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ABSTRACT: The use of 2,7-dimethyl-3,8-dinitrodipyrazolo[1,5-a:1',5'-d]pyrazine-4,9-dione as pre-column reagent for LC analysis of amino acids is proposed. The compound reacts (30 min at 68 degrees C in presence of 0.04 M triethylamine in a dimethylsulfoxide-water mixture) with primary and secondary amino function and the stable resulting adducts can be chromatographed under reversed-phase conditions and detected at lambda=280 nm. The derivatization conditions were optimized by a series of experiments. The effect of temperature, triethylamine concentration and reagent on the reaction was investigated. The yield of the glycine derivative was found to be quantitative at a reagent amino acid molar ratio of about 6 by comparison with an authentic specimen of synthesized glycine adduct. Application of the method to quality control of commercially available oral polyaminoacid formulations is described.
Analytica chimica acta 11/2006; 579(2):152-7. · 4.31 Impact Factor
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ABSTRACT: A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure bearing at the 1 and 3 position n-propyl groups have been synthesized, and their affinities at the four human adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)) have been evaluated. In this case the presence of n-propyl groups seems to induce potency at the A(2A) and A(3) adenosine receptor subtypes as opposed to our previously reported series bearing methyl substituents at the 1 and 3 positions. In particular the non-acylated derivative 17 showed affinity at these two receptor subtypes in the micromolar range. Indeed, preliminary molecular modeling investigations according to the experimental binding data indicate a modest steric and electrostatic antagonist-receptor complementarity.
Il Farmaco 09/2005; 60(8):643-51.
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ABSTRACT: Adenosine, a widely distributed modulator, regulates many physiological functions through specific cell membrane G-protein-coupled receptors classified as A1, A2A, A2B and A3. An intense medicinal chemistry effort made over the last 20 years has led to a variety of selective adenosine receptor agonists and antagonists. In particular, the pyrazolo-triazolo-pyrimidine nucleus has been strongly investigated in the last years by our group. All the modifications performed and a tentative of structure-activity-relationship is reported. In fact, the combination of different substitutions at the N7, N8 and N5 positions afford compounds which showed good affinity and selectivity for the different adenosine receptor subtypes. The data herein summarized, permit to speculate on the use of this nucleus as possible template for the adenosine receptor subtypes.
Current Pharmaceutical Design 11/2002; 8(26):2299-2332. · 3.87 Impact Factor
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ABSTRACT: A series of pyrazolotriazolopyrimidines was previously reported to be highly potent and selective human A(3) adenosine receptor antagonists (Baraldi et al. J. Med. Chem. 2000, 43, 4768-4780). A derivative having a methyl group at the N(8) pyrazole combined with a 4-methoxyphenylcarbamoyl moiety at N(5) position, displayed a K(i) value at the hA(3) receptor of 0.2 nM. We now describe chemically reactive derivatives which act as irreversible inhibitors of this receptor. Electrophilic groups, specifically sulfonyl fluoride and nitrogen mustard (bis-(beta-chloroethyl)amino) moieties, have been incorporated at the 4-position of the aryl urea group. Membranes containing the recombinant hA(3) receptor were preincubated with the compounds and washed exhaustively. The loss of ability to bind radioligand following this treatment indicated irreversible binding. The most potent compound in irreversibly binding to the receptor was 14, which contained a sulfonyl fluoride moiety and a propyl group at the N(8) pyrazole nitrogen. The bis-(beta-chloroethyl)amino derivatives displayed a much smaller degree of irreversible binding than the sulfonyl fluoride derivatives. A computer-generated model of the human A(3) receptor was built and analyzed to help interpret these results. The model of the A(3) transmembrane region was derived using primary sequence comparison, secondary structure predictions, and three-dimensional homology building, using the recently published crystal structure of rhodopsin as a template. According to our model, sulfonyl fluoride derivatives could dock within the hypothetical TM binding domain, adopting two different energetically favorable conformations. We have identified two amino acids, Ser247 and Cys251, both in TM6, as potential nucleophilic partners of the irreversible binding to the receptor.
Journal of Medicinal Chemistry 09/2001; 44(17):2735-42. · 5.25 Impact Factor
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P G Baraldi, B Cacciari,
R Romagnoli,
G Spalluto,
S Moro,
K N Klotz,
E Leung,
K Varani,
S Gessi,
S Merighi,
P A Borea
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ABSTRACT: An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.
