The design, synthesis and structure-activity relationships of 1-aryl-4-aminoalkylisoquinolines: a novel series of CRF-1 receptor antagonists

The design, synthesis and structure-activity relationships of a novel series of CRF-1 receptor antagonist, the 1-aryl-4-alkylaminoisoquinolines, is described. The effects of substitution on the aromatic ring, the amino group and the isoquinoline core on CRF-1 receptor binding were investigated.     

2-Arylpyrimidines: novel CRF-1 receptor antagonists

The design, synthesis and structure-activity relationship studies of a novel series of CRF-1 receptor antagonists, the 2-arylpyrimidines, are described. The effects of substitution on the aromatic ring and the pyrimidine core on CRF-1 receptor binding were investigated. A number of compounds with K(i) values below 10 nM and lipophilicity in a minimally acceptable range for a CNS drug (cLogP<5) were discovered.     

Action of pyrazolopyrimidine derivatives on Trypanosoma rangeli culture forms

The capacity of 54 different pyrazolo(3,4-d)- or pyrazolo(4,3-d)pyrimidine derivatives to inhibit the multiplication of Trypanosoma rangeli culture forms was evaluated. Among pyrazolo(3,4-d)pyrimidines, 14 derivatives showed trypanostatic activity, 4-aminopyrazolo-(3,4-d)pyrimidine (APP) being the most active, with 4-hydroxypyrazolo(3,4-d)pyrimidine (HPP) lacking trypanostatic activity. 7-Hydroxy-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoB) was as active as 7-amino-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoA), both compounds being five-fold less inhibitory than APP. It can be concluded that, regarding T. rangeli, the chemical analogy to hypoxanthine or inosine of pyrazolo(3,4-d)- and pyrazolo(4,3-d)pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.     

Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors

A structure–activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8 phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo[1,5-a]pyrimidine ring were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo[1,5-a]pyridine derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g., plasma t_1/2 = 1.6 h) following intraperitoneal administration in mice. These studies provide useful molecular probes for examining the in vivo pharmacology of BMP signaling inhibition.     

Synthesis and analgesic/anti-inflammatory evaluation of fused heterocyclic ring systems incorporating phenylsulfonyl moiety

A series of pyrazolo[1,5-a]pyrimidine, triazolo[1,5-a]pyrimidine, and pyrimido[1,2-a]benzimidazole ring systems incorporating phenylsulfonyl moiety were synthesized via the reaction of 3-(N,N-dimethylamino)-1-aryl-2-(phenylsulfonyl)prop-2-en-1-one derivatives 2a,b with appropriate nitrogen nucleophiles. The analgesic and anti-inflammatory activities of the newly synthesized compound were investigated in vivo. 3-Bromo-2-phenyl-6-(phenylsulfonyl)-7-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5e) was found to have an excellent analgesic activity in comparison with indomethacin as a reference drug, while the highest anti-inflammatory effect was observed in the case of 2-(4-bromophenyl)-6-(phenylsulfonyl)-5-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5d). From the structure-activity relationship (SAR) point of view, the analgesic/anti-inflammatory activity of pyrazolo[1,5-a]pyrimidine derivatives was found to be much higher than triazolo[1,5-a]pyrimidine and pyrimido[1,2-a]benzimidazole derivatives.     

Ring Opening Reaction of Pyrazolo[3,4-c]Maleimide Nucleosides

Abstract

Synthesis of pyrazolo[3,4-c]maleimide nucleosides was attempted, but ring opening reaction of the maleimide part was observed during ammonolysis of sugar-protected pyrazolo[3,4-c]maleimide nucleosides. The isolated pyrazole nucleosides were characterized by NMR spectra and X-ray analysis.     

Attenuation of corticotropin releasing factor-induced hypotension in anesthetized rats with the CRF antagonist, alpha-helical CRF9-41; comparison with effect on ACTH release.

