Azapyrimidine nucleosides: metabolism and inhibitory mechanisms

AZAPYRIMIDINE NUCLEOSIDES: METABOLISM AND INHIBITORY MECHANISMS ALOIS (2IHAK, JI~'I VESELCf and JAN SI~ODA Institute of Orgamc Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague 6, Czechoslovakia

INTRODUCTION

Various azaanalogs of pyrimidine components of nucleic acids can be used as a useful instrument to study different metabolic processes in the cell and may also be effective in human chemotherapy. This paper describes in chronological order the most important compounds synthesized in this Institute (Fig. 1), their inhibitory mechanisms, biological effects and the present knowledge concerning their action both as chemotherapeutics and biochemical tools.

RESULTS AND DISCUSSION 1. 6-Azauridine

6-Azauridine is produced from 6-azauracil by fermentation and in biological systems is converted by uridine kinase to 6-azauridine 5'monophosphate (1, 2). This intracellularly formed anomalous nucleotide interferes with the decarboxylation of orotidine Y-phosphate to uridine 5'monophosphate, thus inhibiting the de novo biosynthesis of pyrimidines (2-4). 6-Azauridine is not incorporated into animal RNA and DNA. The chemical introduction of 6-azauridine into the trinucleoside diphosphates (i.e. codon triplets) shows that these codons are unable to stimulate the binding of aminoacyl-tRNA to ribosomes (5). According to American (6) and Czechoslovak (7) stu&es, 6-azauridine produces in infant leukemia complete remissions less frequently than 6mercaptopurine but more frequent partial remissions were observed. In several cases of acute leukemia of children and adults, resistant to conventional cytostatics, 6-azauridine administration led to a complete or partial remission. After the administration of 6-azauridine to patients with mycosis fungoides (8--10), malignant chorioeplthelioma (11) and polycythemia vera (12)promising results were also achieved. The oral administration of 6-azauridine m the form of 2',3',5'-triacetate (azaribine) up to a total dose of about 100 g to patients with a viral eye 335

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FIG. 1. Structure of azapynmldlne nucleosldes

infection of herpes simplex virus etiology resulted in rapid healing (13). The patients selected for the therapy suffered from considerable stromal involvement resistant to other therapy, including topical 5-iodo-2'deoxyuridine. Azaribine reduced by 50% the mortality in nonvaccinated patients suffering from smallpox of the ordinary type (14). Azaribine has also been successfully used for the treatment of severe, recalcitrant psoriasis. The daily doses ranged from 50 to 400 mg/kg in more than 1,500 patients. After the approval of azaribine granted by the U. S. Food and Drug Administration, additional psoriatic patients have been treated in the United States. There was no evidence of resistance to azaribine when relapse occurred following withdrawal of the drug. However, in 1976 the U. S. Food and Drug Administration removed azaribine from the market because of the very low incidence of thromboembolic phenomena. The etiology of this side effect is difficult to assess since psoriasis ttself predisposes to occlusive vascular diseases (15). Recent experiments carrted out in animals shed a new light on the possible mechanism of the increased incidence of thromboembolic episodes in man receiving azaribme. It has been shown that the administration of this drug to rabbits by gastric lavage results in a rapid depletion of serum pyridoxal phosphate and the appearance of serum homocystme (16). When pyridoxine was administered concomitantly with azanbine no homocystlne was found. A typical hyperaminoaciduria in man after the administration of high doses of azaribine was described (17), cystine derivatives being the dominating compounds. In a study performed by Cornell and coworkers (private communication), 13 out of 26 psoriatic patients receiving azarlbine showed the presence of urinary homocystine, some of them at the level comparable to that found in congenital homocystinuria. The side effects assocmted with the administration of azanbine thus parallel the symptoms of the deficiency of vitamin Br. Supplementation of azanbine with pyridoxme may thus stimulate new studies of this drug in the management of psortasis.

