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Jul 17, 2018 - Asperversiamides, Linearly Fused Prenylated Indole Alkaloids from the Marine-Derived Fungus Aspergillus versicolor. Huaqiang Li , Weigu...
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Article Cite This: J. Org. Chem. 2018, 83, 8483−8492

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Asperversiamides, Linearly Fused Prenylated Indole Alkaloids from the Marine-Derived Fungus Aspergillus versicolor Huaqiang Li,§ Weiguang Sun,§ Mengyi Deng,§ Qun Zhou, Jianping Wang, Junjun Liu, Chunmei Chen, Changxing Qi, Zengwei Luo, Yongbo Xue,* Hucheng Zhu,* and Yonghui Zhang* Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430030, China

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ABSTRACT: Asperversiamides A−H (1−8), eight linearly fused prenylated indole alkaloids featuring an unusual pyrano[3,2-f ]indole unit, were isolated from the marinederived fungus Aspergillus versicolor. The structures and absolute configurations of these compounds were elucidated by extensive spectroscopic analyses, single-crystal X-ray diffraction, electronic circular dichroism (ECD) calculations, and optical rotation (OR) calculations. The relative configuration of C-21 of iso-notoamide B was herein revised, and a new methodology for preliminarily determining if the relative configuration of the bicyclo[2.2.2]diazaoctane moiety of a spirobicyclo[2.2.2]diazaoctane-type indole alkaloid is syn or anti was developed. The anti-inflammatory activities of the isolated compounds were all tested, and of these compounds, 7 exhibited a potent inhibitory effect against iNOS with an IC50 value of 5.39 μM.

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INTRODUCTION Prenylated indole alkaloids, such as stephacidins,1 paraherquamides,2 notoamides,3 and brevianamides,4 which comprise one or two isoprenyl units in addition to tryptophan and proline moieties, are mostly produced by fungi from the genera Aspergillus and Penicillium. Due to their intriguing structures and appreciable biological activities,1,5 prenylated indole alkaloids have attracted considerable attention from natural product and organic chemists.6 Normally, the isoprenyl group is located at C-7 and is cyclized to form a dimethyldihydropyran ring with the hydroxy group at C-6, and we define these prenylated indole alkaloids with pyrano[2,3-g]indole moieties as angularly fused alkaloids (Figure 1). To date, more than 100 compounds of this class

As part of our ongoing commitment to discover novel, structurally intriguing, and biologically active metabolites from fungi,9 Aspergillus versicolor, a fungus isolated from the mud of the South China Sea, was chemically investigated. A series of linearly fused prenylated indole alkaloids were isolated (Figure 2), and of these compounds, asperversiamides A−C and E (1− 3 and 5) each contains a rare anti bicyclo[2.2.2]diazaoctane ring, and asperversiamide D (4) contains the analogous syn ring (when the C21−C22 and C17−N13 bonds are cofacial, the ring is defined as “syn”, and when the C21−C22 and C17−N13 bonds are on opposite faces, the ring is considered “anti” (Figure S1)).3c,10 Asperversiamide A (1) is the first linearly fused indole alkaloid found to have a rare fused-iminecontaining pyrrole ring system. In addition, compounds 2 and 3 and compounds 4 and 5 are pairs of C-3 and C-21 epimers, respectively. Asperversiamide F (6) is the C-17 epimer of dihydrocarneamide A (9),11 and asperversiamide G (7) possesses an unusual Z-geometry of the double bond between C-10 and C-11. Based on the biosynthesis pathway, compound 8, possessing an isoprenyl unit at C-3, is a pivotal precursor of spiro-bicyclo[2.2.2]diazaoctane type indole alkaloids (2 and 3). Co-isolated deoxybrevianamide E (10)12 could serve as a precursor to a series of structurally related prenylated indole alkaloids by further modification (Scheme S1). Herein, the isolation, structural elucidation, and plausible biosynthetic pathway as well as biological evaluations of new compounds 1−8 are presented.

