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]
BPO-27 racemate directly targets the CFTR protein. CFTR is an ATP-gated anion channel that plays a central role in salt and fluid homeostasis across epithelial tissues. Hyperactivation of this channel is central to the pathophysiology of secretory diarrheas (such as cholera) and polycystic kidney disease. The active (R)-BPO-27 binds near the canonical ATP binding site of CFTR. Whole-cell patch-clamp studies demonstrate that the compound inhibits CFTR chloride current through a voltage-independent blocking mechanism, acting from the cytoplasmic side of the channel. Although early studies suggested an ATP-competitive mechanism, this voltage-independent characteristic indicates a more complex mode of action.

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]

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In vitro, BPO-27 racemate exhibits potent CFTR inhibitory activity with an IC50 of 8 nM. The active R-enantiomer inhibits CFTR chloride conductance with an IC50 of 4 nM, while the S-enantiomer is inactive at the same concentration. In vitro metabolic stability assessments in hepatic microsomes show both enantiomers are highly stable, with less than 5% metabolism observed over 4 hours. Whole-cell patch-clamp studies demonstrate that the R-enantiomer inhibits forskolin-activated CFTR chloride currents in a concentration-dependent manner across all tested voltages, achieving near-complete inhibition at 1 μM, with linear current-voltage relationships indicating a voltage-independent blocking mechanism. In CHO-K1 cells expressing human wild-type CFTR, the R-enantiomer inhibits approximately 50% of current at 0.5 nM. Single-channel patch-clamp experiments reveal that 5 nM R-enantiomer significantly reduces channel open probability from 0.29 to 0.08, substantially increases mean channel closed time, but does not affect single-channel conductance.

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].

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In vivo, the active component of BPO-27 racemate, (R)-BPO-27, demonstrates significant therapeutic efficacy. In a mouse cholera toxin model, intraperitoneal administration of (R)-BPO-27 fully prevents fluid accumulation in intestinal loops in a dose-dependent manner, with an IC50 as low as 0.1 mg/kg. (R)-BPO-27 does not impair intestinal fluid absorption or inhibit other major intestinal transporters, indicating good selectivity. In an embryonic kidney culture model of Autosomal Dominant Polycystic Kidney Disease (ADPKD), BPO-27 racemate demonstrates efficacy in preventing and reversing cyst formation. Furthermore, (R)-BPO-27 blocks fluid secretion in primary cultures of enteroids from human small intestine and inhibits anion current in enteroid monolayers, supporting its potential utility for treating secretory diarrheas in humans.

For mechanistic studies of BPO-27 racemate, cell-free experiments primarily involve metabolic stability assessments using rat hepatic microsomes. The procedure is as follows: BPO-27 enantiomers ((R)-1 and (S)-1) are incubated with NADPH-supplemented rat hepatic microsomes. At specified time points (typically 0, 1, 2, 4 hours), samples are collected and the amount of non-metabolized parent compound is quantified by LC/MS. Results show that both enantiomers exhibit high stability, with less than 5% metabolism observed over 4 hours. Additionally, ATP hydrolysis assays can assess compound effects on CFTR function: in the presence or absence of BPO-27, the change in absorbance at 340 nm using an NADH-coupled system is measured to calculate the Michaelis constant (Km) and maximum reaction rate (Vmax) for ATP hydrolysis. Results demonstrate that (R)-BPO-27 increases the EC50 for ATP activation of CFTR in a concentration-dependent manner.

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]

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In vitro cellular assays typically employ whole-cell patch-clamp techniques. The procedure is as follows: Choose Chinese hamster ovary (CHO-K1) cells or HEK-293T cells stably or transiently expressing human wild-type CFTR. After establishing the whole-cell configuration, perfuse cells with extracellular solution containing BPO-27 (0.5 or 1 µM) and incubate for approximately 5 minutes. Subsequently, add 10 µM forskolin (an adenylyl cyclase activator) to activate CFTR channels. Whole-cell currents are elicited by applying voltage steps from -80 mV to +80 mV in 20 mV increments. Currents are amplified, digitized using an analog-to-digital converter (e.g., Digidata 1440A), filtered at 5 kHz, and recorded/analyzed at room temperature. Alternatively, fluorescence quenching-based assays can indirectly assess CFTR activity by detecting changes in intracellular halide concentration.

