Et₃N/DMSO-supported one-pot synthesis of highly fluorescent β-carboline-linked benzothiophenones via sulfur insertion and estimation of the photophysical properties

Introduction

The pyrido[3,4-b]indole moiety, commonly referred as β-carboline, is the core unit of about one quarter of all natural products [1-4] and pharmacologically active compounds endowed with anticancer [5-9], anti-inflammatory, antioxidant, antimalarial, antifungal, and antileishmanial activities (Figure 1) [10-13]. Notably, this privileged scaffold is incorporated in several marketed drugs such as abecarnil, tadalafil, cipargamin, yohimbine, etc. which are used in the treatment of various ailments [14,15]. Apart from their pharmaceutical properties, β-carboline derivatives also found various applications in fields such as organocatalysts, as ligands, and fluorescent probes [16-18]. Importantly, β-carbolines are also used as fluorescence standards. Recently, a novel β-carboline-based fluorescent chemosensor was developed by Batra and co-workers for the quantitative analysis of fluoride ions (F⁻) at ppb level [19].

Sulfur-containing organic compounds are broadly associated with numerous bioactive natural products and pharmaceutical drugs [20-22]. Thioaurones (2-benzylidene benzo[b]thiophen-3(2H)-one) are sulfur-containing heterocyclic compounds, an important subclass of flavonoids which were first introduced by O’Sullivan in 1977 [23]. Specifically, thioaurones and their analogs show a variety of biological activities such as anticancer [24], inhibition of tyrosine phosphatase 1B, antioxidant properties, etc. [25-27]. Due to their numerous applications, they have found diverse uses such as thioindigo-like dyes, photoresponsive devices, and photoswitchable biomolecules [28-31]. Moreover, these compounds were also used as synthetic intermediates for various sulfur-containing bioactive molecules (Figure 1) [32-34].

In organic synthesis, aromatic compounds having nitro groups play a vital role as building blocks for the synthesis of nitrogen-containing functional groups and aza-heterocyclic frameworks. However, organic transformations in which aromatic nitro groups act as leaving groups are less reported and require the use of transition-metal catalysts such as Cu, Rh, Pd, etc. [35-37]. Though, several elegant methods have been developed for the synthesis of benzothiophenes, however, these methods rely on the use of organosulfur-based substrates [38-41]. Moreover, these methods are associated with some limitations such as using costly metal catalysts, air-sensitive starting materials, malodorous sulfides or thiols, low yields, and multistep syntheses. To overcome these drawbacks, elemental sulfur has emerged as a surrogate approach, where it can be inserted in situ. In this context, several research groups are actively involved in the development of novel and efficient approaches for the synthesis of sulfur-containing heterocycles [42-46].

In our research endeavors, we have been involved in the exploration of the synthetic potential of 1-formyl-9H-β-carboline (an alkaloid, kumujian C) [47] for preparing chemical libraries of β-carboline-substituted [48-54] and N-fused heterocycles [55,56] which were attributed to the presence of an electrophilic as well as a nucleophilic functionality in this natural product [1]. Encouraged by the applications of β-carboline and benzothiophene motifs in medicinal and materials chemistry, it was envisaged to construct a β-carboline-based novel molecular hybrid containing the benzothiophene moiety (Figure 1) [57,58]. The present study was inspired by the recent findings of Nguyen and co-workers [59-61]. As a part of our ongoing project [62,63], we devised a simple and efficient one-pot practical approach for the construction of β-carboline-tethered benzothiophenone derivatives via incorporation of sulfur. To the best of our knowledge, this is the first report of one-pot synthesis of novel β-carboline-tethered benzothiophenones and evaluation of their light-emitting properties. In this regard, detailed studies are presented and discussed herein.

Results and Discussion

The present study commenced with the synthesis of the β-carboline-based 2-nitrochalcone 1bA which was prepared via a Claisen–Schmidt condensation of 1-formyl-β-carboline (1b) with 2-nitroacetophenone (A) in the presence of KOH (1.05 equiv) in dry MeOH at room temperature (Scheme 1). The analytically pure product was obtained in 86% yield by simple filtration of the precipitate followed by washing with dry MeOH.

