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

Abstract

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 (K_i = 4.9 μM), equipotent with the corresponding 6-phenyl derivative 3n (R³ = Ph, K_i = 4.5 μM), but 90-fold more water-soluble.

1. 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].

Previously, we have taken the first steps and succeeded in the construction of allene-containing 3-benzazecines [15]—a new type of allene A (R³ = 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).

2. Results and Discussion

Starting 1-methyl (isopropyl-, benzyl-, phenyl-, tolyl-, p-methoxyphenyl- and p-fluorophenyl)-1-methoxymethylethynyl-1,2,3,4-tetrahydroisoquinolines 2a–h were obtained from 3,4-dihydroisoquinolinium methyl iodide 1 derived via the Bischler–Napieralski reaction [18], followed by alkylation and subsequent methoxymethyl ethynylation in the presence of cuprous bromide in methylene chloride (Scheme 1,

Table 1. Synthesis of 1,2,3,4-tetrahydroisoquinolines 2a–h.

Entry	Isoquinoline	R1	R2	% Yield
1	2a	OMe	Me	50
2	2b	OMe	Bn	80
3	2c	OMe	Ph	72
4	2d	OMe	C6H4-Me-p	77
5	2e	OMe	C6H4-OMe-p	90
6	2f	OMe	C6H4-F-p	83
7	2g	H	i-Pr	40
8	2h	H	Ph	48

) [19].

We continued our study with estimating behavior of isoquinolines 2a–h in reactions with terminal activated alkynes (methyl propiolate and acetylacetylene) in different solvents-trifluoroethanol, hexafluoroisopropanol, isopropanol, acetonitrile, or dichloromethane (Scheme 2,

Table 2. Conditions and products of reactions of isoquinoline 2a–h with activated alkynes.

Entry	Cmpd	R1	R2	X	Solvents	Conditions	Allene 3 (yield, %) a	Benzazecine 4 (yield, %) a
1	2a	OMe	Me	CO2Me	CF3CH2OH	25 °C, 1 day	3a, 80%	4a, -
2	2b	OMe	Bn	CO2Me	CF3CH2OH	25 °C, 1 day	3b, 91%	4b, -
3	2b	OMe	Bn	CO2Me	(CF3)2CHOH	20 °C, 3 days	3b, 40% b	-
4	2b	OMe	Bn	CO2Me	i-PrOH	20 °C, 4 days	3b, 70%	-
5	2c	OMe	Ph	CO2Me	CF3CH2OH	25 °C, 1 day	3c, 30%	4c, 32%
6	2c	OMe	Ph	CO2Me	i-PrOH	20 °C, 10 days	3c, 25% b	-
7	2d	OMe	4-MePh	CO2Me	CF3CH2OH	25 °C, 1 day	3d, 47%	4d, 35%
8	2d	OMe	4-MePh	Ac	CF3CH2OH	7 °C, 4 days	3i, 50%	-
9	2e	OMe	4-MeOPh	CO2Me	CF3CH2OH	25 °C, 1 day	3e, 63%	4e, 28%
10	2e	OMe	4-MeOPh	Ac	CF3CH2OH	7 °C, 2 days	3j, 73%	-
11	2f	OMe	4-FPh	CO2Me	CF3CH2OH	25 °C, 1 day	3f, 24%	4f, 47%
12	2f	OMe	4-FPh	Ac	CF3CH2OH	7 °C, 6 h	3k, 76%	-
13	2g	H	i-Pr	CO2Me	CF3CH2OH	25 °C, 1 day	3g, 87%	4g, -
14	2g	H	i-Pr	Ac	CF3CH2OH	7 °C, 2 days	3l, 44%	-
15	2h	H	Ph	CO2Me	CF3CH2OH	25 °C, 1 day	3h, 64%	4h, -
16	2h	H	Ph	Ac	CF3CH2OH	7 °C, 3 days	3m, 50%	-

^a Products 3 and 4 were isolated via chromatography. ^b A strong tarring was observed.

).

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 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, R³ = 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, IC₅₀s 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 IC₅₀ toward AChE in the low μM range, are reported in

Table 3. Inhibition potency data on human acetyl- and butyrylcholinesterases (AChE and BChE) and monoamine oxidases A and B (MAO A and B) of 10,11-dimethoxy derivatives of allenyl 3-benzazecines (scaffold A, R¹ = OMe).

Entry	Cmpd	R2	R3	X	Enzymes’ Inhibition Data a
					hAChE	hBChE	hMAO-A	hMAO-B
1	3d	4-MePh	CH2OMe	CO2Me	19.3 ± 3.3	n.i.	(30 ± 4)	(30 ± 5)
2	3e	4-OMePh	CH2OMe	CO2Me	12.2 ± 2.6	n.i.	(37 ± 5)	(23 ± 1)
3	3i	4-MePh	CH2OMe	Ac	32.5 ± 4.4	n.i.	(28 ± 1)	(38 ± 4)
4	3j	4-OMePh	CH2OMe	Ac	13.2 ± 0.7	n.i.	(29 ± 5)	(28 ± 5)
5	3n b	4-OMePh	Ph	CO2Me	5.05 ± 0.21	n.i.	(20 ± 5)	(24 ± 5)
6	3o b	4-OMePh	Ph	Ac	(23 ± 4)	n.i.	(34 ± 2)	(16 ± 2)

^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].

