Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1

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Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy Kimberly O. Cameron, Daniel W. Kung, Amit S. Kalgutkar, Ravi G. Kurumbail, Russell Miller, Christopher T. Salatto, Jessica Ward, Jane M. Withka, Samit K. Bhattacharya, Markus Boehm, Kris A Borzilleri, Janice A. Brown, Matthew Calabrese, Nicole L. Caspers, Emily Cokorinos, Edward L. Conn, Matthew S. Dowling, David J. Edmonds, Heather Eng, Dilinie P. Fernando, Richard Frisbie, David Hepworth, James Landro, Yuxia Mao, Francis Rajamohan, Allan R. Reyes, Colin R. Rose, Tim Ryder, Andre Shavnya, Aaron C. Smith, Meihua Tu, Angela C. Wolford, and Jun Xiao J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00866 • Publication Date (Web): 04 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Journal of Medicinal Chemistry

Eng, Heather; Pfizer, Pharmacokinetics, Dynamics and Metabolism Fernando, Dilinie; Pfizer Inc, Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry Frisbie, Richard; Pfizer Global Research and Development, Hepworth, David; Pfizer Inc., Worldwide Medicinal Chemistry Landro, James; Vascumab, LLC Mao, Yuxia; Pfizer Global Research and Development, Pharmaceutical Sciences Rajamohan, Francis; Pfizer Inc, Worldwide Medicinal Chemistry Reyes, Allan; Pfizer Global Research and Development, Cardiovascular, Metabolic and Endocrine Diseases Research Unit Rose, Colin; Pfizer Global Research and Development, Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry Ryder, Tim; Pfizer Global Research and Development, Pharmacokinetics, Dynamics and Metabolism Shavnya, Andre; Pfizer, Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry Smith, Aaron; Pfizer Inc., Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry Tu, Meihua; Pfizer Global Research & Development, Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry Wolford, Angela; Pfizer Global Research and Development, Pharmacokinetics, Dynamics and Metabolism Xiao, Jun; Pfizer Inc., Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry

ACS Paragon Plus Environment


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Discovery

and

Preclinical

Characterization

Page 2 of 48

of

6-Chloro-5-[4-(1-

hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy Kimberly O. Cameron,*,† Daniel W. Kung,*,§ Amit S. Kalgutkar,∞ Ravi G. Kurumbail,ǂ Russell Miller,‡ Christopher T. Salatto,‡ Jessica Ward,‡ Jane M. Withka,ǂ Samit K. Bhattacharya,† Markus Boehm,† Kris A. Borzilleri,ǂ Janice A. Brown,# Matthew Calabrese,ǂ Nicole L Caspers,ǂ Emily Cokorinos,‡ Edward L. Conn,§ Matthew S. Dowling,§ David J. Edmonds,† Heather Eng,# Dilinie P. Fernando,§ Richard Frisbie,# David Hepworth,† James Landro,⊥ Yuxia Mao,∇ Francis Rajamohan,ǂ Allan R. Reyes,‡ Colin R. Rose,§ Tim Ryder,# Andre Shavnya,§ Aaron C. Smith,§ Meihua Tu,† Angela C. Wolford,# Jun Xiao§ †

Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry, ‡Cardiovascular,

Metabolic and Endocrine Diseases Research Unit, and ∞Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research & Development, Cambridge, Massachusetts 02139, United States §

Cardiovascular, Metabolic and Endocrine Diseases Medicinal Chemistry, ⊥Cardiovascular,

Metabolic and Endocrine Diseases Research Unit, ǂWorldwide Medicinal Chemistry, #

Pharmacokinetics, Dynamics and Metabolism, ∇Pharmaceutical Sciences, Pfizer Worldwide

Research & Development, Groton, Connecticut 06340, United States ABSTRACT


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Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. Based on human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound.

