Atul Bhardwaj

In situ click chemistry generation of cyclooxygenase-2 inhibitors

Cyclooxygenase-2 isozyme is a promising anti-inflammatory drug target, and overexpression of this enzyme is also associated with several cancers and neurodegenerative diseases. The amino-acid sequence and structural similarity between inducible cyclooxygenase-2 and housekeeping cyclooxygenase-1 isoforms present a significant challenge to design selective cyclooxygenase-2 inhibitors. Herein, we describe the use of the cyclooxygenase-2 active site as a reaction vessel for the in situ generation of its own highly specific inhibitors. Multi-component competitive-binding studies confirmed that the cyclooxygenase-2 isozyme can judiciously select most appropriate chemical building blocks from a pool of chemicals to build its own highly potent inhibitor. Herein, with the use of kinetic target-guided synthesis, also termed as in situ click chemistry, we describe the discovery of two highly potent and selective cyclooxygenase-2 isozyme inhibitors. The in vivo anti-inflammatory activity of these two novel small molecules is significantly higher than that of widely used selective cyclooxygenase-2 inhibitors.Traditional inflammation and pain relief drugs target both cyclooxygenase 1 and 2 (COX-1 and COX-2), causing severe side effects. Here, the authors use in situ click chemistry to develop COX-2 specific inhibitors with high in vivo anti-inflammatory activity.

Mono-, di-, and triaryl substituted tetrahydropyrans as cyclooxygenase-2 and tumor growth inhibitors. Synthesis and biological evaluation.

Rationally designed tetrahydropyrans (THPs) carrying one, two, or three aryl rings and other substituents were synthesized by the allylation of beta-hydroxy ketones followed by iodocyclization. It has been observed that compounds with one aryl ring on THP are moderate inhibitors of cyclooxygenase-1 (COX-1) (IC(50) = 0.3 microM) and cyclooxygenase-2 (IC(50) = 0.17 microM) with poor selectivity index (SI = 2-3) for COX-2. The presence of two aryl rings enhanced their inhibitory activities for COX-2 (IC(50) = 0.9-5.5 nM). Selectivity for COX-2 over COX-1 also increased (SI = 50-1900), while triaryl substituted THPs, along with high inhibition (IC(50) = 0.57-4.0 nM), also exhibited excellent selectivity for COX-2 over COX-1 (SI = 3200-44000). Similar to the experimental results of increased COX-2 inhibition and selectivity with the increase in the size of the molecule, their docking in the active sites of COX-1 and COX-2 also showed same trend. Seven compounds from the category of di- and triaryl substituted THPs exhibited average GI(50) over all the human tumor cell lines in the range 1.6-3.2 microM and showed in vitro therapeutic indices of 8-17.

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.

Synthesis and Biological Investigations of Nitric Oxide Releasing Nateglinide and Meglitinide Type II Antidiabetic Prodrugs: In-Vivo Antihyperglycemic Activities and Blood Pressure Lowering Studies

A new group of hybrid nitric oxide-releasing type II antidiabetic drugs possessing a 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (13 and 18), 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (14 and 19), or nitrooxyethyl (15 and 20) moiety attached to the carboxylic acid group of the type II antidiabetic drugs nateglinide and meglitinide were synthesized. These prodrugs, based on the beneficial properties of nitric oxide (NO), were designed to reduce the risk of adverse cardiovascular events in diabetic patients. Ester prodrugs (13-15 and 18-20) exhibited appreciable oral antihyperglycemic activity comparable to the parent drugs in nonfasted diabetic rats. Systolic and diastolic blood pressure profiles validated the beneficial hypotensive properties of these prodrugs. These prodrugs released NO (1.3-72.2% range) upon incubation with either phosphate buffer solution at pH 7.4 or in the presence of serum. This new type of hybrid NO donor prodrug represents an attractive approach for the rational design of type II antidiabetic drugs with a reduced risk of contraindicated cardiovascular events.

