Structure-activity relationships of phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitors: Investigations of various 6,5-heterocycles to improve metabolic stability

Article abstract of DOI:10.1021/jm2004442

N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)benzo[d] thiazol-2-yl)acetamide (1) is a potent and efficacious inhibitor of PI3Kα and mTOR in vitro and in vivo. However, in hepatocyte and in vivo metabolism studies, 1 was found to undergo deacet

Full text of DOI:10.1021/jm2004442

ARTICLE
pubs.acs.org/jmc
StructureꢀActivity Relationships of Phosphoinositide 3-Kinase (PI3K)/
Mammalian Target of Rapamycin (mTOR) Dual Inhibitors:
Investigations of Various 6,5-Heterocycles to Improve
Metabolic Stability
Markian M. Stec,*,† Kristin L. Andrews,|| Shon K. Booker,† Sean Caenepeel,‡ Daniel J. Freeman,‡ Jian Jiang,§
Hongyu Liao,† John McCarter,^ Erin L. Mullady,^ Tisha San Miguel,^ Raju Subramanian,§ Nuria Tamayo,†
Ling Wang,‡ Kevin Yang,† Leeanne P. Zalameda,^ Nancy Zhang,‡ Paul E. Hughes,‡ and Mark H. Norman†
†Department of Medicinal Chemistry, ‡Department of Oncology, §Department of Pharmacokinetics and Drug Metabolism,
Department of Molecular Structure, ^Department of High-Throughput Screening/Molecular Pharmacology, Amgen Inc.,
One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States, and 360 Binney Street, Cambridge,
Massachusetts 02142, United States
S Supporting Information
b
ABSTRACT: N-(6-(6-Chloro-5-(4-fluorophenylsulfonami-
do)pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (1) is a potent
and efficacious inhibitor of PI3KR and mTOR in vitro and
in vivo. However, in hepatocyte and in vivo metabolism studies,
1 was found to undergo deacetylation on the 2-amino sub-
stituent of the benzothiazole. As an approach to reduce or
eliminate this metabolic deacetylation, a variety of 6,5-hetero-
cyclic analogues were examined as an alternative to the ben-
zothiazole ring. Imidazopyridazine 10 was found to have similar in vitro potency and in vivo efficacy relative to 1, while only minimal
amounts of the corresponding deacetylated metabolite of 10 were observed in hepatocytes.
’ INTRODUCTION
inhibitors in clinical development.6ꢀ8 These clinical candidates
have varying selectivity profiles toward different class I PI3K
isoforms and mTOR and the optimal selectivity profile for efficacy
against specific tumor types remains to be determined.6ꢀ13
In our efforts to identify novel inhibitors of this pathway, we
identified benzothiazole 1, which was found to be a potent
inhibitor of PI3KR in vitro (Ki = 1.2 nM in the enzymatic assay,
IC50 = 6.3 nM in the U87 MG cellular assay) (Figure 1).14,15 This
compound was also a potent inhibitor of the other class I PI3Ks
β, γ, and δ as well as mTOR and was selective against many
protein kinases. Furthermore, compound 1 showed efficacy in a
variety of tumor xenograft models in mice (e.g., ED50 = 0.26 mg/kg,
once daily (QD), U87 MG tumor xenograft model).
Compound 1 was stable in plasma and exhibited low clearance
(<26 μL/min/mg) in liver microsomes (mouse, rat, dog, mon-
key, and human16) and low (0.2ꢀ1.4% of liver blood flow)
in vivo clearance in mouse, rat, and dog. However, metabolite
profiling in hepatocytes showed that 1 underwent deacetylation
to give compound 2, which itself was a potent inhibitor of PI3KR
(Figure 1). Differential formation of 2 was observed across
species, with dog and human forming more of 2 than rat and
monkey. Analysis of plasma in rat and dog, post oral dosing,
Phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3-hy-
droxyl group of the inositol ring of phosphoinositides. PI3Ks are
grouped into three classes, with PI3KR being in the class IA
subgroup and consisting of a heterodimeric p85 regulatory
subunit and a p110 catalytic subunit. Other class IA PI3Ks
include PI3Kβ and PI3Kδ, while PI3Kγ falls in the class IB
subgroup. Activation of class I PI3Ks results in the initiation of a
signal transduction cascade leading to the phosphorylation of the
serine-threonine kinase Akt (also known as PKB) that in turn
promotes cell growth, survival, and cell cycle progression. An-
other relevant class of kinases is the PI3K-related kinases
(PIKKs) that are protein kinases with a catalytic domain homo-
logous to PI3Ks. One particularly important member of the
PIKK family is mTOR (mammalian target of rapamycin) which
plays an integral key role in regulating class I PI3K activation and
signaling.1ꢀ3
Many human cancers have activated PI3K signaling, often as a
result of gain-of-function mutations in PI3KR4,5 or loss-of-
function mutations in PTEN1 (a phosphatase that performs
the reverse phosphorylation event performed by class I PI3Ks).
These genetic alterations strongly link constitutive PI3K signal-
ing to tumorigenesis and have stimulated an interest in targeting
PI3KR, and other kinases in the PI3K/Akt pathway, for the
treatment of cancer. There are currently at least 15 PI3K
Received: April 13, 2011
Published: June 29, 2011
r
2011 American Chemical Society
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Figure 3. Alternative 6,5-heterocyclic ring systems as derivatives of
compound 1.
observed upon oral administration of 1. Therefore, in humans,
the hepatocyte data suggested that administration of 1 would also
lead to a significant concentration of active metabolite 2 in
plasma. Because formation of this active metabolite would
complicate drug development efforts of 1, an alternative com-
pound was sought.
To avoid this metabolic deacetylation issue, we took one
approach where the benzothiazole moiety was replaced with
various 6,6-bicyclic ring systems that did not contain the N-acetyl
group.17 This work led to the identification of a potent series of
quinoline and quinoxaline PI3K inhibitors with good pharma-
cokinetic properties. An alternative approach, which is described
in this paper, was to replace the benzothiazole ring system with
other 6,5-heterocyclic ring systems (Figure 3) that could either
reduce the rate of deacetylation or retain sufficient activity in the
absence of the N-acetyl group. Toward this end, we prepared and
evaluated several 6,5-heterocyclic analogues of 1 where UꢀY
were replaced with various combinations of heteroatoms to give
imidazopyridazines, imidazopyridines, benzimidazoles, benzoxazoles,
triazolopyridines, and benzoisothiazoles. In addition, we pre-
pared a set of analogues with small variations of the Z substituent
(Z = NHAc, NH2, H) to explore the effect of altering the nature
of the hydrogen bond donor in this area of the molecule.
Figure 1. Deacetylation of 1 in hepatocytes. Reverse-phase HPLC UV
(308 nm) chromatographs of supernatant liquid, post centrifugation,
obtained from incubation of hepatocytes with 1 for 2 h at 37 ꢀC.
’ CHEMISTRY
The majority of the 6,5-heterocyclic analogues of 1 were
prepared as outlined in Scheme 1. The synthesis began with
5-bromo-2-chloropyridin-3-amine (3), which was treated with
4-fluorobenzene-1-sulfonyl chloride (4) under basic conditions
to afford sulfonamide 5.18 Compound 5 was converted to pinacol
boronic ester 7 under palladium-catalyzed borylation conditions
utilizing bis(pinacolato)diboron (6).19 Boronic ester 7 was then
coupled to heterocyclic halides 8 under a variety of palladium-
catalyzed cross-coupling conditions to provide analogues 9, 10,
and 12ꢀ22. Aminoimidazopyridazine 11 was obtained by basic
hydrolysis of the corresponding N-acetyl derivative, 10. The
requisite heterocyclic halides 8 were obtained through commer-
cial suppliers, precedented syntheses, or their syntheses are
described in the Supporting Information.
