PPAR-γ Agonistic Metabolites from the Ascidian Herdmania momus

Nov 28, 2012 - The peroxisome proliferator-activated receptor (PPAR)-γ agonistic activities of the compounds were investigated due to the similarity ...
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PPAR‑γ Agonistic Metabolites from the Ascidian Herdmania momus Jian Lin Li,† Bin Xiao,† Minhi Park,† Eun Sook Yoo,‡ Sook Shin,§ Jongki Hong,⊥ Hae Young Chung,† Hyung Sik Kim,† and Jee H. Jung*,† †

College of Pharmacy, Pusan National University, Busan 609-735, Korea School of Medicine, Jeju National University, Jeju 690-756, Korea § Department of Life Science, Sahmyook University, Seoul 139-742, Korea ⊥ College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea ‡

S Supporting Information *

ABSTRACT: Seven new amino acid derivatives (1−4 and 6− 8) were isolated from MeOH extracts of the marine ascidian Herdmania momus. Planar structures were established on the basis of NMR, IR, and MS spectroscopic analyses. Absolute configurations of these compounds were derived from specific rotation and CD analysis. The peroxisome proliferatoractivated receptor (PPAR)-γ agonistic activities of the compounds were investigated due to the similarity of the structural motif to that of the antidiabetic drug rosiglitazone. Analogues with indoleglyoxyl moieties (5, 6, and 8) showed significant PPAR-γ activation in Ac2F rat liver cells.

T

and purification to yield a series of amino acid-derived metabolites (1−9). Herdmanine E (1) was obtained as a white powder. The (+)-FABMS spectrum of 1 showed isotopic [M + H]+ ion peaks at m/z 534/532 (with peak intensities in a 1:1 ratio), which is characteristic of monobrominated compounds. The molecular formula of 1 was deduced as C22H2279BrN5O6 on the basis of (+)-HRFABMS and NMR data, indicating 14 degrees of unsaturation. Signals due to all 22 carbons were visible in the 13 C NMR spectrum, and HSQC analysis allowed the appropriate assignment of 14 carbon-bound protons (seven aromatic, one methine, and three methylenes). The eight remaining protons observed in the 1H NMR spectrum were considered exchangeable. The 1H NMR spectrum of compound 1 revealed resonances of an AA′XX′ spin system at δH 7.96 (H11/13) and δH 7.34 (H-10/14), attributable to a p-disubstituted phenyl moiety (Table 1). An arginine moiety was suggested by comparison of the NMR data with previously isolated herdmanines A−C.3 The α-proton (H-17) of the arginine moiety showed an HMBC correlation to the amide carbonyl carbon C-15, which in turn showed correlations with the aromatic protons H-11/13. 1H NMR analyses and the correlations observed in the HMBC spectrum (Figure 1) indicated that the molecule contained a 3,5,6-trisubstituted indole moiety. Comparison of the spectroscopic data with those of 6-bromo-5-hydroxyindole3−5 and the typical chemical shift of the quaternary carbons, δC 149.6 (C-5) and 106.9 (C-6), indicated that those positions were substituted with a hydroxy group and bromine atom, respectively. An HMBC correlation

ypical secondary metabolites of ascidians include cyclic peptides, pyridoacridine, β-carboline, and polysulfide alkaloids, which are derived from amino acids.1,2 In many cases, these natural products exhibit biological activities with either human therapeutic or ecological relevance.2 In our previous study investigating the bioactive compounds from the solitary tunicate Herdmania momus, a series of amino acid derivatives (herdmanines A−D) were isolated, and herdmanine D exhibited a moderate suppressive effect on the production of nitric oxide (NO), with IC50 values of 9 μM in LPS-activated murine macrophage cells (RAW 264.7).3 In our continuing search for potential anti-inflammatory molecules from the same tunicate, a series of amino acid-derived metabolites (1−9) were isolated. Planar structures were established on the basis of NMR and MS spectroscopic analyses. Motivated by the similarity of the structural motifs of these novel compounds to that of the antidiabetic drug rosiglitazone, we performed docking simulations of these compounds to the peroxisome proliferator-activated receptor (PPAR)-γ. Congeners with indoleglyoxyl moieties (5, 6, and 8) displayed rosiglitazonelike binding to PPAR-γ. In a subsequent luciferase assay using rat liver cells (Ac2F cells), peroxisome proliferator-activated receptor response element (PPRE) gene expression was confirmed. Here, we describe the structure elucidation and biological evaluation of these compounds.

