Dual targeting of adenosine A2A receptors and monoamine oxidase B by 4H-3,1-benzothiazin-4-ones

Article abstract of DOI:10.1021/jm400336x

Blockade of A_2A adenosine receptors (A_2AARs) and inhibition of monoamine oxidase B (MAO-B) in the brain are considered attractive strategies for the treatment of neurodegenerative diseases such as Parkinson's disease (PD). In the present study, benzothiazinones, e.g., 2-(3-chlorophenoxy)- N-(4-oxo-4H-3,1-benzothiazin-2-yl)acetamide (13), were identified as a novel class of potent MAO-B inhibitors (IC₅₀ human MAO-B: 1.63 nM). Benzothiazinones with large substituents in the 2-position, e.g., methoxycinnamoylamino, phenylbutyrylamino, or chlorobenzylpiperazinylbenzamido residues (14, 17, 27, and 28), showed high affinity and selectivity for A _2AARs (K_i human A_2AAR: 39.5-69.5 nM). By optimizing benzothiazinones for both targets, the first potent, dual-acting A_2AAR/MAO-B inhibitors with a nonxanthine structure were developed. The best derivative was N-(4-oxo-4H-3,1-benzothiazin-2-yl)-4-phenylbutanamide (17, K_i human A_2A, 39.5 nM; IC₅₀ human MAO-B, 34.9 nM; selective versus other AR subtypes and MAO-A), which inhibited A _2AAR-induced cAMP accumulation and showed competitive, reversible MAO-B inhibition. The new compounds may be useful tools for validating the A_2AAR/MAO-B dual target approach in PD.

Full text of DOI:10.1021/jm400336x

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Article
2A
Dual Targeting of the Adenosine A Receptors and
Monoamine Oxidase B by 4H-3,1-Benzothiazin-4-ones
Anne Stößel, Sonja Hinz, Petra Küppers, Jag Heer, Michael Gütschow, and Christa E Müller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400336x • Publication Date (Web): 30 Apr 2013
Downloaded from http://pubs.acs.org on May 13, 2013
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Journal of Medicinal Chemistry
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Dual Targeting of Adenosine A Receptors and Monoamine Oxidase B
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by 4Hꢀ3,1ꢀBenzothiazinꢀ4ꢀones
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Anne Stößel, Sonja Hinz, Petra Küppers, Jag Heer, Michael Gütschow, * and Christa E. Müller *
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PharmaCenter Bonn, University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Bonn,
Germany
†
UCB S.A., Medicinal Chemistry & Lead Generation, CNS Research, Chemin du Foriest, Bꢀ1420
Braine l'Alleud, Belgium
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ABSTRACT
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Blockade of A2A adenosine receptors (A ARs) and inhibition of monoamine oxidase B (MAOꢀB) in
2A
brain are considered attractive strategies for the treatment of neurodegenerative diseases such as
Parkinson’s disease (PD). In the present study benzothiazinones, e.g. 2ꢀ(3ꢀchlorophenoxy)ꢀNꢀ(4ꢀoxoꢀ
4
Hꢀ3,1ꢀbenzothiazinꢀ2ꢀyl)acetamide (13), were identified as a novel class of potent MAOꢀB inhibitors
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(
IC50 human MAOꢀB: 1.63 nM). Benzothiazinones with large substituents in the 2ꢀposition, e.g.
methoxyꢀcinnamoylamino, phenylbutyrylamino, or chlorobenzylpiperazinylbenzamido residues (14, 17,
27, 28) showed high affinity and selectivity for A ARs (K human A AR: 39.5ꢀ69.5 nM). By
2
A
i
2A
optimizing benzothiazinones for both targets the first potent, dualꢀacting A AR/MAOꢀB inhibitors with
2
A
a nonꢀxanthine structure were developed. The best derivative was Nꢀ(4ꢀoxoꢀ4Hꢀ3,1ꢀbenzothiazinꢀ2ꢀyl)ꢀ
4ꢀphenylbutanamide (17, K human A 39.5 nM, IC human MAOꢀB 34.9 nM; selective versus other
i
2A
50
AR subtypes and MAOꢀA), which inhibited A ARꢀinduced cAMP accumulation and showed
2
A
competitive, reversible MAOꢀB inhibition. The new compounds may be useful tools for validating the
A AR/MAOꢀB dual target approach in PD.
2
A
KEYWORDS: A , A , A , A antagonists, adenosine receptors, benzothiazinones, dualꢀacting drugs,
1
2A
2B
3
MAOꢀB inhibitors, neurodegenerative disease, Parkinson’s disease, structureꢀactivity relationships
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INTRODUCTION
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Parkinson’s disease (PD) is a chronic neurodegenerative disorder of the central nervous system,
characterized by the loss of dopaminergic neurons in the substantia nigra and the subsequent depletion
1
of dopamine stores. The consequence is a progressive impairment in motor functions characterized by
1
rigidity, resting tremor and bradykinesia as major symptoms. The current treatment for PD includes the
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administration of levodopa, a metabolic precursor of dopamine (DA) to increase the DA level, as well as
dopamine agonists to mimic the dopamineꢀmediated neurotransmission. This DA replacing therapy
alleviates most of the symptoms especially in the early stage of the disease, but cannot stop the
neurodegenerative processes. In particular, a longꢀterm levodopa treatment leads to the development of
adverse effects, such as dyskinesia, a disorder characterized by involuntary motor movements.
Furthermore, increasing doses are required to maintain the therapeutic effect, and after several years, the
2
drug often loses its effect. For these reasons, the development of novel diseaseꢀmodifying drugs that
stop or at least retard disease progression, delay the onset of levodopa therapy and/or reduce levodopa
3
doses, is urgently needed in PD therapy.
The A2A adenosine receptor (A AR), which is activated by the nucleoside adenosine, represents one
2A
1
,4
promising target for antiꢀParkinsonian drugs. Four subtypes of adenosine receptors (ARs) exist,
referred to as A , A , A and A . They belong to the superfamily of G proteinꢀcoupled receptors
1
2A
2B,
3
(GPCRs) and differ in their affinity for adenosine, in the type of G proteins that they recruit and in the
5
subsequent downstream signaling pathways that are activated in the target cells. While A and A
1
3
receptors preferentially inhibit adenylate cyclase (AC) activity and thereby reduce intracellular cAMP
production, A2A and A ARs stimulate AC and consequently increase cAMP levels. The A AR shows
2B
2A
a restricted expression in the central nervous system being mainly localized in dopamineꢀinnervated
6
areas, such as the nucleus accumbens, the caudateꢀputamen, and the olfactory tubercle. The A AR is
2
A
colocalized and physically associated with the dopamine D receptor, forming heteromeric A /D
2
2
2A
4
,7
receptor complexes. Both receptors have an opposing effect on AC and the cellular production of
cAMP. Inhibition of A ARs leads to enhanced D receptor function and therefore a potentiated
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dopamine neurotransmission, while an activation of A ARs inhibits D receptor signaling. Thus,
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binding of agonists to A ARs reduces the affinity of DA and other DA agonists for their binding site at
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the heteromeric DA receptor.
The blockade of the A AR is considered as an attractive nonꢀdopaminergic treatment for PD. A large
1,9
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number of structurally diverse A AR antagonists has been identified to date (for example see
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,10,11
Figure 1).
These include xanthineꢀlike structures, e.g. istradefylline (1), (E)ꢀ8ꢀ(3ꢀ
chlorostyryl)caffeine (2), and adenineꢀlike compounds, e.g. preladenant (3). While 1 is expected to be
marketed for the treatment of PD in Japan in the near future, 3 is currently being evaluated in phase III
4
,11
clinical trials, and both drugs have demonstrated beneficial antiꢀParkinsonian effects.
Experimental
models of PD indicated that A2A antagonists might be less prone to induce dyskinesias than treatment
1
1
with levodopa. Furthermore, several animal studies indicated neuroprotective effects induced by a
4
blockade of A ARs. Novel A antagonists, such as the benzothiazole derivative tozadenant (SYNꢀ
2
A
2A
1
2
13
1
15, 4) and the benzothiazinone 5, that are structurally neither related to xanthines nor to adenine
have been identified by highꢀthroughput screening. Recently, virtual screening approaches have
10,14
provided further novel scaffolds.
