Gut-Restricted Selective Cyclooxygenase-2 (COX-2) Inhibitors for Chemoprevention of Colorectal Cancer

Article abstract of DOI:10.1021/acs.jmedchem.1c00890

Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC) chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma progression and survival extension in an APCmin/+ mouse model. However, in vitro-in vivo correlation analysis showed their chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.

Full text of DOI:10.1021/acs.jmedchem.1c00890

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Article
Gut-Restricted Selective Cyclooxygenase‑2 (COX-2) Inhibitors for
Chemoprevention of Colorectal Cancer
Zhuming Zhang,* Avijit Ghosh, Peter J. Connolly, Peter King, Thomas Wilde, Jianyao Wang,
Yawei Dong, Xueliang Li, Daohong Liao, Hao Chen, Gaochao Tian, Javier Suarez, William G. Bonnette,
Vineet Pande, Karen A. Diloreto, Yifan Shi, Shefali Patel, Beth Pietrak, Lawrence Szewczuk,
Carlo Sensenhauser, Shannon Dallas, James P. Edwards, Kurtis E. Bachman, and David C. Evans*
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ABSTRACT: Selective cyclooxygenase (COX)-2 inhibitors have been extensively studied for colorectal cancer (CRC)
chemoprevention. Celecoxib has been reported to reduce the incidence of colorectal adenomas and CRC but is also associated
with an increased risk of cardiovascular events. Here, we report a series of gut-restricted, selective COX-2 inhibitors characterized by
high colonic exposure and minimized systemic exposure. By establishing acute ex vivo 18F-FDG uptake attenuation as an efficacy
proxy, we identified a subset of analogues that demonstrated statistically significant in vivo dose-dependent inhibition of adenoma
progression and survival extension in an APCmin/+ mouse model. However, in vitro−in vivo correlation analysis showed their
chemoprotective effects were driven by residual systemic COX-2 inhibition, rationalizing their less than expected efficacies and
highlighting the challenges associated with COX-2-mediated CRC disease chemoprevention.
INTRODUCTION
■
Colorectal cancer (CRC) is the third leading cause of cancer-
related death in both men and women in the United States and
also one of the most preventable malignancies.1,2 It is well
accepted that most CRCs occur via a metachronous adenoma−
carcinoma sequence characterized by well-staged genetic
alterations and oncogenic transformation over many years.2−4
A long-term use of a single or combination of chemoprotective
agents has been shown to impede, arrest, or even reverse the
Figure 1. Chemical structures of celecoxib (1) and etoricoxib (3).
development of adenomas in the colon, and interfere with their
progression from premalignant adenomas to carcinomas.5−7 In
this regard, nonsteroidal anti-inflammatory drugs (NSAIDs),
particularly selective cyclooxygenase COX-2 inhibitors, have
been one of the most studied class of drugs in CRC
chemoprevention.8−10 Recently, extensive epidemiological and
NSAIDs act by inhibiting the synthesis of prostanoids, a
family of biologically active mediators generated by the activity
Received: May 17, 2021
Published: July 19, 2021
randomized clinical studies have unequivocally established the
chemopreventive effects of NSAIDs.11−16 Celecoxib (1, Figure
1), a modestly selective COX-2 inhibitor, was approved by the
FDA for adenoma prevention in patients with familial
adenomatous polyposis (FAP), an inherited genetic predis-
position to CRC.14−16
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Table 1. Inhibitory Potency in Human COX-2/COX-1 Enzymes, and COX-2/COX-1-Driven PGE2 Synthesis in HEK293 Cells of
1, 3−12
a
a
b
b
Cpd
hCOX-2 IC50 (μM)
hCOX-1 IC50 (μM)
PGE2 COX-2 IC50 (μM)
PGE2 COX-1 IC50 (μM)
1
3
4
5
6
7
0.028 0.027
0.43 0.03
0.39 0.04
9.2 0.81
>50
>50
>50
>50
0.057 0.021
0.56 0.19
1.50 0.23
1.89 0.49
0.95 0.16
>50
0.41 0.20
>50
>50
>50
>50
c
6.36
3.24
c
>50
>50
>50
8
9
10
11
12
17.1 1.8
4.10 0.14
0.84 0.06
0.226 0.042
>50
>50
>50
>50
>50
>50
0.60 0.43
0.047 0.036
2.95
>50
>50
>50
14.1 4.2
>50
c
c
18.2
>50
a
Peroxidase fluorescent kinetic activities were measured (IC50 S.D.) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as an electron donor
b
after preincubation with inhibitors for 60 min. Production of PGE2 was measured over time (IC50 S.D.) by tandem MS/MS analysis after
pretreatment with inhibitors for 60 min in 10% FBS. Tested only once. The number of replicates for other tested compounds was at least twice.
c
of COXs.17,18 On the basis of pharmacodynamic (PD) features
at therapeutic doses, NSAIDs are classified as either nonselective
COX-1/COX-2 inhibitors or selective COX-2 inhibitors. The
precise molecular mechanism of colorectal tumorigenesis
inhibition by NSAIDs has not been fully delineated, and likely
involves a combination of COX-dependent and COX-
independent pathways.19−21 A prolonged use of nonselective
NSAIDs is associated with side effects such as gastrointestinal
bleeding and ulceration that is partially attributed to COX-1
inhibition in the cases of SC-560 and indomethacin.22−24
COX-2 and COX-1 are two isoenzymes that catalyze the
initial step of the conversion of arachidonic acid to bioactive
prostaglandins (PGs).18,25,26 Constitutive COX-1 is ubiqui-
tously expressed in almost all tissues for housekeeping PG
synthesis, whereas inducible COX-2 is upregulated via
stimulation of inflammatory cytokines or growth factors.18,27
Increased expression of COX-2 occurs during all stages of the
multistep progression of CRC,28−30 and overexpression of
COX-2 correlates with the colon tumor size, invasiveness, and
increased PG levels.31 It is now understood that proinflamma-
tory and immune-suppressive PGE2 plays a central role in
colorectal tumor growth by promoting proliferation, survival,
motility, angiogenesis, and DNA methylation and potentiating
Wnt signaling.32−36 In preclinical studies, inhibition of COX-2
reduced the size and number of polyps in multiple adenoma and
inflammation mouse models.37−40 In a randomized clinical
study conducted in FAP patients, a significant dose-dependent
reduction in polyp burden was observed in patients who
received celecoxib (1).15 Rofecoxib (2, Figure 1) was also found
to significantly reduce the risk of recurrent adenomas in another
clinical trial focusing on maintaining a polyp-free colon in FAP
patients.13 In the Prevention of Sporadic Adenomatous Polyps
(PreSAP) clinical trial, the use of celecoxib was correlated with a
reduced relative risk (RR of 0.64) for adenomas detected during
a 3 year period.14 However, these studies also showed that
cardiovascular (CV) adverse events increased with the
administration of selective COX-2 inhibitors.16,41,42 The
elevated CV hazard was associated with COX-2 dependent
pathways, putatively attributed to blocked prostacyclin (PGI2)
production.42 This serious CV risk has rendered celecoxib (1)
and rofecoxib (2) unsuitable for a long-term use in a wider
population of patients.12−16,41,42
colon-specific prodrugs of celecoxib have been investigated.43,44
Other approaches to overcome systemic adverse events,
including the use of formulations to deliver active drugs to
different regions of the gastrointestinal tract, have also been
reported.45,46 Instead, we pursued an alternative strategy to
modulate the physicochemical properties of potent and selective
COX-2 inhibitor candidate drugs in order to restrict them to the
intestinal tract. In so doing, local on-target concentrations in the
gastrointestinal tract are maximized while systemic exposure is
minimized.47 Our objective, therefore, was to identify a colon-
targeted, orally administered, COX-2-selective inhibitor that
would suppress local PGE2 production in the target tissue
(lamina propria), reduce adenoma progression for CRC
chemoprevention, and be devoid of systemically mediated CV
adverse effects.
We chose etoricoxib (3, Figure 1) as the starting point based
on two key factors.48,49 First, we recently described the use of
reoptimized MS-based COX enzymatic assays to investigate
previously unpublished time-dependent inhibition kinetics of
COX-2 inhibitors from several chemical series.50 Our studies
confirmed high specificity of COX-2 inhibition associated with
etoricoxib (3) and close analogues (4−12) sharing a common
pyridyl core ring (Table 1). In contrast, celecoxib (1) displayed
only moderate selectivity for COX-2 over COX-1 inhibition
(Tables 1 and 5). Moreover, celecoxib (1) exhibited high overall
thermodynamic potency indicated by the intrinsic inhibition
constant (Ki*), but also associated with a short dissociation half-
life (t1/2(dissociation)). By contrast, etoricoxib (3) and its analogues
10 and 11 (Figure 3), while less potent than 1, showed a longer
kinetic residence time (t1/2(dissociation)) for target occupancy.51
We envisaged that long residence time kinetics would be
important for robust target engagement in local colon tissues by
minimizing systemic drug recirculation resulting from gut-
restricted distribution.
Second, compared to celecoxib (1, clog P 4.57), etoricoxib (3,
clog P 2.35) was a more hydrophilic starting point for our
exploration. Previously, the physicochemical property-based
approach proved to be quite effective in identifying gut-
restricted JAK inhibitors with low intrinsic permeability, which
translated into high colonic retention and low systemic exposure
after oral dosing.47 Our initial plan was centered on lowering
clog P to reduce permeability while balancing potency of COX-2
inhibition, a similar strategy utilized for enteric JAK
inhibitors.47,52 However, an inverse correlation of potency and
To minimize the risk of CV adverse effects, but maintain the
beneficial property of attenuating colon polyp progression,
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clog P counteracted our efforts for further lowering the
permeability of these inhibitors. The high clearance of these
analogues was recognized as a key attribute to minimize systemic
exposure for the small fraction of drug that was absorbed.
Herein, we describe our coordinated multidimensional opti-
mization efforts for a series of potent, selective COX-2 inhibitors
highlighting their gut-restricted pharmacological character-
ization in vitro and in vivo.
endoplasmic reticulum (ER) and the nuclear envelope, we
also established MS-based orthogonal assays to confirm cellular
penetration and uptake as well as COX-1/COX-2 selectivity of
these inhibitors by measuring production of PGE2 from
arachidonic acid in stably transfected human embryonic kidney
(HEK) 293 TRex cells overexpressing COX-1 or COX-2
controlled by an inducible tetracycline promoter.56
The 5-CF3 analogue 4 (clog P 2.06; hCOX-2 IC50 0.39 μM),
with a less planar, gem-dimethyl-substituted glycol replacing its
peripheral pyridinyl ring, was identified to have lower clog P but
still retain similar enzymatic potency and selectivity to etoricoxib
(3, clog P 2.35; hCOX-2 IC50 0.43 μM) (Figure 3, Table 1). The
5-CF3 moiety of 4 was anticipated to occupy a similar region in
the COX-2 active site to 3-CF3 of celecoxib (1). The
nonfluorinated analogues 5 (5-Me; clog P 1.60; hCOX-2 IC50
6.36 μM) and 6 (5-Et; clog P 2.13; hCOX-2 IC50 3.24 μM) lost
enzymatic potency but still maintained cellular COX-2 potency
in the low micromolar range (IC50 1−2 μM). However, by
reducing clog P below 1, analogue 7 (5-CH2OH; clog P 0.51;
hCOX-2 IC50 > 50 μM) became inactive in both enzymatic and
cellular assays (PGE2 COX-2 > 50 μM). Introducing an
additional 6-methoxy (R1 = Me) or 6-isopropoxy (R1 = i-Pr) in 7
led to an improvement in enzymatic activity in 8 (clog P 0.89;
hCOX-2 IC50 17.1 μM) and 9 (clog P 1.73; hCOX-2 IC50 4.10
μM) but did not confer cellular activity (PGE2 COX-2 IC50 > 50
μM). Fortunately, continued SAR development with 6-propoxy
(R1 = n-Pr) and 6-butoxy (R1 = n-Bu) improved both enzymatic
and cellular potencies as shown in analogues 10 (clog P 1.95;
hCOX-2 IC50 0.84 μM; PGE2 COX-2 IC50 0.6 μM) and 11 (clog
P 2.48; hCOX-2 IC50 0.226 μM; PGE2 COX-2 IC50 0.047 μM).
In particular, 11 displayed comparable potency to etoricoxib (3,
PGE2 COX-2 IC50 0.56 μM) and celecoxib (1, PGE2 COX-2
IC50 0.057 μM). Like etoricoxib (3, PGE2 COX-1 IC50/COX-2
IC50 ratio >80), compounds 10 (PGE2 COX-1 IC50 > 50 μM)
and 11 (PGE2 COX-1 IC50 = 14.1 μM) remained more selective
than celecoxib (1, PGE2 COX-1 IC50/COX-2 IC50 ratio ∼ 7).
However, by lowering clog P below 1, compound 12 (R1 =
(CH2)2OMe; clog P 0.78) lost significant potency (hCOX-2
RESULTS AND DISCUSSION
■
Analysis of cocrystal structures of celecoxib (1), close analogues,
and rofecoxib (2) bound to COX-2 enzymes (PDB IDs: 3LN1;
5KIR) indicated the phenylsulfonamide or methylsulfonylphen-
yl groups were optimal and critical in occupying the COX-2-
binding pocket surrounded by key residues, especially Arg499
and Val509.53−55 As etoricoxib (3) was also shown to share a
similar binding orientation to COX-2 (Section S11, Figure S5),
these structures served as the basis for our exploration (Figure
2). We recently reoptimized COX enzymatic assays to evaluate
Figure 2. Docking model of compound 11 (balls and sticks) in complex
with COX-2 (side chains as sticks and binding pocket depicted as an
electrostatic surface mapblue: positive and red: negative potential).
Celecoxib (1, PDB ID: 3LN1) is superimposed as purple sticks for
reference.
COX-2 inhibitors.50 Because COX enzymes are intracellular
targets localized in subcellular compartments such as
Figure 3. Evolution of etoricoxib (3) and analogues 4−12 for local enteric delivery.
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IC50 18.2 μM; PGE2 COX-2 IC50 2.95 μM) compared to
corresponding compound 11 (R1 = n-Bu; clog P 2.48).
compounds 10 and 11 displayed high colonic exposures (>4300
ng/g) and a low systemic exposure (<6 ng/mL) with a
distribution ratio of C/P > 1200 at 4 h (Tmax ∼ 0.25 h) following
cassette 10 mg/kg oral dosing. Both compounds exhibited high
clearance (>90 mL/min/kg) with a volume of distribution
(Vdss) above others (>2 L/kg) after 2 mg/kg IV cassette dosing
(Table 2). The gut-restricted characteristics of compounds 10
and 11 were confirmed by single-dose time-course PK studies
following colon exposures as well as systemic plasma
concentration in expanded time points after an oral dosing
(10 mg/kg) in C57BL Mice (Table 5, also Section S4, Figures
S1 and S2).
The PK results suggested that unique metabolism of
compounds 10 and 11 played a critical role in the observed
high colon/plasma (C/P) ratio distinct from that of compound
6. To study its metabolic fate, compound 11 was incubated with
human and mouse hepatocytes, and metabolites were identified
by liquid chromatography interfaced with tandem mass
spectrometric (LC−MS/MS) analysis and their relative
percentages were quantitatively assessed based on peak areas
in the mass spectral response (Table 3). Compound 11 was
found to undergo extensive metabolism with a turnover of ∼100
and 79.6% in human and mouse hepatocytes, respectively.
Glucuronidation was the major metabolic pathway in both
species with a single O-glucuronide M6 identified as the major
metabolite in human (92.5%) and mouse (24.7%). The hydroxyl
group in 5-CH2OH was the putative site for the formation of O-
glucuronide M6. However, efforts to confirm the structure of
M6 were unsuccessful due to the inability to isolate the putative
M6 or synthesize an authentic sample for authentication. A
subsequent in vitro UDP-glucuronosyltransferase (UGT)
phenotyping screening identified UGT1A9 as the primary
isoform responsible for driving clearance via glucuronidation
(Section S6). Consistent with these findings, the single-dose oral
and IV PK studies confirmed a minimal systemic plasma
exposure and low oral bioavailability in mouse (4.25%), rat
(1.5%), and monkey (4.87%) (Section S5, Table S4). Dog was
the exception, showing high oral bioavailability (74%, Table S4),
possibly resulting from different UGT1A9 expression levels or
substrate affinity, as reported with other human UGT1A9
substrates (Section S7).57
The in vitro metabolism study also identified oxidative
hydroxylation of the 6-butoxy chain as the other major
metabolic clearance pathway in mouse. However, this path
was less significant in human hepatocytes indicated by the
formation of a variable amount of hydroxylated metabolites M4
and M5 (Table 3). Minor metabolites such as o-dealkylated M2
(loss of n-butyl) and carboxylic acid M7 from oxidation of 5-
CH2OH were also observed in human and/or mouse
hepatocytes. These putative metabolites M2, M4, M5, and M7
were synthesized and found to be inactive in the COX-2
enzymatic (COX-2 IC50 > 50 μM) assay and would not
contribute to in vivo pharmacology (Section S3, Table S2). The
metabolite study confirmed that compound 10 was metabolized
similar to compound 11 with glucuronidation representing the
dominant metabolic pathway in both human and mouse species
in vitro. Hydroxylation of the 6-propoxy moiety and oxidation of
5-CH2OH to the carboxylic acid were major pathways in mouse
but less significant in human hepatocytes (Section S2). Like
compound 11, single-dose oral and IV PK studies of compound
10 also generally demonstrated gut-restricted characteristics
across species with low oral bioavailability in mouse (4.25%), rat
Compounds 1, 3, 4, 6, 10, and 11 were screened in cassette
PO dosing PK studies for their systemic (plasma) and colonic
exposures at 2 and 4 h time points following oral dosing of 10
mg/kg in C57BL mice (Tmax ranging from 0.25 to 3 h). In vivo
clearance (CL), volume of distribution (Vdss), and half-life
(T1/2) of these compounds were also determined in cassette IV
dosing PK studies in mice to correlate with in vitro human or
mouse liver microsomal and hepatocyte stability (Table 2).
Table 2. Human/Mouse Liver Microsomal, Hepatic
Clearance, and Mean Cassette Dosing PK Parameters of
Compounds 1, 3, 4, 6, 10, and 11 in C57BL Mice by PO (10
mg/kg) and IV Dosing (2 mg/kg)
Compound
HLM T1/2 (min)
MLM T1/2 (min)
1
3
4
6
10
11
a
a
67.2
59.3
57.4
>184
53.8
>371
>184
>184
>371
68.7
13.2
84.2
>184
19.6
3.1
27.8
2.7
2.4
human Hep
b
T1/2 (min)
mouse Hep
33.1
29.2
>371
3.2
3.0
1.9
b
T
1/2 (min)
c
CL (mL/min/kg)
10.5
0.94
1.27
9460
2553
41.3
1.27
0.78
55.7
36.4
0.54
1.55
38.5
8400
9803
65.6
1.35
0.32
51.6
21.8
92.7
3.05
NA
5336
5.7
101.2
4.92
0.23
4352
3.0
c
V
T
dss (L/kg)
1/2 (h)
c
f
d
colon (ng/g), 4 h
plasma (ng/mL),
d
4 h
ratio colon/plasma,
3.8
1.5
0.9
2.5
1276
1616
e
4 h
a
For HLM/MLM T1/2: high stability > 184 min; 33 min < medium
b
stability <184 min; and low stability < 33 min. For hepatocytes T1/2
:
high stability > 371 min; 60 min < medium stability < 371 min; and
c
low stability < 60 min. IV cassette dosing of 2−4 compounds in each
d
group, solution in 70% PEG/H2O. Oral cassette dosing, solution in
70% PEG/H2O, values shown are the average concentration for
e
individual animals at time point of 4 h. The ratios were calculated as
f
averages of ratios from individual animals. T1/2 was not obtained due
to R-squared (Rsq) < 0.75.
