A new series of neutral 5-substituted 4-anilinoquinazolines as potent, orally active inhibitors of erbB2 receptor tyrosine kinase

Starting from initial lead 1 containing a basic 5-substituent, optimisation of the glycolamide-derived neutral 5-substituent led to potent inhibitors of erbB2 with good pharmacokinetics. Representative compounds 19 and 21 inhibited phosphorylation of erbB2 in a mouse BT474C xenograft model after oral administration.     

Effects of 2-substituted-4-phenylquinolines on uptake of serotonin and norepinephrine by isolated brain synaptosomes

In this present communication, the in vitro inhibition of the uptake of [3H]-L-norepinephrine ([3H] NE) and [3H]-Serotonin ([3H] 5-HT) by eleven synthesized 2-substituted-4-phenyl quinolines were studied using rat brain synaptosomal preparations. Compounds with an open side chain were relatively weak inhibitors of the synaptosomal uptake of [3H] NE and [3H] 5HT. Compounds having a distance of three atoms between the terminal basic nitrogen of the side chain and the quinoline ring were better inhibitors of serotonin uptake than those compounds having a four-atom distance. The replacement of the side chain with a piperazine ring produced compounds which were more potent and selective inhibitors of the uptake of either [3H] 5-HT or [3H] NE. Further structure-activity relationships are also discussed.     

Anti-angiogenic activity of basic-type, selective cyclooxygenase (COX)-1 inhibitors

Indole- and indoline-type basic COX-1-selective competitive inhibitors, 5-amino-1-(3,5-dimethylbenzoyl)-1H-indole (1) and 5-amino-1-(3,5-dimethylphenyl)-2,3-dihydro-1H-indole (2), were found to possess anti-angiogenic activity estimated as a tube formation-inhibition using human umbilical vein endothelial cells (HUVECs).     

Substituted thiophene-anthranilamides as potent inhibitors of human factor Xa

A series of thiophene-containing non-amidine factor Xa inhibitors is described. Simple methyl-substituted thiophene analogs were relatively weak inhibitors. However, introduction of hydrophilic substituents at C-4 or C-5 of the thiophene afforded inhibitors with low nanomolar potency. Optimization of the thiophene substituent at C-4 afforded subnanomolar inhibitors with improved in vitro anticoagulant activity. Incorporating basic amine substituents on the thiophene increased hydrophilicity and improved anticoagulant activity. The pharmacokinetic profile of one inhibitor was evaluated in dogs, and the X-ray crystal structure of this compound bound to factor Xa provides insight into the observed SAR for binding to factor Xa.     

Sialidase inhibitors related to zanamivir. Further SAR studies of 4-amino-4H-pyran-2-carboxylic acid-6-propylamides

SAR investigations of the 4- and 5-positions of a series of 4-amino-4H-pyran-2-carboxylic acid 6-carboxamides are reported. Potent inhibitors of influenza A sialidase with marked selectivity over the influenza B enzyme were obtained when the basic 4-amino substituent was replaced by hydroxyl or even deleted. Modifications at the 5-position exhibited a tight steric requirement, with trifluoroacetamide being optimal.     

Optimization of the indolyl quinolinone class of KDR (VEGFR-2) kinase inhibitors: effects of 5-amido- and 5-sulphonamido-indolyl groups on pharmacokinetics and hERG binding

Modifications to the basic side-chain of early lead structures of the indolyl quinolinone class of KDR kinase inhibitors resulted in improved pharmacokinetic and ancillary profiles. Specifically, compounds bearing 5-amido- and 5-sulphonamido-indolyl substituents exhibited lower plasma clearance and weaker binding affinity for the I(Kr) potassium channel hERG.     

Use of the hydantoin isostere to produce inhibitors showing selectivity toward the vesicular glutamate transporter versus the obligate exchange transporter system x(c)(-)

Evidence was acquired prior to suggest that the vesicular glutamate transporter (VGLUT) but not other glutamate transporters were inhibited by structures containing a weakly basic α-amino group. To test this hypothesis, a series of analogs using a hydantoin (pK(a)～9.1) isostere were synthesized and analyzed as inhibitors of VGLUT and the obligate cystine-glutamate transporter (system x(c)(-)). Of the hydantoin analogs tested, a thiophene-5-carboxaldehyde analog 2l and a bis-hydantoin 4b were relatively strong inhibitors of VGLUT reducing uptake to less than 6% of control at 5mM but few inhibited system x(c)(-) greater than 50% of control. The benzene-2,4-disulfonic acid analog 2b and p-diaminobenzene analog 2e were also good hydantoin-based inhibitors of VGLUT reducing uptake by 11% and 23% of control, respectively, but neither analog was effective as a system x(c)(-) inhibitor. In sum, a hydantoin isostere adds the requisite chemical properties needed to produce selective inhibitors of VGLUT.     