Figure 1. Structures of selected A adenosine receptor antagonists (1ꢀ5) and MAOꢀB inhibitors (6, 7).
2
A
Another therapeutic approach in the treatment of PD is provided by the blocking of monoamine oxidase
1
5
B (MAOꢀB). This flavin adenine dinucleotide (FAD) containing enzyme is one of the two existing
isoforms A and B, which catalyze the oxidative deamination of amine neurotransmitters. Both isoforms
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Journal of Medicinal Chemistry
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are present in the human brain and involved in the catabolic pathway of dopamine in the striatum.
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MAOꢀA is the prevalent isoform in catecholaminergic neurons, whereas MAOꢀB is predominantly
located in glial cells and serotonergic neurons. The activity as well as the expression levels of MAOꢀB
17
in the human brain increase with age, while MAOꢀA activity remains constant. Moreover, the
increased MAOꢀB activity, e.g. under levodopa treatment, is associated with a higher production of
hydrogen peroxide. The occurrence of reactive oxygen species (ROS) may contribute to apoptotic
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signaling causing the death of neuronal cells. Inhibition of the enzyme MAOꢀB improves symptoms of
PD. Selegiline (6) and the related drug rasagiline are successfully used as irreversible inhibitors in PD
1
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monotherapy or in combination with levodopa. For safety reasons and to prevent undesired side
effects, reversible inhibitors (e.g. 7) might be advantageous. Safinamide (7) is currently under
2
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development in phase III clinical trials as an adjuvant therapy for PD.
Considering these two promising approaches, a compound that antagonizes A ARs on the one hand,
2
A
and inhibits MAOꢀB on the other hand may have enhanced therapeutic and neuroprotective potential in
the treatment of PD. Such drugs would possess the combined benefits of addressing two
pharmacological targets with a reduced risk for side effects compared to a combination of two separate
2
1
drugs. Examples for such an approach include dualꢀacting drug candidates for neurodegenerative
2
2
diseases addressing both MAOꢀB and acetylcholinesterase. Moreover, it has been discovered, that the
23
well known A antagonist 2 is also a potent and reversible MAOꢀB inhibitor. A series of analogues of
2
A
2
4
2 has been developed in the course of the continued exploration of the dual targeting concept. One
2
5,26
major drawback of xanthines, such as compound 2, is their low water solubility.
Furthermore, all
recently published dual compounds blocking A ARs and MAOꢀB are styrylꢀ or phenylbutadienylꢀ
2
A
substituted xanthine derivatives, which are not only highly lipophilic, but in addition sensitive to light,
undergoing lightꢀinduced isomerization in solution, or lightꢀinduced dimerization in the solid state,
2
7
respectively.
In the present study we focused on the development of nonꢀxanthineꢀderived, dualꢀacting compounds,
which exhibit an antagonistic effect at A ARs and at the same time inhibit MAOꢀB. Previously
2
A
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reported 4Hꢀ3,1ꢀbenzothiazinꢀ4ꢀones, such as derivative 5, are potent A AR antagonists but lack
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selectivity versus the other AR subtypes. As a continuation of our research, further 4Hꢀ3,1ꢀ
benzothiazinꢀ4ꢀone derivatives have been synthesized and evaluated at all four AR subtypes as well as
MAOꢀB, and optimized for dual A /MAOꢀB blockade.
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Journal of Medicinal Chemistry
RESULTS AND DISCUSSION
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Chemistry. Scheme 1 outlines the synthetic route towards 4Hꢀ3,1ꢀbenzothiazinꢀ4ꢀones 8ꢀ24. Starting
with compound 8, it was intended to systematically vary and extend the spacer between the
heterobicyclic core and the phenyl group and to introduce substituents at the latter. The thioureide 38,
obtained by the reaction of anthranilic acid (37) and benzoyl isothiocyanate, gave the benzothiazinone
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skeleton of 39 in a sulfuric acidꢀpromoted cyclization with a concomitant loss of the benzoyl residue.
Structural diversity of 8ꢀ24 was introduced in the last step of this route by an acylation reaction
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according to a previously described procedure. Because of the low nucleophilicity of the amino group
of 39, several carboxylic acids (40 and 41) were activated as mixed anhydrides using the Yamaguchi
1
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reagent (2,4,6ꢀtrichlorobenzoyl chloride).
a
Scheme 1. Synthesis of 2ꢀAcylaminobenzothiazinones 8ꢀ24
O
O
CO H
CO2H
NH
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38 S
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OCH3
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Cl
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Cl
Cl
Cl
Cl
Cl
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Cl
Br
a
Reagents and conditions: (i) benzoyl isothiocyanate, acetone, RT, 20 min; (ii) 1. concd H SO , 100 °C,
2
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h, 2. NaHCO ; (iii) 1. carboxylic acids 40 or 41 (residues R correspond to those of compounds 8ꢀ24),
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Nꢀmethylmorpholine, 2,4,6ꢀtrichlorobenzoyl chloride, THF, RT, 1 h, 2. 39, pyridine, toluene, reflux,
h.
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Some of the required carboxylic acids 40 and 41 are expensive or not commercially available and were
prepared as follows. The phenylbutenoic acid derivatives 40 were obtained via a Wittig reaction
(Scheme 2), and for this purpose, the phosphonium salt of 3ꢀbromopropionic acid (42) was generated
and reacted with the appropriate aldehydes 43 to form the alkenes 40.
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Scheme 2. Synthesis of Phenylbutenoic Acids 40 and Phenylbutyric Acids 41
a
Reagents and conditions: (i) PPh , acetonitrile, reflux, 5 h; (ii) 1. aldehyde 43, tertꢀBuOK, CH Cl ,
3
2
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°C ꢀ RT, 12 h; (iii) Pd/C, H , EtOH or THF, RT, 1ꢀ2 h.
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A subsequent Pdꢀcatalyzed hydrogenation step provided the saturated phenylbutyric acids 41. 3ꢀ(2ꢀ
Fluorophenyl)propanoic acid (see Scheme 1) was similarly obtained via hydrogenation of the
corresponding cinnamic acid. 3ꢀ(3ꢀMethoxyphenyl)propiolic acid was synthesized according to a
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literature procedure.
In order to increase the compounds’ polarity and water solubility, we planned to attach a basic moiety.
Previous studies indicated an Nꢀbenzylpiperazine residue connected via an amide bond to the
1
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benzoylaminobenzothiazinone as in compound 25 (Scheme 3) to be an advantageous substitution
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pattern with respect to bioactivity and solubility.
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a
Scheme 3. Synthesis of 2ꢀAcylaminobenzothiazinones 26ꢀ28
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Reagents and conditions: (i) 1. 4ꢀformylbenzoic acid, (COCl) , DMF (cat), CH Cl , RT, 2 h, 2.
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pyridine, CH Cl , reflux, 2 h; (ii) oxone, DMF, RT, 12 h; (iii) 1. 1,1’ꢀcarbonyldiimidazole, DMF, RT,
1 h, 2. piperazine 45 (1.25 equiv), imidazole (1.0 equiv), HCl (2.0 equiv), DMF, RT, 2 h.
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To synthesize further analogues, 4ꢀformylbenzoic acid was activated by the conversion to the acyl
3
0
chloride and reacted with 2ꢀaminobenzothiazinone 39. According to a reported method, oxone was
successfully utilized to oxidize the intermediate aldehyde to the corresponding carboxylic acid 44. In
3
1
order to prevent a Dimroth rearrangement of the benzothiazinone system, an imidazole buffer was
applied for the carbonyldiimidazoleꢀpromoted coupling of 43 with different Nꢀbenzylpiperazines 45 to
give the basic carboxamides 26-28. The following syntheses were performed in order to achieve minor
structural modifications of compound 17 (Scheme 1) as this benzothiazinone turned out to be a
particular promising candidate (see afterwards).
a
Scheme 4. Synthesis of NꢀMethylated 2ꢀAcylaminobenzothiazinone 29
O
i
S
O
N
N
H
O
1
7
S
O
N
N
CH3
29
a
Reagents and conditions: (i) 1. CH I, THF, 0 °C, 2. NaH, RT, 24 h.
