Geobarrettin D, a Rare Herbipoline-Containing 6-Bromoindole Alkaloid from Geodia barretti

Geobarrettin D (1), a new bromoindole alkaloid, was isolated from the marine sponge Geodia barretti collected from Icelandic waters. Its structure was elucidated by 1D, and 2D NMR (including 1H-15N HSQC, 1H-15N HMBC spectra), as well as HRESIMS data. Geobarrettin D (1) is a new 6-bromoindole featuring an unusual purinium herbipoline moiety. Geobarrettin D (1) decreased secretion of the pro-inflammatory cytokine IL-12p40 by human monocyte derived dendritic cells, without affecting secretion of the anti-inflammatory cytokine IL-10. Thus, compound 1 shows anti-inflammatory activity.

The marine sponge Geodia barretti is the source of bromoindole alkaloids [14][15][16][17][18] and several N-methylated nucleosides [19]. In our previous study, three new 6-bromoindole derivatives were isolated from G. barretti collected in Icelandic waters [20]; of these, geobarrettins B and C exhibited anti-inflammatory activity [20]. As part of our ongoing investigation to find novel anti-inflammatory compounds, we report, here, the bromoindole geobarrettin D (1) (Figure 1, as the TFA salt) with potential anti-inflammatory effect measured by decreased pro-inflammatory cytokine secretion of human monocyte-derived dendritic cells (DCs).

Structural Elucidation
The HRMS of geobarrettin D (1)   showed the presence of a downfield exchangeable proton (δ H 10.60), which is diagnostic of an NH proton in an indole ring, and confirmed by a weak coupling (J = 2.0 Hz) to the isolated aromatic proton at δ H 7.53 in the pyrrole ring. Correlations observed in the HMBC spectrum ( Figure 2) allowed the definement of the substitution pattern and NMR assignments of the indole: H-4 to C-3 , C-6 , and C-7a , from H-5 to C-3a and C-7 , and from H-7 to C-5 and C-3a . H-2 also showed a correlation to C-3 , C-3a , and C-7a . The NH exchangeable proton H-1 showed correlations to C-2 , C-3 , C-3a , and C-7a . The indole assignment was supported by the presence of several bands in the UV-vis spectrum (λ max 228, 261, and 287 nm).

Anti-Inflammatory Activity
When DCs were matured and activated in the presence of geobarrettin D (1), secretion of the pro-inflammatory cytokine IL-12p40 was diminished by 48%, whereas secretion of the anti-inflammatory cytokine IL-10 was not affected (Figure 3). On balance, these results indicate an overall anti-inflammatory effect of geobarrettin D (1).
Most natural product alkaloids are derived from the aromatic amino acids including tryptophan, tyrosine and phenylalanine. Compound 1 is likely biosynthetically derived from 6-bromotryptamine-an alkaloid known from other marine invertebrates [33]through a fusion of an oxidized tryptamine intermediate and a guanine equivalent (Figure 4) through a 1,4-conjugate addition, followed by extensive methylation reactions involving S-adenosylmethionine (SAM). Oxidation and conjugate addition is necessary and sufficient to explain formation of many C-C and C-heteroatom bonds in alkaloids [34], including 1. For example, one of us (T.F.M) recently reported two cyclic guanidines, aiolochroiamides A and B, whose formation may also be rationalized by an oxidation-conjugate addition mechanism [35]. It is likely that the oxidation reaction is enzyme-mediated as spontaneous autoxidation seems unlikely, however the subsequent conjugate addition may be spontaneous given the low specific rotation (and therefore enantiomeric excess) observed for 1. As with other complex highly-methylated quaternized alkaloids, the ordering of N-methylation and condensation reactions is uncertain. Further biosynthetic studies are necessary for understanding the biosynthesis of 1, but these are beyond the scope of this study.  Purines have found antiviral, antibiotic, and anticancer activities [27,31,36,37], and have the potential to regulate myocardial oxygen supply and cardiac blood flow [38]. In addition, evidence supports their role in biological evolution, differentiation, and ecological processes [31]. Purines are also involved in various inflammatory responses which underscores the significant attention given to purine natural products and their synthetic mimetics for the development of anti-inflammatory agents [20,39,40].
The anti-inflammatory properties of compound 1 were investigated in an in vitro model of human monocyte-derived DCs [20]. DCs matured and activated in the presence of compound 1 secreted less IL-12p40 than DCs cultured without 1, whereas 1 had no effect on their secretion of IL-10. The pro-inflammatory cytokine IL-12p40 (one of the two chains that form the structures of IL-12 and IL-23 cytokines) is a major determinant of the differentiation of naïve T cells into Th1 or Th17 phenotypes [41], whereas the anti-inflammatory cytokine IL-10 drives polarization of naïve T cells into a T regulatory phenotype [42]. Thus, suppression of IL-12p40 secretion by DCs in the presence of 1 indicates that geobarrettin D (1) has anti-inflammatory activity.