Influence of pH on butylylcholinesterase reaction with organophosphorus inhibitors.

The non-covalent enzyme . inhibitor complex dissociation constants and the enzyme phosphorylation rate constants were measured as functions of pH in butyrylcholinesterase (actylcholine acylhydrolase, EC 3.1.1.8) reaction with organophosphorus inhibitors (C2H5O)2P(O)SX, where X = (CH2)3SC2H5 and (CH2)6S+(CH3)C2H5. Two ionizing groups, a basic and an acidic one, were revealed in the overall reaction of the enzyme inhibition within the pH range between 5 and 10.5. In the enzyme phosphorylation step only the acidic group was found, while the basic group appeared in the non-covalent binding step of both the ionic and non-ionic compounds. The results strongly imply the participation of the basic functional group in the conformation transition which affects the ability of butyrylcholinesterase to bind hydrophobic reagents in the acidic pH region.     

Inhibitors of a Rat Brain Enkephalin Aminopeptidase

Abstract: Eight protease inhibitors of microbiological origin were examined as potential inhibitors of a homogeneous rat brain enkephalin aminopeptidase. Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]- l -leucine and analogs of bestatin having basic, acidic, and other neutral amino acids substituted for the Leu residue exhibited inhibition constants ranging from 3.3 ± 10⁻⁵ to 8.3 ± 10⁻⁸ m . The best inhibitor had a positively charged amino acid (Lys) substituted for Leu. A series of phenylalanyl dipeptides were examined as substrates with the aminopeptidase. The amino acid residue on the carboxyl side of the peptide bond undergoing cleavage was varied systematically in the dipeptides to include neutral, acidic, and basic residues. Again, a positively charged amino acid (Arg) adjacent to the bond undergoing scission was kinetically preferred. These results may be used to design highly specific inhibitors of the enkephalin aminopeptidase.     

Functionalized chalcones as selective inhibitors of P-glycoprotein and breast cancer resistance protein

A library of chalcones with basic functionalities were screened for inhibition of P-glycoprotein (Pgp, ABCB1) by the calcein-AM accumulation assay on MDCKII/MDR1 cells. Three members that had ring A substituted with 5-(1-ethylpiperidin-4-yl) and 2,4-dimethoxy groups were found to increase calcein-AM accumulation to a greater extent than verapamil, a Pgp inhibitor. These compounds were subsequently shown to enhance the uptake of doxorubicin by MCF-7 cells that over-expressed Pgp. However, when tested for inhibition of the breast cancer resistance protein (BCRP, ABCG2) by the mitoxantrone uptake assay, the same compounds fared poorly. In comparison, a non-basic chalcone (5-14, 3-(4-chlorophenyl)-1-(2,4-dimethoxyphenyl)prop-2-en-1-one) increased mitoxantrone uptake by BCRP over-expressing MCF-7 cells (MCF-7/MX) by more than 300% at 5 microM. Thus, introducing a basic group on the chalcone template enhanced Pgp inhibition at the expense of BCRP inhibition. The basic chalcones were also better Pgp inhibitors than their non-basic counterparts which may in turn be better BCRP inhibitors. Structure activity analysis showed that lipophilicity of the chalcones was not the overriding factor for Pgp inhibitory activity. Rather, good activity was associated with appropriately placed electron donor atoms, of which the meta-disubstituted dimethoxy motif on either ring A or B was of particular relevance. In spite of differing structural requirements for inhibition of Pgp and BCRP, chalcone 3-100 [3-(2,4-dimethoxyphenyl)-1-(4-(piperazin-1-yl)phenyl)prop-2-en-1-one] inhibited both Pgp and BCRP to a reasonable extent and may be a useful starting point for the design of dual inhibitors.     

The cellular target of histatin 5 on Candida albicans is the energized mitochondrion

Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability of Candida albicans cells was studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion.     

Non-peptidic alpha(v)beta3 antagonists containing indol-1-yl propionic acids

We describe the synthesis and structure/activity relationship of RGD mimetics that are potent inhibitors of the integrin alpha(v)beta3. Indol-1-yl propionic acids containing a variety of basic moieties at the 5-position, as well as substitutions alpha and beta to the carboxy terminus were synthesized and evaluated. Novel compounds with improved potency have been identified.     

