Reactivity of ethyl nitrosoacrylate toward pyrrole, indole and pyrrolo[3,2-c]carbazole: an experimental and theoretical study

Nitrosoalkenes react with 8-methyl-1,6-dihydropyrrolo[3,2-c]carbazole to give both 2- and 3-alkylated products via hetero-Diels-Alder reaction followed by the cycloadduct ring-opening. Quantum chemical calculations, at DFT level of theory, were carried out to investigate the regioselectivity of the cycloaddition of ethyl nitrosoacrylate with 1,6-dihydropyrrolo[3,2-c]carbazoles as well as with pyrrole and indole, allowing a more comprehensive analysis of the reactivity pattern of nitrosoalkenes with five-membered heterocycles. Furthermore, theoretical calculations confirmed that ethyl nitrosoacrylate reacts with these heterocycles via a LUMOheterodiene-HOMOdienophile controlled cycloaddition. The reactivity of one of the oxime-functionalized 1,6-dihydropyrrolo[3,2-c]carbazole was explored and a new hexahydropyrido[4′,3':4,5]pyrrolo[3,2-c]carbazole system was obtained in high yield via a one-pot, two-step procedure.

The reactivity of nitrosoalkenes with electron-rich heterocycles is strongly influenced not only by the nitrosoalkene substituents but also by the type of heterocycle (Nunes et al., 2014;Lopes et al., 2016). The pioneer work of Gilchrist and co-workers showed that the reaction of ethyl nitrosoacrylate (2, R = CO 2 Et), generated from ethyl bromopyruvate oxime 1a by action of base, with pyrrole and indole afforded the open chain oximes 4 and 8, respectively, through hetero-Diels-Alder reactions (Scheme 1) (Gilchrist and Roberts, 1983;Gilchrist and Lemos, 1993). The outcome and mechanistic pathway of the reaction of nitrosoalkenes with pyrrole and pyrrole derivatives depends on the nitrosoalkene substituent, as shown by experimental and theoretical studies (Nunes et al., 2014). In fact, ethyl nitrosoacrylate (2, R = CO 2 Et) reacts via hetero-Diels-Alder reaction, through the formation of the bicyclic 1,2-oxazine 3 followed by 1,2-oxazine ringopening with concomitant rearomatization of the pyrrole unit, giving the open chain oxime 4, as a single isomer. However, aryl nitrosoalkenes 2 (R = Aryl) react with pyrrole by conjugated addition to give two isomeric oximes 5 and 6. On the other hand, the reaction of both nitrosoalkenes 2 with indole affords open chain oximes as single isomers via hetero-Diels-Alder reaction (Lopes et al., 2016). Furthermore, nitrosoalkenes 2 react with pyrrole to give 2-alkylated products, whereas indole undergoes alkylation at the 3-position, as would be expected from the opposite regioselectivity of the hetero-Diels-Alder reaction.
Pyrrolocarbazoles are tetracyclic ring systems containing a pyrrole ring fused to a carbazole unit. Depending on the position of the pyrrole/carbazole ring junction and the relative position of the pyrrole nitrogen to the carbazole moiety, several structural isomers can be found (Giraud et al., 2019). The best known are the pyrrolo [2,3-c]carbazoles (Zhang and Ready, 2017), mainly because their core structure is present in the marine natural product dictyodendrin A, and pyrrolo[2,3-a]carbazoles for the recognized kinase inhibitory activity of some derivatives (Akué-Gédu et al., 2009;Giraud et al., 2012) (Figure 1). Reports on the synthesis and reactivity of pyrrolo[3,2-c]carbazoles are scarce. Recently, however, a few reports describing the synthesis (Benzi et al., 2019), photophysical and biological activity, namely, antioxidant (Bingul et al., 2019), anticancer and antibacterial activity, have been disclosed (Sengul et al., 2016;Saglam et al., 2021;Sinicropi et al., 2021). Compound 9 is an example of a pyrrolo[3,2-c]carbazole with high cytotoxicity against human colon cancer HT29 cells (Sengul et al., 2016).
Compounds containing an oxime moiety have found a wide range of biological applications, displaying anti-inflammatory, antimicrobial, antioxidant and anticancer activity (Surowiak et al., 2020). In this context, and following our interest in the chemistry of nitrosoalkenes, we decided to explore the reactivity of nitrosoalkenes towards 8-methyl-1,6-dihydropyrrolo[3,2-c] carbazole (10) aiming at the synthesis of oxime-functionalized pyrrolo[3,2-c]carbazoles. The combination of these two structural elements in a single molecule would lead to new chemical entities with increased interest in terms of potential biological activity and with the possibility of further structural modulation.

