Synthesis and inhibitory activity of acyl-peptidyl-prolinalderivatives toward post-proline cleaving enzyme as nootropic agents

Several prolinal derivatives were synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain and their possible properties as nootropic agents. Almost all the compounds tested inhibited the activity of both enzymes at low IC50 values of the order of nM, but a specificity difference was observed with alkylacyl-prolinal derivatives which strongly inhibited only the bacterial enzyme. Prolyl-prolinal derivatives were the most effective inhibitors for both enzymes. In the passive avoidance test using amnesic rats experimentally induced with scopolamine, the prolinal derivatives that have potent inhibitory activity toward post-proline cleaving enzymes showed also strong anti-amnesic activities at dose of 10-1000 micrograms/kg, i.p. Some of the compounds showed a bell-shape dose dependency. These results suggest that the post-proline cleaving enzymes play an important role in the regulation of learning and memory consolidation in the brain and inhibitors of these enzymes are suggested as possible candidates for nootropic agents, particularly for an anti-amnesic drug.     

Synthesis and inhibitory activity of acyl-peptidyl-pyrrolidine derivatives toward post-proline cleaving enzyme; a study of subsite specificity.

Several pyrrolidine derivatives have been synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain. Almost all the compounds tested in this study inhibited the activity of both enzymes at low IC50 values (from nM to microM) but a specificity difference was observed with alkylacyl-peptidyl-pyrrolidine derivatives which strongly inhibited only the bacterial enzyme. The most effective inhibitors have a proline residue on their P2 sites and a substituted or unsubstituted phenoxybutyryl moiety on their P3 sites. Thus phenoxybutyryl-prolyl-pyrrolidine is the most effective partial structure of the inhibitors. The best inhibitors found were: 4-(4-benzylphenoxy)butyryl-prolyl-pyrrolidine for bacterial enzyme (IC50 1.4 nM) and 4-phenylbutyryl-thioprolyl-pyrrolidine for bovine brain enzyme (IC50 67 nM). In the passive avoidance test, using amnesic rats experimentally induced with scopolamine, the pyrrolidine derivatives which had potent inhibitory activity toward post-proline cleaving enzymes also showed strong anti-amnesic activities at doses of 1-5 mg/kg, i.p.     

N-Benzyloxycarbonyl-valyl-prolinal, a potent inhibitor of post-proline cleaving enzyme

A peptide aldehyde inhibitor possessing prolinal at the carboxyl terminus was designed as an inhibitor of post-proline cleaving enzyme by analogy with peptide aldehyde inhibitors of serine and thiol proteases. N-Benzyloxycarbonyl-valyl-prolinal was found to be a potent inhibitor of post-proline cleaving enzyme from ascidian sperm with a K1 value of 2.4 nM. The presence of the aldehyde portion of the inhibitor, as well as its prolonged incubation with the enzyme, is indispensable for the potent inhibitory activity of the inhibitor. These results indicate that N-benzyloxycarbonyl-valyl-prolinal functions as a transition-state aldehyde inhibitor of post-proline cleaving enzyme.     

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Comparison of inhibitory effects of prolinal-containing peptide derivatives on prolyl endopeptidases from bovine brain and Flavobacterium

The inhibitory effects of proline-containing peptides and their derivatives on prolyl endopeptidases from Flavobacterium meningosepticum and bovine brain were compared. Replacement of the carboxyl terminal proline in N-blocked peptides with prolinal resulted in remarkable decreases in Ki values for both prolyl endopeptidases. Further reduction of the prolinal to prolinol led to a decrease in their inhibitory effects. Z-Pro-, Z-Val-, and Suc-Pro-prolinals were similarly inhibitory for both the enzymes with Ki values of nM order. However, the inhibitory effects of Z-Pyr-prolinal and Boc-Pro-prolinal on these enzymes were significantly distinguished: they strongly inhibited the mammalian prolyl endopeptidase with Ki values of nM order, while the Ki values of these compounds for the microbial enzyme were only of microM order. These results suggest that there are some structural differences in the S2 and S3 subsites between the two enzymes, though their substrate specificities are apparently indistinguishable.     

Synthesis,biological activity and molecular modelling studies of shikimic acid derivatives as inhibitors of the shikimate dehydrogenase enzyme of Escherichia coli

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC₅₀ 588 and 589?µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.     