Optimization of the core and aryl appendage

improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

INTRODUCTION With the growing global incidence of diabetes and chronic over-nutrition there are increasing numbers of individuals living with metabolic dysregulation known to associate with impaired renal function termed Diabetic Nephropathy (DN). DN is already the leading cause of end stage kidney disease and type II diabetes confers a 6-fold greater risk in the development of chronic kidney disease.1 As the incidence of diabetes rises (projected to rise from 415 million to 642 million adults worldwide by 2040) so will DN, along with a significant health and economic burden for society.2 Although a number of compounds are currently in clinical trials, there are no drugs on the market specifically developed for the treatment of DN. Current therapeutics, including anti-diabetics for glycemic control and angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers to manage hypertension, are beneficial but do not prevent disease progression to end stage renal disease which requires dialysis or transplant.3 Novel mechanisms to improve kidney function and delay the progression of disease in the setting of diabetes are therefore needed.



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DN is a complex disorder that manifests in the impairment of both glomerular filtration and tubular function. While the precise etiology of DN is unclear due to close interaction between these kidney physiological processes, it is clear that hyperglycemia, hyperinsulinemia, and hyperlipidemia associated with diabetes can impair both components of kidney function. In the tubule, hyperglycemia and hyperlipidemia can cause altered mitochondrial metabolism and oxidative stress in addition to increased sodium reabsorption, resulting in engagement of tubular glomerular feedback and glomerular hyperfiltration.

Tubular glomerular feedback induced

hyperfiltration combines with cell autonomous impacts of elevated nutrient and growth factors to cause podocyte and glomerular hypertrophy, resulting in the loss of efficient filtration as evidenced by considerable protein eluent in the urine (albuminuria).

Over time these

impairments in kidney function result in significant inflammation and fibrosis causing declining glomerular filtration rate (GFR).4 In an effort to identify novel regulators of the progression of human kidney disease that could serve as therapeutic drug targets, we examined association studies of common genetic variants with parameters of kidney function.

One locus consistently identified in genome-wide

association studies of kidney function was located around the PRKAG2 gene which encodes the γ2 subunit of AMPK. Single nucleotide polymorphisms in the PRKAG2 locus have reproducible and significant associations with traits of renal function including incidence of chronic kidney disease (p=4.2e-12) and GFR (estimated from serum creatinine, p=5e-11)5 and suggestive associations with urine albumin creatinine ratio (UACR, p=8e-6) and microalbuminuria (p=2e5).6, 7 AMPK is a heterotrimeric serine/threonine protein kinase comprised of a catalytic α-subunit in complex with regulatory β- and γ-subunits that plays a key role in maintaining cellular energy


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homeostasis. As AMP or ADP levels rise during energetic stress, they bind to the regulatory γsubunit of AMPK to modulate its activity. Binding of AMP to the gamma subunit of AMPK promotes phosphorylation of a key threonine (Thr172) on its activation loop by upstream kinases. This activating phosphorylation of Thr172 has been shown to increase the kinase activity of AMPK by 500-1000-fold.8 The protein conformational changes induced by the binding of these two nucleotides also serve to protect Thr172 from dephosphorylation by protein phosphatases, thus increasing the proportion of activated AMPK in vivo.

In addition, the

binding of AMP also allosterically activates AMPK heterotrimers causing an additional enhancement in its catalytic activity by 2-3 fold. Activation of AMPK results in increased phosphorylation of a number of substrate proteins, including Acetyl-CoA Carboxylase, HMGCoA Reductase, Tuberus Sclerosis 2, and Peroxisome Proliferator Activated Receptor-γCoactivator 1α, which control key regulatory steps in lipid, cholesterol, protein, and mitochondrial biosynthesis, respectively.9

Seven subunits (α1, α2, β1, β2, γ1, γ2, γ3) can

theoretically combine to create twelve AMPK isoforms which have differential tissue distribution and function.10,