Compound 4 was highly stable in human and mouse liver
microsomes and hepatocytes in vitro, which was consistent with
its low clearance (CL 0.54 mL/min/kg) and long half-life (T1/2
38.5 h) in a cassette 2 mg/kg IV PK study. Compound 4 also had
good exposure and equal distribution in colon (8400 ng/g) and
plasma (9803 ng/mL) at 4 h (Tmax 3 h) following a cassette 10
mg/kg oral dosing in vivo. Celecoxib (1) showed modest
metabolic stability in vitro but moderate clearance (CL 10.5
mL/min/kg) and half-life (T1/2 1.27 h) in mice in vivo, which
was consistent with a high exposure in colon (9460 ng/g) and a
relatively lower exposure in plasma (2553 ng/mL). Etoricoxib
(3) was metabolically stable in human liver microsomes and
hepatocytes but unstable in mouse and showed higher clearance
(CL 41.3 mL/min/kg) and a short half-life (T1/2 0.78 h), leading
to a low exposure in colon (55.7 ng/g) and plasma (36.4 ng/
mL) in mice. Similarly, compound 6 was metabolically unstable
in mouse in vitro and unsurprisingly showed a low exposure in
colon (51.6 ng/g) and plasma (21.8 ng/mL) consistent with its
higher clearance (CL 65.6 mL/min/kg) and a shorter half-life
(T1/2 0.32 h) in vivo. There were no signs of gut restriction for
compounds 1, 3, 4, and 6 despite a wide range of
physicochemical and ADME properties such as clog P,
metabolic stability, and clearance. Their volume of distribution
(Vdss) was in a narrow range of 1−2 L/kg. Unexpectedly,
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Table 3. In Vitro Metabolite Profiling and Identification from Incubations of Compound 11 with Human and Mouse
a
Hepatocytes
% peak area
metabolite
M1
M2
M3
M4
M5
M6
M7
human
mouse
biotransformation
0.1
3.08
1.15
2.63
0.53
92.51
0.51
7.97
1.56
38.44
2.23
24.76
4.14
O-dealkylation and glucuronidation
O-dealkylation
hydroxylation and glucuronidation
hydroxylation
hydroxylation
O-glucuronidation
alcohol oxidation
compound 11
20.40
a
Final compound 11 concentration of 10 μM and a cell concentration of 1 × 106 cells/mL (1000 μL final incubation volume). Cells were incubated
for 0 and 2 h. The percent compositions of parent drug and its metabolites were based on peak areas from 5 ppm accurate mass measurements and
with assumptions of equal positive ESIs.
Table 4. Inhibitory Potency in COX-2/COX-1-Driven PGE2 Synthesis in HEK293 Cells, and Mean Cassette Dosing PK
Parameters of Compounds 13-20 in C57BL Mice by IV Dosing (2 mg/kg) and PO (10 mg/kg)
b
b
c
d
d
e
cpd
PGE2, COX-2, IC50 (μM)
PGE2, COX-1, IC50 (μM)
CL (mL/min/kg)
colon (ng/g), 4 h
plasma (ng/mL), 4 h
ratio of colon/plasma
11
13
14
15
16
17
18
19
20
0.047 0.036
2.34
1.21 0.30
0.54 0.46
0.049
14.1 4.2
7.69
>50
>50
101.2
48.2
58.8
55.4
32.2
101.5
68.6
89.3
100.4
4352
2810
85.8
478.2
1942
1872
11920
1854
5394
3.0
22.8
35.8
28.7
75.9
6.7
7.7
2
9.9
1616
134.3
2.5
16.5
24.9
402.3
1596
1040
545.2
f
f
0.511
2.46 0.99
>50
0.036 0.009
1.32 0.33
f
f
0.21
>50
0.037 0.027
7.23 2.31
a
Peroxidase fluorescent kinetic activities were measured (IC50 S.D.) using ADHP as an electron donor after preincubation with inhibitors for 60
min. The production of PGE2 and PGD2 was measured over time (IC50 S.D.) by tandem MS/MS analysis after pretreatment with inhibitors for
60 min in 10% FBS. IV cassette dosing, solution in 70% PEG/H2O. Oral cassette dosing, solution in 70% PEG/H2O, values shown are the
average concentration for individual animals at a time point of 4 h. The ratios were calculated as averages of ratios from individual animals. Tested
only once. The number of replicates for other tested compounds was at least twice.
b
c
d
e
f
(2.3%), and monkey (20.0%); dog was an exception (79.5%)
(Section S4, Table S3).
reduced colon/plasma ratio (C/P: 134.3). Adding two geminal
methyl groups (R2 = −CH(OH)Me2) resulted in compound 14
with a diminished colon concentration (85.8 ng/g) and a colon/
To better understand the critical role of the primary hydroxyl
group (5-CH2OH) in compound 10 or 11 leading to high in
vivo clearance and high colon/plasma (C/P) ratios, compounds
13−20 were prepared for characterization (Figure 4, Table 4).
Introducing a methyl group (R2 = −CH2(OH)Me) led to
compound 13 with lower clearance (CL 48.2 mL/min/kg) and a
plasma ratio (C/P: 2.5). Extending compound 11 (R2
=
−CH2OH) with a hydroxyethyl group provided compound 15
(R2 = −CH2O(CH2)2OH) with an attenuated colon/plasma
ratio (C/P: 16.5), presumably attributed to the alteration of
metabolism for hepatic clearance or less dependence on the
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Figure 4. Analogues 13−20 with modifications at R2 or R3 on the pyridine ring sites of compound 11.
glucuronidation pathway. These results provided direct
evidence in support of the primary benzylic hydroxyl moiety
(R2 = −CH2OH) as the major metabolic site for glucuronida-
tion driving clearance and resultant high colon/plasma exposure
ratios.
in this subset of COX-2 inhibitors (Figure 5), we observed a
direct relationship between clog P (log D) and COX-2 activities.
Compounds with low clog P (<1) were unlikely to have
acceptable cellular COX-2 potency (PGE2 COX-2 pIC50 > 6 or
IC50 < 1 μM). This observation was not totally unexpected given
the intracellular localization of the COX-2 enzyme and the
subcellular microenvironment of the COX-2-binding site, which
is surrounded by hydrophobic residues. Low clog P (<1) would
not only attenuate permeability for cellular uptake but also affect
lipophilicity required for biochemical affinity. The correlation
was in line with the weaker potency associated with compounds
8 (clog P 0.89; Figure 3, Table 1), 12 (clog P 0.78; Figure 3,
Table 1), and putative (inactive) metabolites M2, M4, and M5
(clog P < 1; Table 3, also Table S2).
Conversely, an empirically inverse correlation seemed to exist
between a lower clog P (<2) and a higher colon/portal vein
(PV) plasma ratio (>150) at 4 h following oral dosing (10 mg/
kg) cassette PK in C57BL mice, presumably resulting from
limited absorption through reduced permeability (Figure 6).
Careful analysis of single-dose PK studies of compounds 10 (PV
plasma 98.6 ng/mL; systemic plasma 25 ng/mL), 20 (PV
plasma 82.4 ng/mL; systemic plasma 3 ng/mL), 21 (PV plasma
178.4 ng/mL; systemic plasma 3.4 ng/mL), and 22 (PV plasma
230.3 ng/mL; systemic plasma 3.8 ng/mL) revealed the PV
plasma concentrations were consistently higher than systemic
plasma exposures, indicating that some drug was being absorbed
across the gut epithelium but subsequently cleared first pass to
result in apparent gut restriction (Table 5). To minimize this
effect, we incorporated bioisosteric replacements in the phenyl
region (R5) aimed at attenuating clog P (log D) and permeability
(Figure 7). A pyridinyl sulfonamide analogue 23 (clog P 1.94;
PGE2 COX-2 0.23 μM) was identified having acceptable,
though decreased, cellular potency. Compound 23 showed the
lowest MDCK based passive permeability (Papp A>B 8.15 cm/s
×10−6) and the narrowest gap between PV and systemic plasma
exposures (7.1 ng/mL vs 5.5 ng/mL); its similar colon/PV
plasma and colon/plasma ratios (2179 vs 1692) were indicative
of limited absorption via restricted permeability (Table 5).
Turning our attention to the aliphatic gem-dimethyl-
substituted glycol in compound 11, we found that replacing
this group with an aromatic 4-fluorophenyl led to a lower
clearance (CL 32.2 mL/min/kg) and a lower colon/plasma
exposure ratio (C/P: 24.9) as shown in compound 16 (Table 4).
Also, compound 16 showed narrow selectivity in the cellular
COX-1/COX-2 assays (PGE2 COX-1 IC50/COX-2 IC50 ratio ∼
10, Table 4), highlighting that aromatic substitution at R3 was
unfavorable. Compounds 17, 18, and 19, sharing a terminal
oxetanyl group with or without an attached tertiary hydroxyl
moiety, all maintained high colon/plasma ratios (C/P: >400).
This finding indicated the tertiary hydroxyl group might be
additive but not essential for driving the clearance and high
colon/plasma ratio. It should be pointed out that modification of
gem-dimethyl groups to an oxetane ring altered COX-2 potency
or selectivity and colonic exposure. For example, compared to
compound 11 (PGE2 COX-1 IC50/COX-2 IC50 ratio ∼ 300;
colon concentration 6506 ng/g at 2 h) (Tables 1 and 2),
analogue 17 was potent in COX-2 inhibition but 4-fold less
selective (PGE2 COX-2 IC50 0.047 μM; PGE2 COX-1 IC50/
COX-2 IC50 ratio ∼ 70). Compound 18 was relatively less
potent in cellular COX-2 activity (PGE2 COX-2 IC50 1.32 μM).
The colonic exposure of compound 19 was noticeably lower
(1854 ng/g). By contrast, cyclizing the terminal gem-dimethyl
to a cyclobutyl group led to compound 20 with overall in vitro
and in vivo properties comparable with compound 11 (Tables 4
and 5).
To support the selection of compounds for in vivo PD studies,
additional analogues were synthesized. Modifications to the 6-
butoxy (R4) site on compounds 10 and 11 were made by varying
alkoxy length and hydrophobicity, resulting in identification of
the fluorinated analogues 21 (clog P 2.30) and 22 (clog P 2.03)
for further studies (Figure 7). By correlating COX-2 cellular and
enzymatic potency with physicochemical or ADME properties
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Figure 5. Correlation between cellular PGE2 COX-2 potency (pIC50) and clog P with human COX-2 enzymatic potency (pIC50, color coding) [pIC50
= −log10 (IC50 μM)].
Figure 6. Comparison of the mouse exposure colon/plasma ratio to the colon/PV plasma ratio (both measured at 4 h) in cassette PK following an oral
dosing of 10 mg/kg in C57BL mice. All points were color coded in relation to clog P. Logarithmic scale was used on all axes.
Figure 7. Analogues 21−23 as selected representatives with modifications at various sites of compounds 11 (or 10).
To confirm COX-1/COX-2 selectivity in a more physiolog-
ically relevant system, compounds 10, 11, and 20−23 were
evaluated in human whole blood (HWB) for COX-2 inhibition
by measuring PGE2 production stimulated by LPS, and COX-1
inhibition by measuring TXB2 synthesis stimulated by a calcium
ionophore.58,59 All analogues displayed concentration-depend-
ent inhibition of PGE2 and achieved ∼100% inhibition at the
highest tested concentration, with COX-2 IC50 values ranging
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Figure 8. Side-by-side comparison of the [18F]-FDG PET signal (expressed as average injected dose per gram (% ID/g, 20% threshold) in 2-week
curative setting) and polyp area attenuation in APCMin/+ mice in prophylactic setting (12 weeks) for compound 10. Vehicle and celecoxib groups (300
mg/kg/daily in chow diet) served as negative and positive controls, respectively. Analyses were performed in R, version 3.3.2. ANOVA, adjusted for
multiple comparisons in a linear regression model. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.
Figure 9. Compounds 11, 20, and 23 for [18F]-FDG PET in curative setting (dosing in chow diet, mice received daily doses of 3, 10, 30, 100, and 300
mg/kg for 2 weeks from the age of >16 weeks) compared to compound 10. 18F-FDG PET signal expressed as average injected dose per gram (% ID/g,
20% threshold). The percentage injected dose per gram (% ID/g) was calculated as follows: % ID/g = ROI activity divided by injected dose multiplied
by 100%. Vehicle and celecoxib (300 mg/kg/daily) served as negative and positive controls, respectively. Analyses were performed in R, version 3.3.2.
ANOVA, adjusted for multiple comparisons in a linear regression model. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.
from 0.15 to 7.5 μM (Table 5). By contrast, none of these
compounds significantly inhibited COX-1 (IC50 > 60 μM),
consistent with their COX-1/COX-2 selectivities in stably
transfected HEK293 cells. Except for compounds 10 (HWB
COX-2 IC50 2.3 μM) and 23 (HWB COX-2 IC50 7.2 μM),
compounds 11 and 20−22 showed similar potencies to
celecoxib (1, HWB COX-2 IC50 0.44 μM) and etoricoxib (3,
HWB COX-2 IC50 0.611 μM). Compounds 11 and 20−22
= 6.6, 2.5% respectively), fluorinated analogues 21 and 22
displayed much higher unbound fractions (human colon fu % =
22.4, 20.4% respectively) though their human plasma protein-
binding values (human PPB fu %: 0.9−1.6%) were similar. All
analogues showed a narrow range of solubility (FaSSIF, 25−110
μM). It was notable that compound 20 was the most potent
COX-2 inhibitor (hCOX-2 IC50 0.12 μM; PGE2 COX-2 IC50
0.037 μM; and HWB COX-2 IC50 0.146 μM) with low
nanomolar COX-2 enzyme kinetic potency (Ki* 2.9 nM) and a
long residence time (T1/2(dissociation) 39 h) for target occupancy.50
To evaluate the in vivo chemoprotective effects of these gut-
restricted COX-2 inhibitors, we first measured attenuation of ex
vivo local ileum acute glucose (as 18F-FDG) uptake from short-
term treatment (2 weeks) as an indirect PD surrogate and
predictor of long-term efficacy (12 weeks) in a multiple
intestinal neoplasia (MIN) mouse model by utilizing molecular
tomographic imaging.60 This Apc mutant (ApcMin/+) mouse
model, widely used in colorectal carcinogenesis and chemo-
prevention research, simulates human adenoma progression, in
which loss of function (LOF) of tumor suppressor adenomatous
polyposis coli (APC) gene by deletion/mutation leads to the
showed higher selectivity (HWB COX-1 IC50/COX-2 IC50
>
200) than etoricoxib (3, HWB COX-1 IC50/COX-2 IC50
∼
150). The selectivity of compound 10 (HWB COX-1 IC50/
COX-2 IC50 ∼ 80) was still higher than that of celecoxib (1,
HWB COX-1 IC50/COX-2 IC50 ∼ 37). The potency of
compound 23 (HWB COX-2 IC50 7.2 μM) was weaker than
anticipated, presumably attributable in part to high human
plasma/serum protein binding as evidenced by very low
unbound fractions (human PPB fu % = 0.2%) compared to
others in this group (human PPB fu % in a range of 0.5−1.6%).
Interestingly, the unbound fraction of compound 23 was
significantly higher in colon tissue (human colon fu % =
7.5%). In contrast to compounds 10 and 11 (human colon fu %
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Figure 10. Oral in vivo efficacy of compound 21 in prophylactic setting (dosing as a suspension by gavage, mice received doses of 30, 100, and 300 mg/
kg for 10 weeks from the age of 5−6 weeks). Celecoxib (300 mg/kg/daily in a suspension by gavage) served as a positive control. Polyp area/number in
treatment: One-way ANOVA with Dunnett’s multiple comparison test; polyp number vs polyp size: Two-way ANOVA with Dunnett’s multiple
comparison test. GraphPad Prism 7.0.
Table 6. Comparison of In Vitro and In Vivo IC50 Values (nM, Unbound)
a
b
in vivo potency
systemic (IC50 nM)
in vitro potency
compound
gut (IC50 nM)
HWB COX-2 (fu % × IC50 nM)
HWB COX-1 (fu % × IC50 nM)
c
1 (celecoxib)
3.2
35
1.3
1.9
3.2
1.32
37.5
8.77
3.8
44.4
10
21
22
2384
741.8
160
3088
1810
>1980
a
Calculated based on in vivo polyp area TGI (%, 300 mg/kg oral daily dosing) and relevant plasma and ileum exposures 4 h after the last dose in a
b
12 week prophylactic setting in APCmin/+ mice. Calculation (IC50 × fu %) based on measured in vitro human whole blood COX-2/COX-1
potency (IC50) and human plasma protein-binding free faction (fu %) (Table 5). Based on 9 week PK exposure in prophylactic setting (300 mg/
c
kg oral daily dosing), assuming equal exposure in plasma and ileum, fit using Mathematica 11.
initiation of adenomatous polyp formation and growth,
recapitulating clinically relevant CRC tumorigenesis observed
in FAP patients.61−63 Celecoxib (1) has been shown to be
effective in suppressing adenoma formation and growth in this
ApcMin/+ mouse model over a study period of 1−2 months.64 To
shorten the turnaround time, we hypothesized that 18F-FDG
uptake attenuation could represent a sensitive biomarker for
predicting efficacy as it is a common assumption that metabolic
changes precede morphologic responses in the polyp size and/or
number.65 By using compound 10, we first established that there
was a direct correlation between 18F-FDG signals from the
abdominal region in vivo and ileum ex vivo in APC mice (data
not shown). We then confirmed a positive correlation between
short-term dose-dependent reduction in ex vivo ileum 18F-FDG
PET signal from treatment with compound 10 in curative setting
(dosing in chow diet, mice received doses of 30, 100, and 300
mg/kg for 2 weeks starting from the age of 16 weeks with
established adenomas) and long-term does-dependent polyp
area inhibition in ApcMin/+ mice in a prophylactic setting (dosing
of compounds in chow diet, mice received doses of 30, 100, or
300 mg/kg for 12 weeks from the age of 5−6 weeks) using
celecoxib (1, chow diet, 300 mg/kg daily) and vehicle as positive
and negative controls (Figure 8). The correlation established
acute 18F-FDG PET signal attenuation as a prognostic
biomarker to predict efficacy and quickly eliminate ineffective
compounds that failed to reduce glucose uptake within 2 weeks.
