Discovery of 4-[(2R,4R)-4-({[1-(2,2-Difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic Acid (ABBV/GLPG-2222), a Potent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Corrector for the Treatment of Cystic Fibrosis

Article abstract of DOI:10.1021/acs.jmedchem.7b01339

Cystic fibrosis (CF) is a multiorgan disease of the lungs, sinuses, pancreas, and gastrointestinal tract that is caused by a dysfunction or deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an epithelial anion channel that regulates salt and water balance in the tissues in which it is expressed. To effectively treat the most prevalent patient population (F508del mutation), two biomolecular modulators are required: correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Despite approved potentiator and potentiator/corrector combination therapies, there remains a high need to develop more potent and efficacious correctors. Herein, we disclose the discovery of a highly potent series of CFTR correctors and the structure-activity relationship (SAR) studies that guided the discovery of ABBV/GLPG-2222 (22), which is currently in clinical trials in patients harboring the F508del CFTR mutation on at least one allele.

Full text of DOI:10.1021/acs.jmedchem.7b01339

Subscriber access provided by UNIV OF MISSOURI ST LOUIS
Drug Annotation
Discovery of 4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-
yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-
dihydro-2H-chromen-2-yl]benzoic acid (ABBV/GLPG-2222),
a Potent Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR) Corrector for the Treatment of Cystic Fibrosis
Xueqing Wang, Bo Liu, Xenia Searle, Clinton Yeung, Andrew R Bogdan, Stephen Nathaniel
Greszler, Ashvani Singh, Yihong Fan, Andrew M. Swensen, Timothy Vortherms, Corina
Balut, Ying Jia, Kelly E. Desino, Wenqing Gao, Hong Yong, Chris Tse, and Philip R. Kym
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01339 • Publication Date (Web): 18 Dec 2017
Downloaded from http://pubs.acs.org on December 18, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
Discovery of 4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-
yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-
yl]benzoic acid (ABBV/GLPG-2222), a Potent Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) Corrector for the Treatment of Cystic Fibrosis
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Xueqing Wang*, Bo Liu, Xenia Searle, Clinton Yeung, Andrew Bogdan, Stephen Greszler,
Ashvani Singh, Yihong Fan, Andrew M Swensen, Timothy Vortherms, Corina Balut, Ying Jia,
Kelly Desino, Wenqing Gao, Hong Yong, Chris Tse and Philip Kym
†
Research and Development, AbbVie Inc., 1 North Waukegan Road, North Chicago, IL 60064,
United States
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
Cystic fibrosis (CF) is a multiꢀorgan disease of the lungs, sinuses, pancreas, and
gastrointestinal tract that is caused by a dysfunction or deficiency of the cystic fibrosis
transmembrane conductance regulator (CFTR) protein, an epithelial anion channel that regulates
salt and water balance in the tissues in which it is expressed. To effectively treat the most
prevalent patient population (F508del mutation), two biomolecular modulators are required,
correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective
opening of the CFTR channel. Despite approved potentiator and potentiator/corrector
combination therapies, there remains a high need to develop more potent and efficacious
correctors. Herein, we disclose the discovery of a highly potent series of CFTR correctors and
the structure activity relationship (SAR) studies that guided the discovery of ABBV/GLPGꢀ2222
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(22), which is currently in clinical trials in patients harboring the F508del CFTR mutation on at
least one allele.
2
ACS Paragon Plus Environment

