Optimization of Metabolic and Renal Clearance in a Series of Indole

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2018, 61, 2372−2383

Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5′-Adenosine Monophosphate-Activated Protein Kinase (AMPK) David J. Edmonds,*,† Daniel W. Kung,*,‡ Amit S. Kalgutkar,† Kevin J. Filipski,† David C. Ebner,† Shawn Cabral,‡ Aaron C. Smith,‡ Gary E. Aspnes,‡ Samit K. Bhattacharya,† Kris A. Borzilleri,‡ Janice A. Brown,‡ Matthew F. Calabrese,‡ Nicole L. Caspers,‡ Emily C. Cokorinos,† Edward L. Conn,‡ Matthew S. Dowling,‡ Heather Eng,‡ Bo Feng,‡ Dilinie P. Fernando,‡ Nathan E. Genung,‡ Michael Herr,‡ Ravi G. Kurumbail,‡ Sophie Y. Lavergne,‡ Esther C.-Y. Lee,† Qifang Li,‡ Sumathy Mathialagan,‡ Russell A. Miller,† Jane Panteleev,‡ Jana Polivkova,‡ Francis Rajamohan,‡ Allan R. Reyes,† Christopher T. Salatto,† Andre Shavnya,‡ Benjamin A. Thuma,‡ Meihua Tu,† Jessica Ward,† Jane M. Withka,‡ Jun Xiao,‡ and Kimberly O. Cameron† †

Pfizer Worldwide Research and Development, 610 Main Street, Cambridge, Massachusetts 02139, United States Pfizer Worldwide Research and Development, Eastern Point Road, Groton, Connecticut 06340, United States

‡

S Supporting Information *

ABSTRACT: Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphateactivated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

■

(albuminuria). Ultimately, inflammation and fibrosis lead to reduced glomerular filtration rate (GFR) and end-stage renal disease requiring dialysis.2,3 Despite the increasing prevalence of diabetes and its complications, there is no treatment approved specifically for DN. Rather, treatment relies on managing hypertension with angiotensin converting enzyme inhibitors or angiotensin receptor antagonists in addition to standard antidiabetic therapy to manage hyperglycemia.4 As

INTRODUCTION

Diabetic nephropathy (DN), the chronic impairment of renal function resulting from metabolic dysregulation, is a leading cause of end-stage renal disease, and patients with type II diabetes mellitus (T2DM) have a 6-fold increased risk of developing chronic kidney disease.1 The progression of DN is not well understood but involves impairment of glomerular filtration and dysfunction of renal tubule capillaries. The key hallmarks of T2DM, such as hyperglycemia, hyperinsulinemia, and hyperlipidemia, impair both aspects of kidney function. As the disease progresses, glomerular hyperfiltration leads to loss of filtration integrity and excess secretion of urinary protein © 2018 American Chemical Society

Received: November 6, 2017 Published: February 21, 2018 2372

DOI: 10.1021/acs.jmedchem.7b01641 J. Med. Chem. 2018, 61, 2372−2383

Journal of Medicinal Chemistry

Article

such, a novel therapeutic approach to DN would address a considerable unmet medical need. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine kinase that senses and regulates cellular energy levels. AMPK is a heterotrimeric complex consisting of a kinase domain-containing α-subunit and two regulatory subunits (β- and γ-). The seven known AMPK subunits (α1, α2, β1, β2, γ1, γ2, γ3) have differential expression profiles in tissues and can theoretically generate 12 possible trimeric isoforms.5 AMPK is regulated by upstream kinases by phosphorylation of Thr174 (Thr172 on α2) on the activation loop of its α-subunit, leading to a 500−1000-fold increase in activity. Rising cellular AMP and ADP levels during periods of energetic stress activate AMPK by binding to nucleotide binding sites on the γ-subunit and promoting phosphorylation of Thr174. In addition, the binding of AMP to the phosphoThr174 form of AMPK (pAMPK) serves to increase cellular levels of activated AMPK by protecting the protein from dephosphorylation by phosphatases. Finally, a further 2−3-fold allosteric activation of the protein kinase activity of AMPK results from binding of AMP.6 Other reported7 small molecule activators of AMPK activity include AICAR (Figure 1),8 which is phosphorylated in vivo to

Figure 2. Chemical structure of 1 (A) and a view of the co-crystal structure of 1 with α1β1γ1-AMPK (B). Compound 1 (pink ball-andstick) is bound in the ADaM site at the interface of the α1 and β1 subunits (5KQ5.pdb). Some of the key hydrogen bond interactions formed by the ligand are highlighted.19

form hydrogen bonds to protein atoms from the α1 and β1 subunits. From an absorption, distribution, metabolism, and excretion (ADME) perspective, compound 1 is expected to be cleared in humans via a combination of phase II metabolism and renal excretion as an unchanged parent. Metabolism studies in cryopreserved hepatocytes from animals and human indicated that the principal metabolic fate of 1 was its conversion to an acyl glucuronide conjugate by uridine glucuronosyl transferase (UGT) isoforms.19 In addition to glucuronidation, renal excretion of unchanged 1 was observed in intravenous (iv) PK studies in rat, dog, and monkey.19 The unbound renal clearance (CLrenal,u) estimates in animals significantly exceeded the glomerular filtration rate (GFR) in these species (Table 1), indicating an active renal elimination process possibly mediated by organic anion transporter (OAT) proteins expressed at the basolateral membrane of proximal tubules.22−24 Consistent with this hypothesis, subsequent in vitro transporter studies indicated that compound 1 was a substrate for both human OAT3 (SLC22A8) and rat Oat3 with net fold uptake values of 4.7 ± 0.1 and 8.3 ± 0.2, respectively (vide infra). However, 1 was not a substrate for the related human OAT1 (SLC22A6) transporter, which has been reported to exhibit some overlapping substrate specificity with human OAT3.24,25 Given the potential for impaired renal function in the target DN patient population,2,26 including the possibility of impaired transporter function,27−29 we sought to minimize CLrenal in a potential therapeutic agent targeted to treat DN. Described herein is the characterization and optimization of analogues of 1, which achieve an overall decrease in plasma clearance resulting from reduction of both metabolic and renal clearance.

Figure 1. Selected AMPK activators.

form the AMP-mimetic ZMP, and metformin, which does not bind to the protein but works indirectly by increasing intracellular AMP.9 The first non-nucleotide direct activators were disclosed by Abbott Laboratories and come from a series of thienopyridines, exemplified by A-769662.10 More recently, Metabasis and Merck have disclosed both phosphate11 and nonphosphate-containing activators,12 including pan-AMPK activator MK-8722.13,14 AMPK activity is reduced in diseases of caloric excess, and pAMPK levels have been found to be reduced in the glomeruli of diabetic patients as compared to healthy subjects.15 Rodent models of DN also exhibit reduced pAMPK levels. Chronic AMPK activation with AICAR or metformin has been shown to restore pAMPK levels and to improve kidney function in rodent kidney disease models.16−18 We recently reported the identification of an indole-3carboxylic acid containing clinical candidate PF-06409577 (1, Figure 2) for the treatment of diabetic nephropathy.19 Compound 1 is a selective activator of β1-containing AMPK isoforms that allosterically activates the enzyme by binding at the previously described “allosteric drug and metabolite” (ADaM) site at the interface of the α- and β-subunits.20,21 The carboxylic acid, the indole N−H, and the cyclobutanol

■

IN VITRO PHARMACOLOGY AND ADME ASSAYS Functional potency of AMPK activators was evaluated using a time-resolved fluorescence resonance energy transfer (TRFRET) activation/protection assay, which incorporates a protein phosphatase 2a treatment prior to initiation of the kinase reaction.19 Because phosphorylation of Thr174 is required for AMPK kinase activity, this assay is capable of detecting compounds that activate through allosteric activation or through protection of pAMPK from dephosphorylation. All data were normalized relative to the endogenous ligand AMP, which is known to increase AMPK activity via both mechanisms mentioned above. The human α1β1γ1-AMPK isoform, which is the predominant isoform expressed in the kidney,31−33 was evaluated initially, and the same assay format was used to assess the isoform selectivity of key compounds (human α1β2γ1, 2373