With the objective to synthesize β-carboline-linked benzothiophenone frameworks, we set up screening of conditions for the reaction between the β-carboline-based 2-nitrochalcone 1bA and elemental sulfur by testing different activators (Table 1). Recent findings revealed that a combination of an aliphatic amine with DMSO activated elemental sulfur for an electrophilic addition to generate thioaurones and benzothiophenes [59]. At 70 °C, the use of DIPEA in combination with DMF as the solvent was found to be an excellent sulfur activator, leading to the formation of the desired product 2bA in 50% yield after a short silica gel column chromatographic separation (Table 1, entry 1). The structure of 2bA was confirmed on the basis of spectroscopic data. The ¹H NMR spectrum displayed a singlet for one methine proton at δ 8.69 ppm and the presence of additional nine aromatic protons for the β-carboline and benzothiophenone frameworks indicated the formation of the desired product. When elemental sulfur and DIPEA were used in DMSO, a significant increase in the yield (73%) was observed (Table 1, entry 2). At this stage, we realized that DMSO was a better choice for this transformation as the reaction required a shorter time and afforded the product 2bA in a better yield. Then, other amines such as NMP, Et₃N, DBU, and DABCO were also investigated. The use of NMP as an activator in DMSO yielded the desired product in only 65% yield (Table 1, entry 3). Interestingly, Et₃N in combination with DMSO at 70 °C afforded the anticipated product 2bA in 78% yield within a short span of 20 min (Table 1, entry 4). The reaction at 90 °C gave product 2bA in 76% yield, however, the same reaction performed at 30 °C was found to be sluggish and complete conversion could not be achieved even after 12 h (Table 1, entries 5 and 6). We also observed that the reaction in the absence of Et₃N failed to generate the anticipated product 2bA (Table 1, entry 7), which supported the importance of an amine/base with DMSO as an activator. Potassium ethylxanthate [64,65] and sodium sulfide as a sulfur sources in the presence of Et₃N in DMSO (Table 1, entries 8 and 9) also did not furnish the desired product 2bA. A decomposition of the product was observed in the case of Na₂S. Interestingly, the combination of DABCO and DMSO also afforded the desired product via a clean reaction within 20 min, although only 60% yield of the product was obtained (Table 1, entry 10). This promising result using DABCO encouraged us to explore other amines like DBU, ʟ-proline, pyridine, DMAP, and pyrrolidine as an activator but encouraging results were not obtained (Table 1, entries 11–15). Similarly, the use of KI as an activator also failed to promote the reaction (Table 1, entry 16) [62]. Eventually, we came to the conclusion that a combination of Et₃N and DMSO excellently activated elemental sulfur at 70 °C and therefore chose these conditions for the construction of other β-carboline-linked benzothiophenone derivatives.

With the standardized conditions identified, the scope of this domino approach was investigated with diversely substituted β-carboline-based 2-nitrochalcones 1aA-bA, 1dA, and 1hA prepared from aldehydes 1a-b, 1d and 1h in 76–91% yields (Scheme 1). The methodology was found to be general in nature and produced the fluorescent β-carboline-linked benzothiophenone derivatives 2aA-bA, 2dA and 2hA within 15–45 min in DMSO at 70 °C as depicted in Scheme 2. The analytically pure products were obtained in 42–86% yields after a short silica gel column chromatographic purification. It was observed that N-alkyl-β-carboline-based 2-nitrochalcones 1bA, 1dA, and 1hA reacted faster and delivered the products 2bA, 2dA, and 2hA in better yields (78-86%). Conversely, the substrate 1aA bearing a free NH was found to be slow reacting and produced 2aA in a lower yield (42%).

