Absolute configuration and biological properties of enantiomers of CFTR inhibitor BPO-27

Article abstract of DOI:10.1021/ml400069k

We previously reported benzopyrimido-pyrrolo-oxazinedione (BPO) inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and showed their efficacy in a model of polycystic kidney disease. Here, we separated the enantiomers of lead compound BPO-27 (1), which contains a single chiral center, and determined their absolute configuration, activity, and metabolic stability. Following separation by chiral supercritical fluid chromatography, the R enantiomer, as determined by X-ray crystallography, inhibited CFTR chloride conductance with IC₅₀ ≈ 4 nM, while S enantiomer was inactive. In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with <5% metabolism in 4 h. Following bolus interperitoneal administration in mice, serum (R)-1 decayed with t _1/2 ≈ 1.6 h and gave sustained therapeutic concentrations in kidney.

Full text of DOI:10.1021/ml400069k

Letter
pubs.acs.org/acsmedchemlett
Absolute Configuration and Biological Properties of Enantiomers of
CFTR Inhibitor BPO-27
David S. Snyder,†,‡ Lukmanee Tradtrantip,† Sailaja Battula,† Chenjuan Yao,† Puay-wah Phuan,†
James C. Fettinger,‡ Mark J. Kurth,‡ and A. S. Verkman*,†
†Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0521, United States
‡Department of Chemistry, University of California, Davis, California 95616, United States
S
* Supporting Information
ABSTRACT: We previously reported benzopyrimido-pyrrolo-oxazine-
dione (BPO) inhibitors of the cystic fibrosis transmembrane conductance
regulator (CFTR) chloride channel and showed their efficacy in a model of
polycystic kidney disease. Here, we separated the enantiomers of lead
compound BPO-27 (1), which contains a single chiral center, and
determined their absolute configuration, activity, and metabolic stability.
Following separation by chiral supercritical fluid chromatography, the R
enantiomer, as determined by X-ray crystallography, inhibited CFTR
chloride conductance with IC50 ≈ 4 nM, while S enantiomer was inactive.
In vitro metabolic stability in hepatic microsomes showed both enantiomers as stable, with <5% metabolism in 4 h. Following
bolus interperitoneal administration in mice, serum (R)-1 decayed with t1/2 ≈ 1.6 h and gave sustained therapeutic
concentrations in kidney.
KEYWORDS: Cystic fibrosis, polycystic kidney disease, secretory diarrhea, crystallography
he cystic fibrosis transmembrane conductance regulator
(CFTR) protein is a cAMP-regulated chloride channel
chloride conductance with IC50 ≈ 4 nM, while the other
enantiomer was inactive.
T
expressed in secretory and absorptive epithelia.1 Loss of
function mutations in CFTR cause the genetic disease cystic
fibrosis (CF). Excess activation of CFTR in intestinal
enterocytes occurs in secretory diarrheas that are caused by
bacterial enterotoxins, such as cholera.2,3 CFTR is also involved
in fluid secretion into cysts in autosomal dominant polycystic
kidney disease (ADPKD).4,5 CFTR is thus an important drug
target, with CFTR activators (potentiators and correctors) for
cystic fibrosis therapy and CFTR inhibitors for therapy of
secretory diarrheas and ADPKD,6 and for CF research.
Separation of ∼1.0 g racemic ( )-1 was carried out utilizing
chiral supercritical fluid chromatography (SFC) on a RegisCell
3.0 × 25.0 cm column using a combination of CO2 and ethanol
containing 1% 2-propylamine. Two distinct peaks were
detected at 230 nm following elution (Figure 1A). Fraction 1
contained 413 mg with 99.5% e.e. (Figure 1B), and fraction 2
contained 396 mg with 98.6% e.e. (Figure 1C). As a
consequence of the separation process, the isolated material
was not the acid 1, but the 2-proplyamine carboxylic salt 2.
Optical rotation measurements revealed fraction 1 to be (+)-2
and fraction 2 to be (−)-2. When dissolved in aqueous buffer
under physiological conditions, both 2 and 1 convert to the
same carboxylate salt form.
