A sample of a sponge identified as Aphrocallistes beatrix beatrix (Gray) [Phylum: Porifera, Class Hexactinellida, Order Hexactinosida, Family Aphrocallistidae] was collected by manned submersible at a depth of 725.4 m approximately 40 nautical miles east of Fort Pierce on deep-water Lophelia coral mounds (latitude 27° 39.430’N, longitude 79° 34.967’W). The sponge is vasiform with the appearance of folded plates and fingers. It is crispy in texture and friable. It was collected growing in association with yellow zoanthids. A reference sample preserved in ethanol has been deposited in the Harbor Branch Oceanographic Museum (catalog number 002:00022, DBMR number 20-V-04-1-005) and is available for taxonomic evaluation by those skilled in the art.
Isolation of Aphrocallistin
The frozen sponge (106 g) was diced and extracted exhaustively with EtOH (Pharmco 100%). The combined EtOH extracts were concentrated to dryness and the residue partitioned between EtOAc and H2O. The EtOAc partition was concentrated to dryness to yield 0.493 g of an oil residue. The residue from the EtOAc partition was chromatographed by vacuum flash chromatography on a custom prepared RP-18 stationary phase using a step gradient of H2O -CH3CN-IPA as eluent. Column size was 150 mL. The eluent series is as follows: fraction 1: 100 mL of H2O - CH3CN (80:20 v/v); fraction 2: 100 mL of H2O - CH3CN (60:40 v/v); fraction 3: 100 mL of H2O - CH3CN (40:60 v/v); fraction 4: 100 mL of H2O- CH3CN (20:80 v/v); fraction 5: 100 mL of CH3CN; Fraction 6: 100 mL of H2O - CH3CN:TFA (20:80:0.1 v/v); Fraction 7: 100% CH3CN; Fraction 8: 100% IPA. Fractions 6 and 7 (84.4 mg) were combined and further purified by medium pressure liquid chromatography on a C-18 reversed-phase stationary phase using a Combiflash Companion (Isco) with the following gradient program [Solvent A: 5% CH3CN in H2O v/v; Solvent B: 100% CH3CN; t= 0 min, A:B (100:0); t=1 min, A: B (100:0); t=11 min, A: B (70:30); t=17 min, A:B (0:100); flow = 15 mL/min; detected by UV absorption observed at 230 nm] to yield 40 mg of aphrocallistin.
Aphrocallistin (1): light brown oil; UV (EtOH) λmax (log ε) 285 (4.0); 1H NMR (600.2 MHz, CD3OD, see ); 13C NMR data (160.9 MHz, CD3OD see ); HRESIMS: m/z observed 539.042786 [M+H]+ (calculated for C20H25 Br2 N6 O2 539.040571).
NMR Spectroscopic Data (600 MHz, CD3OD) for Aphrocallistin (1)
Preparation of N-(4-Hydroxyphenethyl)-N-methylacetamide (7)
Acetamide 6 (550.0 mg, 2.85 mmol) was placed in a microwave tube and dissolved in DMF (14 mL). Sodium hydride (60% suspension in oil, 230.0 mg, 5.75 mmol, 2.0 equiv) was added at room temperature and the reaction mixture was stirred for 15 min until the bubbling ceased. Iodomethane (888 uL, 14.23 mmol, 5.0 equiv) was added and the vessel was sealed and placed in the microwave reactor. The reaction was heated for 5 min at 130°C with stirring. After the heating was completed the vessel was uncapped and another portion of iodomethane was added to it followed by heating. This process was repeated once to assure full consumption of the starting amide. The reaction was worked up following the standard protocol to afford 590 mg (100%) of the desired tertiary amide as an off-white solid. The compound was used without further purification. N-(4-Methoxyphenethyl)-N-methylacetamide: 1H NMR (400 MHz, CDCl3) δ 7.13 (d, J = 8.6 Hz, 2H, minor), 7.06 (d, J = 8.6 Hz, 2H, major), 6.85 (d, J = 8.6 Hz, 2H, major), 6.83 (d, J = 8.6 Hz, 2H, minor), 3.788 (s, 3H, major), 3.786 (s, 3H, minor), 3.54 (dd, J = 7.6, 7.6 Hz, 2H, minor), 3.46 (t, J = 7.1 Hz, 2H, major), 2.93 (s, 3H, minor), 2.89 (s, 3H, major), 2.79 (t, J = 7.1 Hz, 2H, major), 2.78 (dd, J = 7.6, 7.6 Hz, 2H, minor), 2.06 (s, 3H, major), 1.85 (s, 3H, minor). 13C NMR (100 MHz, CDCl3) δ 170.7 (minor), 170.5 (major), 158.5 (major), 158.2 (minor), 131.3 (major), 130.3 (minor), 129.83 (two carbons, minor), 129.81 (two carbons, major), 114.2 (two carbons, major), 114.0 (two carbons, minor), 55.35 (major), 55.32 (minor), 52.9 (major), 50.0 (minor), 37.0 (minor), 33.9 (major), 33.5 (minor), 32.9 (major), 22.1 (minor), 21.1 (major).