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]

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In vivo experiments commonly use male CD1 background mice as model animals. For the cholera toxin-induced diarrhea model: (R)-BPO-27 is formulated at 1 mg/mL in an aqueous vehicle containing 5% DMSO, 2.5% Tween-80, and 2.5% PEG400. Prior to the experiment, mice receive a single intraperitoneal injection of 300 μL of the (R)-BPO-27 formulation. For pharmacokinetic studies, blood samples are collected by eye bleed at specified time points post-administration (e.g., 0.5, 1, 2, 4 hours). At the 4-hour time point, kidneys are removed following renal arterial perfusion with PBS, weighed, mixed with acetic acid, and homogenized for drug concentration analysis. In intestinal loop fluid secretion models, mice are anesthetized and undergo abdominal surgery to isolate and ligate small intestinal loops. Cholera toxin or heat-stable enterotoxin (STa toxin) is injected into the loops to induce fluid secretion, and after euthanasia, the weight/length ratio of intestinal loops is measured as an indicator of fluid secretion.

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]

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The active component of BPO-27 racemate, (R)-BPO-27, exhibits excellent pharmacokinetic properties. In mouse models, its oral bioavailability exceeds 90%. Following intraperitoneal bolus administration (10 mg/kg), the elimination half-life (t1/2) of serum (R)-BPO-27 is approximately 1.6 hours, with sustained therapeutic serum concentrations for over 4 hours. The compound achieves sustained therapeutic concentrations in the kidneys, with a concentration of 0.6 ± 0.3 μM measured in kidney homogenate at 4 hours post-administration. In vitro metabolism studies demonstrate that BPO-27 enantiomers are highly stable in liver microsomes, with less than 5% metabolism observed over 4 hours. Importantly, no measurable interconversion (racemization) between enantiomers is detected in vivo, indicating configurational stability of the compound in the body. Its basic physicochemical properties are: molecular formula C26H18BrN3O6, molecular weight 548.34, LogP approximately 3.8.

Available toxicological studies indicate that the active component of BPO-27 racemate, (R)-BPO-27, has a favorable safety profile. No significant systemic toxicity was observed with a 7-day administration regimen at a daily dose of 5 mg/kg. BPO-27 racemate and its enantiomers are designated "for research use only, not for human or veterinary use". This low toxicity profile, combined with its high potency and selectivity, provides a significant advantage for clinical development over earlier CFTR inhibitors like CFTRinh-172, which suffers from low solubility and non-specific binding. However, comprehensive assessment of long-term safety and full toxicology still requires support from additional animal studies.

[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]

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Enantiomer Activity Difference: BPO-27 racemate is an equal mixture of R- and S-enantiomers. The biological activity resides almost entirely in the R-enantiomer ((R)-BPO-27), which inhibits CFTR chloride conductance with an IC50 of approximately 4 nM (as low as 0.36 nM in whole-cell patch-clamp recordings). The S-enantiomer shows no significant inhibition even at 1 µM. The absolute configuration of the active enantiomer was determined as R-configuration by X-ray crystallography of its ethyl ester derivative.
Related CAS Numbers: The CAS number for the active monomer (R)-BPO-27 is 1415390-47-4, while the CAS number for the racemate BPO-27 is 1314873-02-3.
Solubility and Storage: The compound is soluble in DMSO (reported ranges from 6-16.67 mg/mL across different suppliers). The powder form is stable for 3 years at -20°C and for 2 years at 4°C; the solution form is stable for 6 months at -80°C and for 1 month at -20°C. Repeated freeze-thaw cycles should be avoided.
Molecular Structure Information: The molecular formula of BPO-27 racemate is C26H18BrN3O6, with a molecular weight of 548.34 g/mol. The SMILES string is: O=C(C1=CC=C2OC(C3=CC=C(Br)O3)C4=C(N(C)C5=O)C(C(N5C)=O)=C(C6=CC=CC=C6)N4C2=C1)O.
Therapeutic Prospects: Due to the potential applications of CFTR inhibitors in polycystic kidney disease and secretory diarrhea, BPO-27 is being investigated as a therapeutic candidate for these conditions. Studies using human small intestinal enteroids further support its potential utility for treating secretory diarrhea in humans.

C26H18BRN3O6

548.3416

547.037

C, 56.95; H, 3.31; Br, 14.57; N, 7.66; O, 17.51

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

BPO-27 racemate; BPO-27 (racemate); 1314873-02-3; BPO27 racemate; BPO-27;

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.)