Practical Total Synthesis of Fuligocandins A and B

Abstract

Fuligocandins A and B, cycloanthranilylproline derivatives isolated from the myxomycete Fuligo candida, were synthesized efficiently by a Meyer-Schuster rearrangement, which gave Z-isomer with high selectivity. The absolute stereochemistry at C-11a of the natural products was determined to be S by comparison of the optical rotation of the natural products with synthetic counterparts derived from l-proline. This report is the first total synthesis of optically active fuligocandin B.

Key words

total synthesis - fuligocandin - Meyer-Schuster rearrangement - myxomycete - TRAIL

References and Notes

• 1 Nakatani S. Yamamoto Y. Hayashi M. Komiyama K. Ishibashi M. Chem. Pharm. Bull.  2004,  52:  368 
• 2 Hasegawa H. Yamada Y. Komiyama K. Hayashi M. Ishibashi M. Sunazuka T. Izuhara T. Sugahara K. Tsuruda K. Masuda M. Takasu N. Tsukasaki K. Tomonaga M. Kamihira S. Blood  2007,  110:  1664 
• 3 For a reported synthesis of fuligocandin A, see: More SS. Shanmughapriya D. Lingam Y. Patel NB. Synth. Commun.  2009,  39:  2058 
• 4 For the synthesis of racemic fuligocandins A and B, see: Pettersson B. Hasimbegovic V. Bergman J. Tetrahedron Lett.  2010,  51:  238 
• 5 For a reported Meyer-Schuster rearrangement of N-Boc lactam, see: Sun C. Lin X. Weinreb S. J. Org. Chem.  2006,  71:  3159 
• 7 A Meyer-Schuster rearrangement of N-alkylated succinimide gave E-isomer as a main product: Mamouni A. Daïch A. Decroix B. Synth. Commun.  1998,  28:  1839 

6

NOE (5.0%) was observed between α-proton of α,β-unsaturated ketone and a proton at C-1 position in five-membered ring.

8

The ee was determined by a chiral phase HPLC; CHIRALPAK AD-RH, MeOH-H₂O = 75:25, flow rate: 1 mL/min, λ = 254 nm detection, t _R = 11.5 min [(ent)-1)], t _R = 14.3 min (1) {or CHIRALPAK IA, hexane-i-PrOH = 4:1, flow rate: 0.5 mL/min, λ = 254 nm detection, t _R = 18.6 min [(ent)-1)], tR = 29.7 min (1)}. Natural product 1: >99% ee.

9

Racemization was checked by comparing retention times of 8 with 9, 10 with 11 by CHIRALPAK AD-RH.

10

Optical rotation of (ent)-1 (52% ee): [α]^¹8 _D -224 (c = 1.4, MeOH).

11

Compound 1 which was synthesized at 0 ˚C was used for the synthesis of 2. Therefore, the ee of 2 was not decreased by this reaction. The ee was determined by a chiral phase HPLC; CHIRALPAK AD-RH, MeOH-H₂O = 1:1, flow rate: 1 mL/min, λ = 370 nm detection, t _R = 56.3 min [(ent)-2], t _R = 68.1 min (2) {or CHIRALPAK IA, hexane- i-PrOH = 4:1, flow rate: 0.5 mL/min, λ = 370 nm detection, t _R = 58.7 min [(ent)-2)], t _R =79.1 min (2)}. Natural product 2: >99% ee.

12

Optical rotation of (ent)-2 (57% ee): [α]^¹8 _D -181 (c = 0.6, MeOH).

