The synthesis of o-NosylOXY (I) was carried out in a similar manner as described in chapter 2, section 2.1. For a model reaction, we took, equivalent mole of phenylacetic acid, o-NosylOXY, and DIPEA were dissolved in ethyl acetate and kept the reaction mixture for pre-activation for 5 min at room temperature, followed by the portion wise addition of o-aminophenol. The reaction mixture was then heated under microwave at 50
°C and 80 Watt in a closed vessel. After completion of first step of the reaction (TLC,
~30 min), the reaction mixture was heated again in presence of catalytic amount of p- TsOH (10 mol%) for another 10 min at 60 °C and 80 Watt, under microwave in a closed vessel. Product was purified by column chromatography with 92% yield.
For optimization, we took benzoic acid (entry 1, Table 4.1.2) as substrate. For solvent optimization of the dehydration step, we screened various solvents, such as toluene, DCM, EtOAc, DMSO, dioxane, CH3CN, CHCl3, and DMF. Out of these solvents, EtOAc was found to be the best (Table 4.1.1). Next, we screened various catalysts and p-TsOH was found to be the best at the optimized condition (entries 8-15, Table 4.1.1).
Table 4.1.1. Optimization of the reaction conditionsa
aPerformed with benzoic acid (1 equiv), o-NosylOXY (1 equiv), DIPEA (1 equiv), and o-aminophenol (1 equiv) under microwave irradiation (upper limit was fixed at 80 Watt). bYields were referred to the isolated yields after column chromatography.
Further, to explore the scope of this methodology, we synthesized various derivatives of benzoxazole (Table 4.1.2) and benzothiazole (Table 4.1.3) using the optimized condition.
The current methodology tolerates variety of functional group, for example methoxy, methyl, nitro, chloro, and bromo. All the substrates underwent the reaction to furnish the desired products with 81-92% yields.
Chapter 4 Benzoxazole and Benzothiazole Synthesis from Carboxylic Acid in Solution and on Resin by Ethyl2- cyano-2-(2-nitro-benzenesulfonyloxyimino)acetate and para-Toluenesulfonic Acid
Table 4.1.2. Synthesis of various benzoxazolesa
aPerformed with carboxylic acid (1 equiv), o-NosylOXY (1 equiv), DIPEA (1 equiv), o-aminophenol (1 equiv), and catalyst II (10 mol%) under microwave for 10 min. bYields were referred to the isolated yields after column chromatography.
Table 4.1.3. Synthesis of benzothiazolesa
aPerformed with carboxylic acid (1 equiv), o-NosylOXY (1 equiv), DIPEA (1 equiv), o-aminothiophenol (1 equiv), and reagent II (10 mol %) under microwave irradiation for 10 min. bYields were referred to the isolated yields after column chromatography
Chapter 4 Benzoxazole and Benzothiazole Synthesis from Carboxylic Acid in Solution and on Resin by Ethyl2- cyano-2-(2-nitro-benzenesulfonyloxyimino)acetate and para-Toluenesulfonic Acid
Figure 4.1.1. X-ray crystallographic structure of the product (entry 4, Table 4.1.2) (ORTEP diagram with ellipsoid of 30% probability, CCDC No. 1422635).
After obtaining the excellent yields with simple carboxylic acids, we tried to explore the application of methodology for the synthesis of benzoxazole and benzothiazole of N- protected amino acids. We took equivalent amount of Cbz-Alanine and o-NosylOXY with DIPEA in ethyl acetate, and treated under previously optimized condition, but the reaction was unsuccessful. Then we went to further optimize the reaction condition and tried various catalysts, solvents and temperatures. Finally, we found that with increasing the reaction temperature to 110 °C (other parameters were same as before), the reaction worked well with reasonably good yields. Next, we synthesized various derivatives of benzoxazoles and benzothiazoles of Cbz-protected (entries 1-3 and 9-12, Table 4.1.4) and Fmoc-protected amino acids (entries 4-8 and 13-14, Table 4.1.4) with 61-72% yields under optimized condition. The yields of table 4.1.2 and 4.1.3 are better than table 4.1.4 due to the aromatic carboxylic acids which form resonance stabilize product.
