Druggability studies of diarylamide E3 as a novel diuretic

Druggability studies of diarylamide E3 as a novel diuretic | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 January 2025 V1 Latest version Share on Druggability studies of diarylamide E3 as a novel diuretic Authors : Hang Zhang , Shuyuan Wang , Nannan Li , Yue Xu , Zhizhen Huang , Yukun Zhang , Jing Li , Yinglin Zuo , Min Li , Rentao Li , and Baoxue Yang [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173651610.01866253/v1 254 views 163 downloads Contents Abstract Introduction 2.1 Chemistry 2.2 Animals 2.3 Blood samples 2.4 Erythrocyte lysis assay for identifying UT-B inhibition activity 2.5 Stopped-flow measurement of erythrocyte urea permeability 2.6 Transwell assay of UT-A1/UT-B inhibition 2.7 Measurement of the diuretic effect in rats and mice 2.8 Rat hyponatremia model caused by SIADH 2.9 Metabolic stability assays 2.10 Pharmacokinetic study in rats 2.11 Tissue distribution study 2.12 Sample preparation 2.13 Toxicological test methods 2.14 Histology 2.15 Western blot analysis 2.16 Statistical analysis 3.1 E3 was identified from structurally optimized diarylamides 3.2 E3 dose dependently inhibited UT-A1 and UT-B 3.3 E3 exerted diuretic effect mainly by inhibiting UT-A1 3.4 Diuretic activity of long-term administration of E3 3.5 E3 had therapeutic effects on hyponatremia of SIADH 3.6 E3 had good metabolic stability and pharmacokinetic characteristics 3.7 Toxicity assay showed the safety of E3 Discussion Conclusion Supplementary Material References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Urea transporters (UTs) play an important role in the urine-concentrating mechanism and have been regarded as a novel drug target for developing salt-sparing diuretics. Our previous studies found that diarylamides 1H and 25a are specific UT inhibitors and have oral diuretic activity. However, these compounds necessitate further optimization and comprehensive druggability studies. Experimental Approach: The optimal compound was identified through structural optimization. Experiments were conducted to investigate its inhibitory activity and evaluate its diuretic effect. Furthermore, disease models were utilized to assess the compound’s efficacy in treating hyponatremia. Pharmacokinetic studies were performed to examine its metabolic stability, and toxicity tests were conducted to evaluate its safety. Key Results: Based on the chemical structure of compound 25a, we synthesized a novel compound, E3, by introducing a benzenesulfonamide group into its side chain. E3 exhibited dose-dependent inhibition of UT at the nanomolar level and demonstrated oral diuretic activity without causing electrolyte excretion disorders in both mice and rats. Experiments involving UT-B-/- and UT-A1-/- mice indicated that E3 enhances the diuretic effect primarily by inhibiting UT-A1 more effectively than UT-B. Furthermore, E3 displayed good metabolic stability and favorable pharmacokinetic characteristics. E3 significantly ameliorated hyponatremia through diuresis in rat model. Importantly, E3 did not induce acute oral toxicity, subacute oral toxicity, genotoxicity, or cardiotoxicity. Conclusion and Implications: Our study confirms that E3 exerts diuretic effect by specifically inhibiting UTs and has good druggability, which offers potential for E3 to be developed into a new diuretic for the treatment of hyponatremia. Submitted to British Journal of Pharmacology Druggability studies of diarylamide E3 as a novel diuretic Hang Zhang 1 , Shu-yuan Wang 1 , Nan-nan Li 1 , Yue Xu 2 , Zhi-zhen Huang 1 , Yu-kun Zhang 3 , Jing Li 4 , Ying-lin Zuo 4 , Min Li 1 , Run-tao Li 5 , Bao-xue Yang 1, *. 1 Department of Pharmacology, School of Basic Medical Sciences, Peking University, Beijing 100191, China 2 Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA 3 Chongqing Key Laboratory of Development and Utilization of Genuine Medicinal Materials in Three Gorges Reservoir Area, Chongqing 404120, China 4 The State Key Laboratory of Anti-Infective Drug Development, Sunshine Lake Pharma Co., Ltd., Dongguan 523871, China 5 School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Runing head: Druggability of E3 as a diuretic Correspondence: Bao-xue Yang, MD, PhD, Department of Pharmacology, School of Basic Medical Sciences, Peking University, Beijing 100191, China. Email: [email protected] Background and Purpose: Urea transporters (UTs) play an important role in the urine-concentrating mechanism and have been regarded as a novel drug target for developing salt-sparing diuretics. Our previous studies found that diarylamides 1H and 25a are specific UT inhibitors and have oral diuretic activity. However, these compounds necessitate further optimization and comprehensive druggability studies. Experimental Approach: The optimal compound was identified through structural optimization. Experiments were conducted to investigate its inhibitory activity and evaluate its diuretic effect. Furthermore, disease models were utilized to assess the compound’s efficacy in treating hyponatremia. Pharmacokinetic studies were performed to examine its metabolic stability, and toxicity tests were conducted to evaluate its safety. Key Results: Based on the chemical structure of compound 25a, we synthesized a novel compound, E3, by introducing a benzenesulfonamide group into its side chain. E3 exhibited dose-dependent inhibition of UT at the nanomolar level and demonstrated oral diuretic activity without causing electrolyte excretion disorders in both mice and rats. Experiments involving UT-B -/- and UT-A1 -/- mice indicated that E3 enhances the diuretic effect primarily by inhibiting UT-A1 more effectively than UT-B. Furthermore, E3 displayed good metabolic stability and favorable pharmacokinetic characteristics. E3 significantly ameliorated hyponatremia through diuresis in rat model. Importantly, E3 did not induce acute oral toxicity, subacute oral toxicity, genotoxicity, or cardiotoxicity. Conclusion and Implications: Our study confirms that E3 exerts diuretic effect by specifically inhibiting UTs and has good druggability, which offers potential for E3 to be developed into a new diuretic for the treatment of hyponatremia. Keywords: Urea transporter inhibitor; Diuretic; Structure optimization; Pharmacokinetic; Safety evaluation; Hyponatremia. Introduction Diuretics are drugs that increase urine output by affecting the reabsorption and secretion of renal tubules, making them commonly used for treatment of hypertension, heart failure, edema and ascites, etc. (Chatur et al., 2023; Inker et al., 2019; B. Wang, Wen, Li, Wang-France, & Sansom, 2017). However, traditional diuretics, such as loop, thiazide, potassium-sparing diuretics, which long term use may induce electrolyte disorders in the body. Hyponatremia is the most common electrolyte disorder diagnosed in the hospital setting, which is a significant independent risk factor for in-hospital mortality(Flahault et al., 2021; J. W. Lee et al., 2018). Vasopressin V2 receptor (V2R) antagonists are effective to alleviating hyponatremia, but their hepatotoxicity was demonstrated in clinical trials (Yottasan, Chu, Chhetri, & Cil, 2024). Therefore, the search for novel diuretic