The Solid-Phase Synthesis Of Methotrexate-α-Phe And Effect On Prostate Cancer

My research can be divided into two parts. The first is to synthesize Methotrexate-a-phenylalanine(MTX-a-Phe) and study the effect of MTX-a-Phe on the prostage cancer .The second is to Synthesize the Taxol side chain precursor using AA reaction.Methotrexat which is used extensively in cancer chemotherapy .The drug is brought into cells by the "reduced folate" or "micromolarfolate" transporter, and polygluta-mylated to enhance its retention in the cell .The cytotoxicity of MTX polygllutamates results primarily from their potent inhibition dihydrofolate reductase (DHFR).Empirical trials have established that MTX is palliative,and in some instances curative, in the treatment of acute lymphocytic leukemia,lymphomas, choriocarcinoma, and some solid tumors;In conventional chemotherapy, introduction of MTX not only killed thetumor cell, but also produces unacceptable damage to normal proliferating cells. People thought that this might be improved by using a prodrug form of MTX (i.e. a derivative that would prevent its uptake, polyglutamylation, or inhibitory activity) and then converting the prodrug toMTX by an activating enzyme linked to the monoclonal antibody. Because of its catalytic power, each enzyme molecule should be able to generate a large number of MTX molecules at the tumor site. This type of chemotherapeutic strategy, sometimes referred to as "antibody-directed enzyme prodrug therapy" In considering how to convert MTX to a prodrug, we focused upon derivatization of the a-carboxyl group to an amide, a procedure known to severely inhibit transport of the drug . It seemed likely, moreover, that the blocking group could be removed by carboxypeptidases, which excise the terminal amino acids from polypeptides . The present report describes preliminary experiments that demonstrated the feasibility of this approach, Several a-amides of MTX (MTX- a -alanine, -aspartate, and -arginine) have been synthesized previously as potential chemotherapeutic agents, but they failed to show significant antitumor activity.MTX-a-phenylalanine (MTX-Phe) was prepared later, And it could be hydrolyzed by carboxypeptidase-A to yield the parent drug MTX.We used the system of Fmoc solid-phase peptide synthesis to synthesize MTX- a- Phe successfully. The product was verified as a single peak by HPLC and its molecular weight was measured by spectrometry without further purification.We took 2,4,5,6-tetraaminopyrimidine as the original material and it was under the reaction of four steps to get 4-amino-4-deoxy-10-methylpteroic acid,which is a versatile intermediate prepaired for an improved approach to analogues of Methotrexate-a-peptides. Wang resin was reacted with protectedamino acids to obtain Glu(OtBu)-Phe-O-Wang-Resin through four steps synthesis manipulation..4-amino-4-deoxy-10-methylpteroic acid was reacted with Glu(OtBu)-Phe-O-Wang-Resin to produce target compound MTX-a-Phe in the presence of active peptide coupling reagen.In Synthesis experiment process, we made some improvements:1. Recently Perron MJ reportrd a series a-amides of MTX were synthesized with the technique of solid-phase peptide synthesis,but the data were not shown in detaile and the scale is small.We used the reactor designed by ourselves,which structure is simple and cost is cheap. It was used easily and made the operation step reduced,the scale extended, the agent economized. We got 5.4g Product in one experiment.2. The ratio of 4-amino-4-deoxy-10-methylperoic acid to protected amino acid was reduced to 2:1 from 7:1 and the other dosages of the original material were reduced owing to adoption of the active peptide coupling reagent and the reactor.3. The overall yields Increased to 90 % from 28% .4. The quantity of resin increased to 10 g from 1 g, it would establish the foundation for the industrialization.We also made the biology experiment of MTX-a-Phe.The effect of MTX-a-Phe on the prostage cancer was studied in vivo and in vitro. It indicated that MTX-a-Phe almost had not the toxic effect on the test cell in vitro.lt was hydrolyzed into MTX by Carboxypeptidase(CP-A).The toxin effect of the products was about 1000 times higher than that of the prodrugs and the former showed an obvious inhibition effects on the prostate cancer cell.The MTX-a-Phe and the CP-A was an ideal ADEPT.In the second part, We synthesized two recoverable and reusable chiral ligands.one-step synthesis of paclitaxel side chain precursor has been achieved...