Side Chain Anchoring of Tryptophan to Solid Supports ... - Springer Link

Int J Pept Res Ther (2012) 18:7–19 DOI 10.1007/s10989-011-9274-8

Side Chain Anchoring of Tryptophan to Solid Supports Using a Dihydropyranyl Handle: Synthesis of Brevianamide F Carolina Torres-Garcıá • Mireia Dıáz • Daniel Blasi • Immaculada Farra`s Irene Fernańdez • Xavier Ariza • Jaume Farra`s • Paul Lloyd-Williams • Miriam Royo • Ernesto Nicola´s

•

Accepted: 15 September 2011 / Published online: 4 October 2011 Ó Springer Science+Business Media, LLC 2011

Abstract The multifunctional character of tryptophan has made it a target for the development of new molecules with therapeutic applications. In this sense the design of alternative solid phase routes would allow the widening of synthetic possibilities to access these molecules through conventional or combinatorial strategies. The present work describes a new strategy for side-chain anchoring of tryptophan to dihydropyranyl-functionalized polystyrene resins and its application to the synthesis of the natural diketopiperazine Brevianamide F. For this study a new handle (4-[(3,4-dihydro-2H-pyran-2-yl)methoxy]benzoic acid) was prepared in order to functionalize aminomethyl or

C. Torres-Garcıá I. Farra`s X. Ariza J. Farra`s P. Lloyd-Williams E. Nicola´s (&) Department of Organic Chemistry, University of Barcelona, Diagonal 647, 08028 Barcelona, Spain e-mail: [email protected] M. Dıáz Institut de Recerca Biome`dica (IRB Barcelona), Parc Cientı`fic de Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain D. Blasi Plataforma Tecnolo`gica Drug Discovery, Parc Cientı´fic de Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain I. Fernańdez Serveis Cientificote`cnics de la Universitat de Barcelona, Diagonal 647, 08028 Barcelona, Spain X. Ariza J. Farra`s Institut de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona, Spain M. Royo Unitat de Quı´mica Combinato`ria, Parc Cientı´fic de Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain

methylbenzhydrylamine resins. A preliminary study in solution using Fmoc-Trp-OR (R = Allyl or Me) and suitable resin models showed that the formation of an hemiaminal linkage with the indole system could be brought about by either conventional or microwave heating in 1,2-dichloroethane and in the presence of pyridine p-toluenesulfonate in yields of 70–95% practically without the formation of subproducts. On the other hand the amino acid could be liberated from the resin at room temperature in yields of up to 90% using trifluoroacetic acid in dichloromethane in the presence of 1,3-dimethoxybenzene as a cation scavenger. The conditions found in solution for the reversible formation of the hemiaminal were only reproducible in solid-phase work using conventional heating. These conditions were used in the synthesis of Brevianamide F, furnishing the diketopiperazine in an overall yield of 56%. These results demonstrate the potential of this strategy for the preparation of new molecules based upon tryptophan as a synthetic precursor. Keywords Dihydropyranyl handle DKPs Side chain anchoring Solid phase Tryptophan

Introduction The amino acid tryptophan has become a focus of attention in the area of Bioorganic Chemistry on account of the structural and chemical properties (the multifunctional chemical nature) of its indole side chain. Thus tryptophan plays a crucial role in the function of proteins on account of indole system giving rise to different types of interactions such as hydrophobic (Mant et al. 2009; Samanta et al. 2000), p-cation (Ma and Dougherty 1997), packing interactions with other aromatic residues (ring-stacking or edgeto-face interactions) (Samanta et al. 1999) or the formation

123

8

of hydrogen bonds (Black et al. 2001). On the other hand tryptophan is a synthetic and biosynthetic precursor to more complex heterocyclic systems with important biological properties (Rodrigues de Sa Alves et al. 2009; RuizSanchis et al. 2011). The multifunctional character of tryptophan makes it a target compound for carrying out synthetic transformations in the search for lead compounds and building blocks in therapeutic applications (Gordon and Kerwin 1998; Sawyer 1997). In this sense the development of synthetic strategies involving the union of the amino acid to solid supports through its side chain are potentially attractive since this would allow chemical modifications in different parts of the carbon skeleton to be carried out in order to, in this way, access new peptide derivatives such as cyclic peptides or peptides modified at the carboxyl terminal, or non-peptide structures such as heterocyclic derivatives from modifications of the indole system. The use of an amino acid side-chain for anchoring to a solid support has proved to be useful for the preparation of peptide derivatives with Asp, Glu, Ser or Lys, trifunctional amino acids that allow union of the peptide chain to the resin through their side-chain functional groups (Spatola and Romanovskis 2000). The sidechain attachment of tryptophan to solid supports, on the other hand, has not been studied to any great extent. In fact, as far as we are aware, the only previous report of such an application is that of Bernhardt et al. (1997), who described the synthesis of a tetrapeptide anilide on a chlorotrityl resin. With the objective of widening the application of the side-chain anchorage of tryptophan to solid supports, we decided to explore the use of the tetrahydropyranyl function as a protecting group for indole nitrogen that would allow its linkage to the polymer through the formation of an hemiaminal. We were particularly interested in adapting this approach to the synthesis of tryptophan containing peptides for further on-resin cyclization (Spatola and Romanovskis 2000). This would then allow cyclic peptides to be cleaved directly from the solid support and would constitute a novel approach to such molecules. Furthermore, the anchorage of other indole derivatives could, in principle, provide a method for the solid-phase synthesis of libraries of small molecules.

Materials and Methods All reagents were synthesis grade or better and were used without purification. Before use DCM was passed through an alumina column, tetrahydrofuran (THF) was distilled ˚ over sodium under N2 and DMF was placed over 4 A

123

Int J Pept Res Ther (2012) 18:7–19

molecular sieves and bubbled with N2 for 30 min. Et2O was stored over sodium under N2. Organic solutions were dried over anhydrous magnesium or sodium sulfate and solvent removal was performed by rotatory evaporation at reduced pressure (water pump). Melting points are uncorrected and were measured with a microscope equipped with a hot stage and a temperature controller. Microwave irradiation was carried out in sealed reaction vessels and reaction temperatures were monitored using an external surface sensor within the cavity of the reactor. Thin-layer chromatography (TLC), was carried out on silica gel plates (20 9 20 cm, 0.2 mm) and were visualized using UV light at 254 nm. The plates were also developed using a phosphomolybdic acid solution. Column chromatography was carried out using silica gel (70–230 mesh, particle size 35–70 lm) neutralized by elution with a 1% (v/v) solution of TEA in DCM. Columns were packed as a suspension of silica gel in the eluent and crude products were loaded as concentrated solutions in the eluent or adsorbed on silica gel. Column elution was performed with the assistance of compressed air. Trial reactions of the reversible union between dihydropyran (DHP) and tryptophan that were performed in solution were monitored using reversed-phase HPLC on an analytical C-18 Nucleosil ˚ ) using mixtures of column (4 9 250 mm, 10 mm, 120 A 0.045% trifluoroacetic acid (TFA) in H2O (eluent A) and 0.036% TFA in MeCN (eluent B). Reversed-phase retention times in the Experimental Sections are given along with the elution gradient expressed as an initial percentage of eluent B in eluent A and a final percentage of eluent B in eluent A over the duration of the gradient. The flow rate was 1 ml min-1 and detection was carried out at 220 and 280 nm. HPLC–MS was performed using mixtures of H2O (containing 0.1% HCOOH) and MeCN (containing 0.07% HCOOH) as eluents and a diode-array detector. In all cases elution was performed for 30 min. HPLC on a chiral stationary phase was carried out using a Chiralpak AS-H column (4.6 9 250 mm, 10 mm) and a mixture of hexanes and i-PrOH (85:5) as eluent. Elution was carried out isocratically for 30 min at 1 ml min-1 and detection was performed at 220 nm. IR spectroscopy was performed using KBr discs or as thin films of the pure products. NMR spectroscopy was carried out on 300, 400, 500 and 600 MHz machines, either using TMS as internal standard or the signals of the deuterated solvent as secondary references. Complete spectroscopic characterization was achieved using COSY, HSQC and HMBC NMR spectroscopy. Signal multiplicities are designated as s (singlet), bs (broad singlet), d (doublet), bd (broad doublet), t (triplet), bt (broad triplet), m (multiplet). Multiplicities designated with an asterisk indicate that duplicate signals corresponding to