CFTR inhibition potency was measured by short-circuit
current analysis in FRT epithelial cells expressing human CFTR
in the presence of a transepithelial chloride gradient and in
which the basolateral membrane was permeabilized with
amphotericin B. Under these conditions, short-circuit current
is proportional to CFTR chloride conductance. Figure 2A
shows no significant inhibition by (−)-2 at 100 nM, whereas
(+)-2 at 100 nM completely inhibited current. Figure 2B shows
the (+)-2 concentration-dependence, giving an IC50 ≈ 4 nM, as
compared to ∼8 nM for ( )-1 as reported previously.16
We previously identified several classes of small-molecule
CFTR inhibitors, including thiazolidinones (e.g., CFTRinh-
172),5,7−9 glycine hydrazides (e.g., GlyH-101),10−14 and
pyrimido-pyrrolo-quinoxalinediones (e.g., PPQ-102),15 and
showed their efficacy in models of polycystic kidney disease
and cholera toxin-induced secretory diarrhea. We recently
reported an analogue of PPQ-102, the benzopyrimido-pyrrolo-
oxazinedione BPO-27 (1) (Scheme 1),16 which inhibited
CFTR with IC50 ≈ 8 nM and had greatly improved in vitro
metabolic stability and aqueous solubility compared to PPQ-
102.15 Compound 1, which was synthesized and tested as a
racemic mixture (R/S 50:50), prevented and reversed renal cyst
formation in an embryonic kidney culture model of ADPKD.16
We separated 1 into its enantiomers with ≥98.6%
enantiomeric excess (e.e.), determined their absolute config-
uration by X-ray crystallography, and measured their CFTR
inhibition activity, metabolic stability, and in vivo pharmacology
in mice. A single enantiomer of 1 strongly inhibited CFTR
Received: July 31, 2012
Accepted: April 8, 2013
Published: April 8, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Transformations of BPO-27
a
Reagents and conditions: (a) RegisCell 4.6 × 100 mm chiral column, 75% CO2, 25% EtOH, 0.1% 2-propylamine, 4 mL/min, 100 bar, 25 °C, yield
73%; (b) (i) HCl, H2O, EtOAc 3× extraction 89% yield; (c) (i) CH2Cl2, EDC, DMAP, EtOH, 24 h, (ii) aq. HCl H2O, 2× extraction, (iii) SiO2 flash
column (2:3 EtOAc/hexane), yield 79%.
Figure 1. Chromatograms of purified BPO-27 enantiomers following chiral HPLC separation. (A) Analytical chromatogram following preparative
separation of ∼1 g ( )-1. (B) Chromatogram of fraction 1. (C) Chromatograph of fraction 2, showing retention time (RT) and % area (A%).
Figure 2. CFTR inhibition by enantiopure (+)-2 and (−)-2. Short-
circuit current was measured in FRT cells expressing human wild-type
CFTR in the presence of a transepithelial chloride gradient and
following permeabilization of the basolateral membrane. CFTR
chloride conductance was activated by 10 μM forskolin. (A) (−)-2
and then (+)-2 (each 100 nM) were added where indicated. (B) (+)-2
added at indicated concentrations, deduced IC50 ≈ 4 nM.
The absolute configuration of the inactive enantiomer was
determined by X-ray crystallography. Attempts to crystallize
Figure 3. ORTEP representation of the crystal structure of (S)-3.
(−)-2 failed to yield X-ray quality crystals, as did the
corresponding carboxylic acid 1, which was isolated by aqueous
acidification and organic extraction (Scheme 1, step b). We
found that the ethyl ester 3 dissolved in multiple solvents and
readily formed large crystals. Chiral ester 3 was thus prepared
from inactive (−)-2 (Scheme 1).
X-ray quality crystals of 3 were obtained by vapor diffusion
crystallization in toluene and hexane. X-ray analysis revealed the
absolute structure to be (S)-3 (Figure 3); therefore, the active
enantiomer has the R configuration. Bioassay of the remaining
(S)-3 crystals confirmed no CFTR inhibition activity at 100
nM, whereas racemic 3 strongly inhibited CFTR.16
Although enantiopure (−)-2 was exposed to both acid and
base conditions during its purification and conversion to (S)-3,
racemization did not occur to any significant extent, as it would
not have been possible to generate chiral crystals from
racemized 3. The absence of activity of the prepared (S)-3
also argues against racemization, as racemic 3 would strongly
inhibit CFTR at 100 nM. The chiral integrity of (−)-2 was
further confirmed by a hydrogen−deuterium exchange experi-
ment. (−)-2 was incubated under relevant physiological
conditions in the presence of deuterium oxide. No reduction
was found in the integrated signal of the singlet peak
corresponding to the proton on the chiral carbon (see
Supporting Information).