Page 3 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
Introduction
Cystic fibrosis (CF) is a multiꢀorgan disease of the lungs, sinuses, pancreas, and
gastrointestinal tract that is caused by a dysfunction or deficiency of the cystic fibrosis
transmembrane conductance regulator protein (CFTR). CFTR is an epithelial anion channel that
regulates salt and water homeostasis in the tissues in which it is expressed. The onset of lung
disease as a result of defects in mucociliary clearance is the primary driver of morbidity in CF
patients.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
There are more than 2000 mutations to the CFTR protein, and among them 281 are
1
diseaseꢀcausing, which can be categorized into six broad classes that result in defects on CFTR
2
ꢀ3
production, trafficking, function, and/or stability. The two most severe mutations lead to
gating or folding defects. Approximately 10% of patients have gating mutations (e.g., G551D),
where wild type levels of CFTR channels on the cell surface are not functional. These patients
4
can be treated with a potentiator such as Ivacaftor, which enhances the channel opening and has
demonstrated remarkable and sustained clinical efficacy represented by the significant
5
improvements of forced expiratory volume in one second (FEV ). For about 50% patients who
1
harbor a homozygous F508del mutation, where the phenylalanine 508 is omitted, the disease is
characterized by both a gating defect and reduced quantity of matured CFTR channels on the cell
2
ꢀ3, 6
surface.
To improve life of these patients, two biomolecular modulators are required,
correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective
7
8
opening of the CFTR channel. Orkambi, a combination of Lumacaftor (VXꢀ809) and
9
Ivacaftor, has been approved for the treatment of this patient population. Despite the approval,
compared to the transformational effects of Ivacaftor on patients with gating mutations, the
effects of Orkambi on F508del homozygous patients are modest (2ꢀ4% improvement of FEV for
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Orkambi vs 10% for Ivacaftor). In the clinic, bronchoconstriction has been reported for
1
0ꢀ12
Lumacaftor treated individuals, likely due to offꢀtarget effects of Lumacaftor.
Additionally,
1
3
Lumacaftor has been reported to be a cytochrome P450 3A4 (CYP3A4) inducer and therefore a
1
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
drugꢀdrug interaction (DDI) perpetrator. More recently, Phase 3 results with the combination
of Tezacaftor (VXꢀ661) and Ivacaftor have been reported that also demonstrated a modest
1
5
improvement in FEV , while the liabilities associated with Lumacaftor (i.e.,
1
bronchoconstriction and CYP3A4 induction) were not observed. The improvement in
tolerability of Tezacaftor/Ivacaftor appears sufficient for an approval submission even though
there is no statistically significant improvement in clinical efficacy. Preclinical characterization
of Tezacaftor suggests that the improvement in safety may have come at the expense of efficacy
given the reduced maximum current restored in F508del homozygous human bronchial epithelial
1
3
(
HBE) cells.
In summary, there is a clear need to continue to develop additional CF therapies to ensure
effective options to the patients. In this regard, we report the discovery of 22 (ABBV/GLPGꢀ
222), a CFTR corrector that is currently in clinical trials and serves as a foundation for
2
combination therapies being investigated for CF patients harboring a F508del mutation on at
least one allele.
Figure1. Structure of known CFTR potentiators and correctors
Potentiators
4
ACS Paragon Plus Environment

Page 5 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
Correctors
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Strategy
Among the correctors that have been disclosed, the most notable correctors are VXꢀ809
Lumacaftor) and VXꢀ661 (Tezacaftor) (Figure 1). We sought to identify a corrector with
(
differentiated structures that can provide improved potency, efficacy and selectivity with the
rationale that this would lead to potential enhanced clinical characteristics. From a strategic
perspective we planned to leverage AbbVie’s large collection of acid and amine monomers to
assemble novel lead compounds through a final amide bond coupling reaction (Figure 2). From
3
a design perspective, we selected acid and amine monomers that contained optimal sp character
and physiochemical properties.
The overall process began with the selection of a large number of monomers, either
amines or acids, based upon physiochemical parameters such as clogP, molecular weight, and sp
content (Figure 2). Virtual libraries were then enumerated using these monomers.
3
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 2. Lead identification approach
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Physicochemical and ADME (absorption, distribution, metabolism, excretion) properties
of the resulting compounds were calculated, from which compounds were selected for library
production depending on the selected cutꢀoff of molecular weight, cLogP, clearance. To ensure
a broad lead set was generated, the property cutoffs for enumerated compounds slightly loosened
(
cLogP< 5.5, and molecular weight < 550). We rationalized that once active compounds were
6
ACS Paragon Plus Environment