Results and discussion
Initially, the reaction of ethyl nitrosoacrylate (2a), generated in situ from ethyl bromopyruvate oxime (1a) by action of sodium carbonate, with pyrrolo[3,2-c]carbazole 10 was explored. The reaction, carried out in dichloromethane at room temperature, gave the 3-and 2-alkylated pyrrolo[3,2-c]carbazoles 11a and 12a, respectively, in 63% overall yield (Scheme 2). The same reactivity pattern was observed in the reaction of the less activated nitrosoalkene 2b with 10 affording the open chain oximes 11b and 12b in 37% and 8%, respectively (Scheme 2). In both cases, the regioisomeric open chain oximes were isolated as single stereoisomers, indicating that these were formed via hetero-Diels-Alder reaction followed by 1,2-oxazine ring-opening and concomitant rearomatization of the pyrrole unit.
The structural assignment of the two regioisomers was established by two-dimensional NMR spectroscopy. From the coupling observed in the COSY spectrum of compound 11a, it was possible to assign the signals corresponding to the protons H-2 (7.14 ppm), H-1 (10.49 ppm), H-4 (4.15 ppm) and the proton of the hydroxyimino moiety (11.35 ppm). The stereochemistry of the C,N-bond was established by the NOESY spectrum data, in which cross peaks were observed between the NOH proton and the H-4 protons, confirming the trans orientation of the OH and ester groups. The NOESY spectra of compounds 11b and 12b also showed a correlation between the proton of the oxime moiety and H-4 protons (see Supplementary Material), suggesting the trans orientation of the OH and ester groups.
Preliminary studies on the reactivity of the new 3-alkylated pyrrolo[3,2-c]carbazoles focused on the interconversion of the oxime-amine functional groups. However, the reduction reaction of pyrrolo[3,2-c]carbazole 11a, using zinc in acetic acid at room temperature, led to an unexpected but interesting result. One product was isolated whose 1 H NMR spectrum features, recorded using acetone-d 6 as solvent, were not those expected for the desired amine 13. From the analysis of the 1 H NMR spectrum it was possible to confirm the presence of the pyrrolo[3,2-c]carbazole scaffold, signals corresponding to the ester group as well as to an ABX system, as it would be for amine 13. It was observed that when the product of the reduction reaction was treated with acetone for 1 h, a compound was isolated in 17% yield with a similar 1 H NMR spectrum but in which the presence of two methyl groups could be observed (Scheme 3).

FIGURE 1
Examples of different pyrrolocarbazoles.

FIGURE 2
Relative stabilities (ΔE in kJ/mol) of the transition states involved in the hetero-Diels-Alder reaction of ethyl nitrosoacrylate (2a) with pyrrole considering the two regioisomers and both endo and exo approaches, both for C2 alkylation (A) and for C3 alkylation (B). All structures were optimized at the B3LYP/6-31G (d,p) level of theory. Color code: grey, carbon; red, oxygen; blue, nitrogen and white, hydrogen.