diastereomers or rotamers were observed. High-resolution mass spectrometry (HRMS) was performed on an electrospray coupled to quadrupole time-of-flight apparatus. 4-[(3,4-Dihydro-2H-pyran-2-yl)methoxy]benzoic acid (3) Methyl 4-hydroxybenzoate (2.51 g, 24.2 mmol) and triphenylphosphine (7.68 g, 29.0 mmol) were dissolved in anhydrous THF (40 ml) under Ar. After stirring for 30 min, (3,4-dihydro-2H-pyran-2-yl)methanol (2.51 ml, 24.2 mmol) and diisopropyl azodicarboxylate (DIPAD) (6.25 ml, 29.0 mmol) were added and the mixture was stirred at rt under Ar for 48 h. The solvent was removed and LiOH (2.66 g, 109 mmol) in H2O/MeOH [(2:1), 75 ml] was added with stirring and the mixture was brought to reflux and was maintained at this temperature for 48 h. On cooling to rt a white precipitate appeared (triphenylphosphine) and the mixture was extracted with DCM (5 9 60 ml). The aqueous phase was cooled in an ice-water bath and aqueous HCl (2 M) was added to pH 2 (pH paper). The white solid formed was recovered by filtration, and was dried in vacuo, furnishing the product, (4.06 g, 72%). Mp = 141–143°C; Rf 0.02 (DCM); IR (KBr) mmax 3420–2554, 1683, 1606, 1428, 1257, 1170 cm-1; 1H-NMR (300 MHz, DMSO-d6) d 1.70 (m, 1H), 1.93 (m, 1H), 1.98 (m, 1H), 2.08 (m, 1H), 4.13 (m, 3H), 4.70 (m, 1H), 6.39 (d, J = 6 Hz, 1H), 7.02 (d, J = 9 Hz, 2H), 7.87 (d, J = 9 Hz, 2H); 13C-NMR (75 MHz, DMSO-d6) d 21.5, 26.3, 72.7, 75.5, 103.3, 145.9, 116.9, 126.1, 134.0, 164.8, 169.9; HRMS calcd for C13H15O4 [M ? H]? 235.0964, found 235.0969. N-Benzyl-4-[(3,4-dihydro-2H-pyran-2yl)methoxy]benzamide (4) 4-[(3,4-Dihydro-2H-pyran-2-yl)methoxy]benzoic acid 3 (234 mg, 1 mmol) was dissolved in DCM (7 ml) and EDCHCl (232 mg, 1.2 mmol), N-hydroxybenzotriazole (HOBt) (186 mg, 1.2 mmol) and benzylamine (134 ll, 1.2 mmol) were added and the mixture was stirred at rt for 22 h. After washing with (0.1 N HCl 3 9 5 ml), 10% NaHCO3 (3 9 5 ml), brine (3 9 5 ml) and H2O (3 9 5 ml), the organic phase was dried. Filtration and solvent removal gave a pale yellow oil that was purified by chromatography [neutralized silica gel, DCM/MeOH (995:5)], affording the product as a pale yellow oil (143 mg, 44%). Mp = 173–175°C; Rf 0.32 [DCM/MeOH (9.9:1)]; IR (KBr) mmax 3310, 3052, 2923, 1634, 1607 cm-1; 1H-NMR (400 MHz, CDCl3) d 1.83 (m, 1H), 1.98 (m, 1H), 2.06 (m, 1H), 2.16 (m, 1H), 4.04 (dd, J1 = 9.8 Hz, J2 = 4.4 Hz, 1H), 4.13 (dd, J1 = 9.8 Hz, J2 = 6.1 Hz, 1H), 4.21 (m, 1H), 4.62 (d, J = 5.7 Hz, 2H), 4.74 (m, 1H), 6.41 (d,

9

J = 5.3 Hz, 1H), 6.42 (bs, 1H), 6.94 (d, J = 9.2 Hz, 2H), 7.30, (m, 5H), 7.75 (d, J = 9.2 Hz, 2H); 13C-NMR (100 MHz, CDCl3) d 19.7, 24.9, 44.6, 70.7, 73.5, 101.2, 114.9, 127.4, 128.0, 128.4, 129.2, 129.3, 138.9, 143.9, 161.8, 167.3; HRMS calcd for C20H22NO3 [M ? H]? 324.1594, found, 324.1597; Reversed-phase HPLC tR 16.2 min, from 40 to 100% eluent B in eluent A over 30 min. (3,4-Dihydro-2H-pyran-2-yl)methyl N-benzylcarbamate (5) (3,4-Dihydro-2H-pyran-2-yl)methanol 2 (441 ll, 4.14 mmol) was dissolved in DCM (8 ml) and stirred under Ar. Solutions of DSC (957 mg, 3.73 mmol) in MeCN (10 ml) under Ar and DMAP (52 mg, 0.42 mmol) in DCM (1 ml) under Ar were then added via canula and the mixture was stirred for 4 h at rt. Benzylamine (102 ll, 0.93 mmol) and DMAP (22 mg, 0.18 mmol) were added and the mixture was stirred at rt for a further 20 h. After washing with 10% HCl (3 9 10 ml), brine (3 9 10 ml) and H2O (3 9 5 ml), the organic phase was dried. Filtration and solvent removal gave a thick, colorless oil that was purified by chromatography [neutralized silica gel, hexanes/EtOAc (1:3)] affording the product as a white solid (185 mg, 79%). Mp = 44–45°C; Rf 0.30 [hexanes/EtOAc (1:3)]; IR(KBr) mmax 3306, 3059, 2885, 1681, 1651, 1532, 1238, 1051, 694 cm-1; 1H-NMR (400 MHz, CDCl3) d 1.68 (m, 1H), 1.85 (m, 1H), 1.99 (m, 1H), 2.10 (m, 1H), 4.04 (m, 1H), 4.16 (dd, J1 = 11.4 Hz, J2 = 7 Hz, 1H), 4.26 (dd, J1 = 11.4 Hz, J2 = 3.6 Hz, 1H), 4.38 (d, J = 6 Hz, 2H), 4.70 (bs, 1H), 5.12 (bs, 1H), 6.38 (d, J = 6.2 Hz, 1H), 7.30 (m, 5H); 13C-NMR (100 MHz, CDCl3) d 19.3, 24.3, 45.4, 67.1, 73.3, 100.9, 127.7 (92), 129.0, 138.6, 143.5, 156.6; HRMS calcd for C14H18NO3 [M ? H]? 248.1281, found, 248.1289. Fmoc-(S)-Trp-OAllyl (6) H-(S)-Trp-OAllylHCl (3.00 g, 10.68 mmol) was dissolved in a mixture of 10% aqueous Na2CO3/THF (2:1, 75 ml) and the mixture was cooled to 0°C. A solution of Fmoc-Cl (3.03 g, 11.73 mmol) in THF (24 ml) was added dropwise with vigorous stirring which, after addition was complete, was continued for a further 30 min at 0°C and then for a further 7 h at rt. The mixture was extracted with EtOAc (3 9 30 ml) and the organic phase was dried. After filtration and solvent removal the yellow solid obtained was purified by chromatography [silica gel, hexanes/EtOAc (3:1)], furnishing the product as a foamy white solid (3.30 g, 86%). Mp = 134–136°C; Rf 0.18 [hexanes/EtOAc (3:1)]; [a]D = ?11.42 (CHCl3, c 1.15); IR (KBr) mmax 3317, 3051, 2912, 1736, 1699, 1522, 1221 cm-1; 1H-NMR (400 MHz, DMSO-d6) d 3.08 (dd, J1 = 14.7 Hz,