Ultrasonic synthesis of tyramine derivatives as novel inhibitors of α-glucosidase in vitro

Tyramine derivatives 3–27 were synthesized by using conventional and environmental friendly ultrasonic techniques. These derivatives were then evaluated for the first time for their α-glucosidase (Sources: Saccharomyces cerevisiae and mammalian rat-intestinal acetone powder) inhibitory activity by using in vitro mechanism-based biochemical assays. Compounds 7, 14, 20, 21 and 26 were found to be more active (IC₅₀?=?49.7?±?0.4, 318.8?±?3.7, 23.5?±?0.9, 302.0?±?7.3 and 230.7?±?4.0?μM, respectively) than the standard drug, acarbose (IC₅₀?=?840.0?±?1.73?μM (observed) and 780?±?0.028?μM (reported)) against α-glucosidase obtained from Saccharomyces cerevisiae. Kinetic studies were carried out on the most active members of the series in order to determine their mode of inhibition and dissociation constants. Compounds 7, 20 and 26 were found to be the competitive inhibitors of α-glucosidase. These compounds were also screened for their protein antiglycation, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Only compounds 20, 22 and 27 showed weak antiglycation activity with IC₅₀ values 505.27?±?5.95, 581.87?±?5.50 and 440.58?±?2.74?μM, respectively. All the compounds were found to be inactive against DDP-IV enzyme. Inhibition of α-glucosidase, DPP-IV enzymes and glycation of proteins are valid targets for the discovery of antidiabetic drugs. Cytotoxicity of compounds 3–27 was also evaluated by using mouse fibroblast 3T3 cell lines. All the compounds were found to be noncytotoxic. The current study describes the synthesis α-glucosidase inhibitory activity of derivatives, based on a natural product tyramine template. The compounds reported here may serve as the starting point for the design and development of novel α-glucosidase inhibitors as antidiabetic agents.     

Synthesis of chiral pyrazolo[4,3-e][1,2,4]triazine sulfonamides with tyrosinase and urease inhibitory activity

A new series of sulfonamide derivatives of pyrazolo[4,3-e][1,2,4]triazine with chiral amino group has been synthesized and characterized. The compounds were tested for their tyrosinase and urease inhibitory activity. Evaluation of prepared derivatives demonstrated that compounds (8b) and (8j) are most potent mushroom tyrosinase inhibitors whereas all of the obtained compounds showed higher urease inhibitory activity than the standard thiourea. The compounds (8a), (8f) and (8i) exhibited excellent enzyme inhibitory activity with IC₅₀ 0.037, 0.044 and 0.042?μM, respectively, while IC₅₀ of thiourea is 20.9?μM.     

Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents

A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC₅₀ of 0.10?mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC₅₀ of 0.18?mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4?μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents.     

Synthesis and biological evaluation of quinazolinone-based hydrazones with potential use in Alzheimer’s disease

Discovering multifunctional agents for the treatment of Alzheimer’s disease (AD) is an attractive therapeutic approach. BACE1 (β-site amyloid precursor protein cleaving enzyme 1) inhibitors may play a pivotal role in treating AD. Therefore, the discovery of novel non-peptide BACE1 inhibitors with desirable blood brain barrier permeability is a favorable approach for treatment. Moreover, the antioxidant potential of a drug could serve as an added value for designing dual-acting therapeutic agents. Here, we report the design, synthesis and biological evaluation of quinazolinone-hydrazone derivatives as new multi-target candidates for the treatment of AD. The compounds were investigated for their in vitro BACE1 inhibitory potential using a FRET-based enzymatic assay and also screened for antioxidant activity using DPPH. Among them, compound 4h bearing a 2,3-dichlorophenyl moiety showed the highest activity with an IC₅₀ value of 3.7 μM against BACE1. In addition, compound 4i with a 2,4-dihydroxyphenyl scaffold demonstrated moderate BACE1 inhibitory activity (IC₅₀ = 27.6 μM) with a significant antioxidant effect (IC₅₀ = 8.4 μM). Furthermore, docking studies revealed strong interaction between compound 4h and the key residues of BACE1 active site. These results demonstrate that quinazolinone-hydrazone derivatives represent a valuable scaffold for the discovery of novel non-peptidic BACE1 inhibitors.     