targets and safer diuretics that avoid electrolyte disturbances is crucial. Urea is a major solute in the hyperosmolar renal medulla and plays an important role in urinary concentration management (Cil, Ertunc, & Onur, 2012; Lang, Lang, Lang, Huber, & Wieder, 2006; Stewart, 2011). Urea transporters (UTs) are membrane channel proteins that are specifically permeable to urea. UTs play an important role in maintaining intrarenal urea recycling, establishing urea concentration gradient in renal medullary tissue (Fenton & Yang, 2014; F. Li et al., 2013; X. Li, Chen, & Yang, 2012; Titko, Perekhoda, Drapak, & Tsapko, 2020; Verkman et al., 2014). Several UT knockout mouse models demonstrate that UT knockout blocks the intrarenal urea recycling and decreases the urine concentration ability, thereby producing a diuretic effect, which suggests UT inhibitors can be developed into diuretics for long-term clinical use without causing electrolyte disorders(Fenton, Chou, Stewart, Smith, & Knepper, 2004; Jiang et al., 2017; Lei et al., 2011; Yang, Bankir, Gillespie, Epstein, & Verkman, 2002). Since 2012, Verkman’s team have identified various small molecule UT inhibitors, mainly including phenylsulfoxyoxazole, benzenesulfonanilide, phthalazinamine, aminobenzimidazole, 8-Hydroxyquinolines, aminothiazolones, benzo-[1,3,5]-triazines, and triazolothienopyrimidine active compounds(Esteva-Font, Phuan, Anderson, & Verkman, 2013; Levin, de la Fuente, & Verkman, 2007; Sridharan, Goel, & Priyakumar, 2022; Yao et al., 2012) (Figure S1a-h). Our research group also identified thienoquinoline compounds PU-14 and PU-48 (Figure S1i, j), thienopyridine compound CB-20 (Figure S1k) (M. Li et al., 2020). However, all these compounds exert diuretic activity with low druggability. Recently, we found that diarylamides 1H (Figure S1l) and 25a (Figure 1a) showed superior diuretic effect in vivo without causing electrolyte imbalance in rats by oral administration(S. Wang et al., 2021; Xu et al., 2022; Zhang et al., 2021). It was found that 25a is effective in treating hyponatremia in the SIADH model and cirrhosis ascites model(N. Li et al., 2024; Ying et al., 2023). However, the oral therapeutic dose of 25a in disease models remain relatively high at 100 mg/kg, indicating a need for improvement in inhibitory activity and metabolic stability. Furthermore, it is crucial to note that safety evaluation data for diarylamide compounds is lacking either. In this study, we derived a new diarylamide UT inhibitor, 5-acetyl- N -(3-(phenylsulfonamido)phenyl)furan-2-carboxamide (E3) basing on structural modification of 25a. E3 exhibited good inhibitory activities and selectivity for UT-A1 in vitro and in vivo . E3 demonstrated oral diuretic and therapeutic effect without significant toxicity. These experimental data suggest that E3 has the potential to be developed as a new diuretic for the treatment of hyponatremia. 2. Materials and Methods 2.1 Chemistry Starting materials, reagents and solvents were all commercially purchased and used without further purification. 1 H spectra and 13 C spectra were recorded on a Bruker AVANCEIII 400 MHz and 100 MHz NMR spectrometer (Bruker, Karlsruhe, Germany), respectively. Chemical shifts are expressed as δ units in ppm (in NMR description, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad peak). HRMS spectra were acquired by electrospray ionization (ESI) in positive ion mode or negative ion mode using Thermo Scientific Orbitrap Elite MS. The general procedure and schemes for the synthesis of intermediates and target compounds E1~E9 are showed in the supplemental materials. 2.2 Animals C 57 BL/6 mice (20~22 g) and male Sprague–Dawley (SD) rats (200~220 g) were purchased from the Laboratory Animal Center of Peking University (Beijing, China). UT-B knockout (UT-B -/- ) and UT-A1 knockout (UT-A1 -/- ) mice with a C 57 BL/6 genetic background were generated by targeted gene disruption as described previously (Geng et al., 2020; Yang et al., 2002). All animals were provided with food and water ad libitum and maintained in a temperature-controlled environment (25 ± 1 °C) in a 12/12 h light–dark cycle. All animal care protocols were approved by the Institutional Animal Care and Use Committee at the Peking University Health Science Center (Approved number: LA220354, 19 May 2020, Peking University, Beijing, China). The study was carried out according to ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines) and the National Research Council’s guide for the Care and Use of Laboratory Animals (https://grants.nih.gov/grants/olaw/guide-for-the-care-anduse-of-laboratory-animals.pdf). 2.3 Blood samples Human venous blood was ethically obtained from healthy adult male volunteers following approval by the Ethics Committee of Peking University (Beijing, China). Rabbit blood was collected from male Japanese white rabbits by ear vein bleeding. Rat blood was collected from male SD rats by orbital venous plexus puncture. Mouse blood was collected from wild-type or UT-B -/- male mice by eyeball extirpating. Blood samples used for erythrocyte lysis assay and stopped-flow light scattering assay were anticoagulated by 0.5% heparin. Erythrocyte was acquired by centrifugation after being washed by PBS (0.01 mol/L, pH = 7.4) for three times. All blood samples were used for experiments within 12 h of collection. 2.4 Erythrocyte lysis assay for identifying UT-B inhibition activity The erythrocyte lysis assay was modified from a method described previously(Levin et al., 2007). Erythrocytes obtained from veins were diluted to a hematocrit value of 2% in hyperosmolar PBS containing 1.25 mol/L urea and 5 mmol/L glucose, and incubated at r.t. for 2 h. Then, 99 μL erythrocyte suspension from a reservoir was added to each well of a 96-well round-bottom microplate, then added 1 μL testing compound (500, 125, 31.25, 7.81, 1.95, 0.488, 0.122, 0.031, 0.0076 μmol/L dissolved in DMSO) to erythrocyte suspension and shook it up with microoscillator for 1 min. After 5 min of incubation, 20 μl of the erythrocyte suspension was rapidly transferred into a 96-well black wall microplate that contained 180 μl isotonic PBS (0.01 mol/L, without urea). Erythrocyte lysis was quantified by absorbance at 710 nm within 6 min. The lysis rate of the erythrocytes was calculated using control values from the same plate as Lysis (%) = 100 × (A neg -A test )/(A neg -A pos ), where A test is the absorbance value from the test well, A neg is from a negative no-lysis control well and A pos is from a positive full-lysis control. 