Rabbit corneal and conjunctival permeability of the novel aldose reductase inhibitors: N-[[4-(benzoylamino)phenyl] sulphonyl]glycines and N-benzoyl-N-phenylglycines.

Corneal and conjunctival permeability has been investigated for novel aldose reductase inhibitors (ARIs) of the N{[4-(benzoylamino)phenyl]sulphonyl}glycine (benzoylaminophenylsulphonylglycine) and N-benzoyl-N-phenylglycine (benzoylphenylglycine) series, compounds developed for prevention of cataract formation in diabetic subjects. Six benzoylaminophenylsulphonylglycines were synthesized with modifications either of the phenyl group or of the glycine structure and three benzoylphenylglycines were synthesized with modification in the phenyl group of the benzoyl moiety. Transport of ARIs in the mucosal to serosal direction was evaluated across rabbit cornea and conjunctiva bathed in glutathione-bicarbonate Ringer's solution maintained at pH 7.4 and 37 degrees C. The permeability coefficients of the novel ARIs across cornea and conjunctiva ranged from 1.87 to 8.95 x 10(-6) cm s(-1) and from 4.6 to 19.15 x 10(-6) cm s(-1), respectively. The ratio of corneal to conjunctival permeability ranged from 0.12 to 0.79. The calculated log partition coefficient (log P) values for the ARIs were in the range 0.84 to 2.78. The log distribution coefficients (log D) were in the range -2.87 to -0.89. There was no apparent relationship between log P or log D and the permeability coefficients of the ARIs for either tissue. Cornea was more resistant to ARI transport than was conjunctiva. Substitution of a phenyl group for hydrogen in the glycine methylene group reduced the permeability coefficient. Permeability coefficients were different for different stereoisomers. Compared with the permeability coefficient of benzoylaminophenylsulphonylglycine, that of 4-fluorobenzoylaminophenylsulphonylglycine was lower in the cornea but similar in the conjunctiva. In both tissues, the permeability coefficient of 2-nitrobenzoylaminophenylsulphonylglycine was less than that of 4-nitrobenzoylaminophenylsulphonylglycine. There was no significant difference between the permeability coefficients of 3-nitro- and 4-nitrobenzoylphenylglycines through either tissue and the permeability coefficients of these compounds were greater than that of the more lipophilic 4-methylbenzoylphenylglycine. The lack of dependence of the permeability coefficients on log P or log D and the different permeabilities of stereoisomers imply the existence of specialized transport processes for the ARIs tested in this study.     

Systemic and ocular pharmacokinetics of N-4-benzoylaminophenylsulfonylglycine (BAPSG), a novel aldose reductase inhibitor

To better develop N-[4-(benzoylamino)phenylsulfonyl]glycine (BAPSG), a potent and selective aldose reductase inhibitor capable of delaying the progression of ocular diabetic complications, the objective of this study was to assess its pharmacokinetics. The plasma pharmacokinetics of BASPG was assessed in male Sprague-Dawley rats following intravenous, intraperitoneal and oral routes of administration and its distribution to various tissues including those of the eye was studied following intraperitoneal administration. In addition, rat plasma protein binding of BAPSG was studied using ultracentrifugation method and its ocular tissue disposition was assessed following topical administration in rabbits. Plasma and tissue levels of BAPSG were analysed using an HPLC assay. BAPSG exhibited dose-proportionate AUC0 --> infinity (area under the plasma concentration-time curve) following both intravenous and intraperitoneal administration over the dose range (5-50 mg kg(-1)) studied and an erratic oral absorption profile with low oral bioavailability. The fraction bioavailability following oral and intraperitoneal administration was 0.06 and 0.7-1, respectively. BAPSG exhibited short plasma elimination half-lives in the range 0.5-1.5 h. BAPSG was bound to rat plasma proteins and the percent protein binding ranged from 83 to 99.8%. BAPSG was better distributed to cornea, lens and retina than to brain, following intraperitoneal administration in rats. However, the distribution was lower compared with kidney and liver. Following topical administration in rabbits, BAPSG delivery to the surface ocular tissues, cornea and conjunctiva was higher compared with intraocular tissues, aqueous humour, iris-ciliary body and lens. Thus, BAPSG was distributed to ocular tissues following systemic and topical modes of administration.     