Page 7 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
identified, an empirical based SAR effort would be devoted to optimizing potency, clearance,
and other drugꢀlike properties.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Assays
Different assays were utilized to evaluate correctors. By definition CFTR correctors rectify misꢀ
folding of mutant CFTR, thereby restoring the channel maturation on the cell surface. A cell
1
6
surface assay, which utilizes an immortalized bronchial epithelial cell (CFBE) transduced with
a F508del CFTR and fused with horseradish peroxidase (HRP) to detect the matured CFTR level
at the cell surface, was established inꢀhouse and referred to as the cell surface expression (CSEꢀ
1
7
HRP) assay. However, the ultimate goal is to improve the function of CFTR channels. To
measure the function of the matured CFTR proteins, a semiꢀautomated HBEꢀTECC (Human
1
8
Bronchial Epithelialꢀ TransꢀEpithelial Current Clamp) assay was established. In this assay,
primary human bronchial epithelial (HBE) cells from F508del CF patients were cultured and
electrophysiology (Eꢀphys) measurement of the CFTR chloride ion current cross the apical
membrane was taken. The magnitude of current measured in this assay is proposed to be a
reasonable predictor of achieving efficacy on clinical endpoints such as FEV , and this
1
1
9
correlation was validated in the clinical development of potentiators.
Results and Discussion
Following the lead identification approach, various amine monomers were coupled with 1ꢀ(2,2ꢀ
difluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid (Scheme 1). Among these
amines, some chromanyl amines were included (Scheme 2). The success of this approach is
illustrated in Table 1, where multiple active compounds were identified (1-5). These 2,2ꢀ
2
0
disubstituted chromanamines could be readily synthesized as enantiomers, and the availability
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of these amine monomers in house allowed us to quickly expand understanding of the SAR and
glean important information. For instance, the C4 position of the chromane ring prefers the (R)ꢀ
configuration (compound 1 and 4 vs compound 2). Substitution at the 8ꢀposition of the
chromane phenyl ring resulted in diminished activity and efficacy (3 vs 1) due to likely
unproductive interactions. Notably, the Cꢀ2 phenyl substituted compound (5) was also active.
Most of these compounds have molecular weight below 500, and HPLCꢀmeasured LogD values
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
eLogD) around 5.
Having established that the chromane compounds are pharmacologically active, we
decided to focus on exploring Cꢀ2 phenyl analogs. In addition to the efficacy that 5 displayed in
CSE assay, we also reasoned that activity and properties would be readily tunable through
modification of substituents on the phenyl ring, providing ready access points for optimization.
An internal collection of some of these phenyl substituted amines enabled us to quickly prepare
analogs (Table 2). The paraꢀfluoro analog 6 maintained activity, and the paraꢀchloro analog 9
was more active, while the activity of metaꢀchloro (8) and orthoꢀchloro (7) analogs were
progressively weaker than 9, indicating a substitution pattern and size preference. The C7ꢀ
methoxy substitution on the chromane phenyl ring provided a significant potency improvement
(
10 vs 5). Also, substitution of the distal phenyl ring with two more methoxy groups (11)
provided an additional boost in potency. Since 11 was a mixture of stereoisomers, the
diastereomers would need to be separated to identify the active component.
8
ACS Paragon Plus Environment

Page 9 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. SAR of Chromane Analogs
CSEꢀHRP
1
b
Compd
R
MW/eLogD
a
(
EC (ꢀM), Max. Act% 15)
50
c
1
2.7, 98%
>20, 9%
419/NT
2
3
4
5
419/5.1
454/5.4
2.3, 51%
4.3,102%
2.5, 108%
c
455/NT
449/5.31
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a.
Assay was run according to experimental description, using compound 15 (3 ꢀM ) as the
b
positive control. All values are the geometric mean of at least two determinations. HPLC
c
measured LogD. Not tested
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. SAR of 2-Phenyl Substituted Chromane Analogs
CSEꢀHRP
1
b
Compd
R
MW/eLogD
449/5.3
a
(EC (ꢀM), Max. Act% 15)
5
0
5
6
2.5, 108%
2.5, 95%
467/5.3
7
8
9
11, 31%
3.9, 83%
0.94, 94%
484/5.6
484/5.6
484/5.6
10
ACS Paragon Plus Environment

Page 11 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
1
0
1
0.45, 112%
479/5.4
539/5.0
O
7
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0.20, 118%
a
Assay was run according to experimental description, using compound 15 (3 ꢀM ) as the
b
positive control. All values are the geometric mean of at least two determinations. HPLC
measured LogD
Compound 11 was purified by chiral supercritical fluid chromatography (SFC), resulting
in two cisꢀisomers as shown as 12 and 13. Xꢀray crystallography confirmed the absolute stereo
configuration (Figure 3) of 13 as having the (R,R)ꢀconfiguration. Upon testing, 13 was
confirmed to be the more active enantiomer, consistent with the early observation that the (R)ꢀ
configuration at 2ꢀposition was preferred, with EC of 130 nM. The corresponding (S,S) isomer
5
0
1
2 was significantly weaker.
Figure 3. Xꢀray Structure of 13 indicating the active enantiomer is the (R,R)ꢀform
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Now equipped with a potent compound in the CSE assay, compound 13 was tested in
human bronchial epithelial (HBE) cells to define its functional potency and efficacy. Active
enantiomer 13 exhibited high potency of 28 nM with efficacy comparable to that of Lumacaftor
(
data now shown). The corresponding (S,S)ꢀisomer 12 was weaker in the assay, highlighting the
concordance of CSEꢀHRP with HBEꢀTECC assay. Based on this data, compounds were
automatically advanced into HBEꢀTECC assay if they were active in the CSE assay (EC50 < 1
ꢀ
M, 50% Max. Act% of positive control). For the discussion of the subsequent compounds, only
HBEꢀTECC data will be reported.
While 13 possesses high potency and efficacy in this HBEꢀTECC assay that assess the
CFTR function, this lead resides in undesirable drugꢀlike space with high molecular weight, and
2
1
high HPLC LogD, indicating high lipophicity. Not surprisingly it suffers from poor passive
permeability measured by parallel artificial membrane permeability assay (PAMPA), and low in
12
ACS Paragon Plus Environment

