Progress toward the total synthesis of mirabalin isomers

Key fragments of the cytotoxic marine macrolide mirabalin have been synthesized, by using a flexible strategy based on asymmetric reductions to control the hydroxy-and carbamate-bearing stereocenters. In particular, ruthenium or rhodium-mediated asymmetric hydrogenation and transfer hydrogenation were used in combination with a dynamic kinetic resolution to control two contiguous stereocenters in a single step


Introduction
Lithistid demosponges are an assemblage of sponges predominantly found in deeper waters. 1,2They are regarded as an excellent source of structurally diverse and bioactive secondary metabolites. 3-5In particular, numerous novel macrolides have been isolated from the Theonellidae family exhibiting excellent biological activities. 6-7Among them, mirabalin, a macrocycle, has been extracted from Siliquariaspongia mirabilis, a lithistid demosponge collected from the archipelago of Chuuk, Micronesia (Figure 1). 8,9This macrocycle exhibits potent cytotoxicity toward human colon tumor cell line HCT-116 with an IC50 of 0.27 µM.Morever, it has been found that the macrocycle ring is crucial to mirabalin antitumor activity.Indeed, no inhibition of the tumor cells growth was observed when treated with the linear polyketide side-chain.From a structural point of view, mirabalin exhibits a 35-membered macrocyclic lactam-lactone possessing a fully conjugated pentaene system along with a tetrasubstituted tetrahydropyran ring.In addition, a linear polyketide side-chain is linked to the macrocyclic ring with an amide linkage.An investigation combining advanced NMR and MS techniques allowed the partial elucidation of the structure of mirabalin.Undeniably, because of the large number of stereocenters and the low amount of material available, the absolute configurations of 12 out of the 25 stereocenters could not be assigned.Nevertheless, the geometry for each of the double bonds was established unambiguously (Figure 1).As part of our ongoing studies on the total synthesis of natural products, 10-17 and intrigued by the structural intricacy of this macrolide, we embarked on a study aiming at developing a flexible, convergent and modular route to one isomer of mirabalin where the configurations of the twelve unknown stereocenters were arbitrarily fixed (Scheme 1).Recently, we reported a synthetic strategy toward the C44−C65 polyketide side chain 18 and toward the C14−C29 fragment of mirabalin, 19 as well as a straightforward method to construct the pentaene moiety C4-C14. 20Scheme 1 shows the mirabalin global retrosynthetic plan with late stage esterification, peptide coupling and C=C bond formation to access the macrocycle by macrolactamization, suggesting A and B as suitable key intermediates for the sake of convergence.Herein, we report a flexible synthesis of fragments A and B but also details about the synthesis of isomers of the C44−C65 side chain of mirabalin.

Results and Discussion
Synthesis of fragment A Our retrosynthetic analysis was based on known methods for the introduction of the phenyltetrazolyl sulfone moiety by a hydroboration/Mitsunobu/oxidation sequence applied to alkene 2. We therefore envisaged that the aldehyde 3 represented a useful intermediate in the synthesis of the unsaturated compound 2, and aldehyde 3 could be obtained from α-amino β-ketoester 4 after asymmetric hydrogenation 21-23 via a dynamic kinetic resolution, 24-26 Claisen condensation and asymmetric hydrogenation (Scheme 3). 27-29  Scheme 2. Retrosynthetic analysis of fragment A (compound A').Our synthesis began with the preparation of the hydrochloride ammonium salt 4 in two steps from methyl acetoacetate 5 after nitrosation followed by hydrogenolysis of the oxime 6 under acidic conditions.To control the anti configuration at C66 and C42, a ruthenium-catalyzed asymmetric hydrogenation through dynamic kinetic resolution of the racemic α-amino β-keto ester hydrochloride 4 was envisaged.Using this method, the corresponding anti-aminoalcohol was successfully prepared with 90% diastereomeric excess and 91% enantiomeric excess.The reaction was performed in CH2Cl2/MeOH (10:1) at 50 o C under 13 bar of H2 using 2 mol% of the in situ prepared [Ru((R)-Synphos)]Br2] catalyst 30 (Scheme 3).Protection of the amino group as a carbamate and conversion of the hydroxy function into a TBS ether delivered compound 8.The ensuing fully protected amino hydroxy ester 8 was then submitted to a Claisen condensation with tert-butyl acetate under basic conditions to deliver β-keto ester 9 in 87% yield (Scheme 3).