Figure 2. In vivo plasma levels of 1 and 2 in dog post administration of a
0.3 mg/kg oral dose of compound 1. Each time-point is a mean plasma
concentration from 3 dogs; vehicle was 20% captisol, 2% HPMC, and 1%
Tween 80.
The final two analogues, benzoxazole 24 and benzisothiazole
27, were synthesized by reversing the coupling partners in the
palladium-catalyzed cross-coupling reaction (Scheme 2). Aryl
bromide 22 underwent palladium-catalyzed borylation,19 fol-
lowed by conversion to the trifluoroborate salt20 to afford 23.
Subsequent coupling with bromopyridine 5 afforded benzoxa-
zole 24. Similarly, aryl bromide 25 was converted to boronic ester
26, which when coupled to bromopyridine 5, provided the final
target, benzoisothiazole 27.
revealed the presence of both 1 and 2 across the pharmacokinetic
profile. For example, in dog, deacetylated metabolite 2 levels
were equal to or higher than that of compound 1 at all time points
greater than 2 h (Figure 2). In dog, the plasma AUC ratio
of compound 2 versus compound 1 was 4.6, while in rat that
ratio was 0.6ꢀ0.8 (data not shown). Across species, there
was a qualitative relationship between the amount of 2 found
from in vitro incubation of 1 and the in vivo plasma level of 2
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Scheme 1a
a Reagents and conditions: (a) NaHMDS, THF, 25 ꢀC; (b) PdCl2(dppf), KOAc, 1,4-dioxane, 100 ꢀC; (c) Pd catalyst, base; (d) aq NaOH, MeOH,
100 ꢀC.
Scheme 2a
a Reagents and conditions: (a) 6, PdCl2(dppf), KOAc, 1,4-dioxane, 100 ꢀC. ;(b) KHF2, CH3CN; (c) 5, PdCl2(dppf), Cs2CO3, THF, 100 ꢀC,
microwave; (d) 6, Pd2(dba)3, PCy3, KOAc, 1,4-dioxane, 110 ꢀC, microwave; (e) 5, FibreCat 1029 triphenylphosphine, aq Na2CO3, 1,4-dioxane,
110 ꢀC, microwave.
’ RESULTS AND DISCUSSION
inhibitors of PI3KR and showed comparable mTOR potencies
(within an order of magnitude) with the exception of compound
21, which was significantly less active toward mTOR. Therefore,
for the purposes of the structureꢀactivity relationship (SAR)
discussion, we will focus on the PI3KR activity. Compound 2, the
nonacetylated derivative of compound 1, showed 5-fold lower
The various 6,5-ring system analogues of 1 were examined in
two enzyme assays to determine the inhibition of PI3KR and
mTOR activity and in a cellular assay that determined Akt (Ser
473) phosphorylation in U87 MG cells.14 The results are shown
in Table 1. In general, the compounds were moderate to potent
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Table 1. In Vitro Potency of PI3Kr Inhibitorsa
a See ref 14 for assay details. b The results without a SD are from a single experiment.
potency in the PI3KR enzyme assay and a 7-fold reduction in
cellular potency relative to 1. Having no substitution on the
2-position of the benzothiazole, compound 9 showed a signifi-
cant reduction in cellular activity relative to 1 and 2.
Imidazopyridazines 10ꢀ12 and imidazopyridines 13ꢀ15
showed good to excellent PI3KR enzyme potencies (1ꢀ23 nM);
however, the majority of these analogues showed a dramatic loss in
cellular potency, with the exception of compound 10 and 12, which
maintained cellular activities of 13 and 50 nM, respectively.
In the benzimidazole series (16ꢀ18), only the 2-acetamide
analogue (16) had single-digit nanomolar potency in the PI3KR
enzyme assay. The other two benzimidazole analogues, 17 and
18, demonstrated decreased PI3KR enzyme activities and all
three benzimidazole analogues gave poor cellular potencies
(>300 nM). Benzoxazole analogues 19, 20, and 24 exhibited
moderate to good potencies in the PI3KR enzyme assay, but a
significant loss in activity was observed in the cellular assay with
values of >100 nM. Interestingly, the nonacetylated aminoben-
zoxazole 19 showed the greatest PI3KR enzyme potency of
the benzoxazoles; however, this did not translate to acceptable
functional activity.
Figure 4. Deacetylation of imidazopyridazine 10 in human, dog, and rat
hepatocytes. Reverse-phase HPLC extracted ion MS chromatographs of
supernatant liquid, post centrifugation, obtained from incubation of
hepatocytes with 10 for 2 h at 37 ꢀC.
was preferred. However, compounds 21 and 27 had similar
PI3KR enzyme activities (within an order of magnitude) relative
to compounds with Z = H, such as 9, 12, 15, 18, and 20.
Taken together, the results outlined in Table 1 show that the
acetylated compounds (R2 = NHAc) generally showed the best
potencies compared to the amino compounds (R2 = NH2) or the
Triazolopyridine 21 and benzoisothiazole 27 precluded sub-
stitution at the 2-position and afforded diminished potency
relative to the 2-acetamide compounds such as 1, 10, and 13
and supported the observation that substitution at the 2-position
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Figure 5. Comparison of calculated bond lengths and energies of formation of tetrahedral intermediates for 2-acetamidobenzothiazole and
2-acetamidoimidazopyridazine.
Figure 6. (A) Effect of compound 10 on Akt (Ser 473) phosphorylation in U87 MG tumors. Asterisk denotes p < 0.01 compared with the vehicle group.
Bars represent the mean ( SD (n = 3). Diamonds represent mean tumor concentration. (B) Plot of plasma concentrations vs percent inhibition of Akt
phosphorylation. The percent inhibition was calculated relative to vehicle from the measured ratios of phosphorylated Akt (pAkt) to total Akt (t-Akt) in
U87 MG tumors in the doseꢀresponse and time course PD experiments. γ is the Hill coefficient, and the solid line represents a nonlinear regression fit
from a sigmoidal doseꢀresponse model.
unsubstituted compounds (R2 = H), with the benzoxazole series
being the exception. Within the acetylated analogues, only
imidazopyridazine 10 showed comparable enzyme and cellular
activities to benzothiazole 1. In addition, compound 10 showed
a similar selectivity profile to 1 in that it was a potent inhibitor
of mTOR (IC50 = 0.4 nM) and other class I PI3Ks (β, γ, and δ;
Ki = 3.5, 1.9, and 0.8 nM, respectively). In a kinase selectivity
screen21 profiling activity on 402 protein and lipid kinases,
compound 10 was only active on PI3Ks (percentage of control
(POC) = 0ꢀ1.4%) and moderately active (POC = 10ꢀ25%) on
three kinases (YSK4, ERK8, and PIK4CB). For all other kinases
in the panel, compound 10 showed little or no kinase activity
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bearing mice were treated with a single oral dose of compound 10
at 0.3, 1, or 3 mg/kg and the tumors were harvested at 3, 8, 16,
and 24 h postdose. Figure 6A illustrates Akt (Ser 473) phos-
phorylation (bar graph) and the observed tumor concentrations
of compound 10 (blue diamonds). Plasma concentrations were
also determined in this study and were found to be 1ꢀ8 times
higher than tumor concentrations.25 Treatment with compound
10 at 1 mg/kg resulted in a significant inhibition (p < 0.01) of Akt
phosphorylation for up to 8 h with a mean tumor to plasma ratio
of 0.13 at that time point. At the 3 mg/kg dose, significant
inhibition of Akt phosphorylation was observed up to 16 h with a
mean tumor to plasma ratio of 0.27. A plot of plasma concentra-
tion versus percent inhibition of Akt phosphorylation in the U87
MG tumors is shown in Figure 6B and a nonlinear regression
analysis revealed a low plasma EC50 (0.5 μM, 232 ng/mL).
