Eur. J. Med. Chem. 2017 Feb;127:470–492. DOI: 10.1016/j.ejmech.2017.01.011

Crystal structures, binding interactions, and ADME evaluation of brain penetrant N-substituted indazole-5-carboxamides as subnanomolar, selective monoamine oxidase B and dual MAO-A/B inhibitors

Nikolay T. Tzvetkov,a* Hans-Georg Stammler,b Beate Neumann,b Silvia Hristova,c Liudmil Antonov,c Marcus Gastreich d

a NTZ Lab Ltd., Krasno selo 198, 1618 Sofia, Bulgaria

b Department of Chemistry, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany

c Bulgarian Academy of Sciences, Institute of Organic Chemistry, Centre of Phytochemistry, Acad. G. Bonchev Str., Bl. 9, Sofia 1113, Bulgaria

d BioSolveIT GmbH, An der Ziegelei 79, 53757 St. Augustin, Germany

 

Abstract

The pharmacological and physicochemical analysis of structurally optimized N-alkyl-substituted indazole-5-carboxamides, developed as potential drug and radioligand candidates for the treatment and diagnosis of Parkinson’s disease (PD) and other neurological disorders, is reported. Recent efforts have been focused on the development of subnanomolar potent, selective MAO-B (N1-alkyl-substituted compounds NTZ-1091, 1441, 1471 and 1446) and dual active MAO-A/B (N2-methylated compounds NTZ-1092, 1442, 1472) inhibitors with nanomolar potency towards MAO-B and moderately active against MAO-A enzyme, respectively. The most promising drug-like derivatives in both series were N-(3-chloro-4-fluorophenyl)-1-methyl-1H-indazole-5-carboxamide (NTZ-1441, IC50 hMAO-B 0.662 nM, >15000-fold selective versus MAO-A) and N-(3-chloro-4-fluorophenyl)-2-methyl-2H-indazole-5-carboxamide (NTZ-1442, IC50 hMAO-B 8.08 nM, IC50 hMAO-A 0.56 µM, SI = 70). Moreover, compounds NTZ-1441 and NTZ-1442 were predicted to cross both the gastrointestinal tract (at pH 2.0, 5.5, and 7,4) and blood-brain barrier (BBB) in vitro with appropriate drug-like properties required for CNS active drugs. Combined single X-ray/molecular modeling studies provided insights into the enzyme–inhibitor interactions within both MAO isoforms and the rationale for their inhibitory activity with controlled MAO-A/B selectivity  – despite their small structural differences. The binding modes of NTZ-1091, 1092 and NTZ-1441, 1442 confirmed that the major interactions with hMAO-B were established via the flexible carbonyl group of the carboxamide linkage and the electron-donating nitrogens N1 or N2 of the indazole moiety, allowing further exploration of the alkyl side chain for next step lead optimization efforts.

KEYWORDS: ADME; MAO inhibitors; Molecular modeling; Indazole-5-carboxamides; Parkinson’s disease; X-ray


Supplement:

Alzheimer´s disease (AD) and Parkinson´s disease (PD) are the most prevalent, aging-related neurodegenerative disorders of the central nervous system (CNS), currently affecting over 8% of individuals aged ≥65 years worldwide. Despite their differences in pathogenesis and symptoms, AD and PD share common underlying features such as chronic, irreversible, and progressive neuronal degradation in the human brain caused by complex pathophysiological processes, including oxidative stress, neuro-inflammation, excitotoxicity, mitochondrial dysfunction, and proteolytic stress [1].

Monoamine oxidases (МАОs, EC 1.4.3.4) are mitochondrial flavoenzymes that are involved in the oxidative deamination of exogenous and endogenous amines, including neurotransmitters in both the peripheral nervous system (PNS) and CNS. Two subtypes of MAOs have been identified in mammals, MAO-A and MAO-B, encoded by distinct genes with opposite orientation on the X-chromosome (Xp11.23-11.4), where they share ~70% identity of the protein sequence [2]. Both MAO isoforms are essential for the inactivation of monoaminergic neurotransmitters but display regional differences in enzyme activity, substrate preference and distribution in the human brain, and therefore, different clinical significances. The expression levels and activity of MAO-B in the human brain, rather than those of MAO-A, increase ~4-fold with aging, leading to a higher production of hydrogen peroxide (H2O2) and other reactive oxygen species (ROS), which are associated with oxidative stress and neuronal cell death [2]. Enhanced activity and overexpression of MAO-B in the brain is thus observed in patients with Alzheimer’s disease (AD) and PD. Considering the beneficial role of reversible MAO inhibition in neuroprotection and modulation of various pathophysiological processes associated with PD, dual and reversible inhibitors of MAO A and B, rather than single MAO-B inhibitors, may have increased therapeutic and neuroprotective potential not only in the treatment of PD but also for AD therapy. Moreover, it has been observed that brain-selective MAO drugs may exhibit the combined benefit of antioxidative and neurorestorative activities with a reduced risk for toxicity and side effects [3].

