Shun Zhang1,2,3, Yan Zhao2,4,5, Shuyuan Wang2,3, Min Li2,3, Yue Xu2,3, Jianhua Ran6, Xiaoqiang Geng2, Jinzhao He2,3, Jia Meng2,3, Guangying Shao2,3, Hong Zhou2,3, Zemei Ge2,4, Guangping Chen7, Runtao Li2,4, Baoxue Yang1,2,3. 1. Department of Pharmacology, School of Basic Medical Sciences, Peking University, Beijing 100191, China. 2. State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China. 3. Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100191, China. 4. School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. 5. College of Pharmacy, Inner Mongolia Medical University, Hohhot 010110, China. 6. Department of Anatomy and Neuroscience Center, Chongqing Medical University, Chongqing 400016, China. 7. Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Abstract
Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.
Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate, N-[4-(acetylamino)phenyl]-5-nitrofuran-2-carboxamide (1H), with excellent in vitro UT inhibitory activity at the submicromolar level. The half maximal inhibitory concentrations of 1H against UT-B in mouse, rat, and human erythrocyte were 1.60, 0.64, and 0.13 μmol/L, respectively. Further investigation suggested that 8 μmol/L 1H more powerfully inhibited UT-A1 at a rate of 86.8% than UT-B at a rate of 73.9% in MDCK cell models. Most interestingly, we found for the first time that oral administration of 1H at a dose of 100 mg/kg showed superior diuretic effect in vivo without causing electrolyte imbalance in rats. Additionally, 1H did not exhibit apparent toxicity in vivo and in vitro, and possessed favorable pharmacokinetic characteristics. 1H shows promise as a novel diuretic to treat hyponatremia accompanied with volume expansion and may cause few side effects.
Diuretics are mainly used for the treatment of edema, heart failure, liver cirrhosis, hypertension, and nephrotic syndrome. Common diuretics, such as loop diuretics, thiazide diuretics, potassium-sparing diuretics, and carbonic anhydrase inhibitors, induce diuresis by increasing the excretion of Na+, thus indirectlycausing electrolyte disorders, including hypokalemia or hyperkalemia1, 2, 3, which subsequently increase the risks of arrhythmia and sudden cardiac death.In addition to electrolytes, urea is also a major solute in thehyperosmolar renal medulla and plays an important role in urinary concentration management,. Urea transporters (UT), a kind of urea-selective membrane channel proteins, facilitate intrarenal urea recycling5, 6, 7, 8, 9. UTs include seven isoforms, 6 UT-As (UT-A1–A6), and a UT-B encoded by genes Slc14a2 and Slc14a110, 11, 12. In human kidney, UT-A1 and UT-A3 are expressed in the principle cells of the inner medullary collecting duct (IMCD), while UT-A2 is expressed in the thin descending limb. UT-B is expressed in the endotheliocyte of the descending vasa recta in the kidney and other tissues, including erythrocyte, brain, heart, colon, testis, bladder, etc. UTs play a critical role in the generation of concentrated urine,. The functional inhibition of UTs causes diuresis with relative salt-sparing,16, 17, 18, 19, 20. Therefore, as potential diuretic therapeutics, UT inhibitors would not disturb the balance of electrolyte metabolism in vivo and can be more suitable for long-term therapy21, 22, 23, 24. UT-A1 null mice showed higher urine output and fewer extrarenal phenotypes than UT-B null mice, which suggests that UT-A1 is a better diuretic target than UT-B.In recent decades, several classes of potent small molecule inhibitors of UTs have been identified25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. Among these inhibitors, thetriazolothienopyrimidine inhibitor UTBinh-14 (Fig. 1A) is the most potent compound targeting UT-B. However, mice showed mild increased urine output and decreased urine osmolality in high vasopressin and fluid-retaining conditions after intraperitoneal administration. Verkman's group reported 1,2,4-triazoloquinoxaline (Fig. 1B) as an UT-A1 inhibitor, which significantly increased the urine output and reduced the urine osmolality after intravenous administration in a rat model.
Figure 1
Chemical structures of reported UT inhibitors.
Chemical structures of reported UT inhibitors.Our previous work identified a class of UT inhibitors with a thienoquinolin scaffold,,, in which PU-48 (Fig. 1C) displayed strong UT inhibition with half maximal inhibitory concentration (IC50) values of 0.32 and 0.22 μmol/L for UT-A1 and -B, respectively,,. Moreover, PU-48 exhibited excellent diuretic effect in a rat model after hypodermic injection without influencing the levels of Na+, K+ and Cl− in the blood. Further optimization of PU-48 yielded a thienopyridine UT inhibitor (Fig. 1D) with improved water solubility and activity almost equal to that of PU-48 in vitro and in vivo,,,. Dimethylthiourea, an urea analogue with millimolar potency for UT inhibition was identified in previous studies (Fig. 1E),. Nevertheless, none of the novel UT inhibitors mentioned above was effective via oral administration, which significantly prevents their clinical application.In the present study, we report the discovery of a new structural type of potent UT inhibitor with a diarylamide scaffold. The compound N-[4-(acetylamino)phenyl]-4-nitrobenzamide (E04) was identified through high-throughput screening of 1040 urea analogues with IC50 of 5.37 μmol/L. Further optimization of E04 and extensive pharmacodynamic and pharmacokinetic investigation led to the identification of compound 1H with more potent inhibitory activity for UT-A1 than UT-B. Also noteworthy is that for the first time, 1H showed highly favorable diuresis after intragastric administration.
Results and discussion
Discovery of the diarylamide compound E04 as an UT inhibitor
According to the principles of medicinal chemistry, inhibitors of a certain protein usually have structural characteristics similar to that of its endogenous substrate,. Thus, urea analogues were considered the most likely small molecules to become UT inhibitors. In fact, some urea analogues with millimolar potency for UT inhibition were identified in previous studies (Fig. 1E),. In addition, a crystal structure of UT-B (PDB ID: 6QD5) suggested that both the surface and the pore that permeate urea were largely hydrophobic. The structure of UT-A1 is not clear but should be similar to that of UT-B, so that theUT-B inhibitor can often have a significant inhibitory effect on UT-A1. Due to the lack of an effective experimental model for high-throughput screening of UT-A1 inhibitors, the active compounds for UT-A inhibition can be found from UT-B inhibitors. Based on these considerations, high-throughput screening of 1040 urea analogues containing hydrophobic structural units was conducted using thehuman erythrocyte lysis model.Fortunately, three molecules, A01, E04 and E06 (Fig. 2), with diarylamide scaffolds were found to exhibit UT-B inhibitory activity at a concentration of 10 μmol/L with inhibition rates at 25%, 99%, and 40%, respectively. Further evaluation of the IC50 showed that E04 was the most potent compound (IC50 = 5.37 μmol/L), which was worth further optimization as a hit compound.
Figure 2
Chemical structures of the hits A01, E04 and E06 by high-throughput screening.
Chemical structures of the hits A01, E04 and E06 by high-throughput screening.
Optimization of E04
According to the structure of E04, its optimization was divided into three steps (Fig. 3). First, the two aromatic rings (Ar1 and Ar2) were modified to identify the optimal structures. Then, the influence of linker between the two aromatic rings on the inhibition potency was explored. Finally, the substituents on the two aromatic rings (R1 and R2) were optimized.
Figure 3
Optimization strategy of the hit E04.
Optimization strategy of the hit E04.
Optimization of Ar1 in part A
Keeping part B and the linker of E04 intact, thebenzene ring (Ar1) in part A was replaced by six- or five-membered heteroaromatic rings, affording compounds 1A–1G. The inhibitory activities of these compounds against UT-B are presented in Table 1. It is clear that three compounds (1C, Ar1 = 2-furan; 1D, Ar1 = 2-pyrrole; 1E, Ar1 = 2-oxazole) exhibited inhibitory activity against human or ratUT-B, and 1C was slightly better than the others. Additionally, it was observed that removing thenitro group of E04 led to a total loss in activity (1A), which suggested that thenitro group at Ar1 was important for the inhibitory activity. Thus, compound 1H was designed and synthesized by the introduction of thenitro group to thefuran ring of 1C. Compound 1H showed an IC50 value of 0.13 μmol/L for humanUT-B, which was over 41 and 211 times the activity of E04 and 1C, respectively.