From the perspective of green chemistry, one-pot reactions are preferred as less waste is generated due to the avoidance of work-up, isolation, and purification of intermediates [66]. Accordingly, the feasibility of a one-pot synthesis of the targeted products was attempted. Therefore, after the formation of the 2-nitrochalcone 1bA, excess of MeOH was decanted, and the crude product was redissolved in 1 mL of DMSO followed by the sequential addition of Et₃N (5 equiv) and elemental sulfur (5 equiv). To our pleasure, the reaction at 70 °C smoothly afforded the corresponding β-carboline-linked benzothiophenone derivative 2bA in less than 30 min. More importantly, a clean reaction was observed during the one-pot strategy which surely avoided the isolation of the intermediate (2-nitrochalcone derivative 1bA), and a significant increment in the overall yield of 2bA (from 67% to 74%) was also noted. Similarly, a remarkable improvement in the yields of 2aA (from 32% to 35%), 2dA (from 78% to 83%), and 2hA (from 69% to 78%) was also observed during the one-pot approach. The comparison of the product yields obtained through both approaches are summarized in Scheme 3.

Having successfully established a one-pot strategy, we next turned our attention to the generality and scope of the method. Interestingly, diversely substituted 1-formyl-β-carbolines 1a–m (except 1k) reacted efficiently with nitroacetophenones A,B in one pot furnishing the anticipated products 2aA–nA, 2bB, and 2hB as depicted in Scheme 4. The synthesized products were purified through silica gel column chromatography and further washed with anhydrous methanol to yield the analytically pure products in 35–83% yields (two-step yield), except for 2kA, which was obtained in trace amounts only. We observed that N-alkyl derivatives 1bA–jA, 1lA–nA, 1bB, and 1hB reacted faster and led to higher product yields. The substrate B bearing a chloro substituent required longer reaction times and afforded the targeted products 2bB and 2hB in slightly lower yields.

Encouraged by the results obtained from the one-pot synthesis of β-carboline C-1 substituted benzothiophenone derivatives, we were interested if the scope of this one-pot strategy could be extended for the synthesis of β-carboline C-3-tethered benzothiophenones (Scheme 5). Thus, the Claisen–Schmidt condensation of 3-formyl-9H-β-carbolines 3a–g [51] with substituted 2-nitroacetophenones (A and B) in the presence of KOH delivered the corresponding 2-nitrochalcones (3aA–gA and 3eB). The in situ-generated β-carboline-based 2-nitrochalcones were further treated with elemental sulfur in the presence of Et₃N in DMSO at 70 °C straightforwardly affording the cyclized products 4aA–gA and 4eB in 48–79% yield as presented in Scheme 5.

It was observed that the reaction time and yields were affected by the nature of the substituent at the C1 (R⁴) and N-9 (R²) position of β-carboline ring. Substrates bearing a dimethoxymethyl group (3c and 3d) reacted smoothly and within shorter reaction time. Similarly, N-alkyl (R²) 3-formyl-β-carbolines (3b, 3d, 3f, and 3g) also reacted faster and delivered higher yields as compared to free NH derivatives (3a, 3c, and 3e). In case of the dihalogenated product 4eB, a slow reaction accompanied with a low yield was detected due to presence of electron-withdrawing substituents in starting compound B. The slightly lower yields obtained for 2bB, 2hB, and 4eB were possibly due to the low reactivity of substrate B during the condensation process (step 1), as in the cyclization process, the presence of the electron-withdrawing substituents in B seemed to favor the anticipated S_NAr mechanism by stabilizing the negatively charged intermediate 10 (Figure 2). Overall, it was noted that the 1-formyl-β-carbolines 1a–m reacted faster and afforded the corresponding products in higher yields as compared to 3-formyl-β-carbolines 3a–g which may be attributed to the higher electrophilicity of the formyl group at C1 position of the β-carboline ring.