N- and 2-substituted N-(phenylsulfonyl)glycines as inhibitors of rat lens aldose reductase

A variety of N-(phenylsulfonyl)-N-phenylglycines 5, N-(phenylsulfonyl)-2-phenylglycines 6, and N-(phenylsulfonyl)anthranilic acids 7 were prepared as analogues of the N-(phenylsulfonyl)glycine 1 aldose reductase inhibitors. In the rat lens assay, several derivatives of 5 display greater inhibitory activity than the corresponding glycines 1, suggesting that N-phenyl substitution enhances affinity for aldose reductase. Enzyme kinetic evaluations of the 4-benzoylamino analogues of 5 and 1 demonstrate that these compounds produce inhibition by the same mechanism. However, the significant differences in relative inhibitory potencies between compounds of series 5 and 1 may indicate that these compounds do not interact with the inhibitor binding site in precisely the same manner. Evaluation of the individual enantiomers of series 6 reveals that the S isomers are substantially more active than the corresponding R isomers. Also, with the exception of the naphthalene analogue 6n, the S stereoisomers of this series display greater inhibitory potencies than the glycines 1. The anthranilates 7 generally are less active than the glycines 1, demonstrating that direct incorporation of an aromatic ring in the glycine side chain may result in a decrease in affinity for aldose reductase.     

Relative structure-inhibition analyses of the N-benzoyl and N-(phenylsulfonyl) amino acid aldose reductase inhibitors

A number of N-benzoyl amino acids were synthesized and tested to compare structure-inhibition relationships with the isosteric N-(phenylsulfonyl) amino acid (PS-amino acid) aldose reductase inhibitors. Inhibition analyses with these series reveals that their kinetic mechanisms of inhibition are similar, but that significant differences in structure-inhibition relationships exist. For example, while the PS-alanines and PS-2-phenylglycines produce enantioselective inhibition (S greater than R), no consistent pattern of enantioselectivity is observed with the isosteric N-benzoylalanines and 2-phenylglycines. Also, N-methyl and N-phenyl substitution in the PS-amino acid series does not substantially alter inhibitory activity, while similar substitutions in the N-benzoyl series (particularly N-phenyl) results in a significant increase in inhibitory activity. Proton NMR analysis of the N-benzoylsarcosines reveals that these compounds exist as a mixture of rotamers in solutions including the enzyme assay buffer and that the preferred conformer is one in which the carboxymethyl moiety is trans to the aromatic ring. Similar analyses with the N-benzoyl-N-phenylglycines demonstrate that these derivatives exist exclusively in the trans rotameric conformation in solution. No such N-substituent effects on conformation were observed in the PS-amino acid series. These results suggest that the differences in structure-inhibition trends between these structurally related series may result from the effect of substituents on preferred conformation.     

N-[2-(4-特戊酰氧基苯磺酰胺基)苯甲酰基]甘氨酸苄酯的合成

目的合成西维来司钠（sivelestat sodium）的关键中间体N—[2-（4-特戊酰氧基苯磺酰胺基）苯甲酰基]甘氨酸苄酯（1）。方法先以特戊酸、氯化亚砜、对羟基苯磺酸为原料，经酯化、苯磺酸成酰氯得到对位特戊酰氧基苯磺酰氯（4）；再以甘氨酸、苄醇、邻硝基苯甲酰氯为原料经酯化、酰氨化、还原得到N-（2-氨基苯甲酰基）甘氨酸苄酯（7）；将4和7两中间体缩合得1。结果及结论本方法原料廉价易得，条件温和易控，收率较高，适合工业化生产。     

(4-[6-(4-isopropoxyphenyl) pyrazolo [1,5-a]pyrimidin- 3-yl] quinoline) is a novel inhibitor of autophagy

Background and Purpose

Autophagy is an important intracellular degradation system, which is related to various diseases. In preliminary experiments we found that D4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline (DMH1) inhibited autophagy responses. However DMH1 also inhibits the signalling pathway activated by bone morphogenetic protein-4 (BMP4). The aim of the present study was to elucidate the inhibitory effects of DMH1 on autophagy and the underlying mechanisms.

Experimental Approach

The effects of DMH1 on autophagy responses were evaluated in cultures of different cell types and with different stimuli to induce autophagy, using Western blots, transmission electron microscopy and fluorescent microscopy.

Key Results

DMH1 inhibited starvation-induced autophagy in cardiomyocytes, HeLa and MCF-7 cells, without involving the signalling pathway of BMP4. DMH1 inhibited aminoimidazole carboxamide ribonucleotide (AICAR)- and rapamycin-induced autophagy in HeLa and MCF-7 cells. DMH1 reversed starvation- and AICAR-induced inhibition of Akt, mammalian target of rapamycin (mTOR) and p70S6 kinase (S6K), and reversed rapamycin-induced inhibition of mTOR and S6K. DMH1 reversed starvation-induced decrease of the phosphorylated form of glycogen synthase kinase-3 in MCF-7 and HT29 cells. Activation of Akt and inhibition of autophagy induced by DMH1 were antagonized by an Akt specific inhibitor or by small interfering RNA for Akt in HeLa cells.

