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1. Introduction

Pyrrolo[1,2-a]pyrazines and indolizines, including their partial or complete saturated analogues, belong to the diverse family of pyrrole-fused aza-bridged heterocyclic compounds, which are abundant in nature. They are known for their strong pharmacological activity. Pyrrolo[1,2-a]pyrazines and structural analogues show a wide range of biological effects [1,2], as illustrated by reports on their anxiolytic [3], anticancer [4,5], and antifungal activity [1,6], as well as their use as insect feeding deterrents [7] and potent and selective non-competitive antagonists of mGluR5 [8]. Antibiotic activity has only been found for the hexahydropyrrolo[1,2-a]pyrazine-1,4-dione analogues, which provides a strong effect against gram-negative bacteria [9,10,11]. Indolizines, on the other hand, have potential as anticancer, herbicidal, anti-inflammatory, antiparasitic, antioxidant, antihistaminic, anticonvulsant, antiviral, and analgesic agents [12,13,14,15,16,17]. In contrast to pyrrolo[1,2-a]pyrazines, antibiotic activity is more widely distributed among indolizines [12,13], in particular against Mycobacterium tuberculosis [18,19]. However, the antifungal effect of indolizines is modest [12]. Although the mechanism of action is not clearly established for either heterocycle, it is probably associated with the nitrogen-bridged heterocyclic scaffold [1,12].

Although pyrrolo[1,2-a]pyrazines have received less attention from researchers than indolizines, their broad spectrum of activity has stimulated the discovery of versatile synthetic strategies [1,2]. One of the most efficient approaches involves pyrrole-containing substrates, which undergo a cyclization process to build the pyrazine ring [20,21,22,23,24] (Scheme 1a). Indolizines have been prepared by following an analogous methodology, starting from functionalized pyrroles as the building block and completing the synthesis by a proper annulation to build the six-membered ring [25,26,27,28,29] (Scheme 1b). Our group has recently described effective procedures for constructing the scaffold of both indolizines and pyrrolo[1,2-a]pyrazines by appropriately substituting pyrrole-based precursors [30] (Scheme 1c).

The capacity of indolizines [31] and pyrrolo[1,2-a]pyrazines [1,6] to treat drug-resistant fungal infections is particularly attractive. Due to the pharmacological value of these heterocycles, the development of short and highly efficient synthetic methods should be a priority.

The aim of the current contribution was to design and test new synthetic approaches for synthesizing pyrrolo[1,2-a]pyrazines and indolizines. For the elaboration of pyrrolo[1,2-a]pyrazines, the potential of the annulation of 2-formylpyrrole-based enaminones (1–3) in the presence of ammonium acetate was evaluated. On the other hand, analogous enaminones were exposed to Lewis acids to prepare indolizines (Scheme 1d). All precursors and the corresponding products were assessed for their in vitro antifungal effect on six strains of Candida species. The most active compounds were analyzed in silico by docking simulations to examine their pharmacological profile as antifungal agents at the active site of the 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) enzyme of the six Candida spp. HMGR is the rate-limiting enzyme for the formation of ergosterol in Candida species, which is generated through the mevalonate pathway. Ergosterol is an essential component in the yeast cell membrane.

2. Results and Discussion

2.1. Chemistry

The design of pyrrole-containing compounds as potential antifungal agents was based on the evidence that benzofuran [32,33], benzothiophene [34], and indole [35,36] frames (including those of several natural products) can be built through intramolecular cyclization of 2-phenoxy-, 2-thiophenoxy-, and 2-anilino-3-(dimethylamino)propenoates, respectively. The latter derivatives have generically been called enaminones and proven to be versatile substrates in heterocyclic chemistry [37,38].

In the current investigation, the nitrogen atom of the 2-formylpyrrole ring was incorporated into the C-2 position of the alkyl (Z)-3-(dimethylamino)propenoate-equivalent (1, Y = O, Z = CO₂R, Scheme 1d), the (Z)-1-aryl-3-(dimethylamino)prop-2-en-1-one-equivalent (2, Y = O, Z = COAr, Scheme 1d), and the (Z)-3-(dimethylamino)acrylonitrile-equivalent (3, Y = O, Z = CN) (Scheme 1d), followed by the cyclization process in the presence of ammonium acetate to provide the corresponding pyrrolo[1,2-a]pyrazines 4. It was assumed that 2-formylpyrrole (6) can serve as a building block for the divergent synthesis of substituted pyrrolizines [30,39] and pyrrolo[1,2-a]pyrazines [30] to furnish the desired pyrrole-fused aza-bridged heterocyclic derivatives.

Thus, the alkylation of 6 with alkyl bromoacetates 7a and 7b under basic conditions produced the series of N-substituted pyrroles 8a [40] and 8b in good yields (Scheme 2). Similarly, the reaction of 6 with the series of bromoacetophenones 7c–f generated 8c–f, and the reaction of 6 with bromoacetonitrile (7g) gave pyrrole 8g. The thermal treatment of 8a–g with N,N-dimethylformamide dimethyl acetal (DMFDMA) afforded the series of enaminones 1a–b and 2a–d in acceptable yields as a single Z isomer, as well as enaminone 3a as an inseparable mixture of Z/E (54:46) isomers (Scheme 2).

For the conversion of 2-formylpyrrole-based enaminones 1a–b, 2a–d, and 3a into the series of 4-substituted pyrrolo[1,2-a]pyrazines 4a–g, a combination of ammonium acetate (as the source of nitrogen) and DBU (as the base) was employed as previously described [30]. However, the yields were low. Similar low yields were obtained with sodium hydride, potassium tert-butoxide, or cesium carbonate as the base. The reaction was improved when applying lithium carbonate as the base and DMF as the solvent at variable temperatures and reaction times (Table 1).

As an alternative route, a multicomponent reaction was explored for the preparation of pyrrolo[1,2-a]pyrazines 4, starting from substituted pyrroles 8a, 8c, and 8g in the presence of DMFDMA and ammonium acetate (Scheme 3). Thus, pyrroles 8a and 8c were reacted with an excess of DMFDMA (5.0 mol eq.) and ammonium acetate (3.0 mol eq.) to provide the corresponding pyrrolo[1,2-a]pyrazines 4a and 4c in 57% and 80% yields, respectively. When comparing this one-step process to that of the previously described two-step procedure, the current overall yield was lower for 4a (57% vs. 74%) and higher for 4c (80% vs. 46%). The reduction of 4c with NaBH₄ resulted in alcohol 4h in moderate yield. The reaction of 8g with DMFDMA generated a complex mixture of products that included the starting material.

Considering the important biological properties of brominated pyrroles [40,41] and pyrrolo[1,2-a]pyrazines [5], the brominated derivatives 8h [40], 1c, and 4i were conceived as potential antifungal compounds. The bromination of 8a with NBS (1.0 mol eq.) afforded 8h as a single regioisomer in high yield (Scheme 3). Treatment of the latter with DMFDMA gave enaminone 1c, which was reacted with ammonium acetate to deliver 7-bromopyrrolo[1,2-a]pyrazine 4i in good yield.

In the same context, pyrrolo[1,2-a]pyrazine 4a was brominated with an equimolar quantity of NBS to yield 6-bromopyrrolo[1,2-a]pyrazine 4j (Scheme 4), and the reaction of 4a with two mol equivalents of NBS furnished 6,7-dibromopyrrolo[1,2-a]pyrazine 4k.

With the aim of expanding the scope of the methodology, the synthesis of 4,6-disubstituted pyrrolo[1,2-a]pyrazine 4l was undertaken. Starting from 6, alkyl 3-(pyrrol-2-yl)acrylate 9a was obtained by a Horner–Wadsworth–Emmons reaction, followed by N-alkylation with 7a to give pyrrole 10a, as reported [42] (Scheme 5). The latter was formylated under the usual Vilsmeier–Haack conditions to give rise to the respective 5-formyl analog 8i. Treatment of 8i with DMFDMA generated a complex mixture of products, from which it was possible to isolate the single Z isomer of enaminone 11 in low yield. Finally, the amination of the latter with ammonium acetate delivered the desired pyrrolo[1,2-a]pyrazine 4l in modest yield, in contrast to the high yields for 4a–g (Table 1).

It was decided to start with similar enaminones to test a new approach for the construction of indolizines. Therefore, 9a or 9b were reacted with the corresponding methyl or ethyl bromoacetates 7a and 7b under basic conditions to afford pyrroles 10a–c [42], which were reacted with DMFDMA to convert them into enaminones 12a–c (Scheme 6). Unfortunately, enaminone 12b was formed in a mixture of methyl esters as a result of transesterification with the methoxy ions in the reaction medium. Hence, this compound was more efficiently obtained in modest yield when using Bredereck’s reagent under MW irradiation.

With the aim of achieving the direct annulation of 12a–c to the corresponding indolizines 5a–c, the reaction was promoted by diverse Lewis acids. By treating 12a with ZnI₂ or CuCl in methylene chloride as the solvent at 0 °C, the starting material was recovered. An increase in the temperature led to the decomposition of the reaction mixture. When utilizing POCl₃ in DMF at 0 °C, the yield of 5a was 28%, and the conversion of 12b to 5b with this Lewis acid was insignificant. The transformation of 12c to 5c with iodine [36] provided a low yield (18%). Finally, a 1.0 M nitrobenzene solution of AlCl₃ in methylene chloride as the solvent at room temperature efficiently furnished the series 5a–c in variable yields (Scheme 6). Disappointingly, when this strategy was started by reacting pyrrole 9a with bromoacetophenones 7c–f, the conversion into the corresponding N-alkyl pyrroles did not proceed efficiently. The structure of each product obtained in all the synthetic schemes was fully established by ¹H and ¹³C NMR, assisted by 2D experiments (ROESY, HSQC, and HMBC) and HRMS.

2.2. Antifungal Activity

According to previous studies, brominated and non-brominated pyrrole-containing compounds structurally related to derivatives 8a, 8h, and 1c inhibit yeast growth and the synthesis of ergosterol by Candida glabrata [40]. Therefore, selected compounds of the present series, consisting of pyrrole-based compounds (8a–i and 10a–c), enaminones (1a–c, 2a–d, 11, and 12a–c), pyrrolo[1,2-a]pyrazines (4a–l), and indolizines (5a–c), were assessed at their minimum inhibitory concentration (MIC) against six Candida spp.: C. albicans, C. glabrata, C. dubliniensis, C. krusei, C. auris, and C. haemulonii. The latter two species are multidrug-resistant. Most of the compounds evaluated afforded substantial yeast growth inhibition. Table 2 summarizes the lowest MIC_50–70 values in regard to the inhibition of the Candida spp. by the test compounds and reference drugs (fluconazole, simvastatin, and atorvastatin). Simvastatin and atorvastatin are well-known inhibitors of human HMGR (hHMGR) and are proposed as inhibitors of HMGR of Candida spp. [40,43,44]. Fluconazole is a reference inhibitor of the enzyme C-14 alpha demethylase of lanosterol (CYP51) that participates in the synthesis of ergosterol [45]. Interestingly, simvastatin and atorvastatin showed a more efficient profile than fluconazole for some of the yeasts, including the two multidrug-resistant species.

Inhibition of yeast growth was observed for all the test compounds, which had lower MICs than the three reference drugs in all cases. Nevertheless, the majority of the MIC_50–70 values for the compounds on C. auris and C. haemulonii were higher than those obtained on C. albicans, C. glabrata, C. dubliniensis, and C. krusei. This can perhaps be explained by the strong resistance of C. auris and C. haemulonii to a variety of traditional antifungal drugs [46,47].

Pyrrole derivatives are reported to inhibit the growth of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Saccharomyces cerevisiae, Aspergillus fumigatus, A. niger, Geotrichum candidum, Syncephalastrum racemosum, and dermatophytes (Trichophyton rubrum, T. mentagrophytes, and Microsporum gypseum), exhibiting in almost all the cases MICs higher than those determined presently [48,49,50,51,52]. These results suggest that the N-alkyl group attached to the pyrrole ring of derivatives 8a–g improves the antifungal effect (especially for N-alkyl 2-formylpyrroles 8a, 8c, and 8g), as seems to be demonstrated for the pyrrole-based drug atorvastatin. Low to modest antifungal activity has also been observed for pyrrole- and/or pyridine-containing fused-bis-heterocyclic compounds [53,54,55].

The few reports on enaminone-containing compounds have evidenced their antibacterial activity against Escherichia coli, Bacillus subtilis, and Salmonella typhi [56] as well as antifungal activity against C. albicans, in each case showing a modest inhibition [57]. In the current contribution, the enaminones 1a, 1c, 2a–d, 11, and 12a–c exhibited good inhibition of the six Candida spp. (Table 2).

Pyrrole-based compounds 8i and 10–12 bear the acrylate moiety as another potentially active pharmacophore group, as does pyrrolo[1,2-a]pyrazine 4l, one of the most active derivatives against the six Candida species. Some acrylate-based heterocyclic derivatives have been proposed as antifungals due to their growth inhibition of Aspergillus, Geotrichum, and various other pathogenic plant fungi [58,59].

