Total synthesis of the potent immunosuppressant ( − )-Pironetin

A convergent and efficient total synthesis of (  -)-pironetin, a compound that shows plant growth regulatory activity, and is immunosuppressive as well as having remarkable antitumoral activity, is described. The synthesis required 19 steps from N - propionyl oxazolidinone ( S )- 9 and produced the desired product in 11% overall yield.


Introduction
The potent immunosuppressor pironetin 1 (1) was isolated independently by two research groups from Streptomyces sp.NK-10958 and from the fermentation broths of Streptomyces prunicolor PA-48153 (Figure 1).Pironetin shows plant growth regulatory activity as well as immunosuppressive and antitumor activities. 2,3The mode of action of pironetin is different from those established for the immunosuppressants cyclosporin A (CsA) and FK506, which inhibit T cell activation. 4Pironetin showed suppressive effects on the responses of T and B lymphocytes to mitogens.Our first approach to fragment C5-C11 started with the aldol reaction between the (Z)-boron enolate of N-propionyl oxazolidinone (R)-9 with propionaldehyde to give aldol adduct 10 in 82% yield and >99:1 diastereoselectivity (Scheme 2). 9 Treatment of 10 with SO 3 -pyr in a mixture of DMSO/CH 2 Cl 2 gave β-ketoimide 7 in 96% yield. 11Treatment of β-ketoimide 7 with Sn(OTf) 2 and Et 3 N in CH 2 Cl 2 followed by addition of aldehyde 8 gave aldol adduct 6 with a modest diastereoselectivity (86:14) in only 50% yield. 11,12ttempts to improve yields and diastereoselectivities for this transformation failed.The next step involved reduction of the ketone function in 6 with Me 4 NHB(OAc) 3 in CH 3 CN/CH 3 CO 2 H to provide diol 11, together with lactone 12 and chiral auxiliary 13, a mixture which was difficult to separate by silica gel column chromatography. 13We were able to isolate only small amounts of lactone 12, and its formation proved that the reduction proceeded with the desired stereochemistry.Coupling constants between H1−H2 (10.3 Hz), H2−H3 (4.4 Hz) and H3−H4 (4.6 Hz), confirmed the relative stereochemistry for lactone 12.
In view of these disappointing results, we decided to use another strategy for the synthesis of fragment C5-C11, which is based in the use of two consecutive asymmetric aldol reactions (Scheme 3).Synthesis of aldehyde 16 began with asymmetric aldol addition of the boron enolate derived from N-propionyloxazolidinone (S)-9 with aldehyde 8 12 to give aldol adduct 14 in 87% yield (ds >95:5) (Scheme 3). 9,106][17] Formation of lactone 12 was accomplished after a three-step sequence (56% overall yield) that involved treatment of aldol 17 with HF/H 2 O/CH 3 CN, cleavage of the oxazolidinone auxiliary with H 2 O 2 /LiOH 14 , and treatment of the carboxylic acid 18 in refluxing benzene.
Methylation of 17 with Me 3 OBF 4 in the presence of a proton sponge at ambient temperature, provided 19 in 60% isolated yield (Scheme 5). 16As this reaction proved to be difficult to reproduce on a larger scale, we decided to promote the conversion of 17 to the corresponding primary alcohol.Treatment of 17 with LiBH 4 in THF/MeOH at 0 o C provided 1,3diol 20 in 89% yield (Scheme 5).The next steps involved tosylation of the primary OH-function in 20 to provide tosylate 21 (95% yield) followed by methylation with Me 3 OBF 4 in the presence of a proton sponge at ambient temperature, providing 5a in 89% isolated yield (Scheme 5). 16