Conclusion and Implications

DMH1 inhibited cellular autophagy responses in a range of cell types and the underlying mechanisms include activation of the Akt pathway.     

A potent aldose reductase inhibitor, (2S,4S)-6-fluoro-2', 5'-dioxospiro[chroman-4,4'-imidazolidine]-2-carboxamide (Fidarestat): its absolute configuration and interactions with the aldose reductase by X-ray crystallography

The absolute configuration of the aldose reductase (AR) inhibitor, (+)-(2S,4S)-6-fluoro-2',5'-dioxospiro?chroman-4, 4'-imidazolidine-2-carboxamide (fidarestat), was established indirectly by single-crystal X-ray analysis of (+)-(2S, 4S)-8-bromo-6-fluoro-2',5'-dioxospiro?chroman-4, 4'-imidazolidine-2-carboxylic acid (1). The crystal structure of human AR complexed with fidarestat was determined, and the specific inhibition activity was discussed on the basis of the three-dimensional interactions between them. The structure clarified that fidarestat was located in the active site by hydrophilic and hydrophobic interactions and that the carbamoyl group of fidarestat was a very effective substituent for affinity to AR and for selectivity between AR and aldehyde reductase (AHR). Explanations for the differences between the observed activities of fidarestat and its stereoisomer 2 were suggested by computer modeling.     

Effects of a novel glutamate transporter blocker, (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), on activities of hippocampal neurons

Glutamate transporters rapidly take up synaptically released glutamate and maintain the glutamate concentration in the synaptic cleft at a low level. (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) is a novel glutamate transporter blocker that potently suppresses the activity of glial transporters. TFB-TBOA inhibited synaptically activated transporter currents (STCs) in astrocytes in the stratum radiatum in rat hippocampal slices in a dose-dependent manner with an IC50 of 13 nM, and reduced them to approximately 10% of the control at 100 nM. We investigated the effects of TFB-TBOA on glutamatergic synaptic transmission and cell excitability in CA1 pyramidal cells. TFB-TBOA (100 nM) prolonged the decay of N-methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs), whereas it prolonged that of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated EPSCs only when the desensitization of AMPARs was reduced by cyclothiazide (CTZ). Furthermore, long-term application of TFB-TBOA induced spontaneous epileptiform discharges with a continuous depolarization shift of membrane potential. These epileptiform activities were mainly attributed to NMDAR activation. Even after pharmacological block of NMDARs, however, TFB-TBOA induced similar changes by activating AMPARs in the presence of CTZ. Thus, the continuous uptake of synaptically released glutamate by glial transporters is indispensable for protecting hippocampal neurons from glutamate receptor-mediated hyperexcitabilities.     

1-(3,5-difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]phenylmethanone as a bioisostere of a carboxylic acid aldose reductase inhibitor

[1-(3,5-Difluoro-4-hydroxyphenyl)-1H-pyrrol-3-yl]phenylmethanone (6) was synthesized as a putative bioisostere of the known aldose reductase (AR) inhibitor (3-benzoylpyrrol-1-yl)acetic acid (I). It was found that 6 is approximately 5 times more potent as an in vitro inhibitor of AR than I, with an IC(50) value in the submicromolar range. Furthermore, 6 showed considerable activity in an in vitro experimental glycation model of diabetes mellitus. Our results support the notion that 6 might become a useful lead structure.     

Determination of tolrestat, a novel aldose reductase inhibitor, in serum and tissues

An ultraviolet spectrophotometric method and a high-performance liquid chromatographic (HPLC) method were developed for the determination of tolrestat, a novel aldose reductase inhibitor, in serum. The limits of detection of the methods are 15 micrograms/ml and 0.2 microgram/ml, respectively. With human serum, a modification of the HPLC method provides sensitivity to 25 ng/ml. The specificity of the methods were compared. The HPLC method can be applied to the lens and the sciatic nerve.     

N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS) is a selective persistent inhibitor of glycine transport.