Figure 1. Two different fusion patterns of pyrane and indole rings.

have been reported.5,7 In contrast, compounds with the isoprenyl substituent located at C-5, which forms a pyrano[3,2-f ]indole moiety, are uncommon, and we call these compounds linearly fused prenylated indole alkaloids (Figure 1). Linearly fused indole alkaloids are quite rare, and only five examples, namely, carneamides A−C, dihydrocarneamide A, and iso-notoamide B, have been previously reported.8 © 2018 American Chemical Society

Received: April 29, 2018 Published: July 17, 2018 8483

DOI: 10.1021/acs.joc.8b01087 J. Org. Chem. 2018, 83, 8483−8492

Article

The Journal of Organic Chemistry

H (Table S1), the presence of rings C and D in 1 was further refined based on the structures of taichunamides A and H. Thus, compound 1 was proposed to have a fused-iminecontaining pyrrole ring (1b). Interestingly, the singlet protons at δH 7.17 (H-4) and 6.83 (H-7) of 1 indicated the presence of a 1,2,4,5-tetrasubstituted benzene ring with two para-protons. The HMBC correlations from H3-28 and H3-29 to C-26 and C-27; from H-25 to C-4, C-5 (δC 118.7), C-6 (δC 153.5), and C-27; from H-26 to C-5; from H-4 to C-25; and from H-7 to C-5 and C-9 confirmed the presence of rings A and B and the linear fusion pattern in 1 (Figure 4). Furthermore, the crucial HMBC correlations from H-4 to C-3 and H-10 to C-9 established the connectivity of rings B and D through C-3, resembled taichunamides A and H.13 Thus, the planar structure of 1 was elucidated as shown. The relative configuration of 1 was confirmed by the examination of its NOESY spectrum (Figure 4). The NOESY correlations of H-21/H-10a and H-21/H3-23 revealed these protons were cofacial and α-oriented. Accordingly, the NOESY correlations of H3-24/3-OH, 3-OH/H-10b, and H-10b/19NH indicated that the 3-OH, 19-NH, and H3-24 groups were β-oriented. Thus, the relative configuration of 1 was determined. To determine the absolute configuration of 1, the calculated ECD were performed using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for 3R,11R,17R,21S (1) and 3S,11S,17S,21R (ent-1). The calculated curve of 1 fit well with its experimental ECD spectrum (Figure 5), which confirmed the absolute configuration of 1 as 3R,11R,17R,21S. Asperversiamide B (2), isolated as colorless crystals, was found to have the chemical formula C26H29N3O4, which indicates 14 degrees of unsaturation, by a positive HRESITOFMS experiment. The 1H NMR data of 2 (Table 1) indicated two singlet aromatic protons (δH 7.09, H-4; and 6.19, H-7) and typical signals of a Z-configured double bond (δH 6.30, J = 9.8 Hz, H-25; 5.58, J = 9.8 Hz, H-26) that closely resembled those of 1, suggesting that 2 and 1 have the same benzopyran motif. The 1H−1H COSY and HMBC spectra confirmed the presence of a bicyclo[2.2.2]diazaoctane ring. In the HMBC spectrum of 2, H3-23 and H3-24 showed correlations with C-3 rather than C-2, which suggested that 2 and notoamide B have the same fusion pattern, bearing a spiro[4.4]nonane ring system.3a Thus, the planar structure of 2 was established. The NOESY correlations (Figure 6) of 19-NH/H3-23 and H-4/H3-23 along with the interaction between H-21 and H324 indicated the α-orientation of 19-NH and β-orientation of H-21. According to the reports by Williams and co-workers, the Cotton effects (CEs) at 200−250 nm are due to the n−π* transition of the diketopiperazine amide bonds, which is diagnostic the absolute configuration of the bicyclo[2.2.2]diazaoctane ring system.4,14 In the case of compound 2, the characteristic positive CE at 223 nm (+25.9) and negative CE at 242 nm (−28.6) in the experimental ECD spectrum (Figure S3) suggested the absolute configuration of 2 was 3S,11S,17S,21R. The single-crystal X-ray diffraction analysis of 2 unambiguously confirmed its structure and absolute configuration (Figure 7). A search using SciFinder indicated the elucidated structure of 2 was identical to that of iso-notoamide B.11 However, the NMR data of 2 and iso-notoamide B were substantially different, which suggested there was a mistake in the structural assignment of iso-notoamide B. After careful analysis of the

Figure 2. Structures of compounds 1−10.

