Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.     

Toxicity and metabolism of the chloral-derived mammalian alkaloid 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) in PC12 cells

Chloral-derived beta-carbolines, which are structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 5), are discussed to contribute to neuronal cell death in idiopathic Parkinson's disease. The cytotoxicity of 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo, 4) to neuronal-like clonal pheochromocytoma PC12 cells was examined by the determination of lactate dehydrogenase (LDH) release. After incubation for 48 h, 4 showed a strong dose-dependent cytotoxic activity towards PC12 cells with an ED50 value of 230 microM. In PC12 cells reductive dehalogenation of 4 was observed giving rise to the formation of 1-dichloromethyl-1,2,3,4-tetrahydro-beta-carboline (6) as a main TaClo metabolite exhibiting a cytotoxic potential comparable to that of TaClo. An X-ray structure analysis, performed for the trifluoroacetyl derivative of 6, revealed the N-substituent of such a highly chlorinated agent to be dramatically pushed out of the beta-carboline ring 'plane' due to the high steric demand of the huge dichloromethyl group at C(1).     

Endogenous alkaloids in man. XXXVIII. "Chiral" and "achiral" determination of the neurotoxin TaClo (1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline) from blood and urine samples by high-performance liquid chromatography--electrospray ionization tandem mass spectrometry

An improved sensitive assay for the determination of the dopaminergic and serotonergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) is presented, based upon on-line coupling of high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS-MS). Applying synthetic [D4]TaClo as a fourfold deuterated internal standard, TaClo was detected and reliably quantified as a trace constituent of blood samples (0.5 up to 70 ng g(-1) of clot) obtained from six patients orally treated with the hypnotic chloral hydrate. Unambiguous identification of this tricyclic "endogenous alkaloid" was achieved by selected reaction monitoring (SRM) experiments. The molecular ion peaks of TaClo, m/z 289 (for [35Cl3]TaClo) and m/z 291 (for its [37Cl35Cl2]isotopomer), were both monitored to undergo a retro-Diels-Alder fragmentation by loss of a CH2=NH portion (-29 u) as typical of a tetrahydropyrido ring system of tetrahydro-beta-carbolines. Detection of the resulting fragments, m/z 260 and m/z 262, with the expected statistical chlorine isotopic intensities of 100:96 confirmed the identity of the TaClo molecule. In addition, an enantiomer-specific device was developed for TaClo, by employing a chiral reversed-phase HPLC column in combination with circular dichroism (CD) spectroscopy and MS-MS analysis (LC-CD and LC-MS-MS coupling). In a human clot sample, both TaClo enantiomers were found in equimolar concentration (i.e., as a racemate) corroborating a spontaneous, non-enzymatic formation of TaClo from biogenic tryptamine and therapeutically administered chloral. In urine samples of TaClo-treated rats, by contrast, the (S)-antipode was found to predominate, hinting at an enantiomer-differentiating metabolism of the compound.     

Synthetic tyrosyl gallate derivatives as potent melanin formation inhibitors

Three tyrosyl gallate derivatives (1-3) with variable hydroxyl substituent at the aromatic ring of tyrosol were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. Among three tyrosyl gallate derivatives, 4-hydroxyphenethyl 3,4,5-trihydroxybenote (1) (IC(50)=4.93 microM), 3-hydroxyphenethyl 3,4,5-trihydroxybenote (2) (IC(50)=15.21 microM), and 2-hydroxyphenethyl 3,4,5-trihydroxybenote (3) (IC(50)=14.50 microM) exhibited significant inhibitory effect on tyrosinase activity. Compound 1 was the most active compound, though it did not show the inhibitory effect on melanin formation in melan-a cells. However, compounds 2 (IC(50)=8.94 microM) and 3 (IC(50)=13.67 microM) significantly suppressed the cellular melanin formation without cytotoxicity. This study shows that the position of hydroxyl substituent at the aromatic ring of tyrosol plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Design, synthesis, and biological evaluation of N-acetyl-2-carboxybenzenesulfonamides: a novel class of cyclooxygenase-2 (COX-2) inhibitors

