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Duo-Qing Xue, Hai-Li Liu, Si-Han Chen, Ernesto Mollo, Margherita Gavagnin, Jia Li, Xu-Wen Li, Yue-Wei Guo. 5-Alkylpyrrole-2-carboxaldehyde derivatives from the Chinese sponge Mycale lissochela and their PTP1B inhibitory activities[J]. Chin. Chem. Lett., 2017, 28(6): 1190-1193  

Duo-Qing Xue^a,1, Hai-Li Liu^a,1, Si-Han Chen^a, Ernesto Mollo^b, Margherita Gavagnin^b, Jia Li^a, Xu-Wen Li^a, Yue-Wei Guo^a    

Marine sponge of the genus Mycale (Carmia) is a rich source of bioactive secondary metabolites of diverse structure features, such as peloruside A [1], mycalamides A and B [2, 3]. Pateamine [4], and a series of pyrrole-containing lipids [5-11], with a wide range of biological activities, including antibacterial, antifungal, antiviral, and antitumoral effects. It is worth to mention that even if pyrrole is a structurally simple five-membered ring, pyrrole-containing lipids have seldom been discovered in the marine organisms, with distribution limited primarily to specific species of algae, sponges, bryozoans, tunicates and mollusks [12]. Among these marine sources, sponges of the Mycale genus have been recognized as one of the main origins of pyrrole-containing lipids. In the last decades, a variety of new 5-alkylpyrrole-2-carbaldehydes have been isolated from this genus of sponges and some of them were found to be of strong antidiabetic and antitumoral activities [8, 10, 11]. In our continuous searching for biologically active secondary metabolites from Chinese marine sponges [13-15], M. lissochela was collected from Lingshui, Hainan, China. The chemical investigation of its acetone extract resulted in the discovery of two new, namely mycalenitrile-15 (1) and mycalenitrile-16 (2), and five known 5-alkylpyrrole-2-carboxaldehyde derivatives (3-7). Herein, we report their isolation, structure determination and PTP1B inhibitory activities.

Fractionation of the Et₂O soluble residue of the acetone extract of the title animals by silica gel column chromatography (CC), followed by further silica gel CC and RP-HPLC, led to the isolation of compounds 1-7 (Fig. 1). All these compounds showed very similar spectroscopic properties. Their typical IR absorptions at v_max around 3260, 1645 cm^-1, and ¹H NMR spectra (a singlet at δ 9.37, a pair of "dd" peak at around δ 6.89 and 6.08, large integration at δ 1.20-1.50) indicated that compounds 1-7 were 5-alkylpyrrole-2-carboxaldehyde derivatives. In addition, their characteristic IR absorption at v_max around 2250 cm^-1, ¹³C NMR datum at δ 119.9, indicated the presence of nitrile functionality. Therefore, compounds 1-7 should be 5-alkylpyrrole-2-carboxaldehyde with a terminal nitrile group. On the basis of the above analysis and by comparing the NMR data with the reported 5-alkylpyrrole-2-carboxaldehyde in the literature, the known isolates were readily identified as mycalenitrile-13 (3) [11], 5-(19'-cyanononadecyl) pyrrole-2-carbaldehyde (4) [7], mycalenitrile-2 (5) [10], mycalenitrile-3 (6) [10], (6'Z)-5-(23'-cyano-6'-tricosenyl)pyrrole-2-carboxaldehyde (7) [9], respectively.

Figure 1

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Fig. 1. The structures of compounds 1-7.

Compound 1 was obtained as an optically inactive white amorphous powder, and its molecular formula, C₂₉H₄₆N₂O, was established from HR-EIMS with the ion peak at m/z 438.3621 (calcd. for C₂₉H₄₆N₂O: 438.3611). Its ¹H NMR spectrum not only comprised an aldehyde proton signal at δ_H 9.37 (s, 1H, H-1), two pyrrole proton signals at δ_H 6.87-6.89 (dd, 1H, J = 3.6, 2.7 Hz, H-3) and 6.06-6.08 (dd, 1H, J = 3.6, 2.7 Hz, H-4), a broad NH signal at δ_H 9.35 (1H, brs), but also four olefinic proton signals at δ_H 5.33-5.38 (m, 4H, H-10, H-11, H-13, and H-14), and signals at δ_H 2.66 (t, 2HJ = 7.5 Hz, H-6), 2.33 (t, 2H, J = 7.2 Hz, H-28), and 2.00-2.10 (m, 4H, H-9 and H-15). The ¹³C NMR data confirmed the presence of the aldehyde [δ_C 178.1 (d, C-1)], pyrrole carbons [δ_C 142.9, 131.8 (s, C-2), 122.4 (d, C-3), 109.4 (d, C-4)], olefin [δ_C 130.4 (d, C-11), 129.3 (d, C-14), 128.6 (d, C-10), 127.8 (d, C-13)], and a nitrile carbon [δ_C 119.9 (s, C-29)] (Table 1). By comparing with the NMR data with those reported for the co-isolated known compounds 3-7, 1 was suggested to be a 5-alkylpyrrole-2-carboxaldehyde derivative with a long unsaturated aliphatic side chain at the C-5 position. Furthermore, the triplet of CH₂-12 at δ 2.77 (t-J = 5.9 Hz) and δ_C 25.6 (C-12), and its strong ¹H-¹H COSY correlation (Fig. 2) with the olefinic protons, inferred that the two double bonds were separated by the methylene-12. The presence of allylic carbon signals at δ_C 27.2 (C-9), 27.7 (C-12) and 27.8 (C-15), with no signals in the range 31-35 ppm, indicated that both two olefinic bonds encompassed Z geometry [5, 9]. The position of the two olefinic bonds was determined from interpretation of the COSY spectrum, leading to the spin-spin system b (C-6 to C-15) (Fig. 2). Such connection was confirmed by the EI-MS analysis (Fig. 2) with the specific signals at m/z 136, 176, 190, and 230, resulted from CH₂ or allylic cleavage of the side chain [5, 9]. In view of the molecular formula of C₂₉H₄₆N₂O, the linkage of the terminal nitrile was determined, and thus compound 1 was characterized as presented in Fig. 1, namely mycalenitrile-15.