Page 13 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
vitro hepatocyte stability (Table 3), which led to in vivo high clearance (>liver blood flow rate
Qh)), low halfꢀlife, and low oral bioavailability (data not shown) in a rat in vivo
pharmacokinetic (PK) study.
Despite the high lipophilicity and undesirable ADME properties of lead 13, the
(
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
observation that dimethoxy substitution was beneficial for the activity suggested that more polar
groups could be tolerated at these positions on the distal phenyl ring. With the objective of
2
2
maintaining potency, minimizing molecular weight increase, and reducing unbound clearance,
we decided to build in polarity by replacing methoxy groups of 13 with an alcohol or acid group.
The primary alcohol analog 14 had slightly improved HPLC LogD and permeability, but
displayed weaker potency with high hepatocyte clearance. Removing both methoxy groups on
the distal phenyl ring, and replacing them with a carboxylic acid group yielded compound 15,
which had comparable molecular weight to 13, and retained high potency and efficacy. The
reduced lipophilicity (HPLC LogD 3.8) resulted in higher permeability and reduced hepatocyte
clearance. Compound 15 was characterized in rat PK studies and demonstrated reasonable
clearance, halfꢀlife, and bioavailability (Table 5). Compound 15 also shows a favorable profile
in the CYP3A4 induction assay (Table 4), exhibiting low induction compared with the positive
control rifampin. In this assay (supplemental material), the compound is tested at 10 ꢀM, and
the response is compared to that of 10 ꢀM rifampin, a CYP3A4 inducer. Compounds with a
response of less than 20% of rifampin are considered to have low induction risk. The potency
and efficacy of 15 in HBEꢀTECC assay are very comparable to that of Lumacaftor (data now
shown), hence 15 was used as the positive control for characterization of additional leads in both
CSEꢀHRP and HBEꢀTECC assays.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. SAR of 2-Phenyl Substituted Chromane Analogs 13-16
CSEꢀHRP
Hepatocyte
HBEꢀTECC
d
(
EC (ꢀM),
MW
PAMPA
Clint,u
5
0
1
Compd
R
(EC (ꢀM), Max.
5
0
c
ꢀ6
Max. Act%
eLogD
(10 cm/s) (human/rat)
b
Act% 15)
a
1
5)
(L/hr/kg)
14
ACS Paragon Plus Environment

Page 15 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
1
2
1.0, 69%
<30% @ 3 ꢀM
540/4.96
0.30
0.56
1250/775
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
0.13, 123%
0.028, 117%
540/4.88
1540/1026
1
4
1.6, 93%
0.75, 103%
0.24, 95%
479/4.82
523/3.84
1.15
14.3
235/471
25/17
15
0.23, 101%
a
Assay was run according to experimental description, using compound 15 (3 ꢀM ) as the
b
positive control. All values are the geometric mean of at least two determinations. Assay was
23
run according to experimental description, using 23 (GLPG1837, 1 ꢀM) as the potentiator,
compound 15 (3 ꢀM ) as positive control. All values are the geometric mean of at least two
c
d
determinations. HPLC measured LogD. Parallel artificial membrane permeability assay
In order to optimize the in vivo clearance of the lead compounds, a common practice was
to assess if there is an in vitro and in vivo correlation (IVIVC) in clearance. A large number of
amide compounds which have 1ꢀ(2,2ꢀdifluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic
acid linked to a wide range of amines, include chromanyl amines, demonstrated a reasonable
2
correlation (R = 0.798) between unbound hepatocyte clearance and unbound in vivo clearance in
rat (Figure 4). This analysis provided support to use in vitro hepatocyte clearance as a surrogate
for in vivo clearance optimization.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To understand if this series of compounds has potential systemic DDI liabilities, a subset
of those compounds which were active in CSE assay (>30% efficacy) was also sampled in drugꢀ
drug interaction (DDI) assays (Figure 5), including CYP3A4 induction and CYP inhibition
assays. Many lead compounds, including chromane analogs, were shown to have low induction
of CYP 3A4 (<20% of rifampin), and low inhibition against various CYP phenotypes, indicating
there is no chemotypeꢀdependent DDI perpetrator liability.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Given the combination of desired biological activity and properties of 15, subsequent
SAR studies were focused on exploring this carboxylic acid analog. To improve potency and
reduce clearance, substituents on the chromane ring were surveyed. Substitutions such as 6ꢀ
methoxy (16) and 7ꢀfluoro (17) maintained potency, and provided improved human hepatocyte
clearance. A methyl substitution at the 7ꢀ position resulted in reduced potency (18), but larger
groups such as trifluoromethyl (19) and difluoromethoxy (20) led to improved potency and
clearance when compared to the methoxy analog 15. Between 19 and 20, the eLogD of 20 is
much smaller and comparable to that of methoxy analog 15. The combination of low LogD,
high potency, and low clearance indicates that difluoromethoxy group may provide optimal
overall benefits. Among these compounds, methoxy substituted analogs (15 and 16) showed low
induction; while fluoroꢀ (17) and trifluoromethylꢀ (19) substituted analogs were found to induce
CYP3A4 mRNA upregulation. These analogs indicated that various substituents on the
chromane phenyl ring provided only modest impact on the potency, prompting us to look at other
parts of the molecule.
Figure 4. In vitro in vivo correlation (IVIVC) of unbound in vivo plasma clearance and unbound
in vitro hepatocyte clearance in rat
16
ACS Paragon Plus Environment

