(MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for

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Development of Novel Monoamine Oxidase-B (MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for the Potential Management of Non-Central Nervous System (CNS) Diseases Ronan GEALAGEAS, Alice Devineau, Pauline P. L. So, Catrina M. J. Kim, Jayakumar Surendradoss, Christian Buchwalder, Markus Heller, Verena Goebeler, EDITH Dullaghan, David S. Grierson, and Edward E. Putnins J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01588 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Journal of Medicinal Chemistry

Development of Novel Monoamine Oxidase-B (MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for the Potential Management of Non-Central Nervous System (CNS) Diseases Ronan Gealageas1, Alice Devineau1, Pauline P.L. So2, Catrina M.J. Kim2, Jayakumar Surendradoss2, Christian Buchwalder1, Markus Heller2, Verena Goebeler3, Edith M. Dullaghan2, David S. Grierson1*, Edward E. Putnins3*

1

Faculty of Pharmaceutical Sciences, The University of British Columbia, 2405 Wesbrook Mall,

Vancouver, BC, V6T 1Z3, Canada

2

Centre for Drug Research and Development, 2405 Wesbrook Mall, Vancouver, BC, V6T 1Z3,

Canada

3

Faculty of Dentistry, The University of British Columbia, 2199 Wesbrook Mall, Vancouver,

BC, V6T 1Z3, Canada

ACS Paragon Plus Environment

1

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ABSTRACT

Studies indicate that MAO-B is induced in peripheral inflammatory diseases. To target peripheral tissues using MAO-B inhibitors that do not permeate the blood-brain barrier (BBB) the MAO-B selective inhibitor deprenyl was remodeled by replacing the terminal acetylene with a CO2H function, and incorporating a para –OCH2Ar motif (compounds 10a-s). Further, in compound 32 the C-2 side chain corresponded to CH2CN. In vitro, 10c, 10j, 10k and 32 were identified as potent reversible MAO-B inhibitors, and all four compounds were more stable than deprenyl in plasma, liver microsomal and hepatocyte stability assays. In vivo, they demonstrated greater plasma bioavailability. Assessment of in vitro BBB permeability showed that compound 10k is a P-glycoprotein (P-gp) substrate and 10j displayed mild interaction. Importantly, compounds 10c, 10j, 10k and 32 displayed significantly reduced BBB permeability after intravenous, subcutaneous and oral administration. These polar MAO-B inhibitors are pertinent leads for evaluation of efficacy in non-central nervous system (CNS) inflammatory disease models.

INTRODUCTION Monoamine oxidases (MAOs, EC 1.4.3.4) are mitochondrial outer membrane enzymes that catalyze the oxidative deamination of various endogenous (neurotransmitters) and dietary amines to form an aldehyde, ammonia (or a substituted amine) and H2O2 (Figure 1a).1 Hydrogen peroxide is an oxidative stress molecule, implicated in normal cellular and pathophysiological roles, which undergoes reduction to highly reactive hydroxyradicals via the iron dependent Fenton reaction.2,

3

The two MAO isoenzymes, MAO-A and MAO-B, contain a covalently

bound flavin adenine dinucleotide (FAD) cofactor. However, they have different tissue


2

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distribution and substrate specificities. MAO-A preferentially catalyzes the oxidation of 5-HT (serotonin), whereas MAO-B oxidizes benzylamine and 2-phenylethylamine. Dopamine, noradrenaline, adrenaline, tryptamine and tyramine are oxidized by both isoenzymes.4 Clorgyline and deprenyl (selegiline)5 are selective MAO-A and MAO-B inhibitors, respectively, whereas phenelzine (phenethylhydrazine) inhibits both MAO isoforms.6 These and related drugs of this class4 were initially developed as antidepressants. More recently their therapeutic value has been extended to the clinical management of neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD).4, 7 Protein and mRNA analysis and Positron Emission Tomography (PET) imaging have shown that MAO-A and B are widely expressed in peripheral tissues such as placenta, duodenum, lung, liver, kidney, thyroid, spleen and heart. However, expression of each enzyme does vary across these tissues.8-11 Human platelets and lymphocytes only express MAO-B, whereas, endothelial cells of lymphatic vessels only express MAO-A.10, 11 Use of MAO-A, MAO-B and MAO-A/B inhibitors has traditionally been prescribed for CNS-associated disorders.4,7 However, a review of patents filed from 2002-2017 outlined how this class of drugs may have the therapeutic potential to manage a variety of non-CNS disorders such as inflammation, obesity/diabetes, hair growth, cancer, cardiovascular damage, ocular diseases and muscle dystrophies.12 Their potential to manage inflammation associated with chronic diseases has a growing body of support. Treatment of patients suffering from Crohn’s disease or rheumatoid arthritis with MAO-A/B inhibitors (phenelzine, tranylcypromine) was associated with disease remission.13,

14

As well,

MAO-B knockout mice showed less apoptosis and fibrosis in cardiac pressure overload experiments, and gene expression profiling in tissues from patients with atrial fibrillation identified MAO-B as one of five significantly induced oxidative stress proteins.15, 16 Treatment


3


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of mice in a diabetic cardiomyopathy model with the MAO-B inhibitor pargyline2 reduced mast cell degranulation, cardiac fibrosis and normalized diastolic function.17 Pertinent to the present study, Ekuni et al.18 identified MAO-B as one of the 10 most significantly up-regulated genes in epithelial tissues harvested from rats with lipopolysaccharide-induced chronic inflammatory periodontal disease. a) NH 2

O2

H 2O 2

CHO HO

HO

HO

HO H 2O

Dopamine

NH 3

Cl

Cl

N

N

O

Clorgyline selective MAO-A inhibitor

b)

Deprenyl selective MAO-B inhibitor

Cl

N

Cl

N

N

N Chlorphenamine

O

Cetirizine

CO 2H

R N OH HO

Terfenadine R = CH 3 Fexofenadine R = CO 2H

c) R N

Cys 397 H N

5 N

Bound FAD

N-

O

NH O

Rasagiline

N Covalent adduct


4

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Figure 1. a) MAO activity and selective MAO-A/B inhibitors; b) incorporation of a CO2H function into drug structures suppresses BBB penetration; c) irreversible MAO inhibition by rasagiline.

Interestingly, inhibition of MAO-A/B activity with phenelzine effectively reduced disease progression. Collectively, these data support that MAO activity is induced in peripheral chronic inflammatory diseases and that MAO inhibition is associated with reduction in disease severity. MAO-A (moclobemide), MAO-B (deprenyl) and MAO-A/B inhibitor (phenelzine) have all been shown to reduce pro-inflammatory or induce anti-inflammatory cytokine expression in a number of cell lines.18-21 These data further supports their development as potential antiinflammatory agents. However, by focusing on selective MAO-B inhibitors the ability of MAOA to degrade dietary tyramine in the gut is preserved. Therefore, the risk of patients experiencing the ‘cheese effect’, which induces hypertensive crisis in patients treated with MAO-A inhibitors, would be reduced.4 Selective targeting of peripheral tissues with MAO-B inhibitors can be achieved by designing polar MAO-B inhibitor-based drugs that do not permeate the BBB. An additional advantage to this strategy is the potential to further reduce a relatively rare CNS side-effect. MAO inhibitors have been associated with serotonin increase in the brain (serotonin syndrome), resulting in mental status changes, autonomic hyperactivity and neuromuscular abnormalities.22 Typically, the MAO-B selective inhibitor deprenyl (selegiline)5 has quite a low incidence of serotonin syndrome, but reduced CNS permeability of novel polar MAO-B inhibitors would further reduce the risk of serotonin syndrome.23 To remodel the MAO-B selective drug deprenyl, such that it no longer crosses the BBB, inspiration was taken from the structures of the 2nd/3rd generation antihistamines cetirizine24-26


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and fexofenadine27 (Figure 1b). By incorporating a CO2H function into the structure of these drugs the peripheral histamine receptors are targeted, without the undesired secondary effects of drowsiness and loss of concentration associated with use of their 1st generation congeners chlorphenamine and terfenadine, which readily penetrate the BBB. Deprenyl, like the related drug rasagiline (Figure 1c), is an irreversible MAO-B inhibitor, since the N-propargyl unit in its structure reacts with the FAD cofactor to form a covalent azadiene-type adduct.28 Importantly, the presence of the acetylene motif indicates that significant space exists in the enzyme active site between the FAD cofactor and the tertiary amine function undergoing redox dealkylation. Consequently, no significant steric clash should occur between FAD and a deprenyl analog in which the acetylene unit is replaced by a CO2H function, as in the L-methamphetamine derivative A (Figure 2). Further, the possibility for H-bonding/salt bridge interactions to occur between the CO2H group in A and the 5-nitrogen of FAD, or to one of the multiple tyrosine residue lining the catalytic site/pocket would, in principle, reinforce binding in the MAO-B active site. Also, as compound A lacks reactive functionality it would be a reversible MAO-B inhibitor. Interestingly, in silico calculations29 predicted that, despite the presence of the terminal CO2H group, compound A would still manage to cross the BBB. However, compound 10a (R1 = R2 = H, R3 = Me) (Figure 2), incorporating an additional O-benzyl motif on the paraposition of the phenyl ring, was predicted to not be BBB permeable.

N N

N

Deprenyl

A

CO 2H

R1 O R2

CO 2H

R3

B (10a R1 = R 2 = H, R 3 = Me)


6

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Figure 2. Structure of deprenyl, the polar deprenyl inspired compounds A and B, and the B-type compound 10a.

Herein, results are presented on the synthesis and evaluation of compound 10a and twentyfour additional L-methamphetamine analogues of general structure B. The results obtained demonstrate that these deprenyl-inspired compounds B maintain selective affinity for MAO-B. Further, the ability of the most active inhibitors to cross the BBB is very significantly attenuated.

RESULTS In Silico Prediction of CO2H Functionalized Methamphetamines Related to Deprenyl to Cross the BBB. In silico prediction of whether compounds in which the acetylene subunit in deprenyl was exchanged for a polar carboxylic acid motif would cross the BBB were made using the Simulations Plus ADMET predictor software (Table 1).29 Two values were determined from the BBB algorithm: a “high” or “low” score predicting that the molecule will or will not enter the CNS, and a numerical LogBBB value indicative of the ratio of compound in blood relative to brain. Although negative values for LogBBB indicate that the concentration of the molecule in the blood is superior to that in the brain, the high/low calculated values are more predictive. The calculations suggested that replacing the acetylene by CO2H would not be sufficient to block BBB passage (Entry 1). Exchanging the amphetamine methyl group by CO2H, so as to have two acid functions in the molecule (Entry 2), or introducing a polar phenolic OH at the para position (Entry 3), also does not tip the scale. However, introducing a benzyl group onto the phenolic OH, as in 10a, provided molecules predicted to not cross the BBB (Entry 4).


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Table 1. In silico prediction of BBB passage for CO2H functionalized methamphetamines.

Chemistry The synthetic route developed to access compounds 10a-s (Scheme 1), bearing different functionality in the -OCH2Aryl ring from L-tyrosine ester carbamate 130, involved, in the first steps, introduction of an O-benzyl group to give 2 (BnBr, acetone, K2CO3; 85%31), LAH reduction of the ester function (known to occur without racemization32), conversion of the derived alcohol 3 to the corresponding tosylate 4, (TsCl, Bu4NHSO4 in benzene-H2O; 67%) and zinc metal reduction of the corresponding iodide generated in situ through iodine exchange of the tosylate group (94%).33 This four-step procedure provided ready access to multigram quantities of the (R)-L-amphetamine derivative 5.


8

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a

Reagents and conditions: (i) BnBr, K2CO3, acetone, RT 16 h then reflux 3 h, 85%; (ii) LAH, THF, RT, 95%; (iii) TsCl, n-Bu4NHSO4, 10%aq NaOH-C6H6, RT, 30 min., 67%; (iv) Zn dust, NaI, THF-H2O, reflux, 2.5 h, 94%; (v) LAH, THF, reflux, 4 h, 54%; (vi) t-butyl bromoacetate, Cs2CO3, DMF, RT, 15 h, 79%; (vii) Pd/C(cat), H2, MeOH, 99%; (viii) ArCH2Br, K2CO3, DMF, RT, 15 h, 36-99%; (ix) (for 9n) 3-OMeBnOH, DIAD, PPh3, THF, RT, 3 min., 62%; (x) conc. HCl, THF, 0°C, 3 h, 35-87%; (xi) Propargyl bromide, Cs2CO3, THF, RT 15h, 42%. Reduction of the carbamate function in 5 using LAH in refluxing THF, followed by alkylation of the derived secondary amino group in O-benzyl methamphetamine 6 with t-butyl bromoacetate in DMF, using K2CO3 as base, produced the pivotal intermediate 7. At this juncture, acid hydrolysis of the t-butyl ester group in 7 gave O-benzyl amphetamine carboxylic acid derivative 10a, whereas hydrogenolysis of the O-benzyl group in 7 provided phenol 8,


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which served as the precursor to intermediates 9b-s. With the exception of the conversion of 8 to 9n under Mitsunobu conditions, compounds 9b-m and 9o-s were obtained by O-alkylation of the phenolic hydroxyl group in 8 through reaction with requisite benzyl halide. Subsequent acid treatment of compounds 9 liberated the acid function in the target compounds 10b-s. Further, in order to have the O-benzyl substituted deprenyl analog 11 as a control/reference compound (see Table 2), it was prepared by reacting intermediate 6 with propargyl bromide using Cs2CO3 as base (42%).

a

Reagents and conditions: (i) BnBr or 3-ClBnBr, K2CO3, acetone, RT 16 h than reflux 3 h, 95% for 13, 100% for 14; (ii) MeI, NaH, THF, RT, 15 h, 35% for 15, 76% for 16; (iii) SO2Cl, MeOH, RT, 15 h, 62% for both 17 and 18; (iv) t-butyl bromoacetate, Cs2CO3, DMF, RT, 15 h, 49% for 19, 54% for 20; (v) conc. HCl, THF, 0°C, 3 h, 86% for 21, 75% for 22.


10

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To access compounds 21 and 22 (Scheme 2), in which the C-2 methyl group in 10a and 10c is replaced by a more polar ester function, Boc-L-tyrosine methyl ester 1134 was converted to the corresponding O-benzyl and O-3-chlorobenzyl derivatives 13 and 14, respectively. NMethylation of the carbamate nitrogen in these intermediates (MeI, NaH, THF)35 gave 15 and 16. Note that in these reactions ester demethylation occurred, presumably during reaction quench and work-up. Of further note was the observation that the presence of rotomers about the amide bond was observed in the 1H NMR spectra for these two intermediates. Reaction of 15 and 16 with SOCl2 - MeOH resulted in both N-Boc deprotection and reintroduction of the methyl ester function. Subsequent reaction of N-methyl esters 17 and 18 with t-butyl bromoacetate, followed by acid treatment provided the desired O-benzylated ester-acids 21 and 22. Scheme 3a N OR1

O 2

CO 2H

25 R1 = H 26 R1 = Bn i

iv

NH BnO

OH

iii N

ii BnO

CO 2t-Bu

OH 24

23

v N BnO

CO 2H

CN

N

R 3O

CO2t-Bu

R2 27 R 2 = Cl, R 3 = Bn

29 ix

vi

28 R 2 = CN, R 3 = Bn vii

30 R 2 = CN, R 3 = H viii N Cl Cl

O

CO 2R 4

CN 31 R 4 = t-Bu 32 R 4 = H

ix


11


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a

Reagents and conditions: (i) LAH, THF, reflux, 3 h, 88%; (ii) t-butyl bromoacetate, Et3N, DMF, RT, 15 h, 80%; (iii) BnBr, NaH, THF, 15 h, then conc HCl, 2 h, 46%; (iv) t-butyl bromoacetate, Cs2CO3, NaI (cat), DMF, 30min, 90°C, µW, 66%; (v) TsCl, Et3N, DMAP, DCM, RT, 15 h, 88%; (vi) KCN, 18-C-6, NaI (cat), DMF, 100°C. 90 min, 80%; (vii) Pd/C, H2 (1 atm), 90%; (viii) 3,4-diClBnBr, K2CO3, DMF, RT, 15 h, 88%; (ix) conc. HCl, THF, 0°C, 3 h, 77% for 29, 66% for 32.

To further obtain polar deprenyl-inspired compounds 25, 26, 29, and 32, (Scheme 3) wherein the C-2 methyl group is functionalized by an OH, OBn and a CN function, both the carbamate and the ester groups in the tyrosine derivative 2 were reduced using LAH in refluxing THF to give alcohol 23. N-alkylation of 23 with t-butyl bromoacetate in the presence of Cs2CO3 and NaI(cat) under microwave heating conditions led to direct formation of carboxylic acid 25. Alternatively, N-Alkylation of 23 (t-butyl bromoacetate, Et3N, DMF, RT) followed by Obenzylation of the derived alcohol 24 (BnBr, NaH, THF, 15 h) and acid treatment gave compound 26. To prepare compounds 29 and 32, alcohol 24 was reacted with TsCl and the derived chloro intermediate 27 was reacted with KCN to give 28. Subsequent reaction of 28 with acid effected ester hydrolysis to give 29, whereas hydrogenolysis of the O-benzyl group in 28 gave phenol 30. Reaction of 30 with 3,4-dichlorobenzyl bromide, followed by treatment of intermediate 31 with acid gave the target compound 32. With the exception of intermediates 9n and 18, where by-products were detected in the 1H NMR spectra, the proton spectra for all compounds showed the presence of a unique product, whose peak positions and multiplicities were consistent with the proposed structures. Note that purification of final product 10n and intermediate 20 by column chromatography enabled removal of the impurity observed in their precursors 9n and 18. For all final compounds tested, purity was established by HPLC (see Supporting Information). The HPLC experiments were conducted on samples that were stored at - 20°C for an extended period of time (>1.5 years) in


12

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water or water-DMSO prior to evaluation. With the exception of compounds 10m (93.6%), 10p (91.8%). 22 (93.4%), and 25 (91.6%), the other twenty-one compounds were established to be > 95% pure.