Table 1

Table 1 1H NMR and 13C NMR data of compounds 1 and 2.a

Table 1 1H NMR and 13C NMR data of compounds 1 and 2.a

Figure 2

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Fig. 2. Selected key 1H-1H COSY (bold lines) correlations of 1 and key EI-MS fragments of 1 and 2.

The molecular formula of compound 2 was also established from HR-EIMS with the ion peak at m/z 412.3461 (calcd. for C₂₇H₄₄N₂O: 412.3453). Its typical IR bands, as well as the specific ¹H NMR data at the downfield [δ_H 9.90 (brs, 1H, NH), 9.35 (s, 1H, H-1), 6.89 (m, 1H, H-3), 6.07 (m, 1H, H-4), 5.33-5.34 (m, 2H, H-11/H-12); δ_C 178.1 (C-1), 131.8 (C-2), 122.6 (C-3), 109.4 (C-4), 143.2 (C-5), 129.4 (C-11), 130.2 (C-12), 119.9 (C-27)] (Table 1), were nearly the same as those of 7, indicated 2 as a 5-alkylpyrrole-2-carboxaldehyde derivative with only one olefinic bond on the middle and a nitrile at the end of the side chain. The only 28 mass units fewer on the MS of compound 2 than that of 7, suggested that the structure of 2 might only a lost of two CH₂ groups on that of 7. The Z geometry of its olefinic bond was also confirmed by the allylic carbon signals at δ_C 27.2 (C-10) and 25.6 (C-13), respectively [5, 9]. The position of the double bond at C-11 and C-12 in the long side chain was determined from the mass spectrum, with the fragment ion peaks at m/z 150 and 204 that resulted from the allylic cleavage (Fig. 2) [5, 9]. From the above spectral evidences, and by comparing with 7, compound 2 was determined as mycalenitrile-16.

All the compounds were assayed for their inhibitory activity against PTP1B, and mostof theminhibited PTP1 B activitiesin a dosedependent manner (Table 2).Among them, the newcompound 1 and the known one 7 exhibited the most potent inhibitory activities, with IC₅₀ values of 8.6 and 3.1 μmol/L, respectively, comparable with the positive control ursolic acid (IC₅₀ = 3.6 μmol/L) [16]. Compounds 3-6, with the presence of a saturated side chain, were less active than 1, 2 and 7, indicating that the unsaturated aliphatic side chain may be helpful for the resultant activity.

Table 2

Table 2 Inhibitory activity of isolated compounds against PTP1B.

Table 2 Inhibitory activity of isolated compounds against PTP1B.

Pyrrole-containing lipids are uncommon secondary metabolites in nature, which mainly distributed in certain marine organisms such as sponges of the Mycale genus. In order to find potential antidiabetic pyrrole-containing lipids [8], we did a systematic chemical investigation of the South China Sea sponge M. lissochela, resulting in the discovery of two new and five known 5-alkylpyrrole-2-carboxaldehyde derivatives with a terminal nitrile on the aliphatic side chain (1-7). All the isolated compounds, except for 3, exhibited PTP1 B inhibitory activities, and those with unsaturated side chain (1, 2 and 7) had a better performance, which gave us some fundamental evidence for the discovery of antidiabetic lead compounds on the specific target protein such as PTP1B. Further pharmacological mechanism study of these compounds should be conducted toward PTP1 B and other targets for the finding of novel antidiabetic drug leads.

Column chromatography: commercial silica gel (Qingdao Haiyang Chemical Group Co.; 200-300 mesh). Reversed-phase HPLC was employed on Agilent 1100 series liquid chromatography with a VWD G1314A detector at 210 nm using a semi-preparative ODS-HG-5 [5 μm, 10 mm (i.d.) × 25 cm] column. TLC: precoated silica gel plates (Yantai Zifu Chemical Group Co.; G60, F-254). Optical rotation: Perkin-Elmer 341 polarimeter. IR spectra: Nicolet Magna FT-IR 750 spectrophotometer; v_max in cm^-1. NMR Spectra: Bruker DRX-400 spectrometer; d in ppm rel. to residual CHCl₃ (δ_H 7.26, δ_C 77.0) as internal standard, J in Hz. MS: Finnigan MAT-95 spectrometer; in m/z.

