Optimizing the Benefit/Risk of Acetyl-CoA Carboxylase Inhibitors through Liver Targeting

Article abstract of DOI:10.1021/acs.jmedchem.0c00640

Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.

Full text of DOI:10.1021/acs.jmedchem.0c00640

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Article
Optimizing the Benefit/Risk of Acetyl-CoA
Carboxylase (ACC) Inhibitors Through Liver Targeting
Kim Huard, Aaron C. Smith, Gregg D Cappon, Robert L. Dow, David J. Edmonds, Ayman El-
Kattan, William P Esler, Dilinie P. Fernando, David A. Griffith, Amit S. Kalgutkar, Trenton Ross,
Scott W. Bagley, David Beebe, Yi-An Bi, Shawn Cabral, Collin Crowley, Shawn D Doran, Matthew
S. Dowling, Spiros Liras, Vincent Mascitti, Mark Niosi, Jeffrey A. Pfefferkorn, Jana Polivkova,
Cathy Preville, David A. Price, Andre Shavnya, Norimitsu Shirai, Andrew H Smith, James A
Southers, David A Tess, Benjamin A. Thuma, Manthena V. S. Varma, and Xiaojing Helen Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00640 • Publication Date (Web): 18 Aug 2020
Downloaded from pubs.acs.org on August 20, 2020
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Optimizing the Benefit/Risk of Acetyl-CoA
Carboxylase (ACC) Inhibitors Through Liver
Targeting
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◊
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†
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Kim Huard, Aaron C. Smith, Gregg Cappon, Robert L. Dow, David J. Edmonds, Ayman El-
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†
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Kattan, William P. Esler, Dilinie P. Fernando, David A. Griffith, Amit S. Kalgutkar, Trenton
◊
†
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†
◊
T. Ross, Scott W. Bagley, David Beebe, Yi-An Bi, Shawn Cabral, Collin Crowley, Shawn D.
†
†
◊
†
†
Doran, Matthew S. Dowling, Spiros Liras, Vincent Mascitti, Mark Niosi, Jeffrey A.
◊
†
†
◊
†
Pfefferkorn, Jana Polivkova, Cathy Préville, David A. Price, Andre Shavnya, Norimitsu
†
†
†
◊
†
Shirai, Andrew H. Smith, James R. Southers, David A. Tess, Benjamin A. Thuma, Manthena
†
†
V. Varma, Xiaojing Yang
◊Pfizer Worldwide Research and Development, 1 Portland Street, Cambridge, Massachusetts
USA 02139
†Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut USA
0
6340
KEYWORDS: Hepatoselective, liver targeting, acetyl CoA carboxylase, inhibitor, organic
anion transporting peptide, CYP, NAFLD, NASH, DNL, PF-05221304.
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ABSTRACT: Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors
have the potential to rebalance disordered lipid metabolism leading to improvements in non-
alcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical
trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis
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(DNL), albeit with concomitant reductions in platelet count, which were attributed to the
inhibition of fatty acid synthesis within bone marrow. Herein we describe the design, synthesis,
and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting
polypeptide (OATP) substrate properties, which facilitated selective distribution of the
compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to
the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in
animals, while demonstrating considerable safety margins against platelet reduction in a non-
human primate model.
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INTRODUCTION
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Acetyl-CoA carboxylase (ACC) is a key enzyme regulating lipid metabolism. There are two
closely related isoforms of ACC that differ in tissue distribution: ACC1 is more highly expressed
in lipogenic tissues such as liver and adipose, while ACC2 is the major isoform expressed in
oxidative tissues such as skeletal and heart muscle. ACC catalyzes the ATP-dependent
condensation of acetyl-CoA with carbonate to produce malonyl-CoA, which is a rate-limiting
substrate for de novo lipogenesis (DNL). In addition, malonyl-CoA acts as an allosteric inhibitor
of the enzyme carnitine palmitoyltransferase 1, which is responsible for catalyzing the rate
limiting transport of medium and long chain fatty acyl-CoAs into the mitochondria for fatty acid
oxidation. Hence, through the intermediacy of malonyl-CoA, ACC serves as a critical switch
regulating the transition from lipogenic to oxidative lipid metabolism.
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Identification of small molecule inhibitors of ACC has been of long-standing interest for treating
multiple indications including type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease
NAFLD) and aspects of the metabolic syndrome,2-6 oncology,7, 8 and acne vulgarus. Pfizer
9
(
2
efforts towards ACC inhibitors identified screening hit CP-610431 (1), which was optimized to
provide pre-candidate 210-12 and first generation clinical candidate PF-05175157 (3), both dual
ACC1 and ACC2 inhibitors (Figure 1).
13
O
O
O
N
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H
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N
N
O
N
N
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H
O
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O
CP-610431 (1)
2
PF-05175157 (3)
Figure 1. Systemic ACC inhibitors identified from the Pfizer program
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Compound 3 inhibited hepatic DNL and lowered the respiratory exchange ratio in healthy human
volunteers.9, 13 These observations indicated that the inhibition of ACC1 and ACC2 in humans
increases dependence on whole-body fatty acid utilization. While 3 was generally well-tolerated
in human clinical studies, development was terminated due to asymptomatic reductions in
platelet count observed during multi-dose studies in human volunteers with type 2 diabetic
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mellitus. Platelet count reductions had not been observed in the regulatory toxicology studies in
rats and dogs which supported the conduct of human clinical trials. However, we subsequently
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found that ACC inhibition reduces platelet count in nonhuman primates (NHPs). Since this
model verified the clinical results observed with 3, it would be useful for predicting the platelet
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lowering effects of other ACC inhibitors in humans. A combination of ex vivo assays and in
vivo toxicology assessments in NHPs demonstrated that reductions in platelet count were
secondary to ACC inhibition within bone marrow, which prevented the normal formation of
platelets due to attenuation of bone marrow DNL and subsequent impairment of megakaryocyte
maturation.14
Recently, there is growing interest in using ACC inhibitors for the treatment of NAFLD and non-
alcoholic steatohepatitis (NASH).15, 16 NASH is a clinical and histological subset of NAFLD that
is associated with increased all-cause mortality, cirrhosis and end-stage liver disease, increased
cardiovascular mortality, and increased incidence of both liver related and non-liver related
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cancers. In the US population, the prevalence of NAFLD and NASH is estimated at 30–40%
and 3–5%, respectively.18
Alterations in lipid metabolism are hypothesized to contribute to the molecular pathogenesis of
NAFLD and NASH. Human subjects with elevated levels of liver fat have more than a three-fold
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higher rate of hepatic DNL relative to subjects with normal amounts of liver fat. ACC
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inhibitors have the potential to alter the balance of hepatic TG accumulation and modulation of
fat production and/or clearance, and this is expected to reduce both liver fat and steatosis in
patients with NASH. In rats, ACC inhibition stimulates fatty acid oxidation, suppresses hepatic
DNL, and consequently reduces steatosis.20, 21 Improvements in steatosis were also observed in
human subjects treated with ACC inhibitors.3, 4, 6, 22, 23 Furthermore, amelioration of steatosis is
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hypothesized to suppress hepatic inflammation and subsequently reduce fibrogenesis. While
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3, 5, 6, 22, 23
the ACC inhibitors MK-4074 and GS-0976
(Figure 2) have been studied in clinical
trials for the treatment of NASH/NAFLD, there are currently no approved therapies for the
treatment of this disease, resulting in a significant unmet medical need.