Page 17 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
A
B
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Figure 5. CYP perpetrator evaluation (A) CYP3A4 induction plot of compounds. (B) CYP3A4, 1A2, 2C9 and 2D6 inhibition of
compounds.
18
ACS Paragon Plus Environment

Page 19 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
Table 4. SAR of Acid Analogs of 15
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hepatocyte
Clint,u
HBE
(EC (ꢀM), Max.
CYP3A4
induction
MW
2
Compd
R
5
0
c
eLogD
(human/rat)
(L/hr/kg)
a
b
Act% 15)
(%rifampin)
15
16
17
18
19
7ꢀOCH
6ꢀOCH
7ꢀF
3
0.24, 95%
0.21, 111%
0.11, 93%
523/3.84
523/3.84
511/4.03
507/4.01
561/5.02
559/4.02
25/17
11/2
26/4
21/8
18/5
14/7
14%
9.4%
40%
3
c
7ꢀMe
< 60% @ 3 uM
0.087, 97%
0.075, 117%
NT
7ꢀCF
7ꢀOCHF
40%
3
c
20
2
NT
a
Assay was run according to experimental description, using 23 (1 ꢀM) as the potentiator,
compound 15 (3 ꢀM ) as positive control. All values are the geometric mean of at least two
b
determinations. CYP3A4 induction was measured at 10 ꢀM, using 10 ꢀM rifampin as positive
c.
control. Not tested
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Upon inspection of the SAR trend from early analogs in this series, it was clear that the
substitution pattern on the distal phenyl ring can be particularly important. For example, paraꢀ
chloro analog 9 was 4ꢀfold more potent than the metaꢀchloro analog 8 (Table 2). Recognizing
that the carboxylic acid is tolerated on the distal phenyl ring, the para carboxylic acid analog 21
was prepared and found to exhibit a potency improvement of more than 20ꢀfold to 9 nM EC in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
HBEꢀTECC assay (Table 6). Compound 21 was also found to have a clean CYP3A4 induction
profile. Additionally, the reduced hepatocyte clearance of 21 translated into lower in vivo
clearance (Table 5).
Table 5. Rat Pharmacokinetic Parameters for Compounds 15 and 21
IV Dose
Oral Dose
Compd
a
b
c
a
Dose
Cl
t1/2
Dose
F (%)
24
d
15
1
1.24
2.8
4.4
1
e
2
1
1
0.62
1
78
a
b
c
Dose (mg/kg). Clearance (L/hr/kg). iv halfꢀlive(h).
Given improved potency of the difluoromethoxy analog over the methoxy analog (20
over 15), the paraꢀcarboxylic acid analog 22 was synthesized and characterized (Table 6).
Compound 22 was highly potent (5 nM) and efficacious in cells from multiple CF patient donors
who have F508del homozygous mutation. In comparison with Lumacaftor, 22 was significantly
24
more potent (>25 fold), and exhibited comparable efficacy. It demonstrated low clearance
20
ACS Paragon Plus Environment

