Zhangjian Huang

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).

Hybrid fluorescent conjugates of COX-2 inhibitors: Search for a COX-2 isozyme imaging cancer biomarker

The observation that the cyclooxygenase-2 (COX-2) isozyme is over-expressed in multiple types of cancer, relative to that in adjacent non-cancerous tissue, prompted this investigation to prepare a group of hybrid fluorescent conjugates wherein the COX inhibitors ibuprofen, (S)-naproxen, acetyl salicylic acid, a chlororofecoxib analog and celecoxib were coupled via a linker group to an acridone, dansyl or rhodamine B fluorophore. Within this group of compounds, the ibuprofen-acridone conjugate (10) showed potent and selective COX-2 inhibition (COX-2 IC(50)=0.67 μM; SI=110.6), but its fluorescence emission (λ(em)=417, 440 nm) was not suitable for fluorescent imaging of cancer cells that over-express the COX-2 isozyme. In comparison, the celecoxib-dansyl conjugate (25) showed a slightly lower COX-2 potency and selectivity (COX-2 IC(50)=1.1 μM; SI>90) than the conjugate 10, and it possesses a better fluorescence emission (λ(em)=500 nm). Ultimately, a celecoxib-rhodamine B conjugate (28) that exhibited moderate COX-2 potency and selectivity (COX-2 IC(50)=3.9 μM; SI>25) having the best fluorescence emission (λ(em)=580 nm) emerged as the most promising biomarker for fluorescence imaging using a colon cancer cell line that over-expresses the COX-2 isozyme.

Rofecoxib Analogues Possessing a Nitric Oxide Donor Sulfohydroxamic Acid (SO2NHOH) Cyclooxygenase-2 Pharmacophore: Synthesis, Molecular Modeling, and Biological Evaluation as Anti-inflammatory Agents

The design of selective cyclooxygenase-2 (COX-2) inhibitors as anti-inflammatory (AI) drugs that preferentially block the inducible COX-2 isozyme, which produces undesirable peripheral inflammation, over the constitutive COX-1 isozyme, which provides desirable gastroprotection and vascular homeostasis, represents an important milestone in the development of nonsteroidal anti-inflammatory drugs (NSAIDs). The discovery that celecoxib (1 a), rofecoxib (2), and valdecoxib (3) show a low risk of gastrointestinal irritation provided validation for this original drug design concept (Figure 1). The subsequent obser-

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.

Acyclic triaryl olefins possessing a sulfohydroxamic acid pharmacophore: synthesis, nitric oxide/nitroxyl release, cyclooxygenase inhibition, and anti-inflammatory studies

Nitric oxide (NO) and its reduced form nitroxyl (HNO), effective vasodilation agents that can inhibit platelet aggregation and adhesion, could suppress adverse cardiovascular effects associated with the use of selective COX-2 inhibitors. In this regard, a sulfohydroxamic acid (SO(2)NHOH) substituent, that can act as a dual NO/HNO donor moiety, was inserted at the para-position of the C2 phenyl ring of acyclic 2-alkyl-1,1,2-triaryl olefins previously shown to be potent and highly selective COX-2 inhibitors. Although this new group of 1,1-diaryl-2-(4-hydroxyaminosulfonylphenyl)alk-1-enes exhibited weak inhibition of the constitutive cyclooxygenase-1 (COX-1) and inducible COX-2 isozymes, in vivo studies showed anti-inflammatory potencies that were generally intermediate between that of the reference drugs aspirin and ibuprofen. All compounds released NO (5.6-13.5% range) upon incubation with phosphate buffer which was increased further (8.3-25.6% range) in the presence of the oxidant K(3)(FeCN(6)).The low release of HNO in MeOH-buffer (< 2% at 24 h incubation) was much higher at alkaline pH (11-37% range). The concept of designing better anti-inflammatory drugs possessing either an effective HNO, or dual NO/HNO, donor moiety that are devoid of adverse ulcerogenic and/or cardiovascular side effects warrants further investigation.

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.

Cardiovascular Properties of a Nitric Oxide Releasing Rofecoxib Analogue: Beneficial Anti-hypertensive Activity and Enhanced Recovery in an Ischemic Reperfusion Injury Model