11

Among the two known β subunits, β1 appears to be the

predominant subunit in kidney as suggested by mRNA levels.12 AMPK exhibits reduced activity in diseases of caloric excess, suggesting dysregulation of cellular metabolism and energetics and the possibility that decreased AMPK activity may play a causal role in disease etiology. Reduced AMPK phospho-Thr172 levels (pAMPK) have been reported in the glomeruli of diabetic subjects as compared to healthy individuals.13 This effect has been duplicated at the level of the whole kidney in multiple rodent models of DN. Chronic treatment with pharmacological activators of AMPK, such as 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin (vide infra, Figure 1), restore pAMPK levels and improve



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kidney function in rodent models of DN.14-17

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These studies have characterized numerous

potential downstream mechanisms leading to improved kidney function of AMPK activation, suggesting the potential for modulation of multiple pathways within different cell types in the kidney that impact lipid metabolism, mTOR (mechanistic target of rapamycin) signaling, and oxidative stress. Many of the biological effects attributed to AMPK in the literature are based on studies using AMPK activators with both AMPK-dependent and AMPK-independent actions, such as metformin, which activates AMPK indirectly by increasing the levels of intracellular AMP,18 or AICAR

which

is

converted

within

cells

to

5-aminoimidazole-4-carboxamide-1-β-D-

ribofuranosyl 5'-monophosphate (ZMP), an AMP mimetic (Figure 1).19,

20

The first small

molecule direct AMPK activator, A-769662, was disclosed by Abbott Laboratories21 and is now known to bind to an allosteric site between the α- and β-subunits of AMPK.22,

23

A recently

disclosed benzimidazole series from Merck and Metabasis, exemplified by compound 991 (Figure 1)24,

25

has also been shown to bind to this allosteric pocket.22 We herein report our

discovery of indazole- and indole acid-based AMPK activators,26,

27

optimization to clinical

candidate 7, and preclinical data that support its potential as a DN therapeutic.


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Figure 1. Direct and indirect AMPK activators

IN VITRO PHARMACOLOGY Known AMPK activators, including AMP and A-769662, have been shown to have dual effects: allosteric activation of AMPK as well as protection of Thr172 of the α-subunit from dephosphorylation.21, 28, 29 In order to identify compounds with different pharmacological modes of action, we developed a novel time-resolved fluorescence resonance energy transfer (TRFRET) activation/protection assay that allowed identification of both allosteric activators and protectors of the phosphorylated protein. In this assay, recombinantly expressed and purified AMPK, fully phosphorylated at Thr172, was incubated with test compounds and with protein



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phosphatase 2a (PP2a) under conditions that had been determined to decrease the basal activity of AMPK by about 50% by dephosphorylation (thus assessing the test compound’s ability to provide “protection” from dephosphorylation).

After quenching the PP2a activity with the

known PP2a inhibitor okadaic acid, ATP and SAMS peptide substrate (derived from ACC1) were added to initiate the kinase reaction (thereby assessing the test compound’s ability to induce “activation” of AMPK). Phosphorylation of the SAMS peptide substrate was monitored using a TR-FRET antibody detection method as described in the Experimental Section. All data were normalized relative to AMP as a 100% control and an increase in signal in this assay relative to the DMSO control indicated that a compound allosterically activated the enzyme, protected Thr172 from dephosphorylation, or both. Initial screening was performed with the human α1β1γ1 AMPK isoform, but the same assay format was also used to assess species and isoform selectivity (rat α1β1γ1, human α1β2γ1, human α2β1γ1, human α2β2γ1, and human α2β2γ3). To account for assay interference including fluorescent artifacts, a similar orthogonal assay was developed using

33

P-ATP, monitoring incorporation of

33

P into the SAMS peptide substrate

using a filter assay. The filter assay format was also used for steady-state enzyme kinetic experiments to determine Km and Vmax parameters for enzyme activation. Additional assays were employed to characterize separately the activation and protection properties of selected test compounds. The TR-FRET activation-only assay was analogous to the TR-FRET activation-protection assay, but without PP2a and okadaic acid. Compounds were incubated with fully phosphorylated AMPK and, following addition of ATP and SAMS peptide, kinase activity was measured using the TR-FRET antibody detection method. The DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay) protection assay was used to assess ‘protection’ afforded by compounds. Fully phosphorylated, and biotionylated AMPK was