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RESULTS AND DISCUSSION Combined MeOH and CH2Cl2 extracts of H. momus were subjected to solvent partition between CH2Cl2 and H2O. The H2O layer was partitioned with n-BuOH, and the n-BuOHsoluble fraction was subjected to chromatographic separation © 2012 American Chemical Society and American Society of Pharmacognosy

Received: June 8, 2012 Published: November 28, 2012 2082

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Chart 1

Table 1. 1H (500 MHz) and 13C (100 MHz) NMR Data for 1,a 6,b and 8b herdmanine E (1) position

δC

1 2 3 3a 4

134.1 105.3 126.8 105.8

5

149.6

6 7

107.0 115.9

7a 8 9 10

131.9 167.3 153.9 122.1

11

128.5

12 13

131.6 128.5

14

122.1

15 16 17 18

163.2

19 20 21 22 23 24 25

24.9 40.2

55.6 30.1

δH (J in Hz) 8.11, s

7.62, s

herdmanine I (6) δC 137.1 111.8 118.4 121.3 111.9

7.63, s

154.1 97.2

δH (J in Hz) 8.56, s

7.96, d (8.5) 6.73, dd (8.5, 1.5) 6.87, d (1.5)

136.9 181.6 163.2 7.34, d (9.0) 7.69, d (9.0) 7.69, d (9.0) 7.34, d (9.0)

m m m m m

δC 138.2 112.9 119.6 122.5 98.4 155.3 113.2

37.9

δH (J in Hz) 11.90, brs 8.53, s

7.93, d (8.5) 6.70, dd (8.5, 1.5) 6.83, d (1.5)

138.1 181.7 163.5 8.62, t (6.5)

28.3 22.2

3.18, dt (6.5, 6.5) 1.48, m 1.36, m

30.4

1.72, m

54.8 169.4 4.51, 2.00, 1.86, 1.70, 3.25,

herdmanine K (8)

1.62, m 3.12, m

53.2

Figure 1. Selected COSY and HMBC data for compounds 1, 7, and 8.

was observed between H-2 and C-8 (δC 167.3). On the basis of the molecular formula of compound 1, we concluded that the (p-oxybenzoyl)arginine and 6-bromo-5-hydroxy-3-carboxyindole moieties were joined via an ester linkage. The arginine moiety is proposed to be in the D-form by comparison of the specific rotation of compound 1 ([α]23D −3.6) with those of the reported analogues.3,6,7 The related herdmanines A−C isolated from this same ascidian also incorporated D-arginine, as determined by Marfey’s analysis.3 Herdmanine F (2) was isolated as a yellow gum. The (+)-FABMS spectrum showed isotopic [M + H]+ ion peaks at m/z 375/373 (with peak intensities in a 1:1 ratio). The molecular formula of compound 2 was deduced as C13H1779BrN4O4 on the basis of the (+)-HRFABMS and NMR data. The 1H NMR spectrum of 2 revealed resonances of an AMX spin system at δH 8.00 (H-3, d, J = 2.0 Hz), 7.69 (H-5, dd, J = 8.5, 2.0 Hz), and 6.89 (H-6, d, J = 8.5 Hz), attributable to a 1,2,4-trisubstituted phenyl moiety. Correlations from H-3 and H-5 of the phenyl moiety to the carbonyl carbon C-7 (δC 167.6) were observed in the HMBC spectrum. The chemical shift of C-1 (δC 158.8) was indicative of oxygenation. An alternative structure, with the switched substitution of hydroxy and bromine groups, was excluded because it would display quite different 13C and 1H NMR data. An arginine moiety was suggested by comparison of the NMR data with those of

8.70, d (7.5) 4.41, m

29.1 117.5

3.00, m

135.2

7.56, s

134.0 173.0

6.79, s

157.4

174.8

a

Spectra were recorded in CD3OD. bSpectra were recorded in d6DMSO.