Chroman amide and nicotinyl amide derivatives: Inhibition of lipid peroxidation and protection against head trauma

A series of chroman amide and nicotinyl amide derivatives was designed and synthesized for the treatment of traumatic and ischemic CNS injury. Five compounds were significantly more potent inhibitors of lipid peroxidation in vitro than the reference antioxidant, trolox (p < 0.01). Quantitative structure activity studies demonstrated that the inhibitory action was related to the ability to donate electrons, charge on hydroxy group and E_LUMO, to scavenging radicals and to the lipophilicity log P, which determines penetration of membrane lipids. ESR study indicated the ability of 12 to scavenge the hydroxyl radicals. The most promising compound, [(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2yl)carbonyl]-3′-(aminoethyl) indole (12), inhibited ex vivo lipid peroxidation in a head injury model and showed potent in vivo neuroprotective efficacy. Improvement of neurological recovery within 1 h of injury (grip test score) by as much as 200% was observed together with significant anti-anoxia activity. Compound 12 was a potent antagonist of methamphetamine-induced hypermotility resulting from dopamine release in the mouse brain. These results support the importance of cerebroprotective radical-scavenging agents for the treatment of traumatic injury and anoxia as well as provide additional evidence for the role of oxygen radicals and dopamine in brain damage.     

Immunological, Physical, and Chemical Evidence for the Identity of Brain and Kidney Post-Proline Cleaving Enzyme

Abstract: Recent studies from this laboratory have suggested a similarity, if not identity, of thyrotropin releasing factor (TRF) deamidase and post-proline cleaving enzyme. Bovine brain TRF deamidase was purified to homogeneity and used to elicit antibodies to the enzyme. These antibodies were used to demonstrate identical immunological reactivity between rat brain TRF deamidase and rat kidney post-proline cleaving enzyme. In addition, both proteins exhibit a molecular weight of 75,000, and have identical K_m values for the synthetic substrate pGlu-( N -benzyl- l -His)-Pro-β-naphthylamide and identical K ₁ values for TRF and luteinizing hormone releasing factor as inhibitors. Finally, the enzymes exhibit the same sensitivities to inhibition by mercury, iodoacetamide, N -ethylmaleimide, and 5, 5'-dithiobis-(2-nitrobenzoic acid). These results strongly suggest that brain TRF deamidase and kidney postproline cleaving enzyme are identical.     

Synthesis of 2-,4,-6-, and/or 7-substituted quinoline derivatives as human dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents: 3D QSAR-assisted design

Following our research for human dihydroorotate dehydrogenase (hDHODH) inhibitors as anticancer agents, herein we describe 3D QSAR-based design, synthesis and in vitro screening of 2-,4,-6-, and/or 7-substituted quinoline derivatives as hDHODH inhibitors and anticancer agents. We have designed 2-,4,-6-, and/or 7-substituted quinoline derivatives and predicted their hDHODH inhibitory activity based on 3D QSAR study on 45 substituted quinoline derivatives as hDHODH inhibitors, and also predicted toxicity. Designed compounds were docked into the binding site of hDHODH. Designed compounds which showed good predictive activity, no toxicity, and good docking score were selected for the synthesis, and in vitro screening as hDHODH inhibitors in an enzyme inhibition assay, and anticancer agents in MTT assay against cancer cell lines (HT-29 and MDA-MB-231). Synthesized compounds 7 and 14 demonstrated IC₅₀ value of 1.56?µM and 1.22?µM, against hDHODH, respectively, and these are our lead compounds for the development of new hDHODH inhibitors and anticancer agents.     

Synthesis and biological evaluation of novel hydrogen sulfide releasing glycyrrhetic acid derivatives

A series of hybrids, which are composed of glycyrrhetic acid (GA) and slowly hydrogen sulfide-releasing donor ADT-OH, were designed and synthesized to develop anticancer and anti-inflammatory agents. Most of the compounds, whose inhibitory rates were comparable to or higher than those of GA and aspirin, respectively, significantly inhibited xylene-induced ear edema in mice. Especially, compound V₄ exhibited the most potent inhibitory rate of 60.7%. Furthermore, preliminary structure–activity relationship studies demonstrated that 3-substituted GA derivatives had stronger anti-inflammatory activities than the corresponding 3-unsubstituted GA derivatives. In addition, anti-proliferative activities of compounds V_1?9 were evaluated in three different human cancer cell lines. Compound V₄ showed the most high potency against all three tumor cell lines with IC₅₀ values ranging from 10.01?μM in Hep G2 cells to 17.8?μM in MDA-MB-231 cells, which were superior to positive GA.     

