Acyl Glucuronide Metabolites of 6-Chloro-5 - ACS Publications

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Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-activated Protein Kinase (AMPK) Tim Ryder, Matthew F. Calabrese, Gregory S Walker, Kimberly O. Cameron, Allan R. Reyes, Kris A. Borzilleri, Jake Delmore, Russell A. Miller, Ravi G. Kurumbail, Jessica Ward, Daniel W. Kung, Janice A. Brown, David J. Edmonds, Heather Eng, Angela C. Wolford, and Amit S. Kalgutkar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00807 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 23, 2018

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Journal of Medicinal Chemistry

Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]1H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphateactivated Protein Kinase (AMPK) Tim F. Ryder,§ Matthew Calabrese,§ Gregory S. Walker,§ Kimberly O. Cameron,† Allan R. Reyes,‡ Kris A. Borzilleri,§ Jake Delmore,‡ Russell Miller,‡ Ravi G. Kurumbail,§ Jessica Ward,‡ Daniel W. Kung,§ Janice A. Brown,§ David J. Edmonds,† Heather Eng,§ Angela C. Wolford,§ and Amit S. Kalgutkar*,† †

Medicine Design and ‡Internal Medicine Research Unit, Pfizer Worldwide Research &

Development, Cambridge, Massachusetts 02139, United States §

Medicine Design, Pfizer Worldwide Research & Development, Groton, Connecticut 06340,

United States

ACS Paragon Plus Environment

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ABSTRACT Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1β1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2) and PF-06679142 (3) as potential clinical candidates. Compounds 1–3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1–3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of β1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Co-crystallization of the AMPK α1β1γ1 isoform with 1–3 and M1–M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.


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INTRODUCTION

Acyl glucuronidation represents a key component in the metabolic clearance of numerous carboxylic acid-containing drugs.1-4 The conjugation reaction is catalyzed by a family of microsomal uridine 5’-diphospho-glucuronosyltransferase (UGT) enzymes in the presence of a co-factor uridine 5’-diphosphoglucuronic acid (UDPGA), which also serves as the donor of the β-D-glucuronic acid residue.5-7 Acyl glucuronides are considerably polar (relative to the parent carboxylic acid precursors) due to the hydrophilic nature of the glucuronic acid moiety and are entirely ionized at physiological pH (7.4), possessing pKa values between 3 and 4.8 Consequently, acyl glucuronide transport between the site of formation (e.g., small intestine and liver) and blood, bile or other tissues is generally restricted due to their inability to passively diffuse across membrane barriers.9 Hepatobiliary and renal transport proteins, therefore, play a crucial role in the elimination of acyl glucuronides via biliary or urinary excretion routes.1,9-13 Because of these aforementioned physicochemical attributes, the process of acyl glucuronidation is generally considered to render the parent carboxylic acid into a pharmacologically inactive compound. Consistent with this notion, scenarios where glucuronide conjugates (including acyl glucuronides) retain primary pharmacology associated with their parent precursors are extremely rare, which is in contrast to the identification of active metabolites generated in the course of oxidative metabolism by cytochrome P450 (CYP) enzymes.14

Frequently discussed cases of pharmacologically active glucuronides include

morphine-6-O-glucuronide and the phenolic glucuronide conjugate of ezetimibe, which retain µopioid receptor agonism and inhibition of intestinal cholesterol absorption noted with the parent compounds morphine and ezetimibe, respectively.14-17

Likewise, the acyl glucuronide

metabolites of the anticoagulant dabigatran,18 the immunosuppresant mycophenolic acid,19 and


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the adenosine A1 receptor antagonist tonapofylline20 appear to be the only known examples where acyl glucuronide conjugates have demonstrated primary pharmacology associated with the parent predecessor drugs. Recently, we disclosed structure-activity relationship studies on a series of indazole- and indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphateactivated protein kinase (AMPK), which culminated in the identification of 6-chloro-5-[4-(1hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577, 1, Figure 1) as a single digit nanomolar activator of the human α1β1γ1 AMPK isoform.21 AMPK is a heterotrimeric serine/threonine kinase that is made of a catalytic α subunit in complex with two regulatory subunits, β and γ. Seven subunits (α1, α2, β1, β2, γ1, γ2, and γ3) can theoretically assemble to generate as many as 12 possible AMPK complexes, which have differential tissue distribution and function.22 Binding of AMP to the γ 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.23 Significant reduction in AMPK activity has been reported in the glomeruli of diabetic subjects as compared to healthy individuals,24 and chronic treatment with AMPK activators such as metformin and 5-aminoimidazole-4-carboxamide ribonucleotide has been shown to restore AMPK activity and improve kidney function in rodent models of diabetic nephropathy.25,26 Among the two known β subunits, β1 appears to be the predominant subunit in kidney as suggested by mRNA levels.27,28 Compound 1 has been advanced into first-in-human clinical trials as a clinical candidate for the treatment of diabetic nephropathy on the basis of its selectivity towards activation of the β1 AMPK isoform, favorable preclinical pharmacokinetics, pharmacodynamics, and safety. As


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outlined in our previous publication,21 1 is resistant to CYP-catalyzed oxidation(s) in NADPHsupplemented liver microsomes from preclinical species (e.g., rat, dog, and monkey) and human. Subsequent studies in UDPGA-supplemented liver microsomes and cryopreserved hepatocytes from preclinical species and human, however, revealed that 1 was metabolized to a single monoglucuronide metabolite by an UGT isoform(s). The present article describes our efforts in the biochemical synthesis, purification, and structural characterization of the glucuronide conjugate of 1 and related analogs PF-06885249 (2) and PF-06679142 (3) (Figure 1) from the indole-3carboxylic acid series of AMPK activators, which have been recently described as potential back-up agents with extended half-lives arising from a net reduction in glucuronidation rates and renal excretion, relative to 1.29 The biotransformation profile of 2 and 3 in human hepatocytes demonstrated that the predominant route of metabolism also involved glucuronidation (similar to the metabolic fate of 1 in human hepatocytes). Considering the similarities in log D values for 1, 2, and 3, we reasoned that the increased resistance of 2 and 3 towards glucuronidation occurred from a reduction in metabolic rate caused by a diminished affinity for the UGT binding site due to unfavorable active site interactions of the 5-aryl (compound 2) or 4-fluoro (compound 3) substituents rather than a straightforward decrease in lipophilicity.29 During the course of this investigation, available analytical (mass and NMR spectra) data and chemical derivatization studies suggested that the glucuronide conjugates obtained upon incubation of the parent compounds 1–3 in UDPGA-supplemented human liver microsomes were acyl glucuronides. In vitro pharmacological evaluation revealed that the acyl glucuronide metabolites were selective activators of the human β1-containing AMPK isoforms, which was consistent with their ability to inhibit de novo lipogenesis in primary human hepatocytes. Solution of the co-crystal structures of the human α1β1γ1 AMPK isoform with the parent


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carboxylic acids and their corresponding acyl glucuronide metabolites provided insights into the structural basis for AMPK activation by these phase II conjugation products.

