Urupocidin A: A New, Inducing iNOS Expression Bicyclic Guanidine Alkaloid from the Marine Sponge <italic>Monanchora pulchra</italic>

Urupocidins A and B (1 and 2), bisguanidine alkaloids with an unprecedented skeleton system, derived from polyketide precursors and containing an unusual N-alkyl-N-hydroxyguanidine moiety, have been isolated from the sponge Monanhora pulchra. The structures of 1 and 2, including absolute configuration, were established using the detailed analysis of 1D and 2D NMR, CD, and mass spectra as well as chemical transformations. Compound 1 increases nitric oxide production in murine macrophages via inducing iNOS expression. T discovery that NO is responsible for an astonishing range of physiological processes in humans presents one of the most exciting findings in biological chemistry. The biosynthesis of NO is catalyzed by nitric oxide synthase (NOS), which is classified into three isoforms: inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Both the endothelial NOS (eNOS) and the neuronal NOS (nNOS) are constitutive enzymes; however, the third isoform is inducible, produced by macrophages as part of the immune response. Therefore, the search for new natural modulators of iNOS may have therapeutic significance at the search for immunoactive compouds. Guanidine alkaloids are known to be characteristic metabolites of some marine sponges. These alkaloids demonstrate a broad spectrum of biological activities, including potent cytotoxic effects, induction of cellular apoptosis, and inhibition of the TRPV1 receptor. During our search for bioactive substances from marine organisms, we have previously isolated some guanidine alkaloids with unusual cyclic skeletons and rare acyclic alkaloids from sponges of the genus Monanchora. Herein, we describe the isolation and structure elucidation of urupocidins A (1) and B (2), possessing a new trisubstituted bicyclic skeleton system with an unprecedented N-alkyl-N-hydroxyguanidine fragment in one of the side chains, from a new collection of the sponge Monanchora pulchra (Figure 1). The induction of endogenous NO in Raw 264.7 cells and iNOS expression in macrophages by 1 is shown (Supporting Information). Urupocidins A and B (1 and 2) were named after one of the biggest Kuril Islands known as Urup Island, near which the sponge was collected. The alkaloids were isolated from the frozen sponge (0.09% and 0.0024% of dry weight, respectively) by extraction with EtOH, evaporation, and partition between H2O and n-BuOH, followed by the partition of the BuOH-soluble materials between aqueous EtOH and hexane, repeated column chromatography of the ethanol-soluble materials on Sephadex LH-20 (EtOH) and HPLC on an YMC-ODS-A column (65% EtOH/0.1% aqueous TFA). Urupocidin A (1), a colorless glass, has molecular formula C29H50N6O4 established by HRESIMS measurement of the [M + H] ion peak at m/z 547.3956. The peak at m/z 274.2015 (calcd for C29H50N6O4 274.2020) in the HRESIMS of 1 corresponds to a doubly charged [M + 2H] ion like similar peaks in MS of the earlier reported monanchomycalins A− C and two-headed sphingolipids. NMR data (DMSO-d6, Table 1) of 1 revealed the presence of two guanidine groups (δH 8.35 (2H), 9.39 (1H) and δC 150.6; δH 7.55 (3H) and δC 157.8), two methyl groups (δH 0.86, 0.85 and δC 13.57, 13.78), two hydroxy groups (δH 10.61, Received: July 10, 2014 Published: August 5, 2014 Figure 1. Structures of urupocidins A and B (1 and 2) and urupocidin A triacetate (1a). Letter pubs.acs.org/OrgLett © 2014 American Chemical Society 4292 dx.doi.org/10.1021/ol502013f | Org. Lett. 2014, 16, 4292−4295 4.70), two disubstituted double bonds (δH 5.33 (2H) and δC 129.5, 129.6; δH 5.36 (2H) and δC 128.5, 130.2), one tetrasubstituted double bond (δC 151.5, 101.0), two Nsubstituted CH groups (δH 4.51, 4.30 and δC 57.5, 48.9), one N-substituted CH2 group (δH 3.55 and δC 50.4), one oxymethine (δH 3.51 and δC 66.8), one carbonyl-linked oxymethylene group (δH 4.11, 4.14 and δC 63.8), and 