Tacrine–propargylamine derivatives with improved acetylcholinesterase inhibitory activity and lower hepatotoxicity as a potential lead compound for the treatment of Alzheimer’s disease

Abstract

A series of tacrine–propargylamine derivatives were synthesised and evaluated as possible anti-Alzheimer’s disease (AD) agents. Among these derivatives, compounds 3a and 3b exhibited superior activities and a favourable balance of AChE and BuChE activities (3a: IC₅₀ values of 51.3 and 77.6?nM; 3b: IC₅₀ values of 11.2 and 83.5?nM). Compounds 3a and 3b also exhibited increased hAChE inhibitory activity compared with tacrine by approximately 5- and 28-fold, respectively, and low neurotoxicity. Importantly, these compounds also had lower hepatotoxicity than tacrine. Based on these results, compounds 3a and 3b could be considered as potential lead compounds for the treatment of AD and other AChE related diseases, such as schizophrenia, glaucoma and myasthenia gravis.     

Design,synthesis and biological evaluation of novel L-isoserine tripeptide derivatives as aminopeptidase N inhibitors

Aminopeptidase N (APN/CD13) is one of the essential proteins for tumour invasion, angiogenesis and metastasis as it is over-expressed on the surface of different tumour cells. Based on our previous work that L-isoserine dipeptide derivatives were potent APN inhibitors, we designed and synthesized L-isoserine tripeptide derivatives as APN inhibitors. Among these compounds, one compound 16l (IC₅₀?=?2.51?±?0.2 µM) showed similar inhibitory effect compared with control compound Bestatin (IC₅₀?=?6.25?±?0.4 µM) and it could be used as novel lead compound for the APN inhibitors development as anticancer agents in the future.     

1-Indol-1-yl-propan-2-ones and related heterocyclic compounds as dual inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgetic and anti-inflammatory drugs. We recently reported that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (10) and related compounds are inhibitors of cPLA₂α. Since cPLA₂α and FAAH possess several common structural features, we now screened this substance series together with some new derivatives for FAAH inhibition. Some of the assayed compounds proved to be selective cPLA₂α inhibitors, while others showed high FAAH and moderate cPLA₂α inhibitory potency. Furthermore, several derivatives were favorably active against both enzymes and, therefore, could represent agents, which have improved analgetic and anti-inflammatory qualities in comparison with selective cPLA₂α and FAAH inhibitors.     

Distribution of post-proline cleaving enzyme in human brain and the peripheral tissues

Summary We have studied the distribution of post-propline cleaving enzyme activity in the various tissues in humans using 7-(succinyl-Gly-Pro)-4-methylcoumarinamide as substrate. The post-propline cleaving enzyme activity was high in muscle, testes, kidney and submandibular gland, but was low in the heart, mesenterium and aorta. In the brain, relatively high post-propline cleaving enzyme activity was observed in the cerebral cortex, but other brain regions showed a very low enzyme activity.On Sephadex G-100 column chromatography, enzyme activity in human kidney showed a major peak and a minor peak. The major peak coincided with the enzyme in human cerebral cortex, but was different from human serum enzyme. Diisopropylfluorophosphate, a serine protease inhibitor, strongly inhibited the enzyme activity of each active fraction. The enzyme in the cerebral cortex and kidney was inhibited by heavy metals and thiol blocking agents. However, inhibition of enzyme activity in the serum was not observed with such inhibitors. Therefore, we suppose that post-proline cleaving enzyme activity in the brain is similar, if not identical, to that in the kidney.     

N-glycosyl-N-hydroxysulfamides as potent inhibitors of Brucella suis carbonic anhydrases

Abstract

We investigated a series of N-hydroxysulfamides obtained by Ferrier sulfamidoglycosylation for the inhibition of two bacterial carbonic anhydrases (CAs, EC 4.2.1.1) present in the pathogen Brucella suis. bsCA I was moderately inhibited by these compounds with inhibition constants ranging between 522 and 958?nM and no notable differences of activity between the acetylated or the corresponding deacetylated derivatives. The compounds incorporating two trans-acetates and the corresponding deprotected ones were the most effective inhibitors in the series. bsCA II was better inhibited, with inhibition constants ranging between 59.8 and 799?nM. The acetylated derivatives were generally better bsCA II inhibitors compared to the corresponding deacetylated compounds. Although these compounds were not highly isoform-selective CA inhibitors (CAIs) for the bacterial over the human CA isoforms, some of them possess inhibition profiles that make them interesting leads for obtaining better and more isoform-selective CAIs targeting bacterial enzymes.