Given the clear pharmacodynamic effect of compound 10, it
was evaluated for its ability to inhibit tumor growth in the mouse
U87 MG glioblastoma xenograft model.14 Treatment with com-
pound10 at 1mg/kgQDresulted insignificantinhibition oftumor
growth of 49% compared to the vehicle control group, while
treatment with 3 mg/kg QD resulted in tumor stasis. The ED50 of
Figure 7. Daily dosing with imidazopyridazine 10 induced tumor stasis
and growth delay in established U87 MG glioblastoma xenograft model.
Arrow denotes the first day of dosing. Data points represent the mean (
SE (n = 10). Statistical significance was evaluated by Repeated Measures
ANOVA followed by Dunnett post hoc test.
(POC = 36ꢀ100%). Kinase activity was measured as a percen-
tage of control (POC) when treated with compound 10 at a
concentration of 1 μM.22
To determine whether the change to an imidazopyridazine
core from a benzothiazole core affected its propensity to deace-
tylate, compound 10 was incubated in microsomes and primary
hepatocytes from different species. The hepatocyte metabolite
profiles are shown in Figure 4. Only trace levels of deacetylated-
10 (compound 11) were detected in vitro.
In attempting to rationalize the increased stability of 10
toward deacetylation, we noted that during our synthetic studies,
imidazopyridazine 10 required stronger conditions for deacety-
lation than benzothiazole 1. For example, after treatment with
Cs2CO3 in MeOH, H2O, and DMF for 3 h at 70 ꢀC, compound 1
underwent complete deacetylation while compound 10 showed
no appreciable deacetylation under the same conditions. The
increased chemical stability of the 2-acetamidoimidazopyridazine
toward deacetylation may be due to the relative strengths of the
amide bonds. The calculated NꢀC amide bond length in the
2-acetamidoimidazopyridazine derivative was 1.377 Å versus
1.383 Å for the 2-acetamidobenzothiazole (Figure 5). This
calculated bond length difference could be attributed to the
availability of an additional tautomer delocalizing the amide bond
into the benzothiazole ring that is not available in the imidazo-
pyridazine case. In addition, quantum mechanics calculations of
the neutral tetrahedral intermediates suggest that higher energy
is required for the 2-acetamidoimidazopyridazine to proceed
from the acetamide to a tetrahedral intermediate versus the
2-acetamidobenzothiazole (ΔΔE = 1.58 kcal/mol), which may
also contribute to the increased hepatocyte stability of the
2-acetamidoimidazopyridazine system.23,24
compound 10 was 0.7 mg/kg (AUC at ED50 of 9.2 μg h/mL)
3
(Figure 7).26 Compound 10 was well tolerated as judged by
minimal body weight loss (<5% compared to predose weight) at
the highest dose.
’ CONCLUSION
In conclusion, a potent and efficacious PI3KR/mTOR inhi-
bitor 1 was found to undergo deacetylation in human hepato-
cytes to form an active metabolite 2; therefore, an alternative
derivative was sought that would not have this liability. Various
6,5-heterocyclic ring systems were explored and imidazopyrida-
zine 10 was found to have comparable in vitro potency and
in vivo efficacy relative to 1 yet it was more metabolically stable
and underwent minimal deacetylation upon incubation in rat,
dog, and human hepatocytes.
’ EXPERIMENTAL METHODS
Chemistry. All reactions were conducted in a well-ventilated fume
hood under N2 using a Teflon-coated magnetic stir bar at the tempera-
ture indicated. Reactions heated by microwave utilized a CEM brand
microwave utilizing 100 W of energy with Powermax (simultaneous
heating while cooling technology) feature turned on. All solvents and
reagents obtained from commercial sources were used without further
purification. Unless otherwise indicated, removal of solvents was con-
ducted by using a rotary evaporator, and residual solvent was removed
from nonvolatile compounds using a vacuum manifold maintained at
approximately 1 Torr. All yields reported are isolated yields.
1H NMR spectra were obtained on a Bruker BioSpin GmbH
1
magnetic resonance spectrometer. H NMR spectra are reported as
chemical shifts in parts-per-million (ppm) relative to an internal solvent
reference. Peak multiplicity abbreviations are as follows: s (singlet), br s
(broad singlet), d (doublet), dd (doublet of doublets), t (triplet), q
(quartet), quin (quintet), and m (multiplet).
Analytical HPLC and mass spectroscopy were conducted using a
reverse-phase Agilent 1100 Series HPLC-mass spectrometer. Purities for
final compounds were measured using UV detection at 254 nm and are
g95.0%.
With its excellent in vitro cellular potency and improved
metabolic stability, imidazopyridazine 10 was further evaluated
in pharmacokinetic and pharmacodynamic (PD) studies. In rats,
intravenous dosing of compound 10 demonstrated a low clear-
ance (0.04 L/h/kg; 1.2% of liver blood flow) and a low volume of
distribution (0.27 L/kg) with an elimination half-life of 7 h.
Compound 10 dosed orally in rats at 2 mg/kg showed good oral
bioavailability (46%) and a high plasma exposure (AUC = 11500
N-(5-Bromo-2-chloropyridin-3-yl)-4-fluorobenzenesulfo-
namide (5). To a 100 mL, round-bottomed flask was added 5-bromo-
2-chloropyridin-3-amine (0.500 g, 2.41 mmol) and THF (10 mL).
ng h/mL). The in vivo activity of compound 10 was evaluated in
3
a mouse pharmacodynamic assay in U87 MG tumors. Tumor
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Sodium bis(trimethylsilyl)amide (1.33 g, 7.23 mmol) was added to the
mixture and stirred for 10 min. 4-Fluorobenzene-1-sulfonyl chloride
(1.41 g, 7.23 mmol) was then added, and the solution was stirred at room
temperature overnight. The reaction mixture was diluted with satd
NaHCO3 and extracted with DCM (4 ꢁ 30 mL). The combined extracts
were dried (Na2SO4), filtered, and concentrated in vacuo. Purification
by silica gel chromatography (1ꢀ30% EtOAc/DCM) afforded 5 as a tan
solid (0.690 g, 78% yield). MS (ESI positive ion) m/z: 366.9 (M + 1). 1H
NMR (400 MHz, DMSO-d6): δ ppm 7.35ꢀ7.48 (m, 2H), 7.79ꢀ7.86
(m, 2H), 7.94 (d, J = 2.5 Hz, 1H), 8.43 (d, J = 2.0 Hz, 1H), 10.65 (br s,
1H).
N-(2-Chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (7). To a 100 mL,
round-bottomed flask was added compound 5 (1.39 g, 3.81 mmol),
bis(pinacolato)diboron (1.26 g, 4.95 mmol), PdCl2(dppf) (0.209 g, 0.286
mmol), potassium acetate (1.12 g, 11.4 mmol), and 1,4-dioxane (20 mL).
The reaction was purged with nitrogen and heated at 100 ꢀC for 3 h. After
cooling to room temperature, themixture was dilutedwith EtOAc, washed
with water and brine and dried. Purification by silica gel chromatography
(15ꢀ40% EtOAc/hexanes) afforded 7 as a white solid (1.24 g, 79% yield).
MS (ESI positive ion) m/z: 331.0 (M (boronic acid) + 1). 1H NMR (400
MHz, CHCl3-d): δ ppm 1.36 (s, 12H), 6.86 (br s, 1H), 7.09ꢀ7.17 (m,
2H), 7.74ꢀ7.82 (m, 2H), 8.30(d, J=1.5 Hz, 1H), 8.45 (d, J =1.5Hz, 1H).