Table 1
In vitro UT-B inhibition of arylamides with modification of Ar1.
Compd.
Ar1
IC50 (μmol/L)a
Mouse
Rat
Human
E04
Image 3
>80b
>80
5.37 ± 1.90
1A
Image 4
>80
>80
>80
1B
Image 5
>80
>80
>80
1C
Image 6
>80
>80
27.41 ± 5.60
1D
Image 7
>80
>80
31.09 ± 10.94
1E
Image 8
>80
20.62 ± 5.98
>80
1F
Image 9
>80
>80
>80
1G
Image 10
>80
>80
>80
1H
Image 11
1.60 ± 0.32
0.64 ± 0.20
0.13 ± 0.01
IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.
“> 80” means the compound shows no activity at concentration of 80 μmol/L.
In vitro UT-B inhibition of arylamides with modification of Ar1.IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.“> 80” means the compound shows no activity at concentration of 80 μmol/L.
Optimization of Ar2 in part B
On the basis of the results of the optimization of Ar1, we next selected compound 1H to optimize its Ar2 in part B. Various five- and six-membered heterocycles containing one or more heteroatoms were used as part B, affording the compounds 2A–2L. The activities summarized in Table 2 showed that all compounds exhibited moderate (2B–2I) to strong (2A) inhibitory activity against UT-B except for thethree compounds (2J–2L) derived from five-membered heterocycles containing multiple heteroatoms. Among these compounds, compound 2A (Ar2 = phenyl) showed the best activity, which suggested that benzene ring was more favorable than theheterocycles. However, the activity of 2A was still approximately two times lower than that of compound 1H. Thus, the effect of the substituents of Ar2 on the activity needed further investigation. Moreover, altering thebenzene ring to an aliphatic ring (2M) led to total loss of activity. This result indicated that the presence of an aromatic structure for Ar2 in 1H is very important for activity.
Table 2
In vitro UT-B inhibition of compounds 2A–2M.
Compd.
Ar2
IC50 (μmol/L)a
Mouse
Rat
Human
1H
Image 13
1.60 ± 0.32
0.64 ± 0.20
0.13 ± 0.01
2A
Image 14
3.68 ± 0.39
2.11 ± 0.90
1.30 ± 0.12
2B
Image 15
5.02 ± 1.84
7.77 ± 1.24
1.96 ± 0.53
2C
Image 16
7.86 ± 1.29
9.93 ± 1.62
5.28 ± 1.05
2D
Image 17
3.72 ± 0.37
8.62 ± 2.65
3.17 ± 0.66
2E
Image 18
38.35 ± 2.56
23.88 ± 9.46
18.38 ± 3.04
2F
Image 19
9.45 ± 0.09
6.83 ± 0.85
3.87 ± 0.39
2G
Image 20
20.19 ± 0.70
20.10 ± 4.72
9.94 ± 2.33
2H
Image 21
12.80 ± 0.66
24.07 ± 6.84
9.05 ± 0.11
2I
Image 22
18.18 ± 4.19
23.14 ± 2.92
6.49 ± 0.60
2J
Image 23
>80b
>80
>80
2K
Image 24
>80
>80
>80
2L
Image 25
>80
>80
>80
2M
Image 26
>80
>80
>80
IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.
“> 80” means the compound shows no activity at concentration of 80 μmol/L.
In vitro UT-B inhibition of compounds 2A–2M.IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.“> 80” means the compound shows no activity at concentration of 80 μmol/L.
Optimization of the linker
According to the results of the previous optimization, we selected 1H as the model to examine the effects of different linkers on the activity. As shown in Table 3, all the changes in the linker, whether inserting methylene between the N atom and aromatic ring (3A) or altering theamide to α,β-unsaturated amide (3B), imine (3C) or amine (3D), dramatically reduced potency. Interesting, replacing thehydrogen on N atom of amide with methyl group (3E) led to the loss of activity. Consequently, amide as a linker is essential for UT-B inhibitory activity, and thehydrogen on the N atom should not be replaced.
Table 3
In vitro UT-B inhibition of compounds 3A–3E.
Compd.
Structure
IC50 (μmol/L)a
Mouse
Rat
Human
1H
Image 27
1.60 ± 0.32
0.64 ± 0.20
0.13 ± 0.01
3A
Image 28
>80b
>80
>80
3B
Image 29
>80
>80
>80
3C
Image 30
>80
>80
>80
3D
Image 31
>80
>80
>80
3E
Image 32
>80
>80
>80
IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.
“> 80” means the compound shows no activity at concentration of 80 μmol/L.
In vitro UT-B inhibition of compounds 3A–3E.IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.“> 80” means the compound shows no activity at concentration of 80 μmol/L.
Optimization of the substituents at aromatic rings
The optimized results above indicated that compound 1H was a potential UT-B inhibitor. However, considering that nitro compounds easily induce mutagenicity or genotoxicity and substituents on thebenzene ring significantly affect activity, we next focused on the optimization of R1 and R2.For the optimization of R1, the 2-nitro group on thefuran in 1H was replaced with various substituents, including methyl (4A), bromine (4B), acetylamino (4C), and methylsulfonyl (4D). All the changes resulted in a marked decrease in activity (Table 4). Thus, it is necessary to retain thenitro group in the molecule.
Table 4
In vitro UT-B inhibition of arylamides with modification of R1.
Compd.
R1
IC50 (μmol/L)a
Mouse
Rat
Human
1H
NO2
1.60 ± 0.32
0.64 ± 0.20
0.13 ± 0.01
4A
CH3
>80b
>80
>80
4B
Br
15.12 ± 0.30
20.55 ± 1.05
19.73 ± 1.10
4C
NHCOCH3
16.92 ± 0.48
1.94 ± 0.48
12.98 ± 0.58
4D
SO2CH3
>80
16.13 ± 2.68
13.62 ± 2.07
IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.
“> 80” means the compound shows no activity at concentration of 80 μmol/L.
In vitro UT-B inhibition of arylamides with modification of R1.IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.“> 80” means the compound shows no activity at concentration of 80 μmol/L.For the optimization of R2, to examine the effects of substituted position on activity, moving the 4-acetylamino at thebenzene ring in 1H to the 2- and 3-position given compounds 5A and 5B, respectively. It was found that the position of theacetylamino group significantly affected activity. Compared with 4-position substitution (1H), 2-substitution (5A) caused a significant decrease in the inhibitory activity against themouse, rat, and humanUT-B by 12-, 18-, and 165-fold, respectively. In contrast, the inhibitory activity of 3-substitution (5B) on themouse, rat, and humanUT-B increased by 227%, 28%, and 31%, respectively. However, it was disappointed that compound 5B at a concentration of 15.6 μmol/L showed substantial toxicity to MDCK cells in a cell counting kit-8 (CCK-8) assay, while 1H did not exhibit significant toxicity to MDCK cells even at a concentration of 62.5 μmol/L (Supporting Information Fig. S1A and B). To verify thetoxicity of the 3-substituted analogues, we designed the compound 3-COCH3 (Fig. S1C), which exhibited significant toxicity to MDCK cells at a concentration of 31.3 μmol/L.Due to thetoxicity profile of 3-substitution (5B) and the poor potency of 2-substitution (5A), further optimization of R2 was focused on the 4-substitution. Compounds 5C–5K were designed and synthesized by replacing acetamido with various substituents. As shown in Table 5, substitution with the alkyl group (5C, 4-Me) resulted in a total loss in activity. 4-methoxy (5D) and 4-hydroxy (5E) substitution caused moderate decreases in potency. Halogen-substituted compounds (5F, 4-F and 5G, 4-Cl) and the 4-CN-substituted compound (5H) were much less potent than 1H. Fortunately, compounds 5I (R2 = 4-acetyl), 5J (R2 = 4-carbethoxy), and 5K (R2 = 4-carbamoyl) with carbonyl groups at R2 exhibited excellent inhibition activities with IC50 values of approximately 1 μmol/L. In particular, 5K showed IC50 values of 1.58, 0.14, and 0.14 μmol/L for mouse, rat, and humanUT-B, respectively, which was similarly potent as 1H (R2 = 4-acetylamino). Therefore, the introduction of carbonyl-containing groups was beneficial to activity. Moreover, to improve the solubility, compounds 5L–5N with hydrophilic amines were also designed and synthesized. As expected, these compounds also showed significant activities, especially5N, with an IC50 value of 0.69 μmol/L for humanUT-B. However, intragastric administration of 5N at a dose of 100 mg/kg did not show any diuretic activity on rats.
