Synthesis and biochemical evaluation of some novel benzoic acid based esters as potential inhibitors of oestrone sulphatase

Oestrone sulphatase is an important target in the fight against hormone-dependent breast cancer. In an effort to investigate the reported definitive pharmacophore for oestrone sulphatase and continue our search for potent inhibitors of this enzyme, we have undertaken extensive synthesis, biochemical evaluation and physicochemical property determination of a range of benzoic acid based esters. Here, we report the initial results of our study into a series of straight chain alkyl esters of 4-sulphonylbenzoic acid. Using these compounds, we have investigated the involvement of two physicochemical properties, namely logP and pK(a). The results of this study show that there was a strong correlation between the inhibitory activity and the logP of the parent compound. Within the series of compounds studied, hydrophobicity appears to be a more important factor than pK(a) in determining the overall inhibitory activity. In a previous report, we showed that pK(a) plays an important role in stabilizing the phenoxide ion resulting from the hydrolysis of the sulphamate group. Here, we propose that although pK(a) is an important factor in determining the overall inhibitory activity when a wide range of compounds are considered, both hydrophobicity and pK(a) need to be considered in the design of potential inhibitors of oestrone sulphatase.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane,tumor-associated isozyme IX with sulfamates including EMATE also acting as steroid sulfatase inhibitors

A series of sulfamates or bis-sulfamates incorporating aliphatic, aromatic, polycyclic (steroidal), and sugar moieties in their molecules has been synthesized and assayed as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more precisely of the cytosolic isozymes CA I andII, and the transmembrane, tumor-associated isozymes CA IX. Some of these compounds were previously reported to act as inhibitors of steroid sulfatases, among which estrone sulfatase (ES) and dehydroepiandrosterone sulfatase (DHEAS) are the key therapeutic targets for estrogen-dependent tumors. Very potent (nanomolar) inhibitors were detected against the three investigated CA isozymes. Best CA I inhibitors were phenylsulfamate and some of its 4-halogeno derivatives, as well as the aliphatic compound n-octyl sulfamate. Against CA II, low nanomolar inhibitors (1.1-5 nM) were phenylsulfamate and some of its 4-halogeno/nitro derivatives, n-octyl sulfamate, and estradiol 3,17beta-disulfamate among others. All the investigated sulfamates showed efficient CA IX inhibitory properties, with inhibition constants in the range of 18-63 nM. The best CA IX inhibitor detected so far was 4-chlorophenylsulfamate. These data are critical for the design of novel antitumor properties, mainly for hypoxic tumors that overexpress CA IX, which are nonresponsive to radiation or chemotherapy. The antitumor properties of the ES/DHEAS inhibitors in clinical trials may on the other hand also be due to their potent inhibitory properties of CA isozymes involved in tumorigenicity, such as CA II and CA IX.     

Synthesis of flavonoids and their effects on aldose reductase and sorbitol accumulation in streptozotocin-induced diabetic rat tissues

Aldose reductase, the key enzyme of the polyol pathway, and oxidative stress are known to play important roles in the complications of diabetes. A drug with potent inhibition of aldose reductase and oxidative stress, therefore, would be a most promising drug for the prevention of diabetic complications. The purpose of this study was to develop new compounds with these dual-effects through synthesis of chalcone derivatives and by examining the structure-activity relationships on the inhibition of rat lens aldose reductase as well as on antioxidant effects. A series of 35 flavonoid derivatives were synthesized by Winget's condensation, oxidation, and reduction of appropriate acetophenones with appropriate benzaldehydes. The inhibitory activity of these derivatives on rat lens aldose reductase and their antioxidant effects, measured using Cu2+ chelation and radical scavenging activities on 1,1-diphenyl-picrylhydrazyl in-vitro, were evaluated. Their effect on sorbitol accumulation in the red blood cells, lenses and sciatic nerves of streptozotocin-induced diabetic rats was also estimated. Among the new flavonoid derivatives synthesized, those with the 2',4'-dihydroxyl groups in the A ring such as 2,4,2',4'-tetrahydroxychalcone (22), 2,2',4'-trihydroxychalcone (11), 2',4'-dihydroxy-2,4-dimethylchalcone (21) and 3,4,2',4'-tetrahydroxychalcone (18) were found to possess the highest rat lens aldose reductase inhibitory activity in-vitro, their IC50 values (concentration of inhibitors giving 50% inhibition of enzyme activity) being 1.6 x 10(-7), 3.8 x 10(-7), 4.0 x 10(-7) and 4.6 x 10(-7) M, respectively. All of the chalcones tested except 3, 18, 23 with o-dihydroxy or hydroquinone moiety showed a weak free radical scavenging activity. In the in-vivo experiments, however, compound 18 with o-dihydroxy moiety in the B ring showed the strongest inhibitory activity in the accumulation of sorbitol in the tissues. It also showed the strongest activity in transition metal chelation and free radical scavenging activity. Of the 35 4,2'-dihydroxyl and 2',4'-dihydroxyl derivatives of flavonoid synthesized, including chalcone, flavone, flavanone, flavonol and dihydrochalcone, some chalcone derivatives synthesized were found to possess aldose reductase inhibition and antioxidant activities in-vitro as well as inhibition in the accumulation of sorbitol in the tissues in-vivo. 3,4,2',4'-Tetrahydroxychalcone (18, butein) was the most promising compound for the prevention or treatment of diabetic complications.     

