Synthesis of pro-apoptotic indapamide derivatives as anticancer agents.

4-Chloro-3-({[(substitutedamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (1-20) and 4-chloro-3-({[3-(substituted)-4-oxo-1,3-thiazolidine-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide derivatives (21-31) were synthesized from 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoylbenzamide (indapamide). 4-Chloro-3-({[(4-chlorophenyl) amino) carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide 12 demonstrated the highest proapoptotic activity among all synthesized compounds on melanoma cell lines MDA-MB-435 with 3.7% growth inhibition at the concentration of 10 µM. Compound 12 (SGK 266) was evaluated in vitro using the MTT colorimetric method against melanoma cancer cell line MDA-MB435 growth inhibition for different doses and exhibited anticancer activity with IC50 values of 85-95 µM against melanoma cancer cell line MDA-MB435. In addition, this compound was investigated as inhibitors of four physiologically relevant human carbonic anhydrase isoforms, hCA I, II, IX and XII. The compund inhibited these enzymes with IC50 values ranging between 0.72 and 1.60 µM.

Degradation kinetics of water-insoluble lauroyl-indapamide in aqueous solutions: prediction of the stabilities of the drug in liposomes.

The aim of this study was to explore the degradation kinetics of water-insoluble lauroyl-indapamide in solutions and predict the stabilities of lauroyl-indapamide encapsulated in liposomes. Buffer-acetone (9:1) was used as the reaction solution and the reaction temperature was maintained at 60 degrees C. The correlation of the apparent degradation constants (k(obs)) of lauroyl-indapamide in liposomes and in buffer-acetone solutions at different pH has been explored. The degradation of lauroyl-indapamide in solutions was found to follow pseudo-first-order kinetics and was significantly dependent on the pH values. Lauroyl-indapamide was the most stable at pH 6.8, increasing or decreasing the pH of the solutions would decrease its stabilities. Buffer concentration had some effects on the stabilities of lauroyl-indapamide. The degradation active energies Ea were 68.19 kJ x mol(-1), 131.75 kJ x mol(-1) and 107.72 kJ x mol(-1) at pH3.6, 6.8 and 12 respectively in acetone-free buffer solutions (0.05M) calculated according to the Arrhenius equation with the extrapolation method. The apparent degradation constants (kobs) of lauroyl-indapamide in liposome and in buffer-acetone (9:1) solutions showed a good correlation at different pH levels, which indicates that the stabilities of the drug that dissolved in acetone-buffer mixture solutions can be used to predict the stabilities of the drug in liposomes as well.

Liposomal encapsulation of lauroyl-indapamide in the presence of divalent or trivalent cations.

Divalent or trivalent cations such as Ca2+, Ba2+, Mg2+, Zn2+, Fe2+, Al3+ and Fe3+ can cause a significant increase in the entrapment efficiency of lauroyl-indapamide in liposomes, from about 5% to more than 90%, which suggests that the presence of these ions plays an important role in the encapsulation of lauroyl-indapamide.

Determination of lauroyl-indapamide in rat whole blood by high-performance liquid chromatography.

A method based on a liquid-liquid extraction procedure followed by high-performance liquid chromatography (HPLC) coupled with UV-visible detection is described and validated for the determination of lauroyl-indapamide in rat whole blood. The blood sample was extracted with diethyl ether after the addition of 10% trifluoroacetic acid (aq.). The chromatographic separation was performed on a Chromasil ODS column, using methanol-acetonitrile-tetrahydrofuran-0.2% trifluoroacetic acid (170:20:15:38, v/v/v/v) as the mobile phase. The UV detection wavelength was set at 240 nm. The extraction recovery of lauroyl-indapamide was ranged from 76.5 to 82.6%, and the calibration curve had a good linearity in the range of 0.048-200 microg/ml (r = 0.9976). The method presents appropriate intra-day and inter-days repeatabilities, showing values below 7.4% in terms of the percentage of relative standard deviation (R.S.D.). The method proposed is simple, rapid and sensitive, being useful for pharmacokinetic studies in rats.

Effect of the location of hydrogen abstraction on the fragmentation of diuretics in negative electrospray ionization mass spectrometry.

The diuretic agents bumetanide, xipamide, indapamide, and related compounds were investigated in order to determine the effect of different ionization sites on their collisionally activated dissociation and the corresponding fragmentation pathways. Therefore, analytes were selectively alkylated, and structural analogues as well as deuterium labeled compounds synthesized, which contain a reduced number of ionizable hydrogen atoms. Thus, specific hydrogen abstractions and their correlated dissociation routes of the negatively charged molecules were eliminated, providing evidence for the influence of the location of ionization on product ion spectra. Fragment ions such as m/z 78 indicate ionization at the commonly present sulfamoyl residue of diuretics but does not exclude additional ionization sites. Product ion spectra of the investigated diuretic agents proved to be composed by fragmentations initiated from different hydrogen abstractions. Moreover, the generation of radical anions by collision-activated dissociation of even-electron precursor ions was observed, the generation of which is discussed by proposed fragmentation pathways.

