Structural basis for selectivity of a small molecule, S1-binding, submicromolar inhibitor of urokinase-type plasminogen activator

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a protease associated with tumor metastasis and invasion. Inhibitors of uPA may have potential as drugs for prostate, breast and other cancers. Therapeutically useful inhibitors must be selective for uPA and not appreciably inhibit the related, and structurally and functionally similar enzyme, tissue-type plasminogen activator (tPA), involved in the vital blood-clotting cascade. RESULTS: We produced mutagenically deglycosylated low molecular weight uPA and determined the crystal structure of its complex with 4-iodobenzo[b]thiophene 2-carboxamidine (K(i) = 0.21 +/- 0.02 microM). To probe the structural determinants of the affinity and selectivity of this inhibitor for uPA we also determined the structures of its trypsin and thrombin complexes, of apo-trypsin, apo-thrombin and apo-factor Xa, and of uPA, trypsin and thrombin bound by compounds that are less effective uPA inhibitors, benzo[b]thiophene-2-carboxamidine, thieno[2,3-b]-pyridine-2-carboxamidine and benzamidine. The K(i) values of each inhibitor toward uPA, tPA, trypsin, tryptase, thrombin and factor Xa were determined and compared. One selectivity determinant of the benzo[b]thiophene-2-carboxamidines for uPA involves a hydrogen bond at the S1 site to Ogamma(Ser190) that is absent in the Ala190 proteases, tPA, thrombin and factor Xa. Other subtle differences in the architecture of the S1 site also influence inhibitor affinity and enzyme-bound structure. CONCLUSIONS: Subtle structural differences in the S1 site of uPA compared with that of related proteases, which result in part from the presence of a serine residue at position 190, account for the selectivity of small thiophene-2-carboxamidines for uPA, and afford a framework for structure-based design of small, potent, selective uPA inhibitors.     

A convenient synthesis of 5-(1,2,4-oxadiazol-5-yl)pyrimidine-2,4(1H,3H)-diones

The synthesis of 5-heteroaryl-substituted uracil derivatives is presented. The 1,3-dipolar cycloaddition reaction was applied for the construction of a heterocyclic ring. The nitrile oxides were obtained from the appropriate 4-substituted benzaldoximes using N-chlorosuccinimide (NCS) under basic conditions. [2+3] Cycloaddition of nitrile oxides with 5-cyanouracil as a dipolarophile gave the corresponding 5-(3-substituited-1,2,4-oxadiazol-5-yl)uracils in satisfactory yields under mild conditions. 5-Substituted uracils having an additional heterocyclic ring were obtained as a result of the [2+3] cycloaddition of 5-cyanouracil to nitrile oxides generated from thiophene-2-carbaldehyde and 5-formyluracil derivatives.     

Novel platinum pyridinehydroxamic acid complexes: Synthesis,characterisation, X-ray crystallographic study and nitric oxide related properties

Herein, we describe the synthesis and characterisation of a novel class of Pt^II and Pt^IV pyridinehydroxamic acid (pyhaH) complexes of general formula cis-[Pt^IICl₂(x-pyhaH)₂] and cis-[Pt^IVCl₄(x-pyhaH)₂], respectively (where x = 3 or 4) in which the pyridinehydroxamic acid is coordinated to the platinum ion via the pyridine nitrogen only leaving the hydroxamic acid free to potentially release cytotoxic nitric oxide (NO). The crystal structure of the Pt^IV derivative, cis-[PtCl₄(4-pyhaH)₂] · 2CH₃OH is reported. To establish the biological effect of the uncoordinated hydroxamic acid moiety in the Pt^II compounds synthesised, the corresponding pyridinecarboxylic acid (pycaH) complexes of general formula cis-[Pt^IICl₂(x-pycaH)₂] (where x = 3 or 4) and the Pt^II pyridine (py) complex, cis-[Pt^IICl₂(py)₂] were synthesised and served as reference standards. The NO-releasing properties of each of the Pt^II compounds, the pyhaH and the pycaH ligands were studied. The Pt^II pyridinehydroxamic acid derivatives were found to induce potent in vitro effects attributable to either NO-release from the hydroxamic acid moiety and/or stimulation of inducible nitric oxide synthase of endothelial cells.     