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RESULTS AND DISCUSSION Asperversiamide A (1), a white amorphous powder, was established to have a molecular formula of C26H29N3O4 by HRESITOFMS from the [M + H]+ ion at m/z 448.2232 (calcd for C26H30N3O4+, 448.2231). The 1H NMR data of 1 (Table 1) included resonances of four olefinic protons (δH 7.17, s; 6.83, s; 6.43, d, J = 9.8 Hz; and 5.71, d, J = 9.8 Hz), two exchangeable protons (δH 7.11, s; and 8.75, s), and four methyl groups (δH 1.16, s; 1.18, s; 1.37, s; and 1.38, s) as well as signals attributable to five methylene moieties and a methine group. The 13C NMR data (Table 2) displayed 26 carbon resonances, namely, four methyl carbons, five methylene carbons, one sp3 and four sp2 methine carbons, and 12 quaternary sp3 carbons including two oxygenated carbons, two in amide groups, and a resonance of unknown origin at δC 189.9. Taken together, the aforementioned data suggested that compound 1 is an indole diketopiperazine alkaloid.1,3a Further analyses of the 1H−1H COSY and HMBC spectra of 1 confirmed the presence of a bicyclo[2.2.2]diazaoctane core including a proline (E/F/G ring system, Figure 2). In view of the uncertainty of the origin of the carbon signal at δC 189.9 (carbonyl or double bond) and δC 80.1 (oxygenated or Nsubstituted), two possible substructures (1a and 1b, Figure 3) were deduced on the basis of the HMBC correlations from H10 to C-2, C-3, and C-9; from H-21 to C-11, C-22, and C-23; and from H3-23 and H3-24 to C-2 and C-21. The proposed substructure (1a) included a unique azetidine unit that was found in the initial structure of taichunamide A.10c Recently, the structure of taichunamide A was revised to have a fusedimine-containing pyrrole ring (1b) based on calculated 13C NMR and ECD data, and the revised structure was further confirmed by X-ray crystallographic analysis of taichunamide H, a diastereomer of taichunamide A.13 Given the 1H and 13C NMR data of 1 were similar to those of taichunamides A and 8484

DOI: 10.1021/acs.joc.8b01087 J. Org. Chem. 2018, 83, 8483−8492

Article

The Journal of Organic Chemistry Table 1. 1H NMR Data of Compounds 1−8 (δ in ppm, J in Hz) no. 1-NH 4 7 10a 10b

1a 7.17, s 6.83, s 1.60, d (14.8) 3.02, d (14.8)

2a

3b

4c

5c

10.25, s 7.09, s 6.19, s 2.06, d (15.1)

10.26, s 6.72, s 6.22, s 2.41, d (15.0)

10.56, s 7.05, s 6.63, s 2.64, d (15.6)

2.78, d (15.1)

2.53, d (15.0)

3.36, d (15.6)

3.28, m 3.39, m 1.84, dt (12.3, 7.3) 1.99, m 1.78, dt (12.3, 7.3) 2.47, dt (12.3, 6.5)

3.25, m 3.33, m 1.85, m

3.35, m 3.40, m 1.81, m

4.15, 3.24, 3.45, 1.52,

2.00, m 1.85, m

1.99, m 1.81, m

2.53, m

2.53, m

11 14a 14b 15a

3.48, m 3.48, m 1.87, m

3.27, m 3.31, m 1.82, m

15b 16a

2.05, m 1.89, m

1.98, m 1.78, m

16b

2.53, m

2.46, m

10.58, s 7.09, s 6.64, s 2.72, d (17.5) 3.59, d (17.5)

17 19NH 20a 20b 21 23 24 25 26 28 29 3-OH

8.75, s 2.00, m

6d

8.72, s

1.64, dd (13.1, 7.1) 2.07, m 1.92, dd (13.1, 10.5) 2.09, m 3.11, dd (10.5, 7.1) 1.18, s 0.98, s 1.16, s 0.68, s 6.43, d (9.8) 6.30, d (9.8) 5.71, d (9.8) 5.58, d (9.8) 1.37, s 1.37, s 1.38, s 1.35, s 7.11, s

8.67, s

8.72, s

8.55, s

1.65, dd (13.1, 7.3) 1.93, dd (13.1, 10.0) 2.58, dd (10.0, 7.3) 1.01, s 0.45, s 6.28, d (9.7) 5.56, d (9.7) 1.33, s 1.33, s

1.99, m

1.90, m

2.02, m 2.42, dd (10.0, 4.9) 1.23, s 0.96, s 6.49, d (9.7) 5.62, d (9.7) 1.35, s 1.35, s