1. The regulation of glycine concentrations within excitatory synapses is poorly understood and it has been proposed that the GLYT1 subtypes of glycine transporters play a critical role in determining resting concentrations of glycine. Selective GLYT1 inhibitors may provide pharmacological tools to probe the dynamics of synaptic glycine concentrations, which may influence the activation properties of NMDA receptor activity. 2. We have characterized the selectivity and mechanism of action of the glycine transport inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS). The glycine transporters, GLYT1a, b and c and GLYT2a were expressed in Xenopus laevis oocytes and two electrode voltage clamp techniques and radiolabelled (3)H-glycine flux measurements were used to characterize the effects of NFPS on glycine transport. 3. NFPS inhibits glycine transport by the GLYT1a, b and c subtypes of glycine transporters, but has no effect on the GLYT2a subtype of transporter. We show that NFPS does not attain its specificity via an interaction with the Na(+), Cl(-) or glycine site, nor does it act at an intracellular site. NFPS inhibition of glycine transport is time and concentration dependent and inhibition of transport by NFPS persists after washout of NFPS from the bath solution, which suggests that inhibition by NFPS is long lasting.     

Pharmacology and expression analysis of glycine transporter GlyT1 with [3H]-(N-[3-(4'-fluorophenyl)-3-(4'phenylphenoxy)propyl])sarcosine

In the central nervous system, re-uptake of the neurotransmitter glycine is mediated by two different glycine transporters, GlyT1 and GlyT2. GlyT2 is found in brainstem and spinal cord, whereas GlyT1 is expressed in rat forebrain regions where it is responsible for most glycine transport activity. Initially, GlyT1 and GlyT2 were pharmacologically differentiated by sarcosine, a weak selective inhibitor of GlyT1. The recently described selective and potent GlyT1 antagonist, NFPS/ALX-5407 provided an important additional tool to further characterize GlyT1 pharmacology. In the present study, we have radiolabeled the racemic form of NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (also known as ALX-5407) to investigate its interaction with GlyT1, as well as define GlyT1 expression in the rat central nervous system. Kinetic studies indicated that [3H]NFPS binds rapidly to rat forebrain membranes and dissociates with a t(1/2) of 28 +/- 5 min. [3H]NFPS labeled a saturable population of sites in rat forebrain with a Kd of 7.1+/-1.3 nM and a B(max) of 3.14 +/- 0.26 pmol/mg protein. Bound [3H]NFPS was fully and potently displaced by unlabeled NFPS, whereas glycine and sarcosine were weak, Na+-dependent inhibitors with IC50 of 1,008 and 190 microM, respectively. Additional saturation experiments indicated that glycine and sarcosine were non-competitive antagonists of [3H]NFPS binding. Functional studies revealed that NFPS was a non-competitive inhibitor of [3H]glycine uptake and does not interact with Na+ and Cl- binding sites of GlyT1. Overall, this work shows that [3H]NFPS is a valuable tool in studying GlyT1 expression and pharmacology and that NFPS interacts with GlyT1 at a site different from the transporter translocation and ion binding sites.     

Pharmacological effects of the novel dopamine uptake inhibitor 1-[2-[bis(4-fluorophenyl)-methoxy]ethyl]-4-(3-phenylpropyl) piperazine dihydrochloride (I-893) on the central nervous system]

The effects of 1-[2-[bis (4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl) piperazine dihydrochloride (I-893) on the central nervous system were behaviorally and electroencephalographically investigated. Intraperitoneally injected I-893 (5-10 mg/kg) dose-dependently increased spontaneous motor activity in mice, but repeated injections did not affect the increase in the locomotor activity. In reserpinized mice, spontaneous motor activity was not increased by oral I-893. In alpha-MPT-treated mice, the motor activity was lower than that in vehicle-treated animals with intermediate doses (10-40 mg/kg, p.o.) of I-893, but there was no difference between the two groups with high doses. In rats with unilaterally 6-OHDA-induced lesion of the nigrostriatal pathway, I-893 induced circling behavior toward the lesioned side. Haloperidol-induced catalepsy in rats was reduced by I-893. Tremorine-induced tremor in mice was inhibited by I-893. The effect was not altered in the mice treated with I-893 for 10 days. Oral I-893 induced stereotypy in rats, but it did not affect methamphetamine-induced stereotypy. Hypnosis induced by barbiturates was antagonized by I-893. In rats treated with I-893 for 6 days, pentobarbital-induced sleep was not different from that in vehicle-treated animals on the day after the final treatment. Intravenous I-893 altered EEGs in the cerebral cortex and amygdala nucleus to low voltage and fast waves and altered hippocampal EEG to theta waves in immobilized rabbits. These results suggest that I-893 inhibits re-uptake of dopamine released by exocytosis and indirectly has dopaminergic effects.     

