Protecting Group-Free Syntheses of (4S,5S,11R)- and (4S,5S,11S)-iso-Cladospolide B and Their Biological Evaluation

Abstract

Short and efficient total syntheses of (4S,5S,11R)- and (4S,5S,11S)-iso-cladospolide B were achieved in five steps each without using any protecting groups. The key steps were an alkyne-zipper reaction, a Suzuki cross coupling, and a Sharpless asymmetric dihydroxylation. The biological activities of both natural products toward various cancer cell lines were tested for the first time.

The natural product iso-cladospolide B (1) was first isolated in 2000 by Ireland and co-workers from fermentations of the marine fungal strain 196S215.[ ¹ ] At the time, they identified its structure as that of a butenolide moiety substituted at the γ-position with an aliphatic substituent bearing two hydroxy groups, but they did not identify the relative or absolute stereochemistry of the three asymmetric carbon atoms. The first synthesis of iso-cladospolide B (1) was achieved by Figadère and co-workers, who proposed the absolute configurations for the three asymmetric carbons as 4S, 5S, and 11R, respectively.[ ² ] Following the announcement of this stereochemistry, another four syntheses were reported.[ ³ ] Later, in 2005, the absolute stereochemistry of a sample of iso-cladospolide B from an ethyl acetate extract of a Cladosporium sp. obtained from a different source, the Red Sea sponge Niphates rowi, was assigned as 4S,5S,11S (2) by Riguera’s method and by studies of its circular dichroism.[ ⁴ ] However, immediately after this second isolation, Trost and Aponick prepared both (4S,5S,11R)-iso-cladospolide and (4S,5S,11S)-iso-cladospolide, and they found that the spectral data and the optical rotations matched those of the two natural products; these authors therefore suggested that the two compounds are diastereomeric natural products and that the (4S,5S,11S)-isomer should be referred as 11-epi-iso-cladospolide B.[ ⁵ ] Since then, five other total syntheses of isomer 2 have been reported.[ ⁶ ] However, all the strategies for preparing isomers 1 and 2 rely on the use of at least one protecting group during the synthesis. Furthermore, most of these syntheses involve the use of tartaric acid derivatives as substrates with a cross metathesis reaction as the key step.

The development of a new strategy that does not require the use of any protecting group is therefore an important challenge. Protecting group-free synthesis is currently gaining prominence as a practical and intellectual challenge in the field of total synthesis.[ ⁷ ] In continuation of our work on the syntheses of bioactive macrolides by alkyne-assisted chemistry,[ ⁸ ] we report short and protecting group-free syntheses of diastereomers 1 and 2 in which an alkyne-zipper reaction, a Suzuki coupling, and a Sharpless asymmetric dihydroxylation serve as the key steps.

We began our synthesis (Scheme [1]) from the (2R)-2-methyloxirane (3), which was transformed into alkyne 5 by means of a modified form of a procedure reported in the literature.[ ^9a ] Treatment of epoxide 3 with hex-1-yne in the presence of butyllithium and hexamethyl­phosphoramide in tetrahydrofuran gave the alkynol 4 in 84% yield. Alkynol 4 was subjected to a potassium 3-aminopropylamide (KAPA)-mediated alkyne-zipper reaction, during which the alkyne functionality moved from the internal position to give the terminal alkyne 5 in 81% yield.[ ⁹ ] Our next task was to prepare (Z,E)-dienoate 8, a precursor of the target compound. Our initial attempts to convert 5 into 8 by means of a Stille coupling provided an inseparable 93:7 mixture of the Z,E- and Z,Z-stereomers.[ ¹⁰ ] In an alternative route, we converted alkyne 5 into the (E)-vinylboronic acid 6 by hydroboration with dibromoborane–dimethyl sulfide in dichloromethane, followed by hydrolysis with water/diethyl ether.[ ¹¹ ] Thallium ethoxide-promoted Suzuki cross coupling of the (E)-vinylboronic acid 6 with the Z-vinyl iodide 7 (obtained in one step from ethyl propiolate)[ ¹² ] gave the (Z,E)-dienoate 8 exclusively in 82% yield.[ ¹³ ] Finally, the dienoate 8 was subjected to Sharpless asymmetric dihydroxylation using AD-mix-α to give the desired (4S,5S,11R)-iso-cladospolide B (1) in 68% yield.[6d] [14] The spectral data for the synthetic compound 1 were identical with those previously reported, and the optical rotation [–91.5 (c 1.0, MeOH)] was comparable with that of the natural product [–91.0 (c 0.23, MeOH)].[1] [3]

