Design and synthesis of fluorogenic substrate-based probes for detecting Cathepsin B activity.

Cathepsin B plays key roles in tumor progression with its overexpression being associated with invasive and metastatic phenotypes and is a primary target of protease activated antibody-directed prodrug therapy. It therefore represents a potential therapeutic and diagnostic target and effort has been made to develop fluorescent probes to report on Cathepsin B activity in cells and animal models of cancer. We have designed, synthesized, and thoroughly evaluated four novel "turn on" probes that employ a lysosomotropic dansylcadaverine dye to report on Cathepsin B activity. Enzyme activity assays using a recombinant human enzyme and cancer cell lysates coupled with confocal microscopy experiments demonstrated that one of the probes, derivatized with the self-immolative prodrug linker p-aminobenzyl alcohol, can selectively report on Cathepsin B in biological samples including live cells.

Suppression of network activity in dorsal horn by gabapentin permeation of TRPV1 channels: implications for drug access to cytoplasmic targets.

The effectiveness of gabapentin (GBP) in the treatment of neuropathic pain depends on access to the α2δ-1 accessory subunit of voltage-gated Ca(2+) channels. Access may be limited by its rate of entry via the neuronal system L-neutral amino acid transporter. The open pore of capsaicin-activated TRPV1 channel admits organic molecules such as local anesthetics and we calculated that GBP entry via this route would be 500× more rapid than via the transporter. Capsaicin should therefore increase GBP effectiveness. We used a quaternary GBP derivative (Q-GBP) as sole charge carrier in whole-cell recording experiments on rat dorsal root ganglion (DRG) neurons. Under these conditions, capsaicin produced a capsazepine-sensitive inward current thereby confirming Q-GBP permeation of TRPV1 channels. We have previously established that 5-6 days exposure to 100 µM GBP decreases excitability of dorsal horn neurons whereas 10 µM is ineffective. Excitability was monitored using confocal Ca(2+) imaging of rat spinal cord slices in organotypic culture. GBP effectiveness was augmented by transient exposures of cultures to capsaicin and robust suppression of excitability was seen with 10 µM GBP. Experiments with an inhibitor of the neutral amino acid transporter, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH 300 µM), showed the actions of GBP seen in the presence of capsaicin were independent of its entry by this route. Capsaicin potentiation of GBP depression of dorsal horn activity may therefore reflect drug permeation of TRPV1 channels. Agonist activation of TRP channels may provide a means for improving drug access to cytoplasmic targets in selective neuronal populations defined on the basis of type of TRP channel expressed.

Creating and virtually screening databases of fluorescently-labelled compounds for the discovery of target-specific molecular probes.

Our group has recently demonstrated that virtual screening is a useful technique for the identification of target-specific molecular probes. In this paper, we discuss some of our proof-of-concept results involving two biologically relevant target proteins, and report the development of a computational script to generate large databases of fluorescence-labelled compounds for computer-assisted molecular design. The virtual screening of a small library of 1,153 fluorescently-labelled compounds against two targets, and the experimental testing of selected hits reveal that this approach is efficient at identifying molecular probes, and that the screening of a labelled library is preferred over the screening of base compounds followed by conjugation of confirmed hits. The automated script for library generation explores the known reactivity of commercially available dyes, such as NHS-esters, to create large virtual databases of fluorescence-tagged small molecules that can be easily synthesized in a laboratory. A database of 14,862 compounds, each tagged with the ATTO680 fluorophore was generated with the automated script reported here. This library is available for downloading and it is suitable for virtual ligand screening aiming at the identification of target-specific fluorescent molecular probes.

Prodrug-inspired probes selective to cathepsin B over other cysteine cathepsins.

Cathepsin B (CTB) is a cysteine protease believed to be an important therapeutic target or biomarker for several diseases including aggressive cancer, arthritis, and parasitic infections. The development of probes capable of assessing CTB activity in cell lysates, living cells, and animal models of disease are needed to understand its role in disease progression. However, discovering probes selective to cathepsin B over other cysteine cathepsins is a significant challenge due to overlap of preferred substrates and binding site homology in this family of proteases. Herein we report the synthesis and detailed evaluation of two prodrug-inspired fluorogenic peptides designed to be efficient and selective substrate-based probes for CTB. Through cell lysate and cell assays, a promising lead candidate was identified that is efficiently processed and has high specificity for CTB over other cysteine cathepsins. This work represents a key step toward the design of rapid release prodrugs or substrate-based molecular imaging probes specific to CTB.

