Improving the pharmacokinetic and CYP inhibition profiles of azaxanthene-based glucocorticoid receptor modulators-identification of (S)-5-(2-(9-fluoro-2-(4-(2-hydroxypropan-2-yl)phenyl)-5 H -chromeno[2,3- b ]pyridin-5-yl)-2-methylpropanamido)-N-(tetrahydr

Article abstract of DOI:10.1021/acs.jmedchem.5b00257

An empirical approach to improve the microsomal stability and CYP inhibition profile of lead compounds 1a and 1b led to the identification of 5 (BMS-341) as a dissociated glucocorticoid receptor modulator. Compound 5 showed significant improvements in pha

Full text of DOI:10.1021/acs.jmedchem.5b00257

Article
pubs.acs.org/jmc
Improving the Pharmacokinetic and CYP Inhibition Profiles of
Azaxanthene-Based Glucocorticoid Receptor
ModulatorsIdentification of (S)‑5-(2-(9-Fluoro-2-(4-(2-
hydroxypropan-2-yl)phenyl)‑5H‑chromeno[2,3‑b]pyridin-5-yl)-2-
methylpropanamido)‑N‑(tetrahydro‑2H‑pyran-4-yl)-1,3,4-thiadiazole-
2-carboxamide (BMS-341)
Michael G. Yang,* T. G. Murali Dhar,* Zili Xiao, Hai-Yun Xiao, James J.-W. Duan, Bin Jiang,
Michael A. Galella, Mark Cunningham, Jinhong Wang, Sium Habte, David Shuster, Kim W. McIntyre,
Julie Carman, Deborah A. Holloway, John E. Somerville, Steven G. Nadler, Luisa Salter-Cid,
Joel C. Barrish, and David S. Weinstein
Research and Development, Bristol-Myers Squibb Company, Princeton, New Jersey 08543-4000, United States
S
* Supporting Information
ABSTRACT: An empirical approach to improve the microsomal stability and CYP inhibition profile of lead compounds 1a and
1b led to the identification of 5 (BMS-341) as a dissociated glucocorticoid receptor modulator. Compound 5 showed significant
improvements in pharmacokinetic properties and, unlike compounds 1a−b, displayed a linear, dose-dependent pharmacokinetic
profile in rats. When tested in a chronic model of adjuvant-induced arthritis in rat, the ED50 of 5 (0.9 mg/kg) was superior to that
of both 1a and 1b (8 and 17 mg/kg, respectively).
induction via GR requires a ∼6-fold higher concentration of
dex than gene repression.11 These studies suggested the
possibility of identifying GR agonists that could achieve a
dissociated pharmacological profile retaining the beneficial anti-
inflammatory effects while minimizing the side effect profile
due to TA.12,13 We recently described the discovery of
azaxanthene-based azole amides (1a) as partially dissociated
GR modulators (Figure 1).14 In our study, cellular assays of
transrepression (AP-1) and transactivation (NP-1) were used
to evaluate GR ligand-dependent activities. The azole amide 1a
displayed excellent transrepression activity (AP-1 EC50 18 nM)
while partial agonism of TA (NP-1 EC50 99 nM, Ymax 57%
relative to the efficacy of dex as 100%) was maintained. Both 1a
and 1b suffered from the risk of clinical drug−drug interactions
(DDI) and nonlinear pharmacokinetics, with greater than dose
proportional increases in exposure. Observed in preclinical
studies, this latter risk brings with it the risk of overexposing
patients to drug with only a small escalation in dose. Both
issues are believed to have a common mechanistic under-
INTRODUCTION
■
Glucocortocoids, such as prednisolone (pred) and dexametha-
sone (dex), have been used for the treatment of autoimmune
and inflammatory diseases for over 60 years.1 Conformational
changes induced on binding of glucocorticoids to the
glucocorticoid receptor (GR) result in its translocation to the
nucleus, where it exerts its transcriptional activity either by
transactivation (TA) or transrepression (TR) pathways. In the
TR pathway, the direct physical contact of the GR with
transcription factors NFκB and AP-1 leads to the repression of
major downstream proinflammatory factors, including proin-
flammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) and
chemokines (e.g., CCL2, CCL19), leading to the observed anti-
inflammatory effects with glucocorticoids.2−5 However, long-
term use of glucocortocoids results in a number of undesirable
side effects, including diabetes/hyperglycemia,6 and osteopo-
rosis, which are attributed to the TA pathway.7,8 As full agonists
of GR, pred and dex activate both TR and TA pathways.
Studies using transgenic GR dimerization-deficient mice have
shown that the transactivation activities (DNA binding) of GR
could be separated from the transrepressive effects (non-DNA
binding) of GR.9,10 In addition, it has been shown that gene
Received: February 18, 2015
© XXXX American Chemical Society
A
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thiadiazole ring in the form of carboxamide may significantly
improve the CYP inhibition profile for this series of
compounds, as outlined in 2−5 (Figure 1). In this paper, we
describe the syntheses, structure−activity relationships (SAR),
in vitro profiles, and in vivo findings associated with 2−5.
In the current study, assays used to evaluate biological
activities of GR ligands, including nuclear receptor binding,
transrepression in an A549 cell line (AP-1 assay), and
transactivation in a HeLa cell line (NP-1 agonist assay), have
been previously reported.14,16,17 The ideal activity profile of a
dissociated GR agonist would be a compound that is a partial
agonist in the in vitro functional assays for transrepression and
one that shows little to no activity in the transactivation
assay.16,18 However, compounds with a low Ymax in the
transrepression AP-1 assay also show reduced potency in the
human whole blood (hWB) assay. On the other hand, if the
efficacy (Ymax value) is too high in the AP-1 transrepression
assay (90−100%), the compound typically brings in potent
transactivation activity (NP-1 assay).19,20 To achieve the goal of
finding a balanced and dissociated GR modulator, we screened
and progressed compounds with a good partial agonist
transrepression activity, i.e. EC50 <50 nM and an agonist
efficacy (Ymax) in the range of 55−85% when normalized to
dexamethasone (100%).
Figure 1. Synthetic targets of thiadiazole carboxamides.
pinning, namely, the inhibition of cytochrome P450 (CYP450)
enzymes. Since these enzymes are responsible for oxidative
metabolism of 1a and 1b, they represent the clearance
mechanism that becomes saturated such that systemic exposure
increases nonlinearly once hepatic exposures are above the Km
for the metabolizing enzymes. The potential for DDI is a
reflection of the requirement of uninhibited CYP450 for the
clearance of most xenobiotics. Making matters more difficult,
both compounds suffered from short half-lives in microsomal
incubations at concentrations likely below the Km for the
metabolizing enzyme(s) (i.e., below saturation).15
We suspected that the metabolic liability of 1a and 1b may
be due to the amide residing on the fluorophenyl ring. Indeed,
biotransformation studies confirmed N-demethylation as the
major metabolic pathway. To improve the metabolic stability
profile and suppress the N-demethylation process, we decided
to synthesize cyclic amides and nonamide functional groups off
the fluorophenyl ring (R3, Figure 1). Although the factor(s)
contributing to the CYP inhibition profile were less clear at this
point, we reasoned that substitution at the 2-position of the
RESULTS AND DISCUSSION
■
A summary of the SAR for compounds 1−5 is shown in Table
1. With the goal of improving metabolic stability, we began our
study with the examination of methyl-substituted thiadiazole
compounds 1c and 1d. Although both compounds had similar
potencies in the transrepression assay (AP-1 EC50 17 vs 27
nM), the fluorine-substituted analog 1d was 3 times more
a
Table 1. Azaxanthene-Based Substituted Thiadiazole Amides
b
c
structure
R
AP-1 repression
EC50
NP-1 agonism
hWB LPS/TNF
EC50
d
e
EC50
(nM)
metabolic stability : % remaining
d
d
compd
X
GR binding: Ki (nM)
(nM)
% dex
% dex
(nM)
% dex
(h/r/m)
dex, 1e
1a
1c
1.4
1.6
5.2
4.3
7.8
4.7
4.1
5.1
3.8
2.9
3.9
2.8
4.6
2.6
1.0
3
18
17
27
52
44
34
54
91
72
42
50
84
24
18
100
79
87
69
71
86
68
77
53
66
59
60
59
86
84
5
99
100
57
50
54
61
62
37
32
27
39
29
50
45
64
53
7
379
100
91
27/24/25
47/65/57
57/62/40
70/79/87
81/84/94
74/56/99
82/71/93
81/47/55
−/−/−
H
F
47
1541
509
112
103
92
1d
2a
2b
2c
107
583
305
486
328
618
376
418
362
584
64
H
H
F
MeNH
6476
1273
517
c-PrNH
MeNH
c-PrNH
NH2
82
83
2d
3a
3b
3c
F
719
84
629
51
MeNH
NMe2
2025
58
50/57/45
68/78/89
−/−/−
3d
3e
4
EtNH
11160
2195
505
112
101
91
c-PrNH
99/81/100
5
102
268
84
92/87/95
a
b
Values are means of two or more experiments performed in triplicate. Activation protein (AP-1) assay was performed in an A549 lung epithelial
c
d
line. GR transactivation NP-1 assay (run in agonist mode) was performed in the HeLa cell line. Efficacy represented as percentage of the maximal
response of dexamethasone (100%). See ref 15.
e
B
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potent in the human whole blood assay (hWB EC50 509 vs
1541 nM). The metabolic stability profiles of 1c and 1d were
improved relative to that of the unsubstituted thiadiazole 1a
(Table 1). Next, we examined a series of compounds (2a−d)
with a 2-substituted carboxamide off the 5-amino-1,3,4-
thiadiazole ring. Noticeably, the metabolic stability profile of
these compounds was further improved compared to that of the
methyl-substituted thiadiazoles 1c,d. Once again, the C-2
fluorinated compounds 2c and 2d exhibited a better human
whole blood activity than the des-fluoro analogs (Table 1, 2a vs
2c and 2b vs 2d).
Changing the morpholine benzamide to a pyrrolidine
resulted in compounds 3a−e. In general, the pyrrolidine
benzamides 3a−e had moderate stability in the liver microsome
assay and exhibited weak potency in the human whole blood
assay; for example, a 4-fold decrease in human whole blood
activity was observed for 3b vs 2c (hWB EC50: 2c, 517 nM vs
3b, 2025 nM; Table 1). However, other structural changes
around 2 proved fruitful. For example, installation of a fluorine
at the C-9 position of the azaxanthene ring of 2b led to the
fluorinated analog 4, which was potent in the transrepression
assay (AP-1 EC50 24 nM, Ymax 86%) while a partial dissociated
profile (NP-1 EC50 64 nM, Ymax 64%) was retained. In addition,
the additional fluorine group at the C-9 position of 4 improved
the human whole blood activity vs 2b, while the in vitro
metabolic stability profile was maintained. The human whole
blood profiles of the fluoro analogs 2d and 4 are similar,
although the in vitro metabolic stability profile is marginally
better for 4 (Table 1).
