BPO-27 is a racemic inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. The activity resides in the R-enantiomer (IC50 ≈ 4 nM for chloride conductance inhibition), while the S-enantiomer is inactive (no significant inhibition at 100 nM). The racemic mixture (±)-BPO-27 was previously reported to have an IC50 ≈ 8 nM. [1]
BPO-27 is an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The activity resides in the (R)-enantiomer, which inhibits CFTR chloride current. The IC50 for (R)-BPO-27 was approximately 0.36 nM when applied extracellularly in whole-cell patch-clamp recordings, and approximately 0.53 nM when applied to the cytosolic side in inside-out membrane patch recordings. The (S)-enantiomer is inactive (no significant inhibition at 1 μM). [2]

Benzopyrimidopyrroloxazinedione BPO-27 is an analog of PPQ-102 and inhibits CFTR with an IC50 of 8 nM. The R enantiomer of BPO-27 inhibits CFTR chloride conductance with an IC50 of 4 nM, while the S enantiomer is inert. In vitro metabolic stability of liver microsomes demonstrated that both enantiomers were stable, with a metabolic rate of less than 5% within 4 hours [1]. (R)-BPO-27 binds at the usual ATP binding site. Whole-cell patch-clamp investigations demonstrated a voltage-independent (R)-BPO-27 blocking mechanism for linear CFTR currents. When (R)-BPO-27 concentration slows CFTR chloride current by 50%, the EC50 of CFTR ATP activation increases from 0.27 mM to 1.77 mM [2].

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The R-enantiomer of BPO-27 ((+)-2, which converts to (R)-1 in physiological buffer) completely inhibited CFTR chloride conductance at 100 nM in a short-circuit current assay using FRT cells expressing human CFTR, while the S-enantiomer ((-)-2) showed no significant inhibition at the same concentration. [1]
The concentration-dependence of the active R-enantiomer gave an IC50 value of approximately 4 nM for CFTR chloride current inhibition. [1]
The previously reported racemic mixture (±)-BPO-27 (compound 1) was shown to prevent and reverse renal cyst formation in an embryonic kidney culture model of autosomal dominant polycystic kidney disease (ADPKD). [1]
In whole-cell patch-clamp recordings using CHO-K1 cells expressing human wild-type CFTR, (R)-BPO-27 inhibited the forskolin-activated CFTR chloride current in a concentration-dependent manner at all voltages tested, showing near-complete inhibition at 1 μM. The current-voltage relationship remained linear in the presence of (R)-BPO-27, indicating a voltage-independent block mechanism. In contrast, (S)-BPO-27 at 1 μM had no effect on the current. [2]
The IC50 of (R)-BPO-27 for CFTR inhibition was approximately 0.36 nM in whole-cell patch-clamp recordings using HEK-293T cells. When applied to the cytoplasmic side in excised inside-out membrane patches, the IC50 was approximately 0.53 nM, indicating the compound acts from the cytoplasmic side and has low membrane permeability. [2]
Single-channel recordings in inside-out patches from HEK-293T cells showed that 5 nM (R)-BPO-27 significantly reduced the channel open probability (NPo) from 0.29 to 0.08, modestly reduced the mean channel open time, and strongly increased the mean channel closed time. The unitary channel conductance was not affected. The same concentration of (S)-BPO-27 had no effect on any of these parameters. [2]
In macroscopic inside-out patch recordings, 0.5 nM (R)-BPO-27 (which inhibits current by ~50% in the presence of 3 mM ATP) significantly increased the EC50 of ATP for CFTR activation from 0.26 mM to 1.77 mM. The same concentration of (S)-BPO-27 did not affect the ATP EC50. The thiazolidinone inhibitor CFTRinh-172 (0.5 μM), which acts at a site distinct from the ATP binding domains, also did not change the ATP EC50. [2]
In the presence of the non-hydrolyzable ATP analog ATPγS (1 mM), the ATP EC50 for CFTR activation increased. Treatment with 0.5 nM (R)-BPO-27 further increased this value, suggesting competition between (R)-BPO-27 and ATPγS at the CFTR ATP binding site. [2]

Serum (R)-1 decays within t1/2 = 1.6 hours after an intraperitoneal bolus is given to mice, giving the kidneys sustained therapeutic concentrations [1].