13 other methylene groups (Table 1). A quaternary carbon signal at δC 164.2 was assigned to the carbonyl group of an ester, which should be conjugated with a double bond according to the IR spectrum data. Substructures a−d were established by COSY, HSQC, and HMBC experiments (Figure 2). Fragment a had never been previously seen in guanidine alkaloids isolated from marine invertebrates. It was revealed starting from the signals of CH212 group (δH 3.05, 3.13 and δC 29.2) and sequentially extended up to end methyl group that showed a higher field methyl triplet CH3-1 in the H NMR spectrum (δH 0.86 and δC 13.64). Interpretation NMR data, starting from the low-field NH23b doublet (δH 9.39) cross-over signals of Δ-olefin (δH 5.36 (2H) and δC 128.5, 130.2) and extending until to the high field CH3-22 triplet (δH 0.85 and δC 13.57), indicated the substructure b (Figure 2). Detailed analysis of NMR data concerning the substructure “c” led to the identification of four methylene groups of an open hydrocarbon chain, linked by one side to an oxygen atom (δH 4.11, 4.14 and δC 63.8) and by another side to a tertiary nitrogen atom of guanidine group (δH 3.55 and δC 50.4). Diagnostic ROESY correlations between the resonances of 29aN-OH (δH 10.61) and 2H-27 (δH 1.67), 2H-28 (δH 3.55), 29bNH, 29c-NH2, as well as the H−N HMBC correlations between 29b-NH, 29c-NH2, 2H-28, and 29a-N indicated the position of N-OH group in substructure c. Moreover, it was confirmed by a downfield shift of proton and carbon signals of CH2-28 group when compared with the corresponding signals in relative guanidine compounds. Substructure d and its connectivity with a−c was assigned by HMBC experiments, which indicated that the H-10 proton at δH 4.51 correlated to C-11 (δC 26.4), C-12 (δC 29.2), C-14 (δC 101.0), and C-23 (δC 150.6) signals. In addition, the H-15 proton at δH 4.30 correlated to C-23 (δC 150.6), C-13 (δC 151.5), C-14 (δC 101.0), C-24 (δC 164.2), C-16 (δC 36.1), and C-17 (δC 21.5), and protons CH2−25 group was also correlated to C-24 (δC 164.2). Moreover, the H−N HMBC correlations between 23b-NH, 16a,b-2H and 23b-N, 23b-NH, 11a,b-2H, 9a,b-2H, and 23c-N indicated that a bicyclyc moiety with two N atoms at positions 23b, 23c and three substitutions at position C-10, C-15, and C-14 presented in the compound 1 (Figure 1). The Z-geometry of double bonds was assigned using the NMR signals of allylic carbons (Table 1). Table 1. H (700 MHz) and C (175 MHz) NMR Data of Urupocidins A (1) and B (2) urupocidin A (DMSO-d6) a urupocidin B (DMSO-d6) a urupocidin A (DMSO-d6) a urupocidin B (DMSO-d6) a position δC δH mult (J in Hz) δC δH mult (J in Hz) position δC δH mult (J in Hz) δC δH mult (J in Hz) 1 13.64* 0.86*, t (7.3) 13.63* 0.86,* t (7.3) 16b 1.50, m 1.47, m 2 22.3* 1.33, sext (7.3) 22.3* 1.33, sext (7.3) 17a 21.5 1.93, m 29.1 1.25, m 3 28.74* 1.97, m 28.7 1.98, m 17b 2.07, m 4 129.5 5.33, m 129.5 5.33, m 18 128.5 5.36, m 30.8 1.24, m 5 129.6 5.33, m 129.6 5.33, m 19 130.2 5.36, m 22.0 1.27, m 6a 23.0 2.00, m 23.0 2.00, m 20 28.68* 1.95, m 13.77* 0.85*, t (7.3) 6b 2.09, m 2.08, m 21 22.2* 1.31*, sext (7.3) 7 38.2 1.39, m 38.1 1.40, m 22 13.57* 0.85*, t (7.3) 8 66.8 3.51, m 66.8 3.51, m 23 150.6 150.5 8-OH 4.70, brs 4.70, brs 23a-NH2 8.35, brs 8.21, brs 9a 39.5 1.62, m 39.7 1.63, m 23b-NH 9.39, d (3.7) 9.17, d (3.9) 9b 1.68, m 1.66, m 24 164.2 164.2 10 57.5 4.51, m 57.5 4.51, m 25a 63.8 4.11, m 63.7 4.10, m 11a 26.4 1.96, m 26.4 1.98, m 25b 4.14, m 4.15, m 11b 2.16, td (8.4; 4.0; 18.6) 2.15, m 26 25.2 1.65, m 25.2 1.63, m 12a 29.2 3.05, m 29.1 3.04, m 27 22.7 1.67, m 22.6 1.66, m 12b 3.13, ddd (8.4; 4.0) 3.13, m 28 50.4 3.55, brt 50.4 3.55, brt (6.6) 13 151.5 151.3 29 157.8 157.7 14 101.0 101.4 29b-NH 7.55, brs 7.52, brs 15 48.9 4.30, dd (4.1; 3.8) 49.0 4.30, dd (4.1; 3.8) 29c-NH2 7.55, brs 7.52, brs 16a 36.1 1.47, m 35.7 1.42, m 29a-NOH 10.61, s 10.58, s 13C NMR assignments supported by HSQC and HMBC data. *Overlapping signals. Figure 2. Partial structures of 1 with selected