Encouraged by our successful synthesis of (4S,5S,11R)-iso-cladospolide B (1), we attempted a synthesis of the second isomer (4S,5S,11S)-iso-cladospolide B (2), with the aim of evaluating the biological activities of the two isomers. (2S)-2-Methyloxirane (3a) was transformed into the desired product 2 by a similar sequence of reactions to that used to prepare its isomer 1 (Scheme [2]). The spectral data for synthetic product 2 were identical with those previously reported, and the optical rotation [–58.2 (c 0.6, MeOH)] was comparable with that of the natural product [–61.6 (c 16.6, MeOH)].[4] [5] [6]

The biological activities of these two compounds had not been previously evaluated. We therefore tested 1 and 2 against a panel of cancer cell lines (A549, Neuro2a, HeLa, MDA-MB-231, and MCF-7) by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.[ ¹⁵ ] Both compounds showed promising inhibition of the proliferation of the A549 cancer cell line (a human alveolar adenocarcinoma). It was noteworthy that (4S,5S,11R)-iso-cladospolide B (1) was a more potent inhibitor (IC₅₀ = 6.8 μM at 24 h) than its (4S,5S,11S)-isomer 2 (IC₅₀ = 21.04 μM at 24 h). Furthermore, isomer 2 showed some inhibitory activity (IC₅₀ = 15.08 μM at 24 h) for the Neuro2a cancer cell line (murine neuroblastoma), whereas 1 showed no such inhibitory activity. Neither 1 nor 2 exhibited cytotoxicity toward HeLa, MDA-MB-231, or MCF-7 cancer cell lines, and neither showed any antibacterial activity or antifungal activity.[ ¹⁶ ]

In summary, we accomplished efficient and protecting-group-free stereoselective total syntheses of (4S,5S,11R)- and (4S,5S,11S)-iso-cladospolide B in five steps each (19% overall yield). The synthesis provides a short route to the natural product and employs an alkyne-zipper reaction, a Suzuki cross coupling, and a Sharpless asymmetric dihydroxylation as key parts of the sequence. Additionally, we performed biological evaluations of both natural products for the first time, and we found they inhibited the proliferation of the A549 cancer cell line. We expect that our alkyne-based strategy will find applications in the synthesis of compounds of a similar class, which we are currently studying.

All the reagents and solvents were of reagent grade and used without further purification unless otherwise stated. Technical-grade EtOAc and PE used for column chromatography were distilled before use. THF, when used as solvent for the reactions, was freshly distilled from sodium benzophenone ketyl. Column chromatography was carried on silica gel (60–120 mesh) packed in glass columns. All the reactions were performed under N₂ in flame- or oven-dried glassware with magnetic stirring. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃, DMSO-d ₆, or CD₃OD solvent on Bruker 300 MHz (Avance) and Varian Unity 500 MHz (Innova) spectrometers at ambient temperature. Chemical shifts are reported in ppm relative to TMS as internal standard. FTIR spectra were recorded on a Perkin-Elmer 683 infrared spectrophotometer, neat or as thin films in KBr. Optical rotations were measured on an Anton Paar MLP 200 modular circular digital polarimeter by using a 2-mL cell with a path length of 1 dm. Low-resolution MS were recorded on an Agilent Technologies LC-MSD trap SL spectrometer and HRMS were recorded on an Agilent technologies 6510 Q-TOF LC-MS spectrometer.