One-pot synthesis of thieno [2,3-d] pyrimidin-4-ol derivatives mediated by polyphosphonic anhydride

An efficient synthesis of thiopyrimidines with different substituents in position 2 is described. A rapid, mild and high yielding microwave-assisted one-pot cyclization of 5-substituted 2-amino thiophene-3-carboxamide derived from Gewald reaction¹ with T3P and different acids gives the corresponding thiopyrimidines. The significant feature of this method includes less reaction time, high purity and reduced toxicity of the reaction.     

A General Procedure for Synthesis of NG-Alkyl,and NG-Aryl-L-Arginines as Potential Nitric Oxide Synthase inhibitors

A general procedure for the synthesis of N^G-alkyl, and N^G-aryl-L -arginines with relatively high overall yield is reported. The key step involved the coupling of protected L -ornithine 4 with isothiourea 7 to give the fully protected N^G-aryl-L -arginine derivative 8 . Subsequent deprotection of 8 in acidic condition provided the final target compound 9 with an overall yield of more than 80%.     

Synthesis and biological evaluation of 2,4-diaminopteridine derivatives as nitric oxide synthase inhibitor

A series of novel 2,4-diamino-pteridines(9a-1)were synthesized and evaluated as inhibitors of inducible nitric oxide synthase (iNOS)in vitro.It was found that 9a,9d,9e,9h,9i and 91 showed potent inhibitory activities similar to that of methotrexate(MTX),while the activities of 9b,9c,9f,9g,9j and 9k ale stronger than MTX.     

A new synthesis of naphtho[2,3-C]pyrroles. Conversion of naphtho[2,3-C]thiophene-4,9-diones to naphtho[2,3-C]pyrrole-4,9-diones

A series of 2-substituted-4,9-dihydronaphtho[2,3-c]pyrrole-4,9-diones was prepared by the reaction of 4,9-dihydronaphtho[2,3-c]thiophene-4,9-diones with primary amines under mild conditions. The presence of halogen in the naphtho[2,3-c]thiophene-4,9-dione and the presence of hydroxyl, ether, or tertiary amine functions in the amine reagent do not interfere with the course of the reaction.     

Effects of Siqi Decoction on Concentration of Nitric Oxide and Activity of Nitric Oxide Synthase in Blood Serum of Rats with Myocardial Ischemia

To study the effects of Siqi decoction on rats with Myocardial Ischemia, the model of Myocardium Ischemia was made for the Wistar rats cured with posterior pituitary injection through vein in tail. Siqi decoction, Diaoxinxuekang（DK） and Fufangdanshenpian（FD）, the latter two drugs of which were effective TCM drugs of anti-Myocardial Ischemia at present, were administrated to the rats with Myocardium Ischemia for 5 days to compare the effect of anti-Myocardium Ischemia as reference drugs by measuring the changes of NO concentration and activity of NOS in the rat blood serum with Myocardial Ischemia. There was a remarkable increase in the NO concentration and activity of NOS in serum in Siqi decoction groups compared with those in control group（p〈0.05）. The results of the prevention group in experiment of Siqi decoction are better than those of the cure group. Siqi decoction was really fit for Myocardium Ischemia via increasing NO concentration by stimulating the activity of NOS in serum. The effect of Siqi decoction against Myocardium Ischemia in preventive group is better than the curative that of Siqi decoction in the curative group.     

Synthesis of polysubstituted benzothiophenes and sulfur-containing polycyclic aromatic compounds via samarium diiodide promoted three-component coupling reactions of thiophene-2-carboxylate