In Vitro MAO Inhibition Assay Results. The twenty-five polar deprenyl-inspired compounds B (10a-s, 21, 22, 25, 26, 29 and 32) described in schemes 1-3 were tested against human recombinant MAO-A and MAO-B in a cellfree biochemical assay to determine to what extent replacement of the terminal acetylene motif in deprenyl by a carboxylic acid function and introduction of a –OCH2Ar motif onto the paraposition of the phenyl ring influenced activity/potency and MAO-B selectivity. In the presence of the serially diluted compounds, each monoamine oxidase reacted with tyramine (MAO-A/B substrate) or benzylamine (MAO-B specific substrate) to produce H2O2. The H2O2 was further oxidized by horseradish peroxidase (HRP) generating fluorescent resorufin, in amounts proportional to MAO-A and MAO-B activities. Clorgyline, a selective MAO-A inhibitor (Figure 1a), and the MAO-B selective inhibitors deprenyl and safinamide (Figure 10) were included as positive controls. The inhibition data for all twenty-five compounds (Table 2) showed that, with the exception of compound 10s, bearing a 3-pyridyl substituent as the “benzyl” motif and 10o, bearing a -CO2Me group at the 4 position of the aromatic ring, all compounds displayed significant ability to inhibit MAO-B (IC50 = 0.203 – 26.607 µM).


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Table 2. In vitro MAO-A/B inhibition resultsa and their comparison to deprenyl, clorgyline, safinamide and reference compound 11. 10 a-s R1 = H (Scheme 1) N Ar

R1

O

Compound

Ar

CO2H

21, 22 R1 = CO 2Me (Scheme 2) 25, 26, and 29/32 R1 = OH, OBn, CN (Scheme 3)

IC50 MAO-A (µM) ± SEM

IC50 MAO-B (µM) ± SEM

Deprenyl

2.089 ± 0.187

0.007 ± 0.001

Safinamide

>50 µM (UTC)b

0.024 ± 0.003

Clorgyline

0.002 ± 0.001

7.647 ± 1.202

11

0.161 ± 0.002

0.005 ± 0.001

10a

>50 µM (UTC)b

0.894 ± 0.013

>50 µM (UTC)b

0.713 ± 0.044

>50 µM (UTC)b

0.264 ± 0.011

217.101 ± 116.14c

0.864 ± 0.107

>50 µM (UTC)b

14.086 ± 1.08

>50 µM (UTC)b

21.267 ± 1.59

Cl

10b

10c

Cl

10d Cl

10e

10f

CN

NC


14

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10g

>50 µM (UTC)b

3.749 ± 0.468

NC

10h

F

>50 µM (UTC)b

0.869 ± 0.104

10i

F

>50 µM (UTC)b

0.911 ± 0.183

>50 µM (UTC)b

0.203 ± 0.004

41.373 ± 20.021

0.218 ± 0.03

>50 µM (UTC)b

1.38 ± 0.109

F

10j

Cl

Br

10k

Cl

Cl

10l

Cl

Cl

10m

O 2N

>50 µM (UTC)b

4.372 ± 0.999

10n

MeO

358.922cd

2.109 ± 0.109

>50 µM (UTC)be

360.579ce

954.993cd

14.219 ± 2.764

>50 µM (UTC)be

26.607e

>50 µM (UTC)b

22.044 ± 1.20

10o MeO 2C

10p

N

10q

10r N


15


10s

>50 µM (UTC)b

>50 µM (UTC)b

21

>50 µM (UTC)b

7.928 ± 2.026

>50 µM (UTC)b

1.26 ± 0.172

25

>50 µM (UTC)b

1.012 ± 0.275

26

>50 µM (UTC)b

8.00 ± 1.24

29

>50 µM (UTC)b

0.532 ± 0.0276

125.996 ± 73.328c

0.206 ± 0.047

22

32

Cl

Cl

Page 16 of 96

Cl

a) All values represent the means of data ± SEM from two independent experiments, each performed in duplicate. b) Compounds did not reach 50% inhibition at the highest compound concentration tested (50 µM), i.e. Prism was unable to calculate an IC50 value within the curvature (>50 µM (UTC) = unable to calculate). c) IC50 values were determined by extrapolating the curvature of log (inhibitor) vs. normalized response with a variable slope in Prism 5.02 (GraphPad Software) resulting in higher than 50 µM IC50 values. d) Two independent experiments were performed but GraphPad Prism was unable to determine an IC50 value for MAO-A for both experiments therefore no SEM shown. e) IC50 values represent data from one independent experiment, performed in duplicate.

Further, at concentrations up to 50 µM the majority of these compounds displayed no activity against MAO-A (>50 µM (UTC)). Thus, even for the least active of these polar inhibitors the selectivity toward MAO-B relative to MAO-A was high. Within the series of compounds 10a-s,


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the three mono/dihalo substituted analogues 10c (IC50 0.264 µM), 10j (IC50 0.203 µM), and 10k (IC50 0.218 µM) were the most potent (Table 2, Figure 3), being approximately 4 X more active than the parent unsubstituted compound 10a (IC50 0.894). Introduction of a polar cyano (10e, 10f, 10g), nitro (10m) or ester function (10o) onto the benzyl motif resulted in a loss in activity, as did exchanging the phenyl by a pyridine (10q, r, s), or replacing the benzyl group by a bicyclic naphthalene ring (10p) (Table 2). Expanding further the scope of the SAR, replacement of the methamphetamine C-2 methyl group by a more polar CO2Me function, as in 21 (IC50 7.928 µM), and 22 (IC50 1.26 µM) also resulted in a decrease in potency (Table 2). In contrast, compound 32 (IC50 0.206 µM), in which the C-2 methyl group was replaced by CH2CN and the CH2Ar motif corresponded to 3,4dichlorophenyl ring was found, along with compound 10j (IC50 0.203 µM), to be the most potent of the polar compound B analogue identified (Table 2). Interestingly, the inhibitory activities of the four most active compounds, 10c, j, k and 32, were approximately 100 times less than the parent irreversible MAO-B inhibitor deprenyl and its O-Bn substituted analogue 11. However, a more informative measure of potency was obtained by comparing their activities to that for the reversible benzylamine-based MAO-B selective inhibitor safinamide. Under the assay conditions used in the present study, it was determined that compounds 10c, j, k and 32 and safinamide have comparable sub-micromolar activities, safinamide being more potent by a factor of 10 (Table 2). Thus, providing the four hit compounds 10c, j, k, and 32 do not enter the BBB to any significant extent, they could be used to selectively target peripheral MAO-B. They were consequently evaluated in assays to measure P-gp interaction, in vitro metabolic stability, and in vivo pharmacokinetic properties after intravenous, oral and subcutaneous administration.


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Figure 3. Inhibition of MAO-A and MAO-B enzyme activity by lead compounds 10c, 10j, 10k and 32. Assessment of P-gp Interaction with MAO-B Inhibitors Prediction and comparison of BBB permeability and P-gp associated efflux in MDCK-WT and MDCK-MDR1 II cell culture model has been widely used in CNS drug discovery. MDCKMDR1 II cells are derived from transfecting the human MDR1 gene encoding P-glycoprotein into MDCK cells36. P-gp is highly expressed in the capillaries of the blood-brain barrier and acts as a major efflux transporter that affects drug permeability into the brain37 . The ability of the selected hit compounds 10c, j, k, and 32 to cross the BBB in bilateral directions could thus be predicted based on efflux ratios in MDCK-WT and MDCK-MDR1 transwell assays (Figure 4).


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Figure 4. Permeability assays and calculated efflux ratios for compounds 10c, j, k, and 32 using wild type MDCK-WT and MDCK-MDR1 II cell lines. Efflux ratio ≥ 2 identifies the compound as a P-gp substrate. Cultures were treated with (white) or without (black) verapamil (an established P-gp inhibitor). The values were indicated as mean ± standard deviation (n=3). Using the efflux ratio of 2 as a cut off, compounds with efflux ratios greater than 2 were categorized as P-gp substrates. In MDCK-WT cells the calculated efflux ratios remained under 2 for the four hit molecules and deprenyl, whereas cetirizine (positive control) showed decreased values from 3.00 to 1.39 in the presence of verapamil (P-gp inhibitor), indicating positive drug efflux by P-gp. The four MAO-B inhibitors were also evaluated in MDCK-MDR1 II cells, which


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is a common cell line used to study in vitro brain uptake and to classify human P-gp substrates. Deprenyl, which is not a P-gp substrate, demonstrated unchanged efflux ratios in MDR1 cells and MDCK-WT cells. In MDCK-MDR1 II cells, cetirizine had efflux ratio of 4.12 and 1.41 without and with verapamil, respectively. The data for both MDCK cell lines confirmed that cetirizine is a P-gp substrate. Compound 10c efflux ratio was not affected by the presence of verapamil. Compound 10j demonstrated mild interactions with P-gp indicated by the decrease in efflux ratio from 2.12 to 1.15 in MDR1 cells. Compound 10k was found to be a P-gp substrate resulting in an efflux ratio of 3.07 without verapamil and 1.22 with verapamil. Compound 32 exhibited no change in efflux ratios after verapamil treatment. Based on the results, compound 10k was classified as the active P-gp substrate and compound 10j displayed very mild efflux transport by P-gp among the four lead compounds, whereas compounds 10c and 32 were not classified as P-gp substrates.

In Vitro Metabolic Stability. In an effort to determine the susceptibility of compounds 10c, j, k and 32 to metabolic clearance38, they were assessed in vitro for metabolic stability in plasma (mouse/human), liver microsomes (mouse/human) and cryopreserved hepatocytes (mouse). As shown in Figure 5A and 5B, all four novel compounds were stable in both mouse and human plasma with ≥ 75% of the parent remaining intact after 4 h incubation at 37°C. In comparison, deprenyl was more rapidly metabolized, with ~ 61% and 55% remaining at the end of the 4 h incubation period in mouse and human plasma, respectively (Figure 5A and 5B).


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Figure 5. In vitro metabolic stability of the novel compounds in plasma (mouse/human), liver microsomes (mouse/human), and hepatocytes (mouse). The positive control compounds were enalapril (mouse plasma), procaine (human plasma), and propranolol (mouse liver microsomes, human liver microsomes, and mouse hepatocytes). Data are shown as mean ± standard deviation (SD). Each experiment was done in triplicate.


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In mouse liver microsomes the four active hits displayed moderate-to-high stability, with compound 32 being the least stable (~ 39% remaining) (Figure 5C). Furthermore, the observed metabolic turnover in mouse liver microsomes was almost entirely β-nicotinamide adenine dinucleotide phosphate (NADPH; cofactor)-dependent, as discerned from the ‘minus NADPH’ condition data. The mean % parent remaining after 60 min incubation without NADPH were respectively 87.3, 93.4, 89.5, and 96.9% for compounds 10c, j, k and 32. In contrast to mouse liver microsomes, all four hit compounds were more stable in human liver microsomes, with ≥ 90% parent remaining at the end of the 60 min incubation period (Figure 5D). These data suggest species-dependent differences in their metabolic stability. Once again, deprenyl was less stable than the four hit compounds in both mouse- (Figure 5C) and human liver microsomes (Figure 5D). Upon incubation with cryopreserved mouse hepatocytes, compounds 10c, j and k were almost completely stable with ≥ 90% parent remaining intact after 60 min incubation, whereas compound 32 showed moderate stability in hepatocytes, with ~ 64% remaining at 60 min (Figure 5E). In all these metabolic stability studies, the observed stability profiles of the positive control compounds enalapril, procaine and propranolol were consistent with our historical data. Overall, the novel MAO-B inhibitors 10c, j, and k were determined to be stable in plasma (mouse/human), liver microsomes (mouse/human), and hepatocytes (mouse), whereas, compound 32 was stable in mouse/human plasma and human liver microsomes, and less stable in mouse liver microsomes and mouse hepatocytes.

In Vivo Acute Tolerability Study. Tolerability tests were carried out to assess whether compounds 10c, j, k and 32 could be safely administered in vivo at the defined therapeutic concentrations and duration. For our


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assessment, animals were clinically observed for any signs of acute toxicity following compound administration. No abnormal clinical findings were noted for the four hit compounds following administration. All animals tolerated the single dose of each compound injected (57.5 µmol/kg), at the specified route of administration, and survived until the termination of the study, at which they were euthanized. In contrast, several animals exhibited lethargy and piloerection after injection with the same dose of deprenyl, which was noted as early as one hour post-injection, but the effects diminished over time.

Pharmacokinetic Study Results from Intravenous Administration. To examine pharmacokinetic properties, the four selected MAO-B inhibitors were administered to mice intravenously (iv) at a single (57.5 µmol/kg) dose. Plasma and brain samples were collected and the concentrations measured were plotted against each time point in a pharmacokinetic curve using GraphPad Prism (Figure 6 and Figure 7). Pharmacokinetic parameters were generated by non-compartmental analysis (NCA) of the data using Phoenix WinNonLin program. Some of the parameters such as half-life (HL Lambda_z), peak plasma concentration (Cmax), time to reach Cmax (Tmax), area under the curve from time zero to the last quantifiable concentration (AUClast) to assess the extent of bioavailability, and the brain to plasma ratio Kp (AUClastbrain/AUClastplasma) as a measure of brain penetration are shown in Table 3.


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Figure 6. Plasma pharmacokinetic curves of deprenyl and compounds 10c, 10j, 10k and 32 following a single 57.5 µmol/kg intravenous dose in C57BL/6 mice. 21 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 7 timepoints (0min, 5min, 15min, 30min, 1h, 4h, 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots.


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Figure 7. Brain pharmacokinetic curves of deprenyl and compounds 10c, 10j, 10k and 32 following a single intravenously 57.5 µmol/kg dose in C57BL/6 mice. 21 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 7 timepoints (0min, 5min, 15min, 30min, 1h, 4h, 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots.


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Table 3.

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Plasma (top) and brain (bottom) pharmacokinetic parameters of deprenyl and

compounds 10c, 10j, 10k and 32 following a single 57.5 µmol/kg dose via intravenous administration in C57BL/6 mice. 21 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 7 timepoints (0min, 5min, 15min, 30min, 1h, 4h, 24h).

Values that were above or below the limit of quantification or were undetected were omitted from the pharmacokinetic calculations.

Following IV administration, the plasma Cmax for deprenyl (568.91 ng/mL) was much lower than that of the other compounds (10c, 16567.55 ng/mL; 10j, 16746.35 ng/mL; 10k, 16191.38 ng/mL and 32, 52103.22 ng/mL). Deprenyl attained a Cmax of 4219.19 ng/mL in brain by 5 min (Tmax), whereas the other compounds took longer to reach Cmax and/or had a significantly lower Cmax (10c, 648.47 ng/mL; 10j, 653.02 ng/mL; 10k, 452.93 ng/mL; and 32, 305.33 ng/mL). These data suggested that deprenyl readily passes into the brain while the other polar compounds remain mostly in the plasma compartment. This is further supported by the brain to


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plasma distribution ratio (Kp) based on AUClast values which is much higher for deprenyl (5.73) than for compounds 10c (0.02), 10j (0.11), 10k (0.09) and 32 (0.004).

Note that the

pharmacokinetic results are preliminary in nature, as one or more of the compounds may undergo enterohepatic recirculation and/or a two-phase elimination (e.g. compound 10j). Additional time points between 4 and 24 hours and/or beyond 24 hours may provide a more descriptive analysis in future studies. Nevertheless, the overall results showed that the polar MAO-B inhibitors 10c, 10j, 10k, and 32 have reduced transport across the blood-brain barrier as predicted.

Pharmacokinetic Study Results from Subcutaneous and Oral Administration Pharmacokinetic studies of subcutaneous and oral administration were carried out for compounds 10j and 32. The pharmacokinetic curves for these studies are illustrated in Figures 8 and 9 and the calculated pharmacokinetic parameters are presented in Table 4.


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Figure 8. Plasma pharmacokinetic curves of compound 10j and compound 32 following a single 57.5 µmol/kg dose via subcutaneous (SC) and oral (PO) administration. 15 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 5 timepoints (1, 2, 4, 6 and 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots.

Figure 9. Brain pharmacokinetic curves of compound 10j and compound 32 following a single 57.5 µmol/kg dose via subcutaneous and oral administration. 15 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 5 timepoints (1, 2, 4, 6 and 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were


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plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots. Table 4. Plasma (top) and brain (bottom) pharmacokinetic parameters of compound 10j and compound 32, following a single 57.5 µmol/kg dose via subcutaneous (SC) or oral (PO) administration. 15 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 5 timepoints (1, 2, 4, 6 and 24h).

Values that were above or below the limit of quantification or were undetected were omitted from the pharmacokinetic calculations.

After subcutaneous dosing the plasma concentrations of compound 10j increased to a Cmax of 7105.06 ng/ml at 2 hours, while compound 32 increased to a Cmax of 13346.31 ng/ml at 1 hour (Table 4). After oral administration the plasma concentration of compound 10j increased to a


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Cmax of 8529.33 ng/ml at 1 hours, while compound 32 increased to a Cmax of 16823.87 ng/ml at 1 hour. Plasma concentrations then declined overtime with a half-life of 4.81 and 3.59 hours for compound 10j, and 3.62 and 2.93 hours for compound 32, respectively, for sc and po. It appears that the Cmax and half-life were relatively comparable for both routes of administration. In terms of bioavailability, the AUClast of compound 10j from sc and po dosing was 66910.31 and 103854.75 h*ng/mL, respectively. Similarly, the AUClast of compound 32 from sc and oral dosing was 35118.27 and 72959.25 h*ng/mL, respectively. These results indicate that both compounds are bioavailable in plasma following either sc or po administration. In the brain, compound 10j took much longer (i.e. 6 hours) to reach the Cmax of 806.41 and 811.55 ng/mL following sc and po dosing, respectively. On the other hand, compound 32 reached Cmax after 1 hour, but the brain concentrations were low at 91.26 ng/ml for sc dosing and 91.94 ng/mL for po dosing. The half-life was non-calculable for compound 10j as it remained in the brain past the 24 hours time point. Likewise, it was also difficult to calculate the half-life for compound 32 as the brain concentrations were at the lower limits of quantification in the later time points. The AUClast is 12913.36 h*ng/mL and 12566.46 h*ng/mL for compound 10j and 176.32 h*ng/mL and 116.43 h*ng/mL for compound 32 under sc and po dosing. The brain to plasma distribution ratio (Kp) based on AUClast values is 0.19 and 0.12 for compound 10j for the sc and po route and 0.005 and 0.002 for compound 32 for the sc and po route, respectively. Therefore, these data support that both compounds do not enter the brain readily for all routes of administration tested. This was expected based on the design of our compounds to not cross the blood-brain barrier. Future studies with additional time points will provide more detailed pharmacokinetic profiles of the compounds.