O
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OH
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O
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O
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HO
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O
S
N
O
O
O
N
N
O
O
O
GS-0976
MK-4074
Figure 2. Liver-targeted ACC inhibitors in clinical development
Although ACC inhibitors have the potential to address multiple pathogenic contributors to
NASH, the importance of DNL within human bone marrow for platelet production limits the
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degree of systemic ACC inhibition safely tolerated during chronic treatment. We hypothesized
that an ACC inhibitor with asymmetric tissue distribution favoring the liver, the site of action for
intervention in NASH, would have an improved therapeutic index for platelet lowering relative
to compound 3 by minimizing ACC inhibition in bone marrow. To test this hypothesis, we
undertook the design, synthesis, and evaluation of ACC inhibitors with the incorporation of
structural features intended for recognition by organic anion transporting polypeptides (OATPs),
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members of the SCLO superfamily of xenobiotic transporters. Of the 11 human OATP
transporters, OATP1B1 and OATP1B3 are expressed on the sinusoidal membrane of hepatocytes
and can facilitate the liver uptake of their respective substrates (e.g., statins such as atorvastatin
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and rosuvastatin). Consequently, we sought ACC inhibitors that were OATP1B1/1B3 transport
substrates to drive hepatoselectivity.17-21 Active transport to the liver by OATPs not only
2
7
influences tissue distribution but also serves as a clearance mechanism of its substrates. Hence,
ensuring that the hepatoselective nature of the ACC inhibitors did not interfere with the
otherwise attractive preclinical (and clinical) pharmacokinetics and disposition attributes
observed with 3 was an important consideration in the design elements of our program. Herein,
2
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we describe the design and evaluation of clinical candidate PF-05221304 (12), a liver-targeted
dual ACC1 and ACC2 inhibitor. The OATP substrate properties of 12 result in pronounced
asymmetrical tissue distribution of 12 in preclinical species. Consistent with its hepatoselectivity,
12 selectively inhibited DNL in the liver relative to bone marrow in rats and had an improved
safety profile for effects on platelet count in NHPs relative to 3. It is interesting to note that the
carboxylic acid-containing ACC inhibitors GS-09763, 5, 6, 22, 23 and MK-4074 (Figure 2) have
also been reported to be OATP substrates, but detailed structure-activity relationship analysis
leading to their identification has not been published.
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RESULTS AND DISCUSSION
Optimization of Acidic ACC Inhibitors. Since OATP substrates are anionic in nature (e.g.,
carboxylates)29-32 with a relatively high molecular weight (MW ≥ 400 Da),29, 33 our approach to
the development of hepatoselective ACC inhibitors began with structural modifications of the
potent systemic inhibitors 2 and 3 to include a carboxylic acid. Initial studies focused on probing
vectors most likely to retain potency against ACC while bearing such a polar and ionizable
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functional group. The x-ray co-crystal structure of compound 2 bound to ACC suggested limited
opportunities to append a carboxylic acid within the central spirocyclic core, however there was
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a likelihood that acidic motifs could be tolerated at either end of the structure. To this end, a set
of carboxylic acid-containing analogs was prepared and a selection of analogs and their in vitro
ACC inhibitory activity is reported in Table 1. As illustrated with compound 4, direct
functionalization of neutral analogs with a carboxylic acid on the pyrazole caused a significant
decrease in potency against both ACC isoforms, and also led to a decrease in passive
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permeability (P ) in Madin-Darby kidney cell line-low efflux (MDCK-LE) cells. On the other
app
hand, substitution of the 6,5-fused heteroaryl at the distal end had modest impacts on potency as
illustrated by indazole 5. Unfortunately, the x-ray co-crystal structure of 2 bound to a humanized
yeast chimeric carboxyltransferase domain of ACC was poorly resolved in the distal region,
preventing the use of structure-based drug design to optimize the placement of the carboxylic
acid or other anionic substituents in order to improve potency. Transforming the 6,5-fused
heteroaryl moiety into an unfused biaryl offered more design opportunities to locate an optimal
position for placement of a carboxylic acid while seeking to improve potency. Substitution of the
carboxylic acid on the terminal phenyl ring (compounds 6 and 7) was then carried out. The para-
substituted carboxylic acid (6) had the greatest potency of the tested ideas but was relatively
modest in comparison to 3.13
Table 1. In vitro inhibitory potency against ACC1 and ACC2 and passive permeability of
carboxylic acid-containing analogs
Pappc (10-6
ACC1
IC50 (µM) IC50 (µM)
ACC2
Compound
Structure
a
a
cm/s)
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O
O
N
H
N
0.111
0.0098
213
313
4
N
13
9.5
N
(7.0 ± 0.2) (8.0 ± 0.2)
N
O
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0
O
N
N
0.098
0.045
N
(
(
7.0 ± 0.4) (7.3 ± 0.5)
N
N
H
O
O
N
H
N
12
1.2
N
0.5
4.9 ± 0.1) (5.9 ± 0.3)
N
HO C
N
2
O
O
N
H
N
0.13
0.033
N
5
<0.4
O
N
(6.9 ± 0.2) (7.5 ± 0.1)
N
CO H
O
2
O
N
N
0.44
0.092
O
6
7
N
2.5
1.3
(6.4 ± 0.1) (7.0 ± 0.2)
O
CO H
2
O
O
N
N
1
.5
0.20
N
CO H
2
(5.8 ± 0.2) (6.7 ± 0.2)
O
a
In vitro potency against human recombinant ACC1 and ACC2 was determined using a
Transcreener® assay as previously described. IC values are reported as geometric mean of at
least three replicates with pIC50 ± SD in parentheses. Apparent passive permeability was
assessed in Madin-Darby canine kidney cell line-low efflux cells with a 30 min pre-incubation
time. ND = not determined.
1
0
5
0
c
34
Acid 6 was further optimized to boost potency and new analogs were evaluated in vitro for
3
5
lipophilicity (logD at pH 7.4 using the shake-flask method), P in the MDCK-LE cellular
app
3
4
assay, with a 30 min pre-incubation time, and metabolic intrinsic clearance (CLint,met) in human
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hepatocytes (HHEPs). Data on a focused set of analogs derived from the most potent biaryl
isomer, compound 6, is presented in Table 2. The phenyl ring proximal to the core of 6 was
modified using parallel medicinal chemistry which led to the identification of 8 with
considerable improvements in ACC inhibitory potency and low metabolic turnover (CLint,met
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4
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4
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4
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4
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5
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5
5
5
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5
6
0
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0
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0
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0
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0
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7
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9
0
<
6
36
3
.0 µL/min/10 cells) in HHEPs. Deletion of the methoxy group (9) led to a large drop off in
ACC inhibitory potency. Replacement of the methoxy group in 8 with a N,N-dimethylamino
substituent (compound 10) maintained inhibitory potency, however CLint,met increased.