Cysteine proteinase inhibitors in human and rat male sex glands

The testis and prostate of men had two types of cysteine proteinase inhibitors of different isoelectric point (pI = 4.5 and 6.3) and molecular masses (90,000 and 12,000). The seminal vesicle contained not only these two inhibitors but also a basic inhibitor with a pI = 9.5 and Mr 11,000. In rats, the inhibitors in the testis and prostate consisted of two types with pI values of 4.3 and 4.8 and Mr 90,000 for both. In addition to these two inhibitors, the seminal vesicle contained large amounts of two basic inhibitors with pI values of 7.5 and 8.3 and Mr of 11,000 for both. We suggest that the basic cysteine proteinase inhibitor found in semen might be excreted from or be modified in the seminal vesicle.     

Isolation and characterization of 5'-nucleotidase inhibitor from rat liver.

5'-Nucleotidase (EC 3.1.3.5) is widely distributed in nature. However, it could not be detected in rat liver, because of the presence of specific inhibitors. Such inhibitors were also found in other tissues of rat, but at lower concentrations than that in the liver. The inhibitor activity was enriched in the membrane fraction and was also present in the cytosol fraction. It was sensitive to treatment with 6M urea and trypsin, while heating in a boiling water bath for 10 min or dialysis reduced the activity only slightly. Gel filtration or Sephadex G-50 yielded two types of inhibitors. Inhibitor I inhibited brain 5'-nucleotidase while inhibitor II inhibited both the brain and liver enzymes. Inhibitor II on further purification on CM Sephadex C-25 yielded five fractions with inhibitor activity of which inhibitor IIC was electrophoretically homogeneous. It had a molecular weight of 8500 by SDS gel electrophoresis, was rich in basic amino acids and had a high proportion of beta structure. Interaction of the inhibitor with 5'-nucleotidase brought about modifications in the secondary structure of the inhibitor as seen from the circular dichroism spectrum.     

Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2'' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr₄-Ile₅ bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.     

Inhibition of rat and bovine trypsins and chymotrypsins by soybean, bovine basic pancreatic, and bovine colostrum trypsin inhibitors.

1. Bovine (Bos taurus) trypsin and trypsin activity in rat (Rattus norvegicus) pancreatic extract were inhibited by soybean trypsin inhibitor and by bovine basic pancreatic and colostrum inhibitors. 2. Bovine alpha-chymotrypsin was inhibited by soybean and bovine basic pancreatic inhibitors but only weakly by colostrum inhibitor. 3. Chymotrypsin activity in rat pancreatic extract was due to at least three different components against all of which the inhibitors were largely ineffective. 4. It is concluded that bovine colostrum inhibitor has a more limited inhibition spectrum than the phylogenetically related basic pancreatic inhibitor which, in turn, is less active against rat than against bovine enzymes.     

Contribution of pi-electrons to the inhibition of S-adenosyl-L-methionine decarboxylase by aromatic tricyclic compounds

Eleven structural analogs with norharmane (9H-pyrido [3, 4-b] indole) as the basic structure were used to study structure-activity relations of adenosyl-methionine decarboxylase inhibition by this type of compounds. All analogs with an intact or 3,4-dihydrogenated pyrido ring inhibited the enzyme competitively with apparent Kis at the micromolar range, but complete hydrogenation of the pyrido moiety abolished the inhibition. This indicates that a suitable arrangement of pi-electrons is an essential structural feature in the process. Strongest inhibitors (Ki approximately 10-5M) were compounds methoxy or hydroxy groups in positions 6 or 7 of the indole moiety, suggesting that polarizing groups containing atoms with free electron pairs also are important in the interaction. The results offer a new viewpoint when action mechanisms of the known adenosylmethionine decarboxylase inhibitors are evaluated and they may prove useful when new inhibitors to the enzyme are designed.     

Synthesis and cytotoxicity of novel indirubin-5-carboxamides

Indirubins have been reported to act as potent inhibitors of protein kinases relevant to tumorigenesis and of tumor cell growth, but their development to antitumor drugs suffer from their poor water solubility. We synthesized a novel class of indirubin derivatives, indirubin-5-carboxamides, carrying amide substituents with basic centers. Quaternization or protonation of these alkylamino substituents provided indirubins with significantly improved solubility without loss of bioactivity.     

Trypsin inhibitors in bovine lung and pancreas during development.

1. The activity of trypsin inhibitors in lung and pancreas increased with age of the animals. 2. In foetal lung the activity of the basic Kunitz-type inhibitor was 1% that, and in calf lung 5-30% that, found in adult animals. 3. In foetal pancreas the basic Kunitz-type inhibitor was not detected, and in calf pancreas its activity was much lower than in adult animals. The activity of the acidic Kazal-type inhibitor in foetal pancreas was significantly lower than in calf or adult animal.     

Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10 600-6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion-exchange chromatography; they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7-4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross-reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.