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DISCUSSION AND CONCLUSION The in vitro data (Table 2) showed that the polar type B methamphetamine derivatives 10a-s, obtained by replacing the acetylene unit in deprenyl by a CO2H function and introducing a p-OCH2Ar motif onto the phenyl ring were selective MAO-B inhibitors. The most potent compounds were the halo substituted analogues 10c, j and k (R and/or R’ = Cl, Br) (IC50 values 0.203 – 0.264 µM) (Figure 10).

Figure 10.

Structural comparison of compounds 10c, 10j and 10k with safinamide, and

compounds 21, 22, 25, 26, 29 and 32 with the coumarin amide 33. The V shaped wedges represent the constriction residues (Tyr 326 and Ile 199) in the MAO-B binding site. These molecules bear a structural resemblance to the reversible and selective MAO-B inhibitor safinamide39, which also contains a -OCH2Ar motif in its structure. Structure-activity studies on safinamide have shown that both potency and MAO-B selectivity are enhanced by incorporating halogen atoms on the O-benzyl phenyl ring.39 Relative to 10a compounds 10c, 10j and 10k show


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the same trend, suggesting that they bind in a similar way to safinamide in the MAO-B site (Figure 11). Indeed, X-ray diffraction data for safinamide and modeling studies carried out on compounds 10c, 10j and 10k (Figure 11 and Figures 11a, b (in the Supplementary Information), and Figure 10) indicate that they all bind in the MAO-B substrate site such that the ether linker straddles the restriction site defined by residues Tyr 326 and Ile 199 and the Ar motif extends toward the exterior cavity, which is globally more hydrophobic than the corresponding pocket in MAO-A.40 In the interior cavity (catalytic site), the polar neutral amide function in safinamide curves away from the FAD co-factor, with the tertiary amine and amide NH forming H-bonds to the side chin C=O in Gln 206. The carboxylic acid functions in compounds 10c, 10k and 10j are also predicted to not interact with the FAD co-factor (O=C….FAD (N5) distance of 5.19-6.56 Å). Instead, in its deprotonated state, the acid function H-bonds to the phenolic hydroxyl group in proximal tyrosine residues (Tyr 435 or 188). Further, being more extended phenethylamine-type compounds, the tertiary nitrogen forms H-bonding or cation-π interactions with proximal tyrosines, rather than H=bonding to Gln 206. These differences in binding mode with residues in the catalytic site may, in part, account for the ten fold difference in measured IC50 values for compounds 10c, 10k and 10j (see Table 2) relative to safinamide (IC50 = 0.024 µM). Although larger in structure (greater volume), a wide range of coumarin-based compounds, such as coumarin amide 33 (Figure 10), are also potent and selective MAO-B inhibitors.41 From structural (X-ray diffraction) studies it has been determined that coumarin 33 bind in the MAO-B substrate site in a similar manner to safinamide.40 This showed that the additional atoms composing the lactone ring are tolerated in this site. Indeed, structures of inhibitor-MAO-B complexes determined for a wide variety of MAO-B inhibitors demonstrate that there is considerable room for structural variation in the sub-elements that occupy the inner substrate


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cavity.42, 43 It was with this knowledge in mind that the polar compounds 21, 22, 25, 26, 29 and 32 (Figure 10 and table 2) were designed and tested. Interestingly, compound 22, where the CO2Me represents a ring-opened version of the lactone motif, and compound 25, in which the ester function has been reduced to the corresponding alcohol, were active in the 1 µM range. However, within this framework it was the cyano compound 29 (IC50 MAO-B 0.532 µM) and 32 (IC50 MAO-B 0.206 µM) that displayed the greatest affinity for the MAO-B binding site (Figure 11, bottom panel). Interestingly, in the predicted model, the CN group is not observed to form strong interactions with the catalytic site residues.


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Figure 11. Binding modes of safinamide (top panels, carbons shown in pink) in human MAO-B (PDB accession code 2v5z), and predicted binding modes of 10j (mid panels, carbons shown in cyan) and 32 (lower panels, carbons shown in cyan). For the docking, the coordinates of the safinamide—hMAO-B co-complex were used (see Material & Methods). Hydrogen bonds and cation—π interactions are indicated by green and magenta dashed lines, respectively. For the cation—π interactions, the center of the aromatic ring is shown as a small sphere. Protein residues are labeled with the 3-letter code followed by the residue number. The N5 of FAD is indicated by an italicized label. The orientation of the structures has been rotated by roughly 180 degrees between the left and right panels in order to illustrate the predicted interactions to the protein for the docked poses.

In terms of their selectivity for MAO-B over MAO-A, the four polar compounds 10c, 10j, 10k and 32 were highly MAO-B selective. Any impact on MAO-A activity can be associated with significant dietary concerns. Approximately 70% of MAO activity in the intestine is due to MAO-A44 and inhibition of its activity can increase the risk of hypertensive crisis due to tyramine uptake and its potentiation of cardiovascular activity by releasing noradrenaline (cheese effect).4 This negative side effect is associated with MAO-A inhibitors.4 Recently long-term use of selective irreversible MAO-B inhibitors (ie. deprenyl) has been suggested to also inhibit MAO-A activity.45 We present evidence that compounds 10j and 32 were well absorbed orally (Table 4), however, they are unlikely to be at risk of the cheese effect because they were reversible inhibitors and more MAO-B selective over MAO-A when compared to deprenyl. MAO-B inhibitors like deprenyl when delivered transdermally (6 mg/24hr) are effective antidepressants46, but they are preferentially used for the management of neurodegenerative


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diseases such as Alzheimer’s and Parkinson’s.7 In this context, they must pass the BBB to produce their therapeutic effect. Our objective has been to develop polar MAO-B inhibitors that have significantly reduced BBB permeability. In the mouse iv pharmacokinetic study (Table 3) the maximum concentration (Cmax) of deprenyl in the brain (4,219.19 ng/ml) far exceeded what was recovered from the brain of animals treated with equimolar doses of compounds 10c, 10j and 10k (452.93-653.02 ng/ml) and compound 32 (305.33 ng/ml). These data support that transport of the novel compounds into the brain has been significantly reduced. The maximal concentrations of drugs recovered from the brains (Cmax) of animals treated with compounds 10c (648.47 ng/ml), and 10j (653.02 ng/ml) were higher than what was found for 10k (452.93 ng/ml). This 30% lower Cmax for compound 10k in the brain when compared to compounds 10c and 10j may reflect that compound 10k like cetirizine47, is an active P-gp substrate (Figure 4). In contrast, compound 10j, a weak P-gp interactor was not associated with a lower Cmax in the brain over compound 10c. Compounds 10c, 10j and 10k also exhibited significantly higher maximum plasma concentrations (Cmax) (16,191.38-16,746.35 ng/ml) when compared to deprenyl (5,689.91 ng/ml) and increased in vivo plasma half-life (3.93-4.51hr) (HL-Lambda-z) when compared to deprenyl (0.56hr) (Table 3). These 3 hit compounds when compared to deprenyl were more stable in the mouse and human plasma stability in vitro assays and the mouse and human liver microsome and mouse hepatocyte assays (Figure 5). This increase in relative metabolic stability of compounds 10c, 10j and 10k is reflected in higher plasma concentrations (Cmax) and longer plasma half-lives (HL-Lambda-z) in the intravenous pharmacokinetic study. Overall compounds 10c, 10j and 10k all demonstrated a much lower brain to plasma ratio (Kp) (range 0.02-0.11) and all were significantly lower than deprenyl (5.73). Compound 10j showed similar AUClast brain/plasma ratios after subcutaneous (0.19)


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and oral (0.12). These data support that compounds 10c, 10j and 10k all had reduced penetration into the brain over deprenyl. Compound 10k had the lowest maximum brain concentration, however, it had the poorest selectivity for MAO-B over MAO-A and therefore would not be the strongest lead. In contrast, compound 10c with a better selectivity for MAO-B over MAO-A and the lowest AUClast brain/plasma ratio of these 3 leads makes it a strong lead. After intravenous administration the in vivo plasma half-life for compound 32 (0.74h) (HLLambda-z) was significantly shorter than the half-lives for compounds 10c, 10j and 10k hits (range 3.93-4.51h) but was longer than deprenyl (0.56h) (Table 3). This shorter in vivo half-life for compound 32 may reflect increased rate of drug metabolism in the mouse liver. Evidence for this was found in the mouse liver microsomal and hepatocyte stability assays (Figure 5). In these metabolic assays compound 32 was more stable then deprenyl but less stable when compared to compounds 10c, 10j and 10k. The human plasma and liver microsome assays did not reflect these findings suggesting species-specific differences. Of the 4 hits tested, compound 32 had the lowest Cmax (305.33 ng/ml) in the brain after intravenous administration (Table 3) and an AUClast brain/plasma ratio of 0.004, 0.005 and 0.002 after intravenous, subcutaneous and oral administration, respectively (Tables 3 and 4). This was two orders of magnitude less that the other 3 hits and was significantly lower than what was determined for deprenyl (5.73) after intravenous administration. The lower brain concentration of compound 32 can not be explained by a P-gp efflux mechanism (Figure 4). Compound 32 exhibited the lowest brain concentration (Cmax, Tables 3 and 4) that was measured for all administration routes, however, its reduced stability in the mouse liver assays (not human liver assays) suggests its bioavailabilty may be reduced in mouse studies. Regardless, compound 32 serves as the second lead for further optimization studies. In future long term disease modelling studies the plasma, tissue and brain


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samples will be assayed for MAO-A and B residual enzyme activity, MAO-B selectivity over MAO-A as well as reduced transport across the BBB. Upregulation of MAO-B protein and gene expression has been identified in samples taken from patients with diseases of the heart and rat periodontium14,15,17. Collectively, the role of MAO-B in the pathogenesis of these inflammatory diseases and the mechanisms by which MAO inhibition may reduce these diseases is not fully understood. However, the MAO B selective inhibtors deprenyl reduced proinflammatory cytokine expression in in vitro airway epithelial cell cultures19 and

pargyline2 reduced mast cell degranulation and fibrosis in a diabetic

cardiomyopathy animal model17. In conclusion, the development of novel polar selective MAOB inhibitors with reduced BBB penetration has positioned our laboratories to test their potential efficacy for the management of non-CNS inflammatory diseases in future studies.

EXPERIMENTAL SECTION

Experimental General: All chemicals were purchased from Sigma Aldrich (Oakville, Canada), Alpha-aesar (Tewksbury, United States), or Oakwood Chemicals (Estill, United States) and they were used without purification unless mentioned. All solvents were dried and kept under N2. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker AC 400 Ultrashield 10 spectrophotometer. Chemical shifts are expressed in ppm, (δ scale). When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), m (multiplet), t (triplet), dd (doublet of doublet), td (triplet of doublet), tt (triplet of triplet), ddd (doublet of doublet of doublet). Coupling constants are reported in Hertz (Hz). High resolution mass spectra for compound 25 was collected on a Waters Xevo G2-S QTof, run in


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resolution mode. 1 µM solution of compound 25 (in 30% ACN, 0.5% FA) were infused at 3 µl/min. Spectra were accumulated for 30 seconds for the mass measurement. High resolution mass spectra for all other compounds were recorded on a AB Sciex UHPLC/MS/MS System and a Thermo Scientific Q Exactive Orbitrap High Resolution Mass Spectrometer, respectively. Flash column chromatography was performed using silica gel (Silicycle, Siliaflash® F60, 4063µm, 230-400 mesh), or on a Biotage Isolera purification system, (PartnerTech Ǻtvidaberg AB) using pre-packed silica gel columns (Biotage, part no. FSKO-1107-0010, FSKO-1107-0025, or FSKO-1107- 0050). A Biotage Initiator 2.5 apparatus was used for experiments using microwave heating. The purity of all final compounds was determined by HPLC, using a Waters Alliance e2696 separations module coupled to a Waters 2489 UV/Vis-detector (λ = 225 nm). The column was a reversed phase C18 Waters Atlantis T3, 100 Å, 5 µm particle size (4.6 × 150 mm), supported by a C18 guard cartridge and was operated in an oven at 40 °C. The column was eluted with the following gradient: A = 0.1% trifluoroacetic acid (TFA) in water; B = methanol; flow rate = 1 mL/min; 0-10 min 10-100% B; 10-13 min 100% B. With the exception of compounds 10m (93.6%), 10p (91.8%), 22 (93.4%), and 25 (91.6%), the other twenty-one compounds were established to be > 95% pure. For the biological assays: Recombinant human MAO A and B proteins, the MAO-B inhibitors deprenyl and safinamide, cetirizine, verapamil, Lucifer yellow, enalapril and propranolol were obtained from Sigma-Aldrich (St. Louis, MO). MDCK-WT cells were obtained from the American Type Culture Collection (Manassas, VA). MDCK-MDR1 II cells were obtained from the Netherlands Cancer Institute (Amsterdam, The Netherlands). Mouse and human plasma were obtained from GeneTex (Irvine, CA) and BioreclamationIVT (Baltimore, MD), respectively. Liver microsomes (mouse and human) were


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obtained from BD Gentest (Woburn, MA) or BioreclamationIVT (Baltimore, MD). Cryopreserved mouse hepatocytes were obtained from BioreclamationIVT (Baltimore, MD).

Chemistry L-(N-Methoxycarbonyl)(O-Benzyl)tyrosine methyl ester (2) Adapting the procedure by Desjardine et al31, a mixture of L-N-(methoxycarbonyl)-tyrosine methyl ester 130 (8.0 g, 32 mmol), benzyl bromide (4.5 mL, 38 mmol) and K2CO3 (5.2 g, 38 mmol) in acetone (125 mL) was stirred for 16 h at room temperature and then refluxed for 3 h. Solids were filtered off and the filtrate was concentrated to dryness in vacuo. The crude product mixture was silica column chromatographed (Hexane/EtOAc 9:1 to 5:5), affording 2 as a translucent wax, which turned into a white solid upon standing (9.3 g, 85% yield). 1H NMR (CDCl3) δ: 7.44-7.37 (m, 4H), 7.357.30 (m, 1H), 7.03 (br d, J = 8.0Hz, 2H), 6.90 (br d, J = 8.0Hz, 2H), 5.13 (br d, J = 7.8Hz, 1H), 5.04 (s, 2H), 4.61 (dt, J = 7.8, 5.8Hz, 1H), 3.72 (s, 3H), 3.67 (s, 3H), 3.09-3.00 (m, 2H). L-(N-Methoxycarbonyl)(O-Benzyl))tyrosinol (3) Following the procedure by Ousmer et al.32, compound 2 (3.44 g, 10 mmol) in THF (25 mL) was added dropwise over two hours to a cold (0°C) suspension of LiAlH4 (0.740 g, 20 mmol) in THF (40 mL) maintained under nitrogen. The reaction was stirred overnight at RT, then cooled to 0°C and quenched by slow dropwise addition of 4N HCl (Caution: production of hydrogen gas). The mixture was subsequently diluted by addition of H2O (100 mL) and extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated. Trace amounts of inorganic salts were removed by filtration through a plug of silica-gel (EtOAc/Hexane 1:1), affording 3 as a white solid (3.0 g, 95% yield). 1H NMR (CDCl3) δ: 7.44-7.31 (m, 5H), 7.12 (d, J = 8.8Hz, 2H), 6.92 (d, J = 8.8Hz, 2H), 5.04 (s, 2H), 4.99 (br s, 1H), 3.88 (br s, 1H), 3.68-3.65 (m, 1H), 3.64 (s, 3H),


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3.58-3.54 (m, 1H), 2.80 (m, 2H), 2.41 (br s, 1H). 13C NMR (CDCl3) δ: 157.5, 157.2, 137.0, 130.2 (2xCH), 129.8, 128.5 (2xCH), 127.9, 127.4 (2xCH), 114.9 (2xCH), 70.0, 64.0, 54.1, 52.2, 36.4. L-(N-Methoxycarbonyl)(O-Benzyl))tyrosinol tosylate (4) Following the procedure by Kohno et al.33, a solution of n-Bu4N.HSO4 (0.470 g, 1.38 mmol) in water (10 mL) was added dropwise over 15 minutes to a rapidly stirred and cooled (0°C) biphasic mixture of compound 3 (2.24 g, 7.11 mmol) and p-TsCl (1.49 g, 7.8 mmol, 1.1 equiv.) in 10% aqueous NaOH (15 mL) and benzene (20 mL). The two-phase mixture was subsequently stirred at RT for 30 min. The layers were then separated, the aqueous layer was washed with benzene, and the combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness in vacuo. The crude product mixture was crystallized from EtOAc-Hexane to provide 4 as colorless crystals (2.23 g, 67% yield). 1H NMR (CDCl3) δ: 7.79-7.76 (m, 2H), 7.44-7.31 (m, 7H), 7.00-6.98 (m, 2H), 6.856.83 (m, 2H), 5.03 (s, 2H), 4.82 (br d, J = 7.7Hz, 1H), 4.03-3.95 (m, 2H), 3.92 (dd, J = 9.5, 2.9Hz, 1H), 3.61 (s, 3H), 2.84-2.71 (m, 2H), 2.46 (s, 3H). (R)-Methyl (1-(4-benzyloxyphenyl)propan-2-yl)carbamate (5) Following the procedure by Kohno et al.33, a mixture of compound 4 (2.00 g, 4.26 mmol), zinc dust (2.78 g, 42.61 mmol) and NaI (3.19 g, 21.3 mmol) in THF (20 mL) - H2O (1 mL) was refluxed for 2.5h. Salts and the remaining zinc were then filtered off and the filtrate was concentrated in vacuo. The residue was taken up in EtOAc/water, the layers were separated and the aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give a bright orange solid. Silica flash column chromatography (EtOAc-Hexane; 3:7) provided 5 as a colorless wax, which turned into a white solid upon standing (1.2 g, 94% yield). 1

H NMR (CDCl3) δ: 7.45-7.43 (m, 2H), 7.41-7.37 (m, 2H), 7.35-7.31 (m, 1H), 7.12-7.09 (m,

2H), 6.94-6.91 (m, 2H), 5.05 (s, 2H), 4.63 (br s, 1H), 3.94 (br s, 1H), 3.65 (s, 3H), 2.80 (dd, J =


40

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13.6, 5.2Hz, 1H), 2.66 (dd, J = 13.6, 7.1Hz, 1H), 1.13 (d, J = 6.6Hz, 3H). 13C NMR (CDCl3) δ: 157.6, 156.4, 137.2, 130.5 (2xCH), 130.4, 128.6 (2xCH), 128.0, 127.5 (2xCH), 114.8 (2xCH), 70.1, 52.0, 48.2, 42.1, 20.3. (R)-1-(4-(Benzyloxy)phenyl)-N-methylpropan-2-amine (6) A solution of 5 (1.15 g, 3.84 mmol) in dry THF (5 mL) was added slowly to a solution of LiAlH4 (583 mg, 15.37 mmol) in dry THF (10 mL) at 0°C. The resulting mixture was stirred at reflux for 4 h and then quenched at 0°C by sequential addition of H2O (600 µL), 10% NaOHaq (600 µL), and H2O (1.2 mL), and the solids were removed by filtration and the filtrate was concentrated. The crude mixture was silica flash column chromatographed (DCM/MeOH 98:2 to 90:10), affording 6 as a colorless oil (529 mg, 54% yield). 1H NMR (CDCl3) δ: 7.45-7.42 (m, 2H), 7.41-7.37 (m, 2H), 7.35-7.30 (m, 1H), 7.12-7.09 (m, 2H), 6.94-6.90 (m, 2H), 5.05 (s, 2H), 2.81 (m, 1H), 2.67 (dd, J = 13.4, 7.0Hz, 1H), 2.58 (dd, J = 13.4, 6.3Hz, 1H), 2.40 (s, 3H), 1.78 (br s, 1H), 1.06 (d, J = 6.1Hz, 3H). N-((R)- (1-(4-(Benzyloxy)phenyl)propan-2-yl)(methyl))glycine tert-butyl ester (7) tertButyl bromoacetate (304 µL, 2.06 mmol) was added dropwise to a vigorously stirred solution of compound 6 (525 mg, 2.06 mmol) in DMF (3 mL). Cs2CO3 (1.34 g, 4.11 mmol) was added after 5 minutes, and the resulting suspension was stirred overnight at room temperature. The crude mixture was diluted with water and extracted with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The crude mixture was silica column chromatographed (Hexane/EtOAc 8:2 to 5:5), affording 7 as a viscous pale yellow oil (603 mg, 79% yield). 1H NMR (CDCl3) δ: 7.45-7.42 (m, 2H), 7.40-7.36 (m, 2H), 7.34-7.30 (m, 1H), 7.117.07 (m, 2H), 6.91-6.88 (m, 2H), 5.04 (s, 2H), 3.22 (br s, 2H), 2.97-2.88 (m, 2H), 2.41 (s, 3H), 2.36-2.29 (m, 1H), 1.48 (s, 9H), 0.93 (d, J = 6.7Hz, 3H).