N-Acetyl-2-carboxybenzenesulfonamide (11), and a group of analogues possessing an appropriately substituted-phenyl substituent (4-F, 2,4-F(2), 4-SO(2)Me, 4-OCHMe(2)) attached to its C-4, or C-5 position, were synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1/COX-2 inhibition studies showed that 11 is a more potent inhibitor (COX-1 IC(50)=0.06microM; COX-2 IC(50)=0.25microM) than aspirin (COX-1 IC(50)=0.35microM; COX-2 IC(50)=2.4microM), and like aspirin [COX-2 selectivity index (S.I.)=0.14], 11 is a nonselective COX-2 inhibitor (COX-2 S.I.=0.23). Regioisomers having a 2,4-difluorophenyl substituent attached to the C-4 (COX-2 IC(50)=0.087microM; COX-2 S.I. >1149), or C-5 (COX-2 IC(50)=0.77microM, SI>130), position of 11 exhibited the most potent and selective COX-2 inhibitory activity relative to the reference drug celecoxib (COX-1 IC(50)=33.1microM; COX-2 IC(50)=0.07microM; COX-2 S.I.=472). N-Acetyl-2-carboxybenzenesulfonamide (11, ED(50)=49 mg/kg), and its C-4 2,4-difluorophenyl derivative (ED(50)=91 mg/kg), exhibited superior antiinflammatory activity (oral dosing) in a carrageenan-induced rat paw edema assay compared to aspirin (ED(50)=129 mg/kg). These latter compounds exhibited comparable analgesic activity to the reference drug diflunisal, and superior analgesic activity compared to aspirin, in a 4% NaCl-induced abdominal constriction assay. A molecular modeling (docking) study indicated that the SO(2)NHCOCH(3) substituent present in N-acetyl-2-carboxy-4-(2,4-fluorophenyl)benzenesulfonamide, like the acetoxy substituent in aspirin, is suitably positioned to acetylate the Ser(530) hydroxyl group in the COX-2 primary binding site. The results of this study indicate that the SO(2)NHCOCH(3) pharmacophore present in N-acetyl-2-carboxybenzenesulfonamides is a suitable bioisostere for the acetoxy (OCOMe) group in aspirin.     

Design of N-acetyl-6-sulfo-beta-d-glucosaminide-based inhibitors of influenza virus sialidase

Biological activity of N-acetyl-6-sulfo-beta-d-glucosaminides (6-sulfo-GlcNAc 1) having a structural homology to N-acetylneuraminic acid (Neu5Ac 2) and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 3) was examined in terms of inhibitory activity against influenza virus sialidase (influenza, A/Memphis/1/71 H3N2). pNP 6-Sulfo-GlcNAc 1a was proved to show substantial activity to inhibit the virus sialidase (IC(50)=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP 6-sulfo-GlcNH(3)(+) 1b without 2-NHAc showed little activity (IC(50) >50 mM). The activity was enhanced nearly 100-fold when the pNP group of 1a was converted to p-acetamidophenyl one 5 (IC(50)=30 microM) or replaced with 1-naphthyl 6 (IC(50)=10 microM) or n-propyl one 8 (IC(50)=11 microM).     

A phosphorylated prodrug for the inhibition of Pin1

Fmoc-pSer-Psi[(Z)CHC]-Pro-(2)-N-(3)-ethylaminoindole 1, showed moderate inhibition towards the mitotic regulator, Pin1 (IC(50)=28.3microM). To improve the cell permeability, the charged phosphate was masked as the bis-pivaloyloxymethyl (POM) phosphate in Fmoc-(bisPOM)-pSer-Psi[(Z)CHC]-Pro-(2)-N-(3)-ethylaminoindole 2. Antiproliferative activity towards A2780 ovarian cancer cells of 1 (IC(50)=46.2microM) was improved significantly in 2 (IC(50)=26.9microM), comparable to the IC(50) of 1 towards Pin1 enzymatic activity.     

