CONVERSION
The present invention provides a process for the in vitro conversion of deoxypodophyllotoxin to podophyllotoxin.
Podophyllotoxin is a naturally occurring lignan which can be isolated from the underground rhizome of Podophyllum species. Podophyllotoxin is an intermediate in the production of the anti-cancer drugs Etoposide and Teniposide. However, the most abundant source of podophyllotoxin is the endangered Indian species Podophyllum hexandrum Royle. Other sources of podophyllotoxin are limited and provide the lignan at low levels.
The present invention relates to a process for the production of podophyllotoxin by the in vitro conversion of deoxypodophyllotoxin to podophyllotoxin.
This process involves in vitro oxidation of the 7-position of deoxypodophyllotoxin as illustrated.
Deoxypodophyllotoxin Podophyllotoxin
The oxidation of deoxypodophyllotoxin can be carried out to produce podophyllotoxin (with the OH in the alpha position), epipodophyllotoxin (with the OH in the beta position) or a mixture thereof. For the purposes of this invention, the term "podophyllotoxin" encompasses podophyllotoxin, epipodophyllotoxin or a mixture thereof unless otherwise stated.
This process provides an alternative and sustainable source of podophyllotoxin and thus allows the continued production of the anticancer drugs derived therefrom (for example, Etoposide and Teniposide).
In one embodiment of the invention, the process involves the in vitro chemical conversion of deoxypodophyllotoxin to podophyllotoxin. This process involves ,the chemical oxidation of the 7-position of deoxypodophyllotoxin.
For the purposes of the present invention, the in vitro conversion of deoxypodophyllotoxin to podophyllotoxin can be carried out by a number of alternative processes, namely direct oxidation at the 7-position, full oxidation at the 7-position followed by stereospecific reduction, displacement, or the formation of a double bond at the 7-position and addition thereto. Each of these processes is considered in more detail below.
The production of podophyllotoxin by direct oxidation involves the selective hydroxylation of the benzylic C-H bond at the 7-position of deoxypodophyllotoxin. This selective hydroxylation can be carried out using a number of different oxidation reagents including but not limited to hydrogen peroxide, 'P^NH, BuLi, [5,10,15,20- tetrakis(pentafluorophenyl)21H,23H porphine] iron(iii) chloride or PhlO.
An alternative process for the production of podophyllotoxin involves full oxidation of the benzylic C-H bond at the 7-position on deoxypodphyllotoxin to a ketone group. This ketone group can then be reduced preferably stereospecifically, to form podophyllotoxin. Oxidation can be carried out by treatment with an oxidizing agent such as pyridinium chlorochromate or KMNO4. Reduction can be carried out for example by hydrogenation using a catalyst such as palladium on activated carbon.
Podophyllotoxin can further be produced by the addition of a leaving group such as halogen onto the 7-position of deoxypodophyllotoxin. The leaving group can be introduced by a free radical reaction, for example using N-bromosuccinimide to introduce bromide as a leaving group. The leaving group can then be displaced by a nucleophile such as water, preferably in the presence of a base such as NaHCO3 or NaOH, to produce podophyllotoxin. The skilled person will appreciate that the displacement of the leaving
group on the 7-position of deoxypodophyllotoxin may result in a further derivative of deoxypodophyllotoxin. This derivative may undergo one or more further substitution reactions at the 7-position to form podophyllotoxin.
Podophyllotoxin can also be produced from deoxypodophyllotoxin substituted at the 7- position, by the elimination of the 7-substitution to form a 7-ene derivative. The elimination can be achieved by treatment with a base such as NaOH. Oxidation of the double bond would provide podophyllotoxin. This step can for example be achieved by hydroboration followed by oxidation using reagents such as borane methylsulfide or trimethylamine-N-oxide.
In an alternative embodiment of the present invention, podophyllotoxin can be produced in vitro from deoxypodophyllotoxin by the enzymatic conversion, in particular the enzymatic oxidation of deoxypodophyllotoxin. In vitro enzymatic oxidation of podophyllotoxin can be carried out by an enzyme that hydroxylates benzylic rings. Examples of such enzymes include eugenol dehydrogenase.
The process for the in vitro production of podophyllotoxin by chemical or enzymatic conversion, uses deoxypodophyllotoxin as a precursor. Deoxypodophyllotoxin is a lignan which accumulates in the roots of Anthriscus sylvestris (wild chervil). It is structurally related to podophyllotoxin and is believed to be a precursor of podophyllotoxin in the biosynthetic pathway. There is however no teaching in the art to indicate that isolated deoxypodophyllotoxin can be used as a precursor in the in vitro production of podophyllotoxin.