N-(5-(Benzo[d]thiazol-6-yl)-2-chloropyridin-3-yl)-4-fluor-
obenzenesulfonamide (9). To a microwave vial equipped with a
magnetic stir bar was added 6-bromobenzo[d]thiazole (0.100 g, 0.467
was 100 bar, column temperature was 35 ꢀC. A = supercritical CO2, B =
methanol with 0.2% diethylamine). A portion of the purified material
(1.20 g) was dissolved in water (100 mL) and treated with AcOH
(0.51 mL). A precipitate formed. The solution was stirred for 15 min and
then allowed to sit for 2 h. The mixture was filtered, and the filter cake was
washed with water (3 ꢁ 15 mL) and then dried by letting air pass through
the fritted funnel. Further drying under vacuum (250 mtor, 45 ꢀC, 16 h)
afforded 10 as a light-brown solid (0.962 g, 18% yield). HRMS calculated
1
for C19H14ClFN6O3S1 (M + H) 461.0592, found 461.0578. H NMR
(400 MHz, DMSO-d6): δ ppm 2.13 (s, 3H), 7.39ꢀ7.48 (m, 2H),
7.80ꢀ7.88 (m, 3H), 8.13 (d, J = 9.4 Hz, 1H), 8.31ꢀ8.36 (m, 2H), 8.92
(d, J = 2.2 Hz, 1H), 10.57 (s, 1H), 10.96 (s, 1H).
N-(5-(2-Aminoimidazo[1,2-b]pyridazin-6-yl)-2-chloropyr-
idin-3-yl)-4-fluorobenzenesulfonamide (11). To a 100 mL,
round-bottomed flask was added 10 (150 mg, 0.33 mmol), methanol
(10 mL), and NaOH (10 N, 5 mL). The mixture was heated at reflux for
2 h. The solution was cooled to 0 ꢀC, and 10 N HCl was added until
pH = 7. The solution was concentrated and purified by silica gel chroma-
tography (10ꢀ50% MeOH/CH2Cl2). Further purification by prepara-
tive HPLC (Phenomenex Synergi 4 μ MAX-RP 150 mm ꢁ 21.2 mm,
40ꢀ90% CH3CN/H2O, 0.1% TFA, over 15 min) (Phenomenex,
Torrance, CA) provided 11 as a yellow solid (20 mg, 15% yield). HRMS
calculated for C17H12ClFN6O2S1 (M + H) 419.0487, found 419.0476.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.28 (t, J = 8.8 Hz, 1H),
7.22ꢀ7.31 (m, 1H), 7.49 (s, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.70ꢀ7.77
(m, 1H), 7.82ꢀ7.90 (m, 2H), 8.52 (d, J = 2.2 Hz, 1H), 8.72 (d, J = 2.2
Hz, 1H).
mmol), compound 7 (0.212 g, 0.514 mmol), PdCl2(dppf) CH2Cl2
3
N-(2-Chloro-5-(imidazo[1,2-b]pyridazin-6-yl)pyridin-3-yl)-
4-fluorobenzenesulfonamide (12). A 15 mL reaction vial was
charged with 6-chloroimidazo[1,2-b]pyridazine (60 mg, 391 μmol),
compound 7 (193 mg, 469 μmol), dioxane (2 mL), and water (1 mL).
The vial was sealed and purged with N2 for several minutes. Then it was
heated at 100 ꢀC for 1 h. After cooling to room temperature, the phases
were separated and the organic phase was dried over Na2SO4 and
concentrated under vacuum. Purification by silica gel chromatography
(1ꢀ4% MeOH/DCM) afforded 12 as a white solid (73 mg, 46% yield).
HRMS calculated for C17H11ClFN5O2S1 (M + H) 404.0378, found
404.0370. 1H NMR (400 MHz, DMSO-d6): δ ppm 7.45 (t, J = 8.8 Hz,
2H), 7.79ꢀ7.87 (m, 2H), 7.88 (d, J = 2.9 Hz, 2H), 8.30 (d, J = 9.4 Hz,
1H), 8.39 (d, J = 2.2 Hz, 1H), 8.43 (s, 1H), 8.92 (d, J = 2.0 Hz, 1H),
10.36ꢀ10.74 (m, 1H).
(0.0191 g, 0.0234 mmol), Cs2CO3 (0.457 g, 1.40 mmol), THF (3 mL),
and water (0.5 mL). The vial was capped and heated by microwave for
10 min at 100 ꢀC. The reaction mixture was diluted with water (10 mL),
DCM (10 mL), and satd NaCl (5 mL). The solution was extracted with
DCM (3 ꢁ 20 mL), and the combined extracts were dried (Na2SO4),
filtered, and concentrated in vacuo. The crude product was recrystallized
from 5:1 DCM/MeOH and Et2O. The crystallized material was
collected by filtration and washed with Et2O and dried under vacuum
to afford 9 as a tan crystalline solid (0.150 g, 77% yield). HRMS
calculated for C18H11ClFN3O2S2 (M + H) 420.0037, found 420.0028.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.42 (t, J = 8.8 Hz, 2H),
7.77ꢀ7.89 (m, 3H), 8.08 (d, J = 2.2 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H),
8.55 (d, J = 1.2 Hz, 1H), 8.63 (d, J = 2.2 Hz, 1H), 9.48 (s, 1H), 10.54 (br
s, 1H).
N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)imidazo[1,2-b]pyridazin-2-yl)acetamide (10). To a 250 mL,
round-bottomed flask was added N-(6-chloroimidazo[1,2-b]pyridazin-
2-yl)acetamide (2.50 g, 11.9 mmol),27ꢀ29 compound 7(5.39 g, 13.1 mmol),
DMSO (50 mL), and aq sodium carbonate (26.7 mL, 53.4 mmol, 2 M).
The flask was carefully evacuated and backfilled with N2. To the mixture
was added PdCl2(PPh3)2 (0.667 g, 0.950 mmol). The flask was carefully
evacuated and backfilled with N2 and then stirred at 90 ꢀC for 3 h. After
cooling to room temperature, the mixture was poured into a solution of
pH 4.8 buffer (300 mL, 3 M sodium acetate solution pH adjusted to 4.8)
and water (300 mL). A precipitate formed, and the solution was allowed
to sit overnight and then was filtered. The filtercake was washed with pH
4.8 buffer and water and then was dried by passing air through the filter.
The resulting solid was dissolved in DMF (125 mL) and treated with
Siliabond-TAAcONa (11.0 g, 5 equiv relative to mmol of Pd catalyst
used, loading = 0.43 mmol/gram, Silicycle brand palladium scavenger,
Silicycle). The mixture was heated at 50 ꢀC for 19 h. The solution was
filtered through Celite (diatomaceous earth), diluted with diethylamine,
filtered through a 0.45 um poly(tetrafluoroethylene) syringe filter, and
purified by preparative HPLC (Chiralpak ASH (21 mm ꢁ 250 mm,
5 μm) column, 25% B, hold for 0.5 min, ramp up to 45% B at 4% B/min
and then ramp up to 60% B at 50% B/min; hold for 2 min and
reequilibrate for 1.1 min. Total flow was 50 mL/min, outlet pressure
N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)imidazo[1,2-a]pyridin-2-yl)acetamide (13). To a 10 mL
round-bottomed flask was added compound 7 (0.14 g, 0.35 mmol),
N-(6-bromoimidazo[1,2-a]pyridin-2-yl)acetamide (0.080 g, 0.31
mmol),27ꢀ29 DMSO (3.0 mL), and aq Na2CO3 (0.94 mL, 1.9 mmol,
2 M). The flask was carefully evacuated and backfilled with N2. To the
mixture was added PdCl2(PPh3)2 (0.022 g, 0.031 mmol). The flask was
carefully evacuated and backfilled with N2 again and then stirred at 95 ꢀC
for 1 h. After cooling to room temperature, the mixture was poured into a
solution of pH 4.8 buffer (25 mL, 3 M sodium acetate pH adjusted to
4.8), and water (25 mL) and filtered. The filter cake was washed with
water (2ꢁ) and DCM (2ꢁ) and then dried under vacuum to afford 13
as
a tan solid (0.102 g, 70% yield). HRMS calculated for
1
C20H15ClFN5O3S1 (M + H) 460.0639, found 460.06445. H NMR
(400 MHz, DMSO-d6): δ ppm 2.08 (s, 3H), 7.16ꢀ7.23 (m, 3H), 7.48
(d, J = 9.2 Hz, 1H), 7.65 (d, J = 2.3 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H),
7.78ꢀ7.82 (m, 2H), 8.14 (s, 1H), 8.72 (s, 1H), 10.69 (s, 1H), 11.95
(br s, 1H).
