Artemisinin derivatives, methods for their preparation and their use as antimalarial agents
Derivatives of the antimalarial agent artemisinin, compositions comprising the derivatives, methods for preparing the derivatives, and their uses in pharmaceutical compositions intended for the treatment of parasitic infections are provided. Methods are provided for the production of artemisinin derivatives via functionalization of positions C7 and C6a, and optionally, in conjunction with modifications at positions C10 and C9, via chemoenzymatic methods. Recombinant cytochrome P450 polypeptides are also provided for use in the methods. The artemisinin derivatives can be used for the treatment of malaria and other parasitic infections, alone or in combination with other antiparasitic drugs.
1. A recombinant cytochrome P450 polypeptide having an improved capability as compared to a P450 enzyme of SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, to hydroxylate at least one substrate from the group consisting of substrates of general formula (I),
wherein:
(i) A represents a carbonyl group (═O), a hydroxy group (—OH), a halogen atom, an unsubstituted or substituted alkyloxy group, an unsubstituted or substituted alkenyloxy group, an unsubstituted or substituted alkynyloxy group, an unsubstituted or substituted aryloxy group, an unsubstituted or substituted heteroaryloxy group, or a group —NR 1 R 2 , wherein:
the R 1 represents a hydrogen atom, an unsubstituted or substituted alkyl group, an unsubstituted or substituted alkenyl group, or an unsubstituted or substituted alkynyl group;
the R 2 represents an unsubstituted or substituted alkyl group, an unsubstituted or substituted alkenyl group, an unsubstituted or substituted alkynyl group, an unsubstituted or substituted aryl group, an unsubstituted or substituted heteroaryl group; or
the R 1 and R 2 are connected together to form an unsubstituted or substituted heterocyclic group;
and wherein the cytochrome P450 polypeptide is SEQ ID NO:8.
2. The polypeptide of claim 1 , wherein the improved capability is an improved capability to hydroxylate position 6a, position 7, or both position 6a and position 7, in the substrate.
3. A method for hydroxylating a substrate of general formula (I),
comprising the steps of:
a. contacting the substrate with a cytochrome P450 polypeptide of SEQ ID NO.:8 under suitable reaction conditions;
b. allowing for the cytochrome P450 polypeptide to catalyze the hydroxylation of the substrate, while preserving the endoperoxide bond therein, thereby producing a hydroxylated derivative of the substrate; and
c. isolating the hydroxylated derivative of the substrate.
4. The method of claim 3 , wherein the substrate is artemisinin and the hydroxylated products comprise at least one compound selected from the group consisting of 6a-hydroxyartemisinin, 7(S)-hydroxyartemisinin, and 7(R)-hydroxyartemisinin.
5. The method of claim 3 , wherein the cytochrome P450 polypeptide is tethered to a solid support.
6. The method of claim 3 , wherein the cytochrome P450 polypeptide is contained in a host cell.