General Procedures
The UV spectrum was recorded on a NanoVueTM spectrophotometer (GE Healthcare Life Sciences, Little Chalfont, UK) with a 0.2 mm path length. Optical rotation was measured on a P-2000 polarimeter (Jasco, Oklahoma City, OK, USA), with a quartz cell (10 mm path length). The infrared spectrum was measured on a Spectrum Two TM FTIR spectrometer (Perkin Elmer ® , Waltham, MA, USA) of samples as thin films. NMR spectra were recorded on a Bruker Avance 600 spectrometer (Billerica, MA, USA) ( 1 H and 13 C frequencies: 600.13 MHz and 150.76 MHz, respectively) in CD3OD and D2O/H2O (1:9). The residual solvent signals were used as internal references: δH 3.30/δC 49.0 ppm (CD3OD) and δH 4.79 (D2O). For 1 H-15 N 2D NMR spectroscopy, the nominal 15 N standard was liquid ammonia, NH3 (l) (δ = 0 ppm). Samples (1.6-6.0 mg) were introduced into Shigemi tubes and their 2D NMR spectra measured as illustrated by the following 1 H{ 13 C} heteronuclear HSQC and HMBC spectra using modifications of the Bruker pulse sequences hsqce- Purines have found antiviral, antibiotic, and anticancer activities [27,31,36,37], and have the potential to regulate myocardial oxygen supply and cardiac blood flow [38]. In addition, evidence supports their role in biological evolution, differentiation, and ecological processes [31]. Purines are also involved in various inflammatory responses which underscores the significant attention given to purine natural products and their synthetic mimetics for the development of anti-inflammatory agents [20,39,40].
The anti-inflammatory properties of compound 1 were investigated in an in vitro model of human monocyte-derived DCs [20]. DCs matured and activated in the presence of compound 1 secreted less IL-12p40 than DCs cultured without 1, whereas 1 had no effect on their secretion of IL-10. The pro-inflammatory cytokine IL-12p40 (one of the two chains that form the structures of IL-12 and IL-23 cytokines) is a major determinant of the differentiation of naïve T cells into Th1 or Th17 phenotypes [41], whereas the anti-inflammatory cytokine IL-10 drives polarization of naïve T cells into a T regulatory phenotype [42]. Thus, suppression of IL-12p40 secretion by DCs in the presence of 1 indicates that geobarrettin D (1) has anti-inflammatory activity.

General Procedures
The UV spectrum was recorded on a NanoVueTM spectrophotometer (GE Healthcare Life Sciences, Little Chalfont, UK) with a 0.2 mm path length. Optical rotation was measured on a P-2000 polarimeter (Jasco, Oklahoma City, OK, USA), with a quartz cell (10 mm path length). The infrared spectrum was measured on a Spectrum Two TM FTIR spectrometer (Perkin Elmer ® , Waltham, MA, USA) of samples as thin films. NMR spec-tra were recorded on a Bruker Avance 600 spectrometer (Billerica, MA, USA) ( 1 H and 13 C frequencies: 600.13 MHz and 150.76 MHz, respectively) in CD 3 OD and D 2 O/H 2 O (1:9). The residual solvent signals were used as internal references: δ H 3.30/δ C 49.0 ppm (CD 3 OD) and δ H 4.79 (D 2 O). For 1 H-15 N 2D NMR spectroscopy, the nominal 15 N standard was liquid ammonia, NH 3 (l) (δ = 0 ppm). Samples (1.6-6.0 mg) were introduced into Shigemi tubes and their 2D NMR spectra measured as illustrated by the following 1 H{ 13 C} heteronuclear HSQC and HMBC spectra using modifications of the Bruker pulse sequences hsqcedetgpsisp2.4 and hmbcgplpndqf, respectively: Spectra were acquired at near ambient temperature (T = 300.0 K) in the specified solvent with an rf pulse calibrated to 1H π/2 = 8.75 µs, with appropriate gradient field strengths, and dwell times corresponding to 1 H (F2) and 13 C (F1) spectral widths of 8417.5 Hz and 33112.6 Hz and centered at δ H 7.01 and δ C 110.4 ppm, respectively. Accumulated scans (n = 8 for HMBC and n = 32 for HSQC) for each T1 increment were averaged between a relaxation delay, D1 =1.5 s. The acquired matrix ( 1 H and 13

