Biocatalysis for pharmaceuticals and fine chemicals

1. Biocatalysis for pharmaceuticals and fine chemicals

2. Chirality and pharmaceuticals Chirality in biologically active molecules is of natural occurrence. Traditionally, it was common practice for a pharmaceutical company to market a chiral drug as the racemate, and as recently as 1985, more than 75% of chiral drugs were sold as the racemate. This policy implied that each dose of a drug is contaminated with an equal amount of an isomer, which usually has no therapeutic value but may have the potential to cause unsuspected deleterious side effects.

3. The Thalidomide case Chiralità e farmaci: La Talidomide è un farmaco con proprietà ipnotico-sedative che è stato commercializzato per la prima volta in Germania nel 1956 per la terapia dell’influenza, e successivamente, in 46 Paesi, per la terapia dell’insonnia. È stato inoltre ampiamente utilizzato in donne in gravidanza nella terapia delle nausee mattutine grazie anche ad una pubblicità che sottolineava la “sicurezza” del prodotto. I test preclinici su roditori ed i trial clinici non avevano infatti evidenziato effetti collaterali. La vendita di Talidomide incrementò drasticamente in pochi anni e, entro il  1960, solo in Germania vennero prodotte circa 15 tonnellate di farmaco. In USA la Talidomide non ottenne l’autorizzazione all’immissione in commercio per una presunta associazione tra il farmaco e lo sviluppo di neuropatie periferiche.

4. La Talidomide Fin dall’inizio degli anni ’60 si osservò un incremento di neonati con malformazioni congenite degli arti e fu ipotizzata una correlazione con l’assunzione materna di Talidomide in corso di gravidanza. Il farmaco venne pertanto ritirato dal commercio nel 1961; l’incidenza di malformazioni degli arti è ritornata nei limiti dopo il ritiro dal commercio, confermando l’effetto teratogeno della Talidomide. È stato stimato che più di 6000 bambini (senza contare i casi di aborto spontaneo e morti fetali endouterine) siano nati con un’embriopatia da Talidomide, caratterizzata da difetti di riduzione degli arti di vario grado ed altri tipi di malformazioni congenite. Studi ad hoc su animali di laboratorio (conigli e primati non umani) hanno inoltre confermato l’effetto teratogeno del farmaco, osservando il medesimo pattern malformativo osservato nell’uomo; il farmaco non è tuttavia risultato teratogeno in alcune specie animali (tra cui topi e ratti). Tale episodio ha dimostrato che l’ambiente gioca un ruolo significativo nel determinare malformazioni congenite, ha demolito l’ipotesi che il feto sia protetto dalla “barriera placentare” rispetto ai farmaci assunti dalla madre ed ha confermato che l’effetto teratogeno è specie-specifico. Le conseguenze pratiche furono un incremento della ricerca di base ed epidemiologica nell’ambito della teratogenesi ambientale e dei controlli per contenere l’introduzione di nuovi agenti chimici, con possibile effetto teratogeno.

5. CHIRALITA’ E FARMACI Recent rulings of the Food and Drug Administration (FDA) in the United States reflect the current situation in “chiral drugs”: pharmaceutical industries will have to provide rigorous justification to obtain the FDA’s approval of racemates.

7. CHIRALITA’ E FARMACI • Dei nuovi farmaci immessi sul mercato tra il 2000 ed il 2004, il 56% è costituito da singoli enantiomeri.

8. Antiinfiammatori non steroidei derivati dell’acido propanoico: configurazione (S) naprossene acido (+)-(S)-2-(6-metossinaftalen-2-il)propanoico

9. Anti-infiammatori non steroidei derivati dell’acido propanoico: configurazione (S) (S)-(+)-Ibuprofen Tuttavia viene venduto come miscela racemica R,S in quanto l’organismo converte l’enantiomero R in S

10. Classical resolution in chemistry is based on formation of diastereomers, by addition of a pure enantiomer (from the chirality pool) to the racemate solution, which can be separated by crystallization. To date it has been the most commonly used industrial technique.

11. B –biotechnol *chyral § not chyral Top drugs 2011-13 § * * § * * * * * § * B * * * B * B * B B * B B * * * B B B

12. Abilify (achiral) ARIPRIPAZOLE: atypical antipsychotic schizophrenia bipolar disorder major depressive disorder irritability associated with autism

13. Nexium S-isomer, esomeprazole At the cellular level, both of these isomers convert to the same inhibitor of the H+,K+-ATPase (proton pump inhibition) and produce the same reduction in gastric acid secretion. Obtained via asymmetric oxidation using Ti based catalyst (94% ee) central sulphur atom, which acts as a chiral centre S-isomer, esomeprazole, is metabolized more slowly and reproducibly than the R-isomer and therefore produces higher plasma concentrations for longer and, as a result, inhibits gastric acid production more effectively and for longer. Thus, esomeprazole has the pharmacological properties of a more effective form of treatment for disorders related to gastric acid secretion

14. Omeprazoleis a racemate. reduction in gastric acid secretion.

15. Cymbalta duloxetine prescribed for major depressive disorder, generalized anxiety disorder, fibromyalgia and neuropathic pain Cymbalta generated sales of nearly $5 billion in 2012 with $4 billion of that in the U.S., but its patent protection terminated January 1, 2014. Lilly received a six-month extension beyond June 30, 2013 after testing for the treatment of depression in adolescents, which may produce $1.5 billion in added sales.

