Analogs of adenosine 3',5'-cyclic phosphate. II. Synthesis and enzymatic activity of derivatives of 1,N6-ethenoadenosine 3',5'-cyclic phosphate

The reactions of chloroacetaldehyde with adenosine 3′,5′-cyclic phosphate, and with several analogs modified at C₈ of the purine ring or C₅, of the sugar, lead to the corresponding 1,N⁶-etheno derivatives^d. Similar reactions using other 2-bromoaldehydes or phenacyl bromide give 1,N⁶-ethenonucleotides substituted at the α- or β-positions of the etheno bridge respectively. The ability of these compounds to activate the protein kinases from rabbit muscle and calf brain has been evaluated over a wide range of concentrations. While no derivative proved to be more active than adenosine 3′,5′-cyclic phosphate itself using the enzyme from rabbit muscle, a wide spectrum of activities was found using that from calf brain.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Reactive column HPLC: rapid analysis on RPC-5 of oligoadenylates that differ at the 3'-terminus.

Oligoadenylates can be analyzed according to the type of 3′-terminus (A_nA, A_nAp, and A_nA > p, oligoadenylates that have at the 3′-terminus no phosphate, a 2′(3′)-monophosphate, and a 2′,3′-cyclic phosphate respectively) by hplc on RPC-5 support using a novel dual-column technique. The first column separates A_nAp plus A_nA > p from A_nA, and at the start of the second column a layer of bacterial alkaline phosphatase enzyme converts the A_nAp into A_nA. Hence this A_nA emerges separately from the original A_nA and from the A_nA > p. The technique can be used to analyze a three-component mixture for a single chain length or a mixture of A_nAp and A_nA > p of mixed chain lengths (n = 3 to 7). The presence of poly(U) does not interfere with the analysis.     

Synthesis and biological activities of cyclic AMP analogs modified in the 1, 2, and 2′-positions

Analogs of adenosine-3′,5′-cyclic phosphate (cAMP) modified in positions 2 (Cl, Br, SCH₃) and 2′ (2,4-dinitrophenyl) and doubly modified in positions 1 and 2 (N¹O and Cl), 2 and 2′ (Cl and 2,4-dinitrophenyl), have been synthesized by convenient methods. These derivatives have been examined as alternative activators of cAMP-dependent protein kinase isolated from bovine muscle and as alternative substrates for a cyclic phosphodiesterase from bovine heart. All analogs activated the kinase, most of them being more effective than cAMP. All were degraded by the diesterase, several at lower rates.     

The cyclic nucleotide phosphodiesterases of spinach chloroplasts and microsomes

Cyclic nucleotide phosphodiesterase was extracted from intact chloroplasts and partially purified. Peak 1_c activity from Sephadex G-200 was resolved by electrophoresis into two major bands (MWs 1.87 × 10⁵ and 3.7 × 10⁵). Both also possessed acid phosphatase, ribonuclease, nucleotidase and ATPase. The chloroplast peak 1_c cyclic nueleotide phosphodiesterase was located in the envelope. Peak 1_m cyclic nucleotide phosphodiesterase obtained from the microsomal fraction had a MW of 2.63 × 10⁵. Electrophoresis separated 1_m into two bands of cyclic nucleotide phosphodiesterase activity (MWs 2.63 × 10⁵ and 1.28 × 10⁵). Both contain ATPase, ribonuclease, nucleotidase, but not acid phosphatase. Peak 1_c has high activity towards 3′:5′-cyclic AMP and 3′:5′-cyclic GMP but little towards 2′:3′-cyclic nucleotides. Peak 1_m showed most activity towards 2′:3′-cyclic AMP, 2′:3′-cyclic GMP and 2′:3′-cyclic CMP with little activity towards 3′:5′-cyclic nucleotides. With 1_c, 3′:5′-cyclic AMP and 3′:5′-cyclic GMP exhibit mixed-type inhibition towards one another. The 2′:3′-cyclic AMP phosphodiesterase 1_m was competitively inhibited by 2′:3′-cyclic GMP. p-Chloromercuribenzoate inhibits 1_c but not 1_m. Electrophoresis after dissociation indicates that 1_c and 1_m are both enzyme complexes. After dissociation, the 1_c complex but not that of 1_m could be reassociated. The ribonuclease of the 1_m complex hydrolyses RNA to yield 2′:3′-cyclic nucleotides as the main products. These results are compatible with the 1_c cyclic nucleotide phosphodiesterase complex being involved in the metabolism of 3′:5′-cyclic AMP, and the 1_m complex being concerned with RNA catabolism.     