By the promotion of samarium diiodide, thiophene-2-carboxylate reacted with 2 equiv of ketones at the C-4 and C-5 positions to give diols such as 2 and 9. Because the intermediary organosamarium species were oxophilic but not too basic, the double hydroxyalkylations with various ketone substrates, including alkyl aryl ketones, acetylthiophenes, cyclohexanone, alpha-tetralone, and alpha-phenylacetophenones, were realized without complication of side reactions. The diol products underwent an acid-catalyzed dehydration to give dienes such as 3 and 10, which were treated with DDQ to give either polysubstituted thiophenes (e.g., 4 and 11) or benzothiophenes (e.g., 5, 13, and 14) depending on the reaction conditions. Oxidative annulations of 4,5-diarylthiophenes 11 and 4,5,6,7-tetraphenylbenzothiophenes 14 were carried out by photochemical or chemical methods to give the sulfur-containing polycyclic aromatic compounds, such as phenanthro[9,10-b]thiophene-2-carboxylate, piceno[13,14-b]thiophene-2-carboxylate, and tribenzo[fg,ij,rst]pentapheno[15,16-b]thiophene-2-carboxylates. This method is applicable to the preparation of polysubstituted thiophenes, benzothiophenes, and the related compounds possessing liquid crystalline, photochromic, and other functional properties.     

What Flow Injection has to Offer in the Environmental Analytical Field

NO plays an important role in the regulation of physiological and pathophysiological mechanisms, and quite a lot of investigations have been focused on this kind of special molecule. It is difficult to achieve reliable ex vivo NO measurements with the existing analytical methods, and developing a reliable in vivo monitoring method for NO is still an urgent task. In this review, the techniques utilized for the determination of endogenous NO formed by enzymatic action and its reactions with other biological substances found in living organisms are discussed with respect to applications both in vivo or in vitro. In addition, various NO (micro)sensors and trap probes based on different principles are presented with their respective advantages and limitations. Finally, an NO monitoring system based on the combination of microdialysis sampling and chemiluminescence is introduced which is considered to be a prospective method for in vivo monitoring.     

Synthese und NMR-spektroskopische Charakterisierung neuer Adenin- und 6-Thioxopurincarboxamidine

Synthesis and NMR-Spectroscopic Characterization of New Adenine- and 6-Thioxopurinecarboxamidines Adenine (1) or 1H-purine-6(7H)-thione ( 4 ) react with carboximidoyl chlorides 2 to yield the adenine-9-carboxamidines 3 or the 6,7-dihydro-6-thioxo-1H-purine-7-carboxamidines 5 and 6,9-dihydro-6-thioxo-1H-purine-9-carboxamidine 6 a , respectively. The structures of the compounds were determined by ¹H- and ¹³C-NMR spectroscopy.     

Reductive decomposition of a diazonium intermediate by dithiothreitol affects the determination of NOS turnover rates

Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure-function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed.     

C-aminoimidoylation and C-thiocarbamoylation of esters, sulfones, and ketones

Esters, sulfones, and ketones were C-aminoimidoylated and C-thiocarbamoylated by benzotriazole-1-carboxamidines 8a-g and 1-(alkyl-or-arylthiocarbamoyl)benzotriazoles 9a-i, respectively. The present work represents the first systematic approach to these compound classes, the few previously known examples of which were obtained by diverse approaches.     

Structure of aminoguanidine hemioxalate. Implications for the synthesis of amidinohydrazones

Summary The crystal and molecular structure of aminoguanidine hemioxalate, a salt in which aminoguanidine exists in the monocation form, was determined by single crystal X-ray diffraction. The salt crystallizes in the monoclinic space group P2(1)/n with unit cell dimensions ofa=4.95,b=10.46,c=10.40 Å, =92.57°, andZ=4. The structure contains one oxalate ion for every two CN₄H ₇ ⁺ ions, the latter being practically planar. The structure of the monocation is largely similar to those of aminoguanidine dications except that the monocation is devoid of one of the protons attached to the terminal hydrazine nitrogen. This result is of interest considering the synthesis of amidinohydrazones, indicating that the concentration of the active nucleophile is nearly maximal even when aminoguanidine exists in the monocation form. Therefore, the synthesis of amidinohydrazones should be performed in thepH range in which aminoguanidine exists mainly in the monocation form,i.e. at apH higher than 2. There is, however, no need to elevate thepH to values at which a considerable proportion of aminoguanidine exists as the free base.