123

10

J2 = 9.4 Hz, 1H), 3.21 (dd, J1 = 14.7 Hz, J2 = 5.4 Hz, 1H), 4.17 (m, 1H), 4.22 (m, 2H), 4.33 (m, 1H), 4.54 (m, 2H), 5.15 (bd, J = 10.8 Hz, 1H), 5.24 (bd, J = 17.6 Hz, 1H), 5.81 (m, 1H), 6.98 (td, J1 = 7.4 Hz, J2 = 0.7 Hz, 1H), 7.06 (td, J1 = 7.6 Hz, J2 = 0.8 Hz, 1H), 7.17 (bd, J = 2.2 Hz, 1H), 7.29 (m, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.40 (m, 2H), 7.53 (d, J = 7.8 Hz, 1H), 7.65 (m, 2H), 7.87 (d, J = 7.7 Hz, 2H), 7.89 (d, J = 8.2 Hz, 1H), 10.9 (bs, 1H); 13C-NMR (100 MHz, CDCl3) d 27.1, 46.7, 55.2, 65.0, 65.9, 109.8, 111.6, 117.8, 118.1, 118.6, 120.3, 121.1, 124.0, 125.4, 127.2 (92), 127.8, 132.5, 136.3, 140.9, 143.9, 156.2, 172.2; HRMS calcd for C29H27N2O4 [M ? H]? 467.1965, found, 467.1962; Reversed-phase HPLC tR 22.0 min, from 40 to 100% eluent B in eluent A over 30 min. Fmoc-(S)-Trp-OMe (7) H-(S)-Trp-OMeHCl (0.99 g, 3.90 mmol) was dissolved in a mixture of 10% aqueous Na2CO3/THF (2:1, 45 ml) and the mixture was cooled to 0°C. A solution of Fmoc-OSu (1.45 g, 4.30 mmol) in THF (15 ml) was added dropwise with vigorous stirring which, after addition was complete, was continued for a further 30 min at 0°C and then for a further 7 h at rt. The mixture was extracted with EtOAc (3 9 30 ml) and the organic phase was dried. After filtration and solvent removal the yellow solid obtained was purified by chromatography [silica gel, hexanes/EtOAc (3:1)], furnishing the product as a foamy white solid (1.64 g, 96%). Mp = 118–120°C; Rf 0.12 [hexanes/EtOAc (3:1)]; [a]D = ?27.75 (CHCl3, c 1.10); IR (KBr) mmax 3385, 3057, 2951, 1741, 1688, 1535, 1218 cm-1; 1H-NMR (400 MHz, DMSO-d6) d 3.05 (dd, J1 = 14.6 Hz, J2 = 5.4 Hz, 1H), 3.17 (dd, J1 = 14.3 Hz, J2 = 9.4 Hz, 1H), 3.60 (s, 3H), 4.17 (m, 1H), 4.20 (m, 2H), 4.30 (m, 1H), 6.98 (td, J1 = 7.5 Hz, J2 = 0.8 Hz, 1H), 7.06 (td, J1 = 7.5 Hz, J2 = 0.7 Hz, 1H), 7.17 (d, J = 2.2, 1H), 7.28 (m, 2H), 7.34 (d, J = 8 Hz, 1H), 7.39 (m, 2H), 7.52 (d, J = 7.7, 1H), 7.64 (t, J = 8 Hz, 2H), 7.86 (d, J = 7.7 Hz, 2H), 7.90 (d, J = 7.6 Hz, 1H), 10.90 (bs, 1H); 13C-NMR (100 MHz, CDCl3) d 28.7, 48.4, 53.7, 56.8, 67.5, 111.5, 113.3, 119.8, 120.2, 121.9, 122.8, 125.6, 127.0, 128.8 (92), 129.4, 137.9, 142.5, 145.5, 157.7, 174.5; HRMS calcd for C27H25N2O4 [M ? H]? 441.1809, found, 441.1807; Reversed-phase HPLC tR 20.3 min, from 40 to 100% eluent B in eluent A over 30 min. Fmoc-Trp(2-THP)-OAllyl (8) Fmoc-Trp-OAllyl (200 mg, 0.43 mmol) was dissolved in DCE (5 ml) and DHP (40 ll, 0.43 mmol) and PPTS (181 mg, 0.68 mmol) were added. The mixture was stirred at 70°C for 8 h. After cooling the solution was washed with H2O (3 9 5 ml) and then dried. Filtration and solvent

123


removal gave a brown oil that was purified by chromatography [neutralized silica gel, hexanes/EtOAc (2:1)] furnishing the product as a white foamy solid (190 mg, 80%). Mp = 58–60°C; Rf 0.30 [hexanes/EtOAc (3:1)]; IR(KBr) mmax 3341, 2943, 1721, 1510, 1461, 1450, 1337, 1232, 1202, 1078, 1036, 758, 739 cm-1; 1H-NMR (400 MHz, DMSO-d6) d 1.51 (m, 2H), 1.71 (m, 1H), 1.82 (m, 1H), 1.91 (m, 1H), 1.99 (m, 1H), 3.05 (m, 1H), 3.17 (dd, J1 = 14.6 Hz, J2 = 5.1 Hz, 1H), 3.69 (m, 1H), 3.88 (m, 1H), 4.17 (m, 1H), 4.22 (m, 2H), 4.32 (m, 1H), 4.54 (m, 2H), 5.16 (dd, J1 = 10.2 Hz, J2 = 4.2 Hz, 1H), 5.25 (dd, J1 = 17.3 Hz, J2 = 11 Hz, 1H), 5.53 (d, J = 10.3 Hz, 1H), 5.82 (m, 1H), 7.05 (t, J1 = 7.3 Hz, J2 = 7.5 Hz, 1H), 7.14 (t, J1 = 7.7 Hz, J2 = 7.6 Hz, 1H), 7.28 (m, 2H), 7.34 (d, J = 6.8 Hz, 1H), 7.40 (m, 2H), 7.50 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.67 (m, 2H), 7.87 (d, J = 7.6 Hz, 2H), 7.94 (d*, J = 5.6 Hz, 1H); 13C-NMR (100 MHz, CDCl3) d 22.9, 25.0, 26.9, 30.4, 46.8, 52.4, 55.1, 65.9, 67.2, 82.5, 110.9, 111.0, 117.9, 118.6, 119.8, 120.4, 121.8, 124.1, 125.7, 127.5, 128.0, 128.2, 132.5, 136.3, 141.1, 144.1, 156.2, 172.0; HRMS calcd for C34H35N2O5 [M ? H]? 551.2540, found 551.2534; Reversed-phase HPLC tR 25.3 min, from 40 to 100% eluent B in eluent A over 30 min. Fmoc-Trp(2-THP)-OMe (9) Fmoc-Trp-OMe (100 mg, 0.22 mmol) was dissolved in DCE (4 ml) and DHP (14 ll, 0.16 mmol) and PPTS (91 mg, 0.36 mmol) were added. This mixture was submitted to microwave irradiation at a temperature of 120°C for 14 min with stirring. After cooling the solution was washed with H2O (3 9 5 ml) and then dried. Filtration and solvent removal gave a brown oil that was purified by chromatography [neutralized silica gel, hexanes/EtOAc (2:1)] furnishing the product as a white foamy solid (80 mg, 69%). Mp = 62.6–65.3°C; Rf 0.20 [hexanes/ EtOAc (2:1)]; IR (KBr) mmax 3321, 2921, 2850, 1715, 1511, 1462, 1203, 1033, 736 cm-1; 1H-NMR (400 MHz, DMSOd6) d 1.51 (m, 2H), 1.70 (m, 1H), 1.82 (m, 1H), 1.90 (m, 1H), 1.99 (m, 1H), 3.03 (dd, J1 = 14.4, J2 = 9.4 Hz, 1H), 3.16 (dd, J1 = 14.7 Hz, J2 = 5 Hz, 1H), 3.61 (s, 3H), 3.68 (m, 1H), 3.88 (m, 1H), 4.17 (m, 1H), 4.21 (m, 2H), 4.28 (m, 1H), 5.53 (d, J = 10.2 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 7.14 (t, J1 = 7.7 Hz, J2 = 7.2 Hz, 1H), 7.29 (m, 2H), 7.33 (d, J = 6.5 Hz, 1H), 7.40 (m, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.67 (m, 2H), 7.87 (d, J = 7.3 Hz, 2H), 7.91 (d*, J = 5.6 Hz, 1H); 13C-NMR (100 MHz, CDCl3) d 23.1, 27.1, 25.4, 30.7, 47.1, 52.4, 55.5, 66.1, 67.4, 82.9, 110.9, 111.0, 118.7, 119.8, 120.4, 121.8, 123.9, 125.6, 127.4, 128.0, 128.1, 136.2, 144.0, 144.1, 157.2, 173.8; HRMS calcd for C32H33N2O5 [M ? H]? 525.2384, found, 525.2381; Reversed-phase