41


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N-((R)- (1-(4-Phenol)propan-2-yl)(methyl))glycine tert-butyl ester (8) Palladium on charcoal (10%, 60 mg) was added to a solution of compound 7 (600 mg, 1.62 mmol) in nitrogen flushed MeOH (20 mL), and the reaction was stirred overnight at room temperature under hydrogen at 1 atmosphere. The mixture was filtered through a 2 layer pad of Celite and alumina to remove the catalyst, and the filtrate was concentrated in vacuo, affording 8 as a light yellow oil (453 mg, 99% yield). 1H NMR (CDCl3) δ: 7.00 (d, J = 8.1Hz, 2H), 6.74 (d, J = 8.1Hz, 2H), 3.23 (s, 2H), 2.95-2.90 (m, 2H), 2.41 (s, 3H), 2.30 (dd, J = 13.3, 11.2Hz, 1H), 1.47 (s, 9H), 0.92 (d, J = 6.1Hz, 3H). N-((R)-(1-(4-(Benzyloxy)phenyl)propan-2-yl)(methyl))glycine

hydrochloride

(10a)

Concentrated HCl (1.5 mL) was added dropwise to a cooled (0°C) solution of tert-butyl ester 8 (200 mg, 0.54 mmol) in THF (1.5 mL), and the mixture was stirred for 3 h. The solvent was then removed in vacuo, and the derived solid material was washed (triturated) with DCM and then C18 reverse phase column chromatographed (H2O/MeOH, 95:5 to 50:50). In this way compound 10a was separated from small quantities (15 mg, 11%) of the contaminant resulting from Odebenzylation. It was isolated as a colorless solid (105 mg, 55% yield). 1H NMR (MeOH-d4) δ: 7.43-7.41 (m, 2H), 7.38-7.34 (m, 2H), 7.32-7.28 (m, 1H), 7.21-7.18 (m, 2H), 6.99-6.96 (m, 2H), 5.07 (s, 2H), 3.70-3.59 (m, 2+1H), 3.13 (dd, J = 13.2, 4.2Hz, 1H), 2.88 (s, 3H), 2.73 (dd, J = 13.2, 10.5Hz, 1H), 1.21 (d, J = 6.7Hz, 3H). 13C NMR (MeOH-d4) δ: 170.0, 159.5, 138.6, 131.5 (2xCH), 129.5 (2xCH), 129.4, 128.9, 128.6 (2xCH), 116.3 (2xCH), 71.0, 64.3, 56.4, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H24NO3+: 314.17507, found: 314.17493. (General

Procedure)

Preparation

of

N-((R)-

(1-(4-(benzyloxy)phenyl)propan-2-

yl)(methyl))glycine tert-butyl esters (9b-s) (step 1) and their conversion to N-((R)- (1(benzyloxy)phenyl)propan-2-yl)(methyl))glycine hydrochlorides (10b-s) (step 2)


42

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Step 1: Compound 8 (1 equiv.), the requisite halide (1.1 equiv.) and K2CO3 (3 equiv.) were added to DMF (2.0 mL for 0.36 mmol of compound 8) and the resulting mixture was stirred overnight at room temperature. Solids in suspension were filtered off and the solvent was evaporated in vacuo. The crude product mixture was column chromatographed (silica gel; Hexane/EtOAc mixtures), to give compounds 9b-s. Step 2: Concentrated HCl (1.5 mL) was added dropwise to a cooled (0°C) solution of the intermediate tert-butyl esters 9b-s in THF (1.5 mL) and the mixture was stirred for 3 h. Removal of the solvent in vacuo and C-18 reverse phase column chromatography (H2O/MeOH mixtures) of the residue provided the desired product 10 b-s. N-((R)-(1-(4-(2-Chlorobenzyloxy)phenyl)propan-2-yl)(methyl))glycine (10b)

via

(9b)

Following

the

two

step

general

procedure

hydrochloride N-((R)-(1-(4-(2-

chlorobenzyloxy)phenyl)propan-2-yl)(methyl)) glycine tert-butyl ester 9b was prepared by reaction of 8 (100 mg, 0.36 mmol) with 2-chlorobenzyl bromide (51 µL, 0.39 mmol). It was isolated after silica chromatography (Hexane/EtOAc 8:2 to 5:5) as a viscous pale yellow oil (83 mg, 57% yield). 1H NMR (CDCl3) δ: 7.57-7.54 (m, 1H), 7.40-7.37 (m, 1H), 7.30-7.22 (m, 2H), 7.12-7.08 (m, 2H), 6.92-6.88 (m, 2H), 5.14 (s, 2H), 3.22 (br s, 2H), 2.97-2.89 (m, 2H), 2.41 (s, 3H), 2.36-2.30 (m, 1H), 1.48 (s, 9H), 0.93 (d, J = 6.7Hz, 3H). Subsequent acid treatment of 9b afforded 10b, isolated as a white solid (69 mg, 87% yield) after C-18 reverse phase chromatography (H2O/MeOH, 95:5 to 50:50). 1H NMR (MeOH-d4) δ: 7.56-7.52 (m, 1H), 7.44-7.40 (m, 1H), 7.33-7.28 (m, 2H), 7.23-7.20 (m, 2H), 6.98-6.96 (m, 2H), 5.13 (s, 2H), 3.70-3.61 (m, 2+1H), 3.15 (dd, J = 13.1, 4.0Hz, 1H), 2.88 (s, 3H), 2.72 (dd, J = 13.1, 10.6Hz, 1H), 1.21 (d, J = 6.7Hz, 3H). 13C NMR (MeOH-d4) δ: 169.9, 159.2, 136.0, 134.0,


43


Page 44 of 96

131.6 (2xCH), 130.4 (3xCH), 129.8, 128.2, 116.3 (2xCH), 68.3, 64.3, 56.4, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H23ClNO3+: 348.13610, found: 348.13620. N-((R)-(1-(4-(3-Chlorobenzyloxy)phenyl)propan-2-yl)(methyl))glycine (10c)

via

(9c)

Following

the

two

step

general

procedure

hydrochloride N-((R)-(1-(4-(3-

chlorobenzyloxy)phenyl)propan-2-yl)(methyl)) glycine tert-butyl ester 9c was prepared by reaction of 8 (400 mg, 1.43 mmol) with 3-chlorobenzyl bromide (226 µL, 1.72 mmol, 1.2 equiv.). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 7:3) as a viscous pale yellow oil (210 mg, 36% yield). 1H NMR (CDCl3) δ: 7.42 (br s, 1H), 7.31-7.27 (m, 3H), 7.117.08 (m, 2H), 6.89-6.85 (m, 2H), 5.00 (s, 2H), 3.21 (s, 2H), 2.96-2.90 (m, 2H), 2.41 (s, 3H), 2.36-2.32 (m, 1H), 1.47 (s, 9H), 0.92 (d, J = 6.8Hz, 3H). Subsequent acid treatment of 9c afforded 10c, isolated as a white solid (145 mg, 73% yield) after C-18 reverse phase chromatography (H2O/MeOH, 95:5 to 3:7). 1H NMR (MeOH-d4) δ: 7.43 (br s 1H), 7.35-7.33 (m, 2H), 7.31-7.27 (m, 1H), 7.20 (d, J = 8.7Hz, 2H), 6.96 (d, J = 8.7Hz, 2H), 5.04 (s, 2H), 3.70-3.55 (m, 2+1H), 3.14 (dd, J = 13.1, 4.0Hz, 1H), 2.88 (s, 3H), 2.71 (dd, J = 13.1, 10.6Hz, 1H), 1.20 (d, J = 6.6Hz, 3H). ).

13

C NMR (MeOH-d4) δ: 169.9, 159.2, 141.1,

135.4 (2xCH), 131.5, 131.1, 129.7, 128.8, 128.3, 126.7, 116.3 (2xCH), 70.0, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H23ClNO3+: 348.13610, found: 348.13617. (R)-N-(1-(4-((4-Chlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine (10d)

via

(9d)

Following

the

two

step

general

procedure

hydrochloride (R)-N-(1-(4-((4-

chlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9d was prepared by reaction of 8 (70 mg, 0.25 mmol) with 4-chlorobenzyl bromide (57 mg, 0.28 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (43 mg, 42% yield). 1H NMR (CDCl3) δ: 7.30-7.27 (m, 4H), 7.04 (d, J = 8.4Hz, 2H), 6.82 (d, J = 8.4Hz, 2H),


44

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4.95 (s, 2H), 3.17 (s, 2H), 2.92-2.85 (m, 2H), 2.37 (s, 3H), 2.28 (dd, J = 14.0, 10.8Hz, 1H), 1.43 (s, 9H), 0.88 (d, J = 6.4Hz, 3H). Subsequent acid treatment of 9d (40 mg, 0.10 mmol) afforded 10d, isolated as a colorless solid (25 mg, 66% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 7.40 (d, J = 8.4Hz, 2H), 7.35 (d, J = 8.4Hz, 2H), 7.20 (d, J = 8.4Hz, 2H), 6.96 (d, J = 8.4Hz, 2H), 5.03 (s, 2H), 3.70-3.59 (m, 2+1H), 3.14 (dd, J = 13.1, 3.8Hz, 1H), 2.88 (s, 3H), 2.72 (dd, J = 13.1, 10.6Hz, 1H), 1.20 (d, J = 6.6Hz, 3H). 13C NMR (MeOH-d4) δ: 169.8, 159.3, 137.5, 134.6, 131.5 (2xCH), 130.1 (2xCH), 129.62, 129.59 (2xCH), 116.4 (2xCH), 70.1, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H23ClNO3+: 348.13610, found: 348.13519. (R)-N-(1-(4-((2-Cyanobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine (10e)

via

(9e)

Following

the

two

step

general

procedure

hydrochloride R)-N-(1-(4-((2-

cyanobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9e was prepared by reaction of 8 (100 mg, 0.36 mmol) with 2-cyanobenzyl bromide (77 mg, 0.39 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless solid (121 mg, 90% yield). 1H NMR (CDCl3) δ: 7.67 (dd, J = 8.4, 0.7Hz, 2H), 7.60 (td, J = 7.6, 1.4Hz, 1H), 7.40 (td, J = 7.6, 1.1Hz, 1H), 7.10 (d, J = 8.6Hz, 2H), 6.90 (d, J = 8.6Hz, 2H), 5.21 (s, 2H), 3.21 (m, 2H), 2.95-2.87 (m, 2H), 2.40 (s, 3H), 2.32 (dd, J = 14.0, 10.8Hz, 1H), 1.46 (s, 9H), 0.91 (d, J = 6.6Hz, 3H). Subsequent acid treatment of 9e afforded 10e, isolated as a colorless solid (88 mg, 77% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 7.77 (br d, J = 7.6, 1H), 7.70-7.66 (m, 2H), 7.54-7.47 (m, 1H), 7.25-7.21 (m, 2H), 7.02-6.99 (m, 2H), 5.21 (s, 2H), 3.71-3.60 (m, 2+1H), 3.16 (dd, J = 13.2, 4.0Hz, 1H), 2.89 (s, 3H), 2.73 (dd, J = 13.2, 10.5Hz, 1H), 1.21 (d, J = 6.7Hz, 3H). 13C NMR (MeOH-d4) δ: 169.9, 159.0, 141.8, 134.3,


45


Page 46 of 96

134.2, 131.6 (2xCH), 130.3, 130.2, 130.0, 118.1, 116.4 (2xCH), 112.9, 69.1, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C20H23N2O3+: 339.17032, found: 339.17056. (R)-N-(1-(4-((3-Cyanobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine (10f)

via

(9f)

Following

the

two

step

general

procedure


cyanobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9f was prepared by reaction of 8 (100 mg, 0.36 mmol) with 3-cyanobenzyl bromide (77 mg, 0.39 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless solid (126 mg, 89% yield). 1H NMR (CDCl3) δ: 7.71 (br s, 1H), 7.64 (br d, J = 8.0Hz, 1H), 7.57 (br d, J = 7.6Hz, 1H), 7.45 (br t, J = 7.6Hz, 1H), 7.10-7.07 (m, 2H), 6.87-6.83 (m, 2H), 5.03 (s, 2H), 3.20 (d, J = 1.4Hz, 2H), 2.94-2.86 (m, 2H), 2.39 (s, 3H), 2.32 (dd, J = 14.1, 10.7Hz, 1H), 1.46 (s, 9H), 0.91 (d, J = 6.5Hz, 3H). Subsequent acid treatment of 9f afforded 10f, isolated as a colorless solid (90 mg, 75% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7).

1

H NMR (MeOH-d4) δ:

7.78 (br s 1H), 7.74 (br d, J = 7.7Hz, 1H), 7.66 (dt, J = 7.7, 1.3Hz, 1H), 7.55 (t, J = 7.7Hz, 1H), 7.24-7.20 (m, 2H), 7.00-6.96 (m, 2H), 5.11 (s, 2H), 3.70-3.58 (m, 2+1H), 3.15 (dd, J = 13.2, 4.2Hz, 1H), 2.89 (s, 3H), 2.73 (dd, J = 13.2, 10.6Hz, 1H), 1.21 (d, J = 6.7Hz, 3H).

13

C NMR

(MeOH-d4) δ: 169.9, 159.0, 140.6, 133.0, 132.6, 131.8, 131.6 (2xCH), 130.7, 129.9, 119.6, 116.3 (2xCH), 113.5, 69.6, 64.3, 56.5, 38.8, 37.5, 13.1. HRMS: m/z calculated for C20H23N2O3+: 339.17032, found: 339.17050. (R)-N-(1-(4-((4-Cyanobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine (10g)

via

(9g)

Following

the

two

step

general

procedure


cyanobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9g was prepared by reaction of 8 (100mg, 0.36 mmol) with 4-cyanobenzyl bromide (77mg, 0.39 mmol). It was


46

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isolated after silica chromatography (Hexane/EtOAc 9:1 to 6:4) as a colorless wax (127 mg, 90% yield). 1H NMR (CDCl3) δ: 7.67 (d, J = 8.4Hz, 2H), 7.54 (d, J = 8.0Hz, 2H), 7.10 (d, J = 8.4Hz, 2H), 6.86 (d, J = 8.4Hz, 2H), 5.09 (s, 2H), 3.22 (s, 2H), 2.97-2.93 (m, 2H), 2.42 (s, 3H), 2.372.31 (m, 1H), 1.47 (s, 9H), 0.93 (d, J = 6.4Hz, 3H). Subsequent acid treatment of 9g (110 mg, 0.28 mmol) afforded 10g, isolated as an off-white solid (51 mg, 49% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 7.72 (d, J = 8.2Hz, 2H), 7.60 (d, J = 8.2Hz, 2H), 7.22 (d, J = 8.6Hz, 2H), 6.97 (d, J = 8.6Hz, 2H), 5.15 (s, 2H), 3.70-3.59 (m, 2+1H), 3.15 (dd, J = 13.1, 4.2Hz, 1H), 2.89 (s, 3H), 2.73 (dd, J = 13.1, 10.6Hz, 1H), 1.21 (d, J = 6.6Hz, 3H). 13C NMR (MeOH-d4) δ: 169.9, 159.0, 144.5, 133.4, 131.76 129.9, 128.9, 119.7, 116.3, 112.5, 69.9, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C20H23N2O3+: 339.17032, found: 339.17065. (R)-N-(1-(4-((3-fluorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine (10h)

via

(9h)

Following

the

two

step

general

procedure


fluorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9h was prepared by reaction of 8 (100 mg, 0.36 mmol) with 3-fluorobenzyl bromide (48 µL, 0.39 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (118 mg, 85% yield). 1H NMR (CDCl3) δ: 7.35-7.30 (m, 1H), 7.19-7.13 (m, 2H), 7.11-7.07 (m, 2H), 6.99 (td, J = 8.5, 2.2Hz, 1H), 6.89-6.86 (m, 2H), 5.01 (s, 2H), 3.21 (s, 2H), 2.97-2.89 (m, 2H), 2.41 (s, 3H), 2.33 (dd, J = 14.1, 10.8Hz, 1H), 1.47 (s, 9H), 0.93 (d, J = 6.5Hz, 3H). Subsequent acid treatment of 9h afforded 10h, isolated as a colorless solid (86 mg, 77% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 7.35 (td, J = 8.0, 6.0Hz, 1H), 7.22-7.18 (m, 3H), 7.16-7.13 (m, 1H), 7.01 (td, J = 8.5, 2.5Hz, 1H), 6.97-6.93 (m, 2H), 5.03 (s, 2H), 3.70-3.59 (m, 2+1H), 3.15 (dd, J = 13.2, 3.9Hz, 1H), 2.88 (s,


47


3H), 2.70 (dd, J = 13.2, 10.6Hz, 1H), 1.19 (d, J = 6.6Hz, 3H).

