The Chemosynthesis And Anti-leukemia Activity Of Demethylated-STI571

The incidence and mortality attributed to malignant tumor has been rising steadily.It has been the main cause of deaths secondary to cardiovascular diseases in developed countries.Moreover it has become the leading mortality in China.To find out effective therapies becomes so urgent and important.The scientists devoted themselves to new and high potent drugs after the first anti-tumor drug on the market in the Second World War.For decades,the hallmark of medical treatment for cancer has been intravenous chemotherapies.The growth and proliferation is inhibited nonspecifically by changing the DNA structure and microtubulin,interfering the duplication and transcription of nucleic acid.The pharmacokinetics abides by first order kinetics,which means that the antineoplasmic activity is proportional.But the toxicity could not be overlooked because these traditional chemotherapeutic agents aim at many ceils,including cancer cells and certain normal tissues.As a result,many patients suffer from the classic toxicities of alopecia,gastrointestinal symptoms,and myelosuppression.Both curative effect and toxicity are in a concentration- and time-dependent manner.Therefore high potent and low toxic drugs are required intensively.Due to the enormous progress that has been made in the past few years in the identification of the human genome,in molecular and cell biology,in structural biology and in bioinformatics,a dramatic shift in cancer therapy occurred.Molecular targeted therapies aim at the special molecular structure of cancer tissue and cells in the forms of antibody or ligand,with selective inhibition on cancer and no effects on normal tissue.Although traditional cytotoxicity chemotherapy remains the main treatment of choice for many malignancies,targeted therapies are now a component of treatment for many types of cancer,including breast cancer,colorectal cancer, lung cancer,and pancreatic cancer,as well as lymphoma,leukemia,and multiple myelomal.The targeted drugs can be sorted into two main types:monoclonal antibody(Rituximab,Heceptin,Erbitux,Avastin)and small molecule inhibitors(Glivec,Iressa,Tarcev).Protein-tyrosine kinases(PTKs),enzymes that catalyze the transfer of theγ-phosphate of ATP to tyrosine residues of protein substrates,are critical components of signaling pathways,including Ras / MAPK,STAT,JNK,PI3K / AKT,which control cellular proliferation and differentiation.PTKs can be subdivided into two large families,receptor tyrosine kinases(RTKs)and non-receptor tyrosine kinases (NRTKs).RTKs span the plasma membrane and contain an extracellular portion, which binds ligand,and an intracellular portion,which possesses catalytic activity and regulatory sequences.The RTK family includes the insulin receptor and the receptors for many growth factors such as epidermal(EGF),platelet-derived(PDGF),fibroblast (FGF),and nerve growth factors.NRTKs contain no extracellular or transmembrane portion but possess modular domains that are responsible for subcellular targeting and regulation of catalytic activity.The NRTK family includes Src,Abl,FAK,and the JAKs among many others.Because of the key roles PTKs play in cellular signaling processes,their catalytic activity is tightly controlled in normal cells by protein-tyrosine phosphatases,by other protein tyrosine or serine/threonine kinases, and by autoregulatory mechanisms.The abnormality of PTKs expression and activity results in tumorigenesis,invasion,metastasis and resistance.The inhibition of tyrosine kinases might therefore provide new therapies for diseases such as cancer and is likely to affect other fields of medicine.In the past decades,the number of receptor and non-receptor tyrosine Mnases that have been identified as valuable molecular targets has greatly increased.Low molecular weight protein tyrosine kinase inhibitors(TKIs)can block the activity of PTKs.Different from antibodies against cancer,Tyrosine kinase inhibitors act inside the cell,competing with adenosine triphosphate(ATP)for binding to the catalytic tyrosine kinase domain.This blocks initiation of downstream signaling.Because TKIs target abnormalities specific to tumor cells,there is hope that they will be free from the generalized toxicities associated with cytotoxic chemotherapy.Currently about 10 ATP-competitive inhibitors targeted against different tyrosine kinases are used in clinical therapy(e.g.,EGFR,VEGFR,PDGFR,Kit).Imatinib,gefitinib,erlotinib and sorafenib have entered the market in China.They are all effective,low-toxic,oral and are popular in patients.However,they are too expensive,so it is necessary