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Methylene blue analogues with marginal monoamine oxidase inhibition retain antidepressant-like activity Anzelle Delport, Brian H Harvey, Anél Petzer, and Jacobus P. Petzer ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00042 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 8, 2018

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ACS Chemical Neuroscience

Methylene blue analogues with marginal monoamine oxidase inhibition retain antidepressant-like activity Anzelle Delport,†,‡ Brian H. Harvey, ‡,§ Anél Petzer, †,‡ Jacobus P. Petzer *,†,‡ †

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

‡

Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa

§

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

Corresponding author *Mailing address: Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom 2520, South Africa. E-mail: [email protected]. Phone: +27 18 2992206.

E-mail addresses: Anzelle Delport: [email protected] Brian H. Harvey: [email protected] Anél Petzer: [email protected] Jacobus P. Petzer: [email protected]

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Abstract Methylene blue (MB) possesses diverse medical applications. Among these MB presents with antidepressant-like effects in animals and has shown promise in clinical trials for the treatment of mood disorders. As an antidepressant, MB may act via various mechanisms which include modulation of the nitric oxide cyclic guanosine monophosphate (NO-cGMP) cascade, enhancement of mitochondrial respiration and antioxidant effects. MB is also, however, a high potency inhibitor of monoamine oxidase (MAO) A, which most likely contributes to its antidepressant effect, but also to its adverse effects profile (eg. serotonin toxicity). The latter has raised the question whether it is possible to design out the MAO inhibition properties of MB yet retaining the clinically useful attributes. This study explores this idea further by characterizing five newly synthesized MB analogues and examining their antidepressant-like properties in the acute forced swim test (FST) in rats, with comparison to imipramine and MB. The results show that all five analogues exhibit antidepressant-like properties in the FST without confounding effects on locomotor activity. The magnitude of these effects is comparable to those of imipramine and MB. Since these newly synthesized MB analogues are markedly less potent MAO-A inhibitors (IC50 = 0.518–4.73 µM) than MB (IC50 = 0.07 µM), we postulate that such lower potency MAO-A inhibitors may present with a reduced risk of adverse effects associated with MAO-A inhibition. While low level MAO-A inhibition still may produce an antidepressant effect, we posit that other MB-related mechanisms may underlie their antidepressant effects, thereby representing a novel group of antidepressant compounds.

Keywords: depression; forced swim test; inhibition; azure B; nitric oxide; mitochondrial electron transport


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Introduction Major depression is multi-factorial in terms of pathogenesis and thus presents with a broad spectrum of symptoms ranging from anhedonia and anxiety to cognitive impairment.1 In this respect, interacting genetic, epigenetic, environmental and developmental factors amongst others provoke the neurochemical and structural dysfunctions that characterizes depressive illness. Depression is furthermore co-morbid with other conditions such as bipolar disorder and schizophrenia which further underscores its multi-factorial origin.2

Based on the above considerations it thus seems likely that depression could be better treated with drugs acting via multiple mechanisms of action.1 The concept of multi-modal drugs that act by two or more complementary mechanisms is an established approach that may potentially be highly effective in major depression, and is exemplified by serotonin–norepinephrine reuptake inhibitor (SNRI) drugs and mirtazapine.3 Although not indicated for the treatment of depression, methylene blue (MB) represents another compound that may harness multiple mechanisms relevant to the treatment of depression, and in this respect MB has shown promise for the treatment of bipolar depression in clinical trials (discussed below).

A noted phenothiazine compound, MB was the first synthetic drug to be used therapeutically against malaria (Figure 1).4 MB continues to be of interest in this regard,5 while it has more recently been investigated in controlled studies of low-back pain.6,7 The compound is a well-known treatment for methemoglobinemia and is used as prophylaxis and reversal of ifosfamide-induced encephalopathy. Clinical studies have shown that MB may slow the cognitive decline in Alzheimer’s disease,8–10 although trials with the MB-related agent, leucomethylthioninium salts (LMTX), have been disappointing. Antidepressant activity in pre-clinical models11,12 has complemented MB’s promise in clinical trials for bipolar disorder,13–17 while its therapeutic value in schizophrenia has also been investigated.18



