Synthesis and biological evaluation of pteridine and pyrazolopyrimidine based adenosine kinase inhibitors

Three new approaches have been tested to modify existing pyridopyrimidine and alkynylpyrimidine classes of nonnucleoside adenosine kinase inhibitors 2 and 3. 4-Amino-substituted pteridines 8a-e were generally less active than corresponding 5- and 6-substituted pyridopyrimidines 2. Pyrazolopyrimidine 13c with IC(50)=7.5 nM was superior to its open chain alkynylpyrimidine analog 13g (IC(50)=22 nM) while pyrrolopyrimidines such as 17a were inactive.     

Synthesis and biological evaluation of 6,7-disubstituted 4-aminopyrido[2,3-d]pyrimidines as adenosine kinase inhibitors

The synthesis and structure-activity relationship of a series of 6,7-disubstituted 4-aminopyrido[2,3-d]pyrimidines as novel non-nucleoside adenosine kinase inhibitors is described. A variety of substituents, primarily aryl, at the C6 and C7 positions of the pyridopyrimidine core were found to yield analogues that are potent inhibitors of adenosine kinase. In contrast to the 5,7-disubstituted and 5,6,7-trisubstituted pyridopyrimidine series, these analogues exhibited only modest potency to inhibit AK in intact cells.     

Nucleosides. 127. Synthesis of Pyrido[2,3-d]pyrimidine Nucleosides from 5-Cyanouridine

Abstract

7-Amino-6-substituted-1-(β-D-ribofuranosyl)pyrido [2,3–d]pyrimidine-2,4 (1H, 3H)-diones were prepared in good yields from 5-cyanouridine by application of a novel ring transformation reaction recently developed in our laboratory. Treatment of 3-benzyloxymethyl-2', 3'-O-isopropylidene-5'-O-trityl-5-cyanouridine with malononitrile, cyanoacetamide or ethyl cyanoacetate in base gave directly the pyridopyrimidine nucleosides bearing a CN, CONH₂ and CO₂ Et at C-6, respectively. The benzyloxymethyl and trityl protecting groups were removed by hydrogenolysis and the isopropylidene group by acid hydrolysis.     

Pyridopyrimidine analogues as novel adenosine kinase inhibitors

A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.     

Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors

The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.     

Novel pyridopyrimidine derivatives as inhibitors of stable toxin a (STa) induced cGMP synthesis

A series of pyridopyrimidine derivatives were synthesized and evaluated for their ability to inhibit cyclic nucleotide synthesis in the presence of stable toxin a of Escherichia coli. The structure activity relationships around the basic core structure were examined and examples with better activity and potentially better pharmacological properties are presented.     

Synthesis of carbazole bearing pyridopyrimidine‐substituted sulfonamide derivatives and studies their carbonic anhydrase enzyme activity

The synthesis of carbazole containing pyridopyrimidine‐substituted sulfonamide derivatives ( 3a‐i ) and their inhibitory effects on human carbonic anhydrase (hCA) I and II were studied. Spectral data and elemental analysis confirmed the structures of the compounds synthesized. The results show that all the synthesized compounds inhibited the CA I and II activities. Among them, 3a was found to be the most active ( K _i: 14 µM) for hCA I and 3f ( K _i: 126 µM) for hCA II.     

Pyridopyrimidine based cannabinoid-1 receptor inverse agonists: Synthesis and biological evaluation

The synthesis, SAR and binding affinities are described for cannabinoid-1 receptor (CB1R) specific inverse agonists based on pyridopyrimidine and heterotricyclic scaffolds. Food intake and pharmacokinetic evaluation of several of these compounds indicate that they are effective orally active modulators of CB1R.     

Rapid assembly of diverse and potent allosteric Akt inhibitors

This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.     

Effects of handling and pair management on reproduction in Japanese quail (Coturnix coturnix )

Three experiments were designed to study the effects of handling, pairing, and frequency of mating opportunities on reproduction in Japanese quail (Coturnix coturnix ) when the sexes were caged separately except during the 90 minute mating periods. In Experiments 1 and 2, 48 females and 48 males were allocated randomly to one of four treatments: 1) continuously paired, 2) paired daily with the same male, 3) paired daily with a different male, and 4) paired every third day with the same male. In Experiment 3, 44 males and 44 females used in Experiments 1 and 2 were again assigned randomly to one of four treatments: 1) continuously paired, 2) paired continuously but the females were handled at the beginning and end of a 90 minute period every third day to simulate the handling associated with moving birds between cages for mating, 3) paired every third day with the same male, and 4) paired every third day with a different male. In Experiments 1 and 3, the females were introduced into the males' cages, while in Experiment 2 males were introduced into the females' cages. In Experiment 1, the females paired every 1 or 3 days with the same males had fewer eggs with embryonic development than continously paired females. In Experiment 2, a reduced number of eggs with embryonic development was observed only in fermales paired every 3 days with the same male. In Experiment 1, more eggs hatched from continuously paired females than from females paired every third day with the same male. In Experiment 3, the females paired every third day with the same males had fewer settable, developing, or hatching eggs. In conclusion, it was found that when the sexes need to be caged separately, it is better to expose the females to different males.     

MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 3: Optimization of potency in the pyridopyrimidine series through the application of a pharmacophore model

The addition of substituents to the pyridopyrimidine scaffold of MexAB-OprM specific efflux pump inhibitors was explored. As predicted by a pharmacophore model, the incorporation substituents at the 2-position improved potency. Piperidines were found to be optimal, and further introduction of polar groups without compromising the activity was shown to be feasible. Careful positioning of the essential acidic moiety of the pharmacophore relative to the scaffold led to the discovery of vinyl tetrazoles with still greater potency.     

Pax: a murine multigene family of paired box-containing genes.

A murine multigene family has been identified that shares a conserved sequence motif, the paired box, with developmental control and tissue-specific genes of Drosophila. To date five murine paired box-containing genes (Pax genes) have been described and one, Pax-1, has been associated with the developmental mutant phenotype undulated. Here we describe the paired boxes of three novel Pax genes, Pax-4, Pax-5, and Pax-6. Comparison of the eight murine paired domains of the mouse, the five Drosophila paired domains, and the three human paired domains shows that they fall into six distinct classes: class I comprises Pox meso, Pax-1, and HuP48; class II paired, gooseberry-proximal, gooseberry-distal, Pax-3, Pax-7, HuP1, and HuP2; class III Pax-2, Pax-5, and Pax-8; class IV Pax-4; class V Pox neuro; and class VI Pax-6. Pax-1 and the human gene HuP48 have identical paired domains, as do Pax-3 and HuP2 as well as Pax-7 and HuP1, and are likely to represent homologous genes in mouse and man. Identical intron-exon structure and extensive sequence homology of their paired boxes suggest that several Pax genes represent paralogs. The chromosomal location of all novel Pax genes and of Pax-3 and Pax-7 has been determined and reveals that they are not clustered.     

S-phase Inhibition of Cell Cycle Progression by a Novel Class of Pyridopyrimidine Tyrosine Kinase Inhibitors

Increased activity of the src family of oncogenic tyrosine kinases is seen in many human tumors and pharmacologic inhibitors of these kinases are investigated as potential anti-tumor agents. A family of pyrido [2, 3-d] pyrimidine compounds (PD) has been characterized as selective inhibitors of Src kinases. We studied the effects of this class of compounds on cancer cell lines and found that they were highly specific inhibitors of cell cycle progression. These compounds inhibit cells either in the mitotic phase or in mid S-phase; these two activities are mutually exclusive: no compound exerts both activities. We undertook experiments to determine the mechanistic basis for these differences and found additional biochemical activities associated with the S-phase inhibitors. Treatment of cells with the S-phase blocker PD179483 causes abnormal and persistent hyperactivation of Cdk2 and Cdc2 due to Tyr-15 dephosphorylation. These effects were associated with hyperphosphorylation of the upstream regulatory kinase Myt1 and Wee1. They were not observed with the anti-mitotic compounds. Furthermore, the S-phase inhibitors PD179483 and PD166326, but not the anti-mitotic compounds, inhibit Wee1 in vitro at concentrations that cause S-phase block in vivo. These data identify a novel subset of pyridopyrimidine compounds which are inhibitors of src and Wee1 kinases and which inhibit tumor cell growth through cell cycle arrest in mid S-phase.     

Opposite base specificity in excision of pyrimidine ring-opened 1,N6-ethenoadenine by thymine glycol-DNA-glycosylases

A highly mutagenic DNA lesion, 1,N6-ethenoadenine ( epsilon A) is chemically unstable and either depurinates or converts to a pyrimidine ring-opened product upon water molecule addition to the C(2)z.sbnd;N(3) bond in epsilon dA (compound B). Compound B subsequently undergoes deformylation to yield compound C, which depurinates in the final step of the epsilon A rearrangement pathway. We have previously shown that epsilon A rearrangement products are not repaired by human N-methylpurine-DNA-glycosylase, which excises parental epsilon A. Compound B was shown to be eliminated from a B:T pair by Escherichia coli formamidopyrimidine-DNA-glycosylase (Fpg protein) and endonuclease III (Nth protein). Fpg protein excised B also from a B:C pair, and much less efficiently from B:A and B:G pairs [J. Biol. Chem. 276 (2001) 21821]. Here we show that efficiency of B excision by the Nth protein also depends on the opposite base in the pair. Most efficient repair is observed when this derivative is paired with dG (Km=18nM, kcat=12) and is less favourable when paired with dC (Km=40nM, kcat=13) and dT (Km=32nM, kcat=11). In physiological conditions, compound B is probably not excised by the Nth-glycosylase from a B:A pair, or from a single-stranded DNA, since kinetic constants in these conditions are an order or two orders of magnitude higher than when B is paired with T, C or G. A similar specificity for B excision was found for Saccharomyces cerevisiae Ntg2-glycosylase. Thus, when paired with A, an epsilon A derivative might be more persistent than when paired with other bases and give rise to AT-->TA transversions.     

