Synthesis of Novel Benzo[b][1,6]naphthyridine Derivatives and Investigation of Their Potential as Scaffolds of MAO Inhibitors

In this work, 2-alkyl-10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridines were obtained and their reactivity was studied. Novel derivatives of the tricyclic scaffold, including 1-phenylethynyl (5), 1-indol-3-yl (8), and azocino[4,5-b]quinoline (10) derivatives, were synthesized and characterized herein for the first time. Among the newly synthesized derivatives, 5c–h proved to be MAO B inhibitors with potency in the low micromolar range. In particular, the 1-(2-(4-fluorophenyl)ethynyl) analog 5g achieved an IC50 of 1.35 μM, a value close to that of the well-known MAO B inhibitor pargyline.


Chemistry
The synthesis of benzonaphthyridines is mainly based on ring-closing methods, which are used in the synthesis of quinolines. The use of Frindler [22], Pfitzinger [23] and Niementowski [24] reactions, as well as synthesis based on 2-ethynylquinolyl-3-carbaldehydes [25] and aminopyridines [26], makes it possible to obtain derivatives containing functional groups in various positions of the tricyclic system.
Previously, we described conversion of tetrahydrobenzo[b] [1,6]naphthyridines, which were obtained by the Pfitzinger reaction, to various derivatives under the action of activated alkynes. The structure of the products directly depended on the nature of the substituent in position 10 and Stevens rearrangement products, ylides, 2-vinylquinolines, and benzopyridonaphthyridines can be formed [27][28][29]. However, the mentioned works were mainly focused on substances with acceptor substituents at position 10 because of their easy synthesis by the Pfitzinger reaction. Thus, it was of interest to perform experiments using substances with electron-donating substituents such as chlorine. N-methyland benzyl-10-chloro-1,2,3,4-tetrahydrobenzo[b] [1,6]naphthyridines 3a-d were synthesized by the Niementowski reaction based on condensation of anthranilic acids 1a-c with the appropriate piperidones 2a,b when heated in a phosphorus oxychloride atmosphere. After alkaline treatment of the reaction mass, compounds 3a-d were obtained in the form of yellow crystals with yields of 68-92% (Scheme 1).  [1,6]naphthyridine.
The biological study combining cheminformatics and biochemical testing focused on AD-related targets. In our ongoing search for biological activities from unconventional chemical scaffolds [18,19], we considered the newly synthesized 1,6-benzonaphthyridine derivatives as worthy of biological investigation. It must be highlighted than piperidinefused naphthyridine derivatives were previously described by others as dual inhibitors of monoamine oxidase subtypes A and B (MAOs A and B) and of acetyl-and butyrylcholinesterase (AChE, BChE) [20]. This feature is also suggested by the shape similarity of this tricyclic scaffold with that of β-carboline, found in the alkaloid harmine and other compounds displaying inhibitory activity toward MAO subtypes [21]. In this study, relying on chemoinformatic predictions, we prioritized the evaluation of inhibitory activity against MAOs A and B.

