Bumepamine, a brain-permeant benzylamine derivative of bumetanide, does not inhibit NKCC1 but is more potent to enhance phenobarbital's anti-seizure efficacy

Abstract

The authors regret that the description of the synthesis of bumepamine in the above-mentioned article lacked an important aspect and is therefore incorrect.The correct synthesis of bumepamine is given below. The authors would like to apologise for any inconvenience caused. Synthesis of bumepamineBumetanide (1) (500 mg, 1.37 mmol) was dissolved in 9 mL dry N, N-dimethylformamide and N,N-diisopropylethylamine (765 μL, 4.39 mmol), followed by addition of aniline (250 μL, 2.74 mmol). The mixture was cooled on an ice-bath. COMU (707 mg, 1.65 mmol) was added in one portion and the mixture was gradually warmed to room temperature and stirred for 16 h. The reaction was quenched with saturated aqueous NaHCO 3 solution and extracted twice with ethyl acetate (EtOAc). The combined organic layers were washed with water, brine and dried over Na 2 SO 4 . The crude product was purified by column chromatography (toluene:EtOAc:Et 3 N, 3:2:0.01). The desired product (2; the phenylamide of bumetanide) (540 mg, 1.22 mmol) was obtained in 89% yield as a slightly yellow solid. 1 H NMR (400 MHz, d 4 -MeOH) δ 7.79 (d, J = 2.1 Hz, 1H), 7.68-7.71 (m, 2H), 7.50 (d, J = 2.1 Hz, 1H), 7.36-7.40 (m, 2H), 7.28-7.32 (m, 2H), 7.14-7.19 (m, 1H), 7.05-7.09 (m, 1H), 6.93-6.96 (m, 2H), 3.17 (t, J = 6.7 Hz, 2H), 1.40-1.48 (m, 2H), 1.13-1.21 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H). 13 C NMR (100 MHz) d 4 -calculated for C 23 H 26 N 3 O 4 S [M+H] + 440.1644, found 440.1646.Next, the phenylamide of bumetanide (2) (250 mg, 0.57 mmol) was dissolved in 12 mL tetrahydrofuran and borane dimethylsulfide complex (90 μL, 0.95 mmol) was added at room temperature. The reaction mixture was stirred for 16 h at 70°C and cooled to room temperature. Since some starting material was still present, an additional amount of the borane dimethylsulfide complex (90 μL, 0.95 mmol) was added and the reaction was stirred for 5 h at 70°C. The reaction mixture was cooled to room temperature and then quenched with half-saturated aqueous NaHCO 3 solution. The mixture was extracted three times with EtOAc and the combined organic layers were dried over Na 2 SO 4 . The crude product was purified by column chromatography (CH 2 Cl 2 :MeOH, 50:1). The obtained oily substance was dried under vacuum for 4 hours and then dissolved in 10 mL dry diethyl ether (Et 2 O). 2M HCl in Et 2 O (135 μL, 0.27 mmol) was added and the flask left to stand for 10 minutes. The salt that was formed was filtered, washed three times with Et 2 O and the desired product (3; bumepamine) (90 mg, 0.21 mmol) was obtained as a slightly beige solid in 37% yield. 1 H NMR (400 MHz, d 4 -MeOH) δ 7.52-7.60 (m, 3H), 7.44-7.46 (m, 2H), 7.33 (d, J = 2.0 Hz, 1H), 7.68-7.30 (m, 2H), 7.04-7.08 (m, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.86-6.89 (m, 2H), 4.64 (s, 2H), 3.00 (t, J = 6.8 Hz, 2H), 1.29-1.37 (m, 2H), 1.06-1.15 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H). 13 C NMR (100 MHz) d 4 -

Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66

“…As shown in Figure 1B, exposure of GL26 glioma cells to 10-60 μM BMT decreased the total Rb + uptake by ~54.3% (p < 0.0001) in isotonic conditions (310 mOsm). No additional inhibition was observed with BMT at >10 μM (p > 0.05, Figure 1B), which is consistent with report showing that BMT (IC 50 of 0.1 μM) with concentrations from 1 to 10 μM completely blocked NKCC1 activity (Brandt et al, 2018). In response to hypertonic osmotic stress (400 mOsm), GL26 glioma cells elevated the total Rb + uptake by ~81.0% (p < 0.0001, Figure 1B).…”

Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders

“…To enhance BBB penetration, some studies have evaluated lipophilic analogs of bumetanide. 36 , 37 However, these studies have no insights about the analogs’ diuretic effect relative to bumetanide or about the establishment of a potential association between their activity and factual targeting of NKCC1. 36 This has suggested the possibility that bumetanide may influence brain-related behaviors not by NKCC1 inhibition but by being a diuretic (i.e., through its well-known osmotic regulation and/or by altering ionic balance).…”

Clearance of activity‐evoked K+ transients and associated glia cell swelling occur independently of AQP4: A study with an isoform‐selective AQP4 inhibitor

“…These experimental findings were all based on AQP4 −/− mice, which were later demonstrated to have secondary changes in basic parameters, such as extracellular space size, brain water content, expression of glutamate transporters and connexins (Haj‐Yasein, Vindedal, et al, 2011; Strohschein et al, 2011; Yao et al, 2008; Zeng et al, 2007), which may have introduced confounding elements in the experimental regime. To validate these findings, extensive searches for AQP4 inhibitors have been conducted (Detmers et al, 2006; Huber, Tsujita, Kwee, et al, 2009; Migliati et al, 2009; Tanimura et al, 2009; Verkman et al, 2017), but mostly failed (Abir‐Awan et al, 2019; Brandt et al, 2018; Sogaard & Zeuthen, 2008). We here illustrate that bumetanide (a well‐established NKCC inhibitor) does not inhibit AQP1 or AQP4 (Brandt et al, 2018), as otherwise proposed (Migliati et al, 2009), and that acetazolamide has no effect on the water permeability of AQP1 or AQP4, as otherwise proposed (Gao et al, 2006; Huber, Tsujita, & Nakada, 2009).…”

“…To validate these findings, extensive searches for AQP4 inhibitors have been conducted (Detmers et al, 2006; Huber, Tsujita, Kwee, et al, 2009; Migliati et al, 2009; Tanimura et al, 2009; Verkman et al, 2017), but mostly failed (Abir‐Awan et al, 2019; Brandt et al, 2018; Sogaard & Zeuthen, 2008). We here illustrate that bumetanide (a well‐established NKCC inhibitor) does not inhibit AQP1 or AQP4 (Brandt et al, 2018), as otherwise proposed (Migliati et al, 2009), and that acetazolamide has no effect on the water permeability of AQP1 or AQP4, as otherwise proposed (Gao et al, 2006; Huber, Tsujita, & Nakada, 2009). While we cannot pin‐point the reasons for the repeated discrepancies between inhibitor efficiencies in the aquaporin field, it is reasonable to suspect that the origin thereof lies within the experimental approach.…”

“…It has, however, proven challenging to develop such an inhibitor, not for want of trying. During the last decade, a few compounds were brought forward as potential inhibitors of AQP4; for example, tetraethylammonium (TEA), acetazolamide, antiepileptic drugs, and bumetanide (and the derivative AqB013) (Detmers et al, 2006; Huber, Tsujita, Kwee, & Nakada, 2009; Migliati et al, 2009; Tanimura, Hiroaki, & Fujiyoshi, 2009; Verkman, Smith, Phuan, Tradtrantip, & Anderson, 2017), but all of these pharmacological agents were later demonstrated to not be effective blockers of AQP4‐mediated water permeability (Abir‐Awan et al, 2019; Brandt et al, 2018; Sogaard & Zeuthen, 2008). The molecule TGN‐020 has been shown to bind to AQP4 (Huber, Tsujita, & Nakada, 2009; Nakamura et al, 2011), act as an inhibitor (Huber, Tsujita, & Nakada, 2012), and promote better outcome in mouse models of ischemic stroke (Igarashi, Huber, Tsujita, & Nakada, 2011; Pirici et al, 2017).…”