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incubated with test compounds and with PP2a to dephosphorylate AMPK. After quenching the PP2a reaction with okadaic acid, the biotin-tagged AMPK protein was captured on a streptavidin coated plate and the amount of phosphorylated AMPK remaining was measured via immunodetection using DELFIA technology from Perkin Elmer. To confirm a direct interaction of compounds with target AMPK isoforms, an in vitro surface plasmon resonance (SPR) binding assay was developed.17

Biotinylated human recombinant AMPK was captured onto a

streptavidin sensor chip and analytes were injected over the surface. Correlation of binding affinities (Kd), and kinetic parameters (kon, koff) where measurable, with the functional response observed in biochemical assays provided additional confidence in the observed SAR.

CHEMISTRY The reported compounds were synthesized by the routes shown in Schemes 1, 2, and 3. Indazoles 2 and 3 were prepared by Suzuki coupling to the commercially available 5-bromoindazoles A (Scheme 1). Amidation of the carboxylate under standard conditions afforded the amide product 1.

Scheme 1. General synthetic route to indazoles (1, 2, and 3).



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Conditions: (a) 4-OMe-PhB(OH)2, PdCl2-dppf, K2CO3, H2O, dioxane or EtOH-toluene, (b) HATU, iPr2NEt, amine, DMF.

The 6-chloro-indole derivatives 4, 5, 6, and 7 were synthesized from 5-bromo-6-chloro-indole B as shown in Scheme 2. Suzuki coupling of boronic acids or boronate esters to the bromide appended the 5-aryl groups to the indole core. The 3-carboxy group was introduced by a Vilsmeier-Haack reaction followed by sodium chlorite oxidation of the resulting aldehyde, or by acylation with trichloroacetyl chloride followed by methanolysis and hydrolysis. The Suzuki reaction at the indole 5-position and the carboxylation at the indole 3-position could be run in either order, as shown in Scheme 2 (B→C, or B→D→E).

Scheme 2. General synthetic route to indole acids (4, 5, 6, and 7).


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Conditions: (a) ArB(OH)2 or ArB(OR)2, PdCl2-dppf, K2CO3, H2O, EtOH-toluene-(THF) or 2MeTHF, (b) ClCHNMe2Cl (POCl3-DMF), CH3CN or DMF; then NaOH, H2O, (c) NaClO2, 2methyl-2-butene, NaH2PO4, H2O-CH3CN-tBuOH, (d) Cl3CCOCl, pyridine, DMAP; NaOMe, MeOH, (e) NaOH, H2O-MeOH.

The aryl boronic acid or boronate derivatives were commercially available, except for the cyclobutyl derivative H required for compound 7.

Scheme 3 shows the synthesis of

hydroxycyclobutylboronate H; selective metal-halogen exchange of 1-bromo-4-iodo-benzene and addition to cyclobutanone afforded the hydroxycyclobutylphenylbromide, which was converted to the corresponding boronate ester for use in the subsequent Suzuki coupling.

Scheme 3. Synthesis of hydroxycyclobutyl boronate ester H.



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I

a

b

HO

HO B

Br

Br

F

G

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O

O H

Conditions: (a) n-BuLi, THF; cyclobutanone, (b) bis(neopentylglycolato)diboron, PdCl2-dppf, KOAc, dioxane.

RESULTS AND DISCUSSION The initial hit (indazole amide 1) was identified in a screen of our corporate file using the TRFRET activation/protection assay, described above. Compound 1 was a weak activator and protector of β1-containing isoforms, with activation reaching 158% at 40 µM (the highest concentration tested), or approximately 1.5-fold higher than the native ligand AMP for the human α1β1γ1 isoform. Activity at β2 containing isoforms was minimal (Figure 2). Although amide 1 demonstrated good metabolic stability in human liver microsomes, the low in vitro passive permeability as measured in Ralph Russ Canine Kidney (RRCK) cells30 and low aqueous apparent solubility31 predicted poor oral absorption as a likely liability (Figure 2). Our lead optimization objectives were therefore to increase potency and to modify physicochemical properties for good oral absorption and cellular penetration.