Table 5
In vitro UT-B inhibition of arylamides with modification of R2.
Compd.
R2
IC50 (μmol/L)a
Mouse
Rat
Human
1H
4-NHCOCH3
1.60 ± 0.32
0.64 ± 0.20
0.13 ± 0.01
5A
2-NHCOCH3
19.95 ± 0.82
11.68 ± 1.36
21.50 ± 7.65
5B
3-NHCOCH3
0.49 ± 0.04
0.50 ± 0.06
0.09 ± 0.01
5C
4-CH3
>80b
>80
>80
5D
4-OCH3
14.65 ± 2.84
4.20 ± 0.92
2.80 ± 0.74
5E
4-OH
11.27 ± 0.89
3.42 ± 0.45
1.70 ± 0.09
5F
4-F
6.07 ± 0.31
4.22 ± 0.76
4.57 ± 1.22
5G
4-Cl
25.17 ± 3.41
10.43 ± 1.18
25.15 ± 5.64
5H
4-CN
8.88 ± 0.98
1.67 ± 0.38
4.25 ± 0.15
5I
4-COCH3
6.66 ± 1.86
1.64 ± 0.14
0.74 ± 0.04
5J
4-COOC2H5
5.76 ± 0.16
3.02 ± 0.74
1.19 ± 0.06
5K
4-CONH2
1.58 ± 0.16
0.14 ± 0.04
0.14 ± 0.01
5L
4-N(CH3)2
3.14 ± 0.26
2.28 ± 0.97
5.45 ± 1.33
5M
4-Image 35
11.89 ± 2.61
2.76 ± 0.57
4.06 ± 0.76
5N
4-Image 36
2.94 ± 0.50
0.78 ± 0.28
0.69 ± 0.11
IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.
“> 80” means the compound shows no activity at concentration of 80 μmol/L.
In vitro UT-B inhibition of arylamides with modification of R2.IC50s are tested with the erythrocyte lysis model. Data are means ± SEM, n = 3.“> 80” means the compound shows no activity at concentration of 80 μmol/L.
Chemistry
Most of the target compounds possess a scaffold of amide, which could be constructed using carboxylic acid and amine as building blocks. Synthesis route of compounds 1A–1H and 4A–4D was described in Scheme 1. Substituted or unsubstituted aromatic carboxylic acids 6A–6L were treated with oxalyl chloride under catalysis of N,N-dimethylformamide (DMF) in CH2Cl2 to give the corresponding aromatic acyl chlorides 7A–7L, which were subsequently reacted with 4-acetylamino phenylamine (8) under basic conditions in tetrahydrofuran (THF) to afford the target compounds 1A–1H and 4A–4D.
Scheme 1
Synthesis method of compounds 1A–1H and 4A–4D. Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2, r.t., 2 h; (b) Et3N, THF, r.t., 2–5 h.
Synthesis method of compounds 1A–1H and 4A–4D. Reagents and conditions: (a) (COCl)2, DMF, CH2Cl2, r.t., 2 h; (b) Et3N, THF, r.t., 2–5 h.The synthesis of substituted furoic acid building blocks 6K and 6L was depicted in Scheme 2. Thenitro in compound 9 was first reduced into amino (10). Then, acetylation of amino and hydrolysis of ester obtained 5-acetylaminofuran-2-carboxylic acid (6K). 5-methylsulfonyl furan-2-carboxylic acid (6L) was prepared from methyl 5-bromofuran-2-carboxylate (12) by methyl sulfonylation and further ester hydrolysis.
Scheme 2
Synthesis route of intermediates 6K and 6L. Reagents and conditions: (a) Pd/C, H2, MeOH, r.t., 2 h; (b) (CH3CO)2O, r.t., 1 h; (c) LiOH, MeOH/H2O, r.t., 15 min; (d) CH3SO2Na, DMSO, 110 °C, 20 h.
Synthesis route of intermediates 6K and 6L. Reagents and conditions: (a) Pd/C, H2, MeOH, r.t., 2 h; (b) (CH3CO)2O, r.t., 1 h; (c) LiOH, MeOH/H2O, r.t., 15 min; (d) CH3SO2Na, DMSO, 110 °C, 20 h.Under the basic conditions, reaction of 7H with different aromatic or aliphatic amines (14A–14M and 15A–15N) yielded the corresponding products 2A–2N and 5A–5N (Scheme 3). Most of theamines could smoothly reacted with 7H at room temperature (r.t.). However, for amines14B–14G, the reactions were performed in dichloroethane at 65 °C for overnight, due to their lower reactivity.
Scheme 3
Synthesis route of 2A–2M and 5A–5N. Reagents and conditions: (a) Et3N, THF, r.t., 2–5 h; (b) DIEA, ClCH2CH2Cl, 60 °C, overnight.
Synthesis route of 2A–2M and 5A–5N. Reagents and conditions: (a) Et3N, THF, r.t., 2–5 h; (b) DIEA, ClCH2CH2Cl, 60 °C, overnight.The synthesis of 3A–3E was the same as that of compounds 1 and 2 (Scheme 4). The building block 18 for 3B was obtained from aldehyde 17 through Knoevenagel reaction, and 19 for 3E was acquired by Borch reduction using paraformaldehyde and amine 8 as starting materials. Thealdehyde 17 condensed with amine 8 to yield imine 3C, which was transferred to 3D by reduction with NaBH4.
Based on the optimization results above, we selected 1H and 5K for further in vivo investigations, as both of these compounds exhibited excellent UT-B inhibition activities and low cell toxicity in vitro (Fig. S1A and D). The diuretic activity of 1H and 5K in mice and rats was determined using metabolic cages. After intragastric administration of a dose of 100 mg/kg, 5K did not show observable diuretic activity in rats (Supporting Information Fig. S2), while 1H showed significant diuretic activity (diuretic activity of 1H will be discussed later). According to our experimental results, 5K showed in vitro inhibition activity on UTs, but no diuretic activity in vivo. We assume that this phenomenon may be due to the higher predicted log P value of 1H than 5K (0.99 vs. 0.21 predicted by Qikprop module from Schrödinger), which results in a better intestinal absorption and a higher oral bioavailability of 1H. Therefore, we further studied the pharmacological characteristics of 1H (Fig. 4A).
Figure 4
Inhibition activity of 1H on UT-B and UT-A1. (A) Structure of 1H. (B) In the erythrocyte lysis assay, inhibition activity of 1H on mouse UT-B. (C) Inhibition activity of 1H on human and rat UT-B. (D) Effect of 1H on UT-B-mediated urea influx measured by stopped-flow light scattering. (E) Effect of 1H on UT-B-mediated urea efflux. (F) Reversibility of UT-B inhibition. 1H was washed out after 5 min of incubation. (G) Inhibition of UT-B-mediated urea influx with different incubation time of 1H. (H) Inhibition of UT-B-mediated urea efflux with different incubation time of 1H. (I) Inhibition activity of 1H on UT-B in MDCK cells. (J) Inhibition activity of 1H on UT-A1 in MDCK cells. (K) Inhibition rate of 1H on UT-B or UT-A1. Data are means ± SEM; n = 3. ∗P < 0.05 indicating that the inhibition rate of 1H on urea transport of MDCK cells transfected with UT-A1 compared with that of MDCK cells transfected with UT-B.
Inhibition activity of 1H on UT-B and UT-A1. (A) Structure of 1H. (B) In the erythrocyte lysis assay, inhibition activity of 1H on mouseUT-B. (C) Inhibition activity of 1H on human and ratUT-B. (D) Effect of 1H on UT-B-mediated urea influx measured by stopped-flow light scattering. (E) Effect of 1H on UT-B-mediated urea efflux. (F) Reversibility of UT-B inhibition. 1H was washed out after 5 min of incubation. (G) Inhibition of UT-B-mediated urea influx with different incubation time of 1H. (H) Inhibition of UT-B-mediated urea efflux with different incubation time of 1H. (I) Inhibition activity of 1H on UT-B in MDCK cells. (J) Inhibition activity of 1H on UT-A1 in MDCK cells. (K) Inhibition rate of 1H on UT-B or UT-A1. Data are means ± SEM; n = 3. ∗P < 0.05 indicating that the inhibition rate of 1H on urea transport of MDCK cells transfected with UT-A1 compared with that of MDCK cells transfected with UT-B.