to develop some molecular target drugs that patients could afford.In 2001 Imatinib mesylate(Gleevec,STI571)entered the market.STI571 is a phenylaminopyrimidine derivative,a tyrosine kinase inhibitor with competitive inhibition of ATP binding.It inhibits the protein tyrosine kinase Bcr-Abl, platelet-derived growth factor receptors(PDGFRalpha and PDGFRbeta)and KIT. Imatinib is approved for the treatment of chronic myeloid leukemia(CML)and c-Kit (CD117)-positive unresectable or metastatic gastrointestinal stromal tumors(GISTs), which have deregulated activity of an imatinib-sensitive kinase as the underlying pathogenetic feature.Pharmacokinetic studies of imatinib in patients with CML,GIST or other cancers and healthy volunteers show that imatinib is approximately 95%bound to human plasma proteins,mainly albumin and alphal-acid glycoprotein.The terminal elimination half-life is approximately 18 hours.Imatinib is extensively metabolized by the cytochrome P450(CYP)isoenzymes in the gut wall and liver;liver CYP3A4 and CYP3A5 is the main enzyme responsible for imatinib metabolism. N-desmethylated STI571(CGP 74588),the main circulating metabolite formed by CYP3A4,is an N-desmethylated piperazine derivative and have a long half-life.The exposure measured as a percentage of the area under the concentration-time curve in plasma of the N-demethylated piperazine derivate is approximately 10%-15%of that of imatinib.Imatinib is a phenylaminopyrimidine derivative which is the active site of the drug,and methylated piperazine portion of imatinib mainly used to increase solubility and bioavailability.We conclude that N-desmethylated STI571 is endowed with high activity comparable to the parent drug and possesses low toxicity.So we try to find a new desmethylated piperazine inhibitor based on the STI571 structure,which is called CGP74588(N-desmethylated STI571).CGP74588 chemosynthesis, anti-leukemia activity and toxicity are reported in this paper.After describing the chemosynthesis of CGP74588,human Ph+ leukemia cell lines K562 and Ph- U937 were tested for their response to a range of doses of STI571 and CGP74588.The results from in vitro studies showed that STI571 and CGP74588 both exhibited markedly potent killing effect on K562 cell line in a concentration- and time-dependent manner,while there was no effect on U937 cell line in the tested concentration range.The result by MTT assay revealed that the IC50 value of CGP74588 on K562 was similar to the value of STI571,which suggesting that CGP74588 activity was comparable to the STI571 in killing the tumor cells grown in vitro.The inhibition rate is almost 50%after exposure for 24hr and near to the top after 48hr.CGP74588 markedly inhibited the PBMCs of CML in a concentration-dependent manner;however there was only nonspecific inhibition of healthy people's PBMCs.The K562 cell was used to examine the apoptosis-inducing effect of CGP74588 and STI571.Morphological analysis after the K562 cells were treated with -the two drugs displayed the hallmarks of apoptosis including chromatin condensation,nuclear shrinkage and fragmentation as well as "apoptotic body";DNA agarose gel electrophoresis showed a characteristic "ladder" of internucleosomal DNA fragmentation in K562 cells following treatment with two drugs;flow cytometric analysis of DNA content disclosed that CGP74588 induced a massive hypodiploid cell population(sub-G1)and G1 arrest,showing significant concentration- and time-dependent manner.These results strongly suggest that CGP74588 is able to induce apoptosis in the K562 cells.Two human Ph- leukemia cell lines(HL-60 and NB4),two solid tumor cell lines(A549 and Ht29)and normal PBMCs were tested for their response to a range of doses of CGP74588 compared with STI571.The ceils were resistant to STI571 and CGP74588.None of the cell lines had any significant antineoplastic response below the dose of 10μM,concentrations that were greater than 13 times the IC50 value for the K562 cells.The in vivo toxicity results demonstrate that CGP74588 was effective in the murine when CGP74588 adds up to 50mg/kg with the maximal murine weight inhibition rate of 24.4%and discomfortableness such as pain,diarrhea,asthenia, while 5mg/kg CGP74588 almost had no effect on weight and no appreciable toxicity during the period of drug administration.All of this indicate that murine were resistant to CGP74588.Taken together,CGP74588 has potent anti-tumor activity both in vitro and in vivo.it can effectively induce apoptosis of K562 cells,with low cell and murine toxicity similar to STI571.So CGP74588 is an effective drug.Pharmacokinetics and pharmacodynamics are on schedule.