Synthesis and Biological Properties of 9-(2, 4-Dihydroxybutyl) Adenine and Guanine: New Analogues Of 9-(2, 3-Dihydroxyprqpyl)adenine (Dhpa) and 9-(2-Hydroxyethoxymethyl)guanine (Acyclovir)

Abstract

New analogues of antiviral agents 9-(2, 3-dihy-droxyproply) adenine (DHPA, 1a.) and 9-(2-hydroxyethoxymethyl) guanine (acyclovir, Ib) - compounds Ic and Id were prepared and their biological activity was investigated. Racemic 1, 2, 4-butanetriol (2) was converted to the corresponding benzylidene derivative (3a) by acetalation with benzalde-hyde and triethyl orthoformate. Acetal 3a and p-toluene- sul-fonyl chloride in pyridine gave the corresponding p-toluenes fonate 3b. Alkylation of adenine 5a via sodium salt of 5a with 3b in dimethylformamide or in the presence of tetra-n-butylammonium fluoride in tetrahydrofuran gave intermediate 6a. Reaction of 2-amino-6-chloropurine (5b) with 3b effected by K₂CO₃ in dimethylsulfoxide gave compound 6b and a smaller amount of 7-alkylated proauct 7. A similar transformation catalyzed by tetra-n-butylammonium fluoride afforded only intermediate 5b. Acid-catalyzed de-protection (hydrolysis) of 6b and 6a gave the title compounds Ic and Id. The S-enantiomer of Ic was deaminated with adenosine deaminase. Our results argue against the presence of a methyl group-binding site of adenosine deaminase. Compounds Ic and Id exhibited little or no activity in antiviral assays with several DNA and RNA viruses.     

Synthesis of 9-(2-deoxy-beta-D-ribofuranosyl)purine-2-thione

A synthesis of 9-(2-deoxy-beta-D-ribofuranosyl)purine-2-thione was performed by desulfurization of 2'-deoxy-6-thioguanine to give 2-amino-9-(2-deoxy-beta-D-ribofuranosyl)purine, diazotization with chloride replacement to give 2-chloro-9-(2-deoxy-beta-D-ribofuranosyl)purine, and the replacement of chloride with sulfur using thiolacetic acid and deacetylation.     

A new synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (AraF-G)

Interesting and very promising antisense properties of 2'-deoxy-2'-fluoroarabinonucleic acids ((a) Wilds, C.J.; Damha, M.J. 2'-Deoxy-2'-fluoroarabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies. Nucl. Acids Res. 2000, 28, 3625-3635; (b) Viazovkina, E.; Mangos, M.; Elzagheid, M.I.; Damha, M.J. Current Protocols in Nucleic Acid Chemistry 2002, 4.15.1-4.15.21) (2'F-ANA) has encouraged our research group to optimize the synthetic procedures for 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides (araF-N). The synthesis of araF-U, araF-T, araF-A and araF-C is straightforward, (Tann, C.H.; Brodfuehrer, P.R.; Brundidge, S.P.; Sapino, C., Jr. Howell H.G. Fluorocarbohydrates in synthesis. An efficient synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (beta-FIAU) and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (beta-FMAU). J. Org. Chem. 1985, 50, 3644-3647; Howell, H.G.; Brodfuehrer, P.R.; Brundidge, S.P.; Benigni, D.A.; Sapino, C., Jr. Antiviral nucleosides. A stereospecific, total synthesis of 2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl nucleosides. J. Org. Chem. 1988, 53, 85-88; Maruyama, T.; Takamatsu, S.; Kozai, S.; Satoh, Y.; Izana, K. Synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine bearing a selectively removable protecting group. Chem. Pharm. Bull. 1999, 47, 966-970) however, the synthesis of the guanine analogue is more complicated and affords poor to moderate yields of araF-G (4) ((a) Elzagheid, M.I.; Viazovkina, E.; Masad, M.J. Synthesis of protected 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. Synthesis of 2'-fluoroarabino nucleoside phosphoramidites and their use in the synthesis of 2'F-ANA. Current Protocols in Nucleic Acid Chemistry 2002, 1.7.1-1.7.19; (b) Tennila, T.; Azhayeva, E.; Vepsalainen, J.; Laatikainen, R.; Azhayev, A.; Mikhailopulo, I. Oligonucleotides containing 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine and -guanine: synthesis, hybridization and antisense properties. Nucleosides, Nucleotides and Nucl. Acids 2000, 19, 1861-1884). Here we describe an efficient synthesis of araF-G (4) that involves coupling of 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (1) with 2-chlorohypoxanthine (2) to afford 2-chloro-beta-araF-I (3) in 52% yield. Nucleoside (3) was transformed into araF-G (4) by treatment with methanolic ammonia (150 degrees C, 6 h) in 67% yield.     