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Die Struktur von Aminoguanidinhemioxalat und ihre Bedeutung für die Synthese von Amidinohydrazonen

Zusammenfassung Die Kristallstruktur von Aminoguanidinhemioxalat, einem Salz, in dem Aminoguanidin als Monokation existiert, wurde mit Einkristallröntgenmethoden aufgeklärt. Das Salz kristallisiert in der monoklinen Raumgruppe P2(1)/n mit den Zellparameterna=4.95,b=10.46,c=10.40 Å, =92.57° undZ=4. In der Einheitszelle kommt ein Oxalation auf je zwei flache CN₄H ₇ ⁺ -Ionen. Die Struktur des Monokations ist bekannten Strukturen des Aminoguanidindikations ähnlich, mit der Ausnahme, daß dem Monokation eines der an das äußere Stickstoffatom der Hydrazingruppe gebundenen drei Protonen fehlt. Dieses Ergebnis ist interessant bezüglich der Synthese von Amidinohydrazonen, da es bedeutet, daß die Konzentration des aktiven Nukleophils auch dann beinahe maximal ist, wenn Aminoguanidin in der Monokationform vorliegt. Synthesen von Amidinohydrazonen sollten daher unter solchen Bedingungen ausgeführt werden, unter denen Aminoguanidin hauptsächlich in der Monokationform existiert (pH höher als 2). Es ist jedoch nicht nötig, beipH-Werten zu arbeiten, die so hoch sind, daß ein bedeutender Teil der Verbindung als freie Base vorliegt.

     

Microwave-assisted Cannizzaro reaction—Optimisation of reaction conditions

The microwave-assisted Cannizzaro reaction was studied in order to develop fully reproducible synthetic protocols for transformation of aldehydes to carboxylic acid and alcohols. Optimised were the following process parameters: power, temperature, and time. Aromatic, heteroaromatic and aliphatic aldehydes were used in the studies. It was found that furfural, thiophene-2-carbaldehyde, pyridinecarboxaldehyde and aromatic aldehydes react under mild conditions, while 1-methyl-pyrrole-2-carboxaldehyde derivatives and aliphatic aldehydes require more drastic reaction conditions and a longer exposure time to microwave radiation.     

Electrochemical Investigation of the Role of Reducing Agents in Copper‐Catalyzed Nitric Oxide Release from S‐Nitrosoglutathione

Studies of nitric oxide (NO) release from S‐nitrosoglutathione (GSNO) decomposition by Cu²⁺ in the presence of reducing agents were performed using a nickel porphyrin and Nafion‐coated microsensor in order to compare the efficiency of sodium hydrosulfite (Na₂S₂O₄) and sodium borohydride (NaBH₄) to that of the most abundant endogenous reducer, glutathione (GSH). When it was mixed to Cu(NO₃)₂ and added to equimolar concentration of GSNO, each reducing agent caused a NO release (measured in terms of oxidation current) but only NaBH₄ induced a proportional rise if its concentration doubled and that of Cu²⁺ remained constant. For Na₂S₂O₄, there was a mild increase and for GSH, no change. Furthermore, when Cu²⁺ concentrations ranging from 0.5 to 5 μM were mixed with 2 μM reducing agent and added to 2 μM GSNO_, the NO oxidation current linearly increased with NaBH₄ and was constant with Na₂S₂O₄. Concerning GSH, Cu²⁺ dose‐dependently increased the NO release from GSNO only if the Cu²⁺‐to‐reducer ratio was ≤1. However, GSH formed the catalytic species Cu⁺ even in excess of Cu²⁺ and GSNO as indicated by suppression of the Cu²⁺/GSH‐induced NO release when the Cu⁺ chelator neocuproine was added to GSNO. This work shows that, among the 3 reducing agents, only NaBH₄ allows Cu²⁺ to dose‐dependently increase the NO release from GSNO for Cu²⁺‐to‐reducer ratios ranging from 0.25 to 2.5. Despite this good effectiveness, excess of NaBH₄ compared to both Cu²⁺ and GSNO seems to be required for optimal NO release.     