O N Me
O O Bn (R)-9  We next moved to the real system (Scheme 7).Treatment of tosylate 5a with Grignard reagent 4b gave bromide 5c in good yields as the sole product, together with starting material.Treatment of tosylate 5a with cuprate 4c (50 equivalents) led to a mixture of primary alcohol 24 (60%) and the desired product 25 in only 15% yield. 18Confirmation that alcohol 24 has been formed in this reaction came from treatment of aldol adduct 19 with LiBH 4 and MeOH in THF providing the same alcohol 24 in excellent yields.Bromide 5c also proved to be unreactive under these conditions with both 4b and 4c.In spite of a series of experimental modifications we were not able to improve the yields for the formation of 25.
After examining several different attempts to couple C12 vinyl cuprates, C11 tosylates and bromides, we turned our attention to the use of a Suzuki coupling approach employing an alkyl iodide with vinyl bromides and vinyl iodides. 19Alkyl iodide 5b was prepared after a four-step sequence starting with diol 20 (Scheme 8).Selective silylation of diol 20, followed by methylation with Me 3 OBF 4 in the presence of a proton sponge at ambient temperature, gave 27 (84% overall yield).Selective removal of the TBS primary group with a solution of HF-pyridine in THF and treatment of the resulting primary alcohol 24 with PPh 3 , I 2 and imidazole gave iodide 5b in 83% overall yield for the two-step sequence.As before, we first did a model study for this coupling (Scheme 9).This was achieved through the use of a Pd-catalyzed coupling of an intermediate boronate derived from alkyl iodide 29 with vinyl bromide 4a (Scheme 9). 19 Scheme 10.Attempts to promote the Suzuki coupling in the real system.
Due to the difficulties in installing the (E)-double bond using these approaches, we abandoned these strategies and altered our synthetic route.At this point, we were attracted to a study by Smith and Beumel, 20 who reported the displacement of tosylates by means of the lithium acetylide-ethylenediamine complex to give alkynes in good yields.On the basis of this precedent, we focused our attention on this synthetic strategy. 21e were very pleased to find that treatment of tosylate 5a with 5 equivalents of lithium acetylide in DMSO at room temperature produced acetylene 31 in 82% yield, together with elimination product 32 in 13% yield (Scheme 11).After treatment of 31 with n BuLi and quenching with methyl iodide we were able to isolate the corresponding alkyne. 22eduction of the alkyne proceeded smoothly with Na and liquid NH 3 providing control for the (E)-geometry of the C12−C13 double bond with concomitant removal of the PMB group at C5, giving primary alcohol 33 in 49% yield for the three-step sequence from 5a (Scheme 11). 23PAP oxidation of 33 under the standard conditions gave the desired aldehyde 2 in 94% yield. 24symmetric aldol addition of the boron enolate derived from N-butanoyloxazolidinone 3 with aldehyde 2 gave aldol adduct 34 in 90% yield (ds >95:5) (Scheme 12).However, all our attempts to prepare the Weinreb amide derivative by treatment of aldol adduct 34 with MeONHMe.HCl and Me 3 Al in THF failed and we decided to prepare primary alcohol 35.Silylation of aldol 34 with TBSOTf and 2,6-lutidine was followed by treatment with LiBH 4 in THF/MeOH to provide alcohol 35 (87% yield, 2 steps).In order to introduce the (Z)-double bond, we prepared phosphonate 36 using two different approaches.The first one involved treatment of o-cresol with PCl 3 in the presence of imidazole in CH 2 Cl 2 as solvent, followed by a sequence involving reaction with water and treatment with ethyl 2-bromoacetate in the presence of Et 3 N to give 36 in 70% over two steps (Scheme 13). 25l 3 Scheme13.Preparation of phosphonate 36.

Me
The second approach to phosphonate 36 started with treatment of ethyl 2-(diethoxyphosphoryl)acetate with PCl 5 under reflux, to give ethyl 2-(chlorophosphonyl)acetate, which, after treatment with o-cresol and Et 3 N in benzene at 0 ºC, gave ethyl 2-((bis(otolyloxy))phosphoryl)acetate (36) in 75% yield over two steps (Scheme 13). 25rimary alcohol 35 was treated with TPAP 24 to provide the aldehyde, which reacted with β-ketophosphonate 36 in the presence of NaH in THF to give (Z)-α,β-unsaturated ester 37 (Z:E >95:05) in 84% yield over two steps (Scheme 14).The (Z)-geometry for ester 37 was confirmed by coupling constant analysis.2][3][4] In summary, a convergent and efficient total synthesis of (-)-pironetin has been accomplished.The synthesis required 19 steps from oxazolidinone (S)-9 and produced the desired product in 11% overall yield.This approach compares very well with other published routes, being one of the shortest approaches to (-)pironetin.As a result, the route presented here is, in principle, readily applicable for the preparation of additional analogues of pironetin. 26

