acetyl - coa carboxylase 
 ( ec6.4.1.2 ) ( acc ) is a biotin carboxylase 
 that catalyzes the atp - dependent condensation of acetyl - coa and carbonate 
 to form malonyl - coa .
it is an essential 
 and rate - limiting substrate for de novo lipogenesis ( dnl ) , and it 
 acts as an allosteric inhibitor of the enzyme carnitine - palmitoyl 
 transferase i ( cpt-1 ) .
cpt-1 is responsible for the transport of long - chain 
 fatty acyl - coas across the mitochondrial membrane into the mitochondria 
 where they become available for fatty acid oxidation .
thus , acc is positioned 
 as a key physiologic switch regulating the transition from oxidative 
 to lipogenic metabolism .
metabolic perturbations , including suppressed 
 fatty acid oxidation and increased hepatic dnl , have been hypothesized 
 to contribute to ectopic accumulation of lipid species in muscle and 
 liver , which in turn have been hypothesized to play a causative role 
 in the molecular pathogenesis of insulin resistance .
inhibition of malonyl - coa production by acc is expected to simultaneously 
 inhibit dnl and increase flux through cpt-1 , leading to increased 
 -oxidation of long - chain fatty acids , and thus may lead to 
 reduced ectopic lipid accumulation and improved insulin sensitivity . 

acc inhibition is therefore an attractive biological target for the 
 treatment of metabolic diseases such as t2 dm and nonalcoholic fatty 
 liver disease .
consistent with this hypothesis , 
 antisense oligonucleotide inhibition of acc significantly reduced 
 diet - induced hepatic steatosis and hepatic insulin resistance .
the two closely related isoforms , acc1 and acc2 , 
 are encoded by separate gene products that differ in tissue and subcellular 
 distribution .
acc1 is primarily located 
 in liver and adipose tissue , while acc2 is the dominant isoform in 
 skeletal and heart muscle .
acc1 is also expressed in multiple human 
 cancers , making it an attractive oncology target .
we 
 sought balanced inhibitors of acc1 and acc2 to gain benefit from inhibition 
 of the enzyme in both liver and muscle .
several acc inhibitors 
 have been disclosed in recent years , with 
 much consideration directed toward understanding whether selective 
 or balanced inhibition of acc1/acc2 is preferable . published results 
 to date
abbott described an acc2-selective 
 thiazole ether ( r = ome ) that elicited dose - dependent reductions in 
 muscle malonyl - coa levels .
however , significant 
 neurological and cardiovascular safety events were observed and attributed 
 to the alkyne - containing structure of the specific compound . using a related acc2-selective compound from 
 the abbott disclosures ( r = me ) , boehringer ingelheim observed reductions 
 in malonyl - coa , stimulation of fatty acid oxidation , improvements 
 in glucose tolerance , and hba1c reductions following chronic treatment 
 of db / db mice .
a phenyl ether from sanofi - aventis , 
 with unselective activity against acc1/acc2 , increased lipid oxidation 
 but failed to decrease hepatic triglycerides or body weight in diet - induced 
 obese ( dio ) mice or in zucker diabetic fatty rats after chronic administration .
takeda described a spiro - pyrazolidinedione with balanced acc1/acc2 
 activity that showed dose - dependent changes in respiratory quotient 
 in rats , providing evidence of increased fatty acid oxidation .
amgen s piperazine oxadiazole with dual acc1/acc2 inhibition 
 decreased malonyl - coa levels but unexpectedly increased plasma glucose 
 and impaired glucose tolerance in dio mice treated for 28 days .
the natural product soraphen a , also an inhibitor 
 of both acc1/acc2 , decreased weight gain and body fat content in mice 
 and improved insulin sensitivity , although a narrow safety window 
 may have confounded the results .
nimbus 
 disclosed favorable impact on weight gain , triglycerides , cholesterol , 
 and insulin sensitivity in dio rats with a compound whose specific 
 structure was not reported .
pfizer described a 
 spiroketone ( 1 ) that decreased malonyl - coa in liver and 
 muscle ; the subject of this paper is 
 follow - up to that disclosure , along with preclinical and human biology 
 data for a lead compound .
the n2-alkyl pyrazole 
 ketones with substitution 
 at the -position to the ketone and the n1-alkyl pyrazole ketones 
 described in this work were synthesized by the general methods shown 
 in schemes 1 and 2 . 

the mono- and dimethyl substituted ketone cores in scheme 1 were synthesized by -alkylation of the respective 
 precursor ketones .
although the desired enolates could be formed at 
 low temperature by treatment of the ketone with lithium diisopropylamide 
 ( lda ) or lithium hexamethyldisilazide ( lhmds ) , the rate of alkylation 
 ( for example , with methyl iodide ) was slow relative to the rate of 
 ring - opening -elimination of the oxy - pyrazole anion .
notably , 
 addition of 1,3-dimethyl-3,4,5,6-tetrahydro-2(1h)-pyrimidinone 
 ( dmpu ) achieved both increased efficiency of enolate formation and 
 increased reactivity of the enolate to the desired alkylation .
the 
 pyrazoloketones in scheme 2 were prepared by 
 regioselective addition of alkyl hydrazines to a cyclohexenone intermediate , 
 followed by functionalization of the olefin .
compound 1 was previously 
 disclosed as a candidate for in vivo efficacy and toxicology evaluation , demonstrating good potency against human acc1 
 and acc2 ( ic50 = 111 and 9.8 nm , respectively ) and low 
 human liver microsomal clearance ( < 4.9 ml
key observations 
 during the preparation for and analysis of those in vivo efficacy 
 and safety studies guided the search for improved properties in subsequent 
 compounds .
first , during synthetic scale - up , a byproduct arising from 
 ring - opening elimination of oxy - pyrazole was observed , presumably 
 via a retro - michael reaction analogous to that shown in scheme 1 , an obstacle that was overcome but which increased 
 concern about potential instability of the -pyrazoloxy - ketone 
 functionality .
second , crystalline compound 1 exhibited low solubility , requiring a spray - dried dispersion 
 formulation to achieve adequate compound exposure in toxicology studies . 

third , the ketone functional group in 1 was reduced in 
 vivo to provide substantial circulating levels of the alcohol metabolite , 1 m .
the alcohol metabolite was 
 formed upon incubation of 1 with rat , dog , and human 
 liver microsomes , with reduction rates highest in dog and lowest in 
 rat .
pharmacokinetic ( pk ) studies in 
 dog showed the alcohol metabolite 1 m had nine - times higher 
 exposure relative to parent compound 1 ( see figure 2 ) .
the metabolite had substantially reduced acc1 
 and acc2 potency ( ic50 = 8.7 and 1.0 m , respectively ; 
 see supporting information , table s1 ) relative 
 to parent , but the high level of ketone reduction was a concern for 
 two main reasons : ( a ) decreased confidence in human pk predictions 
 with ketone reduction as a primary metabolic pathway and ( b ) increased 
 off - target pharmacology risk due to high circulating levels of metabolite , 
 with our attention focused primarily on herg inhibition because ketone 1 and alcohol 1 m had similar potencies versus 
 this ion channel ( ic50 = 34 and 57 m , respectively ) .
plasma 
 concentrations of 1 and its alcohol metabolite 1 m in beagle dogs following oral administration of 1 at 10 mg / kg .
metabolite 1 m had higher exposure ( auc0 = 27800 ngh / ml ) than parent 1 ( auc0 = 3030 ngh / ml ) .
as a result of these observations from the study 
 of compound 1 , a decreased rate of ketone reduction became 
 the primary 
 objective in the design of new compounds .
the initial hypothesis for 
 decreasing ketone reduction was to increase steric hindrance around 
 the ketone , leading to two approaches 
 for substitution proximal to the ketone : adding alkyl substituents 
 on the methylene adjacent to the ketone and altering the pyrazole 
 to the regioisomeric n1-alkyl ( rather than n2-alkyl ) derivative .
consideration of the crystal structure of 1 bound 
 in acc provided support for exploring the -substitution strategy . 

