Novel Thioxothiazolo[3,4- a]quinazolin-5(4 H)-one Derivatives as BKCa Channel Activators for Urinary Incontinence

Abstract

Overactive bladder (OAB) is a syndrome causing a sudden and unstoppable need to urinate with significant global prevalence. Several drugs are used to treat OAB; however, they have various side effects. Therefore, new treatment options for OAB are required. A series of novel 5-oxo-N-phenyl-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide derivatives were synthesized and evaluated for their large-conductance voltage- and Ca²⁺-activated K⁺ channel activation through a cell-based fluorescence assay and electrophysiological recordings. Several compounds, including a 7-bromo substituent on the heterocyclic system, showed increased channel currents. Among the derivatives, compound 12h exhibited potent in vitro activity with a half-maximal effective concentration (EC₅₀) of 2.89 μM, good oral pharmacokinetic properties (area under the curve and half-life), and in vivo efficacy in a spontaneously hypertensive rat model.

Scheme 1. Synthesis of 1-Thioxo-1H-thiazolo[3,4-a]quinazolin-5(4H)-one Derivatives 3–5

Reagents and conditions: (a) thiophosgene, triethylamine, THF, 0 to 25 °C, 1 h, 99%; (b) methyl 2-cyanoacetate, sulfur, triethylamine, DMF, 50 °C, 1 h, 63%; (c) NaOH, THF, H₂O, 25 °C, 12 h, 42%; (d) 7 M NH₃ in MeOH, HATU, 1-hydroxybenzotriazole, DIPEA, DMF, 25 °C, 24 h, 54%; (e) benzylamine, EDCI, 1-hydroxybenzotriazole, DIPEA, CH₂Cl₂, 25 °C, 18 h, 15%.

Scheme 2. Synthesis of Target Compounds 11a–g and 12a–j

Reagents and conditions: (a) EDCI, 1-hydroxybenzotriazole, DIPEA, CH₂Cl₂, 25 °C, 18 h, 35–83%; (b) thiophosgene, triethylamine, THF, 0 to 25 °C, 1 h, 96–99%; (c) sulfur, triethylamine, DMF, 50 °C, 1 h, 5–64%.

Figure 1

. In vitro activities of 1-thioxo-1H-thiazolo[3,4-a]quinazolin-5(4H)-one derivatives in a cell-based fluorescence assay. (A) Initial velocity of channel activation was obtained for the first 4 s after BK_Ca channel stimulation. Compounds were prepared at a concentration of 6 μM. (B) Apparent EC₅₀ was obtained from the initial velocity of channel activation by fitting data in a dose–response curve, y = A_min + (A_max – A_min)/(1 + 10^{(log EC₅₀ – x)p}). NS11021 was used as a positive control. Each bar represents the mean, and each error bar is the SEM (n = 3). NA, not applicable.

Figure 2

Activation effects of compound 12h on BK_Ca channels in a dose-dependent manner. (A) Raw trace of RFU following treatment with 12h at a concentration range of 0.2–6 μM. NS11021 (5 μM) was used as a positive control. (B) Initial velocity of channel activation upon treatment with 12h in a range of concentrations during the first 4 s. Each dot and bar represents the mean, and each error bar is the SEM (n = 3). A Student’s t-test was performed for statistical analysis; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the vehicle group.

Figure 3

Activation effects of compound 12h on macroscopic currents of BK_Ca channels. (A–D) The current was recorded in an outside-out configuration with 3 μM intracellular Ca²⁺ concentration. Ionic currents were elicited with 100 ms voltage step pulses from −80 to 200 mV in 10 mV increments. The holding voltage was 100 mV. Each trace corresponds to a voltage step. (A) Representative current trace after treatment with 10 μM 12h. (B) Conductance (G)–voltage (V) relationship curve after treatment with 10 μM 12h. (C) V_1/2 (voltage at half activation) shift of 12h at 10 μM. (D) The maximum conductance (G_max) increased with 10 μM 12h compared to the vehicle group. (E–H) The current was recorded in an inside-out configuration with 3 μM intracellular Ca²⁺ concentration. Ionic currents were elicited with 100 ms voltage step pulses from 80 to −200 mV in 10 mV decrements. The holding voltage was −100 mV. (E) Representative current trace after treatment with 10 μM 12h. (F) G–V relationship curve after treatment with 10 μM 12h. (G) V_1/2 shift of 12h at 10 μM. (H) The maximum conductance (G_max) increased with 10 μM 12h compared to the vehicle group. G was obtained from the mean outward current for 5 ms after saturation. The values were normalized by the maximum conductance of the vehicle group fitted by the Boltzmann function; G/G_max = {(G_max – G_min)/(1 + exp[(V_1/2 – V)/k])} + G_min, where k is a constant. The dots and bars represent the means, and the error bars are SEMs (n = 4). The Student’s t-test was performed for statistical analysis; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the vehicle group.

Figure 4

Effects of compound 12h on BK_Ca channel gating. (A) Representative current trace following treatment with 10 μM 12h on the outside of the membrane at 170 mV pulse stimulation. (B) Time constant of activation (τ_activation). (C) Time constant of deactivation (τ_deactivation). (D) Representative current trace following treatment with 10 μM 12h on the inside of the membrane. (E) Time constant of activation (τ_activation) for the inside-out patch. (F) Time constant of deactivation (τ_deactivation) for the inside-out patch. The outward current was analyzed for τ_activation on voltage simulation. The tail current after the peak was analyzed for τ_deactivation after the end of the voltage pulse. The time constant (τ) was obtained by fitting every independent data set using the single exponential function (y = A exp(−t/τ) + C). Each dot represents the mean, and each error bar is the SEM (n = 4–5). The Student’s t-test was performed for statistical analysis; *p < 0.05, **p < 0.01, and ***p < 0.001, compared to the vehicle group at the same voltage pulse.

Figure 5

Effects of compound 12h on voiding behavior of spontaneous hypertensive rats. Voiding frequency was measured for 3 h after oral administration of 12h in WKYs and SHRs. The vehicle was composed of DMSO/PEG400/distilled water (v/v, 5:40:55). Each bar represents the mean, and each error bar is the SEM (n = 4–5). One-way ANOVA was performed for statistical analysis; *p < 0.05, **p < 0.01, and ***p < 0.001, compared to the vehicle group of the SHRs; +p < 0.05, ++p < 0.01, and +++p < 0.001, compared to the vehicle group of the WKYs.