Preclinical antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)adenine (Cl-F-ara-A)

Cl-F-ara-A, an analog of fludarabine, was evaluated against a spectrum of tumor systems in culture and in mice. The compound exhibited significant cytotoxicity against a variety of human tumor cell lines. More importantly, the compound showed selectivity in vivo, with excellent activity being demonstrated against human colon and renal tumors. Human nonsmall cell lung and prostate tumors were also sensitive in vivo to the compound, albeit at a reduced level.     

Synthesis of 9-(2-Hydroxyethoxymethyl)guanine (Acyclovir) from Guanosine

Abstract

A convenient synthesis of 9-(2-hydroxyethoxymethyl)guanine (acyclovir) from guanosine by chemical transpurination was developed. The isomerization of the 7-isomer to the desired 9-isomer and the purification of the 9-isomer was achieved simply by concentration, heating and further crystallization.     

Nitric oxide (NO) inhibits prostaglandin E2 9-ketoreductase (9-KPR) activity in human fetal membranes

Nitric oxide (NO) synthesized by fetal membranes may act either directly inhibiting myometrium contractility or indirectly interacting with tocolytic agents as prostaglandins (PGs). Here we examined if NO could modulate prostaglandin E(2) 9-ketoreductase (9-KPR) activity in human fetal membranes (HFM). 9-KPR is the enzyme that converts PGE(2) into PGF(2alpha), the main PGs known to induce uterine contractility at term. Chorioamnion explants obtained from elective caesareans were incubated with aminoguanidine (AG), an iNOS inhibitor, or NOC-18, a NO donor. NOC-18 (2mM) increased PGE(2) production and diminished PGF(2alpha) synthesis in HFM. AG presented the opposite effect. When we evaluated the activity of 9-KPR by the conversion of [(3)H]-PGE(2) into [(3)H]-PGF(2alpha) and 13,14-dihidro-15-keto prostaglandin F(2alpha) (the PGF(2alpha) metabolite), we found that NOC-18 inhibited 9-KPR activity. Interestingly, AG did not elicit any effect on 9-KPR but l-NAME, a non-selective NOS inhibitor, significantly increased its activity. Our data suggests that exogenous NO inhibits 9-KPR activity in HFM, thus modulating the synthesis of important labor mediators as PGF(2alpha).     

Cytogenetic effects of 9-(2-hydroxyethoxymethyl)guanine

Mutageneous activity of a highly-efficient and low toxic antiherpes drug 9-(2-hydroxyethoxymethyl) guanine (Acyclovir) was first shown. The method for the detection of micronuclear polychromatic erythrocytes in mice bone marrow was used. A dose-dependent cytogenetic effect was noted in the dose range of acyclovir of 5-600 mg/kg. The results obtained permitted designation of this preparation to class "B" in accordance with the classification of potential mutagenic danger, adopted for the ready-made drug forms in the USSR. According to the type of cytotoxic effect on hemopoiesis, acyclovir may be referred to antimetabolites of nucleic acids.     

Esophageal cancer proliferation is mediated by cytochrome P450 2C9 (CYP2C9)

Cytochrome P450 epoxygenases (CYP450) have been recently shown to promote malignant progression. Here we investigated the mRNA and protein expression and potential clinical relevance of CYP2C9 in esophageal cancer. Highest expression was detected in esophageal adenocarcinoma (EAC; n=78) and adjacent esophageal mucosa (NEM; n=79). Levels of CYP2C9 in EAC and NEM were significantly higher compared to esophageal squamous cell carcinoma (ESCC; n=105). Early tumor stages and well-differentiated tumors showed a significantly higher CYP2C9 expression compared to progressed tumors. Moreover, CYP2C9 expression was correlated to high Ki-67 labeling indices in EAC and Ki-67 positive tumor cells in EAC and ESCC. Selective inhibition of CYP2C9 decreased tumor cell proliferation (KYSE30, PT1590 and OE19) in vitro, which was abolished by 11,12-epoxyeicosatrienoic acid (11,12-EET). Cell-cycle analysis using FACS revealed that inhibition of CYP2C9 leads to a G0/G1 phase cell-cycle arrest. CYP2C9 seems to be relevant for early esophageal cancer development by promoting tumor cell proliferation. Pharmacological inhibition of CYP2C9 might contribute to a more efficient therapy in CYP2C9 highly expressing esophageal cancers.     