Pyrrolo[1,2-a]pyrazines 4a–l were highly active agents against all the Candida spp. herein tested, particularly derivatives 4b, 4g, and 4l. The conjugated C-4 carbonyl group does not seem to be a key pharmacophore, given that the antifungal activity is comparable for alcohol 4h and precursor 4c. Moderate activity was previously found for analogous heterocycles against Penicillium chrysogenum AUMC 530-15, four species of Aspergillus, Fusarium solani AUMC 2690-6, and Trichothecium roseum AUMC 7410-2 [60]. Interestingly, the brominated pyrrolo[1,2-a]pyrazines 4i–k were more active than their corresponding non-halogenated 4a.

The indolizines 5a–c also significantly inhibited the growth of the majority of the Candida spp., in agreement with recent reports on other analogous compounds [61]. However, these results are in contrast with a series of 7-cyano-1,2-diphenylindolizines, which were not active on C. krusei [18].

2.3. Molecular Docking Study

Molecular docking is a computational tool used to explore the interaction, binding mode (e.g., protein–protein), and affinity between two molecules, generally an organic compound and an endogenous receptor (protein or nucleic acid) of a cell [62,63,64]. Because docking simulations serve to explore the molecular recognition between the active site of a certain therapeutic target (an enzyme) and an organic molecule (ligand), they are widely used to design and develop new drugs that may improve the treatment for diseases or for infections by microorganisms, such as a pathogenic fungus. The binding interaction between two molecules depends on the hydrophilic, electrostatic, and hydrophobic intermolecular forces that take place. The strength of such interactions is expressed in binding energy (ΔG, kcal/mol). A more negative value of ΔG is related to a more stable enzyme–ligand complex and a greater biological activity.

To explore the binding interactions of the test compounds on HMGR, the following compounds were selected among the most active derivatives: enaminone-containing pyrroles 1a, 2a, and 2c, pyrrolo[1,2-a]pyrazines 4b, 4g, and 4l, indolizine 5a, N-alkyl 2-formylpyrroles 8a, 8c, and 8g, and pyrrole-based alkyl acrylates 10a and 12a. The molecular docking study was carried out on the active site of the HMGR enzyme of C. albicans (CaHMGR), C. glabrata (CgHMGR), C. dubliniensis (CdHMGR), C. krusei (CkHMGR), C. auris (CauHMGR), and C. haemulonii (ChaHMGR). The models of the six HMGRs were built (Figure S77, Supplementary Materials) and overlapped with human HMGR, noting a close structural identity between the 3D structures. The high quality of the models employed in the study is evidenced by the fact that over 90% of the amino acid residues were in permitted areas of the Ramachandran diagram (Figures S78–S83, Supplementary Materials), as previously reported for CgHMGR [40,44,65].

The binding energy values of the reference compound atorvastatin were in all cases lower than those for simvastatin (Table 3). A similar pattern of binding energy values was observed for the entire series of compounds. For example, enaminones 2a and 2c and the N-alkyl pyrrole 8c showed more negative values than simvastatin for all the species of Candida spp. herein tested. The pyrrolo[1,2-a]pyrazine 4l had better binding energy values than simvastatin on five of the yeasts. In contrast to the experimental antifungal effect, enaminone 1a only exhibited better binding energy values than simvastatin on CaHMGR and CkHMGR. Overall, the binding energy values found for these series of compounds were better than those previously described for other derivatives proposed as inhibitors of CgHMGR [40,44].

To predict the binding mode of the twelve selected compounds at the active site of the HMGRs, the three most prevalent species (C. albicans, C. glabrata, and C. auris) were selected. The hydrophilic and hydrophobic interactions and the amino acid residues involved are summarized in Table 4 for C. albicans, and in Tables S1 and S2 for C. glabrata, and C. auris, respectively. Likewise, the interactions are illustrated in Figure 1 for C. albicans and in Figures S84 and S85 for C. glabrata and C. auris. Regarding their binding modes to the HMGRs of the three Candida spp., the twelve test compounds share key hydrophilic and hydrophobic interactions with simvastatin and atorvastatin. The residues participating in binding are the following: Glu96, Lys228, and Asp304 in CaHMGR; Glu93, Lys227, and Asp303 in CgHMGR; and Glu96, Lys229, and Asp305 in CauHMGR. Considering the binding interactions of either of the test compounds, at least one of these residues is shared with the binding interactions of the reference compounds, suggesting that all the test compounds and simvastatin and atorvastatin may have a similar mechanism of action. These types of interactions have been previously reported in other works for the HMGR of H. sapiens and CgHMGR [40,44,66].

Hydrophilic interactions involving a conventional hydrogen bond are predominant for the carbonyl groups of compounds 1a, 2a, 2c, and 8a with the amino acids Asp227, Lys228, and Asp304 of CaHMGR as well as with the amino acids Thr92 and Glu93 of CgHMGR. Similar interactions are observed for indolizine 5a and enaminone 13a with residues Gln303, Asn195, and Thr295 of CaHMGR. For more than one of the compounds assessed on CauHMGR, the principal interactions are both conventional hydrogen bond and carbon hydrogen with Thr94, Thr95, Lys229, Asn292, Thr295, and Asp305. In addition, the amino acids that predominate in the hydrophobic interactions with CaHMGR are Glu96 (π-anion), Met192 (π-sigma or π-alkyl), and Met194 (π-sigma or π-alkyl). Compounds 2a and 8g show π-alkyl interactions with Met189 in CgHMGR, and 1a, 2a, 2c, 4l, 8c, and simvastatin display π-alkyl interactions with Pro306 in CauHMGR. The presence of the aromatic rings in the test compounds plays an important role in stabilizing the p interactions with the enzyme residues. According to the results, the test compounds can be considered as inhibitors of the HMGR enzymes of the Candida spp. herein evaluated, because they bind to amino acid residues of the catalytic site and share certain hydrophilic and hydrophobic interactions with simvastatin and atorvastatin.

3. Materials and Methods

3.1. General Information

Melting points were determined on a Krüss KSP 1N (KRÜSS GmbH, Hamburg, Germany) capillary melting point apparatus. IR spectra were recorded on FT-IR 2000 Perkin-Elmer (PerkinElmer, Waltham, MA, USA) and Bruker Vertex 70 (ATR-FT) (Bruker Corporation, Billerica, MA, USA) spectrophotometers. ¹H and ¹³C NMR spectra were recorded on the following instruments: Varian Mercury (300 MHz) (Varian, Inc., Palo Alto, CA, USA), Bruker Ascend 400 (400 MHz) (Bruker Corporation, Billerica, MA, USA), Varian VNMR System (500 MHz) (Varian, Inc., Palo Alto, CA, USA), Bruker 600Avance III (600 MHz), and Bruker Avance III HD (750 MHz) (Bruker Corporation, Billerica, MA, USA), with CDCl₃ as the solvent and TMS or CDCl₃ as the internal standards. Signal assignments were based on 2D NMR spectra (HSQC, HMBC, and ROESY). High-resolution mass spectra (HRMS) (in electron impact mode) were obtained on Jeol JMS-GCMateII and Jeol JMS T100-LC AccuTOF DART (JEOL, Ltd., Tokyo, Japan) spectrometers. MW irradiation was emitted from a CEM MW reactor (CEM Corporation, Matthews, NC, USA). A Multi-Therm Benchmark, Model H5000-HC (Benchmark Scientific, Inc., Sayreville, NJ, USA) was utilized as a heating and cooling shaker in enzymatic stability assays. Yeast growth was quantified in a Multiskan™ GO microplate spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) at 620 nm. Analytical thin-layer chromatography was carried out on 0.25 plates coated with silica gel 60 F254 (E. Merck, Darmstadt, Germany), which were visualized by a long- and short-wavelength UV lamp. Flash column chromatography was performed over silica gel (230–400 mesh, Natland International Co., Morrisville, NC, USA). Commercial reagents were used as received, and anhydrous solvents were obtained by a distillation process. Compounds 1a and 8a, 9a, 9b, and 10b were prepared as reported [40,42].

