Development of acetaminophen proline prodrug

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 13, 2010, Pages 3851-3854

  Wu, Zhiqian ^a   Patel, Ashish ^a   Dave, Rutesh ^a   Yuan, Xudong ^a  

^a LONG ISLAND UNIVERSITY   (United States)

Author keywords

Acetaminophen; Carboxypeptidase A; Pro APAP; Prodrug; Proline

Indexed keywords

CARBOXYPEPTIDASE A; ESTER DERIVATIVE; PARACETAMOL DERIVATIVE; PHOSPHATE BUFFERED SALINE; PRODRUG; PROLINE N ACETYL 4 AMINOPHENOL; UNCLASSIFIED DRUG;

ARTICLE; CELL FREE SYSTEM; CHEMICAL BOND; CONTROLLED STUDY; DIFFERENTIAL SCANNING CALORIMETRY; DRUG HALF LIFE; DRUG HYDROLYSIS; DRUG SCREENING; DRUG STABILITY; DRUG SYNTHESIS; HUMAN; HUMAN CELL; MELTING POINT; PH; PHYSICAL CHEMISTRY;

ACETAMINOPHEN; CACO-2 CELLS; HUMANS; HYDROGEN-ION CONCENTRATION; HYDROLYSIS; MOLECULAR STRUCTURE; PRODRUGS; PROLINE;

EID: 77954309208     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.05.050     Document Type: Article

References (19)

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13. 4, filtered, and concentrated under vacuum. Crude compounds were separated using column chromatography (hexane/ethylacetate, 10:1). The fractions were collected and analyzed by thin layer chromatography (TLC) for purity. Fractions from each spot were concentrated under vacuum separately. The Boc group was removed by treating the residues with 5 mL of trifluoroacetic acid (TFA) and 5 mL of dichloromethane (DCM) for 4 h. After evaporating DCM and most TFA (95%), cold ether was added to precipitate out the pure compounds. After removing ether, the residues were reconstituted with water and lyophilized. White powder was obtained as final product in TFA salt form.
14. +.
15. The hydrolysis study was carried out in 96-well plates. PBS buffer (230 μL) was placed in triplicate wells and the reactions were started with the addition of acetaminophen prodrug and incubated at 37 °C. At various time points, 40 μL aliquots were removed and added to 40 μL of 10% ice-cold TFA. The mixtures were centrifuged and filtered through a 0.45 μm filter for 10 min at 2000 rcf and 4 °C. The filtrate was then analyzed by HPLC.
16. 2 and 90% relative humidity. Caco-2 cells were cultured in 80% minimum essential medium (MEM) with 20% FBS. Split confluent culture 1:4 to 1:6 every 3-5 days using trypsin/EDTA. Cell homogenate were prepared when the cells were 90% confluent. After trypsinization, the cells were washed three times with pH 7.4 PBS buffer and re-suspended in pH 7.4 PBS (10 mM). To prepare cell homogenate, 1% Triton-X 100 was added in PBS solution and vortexed vigorously. The cell suspension was centrifuged at 18,000 rpm for 30 min at 4 °C. The supernatant was used in hydrolysis study and to determine protein content. Total protein was quantified with the BioRad Protein Assay using bovine serum albumin as standard. The protein content was adjusted to approximately 1000 μg/mL by appropriate dilutions before being used in hydrolysis studies.
17. MDSC Q100 series was used in this study to measure melting points of acetaminophen and Pro-APAP. MDSC test of Pro-APAP was repeated up to 3 months to see the stability of the prodrug. About 4-6 mg powder was sealed in aluminum hermetic pan with lid and loaded on MDSC to run this experiment. Melting point of Boc-l-proline was also determined. The following is the procedure for conducting MDSC thermogram of these samples: (1) Data storage: Off; (2) Equilibrate at 20.00 °C; (3) Data storage: On; (4) Ramp 10.00 °C/min to 250.00 °C; (5) End of method. In Step 4, maximum temperature was modified as per the known theoretical melting point of sample to be analyzed. Boc-l-proline has melting point of near to 135 °C, so in this case maximum temperature was set at 200.00 °C.
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