Deuterium Kinetic Isotope Effect Studies of a Potential in Vivo

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Deuterium Kinetic Isotope Effect Studies of a Potential In Vivo Metabolic Trapping Agent for Monoamine Oxidase-B Lindsey R Drake, Allen F Brooks, Anthony J Mufarreh, Jonathan M Pham, Robert A Koeppe, Xia Shao, Peter J. H. Scott, and Michael R. Kilbourn ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00219 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

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ACS Chemical Neuroscience

Deuterium kinetic isotope effect studies of a potential in vivo metabolic trapping agent for Monoamine Oxidase-B Lindsey R. Drake,a,† Allen F. Brooks,b,† Anthony J. Mufarreh,b Jonathan M. Pham,b Robert A. Koeppe,b Xia Shao,b Peter J.H. Scott,a,b,* and Michael R. Kilbournb,* a. The Interdepartmental Program in Medicinal Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States. b. Department of Radiology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, Michigan 48109, United States. †Authors contributed equally to this work.

Abstract

Visualizing the in vivo activity of monoamine oxidase B (MAO-B) is a valuable tool in the ongoing investigation into astrogliosis in neurodegeneration. Existing strategies for imaging changes in MAO enzyme expression or activity have utilized the irreversible suicide inhibitors or high-affinity reversibly binding inhibitors as PET ligands. As an alternative approach, we developed

4-methyl-7-((1-[11C]methyl-1,2,3,6-tetrahydropyridin-4-yl)oxy)-2H-chromen-2-one

([11C]Cou) as a metabolic trapping agent for MAO-B. Trapping of [11C]Cou in rhesus monkey brain demonstrated MAO-B selectivity. In this work we have attempted to improve on the in vivo pharmacokinetics of [11C]Cou by using the deuterium kinetic isotope effect (KIE) to slow the MAO-B mediated oxidation step and thus reduce the rate of trapping in brain tissues. However, in vitro assays of enzyme kinetics and in vivo PET imaging of pharmacokinetics in primate brain showed no effects of deuterium substitution on the tetrahydropyridine ring of [11C]Cou. The results are possibly due to masking of the KIE by a second step in the overall metabolism of the new imaging agent.

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Keywords: neuroimaging, positron emission tomography, carbon-11, kinetic isotope effect monoamine oxidase.

Introduction Monoamine oxidase (MAO) is a flavin-dependent enzyme which metabolizes endogenous monoamines including dopamine, serotonin, epinephrine, norepinephrine, and others. Two isozymes, MAO-A and –B, perform the same oxidation with different substrate preferences and slight structural differences in their respective active sites. In the 1980s, 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was determined as a contaminant in the illicit synthesis of the synthetic opiate desmethylprodine (MPPP), resulting in symptoms of Parkinson’s disease in drug users. It was subsequently shown that MPTP is a substrate and inhibitor of MAO-B, producing dopaminergic cell death via the toxic metabolite 1-methyl-4phenylpyridinium (MPP+).1 The MAO oxidation of MPTP and other tetrahydropyridines has been extensively studied, as has the mechanism of cellular toxicity of MPP+, and MPTP remains a valuable tool in Parkinson’s disease research. Among the many MPTP derivatives and analogues that have been synthesized, Castagnoli and coworkers developed non-toxic derivatives of MPTP by incorporating an ether linkage between the tetrahydropyridine and phenyl rings; this resulted in rapid hydrolytic cleavage of the intermediate 1-methyl-4-phenoxy-2,3-dihydropyridinium species formed by MAO-B mediated oxidation, and no formation of N-methylpyridinium products.2 Intrigued by these substrates and seeking an in vivo imaging agent for MAO enzymatic activity, we recently reported the MAO-B selective PET (positron emission tomography) imaging agent: 4-methyl-7((1-[11C]methyl-1,2,3,6-tetrahydropyridin-4-yl)oxy)-2H-chromen-2-one ([11C]Cou, Fig 1). In vivo PET studies in rhesus macaque brain using [11C]Cou demonstrated that the metabolite formed by MAO-B oxidation and subsequent hydrolysis (Fig 1) was rapidly and efficiently trapped in the brain, which could be blocked by pretreatment with the reversible MAO-B inhibitor lazabemide.3 Although successful as an in vivo metabolic trapping substrate, the rapid trapping rate of [11C]Cou made it difficult to discriminate between regions of high and low MAO enzymatic activity. That limitation is similar to what was observed for the irreversibly-binding MAO-B radiotracer [11C]deprenyl, where the in vivo pharmacokinetics were slowed and sensitivity