3
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To identify the potential role of the amide proton as a hydrogen bond donor for protein binding, the Nꢀ
methylated compound 29 was prepared (Scheme 4).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 5. Synthesis of Substituted 2ꢀAcylaminobenzothiazinones 30ꢀ33 and 2ꢀ
a
Acylaminothienothiazinone 34ꢀ36
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (i) benzoyl isothiocyanate, acetone, RT, 20 min; (ii) 1. concd H SO , 100 °C,
2
4
4 h, 2. NaHCO ; (iii) 1. 4ꢀphenylbutyric acid, Nꢀmethylmorpholine, 2,4,6ꢀtrichlorobenzoyl chloride,
3
THF, RT, 2. amine 48 or 49, pyridine, toluene, reflux, 2 h.
Substituted 2ꢀaminoꢀ4Hꢀ3,1ꢀbenzothiazinꢀ4ꢀones 30ꢀ33 were synthesized (Scheme 5) in order to
implement diversity at the fused benzene ring. Additionally, a bioisosteric replacement by a nonꢀ or
dimethylꢀsubstituted thiophene moiety (compounds 34ꢀ36) increases the electron density of the
thiazinone ring, which may have an impact on its interaction with the target proteins. In all derivatives
the acyl residue of compound 17 was kept constant. The starting reaction was carried out under the
conditions specified in Scheme 1 to yield derivatives 48 and 49, whose acylation with 4ꢀphenylbutyric
acid resulted in the target compounds 30ꢀ36.
Radioligand Binding Studies at Adenosine Receptors. The newly synthesized derivatives (8-36) were
investigated in radioligand binding studies, in most cases initially at rat brain A and A ARs and in
1
2A
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Journal of Medicinal Chemistry
case of measurable affinities subsequently at human recombinant A , A , A and A ARs expressed in
1
2A
2B
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
2
3
6
Chinese hamster ovary (CHO) cells.
For binding assays at A ARs, [ H]2ꢀchloroꢀN ꢀ
1
3
cyclopentyladenosine (CCPA) and at A2A receptors, [ H]3ꢀ(3ꢀhydroxypropyl)ꢀ7ꢀmethylꢀ8ꢀ(mꢀ
methoxystyryl)ꢀ1ꢀpropargylxanthine (MSXꢀ2) were used as selective radioligands in both species. For
3
A AR binding studies, the selective radioligand [ H]8ꢀ(4ꢀ(4ꢀ(4ꢀchlorophenyl)piperazineꢀ1ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2B
sulfonyl)phenyl)ꢀ1ꢀpropylxanthine (PSBꢀ603) and for A AR binding studies, the A ꢀselective
3
3
3
radioligand [ H]2ꢀphenylꢀ8ꢀethylꢀ4ꢀmethylꢀ(8R)ꢀ4,5,7,8ꢀtetrahydroꢀ1Hꢀimidazo[2,1ꢀi]purinꢀ5ꢀone (PSBꢀ
11) were employed. Initial screening was usually performed at two concentrations, 10 ꢁM and 1 ꢁM. In
A and A radioligand binding studies, the highest concentration of test compound was 1 ꢁM because
2
B
3
3
3
higher concentrations often led to precipitation of the radioligands [ H]PSBꢀ603 and [ H]PSBꢀ11. The
results of the AR binding assays are presented in Table 1 together with data for standard ligands for
comparison.
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Table 1. Adenosine Receptor Affinities of 1ꢀ36.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
-Acylaminobenzothiazinones
Substituted 2-acylaminobenzothiazinones and 2-acylaminothienothiazinones
for R see below
29ꢀ36 R = (CH ) Ph
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2 3
a
K ± SEM (nM)
i
rat A1
vs. [ H]CCPA
rat A
vs. [ H]MSXꢀ2
human A1
vs. [ H]CCPA
human A
vs. [ H]MSXꢀ2
human A
2B
vs [ H]PSBꢀ603
human A3
vs [ H]PSBꢀ11
2A
2A
compd
1
3
3
3
3
3
3
b
230
2.2
841
2830
91.2
36
>10000
4470
>3000
c
c
c
c
>1800
2
>10000
28.1 ± 5.5
28
>10000
38.1 ± 12.7
30
>1000
>1000
>1000
d
e
e
d
2
8000
8200
3
68.7 ± 8.7
0.66 ± 0.12
295 ± 10
0.884 ± 0.32
>1000
thiazinones 5, 8-29
R
f
5
25 ± 5
609 ± 61
103 ± 10
294 ± 137
>10000
309 ± 17
91.7 ± 16.9
80.9 ± 21.3
64.9 ± 12.4
n.d.
360 ± 100
365 ± 63
225 ± 33
30.4 ± 6.8
390 ± 69
173 ± 18
8
422 ± 75
367 ± 16
>1500
1050 ± 161
1370 ± 195
9
g
h
h
10
n.d.
>1000
>1000
h
1
1
>10000
>10000
2190 ± 863
>10000
n.d.
n.d.
n.d.
801 ± 139
≥1000
h
h
12
n.d.
n.d.
>1000
>1000
h
h
1
1
1
1
3
4
5
6
>10000
>10000
n.d.
>1000
>1000
h
h
205 ± 19
≥10000
253 ± 37
>10000
>10000
62.4 ± 12.6
>1000
679 ± 88
>1000
h
h
n.d.
>1000
>1000
h
h
>10000
1720 ± 694
423 ± 76
558 ± 67
>10000
n.d.
≥1000
>1000
>1000
h
h
17
229 ± 103
22.5 ± 2.3
898 ± 253
>1500
2500 ± 660
214 ± 11
>10000
n.d.
39.5 ± 5.8
115 ± 24
n.d.
>1000
>1000
1
1
2
2
8
9
0
1
302 ± 30
382 ± 106
h
h
≥1000
>1000
h
h
>10000
n.d.
>1000
>1000
53.0 ± 14.5
11.2 ± 1.5
11.4 ± 1.3
814 ± 167
317 ± 43
≥10000
822 ± 146
500 ± 59
562 ± 96
118 ± 38
91.8 ± 25.9
n.d.
119 ± 23
158 ± 45
115 ± 26
h
22
≥1000
h
h
2
3
>1000
>1000
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Journal of Medicinal Chemistry
h
h
2
2
4
5
≥1500
>1000
>1000
n.d.
n.d.
>1000
>1000
>1000
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
f
285 ± 38
>10000
69.5 ± 14.1
178 ± 41
>1000
2
6
n.d.
n.d.
>10000
>1000
>1000
>1000
>1000
>1000
27
84.7 ± 9.8
58.3 ± 7.6
>1000
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
h
2
2
8
9
n.d.
488 ± 205
>10000
4370 ± 1810
n.d.
44.3 ± 15.9
>10000
>1000
>1000
>1000
h
h
>10000
>1000
thiazinones 30-36
R'
R''
H
3
0
CH
Cl
Br
H
3
98.3 ± 48.0 6060 ± 1940
>1000
n.d.
n.d.
408 ± 31
147 ± 55
h
h
31
H
H
>10000
>10000
>10000
>10000
n.d.
>1000
>1000
h
h
3
3
2
3
n.d.
n.d.
>1000
>1000
h
Cl
ꢀ
64.4 ± 22.9 ≥10000
785 ± 339
n.d.
n.d.
>1000
91.6 ± 14.1
h
34
ꢀ
≥10000
>10000
n.d.
547 ± 167
361 ± 88
>1000
h
3
5
6
H
H
35.2 ± 1.2
>10000
693 ± 141
364 ± 184
82.5 ± 29.5
>1000
h
h
3
CH
3
CH
3
>10000
n.d.
n.d.
>1000
>1000
a
b
c
d
e
f
n = 3, unless otherwise noted. Data from ref. 33. Data from ref. 34. Data from ref. 35. Data from ref. 24b. Data from ref.
g
h
13. n.d., not determined. n = 2.