Compound 10 showed short-term, dose-dependent reduction
of 18F-FDG uptake (35.6%, P < 0.01; IC50, 4.4 μM) at 300 mg/
kg oral daily dosing in a curative setting (2 week), which
translated into a statistically significant polyp area tumor growth
inhibition (TGI 41.9%) for total ileum polyp area (sum all of all
polyp areas with diameter > 1 mm) in a prophylactic setting
(300 mg/kg daily, 12 weeks) over celecoxib (18F-FDG, 31.4% P
< 0.001; TGI 65.9%) (Table 5). Etoricoxib (3, 18F-FDG, 43.5%,
P < 0.01), compounds 11 (18F-FDG, 48.9%, P < 0.01; IC50, 13
μM), 20 (18F-FDG, 61.2%, P < 0.001; IC50, 5.5 μM), and 23
(18F-FDG, 54.8%, P < 0.01; IC50, 14 μM) all showed short-term
dose-dependent 18F-FDG uptake decreases (Table 5), but the
magnitude and ranges of the decrease for compounds 11, 20,
and 23 were smaller compared to compound 10 (Figure 9).
Compound 23 showed much weaker polyp area tumor growth
inhibition (TGI 25.0%, p > 0.05) in a long-term prophylactic
setting (300 mg/kg daily, 12 weeks). Surprisingly, compound 11
was also weaker with respect to polyp area tumor growth
inhibition (TGI 35.1%) despite a significant reduction of 18F-
FDG uptake (48.9%, P < 0.01; IC50, 13 μM). Based on the
results, compound 20 was not considered for long-term studies.
Compounds 21 (TGI 48.7%, Figure 10) and 22 (TGI 39.8%)
showed comparable and statistically significant tumor growth
inhibition in a prophylactic setting (300 mg/kg daily as a
suspension by gavage, 10 weeks). It should be pointed out that
compounds 10, 21, and 22 showed a statistically significant
reduction of the polyp area and polyp number at all doses (30,
100, and 300 mg/kg) driven primarily by reduction in polyps of
size ranging from >2 to <3 mm, as exemplified by compound 21
in Figure 10. Compounds 10 (>82 days, 80% survival, 30 mg/
kg), 21 (74 days, 50% survival, 100 mg/kg), and 22 (62.5 days,
50% survival, 100 mg/kg) all extended survival of Apcmin/+ mice
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a
Scheme 1. Syntheses of Compounds 5, 6, and 7
a
Reagents and Conditions: (a) tert-BuOK, DMF, rt; (b) Pd(dppf)Cl2 (cat) or Pd(dppf)Cl2·DCM (cat), Na2CO3, 1,4-dioxane/H2O, 80 °C; (c)
Red-Al, THF, rt; (d) NBS, DMF, 70 °C; (e) Pd(dppf)Cl2 (cat), Na2CO3, 1,4-dioxane/H2O, 80 °C; and (f) Pd−C (10%), H2, EtOH, rt.
(dosing by gavage P.O., daily starting at age of 4−6 weeks)
compared to positive controls celecoxib (1) (>82 days, 100%
survival, 100 mg/kg) and etoricoxib (3) (>82 days, 80% survival,
100 mg/kg) and negative control (vehicle treatment, 20 days,
50% survival) (Table 5, also Section S8). Nevertheless,
compounds 10, 21, or 22 failed to demonstrate superior in
vivo chemopreventive effects over celecoxib at the highest doses
tested.
To test the hypothesis that GI tissue exposure and COX-2
inhibition drive efficacy rather than systemic exposure/
inhibition, separate IC50s for celecoxib (1) and compounds
10, 21, and 22 were derived using the in vivo tumor (polyp) area
and either systemic or ileum concentrations. These values are
shown for four compounds in Table 6 (plots shown in Section
S9), along with in vitro IC50 values for HWB COX-1 and COX-2
inhibition, adjusted for unbound drug concentrations. Compar-
ison of in vitro with in vivo potencies provides insights into
factors driving efficacy. For example, by looking at the clinical
comparator celecoxib, the systemic and gut in vivo IC50s are
similar to the adjusted in vitro COX-2 IC50, consistent with the
hypothesis that systemic or gut inhibition of COX-2 drives
efficacy. The observation that celecoxib’s in vivo IC50 is 14-fold
lower than its adjusted in vitro COX-1 IC50 suggests a minimal
contribution of COX-1 inhibition driving in vivo efficacy.
In contrast, in vivo gut IC50 values for the gut-restricted
compounds 10, 21, and 22 are 40- to 85-fold higher than the
adjusted in vitro COX-2 potency (IC50). This extreme “right-
shift” of in vivo IC50 and lack of in vitro−in vivo correlation
(IVIVC) suggest that gut COX-2 inhibition is not driving
efficacy. Assuming gut COX-2 inhibition is not driving efficacy,
three other possibilities should be considered: (1) systemic
COX-2 inhibition, (2) systemic COX-1 inhibition, and (3) gut
COX-1 inhibition. Our results suggest systemic COX-2 is the
most likely driver of efficacy both because of good IVIVC (in
vitro and adjusted in vivo potency (IC50s) are within 1.0- to 6.7-
fold of each other) for the gut-restricted compounds and
because celecoxib, with its high systemic exposure, is effective at
reducing the polyp area. Systemic COX-1 inhibition can be ruled
out due to the large disconnect between in vitro and adjusted in
vivo potency (88- to 1,400-fold difference in IC50s for gut-
restricted compounds). Interestingly, gut COX-1 inhibition has
reasonable IVIVC (1.3- to 12.4-fold difference between in vitro
and adjusted in vivo potency IC50s for gut-restricted
compounds), and therefore cannot be ruled out as a driver of
efficacy. However, because celecoxib works systemically with
minimal COX-1 inhibition, systemic COX-2 inhibition is the
more likely driver of tumor growth inhibition in this mouse
model.66,67
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a
Scheme 2. Syntheses of Compounds 8 and 12
a
Reagents and Conditions: (a) SOCl2, DMF, CHCl3, 80 °C; (b) CH3NHOCH3·HCl, TEA, DCM, rt; (c) tert-BuOK, DMF, 60 °C; (d) Br2,
NaOAc, AcOH, 80 °C; (e) NaBH4, MeOH, rt; (f) Pd(dppf)Cl2 (cat), Na2CO3, 1,4-dioxane/H2O, 60 °C; and (g) NaH, DMF, rt.
a
Scheme 3. Synthetic Route for Preparation of Compounds 9, 10, 11, 21, and 22
a
Reagents and Conditions: (a) NaH, DMF or THF; (b) Xphos Pd G3 (cat), Cs2CO3, toluene, 90 °C; (c) NBS, MeCN, rt; (d) Pd(PPh3)4 (cat),
Na2CO3, 1,4-dioxane/H2O, 80 °C; and (e) BH3·THF, THF.
Chemistry. Preparation of compound 4 has been reported.68
The synthesis of compounds 5−7 is outlined in Scheme 1 and
described in detail in Section S1. Commercially available 24a−c
were reacted with 2-methylpropane-1,2-diol in the presence of
potassium tert-butoxide in DMF to afford the corresponding
ethers 25a−c, which were coupled with commercially available
4-(methanesulfonyl)phenylboronic acid under palladium-medi-
ated Suzuki reaction conditions to give compound 5 and
intermediates 26 (R2 = COOEt) and 27 (R2 = H). Intermediate
26 was reduced with sodium bis(2-methoxyethoxy)aluminum
hydride (Red-Al) in THF to provide compound 7. Selective
bromination of 27 with N-bromosuccinimide (NBS) in DMF
led to bromide 28. Subsequent Suzuki−Miyaura coupling of
potassium trifluoro(vinyl)borate with 28 installed a vinyl group
in 29, which was reduced by palladium catalyst-mediated
hydrogenation to generate compound 6.
Several flexible synthetic routes were used to access
compounds 8−23. The initial route outlined in Scheme 2 was
developed for preparation of compounds 8 and 12. Commer-
cially available 2,6-dichloronicotinic acid 30 was converted into
Weinreb amide 31 by reacting with thionyl chloride followed by
coupling with dimethylhydroxylamine hydrochloride under
basic conditions. Treatment of 31 with 2-methylpropane-1,2-
diol in the presence of potassium tert-butoxide generated 32 as
the major product. Selective bromination of 32 with bromine in
acetic acid provided bromide 33. The reduction of Weinreb
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a
Scheme 4. Syntheses of Compounds 13, 14, and 15
a
Reagents and Conditions: (a) BH3·THF, THF; (b) Dess−Martin periodinane, DCM, rt; (c) MeMgCl, THF, rt; (d) TMSCHN2, MeOH, toluene,
rt; (e) MeMgCl, THF, rt; (f) NaH, THF, rt; and (g) LiAlH4, THF, 0 °C.
a
Scheme 5. Synthesis of Compound 16
a
Reagents and Conditions: (a) Pd(PPh3)4 (cat), K2CO3, 1,4-dioxane/H2O, 80 °C; (b) NBS, TFA, MeCN, rt; (c) Pd(PPh3)4 (cat), K2CO3, 1,4-
dioxane/H2O, 80 °C; and (d) BH3·THF, THF.
amide 33 was achieved by using sodium borohydride to provide
34. The coupling reaction of 34 with 4-(methanesulfonyl)-
phenylboronic acid under palladium-mediated Suzuki reaction
conditions gave compound 35. Finally, treatment of 35 with
methanol or 2-methoxyethanol in the presence of sodium
hydride-furnished compounds 8 or 12, respectively.
Analogues 9, 10, 11, 21, and 22 were synthesized by the route
outlined in Scheme 3. In this route, ortho-regioselective
alkoxylation of 2,6-dichloronicotinic acid 30 was achieved
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a
Scheme 6. Synthetic Route for Preparation of Compounds 17, 18, 19, and 20
a
Reagents and Conditions: (a) Xphos Pd G3 (cat), Cs2CO3, toluene, 90 °C; (b) NBS, AcOH, MeCN, rt; (c) Pd(PPh3)4 (cat), K2CO3, 1,4-
dioxane/H2O, 80 °C; and (d) (i) TEA, ClC(O)O−iBu, THF; then NaBH4; or (ii) BH3·THF, THF.
a
Scheme 7. Synthesis of Compound 23
a
Reagents and conditions: (a) BH3·THF, THF; (b) Pd(dppf)Cl2 (cat), KOAc, 1,4-dioxane, 100 °C; and (c) Pd(dppf)Cl2 (cat), Na2CO3, 1,4-
dioxane/H2O, 65 °C.
using various alcohols (R1-OH) in the presence of sodium
hydride under controlled conditions to afford intermediates
36a−e.69 Treatment of 36a−e with 2-methylpropane-1,2-diol
under palladium catalyzed reaction conditions to provide
corresponding 2,6-di-alkoxylated acids 37a−e. Our efforts to
produce 37a−e directly from 30 by combining steps a and b
without purification of 36a−e were unsuccessful due to difficult
separation at the end. Selective bromination of 37a−e with NBS
in acetonitrile led to bromides 38a−e in good yields, followed by
coupling with 4-(methanesulfonyl)phenylboronic acid under
Suzuki reaction conditions to give 39a−e as the major products.
The carboxylic acids 39a−e were reduced by the borane−
tetrahydrofuran complex to afford corresponding analogues 9,
10, 11, 21, and 22.
periodinane gave aldehyde 40 in >90% yield. Subsequent
nucleophilic addition of methylmagnesium chloride led to the
formation of analogue 13. Similarly, gem-dimethyl analogue 14
was obtained through nucleophilic addition of methylmagne-
sium chloride to methyl ester 41, which was prepared by
esterification of acid 39c with TMSCHN2, described previously
in Scheme 2. Selective alkylation of compound 11 was
accomplished using ethyl bromoacetate and sodium hydride
to afford ester 42, which was readily reduced to alcohol 15 using
lithium aluminum hydride under controlled anhydrous
conditions.
Scheme 5 shows the preparation of compound 16.
Intermediate carboxylic acid 36c, described previously in
Scheme 2, was coupled with 4-fluorophenylboronic acid under
palladium-mediated Suzuki conditions to generate intermediate
43 in modest yield (40%). Subsequent transformations
Compounds 13, 14, and 15 were prepared according to
Scheme 4. Oxidation of compound 11 using Dess−Martin
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multiplicity are as follows: s = singlet, d = doublet, t = triplet, q =
quartet, dd = doublet of doublets, ddd = doublet of doublet of doublets,
td = triplet of doublets, dq = doublet of quartet, dt = doublet of triplets,
spt = septet, quin = quintet, m = multiplet, and br = broad. 1H NMR
chemical shifts are expressed in parts per million (δ) downfield from
tetramethylsilane as a standard. Normal-phase silica gel chromatog-
raphy (FCC) was performed on silica gel (SiO2) using prepacked
cartridges. All compounds sent for biological tests were confirmed with
purity >95% in quantitative HPLC analysis [method: Gilson GX-281-
RP-HPLC with Phenomenex Gemini C18 (10 μm, 150 × 25 mm), or
Waters XBridge C18 column (5 μm, 150 × 30 mm), the mobile phase of
5−99% MeCN in water (10 mM NH4HCO3) over 10 min, and then
held at 100% MeCN for 2 min, at a flow rate of 25 mL/min] or
elemental analysis in addition to LCMS and 1H NMR. Celecoxib (1),
etoricoxib (3), and compound 4 were purchased from Combi-Blocks,
Inc (San Diego), Carbosynth Ltd (UK), and Aurora Fine Chemicals
LLC (San Diego), respectively.
Preparation of 2,6-Dichloro-N-methoxy-N-methylnicotina-
mide (31). To a mixture of 2,6-dichloronicotinic acid (30 g, 156
mmol) and DMF (1 mL) in CHCl3 (300 mL) under nitrogen at 0 °C
was added SOCl2 (55.8 g, 469 mmol). The reaction mixture was then
heated and stirred at 60 °C for 1 h. The mixture was cooled to room
temperature and concentrated to give crude 2,6-dichloronicotinoyl
chloride as a gum (35 g), which was used directly in the next step.
To the solution of crude 2,6-dichloronicotinoyl chloride (35 g) in
DCM (300 mL) at 0 °C were added triethylamine (50.9 g, 499 mmol)
and N,O-dimethylhydroxylamine hydrochloride (19.5 g, 200 mmol).
The reaction mixture was stirred at room temperature for 2 h. The
reaction was quenched by the addition of water (300 mL). The mixture
was extracted with DCM (2 × 100 mL). The combined organic extract
was washed with brine (300 mL), dried over anhydrous Na2SO4,
filtered, and concentrated. The crude product (44 g) was recrystallized
in ethyl acetate and petroleum ether (1:5) to give the title compound as
a white solid (35 g, 95% yield). LCMS (ESI): mass calcd for
C8H8Cl2N2O2, 234.0; m/z found, 235.0 [M + H]+.
Preparation of 2-Chloro-6-(2-hydroxy-2-methylpropoxy)-N-
methoxy-N-methylnicotinamide (32). To a solution of 2-
methylpropane-1,2-diol (12.7 g, 140 mmol) in anhydrous DMF (200
mL) at 0 °C was added potassium tert-butoxide (17.2 g, 153 mmol)
under nitrogen. The mixture was stirred for 0.5 h, and then 2,6-
dichloro-N-methoxy-N-methylnicotinamide (30 g, 128 mmol) was
added. The reaction mixture was heated and stirred at 60 °C overnight.
The mixture was cooled to room temperature, quenched with water
(200 mL), and then extracted with ethyl acetate (2 × 200 mL). The
combined organic extract was washed with brine (200 mL), dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was purified
by flash chromatography (50% ethyl acetate in petroleum ether as the
eluent) to give the title compound as a white solid (24.5 g, 67% yield).
LCMS (ESI): mass calcd for C12H16BrClN2O4, 288.1; m/z found,
289.1 [M + H]+.
Preparation of 5-Bromo-2-chloro-6-(2-hydroxy-2-methyl-
propoxy)-N-methoxy-N-methylnicotinamide (33). To a solution
of 2-chloro-6-(2-hydroxy-2-methylpropoxy)-N-methoxy-N-methylni-
cotinamide (12 g, 41.6 mmol) in glacial acetic acid (120 mL) was
added sodium acetate (6.98 g, 83.1 mmol), followed by the dropwise
addition of bromine (6.66 mL, 129 mmol). The reaction mixture was
heated and stirred at 80 °C overnight. The mixture was cooled to room
temperature, quenched with water (400 mL), and then extracted with
ethyl acetate (2 × 400 mL). The combined organic extract was washed
with brine (2 × 400 mL), dried over anhydrous Na2SO4, and
concentrated. The residue was purified by flash chromatography (50%
ethyl acetate in petroleum ether as the eluent) to give the title
compound as a white solid (7.5 g, 49% yield). LCMS (ESI): mass calcd
for C12H17ClN2O4, 366.0; m/z found, 367.0 [M + H]+.
(selective bromination, palladium-mediated Suzuki coupling,
and reduction of the acid moiety) led to the formation of
analogue 16.
In a similar manner to the synthetic route as described in
Scheme 3, compounds 17 and 18 were prepared as illustrated in
Scheme 6. The previously described ortho-alkylated intermedi-
ate 36c was coupled with commercially available oxetanyl or
cyclobutyl reagents 46a−d (X = O or CH2; Y = H, Me, or OH)
to provide corresponding dialkoxylated 47a−d under palla-
dium-mediated reaction conditions. Treatment of 47a−d with
NBS generated bromides 48a−d. Subsequent Suzuki coupling
of 4-(methanesulfonyl)phenylboronic acid with 48a−d installed
a 4-(methanesulfonyl)phenyl group in 49a−d. The carboxylic
acids 49a−c were reduced by first reacting with isobutyl
chloroformate in the presence of triethylamine to generate
mixed anhydrides in situ, followed by treatment with sodium
borohydride, to give analogues 17, 18, and 19. Alternatively, a
reduction of acid 49d with borane tetrahydrofuran complex
directly afforded analogue 20.
Scheme 7 was used to prepare compound 23. The previously
described acid 38c was first reduced to alcohol 50, and then
converted into boronic pinacol ester 51 by reacting with
bis(pinacolato)diboron (Pin2B2)-mediated by Pd(dppf)2Cl2
and potassium acetate under anhydrous and heated conditions.
Suzuki coupling of 51 with commercially available 2-chloro-5-
(methylsulfonyl)pyridine under palladium-mediated reaction
conditions furnished analogue 23.
CONCLUSIONS
■
In summary, we described a series of gut-restricted, selective
COX-2 inhibitors with a central pyridine core as a common
structural feature. These compounds were characterized by high
colonic exposure and minimized systemic exposure mediated by
attenuated permeability and clearance through a glucuronida-
tion pathway via UGT1A9. By establishing short-term ex vivo
18F-FDG uptake attenuation in an acute setting as an indirect,
surrogate readout to predict efficacy, we evaluated a subset of
analogues in long-term in vivo chemopreventive studies in
APCmin/+ mouse models. Compounds 10, 21, and 22 showed
dose-dependent suppression of adenoma formation and growth
as well as extension of survival. Unexpectedly, in vitro potent
analogues such as compounds 11 and 23 showed much weaker
in vivo efficacy in terms of the polyp area and multiplicity
inhibition. An IVIVC demonstrated that in vivo efficacy was not
driven by local gut COX-2 inhibition. Rather, efficacy was
consistent with residual systemic COX-2-driven inhibition,
which may explain why none of these locally targeted inhibitors
demonstrated superior in vivo effects for inhibiting adenoma
progression relative to celecoxib in APCmin/+ mice. Our results
could not rule out that gut COX-1- or COX-independent
mechanisms alternatively play a significant role in driving
adenoma growth inhibition, which warrants further inves-
tigation.