To a solution of N-(4-Methoxyphenethyl)-N-methylacetamide (1.05 g, 5.07 mmol) in CH2Cl2 (50 mL) at 0°C BBr3 (1M, 25.33 mL, 25.33 mmol, 5.0 equiv) was added dropwise. The reaction mixture was slowly brought to room temperature and stirred for 2 h then was quenched with H2O. The aqueous phase was extracted with EtOAc once. The combined organic layers were extracted with 1M NaOH. The aqueous layer was neutralized with 1M HCl and extracted with EtOAc, dried (MgSO4) and concentrated in vacuo to afford 870 mg (89%) of the unprotected phenol 7 as an off-white solid: 1H NMR (400 MHz, CDCl3) δ 8.10 (br s, 1H), 7.03 (d, J = 8.5 Hz, 2H, minor), 6.94 (d, J = 8.5 Hz, 2H, major), 6.740 (d, J = 8.5 Hz, 2H, major), 6.736 (d, J = 8.5 Hz, 2H, minor), 3.59 (dd, J = 7.3, 7.3 Hz, 2H, minor), 3.48 (dd, J = 6.3, 6.3 Hz, 2H, major), 2.97 (s, 3H, major), 2.91 (s, 3H, minor), 2.755 (dd, J = 6.3, 6.3 Hz, 2H, major), 2.752 (dd, J = 7.3, 7.3 Hz, 2H, minor), 2.03 (s, 3H, minor), 1.65 (s, 3H, major). 13C NMR (100 MHz, CDCl3) δ 171.9 (major), 171.1 (minor), 155.9 (major), 155.1 (minor), 129.84 (major, two carbons), 129.75 (minor, two carbons; minor, one carbon), 128.9 (major), 115.8 (major, two carbons), 115.4 (minor, two carbons), 52.9 (major), 49.3 (minor), 36.4 (minor), 33.5 (major), 33.5 (major), 32.7 (minor), 21.7 (minor), 20.7 (major).
Preparation of N-(3,5-Dibromo-4-hydroxyphenethyl)-N-methylacetamide (2)
Phenol 7 (510.0mg, 2.64 mmol) was dissolved in glacial acetic acid (5.30 mL) and then treated with Br2 (340 μL, 6.62 mmol, 2.5 equiv) dropwise at room temperature. The reaction went to completion within 3 h, as monitored by TLC and was quenched with 10% aq. Na2S2O3. The product 2 was extracted with EtOAc, dried (MgSO4) and concentrated in vacuo to provide 820 mg (88%) of tan colored material that was used without further purification: 1H NMR (400 MHz, CD3OD) δ 7.37 (s, 2H, minor), 7.36 (s, 2H, major), 3.54 (t, J = 7.0 Hz, 2H, minor), 3.50 (dd, J = 7.7, 7.7 Hz, 2H, major), 2.98 (s, 3H, major), 2.90 (s, 3H, minor), 2.78 (t, J = 7.0 Hz, minor), 2.71 (dd, J = 7.5, 7.5 Hz, 2H, major), 2.05 (s, 3H, major), 1.87 (s, 3H, minor). 13C NMR (100 MHz, CD3OD) δ 173.3, 151.1 (major), 150.8 (minor), 134.6 (major), 134.1 (minor), 133.9 (two carbons, minor), 133.7 (two carbons, major), 112.3 (two carbons, minor), 112.2 (two carbons, major), 53.3 (minor), 50.5 (major), 37.2 (major), 33.8 (minor), 33.7 (minor), 32.8 (major), 21.7 (major), 21.0 (minor).