Inhibition activity of 1H against UT-B
Using erythrocyte lysis assays, the IC50 of 1H against UT-B-mediated urea transport was 1.60 μmol/L in mouse, 0.64 μmol/L in rat, and 0.13 μmol/L in human (Fig. 4B and C). The maximum inhibition rates of 1H among themouse, rat, and human cells were almost 100%. As a control, erythrocyte from UT-B knockout mice was lysed at approximately 100% due to lack of UT-B in the membrane (Fig. 4B).To determine the inhibitory efficacy of 1H against UT-B, theurea permeability in response to a urea gradient was measured by stopped-flow light scattering. The rapid mixing of rat erythrocyte suspension with 500 mmol/L urea solution led to rapid cell shrinking due to water efflux via thewater channel aquaporin 1 (AQP1), then cell swelling due to urea influx via UT-B and water influx via AQP1, which changed the light scattering rate. 1H significantly reduced theurea influx by inhibiting theUT-B function with obvious dose response (Fig. 4D). After incubating erythrocyte in a 500 mmol/L urea solution for 1 h, the erythrocyte was rapidly mixed with isotonic phosphate buffered saline (PBS). It was found that 1H also dose-dependently inhibited theUT-B-mediated urea efflux (Fig. 4E). However, the inhibitory activity against UT-B disappeared after 1H was washed out (Fig. 4F), suggesting that the binding of 1H with UT-B is reversible.To determine the targeting site of 1H on theUT-B molecule, the time-dependent UT-B inhibition was measured by stopped-flow light scattering. The inhibitory activity against the inward or outward urea transmembrane transport was significantly time dependent after erythrocyte was incubated with 10 μmol/L 1H (Fig. 4G and H). Potent inhibition occurred after incubation with 1H for 5 min, which suggested that 1H took time to enter the erythrocyte and targeted the intracellular region of UT-B. To further confirm, the putative site of 1H binding to theUT-B protein was determined by docking computations after homology modeling of thehumanUT-B structure (accession code, CAB60834) based on crystal structure data of a bacterial UT-B homolog. The predicted binding site was in a pocket of intracellular part of UT-B (Supporting Information Fig. S3A and B). Most of the interactions involved neutral-polar and hydrophobic amino acids, including Phe-301, Phe-176, Phe-71, Leu-364, Leu-121, Thr-368 and so on. We could see that Asn-73 had hydrogen-bonding interactions with oxygen of furan ring and theamide linker, and nitro group built salt bridges with Asp-41 and Lys-43, which proved the structure–activity relationship mentioned above that nitro group of furan and theamide linker is necessary for inhibition effect (Fig. S3C).
Inhibition efficacy of 1H on UT-B and UT-A1
As UT-B is widely expressed in various tissues, some side effects may occur after inhibition of UT-B. UT-A1 knockout mice showed more powerful diuretic effect than UT-B knockout mice and had no significant extrarenal phenotype,,44, 45, 46, 47, 48. Therefore, it was recognized that UT-A1 is a better diuretic target than UT-B. MDCK cells do not normally express any UTs. The inhibitory activity of 1H against UT-B and UT-A1 was assayed using MDCK cell lines stably expressing ratUT-B or UT-A1. Therate of 8 μmol/L 1H inhibition of UT-B-mediated urea transport was 73.9%, while that of UT-A1-mediated urea transport was 86.8% (Fig. 4I–K). The experimental results indicate that 1H significantly inhibited both UT-A1 and UT-B, and the inhibitory activity against UT-A1 was greater than that against UT-B.
Diuretic activity of 1Hin vivo
1H was subcutaneously injected into mice and rats at a dose of 100 mg/kg. Urine was collected every 2 h before and after 1H administration. Urine output significantly increased in both themice (Fig. 5A) and rats (Fig. 5B) treated with 1H compared with the controls. The urine output reached its maximum at the 2nd–4th h after 1H administration and continued to increase the urine output for the 4th–6th h in mice. The diuretic effect in rats was more effective than that in mice. The urinary osmolality was reduced at the 2nd h after 1H administration in mice and continued to decrease for 6 h (Fig. 5C). The variation trends of the urine output and urinary osmolality in rats were similar to those in mice (Fig. 5D). The levels of urine output and urinary osmolality returned to the basal level at the 8th–10th h after 1H administration. The excretion of non-urea solutes was not significantly changed after 1H administration in both mice and rats (Fig. 5E and F), indicating that 1Hcauses diuresis without disturbing the electrolyte metabolism.
Figure 5
Diuretic effect of subcutaneous injection of 1H in mice and rats. (A) Urine output of mice. Mice were adapted in metabolic cages for three days. After collecting 2-h basal urine output (time 0), 1H with a dose of 100 mg/kg was administrated by subcutaneous injection, and then urine samples were collected every 2 h. (B) Urine output of rats after 1H administration. (C) Urinary osmolality of mice. (D) Urinary osmolality of rats. (E) Excretion of non-urea solutes of mice. (F) Excretion of non-urea solutes of rats. Data are means ± SEM; n = 6. 0.05, 0.01, 0.001 compared with control mice or rats; P < 0.05, P < 0.001 compared with basal value.
Diuretic effect of subcutaneous injection of 1H in mice and rats. (A) Urine output of mice. Mice were adapted in metabolic cages for three days. After collecting 2-h basal urine output (time 0), 1H with a dose of 100 mg/kg was administrated by subcutaneous injection, and then urine samples were collected every 2 h. (B) Urine output of rats after 1H administration. (C) Urinary osmolality of mice. (D) Urinary osmolality of rats. (E) Excretion of non-urea solutes of mice. (F) Excretion of non-urea solutes of rats. Data are means ± SEM; n = 6. 0.05, 0.01, 0.001 compared with control mice or rats; P < 0.05, P < 0.001 compared with basal value.When1H (100 mg/kg) was given by gavage, the urine output obviously increased in mice (Fig. 6A) and rats (Fig. 6B) at the 2nd h after 1H administration. The urinary osmolality decreased in mice (Fig. 6C) and rats (Fig. 6D) at the 2nd–4th h after 1H administration. The excretion of non-urea solutes in urine was not significantly different between 1H group and the control group in both mice and rats (Fig. 6E and F), indicating 1H does not influence electrolyte excretion. The experimental results suggest that 1H takes effect more quickly by intragastric administration than by subcutaneous administration.
Figure 6
Diuretic effect of intragastric administration of 1H in mice and rats. (A) Urine output of mice. Mice were adapted in metabolic cages for three days. After collecting 2-h basal urine output (time 0), 1H with a dose of 100 mg/kg was administrated by intragastric injection, and then urine samples were collected every 2 h. (B) Urine output of rats after 1H administration. (C) Urinary osmolality of mice. (D) Urinary osmolality of rats. (E) Excretion of non-urea solutes of mice. (F) Excretion of non-urea solutes of rats. Data are means ± SEM; n = 6. 0.05, 0.01 compared with control mice or rats; P < 0.05, P < 0.01 compared with basal value.
Diuretic effect of intragastric administration of 1H in mice and rats. (A) Urine output of mice. Mice were adapted in metabolic cages for three days. After collecting 2-h basal urine output (time 0), 1H with a dose of 100 mg/kg was administrated by intragastric injection, and then urine samples were collected every 2 h. (B) Urine output of rats after 1H administration. (C) Urinary osmolality of mice. (D) Urinary osmolality of rats. (E) Excretion of non-urea solutes of mice. (F) Excretion of non-urea solutes of rats. Data are means ± SEM; n = 6. 0.05, 0.01 compared with control mice or rats; P < 0.05, P < 0.01 compared with basal value.To observe the pharmacological effects of 1H, mice were treated with 100 mg/kg 1H every 8 h for 7 days by intragastric administration. 1Hcaused continuous diuresis and low urine osmolality (Fig. 7A and B). Meanwhile, dailywater intake also increased from 30.0 ± 1.7 to 40.8 ± 4.1 mL (mean ± SEM) after 1H administration. Unsurprisingly, the excretion of urea and non-urea solutes remained unchanged during long-term 1H intragastric administration (Fig. 7C and D). It was also confirmed that 1H could increase the urine output and decrease urinary osmolality (Fig. 7E and F), and the excretion of urea and non-urea solutes remained unchanged during the long-term intragastric administration of 1H in rats (Fig. 7G and H).
