Chapter 7 Second article Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine oxidase B

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Chapter 7

Second article

Sulfanylphthalonitrile analogues as selective and potent inhibitors of

monoamine oxidase B

Mietha M. Van der Walt,a Gisella Terre’Blanche,a Anna C.U. Lourens,a Anél Petzer,b and Jacobus P. Petzera

a

Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa

b

Unit for Drug Research and Development, School of Pharmacy, North-West University, Private Bag

X6001, Potchefstroom, 2520, South Africa

7.

7.1 Graphical abstract Br S CN CN

6c

Accepted Manuscript: Bioorganic & Medicinal Chemistry Letters Received Date: 27 August 2012

Revised Date: 10 October 2012 Accepted Date: 15 October 2012 Available online: in press

DOI: http://dx.doi.org/10.1016/j.bmcl.2012.10.070 Reference: BMCL 19693

Promising MAO-B inhibitor

High potency IC50 = 0.025 µM

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7.2 Author’s contributions

Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine

oxidase B

a

b

The above mentioned article is part of the doctoral thesis of M.M. van der Walt. As required, the table below indicates the contribution of each author to this research article. Also, the written declaration for consent by each author is provided. This article was submitted for publication in Bioorganic & Medicinal Chemistry Letters and the guidelines to authors are also given.

Author contributions: Author name: Description of contribution

research design: J.P. Petzer Concept of the study was provided.

performed research:

a) Synthetic work M.M. van der Walt Performed all the syntheses.

b) Characterization via NMR and MS

SASOL centre for chemistry, NWU

Carried out by the SASOL Centre for Chemistry, North-West University. NMR spectra was recorded by André Joubert and MS was recorded by Johan Jordaan.

c) Compound purity determination by HPLC

M.M. van der Walt The degree of purity of each

compound was determined by HPLC analysis.

d) IC50 value determination M.M. van der Walt The IC50 values for the inhibition of

MAO-A and MAO-B were measured. e) Recovery of enzyme activity

after dilution study

M.M. van der Walt A. Petzer

The recovery of enzyme activity after dilution was performed for compound

6c. contributed new reagents/ analytic

tools:

a) Synthesis of compounds G. Terre’Blanche Providing the facilities, instrumentation and relevant

reagents for performing the synthetic work.

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the recovery of enzyme activity after dilution study

instrumentation and relevant reagents for performing the IC50

value determintation and the recovery of enzyme activity after dilution study

analyzed data:

a) Characterization by NMR and MS

M.M. van der Walt J.P. Petzer

The analyses of the NMR and MS data were performed by M.M. van der Walt, with critical feedback by J.P. Petzer.

b) Compound purity measurement determination

M.M. van der Walt J.P. Petzer

The analyses of the compound purity data were performed by M.M. van der Walt, with critical feedback by J.P. Petzer.

c) IC50 value measurements M.M. van der Walt

A. Petzer J.P. Petzer

Calculations of the IC50 values were

performed by M.M. van der Walt, with critical feedback by J.P. Petzer and A. Petzer.

d) Recovery of enzyme activity after dilution study

M.M. van der Walt A. Petzer

J.P. Petzer

The analyses and interpretation of the data was performed by M.M. van der Walt with critical feedback by J.P. Petzer and A. Petzer.

manuscript:

a) Writing of manuscript

b) Comments, suggestions and proof reading

M.M. van der Walt J.P. Petzer G. Terre’Blanche A. Petzer

A.C.U. Lourens

Contribution was equally for the mentioned authors.

Contribution was equally for the mentioned authors

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7.3 Declaration and consent of each co-author

Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine

oxidase B

The following is a declaration by the co-authors that confirms their individual roles in the above mentioned article, as described in the table of author’s contributions. The authors hereby give consent that this article may form part of this doctoral thesis.

I declare that I approve the inclusion of above mentioned article in this thesis, that my role in this study is as indicated in the author’s contributions table and is representative of my actual contribution. Herewith, I grant consent that this article may be published as part of the doctoral thesis of Mietha Magdalena van der Walt.

_________________ Prof. J.P. Petzer _________________ Dr. G. Terre’Blanche _________________ Dr. A. Petzer _________________ Dr. A.C.U. Lourens

7.4 Journal publishing agreement

The following assignment of publishing rights was accepted on behalf of all the co-authors of the manuscript published (DOI: http://dx.doi.org/10.1016/j.bmcl.2012.10.070) in Bioorganic & Medicinal Chemistry Letters. Elsevier Ltd has granted the authors retention of these rights for scholarly purposes (section 6.4.2).

7.5 Author’s instructions – Bioorganic & Medicinal Chemistry Letters

The author’s instructions for Bioorganic & Medicinal Chemistry Letters are offered as published in the “Guide for Authors” of this journal. These author’s instructions were provided in detail in section 6.5.

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7.6 Accepted article

Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine

oxidase B

a

b

Abstract―It has recently been reported that nitrile containing compounds frequently act as potent monoamine oxidase B (MAO-B) inhibitors. Modelling studies suggest that this high potency inhibition may rely, at least in part, on polar interactions between nitrile functional groups and polar moieties within the MAO-B substrate cavity. In an attempt to identify potent and selective inhibitors of MAO-B and to contribute to the known structure-activity relationships of MAO inhibition by nitrile containing compounds, the present study examined the MAO inhibitory properties of series of novel sulfanylphthalonitriles and sulfanylbenzonitriles. The results document that the evaluated compounds are potent and selective MAO-B inhibitors with most homologues possessing IC50 values in the nanomolar range. In general, the

sulfanylphthalonitriles exhibited higher binding affinities for MAO-B than the corresponding

sulfanylbenzonitrile homologues. Among the compounds evaluated,

4-[(4-bromobenzyl)sulfanyl]phthalonitrile is a particularly promising inhibitor since it displayed a high degree of selectivity (8720-fold) for MAO-B over MAO-A, and potent MAO-B inhibition (IC50 =

0.025 µM). Based on these observations, this structure may serve as a lead for the development of therapies for neurodegenerative disorders such as Parkinson’s disease.

Keywords: phthalonitrile; benzonitrile; monoamine oxidase; MAO; inhibition; Parkinson’s

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CN CN O CN O 2 1

The enzyme monoamine oxidase B (MAO-B) is considered to be a major dopamine metabolizing enzyme in the human brain and, as such, a target for the treatment of Parkinson’s disease.1–3 Inhibitors of MAO-B are thought to reduce the metabolic degradation of central dopamine and as a result increase dopaminergic neurotransmission.4,5 MAO-B inhibitors are frequently combined with levodopa in the therapy of Parkinson’s disease since this may enhance the dopamine levels derived from levodopa and allow for a reduction of the effective levodopa dose.6 In the initial stages of the disease, MAO-B inhibitors may also delay the emergence of disabilities that require the initiation of levodopa therapy. The inhibition of MAO-B has also been associated with a neuroprotective effect. This effect may, at least in part, depend on blocking the formation of H2O2 and aldehyde species, metabolic by-products of substrate

metabolism by MAO-B.7 These metabolites may be harmful if not rapidly cleared from the central nervous system. Considering that central MAO-B activity increases with age,8–10 inhibition of the MAO-B-catalyzed formation of toxic by-products in the aged parkinsonian brain is of particular relevance. Interestingly, dopamine is also oxidized by the MAO-A isoform in the human brain, and MAO-A inhibitors have been shown to enhance central dopamine levels in primates.4,5 MAO-A inhibitors may, however, lead to serious adverse effects when combined with certain drugs and food. Most notably, when MAO-A inhibitors are used with indirectly acting sympathomimetic amines such as tyramine, which is present is certain foods, a potentially fatal hypertensive response may occur.7,11 MAO-A inhibitors should also be used with caution in combination with levodopa since this may elicit a hypertensive crisis.12 For these reasons, inhibitors that are selective for the MAO-B isoform are more desirable as antiparkinsonian agents.