Page 48 of 96

13

C NMR (MeOH-d4) δ: 169.9,

164.2 (d, J = 244.2Hz), 159.1, 141.5 (d, J = 7.3Hz), 131.5 (2xCH), 131.3 (d, J = 8.1Hz), 129.7, 124.0 (d, J = 2.8Hz), 116.3 (2xCH), 115.4 (d, J = 21.3Hz), 114.9 (d, J = 22.3Hz), 70.0 (d, J = 2.1Hz), 64.2, 56.4, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H23FNO3+: 332.16565, found: 332.16595. (R)-N-(1-(4-((3,5-Difluorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (10i)

via

(9i)

Following

the

two

step

general

procedure

(R)-N-(1-(4-((3,5-

difluorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9i was prepared by reaction of 8 (100 mg, 0.36 mmol) with 3,5-difluorobenzyl bromide (51 µL, 0.39 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (144 mg, 99% yield). 1H NMR (CDCl3) δ: 7.11-7.07 (m, 2H), 6.96-6.92 (m, 2H), 6.87-6.83 (m, 2H), 6.72 (tt, J = 8.9, 2.3Hz, 1H), 4.99 (s, 2H), 3.21 (d, J = 1.3Hz, 2H), 2.96-2.87 (m, 2H), 2.40 (s, 3H), 2.32 (dd, J = 14.1, 10.8Hz, 1H), 1.47 (s, 9H), 0.92 (d, J = 6.5Hz, 3H). Subsequent acid treatment of 9i afforded 10i, isolated as a colorless solid (108 mg, 79% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7).

1

H NMR (MeOH-d4) δ:

7.24-7.20 (m, 2H), 7.07-7.01 (m, 2H), 6.99-6.96 (m, 2H), 6.87 (tt, J = 9.2, 2.3Hz, 1H), 5.09 (s, 2H), 3.70-3.59 (m, 2+1H), 3.15 (dd, J = 13.2, 4.2Hz, 1H), 2.88 (s, 3H), 2.73 (dd, J = 13.2, 10.5Hz, 1H), 1.21 (d, J = 6.7Hz, 3H).

13

C NMR (MeOH-d4) δ: 169.9, 164.6 (dd, J = 247.2,

12.4Hz, 2xCq), 159.0, 143.5 (t, J = 9.1Hz), 131.6 (2xCH), 129.9, 116.3 (2xCH), 110.8 (dd, J = 19.1, 6.9Hz, 2xCH), 103.7 (t, J = 25.9Hz), 69.5 (t, J = 2.2Hz), 64.3, 56.4, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H22F2NO3+: 350.15623, found: 350.15649. (R)-N-(1-(4-((4-Bromo-3-chlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (10j) via (9j) Following the two step general procedure (R)-N-(1-(4-((4-bromo-3-


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chlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9j was prepared by reaction of 8 (200 mg, 0.72 mmol) with 4-bromo-3-chlorobenzyl bromide (224 mg, 0.79 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (246 mg, 71% yield). 1H NMR (CDCl3) δ: 7.60 (d, J = 8.3Hz, 1H), 7.53 (d, J = 1.6Hz, 1H), 7.17 (dd, J = 8.2, 1.6Hz, 1H), 7.10-7.07 (m, 2H), 6.86-6.83 (m, 2H), 4.96 (s, 2H), 3.21 (s, 2H), 2.96-2.88 (m, 2H), 2.40 (s, 3H), 2.33 (dd, J = 14.1, 10.8Hz, 1H), 1.47 (s, 9H), 0.92 (d, J = 6.4Hz, 3H). Subsequent acid treatment of 9j (120 mg, 0.25 mmol) afforded 10j, isolated as a colorless solid (87 mg, 76% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 7.65 (d, J = 8.3Hz, 1H), 7.58 (d, J = 1.7Hz, 1H), 7.26 (dd, J = 8.3, 1.7Hz, 1H), 7.21 (d, J = 8.6Hz, 2H), 6.96 (d, J = 8.6Hz, 2H), 5.02 (s, 2H), 3.69-3.58 (m, 2+1H), 3.14 (dd, J = 13.2, 4.1Hz, 1H), 2.88 (s, 3H), 2.72 (dd, J = 13.2, 10.6Hz, 1H), 1.20 (d, J = 6.7Hz, 3H).

13

C

NMR (MeOH-d4) δ: 169.9, 159.0, 140.3, 135.4, 135.0, 131.5 (2xCH), 130.1, 129.8, 128.2, 122.2, 116.4 (2xCH), 69.4, 64.3, 56.5, 38.6, 37.5, 13.1. HRMS: m/z calculated for C19H20BrClNO3-: 426.02946, found: 426.03046 (main isotope). (R)-N-(1-(4-((3,4-Dichlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (10k) via (9k) Following the two step general procedure (R)-N-(1-(4-((3,4dichlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9k was prepared by reaction of 8 (80 mg, 0.29 mmol) with 3,4-dichlorobenzyl bromide (46 µL, 0.32 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (66 mg, 53% yield). 1H NMR (CDCl3) δ: 7.53 (br s, 1H), 7.52-7.40 (m, 1H), 7.26-7.24 (m, 1H), 7.09 (br d, J = 8.4Hz, 2H), 6.85 (br d, J = 8.4Hz, 2H), 4.98 (s, 2H), 3.22 (s, 2H), 2.97-2.89 (m, 2H), 2.41 (s, 3H), 2.33 (dd, J = 14.0, 10.8Hz, 1H), 1.47 (s, 9H), 0.93 (d, J = 6.4Hz, 3H).


49


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Subsequent acid treatment of 9k afforded 10k, isolated as a colorless solid (43 mg, 68% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 7.58 (d, J = 1.1Hz, 1H), 7.50 (d, J = 8.2Hz, 1H), 7.34 (dd, J = 8.2, 1.1Hz, 1H), 7.21 (d, J = 8.4Hz, 2H), 6.96 (d, J = 8.4Hz, 2H), 5.04 (s, 2H), 3.70-3.59 (m, 2+1H), 3.14 (dd, J = 13.2, 4.1Hz, 1H), 2.88 (s, 3H), 2.72 (dd, J = 13.2, 10.5Hz, 1H), 1.21 (d, J = 6.6Hz, 3H). ). 13C NMR (MeOH-d4) δ: 168.8, 159.0, 139.6, 133.4, 132.5, 131.6, 131.5 (2xCH), 130.3, 129.8, 128.1, 116.4 (2xCH), 69.4, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H20Cl2NO3-: 380.08257, found: 380.08289 (main isotope). (R)-N-(1-(4-((2,5-Dichlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (10l) via (9l) Following the two step general procedure R)-N-(1-(4-((2,5dichlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9l was prepared by reaction of 8 (100 mg, 0.36 mmol) with 2,5-dichlorobenzyl bromide (94 mg, 0.39 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 6:4) as a colorless wax (92 mg, 59% yield). 1H NMR (CDCl3) δ: 7.59 (d, J = 2.4Hz, 1H), 7.32 (d, J = 8.8Hz, 2H), 7.23 (dd, J = 8.4, 2.4Hz, 1H), 7.11 (d, J = 8.4Hz, 2H), 6.90 (d, J = 8.4Hz, 2H), 5.09 (s, 2H), 3.24 (s, 2H), 2.97-2.95 (m, 2H), 2.44 (s, 3H), 2.36 (dd, J = 14.0, 10.8Hz, 1H), 1.48 (s, 9H), 0.94 (d, J = 6.4Hz, 3H). Subsequent acid treatment of 9l (80mg, 0.18 mmol) afforded 10l, isolated as an off-white solid (61 mg, 77% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 5:5). 1H NMR (MeOH-d4) δ: 7.53 (d, J = 1.7Hz, 1H), 7.39 (d, J = 8.4Hz, 1H), 7.30 (dd, J = 8.4, 1.7Hz, 1H), 7.23 (d, J = 7.7Hz, 2H), 6.97 (d, J = 7.7Hz, 2H), 5.08 (s, 2H), 3.71-3.58 (m, 2+1H), 3.16 (br d, J = 12.1Hz, 1H), 2.88 (s, 3H), 2.73 (br t, J = 11.6Hz, 1H), 1.21 (d, J = 5.9Hz, 3H).

13

C NMR

(MeOH-d4) δ: 169.9, 158.9, 138.1, 134.1, 132.1, 131.8, 131.6 (2xCH), 130.2, 130.1, 129.8,


50

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116.3 (2xCH), 67.7, 64.2, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H20Cl2NO3-: 380.08257, found: 380.08325. N-((R)-(1-(4-(3-Nitrobenzyloxy)phenyl)propan-2-yl)(methyl)) (10m)

via

(9m)

Following

the

two

step

general

glycine

procedure

hydrochloride N-((R)-

(1-(4-(3-

nitrobenzyloxy)phenyl)propan-2-yl)(methyl)) glycine tert-butyl ester 9m was prepared by reaction of 8 (95 mg, 0.34 mmol) with 3-nitrobenzyl bromide (81 mg, 0.37 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 7:3) as a colorless wax (55 mg, 80% yield). 1H NMR (CDCl3) δ: 8.30 (br t, J = 2.0Hz, 1H), 8.16 (dd, J = 8.2, 1.4Hz, 1H), 7.76 (ddd, J = 8.2, 1.5, 1.0Hz, 1H), 7.55 (t, J = 8.0Hz, 1H), 7.12-7.08 (m, 2H), 6.90-6.86 (m, 2H), 5.10 (s, 2H), 3.21 (br s, 2H), 2.96-2.87 (m, 2H), 2.39 (s, 3H), 2.32 (dd, J = 13.8, 10.7Hz, 1H), 1.46 (s, 9H), 0.92 (d, J = 6.7Hz, 3H). Subsequent acid treatment of 9m afforded 10m, isolated as a pale yellow solid (34 mg, 65% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 8.30 (br s, 1H), 8.16 (dd, J = 8.0, 1.8Hz, 1H), 7.83 (d, J = 7.6Hz, 1H), 7.61 (t, J = 8.0Hz, 1H), 7.22 (d, J = 8.4Hz, 2H), 7.00 (d, J = 8.4Hz, 2H), 5.18 (s, 2H), 3.66 (br s, 2+1H), 3.15 (dd, J = 13.0, 3.7Hz, 1H), 2.89 (s, 3H), 2.73 (dd, J = 13.0, 10.6Hz, 1H), 1.21 (d, J = 6.6Hz, 3H).

13

C

NMR (MeOH-d4) δ: 169.9, 158.9, 149.7, 141.1, 134.4, 131.6 (2xCH), 130.8, 130.0, 123.7, 122.9, 116.4 (2xCH), 69.6, 64.3, 56.4, 38.7, 37.5, 13.1. HRMS: m/z calculated for C19H21N2O5-: 357.14560, found: 357.14594. (R)-N-(1-(4-((3-Methoxybenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (10n) via (9n) Compound 8 (100 mg, 0.36 mmol), 3-methoxybenzyl alcohol (99 mg, 0.72 mmol), and PPh3 (376 mg, 1.43 mmol) were dissolved in THF (2 mL) under stirring. DIAD (141 µL, 0.72 mmol) was added dropwise and the mixture was stirred for 30 minutes. Solvents were


51


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then evaporated in vacuo and the crude residue was chromatographed through two successive automated flash silica gel columns (Hexane/EtOAc 9:1 to 6:4, then 95:5 to 9:1) in an attempt to separate it from the side product, diisopropyl hydrazine-1,2-dicarboxylate (DIHD). The mixture containing (R)-N-(1-(4-((3-methoxybenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9n was obtained as a colorless wax (87 mg, 62% yield, estimated from the crude 1H NMR spectrum). The mixture was engaged in the next step without further purification. Acid treatment of 9n according to the general procedure afforded 10n, isolated as a colorless solid (57 mg, 68% yield) after C-18 reverse phase chromatography (H2O/MeOH, 6:4 to 3:7). 1H NMR (CDCl3) δ: 7.27 (t, J = 7.8Hz, 1H), 7.13 (d, J = 8.2Hz, 2H), 6.98-6.95 (m, 2H), 6.88 (d, J = 8.2Hz, 2H), 6.84 (dd, J = 8.4, 2.2Hz, 1H), 4.98 (s, 2H), 3.80 (s, 3H), 3.71 (br s, 1H), 3.59-3.48 (m, 2H), 3.24 (br d, J = 13.3Hz, 1H), 2.83 (s, 3H), 2.52 (t, J = 11.8Hz, 1H), 1.17 (d, J = 6.5Hz, 3H).

13

C NMR (CDCl3) δ: 168.0, 160.0, 158.1, 138.6, 130.5, 129.8, 128.4, 119.8, 115.4, 113.7,

113.0, 70.1, 62.5, 55.8, 55.4, 38.3, 37.5, 13.2. HRMS: m/z calculated for C20H26NO4+: 344.18563, found: 344.18469. (R)-N-(1-(4-((4-(Methoxycarbonyl)benzyl)oxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (10o) via (9o)

Following the two step general procedure R)-N-(1-(4-((4-

(methoxycarbonyl)benzyl)oxy)phenyl)propan-2-yl)-N-methylglycine tert-butyl ester 9o was prepared by reaction of 8 (75 mg, 0.27 mmol) with methyl 4-(bromomethyl)benzoate (68 mg, 0.30 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (80 mg, 70% yield). 1H NMR (CDCl3) δ: 8.04 (d, J = 8.4Hz, 2H), 7.49 (d, J = 8.0Hz, 2H), 7.08 (d, J = 8.4Hz, 2H), 6.87 (d, J = 8.4Hz, 2H), 5.09 (s, 2H), 3.09 (s, 3H), 3.21 (br s, 2H), 2.962.88 (m, 2H), 2.40 (s, 3H), 2.32 (dd, J = 14.2, 10.6Hz, 1H),1.47 (s, 9H), 0.92 (d, J = 6.4Hz, 3H).


52

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Acid treatment of 9o according to the general procedure afforded 10o, isolated as a colorless solid (37 mg, 48% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 8.00 (d, J = 8.1Hz, 2H), 7.53 (d, J = 8.1Hz, 2H), 7.20 (d, J = 8.4Hz, 2H), 6.97 (d, J = 8.4Hz, 2H), 5.13 (s, 2H), 3.89 (s, 3H), 3.71-3.59 (m, 2+1H), 3.14 (dd, J = 13.1, 4.0Hz, 1H), 2.88 (s, 3H), 2.72 (dd, J = 13.1, 10.6Hz, 1H), 1.21 (d, J = 6.7Hz, 3H).

13

C NMR

(MeOH-d4) δ: 170.0, 168.4, 159.4, 144.4, 134.5, 131.7, 130.9, 130.8, 129.9, 128.4, 116.5, 70.4, 64.5, 56.7, 52.8, 38.8, 37.7, 13.3. HRMS: m/z calculated for C21H24NO5-: 370.16600, found: 370.16739. (R)-N-Methyl-N-(1-(4-(naphthalen-2-ylmethoxy)phenyl)propan-2-yl)glycine hydrochloride (10p) via (9p) Following the two step general procedure (R)-N-methyl-N-(1-(4(naphthalen-2-ylmethoxy)phenyl)propan-2-yl)glycine tert-butyl ester 9p was prepared by reaction of 8 (70 mg, 0.25 mmol) with 2-(Bromomethyl)naphthalene (61 mg, 0.28 mmol). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (51 mg, 49% yield). 1H NMR (CDCl3) δ: 7.89-7.84 (m, 4H), 7.50-7.48 (m, 3H), 7.12-7.09 (m, 2H), 6.96-6.94 (m, 2H), 5.21 (s, 2H), 3.23 (s, 2H), 2.98-2.94 (m, 2H), 2.43 (s, 3H), 2.37-2.31 (m, 1H), 1.48 (s, 9H), 0.95 (d, J = 6.4Hz, 3H). Acid treatment of 9p afforded 10p, isolated as a beige solid (30 mg, 62% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 7.87-7.81 (m, 4H), 7.52 (br d, J = 8.3Hz, 1H), 7.47-7.45 (m, 2H), 7.18 (d, J = 8.2Hz, 2H), 7.00 (d, J = 8.2Hz, 2H), 5.19 (s, 2H), 3.62 (br s, 2+1H), 3.11 (dd, J = 13.1, 3.3Hz, 1H), 2.85 (s, 3H), 2.69 (dd, J = 13.1, 10.6Hz, 1H), 1.17 (d, J = 6.5Hz, 3H). 13C NMR (MeOH-d4) δ: 169.8, 159.5, 136.1, 134.7, 134.49, 131.5 (2xCH), 129.4, 129.2, 128.9, 128.7, 127.31, 127.27, 127.1, 126.4, 116.4 (2xCH),


53


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71.1, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C23H26NO3+: 364.19072, found: 364.18982. (R)-N-Methyl-N-(1-(4-(pyridin-2-ylmethoxy)phenyl)propan-2-yl)glycine

hydrochloride

(10q) via (9q) Following the two step general procedure (R)-N-methyl-N-(1-(4-(pyridin-2ylmethoxy)phenyl)propan-2-yl) tert-butyl ester 9q was prepared by reaction of 8 (100 mg, 0.36 mmol) with 2-(bromomethyl)-pyridine hydrobromide (100 mg, 0.39 mmol) and K2CO3 (247 mg, 1.79 mmol, 5 equiv.). It was isolated after silica chromatography (Hexane/EtOAc 9:1 to 5:5) as a colorless wax (77 mg, 58% yield). 1H NMR (CDCl3) δ: 8.57 (br d, J = 1.6Hz, 1H), 7.68 (td, J = 7.7, 1.5Hz, 1H), 7.50 (d, J = 7.7Hz, 1H), 7.19 (dd, J = 7.2, 5.1Hz, 1H), 7.08-7.05 (m, 2H), 6.896.87 (m, 2H), 5.16 (s, 2H), 3.20 (br s, 2H), 2.94-2.87 (m, 2H), 2.39 (s, 3H), 2.31 (dd, J = 14.1, 10.8Hz, 1H), 1.45 (s, 9H), 0.91 (d, J = 6.4Hz, 3H). Acid treatment of 9q afforded 10q, isolated as a light brown solid (55 mg, 68% yield) after C18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 8.54 (d, J = 4.9Hz, 1H), 7.86 (td, J = 7.8, 1.4Hz, 1H), 7.59 (d, J = 7.8Hz, 1H), 7.37 (dd, J = 7.8, 4.9Hz, 1H), 7.22 (d, J = 8.4Hz, 1H), 7.00 (d, J = 8.4Hz, 1H), 5.16 (s, 2H), 3.69-3.60 (m, 2+1H), 3.15 (dd, J = 13.1, 3.9Hz, 1H), 2.89 (s, 3H), 2.73 (dd, J = 13.1, 10.6Hz, 1H), 1.21 (d, J = 6.6Hz, 3H).