. Previously reported activities of 3n and 3o are also shown for comparison.

The only noteworthy activity was the AChE inhibition, for which the allene derivatives proved to be more potent than the -ylidene ones. The CO₂Me 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 IC₅₀ 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 CH₂OMe group in 3e, while retaining the same inhibition potency, did improve the water solubility by 90 times. The experimental data (

Table 4. Acetylcholinesterase inhibition constants, aqueous solubility, hydrolytic stability, predicted pharmacokinetics properties, and PAINS alert of 3-benzazecine derivatives 3e and 3n.

Cmpd		3e	3n
AChE inhibition, Ki μM		4.89 ± 0.47	4.45 ± 0.08
Solubility a, μM		17.4 ± 0.7	0.200 ± 0.015
Hydrolytic half-life a, h		4.5	>12
ADME-related properties	GI absorption b	High	High
	BBB permeant c	Yes	Yes
	P-gp substrate	No	No
	CYP2C19 inhib.	No	No
	CYP3A4 inhib.	Yes	Yes
PAINS		No alert	No alert

^a PBS pH 7.4, 0.15 M KCl, 37 °C. Each experiment was performed in triplicate; data expressed as mean ± SD; ^b predicted apparent Caco-2 cell permeability (>4000) [20]; ^c predicted apparent MDCK cell permeability (>2000) [20].

) 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).

The in silico prediction of ADME-related properties for 3e and 3n using the SwissADME 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.

3. Materials and Methods

3.1. Chemistry

3.1.1. 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. ¹H and ¹³C NMR spectra were acquired on a 600 MHz NMR spectrometer (JEOL Ltd., Tokyo, Japan) in CDCl₃ for compounds with a solvent signal as internal standard (7.27 ppm for ¹H nuclei, 77.2 ppm for ¹³C 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 KMnO₄ and H₂SO₄ 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.

3.1.2. Synthesis of Benzazecines 3 and 4

To compounds 2a–h (1.7 mmol) was added 5 mL of 2,2,2-trifluoroethanol (hexafluoroisopropanol, isopropanol), then methyl propiolate or acetylacetylene (2.21 mmol) was added. In the case of methyl propiolate, the reaction proceeded at 25 °C and for acetylacetylene at 7 °C (Table 2). The reaction was carried out under argon atmosphere. The progress of the reaction was monitored by TLC (Sorbfil, 3:2 EtOAc–hexane). The solvent was removed under vacuum and residue was chromatographed on silica gel (1:5 EtOAc–hexane). Compounds 3a–m and 4d, 4f were crystallized from Et₂O.

Methyl 3,8-dimethyl-10,11-dimethoxy-6-(methoxymethyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3a): 0.507 g (80%); beige solid; mp 165–167 °C; R_f 0.60 (3:1, EtOAc–hexane); IR (KBr) ν 1961 (C=C=C), 1690 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.41 (1H, s, H-4), 6.82 (1H, s, H Ar), 6.62 (1H, s, H Ar), 4.36–4.32 (1H, m, 2-CH₂), 4.01 (1H, d, J = 11.6 Hz, CH₂OCH₃), 3.93 (1H, d, J = 11.6 Hz, CH₂OCH₃), 3.88 (3H, s, OCH₃), 3.86 (3H, s, OCH₃), 3.71 (3H, s, OCH₃), 3.38–3.34 (1H, m, 2-CH₂), 3.25 (3H, s, OCH₃), 3.13 (3H, s, N-CH₃), 2.89–2.83 (1H, m, 1-CH₂), 2.75–2.69 (1H, m, 1-CH₂), 2.10 (3H, s, CH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 205.5, 170.0, 147.8, 147.6, 147.5, 131.1, 128.1, 113.1, 110.4, 97.7, 96.8, 94.2, 74.7, 58.7, 56.1, 56.0, 51.6, 51.2, 45.3, 31.4, 19.3; LCMS (ESI) m/z 374 [M + H]⁺; anal. C 67.61, H 7.19, N 3.81%, calcd for C₂₁H₂₇NO₅, C 67.54, H 7.29, N 3.75%.

Methyl 8-benzyl-3-methyl-10,11-dimethoxy-6-(methoxymethyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3b): 0.694 g (91% from CF₃CH₂OH); white solid; mp 168–170 °C; R_f 0.55 (3:2, EtOAc–hexane); IR (KBr) ν 1955 (C=C=C), 1675 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.41 (1H, s, H-4), 7.25–7.23 (4H, m, H Ph), 7.16 (1H, t, J = 7.1 Hz, H Ph), 6.81 (1H, s, H Ar), 6.58 (1H, s, H Ar), 4.39–3.35 (1H, m, 2-CH₂), 4.00 (1H, d, J = 11.9 Hz, CH₂OCH₃), 3.98 (1H, d, J = 11.9 Hz, CH₂OCH₃), 3.84 (3H, s, OCH₃), 3.78 (3H, s, OCH₃), 3.76 (2H, s, CH₂-Ph), 3.64 (3H, s, OCH₃), 3.35–3.31 (1H, m, 2-CH₂), 3.24 (3H, s, OCH₃), 3.13 (3H, s, N-CH₃), 2.88–2.82 (1H, m, 1-CH₂), 2.70–2.64 (1H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 206.4, 170.1, 147.7, 147.5, 139.6, 129.8, 129.0 (3C), 128.6, 128.3 (2C), 126.1, 112.9, 110.7, 101.8, 97.9, 94.1, 74.7, 58.8, 55.9 (2C), 51.6, 51.2, 45.3, 39.8, 31.3; LCMS (ESI) m/z 450 [M + H]⁺; anal. C 72.28, H 6.87, N 3.16%, calcd for C₂₇H₃₁NO₅, C 72.14, H 6.95, N 3.12%.

