New Histamine-Related Five-Membered N-Heterocycle Derivatives as Carbonic Anhydrase I Activators.

A series of histamine (HST)-related compounds were synthesized and tested for their activating properties on five physiologically relevant human Carbonic Anhydrase (hCA) isoforms (I, II, Va, VII and XIII). The imidazole ring of HST was replaced with different 5-membered heterocycles and the length of the aliphatic chain was varied. For the most interesting compounds some modifications on the terminal amino group were also performed. The most sensitive isoform to activation was hCA I (KA values in the low micromolar range), but surprisingly none of the new compounds displayed activity on hCA II. Some derivatives (1, 3a and 22) displayed an interesting selectivity for activating hCA I over hCA II, Va, VII and XIII.

2-(2-Hydroxyethyl)piperazine derivatives as potent human carbonic anhydrase inhibitors: Synthesis, enzyme inhibition, computational studies and antiglaucoma activity.

Targeting Carbonic Anhydrases (CAs) represents a strategy to treat several diseases, from glaucoma to cancer. To widen the structure-activity relationships (SARs) of our series of piperazines endowed with potent human carbonic anhydrase (hCA) inhibition, a new series of chiral piperazines carrying a (2-hydroxyethyl) group was prepared. The Zn-binding function, the 4-sulfamoylbenzoyl moiety, was connected to one piperazine N-atom, while the other nitrogen was decorated with alkyl substituents. In analogy to the approach used for the synthesis of the previously reported series, the preparation of the new compounds started with (R)- and (S)-aspartic acid. A partial racemization occurred during the synthesis. In order to overcome this problem, other chemical strategies were investigated. The inhibitory activity of the new polar derivatives against four hCAs isoforms I, II, IV and IX using a stopped flow CO2 hydrase assay was determined. Some compounds showed potency in the nanomolar range and a preference for inhibiting hCA IX.

Carbonic Anhydrase IV Selective Inhibitors Counteract the Development of Colitis-Associated Visceral Pain in Rats.

Persistent pain affecting patients with inflammatory bowel diseases (IBDs) is still very difficult to treat. Carbonic anhydrase (CA) represents an intriguing pharmacological target considering the anti-hyperalgesic efficacy displayed by CA inhibitors in both inflammatory and neuropathic pain models. The aim of this work was to evaluate the effect of inhibiting CA IV, particularly when expressed in the gut, on visceral pain associated with colitis induced by 2,4-di-nitrobenzene sulfonic acid (DNBS) in rats. Visceral sensitivity was assessed by measuring animals' abdominal responses to colorectal distension. Repeated treatment with the selective CA IV inhibitors AB-118 and NIK-67 effectively counteracted the development of visceral pain induced by DNBS. In addition to pain relief, AB-118 showed a protective effect against colon damage. By contrast, the anti-hyperalgesic activity of NIK-67 was independent of colon healing, suggesting a direct protective effect of NIK-67 on visceral sensitivity. The enzymatic activity and the expression of CA IV resulted significantly increased after DNBS injection. NIK-67 normalised CA IV activity in DNBS animals, while AB-118 was partially effective. None of these compounds influenced CA IV expression through the colon. Although further investigations are needed to study the underlying mechanisms, CA IV inhibitors are promising candidates in the search for therapies to relieve visceral pain in IBDs.

Synthesis and carbonic anhydrase activating properties of a series of 2-amino-imidazolines structurally related to clonidine1.

The Carbonic Anhydrase (CA, EC 4.2.1.1) activating properties of histamine have been known for a long time. This compound has been extensively modified but only in few instances the imidazole ring has been replaced with other heterocycles. It was envisaged that the imidazoline ring could be a bioisoster of the imidazole moiety. Indeed, we report that clonidine, a 2-aminoimidazoline derivative, was found able to activate several human CA isoforms (hCA I, IV, VA, VII, IX, XII and XIII), with potency in the micromolar range, while it was inactive on hCA II. A series of 2-aminoimidazoline, structurally related to clonidine, was then synthesised and tested on selected hCA isoforms. The compounds were inactive on hCA II while displayed activating properties on hCA I, VA, VII and XIII, with KA values in the micromolar range. Two compounds (10 and 11) showed some preference for the hCA VA or VII isoforms.

6,7-Dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline amides and corresponding ester isosteres as multidrug resistance reversers.

