Edward E. Knaus

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.

Electrophysiological characterization of a recombinant human Na+-coupled nucleoside transporter (hCNT1) produced in Xenopus oocytes

Human concentrative nucleoside transporter 1 (hCNT1) mediates active transport of nucleosides and anticancer and antiviral nucleoside drugs across cell membranes by coupling influx to the movement of Na+ down its electrochemical gradient. The two‐microelectrode voltage‐clamp technique was used to measure steady‐state and presteady‐state currents of recombinant hCNT1 produced in Xenopus oocytes. Transport was electrogenic, phloridzin sensitive and specific for pyrimidine nucleosides and adenosine. Nucleoside analogues that induced inwardly directed Na+ currents included the anticancer drugs 5‐fluorouridine, 5‐fluoro‐2′‐deoxyuridine, cladribine and cytarabine, the antiviral drugs zidovudine and zalcitabine, and the novel thymidine mimics 1‐(2‐deoxy‐β‐d‐ribofuranosyl)‐2,4‐difluoro‐5‐methylbenzene and 1‐(2‐deoxy‐β‐d‐ribofuranosyl)‐2,4‐difluoro‐5‐iodobenzene. Apparent Km values for 5‐fluorouridine, 5‐fluoro‐2′‐deoxyuridine and zidovudine were 18, 15 and 450 μm, respectively. hCNT1 was Na+ specific, and the kinetics of steady‐state uridine‐evoked Na+ currents were consistent with an ordered simultaneous transport model in which Na+ binds first followed by uridine. Membrane potential influenced both ion binding and carrier translocation. The Na+–nucleoside coupling stoichiometry, determined directly by comparing the uridine‐induced inward charge movement to [14C]uridine uptake was 1: 1. hCNT1 presteady‐state currents were used to determine the fraction of the membrane field sensed by Na+ (61%), the valency of the movable charge (−0.81) and the average number of transporters present in the oocyte plasma membrane (6.8 × 1010 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.

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).

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.

Second-Generation Aspirin and Indomethacin Prodrugs Possessing an O2-(Acetoxymethyl)-1-(2-carboxypyrrolidin-1-yl)diazenium-1,2-diolate Nitric Oxide Donor Moiety : Design, Synthesis, Biological Evaluation, and Nitric Oxide Release Studies

The carboxylic acid group of the anti-inflammatory (AI) drugs aspirin and indomethacin was covalently linked to the 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate ion via a one-carbon methylene spacer to obtain two new hybrid prodrugs. The aspirin prodrug ( 23) was a 2.2-fold more potent AI agent than aspirin, whereas the indomethacin prodrug ( 26) was about 1.6-fold less potent than indomethacin. Prodrugs 23 and 26 slowly released nitric oxide (NO) upon dissolution in phosphate buffer at pH 7.4 (1.1 mol of NO/mol of compound after 43 h), but the rate and the extent of NO release were higher (1.9 mol of NO/mol of compound in 3 min or less) when the compounds were incubated in the presence of porcine liver esterase. In vivo ulcer index (UI) studies showed that the aspirin prodrug 23 (UI = 0.7) and indomethacin prodrug 26 (UI = 0) were substantially less ulcerogenic than the parent drugs aspirin (UI = 51) and indomethacin (UI = 64).

In vitro antiviral activities of myristic acid analogs against human immunodeficiency and hepatitis B viruses.

A group of myristic acid analogs, designed as alternative substrates for N-myristoyltransferase (NMT), were evaluated against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in vitro. Antiviral potency was increased when S or O was substituted for -CH2- in myristic acid and selectivity was affected by the presence and position of the heteroatoms and phenyl groups. A correlation was established among anti-HIV activity, Log P and Log D7.4 and between anti-HIV activity and carbonyl-heteroatom interatomic distances in the myristoyl analogs. 12-Thioethyldodecanoic acid 6 was moderately active (EC50 = 9.37 microM) against HIV-infected T4-lymphocytes (CEM-SS cell line), and it exhibited in vitro activity (EC50 = 17.8 microM) against HBV-producing 2.2.15 cell cultures derived from a human hepatoblastoma cell line (Hep G2). 12-Methoxydodecanoic acid 1 exhibited in vitro activity (EC50 = 20-30 microM) against hepatitis B in the HBV DNA-transfected 2.2.15 cell line. At a concentration of 10 microg/ml, none of the fatty acids significantly inhibited the replication of DHBV in infected hepatocytes.