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improved using the kinetic isotope effect (KIE) induced by deuterium atoms at the location critical for the rate-determining oxidation step.4,5 The value of the KIE was estimated at 3.8 (using K3’E).5 KIE experiments using deuterium substitutions on the tetrahydropyridine ring have demonstrated that the hydrogen abstraction step is also rate limiting in the MAO-B mediated oxidation of MPTP. The KIE value for MAO oxidation of MPTP-6,6-d2 to the corresponding 1-methyl-4-phenyl-2,3-dihydropyridinium species was 3.55 (using Vmax).6 Given this strong literature precedent, it was reasonable to expect a similar KIE for [11C]Cou and we attempted to apply this concept to further optimize the in vivo pharmacokinetics of this radiotracer. Herein we report the synthesis of two deuterated derivatives of [11C]Cou and a comparison

of

their

in

vitro

kinetics

and

in

vivo

brain

pharmacokinetics.

Figure 1. MAO catalyzed oxidation and subsequent hydrolysis of Cou substrate. The 4aryloxytetrahydropyridine substrate [11C]Cou is oxidized by monoamine oxidase to form an iminium intermediate, which is rapidly hydrolyzed to yield a polar carbon-11 species and a fluorescent coumarin as metabolites.

Results Chemistry The synthesis of COU-d3 is shown in Scheme 1A: 1-methyl-4-((4-methyl-2-oxo-2Hchromen-7-yl)oxy)pyridin-1-ium (1) was prepared by reacting 4-chloro-N-methyl-pyridinium with 4-methylumbeliferone in a solution of sodium methoxide in DMF. Reduction of the pyridinium with sodium borodeuteride in deuterated methanol yielded COU-d3 (2). The carbon11 radiolabeling was accomplished by N-[11C]methylation of pyridine 3 in ethanol-d6, followed by addition of sodium borodeuteride, to form [11C]Cou-d3 (2b), as previously described (Scheme 1B).3 The synthesis of COU-d7 is described in Scheme 2A: Iodocoumarin (4) was prepared via Pechmann condensation by treating 3-iodophenol with ethyl acetoacetate in an acidic solution. Reaction of 4 with p-toluenesulfonic acid and meta-chloroperbenzoic acid (mCPBA) in dichloromethane gave iodonium salt 5, which was converted to the 4-methoxyphenyl substituted iodonium salt (6). A subsequent substitution reaction with 4-phenol-d5 yielded the pyridine



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COU-d7 precursor (7). N-Methylation and reduction of the resulting pyridinium with sodium borodeuteride in deuterated methanol yielded COU-d7 (9). Carbon-11 radiolabeling was conducted by alkylating precursor 7 with [11C]methyl triflate, followed by addition of sodium borodeuteride to form [11C]Cou-d7 (9b), as previously described (Scheme 2B).3

A Cl HO

O

O

O

O

NaBD4 H

MeOH-d4

NaOMe, DMF

N

I

O

I

36%Yield over two steps

1

N

D

O

O

D N

H

D H

O

2

B 1.) [11C]CH3OTf, EtOH-d6 O

O

O

H

2.) NaBD4, EtOH-d6

3

H

N

D

O

O

D N 11CH

D H

O

2b

3

Scheme 1. Synthesis and Radiochemical synthesis of Cou-d3 and [11C]Cou-d3

OMe A

O I

H2SO4

O

40

O

OH

oC,

TsOH, mCPBA 24 hr

10% Yield

DCM, 2 hr, r.t. I

O

O

OTs I OH

98% Yield

4

O

O

CHCl3 , 4 hr, r.t. 57% Yield

5

OD D I

O

O

D

OTs

D N

D , K2CO3

MeCN, 6 hr, 50 OMe

oC

50% Yield

6

O D

O

O

D

D

N

O

MeI D

O

O NaBD , MeOD-d , 4 4

D 1 hr 0

D

D 7

N

D

8

37% Yield over two steps

B 1.) [11C]CH3OTf, EtOH-d6 O D D

O D

N

D

O

D 2.) NaBD4, EtOH-d6

7

D D D

O

O

O

D D D

N 11CH

9b

3

Scheme 2. Synthesis and Radiochemical synthesis of Cou-d7 and [11C]Cou-d7.