OH O

O NH N N H 1


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Molecular weight / LogD (pH 7.4)32 387 / 4.0 Apparent solubility (pH 6.5, µM) 4 HLM CLint,app (µL/min/mg) 100 µM) of the microsomal activities of major human cytochrome P450 isoforms. Finally, 7 was devoid of mutagenic responses in the Salmonella Ames and in vitro micronucleus assays in the absence or presence of CYP activation (ariclorinduced rat liver S9 fraction/NADPH).37


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Table 1. In vitro data for indole and indazole acidsa

Compound

A, R1

(C.I.)

2

3

4

N.A.

N, H

N, Cl

C, Cl

N.A.

Clearance

α1β1γγ1

ability

Solubility

(Human/Rat

(SPR)

(RRCK)

µM

HEP) CLint

Kd nMc

Papp

µL min-1106

10-6 cm/s

cellse

α1β1γγ1 (TR-FRET)

EC50 nM

AMP38

Apparent

Binding

R2 b

LogDd

Perme-

Activation-Protection

%AMP (C.I.)

b

1519

103

3700

(1289-1789)

(98.2-109)

± 200

3391

149

9948

(2700-4259)

(136-162)

± 1083

323

258

1776

(245-427)

(242-274)

± 708

80

259

90.2

N.T.

N.T.

432

N.T.

0.1

5.3

164

9.7 /N.T.

0.9

6.4

376

27 / 27

2.1

18

87

40 / 55



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C, Cl

5

C, Cl

6

C, Cl

7

a

(59.5-108)

(246-273)

± 1.8

1.3

225

22.7

(0.8-2.1)

(214-235)

± 0.7

53

232

149.8

(38.7-72.7)

(219-246)

± 3.5

7.0

226

9.0

(5.0-9.8)

(215-236)

± 0.1

N.T. = not tested; N.A. = not applicable.

intervals (C.I.).

c

b

Page 18 of 48

3.2

2.4

42

40 / N.T.

1.2

3.5

494

70 / >170

2.0

5.8

406

14 / 27

Mean α1β1γ1 EC50 data reported as the mean of at least 4 replicates and 90% confidence

Kd data reported as geometric mean of at least 3 replicates ± standard error of the mean (SEM).

measured at pH 7.4 using the previously described shake-flask method.32

e

d

logD was

CLint refers to total intrinsic clearance obtained from

scaling in vitro half-lives in cryopreserved rat or human hepatocytes (HEP) as previously described.39


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Table 2. Summary of in vitro pharmacology and ADME data for 7

PF-06409577 (7) MW 342, LogD (pH 7.4) 2.0, tPSA 73, pKa = 4.7 α1β1γ1 Kd nM ± SEM a

9.0 ± 0.1

kon (M-1sec-1) / koff (sec-1)

9.4 x 105 / 8.4 x 10-3

α1β1γ1 TR-FRET EC50 nM (C.I.), %AMP (C.I.) b

7.0 (5.0-9.8), 226 (215-236)

α1β1γ1 Filter EC50 nM (C.I.), %AMP (C.I.) b

8.2 (5.0-13.3), 213 (200-226)

α1β1γ1 Activation only EC50 nM (C.I.), %AMP (C.I.) b

3.3 (2.2-5.0), 172 (169-175)

α1β1γ1 Protection only EC50 nM (C.I.), %AMP (C.I.) b

28.2 (14.6-54.4), 102 (92.6-112)

α2β1γ1 TR-FRET EC50 nM (C.I.), %AMP (C.I.) b

6.8 (4.5-10.4), 152 (144-160)

Rat α1β1γ1 TR-FRET EC50 nM (C.I.), %AMP (C.I.) b

7.0 (5.0-9.8), 226 (215-236)