Furthermore, passive permeability decreased in the MDCK-LE assay, potentially hindering oral
(
PO) absorption in pharmacokinetic studies. Given the good potency and metabolic clearance of
8, its human OATP1B1 and OATP1B3 substrate properties were assessed using a differential
uptake format in human embryonic kidney (HEK)293 cells transfected with the relevant
3
7
38
transporter. A normalized OATP1B1- and OATP1B3-mediated uptake ratio of > 2.0,
calculated as the total uptake of compound 8 in OATP-expressing cells divided by uptake
observed in non-transfected parental cells, was observed suggesting that it was an OATP
substrate with the potential to be hepatoselective.
Table 2. In vitro inhibitory potency against ACC1 and ACC2, lipophilicity (logD), metabolic
intrinsic clearance, passive permeability, and OATP uptake ratios of analogs derived from
compound 6
O
N
N
O
N
O
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2
3
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8
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
5
6
HHEP
HEK-
HEK-
ACC1
ACC2
IC50
(µM)
Pappc
d
CLint,met OATP1B1 OATP1B3
a
a
logDb (10
-6
Compound
Structure
IC50
µM)
(µL/min/
Uptake
Uptake
(
cm/s)
6
e
e
1
0 )
Ratio
Ratio
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
0
.022
0.013
(7.9 ±
0.4)
8
(7.7 ±
0.2)
0.5
‒0.4
0.8
2.0
<3.0
ND
7.5
5.0
ND
ND
3.3
ND
ND
CO H
2
N
1
.1
0.19
(6.7 ±
0.1)
9
(6.0 ±
0.1)
ND
0.5f
CO H
2
N
N
0.017
(7.8 ±
0.0094
(8.0 ±
0.2)
10
0
.2)
CO H
2
a
In vitro potency against ACC1 and ACC2 was determined using a Transcreener® assay as
previously described. IC values are reported as geometric mean of at least three replicates
with pIC ± SD in parentheses. logD values were determined using the shake-flask method
with an octanol/water partition at pH 7.4. Apparent permeability was assessed in MDCK-LE
cells with a 30 min pre-incubation time. Metabolic intrinsic clearance obtained from scaling in
vitro half-lives in cryopreserved human hepatocyte incubations conducted for 2 h as previously
described. Ratio of total/passive cellular uptake as measured by compound uptake in OATP-
transfected and parental cells for 5 min at 37 °C, respectively at a dose of 1 µM. A ratio > 2
indicates the compound is a substrate in vitro. No pre-incubation was performed. ND = not
determined.
1
0
5
0
b
50
3
5 c
3
4 d
36 e
3
7
3
8 f
To this point, the structure activity relationship for this series had been mostly developed with
the N-2 t-butyl pyrazole spirocyclic ether found in compound 2. Previous experience from the
13
development of 3 indicated that permeability could be improved by moving the pyrazole
substituent to the N-1 position as found in compound 3 (data not shown), likely due to steric
shielding of the ketone carbonyl group that increases lipophilicity. Additionally, changing from
the cyclic ether to the carbocycle obviates the potential for retro-Michael ring-opening.13
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Combining the N-1 i-propyl pyrazole spirocycle with optimized N,N-dimethylamino- and
methoxy-substituted biaryl acids led to potent analogs 11 and 12 with increased lipophilicity.
This effectively translated to improved passive cell permeability without affecting metabolic
stability (Table 3). Additionally, OATP uptake ratios were sufficiently high to suggest that 11
and 12 were substrates for hepatic transporters.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. In vitro inhibitory potency against ACC1 and ACC2, lipophilicity, metabolic intrinsic
clearance, passive permeability, and OATP uptake ratios of optimized analogs
O
N
N
N
O
HHEP
CLint,met
HEK-
OATP1B1 OATP1B3
HEK-
ACC1 ACC2
Pappc
d
a
a
logDb (10
-6
Compound
Structure
IC50
µM)
IC50
(µM)
(
µL/min/
Uptake
Uptake
Ratio
(
cm/s)
6
e
e
1
0 )
Ratio
N
N
0
.0077 0.0056
1
1
1
2
(8.1 ± (8.3 ±
1.4
1.1
13
<3.0
<3.0
2.1
3.5
1.4
2.5
0.3)
0.2)
CO H
2
O
N
0.033
0.026
(7.5 ± (7.6 ±
.1) 0.2)
14
0
CO H
2
a
In vitro potency against ACC1 and ACC2 was determined using a Transcreener® assay as
1
0
previously described. IC values are reported as geometric mean of at least three replicates
with pIC ± SD in parentheses. logD values were determined using the shake-flask method
with an octanol/water partition at pH 7.4. Apparent permeability was assessed in MDCK-LE
cells with a 30 min pre-incubation time. Metabolic intrinsic clearance obtained from scaling in
vitro half-lives in cryopreserved human hepatocyte incubations conducted for 2 h as previously
5
0
b
50
3
5 c
3
4 d
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described.36 eRatio of total/passive cellular uptake as measured in OATP-transfected and parental
3
7
cells, respectively at doses of 1–3 µM at 0.5–4.0 min. A ratio > 2 indicates the compound is a
3
8
substrate in vitro.
Evaluation of In Vivo Hepatoselectivity in Rats. To confirm the in vitro observations of
OATP-mediated uptake in an in vivo setting, compounds 8, 11, and 12 were administered via PO
gavage to rats and unbound liver-to-plasma concentration ratios (Kpu,u) were determined 6 h
post-dose. Total K values greater than unity and K values ranging from 79 to 376 were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
p
pu,u
observed for 8, 11, and 12 (Table 4), which is consistent with selective distribution of the three
compounds in the liver most likely via hepatic uptake mediated by rat Oatp isoforms.
Table 4. Determination of unbound liver-to-plasma ratios (Kpu,u) for 8, 11, and 12 in animals
Compound Species fu,plasmaa
fu,livera
Dose
Mean total
Kpd
Kpu,ue
(mg/kg)
concentration
c
(ng/mL)
Plasma
Liver
8
Rat
Rat
0.17
0.057
0.07
0.026
0.066
0.15
30b
10 b,f
10b
1g
41.7
57.4
314
88
38100
9920
914
173
78
376
79
1
1
2
2
1
1
Rat
0.0392
0.00956
24600
1300
132
230
NHP
15
All procedures performed on rats and monkeys were in accordance with regulations and
established guidelines and were reviewed and approved by Pfizer Institutional Animal Care and
Use Committee (IACUC).
a
b
39
Fraction unbound (f ) in plasma and liver tissue was determined by equilibrium dialysis.
u
Compounds 8, 11, and 12 were administered as PO suspensions in 0.5% methyl cellulose
c
containing 0.1% Tween 80 to male Wistar-Hannover rats (n = 2 or 3 animals). Total
concentrations in plasma and liver tissue were obtained 6 h post-dose and were converted into
unbound values by multiplying with the corresponding f values in plasma or liver tissue. K
d
u
p
e
refers to the ratio of total liver concentrations divided by total plasma concentrations. Unbound
liver-to-plasma ratio (K ) was determined as the ratio of mean free liver to plasma
pu,u
f
g
concentrations. 11 was administered as a hydrochloride salt. 12 was administered intravenously
IV) as a tris(hydroxymethyl)aminomethane (tris) salt in a solution of 2% N-methylpyrrolidone
(
and 98% sulfobutylether-betacyclodextrin (12.5%) in 50 mM TRIS buffer, pH 8.0 (n = 2).