CFTR inhibition potency was measured using a short-circuit current (Isc) assay in Fischer Rat Thyroid (FRT) epithelial cells stably expressing human wild-type CFTR. Cells were grown on permeable supports. A transepithelial chloride gradient was established. The basolateral membrane was permeabilized with amphotericin B to allow current flow driven by the apical CFTR channel. CFTR was activated by adding forskolin (10 μM) to the basolateral side to increase intracellular cAMP. After establishing a stable forskolin-activated current, the test compounds (enantiomers of BPO-27 as their 2-propylamine salts, compound 2) were added to the apical side. The inhibition of the short-circuit current, which is proportional to CFTR chloride conductance, was measured. Dose-response curves were generated by adding increasing concentrations of the active (+)-2 enantiomer. [1]
For whole-cell patch-clamp recordings, CHO-K1 or HEK-293T cells transiently expressing human wild-type CFTR were used. The bath solution contained N-methyl-D-glucamine chloride, calcium chloride, magnesium chloride, glucose, and HEPES (pH 7.4). The pipette (intracellular) solution contained N-methyl-D-glucamine chloride, EGTA, magnesium chloride, Tris-ATP, and HEPES (pH 7.2). After establishing the whole-cell configuration, cells were incubated with (R)- or (S)-BPO-27 for 5 minutes. CFTR was then activated by adding 10 μM forskolin to the bath in the continued presence of the inhibitor. Whole-cell currents were elicited by applying voltage pulses from a holding potential of 0 mV to potentials between +80 and -80 mV in 20 mV steps. Currents were recorded, digitized, and filtered. [2]
For single-channel and macroscopic inside-out patch recordings, HEK-293T cells co-transfected with CFTR and green fluorescent protein were used. Inside-out membrane patches were excised. The pipette (extracellular) solution contained HCl, N-methyl-D-glucamine, HEPES, magnesium chloride, and EGTA (pH 7.2). The bath (intracellular) solution contained HCl, N-methyl-D-glucamine, HEPES, magnesium chloride, calcium chloride, and glucose (pH 7.4), supplemented with 3 mM MgATP and the catalytic subunit of protein kinase A (10 U/ml) to activate CFTR. For inhibitor studies, (R)- or (S)-BPO-27 was added to the bath solution. Single-channel activity was recorded at a holding potential of -60 mV. Currents were filtered and sampled. For ATP competition studies, macroscopic currents from inside-out patches were measured at varying ATP concentrations in the presence or absence of inhibitors. [2]

The in vivo pharmacokinetics of the active enantiomer (R)-1 was evaluated in mice. (R)-1 was formulated in an aqueous vehicle containing 5% DMSO, 2.5% Tween-80, and 2.5% PEG400. Mice received a single bolus intraperitoneal (i.p.) injection of (R)-1 at a dose of 10 mg/kg body weight. Blood and kidneys were collected at specified time points post-injection for analysis. [1]

The in vitro metabolic stability of two enantiomers of BPO-27 (compounds (R)-1 and (S)-1) was assessed using rat liver microsomes supplemented with NADPH. After incubation with microsomes, the amount of unmetabolized parent compound was quantitatively analyzed over time by liquid chromatography-mass spectrometry (LC/MS). Both enantiomers showed high stability, with a metabolic rate of less than 5% within 4 hours. [1] Serum concentrations were determined by LC/MS after a single intraperitoneal injection of (R)-1 (10 mg/kg) in mice. The disappearance of (R)-1 in serum showed a decaying trend, with an estimated half-life (t1/2) of approximately 1.6 hours. [1] The concentration of (R)-1 in kidney homogenate was determined 4 hours after injection, and the result was 0.6 ± 0.3 μM (mean ± standard error, n=3 mice). [1]
Bioassays were used to distinguish between active (R)-1 and inactive (S)-1. No measurable interconversion (racemification) between enantiomers was detected in serum samples collected 4 hours after administration. [1]

[1]. Absolute Configuration And Biological Properties of Enantiomers of CFTR Inhibitor BPO-27. ACS Med Chem Lett. 2013 May 9;4(5):456-459.