α1β2γ1/α2β2γ1/α2β2γ3 TR-FRET EC50 nM b

> 40,000

CEREP panel (10 µM)

5-HT2b, 76% inhibition c

PDE Panel (1-11) µM

PDE3A IC50 = 14

Human hepatocytes CLint,app (µL/min/million cells)

14

Thermodynamic solubility (pH 6.5) mg/mL

0.325

RRCK Papp [AB] (cm/s)

5.8 x 10-6

fu,p (human/monkey/dog/rat) d

0.017/0.032/0.028/0.0044

a

Kd data reported as geometric mean of at least 5 replicates ± standard error of the mean (SEM).

b

Mean EC50 data reported as the mean of at least 3 replicates and 90% confidence intervals



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(C.I.). c No responses of >50% observed with the exception of 5-HT2b. Follow up studies demonstrated that this activity did not relate to 5-HT2b agonist activity; EC50 > 30,000 nM in a functional cell based assay.

d

Fraction unbound in plasma (fu,p) from preclinical species and

human was determined from equilibrium dialysis method and is a mean value from 3 individual determinations.40

Figure 4. TR-FRET data (90% confidence intervals) for 7 as compared to AMP

Crystal structure of 7 bound to AMPK A crystal structure of 7 bound to the α1β1γ1 isoform of AMPK at ~3.4 Å resolution was obtained (Figure 5). As described previously, AMPK adopts an extended conformation in which the catalytic kinase domain of the α-subunit is sandwiched between the nucleotide-binding γsubunit and the regulatory carbohydrate-binding module (CBM) of the β-subunit.22, 23 Despite modest resolution, clear electron density is present at the interface between the α- and β-subunits permitting modeling of the ligand 7 (Figure 5d). This pocket has recently been termed the ‘ADaM site’ (Allosteric Drug and Metabolite), alluding to the hypothesis that activity may be modulated by an as yet unidentified endogenous ligand.41 The carboxylic acid group of 7 is solvent exposed and is poised to engage lysine 29 from the α-subunit (Figure 5b). Lysine 29,


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together with lysine 31, simultaneously coordinates the phosphomimetic aspartic acid residue that was engineered at position 108 of the β-subunit to aid with protein crystallization. The indole nitrogen donates a hydrogen bond to aspartic acid 88 of the α-subunit at the back of the pocket while the indole core participates in a stacked cation-pi interaction with the side-chain of arginine 83 of the β-subunit. Finally, the orientation of the hydroxyl group of the cyclobutanol appeared most consistent with a pose previously observed for the phenolic hydroxyl of A769772 (Figure 5c). This orientation allows donation of a hydrogen-bond to the backbone carbonyl of glycine 19 as well as acceptance of a hydrogen-bond from the sidechain of lysine 31, both from the α-subunit. Consistent with the observed SAR, the chlorine atom at C6 of the indole binds in a buried lipophilic pocket. A similar trend in SAR was observed with A-769662, where addition of a chlorine atom to the core (2-chloro-4-hydroxy-3-(2'-hydroxy-[1,1'-biphenyl]4-yl)-6-oxo-6,7-dihydrothieno[2,3-b]pyridine-5-carbonitrile (Cl-A-769662)) contributed ~7-fold increase in potency.23 The phenyl ring at C5 of the indole was mostly surrounded by lipophilic groups from the protein. In addition, the phenyl group is anchored in a suitable geometry to form additional cation-pi interaction with lysine 31 from the α-subunit. Comparison of ligands previously crystallized with AMPK reveals some intriguing observations (Figure 5c). A-769662, compound 991, and 7 all function through binding at the ADaM site22, 23 and, despite possessing different bicyclic core rings, the bound conformation of the three scaffolds superimpose well. The conservation of the hydrogen bond between the core of each ligand and aspartic acid 88 is a notable feature shared by all the three ligands. In addition, though all ligands contain an acidic (or acidic hydroxyl) functionality, the slight variation in the location of this group appears tolerated due to the flexibility of lysines 29, 31, and 51. The



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tailpieces of these four ligands show the highest level of divergence consistent with this end of the ligand binding near the solvent front.