Identification of the dopaminergic neurotoxin 1-trichloromethyl-1,2, 3,4-tetrahydro-beta-carboline in human blood after intake of the hypnotic chloral hydrate.

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), a potent toxin toward dopaminergic neurons, readily originates in vitro from the biogenic amine tryptamine and the unnatural aldehyde chloral. For this reason, this heterocycle has been postulated to be formed endogenously in humans after administration of the hypnotic chloral hydrate or after exposure to the industrial solvent trichloroethylene by a spontaneous chemical ring closure reaction. In this paper, we report on the first identification of TaClo in blood samples of patients treated orally with chloral hydrate. Using a specific and sensitive gas chromatographic screening procedure based upon electron-capture and mass-selective detection, TaClo was determined after conversion to its volatile trifluoroacetyl derivative. The identity of TaClo in humans was clearly demonstrated by GC-MS analysis in selected-ion-monitoring mode, by the characteristic chlorine isotopic pattern of the molecular ion.     

Heterocyclic substituted cantharidin and norcantharidin analogues--synthesis, protein phosphatase (1 and 2A) inhibition, and anti-cancer activity

Norcantharidin (3) is a potent PP1 (IC(50)=9.0+/-1.4 microM) and PP2A (IC(50)=3.0+/-0.4 microM) inhibitor with 3-fold PP2A selectivity and induces growth inhibition (GI(50) approximately 45 microM) across a range of human cancer cell lines including those of colorectal (HT29, SW480), breast (MCF-7), ovarian (A2780), lung (H460), skin (A431), prostate (DU145), neuroblastoma (BE2-C), and glioblastoma (SJ-G2) origin. Until now limited modifications to the parent compound have been tolerated. Surprisingly, simple heterocyclic half-acid norcantharidin analogues are more active than the original lead compound, with the morphilino-substituted (9) being a more potent (IC(50)=2.8+/-0.10 microM) and selective (4.6-fold) PP2A inhibitor with greater in vitro cytotoxicity (GI(50) approximately 9.6 microM) relative to norcantharidin. The analogous thiomorpholine-substituted (10) displays increased PP1 inhibition (IC(50)=3.2+/-0 microM) and reduced PP2A inhibition (IC(50)=5.1+/-0.41 microM), to norcantharidin. Synthesis of the analogous cantharidin analogue (19) with incorporation of the amine nitrogen into the heterocycle further increases PP1 (IC(50)=5.9+/-2.2 microM) and PP2A (IC(50)=0.79+/-0.1 microM) inhibition and cell cytotoxicity (GI(50) approximately 3.3 microM). These analogues represent the most potent cantharidin analogues thus reported.     

Modified norcantharidins; synthesis, protein phosphatases 1 and 2A inhibition, and anticancer activity

Fourteen modified norcantharidin analogues have been synthesised and screened for their ability to inhibit the serine/threonine protein phosphatases 1 and 2A. The most potent compounds found were 10 (PP1 IC(50)=13+/-5 microM; PP2A IC(50)=7+/-3 microM) and 16 (PP1 IC(50)=18+/-8 microM; PP2A IC(50)=3.2+/-0.4 microM). Overall, only analogues possessing at least one acidic residue at the former anhydride warhead displayed any PP1 or PP2A inhibitory action. The ability of these analogues to inhibit PP1 and PP2A correlates well with their observed anti-cancer activity against a panel of five cancer cell lines: A2780 (human ovarian carcinoma), G401 (human kidney carcinoma), HT29 (human colorectal carcinoma), H460 (human lung carcinoma) and L1210 (murine leukemia).     