2.5 Stopped-flow measurement of erythrocyte urea permeability Rat erythrocyte urea permeabilities were measured by stopped-flow light scattering using a SX20 instrument (Applied Photophysics, Leatherhead, UK) as described previously(Yang & Verkman, 2002). Erythrocyte was acquired from rat blood and suspended in isotonic PBS (hematocrit 0.5%). Then erythrocyte was incubated with test compounds for 5 min and quickly mixed with 500 mmol/L urea dissolved in PBS. Following an initial osmotic shrinking phase, the kinetics of cell volume increase due to urea influx were measured by monitoring the time course of 90° scattered light intensity at 530 nm. The increase in cell volume resulted in a reduction of scattered light intensity. Keep samples and PBS at 4 °C to reduce the influence of free diffusion. To assay reversibility, compounds were added to erythrocytes for 5 min, and then washed with PBS 3 times by 2,000 r/min centrifugation before stopped-flow measurements. To determine inhibition on urea efflux, erythrocytes were incubated with 500 mmol/L urea in PBS for 2 h, then mixed with PBS without urea. 2.6 Transwell assay of UT-A1/UT-B inhibition MDCK cells stably expressing rat UT-A1 or UT-B were cultured in DMEM supplemented with 10% FBS, and the mRNA levels of UT-A1 and UT-B were measured in our previous research(M. Li et al., 2020). Urea flux was assessed according to previously established methods. MDCK cells (2 × 10 5 cells/cm 2 ) were grown on 12 mm collagen-coated Costar Transwell inserts (0.4 μm pore size, Corning) for 4 d at 37 °C in the presence of 5% CO 2 . Once the cells on the apical side formed a tight monolayer (transepithelial resistance 1 kΩ/cm 2 ), PBS (pH = 7.4, containing 10 μmol/L forskolin) with E3 or DMSO was added to the top (0.25 mL) and bottom (1 mL) compartments, and the cultures were incubated in the absence of urea for 30 min at 37 °C. As UT-B located in the plasma membrane while UT-A1 resides in the cytoplasm, so forskolin was used to stimulate the translocation of UT-A1 from the cytoplasm to the membrane for urea transport. Subsequently, the solution in the bottom compartment was replaced with PBS (pH 7.4, containing 10 μmol/L forskolin and E3 or DMSO) supplemented with 15 mmol/L urea. Apical fluid samples (5 μL) were collected at 0, 1, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min. The samples were subjected to an assay for urea (Urea Colorimetric Assay Kit (Diacetyl Oxime Method), Elabscience) according to the kit procedure. Then, the inhibition rate was calculated as described previously(Zhang et al., 2021). 2.7 Measurement of the diuretic effect in rats and mice In a single-dose administration experiment, male wild-type (WT) mice, UT-B -/- mice, UT-A1 -/- mice, or SD rats were adapted in metabolic cages for 3 d. Food and water were provided ad libitum throughout the experiment. Prior to administration, the bladder of each animal was emptied through gentle abdominal massage, and urine was collected from the metabolic cages every 2 h. Compound E3, suspended in 0.5% CMC-Na at an appropriate concentration, was administered intragastric gavage to the mice or rats (0.16, 0.8, 4 or 20 mg/kg). A 0.5% CMC-Na solution served as the vehicle control. Urine volume was measured by gravimetry, assuming a density of 1 g/mL. Urinary osmolality was measured by freezing point depression (Micro-osmometer, Fisker Associates). Urea concentration was measured with the QuantiChrom urea assay kit (Urea Colorimetric Assay Kit (Diacetyl Oxime Method), Elabscience). In long-term (7 d) diuretic activity experiments, male WT mice, UT-B -/- mice, UT-A1 -/- mice, and SD rats were acclimatized in metabolic cages (Ugo Basile, Comerio) for 3 d. Food and water were provided ad libitum throughout the experiment. E3 was suspended in 0.5% CMC-Na and administered at a dose of 4 or 20 mg/kg by intragastric gavage. Urine was collected by metabolic cages every 24 h. Body weight was measured daily. 4 h after the final administration, a blood sample was obtained by heart puncture. Inner medulla and outer medulla tissue homogenates were prepared, and the supernatant after centrifugation was analyzed for solute concentration and osmolality. Urinary osmolality and urea concentration were measured as above. Serum Na + , K + , Cl − were measured in a clinical chemistry laboratory, while serum creatinine, ALT, AST were measured through specific reagent kits (NJJC Bio, Nanjing). 2.8 Rat hyponatremia model caused by SIADH The protocol for dDAVP-induced hyponatremia was adapted from a previous study(Adler, Verbalis, & Williams, 1994), with tolvaptan was chosen as a positive control. Preliminary experiments determined the dose of dDAVP (0.25 ng/h). Under isoflurane anesthesia, a 0.5 μg/mL dDAVP solution was injected subcutaneously using an osmotic minipump (ALZET 2002, 200 μL, low rate of 0.5 μL/h for 7 d). Rats were housed individually in metabolic cages to collect urine. Before using the pump, give 62.5 mL/d 1.0 kcal/mL liquid food. After pump implantation, rats received 40 mL of 2.1 kcal/mL liquid feed daily. The rats were divided into five groups: control group (sham operation), model group (0.5% poloxamer, solvent), 4 mg/kg tolvaptan group, 4 mg/kg E3 group, and 20 mg/kg E3 group. E3 was administered orally every 8 h, while tolvaptan was given intragastrically once daily. The schedule of the above experiment arrangement is shown in Figure 4a. At the end of the experiment, the rats were sacrificed. 2.9 Metabolic stability assays Plasma from mice, rat, rabbit, human was obtained from blood after centrifugation at 3,000 r/min for 10 min. Simulated gastric fluid (SGF, pH=1, containing pepsin) and simulated intestinal fluid (SIF, pH=6.8, containing trypsin) were configured previously in the experiments(Cao, Wang, Pang, Cheng, & Liu, 2019; Huang, Wu, & Xu, 2022). SD rats and mice were collected, and after removing the mucus layer from the small intestine and colon, 20 times the volume of liquid bacterial culture medium was added. An intestinal microbiota suspension was obtained after anaerobic cultivation on a shaking table at 37 °C for 12 h. In aforementioned systems, E3 was added at a final concentration of 1 μmol/L at 37 °C for different incubation times (0, 10, 20, 30, 60, 120, and 240 min). In liver metabolic stability assays, E3 was incubated with liver microsomes or liver homogenate from mice or rats for 60 min at 37 °C. The stop solution,5 ng/mL tolbutamide (internal standard, IS) in acetonitrile, was added to stop the metabolic process. Then, the mixture was centrifuged at 18,000 g for 10 min at 4 °C, and the supernatant was injected for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The ratio of the peak area of E3 to the internal standard was used to calculate the remaining percentage with the following formula: Remaining% = (E3 peak area /IS peak area at different time) /(E3 peak area /IS peak area at 0 min) × 100%. The hepatic clearance (CL hep ) values were calculated using the amount of microsomal protein (mice: 45 mg/g liver; rat: 45 mg/g liver) and liver weight (mice: 50 g liver/kg; rat: 40 g liver/kg). 2.10 Pharmacokinetic study in rats Male SD rats were allowed free access to food at a controlled environment of 22 ± 2 °C, with a humidity level of 55% ± 5%, and maintained under a 12 h light/dark cycle. In the oral administration groups, E3 was delivered in a 0.5% CMC-Na solution at a dosage of 4 mg/kg body weight. Blood samples were collected by the orbital venous plexus and kept on ice at the following time points: 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. For the intravenous ( i.v. ) group, E3 dissolved in a mixed solvent (DMSO, PEG400, and saline, 10:30:60, v / v / v ) was intravenously injected at 1 mg/kg body weight via the tail vein. Blood was collected at 0.033, 0.083, 0.167, 0.333, 0.5, 1, 2, 4, 8, 12, and 24 h. Plasma was obtained by centrifugation at 3,000 r/min for 10 min at 4 °C. The plasma was stored at −80 °C until analysis. Pharmacokinetic (PK) parameters were calculated by DAS 3.2.8 software (Beijing, China) using the noncompartmental analysis (NCA) model. The oral bioavailability (F) was calculated using the formula F = (AUC p.o. × Dose i.v. )/(AUC i.v. × Dose p.o. ) × 100%. 