Discovery of N-[2-[2-[[3-methoxy-4-(5-oxazolyl)phenyl]amino]-5-oxazolyl]phenyl]-N-methyl-4- morpholineacetamide as a novel and potent inhibitor of inosine monophosphate dehydrogenase with excellent in vivo activity

Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme that is involved in the de novo synthesis of purine nucleotides. Novel 2-aminooxazoles were synthesized and tested for inhibition of IMPDH catalytic activity. Multiple analogues based on this chemotype were found to inhibit IMPDH with low nanomolar potency. One of the analogues (compound 23) showed excellent in vivo activity in the inhibition of antibody production in mice and in the adjuvant induced arthritis model in rats.     

Synthesis, biological evaluation, and structure-activity relationships of 2-[2-(benzoylamino)benzoylamino]benzoic acid analogues as inhibitors of adenovirus replication

2-[2-Benzoylamino)benzoylamino]benzoic acid (1) was previously identified as a potent and nontoxic antiadenoviral compound (Antimicrob. Agents Chemother. 2010, 54, 3871). Here, the potency of 1 was improved over three generations of compounds. We found that the ortho, ortho substituent pattern and the presence of the carboxylic acid of 1 are favorable for this class of compounds and that the direction of the amide bonds (as in 1) is obligatory. Some variability in the N-terminal moiety was tolerated, but benzamides appear to be preferred. The substituents on the middle and C-terminal rings were varied, resulting in two potent inhibitors, 35g and 35j, with EC(50) = 0.6 μM and low cell toxicity.     

In-vitro and in-vivo characterization of JNJ-7925476, a novel triple monoamine uptake inhibitor

Triple reuptake inhibitors, which block the serotonin transporter (SERT), norepinephrine transporter (NET) and dopamine transporter (DAT) in the central nervous system have been described as therapeutic alternatives for classical selective serotonin reuptake inhibitors, with advantages due to their multiple mechanisms of action. JNJ-7925476 (trans-6-(4-ethynylphenyl)-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline) is a selective and potent inhibitor of the SERT, NET, and DAT (K(i)=0.9, 17 and 5.2 nM, respectively). Following subcutaneous dosing in rat, JNJ-7925476 was rapidly absorbed into the plasma, and drug concentrations in the brain tracked with those in the plasma but were 7-fold higher. The ED(50) values for JNJ-7925476 occupancy of the SERT, NET, and DAT in rat brain were 0.18, 0.09 and 2.4 mg/kg, respectively. JNJ-7925476 (0.1-10 mg/kg, s.c.) rapidly induced a robust, dose-dependent increase in extracellular serotonin, dopamine, and norepinephrine levels in rat cerebral cortex. The compound also showed potent antidepressant-like activity in the mouse tail suspension test (ED(50)=0.3 mg/kg, i.p.). These results demonstrate that JNJ-7925476 is a triple reuptake inhibitor with in-vivo efficacy in biochemical and behavioral models of depression.     

N-[2-(4-甲氧基苯基)-1-甲基乙基]苄胺的制备

以1-（4-甲氧基苯基）丙酮-2和N-苄基甲酰胺为原料,采用“一锅法”,经N-苄基化、Leuckart反应和酸性水解制得福莫特罗的重要中间体N-[2-（4-甲氧基苯基）-1-甲基乙基]苄胺,总收率约46%。     

Synthesis and antimicrobial activity of N-[4-(1,3-benzodithiol-2-yl)phenyl]-and N-[4-(4-aryl-1,3-dithiol-2-yl)phenyl]-substituted N-arylmethylamines

Methods for the synthesis of secondary aromatic amines containing 1,3-benzodithiol or 4-aryl-1,3-dithiol cycles have been developed. The antimicrobial activity of the synthesized compounds has been determined. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 10, pp. 28–29, October, 2006.     

N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor

Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer progression. We report here a novel subseries of C-5-substituted anilinoquinazolines that display high affinity and specificity for the tyrosine kinase domain of the c-Src and Abl enzymes. These compounds exhibit high selectivity for SFKs over a panel of recombinant protein kinases, excellent pharmacokinetics, and in vivo activity following oral dosing. N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530) inhibits c-Src and Abl enzymes at low nanomolar concentrations and is highly selective over a range of kinases. AZD0530 displays excellent pharmacokinetic parameters in animal preclinically and in man (t(1/2) = 40 h). AZD0530 is a potent inhibitor of tumor growth in a c-Src-transfected 3T3-fibroblast xenograft model in vivo and led to a significant increase in survival in a highly aggressive, orthotopic model of human pancreatic cancer when dosed orally once daily. AZD0530 is currently undergoing clinical evaluation in man.