HO

O

O

R Cl

F N H F

PF-06409577 (1): R = HO

O

OH

N H

PF-06679142 (3)

PF-06685249 (2): R =

N

N

O

Figure 1. Structures of indole-3-carboxylic acid-based α1β1γ1 AMPK activators. RESULTS AND DISCUSSION Metabolites of Indole-3-carboxylic Acid-based AMPK Activators in Human Hepatocytes Figure 2 depicts a full-scan HPLC-UV chromatogram of reaction mixtures of compounds 1, 2, and 3 (10 µM each) in cryopreserved human hepatocytes incubated for 4 h at 37 °C. Table 1 indicates the retention time (tR), exact mass (MH+), and key diagnostic fragment ions for compounds 1–3 and their metabolites. A single metabolite M1, M2, and M3 was observed when compounds 1, 2, or 3 were incubated in human hepatocytes, respectively. The exact protonated mass (MH+) of M1, M2, and M3 was 176 Da higher than the exact masses of the corresponding carboxylic acid precursors, implying that these metabolites were generated via monoglucuronidation of the parent compounds by a human UGT enzyme(s).

No oxidative

metabolites derived from CYP-mediated catalysis were observed in the human hepatocyte incubations with compounds 1–3. Structural assignments for relevant fragment ions in the collision-induced dissociation spectra of compounds 1–3 and their corresponding metabolites


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M1–M3 are presented in the supporting information (Figures 1–3 in supporting information). Theoretical exact masses of the proposed fragment ion structures in the mass spectra of 1–3 and their metabolites were consistent with the observed accurate masses (< 2 ppm difference) (see Supporting Information).

M1

3.34

(A)

(MH+ = 518.1219)

3.68

200000

7.72

1 (MH+ = 342.0889)

3.85

8.96

0 2

4

6

8

10

12

M2 (MH+ = 522.1268)

7.96

(B) 100000

2 (MH+ = 346.0949)

uAU

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


9.32

50000

3.13 6.70

0 2

3

4

5

6

7

8

9

10

11

12

3

M3

(MH+ = 534.1570) (MH+ = 358.1251)

(C)

100000

9.72

8.37

50000 0 4

5

6

7

8

9

10

11

12

Time (min)

Figure 2. HPLC-UV chromatograms of reaction mixtures of 1 (panel A), 2 (panel B), and 3 (panel C) in cryopreserved human hepatocytes incubated at 37 °C for 4 h. The final concentration of 1–3 in the hepatocyte incubations was 10 µM. Earlier eluting peaks (tR ~ 3.0 – 4.0 min) were also observed in hepatocyte incubations at t = 0 h and are background noise.


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Table 1. Observed mass spectral characteristics of compounds 1, 2, and 3 and their corresponding glucuronide conjugates in cyropreserved human hepatocytes Compound

MH+

1

342.0889

Retention time (tR), min 8.96

Structure

Fragment Ions

HO

HO

Cl

O

324.781, 306.0679, 296.0471, 282.0312, 208.0158

N H

2

346.0949

9.32

328.0850, 296.1031, 278.0925, 251.0815

3

358.1250

9.72

340.1144, 314.1352, 296.1245, 242.0776, 230.0775

M1

518.1219

7.09

500.1105, 482.0997, 464.0891, 324.0784, 306.0677


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M2

522.1268

7.96

346.0952, 328.0847

M3

534.1570

8.37

340.1144


9


Indole-3-carboxylic acid 1 possesses two functional groups (tertiary alcohol and carboxylic acid) capable of undergoing glucuronidation. Hydroxylamine, which selectively transamidates acyl glucuronide metabolites and is inert towards reaction with glucuronide conjugates of alcohols,30 was used as a chemical derivatization reagent to establish the site of glucuronidation in 1. Overnight treatment of the human hepatocyte extract of 1 with hydroxylamine at ambient temperature led to a near quantitative conversion of M1 (tR = 7.10 min, MH+ = 518.1212) to the corresponding hydroxamic acid derivative (4, tR = 7.25 min, MH+ = 357.1000) (Figure 3) suggesting that M1 was a acyl glucuronide metabolite. The mass spectrum of 4 is provided in Figure 4 in the Supporting Information section. On the basis of the available mass spectral and chemical derivatization studies, M1, M2, and M3 were assigned as the corresponding acyl glucuronide conjugates of compounds 1, 2, and 3, respectively. M1 7.10

(A) 0

1

-50000

uAU

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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4

(B)

7.25

0

1

-50000 2

4

6

8 Time (min)

10

12

14

Figure 3. HPLC-UV chromatograms (t = 0 h, panel A and t = 24 h, panel B) of human hepatocytes extract of 1 treated with hydroxylamine at room temperature.


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Biosynthesis of the Glucuronide Metabolites Glucuronide conjugates M1, M2, and M3 were isolated and purified by semipreparative HPLC fraction collection of UDPGA-supplemented human liver microsomal incubations of 1, 2, and 3, respectively, as outlined in the experimental section. The 1H NMR spectrum of purified M1 in DMSO-d6 showed all of the expected 1-phenylcyclobutanol and chloroindole resonances (Figure 4). In addition, proton resonances (δ 5.61, 3.60, 3.37, 3.31 and 3.24 ppm), which are consistent with the addition of a β-D-glucuronic acid residue to the carboxylic acid group in 1 were also present in the 1H NMR spectrum of M1. The position of attachment of the glucuronide residue was further established with heteronuclear multiple-bond correlation spectroscopy (HMBC) data, which provided a correlation from the anomeric 1H resonance at δ 5.61 to a carbon with a chemical shift of δ 163.1 (Figure 5). Such a correlation is only possible if the M1 isolate is an acyl glucuronide.

Correlation spectroscopy (COSY) and heteronuclear single-quantum

correlation spectroscopy (HSQC) data also indicated these 1H resonances are consistent with the addition of a glucuronic acid moiety to 1 (Figures 5 and 6 in Supporting Information). The 1H NMR spectra of the M2 isolate depicted the anticipated aromatic and aliphatic resonances of 2 and also contained proton resonances consistent with the addition of a β-Dglucuronic acid moiety to 2 (δ 5.59, 3.79 and 3.43–3.26 ppm) (Figure 6). COSY and HSQC NMR spectroscopy data also revealed that the 1H resonances are consistent with the addition of a glucuronide residue to 2 (Figures 7 and 8 in the Supporting Information). The 1H NMR spectra of the M3 isolate depicted all of the aromatic and aliphatic resonances of 3 in addition to resonances (δ 5.62, 3.69, 3.42–3.14 ppm) consistent with the addition of a βD-glucuronic

acid residue to 3 (Figure 7). The 1H spectra of the isolated material also depicted

resonances that were attributed to rotameric forms of M3. The position of attachment of the


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glucuronide motif was established on the basis of the HMBC data, which indicated a correlation from the anomeric 1H resonance at δ 5.62 ppm to a carbon with a unique chemical shift of δ 161.8 ppm (Figure 8). Furthermore, COSY and HSQC 1H NMR data are also consistent with the addition of a glucuronide motif to parent carboxylic acid 3 (Figures 9 and 10 in Supporting Information). OH

HO

O d

c

e

HO

21 22

OH 24 23

b

OH

O a

15

O

14

20

O

11 10

16

19

9

13

4

17

a

3

e b, c, d

8

18

2 7

N H

5

Cl 6

12

1

2 21 22

OH 24 23

9

6

15,17 14,18

15

HO

14

20

19

9

13

3

4

17

cyclobutyl

O

11 10

16

8

18

2 7

1

5

Cl 12

6

N H 1

Figure 4. 1H NMR spectra of 1 and biochemically synthesized glucuronide M1.


