Brief Article pubs.acs.org/jmc
Evaluation of Homobivalent Carbolines as Designed Multiple Ligands for the Treatment of Neurodegenerative Disorders Robert Otto,† Robert Penzis,‡ Friedemann Gaube,‡ Oliver Adolph,§ Karl J. Föhr,§ Paul Warncke,† Dina Robaa,∥ Dorothea Appenroth,⊥ Christian Fleck,⊥ Christoph Enzensperger,# Jochen Lehmann,*,† and Thomas Winckler*,‡
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 1, 2015 | http://pubs.acs.org Publication Date (Web): August 17, 2015 | doi: 10.1021/acs.jmedchem.5b00958
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Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, 07743 Jena, Germany ‡ Department of Pharmaceutical Biology, Institute of Pharmacy, University of Jena, Semmelweisstrasse 10, 07743 Jena, Germany § Department of Anesthesiology, University Hospital of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany ∥ Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle/Saale, Germany ⊥ Institute of Pharmacology and Toxicology, University of Jena, Drackendorfer Strasse 1, 07747 Jena, Germany # Institute of Organic and Macromolecular Chemistry, Univerity of Jena, Humboldtstrasse 10, 07743 Jena, Germany S Supporting Information *
ABSTRACT: Neurodegenerative diseases represent a challenge for biomedical research due to their high prevalence and lack of mechanism-based treatments. Because of the complex pathology of neurodegenerative disorders, multifunctional drugs have been increasingly recognized as potential treatments. We identified homobivalent γ-carbolinium salts as potent inihitors of both cholinesterases, N-methyl-D-aspartate receptors, and monoamine oxidases. Homobivalent γ-carbolines displayed similar structure−activity relationships on all tested targets and may present promising designed multiple ligands for the treatment of neurodegenerative disorders.
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INTRODUCTION Neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) pose major challenges to 21st century health systems. Because of their complex pathology, neurodegenerative disorders are unlikely to be mitigated by drugs that address only one clinically relevant target, and the requirement for the development of multifunctional drugs has been increasingly recognized. Several potential drug targets for the treatment of cognition and memory defects in AD patients or the extrapyramidal symptoms in PD patients have been identified. For example, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) degrade acetylcholine in the synaptic clefts of cholinergic neurons. The loss of cholinergic neurotransmission in the frontal cortex and hippocampus is a hallmark of AD, and AChE inhibitors that elevate acetylcholine levels, such as donepezil, have become first-line therapeutics to slow down AD progression.1 Glutamate mediates excitatory synaptic transmission in the brain, which is important for synaptic plasticity involved in long-term potentiation, memory, and learning.2 Pathophysiological conditions resulting in high extracellular concentrations of glutamate favor the excessive activation of N-methyl-D-aspartate receptors (NR), which leads to an overload of cells with calcium that eventually causes the loss of neurons.3−5 This process of NR-mediated, glutamateinduced excitotoxicity is effectively blocked by memantine, the © 2015 American Chemical Society
only approved drug for the treatment of patients suffering from moderate to severe AD.6 Monoamine oxidase (MAO) catalyzes the oxidative deamination of a variety of biogenic amines.7 Two types of MAOs that are encoded by different genes, show different tissue distribution, and display different substrate and inhibitor specificities. MAO-A inhibitors were the first antidepressant drugs developed, whereas MAO-B inhibitors with neuroprotective properties have been found useful for the therapeutic management of PD.8−10 The concept of designed multiple ligands (DMLs), in which distinct pharmacophores of different drugs are combined in the same molecule, is particularly attractive for the treatment of disorders involving multiple pathophysiological processes.11−13 If bifunctional (heterobivalent) molecules are designed in which each pharmacophore binds to different clinically relevant targets, it may be assumed that this single-molecule combination therapy generates additive or synergistic effects that improve therapeutic efficacy. Another approach to creating DMLs would be the use of homobivalent molecules in which each half of the hybrid has identical binding properties but at multiple targets that are not accessible for their monovalent analogues.14 Here, we report the synthesis and evaluation of Received: June 22, 2015 Published: August 17, 2015 6710
DOI: 10.1021/acs.jmedchem.5b00958 J. Med. Chem. 2015, 58, 6710−6715
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 1, 2015 | http://pubs.acs.org Publication Date (Web): August 17, 2015 | doi: 10.1021/acs.jmedchem.5b00958
Journal of Medicinal Chemistry
Brief Article
Figure 1. Overview of the compounds evaluated in this study.
homobivalent γ-carbolines as potential DMLs that address three different targets relevant to the treatment of neurodegenerative disorders: cholinesterases, NR, and MAO.
