Development of new synthetic methodology and studies directed towards the synthesis of cyclothialidine : A new DNA gyrase inhibitor

Abstract

The thesis entitled "Development of New Synthetic Methodology and Studies Directed towards the Synthesis of Cyclothialidine: A New DNA Gyrase Inhibitor" is divided into two parts, PART A and PART B. Each PART has been subdivided into three chapters.

PART A:

Chapter 1 deals with the development of a new protecting group for amines, i.e., the propargyloxy carbonyl (POC) group, and its facile deprotection with tetrathiomolybdate 1 (1 eq.) under sonochemical conditions. The efficacy of this methodology has been demonstrated in the synthesis of a number of peptides, especially in sulfur-containing peptides (Scheme-1), where the removal of the Cbz-group is difficult under hydrogenolytic conditions. It has also been shown that the POC group can be selectively cleaved with tetrathiomolybdate 1 in the presence of t-butyl ester, while selective removal of /e/t-BOC with TFA or HCO2H has been a long-standing problem. The stability of the POC group has also been evaluated in the presence of base as well as acid.

PART A: Chapter 2 deals with the use of the propargyloxy carbonyl (POC) group for the protection of alcohols and phenols and its deprotection upon treatment with tetrathiomolybdate 1 (Scheme-2) under mild conditions. A number of POC-protected alcohols/phenols get cleaved selectively in the presence of other functionalities such as: OMe, OBn, Oallyl, CHO, and OAc, etc., by tetrathiomolybdate 1. This method has also been applied for the protection of the anomeric hydroxyl group of glucose derivatives and has been used mainly on unactivated alcohols where normal protection and deprotection do not give good yields.

PART A: Chapter 3 contains a mechanistic study on the deprotection of the POC group upon treatment with tetrathiomolybdate 1. A plausible mechanism for the deprotection of the POC group, resulting in the formation of an allenyl disulfide as the organic by-product and CO2 and Mo3S92 as the inorganic by-products, has been proposed. Some chemical and spectroscopic data are presented, which seem to support the mechanism proposed (Scheme-3).

PART B:

Chapter 1 deals with studies directed towards the synthesis of “Cyclothialidine” 2: A New class of DNA Gyrase inhibitor. Cyclothialidine 2 is a new DNA gyrase inhibitor isolated from Streptomyces filipinensis NR0484. Its mode of action was shown to be the inhibition of the ATPase activity conferred by the B subunit of DNA gyrase, the target of the coumarin antibiotics novobiocin and coumermycin. Although hardly exhibiting any antibacterial activity against intact bacterial cells, probably due to insufficient penetration of the cytoplasmic membrane, 2 was considered to be a promising lead compound, which, by chemical modification of its structure, might open a route to a new class of antibacterials. To explore and exploit the potential of 2 in this respect, an efficient and flexible synthetic route was developed, allowing the preparation of a great variety of analogues.

As can be seen from the retrosynthetic analysis of cyclothialidine, if the two side chain peptide bonds are disconnected, the new target molecule would be the 12-membered lactone 3. The amino acids that form part of the peptide side chain are easily recognized as alanine, serine, and 3-hydroxy-L-proline, and the synthesis of this unusual amino acid has been discussed in Chapter 3. Further strategic disconnection of the macrolide 3 reveals the presence of two amino acids, cysteine and serine, and a highly functionalized aromatic skeleton, 4.

In principle, starting with commercially available 3,5-dihydroxybenzoic acid, it would be possible to functionalize the aromatic ring by standard procedures to obtain the key intermediate 4.

Synthesis of the Model System: To examine the feasibility of our strategy, the synthesis of the lactone 5 lacking the characteristic phenyl substitution pattern of 2 was first investigated. Phthalide 6 was treated with BBr3 to give ortho-(bromomethyl)benzoic acid, which, on treatment with oxalyl chloride followed by treatment with 4-nitro-benzyl alcohol in the presence of Hunig's base, yielded ortho-(bromomethyl)benzoate 7. This bromide was treated with the dipeptide Boc-Ser-Cys(SH)-OMe (which was obtained from EDC coupling of L-Cys-OMe with Boc-Ser-OH in MeCN) to give methyl nitrobenzyloxycarbonyl]benzyl}-L-cysteinate 8. N-[N-(tert-butoxycarbonyl)-L-seryl]-S-{2-[(4-nitrobenzyl ester was cleaved using 10% Pd/C/H2 in EtOAc, and then lactonization was done under Mitsunobu conditions to give the model lactone 5 in 28% overall yield from phthalide 6 (Scheme-4).

For the synthesis of 2 according to the proposed scheme, we needed the highly functionalized aromatic skeleton 4. First, we introduced a methyl group at the ortho position, starting with N,N-diethyl-3,5-dimethoxybenzamide using H-BuLi/Mel. Then, the second methyl group was introduced in a similar fashion, but finally, the hydrolysis of the amide was difficult and yielded the carboxylic acid in poor yield. Therefore, we tried to incorporate the methyl group directly into the 3,5-dimethoxybenzoic acid (unprotected carboxylic acid) using LIC-KOR, Schlosser's base (n-BuLi/KOBu-/), and Mel. Interestingly, the methylation went to the para position. Within our knowledge, there is no report of para-lithiation of 3-methoxybenzoic acid with organolithium reagents. Therefore, we have explored this methodology further with other electrophiles, and the results are discussed in Chapter 2.

Chapter 2 deals with the "Selective para-metalation of meta-anisic acid using LIC-KOR: Synthesis of 3,5-dimethoxy-4-methyl benzoic acid" 18.

So far, only the ortho-lithiation of benzoic acid derivatives with organolithium reagents has been reported, but direct metalation at the para position of the K-salt of meta-methoxybenzoic acid is possible with n-BuLi/KOBu-/ (where there is no need for protection and deprotection of carboxylic acid), and this methodology has been extended to the synthesis of 3,5-dimethoxy-4-methyl benzoic acid 18 (Scheme-7), which has been used in the synthesis of certain fungal metabolites, e.g., sclerotiorin and mitorubrin.

Chapter 3 deals with the development of a new synthetic route to (2S, 3R)-3-hydroxyproline, known as cis-3-hydroxy-L-proline 19. In this chapter, an asymmetric synthesis of 19 starting with the readily available ?-alanine is described, and the requisite allylic alcohol 20 was prepared from ?-alanine. Sharpless epoxidation of compound 20 with L-(+)-diethyl tartarate/titanium tetraisopropoxide/ t-butyl hydroperoxide yielded the epoxy alcohol 21 in 98% ee. The N-terminal was suitably protected with benzyl and tosyl groups, which are compatible with the asymmetric epoxidation conditions. Next, the tosyl group was cleaved with Mg/MeOH, and under this condition, the epoxide ring was opened intramolecularly to give the corresponding (2S, 3S)-N-(benzyl)-3-hydroxyproline methyl ester 23 (overall yield was 33%, starting from ?-alanine) as a single diastereomer in >97% ee. Finally, the deprotected cis-3-hydroxy-L-proline 24 was obtained after purification through an ion-exchange resin column in >97% ee. Compound 23 could be converted to (2S, 3R)-3-hydroxyproline 19 under Mitsunobu conditions according to the literature methods (Scheme-8).