COX-2 (IC50 = 40 nM)
Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Indomethacin heptyl ester is a non-selective inhibitor of both COX-1 and COX-2, with reported IC50 of 0.04 μM for COX-2. By inhibiting COX enzymes, the compound blocks the conversion of arachidonic acid to prostaglandins, thereby reducing inflammation, pain, and fever. The heptyl ester modification increases the compound's lipid solubility compared to the parent indomethacin.

Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with ICo5 values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 microM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.[1]

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Indomethacin heptyl ester inhibits COX-2 with an IC50 of 0.04 μM and exhibits anti-inflammatory activity. The compound is a non-selective inhibitor of both COX-1 and COX-2. As a lipid-soluble derivative of indomethacin, it retains the anti-inflammatory properties of the parent compound while exhibiting improved lipid solubility. The compound's ability to inhibit prostaglandin synthesis makes it useful for studying inflammation and pain pathways.

In vivo, Indomethacin heptyl ester would be expected to exhibit anti-inflammatory, analgesic, and antipyretic effects similar to other NSAIDs. The heptyl ester modification may affect the compound's absorption, distribution, and metabolism compared to indomethacin. As a research compound, it is used to study the role of COX enzymes in various disease models. The compound is not for human or veterinary use.

Enzymology. [1]
COX-1 was purified from ram seminal vesicles as described earlier.36 The specific activity of the protein was 20 (μMO2/min)/mg, and the percentage of holoprotein was 13.5%. ApoCOX-1 was prepared as described earlier.37 Apoenzyme was reconstituted by the addition of hematin to the assay mixtures. Human COX-2 was expressed in SF-9 insect cells by means of the pVL 1393 expression vector and purified by ion-exchange and gel filtration chromatography. All of the purified proteins were shown by densitometric scanning of a 7.5% SDS PAGE gel to be >80% pure.

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Time- and Concentration-Dependent Inhibition of Ovine COX-1 and Human COX-2 Using Thin Layer Chromatography (TLC) Assay. [1]
Cyclooxygenase activity of ovine COX-1 (44 nM) or human COX-2 (66 nM) was assayed by TLC. Reaction mixtures of 200 μL consisted of hematin-reconstituted protein in 100 mM Tris-HCl, pH 8.0, 500 μM phenol, and [1-14C]arachidonic acid (50 μM, ∼55−57 mCi/mmol). For the time-dependent inhibition assay, hematin-reconstituted COX-1 (44 nM) or COX-2 (66 nM) was preincubated at room temperature for 20 min with varying inhibitor concentrations in DMSO followed by the addition of [1-14C]arachidonic acid (50 μM) for 30 s at 37 °C. Reactions were terminated by solvent extraction in Et2O/CH3OH/1 M citrate, pH 4.0 (30:4:1). The phases were separated by centrifugation at 2000g for 2 min and the organic phase was spotted on a TLC plate. The plate was developed in EtOAc/CH2Cl2/glacial AcOH (75:25:1) at 4 °C. Radiolabeled prostanoid products were quantitated with a radioactivity scanner. The percentage of total products observed at different inhibitor concentrations was divided by the percentage of products observed for protein samples preincubated for the same time with DMSO.

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Enzyme activity assays are used to measure the inhibitory activity of Indomethacin heptyl ester against COX-1 and COX-2. Purified recombinant COX-1 and COX-2 enzymes are incubated with arachidonic acid substrate in the presence of increasing concentrations of the compound. Prostaglandin production is measured by ELISA or radiometric assays to determine IC50 values. The reported IC50 for COX-2 is 0.04 μM.

Inhibition of COX-2 Activity in Activated RAW264.7. [1]
Protocols for COX-2 inhibition in RAW264.7 cells have been previously described.23 Briefly, cells (6.2 × 106 cells/T25 flask) were activated with lipopolysaccharide (1 μg/mL) and γ-interferon (10 U/mL) in serum-free DMEM for 7 h and then treated with inhibitor (0−2 μM) for 30 min at 37 °C. Exogenous arachidonate metabolism was determined by adding [1-14C]arachidonic acid (20 μM) for 15 min at 37 °C. IC50 values are the average of two independent determinations.