EXPERIMENTAL SECTION
■
Chemistry. All commercial reagents and anhydrous solvents were
purchased and used without further purification, unless otherwise
specified. Mass spectra (MS) were obtained on a SHIMADZU LCMS-
2020 MSD or an Agilent 1200\G6110A MSD instrument using
electrospray ionization (ESI) in positive mode unless otherwise
indicated. Calculated (calcd) mass corresponds to the exact mass.
Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker
model AVIII 300 MHz or 400 MHz spectrometer. Definitions for
Preparation of 1-((3-Bromo-6-chloro-5-(hydroxymethyl)-
pyridin-2-yl)oxy)-2-methylpropan-2-ol (34). To a solution of 5-
bromo-2-chloro-6-(2-hydroxy-2-methylpropoxy)-N-methoxy-N-meth-
ylnicotinamide (27 g, 70.7 mmol) in MeOH (200 mL) at 0 °C was
added NaBH4 (16.1 g, 424 mmol). The resulting reaction mixture was
stirred at room temperature for 2 h. The mixture was concentrated
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under reduced pressure, and water was added. The resulting mixture
was extracted with ethyl acetate (2 × 200 mL). The combined organic
extract was washed with brine (100 mL), dried over anhydrous Na2SO4,
and concentrated to give the crude product as a gum (22 g, 100% yield),
which was used directly into the next step. LCMS (ESI): mass calcd for
C10H13BrClNO3, 309.0; m/z found, 310.0 [M + H]+.
mixture was acidified to “pH” 1 using aqueous HCl (1 M) and extracted
with ethyl acetate (3 × 100 mL). The organic layers were combined,
dried over anhydrous Na2SO4, and concentrated to give the title
compound as a yellow solid (5 g, 89% yield). LCMS (ESI): mass calcd
for C9H10ClNO3, 215.0; m/z found, 216.1 [M + H]+.
Preparation of 6-chloro-2-propoxynicotinic acid (36b). To a
solution of 1-propanol (46.4 g, 772 mmol) in anhydrous DMF (2.6 L)
at 0 °C was added NaH (60%, 56.6 g, 1415 mmol). The mixture was
stirred at 0 °C for 1 h, and then 2,6-dichloropyridine-3-carboxylic acid
(130 g, 643 mmol) was added in several portions. The reaction mixture
was stirred at room temperature for 30 h. The mixture was quenched
with iced water (7.8 L). The mixture was acidified to “pH” 1 using
aqueous HCl (1 M). The organic layers were combined, dried over
anhydrous Na2SO4, The precipitate was collected and dried to give the
title compound as a white solid (91 g, 66% yield). LCMS (ESI): mass
calcd. for C9H10ClNO3, 215.0; m/z found, 216.1 [M+H]+.
Preparation of 2-Butoxy-6-chloronicotinic Acid (36c). To a
solution of 1-butanol (63.7 g, 859 mmol) in anhydrous DMF (200 mL)
at 0 °C was added NaH (60%, 65.6 g, 1.64 mol). The mixture was
stirred at 0 °C for 1 h, and then 2,6-dichloropyridine-3-carboxylic acid
(10 g, 52 mmol) was added in several batches. The reaction mixture was
stirred at room temperature overnight. The mixture was quenched with
ice water (2 L). The mixture was acidified to “pH” 5 using aqueous HCl
(1 M) and extracted with ethyl acetate (3 × 2 L). The organic layers
were combined, dried over anhydrous Na2SO4, and concentrated to
give the title compound as a white solid (165 g, 89% yield). LCMS
(ESI): mass calcd for C10H12ClNO3, 229.1; m/z found, 228.0 [M-H]+.
Preparation of 6-Chloro-2-(3,3,3-trifluoropropoxy)nicotinic
Acid (36d). To a solution of 3,3,3-trifluoropropan-1-ol (267 g, 2.34
mol) in anhydrous THF (9 L) was added NaH (60%, 137.5 g, 3.44
mol). The mixture was stirred at room temperature for 1 h, and then
2,6-dichloropyridine-3-carboxylic acid (300 g, 1.56 mmol) was added.
The reaction mixture was stirred at room temperature for 48 h. The
mixture was quenched with water (5 L), acidified to “pH” 1 using
aqueous HCl (1 M), and then extracted with ethyl acetate (3 × 3.5 L).
The organic layers were combined, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by Flash-Prep-HPLC (Column,
C-18, mobile phase, water (a)/MeCN (b), b %: 30%−55%, 30 min;
detector, UV 210 nm) to give the title compound as a white solid (170
g, 40% yield). LCMS (ESI): mass calcd for C9H7ClF3NO3, 269.0; m/z
found, 270.0 [M + H]+.
Preparation of 6-Chloro-2-(4,4,4-trifluorobutoxy)nicotinic
Acid (36e). To a solution of 4,4,4-trifluorobutan-1-ol (150 g, 1.17
mol) in anhydrous THF (4.5 L) was added NaH (60%, 68.7 g, 1.72
mol). The mixture was stirred at 50 °C for 0.5 h, and then 2,6-
dichloropyridine-3-carboxylic acid (150 g, 0.781 mol) was added. The
reaction mixture was heated and stirred at 70 °C for 3 h. The mixture
was cooled to room temperature, quenched with water (2 L), acidified
to “pH” 1 with aqueous HCl (1 M), and then extracted with ethyl
acetate (3 × 2 L). The organic layers were combined, dried over
anhydrous Na2SO4, and concentrated to give the title compound as a
white solid (156 g, 70% yield). LCMS (ESI): mass calcd for
C10H9ClF3NO3, 283.0; m/z found, 284.1 [M + H]+.
Preparation of 1-((6-Chloro-5-(hydroxymethyl)-3-(4-
(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-methylpropan-2-
ol (35). To a mixture of crude 1-(3-bromo-6-chloro-5-
(hydroxymethyl)pyridin-2-yloxy)-2-methylpropan-2-ol (34, 5 g, 16.1
mmol), 4-(methylsulfonyl)phenylboronic acid (3.86 g, 19.3 mmol),
and aqueous Na2CO3 (2.0 M, 16.1 mL, 32 mmol) in 1,4-dioxane (40
mL) was added Pd(dppf)Cl2 (1.18 g, 1.61 mmol) under nitrogen. The
reaction mixture was heated with stirring at 60 °C for 3 h. The mixture
was cooled to room temperature, quenched with water (40 mL), and
extracted with ethyl acetate (2 × 80 mL). The combined organic extract
was washed with brine (40 mL), dried over anhydrous Na2SO4, and
concentrated. The residue was purified by flash chromatography (50%
ethyl acetate in petroleum ether as the eluent) to give the title
compound as a yellow solid (5 g, 80% yield). LCMS (ESI): mass calcd
for C17H20ClNO5S, 385.1; m/z found, 386.2 [M + H]+; 1H NMR (300
MHz, DMSO-d6): δ 8.01 (d, J = 8.7 Hz, 2H), 7.98 (s, 1H), 7.92 (d, J =
8.7 Hz, 2H), 4.54 (s, 2H), 4.10 (s, 2H), 3.84 (br s, 2H), 3.25 (s, 3H),
1.15 (s, 6H) ppm.
Preparation of 1-((5-(Hydroxymethyl)-6-methoxy-3-(4-
(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-methylpropan-2-
ol (8). To a solution of anhydrous methanol (1.25 g, 38.9 mmol) in
DMF (30 mL) at 0 °C was added NaH (60%, 1.56 g, 38.9 mmol). The
mixture was stirred for 0.5 h, and then 1-((6-chloro-5-(hydroxymeth-
yl)-3-(4-(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-methylpropan-2-
ol (1.5 g, 3.89 mmol) was added. The reaction mixture was stirred at
room temperature overnight. The mixture was quenched with water
(40 mL), and then extracted with ethyl acetate (2 × 50 mL). The
combined organic extract was washed with brine (2 × 60 mL), dried
over anhydrous Na2SO4, filtered, and concentrated. The residue was
purified by flash chromatography (60% ethyl acetate in petroleum ether
as the eluent), and the crude product was further purified by prep-
HPLC (10−90% MeCN in water) to give the title compound as a white
solid (280 mg, 19% yield). LCMS (ESI): mass calcd. for C18H23NO6S,
381.1; m/z found, 382.2 [M + H]+; 1H NMR (300 MHz, DMSO-d6): δ
7.95 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 8.7 Hz, 2H), 7.83 (s, 1H), 5.09 (t, J
= 5.7 Hz, 1H), 4.63 (s, 1H), 4.45 (d, J = 5.7 Hz, 2H), 4.16 (s, 2H), 3.94
(s, 3H), 3.25 (s, 3H), 1.17 (s, 6H) ppm.
Preparation of 1-((5-(Hydroxymethyl)-6-(2-methoxye-
thoxy)-3-(4-(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-meth-
ylpropan-2-ol (12). To a solution of anhydrous 2-methoxyethanol
(2.96 g, 38.9 mmol) in DMF (30 mL) at 0 °C was added NaH (60%,
1.56 g, 38.9 mmol). The mixture was stirred for 0.5 h, and then 1-((6-
chloro-5-(hydroxymethyl)-3-(4-(methylsulfonyl)phenyl)pyridin-2-
yl)oxy)-2-methylpropan-2-ol (35, 1.5 g, 3.89 mmol) was added. The
reaction mixture was stirred at room temperature overnight. The
mixture was quenched with water (40 mL), and then extracted with
ethyl acetate (2 × 50 mL). The combined organic extract was washed
with brine (2 × 60 mL), dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was purified by flash chromatography (60%
ethyl acetate in petroleum ether as the eluent), and the crude product
was further purified by prep-HPLC (10−90% MeCN in water) to give
the title compound as a white solid (100 mg, 19% yield). LCMS (ESI):
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-2-isopro-
poxynicotinic Acid (37a). To a mixture of 6-chloro-2-(propan-2-
yloxy)pyridine-3-carboxylic acid (36a, 5 g, 23.2 mmol), Cs2CO3 (18.9
g, 57.8 mmol), and 2-methylpropane-1,2-diol (4.18 g, 46.4 mmol) in
toluene (150 mL) under nitrogen was added XPhos Pd G3 (196 mg,
0.23 mmol). The reaction mixture was heated and stirred at 90 °C
overnight. The mixture was cooled to room temperature and then
quenched with water (200 mL). The organic layer was separated, and
the “pH” of the aqueous layer was adjusted to 1 with aqueous HCl (1
M). The aqueous solution was extracted with ethyl acetate (3 × 150
mL). The organic layers were combined, dried over anhydrous Na2SO4,
and concentrated to give the title compound as a white solid (5.5 g, 88%
yield). LCMS (ESI): mass calcd for C13H19NO5, 269.1; m/z found,
270.1 [M + H]+.
1
mass calcd for C20H27NO7S, 425.2; m/z found, 426.1 [M + H]+; H
NMR (300 MHz, DMSO-d6): δ 7.95 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4
Hz, 2H), 7.84 (s, 1H), 5.11 (t, J = 5.7 Hz, 1H), 4.64 (s, 1H), 4.44−4.49
(m, 4H), 4.13 (s, 2H), 3.69−3.72 (m, 2H), 3.33 (s, 3H), 3.26 (s, 3H, s),
1.16 (s, 6H) ppm.
Preparation of 6-Chloro-2-isopropoxynicotinic Acid (36a).
To a solution of 2-propanol (2.35 g, 39.1 mmol) in anhydrous THF
(150 mL) was added NaH (60%, 2.29 g, 57.3 mmol). The mixture was
stirred at 50 °C for 0.5 h, and then 2,6-dichloropyridine-3-carboxylic
acid (5 g, 26.0 mmol) was added. The reaction mixture was heated and
stirred at 70 °C for 3 h. The mixture was cooled to room temperature,
quenched with water (2 L), and concentrated to a small volume. The
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-2-propoxy-
nicotinic Acid (37b). To a mixture of 6-chloro-2-propoxynicotinic
acid (36b, 91 g, 422 mmol), Cs2CO3 (344 g, 1055 mmol), and 2-
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methylpropane-1,2-diol (57.0 g, 633 mmol) in toluene (270 mL) under
nitrogen was added XPhos Pd G3 (7.1 g, 8.74 mmol). The reaction
mixture was heated and stirred at 90 °C overnight. The mixture was
cooled to room temperature and then quenched with water. The
organic layer was separated, and the “pH” of the aqueous layer was
adjusted to 1 with aqueous HCl (1 M). The aqueous solution was
extracted with ethyl acetate. The organic layers were combined, dried
over anhydrous Na2SO4, and concentrated to give the title compound
as a brown oil (100 g, 88% yield). LCMS (ESI): mass calcd for
C13H19NO5, 269.1; m/z found, 270.2 [M + H]+.
Preparation of 2-Butoxy-6-(2-hydroxy-2-methylpropoxy)-
nicotinic Acid (37c). To a mixture of 2-butoxy-6-chloronicotinic
acid (36c, 5 g, 21.4 mmol), Cs2CO3 (27.9 g, 85.4 mmol), and 2-
methylpropane-1,2-diol (4.8 g, 53.4 mmol) in toluene (150 mL) under
nitrogen were added dicyclohexyl(2′,6′-dimethoxybiphenyl-2-yl)-
phosphine (876 mg, 2.1 mmol) and Pd(OAc)2 (1.03 g, 4.3 mmol).
The reaction mixture was heated and stirred at 90 °C overnight. The
mixture was cooled to room temperature and then quenched with water
(500 mL). The mixture was filtered through a short celite pad, and the
filtrate was extracted with ethyl acetate (200 mL). The “pH” of the
aqueous layer was adjusted to 1 using aqueous HCl (1 M), and then
extracted with ethyl acetate (2 × 200 mL). The organic layers were
combined, dried over anhydrous Na2SO4, and concentrated to give the
title compound as a light yellow oil (5 g, 60% yield). LCMS (ESI): mass
calcd for C14H21NO5, 283.1; m/z found, 282.2 [M − H]+.
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-2-(3,3,3-
trifluoropropoxy)nicotinic Acid (37d). To a mixture of 6-chloro-
2-(3,3,3-trifluoropropoxy)nicotinic acid (36d, 130 g, 482 mmol),
Cs2CO3 (393 g, 1210 mmol), and 2-methylpropane-1,2-diol (86.9 g,
964 mmol) in toluene (3.9 L) under nitrogen was added XPhos Pd G3
(4.08 g, 4.82 mmol). The reaction mixture was heated and stirred at 90
°C overnight. The mixture was cooled to room temperature and then
quenched with water (3 L). The mixture was extracted with ethyl ether
(1 L). The organic layer was separated, and the “pH” of the aqueous
layer was adjusted to 1 with aqueous HCl (1 M). The aqueous solution
was extracted with ethyl acetate (2 × 2 L). The organic layers were
combined, washed with brine (2 L), dried over anhydrous Na2SO4, and
concentrated to give the title compound as a brown oil (130 g, 83%
yield). LCMS (ESI): mass calcd for C13H16F3NO5, 323.1; m/z found,
324.1 [M + H]+.
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-2-(4,4,4-
trifluorobutoxy)nicotinic Acid (37e). To a mixture of 6-chloro-2-
(4,4,4-trifluorobutoxy)nicotinic acid (36e, 156 g, 550 mmol), Cs2CO3
(448 g, 1375 mmol), and 2-methylpropane-1,2-diol (99 g, 1100 mmol)
in toluene (4.68 L) under nitrogen was added XPhos Pd G3 (4.6 g, 5.5
mmol). The reaction mixture was heated and stirred at 90 °C overnight.
The mixture was cooled to room temperature and then quenched with
water (3 L). The organic layer was separated, and the “pH” of the
aqueous layer was adjusted to 1 with aqueous HCl (1 M). The aqueous
solution was extracted with ethyl acetate (3 × 1 L). The organic layers
were combined, dried over anhydrous Na2SO4, and concentrated to
give the title compound as a yellow solid (130 g, 83% yield). LCMS
(ESI): mass calcd for C14H18F3NO5, 337.1; m/z found, 338.2 [M +
H]+.
was concentrated and the residue was purified by flash chromatography
(5% MeOH in DCM) to give the title compound as a white solid (119
g, 92% yield). LCMS (ESI): mass calcd for C13H18BrNO5, 347.0; m/z
found, 348.1 [M + H]+.
Preparation of 5-Bromo-2-butoxy-6-(2-hydroxy-2-
methylpropoxy)nicotinic Acid (38c). To a solution of 2-butoxy-6-
(2-hydroxy-2-methylpropoxy)nicotinic acid (37c, 5.1 g, 14.1 mmol) in
acetonitrile (130 mL) was added NBS (2.8 g, 15.5 mmol). The reaction
mixture was stirred at room temperature for 2 h. Water was added (500
mL). The precipitate was collected by filtration and dried in vacuo to
give the title compound as a white solid (4.5 g, 72% yield). LCMS
(ESI): mass calcd for C14H20BrNO5, 361.1; m/z found, 362.2 [M +
H]+.
Preparation of 5-Bromo-6-(2-hydroxy-2-methylpropoxy)-2-
(3,3,3-trifluoropropoxy)nicotinic Acid (38d). To a solution of 6-
(2-hydroxy-2-methylpropoxy)-2-(3,3,3-trifluoropropoxy)nicotinic
acid (37d, 130 g, 402 mmol) and acetic acid (120.7 g, 2.01 mol) in
acetonitrile (1.3 L) was added NBS (78.7 g, 442 mmol). The reaction
mixture was stirred at room temperature for 2 h. The precipitate was
collected by filtration, washed with cold acetonitrile (200 mL) and
water (1 L), and then dried in vacuo to give the title compound as a
white solid (116 g, 72% yield). LCMS (ESI): mass calcd for
C13H15BrF3NO5, 401.0; m/z found, 402.0 [M + H]+.
Preparation of 5-Bromo-6-(2-hydroxy-2-methylpropoxy)-2-
(4,4,4-trifluorobutoxy)nicotinic Acid (38e). To a solution of 6-(2-
hydroxy-2-methylpropoxy)-2-(4,4,4-trifluoro-butoxy)nicotinic acid
(37e, 180 g, 533 mmol) and acetic acid (160 g, 2.67 mol) in
acetonitrile (1.8 L) was added NBS (114 g, 640 mmol). The reaction
mixture was stirred at room temperature for 2 h, and then quenched
with water (1.5 L). The mixture was concentrated to half of its volume
under reduced pressure. The precipitate was collected by filtration and
recrystallized in acetonitrile to give the title compound as a yellow solid
(140 g, 63% yield). LCMS (ESI): mass calcd for C14H17BrF3NO5,
415.0; m/z found, 416.1 [M + H]+.
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-2-isopro-
poxy-5-(4-(methylsulfonyl)phenyl)nicotinic Acid (39a). To a
mixture of 5-bromo-6-(2-hydroxy-2-methylpropoxy)-2-isopropoxyni-
cotinic acid (38a, 1 g, 2.87 mmol), 4-(methylsulfonyl)phenylboronic
acid (689 mg, 3.44 mmol), and aqueous Na2CO3 (2.0 M, 5.7 mL, 11.4
mmol) in 1,4-dioxane (8 mL) was added Pd(dppf)Cl2 (210 mg, 0.29
mmol) under nitrogen. The reaction mixture was heated with stirring at
80 °C for 3 h. The mixture was cooled to room temperature and
quenched with water (20 mL). The mixture was acidified to “pH” 1 with
aqueous HCl solution (1 M) and then extracted with ethyl acetate (2 ×
50 mL). The combined organic extract was washed with brine (40 mL),
dried over anhydrous Na2SO4, and concentrated. The residue was
purified by reverse-phase Combi-Flash chromatography (column, C18;
eluent, 25−55% MeCN in water; time, 30 min) to give the title
compound as a yellow solid (0.8 g, 66% yield). LCMS (ESI): mass calcd
for C20H25NO7S, 423.1; m/z found, 424.2 [M + H]+.