was subsequently discovered that rofecoxib causes undesirable cardiovascular events such as myocardial infarction and stroke; this triggered its withdrawal from the market. 3] Highly selective COX-2 inhibitors, including rofecoxib, likely alter the biochemical balance in the COX pathway. In this regard, COX-2mediated biosynthesis of beneficial vasodilatory, anti-aggregatory prostacyclin (PGI2), which can improve cardiac function following ischemic reperfusion injury, is suppressed in conjunction with a contraindicated simultaneous increase in the level of the prothrombotic thromboxane A2 (TxA2), which causes vasoconstriction, decreases cardiac function, and induces platelet aggregation). Accordingly, rofecoxib tips the PGI2–TxA2 balance toward TxA2, resulting in elevated blood pressure (BP) and increased risk of adverse prothrombotic effects. In addition to the inhibition of PGI2 biosynthesis in the vascular endothelium, selective COX-2 inhibitors may also block the synthesis of renal PGs and increase sodium reabsorption, which can also contribute to an elevation in blood pressure (hypertension). Nitric oxide (NO) is an efficient vasodilation agent and inhibitor of platelet aggregation and adhesion, and it limits cardiac ischemia-reperfusion injury. 13] Hence, the incorporation of a NO donor moiety onto rofecoxib offers an attractive strategy to circumvent adverse cardiovascular side effects associated with the use of rofecoxib. In a recent investigation, we developed this drug design concept wherein the methanesulfonyl (SO2CH3) substituent in rofecoxib is replaced by a sulfohydroxamic acid (SO2NHOH) dual-function NO donor/COX-2 pharmacophore, and a para-chloro substituent was introduced at the C4 phenyl ring position to prevent formation (obstructive metabolic halogenation) of a para-hydroxy metabolite. This hitherto-unknown sulfohydroxamic acid analogue of rofecoxib (compound 2, Figure 1) showed 1) potent (COX-2 IC50: 0.28 mm) and 2) selective (COX-2 selectivity index >304) COX-2 inhibitory activities, 3) appreciable in vivo anti-inflammatory activity (ED50: 17.7 mg kg 1 po), and 4) a 43 % release of NO for a 24-hour incubation in phosphate buffer at pH 7.4; moreover, 5) a molecular modeling study indicated that N-hydroxy-4-[4(4-chlorophenyl)-5-oxo-2,5-dihydrofuran-3-yl]benzenesulfonamide (2) assumes a favorable orientation inside the COX-2 binding site which allows multiple hydrogen bonding interactions. An illustration of these important biological features is presented in Figure 2. It was therefore of interest to ascertain some cardiovascular properties of this new NO donor rofecoxib analogue 2. Herein we describe the effects of compound 2 on systolic, diastolic, and mean blood pressure (average of systolic and diastolic values), heart rate, and its ability to enhance recovery in a cardiac ischemic reperfusion injury model. Systolic blood pressure (BPsys, mm Hg), diastolic blood pressure (BPdia, mm Hg), and heart rate (HR, beats min ) were measured at 1, 3, 6, and 24 h time intervals following oral adminis-

Aspirin Analogues as Dual Cyclooxygenase‐2/5‐Lipoxygenase Inhibitors: Synthesis, Nitric Oxide Release, Molecular Modeling, and Biological Evaluation as Anti‐Inflammatory Agents

Analogues of aspirin were synthesized through an efficient one‐step reaction in which the carboxyl group was replaced by an ethyl ester, and/or the acetoxy group was replaced by an N‐substituted sulfonamide (SO2NHOR2: R2=H, Me, CH2Ph) pharmacophore. These analogues were designed for evaluation as dual cyclooxygenase‐2 (COX‐2) and 5‐lipoxygenase (5‐LOX) inhibitors. In vitro COX‐1/COX‐2 isozyme inhibition studies identified compounds 11 (CO2H, SO2NHOH), 12 (CO2H, SO2NHOCH2Ph), and 16 (CO2Et, SO2NHOH) as highly potent and selective COX‐2 inhibitors (IC50 range: 0.07–0.7 μM), which exhibited appreciable in vivo anti‐inflammatory activity (ED50 range: 23.1–31.4 mg kg−1). Moreover, compounds 11 (IC50=0.2 μM) and 16 (IC50=0.3 μM), with a sulfohydroxamic acid (SO2NHOH) moiety showed potent 5‐LOX inhibitory activity. Furthermore, the SO2NHOH moiety present in compounds 11 and 16 was found to be a good nitric oxide (NO) donor upon incubation in phosphate buffer at pH 7.4. Molecular docking studies in the active binding site of COX‐2 and 5‐LOX provided complementary theoretical support for the experimental biological structure–activity data acquired.

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.

Isomeric acetoxy analogs of celecoxib and their evaluation as cyclooxygenase inhibitors.

A group of celecoxib analogs having a SO(2)NH(2) (9a-f), or SO(2)Me (12a-f), COX-2 pharmacophore at the para-position of the N-1 phenyl ring in conjunction with a C-5 phenyl ring having a variety of substituents (4-, 3-, 2-OAc; 4-Me,2-OAc, 4-Me,3-OAc, 4-F,2-OAc) was synthesized for evaluation as cyclooxygenase (COX) inhibitors of the COX-1/COX-2 isozymes. Within this group of compounds, 1-(4-aminosulfonylphenyl)-3-trifluoromethyl-5-(2-acetoxy-4-fluorophenyl)pyrazole (9f) emerged as the most potent (COX-1 IC(50)=0.7 μM; COX-2 IC(50)=0.015 μM) and selective (COX-2 selectivity index=47) inhibitor agent that exhibited good anti-inflammatory activity (ED(50)=42.3mg/kg) which was lower than the reference drug celecoxib (ED(50)=10.8 mg/kg), but greater than ibuprofen (ED(50)=67.4 mg/kg) and aspirin (ED(50)=128.7 mg/kg). Molecular modeling studies for 9f showed that the SO(2)NH(2) group assumes a position within the secondary pocket of the COX-2 active site wherein the SO(2)NH(2) oxygen atom is hydrogen bonded to the H90 residue (2.90Å), the SO(2)NH(2) nitrogen atom forms a hydrogen bond with L352 (N⋯O=2.80Å), and the acetyl group is positioned in the vicinity of the S530 residue where the acetyl oxygen atom undergoes hydrogen bonding to L531 (2.99Å).