Novel platinum(II) and palladium(II) complexes with cyclin-dependent kinase inhibitors: synthesis, characterization and antitumour activity

The Pt(II) and Pd(II) complexes of the types cis-[Pt(L(1))(2)Cl(2)].H(2)O (1), cis-[Pt(L(2))(2)Cl(2)].3H(2)O (2), trans-[Pd(L(1))(2)Cl(2)].H(2)O (3), trans-[Pd(L(2))(2)Cl(2)].H(2)O (4), trans-[Pd(L(3))(2)Cl(2)].2DMF (5) and trans-[Pd(L(4))(2)Cl(2)].2DMF (6) (L(1)-L(4)=cyclin-dependent kinase inhibitors derived from 6-benzylamino-9-isopropylpurine) have been prepared and characterized. The complexes have been studied by elemental analyses, conductivity measurements, ES+ MS, FT-IR, (1)H, (13)C and (195)Pt NMR spectra, differential scanning calorimetry and thermogravimetric analysis. The molecular structures of L(1), trans-[Pd(L(3))(2)Cl(2)].2DMF (5) and trans-[Pd(L(4))(2)Cl(2)].2DMF (6) have been determined by single crystal X-ray analysis. The complexes have been tested in vitro due to their presumable anticancer activity against the following human cancer cell lines: K-562, MCF7, G-361 and HOS. Satisfying results were obtained for the complex 1 with IC(50) values of 6 microM acquired against G-361 as well as against HOS cell lines. The lowest values of IC(50) were achieved for the complexes 3 and 4 against MCF 7 cell line with IC(50) 3 microM(for 3) and also 3 microM (for 4).     

Synthesis and antioxidant properties of novel N-methyl-1,3,4-thiadiazol-2-amine and 4-methyl-2H-1,2,4-triazole-3(4H)-thione derivatives of benzimidazole class

Some novel 1-methyl-4-(2-(2-substitutedphenyl-1H-benzimidazol-1-yl)acetyl)thiosemicarbazides (16a-20a), 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl]-N-methyl-1,3,4-thiadiazol-2-amines (17b-20b), and 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl-4-methyl-2H-1,2,4-triazole-3(4H)-thiones (16c-20c) were synthesized and tested for antioxidant properties by using various in vitro systems. Compounds 16a-20a were found to be a good scavenger of DPPH radical (IC(50), 26 microM; IC(50), 30 microM; IC(50), 43 microM; IC(50), 55 microM; IC(50), 74 microM, respectively) when compared to BHT (IC(50), 54 microM). Noteworthy results could not be found on superoxide radical. Compound 19b, which is the most active derivative inhibited slightly lipid peroxidation (28%) at 10(-3)M concentration. Compound 17c inhibited the microsomal ethoxyresorufin O-deethylase (EROD) activity with an IC(50)=4.5 x 10(-4)M which is similarly better than the specific inhibitor caffeine IC(50)=5.2 x 10(-4)M.     

Structure-activity relationship of aza-steroids as PI-PLC inhibitors

A number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The epimeric mixtures 22,25-diazacholesterol (8a) and 3beta-hydroxy-22,25-diazacholestane (8b) were among the most active of these inhibitors, with IC(50) values of 7.4 and 7.5 microM, respectively. The 20alpha epimer, 8a2 (IC(50)=0.64 microM), whose stereochemistry at C-20 coincides with that of cholesterol, was found 50 times more potent than the 20beta epimer, 8a1 (IC(50)=32.2 microM). In diaza-estrone derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC(50)=19.7 microM), while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl group (8a, IC(50)=7.4 microM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the non-aromatic A-ring (26, IC(50)=17.4 microM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent PI-PLC inhibitors.(3) However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of activity. Epimeric mixture 8a showed selective growth inhibition effects in the NCI in vitro tumor cell screen with a mean GI(50) value (MG-MID) of 5.75 microM for 54 tumors.     

