Synthesis of Isomeric 3-Benzazecines Decorated with Endocyclic Allene Moiety and Exocyclic Conjugated Double Bond and Evaluation of Their Anticholinesterase Activity

Transformations of 1-methoxymethylethynyl substituted isoquinolines triggered by terminal alkynes in alcohols were studied and new 3-benzazecine-containing compounds synthesized, such as 6-methoxymethyl-3-benzazecines incorporating an endocyclic C6–C8 allene fragment and the -ylidene derivatives 6-methoxymethylene-3-benzazecines. The reaction mechanisms were investigated and a preliminary in vitro screening of their potential inhibitory activities against human acetyl- and butyrylcholinesterases (AChE and BChE) and monoamine oxidases A and B (MAO-A and MAO-B) showed that the allene compounds were more potent than the corresponding -ylidene ones as selective AChE inhibitors. Among the allenes, 3e (R3 = CH2OMe) was found to be a competitive AChE inhibitor with a low micromolar inhibition constant value (Ki = 4.9 μM), equipotent with the corresponding 6-phenyl derivative 3n (R3 = Ph, Ki = 4.5 μM), but 90-fold more water-soluble.


Introduction
Medium-sized nitrogen-containing heterocycles, i.e., 8-, 9-, 10-, 11-, and 12-membered rings are quite widespread in nature, since a number of alkaloids possess these core cyclic structures [1][2][3][4]. However, the chemical behavior of these heterocycles remains unclear, due to the fact that there are not enough effective methods for their synthesis [5][6][7][8][9] and the available ones are often limited to single examples, complexity of realization, or low group compatibility in substrates. Developing methods with broader applicability to the synthesis of such medium-sized heterocycles should helpfully support drug discovery and structure-activity relationship (SAR) studies. It is well known that the biological properties of compounds with 10-membered rings depend upon the conformation of the cycle [10], which in turn is mainly related to cumulated and conjugated bonds in molecular frameworks ( Figure 1) [11,12] and by the presence of given pharmacophore features. The combination of these factors could open new opportunities for disclosing new medicinal hits targeted to biomolecules (e.g., enzymes, receptors), thus ultimately allowing the modification of the 3-benzazecine scaffold and possibly expanding their applicability in drug-discovery studies.
It should also be noted that heterocyclic nitrogen-containing allenes have not practically been studied. Moreover, while acyclic allenes are well known and successfully used in the syntheses of heterocycles, their cyclic analogues still require further detailed studies [13,14]. It should also be noted that heterocyclic nitrogen-containing allenes have not practically been studied. Moreover, while acyclic allenes are well known and successfully used in the syntheses of heterocycles, their cyclic analogues still require further detailed studies [13,14].
Previously, we have taken the first steps and succeeded in the construction of allenecontaining 3-benzazecines [15]-a new type of allene А (R 3 = Ph)-and later in our ongoing study observed some of their transformations [16,17]. It was shown that 8-alkyl(aralkyl)-substituted allene 3-benzazecines smoothly underwent transformation into 8-ylidene decorated derivatives in acetic acid ( Figure 1). The purposes of this study were to synthesize new 3-benzazecine derivatives and investigate their chemical properties, as well as to preliminarily evaluate their in vitro biological properties as potential inhibitors of enzymes, which are drug targets related to neurological degenerative syndromes (e.g., Alzheimer and Parkinson diseases), namely, acetyl-and butyrylcholinesterases (AChE and BChE) and monoamine oxidases A and B (MAO-A and MAO-B).
Previously, we have taken the first steps and succeeded in the construction of allenecontaining 3-benzazecines [15]-a new type of allene А (R 3 = Ph)-and later in our ongoing study observed some of their transformations [16,17]. It was shown that 8-alkyl(aralkyl)-substituted allene 3-benzazecines smoothly underwent transformation into 8-ylidene decorated derivatives in acetic acid ( Figure 1). The purposes of this study were to synthesize new 3-benzazecine derivatives and investigate their chemical properties, as well as to preliminarily evaluate their in vitro biological properties as potential inhibitors of enzymes, which are drug targets related to neurological degenerative syndromes (e.g., Alzheimer and Parkinson diseases), namely, acetyl-and butyrylcholinesterases (AChE and BChE) and monoamine oxidases A and B (MAO-A and MAO-B).
quinolines 2c-f with aryl substituent in the C-1 position under the same conditions did not proceed so clearly and led to the formation of mixtures of allene-containing benzazecines 3с-f and 6-methoxymethylenebenzazecines 4c-f in different ratios. The latter compounds were unexpected for us, as in previous work [16], we isolated only azecines with -ylidene fragment at C-8. We noticed that the prolongation in the reaction time led to the formation of the second product, compound 4, so we tried to carry out the reactions quickly and immediately isolate target allene 3.