A diazen-1-ium-1,2-diolated nitric oxide donor ester prodrug of 3-(4-hydroxymethylphenyl)-4-(4-methanesulfonylphenyl)-5H-furan-2-one: synthesis, biological evaluation and nitric oxide release studies.

A novel hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrug (NONO-coxib 14) wherein an O(2)-acetoxymethyl 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate (O(2)-acetoxymethyl PROLI/NO) NO-donor moiety was covalently coupled to the CH(2)OH group of 3-(4-hydroxymethylphenyl)-4-(4-methylsulfonylphenyl)-5H-furan-2-one (12), was synthesized. The prodrug 14 released a low amount of NO (4.2%) upon incubation with phosphate buffer (PBS) at pH 7.4 which was significantly higher (34.8% of the theoretical maximal release of two molecules of NO/molecule of the parent hybrid ester prodrug) upon incubation in the presence of rat serum. These incubation studies suggest that both NO and the parent compound 12 would be released from the prodrug 14 upon in vivo cleavage by non-specific serum esterases. The prodrug ester 14 is a selective COX-2 inhibitor that exhibited AI activity (ED(50)=72.2mmol/kg po) between that of the reference drugs celecoxib (ED(50)=30.9µmol/kg po) and ibuprofen (ED(50)=327µmol/kg po). The NO donor compound 14 exhibited enhanced inhibition of phenylephrine-induced vasoconstriction of isolated mesenteric arteries compared with that observed under control conditions. These studies indicate hybrid ester AI/NO donor prodrugs (NONO-coxibs) constitutes a plausible drug design concept targeted toward the development of selective COX-2 inhibitory AI drugs that are devoid of adverse cardiovascular effects.

Ethanesulfohydroxamic acid ester prodrugs of nonsteroidal anti-inflammatory drugs (NSAIDs): synthesis, nitric oxide and nitroxyl release, cyclooxygenase inhibition, anti-inflammatory, and ulcerogenicity index studies.

The carboxylic acid group of the anti-inflammatory (AI) drugs indo-methacin, (S)-naproxen and ibuprofen was covalently linked via a two-carbon ethyl spacer to a sulfohydroxamic acid moiety (CH(2)CH(2)SO(2)NHOH) to furnish a group of hybrid ester prodrugs that release nitric oxide (NO) and nitroxyl (HNO). Biological data acquired for this hitherto unknown class of ethanesulfohydroxamic acid ester prodrugs showed (i) all compounds exhibited superior NO, but similar HNO, release properties relative to arylsulfohydroxamic acids, (ii) the (S)-naproxen and ibuprofen prodrug esters are more potent AI agents than their parent NSAID, (iii) the indomethacin prodrug ester, in contrast to indomethacin which is highly ulcerogenic, showed no visible stomach lesions [ulcer index (UI) = 0 for a 80 µmol/kg oral dose] while retaining potent AI activity, and iv) that the indomethacin prodrug ester, unlike indomethacin which is an ulcerogenic selective COX-1 inhibitor, is a selective COX-2 inhibitor (COX-2 selectivity index = 184) devoid of ulcerogenicity that is attributed to its high COX-2 SI and/or ability to release cytoprotective NO.

Triaryl (Z)-olefins suitable for radiolabeling with iodine-124 or fluorine-18 radionuclides for positron emission tomography imaging of estrogen positive breast tumors.

A group of (Z)-1,2-diphenyl-1-[4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl]but-1-enes were synthesized using methodologies that will allow incorporation of a [(124)I]iodine substituent at the para-position of either the C-1 phenyl ring or the C-2 phenyl ring, or a [(18)F]OCH(2)CH(2)F substituent at the para-position of the C-2 phenyl ring. These [(124)I] and [(18)F] radiotracers are designed as potential radiopharmaceuticals to image estrogen positive breast tumors using positron emission tomography (PET).

Synthesis and biological evaluation of indomethacin analogs possessing a N-difluoromethyl-1,2-dihydropyrid-2-one ring system: a search for novel cyclooxygenase and lipoxygenase inhibitors.

A novel class of indomethacin analogs were synthesized wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety (5-LOX pharmacophore) was attached at its C-4 or C-5 position via either a CO (14a-b) or CH(2) (19a-b) linker to the indole N(1)-position. In this regard, replacement of the 4-chlorobenzoyl group present in indomethacin by N-difluoromethyl-1,2-dihydropyrid-2-one-4-(or 5-)carbonyl and N-difluoromethyl-1,2-dihydropyrid-2-one-4-yl(or 5-yl)methylene moieties furnished compounds showing no inhibitory activities against the COX-2/5-LOX enzymes (except for the weak but selective COX-2 inhibitor 19a, COX-2 IC(50)=31 µM), and moderate in vivo anti-inflammatory activities (except for the methylene compound 19a that was inactive). These structure-activity data indicate replacement of the 4-chlorobenzoyl group present in indomethacin by a N-difluoromethyl-1,2-dihydropyrid-2-one ring system connected by a CO or CH(2) linker is not a suitable approach for the design of dual COX-2/5-LOX inhibitory analogs of indomethacin.