Recently we have discovered a series of selective MAO-B or dual MAO-A/B inhibitors with nanomolar potency [4]; several compounds of structurally related N-unsubstituted indazole-5-carboxamides (designated subclass I), N1-methylated indazole-5-carboxamides (subclass II) and their N2-methyl analogs (subclass III) were identified as best-in-class MAO inhibitors (Figure 1). The compounds were tested at rat and human MAO A and B and, depending on the substitution pattern of the indazole N1- or N2-position, they may act either as selective MAO-B or dual MAO-A/B inhibitors with reversible mode of action.

 

 

Figure 1. Chemical structures and MAO activity of new indazole-5-carboxamide derivatives. Structure optimization process of lead structure NTZ-1034 (Subclass I MAO-B inhibitors) led to N1-methylated indazole-5-carboxamides (Subclass II selective MAO-B inhibitors) and their N2-methyl-substituted analogs (subclass III dual active MAO-A/B inhibitors).

 

Compound NTZ-1441 penetrates the BBB in vitro and effects the neuron survival and neurite network

Structural optimization and SAR analyses led to the discovery of remarkably potent, competitive and reversible selective MAO-B or dual MAO-A/B inhibitors with subnanomolar or even picomolar potency. Therefore, we evaluated the drug-like and in vitro ADME properties of the studied compounds, including their water-solubility, logD, blood-brain-barrier (BBB) penetration, and gastrointestinal (GI) permeability using PAMPA. To achieve an accurate prediction of oral bioavailability, permeability in the PAMPA-GI assay was measured at three different pH values (2.0, 5.5 and 7.4) to mimic the physiological pH gradient of the GI tract. In the PAMBA-BBB model, all compounds were assessed only at pH 7.4 that corresponds to the physiological pH in the blood compartment. On the basis of the PAMPA-BBB results, the N1-methylated indazole-5-caroxamides NTZ-1091 and NTZ-1441 (compounds of subclass II) and their N2-methylated analogues NTZ-1092 and NTZ-1442 (subclass III) can be classified as highly permeable under tested conditions. These compounds are predicted to be able to cross the BBB by passive permeation exhibiting Pe values that are comparable or even better (for NTZ-1441) to the permeability of verapamil measured at different concentrations (Figure 2A). Furthermore, such compounds were found to be GI permeable with predictive oral absorption and highly CNS penetrant (CNS+) in our PAMPA-BBB assay. To further assess the structure-properties relations (SPRs), we plotted the pIC50 values (hMAO-B or hMAO-A) versus the logD7.4 values (LLE score) for all selected compounds as well as reference drugs moclobemide and safinamide (Figure 2B). It appears evident that all evaluated compounds exhibit or are predicted to have optimal drug-like properties suitable for good clearance and oral absorption. Moreover, the compounds show excellent ligand-lipophilicity efficiency values (LLE > 5) and we therefore suggest they should be considered for further drug development.

As next, the ability of the most promising compound NTZ-1441 to penetrate the BBB and its preventive effect on the spontaneous apoptosis of rat cortical neurons was assessed. For this purpose, this compound was incubated for 5 days in the presence (+BBB) or absence (–BBB) of primary human brain microvascular endothelial cells (HBMEC). Compound NTZ-1441 showed a significant effect (by 20%) on cortical neuron survival at a low concentration of 10 nM (Figure 2C). No abolition or decrease of the neuroprotective effect was observed in presence of BBB. As shown in Figure 2D, a significant effect of NTZ-1441 on the neurite outgrowth was still observed after 5 days incubation with HMBEC (+BBB). In contrast, BDNF a large growth factor that is known to not be able to cross the BBB, was inactive in the presence of endothelial cells. 17-b-Etsradiol, used as positive control for good BBB penetrance, was still active on neuron survival and neurite outgrowth in the presence of BBB.