Figure 1. The structures of 4-benzyloxyphthalonitrile (1) and 4-benzyloxybenzonitrile (2).

Based on these considerations, the present study aims to discover novel compounds that bind selectively and with high binding affinities to MAO-B. Among the various types of structures that have been reported to inhibit the MAO enzymes are nitrile containing compounds. Both phthalonitriles and benzonitriles have been found to act as potent and selective MAO-B inhibitors.13,14 For example, 4-benzyloxyphthalonitrile (1) and 4-benzyloxybenzonitrile (2) inhibit

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human MAO-B reversibly with IC50 values of 0.0079 µM and 0.785 µM, respectively (Fig. 1).13

These homologues display 227- and 41-fold selectivities, respectively, for the MAO-B isoform. Based on its good selectivity and high potency, compound 1 may be viewed as a particularly promising inhibitor. The high binding affinities of nitrile containing compounds to MAO-B may be explained by the highly polar nature of this functional group. Modelling studies predict that the nitrile group interacts with the polar regions in the substrate cavity of the MAO-B enzyme.13 This seems to be a prerequisite for potent inhibition since elimination of the nitrile results in a loss of MAO-B inhibitory activity. Literature also documents that phthalonitriles are in general more potent MAO-B inhibitors than benzonitriles, which suggests that the productive interactions between nitrile groups and the MAO-B enzyme are additive. The active site cavity of MAO-B is, however, bipartite and besides the substrate cavity where the phthalonitrile and benzonitrile moieties are predicted to bind, the active site also possesses an entrance cavity.15 The benzyloxy side chains of compounds 1 and 2 are predicted to bind within the entrance cavity of MAO-B, where they are most likely stabilized via Van der Waals interactions. The interactions between the benzyloxy side chain and the entrance cavity are thought to also contribute significantly to the binding affinities of these inhibitors, since modification (e.g. replacement with a phenoxy) leads to a two orders of magnitude loss in MAO-B inhibition activity. In addition, several other potent MAO-B inhibitors possess the benzyloxy side chain. These include safinamide (3) and 8-benzyloxycaffeine (4) (Fig. 2). Crystallographic as well as modelling studies have shown that the benzyloxy side chains of these inhibitors bind within the MAO-B entrance cavity.16,17 Of note is a recent report that the benzylsulfanyl side chain exhibit similar properties to that of the benzyloxy moiety, since a series of 8-(benzylsulfanyl)caffeines (5) possesses similar MAO-B inhibition potencies to those of the 8-benzyloxycaffeines (4).18 In addition, modelling suggests that the binding modes of the benzylsulfanyl and benzyloxy side chains within the entrance cavity of MAO-B are highly comparable. Based on these observations, the present study examines the possibility that benzylsulfanyl substitution on the phthalonitrile and benzonitrile moieties, to yield compounds 6a and 7a, would also lead to highly potent and selective MAO-B inhibition (Fig. 3). To explore the structure-activity relationships (SARs) of MAO inhibition by these compounds, the effects that substitution (Cl, Br, F and OCH3)

on the benzylsulfanyl ring have on MAO inhibition were considered. Halogen substitution on the benzylsulfanyl ring of 5 has been shown to be beneficial for MAO-B inhibition.18 In addition, substitution on the phthalonitrile and benzonitrile moieties with phenylsulfanyl, (2-phenylethyl)sulfanyl, cyclohexylsulfanyl, cyclopentylsulfanyl and (3-methylbutyl)sulfanyl substituents were also considered.

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N H H CH3 NH2 O O F 3 N N N N O O O 4 N N N N S O O 5 CN CN S 6a CN S 7a

Figure 2. The structures of safinamide (3), 8-benzyloxycaffeine (4) and 8-(benzylsulfanyl)caffeine (5).

Figure 3. The general structures of 4-(benzylsulfanyl)phthalonitrile (6a) and

4-(benzylsulfanyl)benzonitrile (7a).

The target sulfanylphthalonitriles (6a–l) and sulfanylbenzonitriles (7a–j) were synthesized according to the procedures described in literature.13,19 The sulfanylphthalonitriles were synthesized in fair to good yields (16–83%) by reacting an appropriate thiol with 4-nitrophthalonitrile in the presence of K2CO3 in dimethyl sulfoxide (DMSO) (Scheme 1). The

crude products were purified by recrystallization from an appropriate solvent as indicated in the supplementary information. The sulfanylbenzonitriles were synthesized in an analogous manner by reacting 4-nitrobenzonitrile with an appropriate thiol (2–72%). The structures of the target nitrile derivatives were verified by 1H NMR, 13C NMR and mass spectrometry, while their purities were estimated by HPLC analysis as cited in the supplementary information.

The MAO inhibitory activities of the sulfanylphthalonitriles and sulfanylbenzonitriles were evaluated by employing recombinant human MAO-A and –B as enzyme sources.20 Kynuramine,

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a mixed MAO–A/B substrate, served as enzyme substrate for these inhibition studies. Kynuramine undergoes MAO-catalyzed oxidation to yield the fluorescent metabolite, 4– hydroxyquinoline, which may be conveniently measured by fluorescence spectrophotometry without interference from the substrate and inhibitors evaluated in this study.21 The inhibitory potencies of the nitriles are expressed as the corresponding IC50 values, and their selectivities

for the MAO-B isoform are expressed as the selectivity index (SI) value [IC50(MAO–

A)]/[(IC50(MAO–B)].

Scheme 1. Synthetic pathway to sulfanylphthalonitriles (6a–l) and sulfanylbenzonitriles (7a–j). Reagents

and conditions: (a) K2CO3, DMSO, argon.

The results document that the sulfanylphthalonitriles (6) are potent MAO-B inhibitors with all homologues, except 6f, possessing IC50 values in the submicromolar range (Table 1). In

accordance to expectation, benzylsulfanyl substitution on the phthalonitrile moiety, to yield 6a (IC50 = 0.167 µM), resulted in potent MAO-B inhibition. Substitution (Cl, Br, F and OCH3) on the

benzylsulfanyl ring further enhanced MAO-B inhibition potency with compounds 6b–e exhibiting IC50 values of 0.014–0.067 µM. In fact, 6b proved to be the most potent MAO-B inhibitor of the

present series. In contrast to the effect of the benzylsulfanyl moiety, phenylsulfanyl substitution on the phthalonitrile moiety, to yield 6f (IC50 = 2.13 µM), resulted in only moderate MAO-B

inhibition. Interestingly, halogen substitution on the phenyl ring of 6f improved MAO-B inhibition with 6g–h exhibiting IC50 values of 0.079–0.114 µM. Substitution on the phthalonitrile moiety

with (2-phenylethyl)sulfanyl, cyclohexylsulfanyl, cyclopentylsulfanyl and (3-methylbutyl)sulfanyl side chains also resulted in potent MAO-B inhibition. These homologues (6i–l) displayed IC50

values of 0.124–0.887 µM. It is noteworthy that the (2-phenylethyl)sulfanyl substituted homologue 6i (IC50 = 0.124 µM) is slightly more potent than the benzylsulfanyl substituted

phthalonitrile 6a. This suggests that extension of the benzylsulfanyl side chain result in slightly improved MAO-B inhibition. Reduction of the length of the benzylsulfanyl side chain of 6a to yield 6f, however, markedly lowers MAO-B inhibition potency.