Methyl 3-methyl-8-phenyl-10,11-dimethoxy-6-(methoxymethyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3c): 0.222 g (30% from CF₃CH₂OH); light yellow oil; R_f 0.53 (2:1, EtOAc–hexane); IR (KBr) ν 1943 (C=C=C), 1683 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.42 (1H, s, H-4), 7.37 (2H, d, J = 8.1 Hz, H Ph), 7.31 (2H, t, J = 7.6 Hz, H Ph), 7.23 (1H, t, J = 7.6 Hz, H Ph), 6.75 (1H, s, H Ar), 6.70 (1H, s, H Ar), 4.44–4.41 (1H, m, 2-CH₂), 4.21 (2H, s, CH₂OCH₃), 3.92 (3H, s, OCH₃), 3.75 (3H, s, OCH₃), 3.70 (3H, s, OCH₃), 3.42–3.39 (1H, m, 2-CH₂), 3.29 (3H, s, OCH₃), 3.15 (3H, s, N-CH₃), 2.94–2.89 (1H, m, 1-CH₂), 2.85–2.80 (1H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 207.1, 169.6, 147.9, 147.6, 147.5, 137.3, 129.6, 128.4 (2C), 128.0 (2C), 127.9, 126.9, 113.1, 112.5, 105.6, 100.5, 93.4, 74.5, 59.0, 56.0, 55.9, 51.5, 51.2, 45.1, 31.5; LCMS (ESI) m/z 436 [M + H]⁺; anal. C 71.55, H 6.89, N 3.14%, calcd for C₂₆H₂₉NO₅, C 71.70, H 6.71, N 3.22%.

Methyl 3-methyl-8-(4-methylphenyl)-10,11-dimethoxy-6-(methoxymethyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3d): 0.359 g (47%); light yellow solid; mp 142–144 °C; R_f 0.53 (2:1, EtOAc–hexane); IR (KBr) ν 1935 (C=C=C), 1680 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.42 (1H, s, H-4), 7.26 (2H, d, J = 8.1 Hz, H Ar), 7.13 (2H, d, J = 7.6 Hz, H Ar), 6.76 (1H, s, H Ar), 6.70 (1H, s, H Ar), 4.45–4.41 (1H, m, 2-CH₂), 4.20 (2H, s, CH₂OCH₃), 3.91 (3H, s, OCH₃), 3.75 (3H, s, OCH₃), 3.70 (3H, s, OCH₃), 3.42–3.39 (1H, m, 2-CH₂), 3.29 (3H, s, OCH₃), 3.14 (3H, s, N-CH₃), 2.94–2.88 (1H, m, 1-CH₂), 2.84–2.79 (1H, m, 1-CH₂), 2.35 (3H, s, CH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 206.8, 169.6, 147.8, 147.5, 147.4, 136.6, 134.2, 129.6, 129.0 (2C), 128.0, 127.9 (2C), 113.1, 112.4, 105.4, 100.3, 93.6, 74.6, 59.0, 56.0, 55.9, 51.5, 51.1, 45.0, 31.4, 21.1; LCMS (ESI) m/z 450 [M + H]⁺; anal. C 72.05, H 6.85, N 3.19%, calcd for C₂₇H₃₁NO₅, C 72.14, H 6.95, N 3.12%.

Methyl 3-methyl-10,11-dimethoxy-6-methoxymethyl-8-(4-methoxyphenyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3e): 0.498 g (63%); orange oil; R_f 0.58 (1:2, EtOAc–hexane); IR (KBr) ν 1939 (C=C=C), 1682 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.42 (1H, s, H-4), 7.28 (2H, d, J = 8.6 Hz, H Ar), 6.85 (2H, d, J = 8.6 Hz, H Ar), 6.75 (1H, s, H Ar), 6.69 (1H, s, H Ar), 4.44–4.40 (1H, m, 2-CH₂), 4.19 (2H, s, CH₂OCH₃), 3.91 (3H, s, OCH₃), 3.82 (3H, s, OCH₃), 3.75 (3H, s, OCH₃), 3.70 (3H, s, OCH₃), 3.43–3.39 (1H, m, 2-CH₂), 3.28 (3H, s, OCH₃), 3.15 (3H, s, N-CH₃), 2.93–2.88 (1H, m, 1-CH₂), 2.84–2.79 (1H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 206.5, 169.6, 158.7, 147.8, 147.5, 147.4, 129.5, 129.4, 129.1 (2C), 128.2, 113.8 (2C), 113.0, 112.4, 105.1, 100.3, 93.7, 74.6, 58.9, 56.0, 55.9, 55.3, 51.5, 51.1, 45.0, 31.4; LCMS (ESI) m/z 466 [M + H]⁺; anal. C 69.60, H 6.65, N 3.07%, calcd for C₂₇H₃₁NO₆, C 69.66, H 6.71, N 3.01%.