Journal of Medicinal Chemistry 01/2001; 43(25):4768-80. · 5.25 Impact Factor
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S Todde,
R M Moresco,
P Simonelli,
P G Baraldi, B Cacciari,
G Spalluto,
K Varani,
A Monopoli,
M Matarrese,
A Carpinelli,
F Magni,
M G Kienle, F Fazio
Journal of Medicinal Chemistry 12/2000; 43(23):4359-62. · 5.25 Impact Factor
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P. G. Baraldi, B. Cacciari,
A. Guiotto,
R. Romagnoli,
G. Spalluto,
A. Leoni,
N. Bianchi,
G. Feriotto,
C. Rutigliano,
C. Mischiati,
Roberto Gambari
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ABSTRACT: The hybrid was designed and synthesized,which was prepd. combining the minor groove binders distamycin A and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 4, related to the natural occurring anthramycin and DC-81. The effects of the hybrid on mol. interactions between DNA and transcription factor Sp1 were studied. Thus, PBD-distamycin hybrid is a powerful inhibitor of Sp1/DNA interactions. [on SciFinder(R)]
Nucleosides Nucleotides & Nucleic Acids 09/2000; 19(8):1219-1229. · 0.90 Impact Factor
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ABSTRACT: The lack of a radiolabeled selective A(3) adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A(3) adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [(3)H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl )pyrazolo [4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). [(3)H]MRE 3008F20 bound specifically to the human adenosine A(3) receptor expressed in CHO cells (hA(3)CHO), and saturation analysis revealed a single high affinity binding site, K(D) = 0.80 +/- 0.06 nM, with a B(max) = 300 +/- 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A(3) versus A(1), A(2A), and A(2B) receptors, respectively, and binds to the rat A(3) receptors with a K(i) > 10 microM. The pharmacological profile of [(3)H]MRE 3008F20 binding to hA(3)CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A(3) adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [(3)H]MRE 3008F20 binding to hA(3)CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A(1) and A(2A) receptors. These results show that [(3)H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A(3) adenosine receptor and may be used to investigate the physiopathological role of A(3) adenosine receptors.
Molecular Pharmacology 05/2000; 57(5):968-75. · 4.88 Impact Factor
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ABSTRACT: Adenosine regulates many physiological functions through specific cell membrane receptors. On the basis of pharmacological studies and molecular cloning, four different adenosine receptors have been identified and classified as A(1), A(2A), A(2B), and A(3). These adenosine receptors are members of the G-protein-coupled receptor family. While adenosine A(1) and A(2A) receptor subtypes have been pharmacologically characterized through the use of selective ligands, the A(3) adenosine receptor subtype is presently under study in order to better understand its physio-pathological functions. Activation of adenosine A(3) receptors has been shown to stimulate phospholipase C and D and to inhibit adenylate cyclase. Activation of A(3) adenosine receptors also causes the release of inflammatory mediators such as histamine from mast cells. These mediators are responsible for processes such as inflammation and hypotension. It has also been suggested that the A(3) receptor plays an important role in brain ischemia, immunosuppression, and bronchospasm in several animal models. Based on these results, highly selective A(3) adenosine receptor agonists and/or antagonists have been indicated as potential drugs for the treatment of asthma and inflammation, while highly selective agonists have been shown to possess cardioprotective effects. The updated material related to this field of research has been rationalized and arranged in order to offer an overview of the topic.
Medicinal Research Reviews 04/2000; 20(2):103-28. · 10.70 Impact Factor
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ABSTRACT: The synthesis and the preliminary biological evaluation of the first high affinity radioligand antagonist for the human A3 adenosine receptor, named [3H]-MRE 3008-F20 are reported. [3H]-MRE 3008-20 bound human A3 receptors expressed in CHO cells with K(D) and Bmax value of 0.82 +/- 0.08 nM and 297 +/- 28 fmol/mg of protein, respectively. [3H]-MRE 3008-F20 represents a useful tool for a further characterization of A3 adenosine receptor subtype.
Bioorganic & Medicinal Chemistry Letters 03/2000; 10(3):209-11. · 2.55 Impact Factor
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ABSTRACT: A novel series of 1H-2-phenyl-substituted-pyrazolo[2,3-d][1,2,4]triazine-3,7-diones (3a-g) as potential inhibitors of Human Leukocyte Elastase (HLE) are reported, the acyl-pyrazole being probably involved in the inhibition mechanism of the serino-protease enzymes. The most potent inhibitor both in vivo and in vitro was 2-o-methoxyphenyl-5-methyl-6-nitro-pyrazolo[2,3-d][1,2,4]triazine-3,7-di one (3e), which significantly suppressed the HLE-induced pulmonary injury in rats when administered orally (100 mg/kg, 3 h prior to HLE administration.