O2N CN CN + R SH a 6al S CN CN R O2N CN + R SH a 7aj S CN R

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CN CN R 1 2 4

Table 1. The IC50 values for the inhibition of recombinant human MAO-A and –B by

sulfanylphthalonitriles 6a–l. IC50 (µM)a R MAO-A MAO-B SIb SIc,d 6a –S–(CH2)-C6H5 9.02 ± 0.896 0.167 ± 0.016 54 33 6b – S–(CH2)-(4-Cl-C6H4) 0.623 ± 0.053 0.014 ± 0.004 45 27 6c – S–(CH2)-(4-Br-C6H4) 218 ± 23.9 0.025 ± 0.003 8720 5334 6d – S–(CH2)-(4-F-C6H4) 2.10 ± 0.218 0.034 ± 0.006 62 38 6e –S–(CH2)-(4-OCH3-C6H4) 129 ± 78.1 0.067 ± 0.021 1925 1178 6f –S–C6H5 3.58 ± 0.592 2.13 ± 0.067 1.7 1.0 6g –S–(4-Cl-C6H4) 11.1 ± 1.17 0.114 ± 0.002 97 59.6 6h – S–(4-Br-C6H4) 19.0 ± 8.27 0.079 ± 0.017 240 147.1 6i –S–(CH2)2-C6H5 7.19 ± 1.35 0.124 ± 0.009 58 35.5 6j –S–C6H11 5.96 ± 0.382 0.223 ± 0.039 27 16.3 6k –S–C5H9 2.78 ± 0.936 0.887 ± 0.072 3.1 1.9 6l –S–(CH2)2-CH(CH3)2 1.45 ± 0.203 0.242 ± 0.035 6.0 3.7 a

All values are expressed as the mean ± SD of triplicate determinations.

b

The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of [IC50

(MAO-A)]/[IC50(MAO-B)]. c

The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of Ki(MAO-A)/Ki(MAO-B).

d

The Ki values were calculated from the experimental IC50 values according to the equation by Cheng

and Prusoff: Ki = IC50/(1 + [S]/Km).For human MAO-A, [S] = 45 µM and Km (kynuramine) = 16.1 µM, while

for human MAO-B, [S] = 30 µM and Km (kynuramine) = 22.7 µM.

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Table 2. The IC50 values for the inhibition of recombinant human MAO-A and –B by sulfanylbenzonitriles 7a–j. CN R 4 1 IC50 (µM)a R MAO-A MAO-B SIb SIc,d 7a –S–(CH2)-C6H5 42.4 ± 3.10 1.58 ± 0.327 27 16 7b – S–(CH2)-(4-Cl-C6H4) 20.3 ± 6.62 0.531 ± 0.114 38 23 7c – S–(CH2)-(4-Br-C6H4) 129 ± 18.5 0.484 ± 0.052 267 163 7d – S–(CH2)-(4-F-C6H4) 54.7 ± 22.8 0.449 ± 0.128 122 75 7e –S–(CH2)-(4-OCH3-C6H4) 5.59 ± 0.744 0.861 ± 0.165 6.5 4.0 7f –S–C6H5 36.9 ± 1.02 11.2 ± 0.584 3.3 2.0 7g –S–(4-Cl-C6H4) 18.2 ± 2.17 4.28 ± 2.09 4.3 2.6 7h – S–(4-Br-C6H4) 8.52 ± 0.869 0.637 ± 0.162 13 8.2 7i –S–(CH2)2-C6H5 54.3 ± 11.9 1.81 ± 0.130 30 18 7j –S–C6H11 9.87 ± 3.03 4.77 ± 0.383 2.1 1.3 a

All values are expressed as the mean ± SD of triplicate determinations.

b

The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of [IC50

(MAO-A)]/[IC50(MAO-B)]. c

The selectivity index is the selectivity for the MAO-B isoform and is given as the ratio of Ki

(MAO-A)/Ki(MAO-B). d

The Ki values were calculated from the experimental IC50 values according to the equation by Cheng

and Prusoff: Ki = IC50/(1 + [S]/Km).For human MAO-A, [S] = 45 µM and Km (kynuramine) = 16.1 µM, while

for human MAO-B, [S] = 30 µM and Km (kynuramine) = 22.7 µM.17,23

The MAO inhibitory properties of the sulfanylbenzonitriles are given in Table 2. The results show that, although several compounds exhibit IC50 values in the submicromolar range, the

sulfanylbenzonitriles exhibited lower binding affinities for MAO-B than the corresponding sulfanylphthalonitrile homologues. The MAO-B inhibitor potencies of the sulfanylbenzonitriles ranged from 0.449–11.2 µM with the most potent inhibitor being compound 7d. As observed for

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the sulfanylphthalonitriles, substitution (Cl, Br, F and OCH3) on the benzylsulfanyl ring enhanced

MAO-B inhibition potency compared to the unsubstituted homologue 7a (IC50 = 1.58 µM). These

substituted homologues, compounds 7b–e, possessed IC50 values of 0.449–0.861 µM.

Similarly, halogen substitution on the phenyl ring of 7f (IC50 = 11.2 µM) improved MAO-B

inhibition with 7g–h exhibiting IC50 values of 0.637–4.28 µM. Among the sulfanylbenzonitriles,

phenylsulfanyl substitution (to yield 7f) resulted in the weakest MAO-B inhibition. This result is similar to that obtained with the sulfanylphthalonitriles. Based on these results it may be concluded that sulfanylphthalonitriles are more favourable for MAO-B inhibition compared to sulfanylbenzonitriles.

The results given in Tables 1 and 2 show that the sulfanylphthalonitriles and sulfanylbenzonitriles also are inhibitors of MAO-A. In all instances, these compounds are, however, selective for MAO-B with SI values ranging from 1.7–8720. Only one compound, 6b (IC50 = 0.623 µM), exhibited an IC50 value in the submicromolar range for the inhibition of

MAO-A. Based on IC50 values of 0.623–218 µM, it may therefore be concluded that

sulfanylphthalonitriles and sulfanylbenzonitriles are in general weak to moderate inhibitors of MAO-A. It is noteworthy that two compounds, 6c and 6e, exhibited SI values in excess of 1000. These compounds, particularly 6c (SI = 8720), may therefore be considered as highly selective for MAO-B. Compounds 6c and 6e are also highly potent MAO-B inhibitors. As mentioned, selective and potent MAO-B inhibitors represent good candidates for antiparkinsonian therapy. It has been reported that phthalonitriles act as reversible MAO-B inhibitors.13 To verify that the sulfanylphthalonitrile class of compounds also interacts reversibly with MAO-B, the reversibility of MAO-B inhibition by 6c was examined. For this purpose 6c, at concentrations of 10 × IC50

and 100 × IC50, was preincubated with MAO-B for 30 min and the extent of enzyme recovery

after dilution of the enzyme-inhibitor complex was measured.22 The results show that, after dilution of the enzyme-inhibitor complexes to concentrations of 6c equal to 0.1 × IC50 and 1 ×