3.2. Chemistry

Ethyl 2-(2-formyl-1H-pyrrol-1-yl) acetate (8b). At 0 °C and under N₂ atmosphere, NaH (60%, 0.051 g, 1.27 mmol) was added to a solution of 6 (0.100 g, 1.05 mmol) and anhydrous DMF (2.0 mL), and the mixture was stirred for 20 min. Then, 7b (0.210 g, 1.26 mmol) was added, and the solution was stirred at room temperature (rt) for 4 h. The reaction mixture was extracted with hexane/EtOAc (1:1, 15.0 mL). The organic layer was washed with brine (5.0 mL × 3) and dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 9:1) to obtain 8b (0.179 g, 94%) as a pale violet oil. R_f 0.64 (hexane/EtOAc, 1:1). IR (film): ῡ 3130, 2993, 2978, 2798, 1735, 1656, 1482, 1402, 1315, 1218, 1027, 752 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 1.28 (t, J = 7.2 Hz, 3H, CO₂CH₂CH₃), 4.22 (q, J = 7.2 Hz, 2H, CO₂CH₂CH₃), 5.05 (s, 2H, CH₂), 6.29 (dd, J = 4.0, 2.4 Hz, 1H, H-4′), 6.90–6.93 (m, 1H, H-5′), 6.98 (dd, J = 4.0, 1.6 Hz, 1H, H-3′), 9.52 (d, J = 0.8 Hz, 1H, CHO). ¹³C NMR (100 MHz, CDCl₃): δ 14.1 (CO₂CH₂CH₃), 50.2 (CH₂), 61.6 (CO₂CH₂CH₃), 110.2 (C-4′), 124.6 (C-3′) 131.7 (C-2′), 132.0 (C-5′), 168.3 (CO₂CH₂CH₃), 179.7 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₉H₁₁NO₃: 181.0739; found: 181.0733.
1-(2-Oxo-2-phenylethyl)-1H-pyrrole-2-carbaldehyde (8c). Following the method described for 8b, a mixture of 6 (0.100 g, 1.05 mmol), NaH (60%, 0.505 g, 1.26 mmol), and 7c (0.251 g, 1.26 mmol) furnished 8c (0.200 g, 89%) as white needles. R_f 0.19 (hexane/EtOAc, 9:1); mp 114–115 °C. IR (film): ῡ 3112, 2938, 2812, 1702, 1648, 1402, 1366, 1330, 1222, 1080, 1019, 1005, 748, 690 cm⁻¹. ¹H NMR (300 MHz, CDCl₃): δ 5.81 (s, 2H, CH₂), 6.36 (dd, J = 4.2, 2.5 Hz, 1H, H-4), 6.94–6.98 (m, 1H, H-5), 7.04 (dd, J = 4.2, 1.5 Hz, 1H, H-3), 7.48–7.55 (m, 2H, H-3″), 7.59–7.67 (m, 1H, H-4″), 7.98–8.03 (m, H-2″), 9.51 (d, J = 0.9 Hz, 1H, CHO). ¹³C NMR (187.5 MHz, CDCl₃): δ 54.7 (CH₂), 110.3 (C-4), 124.7 (C-3), 128.0 (C-2″), 128.9 (C-3″), 131.6 (C-2), 132.5 (C-5), 133.8 (C-4″), 134.8 (C-1″), 179.8 (CHO), 192.9 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₃H₁₁NO₂: 213.0790; found: 213.0790.
1-(2-(3-Methoxyphenyl)-2-oxoethyl)-1H-pyrrole-2-carbaldehyde (8d). Following the method described for 8b, a mixture of 6 (0.060 g, 0.63 mmol), NaH (60%, 0.030 g, 0.76 mmol), and 7c (0.174 g, 0.76 mmol) afforded 8d (0.070 g, 45%) as white needles. R_f 0.48 (hexane/EtOAc, 1:1); mp 106–107 °C. IR (KBr): ῡ 2939, 2799, 1694, 1649, 1591, 1403, 1260, 1192, 1024, 857, 754 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 3.85 (s, 3H, CH₃O), 5.79 (s, 2H, CH₂), 6.35 (dd, J = 3.9, 2.6 Hz, 1H, H-4), 6.95 (br s, 1H, H-5), 7.03 (dd, J = 3.9, 1.5 Hz, 1H, H-3), 7.16 (ddd, J = 8.3, 2.6, 1.1 Hz, H-4″), 7.42 (t, J = 8.3 Hz, 1H, H-5″), 7.50 (dd, J = 2.6, 1.5 Hz, 1H, H-2″), 7.58 (dt, J = 7.5, 1.5 Hz, 1H, H-6″), 9.51 (d, J = 0.8 Hz, 1H, CHO). ¹³C NMR (187.5 MHz, CDCl₃): δ 54.9 (CH₂), 55.5 (CH₃O), 110.3 (C-4), 112.4 (C-2″), 120.4 (C-4″), 120.5 (C-6″), 124.8 (C-3), 129.9 (C-5″), 131.6 (C-2), 132.5 (C-5), 136.1 (C-1″), 160.0 (C-3″), 179.8 (CHO), 192.8 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₄H₁₃NO₃: 243.0895; found: 243.0891.
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-1H-pyrrole-2-carbaldehyde (8e). Following the method described for 8b, a mixture of 6 (0.200 g, 2.10 mmol), NaH (60%, 0.100 g, 2.52 mmol), and 7e (0.577 g, 2.52 mmol) provided 8e (0.413 g, 72%) as white needles. R_f 0.70 (hexane/EtOAc, 1:1); mp 118–119 °C. IR (KBr): ῡ 2939, 1652, 1601, 1406, 1366, 1226, 1178, 1024, 846, 754 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 3.88 (s,1H, CH₃O), 5.77 (s, 2H, CH₂), 6.35 (dd, J = 3.9, 2.4 Hz, 1H, H-4), 6.95 (br s, 1H, H-5), 6.96–6.99 (m, 2H, H-3″), 7.02 (dd, J = 3.9, 1.8 Hz, 1H, H-3), 7.97–8.00 (m, 2H, H-2″), 9.50 (d, J = 0.6 Hz, 1H, CHO). ¹³C NMR (150 MHz, CDCl₃): δ 54.3 (CH₂), 55.5 (CH₃O), 110.2 (C-4), 114.1 (C-3″), 124.7 (C-3), 127.8 (C-1″), 130.4 (C-2″), 131.6 (C-2), 132.6 (C-5), 164.1 (C-4″), 179.8 (CHO), 191.3 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₄H₁₃NO₃: 243.0895; found: 243.0885.
1-(2-(3,4-Dimethoxyphenyl)-2-oxoethyl)-1H-pyrrole-2-carbaldehyde (8f). Following the method described for 8b, a mixture of 6 (0.030 g, 0.32 mmol), NaH (60%, 0.015 g, 0.38 mmol), and 7f (0.098 g, 0.38 mmol) gave 8f (0.070 g, 81%) as white needles. R_f 0.13 (hexane/EtOAc, 1:1); 148–149 °C. IR (film): ῡ 3011, 1707, 1356, 1219, 1417, 762 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 3.93 (s, 3H, CH₃O), 3.97 (s, 3H, CH₃O), 5.79 (s, 2H, CH₂), 6.35 (dd, J = 3.9, 2.7 Hz, 1H, H-4), 6.94 (d, J = 8.1 Hz, 1H, H-5″), 6.96 (br s, 1H, H-5), 7.03 (dd, J = 3.9, 1.8 Hz, 1H, H-3), 7.53 (d, J = 2.1 Hz, 1H, H-2″), 7.66 (dd, J = 8.1, 2.1 Hz, 1H, H-6″), 9.52 (d, J = 0.6 Hz, 1H, CHO). ¹³C NMR (150 MHz, CDCl₃): δ 54.2 (CH₂), 56.0 (CH₃O), 56.1 (CH₃O), 110.2 (C-2″, C-4), 110.3 (C-5″), 122.6 (C-6″), 124.8 (C-3), 128.0 (C-1″), 131.7 (C-2), 132.6 (C-5), 149.3 (C-3″), 154.0 (C-4″), 179.9 (CHO), 191.5 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₅H₁₅NO₄: 273.1001; found: 273.1002.
2-(2-Formyl-1H-pyrrol-1-yl)acetonitrile (8g). Following the method described for 8b, a mixture of 6 (0.050 g, 0.53 mmol), NaH (60%, 0.025 g, 0.63 mmol), and 7g (0.076 g, 0.63 mmol) produced 8g (0.036 g, 51%) as pale violet oil. R_f 0.64 (hexane/EtOAc, 1:1). IR (film): ῡ 3113, 2864, 2253, 1652, 1475, 1406, 1362, 1308, 1222, 1028, 748 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 5.35 (s, 2H, CH₂), 6.34 (dd, J = 3.9, 2.6 Hz, 1H, H-4′), 7.00 (dd, J = 3.9, 1.5 Hz, 1H, H-3′), 7.08 (br s, 1H, H-5′), 9.57 (d, J = 0.8 Hz, 1H, CHO). ¹³C NMR (187.5 MHz, CDCl₃): δ 36.2 (CH₂), 111.5 (C-4′), 114.5 (CN), 125.2 (C-3′), 130.7 (C-5′), 131.0 (C-2′), 179.9 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₇H₆N₂O: 134.0480; found: 134.0477.
Ethyl (Z)-3-(dimethylamino)-2-(2-formyl-1H-pyrrol-1-yl)acrylate (1b). In a threaded ACE glass pressure tube equipped with a magnetic stirring bar and sealed with a Teflon screw cap, a mixture of 8b (0.100 g, 0.55 mmol) and DMFDMA (0.329 g, 2.75 mmol) in anhydrous DMF (2.0 mL) was heated at 100 °C for 20 h. A mixture of CH₂Cl₂/toluene (10.0 mL/1.0 mL) was added, and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 7:3), resulting in 1b (0.057 g, 44%) as a yellow oil. R_f 0.16 (hexane/EtOAc, 1:1). IR (film): ῡ 2934, 1663, 1618, 1303, 1209, 1078, 749 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 1.16 (t, J = 6.8 Hz, 3H, CO₂CH₂CH₃), 2.65 (br, 6H, N(CH₃)₂), 4.04–4.16 (m, 2H, CO₂CH₂CH₃), 6.29 (dd, J = 3.8, 2.3 Hz, H-4′), 6.82 (br t, J = 2.3 Hz, 1H, H-5′), 7.01 (dd, J = 3.8, 1.5 Hz, H-3′), 7.51 (s, 1H, H-3), 9.56 (s, 1H, CHO). ¹³C NMR (187.5 MHz, CDCl₃): δ 14.5 (CO₂CH₂CH₃), 34.9 (N(CH₃)₂), 60.1 (CO₂CH₂CH₃), 97.9 (C-2), 110.2 (C-4′), 120.9 (C-3′), 134.2 (C-5′), 134.9 (C-2′), 146.0 (C-3), 166.9 (CO₂Et), 179.8 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₁₂H₁₆N₂O₃: 236.1161; found: 236.1159.
(Z)-1-(1-(Dimethylamino)-3-oxo-3-phenylprop-1-en-2-yl)-1H-pyrrole-2-carbaldehyde (2a). Following the method described for 1b, a mixture of 8c (0.030 g, 0.14 mmol) and DMFDMA (0.084 g, 0.70 mmol) in anhydrous DMF (1.0 mL) delivered 2a (0.020 g, 53%) as a yellow oil. R_f 0.16 (hexane/EtOAc, 1:1). IR (film): ῡ 3097, 2928, 1637, 1543, 1413, 1388, 1323, 1095, 950, 878, 766, 708 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 2.68 (br, 6H, N(CH₃)₂), 6.33 (dd, J = 3.9, 2.6 Hz, 1H, H-4), 6.89 (br s, 1H, H-5), 7.02 (dd, J = 3.9, 1.5 Hz, 1H, H-3), 7.31–7.36 (m, 3H, H-1′, H-3″), 7.37–7.40 (m, 1H, H-4″), 7.48–7.52 (m, 2H, H-2″), 9.60 (d, J = 0.9 Hz, 1H, CHO). ¹³C NMR (187.5 MHz, CDCl₃): δ 29.6 (br, N(CH₃)₂), 110.6 (C-2′, C-4), 122.6 (C-3), 127.90 (C-2″), 127.93 (C-3″), 129.9 (C-4″), 134.39 (C-2), 134.43 (C-5), 139.9 (C-1″), 150.2 (C-1′), 179.4 (CHO), 190.9 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₆H₁₆N₂O₂: 268.1212; found: 268.1213.
(Z)-1-(1-(Dimethylamino)-3-(3-methoxyphenyl)-3-oxoprop-1-en-2-yl)-1H-pyrrole-2-carbaldehyde (2b). Following the method described for 1b, a mixture of 8d (0.050 g, 0.21 mmol) and DMFDMA (0.125 g, 1.05 mmol) in anhydrous DMF (1.0 mL) formed 2b (0.035 g, 56%) as a pale violet oil. R_f 0.45 (hexane/EtOAc, 1:1). IR (film): ῡ 2925, 1663, 1588, 1424, 1322, 1042, 748 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 2.70 (br, 6H, N(CH₃)₂), 3.79 (s, 3H, CH₃O), 6.31–6.34 (m, 1H, H-4), 6.88 (br s, 1H, H-5), 6.92 (dm, J = 8.0 Hz, 1H, H-4″), 7.01–7.04 (m, 2H, H-2″, H-3), 7.06–7.10 (m, 1H, H-6″), 7.23 (t, J = 8.0 Hz, 1H, H-5″), 7.37 (br s, 1H, H-1′), 9.60 (s, 1H, CHO). ¹³C NMR (125 MHz, CDCl₃): δ 36.5 (br, N(CH₃)₂), 55.2 (CH₃O), 110.6 (C-4), 111.1 (C-2′), 112.7 (C-2″), 116.3 (C-4″), 120.3 (C-6″), 122.5 (C-3), 128.9 (C-5″), 129.5 (C-2), 134.4 (C-5), 141.1 (C-1″), 150.4 (C-1′), 159.2 (C-3″), 179.4 (CHO), 190.6 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₇H₁₈N₂O₃: 298.1317; found: 298.1311.