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Herdmanine J (7) was obtained as a white powder. The (+)and (−)-FABMS spectra showed [M + H]+ and [M + H]− ion peaks at m/z 304 and 302, respectively. The (+)-HRFABMS data supported a molecular formula of C16H21N3O3. 1H and 13 C NMR analyses and the correlations observed in the COSY spectrum suggested that the molecule contained lysine and 3oxoindole moieties (Figure 1). An additional methyl group (δH/C 3.65/33.0) was observed, and the methyl protons showed correlations to C-2 and C-7a in the HMBC spectrum, indicating that compound 7 contained an N-methyl-3oxoindole moiety. On the basis of the molecular formula, we suspected that these two units were joined via an amide linkage. This was confirmed by the HMBC correlation from the H-9 to C-8. The positive specific rotation of compound 7 ([α]23D +11, MeOH) suggested that the lysine moiety was in its common Lform. This was corroborated by hydrolysis of compound 7 and CD analysis [CD (+) (c = 1.2 × 10−4 M, HCl 2 N), λmax = 200 nm, Δε = +0.38]. Herdmanine K (8) was obtained as a yellow powder, and the (+)-HRFABMS data supported the molecular formula C16H21N3O3. NMR data (Table 1) suggested that the molecule contained a 6-hydroxyindole-3-glyoxylyl moiety, similar to compounds 5 and 6. The rest of the signals were attributable to a histidine moiety. The α-proton at δH 4.41 (H-11) was coupled to methylene protons at δH 3.00 (H-12). These methylene protons showed HMBC correlations (Figure 1) to carbon resonances at δC 134.0 (C-13) and 117.5 (C-17). Both of the aromatic proton signals at δH 6.79 (H-17) and 8.53 (H15) showed HMBC correlations to a quaternary carbon at 134.0 (C-13), indicating the presence of an imidazole ring. The negative specific rotation ([α]23D −38, MeOH) of compound 8 suggested that the histidine moiety was in its rare D-form.6,12 This was further confirmed by hydrolysis and Marfey’s method; the acid hydrolysate was allowed to react with Marfey’s reagent to give His-FDAA. RP HPLC analysis showed that it was identical to the compound derived from authentic D-histidine. Herdmanine L (9) was isolated as a white powder, and the (+)-HRFABMS data supported the molecular formula C16H15NO5. The 1H NMR and 13C NMR data of compound 9 showed two sets of AA′XX′ spin systems, suggesting the presence of two p-disubstituted phenyl moieties: δH 7.91 (H-3/ 5) and 6.90 (H-2/6); δH 7.13 (H-9/13) and 7.33 (H-10/12). HSQC and DEPT analyses allowed the assignment of remaining carbon-bound protons (one methine and one methylene), and tyrosine and p-hydroxybenzoyl moieties were suggested. Considering the molecular formula, these two moieties are likely linked via an ester or amide linkage. The signal of the oxygenated phenylic carbon (C-8, δC 150.2) was significantly shifted upfield compared to C-1 (δC 163.6), indicating an ester linkage. The negative specific rotation of compound 9 suggested that the tyrosine moiety was in its common L-form. This compound had previously been synthesized for the evaluation of antioxidant activity, and it exhibited good antioxidant activity in both 1,1-diphenyl-2picrylhydrazine (DPPH) and nitro blue tetrizolium (NBT) assays, with IC50 values of 2.31 and 1.78 μg/mL, respectively.13 Compounds 5, 6, and 8 include the uncommon indoleglyoxylyl moiety. Previous examples of indoleglyoxylate derivatives are polyandrocarpamides A−C from the ascidian Polyandrocarpa sp.,9 didemnidines A and B from the New Zealand ascidian Didemnum sp.,10 leptoclinidamines A and B from the Australian ascidian Leptoclinides durus,7 6-bromo-5-hydroxyindolyl-3-glyoxylate esters from the Far Eastern ascidian Syncarpa

compound 1. On the basis of the molecular formula, we suspected that these two units were joined via an amide linkage. This was confirmed by the HMBC correlation from the αproton (H-9) of arginine to C-7. Comparison of the specific rotation of 2 ([α]23D −15.0) with that of previously isolated analogues suggested a less common D-form of arginine.3,6,7 Herdmanine G (3) was isolated as a white powder. The molecular formula of compound 3 was deduced as C14H20N4O5 from (+)-HRFABMS and NMR data. The 1H NMR spectrum of compound 3 revealed resonances of an AMX spin system at δH 7.43 (H-3, d, J = 2.0 Hz), 7.36 (H-5, dd, J = 8.0, 2.0 Hz), and 6.80 (H-6, d, J = 8.0 Hz), attributable to a 1,2,4trisubstituted phenyl moiety. Comparison of the 1H NMR spectrum of compound 3 with those of compound 2 revealed an additional methoxy group (δH/C 3.88/56.4). The HMBC correlation of methoxy protons to C-2 (δC 148.2) suggested its attachment to C-2. This was further supported by the NOESY correlation between the methoxy protons and H-3. The chemical shift of C-1 (δ C 150.3) was indicative of hydroxylation. An HMBC correlation from the α-proton (H9) of arginine to C-7 was observed, revealing that 3 is an analogue of 2 in which the C-2 bromine is replaced by a methoxy group. Comparison of the specific rotation of compound 3 ([α]23D −8.7) with those of the aforementioned congeners suggested it contained the D-form of arginine.3,6,7 Herdmanine H (4) was isolated as a white powder, and the (+)-HRFABMS data of this compound indicated the molecular formula C13H18N4O4. Comparison of the 1H NMR data of compound 4 with those of compounds 2 and 3 suggested that the 1,2,4-trisubstituted phenyl ring found in compounds 2 and 3 was replaced with a p-hydroxyphenyl moiety. The spectroscopic properties of compound 4 were identical to those previously reported for N-(p-hydroxybenzoyl)-L-arginine ([α]20D +19.5).6 The only difference was the specific optical rotation. Compound 4 exhibited the opposite specific rotation ([α]23D −12.7), suggesting the less-common D-arginine moiety. Compound 5 was isolated as yellow needles. A pseudomolecular ion in the (+)-HRFABMS spectrum at m/z 362.1581 allowed the molecular formula C16H19N5O5 to be assigned to compound 5. The spectroscopic properties of compound 5 were identical to those reported for (+)-leptoclinidamine B ([α]23D +26.0, TFA salt) and (−)-leptoclinidamine B ([α]20D −20.6, TFA salt).7,8 The negative specific rotation value of compound 5 ([α]23D −103) suggested that it is (−)-leptoclinidamine B. Herdmanine I (6) was obtained as a yellow powder, and the (+)-HRFABMS data matched well with the expected molecular formula, C16H19N3O5. 1H and 13C NMR (Table 1) analyses and the correlations observed in the HMBC spectrum suggested that the molecule also contained a 6-hydroxyindole-3-glyoxylyl moiety, and the presence of a 6-hydroxyindolyl-3-glyoxyl moiety was further confirmed by comparison of the NMR data with those of reported analogues.7−11 A COSY spectrum measured in DMSO-d6 showed a contiguous spin system from 10-NH to 15-CH, suggesting a lysine moiety. A correlation between NH-10 and C-9 was also observed. The negative specific rotation of compound 6 ([α]23D −4.7) suggested that the lysine moiety was in its D-form. This was corroborated by hydrolysis of compound 6 and CD analysis6 of the liberated lysine [CD (−) (c = 2.0 × 10−5 M, HCl 2 N), λmax = 196 nm, Δε = −0.13], which displayed a CD spectrum opposite of that of authentic L-lysine [CD (+) (c = 4.2 × 10−5 M, HCl 2 N), λmax = 199 nm, Δε = +0.18]. 2084