Structure-Activity Relationships at Adenosine Receptors. The phenylpropionyl derivative 8 was
potent at the human A AR (K = 80.9 nM) and showed a higher selectivity over the human A and
2
A
i
1
A AR subtypes than the previously described homolog, the benzoyl derivative compound 5. For the
3
purpose of further optimization, a first set of related derivatives (9ꢀ24), characterized by different acyl
residues at the exocyclic 2ꢀamino group, was synthesized. The introduction of a methoxy group in the 3ꢀ
position of the phenyl ring (compound 9) was well tolerated by the A AR. In contrast to 8, the affinity
2
A
for the human subtype (K = 64.9 nM) was more than 4ꢀfold higher compared to the rat A AR (K =
i
2A
i
294 nM). The additional 3ꢀmethoxy substituent did not improve AR subtype selectivity. A 3,4ꢀdichloroꢀ
compound 10) as well as a fluoroꢀ or a bromoꢀsubstitution in the orthoꢀposition (compounds 11 and 12)
(
greatly reduced or abolished A affinity. These halogenꢀsubstituted derivatives showed also no or only
2A
weak affinity for the other AR subtypes. Only derivative 11 was moderately potent at the A AR (K =
2
B
i
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8
01 nM). For compound 13, which contains an aryl ether linkage, no significant affinity for the ARs
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was observed. Replacement of the 3ꢀ(3ꢀmethoxyphenyl)propionyl residue of compound 9 by a
corresponding cinnamoyl moiety (compound 14) led to a similarly high A2A affinity (K = 62.4 nM).
i
Moreover, the double bond was beneficial concerning selectivity for the human A AR, which was 10ꢀ
2
A
fold over the human A AR and more than 16ꢀfold versus the A AR. While compound 14 was inactive
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2B
3
at human A AR, it showed good affinity for the rat A AR (K = 205 nM). An exchange of the methoxy
1
1
i
group of 14 for a chloro substituent (compound 15), or the alkenyl for an alkynyl spacer (compound 16),
respectively, resulted in a loss of affinity.
The extension of the 3ꢀphenylpropionylꢀ (compound 8) to a 4ꢀphenylbutyrylꢀsubstituent (compound 17)
increased the affinity for the human A AR (K = 39.5 nM) and provided the best A antagonist of the
2
A
i
2A
present series. In addition, this spacer elongation had a positive effect on selectivity, which was more
than 60ꢀfold over the human A and more than 25ꢀfold over the human A and A ARs. In contrast, an
1
2B
3
unsaturated spacer, as in derivative 21, decreased the A2A affinity 3ꢀfold at human and 2ꢀfold at rat
A ARs, while increasing affinity for the other three AR subtypes. Introduction of a 3ꢀmethoxy
2
A
substituent at the phenyl ring of 17 leading to compound 18 also reduced the affinity for A ARs (hA :
2
A
2A
K = 115 nM) and increased A , A and A AR affinities. Similar receptor affinities were obtained for
i
1
2B
3
the corresponding 3ꢀmethoxyꢀsubstituted unsaturated derivative 22, in which both modifications were
combined. In the set of compounds containing one or two chloro substituents at the phenyl ring (19, 20,
23, and 24), a complete loss of A2A affinity was observed. These derivatives were also inactive at the
other AR subtypes, except for compound 23, which showed high affinity for the rat A AR (K = 11.4
1
i
nM) and moderate affinity for the human A AR (K = 562 nM). Among the benzothiazinones bearing a
1
i
spacerꢀconnected phenyl ring attached to the 2ꢀposition of the benzothiazinone core structure (i.e. 8-24),
only 3ꢀmethoxyꢀsubstituted, or unsubstituted derivatives possessed good A affinity, whereas halogenꢀ
2
A
substituted compounds proved to be inactive.
As mentioned above, benzylpiperazineꢀsubstituted derivatives 26ꢀ28 were synthesized to enhance water
solubility. Their structure was designed based on the previously reported benzothiazinone derivative
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Journal of Medicinal Chemistry
1
3
2
5. Compound 25 with an unsubstituted benzyl residue was found to be a potent A and A receptor
2A 2B
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
antagonist (hA : K = 69.5 nM; A : K = 178 nM) and exhibited high A ꢀselectivity versus A ARs.
2
A
i
2B
i
2A
1
Apparently, a chloro substituent (compound 26) in the orthoꢀposition was not tolerated by any of the
AR subtypes. In contrast, the A2A affinity of the metaꢀ and the paraꢀchloroꢀsubstituted compounds
(
hA , 27: K = 58.3 nM; 28: K = 44.3 nM) was at least as high as that of the unsubstituted derivative
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2A i i
2
5. Moreover, 28 was more than 22ꢀfold selective for A2A versus the A and the A ARs and nearly
2B 3
1
00ꢀfold versus the A ARs (human species). These results indicated that the binding pocket for the
1
A AR is able to accommodate large and basic substituents and requires a correct orientation of the
2
A
chloro substituent. Thus, Nꢀ(chlorobenzyl)piperazineꢀsubstituted benzothiazinone derivatives like 27
and 28 are interesting new starting points for the development of selective A AR antagonists with an
2
A
expected improved water solubility, at least in slightly acidic media.
Because of the high A AR affinity and selectivity of compound 17, we used the phenylbutyryl residue
2
A
as a fixed motif for further modification of the benzothiazinone core and the amide linker (compound
29ꢀ36). Compound 29, an Nꢀmethylated derivative of 17, was completely inactive at all adenosine
receptor subtypes. This indicates that the amide proton may be required as a hydrogen bond donor for
receptor binding.
Next, we considered the modification of the benzothiazinone core (30-36). Neither methylꢀ, chloroꢀ, or
bromoꢀsubstitution in the 6ꢀpostition, nor a chloroꢀsubstituent in the 7ꢀposition was tolerated by the rat
A AR. Finally, the fused benzene ring of 17 was bioisosterically replaced by thiophene (compounds
2
A
3
4-36). The thieno[3,2ꢀd][1,3]thiazinꢀ4ꢀone 34 was inactive at A , A , and A ARs and had only a
1 2A 3
moderate potency at the A AR subtype (K = 547 nM). In contrast, the isomeric thieno[2,3ꢀ
2
B
i
d][1,3]thiazinꢀ4ꢀone 35 exhibited high affinities for most of the AR subtypes, except for the A AR,
3
comparable to those of the corresponding benzothiazinone analogue 17. Surprisingly the dimethylated
thienothiazinone 36 did not bind to any of the four AR subtypes.
In summary, the benzothiazinones with a methoxyꢀsubstituted cinnamoyl or a phenylbutyryl residue
(
14, 17), as well as representatives with an Nꢀ(chlorobenzyl)piperazine moiety (27, 28) exhibited the
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most favorable properties with regard to A2A affinity and selectivity, being comparable to the standard
ligands 1 and 2.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
With respect to bioactivity, the benzothiazinone scaffold turned out to be versatile. We did not only
obtain potent A AR antagonists, as intended in this study, but also discovered benzothiazinones with
2
A
high affinity for A , A and A ARs, e.g. 33, a relatively potent and selective A antagonist for human
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2B
3
3
A ARs (K = 91.6 nM).
3
i
With the exception of 8, which was equipotent at human and rat A ARs, the affinity for the human
2
A
A AR was consistently higher (2ꢀ10ꢀfold) than for the rat orthologue (plot of 12 rat pK values versus
2
A
i
2
human pK values, linear regression, gave the following results: slope = 0.60, yꢀintercept = 2.16, r =
i
0
.08; for details see Supporting Information). In contrast, all investigated benzothiazinones exhibited
lower affinity for the human than for the rat A AR (plot of 10 rat pK values versus human pK values
1
i
i
2
yielded the following results: slope = 1.39, yꢀintercept = ꢀ1.32, r = 0.58; for details see Supporting
Information). At that receptor subtype, some derivatives (e.g. 22 and 23) had a preference for the rat
species of up to 45ꢀfold. Such extreme species differences have to be taken into account prior to
2
preclinical in vivo studies, which are typically performed in rodents. In view of the low r values of our
analysis, crossꢀspecies extrapolations for benzothiazinones are very limited.