Scheme 2.
Reactions of isoquinoline 2a-h with terminal activated alkynes in protic solvents. Acetylacetylene also smoothly reacted with isoquinolines 2d-h to provide allenes 3im in moderate to high yields (Scheme 2).
Previously, it was shown that 1-alkyl-1-phenylethynyltetrahydroisoquinolines under the action of methyl propiolate in hexafluoroisopropanol produced 8-ylidene-benzazecines [16], but in the case of 1-methoxymethylethynyl-substituted isoquinoline 2b, the same reaction conditions led to the formation of benzazecine 3b with an allene fragment   In trifluoroethanol at 25 • C, isoquinolines 2a, 2b, 2g with alkyl or benzyl substituents in the C-1 position reacted with methyl propiolate, readily forming benzazecines 3a, 3b, 3g with an allene fragment as main products in 80-91% yield. However, reactions of isoquinolines 2c-f with aryl substituent in the C-1 position under the same conditions did not proceed so clearly and led to the formation of mixtures of allene-containing benzazecines 3c-f and 6-methoxymethylenebenzazecines 4c-f in different ratios. The latter compounds were unexpected for us, as in previous work [16], we isolated only azecines with -ylidene fragment at C-8. We noticed that the prolongation in the reaction time led to the formation of the second product, compound 4, so we tried to carry out the reactions quickly and immediately isolate target allene 3.
Acetylacetylene also smoothly reacted with isoquinolines 2d-h to provide allenes 3i-m in moderate to high yields (Scheme 2).
Previously, it was shown that 1-alkyl-1-phenylethynyltetrahydroisoquinolines under the action of methyl propiolate in hexafluoroisopropanol produced 8-ylidene-benzazecines [16], but in the case of 1-methoxymethylethynyl-substituted isoquinoline 2b, the same reaction conditions led to the formation of benzazecine 3b with an allene fragment in 40% yield. The reactions of isoquinolines 2b and 2c with alkynes in less acidic isopropanol proceeded slowly (4-10 days, 20 • C), resulting only in allenes 3b and 3c (Scheme 2, Table 2). The formation of benzazecines with -ylidene moiety was not observed. The low yield of compound 3c can be explained by a prolonged exposure of the reaction mixture in a proton solvent and, as a consequence, its strong tarring.
Acetonitrile and dichloromethane appeared not to be effective solvents for the transformations. Isoquinoline 2 did not react with methyl propiolate in either acetonitrile or dichloromethane. Reflux and MW irradiation could not solve the problem-the reactions in these solvents did not even start.
Based on the obtained experimental data, we presume that the reaction proceeds through the formation of zwitterion I, which exists in equilibrium with zwitterion II (Scheme 3). The equilibrium position depends on the solvation ability of the solvent, substituents in the C-1 position of the isoquinoline, and delocalization of the anionic center.
in 40% yield. The reactions of isoquinolines 2b and 2c with alkynes in less acidic isopropanol proceeded slowly (4-10 days, 20 °C), resulting only in allenes 3b and 3с (Scheme 2, Table 2). The formation of benzazecines with -ylidene moiety was not observed. The low yield of compound 3c can be explained by a prolonged exposure of the reaction mixture in a proton solvent and, as a consequence, its strong tarring.
Acetonitrile and dichloromethane appeared not to be effective solvents for the transformations. Isoquinoline 2 did not react with methyl propiolate in either acetonitrile or dichloromethane. Reflux and MW irradiation could not solve the problem-the reactions in these solvents did not even start.
Based on the obtained experimental data, we presume that the reaction proceeds through the formation of zwitterion I, which exists in equilibrium with zwitterion II (Scheme 3). The equilibrium position depends on the solvation ability of the solvent, substituents in the C-1 position of the isoquinoline, and delocalization of the anionic center.

Scheme 3. A proposed mechanism of the transformations.
In the case of acetylacetylene, the anionic center has greater nucleophilicity in comparison with one formed by methyl propiolate, so the reaction proceeds immediately after the formation of the initial ion I, leading to benzazecines 3i-m.
In the case of methyl propiolate, delocalization of the anionic center promotes the formation of equilibrium and results in formation of a mixture of benzazecines 3c-f and 6-ylidene decorated compounds 4c-f (Scheme 3).
The following step of the research was to study the behavior of obtained allene 3а in acetic acid at 100 °C and microwave irradiation. It was of great interest to see whether the rearrangement in allene 3а proceeds via a previously described route [17] or again prefers to yield 6-methoxymethylene benzazecines. In the abovementioned conditions, allene 3а underwent rearrangement readily to give only 6-methoxymethylene benzazecine 4а in 25% yield (Scheme 4). The poor yield of the product can be explained by the use of more acidic protic solvent, such as AcOH, in which the intensive formation of tar products is observed. The short-term heating of reaction mixtures in an MW reactor does not improve the situation with the yields. We suggest that under the action of acetic acid, the allyl system is protonated, thus producing cation III, after stabilization of which 6-ylidene-substituted compound 4 is formed (Scheme 4).