13

( S )-1-(2- tert -Butoxycarbonylaminobenzoyl)pyrrolidine-2-carboxylic Acid Methyl Ester (6): A solution of 3 (167 mg, 0.70 mmol), PyBOP (474 mg, 0.91 mmol), and DIPEA (0.48 mL, 2.80 mmol) in CH₂Cl₂ (7.0 mL) was cooled to 0 ˚C under an argon atmosphere. The mixture was stirred for 10 min, and then l-proline methyl ester hydrochloride (4, 151 mg, 0.91 mmol) was added. The reaction mixture was allowed to warm to r.t. and stirred for 1 h. The reaction mixture was quenched with 5% aq KHSO₄ solution and extracted with EtOAc. The combined organic layers were washed with sat. aq NaHCO₃ solution, brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was chromatographed on silica gel (hexane-EtOAc, 1:1) to afford 6 (210 mg, 0.60 mmol, 86%) as a white amorphous solid. Data for 6: ^¹H NMR (400 MHz, CDCl₃): δ = 8.37 (br s, 1 H), 8.18 (d, 1 H, J = 8.3 Hz), 7.36-7.40 (m, 2 H), 7.01 (t, 1 H, J = 7.6 Hz), 4.67-4.71 (m, 1 H), 3.79 (s, 3 H), 3.58-3.64 (m, 1 H), 3.48-3.54 (m, 1 H), 2.32-2.41 (m, 1 H), 1.95-2.08 (m, 2 H), 1.85-1.92 (m, 1 H), 1.50 (s, 9 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 172.6, 168.8, 153.0, 137.3, 131.0, 127.2, 123.5, 121.6, 120.2, 80.3, 59.1, 52.5, 50.0, 29.4, 28.3, 25.3. IR (ATR): 3336, 2978, 1730, 1625, 1588, 1522, 1456, 1416, 1242, 1199, 1158 cm^-¹. EIMS: m/z = 348 [M⁺]. HRMS (EI): m/z [M⁺] calcd for C₁₇H₂₄N₂O₅: 348.1685; found: 348.1689.
(11a S )-10- tert -Butoxycarbonyl-2,3-dihydro-1 H -pyrrolo-[2,1- c ][1,4]benzodiazepine-5,11 (10H,11aH)dione (8):
To a stirred solution of 6 (210 mg, 0.60 mmol) in solvent (THF-MeOH-H₂O = 3:1:1, 2.0 mL) was added LiOH×H₂O (50 mg, 1.2 mmol) at 0 ˚C. The solution was stirred for 5 h at r.t. The reaction mixture was acidified with 1 M aq HCl to pH 2, then extracted with EtOAc. The organic layer was washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo to afford (S)-1-(2-tert-butoxycarbonyl-­aminobenzoyl)pyrrolidine-2-carboxylic acid (195 mg) as a white powder. To a solution of the carboxylic acid and PyBOP (406 mg, 0.75 mmol) in CH₂Cl₂ (20 mL) was added DIPEA (0.42 mL, 2.32 mmol) at 0 ˚C under an argon atmosphere. The reaction mixture was allowed to warm to r.t. and stirred for 72 h. The reaction mixture was quenched with 5% aq KHSO₄ and extracted with EtOAc. The organic layer was washed with sat. aq NaHCO₃ solution, brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was chromatographed on silica gel (hexane-EtOAc = 1:5) to afford 8 (148 mg, 0.47 mmol, 79% in 2 steps) as a white amorphous solid. Data for 8: [α]^¹4 _D +157 (c = 1.0, MeOH). ^¹H NMR (400 MHz, CDCl₃): δ = 7.91 (dd, 1 H, J = 1.5, 7.8 Hz), 7.51 (dt, 1 H, J = 1.5, 7.8 Hz), 7.41 (dt, 1 H, J = 1.2, 7.8 Hz), 7.23 (dd, 1 H, J = 1.2, 7.8 Hz), 4.06-4.15 (m, 1 H), 3.82-3.87 (m, 1 H), 3.52-3.59 (m, 1 H), 2.69-2.73 (m, 1 H), 2.09-2.16 (m, 1 H), 1.94-2.07 (m, 2 H), 1.46 (s, 9 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 169.1, 164.9, 150.6, 135.3, 131.2, 130.9, 129.7, 127.4, 126.1, 84.6, 58.8, 46.6, 27.5, 26.3, 23.5. IR (ATR): 2979, 1773, 1748, 1716, 1646, 1457, 1416, 1238, 1144 cm^-¹. EIMS: m/z = 316 [M⁺]. HRMS: (EI): m/z [M⁺] calcd for C₁₇H₂₀N₂O₄: 316.1423; found: 316.1431.
