Stereoselective Synthesis of Southern Fragment of Hantupeptin A

The stereoselective synthesis of the southern fragment (C21–C41) of Hantupeptin A is described. The required stereochemistry of β-hydroxy-α-methyl acid unit was accomplished through the Aldol reaction using Evan’s chiral auxiliary followed by the installation of the terminal alkyne with Ohira–Bestmann reagent.


Introduction
2][3][4][5] Naturally occurring cyclic peptides 6 have come within this class possessing diverse biological activities like immunosuppressant, antibiotic, antifungal, anti-inflammatory and anticancer effects. 7Indeed, marine organisms such as algae, sponges, and coelenterates became an exceptional source of these natural products.Since the discovery of the didemnins, this class of natural products continues to stimulate active research in synthetic and medicinal chemistry, as well as in clinical oncology and cell biology. 8In 2009, Tan and co-workers have isolated a new cyclodepsipeptide, hantupeptin A (1) from the marine cyanobacterium Lyngbya majuscula.The hantupeptin A (1) has exhibited cytotoxicity against MOLT-4 leukemia cells and MCF-7 breast cancer cells with IC 50 values of 32 and 4.0 μM, respectively. 9The extensive spectral studies and advanced chiral techniques have revealed the planar structure as well as the absolute configuration of 1. Structurally, compound 1 is a 19-membered cyclic tetrapeptide, which consists of ?ve α-amino/hydroxy acid residues, including phenyl lactic acid, proline, N-methylvaline, valine, N-methylisoleucine and a α-methyl-β-hydroxy acid unit with an alkyne at the terminal end of the molecule.The stereochemistry at the hydroxyl group attached carbon (C-35) of an unusual hydroxy acid, 3-hydroxy-2methyloctynoic acid (Hmoya) unit 4 was determined as S using the Mosher's analysis.However, the stereochemi-stry at the methyl group attached carbon (C-34) was not reported at that point of time.
Later, in 2010, the same group has isolated hantupeptins B (2) and C (3) along with 1 from the organic extracts of the same marine cyanobacterium. 10The only structural difference among these molecules is the degree of unsaturation in the unusual amino acid part, where 1 is having a terminal alkyne functionality, 2 has an alkene and 3 is without any unsaturation in its structure (Figure 1).Compounds 2 and 3 also showed moderate in vitro cytotoxicity against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.In the study of the re-isolation of compound 1, the relative stereo chemistry at C-34 carbon of Hmoya unit was determined as R by the rigorous NMR experiments.Very recently, the absolute configuration of Hmoya unit of hantupeptin C (3) was assigned as (2R,3S) based on the retention times of the Mosher ester derivative standards by RPLC-MS. 11Till date, no synthetic efforts have been reported in the literature for these molecules.In continuation of our interest on the synthesis of biologically active molecules, 12 we have reported the synthesis of hantupeptin A C21-C41 fragment with the unusual component, Hmoya residue as a part of it.The Hmoya unit 4 is also present in a number of marine-derived compounds, such as onchidin B, kulomo'opunalide-1, kulomo'opunalide-2, and trungapeptin A.

Results and Discussion
From the retrosynthetic outlook (Scheme 1), the desired molecule was envisioned to be obtained from the key intermediate 5, which, in turn may be built from 6 through oxidation followed by Ohira-Bestmann reaction.Compound 6 could be obtained by coupling compound 7 with an amine, derived from compound 8.The stereocentres in compound 7 could be achieved through Evan's syn aldol protocol followed by a reductive etherification.The stereochemistry in compound 8 was achieved from the natural amino acids L-isoleucine and L-valine.
Preparation of the acid 7.The synthesis began with Evan's aldol reaction between the aldehyde 10 and oxazolidinone 9. Aldehyde 10 was prepared in two steps from the inexpensive 1,5-pentanediol following the reported procedure. 13The other, desired (R)-4-benzyl-3-propionyloxazolidin-2-one 9 was also smoothly obtained using well documented literature protocol. 14The di-n-butylboron triflate mediated Aldol reaction between compounds 9 and 10 furnished the syn-product 11 in 83% yield (Scheme 2).Protection of the secondary hydroxy group of the compound 11 as TBS-ether 12 was achieved in 87% yield by exposing to TBSCl/Imidazole in CH 2 Cl 2 at room temperature for 18 h.Compound 12 was then treated with sodium borohydride in THF/pH 7 buffer at room temperature for the reductive removal of the auxiliary to provide alcohol 13.The primary hydroxyl group of compound 13 was oxidized to a carboxylic acid using (bisacetoxyiodo)benzene (BAIB) / 2,2,6,6-tetramethyl-1piperidinyloxy free radical (TEMPO) in CH 2 Cl 2 /pH 7 buffer to obtain the acid fragment 7 in 89% yield.
Preparation of compound 8.The synthesis of compound 8 was commenced by carrying out the preparation of the known N-(tert-butoxycarbonyl)-N-methyl-L-isoleucine 14 from L-isoleucine using the literature procedure. 15sterification of the acid 14 with allyl bromide was clean-

