Spirolactam-based acetyl-CoA carboxylase inhibitors: Toward improved metabolic stability of a chromanone lead structure

Article abstract of DOI:10.1021/jm401033t

Acetyl-CoA carboxylase (ACC) catalyzes the rate-determining step in de novo lipogenesis and plays a crucial role in the regulation of fatty acid oxidation. Alterations in lipid metabolism are believed to contribute to insulin resistance; thus inhibition of ACC offers a promising option for intervention in type 2 diabetes mellitus. Herein we disclose a series of ACC inhibitors based on a spirocyclic pyrazololactam core. The lactam series has improved chemical and metabolic stability relative to our previously reported pyrazoloketone series, while retaining potent inhibition of ACC1 and ACC2. Optimization of the pyrazole and amide substituents led to quinoline amide 21, which was advanced to preclinical development.

Full text of DOI:10.1021/jm401033t

Article
pubs.acs.org/jmc
Spirolactam-Based Acetyl-CoA Carboxylase Inhibitors: Toward
Improved Metabolic Stability of a Chromanone Lead Structure
David A. Griffith, Robert L. Dow, Kim Huard,* David J. Edmonds, Scott W. Bagley, Jana Polivkova,
Dongxiang Zeng, Carmen N. Garcia-Irizarry, James A. Southers, William Esler, Paul Amor,
Kathrine Loomis, Kirk McPherson, Kevin B. Bahnck, Cathy Preville, Tereece Banks, Dianna E. Moore,
́
Alan M. Mathiowetz, Elnaz Menhaji-Klotz, Aaron C. Smith, Shawn D. Doran, David A. Beebe,
and Matthew F. Dunn
Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States
*
S Supporting Information
ABSTRACT: Acetyl-CoA carboxylase (ACC) catalyzes the
rate-determining step in de novo lipogenesis and plays a crucial
role in the regulation of fatty acid oxidation. Alterations in lipid
metabolism are believed to contribute to insulin resistance;
thus inhibition of ACC offers a promising option for
intervention in type 2 diabetes mellitus. Herein we disclose a series of ACC inhibitors based on a spirocyclic pyrazololactam
core. The lactam series has improved chemical and metabolic stability relative to our previously reported pyrazoloketone series,
while retaining potent inhibition of ACC1 and ACC2. Optimization of the pyrazole and amide substituents led to quinoline
amide 21, which was advanced to preclinical development.
INTRODUCTION
mitochondrial membrane and is responsible for regulation of
fatty acid oxidation via inhibition of CPT-I mediated transport
of fatty acids into the mitochondria. Thus, a dual ACC1/2
inhibitor may offer the greatest potential benefit, by
simultaneously reducing lipid production and increasing lipid
oxidation.
■
Type 2 diabetes mellitus (T2DM) is associated with a
fundamental imbalance in lipid metabolism, and these
alterations are hypothesized to contribute to the molecular
pathogenesis of insulin resistance. In the insulin-resistant state,
fatty acid oxidation is suppressed and the rate of hepatic de novo
lipogenesis (DNL) is increased. These alterations are believed
to result in a net ectopic accumulation of lipid species in liver
and skeletal muscle, which in turn have been hypothesized to
7
1
Screening of the Pfizer compound collection and subsequent
structure-based drug design led to the identification o8f
spiroketone-derived ACC inhibitors containing chromanone
1
9
contribute to the pathogenesis of insulin resistance. This
and then ketopyrazole cores. Optimization of the latter series
hypothesis is supported by the inverse relationship observed in
rodent models and humans between insulin sensitivity and both
led to the identification of compound 1, a potent and balanced
10
ACC1/2 inhibitor. The optimization process focused on
2
3
11
hepatic and intramyocellular triglyceride levels. Acetyl-CoA
carboxylase (ACC) serves as a critical switch regulating the
transition from lipogenic to oxidative lipid metabolism.
Inhibition of ACC activity is expected to have a favorable
impact on both skeletal muscle and hepatic insulin sensitivity
by rebalancing fundamental alterations in lipid metabolism
associated with the pathogenesis of insulin resistance.
Consistent with this hypothesis, suppression of ACC activity
lipophilic ligand efficiency (LipE) as a key measure of success,
which facilitated the identification of compounds with desirable
pharmacokinetic properties. Compound 1 has moderate
12
measured lipophilicity (E log D = 1.4), and thus low turnover
13
in human liver microsomal incubation (HLM), good passive
permeability in Ralph Russ canine kidney cells (RRCK, Papp =
−6
14
9
.7 × 10 cm/s), and a clean safety profile as predicted by in
vitro assays. However, during preclinical development, ketone 1
4
5
by knockout, antisense oligonucleotides, or small molecule
15
was observed to undergo species-dependent reduction to the
6
inhibitors has been shown to reduce tissue malonyl-CoA
corresponding alcohol, which complicated human pharmacoki-
netic predictions. Furthermore, the chromanone-like spirocyclic
core is prone to a base-mediated retro-Michael ring-opening
levels, increase fatty acid oxidation or inhibit lipogenesis, reduce
triglyceride accumulation in skeletal muscle or liver or both,
and improve insulin sensitivity in rodents. Consequently,
pharmacologic ACC inhibitors may have utility in the treatment
of T2DM.
Two isoforms of ACC (ACC1 and 2) are found in
mammalian tissue. ACC1 is widely expressed, with higher
levels in the liver and adipose tissue, and supplies the malonyl-
CoA needed for DNL. ACC2 is associated with the
16
reaction, which limits further chemical transformations. We
therefore sought alternative series lacking the ketone functional
group.
Received: July 8, 2013
Published: August 12, 2013
©
2013 American Chemical Society
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1
9
Earlier efforts to replace the ketone group had identified
1. Full details of this work have been reported separately. N1-
and N2-substituted spirolactams were accessed from regioiso-
meric pyrazoles via alkylation of a piperidine carboxylic acid
derivative followed by Curtius rearrangement to install the
required nitrogen-based functional group. Lithiation and ring
closure onto the isocyanate group forged the spirocyclic ring
system, with deprotection and amidation providing the final
analogs.
17
spirocyclic salicylamide derivatives, such as 2, as modest ACC
18
inhibitors (Figure 1). We reasoned that modification of the
19
Pyrazole 5 was prepared to allow direct comparison with
the ketone and aminal analogs (Figure 3). As hypothesized, the
Figure 1. ACC inhibitors 1 and 2 from the spirocyclic ketone and
salicylamide series. IC50 values are the arithmetic mean of at least three
replicates ± SEM. Each individual IC50 value is generated from an 11-
point dose−response curve run in triplicate. A detailed protocol can be
found in the Experimental Section. LipE values are calculated using E
log D.
2
1
Figure 3. Spirolactam ACC inhibitors. The synthesis of 5 has been
previously reported.
aromatic region of 2 to the pyrazole motif of 1 would improve
potency and LipE, as observed in the ketone series. The 5,6-
1
9
9
fused spirocyclic aminal 3 was not isolated when starting from
the corresponding hydroxypyrazole amide, presumably due to
the angle between the hydroxyl and carboxamido substituents
of the 5-membered heterocycle, which imparts unfavorable
strain on the cyclic aminal. We therefore designed spirolactam
structure 4 in which the oxygen linker is replaced by a
methylene group (Figure 2). Co-crystallization of the ACC
t-butylpyrazole system in 5 significantly improved LipE
compared with aminal 2, where improved potency coincided
with reduced lipophilicity. Although we had hypothesized that
the increased H-bond acceptor strength of the amide carbonyl
might improve ACC affinity, the reduced LipE of 5 compared
with ketone 1 suggested that the increased desolvation penalty
for the amide versus the ketone was a dominating effect and
that further optimization was needed.