The effect of pretreatment with the corticotropin releasing factor (CRF-41) antagonist, alpha-helical CRF(9-41), on the hypotensive response obtained on peripheral administration of CRF-41 has been assessed in anesthetized Wistar rats. A single IV bolus dose of rat CRF-41 (2 nmol, at 0 min) produced a hypotensive effect which was rapid in onset (-52 mmHg at +1 min) and sustained throughout the 60-min study period (-42, -40, -26 and -16 mmHg at +3, +10, +30 and +60 min, respectively). The antagonist [alpha CRF(9-41)] was administered in consecutive bolus doses of 12.5, 25 and 50 nmol at -15, -10 and -5 min, respectively. This had no effect on mean arterial blood pressure (MABP) or heart rate, nor did it change significantly the magnitude of the initial rapid fall in MABP when CRF-41 was administered (-45 mmHg at +1 min). However, following pretreatment with alpha CRF(9-41), MABP returned to control values within 3 min and the sustained period of hypotension was completely blocked. Administration of CRF-41 resulted in 44% and 142% increases in norepinephrine and epinephrine measured at +60 min. Pretreatment with the antagonist attenuated the rise in circulating catecholamine levels observed after CRF-41 administration. In comparison, pretreatment with the antagonist did not alter the ACTH response to CRF-41 at +1 and +3 min and only reduced ACTH levels by 28% (p less than 0.05), 43% (p less than 0.001) and 41% (p less than 0.01) at 10, 30 and 60 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of a specific two-site immunoradiometric assay with radioimmunoassay for rat/human CRF-41

We report the development of an immunoradiometric assay (IRMA) for the specific measurement of corticotrophin releasing factor (CRF-41) which uses two antibodies directed to opposite ends of the CRF-41 molecule. In this assay, 125I-labelled affinity purified rabbit anti-(CRF 36-41) immunoglobulin (IgG) and a guinea-pig anti-(CRF 1-20) serum are simultaneously added to 200 microliter volumes of standard or unknown. After 16 h incubation at room temperature, free and CRF-bound guinea-pig antibodies are precipitated using affinity purified sheep anti-(guinea-pig Fc region) IgG coupled to solid phase Dynospheres. Radioactive rabbit anti-(CRF 36-41) is only precipitated in tubes containing CRF-41, since the peptide acts as a link between the 125I-labelled rabbit IgG and the unlabelled guinea-pig CRF-specific antibodies. Precipitated counts are directly proportional to the concentration of CRF-41 in the sample. This CRF IRMA is compared with two radioimmunoassays (RIA) using the N- and C-terminal CRF antisera employed in the IRMA and found to be more sensitive, specific and rapid to perform. The CRF-41 content of rat and human hypothalamic extracts is the same whether measured by IRMA or conventional RIA. Sephadex G50 chromatography of rat hypothalamic extracts reveals two peaks, detected equally by IRMA and RIA, with a main peak in the elution position of synthetic CRF-41, and a smaller void peak. This is the case whether the hypothalamic extracts are prepared from adrenalectomised or sham-operated rats, non-stressed or subjected to ether stress. Re-chromatography of pooled void peaks under dissociating conditions gives the elution profile of synthetic CRF-41, indicating that the large molecular weight 'CRF-41' peak is not a CRF-41 precursor, but is due to CRF-41 associating non-covalently with large molecular weight proteins.     

Exploring pyrazolo[3,4-d]pyrimidine phosphodiesterase 1 (PDE1) inhibitors: a predictive approach combining comparative validated multiple molecular modelling techniques

Phosphodiesterase 1 (PDE1) is a potential target for a number of neurodegenerative disorders such as Schizophrenia, Parkinson’s and Alzheimer’s diseases. A number of pyrazolo[3,4-d]pyrimidine PDE1 inhibitors were subjected to different molecular modelling techniques [such as regression-based quantitative structure-activity relationship (QSAR): multiple linear regression, support vector machine and artificial neural network; classification-based QSAR: Bayesian modelling and Recursive partitioning; Monte Carlo based QSAR; Open3DQSAR; pharmacophore mapping and molecular docking analyses] to get a detailed knowledge about the physicochemical and structural requirements for higher inhibitory activity. The planarity of the pyrimidinone ring plays an important role for PDE1 inhibition. The N-methylated function at the 5th position of the pyrazolo[3,4-d]pyrimidine core is required for interacting with the PDE1 enzyme. The cyclopentyl ring fused with the parent scaffold is necessary for PDE1 binding potency. The phenylamino substitution at 3rd position is crucial for PDE1 inhibition. The N2-substitution at the pyrazole moiety is important for PDE1 inhibition compared to the N1-substituted analogues. Moreover, the p-substituted benzyl side chain at N2-position helps to enhance the PDE1 inhibitory profile. Depending on these observations, some new molecules are predicted that may possess better PDE1 inhibition.     