Pathogenic GLUT9 mutations causing renal hypouricemia type 2 (RHUC2)

Renal hypouricemia (MIM 220150) is an inherited disorder characterized by low serum uric acid levels and has severe complications such as exercise-induced acute renal failure and urolithiasis. We have previously reported that URAT1/SLC22A12 encodes a renal urate-anion exchanger and that its mutations cause renal hypouricemia type 1 (RHUC1). With the large health-examination database of the Japan Maritime Self-Defense Force, we found two missense mutations (R198C and R380W) of GLUT9/SLC2A9 in hypouricemia patients. R198C and R380W occur in highly conserved amino acid motifs in the "sugar transport proteins signatures" that are observed in GLUT family transporters. The corresponding mutations in GLUT1 (R153C and R333W) are known to cause GLUT1 deficiency syndrome because arginine residues in this motif are reportedly important as the determinants of the membrane topology of human GLUT1. Therefore, on the basis of membrane topology, the same may be true of GLUT9. GLUT9 mutants showed markedly reduced urate transport in oocyte expression studies, which would be the result of the loss of positive charges in those conserved amino acid motifs. Together with previous reports on GLUT9 localization, our findings suggest that these GLUT9 mutations cause renal hypouricemia type 2 (RHUC2) by their decreased urate reabsorption on both sides of the renal proximal tubule cells. However, a previously reported GLUT9 mutation, P412R, was unlikely to be pathogenic. These findings also enable us to propose a physiological model of the renal urate reabsorption via GLUT9 and URAT1 and can lead to a promising therapeutic target for gout and related cardiovascular diseases.     

Synthesis of 9-(2-Deoxy-β-D-ribofuranosyl)purine-2-thione

Abstract

A synthesis of 9-(2-deoxy-β-D-ribofuranosyl)purine-2-thione was performed by desulfurization of 2′-deoxy-6-thioguanine to give 2-amino-9-(2-deoxy-β-D-ribofuranosyl)purine, diazotization with chloride replacement to give 2-chloro-9-(2-deoxy-β-D-ribofuranosyl)purine, and the replacement of chloride with sulfur using thiolacetic acid and deacetylation.     

Stereoselective synthesis of 9-beta-d-arabianofuranosyl guanine and 2-amino-9-(beta-d-arabianofuranosyl)purine

9-beta-d-Arabianofuranosyl guanine (6) and 2-amino-9-(beta-d-arabianofuranosyl)purine (8) were prepared from 2-amino-6-chloro-9-(2,3,5-triphenylmethoxyl-beta-d-arabianofuranosyl)purine (4), a key intermediate which was stereoselectively prepared from 2,3,5-triphenylmethoxyl-d-arabianofuranose and 2-amino-6-chloro-purine. The yield of the intermediate was obviously improved and only beta-isomer was formed by using the activated molecular sieve as environmental friendly catalyst, overcoming the defect that a 1:1 mixture of alpha- and beta-isomers was formed, which was difficult to separate, when toxic mercury cyanide was previously used as catalyst.     

Pathogenic GLUT9 Mutations Causing Renal Hypouricemia Type 2 (RHUC2)

Renal hypouricemia (MIM 220150) is an inherited disorder characterized by low serum uric acid levels and has severe complications such as exercise-induced acute renal failure and urolithiasis. We have previously reported that URAT1/SLC22A12 encodes a renal urate-anion exchanger and that its mutations cause renal hypouricemia type 1 (RHUC1). With the large health-examination database of the Japan Maritime Self-Defense Force, we found two missense mutations (R198C and R380W) of GLUT9/SLC2A9 in hypouricemia patients. R198C and R380W occur in highly conserved amino acid motifs in the “sugar transport proteins signatures” that are observed in GLUT family transporters. The corresponding mutations in GLUT1 (R153C and R333W) are known to cause GLUT1 deficiency syndrome because arginine residues in this motif are reportedly important as the determinants of the membrane topology of human GLUT1. Therefore, on the basis of membrane topology, the same may be true of GLUT9. GLUT9 mutants showed markedly reduced urate transport in oocyte expression studies, which would be the result of the loss of positive charges in those conserved amino acid motifs. Together with previous reports on GLUT9 localization, our findings suggest that these GLUT9 mutations cause renal hypouricemia type 2 (RHUC2) by their decreased urate reabsorption on both sides of the renal proximal tubule cells. However, a previously reported GLUT9 mutation, P412R, was unlikely to be pathogenic. These findings also enable us to propose a physiological model of the renal urate reabsorption via GLUT9 and URAT1 and can lead to a promising therapeutic target for gout and related cardiovascular diseases.     