The in vitro metabolic stability of (R)-1 and (S)-1 was
determined using rat hepatic microsomes. Following com-
pound incubation with hepatic microsomes in the presence of
NADPH, the nonmetabolized compound was quantified by
LC/MS. Figure 4A shows little metabolism of either
enantiomer.
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Figure 4. Metabolic stability of compound 1. (A) In vitro metabolic stability of 1 measured in rat hepatic microsomes supplemented with NADPH.
(left) LC/MS chromatograms of nonmetabolized enatiomers of 1 at indicated times. (right) Percentage nonmetabolized 1 remaining (S.E., n = 4).
(B) In vivo pharmacokinetics of (R)-1 in mice following bolus intraperitoneal injection at 10 mg/kg body weight. (left) Reference measurement.
LC/MS chromatograms in which known amounts of (R)-1 were added to blood and then extracted. Inset shows assay linearity. (right) LC/MS
chromatograms of blood at indicated times after bolus intraperitoneal injection. (R)-1 ion chromatograms (m/z = 548 [M + H]+) are shown along
with summary of serum concentration data (mean S.E., n = 3).
The pharmacokinetics of (R)-1 was measured in mice by
bolus intraperitoneal administration in an aqueous formulation
consisting of 5% DMSO, 2.5% Tween-80, and 2.5% PEG400 in
water. The concentration of (R)-1 was measured by LC/MS in
the blood and kidney. Figure 4B (left) shows ion current in
calibration studies in which specified amounts of (R)-1 were
added to mouse blood. The assay was linear with a detection
sensitivity of better than 500 nM (R)-1. Figure 4B (right)
shows serum (R)-1 concentration following bolus intra-
peritoneal administration. The t1/2 for the disappearance of
the original compound was ∼1.6 h. (R)-1 concentration
remained at predicted therapeutic levels (≫IC50 of 4 nM) for
many hours following single dose administration. Using a
CFTR inhibition bioassay to distinguish between active (R)-1
and inactive (S)-1, we confirmed no measurable interconver-
sion of the enantiomers in serum at 4 h. LC/MS analysis of
kidney homogenate at 4 h showed a concentration of 0.6 0.3
μM (3 mice).
In summary, chiral chromatography separated ( )-1 into
nearly pure enantiomers, one of which was ∼2-fold more
potent at inhibiting the CFTR chloride current than the
racemic mixture, while the other enantiomer was inactive. The
absolute configuration of the active enantiomer is R as
determined by X-ray crystallography. The target of (R)-1 and
its analogues is likely CFTR itself, as these compounds inhibit
CFTR chloride current in response to different types of
agonists, including activators that target CFTR directly.15,16
Definitive determination of the (R)-1 binding site will require
mutagenesis, molecular modeling, and/or biochemical studies.
and NMR. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(A.S.V.) Tel: 415-476-8530. Fax: 415-665-3847. E-mail: alan.
verkman@ucsf.edu. Webpage: http://www.ucsf.edu/verklab.
Author Contributions
All authors contributed to writing of this manuscript and
approved the final version.
Funding
Supported by NIH grants DK72517, DK86125, HL73856,
EB00415, DK35124, and EY13574 and a Research Develop-
ment Program grant from the Cystic Fibrosis Foundation. The
dual-source X-ray diffractometer was funded by the National
Science Foundation (grant 0840444)
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ADPKD, autosomal dominant polycystic kidney disease; BPO,
benzopyrimido-pyrrolo-oxazinedione; CFTR, cystic fibrosis
transmembrane conductance regulator; DMAP, dimethylami-
nopyrdine; EDC, 1-ethyl-3-(3-dimethylamino-propyl)-
carbodiimide HCl; EtOAc, ethyl acetate; EtOH, ethanol;
PKD, polycystic kidney disease; PPQ, pyrimido-pyrrolo-
quinoxaline-dione; SiO2, silica gel; SFC, supercritical fluid
chromatography; TLC, thin layer chromatography
ASSOCIATED CONTENT
■
REFERENCES
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* Supporting Information
■
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