Scheme 3. A proposed mechanism of the transformations.
In the case of acetylacetylene, the anionic center has greater nucleophilicity in comparison with one formed by methyl propiolate, so the reaction proceeds immediately after the formation of the initial ion I, leading to benzazecines 3i-m.
In the case of methyl propiolate, delocalization of the anionic center promotes the formation of equilibrium and results in formation of a mixture of benzazecines 3c-f and 6-ylidene decorated compounds 4c-f (Scheme 3).
The following step of the research was to study the behavior of obtained allene 3a in acetic acid at 100 • C and microwave irradiation. It was of great interest to see whether the rearrangement in allene 3a proceeds via a previously described route [17] or again prefers to yield 6-methoxymethylene benzazecines. In the abovementioned conditions, allene 3a underwent rearrangement readily to give only 6-methoxymethylene benzazecine 4a in 25% yield (Scheme 4). The poor yield of the product can be explained by the use of more acidic protic solvent, such as AcOH, in which the intensive formation of tar products is observed. The short-term heating of reaction mixtures in an MW reactor does not improve the situation with the yields. We suggest that under the action of acetic acid, the allyl system is protonated, thus producing cation III, after stabilization of which 6-ylidene-substituted compound 4 is formed (Scheme 4). In previous work [12], the 10,11-dimethoxy derivative of the allene 3-benzazecine 3n (scaffold A, R 3 = Ph), bearing at C-8 the 4-methoxyphenyl group, was found to be the most potent competitive AChE-selective inhibitor (Ki about 4.5 μM). Herein, a number of newly and previously synthesized 3-benzazecine analogs, including either allene (Figure 1, scaffold A) or 6-and 8-ylidene (B and C) derivatives, were firstly assayed as inhibitors of AChE, BChE, and MAOs at 10 μM concentration. For compounds that attained at least 50% inhibition at 10 μM, IC50s were determined from the best-fitting inhibition-concentration curves (five scalar concentrations in the 0.1-50 μM range). The inhibition data only In previous work [12], the 10,11-dimethoxy derivative of the allene 3-benzazecine 3n (scaffold A, R 3 = Ph), bearing at C-8 the 4-methoxyphenyl group, was found to be the most potent competitive AChE-selective inhibitor (K i about 4.5 µM). Herein, a number of newly and previously synthesized 3-benzazecine analogs, including either allene (Figure 1, scaffold A) or 6-and 8-ylidene (B and C) derivatives, were firstly assayed as inhibitors of AChE, BChE, and MAOs at 10 µM concentration. For compounds that attained at least 50% inhibition at 10 µM, IC 50 s were determined from the best-fitting inhibition-concentration curves (five scalar concentrations in the 0.1-50 µM range). The inhibition data only for the allene compounds, which achieved IC 50 toward AChE in the low µM range, are reported in Table 3. Previously reported activities of 3n and 3o are also shown for comparison. a Half-maximal inhibitory concentration or % inhibition at 10 µM in parentheses; values are mean ± SD of three independent measurements; n.i. = no inhibition. b Ref. [12].
The only noteworthy activity was the AChE inhibition, for which the allene derivatives proved to be more potent than the -ylidene ones. The CO 2 Me esters 3d and 3e worked slightly better than the corresponding COMe ketones 3i and 3j. Compound 3e bearing the polar methoxymethyl group at C-6 showed IC 50 just double that of the corresponding 6-Ph analogue 3n.
The Lineweaver-Burk plot of hAChE inhibition kinetics of the most active inhibitor 3e showed a competitive mechanism (Figure 2), with inhibition constant K i equal to 4.89 ± 0.47 µM, suggesting a preferential occupancy of the catalytic cavity of the enzyme by means of noncovalent interactions.    The enzymes' inhibition assays showed that for all the tested compounds, the inhibitory effects toward both MAO isoforms, and BChE as well, were weak to nil in the low micromolar range. Possible antioxidant activities were also explored with the DPPH radical scavenging assay, where all compounds were inactive.
Interestingly, the replacement of the phenyl group at C-6 of 3n with the more polar CH 2 OMe group in 3e, while retaining the same inhibition potency, did improve the water solubility by 90 times. The experimental data (Table 4) showed a solubility in PBS at pH 7.4 for 3e and 3n equal to 17.4 and 0.2 µM, respectively. The hydrolytic stability of 3e was quite good (half-life 4.5 h), though lower than the poorly soluble 3n (half-life > 12 h). Table 4. Acetylcholinesterase inhibition constants, aqueous solubility, hydrolytic stability, predicted pharmacokinetics properties, and PAINS alert of 3-benzazecine derivatives 3e and 3n.  [20]; c predicted apparent MDCK cell permeability (>2000) [20].
The in silico prediction of ADME-related properties for 3e and 3n using the Swis-sADME tool [21] showed high gastrointestinal (GI) absorption, good permeation of the blood-brain barrier (BBB), and poor ability for compounds as P-glycoprotein 1 (P-gp) substrates. Indeed, tested in a P-gp assay, several similar analogues and 3n itself proved to be potent inhibitors of P-gp in the nanomolar range. The two compounds were also predicted to inhibit cytochrome CYP3A4, a key liver enzyme responsible for oxidative detoxification of diverse xenobiotics, while no activity was suggested toward CYP2C19. Furthermore, the computational tool PAINS remover [22] did not alert for any PAINS (pan-assay interference compounds) for 3e or 3n.