Construction of C21-C41 segment from 7 and 8.
With the successful completion of the desired fragments 7 and 8, the attention was turned to couple them to give the di-amide 16.For that, intially the boc protection of compound 8 was removed by using TFA in CH 2 Cl 2 at 0 °C and then coupled with the compound 7 under HATU/HOAt conditions at room temperature, to obtain the required product 16 in 85% yield.The resulting compound 16 was hydrolyzed with potassium carbonate in methanol to give the primary alcohol 6 in 75% yield which upon the oxidation with TPAP/NMO to the aldehyde followed by the treatment with Ohira-Bestmann reagent gave the targeted terminal alkyne product 5 in 92% yield (Scheme 4).

Experimental
NMR spectra were recorded in CDCl 3 on Bruker AM-300 (300 MHz) spectrometer at ambient temperature.Chemical shifts are reported in ppm relative to TMS as internal standard and coupling constants are reported in Hz.FTIR spectra were recorded on a Nicolet FT-IR 400 spectrometer in KBr or as neat.Optical rotations were measured on an Perkin-Elmer 141 polarimeter by using a 2 mL cell with a path length of 1 dm with CHCl 3 or CDCl 3 as solvent.Low resolution mass spectra were obtained on VG 70-70H or LC/MSD trap SL spectrometer operating at 70 eV using direct inlet system.High resolution mass spectra (HRMS) were recorded on an Agilent Technologies 6510 Q-TOF spectrometer.Technical-grade EtOAc and hexanes used for column chromatography were distilled before use.All the reagents and solvents were of reagent grade and used without further purification unless otherwise stated.

(5S,6R)-7-((R)-4-Benzyl-2-oxooxazolidin-3-yl)-5-hydro xy-6-methyl-7-oxoheptyl benzoate (11):
To a stirred solution of acyl oxazolidinone 9 14 (1.04 g, 4.45 mmol) in CH 2 Cl 2 (10 mL) was added dropwise n-Bu 2 BOTf (1.0 M in CH 2 Cl 2 , 4.67 mL, 4.67 mmol) and stirred for 10 min.i-Pr 2 NEt (0.93 mL, 5.34 mmol) was then added dropwise and the reaction was stirred at the same temperature for 1 h.The mixture was cooled to -78 °C before a solution of aldehyde 10 12 (1.02g, 4.95 mmol) in CH 2 Cl 2 (15 mL) was added dropwise via cannula.Stirring was continued at -78 °C for 3 h before gradually warming to 0 °C.The reaction mixture was stirred for additional 3 h at 0 °C and then quenched by the addition of 0.1 M pH 7 phosphate buffer (7.5 mL) followed by MeOH (10 mL) at 0 °C.After stirring for 5 min, a solution of 30% aqueous H 2 O 2 (7.5 mL) in Me-OH (15 mL) was added dropwise and stirred at the same temperature for 1 h before being concentrated under reduced pressure.The residue was diluted with Et 2 O, the phases were separated and the aqueous phase extracted with Et 2 O.The combined organic phase was washed with brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.Flash chromatography over silica gel (35% ethyl acetate in pet.ether) gave 11 (

Conclusions
In conclusion, a practical and stereoselective synthesis of C21-C41 fragment of hantupeptin A having five stereo centers, two amide linkages and one ester linkage was demonstrated with differential protective groups to allow further extensions.The key features of the strategy are the successful utilization of Evan's Aldol reaction, TEMPO mediated oxidation and Ohira-Bestmann homologation.Furthur investigation towards the total synthesis of hantupeptin A are in progress.