Methyl 3-methyl-10,11-dimethoxy-6-methoxymethyl-8-(4-fluorophenyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3f): 0.185 g (24%); light yellow solid; mp 177–180 °C; R_f 0.39 (1:1, EtOAc–hexane); IR (KBr) ν 1941 (C=C=C), 1682 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.42 (1H, s, H-4), 7.33–7.31 (2H, m, H Ar), 7.01–6.98 (2H, m, H Ar), 6.69 (2H, s, H Ar), 4.41–4.37 (1H, m, 2-CH₂), 4.19 (1H, d, J = 11.9 Hz, CH₂OCH₃), 4.17 (1H, d, J = 11.9 Hz, CH₂OCH₃), 3.90 (3H, s, OCH₃), 3.74 (3H, s, OCH₃), 3.69 (3H, s, OCH₃), 3.41–3.37 (1H, m, 2-CH₂), 3.28 (3H, s, OCH₃), 3.14 (3H, s, N-CH₃), 2.95–2.88 (1H, m, 1-CH₂), 2.84–2.79 (1H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 206.8, 169.6, 162.9, 161.2, 148.1, 147.7 (2C), 133.4 (1C, d, J = 2.9 Hz), 129.6 (2C, d, J = 8.7 Hz), 127.9, 115.2 (2C, d, J = 20.2 Hz), 113.0, 112.6, 104.9, 100.8, 93.4, 74.5, 59.1, 56.1, 56.0, 51.5, 51.2, 45.2, 31.5; LCMS (ESI) m/z 454 [M + H]⁺; anal. C 68.80, H 6.32, N 3.15%, calcd for C₂₆H₂₈FNO₅, C 68.86, H 6.22, N 3.09%.

Methyl 3-methyl-8-isopropyl-6-(methoxymethyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3g): 0.505 g (87%); yellow oil; R_f 0.75 (5:1, EtOAc–hexane); IR (KBr) ν 1951 (C=C=C), 1687 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.42 (1H, s, H-4), 7.31 (1H, d, J = 8.1 Hz, H Ar), 7.24–7.21 (1H, m, H Ar), 7.16–7.13 (2H, m, H Ar), 4.44–4.39 (1H, m, 2-CH₂), 4.04 (1H, d, J = 11.1 Hz, CH₂OCH₃), 3.95 (1H, d, J = 11.1 Hz, CH₂OCH₃), 3.72 (3H, s, OCH₃), 3.36–3.32 (1H, m, 2-CH₂), 3.21 (3H, s, OCH₃), 3.13 (3H, s, N-CH₃), 2.88–2.82 (1H, m, CH(CH₃)₂), 2.79–2.75 (2H, m, 1-CH₂), 1.22 (3H, d, J = 6.9 Hz, CH₃), 0.91 (3H, d, J = 6.9 Hz, CH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 204.6, 170.2, 147.4, 138.0, 136.5, 130.0, 127.3, 126.9, 126.4, 109.3, 99.6, 94.6, 75.1, 58.9, 51.6, 51.1, 45.2, 31.8, 31.4, 22.2, 21.6; LCMS (ESI) m/z 342 [M + H]⁺; anal. C 73.76, H 8.11, N 4.19%, calcd for C₂₁H₂₇NO₃, C 73.87, H 7.97, N 4.10%.

Methyl 3-methyl-8-phenyl-6-(methoxymethyl)-benzo[d]-3-aza-cyclodeca-4,6,7-triene-5-carboxylate (3h): 0.408 g (64%); beige solid; mp 148–150 °C; R_f 0.72 (5:1, EtOAc–hexane); IR (KBr) ν 1943 (C=C=C), 1668 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.43 (1H, s, H-4), 7.36 (2H, d, J = 7.6 Hz, H Ar), 7.31 (2H, t, J = 7.6 Hz, H Ar), 7.27–7.25 (1H, m, H Ph), 7.24–7.22 (4H, m, H Ph), 4.45–4.41 (1H, m, 2-CH₂), 4.22 (1H, d, J = 11.9 Hz, CH₂OCH₃), 4.20 (1H, d, J = 11.9 Hz, CH₂OCH₃), 3.71 (3H, s, OCH₃), 3.47–3.43 (1H, m, 2-CH₂), 3.28 (3H, s, OCH₃), 3.15 (3H, s, N-CH₃), 2.95–2.88 (2H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 207.0, 169.6, 147.5, 137.2, 137.0, 136.0, 130.3, 129.8, 128.3 (2C), 128.1 (2C), 127.1, 126.9, 126.5, 105.5, 100.8, 93.1, 74.4, 59.0, 51.3, 51.1, 45.1, 31.8; LCMS (ESI) m/z 376 [M + H]⁺; anal. C 76.65, H 6.82, N 3.88%, calcd for C₂₄H₂₅NO₃, C 76.77, H 6.71, N 3.73%.