Phenol 2 (1.54g, 4.39 mmol) was dissolved in CH3CN (22 mL) and treated with K2CO3 (910 mg, 6.58 mmol, 1.5 equiv) at room temperature. The resulting suspension was heated to 50°C and alkyl bromide 8 (1.04 g, 4.37 mmol, 1 equiv) in CH3CN (22 mL) was added to it. The reaction was allowed to proceed overnight, then cooled to room temperature and worked-up following the standard protocol to give 1.78 g (80%) of the desired alkylated phenol as an off-white solid. In addition, the aqueous phase was neutralized with 1M HCl and further extracted with EtOAc (3 × 50 mL), dried (MgSO4) and concentrated in vacuo to yield 120 mg of the unreacted starting material (8% recovery). The product was used without further purification: 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 2H, major), 7.32 (s, 2H, minor), 4.04 (t, J = 5.8 Hz, 2H, minor), 4.03 (t, J = 5.8 Hz, 2H, major), 3.53 – 3.42 (m, 4H), 2.94 (s, 3H, minor), 2.93 (s, 3H, major), 2.77 (t, J = 7.7 Hz, 2H, minor), 2.75 (dd, J = 7.7, 7.7 Hz, 2H, major), 2.08 (s, 3H, major), 2.04 (m, 2H), 1.99 (s, 3H, minor), 1.44 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 170.7 (major), 170.4 (minor), 156.2, 152.1 (minor), 151.6 (major), 138.1 (major), 137.0 (minor), 133.1 (two carbons, major), 132.9 (two carbons, minor), 118.6 (two carbons, minor), 118.3 (two carbons, major), 79.3, 71.4, 52.2 (minor), 49.5 (major), 38.2, 37.1 (major), 33.8 (minor), 33.6 (minor), 32.6 (major), 30.2, 28.6 (three carbons), 22.1 (major), 21.3 (minor). tert-Butyl-3-(2,6-dibromo-4-(2-(N-methylacetamido)ethyl)-phenoxy)propylcarbamate (1.78 g, 3.50 mmol) was dissolved in 1,4-dioxane (40 mL) and the resulting solution was placed in an ice bath. Concentrated HCl (500 μL, 37%) was added dropwise and the bath was removed. The reaction was completed in 1.5 h as revealed by TLC. The pH of the solution was adjusted to 7 by adding 1M NaOH and extracted with EtOAc (3 × 100 mL), dried (MgSO4) and concentrated in vacuo to yield 1.37 g (96%) of 9: 1H NMR (400 MHz, DMSO-d6) δ 8.40 (br s, 2H), 7.63 (s, 2H, major), 7.53 (s, 2H, minor), 4.95 (br s, 1H), 3.98 (t, J = 5.9 Hz, 2H), 3.44 (m, 2H), 3.01 (t, J = 7.4 Hz, 2H), 2.93 (s, 3H, minor), 2.786 (s, 3H, major), 2.785 (m, 2H, minor), 2.69 (dd, J = 7.5, 7.5 Hz, 2H, major), 2.05 (m, 2H), 1.94 (s, 3H, minor), 1.86 (s, 3H, major). 13C NMR (100 MHz, DMSO-d6) δ 169.5 (minor), 169.3 (major), 150.6 (major), 150.4 (minor), 139.0 (minor), 138.6 (major), 133.3 (two carbons, major), 132.9 (two carbons, minor), 117.3 (two carbons, major), 117.2 (two carbons, minor), 70.59 (major), 70.53 (minor), 51.0 (minor), 47.7 (major), 36.5, 35.7 (minor), 32.6 (major), 32.3 (minor), 31.4 (major), 28.4, 21.6 (minor), 20.9 (major).
