Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, K(i) = 0.23 microM), 10-methanesulfonyl-5-DACTHF (2, K(i) = 0.58 microM), and 10-methylthio-DDACTHF (3, K(i) = 0.25 microM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50 = 100 nM) against the CCRF-CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50 = 1.0 microM and 2, IC50 = 2.0 microM).     

Homonojirimycin analogues and their glucosides from Lobelia sessilifolia and Adenophora spp. (Campanulaceae)

2,6-Dideoxy-7-O-(beta-D-glucopyranosyl) 2,6-imino-D-glycero-L-gulo- heptitol (7-O-beta-D-glucopyranosyl-alpha-homonojirimycin, 1) was isolated from the 50% methanol extract of the whole plant of Lobelia sessilifolia (Campanulaceae), which was found to potently inhibit rice alpha-glucosidase. Adenophorae radix, roots of Adenophora spp. (Campanulaceae), yielded new homonojirimycin derivatives, adenophorine (2), 1-deoxyadenophorine (3), 5-deoxyadenophorine (4), 1-C-(5-amino-5-deoxy-beta-D-galactopyranosyl)butane (beta-1-C-butyl-deoxygalactonojirimycin, 5), and the 1-O-beta-D-glucosides of 2 (6) and 4 (7), in addition to the recently discovered alpha-1-C-ethylfagomine (8) and the known 1-deoxymannojirimycin (9) and 2R,5R-bis(hydroxymethyl)-3R,4R- dihydroxypyrrolidine (DMDP, 10). Compound 4 is a potent inhibitor of coffee bean alpha-galactosidase (IC50 = 6.4 microM) and a reasonably good inhibitor of bovine liver beta-galactosidase (IC50 = 34 microM). Compound 5 is a very specific and potent inhibitor of coffee bean alpha-galactosidase (IC50 = 0.71 microM). The glucosides 1 and 7 were potent inhibitors of various alpha-glucosidases, with IC50 values ranging from 1 to 0.1 microM. Furthermore, 1 potently inhibited porcine kidney trehalase (IC50 = 0.013 microM) but failed to inhibit alpha-galactosidase, whereas 7 was a potent inhibitor of alpha-galactosidase (IC50 = 1.7 microM) without trehalase inhibitory activity.     

Design, synthesis, and biological evaluation of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides as cyclooxygenase isozyme inhibitors

A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring substituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluorobenzenesulfonamide [14, COX-1 IC50 = 2.26 microM; COX-2 IC50 = 0.012 microM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC50 >100 microM; COX-2 IC50 = 0.15 microM; COX-2 SI >667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflammatory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC50 = 4.25 microM; COX-2 IC50 = 0.978 microM; COX-2 SI = 4.3), N-acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)benzenesulfonamide (17c, COX-1 IC50 = 1.02 microM; COX-2 IC50 = 1.00 microM; COX-2 SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC50 = 0.109 microM; COX-2 IC50 = 1.14 microM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflammation 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral dose reduced inflammation by 25% at 3 h postdrug administration.     

N-Acyl-1,2,3,4a,5,10b-hexahydro-[1]benzopyrano-[3,4-b][1,4]oxazine-9-carbonitriles as bladder-selective potassium channel openers

Optically active N-acyl-5,5-dimethyl-1,2,3,4a,5,10b-hexahydro-[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitriles 2-22 were synthesized as rigid analogues of cromakalim. The (4aR, 10bR)-N-benzoyl derivative (-)-11 was identified as a bladder-selective KCO (IC50, bladder = 8.2 microM, C50, portal vein = 34.5 microM). Among the analogues of 11 with substitution on the benzoyl moiety, the 3-methyl analogue (-)-14 showed highly potent and selective activity at portal vein (IC50, bladder = 279 microM, IC50, portal vein = 0.54 microM). The 4-bromo analogue (-)-19 (IC50, bladder = 2.0 microM, IC50, portal vein = 8.1 microM) and the 4-hydroxy analogue (-)-21 (IC50, bladder = 3.8 microM, IC50, portal vein = 75 microM) showed enhanced activity at the bladder, while maintaining unprecedented bladder selectivity in vitro. The N-benzenesulfonyl analogue (-)-22, a bioisoster of (-)-11, showed similar activity at the bladder with enhanced selectivity (IC50, bladder = 11.6 microM, IC50, portal vein = 120 microM).     

