Bromopyrrole Alkaloid Inhibitors of the Proteasome ... - ACS Publications

Jul 1, 2018 - Dictyonella sp. Marine Sponge Collected at the Amazon River Mouth. Renata T. M. P. de Souza,. †,#. Vítor F. Freire,. †,#. Juliana R...
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Bromopyrrole Alkaloid Inhibitors of the Proteasome Isolated from a Dictyonella sp. Marine Sponge Collected at the Amazon River Mouth Renata T. M. P. de Souza,†,# Vítor F. Freire,†,# Juliana R. Gubiani,† Raquel O. Ferreira,‡ Daniela B. B. Trivella,‡ Fernando C. Moraes,§ Wladimir C. Paradas,§ Leonardo T. Salgado,§ Renato C. Pereira,§,⊥ Gilberto M. Amado Filho,§ Antonio G. Ferreira,∥ David E. Williams,▽ Raymond J. Andersen,▽ Tadeusz F. Molinski,△ and Roberto G. S. Berlinck*,† †

Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, CEP 13560-970, São Carlos, SP, Brazil Brazilian Biosciences National Laboratory, National Center for Research in Energy and Material, Giuseppe Maximo Scolfaro, 10000, Pólo II de Alta Tecnologia de Campinas, 13083-970 Campinas, SP, Brazil § Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, R. Pacheco Leão, 915, CEP 22460-030, Rio de Janeiro, RJ, Brazil ⊥ Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense (UFF), P.O.Box 100.644, CEP 24001-970, Niteroi, RJ, Brazil ∥ Departamento de Química, Universidade Federal de São Carlos, Rod. Washington Luiz, km 235 - SP-310, CEP 13565-905, São Carlos, SP, Brazil ▽ Departments of Chemistry and Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, BC V6T 1Z1, Canada △ Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive MC-0358, La Jolla, California 92093-0358, United States

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S Supporting Information *

ABSTRACT: The new pyrrole-imidazole and pyrrole-guanidine alkaloids 4-debromooroidin (1), 4-debromougibohlin (2), 5-debromougibohlin (3), and 5-bromopalau’amine (4), along with the known hymenidin (5) and (+)-monobromoisophakellin (6), have been isolated from a Dictyonella sp. marine sponge, collected at the Amazon River mouth. The bromine-substitution pattern observed for compounds 1, 2 and 4 is unusual among bromopyrrole alkaloids isolated from marine sponges. The 20S proteasome inhibitory activities of compounds 1−6 have been recorded, with 5-bromopalau’amine (4) being the most active in this series.

P

dictyoneolone, a new seco-steroid, was isolated from a Dictyonella sp. obtained in Korea.7 The proteasome is a validated target against cancer. Three drugsbortezomib (Velcade), its derivative ixamib (Ninlaro), and carfilzomib (Kyprolis)are currently available for clinical use based on this mechanism of action. However, these inhibitors display pharmacokinetic limitations due to their peptidic cores and high reactivity.8 Therefore, the discovery and characterization of new chemical scaffolds capable of inhibiting the proteasome are important in the context of nextgeneration proteasome inhibitors. Some of us recently described an extensive reef system in the Amazon River mouth,9 from which a new species of Dictyonella was collected by bottom trawl net in 2014. Analysis by HPLCUV-MS of the MeOH extract of this sponge indicated a

yrrole-imidazole and pyrrole-guanidine alkaloids constitute a group of metabolites found exclusively in marine sponges. The chemical diversity of these secondary metabolites is allied to a variety of potent biological activities. Alkaloids with complex carbon skeletons are often isolated together with biogenetic precursors that have varying degrees of bromination in the pyrrole moieties. Chlorination has also been observed in pyrrole-imidazole and pyrrole-guanidine alkaloids but never on the pyrrole ring.1,2 Although isolated from numerous sponge species, pyrroleimidazole and pyrrole-guanidine alkaloids have not been previously found in sponges belonging to the genus Dictyonella (Dictyonellidae, Bubarida). The reported chemistry of Dictyonella species has been restricted to the occurrence of a highly conjugated ketosteroid,3 degraded sterols,4 and furan fatty acid steroidal esters with inflammatory activity,5 all from D. incisa. Additionally, eryloside W, a glycosylated sterol, has been isolated from D. marsilii, 6 and more recently, © 2018 American Chemical Society and American Society of Pharmacognosy