13

C

NMR (MeOH-d4) δ: 169.9, 159.1, 158.2, 149.8, 139.0, 131.6 (2xCH), 129.9, 124.4, 123.3, 116.3 (2xCH), 71.2, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C18H21N2O3-: 313.15577, found: 313.15601. (R)-N-Methyl-N-(1-(4-(pyridin-4-ylmethoxy)phenyl)propan-2-yl)glycine

hydrochloride

(10r) via (9r) Following the two step general procedure (R)-N-methyl-N-(1-(4-(pyridin-4ylmethoxy)phenyl)propan-2-yl)glycine tert-butyl ester 9r was prepared by reaction of 8 (100 mg, 0.36 mmol) with 4-(bromomethyl)-pyridine hydrobromide (100 mg, 0.39 mmol) and K2CO3 (247


54

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mg, 1.79 mmol, 5 equiv.). It was isolated after silica chromatography (DCM/MeOH 99:1 to 95:5) as a colorless wax (72 mg, 54% yield). 1H NMR (CDCl3) δ: 8.60 (br s, 2H), 7.34 (br d, J = 2.0Hz, 2H), 7.07 (d, J = 8.8Hz, 2H), 6.83 (d, J = 8.8Hz, 2H), 5.03 (s, 2H), 3.19 (s, 2H), 2.94-2.88 (m, 2H), 2.38 (s, 3H), 2.31 (dd, J = 14.0, 10.4Hz, 1H), 1.44 (s, 9H), 0.90 (d, J = 6.4Hz, 3H). Acid treatment of 9r (43 mg, 0.12 mmol) afforded 10r, isolated as an off-white wax (16 mg, 35% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 8.52 (d, J = 4.9Hz, 2H), 7.51 (d, J = 4.9Hz, 2H), 7.23 (d, J = 8.2Hz, 2H), 7.00 (d, J = 8.2Hz, 2H), 5.18 (s, 2H), 3.69-3.57 (m, 2+1H), 3.14 (dd, J = 13.0, 3.5Hz, 1H), 2.88 (s, 3H), 2.74 (dd, J = 13.0, 10.7Hz, 1H), 1.21 (d, J = 6.5Hz, 3H). ).

13


159.0, 150.2 (2xCH), 149.5, 131.6 (2xCH), 130.1, 123.3 (2xCH), 116.3 (2xCH), 70.0, 64.3, 56.6, 38.7, 37.5, 13.1. HRMS: m/z calculated for C18H23N2O3+: 315.17032, found: 315.17029. (R)-N-Methyl-N-(1-(4-(pyridin-3-ylmethoxy)phenyl)propan-2-yl)glycine

hydrochloride

(10s) via (9s) Following the two step general procedure (R)-N-methyl-N-(1-(4-(pyridin-3ylmethoxy)phenyl)propan-2-yl) tert-butyl ester 9s was prepared by reaction of 8 (100 mg, 0.36 mmol) with 3-(bromomethyl)-pyridine hydrobromide (100 mg, 0.39 mmol) and K2CO3 l(247 mg, 1.79 mmol, 5 equiv.). It was isolated after silica chromatography (DCM/MeOH 9:1 to 5:5) as a colorless wax (53 mg, 40% yield). 1H NMR (CDCl3) δ: 8.66 (br s, 1H), 8.56 (br s, 1H), 7.75 (br d, J =7.8Hz, 1H), 7.30 (dd, J = 7.7, 4.9Hz, 1H), 7.10-7.08 (m, 2H), 6.88-6.86 (m, 2H), 5.03 (s, 2H), 3.20 (s, 2H), 2.96-2.87 (m, 2H), 2.39 (s, 3H), 2.32 (dd, J = 14.1, 10.8Hz, 1H), 1.45 (s, 9H), 0.91 (d J = 6.5Hz, 3H). Acid treatment of 9s afforded 10s, isolated as an off-white wax (39 mg, 70% yield) after C-18 reverse phase chromatography (H2O/MeOH, 9:1 to 3:7). 1H NMR (MeOH-d4) δ: 8.84 (br s, 1H), 8.51 (br s,1H), 7.94 (d, J =7.8Hz, 1H), 7.47 (dd, J = 7.0, 4.8Hz, 1H), 7.23 (br d, J = 8.4Hz, 2H),


55


Page 56 of 96

7.01 (br d, J =8.4Hz, 2H), 5.15 (s, 2H), 3.70-3.60 (m, 3H), 3.15 (dd, J =13.2, 3.6Hz, 1H), 2.89 (s, 3H), 2.74 (dd, J = 12.8, 10.6Hz, 1H), 1.21 (d, J = 6.5Hz, 3H).

13


159.1, 149.5, 149.3, 137.6, 135.3, 131.6 (2xCH), 129.9, 125.4, 116.4 (2xCH), 68.4, 64.3, 56.5, 38.7, 37.5, 13.1. HRMS: m/z calculated for C18H21N2O3-: 313.15577, found: 313.15610. (R)-N-(1-(4-(benzyloxy)phenyl)propan-2-yl)-N-methylprop-2-yn-1-amine (11) Propargyl bromide (58 µL, 0.52 mmol) was added dropwise to a suspension of compound 6 (120 mg, 0.47 mmol) and Cs2CO3 (306 mg, 0.94 mmol) in THF (3 mL). The resulting mixture was stirred overnight at room temperature. The crude mixture was diluted with water and extracted with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was flash silica gel column chromatographed (Hexane/EtOAc 90/10 to 70/30), affording compound 11 as a dark yellow oil (58 mg, 42% yield). 1H NMR (CDCl3) δ: 7.44-7.30 (m, 5H), 7.10 (br d, J = 8.6Hz, 2H), 6.90 (br d, J = 8.6Hz, 2H), 5.04 (s, 2H), 3.48 (br s, 2H), 3.04-2.95 (m, 2H), 2.46 (s, 3H), 2.37 (dd, J = 12.6, 9.5Hz, 1H), 2.28 (br t, J = 2.2Hz, 1H), 0.99 (d, J = 6.6Hz, 3H). A small portion of compound 11 (25 mg, 0.09 mmol) was converted to the corresponding hydrochloride salt by treatment with HCl in MeOH for 15 min at 0°C. The residue obtained after removal of solvent in vacuo was partitioned between DCM and water, and the aqueous phase was concentrated to dryness, affording 11-HCl (11 mg, 39% yield) as a slightly beige solid. 1H NMR (MeOH-d4) δ: 7.44-7.42 (m, 2H), 7.38-7.34 (m, 2H), 7.31-7.28 (m, 1H), 7.11 (br d, J = 8.6Hz, 2H), 6.93 (br d, J = 8.6Hz, 2H), 5.00 (s, 2H), 3.60 (br s, 2H), 3.18-3.10 (m, 1H), 2.84 (br s, 1H), 3.02 (dd, J = 13.0, 3.6 Hz, 1H), 2.51 (s, 3H), 2.41 (dd, J = 12.9, 10.4 Hz, 1H), 1.00 (d, J = 6.5Hz, 3H). HRMS: m/z calculated for C20H24NO+: 294.18524, found: 294.18491.


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N-(Boc)-O-Benzyltyrosine methyl ester (13) Following the procedure for O-benzylation of 2, N-(Boc)tyrosine methyl ester 12 (1.0 g, 3.39 mmol) was converted to compound 13, isolated as a colorless solid (1.3 g, 95% yield) after silica flash column chromatography (Hexane/EtOAc 9:1 to 5:5). 1H NMR (CDCl3) δ: 7.46-7.39 (m, 4H), 7.37-7.35 (m, 1H), 7.08-7.05 (m, 2H), 6.94-6.92 (m, 2H), 5.06 (s, 2H), 5.00 (br d, J = 8.2Hz, 1H), 4.58-4.56 (m, 1H), 3.73 (s, 3H), 3.07-3.03 (m, 2H), 1.45 (s, 9H). N-(Boc)-O-(3-Chlorobenzyl))tyrosine methyl ester (14) Following the O-benzylation procedure used to prepare 2, N-(Boc)tyrosine methyl ester 12 (1.0 g, 3.39 mmol) was converted to compound 14, isolated as a colorless solid (1.5 g, 100% yield), after silica flash column chromatography (Hexane/EtOAc 9:1 to 5:5). 1H NMR (CDCl3) δ: 7.43 (br s, 1H), 7.31-7.30 (m, 3H), 7.05-7.03 (m, 2H), 6.89-6.87 (m, 2H), 5.01 (s, 2H), 4.98-4.96 (m, 1H), 4.57-4.52 (m, 1H), 3.71 (s, 3H), 3.07-2.98 (m, 2H), 1.42 (s, 9H). N-(Boc)-N-(Methyl)(O-benzyl)tyrosine (15) To a solution of 13 (1.30 g, 3.37 mmol) and MeI (1.05 mL, 16.86 mmol) in dry THF (15 mL), cooled to 0°C, was added NaH (60% suspension in oil, 674 mg, 16.86 mmol) in portions. The resulting mixture was stirred overnight at room temperature and then cooled to 0°C and quenched with ice water. After removal of the THF in vacuo, the residue was taken up in water and washed twice with hexane. The water layer was then acidified to pH 4 with citric acid and extracted with DCM. The combined organic layers washed with brine and water, dried over Na2SO4, filtered, and concentrated in vacuo. The crude mixture was silica flash column chromatographed (Hexane/EtOAc 6:4), affording 15 as a pale yellow oil (453 mg, 35% yield). 1H NMR (CDCl3) (N-Boc-rotamers) δ: 7.46-7.39 (m, 4H), 7.377.35 (m, 1H), 7.17-7.11 (m, 2H), 6.95-6.93 (m, 2H), 5.07 (s, 2H), 4.83-4.81 (dd, J = 10.8, 5.0Hz,


57


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1H), 4.61-4.57 (dd, J = 10.8, 4.2Hz, 1H), 3.33-3.24 (m, 2H), 3.11- 2.97 (dd, J = 14.3, 11.1, 1H, rotamers), 2.78/2.72 (2s, 3H rotamers), 1.43/1.37 (2s, 9H, rotamers). N-(Boc) N-(Methyl)-O-(3-chlorobenzyl)tyrosine (16) Following the procedure used to prepare 15, compound 16 was obtained from 14 (1.0 g, 2.38 mmol) as a colorless oil (760 mg, 76% yield), after silica flash column chromatography (Hexane/EtOAc 9:1 to 6:4). 1H NMR (CDCl3) (mixture of NBoc-rotamers) δ: δ 7.43 (s, 1H), 7.30-7.26 (m, 3H), 7.15-7.09 (m, 2H), 6.906.88 (m, 2H), 5.01 (s, 2H), 4.82-4.76 (m, 1H), 4.58-4.54 (m, 1H), 3.30-3.22 (m, 2H), 3.09-2.95 (m, 2H), 2.75/2.69 (2s, 3H, rotamers), 1.40/1.34 (2s, 9H, rotamers). N-(Methyl)(O-benzyl)tyrosine methyl ester (17) Compound 15 (450 mg, 1.17 mmol) was dissolved in MeOH (6.0 mL) and cooled to 0°C before thionyl chloride (169 µL, 2.34 mmol) was added dropwise. The resulting solution was stirred overnight at room temperature. The solvent was then evaporated and the residue was taken up in DCM and washed with 5% aqueous NaHCO3, dried over Na2SO4, filtered, and concentrated in vacuo. Compound 17 was obtained as a pale yellow oil (245 mg, 62% yield). 1H NMR (CDCl3) δ: 7.44-7.36 (m, 4H), 7.34-7.30 (m, 1H), 7.10-7.07 (m, 2H), 6.92-6.88 (m, 2H), 5.04 (s, 2H), 3.67 (s, 3H), 3.41 (t, J = 6.7Hz, 1H), 2.94-2.86 (m, 2H), 2.36 (s, 3H), 1.66 (br s, 1H). N-(Methyl)-O-(3-chlorobenzyl)tyrosine methyl ester (18) Following the procedure used to prepare 17, compound 18 was obtained from 16 (750 mg, 1.79 mmol) as a pale yellow oil (368 mg, 62% yield). 1H NMR (CDCl3) δ: 7.43 (br s, 1H), 7.30 (br s, 3H), 7.10-7.08 (m, 2H), 6.896.87 (m, 2H), 5.01 (s, 2H), 3.67 (s, 3H)], 3.43-3.39 (m, 1H), 2.90-2.89 (m, 2H), 2.36 ( br s, 3H). N-(tert-Butyl acetate)-N-(methyl)(O-benzyloxy)tyrosine methyl ester (19) Compound 17 (230 mg, 0.77 mmol) was dissolved in DMF (1.5 mL) and tert-butyl bromoacetate (114 µL, 0.77 mmol) was added dropwise. After 5 minutes of stirring, Cs2CO3 (501 mg, 1.54 mmol) was added


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and the resulting mixture was stirred overnight at room temperature. It was then diluted with a plarge amount of water and extracted with DCM. The organic layers were combined and dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was silica flash column chromatographed (Hexane/EtOAc 9:1 to 7:3), affording 19 as a yellowish wax (157 mg, 49% yield). 1H NMR (CDCl3) δ: 7.44-7.35 (m, 4H), 7.34-7.29 (m, 1H), 7.15-7.11 (m, 2H), 6.90-6.87 (m, 2H), 5.02 (s, 2H), 3.59 (dd, J = 9.4, 5.9Hz, 1H), 3.59 (s, 3H), 3.42 (d, J = 17.0Hz, 1H), 3.26 (d, J = 17.0Hz, 1H), 2.99 (dd, J = 13.4, 9.4Hz, 1H), 2.93 (dd, J = 13.4, 5.9Hz, 1H), 2.48 (s, 3H), 1.47 (s, 9H). N-(tert-Butyl acetate)-N-(methyl)-O-(3-chlorobenzyl)tyrosine methyl ester (20) Following the procedure used to prepare 19, compound 20 was obtained from 18 (350 mg, 1.05 mmol) as a yellowish wax (252 mg, 54% yield) after silica flash column chromatography (Hexane/EtOAc 9:1 to 7:3). 1H NMR (CDCl3) δ: 7.42 (br s, 1H), 7.29-7.27 (m, 3H), 7.14-7.12 (m, 2H), 6.97-6.84 (m, 2H), 4.99 (s, 2H), 3.60-3.56 (m, 3H+1H), 3.42 (d, J = 17.2Hz, 1H), 3.25 (d, J = 16.8Hz, 1H), 3.02-2.90 (m, 2H), 2.47 (s, 3H), 1.46 (s, 9H). N-Carboxymethyl)-N-(methyl-O-benzyl)tyrosine

methyl

ester

hydrochloride

(21)

Concentrated HCl (1.5 mL) was added dropwise to a cooled (0°C) solution of tert-butyl ester 19 (100 mg, 0.24 mmol) in THF (1.5 mL) and the mixture was stirred for 3 h. Removal of the solvent in vacuo and C-18 reverse phase column chromatography (H2O/MeOH 9:1 to 3:7) of the residue provided product 21 as a white powder (82 mg, 86% yield). 1H NMR (MeOH-d4) δ: 7.42-7.39 (m, 2H), 7.37-7.32 (m, 2H), 7.31-7.26 (m, 1H), 7.16-7.12 (m, 2H), 6.96-6.89 (m, 2H), 5.03 (s, 2H), 3.89 (dd, J = 8.4, 6.8Hz, 1H), 3.59 (s, 3H), 3.58 (d, J = 16.9Hz, 1H), 3.48 (d, J = 16.9Hz, 1H)mh, 3.09-2.99 (m, 2H), 2.62 (s, 3H). 13C NMR (MeOH-d4) δ: 172.8, 172.1, 159.2,


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138.7, 131.3 (2xCH), 130.0, 129.5 (2xCH), 128.8, 128.6 (2xCH), 116.1 (2xCH), 70.9, 69.2, 56.7, 52.3, 40.2, 35.4. HRMS: m/z calculated for C20H24NO5+: 358.16490, found: 358.16473. N-Carboxymethyl-N-methyl-O-(3-chlorobenzyl)tyrosine methyl ester hydrochloride (22) Following the procedure for 21, tert-butyl ester 20 (100 mg, 0.22mmol) in THF (1.5 mL) was treated with conc. HCl. Silica C-18 reverse phase column chromatography (H2O/MeOH 9:1 to 3:7) of the crude mixture provided product 22 as a yellow solid (72 mg, 75% yield). 1H NMR (MeOH-d4) δ: 7.43 (br s, 1H), 7.34-7.27 (m, 3H), 7.16-7.13 (m, 2H), 6.92-6.89 (m, 2H), 5.03 (s, 2H), 3.90 (t, J = 7.6Hz, 1H), 3.59 (d, J = 16.9Hz, 1H), 3.59 (s, 3H), 3.49 (d, J = 16.9Hz, 1H), 3.05-3.03 (m, 2H), 2.62 (s, 3H).