Design, synthesis, and biological evaluation of linear 1-(4-, 3- or 2-methylsulfonylphenyl)-2-phenylacetylenes: a novel class of cyclooxygenase-2 inhibitors

A group of regioisomeric 1-(methylsulfonylphenyl)-2-phenylacetylenes possessing a COX-2 SO(2)Me pharmacophore at the para-, meta- or ortho-position of the C-1 phenyl ring, in conjunction with a C-2 phenyl or substituted-phenyl ring substituent (3-F, 3-OMe, 3-OH, 3-OAc, 4-Me), were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. These target linear 1,2-diarylacetylenes were synthesized via a palladium-catalyzed Sonogashira cross-coupling reaction followed by oxidation of the respective 1-(methylthiophenyl)-2-phenylacetylene intermediate. In vitro COX-1/COX-2 isozyme inhibition structure-activity studies identified 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) as a potent COX-2 inhibitor (IC(50) = 0.32 microM) with a high COX-2 selectivity index (SI > 320) comparable to the reference compound rofecoxib (COX-2 IC(50) = 0.50 microM; COX-2 SI > 200). A molecular modeling study where (12d) was docked in the binding site of COX-2 showed that the MeSO(2) COX-2 pharmacophore was positioned in the vicinity of the secondary COX-2 binding site near Val(523). The 1-(4-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (11f, COX-1 IC(50) = 1.00 microM; COX-2 IC(50) = 0.06 microM; COX-2 SI = 16.7) and 1-(3-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (12f, COX-1 IC(50) = 6.5 microM; COX-2 IC(50) = 0.05 microM; COX-2 SI = 130) regioisomers exhibited comparable COX-2 inhibition, and moderately lower selective COX-2 selectivity, relative to the reference drug celecoxib (COX-1 IC(50) = 33.1 microM; COX-2 IC(50) = 0.07 microM; COX-2 SI = 472). The most potent anti-inflammatory agent 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) exhibited moderate oral anti-inflammatory activity (ED(50)= 129 mg/kg) at 3 h postdrug administration relative to the reference drug celecoxib (ED(50) = 10.8 mg/kg) in a carrageenan-induced rat paw edema assay. The structure-activity data acquired indicate that the acetylene moiety constitutes a suitable scaffold (template) to design novel acyclic 1,2-diarylacetylenes with selective COX-2, or dual COX-1/COX-2, inhibitory activities.     

Synthesis, cytotoxicity, and DNA topoisomerase II inhibitory activity of benzofuroquinolinediones

Benzofuroquinolinediones (7c and 7d) were synthesized by base-catalyzed condensation of dichloroquinolinediones with phenolic derivatives. Their dialkylaminoalkoxy derivatives (8i-8p) were prepared by reaction with various dialkylaminoalkyl chlorides. The cytotoxicity of the synthesized compounds was evaluated against eight types of human cancer cell lines, and their topoisomerase II inhibition was assessed. In general, the cytotoxicity of benzofuroquinolinediones (8i-8p) was similar or superior to that of doxorubicin and showed more potent inhibitory activity than naphthofurandiones (8a-8h). Also, most of the compounds exhibited excellent topoisomerase II inhibitory activity at a concentration of 5 microM and two compounds, 8d and 8i, showed IC50 values of 1.19 and 0.68 microM, respectively, and were much more potent than etoposide (IC50=78.4 microM), but similar to doxorubicin (IC50=2.67 microM). However their inhibitory activity on topoisomerase I was lower, and 8d and 8i showed IC50 values of 42.0 and 64.3 microM, respectively.     