Antioxidant properties of indapamide, 5-OH indapamide and hydrochlorothiazide evaluated by oxygen-radical absorbing capacity and electron paramagnetic resonance.

The aim of these experiments was to investigate the radical scavenging properties of three diuretics: indapamide (IND) and its major metabolite, 5-OH indapamide (5-OH IND), compared to a reference diuretic, hydrochlorothiazide (HTZ). Electron Paramagnetic Resonance (EPR) was used to determine the scavenging abilities of these compounds on enzymatically produced superoxide radical anion, with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) used as a spin-trap. These experiments revealed that IND and specially 5-OH IND were effective superoxide radical anion scavengers at 0.2 mg/ml. In the second part of these studies, allophycocyanin was used as an indicator of free radical mediated protein damage. In the assay, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) was used as a peroxyl radical generator, Trolox (a water-soluble analogue of vitamin E) as a control standard, and the loss of allophycocyanin fluorescence was monitored. The antioxidant effects of the diuretics were expressed in oxygen-radical absorbing capacity (ORAC), where one ORAC unit equals the net protection produced by 1 microM Trolox. HTZ showed no protection up to 100 microM final concentration, whereas IND and 5-OH IND showed linear correlation with respect to concentration when expressed in ORAC units: 5-OH IND induced the highest protection against peroxyl radical. The above observations suggested that IND and 5-OH IND are potent radical scavengers, with the metabolite 5-OH IND having a superior antioxidant potency than IND. By contrast, HTZ had no effect. These radical scavenging properties of 5-OH IND may be of clinical interest for vascular protection and may help to protect the heart from oxidative injury.

Vasorelaxant actions of 5-OH-indapamide, a major metabolite of indapamide: comparison with indapamide, hydrochlorothiazide and cicletanine.

5-OH-Indapamide is a principal metabolite of indapamide, and possesses similar antihypertensive and diuretic properties. This study investigated the mechanisms of the acute vasodilator actions of 5-OH-indapamide, indapamide, hydrochlorothiazide and cicletanine and their interaction with ion channels in isolated guinea pig mesenteric arteries. Hydrochlorothiazide, cicletanine and 5-OH-indapamide relaxed noradrenaline-constricted vessels significantly more than K(+)-constricted vessels (P < 0.001) and the relaxations were reduced in the presence of charybdotoxin (P < 0.001). 5-OH-Indapamide-induced relaxation was reduced (by 42% at 30 microM) by glibenclamide (P < 0.001). Hydrochlorothiazide, cicletanine and 5-OH-indapamide (all at 10 microM) were weak Ca2+ antagonists shifting the Ca2+ dose-response curves half a log unit to the right (P < 0.01). Indapamide was a more potent inhibitor, a 10 microM concentration shifting the Ca2+ dose-response curve three log units to the right and reducing maximal-induced Ca2+ contraction by 72% (P < 0.001). Hydrochlorothiazide, cicletanine and 5-OH-indapamide-induced relaxations appear to be partly mediated via Ca(2+)-activated K+ channels; 5-OH-Indapamide-induced relaxation is also partly mediated via ATP-sensitive K+ channels. Indapamide is a potent Ca2+ antagonist.

Diuretic agents related to indapamide. IV--Synthesis and pharmacological activity of N-(4-chloro-3-sulfamoylbenzamido)-methyl-phenyl-pyrrolidines and N1-(4-chloro-3-sulfamoylbenzoyl)-N2-aryl- or aralkyl-alkyl-hydrazines.

A series of N(4-chloro-3-sulfamoylbenzamido) derivatives (6 a-c, 10, 11, 12), structurally related to indapamide and isoindapamide, were synthesized and tested for their diuretic and saluretic activity. In addition, the antihypertensive activity of the most interesting term trans 6a was studied on spontaneously hypertensive rats. All tested compounds with the exception of 10 showed diuretic activity comparable to or higher (trans 6 a) than that of indapamide, taken as reference drug. The antihypertensive activity of trans 6 a was comparable to that of indapamide in potency, but its onset of action was slower.

Antioxidant activity of indapamide and its metabolite.

An antioxidant activity of indapamide (IDP) and its metabolite (OH-IDP) is demonstrated in this study. Both IDP and OH-IDP were found to scavenge 1,1-diphenyl-2-picryl-hydrazyl free radical. The scavenging effect of OH-IDP was stronger than that of IDP. Lipid peroxidation of rat liver microsomes initiated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine diphosphate (ADP)-Fe3+ was inhibited by IDP and OH-IDP with IC50 values of about 6 and 2 microM, respectively. The lipid peroxidation in human erythrocyte membrane, induced by 2,2'-azobis-(2-amidinopropane dihydrochloride) treatment, was also inhibited by 10 microM IDP. The antioxidant capacity of OH-IDP was at almost the same level as that of alpha-tocopherol, tested for comparison. The present data show that IDP and OH-IDP at micromolar concentrations are able to trap the free radicals involved in the lipid peroxidation.

