Design, synthesis, and biological evaluation of N-(3-cyano-1H-indol-5/6-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamides and 5-(6-oxo-1,6-dihydropyrimidin-2-yl)-1H-indole-3-carbonitriles as novel xanthine oxidase inhibitors.

Xanthine oxidase (XO) has been an important target for the treatment of hyperuricemia and gout. The analysis of potential interactions of pyrimidinone and 3-cyano indole pharmacophores present in the corresponding reported XO inhibitors with parts of the XO active pocket indicated that they both can be used as effective fragments for the fragment-based design of nonpurine XO inhibitors. In this paper, we adopted the fragment-based drug design strategy to link the two fragments with an amide bond to design the type 1 compounds 13a-13w,14c, 14d, 14f, 14g, 14j, 14k, and 15g. Compound 13g displayed an evident XO inhibitory potency (IC50 = 0.16 µM), which was 52.3-fold higher than that of allopurinol (IC50 = 8.37 µM). For comparison, type 2 compounds 5-(6-oxo-1,6-dihydropyrimidin-2-yl)-1H-indole-3-carbonitriles (25c-25g) were also designed by linking the two fragments with a single bond directly. The results showed that compound 25c from the latter series displayed the best inhibitory potency (IC50 = 0.085 µM), and it was 98.5-fold stronger than that of allopurinol (IC50 = 8.37 µM). These results suggested that amide and single bonds were applicable for linking the two fragments together to obtain potent nonpurine XO inhibitors. The structure-activity relationship results revealed that hydrophobic groups at N-atom of the indole moiety were indispensable for the improvement of the inhibitory potency in vitro against XO. In addition, enzyme kinetics studies suggested that compounds 13g and 25c, as the most promising XO inhibitors for the two types of target compounds, acted as mixed-type inhibitors for XO. Moreover, molecular modeling studies suggested that the pyrimidinone and indole moieties of the target compounds could interact well with key amino acid residues in the active pocket of XO. Furthermore, in vivo hypouricemic effect demonstrated that compounds 13g and 25c could effectively reduce serum uric acid levels at an oral dose of 10 mg/kg. Therefore, compounds 13g and 25c could be potential and efficacious agents for the treatment of hyperuricemia and gout.

Design, synthesis and biological evaluation of novel FXIa inhibitors with 2-phenyl-1H-imidazole-5-carboxamide moiety as P1 fragment.

Factor XIa, as a blood coagulation enzyme, amplifies the generation of the last enzyme thrombin in the blood coagulation cascade. It was proved that direct inhibition of factor XIa could reduce pathologic thrombus formation without an enhanced risk of bleeding. WSJ-557, a nonpurine imidazole-based xanthine oxidase inhibitor in our previous reports, could delay blood coagulation during its animal experiments, which prompted us to investigate its action mechanism. Subsequently, during the exploration of the action mechanism, it was found that WSJ-557 exhibited weak in vitro factor XIa binding affinity. Under the guide of molecular modeling, we adopted molecular hybridization strategy to develop novel factor XIa inhibitors with WSJ-557 as an initial compound. This led to the identification of the most potent compound 44g with a Ki value of 0.009 µM, which was close to that of BMS-724296 (Ki = 0.0015 µM). Additionally, serine protease selectivity study indicated that compound 44g display a desired selectivity, more 400-fold than those of thrombin, factor VIIa and factor Xa in coagulation cascade. Moreover, enzyme kinetics studies suggested that the representative compound 44g acted as a competitive-type inhibitor for FXIa, and molecular modeling revealed that it could tightly bind to the S1, S1' and S2' pockets of factor XIa. Furthermore, in vivo efficacy in the rabbit arteriovenous shunt model suggested that compound 44g demonstrated dose-dependent antithrombotic efficacy. Therefore, these results supported that compound 44g could be a potential and efficacious agent for the treatment of thrombotic diseases.

Synthesis and biological evaluation of 2-(4-alkoxy-3-cyano)phenylpyrimidine derivatives with 4-amino or 4-hydroxy as a pharmacophore element binding with xanthine oxidase active site.

