RESUMO
Aromatic polyamidines containing two, three or four benzamidine residues inhibit proteinase activity and proliferation of different human tumor cell lines, including leukemic (K562, HEL), melanoma (Colo 38) and B-lymphoid (WI-L2) cell lines. In addition, the benzamidine derivatives analysed in the present study inhibit cell growth of the Chinese hamster FHO6T1-1 cell line, obtained after transfection of primary lung cells with the activated human T24-Ha-ras-1 oncogene. After treatment of FHO6T1-1 cells with benzamidine derivatives, a sharp decrease of the content of Ha-ras-1 mRNA was found, but not of transferrin receptor mRNA. We found that inhibition of cell proliferation by tetra-benzamidine derivatives is not restricted to tumor cells, but concerns also non-tumorigenic cell lines as well as normal primary fibroblasts. Therefore, our analysis was extended to di- and tri-benzamidine derivatives, which could be proposed as useful substrates in the synthesis of drug-conjugated monoclonal antibodies or growth factors. The data obtained demonstrate that these latter compounds and their halo-derivatives also exhibit strong antiproliferative effects on in vitro cultured cells.
Assuntos
Amidinas/farmacologia , Benzamidinas/farmacologia , Inibidores de Proteases/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Cricetulus , Regulação da Expressão Gênica/efeitos dos fármacos , Genes ras , Humanos , Camundongos , Camundongos Nus , RNA Mensageiro/metabolismoRESUMO
In a dual targeting approach, to explore the ability of tretinoin (all-trans-retinoic acid) to behave as a covalent carrier for cytotoxic entities, conjugates of retinoic acid with a few representative molecules, being important examples of antitumor pharmacophores (i.e., nucleoside analogues and alkylating agents), have been synthesized and tested for their cytostatic and differentiating activity. All compounds were stable to in vitro hydrolysis in human plasma and more lipophilic than the parent compounds, thus consenting enhanced uptake into the cells. Among the nucleoside analogues the Ara-C derivatives 3 and 6 and the Ara-A derivative 7 proved the most cytostatic (IC50 < 0.32 microgram/mL) resulting from 25- to > 144-fold more active (Ara-A derivatives) or at least as equally active (Ara-C derivatives) as compared to the parent nucleosides. Compound 3, endowed with a highly lipophilic silyl moiety at the 3' and 5' positions, showed the highest differentiating activity (54% and 44% differentiated HL-60 cells at 0.2 and 0.05 microgram/mL respectively). With regard to the retinoic acid conjugates of alkylating agents, compound 10 was the most cytostatic agent (IC50 < 0.32 microgram/mL) and the most potent differentiating agent (33-34% at 0.32 and 0.08 microgram/mL). These structures may also be regarded as analogs of either retinoic acid or the cytotoxic compound.
Assuntos
Mostarda de Anilina/síntese química , Mostarda de Anilina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Furanos/síntese química , Furanos/farmacologia , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Tretinoína/síntese química , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Citarabina/síntese química , Citarabina/farmacologia , Portadores de Fármacos , Células HL-60/citologia , Células HL-60/efeitos dos fármacos , Humanos , Hidrólise , Tretinoína/análogos & derivados , Vidarabina/síntese química , Vidarabina/farmacologiaRESUMO
In the present paper we have investigated the effects of aromatic tetra-amidines on attachment, oriented migration and in vitro invasiveness of the Chinese Hamster FH06T1-1 fibroblast lung cell line, transformed with the activated human T24-Ha-ras-1 oncogene. The FH06T1-1 cell line is tumorigenic in nude mice and displays growth properties and biological features clearly distinct from those of the FH06N1-1 cell line, obtained after transfection of the same fibroblast cells with the normal Ha-ras-1 proto-oncogene. Attachment, oriented migration and invasiveness were analysed by culturing the cells on a reconstituted extracellular matrix, composed of collagen IV, laminin, entactin and heparan sulphate proteoglycans. The results obtained demonstrate that oriented migration is performed only by FH06T1-1 cells and that tetra-benzamidines are effective inhibitors of oriented migration and "in vitro" invasiveness of these tumorigenic cells. These findings should encourage further studies on the possible antimetastatic effects of these antiproteinase tetra-benzamidines on experimental animals.
