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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 Edward 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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01641 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Withka, Jane; Pfizer Global Research & Development, Xiao, Jun; Pfizer Inc., Worldwide Medicinal Chemistry Cameron, Kimberly; Pfizer, Pfizer Global Research and Development

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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, MA 02139, United States and ‡Eastern Point Road, Groton, CT 06340, United States. KEYWORDS.

5'-Adenosine monophosphate-activated protein kinase, AMPK, diabetic

nephropathy, glucuronidation, uridine glucuronosyl transferase, renal clearance, organic anion transporter, indole-3-carboxylic acid.


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ABSTRACT. Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated 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 (PF06679142) 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 rat and human 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 6-chloroindole 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.

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 six-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 (albuminuria). Ultimately, inflammation and fibrosis lead to reduced glomerular filtration rate (GFR) and end-stage renal disease requiring dialysis.2,3 Despite the increasing



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Page 3 of 43 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 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 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 non-phosphate-containing activators,12 including pan-AMPK


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Page 4 of 43 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

Figure 1. Selected AMPK activators We recently reported the identification of an indole-3-carboxylic 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 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



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Page 5 of 43 as 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

Figure 2. Chemical structure of 1 (A) and a view of the co-crystal structure of 1 with α1β1γ1AMPK (B). Compound 1 (pink ball-and-stick) 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 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


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Page 6 of 43 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. Table 1. CLrenal of unchanged 1 after iv administration to preclinical species

Species

Renal excretion Total CLp Total CLrenal CLrenal,u CLrenal,u/GFR a b c ratiod (% of total dose) (mL/min/kg) (mL/min/kg) (mL/min/kg)

Rat

15.9

22.6

7.64

849

98

Dog

11.8

12.9

2.82

54

14

Monkey

20.6

8.57

2.48

55

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-methyl2-pyrrolidone: 90% of 30% sulfobutylether-β-cyclodextrin solution in water (dogs). b

Total CLrenal in blood was estimated by multiplying the total blood clearance (CLb) with 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). c

CLrenal,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

In vitro Pharmacology and ADME Assays Functional potency of AMPK activators was evaluated using a time-resolved fluorescence resonance energy transfer (TR-FRET) 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.



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Page 7 of 43 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, 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 LogD 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 Due to identification of glucuronidation 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)


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Page 8 of 43 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.



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Page 9 of 43 Scheme 1. Synthetic routes to indole acid AMPK activators

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–t-BuOH; (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. 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 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.


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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 re-absorption 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 Based on 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 less-readily predicted effect on binding to the UGT binding pocket. Approaches (a) and (b) were limited by the balance of α1β1γ1-AMPK 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 α1β1γ1-AMPK but had drastically reduced Papp, presumably due to increased polar surface area. These strategies were thus deprioritized in favor of more productive options involving indole core and 5-aryl substituent modifications.



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Page 11 of 43 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 this pocket.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 α1β1γ1AMPK activation potency and binding affinity.

The cyano group decreased the logD 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 logD of 4 was lower than anticipated. The reduced logD 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.


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Page 12 of 43 Table 2. Indole core substitutions

α1β1γγ1-AMPK 4

6

Cmpd R ,R

c

EC50

%AMP

(nM)a

a

KD

LogD

pKa

d

(nM)b

Pappe

CLintf

(10-6cm/s)

(µL/min/ 106 cells)

hOAT3 uptakeg

1

H,Cl

5.6

210

9.0

2.0

4.7

5.8

14

4.7

2

H,CN

100

180

48

1.1

4.6

1.9

13

N.D.

3

H,F

52

200

48

1.7

4.8

3.3

8.0

3.3

F,F 35 160 20 1.3 4.9 1.9 2.8 17 4 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. cLogD 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. N.D. not determined. a

The core modifications shown in Table 2 led to reductions in both logD 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 solventexposed part of the α1β1γ1-AMPK binding pocket19 and thus was expected to be relatively tolerant of functional group variation. Based on the SAR developed during identification of compound 1, a lipophilicity range of logD ~1.5–2.5 was targeted for balancing AMPK activation potency, Papp and human hepatocyte CLint. To increase Papp, substituents lacking an additional



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Page 13 of 43 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 logD of compounds 6 and 7, and is consistent with an earlier report41 of distal basic groups reducing glucuronidation rates.


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Page 14 of 43 Table 3. Modification of the R5-aryl substituent

α1β1γγ1-AMPK Cmpd

R5

R4,R6

EC50 (nM)a

%AMP

KD (nM)b

a

LogDc

pKad

CLintf Papp(AB)e (µL/min/ (10-6cm/s) 106 cells)

hOAT3 uptakeg

5

H,Cl

47

180

N.D.

1.1

4.8

2.9

16

7.1

6

H,Cl

57

230

N.D.

1.8

5.0

12

35

1.6

7

H,Cl

43

200

22

1.8

5.4, 7.1

6.6

2.0 indicates that the test compound is an OAT3 substrate. N.D. indicates not determined.