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Encouraged by the level of liver selectivity observed, the pharmacokinetic profiles of 8, 11, and
1
2 were examined in rats (Table 5). Relative to compounds 8 and 11, 12 demonstrated a
considerably lower plasma clearance (CL ) and a longer terminal half-life (t ) upon IV
p
1/2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
administration to rats. Moreover, IV administration of 12 to NHPs also led to a very low CL rate
p
and a low steady state distribution volume (V ), which translated in a long terminal half-life (t )
ss
1/2
of ~ 11 h (see Table 5). PO pharmacokinetics studies in rats with the crystalline free form of 8
and 11 revealed that both compounds suffered from low oral bioavailability (F). In the case of 8,
the oral F of ~ 27% was commensurate with its high CL (54 mL/min/kg) in rats. In contrast, 11
p
demonstrated a lower CL relative to 8 in rats but was not systemically exposed when
p
administered orally. Poor PO exposure (assessed by the maximal plasma concentrations (Cmax
)
and area under the plasma concentration-time curve (AUC)) possibly reflected solubility-limited
absorption (in addition to first-pass hepatic uptake/metabolism processes leading to the observed
CL of 31 mL/min/kg) since 11 suffered from low thermodynamic aqueous solubility (pH 1.2
p
(2.0 µM), 6.5 (3.0 µM), and 7.4 (13 µM)). Although, the free form of crystalline 12 was more
soluble (pH 1.2 (8.0 µM), 6.5 (36.0 µM), and 7.4 (234 µM)) than 11, PO pharmacokinetic
studies with 12 were conducted with the corresponding tris(hydroxymethyl)aminomethane (tris)
salt form, which had improved aqueous solubility (pH 1.2 (1324 µM), 6.5 (3981 µM), and 7.4
(4772 µM)) over the free acid form. As shown in Table 5, PO administration of the tris salt of 12
led to rapid absorption (time to reach Cmax (Tmax) ~ 0.5–1.1 h) in rats and NHPs with oral F
4
0
values of 48% and 50%, respectively. The corresponding fraction of the oral dose absorbed was
estimated to be 78% (rats) and 55% (NHPs).
Compound 12 demonstrated negligible renal excretion (< 2% of administered dose) as
unchanged parent in rats upon IV dosing and lack of renal clearance was also noted in the IV
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pharmacokinetic study in NHPs. The potential of 12 to undergo biliary elimination in unchanged
form was also examined in rats. Approximately 12% of the administered IV dose (1 mg/kg) was
recovered in unchanged form in pooled (0–24 h) rat bile, implying that biliary elimination of 12
was minimal in this species. Moreover, IV administration (1 mg/kg) of 12 to NHPs resulted in a
mean Kpu,u value of 166 (Table 4) six hours post-dose, which provided further proof of its
hepatoselective properties mediated through OATP transporters. Given the good balance of ACC
inhibitory potency, desirable preclinical tissue distribution on a compound with high
permeability, and good pharmacokinetics, 12 was selected for further evaluation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Preclinical pharmacokinetics of 8, 11, and 12a
Compound Species Route/
Dose
CLp
Vss
t1/2
Cmax
Tmax
AUC(0-24)
Oral F
(%)
(mL/min/kg) (L/kg) (h) (ng/mL)
(h)
(ng.h/mL)
(mg/kg)
b
8
Rat
Rat
IV/1
54.5
NA
31.3
NA
16
NA
1.3
NA
4.1
NA
5.2
NA
7.2
NA
0.49
NA
3.5
5.7
7.5
NA
8.6
5.6
11
NA
53.9
NA
–
NA
390
NA
0.75
NA
NA
NA
0.54
NA
1.13
299
322
519
–
965
2490
14000
36200
PO/4
27
ND
48
b
1
1
IV/1
f
f
PO/4
c
12
Rat
IV/1
d
PO/5
e
NHP
IV/1
NA
5780
d
PO/5
11
52
a
All experiments involving animals were conducted in our AAALAC-accredited facilities and
were reviewed and approved by Pfizer Institutional Animal Care and Use Committee.
Pharmacokinetic parameters were calculated from plasma concentration–time data and are
reported as average values for n = 2. Maximum plasma concentrations (Cmax) after PO dosing
were determined directly from the experimental data, with Tmax defined as the time of first
occurrence of Cmax. The area under the plasma concentration-time curve from t=0 to 24 h (AUC0-
24)
was estimated using the linear trapezoidal rule. Oral bioavailability (F) was calculated from
dose-normalized AUC /AUC . Systemic plasma clearance was calculated as the IV dose
PO
IV
divided by the AUC obtained after IV dosing. Apparent steady-state volume of distribution (Vss)
was determined as the IV dose divided by the product of AUC and the elimination rate constant.
Male Wistar Hann rats and male cynomolgus monkeys were used for IV (n = 2) and PO (n = 2)
pharmacokinetic analysis. Unless otherwise noted, compounds were orally administered in
crystalline free form as a suspension in 0.5% MC containing 0.1% tween80 and PO
pharmacokinetics were examined in the fasted state in both species.
b
8
and 11 were administered IV as a solution in 10% DMSO/90% of 20% sulfobutylether-
betacyclodextrin in water.
c
12 was administered IV as a solution in 10% DMSO/30% PEG400/60% water.
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9
d
1
2 was administered as a tris(hydroxymethyl)aminomethane (tris) salt
e
1
2 was administered IV as a solution in 2% N-methylpyrrolidone and 98% sulfobutylether-
betacyclodextrin (12.5%) in 50 mM TRIS buffer, pH 8.0.
f
below the lower limit of detection (1 ng/mL). NA = not applicable; ND = not determined.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vitro Characterization of the Pharmacology of 12. A 96–well radioenzymatic assay that
1
4
14
measured incorporation of [ C]-bicarbonate into [ C]-malonyl–CoA was used to examine the
effect of 12 on purified human and rat ACC isozymes (ACC1 and ACC2) as previously
1
3
described. In substrate concentration-dependence experiments, compound 12 was found to be
4
1
uncompetitive with ATP and noncompetitive with carbonate, consistent with previously
2
reported inhibitors binding in this pocket of the protein. Dose-response experiments for
inhibition of human and rat ACC isozymes were performed at saturating concentrations of ATP
where the inhibitory concentration (IC ) approximates the equilibrium dissociation constant for
5
0
an enzyme (K ) for uncompetitive inhibitors. The IC values for inhibition of human ACC1 and
i
50
human ACC2 by 12 were determined to be 12.4  1.4 nM and 8.66  2.45 nM respectively.41
Similarly, 12 inhibited rat ACC1 and ACC2 in a concentration-dependent manner with IC50
4
1
values of 7.53  0.71 nM and 8.24  4.13 nM, respectively. The human ACC1 and ACC2
potencies of 12 in the radiometric assays were ~3-fold more potent than the corresponding values
determined in the Transcreener® assays. Overall, the ACC inhibitory potency and mode of
action of 12 was comparable to the values observed with the non-carboxylic acid derivative 3.