(2R)-Non-4-yn-2-ol (4)

A 1.6 M soln of BuLi (10.7 mL, 17.24 mmol) was added dropwise to a stirred soln of hex-1-yne (2.13 g, 25.9 mmol) in anhyd THF (15 mL) at –40 °C. The mixture was warmed to 0 °C over 30 min and then recooled to –20 °C. Anhyd HMPA (4 mL) was added, followed by a soln of (2R)-2-methyloxirane (1.0 g, 17.24 mmol) in HMPA (4 mL) added over 10 min at –20 °C. The resulting mixture was stirred at –20 °C for 30 min, warmed to r.t. over 4 h, and stirred for 12 h at r.t. The mixture was then poured into ice-cold H₂O (30 mL) and extracted with Et₂O (4 × 50 mL). The organic layers were combined, washed with brine (30 mL), dried (Na₂SO₄), filtered, concentrated, and purified by column chromatography [silica gel, hexane–Et₂O (80:20)] to give a yellow liquid; yield: 2.0 g (84%); [α]_D ²⁰ –10.6 (c 1.0, CHCl₃)

IR (KBr): 3383, 2927, 2857, 1459, 1114, 1082 cm^–1.

¹H NMR (300 MHz, CDCl₃): δ = 3.85 (sextet, J = 6.0 Hz, 1 H), 2.38–2.28 (m, 1 H), 2.28–2.22 (m, 1 H), 2.19–2.12 (m, 2 H), 1.83 (br s, 1 H, OH), 1.59–1.34 (m, 4 H), 1.22 (d, J = 6.8 Hz, 3 H), 0.93 (t, J = 6.8 Hz, 3 H).

¹³C NMR (75 MHz, CDCl₃): δ = 82.8, 76.0, 66.4, 30.9, 29.1, 21.9, 21.7, 18.2, 13.4.

(2R)-Non-8-yn-2-ol (5)

A 35% suspension of KH in mineral oil (7.6 g, 57.1 mmol) was weighed into an oven-dried round-bottomed flask and washed with Et₂O (3 × 5 mL) under N₂. To the oil-free KH was added propane-1,3-diamine (45 mL) at r.t. The mixture was heated for 1 h at 40 °C then cooled to 0 °C. Alcohol 4 (2.0 g, 14.3 mmol) was added at 0 °C and the mixture was allowed to warm to r.t. and stirred for 5 h. When the reaction was complete (TLC), the mixture was cooled to 0 °C and quenched with ice. The aqueous layer was extracted with Et₂O (2 × 50 mL). The organic layers were combined, washed successively with 2 M aq HCI (25 mL) and H₂O (25 mL), dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was purified by column chromatography [silica gel, hexane­–Et₂O (80:20)] to give a colorless oil; yield: 1.62 g (81%); [α]_D ²⁰ –6.4 (c 1.2, CHCl₃).

IR (KBr): 3309, 2929, 2856, 2116, 1461, 1373, 1125 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 3.80–3.71 (m, 1 H), 2.17 (td, J = 7.1, 2.3 Hz, 2 H), 1.84 (t, J = 2.3 Hz, 1 H), 1.58–1.49 (m, 2 H), 1.49–1.36 (m, 6 H), 1.17 (d, J = 6.0 Hz, 3 H).

¹³C NMR (75 MHz, CDCl₃): δ = 84.5, 68.1, 67.8, 39.0, 28.6, 28.3, 25.1, 23.3, 18.2.

[(1E,8R)-8-Hydroxynon-1-en-1-yl]boronic Acid (6)

A 1 M soln of BHBr₂·SMe₂ in CH₂Cl₂ (8.57 mL, 8.57 mmol) was added dropwise to a soln of alkyne 5 (800 mg, 5.7 mmol) in CH₂Cl₂ (6 mL) at 0 °C. The mixture was stirred at r.t. for 4 h then cooled to 0 °C and poured into a mixture of H₂O (10 mL) and Et₂O (30 mL) at 0 °C. The resulting mixture was stirred at r.t. for 30 min. The organic phase was washed successively with H₂O (10 mL) and brine (10 mL), dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (30% EtOAc in hexanes to 5% MeOH in CH₂Cl₂) to give a white foam; yield: 542 mg (51%).