Figure 5. Structure of AMPK bound to 7. (a) Overall topology of AMPK. Position of bound 7 relative to the ATP site and activation loop is indicated. 2D ligand representation shown in inset. (b) Zoom-in of allosteric binding site. A subset of coordinating amino acids are shown as sticks and labelled. (c) Comparison of the bound conformations of 7, Compound 991, and A-769662 within the AMPK allosteric site. Protein is omitted for clarity. (d) Electron density map (2fofc) contoured at 1 σ over 7.


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Table 3. Preclinical pharmacokineticsa of 7

Dose Species

CLp

Route

Cmax Vdss (L/kg)

(mg/kg)

t1/2 (h)

(mL/min/kg)

b

AUC(0-24)

Oral

(ng.h/mL)

F (%)

Tmax (h) (ng/mL)

1.0 Rat

i.v.

22.6 ± 3.05

0.85 ± 0.54

1.06 ± 1.06

N.A.

N.A.

744 ± 103

N.A.

N.A.

N.A.

110 ± 71.9

0.33 ± 0.14

329 ± 57.0

14.7

N.A.

N.A.

N.A.

1900 ± 464

0.33 ± 0.14

1880 ± 258

25.3

(n=3) 3.0 p.o. (n=3) p.o.

10 (n=3)

10500 ± p.o.

30 (n=3)

N.A.

N.A.

N.A.

4020 ± 1860

0.50 ± 0.00

47.0 1910

Dog

0.5

12.9

3.15

6.26

(n=2)

(11.9,13.8)

(3.23,3.06)

(7.64,4.88)

N.A.

N.A.

N.A.

631

i.v.

N.A.

(668,593)

2.0 p.o.

1300

(n=2)

N.A.

0.25

4020 100

(1250,1340)

(0.25,0.25)

(4440,3590)

N.A.

N.A.

1970 ± 577

1.0 Monkey

i.v.

8.57 ± 2.33

2.33 ± 0.99

8.54 ± 2.98

(n=4)



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3.0 p.o.

N.A.

N.A.

N.A.

733 ± 237

1.2 ± 0.45

2690 ± 443

59.4

(n=5) a

All experiments involving animals were conducted in our AAALAC-accredited facilities and were reviewed and approved by Pfizer

Institutional Animal Care and Use Committee. Pharmacokinetic parameters were calculated from plasma concentration–time data and are reported as mean (± S.D. for n=3 or greater and average values for n=2 with individual values provided). Intravenous (i.v.) and oral (p.o.) pharmacokinetics were conducted in male gender of each species (Wistar Han rats, beagle dogs, and/or cynomolgus monkeys). Solution formulation for i.v. pharmacokinetics included 12% sulfobutylether-β-cyclodextrin solution (rats and monkeys) or 10% N-methyl-2-pyrrolidone: 90% of 30% sulfobutylether-β-cyclodextrin solution in water (dogs). Oral pharmacokinetics were conducted using crystalline material as a 0.5% methyl cellulose suspension. N.A. = not applicable.


