This invention concerns an improved synthetic methodology for the preparation of two cyclic aza-amino acid derivates and a new efficient protocol for the application of these compounds for the assembly of melanostatin-based azapeptides. The new azapeptides possess potent pharmacological activity as positive allosteric modulators (PAM) of the dopamine D₂ receptors at nanomolar concentrations, making them applicable in dopamine-related disorders, such as Parkinson's disease.
Melanostatin is a short endogeneous neuropeptide that displays potent PAM activity at the dopamine D₂ receptors, thus being of clinical interest as a therapeutical for dopamine-related disorders such as Parkinson's disease, as corroborated in clinical trials. However, its unfavorable pharmacokinetic properties such as low gastrointestinal absorption and reduced biochemical stability in neuronal tissues hinder its further development. Previous research has shown that the absolute configuration of proline residue in melanostatin neuropeptide is not a requisite for PAM activity, since the replacement of L-proline by D-proline has no significant impact on the pharmacological activity.
Moreover, since the metabolism of melanostatin occurs mainly upon cleavage of the prolyl-leucyl peptide bond, the development of melanostatin azapeptides by the replacement of this peptide bond with a semicarbazide moiety is expected to increase resistance against protease activity without compromising the PAM activity.
The incorporation of aza-amino acids in detriment to canonical amino acids is known to enhance pharmacological activity and selectivity as well as improve pharmacokinetic properties. Aza-proline is being employed as a proline surrogate in bioactive compounds as a chemical strategy to enhance potency and bioavailability. Furthermore, since the peptide bond is replaced by a semicarbazide moiety, the incorporation of aza-proline into bioactive molecules can enhance their biochemical stability towards proteases.
This technology presents a cheap, straightforward, and high-yielding (tri)phosgene-free methodology for the preparation of stable aza-proline and aza-pipecolic acid carbazates that circumvent the limitations of previous protocols compatible with multi-gram scale for the assembly of azapeptides. Moreover, this methodology does not require anhydrous conditions, using NaOH as the base for hydrazine cyclization in the presence of phase-transfer agents.
Furthermore, the methodology also describes a more efficient and milder process for the synthesis of azapeptides, allowing to reduce reaction time and temperature in comparison with other chemical strategies while offering higher yields. This methodology was applied for the assembly of melanostatin azapeptides. In general, melanostatin-based azapeptides exhibited toxicological profiles similar to that of the parental neuropeptide with no significative toxicity up to 100 micromolar concentration. Functional assays at human dopamine D₂ receptors have shown that melanostatin azapeptides are able to enhance dopamine potency at nanomolar concentrations, with the identification and characterization of two azapeptides with superior PAM activity than melanostatin neuropeptide.
The discovery of non-toxic and potent PAM of the dopamine D₂ receptors with expected improved pharmacokinetic profiles offers the possibility to explore the clinical translation of melanostatin-based azapeptides to be used in the early stages of Parkinson's disease delaying the use of levodopa therapy. On the other hand, co-administration of levodopa with PAM molecules of the dopamine D₂ receptors is expected to reduce therapeutic doses of levodopa and, therefore, its side effects.
This invention provides a new method to develop intermediates for azapeptides synthesis, as well as a prospective active pharmaceutical ingredient (API) suitable for the treatment of dopamine-related diseases.