Article

Table 1. CLrenal of Unchanged 1 after iv Administration to Preclinical Species species

renal excretion (% of total dose)a

total CLp (mL/min/kg)

total CLrenal (mL/min/kg)b

CLrenal,u (mL/min/kg)c

CLrenal,u/GFR ratiod

rat dog monkey

15.9 11.8 20.6

22.6 12.9 8.57

7.64 2.82 2.48

849 54 55

98 14 28

a Percentage of 1 recovered unchanged in urine over 24 h following a 1 mg/kg iv dose in male Wistar−Han rats and cynomolgus monkeys or 0.5 mg/ kg in male beagle dogs. Compound 1 was formulated in 12% sulfobutylether-β-cyclodextrin solution (rats and monkeys) or 10% N-methyl-2pyrrolidone: 90% of 30% sulfobutylether-β-cyclodextrin solution in water (dogs). bTotal CLrenal in blood was estimated by multiplying the total blood clearance (CLb) by the fraction of total iv dose of unchanged 1 excreted in urine. CLb was obtained by dividing the observed CLp by the blood to plasma ratio (B:P) of 1 in the individual animal species (rat B:P = 0.470, dog B:P = 0.538, monkey B:P = 0.710). cCLrenal,u in blood was estimated by dividing total CLrenal by the unbound free fraction in blood (rat = 0.009, dog = 0.052, monkey = 0.045), which in turn was obtained by dividing the unbound plasma free fraction by B:P. The unbound fractions of 1 in rat, dog, and monkey plasma were 0.0044, 0.028, and 0.032, respectively. d GFR values in rats, dogs, and monkeys are 8.7, 4.0, and 2.0 mL/min/kg, respectively.30

Scheme 1. Synthetic Routes to Indole Acid AMPK Activatorsa

a Conditions: (a) ClCHNMe2Cl (POCl3-DMF), CH3CN or DMF; then NaOH, H2O; (b) ArB(OH)2 or ArB(OR)2 (pinacol or neopentyl glycol boronate), Pd(dppf)Cl2, K2CO3, H2O, EtOH−toluene−THF−2-MeTHF; (c) NaClO2, 2-methyl-2-butene, NaH2PO4, H2O−CH3CN−THF−tBuOH; (d) Cl3CCOCl, pyridine, DMAP; then NaOMe, MeOH; (e) B2(neop)2, Pd(dppf)Cl2, KOAc, dioxane; (f) ArBr, Pd(dppf)Cl2, Na2CO3 or K2CO3, H2O, EtOH−toluene−dioxane; (g) NaOH, H2O−MeOH.

human α2β1γ1, human α2β2γ1, and human α2β2γ3). The binding affinity of test compounds for α1β1γ1-AMPK was assessed using surface plasmon resonance (SPR) experiments (see Supporting Information). Passive permeability (Papp) was measured in the Ralph Russ canine kidney (RRCK) assay,34 log D was determined at pH 7.4 using the shake-flask method,35 and pKa values were determined by electrophoresis,36 all as described previously. Human OAT/rat Oat transporter substrate properties of the test compounds were assessed using a differential uptake format in HEK293 cells transfected with the relevant transporter,37 details of which are described in the Supporting Information. The net OAT-mediated uptake ratio was calculated as the total uptake in OAT-expressing cells divided by the uptake observed in vector-treated control cells. In this experimental setup, a normalized fold uptake of >2.0 was interpreted to indicate that the test compound is likely to be an OAT substrate in vitro.38 Since glucuronidation was identified as the major metabolic pathway of compound 1, cryopreserved human hepatocytes were employed as the primary screening assay for measurement of in vitro hepatic intrinsic clearance (CLint).39 Hepatic CLint refers to metabolic clearance of the test compounds by the liver and is obtained via scaling of in vitro half-lives of the test compounds following incubation with cryopreserved human hepatocytes. Plasma clearance (CLp) in rats and monkeys was calculated as the iv dose divided by the area under the plasma concentration versus time curve from zero to infinity (AUC0−∞). Blood clearance (CLb) was estimated by dividing the CLp by the measured blood to plasma ratio (B:P) of the

test compounds. Total renal clearance (CLrenal), the rate at which the compound is cleared through the kidneys, was determined by multiplying the CLb by the percentage of the iv dose recovered unchanged in the urine. Unbound renal clearance (CLrenal,u) in blood was estimated by dividing CLrenal by the unbound free fraction in blood, which in turn was obtained by dividing the measured unbound plasma free fraction by the blood to plasma ratio.

■

SYNTHESIS OF COMPOUNDS The reported compounds were synthesized by the routes depicted in Scheme 1. All compounds share a common substituted 5-aryl- or 5-heteroarylindole-3-carboxylic acid structure. The indole−(hetero)aryl bond was formed by Suzuki reaction, with the boronate and halide functional groups possible on either reacting partner, depending on availability, synthetic accessibility, and reactivity of the necessary starting materials. The 3-carboxy group was introduced to the indole either by formylation followed by oxidation or by esterification and subsequent hydrolysis. The Suzuki coupling and the carboxylation steps could be performed in either order, again depending on reactivity and compatibility of other functional groups. Compounds 1, 3, 8, 11, 12, and 13 were synthesized from starting indole A by acylation with trichloroacetyl chloride followed by methanolysis to afford ester C. Suzuki coupling with the appropriate aryl or heteroaryl boronate afforded intermediate D, with ester hydrolysis yielding the final compounds. Compounds 2, 4, 9, and 10 were synthesized 2374



Article

Table 2. Indole Core Substitutions

α1β1γ1-AMPK compd

R4,R6

EC50 (nM)a

%AMPa

KD (nM)b

log Dc

pKad

Pappe (10−6 cm/s)

CLintf (μL/min/106 cells)

hOAT3 uptakeg

1 2 3 4

H,Cl H,CN H,F F,F

5.6 100 52 35

210 180 200 160

9.0 48 48 20

2.0 1.1 1.7 1.3

4.7 4.6 4.8 4.9

5.8 1.9 3.3 1.9

14 13 8.0 2.8

4.7 ND 3.3 17

TR-FRET α1β1γ1-AMPK geometric mean EC50 and mean maximal activation as a percentage of maximal AMP activation, determined from at least four replicates; 90% confidence intervals are included in the Supporting Information. bKD α1β1γ1-AMPK as determined by SPR reported as the global fit of the data using at least three replicates. clog D was measured at pH 7.4 using the previously described shake-flask method.35 dpKa was determined by electrophoresis as previously described.36 ePassive permeability (Papp) from the apical to basolateral (AB) direction was measured in Ralph Russ canine kidney (RRCK) cells as previously described.34 fCLint refers to total intrinsic clearance obtained from scaling in vitro half-lives in cryopreserved human hepatocytes as previously described.39 gRatio of uptake in hOAT3 transfected HEK293 cells to uptake in vector-treated cells; a ratio >2.0 indicates that the test compound is an OAT3 substrate in vitro. ND: not determined. a

deprioritized in favor of more productive options involving indole core and 5-aryl substituent modifications. Previously reported structure−activity relationships (SAR) and AMPK co-crystal structures with indole-3-carboxylic acid derivatives indicated the importance of the C6 substituent for AMPK potency through enhanced hydrophobic interactions and its impact on biaryl torsion angle but also limited space in the pocket that accommodates this substituent.19 Thus, in pursuit of strategy c, we focused on small electron-withdrawing substituents to improve the balance of potency and metabolic clearance (Table 2). The 6-cyano substituted derivative 2 showed both a reduction in α1β1γ1-AMPK activation potency and binding affinity. The cyano group decreased the log D as compared to compound 1; however, human hepatocyte CLint was not significantly improved. The 6-fluoroindole 3 exhibited somewhat reduced potency as compared to 1, albeit with a modest improvement in CLint. The substantially lower CLint for the 4,6-difluoro derivative 4, coupled with a modest decrease in potency, was a notable point of differentiation. The substituent changes on the indole had limited impact on the measured pKa, which was similar (4.6−4.9) for compounds 1−4; however, the log D of 4 was lower than anticipated. The reduced log D of 4 may be due to the influence of the 4-fluoro substituent on the dipole of the core or an (experimentally unverified) interaction between the carboxylate and the proximal (C4) fluorine atom. The proximity of the C4-fluoro group to the carboxylic acid may also reduce the rate of glucuronidation, leading to the observed decrease in CLint of 4 as compared to the isolipophilic cyano analogue 2. As with compound 1, both fluoro analogues 3 and 4 were identified as substrates for hOAT3 in the HEK293 uptake assay. The core modifications shown in Table 2 led to reductions in both log D and Papp. We reasoned that the substituted aryl ring at C5 on the indole ring would provide a simple opportunity for modulation of the overall physicochemical properties of the series. Previous X-ray crystallography studies indicated that the 4-position of this aryl ring pointed toward a solvent-exposed part of the α1β1γ1-AMPK binding pocket19 and thus was expected to be relatively tolerant of functional group variation. On the basis of the SAR developed during identification of compound 1, a lipophilicity range of log D ∼ 1.5−2.5 was targeted for balancing AMPK activation potency, Papp, and

from indole A by a sequence of Vilsmeier−Haack formylation, Suzuki coupling with a substituted phenyl boronate to provide intermediate B, and final step Pinnick oxidation; chiral chromatographic separation afforded the separate enantiomers 9 and 10. Compounds 5, 6, 7, and 14 were synthesized by converting bromo-indole C to the corresponding neopentylglycol-boronate, followed by Suzuki coupling with the appropriate aryl or heteroarylhalide to afford intermediate D, and finally, ester hydrolysis to provide the final compounds.