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Preclinical Disposition of 7 Compound 7 exhibited high plasma protein binding in rat (plasma unbound fraction, fu,p = 0.0044), dog (fu,p = 0.028), monkey (fu,p = 0.032), and human (fu,p = 0.017). No metabolic turnover was detected in standard assays with NADPH-supplemented liver microsomes from rat, dog, monkey, and human, and cryopreserved hepatocytes from dog and monkey. In rat and human hepatocytes, apparent intrinsic clearance (CLint,app) values of 27 and 14 µL/min/106 cells were determined. Metabolite identification studies in hepatocytes from preclinical species and human revealed that 7 is biotransformed to a single acyl glucuronide conjugate; there was no evidence of cytochrome P450 (CYP)-mediated oxidative metabolism of 7 in either liver microsomal or hepatocyte incubations. The in vivo pharmacokinetics of 7 were examined in rats, dogs, and monkeys after intravenous (i.v.) and oral (p.o.) administration (Table 3). Following i.v. administration, indole acid 7 demonstrated moderate plasma clearance (CLp) in rats (22.6 mL/min/kg), dogs (12.9 mL/min/kg), and monkeys (8.57 mL/min/kg), and was well distributed with steady state distribution volumes (Vdss) ranging from 0.846–3.15 L/kg. Renal excretion of unchanged 7 was also examined in animals following i.v. administration. The percentage of dose recovered in urine over 24 h as unchanged 7 was 15.9% (rat), 11.8% (dog), and 20.6% (monkey). Biliary excretion of unchanged 7 was minimal (~3.0% of administered dose) in bile duct cannulated rats upon i.v. administration of 7. Following oral administration of crystalline 7 in 0.5% methylcellulose suspension, 7 was rapidly absorbed (Tmax = 0.25–1.20 h) in rats, dogs, and monkeys.

The corresponding oral

bioavailability (F) values in rats, dogs, and monkeys, were 15%, 100%, and 59%, respectively. Oral F of 7 administered in 0.5% methylcellulose to rats increased in a dose-dependent fashion


24


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(15% (3 mg/kg) → 25% (10 mg/kg) → 47% (30 mg/kg)). Compound 7 was considerably more unstable in UDPGA-supplemented intestinal microsomes from rat (t1/2 = 1.41 min; CLint,app = 492 µL/min/mg) relative to monkey (t1/2 = 35.6 min; CLint,app = 19.5 µL/min/mg) and human (t1/2 = 41.6 min; CLint,app = 18.5 µL/min/mg). These in vitro observations suggested that 7 was subject to a higher degree of first pass intestinal glucuronidation in the rat (relative to other preclinical species and human). Saturation of intestinal glucuronidation in the rat at higher doses resulted in increased oral F. On the basis of its potency and in vivo PK attributes, 7 was progressed to in vivo studies to build confidence in the ability of an AMPK activator to improve function in the diabetic kidney.42 Target engagement in an acute setting was measured by in vivo phosphorylation status of AMPK in kidney tissue after administraton of increasing doses of 7 in normal Wistar Han rats. Dose responsive increases in pAMPK relative to total AMPK (tAMPK) in whole kidney tissue were observed with a maximal 3.8-fold response at 300 mg/kg (Figure 6). A ZSF-1 rat model43 was used to determine if compound 7 would positively impact renal function. The ZSF-1 obese rat develops progressive renal disease as a consequence of metabolic syndrome and diabetes and studies have recapitulated endpoints to those observed in the clinic for two different mechanisms of action.44 ZSF-1 obese rats at 8 weeks of age were treated with a single daily dose of 7 and urine was collected every 2 weeks. The ACE inhibitor, ramipril, was included as a positive control and vehicle-treated ZSF-1 lean rats were included as a model-specific negative control. Figure 7 shows increasing loss of albumin in urine with disease progression over the 6 week study as compared to lean rats; treatment with either 7 or ramipril resulted in drastic decreases in urinary albumin levels. Consistent with the acute studies, significant increases in pAMPK were also observed in kidney tissue (see Supporting Information). In this study, average plasma


25

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concentrations (0 to 24 h) of compound 7 normalized for plasma protein binding revealed adequate systemic exposure of ~10 fold over the α1β1γ1 Kd (free Cavg = 49 nM). These data supported the hypothesis that direct β1-selective AMPK activators have the potential to provide therapeutic benefit for patients with diabetic nephropathy.45

Figure 6. pAMPK/tAMPK after administration of 7 in Wistar Han rats, single dose; pAMPK and tAMPK were measured by quantitative ELISA using recombinant heterotrimeric protein standards. The ratio of pAMPK and tAMPK was calculated and normalized to the vehicle group. **= p < 0.01; ***= p

Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1

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