IC50, the MAO-B catalytic activities are recovered to levels of approximately 76% and 48%,

respectively, of the control value (Fig. 4). This recovery of MAO-B activity is consistent with a reversible interaction between 6c and MAO-B. For comparison, MAO-B was treated in a similar manner with the irreversible inhibitor, (R)-deprenyl, at a concentration of 10 × IC50. After

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No In hibito r 50 [I] = 0 .1 x IC 50 [I] = 1 x IC (R)-D epre nyl 0 25 50 75 100 Ra te ( % ) CN CN O Br 8

Figure 4. The reversibility of the interaction between MAO-B by 6c. Compound 6c at concentrations of 10

× IC50 and 100 × IC50 was preincubated with MAO-B for 30 min. The resulting reactions were diluted

100-fold to yield inhibitor concentrations of 0.1 × IC50 and 1 × IC50, respectively. The control reactions were

conducted in the absence of inhibitor. For comparison, reactions containing (R)-deprenyl, at 10 × IC50,

was also preincubated with MAO-B and diluted to 0.1 × IC50. After dilution of all reactions, the residual

enzyme activities were subsequently measured.

Figure 5. The structure of 4-(4-bromobenzyloxy)phthalonitrile (8).

In conclusion, the present study shows that the sulfanylphthalonitrile and to a lesser extent the sulfanylbenzonitrile classes of compounds are in general highly potent inhibitors of MAO-B. A particularly promising compound among those examined is the para bromo substituted derivative of 4-(benzylsulfanyl)phthalonitrile, compound 6c. This compound displays potent MAO-B inhibition (IC50 = 0.025 µM) and a high degree of selectivity (8720-fold) for MAO-B over

MAO-A. For comparison, (R)-deprenyl exhibits an IC50 value for the inhibition of MAO-B of 0.079

µM under identical experimental conditions.22 Compared to (R)-deprenyl, compound 6c is a threefold more potent MAO-B inhibitor. The MAO-A inhibitor, clorgyline, exhibits an IC50 value

for the inhibition of MAO-A of 0.0026 µM under identical experimental conditions.22 The most potent MAO-A inhibitor of the present series, compound 6b (IC50 = 0.623) is therefore 240-fold

less potent as a MAO-A inhibitor than clorgyline. Compared to the lead compounds of this study, the 4-benzyloxyphthalonitrile (1) class of compounds, the sulfanylphthalonitriles are, however, less potent MAO-B inhibitors. For example, the para bromo substituted homologue of

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4-benzyloxyphthalonitrile, compound 8, inhibits human MAO-B with an IC50 value of 0.0048 µM,

approximately five-fold more potent than 6c (Fig. 5).13 Compound 6c is, however, a considerably (66-fold) more selective MAO-B inhibitor than 8 (SI = 134). From this analysis it may be concluded that among the phthalonitrile and benzonitrile derivatives examined to date,

6c is the most appropriate candidate for Parkinson’s disease therapy.13,14 Acknowledgements

We are grateful to André Joubert and Johan Jordaan of the SASOL Centre for Chemistry, North-West University for recording the NMR and MS spectra, respectively. Financial support for this work was provided by the North-West University, the National Research Foundation and the Medical Research Council, South Africa.

References

1. Nagatsu, T.; Sawada, M. J. Neural Transm. Suppl. 2006, 71, 53. 2. Yamada, M.; Yasuhara, H. Neurotoxicology. 2004, 25, 215.

3. Youdim, M. B. H.; Edmondson, D.; Tipton, K. F. Nat. Rev. Neurosci. 2006, 7, 295.

4. Di Monte, D. A.; DeLanney, L. E.; Irwin, I.; Royland, J. E.; Chan, P.; Jakowec, M. W.; Langston, J. W. Brain. Res. 1996, 738, 53.

5. Finberg, J. P.; Wang, J.; Bankiewich, K.; Harvey-White, J; Kopin, I. J.; Goldstein, D. S. J. Neural Transm. Suppl. 1998, 52, 279.

6. Fernandez, H. H.; Chen, J. J. Pharmacotherapy. 2007, 27, 174S. 7. Youdim, M. B. H.; Bakhle, Y. S. Br. J. Pharmacol. 2006, 147, S287.

8. Nicotra, A.; Pierucci, F.; Parvez, H.; Senatori, O. Neurotoxicology. 2004, 25, 155.

9. Fowler, J. S.; Volkow, N. D.; Wang, G. J.; Logan, J.; Pappas, N.; Shea, C.; MacGregor, R. Neurobiol. Aging. 1997, 18, 431.

10. Kalaria, R. N.; Mitchell, M. J.; Harik, S. I. Brain 1988, 111, 1441. 11. Youdim, M. B.; Weinstock, M. Neurotoxicology. 2004, 25, 243.

12. Zesiewicz, T. A.; Hauser, R. A. In Parkinson's disease: diagnosis and clinical management; Factor, S. A., Weiner, W. J., Eds.; Demos Medical Publishing: New York, 2002; pp 365-378.

13. Manley-King, C. I.; Bergh, J. J.; Petzer, J. P. Bioorg. Chem. 2012, 40, 114.

14. Legoabe, L.; Kruger, J.; Petzer, A.; Bergh, J. J.; Petzer, J. P. Eur. J. Med. Chem. 2011, 46, 5162.

15. Binda, C.; Newton-Vinson, P.; Hubálek, F.; Edmondson, D. E.; Mattevi, A. Nat. Struct. Biol. 2002, 9, 22.

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16. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D. E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.

17. Strydom, B.; Malan, S. F.; Castagnoli, N., Jr.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem. 2010, 18, 1018.

18. Booysen, H. P.; Moraal, C.; Terre'Blanche, G.; Petzer, A.; Bergh, J. J.; Petzer, J. P. Bioorg Med Chem. 2011, 19, 7507.

19. Tau, P.; Nyokong, T. Electrochim. Acta. 2007, 52, 3641.

20. Novaroli, L.; Reist, M.; Favre, E.; Carotti, A.; Catto, M.; Carrupt, P. A. Bioorg. Med. Chem. 2005, 13, 6212.

21. Manley-King, C. I.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem. 2011, 19, 4829. 22. Petzer, A.; Harvey, B. H.; Wegener, G.; Petzer, J. P. Toxicol. Appl. Pharm. 2012, 258,

403.

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7.7 Supplementary material

Sulfanylphthalonitrile analogues as selective and potent inhibitors of monoamine

oxidase B

a

b

7.7.1 Experimental procedures

7.7.1.1 Chemicals and instrumentation

All reagents, unless otherwise stated, were obtained from Sigma-Aldrich and were used without further purification. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance III 600 spectrometer in CDCl3 and DMSO-d6. The chemical shifts are reported in parts

per million (δ) downfield from the signal of tetramethylsilane. Spin multiplicities are abbreviated as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet) or m (multiplet). High resolution mass spectra (HRMS) were recorded with a Bruker micrOTOF-Q II mass spectrometer in atmospheric-pressure chemical ionization (APCI) mode. Melting points (mp) were measured with a Buchi M-545 melting point apparatus and are uncorrected. A Varian Cary Eclipse fluorescence spectrophotometer was employed for fluorescence spectrophotometry. Kynuramine.2HBr and insect cell microsomes containing recombinant human MAO-A and –B (5 mg/mL) were obtained from Sigma-Aldrich.

