Inosine 5'monophosphate dehydrogenase (IMPDH; EC 1：肌苷5单核苷酸脱氢酶（IMPDH；EC 1

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 111126abstractnucleotide coenzymes participate in essential enzyme-catalyzed redox reactions and play a fundamental role in cellular

11、 metabolic 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 an

12、alogues 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 n

13、ad-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 proliferatio

14、n. it was found 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 anticance

15、r chemotherapy.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

16、 tad, sad and/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 ne

17、w dinucleotide 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 recomb

18、inant human impdh 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

19、, as compared 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 si

20、mulations. the 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

21、nad site of both 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

22、cells resistant 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

23、 research was 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.

24、 on the basis 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 modificatio

25、ns (2-me-mzr 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 t

26、o be converted 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 investigat

27、ed the possibility 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

28、 agents that 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

29、 the most active 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

30、 group, the introduction 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

31、deplete both 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 cel

32、l cycle. a simplified 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 s

33、implified cell 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 (

34、doubled). this 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 d

35、ivision) and 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

36、their similarity 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, intesti

37、nal tissue, reproductive 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

38、 platelet counts (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 effect

39、s, like infertility, 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 carci

40、noma, hodgkins 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 availabili

41、ty of numerous 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 compo

42、unds having higher 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, ge

43、ne constitution, 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 essentia

44、l to the replication 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

45、basis of biological 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 nuc

46、leoside and nucleotide 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 ef

47、fective 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 cellular

48、 or viral enzymes is therefore required for the activation of nucleoside analogues. the first step of phosphorylation, catalyzed by nucleoside kinases, is regarded as being rate-limiting for most nucleoside analogues. the triphosphate derivatives of nucleoside analogues can act in the biosynthesis o

49、f nucleic acid in two ways. they can determine cell mutations or chain terminations acting as competitive substrates for cell or viral polymerases and adding to dna or rna. alternatively, they can behave as competitive inhibitors of cell and viral polymerases blocking the addition of endogenous natu

50、ral nucleotides in the nucleic acid extending chain.1.2.2. purine nucleotides biosynthesisboth the salvage and de novo synthesis pathways of purine and pyrimidine biosynthesis are regulated by different enzymes. the activated sugar, 5-phospho-d-ribosyl-1-pyrophosphate (prpp), as precursor of nucleot

51、ides biosynthesis, is generated by the action of prpp synthetase. ribose-5-phosphate + atp -> prpp + amp synthesis of the purine nucleotides begins with prpp and leads to the first fully formed nucleotide, inosine 5'-monophosphate (imp). the synthesis of imp is regulated by the prpp synthetas

52、e and the amidophosphoribosyl transferase through a series of ten reactions utilizing atp, tetrahydrofolate (thf) derivatives, glutamine, glycine and aspartate. imp represents a branch point for purine biosynthesis, because it can be converted into either amp or gmp through two distinct reaction pat

53、hways (figure 1). the amp nucleotide is synthesized starting from the imp through the intermediate adenylosuccinate; the other methabolic pathway of the purinic nucleotides, instead, leads to the conversion of the imp in xmp by the enzyme 5-imp dehydrogenase (impdh). then, the gmp synthase converts

54、the xmp in gmp. the pathway leading to amp requires energy in the form of gtp; that leading to gmp requires energy in the form of atp. the utilization of gtp in the pathway to amp synthesis allows the cell to control the proportions of amp and gmp to near equivalence. the accumulation of excess gtp

55、will lead to accelerated amp synthesis from imp instead, at the expense of gmp synthesis. conversely, since the conversion of imp to gmp requires atp, the accumulation of excess atp leads to accelerated synthesis of gmp over that of amp. fig. 1. biosynthesis of the purinic nucleotides amp and gmp.&#

56、160; the essential rate limiting steps in purine biosynthesis occur at the first two steps of the pathway (figure 2). the synthesis of prpp by prpp synthetase is feed-back inhibited bypurine-5'-nucleotides (predominantly amp and gmp). combinatorial effects of those two nucleotides are greatest, e.g., inhibition is maximal when the correct concentration of both adenine and guanine nucleotides is achieved. fig. 2. purinic nucleotides biosynthesis regulation. since the gtp, in addition to its role as a substrate on the rna polymerisation, takes part on