7.08, s 6.69, s 3.31, m

7e

8d

6.91, s 6.79, s 7.16, s

6.84, s 6.26, s 2.66, dd (14.8, 6.0)

3.38, m t (5.6) m m m

2.82, dd (14.8, 3.0)

3.63, m 3.73, m 2.01, m

4.09, 3.21, 3.28, 1.69,

1.88, m 1.72, m

2.09, m 2.01, m

1.73, m 1.18, m

2.08, m

2.40, m

2.04, m

3.24, m

4.48, dd (10.0, 6.1)

3.91, ddd (11.2, 6.2, 2.4)

5.09, dd (10.4, 1.2) 5.11, dd (17.6, 1.2) 6.09, dd (17.6, 10.4) 1.50, s 1.52, s 6.42, d (9.7) 5.61, d (9.7) 1.39, s 1.39, s

5.00, dd (17.5, 1.3)

5.09, dd (10.6, 1.2) 2.11, m 5.14, dd (17.4, 1.2) 2.10, m 6.17, dd (17.4, 10.6) 1.15, s 1.50, s 1.24, s 1.49, s 6.48, d (9.7) 6.46, d (9.7) 5.61, d (9.7) 5.60, d (9.7) 1.35, s 1.39, s 1.35, s 1.37, s

dt (6.0, 3.0) m m m

5.08, dd (10.8, 1.3) 6.06, dd (17.5, 10.8) 1.00, 1.08, 6.33, 5.56, 1.38, 1.43,

s s d (9.9) d (9.9) s s

a

Recorded at 600 MHz in DMSO-d6. bRecorded at 800 MHz in DMSO-d6. cRecorded at 400 MHz in DMSO-d6. dRecorded at 400 MHz in methanol-d4. eRecorded at 600 MHz in methanol-d4.

chemical shift of C-3 (δC 61.9 for 3; 69.1 for 2), indicating that 3 may be the C-3 epimer of 2. The NOE correlations of 19NH/H-10a and H-10a/H3-23 indicated that 19-NH and Me23 were cofacial, and they were assigned to be α-oriented. The NOE correlations of H-4/H-21, H-4/H3-24, and H3-24/H-21 indicated H-4, H-21, and H3-24 were β-oriented. Thus, C-3 in 3 was confirmed to be in the opposite orientation than it is in 2. The close resembled CEs at 200−250 nm of compounds 3 and 2 indicated that absolute configurations of bicyclo[2.2.2]diazaoctane of compound 3 were 11S,17S. Therefore, according to the relative configurations elucidated above, the absolute configuration of compound 3 was ascertained as 3R,11S,17S,21R. Based on their HRESITOFMS spectra, asperversiamides D (4) and E (5) were found to have the same molecular formula (C26H29N3O3) with 14 degrees of unsaturation. The NMR spectra of 4 and 5 were similar to those of compound 2 except for the presence of two additional olefinic carbons (δC 139.7 and 103.6 for C-2 and C-3 in 4 and δC 139.7 and 102.9 for C-2 and C-3 in 5) (Table 2), indicating the presence of an indole in 4 and 5 rather than a spiro indolone moiety. As the chemical shifts of C-21 of 4 and 5 were considerably different (δC 49.1 in 4 and 45.5 in 5), the compounds were suspected to be C-21 epimers, and this assignment was further confirmed by the key NOESY interactions of 19-NH/H-21 for 4 and 19-NH/H3-23 and H-21/H3-24 for 5 (Figure 6 and Figure S2). The ECD

NOESY spectrum of iso-notoamide B, the H-21 (δH 3.20)/19NH (δH 9.08) cross-peak was observed, indicating that H-21 and 19-NH are cofacial and that the bicyclo[2.2.2]diazaoctane core was in a syn configuration. Therefore, iso-notoamide B was the C-21 epimer of 2, and its structure should be revised (Figure 8). To identify patterns in the syn or anti configurations of the bicyclo[2.2.2]diazaoctanes, the 13C NMR data of previously reported prenylated indole alkaloids with spiro-bicyclo[2.2.2]diazaoctane core were thoroughly analyzed and summarized in Table S4 and Figures S4 and S5.15 We found that for the compounds in the anti-configuration, the Δδ|C11−C21| values (the deviation in chemical shifts of C-11 and C-21) are normally >14 ppm and the Δδ|C21−C22| values are 100 >100 >100 >100 2.95 ± 0.15