Scheme 3. Preparation of b-keto ester 9.
We then turned our attention to the asymmetric hydrogenation of ketone 9 (Scheme 4).Different conditions varying catalyst loading and hydrogen pressure were screened using the binuclear complex [{RuCl((S)-Synphos)}2(μ-Cl)3][Me2NH2] 30 in EtOH at 50 o C. No influence of the catalyst loading was observed on the stereochemical outcome of the reaction.However, changing the hydrogen pressure from 70 to 100 bar afforded a better result in terms of diastereoselectivity (dr switching from 74:26 to >99:1).With these optimized conditions in hand, the synthesis was pursued with the protection of aminoalcohol 10 to the oxazolidine 11.It was found that the starting material 10 was not fully converted during the reaction and the acidic conditions led to the cleavage of the TBS group on both compounds 10 and 11.The yield was modest and variable despite several experimentations.Nevertheless, compound 11 was converted to aldehyde 3 with Dibal-H in 77% yield.Addition of vinylmagnesium bromide to aldehyde 3 at low temperature provided the desired allylic alcohol 12 in 77% yield with a diastereoselectivity of 62:38.The two stereoisomers were separated by flash column chromatography and the synthesis of compound A' was carried out with the major diastereomer, the absolute configuration of which was determined by Mosher analysis (Scheme 4). 31    After protection of alcohol 12 (TBSCl, imid, DMF, rt), hydroboration/oxidation of the obtained compound 2 furnished the desired product 13 in 75% yield.Formation of thioether 14 was accomplished using a Mitsunobu reaction 32,33 applied to alcohol 13 (1-phenyl-1H-tetrazole-5-thiol, DIAD, PPh3, THF, rt) and the thioether 14 was converted to the corresponding sulfone in 68% yield by oxidation with m-CPBA (Scheme 5).Thus, compound A' was obtained in 14 steps in 1.1% overall yield from methyl acetoacetate 5 and three contiguous stereocenters were controlled using asymmetric hydrogenation.To appreciate the feasibility of our envisaged retrosynthetic plan, we performed a Julia-Kocienski olefination 34 between compound A' and the aldehyde generated from alcohol 15 35 (corresponding to the C25-C28 fragment of the macrocycle, Scheme 1).Pleasingly, the desired E-alkene was obtained in 76% yield with tight control of the double bond geometry (E/Z > 95:5) (Scheme 5).

Synthesis of fragment B
The retrosynthetic analysis for the construction of the second major building block B is summarized in Scheme 6 for compound B'.Our strategy focused on the flexibility in term of stereochemistry.For this purpose, asymmetric hydrogenation and transfer hydrogenation 36-40 were chosen as complementary methods to access all four stereoisomers of compound B'.Scheme 6. Retrosynthetic analysis of fragment B (compound B').The synthesis of the α-carbamate β-ketoester 17 proceeded as shown in Scheme 7.After PMBmonoprotection of ethane-1,2-diol 20 and Dess-Martin oxidation of the obtained 21, aldehyde 18 was isolated in 61% yield.The other partner 19 was prepared by simple N-Boc protection of glycine methyl ester hydrochloride 22 in 97% yield.Thereafter, reaction of the zinc derivative of 19 with aldehyde 18 furnished the corresponding α-carbamate β-hydroxy ester whose oxidation afforded the desired β-keto ester 17.It is noteworthy that, after thorough investigation, only the use of Dess-Martin periodinane in the absence of NaHCO3 allowed the oxidation, albeit in low yield; other attempts using IBX, Parikh-Doering, Ley-Griffith, Swern, Fétizon or pyridinium dichromate reagents led only to degradation products.With the desired keto ester 17 in hand, we focused on the asymmetric reduction of 17 via dynamic kinetic resolution.

OTBS
After careful optimization of the reaction parameters, compound syn-23 was obtained in 73% yield with good diastereomeric ratio (dr = 86:14) and enantioselectivity (er = 90:10) by using 0.5 mol% of the complex [RuBr2((S)-Synphos)] prepared in situ from [Ru(COD)(2-methylallyl)2], 30 under 50 bar of hydrogen pressure at 50 o C in dichloromethane.Subsequent TBS protection of the hydroxyl gave syn-24 and the desired compound syn-B' was finally obtained by hydrolysis of the methyl ester to the corresponding carboxylic acid (Scheme 7).

Scheme 7. Synthesis of compound syn-B' through asymmetric hydrogenation.
To obtain the anti-isomer of fragment B, we turned our attention to the use of asymmetric transfer hydrogenation of 17 in combination with dynamic kinetic resolution.Preliminary screening employing different catalysts Cat I, 41 Cat II, 42 and Cat III 43,44 with HCOOH/Et3N (5:2) as the hydrogen donor demonstrated that faster reaction times as well as better diastereoselectivities in favor of compound anti-23 (dr = 78:22) were obtained with catalyst Cat III.Moreover, the catalytic charge could be decreased from 2 mol% to 0.1 mol% without any loss of stereoselectivity and compound 23 was isolated with a comparable 70% yield.It is noteworthy that at this point of the synthesis inseparable mixtures of synand anti-isomers were obtained.To complete the synthesis of fragment B, the newly formed hydroxy-ester 23 was protected by using TBSCl and imidazole in 51% yield before hydrolysis of the ester with TMSOK yielded the fully protected compound anti-B'.Gratifyingly, flash chromatography allowed separation of the synand anti-isomers (Scheme 8).
Scheme 8. Synthesis of compound anti-B' through asymmetric transfer hydrogenation.
Thus, compound anti-B' was synthesized in 7 steps starting from ethylene glycol 20 in 2.2% overall yield.This strategy inherently makes the synthesis flexible because all the four stereoisomers of fragment B can be synthesized by simply changing the configuration of the chiral ligand and by moving from asymmetric hydrogenation to asymmetric transfer hydrogenation.