The low measured lipophilicity of 5 relative to the ketone
series suggested that small lipophilic substituents could be
added to improve potency without adversely increasing HLM
metabolism. The 3,7-dimethylindazole “tail” group, which was
8
,9
consistently among the most potent in related series, was
coupled with the lactam core (Figure 3). The resulting analog 6
had improved potency and LipE, but was found to be a time-
Figure 2. Lactam-based ketone replacements.
20
carboxyltransferase domain and 1 indicated the presence of a
hydrogen bond between the ketone of 1 and the backbone of
dependent inhibitor of CYP3A4, likely due to oxidative
metabolism of the methyl substituents. Both compounds
suffered from poor passive permeability. We therefore sought
to further explore the SAR of the lactam series with the goal of
maintaining a balance between ACC inhibitory potency and
clearance, while removing the liabilities associated with 5 and 6.
Series Optimization. Our investigation first focused on
pyrazole substitution where the synthetic sequence depicted in
Scheme 1 was used to prepare the spirolactam core with
different N-substituents (Table 1). Based on the knowledge
gained from our previous work with similar series, the
8
the enzyme. We hypothesized that, in addition to circum-
venting in vivo ketone reduction, replacement of the ketone
with a lactam would further strengthen this hydrogen bond and
thereby improve affinity for ACC. Here, we describe the
discovery and optimization of this series of ACC inhibitors.
RESULTS
■
Synthesis. The spirolactam cores described herein were
prepared according to the general sequence shown in Scheme
19
Scheme 1. Synthesis of N1- and N2-Susbtituted Spirolactam Cores and Elaboration To Form Final Amides
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Table 1. Pyrazole SAR
a
b
Calculated values. Derived from calculated E log D.
a
Scheme 2. Synthesis of N-Methylated Lactam 14
a
Reagents and conditions: (a) NaH, MeI, DMF, 0 °C, 86%; (b) HCl, dioxane, 95%; (c) T3P, Et N, 3,7-dimethyl-1H-indazole-5-carboxylic acid,
3
3
8%. See Experimental Section for details.
exploration was limited to small secondary or tertiary alkyl
groups. As illustrated with i-propyl and c-butyl substituted
permeability. The N1-substituted regioisomer was synthetically
accessible with the spirolactam core, and we were able to test
9
19
pyrazoles 7 and 8, the replacement of the t-butyl group by a
secondary group proved detrimental to potency. Compared
with 6, the more lipophilic t-pentyl group (9) showed similar
potency and permeability but higher turnover in HLM. The t-
butyl group therefore appeared to be the optimal substituent
for the N-2 lactam regioisomer.
As a way of improving passive permeability, we sought to
sterically mask the inherent polarity of the lactam functionality
with an adjacent alkyl group. This would increase the overall
hydrophobic character of the molecule and thereby improve
this hypothesis. As exemplified with 10 and 11, substitution at
the N1 position led to the expected higher E log D values and
significantly improved permeability. However, the increased
lipophilicity did not provide a corresponding increase in
potency, with the lower LipE values indicating that the N-1
alkyl lactams did not inhibit ACC as efficiently as their
respective N-2 counterparts (6 and 7).
An alternate strategy to increase the passive permeability of
this core was to N-alkylate the lactam to eliminate an H-bond
donor, and lactam 14 was prepared (Scheme 2). Although N-
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Table 2. SAR on the Fused Aryl Bicyclic Portion
methylation led to the expected improvement in permeability,
it was detrimental to ACC inhibitory potency.
To improve permeability in the series, we sought analogues
without an H-bond donor on the heterocyclic tail. In practice,
replacement of the “tailpiece” with a heterocyclic 6,6-fused ring
system such as 20 proved to be a breakthrough for the lactam
series. Although the potency was modest, it provided a
combination of good permeability and low predicted clearance.
Our design efforts in the fused 6,6-ring systems sought to
combine good permeability and low turnover in HLM while
optimizing potency. Unsurprisingly, the lipophilic character of
naphthalene ring systems resulted in high turnover in HLM,
and this scaffold was not pursued. We systematically evaluated
methoxy-substituted regioisomers of quinolines and isoquino-
lines to identify the most efficient ACC inhibitor (data not
shown). Quinoline 21 and isoquinoline 22 were identified as
possessing an optimum profile (Table 3). Compounds 21 and
Co-crystal structures of inhibitors bound to the ACC
carboxyltransferase domain suggest adequate room for the
methyl group, so it seems likely that the reduced affinity is the
result of the increased dipole of the tertiary lactam compared
with the NH lactam. Indeed, E log D was essentially unchanged
by N-methylation.
Having found the N2-t-butyl substituent to be the most
efficient in terms of LipE, we next investigated ways to improve
permeability by modifying the “tailpiece” of the molecule (R
group in Table 2). To this end, a wide range of substituted 5,6-
fused heteroaryl derivatives were investigated, with an emphasis
on small substituents with a low risk of metabolic activation to
form reactive metabolites. Potent indazoles (e.g., compounds
22 potently inhibit ACC, are stable in human liver microsomes
15 and 16, Table 2) with good LipE and low clearance were
(
<8 and 12 μL/(min·mg), respectively), and possess good
identified; however improvements in permeability were modest.
Most other 5,6-fused heteroaryl systems that were evaluated
showed reduced activity. Good potency could be achieved with
the more lipophilic regioisomeric chloroindoles 17 and 18, with
a resulting improvement in permeability. The increased
lipophilicity was not efficiently utilized as reflected by the
lower LipE values of the indole analogues and resulted in
higher microsomal clearance. In an attempt to mitigate this
issue, the more polar azaindole 19 was prepared. While 18 and
−6
−6
passive permeability (11 × 10 and 9.7 × 10 cm/s,
respectively).
Solubilizing functionality was introduced to mitigate the
potential for solubility-limited oral exposure of crystalline
materials at higher doses. Replacement of the methoxy groups
in 21 (pK = 2.2) and 22 with substituted amino groups was
a
investigated to introduce a weakly basic site to promote
solubilization in the stomach and offer the potential for salt
formation (Table 3). As predicted, N-methyl substituted amine
19 are equipotent, the increased polarity resulted in low passive
23 (pK = 6.6) showed improved thermodynamic solubility
a
permeability.
(4.8 ug/mL) compared with its methoxy counterpart 21 (0.49
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Table 3. SAR on the Quinoline and Isoquinoline
ug/mL) as determined with crystalline material at pH 1.2. Both
regioisomeric methyl substituted amines (23 and 24) were
potent ACC inhibitors; however, they possessed low
permeability and low-to-moderate turnover in HLM. With
the aim of improving passive permeability, analogues having a
wide range of amino substituents were prepared by reaction of
chloroquinoline 25 with various amines (Scheme 3). As
illustrated with 26 and 27, longer alkyl chains or disubstitution
on the amino group resulted in modest improvements in
passive permeability, although with poor microsomal stability
line 22 were selected as lead compounds for the project, based
on their overall ADME profiles.