N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea and N'-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]-thiourea as potent inhibitors of multidrug-resistant human immunodeficiency virus-1.

We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.     

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.     

Noradrenaline Antagonizes and Ouabain Potentiates the Effects of iV-Methyl-D-Aspartate on Rat Cerebellar Cyclic GMP Production

Noradrenaline potently antagonizes the effects of N-methyl-D-aspartate (NMDA) (80 microM) on cyclic GMP production in immature rat cerebellar slices in vitro (IC50 = 0.6 microM). The effect is stereospecific (D-noradrenaline, IC50 = 100 microM), and also observed with adrenaline (IC50 = 0.5 microM) and isoprenaline (IC50 = 1.2 microM). The alpha 1-adrenoceptor agonists methoxamine or phenylephrine or the mixed alpha 1/alpha 2 agonists oxymetazoline or xylometazoline (100 microM) do not block the effects of NMDA, but the alpha 2-adrenoceptor agonist clonidine is weakly active (IC50 = 200 microM). Salbutamol and terbutaline were also inactive except at high concentrations (300 microM), as were a number of other catechol and phenylethylamine derivatives. The antagonistic effects of noradrenaline on the NMDA response were insensitive to phentolamine, atenolol, or propranolol (up to 100 microM), but were blocked by the alpha 2 antagonist idazoxan (1-10 microM). The Na+,K+-ATPase inhibitor ouabain (0.1-10 microM) markedly potentiates the effects of NMDA in this model, and also antagonizes and reverses the ability of noradrenaline (10 microM) to block the effects of NMDA. The results suggest that noradrenaline and Na+,K+-ATPase activity have potent modulatory effects on the NMDA response.     

Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.     

Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters

n-Alkyl esters (ethyl, octyl, dodecyl, and cetyl) of gallic acid were evaluated as enzyme inhibitors of recombinant rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. Dodecyl (6) (IC(50) = 0.061 microM) showed the most potent inhibition, which was far more potent than those of previously reported naturally occurring gallocatechins. Octyl gallate (5) (IC(50) = 0.83 microM) and cetyl gallate (7) (IC(50) = 0.59 microM) also showed good inhibition, while gallic acid (IC(50) = 73 microM) itself was not so active. In addition, chemically synthesized galloyl ester of cholesterol (9) (IC(50) = 3.9 microM), farnesol derivative (10) (IC(50) = 0.57 microM), and dodecyl galloyl amide (8) (IC(50) = 3.0 microM) were also potent inhibitors of SE. Inhibition kinetics revealed that dodecyl gallate inhibited SE in competitive (K(I) = 0.033 microM) and no-time-dependent manner. The potent inhibition of the flavin monooxygenase would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the epoxidation reaction.     

Effects of caffeoyl conjugates of isoprenyl-hydroquinone glucoside and quinic acid on leukocyte function

The activity of three prenylhydroquinone glucosides (1-3) and four caffeoylquinic esters (4-7), obtained from Phagnalon rupestre, on elastase release, myeloperoxidase activity and superoxide and leukotriene B(4) production from polymorphonuclear leukocytes was determined. 4,5-Dicaffeoylquinic acid strongly inhibited elastase release with an IC(50) value of 4.8 microM. Methylated caffeoylquinic derivatives were the most potent inhibitors of myeloperoxidase (IC(50) near 60 microM), whereas both methylated and free carboxyl isomers inhibited superoxide production with similar potency (IC(50) between 27 and 42 microM). The monocaffeoyl conjugate of prenylhydroquinone glucoside (3), the most potent inhibitor of leukotriene B(4) production (IC(50) = 33 microM), possesses a mixed hydroquinone-caffeoyl character that could be considered as a potential anti-inflammatory entity.     

Cantharimides: a new class of modified cantharidin analogues inhibiting protein phosphatases 1 and 2A.