(Z)-1-(1-(Dimethylamino)-3-(4-methoxyphenyl)-3-oxoprop-1-en-2-yl)-1H-pyrrole-2-carbaldehyde (2c). Following the method described for 1b, a mixture of 8e (0.100 g, 0.41 mmol) and DMFDMA (0.245 g, 2.05 mmol) in anhydrous DMF (1.0 mL) provided 2c (0.111 g, 90%) as a yellow oil. R_f 0.16 (hexane/EtOAc, 1:1). IR (film): ῡ 2939, 1663, 1600, 1584, 1560, 1386, 1325, 1243, 1096, 1021, 840, 765, 738 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 2.67 (br, 6H, N(CH₃)₂), 3.79 (s, 3H, CH₃O), 6.31 (dd, J = 4.2, 2.4 Hz, 1H, H-4), 6.80 (d, J = 8.4 Hz, 2H, H-3″), 6.87 (br s, 1H, H-5), 7.01 (dd, J = 4.2, 1.8 Hz, 1H, H-3), 7.35 (s, 1H, H-1′), 7.45 (d, J = 8.4 Hz, 1H, H-2″), 9.56 (s, 1H, CHO). ¹³C NMR (125 MHz, CDCl₃): δ 34.8 (br, N(CH₃)₂), 55.2 (CH₃O), 110.1 (C-2′), 110.6 (C-4), 113.2 (C-3″), 122.3 (C-3), 130.0 (C-2″), 132.1 (C-1″), 134.3 (C-2), 134.4 (C-5), 149.7 (C-1′), 161.2 (C-4″), 179.5 (CHO), 189.7 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₇H₁₈N₂O₃: 298.1317; found: 298.1314.
(Z)-1-(3-(3,4-Dimethoxyphenyl)-1-(dimethylamino)-3-oxoprop-1-en-2-yl)-1H-pyrrole-2-carbaldehyde (2d). Following the method described for 1b, a mixture of 8f (0.050 g, 0.18 mmol) and DMFDMA (0.109 g, 0.90 mmol) in anhydrous DMF (1.0 mL) afforded 2d (0.050 g, 83%) as a brown solid. R_f 0.03 (hexane/EtOAc, 1:1); 138–140 °C. IR (film): ῡ 2929, 1738, 1639, 1564, 1509, 1410, 1311, 1096, 1018, 748 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 2.69 (br, 6H, N(CH₃)₂), 3.82 (CH₃O), 3.87 (CH₃O), 6.31 (dd, J = 4.0, 2.1 Hz, 1H, H-4), 6.76 (d, J = 8.1 Hz, 1H, H-5″), 6.87 (br s, 1H, H-5), 7.02 (dd, J = 4.0, 1.8 Hz, 1H, H-3), 7.04 (br s, 1H, H-2″), 7.09 (br d, J = 8.1 Hz, 1H, H-6″), 7.43 (s, 1H, H-1′), 9.58 (CHO). ¹³C NMR (150 MHz, CDCl₃): δ 42.0 (br, N(CH₃)₂₎, 55.8 (CH₃O), 55.9 (CH₃O), 110.00 (C-5″), 110.02 (C-2′), 110.6 (C-4), 111.2 (C-2″), 121.7 (C-6″), 122.2 (C-3), 132.2 (C-1″), 134.3 (C-5), 134.5 (C-2), 148.4 (C-3″), 149.6 (C-1′), 150.1 (C-4″), 179.4 (CHO), 189.4 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₈H₂₀N₂O₄: 328.1423; found: 328.1426.
(Z)-3-(Dimethylamino)-2-(2-formyl-1H-pyrrol-1-yl)acrylonitrile (3a). (E)-3-(Dimethylamino)-2-(2-formyl-1H-pyrrol-1-yl)acrylonitrile (3a’). Following the method described for 1b, a mixture of 8g (0.077 g, 0.57 mmol) and DMFDMA (0.339 g, 2.85 mmol) in anhydrous DMF (2.0 mL) generated an inseparable mixture of 3a/3a’ (54:46, 0.030 g, 28%) as a pale violet oil. R_f 0.16 (hexane/EtOAc, 1:1). IR (film): ῡ 3095, 2922, 2803, 2184, 1650, 1365, 1130, 772 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 2.63 (br, 6H, N(CH₃)₂), 3.16 (s, 6H, N(CH₃)₂), 6.25 (dd, J = 4.2, 2.4 Hz, 1H, H-4′), 6.31 (dd, J = 4.2, 2.4 Hz, 1H, H-4′), 6.68 (s, 1H, H-3), 6.73 (s, 1H, H-3), 6.87–6.89 (m, 1H, H-5′), 6.91–6.93 (m, 1H, H-5′), 6.96 (dd, J = 4.2, 1.8 Hz, 1H, H-3′), 6.99 (dd, J = 4.2, 1.8 Hz, 1H, H-3′), 9.63 (s, 1H, CHO), 9.66 (s, 1H, CHO). ¹³C NMR (150 MHz, CDCl₃): δ 42.3 (br, N(CH₃)₂), 78.6 (C-2), 78.8 (C-2), 110.7 (C-4′), 111.2 (C-4′), 118.5 (CN), 120.24 (CN), 122.5 (2C-3′), 133.0 (C-5′), 133.2 (C-2′), 133.8 (C-5′), 134.5 (C-2′), 147.4 (C-3), 151.0 (C-3), 178.9 (CHO), 179.2 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₁₀H₁₁N₃O: 189.0902; found: 189.0900.
Methyl pyrrolo[1,2-a]pyrazine-4-carboxylate (4a). Method A: In a threaded ACE glass pressure tube equipped with a magnetic stirring bar and sealed with a Teflon screw cap, a mixture of NH₄OAc (0.052 g, 0.675 mmol) and Li₂CO₃ (0.050 g, 0.675 mmol) in anhydrous DMF (1.0 mL) was stirred at rt for 20 min. Then, 1a (0.050 g, 0.225 mmol) was added, and the solution was heated at 70 °C for 4 h. The mixture was diluted with hexane/EtOAc (1:1, 15 mL) and washed with brine (5.0 mL × 3). The organic layer was dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 8:2) to give 4a (0.036 g, 90%) as a yellow solid. R_f 0.58 (hexane/EtOAc, 1:1); mp 93–95 °C.
Method B: In a threaded ACE glass pressure tube equipped with a magnetic stirring bar and sealed with a Teflon screw cap, a mixture of 8a (0.030 g, 0.18 mmol), DMFDMA (0.114 g, 0.90 mmol), and NH₄OAc (0.042 g, 0.54 mmol) was heated at 100 °C for 24 h. The mixture was suspended in CH₂Cl₂/PhMe (10:1, 11 mL), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 1:1) to furnish 4a (0.018 g, 57%) as a yellow solid. R_f 0.58 (hexane/EtOAc, 1:1); mp 93–95 °C. IR (film): ῡ 3168, 2954, 1720, 1621, 1441, 1430, 1306, 1217, 1203, 1177, 1112, 764, 744 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 4.01 (s, 3H, CO₂CH₃), 7.02 (s, 2H, H-7, H-8), 8.46 (s, 1H, H-3), 8.74 (s, 1H, H-6), 8.92 (s, 1H, H-1). ¹³C NMR (125 MHz, CDCl₃): δ 52.4 (CO₂CH₃), 106.7 (C-8), 116.6 (C-7), 118.3 (C-4), 118.9 (C-6), 129.9 (C-8a), 134.8 (C-3), 148.1 (C-1), 163.3 (CO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₉H₈N₂O₂: 176.0586; found: 176.0592.
Ethyl pyrrolo[1,2-a]pyrazine-4-carboxylate (4b). Following method A described for 4a, a mixture of 1b (0.030 g, 0.13 mmol), NH₄OAc (0.030 g, 0.39 mmol), and Li₂CO₃ (0.028 g, 0.39 mmol) was heated at 80 °C for 3 h to obtain 4b (0.016 g, 66%) as a yellow solid. R_f 0.67 (hexane/EtOAc, 1:1); mp 68–70 °C. IR (film): ῡ 3177, 3097, 2848, 1699, 1427, 1287, 1178, 1088, 1033, 734 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 1.45 (t, J = 7.4 Hz, CO₂CH₂CH₃), 4.47 (q, J = 7.4 Hz, CO₂CH₂CH₃), 7.01–7.03 (m, 2H, H-7, H-8), 8.46 (s, 1H, H-3), 8.73–8.74 (m, 1H, H-6), 8.91 (s, 1H, H-1). ¹³C NMR (187.5 MHz, CDCl₃): δ 14.3 (CO₂CH₂CH₃), 61.6 (CO₂CH₂CH₃), 106.6 (C-8), 116.5 (C-7), 118.9 (C-6), 129.9 (C-8a), 134.6 (C-3), 134.8 (C-4), 148.0 (C-1), 162.8 (CO₂CH₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₀H₁₀N₂O₂: 190.0742; found: 190.0742.
Phenyl(pyrrolo[1,2-a]pyrazin-4-yl)methanone (4c). Following method A described for 4a, a mixture of 2a (0.050 g, 0.187 mmol), NH₄OAc (0.043 g, 0.56 mmol), and Li₂CO₃ (0.041 g, 0.56 mmol) was heated at 80 °C for 3 h to give 4c (0.036 g, 87%) as a yellow solid. R_f 0.48 (hexane/EtOAc, 1:1); mp 61–62 °C. Following method B described for 4a, a mixture of 8c (0.030 g, 0.141 mmol), DMFDMA (0.084 g, 0.70 mmol), and NH₄OAc (0.054 g, 0.70 mmol) produced 4c (0.025 g, 80%) as a yellow solid. R_f 0.48 (hexane/EtOAc, 1:1); mp 61–62 °C. IR (film): ῡ 3101, 3040, 2924, 1633, 1467, 1290, 1203, 1052, 882, 741 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 7.10 (d, J = 1.5 Hz, 2H, H-7′, H-8′), 7.53 (t, J = 7.5 Hz, 2H, H-3″), 7.63 (t, J = 7.5 Hz, 1H, H-4″), 7.81 (br d, J = 7.5 Hz, 2H, H-2″), 8.10 (s, 1H, H-3′), 8.92 (br m, 1H, H-6′), 8.94 (s, 1H, H-1′). ¹³C NMR (187.5 MHz, CDCl₃): δ 107.4 (C-8′), 117.1 (C-7′), 119.6 (C-6′), 124.5 (C-8a’), 128.5 (C-3″), 129.6 (C-2″), 130.1 (C-4′), 132.5 (C-4″), 137.8 (C-1″), 139.0 (C-3′), 148 (C-1′), 190.9 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₄H₁₀N₂O: 222.0793; found: 222.0794.
(3-Methoxyphenyl)(pyrrolo[1,2-a]pyrazin-4-yl)methanone (4d). Following method A described for 4a, a mixture of 2b (0.030 g, 0.10 mmol), NH₄OAc (0.023 g, 0.30 mmol), and Li₂CO₃ (0.022 g, 0.30 mmol) provided 4d (0.021 g, 83%) as a yellow solid. R_f 0.39 (hexane/EtOAc, 1:1); mp 88–89 °C. IR (film): ῡ 2970, 1734, 1594, 1424, 1298, 1250, 1171, 1028, 854, 741 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 3.88 (s, 3H, CH₃O), 7.09 (br s, 2H, H-7′, H-8′), 7.17 (dd, J = 8.0, 2.6 Hz, 1H, H-4″), 7.34 (br d, J = 1.2 Hz, 1H, H-2″), 7.36 (br d, J = 8.0 Hz, 1H, H-6″), 7.43 (t, J = 8.0 Hz, 1H, H-5″), 8.13 (s, 1H, H-3′), 8.91 (s, 1H, H-6′), 8.94 (s, 1H, H-1′). ¹³C NMR (150 MHz, CDCl₃): δ 55.4 (CH₃O), 107.5 (C-8′), 114.2 (C-2″), 117.2 (C-7′), 118.9 (C-4″), 119.6 (C-6′), 122.2 (C-6″), 124.6 (C-8a’), 129.5 (C-5″), 130.1 (C-4′), 138.9 (C-3′), 139.0 (C-1″), 147.9 (C-1′), 159.7 (C-3″), 190.6 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₅H₁₂N₂O₂: 252.0899; found: 252.0900.
(4-Methoxyphenyl)(pyrrolo[1,2-a]pyrazin-4-yl)methanone (4e). Following method A described for 4a, a mixture of 2c (0.050 g, 0.168 mmol), NH₄OAc (0.039 g, 0.50 mmol), and Li₂CO₃ (0.037 g, 0.50 mmol) formed 4e (0.036 g, 84%) as a yellow solid. R_f 0.29 (hexane/EtOAc, 1:1); mp 138–139 °C. IR (KBr): ῡ 2928, 1739, 1590, 1507, 1301, 1265, 1167, 1109, 1030, 845, 759, 719 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 3.91 (s, 3H, CH₃O), 7.00–7.03 (m, 2H, H-3″), 7.05 (br s, 1H, H-8′), 7.06 (br s, 1H, H-7′), 7.84–7.87 (m, 2H, H-2″), 8.05 (br s, 1H, H-3′), 8.74 (s, 1H, H-6′), 8.92 (s, 1H, H-1′). ¹³C NMR (150 MHz, CDCl₃): δ 55.6 (CH₃O), 106.9 (C-8′), 113.9 (C-3″), 116.8 (C-7′), 119.0 (C-6′), 124.8 (C-8a’), 130.0 (C-4′), 130.1 (C-1″), 132.2 (C-2″), 137.4 (C-3′), 147.7 (C-1′), 163.6 (C-4″), 189.3 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₅H₁₂N₂O₂: 252.0899; found: 252.0899.
(3,4-Dimethoxyphenyl)(pyrrolo[1,2-a]pyrazin-4-yl)methanone (4f). Following method A described for 4a, a mixture of 2d (0.079 g, 0.24 mmol), NH₄OAc (0.056 g, 0.72 mmol), and Li₂CO₃ (0.053 g, 0.72 mmol) afforded 4f (0.041 g, 60%) as a yellow solid. R_f 0.16 (hexane/EtOAc, 1:1); mp 148–149 °C. IR (KBr): ῡ 3158, 2943, 1632, 1594, 1509, 1414, 1260, 1171, 1014, 772 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 3.95 (s, 3H, CH₃O), 3.97 (s, 3H, CH₃O), 6.94 (d, J = 8.8 Hz, 1H, H-5″), 7.04–7.06 (m, 2H, H-7′, H-8′), 7.44–7.47 (m, H-2″, H-6″), 8.06 (s, 1H, H-3′), 8.69 (br s, 1H, H-6′), 8.91 (s, 1H, H-1′). ¹³C NMR (187.5 MHz, CDCl₃): δ 56.0 (CH₃O), 56.1 (CH₃O), 106.9 (C-8′), 110.0 (C-5″), 111.8 (C-2″), 116.8 (C-7′), 118.9 (C-6′), 124.7 (C-8a’), 124.8 (C-6″), 129.9 (C-4′), 130.1 (C-1″), 137.2 (C-3′), 147.6 (C-1′), 149.2 (C-3″), 153.4 (C-4″), 189.2 (CO). HRMS (EI): m/z [M⁺] calcd. for C₁₆H₁₄N₂O₃: 282.1005; found: 282.1004.