Experimental Section
General Procedure.All reactions were carried out under an atmosphere of argon or nitrogen in flame-dried glassware with magnetic stirring.Dichloromethane, triethylamine, 2,6-lutidine, diisopropylamine, dimethylformamide and N-methylpyrrolidone were distilled from CaH 2 .Dimethyl sulfoxide was distilled under reduced pressure from calcium hydride and stored over molecular sieves.THF and toluene were distilled from sodium/benzophenone ketyl.Oxalyl chloride was distilled immediately prior to use.MeOH was distilled from Mg(OMe) 2 .Petrol refers to the fraction boiling between 40-60 o C. Purification of reaction products was carried out by flash chromatography using silica-gel (230-400 mesh).Analytical thin layer chromatography was performed on silica gel 60 and GF (5-40-µm thickness) plates.Visualization was accomplished with UV light and anisaldehyde, ceric ammonium nitrate stain or phosphomolybdic acid followed by heating or I 2 staining. 1H-NMR spectra were taken in CDCl 3 at 300 MHz or at 500 MHz spectrometer and are reported in ppm using solvent as an internal standard (CDCl 3 at 7.26 ppm) unless otherwise indicated.Data are reported as (ap = apparent, s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, ap t = apparent triplet, m = multiplet, br = broad, td = triplet of doublets, quint d = quintet of doublets, coupling constant(s) in Hz; integration.Proton-decoupled 13 C-NMR spectra were taken in CDCl 3 at 75 MHz spectrometer and are recorded in ppm.

(S)-3-((2S,3R)-5-(4-Methoxybenzyloxy)-3-hydroxy-2-methylpentanoyl)-4-benzyloxazolidin-2-one (14).
Di-n-butylboryltrifluoromethanesulfonate (0.78 mL, 3.15 mmol) was added to a solution of (S)-4-benzyl-3-propionyloxazolidin-2-one 14 (0.61 g, 2.62 mmol) in 7 mL of CH 2 Cl 2 at such a rate to maintain the internal temperature below +3 o C (type K thermocouple thermometer).Triethylamine (0.48 mL, 3.41 mmol) was then added dropwise (internal temperature below +4 o C).The resulting yellow solution was then cooled to -78 o C and aldehyde 8 (0.56 g, 2.89 mmol) in 6 mL of CH 2 Cl 2 was added slowly (internal temperature below -70 o C).After 20 min, the solution was warmed to 0 o C and stirred at that temperature for 1 h.The reaction was quenched by the addition of 3 mL of pH 7.0 aqueous phosphate buffer solution and 9 mL of MeOH (internal temperature below +10 o C, bath temperature = -10 o C).A solution of 12 mL of MeOH and 8 mL of 30% aqueous H 2 O 2 was added carefully (internal temperature below +10 o C) and the resulting yellow solution was stirred at 0 o C for 1 h.The volatiles were removed at aspirator pressure and the residue was extracted with three 15 mL portions of Et 2 O.The combined organic extracts were washed with 20 mL of saturated aqueous NaHCO 3 and 20 mL of brine.The organic solution was dried over anhydrous MgSO 4 and purified by flash column chromatography (30% EtOAc/hexanes) to give 0.974 g of the syn aldol adduct 14 as a colorless oil (87% yield, >95:5 diastereoselectivity).R f 0.34 (50% EtOAc/hexanes); [α]

(2S,3R)-5-(4-Methoxybenzyloxy)-3-hydroxy-N-methoxy-N,2-dimethylpentanamide.
To a suspension of N,O-dimethylhydroxylamine hydrochloride (0.401 g, 4.11 mmol) in 4 mL of THF at 0 °C was added 2.1 mL (4.15 mmol) of a 2.0 M solution of trimethylaluminum in toluene (gas evolution).The resulting solution was stirred at ambient temperature for 30 min, and then cooled to -15 °C.A solution of β-hydroxy imide 14 (0.585 mg, 1.37 mmol) in 3 mL of THF was added by cannula and the resulting mixture was stirred at 0 °C for 2 h.This solution was transferred by cannula to a well-stirred mixture of 15 mL of CH 2 Cl 2 and 30 mL of 0.5 N aq.HCl.After the mixture was stirred at 0 °C for 1 h, the organic phase was separated.The aqueous phase was extracted with three 25 mL portions of CH 2 Cl 2 .The combined organic extracts were dried over anhydrous MgSO 4 , filtered, concentrated and purified by silica gel flash column chromatography (20% EtOAc/hexanes) to give the desired Weinreb amide (0.254 g, 91%) as a colorless oil: R f 0.23 (50% EtOAc/hexanes); [α]