Assuntos
Amidinas/farmacologia , Antineoplásicos , Benzamidinas/farmacologia , Genes ras , Inibidores de Proteases , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fenômenos Químicos , Química , Cricetinae , Cricetulus , Depressão Química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes ras/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Proteína Oncogênica p21(ras)/biossíntese , Proto-Oncogene Mas , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
In this paper, we report the effects of aromatic tetra amidines (TAPP-H) on cell growth and gene expression of a B-lymphoid human tumor cell line, WI-L2. The results obtained give evidence (a) for inhibition of cell proliferation by TAPP-H; (b) for stronger antiproliferative activity of TAPP-halo derivatives; (c) for TAPP-mediated inhibition of accumulation of c-myc RNA sequences but not of HLA-DR alpha mRNA and DR antigens. These results suggest that this class of antiproliferative compounds exhibit differential effects on cell-cycle specific and differentiation specific genes. In addition, also TAPP-related compounds containing 2 (DAPP) or 3 (TAPB) benzamidine residues retain inhibitory activity on the proliferation of WI-L2 cells. These latter compounds might be proposed as useful substrate in the synthesis of drug-conjugated monoclonal antibodies or growth factors.
Assuntos
Amidinas/farmacologia , Benzamidinas/farmacologia , Antígenos HLA-D/genética , Antígenos HLA-DR/genética , Inibidores de Proteases/farmacologia , Proto-Oncogenes , Células Tumorais Cultivadas/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Antígenos HLA-DR/análise , Humanos , RNA/análiseRESUMO
Tetra benzamidine derivatives were found to inhibit proteinase activity, cell proliferation and accumulation of the Ha-ras-1 mRNA in the FHO6T1-1 Chinese Hamster cell line, transformed with the activated human T24-Ha-ras-1 oncogene. Di- and Tri-benzamidine derivatives were also found to be potent inhibitors of proliferation of FHO6T1-1 cells. These latter compounds could be proposed as useful substrates in the synthesis of drug-conjugated monoclonal antibodies or growth factors.
Assuntos
Amidinas/farmacologia , Benzamidinas/farmacologia , Genes ras , RNA Mensageiro/metabolismo , Animais , Anticorpos Monoclonais , Linhagem Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Microscopia Eletrônica de VarreduraRESUMO
We have synthesized N1-substituted benzamidines and poly-benzamidines with the aim to produce antitumor drugs retaining differential biological properties with respect to unsubstituted compounds. Antiproliferative activity on in vitro cultured human leukemic cells was exhibited by N1-substituted poly-benzamidines, while N1-substituted benzamidines were found to retain very low antitumor effects. Furthermore, our results suggest that N1-substituted benzamidines and some of poly-benzamidines exhibit low activity on trypsin and kallikrein. Taken together these data indicate that some N1-substituted poly-benzamidines could be of interest for experimental antitumor therapy, since are likely to retain low side effects due to alteration of proteinase activity.
Assuntos
Antineoplásicos/síntese química , Benzamidinas/síntese química , Inibidores de Proteases/síntese química , Animais , Antineoplásicos/farmacologia , Benzamidinas/farmacologia , Bovinos , Divisão Celular/efeitos dos fármacos , Humanos , Calicreínas/antagonistas & inibidores , Espectroscopia de Ressonância Magnética , Inibidores de Proteases/farmacologia , Espectrofotometria Infravermelho , Relação Estrutura-Atividade , Inibidores da Tripsina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologiaRESUMO
Reaction of pyrazole and 3,5-dimethylpyrazole with methyl vinyl ketone gave 4-(1H-pyrazol-1-yl)-2-butanones which were converted to N-substituted 4-(1H-pyrazol-1-yl)-2-butylamines by reductive amination using ammonium acetate, primary or secondary amines and sodium cyanoborohydride as reducing agent. These new pyrazole derivatives were found to have an inhibitory effect on platelet aggregation in vitro.