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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 logD was higher than our target range, its human 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 logD (~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 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 R5 substituents and the 3-pyridyl analogues 11 and


16


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12 were identified as moderately potent activators of α1β1γ1-AMPK (Table 3). Compounds 11 and 12 demonstrated promising Papp and logD within our target range; however, methoxypyridine analogue 11 was some 15-fold less potent than the previously published methoxyphenyl analogue (1200 nM vs. 80 nM)19 and showed significant turnover in human hepatocytes. The more electron-rich dimethylaminopyridine analogue 12 provided moderate improvements in both parameters. Inspection of the co-crystal structure of compound 1 bound to the ADaM site of α1β1γ1-AMPK indicated that the binding pocket around the R5 aryl substituent is mostly hydrophobic, providing a rationale for the preferred binding of phenyl rings. We sought to improve the potency of these pyridyl analogues through the addition of further substituents. Introduction of a 2'-methoxy substituent had minimal impact on activation potency in the case of compound 13, although its affinity was moderately improved. In contrast, addition of the 2'-methoxy substituent to compound 12 yielded the highly potent analogue 14. The differential effect may be partially explained by the impact of the methoxy group on the pKa of the aminopyridine, which is also reflected in the significant increase in logD for 14 as compared to 12. Compound 14 retained excellent RRCK Papp and had reduced CLint relative to compound 1, despite its relatively high logD. Furthermore, it did not show preferential uptake in the hOAT3 transfected cell line, suggesting that it is not a substrate for that transporter. The co-crystal structure of compound 14 in the allosteric activation site of α1β1γ1-AMPK has been reported previously.33 The structure (Figure 3) shows a similar binding mode to compound 1 at the ADaM site between the α- and β-subunits. The chloroindole acid core makes identical interactions, including electrostatic contacts with lysine 29 of the α-subunit, a hydrogen bond to aspartic acid 88 (also from the α-subunit) and a cation-π interaction with arginine 83 from the βsubunit, and the ligands overlap well. The pyridyl ring is twisted ~60° relative to the plane of the


17

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indole, with the more polar edge oriented ‘up’ toward the β-subunit. The methoxy group is oriented as expected,50 shielding the polarity of the pyridyl ring.

This substituent is

accommodated by a slight adjustment in the position of the carbohydrate binding module motif of the β-subunit proximal to asparagine 111, and fills a small pocket at the top of the binding site tunnel.

Figure 3. A 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. In addition to the evaluation of structure-metabolism relationships, selected indole-3carboxylic acid analogues were examined as substrates for OAT-mediated uptake in HEK293 cells, as described above. From the limited set of compounds studied in these assays, a general trend was observed. Compounds with more overtly polar expressions (such as hydrogen bond donor alcohol OH or the presumably protonated morpholine ring) in the 4-substituent of the R5aryl ring showed increased normalized uptake in the hOAT3 assay, while those with less polar groups (such as an ether or a pyridine nitrogen) showed reduced uptake. The minimal hOAT3mediated uptake observed for compounds 6 and 10 as compared to compounds 1 and 7 indicates


18


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that this effect is not simply determined by logD, nor can it be explained fully by differential passive permeability. These data suggest that an H-bond donor or a weakly basic group (the protonated form of which may function similarly) may be a structural determinant of OAT3 substrate activity in this series. As with compound 1, none of the compounds tested showed normalized uptake ratio in the hOAT1 assay greater than the 2.0-fold substrate threshold. The hypothesis that active excretion by OATs is responsible for the observed renal clearance of compound 1 is supported by the observation that each of the compounds assigned as an OAT3 substrate in vitro demonstrated unbound renal clearance significantly higher than GFR in rats (Table 4). Compounds 10 and 14, which derive their polarity from the indole core modification and a heterocyclic motif at R5, respectively, demonstrated a significant attenuation in the in vitro uptake assays.

The reduced in vitro transporter-mediated uptake was consistent with their

minimal (10) or lack (14) of renal excretion following iv administration in rats.


19

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Table 4. Assessment of CLrenal in rat and OAT substrate properties of selected AMPK activators Total CLrenal b

Unbound CLrenal c

(mL/min/kg)

(mL/min/kg)

Unbound CLrenal/GFR ratiod

22.6

7.64

849

98

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

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

14

778

89

hOAT1 1.95 ± 0.2 hOAT3 7.06 ± 0.2

7

12.0

6.30

1.35

398

46

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

B.L.Q.

0.05

N.A.

N.A.

N.A.