HPLC tR 23.7 min, from 40 to 100% eluent B in eluent A over 30 min. Nin-{6-[4-(N-Benzylcarbamoyl)benzoxymethyl] tetrahydropyran-2-yl}-N-(9-fluorenylmethoxycarbonyl)-(S)-tryptophan allyl ester (10) Fmoc-Trp-OAllyl (150 mg, 0.32 mmol) was dissolved in DCE (4 ml) and N-benzyl-4-[(3,4-dihydro-2H-pyran2-yl)methoxy]benzamide (74 mg, 0.23 mmol) and PPTS (132 mg, 0.51 mmol) were added. The mixture was submitted to microwave irradiation at a temperature of 120°C for 3 h with stirring. After cooling the solution was washed with H2O (3 9 5 ml) and then dried. Filtration and solvent removal gave a brown oil that was purified by chromatography [neutralized silica gel, hexanes/EtOAc (1:1)] furnishing the product as a white foamy solid (90 mg, 50%). 1H-NMR (400 MHz, DMSO-d6) d 1.20 (m, 1H), 1. 38 (m, 1H), 1.72 (m, 1H), 1.81 (m, 1H), 1.86 (m, 1H), 1.98 (m, 1H), 3.07 (dd, J1 = 14.7 Hz, J2 = 5.2 Hz, 1H), 3.19 (dd, J1 = 14.1 Hz, J2 = 9.5 Hz, 1H), 3.93 (m, 1H), 4.02 (m, 1H), 4.14 (m, 1H), 4.17 (m, 1H), 4.22 (m, 2H), 4.33 (m, 1H), 4.45 (d, J = 5.9 Hz, 2H), 4.55 (m, 2H), 5.17 (m, 1H), 5.26 (m, 1H), 5.70 (bt, J = 9.8 Hz, 1H), 5.83 (m, 1H), 6.92–6.97 (d*, J = 8.7 Hz, 2H), 7.08 (m, 1H), 7.17 (m, 1H), 7.22 (t, J = 6.7 Hz, J = 6.4 Hz, 1H), 7.30 (m, 2H), 7.31 (m, 4H), 7.36 (s*, 1H), 7.41 (m, 2H), 7.56 (m, 2H), 7.68 (m, 2H), 7.83–7.84 (d*, J = 8.7 Hz, 2H), 7.89 (d, 7.4 Hz, 2H), 7.97 (m*, 1H), 8.88 (m, 1H); 13C-NMR (100 MHz, CDCl3) d 22.2, 26.5, 26.8, 29.8, 42.5, 46.6, 54.9, 64.9, 65.7, 70.5, 75.3, 82.8, 110.6, 110.8, 114.0, 117.7, 118.4, 119.5, 120.1, 121.5, 123.5, 125.2, 126.6, 127.6, 127.0, 127.1 (92), 127.8, 128.2, 132.3 (92), 135.8, 139.9, 140.7, 143.7, 156.0, 160.7, 165.6, 171.8; HRMS calcd for C49H48N3O7 [M ? H]? 790.3485, found 790.3486. HPLC tR 26.9 min, from 40 to 100% eluent B in eluent A over 30 min. Nin-{6-[4-(N-Benzylcarbamoyl)benzoxymethyl] tetrahydropyran-2-yl}-N-(9-fluorenylmethoxycarbonyl)-(S)-tryptophan methyl ester (11) This product was prepared using similar conditions that those used for the analogue 10. 0.5 g (1.07 mmol) of 7 and 0.25 g (0.77 mmol) of 4 afforded 43 mg of 11 (7%) as a white solid. 1 H-NMR (400 MHz, CDCl3) d 1.56 (m, 2H), 1.84 (m, 1H), 2.02 (m, 2H), 2.12 (m, 1H), 3.29 (d, J = 5.4, 2H), 3.67 (s, 3H), 4.0 (m, 1H), 4.07 (m, 1H), 4.09 (m, 1H), 4.19 (m, 1H), 4.36 (m, 2H), 4.74 (m, 1H), 4.62 (d, J = 5.3 Hz, 2H), 5.36 (m, 1H), 5.54 (dd, J1 = 2.9 Hz, J2 = 2.3 Hz, 1H), 6.27 (bs, 1H), 6.87 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 7.13 (m, 1H), 7.22 (m, 1H), 7.26 (m, 3H), 7.34 (m, 4H), 7.37 (m, 2H), 7.42 (m, 1H), 7.52 (m, 1H), 7.53 (m, 2H), 7.75 (m, 2H), 7.86 (d, J = 8.5 Hz, 2H);

11

HRMS: calcd for C47H45N3O7 [M ? H]? 764.3330, found 764.3337. HPLC tR 25.8 min, from 40 to 100% eluent B in eluent A over 30 min. Fmoc-(S)-Trp(Boc)-OMe (12) Fmoc-Trp-OMe (143 mg 0.32 mmol) was dissolved in anhydrous MeCN (3 ml) under N2 and a solution of DMAP (4.0 mg, 0.033 mmol) and Boc2O (87.0 mg, 0.39 mmol) in anhydrous MeCN (1 ml) was added via canula. The mixture was stirred for 6 h at rt. The mixture was then diluted with H2O (5 ml) and extracted with Et2O (2 9 20 ml). The organic phase was washed with 1 M KHSO4 (3 9 20 ml), saturated NaHCO3 (3 9 20 ml) and brine (3 9 20 ml) and then dried. After filtration and solvent removal the pale yellow crude was purified by chromatography [silica gel, hexanes/EtOAc (4:1)] furnishing the product as a white foamy solid (96 mg, 55%). Mp = 51.3–53.5°C; Rf 0.29 [hexanes/EtOAc (4/1)]; IR (KBr) mmax 3369, 3066, 2951, 1729, 1256 cm-1; 1H-NMR (400 MHz, CDCl3) d 1.65 (s, 9H), 3.27 (m, 2H), 3.71 (s, 3H), 4.21 (t, J = 7 Hz, 1H), 4.38 (m, 2H), 4.76 (m, 1H), 5.4 (d, J = 8 Hz, 1H), 7.22 (td, J1 = 7.4 Hz, J2 = 1 Hz, 1H), 7.29 (m, 2H), 7.31 (td, J1 = 7.7 Hz, J2 = 1.1 Hz, 1H), 7.39 (m, 2H), 7.43 (m, 1H), 7.51 (m, 1H), 7.55 (m, 2H), 7.76 (m, 2H), 8.12, (bd, J = 7.4 Hz, 1H); 13C-NMR (100 MHz, CDCl3) d 28.1, 28.4, 47.4, 52.7, 54.2, 67.5, 84.1, 115.0, 115.5, 119.0, 120.2, 122.9, 124.4, 124.8, 125.3, 127.3, 127.9, 130.7, 135.5, 141.5, 143.9, 149.7, 155.9, 172.2; HRMS calcd for C32H33N2O6 [M ? H]? 541.2333, found, 541.2338; HPLC (chiral stationary phase) tR 18.2 min. Fmoc-Trp(Boc)-OMe (Racemate) As above for the preparation of Fmoc-(S)-Trp(Boc)-OMe but using racemic Fmoc-Trp-OMe (300 mg, 0.68 mmol). The product was obtained as yellow oil (154 mg, 41%). HPLC (chiral stationary phase) tR 13.8 min (R enantiomer), 18.2 min (S enantiomer). N-Benzyl-4-[(6-(2,4-dimethoxyphenyl)tetrahydro-2Hpyran-2-yl)methoxy]benzamide (13) N-Benzyl-4-[(3,4-dihydro-2H-pyran-2-yl)methoxy]benzamide 8 (40 mg, 0.124 mmol) was dissolved in TFA/ mDMB (9:1, 1 ml) and the solution was allowed to stand for 60 min at rt. After solvent removal the reddish oil was purified by chromatography [silica gel, DCM/EtOAc (1:1)] affording the product (34.2 mg, 60%) as a foamy white solid. Mp = 55–59°C; Rf 0.62 [DCM/EtOAc (1:1)]; IR (KBr) mmax 3320, 2928, 2834, 1605, 1500, 1288, 1251, 1205; 1H-NMR (400 MHz, CDCl3) d 1.37 (m, 1H), 1.46 (m, 1H), 1.60 (m, 2H), 1.94 (m, 2H), 3.73 (s*, 3H), 3.75

123

12

(s*, 3H), 3.81 (dd, J1 = 9.3 Hz, J2 = 7.3 Hz, 1H), 3.93 (dd, J1 = 9.5 Hz, J2 = 3.2 Hz, 1H), 3.96 (m, 1H), 4.59 (t, J = 7.8 Hz, 1H), 4.61 (d, J = 5.6 Hz, 2H), 6.40 (m, 1H), 6.41 (m, 2H), 6.87 (d, J = 8.8 Hz, 2H), 7.05 (m, 1H), 7.29 (m, 1H), 7.34 (m, 4H), 7.73 (d, J = 8.8 Hz, 2H); 13C-NMR (100 MHz, CDCl3) d 23.9, 33.1, 34.5, 35.4, 44.2, 55.4, 55.7, 70.1, 72.5, 99.0, 104.0, 114.5, 126.1, 127.1, 127.6, 128.0, 128.6, 128.9 (92), 138.5, 158.3, 158.8, 161.5, 167.0; HRMS for C28H32NO5 [M ? H]? calcd, 462.2275, found, 462.2273; HPLC Rt 19.8 min, from 40 to 100% eluent B in eluent A over 30 min. Synthesis of Brevianamide F (1) in Solution Z-Trp-OH (0.51 g, 1.48 mmol), H-Pro-OMeHCl (257 mg, 1.48 mmol), HOBt (231 mg, 1.48 mmol) and EDCHCl (284 mg, 1.48 mmol) were suspended in MeCN (20 ml). DIEA (383 ll, 2.22 mmol) was added and the mixture was stirred overnight at rt under N2. The solvent was removed and EtOAc (20 ml) was added. The organic phase was washed successively with H2O (2 9 20 ml), aqueous 10% Na2CO3 (2 9 20 ml), H2O (2 9 20 ml), 0.1 M HCl (2 9 20 ml) and brine (2 9 20 ml) and the resulting organic solution was dried. After filtration and solvent removal the residue was dissolved in anhydrous MeOH (50 ml) and was added to 10% Pd/C (54 mg, 10 mol%). This mixture was then stirred vigorously under H2 at stp overnight. After filtration through Celite the solvent was removed affording a foamy white solid that was purified by chromatography [neutralized silica gel, hexanes/Et2O (2:8)] to yield the desired product as a foamy white solid (285 mg, 68%). Mp = 166–168°C; [a]D = -69.5 (MeOH, c 0.44); Rf 0.30 [DCM/EtOAc (2:1)]; 1H-NMR (400 MHz, DMSO-d6) d 1.37 (m, 1H), 1.64 (m, 2H), 1.95 (m, 1H), 3.08 (dd, J1 = 14.9, J2 = 5.7 Hz, 1H), 3.26 (m, 2H), 3.39 (m, 1H), 4.04 (bt, J = 8.3 Hz, 1H), 4.29 (bt, J = 4.9 Hz, 1H), 6.95 (bt, J = 7.5 Hz, 1H), 7.05 (bt, J = 7.5 Hz, 1H), 7.18 (d, J = 2.1 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.72 (s, 1H), 10.9 (s, 1H); 13C-NMR (100 MHz, DMSO-d6) d 21.9, 25.8, 27.7, 44.6, 55.3, 58.4, 109.3, 111.2, 118.2, 118.7, 120.9, 124.4, 127.4, 136.0, 165.5, 169.1; HRMS calcd for C16H18N3O2 [M ? H]? 284.1399, found 284.1391; HPLC: tR 15.8 min, from 5 to 100% eluent B in eluent A over 30 min. Solid-Phase Synthesis of Brevianamide F (1) Aminomethyl-polystyrene resin (0.37 mmol g-1, 249 mg, 0.092 mmol) was introduced into a polypropylene syringe