2.11 Tissue distribution study Male SD rats were randomly divided into four groups and received a single oral dose of E3 at 4 mg/kg. Blood and tissues samples (i.e., heart, liver, kidney, spleen, lung, brain, testis, skin, muscle, leg bone, stomach, small intestine and colon) were collected at 0.5 h, 2 h, 6 h, and 24 h after dosing. The tissue samples were rinsed with cold saline and dried on filter papers, weighed, and homogenized with ten-fold volumes of ultrapure water using a homogenizer (T10, IKA, Staufen) on ice. The right shin bone of the rat was crushed and soaked in methanol for 12 h at 4 °C. Tissue homogenate or bone soak solution was prepared and analyzed by the method above. Samples were stored at −80 °C until analysis. 2.12 Sample preparation The analyte E3 and IS were dissolved in DMSO to generate the stock solutions. The working calibration of E3 was gradient-diluted with MeOH/H 2 O (1:1, v/v ). Three microliters of working solutions were spiked with 27 μL of the blank rat plasma to establish the calibration standards. The final concentrations of the standard samples were 1, 2, 5, 10, 50, 250, 500, and 1,000 ng/mL. The quality controls at three concentrations (3, 100, and 800 ng/mL) were generated similarly. The protein precipitation method was employed for sample preparation. Specifically, 450 μL of stop solution was added to 30 μL of calibration standards and rat biological samples and vortexed for 5 min to facilitate protein precipitation. The mixture was centrifuged at 18,000 g for 10 min at 4 °C, and 2 μL of supernatant was injected for analysis. 2.13 Toxicological test methods 2.13.1 Acute oral toxicity test The acute oral toxicity test was performed according to “Guidance on Single Dose Toxicity Study for Pharmaceuticals” (2014), “Guidance for industry: single dose acute toxicity testing for pharmaceuticals” (1996). The initial dose for preliminary trials was established at 5,000 mg/kg. A total of three female and three male mice were utilized (one of each sex for preliminary experiments and two for subsequent experiments). Following a 12-hour fasting period, one male and one female mouse were selected and administered E3. After 24 h of confirming survival and the absence of abnormal behavior, the remaining four animals were given the same dose. The control group was given solvent (0.5% CMC-Na). Observations of all animals on the day of dosing (d 0) and in the days following were recorded, including general conditions (signs of toxicity, onset of symptoms, recovery period), changes in body weight, and mortality. After 14 d, blood and vital organ tissues were collected, and serum biochemical indicators as well as histopathological examinations were conducted. 2.13.2 Subacute oral toxicity test The subacute oral toxicity test was performed according to “Guidance on Repeated Dose Toxicity Study for Pharmaceuticals” (2014). In the SIADH model, we found that 4 mg/kg of E3 produced good therapeutic effects, so we chose a dose of 1,000 mg/kg, which is greater than 250 times the minimum effective dose, for the subacute toxicity test. 18 male mice were evenly divided into two groups. Mice of the control group were orally administered with solvent (0.5% CMC-Na). Another group was orally administered with E3 1,000 mg/kg. The solvent or E3 was administered once a day for 30 continuous days and withdrawn for 14 d for recovery. After the 30-day treatment, 12 mice (6/group) were chosen at random and sacrificed for necropsy. The remaining 6 mice (3/group) were observed for 14 d after the cessation of treatment and sacrificed for necropsy after the recovery period. During the study, the clinical symptoms, morbidity, and mortality of mice were observed twice a day at the side of the cage. Changes in appearance, behavior, appetite, gait, secretions, and excretions of the rats were meticulously documented. The body weights were measured before the start of the experiment, and the weights were measured every 2 d thereafter. Echocardiography was performed and evaluated every 10 d. Under anesthesia, blood samples were taken before the autopsy for hematological and clinical biochemistry analysis by an automated hematology analyzer (HEMAVET 950FS, USA) and automated biochemical analyzer (BS-180, China). The following organs were weighed: heart, liver, kidney, spleen, lung, thymus gland, testis, brain. Furthermore, the corresponding organ/body weight ratio was computed. A comprehensive histopathological analysis was performed on vital tissues and organs. 2.13.3 Assessment in cardiotoxicity by hERG K + channel The CHO-hERG cells were cultured in F12 medium (Gibco) supplemented with 10% ( v / v ) fetal bovine serum (FBS) and 0.5 mg/mL Geneticin (Invitrogen) at 37 °C in a humidified environment (5% CO 2 /95% air). The cells were seeded out 2 d before reaching 70% confluency. Prior to use, the cells were washed in PBS and incubated with 5 mL Detachin (Genlantis) for 2~3 min at 37 °C to detach cells from the culture dish. The harvested cells were re-suspended in F12 medium at a density of 2 million cells/mL. The cells were transferred to a QPatch instrument (Sophion Bioscience, Denmark) and allowed to recover for 20 min in the Qstir cell preparation station on the Qpatch-16 before experiment. The tail currents of hERG channel were evaluated using the Qpatch automated patch clamp platform (Sophion Bioscience, Denmark). The following solutions were used during patch-clamp recording (compositions in mM): internal solution: KCl 120, CaCl 2 5.374, MgCl 2 1.75, KOH 31.25, EGTA 10, HEPES 10, Na 2 ATP 4, pH 7.2 (KOH); external solution: NaCl 145, KCl 4, MgCl 2 1, CaCl 2 2, HEPES 10, glucose 10, pH 7.4 (NaOH). All solutions were sterile-filtered. Cells were clamped at -80 mV and hyperpolarized to -100 mV to monitor the change of series resistance. The voltage protocol for hERG ion channel started with a short (200 ms) -50 mV step to establish the baseline region. A depolarizing step was applied to the test potential of 20 mV for 2s, and then the cell was depolarized to -50mV to evoke outward tail currents. Currents were filtered using the internal Bessel filter in Qpatch. Recording started in external solution. After this control period, increasing concentrations of the test compounds were applied to record a complete concentration-response curve. The last control period (Saline) is used as baseline for data normalization. The sampling frequency is 2,000Hz. 2.13.4 Mouse sperm malformation assay Male mice were randomly divided into three groups: control group (0.5% CMC-Na), E3 2,000 mg/kg group, positive group (50 mg/kg cyclophosphamide (CP), administered via intraperitoneal injection). E3 and control group were administered once daily by intragastric gavage. All mice received treatment for 5 consecutive days. After the initial dosing period, which lasted 35 d, the mice were euthanized, and evaluation of sperm parameters as follows: Caudal epididymis was cut into pieces in saline and incubated at 37 °C for 10 min in a 5% CO 2 incubator. Then the sperms were gently filtered through nylon gauze. A drop of the sperm suspension was assessed for sperm count and motility parameters by computer assistant semen analysis (CASA). For examining sperm morphology, a 20 μL sperm suspension was spread on a glass slide. Sperm morphology was visualized by the eosin staining. 