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Journal of Medicinal Chemistry
Functional Studies at Adenosine Receptors. Cyclic AMP (cAMP) accumulation studies at human
A ARs were performed with the most potent A AR antagonist of the present series, compound 17, as
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
A
2A
a representative of the benzothiazinone scaffold (Figure 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. cAMP accumulation experiments in CHO cells stably transfected with the human A AR.
2A
The nonꢀselective AR agonist NECA (0.1 and 1 ꢁM) showed a concentrationꢀdependent stimulation of
cAMP formation. Benzothiazinone 17 did not show an effect by itself but inhibited NECAꢀinduced
cAMP accumulation. Graph bars represent results from three independent experiments ± SEM.
Benzothiazinone 17 did not show any agonistic activity at CHO cells transfected with the human
A AR, even at high concentrations up to 20 ꢁM. However, the increase in intracellular cAMP
2
A
production induced by the agonist NECA was significantly blocked in the presence of the compound 17.
We used relatively high concentrations of the agonist NECA, and therefore also had to add high
concentrations of the antagonist 17 to prove significant inhibitory effects (see Figure 2). These results
13
clearly confirm previous findings that the class of benzothiazinones acts as antagonists at ARs.
Monoamine Oxidase Inhibition Studies. All compounds were investigated for inhibition of rat and
human MAOꢀB using mitochondriaꢀenriched rat liver fractions, and human recombinant MAOꢀB
enzyme, respectively. The commercially available Amplex Red monoamine oxidase assay kit was
utilized. MAOꢀA (human) inhibition assays were performed analogously for selected compounds. The
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Page 18 of 53
inhibitors clorgyline for MAOꢀA and selegiline for MAOꢀB were used to block the respective enzyme
in rat liver fractions for determining selective inhibition of one of the isoenzymes. The inhibitory
potencies for the benzothiazinones and for standard inhibitors are collected in Table 2 and 3.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. MAOꢀB Inhibitory Potencies of Benzothiazinones and Standard Compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
IC ± SEM (nM)
IC ± SEM (nM)
50
5
0
compd
2
rat MAO-B
human MAO-B
compd
20
rat MAO-B
human MAO-B
38.5 ± 5.1
18.1 ± 3.3
3300 ± 280
361 ± 38
2020 ± 580
840 ± 135
4640 ± 436
n.d.
3440 ± 1400
39.8 ± 6.4
238 ± 12
>1000
b
96 (baboon enzyme)
2
2
1
2
d
d
6
7
6.74 ± 0.81, 7
25.8 ± 3.0
6.13 ± 0.85, 20
7.67 ± 1.81
23
24
25
26
27
28
29
30
31
32
33
34
35
36
e
5
>10000
n.d.
>10000
8
42.7 ± 19.8
621 ± 39
>10000
17.6 ± 3.7
95.3 ± 8.8
>10000
>10000
9
n.d.
>10000
10
n.d.
>10000
1
1
725 ± 183
64.0 ± 18.4
661 ± 126
11.2 ± 2.4
2.09 ± 0.17
1.63 ± 0.18
470 ± 112
93.3 ± 9.2
43.7 ± 4.2
34.9 ± 2.5
389 ± 122
n.d.
>10000
12
n.d.
53.1 ± 16.5
94.0 ± 8.3
>10000
f
1
1
1
1
1
1
3
4
5
6
7
8
325 ± 28
f
281 ± 178
>10000
815 ± 436
73.9 ± 16.1
186 ± 37
>10000
>10000
1220 ± 452
76.4 ± 14.7
206 ± 42
69.7 ± 6.1
f
298 ± 8
f
1300 ± 80
6880 ± 310
291 ± 107
f
19
>1000
26.6 ± 10.1
9.80 ± 1.97
a
b
c
d
e
f
n = 3, unless otherwise noted. Data from ref. 24b. Data from ref. 36. Data from ref. 37. n.d., not determined. n = 2.
Structure-Activity Relationships at Monoamine Oxidase B. The previously reported benzoylꢀ
1
3
substituted 2ꢀaminobenzothiazinone 5 did not show noticeable activity at rat MAOꢀB. However,
extension of the linker to obtain the phenylpropionyl derivative 8 provided the first potent MAOꢀB
inhibitor. Compound 8 exhibited a slight preference for the human versus the rat isoenzyme (human:
IC = 17.6 nM; rat: IC = 42.7 nM). We therefore considered 8 as a starting point and focused on
50
50
structural variations to explore the potency of this type of inhibitor more precisely. The introduction of a
ꢀmethoxy group at the phenyl ring (compound 9) led to decreased inhibition (human: IC = 95.3 nM;
3
5
0
rat: IC = 621 nM). While a 3,4ꢀdichloro substitution pattern (compound 10) produced no activity at all,
5
0
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Journal of Medicinal Chemistry
an orthoꢀfluoro or orthoꢀbromo substitution resulted in potent MAOꢀB inhibitors at the human enzyme
11: IC = 11.2 nM; 12: IC = 2.09 nM). The number and position of halogen substituents on the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
5
0
50
phenyl ring appeared to be crucial for inhibitory activity towards MAOꢀB. At the rat enzyme, both
compounds were considerably weaker (11: IC = 725 nM, 65ꢀfold; 12: IC = 64.0 nM, 31ꢀfold). An
5
0
50
incorporation of oxygen into the spacer, and the introduction of a 3ꢀchloro substituent (compound 13)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
provided the most potent inhibitor of the entire series (human: IC = 1.63 nM) and exhibited a nearly 4ꢀ
5
0
fold higher inhibitory activity towards human MAOꢀB than the irreversible standard inhibitor 6. Again,
13 was much less potent at the rat MAOꢀB enzyme (IC50 = 661 nM, 400ꢀfold) than at the human
ortholog. The high potency of compounds 11ꢀ13 shows that benzothiazinones represent a new class of
potent MAOꢀB inhibitors. However, all three compounds were inactive at the A AR (see above) and
2
A
therefore not suitable for a dual target approach.
Among the derivatives with an unsaturated spacer (14-16), the 3ꢀmethoxyꢀcinnamoyl derivative 14
showed only a moderate effect, and was about equipotent at both species (human: IC = 470 nM; rat:
50
IC50 = 281 nM). The corresponding 3ꢀchlorinated compound 15 was 5ꢀtimes more potent than the
related methoxy derivative 14 only at the human enzyme (human: IC = 93.3 nM; rat: IC = 815 nM).
5
0
50
Introduction of a (3ꢀmethoxyphenyl)propioloyl residue (compound 16) resulted in a good MAOꢀB
inhibitory potency (human: IC = 43.7 nM; rat: IC = 73.9 nM) without species preference.
5
0
50
In the group with an extended spacer (17-20), the phenylbutyrylꢀsubstituted compound 17 exhibited a
remarkable MAOꢀB inhibitory activity (human: IC = 34.9 nM; rat: IC = 186 nM). As noted above,
50
50
1
7 was identified as a potent and selective A2A antagonist and can thus be considered to be a promising
dualꢀacting candidate. The activity of compound 18 with an additional metaꢀmethoxy group was about
10ꢀfold lower (human: IC = 389 nM) compared to the unsubstituted analogue 17. One or two chloro
50
substituents (19, 20) dramatically decreased the affinity. The same trend was observed for the
unsaturated analogs (21-24), where only the unsubstituted derivative 21 showed notable MAOꢀB
inhibitory potency (human: IC50 = 39.8 nM; rat: IC50 = 361 nM). The introduction of different Nꢀ
benzylpiperazine moieties (25-28) was not tolerated by MAOꢀB and resulted in completely inactive
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compounds. The activity of the Nꢀmethylated compound 29 for human MAOꢀB was only slightly
decreased (IC = 53.1 nM) compared to the corresponding Nꢀunsubstituted derivative 17. This indicates
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
0
that the amide proton, in contrast to the situation at the A AR, is not essential for MAOꢀB binding.