Further structural changes led to the discovery of the tertiary
alcohol 5. Specifically, compound 5 was potent in the
transrepression assay (AP-1 EC50 18 nM, Ymax 84%), while a
partial dissociated profile (NP-1 EC50 102 nM, Ymax 53%) was
maintained. In addition, the tertiary alcohol 5 showed excellent
human whole blood potency (EC50 268 nM) and a promising
in vitro metabolic stability profile (Table 1).
On the basis of the in vitro data outlined in Table 1,
compounds 4 and 5 were clearly the front runners, since they
had potent human whole blood activity, weaker NP-1 activity,
and favorable metabolic stability. Therefore, these compounds
were profiled further for NHR selectivity, CYP inhibition, and
half-life (T1/2) determination in liver microsomes (Table 2).
Both 4 and 5 showed potent GR binding affinity and good
selectivity over AR, PR, and MR.21 Tertiary alcohol 5 showed
improved CYP450 inhibition over compound 4, particularly
against the 2C19 isoform. Furthermore, when midazolam was
used as the substrate, no inhibitory activity for CYP 3A4 was
observed (IC50 > 40 μM), after incubating compounds 4 and 5
for over 30 min. The half-life of 5 upon incubation with human
liver microsomes was similar to that of 4. However, the rat and
dog liver microsomes half-lives of 5 were about 5 and 8 times
better than those of 4 (Table 2). In light of their promising in
vitro pharmacologic profile, the morpholine amide 4 and the
tertiary alcohol 5 were selected for evaluation in in vivo PK and
efficacy models.
The pharmacokinetic properties of the morpholine amide 4
and the tertiary alcohol 5 are summarized in Table 3. A low
clearance (4.8 mL/min/kg) was recorded for 4 after a single 0.5
mg/kg intravenous dose and a bioavailability of 50% was
determined after a single 10 mg/kg oral dose of a solution of 4
to rats. Notably, and consistent with the improved CYP450
inhibition profile, a linear dose-dependent PK profile (both in
terms of Cmax and AUC) was observed in rats when 4 was
dosed at 30 mg/kg orally (Table 3). The pharmacokinetics of 4
was also evaluated in dogs. Given the poor microsomal stability
of 4 in the dog (Table 2), it was not surprising that low
exposures were recorded in the oral leg of the study, and the
related iv study was not pursued (Table 3). For compound 5,
low clearance (2.7 mL/min/kg) and moderate volume of
distribution (1.0 L/kg) were determined after a single 1 mg/kg
intravenous dose administered to rats. At oral doses of 1 and 10
mg/kg, 5 was rapidly absorbed and displayed linear dose-
dependent increase in AUC with an oral bioavailability of 44−
51% (Table 3). The pharmacokinetic study of 5 in dogs
revealed low clearance (1.7 mL/min/kg) after a single 1 mg/kg
intravenous dose, and high exposures with 38% oral
bioavailability were seen after a single 5 mg/kg oral dose.
The efficacy of 4 and 5 in vivo was evaluated in a chronic
model of adjuvant-induced arthritis (Figure 2). Male Lewis rats
were first inoculated with Mycobacterium butyricum in
incomplete Freund’s adjuvant, and then both compounds
were dosed preventatively from day 0 to day 21 (q.d. dosing).
Prednisolone (5 mg/kg) served as the positive control in the
study. A dose-dependent reduction in the onset and
progression of arthritis was observed for both compounds.
The ED50 values of 4 and 5 were found to be 4.6 and 0.9 mg/
kg, respectively. The potency for 5 compares well to that which
had previously been measured for pred (ED50 1.0 mg/kg). The
greater level of efficacy achieved with 5 relative to the standard
dose of pred is notable.
a
Table 2. General Profiles of 1a, 4, and 5
1a
4
5
GR binding Ki, nM
PR binding Ki, nM
AR binding Ki, nM
MR agonist EC50, nM
1.6
2.6
1.0
Table 4 provides a head-to-head comparison of compound 5
with earlier lead compounds 1a and 1b.14 As illustrated in
Table 4, the human whole blood potency, CYP inhibition
profile, and pharmacokinetic properties of compound 5 are
significantly superior to those of compounds 1a and 1b. More
importantly, the improved in vitro potency and PK properties
of 5 translated to a lower ED50 value in the rat AA (adjuvant-
induced arthritis) model.
>1800
>5000
>5000
18 (79)
>4000
>2000
>5000
>5000
18 (85)
−
b
>5000
24 (86)
AP-1 repression EC50, nM
(% dex)
NP-1 agonism EC50, nM (% dex) 99 (57)
64 (64)
102 (53)
268 (84)
hWB LPS/TNF EC50, nM
(% dex)
379 (91)
505 (91)
To understand the potential in vivo benefits of an improved
side effect profile relative to steroids in humans, both 4 and 5
were evaluated in cellular assays of gene products associated
with targets of glucocorticoid-mediated side effects, including
bone-specific alkaline phosphatase (ALP) and glutamine
synthetase (GS). ALP is a marker of osteoblast differentiation22
and glutamine synthetase is a marker of muscle wasting.23 In
the SAOS-2 osteosarcoma cell line, pred was found to induce
human/rat/dog LM T1/2, min
CYP IC50, μM
4.6/5.6/5.3
43/57/6
59/196/48
3A4/2C8
0.4/0.9
2.2/0.5
40/−
3.9/0.5
27/3.2
6.5/7.9
2C9/2C19
a
b
Values are means of at least two experiments. In A549 cell line, EC50
(nM)/(%maximal efficacy) determination (aldosterone as positive
control).
C
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Table 3. Rat and Dog Pharmacokinetic Properties of 4 and 5
compd/species/route/dose
Cmax (nM)
Tmax (h)
AUC0−24 (nM h)
T1/2 (h)
Cl (mL/min/kg)
4.8
Vss (L/kg)
0.3
F (%)
4/rat/iv/0.5 mg/kg
4/rat/po/10 mg/kg
4/rat/po/30 mg/kg
4/dog/po/5 mg/kg
5/rat/iv/1 mg/kg
5/rat/po/1 mg/kg
5/rat/po/10 mg/kg
5/dog/iv/1 mg/kg
5/dog/po/5 mg/kg
2758
27608
100736
370
0.9
8980
23454
206
1.0
2.0
0.8
50
45
9623
4.7
7.9
2.7
1.7
1.0
1.1
625
2.0
1.0
4885
51
44
5345
42071
13643
25566
2883
2.0
38
ALP (68% above background) after a 3-day incubation (Table
5). However, 4 and 5 were significantly less efficacious than
Table 5. Alkaline Phosphatase and Glutamine Synthetase
Induction by Pred, 4, and 5
hGS induction
a
ALP induction
compd
% max SD (n)
EC50 SD, nM (n) % pred SD
pred, 1f
68 16 (31)
20 8 (2)
19 11 (25)
15 3.6 (3)
13 4.8 (7)
100 17
73 17
78 15
4
5
21 21 (10)
a
Induction measured after 3 days of induction at 600 nM.
pred, and the rank order for efficacy was consistent with that
observed in the reporter assays. Similarly, in the MG-63
osteosarcoma cell line, less efficacy than pred for induction of
GS was observed for 4 and 5 (73% and 78% of pred), with a
similar level of potency. Although a direct relationship between
glucocorticoid-mediated induction of gene expression and side
effects in bone and muscle has not been established, the lower
level of transactivation mediated by 4 and 5 suggests that they
will have a differentiated profile compared to prednisolone.
CHEMISTRY
Figure 2. Compounds 4 (top), 5 (bottom), and pred in the rat
adjuvant-induced model of arthritis.
■
The synthetic approach for the preparation of the key
intermediate 16 is outlined in Scheme 1. 2-Chloronicotinic
acid was condensed with 2-fluorophenol (7) using sodium
phenoxide to give the corresponding phenoxypyridine acid 8 in
65% yield. Intramolecular Friedel−Crafts cyclization of
phenoxypyridine acid 8 gave excellent yields of the
azaxanthenone 9. Sodium borohydride reduction of 9 provided
the bis benzyl alcohol 10, which was subjected to a modified
Mukaiyama aldol reaction with the silylketene acetal 11 to
provide the ester 12 in high yields. Oxidation of 12 with
mCPBA gave the N-oxide 13, which on reaction with POCl3
provided the methyl ester 14. Saponification of 14 provided the
acid 15, which was subjected to chiral supercritical fluid
chromatography (SFC) to provide the homochiral acid 16. The
(S) absolute stereoconfiguration of 16 was subsequently
confirmed by an X-ray crystal structure of 5 (vide infra).24
The synthetic procedure for the preparation of tertiary
alcohol 5 is described in Scheme 2. Suzuki−Miyaura coupling
of chloride 16 with aryl boronic acid 17 gave the corresponding
ketone, which on treatment with methylmagnesium bromide in
THF provided the tertiary alcohol 18 in 98% yield (two steps).
The HOBt-promoted coupling reaction between the acid 18
and the amine 19 yielded the ethyl ester 20 in 39% yield. It is
worth mentioning that the coupling reaction was very sluggish
at room temperature and heating was required to achieve the
Table 4. Comparison of Azole Amide 5 to the Previous
Leads 1a and 1b
a
1a
1b
5
GR binding Ki, nM
AP-1 EC50, nM (% dex)
NP-1 EC50, nM (% dex)
hWB EC50, nM (% dex)
CYP IC50, μM
1.6
1.9
1.0
18 (79)
99 (57)
379 (91)
33 (70)
17.9 (85)
102.2 (53)
268 (84)
242 (32)
830 (83)
3A4/2C8
0.4/0.9
2.2/0.5
34.0/0.4
0.6/0.5
4.2/1.3
49.5/0.6
27/3.2
6.5/7.9
2.7/4.7
2C9/2C19
rat/iv/1 mg/kg:
Cl (mL/min/kg)/T1/2 (h)
rat/po/10 mg/kg:
904/17
8
673/20
17
42071/44
0.9
AUC
(nM h)/F (%)
0−24
rat AA ED50, mg/kg
a
Values are means of at least two experiments.
D
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a
properties and revealed a linear dose-dependent PK profile in
rats (Table 3). Furthermore, the ED50 of 5 in the rat adjuvant-
induced arthritis study was 0.9, which is superior to that of both
1a and 1b. The utility of partial agonists of GR such as 5 in
treating human disease while improving on the safety profile of
traditional glucocorticoids needs to be further evaluated in a
clinical setting.