1,000 sperms per sample were observed to identify the abnormality. Sperm shape abnormalities such as banana, headless, without hook, amorphous, double-tailed, and fat head were identified and counted. In addition, the testis was reserved for morphological analysis. The spermatogenesis was graded from 1 to 10 using the Johnsen score(Johnsen, 1970). 2.14 Histology Multiple tissues and organs were fixed with 4% paraformaldehyde and embedded in paraffin. 5 μm paraffin sections were cut and stained with hematoxylin and eosin (H&E). 2.15 Western blot analysis The Inner medulla tissue was homogenized in RIPA lysis buffer, containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor, on ice. The supernatant was collected after centrifugation. Protein was then quantified using a BCA protein assay kit (Thermo Scientific, Massachusetts, USA). Equal amounts of protein were separated by SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Boston, MA, USA). After being blocked with 5% skimmed milk for 2 h, the membranes were washed by TBST (20 mmol/L Tris-HCl, 137 mmol/L NaCl, and 0.1% Tween-20, pH 7.4) and incubated with the relevant primary antibody (UT-A1, UT-A2, UT-A3, UT-B, AQP (aquaporin) 2, AQP3 and β-actin) for 12 h at 4 °C. Following incubation, the membranes were washed 3 times using TBST and probed with horseradish peroxidase-labeled secondary antibody (goat anti-mouse or anti-rabbit IgG) for 2 h at room temperature. The blots were then developed using a super-sensitive ECL luminescence reagent (Meilunbio, Dalian, China). The protein bands were visualized using a chemiluminescence detection system (Syngene, GeneGnome XRQ, Cambridge, Cambridgeshire, UK) and analyzed using ImageJ (ij153-win-java8) software (NIH, Be-thesda, MD, USA). 2.16 Statistical analysis Statistical analyses were carried out using GraphPad Prism 8. All data were presented as mean ± SEM. The difference between the two groups was analyzed by Student’s t -test. Multiple group comparisons were analyzed by a one-way analysis of variance (ANOVA) with Tukey’s correction. A p -value < 0.05 was considered statistically significant. 3. Results 3.1 E3 was identified from structurally optimized diarylamides In conjunction with our prior research, we have determined that the acetylfuran fragment and the amide linker are key structural elements that enhance the bioactivity of the diarylamide compounds(S. Wang et al., 2021). To enhance water solubility, we initially modified compound 25a by introducing ethylpiperazine (E1) and N-(2-(dimethylamino)ethyl)formamide (E2) (Table 1). The inhibitory activities of these derivatives against rat and mouse UT-B were comparable to those of 25a, indicating that the amide bond adjacent to the benzene ring is not a critial functional group. Previous studies have demonstrated that sulfonamides can enhance compound activity; thus, we further designed E3, E4 and E5 by incorporating arylsulfonamides at different positions on the benzene ring. The activity assays revealed that substitution at the meta position yielded the highest activity, followed by the para position, while substitution at the ortho position resulted in the lowest activity. Additionally, to mitigate the potential cytotoxicity associated with sulfonamide acidity, we introduced a hydroxyl group at the ortho position of the sulfonamide in compound E5, with the intention that this hydroxyl group would form a hydrogen bond with the sulfonamide amino group, thereby reducing the ionization of the sulfonamide and leading to the design of compound E6. Concurrently, to prevent the formation of phenylenediamine metabolites, we swapped the sulfonyl and amine groups, resulting in compound E7. Activity assays indicated that compound E7 also exhibited significant UT-B inhibitory activity. Building on E7, we introduced a water-soluble morpholine group at the para position of the benzene ring and designed connecting arms of varying lengths (E8 and E9). The results indicated that the introduction of the morpholine ring resulted in a slight decrease in compound activity; however, it remained superior to the lead compound 25a. Activity studies have demonstrated that the combination of benzene sulfonamide and acetyl furan (E3~E5) significantly enhances the biological activity of the compound. Notably, the activity of benzene sulfonamide at the meta position is the highest, followed by the para position, while the ortho position exhibits the lowest activity. These enhancements prompted the identification of several candidates, with E3 displaying the most potent inhibitory activity. Consequently, E3 was chosen for further extensive preclinical studies. The chemical structure of compound E3, named 5-acetyl-N-(3-(phenylsulfonamido)phenyl)furan-2-carboxamide, is shown in Figure 1a, indicating structural optimization process from 25a to E3. 3.2 E3 dose dependently inhibited UT-A1 and UT-B The IC 50 values of E3 for UT-B-promoted urea transport, as determined by the red blood cell lysis assay, were found to be 47.7 nM in mouse, 7.6 nM in rat, 17.7 nM in rabbit and 25 nM in human respectively (Figure 1b-d), indicating a greater inhibitory activity than the compound 25a (0.48 μM in mouse and 0.14 μM in rat). The maximum inhibition rates of E3 in mouse, rat, rabbit and human cells approached 100%. As a control, the erythrocyte lysis rate in UT-B -/- mouse was approximately 100%, with or without E3 incubation, reflecting the absence of UT-B in the erythrocyte membrane (Figure 1b). Stopped-flow assays were conducted to assess the inhibitory effect of compounds on UT-B. Following the mixing of red blood cells with a high-urea solution, the cells rapidly shrink due to water loss mediated by aquaporin 1 (AQP1), followed by an increase in cell size due to UT-B-mediated urea influx and concurrent water influx via AQP1(Zhang et al., 2021). The changes in cell volume during this process can be monitored through variations in scattered light. After incubation with E3 and subsequent exposure to a high urea solution, it was observed that E3 could dose-dependently inhibit the function of UT-B on the surface of erythrocytes in transporting urea into the cells (Figure 1e). In the efflux experiment, erythrocytes were incubated in a high-urea environment and then mixed with isotonic PBS. It was found that E3 also inhibited UT-B-mediated urea efflux in a dose-dependent manner (Figure 1f). Furthermore, the inhibition was reversible, as demonstrated by exposing erythrocytes to E3 at 1 μM followed by washout with PBS (Figure 1g). We used MDCK cell lines stably expressing rat UT-B or UT-A1 to evaluate the selectivity of E3 towards UT-A1 and UT-B. The experimental results showed that E3 significantly inhibited both UT-A1 and UT-B mediated urea permeabilities, with greater inhibitory effect against UT-A1 (IC 50 = 18 nM) than UT-B (IC 50 = 58 nM) (Figure 1h). 