■

RESULTS AND DISCUSSION Considering our overall goal of reduced CLp relative to compound 1, we sought to minimize both hepatic glucuronidation and renal excretion of the parent compound. To reduce CLrenal, both decreased active excretion and increased Papp, to maximize passive reabsorption in the kidney, were desired. At the outset, there was insufficient data for compounds in this class to show a strong correlation between active CLrenal in rat and rat Oat3 (or human OAT3) transporter activity measured in vitro. Creating a data set to demonstrate the in vitro−in vivo correlation was an integral part of this work.40 On the basis of strategies previously reported in the literature,41−49 several approaches were considered to reduce acyl glucuronidation: (a) increasing three-dimensional shape around the carboxylic acid to create a steric mismatch with the shape of the UGT active site, (b) replacing the carboxylic acid with an isostere, such as a heterocyclic ring, (c) stereoelectronic modulation of the acid motif by optimization of the indole ring, and (d) indirect or longer range effects, such as changing the hydroxycyclobutylphenyl ring, that might have some lessreadily predicted effect on binding to the UGT binding pocket. Approaches a and b were limited by the balance of α1β1γ1AMPK potency and ADME properties that could be achieved. Preliminary efforts to introduce partial saturation to the indole ring led to loss of AMPK activation potency. Several acid isosteres were explored but not pursued due to lack of sufficient potency or undesirable ADME properties. For example, Nsulfonyl amides of 1 retained potent activation of α1β1γ1AMPK but had drastically reduced Papp, presumably due to increased polar surface area. These strategies were thus 2375



Article

Table 3. Modification of the R5-Aryl Substituent

TR-FRET α1β1γ1-AMPK geometric mean EC50 and mean maximal activation as a percentage of maximal AMP activation, determined from at least four replicates; 90% confidence intervals are included in the Supporting Information. bKD α1β1γ1-AMPK as determined by SPR reported as the global fit of the data using at least three replicates. clog D was measured at pH 7.4 using the previously described shake-flask method.35 dpKa was determined by electrophoresis as previously described.36 ePassive permeability (Papp) from the apical to basolateral (AB) direction was measured in Ralph Russ canine kidney (RRCK) cells as previously described.34 fCLint refers to total intrinsic clearance obtained from scaling in vitro half-lives in cryopreserved human hepatocytes as previously described.39 gRatio of uptake in hOAT3 transfected HEK293 cells to uptake in vector-treated cells; a ratio >2.0 indicates that the test compound is an OAT3 substrate. ND: not determined. a

human hepatocyte CLint. To increase Papp, substituents lacking an additional hydrogen bond donor (beyond the indole acid core) were favored. The alcohol and ether compound pair 5 and 6 highlights the differences in RRCK Papp observed with the presence/absence of an alcohol as H-bond donor (Table 3) and the challenge in balancing Papp with low metabolic clearance. Similarly, the Papp of 6 was markedly higher than that of either 1 or 3 despite similar lipophilicity. A weakly basic amine at the terminus of an alkyl ether linker was also tolerated, with morpholine derivative 7 affording a desirable balance of α1β1γ1-AMPK activation potency, Papp, and low CLint. The observed reduction of CLint was notable given the similar log D of compounds 6 and 7, and is consistent with an earlier report41 of distal basic groups reducing glucuronidation rates.

Compounds 5 and 7 were selected for rat PK studies to evaluate the impact of the primary alcohol (5) and weakly basic center (7) on disposition attributes. While both compounds demonstrated low total CLp (relative to 1) and moderate oral F (23 and 68%, respectively), significant renal excretion of unchanged parent was also noted for each compound. In fact, these compounds were identified as substrates for hOAT3, which is consistent with the observation of CLrenal,u in excess of GFR (see Table 4). Compound 6 was not advanced to in vivo PK studies due to its high metabolic turnover in human hepatocytes. Racemic tetrahydropyran 8, which contains a conformationally restrained ether group, demonstrated an attractive profile. Notably, in vitro data suggested 8 was not a hOAT3 substrate and, while its log D was higher than our target range, its human 2376



Article

Table 4. Assessment of CLrenal in Rat and OAT Substrate Properties of Selected AMPK Activators compd

%dose in urinea

total CLp (mL/min/kg)

total CLrenalb (mL/min/kg)

unbound CLrenalc (mL/min/kg)

1

15.9

22.6

7.64

849

98

hOAT1 1.7 ± 0.1 hOAT3 4.7 ± 0.1 rOat3 8.3 ± 0.2

3

20.0

8.07

2.69

280

32

hOAT1 1.0 ± 0.02 hOAT3 3.3 ± 0.1

4

10.7

3.16

0.66

161

19

hOAT3 17 ± 0.7

5

10.0

8.04

778

89

hOAT1 1.95 ± 0.2 hOAT3 7.06 ± 0.2

7

12.0

6.30

1.35

398

46

14

unbound CLrenal/GFR ratiod

normalized OAT fold uptakee

hOAT1 1.2 ± 0.04 hOAT3 6.3 ± 0.2

10

1.60

1.15

0.03

10

1.1

hOAT1 1.5 ± 0.1 hOAT3 2.3 ± 0.1 rOat3 2.3 ± 0.1

14

BLQ

0.05

NA

NA

NA

hOAT1 1.5 ± 0.01 hOAT3 1.4 ± 0.1 rOat3 2.0 ± 0.1

a

Percentage of iv dose recovered in urine over 24 h as unchanged parent compound. bTotal CLrenal in blood was estimated by multiplying the total blood clearance (CLb) with fraction of total iv dose of unchanged compound excreted in urine. CLb was obtained by dividing CLp by the blood to plasma ratio (B:P) of the respective test compounds. cUnbound CLrenal in blood was estimated by dividing total CLrenal by the unbound free fraction in blood, which in turn was obtained by dividing the unbound plasma free fraction by the blood to plasma ratio. dGFR in rats is 8.7 mL/min/kg. e OAT uptake of test compounds (1 μM) was evaluated in triplicate in HEK293 cells transfected with human OAT1/OAT3 or rat Oat3. BLQ: below lower limit of quantification (1 ng/mL). NA: not applicable.

Figure 3. View of compound 14 co-crystallized with α1β1γ1-AMPK (A) and an overlay (B) of the bound structures of 1 (pink) and 14 (cyan). Compound 14 (cyan sticks) is bound in the ADaM site at the interface of the α1 and β1 subunits (5T5T.pdb). Some of the key hydrogen bond interactions are highlighted.

Papp in the RRCK assay relative to 1. Uptake in the in vitro hOAT3 assay was also somewhat attenuated. An alternative method of modulating physical properties was explored through replacement of the phenyl ring at R5 with heteroaryl derivatives. The well-enabled cross-coupling chemistry allowed inspection of a range of heterocyclic R 5 substituents, and the 3-pyridyl analogues 11 and 12 were identified as moderately potent activators of α1β1γ1-AMPK (Table 3). Compounds 11 and 12 demonstrated promising Papp and log D within our target range; however, methoxypyridine analogue 11 was some 15-fold less potent than the previously published methoxyphenyl analogue (1200 vs 80 nM)19 and showed significant turnover in human hepatocytes. The more electron-rich dimethylaminopyridine analogue 12 provided

hepatocyte CLint remained low. The data from the indole core variations described above (Table 2) provided an opportunity to balance the lipophilicity of the tetrahydropyran substituent without adding additional hydrogen bond donors. Such proved to be the case, with difluoro analogues 9 and 10 having a similar reduction in log D (∼1 unit) as compared to chloride 8. As with compounds 1 and 4, the lipophilicity change between the chloro (8, pKa 4.9) and difluoro (10, pKa 5.0) indole acids could not be attributed to modulation of the indole acid pKa. Initial chromatographic separation of enantiomers, and subsequent asymmetric synthesis, led to identification of the (S)-tetrahydropyran 10 as the more active enantiomer. Compound 10 offered favorable in vitro ADME properties including decreased CLint in human hepatocytes and increased 2377



Article

Table 5. Preclinical PK Assessment of Compounds 10 and 14 and Comparison with Compound 1 compd a

1

speciesb rat NHP

10

rat NHP

14

rat NHP

route

CLp (mL/min/kg)

