The Pharmacological Study Of A Series Of Novel Adenosine Receptor Antagonists As Anti-heart Failure Drug Candidate

Heart failure （HF） is the severe stage of various cardiac diseases on behalf of thefinal outcome of many cardiovascular diseases, such as hypertension, coronary heartdisease, cardiomyopathy and valvular heart disease. Heart failure patients havebecome the main source of patients who suffer from cardiovascular disease with highincidence rate. Moreover, the5-year survival rate of heart failure is similar tomalignant tumor. Thus, the treatment of heart failure has become a big burden notonly for global patients and doctors, but also for medical care system.The effective treatments for heart failure play a key role to reduce cardiovasculardisease morbidity and mortality rate. The routine drugs in clinical treatment of heartfailure often combine diuretics with angiotensin converting enzyme inhibitors （ACEI）and beta adrenergic receptor blockers. Diuretics play a crucial role in heart failuretreatment, which control and alleviate the symptoms of heart failure immediately.Diuretic treatment is the foundation of any other effective "biological treatment".Currently, conventional diuretics used in clinic have obvious therapeutic contradiction.They play a diuretic effect at the same time will often cause hypotension, electrolytedisturbances such as hypokalemia, reduced renal blood flow and glomerular filtrationrate （GFR）, eventually causing or aggravating kidney damage. These contradictionsseriously affect the clinical therapeutic effect on heart failure, and even threaten thepatient’s life.Therefore, development of new diuretic drugs is increasingly focusing onenhancing and protecting renal function in patients with heart failure. In recent20years, along with the study of mechanisms in heart failure, the development of newdrug with new mechanisms and strategies in treatment are possible. Adenosinereceptor （AR） antagonists represent a novel class of diuretic agent which exertseffective diuresis and natriuresis accompanied by not influences of renal function as well as electrolyte homeostasis. The innovation pharmacological mechanism ofadenosine receptor antagonists for treating heart failure is the beneficial effects inimproving renal blood flow by block the adenosine A₁receptor （A₁R）. The inhibitionof adenosine receptor do not affect renal tubule function, but directly exert beneficialeffects on the glomerular blood flow and block the tubuloglomerular feedback toimprove glomerular filtration function. Activation of adenosine A₁receptors willinduce vascular relaxation of glomerular and constriction of the afferent arteriole aswell as mediation of tubuloglomerular feedback （TGF）, which increase sodiumdelivery to the proximal tubules of the macula densa leading to glomerular filtrationrate reduction. In animal and human trials, selective A₁receptor blockade reducedthese potential harmful effects. Obvious advantages of adenosine A₁receptorantagonists include improvement of renal blood flow to enhance diuresis, keeping theglomerular filtration rate without causing urinary potassium excretion andmaintaining electrolyte homeostasis.Adenosine receptor antagonists mainly consist of xanthine and non-xanthineantagonists. Currently, most of adenosine receptor antagonists belong to xanthineantagonists. However, the restricted mother nucleus structure of xanthine adenosinereceptor antagonists greatly lead to limitation on the molecular structure diversity,affecting the pharmacokinetic properties and the improvement of selectivity effects ofdrug. The non-xanthine adenosine receptor antagonists with molecular structuraldiversity and specific tissue selective advantages have became the new trend indevelopment of the novel adenosine receptor antagonist agent.In summary, this paper was aimed to screen and evaluate pharmacologicalprofiles of a series of2-（3-fluorophenyl）-4-butyl amino pyrazolo [4,3-c] quinoline-3-ketone derivatives, in order to obtain novel non-xanthine adenosine A₁receptorantagonists with high affinity and high selectivity, as well as better pharmacokineticproperties as a