(3R,4S,5R,6R)-6-(2-(4-Methoxybenzyloxy)ethyl)-4-hydroxy-3,5-dimethyl-tetrahydropyran-2-one (12).
To a solution of 235 mg (0.393 mmol) of aldol adduct 17 in 4 mL of acetonitrile at 0 o C was added 4.2 mL of freshly prepared HF solution (stock solution prepared from 0.50 mL of 48% aqueous HF, 8.6 mL of CH 3 CN, and 0.90 mL of H 2 0).After a total reaction time of 6 h, the solution was poured into 10 mL each of CH 2 C1 2 and saturated aqueous NaHCO 3 .The aqueous layer was extracted with CH 2 C1 2 (3 x 10 mL).The combined organic layers were dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo to afford a yellow oil (0.18 g, 0372 mmol).This product was used immediately without further purification.To a solution of the yellow oil in 6 mL of THF at 0 o C were added 5.3 mL of 30% aqueous hydrogen peroxide and 0.043 g of LiOH (0.744 mmol, 0.2 M in H 2 O).The mixture was stirred for 15 min at 0 o C and was quenched with 5 mL of aqueous 1.5 M Na 2 SO 3 .After 5 min, the reaction mixture was poured into 30 mL each of CH 2 C1 2 and H 2 O.The aqueous layer was acidified to pH 3.0 with aqueous 0.1 M HCl and was extracted with CH 2 C1 2 (3 x 10 mL).The CH 2 C1 2 layers were dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo to give carboxylic acid 18 (0.085 g, 70% over two steps).A solution of carboxylic acid 18 (85 mg) in 5 mL of benzene was maintained under reflux for 6 h.The solvent was removed under reduced pressure and the resulting yellow oil was purified by flash column chromatography (45% EtOAc/hexanes) to give 63 mg of lactone 12 as a colorless oil (56% yield over 3 steps).R f 0.21 (50% EtOAc/hexanes); [α]

(2S,3R,4R,5R)-7-(4-Methoxybenzyloxy)-5-(tert-Butyldimethylsilyloxy)-3-methoxy-2,4dimethylheptyl 4-methylbenzenesulfonate (5a).
To a solution of tosylate 21 (0.176 g, 0.30 mmol) in CH 2 Cl 2 (3 mL) at ambient temperature under argon were added proton sponge (0.382 g, 1.78 mmol) and Me 3 OBF 4 (0.219 g, 1.48 mmol), and the heterogeneous reaction mixture was stirred with protection from light for 12 h.The light brown reaction mixture was poured into CH 2 Cl 2 (10 mL) and was washed with cold aqueous 1 M HCl (2 x 10 mL).The organic layer was dried over anhydrous MgSO 4 , filtered, and concentrated in vacuo.Purification by flash chromatography (15% EtOAc/hexanes) afforded 0.160 g (89%) of 5a as a clear oil: R f 0.56 (30% EtOAc/hexanes); 1 24).Procedure 1.To a solution of 73 mg (0.104 mmol) of the imide 19 and 11 µL (0.26 mmol) of MeOH in 1 ml of THF at 0 °C was slowly added 0.13 mL (0.26 mmol) of a 1.0 M solution of LiBH 4 in THF (gas evolution).After stirring for 1 h at 0 °C the reaction was quenched by the addition of 1.5 mL of 1.0 M aqueous sodium potassium tartrate solution and stirred for an additional 10 min.The mixture was then diluted with 5 mL of CH 2 Cl 2 and 1.5 mL of 1.0 M aqueous sodium potassium tartrate solution.The layers were separated and the aqueous layer was extracted with two 5 mL portions of CH 2 Cl 2 .The combined organic extracts were washed with 5 mL of brine, dried over anhydrous MgSO 4 , and concentrated in vacuo.The crude product was purified by flash chromatography on silica gel (25% EtOAc/hexanes) to give the desired product 24 (39 mg, 84% over two steps) as a viscous oil.R f 0.49 (50% EtOAc/hexanes).Procedure 2. To a solution of 27 (0.457 g, 0.824 mmol) in freshly distilled THF (9 mL) in a plastic vial was added pyridine (2 mL).The reaction mixture was cooled to 0 o C and HF•Pyridine (70:30, 7.1 mL) was added dropwise.After the addition was complete the reaction was let to warm to ambient temperature and stirred for 24 h, and then transferred directly to a pipette column loaded with silica gel.Elution (20% EtOAc/hexanes) gave 24 (0.356 mg, 98% yield) as a colorless oil: IR ν max (film, cm

Scheme 14 .
Scheme 14. Completion of the synthesis of pironetin.
Treatment of alkyl iodide 28 with t BuLi in Et 2 O at -78 o C, followed by addition of 9-MeO-9-BBN, gave a boronate intermediate.Addition of vinyl bromide 4a in the presence of Pd(dppf)Cl 2 , AsPh 3 , K 2 CO 3 and water in DMF gave coupled product 23 in 74% yield.BuLi in Et 2 O at -78 o C followed by addition of 9-MeO-9-BBN, and vinyl bromide 4a in the presence of Pd(dppf)Cl 2 , AsPh 3 , K 2 CO 3 and water in DMF provided only product 29, formed by I/H exchange.We tried the coupling with vinyl iodides 30a and 30b as well, but always isolated only 29, with no signals for the desired product.