Arzneimittel-Forschung 01/2000; 49(12):997-1000. · 0.72 Impact Factor
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P G Baraldi,
G Balboni, B Cacciari,
A Guiotto,
S Manfredini,
R Romagnoli,
G Spalluto,
D E Thurston,
P W Howard,
N Bianchi,
C Rutigliano,
C Mischiati,
R Gambari
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ABSTRACT: The synthesis, biological activity, and DNA-binding properties of a series of four hybrids prepared by combining polypyrrole minor groove binders and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 13, related to the naturally occurring anthramycin (3) and DC-81 (4), have been described, and structure-activity relationships have been discussed. These hybrids 22-25 contain from one to four pyrrole units, respectively. To investigate sequence selectivity and stability of drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on human c-myc oncogene, estrogen receptor gene, and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) gene sequences. The antiproliferative activity of the hybrids has been tested in vitro on human myeloid leukemia K562 and T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1, its tetrapyrrole homologue 17, DC 81 (4), and the PBD methyl ester 12. The results obtained demonstrate that the hybrids 22-25 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 12. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22-25 and stability of drug/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 25 containing the four pyrroles is more active than 22, 23, and 24 both against K562 and Jurkat cell lines.
Journal of Medicinal Chemistry 01/2000; 42(25):5131-41. · 5.25 Impact Factor
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ABSTRACT: The chemical resolution, using N-tosyl-L-proline as a chiral auxiliary, of a racemate of the pyrazole analog (+/-)-N-Boc-CPzI of the left hand segment (CPI) of the antitumor agent CC-1065, and the cytotoxic evaluation of both enantiomers are described. The reported results further validate the direct relationship between chemical solvolytic stability of the cyclopropane ring and cytotoxicity proposed by Boger and coworkers.
Bioorganic & Medicinal Chemistry Letters 12/1999; 9(21):3087-92. · 2.55 Impact Factor
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Journal of Medicinal Chemistry 12/1999; 42(22):4473-8. · 5.25 Impact Factor
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ABSTRACT: Pharmacokinetic and metabolic studies of phytoestrogens of the isoflavone class have been hampered by the lack of suitable stable-isotope-labeled analogs. A method for preparation of a [(13)C]-labeled analog of daidzein is described. [2-(13)C]Daidzein was synthesized by reaction of [(13)C]diethoxydimethylaminomethane with 2,4-dihydroxybenzoin. The final product was purified to more than 99% by reverse-phase high-performance liquid chromatography and structural analysis confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Because [2-(13)C]daidzein is analytically and metabolically stable, it is a suitable analog for use as an internal standard for quantifying daidzein in biological fluids using isotope dilution mass spectrometry. This nonradioactive tracer is also ideal for investigating the pharmacokinetics of daidzein in humans because it is biologically indistinguishable from the unlabeled form.
Journal of Medicinal Food 02/1999; 2(3-4):99-102. · 1.41 Impact Factor
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P G Baraldi, B Cacciari,
A Guiotto,
A Leoni,
R Romagnoli,
G Spalluto,
N Mongelli,
P W Howard,
D E Thurston,
N Bianchi,
R Gambari
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ABSTRACT: We report the synthesis of a new hybrid 13 which is a combination of the naturally occurring antitumor agent distamycin A 1 and the pyrrolo[2,1-c][1,4]benzodiazepine 11, related to the naturally occurring anthramycin 2. The antitumor activity of the hybrid 13 was tested in vitro and compared to the natural product distamycin 1 and the PBD 11.
Bioorganic & Medicinal Chemistry Letters 12/1998; 8(21):3019-24. · 2.55 Impact Factor
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ABSTRACT: Adenosine is a potent coronary vasodilator and causes an increase of coronary blood flow by activation of A2A-adenosine receptors (A2A-AdoRs). The purpose of this study was to test the hypothesis that the high potency of adenosine and adenosine analogues to cause coronary vasodilation is explained by the presence of a large A2A-AdoR reserve ("spare receptors").
A novel, irreversible antagonist of A2A-AdoRs was used to inactivate receptors and reduce the response to agonist. Agonist-induced increases of coronary conductance before and after exposure of hearts to the irreversible antagonist were compared. Three agonists were studied: 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS21680), adenosine, and 2-chloro-N6-cyclopentyladenosine (CCPA). Data were analyzed to determine agonist KA (equilibrium dissociation constant) and EC50 values. Values of KA for activation of A2A-AdoRs by CGS21680, adenosine, and CCPA were 105, 1800, and 2630 nmol/L, respectively. In contrast, values of EC50 for CGS21680, adenosine, and CCPA to increase coronary conductance were 1.5, 85, and 243 nmol/L, respectively. By use of the law of mass action, it was calculated that half-maximal responses to CGS21680, adenosine, and CCPA occurred when only 1.3%, 5%, and 9%, respectively, of A2A-AdoRs were occupied by agonist.