Design and synthesis of 2,6-diprenyl-4-iodophenol TX-1952 with a novel and potent anti-peroxidative activity

The structure-based elucidation of 2,4,6-tri-substituted phenols for their antioxidative and anti-peroxidative effects has been investigated using TX-1952 (2,6-diprenyl-4-iodophenol), TX-1961, TX-1980, BTBP and BHT. In the inhibition of mitochondrial lipid peroxidation, the inhibitory activity of 2,6-di-tert-butyl-4-bromophenol (BTBP) (IC(50)=0.17 microM) was twice as high as that of 2,6-di-tert-butyl-4-methylphenol (BHT) (IC(50)=0.31 microM). This result shows that the 4-halogen group increases inhibitory activity for mitochondrial lipid peroxidation. Besides, TX-1952 (IC(50)=0.60 microM) was the highest inhibitor among 2,6-diprenyl-4-halophenols, followed by TX-1961 (IC(50)=0.93 microM) and TX-1980 (IC(50)=1.2 microM). In 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging experiments, the activity of TX-1952 (IC(0.200)=53.1 microM) was lower than that of BHT (IC(0.200)=33.7 microM) and BTBP (IC(0.200)=16.0 microM), but TX-1952 and BHT showed the same HOMO energy (-8.991 eV). These results suggest that the two prenyl groups at ortho position hinder the phenolic hydrogen abstraction by DPPH radical. These findings demonstrated that TX-1952 was a novel and potent inhibitor for lipid peroxidation.     

Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: potent CYP26 inhibitors and enhancers of all-trans retinoic acid activity in neuroblastoma cells

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine (8), IC(50)=0.5 microM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC(50)=1.0 microM) and heteroaryl imidazole derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC(50)=2.5 microM; benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC(50)=0.9 microM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (17), IC(50)=1.5 microM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure-function studies leading to clinical development are warranted.     

Design, synthesis, and biological evaluation of novel (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids as selective inhibitors for AKR1B1

New substituted (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids were designed as the inhibitor of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa, and twenty derivatives were synthesized and evaluated. The most active compound of the series was (2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7m), which showed comparable inhibitory activity for AKR1B1 (IC(50)=0.15μM) with clinically used epalrestat (IC(50)=0.1μM). In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC(50)=0.17μM) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared with epalrestat.     

Synthesis of N,N',N"-trisubstituted thiourea derivatives and their antagonist effect on the vanilloid receptor

Twenty-seven N,N',N"-trisubstituted thiourea derivatives were prepared. Among them, 1-[3-(4'-hydroxy-3'-methoxy-phenyl)-propyl]-1,3-diphenethyl-thiourea (8l, IC(50)=0.32 microM), showed 2-fold higher antagonistic activity than that of capsazepine (3, IC(50)=0.65 microM) against the vanilloid receptor in a (45)Ca(2+)-influx assay.     

Synthesis, anti-inflammatory, analgesic and kinase (CDK-1, CDK-5 and GSK-3) inhibition activity evaluation of benzimidazole/benzoxazole derivatives and some Schiff's bases

A series of N-(acridin-9-yl)-4-(benzo[d]imidazol/oxazol-2-yl) benzamides has been synthesized by the condensation of 9-aminoacridine derivatives with benzimidazole or benzoxazole derivatives. Condensation of 2-hydroxy naphthaldehyde with functionalized diamines leads to the formation of Schiff's bases and not imidazole derivatives. All these compounds were characterized by correct FT-IR, (1)H NMR, MS and elemental analyses. These compounds were screened for anti-inflammatory, analgesic and kinase (CDK-1, CDK-5 and GSK-3) inhibition activities. Compounds 11 and 7e(f) showed good anti-inflammatory (35.8% at 50 mg/kg po) activity and good analgesic activity (60% at 50 mg/kg po), respectively. Compound 3b showed significant in vitro activity against CDK-5 (IC(50)=4.6 microM) and CDK-1(IC(50)=7.4 microM) and compound 3a showed moderate CDK-5 inhibitory activity (IC(50)=7.5 microM). The other compounds showed moderate anti-inflammatory and analgesic activities.     