Pyrrolo[1,2-a]pyrazine-4-carbonitrile (4g). Following method A described for 4a, a mixture of 3a (0.032 g, 0.169 mmol), NH₄OAc (0.039 g, 0.51 mmol), and Li₂CO₃ (0.038 g, 0.51 mmol) furnished 4g (0.013 g, 53%) as a brown solid. R_f 0.55 (hexane/EtOAc, 1:1); mp 128–130 °C. IR (film): ῡ 3010, 2600, 1741, 1366, 1212 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 7.13 (s, 2H, H-7, H-8), 7.84 (s, 1H, H-6), 8.08 (br s, 1H, H-3), 9.00 (br s, 1H, H-1). ¹³C NMR (100 MHz, CDCl₃): δ 104.5 (CN), 107.9 (C-8), 112.8 (C-4), 116.6 (C-6), 117.0 (C-7), 127.9 (C-8a), 136.3 (C-3), 148.1 (C-1). HRMS (EI): m/z [M⁺] calcd. for C₈H₅N₃: 143.0483; found: 143.0485.
Phenyl(pyrrolo[1,2-a]pyrazin-4-yl)methanol (4h). In a round-bottom flask equipped with a magnetic stirring bar, NaBH₄ (0.051 g, 1.33 mmol) was slowly added to a solution of 4c (0.030 g, 0.13 mmol) in anhydrous THF (5.0 mL). After heating the reaction mixture at 60 °C for 2 h, MeOH (8.0 mL) was added dropwise and stirred at rt for 5.0 min. Then, a saturated aqueous solution of NH₄Cl (8.0 mL) was added and stirred at rt for 20 min. The mixture was extracted with hexane/EtOAc (1:1, 15 mL) and brine (3 × 5.0 mL). The organic layer was dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 7:3), resulting in 4h (0.014 g, 48%) as a yellow solid. R_f 0.10 (hexane/EtOAc, 1:1); mp 150–152 °C. IR (film): ῡ 3042, 2820, 1618, 1451, 1318, 1048, 727, 704 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 3.10 (br, 1H, OH), 5.98 (s, 1H, CHOH), 6.80 (s, 2H, H-7′, H-8′), 7.30–7.39 (m, 4H, H-6′, H-3″, H-4″), 7.43 (br s, 1H, H-3′), 7.44–7.46 (m, 2H, H-2″), 8.58 (s, 1H, H-1′). ¹³C NMR (187.5 MHz, CDCl₃): δ 71.9 (CHOH), 104.5 (C-8′), 114.6 (C-6′), 115.3 (C-7′), 125.5 (C-3′), 126.6 (C-2″), 128.5 (C-4″), 128.83 (C-3″), 128.88 (C-8a’), 130.2 (C-4′), 138.6 (C-1″), 144.3 (C-1′). HRMS (EI): m/z [M⁺] calcd. for C₁₄H₁₂N₂O: 224.0950; found: 224.0948.
Methyl 2-(4-bromo-2-formyl-1H-pyrrol-1-yl)acetate (8h). In a round-bottom flask at 0 °C with a magnetic stirring bar, a solution of NBS (0.080 g, 0.45 mmol) in anhydrous CH₂Cl₂ (5.0 mL) was added dropwise to a solution of 8a (0.05 g, 0.30 mmol) in anhydrous DMF (5.0 mL). The mixture was stirred at rt for 2.0 h before adding a 10% aqueous solution of NaHSO₃ (1.0 mL). It was then washed with brine (3 × 5.0 mL), the organic phase was dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 8:2) to afford 8h (0.066 g, 90%) as a violet oil. R_f 0.74 (hexane/EtOAc, 1:1). IR (film): ῡ 2922, 1692, 1661, 1393, 1215, 998, 768 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 3.76 (s, 3H, CO₂CH₃), 5.01 (s, 2H, CH₂), 6.90 (br s, 1H, H-5′), 6.95 (d, J = 2.0 Hz, 1H, H-3′), 9.45 (d, J = 0.5 Hz, 1H, CHO). ¹³C NMR (125 MHz, CDCl₃): δ 50.1 (CH₂), 52.6 (CO₂CH₃), 97.5 (C-4′), 125.2 (C-3′) 131.2 (C-5′), 131.6 (C-2′), 168.2 (CO₂CH₃), 179.3 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₈H₈BrNO₃: 244.9688; found: 244.9680.
Methyl (Z)-2-(4-bromo-2-formyl-1H-pyrrol-1-yl)-3-(dimethylamino)acrylate (1c). Following the method described for 1b, a mixture of 8h (0.050 g, 0.20 mmol) and DMFDMA (0.121 g, 1.02 mmol) in anhydrous DMF (1.0 mL) gave 1c (0.041 g, 66%) as a pale violet oil. R_f 0.12 (hexane/EtOAc, 7:3). IR (film): ῡ 2921, 1666, 1621, 1212, 1084, 922, 755 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 2.67 (br, 6H, N(CH₃)₂), 3.62 (s, 3H, CO₂CH₃), 6.81 (br s, 1H, H-5′), 6.99 (br s, 1H, H-3′), 7.50 (s, 1H, H-3), 9.49 (s, 1H, CHO). ¹³C NMR (125 MHz, CDCl₃): δ 34.7 (N(CH₃)₂), 51.6 (CO₂CH₃), 96.6 (C-2), 98.2 (C-4′), 121.6 (C-3′), 132.9 (C-5′), 134.8 (C-2′), 146.4 (C-3), 166.9 (CO₂CH₃), 179.1 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₁₁H₁₃BrN₂O₃: 300.0110; found: 300.0108.
Methyl 7-bromopyrrolo[1,2-a]pyrazine-4-carboxylate (4i). Following method A described for 4a, a mixture of 1c (0.030 g, 0.10 mmol), NH₄OAc (0.031 g, 0.40 mmol), and Li₂CO₃ (0.029 g, 0.40 mmol) provided 4i (0.017 g, 74%) as a yellow solid. R_f 0.38 (hexane/EtOAc, 1:1); mp 93–95 °C. IR (film): ῡ 3173, 3126, 2957, 1720, 1442, 1308, 1200, 1175 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 4.01 (s, 3H, CO₂CH₃), 7.10 (br s, 1H, H-8), 8.45 (br s, 1H, H-3), 8.78 (s, 1H, H-6), 8.87 (br s, 1H, H-1). ¹³C NMR (125 MHz, CDCl₃): δ 52.8 (CO₂CH₃), 107.2 (C-7), 109.2 (C-8), 117.7 (C-4), 119.5 (C-6), 129.6 (C-8a), 133.7 (C-3), 145.9 (C-1), 162.5 (CO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₉H₇BrN₂O₂: 253.9691; found: 253.9681.
Methyl 6-bromopyrrolo[1,2-a]pyrazine-4-carboxylate (4j). In a round-bottom flask at 0 °C with a magnetic stirring bar, a solution of NBS (0.040 g, 0.23 mmol) in anhydrous CH₂Cl₂ (5.0 mL) was added dropwise to a solution of 4a (0.040 g, 0.23 mmol) in anhydrous CH₂Cl₂ (5.0 mL). The mixture was stirred at rt for 2.0 h before adding a 10% aqueous solution of NaHSO₃ (1.0 mL). It was then washed with brine (3 × 5.0 mL), the organic phase was dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 1:1) to obtain 4j (0.032 g, 55%) as a pale orange solid. R_f 0.32 (hexane/EtOAc, 1:1); mp 115–117 °C. IR (film): ῡ 3144, 2953, 1724, 1417, 1294, 1175, 957, 857, 773 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 4.00 (s, 3H, CO₂CH₃), 7.03 (d, J = 2.4 Hz, 1H, H-8), 8.46 (s, 1H, H-3), 8.67 (dd, 1H, J = 2.4, 0.6 Hz, H-7), 8.92 (br s, 1H, H-1). ¹³C NMR (150 MHz, CDCl₃): δ 52.6 (CO₂CH₃), 94.3 (C-6), 118.5 (C-8), 118.8 (C-7), 127.1 (C-4), 129.5 (C-8a), 135.2 (C-3), 147.2 (C-1), 162.8 (CO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₉H₇BrN₂O₂: 253.9691; found: 253.9701.
Methyl 6,7-dibromopyrrolo[1,2-a]pyrazine-4-carboxylate (4k). Following the method described for 4j, a mixture of 4a (0.030 g, 0.17 mmol) and NBS (0.063 g, 0.36 mmol) produced 4k (0.023 g, 40%) as an orange solid. R_f 0.61 (hexane/EtOAc, 1:1); mp 106–108 °C. IR (film): ῡ 3123, 2952, 2918, 2850, 1733, 1601, 1464, 1420, 1312, 1294, 1200, 1172, 1095, 862, 753 cm ⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 4.02 (s, 3H, CO₂CH₃), 7.04 (s, 1H, H-8), 8.00 (s, 1H, H-3), 8.80 (s, 1H, H-1). ¹³C NMR (125 MHz, CDCl₃): δ 53.0 (CO₂CH₃), 95.0 (C-6), 99.6 (C-7), 121.1 (C-4), 122.0 (C-8), 128.7 (C-8a), 133.1 (C-3), 145.8 (C-1), 161.6 (CO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₉H₆N₂O₂Br₂: 331.8796; found: 331.8792.
Methyl (E)-3-(1-(2-methoxy-2-oxoethyl)-1H-pyrrol-2-yl)acrylate (10a). At 0 °C and under N₂ atmosphere, NaH (60%, 0.032 g, 0.80 mmol) was added to a solution of 9a (0.100 g, 0.66 mmol) in anhydrous DMF (2.0 mL), and the mixture was stirred for 15 min. Then, 7a (0.124 g, 0.80 mmol) was added, and the solution was stirred at rt for 3 h. The reaction mixture was extracted with a hexane/EtOAc (1:1, 15.0 mL), the organic layer was washed with brine (5.0 mL × 3) and dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 9:1), resulting in 10a (0.088 g, 60%) as a white solid. R_f 0.32 (hexane/EtOAc, 7:3); mp 116–118 °C. IR (film): ῡ 2960, 1753, 1688, 1623, 1467, 1294, 1171, 1080, 987, 745 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 3.76 (s, 3H, =CHCO₂CH₃), 3.77 (s, 3H, CH₂CO₂CH₃), 4.75 (s, 2H, CH₂), 6.15 (d, J = 15.6 Hz, 1H, H-2), 6.24–6.26 (m, 1H, H-4′), 6.72 (dd, J = 3.8, 1.4 Hz, 1H, H-3′), 6.78 (br t, J = 1.4 Hz, 1H, H-5′), 7.43 (d, J = 15.6 Hz, 1H, H-3). ¹³C NMR (150 MHz, CDCl₃): δ 48.2 (CH₂), 51.5 (CH₂CO₂CH₃), 52.7 (=CHCO₂CH₃), 110.4 (C-4′), 112.3 (C-3′), 113.5 (C-2), 126.8 (C-5′), 129.4 (C-2′), 131.5 (C-3), 167.9 (=CHCO₂CH₃), 168.4 (CH₂CO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₁H₁₃NO₄: 223.0845; found: 223.0845.
Methyl (E)-3-(5-formyl-1-(2-methoxy-2-oxoethyl)-1H-pyrrol-2-yl)acrylate (8i). At 0 °C and under N₂ atmosphere, POCl₃ (0.220 g, 1.44 mmol) was added to DMF (0.105 g, 1.44 mmol), and the mixture was stirred for 10 min. Subsequently, 10a (0.200 g, 0.90 mmol) in anhydrous CH₂Cl₂ (6.0 mL) was added dropwise, and the solution was stirred at 0 °C for 3 h. The reaction mixture was quenched with a 2N aqueous solution of NaOH until pH 8.0, and then CH₂Cl₂ (21.0 mL) was added. The mixture was washed with brine (7.0 mL × 3), the organic layer was dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 9:1) to furnish 8i (0.142 g, 63%) as a pale brown solid. R_f 0.67 (hexane/EtOAc, 1:1); mp 108–110 °C. IR (film): ῡ 2960, 1741, 1704, 1663, 1198, 1168, 980, 778 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 3.75 (s, 3H, CH₂CO₂CH₃), 3.77 (s, 3H, =CHCO₂CH₃), 5.24 (s, 2H, CH₂), 6.41 (d, J = 15.8 Hz, 1H, H-2), 6.70 (d, J = 4.0 Hz, 1H, H-3′), 6.97 (d, J = 4.0 Hz, 1H, H-4′), 7.41 (d, J = 15.8 Hz, 1H, H-3), 9.53 (CHO). ¹³C NMR (125 MHz, CDCl₃) δ 46.3 (CH₂), 52.0 (CO₂CH₃), 52.8 (CO₂CH₃), 111.4 (C-3′), 121.1 (C-2), 124.6 (C-4′), 129.7 (C-3), 133.7 (C-5′), 137.7 (C-2′), 166.7 (=CHCO₂CH₃), 168.3 (CH₂CO₂CH₃), 180.2 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₁₂H₁₃NO₅: 251.0794; found: 251.0785.
