Synthesis of azepino[4,5-b]indoles and benzo[f][1,5]diazecine-2,8(1H,3H)diones: promising anti-cancer activity toward cervical and brain cancer

Abstract

The current manuscript comprises (i) the development of a robust protocol for the regio- and stereoselective synthesis of azepino[4,5-b]indoles with wide substrate scope, (ii) the unprecedented formation of tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)diones via a series of oxidation reaction cascades on [4,5-b]indole derivatives and (iii) the biological evaluation of the diazecine compounds for the anti-cancer activity against HeLa and U87MG cell lines. The protocol proceeds via Lewis acid-catalyzed S_N2-type ring-opening of activated aziridines with indoles from the C-3 position, followed by base-mediated propargylation in the same pot under reflux conditions, and subsequent [Au]-catalyzed 7-exo-dig hydroarylation cyclization at room temperature. The synthetic utility of the developed strategy was further demonstrated by a number of important chemical transformations, viz, metathesis, isomerization, oxidation and reduction reactions. The exocyclic azepino[4,5-b]indole compound 6 reacts with mCPBA to furnish an unprecedented formation of 3-hydroxy-4,5,6,7-tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)dione 11, a higher-order di-aza-heterocycle via a series of cascade reactions: Prilezhaev epoxidation, Meinwald rearrangement, Baeyer–Villiger oxidation, Witkop oxidation, followed by hydrolysis (PMBWH). Biological evaluation of our synthesized ten-membered compounds 11 revealed their superior cytotoxic activity compared to cisplatin and temozolomide, which are established marketed drugs for the treatment of cervical and brain cancers, respectively. The significance of the methodology is further demonstrated by the gram-scale synthesis of hexahydroazepino[4,5-b]indoles and the anti-cancer agent tetrahydrobenzo[f][1,5]diazecine. The formation of the products is rationalized by a plausible mechanism, and the mechanistic proposal is substantiated by computational studies (DFT). All these results are reported in the manuscript.

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Introduction

Azepinoindoles are a class of fused di-aza-heterocyclic scaffolds of indoles¹ and azepines,² and both of these compounds are of immense pharmacological significance. There are numerous bioactive alkaloids containing azepinoindole cores³ which contribute to a wide range of interesting biological activities. 1,2,3,4,5,6-Hexahydroazepino[4,5-b]indoles contain [6-5-7]-diazatricyclic skeletons embedded with an indole moiety and an azepane-fused core. Such compounds exhibit promising biological activities, resulting in considerable attention from medicinal chemists. A few examples of bioactive molecules containing the azepino[4,5-b]indole core include kenpaullone, alsterpaullone, and cazpaullone, which act as glycogen synthase kinase-3 inhibitors,⁴ (+)-trigonoliimine G⁵ and pseudocerosine,⁶ which show cytotoxicity against acute myelogenous leukemia (HL-60) or human lung cancer and SKOV-3, a human adenocarcinoma cell line, respectively. Subincanadine F exhibits potent cytotoxicity against murine lymphoma L1210 and human epidermoid carcinoma KB cells,⁷ malassezindoles A and B are responsible for the skin disease pityriasis versicolor,⁸ and ibogamine is effective at reducing withdrawal symptoms and cravings (Fig. 1a).⁹

Fig. 1 (a) Biologically active azepino[4,5-b]indole derivatives; (b) ROC of aziridines with indoles and propargyl bromide.

