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1、CONTENTSAbstractpag. 3CHAPTER 1. Introduction 61.1. Cancer Chemotherapy 71.2. Nucleoside and Nucleotide Analogues 91.2.1. Mechanism of Action 91.2.2. Purine Nucleotides Biosynthesis 101.3. Conformational Issues of Nucleosides and Nucleotides14CHAPTER 2. Inosine 5-Monophosphatate Deydrogenase asTarge
2、t for Chemotherapeutic Agents192.1. Role of IMP Deydrogenase in the nucleotides biosynthesis 202.2. Mechanism of Catalysis of IMPDH212.3. Crystal Structures of IMPDH232.4. IMPDH Inhibitors262.4.1. Reversible Nucleoside Inhibitors to the substrate-binding domain262.4.2. Irreversible Nucleoside Inhibi
3、tors to the substrate-binding domain302.4.3. Non-Nucleoside Inhibitors of IMPDH302.4.4. C-Ribonucleosides Inhibitors of IMPDH: Tiazofurin and Its Analogues322.4.5. Dinucleotide Anabolite of Tiazofurin and Its Analogues: IMPDH Inhibitors in the Cofactor Site382.5. Inhibitors of Human IMPDH in Clinica
4、l Use43CHAPTER 3. Study of New TAD Analogues as IMPDH Inhibitors443.1. Synthesis, Conformational Analysis and Biological Activity of New45Analogues of Thiazole-4-Carboxamide Adenine Dinucleotide (TAD) as IMP Deydrogenase Inhibitors3.1.1. Aim of the Research453.1.2. Synthetic Route for T-2-MeAD (1) a
5、nd T-3-MeAD (2)473.1.3 Conformational Analysis of T-2-MeAD and T-3-MeAD493.1.4. Molecular Modeling of T-2-MeAD and T-3-MeAD503.1.5. Biological Evaluation of T-2-MeAD and T-3-MeAD523.1.6. Stability of T-2-MeAD and T-3-MeAD533.1.7. Conclusions54CHAPTER 4. Study of Modified Mizoribine Analogues as IMPD
6、H Inhibitors554.1. Ribose-Modified Mizoribine Analogue: Synthesis and Biological Evaluation564.1.1. Aim of the Research564.1.2. Synthetic Pathway for the 2-Me-MZR (14) and 3-Me-MZR (15)584.1.3. Conformational Analysis614.1.4. Biological Evaluation62CHAPTER 5. Ribonucleotide Reductase as Target for A
7、ntitumor Agents635.1. Role of Ribonucleotide Reductase in the synthesis of DNA645.2. Mechanism of Catalysis of RR665.3. Cristal Structure of RR685.4. Ribonucleotide Reductase Inhibitors705.4.1. Translation Inhibitors705.4.2. Dimerisation Inhibitors715.4.3. Catalytic Inhibitors71CHAPTER 6. Study of C
8、-methyl-b-D-RibofuranosylAdenine Nucleosides as Ribonucleotide Reductase Inhibitors816.1. Antitumor Activity of C-methyl-b-D-Ribofuranosyladenine Nucleoside Ribonucleotide Reductase Inhibitors826.1.1. Aim of the research826.1.2. Synthesis of Substituted 2 and 3-C-Methyladenosine Analogues (25, 27-31
9、)836.1.3. Biological Evaluation876.1.4. Antitumor Activity of 2-Me-Ado and 3-Me-Ado through RR Inhibition926.1.5. Stability of 2-Me-Ado and 3-Me-Ado946.1.6. Conclusions95CHAPTER 7. Experimental Section967.1. Chemistry977.1.1. Synthesis of T-2-MeAD (1) and T-3-MeAD (2)977.1.2 Synthesis of 2-C-Me-MZR
10、(14) and 3-C-Me MZR (15) 1007.1.3 Synthesis of Substituted 2 and 3-C-Methyladenosine Analogues 1027.2. Biological Section 1047.3. Computational Chemistry 109REFERENCES 111AbstractNucleotide coenzymes participate in essential enzyme-catalyzed redox reactions and play a fundamental role in cellular me
11、tabolic processes such as nicotinamide adenine dinucleotide (NAD) in the case of dehydrogenases. It has been shown that in many living organisms, disturbance of the nucleotide metabolism severely affects cell survival; in fact, altered NAD metabolism has been observed in many cancers. Some NAD analo
12、gues have been identified as active metabolites of nucleosides endowed with antitumor and antiviral potency. These dinucleotides are potent inhibitors of inosine 5-monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo guanine nucleotides biosynthesis. IMPDH, which catalyzes the NAD-
13、dependent conversion of inosine 5-monophosphate (IMP) to xanthosine 5-monophosphate (XMP), was shown to be significantly increased in highly proliferative cells. Inhibition of this enzyme results in a decrease in GTP and dGTP biosynthesis, producing inhibition of tumor cell proliferation. It was fou
14、nd that IMPDH exists in two isoforms, type I which is constitutively expressed, and type II which is up-regulated and predominates in neoplastic and fast replicating cells. Thus, the selective inhibition of IMPDH type II may provide improved selectivity against target cells in anticancer chemotherap
15、y.NAD analogues in which the nicotinamide ring is replaced by the thiazole- and selenazole-4-carboxamide moieties (TAD and SAD, respectively), as active metabolites of the antitumor agents tiazofurin and selenazofurin, proved to be potent noncompetitive inhibitors of IMPDH. Nevertheless TAD, SAD and
16、/or other analogues did not show any isoform specificity. Thus, it was conjectured that ligand modification in the dinucleotide adenosine moiety may provide significant isoform specificity.In order to investigate the subdomain of the enzyme that binds the adenosine moiety of TAD, two new dinucleotid