Compounds 21 and 22 are potent ACC inhibitors with
LipEs comparable to our previous lead 1. At 10 μM, they
showed no significant inhibition of the five major human
CYP450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2D6, and
CYP3A4). Both leads were found to be selective against a set of
1
08 targets including various enzymes, GPCRs, ion channels,
and other transporters (less than 50% inhibition at 10 μM). In
order to avoid future complications in preclinical evaluation and
product development, work was carried out to find more stable
crystalline forms. The more stable crystalline forms identified
for 21 and 22 had reduced but likely acceptable solubility based
on the good passive permeability associated with these leads
(0.05 and 0.13 μg/mL, respectively, at pH 6.5).
In Vivo Evaluation. Based on their respective profiles, 21
and 22 were advanced to pharmacokinetic evaluation. When
dosed in rats at 5 mg/kg using 0.5% methylcellulose as the
(
Table 3). Though a higher LipE could be achieved with amino
substitution, methoxy-substituted quinoline 21 and isoquino-
Scheme 3. Synthesis of ACC Inhibitors with Amino
a
Substituted Quinoline Tailpieces
vehicle, 21 exhibited high oral bioavailability of 71% (Cmax
=
4
03 ng/mL, AUClast = 2070 ng·h/mL). As determined with a 1
mg/kg intravenous dose, 21 demonstrated moderate in vivo
systemic clearance (29 mL/(min·kg)) and volume of
distribution (1.7 L/kg). At the same doses, 22 showed lower
bioavailability (F = 28%, Cmax = 186 ng/mL, AUClast = 713 ng·
h/mL) and higher clearance (41 mL/(min·kg)) compared with
2
1. In acute rat pharmacodynamic studies, 21 dose-propor-
1
4
a
tionally reduced DNL as measured by conversion of C-acetate
to C-lipids after oral dosing, with an ED50 of 1.27 mg/kg
(Figure 4).
Reagents and conditions: (a) Et N, HOBt, EDC, DCM, 100%; (b)
3
14
substituted amine in excess, 80 °C in a sealed vial, 29−92%. See
Experimental Section for details.
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response with final compound concentration ranging from 10 μM to
0
.3 nM. Aliquots of 1 μL were added in replicate to 96 well plates, and
an equal volume of DMSO was added to control wells. The enzyme
solution was activated for 30 min at 37 °C in buffer containing 50 mM
HEPES (pH 7.5), 10 mM MgCl , 10 mM tripotassium citrate, 6 mM
2
DTT, 0.75 mg/mL BSA, and 0.8 μg/mL ACC. After a 10 min
enzyme−compound preincubation, the reaction was initiated at room
temperature in a fume hood by addition of the substrate solution
(
containing 2.4 mM acetyl-CoA, 38.4 mM KHCO , 1.6 mM
3
14
NaH[ C]O , and 8.0 mM ATP). The final assay volume of 100 μL
3
per well consisted of 46 mM HEPES (pH 7.5), 7.5 mM MgCl , 7.5
2
mM tripotassium citrate, 2.8 mM DTT, 0.5 mg/mL BSA, 2.0 mM
ATP, 600 μM acetyl-CoA trilithium salt, 9.6 mM potassium
1
4
bicarbonate, 0.6 μg/mL hACC1 or 2, and 0.4 mM NaH[ C]O (58
3
mCi/mmol). After 20 min, the reaction was terminated by the
addition of a 3 N solution of hydrochloric acid (HCl) with the
concomitant liberation of nonreacted NaHCO as CO . Plates were
Figure 4. Graph of conversion of 14C-acetate to 14C-lipids (percentage
vs vehicle) vs doses of 21 after oral delivery using a SEDDS
formulation composed of DMSO/PEG 400/Polysorbate 80/HPMC
E5LV in water. Data are the arithmetic mean of eight animals ± SEM.
A detailed protocol can be found in the Experimental Section.
3
2
1
4
dried overnight at 50 °C to allow complete [ C]O liberation. The
2
following day, 30 μL of water was added to the dried wells now
14
containing [ C]malonyl-CoA, followed by 95 μL of OptiPhase
Supermix liquid scintillation fluid. The plates were shaken vigorously,
sealed, and transferred to a Microbeta LSC luminescence counter to
quantify the amount of 14C in each assay well.
CONCLUSION
■
In Vivo DNL Study. A single dose of compound 21 was
In summary, the more polar spirolactam core proved a solution
to avoid in vivo ketone reduction observed with our previous
lead 1. Lactam 5 had good metabolic stability but decreased
potency and permeability compared with 1. Optimization of the
lactam series with an aim to improve LipE, HLM stability, and
passive permeability afforded potent ACC inhibitors with good
in vitro ADME and polypharmacological profiles. Excellent oral
bioavailability, moderate systemic clearance, and acceptable
exposure were achieved with lead compound 21 in rat
pharmacokinetic studies. In a preclinical efficacy study, oral
dosing of rats with 21 resulted in potent, dose-proportional
administered via oral gavage to rats 2 h into the light cycle. One
14
hour post dose, C-labeled acetate (0.1 μCi/g) was administered ip.
14
One hour post C-acetate administration, the animals were sacrificed,
and tissues were collected. Liver punches (∼400 mg, bifurcated
median lobe) were collected for DNL determinations as measured by
14
C acetate incorporation to lipid. The tissues were dissolved in NaOH
(1.5 mL of 2.5 M). Following complete degradation, absolute ethanol
(2.5 mL) was added to each sample, and samples were vigorously
mixed. The samples were extracted with petroleum ether (4.8 mL).
Following centrifugation to separate the organic and aqueous phases,
the upper organic phase was removed and discarded. Concentrated
HCl (0.6 mL of 12M) was added to the remaining aqueous phase and
vortexed vigorously. The acidified aqueous phase was subsequently
extracted with petroleum ether (4.8 mL) and then centrifuged to
separate the organic and aqueous phases. The upper organic phase was
collected in an appropriately sized scintillation vial. The remaining
aqueous phase was extracted with petroleum ether as described above
and the organic phase was combined with the previous extract. The
combined organic phases were evaporated to dryness under gentle
flow of nitrogen at room temperature. Compatible scintillation fluid
14
inhibition of ACC activity as measured by incorporation of C
in DNL products. Compound 21 was advanced to exploratory
toxicological studies in rodents and preclinical development for
T2DM.
EXPERIMENTAL SECTION
Materials and Methods. All chemicals, reagents, and solvents
■
were purchased from commercial sources and used without further
1
14
purification unless otherwise noted. H NMR spectra were recorded
was added to each vial, and the level of C in the extraction was
with 400 or 500 MHz spectrometers and are reported relative to
determined.
1
residual undeuterated solvent signals. Data for H NMR spectra are
Synthesis. General Procedure A. To a solution of amine (1 equiv)
reported as follows: chemical shift (δ, ppm), multiplicity, coupling
constant (Hz), and integration. The multiplicities are denoted as
follows: s, singlet; d, doublet; t, triplet; q, quartet; spt, septet; m,
multiplet; br s, broad singlet. High-resolution mass spectrometry
in DMF (0.25 M) were added the carboxylic acid (1−3 equiv), Et N
3
(3−6 equiv), and T3P (1−2 equiv). The resultant mixture was stirred
for 18 h at room temperature (rt). The mixture was diluted with
EtOAc and washed subsequently with water and brine. The aqueous
washes were combined and extracted with additional EtOAc. The
(
HRMS) was performed via electrospray ionization (ESI) source. The
system used was an Agilent 1200 DAD (G1315C), 190−400 nm scan,
nm slit, and Agilent 6220 MS (TOF). Silica gel chromatography was
combined organic extracts were dried over Na
concentrated under reduced pressure.