In Vitro Disposition and Off-Target Activity Characterization of 12. Whether the OATP
substrate properties of 12 translated into active hepatic uptake in primary human cells was
addressed in incubations of 12 (1 µM) in sandwich-cultured human hepatocytes in the absence or
4
2
presence of the OATP inhibitor rifamycin SV. Significant active uptake of 12 was observed
with an active to passive uptake clearance ratio of 8.7 (Table 6). Thus, 12 was likely to have
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
asymmetric liver distribution and an active uptake mediated systemic clearance in humans,
2
7
consistent with its extended clearance classification system (ECCS) class 1B classification. The
OATP isoform OATP2B1 and the sodium-taurocholate cotransporting polypeptide (NTCP) are
also expressed in the sinusoidal membrane of hepatocytes, and are capable of transporting
anionic substrates into the liver.25, 26 Therefore, we also examined the substrate properties of 12
for human OATP2B1 and human NTCP in vitro, using identical experimental conditions as the
ones used for OATP1B1 and OATP1B3. The uptake ratio of 12 (1 µM) by OATP2B1 and NTCP
in their corresponding HEK293 transfected cells was determined from 0.5–4.0 min at 37 °C. The
uptake ratios (mean of ratios from 0.5–4.0 min) of 12, which were obtained by dividing the
uptake in transporter transfected cells by the HEK293-wild type uptake, were 7.0 and 3.2, for
OATP2B1 and NTCP, respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Active Uptake of 12 (1 µM) in Sandwich-Cultured Human Hepatocytesa
CLactive
µL/min/mg) (µL/min/mg)
24 2.7
CLpassive
Active/Passive
Ratio
a
(
1
a
2
8.7
CLactive and CLpassive represent active uptake intrinsic clearance and passive diffusion
clearance, respectively. CLpassive of 12 was assessed in human hepatocyte incubations of 12
in the presence of 0.1 mM rifamycin SV (a pan-inhibitor of hepatic uptake transporters
including OATPs). CLactive and CLpassive were calculated by linear regression fitting of
initial rate analysis in the presence or absence of rifamycin SV in sandwich-cultured human
4
2
hepatocytes as previously outlined.
6
Because 12 was resistant towards metabolic turnover (CLint,met < 3.0 µL/min/10 cells) in a two-
hour human hepatocyte incubation, the CLint,met was reassessed in the human hepatocyte assay
under relay conditions (~ 20 h), which extends drug residence time and provides reliable
4
3
estimates of in vitro CLint,met for slowly metabolized compounds. Using relay conditions, a t
1/2
value of ~ 540 min for the metabolic decline of 12 in human hepatocytes was observed, which
1
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translated into a very low human CLint,met of 2.6 µL/min/10 cells. Preliminary metabolic
profiling of 12 in human hepatocytes revealed trace levels of several metabolites derived from
cytochrome P450 (CYP) mediated oxidations at the isopropyl group and/or the spirocycle region
as well as a secondary alcohol metabolite derived from reduction of the carbonyl moiety. There
were no human unique metabolites of 12; all metabolites of 12 observed in human hepatocytes
were also detected in hepatocytes from rat and monkey, which were the preclinical species for
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
toxicological evaluation of 12. Compound 12 exhibited high plasma protein binding in rat (f =
u,p
0
.0392), monkey (f = 0.00956), and human (f = 0.00600).
u,p u,p
Compound 12 demonstrated no reversible inhibition (IC > 50 µM) of the major human CYP
50
enzymes including CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4
44
in human liver microsomes. In addition, 12 did not cause metabolism-dependent (time-,
concentration- and NADPH-dependency) inhibition of major human CYPs in human liver
microsomes, since there was a < 1.5-fold shift in the IC values for reversible inhibition and <
5
0
2
0% increase in inhibition of CYP activity upon preincubation of 12 in human liver microsomes
4
5
in the presence or absence of the CYP co-factor NADPH. Moreover, treatment of 12 (0.5–200
µM) in individual human hepatocytes lots did not lead to induction of messenger RNA and
enzyme activity of CYP1A2 and CYP3A4, respectively. Reversible inhibition of major human
uridine 5’-diphospho-glucuronosyltransferase (UGT) enzymes by 12 was also investigated in
4
6
human liver microsomes using established protocols. No inhibition (IC > 20 µM) of
50
UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 catalytic activities was observed with
1
2.
The inhibitory potency of 12 for major human drug transporters was assessed in stably
3
8
transfected cell-based transporter systems using well-established protocols. Weak inhibition of
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
human multi-drug resistance protein (p-glycoprotein, ABCB1) and human breast cancer
resistance protein (ABCG2) catalyzed efflux of its respective substrates, pitavastatin and
digoxin, in MDCK-LE cell monolayers was observed with 12 providing IC values of 39 µM
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and 68 µM, respectively. Likewise, 12 demonstrated weak inhibitory effects on OATP1B1 and
OATP1B3 mediated transport of rosuvastatin in HEK293 cells with IC values of 11.7 µM and
5
0
1
37 µM, respectively. Compound 12 did not demonstrate any inhibition (IC > 30 µM) of the
50
uptake of para-aminohippuric acid and metformin by human renal transporters organic anion
transporter 1 (SLC22A6) and 3 (SLC22A8) and organic cation transporter 2 (SLC22A2) in
HEK293 cells. Collectively, these data suggest that 12 is unlikely to cause drug-drug interactions
with drugs that are subject to active transport via intestinal, liver, or kidney transporters.
Compound 12 demonstrated high selectivity for ACC inhibition, with no significant off-target
activity detected upon evaluation of 12 (at 100 µM) against a broad panel of transporters,
receptors, ion channels and enzymes, including the hERG channel. In addition, 12 did not cause
mutagenicity in the Salmonella Ames (strains TA98, TA100, TA1535 and TA1537) and in vitro
micronucleus assays (in Chinese hamster ovary or thymidine kinase heterozygote TK6 cells) in
the absence or presence of metabolic activation (aroclor-induced rat liver S9 fraction/NADPH).