¹H NMR (500 MHz, DMSO-d ₆): δ = 6.42 (dt, J = 18.0, 6.0 Hz, 1 H), 5.30 (d, J = 18.0 Hz, 1 H), 3.59–3.52 (m, 1 H), 2.05 (q, J = 7.0 Hz, 2 H), 1.38–1.15 (m, 8 H), 1.02 (d, J = 6.0 Hz, 3 H).

¹³C NMR (75 MHz, DMSO-d ₆): δ = 150.1, 125.3, 65.8, 39.0, 35.0, 28.8, 28.1, 25.2, 23.6.

Ethyl (2Z,4E,11R)-11-Hydroxydodeca-2,4-dienoate (8)

EtOTl (530 mg, 2.12 mmol) ( CAUTION: toxic) was added dropwise to a soln of boronic acid 6 (220 mg, 1.18 mmol), vinyl iodide 7 (266 mg, 1.18 mmol), and Pd(PPh₃)₄ (136 mg, 0.12 mmol) in degassed THF (6 mL) and H₂O (1.5 mL). The mixture was stirred for 1 h at r.t. then diluted with 1:1 hexane–Et₂O (60 mL) and filtered through a pad of silica. The organic phase was concentrated and the residue was purified by flash column chromatography [silica gel, hexane–EtOAc (85:15)] to give a colorless oil; yield: 232 mg (82%); [α]_D ²⁰ –4.40 (c 1.0, CHCl₃).

IR (KBr): 3415, 2931, 2858, 1715, 1187, 1031 cm^–1.

¹H NMR (500 MHz, CDCl₃): δ = 7.35 (dd, J = 15.0, 12.0 Hz, 1 H), 6.51 (t, J = 12.0 Hz, 1 H), 6.02 (dt, J = 15.0, 7.0 Hz, 1 H), 5.54 (d, J = 12.0 Hz, 1 H), 4.17 (q, J = 7.0 Hz, 2 H), 3.80–3.73 (m, 1 H), 2.21 (q, J = 7.0 Hz, 2 H), 1.51–1.25 (m, 8 H), 1.30 (t, J = 7.0 Hz, 3 H), 1.18 (d, J = 6.0 Hz, 3 H).

¹³C NMR (75 MHz, CDCl₃): δ = 166.5, 145.4, 145.2, 126.8, 115.3, 67.8, 59.7, 39.0, 32.7, 29.0, 28.5, 25.4, 23.3, 14.1.

HRMS (ESI): m/z [M + Na]⁺ calcd for C₁₄H₂₄O₃Na: 263.1623; found: 263.1621.

(5S,6S,11R)-iso-Cladospolide B (1)

A soln of dienoate 8 (140 mg, 0.58 mmol) in 1:1 t-BuOH–H₂O (1 mL) was added to a vigorously stirred suspension of K₃Fe(CN)₆ (575 mg, 1.75 mmol), K₂CO₃ (241 mg, 1.75 mmol), MeSO₂NH₂ (55 mg, 0.58), (3α,9R,3′′′α,4′′′β,9′′′R)-9,9′-[phthalazine-1,4-diylbis(oxy)]bis(6′-methoxy-10,11-dihydrocinchonan) [(DHQ)₂PHAL] (9.5 mg, 0.012 mmol), and OsO₄ (2.9 mg, 0.012 mmol) in 1:1 t-BuOH–H₂O (5 mL), and the mixture was stirred at 0 °C overnight. The reaction was then quenched with solid NaSO₃ (50 mg). EtOAc (20 mL) was added to the mixture, the layers were separated, and the aqueous phase was extracted with EtOAc (2 × 20 mL). The organic layers were combined, washed with brine (10 mL), dried (Na₂SO₄), filtered, and concentrated. The residue was purified by flash column chromatography [silica gel, hexane–EtOAc­ (40:60)] to give a white solid; yield: 94 mg (68%); [α]_D ²⁰ –91.5 (c 1.0, MeOH).