Synthesis and biological evaluation of norcantharidin analogues: towards PP1 selectivity

Simple modifications to the anhydride moiety of norcantharidin have lead to the development of a series of analogues displaying modest PP1 inhibition (low muM IC(50)s) comparable to that of norcantharidin (PP1 IC(50)=10.3+/-1.37 microM). However, unlike norcantharidin, which is a potent inhibitor of PP2A (IC(50)=2.69+/-1.37 microM), these analogues show reduced PP2A inhibitory action resulting in the development of selective PP1 inhibitory compounds. Data indicates that the introduction of two ortho-disposed substituents on an aromatic ring, or para-substituent favours PP1 inhibition over PP2A inhibition. Introduction of a p-morphilinoaniline substituent, 35, affords an inhibitor displaying PP1 IC(50)=6.5+/-2.3 microM; and PP2A IC(50)=7.9+/-0.82 microM (PP1/PP2A=0.82); and a 2,4,6-trimethylaniline, 23, displaying PP1 IC(50)=48+/-9; and PP2A IC(5) 85+/-3 microM (PP1/PP2A=0.56). The latter shows a 7-fold improvement in PP1 versus PP2A selectivity when compared with norcantharidin. Subsequent analysis of 23 and 35 as potential PP2B inhibitors revealed modest inhibition with IC(50)s of 89+/-6 and 42+/-3 microM, respectively, and returned with PP1/PP2B selectivities of 0.54 and 0.15. Thus, these analogues are the simplest and most selective PP1 inhibitors retaining potency reported to date.     

Investigations on the influence of terminal groups at the C2-propyl side chain of 1,1-bis(4-hydroxyphenyl)-2-phenylpent-1-ene and 1,1,2-tris(4-hydroxyphenyl)pent-1-ene on the estrogen receptor binding and the estrogenic/anti-estrogenic properties

1,1-bis(4-Hydroxyphenyl)-2-phenylpent-1-ene (5) and 1,1,2-tris(4-hydroxyphenyl)pent-1-ene (6) derivatives with terminal CN (5a, 6a), NH(2) (5b, 6b), NHCOCH(3) (5c, 6c), NHCOC(2)H(5) (5d, 6d) groups at the C2-propyl chain were synthesized and assayed in vitro for estrogen receptor (ER) binding affinity (RBA) in a competition experiment with [3H]estradiol and for estrogenic and anti-estrogenic properties in a luciferase assay with ER-positive MCF-7-2a cells, stably transfected with the plasmid ERE(wtc)luc. The CN as well as the NH(2) group reduced the RBA-values (5: 2.09%; 5a: 1.50%; 5b: 0.07%; 6: 4.03%; 6a: 0.67%; 6b: 0.20%) and the antagonistic potency (5: IC(50)=0.05 microM; 5a: IC(50)=0.43 microM; 5b: IC(50)=1.50 microM; 6: IC(50)=0.07 microM; 6a: IC(50)=0.60 microM; 6b: IC(50)=2.00 microM). Derivatization of the amino function with acetic anhydride and propionic anhydride did not change the RBA-value but altered the antagonistic profile (5c: IC(50)=2.50 microM; 5d: IC(50)=not detectable; 6c: IC(50)=0.65 microM; 6d: IC(50)=1.00 microM). Agonistic effects were only detected for the amine 6b (34.2% activation of the luciferase expression). These data document that estrogen receptor binding and the antagonistic effects can be modified by terminal groups at the C2-propyl chain of the pure antagonists 5 and 6. The mode of action is unclear. However, it can be assumed that the elongation of the side chain causes a reorientation in the LBD in order to locate the side chain in a side pocket near the ligand binding domain.