Vdss (L/kg)

t1/2 (h)

iv po iv po

22.6

0.85

1.06

8.57

iv po iv po

1.15

iv po iv po

0.05

0.38

0.98

2.33

0.31 0.3

0.1 0.46

Cmax (ng/mL)

tmax (h)

110

0.33

733

1.2

1890

2.7

8180

2.0

10200

5.5

2540

1.5

8.54

4.95 12

27.2 9.14

AUC(0−24) (ng·h/mL) 744 329 1970 2690 16400 12800 35900 91600 254000 160000 15500 31200

oral F (%)

% dose in urine 15.9

14.7 20.6 59.4 1.6 26 2.2 85 BLQ 52 95% purity. (S)-4,6-Difluoro-5-(4-(tetrahydro-2H-pyran-2-yl)phenyl)-1H-indole-3-carboxylic Acid (10). Step 1: 1-(4-Bromophenyl)-5-chloropentan-1-ol. NaBH4 (1.72 g, 45.4 mmol, 5.0 equiv) was added to a solution of 1-(4-bromophenyl)-5-chloropentan-1-one (2.50 g, 9.1 mmol, 1 equiv) in MeOH (200 mL) at 0 °C. The mixture was then warmed to ambient temperature, and after 3 h, a saturated aqueous solution of citric acid was added to bring the pH to 3. The mixture was concentrated to remove MeOH, and the remaining aqueous layer was extracted with EtOAc (3 × 200 mL). The combined organics were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated to afford a colorless oil (2.30 g, 91%) which was used without further purification. 1H NMR (400 MHz, MeOH-d4) δ 7.49 (d, J = 7.6 Hz, 2H), 7.29 (d, J = 7.6 Hz, 2H), 4.61 (dd, J = 7.0, 5.7 Hz, 1H), 3.55 (t, J = 6.6 Hz, 2H), 1.74 (m, 4H), 1.56 (m, 1H), 1.43 (m, 1H). Step 2: 2-(4-Bromophenyl)tetrahydro-2H-pyran. A solution of 1-(4bromophenyl)-5-chloropentan-1-ol (2.0 g, 7.2 mmol, 1 equiv) in THF (100 mL) was added dropwise to NaH (60% dispersion in mineral oil, 432 mg, 10.8 mmol, 1.5 equiv) in THF (100 mL) at 0 °C. The mixture was warmed to ambient temperature and then was stirred for 18 h before being cooled to 0 °C. Excess hydride was quenched by the sequential addition of water (100 mL) and a saturated aqueous solution of NH4Cl (100 mL). The mixture was extracted with EtOAc (3 × 100 mL), and the combined organics were washed with brine

Figure 4. In vivo assessment of AMPK target engagement as measured by increases in pAMPK/tAMPK in kidney pole for compounds 1, 10, and 14 in ZSF-1 rats after 3 days of dosing (*** indicates p < 0.001 relative to vehicle).

(n = 5) were dosed once daily with vehicle or test compound at 30 or 100 mg/kg for 3 days. One hour after the last dose, the animals were euthanized and kidney pole tissue was collected and analyzed for total AMPK (tAMPK) and pAMPK. In each treatment group, the ratio of pAMPK/tAMPK was significantly increased relative to vehicle control, indicating robust engagement of AMPK in the target tissue of interest. On the basis of its favorable potency and in vivo PK attributes, in particular its long half-life and lack of renal clearance in rats, compound 14 was selected for further in vivo characterization. Compound 14 demonstrated improvements in renal function after chronic dosing (30 mg/kg/day for 68 days) in the ZSF-1 rat model, details of which were reported previously.33

■

CONCLUSION In summary, we have identified compounds 10 (PF-06679142) and 14 (PF-06685249), which, relative to compound 1, demonstrated comparable β1-AMPK activation potency and in vivo target engagement, along with significantly increased half-lives and oral bioavailability in preclinical species, decreased intrinsic clearance in human hepatocytes, and markedly 2379



Article

(100 mL), dried over Na2SO4, filtered, and concentrated to afford a residue that was purified by silica gel chromatography (petroleum ether) to afford a yellow oil (1.8 g, >100%; with residual mineral oil). 1 H NMR (400 MHz, MeOH-d4) δ 7.48 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 4.34 (dd, J = 11.0, 2.0 Hz, 1H), 4.09 (m, 1H), 3.65 (m, 1H), 1.95 (m, 1H), 1.84 (m, 1H), 1.63 (m, 4H). Step 3: 5,5-Dimethyl-2-(4-(tetrahydro-2H-pyran-2-yl)phenyl)-1,3,2dioxaborinane. Solid KOAc (2.20 g, 22.4 mmol, 3.0 equiv) was added to a solution of 2-(4-bromophenyl)tetrahydro-2H-pyran (1.8 g, 7.7 mmol, 1 equiv) and 5,5,5′,5′-tetramethyl-2,2′-bi(1,3,2-dioxaborinane) (1.85 g, 8.21 mmol, 1.1 equiv) in dioxane (100 mL). The mixture was sparged with nitrogen for 1 min then Pd(dppf)Cl2 (273 mg, 0.37 mmol, 0.05 equiv) was added and the resulting mixture was stirred at 100 °C for 16 h. The mixture was cooled to ambient temperature and was filtered through Celite, rinsing with EtOAc (2 × 100 mL). The resulting residue was purified by silica gel chromatography (0−10% EtOAc:petroleum ether) to afford the product as a white solid (1.23 g, 60%). 1H NMR (400 MHz, MeOH-d4) δ 7.73 (d, J = 7.6 Hz, 2H), 7.31 (d, J = 7.6 Hz, 2H), 4.36 (dd, J = 11.2, 2.0 Hz, 1H), 4.10 (m, 1H), 3.78 (s, 4H), 3.64 (m, 1H), 1.94 (m, 1H), 1.83 (m, 1H), 1.65 (m, 4H), 1.02 (s, 6H). Step 4: 5-Bromo-4,6-difluoro-1H-indole-3-carbaldehyde. N,N-Dimethylformiminium chloride (1.07 g, 8.34 mmol, 1.5 equiv) was added to a solution of 5-bromo-4,6-difluoro-1H-indole (1.29 g, 5.56 mmol, 1 equiv) in CH3CN (7.0 mL). The mixture was stirred at room temperature for 45 min, and then an aqueous NaOH solution (1N , 15 mL, 15 mmol, 2.7 equiv) was added. The resulting mixture was heated to 100 °C for 1 h and then was cooled to 0 °C. The resulting solids were collected via filtration, washed with water, and dried with vacuum to provide the product (0.90 g). An additional portion of solids formed in the filtrate, which was collected and dried to provide an additional sample of material (1.27 g combined, 88%). MS (M + H)+ 260.3. 1H NMR (400 MHz, DMSO-d6) δ 12.58 (br s, 1H), 9.93 (d, J = 3.9 Hz, 1H), 8.34 (s, 1H), 7.39 (dd, J = 8.5, 1.1 Hz, 1H). Step 5: 4,6-Difluoro-5-(4-(tetrahydro-2H-pyran-2-yl)phenyl)-1Hindole-3-carbaldehyde. A mixture of 5,5-dimethyl-2-(4-(tetrahydro2H-pyran-2-yl)phenyl)-1,3,2-dioxaborinane (444 mg, 1.62 mmol, 1.1 equiv), 5-bromo-4,6-difluoro-1H-indole-3-carbaldehyde (400 mg, 1.54 mmol, 1 equiv), K2CO3 (638 mg, 4.62 mmol, 3.0 equiv), and Pd(dppf)Cl2 (44 mg, 0.06 mmol, 0.04 equiv) in dioxane (4.6 mL) and water (2.3 mL) was stirred at 100 °C for 3 h. After cooling to ambient temperature, the mixture was partitioned between water (20 mL) and EtOAc (3 × 20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated to give a solid residue, which was triturated with 50% petroleum ether−MTBE. Filtration afforded the solid product which was used without further purification (400 mg, 76%). MS (M + H)+ 342.0. 1H NMR (600 MHz, DMSO-d6) δ 12.53 (br s, 1H), 9.97 (s, 1H), 8.32 (s, 1H), 7.41 (m, 4H), 7.31 (d, J = 10.0 Hz, 1H), 4.37 (d, J = 11.2 Hz, 1H), 4.03 (br d, J = 11.2 Hz, 1H), 3.54 (m, 1H), 1.86 (m, 2H), 1.65 (m, 1H), 1.56 (m, 2H), 1.47 (m, 1H). Step 6: 4,6-Difluoro-5-(4-(tetrahydro-2H-pyran-2-yl)phenyl)-1Hindole-3-carboxylic acid. A solution of NaClO2 (1.58 g, 14.0 mmol, 12 equiv) and sodium dihydrogen phosphate (3.20 g, 23.4 mmol, 20 equiv) in water (24 mL) was added to a solution of 4,6-difluoro-5-(4(tetrahydro-2H-pyran-2-yl)phenyl)-1H-indole-3-carbaldehyde (400 mg, 1.17 mmol, 1 equiv) in CH3CN (24 mL), t-BuOH (24 mL), and 2-methyl-2-butene (15.6 mL) at 0 °C. The mixture was then stirred at room temperature for 2 days. To quench excess oxidant, a solution of Na2SO3 (4.40 g, 35.1 mmol, 200 equiv) in water (10 mL) was added. The resulting mixture was stirred for 30 min, then was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated to give a crude residue, which was purified by preparative HPLC to afford the product as an off-white solid (80 mg, 19%). MS (M + H)+ 357.9. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (br s, 1H), 12.01 (br s, 1H), 8.07 (d, J = 2.5 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 9.6 Hz, 1H), 4.38 (d, J = 10.4 Hz,1H), 4.05 (m, 1H), 3.55 (m, 1H), 1.86 (m, 2H), 1.56 (m, 4H).