IR (KBr): 3451, 3370, 2928, 2855, 1746, 1465, 1317, 1170, 1130 cm^–1.

¹H NMR (300 MHz, CD₃OD): δ = 7.64 (dd, J = 5.8, 1.3, 1 H), 6.16 (dd, J = 5.8, 1.9 Hz, 1 H), 5.10–5.04 (m, 1 H), 3.8–3.74 (m, 1 H), 3.74–3.64 (m, 1 H), 1.62–1.50 (m, 2 H), 1.48–1.29 (m, 8 H), 1.13 (d, J = 6.0 Hz, 3 H).

¹³C NMR (75 MHz, CD₃OD): δ = 175.8, 157.1, 122.7, 88.2, 71.6, 68.5, 40.1, 34.2, 30.6, 26.9, 26.8, 23.5.

HRMS (ESI): m/z [M + Na]⁺ calcd for C₁₂H₂₀O₄Na: 251.1259; found: 251.1250.

(5S,6S,11S)-Iso-cladospolide B (2)

The isomer was prepared similarly: yield: 19% (5 steps); [α]_D ²⁰ –58.0 (c 0.6, MeOH).

IR (KBr): 3451, 3370, 2928, 2855, 1746, 1465, 1317, 1170, 1130 cm^–1.

¹H NMR (300 MHz, CDCl₃): δ = 7.47 (d, J = 5.6, 1 H), 6.19 (dd, J = 5.6, 1.9 Hz, 1 H), 5.02–4.96 (m, 1 H), 3.86–3.70 (m, 2 H), 2.20–1.71 (br s, 2 H), 1.66–1.23 (m, 10 H), 1.19 (d, J = 6.0 Hz, 3 H).

¹³C NMR (75 MHz, CDCl₃): δ = 172.8, 153.7, 122.6, 86.1, 71.7, 68.0, 39.0, 33.0, 29.2, 25.5, 25.4, 23.5.

MS (ESI): m/z = 251 [M + Na]⁺.

Acknowledgment

N.N.R. and P.S. thank the Council of Scientific and Industrial Research­, New Delhi, for the award of research fellowships.

• References

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• 9d An improvement in the yield was observed when KH was used instead of NaH
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• 13a Miyaura N, Suzuki A. Chem. Rev. 1995; 95: 2457
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• 16 The antimicrobial activity was tested against seven strains of bacteria (Bacillus subtilis MTCC 121, Staphylococcus aureus MTCC 96, S. aureus MLS16 MTCC 2940, Micrococcus luteus MTCC 2470, Escherichia coli MTCC 739, Klebsiella planticola MTCC 530, and Pseudomonas aeruginosa MTCC 2453) and one fungal strain (Candida albicans MTCC 3017)

