Synthesis of tritium-labeled N-acetyl PGE2 carboxamide (CP-27,987)

The preparation of N-acetyl PGE2 carboxamide (CP-27,987) regioselectively labeled at C-18 and C-19 with tritium is described. The overall radiochemical yield was 8.0% at a specific activity of 0.49 Ci/mmole. The synthesis employed is applicable to the preparation of tritium labeled natural prostaglandins and a variety of analogs.     

Metabolism of N-acetyl PGE2 carboxamide in the rat

After intratracheal administration to rats, the bronchodilator N-acetyl PGE₂ carboxamide was converted rapidly to PGE₂ and 13,14-dihydro-15-keto-PGE₂, the major plasma metabolite. Oxidation of the N-acetyl carboxamide by prostaglandin dehydrogenase and hydrolysis of the imide bond were demonstrated in vitro.     

Synthesis of tritium-labeled N-acetyl PGE2 carboxamide (CP-27,987)

The preparation of N-acetyl PGE₂ carboxamide (CP-27,987) regioselectively labeled at C-18 and C-19 with tritium is described. The overall radiochemical yield was 8.0% at a specific activity of 0.49 Ci/mmole. The synthesis employed is applicable to the preparation of tritium labeled natural prostaglandins and a variety of analogs.     

Characterization of an enantioselective amidase with potential application to asymmetric hydrolysis of (<Emphasis Type="Italic">R</Emphasis>, <Emphasis Type="Italic">S</Emphasis>)-2, 2-dimethylcyclopropane carboxamide

Amidase is a promising synthesis tool for chiral amides and related derivatives. In the present study, the biochemical properties of the Delftia tsuruhatensis CCTCC M 205114 enantioselective amidase were determined for its potential application in chiral amides synthesis. D. tsuruhatensis CCTCC M 205114 amidase was purified 105.2 fold with total activity recovery of 4.26%. The enzyme is a monomer with a subunit of approximately 50 kDa by analytical gel filtration HPLC and SDS–PAGE. It had a broad substrate spectrum and displayed high enantioselectivity against R-2, 2-dimethylcyclopropane carboxamide and R-mandelic amide. The amidase was applied to enantioselective hydrolysis of R-2, 2-dimethylcyclopropane carboxamide from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate S-2, 2-dimethylcyclopropane carboxamide. This enzyme did not require metal ions for the hydrolysis reaction. Its optimal pH and temperature were 8.0 and 35°C, respectively. The K _m and V _max of the amidase for R-2, 2-dimethylcyclopropane carboxamide were 2.54 mM and 8.37 μmol min⁻¹ mg protein⁻¹, respectively. After 60 min of the reaction, R-2, 2-dimethylcyclopropane carboxamide was completely hydrolyzed, generating S-2, 2-dimethylcyclopropane carboxamide with a yield of 45.9% and an e.e. of above 99%. Therefore, this amidase can serve as a promising producer for S-2, 2-dimethylcyclopropane carboxamide and other amides.     

Novel M4 positive allosteric modulators derived from questioning the role and impact of a presumed intramolecular hydrogen-bonding motif in β-amino carboxamide-harboring ligands

This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M₄ PAMs and question if the NH₂ group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH₂, generating des-amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M₄ PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M₄ PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M₄ PAM activity within classical bicyclic M₄ PAM scaffolds.     

Synthesis and structural analysis of oxadiazole carboxamide deoxyribonucleoside analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2'-endo ("south") conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.     

Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2.

A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 mumol.min-1.mg-1 at 30 degrees C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenylethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.     

Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.     

Syntheses and opioid receptor binding properties of carboxamido-substituted opioids

A series of 15 novel opioid derivatives were made where the prototypic phenolic-OH group of traditional opioids was replaced by a carboxamido (CONH₂) group. For 2,6-methano-3-benzazocines and morphinans similar or, in a few instances, enhanced affinity for μ, δ and κ opioid receptors was observed when the OH → CONH₂ switch was applied. For 4,5α-epoxymorphinans, binding affinities for the corresponding carboxamide derivatives were much lower than the OH partner consistent with our pharmacophore hypothesis concerning carboxamide bioactive conformation. The active metabolite of tramadol and its carboxamide counterpart had comparable affinities for the three receptors.     