AZAPYRIMIDINENUCLEOSIDES

337

2. 5-Azacytidine 5-Azacytidine (4-amino-1-/3-o-ribofuranosyl-s-triazin-2)lH)-one) is an analog of cytidine which was prepared (18) in Czechoslovakia by Pis kala and Sorm in 1963. In 1966 5-azacytidine was isolated from Streptoverticillium ladakamus (19). The low stabdity of the drug (20) prompted a study of its hydrolytic products and l-/3-o-ribofuranosyl-3-amidinourea and its formylated precursor were detected in aqueous solutions (21). 5-Azacytidine displays a pronounced b~oiogical activity due to a polyvalent inhibitory mechanism (22, 23). Using Escherichia coli it was originally observed (20) that the drug is deaminated and incorporated into nucleic acids. The incorporation of 5-azacytidine into RNA was later observed in intact mouse liver (24) as well as in the liver of AKR mice with lymphatic leukemia sensitive and resistant to the analog (25). The T~, values of the total RNA isolated from Ehrlich ascites cells were found to be decreased in relation to the amount of incorporated 5-azacytidine (26). In cultured L1210 cells the majority of phosphorylated 5-azacytidine exists as the 5'-triphosphate; a 10-20% portion of the phosphorylated compounds appears to be reduced to deoxyribonucleoside 5'-di and 5'-tnphosphates and subsequently incorporated into DNA (27). The phosphorylation of 5-azacytidine was studied in detail (28-30). Deoxycytidine kinase (EC 2.7.1.74) has no affinity for 5-azacytidine, and the drug has no effect on the phosphorylation of deoxycytidine (31). While the phosphorylatlon of cytidine and uridine is not substantially inhibited by 5azacytidine (28), cytidine markedly blocks the phosphorylation of 5azacytidine (32). However, using a 10-fold higher concentration of the analog (26), the incorporation of cytidme into RNA in Ehrlich ascites cells in vitro was reduced by 75%. A similar inhibition of uridine incorporation was observed in a cell culture line of a murine plasma cell tumor (33). 5-Azacytidine 5'triphosphate can be incorporated into RNA in a reaction catalyzed by RNA polymerase (EC 2.7.7.6) isolated from calf thymus (34). While CTP was a potent competitive inhibitor with respect to the anomalous nucleotide, the incorporation of UTP into RNA was not inhibited at all. These data suggest that the inhibition of RNA synthesis caused by 5-azacytidine is not produced by the inhibition of polymerase reaction. The combination of 5-azacytidine with pyrazofurin, an inhibitor of de novo pyrimidine synthesis causing a depletion of cellular pyrimidine nucleosides, results in a 3-4 times higher incorporation of 5-azacytidine in cultured Novikoff rat hepatoma and P388 mouse leukemia cells (35). Also 3deazauridine pretreatment, resulting in 80% reduction of intracellular cytidine 5'-triphosphate - - the natural feedback inhibitor of uridine kinase (EC 2.7.1.48) and the rate limiting enzyme in the phosphorylation of 5azacytidine, followed by 5-azacytidine demonstrated synergistic cell killing of L5178Y murine leukemia cells (36). Similar effect was seen in human leukemic ER24-L

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myeloblasts. Intracellular levels of 5-azacytidine almost doubled following 3deazauridine treatment (36). In a wild-type strain ofE. coli, a portion of 5-azacytidine is incorporated as 5-azauracil, while strains deficient in cytidine deaminase (EC 3.5.4.5) contain exclusively 5-azacytosine in their RNA (37). In analogy to the bacterial system (20), cytidine deaminase from human leukemic cells also deaminates 5azacytidine (38). The analog was found to be a competitive inhibitor of the deamination of cytidine as well as of cytosine arabinoside whereas tetrahydrouridine inhibited the deamination of 5-azacytidine. 5-Azacytidine blocks in the liver of mice and rats the utilization of orotic acid (39, 40). Simultaneously a higher urinary excretion of orotic acid and orotidine was observed. The administration of 5-azacytidine results (41) in the depression of both orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23). The block of decarboxylase activity is due to the inhibition of the enzyme by the newly formed 5azacytidine 5'-phosphate, while the activity of orotate phosphoribosyltransferase is inhibited by orotidine Y-phosphate accumulated because of its depressed decarboxylation (41). In various biological systems 5-azacyttdine affects the synthesis of DNA. Using L 1210 cells growing in the presence of 5-azacytidine a greater inhibition of thymidine incorporation into DNA than that of uridine into RNA was found (27). 5-Azacytidine strongly inhibits DNA synthesis during the earliest stages of sea urchin development (42). In phytohemagglutinin-stimulated horse lymphocytes the analog completely blocks the induction of DNA polymerase, and in a dose-dependent manner inhibits the incorporation of thymidine into DNA (43). The administration of 5-azacytidine shortly after partial hepatectomy results m a depression of thymidine incorporation into DNA in 24-hr regenerating rat liver (44, 45). Simultaneously the activities of thymidme kinase (EC 2.7.1.75) and thymidylate kinase (EC 2.7.4.9) induced by partial hepatectomy were completely blocked (Fig 2). However, 5-azacytidine administration before partial hepatectomy results in a dramatic increase of mitotic activity in 24-hr regenerating livers (46). In this case the increase of thymidine incorporation into liver DNA was paralleled by increased activity of thymidine and thymidylate kinases. The administration of 5-azacytidine to rats results in a rapid degradation of liver polyribosomes and an accumulation of a fraction containing monosomes and disomes (47). The analog also affects the formation of heavier polyribosomal aggregates which are formed in the liver after dietary Ltryptophan (48). The loss of heavier polynbosomes in the liver after 5azacytidine is dose-dependent and is maximal 4-8 hr after administration of the drug (49). At the time of polyribosome breakdown a decreased binding of leucine-3H charged transfer RNA to polyribosomes was observed (50).