Varicella-zoster virus thymidine kinase gene and antiherpetic pyrimidine nucleoside analogues in a combined gene/chemotherapy treatment for cancer

Ten pyrimidine nucleoside analogues, including (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) and closely related analogues, were evaluated for their cytostatic activity against human osteosarcoma cells transfected with the varicella-zoster virus (VZV) thymidine kinase (tk) (ATP:thymidine 5′ phosphotransferase, EC 2.7.2.21) gene. (E)-5-(2-bromovinyl)-1-β- D-arabinofuranosyluracil (BVaraU), (E)-5-(2-iodovinyl)-2′-deoxy-2′-fluoro-1-β- D-arabinofurano- syluracil (IVFAU) and (E)-5-(2-bromovinyl)-2′-deoxy-4′-thiouridine (S-BVDU) were among the most potent inhibitors of VZVtk gene-transfected cell proliferation. They displayed an inhibitory activity at drug concentrations that were up to four orders of magnitude lower than those required to inhibit the corresponding nontransfected tumor cells. Inhibition of cellular DNA polymerase and/or incorporation of the drugs into cellular DNA may be a likely target for the cytostatic activity of the BVDU derivatives against the VZVtk gene-transfected tumor cells. These compounds were approximately 40- to 80-fold more potent cytostatic agents in VZVtk gene-transfected cells than the anti-VZV compound 6-methoxy-9-β- D-arabinofuranosylpurine (araM), and at least five- to 50-fold more cytostatic than ganciclovir in HSV-1tk gene-transfected murine mammary carcinoma FM3A cells. In addition, the intrinsic resistance of BVaraU, IVFAU and S-BVDU to glycosidic bond cleavage by mammalian dThd phosphorylases makes them promising candidate compounds for the treatment of VZVtk gene-transfected tumors in vivo.

Synthesis of celecoxib analogs that possess a N-hydroxypyrid-2(1H)one 5-lipoxygenase pharmacophore : Biological evaluation as dual inhibitors of cyclooxygenases and 5-lipoxygenase with anti-inflammatory activity

A hitherto unknown class of celecoxib analogs was designed for evaluation as dual inhibitors of the 5-lipoxygenase/cyclooxygenase-2 (5-LOX/COX-2) enzymes. These compounds possess a SO(2)Me (11a), or SO(2)NH(2) (11b) COX-2 pharmacophore at the para-position of the N(1)-phenyl ring in conjunction with a 5-LOX N-hydroxypyrid-2(1H)one iron-chelating moiety in place of the celecoxib C-5 tolyl group. The title compounds 11a-b are weak inhibitors of the COX-1 and COX-2 isozymes (IC(50)=7.5-13.2 microM range). In contrast, the SO(2)Me (11a, IC(50)=0.35 microM), and SO(2)NH(2) (11b, IC(50)=4.9 microM), compounds are potent inhibitors of the 5-LOX enzyme comparing favorably with the reference drug caffeic acid (5-LOX IC(50)=3.47 microM). The SO(2)Me (11a, ED(50)=66.9 mg/kg po), and SO(2)NH(2) (11b, ED(50)=99.8 mg/kg po) compounds exhibited excellent oral anti-inflammatory (AI) activities being more potent than the non-selective COX-1/COX-2 inhibitor drug aspirin (ED(50)=128.9 mg/kg po) and less potent than the selective COX-2 inhibitor celecoxib (ED(50)=10.8 mg/kg po). The N-hydroxypyridin-2(1H)one moiety constitutes a novel pharmacophore for the design of cyclic hydroxamic mimetics capable of chelating 5-LOX iron for exploitation in the design of 5-LOX inhibitory AI drugs.

6-Alkyl, alkoxy, or alkylthio-substituted 3-(4-methanesulfonylphenyl)-4-phenylpyran-2-ones: A novel class of diarylheterocyclic selective cyclooxygenase-2 inhibitors

A novel class of 3-(4-methanesulfonylphenyl)-4-phenylpyran-2-ones possessing a central six-membered lactone (pyran-2-one) ring system, in conjunction with C-6 alkyl (Me, Et or i-Pr), alkoxy (OMe, OEt or O-i-Pr), and alkylthio (SMe, SEt or S-i-Pr) substituents, were designed for evaluation as selective COX-2 inhibitors.

Dinitroglyceryl and diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of aspirin, indomethacin and ibuprofen: Synthesis, biological evaluation and nitric oxide release studies

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) ester prodrugs (NONO-NSAIDs) wherein a 1,3-dinitrooxy-2-propyl (12a-c), or O(2)-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (14a-c), NO-donor moiety is directly attached to the carboxylic acid group of aspirin, indomethacin or ibuprofen were synthesized. NO release from the dinitrooxypropyl, or diazen-1-ium-1,2-diolate, ester prodrugs was increased substantially upon incubation in the presence of l-cysteine (12a-c) or rat serum (14a-c). The ester prodrugs (12a-c, 14a-c), which did not inhibit the COX-1 isozyme, exhibited modest inhibitory activity against the COX-2 isozyme. The NONO-NSAIDs 12a-c and 14a-c exhibited in vivo AI activity that was similar to that exhibited by the parent drug aspirin, indomethacin or ibuprofen when the same oral dose (micromol/kg) was administered. These similarities in oral potency profiles suggest these NONO-NSAIDs act as classical prodrugs that require metabolic activation by esterase-mediated hydrolysis. Hybrid NO-donor/anti-inflammatory prodrugs of this type (NONO-NSAIDs) offer a potential drug design concept targeted toward the development of anti-inflammatory drugs with reduced adverse gastrointestinal effects.