Received: July 1, 2018 Published: October 3, 2018 2296

DOI: 10.1021/acs.jnatprod.8b00533 J. Nat. Prod. 2018, 81, 2296−2300

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of the 1H NMR data for the pyrrole moiety of 1 with that of 5 [for 5 in DMSO-d6, H-1 at δ 12.0 (brs), H-5 at δ 6.86 (dd, 1.2 and 2.5 Hz), and H-4 at δ 6.94 (dd, 1.2 and 2.5 Hz)]10 was clearly distinctive, confirming the position of the Br at C-5 in 1. Thus, the structure of 1 was assigned to that of 4debromooroidin. 4-Debromougibohlin (2) similarly displayed a 1:1 ratio of [M + H]+ ions in the HRESITOFMS spectrum with m/z of 310.0307 and 312.0287, which corresponded to the molecular formula C11H13BrN5O with eight double-bond equivalents (DBEs). While several brominated sponge compounds with this molecular formula were found in the Dictionary of Natural Products, inspection of the 1H and 13C NMR spectra of 2 (Table 1) indicated that the structure of 2 did not correspond to any known metabolite. There was, however, marked similarity between the NMR spectra of 2 and those of the known compound ugibohlin (7) isolated from the sponge Axinella carteri,11 with the molecular formula of the two compounds differing by the replacement of a Br atom in 7 with a hydrogen observed in 2. The NMR assignments of the 2,3dihydroindolizin-5(1H)-one moiety in 2 were very similar to those assigned to 7. The assignments for the pyrrole moiety in 2 were however different from those of 7, suggesting that 2 had a trisubstituted pyrrole with only one bromine substituent. The location of the bromine substituent in 2 was established by analysis of the 1H, 13C, and HMBC NMR spectra. In the 1H NMR spectrum, both the NH hydrogen at δ 12.94 (H-1) and the C−H hydrogen at δ 6.24 (H-4) proved to be singlets, suggesting that these hydrogens were not vicinal to each other, unlike 5-debromougibohlin (3) (see below). In the HMBC spectrum, the hydrogen resonating at δ 6.24 (H-4) correlated with C-2 (δ 130.9), C-3 (δ 123.8), C-5 (δ 110.5), C-6 (δ 103.2), and C-15 (δ 151.8). The pyrrole NH at δ 12.94 (H-1) correlated with C-2 and C-3, while H-7 at δ 9.32 correlated with C-3, C-6, C-8 (δ 156.8), and C-10 (δ 140.9). These correlations supported the presence of a Br atom at C-5, and the structure of 2 could be unambiguously established. 5-Debromougibohlin (3) displayed a [M + H]+ ion in the HRESITOFMS spectrum that was a doubled signal with a 1:1

complex mixture of bromopyrrole alkaloids, which was separated by a series of chromatographic steps using C18derivatized Si gel, Sephadex LH20, BIOGEL P2, and reversedphase HPLC. These separations afforded the new alkaloids 4debromooroidin (1), 4-debromougibohlin (2), 5-debromougibohlin (3), and 5-bromopalau’amine (4), along with the known hymenidin (5) and (+)-monobromoisophakellin (6). Herein we describe the isolation and structure elucidation of 1−4 and the proteasome inhibitory activity of compounds 1− 6.