Scheme 1
EXPERIMENTAL SECTION
■
Chemistry. All commercially available chemicals and solvents were
used without further purification. Reactions are performed under an
atmosphere of nitrogen. All new compounds gave satisfactory 1H
NMR, LC/MS and/or HRMS, and mass spectrometry results. 1H
NMR spectra were obtained on a Bruker 400 MHz or a JEOL 500
MHz NMR spectrometer using the residual signal of deuterated NMR
solvent as internal reference. Electrospray ionization (ESI) mass
spectra were obtained on a Water Micromass ESI-MS single
quadrupole mass spectrometer. High-resolution mass spectral analysis
was performed on an LTQ-FT mass spectrometer interfaced to a
Waters Acquity ultraperformance liquid chromatography. The purity
of tested compounds determined by analytical HPLC was >95%,
except as noted. Analytical HPLC was performed on a Shimadzu
instrument with a YMC Combiscreen ODS-A 4.6 mm × 50 mm
column using a gradient elution of 0−100% B/A over 4 min with 1
min hold (solvent A, 90% water/10% MeOH/0.2% H3PO4; solvent B,
90% MeOH/10% water/0.2% H3PO4), a flow rate of 4 mL/min, and
220 or 254 nm as the detection wavelength, except as noted.
a
Reagents and conditions: (a) step 1, NaOMe; step 2, 168 °C, 3 h,
65%. (b) Step 1, polyphosphoric acid; step 2, 160 °C, 8 h, 97%. (c)
NaBH4, MeOH, 96%. (d) TiCl4, CH2Cl2, 0 °C, 79%. (e) mCPBA,
CH2Cl2, 25 °C, 93%. (f) POCl3, 100 °C → 25 °C, 42%. (g) 4 N aq
KOH, THF, MeOH, 65 °C, 90%. (h) Chiralcel OJ-H (5 μm), CO2/
[IPA:ACN 1:1 w 0.1% TFA] (90:10), 35 °C, 100 bar, peak 1.
a
Scheme 2
Synthesis of (S)-2-Methyl-N-(5-methyl-1,3,4-thiadiazol-2-yl)-
2-(2-(4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]-
pyridin-5-yl)propanamide (1c, Scheme 3). Step 1: Synthesis of
Scheme 3
a
Reagents and conditions: (a) Pd(PPh3)4, DMF, K3PO4 (2 M), 90 °C.
(b) MeMgBr, THF, −78 °C → 25 °C, 98% for two steps. (c) EDC,
HOBt, DIEA, DMF, 40 °C → 60 °C, 39%. (d) LiOH, THF/H2O, 25
°C. (e) Tetrahydro-2H-pyran-4-amine, BOP, DIEA, 25 °C, 96%.
(S)-2-(2-Chloro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-N-(5-
methyl-1,3,4-thiadiazol-2-yl)propanamide (22). The synthesis of
(S)-2-(2-chloro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic
acid (21) was described in ref 14.
desired transformation. Saponification of the ethyl ester 20 with
LiOH gave the corresponding acid, which on coupling with
tetrahydro-2H-pyran-4-amine provided the final compound 5
in 96% yield (two steps).
To a solution of HATU (11.02 g, 29.0 mmol) in MeCN (300 mL)
was added 21 (8 g, 26.3 mmol) and Hunig’s base (13.80 mL, 79
mmol) at room temperature. The mixture was stirred for 30 min. 5-
Methyl-1,3,4-thiadiazol-2-amine (3.34 g, 29.0 mmol) was added and
the mixture was heated to 70 °C for 4.5 h. Additional HATU (1.1 g)
and 5-methyl-1,3,4-thiadiazol-2-amine (1.6 g) were added. The
mixture was heated at 60 °C for additional 17 h. After cooling to
room temperature, the crude material was diluted with EtOAc (100
mL) and acidified with 1 M HCl (126 mL). The resulting suspension
was filtered. The white solid (2.9 g) was confirmed as pure product.
The filtrate was separated into two layers. The organic layer was
washed with water (100 mL), pH 7 buffer solution (2 × 100 mL), sat.
NaHCO3 (50 mL), and brine (50 mL), respectively. It was then dried
over MgSO4, filtered, and concentrated to give a dark brown solid. The
solid was triturated with MeOH and filtered. The solid was washed
with a small amount of MeOH to give 22 as a white solid (8.4 g, 83%
yield). 1H NMR (400 MHz, CDCl3): δ ppm 10.47 (br s, 1H), 7.66 (d,
CONCLUSIONS
■
Efforts aimed at improving metabolic liabilities and CYP
inhibition profiles of the lead chemotype 1a,b led to
compounds of the structural class 2−5 with significantly
improved pharmacokinetic profiles, while the desired dis-
sociated profile was retained. In general, adding an amide motif
at the 2-position off the 5-amino-1,3,4-thiadiazole ring was
found to be beneficial for improving microsomal stability and
CYP inhibition profiles. Our early lead compounds 1a and 1b
exhibited greater than dose-proportional increases in exposure
after oral dosing. However, we demonstrated in this study that
both 4 and 5 showed an improvement in pharmacokinetic
E
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Journal of Medicinal Chemistry
Article
J = 7.8 Hz, 1H), 7.35−7.28 (m, 2H), 7.24 (m, 1H), 7.11−7.05 (m,
2H), 4.73 (s, 1H), 2.76 (s, 3H), 1.21 (br s, 3H), 1.21 (br s, 3H). ESI-
MS: m/z 401.08 ([M + H+]). HPLC: tR = 3.88 min.
(br s, 2H), 1.13 (s, 3H), 1.08 (s, 3H). ESI-MS: m/z 477.17 ([M +
H+]). HPLC: tR = 3.91 min.
Step 3: Synthesis of (S)-2-(2-(3-Fluoro-4-(morpholine-4-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-N-(5-
methyl-1,3,4-thiadiazol-2-yl)propanamide (1d). A MeCN (1 mL)
solution of 26 (19.6 mg, 0.033 mmol, presumed TFA salt), 5-methyl-
1,3,4-thiadiazol-2-amine (18.3 mg, 0.159 mmol), HATU (37.5 mg,
0.099 mmol), and Hunig’s base (30 μL, 0.172 mmol) was stirred at 60
°C for 16 h. The crude material was purified by preparative HPLC
(gradient 70−95% solvent B in 25 min) to give 1d (18 mg, 76% yield).
1H NMR (400 MHz, CDCl3): δ ppm 7.89−7.81 (m, 2H), 7.76 (d, J =
7.8 Hz, 1H), 7.54−7.47 (m, 2H), 7.34 (d, J = 3.8 Hz, 2H), 7.29 (br s,
1H), 7.09 (dt, J = 7.7, 4.1 Hz, 1H), 4.76 (s, 1H), 3.88−3.77 (m, 4H),
3.67 (br s, 2H), 3.38 (br s, 2H), 2.77 (s, 3H), 1.25 (br s, 3H), 1.24 (br
s, 3H). ESI-MS: m/z 574.18 ([M + H+]). HPLC: tR = 3.98 min.
Synthesis of (S)-N-Methyl-5-(2-methyl-2-(2-(4-(morpholine-
4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-
propanamido)-1,3,4-thiadiazole-2-carboxamide (2a, Scheme
5). Step 1: Synthesis of (S)-2-Methyl-2-(2-(4-(morpholine-4-
Step 2: Synthesis of (S)-4-(5-(2-Methyl-1-(5-methyl-1,3,4-thiadia-
zol-2-ylamino)-1-oxopropan-2-yl)-5H-chromeno[2,3-b]pyridin-2-
yl)benzoic Acid (23). A DMF (2 mL) solution of 22 (51.5 mg, 0.128
mmol), 4-boronobenzoic acid (37.5 mg, 0.226 mmol), Pd(Ph3P)4
(21.5 mg, 0.019 mmol), and 2 M K3PO4 (320 μL, 0.640 mmol) was
degassed by N2 for 3 min. The sealed tube was then heated at 100 °C
for 16 h. The crude product was purified by preparative HPLC
(gradient 70−100% solvent B in 30 min) to give 23 (30.7 mg, 40%
yield, presumed TFA salt). 1H NMR (400 MHz, CDCl3): δ ppm
8.11−8.05 (m, 2H), 8.05−7.99 (m, 2H), 7.63−7.57 (m, 1H), 7.55−
7.50 (m, 1H), 7.40−7.31 (m, 2H), 7.22−7.10 (m, 2H), 4.51 (s, 1H),
2.79 (s, 3H), 1.29 (m, 6H). ESI-MS: m/z 487.14 ([M + H+]). HPLC:
tR = 4.08 min.
Step 3: Synthesis of (S)-2-Methyl-N-(5-methyl-1,3,4-thiadiazol-2-
yl)-2-(2-(4-(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]-
pyridin-5-yl)propanamide (1c). A MeCN (1 mL) solution of 23 (15.2
mg, 0.025 mmol, presumed TFA salt), HATU (19.6 mg, 0.052 mmol),
and morpholine (10 μL, 0.115 mmol) was stirred at rt for 16 h. The
crude material was purified by preparative HPLC (gradient 60−90%
Scheme 5
1
solvent B in 30 min) to give 1c (5.5 mg, 32% yield). H NMR (400
MHz, CDCl3): δ ppm 8.08 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 7.8 Hz,
1H), 7.54 (dd, J = 9.4, 7.9 Hz, 3H), 7.38−7.29 (m, 2H), 7.16−7.07
(m, 2H), 4.54 (s, 1H), 3.93−3.42 (m, 8H), 2.76 (s, 3H), 1.27 (s, 3H),
1.23 (s, 3H). ESI-MS: m/z 556.19 ([M + H+]). HPLC: tR = 3.88 min.
Synthesis of (S)-2-(2-(3-Fluoro-4-(morpholine-4-carbonyl)-
phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-N-(5-meth-
yl-1,3,4-thiadiazol-2-yl)propanamide (1d, Scheme 4). Step 1:
Scheme 4
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)propanoic Acid
(28). A mixture of 21 (150 mg, 0.494 mmol), 4-(morpholine-4-
carbonyl)phenylboronic acid (27) (232 mg, 0.988 mmol), 2 M
aqueous solution of potassium phosphate (1.728 mL, 3.46 mmol), and
DMF (5 mL) was bubbled with nitrogen for 5 min before
tetrakis(triphenylphosphine)palladium(0) (57.1 mg, 0.049 mmol)
was added. The mixture was bubbled with nitrogen for an additional
5 min. The reaction mixture was stirred at 90 °C under nitrogen for 3
h and then before being concentrated under reduced pressure. The
solid residue was mixed with 1:1 ethyl acetate/heptane mixture (6
mL). The mixture was extracted with water (3 × 2 mL). The
combined aqueous solutions were decolorized with active charcoal and
neutralized to pH 7 with 6 N aqueous HCl (0.8 mL) and 10% aqueous
citric acid solution. The solid was filtered, washed with water (3 × 1
mL), and dried to give 28 (200 mg, 0.436 mmol, 88% yield) as a white
Synthesis of 3-Fluoro-4-(morpholine-4-carbonyl)phenylboronic
Acid (25). A DMF (1.5 mL) solution of 4-borono-2-fluorobenzoic
acid (24) (134.3 mg, 0.730 mmol), morpholine (128 μL, 1.463
mmol), HOBT (143.5 mg, 0.937 mmol), and EDC (157.4 mg, 0.821
mmol) was stirred at room temperature for 30 min. The crude product
was purified by preparative HPLC (gradient 30−60% solvent B in 30
min) to give 25 (142 mg, 77% yield). 1H NMR (400 MHz, CDCl3): δ
ppm 8.78 (br s, 2H), 8.03−7.69 (m, 1H), 7.65−7.29 (m, 2H), 3.95−
3.30 (m, 8H). ESI-MS: m/z 254.09 ([M + H+]). HPLC: tR = 2.06 min.