Diuretic agents related to indapamide. III--Synthesis and pharmacological activity of N-(4-chloro-3-sulfamoylbenzamido)-1,2,3,4-tetrahydroquinolines and 1,2,3,4-tetrahydroisoquinolines.

A series of N-(4-chloro-3-sulfamoylbenzamido)-1,2,3,4-tetrahydroquinoline++ + (IV-1) and isoquinoline (IV-2) have been synthesized and their diuretic and antihypertensive activities evaluated. While none of the test compounds was found to be provided with antihypertensive properties, most of them displayed a diuretic activity comparable to (IV-2 a) or higher (IV-1 a,b) (IV-2 c) than those of indapamide and clopamide, taken as reference drugs.

[Antihypertensive therapy using metipamide VUFB]. / Antihypertenzívna liecba metipamidom VUFB.

In a group of 17 patients with mild hypertension the antihypertensive effect of Metipamide VUFB was investigated. In the course of fourteen weeks' treatment the mean blood pressure declined from 20.5/13.3 kPa to 17.3/11.2 kPa and at the end of the investigation the diastolic blood pressure readings were normal in all patients. Echocardiography revealed a slight decline of the total peripheral resistance. The natriuretic effect was confirmed which is in the foreground of the antihypertensive action of metipamide. Investigation of side-effects of treatment revealed in 12 patients hypokaliaemia calling for substitution treatment, or when used clinically, a combination with a potassium sparing diuretic. There was a rise of the AST and ALT activity. The renal function did not change. The clinically significant drop of serum uric acid was striking. Glucose tolerance and serum lipids were not affected.

[The effect of metipamide on levels of sodium and potassium in erythrocytes]. / Ucinok metipamidu na koncentráciu sodíka a draslíka v erytrocytoch.

The effect of metipamide on the level of Na+ and K+ in erythrocytes was studied in patients with essential hypertension. The control series consisted of healthy normotonics. In normotonics Nai+ and Ki+ were not affected in vitro, whereas in hypertonics Nai+ was found to be significantly increased (p less than 0.01) and Ki+ decreased (p less than 0.025). In vivo two subgroups of hypertonics were established: a) hypertonics with normal content of Nai+ (6.9 +/- 0.3.10(-3) mol/l) and b) hypertonics with increased content of Nai+ (9.2 +/- 0.3.10(-3) mol/l compared to 7.3 +/- 0.1.10(-3) mol/l, p less than 0.005). With respect to the given heterogeneity of the series, the response to metipamide treatment failed to be uniform. A part of the patients responded to therapy by diminished Nai+, manifesting one of the potential mechanisms of the antihypertensive effect of metipamide. The significant drop of Ki+ recorded in both subgroups of patients in the course of treatment demonstrated the potassium depleting effect of the drug.

Therapeutic monitoring of metipamide during antihypertensive therapy.

The aim of our study was to monitor metipamide during a two-month period of treatment and to determine whether the whole-blood levels estimated by high-performance liquid chromatography provide a relevant indicator of possible accumulation of the drug. We also analysed antihypertensive activity and biochemical changes in the blood of twenty hypertonic patients. The results of our clinical trial showed that metipamide is an effective first-line antihypertensive agent, in that it combines satisfactory reduction of blood pressure with a low frequency of side-effects and a simple once-daily dosage regime.

The diuretic effect of metipamide and its relationship to body weights in rats.

Metipamide [M], a new Czechosclovak diuretic with a hypotensive effect, was administered in a dose of 20 mg/kg (about 500-fold the therapeutic dose) I. for three weeks to rats of both sexes kept under normal conditions in groups of five, and II, for eight days to single male rats in metabolic cages. The animals' body weight and food and water consumption were studied and in the second series their daily faeces, urine and urinary sodium and potassium excretion were measured. Rats kept in individual cages were also given indapamide (I), the first diuretic with a separate hypotensive effect used in other countries, in a dose of 20 mg/kg. The experimental animals' body weight was significantly lower than that of controls with the same food consumption and their water intake and urine flow were much higher, especially after M. Sodium (and to a lesser extent potassium) excretion was raised at the outset of administration of both the test substances and again after the 5th to 8th dose, but only after M. After three weeks' administration of M. SNa, SK and S(osm) values were within normal limits. but after eight days the serum electrolytes and the osmolality of the serum were markedly reduced. After I. these values were normal. We conclude that the strong diuretic effect of M is not the only cause of lower body weight in rats.

Diuretic agents related to indapamide. II. Synthesis and pharmacological activity of 1-alkyl, 1-carboxyalkyl- and 1,3-dialkyl substituted 2-(4-chloro-3-sulfamoylbenzamido)isoindolines.

In pursuing earlier studies, the synthesis of a series of 1 and 1,3- substituted 2-(4-chloro-3-sulfamoylbenzamido)isoindolines (V a-e), related to indapamide, is described. These compounds were evaluated for diuretic and antihypertensive activities.