Synthesis of derivatives of potent antitumor bistramide D and A leading to the first crystal structure of natural bistramide D

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1 SUPPLEMETARY MATERIAL Synthesis of derivatives of potent antitumor bistramide D and A leading to the first crystal structure of natural bistramide D Claude Bauder,* a,c Jean-François Biard b and Guy Solladié c a) Institut de Chimie LC 3, associé au CRS - Université Louis Pasteur, rue Blaise Pascal, Strasbourg, France. b) Groupe SMAB, Université de antes, Pôle Mer et Littoral, Faculté de Pharmacie,. 435 antes Cedex 0, France. c) Laboratoire de Stéréochimie associé au CRS - Université Louis Pasteur, ECPM - 25 rue Becquerel, Strasbourg, Cedex 2, France. cbauder@chimie.u-strasbg.fr Electronic Supplementary Information (ESI) available: 2D MR spectroscopic data of BST Ada 3, BST Dda 4 and BST Dda, 4-TBS 5 Typical experimental procedures for 2, 3, 4 and 5. Table. Comparison of 3 C MR chemical shifts of natural bistramide D, with the hemisynthetic bistramide D (2), derivatives BST Ada (3), BST Dda (4) and BST Dda, 4-TBS (5). Table 2. Comparison of MR chemical shifts of derivatives BST Ada (3), BST Dda (4) and BST Dda, 4-TBS (5). spectroscopic data of and 3C MR for natural bistramide D spectroscopic data of and 3C MR for hemisynthetic bistramide D; 2 spectroscopic data of and 3C MR for bistramide Ada; 3 spectroscopic data of and 3C MR for bistramide Dda; 4 spectroscopic data of and 3C MR for bistramide Dda, 4-TBS; 5

2 2 CSY spectrum of BST Ada : 3 (500 Mz; CDCl 3 ) CSY spectrum of BST Ada : 3 (500 Mz; CDCl 3 )

3 RESY spectrum of BST Ada : 3 (500 Mz; CDCl 3 ) 3

4 RESY spectrum of BST Ada : 3 (500 Mz; CDCl 3 ) 4

5 MQC spectrum of BST Ada : 3 (500 Mz; CDCl 3 ) 5

6 6 MQC spectrum of BST Ada : 3 (500 Mz; CDCl 3 ) MBC spectrum of BST Ada : 3 (500 Mz; CDCl 3 )

7 7 MBC spectrum of BST Ada : 3 (500 Mz; CDCl 3 ) CSY spectrum of BST Dda : 4 (500 Mz; CDCl 3 )

8 CSY spectrum of BST Dda : 4 (500 Mz; CDCl 3 ) 8

9 RESY spectrum of BST Dda : 4 (500 Mz; CDCl 3 )

10 RESY spectrum of BST Dda : 4 (500 Mz; CDCl 3 ) 0

11 SQC spectrum of BST Dda : 4 (500 Mz; CDCl 3 )

12 2 SQC spectrum of BST Dda : 4 (500 Mz; CDCl 3 ) MBC spectrum of BST Dda : 4 (500 Mz; CDCl 3 )

13 3 MBC spectrum of BST Dda : 4 (500 Mz; CDCl 3 ) CSY spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 )

14 CSY spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 ) 4

15 RESY spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 ) 5

16 RESY spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 ) 6

17 SQC spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 ) 7

18 8 SQC spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 ) MBC spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 )

19 MBC spectrum of BST Dda, 4-TBS : 5 (500 Mz; CDCl 3 )

20 20 General Information. Reactions were carried out in oven or flame-dried glassware under an argon atmosphere, unless otherwise noted. All solvents used were of reagent grade. Tetrahydrofuran (TF) was freshly distilled from sodium/benzophenone under argon immediately prior to use. Unless otherwise noted, reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with 0.25 mm Merck pre-coated silica gel plates. Flash chromatography were performed with silica gel 60 (particle size mm) supplied by Merck, Geduran. Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. MR, 3C MR, CSY, RESY, SQC as well as MBC spectra were measured on Bruker ARX-500 spectrometer using an internal deuterium lock at ambient temperature. If not otherwise noted, CDCl 3 (7.26 ppm relative to residual CCl 3 ) is the solvent for all MR experiments. Multiplicities are described using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, ABX = an ABX system. Chemical shift are given in ppm and coupling constant are presented in z. ptical rotations [α] were recorded on a Polarimeter 24MC (Perkin Elmer) at a wavelength of 58 nm and are reported as follows: [α] D, concentration (c in g/00 ml) and solvent. Elemental analysis were collected at the Service de microanalyse of the University Louis Pasteur of Strasbourg. Experimental section. Stereoselective reduction of the bistramide A: BST D 2.

