra were acquired at 100 MHz on a Varian System spectrometer. High-resolution ESI-TOF mass spectra were 1099

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Cultivation and Extraction of SNE-011. Details of the method are the same as our previous report.3 Purification. The extract (3.9 g) was partitioned with EtOAc/H2O. The EtOAc soluble layer (1.6 g) was fractionated by flash column chromatography on ODS (50 μm, 30 g), eluting with a step gradient of MeOH and H2O (10:90−100:0), and 11 fractions (Fr.1−Fr.11) were collected. Fractions 5 (123.9 mg) and 6 (245.4 mg) were combined and separated into eight fractions (Fr.5.1−Fr.5.8) on Sephadex LH-20, eluting with MeOH. Fr.5.3 (157.9 mg) was separated into 15 fractions (Fr.5.3.1−Fr.5.3.15) by flash column chromatography on ODS (50 μm, 30 g), eluting with a step gradient of CH3CN and H2O (10:90−70:30). Fr.5.3.8 (31.7 mg) was purified by HPLC on a EVO C18 column (Phenomenex Kinetex, 100 × 4.6 mm, 2.6 μm, 1.0 mL/min) using 28% CH3CN (0.1% formic acid) to yield compounds 2 (10.0 mg, tR = 10.7 min) and 3 (3.3 mg, tR = 11.3 min). Fr.5.3.11 (4.9 mg) was purified by HPLC on a phenyl-hexyl column (Phenomenex Luna, 250 × 10.0 mm, 5 μm, 2.5 mL/min) using 35% CH3CN (0.1% formic acid) to yield compound 4 (1.8 mg, tR = 22.6 min). Fraction 7 (37.9 mg) was purified by HPLC on a C18 column (Phenomenex Luna, 250 × 10.0 mm, 5 μm, 2.5 mL/min) using a gradient solvent system from 20% to 100% CH3CN (0.1% formic acid) over 15 min to afford compound 1 (13.3 mg, tR = 11.2 min). Fraction 8 (103.1 mg) was separated by HPLC on a C18 column (Phenomenex Luna, 250 × 10.0 mm, 5 μm, 2.5 mL/min) using a gradient solvent system from 20% to 100% CH3CN (0.1% formic acid) over 15 min to afford compound 1 (8.1 mg, tR = 11.2 min) and Fr.8.2 (63.1 mg). Fr.8.2 was separated into 9 fractions (Fr.8.2.1−Fr.8.2.9) by flash column chromatography on ODS (50 μm, 30 g), eluting with a step gradient of CH3CN and H2O (10:90− 70:30). Fr.8.2.3 (5.7 mg) was further purified by HPLC on a phenylhexyl column (Phenomenex Luna, 250 × 10.0 mm, 5 μm, 2.5 mL/min) using 35% CH3CN (0.1% formic acid) to yield compound 5 (2.8 mg, tR = 17.8 min). Daryamide D (1). Yellow oil, [α]D23 − 12 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 280 (4.25) nm; ECD (c 0.4 mM, MeOH) λmax (Δε) 273 (−1.5), 221 (+2.7) nm; IR (NaCl disk) νmax: 3274, 2957, 1652, 1539, 1385, 1257, 1142, 1041, 998, 923, 867 cm−1; 1H and 13C NMR, Table 1; HRESIMS m/z 351.1915 [M + H]+ (calcd for C18H27N2O5, 351.1914). Daryamide E (2). Yellow oil, [α]D23 − 10 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 282 (4.17) nm; ECD (c 0.3 mM, MeOH) λmax (Δε) 270 (−1.4), 220 (+2.1) nm; IR (NaCl disk) νmax: 3282, 2957, 1652, 1539, 1384, 1257, 1142, 1041, 998, 923, 867 cm−1; 1H and 13C NMR, Table 1; HRESIMS m/z 337.1760 [M + H]+ (calcd for C17H25N2O5, 337.1758). Daryamide F (3). Yellow oil, [α]D23 − 17 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 278 (4.31) nm; ECD (c 0.3 mM, MeOH) λmax (Δε) 273 (−1.3), 221 (+2.3) nm; IR (NaCl disk) νmax: 3282, 2958, 1652, 1539, 1385, 1258, 1143, 998, 923, 867 cm−1; 1H and 13C NMR, Table 1; HRESIMS m/z 337.1753 [M + H]+ (calcd for C17H25N2O5, 337.1758). Carpatamide D (4). Yellow oil; UV (MeOH) λmax (log ε): 270 (4.15) nm; IR (NaCl disk) νmax: 3197, 2954, 2925, 2855, 1573, 1414, 1340, 1194, 1068, 998, 859 cm−1; 1H and 13C NMR, Table 2; HRESIMS m/z 378.1913 [M + H]+ (calcd for C20H28NO6, 378.1911). Ornilactam A (5). Yellow oil, [α]D23 − 19 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 264 (4.06) nm; IR (NaCl disk) νmax: 3317, 2954, 1656, 1611, 1535, 1497, 1352, 1335, 1270, 1208, 1160, 1106, 1002, 886 cm−1; 1H and 13C NMR, Table 2; HRESIMS m/z 251.1756 [M + H]+ (calcd for C14H23N2O2, 251.1754). Methylation of 1. To a solution of 1 (3.0 mg) in DMF (anhydrous, 1.0 mL) was added 10.0 mg of NaH. After allowing it to stir at 0 °C for 0.5 h, 50 μL of CH3I was added into the reaction mixture. It was stirred at room temperature (rt) for another 2 h. Then, a saturated solution of NH4Cl (2.0 mL) was added to quench the reaction. The product was extracted with CH2Cl2 (3 × 5.0 mL) and purified by reversed-phase HPLC (Phenomenex Luna, C18, 250 × 10.0 mm, 2.5 mL/min, 5 μm) using 75% CH3CN (0.1% formic acid) to afford compound 1a (2.3 mg, tR = 7.1 min, 64% yield). Compound 1a, white powder; 1H NMR (600 MHz, CDCl3) δH 7.23 (1H, dd, J = 14.8,