Triaryl (Z)-olefins suitable for radiolabeling with carbon-11 or fluorine-18 radionuclides for positron emission tomography imaging of cyclooxygenase-2 expression in pathological disease.

A group of (Z)-1,1-diphenyl-2-(4-methylsulfonylphenyl)alk-1-enes were synthesized using methodologies that will allow incorporation of a [(11)C]OCH(3) substituent at the para-position of the C-1 phenyl ring, a [(11)C]SO(2)CH(3) substituent at the para-position of the C-2 phenyl ring, a [(18)F]OCH(2)CH(2)F substituent at the para-position of the C-1 phenyl ring, and a [(18)F]CH(2)CH(2)F substituent at the C-2 position of the olefinic bond. The [(11)C] and [(18)F] radiotracers are designed as potential radiopharmaceuticals to image cyclooxygenase-2 (COX-2) expression in any organ where COX-2 is upregulated. The COX-1/COX-2 inhibition data acquired suggest that compounds having a [(11)C]OMe or [(18)F]OCH(2)CH(2)F substituent at the para-position of the C-1 phenyl ring may be more suitable for imaging COX-2 expression in view of their ability to exclusively inhibit the COX-2 isozyme.

Celecoxib prodrugs possessing a diazen-1-ium-1,2-diolate nitric oxide donor moiety: synthesis, biological evaluation and nitric oxide release studies.

A new class of anti-inflammatory (AI) cupferron prodrugs was synthesized wherein a diazen-1-ium-1,2-diolato ammonium salt, and its O(2)-methyl and O(2)-acetoxyethyl derivatives, nitric oxide (NO) donor moieties were attached directly to an aryl carbon on a celecoxib template. The percentage of NO released from the O(2)-methyl and O(2)-acetoxyethyl compounds was higher (18.0-37.8% of the theoretical maximal release of one molecule of NO/molecule of the parent compound) upon incubation in the presence of rat serum, relative to incubation with phosphate buffer saline (PBS) at pH 7.4 (3.8-11.6% range). All compounds exhibited weak inhibition of the COX-1 isozyme (IC(50)=5.8-17.0 microM range) in conjunction with weak or modest inhibition of the COX-2 isozyme (IC(50)=1.6-14.4 microM range). The most potent AI agent 5-[4-(O(2)-ammonium diazen-1-ium-1,2-diolato)phenyl]-1-(4-sulfamoylphenyl)-3-trifluoromethyl-1H-pyrazole exhibited a potency that was about fourfold and twofold greater than that observed for the respective reference drugs aspirin and ibuprofen. These studies indicate that use of a cupferron template constitutes a plausible drug design approach targeted toward the development of AI drugs that do not cause gastric irritation, or elevate blood pressure and induce platelet aggregation that have been associated with the use of some selective COX-2 inhibitors.

Synthesis and biological evaluation of N-difluoromethyl-1,2-dihydropyrid-2-one acetic acid regioisomers: dual inhibitors of cyclooxygenases and 5-lipoxygenase.

A new group of acetic acid (7a-c, R(1) = H), and propionic acid (7d-f, R(1) = Me), regioisomers wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety is attached via its C-3, C-4, and C-5 position was synthesized. This group of compounds exhibited a more potent inhibition, and hence selectivity, for the cyclooxygenase-2 (COX-2) relative to the COX-1 isozyme. Attachment of the N-difluoromethyl-1,2-dihydropyrid-2-one ring system to an acetic acid, or propionic acid, moiety confers potent 5-LOX inhibitory activity, that is, absent in traditional arylacetic acid NSAIDs. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid (7c) exhibited the best combination of dual COX-2 and 5-LOX inhibitory activities. Molecular modeling (docking) studies showed that the highly electronegative CHF(2) substituent present in 7c, that showed a modest selectivity for the COX-2 isozyme, is oriented within the secondary pocket (Val523) present in COX-2 similar to the sulfonamide (SO(2)NH(2)) COX-2 pharmacophore present in celecoxib, and that the N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore is oriented close to the region containing the LOX enzyme catalytic iron (His361, His366, and His545). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties suitable for the design of dual COX-2/5-LOX inhibitory drugs.