N-(5-(2-Aminoimidazo[1,2-a]pyridin-6-yl)-2-chloropyri-
din-3-yl)-4-fluorobenzenesulfonamide (14). To a 10 mL,
round-bottomed flask was added compound 7 (0.150 g, 0.363 mmol),
6-bromoimidazo[1,2-a]pyridin-2-amine (0.0700 g, 0.330 mmol),
DMSO (3.0 mL), and aq Na2CO3 (0.990 mL, 1.98 mmol, 2 M). The
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Journal of Medicinal Chemistry
ARTICLE
flask was carefully evacuated and backfilled with N2. To the mixture was
added PdCl2(PPh3)2 (0.0232 g, 0.0330 mmol). Again, the flask was
carefully evacuated and backfilled with N2 and stirred at 95 ꢀC for 3 h.
After cooling to room temperature, the mixture was poured into pH 4.8
buffer (3 M sodium acetate solution pH adjusted to 4.8) and diluted with
water. The solution was filtered, and the filter cake was washed with
water and then 50% CH2Cl2/hexane. The aqueous washings were
extracted with 25% iPrOH/CHCl3, and the extracts were combined
with the filter cake. The combined material was concentrated to remove
all of the solvent and the residue was dissolved in MeOH/DCM and
concentrated onto silica. Purification by silica gel chromatography
(2.0ꢀ9.0% MeOH/DCM) afforded 14 as a yellow solid (0.0307 g,
22% yield). HRMS calculated for C18H13ClFN5O2S1 (M + H)
418.0534, found 418.0525. 1H NMR (400 MHz, DMSO-d6): δ
ppm 7.08 (s, 1H), 7.34 (br s, 3H), 7.38ꢀ7.44 (m, 3H), 7.60 (br s,
2H), 7.78ꢀ7.84 (m, 3 H), 7.97 (d, J = 2.2 Hz, 1H), 8.49 (d, J = 2.0 Hz,
1H), 8.77 (s, 1H).
N-(2-Chloro-5-(imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)-4-
fluorobenzenesulfonamide (15). A 15 mL reaction tube was
charged with compound 7 (130 mg, 315 μmol), 6-bromoimidazo[1,2-
a]pyridine (155 mg, 788 μmol), Pd(PPh3)4 (18 mg, 16 μmol), aq
Na2CO3 (0.47 mL, 0.95 mmol, 2 M), and EtOH (2 mL). The reaction
mixture was purged with N2, sealed and heated at 90 ꢀC for 4 h. After
cooling to room temperature, the solution was treated with satd
NaHCO3 and extracted with DCM (3ꢁ). The combined extracts were
washed with water and brine and then dried (Na2SO4) and concen-
trated. Purification by silica gel chromatography (1ꢀ4% MeOH (2 M in
NH3)/DCM) afforded 15 (40 mg, 32% yield) as an off-white solid.
HRMS calculated for C18H12ClFN4O2S1 (M + H) 403.0425, found
403.0443. 1H NMR (400 MHz, DMSO-d6): δ ppm 7.42 (t, J = 8.9 Hz,
2H), 7.57 (d, J = 9.2 Hz, 1H), 7.68 (s, 1H), 7.72 (d, J = 9.6 Hz, 1H),
7.78ꢀ7.87 (m, 2H), 8.03 (s, 1H), 8.07 (d, J = 2.0 Hz, 1H), 8.58 (s, 1H),
9.03 (s, 1H), 10.69 (br s, 1H).
C20H15ClFN5O3S1 (M + H) 460.0639, found 460.0659. 1H NMR
(400 MHz, DMSO-d6): δ ppm 2.18 (s, 3H), 7.37 (br s, 1H), 7.44 (t, J =
8.8 Hz, 2H), 7.56 (br s, 1H), 7.70 (br s, 1H), 7.83 (dd, J = 8.8, 5.3 Hz,
2H), 7.91 (s, 1H), 8.56 (br s, 1H), 10.44 (br s, 1H), 11.62 (br s, 1H),
12.16 (br s, 1H).
N-(5-(2-Amino-1H-benzo[d]imidazol-6-yl)-2-chloropyri-
din-3-yl)-4-fluorobenzenesulfonamide (17). To a resealable vial
was added 2-amino-5-bromobenzimidazole (0.170 g, 0.802 mmol),
compound 7 (0.430 g, 1.042 mmol), DMF (8.0 mL), and aq Na2CO3
(2.00 mL, 4.01 mmol, 2 M). The flask was carefully evacuated and
backfilled with N2. To the mixture was added PdCl2(PPh3)2 (0.056 g,
0.080 mmol). The flask was carefully evacuated and backfilled with N2
again and then stirred at 90 ꢀC for 2 h. After cooling to room
temperature, the mixture was poured into aq NaHCO3 (100 mL) and
extracted with 25% iPrOH/CHCl3 (3 ꢁ 75 mL). The combined extracts
were dried (Na2SO4) and concentrated in vacuo to a volume of
approximately 10 mL. The material was purified by Prep-HPLC
(Phenomenex Gemini 5 μm, C18, 100 mm ꢁ 30 mm, 5ꢀ60% CH3CN-
(0.1% TFA)/H2O(0.1% TFA) over 20 min then 100% CH3CN(0.1%
TFA) for 3 min at 20 mL/min) with the fractions containing suspected
product concentrated to afford 17 (mono-TFA salt) as an off-white solid
(0.0607 g, 14% yield). HRMS calculated for C18H13ClFN5O2S1 (M +
1
H) 418.0534, found 418.0518. H NMR (400 MHz, DMSO-d6): δ
ppm 7.39ꢀ7.55 (m, 3H), 7.61 (s, 1H), 7.76ꢀ7.85 (m, 2H), 7.98 (d, J =
2.4 Hz, 1H), 8.57 (d, J = 2.4 Hz, 1H), 8.61 (s, 1H), 12.75 (br s, 1H).
N-(5-(1H-Benzo[d]imidazol-5-yl)-2-chloropyridin-3-yl)-4-
fluorobenzenesulfonamide (18). To a resealable vial was added
5-bromo-1H-benzo[d]imidazole (0.0800 g, 0.41 mmol), compound 7
(0.20 g, 0.49 mmol), DMF (3.0 mL), and aq K2CO3 (1.0 mL, 2.0 mmol,
2 M). The flask was carefully evacuated and backfilled with N2, and
PdCl2(PPh3)2 (0.028 g, 0.041 mmol) was added. The flask was carefully
evacuated and backfilled with N2 again and heated at 100 ꢀC for 18 h.