The metabolism of formycin B in Leishmaniadonovani

Formycin B, a pyrazolo(4,3-d)pyrimidine C-nucleoside, inhibited the growth of $\text{Leishmania}\text{donovani}$ promastigotes in culture with an ED₉₀ of 0.2 μg/ml. Promastigotes incubated for 24 hrs with Formycin B at 10 μg/ml were found to convert it to the ribonucleotide, formycin B 5′-monophosphate. The parasites were also capable of aminating formycin B 5′-monophosphate as evidenced by the appearance of formycin A di- and triphosphate. The RNA contained the formycin A moiety in 3′,5′-polynucleotide linkage. Succino-AMP synthetase from these parasites was able to use formycin B 5′-monophosphate as an alternate-substrate with a K'm of 26 μM and a V'm of about 1% the V'm IMP. Formycin B 5′-monophosphate was also a substrate for mammalian succino-AMP synthetase with a V_m' of 40% the V_m' of IMP.     

5′-Halogenated Formycins as Inhibitors of 5′-Deoxy-5′-methylthioadenosine Phosphorylase: Protection of Cells Against the Growth-Inhibitory Activity of 5′-Halogenated Adenosines

Abstract

A series of 5′-halogenated formycins, including the chloro-, bromo- and iodo- derivatives, were synthesized. These compounds are competitive inhibitors of 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAPase) with K_i values in the range of 10^?7 M, making them the most potent inhibitors of MTAPase reported to date. These compounds protect cells from the growth-inhibitory action of 5′-halogenated adenosines, which must be activated by MTAPase. The syntheses of 5′-halogenated formycin B derivatives, which inhibit purine nucleoside phosphorylase, are also described.     

Proton magnetic resonance studies of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate and 9-(β-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate

A detailed ¹H 220-MHz n.m.r. study of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate (3′,5′-xylo-cAMP, 1) and 9-(?-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate (2′,5′-ara-cAMP, 2) in D₂O solution is described. The sugar-ring conformations in 1 and 2 are shown to be ³E and ²E, respectively, and the phosphate rings are in a chair form. An unusual ⁴J_P,H coupling of 2.4 Hz is observed between H-4′ and phosphorus in 1 and a vicinal J_P,H of 30.8 Hz between H-5′ and phosphorus in 2. This latter coupling verifies a similar value found previously in the ara-cytidine analog of 2. A comparison of the conformational properties of cyclic nucleotides having fused phosphate and sugar rings has been made, together with an assessment of the use of the Karplus constants in such ring-systems.     

Effect of O2-monobutyryl guanosine 3', 5'-cyclic monophosphate on hepatic metabolism of free fatty acids.

Livers from fed male rats were perfused $\text{in vitro}$ with O^2′-monobutyryl guanosine 3′,5′-cyclic monophosphate. The output of triglyceride was reduced, while output of ketone bodies and glucose was stimulated by 10^?4M monobutyryl guanosine 3′,5′-cyclic monophosphate. No effect was observed with 10^?5 M nucleotide. Monobutyryl guanosine 3′,5′-cyclic monophosphate did not affect uptake of free fatty acids. In these respects, monobutyryl guanosine 3′,5′-cyclic monophosphate mimics the effects of dibutyryl adenosine 3′,5′-cyclic monophosphate, although the guanylic nucleotide seems to be less potent than the adenosine 3′,5′-cyclic monophosphate derivative.     

Determination of 7-methylguanosine-containing 5'-termini of messenger ribonucleic acid by NaB[3H]4 labeling and high-performance liquid anion-exchange chromatography.

A method is described for the determination of 5′-terminal methylated (cap) structures in unlabeled mRNA based on oxidation with NaIO₄, reduction with NaB[³H]₄, cleavage with P₁ nuclease, and separation on a strong anion-exchange resin by high-performance liquid chromatography (hplc). Model compounds (cap 1 dinucleotides) were used to show that no structural alteration other than cleavage of the ribose ring of 7-methylguanosine occurred under the conditions used for oxidation and reduction. It was shown that the enzyme tobacco acid pyrophosphatase could be used to cleave cap dinucleotides containing unmodified or ring-opened ribose moieties and could also be used to release [³H]pm⁷G′ from NaB[³H]₄-labeled rabbit globin mRNA. All five known cap 1 dinucleotides were resolved by hplc. The cap of rabbit globin mRNA was identified as m⁷Gpppm⁶Am, in agreement with other methods of determination.     