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Figure 5. 1H-13C HMBC spectra of biosynthesized M1. OH

HO

O c d

CH 3

CH 3

24

H 3C

HO

21

N 14 O

N

22

16

23

15

Cl

1

23, 24

21

N

O

N 14 O 16

15

20

19 10

9 17

13 18

1

A

CH3

24

22

a

2

N H

5

CH 3

23

11

3

6

12

H 3C

O

8 7

B

OH

a

19 10

4

13 18

O

O

20 9

17

b

b, c, and d e

Cl 7 12

4

OH

6

11 3

8

21

9 2

5 6

N H

2

18

17

1

Figure 6. 1H NMR spectra of 2 (panel A) and biochemically synthesized M2 (panel B).


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Figure 7. 1H NMR spectra of 3 (A) and biosynthesized M3 (B).

Figure 8. 1H-13C HMBC spectra of biosynthesized M3.


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Determination of the concentrations of M1, M2, and M3 in DMSO-d6. Because of the universal response of hydrogens in an NMR experiment, 1H NMR is an excellent quantitative tool for assessing the concentrations of isolates of small molecules for which there is no synthetic standard available.31-33 The response of an NMR system to a nanomole of a specific hydrogen in a given molecule will be the same as the response to a nanomole of a specific hydrogen in a calibrating compound such as maleic acid when the experimental parameters are identical.34,35

As a consequence, isolated metabolites can be quantitated using calibrating

solutions of organic chemicals that are unrelated to the isolated material. In the present situation, quantitation of the purified isolates in DMSO-d6 (used for NMR characterization) was performed by external calibration against the 1H NMR spectrum of 10 mM maleic acid standard using the electronic reference to access in vivo concentrations 2 function within Topspin as outlined in previous publications from our laboratory.35-37 The final concentrations of the biosynthesized isolates of M1, M2, and M3 in DMSO-d6 were determined to be 2.43 mM, 0.52 mM, and 14.3 mM, respectively, by quantitative NMR spectroscopy. Chemically synthesized38 acyl glucuronide derivatives of 1, 2, and 3 possessed identical tR on HPLC-UV (M1: Supporting Information Figure 11; M2: Supporting Information Figure 13; M3: Supporting Information Figure 15), mass (M1: Supporting Information Figure 12; M2: Supporting Information Figure 14; M3: Supporting Information Figure 16) and

1

H NMR (M1:

Supporting Information Figure 6; M2: Supporting Information Figure 8; M3: Supporting Information Figure 10) spectral characteristics as the biosynthesized metabolites M1, M2, and M3, respectively, which confirmed that M1, M2, and M3 were acyl glucuronides. Details on the methodology utilized to synthesize acyl glucuronides M1, M2, and M3 will be the topic of an independent publication.


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Stability of M1–M3 in Buffer Certain immune-mediated drug toxicities associated with carboxylic acid-based drugs have been linked to the formation of acyl glucuronide metabolites.39 Under physiological conditions, acyl glucuronides can covalently modify proteins via a simple transacylation reaction or through an acyl migration within the β-O-glucuronide unit to a reactive α-hydroxy-aldehyde species.1,40,41 Condensation between the α-hydroxy-aldehyde motif and a lysine residue or an amine group of the N terminus in proteins leads to the formation of a glycated conjugate. A recent Food and Drug Administration guidance on this topic suggests that simple O-glucuronides, O-sulfates, and/or quaternary N+-glucuronides may be considered benign from a human safety perspective, However qualification of circulating acyl glucuronide metabolites in animals and humans is needed because of the aforementioned reactivity concerns.42 Consequently, we examined the degradation kinetics of the biochemically isolated M1 (the principal metabolite of our clinical candidate PF-06409577 (1) in animals and human) in deuteriated phosphate buffer (pH ~ 7.4) at 37 °C using a previously published NMR method based on the disappearance of the anomeric resonance of the β-O-1-acyl glucuronide motif in structurally diverse acyl glucuronide conjugates.43 The analysis was also repeated with acyl glucuronides M2 and M3. Repeated 1H NMR analysis (approximately every 25 min over 21 h) monitoring for the integrated peak area of the anomeric protons (δ ~ 5.7–5.8 ppm) revealed that M1, M2, and M3 isolated from the biochemical incubations was inert towards hydrolysis or acyl migration (t1/2 > 21 h) (Figure 17 in Supporting Information).

Overall, these observations are consistent with structure-toxicity

correlations between covalent binding to liver microsomes and/or serum albumin and acyl glucuronide migration rates. Acyl glucuronides derived from aromatic carboxylic acids (such as 1) and carboxylic acids with a higher degree of alkyl substitution at the α-carbon exhibit lower


16

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reactivity with protein nucleophiles suggesting that inherent electronic and steric effects likely modulate the overall rate of acyl glucuronide rearrangement.43-45 In Vitro Pharmacology A previously described21 time-resolved fluorescence resonance energy transfer (TR-FRET) activation/protection assay was used to assess AMPK activation by 1–3 and their corresponding acyl glucuronide metabolites M1–M3. Incorporating a protein phosphatase 2a (PP2a) treatment prior to initiation of the kinase reaction allowed the identification of both allosteric activators and protectors from dephosphorylation of Thr172. The output from this analysis was normalized relative to AMP as a 100% control and an increase in signal in this assay relative to the vehicle control indicated that a compound allosterically activated the enzyme, protected Thr172 from dephosphorylation, or both. Initial pharmacological evaluation was performed with the human α1β1γ1 AMPK isoform that is predominantly expressed in the kidney,27,28 and the same assay format was also used to assess isoform selectivity (human α1β2γ1, human α2β1γ1, human α2β2γ1, and human α2β2γ3). Consistent with our previous reports,21,29 parent carboxylic acids 1–3 potently activate human AMPK isoforms that contained the β1 subunit (including α1β1γ1) (Table 2 and Figure 9). Under these experimental conditions, acyl glucuronide standards of M1, M2 and M3 also selectively activated human β1 isoforms, albeit with 9-19-fold loss in potency (Table 2). Parent carboxylic acids and the acyl glucuronide conjugates possessed little to no activity against β2-containing isoforms of human AMPK (see Table 2).

Our previously

described in vitro surface plasmon resonance (SPR) binding assay21,46 was also utilized to confirm a direct interaction of the test compounds with the AMPK α1β1γ1 isoform. Biotinylated human recombinant AMPK α1β1γ1 isoform was captured onto a streptavidin sensor chip and analytes were injected over the surface. Correlation of binding affinities (Kd), and kinetic


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parameters (kon, koff) with the functional response observed in biochemical assays provided additional confidence in the observed structure-activity relationships (Table 3).

Figure 9. TR-FRET data (90% confidence intervals) for α1β1γ1 AMPK activation by 1 and M1 (A), 2 and M2 (B), and 3 and M3 (C).


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Table 2. In vitro data for indole-3-carboxylic acids 1–3 and corresponding acyl glucuronide metabolites M1–M3

Compound

AMPK Isoform

Activation-Protection

EC50 nM (C.I.)a 1

M1

(TR-FRET)

Permeability (RRCK)c Papp x 10-6 cm/s

2.0

5.8

-0.14

0.6

%AMP (C.I.)a

1.9

208

(1.1-3.3)

(196-219)

α2β1γ1

11.3 (7.6-16.9)

153 (145-161)

α1β2γ1

>40000

N.D.

α2β2γ1

>40000

N.D.

α2β2γ3

>40000

N.D.

α1β1γ1

38.1

195

(28-52)

(185-204)

α2β1γ1

275 (189-401)

140 (129-152)

α1β2γ1

>40000

N.D.

α2β2γ1

>40000

N.D.

α2β2γ3

>40000

N.D.

