Design, Synthesis And Functional Screening Of Modulators Of KCNQ2/3 Potassium Channels And CaCCs, And Optimization Of Synthesis Conditions For High Activity Compounds

Ion channels in the cell membrane are an important class of membrane proteins regulating cell excitability, and are also the most important drug targets next to the G protein-coupled receptor. Among ion channels, potassium channels have the largest family with many members and subtypes, and have complex functions with great significance in pathology, physiology and pharmacology. The Kv7/KCNQ ion channels belong to the voltage-gated potassium channel superfamily. There are to date five known members,KCNQ1-KCNQ5. KCNQ2/3 heterotetramers are believed to underlie the molecular basis of M-type K+ currents found in neuronal system. Genetic,physiological and pharmacological evidence now exist to support a role for KCNQ channels in controlling of neuronal excitability, suggesting that activation of KCNQ2/3 channels（M channel） might be useful in potential treatment of hyperexcitability-related neuronal disorders, such as hyperspasmia, epilepsy, neuropathic pain. Now, M channel has become one of the most studied targets for development of new drugs.The Ca²⁺ activated Cl- channels（CaCCs） play a variety of physiological roles in many organs and tissues, including transduction of sensory stimulation,regulation of neuronal and cardiac excitability, contraction of smooth muscle,fertilization, and transepithelial Cl- secretion. CaCCs have become the study focus of many scientists since TMEM16 A, a member of the transmembrane protein 16 family, was identified as an important CaCCs subunit. With emerging exciting results of recent studies, TMEM16 A is becoming a new drug target for treatment of diseases such as cystic fiber disease, hypertension,gastrointestinal motility disorder, asthma, tumor, and others.In this project, we determined to identify novel specific modulators ofKv7/M channel and CaCCs, and to establish high throughput screening（HTS）system for modulators of Kv7/M channels and CaCCs channels. The identification of new modulators of these channels is important for further understanding of the basic properties of the channels as well as development of new drug targeting these channels. For this, we designed and synthesized a large number of compounds aiming to be the opners of Kv7/M channel and the blockers of CaCCs channels; we also established and optimized the high throughput screening systems for modulators of M channels and CaCCs channels, using the system of ICR8000, an atomic absorption equipment with automatic sampler. Through high throughput screening of the compounds we synthesized, we identified multiple compounds with high activity for modulation of KCNQ/M channel and CaCCs channels. Furthermore, the synthesis routes for the identified lead compound of the M channel opener were optimized.Part one Design, synthesis, functional screening and structure activity relationship study of pyrazolo[1,5-a]pyrimidin-7（4H）-ones as novel KCNQ2/3 potassium channel activatorsObjective: To design and synthesize a series of pyrazolo[1,5-a]pyrimidin-7（4H）-ones（PPOs） compounds, to screen KCNQ2/3（M） channel openers by high throughput screening（HTS） system, and to perform the structure activity relationship study（SARs） for all synthesized compounds.Methods: â‘  Substitutive 5-amino pyrazoles are important intermediates,which can be prepared through three synthetic routes, two step of condensation and cyclizationreactions, starting from various commercially available substituted acetonitrile as raw material. The synthesis of a library of target PPOs was accomplished via microcrystalline parallel synthesis by reaction of various 5-amino pyrazoles intermediates with respectiveβ-ketoester in acetic acid at reflux. â‘¡ Structural modifications for compounds26 and 58 were done by methylation of 4-position imino and chlorine substitution of 7-position carbonyl. â‘¢ Chinese hamster ovary（CHO） cellsstably expressing KCNQ2/Q3 channels were seeded in 96-well plates at a density of 2 × 105 cells per well. In experimental progress of screening for KCNQ openers, seven concentration gradients of test compounds（100 μM, 30μM, 10 μM, 3 μM, 1 μM, 0.3 μM and 0.1 μM） were set, and each concentration was assayed in triplicate. Retigabine（RTG）, the known KCNQ/M channel opener, was used as a positive control to validate the HTS method of Rb+ efflux assay.Results: About 120 PPOs compounds and 6 derivates of compounds 26 and 58 were synthesized, including more than 110 compounds with novel structures. All synthesized compounds were evaluated for modulation