MB exerts its pharmacological effects through a number of different biochemical mechanisms. Thus, MB acts as an enhancer of mitochondrial respiration19,20 and a redox cycling agent,19,21 as well as a potent chain-breaking antioxidant.19,22 Indeed, mitochondrial and redox dysfunction is well-described in depression,23 while antioxidants such as N-acetylcysteine exert antidepressant effects in animals,24 while also having been investigated in humans for this purpose.25 MB may also mediate some of its effects by interactions with protein receptors and enzymes. For example, MB is recognized as a nonselective inhibitor of nitric oxide (NO) synthase (NOS) and guanylate cyclase.26 Since the NO-cyclic guanosine monophosphate (cGMP) pathway represents a possible biological target in treating depression,27,28 these actions may contribute to its observed antidepressant response.29–32 In support of this, a number of antidepressants are known to inhibit NOS.32,33 MB also is a noteworthy inhibitor of monoamine oxidase (MAO) A,12,34,35 while MAO inhibition is a well-established mechanism of antidepressant action.36

Since MAO-A is a key enzyme in the regulation of central serotonin levels, MAO-A inhibition is associated with an antidepressant effect. MAO-B inhibitors, most notably the selegiline transdermal system, have also been reported to possess antidepressant effects.37 However, it is not clear whether this is due to MAO-B inhibition or whether selectivity is lost at higher inhibitor concentrations, which may result in central MAO-A inhibition.37 A number of MB-related compounds, including MB’s principal metabolite, azure B, have also been reported to potently inhibit the MAO enzymes, and to possess noteworthy antidepressant-like effects.38,39 Azure B inhibits human MAO-A with similar potency to MB, while exhibiting comparatively lower potency MAO-B inhibition.40 Of interest to this study is a report that a synthetic MB analogue, ethylthioninium chloride (ETC), is a weaker albeit reversible and non-selective inhibitor of MAO-A and MAO-B versus MB (IC50 = 0.07 µM).39 Importantly, while presenting with noteworthy antidepressant-like properties comparable to MB, ETC was also found comparable to the reference antidepressant, imipramine.39 Moreover, both azure B and ETC are higher potency MAO-B inhibitors than MB.12 Other MB analogues that have been shown to possess antidepressant-like properties in the preclinical setting include methylene green and thionine, and dye compounds such as toluidine blue O, brilliant cresyl blue and toluylene blue.12,38 4 ACS Paragon Plus Environment

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Given MB’s other molecular targets noted above that may confer psychotropic actions, designing out its MAO inhibitory properties may still confer clinically useful attributes. A strategy to design weakly MAO-A active MB analogues seems feasible since an analysis of the MAO-A inhibition properties of MB and ETC suggests that an increase in the size/steric bulk of the dialkylamine side chains would reduce MAO-A inhibition. In this study steric large dialkylamine side chains (thiomorpholinyl and morpholinyl groups) were selected as a desirable outcome. Interestingly, an increase in the size/steric bulk of the dialkylamine side chains also appears to enhance MAO-B inhibition. MAO-B inhibitors are established therapy for Parkinson’s disease while non-specific MAO inhibitors may be beneficial in the treatment of Parkinson’s disease with comorbid depression.41 In this respect, MAO-A inhibition may improve both the motor and depression symptoms in Parkinson’s disease, while MAO-B inhibition (at low drug concentrations) would alleviate motor dysfunction but most likely would be less effective for depressive symptoms.

Based on the observations that MB and structural analogues thereof inhibit the MAOs and exhibit antidepressant-like activities in animal models, the present study set out to synthesize five new low MAO-A active MB analogues (1–5) with respect to their human MAO inhibition properties and to relate these properties to antidepressant-like actions in vivo (Figure 2).11,12 The aim being to discover MB analogues that display weak or no inhibition of the MAO-A enzyme but that retain potent antidepressant-like properties. The synthesis of these compounds and the assessment of their antidepressant actions in the forced swim test (FST) in rats will be described here and the results compared to that of MB and imipramine, the latter also a tricyclic structure akin to MB, although not a phenothiazine compound.12 Drug effects on locomotor activity may be a significant confounder to interpreting the FST data. Locomotor activity was thus examined to determine if changes in swim behaviour are indeed due to an antidepressant-like response and not to an indirect effect of the test drug on locomotor activity.