Cantharidin and its analogues have been of considerable interest as potent inhibitors of the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A). However, limited modifications to the parent compounds is tolerated. As part of an on-going study we have developed a new series of cantharidin analogues, the cantharimides. Inhibition studies indicate that cantharimides possessing a D- or L-histidine, are more potent inhibitors of PP1 and PP2A (PP1 IC(50)=3.22+/-0.7 microM; PP2A IC(50)=0.81+/-0.1 microM and PP1 IC(50)=2.82+/-0.6 microM; PP2A IC(50)=1.35+/-0.3 microM, respectively) than norcantharidin (PP1 IC(50)=5.31+/-0.76 microM; PP2A IC(50)=2.9+/-1.04 microM) and essentially equipotent with cantharidin (PP1 IC(50)=3.6+/-0.42 microM; PP2A IC(50)=0.36+/-0.08 microM). Cantharimides with non-polar or acidic amino acid residues are only poor inhibitors of PP1 and PP2A.     

Nordihydroguaiaretic acid is a potent in vitro scavenger of peroxynitrite, singlet oxygen, hydroxyl radical, superoxide anion and hypochlorous acid and prevents in vivo ozone-induced tyrosine nitration in lungs

The antioxidant nordihydroguaiaretic acid (NDGA) has recently become well known as a putative anticancer drug. In this paper, it was evaluated the in vitro peroxynitrite (ONOO(-)), singlet oxygen ((1)O(2)), hydroxyl radical (OH(v)), hydrogen peroxide (H(2)O(2)), superoxide anion and hypochlorous acid (HOCl) scavenging capacity of NDGA. It was found that NDGA scavenges: (a) ONOO(-) (IC(50) = 4 +/- 0.94 microM) as efficiently as uric acid; (b) (1)O(2) (IC(50) = 151 +/- 20 microM) more efficiently than dimethyl thiourea, lipoic acid, N-acetyl-cysteine and glutathione; (c) OH(v) (IC(50) = 0.15 +/- 0.02 microM) more efficiently than dimethyl thiourea, uric acid, trolox, dimethyl sulfoxide and mannitol, (d) (IC(50) = 15 +/- 1 microM) more efficiently than N-acetyl-cysteine, glutathione, tempol and deferoxamine and (e) HOCl (IC(50) = 622 +/- 42 microM) as efficiently as lipoic acid and N-acetyl-cysteine. NDGA was unable to scavenge H(2)O(2). In an in vivo study in rats, NDGA was able to prevent ozone-induced tyrosine nitration in lungs. It is concluded that NDGA is a potent in vitro scavenger of ONOO(-), (1)O(2), OH(v), and HOCl and is able to prevent lung tyrosine nitration in vivo.     

Binding to prostaglandin (PG) receptors and activation of adenyl cyclase by 20-isopropylidene-PGS in rabbit platelet membranes

20-Isopropylidene-PGE1 (Isop-PGE1) was about 10 times more potent than PGE1 in inhibition of thrombin-induced aggregation of rabbit washed platelets. Likewise, 20-isopropylidene-17(R)-methyl-carbacyclin (CS-570), a stable PGI2 analogue, was more potent than carbacyclin in the anti-aggregatory activity. In order to define the platelet-prostaglandin interactions, a binding assay was done using platelet membranes with [3H]-PGE1 as a radioligand. Isop-PGE1 (IC50 = 0.18 microM) bound to the PG receptors more potently than PGE1 (IC50 = 2.1 microM). CS-570 (IC50 = 0.39 microM) was more potent than carbacyclin (IC50 = 1.9 microM). These indicate that introduction of an isopropylidene group to the carbon 20 of PGs increases the binding ability to the receptors. These PGE1 and PGI2 analogues activated platelet membrane adenyl cyclase and increased intracellular cAMP levels with the same potency series obtained in the binding experiments. All these results suggest that the binding to the receptors by these PGs is coupled to the activation of adenyl cyclase, followed by the increase in cAMP levels in platelets and the inhibition of platelet aggregation. Thus, the increased anti-aggregatory activity of 20-isop-PGs may be explained by their increased affinity for the PG receptors and stimulation of adenyl cyclase. 15-Epimeric-20-isopropylidene-PGE1 (15-Epi-isop-PGE1), which has an unnatural configuration of the 15-hydroxyl group, was much less potent than isop-PGE1 in the binding experiment and the other three investigations. This indicates that the configuration of the 15-hydroxyl group is important for the binding to the PG receptors and the consequent activities in platelets.     