Structure-activity relationships of 17alpha-derivatives of estradiol as inhibitors of steroid sulfatase

The steroid sulfatase or steryl sulfatase is a microsomal enzyme widely distributed in human tissues that catalyzes the hydrolysis of sulfated 3-hydroxy steroids to the corresponding free active 3-hydroxy steroids. Since androgens and estrogens may be synthesized inside the cancerous cells starting from dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E(1)S) available in blood circulation, the use of therapeutic agents that inhibit steroid sulfatase activity may be a rewarding approach to the treatment of androgeno-sensitive and estrogeno-sensitive diseases. In the present study, we report the chemical synthesis and biological evaluation of a new family of steroid sulfatase inhibitors. The inhibitors were designed by adding an alkyl, a phenyl, a benzyl, or a benzyl substituted at position 17alpha of estradiol (E(2)), a C18-steroid, and enzymatic assays were performed using the steroid sulfatase of homogenized JEG-3 cells or transfected in HEK-293 cells. We observed that a hydrophobic substituent induces powerful inhibition of steroid sulfatase while a hydrophilic one was weak. Although a hydrophobic group at the 17alpha-position increased the inhibitory activity, the steric factors contribute to the opposite effect. As exemplified by 17alpha-decyl-E(2) and 17alpha-dodecyl-E(2), a long flexible side chain prevents adequate fitting into the enzyme catalytic site, thus decreasing capacity to inhibit the steroid sulfatase activity. In the alkyl series, the best compromise between hydrophobicity and steric hindrance was obtained with the octyl group (IC(50) = 440 nM), but judicious branching of side chain could improve this further. Benzyl substituted derivatives of estradiol were better inhibitors than alkyl analogues. Among the series of 17alpha-(benzyl substituted)-E(2) derivatives studied, the 3'-bromobenzyl, 4'-tert-butylbenzyl, 4'-butylbenzyl, and 4'-benzyloxybenzyl groups provided the most potent inhibition of steroid sulfatase transformation of E(1)S into E(1) (IC(50) = 24, 28, 25, and 22 nM, respectively). As an example, the tert-butylbenzyl group increases the ability of the E(2) nucleus to inhibit the steroid sulfatase by 3000-fold, and it also inhibits similarly the steroid sulfatase transformations of both natural substrates, E(1)S and DHEAS. Interestingly, the newly reported family of steroid sulfatase inhibitors acts by a reversible mechanism of action that is different from the irreversible mechanism of the known inhibitor estrone sulfamate (EMATE).     