Step 7: Chiral SFC purification afforded the separate enantiomers: Chiralcel OJ-H 250 mm × 4.6 mm, 5 μm, 5−40% methanol (0.05% Et2NH) in CO2, 2.35 mL/min, 220 nm. (R)-4,6-Difluoro-5-(4-(tetrahydro-2H-pyran-2-yl)phenyl)-1H-indole-3-carboxylic acid (9). Retention time 8.1 min (22 mg); 94% ee. MS (M + H)+ 358. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (br s, 2H), 8.07 (s, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 9.5 Hz, 1H), 4.39 (dd, J = 11.5, 1.0 Hz, 1H), 4.06 (m, 1H), 3.58 (m, 1H), 1.87 (m, 2H), 1.58 (m, 4H). (S)-4,6-Difluoro-5-(4-(tetrahydro-2H-pyran-2-yl)phenyl)-1H-indole-3-carboxylic acid (10). Retention time = 7.5 min (30 mg), 97% ee. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (br s, 2H), 8.07 (s, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H) 7.25 (d, J = 9.5 Hz, 1H), 4.39 (d, J = 10.5 Hz,1H), 4.06 (m, 1H), 3.56 (m, 1H), 1.87 (m, 2H), 1.59 (m, 4H). Characterization from a subsequent batch. HRMS (ESI): calcd for C20H18F2NO3 (M + H)+ 358.1249, found 358.1244. Analytical (%) calcd: C, 67.22; H, 4.80; N, 3.92. Found: C, 66.86; H, 4.51; N, 3.84. 13C NMR (101 MHz, DMSO-d6) δ 164.2, 155.8, 151.7, 143.0, 137.2, 134.4, 130.3, 128.7, 125.5, 110.9, 110.5, 107.3, 95.1, 78.7, 68.0, 33.8, 25.5, 23.5. 6-Chloro-5-(6-(dimethylamino)-2-methoxypyridin-3-yl)-1H-indole-3-carboxylic Acid (14). Step 1: Methyl 6-chloro-5-(5,5-dimethyl1,3,2-dioxaborinan-2-yl)-1H-indole-3-carboxylate. A mixture of methyl 5-bromo-6-chloro-1H-indole-3-carboxylate19 (10.5 g, 36.4 mmol, 1 equiv), 5,5,5′,5′-tetramethyl-2,2′-bi-1,3,2-dioxaborinane (9.04 g, 40.0 mmol, 1.1 equiv), and oven-dried KOAc (17.9 g, 182 mmol, 5.0 equiv), in 1,4-dioxane (170 mL) was sparged with nitrogen for 10 min, then was treated with Pd(dppf)Cl2 (1.60 g, 2.18 mmol, 0.06 equiv). The mixture was heated at 110 °C for 3 h. The mixture was then cooled to ambient temperature and was filtered through Celite, rinsing with EtOAc. The filtrate was concentrated and then was eluted from a pad of silica gel (EtOAc). After concentration, the residue was loaded onto a silica gel column with a minimal amount of CH2Cl2 and was eluted with EtOAc:heptane (1:4 to 1:1) to provide a tan solid (5.6 g, 48%). MS (M + H)+ 254.1 (M = RB(OH)2 on LCMS). 1H NMR (500 MHz, DMSO-d6) δ 11.98 (br s, 1H), 8.29 (s, 1H), 8.10 (d, J = 2.93 Hz, 1H), 7.46 (s, 1H), 3.80 (s, 3H), 3.79 (s, 4H), 1.01 (s, 6H). Step 2: 5-Bromo-6-fluoro-N,N-dimethylpyridin-2-amine. N-Bromosuccinimide (4.60 g, 25.9 mmol, 0.50 equiv) was added to a solution of 6-fluoro-N,N-dimethylpyridin-2-amine (7.25 g, 51.7 mmol, 1 equiv) in CH3CN (345 mL) at 0 °C. After 1 h, a second portion of Nbromosuccinimide (4.60 g, 25.9 mmol, 0.50 equiv) was added and the resulting mixture was warmed to ambient temperature. After 16 h, the mixture was partitioned between CH2Cl2 (500 mL) and water (300 mL). The aqueous layer was further extracted with CH2Cl2 (3 × 200 mL), and the combined organics were dried over MgSO4, filtered, and concentrated to afford a yellow solid (10.8 g, 95%). MS (M)+ 218, 220. 1H NMR (400 MHz, CDCl3) δ 7.50 (t, J = 8.8 Hz, 1H), 6.14 (dd, J = 8.8, 2.0 Hz, 1H), 2.98 (s, 6H). Step 3: 5-Bromo-6-methoxy-N,N-dimethylpyridin-2-amine. 5Bromo-6-fluoro-N,N-dimethylpyridin-2-amine (10.8 g, 50.8 mmol, 1 equiv) was added to a 25% solution of MeOK in MeOH (45 mL, 152 mmol, 3.0 equiv), and the resulting mixture was heated at reflux for 2.5 h. After cooling to ambient temperature, saturated aqueous NH4Cl solution (400 mL) was added, and the mixture was extracted with EtOAc (2 × 300 mL). The combined organics were washed with brine (300 mL), dried over MgSO4, filtered, and concentrated to afford a solid that was used without further purification (11.2 g, 95%). MS (M)+ 230, 232. 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.6 Hz, 1H), 5.96 (d, J = 8.6 Hz, 1H), 3.97 (s, 3H), 3.06 (s, 6H). Step 4: Methyl 6-chloro-5-(6-(dimethylamino)-2-methoxypyridin-3yl)-1H-indole-3-carboxylate. A mixture of methyl 6-chloro-5-(5,5dimethyl-1,3,2-dioxaborinan-2-yl)-1H-indole-3-carboxylate (9.25 g, 28.8 mmol, 1.5 equiv), 5-bromo-6-methoxy-N,N-dimethylpyridin-2amine (4.44 g, 19.2 mmol, 1 equiv), PdCl2(dppf) (1.12 g, 1.53 mmol, 0.08 equiv), an aqueous solution of K2CO3 (7.95 g, 57.5 mmol, 3.0 equiv, in 29 mL water), EtOH (630 mL), and toluene (60 mL) was sparged with nitrogen for 20 min and then was heated at 120 °C for 16 h. The mixture was cooled to ambient temperature and was diluted with EtOAc (800 mL) and was filtered through a pad of Celite. The 2380