4-Debromooroidin (1) displayed a [M + H]+ ion in the HRESITOFMS spectrum with a 1:1 isotopic pattern at m/z 310.0286 and 312.0266, corresponding to the formula C11H13BrN5O, the same as hymenidin (5).10 Inspection of the 1H and 13C NMR data of 1 (Table 1) suggested a close relationship with 5, likely only differing at the position of the bromine on the pyrrole ring. Analysis of HSQC, COSY, and HMBC data confirmed this hypothesis. A correlation in the COSY spectrum between H-3 at δ 6.79 (dd, 2.6 and 3.9 Hz) and H-4 at δ 6.13 (dd, 2.5 and 3.8) suggested that H-3 and H4 were vicinal and the bromine group was at C-5. Comparison

Table 1. NMR Data for Compounds 1−3 in DMSO-d6 (1H: 600 MHz, 13C: 150 MHz) 4-debromooroidin (1) position NH-1 2 3 4 5 6 NH-7 8 9 10 11 12 13 14 15 NH-16

δC, type

δH (J in Hz)

4-debromougibohlin (2) δC, type

12.21, s 127.8, 111.7, 110.8, 102.3, 159.4,

C CH CH C C

39.9, CH2 116.1, CH 126.9, CH 124.8, C

6.79, dd (2.6, 3.9) 6.13, dd (2.5, 3.8)

8.30, 3.95, 6.10, 6.20,

t (5.9) t (5.4) dt (5.3, 16.1) d (16.3)

12.68, brs 147.7, C 111.0, CH

12.05 6.90, s 7.62, s

δH (J in Hz)

5-debromougibohlin (3) δC, type

12.94, s 130.9, 123.8, 102.2, 110.5, 103.2,

C C CH C C

6.24, s

δH (J in Hz) 12.53, s

123.1, C 126.4, C 86.8, C 127.4, CH 102.8, C

7.46, d (1.6) 9.26, s

9.32, s 156.8, C 140.9, C 28.6, CH2 21.6, CH2 48.3, CH2

157.5, C

2.92, m 2.13, qui (7.4) 4.03, s

151.8, C

142.2, C 28.4, CH2 21.6, CH2 48.3, CH2

3.00/2.87, m 2.14, sex (7.6)a 4.11, m/3.97, m

152.6, C 7.49, s

7.57, s

a

The unexpected coupling pattern of CH2-12 in 3 is probably due to magnetic inequivalences of the 11-, 12-, and 13-CH2 protons, which complicate the splitting patterns through second-order effects. 2297

DOI: 10.1021/acs.jnatprod.8b00533 J. Nat. Prod. 2018, 81, 2296−2300

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NMR data with the literature.13 The specific rotation of 6, herein determined to be [α]D +14 (c 0.078, MeOH), has not been previously reported.13 The absolute configuration of (+)-monobromoisophakellin has been assigned as 6(R),10(S) based on analysis of its circular dichroism spectrum (Figure S35) and comparison with the same compound previously isolated from Agelas sp.13 The isolation of new derivatives of oroidin, ugibohlin, and palau’amine, bearing pyrrole groups substituted at C-5, raised a question about the diversity of bromopyrrole alkaloids within marine sponges. Among a total of 230 bromopyrrole isolated from marine sponges,1,2 bromination of pyrroles is prevalent in both positions 4 and 5 (48.2%) and position 4 alone (34.5%). Pyrroles brominated exclusively at position 5 are observed in only 9.2% of the sponge bromopyrrole alkaloids. Our present investigation of Dictyonella sp. led to the isolation of six brominated pyrroles, three being 4-bromosubstituted (3, 5, and 7) and three 5-bromo-substituted (1, 2, and 4). The substitution pattern of Dictyonella sp. bromopyrroles is quite distinct from the pattern observed for these metabolites isolated from other sponges. This uneven pattern of bromopyrrole substitution may be related to the associated microbiota of the marine sponges and other marine organisms,18 but such an assumption still requires experimental evidence. Proteasome Inhibition Activity. Palau’amine (8) and the enantiomeric mixtures of 4,5-dibromophakellin and 4,5dibromophakellstatin have been reported to be inhibitors of the 20S human proteasome, with IC50 values of 2.3 ± 0.01, 18.7, and 6.2 ± 1.6 μM, respectively.19 Derivatives of phakellins were also tested as specific inhibitors of β5 activity of the human constitutive core particle of the 20S proteasome.8 The brominated indole moiety of indolo-phakellin appeared essential for its bioactivity (IC50 of 3.5 μM), because the absence of bromine reduced the inhibitory potency by 7-fold.8 The rigid scaffold of this compound allows for the formation of a strong halogen bond with the proteasome residue Thr21.8 As the compounds isolated from the sponge Dictyonella sp. belong to the class of pyrrole-imidazole and pyrrole-guanidine alkaloids, we screened 1−6 for inhibition of the 20S yeast proteasome. Purified compounds 1−6 were incubated with the 20S proteasome core particle in concentrations ranging from 0.1 to 100.0 μM. Concentration−response curves were constructed, and the IC50 inhibitory values of 1−6 to the ChTL subunit of the proteasome were recorded. 5Bromopalau’amine (4) was the most active (IC50 9.2 ± 3.2 μM). 4-Debromooroidin (1) and hymenidin (5) displayed comparable inhibition of the proteasome (IC50 values of 27 ± 6 and 23 ± 5 μM, respectively). Monobromoisophakellin (6) displayed only weak inhibitory activity (IC50 69 ± 10 μM), while 4-debromo-seco-isophakellin (2) and 5-debromo-secoisophakellin (3) were inactive at the concentrations tested (IC50 > 100 μM) (Supporting Information Figure S37). Both bromination and the position of the bromine substituent in the pyrrole ring seem to influence the inhibitory activity of the proteasome, since 5-bromopalau’amine (4) is 4fold less active than palau’amine (8).19 The only difference between 6 and 3 is the cyclization of the guanidine group. Compound 3 was inactive in the concentration range tested, whereas cyclic guanidine 6 displayed a clear concentration− response curve (Figure S37). Both (±)-4,5-dibromophakellin and (±)-4,5-dibromophakellstatin (Figure S36) were more active than 6,19 where the difference lies in the orientation and