Assuntos
Butilaminas/síntese química , Inibidores da Agregação Plaquetária/síntese química , Pirazóis/síntese química , Difosfato de Adenosina/farmacologia , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Butilaminas/farmacologia , Fenômenos Químicos , Química , Colágeno/farmacologia , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Agregação Plaquetária/efeitos dos fármacos , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
The inhibitory effect of bis-, tris- and tetra-benzamidine derivatives (DAPP, TAPB and TAPP, respectively) on the catalytic properties of bovine beta-trypsin (beta-trypsin), human alpha-thrombin (alpha-thrombin) and porcine pancreatic beta-kallikrein-B (beta-kallikrein-B) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, benzamidine, DAPP, TAPB and TAPP, show the same value of the association inhibition constant (Ki, M-1) for beta-trypsin; at variance, the affinity of DAPP, TAPB and TAPP for alpha-thrombin and beta-kallikrein-B is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to beta-trypsin, alpha-thrombin and beta-kallikrein-B as well as for DAPP, TAPB and TAPP association to beta-trypsin reflects the acidic-pK shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for DAPP, TAPB and TAPP binding to alpha-thrombin and beta-kallikrein-B appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues. Considering the X-ray three dimensional structures and the computer-generated molecular models of the serine proteinase inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to beta-trypsin, alpha-thrombin and beta-kallikrein-B has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).
Assuntos
Benzamidinas/síntese química , Benzamidinas/farmacologia , Inibidores de Serina Proteinase/síntese química , Animais , Bovinos , Humanos , Concentração de Íons de Hidrogênio , Calicreínas/antagonistas & inibidores , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pâncreas/enzimologia , Inibidores de Serina Proteinase/farmacologia , Suínos , Termodinâmica , Trombina/antagonistas & inibidores , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/farmacologia , Difração de Raios XAssuntos
Amidinas/farmacologia , Inibidores de Proteases , Amidinas/metabolismo , Benzamidinas/farmacologia , Sítios de Ligação , Quimotripsina/antagonistas & inibidores , Endopeptidases/metabolismo , Calicreínas/antagonistas & inibidores , Elastase Pancreática/antagonistas & inibidores , Serina Endopeptidases , Trombina/antagonistas & inibidores , Inibidores da Tripsina/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresAssuntos
Bradicinina/síntese química , Pirazóis , Aminoácidos , Animais , Bradicinina/farmacologia , Cães , Duodeno/efeitos dos fármacos , Feminino , Cobaias , Íleo/efeitos dos fármacos , Iminas , Técnicas In Vitro , Métodos , Contração Muscular/efeitos dos fármacos , Ratos , Útero/efeitos dos fármacosAssuntos
Amidinas/farmacologia , Plaquetas/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/antagonistas & inibidores , Benzamidinas/farmacologia , Plaquetas/metabolismo , Calcimicina/antagonistas & inibidores , Colágeno/antagonistas & inibidores , Depressão Química , Humanos , Ristocetina/antagonistas & inibidores , Taxa Secretória/efeitos dos fármacos , Serotonina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Trombina/antagonistas & inibidoresRESUMO
The synthesis of the amino-protected decapeptide tert-butyloxycarbonylhydrazide corresponding to positions 15-24 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal inhibitor) is described. The tripeptide free base threonyl-beta-tert-butylaspartylglycine tert-butyloxycarbonylhydrazide (sequence 22-24) was acylated with 1-succinimidyl o-nitrophenylsulfenylvalyl-S-acetamidomethylcysteinylglycinate (sequence 19-21). Removal of the amino protecting group from the resulting hexapeptide followed by acylation of the free base with either benzyloxycarbonylisoleucyl-O-tert-butyltyrosylasparaginylproline or O-nitrophenylsulfenylisoleucyl-O-tert-butyltyrosylasparaginylproline, via the pyrazoline active ester method, yielded the decapeptide tert-butyloxycarbonylhydrazide (sequence 15-24) in the form of Nalpha-benzyloxycarbonyl or Nalpha-O-nitrophenylsulfenyl derivative. The stereochemical homogeneity of the two decapeptides was assessed, after partial deprotection with liquid hydrogen fluoride, or thioacetamide and aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.