SO , filtered, and
2 4
4
performed using a medium pressure Biotage or ISCO system and
columns prepackaged by various commercial vendors including
Biotage and ISCO. The terms “concentrated” and “evaporated” refer
to the removal of solvent at reduced pressure on a rotary evaporator
with a water bath temperature not exceeding 60 °C. All tested
compounds were determined to be >95% pure by high-performance
liquid chromatography (HPLC).
General Procedure B. To a solution of the carboxylic acid (1 equiv)
in DCM (0.1 M) were added DMAP (0.3 equiv), T3P (1.25 equiv),
and Et
a solution of amine (1 equiv) and Et
N (2−3 equiv). The mixture was stirred for 10 min at rt before
3
N (1.25 equiv) in DCM (0.1 M)
3
was added, and the resultant mixture was stirred at rt for 18 h. A
saturated aqueous solution of NaHCO was added and stirred for 10
3
min then extracted with DCM (2×). The combined organic layers
Preparative HPLC Conditions for Combinatorial Chemistry.
Compounds prepared via combinatorial chemistry were purified by
preparative HPLC using the following conditions: column, Waters
Atlantis dC , 4.6 mm × 50 mm, 5 μm; solvents A and B are water
were dried over Na SO , filtered, and concentrated.
2
4
General Procedure C. To a solution of the carboxylic acid (1 equiv)
in DCM (0.1 M) were added the amine (1 equiv), Et N (3 equiv), and
3
HATU (1 equiv). The resultant mixture was stirred at rt for 18 h. The
mixture was washed with a saturated aqueous solution of NaHCO3
and brine. The organic layer was dried over Na SO , filtered, and
18
with 0.05% TFA and MeCN with 0.05% TFA, respectively. A gradient
of 5−95% of solvent B in A was used over 4 min at 2 mL/min.
In Vitro ACC1 and ACC2 Inhibition Assay. For ACC inhibition
studies, test compounds were dissolved in dimethyl sulfoxide (DMSO)
and serially diluted in DMSO in order to run in a 11-point dose−
2
4
concentrated.
General Procedure D. To a suspension of carboxylic acid (1 equiv)
in DMF (0.3 M) were added the amine (1 equiv), EDC (1−1.5 equiv),
7
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and HOBt (1−1.5 equiv) followed by Et N (3−4 equiv). The reaction
crude material was purified by flash chromatography (10 g of silica gel,
3
was warmed to 50 °C and stirred at that temperature for 3.5 h. The
reaction was allowed to cool to rt and partitioned between water and
DCM. The aqueous layer was extracted with further DCM (3×), and
the combined organic solutions were dried over Na SO , filtered, and
1−10% MeOH/DCM gradient) to yield 10 (20 mg, 36%) as a white
1
solid: H NMR (400 MHz, CDCl ) δ 10.34 (br s, 1H), 7.57 (s, 1H),
3
7.23 (s, 1H), 7.15 (s, 1H), 6.59 (br s, 1H), 3.68−3.59 (m, 4H), 2.83
(s, 2H), 2.55 (s, 3H), 2.49 (s, 3H), 1.86 (br s, 4H), 1.70 (s, 9H); m/z
2
4
+
concentrated.
= 435.2 [M + H] .
1
′-(1H-Indazole-5-carbonyl)-6-methylspiro[benzo[e][1,3]oxazine-
2′-(tert-Butyl)-6′-methyl-4′,6′-dihydrospiro[piperidine-4,5′-
2
6
,4′-piperidin]-4(3H)-one (2). The title compound was prepared from
pyrazolo[3,4-c]pyridin]-7′(2′H)-one hydrochloride salt (13). Inter-
1
9
-methylspiro[1,3-benzoxazine-2,4′-piperidin]-4(3H)-one hydrochlor-
ide salt (28) (35 mg, 0.15 mmol), 1H-indazole-5-carboxylic acid
29) (86 mg, 0.45 mmol), Et N (73 μL, 0.52 mmol), and T3P (0.15
mL, 50% EtOAc, 0.19 mmol) according to general procedure A. The
crude product was purified by flash chromatography (12 g of silica gel,
−20% MeOH/DCM gradient) to yield 2 (19 mg, 39%) as a white
solid: H NMR (400 MHz, DMSO-d ) δ 13.25 (br s, 1H), 8.69 (s,
H), 8.15 (s, 1H), 7.86 (s, 1H), 7.59 (d, J = 8.59 Hz, 1H), 7.55 (s,
H), 7.39 (d, J = 8.59 Hz, 1H), 7.33 (d, J = 8.20 Hz, 1H), 6.96 (d, J =
.20 Hz, 1H), 3.62−4.32 (m, 2H), 3.34−3.45 (m, 2H), 2.29 (s, 3H),
.96−2.15 (m, 2H), 1.72−1.88 (m, 2H); m/z = 377.2 [M + H] .
2
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one (6).
The title compound was prepared from 3,7-dimethyl-1H-indazole-5-
carboxylic acid (30) (22 mg, 0.13 mmol), DMAP (4.7 mg, 0.04
mediate 12 (235 mg, 0.65 mmol) was dissolved in DMF (8 mL) and
cooled to 0 °C. Sodium hydride (78 mg, 60% in mineral oil, 1.9 mmol)
was added, and the suspension was stirred in the ice bath for 10 min.
MeI (0.12 mL, 1.9 mmol) was added, the ice bath was removed, and
the reaction was stirred at rt for 4 h. The reaction mixture was diluted
with water (50 mL), and the resulting suspension was extracted with
MTBE (2 × 20 mL). The combined organics were washed with brine
(50 mL), dried over Na SO , filtered, and concentrated under reduced
22
(
3
0
1
6
1
1
8
1
2
4
pressure. The crude material was purified by flash chromatography (12
g silica gel, 20−100% EtOAc/heptane gradient) to yield the
corresponding N-methylated lactam (209 mg, 86%) as a white solid:
+
1
′-tert-Butyl-1-(3,7-dimethyl-1H-indazole-5-carbonyl)-4′,6′-
H NMR (500 MHz, CDCl ) δ ppm 7.35 (s, 1H), 4.07 (br s, 2H),
3
3
(
.07 (s, 3H), 2.97 (s, 2H), 2.89 (br s, 2H), 1.95 (br s, 2H), 1.74−1.68
+
m, 2H), 1.62 (s, 9H), 1.47 (s, 9H); m/z = 377.3 [M + H] . The
product isolated from the previous step (200 mg, 0.53 mmol) was
suspended in EtOAc (5 mL) and treated with 4 N HCl in dioxane (1
mL). The reaction was stirred at rt for 18 h. Volatiles were removed
under reduced pressure, and the resultant white solid was triturated
mmol), T3P (0.10 mL, 50% in EtOAc, 0.15 mmol), Et N (0.11 mL,
3
0
.76 mmol), and 2′-tert-butyl-4′,6′-dihydrospiro[piperidine-4,5′-
19
pyrazolo[3,4-c]pyridin]-7′(2′H)-one hydrochloride salt (31) (38
mg, 0.13 mmol) according to general procedure B. The resultant crude
product was purified by flash chromatography (10 g silica gel, 1−10%
with heptanes (10 mL) and concentrated to yield 13 (157 mg, 95%) as
1
a white solid: H NMR (500 MHz, DMSO-d ) δ 9.40 (br s, 1H), 9.17
6
1
MeOH/DCM gradient) to yield 6 (33 mg, 76%) as a white solid: H
NMR (400 MHz, CDCl ) δ 10.93 (br s, 1H), 7.62 (s, 1H), 7.40 (s,
(
br s, 1H), 7.69 (s, 1H), 3.20 (d, J = 12.44 Hz, 2H), 2.98−3.09 (m,
3
4H), 2.96 (s, 3H), 2.32 (dt, J = 4.27, 13.60 Hz, 2H), 1.68 (d, J = 13.66
1
1
4
H), 7.21 (s, 1H), 6.74 (br s, 1H), 3.76−4.12 (m, 2H), 3.64 (t, J =
+
Hz, 2H), 1.52 (s, 9H); m/z = 277.3 [M + H] .