Materials and General Procedures
IR spectra were recorded on an Infralum FT-801 FTIR spectrometer in KBr tablets for crystalline compounds or in a film for amorphous compounds (ISP SB RAS, Novosibirsk, Russia). Elemental analysis was carried out on a Euro Vector EA-3000 elemental Analyzer (Eurovector, S.p.A., Milan, Italy) for C, H and N; experimental data agreed to within 0.04% of the theoretical values. 1 H and 13 C NMR spectra were acquired on a 600 MHz NMR spectrometer (JEOL Ltd., Tokyo, Japan) in CDCl 3 for compounds with a solvent signal as internal standard (7.27 ppm for 1 H nuclei, 77.2 ppm for 13 C nuclei); peak positions were given in parts per million (ppm, δ). Mass spectra (LC-MS) of compounds were acquired on an Agilent 1100 LC/MSD VL system (electrospray ionization) (Agilent Technologies Inc., Santa Clara, CA, USA). Melting points were determined on an SMP-10 apparatus (Bibby Sterilin Ltd., Stone, UK) in open capillary tubes. Sorbfil PTH-AF-A-UF plates (Imid Ltd., Krasnodar, Russia) were used for TLC, visualization in an iodine chamber, or using KMnO 4 and H 2 SO 4 solutions. Silica gel (40-60 µm, 60 Å) Macherey-Nagel GmbH&Co (Loughborough, UK) was used for column chromatography. MW-assisted reactions were carried out in a Monowave 400 reactor from Anton Paar GmbH (Graz, Austria); the reaction temperature was monitored by an IR sensor; standard 10 mL G10 reaction vials, sealed with silicone septa, were used for the MW irradiation experiments. All reagents (Sigma-Aldrich, St. Louis, MO, USA; Merck, Darmstadt, Germany; J.T. Baker, Phillipsburg, NJ, USA), and fluorinated solvents (SIA P&M-Invest Ltd., Moscow, Russia) were used without additional purification.

Conclusions
The conversion of 1-methoxymethylethynyl-substituted isoquinolines under the action of terminal alkynes in various alcohols was studied. It was shown that under the same reaction conditions, the transformations of the allene fragment depends on the substituent at C6 position in 3-benzazecines. A decrease in the yield of 6-methoxymethyl decorated allenes was observed in long-term and/or high-temperature reactions in protic solvents. A protocol for the synthesis of new 6-methoxymethyl substituted 3-benzazecines with an allene fragment and 6-methoxymethylene-3-benzazecines was developed.
A preliminary in vitro evaluation of the inhibition activity against the main target enzymes related to neurodegeneration revealed that the allene 3-benzazecine derivative 3e, bearing the 6-methoxymethyl polar group, competitively inhibits AChE with a singledigit micromolar K i . Compound 3e resulted in an inhibitor equipotent with the 6-phenyl analogue 3n, but 90-fold more soluble in buffered aqueous solution at pH 7.4. This higher water-solubility property, joined with the potential of the core structure to inhibit P-gp efflux pumps and consequently to favor brain disposition [20], makes us confident that 3e can be a candidate for further optimization of novel brain-permeant AChE inhibitors.