Stilbene-based inhibitors of estrone sulfatase with a dual mode of action in human breast cancer cells

Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to target cells, after hydrolysis by the enzyme estrone sulfatase, which is active in various tissues including hormone-dependent breast cancer. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation. In this study, a number of sulfamoyloxy-substituted stilbenes with side chains that guarantee antiestrogenic activity were synthesized and evaluated as inhibitors of estrone sulfatase. They inhibited this enzyme in human MDA-MB 231 breast cancer cells, with IC(50) values in the submicromolar range. The effects of both the free hydroxy derivatives and the sulfamates on gene activation were determined in transfected MCF-7/2a breast cancer cells stimulated either with estradiol or with estrone sulfate. The analysis of data revealed a dual mode of action of the majority of compounds. They blocked gene expression by inhibition of estrone sulfatase and by antiestrogenic action. This pharmacological profile was also observed in assays on antiproliferative activity. The most potent derivative 8 g inhibited the growth of wild-type human MCF-7 cells with an IC(50) value of 13 nM.     

Stability and hydrolysis kinetics of spirosuccinimide type inhibitors of aldose reductase in aqueous solution and retardation of their hydrolysis by the target enzyme

The stability and the hydrolysis kinetics of spirosuccinimide type aldose reductase (AR) inhibitors, SX-3030 (racemate) and its optical enantiomers (R- and S-isomers), were investigated in aqueous solution. The hydrolysis followed pseudo-first-order kinetics and showed significant pH dependence. Maximum solution stability was observed below pH 2.4, whereas the hydrolysis was gradually catalyzed by hydroxide ion at neutral to alkaline pH while the compounds exhibiting moderate pH-independent stability at acidic to neutral conditions (pH 4-7) to enable oral administration. A pK of 3.7 was obtained from the pH-rate profile, but this kinetically derived pK is approximately 2 pH units below the pK of the parent compounds, suggesting the presence of an acidic intermediate involved in the hydrolysis process. These findings, together with structural analysis, support the notion that the hydrolysis would proceed via nucleophilic attack of a water molecule or hydroxide ion on the scissile carbonyl bond of the succinimide ring to form a succinamic acid intermediate that has a beta-keto acid structure, followed by decarboxylation to give a racemized succinimide ring-opened product. On the other hand, the interconversion of the R- and S-isomers did not occur during hydrolysis; however, the hydrolysis of the R-isomer was markedly suppressed by the target enzyme AR whereas that of the S-isomer was not, indicating a high degree of complementarity of interacting surfaces between the R-isomer and the enzyme. The results in the present study could provide useful clues for facilitating the appropriate stabilization strategies as well as for evaluating the pharmacological effects on target tissues in vivo, and suggested that the R-isomer may be a suitable candidate as AR inhibitor.     

Antitumor AHMA linked to DNA minor groove binding agents: synthesis and biological evaluation

DNA minor groove binder hybrid molecules, netropsin derivatives such as N-[2-(dimethylamino)ethyl]-1-methyl-4-aminopyrrolo-2-carboxamide (MePy) or its derivatives containing two units of N-methylpyrrolecarboxamide (diMePy) and bisbenzimidazole (Ho33258), were linked to the NH(2) function of AHMA or to the CH(2)OH group of AHMA-ethylcarbamate to form AHMA-N-netropsins (13-16) and AHMA-ethylcarbamate-O-netropsins (19-22), and AHMA-bisbenzimidazole (AHMA-Ho33258, 25), respectively. These conjugates' in vitro antitumor activity, inhibition of a variety of human tumor cell growth, revealed that AHMA-ethylcarbamate-O-netropsin derivatives were more cytotoxic than AHMA-N-netropsin compounds. In the same studies, all compounds bearing MePy were more potent than those compounds linked with diMePy. Moreover, AHMA-netropsin derivatives bearing a succinyl chain as the linking spacer were more potent than those compounds having a glutaryl bridge. Among these hybrid molecules, AHMA-ethylcarbamate-O-succinyl-MePy (19) was 2- to 6-fold more cytotoxic than the parent compound AHMA (5) in various cell lines, whereas compound 25 had very poor solubility and was inactive. Studies on the inhibitory effect against topoisomerase II (Topo II) and DNA interaction of these conjugates showed no correlation between the potency of DNA binding and inhibitory activity against Topo II.     