Chemistry
The synthesis of benzonaphthyridines is mainly based on ring-closing methods, which are used in the synthesis of quinolines. The use of Frindler [22], Pfitzinger [23] and Niementowski [24] reactions, as well as synthesis based on 2-ethynylquinolyl-3-carbaldehydes [25] and aminopyridines [26], makes it possible to obtain derivatives containing functional groups in various positions of the tricyclic system.
The study of the reactivity of 10-chloro-tetrahydrobenzo[b] [1,6]naphthyridines showed that 2-methyl-10-chloro-1,2,3,4-tetrahydrobenzo[b] [1,6]naphthyridine 3c was completely inactive in reactions with activated alkynes. Extended boiling and microwave activation in various solvents did not lead to the formation of products, whereas only the initial compound was released back from the reaction mass. However, the presence of an acceptor substituent at C-8 in compound 3d made it possible to obtain product 4c. The reaction of  [1,6]naphthyridines 3a,b with methyl propiolate proceeded at room temperature in methanol under acid catalysis conditions. The N-vinyl derivatives 4a,b were obtained as a result of debenzylation (Scheme 2). We previously described such transformations for N-benzyl-chromenopyridines [30].
The study of the reactivity of 10-chloro-tetrahydrobenzo[b] [1,6]naphthyridines showed that 2-methyl-10-chloro-1,2,3,4-tetrahydrobenzo[b] [1,6]naphthyridine 3c was completely inactive in reactions with activated alkynes. Extended boiling and microwave activation in various solvents did not lead to the formation of products, whereas only the initial compound was released back from the reaction mass. However, the presence of an acceptor substituent at C-8 in compound 3d made it possible to obtain product 4c. The reaction of 2-benzyl-10-chloro-1,2,3,4-tetrahydrobenzo[b] [1,6]naphthyridines 3a,b with methyl propiolate proceeded at room temperature in methanol under acid catalysis conditions. The N-vinyl derivatives 4a,b were obtained as a result of debenzylation (Scheme 2). We previously described such transformations for N-benzyl-chromenopyridines [30]. We supposed that the functionalization of this system by introducing indole or phenylethynyl fragments onto the tetrahydropyridine ring would significantly expand the potential of tetrahydrobenzo[b] [1,6]naphthyridines as biologically active compounds and reveal new ways of further achieving chemical modifications. The introduction of a substituent to the nearest position of the nitrogen atom in the tetrahydropyridine fragment was performed by imine salt formation. Such reactions are well described for tetrahydroisoquinolines [31][32][33][34], but they never have been used in case of tetrahydrobenzonaphthyridines. 1-Phenylethynylated benzonaphthyridines 5a-h were obtained as result of the cross-combination of compounds 3 with phenylacetylene in the presence of CuI and diisopropyldiazodicarboxylate (DIAD) (Scheme 3 and Table 1).  We supposed that the functionalization of this system by introducing indole or phenylethynyl fragments onto the tetrahydropyridine ring would significantly expand the potential of tetrahydrobenzo[b] [1,6]naphthyridines as biologically active compounds and reveal new ways of further achieving chemical modifications. The introduction of a substituent to the nearest position of the nitrogen atom in the tetrahydropyridine fragment was performed by imine salt formation. Such reactions are well described for tetrahydroisoquinolines [31][32][33][34], but they never have been used in case of tetrahydrobenzonaphthyridines. 1-Phenylethynylated benzonaphthyridines 5a-h were obtained as result of the cross-combination of compounds 3 with phenylacetylene in the presence of CuI and diisopropyldiazodicarboxylate (DIAD) (Scheme 3 and Table 1).
The study of the reactivity of 10-chloro-tetrahydrobenzo[b] [1,6]naphthyridines showed that 2-methyl-10-chloro-1,2,3,4-tetrahydrobenzo[b] [1,6]naphthyridine 3c was completely inactive in reactions with activated alkynes. Extended boiling and microwave activation in various solvents did not lead to the formation of products, whereas only the initial compound was released back from the reaction mass. However, the presence of an acceptor substituent at C-8 in compound 3d made it possible to obtain product 4c. The reaction of 2-benzyl-10-chloro-1,2,3,4-tetrahydrobenzo[b] [1,6]naphthyridines 3a,b with methyl propiolate proceeded at room temperature in methanol under acid catalysis conditions. The N-vinyl derivatives 4a,b were obtained as a result of debenzylation (Scheme 2). We previously described such transformations for N-benzyl-chromenopyridines [30]. We supposed that the functionalization of this system by introducing indole or phenylethynyl fragments onto the tetrahydropyridine ring would significantly expand the potential of tetrahydrobenzo[b] [1,6]naphthyridines as biologically active compounds and reveal new ways of further achieving chemical modifications. The introduction of a substituent to the nearest position of the nitrogen atom in the tetrahydropyridine fragment was performed by imine salt formation. Such reactions are well described for tetrahydroisoquinolines [31][32][33][34], but they never have been used in case of tetrahydrobenzonaphthyridines. 1-Phenylethynylated benzonaphthyridines 5a-h were obtained as result of the cross-combination of compounds 3 with phenylacetylene in the presence of CuI and diisopropyldiazodicarboxylate (DIAD) (Scheme 3 and Table 1). Scheme 3. Phenylethynylation of compounds 3a,c. The nucleophilic addition of benzonaphthyridine tertiary nitrogen to DIAD led to the formation of a zwitterion, which then turned into an iminium salt. The target products 5 were obtained after further alkynylation of the salt with copper acetylenide. The isolation of the reaction products was hampered by the presence of substituted hydrazine in the reaction mixture. This compound was obtained from DIAD and crystalized simultaneously with the target compounds, so it was necessary to use column chromatography. The structure of compound 5a was determined by single crystal X-ray analysis (CCDC 2224256, Figure 2).
The nucleophilic addition of benzonaphthyridine tertiary nitrogen to DIAD led to the formation of a zwitterion, which then turned into an iminium salt. The target products 5 were obtained after further alkynylation of the salt with copper acetylenide. The isolation of the reaction products was hampered by the presence of substituted hydrazine in the reaction mixture. This compound was obtained from DIAD and crystalized simultaneously with the target compounds, so it was necessary to use column chromatography.
The structure of compound 5a was determined by single crystal X-ray analysis (CCDC 2224256, Figure 2). The phenylethynyl derivatives of benzo[b] [1,6]naphthyridines 5 turned out to be much more reactive towards activated alkynes. The reaction with methyl propiolate in trifluoroethanol and acetonitrile took place at room temperature after 10 min with the formation of complex separable mixtures. After selecting the reaction conditions, we obtained satisfactory results using subzero temperatures and isopropanol as a solvent. As a result of the interaction of compounds 5d,g with methyl propiolate under these conditions, two products were formed: the Stevens rearrangement products 6a,b and 2-vinylquinoline 7. The reaction of compounds 5g and 5d also yielded quinoline 7. Under the same conditions, the interaction of compounds 5c,d,f,g with acetylacetylene led to the formation of products 6c-f, the only products with good yields (Scheme 4 and    The phenylethynyl derivatives of benzo[b] [1,6]naphthyridines 5 turned out to be much more reactive towards activated alkynes. The reaction with methyl propiolate in trifluoroethanol and acetonitrile took place at room temperature after 10 min with the formation of complex separable mixtures. After selecting the reaction conditions, we obtained satisfactory results using subzero temperatures and isopropanol as a solvent. As a result of the interaction of compounds 5d,g with methyl propiolate under these conditions, two products were formed: the Stevens rearrangement products 6a,b and 2-vinylquinoline 7. The reaction of compounds 5g and 5d also yielded quinoline 7. Under the same conditions, the interaction of compounds 5c,d,f,g with acetylacetylene led to the formation of products 6c-f, the only products with good yields (Scheme 4 and Table 2).
formation of a zwitterion, which then turned into an iminium salt. The target products 5 were obtained after further alkynylation of the salt with copper acetylenide. The isolation of the reaction products was hampered by the presence of substituted hydrazine in the reaction mixture. This compound was obtained from DIAD and crystalized simultaneously with the target compounds, so it was necessary to use column chromatography.
The structure of compound 5a was determined by single crystal X-ray analysis (CCDC 2224256, Figure 2). The phenylethynyl derivatives of benzo[b] [1,6]naphthyridines 5 turned out to be much more reactive towards activated alkynes. The reaction with methyl propiolate in trifluoroethanol and acetonitrile took place at room temperature after 10 min with the formation of complex separable mixtures. After selecting the reaction conditions, we obtained satisfactory results using subzero temperatures and isopropanol as a solvent. As a result of the interaction of compounds 5d,g with methyl propiolate under these conditions, two products were formed: the Stevens rearrangement products 6a,b and 2-vinylquinoline 7. The reaction of compounds 5g and 5d also yielded quinoline 7. Under the same conditions, the interaction of compounds 5c,d,f,g with acetylacetylene led to the formation of products 6c-f, the only products with good yields (Scheme 4 and    Similarly, the Stevens rearrangement occurred in the case of 1-phenylethynyl-substitutedβ-carbolines reacted with activated alkynes [35]. The formation of products 6 and 7 started with the Michael addition of nitrogen of the tetrahydropyridine fragment to the activated alkyne leading to the formation of the zwitterion A. Then, either a Stevens rearrangement (route a) took place with the formation of compound 6, or further attack on the triple bond of the phenylethynyl fragment yielded adduct C (route b) and then proton migration and Hoffmann elimination completed this cascade of transformations to give minor product 7 (Scheme 5).
The formation of products 6 and 7 started with the Michael addition of nitrogen of the tetrahydropyridine fragment to the activated alkyne leading to the formation of the zwitterion A. Then, either a Stevens rearrangement (route a) took place with the formation of compound 6, or further attack on the triple bond of the phenylethynyl fragment yielded adduct C (route b) and then proton migration and Hoffmann elimination completed this cascade of transformations to give minor product 7 (Scheme 5).