Finally compound 12 demonstrated very weak inhibition (IC = 94 µM) of the human bile salt
5
0
4
7
export pump mediated transport of taurocholic acid in a membrane vesicle assay, suggesting
that 12 does not pose a risk of cholestatic liver injury due to impaired transport of cytotoxic bile
salts.48
In Vivo Pharmacodynamics of 12 in Rats. Sprague-Dawley rats were utilized to quantify the
1
4
effect of 12 on DNL in a tissue-specific manner by comparing the local incorporation of C-
labelled acetate into lipids, relative to vehicle treated animals. Single dose, PO administration of
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1
2 resulted in dose-dependent reductions in liver DNL with a maximal reduction of 82% (EC =
5
0
5
.4 nM (1.6-16.9 nM for 95% confidence interval)) at 300 mg/kg (corresponding to an unbound
plasma concentration of 119.4 nM ± 57.2 at 300 mg/kg). In contrast, the maximal DNL
inhibition observed in lung and bone marrow at this dose was 29 and 33%, respectively (Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
A). Collectively, these data demonstrate that the hepatoselective distribution of 12 results in
greater DNL inhibition in liver relative to other tissues.
A
B
n.s.
p<0.0001
p<0.0001
Liver
n.s.
n.s.
Bone Marrow
Lung
80
60
125
100
7
5
2
5
0
5
0
4
0
20
0
1 -
0
10
10-9
10-8
10-7
Free Plasma Conc. of 12 (M)
Vehicle
Vehicle
1
mg/kg
3 mg/kg
10 mg/kg
Chow
High-fat diet
Figure 3. Pharmacodynamic evaluation of 12 in rats. (A) Dose dependent effect of orally
administered 12 on DNL in liver, bone marrow, and lungs in rats relative to vehicle-treated
animals (n = 16 animals per dose group, data reported as mean ± SEM) two hours post-dose.
Administered PO doses of 12 were 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 mg/kg, respectively.
Vehicle was 0.5% methyl cellulose. (B) Dose dependent effect of chronic administration of 12
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
on hepatic TG levels in rats fed a high-fat diet (n = 10 animals per dose group, data reported as
mean ± SEM). n.s. = non-significant. SEM = standard error of the mean.
The effect of 12 on hepatic TG accumulation was also evaluated in Sprague-Dawley rats.
Relative to chow fed rats, vehicle treated rats fed a high-fat diet showed an approximately six-
fold increase in hepatic TG accumulation (Figure 3B). PO administration of 12 for 42 days to
high-fat diet fed rats produced dose dependent reductions in hepatic TG levels, with the 10
mg/kg dose reducing hepatic TG to levels that are statistically similar to the hepatic TG levels
observed in chow fed rats. Lower doses of 12 (1 and 3 mg/kg) demonstrated a trend towards
lowering of hepatic TG levels by ~30% but this did not reach statistical significance (p>0.05).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Vivo Pharmacodynamics and Safety Evaluation of 12 in NHPs. In order to measure the
effect of 12 on hepatic DNL in NHPs, male cynomolgus macaques were administered D O and
2
the correlation between plasma deuterium oxide and deuterium incorporation into plasma TG
palmitate was measured. PO fructose administration has been found to increase both the rate of
49
hepatic DNL and the reproducibility of DNL measures made between study periods. For these
reasons, the effect of 12 on hepatic DNL inhibition was assessed in NHPs concurrent to PO
fructose administration. Following an overnight fast and enrichment of plasma deuterium oxide,
fructose-loading resulted in significantly increased DNL in vehicle-treated animals (Figure 4).
This increase in DNL was fully attenuated by PO administration of 10 mg/kg of 12 and complete
inhibition of DNL persisted for the remainder of the nine-hour study.
2
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0
Vehicle
Compound 12
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
*
*
0
2
4
6
8
10
Hours
p <0.05 vs vehicle control
*
Figure 4. Effect of a single 10 mg/kg PO dose of 12 on fractional palmitate synthesis during
fructose-simulated DNL in NHPs (n = 12 animals, data reported as MEAN ± SEM). *p <0.05 vs
vehicle control. SEM = standard error of the mean. Fructose was given orally every 30 minutes
beginning with dosing of 12 (t = 0). One banana was also given at hour 2 and 3.
Compound 12 was also orally administered to NHPs (n = 3/sex/dose) at doses of 3, 10, 30, and
9
0 mg/kg/day for 2 weeks and platelet counts were evaluated prior to the initiation of dosing and
on dosing day 13. Administration of 12 at 3 or 10 mg/kg did not alter platelet count. However, at
doses ≥30 mg/kg/day, mild decreases in platelet counts were observed in animals (Figure 5). The
lowest absolute platelet count was 193,000/µL compared with concurrent control values ranging
from 218,000 to 442,000/µL. Overall, these observations were consistent with the
hepatoselectivity of 12, where fructose-stimulated increase in hepatic DNL was blunted with
administration of 12 at 10 mg/kg without any concomitant reduction in platelet count. The
hypothesis that the hepatoselective attribute of 12 is expected to provide an improved therapeutic
index in comparison to 3 for treating a liver disease such as NASH has been confirmed in a first-
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9
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in-human safety, tolerability, and pharmacokinetics study in healthy human subjects.50, 51 In this
study, compound 12 was well-tolerated in humans and doses that were associated with ~ 80%
inhibition of liver DNL did not lead to statistically significant decreases in platelet counts
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
following two weeks of daily dosing.50, 51
Compound 12
1
1
1
50
25
00
7
5
2
5
0
5
0
l
/kg
/kg
/kg
/kg
o
r
t
n
g
g
g
g
o
m
m
m
m
c
3
0
0
0
1
3
9
Figure 5. Change in platelets following 14 days of administration of compound 12 in NHPs.14
CHEMISTRY
Scheme 1. Synthesis of compound 4a
O
O
O
N
a
b
N
c
H
N
N
N
N
N
N
N
N
NH
HO C
N
2
NBoc
NBoc
O
Br
O
O
13
14
15
4
2
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a(a) i. NBS, MeOH, 44%; ii. KOt-Bu, THF, 80 °C; then 2 M HCl, quant.; (b) i. Pd(dppf)Cl , CO
2
(
1 mPa), MeOH, 100 °C, 78%; ii. TFA, CH Cl , 88%; (c) i. 2-methylbenzimidazole-5-carboxylic
2 2
acid, HATU, NMM, THF, 37%; ii. Aq. LiOH, MeOH, 62%.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
52
To synthesize pyrazole acid 4, the previously-described olefin 13 was treated with excess N-
bromosuccinimide (NBS) in methanol which led to both bromination of the pyrazole and O-
methyl bromohydrin formation (Scheme 1). Elimination to the enol ether with potassium t-
butoxide followed by treatment with acid led to ketone 14. The methyl ester was installed via Pd-
catalyzed carbonylation and subsequent Boc removal led to amine 15. Amide coupling followed
by ester hydrolysis provided pyrazole acid 4.
Scheme 2. Synthesis of compound 5a
O
H
H
N
N
N
a
b
H
N
N
N
N
HO
O
O
O
N
N
N
O
NH2
O
N
O
CO H
2
O
16
17
O
5
NH
18
a(a) i. K CO , I , DMF, 69%; ii. Zn(CN) , Zn, Pd(dppf)Cl , CuI, DMAc, 120 °C, quant.; iii.