1-(3-Methyl-8-(4-methylphenyl)-10,11-dimethoxy-6-methoxymethyl-benzo[d]-3-aza-cyclodeca-4,6,7-trien-5-yl)ethanone (3i): 0.368 g (50%); yellow solid; mp 156–159 °C; R_f 0.30 (EtOAc); IR (KBr) ν 1950 (C=C=C), 1641 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.38 (1H, s, H-4), 7.26 (2H, d, J = 8.1 Hz, H Ar), 7.13 (2H, d, J = 8.1 Hz, H Ar), 6.73 (1H, s, H Ar), 6.69 (1H, s, H Ar), 4.43–4.39 (1H, m, 2-CH₂), 4.13 (1H, d, J = 11.6 Hz, CH₂OCH₃), 4.11 (1H, d, J = 11.6 Hz, CH₂OCH₃), 3.90 (3H, s, OCH₃), 3.73 (3H, s, OCH₃), 3.42–3.38 (1H, m, 2-CH₂), 3.29 (3H, s, OCH₃), 3.18 (3H, s, N-CH₃), 2.96–2.90 (1H, m, 1-CH₂), 2.83–2.78 (1H, m, 1-CH₂), 2.34 (3H, s, COCH₃), 2.23 (3H, s, CH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 206.1, 195.4, 148.0, 147.9, 147.5, 136.8, 134.1, 129.3, 129.1 (2C), 127.93, 127.90 (2C), 113.0, 112.5, 106.7, 105.7, 101.0, 75.0, 59.1, 55.9, 55.8, 51.6, 45.5, 31.2, 26.6, 21.1; LCMS (ESI) m/z 434 [M + H]⁺; anal. C 74.75, H 7.29, N 3.31%, calcd for C₂₇H₃₁NO₄, C 74.80, H 7.21, N 3.23%.

1-(3-Methyl-10,11-dimethoxy-6-methoxymethyl-8-(4-methoxyphenyl)-benzo[d]-3-aza-cyclodeca-4,6,7-trien-5-yl)ethanone (3j): 0.557 g (73%); beige solid; mp 137–139 °C; R_f 0.26 (EtOAc); IR (KBr) ν 1938 (C=C=C), 1649 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.38 (1H, s, H-4), 7.30 (2H, d, J = 9.1 Hz, H Ar), 6.86 (2H, d, J = 9.1 Hz, H Ar), 6.74 (1H, s, H Ar), 6.69 (1H, s, H Ar), 4.44–4.40 (1H, m, 2-CH₂), 4.13 (1H, d, J = 11.9 Hz, CH₂OCH₃), 4.11 (1H, d, J = 11.9 Hz, CH₂OCH₃), 3.91 (3H, s, OCH₃), 3.82 (3H, s, OCH₃), 3.74 (3H, s, OCH₃), 3.43–3.40 (1H, m, 2-CH₂), 3.30 (3H, s, OCH₃), 3.20 (3H, s, N-CH₃), 2.96–2.90 (1H, m, 1-CH₂), 2.84–2.78 (1H, m, 1-CH₂), 2.24 (3H, s, COCH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 206.0, 195.5, 158.9, 148.2, 148.0, 147.6, 129.5, 129.4, 129.3 (2C), 128.2, 114.0 (2C), 113.1, 112.7, 107.1, 105.6, 101.1, 75.2, 59.3, 56.1, 56.0, 55.4, 51.7, 45.7, 31.3, 26.7; LCMS (ESI) m/z 450 [M + H]⁺; anal. C 72.05, H 6.75, N 3.04%, calcd for C₂₇H₃₁NO₅, C 72.14, H 6.95, N 3.12%.

1-(3-Methyl-10,11-dimethoxy-6-methoxymethyl-8-(4-fluorophenyl)-benzo[d]-3-aza-cyclodeca-4,6,7-trien-5-yl)ethanone (3k): 0.565 g (76%); light yellow solid; mp 164–166 °C; R_f 0.35 (EtOAc); IR (KBr) ν 1940 (C=C=C), 1650 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.37–7.34 (3H, m, H-4, H Ar), 7.01 (2H, t, J = 8.6 Hz, H Ar), 6.70 (1H, s, H Ar), 6.69 (1H, s, H Ar), 4.42–4.38 (1H, m, 2-CH₂), 4.13 (1H, d, J = 11.9 Hz, CH₂OCH₃), 4.10 (1H, d, J = 11.9 Hz, CH₂OCH₃), 3.92 (3H, s, OCH₃), 3.74 (3H, s, OCH₃), 3.42–3.38 (1H, m, 2-CH₂), 3.30 (3H, s, OCH₃), 3.20 (3H, s, N-CH₃), 2.97–2.92 (1H, m, 1-CH₂), 2.85–2.79 (1H, m, 1-CH₂), 2.23 (3H, s, COCH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 206.1, 195.3, 162.1 (1C, d, J = 247.1 Hz), 148.4, 148.2, 147.8, 133.3 (1C, d, J = 2.9 Hz), 129.7 (2C, d, J = 8.7 Hz), 129.4, 127.9, 115.4 (2C, d, J = 21.7 Hz), 113.0, 112.7, 106.9, 105.2, 101.5, 74.9, 59.3, 56.1, 56.0, 51.7, 45.7, 31.3, 26.5; LCMS (ESI) m/z 438 [M + H]⁺; anal. C 71.46, H 6.54, N 3.26%, calcd for C₂₆H₂₈FNO₄, C 71.38, H 6.45, N 3.20%.