α1β1γ1

Log Db


19


2

M2

3

7.0

206

(4.0-12)

(192-219)

α2β1γ1

27 (19-38)

152 (144-160)

α1β2γ1

>40000

N.D.

α2β2γ1

>40000

N.D.

α2β2γ3

>40000

N.D.

α1β1γ1

76.0

193

(55.3-104)

(182-203)

α2β1γ1

353 (255-491)

154 (143-165)

α1β2γ1

>40000

N.D.

α2β2γ1

>40000

N.D.

α2β2γ3

>40000

N.D.

α1β1γ1

8.0

179

(5.0-12.2)

(168-189)

α2β1γ1

6.0 (4.5-8.1)

145 (140-150)

α1β2γ1

>40000

N.D.

α2β2γ1

>40000

N.D.

α2β2γ3

>40000

N.D.

α1β1γ1

Page 20 of 54

2.3

12

-0.05

0.3

1.8

14


20

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M3

59

193

(41-85)

(181-204)

α2β1γ1

332 (239-461)

147 (137-157)

α1β2γ1

>40000

N.D.

α2β2γ1

>40000

N.D.

α2β2γ3

>40000

N.D.

α1β1γ1

0.08

0.3

a

Mean EC50 data reported as the mean of at least 3 replicates and 90% confidence intervals (C.I.). %AMP is the amount of activation relative to the AMP control at maximum concentration of the test compound. b logD was measured at pH 7.4 using the previously described shake-flask method.48 c Passive absorptive permeability (Papp) was measured in Ralph Russ Canine Kidney (RRCK) cells as previously described.47 N.D. Not determined.


21


Table 3.

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In vitro SPR data for direct binding of indole-3-carboxylic acids 1–3 and

corresponding acyl glucuronide metabolites M1–M3 to human AMPK α1β1γ1 isoform Dissociation

kon x 105 ± SE

koff x 10-3 ± SE

Binding KD

t1/2 (s)

(M-1s-1)

(s-1)

± SE (nM)a

1

82.11 ± 0.16

9.42 ± 0.09

8.44 ± 0.06

8.96 ± 0.11

M1

19.80 ± 0.13

3.10 ± 0.04

35.0 ± 0.2

112.9 ± 1.6

2

103.74 ± 2.10

4.92 ± 0.09

6.68 ± 0.1

13.6 ± 0.4

M2

30.13 ± 0.25

1.10 ± 0.02

23.0 ± 0.2

209.1 ± 3.5

3

67.94 ± 0.64

7.08 ± 0.09

10.2 ± 0.1

14.4 ± 0.2

M3

15.30 ± 0.06

2.29 ± 0.02

45.3 ± 0.2

197.8 ± 3.5

Compound

a

Dissociation t1/2 = ln(2)/koff; kinetic parameters (kon, koff) were obtained by a global fit using at least three concentrations and at least three replicates. Kd was determined by the standard equation, Kd = koff/kon ; SE = Standard Error.

AMPK Co-crystal Structures with Activators We had previously described the crystal structure of 1 and 2 bound to the α1β1γ1 isoform of AMPK at ~ 3.4 and 3.5 Å, respectively.21,29,49 AMPK adopts an elongated conformation in which the regulatory carbohydrate-binding module motif of the β-subunit sits atop the catalytic kinase domain of the α-subunit.50,51 The interface between these two domains generates a novel site termed the ‘Allosteric Drug and Metabolite (ADaM)’ site and it is within this binding pocket that indole-3-acid activator 1 binds (Figure 10, panel A). The carboxylate moiety in 1 forms an ion-pair with Lys29 while the indole NH donates a hydrogen bond to Asp88 of the kinase module. The chlorine and the phenyl substituents form lipophilic interactions with both nonpolar amino acids from both the α and β subunits. In addition, the indole core forms a pi-cation interaction with the side chain guanidinium group of Arg83.


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In order to better understand the binding of the acyl glucuronide metabolites, we determined the crystal structure of the α1β1γ1 isoform with the biochemical isolate of M1 in DMSO-d6 (Figure 10, panel B). Despite glucuronidation of the C-3 carboxyl acid moiety, M1 retains binding at the ADaM site with a binding pose that is remarkably similar to the parent indole acid. The glucuronic acid moiety projects toward solvent and is easily accommodated within the exit tunnel of the α/β interface. The acidic moiety in the acyl glucuronide metabolite can potentially adopt multiple poses – a suggestion that we cannot rule out with certainty based on electron density, however, a pose with the acid directed ‘up’ toward the β-subunit gave the best fit to electron density and was chosen as the modeled conformation (Figure 18 in Supporting Information). Regardless of binding pose, the ion-pair interaction with Lys29 of the α-subunit is lost with M1, which may be the potential cause for the loss in potency of the aglycone derivative. However the glucuronide does remain in the vicinity of polar residues contributed by both α and β subunits, including Arg83 of β, which could partially compensate for this loss. In addition, we also solved the crystals structures of AMPK with the acyl glucuronide metabolites M2 and M3 (isolated and purified from human liver microsomes) and observed similar results (Figure 10, panel C). Several key interactions between the indole NH and protein, including a hydrogen bond to α-Asp88, are preserved while the glucuronide motif in all three compounds occupies a similar space at the border of the binding site.


23


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Figure 10. Crystal structures of AMPK α1β1γ1 with acyl glucuronide conjugates M1-M3. A) Overall topology of AMPK α1β1γ1 illustrating compound 1 bound to the ADaM site. Structure derived from 5KQ5.pdb.21 B) Overlay of bound pose of compounds 1 and M1 (top) together with a surface representation of the ADaM site from the M1 structure (gray surface). C) Overlay of bound poses of M1, M2, and M3 along with electron density over ligand from each crystal structure (2fo-fc map contoured at 1 σ).

Activation of AMPK Activity in Human Hepatocytes.

AMPK activation in primary

hepatocytes leads to an acute increase in phosphorylation of Ser79 in Acetyl-CoA Carboxylase (ACC), and a concomitant decrease in de novo production of fatty acids.52 We elected to examine activation of cellular AMPK by representative parent carboxylic acid derivatives 1 and 2 and their respective acyl glucuronide metabolites M1 and M2 by treating primary human


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hepatocytes with the test compounds for 1 h and measuring the phosphorylation of the AMPK substrate ACC and the rate of [14C]-acetate incorporation into lipid species. Parent carboxylic acids 1 and 2 and their corresponding acyl glucuronide derivatives M1 and M2 caused a dose dependent increase in the phosphorylation status of ACC at Ser79, with an EC50 of 0.11 µM (compound 1) and 0.28 µM (compound 2), 3.27 µM (M1), and 2.11 µM (M2), respectively (Figure 11). Additionally, Parent carboxylic acids 1 and 2 as well as the corresponding acyl glucuronide derivatives M1 and M2 demonstrated a marked reduction in [14C]-acetate incorporation (Figure 19 in Supporting Information), with apparent IC50 values of 0.40 µM (compound 1) and 0.42 µM (compound 2), 11 µM (M1), and 25 µM (M2), respectively. The estimated IC50 for inhibition of de novo lipogenesis and ACC phosphorylation with 1 is consistent with the value obtained in our recent study examining ACC phosphorylation and suppression of lipid synthesis in primary hepatocytes from animals and human.53 The apparent right-shift in cellular IC50 values (relative to the values measured with the recombinant human β1 kinase) for AMPK activation by the parent carboxylic acids and their respective acyl glucuronide conjugates possibly reflects their non-specific binding to hepatocytes/media. Furthermore, in the case of the acyl glucuronide derivatives M1 and M2, it is likely that their impaired passive permeability (Papp) relative to the parent carboxylic acid derivatives as measured in Ralph Russ Canine Kidney (RRCK) cells47 (see Table 1) further impedes access to AMPK expressed intracellularly in the hepatocyte. Determination of intracellular unbound concentrations of the test compounds in this assay could potentially shed additional light on this discrepancy. As such, it is noteworthy to point out that incubation of the individual acyl glucuronide metabolites M1 and M2 (at a concentration of 10 µM each) with primary human hepatocytes revealed complete


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resistance (t1/2 > 400 min, intrinsic clearance (CLint) < 3.0 µL/min/106cells) towards metabolism (including potential hydrolytic cleavage to the parent compounds 1 and 2, respectively).