of KCNQ2/Q3 channel using the same Rb+ efflux assay, and preliminary SARs were finished successfully. Retigabine increased Rb+ efflux by activating KCNQ2/Q3 channel stably expressed in CHO cells, with an EC50 in the range of 0.30- 0.50 μM. EC50 values of lead compounds 26 and 28 are 2.27 ± 1.02μM and 1.46 ± 0.36 μM, respectively. The preferred compounds 58（QO-58）and 60 by SARs showed similar efficacy when compared with retigabine, with EC50 values of 0.06 ± 0.01 μM and 0.15 ± 0.02 μM, respectively. The EC50 value of compound 58 for KCNQ1 open activity is 49.29 ± 7.04 mM.Conclusion: Most of synthesized compounds are new compounds with novel structures. The structures of all active compounds from HTS were identified by HPLC-MS and NMR, and purities were detected by HPLC-DAD.The compound 58 which was produced from structural optimization of lead compounds, showed stronger potency of and similar efficacy in activating KCNQ2/Q3 channel in comparison with retigabine. Compound 58 showed lower potency in activating KCNQ1 channels. Thus compound 58 is a novel,high active and selective KCNQ/M channel opener. PPOs mother nucleus compounds have been protected by China patent and Patent Cooperation Treaty（PCT）,and the petition for America patent is under reviewing. The present results suggest that the PPOs as a new class of KCNQ/M channel openers have great potential for further investigation for their possible treatment of diseased conditions such as epilepsy and neuropathic pain.Part two Optimization of synthesis conditions for QO-58LObjective: To synthesize QO-58 salt（Lysine, QO-58L）, and to optimize the synthesis conditions for QO-58 and QO-58 L according to the standard of new drug preclinical studies; to supply sufficient amounts of above compounds for pharmacology and toxicology studies.Method: â‘  The first step reaction（synthesis of sodium salt）:Compared the results of using freshly prepared ethylate sodium with the commercial industrial grade ethylate sodium through single factor experiments,and optimized reaction conditions through L9（34） orthogonal experiment. â‘¡The second step reaction（synthesis of substitutive 5-amino pyrazoles）: Effects of trifluoroacetic acid and molecular sieve were envaluated by single factor experiments with HPLC online monitoring, and reaction time and reaction temperature were determined by the same process. â‘¢ The third step reaction（cyclization of QO-58）: Envaluated the effects of reaction time and solvent volume, and optimized the processing of reaction products. â‘£ The fourth step reaction（synthesis of QO-58L）: Select the solvent and its amoumt for QO-58 salt（lysine） forming reaction. Scale up the optimized synthesis conditions for 10 times.Result: â‘  2-Phenylacetonitrile was added to a fresh solvent of Na in absolute alcohol, keeping 60℃ for 5 min, and then ethyl trifluoroacetate（Na :2-phenylacetonitrile : trifluoroacetate = 1 : 1 : 1.2, equiv） was added dropwise at the same temperature. After dropping, the mixture of reaction was slightly refluxed for 2 h. After this period, the reaction product was cooled to room temperature and solvent was removed under reduced pressure. The solid was dissolved in water, washed by CH2Cl2 four times, and then the solution was concentrated to dryness under reduced pressure. â‘¡ A mixture of intermediate2, hydrazine sulfate（intermediate 2 : hydrazine = 1 : 2, equiv）, 3A molecular sieve and dimethyl carbonate（DMC） was slightly refluxed for 3.5 h. The solid was filtered off under reduced pressure and washed with small amount DMC.The solvent of filtrate was reclaimed for reuse under reduced pressure, to obtain intermediate 3, which was used in the next step without furtherpurification. â‘¢ Intermediate 3（all the previous step product without further purification） and ethyl 3-（2,6-dichloro-5-fluoropyridin-3-yl）-3-oxopropanoate（1 : 1, equiv） were dissolved in acetic acid, and refluxed for 4 h. After this period, part of solvent was removed under reduced pressure, and then cooled to room temperature for filtering. The filter cake was washed to pH 7 with acetic acid and water in order, and absolute alcohol was added subsequently to reflux for 10 min. The mixture was sufficiently cooled and filtered. Filter cake was washed by water and dried under vacuum. â‘£ L-lysine and QO-58（1 : 1）were dissolved in 95% hot ethanol（20 folds of QO-58 weighs）, filtered under reduced pressure, and solvent was reclaimed to gain QO-58 L with an overall yield about 35%.Conclusion: The optimized synthesis condition is easy to operate, has minimum requirement for device requirements and no toxic solvents is involved. Furthermore, the raw