Despite the immense pharmacological significance of hexahydroazepino[4,5-b]indoles,¹⁰ only a few reports are known for their syntheses that include formic acid-catalyzed cyclization of phenylhydrazones of 1-benzoylhexahydroazepin-4-one,¹¹ Pd/C-catalyzed hydrogenation of 2-(2-benzylaminoethyl)-3-cyanomethyl indoles,¹² an [Au]-catalyzed intramolecular hydroarylation reaction of indoles with alkynes and an intramolecular cyclization reaction of indolylcyclopropenes,¹³ an [Ir]-catalyzed allylic dearomatization/retro-Mannich/hydrolysis cascade reaction of (tetrahydro-2H-pyrido[4,3-b]indol-2-yl)propenylcarbonate derivatives,¹³ [Ir]-catalyzed asymmetric allylic dearomatization of indoles followed by a p-TSA-catalyzed ring-expansion reaction,¹⁴ Zn(OTf)₂-mediated hydroarylation followed by a retro-Mannich reaction of N-propargylated tetrahydro-β-carbolines,¹⁵etc. Some of the existing strategies typically rely on the use of expensive catalysts, sensitive reaction conditions, are limited to tryptamine derivatives, and require multistep synthesis of the starting materials. To overcome the shortcomings of the existing methodologies, we realized that the development of an expedient route to access azepino[4,5-b]indoles with wide substrate scope is highly desirable. Aziridines are very versatile building blocks in organic chemistry,¹⁶ and for more than two decades, we have been involved in S_N2-type nucleophilic ring-opening transformations of small ring aza/oxa and carbacycles, following ROC (ring-opening cyclization) or DROC (domino ring-opening cyclization) protocols to construct bioactive targets of contemporary interest.¹⁷

During the study of the ROC of activated aziridines with indole/propargyl carbonates,¹⁸ aniline/propargyl carbonate,¹⁹ propargyl alcohols,²⁰ and DROC of activated aziridines with propargyl aniline,²¹ we anticipated that azepino[4,5-b]indoles could easily be synthesized via S_N2-type nucleophilic ring-opening of activated aziridines with indoles from the C-3 position, followed by base-mediated propargylation, and subsequently, metal-catalyzed 7-exo-dig hydroarylation reactions (Fig. 1b). In continuation of our research activities in this area, we have developed a simple strategy for the synthesis of hexahydroazepino[4,5-b]indole derivatives via ROC of aziridines with indoles/propargyl bromide for the first time, the oxidation of such azepino[4,5-b]indoles leading to the unprecedented formation of tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)diones for the first time and studied the biological activities of those diazecine compounds, which exhibited excellent anti-cancer activities against HeLa and U87MG cell lines. Herein, we are pleased to report our results as a research article.

Results and discussion

Our study commenced with the S_N2-type nucleophilic ring-opening of (±)-2-phenyl-N-tosylaziridine (1a) with N-methyl indole (2a) through its C-3 site in the presence of 20 mol% of LiClO₄ as the Lewis acid catalyst in CH₃CN solvent at 85 °C, to give the corresponding ring-opening product. Furthermore, the crude product was subjected to propargylation employing propargyl bromide (3a) and K₂CO₃ in the same pot at 80 °C in the CH₃CN medium to afford the N-propargylated product 4a in 98% overall yield (Scheme 1). Next, the product 4a was subjected to intramolecular hydroarylation cyclization conditions in the presence of 10 mol% of Zn(OTf)₂ and 1.0 equiv. of Bu₄N⁺OTf⁻ at 85 °C in DCE solvent to afford the products 5a (minor) and 6a (major) as an inseparable mixture of regioisomers along with some unwanted impurities in 85% overall yield. The structures of the compounds 5a and 6a were confirmed by single-crystal X-ray analysis.

Scheme 1 ROC of activated aziridine with N-Me indole and propargyl bromide.

Our attempts, such as temperature, solvent screening (e.g., DCM, DCE, toluene, dioxane, THF, DMF, etc.), and metal salt (Cu(OTf)₂, Sc(OTf)₃, Yb(OTf)₃, AgOTf, etc., in combination with Bu₄NOTf at 85 °C) variations, to synthesize the regioselective single product 6a and separate it from unwanted impurities were unsuccessful (SI, Table S7). Although the synthesis of similar compounds from tryptamine derivatives using [Au]-catalysts is known, the major issues associated with these approaches are as follows: limited substrate scope (only tryptamine derivatives were used) and problems of regioselectivity in the cyclization step (leading to the formation of either seven- or eight-membered rings or a mixture of both and different products in some cases).¹² Only one example was reported for an N-protected indole derivative, where Au-catalyzed cyclisation gave the seven-membered ring selectively. We wanted to explore whether [Au]-catalysis can solve our problem of regioselectivity with wider substrate scope.