Figure 11: Increase in Ser79 phosphorylation of the specific AMPK substrate ACC in primary human hepatocytes following treatment with compounds 1, 2, M1, and M2.

Intravenous Pharmacokinetics of M1 in Animals The preclinical disposition of the acyl glucuronide metabolite M1 was further investigated as part of the regulatory package supporting human safety and pharmacokinetics studies with 1. Like the parent compound 1,21 M1 exhibited high plasma protein binding in rat (plasma unbound fraction, fu,p = 0.0044 (1), 0.0018 (M1)), monkey (fu,p = 0.032 (1), 0.028 (M1)), and human (fu,p = 0.017 (1), 0.019 (M1)). The intravenous pharmacokinetics of M1 (and the previously reported values for the parent compound 1) in rats and monkeys are shown in Table 4. Following intravenous administration, M1 demonstrated low plasma clearance (CLp) in rats (0.99


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mL/min/kg), and monkeys (9.85 mL/min/kg), and was well distributed with steady state distribution volumes (Vdss) ranging from 0.84–4.05 L/kg. Relative to 1, the CLp values of M1 were considerably lower in rats and similar in monkeys. The percentage of dose recovered in urine over 24 h as unchanged M1 was ~ 1.0 % (rat), and 9.8% (monkey), which is considerably lower than the values noted with 1 (rats, 15.9% and monkeys, 20.6%). The in vivo conversion of M1 to 1 was also studied by monitoring the plasma samples for the formation of 1. Comparison of the unbound area under the plasma–time concentration (AUC) curve for M1 and 1 revealed M1/1 ratios of 0.035 and 0.22, in rats and monkeys, respectively, suggestive of low potential for the enterohepatic recirculation54,55 of 1 potentially via β-glucuronidase mediated hydrolysis of M1 in the small intestine of preclinical species. In rat, monkey, and human hepatocytes, M1 was resistant towards metabolic turnover (t1/2 > 240 min, CLint < 6 µl/min/106cells).

Finally,

qualitative HPLC-UV examination of pooled (0–7 h) rat bile revealed substantial quantities of M1 following i.v. administration of 1 to bile-duct cannulated rats (data not shown), implying that the acyl glucuronide derivative generated through the glucuronidation of 1 is predominantly eliminated via biliary excretion possibly mediated by the efflux transport proteins (e.g., multidrug resistance-associated protein 2 and breast cancer resistant protein1,56,57).


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Table 4. Intravenous pharmacokineticsa of 1 and M1 in rats and monkeys

Compound

Dose

Species

Analyte

(mg/kg)

CLp

AUC(0-24)

(mL/min/kg)

(ng.h/mL)

Vdss (L/kg)

t1/2 (h)

Unbound metabolite/parent AUC ratio

1

1.0

Rat

1

22.6 ± 3.05

744 ± 103

0.85 ± 0.54

1.06 ± 1.06

Rat

M1

0.99

8850

0.4

6.96

(0.75,1.24)

(11000,6700)

(0.27,0.52)

(7.11,6.81)

(n=3) M1

0.5 (n=2)

101

1

1

1.0

0.035

Monkey

1

8.57 ± 2.33

1970 ± 577

2.33 ± 0.99

8.54 ± 2.98

Monkey

M1

9.85

1750

4.52

18

(7.09,12.6)

(2240,1260)

(3.13,5.91)

(17.3,18.7)

(n=4) M1

1.0 (n=2)

1

0.22

303 (257,349)

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


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pharmacokinetics were conducted in male gender of each species (Wistar Han rats and cynomolgus monkeys). Solution formulation for the intravenous dose included 10% DMSO/30% PEG400/60% water.


29


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CONCLUSION Taking into account the hurdles associated with the synthesis of acyl glucuronide metabolites,58,59 we initially utilized UDPGA-supplemented human liver microsomes to rapidly biosynthesize acyl glucuronide conjugates (M1–M3) of AMPK activators 1–3. Determination of the concentrations of the purified acyl glucuronide conjugate stock solutions in DMSO-d6 by quantitative NMR spectroscopy allowed preliminary evaluation of their potential to activate human AMPK α1β1γ1 in recombinant kinase assays and human hepatocytes.

The acyl

glucuronide stock solutions were also utilized in the solution of the corresponding AMPK:acyl glucuronide co-crystal structures, which provided insights into the structural basis for AMPK activation by the polar aglycones relative to their parent carboxylic acid precursors. The potential for synergistic contribution of the acyl glucuronides towards activation of kidney AMPK in rodents (and potentially humans) administered with the parent carboxylic acid precursors21,29 remains unclear at the present time. The lower passive permeability of the acyl glucuronides (relative to the parent precursors) in the RRCK assay would suggest that the conjugates (relative to the parent carboxylic acid precursors) will demonstrate minimal activation of AMPK in rodent models of diabetic nephropathy. However, as noted in the literature,1,9-13 active transport of the acyl glucuronide conjugates generated in the small intestine and/or liver by intestinal, hepatic (e.g., organic anion transporting polypeptides 1A2, 1B1 and 1B3, multidrug resistant protein 3 and 4) and renal (e.g., organic anion transporters 1 and 3) transporters across cell membranes will ultimately govern their disposition and their potential to activate of AMPK in the site of action.

As demonstrated in our pharmacokinetic studies on M1 in rats and

monkeys, pharmacological evaluation of the acyl glucuronide conjugates M1–M3 in rodent models of diabetic nephropathy is likely to be compromised to some degree given their


30

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propensity to undergo β-glucuronidase-mediated hydrolysis to the parent carboxylic acid precursors. In addition, the potential for translatability of animal pharmacology data to humans will be confounded due to the marked species differences in the expression of β-glucuronidases (e.g., rat >> human).60 Finally, considering the physicochemical properties of acyl glucuronide conjugates, it is noteworthy to point out that assessment of in vivo animal pharmacology would only be possible via intravenous administration of the preformed acyl glucuronide standard, which presents the risk of producing misleading data compared to the “real world” situation where conjugation of the parent carboxylic acid occurs in the gut wall and/or liver and the glucuronide conjugate relies upon active transport proteins for passage across tissues.61,62 As such, the complexities surrounding in vivo pharmacological assessments with glucuronide conjugates is further exemplified by the fact that it has taken over three decades to provide concrete evidence that the the analgesic action of morphine in humans is mediated by the morphine-6-glucuronide conjugate, formed in vivo.63–66 Apart from the prerequisite regulatory MIST (Metabolites in Safety Testing) guidance on safety assessment (reactivity) of acyl glucuronide metabolites,61,67 our present case study with the indole-3-carboxylic acid-based AMPK activators suggests that additional diligence may be warranted towards studying on-target pharmacology of acyl glucuronide metabolites that are derived from carboxylic acid-based drug candidates. This is of particular importance when acyl glucuronidation constitutes the primary clearance mechanism and/or in cases (e.g., dabigatran) where the pharmacological target resides in the extracellular matrix and does not require cell penetration by an acyl glucuronide conjugate. Finally, detailed characterization of the human UGT enzyme(s) responsible for the glucuronidation of 1 to M1 is currently in progress as part of studies to support the clinical


