Design, Synthesis And Biological Activity Of Piperazine α_1A Adrenoceptor Antagonists

Background:Benign prostatic hyperplasia （BPH） is a common and frequently-occurring diseaseamong middle-aged men. It was usually accompanied with the symptoms of frequenturination, urinary urgency, urinary retention and other lower urinary tract symptomson clinical, which must affect the quality of life. Thus, BPH has become a publichealth issue of global concern. More and more selective α₁-AR antagonists have usedin the treatment of BPH. The bladder neck, prostatic capsule and the smooth muscleprostatic stromal hyperplasia mainly contained the α₁-AR, the vast majority was α_1Aand α₁Dsubtype. The selective α_1A/1Dsubtype antagonists have become an effectivemethod to treat BPH. The traditional antagonist used in BPH would produceorthostatic hypotension and other serious side effects, so, avoiding blocking of theα₁B-AR would help to reduce the cardiovascular side effects. Therefore, the researchfocus of current design on α₁-AR antagonists for BPH was how to improve theselectivity of α₁-AR subtypes, that was to super-selectively block α_1Aand α₁Dand toavoid or reduce the block on α₁Bsubtype. The clinical application of super-selective α₁-AR antagonists were only a few drugs, such as tamsulosin, naftopidil and so on.Such super-selective α₁-AR antagonists have become the new hot focus because oftheir high therapeutic value, lower adverse reactions and lower side effects withpotential applications.Research contents:This paper was around with finding a candidate drug for the treatment of benignprostatic hyperplasia, the work was done as follows:1. Using the synthetic method of chiral source, the indole derivatives andbenzotriazole derivatives, named HJ-1^HJ-6, eighteen α₁-receptor antagonists,including twelve optical isomers and six racemics were synthesized. These18compounds were carried out on the dual luciferase reporter gene activity screeningtest. The two groups of compounds, named HJ-1-R, the HJ-1-rac, HJ-1-S, HJ-4-R,HJ-4-rac, HJ-4-S were then carried through the in vitro isolated rat muscle stripsbiological functional assays, which could study the chiral pharmacology and explorewhether the chiral factor will effect the α₁subtype receptor antagonism andsubtype-selectivity.2. Refering to the extensive literature, combined with computer-aided drug designmodule, according to the design principles of skeleton replacement andbio-electronics groups, a series of indole new amide structure of α₁-receptorantagonist compounds HJ-Z-1^HJ-Z-11were synthesized. Dual-luciferase reportergene activity assay was screened on the synthetic compounds, the structure-activityrelationship was summarized, which could provide the basis for further design andsynthesis of newα₁subtype receptor antagonists for the treatment of benign prostatichyperplasia.3. From the level of tissues and organs, the biological functional assays wassuccessfully established for screening the α₁subtype receptor antagonist activity.Through measuring the relaxation effect of every compound on norepinephrineinduced different rats organizations (vas deferens: α_1A, spleen: α₁B, aorta: of α₁D)contraction, based on the classic Schild Plot method using Graphpad Prism5.0 software to calculate the pA2values of every compound on every subtype receptor.The aim was to compare activity differences and selectivity differences among thetarget compounds and two isomers.4. The separation method of racemic compounds of aryl piperazines on supercriticalfluid chromatography （SFC） was established. This method was superior tohigh-performance liquid chromatography （HPLC）. The effect of three kinds of chiralstationary phase and six kinds of organic modifier, the column temperature, backpressure, and the proportion of modifier was described, which provided the basis forfurther establishment of semi-preparative.Reaearch results:1. Indole derivatives and benzotriazole derivatives, together eighteen compoundswere identified through mass spectrometry,1H NMR,13C NMR, IR and chiral HPLC.The purity was greater than99%and ee value was85-99%. Through thedual-luciferase reporter gene testing of18compounds, compounds except HJ-3-R, theHJ-4-R, HJ-4-S, other’s activity was higher than the positive control drug prazosin.Comparing the differences between three