7.7.1.2 Synthesis of the sulfanylphthalonitrile analogues (6a–l)

The C4-substituted phthalonitrile derivatives, compounds 6a–l, were synthesized according to a previously reported protocol.1 In brief, an appropriate thiol (6 mmol) was dissolved in 4.3 mL dimethyl sulfoxide (DMSO) and the reaction was placed under an atmosphere of argon. Subsequently, 4–nitrophthalonitrile (5 mmol) was added to the reaction and stirring was continued for 15 min at room temperature. Potassium carbonate (15 mmol) was added portionwise to the reaction over a period of 2 h and the resulting mixture was stirred for a further 48 h. The reaction was subsequently poured into 50 mL ice cold water and stirred for another 30 min, yielding a precipitate. The precipitate was collected by filtration, washed with 60 mL

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water and finally dried at 50 ºC. The products were recrystallized form the appropriate solvent as cited below.

4-(Benzylsulfanyl)phthalonitrile (6a)

The title compound (yellow crystals) was prepared from 4-nitrophthalonitrile and benzyl mercaptan in a yield of 83%: mp 140–141 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3)

δ 4.23 (s, 2H), 7.28–7.36 (m, 5H), 7.48 (dd, 1H, J = 1.9, 8.7 Hz), 7.55 (d, 1H, J = 1.9 Hz), 7.59 (d, 1H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 36.8, 111.2, 115.0, 115.4, 116.1,

128.1, 128.6, 128.9, 130.3, 130.6, 133.2, 134.4. 146.5; APCI-HRMS m/z: calcd for C15H11N2S

(MH+), 251.0643, found 251.0646; Purity (HPLC): 100%.

4-[(4-Chlorobenzyl)sulfanyl]phthalonitrile (6b)

The title compound (yellow crystals) was prepared from 4-nitrophthalonitrile and 4-chlorobenzyl mercaptan in a yield of 27%: mp 145.1–148 ºC (ethyl acetate\methanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 4.19 (s, 2H), 7.29 (s, 4H), 7.46 (dd, 1H, J = 1.9, 8.3 Hz), 7.55 (d, 1H, J

= 1.9 Hz), 7.61 (d, 1H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 36.2, 111.5,

115.0, 115.3, 116.3, 129.2, 130.0, 130.4, 130.6, 132.9, 133.3, 134.0. 145.9; APCI-HRMS m/z: calcd for C15H10ClN2S (MH+), 285.0253, found 285.0242; Purity (HPLC): 100%.

4-[(4-Bromobenzyl)sulfanyl]phthalonitrile (6c)

The title compound (yellow crystals) was prepared from 4-nitrophthalonitrile and 4-bromobenzyl mercaptan in a yield of 38%: mp 150.0–150.2 ºC (ethyl acetate); 1H NMR (Bruker Avance III 600, CDCl3) δ 4.18 (s, 2H), 7.23 (d, 2H, J = 8.3 Hz), 7.45–7.47 (m, 3H), 7.55 (d, 1H, J = 1.9 Hz),

7.61 (d, 1H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 36.2, 111.5, 114.9, 115.3,

116.3, 122.1, 130.3, 130.4, 130.6, 132.1, 133.3, 133.5. 145.9; APCI-HRMS m/z: calcd for C15H10BrN2S (MH+), 328.9748, found 328.9745; Purity (HPLC): 100%.

4-[(4-Fluorobenzyl)sulfanyl]phthalonitrile (6d)

The title compound (dark yellow crystals) was prepared from nitrophthalonitrile and 4-fluorobenzyl mercaptan in a yield of 16%: mp 173.6–173.7 ºC (ethyl acetate\methanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 4.11 (s, 2H), 6.93 (t, 2H, J = 8.7 Hz), 7.22–7.24 (m, 2H), 7.37

(dd, 1H, J = 1.9, 8.3 Hz), 7.46 (d, 1H, J = 1.9 Hz), 7.51 (d, 1H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 36.2, 111.5, 115.0, 115.3, 115.9, 116.1, 116.3, 130.1, 130.3, 130.4,

130.6, 133.2 146.1, 161.6, 163.2; APCI-HRMS m/z: calcd for C15H10FN2S (MH+), 269.0549,

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4-[(4-Methoxybenzyl)sulfanyl]phthalonitrile (6e)

The title compound (dark yellow crystals) was prepared from nitrophthalonitrile and 4-methoxybenzyl mercaptan in a yield of 36%: mp 176.7–176.9 ºC (ethanol); 1H NMR (Bruker Avance III 600, DMSO-d6) δ 3.71 (s, 3H), 4.39 (s, 2H), 6.88 (d, 2H, J = 8.7 Hz), 7.34 (d, 2H, J = 8.7 Hz), 7.76 (dd, 1H, J = 1.9, 8.3 Hz), 7.96 (d, 1H, J = 8.3 Hz), 8.08 (d, 1H, J = 1.9 Hz); 13C NMR (Bruker Avance III 600, DMSO-d6) δ 34.5, 55.1, 79.17, 109.9, 114.1, 115.0, 115.6, 116.1, 127.3, 130.2, 130.6, 133.7, 146.5, 158.7; APCI-HRMS m/z: calcd for C16H13N2OS (MH+),

281.0749, found 281.0741; Purity (HPLC): 100%.

4-(Phenylsulfanyl)phthalonitrile (6f)

The title compound (light yellow crystals) was prepared from 4-nitrophthalonitrile and thiophenol in a yield of 60%: mp 166.1–168.6 ºC (ethyl acetate\methanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 7.22–7.24 (m, 2H), 7.39–7.44 (m, 5H), 7.48 (dd, 1H, J = 0.8, 7.9 Hz); 13C NMR (Bruker

Avance III 600, CDCl3) δ 111.2, 115.1, 115.4, 116.3, 128.3, 129.9, 130.0, 130.5, 130.6, 133.3,

135.2. 148.3; m/z: calcd for C14H9N2S (MH+), 237.0486, found 237.0499; Purity (HPLC): 99%.

4-[(4-Chlorophenyl)sulfanyl]phthalonitrile (6g)

The title compound (light orange crystals) was prepared from nitrophthalonitrile and 4-chlorothiophenol in a yield of 57%: mp 147.5–147.9 ºC (ethyl acetate\methanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 7.22–7.25 (m, 2H), 7.37 (s, 4H), 7.50 (d, 1H, J = 8.3 Hz); 13C

NMR (Bruker Avance III 600, CDCl3) δ 111.7, 114.9, 115.3, 116.5, 126.9, 130.0, 130.2, 130.8,

133.4, 136.4, 137.2. 147.5; APCI-HRMS m/z: calcd for C14H8ClN2S (MH+), 271.0097, found

271.0106; Purity (HPLC): 100%.