1-(3-Methyl-6-methoxymethyl-8-isopropyl-benzo[d]-3-aza-cyclodeca-4,6,7-trien-5-yl)ethanone (3l): 0.243 g (44%); colorless solid; mp 130–132 °C; R_f 0.45 (5:1, EtOAc–hexane). IR (KBr) ν 1941 (C=C=C), 1580 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.45 (1H, s, H-4), 7.33 (1H, d, J = 7.6 Hz, H Ar), 7.25 (1H, td, J = 6.9, 1.7 Hz, H Ar), 7.16–7.13 (2H, m, H Ar), 4.39–4.35 (1H, m, 2-CH₂), 3.96 (2H, s, CH₂OCH₃), 3.34–3.30 (1H, m, 2-CH₂), 3.22 (3H, s, OCH₃), 3.19 (3H, s, N-CH₃), 2.90–2.86 (1H, m, CH(CH₃)₂), 2.85–2.83 (1H, m, 1-CH₂), 2.82–2.77 (1H, m, 1-CH₂), 2.29 (3H, s, COCH₃), 1.27 (3H, d, J = 6.6 Hz, CH₃), 0.93 (3H, d, J = 6.6 Hz, CH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 204.1, 195.9, 147.9, 137.6, 136.1, 130.0, 127.2, 127.0 (2C), 126.5, 109.7, 100.6, 75.6, 59.1, 51.7, 45.6, 31.7, 31.4, 26.7, 22.3, 22.0; LCMS (ESI) m/z 326 [M + H]⁺; anal. C 77.61, H 8.25, N 4.25%, calcd for C₂₁H₂₇NO₂, C 77.50, H 8.36, N 4.30%.

1-(3-Methyl-6-methoxymethyl-8-phenyl-benzo[d]-3-aza-cyclodeca-4,6,7-trien-5-yl)ethanone (3m): 0.305 g (50%); beige solid; mp 183–185 °C; R_f 0.47 (1:3, EtOAc–hexane); IR (KBr) ν 1942 (C=C=C), 1580 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.39–7.37 (3H, m, H Ar and H-4), 7.32 (2H, t, J = 8.1 Hz, H Ar), 7.25–7.22 (5H, m, H Ph), 4.45–4.41 (1H, m, 2-CH₂), 4.15 (1H, d, J = 11.9 Hz, CH₂OCH₃), 4.13 (1H, d, J = 11.9 Hz, CH₂OCH₃), 3.46–3.42 (1H, m, 2-CH₂), 3.29 (3H, s, OCH₃), 3.20 (3H, s, N-CH₃), 2.98–2.88 (2H, m, 1-CH₂), 2.24 (3H, s, COCH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 206.4, 195.4, 148.1, 137.3, 136.9, 136.2, 130.4, 130.1, 128.5 (3C), 128.3 (2C), 127.3, 127.2, 126.8, 106.0, 101.6, 74.9, 59.3, 51.6, 45.7, 31.7, 26.7; LCMS (ESI) m/z 360 [M + H]⁺; anal. C 80.03, H 7.15, N 3.80%, calcd for C₂₄H₂₅NO₂, C 80.19, H 7.01, N 3.90%.

Methyl (4E,6E,7Z)-10,11-dimethoxy-6-(methoxymethylidene)-3-methyl-8-phenyl-1,2,3,6-tetrahydro-3-benzazecin-5-carboxylate (4c): 0.237 g (32% from CF₃CH₂OH); yellow oil; R_f 0.52 (2:1, EtOAc–hexane); IR (KBr) ν 1685 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.38 (1H, s, H-4), 7.28 (1H, s, H Ph), 7.24 (3H, t, J = 7.9 Hz, H Ph), 7.17 (1H, t, J = 7.1 Hz, H Ph), 6.64 (2H, br. s, H Ar and =CH-OCH₃), 6.44 (1H, s, H Ar), 5.99 (1H, s, H-7), 4.15–4.08 (1H, m, 2-CH₂), 3.91 (3H, s, OCH₃), 3.75 (3H, s, OCH₃), 3.74 (3H, s, OCH₃), 3.50 (3H, s, OCH₃), 2.96 (3H, s, N-CH₃), 2.94–2.91 (1H, m, 2-CH₂), 2.65–2.63 (1H, m, 1-CH₂), 2.52–2.50 (1H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 170.1, 152.9, 150.0, 148.0, 147.7, 142.5, 135.7, 134.6, 128.5, 128.1 (2C), 126.5, 126.2 (2C), 122.6, 113.8, 113.6, 112.0, 94.3, 60.3, 56.2 (2C), 55.8 (2C), 50.7, 32.3; LCMS (ESI) m/z 436 [M + H]⁺; anal. C 71.57, H 6.84, N 3.28%, calcd for C₂₆H₂₉NO₅, C 71.70, H 6.71, N 3.22%.