Scheme 5.
Mechanism of interaction of 5c,d,f,g with activated alkynes.
Besides phenylethynylation, we introduced an indole fragment at position C1 of benzonaphthyridines 3. At the first stage, iminium salts were obtained by interaction of benzonaphthyridines 3 with DIAD, and these salts reacted with substituted indoles at the second stage. The reaction was carried out in absolute THF and benzonaphthyridines 8 were isolated by column chromatography. The isolation of the products was hampered by the presence of substituted hydrazine as in the case of phenethynylation. Thus, only Besides phenylethynylation, we introduced an indole fragment at position C1 of benzonaphthyridines 3. At the first stage, iminium salts were obtained by interaction of benzonaphthyridines 3 with DIAD, and these salts reacted with substituted indoles at the second stage. The reaction was carried out in absolute THF and benzonaphthyridines 8 were isolated by column chromatography. The isolation of the products was hampered by the presence of substituted hydrazine as in the case of phenethynylation. Thus, only compound 8a was isolated in its individual form, whereas compounds 8b-d were isolated in mixture with hydrazine 9 (Scheme 6).  Here, we describe the first example of the formation of tetrahydroazocino[4,5-b]quinolines; however, such transformations have been studied for other heterocyclic systems annulated with the tetrahydropyridine fragment and the mechanism of azocine fragment formation has been presented [36][37][38][39][40][41].