To realize our idea, the product 4a was treated with 2.0 mol% of Ph₃PAuCl as a precatalyst along with 3.0 mol% of AgOTf as a cocatalyst in toluene at room temperature for 24 h, and to our delight, the desired product 6a was formed in 84% yield as a single regioisomer (entry 1, Table 1). It is worth noting that in the reported methods, the formation of mixtures of regioisomers or eight-membered rings was observed.¹² To further optimize the reaction conditions with respect to yield, reaction time, and selectivity, we systematically investigated different combinations of co-catalysts with catalysts and solvents. The outcomes of these experiments are summarized in Table 1. The best results were obtained in the case of CH₃CN (entry 2), DCM and DCE (entries 6 and 11) as solvents. To promote sustainability by avoiding chlorinated solvents, we identified 2 mol% of Ph₃PAuCl as the precatalyst and 3 mol% of AgOTf as the co-catalyst in CH₃CN as the optimal catalyst system for the 7-exo-dig hydroarylation (cyclization) reaction (entry 2). Slightly lower yields were obtained when THF and 1,4-dioxane were used as solvents (entries 3 and 4), and no reaction was observed in the case of DMF (entry 7).

Table 1 Optimization study for 7-exo-dig hydroarylation of 4a ^a

S. no.	Catalyst (mol%), cocatalyst (mol%)	Solvent	Time (h)	% Yield^b (6a)
a Unless otherwise stated, all the reactions were carried out with 1.0 equiv. (0.226 mmol) of 3a, in the appropriate solvent (1.0 mL) at room temperature under an Ar environment. b Yield of the isolated product.
1	Ph3PAuCl (2.0), AgOTf (3.0)	Toluene	24	84
2	Ph3PAuCl (2.0), AgOTf (3.0)	CH3CN	10	96
3	Ph3PAuCl (2.0), AgOTf (3.0)	THF	12	90
4	Ph3PAuCl (2.0), AgOTf (3.0)	1,4-Dioxane	10	83
5	Ph3PAuCl (2.0), AgOTf (3.0)	DCM	2	93
6	Ph3PAuCl (2.0), AgOTf (5.0)	DCM	2.5	95
7	Ph3PAuCl (2.0), AgOTf (3.0)	DMF	24	—
8	Ph3PAuCl (2.0), AgSbF6 (5.0)	DCM	2	94
9	Ph3PAuCl (2.0), Bu4NOTf (5.0)	DCM	24	—
10	Ph3PAuCl (2.0), SbCl5 (5.0)	DCM	24	—
11	Ph3PAuCl (2.0), AgOTf (5.0)	DCE	2	94
12	Ph3PAuCl (2.0), —	DCM	24	—
13	—, AgOTf (5.0)	DCM	24	—

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We carried out separate experiments to ensure that AgOTf and Ph₃PAuCl alone cannot catalyse the reaction (entries 12 and 13). No hydroarylation reaction was observed when AgOTf was replaced with Bu₄NOTf and SbCl₅ as the cocatalyst (entries 9 and 10, Table 1). Unfortunately, our attempts to perform the reaction under one-pot, three-step conditions (ring opening, propargylation and cyclisation) for the synthesis of azepino[4,5-b]indole were unsuccessful.