Xanthine oxidase is the rate-limiting enzyme critical for the synthesis of uric acid, and therefore xanthine oxidase inhibitors are considered as one of the promising therapies for hyperuricemia and gout. In our previous study, series of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids and 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids were synthesized that presented excellent in vitro xanthine oxidase inhibitory potency. Interestingly, molecular docking studies revealed that the interaction behavior of these compounds with xanthine oxidase was changed after the conversion from a hydroxy group to amine group. To further investigate the structure-activity relationships of these pyrimidine-containing xanthine oxidase inhibitors and explore the contribution of amino or hydroxy group on xanthine oxidase inhibitory potency, several 2-phenylpyrimidine derivatives with amino or hydroxy functional group were designed and synthesized. Thereafter, the structure-activity research and molecular modeling study proved that hydroxy and amino groups could be used as pharmacophore elements for the design of 2-phenylpyrimidines xanthine oxidase inhibitors. Particularly, the optimized compound, 2-(3-cyano-4-isopentoxy)phenylpyrimidine-4-ol, emerged the strongest xanthine oxidase inhibitor potency, with an IC50 value of 0.046 µM, which was approximately 120-fold more potent than that of allopurinol (IC50 = 5.462 µM). Additionally, Lineweaver-Burk plot analysis revealed that the optimized compound acted as a mixed-type inhibitor. Furthermore, the in vivo hypouricemic effect of the optimized compound was investigated in a hyperuricemia rat model induced by potassium oxonate, and the results showed that the optimized compound could effectively reduce serum uric acid levels at an oral dose of 30 mg/kg.

Switching a Xanthine Oxidase Inhibitor to a Dual-Target Antagonist of P2Y1 and P2Y12 as an Oral Antiplatelet Agent with a Wider Therapeutic Window in Rats than Ticagrelor.

ADP-mediated platelet aggregation is signaled through G protein-coupled receptors P2Y1 and P2Y12 on the platelet. The clinical effectiveness of inhibiting P2Y12 has been well established, and preclinical studies indicated that the inhibition of P2Y1 could provide equivalent antithrombotic efficacy as P2Y12 antagonists and reduce bleeding risks. On the basis of the 2-phenyl-1H-imidazole scaffold of our previously reported xanthine oxidase inhibitor WSJ-557, we first achieved the transition from the xanthine oxidase inhibitors to dual-target antagonists against P2Y1 and P2Y12. We described the structure-activity relationships of the 2-phenyl-1H-imidazole compounds, which led to the identification of the most potent antiplatelet agents, 24w and 25w, both showing a rapid onset of action in pharmacokinetic study. Furthermore, the rat model suggested that 24w demonstrated a wider therapeutic window than ticagrelor, displaying equivalent and dose-dependent antithrombotic efficacy with lower blood loss compared to ticagrelor at same oral dose. These results supported that 24w and 25w could be promising drug candidates.

Design, synthesis and biological evaluation of 1-alkyl-5/6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles as novel xanthine oxidase inhibitors.

Xanthine oxidase (XO) has emerged as an important target for the treatment of hyperuricemia and gout. In this study, to obtain novel nonpurine XO inhibitors, a series of 1-alkyl-5/6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles (1a-1u, 2c, 2e, 2h and 2n) were designed using a bioisosteric replacement strategy and were synthesized through a five-step procedure with good yields. Thereafter, the in vitro XO inhibitory potencies of these compounds were evaluated by spectrophotometry, showing inhibitory profiles in the micromolar/submicromolar range. Particularly, compound 1h emerged as the strongest XO inhibitor, with an IC50 value of 0.36 µM, which was approximately 21-fold more potent than the positive control allopurinol. Additionally, the structure-activity relationships revealed that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety linked at the 5-position of the indole scaffold was more preferable than the 6-position for the XO inhibitory potency. Enzyme kinetic studies indicated that compound 1h acted as a mixed-type XO inhibitor. Moreover, molecular modeling studies were performed on compound 1h to gain insights into its binding modes with XO. The results showed that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety could interact with Arg880 and Thr1010 in the innermost part of the active pocket through hydrogen bonds, while the cyano group could form hydrogen bonds with Asn768 and Lys771 in the subpocket. Furthermore, the in vivo hypouricemic effect of compound 1h was further investigated in a hyperuricemia rat model induced by potassium oxonate. The results suggested that compound 1h could effectively reduce serum uric acid levels at an oral dose of 10 mg/kg. Therefore, compound 1h could be a promising lead compound for the treatment of hyperuricemia and gout.

Novel 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones as promising xanthine oxidase inhibitors: Design, synthesis and biological evaluation.