Protective effects of N-acetyl cysteine on lipid peroxide metabolism on isoproterenol-induced myocardial infarcted rats

The present study was designed to evaluate the preventive effects of N-acetyl cysteine on lipid peroxide metabolism in isoproterenol (ISO) induced myocardial infarcted rats. Male albino Wistar rats were pretreated with N-acetyl cysteine (5 and 10 mg/kg) daily for a period of 14 days. After the pretreatment period, ISO (100 mg/kg) was subcutaneously injected to rats twice at an interval of 24 h. Increased activities of serum creatine kinase, creatine kinase-MB, lactate dehydrogenase, and increased intensities of serum lactate dehydrogenase-isoenzyme bands (LDH-1, LDH-2) were observed in ISO-induced rats. The heart lipid peroxidation products were significantly increased, and the antioxidant system was significantly reduced in ISO-induced rats. Pretreatment with N-acetyl cysteine (5 and 10 mg/kg) to ISO-induced rats showed significant effects on all the biochemical parameters studied. Histopathological findings of the myocardium also showed the protective role of N-acetyl cysteine in ISO-induced rats. Furthermore, in vitro study confirmed the potent-free radical scavenging activity of N-acetyl cysteine. The effect at a dose of 10 mg/kg of N-acetyl cysteine was more pronounced than the dose, 5 mg/kg. The results of our study show that N-acetyl cysteine protects the heart against ISO-induced myocardial infarction by its free radical scavenging effect.     

EPR and magnetic studies of copper amino carboxylates

Metal complexes of N-substituted amino acids provide simple but appropriate model compounds for the understanding of metal protein interactions. Seven complexes of copper viz. Cu(II) N-acetyl glycinate mono-hydrate, Cu(II) N-acetyl methioninate, Cu(II) N-acetyl alaninate, Cu(II) N-acetyl valinate, Cu(II) N-acetyl glutamate, Cu(II) cyanoacetate, and Cu(II) thio-dipropionate, have been investigated by EPR measurements. The spectra appear to arise from dimeric coppers (s=1) coupled by anti-ferromagnetic exchange. The exchange coupling constant ‘2J’ and the CuCu separation ‘r’ have been evaluated from the spectral data. Although the existence of bridged structure is confirmed, super-exchange via the ligands appears to be the dominant mechanism. All of these complexes exist as monomers in strongly coordinating solvents.     

N-Acetyl-L-Tyrosine (NAT) as a Substrate for Mushroom Tyrosinase

N-acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N-acetyl dopa formed is oxidized by the enzyme to N-acetyl dopaquinone (λ_max= 390 ± 10 nm). H₂O₂ and NH₂OH each shortened the lag period of NAT hydroxylation by the enzyme. H₂O₂ had an effect on the changes with time in the spectrum of product(s) formed and on the spectrum of the final product(s) obtained when NAT was hydroxylated by mushroom tyrosinase, in a manner suggesting that H₂O₂ converts N-acetyl dopaquinone to a pink-violet product(s) (λ_max= 490 nm), whereas such a product(s) was not formed in the absence of H₂O₂. A pink-violet product(s) (λ_max 490 ± 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH₂OH or para amino benzoic acid (PABA), probably as a result of an interaction between N-acetyl dopaquinone and NH₂OH or PABA forming mono- or di-oximes. Kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) inhibited effectively the rate of NAT hydroxylation by mushroom tyrosinase in the absence or presence of H₂O₂. When NAT was oxidized by the enzyme in the absence of kojic acid, N-acetyl dopaquinone was formed at once and a shoulder at 490–530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a peak at 320±10 nm, was detected, suggesting that N-acetyl dopaquinone oxidizes kojic acid to the yellow product(s). Maltol(3-hydroxy-2-methyl-4H-pyran-4-one), a γ-pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroom tyrosinase. The addition of maltol at the plateau phase of the reaction resulted in an immediate decline in absorbance at 400 nm, suggesting that maltol conjugates with N-acetyl dopaquinone, yielding a product(s) characterized by a lower extinction coefficient at 400 nm than that of N-acetyl dopaquinone alone. The final brown-red product(s) formed when NAT was hydroxylated by mushroom tyrosinase was bleached in the presence of ascorbic acid or H₂O₂.     

Synthesis, fluorine-18 radiolabeling, and in vitro characterization of 1-iodophenyl-N-methyl-N-fluoroalkyl-3-isoquinoline carboxamide derivatives as potential PET radioligands for imaging peripheral benzodiazepine receptor

The isoquinoline carboxamide derivative 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) has been shown to bind strongly and selectively to the peripheral benzodiazepine receptor (PBR) binding sites. A series of PK11195 analogues have been synthesized and biologically characterized. The affinities of the analogues for the PBR were determined using in vitro competitive binding assays with [(3)H]PK11195 in rat kidney mitochondrial membranes. The results showed that the 1-(2-iodophenyl)-N-methyl-N-(3-fluoropropyl)-3-isoquinoline carboxamide (9a) was the most potent compound (K(i)=0.26nM) of this series and is an excellent lead ligand for additional studies for labeling with fluorine-18 to determine whether it possesses the desired in vivo performance in non-human primates by PET imaging. Thus, radiolabeling of 9a with fluorine-18 was developed.     