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development program of 1. These results will be reported in due course alongside the first-inhuman oral single ascending dose pharmacokinetics for the clinical candidate 1, wherein circulating concentrations of its glucuronide conjugate M1 were also measured, and found to be comparable to that of the parent compound. EXPERIMENTAL SECTION

General Experimental Methods. All experiments involving animals were conducted in our AAALAC-accredited facilities and were reviewed and approved by Pfizer Institutional Animal Care and Use Committee. Indole-3-carboxylic acid derivatives 1–3 (chemical and isomeric purity >99% by HPLC and NMR) were synthesized at Pfizer Worldwide Research and Development (Groton, CT) using previously published protocols.21,29 Cryopreserved human hepatocytes (lot RHT; pooled, n = 5 male livers and n = 5 female livers) were obtained from BioreclamationIVT (Baltimore, MD) and pooled (pool of 50 livers from male/female) human liver microsomes were purchased from BD Gentest (Woburn, MA). Alamethicin, UDPGA, MgCl2, and DMSO were purchased from Sigma Aldrich (St. Louis, MO). Solvents used for bioanalysis were of analytical or HPLC grade (Thermo Fisher Scientific, Waltham, MA). Purity of biosynthesized acyl glucuronide metabolites was assessed by reversed-phase HPLC with UV detection at λ 290 nm (see supporting information); all tested compounds demonstrated >95% purity. Metabolite Identification Studies in Human Hepatocytes. Cryopreserved human hepatocytes were thawed and suspended in Williams' E medium (ThermoFisher Scientific, Waltham, MA) supplemented with 24 mM NaHCO3 and 10% fetal bovine serum at 2 × 106 viable cells/mL. Compounds 1–3 were individually incubated (10 µM final concentration) with hepatocytes at 37 °C for 4 h with gentle agitation. A gas mixture of O2:CO2 (95:5) maintained at ∼2.5 kPa for ∼5


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s was passed through this mixture at every hour of incubation. Flasks were corked immediately after gassing.

Reactions were stopped by addition of ice-cold acetonitrile (10 mL) and

centrifuged (3000 x g, 15 min). The supernatants were dried under a steady stream of nitrogen, reconstituted with 25% aqueous acetonitrile (250 µL), and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for metabolite formation. Qualitative assessment of metabolites was conducted using a Thermo Orbitrap Elite mass spectrometer (ThermoFisher Scientific, Waltham, MA) operating in positive ion electrospray mode. The HPLC system consisted of an Accela quaternary solvent delivery pump, an Accela autoinjector, a Surveyor PDA Plus photodiode array detector. The autoinjector was programmed to inject 50 µL of reconstituted sample onto a Phenomenex Kinetex column (2.1 mm x 100 mm, 2.6 µm) using a gradient consisting of a mixture of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at a flow rate of 400 µL/min. The binary gradient was as follows: solvent A to solvent B ratio was held at 95:5 (v/v) for 0.5 min and then adjusted to 35:65 (v/v) from 0.0 to 11 min and 5:95 (v/v) from 11 to 13 min where it was held for 1.0 min and then returned to 95:5 (v/v) for 1.0 min before the next analytical run. Postcolumn flow was split such that the mobile phase was introduced into the mass spectrometer via an ion spray interface at a rate of 50 µL/min. The remaining flow was diverted to the photodiode array detector positioned in line to provide simultaneous UV detection and a total ion chromatogram. Xcalibur software version 2.2 SP1.48 was used to control the HPLC/MS system. Full scan data were collected at 15,000 resolution. Data dependent product ion scans of the two most intense ions found in the full scan were obtained at 15,000 resolution. The dynamic exclusion function was used with a 10 second exclusion duration after 3 successive product ion scans with an early exclusion if the precursor ion felt below a signal to noise ratio of 20. Initial full scans were performed between m/z 50 and


33


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1000. Parent carboxylic acid derivatives and identifiable metabolites eluted in the first 30 min. Metabolites were identified in the full-scan mode (from m/z 100 to 850) by comparing t = 0 h samples with t = 4 h human hepatocytes samples, and structural information was generated from the mass spectra of the molecular ions (MH+). Biosynthesis of Glucuronide Analogs.

Acyl glucuronides M1, M2, and M3 were

biosynthesized as follows. Individual parent carboxylic acid derivatives 1–3 (500 µM) were incubated in UDPGA-supplemented human liver microsomes (four separate 10 mL incubations). Phosphate buffer (0.1 M pH 7.4) was aliquoted into a 50 mL centrifuge tube followed by the addition of MgCl2 (3.3 mM), human liver microsomes (1 mg/mL), alamethicin (10 µg/mL), and UDPGA (2 mM). The reactions were initiated with the addition of the parent carboxylic acid derivatives and allowed to incubate in a shaking water bath for 2 h at 37 °C. Reactions were quenched by the addition of 30 mL of ice-cold acetonitrile per incubation. Samples were concentrated using an evaporative centrifuge at 37 °C then reconstituted with 2 mL of 5% acetonitrile in water (total volume for all incubation) combining incubates.

The four

reconstituted samples were combined and injected onto a semi-prep HPLC system in six separate injections (300 µL). The semi-prep HPLC consisted of a Shimadzu SiL-HTC autosampler, two LC-20AD solvent pumps, an SPD-M20A diode array detector, and a FRC-10 A fraction collector. Separation was performed on a Zorbax RX C8, 9.4 x 250 mm, 5 µM semi preparative HPLC column. For M1 and M3, the mobile phase consisted of 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 4 mL/min. The mobile phase composition commenced at 95% A/5% B, was held at that composition for 5 min, followed by a linear gradient to 35% A/65% B at 35 min. A second gradient was then executed to 5% A/95% B at 45 min, these conditions were held at this composition for 5 min, and the system was re-equilibrated


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at initial conditions for 5 min prior to the next injection. For M2, the mobile phase composition commenced at 95% A/5% B, was held at that composition for 5 min, followed by a linear gradient to 30% A/70% B at 35 min. A second gradient was then executed to 5% A/95% B at 45 min, these conditions were held at this composition for 5 min, and the system was re-equilibrated at initial conditions for 5 min prior to the next injection. From all chromatographic separations, fractions were collected at 0.5 min intervals. Aliquots (25 µL) of fractions at tR of UV peaks believed to be the acyl glucuronide metabolite and the parent carboxylic acid were verified by LC-MS/MS analysis. Fractions containing the isolated acyl glucuronides were combined and concentrated to dryness using evaporative centrifugation under nitrogen gas. Derivatization of Acyl Glucuronide Metabolite M1 with Hydroxylamine.

