# US 2021/0330822 A1 · Fluorescent probe for cyclooxygenase-2

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- Number: US 2021/0330822 A1
- Document ID: US20210330822A1
- Status: Published application
- Kind: A1
- Application number: 17/240,427
- Filed: 2021-04-26
- Published: 2021-10-28
- Earliest claimed: 2020-04-24
- Assignees: The Board of Trustees of the University of Illinois
- Applicants: The Board of Trustees of the University of Illinois
- Inventors: Christopher J. Reinhardt; Anuj K. YADAV; Jefferson CHAN
- Web page: https://patentgrep.com/patent/US20210330822A1

## CPC

- A61K 51/0474: {complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group}
- C07D 311/72: 3,4-Dihydro derivatives having in position 2 at least one methyl radical and in position 6 one oxygen atom, e.g. tocopherols
- C09B 19/00: Oxazine dyes
- G01N 33/582: {with fluorescent label}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Cyclooxygenase-2 (COX-2) over-expression is prominent in inflammatory diseases, neurodegenerative disorders, and cancer. Directly monitoring COX-2 activity within its native environment poses an exciting approach to account for and illuminate the effect of the local environments on protein activity. Herein, we report the development of CoxFluor, the first activity-based sensing approach for monitoring COX-2 within live cells with confocal microscopy and flow cytometry. CoxFluor strategically links a natural substrate with a dye precursor to engage both the cyclooxygenase and peroxidase activities of COX-2. This catalyzes the release of resorufin and the natural product, as supported by molecular dynamics and ensemble docking. CoxFluor enabled the detection of oxygen-dependent changes in COX-2 activity that are independent of protein expression within live macrophage cells.

## Claims

### Claim 1

A compound of Formula I:
wherein
X1 and X2 are independently OH, SH, or OC(═O)(C1-C6)alkyl;
X3 is O, S, SiR2, SiPhs, CR2, CPh2, C(fluorene), C(═O), C(═S), P(═O)R, or P(═O)OR wherein R is —(C1-C6)alkyl;
X4 is O or S;
R1 and R2 are independently H, halo, amino, nitro, cyano, or —(C1-C6)alkyl; and
R3 is the alkyl moiety of arachidonic acid wherein the alkyl moiety is unsaturated or saturated; or
R3 is the alkyl moiety of a prostaglandin wherein the alkyl moiety is substituted with heteroatoms.

### Claim 2 (depends on 1)

The compound of claim 1 wherein the compound is a compound of Formula II:
wherein each is independently a double bond or a single bond.

### Claim 3 (depends on 2)

The compound of claim 2 wherein each is a double bond.

### Claim 4 (depends on 3)

The compound of claim 3 wherein the compound is a selective substrate for cyclooxygenase-2 (COX-2) over other enzymes.

### Claim 5 (depends on 2)

The compound of claim 2 wherein each is a single bond.

### Claim 6 (depends on 2)

The compound of claim 2 wherein the compound is not fluorescent or emits less fluorescence than Resorufin.

### Claim 7 (depends on 1)

The compound of claim 1 wherein X1 and X2 are OH, and X3 and X4 are O.

### Claim 8 (depends on 1)

The compound of claim 1 wherein R1 and R2 are H.

### Claim 9 (depends on 1)

The compound of claim 1 wherein the compound is a compound of Formula III:
wherein X5 is OH, SH, or OOH.

### Claim 10 (depends on 9)

The compound of claim 9 wherein X4 is O.

### Claim 11 (depends on 10)

The compound of claim 10 wherein the amide bond is cleavable selectively over other enzymes at heme B of the native COX-2 peroxidase active site.

### Claim 12 (depends on 1)

The compound of claim 1 wherein the compound is:

### Claim 13 (depends on 12)

The compound of claim 12 wherein the compound is CoxFluor.

### Claim 14 (depends on 1)

The compound of claim 1 wherein the compound has a quantum yield of about 0.29 and a molar extinction coefficient of about 820 M−1cm−1 at 572 nm.

### Claim 15

A method for imaging cyclooxygenase-2 (COX-2) activity comprising:
a) contacting a substrate and a compound of Formula IIB:
wherein
X1 and X2 are independently OH, SH, or OC(═O)(C1-C6)alkyl;
X3 is O, S, SiR2, SiPhs, CR2, CPh2, C(fluorene), C(═O), C(═S), P(═O)R, or P(═O)OR wherein R is —(C1-C6)alkyl;
X4 is O or S; and
R1 and R2 are independently H, halo, amino, nitro, cyano, or —(C1-C6)alkyl; and
b) quantifying the fluorescent intensity of the contacted substrate by comparing the fluorescent intensity of the contacted substrate to a control;
thereby imaging the COX-2 activity of the substrate.

### Claim 16 (depends on 15)

The method of claim 15 wherein the substrate is COX-2 or a cell.

### Claim 17 (depends on 15)

The method of claim 15 wherein the substrate is contacted with a second agent prior to step a).

### Claim 18 (depends on 17)

The method of claim 17 wherein the second agent depletes glutathione.

### Claim 19 (depends on 15)

The method of claim 15 wherein the substrate and the compound are contacted with a second agent at step a).

### Claim 20 (depends on 19)

The method of claim 19 wherein the second agent is a cyclooxygenase inhibitor or glutathione.

### Claim 21 (depends on 15)

The method of claim 15 wherein quantifying the fluorescent intensity is performed by flow cytometry.