After cooling to room temperature, the reaction mixture was poured into
a solution of pH 7 buffer (3.0 M NaCl and 0.3 M Sodium Citrate) and
water. The solution was filtered, and the filtrate was partially concen-
trated and extracted with 25% iPrOH/CHCl3 (3 ꢁ 30 mL). The
combined extracts were washed with satd aq NaCl (25 mL) and then
dried (Na2SO4) and concentrated onto silica. Purification by silica gel
chromatography (1ꢀ10% MeOH/CH2Cl2) afforded 18 as a white solid
(0.034 g, 21% yield). HRMS calculated for C18H12ClFN4O2S1 (M + H)
403.0425, found 403.0438. 1H NMR (400 MHz, DMSO-d6): δ
ppm 7.40ꢀ7.49 (m, 3H), 7.72 (d, J = 8.4 Hz, 1H), 7.79ꢀ7.86 (m,
2H), 7.87 (s, 1H), 7.97 (d, J = 2.4 Hz, 1H), 8.33 (s, 1H), 8.60 (d, J = 2.4
Hz, 1H), 11.60 (br s, 1H).
N-(5-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)-1H-benzo[d]imidazol-2-yl)acetamide (16). N-(6-Bromo-
1H-benzo[d]imidazol-2-yl)acetamide. To a 250 mL, round-bottomed
flask was added 6-bromo-1H-benzo[d]imidazol-2-amine (2.00 g, 9.43
mmol), DCM (100 mL), pyridine (1.0 mL, 12 mmol), and acetic
anhydride (0.979 mL, 10.4 mmol). The mixture was stirred at 25 ꢀC for
18 h. The solution was concentrated and then diluted with 10% MeOH
(2 M in NH3)/CH2Cl2 until homogeneous and then concentrated onto
silica. Purification by silica gel chromatography (0.5ꢀ5.0% MeOH (2 M
in NH3)/CH2Cl2) afforded the title compound as a white solid (0.676 g,
28% yield). MS (ESI, positive ion) m/z: 254 (M (79Br) + 1).
N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)benzo[d]oxazol-2-yl)acetamide (24). N-(Benzo[d]oxazol-2-
yl)acetamide. To a round bottomed flask was added benzo[d]oxazol-
2-amine (0.50 g, 3.7 mmol) and DCM (15 mL). Pyridine (0.30 mL, 3.7
mmol), DMAP (0.023 g, 0.19 mmol), and acetic anhydride (0.37 mL,
3.9 mmol) were added to the mixture. After stirring overnight at room
temperature, the mixture was diluted with water and extracted with
DCM (4 ꢁ 30 mL). The combined organic extracts were dried
(Na2SO4), filtered, and concentrated in vacuo. The crude product was
recrystallized from DCM/hexanes to afford the title compound as a
light-yellow crystalline solid (0.450 g, 69% yield). MS (ESI, positive ion)
m/z: 177.1 (M + 1). 1H NMR (400 MHz, DMSO-d6): δ ppm 2.23 (s,
3H), 7.23ꢀ7.34 (m, 2H), 7.54ꢀ7.63 (m, 2H), 11.62 (s, 1H).
N-(5-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)-1H-benzo-
[d]imidazol-2-yl)acetamide. To a 25 mL round-bottomed flask was
added compound 5 (0.150 g, 0.410 mmol), bis(pinacolato)diboron
(0.156 g, 0.615 mmol), potassium acetate (0.161 g, 1.64 mmol), and 1,4-
dioxane (4.0 mL). The flask was carefully evacuated and backfilled with
N2. To the solution was added PdCl2(dppf) CH2Cl2 (0.030 g, 0.041
3
mmol). The flask was carefully evacuated and backfilled with N2 again
and stirred at 90 ꢀC for 6 h. The reaction mixture was allowed to cool to
room temperature and to the mixture was added DMF (4.0 mL), N-(5-
bromo-1H-benzo[d]imidazol-2-yl)acetamide (0.080 g, 0.31 mmol), and
aq Na2CO3 (0.79 mL, 1.6 mmol, 2 M). The flask was carefully evacuated
and backfilled with N2. To the mixture was added PdCl2(PPh3)2 (0.022
g, 0.031 mmol). The flask was carefully evacuated and backfilled with N2
again and was stirred at 90 ꢀC for 16 h. After cooling to room
temperaure, the solution was poured into satd NaCl (100 mL) and
extracted with 25% iPrOH/CHCl3 (3 ꢁ 75 mL). The combined extracts
were dried (Na2SO4) and concentrated. The material was dissolved in
MeOH/CH2Cl2, concentrated onto silica, and purified by silica gel
chromatography (0.5ꢀ5.0% MeOH (2 M in NH3)/CH2Cl2) to afford
16 as a white solid (0.0217 g, 15% yield). HRMS calculated for
N-(6-Bromobenzo[d]oxazol-2-yl)acetamide (22). To a round-bot-
tomed flask was added 50% acetic acid (8 mL, 4 mmol), followed by
N-(benzo[d]oxazol-2-yl)acetamide (0.800 g, 5 mmol). DCM (5 mL)
was added to the mixture, and the mixture was stirred until homo-
geneous. Bromine (0.5 mL, 9 mmol) was then added slowly while
stirring under an inert atmosphere at room temperature. After 1.5 h, the
mixture was diluted with DCM (5 mL) and water (5 mL), and then
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Journal of Medicinal Chemistry
ARTICLE
sodium thiosulfate was added. After stirring for 15 min, the solution was
extracted with DCM (3 ꢁ 20 mL). The combined extracts were dried
(Na2SO4), filtered, and concentrated in vacuo. The crude product was
recrystallized from DCM/hexanes to afford 22 as an off-white crystalline
solid (0.85 g, 73% yield). MS (ESI, positive ion) m/z: 257.0 (M + 1). 1H
NMR (400 MHz, DMSO-d6): δ ppm 2.22 (s, 3H), 7.45ꢀ7.54 (m, 2H),
7.94 (s, 1H), 11.73 (s, 1H).
Potassium trifluoroborate salt 23. To a round-bottomed flask was
added compound 22 (0.635 g, 2.49 mmol), bis(pinacolato)diboron
(3.16 g, 12.4 mmol), potassium acetate (0.733 g, 7.47 mmol) and 1,4-
dioxane (15 mL). Argon was bubbled into the mixture for 10 min, and
7.60 (s, 2H), 7.68 (s, 1H), 7.78ꢀ7.83 (m, 2H), 7.92 (d, J = 2.4 Hz, 1H),
8.55 (d, J = 1.8 Hz, 1H), 10.44 (br s, 1H).