Xanthine oxidase (XO) is a critical target for the therapy of hyperuricemia and gout. In this study, a number of 3-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-1,2,4-oxadiazol-5(4H)-ones (3a-3w) were newly designed by a bioisosteric replacement and hybrid strategy with the hope of obtaining novel and effective nonpurine XO inhibitors. Subsequently, these compounds were synthesized through a three-step procedure, with good yields. In addition, the in vitro bovine XO inhibitions were measured by spectrophotometric determination of uric acid formation at 295 nm using allopurinol as a positive control. As a result, compound 3j was found to be the most potent XO inhibitor, with an IC50 value of 0.121 µM, which was approximately 63-fold more potent than allopurinol, and the analysis of the structure-activity relationships indicated that the hydrophobic group at 4'-position was essential for inhibitory potency. Additionally, the molecular modeling results showed that the 1,2,4-oxadiazol-5(4H)-one moiety binds to XO active site via various hydrogen bonds with Arg880 and Thr1010. Moreover, the compound 3j was demonstrated to be a mixed-type nonpurine XO inhibitor. Furthermore, the hypouricemic studies on a rat model, induced by potassium oxonate, demonstrated that serum uric acid levels could be effectually reduced by compound 3j at an oral dose of 15 mg/kg. Therefore, compound 3j could be a promising lead compound for the treatment of hyperuricemia and gout.

Design, synthesis and biological evaluation of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives as novel xanthine oxidase inhibitors.

In our previous study, we reported a series of 1-hydroxy-2-phenyl-1H-imidazole-5-carboxylic acid derivatives that presented excellent in vitro xanthine oxidase (XO) inhibitory potency. To further investigate the structure-activity relationships of these compounds, the imidazole ring was transformed to a pyrimidine ring to design 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (8a-8j), 2-(4-alkoxy-3-cyano)phenyl-4-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (9c, 9e, 9j, 9l) and 2-(4-alkoxy-3-cyano)phenyl-6-imino-1,6-dihydropyrimidine-5-carboxylic acids (10c, 10e, 10j, 10l). These compounds exhibited remarkable in vitro XO inhibitory potency with IC50 values ranging from 0.0181 µM to 0.5677 µM. Specifically, compounds 10c and 10e, with IC50 values of 0.0240 µM and 0.0181 µM, respectively, emerged as the most potent XO inhibitors, and their potencies were comparable to that of febuxostat. Structure-activity relationship analysis revealed that the methyl group at 4-position of pyrimidine ring could damage the potency, and the XO inhibitory potency was maintained when carbonyl group was changed to an imino group. Lineweaver-Burk plot analysis revealed that the representative compound 10c acted as a mixed-type inhibitor. A potassium oxonate induced hyperuricemia model in rats was chosen to further confirm the hypouricemic effect of compound 10c, and the results showed that compound 10c (5 mg/kg) was able to significantly lower the serum uric acid level. Furthermore, in acute oral toxicity study, no sign of toxicity was observed when the mice were administered with a single 2000 mg/kg oral dose of compound 10c. These results suggested that compound 10c was a potent and promising uric acid-lowing agent for the treatment of hyperuricemia.

Targeting the subpocket in xanthine oxidase: Design, synthesis, and biological evaluation of 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives.

Xanthine oxidase is an important target for the treatment of hyperuricemia, gout and other related diseases. Analysis of the high-resolution structure of xanthine oxidase with febuxostat identified the existence of a subpocket formed by the residues Leu648, Asn768, Lys771, Leu1014 and Pro1076. In this study, we designed and synthesized a series of 2-[4-alkoxy-3-(1H-tetrazol-1-yl) phenyl]-6-oxo-1,6-dihydropyrimidine-5-carboxylic acid derivatives (8a-8z) with a tetrazole group targeting this subpocket of the xanthine oxidase active site, and they were further evaluated for their inhibitory potency against xanthine oxidase in vitro. The results showed that all the tested compounds (8a-8z) exhibited an apparent xanthine oxidase inhibitory potency, with IC50 values ranging from 0.0288 µM to 0.629 µM. Among them, compound 8u emerged as the most potent xanthine oxidase inhibitor, with an IC50 value of 0.0288 µM, which was comparable to febuxostat (IC50 = 0.0236 µM). The structure-activity relationship results revealed that the hydrophobic group at the 4'-position was indispensable for the inhibitory potency in vitro against xanthine oxidase. A Lineweaver-Burk plot revealed that the representative compound 8u acted as a mixed-type inhibitor for xanthine oxidase. Furthermore, molecular modeling studies were performed to gain insights into the binding mode of 8u with xanthine oxidase and suggested that the tetrazole group of the phenyl unit was accommodated in the subpocket, as expected. Moreover, a potassium oxonate-induced hyperuricemia model in rats was chosen to further confirm the hypouricemic effect of compound 8u, and the result demonstrated that compound 8u could effectively reduce serum uric acid levels at an oral dose of 5 mg/kg. In addition, acute oral toxicity study in mice indicated that compound 8u was nontoxic and tolerated at a dose up to 2000 mg/kg. Thus, compound 8u could be a potential and efficacious agent in treatment of hyperuricemia with low toxicity.