as described previously , the acc ct - domain 
 is an obligate homodimer , with the active site composed of residues 
 from each monomer ( denoted a and b ) . an x - ray cocrystal structure 
 of 1 bound in a 
humanized yeast  chimeric 
 ct - domain of acc demonstrated binding 
 to the putative biotin binding site consistent with previous structures 
 ( figure 3 ) .
compound 1 bound in a generally hydrophobic cleft , with all of its polar atoms 
 satisfied and sequestered from competing solvent interactions .
the 
 ketone and amide carbonyls made key hydrogen - bond contacts to the 
 protein , orienting the rigid spirocycle in a low - energy conformation 
 that allowed for ideal placement of polar and hydrophobic interactions 
 with a minimal loss of conformational entropy .
there appeared to be 
 sufficient space adjacent to the ketone to accommodate substitution 
 with alkyl groups , though specific interactions with the protein were 
 not expected with small substituents .
a crystallographic water 
 was hydrogen - bonded to n1 , but the water was calculated using hydrosite to be low energy and easily displaced by a potential 
 n1-substituent . however , there was also potential that the increased 
 steric congestion would negatively impact the hydrogen - bond between 
 the ketone and the backbone nh .
co - crystal structure of 1 bound in the ct - domain binding 
 domain of acc .
compound 1 was oriented in the channel 
 by hydrogen bonds between the ketone and nh - gly - b1958 and between 
 the amide carbonyl and nh - glu - b2026 .
the pyrazolopyranone group was 
 sandwiched in a hydrophobic cleft lined with side chains of leu - a1762 , 
 val - a1765 , leu - a1766 , ala - b1920 , val - b1923 , and phe - b1925 .
the hydrogen 
 bonding potential of the pyranone ether oxygen was unsatisfied , but 
 this region of the active site was capped by the side chain of arg - b1954 . 

the distance between the ether oxygen and the guanidinium group of 
 the arginine ( 3.8  ) was consistent with a weak electrostatic 
 interaction , which likely alleviated the thermodynamic penalty associated 
 with burying an unsatisfied hydrogen bonding group .
the results of introducing substituents on the 
 carbon adjacent 
 to the ketone are shown in table 1 .
for routine 
 screening , potencies were assessed versus human acc1 and acc2 using 
 recombinant enzymes in a transcreener format .
consistent with the strategy employed in the identification of compound 1 , lipophilic efficiency ( lipe ) was a key parameter that was 
 used to evaluate the quality of new compounds .
unfortunately , the monomethyl stereoisomers , 2 and 3 , and the dimethyl derivative , 4 , 
 demonstrated insufficient potency as acc inhibitors .
the additional 
 lipophilicity relative to parent compound 1 also increased 
 intrinsic clearance in human liver microsomes ( hclint , 
 table 1 ) . on the specific property of ketone 

reduction , the qualitative rate of ketone reduction as assessed by 
 rate of alcohol formation in human liver microsomes was 1 > 2  3 > 4 ( data 
 not shown ) .
evaluation of the dimethyl derivative 4 in 
 a dog pk study ( figure 4 ) showed a marked decrease 
 in the amount of alcohol metabolite 4 m relative to parent 
 ketone ( 23-fold greater exposure for 4 ) .
although the 
 -alkylation approach did not seem likely to lead directly to 
 a candidate , these adme data provided strong support for the hypothesis 
 that increased steric hindrance was a viable strategy for decreasing 
 the rate of reduction .
human 
 acc1 and acc2 potencies 
 are the geometric mean of at least three replicates ( 95% confidence 
 interval ) .
hclint = intrinsic clearance in human liver 
 microsomes ; herg = inhibition of the herg channel in a patch - clamp 
 assay ; nd = not determined .
plasma concentrations 
 of 4 and its alcohol metabolite 4 m in beagle 
 dogs following oral administration of 4 at 1 mg / kg .
metabolite 4 m had lower exposure ( auc0 = 14 
 ngh / ml ) than parent 4 ( auc0- = 321 ngh / ml ) .
the regioisomeric n1-alkyl pyrazole derivatives were the 
 alternative 
 structural change designed to increase steric hindrance around the 
 ketone .
the synthetic route developed for these compounds also provided 
 the opportunity to replace the cyclic ether in compound 1 with a carbocycle , thus obviating the potential for retro - michael 
 ring - opening .
the potency of the corresponding cyclic ether and carbocyclic 
 analogues was similar for both the n2- and n1-alkyl series across 
 a range of analogues ( data not shown ) .
although there was some concern 
 that removing the electron - donating ether might increase electrophilicity 
 of the pyrazoloketone and the associated rate of ketone reduction , 
 we believed that the steric effects of the n1-alkyl substituent would 
 dominate any change in electronics .
compounds 5 , 6 , and 7 with varying steric 
 bulk of the n1-substituent showed an 
 encouraging profile .
importantly , the structural changes from 1 to 5/6 retained both acc potency 
 and selectivity versus herg .
the relatively high lipe of compound 6 , which was reflected in the desirable balance of acc potency 
 and human liver microsomal ( hlm ) stability , led to selection of r 
 = isopropyl as the preferred pyrazole n1-substituent . having made 
 structural changes to the core of the molecule that increased steric 
 hindrance around the ketone and removed the potentially labile cyclic 
 ether linkage , we focused on the piperidine amide group to improve 
 solubility . within the scope of 5,6-bicycloheteroaromatic groups ( a 
 topology found to be important for potency in the discovery of compound 1 ) ,
weakly basic rings were targeted to achieve the most desirable 
 balance between solubility and herg potency .
pyrrolopyridine 8 and benzimidazole 9 were two of the most promising 
 derivatives , with the benzimidazole 9 favorably differentiating 
 on both hlm stability and herg activity .
furthermore , the solubility 
 at ph 1.2 of crystalline compounds 8 ( 2320 g / ml ) 
 and 9 ( 1980 g / ml ) increased substantially as compared 
 to crystalline compound 1 ( 20 g / ml ) . to assess 
 the impact of pyrazole n1-substitution on the rate of ketone reduction , 

the benzimidazole 9 was tested in a dog pk study ; the 
 data depicted in figure 5 demonstrated a 24-fold 
 lower exposure to alcohol metabolite 9 m as compared to 
 parent compound 9 .
human 
 acc1 and acc2 potencies 
 are the geometric mean of at least three replicates ( 95% confidence 
 interval ) .
plasma concentrations 
 of 9 and its alcohol metabolite 9 m in beagle 
 dogs following oral administration of 9 at 3 mg / kg .
plasma 
 levels of 9 m were below the lower 
 level of quantitation beyond the 4 h time point .
metabolite 9 m had lower exposure ( auc0 = 330 
 ngh / ml ) than parent 9 ( auc0 = 7930 ngh / ml ) .
comparison of the x - ray cocrystal structures of 1 and 6 bound in the ct - domain of acc showed that both 
 inhibitors 
 maintained hydrogen bonds between the two carbonyls and protein backbone 
 nhs ( figure 6 ) .
as predicted , the n1 substituent 
 of 6 displaced the crystallographic water that was hydrogen - bonded 
 to the ketone and to the pyrazole n1 in the bound structure of 1 .
notably , however , the pyrazoloketone group of compound 6 shifted by 17 relative to 1 such 
 that the n1-ipr group bound in a similar position 
 to the n2-tbu group .
the concomitant shift in the 
 ketone position resulted in a less favorable geometry but a nearly 
 identical hydrogen bond length for 6 as compared to 1 , suggesting comparable binding energies for the two interactions . 

the piperidine and indazole groups were essentially undisturbed by 
 the n1 substitution and made similar interactions with the protein 
 as in the structure of 1 .
co - crystal structure 
 of 6 ( magenta ) bound in the ct - domain 
 of acc , overlaid with the bound conformation of 1 ( orange ) .
compound 9 met our 
 initial project objectives with 
 its attractive overall profile of acc inhibitory potency , relatively 
 low extent of ketone reduction , increased solubility , diminished herg 
 activity , and good hlm stability .
compound 9 exhibited low protein binding in both rat and dog plasma but approximately 
 10-fold higher binding in both monkey and human plasma .
the in vitro 
 metabolism of 9 was evaluated in microsomes from rat , 
 dog , and human hepatocytes .
compound 9 was not metabolized 
 ( as assessed by disappearance of parent ) in rat , dog , or human microsomes . 