Preparation of 3-Methyl-3H-purin-6(9H)-one (12)
Sodium metal (1.47 g, 64.0 mmol, 2 equiv.) was dissolved in absolute EtOH (60.0 mL) to prepare 1.0M sodium ethoxide in EtOH. Compound 10 (4.55 g, 32.0 mmol) was added to the solution at room temperature and stirred until all material dissolved. A solution of thiourea 11 (2.88 g, 32.0 mmol) in 42 mL of 2-methoxyethanol was combined with the reaction mixture and the resulting mix was refluxed for 5.5 h. After cooling to 0 °C, 1.0 M HCl was added dropwise until a pH of 2 was obtained. A blue-grey solid formed which was filtered, washed with small portion of ice-cold H2O, and dried in oven overnight to yield 5.10 g (86%) of 6-amino-2-mercapto-1-methyl-5-nitrosopyrimidin-4(1H)-one. The compound was used without purification.
6-Amino-2-mercapto-1-methyl-5-nitrosopyrimidin-4(1H)-one (5.10g, 27.4 mmol) was dissolved in 100 mL 1.0 M NaOH and sodium dithionite (17.65 g, 101.4 mmol, 3.7 equiv.) was added. After 1.5 h at room temperature, the solids were filtered off and dried in oven to obtain 4.7 g of 5,6-diamino-2-mercapto-1-methylpyrimidin-4(1H)-one in quantitative yield. These steps were repeated to provide sufficient material for the last stage of the synthesis of 12.
5,6-Diamino-2-mercapto-1-methylpyrimidin-4(1H)-one (7.74 g, 44.95 mmol) was dissolved in DMF (10 mL) and trimethyl orthoformate (73.76 mL, 674.18 mmol, 15.0 equiv.). The mixture was heated at reflux for 5 days. After full conversion of the starting material was achieved (LC-MS analysis) the reaction mixture was concentrated and the residual material was recrystallized from water to obtain 8.19 g (100%) of compound 12: 1H NMR (400 MHz, DMSO-d6) δ 13.84 (br s, 1H), 12.47 (br s, 1H), 8.17 (s, 1H), 3.78 (s, 3H).
Raney nickel (16.0 g) was washed three times with 2-methoxyethanol, then suspended in minimal H2O. A solution of purinone 12 (8.20 g, 45.0 mmol) in 1.0 M NaOH (55.0 mL) was added to the Raney nickel slurry. The reaction mixture was heated to 80 °C for 3 h until all starting material was consumed as determined by TLC. The room temperature mixture was filtered through a pad of Celite which was washed with H2O and 2-methoxyethanol. The filtrate was concentrated in vacuo. 6.76 g of 13 was obtained which was used without purification: 1H NMR (400 MHz, DMSO-d6) δ 7.86 (s, 1H), 7.32 (s, 1H), 3.66 (s, 3H).
6-Chloro-3-methyl-9H-purin-3-um chloride (14)
To purinone 13 (100 mg, 666 μmol) was added pyridine (5.45 mL) and phosphorus pentasulfide (454.5 mg, 2.04 mmol, 3.1 equiv.) and the reaction was refluxed for 4 h and then concentrated in vacuo. To the resulting brown solid, boiling water was added. Suspension formed which was allowed to cool to room temperature. The solids were filtered off and dried in oven overnight to deliver 56 mg (51%) of the corresponding purine-6(9H)-thione: 1H NMR (400 MHz, DMSO-d6) δ 13.65 (br s, 1H), 8.42 (s, 1H), 8.36 (s, 1H), 3.86 (s, 3H).
3-Methyl-3H-purine-6(9H)-thione (5.34 g, 32.13 mmol) was combined with 2.5 % aqueous NaOH (66.74 mL, 1.3 equiv.) and iodomethane (4.00 mL, 64.26 mmol, 2.0 equiv.) at room temperature. The resulting mixture was stirred for 2 h at room temperature during which time a white solid precipitated. The precipitate was filtered off and dried to deliver 3.56 g (61%) of the methylthio product: 1H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H), 8.15 (s, 1H), 4.11 (s, 3H), 2.74 (s, 3H).