Inspired by the results of the above study, it was envisaged to check the generality of this strategy for the synthesis of β-carboline linked benzothiophene derivatives. Accordingly, we employed 1-acetyl-β-carboline 5 [67,68] for Claisen–Schimdt condensation with 2-nitrobenzaldehyde (C) in the presence of Cs₂CO₃ and anhydrous THF at room temperature. Product 5C was obtained as a major product along with some unidentified impurities. The evaporation of excess solvent (THF) followed by the treatment of the resultant crude, 2-nitrochalcone 5C with Et₃N and elemental sulfur in DMSO at 70 °C furnished the expected product 6C, albeit in a low yield (39%) as shown in Scheme 6. It is important to mention that the Claisen–Schmidt condensation of 5 with 2-nitrobenzaldehyde (C) could not be achieved with KOH in MeOH or Cs₂CO₃ in DMSO.

To probe the reaction mechanism, a control experiment was conducted with model substrate 1bA in the presence of a radical scavenger (TEMPO) to check the possibility of a radical mechanism vs an electrophilic addition of sulfur [69] (Scheme 7). It was observed that the reaction could not be completed even after 24 h in the presence of TEMPO whereas only 20 min were required for completion under standard conditions. Thus, it is assumed that the reaction proceeds through a radical pathway.

Based on our observations during the present study and previous reports [69], a plausible mechanism for the formation of the benzothiophenone ring is depicted in Figure 2. It is anticipated that an initial formation of trisulfur radical anion (S₃^·−) occurs via the reaction of elemental sulfur with triethylamine in DMSO. The addition of the trisulfur radical anion to the double bond in 2-nitrochalcone (1bA) may yield the intermediate 7. The further abstraction of hydrogen in intermediate 7 may result in formation of intermediate 8. The cleavage of the S–S bond in 8 under basic conditions may generate sulfur anion 9. The nucleophilic substitution reaction (S_NAr) by transit the sulfur anion in 9 followed by dismissal of the nitrite ion may result in the formation of the β-carboline-tethered benzothiophenone derivative 2bA. It is anticipated that the role of DMSO is to stabilize the ionic intermediates, specifically 10 and to accelerate the transformation.

Photophysical studies

Fluorescence, offering a nondestructive exceptional technique to monitor a system of interest at the molecular level [70-72], has found wide-ranging applications in several research areas such as medicine, pharmaceutics, biology, environment, and food science [73,74]. Therefore, the light-emitting properties of the novel β-carboline C1 as well as C3-substituted benzothiophenone derivatives 2aA–nA, 2bB, 2hB, 4aA–gA, 4eB, and 6C were evaluated to stimulate their further exploration for possible applications in the field of materials science as chemosensors, ligands, and fluorescent probes. In order to investigate the fluorescence properties, compound 2bA was chosen as the model substrate for optimization of various parameters like time, concentration, and solvent. In order to obtain the maximum emission, various solvents were screened. The synthesized compounds showed best solubility and displayed a maximum intensity in CHCl₃ (Supporting Information File 1) as compared to other solvents (DMSO, DMF, and MeOH). No considerable change in the fluorescence intensity of was observed even after 5 h of sample preparation. Next, after careful analysis of concentrations, a 4 µM concentration in CHCl₃ was found to be optimal for the photophysical studies of the synthesized derivatives.

The fluorescence quantum efficiency (Φ_F) was measured relative to quinine sulfate (Φ_R = 0.546 in 0.1 M H₂SO₄ under 350 nm excitation) as a reference compound. For the measurement of UV–vis absorption and fluorescence emission of the samples, stock solutions of 1.0 mM concentration were prepared using analytical grade CHCl₃ as the solvent, and diluted to the final concentration of 4.0 μM. Next, we carefully measured the photophysical properties at room temperature including absorption, excitation, emission, Stokes shift, fluorescence quantum efficiency, molar extinction coefficient, and brightness. The photophysical data of the β-carboline C1 or C3-tethered benzothiophenone derivatives are summarized in Table 2, and their graphical data are depicted in Figure 3 and Figure 4. The quantum yields were calculated based on Equation 1.

where R means reference and S sample, respectively.