Step 2: Synthesis of (S)-2-(2-(3-Fluoro-4-(morpholine-4-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropa-
noic Acid (26). A DMF (1.5 mL) solution of 21 (36.1 mg, 0.119
mmol), 25 (55.5 mg, 0.219 mmol), Pd(Ph3P)4 (17.6 mg, 0.015
mmol), and 2 M K3PO4 (300 μL, 0.600 mmol) was degassed by N2 for
3 min. The sealed tube was then heated at 100 °C for 16 h. The crude
material was purified by preparative HPLC (gradient 70−100% solvent
B in 30 min) to give 26 (32.5 mg, 46%, presumed TFA salt). 1H NMR
(400 MHz, CDCl3): δ ppm 9.66 (br s, 1H), 7.89−7.82 (m, 2H), 7.78
(d, J = 7.8 Hz, 1H), 7.58−7.50 (m, 2H), 7.40−7.28 (m, 3H), 7.20−
7.14 (m, 1H), 4.50 (s, 1H), 3.89−3.84 (m, 4H), 3.71 (br s, 2H), 3.42
1
solid. H NMR (400 MHz, CDCl3): δ ppm 0.99 (s, 3 H), 1.06 (s, 3
H), 3.28−3.85 (m, 8 H), 4.42 (s, 1 H), 7.05−7.11 (m, 1 H), 7.21−7.29
(m, 3 H), 7.44−7.50 (m, 3 H), 7.67 (d, J = 7.81 Hz, 1 H), 8.03 (d, J =
8.56 Hz, 2 H). ESI-MS: m/z 459.18 ([M + H+]). HPLC: tR = 3.10
min.
Step 2: Synthesis of 5-Amino-N-methyl-1,3,4-thiadiazole-2-
carboxamide (29). To a suspesion of ethyl 5-amino-1,3,4-
thiadiazole-2-carboxylate (19) (106 mg, 0.612 mmol) in methanol
(1.5 mL) was added methylamine solution (0.5 mL) (33 wt %) in
ethanol. The mixture was stirred at rt for 6 h, heated to 70 °C, and
then cooled. The mixture was concentrated. Lyophilization gave 29
1
(99 mg, 0.626 mmol, 100% yield) as a white solid. H NMR (400
MHz, DMSO-d6): δ ppm 8.69 (q, J = 4.8 Hz, 1H), 7.70 (s, 2H), 2.73
(d, J = 4.78 Hz, 3H). ESI-MS: m/z 159.03 ([M + H+]). HPLC: tR =
0.44 min.
F
DOI: 10.1021/acs.jmedchem.5b00257
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Journal of Medicinal Chemistry
Article
Step 3: Synthesis of (S)-N-Methyl-5-(2-methyl-2-(2-(4-(morpho-
line-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-
propanamido)-1,3,4-thiadiazole-2-carboxamide (2a). A mixture of
28 (20 mg, 0.044 mmol), PyBOP (35 mg, 0.067 mmol), DIPEA (0.03
mL, 0.172 mmol), and anhydrous DMF (0.3 mL) was stirred at rt
under nitrogen for 15 min before 29 (15 mg, 0.095 mmol) was added.
The mixture was then sitrred at 100 °C for 1 h. Purification using
reverse-phase HPLC (YMC S5 20 × 100 mm, 10 min run; solvent A,
10% MeOH:90% H2O:0.1% TFA; solvent B, 90% MeOH, 10% H2O,
0.1% TFA) gave 2a (16 mg, 0.022 mmol, 51.5% yield) as a yellow
Scheme 7
1
solid. H NMR (400 MHz, CDCl3): δ ppm 8.13 (d, J = 8.3 Hz, 2H),
7.75−7.66 (m, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.37 (t, J = 8.1 Hz, 1H),
7.30−7.22 (m, 2H), 7.18−7.09 (m, 1H), 4.63 (s, 1H), 3.83−3.47 (m,
8H), 2.97 (s, 3H), 1.19 (s, 3H), 1.16 (s, 3H). ESI-MS: m/z 599.20
([M + H+]). HPLC: tR = 3.15 min.
Synthesis of (S)-N-Cyclopropyl-5-(2-methyl-2-(2-(4-(mor-
pholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-
propanamido)-1,3,4-thiadiazole-2-carboxamide (2b, Scheme
6). Step 1: Synthesis of 5-Amino-N-cyclopropyl-1,3,4-thiadiazole-2-
(s, 1H), δ 9.12 (s, 1H), 7.89−8.05 (m, 3H), 7.64−7.66 (d, J = 7.9 Hz,
1H), 7.52−7.57 (t, J = 7.76 Hz, 1H), 7.36−7.39 (m, 1H), 7.30−7.32
(d, J = 7.96 Hz, 1H), 7.15−7.16 (m, 2H), 4.81(s, 1H), 3.66 (s, 4H),
3.55−3.56 (t, J = 4.0 Hz, 2H), 3.27−3.30 (m, 2H), 2.81−2.82 (m,
3H), 2.46 (m, 2H), 1.0−1.1 (m, 6H). ESI-MS: m/z 617.19 ([M +
H+]). HPLC: tR = 15.14 min (SunFire C18 3.5u column 4.6 × 150
mm; solvent A, 5% MeCN:95% H2O:0.05% TFA; solvent B, 95%
MeCN, 5% H2O, 0.05% TFA; gradient, 10−100% of solvent B in
solvent A over 30 min; flow rate, 1 mL/min; monitoring at 220 nm).
Synthesis of (S)-N-Cyclopropyl-5-(2-(2-(3-fluoro-4-(morpho-
line-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-
methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (2d,
Scheme 8). A procedure similar to that described in step 3 of
Scheme 6
Scheme 8
carboxamide (30). To a suspesion of 19 (100 mg, 0.577 mmol) in
methanol (1.5 mL) was added cyclopropylamine (0.3 mL, 4.28 mmol).
The mixture was stirred at rt for 6 h, heated to 70 °C, and
concentrated to 0.5 mL in total volume. After water (0.5 mL) was
added, the mixture was heated to 70 °C (clear solutioin) and cooled.
The solid was filtered, washed with cold methanol/water (1/1)
mixture, and dried to give 30 (66 mg, 0.358 mmol, 62.0% yield) as a
yellow solid. 1H NMR (400 MHz, DMSO-d6): δ ppm 8.90 (d, J = 4.5
Hz, 1H), 7.80 (s, 2H), 2.96−2.81 (m, 1H), 0.81−0.61 (m, 4H). ESI-
MS: m/z 185.04 ([M + H+]). HPLC: tR = 0.82 min.
Step 2: Synthesis of (S)-N-Cyclopropyl-5-(2-methyl-2-(2-(4-
(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-
propanamido)-1,3,4-thiadiazole-2-carboxamide (2b). To a stirred
mixture of 28 (600 mg, 1.309 mmol), 30 (723 mg, 3.93 mmol),
HOBT (200 mg, 1.309 mmol), anhydrous MeCN (15 mL), and
DIPEA (0.594 mL, 3.40 mmol) was added WSCDI (753 mg, 3.93
mmol) at rt under nitrogen. The mixture was stirred at rt under
nitrogen for 10 min and at 80 °C for 3 h. The reaction mixture was
concentrated in vacuo and mixed with water (10 mL) and ethyl acetate
(10 mL). The aqueous layer was separated and extracted with ethyl
acetate (2 × 2 mL). The combined organic solutions were dried over
Na2SO4 and concetrated in vacuo. The residue was dissolved in a
mixture of ethyl acetate, dichloromethane, and methanol (the solid
formed was filtered off). Flash chromatography purification using
ISCO (12 g silica gel column, 20−100% ethyl acetate in hexanes)
afforded 2b (480 mg) as a foam solid. 1H NMR (400 MHz, CDCl3): δ
ppm 8.13 (d, J = 8.3 Hz, 2H), 7.76−7.66 (m, 2H), 7.55 (d, J = 8.3 Hz,
2H), 7.40−7.34 (m, 1H), 7.30−7.22 (m, 2H), 7.16−7.08 (m, 1H),
4.63 (s, 1H), 3.85−3.44 (m, 8H), 2.94−2.85 (m, 1H), 1.20 (s, 3H),
1.16 (s, 3H), 0.90−0.81 (m, 2H), 0.75−0.68 (m, 2H). ESI-MS: m/z
625.22 ([M + H+]). HPLC: tR = 3.28 min.
Scheme 4 was used to prepare 2d. 1H NMR (400 MHz, DMSO-d6): δ
ppm 12.96 (s., 1H), δ 9.27 (s, 1H), 7.91−8.05 (m, 3H), 7.64−7.66 (d,
J = 7.9 Hz, 1H), 7.53−7.57 (t, J = 7.2 Hz, 1H), 7.31−7.53 (m, 2H),
7.15−7.16 (m, 2H), 7.15−7.16 (m, 2H), 4.81(s, 1H), 3.74−3.76 (m,
6H), 3.55 (s, 2H), 3.28 (s, 2H), 2.88−2.91 (m, 1H), 1.04−1.07 (m,
5H), 0.70−0.72 (m, 3H). ESI-MS: m/z 643.21 ([M + H+]). HPLC: tR
= 10.28 min (SunFire C18 3.5u column 4.6 × 150 mm; solvent A, 5%
MeCN:95% H2O:0.05% TFA; solvent B, 95% MeCN, 5% H2O, 0.05%
TFA; gradient, 10−100% of solvent B in solvent A over 15 min; flow
rate, 1 mL/min; monitoring at 220 nm).
Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)-
phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanami-
do)-1,3,4-thiadiazole-2-carboxamide (3a, Scheme 9). Step 1:
Synthesis of (S)-2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)phenyl)-
5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic Acid (32). A
mixture of 21 (800 mg, 2.63 mmol), 3-fluoro-4-(pyrrolidine-1-
carbonyl)phenylboronic acid (31) (1.7 g, 7.17 mmol), 2 M aqueous
solution of potassium phosphate (9 mL, 18.00 mmol), and DMF (24
mL) was bubbled with nitrogen for 5 min before tetrakis-
(triphenylphosphine)palladium(0) (300 mg, 0.260 mmol) was
added. The mixture was bubbled with nitrogen for an additional 5
min. The reaction mixture was stirred at 90 °C under nitrogen for 3 h
and then concentrated under reduced pressure. The residue was mixed
with 1:1 ethyl acetate/heptane mixture (30 mL). The mixture was
extracted with water (50 mL, 3 × 15 mL). The combined aqueous
solutions were decolorized with active charcoal and neutralized to pH
6−7 with 6 N aqueous HCl (4 mL) and then 10% aqueous citric acid
solution. Ethyl acetate (15 mL) was added and the mixture was stirred
for 1 h. The solid was filtered, washed with water (3 × 2 mL) and ethyl
acetate (2 × 1 mL), and dried to give 32 (1.07 g, 2.324 mmol, 88%
yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ ppm 11.42 (br
Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(morpholine-4-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-
propanamido)-N-methyl-1,3,4-thiadiazole-2-carboxamide (2c,
Scheme 7). A procedure similar to that shown in step 3 of Scheme 4
was used to prepare 2c. 1H NMR (400 MHz, DMSO-d6): δ ppm 12.92
G
DOI: 10.1021/acs.jmedchem.5b00257
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Journal of Medicinal Chemistry
Article
do)-N-methyl-1,3,4-thiadiazole-2-carboxamide (3b, Scheme
10). A procedure similar to that described in step 3 of Scheme 9
Scheme 9
Scheme 10
was used to prepare 3b. 1H NMR (400 MHz, CD3OD): δ ppm 8.01−
7.89 (m, 2H), 7.80−7.66 (m, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.44−7.33
(m, 1H), 7.32−7.22 (m, 2H), 7.19−7.08 (m, 1H), 4.64 (s, 1H), 3.63
(t, J = 6.8 Hz, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.97 (s, 3H), 2.08−1.89
(m, 4H), 1.20 (s, 3H), 1.16 (s, 3H). ESI-MS: m/z 601.20 ([M + H+]).
HPLC: tR = 3.41 min.
Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)-
phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanami-
do)-N,N-dimethyl-1,3,4-thiadiazole-2-carboxamide (3c,
Scheme 11). A procedure similar to that described in step 3 of
s, 1H), 7.89−7.80 (m, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.57−7.46 (m,
2H), 7.36−7.28 (m, 3H), 7.18−7.09 (m, 1H), 4.49 (s, 1H), 3.70 (t, J =
6.9 Hz, 2H), 3.37 (t, J = 6.7 Hz, 2H), 2.06−1.86 (m, 4H), 1.11 (s,
3H), 1.02 (s, 3H). ESI-MS: m/z 461.18 ([M + H+]). HPLC: tR = 3.41
min.
Scheme 11
Step 2: Synthesis of (S)-Ethyl 5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropa-
namido)-1,3,4-thiadiazole-2-carboxylate (33). To a stirred mixture
of 32 (400 mg, 0.869 mmol), 19 (400 mg, 2.310 mmol), HOBT (140
mg, 0.914 mmol), and anhydrous MeCN (10 mL) was added 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (340 mg,
1.774 mmol) at rt under nitrogen, followed by DIPEA (0.2 mL, 1.145
mmol). The mixture was stirred at rt for 1 h and at 80 °C for 4.5 h and
then concentrated. The residue was mixed with water (15 mL). The
mixture was extracted with ethyl acetate (15 mL, 3 × 10 mL). The
combined organic solutions were dried over Na2SO4 and concentrated.
Flash chromatography (12 g silica gel column, 20−100% ethyl acetate
in hexanes) gave 33 (411 mg, 0.668 mmol, 77% yield) as a yellow
solid. 1H NMR (400 MHz, CDCl3): δ ppm 8.05 (br s, 1H), 7.91−7.83
(m, 2H), 7.59−7.51 (m, 2H), 7.34 (d, J = 3.3 Hz, 2H), 7.24 (d, J = 7.6
Hz, 1H), 7.12−7.00 (m, 1H), 4.88 (br s, 1H), 4.58 (q, J = 7.1 Hz, 2H),
3.70 (t, J = 6.8 Hz, 2H), 3.38 (t, J = 6.3 Hz, 2H), 2.05−1.91 (m, 4H),
1.50 (t, J = 7.2 Hz, 3H), 1.37 (br s, 3H), 1.27−1.22 (m, 3H). ESI-MS:
m/z 616.20 ([M + H+]). HPLC: tR = 3.70 min.
1
Scheme 9 was used to prepare 3c. H NMR (400 MHz, CD3OD): δ
ppm 7.99−7.90 (m, 2H), 7.78−7.70 (m, 2H), 7.53 (t, J = 7.4 Hz, 1H),
7.42−7.35 (m, 1H), 7.32−7.22 (m, 2H), 7.17−7.10 (m, 1H), 4.64 (s,
1H), 3.63 (t, J = 6.9 Hz, 2H), 3.55 (s, 3H), 3.39 (t, J = 6.7 Hz, 2H),
3.18 (s, 3H), 2.08−1.90 (m, 4H), 1.20 (s, 3H), 1.16 (s, 3H). ESI-MS:
m/z 615.21 ([M + H+]). HPLC: tR = 3.44 min.
Synthesis of (S)-N-Ethyl-5-(2-(2-(3-fluoro-4-(pyrrolidine-1-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methyl-
propanamido)-1,3,4-thiadiazole-2-carboxamide (3d, Scheme
12). A procedure similar to that described in step 3 of Scheme 9 was
Step 3: Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropa-
namido)-1,3,4-thiadiazole-2-carboxamide (3a). To a stirred solution
of 33 (183 mg, 0.297 mmol) in MeOH (1.5 mL) was added 1 M
aqueous NaOH (1.040 mL, 1.040 mmol). The reaction mixture was
stirred at rt for 1.5 h and then concentrated under reduced pressure to
remove methanol. Lyophilization gave a yellow solid (207 mg; 0.297/
0.207 = 1.4 mmol/g) which was then used as such without further
purification. To a dry flask were added ammonium chloride (18 mg,
0.337 mmol), the sodium salt of (S)-5-(2-(2-(3-fluoro-4-(pyrrolidine-
1-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropa-
namido)-1,3,4-thiadiazole-2-carboxylic acid (0.029 mmol, generated
from the above procedure), DIPEA (50 μL, 0.286 mmol), and
anhydrous DMF (0.3 mL) at rt under nitrogen. The mixture was
stirred at rt for 2 min before PyBOP (20 mg, 0.038 mmol) was added.
The mixture was stirred at rt for 1 h. Purification using reverse-phase
HPLC (YMC S5 20 × 100 mm, 10 min run; solvent A, 10%
MeOH:90% H2O:0.1% TFA; solvent B, 90% MeOH, 10% H2O, 0.1%
Scheme 12
used to prepare 3d. 1H NMR (400 MHz, CD3OD): δ ppm 7.98−7.90
(m, 2H), 7.77−7.69 (m, 2H), 7.53 (t, J = 7.4 Hz, 1H), 7.42−7.34 (m,
1H), 7.31−7.23 (m, 2H), 7.16−7.10 (m, 1H), 4.64 (s, 1H), 3.63 (t, J =
7.1 Hz, 2H), 3.46 (q, J = 7.2 Hz, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.08−
1.90 (m, 4H), 1.26 (t, J = 7.2 Hz, 3H), 1.20 (s, 3H), 1.16 (s, 3H). ESI-
MS: m/z 615.21 ([M + H+]). HPLC: tR = 3.49 min.
Synthesis of (S)-N-Cyclopropyl-5-(2-(2-(3-fluoro-4-(pyrroli-
dine-1-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-
methylpropanamido)-1,3,4-thiadiazole-2-carboxamidexamide
(3e, Scheme 13). A procedure similar to that described in step 3 of
Scheme 9 was used to prepare 3e. 1H NMR (400 MHz, DMSO-d6): δ
ppm 7.98−7.89 (m, 2H), 7.76−7.68 (m, 2H), 7.52 (t, J = 7.3 Hz, 1H),
7.41−7.34 (m, 1H), 7.31−7.21 (m, 2H), 7.16−7.09 (m, 1H), 4.63 (s,
1H), 3.66−3.59 (m, 2H), 3.43−3.36 (m, 2H), 2.94−2.85 (m, 1H),
2.07−1.90 (m, 4H), 1.19 (s, 3H), 1.16 (s, 3H), 0.89−0.80 (m, 2H),
1
TFA) gave 3a (16 mg, 0.023 mmol, 79% yield) as a white solid. H
NMR (400 MHz, CD3OD): δ ppm 7.98−7.90 (m, 2H), 7.77−7.69
(m, 2H), 7.52 (t, J = 7.4 Hz, 1H), 7.41−7.34 (m, 1H), 7.31−7.22 (m,
2H), 7.17−7.10 (m, 1H), 4.63 (s, 1H), 3.63 (t, J = 6.9 Hz, 2H), 3.39
(t, J = 6.5 Hz, 2H), 2.07−1.90 (m, 4H), 1.20 (s, 3H), 1.16 (s, 3H).
ESI-MS: m/z 587.18 ([M + H+]). HPLC: tR = 3.34 min.
Synthesis of (S)-5-(2-(2-(3-Fluoro-4-(pyrrolidine-1-carbonyl)-
phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanami-
H
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Journal of Medicinal Chemistry
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(400 MHz, CD3OD): δ ppm 8.33 (1 H, d, J = 8.06 Hz), 7.09−7.56 (5
H, m). ESI-MS: m/z 234.05 ([M + H+]). HPLC: tR = 2.18 min (4 min
gradient, YMC S5 ODS 4.6 × 50 mm, 4 mL/min, 0% B to 100% B, B
= 90% MeOH/10% H2O, 0.2% H3PO4, 4 mL/min).