10.0 Hz, H-3′), 6.12 (1H, d, J = 14.9 Hz, H-2′), 6.06 (2H, m, H-4′ and H-5′), 5.46 (1H, d, J = 2.0 Hz, H-6), 4.02 (1H, s, H-4), 3.48 (3H, s, 4-OCH3), 3.40 (1H, dd, J = 4.0, 1.9 Hz, H-3), 3.36 (3H, s, 1-OCH3), 3.32 (1H, dd, J = 4.0, 2.2 Hz, H-2), 3.13 (3H, s, 5-NCH3), 2.95 and 2.94 (6H, s, 9-NCH3), 2.50 and 2.30 (2H, m, H2-8), 2.11 and 2.04 (2H, m, H2-7), 2.00 (2H, t, J = 6.8 Hz, H-6′), 1.67 (1H, m, H-7′), 0.88 (6H, d, J = 6.7 Hz, H-8′/H-9′); 13C NMR (100 MHz, CDCl3) δC 172.1 (C-9), 166.9 (C-1′), 142.6 (C-3′), 142.3 (C-5′), 139.3 (C-5), 130.5 (C-6), 129.9 (C-4′), 120.0 (C-2′), 76.0 (C-1), 75.8 (C-4), 59.5 (4-OCH3), 56.0 (C-2), 53.5 (1-OCH3), 49.3 (C-3), 42.4 (C-6′), 37.1 and 35.7 (9-NCH3), 34.9 (5-NCH3), 34.3 (C-7), 28.5 (C-7′), 27.3 (C-8), 22.4 and 22.5 (C-8′/C-9′); HRESIMS m/z 421.2698 [M + H]+ (calcd for C23H37N2O5, 421.2697). Preparation of the (S)-and (R)-MTPA Esters of 1 by Modified Mosher’s Method. Compound 1 (1.0 mg) and dimethylaminopyridine (1.0 mg) were dissolved in 500 μL of pyridine, and (R)-MTPACl (20 μL) was then added. The reaction mixture was stirred for 22 h at rt, and 4 mL of H2O was then added. The solution was extracted by 5 mL of CH2Cl2, and the organic phase was washed for three times (5 mL each) with H2O. Then, the organic layer was concentrated under reduced pressure to afford (S)-MTPA ester 1b (0.7 mg). By the same procedure, (R)-MTPA ester 1c (0.8 mg) was obtained from the reaction of 1 (1.0 mg) with (S)-MTPACl (20 μL). (S)-MTPA ester (1b): 1H NMR (CDCl3, 600 MHz) δH 7.21 (1H, dd, J = 14.9, 10.0 Hz, H-3′), 6.47 (1H, d, J = 2.0 Hz, H-6), 6.14 (2H, m, H-4′/H-5′), 5.79 (1H, d, J = 1.7 Hz, H-4), 5.71 (1H, d, J = 14.8 Hz, H-2′), 3.41 (1H, dd, J = 3.8, 2.3 Hz, H-3), 3.30 (1H, dd, J = 3.7, 2.1 Hz, H-2), 2.33 and 2.19 (2H, m, H-8), 2.02 and 1.86 (2H, m, H-7), 2.07 (2H, t, J = 6.2 Hz, H-6′), 1.72 (1H, m, H-7′), 0.91 (6H, d, J = 6.7 Hz, H-8′/H-9′); 1H NMR (DMSO-d6, 400 MHz) δH 9.47 (1H, s, 5-NH), 7.10 (1H, dd, J = 15.6, 10.5 Hz, H-3′), 6.21 (2H, m, H-4′/H-5′), 6.12 (1H, brs, H-4), 5.96 (1H, d, J = 15.6 Hz, H-2′), 5.95 (1H, brs, H-6), 5.61 (1H, s, 1-OH), 3.54 (1H, brs, H-3), 3.24 (1H, brs, H-2), 2.18 (2H, m, H-8), 2.05 (2H, t, J = 6.8 Hz, H-6′), 1.68 (1H, m, H-7′), 1.61 (2H, m, H-7), 0.88 (6H, d, J = 6.8 Hz, H-8′/H-9′); ESIMS