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oviformis,5 and coscinamides A−C from the sponge Coscinoderma sp.11 Biological Activity. PPAR-γ has been the focus of intense research during the past decade, as ligands for this receptor have emerged as potent agents in the treatment of type II diabetes,14 inflammatory disorders of the central nervous system,15 cardiovascular system diseases,16 and tissue injury associated with ischemia and reperfusion.17 PPAR-γ can be activated by a number of ligands, including polyunsaturated fatty acids (docosahexaenoic acid and linoleic acid), prostaglandin (15-deoxy-Δ12,14-PGJ2), lipoxygenase products, oxidized linoleic acid products, oxidized low-density lipoprotein, flavonoids, and synthetic thiazolidinedione derivatives.18−21 Rosiglitazone is a synthetic thiazolidinedione derivative that has been extensively used for the treatment of diabetes. The structural motif of rosiglitazone is composed of an H-bondforming headgroup, a linker chain, and a hydrophobic tail (Figure 2). 22 Our new ascidian metabolites resemble

compound 8/PPAR-γ complex, the 6-hydroxy group of the indoleglyoxylyl headgroup participated in H-bonding with Tyr473 and His323; these H-bonds are observed for most PPAR-γ ligands and are essential for PPAR-γ activation (Figure 3). Other indoleglyoxyl derivatives (compounds 5 and 6) also showed significant binding affinity. The binding of agonists to PPAR-γ results in expression of mRNA encoded by PPAR-γ target genes. This process is known as transactivation, and cell-based assays have been developed that monitor this functional activity. Transactivation assays use cells that have been transfected with a vector expressing the receptor as well as a second vector containing PPRE and a reporter gene, which encodes an enzyme such as firefly luciferase.23 Activation of the receptor by agonists leads to induction of reporter gene expression, which can be conveniently monitored. The PPAR-γ agonistic activities of indoleglyoxylyl derivatives 5, 6, and 8 were evaluated in a luciferase assay using rat liver Ac2F cells. Ac2F cells were cotransfected with a reporter PPAR-γ expression vector and PPRE-driven luciferase reporter gene construct. As shown in Figure 4, the indoleglyoxylyl derivatives, especially compound

Figure 2. Graphical illustration of the key pharmacophore of rosiglitazone.22 The rosiglitazone skeleton is divided into the head, linker, and tail by dashed line boxes. Key helices and sheets (light blue), amino acid residues (purple), hydrogen bonds (gray dashed line), and water molecule (green) of PPAR-γ LBD are shown.