Synthesis of 2-pyridyl-substituted derivatives of 7-benzyl-5,6,7,8-tetra-hydropyrido[3,4-<Emphasis Type="Italic">d</Emphasis>]pyrimidine

A method has been developed for the synthesis of 2-pyridyl-substituted derivatives of 7-benzyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine based on the condensation of ethyl 1-benzyl-3-oxopiperidine-4-carboxylate with pyridyl-2-, pyridyl-3-, and pyridyl-4-carboxamidines and subsequent reactions of 7-benzyl-2-pyridyl-5,6,7,8-tetrahydro-3H-pyrido[3,4-d]pyrimidin-4-ones with trifluoromethanesulfonic anhydride and secondary amines. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, 1556–1561, October, 2007.     

Calreticulin Transacetylase Mediates the Acetylation of Nitric Oxide Synthase by Polyphenolic Acetate

Our earlier investigations identified acetoxy drug: protein transacetylase (TAase), a unique enzyme in the endoplasmic reticulum (ER) catalyzing the transfer of acetyl groups from polyphenolic acetates (PA) to certain functional proteins. Recently we have established the identity of TAase with ER protein calreticulin (CR) and subsequently transacetylase function of CR was termed calreticulin transacetylase (CRTAase). CRTAase was purified and characterized from human placenta. CRTAase catalyzed the acetylation of a receptor protein nNOS, by a model PA 7, 8-diacetoxy-4-methylcoumarin (DAMC), which was visually confirmed by using antiacetyl lysine. The aim of this report was to provide tacit proof by providing mass spectrometry evidence for CRTAase catalyzed acetylation of purified nNOS by DAMC. For this purpose, purified nNOS was incubated with DAMC and CRTAase, the modified nNOS was analyzed by nanoscale LC-MS/MS, which recorded 11 distinct peptides with a significant score as acetylated on lysine residues. The distribution was in order: lysines-24, -33, -38, -131, and -229 of the PDZ domain, Lys-245 of the oxygenase domain, Lys-754 and -856 of FMN binding domain, Lys-989 of connecting domain and Lys-1300, -1321, and -1371 of the NADPH-binding domain were acetylated. The results documented in this paper highlighted for the first time modification of nNOS by way of acetylation. Our earlier work recorded the profound activation of platelet NADPH cytochrome P-450 reductase and the acetylation of the reductase protein by DAMC, which also remarkably enhanced intracellular levels of nitric oxide. The results reported here coupled with the aforementioned previous observations strongly implicate the possible role of the acetylation of the reductase domain of nitric oxide synthase (NOS) in the NOS activation. In addition, the acetylation of nNOS can be expected to potentiate the interaction with CR, eventually leading to the augmented catalytic activity of NOS and expression of the related biological effects.     

Some heterocyclic sulfonyl chlorides and derivatives

The syntheses of 4- and 5-chlorosulfonylfuran-2-carboxylic acid (Ia,IIa), 4-chlorosulfonylfuran-2-carboxamide (Ib), 3,5-dimethylpyrazole and isoxazole-4-sulfonyl chlorides (IIIa,IVa) and 2,4-dimethylthiazole-5-sulfonyl chloride (Va) are described. The sulfonyl chlorides were converted into a range of amides, hydrazides and azides. Condensation of the sulfonohydrazides with β-dicarbonyl compounds, gave the corresponding β-ketohydrazones (VII), which, with the exception of the derivatives (VIIe,f,g,i), were converted to the sulfonylpyrazoles (VIII). The structures and spectral data of these compounds are briefly discussed. The reaction of the sodio derivative of acetylacetone with thiophene-2-sulfonyl chloride (VIc) gave 3-(thiophene-2-sulfonyl)pentane-2,4-dione (XII), which with hydrazine gave 4-(thiophene-2-sulfonyl)-3,5-dimethylpyrazole (XIII). However, the analogous reaction with thiophene-2-sulfonohydrazide (VIa) failed to give the expected 1,4-bisthiophenesulfonylpyrazole.     

Synthesis and Some Reactions of 2-(Thien-2-yl)naphtho[1,2-d]thiazole

Russian Journal of General Chemistry - Condensation of 1-aminonaphthalene with thiophene-2-carbonyl chloride in 2-propanol furnished N-(1-naphthyl)thiophene-2-carboxamide, the treatment of which...