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FIG 2. Changes m the synthesis of DNA m regenerating rat hvers following 5-azacytldme (25 mg/kg). A" Time-course of thymidine kinase enhancement in control (dotted) and 5azacytidine-treated regenerating rat hvers The analog was injected immediately after partial hepatectomy. B: Thymldine 5'-tnphosphate formed m vttro from thymldme and ATP using the cell-free liver extracts from 24-hr regenerating hvers or rats treated w~th 5-azacytldme at various ttme intervals before partial hepatectomy; dotted, synthesis of TTP in control 24-hr regeneratmg hvers. C. DNA synthesis m regenerating livers of control (dotted) and 5-azacyudme-treated rats recewmg the drug immediately after partial hepatectomy. D The enhancement of DNA synthesis and of mitotic actwlty C~, ~<) m 24-hr regenerating hvers of rats exposed to the action of 5azacyUdlne 48, 36 and 24 hr before partial hepatectomy; dotted, DNA synthesis m control 20-hr regenerating rat hvers. According to (46) and (47), mo&fied. F o r the stability o f p o l y r i b o s o m e s a c o n t i n u o u s synthesis o f R N A is essential. 5 - A z a c y t i d i n e causes a b r e a k d o w n of p o l y n b o s o m e s a n d an a b e r r a n t processing o f r i b o s o m a l R N A p r e c u r s o r s (51). Using N o v i k o f f h e p a t o m a cells in culture it was o b s e r v e d (Fig. 3) t h a t 5-azacytidine c o m p l e t e l y i n h i b i t e d the f o r m a t i o n o f the m a t u r e 28S a n d 18S r i b o s o m a l R N A (52, 53). 45S a n d 32S r i b o s o m a l R N A p r e c u r s o r s isolated f r o m 5-azacytidinet r e a t e d N o v i k o f f h e p a t o m a cells d i s p l a y e d different e l e c t r o p h o r e t i c mobilities a n d r e d u c t i o n in the rate a n d degree o f m e t h y l a t i o n (54). H o w e v e r , the s t r u c t u r a l changes o f the r i b o s o m a l p r e c u r s o r R N A s a n d the s u b s e q u e n t i n h i b i t i o n o f their processing caused b y 5-azacytidine were n o t consequences o f altered methylat~on since o t h e r analogs strongly inhibiting m a t u r a t i o n of r i b o s o m a l R N A were n o t f o u n d to affect r i b o s o m a l R N A m e t h y l a t i o n (54). The a c c e p t o r activity o f transfer R N A s isolated f r o m the liver o f mice after i n v i v o t r e a t m e n t with 5 - a z a c y t i d m e was f o u n d to be significantly decreased (55). Also transfer R N A isolated f r o m 5 - a z a c y t t d i n e - t r e a t e d H e L a cells of h a m s t e r f i b r o s a r c o m a cells was less active t h a n n o r m a l f i b r o s a r c o m a transfer R N A (56). A f t e r e x p o s u r e to 5-azacytidine, the i n t r a c e l l u l a r methylat~on of 4 - 5 S R N A in H e L a cells was r a p i d l y d i m i n i s h e d (57). A l s o , in the liver a m a r k e d r e d u c t i o n m the 5-methylcytidine c o n t e n t o f t r a n s f e r R N A was