4-[(4-Bromophenyl)sulfanyl]phthalonitrile (6h)

The title compound (yellow crystals) was prepared from nitrophthalonitrile and 4-bromothiophenol in a yield of 55%: mp 139.2–139.5 ºC (ethyl acetate\methanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 7.33 (dd, 1H, J = 1.9, 8.3 Hz), 7.35 (m, 1H), 7.38 (d, 2H, J =

6.8 Hz), 7.59 (d, 1H, J = 8.3 Hz), 7.61 (d, 2H, J = 6.8 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 111.7, 114.9, 115.3, 116.5, 125.3, 127.6, 130.1, 130.3, 133.4, 133.7, 136.5. 147.3;

APCI-HRMS m/z: calcd for C14H8BrN2S (MH+), 314.9592, found 315.9513; Purity (HPLC):

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4-[(2-Phenylethyl)sulfanyl]phthalonitrile (6i)

The title compound (dark purple crystals) was prepared from 4-nitrophthalonitrile and 2-phenylethanethiol in a yield of 57%: mp 132.8–133.1ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 3.00 (t, 2H, J = 7.15 Hz), 3.27 (t, 2H, J = 7.53 Hz), 7.20 (d, 2H, J = 7.15 Hz),

7.23–7.26 (m, 1H), 7.30–7.32 (m, 2H), 7.45 (dd, 1H, J = 1.9, 8.3 Hz), 7.48 (d, 1H, J = 1.9 Hz), 7.60 (d, 1H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 33.4, 34.7, 110.8, 115.1,

115.5, 116.1, 127.0, 128.5, 128.8, 129.9, 130.1, 133.1, 138.7, 146.8; APCI-HRMS m/z: calcd for C16H13N2S (MH+), 265.0799, found 265.0795; Purity (HPLC): 100%.

4-(Cyclohexylsulfanyl)phthalonitrile (6j)

The title compound (emerald-green crystals) was prepared from 4-nitrophthalonitrile and cyclohexanethiol in a yield of 39%: mp 85.2–85.3 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 1.27–1.34 (m, 1H), 1.37–1.48 (m, 4H), 1.65–1.67 (m, 1H), 1.78–1.81 (m, 2H), 1.99–

2.02 (m, 2H), 3.31–3.35 (m, 1H), 7.49 (dd, 1H, J = 1.9, 8.3 Hz), 7.56 (d, 1H, J = 1.9 Hz), 7.61 (d, 1H, J = 8.7 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 25.4, 25.7, 32.6, 44.8, 110.9, 115.2,

115.5, 116.2, 131.2, 131.3, 133.2, 146.4; APCI-HRMS m/z: calcd for C14H15N2S (MH+),

243.0956, found 243.0978; Purity (HPLC): 100%.

4-(Cyclopentylsulfanyl)phthalonitrile (6k)

The title compound (green powder) was prepared from 4-nitrophthalonitrile and cyclopentanethiol in a yield of 51%: mp 54.7–54.8 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 1.61–1.69 (m, 4H), 1.78–1.79 (m, 2H), 2.15–2.19 (m, 2H), 3.67–3.71 (m, 1H), 7.48

(dd, 1H, J = 1.9, 8.3 Hz), 7.55 (d, 1H, J = 1.9 Hz), 7.60 (d, 1H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 24.9, 33.2, 43.9, 110.4, 115.2, 115.6, 116.1, 130.3, 130.4, 133.1,

148.1; APCI-HRMS m/z: calcd for C13H13N2S (MH+), 229.0799, found 229.0812; Purity (HPLC):

100%.

4-[(3-Methylbutyl)sulfanyl]phthalonitrile (6l)

The title compound (turquoise needles) was prepared from 4-nitrophthalonitrile and 3-methyl-1-butanethiol in a yield of 71%: mp 63.9–64.0 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 0.94 (d, 6H, J = 6.8 Hz), 1.56–1.60 (m, 2H), 1.70–1.77 (m, 1H), 2.97–2.99 (m, 2H),

7.46 (dd, 1H, J = 1.9, 8.3 Hz), 7.52 (d, 1H, J = 1.9 Hz), 7.62 (d, 1H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 22.1, 27.5, 29.9, 36.8, 110.6, 115.2, 115.6, 116.2, 129.7,

129.9, 133.1, 147.5; APCI-HRMS m/z: calcd for C13H15N2S(MH+), 231.0956, found 231.0962;

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7.7.1.3 Synthesis of the sulfanylbenzonitrile analogues (7a–j)

The C4-substituted benzonitrile derivatives (7a–j) were synthesized according to a previously described protocol.1 The appropriate thiol (6 mmol) was dissolved in 4.3 mL dimethyl sulfoxide (DMSO) and the atmosphere was replaced with argon. 4–Nitrobenzonitrile (5 mmol) was added to the reaction and stirring continued for 15 min at room temperature. Potassium carbonate (15 mmol) was added portionwise over a period of 2 h and stirring was continued for 48 h. The reaction was poured into 50 mL ice cold water and stirred for another 30 min. For the synthesis of compounds 7a–e, 7g–h and 7j a precipitate was obtained. The precipitate was collected by filtration, washed with 60 mL water and dried at 50 ºC. These compounds were recrystallized from an appropriate solvent as cited below. For the synthesis of compounds 7f and 7i, an oil was obtained which was extracted to ethyl acetate (30 mL). The ethyl acetate phase was dried over anhydrous MgSO4 and concentrated under reduced pressure to yield the product.

4-(Benzylsulfanyl)benzonitrile (7a)

The title compound (white needles) was prepared from 4-nitrobenzonitrile and benzyl mercaptan in a yield of 35%: mp 84.5–84.7 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 4.19 (s,

2H), 7.24–7.35 (m, 7H), 7.48 (d, 2H, J = 8.7 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ

37.0, 108.5, 118.8, 127.2, 127.7, 128.7, 128.7, 132.2, 135.6, 144.4; APCI-HRMS m/z: calcd for C14H12NS (MH+), 226.0690, found 226.0686; Purity (HPLC): 100%.

4-[(4-Chlorobenzyl)sulfanyl]benzonitrile (7b)

The title compound (white crystals) was prepared from 4-nitrobenzonitrile and 4-chlorobenzyl mercaptan in a yield of 71%: mp 106.4–108.5 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 4.14 (s, 2H), 7.26–7.27 (m, 6H), 7.48 (d, 2H, J = 8.3 Hz); 13C NMR (Bruker Avance III

600, CDCl3) δ 36.4, 108.8, 118.7, 127.5, 128.9, 130.0, 132.2, 133.5, 134.3, 143.8; APCI-HRMS

m/z: calcd for C14H11ClNS (MH+), 260.0301, found 260.0291; Purity (HPLC): 100%.

4-[(4-Bromobenzyl)sulfanyl]benzonitrile (7c)

The title compound (light orange crystals) was prepared from nitrobenzonitrile and 4-bromobenzyl mercaptan in a yield of 62%: mp 116.6–116.9 ºC (ethyl acetate/methanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 4.12 (s, 2H), 7.21 (d, 2H, J = 8.3 Hz), 7.26 (d, 2H, J = 8.3

Hz), 7.42 (d, 2H, J = 6.8 Hz), 7.48 (d, 2H, J = 6.8 Hz); 13C NMR (Bruker Avance III 600, CDCl3)

δ 36.5, 108.9, 118.7, 121.6, 127.5, 130.3, 131.9, 132.3, 134.8, 143.7; APCI-HRMS m/z: calcd for C14H11BrNS (MH+), 303.9796, found 303.9778; Purity (HPLC): 100%.