3.3 E3 exerted diuretic effect mainly by inhibiting UT-A1 The diuretic effect of E3 was determined in mice and rats using metabolic cages. Following 3 d acclimatization period in the metabolic cages, the animals were provided a standard diet and had free access to drinking water. The mice and rats received were intragastric gavage varying doses of E3 or a solvent control, with urine being collected every 2 h both before and after administration. Urine output significantly increased in both mice (Figure 2a) and rats (Figure 2b) in a dose-dependent manner after E3 intragastric gavage compared to the vehicle control. Concurrently, urinary osmolality (Figure 2c, d) and urea (Figure 2e, f) decreased in the same experiment. The excretion of non-urea solutes was not significantly different between E3 groups and the control group in both mice and rats (Figure 2g, h), indicating that E3 does not influence electrolyte excretion. The inhibitory selectivity of E3 on UT-A1 and UT-B was assessed in UT-A1 -/- mice and UT-B -/- mice administered 20 mg/kg E3. In UT-B -/- mice, urine output markedly increased 2 h post-E3 administration (Figure 2i), the urinary osmolality (Figure 2j) and urea (Figure 2k) decreased correspondingly. However, there was no significant change in urine output, urinary osmolality and urea concentration in UT-A1 -/- mice. These data suggest that E3 exerts a significant diuretic effect by mainly inhibiting UT-A1. The excretion of non-urea solutes (Figure 2l) was not significantly changed in both colonies. 3.4 Diuretic activity of long-term administration of E3 Clinically, diuretic drugs are typically employed for long-term treatment. Therefore, we further studied the pharmacological characteristics of E3 for long-term diuretic effects. UT-A1 -/- mice, UT-B -/- mice, and rats received intragastric administration of E3 for 7 d. Following administration of 4 mg/kg and 20 mg/kg E3 to rats, urine output continuously increased (Figure 3a), urine osmolality significantly decreased (Figure 3b), excretion of non-urea solutes did not change (Figure 3c). Compared to the control group, the osmolality (Figure 3d) and urea concentration (Figure 3e) significantly decreased, while non-urea solutes did not change (Figure 3f) in the inner medullary tissue of E3-treated rats. However, there were no differences in outer medullary osmolality, urea concentration, and non-urea solute concentration between E3-treated rats and control rats (Figure 3d-f). These results indicate that E3 exerts a diuretic effect by blocking the intrarenal urea recycling without interfering with the excretion of Na + , K + , and Cl - . In addition, after 7 d of E3 treatment in rats, there was no significant difference in body weight, organ indexes or various blood biochemical parameters (including serum Na + , K + , and Cl - ) compared to the control rats (Table S1). H&E staining showed that continuous administration of E3 did not induce morphological abnormalities in the cortex and medulla of rat kidneys, except dilatation of collecting duct in medullary tissue due to polyuria (Figure 3g). Western blot analysis of UTs and aquaporins (AQPs) in the medullary tissue of rats revealed that E3 did not significantly alter the expression levels of UT-A1, UT-A2, UT-A3 and UT-B and AQP3 (Figure 3h, i). The AQP2 expression in 20 mg/kg E3 treated rats was lower than control rats, which may be due to long term polyuria and low urinary osmolality that decreased AQP2 protein trafficking to apical membrane. All these data suggest that E3 had a diuretic effect by selectively inhibiting UT-A1, without disturbing electrolyte balance, normal metabolism, or renal function. Subsequent experiments were conducted in mice. Following oral administration of 20 mg/kg E3, the urine output significantly increased (Figure 3j), urine osmolality (Figure S2a) and urea concentration were significantly decreased (Figure S2b). There was no significant difference in the excretion of non-urea solutes (Figure S2c). After long-term treatment of E3, urine output increased (Figure 3k) and urine osmolality decreased (Figure 3l) in UT-B -/- mice, whereas there was no significant diuretic effect in UT-A1 -/- mice, which suggests that the diuretic effect of E3 mainly based on UT-A1 inhibition. 3.5 E3 had therapeutic effects on hyponatremia of SIADH The therapeutic effect of E3 on hyponatremia was subsequently evaluated in a rat model with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), as illustrated in Figure 4a. Throughout the experiment, all rats showed moderate weight gain (Figure 4b). Following the infusion of dDAVP via a pump, the urine output in the model rats was significantly reduced (Figure 4c), while urine osmolality was increased in comparison to the control rats (Figure 4d). Concurrently, serum osmolality and sodium levels decreased in the model rats (Figure 4e, f), confirming the successful establishment of the SIADH hyponatremia model. After the treatment of E3 or tolvaptan (the positive control), the hyponatremia in SIADH rats was significantly alleviated, with significantly increased serum osmolality and sodium concentration compared to the model rats. There was no significant difference in blood urea levels between the experimental groups before and after treatment (Figure 4g). In addition，H&E staining also showed normal tissue structures in kidney, except for dilatation of collecting ducts, and liver of E3 or tolvaptan-treated rats (Figure 4h, i). These results indicate that E3 alleviates hyponatremia through its diuretic mechanism. 3.6 E3 had good metabolic stability and pharmacokinetic characteristics To characterize the metabolism of E3, we assessed its metabolic stability in blood and plasma, liver microsomes, and liver homogenate. After incubating E3 in the plasma of various species at 37 °C for 4 h, we found that over 81% of E3 remained, indicating that E3 exhibits significant stability in the plasma of these species (Figure 5a). The remaining amount after blood incubation has slightly decreased (Table S2). Oral administration offers better patient compliance and facilitates long-term use. Consequently, we examined the stability of compound E3 in simulated gastric fluid (SGF) at pH 1.0 and simulated intestinal fluid (SIF) at pH 6.8 (Figure 5b). More than 90% of E3 remained after 4 h. In addition, after incubating with intestinal microbiota (colon and small intestine), over 82% of E3 persisted after 4 h (Table S2). Following 60 min of incubation in mouse and rat liver microsomes and liver homogenate, the remaining amounts were 36.8%, 39.8%, 61.1%, and 69.0%, respectively, suggesting varying degrees of metabolism and notable species differences (Figure 5c) (Table S2). These results suggest that liver metabolism may be the primary elimination pathway for E3, while its metabolic stability in the gastrointestinal tract is notably high. The validated LC–MS/MS method was utilized in the pharmacokinetic study of E3 in rats following a single intravenous administration at a dose of 1 mg/kg and single oral ( p.o. ) doses of 4 mg/kg. The mean plasma concentration–time profiles for both