compound 9 was also stable in human hepatocyte incubations 
 ( data not shown ) , but was minimally metabolized by recombinant human 
 cyp3a4 and cyp3a5 , suggesting it is a substrate for cyp3a4 and cyp3a5 .
clh microsomes estimated 
 from well - stirred model , including blood : plasma ratio and plasma and 
 microsomal binding . fed , 
 vehicle : 20% sbecd .
fed , vehicle : 40% 
 2-pyrrolidinone/60% 
 ph 4.5 citrate buffer , solution , sterile filtered , ph = 5.41 .
fed , 
 vehicle : 10% dmso/20% 2-pyrrolidinone/70% 
 of 20% sbecd in water . in 
 vivo , the plasma clearance of 9 was low following 
 intravenous ( iv ) administration ( 1 mg / kg ) to rats , dogs , and monkeys 
 ( table 4 ) .
oral ( po ) administration ( 3 mg / kg ) 
 to rats and dogs showed bioavailability of 40% and 54% , respectively , 
 consistent with the low microsomal clearance and good solubility at 
 low ph .
the bioavailability following a 50 mg / kg oral dose in rats 
 was 106% , suggesting saturation of clearance .
in vitro potencies are the geometric 
 mean of at least 3 replicates  the standard error of the mean 
 ( sem ) .
a 96-well radioenzymatic assay 
 using both recombinant human and 
 purified rat acc isozymes was used to confirm in vitro inhibitory 
 potency for the determination of pharmacokinetic / pharmacodynamic ( pk / pd ) 
 relationships .
this assay format was also used with recombinant human 
 acc2 to determine the mode and reversibility of inhibition .
compound 9 was found to be uncompetitive with atp and noncompetitive 
 with carbonate and acetyl - coa ( see lineweaver 
burk plots in 
 the supporting information ) , consistent 
 with previously reported inhibitors binding in this pocket of the 
 protein . dose 
response experiments 
 were performed at saturating concentrations of atp and acetyl - coa 
 where the inhibitory concentration ( ic50 ) approximated 
 the equilibrium dissociation constant for an enzyme ( ki ) for uncompetitive inhibitors .
compound 9 was shown to inhibit both rat and human acc1 and acc2 in vitro with 
 similar potencies ( table 4 and supporting information ) .
the human acc1 and acc2 
 potencies in the radiometric assays were 3- and 2-fold more potent , 
 respectively , than values determined in the transcreener assays .
ex vivo , the effect of 9 on the acc / malonyl - coa axis 
 was assessed in primary rat hepatocytes ( figure 7 ) .
compound 9 inhibited formation of malonyl - coa in 
 a concentration - dependent manner with a potency ( ec50 = 
 30 nm ) in rat hepatocytes consistent with its potency against rat 
 acc1 ( 24 nm ) .
cryopreserved rat hepatocytes were plated on collagen - coated 96-well 
 plates and overlaid with matrigel .
the cultured hepatocytes were incubated 
 in the presence and absence of a range of concentrations of 9 . following incubation , malonyl - coa levels in lysates from 
 the treated and untreated hepatocytes were analyzed using high - throughput 
 mass spectrometry .
on the basis of the robust 
 enzyme and cellular activity of 9 , the effects of the 
 compound on modulation of acc activity 
 in vivo was assessed first as changes in levels of malonyl - coa , and 
 subsequently as the downstream impact on dnl and fuel substrate utilization . 

the low plasma clearance and high exposure following oral administration 
 in rats ( see table 3 ) enabled the ready examination 
 of in vivo pharmacology .
formation of the direct product of 
 acc , malonyl - coa , in the skeletal 
 muscle ( quadriceps ) and liver of lean sprague 
dawley ( sd ) rats 
 was assessed 1 h following an acute oral dose of 9 , showing 
 concentration - dependent reductions in both skeletal muscle and liver 
 malonyl - coa ( figure 8) . at the nadir ,
quadriceps 
 and liver malonyl - coa levels were reduced by 76% and 89% , respectively . 

the ec50s for inhibition of quadriceps and liver malonyl - coa 
 were 870 and 540 nm , respectively , determined from unbound plasma 
 concentrations of 9 .
effect 
 relationship of orally 
 dosed 9 and malonyl - coa tissue levels in liver and quadriceps 
 from male sd rats .
rats were dosed by oral gavage with vehicle or 
 a range of doses of 9 .
one hour postdose , rats were euthanized , 
 plasma was collected for compound exposure measurements , and liver 
 and skeletal muscle ( quadriceps ) were rapidly removed and freeze - clamped 
 in liquid nitrogen .
malonyl - coa content of tissue extracts was determined 
 by liquid chromatography mass spectrometry ( lc - ms ) .
tissue concentrations 
 of malonyl - coa for each dose group were plotted against the corresponding 
 free plasma concentrations of 9 measured from the same 
 animals . to confirm that the observed malonyl - coa 
 biomarker reduction led 
 to the anticipated changes in lipid metabolism , we examined the impact 
 of 9 on dnl and respiratory exchange ratio ( rer ) , the 
 latter being an indication of whole - body fuel substrate utilization . 

acute oral administration of 9 inhibited hepatic dnl 
 in rats in an unbound plasma drug concentration - dependent manner . 

compound 9 inhibited up to 82% of the incorporation of 
 [ c]acetate into [ c]lipids with an ec50 of 326 nm ( figure 9 ) .
lean , male sd rats fed ad libitum with 
 standard chow were dosed by oral gavage with vehicle or a range of 
 doses of 9 .
one hour later , animals 
 were euthanized and liver punch samples and plasma were collected . 

the [ c]lipid generated from the [ c]acetate 
 through dnl was isolated and measured from the liver samples by organic 
 extraction with ethanol and petroleum ether and quantified by scintillation 
 counting .
consistent with the 
 role of acc in modulating fatty acid oxidation , 
 oral treatment with 9 also produced acute reductions 
 in whole - body rer .
to drive the basal fuel substrate utilization toward 
 increased dependence on carbohydrate usage ( increased rer ) , rats were 
 fasted for 24 h and then re - fed a high sucrose diet for 48 h prior 
 to the experiment . following baseline rer measurement ,
the rats were 
 dosed by oral gavage with a range of doses of 9 or vehicle . 
 immediately following dosing ,
animals were returned to the metabolic 
 chambers and rer was monitored for an additional 105 min .
following 
 the last rer measurement , animals were euthanized and skeletal muscle 
 and plasma were collected for malonyl - coa and drug exposure measurements , 
 respectively . within 15 min of oral administration of 9 ,
dose - dependent 
 reductions in rer indicated a shift in fuel substrate utilization 
 toward increased net whole - body dependence on fatty acid utilization 
 ( figure 10a ) . at the nadir ,
rer values in the 
 highest two doses of 9 dropped 0.19 units and at 120 
 min postdose the ec50 was 119 nm ( figure 10b ) .
further , the change in rer from predose was found to be 
 proportional to the quadriceps malonyl - coa levels measured from the 
 same animals ( figure 10c ) .
respiratory exchange 
 ratio ( rer ) in male sd rats dosed orally with 9 .
rats were 
 treated with a single oral dose of 9 at 100 mg / kg ( red 
 circle ) , 40 mg / kg ( blue square ) , 20 mg / kg ( green inverted triangle ) , 
 10 mg / kg ( light - blue triangle ) , 5 mg / kg ( orange diamond ) , 2 mg / kg 
 ( green star ) , 1 mg / kg ( purple square ) , or vehicle ( black open circle ) 
 at time zero , immediately following the baseline measurement period 
 shown .
quadriceps 
 were collected and freeze - clamped for quantification of malonyl - coa 
 levels ; plasma was collected for drug exposure measurements .
( b ) unbound 
 plasma concentration - change in rer relationship at steady state following 
 treatment .
( c ) relationship between quadriceps malonyl - coa levels 
 and change in rer measured in the same animal .
overall , the mechanistic studies with compound 9 in 
 rats demonstrated that decreases in liver and skeletal muscle malonyl - coa 
 lead to downstream decreases in dnl and increases in the use of fatty 
 acids as fuel substrate .
secondary in vitro pharmacology studies did 
 not identify any significant off - target activity for 9 . in vivo safety pharmacology studies of 9
were conducted 
 in rat and dog ; doses with no observed adverse effects were identified 
 in both species .
the effects 
 described above 
 for liver and muscle malonyl - coa , dnl , and rer in rats were the foundation 
 for a rat pk / pd model that was then applied to predict human dosing 
 for 9 .
quadriceps muscle malonyl - coa level ( qmcoa ) in 
 rat was chosen as the primary mechanistic biomarker .
on the basis 
 of the rapid formation and elimination ( seconds to min ) of malonyl - coa 
 in muscle tissue , a direct response model was selected as a reasonable 
 approximation to describe the pk / pd relationship . using the resulting 
 rat qmcoa pk / pd model and the predicted human pk of 9 ,
gastroplus was used to project the steady - state dose necessary 
 to achieve 50% inhibition of human quadriceps acc2 at the steady - state 
 average concentration ( cave ) .
these projections , 
 after correction for species differences in acc2 enzyme potency ( radiometric 
 assay ) and plasma / tissue protein binding , were based on assumptions 
 of : ( a ) a 1:1 translation in qmcoa response between rats and humans , 
 and ( b ) as observed in rat , a 1:1 relationship between human free 
 muscle and free plasma drug concentrations . on the basis of these 
 assumptions and models , the clinical plasma concentration of 9 to achieve 50% inhibition of human quadriceps acc2 was predicted 
 to be 14800 ng / ml ( 473 nm free ) , which would be achieved by a dose 
 of 125 mg once - daily . in the first - in - human study , the safety , 
 tolerability , and pharmacokinetics of single doses of 9 were tested according to a randomized , placebo - controlled , double - blind , 
 parallel - group design .
seven sequential cohorts of healthy volunteers 
 were randomized to receive either 9 or placebo ( six and 
 three subjects per cohort , respectively ) .
compound 9 was 
 administered at escalating doses ranging from 10 to 800 mg ; the 800 
 mg dose was provided as split doses of 400 mg in the a.m. and p.m. overall , single ascending doses of 9 were safe and 
 well - tolerated , exhibiting a benign adverse event profile similar 
 to placebo - treated subjects .
five adverse events were considered to 
 be potentially treatment related , including nightmare in two subjects 
 [ one treated with placebo and one with 9 ( 800-mg split 
 dose ) ] , gastroesophageal reflux disease in one subject treated with 9 ( 300 mg ) , dyspepsia in one subject treated with 9 ( 600 mg ) , and dry mouth in one subject treated with 9 ( 300 mg ) .
all potentially treatment - related adverse events were 
 mild in intensity . in the fasted state ,
rapid absorption was noted 
 with median peak concentrations observed 12 h postdose ( figure 11 ) .
the pharmacokinetic profiles exhibited low - to - moderate 
 interindividual variability in auc and cmax .
auc was approximately proportional to dose over the 10800 
 mg range , while cmax was observed to be 
 slightly less than dose - proportional .
dosing in the presence of food 
 ( 300 mg dose ) resulted in a delayed tmax ( 3.5 h ) , with marginal differences in peak and total exposure .
terminal 
 half - life values were consistent across doses and were independent 
 of dosing in the presence of food , with mean values of 1013 
 h ( data not shown ) .
time - course of plasma concentration of 9 in 
 healthy 
 human volunteers at a range of doses .
compound 9 was 
 administered orally at 10 ( blue circles ) , 30 ( red squares ) , 100 ( green 
 triangles ) , 300 ( purple triangles ) , 600 ( orange circles ) , and 800 
 ( as 2  400 , pink circles ) mg doses .
the pharmacodynamic effects of 9 on metabolic 
 parameters 
 were evaluated at the top ( nonsplit ) dose in a double - blinded , placebo - controlled 
 crossover study in healthy volunteers in each of two study periods . 