High-performance liquid chromatographic assay for the determination of sulfadoxine and N-acetyl sulfadoxine in plasma from patients infected with sensitive and resistant Plasmodium falciparum malaria

A reversed-phase high-performance liquid chromatographic method using a mobile phase of acetonitrile-methanol-trifluoroacetic acid-water (16.1:7.2:0.1:76.6, v/v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher RP-18 column with UV (254 nm) detection has been developed for the separation of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma. No interferences due to endogenous compounds or common antimalarial drugs were noticed. The limit of detection for sulfadoxine and N-acetyl sulfadoxine was 0.01 microg ml(-1) with a signal-to-noise ratio of 5:1 while the limit of quantification was 2.5 microg ml(-1). Intra-day mean relative standard deviations (RSD's) for sulfadoxine and N-acetyl sulfadoxine were 2.6 and 2.8%, respectively, while mean inter-day RSD's for sulfadoxine and N-acetyl sulfadoxine were 2.4 and 2.8%, respectively. Extraction recoveries averaged 90.6% for sulfadoxine and 86.9% for N-acetyl sulfadoxine. The method was applied for the assay of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma from Plasmodium falciparum malaria patients. Mean plasma sulfadoxine concentrations on day 2 (51 h) from samples collected from sensitive and resistant P. falciparum patients treated with three tablets of Fansidar were 62.8 and 60.5 microg ml(-1), respectively. Mean ratio of N-acetyl sulfadoxine to sulfadoxine was 9.1% for responders and 13.9% for non-responders which revealed that higher amounts of the metabolite N-acetyl sulfadoxine were present in non-responders. The method described should find an application in the therapeutic monitoring of malaria patients.     

Synthesis of undecagold cluster molecules as biochemical labeling reagents. 2. Bromoacetyl and maleimido undecagold clusters

Derivatives of heptakis[4,4',4"-phosphinidynetris(benzenemethanamine) ]undecagold, 1, molecular formula Au11(CN)3[P(C6H4CH2NH3)]7, are described. These include undecagold complexes with a single free primary amino group, a single bromoacetyl group, and a single maleimido group per molecule. Hydrolysis of mono(N-phthalyl)icosa(N-acetyl)-1 at pH 3.2 and 46 degrees C under anaerobic conditions and in the presence of NaBH3CN produces icosa(N-acetyl)-1. Partial acylation of 1 with 1.3 equiv of 2,3-dimethylmaleic anhydride followed by complete acetylation with acetic anhydride produces a mixture consisting largely of mono- and bis(dimethylmaleyl)peracetyl-1. Hydrolysis of 2,3-dimethylmaleimides at pH 3.2 for 1 at 25 degrees C produces a mixture of icosa(N-acetyl)-1, with a single free amino group, and nondea(N-acetyl)-1. This mixture can be quantitatively separated by cation-exchange chromatography at pH 7, giving homogeneous icosa(N-acetyl)-1 in an overall yield of 55%. Icosa(N-acetyl)-1 serves as the starting material for the synthesis of the alkylating derivatives mono(N-bromoacetyl)icosa(N-acetyl)-1 and mono[N-(p-maleimidobenzoyl)]icosa(N-acetyl)-1. These derivatives can be used for alkylating proteins in preparation for electron microscopy.     

C-nucleoside analogues of furanfurin as ligands to A1 adenosine receptors

Furanfurin (2-beta-D-ribofuranosylfuran-4-carboxamide) derivatives and analogues were synthesized and their affinity for adenosine receptors was determined. The agonistic behavior of furanfurin against A1 receptors is preserved only when the furan ring is substituted with isosteric pentatomic ring systems such as oxazole, thiazole or thiophene, and the carboxamide group is unsubstituted. Replacement of the hydrogen atoms of the carboxamide group with alkyl, cycloalkyl or arylalkyl groups generates compounds endowed with moderate antagonistic activity.     

Fatty acid binding protein (FABP) modulates prostaglandin E binding to rat epididymal adipocyte membrane similarly to albumin

Albumin enhances prostaglandin E2 (PGE2) binding to isolated epididymal adipocyte membrane and also binds PGE2 with low affinity. On the other hand, S-100, ovalbumin and albumin-stearate failed to bind PGE2, as shown by ultrafiltration, and also failed to enhance PGE2 binding to the isolated adipocyte membranes. These results suggested that albumin enhances PGE2 binding possibly by serving as a carrier for the prostaglandin molecules. 3 mM warfarin or 1 mM phenylbutazone inhibited PGE2 binding to albumin by 70% and 95%, respectively, but both drugs failed to affect the enhancement of PGE2 binding to the isolated adipocyte membrane in the presence of albumin. These results exclude the possibility that PGE2 bound to albumin is more accessible to the prostaglandin receptor than free PGE2 in solution. Finally it is shown that fatty acid binding protein (FABP), a cytosolic protein which binds specifically PGE1 but not PGE2, enhances PGE1 and PGE2 binding to isolated adipocyte membranes similarly to albumin. The physiological implications of these findings are discussed.     