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-5-(4-
(methylsulfonyl)phenyl)-2-propoxynicotinic Acid (39b). To a
mixture of 5-bromo-2-butoxy-6-((1-hydroxycyclobutyl)methoxy)-
nicotinic acid (38b, 115 g, 342 mmol), 4-(methylsulfonyl)-
phenylboronic acid (82 g, 410 mmol), and aqueous Na2CO3 (2.0 M,
513 mL, 1.03 mol) in 1,4-dioxane (1.2 L) was added Pd(dppf)Cl2 (25 g,
34.2 mmol) under nitrogen. The reaction mixture was heated with
stirring at 100 °C for 2 h. The mixture was cooled to room temperature
and quenched with water. The mixture was acidified to “pH” 1 with
aqueous HCl solution (1 M) and then extracted with a mixture of ethyl
acetate and THF (1:1). The combined organic extract was washed with
brine, dried over anhydrous Na2SO4, and concentrated. The residue
was purified by flash chromatography (4% MeOH in DCM as the
eluent) to give the title compound as a light yellow solid (70 g, 48%
yield). LCMS (ESI): mass calcd for C20H25NO7S, 423.1; m/z found,
424.2 [M + H]+; 1H NMR (300 MHz, CDCl3): δ 8.48 (s, 1H), 8.02 (d,
J = 8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 4.61 (d, J = 6.6 Hz, 2H), 4.30
(s, 2H), 3.13 (s, 3H), 1.91−2.01 (m, 2H), 1.33 (s, 6H), 1.13 (t, J = 7.5
Hz, 6H) ppm.
Preparation of 5-Bromo-6-(2-hydroxy-2-methylpropoxy)-2-
isopropoxynicotinic Acid (38a). To a solution of 6-(2-hydroxy-2-
methylpropoxy)-2-isopropoxynicotinic acid (37a, 5.5 g, 20.4 mmol)
and acetic acid (6.13 g, 102 mmol) in acetonitrile (55 mL) was added
NBS (4.36 g, 74.1 mmol). The reaction mixture was stirred at room
temperature for 2 h, quenched by the addition of water (50 mL), and
then extracted with ethyl acetate (3 × 100 mL). The organic layers were
combined, dried over Na2SO4, and concentrated. The residue was
recrystallized in acetonitrile to give the title compound as a white solid
(3.3 g, 46% yield). LCMS (ESI): mass calcd for C13H18BrNO5, 347.0;
m/z found, 348.1 [M + H]+.
Preparation of 5-Bromo-6-(2-hydroxy-2-methylpropoxy)-2-
propoxynicotinic Acid (38b). To a solution of 5-bromo-6-(2-
hydroxy-2-methylpropoxy)-2-propoxynicotinic acid (37b, 109 g, 340
mmol) in acetonitrile (2.7 L) was added NBS (72.9 g, 410 mmol). The
reaction mixture was stirred at room temperature for 3 h. The mixture
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Preparation of 2-Butoxy-6-(2-hydroxy-2-methylpropoxy)-5-
(4-(methylsulfonyl)phenyl)nicotinic Acid (39c). To a mixture of
5-bromo-2-butoxy-6-(2-hydroxy-2-methylpropoxy)nicotinic acid (38c,
2 g, 4.5 mmol), 4-(methylsulfonyl)phenylboronic acid (1.2 g, 5.9
mmol), and K2CO3 (2.8 g, 20.4 mmol) in 1,4-dioxane (40 mL) and
water (4 mL) was added Pd(Ph3P)4 (524 mg, 0.45 mmol) under
nitrogen. The reaction mixture was heated with stirring at 80 °C
overnight. The mixture was cooled to room temperature and quenched
with water. The mixture was acidified to “pH” 1 using aqueous HCl
solution (1 M) and then extracted with ethyl acetate (3 × 90 mL). The
combined organic extract was washed with brine, dried over anhydrous
Na2SO4, and concentrated. The residue was purified by flash
chromatography (3−10% MeOH in DCM as the eluent) to give the
title compound as a white solid (1.2 g, 52% yield). LCMS (ESI): mass
calcd for C21H27NO7S, 437.2; m/z found, 438.1 [M + H]+; 1H NMR
(300 MHz, CDCl3): δ 8.48 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.78 (d, J =
8.4 Hz, 2H), 4.65 (d, J = 6.6 Hz, 2H), 4.30 (s, 2H), 3.13 (s, 3H), 1.88−
2.02 (m, 2H), 1.49−1.60 (m, 2H), 1.33 (s, 6H), 1.05 (t, J = 7.5 Hz, 6H)
ppm.
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-5-(4-
(methylsulfonyl)phenyl)-2-(3,3,3-trifluoropropoxy)nicotinic
Acid (39d). To a mixture of 5-bromo-6-(2-hydroxy-2-methylpro-
poxy)-2-(3,3,3-trifluoropropoxy) nicotinic acid (38d, 116 g, 288
mmol), 4-(methylsulfonyl)phenylboronic acid (86.5 g, 433 mmol),
and Na2CO3 (91.7 g, 865 mmol) in 1,4-dioxane (928 mL) and water
(232 mL) was added Pd(dppf)Cl2 (21.1 g, 28.8 mmol) under nitrogen.
The reaction mixture was heated with stirring at 100 °C for 5 h. The
mixture was cooled to room temperature and quenched with water. The
mixture was acidified to “pH” 1 using aqueous HCl solution (1 M) and
then extracted with ethyl acetate (2 × 1 L). The combined organic
extract was washed with brine, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by reverse-phase prep-HPLC
(column, C18; eluent, 25−55% MeCN in water; time, 30 min; detector,
UV 210 nm) to give the title compound as a white solid (100 g, 73%
yield). LCMS (ESI): mass calcd for C20H22F3NO7S, 477.1; m/z found,
478.2 [M + H]+.
Preparation of 6-(2-Hydroxy-2-methylpropoxy)-5-(4-
(methylsulfonyl)phenyl)-2-(4,4,4-trifluorobutoxy)nicotinic
Acid (39e). To a mixture of 5-bromo-6-(2-hydroxy-2-methylpropoxy)-
2-(4,4,4-trifluorobutoxy)nicotinic acid (38e, 140 g, 336 mmol), 4-
(methylsulfonyl)phenylboronic acid (80.7 g, 404 mmol), and aqueous
Na2CO3 (2 M, 504 mL, 1.0 mol) in 1,4-dioxane (1.4 L) was added
Pd(dppf)Cl2 (24.6 g, 33.6 mmol) under nitrogen. The reaction mixture
was heated with stirring at 100 °C for 1 h. The mixture was cooled to
room temperature and quenched with water. The mixture was acidified
to “pH” 1 using aqueous HCl solution (1 M) and then extracted with
ethyl acetate (3 × 1 L). The combined organic extract was washed with
brine, dried over anhydrous Na2SO4, and concentrated. The residue
was purified by reverse-phase Combi-Flash (column, C18; eluent, 25−
55% MeCN in water) to give the title compound as a white solid (90 g,
54% yield). LCMS (ESI): mass calcd for C21H24F3NO7S, 491.1; m/z
found, 492.2 [M + H]+.
Preparation of 1-((5-(Hydroxymethyl)-6-isopropoxy-3-(4-
(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-methylpropan-2-
ol (9). To a solution of 6-(2-hydroxy-2-methylpropoxy)-2-isopropoxy-
5-(4-(methylsulfonyl)phenyl)nicotinic acid (39a, 0.8 g, 1.89 mmol) in
anhydrous THF (24 mL) at 0 °C was added a THF solution (1 M) of
BH3·THF (5.7 mL, 5.7 mmol). The reaction mixture was stirred at
room temperature for 1 h. The mixture was quenched with MeOH (5
mL) and brine (10 mL), and the organic layer was separated. The
aqueous layer was extracted with ethyl acetate (3 × 30 mL). The
combined organic extract was washed with brine (2 × 30 mL), dried
over anhydrous Na2SO4, filtered, and concentrated. The residue was
purified by reverse-phase Combi-Flash (column, C18; eluent, 25−60%
MeCN in water) to give the title compound as a white solid (435 mg,
56% yield). LCMS (ESI): mass calcd for C20H27NO6S, 409.2; m/z
found, 410.1 [M + H]+; 1H NMR (300 MHz, DMSO-d6): δ 7.95 (d, J =
8.7 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.82 (s, 1H), 5.27 (sept, J = 6.3
Hz, 1H), 5.05 (t, J = 5.7 Hz, 1H), 4.63 (s, 1H), 4.42 (d, J = 5.7 Hz, 2H),
4.11 (s, 2H), 3.25 (s, 3H), 1.34 (d, J = 6.3 Hz, 6H), 1.17 (s, 6H) ppm.
P r e p a r a t i o n o f 1 - ( ( 5 - ( H y d r o x y m e t h y l ) - 3 - ( 4 -
(methylsulfonyl)phenyl)-6-propoxypyridin-2-yl)oxy)-2-meth-
ylpropan-2-ol (10). To a solution of 6-(2-hydroxy-2-methylpro-
poxy)-5-(4-(methylsulfonyl)phenyl)-2-propoxynicotinic acid (39b,
72.8 g, 163 mmol) in anhydrous THF (1.4 L) at 0 °C was added a
THF solution (1 M) of BH3·THF (653 mL, 653 mmol). The reaction
mixture was stirred at room temperature for 3 h. MeOH (1 L) and
SiliaMrtS DMT (7 g) were added. The mixture was stirred at room
temperature for 12 h, filtered, and concentrated. The residue was
purified by reverse-phase Combi-Flash (column, C18; eluent, 25−55%
MeCN in water; time, 40 min), followed by recrystallization in
acetonitrile to give the title compound as a white solid (45.1 g, 67%
yield). LCMS (ESI): mass calcd for C20H27NO6S, 409.2; m/z found,
410.2 [M + H]+; 1H NMR (400 MHz, DMSO-d6): δ 7.95 (d, J = 8.8 Hz,
2H), 7.88 (d, J = 8.8 Hz, 2H), 7.83 (s, 1H), 5.07 (t, J = 5.6 Hz, 1H), 4.62
(s, 1H), 4.46 (d, J = 5.6 Hz, 2H), 4.31 (t, J = 6.6 Hz, 2H), 4.13 (s, 2H),
3.25 (s, 3H), 1.79−1.74 (m, 2H), 1.17 (s, 6H), 0.99 (t, J = 7.6 Hz, 3H)
ppm; Anal. Calcd for C20H27NO6S: C, 58.66; H, 6.65; N, 3.42. Found:
C, 58.63; H, 6.68; N, 3.37; Pd level < 1 ppm.
P r e p a r a t i o n o f 1 - ( ( 5 - ( H y d r o x y m e t h y l ) - 3 - ( 4 -
(methylsulfonyl)phenyl)-6-propoxypyridin-2-yl)oxy)-2-meth-
ylpropan-2-ol (11). To a solution of 2-butoxy-6-(2-hydroxy-2-
methylpropoxy)-5-(4-(methylsulfonyl)phenyl)nicotinic acid (39c, 1.2
g, 2.74 mmol) in anhydrous THF (24 mL) at 0 °C was added a THF
solution (1 M) of BH3·THF (7.05 mL, 7.05 mmol). The reaction
mixture was stirred at room temperature for 2 h. The mixture was
quenched with MeOH (10 mL) and water (60 mL), and the organic
layer was separated. The aqueous layer was extracted with ethyl acetate
(3 × 30 mL). The combined organic extract was washed with saturated
aqueous NaHCO3 (30 mL), brine (30 mL), dried over anhydrous
Na2SO4, filtered, and concentrated. The residue was recrystallized in
acetonitrile to give the title compound as a white solid (860 mg, 74%
yield). LCMS (ESI): mass calcd for C21H29NO6S, 423.2; m/z found,
424.2 [M + H]+; 1H NMR (300 MHz, DMSO-d6): δ 7.95 (d, J = 8.7 Hz,
2H), 7.88 (d, J = 8.7 Hz, 2H), 7.82 (s, 1H), 5.08 (t, J = 5.7 Hz, 1H), 4.63
(s, 1H), 4.45 (d, J = 5.4 Hz, 2H), 4.36 (t, J = 6.6 Hz, 2H), 4.13 (s, 2H),
3.25 (s, 3H), 1.68−1.78 (m, 2H), 1.40−1.50 (m, 2H), 1.16 (s, 6H),
0.99 (t, J = 7.5 Hz, 3H) ppm; Anal. Calcd for C21H29NO6S: C, 59.56; H,
6.90; N, 3.31. Found: C, 59.54; H, 7.04; N, 3.32; Pd level 1 ppm.
P r e p a r a t i o n o f 1 - ( ( 5 - ( H y d r o x y m e t h y l ) - 3 - ( 4 -
(methylsulfonyl)phenyl)-6-(3,3,3-trifluoropropoxy)pyridin-2-
yl)oxy)-2-methylpropan-2-ol (21). To a solution of 6-(2-hydroxy-2-
methylpropoxy)-5-(4-(methylsulfonyl)phenyl)-2-(3,3,3-trifluoro-
propoxy)nicotinic acid (39d, 100 g, 209 mmol) in anhydrous THF (3
L) at 0 °C was added a THF solution (1 M) of BH3·THF (628 mL, 628
mmol). The reaction mixture was stirred at room temperature for 3 h.
The mixture was quenched with MeOH (100 mL) and concentrated.
The residue was triturated in acetonitrile (500 mL) and water (1 L).
The precipitate was collected by filtration, washed with water, and then
recrystallized in acetonitrile (200 mL) to give the title compound as a
white solid (71.3 g, 73% yield). LCMS (ESI): mass calcd for
1
C20H24F3NO6S, 463.1; m/z found, 464.4 [M + H]+; H NMR (400
MHz, DMSO-d6): δ 7.96 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H),
7.86 (s, 1H), 5.10 (br s, 1H), 4.61 (s, 1H), 4.60 (t, J = 6.0 Hz, 2H), 4.45
(s, 2H), 4.14 (s, 2H), 3.25 (s, 3H), 2.78−2.90 (m, 2H), 1.17 (s, 6H)
ppm; 19F NMR (376 MHz, DMSO-d6): δ −63.05 ppm; Anal. Calcd for
C20H24F3NO6S: C, 51.83; H, 5.22; N, 3.02. Found: C, 51.82; H, 5.24;
N, 2.96; Pd level < 5.4 ppm.
P r e p a r a t i o n o f 1 - ( ( 5 - ( H y d r o x y m e t h y l ) - 3 - ( 4 -
(methylsulfonyl)phenyl)-6-(4,4,4-trifluorobutoxy)pyridin-2-
yl)oxy)-2-methylpropan-2-ol (22). To a solution of 6-(2-hydroxy-2-
methylpropoxy)-5-(4-(methylsulfonyl)phenyl)-2-(4,4,4-
trifluorobutoxy)nicotinic acid (39e, 90 g, 183 mmol) in anhydrous
THF (2.5 L) at 0 °C was added a THF solution (1 M) of BH3·THF
(550 mL, 550 mmol). The reaction mixture was stirred at room
temperature for 3 h. The mixture was slowly quenched with MeOH
(500 mL) dropwise and concentrated. The residue was diluted with
water (3 L). The precipitate was collected by filtration, washed with
water, and then recrystallized in acetonitrile (150 mL) and ethanol (150
mL) to give the title compound as a white solid (62.1 g, 70% yield).
LCMS (ESI): mass calcd for C21H26F3NO6S, 477.1; m/z found, 478.1
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[M + H]+; 1H NMR (400 MHz, DMSO-d6): δ 7.95 (d, J = 8.4 Hz, 2H),
7.89 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 5.06 (t, J = 5.6 Hz, 1H), 4.60 (s,
1H), 4.46 (d, J = 5.6 Hz, 2H), 4.31 (t, J = 6.4 Hz, 2H), 4.13 (s, 2H), 3.25
(s, 3H), 2.41−2.46 (m, 2H), 1.95−2.02 (m, 2H), 1.16 (s, 6H) ppm; 19F
NMR (376 MHz, DMSO-d6): δ −64.70 ppm; Anal. Calcd for
C21H26F3NO6S: C, 52.82; H, 5.49; N, 2.93. Found: C, 52.76; H,
5.50; N, 2.89; Pd level < 4.1 ppm.
Preparation of 2-Butoxy-6-(2-hydroxy-2-methylpropoxy)-5-
(4-(methylsulfonyl)phenyl)nicotinaldehyde (40). To a solution
of 1-[[6-butoxy-5-(hydroxymethyl)-3-(4-methanesulfonylphenyl)-
pyridin-2-yl]oxy]-2-methylpropan-2-ol (11, 860 mg, 2.03 mmol) in
dichloromethane (20 mL) at 0 °C was added Dess−Martin periodinane
(2.34 g, 5.52 mmol) in several batches. The resulting reaction mixture
was stirred at room temperature overnight. The reaction was then
quenched by aqueous KHCO3 solution (60 mL). The resulting solution
was extracted with ethyl acetate (3 × 30 mL). The combined organic
extract was washed with brine (30 mL), dried over anhydrous Na2SO4,
filtered, and concentrated. The residue was purified by chromatography
(33% ethyl acetate in petroleum ether as the eluent) to give the title
compound as a white solid (810 mg, 95% yield). LCMS (ESI): mass
calcd for C21H27NO6S, 421.2; m/z found, 422.3 [M + H]+.
Preparation of 1-((6-Butoxy-5-(1-hydroxyethyl)-3-(4-
(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-methylpropan-2-
ol (13). To a solution of 2-butoxy-6-(2-hydroxy-2-methylpropoxy)-5-
(4-(methylsulfonyl)phenyl)nicotinaldehyde (40, 810 mg, 1.83 mmol)
in anhydrous THF (20 mL) at 0 °C was added a THF solution (3 M) of
methylmagnesium chloride (1.8 mL, 5.4 mmol). The reaction mixture
was stirred at 0 °C for 2 h. The reaction was then quenched by aqueous
saturated NH4Cl solution (40 mL). The resulting solution was
extracted with ethyl acetate (3 × 20 mL). The combined organic
extract was washed with brine (40 mL), dried over anhydrous Na2SO4,
filtered, and concentrated. The residue was purified by reverse-phase
prep-HPLC [Column, XBridge Prep C18 OBD 19 × 150 mm 5 μm C-
0013; mobile phase, phase A: water(0.1% NH4HCO3+0.1%NH3.H2O),
phase B: MeCN from 43 to 54; detector, 254 nm] to give the title
compound as a white solid (423 mg, 53% yield). LCMS (ESI): mass
calcd for C22H31NO6S, 437.2; m/z found, 438.1 [M + H]+; 1H NMR
(300 MHz, DMSO-d6): δ 7.96 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz,
2H), 7.86 (s, 1H), 5.12 (d, J = 4.2 Hz, 1H), 4.84−4.90 (m, 1H), 4.62 (s,
1H), 4.36 (dq, J = 4.2, 6.6 Hz, 2H), 4.13 (s, 2H), 3.25 (s, 3H), 1.70−
1.79 (m, 2H), 1.39−1.46 (m, 2H), 1.32 (d, J = 6.3 Hz, 3H), 1.17 (s,
6H), 0.93 (t, J = 6.6 Hz, 3H) ppm.