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F I G 3. Inhibition of processing of ribosomal R N A maturation by 5-azacytldme m cultured Novlkoff hepatoma cells Cells were treated with 10-4 M cytidme or 10-4 M 5-azacyUdme m the presence of guanosine-3H. R N A was isolated after 2 hr oflabelhng and analyzed by acrylamldeagarose gel electrophoresls According to (53); modified

observed (58). It was proposed that 5-azacytidine or its metabolite selectively inhibit 5-methylcytosine methyltransferase (58). A large body of recent evidence supports the role for DNA methylatton in the control of gene expression in eukaryotes (59). 5-Azacytidine inhibits the methylation of cytosine residue not only m RNA but also in a newly replicated DNA (60). It was suggested that incorporation of 5-azacytidine into DNA inhibits the progress of DNA methyltransferase (EC 2.1.1.37) along the duplex by the formation of a tight-binding complex (61). 5-Azacytidineinduced changes in gene expression have been documented in a number of different systems (62-69) and led to a proposal of a cause and effect relationship between DNA methylation and cellular differentiation. The administration of 5-azacytidine to rats at non-toxic doses markedly affects different induced liver enzymes (70, 71). The available data indicate that the drug interferes with mechanisms operating at both the transcriptional as well as post-translational levels. 5-Azacytidine administration in vivo also results in an increase of liver uridine kmase activity (72, 73). In Webb's laboratory, two different forms ofuridine kinase were observed in normal and neoplastic cells which were independently modified following 5-azacytidine treatment (74, 75). 5-Azacytidine causes a variety of biological effects (22, 23). Of these, the cytostatlc action is the important one. The first report on the antileukem~c activity of 5-azacytldine appeared in 1964 (76). The treatment schedule dependency of 5-azacytidine has been investtgated by Venditti (77). It was concluded that the drug does not exhibit schedule dependency m the L 1210

AZAPYRIMIDINENUCLEOSIDES

341

system; daily and intermittent treatments were equally effective and superior to treatment on day one only. This observation was later confirmed in AKR leukemia (78, 79). The first clinical trial of 5-azacytidine in 19 children with acute lymphatic leukemia showed that the drug was active in inducing partial remissions (80). In a clinical report that appeared 2 years later (81) 37 children with acute leukemia were treated with 5-azacytidine in 5-day courses given every 2 weeks; 6 out of 14 children with acute myelogenous leukemia achieved an MI marrow, and 5 of these developed a complete remission lasting 2-8 months. A confirmatory finding was reported using a group of 45 previously treated patients (82). Further trials were designed to test the efficacy of 5-azacytidine when used alone in the treatment of acute leukemia, after patients had become refractory to chemotherapy with other cytostatics. The best results were achieved in patients with acute myelogenous leukemia, complete remission being achieved in 39% of these individuals (83). The results obtained with 5-azacytidine in solid tumors have been rather disappointing although some activity has been observed in embryonal carcinoma of testis, Hodgkin's disease, lymphoma and breast cancer. However, the beneficial effects of the drug have already been shown in acute myelogenous leukemia (20-39% of complete remissions), and, to a lesser degree, in acute lymphatic leukemia. The major obstacle to the use of 5azacytidine in the clinic is the toxicity of the drug and its low stability. A slow infusion lasting 18-24hr helps substantially in overcoming nausea and vomiting. During a 120-hr continuous intravenous infusion repeated at 28day intervals the toxicity has been stated, nevertheless, to be safe and manageable (84).