Flexural instability and sound amplification of a membrane-cavity configuration in shear flow.

The scattering of sound by a membrane-covered cavity in a duct with shear flow is calculated with a linear model based on the multimodal method. The model is verified by comparison against the previous experiments focused on sound suppression of a stable system with high-tension membranes and a low-speed flow. It is shown in this paper that such a situation is drastically changed when the flow velocity is larger than the in vacuo flexural wave speed of the membrane. One of the neutral hydrodynamic modes can be destabilized under certain conditions, and this flexural instability can lead to sound amplification. For a given flow profile, the axial growth rate of the instability increases with the mean flow velocity but saturates at high velocities. For a given mean flow velocity, there is an optimum boundary layer thickness for the instability. Increasing the structural damping tends to stabilize the instability and thus inhibit the sound amplification.

Beneficial effects of Fu-Zheng-Qu-Zhuo oral liquid combined with standard integrated therapy in patients with chronic kidney disease (stage 3-4): A randomized placebo-controlled clinical trial.

BACKGROUND: The high worldwide prevalence of chronic kidney disease (CKD) is a critical health problem and the development of more effective therapies is urgently needed. We conducted a randomized, double-blinded, placebo-controlled clinical trial from October 2010 to December 2012 to assess whether Fu-Zheng-Qu-Zhuo oral liquid (FZQZ) has a beneficial effect in preventing CKD progression when added to standard integrated therapies. METHODS: Patients with CKD stage 3 to 4 from 3 hospitals in Beijing, China were enrolled. Patients were randomly assigned to the FZQZ or placebo groups and were treated with standard integrated therapy plus FZQZ or placebo (20 mL each time, 3 times/d) for 12 months. Patients received post-trial follow-up until October 2014. The primary outcome was the estimated glomerular filtration rate (eGFR)-Slope (mL/min per 1.73 m2 per month) during the in-trial time, which was calculated by the eGFR regression curve estimated from each serum creatinine measurement during the in-trial period. Secondary outcomes were changes in 24-h urine protein excretion (24-h UP) and albumin and hemoglobin levels from baseline during the in-trial period. Time to composite endpoint events (initiation of long-term dialysis, doubling of serum creatinine, or CKD-related death during the in-trial and post-trial phases) was assessed as a secondary outcome. RESULTS: A total of 68 patients (43 in the FZQZ group and 25 in the placebo group) completed the in-trial and post-trial phases, with an average follow-up time of 31.6 ±â€Š9.6months. The FZQZ group had amean eGFR-Slope of 0.25 ±â€Š1.44 as compared with -0.72 ±â€Š1.46 (mL/min per 1.73m2 per month) in the placebo group during the in-trial period (P = .003). The FZQZ group showed decreased 24-h UP, with a change from baseline of -0.08 (interquartile range [IQR], -0.33 to 0.01) versus 0.01 (IQR, -0.19 to 0.33) g/24h in the placebo group (P = .049). Decreased risk of composite endpoint events was observed only in the post-trial phase (hazard ratio = 0.42, 95% confidence interval: 0.16-1.11, P = .038). No significant differences in albumin and hemoglobin level changes were observed. CONCLUSION: Adding FZQZ oral liquid to standard integrated therapies may aid in attenuating CKD progression.

Acoustic of a perforated liner with grazing flow: Floquet-Bloch periodical approach versus impedance continuous approach.