To investigate the substrate scope of our approach, a range of 2-phenyl-N-aryl/alkyl sulfonyl aziridines 1b–h were studied with N-methyl indole (2a) and propargyl bromide (3a) using the optimized ROC protocol, and in all cases, the corresponding 5-methylene-1,2,3,4,5,6-hexahydroazepino[4,5-b]indoles (6b–h) were obtained in good to excellent yields. The results are described in Scheme 2. The S_N2-type nucleophilic ring-opening of 2-phenyl-N-arylsulfonyl aziridines having EWG-substituents like p-NO₂ (1b) and p-F (1c) has been found to be faster in comparison with EDG-substituents like p-OMe (1e), p-t-Bu (1f), and 2,4,6-tri-Me (1g) substituted aziridines. The strategy was further generalized by engaging several 2-aryl/alkyl-N-tosyl aziridines 1i–t with indole as the nucleophile and propargyl bromide as the alkylating agent. The results are described in Scheme 2. In all cases with 2-aryl-N-tosyl aziridines in the 7-exo-dig hydroarylation step, the corresponding products were obtained in 65–99% yields. In the case of 2-methyl aziridine 1q and 5-membered bicyclic aziridine 1r, nucleophilic ring-opening followed by propargylation afforded the products 4q and 4r in 65% and 48% yields, respectively. These products after the cyclization afforded the corresponding products 6q and 6r in good to excellent yields. In the case of the diastereomeric pair of 2-phenyl-3-propyl-N-tosylaziridine (1s), the corresponding product 6s was obtained as an inseparable mixture of diastereomers. In the case of bulky naphthyl-substituted aziridine 1t, the reaction took 40 h for the nucleophilic ring-opening, which after the propargylation reaction afforded 4t in 96% overall yield. Compound 4t after the hydroarylation cyclization afforded the corresponding product 6t in 66% yield.

Scheme 2 ROC^a of (±)-aziridines 1b–t with indole (2a) and propargyl bromide (3a). ^a Unless otherwise stated, all the ring-opening reactions were carried out with 1.0 equiv. (0.366 mmol) of 1b–t, 1.2 equiv. (0.439 mmol) of 2a, 20 mol% (0.073 mmol) of LiClO₄ in CH₃CN (20 μL) at 85 °C under an Ar environment. Propargylation reactions were carried out with 3.0 equiv. (0.798 mmol) of propargyl bromide (3a) in the presence of 3.0 equiv. (0.798 mmol) of K₂CO₃ in CH₃CN (2.0 mL) at 80 °C temperature. All the hydroarylation reactions of 4 (1.0 equiv.) were carried out in the presence of 2.0 mol% of Ph₃PAuCl and 3.0 mol% of AgOTf in CH₃CN solvent at room temperature under an Ar atmosphere. ^b The ring-opening reaction was carried out in the presence of 30 mol% of LiClO₄. ^c Inseparable mixture of products. The diastereomeric ratio of 1s is 1.6 : 1.

The efficiency of the developed methodology was examined by using substituted indoles 2b–j, as nucleophiles with varying electronic nature, and the propargyl bromide derivative 3b. The results are shown in Scheme 3. In the case of N-benzyl indole (2b) and N-homoallyl indole (2e), the corresponding nucleophilic ring-opening followed by propargylation furnished the corresponding products 4w and 4z in 60% and 75% yields, respectively. No ring-opening reaction took place in the case of N-Ts indole (2f), probably because of the low nucleophilicity of the indole moiety.

Scheme 3 ROC of aziridine 1a, with indoles 2b–f and propargyl bromides 3a–b. Unless otherwise stated, all the ring-opening reactions were carried out with 1.0 equiv. (0.366 mmol) of 1a, 1.2 equiv. (0.439 mmol) of 2b–j, 20 mol% (0.073 mmol) of LiClO₄ in CH₃CN (20 μL) at 85 °C under an Ar environment. Propargylation reactions were carried out with 3.0 equiv. (0.798 mmol) of propargyl bromides (3a–b) in the presence of 3.0 equiv. (0.798 mmol) of K₂CO₃ in CH₃CN (2.0 mL) at 80 °C temperature. All the hydroarylation reactions of 4 (1.0 equiv.) were carried out in the presence of 2.0 mol% of Ph₃PAuCl and 3.0 mol% of AgOTf in CH₃CN solvent at room temperature under an Ar environment. ^a The reaction temperature was screened up to 85 °C. ^b The reaction was carried out with Zn(OTf)₂ (10 mol%) and Bu₄NOTf (1.0 equiv.) in DCE solvent at 85 °C under an Ar environment. cm = complex mixture.

The formation of a complex mixture of products was observed in the case of 1-(pyridin-2-ylmethyl)-1H-indole (2b) and N-H free indole (2d). No significant effect of the substituent, whether it is an EDG or EWG, present on the indole core on the ring-opening followed by propargylation product yield was observed. Although the hydroarylation process afforded the corresponding products 6z–ac in 62–98% yields, no hydroarylation reaction occurred in the case of the 1-bromo-2-butyne (4ad) derivative, which indicates the limitations of the reaction to terminal alkynes (Scheme 3).