Novel pyrazole derivatives: Synthesis and evaluation of anti-angiogenic activity

The synthesis of a series of novel trisubstituted pyrazole derivatives and their PIFA-mediated conversion to molecules bearing the fused pyrazolo[4,3-c]quinoline ring system is reported. The anti-angiogenic activity of these compounds was evaluated by using in vitro assays for endothelial cell proliferation and migration, and in the chicken chorioallantoic membrane (CAM) assay. Compounds containing the fused pyrazolo[4,3-c]quinoline motifs emerged as potent anti-angiogenic compounds, which also had the ability to inhibit the growth of human breast (MCF-7) and cervical (Hela) carcinoma cells in vitro.     

Inhibition of CRF- and urotensin I-stimulated ACTH release from goldfish pituitary cell columns by the CRF analogue alpha-helical CRF-(9-41)

alpha-Helical CRF-(9-41) is an analogue of corticotropin-releasing factor (CRF) that antagonizes CRF-stimulated ACTH release in rats. In the present study, alpha-helical CRF-(9-41) was tested to determine whether it would antagonize the ACTH-releasing activity of CRF or urotensin I (UI) observed with superfused, dispersed goldfish anterior pituitary cells. At a concentration of 4 microM, alpha-helical CRF-(9-41) completely blocked the ACTH-releasing activity of 100 nM CRF or 100 nM UI. The inhibitor by itself showed little intrinsic ACTH-releasing activity. This investigation reveals similarities in the CRF-antagonism of alpha-helical CRF-(9-41) in the teleost and mammalian pituitary in vitro. It also provides are similar and suggests that alpha-helical CRF-(9-41) may be useful as a tool to investigate the effects of CRF-like and UI-like peptides in teleost fishes.     

CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal Ca(2+) imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine 10(-6) M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM K(+)-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF 10(-6) M and CRF 10(-7) M during 30 s induced a Ca(2+) response in 22.4% of the myenteric neurons (n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin (n = 55). The selective CRF-1 receptor antagonist CP 154,526 (n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at 10(-7) M, and its effect at 10(-6) M (15.3%, n = 59) was completely blocked by CP 154,526. TTX 10(-6) M (n = 70) could not block the CRF-induced Ca(2+) transients but reduced the amplitude of the signals significantly. Removal of extracellular Ca(2+) blocked all responses to CRF (n = 47). L-type channels did not contribute to the CRF-induced Ca(2+) transients. Blocking N- or P/Q-type Ca(2+) channels did not reduce the responses significantly. Combined L- and R-type Ca(2+) channel blocking (SNX-482 10(-8) M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces Ca(2+) transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These Ca(2+) transients highly depend on somatic calcium influx through voltage-operated Ca(2+) channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the Ca(2+) signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level.     

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.     

Synthesis and biological evaluation of novel pyrazolo[4,3-b]oleanane derivatives as inhibitors of glycogen phosphorylase

Eighteen pyrazolo[4,3-b]oleanane derivatives have been synthesized and biologically evaluated as inhibitors of rabbit muscle GPa. Key compound 5 was readily obtained in four steps starting from oleanolic acid (OA; 1). Further modification based on pyrazolo triterpene 5 resulted in 17 novel pyrazolo pentacyclic triterpenes. All of the synthesized pyrazolo[4,3-b]oleanane derivatives were biologically assayed against rabbit muscle GPa. Within this series of compounds, pyrazole triterpene 19 (IC(50)=9.9 microM) exhibited more potent activity than the parent compound 1. Preliminary structure-activity relationship analysis of the pyrazolo[4,3-b]oleanane derivatives as GPa inhibitors is discussed.     

Synthesis and antitumor activity of a new class of pyrazolo[4,3-e]pyrrolo[1,2-a][1,4]diazepinone analogs of pyrrolo[1,4]benzodiazepines (PBDs)

A new class of Pyrrolo[1,4]benzodiazepines (PBDs) analogs featuring a pyrazolo[4,3-e]pyrrolo[1,2-a][1,4]diazepinone ring system has been designed and synthesized. In these compounds the A-benzene ring, characteristic of PBDs, has been replaced by a dimethylpyrazole ring, a modification suggested by modelling studies performed on the PBD base structure. Biological evaluation releaved appreciable antitumor activity for compounds 14 and 15 (8.84–22.4 μM) which encourages further investigation of the N⁶ and N⁷ alkyl pyrazole analogs.     