Scheme 13
Step 2: Synthesis of 9-Fluoro-5H-chromeno[2,3-b]pyridin-5-one
(9). Compound 8 and polyphosphoric acid were combined in a 500
mL round-bottom flask, which was stirred slowly on a rotary
evaporator (open to the atmosphere) until a homogeneous paste
was formed, and then heated at 160 °C for 8 h. Ice (∼500 g) was
added portionwise with agitation to the reaction mixture to give a tan-
colored suspension. In a 1 L beaker, the suspension (still containing
ice) was stirred while 10 N NaOH was added until the mixture
reached about pH 10. The mixture was filtered through a medium-
porosity fritted disc filter, washed with water three times, and dried
overnight on the pump to give 9 (8.95 g, 97% yield) as a gray solid. 1H
NMR (400 MHz, methanol-d4): δ ppm 8.86−8.66 (m, 2H), 8.08 (dt, J
= 8.1, 1.4 Hz, 1H), 7.75 (ddd, J = 10.7, 8.0, 1.5 Hz, 1H), 7.64 (dd, J =
7.8, 4.7 Hz, 1H), 7.49 (td, J = 8.1, 4.5 Hz, 1H). ESI-MS: m/z 216.04
([M + H+]). HPLC: tR = 2.16 min (4 min gradient, YMC S5 ODS 4.6
× 50 mm, 4 mL/min, 0% B to 100% B, B = 90% MeOH/10% H2O,
0.2%H3PO4, 4 mL/min).
0.75−0.67 (m, 2H). ESI-MS: m/z 627.21 ([M + H+]). HPLC: tR =
3.52 min.
Synthesis of (S)-N-Cyclopropyl-5-(2-(9-fluoro-2-(4-(morpho-
line-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-
methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (4,
Scheme 14). Step 1: Synthesis of 2-(2-Fluorophenoxy)nicotinic
Scheme 14
Step 3: Synthesis of 9-fluoro-5H-chromeno[2,3-b]pyridin-5-ol
(10). NaBH4 (4.92 g, 130 mmol) was added to a slurry of 9 (4.0 g,
18.59 mmol) in MeOH (90 mL) and CH2Cl2 (40 mL) which was
stirring at 0 °C. The reaction stirred for 2 h at 0 °C. The reaction
mixture was concentrated in vacuo to remove all the methanol. The
reaction was partitioned between brine and chloroform and the aq
layer was backwashed with chloroform twice more until there was no
product left. All the organic layers were combined, dried over sodium
sulfate, filtered, and concentrated in vacuo to afford 10 (3.87 g, 17.82
mmol, 96% yield) as a light yellow solid. 1H NMR (400 MHz,
methanol-d4): δ ppm 8.32 (dd, J = 4.8, 2.0 Hz, 1H), 8.16 (ddd, J = 7.5,
1.9, 0.7 Hz, 1H), 7.48−7.42 (m, 1H), 7.38 (dd, J = 7.5, 4.8 Hz, 1H),
7.30−7.20 (m, 2H), 5.90 (s, 1H). ESI-MS: m/z 218.05 ([M + H+]).
HPLC: tR = 2.08 min.
Step 4: Synthesis of Methyl 2-(9-Fluoro-5H-chromeno[2,3-b]-
pyridin-5-yl)-2-methylpropanoate (12). To a suspension of the crude
alcohol 10 (5.0 g, 23.02 mmol) in dichloromethane (125 mL) at 0 °C
was added TiCl4 (23.02 mL, 23.02 mmol) dropwise. The resulting tan-
colored suspension stirred for 10 min at 0 °C before (1-methoxy-2-
methylprop-1-enyloxy)trimethylsilane (11) (10.76 mL, 52.9 mmol)
was added to the mixture dropwise. The resulting dark, homogeneous
solution was allowed to stir for 3 h at 0 °C and then was quenched
with the addition of saturated aqueous sodium bicarbonate, yielding
gas evolution and formation of a precipitate. The mixture was filtered
over Celite, and the resulting organic layer of the filtrate was separated,
dried over sodium sulfate, and concentrated to give 12 (5.50 g, 18.25
mmol, 79% yield). 1H NMR (400 MHz, chloroform-d): δ ppm 8.27 (1
H, dd, J = 4.78, 1.76 Hz), 7.56 (1 H, dd, J = 7.55, 1.76 Hz), 7.00−7.15
(3 H, m), 6.90−6.95 (1 H, m), 4.41 (1 H, s), 3.66 (3 H, s), 1.03 (3 H,
s), 1.00 (3 H, s). ESI-MS: m/z 302.17 ([M + H+]). HPLC: tR = 3.03
min.
Step 5: Synthesis of 9-Fluoro-5-(1-methoxy-2-methyl-1-oxopro-
pan-2-yl)-5H-chromeno[2,3-b]pyridine 1-Oxide (13). A solution of
12 (4.0 g, 13.28 mmol) in CH2Cl2 (100 mL) stirring at room
temperature was treated with mCPBA (containing 30% m-CBA) (9.37
g, 54.3 mmol). The reaction (light yellow solution) stirred overnight
for 14 h. The reaction was washed sequentially with 10% aqueous
sodium sulfite and 1 N aqueous sodium hydroxide and then dried over
sodium sulfate, filtered, and concentrated to give 13 (4.37 g, 12.39
mmol, 93% yield). The crude product was taken directly to the next
step. ESI-MS: m/z 318.19 ([M + H+]). HPLC: tR = 2.09 min.
Step 6: Synthesis of Methyl 2-(2-Chloro-9-fluoro-5H-chromeno-
[2,3-b]pyridin-5-yl)-2-methylpropanoate (14). A solution of 13 (4.1
g, 12.92 mmol) in POCl3 (50 mL, 536 mmol) was heated at 100 °C
for 1.5 h. The reaction was cooled down to rt and stirred for 16 h. The
solvent was carefully removed by distilling off the excess solvent. The
excess solvent was quenched by pouring onto ice and cautiously
neutralized with base. The concentrated reaction was poured slowly in
small increments onto ice. With stirring, the icy slurry was neutralized
Acid (8). Sodium methoxide in MeOH (65.3 mL, 286 mmol) was
added to 2-fluorophenol (7) (80 g, 714 mmol), followed by 2-
chloronicotinic acid (22.5 g, 143 mmol). The reaction mixture was
concentrated in vacuo and then dried on the pump for 2 h. The flask
containing the dried material was then heated in an oil bath at 168 °C
for 3 h. The reaction mixture was cooled to rt and sat under nitrogen
gas over one weekend. Water and diethyl ether were added to the
solid, which partitioned, upon agitation, between the two layers. The
purple aqueous layer was acidified with glacial acetic acid (to pH ∼5)
and chilled, giving a precipitate. The solid (colorless needles) was
filtered, washed with water, then dried on the pump to give 8 (18 g).
The aqueous layer was rendered acidic (to pH ∼ 3) with the dropwise
addition of concentrated HCl. The cloudy mixture was washed with
ethyl acetate (150 mL). The organic layer was washed with brine,
dried (sodium sulfate), and concentrated to give a second crop of 8
1
(3.75 g). The total amount of 8 was 21.75 g, 65.3% yield. H NMR
I
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Journal of Medicinal Chemistry
Article
by adding solid potassium carbonate in small portions. Each addition
of the salt generated gas that needed to subside before more salt was
introduced into the flask. Once the reaction was not acidic anymore, it
was diluted with methylene chloride. The layers were separated, and
the aq layer was backwashed with fresh DCM twice. All the DCM
layers were combined and dried over sodium sulfate, filtered, and
concentrated in vacuo to afford the crude material as a caramel-colored
gum. The reaction mixture was placed on a silica gel column. The
product was eluted using 10% ethyl acetate in hexane, and the
collected organic solution was concentrated in vacuo to afford 14 as a
mixture was stirred at rt for 10 min and at 80 °C for 6 h. The reaction
mixture was cooled, concentrated, and mixed with water (30 mL) and
ethyl acetate (50 mL). The organic solution was separated, dried over
Na2SO4, and concentrated. The residue was dissolved in ethyl acetate
and acetone, mixed with Celite (16 g) and DMF (1.6 mL), and
concentrated to give an almost dry solid. Flash chromatography
purification of the solid (80 g silica gel column, 50 → 100% ethyl
acetate in hexanes) afforded 36 (3.15 g, 4.99 mmol, 83% yield) as a
yellowish foam solid. 1H NMR (400 MHz, CD3OD): δ ppm 8.12 (d, J
= 8.03 Hz, 2 H), 7.86 (br s, 1 H), 7.59 (d, J = 7.78 Hz, 1 H), 7.53 (d, J
= 8.03 Hz, 2 H), 7.14 (ddd, J = 9.85, 7.84, 1.88 Hz, 1 H), 6.95−7.04
(m, 2 H), 4.77 (br s, 1 H), 4.56 (q, J = 7.28 Hz, 2 H), 3.42−3.92 (m, 8
H), 1.49 (t, J = 7.15 Hz, 3 H), 1.32 (s, 3 H), 1.25 (s, 3 H). ESI-MS: m/
z 632.19 ([M + H+]). HPLC: tR = 3.48 min.
1
white solid (1.8 g, 5.36 mmol, 41.5% yield). H NMR (400 MHz,
methanol-d4): δ ppm 7.77 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 7.9 Hz,
1H), 7.27−7.14 (m, 2H), 7.12−7.03 (m, 1H), 4.49 (s, 1H), 3.69 (s,
3H), 1.04 (d, J = 4.0 Hz, 6H). ESI-MS: m/z 336.07 ([M + H+]).
HPLC: tR = 3.35 min.
Step 11: Synthesis of (S)-N-Cyclopropyl-5-(2-(9-fluoro-2-(4-
(morpholine-4-carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-
2-methylpropanamido)-1,3,4-thiadiazole-2-carboxamide (4). To a
solid−liquid mixture of 36 (2.1 g, 3.32 mmol) and anhydrous MeOH
(10 mL) was added cyclopropylamine (6 mL, 87 mmol). The solution
was stirred at 40 °C under nitrogen for 18 h. The mixture was
concentrated. The residue was partitioned between water (10 mL) and
ethyl acetate (4 mL). The aqueous layer was separated and extracted
with dichloromethane (2 × 3 mL). The combined organic solutions
were dried over Na2SO4. Flash chromatography purification (40 g
silica gel column, 50 → 100% ethyl acetate in hexanes) afforded a
glassy solid. The solid was dissolved in ethyl acetate (15 mL) and
precipitated out with hexanes (30 mL). The solid was filtered, washed
with a mixture of ethyl acetate and hexanes (1:2, 2 × 10 mL), and
Step 7: Synthesis of 2-(2-Chloro-9-fluoro-5H-chromeno[2,3-b]-
pyridin-5-yl)-2-methylpropanoic Acid (15). To a pale yellow solution
of 14 (2.33 g, 6.94 mmol) in THF (13 mL)/MeOH (22 mL) was
added 4 N aqueous potassium hydroxide (17.35 mL, 69.4 mmol). The
mixture was stirred at 65 °C for 10 h and then at rt for another 6 h.