21 BST A 28 BST D LiB(Et) A solution of Super-ydride (M in TF; 2.7 ml; 5.3 eq) was added dropwise at -78 C to a precooled solution of bistramide A (358 mg, mmol) in dry TF ( ml). The reaction was stirred for h at this temperature and then quenched with a saturared aqueous solution of 4 Cl (5 ml) and water (20 ml). The mixture was stirred at ambient temperature during 0.5 h. The organic layer was extracted with C 2 Cl 2 (2 x 5 ml). The combined organic extracts were washed with water (5 ml), brine (5 ml) and then dried (MgS 4 ), filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (acetone / C 2 Cl 2 ; 7:3) to afford the pure bistramide D 2 (254.4 mg; 70.%). The spectroscopic data ( and 3 C MR) are in total agreement with the literature (J.F. Biard, C. Roussakis, J.M. Kornprobst, D. Gouiffes-Barbin, J.F. Verbist, P. Cotelle, M.P. Foster, C.M. Ireland and C. Debitus, J. at. Prod., 4, 57 (0), ). See table (below) for comparison of the 3 C MR values. PLC chromatogram of natural bistramide D (A) and a mixture (50 / 50) of natural and synthetic bistramide D (B). Each injection has a concentration of 250 µg / 25 µl in CCl 3 : Me (6 : 4) as solvant and eluant ( ml / min). Silicagel column (Interchim ucleosil 5 µm, 250 x 4.6 mm) was used and absorbance was monitored with a refractometer detector. Acetylation of bistramide A into diacetylated product: BST Ada 3.

22 BST A 28 BST Ada 28 Ac Ac Ac Anhydride acetic (0.7 ml; 24 eq.) was added dropwise under argon to a solution of bistramide A (27.5 mg; mmol) in pyridine (3 ml). The resulting yellow mixture was stirred during 4 h at room temperature. Tlc analysis (acetone/ C 2 Cl 2 6:4) showed no more starting material so the reaction was diluted with diethylether (20 ml) and then quenched with a saturated aqueous solution of CuS 4 (25 ml) and stirring was continue during 5 h at room temperature. The organic layer was washed with water (4 x 20 ml), brine (0 ml), dried (MgS 4 ), filtered and concentrated under reduced pressure to furnish 3 (2 mg;. %) as a white foam. Elemental analysis for C (78.050): calcd. C, 66.8;,.20;, Found: C, 66.54;,.26;, 3.2. For comparison with the other derivatives of the bistramides, the MR spectroscopy data are given below in table (for 3 C MR) and in table 2 (for MR). Stereoselective reduction of the diacetylated bistramide A: BST Dda Ac 4 6 BST Ada 28 BST Dda LiB(Et) 3 3 Ac Ac Ac A solution of bistramide Ada 3 (27. mg; 0.62 mmole) in anhydrous TF (3.5 ml) was left under argon and stirred at -75 C during 30 min before adding dropwise the Super-ydride (M in TF; 0.65;.02 eq). After 25 min at this temperature no starting material was observed (monitored by tlc, acetone/c 2 Cl 2 6:4). Thus the reaction was quenched by a saturated aqueous solution of 4 Cl (0 ml) and the reaction flask was brought to room temperature for more 4 h. If no good separation is

23 observed, it is recommanded to add a small quantity of Cl 5 % until p 3-4, specially for gramme amount of starting material. The organic layer was concentrated under reduced pressure and the aqueous layer was extracted with AcEt (4 x 5 ml). The combinated organic layers were washed with brine (0 ml), dried over MgS 4, filtered and concentrated under reduced pressure to afford a white foam. The residue was purified by silica gel chromatography (eluant gradian, Acetone/C 2 Cl 2 :/3 to /) to get 4 (2. mg; 72.5 %) as a white foam after evaporation of all the solvants. [α] 20 D + 0 (c.0 in C 2 Cl 2 ). Elemental analysis for C (7.066): calcd. C, 66.80;,.43;, Found: C, 65.6;,.50;, For comparison with the other derivatives of the bistramides, the MR spectroscopy data are given below in table (for 3 C MR) and in table 2 (for MR). Regioselective hydrolysis of bistramide Dda: BST D BST Dda Ac LiAl Ac BST D A solution of LiAl 4 (M in Et 2 ; 0.33 ml; 3.48 eq) was dropwise added at 0 C into a solution of bistramide Dda 4 (75 mg; 0.05 mmol). A white precipitate was observed and the mixture was left for 6 h at room temperature. The reaction was then quenched with a saturated aqueous solution of 4 Cl (3 ml) and water (3 ml) and stirring was continue during 6 h at ambient temperature. The aqueous layer was brought to p 5 with Cl 0% and then extracted with Et 2 (3 x 0 ml). The combined organic extracts were washed with water (2 x 0 ml), brine (0 ml), dried (MgS 4 ), filtered and concentrated under reduced pressure to afford the bistramide D 2 (54. mg; 80.%) which exhibited the same MR characteristics as the bistramide D obtained above in the stereoselective reduction of bistramide A. The spectroscopic data ( and 3 C MR) are in total agreement with the literature (J.F. Biard, C. Roussakis, J.M. Kornprobst, D. Gouiffes-Barbin, J.F. Verbist, P. Cotelle, M.P. Foster, C.M. Ireland and C. Debitus, J. at. Prod., 4, 57 (0), ). Silylation of diacetylated bistramide Dda: BST Dda 4-TBS 5.