Preparation of Methyl 2-Butoxy-6-(2-hydroxy-2-methylpro-
poxy)-5-(4-(methylsulfonyl)phenyl)nicotinate (41). To a stirring
solution of 2-butoxy-6-(2-hydroxy-2-methylpropoxy)-5-(4-
(methylsulfonyl)phenyl)nicotinic acid (39c, 1.2 g, 2.4 mmol) in
MeOH (12 mL) and toluene (12 mL) at 0 °C was added a hexane
solution (1 M) of (trimethylsilyl)diazomethane (3.5 mL, 3.5 mmol)
dropwise. The resulting reaction mixture was stirred at 0 °C for 0.5 h.
The reaction was then quenched by water (60 mL). The resulting
solution was extracted with ethyl acetate (3 × 30 mL). The combined
organic extract was washed with brine (30 mL), dried over anhydrous
Na2SO4, and concentrated. The residue was purified by flash
chromatography (25% ethyl acetate in petroleum ether as the eluent)
to give the title compound as a white solid (1.1 g, 71%). LCMS (ESI):
mass calcd for C22H29NO7S, 451.2; m/z found, 452.3 [M + H]+.
Preparation of 1-((6-Butoxy-5-(2-hydroxypropan-2-yl)-3-(4-
(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-methylpropan-2-
ol (14). To a solution of methyl 2-butoxy-6-(2-hydroxy-2-methyl-
propoxy)-5-(4-(methylsulfonyl)phenyl)nicotinate (41, 1.3 g, 2.88
mmol) in anhydrous THF (20 mL) at 0 °C was added a THF solution
(3 M) of methylmagnesium chloride (10 mL, 30 mmol). The reaction
mixture was stirred at room temperature overnight. The reaction was
then quenched by aqueous saturated NH4Cl solution (50 mL). The
resulting solution was extracted with ethyl acetate (3 × 20 mL). The
combined organic extract was washed with brine (20 mL), dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was purified
by flash chromatography (25% ethyl acetate in petroleum ether as the
eluent) to give the title compound as a white solid (330 mg, 25% yield).
LCMS (ESI): mass calcd for C23H33NO6S, 451.2; m/z found, 452.2 [M
+ H]+; 1H NMR (300 MHz, DMSO-d6): δ 8.00 (s, 1H), 7.95 (d, J = 8.7
Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 5.11 (s, 1H), 4.61 (s, 1H), 4.38 (t, J =
6.3 Hz, 2H), 4.12 (s, 2H), 3.25 (s, 3H), 1.71−1.81 (m, 2H), 1.54 (s,
6H), 1.41−1.54 (m, 2H), 1.17 (s, 6H), 0.97 (t, J = 7.5 Hz, 3H) ppm.
Preparation of Ethyl 2-((2-Butoxy-6-(2-hydroxy-2-methyl-
propoxy)-5-(4-(methylsulfonyl)phenyl)pyridin-3-yl)methoxy)-
acetate (42). To a solution of 1-[[6-butoxy-5-(hydroxymethyl)-3-(4-
methanesulfonylphenyl)pyridin-2-yl]oxy]-2-methylpropan-2-ol (11,
1.1 g, 2.60 mmol) in anhydrous THF (22 mL) at 0 °C was added
NaH (60%, 417 mg, 10.4 mmol) in several batches. The mixture was
stirred at 0 °C for 1 h, and then ethyl 2-bromoacetate (1.18 g, 7.06
mmol) was added dropwise. The reaction mixture was stirred at room
temperature overnight. The mixture was quenched with acetic acid (10
mL) and water (50 mL). The mixture was extracted with ethyl acetate
(3 × 30 mL). The organic layers were combined, dried over anhydrous
Na2SO4, and concentrated to give the title compound as a colorless oil
(910 mg, 69% yield). LCMS (ESI): mass calcd for C25H35NO8S, 509.2;
m/z found, 510.3 [M − H]+.
Preparation of 1-((6-Butoxy-5-((2-hydroxyethoxy)methyl)-
3-(4-(methylsulfonyl)phenyl)pyridin-2-yl)oxy)-2-methylpro-
pan-2-ol (15). To a solution of ethyl 2-((2-butoxy-6-(2-hydroxy-2-
methylpropoxy)-5-(4-(methylsulfonyl)phenyl)pyridin-3-yl)methoxy)-
acetate (42, 910 mg, 1.34 mmol) in anhydrous THF (20 mL) at 0 °C
was added LiAlH4 (102 mg, 2.7 mmol). The reaction mixture was
stirred at 0 °C for 2 h. The mixture was quenched by Fieser workup
(water, 0.1 mL, followed by 15% aqueous NaOH, 0.3 mL; and then
stirred for 10 min). The mixture was filtered, and the filtrate was
concentrated. The residue was purified by flash chromatography (33−
50% ethyl acetate in petroleum ether as the eluent) to give the title
compound as a white solid (441 mg, 53% yield). LCMS (ESI): mass
calcd for C23H33NO7S, 467.2; m/z found, 468.1 [M + H]+; 1H NMR
(300 MHz, DMSO-d6): δ 7.95 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 8.7 Hz,
2H), 7.86 (s, 1H), 4.62 (s, 2H), 4.45 (s, 2H), 4.35 (d, J = 6.0 Hz, 2H),
4.14 (s, 2H), 3.49−3.54 (m, 4H), 3.25 (s, 3H), 1.70−1.79 (m, 2H),
1.39−1.52 (m, 2H), 1.17 (s, 6H), 0.93 (d, J = 7.5 Hz, 3H) ppm.
Preparation of 2-Butoxy-6-(4-fluorophenyl)nicotinic Acid
(43). To a mixture of 2-butoxy-6-chloronicotinic acid (36c, 4 g, 17.1
mmol), K2CO3 (10.6 g, 76.9 mmol), and 4-fluorophenylboronic acid
(3.6 g, 25.6 mmol) in 1,4-dioxane (80 mL) and water (16 mL) under
nitrogen was added Pd(Ph3P)4 (2.0 g, 1.7 mmol). The reaction mixture
was heated and stirred at 80 °C overnight. The mixture was cooled to
room temperature and then quenched with water (200 mL). The
mixture was acidified to “pH” 1 using aqueous HCl solution (1 M), and
then extracted with ethyl acetate (3 × 50 mL). The organic layers were
combined, dried over anhydrous Na2SO4, and concentrated. The
residue was purified by flash chromatography (50−70% ethyl acetate in
petroleum ether as the eluent) to give the title compound as a white
solid (2.1 g, 40% yield). LCMS (ESI): mass calcd for C16H16FNO3,
289.1; m/z found, 288.2 [M − H]+.
Preparation of 5-Bromo-2-butoxy-6-(4-fluorophenyl)-
nicotinic Acid (44). To a solution of 2-butoxy-6-(4-fluorophenyl)-
nicotinic acid (2.1 g, 6.9 mmol) in acetonitrile (40 mL) and
trifluoroacetic acid (40 mL) was added NBS (1.6 g, 9.0 mmol). The
reaction mixture was stirred at room temperature overnight. Water
(500 mL) was added. The precipitate was collected by filtration, washed
with water (2 × 40 mL), and dried in vacuo to give the title compound
as a white solid (1.5 g, 57% yield). LCMS (ESI): mass calcd for
C16H15BrFNO3, 367.0; m/z found, 368.2 [M + H]+.
Preparation of 2-Butoxy-6-(4-fluorophenyl)-5-(4-
(methylsulfonyl)phenyl)nicotinic Acid (45). To a mixture of 5-
bromo-2-butoxy-6-(4-fluorophenyl)nicotinic acid (44, 1.1 g, 2.87
mmol), K2CO3 (1.8 g, 12.9 mmol), and 4-(methylsulfonyl)-
phenylboronic acid (630 mg, 3.15 mmol) in 1,4-dioxane (20 mL)
and water (4 mL) was added Pd(Ph3P)4 (331 mg, 0.29 mmol) under
nitrogen. The reaction mixture was heated with stirring at 80 °C
overnight. The mixture was cooled to room temperature and quenched
with water (60 mL). The mixture was acidified to “pH” 1 using aqueous
HCl solution (1 M) and then extracted with ethyl acetate (3 × 20 mL).
The combined organic extract was washed with brine (30 mL), dried
over anhydrous Na2SO4, and concentrated. The residue was purified by
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flash chromatography (70% ethyl acetate in petroleum ether as the
eluent) to give the title compound as a white solid (810 mg, 56% yield).
LCMS (ESI): mass calcd for C23H22FNO5S, 443.1; m/z found, 444.2
[M + H]+.
mmol). The reaction mixture was heated and stirred at 90 °C overnight.
The mixture was cooled to room temperature and then quenched with
water (3 L). The organic layer was separated, and the “pH” of the
aqueous layer was adjusted to 1 using aqueous HCl solution (1 M). The
aqueous solution was extracted with ethyl acetate. The organic layers
were combined, dried over anhydrous Na2SO4, and concentrated to
give the title compound as a brown oil (15 g, 45% yield). LCMS (ESI):
mass calcd for C15H21NO5, 295.1; m/z found, 296.0 [M + H]+.
Preparation of 5-Bromo-2-butoxy-6-(oxetan-3-ylmethoxy)-
nicotinic Acid (48a). To a solution of 2-butoxy-6-(oxetan-3-
ylmethoxy)nicotinic acid (47a, 19 g, 60.8 mmol) and acetic acid
(18.3 g, 305 mmol) in acetonitrile (400 mL) was added NBS (13 g, 73
mmol). The reaction mixture was stirred at room temperature for 3 h.
The mixture was concentrated under reduced pressure. The residue was
triturated in ethyl acetate to give the title compound as a white solid
(19.5 g, 85% yield). LCMS (ESI): mass calcd for C14H18BrNO5, 359.0;
m/z found, 360.0 [M + H]+.
Preparation of 5-Bromo-2-butoxy-6-((3-hydroxyoxetan-3-
yl)methoxy)nicotinic Acid (48b). To a solution of 2-butoxy-6-((3-
hydroxyoxetan-3-yl)methoxy) nicotinic acid (47b, 1.4 g, 4.7 mmol) in
acetonitrile (25 mL) and acetic acid (1.4 g, 23 mmol) was added NBS
(1 g, 5.6 mmol). The reaction mixture was stirred at room temperature
for 2 h. The mixture was concentrated. The residue was purified by
reversal-phase Combi-Flash [MeCN/water (0.05% NH4HCO3) from
10% to 60% in 45 min] to give the title compound as a white solid (1.3 g,
73% yield). LCMS (ESI): mass calcd for C14H18BrNO6, 375.0; m/z
found, 376.2 [M + H]+.
Preparation of 5-Bromo-2-butoxy-6-((3-methyloxetan-3-yl)-
methoxy)nicotinic Acid (48c). To a solution of 2-butoxy-6-((3-
methyloxetan-3-yl)methoxy) nicotinic acid (47c, 4.1 g, 13.9 mmol) and
acetic acid (4.2 g, 69.4 mmol) in acetonitrile (80 mL) was added NBS
(3 g, 16.9 mmol). The reaction mixture was stirred at room temperature
for 3 h. The mixture was concentrated under reduced pressure. The
residue was triturated in ethyl acetate to give the title compound as a
white solid (4.5 g, 87% yield). LCMS (ESI): mass calcd for
C15H20BrNO5, 373.0; m/z found, 374.2 [M + H]+.
Preparation of 5-Bromo-2-butoxy-6-((1-hydroxycyclobutyl)-
methoxy)nicotinic Acid (48d). To a solution of 2-butoxy-6-((1-
hydroxycyclobutyl)methoxy)nicotinic acid (47d, 15 g, 39.1 mmol) in
acetonitrile (375 mL) was added NBS (7.66 g, 43.0 mmol). The
reaction mixture was stirred at room temperature for 3 h. The mixture
was concentrated under reduced pressure. The residue was triturated in
ethyl acetate to give the title compound as a white solid (8 g, 50% yield).
LCMS (ESI): mass calcd for C15H20BrNO5, 373.0; m/z found, 374.1
[M + H]+.
Preparation of 2-Butoxy-5-(4-(methylsulfonyl)phenyl)-6-
(oxetan-3-ylmethoxy)nicotinic Acid (49a). To a mixture of 5-
bromo-2-butoxy-6-(oxetan-3-ylmethoxy)nicotinic acid (48a, 10 g, 27.8
mmol), 4-(methylsulfonyl)phenylboronic acid (6.1 g, 30.5 mmol), and
K2CO3 (17.3 g, 125 mmol) in 1,4-dioxane (200 mL) and water (20 mL)
was added Pd(Ph3P)4 (3.2 g, 2.76 mmol) under nitrogen. The reaction
mixture was heated with stirring at 80 °C overnight. The mixture was
cooled to room temperature and quenched with water. The mixture was
filtered to remove the solids. The filtrate was acidified to “pH” 1 using
aqueous HCl solution (1 M), and then extracted with ethyl acetate. The
combined organic extract was washed with brine, dried over anhydrous
Na2SO4, and concentrated. The residue was triturated in ethyl acetate
to give the title compound as a white solid (8 g, 66% yield). LCMS
(ESI): mass calcd for C21H25NO7S, 435.1; m/z found, 436.4 [M + H]+.
Preparation of 2-Butoxy-6-((3-hydroxyoxetan-3-yl)-
methoxy)-5-(4-(methylsulfonyl)phenyl)nicotinic Acid (49b).
To a mixture of 5-bromo-2-butoxy-6-((3-hydroxyoxetan-3-yl)-
methoxy)nicotinic acid (48b, 1.3 g, 3.5 mmol), (4-(methylsulfonyl)-
phenyl)boronic acid (760 mg, 3.80 mmol), and K2CO3 (2.15 g, 15.6
mmol) in 1,4-dioxane (30 mL) and water (3 mL) was added Pd(Ph3P)4
(400 mg, 0.346 mmol) under nitrogen. The reaction mixture was
heated with stirring at 80 °C overnight. The mixture was cooled to
room temperature and quenched with water. The mixture was filtered
to remove the solids. The filtrate was acidified to “pH” 1 using aqueous
HCl solution (1 M), and then extracted with ethyl acetate. The
Preparation of (2-Butoxy-6-(4-fluorophenyl)-5-(4-
(methylsulfonyl)phenyl)pyridin-3-yl)methanol (16). To a sol-
ution of 2-butoxy-6-(4-fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-
nicotinic acid (45, 810 mg, 1.6 mmol) in anhydrous THF (16 mL) at 0
°C was added a THF solution (1 M) of BH3·THF (4.7 mL, 4.7 mmol).
The reaction mixture was stirred at room temperature for 3 h. The
mixture was quenched with MeOH (10 mL) and water (50 mL), and
the organic layer was separated. The aqueous layer was extracted with
ethyl acetate (3 × 20 mL). The combined organic extract was washed
with saturated aqueous NaHCO3 (20 mL), brine (20 mL), dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was purified
by reverse-phase prep-HPLC [Column, XBridge Prep C18 OBD
column 19 × 150 mm 5umC-0013; mobile phase, phase A: water (10
mmol NH4HCO3 solution; phase B: MeCN from 56 to 74% in 7 min;
detector, 254 nm)] to give the title compound as a white solid (416 mg,
61% yield). LCMS (ESI): mass calcd for C23H24FNO4S, 429.1; m/z
found, 430.1 [M + H]+; 1H NMR (300 MHz, DMSO-d6): δ 7.86 (d, J =
8.4 Hz, 2H), 7.75 (s, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.31−7.37 (m, 2H),
7.10−7.17 (m, 2H), 5.30 (t, J = 5.4 Hz, 1H), 4.56 (d, J = 5.4 Hz, 2H),
4.40 (t, J = 6.3 Hz, 2H), 3.23 (s, 3H), 1.70−1.79 (m, 2H), 1.42−1.49
(m, 2H), 0.95 (t, J = 7.2 Hz, 3H) ppm.
Preparation of 2-Butoxy-6-(oxetan-3-ylmethoxy)nicotinic
Acid (47a). To a mixture of 2-butoxy-6-chloronicotinic acid (36c, 20
g, 85 mmol), Cs2CO3 (83.1 g, 255 mmol), and oxetan-3-ylmethanol
(18.7 g, 212 mmol) in toluene (600 mL) under nitrogen was added
XPhos Pd G3 (1.4 g, 1.65 mmol). The reaction mixture was heated and
stirred at 90 °C for 3 h. The mixture was cooled to room temperature
and then quenched with water. The organic layer was separated, and the
“pH” of the aqueous layer was adjusted to 1 using aqueous HCl solution
(1 M). The aqueous solution was extracted with ethyl acetate. The
organic layers were combined, dried over anhydrous Na2SO4, and
concentrated to give the title compound as a white solid (19 g, 80%
yield). LCMS (ESI): mass calcd for C14H19NO5, 281.1; m/z found,
282.1 [M + H]+.
Preparation of 2-Butoxy-6-((3-hydroxyoxetan-3-yl)-
methoxy)nicotinic Acid (47b). To a mixture of 2-butoxy-6-
chloronicotinic acid (36c, 2 g, 8.7 mmol), Cs2CO3 (8.6 g, 26.4
mmol), and 3-(hydroxymethyl)oxetan-3-ol (1.4 g, 13.4 mmol) in
toluene (60 mL) under nitrogen was added XPhos Pd G3 (148 mg,
0.175 mmol). The reaction mixture was heated and stirred at 90 °C for
3 h. The mixture was cooled to room temperature and then quenched
with water. The organic layer was separated, and the “pH” of the
aqueous layer was adjusted to 1 using aqueous HCl solution (1 M). The
aqueous solution was extracted with ethyl acetate. The organic layers
were combined, dried over anhydrous Na2SO4, and concentrated to
give the title compound as a white solid (1.4 g, 54% yield). LCMS
(ESI): mass calcd for C14H19NO6, 297.1; m/z found, 298.2 [M + H]+.
Preparation of 2-Butoxy-6-((3-methyloxetan-3-yl)methoxy)-
nicotinic Acid (47c). To a mixture of 2-butoxy-6-chloronicotinic acid
(36c, 4 g, 17.4 mmol), Cs2CO3 (17 g, 52.2 mmol), and (3-
methyloxetan-3-yl) methanol (4.4 g, 43.1 mmol) in toluene (120
mL) under nitrogen was added XPhos Pd G3 (295 mg, 0.349 mmol).
The reaction mixture was heated and stirred at 90 °C for 3 h. The
mixture was cooled to room temperature and then quenched with
water. The organic layer was separated, and the “pH” of the aqueous
layer was adjusted to 1 using aqueous HCl solution (1 M). The aqueous
solution was extracted with ethyl acetate. The organic layers were
combined, washed with brine, dried over anhydrous Na2SO4, and
concentrated to give the title compound as a white solid (4.1 g, 80%
yield). LCMS (ESI): mass calcd for C15H21NO5, 295.1; m/z found,
296.1 [M + H]+.