The synthetic significance of our developed strategy was demonstrated by the synthesis of a nonracemic azepino[4,5-b]indole derivative. The enantiopure (R)-2-phenyl-N-tosyl aziridine (R-1a, ee >99%) was treated with 2a to give the corresponding ring-opening product, which upon treatment with propargyl bromide afforded the corresponding N-propargylated product (R)-4a in 99% yield. Subsequent intramolecular hydroarylation cyclization of (R)-4a furnished the desired azepino[4,5-b]indole (R)-6a in 96% yield and 98% ee (Scheme 4). To further demonstrate the practical utility of our protocol, we carried out some useful post-transformations including metathesis, isomerization, reduction, and oxidation reactions on the synthesized hexahydroazepino[4,5-b]indole derivatives to convert them into other pharmaceutically valuable compounds. We performed [Au]-catalyzed intramolecular hydroarylation of 4a in the CH₃CN medium followed by p-TSA-mediated isomerization in the same pot, which afforded the corresponding highly conjugated tetrahydroazepino[4,5-b]indole product 7a in 86% overall yield (Scheme 5a).²²

Scheme 4 Synthesis of (R)-5-methylenehexahydroazepino [4,5-b]indoles.

Scheme 5 Synthetic applications.

Pd/C-catalyzed hydrogenation of 6a gave the corresponding azepine ring saturated product 5,6-dimethyl-1-phenyl-3-tosyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole (8a) in 92% yield (Scheme 5b).²³ Grubbs-II-catalyzed alkene metathesis of 6x afforded the corresponding 1-phenyl-3-tosyl-1,2,3,4,6,7-hexahydroazepino[3,4,5-hi]benzo[b]indolizine (9) in 71% yield,²⁴ which in the presence of p-TSA undergoes exo–endo isomerization to generate the corresponding 1-phenyl-3-tosyl-1,2,3,5,6,7-hexahydroazepino[3,4,5-hi]benzo[b]indolizine (10) with 80% yield (Scheme 5c), and this reaction was successfully transformed into a one pot, two-step process to give the corresponding product 10 in 79% overall yield.

Next, we wanted to oxidise the azepino[4,5-b]indole 6a to the corresponding epoxide 11a′. When the oxidation of azepino[4,5-b]indole 6a was performed in the presence of m-CPBA, to our utter surprise, the corresponding epoxide compound 11a′ was not formed; instead, the tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)-dione 11a was obtained in 13% yield unprecedentedly. The formation of the product 11a was rationalized by a series of cascade reactions: the compound 6a undergoes Prilezhaev epoxidation²⁵ followed by Meinwald rearrangement,²⁶ subsequently, Baeyer–Villiger oxidation,²⁷ Witkop oxidation,²⁸ and finally a hydrolysis reaction (PMBWH) to afford the tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)-dione 11a (Scheme 6). Here, a single reagent triggers a series of cascade reactions, representing a unique and unprecedented transformation. The structure of the compound 11a was confirmed by single-crystal X-ray analysis. The compound 11a appears to be highly impressive in terms of the presence of several functional groups and certainly it is worth pursuing its biological evaluation.

Scheme 6 Synthetic application: mCPBA-mediated oxidation reaction.

Furthermore, to optimize the reaction conditions in terms of yield, shorter reaction time and selectivity, several solvents, amounts of oxidants, mild bases and temperatures were screened (Table 2). We found that 3.0 equiv. of m-CPBA and NaHCO₃ each in 1,4-dioxane solvent at rt were the best conditions for the oxidation reaction (Table 2, entry 8). With the optimized conditions in hand, we explored structural variations on the azepino[4,5-b]indole framework. In the case of azepino[4,5-b]indoles 6a (Ar¹ = C₆H₅) and 6o (Ar¹ = p-CH₃C₆H₄), the corresponding products 11a and 11b were obtained in 95% and 91% yields, respectively (Scheme 7). In contrast, in the case of o-Br (6i), p-Cl (6l) and p-CF₃ (6n) substituted derivatives, the corresponding products 11c–e were obtained in 61–80% yields. The reaction rate was found to be a little bit slower in the case of the 5-Br (6aa) substituted derivative to give the corresponding product 11g in 89% yield. However, in the case of the N-Ns (6c) substituted derivative, the reaction was completed in 3 h to give the corresponding product 11h in 90% yield.