hepatic dnl was assessed by measuring the incorporation of c - labeled acetate into very low density lipoprotein triglyceride 
 ( vldl - tg ) , quantified using mass isotopomer distribution analysis 
 ( mida ) .
whole - body fuel substrate utilization 
 was assessed by measuring rer using indirect calorimetry .
subjects 
 were randomized to receive a single oral dose of 9 ( 600 
 mg ) or placebo in the first period and were crossed over to receive 
 the other treatment at least 1 week apart .
oral fructose loading was 
 used during the 10 h duration of dnl and rer assessments to provide 
 reproducible fractional contribution of dnl to vldl - tg as well as elevated and reproducible rer measures 
 from one assessment period to the other .
oral fructose administration 
 increased the fractional contribution 
 of dnl over fasting values to a peak fractional dnl contribution of 
 approximately 27% ( figure 12a ) . in subjects 
 treated with compound 9 ,
fructose stimulated dnl was 
 inhibited over this time course ; peak fructose - stimulated fractional 
 dnl was reduced by 63.6% ( 90% ci = 75.152.0% ) relative to 
 placebo treatment .
all subjects responded to treatment with similar 
 reductions in peak fractional dnl ( figure 12b ) .
( a ) fructose - stimulated fractional dnl in subjects treated 
 with 
 placebo ( pbo , black circles ) or a single dose of compound 9 ( 600 mg , blue squares ) using a crossover design .
( b ) treated subjects 
 had significantly lower peak fractional dnl ( 10.5% ; 90% ci = 7.613.5% ) 
 compared with placebo ( 26.5% ; 90% ci = 20.932.1% ) . all data 
 were corrected for baseline dnl .
the effect of 9 on rer was assessed by indirect 
 calorimetry 
 in the same subjects concurrent with the above dnl determinations . 
 over the course of the study ,
the auc0.510h for 
 rer values in subjects treated with 9 were significantly 
 lower than for subjects treated with placebo ( ratio = 0.86 , 90% ; ci 
 0.810.92 ) , indicating an increase in net whole - body fatty 
 acid utilization in the 9 subjects compared with placebo 
 subjects ( figure 13 ) .
baseline corrected respiratory 
 exchange ratio ( rer ) over time in 
 subjects treated by crossover design with placebo or a single dose 
 of compound 9 ( 600 mg ) .
the 
 design , synthesis , and biological characterization of a dual 
 acc1/acc2 inhibitor with low clearance and high off - target selectivity 
 have been described .
metabolic ketone reduction was greatly attenuated 
 through introduction of steric hindrance adjacent to the ketone carbonyl . 

phase i clinical studies demonstrated dose - proportional increases 
 in exposure , a pharmacokinetic profile suitable for once - daily dosing , 
 single - dose inhibition of dnl , and increased net whole - body fatty 
 acid utilization that support its further clinical evaluation in type 
 2 diabetes patients .
all chemicals , reagents , 
 and solvents were purchased from commercial sources and used without 
 further purification .
data for h nmr spectra are 
 reported as follows : chemical shift (  ppm ) , multiplicity , coupling 
 constant ( hz ) , and integration .
the multiplicities are denoted as 
 follows : s , singlet ; d , doublet ; t , triplet ; q , quartet ; sept , septet ; 
 m , multiplet ; ( v ) br s , ( very ) broad singlet ; app , apparent .
silica 
 gel chromatography was performed using a medium pressure biotage or 
 isco system and columns prepackaged by various commercial vendors 
 including biotage and isco .
whatman precoated silica gel plates ( 250 
 m ) were used for analytical thin - layer chromatography ( tlc ) . 

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 .
purity of 
 final compounds was assessed by reversed - phase hplc with uv detection 
 at 215 nm ; all tested compounds were > 95% purity , unless otherwise 
 noted .
sodium borohydride ( 0.37 g , 10 mmol , 2.5 equiv ) 
 was added to a suspension of 2-(tert - butyl)-1-(1h - indazole-5-carbonyl)-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7(6h)-one ( 1.67 g , 4.10 mmol , 1 equiv ) in methanol ( 24 
 ml ) . after 1 h , the mixture was partitioned between water and ethyl 
 acetate ( 2 ) .
the combined organic layers were washed sequentially 
 with water and with brine , and the organics were dried over sodium 
 sulfate , filtered , and concentrated to afford a foam that was triturated 
 with ether to afford an off - white solid that was dried under vacuum 
 ( 1.0 g , 67% ) .
h nmr ( 400 mhz , dmso - d6 )  13.19 ( s , 1h ) , 8.10 ( br s , 1h ) , 7.82 ( s , 1h ) , 7.54 
 ( d , j = 8.6 hz , 1h ) , 7.36 ( s , 1h ) , 7.36 ( dd , j = 8.6 , 1.2 hz , 1h ) , 5.19 ( d , j = 5.4 
 hz , 1h ) , 4.66 ( app q , j = 5.5 hz , 1h ) , 4.08 ( v br 
 s , 1h ) , 3.51 ( br s , 1h ) , 3.23 ( br s , 2h ) , 1.98 ( dd , j = 14.1 , 5.9 hz , 1h ) , 1.91 ( br s , 1h ) , 1.81 ( dd , j = 14.1 , 6.1 hz , 1h ) , 1.70 ( m , 3h ) , 1.42 ( s , 9h ) .
+ esi ms ( m + h ) 
 410.2 . a solution of hydrogen chloride in 
 dioxane ( 4 m
, 3 ml , 12 mmol , 12 equiv ) was added to a solution of tert - butyl 2-(tert - butyl)-6-methyl-7-oxo-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 400 mg , 1.1 
 mmol , 1 equiv ) in dioxane ( 4 ml ) .
after 2 h , the mixture was concentrated 
 and the residue was triturated with ether .
the resulting solid was 
 partitioned between aqueous sodium hydroxide solution ( 3 ml ) and ethyl 
 acetate ( 2  3 ml ) .
the combined organics were dried over magnesium 
 sulfate , filtered , and concentrated to afford the amine as an oil 
 ( 215 mg , 65% ) , which was used without further purification . a solution of 1h - indazole-5-carboxylic acid ( 111 
 mg , 0.69 mmol , 1.0 equiv ) , 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 120 
 mg , 0.69 mol , 1.0 equiv ) , and n - methylmorpholine 
 ( 0.15 ml , 1.4 mmol , 2.0 equiv ) in dmf ( 2 ml ) was stirred for 1 h , 