new mechanism of diuretic agent.The main findings of this project are as follows:1. A series of novel non-xanthine adenosine receptor antagonists wereevaluated, some of which displayed inverse agonistic and antagonistic activity.First, we have established stably transfected human A₁R on CHO-K1cell lineand human A₂AR on HEK-293cell line in vitro. Through radioligand binding on rat brain tissues and CHO-A₁R and HEK293-A₂AR cell lines,302-（3-fluorophenyl）-4-butyl amino pyrazolo [4,3-c] quinoline-3-ketone derivatives were evaluated in total,and8candidate ligands with high affinity to A₁R were identified on10-5M and10-7M,respectively. The Kivalues for A₁R of all compounds are as follows: PQ-69（0.955nM）、 PQ-81（46.5nM）、PQ-83（0.9nM）、PQ-85（1.57nM）、PQ-86（7.07nM）、PQ-88（55.8nM）、 PQ-103（1.54nM）、 PQ-107（1.07nM）； the Kivaluesfor A₂AR were PQ-69（208nM）、PQ-81（67.8nM）、PQ-83（109nM）、PQ-85（52.6nM）、PQ-86（247nM）、PQ-88（1240nM）、PQ-103（154nM）、PQ-107（170nM）.The ratio of A₁R/A₂AR were PQ-69（217）、PQ-81（1.46）、PQ-83（120）、PQ-85（33.6）、PQ-86（35）、PQ-88（22.2）、PQ-103（99.9）、PQ-107（159） respectively. Furthermore,the affinity of these compounds were evaluated on a variety of other G-proteincoupled receptors （GPCRs） in stable cell lines established in our laboratory, findingthat these compounds had low affinity with dopamine D1, D2, D3receptor, cholineM4, M5receptor, adrenaline α1A,α1Breceptor with more than1000-fold selectivity onA₁R.Thus, compared with the reported high-affinity A₁R antagonist DPCPX, the Kivalues of PQ-69, PQ-83on A₁R were0.955and0.9nM, A₁R/A₂AR were217-foldand120-fold. PQ-69and PQ-83possessed high affinity and selectivity for A₁R overA₂AR and they expected to become the high selectivity and high affinity ligand for thefunctional research of A₁Rs. Compared with non-xanthine adenosine receptorsantagonists that reported in the literature, these compounds belonged to the class ofhigh affinity ligands.Second, using [35S]-GTPγS （guanosine-5-O-（3-[35S] thio） triphosphate） bindingassay, the antagonistic activity were evaluated. It was found that these compounds cannot activate A₁R by itself. However, these compounds displayed inverseagonistic activity by reducing basal [35S]-GTPγS binding, Using whole cell cyclicAMP （cyclic adenosine monophosphate, cAMP） assay, it was further confirmed thatPQ-69, PQ-83possessed A₁R antagonistic and inverse agonistic activity.Third, using the guinea pig tracheal strip, the antagonistic activity of PQ-69,PQ-83on antagonizing the A₁R agonist2-Chloro-N6-cyclopentyladenosine （CCPA）,was evaluated. Compared with selective A₁R antagonist1,3-dipropyl-8-cyclopentylxanthine （DPCPX）, PQ-69, PQ-83strongly antagonized the effect on CCPA inducedtracheal strip contraction. The pA₂values of PQ-69, PQ-83were8.99,9.54, respectively. Accordingly, the SchildSlope values were1.3,1.1, respectively. It is alsofound that PQ-69concentration-dependently relaxed tonic contractions of guinea-pigtracheal strips （10-10-10-6M）. This indicates that PQ-69and PQ-83represented potentcompetitive antagonists in vivo.2. PQ-69and PQ-83showed better pharmacokinetic profiles compared withselective A₁R antagonist DPCPX.Early pharmacokinetic properties obtained from software prediction showed that,the solubility and absorption properties of PQ compounds were good. We then furtherevaluated the pharmacokinetics of PQ-69, PQ-83and compared them withDPCPX.PQ-69displayed longer terminal elimination half life （t1/2） with7.92hrcompared with2.36hr of DPCPX. In contrast, PQ-69displayed better metabolicstability and broader tissue distribution than those of DPCPX. Therefore, the largetissue distribution balanced the slow metabolism. The difference of liver metabolismwas also evaluated using in vitro microsomal stability assay. After60min incubationin rat liver microsomes, the percentages of remaining parent drugs were significantlydifferent between PQ-69（73.5±2.6%） and DPCPX （30.1±3.4%）. Therefore, we haveshown that PQ-69displayed better metabolic stability in rat liver microsomes thanDPCPX. The absorptive permeability （Papp） of PQ-69was measured using Caco-2transport assay. The results indicated that both PQ-69and DPCPX