Inheritance and meiotic behaviour of a de novo chromosome fusion in the aphid Myzus persicae (Sulzer)

A de novo tandem fusion between autosomes 2 and 3 (A2+3), arising in the course of laboratory crosses of sexual morphs of two clones of the aphid Myzus persicae, was stable through more than 180 generations of parthenogenetic (clonal) reproduction. Studies of its inheritance through the sexual phase, and segregation from an amplified esterase marker gene, showed that crossing over occurred during oogenesis, but not in spermatogenesis, confirming previous cytological observations. Only a small number of progeny resulted from attempts at selfing fusion heterozygotes, and none of these was homozygous for the fusion. A2+3 paired in parallel alignment with the separate A2 and A3 to form a trivalent at prophase I of spermatogenesis. Fusion heterozygotes had a segregation problem at anaphase I of meiosis, A2+3 forming a chromatin bridge between the daughter spermatocytes in about 42% of dividing cells, which could be attributed to alternate orientation in the trivalent (A2 and A3 paired with opposite sides of A2+3) in the preceding metaphase I. Males heterozygous for an A2 dissociation were also studied and found to have much less of a segregation problem, despite showing similar orientation patterns at metaphase I. Possible reasons for this difference and the significance of the findings in relation to karyotype evolution in aphids are discussed.     

Instrumental outcome devaluation with representation-mediated conditioning

In three experiments, rats were trained to perform two instrumental behaviours (R1 and R2) in the presence of discriminative stimuli (Sd1 and Sd2, respectively) to obtain a common food outcome (O1). Acquisition of the two discriminations was followed by switching the outcome accompanying R2 performance from O1 to a new one (O2). Experiment 1 showed paired presentations of O2 with a lithium chloride (LiCl) injection resulted in a reduction in the R2 performance. In the subsequent two experiments, each Sd was paired with LiCl injection and its effects on outcome consumption and instrumental performance were investigated. A reduction in the O2 consumption subsequent to the Sd devaluation was found in Experiments 2 and 3. Experiment 3 revealed a reduced R2 performance in an extinction test, following the animals’ consummatory access to the outcomes in training context. These results demonstrate representation-mediated outcome devaluation in the course of the Sd devaluation.     

US specificity of occasion setting: Hierarchical or configural learning?

Four experiments in rats examined whether occasion setters and target CSs play qualitatively different roles in occasion-setting discriminations. Two visual occasion setters, A and B, signalled reinforcement of two auditory target CSs, x and y, with sucrose and oil (A…x→suc, B…y→oil, A-, B-, x-, y-); in addition two transfer CSs w and z were paired with sucrose and oil (w→suc, z→oil). When w and z were substituted for x and y (A…w, B…w, A…z, B…z) more responding was observed when both stimuli had been paired with the same outcome (Experiments 1 and 3a). No effect was observed when two visual "pseudo-occasion setters", C and D (paired with sucrose and oil in a trace relation to the US:C…→suc, D…→oil), were substituted for the occasion setters A and B (C…x, D…x, C…y, D…y; Experiments 2, 3b and 4). These results could not be explained in terms of Pavlovian summation: responding to combinations of Pavlovian CSs paired with same or different outcomes was either the same, or lower when both stimuli had been paired with the same outcome (Experiment 4). Implications of these results for theories of occasion setting and configural learning are discussed.     

Pavlovian biconditional discrimination learning in the C57BL/6J mouse

Four experiments using mice examined acquisition of Pavlovian biconditional discriminations in which two stimulus compounds were paired with food (AX+ and BY+) and two were not (AY- and BX-). Temporally asynchronous compounds were generated by using contextual stimuli (Experiment 1) and 15-s discrete visual cues (Experiments 2A, 2B and 3) to disambiguate when embedded noise or tone stimuli would be paired with food. When food pellets followed both reinforced compounds, successful acquisition was obtained in Experiment 1 but not in Experiments 2A and 2B even though the order of trials was modeled after that used in Experiment 1. However, when differential outcomes followed the reinforced compounds in Experiment 3, acquisition was obtained with discrete cue stimulus compounds. The implications of these results for modulatory models of conditional discrimination learning in animals are discussed.     

Functional Hypermethylation of ALDH1L1 , PLCL2 , and PPP2R3A in Colon Cancer

DNA hypermethylation and mutations are key mechanisms for the downregulation of tumor suppressor genes. NotI-microarrays allowed us to detect hypermethylation and/or deletions in 180 NotI sites associated with 188 genes of human chromosome 3, in 24 paired (tumor/normal) colon samples. The most frequent aberrations (in more than 20% of tumor samples) were detected in the promoter regions of 20 genes. Expression and promoter methylation of these genes were analyzed using the data for paired colon samples from The Cancer Genome Atlas project. Three genes—ALDH1L1, PLCL2, and PPP2R3A—revealed a more than two-fold average decrease in expression and a negative correlation between mRNA level and promoter hypermethylation. The expression of these three genes was then evaluated in 30 paired colon samples by quantitative PCR. Frequent (in more than 60% of cases) and significant (5–9-fold on average) mRNA level decrease was found for each of the genes in the tumor samples. The results indicate a suppressor role of the ALDH1L1, PLCL2, and PPP2R3A genes in colon cancer, as well as functional significance of hypermethylation in the downregulation of these genes.