Chlorine gas was bubbled through ice-cold anhydrous MeOH (2.5 mL). To this saturated solution, 3-methyl-6-(methylthio)-3H-purine (215.0 mg, 1.19 μmol) in MeOH (1.5 mL) was slowly added, after which Cl2 was bubbled through the solution for an additional 10 min. The resultant precipitate was filtered, washed with cold benzene and dried in vacuo to afford a light yellow solid salt 14 (70 mg, 29%).
Preparation of Aphrocallistin (1)
A solution of amine 9 (139 mg, 341 μmol) in 1-butanol (5 mL) and Hünig’s base (130 μL, 751 μmol) was cooled to 0 °C. To this solution, salt 14 (70 mg, 341 μmol) was added and stirred at 0 °C for 1 h after which the solution was warmed to room temperature and stirred for 16 h. The reaction was quenched with brine and the pH was adjusted to 9 with saturated Na2CO3 solution and extracted with EtOAc (3 × 20mL), dried (MgSO4), and concentrated in vacuo.
was dissolved into anhydrous methanol (5 mL) and TFA (100 μL) was added. The solution was stirred for 30 min at room temperature. The reaction was concentrated in vacuo
, redissolved in MeOH (5 mL) and concentrated in vacuo
(2×). The resulting yellow oil was purified via reverse phase HPLC to obtain 70 mg of 1
(31% unoptimized yield). Spectral analysis (1
H-NMR and LCMS- See Supporting Information
) confirmed the synthetic material to be identical with the natural product.
Aphrocallistin was analyzed as to its effects on proliferation of A549 human lung adenocarcinoma, AsPC-1 human pancreatic adenocarcinoma, PANC-1 human pancreatic carcinoma, BxPC-3 pancreatic adenocarcinoma, MIA PaCa2 human pancreatic carcinoma, DLD-1 human colorectal adenocarcinoma, NCI-ADR-RES (formerly MCF-7/ADR) human ovarian carcinoma and the Vero monkey kidney cell lines. The A549 (ATCC# CCL-185), PANC-1 (ATCC# CRL-1469), DLD-1 (ATCC# CCL-221), AsPC-1 (ATCC# CRL-1682), BxPC-3 (ATCC# CRL-1687), MiaPaca2 (ATCC# CRL-1420) and Vero (ATCC# CCL-81) cell lines were obtained from the American Type Culture Collection (Rockville, MD). The NCI/ADR-RES cell line was obtained from the NCI Cancer Cell Repository. Assays were run using protocols described previously.13
All samples were assayed a minimum of three times to derive the final IC50
Cell Cycle Analysis
PANC-1 human pancreatic carcinoma cells were used as targets to observe the effects of aphrocallistin on the cell cycle. Cell cycle analysis was performed as follows: PANC-1 cells were incubated in tissue culture media (TCM = Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, 60 mg/mL L-glutamine, 18 mM HEPES, 0.05 mg/mL gentamicin, 100 μg/mL sodium pyruvate, 2.5 mg/mL glucose and 10% fetal bovine serum) at 37°C in 5% CO2 in air in the presence or absence of varying concentrations of aphrocallistin, methanol (vehicle control) or paclitaxel (positive control) for 24 h. At the end of this incubation, cells were harvested, fixed in ethanol and stained with 0.02 mg/mL of propidium iodide (P.I.) together with 0.1 mg/mL of RNAse A. Stained preparations were analyzed on a BD FACSCanto. 10,000 events were collected per sample. Raw histogram data was further analyzed using the DNA analysis software ModFit (Verity, Topsham, ME).
The antimicrobial activity of 1 was determined through the use of broth dilution assays using the following strains identified by their ATCC catalog number: Candida albicans 44506; methicillin-resistant Staphylococcus aureus 700787; and Pseudomonas aeruginosa 27853. Assays were performed as standard microtiter assays in a total volume of 50μL at a seeding density of 103/mL. C. albicans was grown in Sabouraud Dextrose Broth. The bacteria were grown in cation-supplemented Mueller-Hinton broth. Assays were performed at 37°C. The level of growth was determined after 24 h with the minimum inhibitory concentration being defined as the lowest concentration of the compound which completely inhibited growth of the test strain. 1 gave MICs of 250 μg/mL against C. albicans and methicillin-resistant S. aureus and >500 μg/mL against P. aeruginosa.