1a/NR2B, and monoamine oxidases MAO-A and MAO-B. The obtained data are summarized in Table 1 and will be described below in detail. Homobivalent β-carbolines are nanomolar cholinesterase inhibitors with a preference for AChE over BChE.14 For example, the β-carbolinium salt 2 displayed IC50 values for AChE and BChE of 0.52 and 5.3 nM, respectively (Table 1). The potency of the bivalent β-carbolinium as an AChE inhibitor of cholinesterase was substantially decreased upon removal of the permanent positive charge (compound 1; IC50 = 753.2 nM) or the hydrogenation of the pyridine ring, as in 3 (IC50 = 17.3 nM). Homobivalent γ-carbolines displayed some interesting differences to the β-carboline analogues (Table 1). The bivalent quaternary γ-carboline with the nonylene linker (5d) showed similar AChE-inhibitory activity compared with the β-carbolinium 2, yet it showed an approximately 34-fold specificity for AChE over BChE (IC50 values 0.45 versus 15.7 nM). The unmethylated pyridyl derivative 4d served as a moderate inhibitor of AChE (IC50 = 40.0 nM). Although 4d was approximately 39-fold more active as an AChE inhibitor compared with its β-carboline analogue, it was still 100-fold less active than the quaternary γ-carboline 5d (Table 1). On the other hand, the N-methylated tetrahydro γ-carboline 6d and its β-carboline counterpart 3 were similarly active as AChE inhibitors, yet they were ∼37-fold less active than the γcarbolinium 5d. In summary, homobivalent γ-carbolinium molecules with linker lengths of 7−9 carbons (5b−d) were the most potent cholinesterase inhibitors, with activities similar to the previously described β-carbolinium analogue 2. Both the removal of the permanent charge from the pyrido-N and the hydrogenation of the pyridine ring significantly reduced cholinesterase inhibition, as did extending the pyrido-N-linked alkyl chain (5f−h) or attachment of a methoxy group at position C6 (5e) (Table 1). Several studies have evaluated the potencies of homo- and heterodimeric AChE inhibitors.15−17 A recurrent observation in these studies was that the monomeric compounds were less active than their bifunctional analogues. For example, a homobivalent tacrine with a heptylene tether, bis(7)-tacrine, was shown to exhibit a more than 1000-fold higher potency as
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RESULTS AND DISCUSSION In this study, we investigated new homobivalent γ-carbolines as potential drugs for the treatment of neurodegenerative disorders. For convenience, we will use the term “indolo-N” to assign the positions of linkers that connect the carboline moieties in homobivalent structures. Indolo-N linker positions refer to N9 in β-carbolines and N5 in γ-carbolines, as outlined in Figure 1. The term “pyrido-N” will be used to address βcarbolines or γ-carbolines carrying alkyl groups at N2. Recently described bivalent β-carbolines14 served as lead structures for our investigation. The bivalent β-carboline 1 was pyrido-N-methylated to give the quaternary salt 2, which was then hydrogenated to generate the 1,2,3,4-tetrahydro derivative 3 (Figure 1). We synthesized 18 new, bivalent γ-carboline derivatives and focused on compounds with 6−9 carbon linkers. According to general procedure 1 (see Experimental Section), two identical γ-carboline (5H-pyrido[4,3-b]indole) moieties (Figure 1) were connected at the indolo-N by an alkyl chain using alkyl halides to create a set of bivalent substances that differed in the