3. a- and [3-Anomers of 5-Aza-2'-Deoxycyttdine 1-fl-D-5-Aza-2'-deoxycytidine is a nucleoside analog (85) whose antileukemic action can be reversed by deoxycytidine (86). The biologically active metabolite is the corresponding nucleotide since cells deficient in deoxycytidine kinase are resistant to this compound (87). In AKR mice with lymphatic leukemia (Table 1) the drug is preferentially incorporated into the spleen and thymus DNA (88) and markedly depresses DNA synthesis in the lymphatic system (89). In mammalian tissues, 5-aza-2'-deoxycytidine is deaminated (90) and readily phosphorylated by deoxycytidine klnase (91,92). It has been proposed that the lethal action of 5-aza-2'-deoxycytidine is due to its incorporation into DNA (93) possibly producing an interference m DNA function on account of ~ts chemical instability. Recent reports indicate that the drug incorporation into DNA can induce cellular differentiation by producing hypomethylation of the cytosine residue in DNA (60, 94--96). EnzymaUcally prepared 5-aza-2'-deoxycytidine 5'-triphosphate was incor-

342

ALOIS (21HAK, et al. TABLE 1. INCORPORATION OF THYMIDINE-3H AND 5-AZA-2 DEOXYCYTIDINE-3H IN VARIOUS TISSUES OF AKR MICE WITH LYMPHATIC LEUKEMIA* Incorporation

Tissue Lwer K~dney Spleen Thymus

Thymi&ne-3H d p m / m g tissue ___S E. 640 60 501 158

_+ 64 + 5 -+ 63 + 21

5-Aza-2-deoxycytldme-3H d p m / m g tissue + S.E. 753 _+ 93 309 + 33 6 120 _+ 510 2 720 + 304

*Groups of 5 male AKR mice (25 g) 7 days after s.c. transplantation with 2 x 107 leukemic cells recewed i.p. 2 hr before killing thymldine-3H (50 #C1/0.2 #mol per animal) or 5-aza-2-deoxycytldme-3H (125 vC1/0.2 #mol per ammal).

porated into DNA in the presence of DNA polymerase (EC 2.7.7.7), and its incorporation into hemimethylated DNA resulted in a significant inhibition of DNA methylase (97). In another experiment, the DNA of L1210 leukemia cells was isolated from 5-aza-2'-deoxycytidine-treated mice given radioactive uridine. Analysis of the labelled pyrimidine bases showed that the drug produced a dose-dependent reduction in 5-methylcytosine content of the DNA (98). The stability profile of 5-aza-2'-deoxycytidine in aqueous buffer solutions shows that the drug is most stable at neutral pH and low temperature. At higher pH the triazine ring is opened (Fig. 4) giving rise to N-(formylamidino)N-deoxy-/3-D-ribofuranosylurea, which by the irreversible loss of the formyl group forms the N-amidino derivative (99, 100). It has been reported (101) that also o~-anomer of 5-aza-2'-deoxycytidine is effective in preventing the growth of LI210 cells in wtro. The c~-anomer, when kept at 23°C for 4 days, inhibtted the uptake of deoxycytidine by L1210 cells in vitro as well as the phosphorylation of deoxycytidine by the cell-free extract. However, these effects could not be obtained with the fresh solution of the drug. Recently it has been proved that this action is due (Fig. 4) to the conversion of inactive t~anomer to active/3-anomer (102). 5-Aza-2'-deoxycytldine is a potent antileukemic agent m mice (86, 103) and acts also in combination with different cytostatic agents. With 3-deazauridine it acted synergistically since 3-deazauridine reduces the intracellular pool of cytosine nucleotides (104) stimulating thus the incorporation of 5-aza-2'deoxycytidine into DNA (105). The combinations of 5-aza-2'-deoxycytidme with 2'-deoxythioguanosine (106) or with cis-dichlorodiammoneplatinum (107) had in mlce with L1210 leukemia remarkable antineoplastlc action producing long-term survivors and a clear synergistic effect. The combination of the drug with thymidine m HL-60 human promyelocytic leukemia cells led