Methyl (4E,6E,7Z)-3-methyl-10,11-dimethoxy-6-(methoxymethylidene)-8-(4-methylphenyl)-1,2,3,6-tetrahydro-3-benzazecin-5-carboxylate (4d): 0.267 g (35%); light yellow solid; mp 153–155 °C; R_f 0.52 (2:1, EtOAc–hexane); IR (KBr) ν 1680 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.34 (1H, s, H-4), 7.17 (2H, d, J = 8.1 Hz, H Ar), 7.05 (2H, d, J = 8.1 Hz, H Ar), 6.63 (2H, br. s, H Ar and =CH-OCH₃), 6.43 (1H, s, H Ar), 5.96 (1H, s, H-7), 4.15–4.08 (1H, m, 2-CH₂), 3.91 (3H, s, OCH₃), 3.75 (3H, s, OCH₃), 3.74 (3H, s, OCH₃), 3.49 (3H, s, OCH₃), 2.96 (3H, s, N-CH₃), 2.93–2.90 (1H, m, 2-CH₂), 2.64–2.62 (1H, m, 1-CH₂), 2.51–2.49 (1H, m, 1-CH₂), 2.31 (3H, s, CH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 170.1, 152.5, 149.9, 148.0, 147.6, 139.6, 136.2, 135.5, 134.8, 128.8 (2C), 128.4, 126.1 (2C), 121.6, 113.7, 113.6, 112.0, 94.3, 60.2, 56.2, 55.7 (2C), 50.6 (2C), 32.2, 21.0; LCMS (ESI) m/z 450 [M + H]⁺; anal. C 72.01, H 7.08, N 3.18%, calcd for C₂₇H₃₁NO₅, C 72.14, H 6.95, N 3.12%.

Methyl (4E,6E,7Z)-10,11-dimethoxy-6-(methoxymethylidene)-8-(4-methoxyphenyl)-3-methyl-1,2,3,6-tetrahydro-3-benzazecin-5-carboxylate (4e): 0.221 g (28%); orange oil; R_f 0.53 (2:1, EtOAc–hexane); IR (KBr) ν 1679 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.27 (1H, s, H-4), 7.19 (2H, d, J = 8.6 Hz, H Ar), 6.78 (2H, d, J = 8.6 Hz, H Ar), 6.63 (2H, br. s, H Ar and =CH-OCH₃), 6.43 (1H, s, H Ar), 5.95 (1H, s, H-7), 3.91 (3H, s, OCH₃), 3.78 (3H, s, OCH₃), 3.74 (6H, s, OCH₃), 3.72–3.70 (1H, m, 2-CH₂); 3.49 (3H, s, OCH₃), 2.96 (3H, s, N-CH₃), 2.93–2.90 (1H, m, 2-CH₂), 2.65–2.62 (1H, m, 1-CH₂), 2.51–2.47 (1H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 170.1, 158.6, 152.3, 149.9, 148.0, 147.7, 147.6, 135.3, 134.9, 129.2, 128.4, 127.3 (2C), 120.8, 113.6, 113.5 (2C), 112.1, 94.4, 60.2, 56.2, 55.8 (2C), 55.3 (2C), 50.6, 32.2; LCMS (ESI) m/z 466 [M + H]⁺; anal. C 69.55, H 6.85, N 3.23%, calcd for C₂₇H₃₁NO₆, C 69.66, H 6.71, N 3.01%.

Methyl (4E,6E,7Z)-3-methyl-10,11-dimethoxy-6-(methoxymethylidene)-8-(4-fluorophenyl)-1,2,3,6-tetrahydro-3-benzazecin-5-carboxylate (4f): 0.362 g (47%); light yellow solid; mp 177–179 °C; R_f 0.38 (1:1, EtOAc–hexane); IR (KBr) ν 1682 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 7.30 (1H, s, H-4), 7.23–7.21 (2H, m, H Ar), 6.94–6.91 (2H, m, H Ar), 6.64 (2H, br. s, H Ar and =CH-OCH₃), 6.41 (1H, s, H Ar), 5.98 (1H, s, H-7), 4.16–4.11 (1H, m, 2-CH₂), 3.91 (3H, s, OCH₃), 3.75 (3H, s, OCH₃), 3.74 (3H, s, OCH₃), 3.49 (3H, s, OCH₃), 2.96 (3H, s, N-CH₃), 2.93–2.91 (1H, m, 2-CH₂), 2.62–2.60 (1H, m, 1-CH₂), 2.52–2.50 (1H, m, 1-CH₂); ¹³C NMR (CDCl₃, 150 MHz) δ 170.1, 162.8, 161.1, 152.9, 150.0, 148.2, 147.9, 138.8, 134.7, 134.5, 127.7 (2C, d, J = 7.2 Hz), 122.3, 114.9 (2C, d, J = 21.7 Hz), 113.7 (2C), 112.0, 94.3, 60.4, 56.3, 55.9 (2C), 50.7 (2C), 32.3; LCMS (ESI) m/z 454 [M + H]⁺; anal. C 68.75, H 6.17, N 3.15%, calcd for C₂₆H₂₈FNO₅, C 68.86, H 6.22, N 3.09%.

3.1.3. Transformation of Allene 3a into 6-Methoxymethylidenebenzazecin 4a

A solution of allene 3a (0.4 mmol) in glacial acetic acid was placed into microwave reactor. The reaction was carried out for 20 min at 100 °C. The progress of the reaction was monitored by TLC (Sorbfil, 3:2 EtOAc-hexane). The solvent was removed under vacuum and the residue chromatographed on silica gel (1:5 EtOAc-hexane).