then a solution of 2-(tert - butyl)-6-methyl-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7(6h)-one ( 215 mg , 0.69 
 mmol , 1 equiv ) in dmf ( 3 ml ) was added and the resulting solution 
 was stirred for another 2 h. the product mixture was partitioned between 
 saturated aqueous ammonium chloride solution ( 25 ml ) and ethyl acetate 
 ( 2  15 ml ) .
the combined organics were concentrated , and the 
 resulting residue was purified by column chromatography twice , first 
 using 4% methanol in dichloromethane as eluent , then using 50100% 
 ethyl acetate in heptanes , to afford the racemic product ( 144 mg , 
 50% ) .
the enantiomers were separated by sfc ( chiralpak ad - h , 65:35 
 co2:methanol ) to afford compounds 2 ( retention 
 time 3.61 min , 51 mg , 18% , chiral purity > 99% ) and 3 ( retention 
 time 5.63 min , 40 mg , 14% , chiral purity > 99% ) .
2 : h nmr ( 400 mhz , dmso - d6 )  
 13.21 ( s , 1h ) , 8.12 ( s , 1h ) , 7.84 ( s , 1h ) , 7.77 ( s , 1h ) , 7.55 ( d , j = 8.5 hz , 1h ) , 7.38 ( d , j = 8.5 hz , 1h ) , 
 4.30 ( v br s , 1h ) , 3.59 ( v br s , 1h ) , 3.19 ( v br s , 2h ) , 2.61 ( q , j = 7.0 hz , 1h ) , 1.90 ( br s , 2h ) , 1.70 ( m , 2h ) , 1.49 ( s , 
 9h ) , 1.06 ( d , j = 7.0 hz , 3h ) ; + esi ms ( m + h ) 422.4 . 3 : h nmr ( 400 mhz , dmso - d6 )  13.21 ( s , 1h ) , 8.12 ( s , 1h ) , 7.84 ( s , 1h ) , 7.77 
 ( s , 1h ) , 7.55 ( d , j = 8.5 hz , 1h ) , 7.38 ( d , j = 8.5 hz , 1h ) , 4.29 ( v br s , 1h ) , 3.60 ( v br s , 1h ) , 3.19 
 ( v br s , 2h ) , 2.61 ( q , j = 7.0 hz , 1h ) , 1.89 ( br 
 s , 2h ) , 1.71 ( m , 2h ) , 1.49 ( s , 9h ) , 1.06 ( d , j = 
 7.0 hz , 3h ) ; + esi ms ( m + h ) 422.2 .
a solution of hydrogen chloride in dioxane ( 4 m , 0.86 ml , 
 3.5 mmol , 15 equiv ) was added to a solution of tert - butyl 2-(tert - butyl)-6,6-dimethyl-7-oxo-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 90 mg , 0.23 
 mmol , 1 equiv ) in dioxane ( 2.5 ml ) .
after 18 h , the mixture was concentrated 
 and the resulting residue was concentrated from heptanes .
the resulting 
 solid ( 81 mg ) was used without further purification . a solution 
 of 1h - indazole-5-carboxylic acid ( 40 mg , 0.25 mmol , 
 1.0 equiv ) , 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 43 mg , 0.25 mol , 
 1.0 equiv ) , and n - methylmorpholine ( 82 l , 
 0.74 mmol , 3.0 equiv ) in dmf ( 3 ml ) was stirred for 1 h , then a solution 
 of 2-(tert - butyl)-6,6-dimethyl-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7(6h)-one hydrochloride 
 ( 81 mg , 0.25 mmol , 1 equiv ) in dmf ( 3 ml ) and n - methylmorpholine 
 ( 54 l , 0.50 mmol , 2.0 equiv ) were added sequentially and the 
 resulting solution was stirred for another 1.5 h. the product mixture 
 was diluted with saturated aqueous ammonium chloride solution ( 1 ml ) , 
 and the resulting mixture was partitioned between water ( 5 ml ) and 
 ethyl acetate ( 30 ml + 20 ml ) .
purification of the 
 residue by column chromatography ( 05% methanol in dichloromethane ) 
 afforded the product as a solid ( 74 mg , 69% ) .
h nmr ( 400 
 mhz , dmso - d6 )  13.19 ( s , 1h ) , 8.10 
 ( br s , 1h ) , 7.84 ( s , 1h ) , 7.76 ( s , 1h ) , 7.54 ( app d , j = 8.6 hz , 1h ) , 7.38 ( dd , j = 8.1 , 1.6 hz , 1h ) , 
 4.32 ( v br s , 1h ) , 3.84 ( br s , 1h ) , 3.12 ( v br s , 2h ) , 1.84 ( m , 2h ) , 
 1.73 ( m , 2h ) , 1.48 ( s , 9h ) , 1.07 ( s , 6h ) . + esi ms ( m + h ) 436.6 .
sodium borohydride ( 11 mg , 0.28 mmol , 1.1 equiv ) 
 was added to tert - butyl 2-(tert - butyl)-6,6-dimethyl-7-oxo-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 100 mg , 0.26 
 mmol , 1 equiv ) in methanol ( 2 ml ) at 0 c .
after 1 h at ambient 
 temperature , the mixture was again cooled to 0 c and an additional 
 portion of sodium borohydride was added ( 10 mg , 0.26 mmol , 1.0 equiv ) . 

after an additional 2 h at ambient temperature , the mixture was cooled 
 to 0 c and water ( 1 ml ) was added .
the mixture was concentrated , 
 and the resulting residue was partitioned between water ( 5 ml ) and 
 ethyl acetate ( 2  30 ml ) .
the combined organic layers were dried 
 over sodium sulfate , filtered , and concentrated to afford a solid 
 ( 98 mg , 98% ) , which was used without further purification .
a 
 solution of hydrogen chloride in dioxane ( 4 m , 1.8 ml , 7.2 mmol , 30 
 equiv ) was added to a solution of tert - butyl 2-(tert - butyl)-7-hydroxy-6,6-dimethyl-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazole]-1-carboxylate ( 95 mg , 0.24 mmol , 1 equiv ) in dioxane ( 2 
 ml ) .
after 6 h , the mixture was concentrated to afford an oil which 
 was used without further purification .
a solution of 1h - indazole-5-carboxylic acid ( 39 
 mg , 0.24 mmol , 1.0 equiv ) , 2-chloro-4,6-dimethoxy-1,3,5-triazine ( 43 
 mg , 0.24 mol , 1.0 equiv ) , and n - methylmorpholine 
 ( 80 l , 0.73 mmol , 3.0 equiv ) in dmf ( 5 ml ) was stirred for 
 1 h , then a solution of 2-(tert - butyl)-6,6-dimethyl-6,7-dihydro-2h - spiro[piperidine-4,5-pyrano[3,2-c]pyrazol]-7-ol hydrochloride ( 80 mg , 0.24 mmol , 1 equiv ) 
 in dmf ( 3 ml ) and n - methylmorpholine ( 52 l , 
 0.50 mmol , 2.0 equiv ) were added sequentially and the resulting solution 
 was stirred for another 18 h. the product mixture was diluted with 
 saturated aqueous ammonium chloride solution ( 3 ml ) , and the resulting 
 mixture was partitioned between water ( 10 ml ) and ethyl acetate ( 2 
  50 ml ) . the combined organics were dried over magnesium sulfate , 
 filtered , and concentrated .
purification of the residue by column 
 chromatography ( 08% methanol in dichloromethane ) afforded 
 the product as a solid ( 67 mg , 63% ) .
h nmr ( 400 mhz , dmso - d6 )  13.19 ( s , 1h ) , 8.10 ( br s , 1h ) , 7.82 
 ( s , 1h ) , 7.53 ( d , j = 8.6 hz , 1h ) , 7.36 ( dd , j = 8.6 , 1.6 hz , 1h ) , 7.35 ( s , 1h ) , 5.25 ( d , j = 6.3 hz , 1h ) , 4.30 ( v br s , 1h ) , 4.19 ( br s , 1h ) , 3.60 ( v br s , 
 1h ) , 3.09 ( v br s , 2h ) , 1.99 ( br s , 1h ) , 1.68 ( m , 3h ) , 1.42 ( s , 9h ) , 
 0.89 ( br s , 6h ) .
+ esi ms ( m + h ) 438.5 . a suspension of 1-(tert - butyl)-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 158 mg , 
 0.53 mmol , 1 equiv ) , 1h - indazole-5-carboxylic acid 
 ( 86 mg ,
0.53 mmol , 1.0 equiv ) , hatu ( 207 mg , 0.53 mmol , 1.0 equiv ) , 
 and triethylamine ( 0.15 ml , 1.1 mmol , 2.0 equiv ) were combined in 
 dmf ( 7 ml ) and stirred at room temperature for 16 h. the mixture was 
 partitioned between water ( 5 ml ) and ethyl acetate ( 3  15 ml ) . 