17、e analogues in which the ribose ring in the adenosine portion was replaced by 2-C-methyl ribose (T-2-MeAD) or 3-C-methyl ribose (T-3-MeAD) were synthesized, and their conformation was investigated in relation to their inhibitory activity against human IMPDH isoforms.Inhibition of recombinant human I
18、MPDH type I and type II isoenzymes by T-2-MeAD and T-3-MeAD proved to be noncompetitive with respect to NAD substrate. Binding of T-3-MeAD was slightly inferior to that of the parent compound TAD, while T-2-MeAD proved to be a weaker inhibitor. The higher inhibitory activity of T-3-MeAD, as compared
19、 to that of T-2-MeAD, may be explained by the preference of the first dinucleotide for a conformation of the adenosine moiety South (2T3) very close to that of TAD, as determined by molecular modeling techniques of energy minimization, conformational searching, and molecular dynamics simulations. Th
20、e decrease in activity is more remarkable in the case of the 2-C-substitution in T-2-MeAD. It appears that the variation of conformation of the adenosine moiety in T-2-MeAD and T-3-MeAD impairs the ability of the 2- and 3-hydroxy group of the ribose and/or the purine moiety to bind the NAD site of b
21、oth IMPDH isoforms.T-2-MeAD and T-3-MeAD were also tested for their cytotoxicity against human myelogenous leukemia K562 in culture. T-2-MeAD and T-3-MeAD were found to be less cytotoxic than TAD. Interestingly, T-3-MeAD was found to be active, although to a lesser degree, against K562 cells resista
22、nt to tiazofurin. It was found that the activity of T-3-MeAD was due to 3-C-methyl-adenosine (3-Me-Ado), that was formed in culture medium by the hydrolysis of the heterodinucleotide. In fact, 3-Me-Ado proved to be active against a broad spectrum of tumor cell lines. Another part of the research was
23、 addressed to the analogues of mizoribine (MZR), a immunosuppressive agent that behaves as a transition-state analogue inhibitor, able to bind the active site of IMPDH adopting the C3-endo North (3T2) conformation sugar pucker as the xanthosine monophosphate (XMP) in the E-XMP* complex. On the basis
24、 of the knowledge that substitution of hydrogen atoms at the 2-, and 3-position of the sugar moiety of ribonucleosides with a methyl group induces the stabilization of the conformation into the C3-endo and C2-endo forms respectively, two mizoribine analogues containing these modifications (2-Me-MZR
25、and 3-Me-MZR, respectively) were synthesized.Surprisingly, mizoribine and its 2- and 3-C-methyl derivatives, evaluated for their ability to inhibit the growth of human myelogenous leukemia K562, proved to be inactive. The inactivity of these nucleosides might be due to their inability to be converte
26、d into the corresponding 5-monophosphate derivative in K562 cells. Further experiments are underway to check this hypothesis.On the basis of the activity of T-3-MeAD against K562 cells resistant to tiazofurin, probably due to 3-Me-Ado that formed in the culture medium, it was investigated the possib
27、ility that this nucleoside could be responsible of the antitumor activity through the inhibition of ribonucleotide reductase (RR). RR catalyzes the conversion of nucleotides to deoxynucleotides in all organisms and thus plays a central role in nucleic acid metabolism. The development of agents that
28、inhibit RR activity is an established strategy in cancer therapy.In this respect, a series of 1-, 2-, and 3-C-methylsubstituted adenosine and 2-chloroadenosine analogues and N6-substituted derivatives were synthesized and evaluated as antitumor agents.From this study 3-Me-Ado emerged as the most act
29、ive compound, showing activity against both human leukemia and carcinoma cell lines. Structure-activity relationship studies showed that the structure of 3-Me-Ado is crucial for the activity. In fact, substitution of a hydrogen atom of the N6-amino group with a small alkyl or cycloalkyl group, the i
30、ntroduction of a chlorine atom in the 2-position of the purine ring or the moving of the methyl group from the 3-position to other ribose positions brought about a decrease or loss of antitumor activity. The antiproliferative activity of 3-Me-Ado appears to be related to its ability to deplete both
31、intracellular purine and pyrimidine deoxynucleotides through ribonucleotide reductase inhibition.CHAPTER 1. Introduction1.1. Cancer Chemoteraphy The term chemotherapy is used for cancer treatment methods which use cytotoxic drugs and substances. All growing cells are going through a cell cycle. A si