had a moderatepermeability across epithelial cells.3. PQ-69showed low toxicity by evaluation of acute toxicity test.Oral administration of PQ-69（2100mg/kg） did not lead to death in both maleand female mice.4. PQ-69showed potent diuresis in normal rats and protective effect againstrenal function in cisplatin induced renal failure rats.In normal rats, PQ-69（30mg/kg, p.o.） could significantly increase the urinevolume of rats （P <0.001）, PQ-69（10mg/kg, p.o.） could significantly increase thesodium excretion in rats （P <0.05）. PQ-69（0.03,0.3,1,3mg/kg, i.v.） candose-dependently increased urine volume. PQ-69（0.3mg/kg, i.v., P <0.001）possessed equal diuretic effect with hydrochlorothiazide （HCTZ）（1mg/kg, i.v., P < 0.001） and DPCPX （1mg/kg, i.v., P <0.001）. PQ-69（0.03,0.3,1,3mg/kg, i.v.）significantly increased the sodium and chlorine excretion. Intravenous and oraladministration has no effect on potassium excretion, suggesting that PQ-69played thediuretic effect with increasing sodium excretion and chlorine ion excretion andwithout affecting on potassium ion excretion, maintaining the balance of electrolyte.In acute renal failure rats induced by cisplatin （5mg/kg, i.v.）, PQ-69（10,30mg/kg, p.o.） increased urine volume compared with the saline group. PQ-69alsoincreased creatinine clearance rate to saline group level, suggesting that PQ-69showed better diuretic and renal function protective effect.In acute renal failure rats induced by cisplatin （4mg/kg, i.v.）, PQ-69（0.01,0.1,1mg/kg, i.v） can dose-dependently increased urine volume and sodium, chlorineexcretion PQ-69（0.1mg/kg, P <0.05） significantly increased creatinine clearancerate when compared to the solvent model group, suggesting that PQ-69playeddiuretic action accompied by renal protective effect. Intravenous and oraladministration of PQ-69had no effect on potassium metabolism, suggesting thatPQ-69play a diuretic effect without affect potassium excretion to maintaining thebalance of electrolyte. Immunohistochemistry in acute renal failure rats induced bycisplatin （4mg/kg, i.v.）: The pathological damage was not very obvious. PQ-69（0.1mg/kg, i.v） can improve the thinning renal cortex and swelling proximal tubularepithelial cell injury in which the effect is better than that of DPCPX （0.1mg/kg, i.v.）.In accord with the histological observation demonstrating the cortex wereevidently affected accompanied by tubular dilation, protein cast in the kidney ofcis-induced renal failure rats compared with that in normal rats. However,glomerulous medulla and papilla were histologically intact in the kidney ofcis-induced renal failure rats. PQ-69attenuated the damage of renal function withoutaffecting on the cortex in the kidney. Renal mRNA expression and A₁R receptorexpression were detected by Q-PCR assay and western botting assay on Cisplatin-induced renal failure rat. Cisplatin （4mg/kg, i.v.） significantly increased A₁R （P <0.001） and A₂AR （P <0.05） mRNA expression. PQ-69（0.1mg/kg, i.v.） had no effecton the increased expression of A₁R. Cisplatin （4mg/kg, i.v.） increased the proteincontent of A₁R and A₂AR. PQ-69（0.1mg/kg, i.v.） did not affect the expression ofA₁R.In conclusion, a series of2-（3-fluorophenyl）-4-butyl amino pyrazolo [4,3-c]quinoline-3-ketone derivatives were screened and the pharmacological profiles of PQ-69were evaluated through various in vivo and in vitro experiments. PQ-69is oneof the most potent and selective non-xanthine A₁R antagonist yet described. Theselectivity profile of PQ-69is superior to that of DPCPX, which was A₁vs A₂Aselectivity of52-fold compared to217-fold human A₁R selectivity for PQ-69.Moreover, PQ-69displayed more potent inverse agonistic activity for A₁R thanDPCPX. We have demonstrated that PQ-69has similar diuretic pharmacologicalproperties to that of thiazide diuretics and DPCPX under normal condition. Thus,PQ-69represents a novel, potent and selective non-xanthine A₁R antagonist withpronounced inverse agonist activity and in vivo bioactivity. It will be useful tool tofunctionally characterize physiological functions and complicated nature inpathological condition in relation to responses mediated by A₁R.