Article

filtrate was washed with saturated aqueous NH4Cl solution (2 × 400 mL). The combined organics were dried over MgSO4, filtered, and concentrated. The resulting solids were suspended in MeOH (200 mL) and were heated to reflux then were cooled to ambient temperature and were filtered to afford a tan solid which contained residual 2,2-dimethylpropane-1,3-diol. The mother liquor was concentrated and crystallized from MeOH (100 mL) at reflux to afford another batch of solid. The combined solids were slurried in MeOH (40 mL) at ambient temperature for 30 min then were filtered to afford the desired product (5.15 g, 75%). MS (M + H)+ 360.1. 1H NMR (400 MHz, DMSO-d6) δ 12.00 (br s, 1H), 8.14 (s, 1H), 7.84 (s, 1H), 7.58 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 6.23 (d, J = 8.0 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 3.08 (s, 6H). Step 5: 6-Chloro-5-(6-(dimethylamino)-2-methoxypyridin-3-yl)1H-indole-3-carboxylic acid (14). Aqueous NaOH solution (2 N, 10 mL, 20 mmol, 10 equiv) was added to a solution of methyl 6-chloro-5(6-(dimethylamino)-2-methoxypyridin-3-yl)-1H-indole-3-carboxylate (700 mg, 1.94 mmol, 1 equiv) in MeOH−THF (6 mL each) at 70 °C. After 24 h, the mixture was cooled to 0 °C, and aqueous HCl solution (1 N, ∼20 mL) was added dropwise to bring the pH to ∼5−6. After extraction with EtOAc (3 × 75 mL), the combined organics were washed with brine (50 mL), dried over MgSO4, filtered, and concentrated to afford an off-white solid (670 mg), which was suspended in MeOH (5 mL) and stirred at 40 °C for 16 h. After cooling to ambient temperature and filtration, the product was isolated as a white solid (500 mg, 74%). HRMS (ESI): calcd for C17H16ClN3O3 (M + H)+ 346.0953, found 346.0953. Analytical (%) calcd: C, 59.05; H, 4.66; N, 12.15. Found: C, 59.01; H, 4.64; N, 12.00. 1H NMR (400 MHz, DMSO-d6) δ 12.03 (br s, 1H), 11.86 (br s, 1H), 8.04 (d, J = 2.7 Hz, 1H), 7.83 (s, 1H), 7.55 (s, 1H), 7.32 (d, J = 8.2 Hz, 1H), 6.21 (d, J = 8.2 Hz, 1H), 3.76 (s, 3H), 3.07 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 165.7, 159.0, 157.4, 141.2, 135.9, 133.3, 129.4, 127.9, 125.0, 123.3, 112.2, 108.9, 107.5, 96.7, 52.4, 37.5.

■

Notes

The authors declare the following competing financial interest(s): All authors were employed by Pfizer, Inc. at the time this work was done.

■

ACKNOWLEDGMENTS We thank Yuxia Mao for compound formulation support, Marina Shalaeva for pKa determinations, Derek Vrieze and Yingxin Zhang for synthesis contributions, Chris Limberakis for chemistry outsourcing support, and Jared Milbank and Laura McAllister for helpful discussions. Crystallographic data were collected at IMCA-CAT (17-ID) and GMCA-CAT at the Advanced Photon Source. IMCA was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman−Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357.

■

ABBREVIATIONS ADaM, allosteric drug and metabolite; ADME, absorption, distribution, metabolism and excretion; ADP, 5′-adenosine diphosphate; AMP, 5′-adenosine monophosphate; AMPK, 5′adenosine monophosphate-activated protein kinase; CLint, intrinsic clearance; CLp, plasma clearance; CLrenal, renal clearance; CLb, blood clearance; DMAP, 4-dimethylaminopyridine; DN, diabetic nephropathy; ee, enantiomeric excess; equiv, equivalents; ESI, electrospray ionization; F, oral bioavailability; GFR, glomerular filtration rate; HRMS, high resolution mass spectrometry; neop, neopentyl; OAT, organic anion transporter; Papp, apparent passive permeability; pAMPK, phospho-AMPK; Pd(dppf)Cl2, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II); PK, pharmacokinetics; RRCK, Ralph Russ canine kidney; SAR, structure−activity relationship; SPR, surface plasmon resonance; tAMPK, total AMPK; T2DM, type II diabetes mellitus; MTBE, t-butylmethyl ether; THF, tetrahydrofuran; TLC, thin layer chromatography; TR-FRET, time-resolved fluorescence resonance energy transfer; UGT, uridine 5′-diphospho-glucuronosyltransferase

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01641. Additional experimental and characterization data for compound synthesis; experimental details for the SPR assay, the in vitro OAT uptake assays, the in vivo PK studies, and the in vivo ZSF-1 rat study; additional AMPK isoform pharmacology data including dose response curves and confidence intervals for the FRET and SPR assays; plasma protein binding data for compounds tested in vivo (PDF) Molecular formula strings (CSV)

■

(1) Gallagher, H.; Suckling, R. J. Diabetic nephropathy: where are we on the journey from pathophysiology to treatment? Diabetes, Obes. Metab. 2016, 18, 641−647. (2) Bjornstad, P.; Cherney, D. Z.; Maahs, D. M. Update on estimation of kidney function in diabetic kidney disease. Curr. Diabetes Rep. 2015, 15, 57. (3) Vallon, V.; Komers, R. Pathophysiology of the diabetic kidney. Compr. Physiol. 2011, 1, 1175−1232. (4) Pofi, R.; Di Mario, F.; Gigante, A.; Rosato, E.; Isidori, A. M.; Amoroso, A.; Cianci, R.; Barbano, B. Diabetic nephropathy: focus on current and future therapeutic strategies. Curr. Drug Metab. 2016, 17, 497−502. (5) Ross, F. A.; MacKintosh, C.; Hardie, D. G. AMP-activated protein kinase: a cellular energy sensor that comes in 12 flavours. FEBS J. 2016, 283, 2987−3001. (6) Mihaylova, M. M.; Shaw, R. J. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat. Cell Biol. 2011, 13, 1016−1023. (7) Cameron, K. O.; Kurumbail, R. G. Recent progress in the identification of adenosine monophosphate-activated protein kinase (AMPK) activators. Bioorg. Med. Chem. Lett. 2016, 26, 5139−5148.

Accession Codes

Crystal structure coordinates for compounds 1 and 14 have been deposited to the RCSB Protein Data Bank (www.rcsb.org, accession codes 5KQ5 and 5T5T, respectively). Authors will release the atomic coordinates and experimental data upon article publication.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*For D.J.E.: phone, (617) 441-3263; E-mail, David.Edmonds@ pfizer.com. *For D.W.K.: E-mail, Daniel.W.Kung@pfizer.com. ORCID

David J. Edmonds: 0000-0001-9234-8618 Daniel W. Kung: 0000-0002-5019-1939 Kevin J. Filipski: 0000-0003-0845-5723 Aaron C. Smith: 0000-0001-6796-5155 2381