Figure 7
Long-term diuretic effect of 1H in mice and rats. Mice and rats were adapted in the metabolic cage for three days, and then urine was collected for 1 day as the basal level. 1H with a dose of 100 mg/kg was given to the experimental group by gavage 3 times a day (the first dose was doubled) for consecutive 7 days. After the last dose, the renal inner medulla and outer medulla were acquired for experiments. (A) Urine output of mice. (B) Urine osmolality of mice. (C) Urea excretion of mice. (D) Excretion of non-urea solutes of mice. (E) Urine output of rats. (F) Urine osmolality of rats. (G) Urea excretion of rats. (H) Excretion of non-urea solutes of rats. (I) Osmolality of the inner medulla (IM) and outer medulla (OM) interstitial fluid of rats. (J) Urea concentration of rats. (K) Concentration of non-urea solutes of rats. Data are means ± SEM; n = 8. ∗P < 0.05, ∗∗P < 0.01 compared with control.
Long-term diuretic effect of 1H in mice and rats. Mice and rats were adapted in the metabolic cage for three days, and then urine was collected for 1 day as the basal level. 1H with a dose of 100 mg/kg was given to the experimental group by gavage 3 times a day (the first dose was doubled) for consecutive 7 days. After the last dose, the renal inner medulla and outer medulla were acquired for experiments. (A) Urine output of mice. (B) Urine osmolality of mice. (C) Urea excretion of mice. (D) Excretion of non-urea solutes of mice. (E) Urine output of rats. (F) Urine osmolality of rats. (G) Urea excretion of rats. (H) Excretion of non-urea solutes of rats. (I) Osmolality of the inner medulla (IM) and outer medulla (OM) interstitial fluid of rats. (J) Urea concentration of rats. (K) Concentration of non-urea solutes of rats. Data are means ± SEM; n = 8. ∗P < 0.05, ∗∗P < 0.01 compared with control.After treatment with 100 mg/kg 1H every 8 h for 7 days by intragastric administration, the osmolality and solutes in the inner and outer medullary tissues of rats were measured. The results showed that the osmolality and urea concentration were significantly lower in the inner medullary tissue of 1H-treated rats compared with control-treated rats (Fig. 7I and J). The non-urea solutes exhibited no significant difference between the1H-treated rats and control rats (Fig. 7K). However, the osmolality, urea concentration, and non-urea solutes concentration in the outer medulla were not different between the1H-treated rats and control rats. These results indicate that 1H plays a diuretic role by blocking intrarenal urea recycling without interfering with the metabolism of Na+, K+, and Cl−.
Toxicity analysis of 1H
After 7-day intragastric administration of 1H, there was no significant difference in body weight and kidney index (ratio of kidney weight to body weight) compared with the control rats (Table 6) or mice (Fig. 8A). Meanwhile, no abnormality was observed in levels of blood urea and creatinine, suggesting 1H did not impair renal function (Table 6). No obvious change was occurred in levels of blood Na+, K+ and Cl− in rats after 1H administration, suggesting that 1H did not influence the electrolyte disturbance that traditional diuretics may lead to (Table 6). Meanwhile, levels of glucose, cholesterol, triglyceride, high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) kept normal after 1H administration, indicating that 1H did not influence the glycometabolism, lipid metabolism. HE-staining did not show morphological abnormality in kidney (data not shown). Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. We detected whether 1H influenced GFR on mice and did not find any abnormality after 1H administration (Fig. 8B).
Table 6
Body weight, kidney index and blood chemistry in control or 1H-treated rats.
Measured parameters
Control
1Ha
Body weight (g)
276.2 ± 18.7
281.9 ± 5.0
Kidney index (%)
0.82 ± 0.09
0.85 ± 0.03
Serum urea (mmol/L)
7.03 ± 0.34
7.05 ± 0.43
Serum creatinine (μmol/L)
33.9 ± 2.9
34.6 ± 8.3
Serum Na (mmol/L)
146.7 ± 2.0
147.3 ± 2.0
Serum K (mmol/L)
4.6 ± 0.1
4.8 ± 0.2
Serum Cl (mmol/L)
102.0 ± 1.5
101.7 ± 1.3
Serum triglyceride (mmol/L)
0.54 ± 0.05
0.68 ± 0.07
Serum HDL-C (mmol/L)
0.70 ± 0.02
0.71 ± 0.03
Serum LDL-C (mmol/L)
0.35 ± 0.03
0.34 ± 0.03
Serum glucose (mmol/L)
9.0 ± 0.3
9.4 ± 0.3
Serum cholesterol (mmol/L)
1.63 ± 0.07
1.59 ± 0.06
P < 0.05 compared with control rats.
Data are means ± SEM, n = 8.
Figure 8
Toxicity assessments of 1H. Mice were adapted in the metabolic cage for three days. 1H with a dose of 100 mg/kg was given to the experimental group by gavage 3 times a day (the first dose was doubled) for consecutive 7 days. The body weight and the GFR were measured. (A) Body weight, n = 8. (B) glomerular filtration rate (GFR), n = 4. Data are means ± SEM. ∗P < 0.05 compared with control mice.
Body weight, kidney index and blood chemistry in control or 1H-treated rats.P < 0.05 compared with control rats.Data are means ± SEM, n = 8.Toxicity assessments of 1H. Mice were adapted in the metabolic cage for three days. 1H with a dose of 100 mg/kg was given to the experimental group by gavage 3 times a day (the first dose was doubled) for consecutive 7 days. The body weight and the GFR were measured. (A) Body weight, n = 8. (B) glomerular filtration rate (GFR), n = 4. Data are means ± SEM. ∗P < 0.05 compared with control mice.Comparing with common diuretics, 1H did not change the non-urea solutes excretion in the urine, and not influence the Na+, K+ and Cl− in blood, indicating less side effect, such as hypokalemia, hyponatremia, hyperuricemia, caused by common diuretics. Meanwhile, 1H did not influence normal renal function and glycometabolism, lipid metabolism in the body, suggesting that 1H is an ideal candidate drug for clinical applications in the future.
UT-A1 selective inhibition activity of 1H
To measure the IC50 against ratUT-A1, MDCK cell lines stably expressing ratUT-A1 were used. The IC50 of 1H against ratUT-A1 was 0.09 ± 0.02 μmol/L (Fig. 9A). Compared with the IC50 of 1H against ratUT-B (0.64 ± 0.20 μmol/L), we concluded that 1H exhibited higher inhibitory activity against UT-A1 than UT-B (Fig. 9B). To further confirm this conclusion, UT-A1 knockout mice and UT-B knockout mice were treated with 100 mg/kg 1H by intragastric administration. The urine output obviously increased (Fig. 9C) at 2nd h after 1H administration in UT-B knockout mice, and the urinary osmolality correspondingly decreased in UT-B knockout mice (Fig. 9D). However, the urine output and urinary osmolality did not change significantly in UT-A1 knockout mice (Fig. 9C and D). As UT-A1 was more suitable as a diuretic target than UT-B, 1H was more likely to be developed as a novel diuretic.
Figure 9
Selective inhibition activity of 1H on UT-A1. (A) Inhibition activity of 1H on rat UT-A1 determined with MDCK cells expressing rat UT-A1. (B) Calculated IC50 of 1H on rat UT-B and UT-A1. (C) Urine output of UT-A1 and UT-B knockout mice. Mice were adapted in the metabolic cage for three days. After collecting 2-h basal urine output (time 0), 1H with a dose of 100 mg/kg was administrated by intragastric injection, and then urine samples were collected every 2 h. (D) Urinary osmolality of UT-A1 and UT-B knockout mice, n = 6. Data are means ± SEM. ∗P < 0.05, ∗∗P < 0.01 compared with control mice.
Selective inhibition activity of 1H on UT-A1. (A) Inhibition activity of 1H on ratUT-A1 determined with MDCK cells expressing ratUT-A1. (B) Calculated IC50 of 1H on ratUT-B and UT-A1. (C) Urine output of UT-A1 and UT-B knockout mice. Mice were adapted in the metabolic cage for three days. After collecting 2-h basal urine output (time 0), 1H with a dose of 100 mg/kg was administrated by intragastric injection, and then urine samples were collected every 2 h. (D) Urinary osmolality of UT-A1 and UT-B knockout mice, n = 6. Data are means ± SEM. ∗P < 0.05, ∗∗P < 0.01 compared with control mice.