Page 21 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
across multiple preclinical species, did not inhibit CYP enzymes, was not a CYP3A4 inducer,
and therefore presented a low DDI perpetrator liability.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6. SAR of Para-substituted Acid Analogs
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HBE
Hepatocyte Clint,u
(human/rat)
(L/hr/kg)
CYP3A4
induction
2
Compd
R
(EC (ꢀM), Max.
5
0
b
Act% 15)
(%rifampin)
2
2
1
2
7ꢀOCH
3
0.009, 94%
15/3
7/3
0%
7ꢀOCHF
0.005, 87%
6.4%
2
a.
Assay was run according to experimental description, using 23 (1 ꢀM) as the potentiator,
compound 15 (3 ꢀM ) as positive control. All values are the geometric mean of at least two
b
determinations. CYP3A4 induction was measured at 10 ꢀM, using 10 ꢀM rifampin as positive
control.
The pharmacokinetic profile of 22 is provided in Table 7. Compound 22 is primarily
cleared via glucoronidation in human plasma, presenting a low DDI victim liability. Thorough
preclinical characterization including DMPK, and safety pharmacology and toxicology of
compound 22 in rat and dog support this compound as a candidate for clinical development. In a
firstꢀinꢀhuman phase I study, compound 22 was given to healthy volunteers at single doses up to
8
00 mg and multiple doses up to 600 mg daily for 14 days. The drug was well tolerated and had
22
ACS Paragon Plus Environment

Page 23 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
a favorable safety profile, with no early discontinuations of study drug and no severe adverse
2
5
events.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 7. Rat and Dog Pharmacokinetic Parameters for Compound 22
IV Dose
Oral Dose
Species
a
b
c
a
Dose
Cl
t1/2
Dose
F (%)
74
rat
1
0.55
2.7
5.0
1
dog
2
0.83
5
40
a
b
c
Dose (mg/kg). Clearance (L/hr/kg). iv halfꢀlive (h).
Conclusion
By utilizing an internal collection of proprietary monomers, we were able to quickly identify
chromane substituted amides as CFTR correctors. The initial collection of enriched analogs
greatly facilitated early SAR work of identifying active compounds. Introduction of a carboxylic
acid group at the distal phenyl ring maintained the desired pharmacological activity, and
optimized the ADME properties significantly, especially permeability and unbound clearance.
Good in vitroꢀin vivo correlation in clearance helped guide the effort for in vivo pharmacokinetic
evaluation. Through empirical and iterative SAR investigation the paraꢀcarboxylic acid analog,
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2, a highly potent and efficacious CFTR corrector, was identified and advanced into clinical
trials.
Chemistry
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of amides were by coupling of acid and amine, either through use of coupling reagent,
or by preparing the corresponding carbonyl chloride followed by reacting with the amines
(Scheme 1).
2
0
For analogs 1-11, chiral chromanyl amines and racemic 2ꢀphenyl chromanyl amines
were coupled to 1ꢀ(2,2ꢀdifluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid. For
compounds with chiral 2ꢀphenyl substituted chromanyl amines (14-22), synthesis from the
appropriated substituted chromenones (14aꢀ22a) is described (Scheme 3). The corresponding
chromenone were either prepared by condensing substituted hydroxyacetonphenone with DMFꢀ
DMA, or could be purchased. Palladium catalyzed conjugated addition of aromatic boronic acid
was carried out using (S)ꢀtꢀbutyl PyꢀOX as chiral ligand, affording product 14b-22b in modest
2
6
yield and good enantioselectivity. The resulting chromanones 14b-22b were condensed with
hydroxylamine or alkoxyamine to form the oxime, which underwent hydrogenation. Raney
Nickel provided a mixture of trans:cis substituted compounds, while platinum yielded cisꢀ
selectivity, enriching desired diastereomers. The resulting amine can be coupled with acid or
carbonyl chloride as shown for compounds 14e-22e. For 15e-21e, and 22f, saponification
provided the final acid analogs 15-22.
a
Scheme 1. Amide coupling of early libraries
24
ACS Paragon Plus Environment