• References

• 1 Cameron JS, Abbanat D, Bernan VS, Maiese WM, Greenstein M, Jompa J, Tahir A, Ireland CM. J. Nat. Prod. 2000; 63: 142
  CrossrefSearch in Google Scholar
• 2 Franck X, Vaz Araujo ME, Jullian J.-C, Hocquemiller R, Figadère B. Tetrahedron Lett. 2001; 42: 2801
  CrossrefSearch in Google Scholar
• 3a Si D, Sekar NM, Kaliappan KP. Org. Biomol. Chem. 2011; 9: 6988
  CrossrefSearch in Google Scholar
• 3b Srihari P, Bhasker EV, Harshavasdhan SJ, Yadav JS. Synthesis 2006; 4041
  Thieme Connect
• 3c Sharma GV. M, Reddy JJ, Reddy KL. Tetrahedron Lett. 2006; 47: 6531
  CrossrefSearch in Google Scholar
• 3d Pandey SK, Kumar P. Tetrahedron Lett. 2005; 46: 6625
  CrossrefSearch in Google Scholar
• 4 Gesner S, Cohen N, Ilan M, Yarden O, Carmeli S. J. Nat. Prod. 2005; 68: 1350
  CrossrefSearch in Google Scholar
• 5 Trost BM, Aponick A. J. Am. Chem. Soc. 2006; 128: 3931
  CrossrefSearch in Google Scholar
• 6a Yadav JS, Mandal SS. Synlett 2011; 2803
  Thieme Connect
• 6b Prasad KR, Gandi VR. Tetrahedron: Asymmetry 2011; 22: 499
  CrossrefSearch in Google Scholar
• 6c Prasad KR, Gandi VR. Tetrahedron: Asymmetry 2010; 21: 275
  CrossrefSearch in Google Scholar
• 6d Ferrié L, Reymond S, Capdevielle P, Cossy J. Synlett 2007; 2891
  Thieme Connect
• 6e Sharma GV. M, Reddy JJ, Reddy KL. Tetrahedron Lett. 2006; 47: 6537
  CrossrefSearch in Google Scholar
• 7a Roulland E. Angew. Chem. Int. Ed. 2011; 50: 1226
  CrossrefPubMedSearch in Google Scholar
• 7b Chen DY.-K. Synlett 2011; 2459
  Thieme Connect
• 7c Young IS, Baran PS. Nat. Chem. 2009; 1: 193
  CrossrefPubMedSearch in Google Scholar
• 7d Hoffmann RW. Synthesis 2006; 3531
  Thieme Connect
• 8a Reddy ChR, Rao NN, Sujitha P, Kumar CG. Eur. J. Org. Chem. 2012; 1819
• 8b Reddy ChR, Suman D, Rao NN. Synlett 2012; 23: 272
  Thieme ConnectSearch in Google Scholar
• 8c Reddy ChR, Srikanth B. Synlett 2010; 1536
  Thieme Connect
• 8d Reddy ChR, Rao NN. Tetrahedron Lett. 2009; 50: 2478
  CrossrefSearch in Google Scholar
• 9a Schweitzer S, Voss G, Gerlach H. Liebigs Ann. Chem. 1994; 189
• 9b Kimmel T, Becker D. J. Org. Chem. 1984; 49: 2494
  CrossrefSearch in Google Scholar
• 9c Brown CA, Yamashita A. J. Am. Chem. Soc. 1975; 97: 891
  CrossrefSearch in Google Scholar
• 9d An improvement in the yield was observed when KH was used instead of NaH
• 10 Conversion of alkyne 5 into dienoate 8 gave an inseparable mixture of isomers (Scheme 3)
• 11 Brown HC, Bhat NG, Somayaji V. Organometallics 1983; 2: 1311
  CrossrefSearch in Google Scholar
• 12 Marek I, Meyer C, Normant J.-F. Org. Synth. Coll. Vol. IX . John Wiley & Sons; London: 1998: 510
  Search in Google Scholar
• 13a Miyaura N, Suzuki A. Chem. Rev. 1995; 95: 2457
  Search in Google Scholar
• 13b Frank SA, Chen H, Kunz RK, Schnaderbeck MJ, Roush WR. Org. Lett. 2000; 2: 2691
  Search in Google Scholar
• 14a Xu D, Crispino GA, Sharpless KB. J. Am. Chem. Soc. 1992; 114: 7570
  CrossrefSearch in Google Scholar
• 14b Kolb HC, VanNieuwenhze MS, Sharpless KB. Chem. Rev. 1994; 94: 2483
  Search in Google Scholar
• 15 Mosmann T. J. Immunol. Methods 1983; 65: 55
  CrossrefPubMedSearch in Google Scholar
• 16 The antimicrobial activity was tested against seven strains of bacteria (Bacillus subtilis MTCC 121, Staphylococcus aureus MTCC 96, S. aureus MLS16 MTCC 2940, Micrococcus luteus MTCC 2470, Escherichia coli MTCC 739, Klebsiella planticola MTCC 530, and Pseudomonas aeruginosa MTCC 2453) and one fungal strain (Candida albicans MTCC 3017)