m/z 549.2 [M + H]+. (R)-MTPA ester (1c): 1H NMR (CDCl3, 600 MHz) δH 7.23 (1H, dd, J = 14.9, 10.1 Hz, H-3′), 6.47 (1H, d, J = 1.9 Hz, H-6), 6.16 (2H, m, H-4′/H-5′), 5.73 (1H, d, J = 1.9 Hz, H-4), 5.73 (1H, d, J = 14.9 Hz, H-2′), 3.48 (1H, dd, J = 3.8, 2.2 Hz, H-3), 3.34 (1H, dd, J = 3.8, 2.0 Hz, H-2), 2.19 and 1.99 (2H, m, H-8), 2.08 (2H, t, J = 6.3 Hz, H-6′), 1.94 and 1.81 (2H, m, H-7), 1.73 (1H, m, H-7′), 0.92 (6H, d, J = 6.7 Hz, H-8′/H-9′); 1H NMR (DMSO-d6, 400 MHz) δH 9.30 (1H, s, 5-NH), 7.06 (1H, dd, J = 15.1, 10.5 Hz, H-3′), 6.22 (1H, d, J = 1.6 Hz, H-4), 6.20 (2H, m, H-4′/H-5′), 5.91 (1H, d, J = 15.2 Hz, H-2′), 5.86 (1H, d, J = 1.8 Hz, H-6), 5.64 (1H, s, 1-OH), 3.58 (1H, dd, J = 3.7, 2.3 Hz, H-3), 3.31 (1H, brs, H-2), 2.34 (2H, m, H-8), 2.05 (2H, t, J = 6.8 Hz, H-6′), 1.77 (2H, m, H-7), 1.68 (1H, m, H-7′), 0.88 (6H, d, J = 6.8 Hz, H-8′/H-9′); ESIMS m/z 549.2 [M + H]+. Dess−Martin Oxidation of 1. A mixture of compound 1 (4.0 mg), CH2Cl2 (1.0 mL), and 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol3(1H)-one (12.0 mg) was stirred at rt for 3 h. The reaction mixture was diluted with CH2Cl2 (50 mL), washed successively with 5% aq Na2S2O3 solution (10 mL), sat. aq NaHCO3 solution (10 mL), and H2O (10 mL). Drying (MgSO4) and evaporation gave the reaction product, which was further purified by reversed-phase HPLC (Phenomenex Luna, C18, 250 × 10.0 mm, 2.5 mL/min, 5 μm) using a gradient solvent system from 30% to 100% CH3CN (0.1% formic acid) over 15 min to afford compound 1d (1.3 mg, tR = 11.6 min, 33% yield). Compound 1d, white powder; ECD (c 0.6 mM, MeOH) λmax (Δε) 353 (+0.9), 307 (−4.6), 251 (+4.9), 228 (−7.3) nm; 1H NMR (600 MHz, CD3OD) δ 7.25 (1H, d, J = 2.7 Hz, H-6), 7.21 (1H, dd, J = 14.8, 10.6 Hz, H-3′), 6.25 (1H, dd, J = 15.2, 10.1 Hz, H-4′), 6.17 (1H, dt, J = 15.0, 7.2 Hz, H-5′), 6.15 (1H, d, J = 15.0 Hz, H-2′), 3.66 (1H, dd, J = 3.8, 2.9 Hz, H-2), 3.56 (1H, d, J = 4.0 Hz, H-3), 2.34 (2H, m, H2-8), 2.08 (2H, m, H2-7), 2.08 (2H, t, J = 6.6 Hz, H-6′), 1.72 (1H, m, H-7′), 0.93 (6H, d, J = 6.7 Hz, H-8′ and H-9′); ESIMS m/z 349.2 [M + H]+. Absolute Configuration Determination of Ornithine of 5 by Marfey’s Method. A solution of 5 (0.5 mg) in 6 M HCl (0.5 mL) was heated at 110 °C for 19 h. The solution was then evaporated to 1100