Scheme 7 Substrate scope of oxidation products. Unless otherwise stated, all the reactions were carried out with 1.0 equiv. (0.113 mmol) of 6, 3.0 equiv. of m-CPBA and 3.0 equiv. of NaHCO₃ in 1,4-dioxane solvent (2.0 mL) at room temperature.

Table 2 Optimization study for the synthesis of tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)dione^a

S. no.	Reaction conditions	Time (h)	% Yield^b
a Unless otherwise stated, all the reactions were carried out with 0.113 mmol (50 mg, 1.0 equiv.) of 6a at room temperature in the appropriate solvent (1.0 mL). b Yield of the isolated product.
1	m-CPBA (1.0), NaHCO3 (1.0), DCM, rt	24	13
2	m-CPBA (2.0), NaHCO3 (2.0), DCM, rt	24	30
3	m-CPBA (3.0), NaHCO3 (3.0), DCM, rt	2	60
4	m-CPBA (3.0), DCM, rt	2	65
5	m-CPBA (3.0), NaHCO3 (3.0), DCE, rt	2	67
6	m-CPBA (3.0), NaHCO3 (3.0), toluene, rt	3	42
7	m-CPBA (3.0), NaHCO3 (3.0), THF, rt	2	57
8	m-CPBA (3.0), NaHCO 3 (3.0), 1,4-dioxane, rt	2	95
9	m-CPBA (3.0), NaHCO3 (3.0), 1,4-dioxane, 0 °C	2	85
10	m-CPBA (3.0), NaHCO3 (3.0), DMF, rt	10	20
11	m-CPBA (3.0), NaHCO3 (3.0), DMSO, rt	10	—
12	m-CPBA (3.0), NaHCO3 (3.0), THF : H2O (4 : 1), rt	5	70

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Furthermore, to increase the synthetic utility of our developed methodology, the protocol was applied to the gram-scale synthesis of azepino[4,5-b]indole 6a and tetrahydrobenzo[f][1,5]diazecine 11avia ROC of aziridine 1a with N-Me indole and propargyl bromide to give the corresponding products in 94% and 64% yields, respectively (Scheme 8a and b).

Scheme 8 Gram-scale synthesis.

Mechanistic considerations

To gain insight into the reaction mechanism, we conducted a control experiment. The formation of 1-vinyl-tetrahydropyrido[3,4-b]indole in 91% yield starting from allene compound 12 under the optimized conditions suggests that the reaction does not proceed via an allene intermediate (Scheme 9).

Scheme 9 Control experiment.

In this study, a plausible reaction mechanism has been proposed based on the literature reports and our experimental findings (Scheme 10). Detailed DFT calculations have been performed to substantiate our experimental results and mechanistic proposal (Fig. 2). The optimised geometries of intermediates and transition states and their respective energetics and coordinates are given in the SI (Fig. S93–105). At first, A (aziridine) interacts with the Lewis acid (LiClO₄) catalyst to form an activated aziridine intermediate, which reacts with B (indole) to generate intermediate C.^17d Furthermore, C get rearomatized to generate D which undergoes propargylation in the presence of K₂CO₃ to form the species E (C1⋯C2 = 1.21 Å). In the next step, the precatalyst K (Ph₃PAuCl) upon reaction with AgOTf generates the active catalyst J (Ph₃PAuOTf) with the formation of AgCl.²⁹ The active catalyst J further reacts with E (C1⋯C2 = 1.21 Å) to form species E-i (activated π-Au^I species, C1⋯C2 = 1.30 Å, C2⋯Au = 2.98 Å and C1⋯Au = 2.05 Å) and –OTf (L).¹²

Scheme 10 Plausible mechanism.