123


fitted with a porous polystyrene frit and was washed successively with DCM (10 9 30 s), TFA (40% v/v) in DCM (1 9 1 min and 2 9 10 min), DCM (5 9 30 s), DIEA (5% v/v) in DCM (6 9 2 min), DCM (5 9 30 s), DMF (5 9 30 s) and DCM (5 9 30 s). 3 (65 mg, 0.28 mmol), N,N0 -diisopropylcarbodiimide (DIPCDI) (43 ll, 0.28 mmol) and ethyl cyanoglyoxyl-2-oxime (39 mg, 0.28 mmol) in DCM (1 ml) were then added and the mixture was allowed to stand for 1 h at rt with occasional manual stirring. The resin was then washed with DCM (5 9 30 s). 6 (64 mg, 0.14 mmol) and PPTS (53 mg, 0.21 mmol) in DCE (0.5 ml) were then added to the handle-resin and the suspension was shaken at 80°C for 16 h in an Advanced Chemtech PLS 4 9 4 organic synthesizer. After cooling to rt the aminoacyl-resin was washed successively with DCM (5 9 30 s), DMF (5 9 30 s) and MeOH (5 9 30 s). Spectrophotometric quantification of the dibenzofulvene-piperidine adduct from a portion of the resin indicated a 76% yield for amino acid coupling. After washing with DMF (5 9 30 s) and DCM (5 9 30 s) this resin was placed under Ar and Pd(PPh3)4 (43 mg, 0.037 mmol) and PhSiH3 (544 ll, 4.4 mmol) in DCM (2 ml) were added. The mixture was shaken for 30 min at rt, filtered and washed with DCM (8 9 30 s). A second treatment with Pd(PPh3)4 (43 mg, 0.037 mmol) and PhSiH3 (544 ll, 4.4 mmol) in DCM (2 ml) was then carried out. After filtration the resin was washed successively with DCM (8 9 30 s), diethyl dithiocarbamate (5% v/v) in DMF (2 9 5 min), DMF (5 9 1 min), DCM (5 9 30 s) and DMF (5 9 30 s). The resin (140 mg) was then treated with H-Pro-OMeHCl (19 mg, 0.28 mmol), PyBOP (76 mg, 0.28 mmol) and DIEA (77 ll, 0.83 mmol) in DMF (0.5 ml) for 60 min with occasional manual stirring. The resin was washed with DCM (5 9 30 s) and DMF (5 9 30 s). This coupling reaction and washing cycle was then repeated twice using the same quantities of reagents and solvents. The resulting resin was treated with piperidine (20% v/v) in DMF (2 9 10 min), was washed with DMF (5 9 30 s) and DCM (5 9 30 s) and dried. Cleavage of the product from the resin was brought about by treatment with TFA/ mDMB/DCM (5:5:90 v/v) (3 9 10 min) and the collected washings were submitted to solvent removal. The crude product was precipitated with hexanes and the remaining solid was centrifuged (10 min at 6000 rpm) and filtered, affording 14 mg of a foamy white solid that co-eluted with an authentic sample of cTrpPro. HPLC analysis indicated a cleavage yield of 74% for this process. HRMS calcd for C16H18N3O2 [M ? H]? 284.1399, found 284.1394.


Assays of Conventional and Microwave Heating in the Reaction of Fmoc-Trp-OR (6 for R = Allyl or 7 for R = Me) with 3,4-Dihydro-2H-pyran and with N-Benzyl-4-[(3,4-dihydro-2H-pyran-2yl)methoxy]benzamide (4) Conventionally heated reactions were carried out in 2 ml Wheaton sealable vials with magnetic stirring. In order to ensure homogeneous heating of the vials they were inserted into an aluminium block that incorporated a heating sensor. Reaction time was 16 h and the temperature was 70°C in all cases. Product yields were determined by measuring the peak areas in reversed-phase chromatography (see ‘‘Materials and Methods’’). Microwave heated reactions were carried out with magnetic stirring using 5 ml vials (see ‘‘Materials and Methods’’). Reaction times were 30 min when DHP was used, and 30 and 150 min for reactions involving 4. The temperature was 120°C in all cases. For reactions involving DHP (9.5 ll, 0.104 mmol, 1 eq), the relevant amino acid derivative (76.2 mg of 6 and 72.3 mg of 7, 0.156 mmol, 1.5 eq) and PPTS (60.5 mg, 0.239 mmol, 2.3 eq) were dissolved in DCE (1.5 ml). For those reactions involving 4 (20.0 mg, 61.7 mmol, 1 eq), the relevant amino acid derivative (45.3 mg of 6 and 42.9 mg of 7, 92.6 mmol, 1.5 eq) and PPTS (35.7 mg, 0.142 mmol, 2.3 eq), were dissolved in DCE (1 ml). Yields were 68–70% with conventional heating and 63–67% under microwave heating for DHP, and 95–96% with conventional heating and 97–99% under microwave heating in the case of 4. Assays of Anchoring Fmoc-Trp-OAllyl (6) to the Solid Support Using Conventional Heating Aminomethylpolystyrene-resin (batches of 59–65 mg) and MBHA-resin (batches of 61–68 mg), with functionalizations 0.37 and 1.00 mmol g-1, respectively were used. The resin batches were introduced into polypropylene syringes fitted with porous polystyrene frits and were treated with, successively, DCM (10 9 30 s), 40% TFA in DCM (1 9 1 and 2 9 10 min), DCM (5 9 30 s), 5% DIEA in DCM (6 9 2 min), DCM (5 9 30 s), DMF (5 9 30 s) and DCM (5 9 30 s). The aminomethylpolystyrene-resin and MBHA-resin batches were then subjected to treatment with the handle 3 (3 eq), DIPCDI (3 eq) and ethyl cyanoglyoxyl-2-oxime (3 eq) in DCM for 3 h. (After treatment the ninhydrin test was negative in all cases). Excess reagents were filtered and the resulting resins were washed with DCM (5 9 30 s), DMF (5 9 30 s) and DCE (3 9 30 s). Batches of both resins in DCE were then introduced into 2 ml screw-cap vials (Wheaton) and 6, PPTS and DCE

13

were added. The tubes were closed and heated to the desired temperature with mechanical stirring in an Advanced Chemtech PLS 4 9 4 organic synthesizer. Optimum heating of the vials was achieved using suitablysized aluminium adaptors. The reaction conditions assayed were: 1.5 and 3 eq for 6; 1, 2 and 6 eq for PPTS; 100, 200 and 300 ll for DCE and 70, 80 and 90°C (the reaction time was 16 h in all cases). The yields of amino acid anchoring were between 67 and 90% for aminomethylpolystyrene-resin, and between 64 and 84% for MBHA-resin. The final functionalization levels and reaction yields were determined by spectrophotometric quantification of the dibenzofulvene adduct formed on elimination of the Fmoc group. Assays of Anchoring Fmoc-Trp-OAllyl (6) to the Solid Support Using Microwave Heating Standard protocol: aminomethylpolystyrene resin (59.6 mg, 0.37 mmol g-1, 0.022 mmol) was introduced into a polystyrene syringe fitted with a porous polystyrene frit and was washed with, successively, DCM (10 9 30 s), 40% TFA in DCM (1 9 1 min and 2 9 10 min), DCM (5 9 30 s), 5% DIEA in DCM (6 9 2 min), DCM (5 9 30 s), DMF (5 9 30 s) and DCM (5 9 30 s). The handle 3 (15 mg, 3 mmol) in DCM (1 ml) was added together with DIPCDI (10.22 ll, 3 mmol) and ethyl cyanoglyoxyl-2-oxime (9 mg, 3 mmol) in DCM (1 ml) and the mixture was allowed to stand for 3 h at rt with occasional manual stirring. The resulting resin was then washed with DCM (5 9 30 s), DMF (5 9 30 s) and DCE (5 9 30 s) and added to a solution of 6 (15 mg, 1.5 mmol) and PPTS (13 mg, 2.3 mmol) in anhydrous DCE (0.3 ml). The suspension was then subjected to microwave irradiation for 2 h at 120°C. After cooling it was washed with DMF (5 9 30 s), DCM (5 9 30 s), MeOH (5 9 30 s) and DCM (5 9 30 s). The yield of amino acid anchoring was judged to be 47% as determined by spectrophotometric quantification of the dibenzofulvene adduct formed on elimination of the Fmoc group. Other trial reactions carried out with an aminomethylpolystyrene resin with a functionalization level of 0.55 mmol g-1 gave yields between 30 and 58%. Assays of Stability of Nim-{6-[4-(N-Benzylcarbamoyl) phenoxymethyl]tetrahydropyran-2-yl}N-(9-fluorenylmethoxycarbonyl)-(S)-tryptophan Allyl Ester (10) Under Acidic Conditions Reactions were carried out at rt in polypropylene vials equipped with a magnetic stirrer bar. Between 4.8 and 5.5 mg of tryptophan derivative (6.1–11.2 mmol) was dissolved in 1 ml of acidolytic mixture. Product yields shown in Table 1 were determined by measuring the peak