2
A
Next, modified 6ꢀ or 7ꢀsubstituted benzothiazinones (30-33) and thiophene analogues (34-36) were
considered. In position 6, only a methyl group (compound 30) was tolerated by MAOꢀB (human: IC =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
9
4.0 nM) whereas the derivatives with a 6ꢀchloro or a 6ꢀbromo substituent (31 and 32) were inactive. In
contrast, the 7ꢀchlorinated compound 33 was potent towards MAOꢀB (human: IC = 76.4 nM). The
5
0
thieno[3,2ꢀd][1,3]thiazinꢀ4ꢀone 34 had a moderate inhibitory effect (human: IC = 206 nM; rat: IC =
5
0
50
2
98 nM) and showed, as one of only a few examples in this series, no major species differences. The
isomeric thieno[2,3ꢀd][1,3]thiazinꢀ4ꢀone 35 exhibited slightly better MAOꢀB inhibition (human: IC =
5
0
69.7 nM; rat: IC50 = 291 nM) but was still weaker than the benzothiazinone lead structure 17.
Dimethylation of 35 to compound 36 increased potency at both, human and rat MAOꢀB, leading to a
potent MAOꢀB inhibitor (human: IC = 9.80 nM; rat: IC = 26.6 nM) that had a 3ꢀfold higher potency
50
50
than the lead compound 17.
Considering the whole series (8-36), more than half of the compounds were found to be active at MAOꢀ
B. The highest inhibition potencies were achieved with the dimethylated thienothiazinone derivative 36
and benzothiazinones bearing an orthoꢀhalogenꢀsubstituted phenylpropionyl residue (compound 11 and
1
2) or a metaꢀchloroꢀsubstituted phenoxyethyl moiety (compound 13). All of these compounds inhibited
MAOꢀB in the low nanomolar range, comparable or even superior to the standard inhibitors (6 and 7).
For the established inhibitor 2 as well as related xanthine derivatives and analogues, the styryl moiety
2
3,24
had been identified as an essential unit to achieve MAOꢀB inhibition.
However, in the present study
we could not observe a marked difference between benzothiazinones with a saturated and those with an
unsaturated spacer.
A comparison of the IC50 values determined at the human and the rat MAOꢀB enzyme revealed
significant species differences. For the present benzothiazinone series (except for 14, 20, and 24) the
inhibitory potency at the human MAOꢀB was always higher (1.4ꢀ400ꢀfold) than at the rat enzyme (plot
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Journal of Medicinal Chemistry
of 18 rat pIC values versus human pIC values, linear regression gave the following results: slope =
5
0
50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
0
.40, yꢀintercept = 3.54, r = 0.33; for details see Supporting Information). As these differences did not
follow a clear trend with respect to the benzothiazinones’ structures, it will always be required to
determine both, human and rat enzyme inhibition constants, prior to planning preclinical and clinical in
vivo studies. Similar findings of moderate correlations between human and rat IC50 values were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
8
previously reported for other classes of MAOꢀB inhibitors.
Mechanism of Monoamine Oxidase B Inhibition. It has already been mentioned that, from a
therapeutic point of view, reversible inhibition of MAOꢀB may have significant advantages over the
irreversible inactivation of the enzyme. Therefore, it was investigated whether four selected compounds,
8, 9, 17, and 35, act as reversible or irreversible inhibitors of MAOꢀB. For this purpose, human MAOꢀB
was treated with a test compound or with the irreversible inhibitor 6, or the reversible inhibitor 7 as
reference compounds, respectively (Figure 3).
Figure 3. Reactivation of MAOꢀB. Human MAOꢀB was treated under assay conditions with several
inhibitors (each at the concentration that represents its IC value) in the presence of the substrate pꢀ
8
0
tyramine. After 22 min, the substrate concentration was increased from 150 ꢁM to 1 mM, and
fluorescence was measured over a period of 5 h.
After preincubation, the substrate concentration was increased and the course of the reaction was
observed to assess reactivation of MAOꢀB. In the case of reversible inhibition, the inhibitor will be
replaced by the competing excess substrate. The measurements with the reference compound 7 and the
inhibitors 8, 9, 17, and 35, clearly indicated the expected reversible mode of interaction because an
ACS Pa gon P s Environment
elevated fluorescence could be detected aft er ar incre al us ing the substrate concentration. In contrast, in the

Journal of Medicinal Chemistry
Page 22 of 53
experiment with the irreversible inhibitor 6, the residual activity was not enhanced, as expected. These
results clearly showed that the benzothiazinones are reversible inhibitors of MAOꢀB.
To further characterize the interaction of the benzothiazinones with MAOꢀB, the type of enzyme
inhibition was determined by MichaelisꢀMenten kinetics. Thus, the initial rates of the MAOꢀBꢀcatalyzed
oxidation of the substrate pꢀtyramine, applied at six different concentrations, were measured in the
presence of different concentrations of inhibitor 17. The results are depicted as double reciprocal
LineweaverꢀBurk plots in Figure 4.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. LineweaverꢀBurk plot of the inhibition of human MAOꢀB in the presence of different
concentrations of benzothiazinone 17 (0, 50, 100, and 300 nM) with pꢀtyramine (0.05, 0.1, 0.5, 1.0, 1.5,
and 3.0 mM) as substrate. The reciprocal MAOꢀB activity was plotted against the reciprocal substrate
concentration (n = 2).
The plots for compound 17 were linear and intersected at the yꢀaxis with the plot for the uninhibited
enzyme. These results led us to conclude that the benzothiazinones are competitive inhibitors, which
occupy the substrate binding site of MAOꢀB, in agreement with their reversible mode of interaction.
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Journal of Medicinal Chemistry
Identification of Dual-Acting Drugs. With regard to the dual target approach to develop candidates
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that simultaneously antagonize A ARs and inhibit the activity of MAOꢀB, four derivatives have to be
2
A
highlighted (8, 9, 17, and 35). The corresponding data are summarized in Table 3. We have additionally
determined the inhibitory potency of theses compounds towards the human MAOꢀA. They did not
inhibit MAOꢀA even at high concentrations and could therefore be characterized as selective inhibitors
for the isoenzyme MAOꢀB.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Selected Dualꢀacting 2ꢀAcylaminobenzothiazinones and 2ꢀAcylaminothienothiazinones
a
a
K ± SEM (nM)
i
A2A selectivity
IC ± SEM (nM)
50
A2A
compd
8
3
hA
1
/hA2A hA2B/hA2A
hA
3
/hA2A
5
MAO-B
MAO-A
vs. [ H]MSXꢀ2
O
N
b
8
0.9 ± 21.3 (h)
103 ± 10 (r)
13
5
4
17.6 ± 3.7 (h)
42.7 ± 19.8 (r)
>10000 (h)
S
O
c
N
H
6
4.9 ± 12.4 (h)
294 ± 137 (r)
21
63
4
3
95.3 ± 8.8 (h)
621 ± 39 (r)
>10000 (h)
≥10000 (h)
≥10000 (h)
9
39.5 ± 5.8 (h)
423 ± 76 (r)
>25
4
>25
12
34.9 ± 2.5 (h)
186 ± 37 (r)
1
7
5
82.5 ± 29.5 (h)
93 ± 141 (r)
69.7 ± 6.1 (h)
291 ± 107 (r)
3
6
a
b
c
n = 3. (h) Human enzyme. (r) Rat enzyme.
The most potent dualꢀacting compounds 8, 9, 17, and 35 are characterized by a high shape similarity.
Considering the benzothiazinone core, an unsubstituted fused benzene ring provided the best activities
towards both targets. While the introduction of halogen atoms or methyl groups was not tolerated, the
benzene moiety could be replaced by a thiophene isostere retaining the duality of action. An
unsubstituted phenylpropionyl or phenylbutyryl moiety turned out to be the most beneficial acyl
substituent at the exocyclic 2ꢀamino group. Only for the shorter phenylpropionyl side chain, the
attachment of an additional methoxy substituent was allowed without losing dual activity. Compound 17
was the most potent human A2A antagonist (K = 39.5 nM) of the whole series and showed high
i
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Page 24 of 53
selectivity over the other adenosine receptor subtypes. Furthermore, 17 had the capability to selectively
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
inhibit human MAOꢀB in the same nanomolar range (IC = 34.9 nM). A reduced spacer length in the
5
0
phenylpropionyl derivative 8 resulted in a better human MAOꢀB inhibition, but a slightly reduced
human A affinity and selectivity.