the combined organics were washed sequentially with saturated aqueous 
 sodium bicarbonate solution ( 5 ml ) and brine ( 5 ml ) . after concentration , 
 the resulting residue was partitioned between dichloromethane ( 20 
 ml ) and 10% aqueous citric acid solution ( 5 ml ) to remove residual 
 triethylamine hydrochloride .
the resulting residue was purified 
 by column chromatography ( 38% methanol in dichloromethane ) 
 to afford the title compound as a solid ( 69 mg , 32% ) .
h nmr ( 400 mhz , cdcl3 )  10.23 ( br s , 1h ) , 8.10 
 ( s , 1h ) , 7.82 ( br s , 1h ) , 7.49 ( d , j = 8.6 hz , 1h ) , 
 7.43 ( dd , j = 8.6 , 1.4 hz , 1h ) , 7.29 ( s , 1h ) , 3.46 
 ( m , 4h ) , 2.81 ( s , 2h ) , 2.61 ( s , 2h ) , 1.64 ( s , 9h ) , 1.57 ( m , 4h ) .
1-isopropyl-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 30.3 
 g , 94.6 mmol , 1 equiv ) and 1h - indazole-5-carboxylic 
 acid ( 16.96 g , 104.6 mmol , 1.1 equiv ) were suspended in n , n - dimethylacetamide ( 430 ml ) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide 
 hydrochloride ( 22.3 g , 115 mmol , 1.2 equiv ) was added , followed by 
 the dropwise addition of triethylamine ( 65 ml , 475 mmol , 5.0 equiv ) . 

1-hydroxybenzotriazole hydrate ( 16.2 g , 106 mmol , 1.1 equiv ) was then 
 added , and the reaction mixture was stirred at 60 c for 2 h. 
 the reaction was poured into half - saturated aqueous ammonium chloride 
 solution ( 500 ml ) and extracted with ethyl acetate ( 1  1 l , 
 2  500 ml ) .
the combined organic layers were washed with aqueous 
 sodium bicarbonate solution ( 2  500 ml ) , water ( 3  500 
 ml ) , and aqueous saturated sodium chloride solution ( 500 ml ) .
the 
 organic layer was dried over sodium sulfate , filtered , and concentrated 
 under reduced pressure to an oil .
the oil was purified via column 
 chromatography ( 16% methanol in dichloromethane ) to afford 
 the desired product ( 27.1 g ) .
a small amount was crystallized from 
 ethyl acetate / heptanes , which was then used to seed the following 
 crystallization .
the product was dissolved in ethyl acetate ( 100 ml ) 
 and heated to reflux until the solution turned hazy . a small amount 
 of seed crystal was added .
the mixture was cooled to room temperature , 
 and a precipitate formed and was stirred for 80 h. the precipitate 
 was collected by filtration and washed with cold ethyl acetate ( 2 
  30 ml ) .
the material was air - dried and then further dried under 
 high vacuum to afford an off - white solid ( 23 g , 62% ) .
h nmr ( 400 mhz , dmso - d6 )  13.19 
 ( s , 1h ) , 8.10 ( m , 1h ) , 7.79 ( m , 1h ) , 7.53 ( m , 1h ) , 7.43 ( s , 1h ) , 7.34 
 ( m , 1h ) , 5.24 ( sept , j = 6.6 hz , 1h ) , 3.45 ( v br 
 s , 4h ) , 2.78 ( s , 2h ) , 2.59 ( s , 2h ) , 1.48 ( br s , 4h ) , 1.32 ( d , j = 6.6 hz , 6h ) . + esi ms ( m + h ) 392.5 .
a mixture of benzyl 1-ethyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole-5,4-piperidine]-1-carboxylate ( 358 mg , 0.97 mmol , 1 equiv ) and palladium on 
 carbon ( 50% wet , 60 mg ) in ethanol ( 20 ml ) was treated with 50 psi 
 hydrogen gas for 4 h. the mixture was filtered through celite , rinsing 
 with ethanol ( 50 ml ) .
the filtrate was concentrated , then the resulting 
 residue was slurried in heptanes ( 20 ml ) and concentrated .
the resulting 
 solid was dissolved in dichloromethane ( 5 ml ) and was treated with 
 triethylamine ( 0.40 ml , 2.9 mmol , 3.0 equiv ) and 1h - indazole-5-carbonyl chloride ( 211 mg , 0.97 mmol , 1.0 equiv ) for 
 18 h at ambient temperature .
the mixture was partitioned between water 
 ( 50 ml ) and ethyl acetate ( 2  50 ml ) .
purification 
 by column chromatography ( 210% methanol in dichloromethane ) 
 afforded the title compound as a solid ( 172 mg , 47% ) .
h nmr ( 400 mhz , cdcl3 )  8.13 ( s , 1h ) , 7.85 ( s , 
 1h ) , 7.51 ( d , j = 8.6 hz , 1h ) , 7.46 ( d , j = 8.6 hz , 1h ) , 7.37 ( s , 1h ) , 4.52 ( q , j = 7.2 hz , 
 2h ) , 3.64 ( v br s , 4 h ) , 2.82 ( s , 2h ) , 2.61 ( s , 2h ) , 1.63 ( br s , 4h ) , 
 1.40 ( t , j = 7.2 hz , 3h ) .
+ esi ms ( m + h ) 378.2 . a suspension of 1-isopropyl-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 100 mg , 
 0.35 mmol , 1 equiv ) , 1h - pyrrolo[3,2-b]pyridine-6-carboxylic acid ( 57 mg , 0.35 mmol , 1.0 equiv ) , hatu ( 83 
 mg , 0.35 mmol , 1.0 equiv ) , and triethylamine ( 0.10 ml , 0.70 mmol , 
 2.0 equiv ) were combined in dmf ( 3 ml ) and stirred at room temperature 
 for 16 h. the mixture was partitioned between saturated aqueous sodium 
 bicarbonate solution ( 10 ml ) and ethyl acetate ( 2  5 ml ) .
the 
 combined organics were washed sequentially with water ( 5 ml ) and brine 
 ( 5 ml ) .
after concentration , the resulting residue was purified by 
 column chromatography ( 5% methanol in ethyl acetate ) to afford the 
 title compound as a solid ( 65 mg , 47% ) .
h nmr ( 400 mhz , 
 dmso - d6 )  11.76 ( br s , 1h ) , 8.43 
 ( d , j = 1.8 hz , 1h ) , 7.95 ( br s , 1h ) , 7.86 ( t , j = 2.9 hz , 1h ) , 7.46 ( s , 1h ) , 6.65 ( m , 1h ) , 5.27 ( sept , j = 6.6 hz , 1h ) , 3.56 ( m , 4h ) , 2.82 ( s , 2h ) , 2.63 ( s , 2h ) , 
 1.53 ( m , 4h ) , 1.36 ( d , j = 6.6 hz , 6h ) . + apci ms 
 ( m + h ) 392.2 .
n , n - dimethylformamide ( 0.33 ml , 4.3 mmol , 0.05 equiv ) and 
 oxalyl chloride ( 22.1 ml , 257 mmol , 3.0 equiv ) were added to a solution 
 of 2-methyl-1h - benzimidazole-5-carboxylic acid ( 15 
 g , 85 mmol , 1 equiv ) in tetrahydrofuran ( 500 ml ) .
the reaction solution 
 was stirred at ambient temperature for 16 h. the solution was concentrated , 
 and the resulting residue was twice taken up in dichloromethane and 
 concentrated under reduced pressure . to the resulting acid chloride 
 was added tetrahydrofuran ( 500 ml ) , 1-isopropyl-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one hydrochloride ( 25.9 
 g , 91 mmol , 1.1 equiv ) , and triethylamine ( 71.2 ml , 510 mmol , 6.0 
 equiv ) .
the solution was stirred at room temperature for 16 h , then 
 saturated aqueous sodium bicarbonate solution ( 250 ml ) was added and 
 the resulting mixture was stirred for 5 min .
the layers were separated , 
 and the aqueous layer was extracted with 1:1 ethyl acetate  tetrahydrofuran . 