Aiming to deepen the structure-activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar, a new series of amide and ester derivatives carrying a 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline scaffold linked to different methoxy-substituted aryl moieties were synthesised. The obtained compounds were evaluated for their P-gp interaction profile and selectivity towards the two other ABC transporters, multidrug-resistance-associated protein-1 and breast cancer resistance protein, showing to be very active and selective versus P-gp. Two amide derivatives, displaying the best P-gp activity, were tested in co-administration with the antineoplastic drug doxorubicin in different cancer cell lines, showing a significant sensitising activity towards doxorubicin. The investigation on the chemical stability of the derivatives towards spontaneous or enzymatic hydrolysis, showed that amides are stable in both models while some ester compounds were hydrolysed in human plasma. This study allowed us to identify two chemosensitizers that behave as non-transported substrates and are characterised by different selectivity profiles.

Sulfonamides incorporating piperazine bioisosteres as potent human carbonic anhydrase I, II, IV and IX inhibitors.

Starting from the molecular simplification of (R) 4-(3,4-dibenzylpiperazine-1-carbonyl)benzenesulfonamide 9a, a compound endowed with selectivity for human Carbonic Anhydrase (hCA) IV, a series of piperazines and 4-aminopiperidines carrying a 4-sulfamoylbenzamide moiety as Zn-binding group have been designed and tested on human isoforms hCA I, II, IV and IX, using a stopped flow CO2 hydrase assay. The aim of the work was to derive structure-activity relationships useful for designing isoform selective compounds. These structural modifications changed the selectivity profile of the analogues from hCA IV to hCA I and II, and improved potency. Several of the new compounds showed subnanomolar activity on hCA II. X-ray crystallography of ligand-hCAII complexes was used to compare the binding modes of the new piperazines and the previously synthesized 2-benzyl-piperazine analogues, explaining the inhibition profiles.

Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to the discovery of a series of highly potent P-glycoprotein-based multidrug resistance (MDR) modulators.

In this study, a new series of N,N-bis(alkanol)amine aryl ester heterodimers was synthesized and studied. The new compounds were designed based on the structures of our previous arylamine ester derivatives endowed with high P-gp-dependent multidrug resistance reversing activity on a multidrug-resistant leukemia cell line. All new compounds were active in the pirarubicin uptake assay on the doxorubicin-resistant erythroleukemia K562 cells (K562/DOX). Compounds bearing a linker made up of 10 methylenes showed unprecedented high reversal activities regardless of the combination of aromatic moieties. Docking results obtained by an in silico study supported the data obtained by the biological tests and a study devoted to establish the chemical stability in phosphate buffer solution (PBS) and human plasma showed that only a few compounds exhibited a significant degradation in the human plasma matrix. Ten selected non-hydrolysable derivatives were able to inhibit the P-gp-mediated rhodamine-123 efflux on K562/DOX cells, and the evaluation of their apparent permeability and ATP consumption on other cell lines suggested that the compounds can behave as unambiguous or not transported substrates. The activity of these the compounds on the transport proteins breast cancer resistance protein (BCRP) and multidrug resistance associated protein 1 (MRP1) was also analyzed. All tested derivatives displayed a moderate potency on the BCRP overexpressing cells; while only four molecules showed to be effective on MRP1 overexpressing cells, highlighting a clear structural requirement for selectivity. In conclusion, we have identified a new very powerful series of compounds which represent interesting leads for the development of new potent and efficacious P-gp-dependent MDR modulators.

New Rigid Nicotine Analogues, Carrying a Norbornane Moiety, Are Potent Agonists of α7 and α3* Nicotinic Receptors.

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [3H]-cytisine and [3H]-methyllycaconitine to measure their affinity for α4ß2* and α7* nicotinic receptors. The new derivatives showed some preference for the α4ß2* over the α7* subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest Ki values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4ß2, α7, and α3ß2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4ß2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3ß2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC50 = 0.43 µM). The primary amines described here represent new chemotypes for the α7 and α3* receptor subtypes.

Amino Acids as Building Blocks for Carbonic Anhydrase Inhibitors.

Carbonic anhydrases (CAs) are a superfamily of metalloenzymes widespread in all life, classified into seven genetically different families (α⁻θ). These enzymes catalyse the reversible hydration of carbonic anhydride (CO2), generating bicarbonate (HCO3-) and protons (H⁺). Fifteen isoforms of human CA (hCA I⁻XV) have been isolated, their presence being fundamental for the regulation of many physiological processes. In addition, overexpression of some isoforms has been associated with the outbreak or progression of several diseases. For this reason, for a long time CA inhibitors (CAIs) have been used in the control of glaucoma and as diuretics. Furthermore, the search for new potential CAIs for other pharmacological applications is a very active field. Amino acids constitute the smallest fundamental monomers of protein and, due to their useful bivalent chemical properties, are widely used in organic chemistry. Both proteinogenic and non-proteinogenic amino acids have been extensively used to synthesize CAIs. This article provides an overview of the different strategies that have been used to design new CAIs containing amino acids, and how these bivalent molecules influence the properties of the inhibitors.