Phenylacetic acid regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore: evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity.

A novel class of phenylacetic acid regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore attached to its C-2, C-3 or C-4 position was designed for evaluation as anti-inflammatory (AI) agents. A number of compounds exhibited a combination of potent in vitro cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitory activities. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9a) exerted the most potent AI activity among this group of compounds. Molecular modeling studies showed that the N-difluoromethyl-1,2-dihydropyridin-2-one moiety present in 9a inserts into the secondary pocket present in COX-2 to confer COX-2 selectivity, and that the N-difluoromethyl-1,2-dihydropyrid-2-one group (9a) binds close to the region of the 15-LOX enzyme containing catalytic iron (His361, His366). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties that make it an attractive pharmacophore suitable for the design of dual COX-2/5-LOX inhibitory AI drugs.

Celecoxib analogs possessing a N-(4-nitrooxybutyl)piperidin-4-yl or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl nitric oxide donor moiety: synthesis, biological evaluation and nitric oxide release studies.

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs) wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl 15a-b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a-b, NO-donor moiety was synthesized. All compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH 7.4 (2.4-5.8% range). In comparison, the percentage NO released was higher (3.1-8.4% range) when these nitrate prodrugs were incubated in the presence of L-cysteine. In vitro COX-1/COX-2 isozyme inhibition studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of the COX-2 relative to the COX-1 enzyme. AI structure-activity relationship data acquired showed that compounds having a MeSO2 COX-2 pharmacophore exhibited superior AI activity compared to analogs having a H2NSO2 substituent. Compounds having a MeSO2 COX-2 pharmacophore in conjunction with a N-(4-nitrooxybutyl)piperidyl (ED50=132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl (ED50=118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin (ED50=128.7 mg/kg po) but lower than ibuprofen (ED50=67.4 mg/kg po).

Synthesis and biological evaluation of salicylic acid and N-acetyl-2-carboxybenzenesulfonamide regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore: dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity.

A novel class of salicylic acid and N-acetyl-2-carboxybenzenesulfonamide regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore attached to its C-4 or C-5 position was designed for evaluation as anti-inflammatory (AI) agents. Replacement of the 2,4-difluorophenyl ring in diflunisal by the N-difluoromethyl-1,2-dihydropyrid-2-one moiety provided compounds showing dual selective cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LOX) inhibitory activities. AI structure-activity studies showed that the C-4 (14a) and C-5 (14b) salicylate regioisomers were 1.4- and 1.6-fold more potent than aspirin, and the C-5 N-acetyl-2-carboxybenzenesulfonamide regioisomer (22b) was 1.3- and 2.8-fold more potent than ibuprofen and aspirin, respectively. In vivo ulcer index (UI) studies showed that the 4- and 5-(N-difluoromethyl-1,2-dihydropyrid-2-one-4-yl)salicylic acids (14a and 14b) were completely non-ulcerogenic since no gastric lesions were present (UI=0) relative to aspirin (UI=57) at an equivalent mumol/kg oral dose. The N-difluoromethyl-1,2-dihydropyridin-2-one moiety provides a novel 5-LOX pharmacophore for the design of cyclic hydroxamic mimetics for exploitation in the development of dual COX-2/5-LOX inhibitory AI drugs.

Synthesis of celecoxib analogues possessing a N-difluoromethyl-1,2-dihydropyrid-2-one 5-lipoxygenase pharmacophore: biological evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity.

A novel class of 1-(4-methanesulfonylphenyl and 4-aminosulfonylphenyl)-5-[4-(1-difluoromethyl-1,2-dihydropyrid-2-one)]-3-trifluoromethyl-1H-pyrazole hybrid cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LOX) inhibitory anti-inflammatory agents was designed. Replacement of the tolyl ring present in celecoxib by the N-difluoromethyl-1,2-dihydropyrid-2-one moiety provided compounds showing dual selective COX-2/5-LOX inhibitory activities. 1-(4-Aminosulfonylphenyl)-5-[4-(1-difluoromethyl-1,2-dihydropyrid-2-one)]-3-trifluoromethyl-1H-pyrazole exhibited good anti-inflammatory (AI) activity (ED(50) = 27.7 mg/kg po) that compares favorably with the reference drugs celecoxib (ED(50) = 10.8 mg/kg po) and ibuprofen (ED(50) = 67.4 mg/kg po). The N-difluoromethyl-1,2-dihydropyridin-2-one moiety provides a novel 5-LOX pharmacophore for the design of cyclic hydroxamic mimetics for exploitation in the development of COX-2/5-LOX inhibitory AI drugs.