Effects of prenylated flavonoids and biflavonoids on lipopolysaccharide-induced nitric oxide production from the mouse macrophage cell line RAW 264.7

Certain flavonoid derivatives possess anti-inflammatory activity in vitro and in vivo. Besides their antioxidative properties and effects on the arachidonic acid metabolism including cyclooxygenase/lipoxygenase inhibition, some flavones and flavonols were previously found to show inhibitory activity on nitric oxide production by inducible nitric oxide synthase (iNOS; NOS type 2) through suppression of iNOS induction. As part of our continuing investigations, the effects of unique and minor flavonoids (prenylated flavonoids and biflavonoids) on nitric oxide production from lipopolysaccharide-induced macrophage cell line (RAW 264.7) were evaluated in order to establish their inhibitory activity on NO production and correlate this action with their in vivo anti-inflammatory potential. Among the derivatives tested, prenylated compounds including morusin, kuwanon C, and sanggenon D and biflavonoids such as bilobetin and ginkgetin were found to inhibit NO production from lipopolysaccharide (LPS)-induced RAW 264.7 cells at > 10 microM. Inhibition of nitric oxide production was mediated by suppression of iNOS enzyme induction but not by direct inhibition of iNOS enzyme activity. An exception was echinoisoflavanone that inhibited iNOS enzyme activity (IC50 = 83 microM) and suppressed iNOS enzyme induction as well. While most prenylated derivatives showed cytotoxicity to RAW cells at 10-100 microM, all biflavonoids tested were not cytotoxic. Since nitric oxide (NO) produced by inducible NO synthase (iNOS) plays an important role in inflammatory disorders, inhibition of NO production by these flavonoids may contribute, at least in part, to their anti-inflammatory and immunoregulating potential in vivo.     

1-[(Benzofuran-2-yl)phenylmethyl]triazoles as steroidogenic inhibitors: synthesis and in vitro inhibition of human placental CYP19 aromatase

Hormone-dependent breast cancer is stimulated by the female hormones oestrone and oestradiol, therefore compounds which inhibit the specific enzymes involved in the formation of the nitogenic hormones, namely CYP19 aromatase (P450arom) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1, are targets of therapeutic interest for the treatment of breast cancer. A series of novel 1-[(benzofuran-2-yl)phenylmethyl]1,2,4-triazoles were prepared using a three-step synthesis and evaluated for their inhibitory activity against human placental aromatase in vitro, using [1,2,6,7-3H]androstenedione as the substrate for the aromatase enzyme. Inhibitory activity was dependent on both substituent and position of substitution, with introduction of small electron-withdrawing groups in the phenyl ring showing optimum activity (IC50 ranging from 0.065 to 2.02 microm). Substitution in the benzofuran ring resulted in a loss of activity when substituted at C-5 (IC50 > 20 microm). The compounds were all shown to exhibit weak inhibitory activity against rat testes P450 17 (17,20-lyase), indicating good selectivity towards P450arom.     

Synthesis and biological evaluation of thiophosphate tricyclic coumarin derivatives as steroid sulfatase inhibitors

Steroid sulfatase (STS) enzyme inhibition is an important approach to the management of hormone-dependent breast cancer. In this paper, we report convenient methods for the synthesis and biological evaluation of thiophosphate tricyclic coumarin analogs exhibiting STS activity. The described methods are based on the straightforward preparation of 7-hydroxy-2,3-dihydro-1H-cyclopenta[c]chromen-2-one, 3-hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one, and 3-hydroxy-8,9,10,11-tetrahydro-7H-cyclohepta[c]chromen-6-one and their further modification by the introduction of various thiophosphate moieties. The inhibition properties of the synthesized compounds were tested toward STS isolated from human placenta. Most of the new STS inhibitors possessed good to moderate activity toward STS. During the course of our investigation, the largest inhibitory effects in the STS enzyme assays were observed for the two compounds 3f and 4r, with IC₅₀ values of 13.3 and 30.3 μM, respectively (the IC₅₀ value of 1 μM for the 665-COUMATE was used as a reference). The structure–activity relationships of the synthesized coumarin derivatives toward STS enzymes are discussed.     