Article

(8) Hasenour, C. M.; Ridley, D. E.; Hughey, C. C.; James, F. D.; Donahue, E. P.; Shearer, J.; Viollet, B.; Foretz, M.; Wasserman, D. H. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) effect on glucose production, but not energy metabolism, is independent of hepatic AMPK in vivo. J. Biol. Chem. 2014, 289, 5950−5959. (9) Foretz, M.; Hebrard, S.; Leclerc, J.; Zarrinpashneh, E.; Soty, M.; Mithieux, G.; Sakamoto, K.; Andreelli, F.; Viollet, B. Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/ AMPK pathway via a decrease in hepatic energy state. J. Clin. Invest. 2010, 120, 2355−2369. (10) Cool, B.; Zinker, B.; Chiou, W.; Kifle, L.; Cao, N.; Perham, M.; Dickinson, R.; Adler, A.; Gagne, G.; Iyengar, R.; Zhao, G.; Marsh, K.; Kym, P.; Jung, P.; Camp, H. S.; Frevert, E. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 2006, 3, 403−416. (11) Gomez-Galeno, J. E.; Dang, Q.; Nguyen, T. H.; Boyer, S. H.; Grote, M. P.; Sun, Z.; Chen, M.; Craigo, W. A.; van Poelje, P. D.; MacKenna, D. A.; Cable, E. E.; Rolzin, P. A.; Finn, P. D.; Chi, B.; Linemeyer, D. L.; Hecker, S. J.; Erion, M. D. A Potent and Selective AMPK Activator That Inhibits de Novo Lipogenesis. ACS Med. Chem. Lett. 2010, 1, 478−482. (12) Lan, P.; Romero, F. A.; Wodka, D.; Kassick, A. J.; Dang, Q.; Gibson, T.; Cashion, D.; Zhou, G.; Chen, Y.; Zhang, X.; Zhang, A.; Li, Y.; Trujillo, M. E.; Shao, Q.; Wu, M.; Xu, S.; He, H.; MacKenna, D.; Staunton, J.; Chapman, K. T.; Weber, A.; Sebhat, I. K.; Makara, G. M. Hit-to-lead optimization and discovery of 5-((5-([1,1’-Biphenyl]-4-yl)6-chloro-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic acid (MK3903): a novel class of benzimidazole-based activators of ampactivated protein kinase. J. Med. Chem. 2017, 60, 9040−9052. (13) Myers, R. W.; Guan, H. P.; Ehrhart, J.; Petrov, A.; Prahalada, S.; Tozzo, E.; Yang, X.; Kurtz, M. M.; Trujillo, M.; Gonzalez Trotter, D.; Feng, D.; Xu, S.; Eiermann, G.; Holahan, M. A.; Rubins, D.; Conarello, S.; Niu, X.; Souza, S. C.; Miller, C.; Liu, J.; Lu, K.; Feng, W.; Li, Y.; Painter, R. E.; Milligan, J. A.; He, H.; Liu, F.; Ogawa, A.; Wisniewski, D.; Rohm, R. J.; Wang, L.; Bunzel, M.; Qian, Y.; Zhu, W.; Wang, H.; Bennet, B.; LaFranco Scheuch, L.; Fernandez, G. E.; Li, C.; Klimas, M.; Zhou, G.; van Heek, M.; Biftu, T.; Weber, A.; Kelley, D. E.; Thornberry, N.; Erion, M. D.; Kemp, D. M.; Sebhat, I. K. Systemic pan-AMPK activator MK-8722 improves glucose homeostasis but induces cardiac hypertrophy. Science 2017, 357, 507−511. (14) Feng, D.; Biftu, T.; Romero, F. A.; Kekec, A.; Dropinski, J.; Kassick, A.; Xu, S.; Kurtz, M. M.; Gollapudi, A.; Shao, Q.; Yang, X.; Lu, K.; Zhou, G.; Kemp, D.; Myers, R. W.; Guan, H. P.; Trujillo, M. E.; Li, C.; Weber, A.; Sebhat, I. K. Discovery of MK-8722: a systemic, direct pan-activator of AMP-activated protein kinase. ACS Med. Chem. Lett. 2018, 9, 39−44. (15) Dugan, L. L.; You, Y. H.; Ali, S. S.; Diamond-Stanic, M.; Miyamoto, S.; DeCleves, A. E.; Andreyev, A.; Quach, T.; Ly, S.; Shekhtman, G.; Nguyen, W.; Chepetan, A.; Le, T. P.; Wang, L.; Xu, M.; Paik, K. P.; Fogo, A.; Viollet, B.; Murphy, A.; Brosius, F.; Naviaux, R. K.; Sharma, K. AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function. J. Clin. Invest. 2013, 123, 4888−4899. (16) Lee, M. J.; Feliers, D.; Mariappan, M. M.; Sataranatarajan, K.; Mahimainathan, L.; Musi, N.; Foretz, M.; Viollet, B.; Weinberg, J. M.; Choudhury, G. G.; Kasinath, B. S. A role for AMP-activated protein kinase in diabetes-induced renal hypertrophy. Am. J. Physiol. Renal Physiol. 2007, 292, F617−627. (17) Decleves, A. E.; Mathew, A. V.; Cunard, R.; Sharma, K. AMPK mediates the initiation of kidney disease induced by a high-fat diet. J. Am. Soc. Nephrol. 2011, 22, 1846−1855. (18) Kim, D.; Lee, J. E.; Jung, Y. J.; Lee, A. S.; Lee, S.; Park, S. K.; Kim, S. H.; Park, B. H.; Kim, W.; Kang, K. P. Metformin decreases high-fat diet-induced renal injury by regulating the expression of adipokines and the renal AMP-activated protein kinase/acetyl-CoA carboxylase pathway in mice. Int. J. Mol. Med. 2013, 32, 1293−1302.

(19) Cameron, K. O.; Kung, D. W.; Kalgutkar, A. S.; Kurumbail, R. G.; Miller, R.; Salatto, C. T.; Ward, J.; Withka, J. M.; Bhattacharya, S. K.; Boehm, M.; Borzilleri, K. A.; Brown, J. A.; Calabrese, M.; Caspers, N. L.; Cokorinos, E.; Conn, E. L.; Dowling, M. S.; Edmonds, D. J.; Eng, H.; Fernando, D. P.; Frisbie, R.; Hepworth, D.; Landro, J.; Mao, Y.; Rajamohan, F.; Reyes, A. R.; Rose, C. R.; Ryder, T.; Shavnya, A.; Smith, A. C.; Tu, M.; Wolford, A. C.; Xiao, J. Discovery and preclinical characterization of 6-chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1Hindole-3-carboxylic acid (PF-06409577), a direct activator of adenosine monophosphate-activated protein kinase (AMPK), for the potential treatment of diabetic nephropathy. J. Med. Chem. 2016, 59, 8068−8081. (20) Xiao, B.; Sanders, M. J.; Carmena, D.; Bright, N. J.; Haire, L. F.; Underwood, E.; Patel, B. R.; Heath, R. B.; Walker, P. A.; Hallen, S.; Giordanetto, F.; Martin, S. R.; Carling, D.; Gamblin, S. J. Structural basis of AMPK regulation by small molecule activators. Nat. Commun. 2013, 4, 3017. (21) Calabrese, M. F.; Rajamohan, F.; Harris, M. S.; Caspers, N. L.; Magyar, R.; Withka, J. M.; Wang, H.; Borzilleri, K. A.; Sahasrabudhe, P. V.; Hoth, L. R.; Geoghegan, K. F.; Han, S.; Brown, J.; Subashi, T. A.; Reyes, A. R.; Frisbie, R. K.; Ward, J.; Miller, R. A.; Landro, J. A.; Londregan, A. T.; Carpino, P. A.; Cabral, S.; Smith, A. C.; Conn, E. L.; Cameron, K. O.; Qiu, X.; Kurumbail, R. G. Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms. Structure 2014, 22, 1161−1172. (22) Burckhardt, G. Drug transport by organic anion transporters (OATs). Pharmacol. Ther. 2012, 136, 106−130. (23) Muller, F.; Fromm, M. F. Transporter-mediated drug-drug interactions. Pharmacogenomics 2011, 12, 1017−1037. (24) VanWert, A. L.; Gionfriddo, M. R.; Sweet, D. H. Organic anion transporters: discovery, pharmacology, regulation and roles in pathophysiology. Biopharm. Drug Dispos. 2010, 31, 1−71. (25) Liu, H. C.; Goldenberg, A.; Chen, Y.; Lun, C.; Wu, W.; Bush, K. T.; Balac, N.; Rodriguez, P.; Abagyan, R.; Nigam, S. K. Molecular properties of drugs interacting with SLC22 transporters OAT1, OAT3, OCT1, and OCT2: a machine-learning approach. J. Pharmacol. Exp. Ther. 2016, 359, 215−229. (26) Radcliffe, N. J.; Seah, J. M.; Clarke, M.; MacIsaac, R. J.; Jerums, G.; Ekinci, E. I. Clinical predictive factors in diabetic kidney disease progression. J. Diabetes Invest. 2017, 8, 6−18. (27) Thomas, M. C.; Tikellis, C.; Burns, W. C.; Thallas, V.; Forbes, J. M.; Cao, Z.; Osicka, T. M.; Russo, L. M.; Jerums, G.; Ghabrial, H.; Cooper, M. E.; Kantharidis, P. Reduced tubular cation transport in diabetes: prevented by ACE inhibition. Kidney Int. 2003, 63, 2152− 2161. (28) Enomoto, A.; Niwa, T. Roles of organic anion transporters in the progression of chronic renal failure. Ther. Apheresis Dial. 2007, 11, S27−S31. (29) Kunin, M.; Holtzman, E. J.; Melnikov, S.; Dinour, D. Urinary organic anion transporter protein profiles in AKI. Nephrol., Dial., Transplant. 2012, 27, 1387−1395. (30) Lin, J. H. Species similarities and differences in pharmacokinetics. Drug Metab. Dispos. 1995, 23, 1008−1021. (31) GTEx Consortium. The Genotype-Tissue Expression (GTEx) project. Nat. Genet. 2013, 45, 580−585. (32) Wu, J.; Puppala, D.; Feng, X.; Monetti, M.; Lapworth, A. L.; Geoghegan, K. F. Chemoproteomic analysis of intertissue and interspecies isoform diversity of AMP-activated protein kinase (AMPK). J. Biol. Chem. 2013, 288, 35904−35912. (33) Salatto, C. T.; Miller, R. A.; Cameron, K. O.; Cokorinos, E.; Reyes, A.; Ward, J.; Calabrese, M.; Kurumbail, R.; Rajamohan, F.; Kalgutkar, A. S.; Tess, D. A.; Shavnya, A.; Genung, N. E.; Edmonds, D. J.; Jatkar, A.; Maciejewski, B. S.; Amaro, M.; Gandhok, H.; Monetti, M.; Cialdea, K.; Bollinger, E.; Kreeger, J. M.; Coskran, T. M.; Opsahl, A. C.; Boucher, G. G.; Birnbaum, M. J.; DaSilva-Jardine, P.; Rolph, T. Selective activation of AMPK β1-containing isoforms improves kidney function in a rat model of diabetic nephropathy. J. Pharmacol. Exp. Ther. 2017, 361, 303−311. 2382