Preparation of 2-Butoxy-6-((1-hydroxycyclobutyl)-
methoxy)nicotinic Acid (47d). To a mixture of 2-butoxy-6-
chloronicotinic acid (36c, 20 g, 87.0 mmol), Cs2CO3 (85.1 g, 261
mmol), and 1-(hydroxymethyl) cyclobutan-1-ol (17.8 g, 174 mmol) in
toluene (600 mL) under nitrogen was added XPhos Pd G3 (1.47 g, 1.74
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combined organic extract was washed with brine, dried over anhydrous
Na2SO4, and concentrated. The residue was triturated in ethyl acetate
to give the title compound as a white solid (660 mg, 42% yield). LCMS
(ESI): mass calcd for C21H25NO8S, 451.1; m/z found, 452.3 [M + H]+.
Preparation of 2-Butoxy-6-((3-methyloxetan-3-yl)methoxy)-
5-(4-(methylsulfonyl)phenyl)nicotinic Acid (49c). To a mixture of
5-bromo-2-butoxy-6-((3-methyloxetan-3-yl)methoxy)nicotinic acid
(48c, 4.5 g, 12.0 mmol), 4-(methylsulfonyl)phenylboronic acid (2.6
g, 13.0 mmol), and K2CO3 (7.5 g, 54.3 mmol) in 1,4-dioxane (90 mL)
and water (9 mL) was added Pd(Ph3P)4 (1.4 g, 1.21 mmol) under
nitrogen. The reaction mixture was heated with stirring at 80 °C
overnight. The mixture was cooled to room temperature and quenched
with water. The mixture was filtered to remove the solids. The filtrate
was acidified to “pH” 1 using aqueous HCl solution (1 M), and then
extracted with ethyl acetate. The combined organic extract was washed
with brine, dried over anhydrous Na2SO4, and concentrated. The
residue was purified by reverse-phase Combi-Flash [MeCN/water
(0.05% NH4HCO3) from 10% to 40% in 45 min] to give the title
compound as a white solid (2.2 g, 41% yield). LCMS (ESI): mass calcd
for C22H27NO7S, 449.2; m/z found, 450.3 [M + H]+.
Preparation of 2-Butoxy-6-((1-hydroxycyclobutyl)-
methoxy)-5-(4-(methylsulfonyl)phenyl)nicotinic Acid (49d).
To a mixture of 5-bromo-2-butoxy-6-((1-hydroxycyclobutyl)-
methoxy)nicotinic acid (48d, 8 g, 19.5 mmol), 4-(methylsulfonyl)-
phenylboronic acid (4.28 g, 21.4 mmol), and K2CO3 (12.1 g, 87.5
mmol) in 1,4-dioxane (160 mL) and water (16 mL) was added
Pd(Ph3P)4 (2.25 g, 1.95 mmol) under nitrogen. The reaction mixture
was heated with stirring at 80 °C overnight. The mixture was cooled to
room temperature and quenched with water. The mixture was filtered
to remove the solids. The filtrate was acidified to “pH” 1 using aqueous
HCl solution (1 M), and then extracted with ethyl acetate. The
combined organic extract was washed with brine, dried over anhydrous
Na2SO4, and concentrated. The residue was purified by reverse-phase
Combi-Flash [MeCN/water (0.05% NH4HCO3) from 10% to 40% in
45 min] to give the title compound as a white solid (4.1 g, 45% yield).
LCMS (ESI): mass calcd for C22H27NO7S, 449.2; m/z found, 450.2 [M
+ H]+.
Preparation of (2-Butoxy-5-(4-(methylsulfonyl)phenyl)-6-
(oxetan-3-ylmethoxy)pyridin-3-yl)methanol (17). To a solution
of 2-butoxy-5-(4-(methylsulfonyl) phenyl)-6-(oxetan-3-ylmethoxy)-
nicotinic acid (49a, 8 g, 18.4 mmol) and triethylamine (2.3 g, 22.7
mmol) in anhydrous THF (200 mL) at 0 °C was added isobutyl
chloroformate (3 g, 22 mmol). The mixture was stirred at 0 °C for 0.5 h,
and then an aqueous suspension (2 mL) of NaBH4 (1.4 g, 37.0 mmol)
was added. The reaction mixture was stirred at room temperature for 2
h. The mixture was concentrated. The residue was purified by reverse-
phase prep-HPLC (column, C-18, mobile phase, water (a)/MeCN (b),
b %: 30%−80%, 35 min; detector, UV 210 nm) to give the title
compound as a white solid (3.41 g, 44% yield). LCMS (ESI): mass
calcd for C21H27NO6S, 421.2; m/z found, 422.2 [M + H]+; 1H NMR
(300 MHz, DMSO-d6): δ 7.94 (d, J = 8.4 Hz, 2H), 7.82 (s, 1H), 7.82 (d,
J = 8.4 Hz, 2H), 5.08 (t, J = 5.6 Hz, 1H), 4.65−4.74 (m, 2H), 4.57 (d, J =
6.4 Hz, 2H), 4.41−4.50 (m, 4H), 4.37 (t, J = 6.4 Hz, 2H), 3.32−3.49
(m, 1H), 3.24 (s, 3H), 1.65−1.80 (m, 2H), 1.36−1.53 (m, 2H), 0.95 (t,
J = 7.4 Hz, 3H) ppm.
Preparation of 3-(((6-Butoxy-5-(hydroxymethyl)-3-(4-
(methylsulfonyl)phenyl)pyridin-2-yl)oxy)methyl)oxetan-3-ol
(18). To a solution of 2-butoxy-6-((3-hydroxyoxetan-3-yl)methoxy)-5-
(4-(methylsulfonyl)phenyl)nicotinic acid (49b, 660 mg, 1.46 mmol)
and triethylamine (178 mg, 1.76 mmol) in anhydrous THF (18 mL) at
0 °C was added isobutyl chloroformate (240 mg, 1.76 mmol). The
mixture was stirred at 0 °C for 0.5 h, and then an aqueous suspension
(0.2 mL) of NaBH4 (111 mg, 2.93 mmol) was added. The reaction
mixture was stirred at room temperature for 2 h. The mixture was
concentrated. The residue was purified by reverse-phase prep-HPLC
(column, C-18, mobile phase, water (a)/MeCN (b), b %: 20−50%, 35
min; detector, UV 210 nm) to give the title compound as a white solid
(350 mg, 54% yield). LCMS (ESI): mass calcd for C21H27NO7S, 437.2;
m/z found, 438.2 [M + H]+; 1H NMR (300 MHz, DMSO-d6): δ 7.97−
7.87 (m, 4H), 7.85 (s, 1H), 4.57−4.48 (m, 8H), 4.38 (t, J = 6.5 Hz, 2H),
3.46 (br s, 2H), 3.25 (s, 3H), 1.80−1.68 (m, 2H), 1.54−1.36 (m, 2H),
0.95 (t, J = 7.4 Hz, 3H) ppm.
Preparation of (2-Butoxy-6-((3-methyloxetan-3-yl)-
methoxy)-5-(4-(methylsulfonyl)phenyl)pyridin-3-yl)methanol
(19). To a solution of 2-butoxy-6-((3-methyloxetan-3-yl)methoxy)-5-
(4-(methylsulfonyl)phenyl)nicotinic acid (49c, 2.2 g, 4.9 mmol) and
triethylamine (594 mg, 5.87 mmol) in anhydrous THF (55 mL) at 0 °C
was added isobutyl chloroformate (802 mg, 5.87 mmol). The mixture
was stirred at 0 °C for 0.5 h, and then an aqueous suspension (0.6 mL)
of NaBH4 (370 mg, 9.78 mmol) was added. The reaction mixture was
stirred at room temperature for 2 h. The mixture was concentrated. The
residue was purified by reverse-phase prep-HPLC (column, C-18,
mobile phase, water (a)/MeCN (b), b %: 30−60%, 35 min; detector,
UV 210 nm) to give the title compound as a white solid (746 mg, 35%
yield). LCMS (ESI): mass calcd for C22H29NO6S, 435.2; m/z found,
436.2 [M + H]+; 1H NMR (300 MHz, DMSO-d6): δ 7.95 (d, J = 8.7 Hz,
2H), 7.84 (s, 1 H), 7.82 (d, J = 8.7 Hz, 2H), 5.10 (t, J = 5.7 Hz, 1H),
4.50−4.48 (m, 6H), 4.46 (t, J = 6.5 Hz, 2H), 4.29 (d, J = 5.7 Hz, 2H),
3.25 (s, 3H), 1.80−1.65 (m, 2H), 1.51−1.39 (m, 2H), 1.29 (s, 3H),
0.95 (t, J = 7.4 Hz, 3H) ppm.
Preparation of 1-(((6-Butoxy-5-(hydroxymethyl)-3-(4-
(methylsulfonyl)phenyl)pyridin-2-yl)oxy)methyl)cyclobutan-
1-ol (20). To a solution of 2-butoxy-6-((1-hydroxycyclobutyl)-
methoxy)-5-(4-(methylsulfonyl) phenyl) nicotinic acid (49d, 4.1 g,
8.8 mmol) in anhydrous THF (82 mL) at 0 °C was added a THF
solution (1 M) of BH3·THF (27 mL, 27 mmol). The reaction mixture
was stirred at room temperature for 5 h. The mixture was slowly
quenched with MeOH (5 mL) dropwise and concentrated. The residue
was purified by reverse-phase Combi-Flash chromatography [MeCN/
water (0.05% NH4HCO3) from 45 to 65% in 30 min] to give the crude
product (3.2 g). Then, SiliaMrtS DMT (0.5 g) and MeCN (100 mL)
were added to the crude product. The mixture was stirred at room
temperature for 2 h, and then filtered. The filtrate was concentrated to
give the title compound as a white solid (2.85 g, 74% yield). LCMS
(ESI): mass calcd for C22H29NO6S, 435.2; m/z found, 436.2 [M + H]+;
1H NMR (300 MHz, DMSO-d6): δ 7.94−7.82 (m, 4H), 7.82 (s, 1H),
5.21 (s, 1H), 5.07 (t, J = 4.2 Hz, 1H), 4.45 (d, J = 4.2 Hz, 2H), 4.39−
4.34 (m, 2H), 4.34 (s, 2H), 3.24 (s, 3H), 2.13−1.94 (m, 4H), 1.78−
1.63 (m, 3H), 1.55−1.39 (m, 3H), 0.95 (t, J = 6.8 Hz, 3H) ppm; Anal.
Calcd for C22H29NO6S: C, 60.67; H, 6.71; N, 3.22. Found: C, 60.32; H,
7.04; N, 3.17; Pd level 4.8 ppm.
Preparation of 1-((3-Bromo-6-butoxy-5-(hydroxymethyl)-
pyridin-2-yl)oxy)-2-methylpropan-2-ol (50). To a solution of 5-
bromo-2-butoxy-6-(2-hydroxy-2-methylpropoxy)nicotinic acid (38c,
100 g, 255 mmol) in anhydrous THF (2 L) at 0 °C was added a THF
solution (1 M) of BH3·THF (575 mL, 575 mmol). The reaction
mixture was stirred at room temperature for 5 h. The mixture was
quenched with MeOH (300 mL) and water (2 L), and then extracted
with ethyl acetate (3 × 600 mL). The combined organic extract was
washed with saturated aqueous NaHCO3 (600 mL) and brine (600
mL), dried over anhydrous Na2SO4, filtered, and concentrated to give
the title compound as a white solid (82 g, 81% yield). LCMS (ESI):
mass calcd for C14H22BrNO4, 347.1; m/z found, 348.0 [M + H]+.
Preparation of 1-((6-Butoxy-5-(hydroxymethyl)-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)oxy)-2-
methylpropan-2-ol (51). To a mixture of 1-((3-bromo-6-butoxy-5-
(hydroxymethyl)pyridin-2-yl)oxy)-2-methylpropan-2-ol (50, 82 g, 208
mmol), bis(pinacolato)diboron (105.6 g, 416 mmol), and potassium
acetate (40.8 g, 416 mmol) in anhydrous 1,4-dioxane (800 mL) was
added Pd(dppf)Cl2 (15.2 g, 20.8 mmol) under nitrogen. The reaction
mixture was heated with stirring at 100 °C overnight. The mixture was
cooled to room temperature and diluted with water (1 L), and then
extracted with ethyl acetate (3 × 400 mL). The combined organic
extract was washed with brine (400 mL), dried over anhydrous Na2SO4,
and concentrated to give the title compound as a crude product: a black
solid (90 g, 67% yield). LCMS (ESI): mass calcd for C20H34BNO6,
395.2; m/z found, 396.2 [M + H]+.
Preparation of 6′-Butoxy-2′-(2-hydroxy-2-methylpropoxy)-
5′-(hydroxymethyl)-[2,3′-Bipyridine]-5-sulfonamide (23). To a
mixture of 1-(6-butoxy-5-(hydroxymethyl)-3-(4,4,5,5-tetramethyl-
1,3,2-dioxa-borolan-2-yl)pyridin-2-yloxy)-2-methylpropan-2-ol (51,
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70 g, 115 mmol), 6-chloropyridine-3-sulfonamide (26.5 g, 138 mmol),
and Na2CO3 (24.3 g, 229.5 mmol) in 1,4-dioxane (700 mL) and water
(70 mL) was added Pd(dppf)Cl2 (8.4 g, 11.5 mmol) under nitrogen.
The reaction mixture was heated with stirring at 65 °C overnight. The
mixture was cooled to room temperature and concentrated. The residue
was diluted with water (500 mL), and then extracted with ethyl acetate
(3 × 200 mL). The combined organic extract was washed with brine
(200 mL), dried over anhydrous Na2SO4, and concentrated. The
residue was purified by flash chromatography (50−80% ethyl acetate in
petroleum ether as the eluent) to give a crude product. The crude
product was dissolved in THF (200 mL) and SiliaMrtS DMT (3 g) was
added. The mixture was stirred at room temperature overnight. The
mixture was filtered, and the filtrate was concentrated. The residue was
purified by reverse-phase prep-HPLC [Column, C-18, mobile phase,
water (a, 0.1%TFA)/MeCN (b), b %: 20−57%, 12 min; detector, UV
254 nm] followed by recrystallization in acetonitrile to give the title
compound as a white solid (15 g, 30% yield). LCMS (ESI): mass calcd
for C19H27N3O6S, 425.2; m/z found, 426.2 [M + H]+; 1HNMR (400
MHz, DMSO-d6): δ 9.00 (d, J = 2.0 Hz, 1H), 8.46 (s, 1H), 8.34 (d, J =
8.4 Hz, 1H), 8.18 (dd, J = 2.4, 8.4 Hz, 1H), 7.58 (s, 2H), 5.17 (t, J = 6.0
Hz, 1H), 4.73 (s, 1H), 4.46 (d, J = 5.6 Hz, 2H), 4.38 (t, J = 6.4 Hz, 2H),
4.21 (s, 2H), 1.71−1.78 (m, 2H), 1.40−1.49 (m, 2H), 1.22 (s, 6H),
0.95 (t, J = 6.8 Hz, 3H) ppm; Anal. Calcd for C19H27N3O6S: C, 53.63;
H, 6.40; N, 9.88. Found: C, 53.45; H, 6.41; N, 9.95; Pd level < 4.4 ppm.
Human COX-2/COX-1 Enzymatic Assays. COX-2 is a bifunc-
tional enzyme that catalyzes two sequential reactions starting with a
COX reaction that oxidizes arachidonic acid to PGG2 and followed by a
peroxidase reaction that reduces PGG2 to PGH2. COX-2 activity was
assessed in an in vitro assay through coupling of the peroxidase activity
with oxidation of ADHP to form resurofin, which is highly fluorescent
with an excitation wavelength between 530 and 540 nm and an
emission wavelength between 585 and 595 nm. The final assay buffer
consisted of 30 nM recombinant human COX-2 and 25 mM
arachidonic acid in 100 mM Tris−HCl, pH 8, containing 2 mM
heme, 20 μM ADHP, 10 nM catalase, 0.1 mg/mL BSA, and 0.002%
Tween-20. The Km value under these conditions was determined to be
13 3 μM. Test compound concentrations ranged from 50 μM to 0.85
nM prepared using a 11-step serial dilution (1:3) in pure DMSO. The
serially diluted test compounds were spotted per well of a black 384-
Proxiplate (Perkin Elmer) with DMSO maintained at 1% of the total
assay volume. The background signal was measured from control wells
containing assay buffer without arachidonic acid, which functions as
low control (LC). High control (HC) values were generated using the
reaction with enzyme but no compound treatment. Compounds were
preincubated with enzyme for 60 min at room temperature. The
substrate was added to initiate the reaction and the reaction mixture was
incubated for 20 s. The reaction was quenched by adding 10 mM
ascorbic acid (diverting oxidation of ADHP to that of ascorbic acid).
Fluorescence was then measured in PheraStar with excitation at 540 nm
and emission at 590 nm.
200 μL of tetracycline-free media (Life-Technologies 10566016) was
dispensed into a 384-well deep well microplate (Greiner#781270)
using the Combi. The Janus liquid handler was used to transfer 1 μL of
each well from the compound plate (100% DMSO) and transfer it to
the 200 μL of media and mix, creating 0.5% DMSO final concentrations
of compounds in cell media. Spent media were removed from the
overnight-induced PDL plates via plate flicking, and 30 μL of
compound titrations in cell media were added atop PDL-attached
cells. The plates were covered with a lid, centrifuged briefly (250g, 1
min), and incubated for 1 h at 37 °C. A MultiDrop Combi was used to
dispense 30 μL/well of deuterated substrate (40 μM arachidonic acid-
D11, AA-D11, Cayman Chemicals #10006758), which was diluted in
tetracycline-free media. AA-D11 was dispensed with a small tube
cassette due to the smaller tubing diameter and thus more vigorous
mixing upon addition. Assay plates were covered with a lid, centrifuged,
and incubated for 1 h at 37 °C. The Janus liquid handler was used to
gently mix the media atop the cells now containing deuterated product
PGD2-D11 and transfer 10 μL into 90 μL of Agilent RapidFire effluent
containing internal standard PGD2-D4 (Cayman Chemicals #312010)
(0.01% Formic Acid, 0.1% Tween 20, 0.025 μg/mL PGD2-D4). The
assay plates were analyzed using a RapidFire high-throughput solid-
phase extraction system (Agilent) coupled to a triple quadrupole mass
spectrometer (AB SCIEX) to measure relative peak areas of PGD2-D11
and PGD2-D4. Due to the variable sip volume of the RF-MS system,
quantification of product (PGD2-D11) via a relative peak area was
divided by the relative peak area of internal control (PGD2-D4), for
internal assay normalization. Peaks were integrated using the RapidFire
integrator software. RF-MS sample loading was done on a miniature C4
column, Agilent “Cartridge A” (Agilent #G9303A). The RF-MS was
tuned to capture the PGD2-D11 product at XIC 362.2/282.2 and the
PGD2-d4 spiked in product at XIC 354.9/275.2 on a Sciex Triple Quad
4000 QTRAP.
Human Whole Blood COX-2/COX-1 Assays. The whole blood
assay and related analytical procedure were previously described.59
Briefly, DMSO solutions of test compounds (50,000, 33,300, 10,000,
3,000, 900, 270, 81.4, 24.4, 7.34, 2.20, 0.662, 0.199, and 0.0597 μM)
were prepared. The test compound (1.8 μL) for each concentration was
added into a 96 deep-well plate. Each of 300 μL freshly heparinized
blood was used for COX-2 and COX-1 assays. The final compound
concentrations ranged from 600 to 0.4 μM. The plate was covered with
foil, vortexed at 800 rpm for 1 min, and then incubated at 37 °C/5%
CO2 for 1 h.