0.2 Hz, 2H), 2.86 (s, 2H), 2.60 (s, 3H), 2.55 (s, 3H), 1.69−1.87 (m,
2
′-(tert-Butyl)-1-(3,7-dimethyl-1H-indazole-5-carbonyl)-6′-meth-
+
H), 1.63 (s, 9H); m/z = 435.1 [M + H] .
yl-4′,6′-dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-
1
-(3,7-Dimethyl-1H-indazole-5-carbonyl)-2′-isopropyl-4′,6′-
one (14). The title compound was prepared from amine 13 (125 mg,
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one (7).
0
0
.45 mmol), carboxylic acid 30 (86 mg, 0.45 mmol), Et N (0.10 mL,
3
The title compound was prepared from carboxylic acid 30 (22 mg,
.90 mmol), and T3P (0.58 mL, 50% ethyl acetate, 0.87 mmol)
0
.13 mmol), DMAP (4.7 mg, 0.04 mmol), T3P (0.10 mL, 50% in
according to general procedure A. The crude material was purified by
flash chromatography (12 g of silica gel, 0−20% MeOH/DCM) to
EtOAc, 0.15 mmol), Et N (0.11 mL, 0.76 mmol), and 2′-isopropyl-
3
4
′,6′-dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-
1
yield 14 (79 mg, 38%) as a white solid: H NMR (500 MHz, DMSO-
23
one hydrochloride salt (32) (38 mg, 0.13 mmol) according to
general procedure B. The resultant crude product was purified by flash
d6) δ ppm 12.87 (s, 1H) 7.71 (s, 1H) 7.60 (s, 1H) 7.16 (s, 1H) 3.31
(
(
s, 2H) 3.05 (br s, 2H) 2.96 (s, 3H) 2.45−2.52 (m, 10H) 1.85−2.00
chromatography (10 g silica gel, 1−10% MeOH/DCM gradient) to
yield 7 (31 mg, 76%) as a white solid: H NMR (400 MHz, CDCl ) δ
0.81 (br s, 1H), 7.59 (s, 1H), 7.24 (s, 1H), 7.18 (s, 1H), 6.77 (s, 1H),
.55 (spt, J = 6.7 Hz, 1H), 3.62 (t, J = 10.1 Hz, 2H), 2.84 (s, 2H), 2.58
s, 3H), 2.53 (s, 3H), 1.91−1.57 (m, 6H), 1.52 (d, J = 8.0 Hz, 6H); m/
z = 421.3 [M + H] .
2
+
m, 2H) 1.52 (s, 9H); m/z = 449.4 [M + H] .
1
3
2′-(tert-Butyl)-1-(3-(trifluoromethyl)-1H-indazole-5-carbonyl)-
1
4
(
4
′,6′-dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-
19
one (15). The title compound was prepared from amine 31 (28 mg,
0.083 mmol), 3-(trifluoromethyl)-1H-indazole-5-carboxylic acid (36)
+
(19 mg, 0.083 mmol), Et N (0.035 mL, 0.25 mmol), and HATU (32
3
′-Cyclobutyl-1-(3,7-dimethyl-1H-indazole-5-carbonyl)-4′,6′-
mg, 0.083 mmol) according to general procedure C via combinatorial
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one (8).
The title compound was prepared from 2′-cyclobutyl-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one hy-
+
chemistry (2.9 mg, 7%): m/z = 475.2 [M + H] .
2
′-(tert-Butyl)-1-(3-chloro-1H-indazole-5-carbonyl)-4′,6′-
19
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
23
drochloride salt (33) (20 mg, 0.060 mmol), carboxylic acid 30 (14
(16). The title compound was prepared from amine 31 (50 mg, 0.15
mg, 0.072 mmol), Et N (0.045 mL, 0.32 mmol), and T3P (0.07 mL of
mmol), 3-chloro-1H-indazole-5-carboxylic acid (37) (45 mg, 0.23
3
5
0% in EtOAc, 0.1 mmol) using general procedure A via combinatorial
mmol), Et N (0.13 mL, 0.90 mmol), and T3P (0.18 mL of 50% in
3
+
chemistry (12.5 mg, 48%): m/z = 433.1 [M + H] .
EtOAc, 0.30 mmol) using general procedure A via combinatorial
+
1
-(3,7-Dimethyl-1H-indazol-5-carbonyl)-2′-(tert-pentyl)-4′,6′-
chemistry (11.7 mg, 18%): m/z = 441.1 [M + H] .
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one (9).
The title compound was prepared from 2′-tert-pentyl-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one hy-
2′-(tert-Butyl)-1-(3-chloro-1H-indole-5-carbonyl)-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
1
9
(17). The title compound was prepared from amine 31 (36 mg, 0.10
mmol), 3-chloro-1H-indole-5-carboxylic acid (38) (20 mg, 0.10
23
drochloride salt (34) (20 mg, 0.064 mmol), carboxylic acid 30 (15
mg, 0.077 mmol), Et N (0.045 mL, 0.32 mmol), and T3P (0.08 mL of
mmol), Et N (0.043 mL, 0.31 mmol), and HATU (39 mg, 0.10
3
3
5
0% in EtOAc, 0.1 mmol) using general procedure A via combinatorial
mmol) according to general procedure C via combinatorial chemistry
(18.7 mg, 42%): m/z = 440.1 [M + H] .
+
+
chemistry (12.9 mg, 45%): m/z = 449.2 [M + H] .
′-(tert-Butyl)-1-(3,7-dimethyl-1H-indazole-5-carbonyl)-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(1′H)-one
10). The title compound was prepared from carboxylic acid 30 (24
1
2′-(tert-Butyl)-1-(3-chloro-1H-indole-6-carbonyl)-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
1
9
(
(18). The title compound was prepared from amine 31 (36 mg, 0.10
mmol), 3-chloro-1H-indole-6-carboxylic acid (39) (20 mg, 0.10
mg, 0.13 mmol), DMAP (5 mg, 0.04 mmol), T3P (0.10 mL, 50% in
EtOAc, 0.15 mmol), Et N (53 μL, 0.38 mmol), and 1′-tert-butyl-4′,6′-
mmol), Et N (0.043 mL, 0.31 mmol), and HATU (39 mg, 0.10
3
3
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]-7′(1′H)-one hydrochlor-
mmol) according to general procedure C via combinatorial chemistry
23
+
ide salt (35) (38 mg, 0.13 mmol) using general procedure B. The
(11.5 mg, 26%): m/z = 440.1 [M + H] .