Methyl (Z)-3-(dimethylamino)-2-(2-formyl-5-((E)-3-methoxy-3-oxoprop-1-en-1-yl)-1H-pyrrol-1-yl)acrylate (11). Following the method described for 1b, a mixture of 8i (0.090 g, 0.36 mmol) and DMFDMA (0.213 g, 1.79 mmol) in anhydrous DMF (1.0 mL) afforded 11 (0.031 g, 28%) as a pale violet oil. R_f 0.16 (hexane/EtOAc, 1:1). IR (film): ῡ 2957, 1619, 1435, 1164, 1103, 1037, 762 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 2.46 (br, 6H, N(CH₃)₂), 3.63 (s, 3H, CO₂CH₃-1), 3.78 (s, 3H, =CHCO₂CH₃), 6.41 (d, J = 16.1 Hz, 1H, H-2″), 6.74 (d, J = 3.8 Hz, 1H, H-4′), 7.05 (d, J = 3.8 Hz, 1H, H-3′) 7.41 (d, J = 16.1 Hz, 1H, H-1″), 7.67 (s, 1H, H-3), 9.61 (s, 1H, CHO). ¹³C NMR (187.5 MHz, CDCl₃): δ 36.4 (br, N(CH₃)₂), 51.6 (CO₂CH₃-1), 51.8 (=CHCO₂CH₃), 93.4 (C-2), 111.2 (C-4′), 119.5 (C-2″), 120.6 (C-3′), 131.6 (C-1″), 136.8 (C-2′), 139.0 (C-5′), 147.4 (C-3), 166.8 (CO₂CH₃-1), 167.0 (=CHCO₂CH₃), 180.0 (CHO). HRMS (EI): m/z [M⁺] calcd. for C₁₅H₁₈N₂O₅: 306.1216; found: 306.1214.
Methyl (E)-6-(3-methoxy-3-oxoprop-1-en-1-yl)pyrrolo[1,2-a]pyrazine-4-carboxylate (4l). Following method A described for 4a, a mixture of 11 (0.030 g, 0.10 mmol), NH₄OAc (0.023 g, 0.30 mmol), and Li₂CO₃ (0.022 g, 0.30 mmol) was heated at 70 °C for 7 h to give 4l (0.015 g, 61%) as a yellow solid. R_f 0.25 (hexane/EtOAc, 1:1); mp 100–102 °C. IR (film): ῡ 2917, 2852, 1717, 1623, 1717, 1623, 1427, 1330, 1262, 1171, 1099, 1066 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 3.82 (s, 3H, =CHCO₂CH₃), 4.04 (s, 3H, CO₂CH₃-4), 6.39 (d, J = 15.6 Hz, 1H, H-2′), 7.18 (d, J = 4.2 Hz, 1H, H-8), 7.35 (d, J = 4.2 Hz, 1H, H-7), 7.70 (d, J = 15.6 Hz, 1H, H-1′), 8.24 (s, 1H, H-3), 8.93 (br s, 1H, H-1). ¹³C NMR (187.5 MHz, CDCl₃): δ 51.8 (=CHCO₂CH₃), 53.0 (CO₂CH₃-4), 108.5 (C-8), 115.9 (C-2′), 118.1 (C-7), 120.9 (C-4), 126.9 (C-6), 132.01 (C-8a), 133.3 (C-1′), 135.2 (C-3), 147.9 (C-1), 163.3 (CO₂CH₃-4), 167.1 (=CHCO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₃H₁₂N₂O₄: 260.0797; found: 260.0795.
Ethyl (E)-3-(1-(2-methoxy-2-oxoethyl)-1H-pyrrol-2-yl)acrylate (10c). Following the method described for 10a, a mixture of 9b (0.100 g, 0.60 mmol), NaH (60%, 0.029 g, 0.72 mmol), and 7a (0.110 g, 0.72 mmol) formed 10c (0.10 g, 70%) as a pale brown solid. R_f 0.67 (hexane/EtOAc, 1:1); mp 56–57 °C. IR (film): ῡ 2997, 2949, 1742, 1692, 1626, 1474, 1290, 1171, 1080, 983, 745 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 1.31 (t, J = 7.2 Hz, 3H, CO₂CH₂CH₃), 3.77 (s, 3H, CO₂CH₃), 4.22 (q, J = 7.2 Hz, 2H, CO₂CH₂CH₃), 4.75 (s, 2H, CH₂), 6.16 (d, J = 15.6 Hz, 1H, H-2), 6.26–6.26 (ddd, J = 4.0, 3.0, 0.8 Hz, 1H, H-4′), 6.71 (dd, J = 4.0, 1.4 Hz, 1H, H-3′), 6.78 (dd, J = 3.0, 1.4 Hz, 1H, H-5′), 7.44 (d, J = 15.6 Hz, 1H, H-3). ¹³C NMR (100 MHz, CDCl₃): δ 14.3 (CO₂CH₂CH₃), 48.2 (CH₂), 52.7 (CO₂CH₃), 60.2 (CO₂CH₂CH₃), 110.4 (C-4′), 112.1 (C-3′), 114.0 (C-2), 126.7 (C-5′), 129.4 (C-2′), 131.3 (C-3), 167.5 (CO₂CH₂CH₃), 168.4 (CO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₂H₁₅NO₄: 237.1001; found: 237.1001.
Methyl (Z)-3-(dimethylamino)-2-(2-((E)-3-methoxy-3-oxoprop-1-en-1-yl)-1H-pyrrol-1-yl)acrylate (12a). Following the method described for 1b, a mixture of 10a (0.119 g, 0.53 mmol) and DMFDMA (0.318 g, 2.67 mmol) in anhydrous DMF (1.0 mL) provided 12a (0.134 g, 90%) as a pale violet oil. R_f 0.33 (hexane/EtOAc, 7:3). IR (film): ῡ 2949, 1690, 1615, 1308, 1208, 1164, 1103, 1079, 1034 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 2.70 (br, 6H, N(CH₃)₂), 3.62 (s, 3H, =CHCO₂CH₃), 3.73 (s, 3H, CO₂CH₃-1), 6.15 (d, J = 15.8 Hz, 1H, H-2″), 6.23 (ddd, J = 3.7, 2.4, 0.4 Hz, 1H, H-4′), 6.78 (dd, J = 3.7, 1.5 Hz, 1H, H-3′), 6.71 (dd, J = 2.4, 1.5 Hz, H-5′), 7.37 (d, J = 15.8 Hz, 1H, H-1″), 7.57 (s, 1H, H-3). ¹³C NMR (100 MHz, CDCl₃): δ 35.2 (N(CH₃)₂), 46.7 (N(CH₃)₂), 51.3 (CO₂CH₃-1), 51.5 (=CHCO₂CH₃), 96.3 (C-2), 110.1 (C-4′), 111.7 (C-3′), 112.5 (C-2″), 130.1 (C-5′), 132.3 (C-2′), 133.6 (C-1″), 147.1 (C-3), 167.6 (CO₂CH₃-1), 168.2 (=CHCO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₄H₁₈N₂O₄: 278.1267; found: 278.1263.
Ethyl (Z)-3-(dimethylamino)-2-(2-((E)-3-ethoxy-3-oxoprop-1-en-1-yl)-1H-pyrrol-1-yl)acrylate (12b). In a MW glass vial equipped with a magnetic stirring bar and sealed with a cap, a mixture of 10b (0.300 g, 1.20 mmol) and tert-butoxy bis(dimethylamino)methane (0.625 g, 3.60 mmol) was heated at 125 °C for 2.0 h under MW irradiation (200 W) and a N₂ atmosphere. The crude mixture was suspended and stirred in CH₂Cl₂/toluene (10:1, 11 mL), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 7:3), resulting in 12b (0.150 g, 41%) as a pale brown oil. R_f 0.52 (hexane/EtOAc, 1:1). IR (film): ῡ 2977, 1690, 1615, 1301, 1212, 1161, 1079, 1034 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 1.16 (t, J = 7.0 Hz, 3H, CO₂CH₂CH₃), 1.29 (t, J = 7.0 Hz, 3H, CO₂CH₂CH₃), 2.29 (br, 3H, N(CH₃)₂), 3.01 (br, 3H, N(CH₃)₂), 4.05–4.17 (m, 2H, CO₂CH₂CH₃), 4.17–4.23 (m, 2H, CO₂CH₂CH₃), 6.15 (d, J = 15.5 Hz, 1H, H-2″), 6.23 (dd, J = 4.0, 2.5 Hz, 1H, H-4′), 6.67 (dd, J = 4.0, 1.5 Hz, 1H, H-3′), 6.71 (dd, J = 2.5, 1.5 Hz, 1H, H-5′), 7.37 (d, J = 15.5 Hz, 1H, H-1″), 7.56 (s, 1H, H-3). ¹³C NMR (125 MHz, CDCl₃): δ 14.4 (CO₂CH₂CH₃), 14.5 (CO₂CH₂CH₃), 38.6 (N(CH₃)₂), 59.98 (CO₂CH₂CH₃), 60.03 (CO₂CH₂CH₃), 96.3 (C-2), 110.0 (C-4′), 111.6 (C-3′), 112.7 (C-2″), 130.1 (C-5′), 132.4 (C-2′), 133.4 (C-1″), 146.9 (C-3), 167.2 (CO₂CH₂CH₃-1), 168.9 (=CHCO₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₆H₂₂N₂O₄: 306.1580; found: 306.1573.
Methyl (Z)-3-(dimethylamino)-2-(2-((E)-3-ethoxy-3-oxoprop-1-en-1-yl)-1H-pyrrol-1-yl)acrylate (12c). Following the method described for 1b, a mixture of 10c (0.060 g, 0.25 mmol) and DMFDMA (0.151 g, 1.25 mmol) in anhydrous DMF (2.0 mL) afforded 12c (0.068 g, 92%) as a pale violet oil. R_f 0.48 (hexane/EtOAc, 1:1). IR (film): ῡ 2923, 1697, 1618, 1308, 1212, 1158, 1079, 1308, 1034 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 1.29 (t, J = 7.2 Hz, 3H, CO₂CH₂CH₃), 2.26 (br, 3H, N(CH₃)₂), 3.05 (br, 3H, N(CH₃)₂), 3.63 (s, 3H, CO₂CH₃), 4.20 (q, J = 7.2 Hz, 2H, CO₂CH₂CH₃), 6.15 (d, J = 16.0 Hz, 1H, H-2″), 6.24 (br d, J = 3.3 Hz, 1H, H-4′), 6.68 (br d, J = 3.3 Hz, 1H, H-3′), 6.71 (br t, J = 1.7 Hz, 1H, H-5′), 7.38 (d, J = 16.0 Hz, 1H, H-1″), 7.57 (s, 1H, H-3). ¹³C NMR (125 MHz, CDCl₃): δ 14.4 (CO₂CH₂CH₃), 35.0 (N(CH₃)₂), 47.4 (N(CH₃)₂), 51.5 (CO₂CH₃), 60.1 (CO₂CH₂CH₃), 96.3 (C-2), 110.1 (C-4′), 111.6 (C-3′), 113.0 (C-2″), 130.1 (C-5′), 132.4 (C-2′), 133.3 (C-1″), 147.1 (C-3), 167.7 (CO₂CH₃), 167.8 (CO₂CH₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₅H₂₀N₂O₄: 292.1423; found: 292.1414.
Dimethyl indolizine-5,7-dicarboxylate (5a). At rt and under N₂ atmosphere, a solution of AlCl₃ (0.043 g, 0.321 mmol) in nitrobenzene (1.0 M) was added to a solution of 12a (0.030 g, 0.17 mmol) in anhydrous CH₂Cl₂ (5.0 mL). The mixture was stirred at rt for 2 h before adding CH₂Cl₂ (15 mL). It was then washed with brine (5.0 mL × 3) and dried (Na₂SO₄), and the solvent was removed under vacuum. The residue was purified by column chromatography over silica gel (hexane/EtOAc, 9:1) to give 5a (0.024 g, 66%) as a yellow solid. R_f 0.80 (hexane/EtOAc, 1:1); mp 110–112 °C. IR (film): ῡ 2919, 1707, 1625, 1434, 1232, 1195, 1161, 1082, 758, 730 cm⁻¹. ¹H NMR (750 MHz, CDCl₃): δ 3.94 (s, 3H, CO₂CH₃-5), 4.00 (s, 3H, CO₂CH₃-7), 6.95 (dd, J = 3.8, 0.9 Hz, 1H, H-1), 7.02 (dd, J = 3.8, 1.9 Hz, 1H, H-2), 8.17 (br d, J = 1.5 Hz, 1H, H-6), 8.41 (br d, J = 1.5 Hz, 1H, H-8), 8.84 (br s, 1H, H-3). ¹³C NMR (187.5 MHz, CDCl₃): δ 52.2 (CO₂CH₃-7), 52.4 (CO₂CH₃-5), 107.6 (C-1), 116.0 (C-5), 116.7 (C-2), 118.0 (C-6), 119.4 (C-3), 123.0 (C-7), 127.2 (C-8), 133.5 (C-8a), 163.1 (CO₂CH₃-5), 165.9 (CO₂CH₃-7). HRMS (EI): m/z [M⁺] calcd. for C₁₂H₁₁NO₄: 233.0688; found: 233.0687.
Diethyl indolizine-5,7-dicarboxylate (5b). Following the method described for 5a, a mixture of 12b (0.060 g, 0.20 mmol) and AlCl₃ (0.078 g, 0.39 mmol) in anhydrous CH₂Cl₂ (2.0 mL) was stirred at rt for 2h. After the further addition of AlCl₃ (0.078 g, 0.39 mmol), the mixture was stirred for 2 h to obtain 5b (0.016 g, 31%) as a yellow solid. R_f 0.81 (hexane/EtOAc, 7:3); mp 55–57 °C. IR (film): ῡ 2923, 2851, 1707, 1226, 1198, 1182, 1024 cm⁻¹. ¹H NMR (500 MHz, CDCl₃): δ 1.42 (t, J = 7.0 Hz, 3H, CO₂CH₂CH₃), 1.45 (t, J = 7.0 Hz, 3H, CO₂CH₂CH₃), 4.39 (q, J = 7.0 Hz, 2H, CO₂CH₂CH₃), 4.46 (q, J = 7.0 Hz, 2H, CO₂CH₂CH₃), 6.94 (br d, J = 4.0 Hz, 1H, H-1), 7.02 (dd, J = 4.0, 3.0 Hz, 1H, H-2), 8.17 (d, J = 1.5 Hz, 1H, H-6), 8.41 (d, J = 1.5 Hz, 1H, H-8), 8.85 (br s, 1H, H-3). ¹³C NMR (125 MHz, CDCl₃): δ 14.33 (CO₂CH₂CH₃), 14.41 (CO₂CH₂CH₃), 61.1 (CO₂CH₂CH₃), 61.6 (CO₂CH₂CH₃), 107.4 (C-1), 116.4 (C-5), 116.6 (C-2), 117.8 (C-6), 119.4 (C-3), 123.2 (C-7), 127.0 (C-8), 133.5 (C-8a), 162.7 (CO₂Et), 165.6 (CO₂Et). HRMS (EI): m/z [M⁺] calcd. for C₁₂H₁₁NO₄: 233.0688; found: 233.0687.
7-Ethyl 5-methyl indolizine-5,7-dicarboxylate (5c). Following the method described for 5a, a mixture of 12c (0.030 g, 0.10 mmol) and AlCl₃ (0.028 g, 0.20 mmol) in anhydrous CH₂Cl₂ (2.0 mL) furnished 5c (0.013 g, 52%) as a yellow solid. R_f 0.87 (hexane/EtOAc, 1:1); mp 133–135 °C. IR (film): ῡ 2987, 1704, 1256, 1232, 1198, 1168, 1018, 751 cm⁻¹. ¹H NMR (600 MHz, CDCl₃): δ 1.42 (t, J = 7.2 Hz, 3H, CO₂CH₂CH₃), 4.00 (s, 3H, CO₂CH₃), 4.40 (q, J = 7.2 Hz, 2H, CO₂CH₂CH₃), 6.94 (br d, J = 4.2 Hz, 1H, H-1), 7.02 (dd, J = 4.2, 2.0 Hz, 1H, H-2), 8.16 (br d, J = 1.8 Hz, H-6), 8.41 (s, 1H, H-8), 8.84 (br s, 1H, H-3). ¹³C NMR (150 MHz, CDCl₃): δ 14.4 (CO₂CH₂CH₃), 52.4 (CO₂CH₃), 61.1 (CO₂CH₂CH₃), 107.5 (C-1), 116.4 (C-5), 116.6 (C-2), 118.1 (C-6), 119.4 (C-3), 123.0 (C-7), 127.1 (C-8), 133.5 (C-8a), 163.1 (CO₂CH₃), 165.5 (CO₂CH₂CH₃). HRMS (EI): m/z [M⁺] calcd. for C₁₃H₁₃NO₄: 247.0845; found: 247.0840.