Figure 4. PPAR-γ transactivation activity of compounds 5, 6, and 8 in rat liver cells (Ac2F). Ac2F cells (1 × 105) were transiently transfected with pcDNA or PPRE with the pFlag-PPAR-γ1 vector. NRC (no receptor control, without transfection of plasmid), NC (negative control, transfected with plasmid containing PPRE and pcDNA), and Rosi (rosiglitazone) were used as controls to monitor luciferase reporter activity. Luciferase activity was quantified after a 6 h incubation. The results are the mean ± SE of triplicate cultures. RLU; relative light unit.

rosiglitazone in terms of their structural motifs. Most PPAR-γ agonists show similar H-bonding with key amino acid residues (Tyr473, His449, His323, Ser289, and Glu286), which are crucial for the activation of PPAR-γ. In our docking simulation using AutoDock Vina, compound 8 formed strong H-bonds with key amino acids (Tyr473 and His323) and exhibited a strong binding affinity (−7.8 kcal/mol) comparable to that of rosiglitazone (−8.2 kcal/mol). In the docking model of the

8, exhibited strong PPAR-γ activation at 1 and 10 μg/mL concentrations, with higher potency than rosiglitazone. Further refinement of structure−activity relationships and optimization

Figure 3. Binding model of rosiglitazone and herdmanine K (8) with PPAR-γ. H-bonds are shown as yellow dotted lines. (A) Rosiglitazone interacts with key amino acid residues (Tyr473, His449, His323, Ser289, and Glu286) in the PPAR-γ binding pocket (−8.2 kcal/mol).22 (B) Binding of 8 with key amino acid residues (Tyr473 and His323; −7.8 kcal/mol). 2085

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Table 2. 1H (500 MHz, CD3OD) and 13C (100 MHz) NMR Data for 2, 7, and 9 herdmanine F (2) position

δC

1 2 3 3a 4 5 6 7 7a 8 9 10

158.8 110.8 133.7

11 12

26.2 42.1

127.6 128.9 116.7 167.6

55.9 31.2

δH (J in Hz)

8.00, d (2.0)

7.69, dd (8.5, 2.0) 6.89, d (8.5)

4.45, 1.96, 1.83, 1.66, 3.24, 3.18,

m m m quintet (7.0) dt (14.0, 7.0) dt (14.0, 7.0)

13

a

herdmanine J (7) δ Ca 123.8 108.4 124.0 118.2 116.8 121.5 111.3 137.0 165.8 38.8

30.3 158.6

54.3

15 −COOH N−CH3

178.8

173.5 33.0

δC

7.18, s

163.6 116.4 132.8

7.53, 7.02, 7.10, 7.35,

119.8 132.8 116.4 164.8

3.21, 3.16, 1.50, 1.39,

28.7 22.4

14

herdmanine L (9)

δH (J in Hz)

dd (8.0, 2.0) ddd (8.0, 7.0, 2.0) ddd (8.0, 7.0, 1.5) dd (7.0, 1.5)

dt (14.0, 7.0) dt (14.0, 7.0) quintet (7.0) m

1.82, m 1.75, m 3.63, m

δH (J in Hz) 6.90, d (8.5) 7.95, d (8.5)

7.95, d (8.5) 6.90, d (8.5)

150.2 122.4 131.1

7.13, d (8.5) 7.33, d (8.5)

135.7 131.1

7.33, d (8.5)

122.4

7.13, d (8.5)

37.5 56.2 170.0

3.14, dd (14.0, 4.0) 2.90, dd (14.0, 8.0) 3.43, dd (8.0, 4.0)