13

C NMR (MeOH-d4) δ: 172.8, 172.1, 158.9, 141.2, 135.3,

131.4 (2xCH), 131.0, 130.2, 128.8, 128.3, 126.7, 116.1 (2xCH), 70.0, 69.1, 56.7, 52.3, 40.2, 35.4. HRMS: m/z calculated for C20H24NO5+: 392.12593, found: 392.12552. (S)-N-(1-(4-(Benzyloxy)phenyl)-3-hydroxypropan-2-yl)-N-methylamine (23) Compound 2 (5.0 g, 14.6 mmol) was dissolved in dry THF (75 mL) and LiAlH4 (2.2 g, 58.2 mmol) was added in portions. The resulting suspension was stirred for 3h at reflux and then quenched by successive dropwise additon of water (2.2 mL), 10% aqueous NaOH (2.2 mL) and water (2.2 mL). The solids were collected by filtration and washed with DCM and THF. The combined filtrate and washings were concentrated in vacuo and the residue was silica flash column chromatographed (Hexane/EtOAC 1:1 to EtOAc, then DCM/MeOH 7:3) affording compound 23 as a colorless solid (3.48 g, 88% yield). 1H NMR (CDCl3) δ: 7.44-7.31 (m, 5H), 7.10 (d, J = 8.4Hz, 2H), 6.92 (d, J = 8.4Hz, 2H), 5.05 (s, 2H), 3.64 (dd, J = 10.8, 3.6Hz, 1H), 3.34 (dd, J = 10.8, 4.8Hz, 1H), 2.78-2.64 (m, 2H+1H), 2.41 (s, 3H), 2.06 (br s, 2H).

13

C NMR (CDCl3) δ:

158.1, 137.5, 130.7, 130.6 (2xCH), 129.0 (2xCH), 128.4, 127.9 (2xCH), 115.4 (2xCH), 70.1, 62.1, 61.4, 35.8, 33.0.


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tert-Butyl (S)-N-(1-(4-(Benzyloxy)phenyl)-3-hydroxypropan-2-yl)-N-methylglycinate (24) To a solution of 23 (1.14 g, 4.20 mmol) in DMF (20 mL) was added Et3N (615 µL, 4.41 mmol) and tert-butyl bromoacetate (665 µL, 4.41 mmol). The mixture was stirred at room temperature overnight and then concentrated in vacuo. The residue was taken up in DCM and washed with an aqueous solution of citric acid. The aqueous layer was extracted three times with DCM. Then the combined organic layers were successively washed with satd. NaHCO3, brine and water. The organic layer was dried over Na2SO4, filtered and concentrated. The crude mixture was silica flash chromatographed (Hexane/EtOAc 7:3 to 5:5) affording 24 as a colorless oil (1.30 g, 80% yield). 1H NMR (CDCl3) δ: 7.44-7.30 (m, 5H), 7.06 (d, J = 8.4Hz, 2H), 6.90 (d, J = 8.4Hz, 2H), 5.04 (s, 2H), 3.44 (dd, J = 10.4, 4.4Hz, 1H), 3.32 (br t, J = 10.4Hz, 1H), 3.28 (d, J = 16.4Hz, 1H), 3.13 (d, J = 16.4Hz, 1H), 3.01-2.94 (m, 1H), 2.78 (dd, J = 13.6, 5.6Hz, 1H), 2.43 (s, 3H), 2.35 (dd, J = 13.6, 8.8Hz, 1H), 1.47 (s, 9H). (S)-N-(1-(4-(Benzyloxy)phenyl)-3-hydroxypropan-2-yl)-N-methylglycinine (25) A mixture of compound 23 (140 mg, 0.52 mmol), t-butyl bromoacetate (76 µL, 0.52 mmol), Cs2CO3 (336 mg, 1.03 mmol) and a catalytic amount of NaI in DMF (2.0 mL) was heated at 90°C under microwave irradiation for 30 min. The mixture was then diluted with water, extracted with DCM and the DCM extract was washed with brine and concentrated in vacuo. The residue was taken up in minimum DCM and the insoluble material was removed by suction filtration. Concentration of the filtrate afforded 25 as a white powder (120 mg, 66% yield). 1H NMR (MeOH-d4) δ: 7.43-7.27 (m, 5H), 7.09 (d, J = 8.6Hz, 2H), 6.91 (d, J = 8.6Hz, 2H), 5.05 (s, 2H), 3.47 (dd, J = 11.4, 10.0Hz, 1H), 3.39 (dd, J = 11.4, 3.8Hz, 1H), 3.27 (d, J = 15.5Hz, 1H), 3.12 (d, J = 15.5Hz, 1H), 2.95 (br s, 1H), 2.84 (dd, J = 13.4, 3.8Hz, 1H), 2.39 (s, 3H), 2.28 (dd, J = 13.4, 10.7Hz, 1H).

13

C NMR (DMSO-d6) δ: 175.3, 156.5, 137.2, 132.4, 129.9, 128.4 (2xCH),


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127.8, 127.7 (2xCH), 114.6 (2xCH), 69.1, 66.7, 59.7, 58.4, 37.1, 30.4. HRMS: m/z calculated for C19H23NO4 [M+H]+ = 330.1705, found: 330.1703. (S)-N-(1-(benzyloxy)-3-(4-(benzyloxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (26) Sodium hydride (60% suspension in oil, 42 mg, 1.04 mmol) was added to a cold (0°C) solution of 24 (200 mg, 0.52 mmol) in THF (3.0 mL). After stirring for 10 min, benzyl bromide (93 µL, 0.78 mmol) was added dropwise and the resulting mixture was stirred overnight at RT. After cooling to 0°C the reaction was stopped by dropwise addition of conc. HCl (2.0 mL). The resulting mixture was stirred for an additional 2 hours and concentrated in vacuo. The residue was column chromatographed twice (C18 reverse phase (first column H2O/MeOH 90:10 to 5:95 and second column 50:50 to 20:80)), affording 26 as a white solid (108 mg, 46% yield). 1H NMR (MeOH-d4) δ: 7.43-7.42 (m, 2H), 7.38-7.27 (m, 8H), 7.15 (d, J = 8.6Hz, 2H), 6.94 (d, J = 8.6Hz, 2H), 5.06 (s, 2H), 4.55 (d, J = 11.7Hz, 1H), 4.45 (d, J = 11.7Hz, 1H), 3.83-3.68 (m, 3H), 3.64 (dd, J = 11.7, 2.9Hz, 1H), 3.55 (dd, J = 11.7, 7.1Hz, 1H), 3.07 (dd, J = 13.3, 4.6Hz, 1H), 2.93 (s, 3H), 2.90 (dd, J = 13.3, 10.9Hz, 1H).

13

C NMR (MeOH-d4) δ:

169.9, 159.5, 138.6, 138.4, 131.4 (2xCH), 129.59 (2xCH), 129.51 (2xCH), 129.3 (2xCH), 129.1, 128.9, 128.8, 128.5 (2xCH), 116.4 (2xCH), 74.4, 71.0, 67.3, 66.4, 57.9, 39.8, 31.9. HRMS: m/z calculated for C26H30NO4+:420.21693, found: 420.21628. tert-Butyl (S)-N-(1-(4-(benzyloxy)phenyl)-3-chloropropan-2-yl)-N-methylglycinate (27) To a solution of 24 (1.23 g, 3.20 mmol) in dry DCM (20 mL) was added Et3N (900 µL, 6.40 mmol) at 0°C followed by TsCl (1.22 g, 6.40 mmol) and DMAP (117 mg, 0.96 mmol). The mixture was stirred at room temperature overnight. The mixture was then diluted with DCM and washed with a 10% citric solution. The aqueous phase was extracted with DCM (3x), and the combined organic layers were washed with satd. NaHCO3 and brine, dried over Na2SO4, filtered and


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concentrated in vacuo. Flash silica gel column chromatography of the residue (Hexane to Hexane/EtOAc 85:15) afforded 27 as a pale yellow oil (1.14 g, 88% yield). 1H NMR (CDCl3) δ: 7.45-7.31 (m, 5H), 7.18 (d, J = 8.4Hz, 2H), 6.93 (d, J = 8.4Hz, 2H), 5.05 (s, 2H), 4.16-4.10 (m, 1H), 3.31 (s, 2H), 3.20 (dd, J = 14.4, 4.8Hz, 1H), 2.90-2.85 (m, 3H), 2.49 (s, 3H), 1.47 (s, 9H). 13

C NMR (CDCl3) δ: 170.3, 157.8, 137.2, 130.6 (2xCH), 130.3, 128.7 (2xCH), 128.0, 127.6

(2xCH), 114.8 (2xCH), 81.2, 70.1, 62.5, 61.5, 59.5, 42.5, 41.5, 28.3 (3xCH3). tert-Butyl (S)-N-(1-(4-(benzyloxy)phenyl)-3-cyanopropan-2-yl)-N-methylglycinate

(28)

To a solution of 27 (235 mg, 0.58 mmol) in dry DMF (2.5 mL) was added KCN (379 mg, 5.82 mmol), 18-crown-6 (31 mg, 0.12 mmol) and a catalytic amount of NaI. The mixture was stirred at 100°C for 90 minutes in a microwave reactor, then diluted with DCM and washed with satd. NaHCO3 and brine, dried over Na2SO4, filtered and concentrated in vacuo. Flash silica gel column chromatography of the solid residue (Hexane to Hexane/EtOAc 8:2) afforded 28 as a pale yellow oil (183 mg, 80% yield). 1H NMR (CDCl3) δ: 7.45-7.31 (m, 5H), 7.14 (d, J = 8.0Hz, 2H), 6.93 (d, J = 8.0Hz, 2H), 5.05 (s, 2H), 3.39 (d, J = 16.8Hz, 1H), 3.33 (d, J = 16.8Hz, 1H), 3.26-3.20 (m, 1H), 3.02 (dd, J =13.6, 4.8Hz, 1H), 2.67 (dd, J =13.6, 9.6Hz, 1H), 2.53 (s, 3H), 2.46 (dd, J =17.2, 4.8Hz, 1H), 2.37 (dd, J = 17.2, 6.4Hz, 1H), 1.48 (s, 9H). 13C NMR (CDCl3) δ: 170.5, 157.7, 137.1, 130.4, 130.1 (2xCH), 128.7 (2xCH), 128.0, 127.5 (2xCH), 118.8, 115.2 (2xCH), 81.3, 70.1, 61.7, 56.6, 38.0, 36.4, 28.2 (3xCH3), 19.0. (S)-N-(1-(4-(Benzyloxy)phenyl)-3-cyanopropan-2-yl)-N-methylglycine hydrochloride (29) Following the procedure for 21, tert-butyl ester 28 (74 mg, 0.19 mmol) in THF (1.5 mL) was treated with conc. HCl. Reverse phase C-18 column chromatography (H2O/MeOH 9:1 to 3:7) of the crude mixture provided product 29 as a colorless solid (54 mg, 77% yield). 1H NMR (MeOHd4) δ: 7.41-7.27 (m, 5H), 7.20 (d, J = 8.3Hz, 2H), 6.95 (d, J = 8.3Hz, 2H), 5.03 (s, 2H), 3.60-


63


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3.50 (m, 3H), 3.10 (dd, J = 13.3, 5.0Hz, 1H), 2.79 (dd, J = 13.3, 9.8Hz, 1H), 2.75-2.62 (m, 2H), 2.69 (s, 3H). 13C NMR (MeOH-d4) δ: 172.4, 159.4, 138.6, 131.4 (2xCH), 130.0, 129.5 (2xCH), 128.9, 128.6 (2xCH), 119.0, 116.4 (2xCH), 71.0, 63.4, 57.0, 38.6, 36.1, 18.6. HRMS: m/z calculated for C20H23N2O3+: 339.17032, found: 339.17044. tert-butyl

(S)-N-(1-cyano-3-(4-hydroxyphenyl)propan-2-yl)-N-methylglycinate

(30)

Palladium on charcoal (10%, 25 mg) was added to a solution of compound 28 (213 mg, 0.54 mmol) in nitrogen flushed MeOH (5.0 mL), and the reaction was stirred overnight at room temperature under hydrogen at 1 atmosphere. The mixture was then filtered through a Celite pad, followed by an alumina pad, to remove the catalyst, and the filtrate was concentrated in vacuo, affording 30 as a colorless oil, which solidified on standing (148 mg, 90% yield). 1H NMR (CDCl3) δ: 7.06 (d, J = 8.0Hz, 2H), 6.77 (d, J = 8.0Hz, 2H), 5.43 (br s, OH), 3.38 (d, J = 16.8Hz, 1H), 3.33 (d, J = 16.8Hz, 1H), 3.22-3.17 (m, 1H), 2.99 (dd, J =13.6, 4.8Hz, 1H), 2.64 (dd, J =13.6, 9.6Hz, 1H), 2.52 (s, 3H), 2.46 (dd, J =17.0, 4.8Hz, 1H), 2.36 (dd, J = 17.0, 6.2Hz, 1H), 1.47 (s, 9H).

13

C NMR (CDCl3) δ: 170.7, 154.8, 130.3 (2xCH), 129.9, 118.8, 115.8 (2xCH),

81.7, 61.9, 56.6, 38.1, 36.4, 28.3 (3xCH3), 19.0. tert-Butyl-(S)-N-(1-cyano-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)propan-2-yl)-Nmethylglycinate (31) To a solution of 30 (131 mg, 0.43 mmol) in dry DMF (2 mL) were successively added K2CO3 (179 mg, 1.29 mmol) and 3,4-dichlorobenzyl bromide (83 µL, 0.56 mmol). The reaction was stirred at room temperature under nitrogen overnight. The mixture was then filtered through a pad of Celite using DCM as solvent and concentrated in vacuo. Flash silica gel column chromatography (Hexane/EtOAc 90:10 to 75:25) of the concentrate afforded 31 as a colorless oil (175 mg, 88 % yield). 1H NMR (CDCl3) δ: 7.53 (s, 1H), 7.45 (d, J = 8.4Hz, 1H), 7.25 (d, J = 8.4Hz, 1H), 7.14 (d, J = 8.4Hz, 2H), 6.88 (d, J = 8.4Hz, 2H), 4.99 (s, 2H), 3.38


64

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(d, J = 17.0 Hz, 1H), 3.32 (d, J = 17.0 Hz, 1H), 3.26-3.19 (m, 1H), 3.02 (dd, J = 13.6, 5.2Hz, 1H), 2.68 (dd, J = 13.6, 9.2Hz, 1H), 2.52 (s, 3H), 2.46 (dd, J = 17.2, 4.8Hz, 1H), 2.37(dd, J = 17.2, 6.0Hz, 1H), 1.47 (s, 9H).

13

C NMR (CDCl3) δ: 170.5, 157.3, 137.4, 132.9, 132.1, 131.0,

130.7, 130.3 (2xCH), 129.4, 126.6, 118.8, 115.2 (2xCH), 81.5, 68.7, 61.8, 56.6, 38.0, 36.5, 28.3 (3xCH3), 19.1. (S)-N-(1-Cyano-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)propan-2-yl)-N-methylglycine hydrochloride (32) Following the procedure for 21, tert-butyl ester 31 (162 mg, 0.44 mmol) in THF (1.5 mL) was treated with conc. HCl. Reverse phase C-18 column chromatography (H2O/MeOH 9:1 to 3:7) of the crude mixture provided product 32 as a colorless solid (120 mg, 66% yield). 1H NMR (MeOH-d4) δ: 7.58 (s, 1H), 7.49 (d, J = 8.2Hz, 1H), 7.33 (d, J = 8.2Hz, 1H), 7.22 (d, J = 8.2Hz, 2H), 6.95 (d, J = 8.2Hz, 2H), 5.03 (s, 2H), 3.57-3.48 (m, 3H), 3.10 (dd, J = 13.3, 3.8Hz, 1H), 2.79 (dd, J = 13.3, 9.9Hz, 1H), 2.74-2.62 (m, 2H), 2.66 (s, 3H).

13

C NMR

(MeOH-d4) δ: 173.0, 159.9, 139.6, 133.4, 132.5, 131.6, 131.5 (2xCH), 130.7, 130.3, 128.1, 119.1, 116.3 (2xCH), 69.4, 63.4, 57.1, 38.6, 36.2, 18.7. HRMS: m/z calculated for C20H20Cl2N2O3-: 405.07782, found: 405.07837. Docking Studies. Docking of compounds was performed using applications of the software suite offered by OpenEye, Inc. The compounds were obtained as isomeric SMILES strings. Protonation states were adjusted to pH 7.4 using FIXPKA from QUACPAC48, followed by conformer generation of up to 800 conformers using OMEGA249, 50 and standard parameters. In addition, stereochemistry of amine nitrogens was enumerated, yielding a total of 8 molecules for docking. Docking was performed using HYBRID51-53 and the coordinates of the co-crystal structure of human MAO-B complexed with Safinamide (PDB accession code 2v5z), keeping 20 poses for each docked molecule. Optimization of the docked poses was carried out using a two-


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step protocol and SZYBKI54: first, optimization of ligand Cartesian coordinates was performed using a constraint of 1 kcal/(mol Å2), followed by optimization of the complex using with MMFF94s as forcefield, Poisson-Boltzmann model, and AM1BCC charges for the ligands. Protein side chains within 5 Å of the ligand were kept flexible. The resulting poses were ranked according to the predicted Ligand—Protein Energy (LPE), and the pose with the lowest score was selected as best pose for each molecule. Biological Assays MAO-A/B Inhibition Assay. MAO-A and B activities were measured using the Fluoro MAOTM Monoamine Oxidase A & B Detection Kit (Cell Technology Inc., Fremont, United States). Procedures were according to manufacturer’s recommendations with modifications. Human MAO-A and MAO-B recombinant proteins (Sigma-Aldrich) were incubated at 2 nmol per min per ml in a volume of 50 µl with serially diluted novel compounds from concentrations of 0 to 50 µM in 96-well plates at room temperature for 1h. Positive controls included deprenyl (MAO-B specific irreversible inhibitor), safinamide (MAO-B specific reversible inhibitor), and clorgyline (MAO-A specific irreversible inhibitor). All positive controls or compounds were preincubated with recombinant human MAO-A or MAO-B proteins for 1h at room temperature to achieve maximal binding and to ensure the reaction reached its equilibrium state. A comparison study of pre-incubation and immediate binding showed that 1h pre-incubation is critical in generating consistent IC50 values in the enzyme inhibition assay (data not shown). In addition, we compared the IC50 values generated using either subsaturating (2x Michaelis constant (Km)) or saturating (2.5 mM) substrate concentrations and determined the same IC50 values under both conditions (data not shown). Substrates (tyramine (final concentration 2.5 mM) for MAO-A and benzylamine (final concentration 2.5 mM) for MAO-B) were metabolized by recombinant