[2]. Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP. Mol Pharmacol. 2015 Oct;88(4):689-96.

BPO-27 is a benzopyrimidine-pyrroloxazinedione (BPO) compound, an analogue of the early CFTR inhibitor PPQ-102. [1]
The racemic BPO-27 (compound 1) was initially synthesized and tested as a mixture of R and S enantiomers in a 50:50 ratio. [1]
H-deuterium exchange experiments showed that the chiral center in BPO-27 was conformably stable under physiological conditions, and no protons were lost from the chiral carbon. [1]
The absolute configuration of the inactive enantiomer was determined by X-ray crystallography analysis of the ethyl ester derivative (S)-3, confirming that the active enantiomer has the R configuration. [1] The target of (R)-BPO-27 is likely CFTR itself, as it can inhibit the chloride ion current of CFTR activated by various agonists. [1] Potential therapeutic applications of CFTR inhibitors, such as BPO-27, include autosomal dominant polycystic kidney disease (ADPKD) and secretory diarrhea. [1] BPO-27 is a benzopyrimidine-pyrroloxazinidone CFTR inhibitor. [2] The active enantiomer is (R)-BPO-27, while the (S)-enantiomer is inactive. [2] Computer docking using a homology model of the closed conformation of CFTR revealed five potential binding sites. Scoring function and stereoselectivity analysis indicated that the most likely binding site for (R)-BPO-27 is the typical ATP binding site (labeled C site) at the interface of nucleotide-binding domain (NBD) 1 and NBD2. [2] Molecular dynamics simulations were performed to dock (R)- and (S)-BPO-27 to the typical ATP binding site. The calculated binding energy (ΔG_binding) of the (R)-enantiomer was significantly lower than that of the (S)-enantiomer (which was more favorable), consistent with experimental activity data. [2] The mechanism of action of (R)-BPO-27 may be competitive inhibition of ATP at the typical ATP-binding site of CFTR, thereby interfering with ATP-dependent channel gating. This mechanism is different from other types of CFTR inhibitors, such as thiazolidinones (e.g., CFTRinh-172) and glycine hydrazides (e.g., GlyH-101). [2] Potential therapeutic applications of CFTR inhibitors (e.g., BPO-27) include secretory diarrhea and autosomal dominant polycystic kidney disease (ADPKD). [2]

C26H18BRN3O6

548.3416

547.037

1314873-02-3

(R)-BPO-27;1415390-47-4

53387352

White to off-white solid powder

3.8

1

6

3

36

914

0

BrC1=C([H])C([H])=C(C2([H])C3=C4C(C(N(C([H])([H])[H])C(N4C([H])([H])[H])=O)=O)=C(C4C([H])=C([H])C([H])=C([H])C=4[H])N3C3C([H])=C(C(=O)O[H])C([H])=C([H])C=3O2)O1

GNHIGSRGYXEQEP-UHFFFAOYSA-N

InChI=1S/C26H18BrN3O6/c1-28-21-19(24(31)29(2)26(28)34)20(13-6-4-3-5-7-13)30-15-12-14(25(32)33)8-9-16(15)36-23(22(21)30)17-10-11-18(27)35-17/h3-12,23H,1-2H3,(H,32,33)

9-(5-bromofuran-2-yl)-12,14-dimethyl-13,15-dioxo-17-phenyl-8-oxa-1,12,14-triazatetracyclo[8.7.0.02,7.011,16]heptadeca-2(7),3,5,10,16-pentaene-4-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~16.67 mg/mL (~30.40 mM)

Solubility in Formulation 1: 1.67 mg/mL (3.05 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (3.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)