Jatinder Kaur

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.

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-

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.

Synthesis of three 18F-labelled cyclooxygenase-2 (COX-2) inhibitors based on a pyrimidine scaffold

Cyclooxygenase (COX) is the key enzyme within the complex conversion of arachidonic acid into prostaglandins (PGs). Inhibitors of this enzyme represent a particularly promising class of compounds for chemoprevention and cancer therapy. The experimental data on the involvement of COX isoform COX-2 in tumour development and progression, as well as the observed overexpression of COX-2 in a variety of human cancers provide the rationale for targeting COX-2 for molecular imaging and therapy of cancer. A series of trifluoromethyl-substituted pyrimidines was prepared as a novel class of selective COX-2 inhibitors, based on the lead structure 1a. All compounds were tested in cyclooxygenase (COX) assays in vitro to determine COX-1 and COX-2 inhibitory potency and selectivity. Molecular docking studies using the catalytic site of COX-1 and COX-2, respectively, provided complementary theoretical support for the obtained experimental biological structure–activity relationship data of three highly potent and selective fluorobenzyl-containing COX-2 inhibitors. Selected fluorobenzyl-substituted pyrimidine derivatives were further developed as (18)F-labelled radiotracers ([(18)F]1a, [(18)F]2a, [(18)F]3a). Radiotracers [(18)F]1a and [(18)F]2a were radiolabelled using 4-[(18)F]fluorobenzylamine ([(18)F]FBA) as a building block. Radiotracer [(18)F]3a was radiofluorinated directly using a nucleophilic aromatic substitution reaction with no-carrier-added (n.c.a.) [(18)F]fluoride on an iodylaryl compound as a labelling precursor.

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.

1,4-Diaryl-substituted triazoles as cyclooxygenase-2 inhibitors: Synthesis, biological evaluation and molecular modeling studies

A novel group of 1,4-diaryl-substituted triazoles was designed and synthesized by introducing the cyclooxygenase-2 (COX-2) pharmacophore SO2NH2 attached to one aryl ring and various substituents (H, F, Cl, CH3 or OCH3) attached to the other aryl ring. The effects of size and flexibility of the compounds upon COX-1/COX-2 inhibitory potency and selectivity was studied by increasing the size of an alkyl linker chain [(-CH2)n, where n=0, 1, 2]. In vitro COX-1/COX-2 inhibition studies showed that all compounds (14-18, 21-25 and 28-32) are more potent inhibitors of COX-2 isozyme (IC50=0.17-28.0μM range) compared to COX-1 isozyme (IC50=21.0 to >100μM range). Within the group of 1,4 diaryl-substituted triazoles, 4-{2-[4-(4-chloro-phenyl)-[1,2,3]triazol-1-yl]-ethyl}-benzenesulfonamide (compound 30) displayed highest COX-2 inhibitory potency and selectivity (COX-1: IC50=>100μM, COX-2: IC50=0.17μM, SI >588). Molecular docking studies using the catalytic site of COX-1 and COX-2, respectively, provided complementary theoretical support for the obtained experimental biological structure-activity relationship data. Results of molecular docking studies revealed that COX-2 pharmacophore SO2NH2 in compound 30 is positioned in the secondary pocket of COX-2 active site; with the nitrogen atom of the SO2NH2 group being hydrogen bonded to Q192 (N⋯OC=2.85Å), and one of the oxygen atoms of SO2NH2 group forming a hydrogen bond to H90 (SO⋯N=2.38Å).

Fluorescent Hexose Conjugates Establish Stringent Stereochemical Requirement by GLUT5 for Recognition and Transport of Monosaccharides

The specificity characteristics of transporters can be exploited for the development of novel diagnostic therapeutic probes. The facilitated hexose transporter family (GLUTs) has a distinct set of preferences for monosaccharide substrates, and while some are expressed ubiquitously (e.g., GLUT1), others are quite tissue specific (e.g., GLUT5, which is overexpressed in some breast cancer tissues). While these differences have enabled the development of new molecular probes based upon hexose- and tissue-selective uptake, substrate design for compounds targeting these GLUT transporters has been encumbered by a limited understanding of the molecular interactions at play in hexose binding and transport. Four new fluorescently labeled hexose derivatives have been prepared, and their transport characteristics were examined in two breast cancer cell lines expressing mainly GLUTs 1, 2, and 5. Our results demonstrate, for the first time, a stringent stereochemical requirement for recognition and transport by GLUT5. 6-NBDF, in which all substituents are in the d-fructose configuration, is taken up rapidly into both cell lines via GLUT5. On the other hand, inversion of a single stereocenter at C-3 (6-NBDP), C-4 (6-NBDT), or C-5 (6-NDBS) results in selective transport via GLUT1. An in silico docking study employing the recently published GLUT5 crystal structure confirms this stereochemical dependence. This work provides insight into hexose-GLUT interactions at the molecular level and will facilitate structure-based design of novel substrates targeting individual members of the GLUT family and forms the basis of new cancer imaging or therapeutic agents.