The present invention provides a process for the in vitro production of podophyllotoxin using the precursor deoxypodophyllotoxin. Deoxypodophyllotoxin can be isolated in high yield and with a straightforward purification procedure from a sustainable source, such as Anthriscus sylvestris. The present invention therefore provides a process for the sustainable production of podophyllotoxin and the cancer drugs derived therefrom.
Deoxypodophyllotoxin for the present application can be isolated using conventional methods (Koulman et al, (2001), Planta Medica, Nol 67(9): 858-862; Lim et al (1999),
Archives of Pharmacal Research, Nol 22(2): 208-212; Ikeda et al (1998), Chemical & Pharmaceutical Bulletin, Nol 46(5): 871-874; Ikeda et al (1998) Chemical & Pharmaceutical Bulletin, Nol 46(5): 875-878; NanUden et al, (1997), Journal of Natural Products, Nol 60(4): 401-403). For example, deoxypodophyllotoxin can be extracted from the powdered roots of Anthriscus sylvestris with an organic solvent and then partitioned against a water immiscible organic solvent and water. The organic fraction containing deoxypodophyllotoxin can be further purified by crystallisation or column chromatography.
Podophyllotoxin produced by the present invention can be used as an intermediate in the production of anti-cancer drugs such as Etoposide and Teniposide.
The invention will now be illustrated by the following non-limiting examples.
Example 1 - Production of podophyllotoxin by Direct Oxidation
Deoxypodophyllotoxin (0.4 mmol) was dissolved in CH2C12 (10ml) at RT, and [5,10,15,20-tetrakis (pentafluorophenyl)-21H,23H-porphine] iron(III) chloride (30mg) was added, followed by iodosobenzene (O.lόg). The resulting reaction was stirred at RT for lh and then filtered. The filtrate was concentrated and the resulting crude product was purified by column chromatography.
Example 2 - Production of podophyllotoxin by Oxidation and Reduction
2.1 Oxidation
To a stirred suspension of PCC (7.5 mmol) in dry CH2C12 (10ml) was added a solution of deoxypodophyllotoxin (5mmol) in dry CH2C12 (10ml). The reaction mixture was allowed to react at RT for 1.5h followed by addition of dry ether (20ml). The supernatant liquid was decanted and the black residue washed thoroughly with warm ether (20ml x 3), and combine the ether washings combined with the previous organic layer. Evaporation and purification by column chromatography provide the desired product.
2.2 Reduction
Ketone (lmmol, from above reaction), 10%Pd/C(en) (Pd/C-ethylenediamine complex) (10% of the weight of the substrate) and methanol (2ml) was stirred at RT under a hydrogen atmosphere for 24h. The reaction mixture was filtered using a membrane filter (Advantec DISMIC-13CP) and the filtrate was concentrated in vacuo to give a crude product which can be purified by column chromatography.
Example 3 - Production of podophyllotoxin by Substitution and Displacement
3.1 Substitution
To a solution of deoxypodophyllotoxin (2mmol) and N-bromosuccinimide (2 mmol) in chloro-benzene (3ml) was added benzoyl peroxide (20mg). The resulting solution was allowed to reflux gently for lh and then cooled down to RT. The precipitate was filtered and washed thoroughly with ether (2 x 5ml). The filtrate was concentrated and the crude product was purified by column chromatography.
3.2 Displacement
The 7-bromo-deoxypodophyllotoxin from above experiment was dissolved in a mixture of 1:1 H2O-THF (3ml). To the solution was added KOH (2mmol). The resulting reaction mixture was allowed to be stirred at RT for 2h. Solvents were removed under reduced pressure and the crude product was purified by column chromatography.
Example 4 - Formation of 7-ene product and addition across double bond.
4.1 The formation of 7-Ene product
The 7-bromo deoxypodophyllotoxin from above experiment was dissolved in a mixture of 1:1 H2O-THF (3ml). To the solution was added KOH (2mmol). The resulting reaction mixture was allowed to be gently reflux for 2h. The reaction mixture was cooled down to RT. Solvents were removed under reduced pressure and the crude product was purified by column chromatography.
4.2 Addition across the double bond
To an ice-cooled solution of the 7-ene product (lmmol) in diglyme (1ml) was added borane methysulfide under nitrogen. The reaction was allowed to warm up to RT and stirred for lh before addition of water followed by trimethylamine-N-oxide (lmmol). The resulting reaction mixture was then gently refluxed for lh and cooled to RT followed by addition of ether (10 ml). The organic phase was washed with water and dried, filtered. The filtrate was concentrated down and the crude product purified by column chromatography.