Pharmacokinetics of 1H
The pharmacokinetic parameters of 1H were measured both in vitro and in vivo. Membrane permeability is a key parameter to assess the absorption of oral drugs in the intestine49, 50, 51, 52, and the apparent permeability coefficient (Papp) in theCaco-2 cell model corresponds to the fraction absorbance (Fa) of oral drug administration in thehuman intestine. As shown in Fig. 10A, thePapp (A to B) of 1H was (10.46 ± 1.36) × 10−6 cm/s in theCaco-2 bidirectional transport assay, which was comparable to the high permeability compound metoprolol [(13.08 ± 0.65) × 10−6 cm/s]. The high Papp (A to B) value demonstrated that 1H obtained a better membrane permeability that led to adequate oral absorption in the gastrointestinal tract. The efflux ratio of 1H was 2.00 ± 0.14 in Caco-2 cell monolayers, and the addition of the efflux transporter inhibitor GF120918 did not significantly alter the efflux ratio (Fig. 10B), which indicates that 1H may not be a sensitive substrate for efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP).
Figure 10
Pharmacokinetics of 1H. (A) The apparent permeability of 1H in the Caco-2 cell. (B) The efflux ratio of 1H in Caco-2 cell monolayers. (C) Plasma concentration–time profiles of 1H in rats. Data are means ± SEM, n = 3.
Pharmacokinetics of 1H. (A) The apparent permeability of 1H in theCaco-2 cell. (B) The efflux ratio of 1H in Caco-2 cell monolayers. (C) Plasma concentration–time profiles of 1H in rats. Data are means ± SEM, n = 3.The mean plasma concentration–time profiles of 1H in rats are shown in Fig. 10C. 1H was quickly absorbed after oral gavage at a 100 mg/kg dose, and the time to reach the maximum concentration (Cmax=0.26 ± 0.12 μmol/L) was 0.31 ± 0.13 h. Then, the plasma concentrations slightly decreased, and the plasma half-life (t1/2) was 0.93 ± 0.09 h. The results demonstrated that 1H obtained a good gastric-intestinal absorption in rats. The oral bioavailability of 1H was 4.38 ± 1.30%, so it needs a higher dose (100 mg/kg) to exert an obvious oral activity, despite a potent UT inhibitory activity of 1H
in vitro. In the following project, it is necessary to further optimize the structure or improve the dosage form to increase the oral bioavailability of 1H for its clinical applicability. Eight hours after oral administration, the mean plasma concentrations decreased to levels below the lower limit of quantification (1 ng/mL), which suggests that 1H was rapidly and completely cleared from therat plasma and may have other metabolism-related issues in the in vivo deposition process. The Cmax was 0.26 ± 0.12 μmol/L, which was more than the IC50 against UT-A1 (0.09 ± 0.02 μmol/L) and less than the IC50 against UT-B (0.64 ± 0.20 μmol/L) and reached the effective concentration measured in vitro. Inhibiting UT-A1rather than inhibiting UT-B is a great merit of the development of 1H as a diuretic.
Conclusions
In this study, we discovered 1H as a novel small molecule that inhibited both UT-A and UT-B with IC50 values at the submicromolar level and inhibited UT-A more than UT-B. For the first time, 1H showed significant diuretic activity by oral administration without causing electrolyte imbalance. 1H did not exhibit apparent toxicity either in vitro or in vivo. All these results suggest that 1H might be developed as a novel diuretic to treat hyponatremia accompanied with volume expansion, such as hepatic cirrhosis, congestive heart failure, and nephrotic syndrome.
Experimental
Starting materials, solvents, and reagents were commercially available and used without further purification. 1H spectra and 13C 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). Melting points were determined on X4 microscope (Beijing, China) and uncorrected. The maximum temperature was 300 °C. HR-MS spectra were acquired by electrospray ionization (ESI) in positive ion mode using Bruker Solarix XR FTMS (Bruker).
Purity of primary compounds
Purity of all compounds tested in biological assays was determined to be >95% by HPLC analysis. The following methods were used: HPLC-Agilent 1260 (Agilent Technologies Inc., Palo Alto, CA, USA), Agilent zorbax eclipse ZOEBAX SB-C18 (150 mm × 4.6 mm, I.D. 5 μm), DAD (254 or 260 nm) detector, water (mobile phase A), methanol (mobile phase B), 0 min 20% B, 8 min 55% B, 20 min 80% B; or Waters e2695 (Waters, Milford, MA, USA), shim-pack VP-ODS (150 mm × 4.6 mm, I.D. 5 μm), DAD (254 or 260 nm) detector, water (mobile phase A), methanol (mobile phase B), 0–8 min 20% B; 9 min 55% B; 20 min 80% B.
General synthetic procedure A
Compounds 6A–6L (2.0 mmol) in CH2Cl2 (5 mL) were cooled to 0 °C. Three drops of DMF was added followed by dropping oxalyl chloride (3 mmol, 380 mg) slowly to the suspension. After 2 h of stirring at r.t., the reaction mixture was concentrated under reduced pressure to remove solvent and excessive oxalyl chloride to afford crude material of 7A–7L without further purification.Compound 8 (1.0 mmol, 150 mg) and Et3N (1.5 mmol, 152 mg) in THF (5 mL) were cooled to 0 °C and then 7A–7L prepared above in THF (2 mL) were dropped slowly to the solution. The reaction mixture was stirred for 2–5 h at r.t. After the reaction completed, water was added and the mixture was stirred for further 10 min. The precipitate was filtered, washed with water and recrystallized with ethanol or methanol to afford the pure product.
N-(4-Acetamidophenyl)benzamide (1A)
Prepared from 6A following general synthetic procedure A. White solid; Yield 75%; m.p. 211–213 °C.
Amines (14A, 14H–14M, 15A–15N, 16 or 19, 1.0 mmol) and Et3N (1.5 mmol, 152 mg) in THF (5 mL) were cooled to 0 °C, and then 7H (1.0 mmol, 175 mg) in THF (2 mL) was dropped slowly to the solution. The reaction mixture was stirred for 2–5 h at r.t. After the reaction completed, water was added and the mixture was stirred for further 10 min. The precipitate was filtered, washed with water and recrystallized with ethanol or methanol to afford the pure products.
NaBH4 was added in batches to a solution of compound 3C (180 mg, 0.66 mmol) and methanol (5 mL). The mixture was stirred at r.t. overnight. Water (5 mL) was added. The resulted mixture was stirred for further 30 min and filtered. The filtrate was extracted with ethyl acetate (5 mL × 3). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated. Ethanol (2 mL) following by 5 drops of 36% hydrochloric acid to the residue were added. After cooled at 4 °C for 2 h, yellow crystals were precipitated as thehydrochloride of compound 3D which was afforded by filtration. Yield 60%; m.p. 159–161 °C. 1H NMR (400 MHz, MeOD) δ 7.69 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 3.6 Hz, 1H), 7.29 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 3.6 Hz, 1H), 4.77 (s, 2H), 2.13 (s, 3H). 13C NMR (101 MHz, MeOD) δ 170.45, 152.68, 149.02, 138.87, 131.34, 123.10, 121.72, 120.88, 120.74, 115.29, 111.71, 45.92, 22.40. HR-MS m/z: Calcd. C13H14N3O4 [M+H]+: 276.0979; Found: 276.0979.