Assuntos
Peptídeos/síntese química , Inibidor da Tripsina Pancreática de Kazal/síntese química , Inibidores da Tripsina/síntese química , Animais , Asparagina , Ácido Aspártico , Cisteína , Glicina , Isoleucina , Peptídeos/análise , Prolina , Suínos , Treonina , Inibidor da Tripsina Pancreática de Kazal/análise , Tirosina , ValinaRESUMO
Arginine residues (5.5 out of 6) of the trypsin-kallikrein inhibitor from bovine organs (Kunitz inhibitor) were selectively modified by reaction with 1, 2-cyclohexanedione in sodium borate buffer, pH 9.0. The modified inhibitor is still highly active in inhibiting trypsin and chymotrypsin at 1:1 inhibitor: enzyme molar ratio and full inhibition was achieved at slightly higher molar ratio. The extent of correct refolding, upon reoxidation, of the reduced, arginine-modified inhibitor is diminished and regeneration of two arginines occurred under the reduction conditions. The stability constants and the standard-free energies of binding of the complexes between trypsin, or chymotrypsin, and the native, the arginine-modified and the reduced and reoxidized arginine-modified inhibitor have been determined from inhibitory assays.
Assuntos
Aprotinina , Arginina , Cicloexanos , Aminoácidos , Animais , Soluções Tampão , Bovinos , Quimotripsina , Hidrólise , Temperatura , Inibidores da TripsinaRESUMO
Since oximinyliminopyrrazoline ester (OPmp) present a peculiar characteristic in the omogenous peptide synthesis, the use of them, also in the synthesis of bradikinine by the solid phase technique, was duly taken into consideration and tried out. This synthesis was achieved by employing N-protected amino acid OPmp esters containing a methoxycarbonyl or a benzylaminocarbonyl as the acyl function in position 5 of the pyrazoline ring. No significant differences were found in the reactivity of such derivatives in respect of OPmp esters containing the benzyloxycarbonylglycyl moiety. The biological activity of the synthetized braikinine is similar to that of a sample of natural "standard".
Assuntos
Bradicinina/síntese química , Pirazóis , Ésteres , Iminas , Indicadores e Reagentes , OximasRESUMO
The stability of native and reduced bovine pancreatic trypsin inhibitor (Kunitz inhibitor) in anhydrous hydrogen fluoride and their reaction with acetamidomethanol, in the same solvent, have been investigated. The bovine Kunitz inhibitor appears to be stable in liquid hydrogen fluoride but the reduced molecule loses about 50% of its ability to regain inhibitory power, upon air oxidation, by exposure to this solvent. Tyrosine residues appear to be affected by acetamidomethylation of the native protein to give a modified inhibitor which is still highly active in inhibiting trypsin. The extent of correct refolding, upon reoxidation, of the reduced tyrosine modified-inhibitor is greatly diminished. Tyrosine modification can be prevented by carrying out the acetamidomethylation reaction in the presence of excess anisole. The stability constants and the standard free energies of binding of the complexes between trypsin and the native and the tyrosine modified-inhibitor have been determined.
Assuntos
Acetamidas/farmacologia , Inibidor da Tripsina de Soja de Kunitz/metabolismo , Inibidores da Tripsina/metabolismo , Animais , Bovinos , Depressão Química , Metanol/farmacologia , Metilação , Espectrofotometria Ultravioleta , Tripsina/farmacologia , Inibidor da Tripsina de Soja de Kunitz/análise , Tirosina/metabolismoRESUMO
The inhibitory effect of benzamidine as well as of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H) and TAPP-halo derivatives (with Cl, Br or I) on (i) the catalytic properties of purified plasma serine proteinases (notably, factor Xa, factor VIIa, thrombin and plasmin), (ii) blood coagulation, and (iii) platelet aggregation was investigated in vitro. For all the enzyme/inhibitor systems examined, the inhibition patterns were strictly competitive, and titrations conformed to simple equilibria. The inhibitory effect of TAPP-H and TAPP-halo derivatives is higher, by at least 10-fold, than that of benzamidine, which binds at the primary specificity subsite (S1) of serine proteinases and is commonly taken as a molecular inhibitor model. The high inhibitory effect of aromatic tetraamidines has been interpreted taking into account an additional productive binding for a second benzamidine or halo-benzamidine moiety to the enzyme surface. As a whole, the data reported here indicate that aromatic amidines inhibit the plasma serine proteinases involved in different steps of haemostasis (coagulation and platelet aggregation) as well as clot lysis under physiological-like conditions in vitro.