ratio at m/z 310.0309 and 312.0289, corresponding to the molecular formula C11H13BrN5O, isomeric to compound 2. Analysis of its 1H, 13C, HSQC, COSY, and HMBC data and a COSY correlation between H-5 at δ 7.46, (d, 1.6 Hz) and the pyrrole NH at δ 12.53 suggested that 3 was an isomer of 2 with a bromine at C-4. Confirmation of this hypothesis was obtained by analysis of the HMBC spectrum, in which H-5 at δ 7.46 correlated with C-2 (δ 123.1), C-3 (δ 126.4), C-4 (δ 86.8), C-6 (δ 102.8), and C-15 (δ 152.6). Additional differentiation between the structures of compounds 2 and 3 was the comparison of 1H and 13C NMR data of 3 (Table 1) with those of monobromoisophakellin (6)12,13 and of 3bromostyloguanidine,14 indicating the position of bromine at C-4. 5-Bromopalau’amine (4) gave [M + H] + ions by HRESITOFMS at m/z 498.0783, 500.0768, and 502.0735, with an isotopic pattern appropriate for the presence of one Br and one Cl, corresponding to the formula C17H22BrClN9O2, identical to that of 3-bromostyloguanidine14 and 4-bromopalau’amine.15 Detailed analysis of the NMR data of 4 (Table 2) Table 2. NMR Data for 5-Bromopalau’amine (4) in DMSOd6 (1H: 600 MHz, 13C: 150 MHz) position

4 δC, type

2 3 4 5 6 N-H 8 10 11 12 13 15 16 17 18 19 20 N-H 22 23 O-H

124.9, C 114.7, CH 114.9, CH 104.5, C 67.9, CH 157.3, C 80.0, C 56.0, CH 41.2, CH 45.0, CH2 155.4, C 71.0, C 73.9, CH 47.7, CH 40.6, CH2 82.7, CH

4 δH (J in Hz) 6.85, d (3.9) 6.52, d (4.0) 6.48, s 9.81, s

3.15, d (14.5) 2.55, m 4.10, dd (7.6, 10.3)/3.10, t (10.3)