A mixture of compound 8 (300 mg, 2 mmol), paraformaldehyde (300 mg, 10 mmol) and MeONa (540 mg, 10 mmol) in methanol (30 mL) was refluxed. The reaction was monitored by TLC (CH2Cl2/MeOH = 10:1, v/v) until compound 8 was reacted completely. The reaction mixture was cooled to r.t. And NaBH4 (152 mg, 4 mmol) was added in batches. The resulted mixture was stirred overnight at ambient temperature. Solvent was removed. The residue was diluted with 2 mol/L hydrochloric acid (5 mL) and ethyl acetate (5 mL). The two layers were separated. The aqueous layer was adjusted with saturated Na2CO3 solution to pH 9 and extracted with ethyl acetate (5 mL × 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography (silica gel, CH2Cl2/MeOH = 80:1, v/v) to afford compound 19 as white solid (250 mg, 76% Yield). The title compound 3E was prepared from compound 19 following general synthetic procedure B. Yield 72%; m.p. 81–83 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 2.7 Hz, 1H), 7.30 (d, J = 8.7 Hz, 2H), 5.91 (s, 1H), 3.37 (s, 3H), 2.06 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.01, 157.02, 151.44, 147.87, 139.76, 137.59, 128.18, 120.06, 117.90, 113.07, 38.61, 24.51. HR-MS m/z: Calcd. C14H14N3O5 [M+H]+: 304.0928; Found: 304.0929.
Amines (14B−14G, 1.0 mmol) and DIEA (1.5 mmol, 194 mg) in dichloroethane (5 mL) were cooled to 0 °C, and then 7H (1.0 mmol, 175 mg) in dichloroethane (2 mL) was dropped to the suspension. The reaction mixture was stirred at 65 °C for 3 h. After cooled to r.t., the reaction mixture was concentrated and diluted with water (15 mL) and EtOAc (15 mL). The two layers were separated and the aqueous layer was extracted with EtOAc (15 mL × 2). The combined organic layer was washed successively with saturated NaHCO3 solution, 10% citric acid and brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography (silica gel, petroleum ether/ethyl acetate = 5:1, v/v) to afford the pure products.
C57BL/6 mice (body weight: 20–21 g, male) and adult male Sprague–Dawley (SD) rats (body weight: 190–210 g) were supplied by the Experimental Animal Center, Peking University (Beijing, China). UT-B knockout mice in a C57BL/6 genetic background were introduced from the University of California, San Francisco (UCSF, San Francisco, CA, USA). UT-A1 knockout mice in a C57BL/6 genetic background were generated by targeted gene disruption. All animal protocols were approved by the Ethics Committee of Peking University (Beijing, China).
Blood samples
Human venous blood was taken from a healthy adult male volunteer, which was approved by the Ethics Committee of Peking University, (Beijing, China). Rat blood was collected from male SD rats (210–230 g) by orbital venous plexus puncture. Mouse blood was collected from male (20–21 g) wild-type or UT-B-null mice on a C57BL/6 genetic background 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 (TDZ5-WS, Changsha, China, 2000 r/min) after washed by PBS (0.01 mol/L, pH = 7.4) for three times. Then the erythrocyte was used for experiments in 12 h.
Compounds
Compounds for preliminary screening were purchased (Selleck, Shanghai, China) or gifted from of Peking University School of Pharmaceutical Sciences (Beijing, China) and were dissolved in DMSO.
Erythrocyte lysis assay for determining UT-B inhibition activity
The erythrocyte lysis assay was adopted after modified from a method described previously. Erythrocyte was diluted to a hematocrit value of 2% (200 μL erythrocyte diluted in 10 mL PBS) in PBS (0.01 mol/L, pH = 7.4, r.t.) containing 1.25 mol/L urea and 5.0 mmol/L glucose. Erythrocyte was incubated at r.t. for 2 h. Moving 100 μL of the erythrocyte suspension to a 96-well microplate, then added 1 μL testing compound (8, 2, 0.5, 0.12, 0.03 and 0.007 mmol/L dissolved in DMSO) to erythrocyte suspension and shook it up with microoscillator (QILINBEIER, Haimen, China) for 1 min. After 5 min of incubation, 20 μL of the erythrocyte suspension was rapidly transferred to a 96-well black wall microplate that contained 180 μL isotonic PBS (0.01 mol/L PBS without urea). Following quickly and sufficiently mixing (blow and suck with the pipettor for ten times), erythrocyte lysis was quantified by measuring absorbance at 710 nm wavelength with a plate reader (BioTek, Winooski, VT, USA) within 5 min. Each assay plate included negative no-lysis controls (1.25 mol/L urea + isotonic PBS with 1% DMSO) and positive full-lysis controls (distilled H2O with 1% DMSO) that were mixed with vehicle-treated erythrocyte suspension.The percentage of erythrocyte lysis in each test well was calculated using control values from the same plate as Eq. (1):where Atest, Aneg and Apos are the absorbance values from a test well, a negative no-lysis control well and a positive full-lysis control respectively. IC50s are calculated by the software Graphpad Prism 5 with a log (inhibitor) vs. response analysis.
Measurement of urea permeability in erythrocyte by stopped-flow light scattering
Erythrocyte was acquired from rat blood and suspended in PBS (0.01 mol/L, pH = 7.4, r.t.) to 0.5% (50 μL erythrocyte diluted in 10 mL PBS). Then erythrocyte was incubated with targeted compounds for 5 min and quickly mixed with 500 mmol/L urea dissolved in PBS in an SX20 instrument (Applied Photophysics, Leatherhead, UK) with dead time of ∼1.2 ms as described previously,. The stopped-flow light scattering was measured as the time course of 90° scattered light intensity at 530 nm. Keep samples and PBS at 4 °C to reduce the influence of free diffusion. To test reversibility, compounds were added to erythrocyte for 5 min, then washed with PBS [1 mL erythrocyte was washed out by 10 mL PBS, and erythrocyte was acquired by 2000 r/min centrifugation (TDZ5-WS)] for 3 times before stopped-flow measurements. To determine inhibition on urea efflux, erythrocyte was incubated with 500 mmol/L urea dissolved in PBS for 2 h, then mixed with PBS without urea.
Cytotoxicity assay
MDCK cells (passage numbers: 4–5) were cultured at 37 °C in a humidified 95% air/5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin. MDCK cells were cultured in a 96-well plate (5000 cells per well). When cells were grown to 50% confluence, synchronized by DMEM without fetal bovine serum for 12 h. Then cells were exposed to the compounds (1H, 5B and 5K) at 0, 15.6, 31.3, 62.5, 125 and 250 μmol/L for 24 h. A cell counting kit-8 (Dojindo, Kumamoto, Japan) was used to measure cytotoxicity of compounds with different concentrations. The absorbance at 450 nm was measured 1–2 h after CCK-8 solution (10%, 100 μL per well) was added to each well. Cytotoxicity was expressed as cell viability rate. The cell viability rate was calculated using control values from the same plate as Eq. (2):where ODtest, ODblank and ODcontrol are the absorbance values from a test well, a blank control well and a solvent control well respectively.
Assay of UT-A1-mediated and UT-B-mediated urea permeability
MDCK cells steadily expressing theUT-A1 or UT-B were used. The mRNA level of UT-A1 and UT-B was measured in our previous research. Measurement of urea flux was as described previously. MDCK cells (2 × 105 cells/cm2) that stably expressed ratUT-A1 were grown on 12 mm collagen-coated Costar Transwell inserts (0.4 μm pore size; Corning, NY, USA) for 4 days at 37 °C in the presence of 5% CO2. When cells in the apical side grew to become tight monolayer (transepithelial resistance 1 kΩ/cm2), PBS (pH = 7.4, containing 10 μmol/L forskolin) with 1H or DMSO was added into top (0.25 mL) and bottom (1 mL) and cultures were incubated in the absence of urea for 30 min at 37 °C. As UT-B is located in the plasma membrane while UT-A1 is located in the cytoplasm, so forskolin was used to stimulate the transport of UT-A1 from cytoplasm to membrane to transport urea. Then, the solution in the bottom was replaced by PBS (pH = 7.4, containing 10 μmol/L forskolin, and containing 1H or DMSO) with 15 mmol/L urea. The solution on the top (5 μL) was collected at 0, 1, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min to test theurea concentration by QuantiChrom Urea Assay kit (BioAssay Systems, Hayward, CA, USA). The initial slope of urea concentration curve was calculated by absorbance values at 520 nm by Graphpad Prism 5. Inhibition of UT-A1-mediated urea permeability was calculated as Eq. (3):where 1Hinitial slope and DMSOinitial slope are initial slope values of cultures treated with 1H or DMSO. controlinitial slope is initial slope values of MDCK cells without UT expression.The method to measure theUT-B-mediated urea permeability was the same as the method to measure theUT-A1-mediated urea transport except thePBS without forskolin because UT-B was highly expressed in theMDCK cell membrane.The method to measure the IC50 on ratUT-A1 was the same as the method to measure theUT-A1-mediated urea transport except the concentration of 1H was 5, 1.25, 0.31, 0.08, 0.02, and 0.005 μmol/L. IC50 was calculated by the software Graphpad Prism 5 with a log (inhibitor) vs. response analysis.