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-

O2-Sulfonylethyl Protected Isopropylamine Diazen-1-ium-1,2-diolates as Nitroxyl (HNO) Donors: Synthesis, β-Elimination Fragmentation, HNO Release, Positive Inotropic Properties, and Blood Pressure Lowering Studies

New types of nonexplosive O(2)-sulfonylethyl protected (-CH(2)CH(2)SO(2)R; R = OMe, NHOMe, NHOBn, Me) derivatives of isopropylamine diazen-1-ium-1,2-diolate (IPA/NO) (2-5) were developed that are designed to act as novel HNO donors. These compounds, with suitable half-lives (6.6-17.1 h) at pH 7.4, undergo a base-induced β-elimination reaction that releases a methyl vinyl sulfone product and the parent IPA/NO anion which subsequently preferentially releases HNO (46-61% range). Importantly, the O(2)-methylsulfonylethyl compound 5 exhibited a significant in vitro inotropic effect up to 283% of the baseline value and increased the rates of contraction and relaxation but did not induce a chronotropic effect. Furthermore, compound 5 (22.5 mg/kg po dose) provided a significant reduction in blood pressure up to 6 h after drug administration. All these data suggest that O(2)-sulfonylethyl protected derivatives of IPA/NO, which are efficient HNO donors, could have potential applications to treat cardiovascular disease(s) such as congestive heart failure.

Fluorophore‐Labeled Cyclooxygenase‐2 Inhibitors for the Imaging of Cyclooxygenase‐2 Overexpression in Cancer: Synthesis and Biological Studies

A group of cyclooxygenase‐2 (COX‐2)‐specific fluorescent cancer biomarkers were synthesized by linking the anti‐inflammatory drugs ibuprofen, (S)‐naproxen, and celecoxib to the 7‐nitrobenzofurazan (NBD) fluorophore. In vitro COX‐1/COX‐2 inhibition studies indicated that all of these fluorescent conjugates are COX‐2 inhibitors (IC50 range: 0.19–23.0 μM) with an appreciable COX‐2 selectivity index (SI≥4.3–444). In this study the celecoxib–NBD conjugate N‐(2‐((7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)amino)ethyl)‐4‐(5‐(p‐tolyl)‐3‐(trifluoromethyl)‐1H‐pyrazol‐1‐yl)benzenesulfonamide (14), which displayed the highest COX‐2 inhibitory potency and selectivity (COX‐2 IC50=0.19 μM; SI=443.6), was identified as an impending COX‐2‐specific biomarker for the fluorescence imaging of cancer using a COX‐2‐expressing human colon cancer cell line (HCA‐7).

Design, Synthesis, and Evaluation of an (18)F-Labeled Radiotracer Based on Celecoxib-NBD for Positron Emission Tomography (PET) Imaging of Cyclooxygenase-2 (COX-2).

A series of novel fluorine‐containing cyclooxygenase‐2 (COX‐2) inhibitors was designed and synthesized based on the previously reported fluorescent COX‐2 imaging agent celecoxib–NBD (3; NBD=7‐nitrobenzofurazan). In vitro COX‐1/COX‐2 inhibitory data show that N‐(4‐fluorobenzyl)‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide (5; IC50=0.36 μM, SI>277) and N‐fluoromethyl‐4‐(5‐p‐tolyl‐3‐trifluoromethylpyrazol‐1‐yl)benzenesulfonamide (6; IC50=0.24 μM, SI>416) are potent and selective COX‐2 inhibitors. Compound 5 was selected for radiolabeling with the short‐lived positron emitter fluorine‐18 (18F) and evaluated as a positron emission tomography (PET) imaging agent. Radiotracer [18F]5 was analyzed in vitro and in vivo using human colorectal cancer model HCA‐7. Although radiotracer uptake into COX‐2‐expressing HCA‐7 cells was high, no evidence for COX‐2‐specific binding was found. Radiotracer uptake into HCA‐7 tumors in vivo was low and similar to that of muscle, used as reference tissue.

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.