9.95 16.58 13.86 5.39 0.17

0.46 0.57 1.22 0.27 0.02

EXPERIMENTAL SECTION

General Experimental Procedures. The NMR spectra were recorded on Bruker AM-400, 600, and 800 spectrometers (Bruker, Karlsruhe, Germany). The 1H and 13C NMR chemical shifts were referenced to the solvent or solvent impurity peaks for methanol-d4 (δH 3.31 and δC 49.0), CDCl3 (δH 7.26 and δC 77.0), and DMSO-d6 (δH 2.50 and δC 39.5). The UV, ECD, and FT-IR spectra were measured using a PerkinElmer Lambda 35 UV spectrophotometer (PerkinElmer, Inc., Fremont, CA, USA), a JASCO-810 ECD spectrometer (JASCO Co., Ltd., Tokyo, Japan), and a Bruker Vertex 70 instrument (Bruker, Karlsruhe, Germany), respectively. Optical rotations were determined with an AUTOPOL IV-T Automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). HRESITOFMS data were obtained in the positive ion mode on a Thermo Fisher LTQ XL spectrometer (Thermo Fisher, Palo Alto, CA, USA). Crystal X-ray diffraction data were measured on a Rigaku XtaLAB PRO MM007HF (Rigaku, Tokyo, Japan). Semipreparative HPLC was carried out using an Agilent 1200 quaternary system with a UV detector, using a reversed-phase C18 column (5 μm, 10 × 250 mm, Welch Ultimate XB-C18). Column chromatography (CC) was performed using silica gel (100−200 and 200−300 mesh; Qingdao Marine Chemical Inc., China), ODS (50 μm, YMC, Kyoto, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Yantai Chemical Industry Research Institute, Yantai, China) and RP-C18 F254 plates (Merck, Germany). Fungal Material. The fungus Aspergillus versicolor was isolated from the mud of the South China Sea. The sequence data for this

Data are reported as the mean ± SD, n = 3. bPositive control.

a

of these compounds, 7 showed a significant inhibitory effect against iNOS with an IC50 value of 5.39 μM. Considering their weak activities against Raw264.7 cells, the inhibitory effects of these compounds should be cell viability independent. Molecular docking studies between compound 7 and iNOS were performed to better understand their interaction.22 As shown in Figure 10, compound 7 adopted an extended conformation and fit well into the ligand binding site of mutant iNOS. In addition, hydrogen bonds were predicted between the carbonyl (C-12) and Asn115 and the carbonyl (C-18) and Gln257. Moreover, a possible π−π stacking interaction 8488