16. Process for preparation of duloxetine hydrochloride patent US 8269023 B2 • An improved process for synthesis of duloxetine hydrochloride having chiral purity greater than 99.9% that is characterized by the following: (i) preparation of racemic condensed compound (RS)—N,N-di methyl-3-(1-naphthyloxy)-3-(2-thienyl)propanamine by reaction of racemic hydroxy compound with 1-fluoronaphthalene in presence of a base such as sodamide, potassium amide or potassium bis(trimethylsilyl)amide (KHDMS) in polar aprotic solvent, • (ii) optical resolution of racemic condensed compound with di-benzoyl-L-tartaric acid or di-para-anisoyl-L-tartaric acid to obtain crude (S)—N.N-dimethyl-3-(1-naphthyloxy)-3-(2-thienyl)propanamine dibenzoyl tartarate salt (8a) or (S)—N.N-dimethyl-3-(1-naphthyloxy)-3-(2-thienyl)propanamine di-p-anisoyl tartarate salt (9a) respectively, • (iii) optionally purification of crude tartarate salts by crystallization, • (iv) optionally purification of duloxetine hydrochloride by crystallization and • (v) racemization of undesired (R)—N,N-di methyl-3-(1-naphthyloxy)-3-(2-thienyl)propanamine by treatment with base potassium bis(trimethylsilyl)amide (KHDMS) to obtain racemic mixture of condensed compounds

17. Humira Adalimumab  is a monoclonal antibody used to treat the symptoms and prevent the progression of active rheumatoid arthritis and ankylosing spondylitis. It is used in children 2 years of age and older for juvenile idiopathic arthritis. This medicine is also used to treat psoriatic arthritis, which is a type of arthritis that causes pain and swelling  It binds to tumor necrosis factor-alpha (TNFα), which normally binds to TNFα receptors, leading to the inflammatory response of autoimmune diseases. By binding to TNFα, adalimumab reduces this inflammatory response. Because TNFα is also part of the immune system, which protects the body from infection, treatment with adalimumab may increase the risk of infections. Humira costs approximately $4370 per month (2017). From 2012 until the US patent expiry in 2016, Humira led the list of top-selling pharmaceutical products, and in 2016, it had $16 billion of global sales. In 2014, in India, the first adalimumab biosimilar came to market at a price of $200.

18. Crestor Rosuvastatina Statins Inhibitors of cholesterol synthesis

19. In 2005, sales were estimated at US$ 18.7 billion in the United States. The best-selling statin is atorvastatin, which in 2003 became the best-selling DRUG in history. The manufacturer Pfizer reported sales of US$ 12.4 billion in 2008. Due to patent expirations, several statins are nowavailable as less expensive generics. LEVOSTATIN ( R ) – mevalonic acid (intermediate in cholesterol synthesis) ATORVASTATIN (Lipitor)

20. Biosintesi del colesterolo

26. Retrosynthesis

32. Alternative routes to Pravastatina

33. Asymmetric synthesis for the production of pravastatin hydroxynitrile intermediate (1.) 1. Ketoreductase + Glucosedehydrogenase for cofactorrecycling

34. Asymmetric synthesis for the production of statin intermediates (2.) 2. The halohydrin dehalogenase (HHDH) is employed to catalyse the replacement of the chloro substituent with cyano by reaction with HCN at neutral pH and ambient temperature

35. Classification of enzymes used in organic synthesis.

36. Steroids synthesis DHEA (deidroepiandrosterone), natural steroid (max in humans 15-20 anni) Precursor of estrogens and androgens produced from natural vegetal sources (tropical plant) used as a radiation countermeasure: stimulates white cells and platelets production

39. Main coenzymes required by enzymes used in biocatalysis Several cofactors can be recycled effectively, including nucleoside triphosphates such as ATP in phosphoryl transfer reactions, (NAD+/NADH and NADP+/NADPH) in oxidoreductions, acetylCoA in acyl transfer reactions, and sugar nucleotides in glycosyl transfer reactions. Many cofactor dependent reactions have been applied on preparative or industrial scales. Nevertheless, hydrolases still remain the most widely employed enzymes due to their large availability and to the fact that they do not need organic coenzymes.

40. CHIRALITY AND PHARMACEUTICALS Chirality in biologically active molecules is of natural occurrence. Traditionally, it was common practice for a pharmaceutical company to market a chiral drug as the racemate, and as recently as 1985, more than 75% of chiral drugs were sold as the racemate. This policy implied that each dose of a drug is contaminated with an equal amount of an isomer, which usually has no therapeutic value but may have the potential to cause unsuspected deleterious side effects.

42. Some classical examples of aplications of biocatalysts in the synthesis of drugs at industrial scale

44. Playing with nitrile group

45. Playing with epoxides

48. Halohydrin dehalogenases mechanistically cleaves the carbon-halogen bond through the formation of an epoxide from a vicinal hydroxyl group Halohydrin dehalogenase is  involved in the  degradation of  vicinal halohydrines. In several species of bacteria, it catalyses the dehalogenation of halohydrins to produce the corresponding epoxides.

49. Citocromi Modello della struttura tridimensionale del citocromo P450 BM3 (CYP102A1) da Bacillus megaterium. E’ un citocromo estremamente efficiente, con un numero di turnover pari a 17.000 min-1 per la trasformazione dell’acido arachidonico. Il gruppo di ricerca di Frances Arnold ha sviluppato numerosi mutanti di questo citocromo in grado di catalizzare nuove reazioni (destra), tra le quali l’idrossilazione di alcani gassosi a catena breve per la sintesi di alcoli, la deprotezione selettiva di zuccheri, la sintesi in vitro di metaboliti di farmaci che vengono fisiologicamente prodotti nell’organismo e l’epossidazione stereoselettiva di alcheni.

50. Selectivity of enzymes is not always the key factor for selecting a synthetic route