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MAOs to generate H2O2. The coupling enzyme, HRP, uses the H2O2 formed to convert resazurin into highly fluorescent resorufin in a concentration-dependent manner. The reaction cocktails (50 µl) contained 1x HRP, 5 mM substrate (tyramine or benzylamine), 1x (w/v) detection reagent in 1x reaction buffer supplied from the assay kit. After the 1h pre-incubation step, reaction cocktails were added to pre-incubated reactions (total volume of 100 µl) and allowed to incubate for an additional 30 min at room temperature in the dark. The fluorescent signals were measured with a fluorescence plate reader with excitation and emission wavelengths of 570 nm and 590 nm. Signals were subtracted from background and normalized to 0 µM of compound (or non-treated) wells to calculate % inhibition. IC50 values were determined using the curvatures of log (inhibitor) vs. normalized response with a variable slope in Prism 5.02 (GraphPad Software). Initial compound concentrations of 0 to 50 µM were used for the IC50 calculations because equilibrium between the compounds and MAO-A/B enzymes was reached during the 1h preincubation step. P-gp Efflux Evaluation in MDCK-WT and MDCK-MDR1 II Cells. Permeability assays were carried out as described in Feng et al.36. MDCK-WT cells were grown in MEM-α (Gibco) supplemented with 10% FBS (Gibco). MDCK-MDR1 II cells maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco). Cells were seeded at a density of 5 x 105 cells/ml on the apical or basolateral compartment of 12-well transwell plates (Costar). Cells were incubated in standard growth media at 37°C with 95% humidity and 5% CO2 and maintained for three days. Transepithelial electrical resistance (TEER) measurement were taken before and after the treatment to confirm the integrity and permeability of the monolayer established. The epithelial integrity of each well was also confirmed by addition of Lucifer yellow solution at 100 µM. Cells in both apical and basolateral compartments were washed with HBSS solution containing


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10 mM HEPES for 30 min at 37°C with 95% humidity and 5% CO2. Deprenyl, cetirizine, compounds 10c, 10j, 10k and 32 were added at a concentration of 10 µM to the apical (A) or basal (B) compartments in triplicates in each cell line. Additionally, a known P-gp inhibitor, verapamil, was used at 100 µM. At 1 hour post-treatment, the supernatant of the samples were collected and analyzed by UPLC-MS/MS. The Papp values were calculated for both A-to-B and B-to-A transport directions. The ratio of Papp values was defined as efflux ratio. The efflux ratio was calculated for each cell line in the presence and absence of verapamil. All calculations were determined using the formulas described below. P =

V dC × (A × C ) dT

VR = volume in the receiver compartment (mL) = 0.5 (in apical compartment) or 1.5 (in basal compartment) A = filter surface area (cm2) = 1.12 cm2 for 12-well Transwell plate Co = initial concentration of test compound in the donor compartment dC/dT = change in compound concentration in receiver compartment over time (sec) = 3600 seconds Efflux ratio =

P B to A P A to B

Papp = Apparent permeability coefficient B = Basolateral compartment A = Apical compartment Plasma Stability. Mouse and human plasma stability was assessed using “loss of parent” approach by incubating the test compounds at 5 µM concentration in mouse or human plasma (100 µL) for up to 4 h at 37°C. The reactions were initiated by the addition of the test compound


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to plasma followed by gentle vortexing and incubation at 37oC with shaking at 100 rpm. At 0, 5, 15, 30, 60, 120, and 240 min of incubation at 37°C, the reactions were terminated by the addition of 400 µL of ice-cold stop solution and vortexing briefly. The stop solution was acetonitrile containing 0.1% (v/v) formic acid and 0.5 µM compound 11 (internal standard). Only for the 0 min samples, the stop solution was added to plasma prior to the addition of the test compound. At the end of the assay, the samples were centrifuged and the supernatants were subjected to UPLC-MS/MS analysis. The positive control compounds for the plasma stability assays were enalapril and procaine in mouse and human plasma, respectively. Metabolic Stability in Liver Microsomes. Mouse and human liver microsomal stability assays were performed by incubating the test compounds at 1 µM concentration in 100 mM potassium phosphate buffer (pH 7.4) containing 3.3 mM MgCl2, 0.5 mg/mL microsomal protein (mouse/human), and 1 mM NADPH (cofactor) in a 100 µL volume at 37°C. The reactions were initiated by the addition of NADPH followed by gentle vortexing and incubation at 37oC with shaking at 100 rpm. At 0, 5, 15, 30, 45, and 60 min after incubation at 37°C, the reactions were terminated by the addition of an equal volume (100 µL) of ice-cold stop solution and brief vortexing. The stop solution was acetonitrile containing 0.1% (v/v) formic acid and 0.5 µM of compound 11 (internal standard). In the case of 0 min samples, the stop solution was added prior to the addition of NADPH. The microsomal stability studies also included an additional “minus NADPH” control group for the 60 min time point. After centrifugation, the supernatants of the samples were subjected to UPLC-MS/MS analysis. Propranolol served as a positive control in both mouse and human liver microsomal stability assays. Metabolic Stability in Cryopreserved Mouse Hepatocytes. Mouse hepatocyte stability assays were conducted by incubating the test compounds with mouse hepatocytes in a modified


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Krebs-Henseleit buffer (InVitroGROTM KHB from BioreclamationIVT, Baltimore, MD) at 37°C for up to 60 min.

The final incubation mixture consisted of the test compound at 1 µM

concentration and 2.5 X 105 hepatocytes in 0.5 mL of InVitroGROTM KHB. The reactions were initiated by the addition of hepatocyte suspension to solutions of test compounds in InVitroGROTM KHB followed by gentle mixing and incubation at 37°C with shaking at 100 rpm. At 0, 5, 15, 30, 45, and 60 min after incubation at 37°C, 50 µL aliquots of the homogenous incubation mixture, which included both the incubation buffer and cells, were obtained and transferred to the corresponding wells of another 96-well plate containing 2× volume (100 µL) of ice-cold stop solution and vortexed gently. The stop solution was acetonitrile containing 0.1% (v/v) formic acid and 0.5 µM of compound 11 (internal standard). The samples were centrifuged and the supernatants were subjected to UPLC-MS/MS analysis. Propranolol was included as a positive control in hepatocyte stability assays. Tolerability and Pharmacokinetics Study. 8-12 weeks old C57BL/6 mice (Charles River) were housed at 3 animals per cage in a 12-hour light/dark cycle, and were given sterile food and water ad libitum.

Prior to injections, animals were individually weighed to determine the

injection dose at 57.5 µmol/kg (equivalent to 10.8 mg/kg for deprenyl, 24.5 mg/kg for compound 10j, 23.4 mg/kg for compound 32, 20.0 mg/kg for compound 10c and 22.0 mg/kg for compound 10k), and were then injected intravenously (iv), subcutaneously (sc) or orally (po) as indicated. Animals were monitored for acute toxicity effects to assess tolerability of compounds.

At the

end of each indicated time points (5, 15, 30 min, 1, 4, and 24 hours for iv; and 1, 2, 4, 6, and 24 hours for sc and po), animals were anesthetized with isofluorane and euthanized with CO2 asphyxiation. Blood were collected in EDTA tubes by cardiac puncture and placed on ice. Plasma was generated by centrifuging blood samples at 1,200 rcf for 15 min at 40C. The brains


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of animals were also harvested and flash frozen. Both plasma and brain samples were stored at 800C until further analysis. The methodology described have been reviewed and approved by the Institutional Animal Care Committee (IACC) at the University of British Columbia (UBC). The care, housing and use of animals were also performed in accordance with the Canadian Council on Animal Care Guidelines. Compound Measurement in Plasma and Brain Samples. For the determination of compound concentrations in plasma, compounds were first extracted from plasma using the Ostro protein precipitation and phospholipid removal plate (Waters), followed by measurements using a Waters UPLC system equipped with an Acquity TQD mass spectrometer detector operated in multiple reaction monitoring (MRM) mode. For the determination of compound concentration from brain tissues, the brains were first weighed and then a portion of the brain was homogenized using a bead beater with Si/Zr beads in 50 mM ammonium formate buffer followed by extraction in acidified organic solvent. Quantitation was also performed using UPLC-MS/MS.

Blood volume accounted for less than 1% changes in total compound

measurements in the brain. Pharmacokinetic Analysis. Pharmacokinetic parameters were determined using a noncompartmental analysis with the Phoenix WinNonLin PK/PD Modeling and Simulation software. Pharmacokinetic curves were generated using GraphPad Prism. For better comparison between plasma and brain parameters, brain values were converted from the measured ng/g values to the reported ng/mL values, with the assumption of 1 g is approximately equal to 1 mL.

ASSOCIATED CONTENT


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Supporting Information.

The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Molecular modeling (Docking) studies (pages S2-S3) and 1H and

13

C NMR spectral

data for the 25 final compounds 10a-s, 21, 22, 25, 26, 29, 32 and all synthetic intermediates described in the Experimental Section (pages S4-S101) (PDF) HPLC data to establish final product purity (pages S102-S129) (PDF) SMILES string for the 25 final compounds described in Table 2 (XLS)

AUTHOR INFORMATION Corresponding Authors Chemistry: *Phone +1 604 8273353. E-mail: [email protected]. Biology: *Phone +1 604 8221734. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.


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ACKNOWLEDGMENT The authors would like to thank Canadian Institutes of Health Research (CIHR) (CIHR MOP123397 and CIHR PPP-136713) and The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital (NKI-AVL) for generously providing the MDCK-MDR1 II cell line. The authors acknowledge the Centre for Drug Research and Development (CDRD), and the PfizerCDRD Innovation Fund for financial support. The authors would like to thank Elaine Tung for technical support.

ABBREVIATIONS USED BnBr, benzyl bromide, TsCl, tosyl chloride, DIAD, diisopropyl azodicarboxylate; MAO, monoamine oxidase; BBB, blood-brain barrier; CNS, central nervous system; FAD, Flavin adenine

dinucleotide;

5-HT,

5-Hydroxytryptamine;

NADPH,

β-nicotinamide

adenine

dinucleotide phosphate; ADMET, absorption, distribution, metabolism, excretion, toxicity; po, per os (oral); sc, subcutaneous; iv, intravenous; TNF-α, tumor necrosis factor-alpha; MDCK, Madin-Darby Canine Kidney; MDR1, Multi drug resistance gene; HBSS, Hanks’ Balanced Salt Solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP, Horseradish peroxidase; MEM, Minimum essential Eagle’s medium; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, Fetal Bovine Serum; P-gp, P-glycoprotein; Papp, Apparent permeability coefficient; WT, wilde-type; Km, Michaelis constant.

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44. Hasan, F.; McCrodden, J. M.; Kennedy, N. P.; Tipton, K. F. The Involvement of Intestinal Monoamine Oxidase in the Transport and Metabolism of Tyramine. J Neural Transm Suppl 1988, 26, 1-9. 45. Riederer, P.; Muller, T. Use of Monoamine Oxidase Inhibitors in Chronic Neurodegeneration. Expert Opin Drug Metab Toxicol 2017, 13, 233-240. 46. Wimbiscus, M.; Kostenko, O.; Malone, D. MAO Inhibitors: Risks, Benefits, and lore. Cleve Clin J Med 2010, 77, 859-882. 47. Polli, J. W.; Baughman, T. M.; Humphreys, J. E.; Jordan, K. H.; Mote, A. L.; Salisbury, J. A.; Tippin, T. K.; Serabjit-Singh, C. J. P-Glycoprotein Influences the Brain Concentrations of Cetirizine (Zyrtec), a Second-Generation Non-Sedating Antihistamine. J Pharm Sci 2003, 92, 2082-2089. 48. QUACPAC, version 1.7.0.2; OpenEye Scientific Software: Santa Fe, NM, 2016. 49. OMEGA, version 2.5.1.4; OpenEye Scientific Software: Santa Fe, NM, 2013. 50. Hawkins, P. C.; Skillman, A. G.; Warren, G. L.; Ellingson, B. A.; Stahl, M. T. Conformer Generation with OMEGA: Algorithm and Validation Using High Quality Structures from the Protein Databank and Cambridge Structural Database. J Chem Inf Model 2010, 50, 572-584. 51. HYBRID, version 3.2.0.2; OpenEye Scientific Software: Santa Fe, NM, 2015. 52. McGann, M. FRED Pose Prediction and Virtual Screening Accuracy. J Chem Inf Model 2011, 51, 578-596.


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53. McGann, M. FRED and HYBRID Docking Performance on Standardized Datasets. J Comput Aided Mol Des 2012, 26, 897-906. 54. SZYBKI, version 1.9.0.3; OpenEye Scientific Software: Santa Fe, NM, 2016.

Table of Contents Graphic


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Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

N N

Deprenyl

R1

O

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CO2H

R3

R2 10c,j,k R 3 = H, R1 /R2 = halogen or H 32 R 3 = CN, R1 /R2 = Cl


Reduced BBB Permeability

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Figure 1. a) MAO activity and selective MAO-A/B inhibitors; b) incorporation of a CO2H function into drug structures suppresses BBB penetration; c) irreversible MAO inhibition by rasagiline. 243x585mm (300 x 300 DPI)



Figure 2. Structure of deprenyl, the polar deprenyl inspired compounds A and B, and the B-type compound 10a. 34x6mm (300 x 300 DPI)


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a Reagents and conditions: (i) BnBr, K2CO3, acetone, RT 16 h then reflux 3 h, 85%; (ii) LAH, THF, RT, 95%; (iii) TsCl, n-Bu4NHSO4, 10%aq NaOH-C6H6, RT, 30 min., 67%; (iv) Zn dust, NaI, THF-H2O, reflux, 2.5 h, 94%; (v) LAH, THF, reflux, 4 h, 54%; (vi) t-butyl bromoacetate, Cs2CO3, DMF, RT, 15 h, 79%; (vii) Pd/C(cat), H2, MeOH, 99%; (viii) ArCH2Br, K2CO3, DMF, RT, 15 h, 36-99%; (ix) (for 9n) 3-OMeBnOH, DIAD, PPh3, THF, RT, 3 min., 62%; (x) conc. HCl, THF, 0°C, 3 h, 35-87%; (xi) Propargyl bromide, Cs2CO3, THF, RT 15h, 42%. 203x168mm (300 x 300 DPI)



a Reagents and conditions: (i) BnBr or 3-ClBnBr, K2CO3, acetone, RT 16 h than reflux 3 h, 95% for 13, 100% for 14; (ii) MeI, NaH, THF, RT, 15 h, 35% for 15, 76% for 16; (iii) SO2Cl, MeOH, RT, 15 h, 62% for both 17 and 18; (iv) t-butyl bromoacetate, Cs2CO3, DMF, RT, 15 h, 49% for 19, 54% for 20; (v) conc. HCl, THF, 0°C, 3 h, 86% for 21, 75% for 22. 95x126mm (300 x 300 DPI)


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a Reagents and conditions: (i) LAH, THF, reflux, 3 h, 88%; (ii) t-butyl bromoacetate, Et3N, DMF, RT, 15 h, 80%; (iii) BnBr, NaH, THF, 15 h, then conc HCl, 2 h, 46%; (iv) t-butyl bromoacetate, Cs2CO3, NaI (cat), DMF, 30min, 90°C, µW, 66%; (v) TsCl, Et3N, DMAP, DCM, RT, 15 h, 88%; (vi) KCN, 18-C-6, NaI (cat), DMF, 100°C. 90 min, 80%; (vii) Pd/C, H2 (1 atm), 90%; (viii) 3,4-diClBnBr, K2CO3, DMF, RT, 15 h, 88%; (ix) conc. HCl, THF, 0°C, 3 h, 77% for 29, 66% for 32. 167x271mm (300 x 300 DPI)



Figure 3. Inhibition of MAO-A and MAO-B enzyme activity by lead compounds 10c, 10j, 10k and 32. 177x91mm (300 x 300 DPI)


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Figure 4. Permeability assays and calculated efflux ratios for compounds 10c, j, k, and 32 using wild type MDCK-WT and MDCK-MDR1 II cell lines. Efflux ratio ≥ 2 identifies the compound as a P-gp substrate. Cultures were treated with (white) or without (black) verapamil (an established P-gp inhibitor). The values were indicated as mean ± standard deviation (n=3). 113x151mm (600 x 600 DPI)



Figure 5. In vitro metabolic stability of novel compounds in plasma (mouse/human), liver microsomes (mouse/human), and hepatocytes (mouse). The positive control compounds were enalapril (mouse plasma), procaine (human plasma), and propranolol (mouse liver microsomes, human liver microsomes, and mouse hepatocytes). Data are shown as mean ± standard deviation (SD). Each experiment was done in triplicate. 114x143mm (300 x 300 DPI)


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Figure 6. Plasma pharmacokinetic curves of deprenyl and compounds 10c, 10j, 10k and 32 following a single 57.5 µmol/kg intravenous dose in C57BL/6 mice. 21 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 7 timepoints (0min, 5min, 15min, 30min, 1h, 4h, 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots. 93x104mm (600 x 600 DPI)



Figure 7. Brain pharmacokinetic curves of deprenyl and compounds 10c, 10j, 10k and 32 following a single intravenously 57.5 µmol/kg dose in C57BL/6 mice. 21 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 7 timepoints (0min, 5min, 15min, 30min, 1h, 4h, 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots. 84x97mm (300 x 300 DPI)


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Figure 8. Plasma pharmacokinetic curves of compound 10j and compound 32 following a single 57.5 µmol/kg dose via subcutaneous (SC) and oral (PO) administration. 15 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 5 timepoints (1, 2, 4, 6 and 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots. 84x65mm (300 x 300 DPI)



Figure 9. Brain pharmacokinetic curves of compound 10j and compound 32 following a single 57.5 µmol/kg dose via subcutaneous and oral administration. 15 animals were injected at 0min, with 3 animals sacrificed at each time point, for a total of 5 timepoints (1, 2, 4, 6 and 24h). For the plotting of pharmacokinetic curves, values that were below the limit of quantification were plotted as half of limit of quantification while values that were above the limit of quantification or non-detected were omitted from the plots. 84x65mm (300 x 300 DPI)


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Figure 10. Structural comparison of compounds 10c, 10j and 10k with safinamide and compounds 21, 22, 25, 26, 29 and 32 with the coumarin amide 33. The V shaped wedges represent the constriction residues (Tyr 326 and Ile 199) in the MAO-B binding site. 144x107mm (300 x 300 DPI)



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(MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for

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