24 Ac 0 TBS Ac BST Dda 4 Ac TBSCl BST Dda, 4-TBS 5 Ac Bistramide Dda 4 (270.8 mg; mmole) and imidazole (76 mg; 3.26 eq) were dissolved under argon in DMF (5 ml) and then TBDMSCl (77 mg; 3.43 eq) was added int one portion. The mixture was stirred 6 h at room temperature before hydrolysis by water (30 ml) and diluted with diethylether (50 ml). Vigorous stirring was continued for.5 h and then the aqueous layer was extracted with diethylether (2 x5 ml). The combined organic layers were washed with water (6 x 50 ml), brine (5 ml), dried (MgS 4 ), filtered and concentrated under reduced pressure to obtain a crude product. The residue was purified by column chromatography on silica gel (AcEt/C 2 Cl 2 :) to get 5 ( mg; 74.6 %) as a white solid. In order to have some crystals of this compound for X-ray analysis we recrystallized it in hot pentane. Mp = C (pentane). [α] D + 84 (c. in C 2 Cl 2 ). Elemental analysis for C Si (05.327): calcd. C, 66.33;,.80;, 3.0. Found: C, 66.20;,.2;, For comparison with the other derivatives, the MR spectroscopy data are given below in table (for 3 C MR) and in table 2 (for MR). Table : 3 C RM (50 Mz, CDCl 3 ) for natural BST D and BST D (2) 3 C RM (25 Mz, CDCl 3 ) for BST Ada (3), BST Dda (4) and BST Dda, 4-TBS (5)

25 25 natural BST D BST D (2) BST Ada (3) BST Dda (4) BST Dda, 4-TBS (5) 75.6; C ; C 72.5; C ; C ; C ; C ; C ; C-8 37.; C ; C ; CMe 70.5; CMe ; C ; CMe 70.4; CMe 70.2; CMe-5 3.; C ; C2 34.2; C3 34.2; C ; C ; C ; C ; C3 5.5; C ; C ; C37 32.; C ; C ; C3 26.0; C2 25.; C2 74.2; C ; C27 5.5; C27 5.4; C ; C ; C3 75.6; C3 75.6; C3 73.; C ; C 75.2; C5 74.3; C5 and C22 7.; C ; C5 and C ; C ; C 6.4; C ; C3 74.0; C 70.6; C4 6.; C ; C6 72.4; C4 6.0; C3 43.8; C ; C5 6.6; C6 66.; C6 43.2; C ; C6 6.0; C3 43.4; C5 42.; C ; C4 42.; C5 42.3; C6 3.4; C ; C 4.8; C6 3.; C4 36.; C ; C26.0; C4 3.8; C 35.4; C ; C28 3.7; C 36.; C ; C ; C 36.2; C ; C ; C ; C ; C ; C 33.5; C ; C33 34.; C 34.; C32 33.; C ; C 34.; C ; C2 33.0; C ; C2 33.4; C ; C33 3.8; C ; C ; C 33.0; C 3.2; C ; C30 32.; C2 3.; C34 3.; C ; C2 32.0; C34 3.3; C ; C ; C7 3.4; C ; C2 27.8; C ; C25 3.3; C7 2.0; C7 26.4; C ; C8 30.6; C2 27.8; C ; C ; C20 27.; C ; C8 2.8; Me ; CMe 26.4; C8 25.; C20 20.; Me ; CMe 25.; C ; tbu-si.2; C ; Me ; CMe 2.5; CMe 7.; Me ; Me- 2.2; CMe 2.; CMe 7.6; Me ; C2 20.8; Me ; Me ; Me ; Me-.3; Me-.2; Me- 5.8; Me ; Me-24.2; C2.; C2.; Me ; Me-0 8.0; Me-24 8.; C-Si 4.3; Me-7 7.7; Me- 8.0 Me-24.; Me ; Me-0 7.6; Me- 4.5; Me-7 6.2; Me ; Me ;Me-7 natural BST D BST D (2) BST Ada (3) BST Dda (4) BST Dda, 4-TBS (5) 2.; Me-38