DOI: 10.1021/acs.joc.8b01087 J. Org. Chem. 2018, 83, 8483−8492

Article

The Journal of Organic Chemistry

Figure 10. Low-energy binding conformations of compound 7 bound to iNOS generated by virtual ligand docking. Black and red balls indicate the hydrogen bonding and π−π interactions, respectively. and 317 (3.77) nm; IR (KBr) νmax 3427, 1678, 1468, 1388, 1275, 1209, 1142, 1026 cm−1; ECD (MeOH) λmax (Δε) = 223 (+11.5), 244 (−13.5), and 311 (+2.36) nm. For 1H NMR (800 MHz) and 13C NMR (200 MHz) data, see Tables 1 and 2. HRESITOFMS [M + Na]+ m/z 470.2064 (calcd for C26H29N3O4Na+, 470.2050). Asperversiamide D (4). C26H29N3O3; colorless crystals; mp 198− 199 °C; [α]25 D +104.1 (c 0.34, MeOH); UV (MeOH) λmax (log ε) 236 (4.36) and 317 (3.79) nm; IR (KBr) νmax 3363, 1668, 1462, 1426, 1378, 1255, 1216, 1153, 1114 cm−1; ECD (MeOH) λmax (Δε) = 203 (−4.58), 278 (+10.41), 239 (−2.15), and 257 (+2.45) nm. For 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2. HRESITOFMS [M + Na] + m/z 454.2092 (calcd for C26H29N3NaO3+, 454.2101). Asperversiamide E (5). C26H29N3O3; white amorphous powder; [α]25 D +27.5 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 232 (4.20) and 281 (3.93) nm; IR (KBr) νmax 3421, 1688, 1469, 1443, 1385, 1258, 1217, 1148, 1127 cm−1; ECD (MeOH) λmax (Δε) = 207 (+0.81), 222 (+17.30), 240 (−4.78), and 256 (+10.71) nm. For 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2. HRESITOFMS [M + H]+ m/z 432.2296 (calcd for C26H30N3O3+, 432.2282). Asperversiamide F (6). C26H31N3O3; white amorphous powder; [α]25 D +91.2 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 257 (4.77) and 330 (3.82) nm; IR (KBr) νmax 3434, 1693, 1628, 1490, 1400, 1333, 1274, 1202, 1139 cm−1; ECD (MeOH) λmax (Δε) = 200 (+24.32), 215 (−19.44) and 259 (+37.38) nm. For 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2. HRESITOFMS [M + Na]+ m/z 456.2254 (calcd for C26H31N3NaO3+, 456.2258). Asperversiamide G (7). C26H29N3O3; light yellow power; [α]25 D −1.23 (c 0.24, MeOH); UV (MeOH) λmax (log ε) 253 (4.41) and 334 (3.98) nm; IR (KBr) νmax 3364, 1690, 1664, 1629, 1431, 1384, 1256, 1116 cm−1; ECD (MeOH) λmax (Δε) = 221 (+33.28), 251 (−17.05), 297 (+3.97) and 356 (+5.71) nm. For 1H NMR (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2. HRESITOFMS [M + H]+ m/z 432.2277 (calcd for C26H30N3O3+, 432.2282). Asperversiamide H (8). C26H31N3O4; colorless cubic crystals; mp 201−202 °C; [α]25 D +83.4 (c 0.55, MeOH); UV (MeOH) λmax (log ε) 237 (4.51), 316 (3.93); IR (KBr) νmax 3363, 3196, 2972, 2925, 2880, 1703, 1664, 1626, 1444, 1331, 1213, 1138 cm−1; ECD (MeOH) λmax (Δε) = 200 (−37.46) and 232 (+38.70) nm. For 1H NMR (400 MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2. HRESITOFMS [M + Na]+ m/z 472.2208 (calcd for C26H31N3NaO4+, 472.2207). Dihydrocarneamide A (9). C26H31N3O3; colorless crystals; mp 1 201−202 °C; [α]25 D −39.9 (c 0.38, MeOH). For H NMR (400 MHz) 13 and C NMR (100 MHz) data, see Tables S3. HRESITOFMS [M + Na]+ m/z 456.2227 (calcd for C26H31N3NaO3+, 456.2258). Deoxybrevianamide E (10). C21H25N3O2; white amorphous 1 powder; [α]25 D −45.9 (c 0.44, MeOH); H NMR (600 MHz, CDCl3) data: δH 8.06 (s, 1H, 19-NH), 7.48 (d, J = 7.9 Hz, 1H, H-4), 7.33 (d, J = 8.1 Hz, 1H, H-7), 7.17 (t, J = 7.6 Hz, 1H, H-6), 7.11 (t, J = 7.5 Hz, 1H, H-5), 6.14 (dd, J = 17.4, 10.6 Hz, 1H, H-21), 5.70 (s, 1H, 1-NH), 5.20 (brs, 1H, H-20b), 5.18 (d, J = 6.7 Hz, 1H, H-20a), 4.44 (dd, J = 11.9, 4.0 Hz, 1H, H-11), 4.07 (dd, J = 9.0, 6.9 Hz, 1H,