Evaluation of Monoamine Oxidase (MAO) Inhibition
Taking advantage of PLATO, a free online platform for structure-based target prediction [42] that relies on a multi-fingerprint similarity search algorithm [43,44], we submitted derivatives 3a-d, 5a-h, 8a-b, and 10a-c for bioactivity prediction. Interestingly, MAOs were found among the targets predicted with higher probability along with binding affinities for dopamine and opioid receptor subtypes only for the N(2)-methyl analogues 5c-h bearing phenylethynyl groups at C1. In contrast, N(2)-benzyl analogs 5a-b and compounds 3, 8, and 10 were unpredicted as MAO ligands.
Compounds 5c-h were then tested on human (h) recombinant MAO A and B using previously reported assays [16,17]. The MAO-B-selective inhibitor, pargyline, was used as the positive control. Each compound was first tested at a concentration of 10 μM and then lower scalar concentrations were tested when >60% inhibition was achieved at 10 μM. The IC50 values were calculated from the best-fitting inhibition-concentration curves. The MAO A and B inhibition data are summarized in Table 3. Here, we describe the first example of the formation of tetrahydroazocino[4,5-b]quinolines; however, such transformations have been studied for other heterocyclic systems annulated with the tetrahydropyridine fragment and the mechanism of azocine fragment formation has been presented [36][37][38][39][40][41].

Evaluation of Monoamine Oxidase (MAO) Inhibition
Taking advantage of PLATO, a free online platform for structure-based target prediction [42] that relies on a multi-fingerprint similarity search algorithm [43,44], we submitted derivatives 3a-d, 5a-h, 8a-b, and 10a-c for bioactivity prediction. Interestingly, MAOs were found among the targets predicted with higher probability along with binding affinities for dopamine and opioid receptor subtypes only for the N(2)-methyl analogues 5c-h bearing phenylethynyl groups at C1. In contrast, N(2)-benzyl analogs 5a-b and compounds 3, 8, and 10 were unpredicted as MAO ligands.
Compounds 5c-h were then tested on human (h) recombinant MAO A and B using previously reported assays [16,17]. The MAO-B-selective inhibitor, pargyline, was used as the positive control. Each compound was first tested at a concentration of 10 µM and then lower scalar concentrations were tested when >60% inhibition was achieved at 10 µM. The IC 50 values were calculated from the best-fitting inhibition-concentration curves. The MAO A and B inhibition data are summarized in Table 3. All of the tested compounds showed a certain selectivity toward MAO B, with most of them achieving IC 50 values in the low micromolar range. The 4-F derivative 5g showed a noteworthy IC 50 (1.35 µM), a value that is even lower than that of the reference pargyline. The absence of chemical groups able to create covalent bonds, such as the propargylamine fragment in pargyline, suggested a tight, yet reversible, interaction at the binding site of the enzyme for 5g. Compounds 5c-h were also assayed as inhibitors of human cholinesterases [45], but they proved to be inactive as AChE inhibitors and scarcely active toward BChE at 10 µM. Meanwhile, the 4-Cl derivative 5h displayed less than 44% antiaggregating activity against amyloidogenic Aβ  peptide at 100 µM.