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Cellular assays using COX-expressing cell lines are employed to evaluate the anti-inflammatory activity of Indomethacin heptyl ester. Cells (e.g., RAW 264.7 macrophages) are stimulated with LPS to induce COX-2 expression and prostaglandin production. Cells are treated with increasing concentrations of the compound, and PGE2 levels in the culture supernatant are measured by ELISA to assess inhibition of prostaglandin synthesis. Cytotoxicity is assessed to determine the compound's safety profile.

In vivo efficacy of Indomethacin heptyl ester can be evaluated in standard animal models of inflammation. The compound is typically administered orally or intraperitoneally in murine models such as carrageenan-induced paw edema or acetic acid-induced writhing test. Efficacy endpoints include reduction of paw swelling, pain response, and inflammatory cytokine levels. The heptyl ester modification may affect the compound's oral bioavailability and tissue distribution compared to indomethacin.

As a lipid-soluble derivative of indomethacin with molecular weight of 455.97 g/mol, Indomethacin heptyl ester has a molecular formula of C26H30ClNO4. The heptyl ester modification increases lipophilicity compared to the parent compound, which may affect absorption and tissue distribution. The compound is soluble in DMSO (45 mg/mL). Powder stability is 3 years at -20°C. Pharmacokinetic properties would be expected to differ from indomethacin due to the ester modification.

No specific toxicity data has been reported for Indomethacin heptyl ester. As an NSAID derivative, potential toxicities include gastrointestinal ulceration, renal toxicity, and cardiovascular effects, which are class effects of COX inhibitors. The compound is not for human or veterinary use. Standard toxicity assessments would include evaluation of cytotoxicity, genotoxicity, and organ toxicity in animal models. The compound is for research use only.

[1]. Ester and amide derivatives of the nonsteroidal antiinflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors. J Med Chem. 2000 Jul 27;43(15):2860-70.

Indomethacrylheptaester is an N-acylindole.

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Indomethacin heptyl ester (CAS#: 282728-47-6) is a lipid-soluble derivative of indomethacin, a nonsteroidal anti-inflammatory drug. It is a non-selective inhibitor of both COX-1 and COX-2 with an IC50 of 0.04 μM for COX-2. The compound has a molecular formula of C26H30ClNO4 and molecular weight of 455.97. It is supplied as a solution in methyl acetate with ≥98% purity. Indomethacin heptyl ester exhibits anti-inflammatory activity and is used for research purposes only. CAS: 282728-47-6.

C26H30CLNO4

455.97

455.186

C, 68.49; H, 6.63; Cl, 7.77; N, 3.07; O, 14.03

282728-47-6

10389320

Typically exists as solid at room temperature

1.2±0.1 g/cm3

533.6±50.0 °C at 760 mmHg

276.5±30.1 °C

0.0±1.4 mmHg at 25°C

1.565

6.75

0

4

11

32

607

0

CCCCCCCOC(=O)CC1=C(C)N(C2=C1C=C(C=C2)OC)C(=O)C3=CC=C(C=C3)Cl

PYBCHCVNKGZCOH-UHFFFAOYSA-N

InChI=1S/C26H30ClNO4/c1-4-5-6-7-8-15-32-25(29)17-22-18(2)28(24-14-13-21(31-3)16-23(22)24)26(30)19-9-11-20(27)12-10-19/h9-14,16H,4-8,15,17H2,1-3H3

heptyl 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetate

Indomethacin heptyl ester; 282728-47-6; INDOMETHACIN ESTER, N-HEPTYL-; heptyl 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetate; CHEMBL330194; CHEBI:184056; HMS3649K19; BDBM50090775;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)