strain have been submitted to DDBJ/EMBL/GenBank under accession no. 2081031. A voucher sample has been deposited in the culture collection of Tongji Medical College, Huazhong University of Science and Technology, P. R. China. Fermentation and Isolation. The A. versicolor strain was cultured on potato dextrose agar (PDA) at 28 °C for 7 days to prepare the seed culture. Then, the agar plugs were cut into small pieces and inoculated into 250 Erlenmeyer flasks (1 L), previously sterilized by autoclaving, each containing 200.0 g of rice and 160.0 mL of distilled water. All flasks were incubated at 28 °C for 28 days. Following incubation, the growth of cells was stopped by adding 300 mL of 95% EtOH to each flask, collected the culture with the rice in buckets, and then soaked with 95% EtOH many times until the solvent was near colorless at room temperature. The culture was removed under reduced pressure to yield a brown extract, suspended in water (2 L), and then extracted with EtOAc (1:1) for three times. The EtOAc was removed under reduced pressure. The extracts (170.0 g) were subjected to a silica gel chromatography column (CC) eluting with PE/EtOAc (20:1−0:1) progressively to obtain six fractions (Fr. 1−Fr. 6). Fr. 3 was further separated by silica gel CC to yield four subfractions (Fr. 3.1−Fr. 3.4). The subfraction Fr. 3.2 was subjected to a Sephadex LH-20 (MeOH) to afford three parts (Fr. 3.2a−Fr. 3.2c). The second part (Fr. 3.2b) was chromatographed on ODS (MeOH−H2O, 30:70−100:0, v/v) to yield a mixture (1, 4, 9). The mixture was purified by repeated semipreparative HPLC (MeCN− H2O, 40:60−60:40, v/v, 1h, 2 mL/min) to yield 1 (7.6 mg, tR 25.5 min, 45:55, v/v, 2 mL/min), 4 (48.9 mg, tR 27.2 min, 45:55, v/v, 2 mL/min), and 9 (400 mg, tR 21.0 min, 40:60, v/v, 2 mL/min). Fr. 4 was further separated by repeated silica gel CC to yield four subfractions (Fr. 4.1−Fr. 4.4). Fr. 4.2 and Fr. 4.3 were subjected by Sephadex LH-20 (MeOH), medium-pressure ODS, and silica gel chromatography columns alternately before purified by semipreparative HPLC (MeCN-H2O, 40%−60%, t = 1 h, 2 mL/min). Then, six compounds were isolated: 2 (12.5 mg, tR 26.5 min, 48:52, v/v, 2 mL/ min), 3 (1.1 mg, tR 21.5 min, 40:60, v/v, 2 mL/min), 5 (3.4 mg, tR 22.0 min, 40:60, v/v, 2 mL/min), 6 (18.7 mg, tR 23.0 min, 50:50, v/v, 2 mL/min), 7 (3.2 mg, tR 32.0 min, 50:50, v/v, 2 mL/min), 8 (20.5 mg, tR 13.4 min, 57:43, v/v, 2 mL/min), and 10 (5.1 mg, tR 30.0 min, 50:50, v/v, 2 mL/min). Asperversiamide A (1). C26H29N3O4; white amorphous powder; [α]25 D −95.3 (c 0.28, MeOH); UV (MeOH) λmax (log ε) = 243 (4.46) and 338 (3.68) nm; IR (KBr) νmax 3419, 3260, 1702, 1659, 1461, 1261, 1161, 1111 cm−1; ECD (MeOH) λmax (Δε) = 221 (+13.1), 246 (+15.5), and 268 (−22.7) nm. For 1H NMR (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2. HRESITOFMS [M + H]+ m/z 448.2232 (calcd for C26H30N3O4+, 448.2231). Asperversiamide B (2). C26H29N3O4; colorless crystals; mp 198− 199 °C; [α]25 D −10.9 (c 0.73, MeOH); UV (MeOH) λmax (log ε) 236 (4.54) and 315 (3.89) nm; IR (KBr) νmax 3434, 1693, 1400, 1274, 1202, 1139 cm−1; ECD (MeOH) λmax (Δε) = 223 (+25.9), 243 (−28.7), and 266 (+0.6) nm. For 1H NMR (600 MHz) and 13C NMR (150 MHz) data, see Tables 1 and 2. HRESITOFMS [M + Na]+ m/z 470.2074 (calcd for C26H29N3O4Na+, 470.2050). Asperversiamide C (3). C26H29N3O4; white amorphous powder; [α]25 D +15.8 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 236 (4.40) 8489