the organic layers were combined , diluted with ethyl acetate ( 1 l ) , 
 and washed sequentially with saturated aqueous sodium bicarbonate 
 solution ( 200 ml ) and saturated aqueous sodium chloride solution . 

the organic layer was dried over sodium sulfate , filtered , and concentrated 
 to afford a light - yellow solid .
the solid was dissolved in hot methanol 
 ( 300 ml ) and then heated to reflux . to the solution
was added 350 
 ml of ethyl acetate , and 300 ml of solvent was then removed by distillation . 

additional ethyl acetate was added dropwise until an internal temperature 
 of 70 c was reached .
the solution was cooled to ambient temperature 
 over 3 h. the resulting solids were collected by filtration and dried 
 in a vacuum oven ( 40 c ) for 16 h to afford a white solid ( 20.5 
 g , 59% ) .
h nmr ( 400 mhz , dmso - d6 )  12.29 ( br s , 1h ) , 7.43 ( m , 3h ) , 7.11 ( m , 1h ) , 5.24 ( sept , j = 6.5 hz , 1h ) , 3.45 ( m , 4h ) , 2.77 ( s , 2h ) , 2.57 ( s , 2h ) , 
 2.45 ( s , 3h ) , 1.46 ( m , 4h ) , 1.32 ( d , j = 6.5 hz , 
 6h ) . + esi ms ( m + h ) 406.5 .
sodium borohydride ( 27 mg , 0.76 mmol , 2.2 equiv ) 
 was added to 1-isopropyl-1-(2-methyl-1h - benzo[d]imidazole-6-carbonyl)-1,4-dihydrospiro[indazole-5,4-piperidin]-7(6h)-one ( 139 mg , 0.34 mmol , 1 equiv ) in methanol . after 1 
 h , water ( 1 ml ) was added dropwise and the resulting mixture was partitioned 
 between ethyl acetate ( 20 ml ) and water . the organic layer was washed 
 with brine , dried over sodium sulfate , and concentrated .
the resulting 
 solid was purified by column chromatography ( 220% methanol 
 in dichloromethane ) to afford a white solid ( 75 mg , 54% ) .
h nmr ( 400 mhz , dmso - d6 )  12.32 
 ( br s , 1h ) , 7.43 ( m , 2h ) , 7.13 ( s , 1h ) , 7.11 ( dd , j = 8.2 , 1.6 hz , 1h ) , 5.19 ( d , j = 6.6 hz , 1h ) , 4.71 
 ( m , 2h ) , 3.47 ( v br s , 4h ) , 2.46 ( s , 3h ) , 2.41 ( d , j = 15.3 hz , 1h ) , 2.32 ( d , j = 15.3 hz , 1h ) , 1.96 
 ( dd , j = 13.4 , 5.8 hz , 1h ) , 1.66 ( dd , j = 13.4 , 6.8 hz , 1h ) , 1.56 ( br s , 1h ) , 1.43 ( m , 3h ) , 1.34 ( d , j = 6.4 hz , 3h ) , 1.28 ( d , j = 6.6 hz , 3h ) . 
 + esi ms
the preparation 
 and crystal structure 
 analysis of  humanized  acc ct domain has been described 
 previously .
compounds 1 or 6 were soaked for 24 h into humanized acc ct domain crystals 
 at a concentration of 3 mm , then flash cooled in liquid propane prior 
 to data collection .
data for compound 1 was collected 
 at beamline x29a at the national synchrotron light source ( brookhaven 
 national laboratory , upton , ny , usa ) . data for compound 6 was collected at sector 17bm of the industrial macromolecular crystallography 
 association collaborative access team ( imca - cat ) ( advanced photon 
 source , argonne , il ) .
the structures of acc - ct were determined by rigid 
 body refinement of a reference structure against the isomorphous data . 

compound 1 was refined using the program autobuster ; compound 6 was refined using refmac .
the intensity 
 of reflection hkl , and ihkl is the average intensity 
 of multiple observations .
rwork = |fo  fc|/fo , where fo and fc are the 
 observed and calculated structure factor amplitudes , respectively .
rfree is the r - factor for a 
 randomly selected 5% of reflections which were not used in the refinement .
% ( preferred ) + % ( allowed ) recombinant 
 human acc1 ( rhacc1 ) was utilized in enzyme 
 inhibition assays as previously described .
recombinant human acc2 ( rhacc2 ) was 
 prepared , purified , and utilized in enzyme inhibition assays as previously 
 described .
rat acc1 
 and acc2 were purified from rat liver and muscle , respectively , as 
 previously described .
cryopreserved male wistar rat hepatocytes from 
 xenotech were thawed 
 and isolated using a percol rat hepatocyte isolation kit .
hepatocytes 
 were plated in seeding media containing high glucose dmem , 5% fbs , 
 1 m dexamethasone , 4 g / ml insulin , 1% pen / strep , and 
 1% glutamine .
hepatocytes were plated in 48-well collagen coated type 
 i plates using a viable seeding density of 125000 cells / well in seeding 
 media to result in 8090% confluency at 5% co2 and 
 37 c .
media was aspirated 46 h later and fresh hepatocyte 
 modified eagle medium , dr .
fresh cold mcm media containing a 1:40 dilution of matrigel , 250 ng / ml , 
 200 l / well was added 24 h post seeding .
the assay was run 72 
 h post seeding with a mcm media change the night before the experiment . on the day of the study
, media was aspirated and cells were treated 
 with fresh mcm media containing dmso vehicle or varying concentrations 
 of 9 as indicated .
an aliquot of this solution 
 was added to the fresh mcm in each well , further diluting the compound 
 by 1:10 .
this dilution progression ensured that all wells had a final 
 dmso concentration of 0.1% .
after 5 h at 37 c , incubation media 
 was removed and the experiment was terminated by washing the cells 
 with ice - cold pbs .
cells were lysed with the addition of 70 l 
 of 10% trichloroacetic acid ( tca ) .
plates were mixed for 20 min at 
 4 c and centrifuged at 2200 g for 5 min .
supernatant 
 ( 48 l ) was transferred to a screenmates clear 384 well v bottom 
 polypropylene microplates and frozen .
tca , ammonium 
 acetate , methanol , acetone , acetonitrile , and water were used to make 
 the necessary mobile phases .
then 2 l of malonyl - c3-coa internal standard ( 0.4 pmol/l final ) and 
 2 mm final atp in 10% tca were added to the reaction and mixed .
the 
 samples were analyzed on a rapidfire platform coupled to a sciex api4000 
 triple quadrupole mass spectrometer ( rf - ms ) .
malonyl - coa and malonyl - c3-coa were monitored in positive ion mode following 
 mrm transitions at 854.1/347.1 and 857.1/350.1 , respectively .
sample 
 ( 10 l ) was aspirated directly from assay plates quenched with 
 tca ( 10% final ) .
the aspirated sample was loaded onto the rf ms microscale 
 solid - phase hypercarb extraction cartridge ( d ) and washed with hplc 
 grade h2o in a 3 s wash cycle to remove the nonvolatile 
 assay components .
the product and internal standard were coeluted 
 to the mass spectrometer in 3 s with 50% h2o , 25% mecn , 
 25% acetone , and 5 mm ammonium acetate .
peak area ratios ( malonyl - coa / malonyl - c3-coa ) were compared against a standard curve 
 prepared in 10% aqueous tca to determine sample concentrations of 
 malonyl - coa .
malonyl - coa values were expressed as pmol/l and 
 compared against a standard curve .
dose response curves of malonyl - coa 
 inhibition by acc inhibitor compound 9 were generated 
 by plotting percent of control .
male sd rats 
 were weighed and randomized by body weight into treatment groups consisting 
 of vehicle , 0.25 , 0.5 , 1 , 2 , 4 , 8 , 15 , 25 , 50 , and 100 mg / kg of 9 . immediately prior to initiation of the study ,
the dosing 
 solutions were prepared in dosing vehicle ( 0.5% methyl cellulose:0.1% 
 polysorbate 80 ) , giving a final dosing volume of 5 ml / kg .
animals 
 were orally dosed 2 h into the light cycle with their respective treatments 
 and fed ad libitum .
one hour postdose , the animals were sacrificed 
 via co2 asphyxiation followed by cervical dislocation . 

