A formal synthesis of Balgacyclamide A using solution phase fragments condensation

A formal total synthesis of Balgacyclamide A as an antimalarial cynobactin of Microcystis aeruginosa (EAWAG 251) has been described. The synthesis of titled cyclamide was accomplished by the solution phase fragment synthesis using protection, deprotection and macrocylization process. Four common amino acids such as d-alanine, l-threonine, lvaline and d-allo-isoleucine, has been used for the construction of Balgacyclamide A. Including, the oxazoline and thiazole are the core structures was successfully achieved by using Burgess reagent and Hantzsch methods. The overall yield of the synthesized balgacyclamide A was found to be 2.03%, also structure was confirmed byH-NMR, CNMR and HRMS spectral data.


Introduction
Cyclamides are the cyclic structure of well-defined macrocyclic hexapeptides in which hydrophobic residues flanked by heterocyclic rings of oxazol(ine) or thiazol(ine) residues. In recent years, cyclamide and related macrocycles have gain substantial attention in the field of biosynthesis as well as drug discovery. 1 These cyclamides are preferably suitable candidates to interact with many receptors or interferes with protein-protein interactions. 2 Due to the presence of well-defined conformational space outlined by systematically linked amino acids through peptide bond, cyclamides are emerging as future drug candidates. Moreover, these cyclamides are found in organisms as well as in animals and suggested to be symbiotic or dietary origin. 3 In particular, macrocyclic hexapeptides like balgacyclamides are having well defined structure in which hydrophobic residues are flanked by heterocyclic cores such as oxazolineor thiazoline. [3][4][5][6] Balgacyclamide A is the class of cyclamides, was isolated from aqueous methanolic extracts of Microcystis aeruginosa (EAWAG 251) by Karl Gademann et al 7 in 2013 composed with two oxazoline and one thiazole cores exhibited antiparasite activity against Plasmodium falciparum K1 strain (IC50= 9.0 μM). Efforts undertaken towards the synthesis of balgacyclamide A including the individual synthesis of oxazoline cores using methoxycarbonylsulfamoyl)-triethylammonium hydroxide (Burgess reagent) or diethylamino sulphur trifluoride (DAST) followed by coupling reaction but it has been arrested under the selected reaction pathway. To overcome the difficulties raised for achieving the goal of total synthesis, the key fragment building blocks were assembled by convergent reaction pathway.

Result and discussion
The retrosynthetic approach of Balgacyclamide A is depicted in scheme 1. The late-stage cyclization formation of oxazoline ring could achieved followed by coupling reaction. The oxazoline moiety is acid and base sensitive, which can be easily opened to corresponding amino alcohol derivative. 8a-d Moreover, the construction of heterocyclic cores after macrocyclization leads to stained conformational transition state. Furthermore, angular strain could arise from the isopropyl and methyl substituents makes difficulties in cyclisation of final core with heterocyclic rings. 9 Therefore, in the total synthesis, the construction of oxazoline core was conducted in the final step by convergent method. The macromolecule 3 could be formed from condensation of peptide fragments 4 and thiazole 8. However, compound 4 and 8 could be prepared from commercially available starting substrates (scheme 1).

Scheme 1. Retrosynthetic planning for the synthesis of Balgacyclamide A.
In continuation to our ongoing research on development of new methodologies for the synthesis of biologically active compounds 10 , the total synthesis of balgacyclamide A was conducted by coupling of the different fragments. Our synthetic strategy was divided into three parts, (a) synthesis of fragment 4, (b) synthesis of fragment 8, and (c) convergent coupling of fragments. In the first part, L-valine 5 was protected by Boc-anhydride. 11a The boc-L-valine (11) was coupled with L-threonine methyl ester using HATU/DIPEA in N-Ndimethylformamide afforded compound 12 in 88% yield. 11a

Scheme 4. Synthesis of peptide 4
In the second part, synthesis of thiazole fragment 8 was conducted by a series of reactions as mentioned in Scheme 5. The d-allo-isoleucine (9) was protected using di-tert-butyl carbonate in THF to furnish 18, followed by the treatment with HATU/NH4OH afforded compound 19 in 94% yields. 12 The compound 19 on treatment with Lawesson's reagent afforded thiomide 20 in 80% yield. In order to transfer compound 20 to 8, different reaction conditions were employed. However, the best condition was obtained by Ethyl bromopyruvate in DMF at RT for 4 h (condition e), afforded the desired product 8 in 84% yield with retention in stereochemistry. 12b-c,13 Furthermore, the deprotection of compound 8 with trifluoroacetic acid (TFA) in CH2Cl2 afforded compound 21 as salt in 96% yield (Scheme 5). Scheme 5. Synthesis of thiazole core 21 In the final part of the synthesis, coupling of peptide 4 and 20 was carried in presence of HATU/DIPEA to afforded hexapeptide 3. The hydrolysis of 3 with lithium hydroxide followed by deprotection with trifluoroacetic acid gave 21. The requisite 22 was used as a crude product undergo internal coupling by using HBTU, N,N-diisopropylethylamine (DIPEA) in DMF furnished peptide macrocylisation 2. 12b The final step for formation of oxazoline ring formation was optimized by different conditions. 14 The best condition was obtained by Burgess reagemt in THF at 80 °C in 66% yield (Scheme 6). 11b,14a-b The spectroscopic data of the synthesized product was in agreed with the reported data of balgacyclamide A (Table 3).

Conclusion
In conclusion, we have developed a total synthesis of balgacyclamide A as a natural animalarial cyclamide. The synthesis has been accomplished by coupling of peptides and thiazole heterocyclic as building blocks. The optimized the reaction condition for the synthesis of thiazole core unit was found to be proficient over reported methods. This is first total synthesis which was conducted by solution phase fragment synthesis using readily 8 available amino acids. The developed total synthesis was found to be adventurous for researchers to develop new analogues of this class of compounds as drug candidates. 4.2 Experimental procedure and data for synthetic compounds.

Synthesis of the compound 11.
To a stirred solution of l-valine (10g, 85mmol, 1eq) in 1,4-dioxane (100 mL), NaOH(1M, 150mL) was added at 0 o C. The reaction mixture was stirred at room temperature for 10 min, followed by addition of di-tert-butyl carbonate (23.53mL, 102 mmol) drop wise at 0 o C.The reaction mixture was allowed to stir at room temperature for 12h. After complete consumption of staring material (as indicated by TLC), reaction mixture was quenched by water (250mL)

Synthesis of the Compound 16.
To a solution of 15 (4 g, 13 mmol, 1 eq) in THF: water: MeOH (3:2:1, 50mL) was added lithium hydroxide (0.378 g, 15.6 mmol, 1.2 equiv) at 0 o C and reaction was then stirred at room temperature for 4h. The resulting reaction mixture was concentrated and acidified with HCl(1N) to pH= 2. The reaction mixture was extracted with ethyl acetate (3x100mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated to afford 16 as a yellowish semisolid (3.6g, 94%) which was forwarded for next step without purification.

Synthesis of Compound 2.
To a stirred solution of compound 21 (0.380 g, 0.648 mmol, 1 equiv) in DMF(10 mL), DIPEA(0.345 mL, 1.94 mmol, 3 equiv) was added at 0 o C and reaction mixture was stirred for 10 min. To this mixture, HBTU (0.369 g, 0.972 mmol, 1.5 equiv) was added and reaction mixture was stirred at room temperature for 12h. The reaction mixture was quenched by water (50 mL) and extracted with ethyl acetate(3x50mL).