Animal Materials
In short, the sponge material Geodia barretti was collected in Iceland, identified by. Hans Tore Rapp, University of Bergen (Norway), and deposited at University of Iceland. For a complete description of the samples, see Xiaxia Di et al. [20].

Extraction and Isolation
Frozen sponge was cut into approximately 1 cm 3 pieces and freeze-dried. The dried tissue was extracted with a mixture of CH 2 Cl 2 :CH 3 OH (v/v, 1:1) for 3 times (2 L, each for 24 h) at room temperature. The combined CH 2 Cl 2 -MeOH extracts were dried under reduced pressure then the residue (1.8 g) was suspended in MeOH:H 2 O (v/v, 9:1) and subjected to a modified Kupchan partition, as previously described [21,22], to yield five fractions: hexane

Maturation and Activation of DCs
DCs were differentiated from human monocytes as previously described [20]. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood obtained from healthy human donors (approval #06-068-V1 by National Bioethics Committee of Iceland (Visindasidanefnd) from 15 th of December 2015) by density centrifugation using Histopaque-1077 (Sigma-Aldrich, Munich, Germany). Then CD14 + monocytes were isolated from the PBMCs using magnetic cell sorting and CD14 Microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). The CD14 + monocytes were cultured at 5 × 10 5 cells/mL in RPMI 1640 medium, supplemented with 10% fetal calf serum and 5% penicillin/streptomycin (all from Gibco ® , Thermo Fisher Scientific, UK) in 48 well tissue culture plates for seven days. In order to differentiate CD14 + monocytes into immature DCs, IL-4 at 12.5 ng/mL and GM-CSF at 25 ng/mL (both from R&D Systems, Bio-Techne, Abingdon, England) were added to the cells. After seven days the monocytes had differentiated into immature DCs which were harvested and cultured for 24 h in 48 well tissue culture plates at 2.5 × 10 5 cells/mL. The immature DCs were matured and activated by culturing them with IL-1β at 10 ng/mL, TNF-α at 50 ng/mL (both from R&D Systems), and lipopolysaccharide (LPS) at 500 ng/mL (Sigma-Aldrich, Munich, Germany). Geobarrettin D (1) was dissolved in DMSO and added to the DCs at 10 µg/mL at the same time as the cytokines and LPS. DMSO was used as a control. After 24 h the mature and activated DCs were harvested and the concentrations of IL-12p40 and IL-10 in the supernatants were measured by sandwich ELISA using DuoSets from R&D Systems according to the manufacturer's protocol. The results are expressed as a secretion index (SI). Data are presented as the mean values ± SEM, n = 3. As the data were not normally distributed, Mann-Whitney U test was used to determine statistical differences between the groups (SigmaStat 3.1, Systat Software, San Jose, CA, USA) and p < 0.05 was considered as statistically significant.

Conclusions
Geobarrettin D (1) is a newly detected bromoindole alkaloid possessing an unusual brominated and fused-herbipoline-dimethylguaninium heterocycle. Geobarrettin D (1) decreased DC secretion of the pro-inflammatory cytokine IL-12p40, indicating its potential as an anti-inflammatory agent.

Acknowledgments:
The authors would like to thank Hans Tore Rapp at the University of Bergen for the identification of the animal material and Caroline Rouger and Deniz Tasdemir at GEOMAR Helmholtz Center for Ocean Research for NMR spectrometry. The parts of the results for the manuscript are the doctoral thesis "Searching for immunomodulatory compounds from Icelandic marine invertebrates" of the author Xiaxia Di.