In silico design of novel hERG-neutral sildenafil-like PDE5 inhibitors

Cyclic nucleotide phosphodiesterase enzymes (PDEs) have functions in regulating the levels of intracellular second messengers, 3′, 5′-cyclic adenosine monophosphate (cAMP) and 3′, 5′-cyclic guanosine monophosphate (cGMP), via hydrolysis and decomposing mechanisms in cells. They take essential roles in modulating various cellular activities such as memory and smooth muscle functions. PDE type 5 (PDE5) inhibitors enhance the vasodilatory effects of cGMP in the corpus cavernosum and they are used to treat erectile dysfunction. Patch clamp experiments showed that the IC₅₀ values of the human ether-à-go-go-related gene (hERG1) potassium (K) ion channel blocking affinity of PDE5 inhibitors sildenafil, vardenafil, and tadalafil as 33, 12, and 100 μM, respectively. hERG1 channel is responsible for the regulation of the action potential of human ventricular myocyte by contributing the rapid component of delayed rectifier K⁺ current (I_Kr) component of the cardiac action potential. In this work, interaction patterns and binding affinity predictions of selected PDE5 inhibitors against the hERG1 channel are studied. It is attempted to develop PDE5 inhibitor analogs with lower binding affinity to hERG1 ion channel while keeping their pharmacological activity against their principal target PDE5 using in silico methods. Based on detailed analyses of docking poses and predicted interaction energies, novel analogs of PDE5 inhibitors with lower predicted binding affinity to hERG1 channels without loosing their principal target activity were proposed. Moreover, molecular dynamics (MD) simulations and post-processing MD analyses (i.e. Molecular Mechanics/Generalized Born Surface Area calculations) were performed. Detailed analysis of molecular simulations helped us to better understand the PDE5 inhibitor–target binding interactions in the atomic level. Results of this study can be useful for designing of novel and safe PDE5 inhibitors with enhanced activity and other tailored properties.     

Piperidinyl-nicotinamides as potent and selective somatostatin receptor subtype 5 antagonists

Nicotinamides of benzyl-substituted 4-aminopiperidines and their seven-membered analogs of generic structure 2 and 2′ have been discovered as potent and selective SST5 antagonists. The activity (K_i) ranges from 2.4 to 436 nM. Most compounds exhibit decent physicochemical properties and follow a clear SAR pattern. Interestingly enough, the receptor is strongly enantiodiscriminating and binds in the amino-azepane-series only the (R)-enantiomer.     

Discovery of imidazo[1,2-a]-, [1,2,4]triazolo[4,3-a]-, and [1,2,4]triazolo[1,5-a]pyridine-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5

Based on a hypothesis that an intramolecular hydrogen bond was present in our lead series of picolinamide mGlu₅ NAMs, we reasoned that an inactive nicotinamide series could be modified through introduction of a fused heterocyclic core to generate potent mGlu₅ NAMs. In this Letter, we describe the synthesis and evaluation of compounds that demonstrate the viability of that approach. Selected analogs were profiled in a variety of in vitro assays, and two compounds were evaluated in rat pharmacokinetic studies and a mouse model of obsessive-compulsive disorder. Ancillary pharmacology screening revealed that members of this series exhibited moderate inhibition of the dopamine transporter (DAT), and SAR was developed that expanded the selectivity for mGlu₅ versus DAT.     

Cyclic nucleotide esterification: relationship to phosphate ring geometry and growth regulation in synchronized human lymphocytes.

Infrared spectra of neutral aqueous solutions of nucleoside 3′,5′-cyclic monophosphates indicate an increase in the antisymmetric phosphoryl stretching frequency to 1236 cm^?1 from 1215 cm^?1 in trimethylene cyclic phosphates. A further increase to 1242 cm^?1 accompanies esterification of the 2′-ribose hydroxyl. The O²′-esterified and 2′-deoxy cyclic nucleotides examined display both reduced kinase binding and altered phosphoryl stretching frequencies, suggesting that modification of the phosphate ring represents a common feature in decreased kinase activation. Reversible inhibition of mitosis in thymidine-synchronized human lymphocytes by 2 mmN⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate and N⁶-monobutyryladenosine 3′,5′-cyclic monophosphate was observed. However, adenosine 3′,5′-cyclic monophosphate, O²′-monobutyryladenosine 3′,5′-cyclic monophosphate, butyric acid, and ethyl butyrate had no effect on mitosis when present at 2 mm concentrations during S and G2. These results are consistent with hydrolysis of O²′-monobutyryladenosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyclic monophosphate by esterase and phosphodiesterase enzymes and suggest that modification of the N⁶ amino group is necessary for the antimitotic activity of N⁶,O²′-dibutyryladenosine 3′, 5′-cyclic monophosphate.     