4.42, 2.29, 2.97, 5.74, 9.47,

d (8.5) m dd (8.8, 13.9)/3.15, m d (5.0) s

157.6, C 8.29, s 7.92, s

and comparison with the data reported for the revised structure of palau’amine (8)16 indicated the structure of 4 to be that of the 5-bromo derivative of 8. A detailed analysis of the 1D NOEDiff and ROESY spectra of 4 (Figures S29−S33) confirmed that the relative configuration was identical to that of revised 8.16 Moreover, both the specific rotation of [α]D −81 (c 0.12, MeOH) and ECD spectrum (Figure S34) are consistent with data previously reported for palau’amine (8),17 indicating the same absolute configuration for 4. Although the longer-wavelength Cotton effects (CEs) of 4 are weak [e.g., λ 286 nm (Δε +0.6)], we observed that the stronger, shorterwavelength CE of 4, when recorded in CF3CH2OH [206 nm (−6.8)], may be more generally diagnostic of the absolute configuration of the palau’amines. (+)-Monobromoisophakellin (6) isolated by us from Dictyonella sp. was identified by comparing its 1H and 13C 2298

DOI: 10.1021/acs.jnatprod.8b00533 J. Nat. Prod. 2018, 81, 2296−2300

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Extraction and Isolation. The sponge EtOH extract (11.0 g) was partitioned between MeOH and hexane. The MeOH fraction (8.5 g) was submitted to chromatographic separations, including C18 reversed-phase column chromatography, Sephadex LH-20, and Biogel P-2 gel permeation chromatography and C8 HPLC purification. The procedure led to the isolation of six compounds: 4-debromooroidin (1, 48.0 mg), 4-debromougibohlin (2, 7.3 mg), 5-debromougibohlin (3, 16.0 mg), 5-bromopalau’amine (4, 3.7 mg), hymenidin (5, 9.1 mg), monobromoisophakellin (6, 1.1 mg). A detailed isolation and purification protocol is described in the Supporting Information. 4-Debromooroidin (1): brown, glassy solid; UV (MeOH) λmax (log ε) 213 (3.97), 274 (4.37) nm; IR (film) νmax 3294, 3167, 1681, 1199, 1133, 796, and 719 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 310.0286 and 312.0266 [M + H]+ (calcd for C11H13BrN5O+, 310.0298 and 312.0278). 4-Debromo-seco-isophakellin (2): white powder; UV (MeOH) λmax (log ε) 238 (4.36), 265 (3.70) nm; IR (film) νmax 3132, 1660, 1589, 1433, 1193, and 1134 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 310.0307 and 312.0287 [M + H]+ (calcd for C11H13BrN5O+, 310.0298 and 312.0278). 5-Debromo-seco-isophakellin (3): white powder; UV (MeOH) λmax (log ε) 238 (4.35), 294 (3.64) nm; IR (film) νmax 3338, 3155, 1670, 1593, 1197, and 1141 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 310.0309 and 312.0289 [M + H]+ (calcd for C11H13BrN5O+, 310.0298 and 312.0278). 5-Bromopalau’amine (4): colorless, glassy solid; [α]D −80.7 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 280 (4.41) nm; IR (film) νmax 3388, 1676, 1421, 1197, 1134, and 717 cm−1; ECD (5.8 × 10−4 M, CF3CH2OH, 23 °C) λmax (Δε) 286 (+0.6), 257 (+0.2, sh), 225 (−1.4, sh), and 206 (−6.8) nm; 1H and 13C NMR data, Table 2; HRESIMS m/z 498.0783, 500.0768, and 502.0735 [M + H]+ (calcd for C17H22BrClN9O2+, 498.0763, 500.0743, and 502.0713). Monobromoisophakellin (6): ECD (4.33 × 10−4 M, MeOH) λmax (Δε) 241 (+0.65) and 201 (−4.23) nm; (4.84 × 10−4 M, CF3CH2OH) λmax (Δε) 234 (+1.64) and 197 (−10.0) nm. Bioassay Procedures. The 20S proteasome complex was purified in-house from Saccharomyces cerevisiae, using affinity, ion exchange, and size exclusion chromatography. Purification was guided by ChTL activity using the fluorogenic substrate suc-LLVY-AMC (R&D Biosciences). Briefly, S. cerevisiae cells (50 g) were suspended in buffer A (Tris 25 mM pH 7.5, MgCl2 10 mM, DTT 1 mM), fast frozen in liquid nitrogen drops, and lysed. After centrifugation (45000g for 1 h at 4 °C) and filtration, the lysate (200 mL) was incubated with DEAE AffiGel resin (BioRad) previously equilibrated with buffer A. The proteasome was eluted in a gradient of buffer A supplemented with increasing concentrations of NaCl (up to 150 mM). Next, the ChTL active fractions were subjected to ion exchange chromatography, using a HiTrap Q XL 5 mL column (GE Life Sciences) under a gradient of buffers B (Tris 20 mM pH 7.4, NaCl 100 mM, MgCl2 10 mM, DTT 1 mM) and C (Tris 20 mM pH 7.4, NaCl 500 mM, MgCl2 10 mM, DTT 1 mM). The ChTL active fractions were concentrated to 1 mL and applied to a HiPrep 16/60 Sephacryl S-400 HR size exclusion column (GE Life Sciences) equilibrated with buffer D (Tris 25 mM pH 7.4, NaCl 150 mM, MgCl2 10 mM, DTT 1 mM). After elution, the active fractions were concentrated to 1 mg/mL and used in the inhibition assays. Protein concentration was accessed by absorbance at 280 nm, using the extinction coefficient of ε = 707 355 M−1 cm−1 and the NanoDrop reader (Thermo Scientific). Purified samples were further analyzed by dynamic light scattering (Zetasizer Nano, Malvern), PAGE-SDS, and native PAGE electrophoresis (PhastGel GE Life Sciences) for quality control. Inhibitory curves were constructed by pipetting 1 μL of inhibitor stocks in DMSO in 384-well black V-shape microplates containing 19 μL of the yeast proteasome at 1 μg/mL, in Tris 10 mM pH 7.5/SDS 0.01%. Eleven inhibitory concentrations were used in each curve, with final concentrations ranging from 0.1 to 100 μM, in a dilution factor of 0.3 log. The enzyme was incubated for 1 h with the inhibitor before the addition of 10 μL of the fluorogenic proteasome substrate SucLLVY-Amc in a final reaction concentration of 40 μM. The