the single intravenous dose groups and the single oral dose are presented (Figure 5d, e). The pharmacokinetic parameters derived from noncompartmental analysis (NCA) are summarized in Table 2. Notably, the concentration of E3 in rat plasma reached its maximum concentration ( C max ) within 2 h after oral administration, indicating moderate oral absorption in rats. The half-life of E3 in rats following single oral doses was approximately 4.9 h, while the mean residence time (MRT) was around 6.1 h. The bioavailability was measured at 21.1%, which may be attributed to the limited water solubility of E3 and potential absorption saturation, warranting further investigation. The tissue distribution experiment demonstrated the concentrations of E3 in plasma and various tissue samples at 0.5, 2, 6, and 24 h post a single oral administration of 4 mg/kg E3 in rats (Figure 5d). Post-administration, E3 exhibited widespread distribution across the rat tissues. Notably, the E3 concentrations were elevated in organs with high blood perfusion, such as the kidney, heart, lung, and spleen, with the kidney exhibiting the highest concentration (Figure 5d). Conversely, E3 showed relatively low distribution in the brain, testis, indicating that E3 has lower permeability to the blood-brain barrier (BBB) and blood-testis barrier (BTB). The drug concentration in most tissues peaked at 2 h post-administration, then eliminated gradually. 3.7 Toxicity assay showed the safety of E3 In the 14-day acute toxicity assay conducted on mice, all animals survived and exhibited normal behavior and good mental state, indicating LD 50 > 5,000 mg/kg (Figure S3a). During the test, the weight of the mice treated with E3 increased steadily, and the representative blood biochemical indicators were not statistically altered (Figure S3b, c-f). Furthermore, the organ indexes of E3-treated groups showed no significant difference compared to the control group (Table S3). H&E staining results indicated that the heart, kidney, liver, spleen, and brain of the mice were structurally intact, with clear outlines and no apparent abnormalities (Figure S3g). During 30-day of continuous 1,000 mg/kg E3 daily administration, there was no apparent abnormality in the appearance, behavior, secretions, and excretions of all treated mice. There was no significant change in body weight, or organ indexes (Figure 6a, b). In addition, the results of cardiac ultrasound showed no significant abnormality in the left ventricular ejection fractions (LVEF) and left ventricular fractional shortening (LVSF) indicators of mice during and after the administration of E3 (Figure 6c-e). No histopathological toxic lesion related to the treatment with E3 was found in brain, lung, heart, spleen, liver, kidney, testis (Figure 6f). The analysis of blood samples showed that there were no significant changes in blood parameters and blood biochemical indexes between E3 treated and control groups (Figure 6g) (Table S4). Observing mice during the recovery period, it was found that compared with the control group, all mice in the E3 group survived without any abnormal changes in body weight, and organ indexes, indicating that E3 had no delayed toxicity (Figure S3 a, b). Blockade of the hERG (human ether-a-go-go-go related gene) K + channel and the consequent prolongation of the QT interval on the ECG have been considered the gold standard in non-clinical development studies aimed at supporting regulatory guidance (ICHS7) to predict the arrhythmogenic risk of drugs(Vijayakumar et al., 2014). Therefore, we evaluated the effect of compound E3 on the hERG K + channel. Encouragingly, E3 showed a low inhibitory effect on the hERG K + channel with an IC 50 value greater than 33.3μM (Table S2)，indicating low potential cardiac safety issues. Mouse sperm malformation assay is a common type of genotoxicity assays(Juntao et al., 2024). On the 35th day following the oral administration of 2000 mg/kg/day of E3 for five consecutive days, no significant differences were observed in the testicular and epididymal indices (Figure S5a), sperm motility (Figure S5b), the percentage of sperms with abnormal morphology (Figure S5c), sperm counts (Figure S5d, e), or Johnsen score (Figure S5g) when compared to the control group. Histological examination using H&E staining revealed no significant differences in testicular tissue between the E3 mice and the control group (Figure S5f). In contrast, the reproductive toxic compound cyclophosphamide (CP) caused abnormalities in male reproductive indexes (Figure S5a-g). Discussion Our previous study found that the UT inhibitor diarylamides 1H and 25a had oral diuretic activity with the potential to be developed into novel diuretics(S. Wang et al., 2021; Zhang et al., 2021). However, these diarylamide compounds have some limitations that restrict their suitability for further development. Hence, the motivation of this study is to discover the new diarylamides with strong diuretic activity and good druggability, and to develop them into candidate drugs. In this study, we conducted a series of structural optimizations on the primary compound 25a. The 5-acetylfuran fragment and the benzenesulfonamide fragment can enhance UT inhibitory activity. Therefore, we spliced these two fragments using amide bonds to design compounds E3 to E5. Additionally, to address the potential cytotoxicity associated with the acidity of sulfonamide, we introduced a hydroxyl group into the ortho position of the amide, aiming for this hydroxyl group to form a hydrogen bond with the sulfonamide amino group, thereby reducing the ionization of the sulfonamide, which led to the design of compound E6. Furthermore, considering of the potential toxic metabolites of phenylenediamines, we also designed compound E7. To improve the water solubility and molecular weight of the compounds, we incorporated ethoxymorpholine (E8) and propoxymorpholine (E9) into the sulfonamide benzene ring, resulting in a total of nine compounds. The analysis of the structure-activity relationship indicates that the combination of benzene sulfonamide and acetyl furan (E4-E7) significantly enhances the UT inhibitory activity of the compounds. The IC 50 in rat erythrocytes was < 0.1 μmol/L, representing an increase of two orders of magnitude, with the activity ranking as meta- < para- < ortho- position. The ortho position of the phenyl group on the aniline side can accommodate small groups (E6), allowing for the subsequent introduction of additional groups to enhance the water solubility and metabolic stability of the compound. Moreover, based on E7, we introduced morpholine through a connecting arm (E8, E9). Although this modification slightly reduced activity, the inhibitory efficacy did not improve significantly comparaed to 25a. The water solubility of this class of compounds is significantly improved, potentially leading to higher bioavailability. Subsequent in vitro and in vivo studies were conducted to assess the efficacy of the small molecule inhibitor. In vitro experiments revealed that E3 exhibited a much lower IC 50 against UT-B of multiple species than other diarylamide compounds. It was found that E3 had stronger inhibitory activity on UT-A1 