26 26-3.; Me-Si -4.; Me-Si Table 2 : RM (500 Mz, CDCl 3 ). δ (ppm) BST Ada (3) BST Dda (4) BST Dda, 4-TBS (5) mult. 3 J, (z) dd 6.8,.6 dq 5.8, 6. dq 5.8,.6 δ (ppm) mult. 3 J, (z) dd 6.4,.4 dq 5.2, 6.4 ddq 5.2, 6.4,.5 δ (ppm) m 4.0 mult. 3 J, (z) dd 6.0,.6 ddq 5.4, 6.6,. ddq 5.4, 7.,.6 dt 7.5, , 2.50 ABX 6.7,.0, 2.7.7,.56 m, m.77,.54 m, m m 3.86 m 3.76 m 7.65,.34 m, m.4,. m, m.77,.27 m, m 8.58,.33 m, m.62,.3 m, m.66,.28 m, m. m.3 m.0 m d d ddd.4, 4., m 4.3 m 2 ABX 2.7, ,.5, , 2.4 ABX 5.7,.8, , dt and dt , , , 3.43 m, m 3.53 and 5., 6.3 d 7.5 ABX 6.4, 4.4,. dd 6.05 (c) ddd dt 4.88 m (d) 7., dq dq dq (a) 6. (a) 7. (a) 7.8 d d d m 3.2, 3.20 m, m 3.2 m 20.8,.50 m, m.80,.46 m, m.82,.52 m, m 2.66,.32 m.33,.65 m, m.67,.33 m, m ddd ddd ddd (b), 2..6 (b), (b), 2.2 BST Ada (3) BST Dda (4) BST Dda, 4-TBS (5)

27 27 mult. mult. δ (ppm) δ (ppm) δ (ppm) 3 J, (z) 3 J, (z).26 m.24 m.27 m mult. 3 J, (z) d d d ,.42 m, m.52,.4 m, m.55,.45 m, m 26.57,. m, m.58,.42 m, m.5,.43 m, m ,.32 m, m.52,.32 m, m.54,.35 m, m 2.7,.48 m, m.77,.48 m, m.85,.54 m, m 30.48,.0 m, m.4,. m, m.50,.4 m, m m 3.43 m 3.4 m 32.3,.24 m, m.33,.27 m, m.36,.2 m, m 33.35,.27 m, m.35,.28 m, m.37,.30 m, m m 2.32 m 2.32 m d d d bd bd m d d d q q dq , d d d br t br t br t br t br t br t Ac. S (6) 2.04, x s 2.05, x s or TBDMS 3.56 br s 0.86 (tbu) 0.03 (MeSi) 0.00(MeSi) 3 x s (a) : (J6-5 = J6-7) (b) : (J22-2 = J22-) (c) : (J4-5 = J4-) (d) : (J5-6 = J5-4a = J5-4b) atural bistramide D ( MR, 200 Mz, CDCl 3 )

28 BST D emisynthetic bistramide D, BST D : 2 ( MR, 200 Mz, CDCl 3 ) BST A 28 2 BST D LiB(Et) BST D : 2 ( MR, 200 Mz, CDCl 3 ) BST Dda 5 7 Ac LiAl Ac BST D atural bistramide D ( 3 C MR, 50 Mz, CDCl 3 )

29 emisynthetic bistramide D, BST D : 2 ( 3 C MR, 50 Mz, CDCl 3 ) BST A 28 LiB(Et) BST D BST D : 2 ( 3 C MR, 50 Mz, CDCl 3 ) BST D via reduction of BST Dda

30 BST Dda 5 7 Ac LiAl Ac BST D

31 BST A 28 BST Ada 28 Ac Ac Ac BST Ada : 3 ( MR, 500 Mz, CDCl 3 ) BST Ada : 3 ( 3 C MR, 25 Mz, CDCl 3 )

32 BST Ada Ac Ac 3 34 LiB(Et) BST Dda Ac Ac 3 34 BST Dda : 4 ( MR, 500 Mz, CDCl 3 ) BST Dda : 4 ( 3 C MR, 25 Mz, CDCl 3 )

33 BST Dda Ac Ac 3 34 TBSCl TBS 4 6 BST Dda, 4-TBS 5 0 Ac Ac BST Dda, 4-TBS : 5 ( MR, 500 Mz, CDCl 3 ) BST Dda, 4-TBS : 5 ( 3 C MR, 25 Mz, CDCl 3 )

34 34 MR details of bistramide BST Ada : C MR details of bistramide BST Ada :

35 35 MR details of bistramide BST Dda : C MR details of bistramide BST Dda :

36 36 MR details of bistramide BST Dda, 4-TBS : C MR details of bistramide BST Dda, 4-TBS :