Fig. 2 Free energy reaction profile for the homogeneous [Au]-catalyzed 7-exo-dig hydroarylation cyclization reaction at rt and 1 atm pressure. The optimized transition state structure *FGTS1 with important bond lengths (in Å) and bond angles (°) labelled. For clarity, hydrogen atoms attached to carbon atoms are not shown. The following color codes have been used to represent various elements Au (light yellow), C (grey), N (blue), O (red), H (white), P (orange) and S (yellow).

The formation of E-i from E is an endothermic process which requires 14.2 kcal mol⁻¹ of energy barrier. The C1⋯C2 bond length stretches from 1.21 Å (E) to 1.30 Å (E-i), which indicates the formation of E-i. The species E-i undergoes intramolecular spiro-hydroarylation to generate the spiro-cyclic cation intermediate F (C1⋯C2 = 1.34 Å, C1⋯Au = 2.04 Å, 2.0 kcal mol⁻¹) in an exothermic process (losing 12.2 kcal mol⁻¹ of energy). In this step, the C1⋯C2 bond length stretched from 1.30 (E) to 1.34 (E-i), and the C1⋯Au bond length shortens from 2.05 (E) to 2.04 (E-i). Furthermore, the formation of the cationic species G (C1⋯C2 = 1.46 Å, C1⋯Au = 2.02 Å) takes place from F (C1⋯C2 = 1.34 Å, C1⋯Au = 2.04 Å) via a 1,2-alkenyl shift mechanism. In these transformations, the C1⋯C2 bond length in G stretched from 1.34 Å (F) to 1.46 Å (G), and there is a slight change (shortening) in the C1⋯Au bond length.

The elongation in the C1⋯C2 bond length is responsible for the shift in electron density and the formation of the seven-membered ring. This is an exothermic step. As per the literature reports and experimental evidence, the formation of G from F could be possible via the transition states *FGTS1 and *FGTS2. Therefore, detailed DFT calculations were also performed to confirm which transition state is involved or favourable in this step.

The DFT calculations confirm that the formation of G from F could be possible only via*FGTS1 (C1⋯C2 = 1.41 Å, C1⋯Au = 2.03 Å, C5–C6–C7 = 74.9°, C6–C7–C2 = 113.2°, C2–C3–N1 = 112.2°, C3–N1–C4 = 112.9°, and C1–C4–C5 = 111.41°) which requires 14.0 kcal mol⁻¹ energy barrier (an endothermic step). We tried to find another possible transition state *FGTS2, but we could not get the desired result, although the possibility of *FGTS2 cannot be ruled out.³⁰ Moreover, re-aromatization of G takes place to generate species H (7-exo-dig hydroarylation product) through an exothermic (−25.1 kcal mol⁻¹) process. Furthermore, catalyst regeneration takes place (M (AuPPh₃) which further reacts with ⁻OTf to the catalyst J (Ph₃PAuOTf)) with the formation of I involving a protodemetalation step in an exothermic process.

Biological studies

The unprecedented formation of the diazecine compounds inspired us to carry out their biological evaluation. The synthesized diazecine compounds were systematically evaluated for their cytotoxic activity against U87MG glioblastoma and HeLa cervical carcinoma cell lines. An initial screening was conducted with two representative analogues 6i and 11c. Notably, compound 11c exhibited pronounced cytotoxic effects against both the cancer cell lines, whereas 6i demonstrated no discernible toxicity (Fig. 3). This prompted us to conduct a comprehensive assessment of all the synthesised derivatives of 11 (Table 3).

Fig. 3 Cell viability curves of compounds 6i, 11c, cisplatin and temozolomide in HeLa and U78MG cell lines.