Synthesis of stereoisomers of the C44−C65 side chain
As outlined above, flexibility of our synthetic plan was essential as the configurations of several stereogenic centers remain unknown.We decided to prepare other isomers of the north fragment of the targeted molecule (C44-C65 fragment, the two relevant stereocenters are highlighted with a star on Scheme 9).As such, it was shown previously that the C44-C65 part of the molecule could easily be synthesized after assembly of fragments C1, C2 and C3 (Scheme 9). 18Herein, we focused our attention to the modifications of the two C46 and C49 stereocenters of fragment C3.The stereogenic center at C46 would be easily modulated by simply changing the configuration of the upstream Roche ester, while modification of the configuration at the C49 stereocenter would be achieved by switching the ligand configuration during the asymmetric hydrogenation, thus allowing access to new isomers of the side chain.Scheme 9. Retrosynthetic analysis of mirabalin side chain C44-C65.
The synthesis began with the benzyl protection of (R)-Roche ester 25 followed by reduction of the resulting ester 26 to 27 using LiAlH4.Garegg iodination 45 (I2, PPh3, imid, CH2Cl2, rt) of the latter afforded iodide 28, which was used for the alkylation of methyl acetoacetate 46 to provide the key β-keto ester 29 (Scheme 10).With convenient amounts of compound 29 in hand, ruthenium-catalyzed asymmetric hydrogenation of the ketone was tackled.This transformation was accomplished using the in situ generated chiral complexes either [RuBr2((S)-Synphos)] or [RuBr2((R)-Synphos)].The stereochemical outcome of the reaction was controlled by choosing the appropriate configuration of the Synphos ligand and the reaction afforded (S,S)-30 and (R,S)-30 in 87% and 93% yields, respectively, as single diastereomers.The protection of these alcohols (TBSOTf, 2,6lutidine, CH2Cl2, 0 o C) yielded (S,S)-31 and (R,S)-31 which were subjected to debenzylation conditions (H2, Pd/C) to give (S,S)-32 and (R,S)-32, and subsequent Dess-Martin oxidation afforded aldehydes (S,S)-33 and (R,S)-33 required for the Nozaki-Hiyama-Kishi 47,48 coupling with (E)-iodoalkene 34. 18This reaction was performed in DMSO using CrCl2 (14 equiv) in the presence of a catalytic amount of NiCl2(dppe) (6 mol%).Under these conditions, alcohols 35 and 36 were obtained in 25% and 34% yields, respectively (quantitative yields based on the recovered starting material) as a mixture of diastereomers (dr = 75:25 for 35, dr = 71:29 for 36) which were separated by flash chromatography on silica gel.In summary, the facile synthesis of two new isomers 35 and 36 of the C44-C65 part of mirabalin demonstrated the high flexibility of the synthetic plan offered by using asymmetric reduction and one can envisage to access other stereoisomers using this versatile reduction.

Conclusions
In conclusion, the synthesis of several key fragments of mirabalin 1 was achieved using flexible and robust methods based on asymmetric hydrogenation and transfer hydrogenation.The C30-C67 fragment (compound A') as well as two isomers of the C1-C36 subunits (compounds syn-B' and anti-B') have been prepared.Moreover the synthesis of two isomers of the C44-C65 part of mirabalin has been achieved.Most importantly, the flexibility of this approach provides a platform for modification of several unknown stereocenters.The assembly of the different fragments is currently being pursued in our laboratory.

Experimental Section
General.All air and/or water sensitive reactions were carried out under an argon atmosphere.Enantiomeric excesses were determined by HPLC using a chiral stationary phase column (Chiralpak IA) and eluting with a hexane/isopropanol mixture as indicated.(6).A solution of methyl acetoacetate 5 (15 g, 129 mmol, 1 equiv) in acetic acid (100 mL) was cooled to 0 o C and a suspension of NaNO2 (22.3 g, 323 mmol, 2.5 equiv) in water (75 mL) was added dropwise, the temperature of the reaction mixture being kept below 5 o C.After evolution of the brown fumes, the stirring was maintained 1.5 h at 0 o C and then at rt until completion of the reaction, monitored by TLC (1 h).The reaction mixture was then diluted and extracted with Et2O.The combined organic layers were washed with saturated aqueous NaHCO3, dried over MgSO4, concentrated under reduced pressure and the crude mixture was purified by silica gel column chromatography (cyclohexane/EtOAc: 6/4) to give the desired product 6 (9.54 g, 51%) as a white solid.