The effect of a shear flow on an acoustic liner consisting of a perforated plate backed by cavities is studied. Two different approaches are investigated: First, the duct and the liner are considered as a periodic system while in the second approach the liner is considered as homogeneous and described by an impedance. Those two approaches coincide perfectly without flow for a small hole spacing compared to the acoustic wavelength. This work demonstrates that those two approaches are not wholly consistent when a shear flow is present and reveals some problems in the use of the local impedance with flow. The no-flow impedance cannot be used to describe the liner when a shear flow is present. An equivalent impedance with flow can be defined but it depends on the direction of the incident waves and loses its local characteristic.

Discrete vortex model of a Helmholtz resonator subjected to high-intensity sound and grazing flow.

In this paper, a theoretical model is developed to study the acoustical response of a Helmholtz resonator as a duct-branched acoustic absorber subjected to both high-intensity sound and grazing flow. The present model is comprised of a discrete vortex model in combination with a one-dimensional duct sound propagation model. The present work is to study the overall effect of incident sound interacting with grazing flow but putting emphasis on the nonlinear or intermediate regime where the sound intensity has a marked or non-negligible influence on the acoustic behavior of the Helmholtz resonator. The numerical results reveal that the flow field around the orifice is dominated by the evolution of the vortex sheet and the flow pattern is influenced by the ratio of the orifice flow velocity to the grazing flow velocity. When the incident sound pressure is high or the resonance occurs, the resonator shows nonlinearity, i.e., the acoustic impedance and absorption coefficient vary not only with duct flow Mach number buy also with incident frequency and incident sound pressure level.

Study on the relationship between blood stasis syndrome and clinical pathology in 227 patients with primary glomerular disease.

OBJECTIVE: To investigate the relationship between the severity of Chinese medicine (CM) blood stasis syndrome (BSS) with clinical features and renal lesion indexes of the primary glomerular disease. METHODS: An epidemiological survey was conducted to collect the data of 227 patients diagnosed as chronic primary glomerular diseases, and their severity of BSS were scored three days before renal biopsies were performed. The following clinical indexes were analyzed: age, course of glomerular diseases, 24-h urine protein ration (Upro), hypertension and blood pressure (BP) progress, serum creatinine levels (Scr), estimation of glomerular filtration rate based on the predigesting equation of MDRD (eGFR), blood urea nitrogen (BUN), uric acid (UA), triglyceride (TG), cholesterol (CHO), haematoglobin (HGB), albumin (ALB), and the correlation among renal pathological types, pathology lesion indexes, and BSS scores. RESULTS: (1) Among the 227 patients, 207 (91.19%) were diagnosed as BSS, in which 95 cases were considered as moderate and the rest 112 cases as severe. (2) There was a negative correlation between age, gender, grades of the hypertension, and the BSS score. Multiple stepwise regression analysis showed that Upro, CHO, TG, and eGFR were positively related to the BSS score (P<0.05). (3) The BSS score has a positive correlation with indexes of chronic renal pathology, especially the tubular atrophy and interstitial fibrosis. The severity of proliferation and glomerular sclerosis was accompanied with higher BSS scores with a significant difference (P<0.05). CONCLUSIONS: BSS is one of the most common CM syndromes among patients with the primary glomerular diseases; the BSS score has a positive correlation with Upro, CHO, TG, eGFR, as well as the index of chronic renal pathology. Based on these observations, the BSS may be used as an indicator of the development of renal diseases. Being positively diagnosed as BSS could indicate the beginning of the chronic phase of the primary glomerular diseases.

[Surrogate end points for clinical trials on chronic kidney disease and research of Chinese medicine].

Chronic kidney disease is a kind of disease with the condition always worsening over time passing through a sequence of stages, and the evaluation on its clinical treatment is mainly by observing the speed of renal function deteriorating and the time of terminal renal failure occurrence. In order to conduct the trial go on wheels, the authors proposed that the "surrogate end points (SEP)" should be introduced. It is the biologic mark for substitute the clinical terminal point (event), formed depending upon the scientific evidences of epidemiology, pathophysiology, drug-therapy and other scientific evidence, which could be used for predicting the efficacy or damage of a certain measure, present or absent. This article aimed to explain the definition of SEP and to discuss the usable SEP for clinical trial on chronic kidney disease, such as proteinuria, declination of glomerular filtration rate and its slope coefficient as well as the time of terminal occurrence. Moreover, through analyzing the existent problems in clinical researches concerning TCM treatment of chronic kidney disease, the authors suggested that some improvements, chiefly the utilization of SEP for efficacy evaluation, are necessary in the clinical observation methodologies for chronic kidney disease.