3.3. Evaluation of Antifungal Activity

The pyrrole derivatives herein synthesized were submitted to the CLSI M27-A3 microdilution method, and an evaluation of the antifungal sensitivity of the various concentrations was carried out against Candida spp. (C. albicans ATCC 10231, C. glabrata CBS138, C. dubliniensis CD36, C. krusei ATCC 14423, C. auris Monterrey, and C. haemulonii ENCB87) [67]. The compounds were used at 187–0.01 μg/mL. The references were fluconazole, simvastatin, and atorvastatin. The inoculum of Candida spp. was adjusted in a spectrophotometer to 620 nm. Subsequently, a 1:1000 dilution was made with RPMI medium. The 96-well microplates were inoculated with 75 µL of yeast suspension and 75 µL of the concentration of the compound to be tested. RPMI served as the sterility control, and DMSO in the absence of an antifungal agent as the growth control. The microplates were incubated at 37 °C for 24 h, and growth was quantified in a microplate spectrophotometer at 620 nm. The values of yeast growth are expressed as the average of three independent assays.

3.4. Docking Studies

The sequences of the HMGR enzyme from each Candida spp., including Candida albicans (CaHMGR), C. glabrata (CgHMGR), C. dubliniensis (CdHMGR), C. krusei (CkHMGR), C. auris (CauHMGR), and C. haemulonii (ChaHMGR), were downloaded from the NCBI database [68]. The HMGR sequences were processed by the homology modeling technique to generate 3D models on the Modeller program version 10.2 [69]. The crystallized structure of the catalytic portion of human HMG-CoA reductase with simvastatin (PDB: 1HW9), deposited in the protein data bank (PDB) [70], served as a template. These 3D models were validated with the Procheck program [71] before being utilized in the docking studies, which were run on Autodock4 with the series of enaminones, N-alkyl pyrroles, pyrrolo[1,2-a]pyrazines, 2-methyl acrylates, and indolizines. The 2D structure of each ligand was sketched in the chemical editor ChemDraw [72] and converted to 3D in mol2 format with the Open Babel GUI program [73]. The structures of the reference compounds (simvastatin and atorvastatin) were downloaded from ZINC 20 [74] and optimized with Gaussian 16W software [75]. The instruction files were prepared in AutoDock Tools [76], setting a grid box of 80 × 62 × 58 Å, centered at: X = 23.123, Y = 9.113, and Z = 1.802, with a grid spacing of 0.375 Å³. In Autodock, the Lamarckian Genetic Algorithm was chosen, considering 100 docking trials, a population size of 150, a maximum number of energy evaluations of 25,000, a maximum number of generations of 27,000, and a mutation rate of 0.02. After docking, the coupling with the lowest binding energy (expressed in kcal/mol) was selected and analyzed with Biovia Discovery Studio 2017 R2 software [77].

4. Conclusions

New synthetic strategies are described for the preparation of pyrrolo[1,2-a]pyrazines and indolizines. The design of these approaches is centered on the cyclization of pyrrole-based enaminone precursors through treatment with ammonium acetate to afford pyrrolo[1,2-a]pyrazines, and on the application of a Lewis acid to enaminone precursors to furnish indolizines. All the intermediates and precursors were synthesized starting from 2-formylpyrrole (6), following short and efficient routes. The stereoselective introduction of the (Z)-enaminone moiety was achieved by reacting the N-alkyl pyrrole with DMFDMA under conventional heating or with Bredereck’s reagent under MW irradiation. The following series of the intermediates and cyclization products were: enaminone-containing pyrroles 1a, 1c, and 2a–d, pyrrolo[1,2-a]pyrazines 4a–l, indolizines 5a–c, N-alkyl 2-formylpyrroles 8a–h, and pyrrole-based alkyl acrylates 8i, 10a–c, 11, and 12a–c, which were evaluated for their in vitro growth inhibition of six Candida spp. (C. albicans, C. glabrata, C. dubliniensis, C. krusei, C. auris, and C. haemulonii). The majority of these compounds demonstrated good inhibition of yeast growth. Docking simulations were carried out between the twelve most active compounds and the three most prevalent Candida spp. (C. albicans, C. glabrata, and C. auris). Insights were provided into the possible mechanism of action of the compounds evaluated. Polar and non-polar interactions between the functional groups of the derivatives and the active site of the HMGR enzyme of the three selected yeasts are involved, which have also been established for simvastatin and atorvastatin (the reference drugs). The lead compounds bear plausible pharmacophore groups responsible for the antifungal activity, which was found against all six Candida spp. analyzed, including the two multidrug-resistant species. Hence, the structural requirement is presently established in order that the most active analogues can be used in future studies on drug design and development.

Author Contributions

Conceptualization, J.T.; writing—original draft preparation, J.T.; methodology, D.M.-S., C.H.E., D.A.-P., E.B. and L.V.-T.; software, O.G.-G.; formal analysis, F.D. and J.T., study investigation, D.M.-S., D.A.-P. and E.B.; data curation, O.G.-G., C.H.E. and E.B.; interpretation, D.M.-S., D.A.-P. and C.H.E.; supervision, J.T., F.D. and O.G.-G. All authors contributed to the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the Supplementary Materials.

Acknowledgments

We thank Elvia Becerra, Eder I. Martínez, Julio C. López, and the CNMN-IPN for their assistance in spectrometric measurements, and Bruce A. Larsen for proofreading. D.M.-S., C.H.E., and E.B. are grateful to CONACYT for graduate scholarships awarded and to SIP-IPN (PIFI), and to the Ludwig K. Hellweg Foundation for scholarship complements and postdoctoral fellowships. O.G.-G., L.V.-T., F.D. and J.T. are fellows of the Estímulos al Desempeño de los Investigadores (EDI)-IPN and Comisión de Operación y Fomento de Actividades Académicas (COFAA)-IPN programs.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not available.

Footnotes

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Figure, Schemes and Tables

View Image - Scheme 1. Previously reported synthetic procedures: (a) a Pd(II)-promoted vinyl cyclization to achieve pyrrolo[1,2-a]pyrazines; (b) a condensation process to afford indolizines; and (c) the use of 2-formylpyrroles as the starting material to obtain pyrrolo[1,2-a]pyrazines and indolizines. (d) With the current strategy, pyrrole-based enaminones (1–3) were the building block for the preparation of pyrrolo[1,2-a]pyrazines (4) and indolizines (5) [23,25,30].

Scheme 1. Previously reported synthetic procedures: (a) a Pd(II)-promoted vinyl cyclization to achieve pyrrolo[1,2-a]pyrazines; (b) a condensation process to afford indolizines; and (c) the use of 2-formylpyrroles as the starting material to obtain pyrrolo[1,2-a]pyrazines and indolizines. (d) With the current strategy, pyrrole-based enaminones (1–3) were the building block for the preparation of pyrrolo[1,2-a]pyrazines (4) and indolizines (5) [23,25,30].

Scheme 2. Preparation of 2-formylpyrrole-based enaminones 1a–b, 2a–d, and 3a.

View Image - Scheme 3. Preparation of pyrrolo[1,2-a]pyrazines 4a and 4c via a multicomponent procedure, and of 4h and 4i by the transformation of 4c and 8h, respectively.

Scheme 3. Preparation of pyrrolo[1,2-a]pyrazines 4a and 4c via a multicomponent procedure, and of 4h and 4i by the transformation of 4c and 8h, respectively.

Scheme 4. Preparation of pyrrolo[1,2-a]pyrazines 4j and 4k by bromination of 4a.

Scheme 5. Preparation of pyrrolo[1,2-a]pyrazine 4l by transformation of pyrrole 9a.

Scheme 6. Conversion of the series 10a–c into indolizines 5a–c.

View Image - Figure 1. Representation of the interactions between the active site of HMGR of C. albicans and simvastatin, atorvastatin, and enaminone-containing pyrroles 1a, 2a, and 2c, pyrrolo[1,2-a]pyrazines 4b, 4g, and 4l, indolizine 5a, N-alkyl 2-formylpyrroles 8a, 8c, and 8g, and pyrrole-based alkyl acrylates 10a and 12a. The 3D model shows the amino acid residues involved in ligand binding to the active site of the enzyme. In the 2D model, the following interactions are depicted with dotted lines: conventional hydrogen bond (dark green), carbon hydrogen (light green), π–sigma (purple), π–π T-shaped and π–π stacked (fuchsia), π–alkyl and alkyl (pink), π–anion (orange), and π–cation (yellow). The amino acids are represented in pink circles (for basic amino acids), orange (for acid), cyan (for polars), and yellow (for non-polar).

Figure 1. Representation of the interactions between the active site of HMGR of C. albicans and simvastatin, atorvastatin, and enaminone-containing pyrroles 1a, 2a, and 2c, pyrrolo[1,2-a]pyrazines 4b, 4g, and 4l, indolizine 5a, N-alkyl 2-formylpyrroles 8a, 8c, and 8g, and pyrrole-based alkyl acrylates 10a and 12a. The 3D model shows the amino acid residues involved in ligand binding to the active site of the enzyme. In the 2D model, the following interactions are depicted with dotted lines: conventional hydrogen bond (dark green), carbon hydrogen (light green), π–sigma (purple), π–π T-shaped and π–π stacked (fuchsia), π–alkyl and alkyl (pink), π–anion (orange), and π–cation (yellow). The amino acids are represented in pink circles (for basic amino acids), orange (for acid), cyan (for polars), and yellow (for non-polar).

Table 1

Reaction conditions and yields for the preparation of 4-substituted pyrrolo[1,2-a]pyrazines 4a–g ^a.

[Image omitted. Please see PDF.]
Entry	1–3	Z	T (°C)	t (h)	4 (%) ^b
1	1a	CO₂Me	70	4	4a (90)
2	1b	CO₂Et	80	3	4b (66)
3	2a	COPh	80	3	4c (87)
4	2b	COC₆H₄-3-OMe	70	4	4d (83)
5	2c	COC₆H₄-4-OMe	70	4	4e (84)
6	2d	COC₆H₃-3,4-(OMe)₂	70	4	4f (60)
7	3a	CN	70	4	4g (53)

^a The reactions were carried out with the enaminones 1–3 (1.0 mol eq.), NH₄OAc (3.0 mol eq.), and Li₂CO₃ (3.0 mol eq.) in anh. DMF. ^b After purification by column chromatography.

Table 2

MIC₅₀ and MIC₇₀ values for the inhibition of Candida spp. by the following compounds: enaminone-containing pyrroles 1a, 1c, and 2a–d, pyrrolo[1,2-a]pyrazines 4a–l, indolizines 5a–c, N-alkyl 2-formylpyrroles 8a–h, and pyrrole-based alkyl acrylates 8i, 10a–c, 11, and 12a–c ^a.