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4-[(4-Fluorobenzyl)sulfanyl]benzonitrile (7d)

The title compound (light yellow needles) was prepared from nitrobenzonitrile and 4-fluorobenzyl mercaptan in a yield of 61%: mp 105.0–105.1 ºC (ethyl acetate\methanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 4.15 (s, 2H), 6.98 (t, 2H, J = 8.7 Hz), 7.27–7.31 (m, 4H), 7.49

(d, 2H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 36.4, 108.8, 115.6, 115.7, 118.7,

127.4, 130.2, 130.3, 131.4, 131.4, 132.2, 144.0, 161.3, 162.9; APCI-HRMS m/z: calcd for C14H11FNS (MH+), 244.0596, found 244.0593; Purity (HPLC): 100%.

4-[(4-Methoxybenzyl)sulfanyl]benzonitrile (7e)

The title compound (white needles) was prepared from 4-nitrobenzonitrile and 4-methoxybenzyl mercaptan in a yield of 72%: mp 114.8–114.9 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 3.77 (s, 3H), 4.14 (s, 2H), 6.83 (d, 2H, J = 6.8 Hz), 7.25 (d, 2H, J = 8.7 Hz), 7.28 (d,

2H, J = 8.3 Hz), 7.47 (d, 2H, J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 36.5, 55.2,

108.4, 114.1, 118.8, 127.2, 127.4, 129.8, 132.1, 144.6, 159.0; APCI-HRMS m/z: calcd for C15H14NOS (MH+), 256.0796, found 256.0778; Purity (HPLC): 100%.

4-(Phenylsulfanyl)benzonitrile (7f)

The title compound (beige oil) was prepared from 4-nitrobenzonitrile and thiophenol in a yield of 9% (oil); 1H NMR (Bruker Avance III 600, CDCl3) δ 7.14 (d, 2H, J = 8.7 Hz), 7.41–7.43 (m, 3H),

7.45 (d, 2H, J = 8.7 Hz), 7.49–7.50 (m, 2H); 13C NMR (Bruker Avance III 600, CDCl3) δ 108.6,

118.8, 127.3, 129.4, 129.9, 130.8, 132.3, 134.5, 145.7; APCI-HRMS m/z: calcd for C13H10NS

(MH+), 212.0534, found 212.0552; Purity (HPLC): 93%.

4-[(4-Chlorophenyl)sulfanyl]benzonitrile (7g)

The title compound (light yellow crystals) was prepared from nitrobenzonitrile and 4-chlorothiophenol in a yield of 7%: mp 89.6–89.9 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 7.15 (d, 2H, J = 8.3 Hz), 7.38 (d, 2H, J = 6.8 Hz), 7.41 (d, 2H, J = 6.8 Hz), 7.48 (d, 2H,

J = 8.3 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 109.1, 118.6, 127.5, 129.5, 130.1, 132.5,

135.6, 135.7, 144.9; APCI-HRMS m/z: calcd for C13H9ClNS (MH+), 246.0144, found 246.0155;

Purity (HPLC): 100%.

4-[(4-Bromophenyl)sulfanyl]benzonitrile (7h)

The title compound (white powder) was prepared from nitrobenzonitrile and 4-bromothiophenol in a yield of 2%: mp 100.2–100.5 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 7.16 (d, 2H, J = 8.3 Hz), 7.34 (d, 2H, J = 6.4 Hz), 7.48 (d, 2H, J = 6.4 Hz), 7.53

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(d, 2H, J = 8.7 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 109.2, 118.6, 123.8, 127.7,

130.3, 132.5, 133.1, 135.7, 144.7; APCI-HRMS m/z: calcd for C13H9BrNS (MH+), 289.9639,

found 289.9644; Purity (HPLC): 97%.

4-[(2-Phenylethyl)sulfanyl]benzonitrile (7i)

The title compound (light yellow oil) was prepared from 4-nitrobenzonitrile and 2-phenylethanethiol in a yield of 25%: 1H NMR (Bruker Avance III 600, CDCl3) δ 2.96 (t, 2H, J =

7.5 Hz), 3.22 (t, 2H, J = 7.5 Hz), 7.21 (d, 2H, J = 6.8 Hz), 7.23–7.26 (m, 1H), 7.28–7.33 (m, 4H), 7.50 (dd, 2H, J = 1.9, 6.8 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 33.2, 34.9, 108.1,

118.8, 126.7, 126.8, 128.4, 128.6, 132.2, 139.3, 144.5; APCI-HRMS m/z: calcd for C15H14NS

(M+), 240.0847, found 240.0855; Purity (HPLC): 100%.

4-(Cyclohexylsulfanyl)benzonitrile (7j)

The title compound (white crystals) was prepared from 4-nitrobenzonitrile and cyclohexanethiol in a yield of 35%: mp 49.8–51.3 ºC (ethanol); 1H NMR (Bruker Avance III 600, CDCl3) δ 1.23–

1.28 (m, 1H), 1.33–1.44 (m, 4H), 1.62–1.64 (m, 1H), 1.76–1.79 (m, 2H), 1.99–2.01 (m, 2H), 3.25–3.29 (m, 1H), 7.31 (d, 2H, J = 6.8 Hz), 7.49 (d, 2H, J = 6.4 Hz); 13C NMR (Bruker Avance III 600, CDCl3) δ 25.7, 25.8, 32.9, 44.8, 108.4, 118.9, 128.5, 132.2, 143.9; APCI-HRMS m/z:

calcd for C13H16NS (MH+), 218.1003, found 218.1022; Purity (HPLC): 100%.

7.7.1.4 IC50 values for the inhibition of MAO

Recombinant human MAO-A and –B from insect cell microsomes (5 mg protein/mL) were used for the inhibition studies. The incubations were carried out in potassium phosphate buffer (K2HPO4/KH2PO4 100 mM, made isotonic with KCl, pH 7.4) to a final volume of 500 µL. The

reactions contained various concentrations of the test inhibitor (0, 0.003–100 µM), kynuramine (a mixed MAO-A/B substrate) and DMSO as co-solvent (4%). The final concentrations of kynuramine in the reactions were 45 µM and 30 µM for MAO-A and MAO-B, respectively. The reactions were initiated with the addition of MAO-A or MAO-B (0.0075 mg protein/mL), allowed to incubate for 20 min at 37 ºC and finally terminated with the addition of 400 µL NaOH (2 N) and 1000 µL water. The concentrations of the MAO generated 4-hydroxyquinoline were measured by fluorescence spectrophotometry (λem = 310; λex = 400 nm) employing a linear

calibration curve (4-hydroxyquinoline: 0.047–1.56 µM).2,3 The enzyme catalytic rates were fitted to the one site competition model incorporated into the Prism 5 software package (GraphPad). The IC50 values were determined in triplicate and are expressed as mean ± standard deviation

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7.7.1.5 Recovery of enzyme activity after dilution

The test inhibitor 6c [IC50(MAO-B) = 0.025 µM], at concentrations equal to 0 × IC50, 10 × IC50

and 100 × IC50, was preincubated with recombinant human MAO-B (0.75 mg/ml) for 30 min at