123

14


Table 1 Stability of Nim-{6-[4-(N-benzylcarbamoyl)phenoxymethyl]tetrahydropyran-2-yl}-N-(9-fluorenylmethoxycarbonyl)tryptophan allyl ester (10) under different acidolytic conditions Entry

Acidolytic mixture

Time (min)

10 (%)a

6 (%)b

1

TFA/DCM (9:1)

30

50

14 c

21 (16)c

2

TFA/DCM (1:9)

30

65 (41)

3

TFA/TIS/DCM (1:1:8)

60

–

13

4

TFA/H2O (9:1)

60

34

8

5

TFA/mDMB/DCM (0.2:1:8.8)

60

75

17

6

TFA/mDMB/DCM (0.5:1:8.5)

30

–

90d

7

TFA/mDMB/DCM (1:0.2:8.8)

15

–

68 (56)e

8

TFA/mDMB/DCM (1:1:8)

60

–

72

9

TFA/mDMB/DCM (4:1:5)

60

–

40

10

TFA/mDMB (9:1)

60

–

17

11

TFA/mDMB/THF (4:1:5)

60

34 (25)f

21 (7)f

a

Percentage of unreacted starting material

b

Based on integration of chromatographic peaks

c

After 1 h

d

A similar result was obtained after 1 h reaction time

e

After 30 min

f

After 3 h

areas in reversed-phase chromatography (see ‘‘Materials and Methods’’). Assays of Cleavage of Fmoc-Trp-OAllyl (6) from the Resin The aminoacyl resins obtained by conventional heating and by microwave irradiation were introduced into polystyrene syringes fitted with porous polystyrene frits and were washed with DCM (5 9 30 s). They were then treated with a mixture of TFA/mDMB/DCM (5:10:85) for 1 h at rt and were washed with DCM (5 9 30 s). Cleavage yields of 80% for the resin subjected to conventional heating and 33% for that subjected to microwave heating were obtained as judged by spectrophotometric quantification of the dibenzofulvene adduct formed on elimination of the Fmoc group.

Results and Discussion The functionalization of a resin with tetrahydropyranyl groups was first described by Thompson and Ellman (1994) for the anchoring of molecules to solid supports through their hydroxyl groups. Such supports have been shown to be useful for a range of organic molecules (Kitade et al. 2006; Liu et al. 2005; Nam et al. 2003; Tanaka et al. 2006; Wallace 1997). More specifically, they have been used for

123

the side-chain anchoring of suitably protected trifunctional amino acids such as Thr (Graham et al. 2002), Ser (Villorbina et al. 2007) and 4-hydroxyproline (Hyp) (Anderson et al. 2005). However, the solid-phase protection of nitrogen-based functional groups as their tetrahydropyranyl derivatives has not been extensively studied. The reversible union of purines (Nugiel et al. 1997; Chang et al. 2005) and benzimidazoles (Wang et al. 2002) through one of the imidazole nitrogens has been reported. We were interested by Smith et al. (1998) report describing a similar approach for indoles using a tetrahydropyranyl-functionalized resin. Typical anchoring conditions involved submitting the indole derivative (1.4 eq with respect to resin functionalization) and a sulfonic acid as catalyst in 1,2-dichloroethane (DCE) as solvent to moderately high temperatures (60–70°C) and long reaction times (12–30 h) in the presence of the solid support. On the other hand, cleavage of the hemiaminal linkage between the heterocycle and the resin is usually carried out using TFA in DCM at rt and requires only short reaction times. These results encouraged us to study the reversible linkage of suitably protected tryptophan derivatives to a tetrahydropyranyl-functionalized resin with a view to developing new synthetic strategies based on tryptophan as the synthetic precursor. As a first approximation, in this communication we report on our in-depth study of the anchoring of tryptophan to tetrahydropyranyl-functionalized resins and on its application to the synthesis of the naturally occurring diketopiperazine Brevianamide F 1. Cyclic peptides such as this have interesting biological properties and potential applications in a range of fields (Milne and Kilian 2010). Our study demonstrates the viability of our strategy and its potential use in the preparation of larger cyclic peptides or other tryptophan containing derivatives. Commercial polystyrene-based resins functionalized with tetrahydropyranyl groups are available. Nevertheless, greater versatility would be achieved by using a handle that could be incorporated into different aminomethylated resins (polyestyrene, polyethyleneglycol, etc.) in this way extending application to other types of solid support. Basso and Ernst (2001) have described the use of (3,4-dihydro2H-pyran-2-yl)methoxyacetic acid in the functionalization of aminomethylated ‘Syn-PhaseTM-lanterns’ for the synthesis of cyclic peptides in which the peptide was attached to the solid support via the hydroxyl group of Hyp. In our laboratory this particular handle was obtained as an hygroscopic, low melting-point and difficult to manipulate solid. Consequently, in the present study we used 4-[(3, 4-dihydro-2H-pyran-2-yl)methoxy]benzoic acid as an alternative, which is a white solid with mp 141–143°C. This product 3 (see Scheme 1) was obtained by Mitsunobu reaction between (3,4-dihydro-2H-pyran-2-yl)methanol 2


15 O CO2H

O O

O 5

N H

Bz

c

O

OH

a

O

2

b

O 3

O

NH Bz

O 4

Scheme 1 Reagents and conditions: (a) (i) methyl p-hydroxybenzoate (1 eq), PPh3 (0.8 eq), THF, Ar (30 min); then 2 (1 eq) and DIPAD (0.8 eq), 48 h, (ii) LiOH (4.5 eq), MeOH (2)/H2O (1) (72%, two steps); (b) benzylamine (1.2 eq), EDCHCl (1.2 eq), HOBt (1.2 eq),

DCM, 22 h (44%); (c) (i) DSC (0.9 eq), DMAP (0.1 eq), DCM (10)/ MeCN (1), Ar, 4 h, (ii) benzylamine (0.22 eq), DMAP (0.04 eq), 20 h (79%, two steps)

and methyl 4-hydroxybenzoate followed by ester hydrolysis. Isolation of the intermediate ester led to low overall yields (25%) but preparation of the acid directly without isolating the ester gave the desired handle in 72% yield. As a prior step to the solid phase, it was considered desirable to find suitable conditions for the formation of the hemiaminal using amide 4 as a soluble model of an aminomethyl polystyrene functionalized with tetrahydropyranyl groups (Scheme 1). Thus, this product was easily obtained from handle 3 and benzylamine under similar conditions to those used in solid phase work. On the other hand, alcohol 2 was used in order to obtain carbamate model 5 with the objective of exploring an alternative functionalization of the resin. This product was obtained from 2 and benzylamine using N,N0 -disuccinimidyl carbamate (DSC) in the presence of 4-dimethylaminopyridine (DMAP). In order to attach tryptophan to a tetrahydropyranfunctionalized resin via its indole ring, the a-amino and carboxyl functions must be protected with groups that are compatible with the conditions necessary for the formation of the hemiaminal linkage and with the elongation of the peptide chain itself. We considered that the threedimensional orthogonal strategy based on the 9-fluorenylmethoxycarbonyl (Fmoc), t-Bu and allyl groups (Kates et al. 1993; Trzeciak and Bannwarth 1992) had much to recommend it, since the reversible union of tryptophan to the resin requires acidic conditions and the resulting hemiaminal linkage is stable to bases such as piperidine, used for removal of the Fmoc group, or the nucleophiles used to remove the allyl group under neutral conditions (Guibe 1998). Nevertheless, we also considered protecting the carboxyl group as a methyl ester since this could be cleanly removed if required by basic treatment (LiOH) in the presence of the hemiaminal linkage (Cabrele et al. 1999; Gu and Silverman 2003; Liu et al. 2005). Therefore the amino acid derivatives used in this study were FmocTrp-OAllyl 6 and Fmoc-Trp-OMe 7, that were prepared using slightly modified classical methods starting from the commercially available products H-Trp-OAllyl and H-Trp-OMe (Kuriyama and Kitahara 2001; Richard et al. 2004).