than UT-B. Oral E3 administration to rats and mice exhibited stronger diuretic activity than 25a. Furthermore, single and long-term administration of E3 significantly increased urine output with a corresponding decreased urine osmolarity in UT-B knockout mice, but not in UT-A1 knockout mice, indicating that the diuretic effect of E3 based on its highly selective UT-A1 inhibition. Our previous studies confirmed that UT inhibitor 25a alleviated cirrhotic ascites and SIADH caused hyponatremia by exerting a diuretic effect(N. Li et al., 2024; Ying et al., 2023). Compared to 25a (100 mg/kg), E3 significantly improved SIADH hyponatremia at a lower dosage (20 mg/kg) due to its stronger diuretic activity. These data indicate that structurally optimized E3 has promising therapeutic potential to be developed into a ovel diuretic. The metabolic stability is a crucial aspect of new drug development (D. Wang et al., 2019; Yuan et al., 2021). Our study evaluated the pharmacokinetic profile of E3. In plasma metabolic stability tests across multiple species, E3 demonstrated high stability with over 80% of the compound remaining. As we aim to advance E3 as an oral diuretic, its robust stability in SGF, SIF, and intestinal microbiota gave more confidence on its further development. Furthermore, analysis of liver microsomes and homogenates indicated alternative metabolic pathways for E3, beyond phase I metabolism. Pharmacokinetic assessments in rats showed a C max of 170.3 ng/mL (443.0 nmol/L) for E3, surpassing the IC 50 of UT-A1 (18 nmol/L), aligning with efficacy outcomes. Following intragastric gavage administration, plasma concentrations of E3 dropped to less than 2 ng/mL within 24 h, indicating rapid and complete elimination in rats. Notably, E3 exhibited prolonged t max time (2 h vs 0.25 h) and half-life (4.9 h vs 2.86 h) compared to 25a, suggesting slow absorption and favorable long-term effects(Xu et al., 2022). Tissue distribution results indicate that a high distribution of E3 in the kidney is a favorable characteristic for targeting renal UTs. Furthermore, E3 exhibits low permeability to both the blood-brain barrier and the blood-testis barrier, suggesting that the potential risks to the central nervous system and male reproductive system associated with E3 are likely negligible. Subsequent genotoxicity-related experiments have corroborated this finding. Preclinical drug safety evaluation is a crucial step in the initial phase of new drug research and development(N. H. Lee et al., 2018). The safety of E3 was assessed by acute, subacute, genotoxicity and cardiotoxicity studies. In the acute toxicity experiment, mice were given a dose of 5,000 mg/kg/day (>200 times the therapeutic dose). There were no animal death, significant adverse reactions or tissue lesions during the 2-week period after E3 administration, indicating that the E3 has not acute toxicity and has a broad safety margin. The results of the subacute toxicity test showed that there was no death, abnormal body weight, obvious abnormalities in vital organ indexes or pathological change. Hematological and clinical biochemistry analysis showed relatively normal. Moreover, during the 2 weeks observation period after E3 administration for 30 d, no abnormal phenomena were found in the mice, indicating that E3 has no reversible toxic reaction and delayed toxicity, establishing 1,000 mg/kg/day as the no observed adverse effect level (NOAEL). Cardiotoxicity hERG testing showed that E3 had a lower inhibitory effect on the hERG K + channel，indicating a potential safety. Regarding genotoxicity toxicity, results from sperm abnormality tests in mice at a dose of 2,000 mg/kg/day for 5 consecutive days were all negative, indicating that E3 has a low risk of genotoxicity toxicity. Conclusion In this study, a new urea transporter inhibitor E3 was obtained through structural modification of the diarylamide compound 25a. Compared to the lead compound, E3 exhibits high inhibitory activity against both UT-B and UT-A1, with greater selectivity towards UT-A1. E3 exhibits significant diuretic activity in rats and mice following oral administration without causing electrolyte imbalances, and improved SIADH at a lower dosage due to its stronger diuretic activity, confirming its efficacy in therapeutic potential. E3 significantly. Furthermore, E3 shows good metabolic stability in vitro and in vivo , with no apparent cytotoxicity, acute toxicity, subacute toxicity, genotoxicity, or cardiotoxicity observed. These preclinical data suggest that E3 possesses favorable druggability as a new diuretic. This study provides a proof of concept that the diarylamide E3 has the potential to be developed into a novel diuretic for treating hyponatremia associated with volume expansion. Acknowledgements This work was supported by the National Natural Science Foundation of China grants (82273999, 81974083), the Beijing Natural Science Foundation grant (7212151) and the grant from the non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2022-JKCS-15). Author contribution HZ, SYW, NNL, YX, RTL and BXY conceived and designed the research. HZ, SYW, NNL, YX, ZZH, YKZ, JL, YLZ and ML conduct the research. HZ, SYW, NNL and YX analyzed data. HZ wrote the manuscript. BXY revised the revised the manuscript. All authors have read and agreed to the published version of the manuscript. Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary Material File (figures.docx) Download 11.91 MB File (tables.docx) Download 118.87 KB References 1. Adler, S., Verbalis, J. G., & Williams, D. (1994). 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Keywords drug discovery/target validation pharmacodynamics renal pharmacology small molecules systems pharmacology Authors Affiliations Hang Zhang Peking University School of Basic Medical Sciences View all articles by this author Shuyuan Wang Peking University School of Basic Medical Sciences View all articles by this author Nannan Li Peking University School of Basic Medical Sciences View all articles by this author Yue Xu The Ohio State University College of Pharmacy View all articles by this author Zhizhen Huang Peking University School of Basic Medical Sciences View all articles by this author Yukun Zhang Chongqing Key Laboratory of Development and Utilization of Genuine Medicinal Materials in Three Gorges Reservoir Area View all articles by this author Jing Li Sunshine Lake Pharma Co Ltd View all articles by this author Yinglin Zuo Sunshine Lake Pharma Co Ltd View all articles by this author Min Li Peking University School of Basic Medical Sciences View all articles by this author Rentao Li Peking University School of Pharmaceutical Sciences View all articles by this author Baoxue Yang [email protected] Peking University School of Basic Medical Sciences View all articles by this author Metrics & Citations Metrics Article Usage 254 views 163 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Hang Zhang, Shuyuan Wang, Nannan Li, et al. Druggability studies of diarylamide E3 as a novel diuretic. Authorea . 10 January 2025. DOI: https://doi.org/10.22541/au.173651610.01866253/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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