N-(5-(Benzo[d]oxazol-6-yl)-2-chloropyridin-3-yl)-4-fluor-
obenzenesulfonamide (20). To a microwave vial was added
6-bromobenzo[d]oxazole (0.050 g, 0.25 mmol), Cs2CO3 (0.25 g, 0.76
mmol), PdCl2(dppf) DCM (0.037 g, 0.045 mmol), compound 7 (0.10
3
g, 0.25 mmol), THF (3 mL), and water (0.5 mL). The vial was capped
and then stirred and heated by microwave for 10 min at 100 ꢀC. The
mixture was diluted with water and extracted with DCM. The combined
organic extracts were dried (Na2SO4), filtered, and concentrated. The
crude product was recrystallized from 5:1 DCM/MeOH and hexanes to
afford 20 as a tan solid (0.040 g, 39% yield). HRMS calculated for
C18H11ClFN3O3S1 (M + H) 404.0265, found 404.0270. 1H NMR (400
MHz, DMSO-d6): δ ppm 7.44 (t, J = 8.8 Hz, 2H), 7.71 (d, J = 8.0 Hz,
1H), 7.82 (dd, J = 5.5, 8.0 Hz, 2H), 7.95 (d, J = 8.0 Hz, 1H), 8.07 (s, 1H),
8.17 (s, 1H), 8.66 (s, 1H), 8.85 (s, 1H), 10.53 (br s, 1H).
then PdCl2(dppf) DCM (1.02 g, 1.24 mmol) was added. The solution
3
was stirred at 100 ꢀC for 2 days. After cooling to room temperature, the
reaction mixture was diluted with satd NaCl and extracted with DCM
(4 ꢁ 50 mL). The combined extracts were dried (Na2SO4), filtered, and
concentrated. Purification by silica gel chromatography (10ꢀ100%
EtOAc/hexanes) afforded a tan oil. This material was dissolved in
acetonitrile (10 mL), and potassium hydrogen fluoride (0.731 g, 9.4
mmol) was added and the mixture was stirred at room temperature
overnight. The solution was then concentrated in vacuo to afford 23 as a
light-yellow solid (0.100 g, 14% yield). MS (ESI, positive ion) m/z: 221
(M (boronic acid) + 1). 1H NMR (400 MHz, MeOH-d4): δ ppm 2.24
(s, 3H), 7.30ꢀ7.43 (m, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.53 (s, 1H).
N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)benzo-
[d]oxazol-2-yl)acetamide (24)To a microwave vial was added com-
pound 23 (0.16 g, 0.57 mmol), compound 5 (0.140 g, 0.38 mmol),
N-(5-([1,2,4]Triazolo[4,3-a]pyridin-6-yl)-2-chloropyridin-
3-yl)-4-fluorobenzenesulfonamide (21). To a 15 mL resealable
vial was added 6-bromo-[1,2,4]triazolo[4,3-a]pyridine (60 mg,
303 μmol), compound 7 (150 mg, 364 μmol), PdCl2(dppf) DCM
3
(17 mg, 23 μmol), Na2CO3 (96 mg, 909 μmol), 1,4-dioxane (2 mL), and
water (1 mL). The vial was purged with nitrogen for several minutes and
sealed. The reaction mixture was then heated at 100 ꢀC for 1 h. After
cooling to room temperature, the organic phase was dried (Na2SO4) and
concentrated. Purification by silica gel chromatography (1ꢀ4% MeOH/
DCM) afforded 21 as a light-yellow solid (70 mg, 57% yield). HRMS
calculated for C17H11ClFN5O2S1 (M + H) 404.0378, found 404.0382.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.43 (t, J = 8.8 Hz, 2H), 7.7 (d,
J = 9.8 Hz, 1H), 7.82 (dd, J = 5.1, 8.8 Hz, 2H), 7.93 (d, J = 10.0 Hz, 1H),
8.14 (d, J = 2.2 Hz, 1H), 8.64 (s, 1H), 9.03 (s, 1H), 9.32 (s, 1H), 10.56
(br s, 1H).
PdCl2(dppf) DCM (0.016 g, 0.019 mmol), Cs2CO3 (0.37 g, 1.1 mmol),
3
THF (3 mL), and water (0.5 mL). The vial was capped and then stirred
and heated by microwave for 10 min at 100 ꢀC. The mixture was diluted
with water (10 mL) and satd NaCl (5 mL). The solution was extracted
with DCM (4 ꢁ 25 mL), and the combined extracts were dried
(Na2SO4), filtered, and concentrated. Purification by reverse-phase
HPLC afforded 24 (TFA salt) as a tan solid (0.019 g, 11% yield).
HRMS calculated for C20H14ClFN4O4S1 (M + H) 461.0479, found
461.0485. 1H NMR (400 MHz, DMSO-d6) δ ppm 3.10 (s, 3H), 7.12 (s,
1H), 7.42ꢀ7.45 (m, 2H), 7.81ꢀ7.85 (m, 3 H), 8.23 (d, J = 7.2 Hz, 1H),
8.44 (s, 1H), 10.40ꢀ10.48 (m, 2H), 10.70 (s, 1H), 10.91 (br s, 1H).
N-(5-(2-Aminobenzo[d]oxazol-6-yl)-2-chloropyridin-3-yl)-
4-fluorobenzenesulfonamide (19). 6-Bromobenzo[d]oxazol-2-
amine. To a round-bottomed flask was added benzo[d]oxazol-2-amine
(0.59 g, 4.0 mmol) followed by 50% acetic acid (2 mL, 1 mmol).
Bromine (0.11 mL, 2 mmol) was added slowly, and the mixture was
stirred for 1.5 h at room temperature. The mixture was diluted with
DCM (5 mL) and water (5 mL), and then sodium thiosulfate was added.
After stirring for 15 min, the solution was extracted with DCM (3 ꢁ
20 mL), and the combined organic extracts were dried (Na2SO4),
filtered, and concentrated in vacuo. Purification by silica gel chroma-
tography (5ꢀ50% EtOAc/DCM) afforded the title compound as a light-
yellow solid (0.60 g, 64% yield). MS (ESI, positive ion) m/z: 214.9 (M + 1).
1H NMR (400 MHz, DMSO-d6): δ ppm 7.07ꢀ7.20 (m, 1H),
7.23ꢀ7.36 (m, 1H), 7.55 (s, 2H), 7.60 (d, J = 2.0 Hz, 1H).
N-(5-(Benzo[d]isothiazol-5-yl)-2-chloropyridin-3-yl)-4-fluoro-
benzenesulfonamide (27). 5-(4,4,5,5-Tetramethyl-1,3,2-dioxabor-
olan-2-yl)benzo[d]isothiazole (26). To a 250 mL, round-bottomed flask
was added bis(pinacolato)diboron (5.6 g, 22 mmol), 5-bromobenzo-
[d]isothiazole (25, 3.63 g, 17 mmol),30 potassium acetate (2.5 g, 25
mmol), 1,4-dioxane (100 mL), tricyclohexylphosphine (0.68 g, 2.4
mmol), and tris(dibenzylideneacetone)dipalladium (0) (0.93 g, 1.0
mmol). The mixture was stirred at 80 ꢀC overnight. After cooling to
room temperature, the solution was concentrated and purified by silica
gel chromatography (0ꢀ40% EtOAc/hexane) to afford 26 (3.2 g, 72%
yield). 1H NMR (400 MHz, CDCl3): δ ppm 1.41 (s, 12H), 7.91ꢀ8.02
(m, 2H), 8.58 (s, 1H), 8.95 (s, 1H).
N-(5-(Benzo[d]isothiazol-5-yl)-2-chloropyridin-3-yl)-4-fluoroben-
zenesulfonamide (27). To a 5 mL microwave vial was added boronic
ester 26 (0.070 g, 0.30 mmol), compound 5 (0.10 g, 0.30 mmol), aq
Na2CO3 (0.4 mL, 0.8 mmol, 2 M), FibreCat 1029 triphenylphosphine
(20 wt %, 15 mg), and 1,4-dioxane (3 mL). The vial was sealed and then
stirred and heated by microwave for 20 min at 120 ꢀC. The reaction
mixture was diluted with water and extracted with DCM (3 ꢁ 10 mL).