The reaction conditions necessary for the reversible union of tryptophan via its side-chain to the model compound 4, were first explored using DHP as the simplest hemiaminal precursor. A comparison was made between results obtained with conventional heating and with microwave radiation as an alternative to the conditions described by Smith et al. (1998), in order to reduce reaction times. Thus, when mixtures of DHP (1 eq) and 6 or 7 (1.5 eq) were heated (70°C for 16 h) or irradiated (120°C for 30 min) in the presence of pyridinium 4-toluenesulfonate (PPTS) (2.3 eq) in DCE (Scheme 2), diastereomeric Fmoc-Trp(THP)-OAllyl 8 or Fmoc-Trp(THP)-OMe 9 were obtained, respectively. These diastereomers were practically indistinguishable by NMR spectroscopy and HPLC. Conventional heating and microwave irradiation afforded similar yields of 65–70% and no other products were detected, indicating that a-amino and carboxyl protecting groups were stable to the conditions employed. When monomeric analog of dihydropyranyl functionalized aminomethylated resin 4 was reacted with tryptophan derivatives 6 and 7 (70°C, 16 h), HPLC monitoring showed the disappearance of 4 and the appearance of two new products 10 and 11, respectively. Both of these were formed as diastereomeric mixtures but, like 8 and 9, only one chromatographic peak was detected by HPLC. On the other hand a precise analysis of derivative 10 by NMR revealed a single set of signals, indicating very probably, the presence of one very major diastereomer. The purities of 10 and 11, as determined by integration of the HPLC peak areas were around 95% and no other products were observed. Similar results were obtained when the same reactions were subjected to microwave radiation (120°C, 3 h). We also addressed the issue of the possible racemization of these tryptophan derivatives using HPLC on a chiral stationary phase. This required conversion of a racemic mixture of 7 into its Nin-t-butoxycarbonyl (Boc) derivative 12 by treatment with Boc2O/DMAP, in order to achieve optimal peak separation (Scheme 2). Optically pure amino acid 7 was then subjected to the conditions necessary for the anchorage of tryptophan, and was further transformed to the corresponding Nin-Boc derivative. HPLC analysis on

123

16


Scheme 2 Reagents and conditions: (a) from 6: DHP (1 eq), PPTS (1.6 eq), DCE, 70°C, 8 h; from 7: DHP (0.7 eq), PPTS (1.6 eq), DCE, 120°C (MW), 14 min (69%); (b) 4 (0.72 eq), PPTS (1.6 eq), DCE, 120°C (MW), 3 h (10: 50%, 11: 7%); (c) from 7: Boc2O (1.22 eq), DMAP (0.1 eq), MeCN, N2, 6 h (55) % (similar conditions were used for a racemic mixture of 7, 40%); (d) TFA (0.5)/mDMB (1)/DCM (8.5), 30 min (90%)

a chiral stationary phase showed that chiral integrity had been maintained. The coupling of tryptophan to an aminomethyl resin functionalized by direct union of DHP derivative 2 to the polymer via a carbamate linkage was also assayed using the dihydropyranyl model 5 (Scheme 1). Unfortunately, HPLC showed that product 5 was unstable to the coupling conditions and this route was not pursued further. After completion of our studies in solution, we next turned to the union of the amino acid derivative to a polymeric solid support. Thus, handle 3 was attached to aminomethyl-polystyrene and benzhydrylamino (MBHA)polystyrene resins using benzotriazol-1-yl-oxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP) with N,N-diisopropylethylamine (DIEA) in N,N0 -dimethylformamide (DMF). Microwave irradiation of these resins in the presence of 3 eq of protected amino acid 6 and 1.6 eq of PPTS in DCE gave variable results with yields in the range 30–60%. On the other hand, conventional heating reproducibly gave yields above 80% making it the method of choice for the anchorage of tryptophan to these resins. Final amino acid functionalization levels were determined by spectrophotometric quantification of the Fmoc group (Ma and Sonveaux 1989). As with the anchoring of the amino acid to the solid support, the cleavage of the amino acid from the resin was first studied in solution with monomeric analogs and was monitored by HPLC. Table 1 summarizes the results obtained with 10 under different acidolytic conditions.

123

The use of TFA in the absence of cation scavengers furnished tryptophan derivative 6 in low yields and different reaction sub products (entries 1 and 2). These results were not improved by adding TIS (entry 3) or H2O (entry 4) to the mixture as cation scavengers. However, 3,5-dimethoxybenzene (mDMB) (Stathopoulos et al. 2006) was found to be an efficient scavenger since it notably improved the yield of 6 using similar conditions of TFA (compare entry 8 with entries 2 and 3). Higher concentrations of acid (entries 9 and 10) or other solvents (compare entries 9 and 11) did not lead to improvements in the yields of 6; nevertheless, the reduction of the TFA concentration to 5% furnished the desired tryptophan derivative in excellent yield (entry 6). HPLC analysis of this reaction showed the protected amino acid 6, an unidentified by-product (10%) and product 13 resulting from electrophilic aromatic substitution of the scavenger by the pyranyl carbocation derived from monomeric analog 4 under acidic conditions (Scheme 2). The identity of 13 was established by mass spectrometry and by chromatographic comparison with a pure sample of this product obtained by reaction of 4 with mDMB in TFA. Finally, treatment of 10 with lower concentrations of TFA (entry 5), lower concentrations of mDMB (compare entries 7 and 8) or longer reaction times (entry 7) led to lower cleavage yields of 6. The application of these optimized cleavage conditions to tryptophanyl-polystyrene resins obtained from anchoring reactions carried out using conventional heating gave good


17 O

O N H

a

H2N

Fmoc

N O

b

O

O

N N H

N H

O

N H Brevianamide F 1

N

c

O

N O

Scheme 3 Reagents and conditions: (a) (i) 3 (3 eq), DIPCDI (3 eq), OxymeTM (3 eq), DCM, 1 h, (ii) 6 (1.5 eq), PPTS (2.3 eq), DCE, 80°C, 16 h (76%, two steps); (b) (i) Pd(PPh3)4 (0.4 eq), PhSiH3 (48 eq), DCM, 30 min (two treatments), (ii) H-Pro-OMeHCl (3 eq), PyBOP (3 eq), DIEA (9 eq), DCM, 1 h (three treatments), (iii) 20% of piperidine in DMF, 10 min (two treatments); (c) TFA (0.5)/mDMB (0.5)/DCM (9), 10 min (three treatments) (74% from amino-acyl resin)

yields although longer reaction times were required (1 h, 80%). However with tryptophanyl-polystyrene resins prepared using microwave irradiation cleavage yields were much lower (around 30%) although they could be improved to around 50% by adding TIS (3%) and H2O (2%) to the cleavage cocktails. We believe that this difference in behavior may be explained by non-specific union of the amino acid to the resins or by structural changes produced in the polymers at the high temperatures generated on microwave irradiation. In order to demonstrate the usefulness of this new approach to the solid-phase synthesis of peptide derivatives

we chose to apply it to Brevianamide F 1, a natural diketopiperazine with antibiotic properties isolated from certain bacteria (Mehdi et al. 2009, and references therein) (Scheme 3). However, in order to facilitate the identification of the product synthesized by solid phase methods, we elected to synthesize the molecule in solution first in order to provide material for HPLC comparison. From among the different methods described in the literature for the synthesis of Brevianamide F (Ashnagar et al. 2007; Caballero et al. 2003; Jhaumeer-Laulloo et al. 2003; Steyn 1973), that of Ashnagar et al. (2007) was used for various reasons: the product is obtained in very good yields, the strategy developed employs protecting groups that are removed under mild conditions and that allows it to be adapted to a solid phase synthesis, and the synthetic protocol is based on coupling proline to the a-carboxyl of tryptophan, an approximation that is compatible with the union of this amino acid to the polymer through its side chain. Thus, Z-Trp-OH and H-Pro-OMe were coupled using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in DCM and hydrogenolysis of the Z group at rt using 10% Pd/C then led to spontaneous cyclization (68% yield). NMR spectroscopic data of the crude product obtained in this way indicated no significant impurities and further purification was not required. Solid-phase synthesis of 1 was then undertaken starting from an aminomethyl-polystyrene resin to which handle 3 had been attached (Scheme 3). Tryptophan derivative 6 was anchored (76% yield) as described above using conventional heating (80°C, 16 h). The allyl group was removed using Pd(PPh3)4 in the presence of PhSiH3 and H-Pro-OMeHCl was coupled using PyBOP/DIEA. Removal of the Fmoc group with piperidine then led to spontaneous cyclization as demonstrated by a negative ninhydrin test on the resin at this point. Cleavage from the resin was effected using TFA/mDMB/DCM (5:5:90 v/v),

Fig. 1 Solid-phase synthesis of Brevianamide F: a HPLC of the acidolytic crude; b HPLC of the same crude after washing with hexanes (see experimental details for HPLC conditions)

123

18

giving the essentially pure cyclic peptide in an overall yield of 56%, as determined by HPLC analysis (Fig. 1). Cyclization of the dipeptide resulting from coupling of FmocPro-OH to the a-amino group of tryptophan required longer reaction times.