bromination of the pyrrole ring. (±)-4,5-Dibromophakellstatin is an urea derivative, the most active in the series that includes alkaloids 2, 3, and 6. It seems that densely cyclized 4, 8, and (±)-4,5-dibromophakellstatin have more pronounced activity. Our structure−activity relationship findings agree with previous investigations on the proteasome inhibitory activity of pyrrole-imidazole and pyrrole-guanidine alkaloids and illustrates that bromination at the pyrrole ring negatively influences the proteasome inhibitory activity.8,19 In addition to the presence of the pyrrole moiety and the polycyclic framework, the cyclic guanidine and urea functionalities appear to be relevant to inhibition of the proteasome. Considering the relevance in finding new inhibitors of the proteasome, this class of secondary metabolites appear to be a valuable model for the elaboration of further proteasome inhibitors. In conclusion, the present investigation reports the isolation of four new monobrominated pyrrole alkaloids from a Dictyonella sp. sponge, three of which possess a bromine substituent at C-5 of the pyrrole moiety, a rather unusual substitution for this class of metabolites isolated from marine sponges. The isolation of 4-debromooroidin adds a new member to this family, the first since 1986, and the isolation of 5-bromopalau’amine is the first new palau’amine since 1996. Both ugibohlin isomers 2 and 3 are the first new analogues isolated since 2001. New and known bromopyrrole alkaloids were assayed as proteasome inhibitors, of which 5bromopalau’amine (4) displayed the highest inhibitory activity. Our results indicate that brominated pyrrole-imidazole and pyrrole-guanidine alkaloids clearly constitute an interesting class of compounds for further investigation as proteasome inhibitors.



EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Polartronic H Schmidt+Haensch polarimeter. Ultraviolet spectra were obtained on a UV-3600 Shimadzu UV spectrophotometer. The samples were diluted in MeOH at a concentration of 0.01 mg mL−1. Circular dichroism spectra were measured on a JASCO J-810 spectropolarimeter using quartz cells (1, 2, or 5 mm path length) at 23 °C. Infrared (IR) spectra were obtained using the Shimadzu model IRAffinity on a silica plate. NMR spectra were obtained at 25 °C, with tetramethylsilane as an internal standard. NMR spectra were recorded on a Bruker AVANCE III spectrometer (9.4 T) operating at either 600 MHz (1H) or 150 MHz (13C). The 1H chemical shifts are referenced to the residual DMSO-d6 (δ 2.49), whereas 13C chemical shifts are referenced to the DMSO-d6 solvent peak (δ 39.5). HRMS measurements were made on a Waters Acquity UPLC H-class liquid chromatograph coupled to a Waters Xevo G2XS QToF mass spectrometer with electrospray interface (ESI) (Waters Corporation). Animal Material. The Dictyonella sp. sponge was collected during an expedition aboard the NHo Cruzeiro do Sul ship (Brazilian Navy) along the equatorial margin of the Amazon River mouth (Pará State) and identified by F. C. Moraes and C. Leal. Dictyonella sp. is a massive erect species, soft and elastic in consistency, with a bright orange color in vivo and circular oscules scattered over the conulose surface. Spicules are abundant, long, and slightly curved subtylostyles/ tylostyles and less abundant styles, arranged in plumose tracts in the choanosome. Taxonomic identification is based on the resemblance of the studied specimens to Dictyonella diagnosis,21 particularly to the external morphology and abundance of the same megasclere spicules in the East Atlantic D. madeirensis.21,22 Vouchers of the collected sponge have been deposited at the Museu Nacional, Universidade Federal do Rio de Janeiro, under registry numbers MNRJ 18802 and MNRJ 18810. This species was previously identified as Pseudosuberites sp.20 2299

DOI: 10.1021/acs.jnatprod.8b00533 J. Nat. Prod. 2018, 81, 2296−2300

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Note

fluorescence was monitored in each well for 1 h, every 2 min, using a Clario (BMG Labtech) or Enspire (PerkinElmer) plate reader. Liquid handling was assisted by the Versette (Thermo Scientific) automated pipetting system. Each inhibitory curve was carried out in three replicates. At least three independent experiments were performed. The percent of remaining enzyme activity was calculated in comparison to a control carried out in the same plate, but in the absence of the inhibitor (DMSO controls). The data were plotted as average ± SEM of the percent of enzyme activity, as a function of the logarithmic concentration of the inhibitor. Curves were fitted using the normalized four-parameter concentration−response equation with variable slope implemented in Prism (Graph Pad, San Diego, v. 7). The IC50 values were extracted from each curve and reported as average and ±SEM of the three independent experiments.



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00533. General experimental procedures, isolation procedures, HRMS, IR, NMR spectra of compounds 1−4, ECD spectra of 4 and 6, SAR of bromopyrrole alkaloids inhibitors of the proteasome, graph of proteasome inhibition and photograph of Dictyonella sp. (PDF)



AUTHOR INFORMATION

Corresponding Author

*Tel: +55-16-33739954. Fax: +55-16-33739952. E-mail: rgsberlinck@iqsc.usp.br. ORCID

Vítor F. Freire: 0000-0001-6565-1830 David E. Williams: 0000-0001-5824-6415 Raymond J. Andersen: 0000-0002-7607-8213 Tadeusz F. Molinski: 0000-0003-1935-2535 Roberto G. S. Berlinck: 0000-0001-8883-6897 Author Contributions #

R. T. M. P. de Souza and V. F. Freire contributed equally.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Financial support was provided by a FAPESP BIOTA/ BIOprospecTA grant (2013/50228-8) to R.G.S.B., by the P&D Program ANP/Brasil (48610.011015/2014-55) to G.M.A.F., and FAPERJ and CNPq grants to G.M.A.F., L.T.S., and R.C.P. D.B.B.T. and R.O.F. thank LBE, LPP, and LEC facilities of LNBio-CNPEM for infrastructure. R.T.M.P.S. and V.F.F. thank CNPq for MSc and PhD scholarships, respectively. R.O.F. thanks CAPES for the MSc scholarship.



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DOI: 10.1021/acs.jnatprod.8b00533 J. Nat. Prod. 2018, 81, 2296−2300