configurations, it could be found that theorder of antagonistic activity targeting α_1Ain the indole structures was Sconfiguration> rac configuration> R-configuration, ortho-methoxy substituted≈ortho-ethoxy substituted> ortho-chlorine substituted. The three configurations ofortho-methoxy substituted benzotriazole compounds had large effort on theantagonistic activity targeting α_1A, and the activity of enantiomers was weaker thanthe racemic. Otherwise, the three configurations of the ortho-ethoxy substituted andortho-chlorine substituted showed small difference on the antagonistic activitytargeting α_1A. This fully exhibited the pharmacological effects of differentenantiomers of chiral compounds. In the process of studying the chiral compounds,the activity differences between enantiomers and racemic must be in-depth study.2. New amide compounds named HJ-Z were identified through mass spectrometry,1H NMR,13C NMR, infrared. The purity was greater than99%. By the dual luciferasereporter gene testing, all compounds except HJ-Z-10（5-chloro-2-carboxylic acid substituent） and HJ-Z-1（2-carboxylic acid substituent） had higher antagonisticactivity of α_1Athan prazosin. Comparing of length of carbon, the longer length, thehigher activity: HJ-Z-5（3-butyl carboxylic acid substituent）> HJ-Z-4（3-propylcarboxylic acid derivative）> HJ-Z-3（3-ethyl carboxylic acid substituent）> HJ-Z-2（5-carboxylic acid substituent）> HJ-Z-1（2-carboxylic acid substituent）.5and7-position of the right side of indole group should not introduce electron-withdrawinggroups （HJ-Z-10）, while the introduction of electron-donating group （HJ-Z-6andHJ-Z-7） on5-position could strengthen the activity. The6-position of indolyl groupcould introduction electron withdrawing groups （HJ-Z-9）.3. From the level of tissues and organs, the biological functional assays wassuccessfully established for screening the α₁subtype receptor antagonist activity. ThepA2value of positive control drug terazosin was close with the literature valuethrough this test, which further confirmed the stability and feasibility of this method.Two groups of compounds, named HJ-1-R, the HJ-1-rac, the HJ-1-S, the HJ-4-R, theHJ-4-rac, the HJ-4-S were selected on screening their biological activity. It was foundthat the six compounds were stronger or similar to the positive control drug naftopidil.HJ-4-S had the weakest antagonism activity. The indole ring structures had strongerpharmacological activity than benzotriazole derivatives. Rac configuration of theindole derivatives had better α_1A/1Dselectivity than other two configurations. HJ-1-racexhibited6.46-and16.22-fold more potent α₁Dand α_1Arelative to the α₁Bsubtype.The three kinds of configurations of the benzotriazole had certain subtype selectivity,among them, rac configuration had the weakest selectivity, while the R configurationexhibited11.48-and15.49-fold more potent α₁Dand α_1Arelative to the α₁Bsubtype.Thus, the compounds HJ-1-rac and HJ-4-R are two potential α_1A/1Dantagonists, withhigher pharmacological activity than NAF, and similar α_1A/1Dsubtype selectivity.4. The separation method of racemic compounds of aryl piperazines on supercriticalfluid chromatography （SFC） was established. The effect of three kinds of chiralstationary phase （Chiralpak IA, Chiralpak IB, Chiralpak IC column） and six kinds oforganic modifier （methanol, ethanol, isopropyl alcohol, tetrahydrofuran, acetonitrile,dichloromethane）, the column temperature （20-40°C）, back pressure （100-200bar）, and the proportion （20%-35%） of modifier was described. The ultimately optimumseparation conditions was as follows: mobile phase CO2: isopropanol: diethylamine=65:35:0.1, the column temperature35°C, back pressure120bar, and detectionwavelength of283nm, and a flow rate of3mL/min, the resolution factors of allcompounds were greater than4.00. Compared with high-performance liquidchromatography （HPLC） under the same conditions, it was found that SFC separationcould give shorter elution time and higher enantioselectivity and resolution. The SFCmethod could be used in monitoring for the asymmetric chiral synthesis reaction ofthis type, and the measuring of ee values of chiral compounds. Next step, thesemi-preparative method of enantiomers will be established on this basis, which couldprovide more samples for further bioassay experiments.