Marc A. Ilies

Carbonic anhydrase inhibitors - Part 49: Synthesis of substituted ureido and thioureido derivatives of aromatic/heterocyclic sulfonamides with increased affinities for isozyme I

Reaction of nine aromatic/heterocyclic sulfonamides containing a free amino group with aryl isocyanates/isothiocyanates or allyl isothiocyanate afforded the corresponding urea/thiourea derivatives, which were characterized by standard physico-chemical procedures and assayed as inhibitors of three isozymes of carbonic anhydrase (CA), i.e. hCA I, hCA II and bCA IV (h = human, b = bovine isozyme). Another series of compounds, 1,5-disubstituted-2-thiobiuret derivatives, were prepared by reaction of 3,4-dichlorophenyl isocyanate with thioureido-containing aromatic/heterocyclic sulfonamides. Good inhibition of all these three CA isozymes was observed with the new compounds, but an exciting finding was that the ureas/thioureas and especially the above-mentioned thiobiurets reported here have an increased affinity to the slow isozyme hCA I, generally less susceptible to inhibition by sulfonamides, as compared to the rapid isozymes hCA II and bCA IV. Some of the new compounds might constitute good lead molecules for developing more selective CA I inhibitors.

Carbonic anhydrase inhibitors — Part 29 1: Interaction of isozymes I, II and IV with benzolamide-like derivatives

Reaction of 5-amino-1,3,4-thiadiazole-2-sulfonamide and 5-imino-4-tnethyl-2-sulfonamido-δ2-1,3,4-thiadiazoline with sulfonyl halides/sulfonic acid anhydrides afforded benzolamide-like derivatives possessing very strong inhibitory effects towards three isozymes of carbonic anhydrase (CA), CA I, II and IV. Some of the compounds were designed in such a way to possess good leaving groups (such as nitro-; 2,4,6-triphenyl-pyridinium, etc.) for aromatic nucleophilic substitution reactions with fluoride, in order to introduce positron-emitting isotopes in their molecule, such as 18F. Reactions done initially with the stable isotope of fluorine were not very effective, as the yields in the desired fluro deriatives were low, and a complex reaction mixture was obtained. By using this type of approach, and optimizing the synthetic procedure, CA inhibitors for positron emission tomography (PET) applications might be obtained (in the case of utilizing a carrier, which is the non-radioactive derivative itself, since the affinities of such derivatives for the receptor are in the nanomolar range). Further improving of such synthetic procedures might lead to better yields and the respective compounds should be used as selective ligands (also in carrier-free systems) in assessing the role of membrane bound CA isozymes or for new diagnostic tools based on PET.

Carbonic anhydrase inhibitors : Water-soluble 4-sulfamoylphenylthioureas as topical intraocular pressure-lowering agents with long-lasting effects

A series of sulfonamides has been obtained by reaction of 4-isothiocyanatobenzenesulfonamide with amines, amino acids, and oligopeptides. The new thiourea derivatives showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA, EC 4.2.1.1). In vitro inhibitory power was good (in the low-nanomolar range) for the derivatives of beta-phenylserine and alpha-phenylglycine, for those incorporating hydroxy and mercapto amino acids (Ser, Thr, Cys, Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr, DOPA), and dicarboxylic amino acids as well as di/tri/tetrapeptides among others. Such CA inhibitors displayed very good water solubility (in the range of 2-3%) mainly as sodium (carboxylate) salts, with pH values of the obtained solutions being 6.5-7.0. Some of these preparations (such as the derivatives of Ser, beta-Ph-Ser, Leu, Asn, etc.) strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. It is interesting to note that not all the powerful CA inhibitors designed in the present study showed topical IOP-lowering effects (such as, for instance, the Cys and Lys derivatives, devoid of such properties) whereas the Pro, Arg, and oligopeptidyl thiourea derivatives showed reduced efficacy when administered topically. This may be due to the very hydrophilic nature of some of these compounds, whereas inhibitors with balanced hydro- and liposolubility also showed optimal in vivo effects. The interesting pharmacological properties of this new type of CA inhibitors, correlated with the neutral pH of their solutions used in ophthalmologic applications, make them attractive candidates for developing novel antiglaucoma drugs devoid of major ocular side effects.