Article

(34) Di, L.; Whitney-Pickett, C.; Umland, J. P.; Zhang, H.; Zhang, X.; Gebhard, D. F.; Lai, Y.; Federico, J. J., 3rd; Davidson, R. E.; Smith, R.; Reyner, E. L.; Lee, C.; Feng, B.; Rotter, C.; Varma, M. V.; Kempshall, S.; Fenner, K.; El-Kattan, A. F.; Liston, T. E.; Troutman, M. D. Development of a new permeability assay using low-efflux MDCKII cells. J. Pharm. Sci. 2011, 100, 4974−4985. (35) Stopher, D.; McClean, S. An improved method for the determination of distribution coefficients. J. Pharm. Pharmacol. 1990, 42, 144. (36) Shalaeva, M.; Kenseth, J.; Lombardo, F.; Bastin, A. Measurement of dissociation constants (pKa values) of organic compounds by multiplexed capillary electrophoresis using aqueous and cosolvent buffers. J. Pharm. Sci. 2008, 97, 2581−2606. (37) Yuan, H.; Feng, B.; Yu, Y.; Chupka, J.; Zheng, J. Y.; Heath, T. G.; Bond, B. R. Renal organic anion transporter-mediated drug-drug interaction between gemcabene and quinapril. J. Pharmacol. Exp. Ther. 2009, 330, 191−197. (38) Tweedie, D.; Polli, J. W.; Berglund, E. G.; Huang, S. M.; Zhang, L.; Poirier, A.; Chu, X.; Feng, B. Transporter studies in drug development: experience to date and follow-up on decision trees from the International Transporter Consortium. Clin. Pharmacol. Ther. 2013, 94, 113−125. (39) Di, L.; Trapa, P.; Obach, R. S.; Atkinson, K.; Bi, Y. A.; Wolford, A. C.; Tan, B.; McDonald, T. S.; Lai, Y.; Tremaine, L. M. A novel relay method for determining low-clearance values. Drug Metab. Dispos. 2012, 40, 1860−1865. (40) Soars, M. G.; Barton, P.; Elkin, L. L.; Mosure, K. W.; Sproston, J. L.; Riley, R. J. Application of an in vitro OAT assay in drug design and optimization of renal clearance. Xenobiotica 2014, 44, 657−665. (41) Borzilleri, R. M.; Cai, Z. W.; Ellis, C.; Fargnoli, J.; Fura, A.; Gerhardt, T.; Goyal, B.; Hunt, J. T.; Mortillo, S.; Qian, L.; Tokarski, J.; Vyas, V.; Wautlet, B.; Zheng, X.; Bhide, R. S. Synthesis and SAR of 4(3-hydroxyphenylamino)pyrrolo[2,1-f][1,2,4]triazine based VEGFR-2 kinase inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 1429−1433. (42) Bouska, J. J.; Bell, R. L.; Goodfellow, C. L.; Stewart, A. O.; Brooks, C. D.; Carter, G. W. Improving the in vivo duration of 5lipoxygenase inhibitors: application of an in vitro glucuronosyltransferase assay. Drug Metab. Dispos. 1997, 25, 1032−1038. (43) Crawford, T. D.; Romero, F. A.; Lai, K. W.; Tsui, V.; Taylor, A. M.; de Leon Boenig, G.; Noland, C. L.; Murray, J.; Ly, J.; Choo, E. F.; Hunsaker, T. L.; Chan, E. W.; Merchant, M.; Kharbanda, S.; Gascoigne, K. E.; Kaufman, S.; Beresini, M. H.; Liao, J.; Liu, W.; Chen, K. X.; Chen, Z.; Conery, A. R.; Cote, A.; Jayaram, H.; Jiang, Y.; Kiefer, J. R.; Kleinheinz, T.; Li, Y.; Maher, J.; Pardo, E.; Poy, F.; Spillane, K. L.; Wang, F.; Wang, J.; Wei, X.; Xu, Z.; Xu, Z.; Yen, I.; Zawadzke, L.; Zhu, X.; Bellon, S.; Cummings, R.; Cochran, A. G.; Albrecht, B. K.; Magnuson, S. Discovery of a potent and selective in vivo probe (GNE-272) for the bromodomains of CBP/EP300. J. Med. Chem. 2016, 59, 10549−10563. (44) Henderson, J. L.; Sawant-Basak, A.; Tuttle, J. B.; Dounay, A. B.; McAllister, L. A.; Pandit, J.; Rong, S.; Hou, X.; Bechle, B. M.; Kim, J.Y.; Parikh, V.; Ghosh, S.; Evrard, E.; Zawadzke, L. E.; Salafia, M. A.; Rago, B.; Obach, R. S.; Clark, A.; Fonseca, K. R.; Chang, C.; Verhoest, P. R. Discovery of hydroxamate bioisosteres as KAT II inhibitors with improved oral bioavailability and pharmacokinetics. MedChemComm 2013, 4, 125−129. (45) Mascitti, V.; Maurer, T. S.; Robinson, R. P.; Bian, J.; BoustanyKari, C. M.; Brandt, T.; Collman, B. M.; Kalgutkar, A. S.; Klenotic, M. K.; Leininger, M. T.; Lowe, A.; Maguire, R. J.; Masterson, V. M.; Miao, Z.; Mukaiyama, E.; Patel, J. D.; Pettersen, J. C.; Preville, C.; Samas, B.; She, L.; Sobol, Z.; Steppan, C. M.; Stevens, B. D.; Thuma, B. A.; Tugnait, M.; Zeng, D.; Zhu, T. Discovery of a clinical candidate from the structurally unique dioxa-bicyclo[3.2.1]octane class of sodiumdependent glucose cotransporter 2 inhibitors. J. Med. Chem. 2011, 54, 2952−2960. (46) Rose, K.; Yang, Y. S.; Sciotti, R.; Cai, H. Structure-activity relationship (SAR): effort towards blocking N-glucuronidation of indazoles (PF-03376056) by human UGT1A enzymes. Drug Metab. Lett. 2009, 3, 28−34.

(47) Takenaga, N.; Ishii, M.; Kamei, T.; Yasumori, T. Structureactivity relationship in O-glucuronidation of indolocarbazole analogs. Drug Metab. Dispos. 2002, 30, 494−497. (48) Zimmermann, S. C.; Rais, R.; Alt, J.; Burzynski, C.; Slusher, B. S.; Tsukamoto, T. Structure−metabolism relationships in the glucuronidation of d-amino acid oxidase inhibitors. ACS Med. Chem. Lett. 2014, 5, 1251−1253. (49) Stewart, A. O.; Bhatia, P. A.; Martin, J. G.; Summers, J. B.; Rodriques, K. E.; Martin, M. B.; Holms, J. H.; Moore, J. L.; Craig, R. A.; Kolasa, T.; Ratajczyk, J. D.; Mazdiyasni, H.; Kerdesky, F. A.; DeNinno, S. L.; Maki, R. G.; Bouska, J. B.; Young, P. R.; Lanni, C.; Bell, R. L.; Carter, G. W.; Brooks, C. D. Structure-activity relationships of N-hydroxyurea 5-lipoxygenase inhibitors. J. Med. Chem. 1997, 40, 1955−1968. (50) Brameld, K. A.; Kuhn, B.; Reuter, D. C.; Stahl, M. Small molecule conformational preferences derived from crystal structure data. A medicinal chemistry focused analysis. J. Chem. Inf. Model. 2008, 48, 1−24. (51) Solubility was measured at Analiza, Ohio, USA; compounds were added to 50 mM sodium phosphate buffer (pH 7.4), stirred for 24 h, filtered, and the dissolved concentration determined by nitrogen detection. (52) Bilan, V. P.; Salah, E. M.; Bastacky, S.; Jones, H. B.; Mayers, R. M.; Zinker, B.; Poucher, S. M.; Tofovic, S. P. Diabetic nephropathy and long-term treatment effects of rosiglitazone and enalapril in obese ZSF1 rats. J. Endocrinol. 2011, 210, 293−308. (53) Boustany-Kari, C. M.; Harrison, P. C.; Chen, H.; Lincoln, K. A.; Qian, H. S.; Clifford, H.; Wang, H.; Zhang, X.; Gueneva-Boucheva, K.; Bosanac, T.; Wong, D.; Fryer, R. M.; Richman, J. G.; Sarko, C.; Pullen, S. S. A soluble guanylate cyclase activator inhibits the progression of diabetic nephropathy in the ZSF1 rat. J. Pharmacol. Exp. Ther. 2016, 356, 712−719.

2383


Optimization of Metabolic and Renal Clearance in a Series of Indole

Recommend Documents