Tacrolimus EP Impurity A

The antascomicins A to E (7–11) have been isolated from a strain of Micromonospora found in a soil sample from China and have a similar structure to ascomycin 12. The former compounds bind strongly to FKBP12 and antagonise the immunosuppressant activity of both rapamycin and FK506 but do not themselves exhibit immunosuppressant activity. Ascomycin itself only differs in structure from FK506 at the C-21 position where an ethyl group replaces an allyl group. It has similar in vitro potency to FK506 and several analogues have been prepared. Of particular interest was the fact that 24-deoxyascomycin 13 had a similar potency to ascomycin both as an immunosuppressant and also in its interaction with FKBP-12, suggesting that H-bonding with the C24 hydroxy of the natural product is not important for binding to FKBP12.

This section covers the three potent immunosuppressants rapamycin 324, FK506 328 and FK520 (also called ascomycin and immunomycin) that prevent T-cell proliferation by binding to immunophilins, being particularly effective at preventing graft rejection after organ transplantation. These potent immunosuppressants act at very much lower concentrations than cyclosporin. They are all structurally and biosynthetically closely related, having a (1R,3R,4R)-3,4-dihydroxycyclohex-1-enyl (or -hexanyl-)carbonyl starter unit; followed by PKS extension (14 extender units (7 × C₂ and 7 × C₃) for Rap, or ten (4 × C₂, 5 × C₃, 1 × C₅) for FK 506), followed by a lysine derived pipecolate (piperidine-2-carboxylate) termination unit (as its adenosylmonophosphate), without glycosylation, but some O-methylation and P-450 oxidation occurring. Rapamycin is assembled in Streptomyces hygroscopicus from a (1R,3R,4R)-3,4-dihydroxycyclohexanecarbonyl CoA (DHCHC CoA) starter unit followed by seven acetate and seven propionate extender units, and terminate with a pipecolic acid unit to form a large macrocyclic ring (Scheme 28).

Replacement of AT8 with a methylmalonyl-specific AT (Rap AT3 or DEBS AT2) led to the production of 13-methyl-13-desmethoxyascomycin (13-MDMA) 326 (Fig. 73), and with a malonyl-specific AT (rapamycin AT12) gave 13-desmethoxyascomycin 325, clearly demonstrating that ascomycin AT8 does not use malonyl or methylmalonyl-CoA in the native organism. However, replacement of the methylmalonyl-specific DEBS AT6 with ascomycin AT8 and expression in Streptomyces lividans led to the production of 6-dEB 15 and 2-desmethyl-6- dEB 296, demonstrating that ascomycin AT8 can accept both malonyl and methylmalonyl-CoA in a heterologous host. No 2-methoxy-2-desmethyl-6-dEB 297 was produced, as the strain lacked the methoxymalonyl precursor. Note that co-expression of the ansamitocin methoxymalonate genes in this strain did result in the production of 2-methoxy-2-desmethyl-6-dEB 297 (see Section 6.3.2 for more details).