2
A
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
We investigated the stability of compound 17 dissolved in DMSO by LCMS and H NMR spectroscopy
and compared it to that of standard compound 2 (for details see Figures S2, S3, S4, S5, and Table S1,
Supporting Information). In accordance with previously published observations for styrylxanthine
2
7
derivatives, compound 2 was unstable when exposed to daylight at room temperature, which was
already seen after 24 h of incubation. In contrast, 17 was found to be completely stable even after 7 days
of incubation under the same conditions.
Our findings indicate the restricted opportunities for scaffold modifications, when addressing two
diverse and unrelated protein targets. In general, the development of a dualꢀacting compound requires a
complex design and optimization process to adjust the ratio of activities at the two diverse targets.
However, it may be well worth while developing such dual or multiple targetꢀdirected drugs. A multiple
ligand might be able to enhance efficacy and improve safety compared to a mixture of two compounds
each of which addresses only a single target. There is an increasing interest in modulating multiple
targets, especially in complex diseases such as PD.
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Journal of Medicinal Chemistry
CONCLUSIONS
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In conclusion, we discovered 4Hꢀ3,1ꢀbenzothiazinꢀ4ꢀones and thienothiazinone analogues as a
structurally novel class of nonꢀxanthine and nonꢀadenine related compounds with a dual mode of action,
targeting A ARs and MAOꢀB at the same time. Twenty eight new compounds were synthesized and
2
A
investigated in radioligand binding studies at all four adenosine receptor subtypes and in enzyme
inhibition studies at MAOꢀB and MAOꢀA. Structural optimization and SAR analyses led to the
development of potent and selective A AR antagonists and the discovery of the first MAOꢀB inhibitors
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
A
within the class of benzothiazinones. We were able to identify several ligands potently addressing both
targets at similar concentrations. Species differences were determined between the human and the rat
ARs. All derivatives showed a preference for the human over the rat A AR, while the opposite trend
2
A
was observed for the A AR. A similar observation was made for MAOꢀB, where in most cases a strong
1
preference for the human enzyme was found. 4Hꢀ3,1ꢀBenzothiazinꢀ4ꢀone 17 was characterized as a
potent and selective A AR antagonist and a reversible, competitive MAOꢀB inhibitor. This compound
2A
is a promising new lead structure for dualꢀacting drugs and may serve as a pharmacological tool for
proofꢀofꢀconcept in vivo studies to validate this dual target approach as a novel strategy for the
treatment of PD.
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Journal of Medicinal Chemistry
Page 26 of 53
EXPERIMENTAL SECTION
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Chemistry. All reagents were obtained from various producers (Acros, Sigma Aldrich, Alfa Aesar, and
Fluorochem) and used without further purification. Solvents were used without additional purification
or drying unless otherwise noted. Reactions were monitored by thin layer chromatography (TLC) using
aluminum sheets coated with silica gel 60 F254 (Merck). Compounds were visualized under UV light
0
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0
(254 nm). Preparative column chromatography was performed on silica gel 60 (Acros Organics) 0.060–
0
.200 mm. Mass spectra were recorded on an API 2000 mass spectrometer (electron spray ion source,
Applied Biosystems, Darmstadt, Germany) coupled with an Agilent 1100 HPLC system using a
Phenomenex Luna HPLC C18 column (50 × 2.00 mm, particle size 3 ꢁm). The purity of the tested
compounds was determined by HPLCꢀUV obtained on an LCꢀMS instrument (Applied Biosystems API
2000 LCꢀMS/MS, HPLC Agilent 1100) using the procedure as follows: dissolving of the compounds at
a concentration of 1.0 mg/mL in methanol and if necessary sonicated to complete dissolving. Then, 10
ꢁ
L of the substance solution was injected into a Phenomenex Luna C18 HPLC column (50 × 2.00 mm,
particle size 3 ꢁm) and elution performed with a gradient of water/methanol either containing 2 mM
ammonium acetate and 0.1% formic acid (chrom. system A) or 2 mM ammonium acetate (chrom.
system B) from 90:10 up to 0:100 for 30 min at a flow rate of 250 ꢁL/min, starting the gradient after 10
min. UV absorption was detected from 220 to 400 nm using a diode array detector. All tested
1
13
compounds possessed a purity of not less than 95%. H and C NMR spectra were recorded on a
Bruker Avance 500 MHz NMR spectrometer. DMSOꢀd , CDCl , acetoneꢀd was used as solvents as
6
3
6
indicated below. NMR spectra were recorded at room temperature. Chemical shifts are given in parts
per million (ppm) relative to the remaining protons of the deuterated solvent used as internal standard.
Coupling constants J are given in Hertz, and spin multiplicities are given as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), br (broad).
Melting points were determined on a Büchi Bꢀ545 melting point apparatus and were uncorrected. The
1
3
28
benzothiazinones 25 and 44, 39 and 48 (R’ = CH , R’’ = H and R’ = Cl, R’’ = H), and the
3
28
thienothiazinones 49 were prepared as described.
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Journal of Medicinal Chemistry
(
2-Carboxyethyl)triphenylphosphonium Bromide. 3ꢀBromopropanoic acid 42 (25.4 g, 165.5 mmol) and
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triphenylphosphine (45.7 g, 174 mmol) were suspended in acetonitrile (125 mL) and refluxed for 5 h.
After stirring for additional 12 h at room temperature, diethylether (200 mL) was added and the mixture
was kept at ꢀ18 °C for 2 h. The formed precipitate was collected by suction filtration, washed with
diethylether and dried in vacuo to obtain the product (54.1 g, 79%) as a colorless powder. The crude
product was used in the next step without further purification.
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General Procedure for the Synthesis of Phenylbutenoic Acids 40.
(2ꢀCarboxyethyl)triphenylphosphonium bromide (1.2 equiv) was suspended in CH Cl (1 mL/mmol)
2
2
cooled to 0 °C and the appropriate aldehyde 43 (1.0 equiv) was added. After addition of tertꢀBuOK (2.5
equiv) in portions, the reaction mixture was stirred for 12 h at room temperature. The reaction was
quenched with water (2 mL/mmol), washed two times with CH Cl and adjusted to pH 1. After
2
2
extraction with diethylether the combined organic layers were washed with brine, dried (MgSO ) and
4
the solvent was rotary evaporated. The crude material was purified by silica gel chromatography as
indicated below.
(E)-4-Phenylbut-3-enoic Acid. (40, R’’’ = H). Purification by silica gel chromatography
1
(CH Cl /MeOH, 15:1 v/v) to obtain a colorless solid (790 mg, 53%); H NMR (500 MHz, DMSOꢀd ) δ
2
2
6
=
3.18 (dd, J = 1.5, 7.1 Hz, 2H), 6.32 (dt, J = 7.1, 15.9 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H), 7.20 – 7.24
13
(m, 1H), 7.28 – 7.33 (m, 2H), 7.42 – 7.38 (m, 2H), 12.35 (br, 1H); C NMR (125 MHz, DMSOꢀd ) δ =
6
38.0, 123.3, 126.1 (2C), 127.5, 128.7 (2C), 132.4, 136.9, 172.7.