Biology


oC

D D D D

O

O D N

D D

9

O

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In vitro kinetic parameters were determined by incubation of substrates (Cou, Cou-d3, and Cou-d7) with human MAO-B Supersomes and monitoring the appearance of the fluorescent coumarin product (ex: 360 nm/ em: 460 nm) released after oxidation and hydrolysis. Reactions proceeded at 37 °C for 60 minutes in borate buffer (pH 8.4 at room temperature). All three substrates displayed well-behaved Michaelis-Menten kinetics (Fig 2). There were no significant observed kinetic isotope effects based on the turnover (Vmax) or enzymatic efficiency (Vmax/KM) (Table

1).

Figure 2. In vitro kinetics for Cou and deuterium-substituted substrates. The velocity of fluorescent product appearance was plotted against concentration of substrates. Michaelis-Menten Kinetic parameters (Table 1) were calculated in GraphPad Prism (n= 6).

Table 1. Michaelis-Menten Kinetic parameters for Cou and deuterium-substituted substrates.

Cou

Cou-d3

Cou-d7

K (µM)

1.35 ± 0.12

1.55 ± 0.12

1.56 ± 0.15

V

(nM/min)

21.1 ± 0.56

20.5 ± 0.48

19.9 ± 0.65

/K

15.63 ± 1.44

13.23 ± 1.08

12.75 ±1.51

---

1.03 ± 0.036

1.06 ± 0.045

---

1.18 ± 0.15

1.23 ± 0.16

M

V

max max

M

D

(V

max

)

D

(V

/K )

max

M

The in vivo pharmacokinetics of the three MAO-B substrates were evaluated in rhesus monkey brain using PET imaging. The brain uptake and distribution of these MAO-B substrates were imaged in an adult female rhesus macaque (n=2 per substrate). Radiolabeled compounds



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were injected intravenously and the regional brain distributions of radioactivity were determined using dynamic pre-clinical PET imaging. Tissue time-radioactivity curves were generated for cerebellum, basal ganglia, and cortex (Fig 3), and data are presented as % injected dose per gram (%ID/g). Curves for [11C]Cou were consistent with those previously reported,3 and brain uptake was an order of magnitude higher than that previously reported for [11C]deprenyl and [11C]deprenyl-d2.4 There were no significant changes in the shapes of the curves in any brain region

for

either

[11C]Cou-d3

or

[11C]Cou-d7.

Figure 3. Regional tissue time-radioactivity curves for PET imaging of [11C]Cou, [11C]Cou-d3, and [11C]Cou-d7 in the rhesus monkey brain.

Discussion Kinetic isotope effects have been clearly demonstrated for a number of amine substrates of the monoamine oxidases, leading to the conclusion that the abstraction of an allylic hydrogen at the position alpha to the nitrogen is the rate limiting step.6-9 This has been exploited in the design of PET imaging agents for monoamine oxidases, where incorporation of deuterium has been used to slow down the rate of irreversible trapping of [11C]deprenyl, resulting in improved in vivo pharmacokinetics in animals and humans.4,5 In vitro kinetic isotope effects have also been demonstrated for MAO oxidation of MPTP and related analogues,6,7 prompting our expectation that deuterium substitution could be employed to modify the in vivo pharmacokinetics of our newly developed MAO substrate, [11C]Cou. It was therefore surprising that our in vitro assays of MAO-B activity showed no isotope effect for deuterium substitution of Cou (Table 1); in comparison, the KIE for [11C]deprenyl-d2 was estimated to be 3.8 (using K3’E),5 and values for MAO oxidation of MPTP-6,6-d2 to the corresponding 1-methyl-4-phenyl-2,3-dihydropyridinium species were 3.55 (Vmax) and 8.01 (Vmax/ Km).6 The in vivo studies in primate brain for [11C]Coud3 and [11C]Cou-d7 were supportive of the lack of a deuterium substitution effect as the