Methyl (4E,6E,7Z)- 10,11-dimethoxy-6-(methoxymethylidene)-3,8-dimethyl-1,2,3,6-tetrahydro-3-benzazecin-5-carboxylate (4a): 0.037 g (25%); brown oil; R_f 0.52 (2:1, EtOAc–hexane); IR (KBr) ν 1683 (C=O) cm⁻¹; ¹H NMR (CDCl₃, 600 MHz) δ 6.71 (2H, s, H-4 and =CH-OCH₃), 6.59 (1H, s, H Ar), 6.40 (1H, s, H Ar), 5.71 (1H, s, H-7), 3.86 (3H, s, OCH₃), 3.76 (3H, s, OCH₃), 3.68 (3H, s, OCH₃), 3.47 (3H, s, OCH₃), 2.94 (3H, s, N-CH₃), 2.87–2.82 (2H, m, 1-CH₂, 2-CH₂), 2.66–2.62 (2H, m, 1-CH₂), 2.03 (3H, s, CH₃); ¹³C NMR (CDCl₃, 150 MHz) δ 170.1, 150.8, 149.3, 148.0, 147.1, 137.2, 134.0, 126.1, 122.1, 113.8, 111.8, 111.6, 94.3, 60.0, 56.2 (2C), 55.9 (2C), 50.6, 31.7, 28.0; LCMS (ESI) m/z 374 [M + H]⁺; anal. C 67.41, H 7.08, N 3.38%, calcd for C₂₁H₂₇NO₅, C 67.54, H 7.29, N 3.75%.

3.2. Inhibition of Cholinesterases and Inhibition of Monoamine Oxidases

3.2.1. Inhibition of Cholinesterases

Inhibition of human recombinant AChE (2770 U/mg) or BChE from human serum (50 U/mg) was determined as described [23] using the Ellman spectrophotometric method in a 96-well plate procedure. Briefly, test compounds were incubated in phosphate buffer pH 8.0 in the presence of the enzyme and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) as the chromophoric reagent. Incubation samples were made in 96-well, flat-bottomed transparent polystyrene plates (Greiner Bio-One, Kremsmünster, Austria), at 25 °C for 20 min, and read at 412 nm using an Infinite M1000 Pro plate reader (Tecan, Cernusco s.N., Italy). For inhibition kinetics, four concentrations of compound 3e (ranging from 0 to 15 μM), and six concentrations of acetylthiocholine (from 33 to 200 μM) were used. Inhibition data and kinetics were obtained as means ± SD from 3 independent experiments, using GraphPad Prism (version 5.00 for Windows; GraphPad Software, San Diego, CA, USA).

3.2.2. Inhibition of Monoamine Oxidases

Inhibition of human recombinant monoamine oxidases A (250 U/mg) and B (59 U/mg; microsomes from baculovirus infected insect cells; Sigma Aldrich) was determined as already described [24], measuring the fluorescence of 4-hydroxyquinoline produced by MAOs in the oxidative deamination of substrate kynuramine. Briefly, compounds were tested in coincubation with MAO and kynuramine in phosphate buffer 390 mOsm pH 7.4, at 37 °C for 30 min. Assays were performed in 96-well black polystyrene plates (Greiner) using the Infinite M1000 Pro plate reader (Tecan). Inhibition data were obtained as means ± SD using GraphPad Prism.

3.3. Solubility and Hydrolytic Stability of 3e and 3n

3.3.1. Aqueous Solubility Measurement and U-HPLC Analytical Condition

The determination of kinetic solubility in aqueous buffer solution (50 mM phosphate buffer, pH 7.4, 0.15 M KCl) at 37 °C by U-HPLC was obtained as described [25], using a stock solution 10 mM in DMSO of compound (3e and 3n) solubilized in PBS (50 mM) to final concentration of 200 μM. Following shaking of the suspension in an orbital shaker at 250 rpm for 2 h, the solution was separated by centrifugation (2500 rpm, 3 min) and filtered. Equal volume of solution was transferred into 1:1 (v/v) mixture of DMSO/PBS. The concentration of compound was determined by U-HPLC and UV detector (255 nm) comparing the peak area of external standard solution. All data were means of 3 independent experiments (± SEM). Analytical condition: mobile phase: MeOH/Ammonium formate 10 mM pH 4.5 (72:28); column: Kinetex C18, 150 × 2.1 mm, 2.6 µm; flow: 0.3 mL/min; injection: 2 µL (3e) and 5 µL (3n). HPLC analyses were performed on an Agilent U-HPLC 1260 Infinity Quaternary LC system (Agilent Technologies, Milan, Italy) (Table 4).

3.3.2. Hydrolytic Stability in Water-Buffered Solution and U-HPLC Analytical Condition

Hydrolytic stability of compounds 3e and 3n was determined as described [26], using 10 mM stock solution in MeOH, solubilized in MeOH and aqueous buffer solution (50 mM phosphate buffer, pH 7.4 in 0.15 M KCl) to 25 µM final concentration, and incubated with shaking at 25 ± 0.5 °C. At appropriate time intervals, samples were withdrawn and analyzed by U-HPLC using a 1290 Infinity Quaternary LC system (Agilent Technologies, Milan, Italy) equipped with autosampler and photodiode array detector. A Phenomenex Kinetex C18 column 2.6 µm (150 × 2.1 mm i.d.) was used as stationary phase. The analyte was eluted with 8 min in isocratic mobile phase: MeOH/ammonium formate (10 mM, pH 4.5)/(68:32, v/v) at constant flow rate of 0.3 mL/min, injection volume: 2 µL (3e) and 5 µL (3n), UV detector: 255 nm. Pseudo-first-order rate constants (kobs) for the hydrolysis of the compound were calculated from the slopes of the linear plots of log (% remaining compound) against time. Each kinetic experiment was performed in triplicate (Table 4).

4. 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 single-digit 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.