32、mplified cell cycle would start with an increased production of cellular structures like proteins and enzymes, followed by a replication of genetic material. After this material (genome) is doubled, every genome copy is moved to each cell pole and the cell starts to divide. After this simplified cel
33、l cycle there are two daughter cells with equal genetic material. Almost every step of this cell cycle can be altered or stopped by chemical substances by following mechanisms:-Alkylation of genetic material - causes stable bonds in genome and the genetic material cannot be replicated (doubled). Thi
34、s mechanism is seen in drugs like cisplatin, busulfan and cyclophosphamide. -Enzyme inhibition - causes a stop of replicating enzyme activity. This mechanism is seen in drugs like lomustine and carmustine.-Mitosis protein inhibition - causes a stop of the polar genome movement (before division) and
35、division due to an alteration of intracellular movement proteins (microtubuli). This mechanism is seen in alcaloid drugs like vincristine, vinblastine and paclitaxel.-Component competition or false components for genome synthesis causes, after their implantation into genome (because of their similar
36、ity to normal genome components), production of false or irreplicable genetic material. This mechanism is seen in drugs like 5-fluorouracil and cytarabine. The mentioned mechanisms affect all fast growing cells in the body, fast growing normal cells included (bone marrow tissue, intestinal tissue, r
37、eproductive system, hair follicles, .), what may result in a couple of side effects: bone marrow (produces blood cells) supression can result in low white blood cell counts (lower resistance to infection), low red cell counts (fatigue, headaches, shortness of breath, anaemia) or/and low platelet cou
38、nts (problems to stop bleeding). The possible damaging or irritating effect on intestinal or digestive tissue may cause changes of taste, esophagitis, vomiting or loss of weight or appetite. The damaging effect on hair follicles may cause a loss of hair. There are also some other effects, like infer
39、tility, allergic reactions, lethargy and changes in nervous system, but most effects are only temporary, during the treatment period. Scientific research into cancer has made considerable progress in recent years. Death rates for some types of tumor such as for example, testicular carcinoma, Hodgkin
40、s disease, cancer of the colon, rectum, breast and prostate, as well as gynecological tumors, are constantly diminishing. Instead, death due to certain tumors, in particular pulmonary cancer in women, and lymphomas in both men and women continues to rise. However, despite the availability of numerou
41、s antineoplastic agents, this type of therapy cannot yet be considered satisfactory. In fact, serious toxicity, numerous side effects common to the majority of antitumor drugs, and the onset of resistance, all make it necessary to carry forward the research efforts to identify new compounds having h
42、igher efficacy and less toxicity. Numerous research works have been carried out on the cancerogenic process with the aim of identifying differences between cancer cells and normal ones. At the molecular level, differences have been found in the enzymatic activity, surface properties, gene constituti
43、on, and growth kinetics, merely to mention some of the intensely researched areas. Unfortunately, none of these changes is exclusive or common to all neoplastic pathologies. Many chemotherapeutic drugs act by interfering with the molecular processes in the cancer cells that are essential to the repl
44、ication process. However, any biological or chemical event that is essential for the fuctioning of a normal cell is a potential target for a cytotoxic drug. Thus, there is a need for new structures that flank the classical study of already know structures, which will be modified on the basis of biol
45、ogical activity analysis. The need for molecules with new pharmacological properties which are able to overcome cellular resistance problems has led researchers to develop methods based on the study of the targets three-dimensional structure. 1.2. Nucleoside and Nucleotide Analogues Nucleoside and n
46、ucleotide analogues have similar chemical structures to natural purinic andpirimidinic nucleosides, with modified base and/or sugar moiety. Several syntheses of nucleoside and nucleotide analogues have been developed in the last decades, which held to many molecules that proved to be effective in the antitumor and/or antiviral chemotherapy.1.2.1. Mechanism of Action The main mechanism of action of nucleoside analogues is the incorporation of the corresponding triphosphates into DNA where their interfere with DNA replication. The phosphorylation to the triphosphate form by cellula