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pharmacokinetic curves for radiotracer trapping were similar for all three substrates, showing a rapid uptake to a plateau with very little washout of radioactivity, consistent with irreversible trapping. This is markedly different than the effect seen for [11C]deprenyl-d2, where incorporation of deuterium resulted in a significant egress of radioactivity after peak uptake and a lower plateau level of trapped radioactivity, and thus very different shapes for the brain timeradioactivity curves.4 Based on the well-established mechanism of MAO-mediated oxidation of amines, and the known effect of deuterium substitution on MPTP oxidation kinetics, it is reasonable to assume the oxidation of [11C]Cou should also proceed via abstraction of the allylic C-6 proton alpha to the ring nitrogen. The lack of a kinetic isotope effect both in vitro and in vivo suggests that the proton abstraction step is not rate limiting. A possible explanation lies in the observation that our studies actually detect the final metabolites, the radioactive N-[11C]-methylpyridinone (in vivo PET) or the fluorescent coumarin (in vitro assays; Fig. 1) that form after a second step of hydrolysis of the intermediate 1-methyl-4-phenoxy-2,3-dihydropyridinium species (in contrast, the in vitro kinetic studies of deuterated MPTP measured the dihydropyridinium species). Thus, it is possible that the hydrolysis step is partially rate limiting and obscuring any effect from the deuterium substitution, an example of a masked kinetic isotope effect.8 Future work aimed at testing this hypothesis will require more sophisitcated physicochemical methods for probing enzyme kinetics that are capable of detecting the 1-methyl-4-phenoxy-2,3-dihydropyridinium intermediate.

Conclusion The objective of this study was to utilize deuterium substitution to alter the in vivo pharmacokinetics of [11C]Cou a metabolically-trapped PET imaging agent for MAO-B that shows rapid and essentially irreversible retention in the primate brain. From a compartmental modeling perspective, the goal was to reduce the rate of trapping (k3) such that a portion of the radiotracer taken up into the brain would flow back out (k2): this was exactly what had been achieved with deuteration of the irreversible radioligand [11C]deprenyl. Unfortunately, deuterium substitution of [11C]Cou failed to produce any significant change of in vitro kinetics or in vivo pharmacokinetics. Efforts to improve the in vivo behavior of metabolically-trapped MAO substrates for PET imaging will likely require further exploration of alternative 4-aryloxy



substituents.2 As we undertake this work and validate the new radiotracers, future experiments could involve evaluation in animal models of disease, such as the recently reported MAO-B mouse model of Parkinson’s disease,10 before anticipated translation into clinical studies.

Experimental Section Detailed experimental procedures are provided in the Supporting Information. Associated Content The supporting information is available on the ACS Publications website at DOI. 10.XXXX. Synthetic and radiochemistry procedures, including spectra for all novel compounds synthesized and chromatographic data for radiotracer purifications; procedures for in vitro assays and preclinical PET imaging. Author Information Corresponding Authors Peter J. H. Scott; E-mail: [email protected]; Tel: +1 (734) 615-1756 Michael R. Kilbourn; E-mail: [email protected]; Tel: +1 (734) 763-9246 ORCID Lindsey R. Drake: 0000-0001-8594-8922. Allen F. Brooks: 0000-0003-3773-3024. Anthony J. Mufarreh: 0000-0003-0611-1185. Jonathan Pham: 0000-0002-4617-5143. Robert A. Koeppe: 0000-0003-0643-577X. Xia Shao: 0000-0002-7291-2477. Peter J. H. Scott: 0000-0002-6505-0450. Michael R. Kilbourn: 0000-0003-4100-4676.