3.65, s

Chemical shifts obtained from gHSQC and gHMBC spectra. FAB-CID-MS/MS m/z 417 (100), 398 (1.2), 356 (0.7), 294 (2.0), 241 (3.4), 121 (1.8); (+)-FABMS m/z 375/373 [M + H]+; (+)-HRFABMS m/z 373.0508 (calcd for C13H1879BrN4O4, 373.0511). Herdmanine G (3): white powder; [α]23D −8.7 (c 0.09, MeOH); 1H NMR (CD3OD, 500 MHz) δ 7.43 (1H, d, J = 2.0 Hz, H-3), 7.36 (1H, dd, J = 8.0, 2.0 Hz, H-5), 6.80 (1H, d, J = 8.0 Hz, H-6), 4.58 (1H, m, H-9), 3.88 (3H, s, -OCH3), 3.25 (1H, dt, J = 13.0, 6.5 Hz, H-12a), 3.19 (1H, dt, J = 13.0, 6.5 Hz, H-12a), 1.98 (1H, m, H-10a), 1.80 (1H, m, H-10b), 1.68 (2H, m, H-11); 13C NMR (CD3OD, 100 MHz) δ 177.0 (C-17), 168.3 (C-7), 157.8 (C-14), 150.3 (C-1), 148.2 (C-2), 125.2 (C-6), 125.0 (C-4), 121.7 (C-5), 111.0 (C-3), 56.4 (−OCH3), 55.4 (C-9), 41.2 (C-12), 30.8 (C-10), 25.7 (C-11); (+)-FABMS m/z 325 [M + H]+; (+)-HRFABMS m/z 325.1525 (calcd for C14H21N4O5, 325.1512). Herdmanine H (4): white powder; [α]23D −12.7 (c 0.30, MeOH); 1 H NMR (CD3OD, 500 MHz) δ 7.73 (2H, d, J = 8.5 Hz, H-3, H-5), 6.79 (2H, d, J = 8.5 Hz, H-2, H-6), 4.47 (1H, m, H-9), 3.24 (1H, dt, J = 13.0, 6.5 Hz, H-12a), 3.19 (1H, dt, J = 13.0, 6.5 Hz, H-12a), 1.97 (1H, m, H-10a), 1.83 (1H, m, H-10b), 1.68 (2H, m, H-11); 1H NMR (D2O, 500 MHz) δ 7.44 (2H, dd, J = 8.0, 1.0 Hz, H-3, H-5), 6.65 (2H, dd, J = 8.0, 1.0 Hz, H-2, H-6), 4.08 (1H, m, H-9), 2.93 (2H, m, H-12), 1.67 (1H, m, H-10a), 1.56 (1H, m, H-10b), 1.40 (2H, quintet, J = 7.5 Hz, H-11); 13C NMR (CD3OD, 100 MHz) δ 176.4 (C-17), 168.2 (C7), 161.0 (C-1), 157.6 (C-14), 129.0 (C-3, C-5), 125.2 (C-4), 114.8 (C-2, C-6), 54.8 (C-9), 41.0 (C-12), 30.0 (C-10), 25.7 (C-11); (+)-FABMS m/z 295 [M + H]+; (+)-HRFABMS m/z 295.1401 (calcd for C13H19N4O4, 295.1406). (−)-Leptoclinidamine B (5): yellow needles; [α]23D −103 (c 0.20, MeOH); (+)-FABMS m/z 415 [M + H]+; (+)-HRFABMS m/z 415.1595 (calcd for C20H23N4O6, 415.1618); FAB-CID-MS/MS m/z 417 (100), 398 (1.2), 356 (0.7), 294 (2.0), 241 (3.4), 121 (1.8); (+)-FABMS m/z 362 [M + H]+; (+)-HRFABMS m/z 362.1447 (calcd for C16H20N5O5, 362.1464). Herdmanine I (6): yellow powder; [α]23D −4.7 (c 0.01, MeOH); 1H and 13C NMR (see Table 1); (+)-FABMS m/z 334 [M + H]+; (+)-HRFABMS m/z 334.1414 (calcd for C16H20N3O5, 334.1403).

of in vitro and in vivo PPAR-γ agonistic activity of these indoleglyoxyl derivatives would yield valuable data.

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EXPERIMENTAL SECTION

General Experimental Procedures. General instrumentation was the same as described in an earlier study.3 CD spectra were recorded using a JASCO J-715 spectroplarimeter (sensitivity 50 mdeg, resolution 0.2 nm). HPLC was performed using a YMC-packed C-8 column (250 × 10 mm, 5 μm, 12 nm), a YMC-packed J’sphere ODSH80 column (250 × 4.6 mm, 4 μm, 80 Å), and a Shodex-packed NH5E column using Shodex RI-71 and JASCO UV-975 detectors. Animal Material. H. momus was collected and identified as previously described.3 Extraction and Isolation. Initial extraction and isolation steps have been reported.3 Fraction 10 (37.3 mg), which gave interesting signals in the 1H NMR spectrum, was subjected to reversed-phase HPLC (eluent: 50% MeOH) to afford compound 1 (0.9 mg). Fraction 8 (136.9 mg), which gave 1H NMR signals similar to those given by fraction 10, was first purified on a Shodex-packed NH-5E column using 85% MeOH as the eluent and further purified on a YMC-packed J’sphere ODS-H80 column using 35% MeOH as the eluent to afford compound 9 (8.0 mg). Fraction 5 (120.3 mg) was first purified on an NH-5E column using 85% MeOH as the eluent and further purified on a YMC-packed C-8 column using 15% MeOH as the eluent to afford compounds 2 (1.7 mg), 5 (16.0 mg), 6 (5.0 mg), and 7 (0.7 mg). Fractions 3 (136.9 mg) and 4 (136.9 mg), which gave 1H NMR signals similar to those given by fraction 5, were first purified on a Shodex-packed NH-5E column using 90% MeOH as the eluent and further purified on a YMC-packed C-8 column using 15% MeOH as the eluent to afford compounds 3 (0.9 mg) and 4 (3.0 mg), and 8 (4.0 mg), respectively. Herdmanine E (1): white powder; [α]23D −3.6 (c 0.09, MeOH); 1H and 13C NMR (see Table 1); (+)-FABMS m/z 534/532 [M + H]+; (+)-HRFABMS m/z 532.0826 (calcd for C22H2379BrN5O6, 532.0832). Herdmanine F (2): yellow gum; [α]23D −15.0 (c 0.15, MeOH); 1H and 13C NMR (see Table 2); (+)-FABMS m/z 415 [M + H]+; (+)-HRFABMS m/z 415.1595 (calcd for C20H23N4O6, 415.1618); 2086