Table 4.1.4. Synthesis of benzoxazole and benzothiazole of N-protected amino acida
Chapter 4 Benzoxazole and Benzothiazole Synthesis from Carboxylic Acid in Solution and on Resin by Ethyl2- cyano-2-(2-nitro-benzenesulfonyloxyimino)acetate and para-Toluenesulfonic Acid
aPerformed with N-protected amino acid (1 equiv), reagent I (1 equiv), DIPEA (1 equiv), o-aminophenol or o-aminothiophenol (1 equiv) for 30 min and catalyst II (10 mol%) under microwave irradiation for 30-40 min. bYields were referred to the isolated yields after column chromatography.
After successful synthesis of the benzoxazoles and benzothiazoles of N-protected amino acids in solution phase, we extended the methodology further for the synthesis of benzoxazoles on solid support. We have synthesized benzoxazole derivatives of two biologically active molecules, hIAPP22-27 (human islet amyloid polypeptide, Scheme 4.1.1), which is known to be the core sequence responsible for the initiation of aggregation of the amylin peptide that leads to type 2 diabetes and Aβ18-21 (amyloid β, Figure 4.1.2) that is known as key sequence of Aβ aggregation responsible for Alzheimer’s disease. Both are known as difficult sequence for solid phase peptide synthesis (SPPS).
Figure 4.1.2. Benzoxazole derivative of side chain of aspartic acid of Fmoc-DVFFAG-NH2
Scheme 4.1.1. Synthesis of benzoxazole of adipoyl hIAPP22-27 (NFGAILG) using Fmoc/tBu protection strategy
For the synthesis of benzoxazole of hIAPP22-27, we first synthesized hIAPP22-27 on Rink Amide MBHA resin using Fmoc/tBu based SPPS chemistry. Then, we attached an adipic acid at the N-terminal of hIAPP22-27 and the attachment was confirmed by HPLC and ESI- MS (Figure S9 and S10). Further, the free carboxylic acid of adipic acid was coupled with o-aminophenol (3 equiv) in presence of reagent I (3.5 equiv) and DIPEA (4 equiv) at
Chapter 4 Benzoxazole and Benzothiazole Synthesis from Carboxylic Acid in Solution and on Resin by Ethyl2- cyano-2-(2-nitro-benzenesulfonyloxyimino)acetate and para-Toluenesulfonic Acid
50 °C in microwave on solid support. Special feature of this synthesis was, all the coupling reactions including the coupling of o-aminophenol could be performed by reagent o-NosylOXY. Finally, cyclization was performed in presence of p-TsOH (10 mol%) at 110 °C under microwave on resin. Final product was cleaved from the resin using the cocktail solution TFA/DCM/H2O (9/0.5/0.5) for nearly 3 h. The cleaved product was precipitated by cold diethyl ether and centrifuged to get crude solid product. The crude product was purified by semi-preparative HPLC and characterized by ESI-MS (Figure S11 and S12). Another interesting feature was that the yield of the peptide was good (59% crude and 21% after purification by HPLC w.r.t. resin loading) indicating practically no significant loss despite of using p-TsOH and temperature as high as 110 °C on acid labile Rink Amide MBHA resin.
Similarly, for the synthesis of benzoxazole derivative of Aβ18-21, first we attached a Fmoc-(OtBu)Asp-OH at the N-terminal of VFFAG by above described method on solid support. Then the side chain protecting group, tBu of Fmoc-Asp(OtBu)-OH was de- protected by ZnCl2/DCM in 48 h on solid support (Figure S13 and S14). Then the generated side chain carboxylic acid of aspartic acid residue was coupled with o- aminophenol (3 equiv) using reagent o-NosylOXY (3.5 equiv) and DIPEA (4 equiv) at 50
°C in microwave, after that cyclization was performed with p-TsOH (10 mol%) at 110 °C in microwave. Final product was cleaved from the resin using the cocktail of TFA/DCM/H2O (9/0.5/0.5) for nearly 3 h. The final product was purified by semi- preparative HPLC and characterized by ESI-MS (Figure S15 and S16). Yield of the crude peptide was 54% (16% w.r.t. the resin loading after purification by semi-preparative HPLC). The Gly residue was attached at the C-terminal of both the peptides to serve as
spacer. Thus, the syntheses of benzoxazole derivatives of biologically important peptides were achieved on solid support.