The reaction mixture was rendered neutral with the dropwise addition
of concentrated HCl and then concentrated to half its volume in
vacuo. The remaining solution was then partitioned between ethyl
acetate and 1 N HCl. The organic layer was dried over sodium sulfate
and concentrated to give 15 (2.0 g, 6.22 mmol, 90% yield) as a tan
1
solid. H NMR (400 MHz, CD3OD): δ ppm 7.82 (d, J = 8.06 Hz, 1
H), 7.29 (d, J = 7.81 Hz, 1 H), 7.25−2.11 (m, 3 H), 4.51 (s, 1 H), 1.01
(s, 3 H), 0.96 (s, 3 H). ESI-MS: m/z 322.06 ([M + H+]). HPLC: tR =
3.11 min.
1
dried to give 4 (1.2 g, 1.848 mmol, 56% yield) as a white solid. H
NMR (500 MHz, DMSO-d6): δ ppm 12.92 (s, 1 H), 9.25 (d, J = 4.67
Hz, 1 H), 8.18 (d, J = 8.25 Hz, 2 H), 7.92 (d, J = 7.97 Hz, 1 H), 7.69
(d, J = 7.97 Hz, 1 H), 7.54 (d, J = 8.25 Hz, 2 H), 7.32−7.38 (m, 1 H),
7.15 (td, J = 8.04, 5.09 Hz, 1 H), 6.96 (d, J = 7.70 Hz, 1 H), 4.86 (s, 1
H), 3.34−3.74 (m, 8 H), 2.86−2.94 (m, 1 H), 1.08 (br s, 3 H), 1.07
(br s, 3 H), 0.68−0.73 (m, 4 H). ESI-MS: m/z 643.21 ([M + H+]).
HPLC: tR = 3.26 min.
Synthesis of (S)-5-(2-(9-Fluoro-2-(4-(2-hydroxypropan-2-yl)-
phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanami-
do)-N-(tetrahydro-2H-pyran-4-yl)-1,3,4-thiadiazole-2-carboxa-
mide (5, Scheme 15). Step 1: Synthesis of (S)-2-(9-Fluoro-2-(4-(2-
Step 8: Synthesis of (S)-2-(2-Chloro-9-fluoro-5H-chromeno[2,3-
b]pyridin-5-yl)-2-methylpropanoic Acid (16). Chiral resolution
[preparative column, Chiralcel OJ-H (3 × 25 cm, 5 μm); BPR
pressure, 100 bar; temperature, 35 °C; flow rate, 70 mL/min; mobile
phase, CO2/(IPA:ACN 1:1, 0.1% TFA) (90/10); detector wavelength,
1
212 nm] gave 16 (peak 1). H NMR (400 MHz, CD3OD): δ ppm
7.82 (d, J = 8.06 Hz, 1 H), 7.29 (d, J = 7.81 Hz, 1 H), 7.25−2.11 (m, 3
H), 4.51 (s, 1 H), 1.01 (s, 3 H), 0.96 (s, 3 H). ESI-MS: m/z 322.06
([M + H+]). HPLC: tR = 3.11 min. The (S) absolute stereo-
configuration of 16 was subsequently confirmed by an X-ray crystal
structure of 5 (vide infra).
Step 9: Synthesis of (S)-2-(9-Fluoro-2-(4-(morpholine-4-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropa-
noic Acid (35). A mixture of 16 (2.85 g, 5.98 mmol), morpholino(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanone (34)
(3.55 g, 11.19 mmol), potassium phosphate (21.76 mL, 43.5 mmol),
and DMF (60 mL) was bubbled with nitrogen for 5 min before
Pd(Ph3P)4 (0.718 g, 0.622 mmol) was added. The mixture was
bubbled with nitrogen for an additional 5 min and then stirred at 100
°C under nitrogen for 3 h. The mixture was concentrated under
reduced pressure. The solid residue was mixed with water (40 mL)
and diethyl ether (100 mL). The organic mixture was extracted with
water (3 × 15 mL). The combined aqueous solutions were filtered
through a pad of Celite, washed with diethyl ether (30 mL), and
acidified to pH 5−6 with concd aq HCl. Ethyl acetate (17 mL) was
added. The mixture was stirred at rt for 20 min before heptanes (34
mL) were added. The mixture was stirred at rt for 30 min. The solid
was filtered, washed with water and a mixture of ethyl acetate and
heptanes (1:2), and dried to give 35 (2.67 g, 5.60 mmol, 90% yield) as
Scheme 15
1
a brownish solid. H NMR (400 MHz, CD3OD): δ ppm 8.17 (d, J =
8.6 Hz, 2H), 7.94 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.57
(d, J = 8.3 Hz, 2H), 7.22−7.13 (m, 3H), 4.57 (s, 1H), 3.83−3.46 (m,
8H), 1.04 (s, 3H), 1.00 (s, 3H). ESI-MS: m/z 477.17 ([M + H+]).
HPLC: tR = 3.07 min.
Step 10: Synthesis of (S)-Ethyl 5-(2-(9-Fluoro-2-(4-(morpholine-4-
carbonyl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropa-
namido)-1,3,4-thiadiazole-2-carboxylate (36). To a stirred mixture
of 35 (2.85 g, 5.98 mmol), 19 (2.072 g, 11.96 mmol), HOBT (0.916 g,
5.98 mmol), and anhydrous MeCN (15 mL) was added WSCDI
(2.293 g, 11.96 mmol) at rt under nitrogen. The mixture was stirred at
rt for 5 min before DIPEA (1.358 mL, 7.78 mmol) was added. The
hydroxypropan-2-yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-
methylpropanoic Acid (18). The mixture of 16 (250 mg, 0.777
mmol), 4-acetylphenylboronic acid (17) (255 mg, 1.55 mmol), and
potassium phosphate (2.72 mL, 5.44 mmol) in DMF (6 mL) was
flushed with nitrogen for 5 min. Then tetrakis(triphenylphosphine)-
palladium(0) (90 mg, 0.078 mmol) was added and the mixture was
flushed with nitrogen for another 5 min. The mixture was stirred at 90
°C for 8 h. After cooling, the mixture was filtered and the filtrate was
added to water (15 mL) and washed with diethyl ether (2 × 20 mL).
The aqueous layer was acidified with 4 N HCl to pH 5−6 and
extracted with AcOEt (35 mL), which was washed with saturated
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NH4Cl (2 × 20 mL), dried over Na2SO4, and concentrated under
vacuo to give the corresponding ketone {ESI-MS: m/z 406.14 ([M +
H]+)}, which was used in the next reaction without further
purification. The above intermediate was taken up in THF (5 mL),
methylmagnesium bromide (2.59 mL, 7.77 mmol) was added
dropwise at −78 °C, and the mixture stirred at rt for 1 h. The
mixture was quenched with saturated NH4Cl (3 mL), diluted with
NH4Cl (20 mL), and mixed with AcOEt (60 mL). The pH was
adjusted with 4 N HCl to 5−6. The entire mixture was filtered through
a pad of Celite and the organic phase was separated and washed with
saturated NH4Cl (2 × 30 mL), dried over Na2SO4, and concentrated
under vacuo to give the crude compound 18, which was used in the
next reaction without further purification. The crude yield for the two
steps was 98% yield (320 mg). 1H NMR (400 MHz, CD3OD): δ ppm
8.08−7.96 (m, 2H), 7.89 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H),
7.68−7.59 (m, 2H), 7.28−7.12 (m, 3H), 4.56 (s, 1H), 1.59 (s, 6H),
1.04 (d, J = 7.5 Hz, 6H). ESI-MS: m/z 422.17 ([M + H+]). HPLC: tR
= 3.51 min.
Step 2: Synthesis of (S)-Ethyl 5-(2-(9-Fluoro-2-(4-(2-hydroxypro-
pan-2-yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropa-
namido)-1,3,4-thiadiazole-2-carboxylate (20). A mixture of 18 (160
mg, 0.380 mmol), 19 (131 mg, 0.759 mmol), EDC (146 mg, 0.759
mmol), HOBt (116 mg, 0.759 mmol), and DIEA (0.332 mL, 1.898
mmol) in DMF (10 mL) was stirred at 40 °C for 16 h and then at 60
°C for 3 h. The solvent was removed and the residue was taken up in
AcOEt (50 mL), washed with saturated NH4Cl (2 × 20 mL) and
NaHCO3 (2 × 20 mL), dried (Na2SO4), and concentrated under
vacuo to give the crude product, which was purified with ISCO flash
column chromatography (12 g, eluting with AcOEt/hexane = 0−50%
gradient, 15 min). The yield of 20 was 85 mg (38.8% yield). 1H NMR
(400 MHz, CD3OD): δ ppm 8.05 (d, J = 8.5 Hz, 2H), 7.88−7.81 (m,
1H), 7.62−7.56 (m, 3H), 7.16−7.08 (m, 1H), 6.97 (d, J = 3.5 Hz,
2H), 4.73 (s, 1H), 4.55 (d, J = 7.3 Hz, 2H), 1.65−1.61 (m, 6H), 1.51−
1.46 (m, 3H), 1.31 (s, 3H), 1.24 (d, J = 3.0 Hz, 3H). ESI-MS: m/z
577.18 ([M + H+]). HPLC: tR = 4.22 min.
1.60−1.54 (m, 6H), 1.18 (d, J = 1.5 Hz, 6H). ESI-MS: m/z 632.23
([M + H+]). HPLC: tR = 4.03 min.
Nuclear Receptor Binding Assays. The GR ligand binding assay
was conducted in fluorescence polarization format, which measures the
competition between a test compound and a fluorescently labeled
ligand (GS-red) for binding to the full length or ligand binding domain
of GRα (Invitrogen, catalog no. P2893). Compounds were tested in
concentrations ranging from 5 μM to 85 pM. IC50 values were
determined by fitting the fluorescence polarization signal data using a
four-parameter logistic equation. The Ki values were determined by
application of the Cheng−Prusoff equation to the IC50 values, where
Ki = IC50/(1 + ligand concentration/Kd) (Cheng, Y.; Prusoff, W. H.
Biochem. Pharmacol. 1973, 22, 3099−3108). The Kd used for GR was
0.3 nM, as supplied by the assay manufacturer (Invitrogen, catalog no.
P2893). Data shown represent the mean values of two or more
experiments. PR ligand binding assay was also conducted in
fluorescence polarization format, which measures the competition
between a test compound and a fluorescently labeled ligand for
binding to the full length or ligand binding domain of the nuclear
hormone receptor.
Transrepression Assays. AP-1 activity is measured using an AP-1
response element (five copies) cloned into a luciferase reporter vector.
This reporter is stably transfected into the human A549 lung epithelial
cell line. AP-1 activity is induced by addition of phorbol myristate
acetate (PMA) (15 ng/mL), and inhibition of induction by
compounds is quantitated by measuring decreased luciferase activity.