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Journal of Natural Products

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Herdmanine J (7): white powder; [α]23D +11 (c 0.07, MeOH); 1H and 13C NMR (see Table 2); (+)-FABMS m/z 304 [M + H]+; (−)-FABMS m/z 302 [M − H]+; (+)-HRFABMS m/z 304.1637 (calcd for C16H22N3O3, 304.1661). Herdmanine K (8): yellow powder; [α]23D −38 (c 0.04, MeOH); 1H and 13C NMR (see Table 1); (+)-FABMS m/z 343 [M + H]+; (+)-HRFABMS m/z 343.1050 (calcd for C16H22N3O3, 343.1042). Herdmanine L (9): white powder; [α]23D −8.4 (c 0.10, MeOH); 1H and 13C NMR (see Table 2); (+)-FABMS m/z 302 [M + H]+; (+)-HRFABMS m/z 302.1026 (calcd for C16H16NO5, 302.1028). Acid Hydrolysis and CD Analysis of Compounds 6 and 7. Compound 6 (0.5 mg) and 7 (0.5 mg) was dissolved in 6 N HCl (1 mL) and hydrolyzed at 110 °C for 24 h. The acid hydrolysate was partitioned between H2O and CH2Cl2. The H2O layer was then dried in vacuo to give the crude residue, and TLC analysis showed the same Rf value as standard lysine. The residue was then dissolved in 2 N HCl for further CD analysis. Acid Hydrolysis and Marfey Analysis of 8. Compound 8 (0.5 mg) was dissolved in 6 N HCl (1 mL) and hydrolyzed at 110 °C for 24 h.The acid hydrolysate was dried under N2. To the acid hydrolysate were added 0.1% FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) solution in acetone (100 μL) and 1 M NaHCO3 (20 μL), and the mixture was heated at 40 °C for 2 h. After cooling to room temperature, the reaction mixture was neutralized with 2 M HCl (10 μL) and diluted for HPLC analysis using TFA mobile phase (0.1%) in both A and B, with 5% MeOH in A and 90% MeOH in B (linear gradients started with 100% A and finished with 100% B in 60 min, flow rate 0.5 mL/min; UV, 340 nm). (D-His-FDAA and L-His-FDAA showed tR values of 14.13 and 12.42 min, respectively.) Evaluation of PPAR-γ Agonistic Activity. Cell Culture. Rat liver Ac2F cells were obtained from American Type Culture Collection (ATCC). Ac2F cells were grown in Dulbecco’s modified Eagle’s medium (Nissui) containing 2 mM L-glutamine, 100 mg/mL streptomycin, 2.5 mg/L amphotericin B, and 10% heat-inactivated fetal bovine serum. Cells were maintained at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. Plasmids, Transfections, and Luciferase Assays. A 3× AOX-TKluciferase reporter plasmid containing three copies of the PPAR response element present in the acyl CoA oxidase promoter was kindly provided by Dr. Christopher K. Glass (University of California at San Diego). The pcDNA3 expression vector and full-length human PPARγ expression vector (pFlag-PPAR-γ) were provided by Dr. Chatterjee (University of Cambridge, Addenbrooke’s Hospital). Lipofectamine 2000 transfection reagent was obtained from Invitrogen Co. OptiMEM was obtained from Gibco. For luciferase assays, 0.02 mg of plasmids was transfected into 1 × 105 Ac2F cells in a 48-well plate with proper combinations of effector plasmids, 3× AOX-TK-luciferase reporter plasmid, pcDNA3, and pFlag-PPAR-γ, using Lipofectamine 2000. After transfection for 24 h, the conditioned medium was removed, and cells were treated with test compounds at different concentrations (1 and 10 μg/mL), after the growth medium was replaced with serum-free media for all wells. After an additional incubation for 6 h, cells were washed with PBS, and the luciferase assay was carried out using the Steady-Glo Luciferase Assay System (Promega). Resulting reporter gene activity was measured in a GloMax-Multi Microplate Multimode Reader (Promega Corporation). Molecular Modeling. Docking simulations were performed using AutoDock Vina 1.1.2 software. The default settings and scoring functions of AutoDock Vina were applied. For ligand preparation, Chem3D Ultra 8.0 software was used to convert the 2D structures of the candidates into 3D structural data. Protein coordinates were downloaded from the Protein Data Bank, accession code 2PRG. Chain A was prepared for docking using the molecular modeling software package UCSF Chimera 1.5.3 by removing chain B. A four-stage protocol was set up for energy minimizations of the protein-inhibition complex. Polar hydrogens were added and grid box parameters were set using MGL Tools 1.5.4. After the optimization, the protein−ligand complexes were visualized and studied using PyMol v 1.5.