length of the connecting alkyl chain, which ranged from 6 to 9 carbon atoms (series 4a−d). In addition, the effect of a methoxy group attached to the γ-carboline scaffold was investigated (4e). These substances were also treated according to general procedures 2 and 3 (see Experimental Section) to obtain quaternary salts (series 5a− h) and partially reduced compounds (series 6a−e) (Figure 1). Considering the most active bivalent β-carboline with a nonylene linker14 as lead structure for further investigations, we were able to compare the properties of an analogous set of bivalent β-carbolines and γ-carbolines consisting of pyridineunmethylated (1 vs 4d), pyrido-N-methylated (2 vs 5d), and N-methylated 1,2,3,4-tetrahydro derivatives (3 vs 6d) in parallel pharmacological testing. All compounds were characterized as DMLs for the three structures relevant to the treatment of neurodegenerative diseases: cholinesterases AChE and BChE, NMDA receptors of the composition NR1-1a/NR2A or NR16711
DOI: 10.1021/acs.jmedchem.5b00958 J. Med. Chem. 2015, 58, 6710−6715
Journal of Medicinal Chemistry
Brief Article
Table 1. Properties of the Carbolines Described in This Study IC50 [nM] ± SD
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 1, 2015 | http://pubs.acs.org Publication Date (Web): August 17, 2015 | doi: 10.1021/acs.jmedchem.5b00958
compd
melting points [°C]
AChE
BChE
130−131
753.2 ± 62.4
288.6 ± 39.0
1 2 3
250−251 131−132
0.52 ± 0.07 17.3 ± 3.7
5.3 ± 0.3 23.6 ± 2.2
4a
168−169
473.3 ± 118.8
50.2 ± 5.7
4b
139−140
88.8 ± 34.6
16.2 ± 2.7
4c
200−203
16.3 ± 8.3
13.2 ± 6.7
4d
124−126
40.0 ± 7.9
13.1 ± 1.2
4e
128−130
12.5 ± 2.4
6.5 ± 0.9
5a 5b 5c 5d 5e 5f 5g 5h 6a
>350 325−330 312−315 294−297 212−214 147−148 220−221 90−91 f
14.8 ± 1.1 0.50 ± 0.27 0.64 ± 0.03 0.45 ± 0.17 4.1 ± 0.9 2.3 ± 0.3 7.2 ± 2.5 3.1 ± 0.7 76.7 ± 23.0
93.5 ± 13.0 46.9 ± 1.4 31.4 ± 6.0 15.7 ± 1.1 47.8 ± 2.6 32.5 ± 2.0 85.4 ± 12.1 55.8 ± 3.3 80.1 ± 11.4
6b
f
31.7 ± 3.0
37.9 ± 11.1
6c
f
35.1 ± 8.4
42.5 ± 12.5
6d
f
16.8 ± 4.6
52.4 ± 8.4
6e
f
7.4 ± 1.5
43.5 ± 6.1
27.2 ± 3.5 nd nd nd
4.4 ± 0.4 nd nd nd
tacrine memantine clorgyline selegiline
IC50 [μM] ± SD (excitotoxicity [%] at 50 μM) NR1-1a/NR2A a,b
nd (92.6 ± 4.3) 1.4 ± 0.2c ndc (91.0 ± 11.0) ndd (84.8 ± 15.5) nde (>150) ndd (94.7 ± 1.9) nde (>150) nde (125.0 ± 19.7) 13.8 ± 2.8 3.8 ± 1.4 0.57 ± 0.10 0.40 ± 0.05 2.4 ± 0.2 0.59 ± 0.02 0.74 ± 0.003 1.0 ± 0.1 nde (>150) nde (>150) nde (>150) nde (>150) nde (>150) 4.9 ± 1.3c 4.4 ± 2.1 nd nd
NR1-1a/NR2B b
nd (111.3 ± 4.0) 2.9 ± 1.1c ndc (89.3 ± 10.2) ndd (98.2 ± 6.3) nde (146.5 ± 10.9) ndd (97.9 ± 3.0) nde (133.1 ± 30.9) nde (126.4 ± 7.1) 30.8 ± 5.8 7.2 ± 4.8 1.5 ± 0.7 0.78 ± 0.09 7.4 ± 0.8 2.6 ± 0.5 2.8 ± 0.6 2.0 ± 0.1 nde (>150) nde (>150) nde (>150) nde (>150) nde (>150) 44.0 ± 2.1c 5.2 ± 1.7 nd nd
IC50 [μM] ± SD MAO-A
MAO-B
>150
>150
2.7 ± 1.0 >150
1.0 ± 0.3 >150
67.3 ± 5.3
7.1 ± 2.6
1.5 ± 0.4
1.6 ± 0.4
4.9 ± 0.5
14.2 ± 6.6
5.1 ± 0.8
3.9 ± 0.5
1.6 ± 0.5
0.36 ± 0.06
1.7 ± 0.1 0.12 ± 0.023 0.25 ± 0.009 0.22 ± 0.04 3.2 ± 0.5 7.3 ± 0.7 14.6 ± 2.4 22.5 ± 3.8 4.5 ± 0.98
0.097 ± 0.002 0.051 ± 0.008 0.066 ± 0.008 0.06 ± 0.02 5.1 ± 1.4 2.9 ± 0.5 23.1 ± 0.7 20.8 ± 1.2 0.32 ± 0.04
1.2 ± 0.2
0.65 ± 0.11
0.51 ± 0.06
0.69 ± 0.12
3.5 ± 0.1
3.9 ± 1.1
6.3 ± 0.31
9.8 ± 1.7
nd nd 0.003 ± 0.001 47.9 ± 13.3
nd nd 54.3 ± 3.9 0.27 ± 0.03
nd: not determined. bExcitotoxicity determined at 25 μM due to solubility problems at 50 μM. cDetermined previously.14 dIC50 not determined; inhibition