Page 25 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
H
N
F
O
O
OH
iii
F O
O
R1
+
H N
2
F
R1
F
O
O
i or ii
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F O
O
Cl
+
H N
2
F
O
R1
a
Reagents and conditions: (i) oxalyl chloride, cat. DMF, DCM; (ii) thionyl chloride, reflux; (iii)
HATU, DIPEA, DMA.
a
Scheme 2. Amide coupling of chromanylamines
a
Reagents and conditions: (i) HATU, DIPEA, DMA, 7ꢀ98%.
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 53
`
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
a
Scheme 3. Synthesis of amides with chiral 2-phenyl substituted chromanyl amines
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
a
o
3
Reagents and conditions: (i) DMFꢀDMA, 115ꢀ120 C, 64ꢀ80%; (ii) Pd(TFA) , (S)ꢀtꢀbutyl PyOX, R ꢀPhB(OH) , DCE, 34ꢀ83%; (iii)
2
2
MeONH HCl, pyridine or NaOAc, 77%ꢀquantitative; (iv) Raney Ni, H , MeOH, 94ꢀ99%; (v) Pt/C, H , AcOH, 34ꢀ55%, 10ꢀ20:1 dr;
2
2
2
(
vi) 1ꢀ(2,2ꢀdifluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarbonyl chloride, Hunig's base, DCM, 30ꢀ85%; (vii) 1ꢀ(2,2ꢀ
difluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid, HATU, Hunig's base, DMF, 28ꢀ76%; (viii) NaOH, LiOH or KOTMS,
1ꢀ90%; (ix) NaBH , THF/MeOH, 85%; (x) diethyl (bromodifluoromethyl)phosphonate, KOH, CH CN/H O, 50ꢀ72%.
7
4
3
2
26
ACS Paragon Plus Environment

Page 27 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
Experimental section
Chemistry
Synthetic Materials and Methods. Unless otherwise specified, reactions were performed under
an inert atmosphere of nitrogen and monitored by thinꢀlayer chromatography (TLC) and/or
LCMS. All reagents were purchased from commercial suppliers and used as provided. Flash
column chromatography was carried out on preꢀpacked silica gel cartridges. Reverse phase
chromatography samples were purified by preparative HPLC on a Phenomenex Luna C8(2) 5 um
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00Å AXIA column (30mm × 75mm). A gradient of acetonitrile (A) and 0.1% trifluoroacetic
acid in water (B) was used, at a flow rate of 50 mL/min (0ꢀ0.5 min 10% A, 0.5ꢀ7.0 min linear
gradient 10ꢀ95% A, 7.0ꢀ10.0 min 95% A, 10.0ꢀ12.0 min linear gradient 95ꢀ10% A). Samples
were injected in 1.5mL DMSO:methanol (1:1). A custom purification system was used,
consisting of the following modules: Waters LC4000 preparative pump; Waters 996 diodeꢀarray
detector; Waters 717+ autosampler; Waters SAT/IN module, Alltech Varex III evaporative lightꢀ
scattering detector; Gilson 506C interface box; and two Gilson FC204 fraction collectors. The
system was controlled using Waters Millennium32 software, automated using an AbbVie
developed Visual Basic application for fraction collector control and fraction tracking. Fractions
were collected based upon UV signal threshold and selected fractions subsequently analyzed by
flow injection analysis mass spectrometry using positive APCI ionization on a Finnigan LCQ
using 70:30 methanol:10 mM NH OH (aq) at a flow rate of 0.8 mL/min. Loopꢀinjection mass
4
spectra were acquired using a Finnigan LCQ running LCQ Navigator 1.2 software and a Gilson
2
15 liquid handler for fraction injection controlled by an AbbVie developed Visual Basic
application. All NMR spectra were recorded on 300ꢀ500 MHz instruments as specified with
chemical shifts given in ppm (δ) and are referenced to an internal standard of tetramethylsilane
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 53
`
(
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1
δ 0.00). H – H couplings are assumed to be firstꢀorder and peak multiplicities are reported in
the usual manner. HPLC purity determinations were performed on a Waters e2695 Separation
Module / Waters 2489 UV/Visible Detector. Column types and elution methods are described in
the Supporting Information section. The purity of the biologically evaluated compounds was
determined by HPLC to be >95%. Solvents used for HPLC analysis and sample preparation
were HPLC grade.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[(4R)-7-fluoro-2,2-dimethyl-3,4-dihydro-2H-
chromen-4-yl]cyclopropanecarboxamide (1) A stock solution of 1ꢀ(2,2ꢀ
difluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid and diisopropylethylamine (0.218
M and 0.654 M in dimethylacetamide, respectively), 284 ꢁL, 0.061 mmol 1ꢀ(2,2ꢀ
difluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid (1.0 eq) and 0.18 mmol
diisopropylethylamine (3.0 eq)), 2ꢀ(3Hꢀ[1,2,3]triazolo[4,5ꢀb]pyridinꢀ3ꢀyl)ꢀ1,1,3,3ꢀ
tetramethylisouronium hexafluorophosphate(V) (0.26 M in dimethylacetamide, 284 ꢁL, 0.074
mmol, 1.2 eq), and (R)ꢀ7ꢀfluoroꢀ2,2ꢀdimethylchromanꢀ4ꢀamine (2S,3S)ꢀ2,3ꢀdihydroxysuccinate
(0.40 M in dimethylacetamide, 232 ꢁL, 0.093 mmol, 1.5 eq) were aspirated from their respective
source vials, mixed through a PFA mixing tube (0.2 mm inner diameter), and loaded into an
injection loop. The reaction segment was injected into the flow reactor (Hastelloy coil, 0.75 mm
inner diameter, 1.8 mL internal volume) set at 100 °C, and passed through the reactor at 180 ꢁL
ꢀ1
min (10 minute residence time). Upon exiting the reactor, the reaction was loaded directly into
an injection loop and purified using prep LC method TFA1 to yield the title compound (4.1 mg,
1
1
6% yield). H NMR (400 MHz, DMSOꢀd :D O = 9:1 (v/v)) δ 7.39 (d, J = 1.7 Hz, 1H), 7.31 (d,
6
2
J = 8.3 Hz, 1H), 7.22 (dd, J = 8.3, 1.7 Hz, 1H), 7.09 – 6.97 (m, 1H), 6.69 (td, J = 8.6, 2.7 Hz,
1H), 6.52 (dd, J = 10.6, 2.6 Hz, 1H), 5.07 (t, J = 8.8 Hz, 1H), 1.83 (d, J = 9.0 Hz, 2H), 1.56 –
28
ACS Paragon Plus Environment