Carbonic anhydrase inhibitors — Part 52. Metal complexes of heterocyclic sulfonamides: A new class of strong topical intraocular pressure-lowering agents in rabbits†

Abstract A new sulfonamide possessing strong carbonic anhydrase (CA) inhibitory properties has been prepared by reaction of 5-amino-1,3,4-thiadiazole-2-sulfonamide with adamantyl-1-carboxylic acid chloride. Metal complexes of the adamantyl derivative, containing di- and trivalent cations, were also obtained and characterized by standard procedures. Although the parent sulfonamide does not possess topical pressure-lowering effects in rabbits when applied as a 2% solution directly into the eye, some of its metal complexes, such as the Zn(II) and Cu(II) derivatives, lower intraocular pressure (IOP) in experimental animals better than dorzolamide, the topical sulfonamide with strong antiglaucoma action, recently introduced in clinical medicine. We propose here that the whole class of metal complexes of heterocyclic sulfonamides (which generally possess much stronger CA inhibitory properties as compared to the original sulfonamides from which they were prepared) might be used for lowering IOP with enhanced efficiencies as compared to those of the simple sulfonamides. Thus, metal complexes of sulfonamides possessing themselves topical antiglaucoma properties might lead to more effective pharmacological agents of this type.

Carbonic anhydrase inhibitors: synthesis of membrane-impermeant low molecular weight sulfonamides possessing in vivo selectivity for the membrane-bound versus cytosolic isozymes.

Aromatic/heterocyclic sulfonamides act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA; EC 4.2.1.1), but the presently available compounds do not generally discriminate between the 14 isozymes isolated in higher vertebrates. Thus, clinically used drugs from this class of pharmacological agents show many undesired side effects due to unselective inhibition of all CA isozymes present in a tissue/organ. Here we propose a new approach for the selective in vivo inhibition of membrane-bound versus cytosolic CA isozymes with a new class of positively charged, membrane-impermeant sulfonamides. This approach is based on the attachment of trisubstituted-pyridinium-methylcarboxy moieties (obtained from 2,4, 6-trisubstituted-pyrylium salts and glycine) to the molecules of classical aromatic/heterocyclic sulfonamides possessing free amino, imino, hydrazino, or hydroxyl groups in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes: i.e., hCA I, hCA II (cytosolic forms), and bCA IV (membrane-bound isozyme) (h = human isozyme; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, and ethoxzolamide), are unable to penetrate through biological membranes, as shown by ex vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors undoubtedly proved that: (i) when using the new type of positively charged sulfonamides, only the membrane-bound enzyme (CA IV) was inhibited, whereas the cytosolic isozymes (CA I and II) were not affected; (ii) in the experiments in which the classical compounds (acetazolamide, benzolamide, etc.) were used, unselective inhibition of all CA isozymes (I, II, and IV) has been evidenced.

Endothelial targeting of antibody-decorated polymeric filomicelles.

The endothelial lining of the lumen of blood vessels is a key therapeutic target for many human diseases. Polymeric filomicelles that self-assemble from polyethylene oxide (PEO)-based diblock copolymers are long and flexible rather than small or rigid, can be loaded with drugs, and--most importantly--they circulate for a prolonged period of time in the bloodstream due in part to flow alignment. Filomicelles seem promising for targeted drug delivery to endothelial cells because they can in principle adhere strongly, length-wise to specific cell surface determinants. In order to achieve such a goal of vascular drug delivery, two fundamental questions needed to be addressed: (i) whether these supramolecular filomicelles retain structural integrity and dynamic flexibility after attachment of targeting molecules such as antibodies, and (ii) whether the avidity of antibody-carrying filomicelles is sufficient to anchor the carrier to the endothelial surface despite the effects of flow that oppose adhesive interactions. Here we make targeted filomicelles that bear antibodies which recognize distinct endothelial surface molecules. We characterize these antibody targeted filomicelles and prove that (i) they retain structural integrity and dynamic flexibility and (ii) they adhere to endothelium with high specificity both in vitro and in vivo. These results provide the basis for a new drug delivery approach employing antibody-targeted filomicelles that circulate for a prolonged time yet bind to endothelial cells in vascular beds expressing select markers.