2
3
2
2
2
UHP, 2.5 M NaOH, MeOH, 77%. (b) i. 18, EDC, HOBT, DMF, 70%; ii. NOPF , CH NO ; then
6
3
2
H O, 15%.
2
To access compound 5, indazole acid 17 was first synthesized in a three-step sequence (Scheme
). Indazole 16 was iodinated at the 3-position, copper-catalyzed cyanation was used to install
2
the nitrile, and urea-hydrogen peroxide (UHP) and sodium hydroxide were used to hydrolyze the
ester and nitrile to deliver indazole acid 17. Standard amidation conditions were used to couple
1
1
1
7 to amine 18. Oxidative hydrolysis with nitrosonium hexafluorophosphate was used to
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
provide 5 (Scheme 2) as the indazole amide intermediate was resistant to base-mediated
hydrolysis. Additionally, this method avoided a problematic opening of the spirochromanone
that was observed upon exposure to aqueous strong base and competing hydrolysis of the central
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
amide linkage.52
Scheme 3. Synthesis of compounds 6 and 7a
O
O
N
N
a
b or c
N
N
HO
I
O
O
N
N
O
I
19
20
O
O
R
6
, R = p-CO H
2
7
, R = m-CO H
2
a
(a) i. (COCl) , DMF, CH Cl ; ii. 18, i-Pr NEt, CH Cl , 71% two steps; (b) 4-
2
2
2
2
2
2
Carboxyphenylboronic acid, Pd(PPh ) , aq. Na CO , DMF, , 22%; (c) 3-Carboxyphenylboronic
3
4
2
3
acid, Pd(PPh ) , aq. Na CO , DMF, , 33%.
3 4
2
3
In order to access the substituted biphenyl acids 6 and 7, m-iodocarboxylic acid 19 was first
coupled to spirocylic amine 18 via acid chloride formation to provide 20. Suzuki coupling of
iodide 20 with 4- and 3-carboxylphenylboronic acids directly led to 6 and 7, respectively
(Scheme 3).
Scheme 4. Synthesis of compounds 8–12a
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9
R
N
O
O
Cl
R
R
N
a or b
c, d, or e
HO
f, g, or h
N
N
N
N
HO
HO
N
Cl
X
O
O
O
O
O
2
1
CO H
2
Ot-Bu
2
2
2, R = OMe, X = Cl
4, R = H,
X = Br
26, R = NMe , X = Cl
23, R = OMe
25, R = H
27, R = NMe2
8, R = OMe
9, R = H
10, R = NMe2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
O
O
R
N
N
N
i or j
N
N
NH
N
2
8
O
CO H
2
1
1
1, R = NMe /HCl salt
2
2, R = OMe/Tris salt
a(a) 32% aq. NaOH, MeOH, 93%; (b) Me NH, THF, 80 °C, 90% (c) 22, 4-(tert-
2
butoxycarbonyl)phenylboronic acid, pinacol ester, Pd(PPh ) , aq. Na CO , dioxane, 110 °C,
3
4
2
3
6
6%; (d) 24, 4-(tert-butoxycarbonyl)phenylboronic acid, Pd(dppf)Cl , aq. K PO , dioxane, 70
2 3 4
°
C, 20%; (e) 26, 4-(tert-butoxycarbonyl)phenylboronic acid, Pd(OAc) , SPhos, aq. Na CO ,
2 2 3
dioxane, 95 °C, 65% or 26, 4-(tert-butoxycarbonyl)phenylboronic acid, aq. Na CO ,
2
3
Pd(dppf)Cl ·CH Cl , DME, 100–120 °C; (f) i. 18, 23, HATU, i-Pr NEt, CH Cl , quant.; ii. TFA,
2
2
2
2
2
2
CH Cl , 92%; (g) 18, 25, EDC, HOBT, i-Pr NEt, DMF, 23–70 °C; ii. 4 M HCl in dioxane, 46%
2
2
2
two steps, (h) i. 18, 27, EDC, HOBT, Et N, CH Cl ; ii. TFA, CH Cl , 66% two steps; (i) i. 27,
3
2
2
2
2
EDC, HOBT, iPr NEt, CH Cl , 48% over two steps; ii.4 M HCl in dioxane, CH Cl , MeOH,
2
2
2
2
2
5
5%; (j) i. CDI, THF; 23, CH Cl , 50 °C, 82%; ii. H PO , PhCH , 50 °C, 52%; iii.
2 2 3 4 3
(
HOCH ) CNH , EtOH, heptane, 92%.
2 3 2
The syntheses of pyridyl analogs 8–11 were completed through a three-step sequence (Scheme
): (1) Suzuki coupling of halo-carboxylic acids 22, 24, or 26, with the para-ester substituted
4
5
2
phenylboronic acid or pinacol ester, (2) amidation with 18 or 28, and (3) acid-catalyzed
hydrolysis of the t-butyl ester to reveal the carboxylic acid. To synthesize 12 on larger scale, acid
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
intermediate 22 was prepared from dichloropyridine 21 via S Ar with sodium methoxide. To
N
improve practicality, CDI was employed for the amide coupling between 23 and 28 and
phosphoric acid was used for the ester hydrolysis to produce 12.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Tris(hydroxymethyl)aminomethane (Tris) was then used to produce the salt of 12.
CONCLUSIONS
Reductions in platelet count were observed in previous clinical studies evaluating ACC inhibitor
3
. These reductions were demonstrated to result from ACC inhibition in bone marrow leading to
impaired platelet production. The design of hepatoselective compounds was pursued as a
strategy to improve the safety profile of ACC inhibitors. Starting with ACC inhibitors 2 and 3, a
series of analogs was prepared by incorporating a biaryl motif appended with a carboxylic acid
to infer OATP recognition and active transport into hepatocytes. This effort led to the
identification of ACC inhibitor PF-05221304 (12) which exhibited both a high level of
hepatoselectivity and passive permeability, and good oral bioavailability in animals. The good
oral absorption of 12 in animals when coupled with its very low metabolic clearance in human
hepatocytes suggest that 12 may be suitable for administration to humans in a once daily dosing
paradigm with little-to-no risks of drug-drug interactions with concomitantly administered
medications. The liver exposure of 12 led to significant and selective DNL inhibition in the liver
relative to other tissues, providing an efficacious and safe profile in preclinical species. Based on
its overall disposition and safety profile, 12 was progressed to clinical trials and demonstrated an
attractive safety, tolerability, pharmacokinetic, and pharmacodynamic profile in humans
following administration of single- and multiple-ascending doses.51
EXPERIMENTAL SECTION
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General. Unless otherwise noted, the reported experiments followed these considerations: All
chemicals, reagents, and solvents were purchased from commercial sources when available and
used without further purification. Reactions were magnetically stirred, and monitored by thin
layer chromatography, liquid chromatography-mass spectrometry (LCMS), and/or high
performance liquid chromatography (HPLC). Reactions were carried out at room temperature
(∼23 °C) unless otherwise noted. Air and moisture-sensitive were carried out under an
atmosphere of nitrogen. Silica gel chromatography was performed using an Isco or Biotage
automated purification system with pre-packaged columns. Concentration under reduced
pressure (in vacuo) was performed by rotary evaporation at 25–45 °C. Purified compounds were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
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5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
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9
0
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9
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1
further dried under high vacuum with or without heat to remove residual solvent. H NMR
spectra were obtained with a spectrometer at 600 MHz or 400 MHz. Chemical shifts were
1
referenced to the residual H solvent signals (CDCl , δ 7.27; DMSO-d , δ 2.50; CD OD, δ 3.31).