Receptor reserves for 3 A2A-AdoR agonists were large. The receptor reserve for A2A-AdoRs to cause an increase of coronary conductance can explain both the high potency of adenosine to cause coronary vasodilation and the observation that an A2A-AdoR agonist can cause coronary vasodilation without systemic effects.
Circulation 09/1998; 98(7):711-8. · 14.74 Impact Factor
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P G Baraldi, B Cacciari,
M J Pineda de Las Infantas,
R Romagnoli,
G Spalluto,
R Volpini,
S Costanzi,
S Vittori,
G Cristalli,
N Melman,
K S Park,
X D Ji,
K A Jacobson
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ABSTRACT: A new series of 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ +-b earing N-arylureas or N-arylcarboxamido groups at the purine 6 position and N-arylureas combined with halogens or alkynyl chains at the 2 position have been synthesized and tested for affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors expressed in CHO cells. The derivatives contained the 5' substituent found in the potent, nonselective agonist 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ + (NECA). While the carboxamido derivatives (9-13) showed affinity for A1 receptors, the urea derivatives (30-45) showed different degrees of affinity and selectivity for the A3 adenosine receptor subtype. In particular the derivative bearing a p-sulfonamidophenyl-urea at the 6 position, 31 showed a high affinity (Ki = 9 nM) and selectivity for the A3 receptors compared to that of the reference compound 1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-be ta-D-ribofuranuronamide (IB-MECA). Furthermore, the importance of the stereochemistry in the interaction of these ligands at the rat A3 adenosine receptors has been evaluated by introducing a chiral chain at the 6 position. The introduction of halogens or alkynyl chains at the purine 2 position of selected ureas did not give the expected enhancement of potency at A2A and/or A3 receptors but rather showed a dramatic reduction of A2A affinity, resulting in compounds with good A2A/A3 selectivity. For example, the 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)-6-(4-methoxyphenylurea) derivative 61 showed the capability to bind simultaneously to A1 and A3 receptor subtypes, excluding the A2A receptor. Compound 31 was shown to be an agonist, 9-fold more potent than NECA, at A3 receptors in rat RBL-2H3 mast cell membranes through stimulation of binding of [35S]GTP-gamma-S.
Journal of Medicinal Chemistry 09/1998; 41(17):3174-85. · 5.25 Impact Factor
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ABSTRACT: New A2A adenosine receptor antagonists in the series of pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidines, bearing oxygenated substituents on the phenylalkyl chains on the 7-position, have been synthesized. The compounds were tested in binding and functional assays to evaluate affinity, potency, and selectivity for rat A2A compared to rat A1 and human A3 receptor subtypes. The most interesting compounds (5d,e,h) were tested also in binding to human A1 and A2A adenosine receptors. They showed very good affinity (Ki = 0.94 nM for compound 5h) and interesting selectivity with respect to both rA1 and hA3 (compound 5h: rA1/rA2A = 787, hA3/rA2A > 10 000). These important findings make this new series of compounds the first really selective for A2A adenosine receptors. Thermodynamic parameters were evaluated; all the tested compounds displayed an enthalpy-driven binding as expected for antagonists. Moreover, compound 5h showed a negative entropy value. The highly negative enthalpic and entropic contributions could mean that 5h fits very well in the binding site where, probably, an electrostatic interaction is present associated to a scarce solvent reorganization around the receptor binding site. These compounds deserve to be further developed to assess their potential for treatment of neurodegenerative disorders such as Parkinson's disease.
Journal of Medicinal Chemistry 07/1998; 41(12):2126-33. · 5.25 Impact Factor
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ABSTRACT: It is generally accepted that neoplastic transformation is related to genes alteration or oncogene activation. In particular, DNA minor groove binding drugs have been extensively studied through the years in order to influence the regulation of gene expression by means of specific interactions with DNA bases moieties. Pyrrolo[2,1-c],[1,4].benzodiazepines (PBDs), CC-1065 and distamycins are three classes of minor groove binders which showed interesting cytotoxicity profiles, refined through already reviewed processes of SAR studies. Among the modifications to the three families of antitumor compounds, heterocyclic substitutions have been extensively applied by many groups in order to either modify the reactivity profile or introduce extra interactions within the minor groove, thus changing the binding site or modulating the binding sequence. The updated material related to these modifications has been rationalised and ordered to offer an overview of the argument.
Current Pharmaceutical Design 07/1998; 4(3):249-76. · 3.87 Impact Factor