Formation and Colloidal Stability of DMPC Supported Lipid Bilayers on SiO2 Nanobeads

Supported lipid bilayers (SLBs) of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were formed on 20-100 nm silica (SiO(2)) nanobeads, and the formation was accompanied by an 8 nm increase in diameter of the SiO(2), consistent with single nanobeads surrounded by a DMPC bilayer. Complete SLBs were formed when the nominal surface areas of the DMPC matched that of the silica, SA(DMPC)/SA(SiO2) = 1, and required increasing ionic strength and time to form on smaller size nanobeads, as shown by a combination of nano-differential scanning calorimetry (nano-DSC), dynamic light scattering (DLS), and zeta potential (zeta) measurements. For 5 nm SiO(2), where the nanoparticle and DMPC dimensions were comparable, DMPC fused and formed SLBs on the nanobeads, but it did not form single bilayers around them. Instead, stable agglomerates of 150-1000 nm were formed over a wide surface ratio range (0.25 < or = SA(DMPC)/SA(SiO2) < 2) in 0.75 mM NaCl. At ionic strengths > 1 mM NaCl, charge shielding, as measured by zeta potential measurements (zeta --> 0), resulted in precipitation of the SLBs.

Carbonic anhydrase inhibitors - Part 53?. Synthesis of substituted-pyridinium derivatives of aromatic sulfonamides: The first non-polymeric membrane-impermeable inhibitors with selectivity for isozyme IV

Abstract Reaction of three aromatic sulfonamides containing a free amino group, i.e., sulfanilamide, homosulfanilamide and 4-(2-aminoethyl)-benzenesulfonamide with di-, tri- or tetra-substituted pyrylium salts afforded three series of cationic sulfonamides, containing a large variety of moieties substituting the pyridinium ring. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form). Efficient inhibition was observed against all three isozymes, but due to the cationic nature of these inhibitors, in vivo and ex vivo experiments showed that only CA IV is selectively inhibited to a high degree, without affecting the cytosolic isozymes, present in appreciable concentrations in the experimental model used. This is the first example of selective in vivo inhibition of only one physiologically relevant CA isozyme with non-polymeric inhibitors and might lead to more selective drugs from this class of pharmacological agents.

Heterocyclic cationic gemini surfactants: a comparative overview of their synthesis, self-assembling, physicochemical, and biological properties.

Gemini surfactants (GS) are presently receiving substantial attention due to their special self‐assembling properties and unique interfacial activity. This comprehensive review is focused on positively charged heterocyclic GS, presenting their major synthetic access routes and examining the impact of structural elements on physicochemical and aggregation properties of this class of amphiphiles. Interaction of geminis surfactants with cells and their biological properties as novel transfection agents are emphasized through a detailed structure–activity relationship analysis. Throughout the review we have also presented the properties of selected ammonium GS, simple surfactants and lipid congeners, in order to emphasize the advantages conferred by using heterocyclic polar heads in GS design.

Cationic Lipids in Gene Delivery: Principles, Vector Design and Therapeutical Applications

Gene therapy will change medicine by treating the diseases at their core levels revolutionizing the way to deliver functional proteins. The development of this technology relies in designing optimal systems for DNA transfer and expression (transfection), cationic lipids being a promising alternative. Being safer than viral vectors, they also allow the delivery of larger plasmids and can be easily GMP-manufactured and stored. The main problem associated with the use of these vectors is their transfection efficiency, which is still inferior to viral methods. In this paper we present an overview of the correlations between the chemical structure and biological activity for the principal classes of cationic lipids. Key issues in the design of this class of transfection agents are presented, as well as the future trends.