Aliquots of

collected fractions containing M1 were treated with hydroxylamine as previously described30 to examine the potential for in situ aminolysis of the acyl glucuronide moiety to the corresponding hydroxamic acid derivatives in the presence of hydroxylamine. The aliquots were split into two equal fractions using one as a control. A 5 µL aliquot of hydroxylamine was added to the test sample and a 5 µL aliquot of water was added to the control sample. Both samples were allowed to remain at room temperature for 1 h before re-analysis by LC-MS/MS. NMR Sample Analysis. Isolated samples were reconstituted in 0.1 mL of DMSO-d6 “100%” (Cambridge Isotope Laboratories, Andover, MA) and placed in a 1.7 mm NMR tube which had been stored in a dry argon atmosphere. The 1H and

13

C spectra were referenced using residual

DMSO-d6 (1H δ = 2.50 ppm relative to tetramethylsilane, δ = 0.00, 13C δ = 39.50 ppm relative to tetramethylsilane, δ = 0.00). NMR spectra were recorded on a Bruker Avance 600 MHz (Bruker BioSpin Corporation, Billerica, MA) controlled by Topspin V3.2 and equipped with a 1.7 mm TCI Cryo probe. 1D spectra were recorded using an approximate sweep width of 8400 Hz and a


35


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total recycle time of approximately 7 s. The resulting time-averaged free induction decays were transformed using an exponential line broadening of 1.0 Hz to enhance signal to noise. The 2D data were recorded using the standard pulse sequences provided by Bruker. At minimum a 1K x 128 data matrix was acquired using a minimum of 2 scans and 16 dummy scans with a spectral width of 10000 Hz in the f2 dimension. The 2D data sets were zero-filled to at least 1k data point. Post-acquisition data processing were performed with either Topspin V3.2 MestReNova Mnova 9.1. Quantitation of NMR isolates was performed by external calibration against the 1H NMR spectrum of an authentic 10 mM maleic acid standard compared to that of the isolated glucuronides using the electronic reference to access in vivo concentrations 2 function within Topspin V3.2 as outlined in our previous publications.36,37 (2S,3S,4S,5R,6S)-6-((6-Chloro-5-(4-(1-hydroxycyclobutyl)phenyl)-1H-indole-3carbonyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (M1). 1H NMR (600 MHz, DMSO-d6) δ 12.37 (s, 1H), 8.25 (d, J = 5.0 Hz, 3H), 7.96 (s, 1H), 7.69 (s, 1H), 7.57 (d, J = 7.8 Hz, 2H), 7.43 (d, J = 7.8 Hz, 2H), 5.61 (d, J = 8.0 Hz, 1H), 3.60 (d, J = 10.1 Hz, 1H), 3.37 (t, J = 9.5 Hz, 1H), 3.31 (t, J = 8.8 Hz, 1H), 3.24 (t, J = 8.5 Hz, 1H), 2.46 (t, J = 4.3 Hz, 1H), 2.30 (ddd, J = 6.8, 9.2, 11.9 Hz, 2H), 1.95 (tt, J = 4.8, 9.8 Hz, 1H), 1.70 (dt, J = 8.0, 10.5 Hz, 1H). (2S,3S,4S,5R,6S)-6-((6-Chloro-5-(6-(dimethylamino)-2-methoxypyridin-3-yl)-1H-indole-3carbonyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (M2). 1H NMR (600 MHz, DMSO-d6) δ 12.13 (d, J = 3.1 Hz, 1H), 8.22 (d, J = 3.0 Hz, 1H), 7.84 (s, 1H), 7.60 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 6.22 (d, J = 8.2 Hz, 1H), 5.59 (d, J = 7.7 Hz, 1H), 5.42 (s, 1H), 5.35 (s, 1H), 5.27 (s, 1H), 3.79 (d, J = 9.5 Hz, 1H), 3.77 (s, 3H), 3.43–3.26 (m, 3H), 3.07 (s, 6H). (2S,3S,4S,5R,6S)-6-((4,6-Difluoro-5-(4-((S)-tetrahydro-2H-pyran-2-yl)phenyl-1H-indole-3carbonyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (M3). 1H NMR (600


36

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MHz, DMSO-d6) δ 12.56 (s, 1H), 8.26 (d, J = 11.5 Hz, 1H), 7.50–7.35 (m, 4H), 7.30 (d, J = 9.4 Hz, 1H), 5.62 (d, J = 8.1, 1H), 4.38 (dd, J = 2.1, 11.1 Hz, 1H), 4.04 (d, J=11.8 Hz, 1H), 3.70 (d, J= 10.53 Hz, 1H), 3.63–3.50 (m, 2H), 3.42–3.14 (m, 3H), 1.88 (dt, J = 3.1, 4.1, 10.7 Hz, 2H), 1.74–1.44 (m, 4H). Stability of M1 in Phosphate Buffer. The stability of M1, M2, and M3 in phosphate buffer (pH = 7.4) was examined by 1H NMR spectroscopy. A 5 µL aliquot of the pure NMR isolate of M1, M2, or M3 (in DMSO-d6) was diluted with 45 µL of the 10:90 D2O:0.1 mM phosphate buffer (pH = 7.4) mixture. The samples were placed in a 1.7 mM NMR tube and were repeatedly scanned for ~ 21 h with a 1H pre-saturation NMR experiment designed to suppress the signal due to water with the probe temperature set to 37 °C. In Vitro Pharmacology Studies. Recombinant AMPK complexes and human PP2A alpha1 (Uniprot P67775-1) were expressed and purified as previously described.21,50,51 The TR-FRET activation assay was performed as previously described.21 SPR Studies. Experiments were performed on a BiacoreTM 3000 instrument (GE Healthcare). Bap-tagged AMPK α1β1γ1 was captured onto a streptavidin sensor chip to levels ranging from 4000-6000 RU. Compound binding experiments were performed in 25mM Tris, pH 7.5, 150 mM NaCl, 250 µM Tris(2-carboxyethyl)phosphine hydrochloride, 0.01% P20, 0.5 mg/mL BSA, 2% DMSO and 150 µM AMP at 25 ºC. Binding responses were processed using Scrubber 2 (BioLogic Software Pty Ltd) to zero, x-align, double reference and corrected for excluded volume effects of DMSO in the data. Experiments were carried out with at least n = 3 for three or more compound concentrations with 3-fold dilutions. The compound concentrations were based on the EC50 with the top concentration chosen to be several fold over the EC50 to achieve saturation. Compounds were injected at a flow rate of 50 µL/min, total contact time of 120 s and


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dissociation time of 600 s. Rate parameters (kon, koff,) and corresponding affinity constant (KD = koff/kon ) were determined by globally fitting the experimental data to a simple 1:1 interaction model using Biaeval (GE Healthcare). Cocrystallization of AMPK with Acyl Glucuronide Metabolites.

AMPK was expressed,

purified, and crystallized as previously reported50 with the exception that native Ser108 of the βsubunit was reinstated in place of the phosphomimetic aspartic acid. Ser108 was phosphorylated in vitro upon AMPK activation with Camkkb as confirmed by mass spectrometry. Data were collected at the IMCA-CAT beamlines at the Advanced Photon Source and each structure was solved from a single dataset using our previous α1β1γ1 model for molecular replacement.49 Apo crystals were soaked overnight in mother liquor supplemented with 10% glycerol in the presence of 200 µM Staurosporine, 400 µM AMP, and 500 µM of individual acyl glucuronide metabolite isolates and frozen in a matched solution containing 25-30% glycerol for cryoprotection. Refinement was carried out principally using autoBUSTER.68,69

Nucleotide content was

modeled as previously described including the tentative modeling of ADP at site 1 – and all caveats and limitations described previously apply here as well.50 Xray data collection and refinement statistics are provided in Supporting Information Table 1. AMPK Activation and Inhibition of De Novo lipogenesis in Primary Human Hepatocytes. Cryopreserved human hepatocytes (BD Biosciences, lot # BD310) were thawed rapidly at 37 °C and poured into 45 mL of pre-warmed BD-Biosciences CryoHepatocyte Recovery Media (BD Biosciences #454560).

