Discovery of Quinic Acid Derivatives as Oral Anti-inflammatory Agents
Quinic acid (QA) esters found in hot water extracts of Uncaria tomentosa (a.k.a. Cat’s claw) exert anti-inflammatory activity through mechanisms involving inhibition of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB). Herein, we described the synthesis and biological testing of novel QA derivatives. Inhibition of NF-κB was assessed using A549 (Type II alveolar epithelial-like) cells that stably express a secreted alkaline phosphatase (SEAP) reporter driven by an NF-κB response element. A549- NF-κB cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of QA derivative for 18 hours followed by measurement of SEAP activity. Amide substitution at the carboxylic acid position yielded potent inhibitors of NF-κB. A variety of modifications to the amide substitution were tolerated with the N-propyl amide derivative 3 being the most potent. Compound 3 was named as KZ-41. The NF-kB inhibitory potency (IC50) of our most active analog KZ-41 was determined to be 2.83±1.76 mM. Further examination of the structure and activity relationship (SAR) demonstrated that acetylation of the hydroxyl groups reduced NF-κB inhibitory activity. QA amide derivatives lacked anti-oxidant activity and were found to be neither anti-proliferative nor cytotoxic at concentrations up to 100 mM. The dehydroxyl QA amides 18 and 20 were synthesized to retain anti-inflammatory activity while having enhanced resistance to microbial degradation. They showed NF-kB inhibition at concentration 1 mM. The extent of NF-kB inhibition was close to positive control drug dexamethasone. The dehydroxyl QA amide with a double bond 38 was also synthesized. The QA amide esters 24, 25, 29 and 33 were designed and synthesized to retain anti-inflammatory activity with additional antioxidant properties. As expected, compound 25 showed strong anti-oxidant activity. Furthermore, they were hydrolyzed by the microflora, for an example, 25 was hydrolyzed into sinapic acid (SA) and 3. Both SA and 3 will not be consumed by gut bacteria, and are easily absorbed in animal digestive tract. This was done by other researchers, and not reported here. To facilitate pre-clinical biopharmaceutic and pharmacokinetic (B/PK) studies of our lead QA amide analog KZ-41, we developed and validated a novel hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) analytical assay. An analog of KZ-41 was used as internal standard (IS). KZ-41 and the IS were obtained by protein precipitation and separated by HILIC chromatography using acetonitrile and water. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect KZ-41 and IS transitions of m/z 232 → 178 and 272 → 218 respectively. The lower limit of quantification (LLOQ) was 0.5 ng/mL in plasma. The method was validated for selectivity, linearity, accuracy and precision in rat plasma. The ion suppression, recovery and stability of the analyte in the biological matrix were also tested. The assay developed is rapid, sensitive and robust enough to support preclinical B/PK studies of KZ-41. The study was also conducted to characterize the biopharmaceutics and pre-clinical pharmacokinetics of the lead QA amide analog, KZ-41. Rats (n=6/group) received a dose of either an i.v. or p.o. dose (10 mg/kg) of KZ-41. Pharmacokinetic parameters were determined from concentration - time profiles by non-compartmental analysis. Bacterial stability study was conducted in cultured bacterial gluconobacter oxydans. Plasma protein binding and metabolic stability were determined using equilibrium dialysis and rat liver microsomes respectively. Following i.v. administration, KZ-41 demonstrated a medium clearance (15.1±4.8 mL/min/kg), medium volume of distribution (3.3±1.1 L/kg), and a terminal half-life of 2.6±0.4 hrs. KZ-41 was rapidly absorbed with complete oral bioavailability (F≈1), which was consistent with the fact that KZ-41 was not susceptible to degradation in bacterial and liver microsomal studies. KZ-41 binding to plasma proteins was about 30%. These studies demonstrate that KZ-41 is a potential new orally active anti-inflammatory agent. In summary, we have discovered a novel series of non-toxic QA amides that potently inhibit NF-κB. The NF-kB inhibitory potency (IC50) of our most active analog KZ-41 was determined as 2.83±1.76 mM. It was rapidly absorbed with complete oral bioavailability (F≈1). It could be a potential new orally active anti-inflammatory agent. Mechanistic studies and pre-clinical efficacy studies of these newly designed compounds in various in vitro and in vivo models are on-going by other researchers, and are not reported in this dissertation.
anti-inflammation, orally active drug, quinic acid derivatives, Alternative and Complementary Medicine, Chemicals and Drugs, Medicinal and Pharmaceutical Chemistry, Medicine and Health Sciences, Natural Products Chemistry and Pharmacognosy, Pharmaceutical Preparations, Pharmaceutics and Drug Design, Pharmacy and Pharmaceutical Sciences
University of Tennessee Health Science Center
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