7
116
dx.doi.org/10.1021/jm401033t | J. Med. Chem. 2013, 56, 7110−7119

Journal of Medicinal Chemistry
Article
2
′-(tert-Butyl)-1-(3-chloro-1H-pyrrolo[3,2-b]pyridine-6-carbonyl)-
0.033 mmol), and T3P (1.6 mL, 50% in EtOAc, 2.7 mmol) according
to general procedure B. The crude material was purified by flash
chromatography (40 g of silica gel, 0−20% MeOH/EtOAc gradient)
4
′,6′-dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-
1
9
one (19). The title compound was prepared from amine 31 (150 mg,
.64 mmol), 3-chloro-1H-pyrrolo[3,2-b]pyridine-6-carboxylic acid
40) (220 mg, 0.66 mmol), EDC (156 mg, 0.77 mmol), HOBt
110 mg, 0.71 mmol), and Et N (0.45 mL, 3.2 mmol) according to
general procedure D. The crude material was purified by flash
chromatography (25 g of silica gel, 0−10% MeOH/DCM gradient) to
yield 19 (190 mg, 67%) as a white solid: H NMR (400 MHz, DMSO-
d6) δ 11.81 (d, J = 2.73 Hz, 1H), 8.42 (d, J = 1.76 Hz, 1H), 7.96 (d, J =
0
(
(
1
to provide 24 (294 mg, 40%) as a white solid: H NMR (400 MHz,
DMSO-d ) δ 8.23 (s, 1H), 7.92 (d, J = 5.87 Hz, 1H), 7.69−7.78 (m,
6
3
3H), 7.57 (dd, J = 8.31, 1.47 Hz, 2H), 6.89 (d, J = 5.87 Hz, 1H), 3.33−
3
4
.85 (m, 4H), 2.95 (d, J = 4.30 Hz, 3H), 2.82 (s, 2H), 1.55−1.79 (m,
+
H), 1.52 (s, 9H); m/z = 447.4 [M + H] .
1
2
′-(tert-Butyl)-1-(2-chloroquinoline-7-carbonyl)-4′,6′-
19
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
2
1
9
.93 Hz, 1H), 7.84 (d, J = 1.76 Hz, 1H), 7.74−7.79 (m, 1H), 7.72 (s,
(25). The title compound was prepared from amine 31 (1.19 g, 3.54
mmol), 2-chloroquinoline-7-carboxylic acid (45) (700 mg, 3.37
H), 3.42−3.87 (m, 4H), 2.83 (s, 2H), 1.70−1.58 (m, 4H), 1.53 (s,
+
H); m/z = 441.3 [M + H] .
mmol), Et N (1.9 mL, 14 mmol), HOBt (547 mg, 4.05 mmol), and
3
2
′-(tert-Butyl)-1-(3-methoxyisoquinoline-6-carbonyl)-4′,6′-
EDC (776 mg, 4.05 mmol) according to general procedure D. The
crude material was purified by flash chromatography (100 g of silica
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
23
(
20). 6-Bromo-3-methoxyisoquinoline (41) (90 mg, 0.38 mmol) was
gel, 0−20% MeOH/DCM gradient) to provide 25 (1.5 g, quant.) as a
placed into a microwave vial and dissolved in dioxane (6 mL). Amine
1
white solid: H NMR (400 MHz, CDCl ) δ 8.12 (d, J = 8.36 Hz, 1H),
19
3
3
1
(244 mg, 0.727 mmol) and sodium acetate (131 mg, 1.5 mmol)
7
1
.98 (br s, 1H), 7.88 (dd, J = 8.36 Hz, 1H), 7.61 (dd, J = 8.36, 1.59 Hz,
H), 7.44 (d, J = 1.59 Hz, 1H), 7.39 (s, 1H), 5.91 (br s, 1H), 4.06−
were then added. Nitrogen was bubbled into the reaction for 15 min,
Pd(dppf)Cl /DCM (102 mg, 0.125 mmol) was added, and carbon
2
4.22 (m, 1H), 3.38−3.64 (m, 3H), 2.85 (br s, 2H), 1.67−1.97 (m,
monoxide from a tank was bubbled in the reaction for 5 min. The
reaction mixture was warmed to 80 °C in the microwave and left
stirring for 8 h. The reaction was allowed to cool to rt and was diluted
with EtOAc. The solids were filtered on a pad of Celite, and the
solvent was removed under reduced pressure. This reaction was run
via combinatorial chemistry, providing 4.1 mg (2%) of compound 20:
+
4
H), 1.61 (s, 9H); m/z = 452.3 [M + H] .
2
′-(tert-Butyl)-1-(2-(propylamino)quinoline-7-carbonyl)-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
(26). A mixture of chloroquinoline 25 (100 mg, 0.221 mmol) and n-
propylamine (13.1 mg, 0.221 mmol) was heated in a sealed vial at 80
°C for 18 h. The reaction mixture was concentrated to dryness and the
crude product was purified by flash chromatography (12 g silica gel,
+
m/z = 448.1 [M + H] .
2
′-(tert-Butyl)-1-(2-methoxyquinoline-7-carbonyl)-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
21). The title compound was prepared from 2-methoxyquinoline-7-
0−10% MeOH/DCM gradient) to provide 26 (82 mg, 78%) as a
1
white foam: H NMR (400 MHz, CDCl ) δ 7.79 (d, J = 8.81 Hz, 1H),
3
(
7
=
.61−7.62 (m, 1H), 7.59 (d, J = 8.21 Hz, 1H), 7.37 (s, 1H), 7.19 (dd, J
22
19
carboxylic acid (42) (1.4 g, 6.8 mmol), amine 31 (2.7 g, 8.2
mmol), Et N (4.8 mL, 34 mmol), and T3P (4,7 mL of 50% in EtOAc,
.1 mmol) according to general procedure A. The material was
purified by flash chromatography (100 g silica gel, 2−20% MeOH/
DCM) to provide 21 (2.94 g, 96%) as a white solid: H NMR (400
MHz, CDCl ) δ 8.00 (d, J = 8.97 Hz, 1H), 7.90 (s, 1H), 7.76 (d, J =
.19 Hz, 1H), 7.41 (dd, J = 1.56, 8.19 Hz, 1H), 7.39 (s, 1H), 6.96 (d, J
8.17, 1.57 Hz, 1H), 6.65 (d, J = 8.81 Hz, 1H), 5.79 (s, 1H), 4.69−
3
4
1
4
.73 (m, 1H), 4.00−4.15 (m, 1H), 3.41−3.63 (m, 5H), 2.83 (s, 2H),
7
.64−1.92 (m, 6H), 1.61 (s, 9H), 1.02 (t, J = 7.41 Hz, 3H); m/z =
+
75.6 [M + H] .
1
2
′-(tert-Butyl)-1-(2-(dimethylamino)quinoline-7-carbonyl)-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
27). A mixture of chloroquinoline 25 (100 mg, 0.221 mmol) and
dimethylamine (2.2 mL, 4.4 mmol) was heated in a sealed vial at 80
C for 18 h. The reaction mixture was then concentrated to dryness,
3
8
=
(
(
8.78 Hz, 1H), 6.18 (s, 1H), 4.09 (s, 3H), 3.41−3.75 (m, 4H), 2.86
+
s, 2H), 1.70−1.99 (m, 4H), 1.62 (s, 9H); m/z = 448.5 [M + H] .