(11a S )-10- tert -Butoxycarbonyl-2,3-dihydro-11-hydroxy-11-prop-1-ynyl-1 H -pyrrolo[2,1- c ][1,4]benzodiazepine-5,11 (10H,11aH)dione (10): To a solution of prepared LDA (9.0 mmol) in THF (90 mL) was added 1,2-dibromopropane (0.31 mL, 3.0 mmol) at -78 ˚C under an argon atmosphere. The solution was stirred for 10 min at this temperature, and another 20 min at 0 ˚C. The resulting lithium acetylide solution was then transferred to a THF solution of 8 (624 mg, 2.0 mmol) via cannula at -78 ˚C. The mixture was then stirred at -78 ˚C for 40 min and sat. aq NH₄Cl solution was added slowly. The mixture was extracted with EtOAc and the organic extracts were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was chromatographed on silica gel (hexane-EtOAc, 2:1) to afford a single isomer 10 (329 mg, 0.92 mmol, 46%) as a white amorphous solid. Data for 10: ^¹H NMR (400 MHz, CDCl₃): δ = 8.44 (br s, 1 H), 8.21 (d, 1 H, J = 7.6 Hz), 7.38 (t, 1 H, J = 7.6 Hz), 7.02 (t, 1 H, J = 7.6 Hz), 4.80-4.84 (m, 1 H), 3.52-3.66 (m, 2 H), 2.30-2.40 (m, 1 H), 2.06 (s, 3 H), 1.83-2.11 (m, 3 H), 1.51 (s, 9 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 185.6, 168.9, 153.0, 137.6, 131.2, 127.3, 123.1, 121.5, 120.2, 92.9, 80.4, 66.5, 29.7, 28.5, 28.3, 25.2, 4.3. IR (ATR): 3336, 2926, 2220, 1727, 1521, 1409, 1340, 1157, 756 cm^-¹. EIMS: m/z = 356 [M⁺]. HRMS (EI): m/z [M⁺] calcd for C₂₀H₂₄N₂O₄: 356.1736; found: 356.1730.
Fuligocandin A (1): To a solution of 10 (10 mg, 33.0 mmol) in CH₂Cl₂ (0.8 mL) was added TFA (0.8 mL) at -20 ˚C. The reaction mixture was stirred for 4 h at -20 ˚C. After addition of toluene, TFA was carefully evaporated in vacuo at r.t. To this mixture was added sat. aq NaHCO₃ solution, then the mixture was stirred at r.t. The mixture was extracted with CH₂Cl₂ and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was chromatographed on silica gel (hexane-EtOAc = 1:1) to afford 1 (7.6 mg, 29.7 µmol, 90%, 70% ee). The ee value was determined by CHIRALPAK AD-RH (46 × 150 mm); eluent: 75% MeOH; flow rate: 1.0 mL/min; t _R: (ent)-1 (14.4 min), 1 (22.6 min). Data for 1: [α]^¹8 _D +225 (c = 1.4, MeOH). ^¹H NMR (400 MHz, CDCl₃): δ = 12.6 (br s, 1 H), 7.97 (dd, 1 H, J = 1.5, 7.7 Hz), 7.45 (dt, 1 H, J = 1.5, 7.9 Hz), 7.21 (dt, 1 H, J = 1.5, 7.9 Hz), 7.02 (d, 1 H, J = 7.9 Hz), 5.29 (s, 1 H), 4.30 (d, 1 H, J = 7.9 Hz), 3.78-3.85 (m, 1 H), 3.61-3.69 (m, 1 H), 2.39-2.41 (m, 1 H), 2.20 (s, 3 H), 2.06-2.16 (m, 3 H). ^¹³C NMR (100 MHz, CDCl₃): δ = 198.4, 165.7, 158.8, 137.1, 132.4, 131.1, 127.2, 124.4, 122.1, 91.1, 55.3, 46.9, 30.0, 27.0, 23.4. IR (ATR): 2987, 2877, 1629, 1591, 1557, 1406, 1262, 1250, 754, 733, 696, 683 cm^-¹. EIMS: m/z = 256 [M⁺]. HRMS (EI): m/z [M⁺] calcd for C₁₅H₁₆N₂O₂: 256.1212; found: 256.1211.