blood for plasma exposure of compound 9 was collected 
 via cardiac puncture , transferred to bd microtainers tubes with k2edta , centrifuged at 4 c , and the plasma transferred 
 to a 96-well microtiter plate and stored at 20 c .
liver 
 and quadriceps were rapidly removed , freeze - clamped in a wollenberg 
 clamp ( precooled in liquid nitrogen ) , and subsequently stored at 80 
 c .
approximately 200 mg of pulverized tissue were added 
 to the lysing matrix tube ( mp bio , lysing matrix tube a for quadriceps 
 and lysing matrix tube d for liver ) containing ice - cold 10% tca , giving 
 a final ratio of 1:5 , tissue to acid .
the extracted samples were then centrifuged 
 at 4 c and 20000 g for 15 min ( liver ) and 30 
 min ( muscle ) .
the supernatants containing malonyl - coa were frozen 
 at 80 
 c until analysis was completed via lc - ms ms using an abi sciex 
 api-3000 triple quadrupole mass spectrometer with turbo ion spray . 

tissue concentrations for malonyl - coa were calculated from a standard 
 curve prepared in a 10% solution of tca in water and ranged from 0.01 
 to 1 pmol/l .
malonyl - c3-coa ( final 
 concentration of 0.4 pmol/l ) was added to each standard curve 
 component and served as an internal standard , and the resulting chromatograms 
 were integrated using analyst software ( applied biosystems ) .
this procedure was derived from 
 a published method for the examination of rat liver de novo lipid 
 synthesis . on the day of the study , male 
 sd rats , previously randomized into groups of seven based on body 
 weight ,
were administered a single dose of compound 9 or vehicle via oral gavage 2 h into the light cycle ( 8 a.m. ) .
[ 2-c]-acetic acid ( amersham ) was diluted to 64 ci / ml 
 with saline prior to being administered intraperitoneally at 2.5 ml / kg 
 1 h postdose of compd / vehicle . then 1 h following c - acetic 
 acid administration , the animals were sacrificed ( co2 euthanasia ) 
 and liver samples collected ( 400 mg , bifurcated median lobe ) 
 using disposable tissue biopsy punches .
briefly , tissues were saponified in aqueous naoh ( 1.5 ml of 
 2.5 m ) .
following complete degradation , absolute ethanol ( 2.5 ml ) 
 was added to each sample and vigorously mixed .
following centrifugation to separate 
 the organic and aqueous phases , the upper organic phase was removed 
 and discarded .
concentrated hcl ( 0.6 ml of 12 m ) 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 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 was added to each vial , and the level of c in the 
 extraction was determined .
the data were analyzed with microsoft 
 excel and plotted using graphpad 
 prism 5 . statistical analysis ( one - way anova , dunnett s posttest ) 

male sd rats were fasted for 24 
 h and then re - fed a diet high in sucrose ( d10001 , research diets ) 
 for 2 days prior to study initiation to elevate baseline respiratory 
 exchange ratio ( rer ) . on the day of experiment , rats were removed 
 from their home cage , weighed , and individually placed into the calibrated 
 indirect calorimetery chambers ( oxymax , columbus instruments , columbus , 
 oh ) with free access to water and diet ( d10001 ) .
baseline oxygen consumption 
 and carbon dioxide production rates were measured every 15 min for 
 75 min before treatment . after collecting baseline calorimetery data
, 
 rats were dosed orally with either vehicle control ( 0.5% methylcellulose/0.1% 
 tween 80 ) or 9 ( 1 , 2 , 5 , 20 , 40 , or 100 mg / kg ) and then 
 returned to the calorimetry chambers .
oxygen consumption and carbon 
 dioxide production were measured for an additional 2.25 h after being 
 placed back in the calorimetry chamber . immediately following completion 
 of the calorimetry measurement period ,
plasma 
 for determining plasma exposure of compound 9 was collected 
 via cardiac puncture , and quadriceps muscles were rapidly removed , 
 freeze - clamped as described above , and stored at 80 c . 

studies involving human 
 subjects were conducted in compliance with the ethical principles 
 originating in or derived from the declaration of helsinki and in 
 compliance with all international conference on harmonisation ( ich ) 
 good clinical practice ( gcp ) guidelines .
in addition , all local regulatory 
 requirements were followed , in particular , those affording greater 
 protection to the safety of trial participants .
final study protocols 
 and informed consent document were reviewed and approved by the investigational 
 centers participating in the studies and by an independent institutional 
 review board ( irb ) .
the investigator was required to inform the irb 
 of the study s progress and occurrence of any serious and/or 
 unexpected adverse events .
a signed and dated informed consent was 
 required before any screening procedures were initiated . the investigator 
 or his / her delegate explained the nature , purpose , and risks of the 
 study to each subject .
each subject was informed that he / she could 
 withdraw from the study at any time and for any reason .
each subject 
 was given sufficient time to consider the implications of the study 
 before deciding whether to participate .
safety , tolerability , and 
 pharmacokinetics were evaluated in the first - in - human study .
a total 
 of 63 healthy volunteers ( all male ; mean age 32.6 years [ range 2045 
 years ] ; mean body mass index ( bmi ) 27.0 kg / m ; [ range 21.335.1 
 kg / m ] ) participated in the study .
seven sequential cohorts 
 of volunteers were randomized to receive either 9 or 
 placebo ( six and three per cohort , respectively ) .
compound 9 was administered at escalating doses ranging from 10 to 800 mg ; 
 the 800 mg dose was provided as split doses of 400 mg in the a.m. 
 and p.m. due to the prediction that cmax might exceed the exposure limits .
compound 9 was administered 
 after 10 h of fasting in a 10 , 50 , and 100 mg powder - in - capsule formulation . 

the effect of food on the pharmacokinetics of 9 was evaluated 
 at the 300 mg dose .
safety and tolerability were assessed by adverse 
 event monitoring , laboratory values , and cardiovascular parameters 
 including blood pressure , heart rate , and electrocardiogram .
the effects of 9 on inhibition of dnl and on whole - body 
 fuel substrate utilization was assessed in a randomized , double - blinded , 
 placebo - controlled crossover study in healthy volunteers ( mean age 
 35.4 years [ range 2050 years ] ; mean body mass index ( bmi ) 
 29.9 kg / m ; [ range 25.835.5 kg / m ] ) . 

substrate utilization , assessed by rer , was measured in each of two 
 study periods .
subjects were randomized to receive a single oral dose 
 of 9 ( 600 mg ) or placebo in the first period . in the 
 second period , subjects were crossed over to receive the other treatment 
 ( placebo or 9 ) at least 1 week apart .
oral fructose loading 
 was used during the dnl and rer assessments to provide reproducible 
 fractional contribution of dnl to vldl - tg from one assessment period 
 to the other .
a continuous infusion of c - acetate ( 99.5 mg c - acetic acid sodium 
 salt per min via an infusion pump ) was started at approximately 10:00 
 a.m. on the evening prior to each treatment period and continued until 
 approximately 6:30 p.m. on the study day .
blood samples for assessment 
 of the fractional contribution of dnl to vldl - tg were collected and 
 rer measures made hourly for 10 h. subjects received study medication 
 ( 9 or matching placebo ) at approximately 08:00 h ( plus 
 or minus 2 h ) .
subjects received a bolus of 0.25 g fructose / kg body 
 weight every 30 min starting at approximately 08:30 h for approximately 
 9.5 h ( total of 20 fructose administrations ) during each of the two 
 study periods .
urine samples for purpose of urinary nitrogen determination 
 were collected during each study period .
subjects refrained from eating 
 and drinking beverages other than water and the fructose drinks administered 
 as part of study procedures .
dnl was measured by c - incorporation 
 ( derived from intravenous c - acetate infusion ) into vldl 
 palmitate using mass isotopomer distribution analysis ( mida ) .
rer values reported are nonprotein rer as no 
 changes in urinary nitrogen were observed during or between the study 
 periods .
 acetyl - coa 
 carboxylase ( acc ) inhibitors offer significant potential 
 for the treatment of type 2 diabetes mellitus ( t2 dm ) , hepatic steatosis , 
 and cancer . 
 however , the identification of tool compounds suitable 
 to test the hypothesis in human trials has been challenging . an advanced 
 series of spirocyclic ketone - containing acc inhibitors recently reported 
 by pfizer were metabolized in vivo by ketone reduction , which complicated 
 human pharmacology projections . 
 we disclose that this metabolic reduction 
 can be greatly attenuated through introduction of steric hindrance 
 adjacent to the ketone carbonyl . 
 incorporation of weakly basic functionality 
 improved solubility and led to the identification of 9 as a clinical candidate for the treatment of t2 dm . 
 phase i clinical 
 studies demonstrated dose - proportional increases in exposure , single - dose 
 inhibition of de novo lipogenesis ( dnl ) , and changes in indirect calorimetry 
 consistent with increased whole - body fatty acid oxidation . 
 this demonstration 
 of target engagement validates the use of compound 9 to 
 evaluate the role of dnl in human disease . 
Introduction
Results and Discussion
Conclusion
Experimental Section