Conclusions With the synthesis of Brevianamide F, this work demonstrates the promise of anchoring tryptophan through its side chain to a DHP functionalized solid support as an approach to the synthesis of peptide derivatives containing tryptophan such as cyclic peptides. In principle, it would be also applicable to the solid-phase synthesis of other tryptophancontaining molecules with potential pharmacologic interest and libraries of such compounds. On the other hand, 4-[(3,4-dihydro-2H-pyran-2-yl)methoxy]benzoic acid has proved to be a suitable handle to functionalize aminomethyl- or MBHA-polystyrene resins with dihydropyranyl groups. Acknowledgment We are grateful for financial support from Ministerio de Ciencia e Innovacioń (CTQ2006-12460).

References Anderson MO, Shelat AA, Guy RK (2005) Solid-phase approach to the phallotoxins: total synthesis of [Ala7]-phalloidin. J Org Chem 70:4578–4584 Ashnagar A, Bailey PD, Cochrane PJ, Mills TJ, Price RA (2007) Unusual rearrangements and cyclizations involving polycyclic indolic systems. Arkivoc 11:161–171 Basso A, Ernst B (2001) Solid-phase synthesis of hydroxyprolinebased cyclic hexapeptides. Tetrahedron Lett 42:6687–6690 Bernhardt A, Drewello M, Schutkowski M (1997) The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides. J Pept Res 50:143–152 Black KM, Clark-Lewis I, Wallace CJA (2001) Conserved tryptophan in cytochrome c: importance of the unique side-chain features of the indole moiety. Biochem J 359:715–720 Caballero E, Avendanõ C, Meneńdez JC (2003) Brief total synthesis of the cell cycle inhibitor tryprostatin B and related preparation of its alanine analogue. J Org Chem 68:6944–6951 Cabrele C, Langer M, Beck-Sickinger AG (1999) Amino acid side chain attachment approach and its application to the synthesis of tyrosine-containing cyclic peptides. J Org Chem 64:4353–4361 Chang J, Dong C, Guo X et al (2005) A solid-phase approach to novel purine and nucleoside analogs. Bioorg Med Chem 13:4760–4766 Gordon EM, Kerwin JF (1998) Combinatorial chemistry and molecular diversity in drug discovery. Wiley-Liss, New York Graham KAN, Wang Q, Eisenhut M, Haberkorn U, Mier W (2002) A general method for functionalising both the C- and N-terminals of Tyr3-octreotate. Tetrahedron Lett 43:5021–5024 Gu W, Silverman RB (2003) Solid-phase total synthesis of scytalidamide A. J Org Chem 68:8774–8779 Guibe F (1998) Allylic protecting groups and their use in a complex environment. Part II: allylic protecting groups and their removal

123

Int J Pept Res Ther (2012) 18:7–19 through catalytic palladium pi-allyl methodology. Tetrahedron 54:2967–3042 Jhaumeer-Laulloo S, Khodabocus A, Jugoo A, Jheengut D, Sobha S (2003) Synthesis of diketopiperazines containing prolinyl unit— cyclo(L-prolinyl-L-leucine), cyclo(L-prolinyl-L-isoleucine) and cyclo(L-tryptophyl-L-proline). J Indian Chem Soc 80:765–768 Kates SA, Sole NA, Johnson CR, Hudson D, Barany G, Albericio F (1993) A novel, convenient, three-dimensional orthogonal strategy for solid-phase synthesis of cyclic peptides. Tetrahedron Lett 34:1549–1552 Kitade M, Tanaka H, Oe S, Iwashima M, Iguchi K, Takahashi T (2006) Solid-phase synthesis and biological activity of a combinatorial cross-conjugated dienone library. Chem Eur J 12:1368–1376 Kuriyama W, Kitahara T (2001) Synthesis of apicidin. Heterocycles 55:1–4 Liu S, Gu W, Lo D et al (2005) N-Methylsansalvamide and peptide analogues. Potent new antitumor agents. J Med Chem 48: 3630–3638 Ma JC, Dougherty DA (1997) The cation-p interaction. Chem Rev 97:1303–1324 Ma Y, Sonveaux E (1989) The 9-fluorenylmethyloxycarbonyl group as a 50 -OH protection in oligonucleotide synthesis. Biopolymers 28:965–973 Mant CT, Kovacs JM, Kim HM, Pollock DD, Hodges RS (2009) Intrinsic amino acid side-chain hydrophilicity/hydrophobicity coefficients determined by reversed-phase high-performance liquid chromatography of model peptides: comparison with other hydrophilicity/hydrophobicity scales. Biopolymers 92: 573–595 Mehdi RBA, Shaaban KA, Rebai IK, Smaoui S, Bejar S, Mellouli L (2009) Five naturally bioactive molecules including two rhamnopyranoside derivatives isolated from the Streptomyces sp. strain TN58. Nat Prod Res B 23:1095–1107 Milne PJ, Kilian G (2010) The properties, formation, and biological activity of 2,5-diketopiperazines. In: Mander L, Liu HW (eds) Comprehensive natural products II: chemistry and biology, vol 5. Elsevier Science, Amsterdam, pp 657–698 Nam N-H, Sardari S, Parang K (2003) Reactions of solid-supported reagents and solid supports with alcohols and phenols through their hydroxyl functional group. J Comb Chem 5:479–546 Nugiel DA, Cornelius LAM, Corbett JW (1997) Facile preparation of 2,6-disubstituted purines using solid-phase chemistry. J Org Chem 62:201–203 Richard DJ, Schiavi B, Joullie MM (2004) Synthetic studies of roquefortine C: synthesis of isoroquefortine C and a heterocycle. Proc Natl Acad Sci USA 101:11971–11976 Rodrigues de Sa Alves F, Barreiro EJ, Fraga CAM (2009) From nature to drug discovery: the indole scaffold as a ‘Privileged Structure’. Mini Rev Med Chem 9:782–793 Ruiz-Sanchis P, Savina Svetlana A, Albericio F, Alvarez M (2011) Structure, bioactivity and synthesis of natural products with hexahydropyrrolo[2,3-b]indole. Chemistry 17:1388–1408 Samanta U, Pal D, Chakrabarti P (1999) Packing of aromatic rings against tryptophan residues in proteins. Acta Crystallogr D D55:1421–1427 Samanta U, Pal D, Chakrabarti P (2000) Environment of tryptophan side chains in proteins. Proteins Struct Funct Genet 38:288–300 Sawyer TK (1997) Peptidomimetic and nonpeptide drug discovery: impact structure-based drug design. In: Veerapandian P (ed) Structure based drug design: disease, targets, techniques and development. Marcel Dekker, New York, pp 559–634 Smith AL, Stevenson GI, Swain CJ, Castro JL (1998) Traceless solid phase synthesis of 2,3-disubstituted indoles. Tetrahedron Lett 39:8317–8320

Int J Pept Res Ther (2012) 18:7–19 Spatola AF, Romanovskis P (2000) Head-to-tail cyclic peptides and cyclic peptide libraries. In: Greenberg A, Breneman CM, Liebman JF (eds) The amide linkage: selected structural aspects in chemistry, biochemistry and materials science. Wiley, New York, pp 519–564 Stathopoulos P, Papas S, Tsikaris V (2006) C-terminal N-alkylated peptide amides resulting from the linker decomposition of the Rink amide resin. J Pept Sci 12:227–232 Steyn PS (1973) Structure of five dioxopiperazines from Aspergillus ustus. Tetrahedron 29:107–120 Tanaka H, Ishida T, Matoba N, Tsukamoto H, Yamada H, Takahashi T (2006) Efficient polymer-assisted strategy for the deprotection of protected oligosaccharides. Angew Chem Int Ed 45:6349–6352

19 Thompson LA, Ellman JA (1994) Straightforward and general method for coupling alcohols to solid supports. Tetrahedron Lett 35:9333–9336 Trzeciak A, Bannwarth W (1992) Synthesis of head-to-tail cyclized peptides on solid support by Fmoc [9-fluorenylmethoxycarbonyl] chemistry. Tetrahedron Lett 33:4557–4560 Villorbina G, Canals D, Carde L et al (2007) Solid-phase synthesis of a combinatorial library of dihydroceramide analogues and its activity in human alveolar epithelial cells. Bioorg Med Chem 15:50–62 Wallace OB (1997) Solid phase synthesis of ketones from esters. Tetrahedron Lett 38:4939–4942 Wang X, Choe Y, Craik CS, Ellman JA (2002) Design and synthesis of novel inhibitors of gelatinase B. Bioorg Med Chem Lett 12:2201–2204

123