materials are cheap and easy to get. This synthesis process is fully in accordance with the evaluation criteria for synthesis process of the new drugs. Stability and reliability of the optimized conditions were confirmed by enlarged verification test. Purity of QO-58 and QO-58 L synthesized by this technology can meet basic quality requirements for bulk drugs.Part three Design, synthesis and functional screening of modulators for the Ca²⁺ activated Cl- channelsObjective: To design and synthesize three series of CaCCs blockers; to establish high throughput screening technology for CaCCs modulators and to screen all synthesized compounds.Methods:（1） Synthesis of carboxylic acid derivates: Some analogs of anthranilate class NSAIDs were used to produce the derivates by condensation reaction with various amines or alcohols in the presence of hydrochloric acid EDC and DMAP. Synthesis of celecoxib analogues: The substituted acetophenone was used for condensation reaction with ethyl trifluoroacetate in the presence of sodium methylate, and was subsequently cyclized withsubstituted hydrazinium. Synthesis of carboxylic acid compounds:Halogenated aromatic acid or chlorinated aromatic estesr were used for nucleophilic substitution reaction with substituted aniline in various conditions;Schiff bases from amino aromatic acid by reaction with substituted benzaldehyde were reduced by sodium cyanoborohydride.（2） Establishment of HTS system for CaCCs: Analytical method validation for ICR8000 detecting Ag+（Including linearity, precision and accuracy） was performed; the influence on chloride efflux（Cl- concentration） by various factors, including cell density, reaction time, buffer composition and so on was envaluated, to determine the optimal assay conditions. Positive control of CaCC blockers were used for method validation of the established HTS system.（3） The synthesized compounds were screened by the newly established HTS system first with three concentration gradients（300 μM, 100 μM and 30 μM）, and concentration-response curves of active compounds were drawn by adding more concentration gradients.Results:（1） Total 58 compounds（including 38 carboxylic acid derivates,10 celecoxib analogues and 10 carboxylic acid compounds） were synthesized.（2） HTS system for screening of CaCC blockers was established successfully:The standard curve showed good linearity（r2 = 0.9979）; intra-day and inter-day precision were less than 5.68% and 6.15%, respectively, and accuracy error were all less 20%. Thus the method using ICR8000 for Ag+（indirect indicator of Cl-） detection was accurate and sensitive, and the method can can be used for the screening. Screening Process: â‘  Cell incubation: The F-12 K medium which has been used to incubate CHOs which has TMEM16A（CaCC） overexpressed was removed quickly, and 200 μl incubation buffer(containing Eact 25 μM,KCl 150 mM,Ca²⁺ 4 mM and test compound) was added, and was then placed for 15 min at 37℃. â‘¡ Wash: the incubation buffer was revmoved and washed with wash buffer（200 μl×3） quickly and carefully. â‘¢ Activation of CaCC（TMEM16A） channel:After wash, 200 μl open buffer(Containing Ionomycin 10 μM, K gluconate 100 mM and Ca²⁺ 4mM) was added and then placed for 12 min at 37℃. â‘£ Measureing of Ag+:190 μl open buffer from the step 3 was transferred to another 96-well plates,and 30 μl AgNO3（50 ppm） was added in each well and mixed sufficiently.The mixture was placed for 24 h at the condition of dark and still. The next day, 100 μl supernatant was drawn into ICR8000 to measure Ag+concentration and next to calculate Cl- efflux.（3） Screening using the new HTS system for CaCCs blockers. Positive control drugs included NPPB,CaCCinh-A01（C38） and flufenamic acid（Flu） which all showed good concentration-dependent effects. Inhibition ratio of NPPB and CaCCinh-A01 for TMEM16A are both 100% at 300 μM, with IC50 values of 39.35±4.72μM and 6.35±0.27 μM, respectively. The summary of inhibitory activities of synthesized carboxylic acid compounds showed C56 > C58 > C49 ≈ C50 >C54 > C57, and celecoxib analogues（C39-C42）, carboxylic acid derivates（C15, C31 and C34） showed stronger inhibitory activities.Conclusion: All synthesized compounds had high purities measured by HPLC-DAD, and were properly used for HTS. The newly established HTS system for CaCCs modulator has the features of high-throughput, low consumption, high efficiency, high sensitivity and accuracy. We identified some active compounds in all of three designed series of compounds. These active compounds can be used as the lead compounds for further development of CaCC modulators, which has great pharmacological significance.