Separation of a population of human T lymphocytes that bind prostaglandin E2 and exert a suppressor activity

Prostaglandin E2 (PGE2) is a potent inhibitor of immune functions. Two possible mechanisms of PGE2-mediated suppression have been proposed: one is a direct inhibitory effect exerted on interleukin 2-producing T cells; the second is mediated by the activation of nonspecific suppressor T lymphocytes. We previously showed that PGE2 can directly activate human T lymphocytes to suppress lymphocyte proliferation and B lymphocyte maturation. Herein is described the binding of 10 to 30% of human peripheral blood T lymphocytes to insolubilized PGE2 coated to albumin-Sepharose. The T lymphocytes that bound PGE2 (PGE2(+] could be eluted by the addition of serum and gentle shaking of the beads. The following data indicated the specificity of the binding: i) T lymphocytes after an overnight incubation, a condition known to abolish sensitivity to PGE2, lost their affinity for PGE2; ii) preincubation of T lymphocytes with PGE2 blocked the binding; iii) PGE2(+) T cells bound PGE after a 24-hr incubation, whereas PGE2(-) T cells did not. Few T cells bound albumin, and only a small percentage (7 to 9%) bound 6-keto-prostaglandin F1 alpha-coated beads. Among PGE2(+) T cells, there was a slight increase in the percentage of OKT8+ cells. Although T cells that had no affinity for PGE2 (PGE2(-] proliferated as well as unseparated T lymphocytes when stimulated with mitogens or antigens, the proliferative response of the PGE2(+) subset was poor. Moreover, PGE2(+) T lymphocytes did exert a strong suppressor activity on mitogen- or allogeneic cell-induced lymphocyte proliferation as well as on pokeweed mitogen-driven B cell maturation into Ig-containing cells. PGE2(-) T lymphocytes were shown not to exert a significant suppressor activity in these assays. The PGE2(+) subset-mediated suppression was not secondary to a carry-over of PGE2 released from the beads, because its suppressor activity was not altered by the addition of an anti-PGE2 serum. Moreover, PGE2(-) T lymphocytes were not sensitive to the inhibitory activity on cell proliferation of PGE2. These results indicate that a given functional subset of peripheral blood T lymphocytes binds PGE2, and that at least some of them are activated into suppressor T cells. The relationship between the PGE2-activatable T suppressor subset and other functionally defined suppressor T cells remains to be clarified; it is suggested, however, that PGE2 can act as an immunoregulator through the activation of identifiable suppressor T cells.     

Structure-based design of 2-arylamino-4-cyclohexylmethyl-5-nitroso-6-aminopyrimidine inhibitors of cyclin-dependent kinases 1 and 2

A series of O(4)-cyclohexylmethyl-5-nitroso-6-aminopyrimidines bearing 2-arylamino substituents was synthesised and evaluated for CDK1 and CDK2 inhibitory activity. Consistent with analogous studies with O(6)-cyclohexylmethylpurines, 2-arylaminopyrimidines with a sulfonamide or carboxamide group at the 4'-position were potent inhibitors, with IC(50) values against CDK2 of 1.1+/-0.3 and 34+/-8 nM, respectively. The crystal structure of the 4'-carboxamide derivative, in complex with phospho-Thr160 CDK2/cyclin A, confirmed the expected binding mode of the inhibitor, and revealed an additional interaction between the carboxamide function and an aspartate residue.     

Signaling pathways leading to prostaglandin E(2) production by rat cerebral frontal cortex

In this paper, we have determined the effect of both muscarinic acetylcholine receptor (mAChR) and exogenous prostaglandin E(2) (PGE(2)) on PGE(2) production and cyclooxygenases (COX) mRNA gene expression on rat cerebral frontal cortex. Carbachol and PGE(2) increase endogenous PGE(2) production and the COX-1 mRNA levels by activation of PLA(2)s. The COX-1 and COX-2 activity participated in the production of PGE(2) triggered by exogenous PGE(2). While in carbachol-PGE(2) only COX-1 activity is affected. The specific inhibition of PGE(2) receptor was able to impair the increase of endogenous PGE(2) production triggered by both carbachol and exogenous PGE(2). These results suggest that carbachol-activation mAChR increased PGE(2) production that in turn interacting with its own receptor triggers an additional production of PGE(2). Both mechanisms appear to occur by using PLA(2) signaling system. This data should be able to contribute to understand the involvement of PGE(2) in normal brain function and its participation in neuroinflammatory processes.