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to a 3-fold increase in DNA incorporation of 5-aza-2'-deoxycytidine to compare with control, producing a dose and schedule-dependent synergism between the two compounds (108). A climcal report on a Phase I study with 5-aza-2'-deoxycytidine performed in children with acute leukemia resistant to conventional chemotherapy is available (109). At doses of 36-80 mg/kg administered as i.v. infusion two of nine patients obtained M t marrow and one M2 marrow. However, drug resistance developed relatively rapidly. In rats the drug strongly affects the ability of the immune system to synthesize IgG antibodies without affecting IgM antibody formation (110). As already mentioned, 5-azacytosine incorporated into DNA inhibits DNA methylation and affects gene expression and differentiation (59, 61). DNAs containing 5-azacytosine are potent inhibitors of DNA-cytosine methyltransferase. Using mouse-human hybrid clone 37-26R-D with structurally normal inactive X chromosome and deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) it has been shown (94) that this enzyme is induced by 5-aza-2'-deoxycytidine. In Friend erythroleukemia cells the analog decreases DNA methyltransferase activity and induces the synthesis of undermethylated DNA. The differentiation of erythroleukemic cells occurred in less than 15% of the population (95). However, the direct relation between the undermethylation of the DNA produced by the drug and the expression of the gene acUvlty ~s not yet clear. The differentiative changes of the cell and the expresmon of the hitherto repressed enzyme due to the presence of 5-

344

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azacytosine in cellular DNA may also be due to the altered interactions of proteins with the substituted DNA (96).

4. 5-Azacytosine Arabinoside

The first reported synthesis of 1-/3-o-arabinofuranosyl-5-azacytosine was accomplished by Beisler and coworkers (111, 112) who have also established its antileukemic activity. These findings prompted us to reinvestigate our earlier results concerning the synthesis of this drug (113) which we have originally carried out in 1968. It was found at that time that the drug had only negligible bacteriostatic activity and consequently no further study of this analog was attempted. The behavior of 5-azacytosine arabinoside m aqueous solution ~s characterized by an increase in optical density due to the formation of the ring-opened N-formyl hydrolysis product (114) generally observed in sym. triazines (21, 115). The antileukemic effect of 5-azacytosine arabinoside in L1210 mouse leukemia is nearly completely reversed by deoxycytidine (114). From the measurement of the apparent Michaelis constants for the phosphorylatlon of 5-azacytosine arabinoside by the cell-free extract from L 1210 cells it is evident that the affinity of deoxycytidine kinase, which is regarded as a rate limiting enzyme in the phosphorylation of deoxycytidine and of its analogs (116), is considerably lower towards this compound than towards 5-aza-2'deoxycytidine or cytosine arabinoside (114). Using murine LI210 leukemia, Beisler and coworkers (112) evaluated 5azacytosine arabinoside for antitumor properties in parallel determinations with 5-azacytidine and cytosine arabinoside. Although higher dose levels were necessary, 5-azacytosine arabinoside demonstrated a reproducibly greater efficacy than that shown by 5-azacytidine or cytosine arabinoside. Its antitumor activity exhibited an apparent schedule dependency in the L1210 system similar to cytosine arabinoside. However, we were not able to confirm the latter finding (Table 2) since in L1210 leukemia 5-azacytosme arabinoslde was effective regardless whether the mice received a single total dose or five daily doses of the drug (114). The clinical trials with 5-azacytosine arabinoslde are in progress in Czechoslovakia. 5. Substituted Derivatives of 5-Azacytidine

6-Amino derivatives of 5-azacytidine and 5-aza-2'-deoxycytidine (117) differ considerably in their inhibitory mechanism from unsubstituted molecules. While 6-amino-5-azacytosine (ammeline) and its derivatives (they are used as herbicides - - atrazin, slmazin, etc.) have only low antibacterial activity, the corresponding riboslde and 2'-deoxyriboside effectively inhibit the growth of E. coli (I 18). The growth-inhibitory effect is displayed by/3-

345

AZAPYRIMIDINE NUCLEOSIDES

T A B L E 2. C O M P A R I S O N O F THE S C H E D U L E - D E P E N D E N T A N T I L E U K E M I C E F F E C T OF CYTOSINE A R A B I N O S I D E (Ara C), 5-AZACYTOSINE A R A B I N O S I D E (Ara-AzC) A N D 5 - A Z A - 2 - D E O X Y C Y T I D I N E (5-AzCdR) IN D B A / 2 MICE W I T H L1210 LEUKEMIA* Daily dose Number (mg/kg) of doses (mg/kg)

Drug None Ara C Ara 5-AzC Ara 5 - A z C 5-AzCdR 5-AzCdR

0 200 200 40 10 2

Total dose Average life span (days +- S E ) (%)