For the COX-2 assay, 10 μL of 300 μg/mL LPS solution was added
to COX-2 plate for a 10 μg/mL LPS final concentration. The plate was
covered with foil, vortexed at 800 rpm for 1 min, and then incubated at
37 °C/5% CO2 overnight. 100 μL ice-cold PBS buffer was added to the
wells and mixed by using a pipet 10 times. The plates were centrifuged
at 1400 rpm at 4 °C for 10 min. The supernatant aliquot (50 μL) was
transferred into 150 μL ELISA buffer and vortexed. The samples were
analyzed using PGE2 ELISA kits from Cayman Chemical Inc.
For the COX-1 assay, 2 μL of 2.25 mM calcium ionophore solution
was added to the COX-1 plate for a 15 μM calcium ionophore final
concentration. The plate was covered with foil, vortexed at 800 rpm for
1 min, and then incubated at 37 °C/5% CO2 for 1 h with continuous
shaking at 175 rpm. 100 μL ice-cold PBS buffer was added to the wells
and mixed by using a pipet 10 times. The plates were centrifuged at
1400 rpm at 4 °C for 10 min. The supernatant aliquot (20 μL) was
transferred into 180 μL ELISA buffer and vortexed. The samples were
analyzed using TXB2 ELISA kits from Cayman Chemical Inc.
Mouse Cassette PK Studies Following IV or Oral Dosing. The
selected compounds were formulated together for the cassette dosing.
Three fasted male C57BL/6J mice were administered a single
intravenous (IV) bolus injection of 2 mg/kg in a solution of
PEG400/water (70:30) at a dose volume of 2 mL/kg. Blood samples
were collected at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose via dorsal
metatarsal vein. Six fasted male C57BL/6J mice were administered a
single oral dose of 10 mg/kg in a solution of PEG400/water (70:30) at a
dose volume of 10 mL/kg. Blood samples were collected at 0.25, 0.5, 1,
2, 3, and 4 h postdose. Colon tissue was collected at 2 and 4 h postdose.
For both studies, blood samples were collected into tubes containing
The COX-1 enzyme assay was identical to the COX-2 enzyme assay
as described above with the exception that the arachidonic acid
concentration in the COX-1 enzyme assay was maintained at 10 μM
rather than at 25 μM as in the COX-2 assay. The Km value of COX-1
was determined to be 2.8 0.4 μM under the assay conditions.
COX-2/COX-1 RapidFire High-Throughput Mass Spectrom-
etry Cellular Assay. The COX-2/COX-1 RapidFire mass spectro-
metric cellular assay monitors the conversion of arachidonic acid to
PGD2 by COX1 and COX2. Tetracycline-inducible HEK 293 T-Rex
cells (Thermo) overexpressed COX-2 or COX-1 via overnight
promotion. Cells were adjusted to a given concentration (COX-2,
80,000 cells/mL; Cox-1, 30,000 cells/mL) in cell media supplemented
with 1 μg/mL tetracycline (Sigma, T7660) and dispensed into Poly-D
lysine (PDL)-coated 384-well cell culture plates (Greiner #655940),
100 μL/well, using a ThermoFisher MultiDrop Combi liquid handler.
The plates were incubated overnight at 37 °C, 5% CO2, 95% RH to
induce COX-2 or COX-1 protein. Compounds were diluted fresh in
DMSO (Sigma, 276855) and plated in Greiner 384-well PP plates
(Greiner#781280). Using a Janus MDT liquid handler, a 3-fold
titration was conducted for each compound (11pt. + vehicle control).
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the K2EDTA anticoagulant and placed on wet ice. The plasma fraction
was separated by centrifugation and frozen at −20 °C or lower until
analysis. Colon tissue was collected at 2 and 4 h postdose. For both
studies, blood samples were collected into tubes containing K2EDTA
anticoagulant and placed on wet ice. The plasma fraction was separated
by centrifugation and frozen at −20 °C or lower until analysis. Colon
tissue was homogenized with PBS at a ratio of 1:5 [colon weight (g) to
PBS volume (mL)]. Plasma and colon samples were processed for
analysis by protein precipitation using acetonitrile. The final compound
concentration in colon (ng/mL) was corrected by multiplying by 6.
Concentrations of the compound in plasma and colon were determined
using a liquid chromatographic triple-quadrupole mass spectrometric
method (Shimadzu HPLC coupled to an AAB API 5500 instrument;
Quantification: MRM, Positive ion).
Metabolite Identifications from Incubations of Compound
11 with Human and Mouse Hepatocytes. The cryopreserved
hepatocytes were thawed on ice and vials were placed in a 37 °C water
bath for approximately 90 s and then immediately added to a
prewarmed hepatocyte-thawing media. The cells were resuspended
with the media and then centrifuged at 700g for 7 min at room
temperature. The supernatant was discarded, and the cells were
resuspended in 2 mL of Krebs−Henseleit buffer (KHB) containing
12.5 mM HEPES pH 7.4. Cell viability and yield were determined by
Trypan Blue exclusion. The hepatocytes were then diluted in KHB to a
cell concentration of 2 × 106 cells/mL (500 μL/well in 10 mL glass
tubes). The tubes were placed on a rotary shaker in a humidifier
incubator supplied with 5% CO2 and allowed for 15 min acclimation at
37 °C prior to use. Compound 11 or positive control, diclofenac (10
μM), in KHB was then added to the hepatocytes to achieve a final
compound concentration of 10 μM and a cell concentration of 1 × 106
cells/mL (1000 μL final incubation volume). Cells were incubated for 0
and 2 h. At the end of the incubation, cells were quenched with 3
volumes of ice-cold acetonitrile containing 0.02% formic acid, vortexed,
and sonicated (3 min). Precipitated proteins were pelleted by
centrifugation (3000 rpm, 10 min, 4 °C) and the supernatants were
evaporated to dryness under a gentle stream of nitrogen. The resulting
residues were reconstituted in 250 μL of water: acetonitrile (9:1)
containing 0.01% formic acid with sonication and vortexing to aid
solubilization of drug-derived materials prior to filtration using 0.45 μm
nylon filter at 10,000g at room temperature for 10 min. The filtrate was
transferred to a 96-well plate for liquid chromatography/tandem mass
spectrometric (LC−MS/MS) analysis.
imaging), for the [18F]-FDG imaging, mice were euthanized with CO2,
death confirmed by no response of paw-pinch reflex. Cardiac puncture
was carried out to draw blood, which was transferred into EDTA
containing tubes that were placed onto a rocker for 10 min. Mice were
opened up and GI-tract was removed and placed onto paper towel.
Kidneys, liver, heart, and stomach (opened and pinned) were taken out
and placed in formalin (10% neutral buffer saline; NBF) for
histopathology. GI was sectioned in duodenum, jejunum, ileum, and
colon and opened up longitudinally. One section at a time was washed
with clean cold PBS (∼300 mL) in a glass beaker (a separated glass
beaker for each mouse). The tissue was placed onto a black-coated cork
board and flattened out (proximal at top, lumen surface facing
upwards). Approximately 0.5−1.0 cm of the proximal portions of each
tissue were cut and placed into separate reweighed Eppendorf tubes.
The remainder was photographed. Fresh PBS was squirted over top of
sections to keep them from drying out. These were imaged using a PET
scanner20 min static scan on an ice pack/cold cork board. During
scan, instruments were cleaned with PBS and EtOH, wiped dry.
Contents of the glass beakers were placed in large plastic containers, as
all radioactive ([18F]) was kept for 24 h until it had decayed (10× half-
life). Beakers were washed briefly with water, wiped, rinsed again, and
wiped dry. After scanning, sections on board were carefully blotted to
remove PBS, and formalin was added onto the sections (15−20 mL
10% NBF). After 3 h, each GI tract portion was swiss-rolled, pinned,
and placed into a large white cassette and into formalin. Analyses were
performed in R, version 3.3.2. ANOVA, adjusted for multiple
comparisons in a linear regression model.
In Vivo Polyp Growth/Formation Inhibition within the GI-
Tract of 4−5 weeks of Age Apcmin/+ Mice in a Prophylactic
Setting. Fifty 4- to 5-week-old female C57BL/6J-ApcMin/J mice were
sourced from JAX labs and acclimated for 5−7 days. Mice were group
housed in IVC-cages under a 12 h light: dark cycle at a temperature of
19 to 22 °C. Mice were fed an autoclaved powdered diet laboratory
chow (5008) and water ad libitum. Mice were ear-tagged and tailed
tattooed, which will be placed 5−7 days prior to the start of the study to
identify each animal. The spontaneous GI-tract tumor Apcmin (C57BL/
6J-ApcMin/J) was used that was approximately 5+ weeks of age. Day 1
(start of experiment), diet was changed to chow containing testing
compound for 84 days (12 weeks, with hoppers changed/topped off a
minimum of 1× weekly). Body weights and food intake were measured
at a minimum once weekly. On day 83, mice were fasted overnight for
12+ hour (for [18F]-FDG imaging). On day 84 (ex vivo imaging), for
the [18F]-FDG imaging, mice were euthanized with CO2, with death
confirmed by no response of paw-pinch reflex. Cardiac puncture was
carried out to draw blood, which was transferred into EDTA-containing
tubes that were placed onto a rocker for 10 min. Mice were opened up
and GI-tract was removed and placed onto paper towel. Kidneys, liver,
heart, and stomach (opened and pinned) were taken out and placed in
formalin (10% neutral buffer saline; NBF) for histopathology. GI was
sectioned in duodenum, jejunum, ileum and colon, and opened up
longitudinally. One section at a time was washed in clean cold PBS
(∼300 mL) in a glass beaker (separated glass beaker for each mouse).
The tissue was placed onto a black-coated cork board and flattened out
(proximal at the top, lumen surface facing upwards). Approximately
0.5−1.0 cm of the proximal portions of each tissue (duodenum,
jejunum, ileum, and colon) were cut and placed into separate reweighed
Eppendorf tubes. The remainder was photographed. Fresh PBS was
squirted over top of sections to keep them from drying out. These were
imaged using a PET scanner20 min static scan on ice pack/cold cork
board. During scan, instruments were cleaned with PBS and EtOH, and
wiped dry. Contents of the glass beakers were placed in large plastic
containers, as all radioactive ([18F]) was kept for 24 h until it had
decayed (10 × half-life). Beakers were briefly washed with water, wiped,
rinsed again, and wiped dry. After scan, sections on board were carefully
blotted to remove PBS, and formalin was added onto of sections (15−
20 mL 10% NBF). After 3 h, each GI tract portion was swiss-rolled,
pinned, and placed into a large white cassette and into formalin.
Weight and tumor assessment: body weights were measured on a
balance a minimum of once weekly. Mice were monitored daily for
clinical signs of toxicity for the duration of the treatment. A sustained
LC−MS/MS analysis: Accela HPLC coupled to LTQ-Orbitrap XL
and operated in a positive ESI mode. Column: Betasil PHENYL-
HEXYL (100 × 2.0 mm ID, 3 μm). Mobile phase A: 0.1% formic acid;
mobile phase B: acetonitrile containing 0.1% formic acid. A nonlinear
gradient with a flow rate of 0.2 mL/min was used throughout the
analysis. Compound 11 underwent extensive metabolism in both
species. The turnover at a 10 μM substrate concentration was 79.6%
and ∼100% in mouse and human hepatocytes, respectively. The
percentages of compound 11 and its derived metabolites in the
incubation extracts were obtained based on the peak areas in LC/UV
chromatograms.
Uptake of [18F]-FDG in Established Polyps/Adenomas
Present in the GI-Tract of Aged Apcmin/+ Mice Using Ex Vivo
PET Imaging. The uptake of [18F]-FDG by tissues is a marker for the
tissue uptake of glucose, which in turn is closely correlated with certain
types of tissue metabolism. After [18F]-FDG is injected into a patient
(or animal), a PET scanner can form two-dimensional or three-
dimensional images of the distribution of [18F]-FDG within the body.
The spontaneous GI-tract tumor Apcmin (C57BL/6J-ApcMin/J) was
used that was approximately 16+ weeks of age. All mice were
individually housed in IVC cages with 2 oz glass food hoppers (with
metal food follower and lids) containing standard diet 5008
(powdered/meal form) and ad libitum water 4−5 days prior to the
start of experiment. Day 1 (start of experiment), diet was changed to
chow containing testing compound for 14 days (changed/topped off a
minimum of 1× weekly). Body weights and food intake were measured
at a minimum once weekly (more often if possible). Day 13, mice were
fasted overnight for 12+ hour (for [18F]-FDG imaging). Day 14 (ex vivo
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body weight loss greater than 15% of the initial body weight was
considered as clinical toxicity, with the animal being humanely
sacrificed. Tumor growth inhibition (TGI) values were determined
from total ileum polyp area (sum of all polyp areas with diameter > 1
mm) following a daily oral dosing of 300 mg/kg as a suspension for 10−
12 weeks, % TGI = 1 − (Tt/Ct), Tt = median tumor volume of treated at
time t, and Ct = median tumor volume of control at time t. Analyses
were performed in One-way ANOVA with Dunnett’s Multiple
comparisons test; or Two-way ANOVA with the Dunnett’s Multiple
Comparisons test using GraphPad Prism 7.0..
Gaochao Tian − Discovery Technology and Molecular
Pharmacology, Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
Javier Suarez − Discovery Technology and Molecular
Pharmacology, Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
William G. Bonnette − Discovery Technology and Molecular
Pharmacology, Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
Vineet Pande − Discovery Chemistry, Janssen Research and
Development, B-2340 Beerse, Belgium
ASSOCIATED CONTENT
■
Karen A. Diloreto − Drug Metabolism and Pharmacokinetics,
Janssen Research and Development, Spring House,
Pennsylvania 19477, United States
Yifan Shi − Drug Metabolism and Pharmacokinetics, Janssen
Research and Development, Spring House, Pennsylvania
19477, United States
Shefali Patel − Drug Metabolism and Pharmacokinetics, Janssen
Research and Development, Spring House, Pennsylvania
19477, United States
Beth Pietrak − Discovery Technology and Molecular
Pharmacology, Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00890.
Molecular modeling; molecular formula strings (PDB)
(CSV)
Assays and protocols: chemistry; metabolite identifica-
tions; PK; UGT phenotyping; glucuronidation Km
determination; survival extension; plots for derived free
unbound fraction (fu %) in vivo potency; and HPLC
traces of compounds 10, 11, 20, 21, 22, and 23 (PDF)
Lawrence Szewczuk − Discovery Technology and Molecular
Pharmacology, Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
Carlo Sensenhauser − Drug Metabolism and
Pharmacokinetics, Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
Shannon Dallas − Drug Metabolism and Pharmacokinetics,
Janssen Research and Development, Spring House,
Pennsylvania 19477, United States
James P. Edwards − Discovery Chemistry, Janssen Research and
Development, Spring House, Pennsylvania 19477, United
States
AUTHOR INFORMATION
■
Corresponding Authors
Zhuming Zhang − Discovery Chemistry, Janssen Research and
Development, Spring House, Pennsylvania 19477, United
States; Present Address: Deerfield Discovery &
Development, New York, NY, USA; orcid.org/0000-
0002-5638-5320; Email: zzzhang_3000@yahoo.com
David C. Evans − Drug Metabolism and Pharmacokinetics,
Janssen Research and Development, Spring House,
Pennsylvania 19477, United States; Email: DCEvans@
its.jnj.com
Kurtis E. Bachman − Oncology Discovery, Janssen Research and
Development, Spring House, Pennsylvania 19477, United
States
Authors
Avijit Ghosh − Drug Metabolism and Pharmacokinetics,
Janssen Research and Development, Spring House,
Pennsylvania 19477, United States; Present Address: Amgen
Corporation, South San Francisco, CA, USA.
Peter J. Connolly − Discovery Chemistry, Janssen Research and
Development, Spring House, Pennsylvania 19477, United
States
Peter King − Drug Metabolism and Pharmacokinetics, Janssen
Research and Development, Spring House, Pennsylvania
19477, United States; Present Address: Immuneering
Corporation, San Diego, CA, USA.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00890
Author Contributions
This manuscript was written through contributions of all
authors. Z.Z., A.G., and P.K. contributed to this work while
employed at Janssen Research and Development.
Notes
The authors declare no competing financial interest.
Thomas Wilde − Drug Metabolism and Pharmacokinetics,
Janssen Research and Development, Spring House,
Pennsylvania 19477, United States
Jianyao Wang − Drug Metabolism and Pharmacokinetics,
Janssen Research and Development, Spring House,
Pennsylvania 19477, United States; orcid.org/0000-0003-
2559-8849
Yawei Dong − Chemistry, Pharmaron Beijing, Co. Ltd., Beijing
100176, P. R. China
Xueliang Li − Chemistry, Pharmaron Beijing, Co. Ltd., Beijing
100176, P. R. China
ACKNOWLEDGMENTS
■
The authors would like to thank Christopher Teleha (Janssen
Research and Development) for his valuable insights into
chemistry, and Karine Smans (Janssen Research and Develop-
ment), Heather Murrey (Janssen Research and Development),
and Kay Ahn (Janssen Research and Development) for their
valuable insights into biology. We are also grateful to Wenqiong
Wu (Pharmaron, Beijing, China) for her contributions to the
synthesis of compounds described herein.
ABBREVIATIONS
Daohong Liao − Chemistry, Pharmaron Beijing, Co. Ltd.,
Beijing 100176, P. R. China
Hao Chen − Chemistry, Pharmaron Beijing, Co. Ltd., Beijing
100176, P. R. China
■
COX, cyclooxygenase; NSAID, nonsteroidal anti-inflammatory
drug; CRC, colorectal cancer; FDG, fluorodeoxyglucose; PET,
positron emission tomography; PD, pharmacodynamics; APC,
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adenomatous polyposis coli; MIN, multiple intestinal neoplasia;
IVIVC, in vitro−in vivo correlation; FAP, familial adenomatous
polyposis; PG, prostaglandin; PGE2, prostaglandin E2; RR,
relative risk; CV, cardiovascular; PGI2, prostacyclin; PDB,
Protein Data Bank; ER, endoplasmic reticulum; HEK, human
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simulated intestinal fluid; ADME, absorption, distribution,
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MLM, mouse liver microsomes; MDCK-MDR1, Madin Darby
canine kidney (MDCK) cells with the MDR1 gene (ABCB1);
ADHP, 10-acetyl-3,7-dihydroxyphenoxazine; UGT, UDP-glu-
curonosyltransferases; PV, portal vein; HWB, human whole
blood; PPB, plasma protein binding; LPS, lipopolysaccharide;
DMSO, dimethyl sulfoxide; NBS, N-bromosuccinimide; ACN
or MeCN, acetonitrile; AcOH, acetic acid; XPhos Pd G3, (2-
dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-
(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate;
Cy2NMe, N,N′-dicyclohexyl-carbodiimide; ESI, electrospray
ionization; LiHMDS, lithium bis(trimethylsilyl)amide; EtOAc,
ethyl acetate; DCM, dichloromethane; Red-Al, sodium bis(2-
methoxyethoxy)aluminum hydride, boron trichloride; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; CHCl3, chloroform;
Pd(dppf)Cl2, [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II); MeOH, methanol; Pd(Ph3P)4, tetrakis-
(triphenylphosphine)palladium(0)
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