Assuntos
Amidinas/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores de Serina Proteinase , Animais , Benzamidinas/farmacologia , Cobaias , Humanos , Técnicas In Vitro , CinéticaRESUMO
Amidinosemicarbazido derivatives of pyrazole having various substituents on pyrazole ring were prepared and their effect on platelet function and blood coagulation was determined in vitro. Platelet aggregation and serotonin release induced by ADP, collagen, arachidonic acid and thrombin were markedly inhibited by pyrazole derivatives at mM concentrations. Compounds with a hydrophobic nucleus at position 1 of the pyrazole ring showed the most potent antiplatelet activity. On the contrary, their effect on blood coagulation was faintly inhibitory.
Assuntos
Anticoagulantes/síntese química , Inibidores da Agregação Plaquetária/síntese química , Pirazóis/síntese química , Humanos , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
Ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids (namely, glycine, alanine, valine, leucine and phenylalanine) were synthesized and their inhibitory effect on the bovine trypsin and porcine pancreatic kallikrein catalyzed hydrolysis of p-nitroanilides of amino acids was investigated, at pH 8.1 and 37 degrees, in parallel with the effect of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids and benzamidine. For both proteinases, the inhibitory effect of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids is independent of the aminoacidic side chain, is closely similar to that of benzamidine (which binds at the S1 subsite of the proteinases examined and is commonly taken as a molecular inhibitor-model), and is higher by at least 10-fold than that of ethyl and/or methyl esters of N-benzoyl(-L-)amino acids (depending on the aminoacidic residue). On structural grounds, the peculiar inhibitory behaviour of ethyl esters of N-(3- and N-(4-amidinobenzoyl)(-L-)amino acids has been related to the interaction of the positively charged substituent at the N-position with the Asp189 residue present in the primary specificity subsite (S1) of bovine trypsin and porcine pancreatic kallikrein. The consistently lower affinity for porcine pancreatic kallikrein of all the inhibitors considered, as compared to bovine trypsin, may be related to the marked structural differences of the primary specificity subsite of these two serine proteinases.
Assuntos
Aminoácidos/síntese química , Calicreínas/síntese química , Pâncreas/enzimologia , Inibidores da Tripsina/síntese química , Amidinas/síntese química , Amidinas/farmacologia , Aminoácidos/farmacologia , Animais , Benzoatos/síntese química , Benzoatos/farmacologia , Bovinos , Fenômenos Químicos , Química , Cristalização , SuínosRESUMO
Guanidinophenyl derivatives of pyrazole have been synthesized. Their inhibitory effects on (i) bovine trypsin, bovine thrombin, porcine pancreatic kallikrein catalyzed hydrolysis of p-nitro anilide of N alpha -benzoyl-arginine and (ii) blood coagulation and platelet aggregation, were investigated. The kinetic behaviour of all compounds conformed to that of a reversible competitive inhibition pattern, and they were also found to act in vitro as inhibitors of platelet aggregation induced by ADP.
Assuntos
Anticoagulantes/síntese química , Guanidinas/síntese química , Agregação Plaquetária/efeitos dos fármacos , Inibidores de Proteases , Pirazóis/síntese química , Animais , Bovinos , Fenômenos Químicos , Química , Endopeptidases , Guanidinas/farmacologia , Humanos , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Pirazóis/farmacologia , Serina EndopeptidasesRESUMO
A series of N-[(1H-pyrazol-1-yl)alkyl]benzoylamides was synthesized and tested in vitro for their inhibitory effects on adenosine diphosphate-, collagen-, arachidonic acid- and thrombin-induced aggregation of human platelets. Among them, N-[(1H-pyrazol-1-yl)butyl]benzoylamide (Ve) was found to have the most potent inhibitory activity. The structure-activity relationships are reported. The biological activity of the title compounds is reported in parallel with that of a known inhibitor of thromboxane A2 synthetase.