FIGURE 3
Relative stabilities (ΔE in kJ/mol) of transition states involved in the hetero-Diels-Alder reaction of ethyl nitrosoacrylate (2a) with indole considering the two regioisomers and both endo and exo approaches, both for C3 alkylation (A) and C2 alkylation (B). All structures were optimized at the B3LYP/6-31G (d,p) level of theory. Color code: grey, carbon; red, oxygen; blue, nitrogen and white, hydrogen.
The formation of the pentaheterocyclic system 14 can be explained considering the initial reduction of the oxime moiety to give amine 13 followed by its Pictet-Spengler condensation with acetone to give the final product.
The condensation of tryptamines with aldehydes and ketones, known as the Pictet-Spengler reaction, has been extensively explored for the synthesis of ring-fused indole derivatives, including tetrahydro-β-carbolines a core structure of various indole alkaloids (Stöckigt et al., 2011;Maity et al., 2019;Panice et al., 2019;Ribeiro et al., 2022). The mechanism of this transformation has been a research topic of some controversy as two pathways can be considered: the formation of spiroindolenines via the attack of indole's C3 to the initially formed imine followed by a 1,2-migration/elimination sequence to restore aromaticity, or the direct C2 attack. However, there are several experimental studies where the spiroindolenines, intermediates of Pictet−Spengler-type reactions, were captured (Williams and Unger, 1970;Stöckigt et al., 2011;Chambers et al., 2016;Zheng and You, 2020). Furthermore, in recent years the interrupted Pictet-Spengler reaction is being explored as a strategy for the dearomatisation of indoles (James et al., 2016). Several successful syntheses of spirocyclic indolenine are known  This interesting result justified the optimization of the synthetic procedure, as a one-pot two-step procedure. Thus, a solution of compound 11a in acetic acid and acetone was treated with zinc powder at room temperature for 48 h. After the neutralization of the reaction medium and purification, the target compound 14 was isolated in 85% yield (Scheme 3).

Rationalization of the hetero-Diels-Alder reactions outcome
In order to investigate the observed and diverse regioselectivity, calculations at the DFT level of theory using the B3LYP hybrid functional (Becke, 1988;Lee et al., 1988;Becke, 1993) and the standard 6-31G (d,p) basis set were carried out for the hetero-Diels-Alder reaction of ethyl nitrosoacrylate (2a) with pyrrole, indole and 8-methyl-1,6-dihydropyrrolo[3,2-c] carbazole (10). For each heterocycle, relative stabilities of the different transition states (TS) involved in the hetero-Diels-Alder reactions were calculated, considering the two possible regioisomers and both endo and exo approaches (Figures 2-4). The individual contributions to the energy barriers associated with all the transition states studied for the reactions of ethyl nitrosoacrylate (2a) with pyrrole, indole and 8-methyl-1,6dihydropyrrolo[3,2-c]carbazole (10) are reported in Tables 1-3, considering both zero-point-energy (ZPE) correction, and basis set superposition error (BSSE) correction.
In the reaction between nitrosoalkene 2a and pyrrole, the computational results showed that the energy barrier associated with the formation of cycloadduct 3 by an endo approach is lower (about 25 kJ/mol) than the energy required for the formation of cycloadduct 3', which is in agreement with the regioselectivity observed experimentally ( Figure  2). Furthermore, the open-chain oxime 4 was obtained as single product, which is more stable than the primarily formed bicyclic 1,2-oxazine 3, as confirmed by DFT calculations (about 63 kJ/mol) (Figure 2; Table 1).
The 3-alkylated indole 8a is obtained from the reaction of nitrosoalkene 2a with indole via the hetero-Diels-Alder reaction by an exo approach. Indeed, this mechanistic pathway involves a lower energy transition state (ΔE = 41.5 kJ/mol) than that which would lead to the C2 alkylation product (Figure 3). Once again, the theoretical predictions of the regioselectivity are in agreement with the experimental results. In addition, the cycloadduct 7a, involved in C3 alkylation pathway, is more stable (about 16 kJ/ mol) than the homologue leading to C2 alkylation product ( Figure 3; Table 2).
The energy barriers calculated for the transition states of the hetero-Diels-Alder reaction of nitrosoalkene 2a with pyrrolo[3,2-c] carbazole 10 are very similar for the formation of both alkylated  Frontiers in Chemistry frontiersin.org 06 products (Figure 4). The DFT calculations showed that the formation of the 2-alkylated product proceeded via the endo transition state (TS endo ), whereas the exo transition state (TS exo ) was involved in the formation of the 3-alkylated product. The cycloadduct precursors of the 3-alkylated pyrrolo[3,2-c]carbazole 11a are more stable than the precursors of the 2-alkylated pyrrolo [3,2-c]carbazole 12a (about 13 kJ/mol), explaining the predominance of the 3-alkylated product. However, the stability of the open chain oximes 11a and 12a is very similar (Figure 4; Table 3). These computational results explain the isolation of both 2-alkylated and 3-alkylated products from the reaction between nitrosoalkene 2a and pyrrolo[3,2-c]carbazole 10.
Frontier Molecular Orbital (FMO) analysis of the hetero-Diels-Alder reaction of ethyl nitrosoacrylate (2a) with pyrrole, indole and pyrrolo[3,2-c]carbazole 10 was carried out. The relative energy values of the HOMO and LUMO orbitals of the reactants were obtained at the HF/6-31G (d,p) level of theory ( Figure 5). The results show that the energy difference between the LUMO of the nitrosoalkene and the HOMO of the heterocycles is smaller (between 7.99 and 9.08 eV) than that calculated for the HOMO nitrosoalkene -LUMO heterocycle pair (between 14.03 and 16.51 eV). Thus, the results show that these reactions are LUMO nitrosoalkene -HOMO heterocycle controlled and confirm that pyrrole, indole and 8-methyl-1,6-dihydropyrrolo [3,2-c]carbazole participate in inverse electron-demand hetero-Diels-Alder reaction with nitrosoalkene 2a acting as electron-rich 2π component. Furthermore, FMO analysis indicates that the molecular orbital energy profile of 8-methyl -1,6-dihydropyrrolo[3,2-c]carbazole is closer to the one of indole than to pyrrole.
The results of the Frontier Molecular Orbital interactions are in accordance with the observed regioselectivity, pointing generally to the formation of the product that stems from the interaction sites corresponding to the larger orbital coefficients. For indole the calculated orbital coefficients do not allow to distinguish between the two possible regiochemistries (Figure 6).
Scheme 4 summarizes the mechanistic pathways leading to oxime-functionalized pyrrolo[3,2-c]carbazoles 11a and 12a. The 3-alkylated pyrrolo[3,2-c]carbazole is obtained via hetero-Diels-Alder reaction of nitrosoalkene 2a with an exo approach followed by 1,2-oxazine ring-opening reaction. The synthesis of the 2alkylated derivative takes place with the initial endo cycloaddition reaction and subsequent conversion into the final oxime 12a. The selectivity towards the 3-alkylated pyrrolo[3,2-c]carbazole 11a is determined by the more exothermic formation of the hetero-Diels-Alder cycloadduct than that derived from the opposite regiochemistry. It should be noted that the chemical behaviour of  Orbital interaction diagrams for the hetero-Diels-Alder reactions with the indication of the orbital coefficients for the interacting orbitals obtained through NBO analysis at the HF/6-31G (d,p) level of theory.