Page 29 of 53
Journal of Medicinal Chemistry
`
1
2
3
4
5
6
7
8
9
1
.45 (m, 1H), 1.44 – 1.35 (m, 1H), 1.33 (s, 3H), 1.21 (s, 3H), 1.19 – 0.99 (m, 2H); MS (APCI+)
+
m/z 420.1 (M+H) .
1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[(4S)-6-fluoro-2,2-dimethyl-3,4-dihydro-2H-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chromen-4-yl]cyclopropanecarboxamide (2). A stock solution of 1ꢀ(2,2ꢀ
difluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid and diisopropylethylamine (0.218
M and 0.654 M in dimethylacetamide, respectively, 284 ꢁL, 0.061 mmol 1ꢀ(2,2ꢀ
difluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid (1.0 eq) and 0.18 mmol
diisopropylethylamine (3.0 eq)), 2ꢀ(3Hꢀ[1,2,3]triazolo[4,5ꢀb]pyridinꢀ3ꢀyl)ꢀ1,1,3,3ꢀ
tetramethylisouronium hexafluorophosphate(V) (0.26 M in dimethylacetamide, 284 ꢁL, 0.074
mmol, 1.2 eq), and ((S)ꢀ6ꢀFluoroꢀ2,2ꢀdimethylꢀchromanꢀ4ꢀylamine with (2R,3R)ꢀ2,3ꢀdihydroxyꢀ
succinic acid (0.40 M in dimethylacetamide, 232 ꢁL, 0.093 mmol, 1.5 eq) were aspirated from
their respective source vials, mixed through a PFA mixing tube (0.2 mm inner diameter), and
loaded into an injection loop. The reaction segment was injected into the flow reactor (Hastelloy
coil, 0.75 mm inner diameter, 1.8 mL internal volume) set at 100 °C, and passed through the
ꢀ1
reactor at 180 ꢁL min (10 minute residence time). Upon exiting the reactor, the reaction was
loaded directly into an injection loop and purified using prep LC method TFA1 to yield the title
1
compound (1.9 mg, 7% yield). H NMR (400 MHz, DMSOꢀd :D O = 9:1 (v/v)) δ 7.41 (d, J =
6
2
1
1
.7 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.23 (dd, J = 8.3, 1.7 Hz, 1H), 6.94 (td, J = 8.6, 3.3 Hz,
H), 6.78 – 6.66 (m, 2H), 5.14 – 5.01 (m, 1H), 1.87 – 1.73 (m, 2H), 1.56 – 1.44 (m, 1H), 1.43 –
+
1.36 (m, 1H), 1.32 (s, 3H), 1.20 (s, 3H), 1.14 – 1.03 (m, 2H); MS (APCI+) m/z 420.1 (M+H) .
1
-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(7-methoxy-2-phenyl-3,4-dihydro-2H-
chromen-4-yl)cyclopropanecarboxamide (10) In a 4 mL vial, 300 ꢁL of a stock solution
containing 1ꢀ(2,2ꢀdifluorobenzo[d][1,3]dioxolꢀ5ꢀyl)cyclopropanecarboxylic acid (0.25 M, 0.073
29
ACS Paragon Plus Environment