DOI: 10.1021/acs.joc.8b01087 J. Org. Chem. 2018, 83, 8483−8492

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Optical Rotation Calculations of Compound 6. Monte Carlo conformational searches were carried out by means of the Spartan 10 software using Merck molecular force field (MMFF). The conformers with Boltzmann population of over 5% were chosen for OR calculations, and then the conformers were initially optimized at B3LYP/6-31+g (d, p) level in MeOH using the CPCM polarizable conductor calculation model. The theoretical calculation of OR was conducted in MeOH using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of compound 6a (11S,17R). ECD Calculations of Compound 7. The conformations generated by BALLOON were subjected to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program. Duplicate conformations were identified and removed when the rootmean-square (RMS) distance was 2σ (I)). The final R1 values were 0.0267, and wR(F2) values were 0.0711. The goodness of fit on F2 was 1.038. Flack parameter = 0.09(7). Crystal Data for Asperversiamide D (4). C26H29N3O3, M = 431.52, a = 11.02750(10) Å, b = 11.02750(10) Å, c = 38.1512(5) Å, α = 90°, β = 90°, γ = 90°, V = 4639.41(10) Å3, T = 100.00(10) K, space group P43212, Z = 8, μ (Cu Kα) = 0.653 mm−1, Dcalc = 1.236 g/cm3, 29747 reflections measured, (Rint = 0.0622, Rσ = 0.0316). The final R1 values were 0.0420 and wR2 values were 0.1071 (I > 2σ (I)). The final R1 values were 0.0435 and wR(F2) values were 0.1083. The goodness of fit on F2 was 1.054. Flack parameter = −0.09(11). Crystal Data for Asperversiamide H (8). C26H31N3O4, M = 449.54, a = 8.14900 (10) Å, b = 9.85930 (10) Å, c = 28.6560 (2) Å, α = 90°, β = 90°, γ = 90°, V = 2302.32(4) Å3, T = 100.01(10) K, space group P212121, Z = 4, μ (Cu Kα) = 0.711 mm−1, Dcalc = 1.297 g/ cm3,10122 reflections measured, and 4507 independent reflections (Rint = 0.0208, Rσ = 0.0219). The final R1 values were 0.0738 and wR2 values were 0.1731 (I > 2σ (I)). The final R1 values were 0.0756 and wR(F2) values were 0.1749. The goodness of fit on F2 was 1.034. Flack parameter = 0.07(7). Crystal Data for Dihydrocarneamide A (9). C26H31N3O3, M = 433.54, a = 21.9246(7) Å, b = 15.7608(5) Å, c = 6.6049(2) Å, α = 90°, β = 90°, γ = 90°, V = 2282.32(13) Å3, T = 100.00(10) K, space group P212121, Z = 4, μ (Cu Kα) = 0.664 mm−1, Dcalc = 1.262 g/cm3, 14262 reflections measured, and 4495 independent reflections (Rint = 0.0403, Rσ = 0.0371). The final R1 values were 0.0530 and wR2 values were 0.1627 (I > 2σ (I)). The final R1 values were 0.0556 and wR(F2) values were 0.1646. The goodness of fit on F2 was 1.033. Flack parameter = 0.06(15). Quantum Mechanical Calculation. ECD Calculations of Compound 1. The preliminary conformational distribution search was performed by Spartan 10 software (Wave function, Inc., Irvine, CA, USA) using the MMFF94S force field. The corresponding minimum geometries were further fully optimized with the Gaussian 03 (Gaussian, Wallingford, CT, USA) program package at the B3LYP/6-31G (d) computational level as frequency calculations. The obtained stable conformers were submitted to CD calculation by the TDDFT B3LYP/6-311+G(d) method. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and OriginPro 8.5 (OriginLab, Ltd., Northampton, MA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with σ = 0.19 eV. 8490

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The Journal of Organic Chemistry ligands were extracted from the active site and the designed ligands were modeled. All hydrogen atoms were added to define the correct ionization and tautomeric states, and the carboxylate, phosphonate, and sulfonate groups were considered in their charged form. In the docking calculation, the default FlexX scoring function was used for exhaustive searching, solid body optimizing, and interaction scoring. The pose with the most favorable score remained.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b01087. Spectroscopic data including NMR, HRESITOFMS, UV, and IR spectra of 1−8 (PDF) X-ray crystallographic data of 2 (CIF) X-ray crystallographic data of 4 (CIF) X-ray crystallographic data of 8 (CIF) X-ray crystallographic data of 9 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Junjun Liu: 0000-0001-9953-8633 Yongbo Xue: 0000-0001-9133-6439 Yonghui Zhang: 0000-0002-7222-2142 Author Contributions §

H.L., W.S., and M.D. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Program for the Changjiang Scholars of Ministry of Education of the People’s Republic of China (no. T2016088); the National Science Fund for Distinguished Young Scholars (no. 8172500151); the National Natural Science Foundation of China (nos. 81573316, 31770379, and 31670354); the Academic Frontier Youth Team of HUST; the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST); and Innovative Research Groups of the National Natural Science Foundation of China (no. 81721005). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in testing of ECD, UV, and IR spectra.

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REFERENCES

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