Table 3 Cell viability of compounds assessed by the MTT assay after 48 h of incubation in glioma (U87MG) and cervical cancer (HeLa) cell lines

IC50 (µM, 48 h)
S. no.	Compound	U87MG	HeLa
1	11a	2.54 ± 0.48	4.11 ± 0.67
2	11b	2.64 ± 0.76	2.76 ± 0.28
3	11c	2.30 ± 0.43	7.85 ± 6.21
4	11d	3.30 ± 2.71	7.75 ± 0.51
5	11e	6.05 ± 2.35	4.36 ± 0.77
6	11f	3.91 ± 0.25	8.35 ± 2.12
7	11g	5.55 ± 1.28	23.91 ± 18.23
8	11h	>50	>50
9	6i	>50	>50
10	Temozolomide	15.01 ± 6.78	—
11	Cisplatin	3.25 ± 0.56	23.16 ± 3.86

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It was observed that all compounds incorporating a 10-membered ring structure exhibited significant cytotoxicity toward both U87MG and HeLa cells, with the exception of compound 11g. The observed cytotoxicity is probably due to the presence of >C=O, –CON<, OH and >NSO₂–, which collectively enhance the molecular hydrophilicity and facilitate hydrogen bonding interactions, potentially influencing cellular uptake and intracellular target engagement. Conversely, compound 6i, characterized by a 7-membered ring, lacks these critical carbonyl groups (hydrogen bond acceptors) and hydroxyl groups (donors and acceptors both), which may account for its lack of cytotoxic activity.

Furthermore, the cytotoxic potential of all tested compounds was markedly higher in U87MG glioma cells, with IC₅₀ values consistently below 7 µM, compared to HeLa cells following 48 hours of incubation. This differential sensitivity suggests a potential selective vulnerability of U87MG cells to this class of compounds, warranting further mechanistic investigations.

Conclusions

In summary, we have developed a two-pot, three-step protocol for the synthesis of 5-methylene-1,2,3,4,5,6-hexahydroazepino[4,5-b]indoles, via LiClO₄-catalyzed S_N2-type nucleophilic ring-opening of activated aziridines, followed by a base-mediated propargylation reaction in the same pot to give the corresponding N-propargylated products in good to excellent yields. The N-propargylated products were subjected to [Au]-catalyzed homogeneous catalysis to undergo a 7-exo-dig hydroarylation to give the corresponding hexahydroazepino[4,5-b]indoles with up to 99% yield and 98% ee (for non-racemic aziridines). p-TSA-mediated exo–endo isomerization in the same pot gives the corresponding 1,2,3,6-tetrahydroazepino[4,5-b]indoles in high yield. Interestingly, the m-CPBA-mediated oxidation of the resulting hexahydroazepino[4,5-b]indoles led to the unprecedented formation of the corresponding tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)-diones via a Prilezhaev epoxidation, followed by a Meinwald rearrangement and a Baeyer–Villiger oxidation, culminating in a Witkop oxidation and hydrolysis cascade. Such diazecine compounds exhibit potent anticancer activity. The key advantage of our synthesized ten-membered tetrahydrobenzo[f][1,5]diazecine-2,8(1H,3H)-diones 11 is their superior cytotoxic activity compared to cisplatin and temozolomide, which are established drugs for the treatment of cervical and brain cancers, respectively. Further study of Au-catalyzed hydroarylation reactions including aziridine chemistry is in progress in our laboratory. Additionally, we are investigating the mechanistic profile of these compounds, focusing on their effects on 3D tumor spheroids, cell death mechanisms, cellular uptake pathways, and interactions with various cellular organelles.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: (a) experimental procedures, (b) material/compound characterization, (c) DFT study, (d) NMR spectra, (e) HRMS data, (f) IR-data, (g) HPLC data and chromatograms, (h) biological studies and supplementary figures. See DOI: https://doi.org/10.1039/d5qo01306b.

CCDC 2356344 ((±)-5a), 2356345 (6a), 2356346 (6c), 2464359 (11a), and 2356348 (11c) contain the supplementary crystallographic data for this paper.^31a–e

Acknowledgements

M. K. G. is grateful to IIT Kanpur and ANRF (SERB), India, for the financial support. R. G. S. is grateful to IIT Kanpur, India, for the financial support. B. S. and S. K. thank CSIR, India, and G. G., P. C., P. R., S. S. and R. M. thank IIT Kanpur, India. A. S. thanks the PMRF scheme for a fellowship. M. K. G. specially thanks Mr Indresh Verma for the important crystal analysis. We thank the IIT Kanpur for providing high-performance computing (HPC) facilities.

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Footnote

† These authors contributed equally.

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