(E)-4-(3-Methoxyphenyl)but-3-enoic Acid (40, R’’’ = 3-OMe). Purification by silica gel
1
chromatography (CH Cl /MeOH, 10:1 v/v) to obtain a colorless solid (2.12 g, 75%); H NMR (500
2
2
MHz, DMSOꢀd ) δ = 3.17 (dd, J = 1.3, 7.1 Hz, 2H), 3.75 (s, 3H), 6.31 (dt, J = 7.1, 15.9 Hz, 1H), 6.45
6
(d, J = 16.0 Hz, 1H), 6.80 (dd, J = 2.6, 8.2 Hz, 1H), 6.94 – 6.96 (m, 1H), 7.22 (t, J = 7.9 Hz, 1H), 6.97
1
3
(d, J = 7.7 Hz, 1H), 12.32 (br, 1H); C NMR (125 MHz, DMSOꢀd ) δ = 38.0, 55.2, 111.3, 113.4, 118.7,
6
1
23.7, 129.8, 132.4, 138.4, 159.7, 172.7.
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Page 28 of 53
(
E)-4-(3-Chlorophenyl)but-3-enoic Acid (40, R’’’ = 3-Cl). Purification by silica gel chromatography
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9
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(CH Cl /MeOH, 15:1 v/v) to obtain a colorless solid (12.6 g, 41%); H NMR (500 MHz, DMSOꢀd ) δ =
2
2
6
3.19 (dd, J = 1.2, 6.9 Hz, 2H), 6.38 (dt, J = 6.9, 16.0 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H), 7.27 (ddd, J =
1
1
1
.4, 2.0, 7.7 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 7.37 (dt, J = 1.4, 7.7 Hz, 1H), 7.46 (t, J = 1.8 Hz, 1H),
1
3
2.45 (br, 1H); C NMR (125 MHz, DMSOꢀd ) δ = 38.0, 124.8, 125.4, 125.9, 127.2, 130.6, 131.0,
0
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33.6, 139.2, 172.5.
(E)-4-(3,4-Dichlorophenyl)but-3-enoic Acid (40, R’’’ = 3,4-Cl). Purification by silica gel
1
chromatography (CH Cl /MeOH, 10:1 v/v) to obtain a colorless solid (1.61 g, 61%); H NMR (500
2
2
MHz, DMSOꢀd ) δ = 3.18 (d, J = 6.0 Hz, 2H), 6.42 (dt, J = 6.3, 16.0 Hz, 1H), 6.47 (d, J = 16.1 Hz, 1H),
6
1
3
7
.41 (dd, J = 2.1, 8.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 12.28 (br, 1H); C
NMR (125 MHz, DMSOꢀd ) δ = 38.0, 123.0, 126.2, 126.3, 128.0, 129.7, 130.8, 131.6, 137.8, 172.4.
6
General Procedure for the Synthesis of Phenylbutyric Acids 41. 10% Pd/C (10 wt%) was added to a
solution of the corresponding olefins (40 or commercially available reagent) in EtOH or THF (3
mL/mmol) and stirred under H atmosphere for 1 h. After filtration through a pad of Celite, the filter
2
cake was rinsed with EtOH or THF and the solvent of the filtrate was removed to obtain the desired
product. In some cases purification by silica gel chromatography was necessary as indicated below.
1
4
-(3-Methoxyphenyl)butanoic Acid (41, R’’’ = 3-OMe). Light beige solid (587 mg, 97%); H NMR (500
MHz, DMSOꢀd ) δ = 1.83 – 1.73 (m, 2H), 2.19 (t, J = 7.4 Hz, 2H), 2.55 (t, J = 7.5 Hz, 2H), 3.72 (s, 3H),
6
1
3
6.71 – 6.78 (m, 3H), 7.01 – 7.27 (m, 1H), 12.13 (br, 1H); C NMR (125 MHz, DMSOꢀd ) δ = 26.3,
6
3
3.2, 34.6, 55.0, 111.4, 114.1, 120.7, 129.4, 143.3, 159.5, 174.4.
4
-(3-Chlorophenyl)butanoic Acid (41, R’’’ = 3-Cl). Purification by silica gel chromatography
1
(CH Cl /MeOH, 15:1 → 10:1 v/v) to obtain a colorless oil (345 mg, 38%); H NMR (500 MHz, CDCl )
2
2
3
δ = 1.94 (m, 2H), 2.36 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 7.4 Hz, 2H), 7.04 (d, J = 7.4 Hz, 1H), 7.14 – 7.19
1
3
(m, 3H), 12.10 (br, 1H); C NMR (125 MHz, DMSOꢀd ) δ = 26.0, 33.6, 33.8, 126.0, 127.2, 128.4,
6
1
30.2, 133.0, 144.6, 174.3.
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Page 29 of 53
Journal of Medicinal Chemistry
4
-(3,4-Dichlorophenyl)butanoic Acid (41, R’’’ = 3,4-Cl). Purification by silica gel chromatography
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(CH Cl /MeOH, 15:1 → 10:1 v/v) to obtain a colorless oil (513 mg, 57%); H NMR (500 MHz,
2
2
DMSOꢀd ) δ = 1.73 – 1.83 (m, 2H), 2.20 (t, J = 7.4 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 7.19 (dd, J = 2.1,
6
13
8
.2 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 12.10 (br, 1H); C NMR (125 MHz,
DMSOꢀd ) δ = 26.0, 33.1, 33.5, 128.5, 128.9, 130.48, 130.50, 130.9, 143.1, 174.2.
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-(2-Fluorophenyl)propanoic Acid (41, R’’’ = 2-F). Colorless solid (506 mg, quantitative); H NMR
(500 MHz, CDCl ) δ = 2.68 (t, J = 7.8 Hz, 2H), 2.97 (t, J = 7.7 Hz, 2H), 6.98 – 7.03 (m, 1H), 7.03 –
3
1
3
7.07 (m, 1H), 7.15 – 7.23 (m, 2H), 10.09 (br, 1H); C NMR (125 MHz, DMSOꢀd ) δ = 24.3, 34.1,
6
1
15.4 (d, J(C,F) = 21.8 Hz), 124.1. (d, J(C,F) = 16.0 Hz), 127.0 (d, J(C,F) = 15.5 Hz), 128.2 (d, J(C,F)
8.1 Hz), 130.6 (d, J(C,F) = 4.6 Hz), 161.2 (d, J(C,F) = 256 Hz), 178.8.
2-Amino-6-bromo-4H-3,1-benzothiazin-4-one (48, R’ = Br, R’’ = H). Benzoyl isothiocyanate (2.00 mL,
5.0 mmol) was added dropwise to a solution of methyl 2ꢀaminoꢀ5ꢀbromobenzoate (3.00 g, 13.0 mmol)
=
1
in acetone (10 mL). After stirring for 20 min at room temperature the formed precipitate was filtered by
suction filtration and recrystallized from toluene to obtain the thioureide as a light yellow solid (4.48 g,
8
7%). The intermediate (1.00 g, 2.54 mmol) was treated with 10 drops water and concentrated H SO
2 4
(7 mL) and stirred for 4 h at 100 °C. After cooling to room temperature the reaction mixture was put
carefully into ice water and neutralized by addition of solid NaHCO . The formed precipitate was
3
filtered by suction filtration and dried in vacuo. After recrystallization from toluene the product 48
1
(R’ = Br, R’’ = H) was obtained as a light yellow solid (510 mg, 78%); H NMR (500 MHz, CDCl ) δ =
3
1
3
7
.22 (d, J = 8.8 Hz, 1H), 7.77 (dd, J = 2.5, 8.8 Hz, 1H), 7.93 (d, J = 2.5 Hz, 1H), 8.04 (br, 2H); C
NMR (125 MHz, DMSOꢀd ) δ = 115.0, 117.8, 126.3, 129.9, 138.6, 150.4, 157.5, 183.1; LC/ESIꢀMS
6
ꢀ
+
(m/z): negative mode 257 [MꢀH] , positive mode 259 [M+H] .
2
-Amino-7-chloro-4H-3,1-benzothiazin-4-one (48, R’ = H, R’’ = Cl). Benzoyl isothiocyanate (0.90 mL,
6
.70 mmol) was added dropwise to a solution of 6ꢀchloroanthranilic acid (1.00 g, 5.80 mmol) in acetone
(34 mL). After stirring for 20 min at room temperature the formed precipitate was filtered by suction
filtration and recrystallized from MeOH to obtain the thioureide as a light yellow solid (1.55 g, 79%).
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