Hydrolysis of 2',3'-cyclic adenosine monophosphate and 3',5'-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic mouse spleen

A comparison has been made between the capacity to hydrolyse 2′,3′-cyclic adenosine monophosphate and 3′,5′-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic (lymphosarcoma) spleen of C₅₇BL mice. The effect of X-irradiation on these activities was tested. Subcellular fractionation of normal and lymphosarcoma spleen points to a different overall localization of the enzymes. The 2′,3′-cyclic nucleotide phosphodiesterase (2′,3′-cAMPase) has its highest specific activity in the particulate fractions of the cell, while the data on 3′,5′-cyclic nucleotide phosphodiesterase (3′,5′-cAMPase) show the highest activity in the soluble fraction. The 2′,3′-cAMPase activity is higher in the tumor as compared to the normal tissue, while the opposite holds for 3′,5′-cAMPase. Total body irradiation of normal mice with a dose of 600 rads of X-rays, results in a clear drop in 2′,3′-cAMPase 48 hours after the exposure. The 3′,5′-cAMPase is hardly affected at this time. Neither imidazol nor Mg⁺⁺ has any influence on the 2′,3′-cAMPase. The pH optimum for 3′,5′-cAMPase and 2′,3′-cAMPase appears to be 7.7 and 6.2 respectively. This report suggests a no-identity of the two enzymes in mouse spleen, a situation different from that found in certain plants.     

The role of adenosine 3':5'-cyclic monophosphate in relation to the diuretic hormone of Rhodnius prolixus.

The relationship between diuretic hormone (DH) and adenosine 3′:5′-cyclic monophosphate (cyclic AMP) in Rhodnius Malpighian tubules has been investigated. Direct measurement of cyclic AMP levels during stimulation of the tubules by DH supports the view that cyclic AMP is a ‘second messenger’ in this system.Also, the activity of endogenous cyclic AMP phosphodiesterase and its inhibition by theophylline has been investigated briefly. Certain other 3′:5′-cyclic nucleotides have been examined for diuretic activity on Rhodnius Malpighian tubules.     

Purification and properties of a high affinity guanosine 3′: 5′-cyclic monophosphate-binding phosphatase from silver beet leaves

A cyclic nucleotide-binding phosphatase was purified from silver beet leaves by a procedure involving chromatography on CM-Sepharose CL-6B, DEAE-Sephacel, casein-Sepharose 4B, concanavalin A-agarose and Ultrogel AcA44. The enzyme is eluted from concanavalin A-agarose by 0.5 M α-methylglucoside at high ionic strength. The enzyme is monomeric, having a subunit molecular weight (M_r) of 28 000; the native M_r is 31 000 as determined from gel filtration. The enzyme catalyzes the hydrolysis of a range of phosphomonoesters including various nucleotides and O-phosphotyrosine but not O-phosphoserine or O-phosphothreonine. The leaf phosphatase is competitively inhited by guanosine 3′ : 5′-cyclic monphosphate (cGMP) and adenosine 3′ : 5′-cyclic monophosphate (cAMP) (K_i-values: 0.4 μM and 3.3 μM, respectively). The leaf phosphotase has the highest affinity for cGMP yet reported for a plant protein.     

The Riddle of Formycin A Insulinotropic Action

Formycin A augments insulin release evoked by glucose (5.6 mmor more), this effect not being rapidly reversible. The mechanism responsible for the insulinotropic action of formycin A was investigated in isolated pancreatic islets. It could not be ascribed to facilitation of glucose metabolism. On the contrary, formycin A inhibited glucose oxidation, lowered ATP content, and impaired glucose-stimulated protein biosynthesis. The insulinotropic action of formycin A was apparently attributable to its conversion to formycin A 5′-triphosphate, both this process and the secretory response to formycin A being abolished by the inhibitor of adenosine kinase 5-iodotubercidin. In agreement with the latter view, adenosine receptor antagonists such as 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine failed to suppress and, instead, augmented the insulinotropic action of formycin A. Unexpectedly, however, formycin A failed to decrease⁸⁶Rb efflux, this coinciding with a low efficiency of formycin A 5′-triphosphate to inhibit K_ATP-channel activity in excised membranes and with the fact that formycin A increased gliben-clamide-stimulated insulin release. The secretory response to formycin A represented a Ca²⁺-dependent process suppressed in the absence of extracellular Ca²⁺or presence of verapamil and associated with an increased net uptake of⁴⁵Ca. Nevertheless, the view that formycin A exerts any major effect upon intracellular Ca²⁺redistribution, protein kinase C activity, or cyclic AMP net production also met with objections such as the minor secretory effect of formycin A in islets exposed to a high concentration of K⁺in the presence of a diazoxide analog, the resistance of formycin A insulinotropic action to bisindolylmaleimide, the poor increase of cyclic AMP content in formycin A-stimulated islets, and the pronounced enhancement by forskolin or theophylline of insulin release from islets exposed to formycin A. It is concluded, therefore, that the mechanism of action of formycin A in the pancreatic β-cell remains to be elucidated.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.