Formation of thiol conjugates of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2

Albumin catalyzes the transformation of prostaglandin D2 to 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and to isomeric prostaglandin D2 compounds including delta 12(E)-prostaglandin D2. Both of these compounds are alpha,beta-unsaturated ketones, which should render them susceptible to nucleophilic addition. We therefore examined the ability of the compounds to form conjugates with thiols glutathione and cysteine. During incubation with excess glutathione, both 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and delta 12(E)-prostaglandin D2 formed a conjugate. Conjugation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 occurred very rapidly; approximately 70% was conjugated within 2 min. In contrast, conjugation of delta 12(E)-prostaglandin D2 with glutathione proceeded at a much slower rate; only 38% was conjugated at 60 min. The formation of both conjugates was enhanced by glutathione S-transferase. Conjugation of both compounds with cysteine was found to occur more rapidly than with glutathione. This effect was more pronounced with delta 12(E)-prostaglandin D2 in which 60% conjugated with cysteine within 2 min. These differences are likely attributed to greater steric hindrance for conjugation across the delta 12 double bond compared to that across the delta 9 bond. Analysis by fast atom bombardment mass spectrometry confirmed the formation of the glutathione conjugate of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2. Following prolonged incubation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 with excess glutathione in the presence of glutathione S-transferase, a small quantity of a bis conjugate of this compound was also detected by mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereospecific syntheses of 9-(S)-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA)

9-(S)-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) was prepared from 9-(S)-(2,3-dihydroxypropyl)adenine (DHPA) via its 3-O-chloromethanephosphonate. The latter compound is obtained by treatment of DHPA with chloromethanephosphonyl dichloride and the 3'-isomer separated from its 2'-congener by ion-exchange chromatography. The 3'-isomer is prepared selectively by the same method starting from 2',6-dibenzoyl derivative of DHPA. The 3'-ester is transformed to HPMPA by treatment with aqueous alkali. Alternatively, 9-(S)-(2-hydroxy-3-triphenylmethoxypropyl)-N6-benzoyladenine can be converted to HPMPA by reaction with dialkyl p-tolylsulfonyloxymethane-phosphonates in the presence of NaH followed by successive acid and alkaline treatment.     

Trisomy 9p with i(9p) and t(9q18p)

Summary The trisomy 9p syndrome in a 2-year-old girl with moderate mental retardation is presented. She has a unique karyotype with a de novo isochromosome 9p and a translocation between 9q and 18p.     

The C. elegans B-cell lymphoma 2 (Bcl-2) homolog cell death abnormal 9 (CED-9) associates with and remodels LIPID membranes

Bcl-2 proteins associate with and remodel mitochondria to regulate apoptosis. While the C. elegans Bcl-2 homolog CED-9 constitutively associates with mitochondria, it is unclear whether or not this association reflects an innate ability of CED-9 to directly remodel mitochondrial membranes. To address this question, we have characterized the effects of recombinantly expressed and purified CED-9 on synthetic lipid vesicles. We found that CED-9 associates with anionic lipid vesicles at neutral pH, and that association can occur independently of the C-terminal transmembrane domain. Membrane association changes the environment of CED-9 tryptophans and results in an apparent increase in α-helical structure. Upon association, CED-9 alters the permeability of membranes resulting in leakage of encapsulated dyes. Furthermore, this membrane remodeling promotes membrane fusion upon protonation of CED-9. Bypass of this protonation trigger can be achieved by mutating two conserved glutamates (E187K/E190K) or removing the N-terminal 67 residues. Together, these in vitro results suggest that CED-9 retains the amphitropic ability of mammalian Bcl-2 proteins to associate with cellular membranes. We therefore discuss the possibility that CED-9 and other Bcl-2 homologs localize at mitochondria to regulate mitochondrial homeostasis by either modulating mitochondrial membrane permeability or fusion.     

Expression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and -9) in human trophoblast invasion

Background  

The aim of this study was to examine the invasiveness of first trimester trophoblasts according to the secretion profile of MMP-2 and -9 at different gestational stages, and to test the similarity between primary trophoblast cell-culture and the JAR choriocarcinoma cell-line.