Generally weak fluorescence emissions were observed for the thiophene-based chromophores due to a remarkable spin–orbit coupling which is originating from the heavy atom effect of sulfur [73,74]. It is worth mentioning that the majority of the β-carboline-substituted benzothiophenone derivatives showed good fluorescence. The emission maxima of the fluorophores showed a wide region for fluorescent emissions (λ_em, 490–582 nm in CHCl₃) along with large Stokes shifts (up to 293 nm), excellent quantum yields (up to 47%), and high brightness (up to 11196). The brightness of the fluorophores was calculated by multiplication of the quantum yield (Φ) with its molar extinction coefficient (ε).

In case of the β-carboline C1-substituted benzothiophenones 2aA–nA, 2bB, and 2hB, the substituents R¹ and R² significantly affected the fluorescence of the compounds. It was noted that the fluorescence increased with lengthening of the alkyl chain at N-9 (R²) and followed the order n-Bu > n-Pr > Et > Me > H. The β-carboline derivative with free N–H (N-9), 2aA, showed a low fluorescence quantum yield (Φ_F = 18%) in this series (Table 2 and Figure 3). The presence of a benzyl group at R² (2hA), improved the photophysical properties including a higher quantum yield (Φ_F = 47%). With variation of the substituents at the R¹ position, a regular pattern of fluorescence was observed, i.e., CO₂iPr > CO₂Me > H which may be attributed to the electron-withdrawing nature of the substituents (ester group) at C3 position of the β-carboline ring.

Interestingly, a similar trend was observed in case of β-carboline C3-substituted benzothiophenone derivatives (4aA–gA and 4eB). Compared to 4aA and 4aC bearing aliphatic substituents at C1 (R⁴), compound 4eA with aromatic substituent exhibited better fluorescence due to extended conjugation. The effect of R² substituent in these derivatives (4bA, 4dA, 4fA and 4gA) was also investigated and it was found that N-alkylation improved the photophysical properties along with higher quantum yields (Figure 4). With regard to the impact of R³ substituent, thiopheneone derivatives with chloro substitution (2bB and 4eB) displayed a higher quantum yield than unsubstituted derivatives (2bA and 4eA) as evident from Table 2. Overall, it can be concluded that β-carboline C1 substituted benzothiophenone derivatives exhibited better photophysical properties including high quantum yield, brightness and significant bathochromic shift in the emission wavelengths. In short, β-carboline-substituted benzothiophenone derivatives emerged as excellent fluorophores and displayed remarkable photophysical properties with quantum yield (Φ_F) up to 47%. It is believed that these compounds may find applications in materials science and biomedical investigations.

Conclusion

In summary, an efficient synthesis of highly fluorescent β-carboline-linked benzothiophenone derivatives was successfully accomplished through a one-pot metal-free approach for the first time. The transformation could be executed from β-carboline-based 2-nitrochalcones via a one-pot, two-step procedure starting from 1(3)-formyl-β-carbolines (a framework represented by alkaloid kumujian C). The combination of Et₃N and DMSO played a vital role in the activation of sulfur resulting in the formation of two C–S bonds in a single operation. This strategy offers several advantages, such as one-pot procedure, operational simplicity, easy purification, use of inexpensive reagents, and wide functional group compatibility. Importantly, the presence of two important pharmacophores along with the exocyclic double bond with Michael acceptor properties in the title compounds offers the opportunity to explore their biological potential. Moreover, these β-carboline-linked benzothiophenones displayed excellent fluorescence properties with quantum yields (Φ_F) of up to 47%. Detailed studies to synthesize novel fluorophores with improved optical properties which can easily find application in materials science are underway in our laboratory

Experimental

General experimental procedure for the synthesis of β-carboline-based 2-nitrochalcone derivatives (1aA, 1bA, 1dA and 1hA) as exemplified for compound 1bA. To a stirred solution of KOH (0.033 g, 0.587 mmol) in dry MeOH (4 mL), 2-nitroacetophenone (A, 0.080 mL, 0.587 mmol) was added at room temperature, and the reaction mixture was stirred for 15 min. Thereafter, 1b (0.15 g, 0.560 mmol) was added portionwise and the reaction mixture was allowed to stir for an additional 1 h at room temperature. After completion of the reaction (as monitored by TLC), the precipitate was filtered through a sintered funnel, washed twice with anhydrous MeOH, and dried in vacuum to obtain the analytically pure product 1bA, 0.20 g (86%) as yellow solid.