°
2
′-(tert-Butyl)-1-(1-methoxyisoquinoline-7-carbonyl)-4′,6′-
and the crude product was purified by flash chromatography (12 g of
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
silica gel, 0−10% MeOH/DCM gradient) to provide 27 (30.3 mg,
(
7
2
22). The title compound was prepared from 1-methoxyisoquinoline-
1
3
9
7
1
4
0%) as a white foam: H NMR (400 MHz, CDCl ) δ 7.87 (d, J =
22
19
3
-carboxylic acid (43) (400 mg, 2.00 mmol), amine 31 (660 mg,
.00 mmol), Et N (1.4 mL, 9.9 mmol), and T3P (1.36 mL of 50% in
EtOAc, 2.04 mmol) according to general procedure A. The material
was purified by flash chromatography (100 g silica gel, 2−20%
MeOH/DCM gradient) to provide 22 (765 mg, 87%) as a white solid:
.17 Hz, 1H), 7.69 (br s, 1H), 7.62 (d, J = 8.00 Hz, 1H), 7.39 (s, 1H),
.17−7.22 (m, 1H), 6.94 (d, J = 9.17 Hz, 1H), 5.82 (s, 1H), 4.08 (s,
H), 3.45−3.68 (m, 3H), 3.25 (s, 6H), 2.86 (s, 2H), 1.70−1.99 (m,
3
+
H), 1.63 (s, 9H); m/z = 461.4 [M + H] .
1
H NMR (400 MHz, CDCl ) δ 8.28−8.33 (m, 1H), 8.06 (d, J = 5.85
3
ASSOCIATED CONTENT
Hz, 1H), 7.75−7.80 (m, 1H), 7.67−7.72 (m, 1H), 7.40 (s, 1H), 7.23
■
(
3
4
d, J = 5.46 Hz, 1H), 6.20 (s, 1H), 4.14 (s, 3H), 3.99−4.12 (m, 1H),
* Supporting Information
S
.59 (br s, 3H), 2.87 (s, 2H), 1.68−2.05 (m, 4H), 1.62 (s, 9H); m/z =
+
Experimental details for the synthesis of key intermediates as
well as compounds 1−27. This material is available free of
charge via the Internet at http://pubs.acs.org.
48.3 [M + H] .
2
′-tert-Butyl-1-{[2-(methylamino)quinolin-7-yl]carbonyl}-2′,4′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(6′H)-one
19
(
23). The title compound was prepared from amine 31 (87 mg, 0.25
23
mmol), 2-(methylamino)quinoline-7-carboxylic acid (50 mg, 0.26
mmol), Et N (0.11 mL, 0.74 mmol), HOBt (40 mg, 0.30 mmol), and
EDC (52 mg, 0.27 mmol) using general procedure D. The crude
material was purified by flash chromatography (12 g silica gel, 0−10%
MeOH/DCM gradient) to provide 23 (84 mg, 76%) as a white
powder: H NMR (400 MHz, DMSO-d ) δ 7.82 (d, J = 8.97 Hz, 1H),
AUTHOR INFORMATION
3
■
Corresponding Author
*
Tel: 617-551-3302. E-mail: kim.huard@pfizer.com.
1
Notes
6
7
1
8
.72 (s, 1H), 7.63 (d, J = 8.04 Hz 1H), 7.66 (br s, 1H), 7.39−7.43 (m,
H), 7.08−7.13 (m, 1H), 7.06 (dd, J = 8.10, 1.66 Hz, 1H), 6.75 (d, J =
.97 Hz, 1H), 3.77 (br s, 1H), 3.58 (br s, 1H), 3.46 (br s, 1H), 3.34
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(
4
br s, 1H), 2.87 (d, J = 4.68 Hz, 3H), 2.80 (br s, 2H), 1.55−1.77 (m,
■
+
H), 1.49 (s, 9H); m/z = 447.4 [M + H] .
The authors would like to thank Kristen Ford, Kristin L.
Rockwell, and Fabien Vincent for their work on the biological
screening, Margaret S. Landis and Brenda Ramos for the
formulation work, and Mark Niosi for his work on the
pharmacokinetic studies.
2
′-(tert-Butyl)-1-(1-(methylamino)isoquinoline-7-carbonyl)-4′,6′-
dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one
1
9
(24). The title compound was prepared from amine 31 (657 mg,
2
2
2
.00 mmol), 1-(methylamino)isoquinoline-7-carboxylic acid (44)
(360 mg, 1.78 mmol), Et N (1.5 mL, 11 mmol), DMAP (4.5 mg,
3
7
117
dx.doi.org/10.1021/jm401033t | J. Med. Chem. 2013, 56, 7110−7119

Journal of Medicinal Chemistry
Article
Coffey, S. B.; Buzon, L.; Minich, M. L.; Dirico, K. J.; Tapley, S.;
McPherson, R. K.; Sugarman, E.; Harwood, H. J., Jr.; Esler, W.
Discovery of Small Molecule Isozyme Non-Specific Inhibitors of
Mammalian Acetyl-CoA Carboxylase 1 and 2. Bioorg. Med. Chem. Lett.
ABBREVIATIONS
■
T2DM, type 2 diabetes mellitus; DNL, de novo lipogenesis;
ACC, acetyl-CoA carboxylase; CPT-I, carnitine palmitoyltrans-
ferase 1; LipE, lipophilic ligand efficiency; HLM, human liver
microsome; CYP, cytochrome P450; NaH, sodium hydride;
MeI, iodomethane; HCl, hydrochloric acid; T3P, propylphos-
2
010, 20, 2383−2388.
(9) Freeman-Cook, K. D.; Amor, P.; Bader, S.; Buzon, L. M.; Coffey,
S. B.; Corbett, J. W.; Dirico, K. J.; Doran, S. D.; Elliott, R. L.; Esler, W.;
Guzman-Perez, A.; Henegar, K. E.; Houser, J. A.; Jones, C. S.;
Limberakis, C.; Loomis, K.; McPherson, K.; Murdande, S.; Nelson, K.
L.; Phillion, D.; Pierce, B. S.; Sugarman, E.; Tapley, S.; Tu, M.; Zhao,
Z. Maximizing Lipophilic Efficiency: The Use of Free-Wilson Analysis
in the Design of Inhibitors of Acetyl-CoA Carboxylase. J. Med. Chem.
phonic anhydride; Et N, triethylamine; HOBt, N-hydroxyben-
3
zotriazole; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; Cmax, maximum concentration; AUC, area
under the curve; SEDDS, self-emulsifying drug delivery system;
ESI-MS, electrospray ionization coupled with mass spectrom-
etry; HPLC, high-performance liquid chromatography; TFA,
trifluoroacetic acid; MeCN, acetonitrile; DMSO, dimethylsulf-
oxide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; DTT, dithiothreitol; BSA, bovine serum albumin; LSC,
liquid scintillation counting; NaOH, sodium hydroxide; DMF,
N,N-dimethylformamide; rt, room temperature; DCM, di-
chloromethane; DMAP, N,N-dimethyl-4-aminopyridine;
MTBE, methyl tert-butyl ether
2
012, 55, 935−942.
10) Experimental details for the assay used to determine IC50 values
are reported in the Experimental Section.