2-Benzylpiperazine: A new scaffold for potent human carbonic anhydrase inhibitors. Synthesis, enzyme inhibition, enantioselectivity, computational and crystallographic studies and in vivo activity for a new class of intraocular pressure lowering agents.

Two series of 2-benzylpiperazines have been prepared and tested for the inhibition of physiologically relevant isoforms of human carbonic anhydrases (hCA, EC 4.2.1.1). The new compounds carry on one nitrogen atom of the piperazine ring a sulfamoylbenzamide group as zinc-binding moiety, and different alkyl/acyl/sulfonyl groups on the other nitrogen. Regio- and stero-isomers are described. The majority of these compounds showed Ki values in the low-medium nanomolar range against hCA I, II and IV, but not IX. In many instances interaction with the enzyme was enantioselective. The binding mode has been studied by means of X-ray crystallography and molecular modelling. Two compounds, evaluated in rabbit models of glaucoma, were able to significantly reduce intraocular pressure, making them interesting candidates for further studies.

Designing selective modulators for the nicotinic receptor subtypes: challenges and opportunities.

Nicotinic receptors are membrane proteins involved in several physiological processes. They are considered suitable drug targets for various CNS disorders or conditions, as shown by the large number of compounds which have entered clinical trials. In recent years, nonconventional agonists have been discovered: positive allosteric modulators, allosteric agonists, site-specific agonists and silent desensitizers are compounds able to modulate the receptor interacting at sites different from the orthodox one, or to desensitize the receptor without prior opening. While these new findings can further complicate the pharmacology of these proteins and the design and optimization of ligands, they undoubtedly offer new opportunities to find drugs for the many therapeutic indications involving nicotinic receptors.

Investigation of piperazines as human carbonic anhydrase I, II, IV and VII activators.

Four human (h) carbonic anhydrase isoforms (CA, EC 4.2.1.1), hCA I, II, IV, and VII, were investigated for their activation profile with piperazines belonging to various classes, such as N-aryl-, N-alkyl-, N-acyl-piperazines as well as 2,4-disubstituted derivatives. As the activation mechanism involves participation of the activator in the proton shuttling between the zinc-coordinated water molecule and the external milieu, these derivatives possessing diverse basicity and different scaffolds were appropriate for being investigated as CA activators (CAAs). Most of these derivatives showed CA activating properties against hCA I, II, and VII (cytosolic isoforms) but were devoid of activity against the membrane-associated hCA IV. For hCA I, the KAs were in the range of 32.6-131 µM; for hCA II of 16.2-116 µM, and for hCA VII of 17.1-131 µM. The structure-activity relationship was intricate and not easy to rationalize, but the most effective activators were 1-(2-piperidinyl)-piperazine (KA of 16.2 µM for hCA II), 2-benzyl-piperazine (KA of 17.1 µM for hCA VII), and 1-(3-benzylpiperazin-1-yl)propan-1-one (KA of 32.6 µM for hCA I). As CAAs may have interesting pharmacologic applications in cognition and for artificial tissue engineering, investigation of new classes of activators may be crucial for this relatively new research field.

Design and synthesis of aminoester heterodimers containing flavone or chromone moieties as modulators of P-glycoprotein-based multidrug resistance (MDR).

In this study, a new series of heterodimers was synthesized. These derivatives are N,N-bis(alkanol)amine aryl esters or N,N-bis(ethoxyethanol)amine aryl esters carrying a methoxylated aryl residue combined with a flavone or chromone moiety. The new compounds were studied to evaluate their P-gp modulating activity on a multidrug-resistant leukemia cell line. Some of the new compounds show a good MDR reversing activity; interestingly this new series of compounds does not comply with the structure-activity relationships (SAR) outlined by previously synthesized analogs carrying different aromatic moieties. In the case of the compounds described in this paper, activity is linked to different features, in particular the characteristics of the spacer, which seem to be critical for the interaction with the pump. This fact indicates that the presence of a flavone or chromone residue influences the SAR of these series of products, and that flexible molecules can find different productive binding modes with the P-gp recognition site. These results support the synthesis of new compounds that might be useful leads for the development of drugs to control P-gp-dependent MDR.