DOI: 10.1021/acs.jnatprod.7b00011 J. Nat. Prod. 2017, 80, 1096−1101

Journal of Natural Products

Article

dryness and redissolved in H2O (250 μL). Then 100 μL of the acid hydrolysate solution was placed in a 1.5 mL vial and treated with 1% solution of L-FDVA (100 μL) in acetone followed by 1.0 M NaHCO3 (40 μL). The reaction mixture was heated at 45 °C for 1 h, cooled to rt, and then acidified with 1.0 M HCl (40 μL). In a similar fashion, standard L-ornithine was derivatized with L-FDVA and D-FDVA. The derivatives of the hydrolysates and standard amino acid were subjected to HPLC analysis (Phenomenex Luna C18 column; 5 μm, 4.6 × 150 mm; 0.7 mL/min) using the following gradient program: solvent A, H2O + 0.1% formic acid; solvent B, CH3CN + 0.1% formic acid; linear gradient: 0 min 25% B, 40 min 60% B; UV detection at 340 nm. The retention times for the L-FDVA derivatives of hydrolysates of 5 and standard L-ornithine were all 21.6 min, while the retention time for the D-FDVA derivative of standard L-ornithine was 19.8 min (Figure S3).

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Org. Chem. 2012, 2012, 6722−6728. (c) Nugroho, A. E.; Morita, H. J. Nat. Med. 2014, 68, 1−10. (7) Sekiguchi, J.; Gaucher, G. M. Biochem. J. 1979, 182, 445−453. (8) Amagata, T.; Doi, M.; Ohta, T.; Minoura, K.; Numata, A. J. Chem. Soc., Perkin Trans. 1 1998, 3585−3599. (9) Carreno, M. C.; Merino, E.; Ribagorda, M.; Somoza, A.; Urbano, A. Chem. - Eur. J. 2007, 13, 1064−1077. (10) (a) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096. (b) Kusumi, T.; Fujita, Y.; Ohtani, I.; Kakisawa, H. Tetrahedron Lett. 1991, 32, 2923−2926. (c) Seco, J. M.; Quinoa, E.; Riguera, R. Chem. Rev. 2004, 104, 17−117. (11) Jotterand, N.; Vogel, P.; Schenk, K. Helv. Chim. Acta 1999, 82, 821−847. (12) (a) Djerassi, C.; Klyne, W.; Norin, T.; Ohloff, G.; Klein, E. Tetrahedron 1965, 21, 163−178. (b) Kis, Z.; Closse, A.; Sigg, H. P.; Hruban, L.; Snatzke, G. Helv. Chim. Acta 1970, 53, 1577−1597. (c) Shen, B.; Whittle, Y. G.; Gould, S. J.; Keszler, D. A. J. Org. Chem. 1990, 55, 4422−4426. (d) Mohamed, I. E.; Gross, H.; Pontius, A.; Kehraus, S.; Krick, A.; Kelter, G.; Maier, A.; Fiebig, H.; Konig, G. M. Org. Lett. 2009, 11, 5014−5017. (13) Stephens, P. J.; Pan, J. J.; Krohn, K. J. J. Org. Chem. 2007, 72, 7641−7649. (14) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. (15) Rui, Z.; Petrickova, K.; Skanta, F.; Pospisil, S.; Yang, Y.; Chen, C.; Tsai, S.; Floss, H. G.; Petricek, M.; Yu, T. J. Biol. Chem. 2010, 285, 24915−24924. (16) Keller, M. D.; Selvin, R. C.; Claus, W.; Guillard, R. R. L. J. Phycol. 1987, 23, 633−638.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00011. NMR spectra for compounds 1−5 and 1a−1d, a description of the bioassay protocols used (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +1-214648-8653. Fax: +1-214-648-8856. ORCID

John B. MacMillan: 0000-0003-1430-1077 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the following grants for funding this project: Welch Foundation I-1689 and NIH R01 CA149833. J.B.M. is a Chilton/Bell Foundation Endowed Scholar.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00011 J. Nat. Prod. 2017, 80, 1096−1101