11) Edwards, M. P.; Price, D. A. Role of Physicochemical Properties
(
(
and Ligand Lipophilicity Efficiency in Addressing Drug Safety Risks.
Annu. Rep. Med. Chem. 2010, 45, 380−391.
(12) Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F. ElogDoct:
A Tool for Lipophilicity Determination in Drug Discovery. J. Med.
Chem. 2001, 44, 2490−2497.
(13) Obach, R. S. The Prediction of Human Clearance from Hepatic
Microsomal Metabolism Data. Curr. Opin. Drug Discovery Dev. 2001, 4,
6−44.
14) Di, L.; Whitney-Pickett, C.; Umland, J. O.; Zhang, H.; Zhang,
REFERENCES
■
3
(
(
1) Savage, D. B.; Petersen, K. F.; Shulman, G. I. Disordered Lipid
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2) (a) Hwang, J.-H.; Stein, D. T.; Barzilai, N.; Cui, M.-H.; Tonelli, J.;
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2
(
(15) Eng, H.; Niosi, M.; Tan, B.; Doran, S.; Garcia-Irizarry, C. N.;
2
93, E1663−E1669. (b) Korenblat, K. M.; Fabbrini, E.; Mohammed,
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Sterically Hindered Spiro[pyrazolopyranpiperidine]ketone. Tetrahe-
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(
3) Krssak, M.; Peterson, K. F.; Dresner, A.; DiPietro, L.; Vogel, S.
M.; Rothman, D. L.; Shulman, G. I.; Roden, M. Intramyocellular Lipid
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(17) Compound 2 was prepared by condensation of N-protected 4-
1
piperidinone with salicylamide under previously reported conditions:
Gammill, R. B. A New Amine Catalyzed Synthesis of 2-Substituted
(
Kulkarni, A.; Distefano, A.; Hwang, Y.-J.; Reznick, R. M.; Codella, R.;
Zhang, D.; Cline, G. W.; Wakil, S. J.; Shulman, G. I. Continuous Fat
Oxidation in Acetyl-CoA Carboxylase 2 Knockout Mice Increases
Total Energy Expenditure, Reduces Fat Mass, and Improves Insulin
Sensitivity. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16480−16485.
2
3
,3-Dihydro-4H-1,3-benzoxazin-4-ones. J. Org. Chem. 1981, 46, 3340−
342. See Supporting Information for more details.
(18) For examples of aminal ACC inhibitors, see: Anderson, R.;
Breazeale, S.; Elich, T.; Lee, S.-F. Modulators of acetyl-coenzyme A
carboxylase and methods of use thereof. U.S. Patent US20100009982,
(
5) Savage, D. B.; Choi, C. S.; Samuel, V. T.; Liu, Z.-X.; Zhang, D.;
2
010.
19) For experimental details and synthesis of compounds 5, 11, 12,
and 31, see Huard, K.; Bagley, S. W.; Menhaji-Klotz, E.; Preville, C.;
Wang, A.; Zhang, X.-M.; Cline, G. W.; Yu, X. X.; Geisler, J. G.; Bhanot,
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Steatosis and Hepatic Insulin Resistance by Antisense Oligonucleotide
Inhibitors of Acetyl-CoA Carboxylases 1 and 2. J. Clin. Invest. 2006,
(
́
Southers, J. A.; Smith, A. C.; Edmonds, D. J.; Lucas, J. C.; Dunn, M. F.;
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Griffith, D. A.; Dow, R. L. Synthesis of Spiropiperidine Lactam Acetyl-
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1
(
16, 817−824.
6) (a) Harwood, H. J., Jr.; Petras, S. F.; Shelly, L. D.; Zaccaro, L. M.;
Perry, D. A.; Makowski, M. R.; Hargrove, D. M.; Martin, K. A.; Tracey,
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itors Reduce Tissue Malonyl-CoA Concentrations, Inhibit Fatty Acid
Synthesis, and Increase Fatty Acid Oxidation in Cultured Cells and in
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(20) Compound 6 was tested for cytochrome P450 time-dependant
inhibition at 60 μM in human liver microsomes and caused a 38%
decrease activity of CYP3A4 after 30 minutes of incubation, as
determined by midazolam metabolism. Other examples of formation
of reactive metabolites associated with 3-methylindazole scaffold were
previously reported in the literature: Jones, L. H.; Allan, G.; Barba, O.;
̈
Burt, C.; Corbau, R.; Dupont, T.; Knochel, T.; Irving, S.; Middleton,
(
b) Schreurs, M.; van Dijk, T. H.; Gerding, A.; Havinga, R.;
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(21) IC50 values for ACC1 and ACC2 were obtained using a
radiometric assay, as described in the Experimental Section and are
reported as the geometric mean based on n ≥ 3 (except for compound
5, n = 1). Compounds 1, 5, and 6 were also compared using a
previously reported assay format (see ref 9). IC50 values (n ≥ 3) for
Reijngoud, D.-J.; Kuipers, F. Soraphen, an Inhibitor of the Acetyl-
CoA Carboxylase System, Improves Peripheral Insulin Sensitivity in
Mice Fed a High-Fat Diet. Diabetes, Obes. Metab. 2009, 11, 987−991.
(
7) Kusunoki, J.; Kanatani, A.; Moller, D. E. Modulation of Fatty
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and the Metabolic Syndrome. Endocrine 2006, 29, 91−100.
(
8) Corbett, J. W.; Freeman-Cook, K. D.; Elliott, R.; Vajdos, F.;
Rajamohan, F.; Kohls, D.; Marr, E.; Zhang, H.; Tong, L.; Tu, M.;
Murdande, S.; Doran, S. D.; Houser, J. A.; Song, W.; Jones, C. J.;
7
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Journal of Medicinal Chemistry
Article
ACC1 and ACC2, respectively, were as follows: 1 (111 ± 21 nM, 11 ±
1
(
4
nM); 5 (164 ± 40 nM, 67 ± 35 nM); 6 (7 ± 1 nM, 2.1 ± 0.6 nM).
22) The synthesis of 6-methylspiro[1,3-benzoxazine-2,4′-piperidin]-
(3H)-one hydrochloride salt (28), 2-methoxyquinoline-7-carboxylic
acid (42), 1-methoxyisoquinoline-7-carboxylic acid (43), and 1-
methylamino)isoquinoline-7-carboxylic acid (44) is described in the
Supporting Information.
(
(
23) The synthesis of 2′-isopropyl-4′,6′-dihydrospiro[piperidine-4,5′-
pyrazolo[3,4-c]pyridin]-7′(2′H)-one hydrochloride salt (32), 2′-cyclo-
butyl-4′,6′-dihydrospiro[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-
7′(2′H)-one hydrochloride salt (33), 2′-tert-pentyl-4′,6′-dihydrospir-
o[piperidine-4,5′-pyrazolo[3,4-c]pyridin]-7′(2′H)-one hydrochloride
salt (34), 1′-tert-butyl-4′,6′-dihydrospiro[piperidine-4,5′-pyrazolo[3,4-
c]-7′(1′H)-one hydrochloride salt (35), 6-bromo-3-methoxyisoquino-
line (41), and 2-(methylamino)quinoline-7-carboxylic acid was
previously described in the literature: Bagley, S. W.; Dow, R. L.;
Griffith, D. A.; Smith, A. C. N1/N2-Lactam Acetyl-CoA Carboxylase
Inhibitors. International Patent WO12056372; 2012.
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