3
6
3
Signals are listed as follows: chemical shift in ppm; multiplicity identified as br, broad; s, singlet;
d, doublet; t, triplet; q, quartet; spt, septet. m, multiplet; coupling constants (J) in Hz.
All compounds evaluated were > 95% pure. Specific interaction between this chemical series and
ACC is supported by co-crystal structures such as the one reported with compound 2.13
Synthesis of analogs and intermediates
tert-Butyl 3-bromo-1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-
52
carboxylate (14). Step 1: 13 (1 g, 3 mmol) in MeOH (20 mL) was added NBS (1.6 g, 9.0
mmol). The mixture was stirred at rt for 1 h then concentrated in vacuo. The residue was
dissolved in ethyl acetate, then washed with saturated sodium thiosulfate, 0.5 M NaOH (20 mL),
brine, and concentrated in vacuo. The resulting oil was purified by flash column chromatography
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(
petroleum ether:ethyl acetate, 10:1) to provide tert-butyl 3,6-dibromo-1-isopropyl-7-methoxy-
1
,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1'-carboxylate (730 mg, 44%) as a colorless oil.
1
H NMR (400 MHz, CDCl ) δ 4.70 (d, J=1.5 Hz, 1H), 4.40 (s, 1H), 4.29 (spt, J=6.7 Hz, 1H),
3
0
1
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5
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0
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.77–3.66 (m, 1H), 3.63-3.53 (m, 4H), 3.25 (ddd, J=3.5, 10.3, 13.3 Hz, 1H), 3.14 (ddd, J=3.5,
.8, 13.8 Hz, 1H), 2.57 (d, J=16.1 Hz, 1H), 2.35 (d, J=16.1 Hz, 1H), 1.80-1.63 (m, 4H), 1.51 (d,
9
J=6.5, 3H), 1.48 (d, J=6.5, 3H), 1.46 (s, 9H).
Step 2: To a solution of tert-butyl 3,6-dibromo-1-isopropyl-7-methoxy-1,4,6,7-
tetrahydrospiro[indazole-5,4'-piperidine]-1'-carboxylate (760 mg, 1.46 mmol) in THF (20 mL)
was added KOtBu (340 mg, 3.03 mmol). The mixture was stirred at 80 °C for 15 h. HCl (2 M, 20
mL) was added to the solution and the mixture was stirred at rt for 3 h. The reaction was
concentrated in vacuo. The resulting residue was dissolved in ethyl acetate and washed with
water (20 mL) and brine then concentrated in vacuo. Flash column chromatography (petroleum
1
ether:ethyl acetate, 10:1) was used to provide 14 (620 mg, quant.) as a colorless oil. H NMR
(400 MHz, CDCl ) δ 5.36 (spt, J=6.6 Hz, 1H), 3.55–3.44 (m, 2H), 3.44–3.30 (m, 2H), 2.65 (s,
3
2H), 2.54 (s, 2H), 1.71–1.38 (m, 19H).
Methyl 1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4’-piperidine]-3-carboxylate-TFA
(15). Step 1: To a solution of 14 (300 mg, 0.70 mmol) in MeOH (10 mL) was added Pd(dppf)Cl2
(20 mg, 0.027 mmol). The mixture was degassed for 10 min, filled with carbon monoxide (1
mPa), and heated to 100 °C for 15 h. The reaction was concentrated in vacuo and purified via
flash column chromatography (petroleum ether:ethyl acetate, 15:1) to provide 1'-(tert-butyl) 3-
methyl 1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-1',3-dicarboxylate
2
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(
220 mg, 78%) as a colorless oil. H NMR (400 MHz, CDCl ) δ 5.45 (spt, J=6.6 Hz, 1H), 3.94
3
(
s, 3H), 3.51–3.35 (m, 4H), 3.05 (s, 2H), 2.55 (s, 2H), 1.53–1.48 (m, 10H), 1.44 (s, 9H).
Step 2: 1'-(tert-butyl) 3-methyl 1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-
piperidine]-1',3-dicarboxylate (220 mg, 0.540 mmol) was added to 1:1 TFA:CH Cl (10 mL) and
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2
the mixture was stirred at rt for 3 h. The reaction was concentrated in vacuo to provide 15 (200
mg, 88%) which was used into next step without further purification.
1
-Isopropyl-1'-(2-methyl-1H-benzo[d]imidazole-5-carbonyl)-7-oxo-1,4,6,7-
tetrahydrospiro[indazole-5,4'-piperidine]-3-carboxylic acid (4). Step 1: To a solution of 2-
methylbenzimidazole-5-carboxylic acid (200 mg, 1.13 mmol) in THF (10 mL) was added HATU
(570 mg, 1.50 mmol) and the mixture was stirred at rt for 30 min. Compound 15 (300 mg, 1
mmol) and NMM (300 mg, 1 mmol) were then added and the reaction stirred for 3 h. The
reaction was concentrated in vacuo and reverse-phase preparative HPLC was used to provide
methyl 1-isopropyl-1'-(2-methyl-1H-benzo[d]imidazole-5-carbonyl)-7-oxo-1,4,6,7-
1
tetrahydrospiro[indazole-5,4'-piperidine]-3-carboxylate (170 mg, 37%) as a white solid. H NMR
(
400 MHz, CD OD) δ 7.68–7.42 (m, 2H), 7.27 (dd, J=1.5, 8.5 Hz, 1H), 5.46 (spt, J=6.7 Hz, 1H),
3
3
1
.93 (s, 3H), 3.91–3.65 (m, 2H), 3.64–3.44 (m, 2H), 3.13 (s, 2H), 2.70 (s, 2H), 2.58 (s, 3H),
.78–1.50 (m, 4H), 1.46 (d, J=7.0 Hz, 6H).
Step 2: A round bottom flask was charged with methyl 1-isopropyl-1'-(2-methyl-1H-
benzo[d]imidazole-5-carbonyl)-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4'-piperidine]-3-
carboxylate (120 mg, 0.26 mmol) and 1:1 MeOH:water (6 mL). Lithium hydroxide (100 mg, 2.6
mmol) was then added and the reaction was stirred at rt for 15 h. The reaction was concentrated
in vacuo. The residue was acidified to pH 5 and dried in vacuo to give a residue, which was
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