The combined extracts were washed with water and satd aq NaCl and
then dried (MgSO4) and concentrated. Purification by silica gel
chromatography (15% acetone/hexane) afforded 27 as a light-yellow
solid (60.0 mg, 48%). HRMS calculated for C18H11ClFN3O2S2 (M + H)
420.0037, found 420.0042. 1H NMR (300 MHz, CHCl3-d) δ ppm 7.03
(br s, 1H), 7.13ꢀ7.23 (m, 2H), 7.73 (dd, J = 8.5, 1.8 Hz, 1H), 7.80ꢀ7.89
(m, 2H), 8.12 (d, J = 8.5 Hz, 1H), 8.25 (s, 1H), 8.30 (s, 1H), 8.47 (d, J =
2.3 Hz, 1H), 9.04 (s, 1H).
N-(5-(2-Aminobenzo[d]oxazol-6-yl)-2-chloropyridin-3-yl)-4-fluoro-
benzenesulfonamide (19). To a microwavevial wasadded 6-bromobenzo-
[d]oxazol-2-amine (0.075 g, 0.35 mmol), Cs2CO3 (0.34 g, 1.1 mmol),
PdCl2(dppf) DCM (0.029 g, 0.035 mmol), compound 7 (0.17 g, 0.42
3
mmol), THF (3 mL), and water (0.2 mL). The vial was capped and then
stirred and heated by microwave for 10 min at 100 ꢀC. The mixture was
diluted with water and extracted with DCM (4 ꢁ 25 mL). The combined
extracts were dried (Na2SO4), filtered, and concentrated. The crude
product was recrystallized from MeOH and then washed with Et2O to
afford 19 as a tan solid (0.030 g, 20% yield). HRMS calculated for
C18H12ClFN4O3S1 (M + H) 419.0374, found 419.0376. 1H NMR (400
MHz, MeOH-d): δ ppm 7.30 (d, J = 8.2 Hz, 1H), 7.36ꢀ7.47 (m, 3H),
Hepatocyte Incubation Conditions. Compounds (20 μM)
were incubated in freshly isolated hepatocytes at 2 million cells/mL in
KrebsꢀHenseleit buffer for 0 h (control) or 2 h at 37 ꢀC in a shaking
water bath saturated with 95% oxygen/5% carbon dioxide. Reactions
were quenched with an equal volume of acetonitrile containing 0.3%
formic acid, vortex-mixed, and centrifuged at 14000 rpm for 10 min.
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Journal of Medicinal Chemistry
ARTICLE
Supernatants were separated by reverse phase HPLC (Agilent 1100,
Delaware, DE) on a C18 column (Waters C18, 2.0 mm ꢁ 150 mm,
3 μm) in-line with an ion-trap mass spectrometer (LTQ; Thermo
Fisher Inc., San Jose, CA) employing electrospray ionization in positive
ion mode.
’ ASSOCIATED CONTENT
S
Supporting Information. Plasma and tumor concentra-
b
tions from in vivo PD assay, calculation methods of ED50 and
AUC at EC50 and AMBIT KINOMEscan selectivity data for
compound 10. This material is available free of charge via the
Internet at http://pubs.acs.org.
Akt Phosphorylation in the U87 MG Tumor Xenograft
Model: Dose Response and Time Course PD Assay. The effect
of compound 10 on Akt phosphorylation was evaluated in the U87 MG
glioblastoma tumor model grown in female CD1 NU/NU mice. Mice
were implanted subcutaneously with 5 ꢁ 106 U87 MG cells. Treatment
with compound 10 at 0.3, 1, or 3 mg/kg by oral administration was
initiated when tumor size achieved ∼300ꢀ400 mm3. Mice were
sacrificed at 3, 8, 16, and 24 h after a single dose and tumors were
immediately removed and snap frozen. Levels of phosphorylated Akt
versus total Akt were determined by a quantitative MSD assay. Akt
phosphorylation measured in tumors from mice treated with vehicle
served as a negative control and indicated the background level of
phosphorylation.
U87 Tumor Xenograft pAkt (S473) MSD Assay Protocol.
Each U87 tumor xenograft was homogenized in 1 mL of ice-cold MSD
lysis buffer (containing 150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM
EDTA, 1 mM EGTA, 1% Triton-X-100, 1/10 protease inhibitor tablet
(Roche, catalogue no. 1836170), 20 μL of phosphatase inhibitor I
(Sigma-Aldrich, Inc., catalogue no. P-2850), 20 μL of phosphatase
inhibitor II (Sigma-Aldrich, Inc., catalogue no. P-5726), and 40 mM
NaF) with GenoGrinder 2000 (BT&C Inc., product no. SP2100ꢀ115)
at 350 strokes per min for 2 min at room temperature. The lysate was
centrifuged twice in a 1.5 mL Eppendorf microcentrifuge tube at
14000 rpm for 15 min at 4 ꢀC. The supernatant was precleared with
Protein A/G beads (PIERCE, product no. 20421) in an Eppendorf
microcentrifuge tube at 4 ꢀC for 1 h. The beads were centrifuged at
14000 rpm for 15 min at 4 ꢀC, and the supernatant was transferred to a
clean Eppendorf microcentrifuge tube. The protein concentration of
each sample was determined using Bio-Rad DC protein assay (Bio-Rad,
catalogue no. 500ꢀ0116) and normalized to a final concentration of
1 mg/mL.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 805-447-8006. E-mail: mstec@amgen.com. Address:
One Amgen Center Drive, Thousand Oaks, California 91320-
1799, United States
’ ACKNOWLEDGMENT
We thank Yihong Zhou and Shuguang Ma for the metabolite
profiling studies, Mike Hayashi for the hepatocyte incubations,
Paul Andrews for his assistance with the in vitro U87 MG cellular
assay, and Paul Schnier for HRMS analyses.
’ ABBREVIATIONS USED
ANOVA, analysis of variance; AUC, area under the curve; dba,
dibenzylideneacetone; DCM, dichloromethane; DMAP, 4-dimethyl-
aminopyridine; dppf, 1,10-bis(diphenylphosphino)ferrocene; EC,
effective concentration; ED, effective dose; EGTA, ethylene gly-
col bis(2-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; HPMC,
hydroxypropylmethylcellulose; IC, inhibitory concentration;
MSD, mesoscale discovery; mTOR, mammalian target of rapa-
mycin; NaHMDS, sodium hexamethyldisilazide (sodium bis(tri-
methylsilyl)amide); PD, pharmacodynamic; PI3K, phosphoinositide
3-kinase; PIKK, PI3K-related kinases; POC, percentage of con-
trol; PTEN, phosphotase and tensin homologue; QD, once daily;
SAR, structureꢀactivity relationship; SD, standard deviation; SE,
standard error; TFA, trifluoroacetic acid
The levels of phosphorylated Akt (S473) and total Akt in tumor
xenograft lysates were detected using Meso Scale Discovery phospho-
Akt (S473) (catalogue no. K151CAD-2) and total Akt (catalogue no.
K151CBD-2) whole cell lysate kits: pAkt (S473) MSD plate and total
Akt MSD plate were blocked with 150 μL MSD blocking solution
(containing 3% bovine serum albumin, 50 mM Tris, pH 7.5, 150 mM
NaCl and 0.02% Tween-20) and incubated at room temperature for 1 h.
The plates were then washed four times with 300 μL of MSD Tris wash
buffer (containing 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.02%
Tween-20). Tumor xenograft lysate (25 μL, 1 mg/mL) was dispensed to
each well of the MSD plate. The plate was incubated with shaking in an
Eppendorf thermomixer R (Eppendorf, product no. 22670107) at
600 rpm at room temperature for 2 h. The plate was then washed four
times with 300 μL of MSD Tris wash buffer. Anti-pAkt (S473) or
antitotal Akt detection antibody solution (25 μL, 1.5 μg/mL) was added
to each well of the MSD plate. The plate was incubated with shaking in
an Eppendorf thermomixer R at 600 rpm at room temperature for 1 h.
The plate was then washed four times with 300 μL of MSD Tris wash
buffer. MSD read buffer (150 μL, 1ꢁ) was added to each well of the
MSD plate. The intensity of pAkt (S473) or total Akt signal was
measured using MSD sector imager 6000.
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