Figure 1. The structures and human MAO inhibition potencies of MB, azure B and ETC.

Figure 2. The structures of the five synthetic MB analogues, compounds 1–5, examined in this study.

Results and discussion Chemistry The synthesis of analogues of MB is complex since the products obtained often are mixtures that are challenging to purify by chromatographic techinques.42–44 Other synthetic strategies employ metals as reactants. However, this has inherent disadvantages as MB and its analogues may form metal chelates.45 Separating the reaction products from metal ions may be difficult, but is essential since metals may affect the outcomes of biological studies. The most useful route for the synthesis of MB analogues was first reported by Strekowski and colleagues (1993) (Figure 3).46 In this method, phenothiazine is converted to phenothiazin-5-ium tetraiodide hydrate (6) by reaction with iodine. 6 ACS Paragon Plus Environment

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Phenotiazinium 6 is then reacted with an appropriate amine to yield the disubstituted or monosubstituted MB analogue. The formation of the disubstituted or monosubstituted analogues depends on the stoichiometry of the amine and phenothiazinium reactants. Using this method, and after extensive experimentation with a number of amine reactants, and trial-and-error with respect to the synthetic protocol, this study synthesized five MB analogues, 1–5. For this purpose, morpholine and thiomorpholine served as amine substrates and the products were obtained as the triiodide or iodide salts. The structures of the MB analogues were characterized by mass spectrometry and NMR analyses. As cited in the experimental section, the 1H NMR and 13C NMR spectra correspond to the proposed structures of compounds 1–5 and the experimentally determined mass of each MB analogue corresponds with the calculated mass. For the disubstituted analogues, 1 and 2, two doublets and a doublet of doublets were observed for the aromatic protons, as expected, while a single signal for each carbon type was observed on the 13C NMR. Two signals were thus observed for the aliphatic carbons. Interestingly, for the monosubstituted analogues 3 and 4, four signals were observed for the aliphatic carbons, which suggests restricted rotation of the N-C bond between the morpholinyl and thiomorpholinyl moieties, and the phenothiazinium ring system, presumably due to the double bond character of this bond.



Figure 3. Synthetic route MB analogues 1–5.46 Key: (i) I2/CH3Cl; (ii) dialkylamine, 2 equiv; (iii) dialkylamine, 6 equiv.

MAO inhibition activities To determine IC50 values for the inhibition of MAO-A and MAO-B by the MB analogues, the recombinant human enzymes were used. Kynuramine served as substrates for both MAO isoforms. By measuring the MAO catalytic rates in the presence of a concentration range of the test inhibitors, sigmoidal plots of enzyme rate versus logarithm of inhibitor concentrations were constructed. From these plots (Figure 4), the IC50 values were estimated.


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Figure 4. Sigmoidal curves constructed for the measurement of IC50 values for human MAO-A and MAO-B inhibition by compounds 1–5. Data points are shown as mean ± SD.

The IC50 values for the inhibition of recombinant human MAO-A and MAO-B by the MB analogues are given in table 1. All the synthesized MB analogues are significantly weaker as MAO-A inhibitors compared to MB (IC50 = 0.07 µM) and azure B (IC50 = 0.01 µM), which were evaluated under identical experimental conditions.12,40 The most potent MAO-A inhibitor of the series is compound 3, the thiomorpholinyl mono-substituted compound, with an IC50 value of 0.518 µM. This compound also is the most potent MAO-B inhibitor (IC50 = 0.569 µM) of the series. The inhibition profile of 3 is thus similar to that of ETC, and like ETC this compound does not display specificity for either MAO



isoform. The other mono-substituted analogue, 4, is the only compound besides 3 that inhibits both MAO isoforms with IC50

Methylene blue analogues with marginal ... - ACS Publications

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