Cells were pelleted at 1000 x g for 10 min at room temperature.

Supernatent was aspirated and cells were resuspended in 5 mL of Plating Media (Celsis InVitro Technologies #Z99029).

Cells were counted using a hemocytometer and trypan blue to

determine viable cell concentration. Cells were then plated in 48 well collagen I coated plates


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(BD Biocoat #356505) at a density of 150,000 cells/well, and allowed to attach to the plates overnight. After overnight incubation, the media was replaced with Williams E (16.5 mM glucose) for a 1 h serum starvation followed by replacement with Williams E media containing test compounds. Following a 1 h incubation with varying concentrations of the test compounds (1, 2, M1, and M2), 10 µL of

14

C-2-Acetic acid (0.1 µCi/µL) was added and the mixture was

allowed to incubate for an additional hour. Media was then removed and the cells washed once with warm Williams E media, and lysed with 0.25 mL of MPER lysis buffer (ThermoScientific #78505). Cells lysates were stored at 4 °C overnight and 0.24 mL of the lysis solution was transferred to a glass scintillation vial containing 2 mL of Microscint E Scintillation fluid (Perkin Elmer 6013661), vortexed, and centrifuged at 2000 x g for 3 min. Radioactivity was counted on a TriCarb counter. For measurement of ACC phosphorylation, cells were treated with test compounds for 1 hour and lysed in cell lysis buffer for subsequent MSD ELISA measurements. For ACC MSD assays, MSD Streptavidin gold plates (Meso-Scale Devices, L15SA-2) were blocked in 1% Blocker A (Meso-Scale Diagnostics, R3AA-2), diluted in 1X Tris Wash Buffer, and incubated at 37 °C for one hour. Following incubation, wells were washed three times with 200 µL of 1X Tris Wash Buffer, and 25 µg of the protein samples of interest were added to each well, then incubated at 37 °C for two hours. Wells were then washed three times with 200 µL of 1X Tris Wash Buffer, then either phospho-ACC antibody (Millipore, 07-303) at 50 ng/well or total ACC antibody (Cell Signaling, 3676) at 25 ng/well, diluted in 1% Blocker A, were added to respective wells and incubated for 1.5h at 37 °C. Wells were then washed with 1X Tris Wash Buffer, once, then 50 ng of MSD Sulfo-Tag antibody (Meso-Scale Diagnostics, R32AB-1) was added to each well and incubated at 37 °C for one hour. Wells were washed three times with 1X Tris Wash Buffer, 150 uL of 1X MSD Read Buffer (Meso-Scale Diagnostics, R92TC-2) was


39


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added to each well and the plate was read on an MSD plate reader (Meso-Scale Diagnostics, Sector S600). Pharmacokinetics studies in rats and monkeys with M1. All experiments involving animals were conducted in our AAALAC-accredited facilities and were reviewed and approved by Pfizer Institutional Animal Care and Use Committee.

Male jugular vein/carotid artery double

cannulated Wistar-Han rats (250 g), obtained from Charles River Laboratories (Wilmington, MA) and male cynomolgus monkeys (7 kg) were used for these studies. Animals were fasted overnight and through the duration of the study (1.0 or 2.0 h), whereas access to water was provided ad libitum. M1 was administered intravenously in 10% DMSO/30% PEG400/60% water via the carotid artery (rats, n=2) or femoral vein (monkeys, n=2) at a dose of 0.5 (rats) or 1.0 (monkeys) mg/kg in a dosing volume of 1 mL/kg. Serial blood samples were collected in heparinized tubes before dosing and 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 7, 24, and 48 h after dosing. Blood samples from the various pharmacokinetic studies were centrifuged to generate plasma and treated with 1 M citric acid to prevent any potential ex vivo hydrolysis of M1 to 1. All plasma samples were kept frozen until analysis. Urine samples (0–7.0 h, 7.0–24 h, and 24–48 h) were also collected after intravenous administration to rats and monkeys. Aliquots of plasma or urine (20-25 µL) were transferred to 96-well blocks and acetonitrile (150-200 µL) containing internal standard was added to each well.

Supernatant was dried under nitrogen and

reconstituted with 100 µl acetonitrile/water and analyzed by liquid chromatography tandem mass spectrometry and concentrations of M1 and 1 in plasma and/or urine were determined by interpolation from a standard curve. M1 and 1 was detected using electrospray ionization in the multiple reaction monitoring mode monitoring for mass-to-charge (m/z+) transition 518→500 and 342→324, respectively.


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Determination of Pharmacokinetic Parameters

of M1 in Rats and Monkeys.

Pharmacokinetic parameters of M1 in rats and monkeys were determined using noncompartmental analysis (Watson v.7.4, Thermo Scientific, Waltham, MA. The area under the plasma concentration-time curve from t = 0 to 24 h (AUC0-24) was estimated using the linear trapezoidal rule.

Systemic plasma clearance (CLp) was calculated as the intravenous dose

divided by AUC. The terminal rate constant (kel) was calculated by a linear regression of the log-linear concentration-time curve, and the terminal elimination t1/2 was calculated as 0.693 divided by kel. Apparent steady state distribution volume (Vdss) in animals were determined as the intravenous dose divided by the product of AUC and kel. Percentage of unchanged M1 excreted in urine over 24 h was calculated using the following equation: amount (in mg) of M1 in urine over the 24 h interval post dose/actual amount of M1 dose administered (mg) x 100%. ASSOCIATED CONTENT Supporting Information Additional experimental information for: Procedure for plasma protein binding, mass spectra of 1–3 and M1–M3, comparative HPLC-UV chromatograms and mass spectra for the biochemical and synthetic acyl glucuronides M1–M3, COSY NMR spectra of M1–M3, comparative HSQC NMR spectra of biochemical and synthetic acyl glucuronides M1–M3, degradation kinetics of M1–M3 in deuteriated phosphate buffer (pH 7.4), inhibition of de novo lipogenesis, X-ray Data Collection and Refinement Statistics, Crystallization and Refinement Methods. Molecular formula strings. Accession Code


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Crystal structure coordinates for compounds M1, M2, and M3 will be deposited to the RCSB protein data bank (www.rcsb.org). Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding Author *Phone (+1-617-551-3336). E-mail: [email protected] ORCID Amit S. Kalgutkar: 0000-0001-9701-756X Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. 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. Crystallographic data were collected at IMCA-CAT (17-ID) 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 USED


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ACC, Acetyl-CoA Carboxylase; ADaM, allosteric drug and metabolite; AMPK, adenosine monophosphate-activated protein kinase; AUC, area under the plasma concentration–time curve; CLint, intrinsic clearance; CLp, plasma clearance; COSY, correlation spectroscopy; CYP, cytochrome P450; fu,p, plasma unbound fraction; HMBC, heteronuclear multiple bond-bond correlation spectroscopy; HSQC, heteronuclear single-quantum correlation spectroscopy; LCMS/MS, liquid chromatography tandem mass spectrometry; Papp, apparent passive permeability; PP2a, protein phosphatase 2a; SPR, surface plasmon resonance; RRCK, Ralph Russ canine kidney cells; tR, retention time; TR-FRET, time-resolved fluorescence resonance energy transfer; UDPGA,

uridine

5’-diphosphoglucuronic

acid;

UGT,

uridine

5’-diphospho-

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Acyl Glucuronide Metabolites of 6-Chloro-5 - ACS Publications

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