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

%Dose in urinea

(mL/min/kg)

1

15.9

3

Cmpd

Total CLp

Normalized OAT Fold Uptakee

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 in the individual animal species. c Unbound CLrenal in blood was estimated by dividing total CLrenal with 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. eOAT uptake of test compounds (1 µM) was evaluated in triplicate in HEK293 cells transfected with human OAT1/OAT3 or rat Oat3. B.L.Q. below lower limit of quantification (1 ng/mL). N.A. not applicable. The pharmacokinetics of 10 and 14 were studied in rats and non-human primates after iv and oral administration (Table 5). For the purposes of comparison, the PK properties of 119 in rats and monkeys are also provided in Table 5. Relative to compound 1, 10 and 14 demonstrated significantly lower CLp in rats (10: 1.15 mL/min/kg; 14: CLp = 0.05 mL/min/kg) and monkeys (10: 0.38 mL/min/kg; 14: 0.98 mL/min/kg) and improvements in oral F in both species. Gratifyingly, both compounds were well absorbed following administration at the 3 mg/kg dose, despite low to moderate thermodynamic solubility (pH 7.4: 10, 14 µM; 14, 120 µM).51


20

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Examination of the metabolic fate of 10 and 14 in human and rat hepatocytes revealed that the principal route of biotransformation also involved glucuronidation of their respective carboxylic acid groups (similar to metabolism of 1). Considering the similarity in logD values for 1, 10 and 14, we speculate that the increased metabolic stability (↓ CLint) of 10 and 14 in human hepatocytes (relative to 1) results from a slower rate of metabolism caused by a diminished affinity for the UGT binding site due to unfavorable active site interactions of the 4-fluoro or 5aryl substituents rather than a decrease in lipophilicity. Table 5. Preclinical PK assessment of compounds 10 and 14 and comparison with compound 1.

Cmpd Speciesb Route 1

a

Rat

iv

CLp

Vdss

(mL/min/kg) (L/kg)

22.6

0.85

t1/2

Cmax

tmax

AUC(0–24) Oral F

(h)

(ng/mL)

(h)

(ng.h/mL)

1.06

po NHP

iv

2.33

Rat

iv

0.31

NHP

iv

0.3

14

Rat

iv

0.1

NHP

iv po

0.46

59.4

12800

1.6 26

91600

2.2 85

254000 10200

0.98

2.0

27.2

po

2690

20.6

35900 8180

0.05

2.7

12

po

14.7

16400 1890

0.38

1.2

4.95

po

329

5.5

9.14

160000

B.L.Q. 52

15500 2540

a

1.5

31200

% Dose in urine 15.9

1970 733

1.15

0.33

8.54

po 10

744 110

8.57

(%)

100%; with residual mineral oil). 1H NMR (400 MHz, MeOH-d4)


25

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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)-1,3,2-dioxaborinane.

Solid

KOAc (2.20 g, 22.4 mmol, 3.0 equiv) was added to a solution of 2-(4-bromophenyl)tetrahydro2H-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%).

1

H 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, 8.34mmol, 1.5 equiv) was added to a solution of 5-bromo-4,6-difluoro-1Hindole (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).


26


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Step 5: 4,6-Difluoro-5-(4-(tetrahydro-2H-pyran-2-yl)phenyl)-1H-indole-3-carbaldehyde.

A

mixture of 5,5-dimethyl-2-(4-(tetrahydro-2H-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)-1H-indole-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-(tetrahydro2H-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.

1

H NMR (400 MHz, DMSO-d6) δ 12.15 (br s, 1H), 12.01 (br s, 1H), 8.07 (d,


27

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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 250×4.6 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% e.e. 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% e.e. 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): calc. for C20H18F2NO3 (M+H)+: 358.1249; found: 358.1244. Analytical (%) calc.: C, 67.22; H, 4.80; N, 3.92; found: C, 66.86; H, 4.51; N, 3.84.

13

C 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-dimethyl-1,3,2-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


28

Page 29 of 43


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).

1

H 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 N-

bromosuccinimide (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.

5-Bromo-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).


29

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Step

4:

Methyl

Page 30 of 43

6-chloro-5-(6-(dimethylamino)-2-methoxypyridin-3-yl)-1H-indole-3-

carboxylate. A mixture of methyl 6-chloro-5-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-1H-indole3-carboxylate (9.25 g, 28.8 mmol, 1.5 equiv), 5-bromo-6-methoxy-N,N-dimethylpyridin-2-amine (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, 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 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 2,2dimethylpropane-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 (2N, 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 (1N, ~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


30

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(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): calc. for C17H16ClN3O3 (M+H)+: 346.0953; found: 346.0953. Analytical (%) calc.: 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).

13

C 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.

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; e.e.: 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


31

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Page 32 of 43

resonance energy transfer; UACR: urine albumin creatinine ratio; UGT: Uridine 5'-diphosphoglucuronosyltransferase.

Acknowledgements 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.

Author Information Corresponding Authors *For D.J.E.: e-mail, [email protected]. *For D.W.K.: e-mail, [email protected]. Note: The authors declare the following competing financial interest(s): All authors were employed by Pfizer, Inc. at the time this work was done.

Associated Content Supporting Information Additional experimental and characterization data for compound synthesis. Molecular formula strings. Experimental details for the SPR assay, the in vitro OAT uptake assays, the in vivo PK


32


studies, and the in vivo ZSF-1 rat study.

Page 33 of 43

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. 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.

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Hasenour, C. M.; Ridley, D. E.; Hughey, C. C.; James, F. D.; Donahue, E. P.; Shearer, J.;

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Optimization of Metabolic and Renal Clearance in ... - ACS Publications

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