Retinoic acid conjugates as potential antitumor agents: synthesis and biological activity of conjugates with Ara-A, Ara-C, 3(2H)-furanone, and aniline mustard moieties.

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.

Cholinergic agents structurally related to furtrethonium. 3. Synthesis and antimuscarinic activity of a series of N-(5-methyl-2-furfuryl and-tiophenyl)-dialkyl amines and of 2-dimethylamino methyl-4-(1'-substituted-acetoxy-methyl)-1,3-dioxolanes.

The introduction of an homologous series of cyclic amines at position 2 of 5-methylfurane and its isoster 5-methylthiophene induced a weak antagonist behaviour, probably depending on the steric hindrance of the substituents at the nitrogen, in the case of the cardiac tissue. Surprisingly, when evaluated on guinea-pig ileum preparatons, these compounds showed non-muscarinic effects, not-related either to nicotinic or istaminergic effects, the nature of which awaits to be explained. Substitution of the furane ring on the structure of the lead 2a, b, obtained in a previous study, with the bioisoster 1,3-dioxolane moiety gave potent but not selective analogues (17 and 18).

Synthesis of N1-substituted benzamidines. Effects on blood coagulation, platelet aggregation and antiarrhythmic activity.

A series of N1-substituted-4-alkoxybenzamidines was synthesized and tested in vitro for their inhibitory effects on blood coagulation and agonist induced platelet aggregation. The antiarrhythmic activity against chloroform-induced arrhythmias in mice was also evaluated. The biological activity of the title compounds is reported in comparison with that of procainamide; among the new products described, IVi and IVe were found to have the most potent anti-platelet and antiarrhythmic activity, respectively. The structure-activity relationships are discussed.

New isoxazole derivatives of retinoids: synthesis and activity on growth and differentiation of tumor cells.

The effects of several newly synthesized isoxazole analogues of retinoids on differentiation and proliferation of 'in vitro' cultured tumor cell lines are reported. Some of the tested compounds exhibit significative differentiating action, inducing adipogenic conversion of the Chinese hamster FH06T1-1 cell line in a range of 2-10 times the activity of retinoic acid and retinol. In addition, most of the compound tested display antiproliferative activity comparable to that of natural retinoids. The reported data could be of interest for experimental anticancer therapy.

Inhibition of bovine beta-trypsin, human alpha-thrombin and porcine pancreatic beta-kallikrein-B by benzamidine and its bis-, tris- and tetra-derivatives: thermodynamic and molecular modeling study.

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).

N1-substituted benzamidines: synthesis, antiproteinase activity and inhibition of tumor cell growth.

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.

Cyclic guanidines: synthesis and antiplatelet activity of 4,6,7,8-tetrahydro-1H-imidazo[1,2-a]pyrazolo[3,4-d]pyrimidin-7-ones and 1,4,6,7,8,9-hexahydropyrazolo [3',4':4,5]pyrimido[2,1-c] [1,2,4]triazin-7-ones.

A series of 4,6,7,8-tetrahydro-1H-imidazo[1,2-a]pyrazolo [3,4-d]pyrimidin-7-ones (1b-n) and 1,4,6,7,8,9-hexahydropyrazolo [3',4':4,5]pyrimido [2,1-c][1,2,4]triazin-7-ones (2a-d) has been synthesized. In view of their potential anti-aggregating activity the compounds were tested in vitro for inhibitory activity towards ADP- and collagen-induced aggregation of human platelets. Among the compounds studied, 8-benzyl-1-(2,5-dichlorophenyl)-4,6,7,8-tetrahydro-1H-imidazo [1,2-a]pyrazolo[3,4-d]pyrimidin-7-one (1n) exhibited the most favorable activity. The 2,5-dichlorophenyl side chain is an important lipophilic and/or steric pharmacophore.

Synthesis and platelet aggregation inhibitory effects of N-[(1H-pyrazol-1-yl)alkyl]benzoylamides.

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.

Tetra-amidines exhibiting anti-proteinase activity: effects on oriented migration and in vitro invasiveness of a Chinese hamster cell line transfected with the activated human T24-Ha-ras-1 oncogene.

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.

Induction of murine erythroleukemia cell differentiation by proteinases is inhibited by aromatic poly-amidines.

The effects of the tetra benzamidine serine-proteinase inhibitor 1,3-di-(p-amidinophenoxy) -2,2- bis- (p-amidinophenoxymethyl)propane (TAPP-H) and related compounds, including halo-derivatives, were determined on the erythroid differentiation of murine erythroleukemic cells induced by trypsin and kallikrein. These aromatic poly-amidines and their halo derivatives were found to be strong inhibitors of both trypsin and kallikrein mediated induction of commitment of MEL cells to erythroid differentiation, hemoglobin synthesis and accumulation, globin mRNA production. No inhibitory effects were detected by treating proteinase-induced MEL cells with benzamidine. Only slight inhibitory activity was found after treatment of trypsin-induced MEL cells with other antiproteinase compounds widely used in the control of proteinase-dependent functions, including leupeptin, antipain and Bowman-Birk proteinase inhibitor. MEL cells induced to erythroid differentiation by proteinases could be proposed as an experimental system to test the biological activity of proteinase inhibitors.

4-(1H-pyrazol-1-yl)-2-butylamine derivatives as inhibitors of blood platelet aggregation.

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.

Inhibition of 'in vitro' tumor cell growth by aromatic polyamidines exhibiting antiproteinase activity.

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.

Amidinosemicarbazido derivatives of pyrazole: synthesis and inhibitory effect on blood coagulation and platelet aggregation.

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.

Effects of benzamidine derivatives on Ha-ras-1 mRNA accumulation in a Chinese hamster cell line transformed with the activated human T24 Ha-ras-1 oncogene.

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.

Differential effects of benzamidine derivatives on the expression of c-myc and HLA-DR alpha genes in a human B-lymphoid tumor cell line.

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.

Aromatic amidines: inhibitory effect on purified plasma serine proteinases, blood coagulation and platelet aggregation.

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.

[Ethyl esters of N-amidinobenzoyl aminoacids: inhibitory effect on thrombin, blood coagulation and platelet aggregation]. / Esteri etilici di N-amidinobenzoil aminoacidi: effetto inibitore sulla trombina sulla coagulazione del sangue e sull'aggregazione piastrinica.

In a previous paper we described the synthesis and the antiproteolytic activity of N-(3- and N-(4-amidinobenzoyl)-L-amino acids. As an extension of these studies we examined the possible inhibitory effect of these compounds on thrombin, blood coagulation and platelet aggregation. Values of Ki for the binding to thrombin are independent of the nature of the amino acids side chain and superimposable with those obtained for the formation of benzamidine-thrombin adduct. These data suggest slight effect on blood coagulation. The benzamidine derivatives are more active on platelets.