 

 

Figure 2. (A) PAMPA GI (at pH 2.0, 5.5, and 7.4) and BBB mean permeability of standard drugs and new MAO inhibitors. Verapamil and theophylline were used as high and low permeability standard, respectively. Each data point represents the mean ± SD (n = 3). The initial test concentrations are indicated. (B) Square plot of potency (pIC50 at hMAO-B for safinamide and new inhibitors, and at hMAO-A for moclobemide) vs. logD7.4 values representing the excellent lipophilic ligand efficiency (LLE ≥ 5) for all selected indazole-5-carboxamides, which lie the target area of 0.5 ≤ logD7.4 ≤ 3.0 and pIC50 ≥ 8.0 (green square). (C) Effect of NTZ-1441 (10 nM) on rat cortical neurons survival after 5 days incubation in the presence (+BBB) or absence (–BBB) of primary human brain microvascular endothelial cells (HBMEC). (D) Effect of NTZ-1441 (10 nM) on neurite network after 5 days incubation in the presence (+BBB) or absence (–BBB) of HBMEC. Data are expressed as percentage of control from the mean ± SEM (100% = no treatment). Brain-derived neurotrophic factor (BDNF) and 17-b-estradiol were used as negative and positive control, respectively.

 

Binding modes of NTZ-1441 and 1442 to hMAO-B

To explain their exceedingly high MAO-B affinities and the observed regioisomeric-based differences in selectivity, docking experiments were performed using a well-validated NTZ®-modeling platform utilizing the availability of single X-ray structures of selected compounds. Furthermore, we used the novel free energy approximation concept “HYDE” for estimation, visualization, and quantification of effects of (de)hydration and hydrogen bonding. In particular, the binding modes of NTZ-1441 and 1442 within the binding pocket of the hMAO-B enzyme were investigated (Figure 3). The molecular modeling studies provided insights into the main interactions and structural requirements of enzyme-inhibitor binding and broadened our understanding of the compounds’ requirements for CNS activity.

 

 

Figure 3. SeeSAR visualization of binding, desolvation effects, and interactions for NTZ-1441 (A) and NTZ-1442 (B) to human MAO-B (PDB: 2V5Z). HYDE visual affinity assessment: green = favorable, red = unfavorable and non-colored = no relevant for affinity (SeeSAR v.5.6, BioSolveIT, 2017, http://www.biosolveit.de/SeeSaR).

 

Thus, indazole-5-carboxamide analogues with different substituents at the phenyl 3- and 4-positions and an N1- or N2-methylated indazole moiety were identified that may serve as promising drug candidates e.g., for the treatment of PD or AD. Moreover, they will be highly useful as pharmacological tools for in vitro and in vivo studies, and may be suitable for the development of radioligands, including diagnostics for positron emission tomography (PET). As an example, compound NTZ-1441 can be highlighted because of its remarkable in vitro MAO-B inhibitory activity and its well-balanced physicochemical profile, which is predictive of CNS bioavailability. In addition, NTZ-1441 exhibits in vitro neroprotective or even neurorestorative properties combined with excellent BBB permeability through human brain microvascular endothelial cells (HBMEC).

 

References

[1]   A.A. Belaid, A.I. Bush. Iron neurochemistry in Alzheimer´s disease and Parkinson´s disease: targets for therapeutics. J. Neurochem. 139(Suppl. I) (2016) 179–97.

[2]   N.T. Tzvetkov, S. Hinz, P. Küppers, M. Gastreich, C.E. Müller. Indazole- and indole-5-carboxamides: selective and reversible monoamine oxidase B inhibitors with subnanomolar potency. J. Med. Chem. 57 (2014) 6679–6703.

[3]   M.B.H. Youdim, L. Kupershmidt, T. Amit, O. Weinreb, Promises of novel multi-target neuroprotective and neurorestorative drugs for Parkinson’s disease, Parkinsonism Relat. Disord. 20S1 (2014) S132–S136.

[4]   N.T. Tzvetkov. Substituted indazole or indole derivatives as in vitro MAO-B inhibitors, 2014. WO2014/107771.

[5]   Binda C, Newton-Vinson P, Hubálek F, Edmondson DE, Mattevi A. Structure of human monoamine oxidase B, a drug target for the treatment of neurological disorders. Nat. Struct. Biol. 9(1): 22–26.(2002).

 

Contact

Dr. Nikolay T. Tzvetkov

Department of Drug Research & Development

NTZ Lab Ltd.

Krasno selo 198, 1618 Sofia, Bulgaria

E-mail: ntzvetkov@ntzlab.com