COSY, HMBC, and key ROESY correlations. Organic Letters Letter dx.doi.org/10.1021/ol502013f | Org. Lett. 2014, 16, 4292−4295 4293 When reacted with Ac2O, urupocidin A gave the triacetate 1a, the NMR spectrum of which showed, along with the expected signals of the urupocidin skeleton system, the characteristic singlets at δH 2.07 (3H) (δC 171.0), δH 2.17 (3H) (δC 181.5), and δH 2.20 (3H) (δC 168.7) attributable to two O-acetyl and one acetamide groups, respectively (Supporting Information, Table S2). A determination of a relative stereochemistry of C-10 and C15 by NOE experiments using 1 was unsuccessful as a consequence of the overlapping of 2H-9, 2H-26, and 2H-27 signals in the H NMR spectrum (DMSO-d6, Table 1) as well as of H-10 with H-15 and allylic signals with the H-11a signal in the H NMR spectrum in CD3OD (Supporting Information, Table S2). In order to determine their relative stereochemistry, the NOESY analysis in CD3OD was applied to a tetrahydro derivative 1b prepared from 1 (Figure 3). Diagnostic NOEs correlations between the resonances of H-9a (δH 1.68, ddd, 2.4, 9.4, 12.4), H-15 (δH 4.41, dd, 4.1, 7.1), and H-11a (δH 2.04, dddd, 3.8, 5.1, 8.2, 13.0) were indicative of the 10R* and 15S* relative configurations in 1 (Figure 3). Absolute 15S configuration were proposed using CD (n → π* transition of the enone) and comparison with crambescin A2 (see the Supporting Information). In order to determine the absolute configuration of C-8 asymmetric center in 1, the modified Mosher’s method was applied to the compound 3 prepared from 1 (Figure 4). Urupocidin A (1) was treated with NaOMe in methanol, and subsequent purification of the obtained products using reversed-phase HPLC gave the methyl ester (3). Esterification of 3 with (R)or (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chlorides (MTPA-Cl) in an NMR tube gave (S)and (R)-MTPA esters 4 and 5, respectively. Positive ΔδH values for the left-hand protons and negative ΔδH values for the most right-hand protons were observed in the H NMR spectra of 4 and 5 (Figure 4). These data established the absolute configuration at C-8 as R. Therefore, the 8R,10R,15S absolute configuration in 1 was assigned. Urupocidin B (2), as a colorless glass, has the molecular formula of C27H48N6O4 established by HRESIMS measurement of the [M + H] ion peak at m/z 521.3816. The H NMR data (Table 1) of 2 closely coincided with those of urupocidin A (1), maintaining the Δ-olefin signals. In addition, MS data showed the molecular mass of 2 to be 26 amu less than that of 1, suggesting that the hydrocarbon chain at C-15 is shortened by two CH2 group in 2. Comparison of the H and C NMR, and CD spectra and optical rotation data of urupocidin B (2) with those of urupocidin A (1) and their complete coincidence suggest the same absolute configurations of 2 as for 1. The structures of urupocidins A and B (1 and 2) possess uncommon structural features, including a trisubstituted bicyclic system and an N-alkyl-N-hydroxyguanidine moiety unprecedented in comparison with other marine guanidine alkaloids. So far, natural N-alkyl-N-hydroxyguanidines have been detected in some terrestrial microorganisms only. Compound 1 induced an expression of iNOS with increase in NO production in macrophages at 10.0 and 1.0 μM concentrations, respectively (see the Supporting Information). Urupocidin A is the first marine alkaloid stimulator of NO production. To date, a few natural products have been reported to up-regulate NO level in cells; examples include polysaccharides from green alga Capsosiphon fulvescens and Strongylocentrotus nudus eggs and triterpene glycosides from Panax ginseng. ■ ASSOCIATED CONTENT *S Supporting Information Full experimental details, copies of 1D and 2D NMR, CD spectra for compounds 1 and 2, tabulated NMR data for compounds 1a, 1b, 3, 4, and 5, and bioassay results. This material is available free via the Internet at http://pubs.acs.org. ■ AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].