3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides, a new class of synthetic histone deacetylase inhibitors

Novel 3-(4-aroyl-2-pyrrolyl)-N-hydroxy-2-propenamides are disclosed as a new class of histone deacetylase (HDAC) inhibitors. Three-dimensional structure-based drug design and conformational analyses into the histone deacetylase-like protein (HDLP) catalytic core suggested the synthesis and biological evaluation of compounds 7a-h. Experimental pK(i) values are in good agreement with VALIDATE predicted pK(i) values of new derivatives. All compounds 7a-h show HDAC inhibitory activity in the micromolar range, with 7e as the most potent derivative (IC(50) = 1.9 microM). The influence of the 4'-substituent in the aroyl moiety is not significant for the inhibitory activity, as all compounds 7a-g show IC(50) values between 1.9 and 3.9 microM. Otherwise, the unsaturated chain linking the pyrrole ring to the hydroxamic acid group is clearly important for the anti-HDAC activity, the saturated analogue 7h being 10-fold less active than the unsaturated counterpart 7a.     

Antitumor agents. 123. Synthesis and human DNA topoisomerase II inhibitory activity of 2'-chloro derivatives of etoposide and 4 beta-(arylamino)-4'-O-demethylpodophyllotoxins.

The 2'-chloro derivatives of etoposide and 4 beta-(arylamino)-4'-O-demethylpodophyllotoxins have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. The results showed that none of the compounds are active as a result of the C-2' chloro substitution on ring E. This would suggest that the free rotation of ring E is essential for the aforementioned enzyme inhibitory activity. In addition, these 2'-chloro derivatives showed no significant cytotoxicity (KB).     

Selective acetylenic 'suicide' and reversible inhibitors of monoamine oxidase types A and B.

1 A number of aromatic-N-propargyl (acetylenic) compounds and indoleamines were tested for their inhibitory action on monoamine oxidase (MAO) type A and type B using the substrates 5-hydroxytryptamine (5-HT), beta-phenylethylamine (PEA) and dopamine. 2 Structure activity studies with aromatic-N-propragyl (acetylenic) derivatives have shown that MAO inhibitory potency is least dependent on the aromatic portion of the compounds. N-methylated propargyl derivatives are the most active and replacement of the methyl group with a higher alkyl or aromatic group results in significant reduction of activity. The triple bond in the N-propargyl portion is absolutely essential for activity and must be beta-to the nitrogen. It is the acetylenic group that gives these compounds their irreversible MAO inhibitory property. 3 The present study has indicated that since the acetylenic compounds resemble the enzyme substrates the distance between the aromatic ring and the N-propargyl terminal is crucial in designating the type A or type B MAO inhibitory property. For MAO type A inhibition, a distance equivalent to at least three carbon units is required, while for the inhibition of the B type enzyme this distance can be 1 or 2 carbon units. 4 The compounds AGN-1133 and AGN-1135 show most promise in Parkinson's disease or as anti-depressants because of their irreversible selective type B MAO inhibition in vitro and in vivo. 5 A number of indoleamine derivatives were found to be reversible selective type A inhibitors.     

Inhibition of ribonucleotide reductase by antitumor agents related to levodopa and dopamine

Using partially purified enzyme from L1210 cells, dihydroxybenzene derivatives related structurally to dopamine were shown to reversibly inactivate ribonucleotide reductase. A structure-activity analysis revealed that derivatives with side-chains, which contain a negatively-charged group, had significantly reduced inhibitory activity. The ability of these compounds to inhibit ribonucleotide reductase was dependent on the hydroxyl groups being in the ortho position and did not correlate with free radical inhibitory activity. A kinetic analysis by the method of Lineweaver-Burk indicated that the inhibition of ribonucleotide reductase by the derivative 3,4-dihydroxybenzylamine was competitive with the reducing substrate dithioerythritol. This analog, in combination with hydroxyurea, gave synergistic inhibition or ribonucleotide reductase, suggesting different sites of action. Using Tween 80-treated L1210 cells, it was found that these drugs had an immediate inhibitory effect on ribonucleotide reductase activity in intact, reversibly permeabilized cells. Furthermore, although these drugs had no immediate effect on DNA polymerase, in permeabilized L1210 cells (when the cells were preincubated with the dihydroxybenzene derivatives for 1 hr prior to permeabilization), there was significant inhibition of DNA polymerase activity. The two key enzymes for DNA synthesis appear to be sequentially inhibited by these analogs, with the reduced form (quinol) inhibiting ribonucleotide reductase and the oxidized form (quinone) inhibiting DNA polymerase.     