The specimen of M. cecilia, identified by Prof. R.-L. Zhou of the South China Sea Institute of Oceanology, Chinese Academy of Sciences, were collected in February 2004 by scuba techniques at a depth of -10 m off Lingshui, Hainan Province, China, in the South China Sea. A voucher specimen is available for inspection at the Shanghai Institute of Materia Medica, CAS, under the registration No.04LS-247.

The frozen animals (120 g dry weight) were cut into small pieces and exhaustively extracted with acetone (1.0 L×3) at r.t. The extract was concentrated under reduced pressure, and the crude residue was partitioned between Et₂O and H₂O. The Et₂O-soluble portion was fractionated by CC (silica gel, light petroleum ether with increasing amounts of ethyl acetate): 10 fractions (A-J). The fraction D, which performed obvious reactions with dragendorff reagent, was subjected to repeated reversed HPLC separations using MeOH-H₂O (90:10) as eluent to afford 1 (2.8 mg) with retention time of 67.4 min, 2 (12.0 mg, with retention time of 65.1 min), 3 (3.6 mg, with retention time 62.9 min), 4 (22.0 mg, with retention time of 64.3 min), 5 (4.0 mg, with retention time of 66.7 min), 6 (24.0 mg, with retention time of 68.3 min), 7 (28.0 mg, with retention time of 70.3 min).

Mycalenitrile-15 (1): Optically inactive white amorphous powder; IR (KBr, cm^-1): v_max 3260, 2924, 2853, 2245, 1643, 1495, 1412, 1195, 1132, 1077, and 1042; ¹H NMR (CDCl₃, 400 MHz): see Table 1; ¹³C NMR (CDCl₃, 100 MHz): see Table 1; EI-MS (70 eV) m/z 438 (60.8) (M)⁺, 412 (8.0) (M-CN)⁺, 409 (24.0) (M-CHO)⁺, 230 (8.0) (M-C₁₄H₂₆N)⁺, 190 (12) (M-C₁₇H₃₀N)⁺, 176 (12) (M-C₁₈H₃₂N)⁺, 150 (24) (M-C₂₀H₃₄N)⁺, 136 (12) (M-C₂₁H₃₆N)⁺, 122 (100) (M-C₂₂H₃₈N)⁺, and 108 (90.9) (M-C₂₃H₄₀N)⁺; HR-EI-MS: m/z 438.3621 (calcd. for C₂₉H₄₆N₂O: 438.3611).

Mycalenitrile-16 (2): Optically inactive white amorphous powder; IR (KBr, cm^-1): v_max 3255, 2924, 2853, 2248, 1644, 1496, 1411, 1195, 1132, 1077, and 1042; ¹H NMR (CDCl₃, 400 MHz): see Table 1; ¹³C NMR (CDCl₃, 100 MHz): see Table 1; EI-MS (70 eV) m/z 412 (92.0) (M)⁺, 386 (11.0) (M-CN)⁺, 150 (21.0) (M-C₁₈H₃₂N)⁺, 136 (8.0) (M-C₁₉H₃₄N)⁺, 122 (100) (M-C₂₀H₃₆N)⁺, and 108 (68.0) (M-C₂₁H₃₈N)⁺; HR-EI-MS: m/z 412.3461 (calcd. for C₂₇H₄₄N₂O: 412.3453).

PTP1B (human, recombinant) was purchased from BIOMOL^® International LP (USA) and the enzyme activity was measured using pNPP as previously described [17]. To each well of a 96-well plate (final volume: 200 μL) was added 2 mmol/L pNPP and PTP1 B (0.05-0.1 μg) in a buffer containing 50 mmol/L citrate (pH 6.0), 0.1 mol/L NaCl, 1 mmol/L EDTA and 1 mmol/L dithiothreitol (DTT) with or without test compounds. Following incubation at 37 ℃ for 30 min, the reaction was terminated with 10 mol/L NaOH. The amount of produced p-nitrophenol was estimated by measuring the absorbance at 405 nm. The non-enzymatic hydrolysis of 2 mmol/L pNPP was corrected for by measuring the increase in absorbance at 405 nm obtained in the absence of PTP1 B enzyme.

This research work was financially supported by the National Natural Science Foundation of China (Nos. 41476063, 81520108028, 81273430, 41306130 81302692, 41676073, 81603022), SCTSM Project from Shanghai Science and Technology Committee, Shanghai, China (Nos. 14431901100, 15431901000), The project from Institutes for Drug Discovery and Development, Chinese Academy of Sciences, China (No. CASIMM0120152039), the SKLDR/SIMM Projects from Shanghai Institute of Materia Medica, China (No. SIMM 1501ZZ-03). X.-W. Li acknowledges the financial support of "Youth Innovation Promotion Association" (No. 2016258) from Chinese Academy of Sciences, "Young Talent Supporting Project" from China Association for Science and Technology (No. 2016QNRC001), and Shanghai "Pujiang Program"(No. 16PJ1410600).