37 ºC (K2HPO4/KH2PO4 100 mM, made isotonic with KCl, pH 7.4). DMSO (4%) was added as

co-solvent to all preincubations. The reactions were subsequently diluted 100-fold with the addition of a solution of kynuramine to yield final concentrations of the test compound equal to 0 × IC50, 0.1 × IC50 and 1 × IC50. After dilution, the final concentration of kynuramine was 30 µM,

and the final concentration of MAO-B was 0.0075 mg/mL. The reactions were incubated for a further 20 min at 37 °C, terminated and the residual rates of 4-hydroxyquinoline formation were measured as described above. For comparison, (R)-deprenyl (IC50 = 0.079 µM), at a

concentration of 10 × IC50 was similarly preincubated with MAO-B, diluted to 0.1 × IC50 and the

residual MAO-B activity was measured as above.4 7.7.2 References

1. Manley-King, C. I.; Bergh, J. J.; Petzer, J. P. Bioorg. Chem. 2012, 40, 114.

2. Strydom, B.; Malan, S. F.; Castagnoli, N.; Bergh, J. J.; Petzer, J. P. Bioorg. Med. Chem.

2010, 18, 1080.

3. Novaroli, L.; Reist, M.; Favre, E.; Carotti, A.; Catto, M.; Carrupt, P. A. Bioorg. Med. Chem. 2005, 13, 6212.

4. Petzer, A.; Harvey, B. H.; Wegener, G.; Petzer, J. P. Toxicol. Appl. Pharm. 2012, 258, 403.

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7.7.3 NMR spectra

7.7.3.1 NMR spectra of the sulfanylphthalonitrile analogues (6a–l)

1_{H NMR (CDCl}

3): 4-(Benzylsulfanyl)phthalonitrile (6a)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(4-Chlorobenzyl)sulfanyl]phthalonitrile (6b)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(4-Bromobenzyl)sulfanyl]phthalonitrile (6c)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(4-Fluorobenzyl)sulfanyl]phthalonitrile (6d)

13_{C NMR (CDCl}

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1_{H NMR (DMSO-d6): 4-[(4-Methoxybenzyl)sulfanyl]phthalonitrile (6e)}

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1_{H NMR (CDCl}

3): 4-(Phenylsulfanyl)phthalonitrile (6f)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(4-Chlorophenyl)sulfanyl]phthalonitrile (6g)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(4-Bromophenyl)sulfanyl]phthalonitrile (6h)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(2-Phenylethyl)sulfanyl]phthalonitrile (6i)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-(Cyclohexylsulfanyl)phthalonitrile (6j)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-(Cyclopentylsulfanyl)phthalonitrile (6k)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(3-Methylbutyl)sulfanyl]phthalonitrile (6l)

13_{C NMR (CDCl}

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7.7.3.2 NMR spectra of the sulfanylbenzonitrile analogues (7a–j)

1

H NMR (CDCl3): 4-(Benzylsulfanyl)benzonitrile (7a)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3) :4-[(4-Chlorobenzyl)sulfanyl]benzonitrile (7b)

13_{C NMR (CDCl}

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1_{H NMR (CDCl}

3): 4-[(4-Bromobenzyl)sulfanyl]benzonitrile (7c)

13

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1_{H NMR (CDCl}

3): 4-[(4-Fluorobenzyl)sulfanyl]benzonitrile (7d)

13

(40)

1_{H NMR (CDCl}

3): 4-[(4-Methoxybenzyl)sulfanyl]benzonitrile (7e)

13_{C NMR (CDCl}

(41)

1_{H NMR (CDCl}

3): 4-(Phenylsulfanyl)benzonitrile (7f)

13_{C NMR (CDCl}

(42)

1_{H NMR (CDCl}

3): 4-[(4-Chlorophenyl)sulfanyl]benzonitrile (7g)

13_{C NMR (CDCl}

(43)

1_{H NMR (CDCl}

3): 4-[(4-Bromophenyl)sulfanyl]benzonitrile (7h)

13_{C NMR (CDCl}

(44)

1_{H NMR (CDCl}

3): 4-[(2-Phenylethyl)sulfanyl]benzonitrile (7i)

13

(45)

1_{H NMR (CDCl}

3): 4-(Cyclohexylsulfanyl)benzonitrile (7j)

13_{C NMR (CDCl}

(46)

7.7.4 HPLC traces

An Agilent 1100 HPLC system equipped with a quaternary pump and an Agilent 1100 series diode array detector was used to determine the purities for the synthesized compounds. Milli-Q water (Millipore) and HPLC grade acetonitrile (Merck) were used for the chromatography. Separation was achieved with a Venusil XBP C18 column (4.60  150 mm, 5 µm) with a mobile phase which consisted initially of 30% acetonitrile and 70% MilliQ water. The flow rate was set to 1 mL/min. At the start of each HPLC run a solvent gradient program was initiated by linearly increasing the composition of the acetonitrile in the mobile phase to 85% over a period of 5 min. Each HPLC run lasted 15 min and a time period of 5 min was allowed for equilibration between runs. A volume of 20 µL of solutions of the test compounds in acetonitrile (1 mM) was injected into the HPLC system and the eluent was monitored at a wavelength of 254 nm.

7.7.4.1 HPLC traces of the sulfanylphthalonitrile analogues (6a–l)

4-(Benzylsulfanyl)phthalonitrile (6a)

(47)

4-[(4-Chlorobenzyl)sulfanyl]phthalonitrile (6b)

4-[(4-Bromobenzyl)sulfanyl]phthalonitrile (6c)

(48)

4-[(4-Fluorobenzyl)sulfanyl]phthalonitrile (6d)

4-[(4-Methoxybenzyl)sulfanyl]phthalonitrile (6e)

(49)

4-(Phenylsulfanyl)phthalonitrile (6f)

Purity (HPLC): 99%.

4-[(4-Chlorophenyl)sulfanyl]phthalonitrile (6g)

(50)

4-[(4-Bromophenyl)sulfanyl]phthalonitrile (6h)

4-[(2-Phenylethyl)sulfanyl]phthalonitrile (6i)

(51)

4-(Cyclohexylsulfanyl)phthalonitrile (6j)

4-(Cyclopentylsulfanyl)phthalonitrile (6k)

(52)

4-[(3-Methylbutyl)sulfanyl]phthalonitrile (6l)

7.7.4.2 HPLC traces of the sulfanylbenzonitrile analogues (7a–j)

4-(Benzylsulfanyl)benzonitrile (7a)

(53)

4-[(4-Chlorobenzyl)sulfanyl]benzonitrile (7b)

4-[(4-Bromobenzyl)sulfanyl]benzonitrile (7c)

(54)

4-[(4-Fluorobenzyl)sulfanyl]benzonitrile (7d)

4-[(4-Methoxybenzyl)sulfanyl]benzonitrile (7e)

(55)

4-(Phenylsulfanyl)benzonitrile (7f)

Purity (HPLC): 93%.

4-[(4-Chlorophenyl)sulfanyl]benzonitrile (7g)

(56)

4-[(4-Bromophenyl)sulfanyl]benzonitrile (7h)

Purity (HPLC): 97%.

4-[(2-Phenylethyl)sulfanyl]benzonitrile (7i)

(57)

4-(Cyclohexylsulfanyl)benzonitrile (7j)