Synthesis and biochemical evaluation of analogues of aminoglutethimide based on phenylpyrrolidine-2,5-dione

A series of (aminophenyl)pyrrolidine-2,5-diones has been prepared that bear structural similarities to aminoglutethimide (1, 3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione). The inhibitory activity of these compounds was evaluated toward human placental aromatase and bovine adrenal cholesterol side chain cleavage (CSCC) enzyme assay systems. Selective, competitive inhibition of the aromatase enzyme system was demonstrated by 5 (3-(4-aminophenyl)-1-methyl-pyrrolidine-2,5-dione, Ki = 1.75 microM), 6 (3-(4-aminophenyl)-1,3-dimethylpyrrolidine-2,5-dione, Ki = 1.75 microM), 7 (3-(4-aminophenyl)-3-methylpyrrolidine-2,5-dione, Ki = 0.8 microM), and 8 (3-(4-aminophenyl)-3-ethylpyrrolidine-2,5-dione, Ki = 1.0 microM). Compound 15 (3-(4-aminophenyl)pyrrolidine-2,5-dione) proved unexpectedly difficult to prepare following standard methods and was only moderately inhibitory toward aromatase (IC50 = 20 microM). Compound 16 (3-(4-aminophenyl)-3-ethyl-1-methylpyrrolidine-2,5-dione) was weakly inhibitory toward testosterone aromatization and totally inactive toward androstenedione aromatization. These compounds were either weak or ineffective inhibitors of the CSCC enzyme systems, while 1 gave Ki values toward aromatase and CSCC enzymes of 0.68 and 14 microM, respectively. The unsubstituted phenylpyrrolidinediones were inactive in either system, and the 4-nitrophenyl derivatives exhibited weak, nonselective inhibition, indicating the importance of the primary amine moiety for potent inhibition of aromatase activity.     

Compounds for the treatment of oestrogen-dependent illnesses

Compounds that interfere with the enzymes involved in oestrogen biosynthesis are useful in the treatment of oestrogen-dependent breast cancers. Oestrone sulphatase is the critical enzyme in the biosynthesis of oestradiol in breast cancer cells and high levels of this enzyme, as well as its substrate oestradiol sulphate, were detected in the cancerous, but not normal, tissues. This enzyme is inhibited by sulphamates, which act as transition state analogues in the steroid sulphate hydrolysis. Duquesne University of the Holy Ghost discloses in this patent a series of sulphamates also possessing a moiety that assures affinity for the oestrogen receptor, so that these derivatives possess a dual mechanism of action: inhibition of oestradiol biosynthesis (due to inhibition of oestrone sulphatase enzymatic activity) and binding to the oestrogen receptor, thus impeding the access of the hormone to its cellular site of action. The disclosed compounds were synthesised by simple procedures, for instance, the reaction of (Z)-4-hydroxytamoxifen with sulphamoyl chloride, and were assayed in vitro and ex vivo for the inhibition of liver microsome sulphatase and MDA-MB-231 breast cancer cell sulphatase, respectively. The compound (Z)-4-hydroxytamoxifen represents an interesting lead for the development of novel types of anticancer drugs.     

Influence of subchronic administration of oestrone-3-O-sulphamate on oestrone sulphatase activity in liver, spleen and white blood cells of ovariectomized rats