Compound	C. albicans		C. glabrata		C. dubliniensis		C. krusei		C. auris		C. haemulonii
	MIC₅₀	MIC₇₀	MIC₅₀	MIC₇₀	MIC₅₀	MIC₇₀	MIC₅₀	MIC₇₀	MIC₅₀	MIC₇₀	MIC₅₀	MIC₇₀
	µg/mL		µg/mL		µg/mL		µg/mL		µg/mL		µg/mL
fluconazole	1.40	1.80	5.60	7.20	1.40	1.80	5.60	7.20	>44.8	>57.6	>44.8	>57.6
simvastatin	1.25	1.75	15.00	21.00	1.25	1.75	40.00	56.00	10.00	14.00	20.00	28.00
atorvastatin	3.77	5.27	1.71	2.39	2.77	3.87	4.80	6.72	15.3	21.42	8.00	11.20
1a	0.11	0.15	0.09	0.12	0.07	0.09	0.05	0.07	4.17	5.83	4.45	6.23
1c	4.20	5.88	0.65	0.91	4.00	5.60	0.36	0.50	2.59	3.62	2.00	2.80
2a	0.13	0.18	0.09	0.12	0.06	0.08	0.03	0.04	5.57	7.79	3.74	5.23
2b	0.13	0.18	0.11	0.15	0.07	0.09	0.05	0.07	5.31	7.43	5.20	7.28
2c	0.10	0.14	0.07	0.09	0.06	0.08	0.06	0.08	4.50	6.30	4.45	6.23
2d	0.12	0.16	0.12	0.16	0.09	0.12	0.04	0.06	4.68	6.55	12.48	17.47
4a	0.18	0.25	1.56	2.18	0.73	1.02	0.20	0.28	5.85	8.19	11.70	16.30
4b	0.15	0.21	0.11	0.15	0.07	0.09	0.09	0.12	3.07	4.29	3.90	5.46
4c	0.33	0.46	0.15	0.21	0.09	0.12	0.01	0.02	1.46	2.04	18.72	26.20
4d	0.45	0.63	0.12	0.16	0.16	0.22	0.18	0.36	1.62	2.25	11.70	16.38
4e	0.33	0.46	0.13	0.18	0.36	0.50	0.18	0.36	1.46	2.04	7.80	10.92
4f	0.18	0.25	0.16	0.22	0.09	0.12	0.36	0.50	10.63	14.88	11.70	16.38
4g	0.07	0.09	0.18	0.25	0.09	0.12	0.14	0.19	6.15	8.61	6.88	9.63
4h	0.23	0.32	0.12	0.16	0.09	0.12	0.12	0.16	2.20	3.08	4.92	6.88
4i	0.08	0.11	0.15	0.21	0.01	0.02	0.28	0.39	11.68	16.35	11.68	16.35
4j	0.22	0.30	0.11	0.15	0.22	0.04	0.05	0.07	11.68	16.35	11.68	16.35
4k	0.10	0.14	0.08	0.11	0.07	0.09	0.18	0.36	3.90	5.46	18.72	26.20
4l	0.04	0.05	0.11	0.15	0.06	0.08	0.04	0.06	5.57	7.79	4.92	6.88
5a	0.25	0.17	0.12	0.16	0.12	0.16	0.12	0.16	4.00	5.60	3.12	4.36
5b	0.50	0.70	1.82	2.54	1.58	2.21	4.00	5.60	3.10	4.34	2.55	3.57
5c	0.50	0.70	2.00	2.80	2.45	3.43	0.45	0.63	2.60	3.64	3.24	4.53
8a	0.10	0.14	0.24	0.33	0.03	0.04	0.06	0.08	1.46	2.04	7.20	10.08
8b	0.19	0.26	0.12	0.16	0.14	0.19	0.06	0.08	2.96	4.14	18.72	26.20
8c	0.07	0.09	0.10	0.14	0.07	0.09	0.04	0.06	8.35	11.69	11.70	16.38
8d	0.20	0.28	0.03	0.04	0.05	0.07	0.05	0.07	6.15	8.61	11.70	16.38
8e	0.28	0.39	0.09	0.12	0.06	0.08	0.04	0.06	9.00	12.60	10.63	14.88
8f	0.20	0.28	0.05	0.07	0.05	0.07	0.05	0.07	4.68	6.55	10.63	14.88
8g	0.12	0.16	0.08	0.11	0.16	0.22	0.04	0.06	5.57	7.79	16.71	23.39
8h	4.00	5.60	0.82	1.14	2.43	3.40	1.00	1.40	2.38	3.33	2.27	3.17
8i	0.14	0.19	0.45	0.63	0.32	0.44	0.20	0.28	6.88	9.63	10.63	14.88
10a	0.50	0.70	0.50	0.70	0.55	0.77	0.88	1.23	3.57	4.99	4.00	5.60
10b	0.58	0.81	0.26	0.36	4.00	5.60	4.66	6.52	2.82	3.94	2.38	3.33
10c	3.74	5.23	1.42	1.98	1.08	1.51	1.02	1.42	2.56	3.58	2.30	3.22
11	0.18	0.25	0.84	1.17	0.73	1.02	0.16	0.22	4.87	6.81	5.85	8.19
12a	0.10	0.14	0.13	0.18	0.10	0.70	0.14	0.19	4.00	5.60	2.00	2.80
12b	0.12	0.17	0.91	1.27	2.14	2.99	0.88	1.20	2.80	3.92	2.27	3.17
12c	1.77	2.47	0.85	1.19	2.38	3.33	1.30	1.82	2.85	3.99	2.70	3.78

^a MIC₅₀ is the 50% inhibition of yeast growth. MIC₇₀ is the 70% inhibition of yeast growth.

Table 3

Binding energy values (DG, kcal/mol) at the active site of the HMGR enzymes of six Candida species for simvastatin and atorvastatin and for the test compounds: enaminone-containing pyrroles 1a, 2a, and 2c, pyrrolo[1,2-a]pyrazines 4b, 4g, and 4l, indolizine 5a, N-alkyl 2-formylpyrroles 8a, 8c, and 8g, and pyrrole-based alkyl acrylates 10a and 12a ^a.

Compound	HMGR Enzymes of Candida spp.
Compound	C. albicans	C. glabrata	C. dubliniensis	C. krusei	C. auris	C. haemulonii
simvastatin	−6.12	−6.30	−6.57	−6.06	−6.51	−6.18
atorvastatin	−3.82	−4.63	−4.64	−4.97	−2.14	−4.66
1a	−6.21	−5.26	−5.42	−6.55	−5.47	−5.34
2a	−7.19	−7.48	−8.00	−8.73	−8.34	−6.89
2c	−7.14	−6.34	−8.05	−6.88	−7.98	−7.35
4b	−6.41	−5.53	−5.74	−7.02	−5.69	−7.01
4g	−5.74	−5.18	−5.18	−6.07	−5.18	−6.22
4l	−7.70	−6.44	−6.48	−7.58	−7.31	−6.43
5a	−7.22	−6.65	−6.73	−7.04	−6.71	−5.57
8a	−5.33	−4.74	−4.49	−5.55	−5.55	−5.92
8c	−8.24	−6.77	−6.81	−8.32	−7.06	−7.49
8g	−5.21	−5.00	−4.56	−5.28	−4.93	−5.58
10a	−6.42	−5.88	−5.92	−6.52	−6.11	−4.93
12a	−7.09	−7.20	−7.15	−5.42	−6.70	−5.50

^a C. albicans (CaHMGR), C. glabrata (CgHMGR), C. dubliniensis (CdHMGR), C. krusei (CkHMGR), C. auris (CauHMGR), and C. haemulonii (ChaHMGR).

Table 4

Data on the interactions of enaminones 1a, 2a, and 2c, pyrrolo[1,2-a]pyrazines 4b, 4g, and 4l, indolizine 5a, N-alkyl 2-formylpyrroles 8a, 8c, and 8g, and pyrrole-based alkyl acrylates 10a and 12a at the active site of the HMGR enzyme of C. albicans (CaHMGR).

Compound	Residues of the Enzyme Interacting with the Ligand	Polar Interactions	Hydrophobic Interactions
simvastatin	Ala62, Thr95, Glu96, Gly97, Cys98, Arg127, Met192, Met194, Asn195, Asp227, Lys228, Gly302, Gln303, Asp304, Gln307, Gly343, Gly344	Asp227, Lys228, Asp304	-
atorvastatin	Thr95, Glu96, Gly97, Met192, Met194, Asn195, Asp227, Lys228, Asn292, His289, Gly302, Gln303, Asp304, Gln307, Gly342, Gly343, Gly344, Leu389	Asn195, Asp304	Glu96, Met192, Met194, Lys228, Asp304
1a	Ala62, Cys63, Thr95, Glu96, Gly97, Met192, Gly302, Gln303, Asp304, Pro305, Gly343, Gly344	Asp304	Cys63, Glu96
2a	Glu96, Leu99, Arg127, Met194, Asn195, Ser221, Asp227, Lys228, Lys272, Ala288, His289, Ans292, Leu389	Asp227, Lys228, Asn292	Glu96, Met194, His289, Leu389
2c	Thr95, Glu96, Met192, Met194, Asn195, Asp227, Lys228, Gln303, Asp304, Pro305, Gly344	Lys228	Met192, Met194
4b	Thr95, Glu96, Met192, Gly302, Gln303, Asp304, Gln307, Glu337, Val338, Gly339, Ile341, Gly342, Gly343, Gly344, Thr345	Gly339, Ile341, Gly344	Met192, Asp304, Gly342, Gly343
4g	Leu73, Ala93, Thr94, Thr95, Glu96, Thr295, Ala 296, Leu299, Gln303, Asp304, Pro305	Thr95	Ala296, Pro305
4l	Leu73, Ala93, Thr94, Thr95, Glu96, Leu99, Asn292, Thr295, Ala296, Leu299, Gly302, Gln303, Asp304, pro305, Gly344	Ala93, Thr94	Leu73, Thr94, Ala296, Leu299, Pro305
5a	Thr95, Glu96, Met192, Met194, Asn195, Gly302, Gln303, Asp304, Gly339, Ile341, Gly342, Gly343, Gly344, Thr345	Thr95, Asn195, Gly302, Gly343, Gly344	Met192, Gly339, Gly342, Thr345
8a	Thr95, Glu96, Met192, Gly302, Gln303, Asp304, Gln307, Glu337, Val338, Gly339, Ile341 Gly342, Gly343, Gly344, Thr345	Asp304, Thr95, Val338, Gly344	Met192
8c	Thr95, Glu96, Met192, Met194, Asn195, Lys228, Gly302, Gln303, Asp304, Gly339, Ile341 Gly342, Gly343, Gly344, Thr345	Thr95, Gly344	Glu96, Met192
8g	Thr94, Thr95, Ala93, Glu96, Leu99, Asn292, Thr295, Ala296, Gln303, Pro305	Thr94, Thr95, Thr295	Ala296, Pro305
10a	Leu73, Ala93, Thr94, Thr95, Glu96, Leu99, Asn292, Thr295, Ala296, Leu299, Gln303, Asp304, Pro305, Ala306	Asn292, Thr295, Asp304	Leu73, Thr94, Ala296, Leu299, Pro305
12a	Ala62, Thr95, Glu96, Gly97, Met192, Gly193, Met194, Asn195, Gly302, Gln303, Asp304, Ile341, Gly342, Gly343, Gly344	Ala62, Gly97, Ile341	Met192, Met194

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28207223/s1. Figures S1–S76: ¹H and ¹³C NMR spectra of all synthesized compounds; Figure S77: Acquisition and evaluation of the models of the HMGRs of the Candida spp; Figures S78–S83: Ramachandran plots of HMGRs; Tables S1 and S2: Data of the interactions between the simvastatin, atorvastatin, and compounds 1a, 2a, 2c, 4b, 4g, 4l, 5a, 8a, 8c, 8g, 10a, and 12a at the active site of the enzyme HMGR of C. glabrata (CgHMGR) and C. auris (CauHMGR); Figures S84 and 85: Representation of the interactions between simvastatin, atorvastatin, 1a, 2a, 2c, 4b, 4g, 4l, 5a, 8a, 8c, 8g, 10a, and 12a at the active site of CgHMGR and CauHMGR, respectively.

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As a new approach, pyrrolo[1,2-a]pyrazines were synthesized through the cyclization of 2-formylpyrrole-based enaminones in the presence of ammonium acetate. The enaminones were prepared with a straightforward method, reacting the corresponding alkyl 2-(2-formyl-1H-pyrrol-1-yl)acetates, 2-(2-formyl-1H-pyrrol-1-yl)acetonitrile, and 2-(2-formyl-1H-pyrrol-1-yl)acetophenones with DMFDMA. Analogous enaminones elaborated from alkyl (E)-3-(1H-pyrrol-2-yl)acrylates were treated with a Lewis acid to afford indolizines. The antifungal activity of the series of substituted pyrroles, pyrrole-based enaminones, pyrrolo[1,2-a]pyrazines, and indolizines was evaluated on six Candida spp., including two multidrug-resistant ones. Compared to the reference drugs, most test compounds produced a more robust antifungal effect. Docking analysis suggests that the inhibition of yeast growth was probably mediated by the interaction of the compounds with the catalytic site of HMGR of the Candida species.

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Pyrrole-Based Enaminones as Building Blocks for the Synthesis of Indolizines and Pyrrolo[1,2-a]pyrazines Showing Potent Antifungal Activity

Author

Miranda-Sánchez, Diter¹; Escalante, Carlos H¹

; Andrade-Pavón, Dulce²; Gómez-García, Omar¹

; Barrera, Edson¹; Villa-Tanaca, Lourdes³

; Delgado, Francisco¹; Tamariz, Joaquín¹

¹ Departamento de Química Orgánica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Mexico City 11340, Mexico; [email protected] (D.M.-S.); [email protected] (C.H.E.); [email protected] (O.G.-G.); [email protected] (E.B.); [email protected] (F.D.)
² Departamento de Fisiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Av. Wilfrido Massieu S/N, Mexico City 07738, Mexico; [email protected]; Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Mexico City 11340, Mexico; [email protected]
³ Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Mexico City 11340, Mexico; [email protected]

First page

7223

Publication year

2023

Publication date

2023

Publisher

MDPI AG

e-ISSN

14203049

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.3390/molecules28207223

ProQuest document ID

2882603762

Pyrrole-Based Enaminones as Building Blocks for the Synthesis of Indolizines and Pyrrolo[1,2-a]pyrazines Showing Potent Antifungal Activity

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