Author Contributions L.R.D, A.F.B, R.A.K, P.J.H.S, and M.R.K contributed to design of experiments, analysis of data, and composition of the manuscript. L.R.D, A.F.B, A.M, X.S, and J.M.P performed the experiments. Funding Financial support for this work from the NIH (NIGMS: Pharmacological Sciences Training Program T32GM07767 (L.R.D., P.J.H.S); NINDS: R21-NS075553 (M.R.K, P.J.H.S, A.F.B) and NIBIB: T32EB005172 (M.R.K, P.J.H.S, A.F.B)) is gratefully acknowledged. The content of this article is solely the


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responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Notes The authors declare no competing financial interest.



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Acknowledgments We thank Phillip Sherman, Janna Arteaga, and Jenelle Stauff for conducting pre-clinical PET imaging.

Abbreviations Used Cou,

4-methyl-7-((1-methyl-1,2,3,6-tetrahydropyridin-4-yl)oxy)-2H-chromen-2-one;

MAO-B,

monoamine oxidase-B; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PET, positron emission tomography; VOI, volumes of interest.

References 1. Maret, G., Testa, B., Jenner, P., Tayar, N. E., and Carrupt, P.-A. (1990) The MPTP Story: MAO Activates Tetrahydropyridine Derivatives to Toxins Causing Parkinsonism, Drug Metab. Rev. 22, 291332. 2. Kalgutkar, A. S., Castagnoli, K., Hall, A., and Castagnoli, N. (1994) Novel 4(Aryloxy)tetrahydropyridine Analogs of MPTP as Monoamine Oxidase A and B substrates, J. Med. Chem. 37, 944-949. 3. Brooks, A. F., Shao, X., Quesada, C. A., Sherman, P., Scott, P. J. H., and Kilbourn, M. R. (2015) In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and -B Enzymatic Activity, ACS Chem. Neurosci. 6, 1965-1971. 4. Fowler, J. S., Wang, G.-J., Logan, J., Xie, S., Volkow, N. D., MacGregor, R. R., Schlyer, D. J., Pappas, N., Alexoff, D. L., Patlak, C., and Wolf, A. P. (1995) Selective Reduction of Radiotracer Trapping by Deuterium Substitution: Comparison of Carbon-11-L-Deprenyl and Carbon-11-Deprenyl-D2 for MAO B Mapping, J. Nucl. Med. 36, 1255-1262. 5. Fowler, J. S., Wolf, A. P., MacGregor, R. R., Dewey, S. L., Logan, J., Schlyer, D. J., Langstron, B. (1988) Demonstration of a Deuterium Isotope Effect in the Monoamine Oxidase-Catalyzed Binding of [11C]L-Deprenyl in Living Baboon Brain. J. Neurochem., 51, 1524-1534. 6. Ottoboni, S., Caldera, P., Trevor, A., and Castagnoli, N., Jr. (1989) Deuterium Isotope Effect Measurements on the Interactions of the Neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine with Monoamine Oxidase B, J. Biol. Chem. 264, 13684-13688. 7. Pretorius, A., Ogunrombi, M. O., Terre'Blanche, G., Castagnoli, N., and Bergh, J. J. (2008) Deuterium Isotope Effects for the Oxidation of 1-Methyl-3-phenyl-3-pyrrolinyl Analogues by Monoamine Oxidase B, Bioorg. Med. Chem. 16, 8813-8817. 8. Wang, Z., Roston, D., and Kohen, A. (2012) Chapter 6 - Experimental and Theoretical Studies of Enzyme-Catalyzed Hydrogen-Transfer Reactions. In Advances in Protein Chemistry and Structural Biology (Christov, C., and Karabencheva-Christova, T., Eds.), pp 155-180, Academic Press, Cambridge MA. 9. Mavri, J., Matute, R. A., Chu, Z. T., and Vianello, R. (2016) Path Integral Simulation of the H/D Kinetic Isotope Effect in Monoamine Oxidase B Catalyzed Decomposition of Dopamine, J. Phys. Chem. B 120, 3488-3492. 10. Chamoli, M., Chinita, S. J., and Andersen, J. K. (2018) An Inducible MAO-B Mouse Model of Parkinson’s Disease: a Tool Towards Better Understanding Basic Disease Mechanisms and Developing Novel Therapeutics. J. Neural Transm. https://doi.org/10.1007/s00702-018-1887-z.


Deuterium Kinetic Isotope Effect Studies of a Potential in Vivo

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