General experimental procedure for the synthesis of β-carboline C-1-substituted benzothiophenone derivatives (2aA, 2bA, 2dA, and 2hA) as exemplified for compound 2bA. A 10 mL round-bottomed flask was charged with 2-nitrochalcone 1bA (0.20 g, 0.482 mmol), Et₃N (0.336 mL, 2.41 mmol), sulfur powder (0.077 g, 2.41 mmol), DMSO (1 mL), and the reaction mixture was stirred at 70 °C for 20 min. After completion of the reaction, as analyzed by TLC, the crude product was directly purified by silica gel column chromatography (CHCl₃/MeOH 95:5, v/v) without aqueous treatment to afford 0.15 g of 2bA (78%) as orange solid.

One-pot experimental procedure for the synthesis of β-carboline C-1(3)-substituted benzothiophenone derivatives (2aA–nA, 2bB, 2hB, 4aA–gA, and 4eB) as exemplified for compound 2bA. To a stirred solution of KOH (0.033 g, 0.587 mmol) in dry MeOH (4 mL) in a 10 mL round-bottomed flask; 2-nitroacetophenone (0.080 mL, 0.587 mmol) was added at room temperature and the reaction mixture was stirred for 15 min. Thereafter, methyl 1-formyl-9-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (1b, 0.15 g, 0.560 mmol) was added portionwise and the reaction mixture was allowed to stir for an additional 1 h at room temperature. After completion of the reaction (as detected by TLC), the reaction content was allowed to settle for 5 min, MeOH was decanted, and evaporated under reduced pressure. Thereafter, DMSO (1.5 mL) was added to the crude product 1bA (nitrochalcone) followed by the sequential addition of sulfur powder (0.089 g, 2.80 mmol) and Et₃N (0.390 mL, 2.80 mmol) at room temperature. The reaction mixture was stirred at 70 °C for 20 min. After completion of the reaction (as analyzed by TLC), the product 2bA was directly purified through column chromatography on silica gel (CHCl₃/MeOH 95:5, v/v) to afford the analytically pure product 2bA as orange solid in 74% yield (two step yield).

One-pot experimental procedure for the synthesis of methyl 1-(benzo[b]thiophene-2-carbonyl)-9H-pyrido[3,4-b]indole-3-carboxylate (6C). To a stirred suspension of Cs₂CO₃ (0.182 g, 0.560 mmol) in dry THF (4 mL) in a 10 mL round-bottomed flask, methyl 1-acetyl-9-benzyl-9H-pyrido[3,4-b]indole-3-carboxylate (5, 0.10 g, 0.373 mmol) was added and the mixture was stirred for 10 min. Thereafter, 2-nitrobenzaldehyde (C, 0.062 g, 0.410 mmol) was added and the reaction mixture was stirred for additional 2 h at room temperature. After completion of the reaction (TLC), THF was evaporated under reduced pressure. Next, the crude nitrochalcone 5C was re-dissolved in DMSO (1 mL) followed by the addition of sulfur powder (0.060 g, 1.86 mmol) and Et₃N (0.260 mL, 1.86 mmol) at room temperature. The reaction mixture was stirred at 70 °C for 1 h. After completion of the reaction, the product was directly purified by silica gel column chromatography (hexane/EtOAc 60:40, v/v) to afford 0.056 g (39%) of 6C as light brown solid (two step yield).