The synthesis and biological activity of certain pentaazaacenaphthylenes, hexaazaacenaphthylenes and their corresponding nucleosides

Synthesis of the tricyclic nucleoside 8-amino-6-N-methyl-2-(beta-D-ribo-furanosyl)-1,2,3,5,6,7-hexaazaacena phthylene (5) has been accomplished by the ring closure of an appropriately substituted pyrazolo[3,4-d]-pyrimidine nucleoside followed by the requisite chemical conversions. The formation, isolation and structural elucidation of two unexpected nucleosides formed by a reductive ring cleavage of the hexaazaacenaphthylene ring system is discussed. A comparison of the antitumor and biological activity of 5 with the structurally related tricyclic pentaazaacenaphthylene nucleoside which is currently in phase II clinical trials at the 5'-phosphate pro-drug is also presented.     

逍遥散对慢性束缚应激模型大鼠相关脑区CRF基因表达的影响

目的:观察慢性束缚应激大鼠相关脑区CRF mRNA(下丘脑、垂体、海马、皮层)含量变化以及逍遥散对其影响.方法:用RT-PCR和图像分析方法测定相关脑区CRF mRNA含量变化.结果:应激组较正常对照组在下丘脑CRF-1基因表达下调(P<0.01).在下丘脑逍遥散组较应激组CRF-1基因表达显著下调(P<0.01),CRF-2基因表达显著上调(P<0.01);在海马区逍遥散组CRF-2基因表达较模型组上调(P<0.05);在皮层逍遥散组CRF-1基因表达较应激组则显著上调(P<0.01).结论:逍遥散组对慢性束缚应激中枢神经肽CRF的调节位点在下丘脑、垂体、海马和皮层,充分证实逍遥散的调节靶点与下丘脑、边缘系统及皮层中枢有关.     

In vivo potentiation of corticotropin releasing factor activity by vasopressin analogues

The ability of the neurohypophyseal hormones and related synthetic peptides to potentiate the action of synthetic ovine corticotropin releasing factor (CRF-41) was examined using male rats whose endogenous CRF release was blocked with chlorpromazine, morphine and nembutal. CRF potency was clearly related to the pressor activity of the analogues. However, the threshold dose for eliciting a significant corticosterone response with the neurohypophyseal hormones was greater than with CRF-41. When arginine vasopressin (AVP) was coadministered with CRF-41 at subthreshold doses of both peptides, a significant corticosterone response was observed. When the neurohypophyseal hormone analogues were compared with regard to intrinsic CRF bioactivity, it was observed that an L-basic residue in sequence position 8 is necessary for high intrinsic activity. With one exception, l-Deamino-(9-D-Ala) arginine vasopressin, the ability to potentiate the effect of CRF-41 was related to the intrinsic CRF potency of the analogues. These results support previous reports of in vitro potentiation of CRF-41 by AVP and point out the complexity of CRF-neurohypophyseal hormone interaction in vivo.     

Discovery of pyrazolo[3,4-d]pyrimidine derivatives as GPR119 agonists

We designed and synthesized a novel series of phenylamino- and phenoxy-substituted pyrazolo[3,4-d]pyrimidine derivatives as GPR119 agonists. SAR studies indicated that electron-withdrawing substituents on the phenyl ring are important for potency and full efficacy. Compound 26 combined good potency with a promising pharmacokinetic profile in mice, and lowered the glucose excursion in mice in an oral glucose-tolerance test.     

Phosphodiesterase inhibitors. Part 4: Design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-4,4-dimethylpyrazolones

(-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. Here we show that a 4,4-dimethylpyrazolone subunit serves as an effective surrogate for the 5-methyl-4,5-dihydropyridazin-3(2H)-one ring of KCA-1490 whilst lacking a stereogenic centre. The 2- and 7-substituents in the pyrazolo[1,5-a]pyridine subunit markedly influence the PDE-inhibitory profile and can be adjusted to afford either potent PDE4-selective inhibitors or dual PDE3/4 inhibitors. A survey of bicyclic heteroaromatic replacements for the pyrazolo[1,5-a]pyridine allowed further refinement of the inhibitory profile and identified 3-(8-methoxy-2-(trifluoromethyl)imidazo[1,2-a]pyridin-5-yl)-4,4-dimethyl-1H-pyrazol-5(4H)-one as an orally active, achiral KCA-1490 analog with well-balanced dual PDE3/4-inhibitory activity.