NFκB is measured using a truncated, NFκB-dependent E-selectin
promoter (−383 bp from transcriptional start) cloned into a luciferase
reporter vector. This reporter is stably transfected into the human
A549 lung epithelial cell line. NFκB activity is induced using IL-1β (0.5
ng/mL), and inhibition of induction by compounds is quantitated by
measuring decreased luciferase activity.
Transactivation Assays: NP-1 Reporter Assay. The direct
transcriptional activity is measured using a chimera between the GAL4
DNA-binding domain and the human GR ligand-binding domain
(GR-LBD) cloned into a GAL4 luciferase reporter system. This
reporter system is stably transfected into a NP-1 HeLa cell line
(Webster, N. J. G.; Green, S.; Jin, J. R.; Chambon, P. Cell 1988, 54 (2),
199−207). The hormone-binding domains of the glucocorticoid
receptor contain an inducible transcription activation function.
Response to ligand/compound induced binding is quantitated by
measuring luciferase activity. Direct activation of the GR-LBD by
compounds (agonist) can be measured as increased luciferase activity
after a 20 h incubation at 37 °C/5% CO2.
Human Whole Blood Assays for TNFα and IL-1β. Human
whole blood (two donors per experiment) was drawn into syringes
containing ACD-A and kept at rt until initiation of the experiment
(about 1 h). The blood was plated into 96-well flat-bottom,
polystyrene, tissue-culture-treated plates (BD Falcon) at 180 μL/
well. An amount of 10 μL of 20× test compound dilutions was added
per well and mixed, and the plates were incubated overnight (about 20
h) at 37 °C with 5% CO2. The next day, 10 μL of 20× LPS (Sigma-
Aldrich Escherichia coli 055/B5 LPS; for TNFα induction, the final
concentration of LPS was 100 ng/mL) or 20× human recombinant IL-
1β (R & D Systems; for IL-8 induction, the final concentration of IL-
1β was 10 ng/mL) was added per well. The plates were incubated for
an additional 5 h and centrifuged at low speed, and the plasmas were
harvested for immediate analysis by ELISA or were frozen at −20 °C.
TNFα and IL-8 induction was quantitated by ELISA (R & D
Systems).
Step 3: Synthesis of (S)-5-(2-(9-Fluoro-2-(4-(2-hydroxypropan-2-
yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanami-
do)-N-(tetrahydro-2H-pyran-4-yl)-1,3,4-thiadiazole-2-carboxamide
(5). To a mixture of 20 (7.0 g, 12.14 mmol) in THF (70 mL) was
added water (5 mL) followed by lithium hydroxide hydrate (2 g, 47.7
mmol). The mixture was stirred at rt for 7 h. The mixture was
concentrated under vacuo to dryness. Ethanol (50 mL) was added and
the solvent was removed under vacuo (repeated twice) to give the
lithium salt, which was used in the next step {ESI-MS: m/z 549.15
([M + H+])}. To a solution of (S)-5-(2-(9-fluoro-2-(4-(2-hydrox-
ypropan-2-yl)phenyl)-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpro-
panamido)-1,3,4-thiadiazole-2-carboxylic acid (5 g, 9.11 mmol) lithium
salt in DMF (60 mL) was added tetrahydro-2H-pyran-4-amine (1.383
g, 13.67 mmol), followed by BOP (8.06 g, 18.23 mmol) and DIEA
(3.98 mL, 22.79 mmol), and the mixture was cooled with an ice water
bath. After the addition, the ice bath was removed and the mixture was
stirred at rt for 6 h. The mixture was diluted with AcOEt (300 mL);
washed with saturated NH4Cl (200 mL, 2 × 120 mL), saturated
NaHCO3 (3 × 120 mL), and brine (120 mL); dried over MgSO4; and
concentrated under vacuo to 80 mL of volume. The solid was formed
when standing at rt after 5 h. The solid was collected by filtration,
washed with EtOAc (50 mL), and dried under vacuo at 60 °C for 16 h
to get the crystals (4.2 g). The first 200 mL of NH4Cl wash was
extracted with AcOEt (120 mL), which was washed with saturated
NH4Cl (3 × 50 mL), saturated NaHCO3 (3 × 50 mL), and brine (50
mL), dried over MgSO4, and concentrated under vacuo to 15 mL of
volume. The solid was formed when standing at rt for 5 h. The solid
was collected by filtration, washed with EtOAc (50 mL), and dried
under vacuo to get the second crop of product (0.6 g). An extra 0.7 g
product was recovered from the mother liquor. The total yield of 5
Adjuvant-Induced Arthritis. Male Lewis rats (Harlan; ∼200 g)
were injected intradermally at the shaved based of the tail on day 0
with 100 μL of 10 mg/mL freshly ground Mycobacterium butyricum in
incomplete Freund’s adjuvant and then randomized into dose groups
(n = 8/group). Test compounds were orally (po) dosed in solution
(EtOH/TPGS/PEG300, 5:5:90) once daily. Baseline paw measure-
ments were taken between days 7 and 10, and measurements were
taken three times per week after disease developed (between days 11
and 14). All studies involving animals were reviewed and approved by
the BMS Institutional Animal Care and Use Committee.
1
was 5.5 g (96% yield). H NMR (400 MHz, CD3OD): δ ppm 8.05−
7.95 (m, 2H), 7.79−7.52 (m, 4H), 7.27−6.96 (m, 3H), 4.65 (s, 1H),
4.21−4.06 (m, 1H), 4.04−3.92 (m, 2H), 3.53 (td, J = 11.8, 2.1 Hz,
2H), 1.91 (dd, J = 12.4, 2.3 Hz, 2H), 1.74 (qd, J = 12.0, 4.5 Hz, 2H),
K
DOI: 10.1021/acs.jmedchem.5b00257
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Glutamine Synthetase (GS) Induction Assay. An enzymatic
assay was used to measure the induction of GS activity in the human
osteosarcoma cell line MG-63. The cell line was maintained in log
phase in growth medium [Dulbecco’s modified Eagle’s medium
(DMEM, Invitrogen) with 10% charcoal−dextran-treated fetal calf
serum (FCS, Hyclone), penicillin/streptomycin (10 000 U/mL and 10
mg/mL, respectively), and 2 mM L-glutamine]. Briefly, on day 1 MG-
63 cells were plated in assay medium (growth medium but without
phenol red and L-glutamine) at a density of 50 000 cells/well (200 μL/
well) in 96-well flat-bottom tissue-culture-treated plates and incubated
overnight at 37 °C with 5% CO2. On day 2, the assay medium was
removed from the wells and replaced with 100 μL/well induction
medium (DMEM without L-glutamine, 1% FCS, and penicillin/
streptomycin) and test compounds followed by incubation overnight
at 37 °C with 5%CO2. On day 3, the induction medium was removed,
and the wells were washed three times with 200 μL/well PBS. After
the last wash, PBS was removed, and the cells were lysed at room
temperature with 30 μL of 50 mM imidazole (pH 6.8)/0.1% Trixon-
100 per well. Then 30 μL of GS assay mix (imidazole, 50 mM; arsenic
acid, 25 mM; ADP, 0.16 mM; L-glutamine, 50 mM; MnCl2, 2 mM;
hydroxylamine, 25 mM) was added per well, and the plates were
incubated at 37 °C, 5% CO2 for 90 min. At the end of the incubation
period, 120 μL of ferric chloride stop solution (ferric chloride, 2.42%;
TCA, 1.45%; HCl, 1.82%) was added per well, and the absorption was
read at 540 nm.
ACKNOWLEDGMENTS
■
The authors are grateful to the following colleagues for their
support of the project and their help in the preparation of this
paper: Mary Ellen Cvijic, Raymond Scaringe, Ding Ren Shen,
Meliisa Yarde, Dauh-Rurng Wu, Yi Tao, Ling Gao, and Peng Li.
ABBREVIATIONS USED
■
AA, adjuvant-induced arthritis; ALP, alkaline phosphatase; AP-
1, activator protein 1; AR, androgen receptor; DDI, drug−drug
interactions; dex, dexamethasone; ER, estrogen receptor; GR,
glucocorticoid receptor; GS, glutamine synthetase; HATU, (2-
(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hex-
afluorophosphate); HOBT, hydroxybenzotriazole; iv, intra-
venous; mCPBA, m-chloroperoxybenzoic acid; MR, mineralo-
corticoid receptor; NFκB, nuclear factor kappa B; NP-1,
neuropilin-1; PK, pharmacokinetic; PD, pharmacodynamic;
PAMPA, parallel artificial membrane permeability assay; PR,
progesterone receptor; pred, prednisolone; rt, room temper-
ature; SAR, structure−activity relationships; SFC, supercritical
fluid chromatography; TA, transactivation; TNFα, tumor
necrosis factor α; WSCDI, 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride
Alkaline Phosphatase Induction Assay. An enzymatic assay was
used to measure the induction of alkaline phosphatase activity in the
human osteosarcoma cell line SAOS-2. The cell line was maintained in
log phase in growth medium [McCoy’s 5A (Invitrogen) with 15% fetal
bovine serum (FBS, Summit) and penicillin/streptomycin (10 000 U/
mL and 10 mg/mL, respectively)]. Briefly, on day 1, SAOS-2 cells
were plated in assay medium (growth medium except using 15%
charcoal−dextran-treated FCS) at a density of 3000 cells/well (100
μL/well) in 96-well flat-bottom tissue-culture-treated plates and
incubated overnight at 37 °C with 5% CO2. On day 2, the assay
medium was removed from the wells and replaced with 200 μL/well
induction medium (assay medium except using 1.5% charcoal−dextran
treated FCS) and test compounds followed by incubation for 3 days at
37 °C with 5% CO2. After the third day of compound treatment, the
induction medium was removed, and the wells were washed three
times with 200 μL/well cold PBS. After the last wash, PBS was
removed, and the cells were lysed at room temperature with 100 μL
per well of 12.5 mM NaHCO3, 12.5 mM Tris, 0.01% sodium azide,
and 0.05% Triton X-100. Then 25 μL of cell lysate was added to 100
μL of alkaline phosphatase assay mix (p-NPP diluted in 1-KPL DEA
substrate buffer to 3 mg/mL), and the plates were incubated in the
dark at room temperature for 15 min. At the end of the incubation
period, 100 μL per well of 1 N HCL stop solution was added per well,
and the absorbance was read at 405 nm.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The X-ray crystal structure of 5 is also available. The
Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00257.
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AUTHOR INFORMATION
■
Corresponding Authors
*M.G.Y.: phone, 609-252-3234; e-mail, michael.yang@bms.
com.
*T.G.M.D.: phone, 609-252-4158; e-mail, murali.dhar@bms.
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Notes
The authors declare no competing financial interest.
Schutz, G. Repression of inflammatory responses in the absence of
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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DOI: 10.1021/acs.jmedchem.5b00257
J. Med. Chem. XXXX, XXX, XXX−XXX