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11.4 + 0.6 12.8 + 0.8 24.3 + 5 9 22.7 + 7 2 22 3 +- 7 4 10.8 + 0,6

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*Each group included 10 male mice (25 g) Therapy was given on day 4 following i.p inoculation of 104 leukemic ceils or in case of repeated administrations it started on day 1.

anomers; tx-anomers and 6-amino-1-/3-D-glucopyranosyl-5-azacytosine are without antibacterial activity. The growth-inhibitory effect of 6-amino-5-azacytidine and its 2'-deoxy compartment can be partially reversed by high doses of uridine and cytidine and almost completely by purines (118). Even low levels of adenine, guanine and inosme effectively prevent the inhibition (Fig. 5). Accordingly, 6-amino derivatives of 5-azacytidine and 5-aza-2'-deoxycytidme did not affect the activity of uridine, deoxycytidine and thymidine kinases and depressed the activity of adenosine kmase (EC 2,7.1.20). The measurements of ~H-NMR spectra of 6-amino-5-azacytidine revealed a marked preference for the gauche-gauche rotamer about C4-C5 bond and a i

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346

ALOIS ~?IH,~K, et al

preponderance of C2-endo conformation for the rlbose ring (119). In this respect 6-amino-5-azacytidine resembles adenosine but differs from uridine and other pyrimidine nucleosides. The physico-chemical measurements clearly indicate a conformational resemblance of 6-ammo-5-azapyrimidine nucleosides to purine nucleosides. Therefore, the 6-amino substituted 5azapyrimidine analogs can be considered as purine antagonists. N-Substituted derivatives of 5-azacytidine represent another group of compounds which similarly to 5-azacytidine (120) affect gastric secretion. Especially N4-methyl and N4,N4-dimethyl-5-azacytidine block (121)gastric acidity, the extent of hemorrhage and the number and size of gastric defects (Table 3). Some of the N4-alkyl substituted derivatives of 5-azacytidine result also in the enhancement of orotic acid incorporation into liver RNA and block the evacuation of the stomach. T A B L E 3. E F F E C T O F 5 - A Z A C Y T I D I N E A N D S T R U C T U R E R E L A T E D C O M P O U N D S ON G A S T R I C SECRETION A N D THE D E V E L O P M E N T O F E X P E R I M E N T A L G A S T R I C ULCERS IN RATS* Drug (15 m g / k g ) None 5-Azacytldme N4-Methyl-5-azacytidine N4,N4-Dlmethyl-5-azacytldine N4-n-Butyl-5-azacytldme 5-Azacytosme 1-Methyl-5-azacytosme

Secretion (ml + S.E.) 17.4+ 1.2 3.1 + 0.8 10.2 + 1.3 15.1 __. 1.4 18.9 _+ 2.3 15.4 __. 1.1 14.0 + 2.6

Acidity Gastric defects (mequiv.H+/ml + S.E.) (points + S.E. (%)) 1.28 + 0.14 0.10 + 0.01 0.63 + 0.09 1.14 + 0.14 1.31 _+ 0 27 0.66 + 0.17 0.60 + 0.08

2 7 . 4 + 4.1 (100) 3.8 _+ 1.7 (13.8) 8.9 + 2. 6 (32.5) 10. 4 + 4.3 (37.9) 8.3 + 1.5 (30.3) 5.4 + 1.1 (19.7) 8 2 + 1 6 (29.9)

*Groups of 10 male Wistar rats (200 g) starved 18 hr were subjected to pylonc ligatlon and lmmed,ately reJected Lp. with the drug The ammals were killed 22 hr later.