Conclusion
The reactivity of nitrosoalkenes towards 8-methyl-1,6dihydropyrrolo[3,2-c]carbazole was studied leading to the synthesis of oxime-functionalized pyrrolo[3,2-c]carbazoles. The mechanistic pathway involves a hetero-Diels-Alder reaction leading to the construction of a 1,2-oxazine ring which undergoes a ring-opening reaction to give openchain oximes. Calculations at the DFT level of theory were carried out to investigate the regioselectivity of the hetero-Diels-Alder reaction of ethyl nitrosoacrylate with 8-methyl-1,6-dihydropyrrolo[3,2-c]carbazole as well as with pyrrole and indole, allowing a comparison between these three types of heterocycles. The computational results allowed the rationalization of the regioselectivity observed in the cycloaddition reaction and the formation of the more stable open chain oximes as the final products. The relative energy values of the Frontier HOMO and LUMO molecular orbitals for the ethyl nitrosoacrylate and the studied heterocyclic dienophiles were also calculated, substantiating that the cycloadditions are LUMO heterodiene -HOMO dienophile controlled.
General procedure for the hetero-Diels-Alder reactions Sodium carbonate (0.75 mmol) was added to a solution of a-bromooxime 1 (0.15 mmol) and 8-methylpyrrolo[3,2-c] carbazole 10 (0.22 mmol) in dry dichloromethane (10 mL). The reaction mixture was stirred at room temperature for the time indicated in each case, monitored by TLC. Upon completion, the mixture was filtered through a Celite pad, which was washed with ethyl acetate (2 × 10 mL). The solvent was evaporated, and the products were purified by flash chromatography.

Funding
The Coimbra Chemistry Centre-Institute of Molecular Sciences (CQC-IMS) is supported by Portuguese Foundation for Science and Technology (FCT) through projects UIDB/00313/2020 and UIDP/ 00313/2020 (National Funds) and the IMS special complementary funds provided by FCT. This work was also supported by Project PTDC/QUI-QOR/0103/2021, funded by national funds (PIDDAC) via FCT.