Chemistry
Materials and general procedures. All reagents and solvents were purchased from Merck (Darmstadt, Germany), J.T. Baker (Phillipsburg, NJ, USA), or Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and, unless specified, used without further purification. The melting points (m.p.) of all of the compounds were determined using a SMELTING POINT 10 apparatus in open capillaries (Bibby Sterilin Ltd., Stone, UK). IR spectra were recorded using an Infralum FT-801 FTIR spectrometer (ISP SB RAS, Novosibirsk, Russia). The samples were analyzed as KBr disk solids and the more important frequencies are shown in cm −1 . 1 H and 13 C NMR spectra were recorded in chloroform-d 3 (CDCl 3 ) or dimethylsulfoxide-d 6 (DMSO-d 6 ) solutions at 25 • C with a 600-MHz NMR spectrometer (JEOL Ltd., Tokyo, Japan). Peak positions were given in parts per million (ppm), referenced to the appropriate solvent residual peak, and signal multiplicities were collected by: s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), t (triplet), q (quartet), br.s (broad singlet), and m (multiplet). MALDI mass spectra were recorded using a Bruker autoflex speed instrument operating in positive-ion reflectron mode (Bremen, Germany). The data of compound 5a were collected at room temperature using an STOE diffractometer Pilatus100K detector, focusing on mirror collimation Cu Kα (1.54086 Å) radiation, in rotation method mode. STOE X-AREA software was used for cell refinement and data reduction. Data collection and image processing were performed with X-Area 1.67 (STOE & Cie GmbH, Darmstadt, Germany, 2013). Intensity data were scaled with LANA (part of X-Area) in order to minimize the differences in intensities of symmetry-equivalent reflections (multiscan method).

Synthesis of 2-Alkyl-10-chloro-1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridines 3a-d
Phosphorus chloride was added dropwise in a volume of 10 mL to anthranilic acids 1a-c (0.0146 mol), which was then added in 1 equivalent excess of (0.0146 mol) 1-alkylpiperidine-4-one 2a,b. Next, the reaction was stirred for 4 h at 100 • C and controlled by TLC in an ethyl acetate-hexane (1:1) system on Silufol plates. After cooling, the resulting solution was neutralized with dilute NaOH solution to pH = 9-10, and the product was extracted with CH 2 Cl 2 . The organic phase was dried over anhydrous sodium sulfate and concentrated on a rotary evaporator. The product was obtained by crystallization from diethyl ether.  To a solution of 0.2 g of benzonaphthyridines 3a,b in 5 mL of methanol with 0.5 mL of formic acid was added a 1.2 equivalent of activated alkyne. The reaction was kept at room temperature for 10 days. Compounds 4a,b spontaneously fell out of the reaction mass in the form of crystals and were released by filtration.
To a solution of 0.2 g of benzonaphthyridines 3d in 5 mL of trifluoroethanol with 0.5 mL was added a 1.2 equivalent of activated alkyne. The reaction was kept at room temperature for 15 days. The product was obtained by crystallization from diethyl ether.  [1,6] naphthyridines 5a-h A solution of 3a,c (0.5 g) in 10 mL of THF was cooled to 0 • C, then a 1.5 equivalent excess of DIAD (diisopropylazodicarboxylate) was added. The mixture was stirred at room temperature for 1 h. After cooling it again to 0 • C, a 3 equivalent excess of the appropriate phenylacetylene and CuI catalyst were added. The reaction was stirred at room temperature and controlled by TLC in an ethyl acetate-hexane (1:5) system on Silufol plates. The product was separated by column chromatography.

Biochemical Assays MAO Inhibition
All reagents were purchased from Sigma Aldrich (Milan, Italy). The fluorometric assay was performed as previously described [17] using human recombinant enzymes from baculovirus-infected insect cells, following the formation of fluorescing 4-hydroxyquinoline from the MAO substrate, kynuramine. Assays were performed in triplicate in 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) using an Infinite M1000 multiplate reader (Tecan, Cernusco sul Naviglio (MI), Italy). Results were expressed as mean ± SEM. IC 50 values were obtained by nonlinear regression using Prism software (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA).

Conclusions
As a major outcome of this study, novel functionalized 2-alkyl-10-chloro-1,2,3,4tetrahydrobenzo[b] [1,6]naphthyridines 3 were synthesized for the first time, specifically 1-phenylethynyl derivatives 5 and 1-indol-3-yl derivatives 8. Moreover, the interaction of these compounds with activated alkynes was studied, revealing that the substituent in the first position played a key role in these reactions and either Stevens rearrangement products or azocino [4,5-b]quinolines were formed.
The 1-phenylethynyl derivatives 5c-h were discovered as MAO inhibitors, showing selectivity toward the human MAO B isoform and potency in the low micromolar range. In particular, the 4-F derivative 5g achieved an IC 50 of 1.35 µM in vitro, which was almost equipotent with pargyline (IC 50 2.69), a known MAO B irreversible inhibitor that was taken as the positive control. MAO B inhibitors are typically used in the treatment of early symptoms of PD [47], while their efficacy in decreasing oxidative stress may provide neuroprotective effects in the treatment of AD [48]. In this context, compound 5g deserves further optimization studies for improving its pharmacological potential as an effective agent for the treatment of neurodegenerative syndromes.