Article

ASSOCIATED CONTENT

S Supporting Information *

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H, 13C, and 2D NMR spectra for compounds 1−9. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 82-51-510-2803. Fax: 82-51-513-6754. E-mail: jhjung@ pusan.ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Research Foundation of Korea (grant nos. 20090400000 and 20090083538). J.L.Li acknowledges the financial support from the 2012 Post-Doc. Development program of Pusan National University.

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REFERENCES

(1) Davidson, B. S. Chem. Rev. 1993, 93, 1771−1991. (2) Wang, W.; Namikoshi, M. Heterocycles 2007, 74, 53−88. (3) Li, J. L.; Han, S. C.; Yoo, E. S.; Shin, S.; Hong, J.; Cui, Z.; Li, H.; Jung, J. H. J. Nat. Prod. 2011, 74, 1792−1797. (4) Ochi, M.; Kataoka, K.; Ariki, S.; Iwatsuki, C.; Kodama, M.; Fukuyama, Y. J. Nat. Prod. 1998, 61, 1043−1045. (5) Santalova, E. A.; Denisenko, V. A.; Berdyshev, D. V.; Aminin, D. L.; Sanamyan, K. E. Nat. Prod. Commun. 2008, 3, 1617−1620. (6) García, A.; Vázquez, M. J.; Quiñoá, E.; Riguera, R.; Debitus, C. J. Nat. Prod. 1996, 59, 782−785. (7) Carroll, A. R.; Avery, V. M. J. Nat. Prod. 2009, 72, 696−699. (8) Yamazaki, H.; Wewengkang, D. S.; Nishikawa, T.; Rotinsulu, H.; Mangindaan, R. E. P.; Namikoshi, M. Mar. Drugs 2012, 10, 349−357. (9) Lindquist, N.; Fenical, W. Tetrahedron Lett. 1990, 31, 2521− 2524. (10) Finlayson, R.; Pearce, A. N.; Page, M. J.; Kaiser, M.; BourguetKondracki, M. L.; Harper, J. L.; Webb, V. L.; Copp, B. R. J. Nat. Prod. 2011, 74, 888−892. (11) Bokesch, H. R.; Pannell, L. K.; McKee, T. C.; Boyd, M. R. Tetrahedron Lett. 2000, 41, 6305−6308. (12) Zaharenko, A. J.; Picolo, G.; Ferreira, W. A.; Murakami, T.; Kazuma, K.; Hashimoto, M.; Cury, Y.; Freitas, J. C.; Satake, M.; Konno, K. J. Nat. Prod. 2011, 74, 378−382. (13) Venkateswara Rao, B.; Ramanjaneyulu, K.; Rambabu, T.; Sundari Devi, Ch. B. T. Int. J. Pharm. Biol. Sci. 2011, 2 (4), 39−44. (14) Rangwala, S. M.; Lazar, M. A. Trends Pharmacol. Sci. 2004, 25, 331−336. (15) Sundararajan, S.; Jiang, Q.; Heneka, M.; Landreth, G. Neurochem. Int. 2006, 49, 136−144. (16) Robinson, E.; Grieve, D. J. Pharmacol. Ther. 2009, 122, 246− 263. (17) Liliane, M.; Walter, W. J. Clin. Invest. 2006, 116, 598−606. (18) Nosjean, O.; Boutin, J. A. Cellular Signal 2002, 14, 573−583. (19) Rosen, E. D.; Spiegelman, B. M. J. Biol. Chem. 2001, 41, 37731− 37734. (20) Nagy, L.; Tonzonoz, P.; Alvarez, J. G. A.; Chen, H.; Evans, R. M. Cell 1998, 2, 229−240. (21) Lee, J.-Y.; Kim, J.-K.; Cho, M.-C.; Shin, S.; Yoon, D.-Y.; Heo, Y. S.; Kim., Y. J. Nat. Prod. 2010, 73, 1261−1265. (22) Xiao, B.; Yin, J.; Park, M.; Liu, J.; Li, J. L.; Kim, E. L.; Hong, J.; Chung, H. Y.; Jung, J. H. Bioorg. Med. Chem. 2012, 20, 4954−4961. (23) McDonnell, D. P.; Vegeto, E.; Gleeson, M. A. G. Nat. BioTechnol. 1993, 11, 1256−1261.

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