Inhibition of poly(ADP-ribose) synthetase by unsaturated fatty acids, vitamins and vitamin-like substances

Various vitamins and vitamin-like substances inhibited the activity of poly(ADP-ribose) synthetase in vitro. The most potent were essential fatty acids, i.e. arachidonic acid, linoleic acid, and linolenic acid; their 50% inhibitory concentrations (IC50) were 44-110 microM, indicating a higher potency than nicotinamide, a well-known vitamin inhibitor (IC50 = 210 microM). Vitamins K3, K1, and retinal were the next strongest inhibitors, followed by alpha-lipoic acid, coenzyme Q0, and pyridoxal 5-phosphate. Nicotinamide and vitamin K3 exhibited mixed-type inhibition with respect to NAD+, while arachidonic acid exhibited dual inhibitions, competitive at 50 microM and mixed-type at 100 microM.     

3Beta-hydroxy-6-aza-cholestane and related analogues as phosphatidylinositol specific phospholipase C (PI-PLC) inhibitors with antitumor activity

6-Aza steroid analogues were synthesized as PI-PLC inhibitors. The most active compound, 3beta-hydroxy-6-aza-cholestane (1) showed potent PI-PLC inhibition (IC50 = 1.8 microM), similar to that of the commercially available steroid analogue U73122 (IC50 = 1-2.1 microM). Compound 1 exhibited significant growth inhibition effects (IC50 = 1.3 microM in each case) against MCF-7 and HT-29 cancer cells in in vitro cell culture. Compound 1 also inhibited the in vitro adhesion and transmigration of HT-1080 fibrosarcoma cells at 2.5 and 5.0 microM, respectively. In vivo, compound 1, at 1 mg/kg/day, reduced the volume of MCF-7 tumors in xenograft models, without weight loss in mice. Structure activity relationships of this series of compounds revealed that a hydrophobic cholesteryl side chain, 3beta-hydroxy group and a C-6 nitrogen containing a hydrogen atom at position-6 are crucial for activity. N-Maleic amidoacid derivative 11 also exhibited weak inhibition (IC50 = 16.2 microM).     

Discovery of cyclin-dependent kinase inhibitor, CR229, using structurebased drug screening

To generate new scaffold candidates as highly selective and potent cyclin-dependent kinase (CDK) inhibitors, structure-based drug screening was performed utilizing 3D pharmacophore conformations of known potent inhibitors. As a result, CR229 (6-bromo-2,3,4,9-tetrahydro-carbolin-1-one) was generated as the hit-compound. A computational docking study using the X-ray crystallographic structure of CDK2 in complex with CR229 was evaluated. This predicted binding mode study of CR229 with CDK2 demonstrated that CR229 interacted effectively with the Leu83 and Glu81 residues in the ATP-binding pocket of CDK2 for the possible hydrogen bond formation. Furthermore, biochemical studies on inhibitory effects of CR229 on various kinases in the human cervical cancer HeLa cells demonstrated that CR229 was a potent inhibitor of CDK2 (IC50: 3 microM), CDK1 (IC50: 4.9 microM), and CDK4 (IC50: 3 microM), yet had much less inhibitory effect (IC50: >20 microM) on other kinases, such as casein kinase 2-1 (CK2- alpha1), protein kinase A (PKA), and protein kinase C (PKC). Accordingly, these data demonstrate that CR229 is a potent CDK inhibitor with anticancer efficacy.