Inhibition of oestrone sulphatase followed by oestrogen removal from tumour cells may be a new form of endocrine therapy of breast cancer in women. We investigated the inhibitory effect of the subchronic administration of oestrone-3-O-sulphamate (EMATE), a steroid sulphatase inhibitor, to ovariectomized rats, to evaluate this method for testing new nonsteroidal inhibitors. EMATE in DMSO was administered both orally and subcutaneously (s.c.) for 7 days at doses of 0.5 and 2.5 mg/kg. In addition the rats were injected s.c. with 0.5 mg oestrone sulphate/kg 26 and 2 h before decapitation under ether anaesthesia. Oestrone sulphatase activity (ESA) was measured radiometrically using [3H]oestrone sulphate as substrate for desulphuration in white blood cells, liver homogenate, microsomes and spleen homogenate. ESA in liver microsomes was found to be nearly 40 times higher than in white blood cells while in spleen ESA was nearly half of that found in liver homogenates and white blood cells. ESA can be inhibited by EMATE down to 50-1.5% of control activity depending on the dose and administration route. The inhibition was in the order, liver homogenate < spleen < liver microsomes < white blood cells, and was more pronounced after s.c. administration of the inhibitor than after oral administration. Ovariectomy was found to be not necessary for oestrone sulphatase-inhibiting studies. Two sequential s.c. injections of oestrone sulphate enhanced the enzyme activities significantly in liver and white blood cells, but not in spleen. In conclusion, white blood cells and liver microsomes of intact female rats can be used for ESA-inhibiting studies. Sulphate-conjugated oestrone can induce oestrone sulphatase in vivo in liver and white blood cells thereby enhancing oestrogen supply in the peripheral organs.     

Synthesis and aldose reductase inhibitory activity of pyridazine derivatives possessing acetic acid group.

N-acetic acid and S-acetic acid derivatives of 5-arylidene pyridazines were synthesized for evaluation as new aldose reductase inhibitors. Intrinsic activity for each compound was assessed by measuring inhibition of enzymatic activity in an isolated pig lens enzyme preparation. All prepared compounds exhibited a significant in vitro aldose reductase inhibitory effect (10(-5) M less than or equal IC50 less than or equal to 10(-4) M). It was found that lipophilicity was important in increasing activity. Furthermore, this activity (log 1/IC50) could be correlated directly to a lipophilic parameter (log kw) for the whole data set.     

Structure-activity relationships in 4-aminoquinoline antiplasmodials. The role of the group at the 7-position

Antiplasmodial activities versus the chloroquine sensitive D10 strain of Plasmodium falciparum of a series of N(1),N(1)-diethyl-N(2)-(4-quinolinyl)-1,2-ethanediamines with 11 different substituents at the 7-position on the quinoline ring have been investigated in vitro. Electron-withdrawing groups at the 7-position have been shown to lower the pK(a) of both the quinoline ring nitrogen atom and the tertiary amino nitrogen in the alkyl side chain. The quinoline nitrogen pK(a) ranges from 6.28 in the nitro derivative to 8.36 in the amino derivative, while the tertiary amino nitrogen has a pK(a) ranging between 7.65 in the trifluoromethyl derivative and 10.02 in the amino derivative. Calculation suggests that the resulting pH trapping of these compounds in the parasite food vacuole ranges between about 7% of that observed in chloroquine for the NO(2) derivative and 97% in the amino derivative. A direct proportionality between antiplasmodial activity normalized for pH trapping and beta-hematin inhibitory activity was observed. Activity could not be correlated with any other observed physical parameter. The beta-hematin inhibitory activity of these derivatives appears to correlate with both the hematin-quinoline association constant and the electron-withdrawing capacity of the group at the 7-position (Hammett constant). For the compounds under investigation, the hematin association constant is in turn influenced by the lipophilicity of the group at the 7-position.     

Inhibitory activity and protein binding of L-lysine derivatives as angiotensin converting enzyme inhibitors

Four compounds 1a-4a containing one L-lysine residue in the molecule including a diastereomer mixture of lisinopril (N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) and N epsilon-carbobenzoxy-L-lysine derivatives 1b-4b of each of the four compounds were synthesized to compare the angiotensin converting enzyme (ACE) inhibitory activities in vitro and in vivo. They all showed high ACE inhibitory activity in vitro (IC50 = 0.14-42 nmol/l). A marked difference, however, was observed in inhibition of the pressor response to angiotensin I between 1a-4a (high activity) and 1b-4b (low activity). The binding of these compounds to human serum proteins in vitro was investigated by means of equilibrium dialysis and ultracentrifugation. Compounds 1b-4b showed higher levels of binding to serum albumin than that of the corresponding compounds 1a-4a, and the percentage of binding ranged from 20.1 to 89.1%. Furthermore, the inhibitory activity of compounds 1b-4b in vitro was decreased by the addition of albumin in a concentration-dependent manner. These results suggested that the difference in the protein binding rate of compounds is one of the important factors influencing the inhibitory activity in vivo.