Since the biological effect and toxicity of 5-azacytidine are associated with its incorporation into RNA or DNA, other interesting agents are 5azacytosine and 1-methyl-5-azacytosine. Both drugs in mammalian systems are not incorporated into nucleic acids and also block gastric secretion and the development of gastric ulcers (123). The derivatives of 5-azacytosine depressed in rats the development of acute pancreatitis mediated by interstitial administration of 7.5% natrium cholate into the pancreas in vivo. They affected the amount of abdominal fluid and 6 hr after their administration pathological changes evaluated in the pancreas and abdominal cavity were significantly decreased (123). CONCLUSION

The inhibitory mechanism of pyrimidine azaanalogs with the apparent exception of 6-azauridine (4) is widely polyvalent and individual drugs affect

347

AZAPYRIMIDINE NUCLEOSIDES

simultaneously different cellular processes (22, 124). At present, the interference of 5-azacytosine residue incorporated into DNA with DNA methylation is regarded as an important inhibitory site, at least in case of 5aza-2'-deoxycytidine (59, 61). During the study of azapyrimidine nucleosides several apparently paradoxical findings have appeared: 1. The development of resistance of leukemic cells towards 5-azacytidine is paralleled by the depression of uridine kinase (87); the same drug results also in the enhancement of liver uridine kinase activity (72, 74). 2. 5-Azacytidine blocks in rats the formation of 7S antibodies (125); the same drug (Fig. 6) using the same antigen results under changed schedule of treatment in the enhancement of antibody production (126). In this connection the finding that 5-azacytidine is able to establish constitutive interleukin 2-producing clones of the EL-4 thymoma is of special interest (127). 3. A similar stimulatory effect has been observed during the study of the effect of 5-azacytidine on the activity of induced liver enzymes, tyrosine aminotransferase (EC 2.6.1.5) being a typical example of an overinduced enzyme (71, 128). To explain the inhibitory mechanism underlying a number of biological effects elicited by 5-azacytidine, 5-aza-2'-deoxycytidine and their derivatives requires better understanding of basic biochemical and regulatory processes operating in the cell. The changes in DNA methylat~on caused by the presence of 5-azacytosine residue m DNA seem to be responsible for some of these effects. Notwithstanding recent theoretical as well as experimental limitations 6-azauridine and 5-azacytidine have been used with certain success in human clinic.

N o~

o

o

<[

5

10 5 D0ys after primary it~rrlurllzatl0r~

10

FIG. 6. Changes by 5-azacytldme of 7S antibody production in rats immumzed wtth sheep red blood cells 5-Azacytldme (16 mg/kg) was administered simultaneously (1), 48 hr (2) or 96 hr (3) after lmmumzation. According to (126), modified.

348

ALOIS ~21HAK, et al

SUMMARY Triazine nucleosides represent highly acttve c o m p o u n d s affecting different cellular processes. W h i l e 6-azauridine displays a r a t h e r selective i n h i b i t o r y effect, biological a c t i o n o f 5-azacytidine reflects the p o l y v a l e n t i n h i b i t o r y m e c h a n i s m o f the d r u g (interaction with p y r i m i d i n e synthesis de novo, i n c o r p o r a t i o n into R N A a n d D N A , depressed m a t u r a t i o n o f r i b o s o m a l R N A , i n h i b i t i o n o f R N A a n d D N A m e t h y l a t i o n , etc.) a n d the a n a l o g dtsplays p r o n o u n c e d cytostatic a n d i m m u n o s u p p r e s s t v e activity. 5 - A z a - 2 ' - d e o x y cytidine a c t i o n is directed a g a i n s t D N A synthesis s i m i l a r to that o f 5a z a c y t o s i n e a r a b i n o s i d e . N4-Substituted derivatives o f 5-azacytidine affect gastric secretion a n d t o g e t h e r with 5-azacytosine a n d 5-azacytidine represent a n e w type o f drugs with a n t i u l c e r activity. 6 - A m i n o - 5 - a z a c y t o s i n e nucleosides interfere with the m e t a b o l i s m o f p u r i n e s r a t h e r t h a n p y r i m i d i n e s as evidenced b y the c h a r a c t e r o f their i n h i b i t o r y m e c h a n i s m a n d m e a s u r e m e n t of conformation. 6 - A z a u r i d i n e (as 2 ' , Y , 5 ' - t r i a c e t a t e ) a n d 5-azacytidine were used with certain success in h u m a n c h e m o t h e r a p y , the first one as a d r u g affecting recalcitrant psoriasis, the s e c o n d one f o r the t r e a t m e n t o f different f o r m s o f leukemia. The i n h i b i t o r y m e c h a n i s m s o f i n d i v i d u a l a z a p y r i m i d i n e nucleosides are discussed in r e l a t i o n to their k n o w n b i o l o g i c a l effects.

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