Potent, metabolically stable benzopyrimido-pyrrolo-oxazine-dione (BPO) CFTR inhibitors for polycystic kidney disease

Abstract

We previously reported the discovery of pyrimido-pyrrolo-quinoxalinedione (PPQ) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in an organ culture model of polycystic kidney disease (PKD) (J. Med. Chem. 2009, 52, 6447-6455). Here, we report related benzopyrimido-pyrrolo-oxazinedione (BPO) CFTR inhibitors. To establish structure-activity relationships and select lead compound(s) with improved potency, metabolic stability, and aqueous solubility compared to the most potent prior compound 8 (PPQ-102, IC(50) ∼ 90 nM), we synthesized 16 PPQ analogues and 11 BPO analogues. The analogues were efficiently synthesized in 5-6 steps and 11-61% overall yield. Modification of 8 by bromine substitution at the 5-position of the furan ring, replacement of the secondary amine with an ether bridge, and carboxylation, gave 6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid 42 (BPO-27), which fully inhibited CFTR with IC(50) ∼ 8 nM and, compared to 8, had >10-fold greater metabolic stability and much greater polarity/aqueous solubility. In an embryonic kidney culture model of PKD, 42 prevented cyst growth with IC(50) ∼ 100 nM. Benzopyrimido-pyrrolo-oxazinediones such as 42 are potential development candidates for antisecretory therapy of PKD.

Figure 1

Chemical structures of CFTR inhibitors.

Figure 2

Metabolism of compound 8 in hepatic microsomes. A. LC/MS showing disappearance over 30 min during incubation with microsomes in the presence of NADPH. B. Appearance of metabolites at +14 and +16 daltons. C. Schematic of potential sites of metabolism.

Figure 3

Characterization of PPQ analogs. A. Fluorescence plate-reader assay of CFTR inhibition. FRT cells expressing human wildtype CFTR and iodide-sensing YFP fluorescent dye were incubated with test compound and CFTR agonists, and then subjected to an inwardly directed iodide gradient. (top) Representative data showing kinetics of fluorescence decrease following iodide addition (causing YFP fluorescence quenching) in the absence of cAMP agonists, and in the presence of cAMP agonists and indicated concentrations of 18. (bottom) Summary of concentration-inhibition data for indicated compound (S.E. n=4). See Table 1 for IC₅₀ values. B. Short-circuit current analysis of CFTR inhibition in CFTR-expressing FRT cells in the presence of a transepithelial chloride gradient and basolateral membrane permeabilization. Where indicated, forskolin (20 μM) was added to activate CFTR chloride conductance, following by indicated concentrations of 18. C. (top) LS/MS analysis showing disappearance of 18 and 32 in hepatic microsomes in the presence of NADPH. (bottom) Summary of kinetics of compound disappearance (SEM, n=3). Data for 8 shown for comparison.

Figure 4

BPO CFTR inhibitors with high potency, metabolic stability and water solubility. A. Short-circuit current analysis showing CFTR inhibition by 36 and 42. B. Compound stability in hepatic microsomes in the presence of NADPH.

Figure 5

Compound 42 reduces renal cytogenesis. A. Transmission light micrographs of E13.5 embryonic kidneys cultured for indicated days without or in the presence of 100 μM 8-Br-cAMP, and with 0, 0.1 or 1 μM 42. B. Summary of percentage cyst areas at 5 days in cultures (SEM, n=4–6, * P < 0.001

Scheme 1

Synthesis of PPQ analogs Reagents: (a) NaOH, H₂O, dimethylsulfate, r.t. 3 days; (b) benzoyl chloride, ZnCl₂, chlorobenzene, reflux; (c) Br₂, CH₂Cl₂, cat. H₂O, reflux; (d) 4-R²-1,2-phenylenediamine, EtOH, reflux; (e) R²=H, 4-R⁵-5-R⁶-furfural, 1,2-dichloroethane, TsOH, reflux; X=S, 5-methylthiophene carbaldehyde, 1,2-dichloroethane, TsOH, reflux; R²=NO₂, 5-bromo-2-furaldehyde CHCl₃, TFA reflux; (f) R⁴= CH₃SO₂-, methanesulfonyl chloride, DCM, Et₃N; R⁴=ClCH₂CO-, Chloroacetyl chloride, DCM, Et₃N; R⁴=CH₃CO-, acetic anhydride, DMAP, 100 °C; R⁴=NO, t-butyl nitrite, DCM; (g) N-methyl-1,2-phenylenediamine, EtOH, reflux; (h) 5-methylfurfural, 1,2-dichloroethane, TsOH, reflux.

Scheme 2

Synthesis of BPO analogs. Reagents: (a) R¹=H, benzoyl chloride, ZnCl₂, chlorobenzene, reflux; R¹=Me, m-tolyl chloride, ZnCl₂, chlorobenzene, reflux; (b) Br₂, CH₂Cl₂, cat. H₂O, reflux; (c) 2-amino-3-R²-4-R³-phenol, EtOH, reflux; (d) 5-R⁶-furfual, TFA, CHCl₃ or 1,2-dichloroethane, 150 °C; (e) KOH, THF, H₂O, HCl workup.