Measurement of diuretic activity
Male wild-type mice, UT-B knockout mice (acquired by CRISPR/Cas9 gene-editing technique), UT-A1 knockout mice (acquired by CRISPR/Cas9 gene-editing technique), or SD rats were adapted in metabolic cages (Ugo Basile, Comerio, VA, Italy) for 3 days. Water and food were provided ad libitum. Bladder was emptied by gentle abdominal massage and urine was collected by metabolic cages every 2 h. 1H (10 mg/mL) in corn oil (Yuanye, Shanghai, China) was administered by subcutaneous injection or in carboxymethylcellulose sodium (CMC-Na, Sigma, St. Louis, MO, USA) was administered by gavage on mice or rats (100 mg/kg). Corn oil or CMC-Na was used as a 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, Norwood, MA, USA). Urea concentration was measured with the QuantiChrom urea assay kit (BioAssay Systems).In long-term diuretic activity experiments, 1H (10 mg/mL) dissolved in CMC-Na was administered to rats at a dose of 100 mg/kg (the first dose was double in order to reach an effective concentration faster) by gavage every 8 h. CMC-Na was used as a vehicle control. Urine was collected every 24 h by metabolic cages. Body weight and water intake were measured every day. Two hours after the last administration, a blood sample was collected by heart puncture. Inner medulla and outer medulla tissue homogenates were obtained, and the supernatant after centrifugation was assayed for solute concentration and osmolality. Urinary osmolality and urea concentration were measured as above. Serum Na+, K+, Cl−, glucose, cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein were measured in a clinical chemistry laboratory. Serum creatinine was measured through specific reagent kits (NJJC Bio, Nanjing, China).
Measurement of GFR
We measure the GFR in mice using an optical device (Biotimestech, Hong Kong, China) and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin,. After 1H intragastric administration every 8 h for 7 days, wild-type mice were anesthetized by 3% isoflurane and then remove thefur by depilatory paste from the flank of the back. Fixation of the device on the animal, then inject through tail vein theFITC-sinistrin stock solution (15 mg/mL) at a dose of 50 mg/kg. Mice were placed in a calm place to avoid being disturbed. The measurement was performed during at least 1 h.
Histology
Kidney and liver were fixed with paraformaldehyde and embedded in paraffin. 5 μm paraffin sections were cut and stained with hematoxylin and eosin.
Transport assay in Caco-2 cell monolayer
Caco-2 cells (passage numbers: 42–50) were obtained from the American Type Cell Culture (ATCC; HTB-37, Manassas, VA, USA). Caco-2 cells were maintained in the sterile cell culture flasks (Corning Life Science) at 37 °C cell culture incubators (Thermo, Waltham, MA, USA) with 5% CO2, and saturated humidity in MEM with 10% FBS for cell culture. Caco-2 cells were seeded at a density of 50,000 cells/cm2 into the apical chamber of Transwell® system (0.0804 cm2, 1 μm pore PC insert, Corning Life Science).The transport assay in Caco-2 cell monolayer was conducted as previous reported. TheLucifer Yellow (LY) rejection assay was conducted to determine the cell monolayer integrity at the same duration of test compounds in the assay. The solutions added into apical and basolateral wells were 75 and 250 μL respectively. The final concentration of all test compounds was 2 μmol/L in transport buffer. The plates of Caco-2 were incubated for 120 min in CO2 incubator at 37 °C, with 5% CO2 at saturated humidity. The initial dosing solution was mixed with stop solution (acetonitrile containing 250 ng/mL tolbutamide as the internal standard) as the T0 sample. After reaching the incubation time, 50 μL terminal samples were collected from the donor and receiver sides of each well, and mixed with 250 μL stop solution for LC−MS/MS analysis. The apparent permeability (Papp, cm/s), efflux ratio (ER) and recovery parameters were calculated for drug transport assay using Eqs. (4), (5):where VR is the solution volume in the receiver chamber; Area is the surface area for the insert membrane, i.e., 0.0804 cm2 for the area of the monolayer; Time is incubation time, expressed in s; C0 is the initial peak area ratio (PAR) of control compounds in the donor chamber.The high permeability compound metoprolol (TargetMol, Boston, USA) and a P-gp substrate digoxin (Sigma–Aldrich, St. Louis, USA) were used as controls in the assay, and the inhibitor of multiple efflux transporters GF120918 was added to determine that whether 1H is a substrate of efflux transporters.
Rat pharmacokinetics
Male SD rats (7–9 weeks, 250–270 g) were purchased from Vital River (Beijing, China) and fed with standard food and water at a stable temperature (22 ± 2 °C) and humidity (55 ± 5%) with a 12 h light–dark cycle. A 10.0 mg/mL 1H suspension resolved in 0.5% CMC-Na was administrated at 100 mg/kg in oral routine. Blood samples (250 μL) were collected via tail vein puncture at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h in tubes with anticoagulant (K2EDTA), and the plasma samples were obtained by centrifugation at 4000 rpm (ThermoScientific, Shanghai, China) for 15 min. The plasma samples were spiked with 200 μL of 5 ng/mL terfenadine (internal standard, IS) in MeOH/acetonitrile (1:1, v/v), vortexed for 1 min and centrifuged at 4000 rpm (ThermoScientific) for 15 min. The supernatant was diluted 10-fold with MeOH/water (1:1, v/v, with 0.1% formic acid) for injection, and the injection volume was 6 μL. Thecalibration standards were prepared by spiking the working solution into untreated rat plasma at the final concentrations of 1–1000 ng/mL. Thecalibration curves were fitted with a linear regression model (r > 0.99) weighted by 1/(x × x). The oral bioavailability was calculated by (AUC × Dosei.v.)/(AUCi.v. × Dose).
Sample analysis
Analysis of rat plasma was performed on a high-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) system consisting of an API 5500 Mass Spectrometer (AB Sciex, Foster City, CA, USA), Shimadzu LC-20AD and Shimadzu SIL-20 A C (Shimadzu, Japan). The MS acquisition was operated in the electrospray (ESI) positive mode. Chromatographic separation was performed on a Kinetex 2.6 μm C18 100 Å column, 50 mm × 3.00 mm (Phenomenex, Torrance, CA, USA) at r.t. using a mobile phase of 5 mmol/L NH4OAc supplemented with 0.05% (v/v) formic acid (solvent A) and acetonitrile supplemented with 0.1% (v/v) formic acid (solvent B). The gradient was performed with a total flow at 0.7 mL/min as follow: 0–0.40 min 5% (B), 0.40–2.20 min 5%–95% (B), 2.20–2.30 min 95% (B), 2.30–2.31 min 95%–5% (B), 2.31–3.00 min 5% (B). Quantification was achieved by multiple reaction monitoring to identify the analytes (1H) and IS (terfenadine). The retention times of 1H and IS were 1.73 and 2.08 min, respectively. The declustering potential (DP) and collision energy (CE) were optimized as followed: DP: 61V CE: 29V for 1H, DP: 66V CE: 50V for terfenadine. The selected mass transitions were m/z 290.1 → 231.2 for 1H, m/z 472.4 → 436.4 for terfenadine, respectively. AB SCIEX Analyst® software (version 1.6.1) was used for data acquisition and analysis.
Molecular docking
A homology model of humanUT-B was generated using the SWISS MODEL online utility (http://swissmodel.expasy.org) in automated mode, using the sequence of the full humanUT-B protein (accession code, CAB60834). 1H was drawn in ChemDraw (CambridgeSoftware, Cambridge, MA, USA). TheUT-B protein was prepared for docking using the Protein Preparation (maestro, Schrödinger), using the homology model of humanUT-B. 1H was prepared using the LigPrep (maestro, Schrödinger). Docking was performed using Ligand Docking.
Data analysis
Statistical analysis was performed using Graphpad Prism 5 software. All of the quantitative data are expressed as means ± SEM. Statistical analysis was performed using Student's t-test, one-way ANOVA followed by Fisher's least significant difference analysis for multiple comparisons. P < 0.05 was considered statistically significant.
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