Fuligocandin B (2): To a solution of prepared LDA (0.79 mmol) in THF (4.0 mL) was added 1 (51 mg, 0.20 mmol) in THF via cannula at -78 ˚C under an argon atmosphere. The solution was stirred for 30 min at this temperature. The resulting solution was then transferred to a THF solution of 20 (298 mg, 1.00 mmol) via cannula at -78 ˚C. The solution was stirred for 20 min at this temperature. The reaction mixture was allowed to warm to 0 ˚C. The reaction mixture was stirred for 1 h at 0 ˚C and H₂O was then added. The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was chromatographed on silica gel (hexane-EtOAc = 10:1 → 1:1) to afford a white solid. The solution of white solid in solvent (THF-1 N aq HCl = 1:1, 5.0 mL) was stirred for 17 h at r.t. and sat. aq NaHCO₃ was then added to it and the mixture was then extracted with EtOAc. The organic layer was washed with H₂O, brine, dried over Na₂SO₄, filtered and concentrated
in vacuo. The residue was chromatographed on silica gel (hexane-EtOAc = 1:2 → 0:1) to afford 2 (48 mg, 0.12
mmol, 63%, 61% ee). The ee value was determined by CHIRALPAK AD-RH (46 × 150 mm); eluent: 50% MeCN; flow rate: 1.0 mL/min; t _R: (ent)-2 (56.3 min), 2 (68.1 min). Data for 2: [α]^¹8 _D +248 (c = 0.6, MeOH). ^¹H NMR (400 MHz, acetone-d ₆): δ = 13.4 (br s, 1 H), 10.9 (br s, 1 H), 8.02 (d, 1 H, J = 8.3 Hz), 7.91 (d, 1 H, J = 15.8 Hz), 7.89 (dd, 1 H, J = 1.7, 7.8 Hz), 7.83 (d, 1 H, J = 2.7 Hz), 7.49-7.54 (m, 2 H), 7.17-7.24 (m, 3 H), 7.13 (d, 1 H, J = 8.1 Hz), 7.02 (d, 1 H, J = 15.8 Hz), 5.81 (s, 1 H), 4.44 (d, 1 H, J = 7.6 Hz), 3.67-3.72 (m, 1 H), 3.55-3.62 (m, 1 H), 2.56-2.63 (m, 1 H), 2.22-2.32 (m, 1 H), 2.05-2.16 (m, 2 H). ^¹³C NMR (100 MHz, acetone-d ₆): δ = 190.2, 165.8, 160.4, 138.7, 138.6, 134.9, 133.1, 131.9, 128.2, 126.4, 124.3, 123.8, 123.4, 122.6, 121.6, 121.2, 114.4, 113.0, 93.4, 56.1, 47.4, 27.6, 24.1. IR (ATR): 3250, 1590, 1567, 1270, 1247, 1137, 1106, 754, 738 cm^-¹. HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₄H₂₁O₂N₃Na: 406.1514; found: 406.1526.
TRAIL-Resistance-Overcoming Activity: To assess the sensitivity to TRAIL, cell viability was evaluated using a fluorometric microculture cytotoxicity assay (FMCA). Briefly, AGS cells were seeded in a 96-well plate (6 × 10^³ cells per well) in 200 µL of RPMI medium containing 10% FBS. Cells were cultured in 24 h and then treated with test samples in the absence or presence of TRAIL (100 ng/mL), and control cells were treated with culture medium containing 0.1% DMSO. After 24 h incubation, the cells were washed with PBS, and 200 µL of PBS containing fluorescein diacetate (10 µg/mL) was added to each well. After 1 h incubation at 37 ˚C, fluorescence was measured in a 96-well scanning spectrofluorometer (Thermo Electron Corporation) at 538 nm, following excitation at 485 nm.