Structure–Activity Relationship Studies of Tetracyclic Pyrrolocarbazoles Inhibiting Heterotetrameric Protein Kinase CK2

Abstract

The serine/threonine kinase CK2 (formerly known as casein kinase II) plays a crucial role in various CNS disorders and is highly expressed in various types of cancer. Therefore, inhibiting this key kinase could be promising for the treatment of these diseases. The CK2 holoenzyme is formed by the recruitment of two catalytically active CK2α and/or CK2α′ subunits by a regulatory CK2β dimer. Starting with the lead furocarbazole W16 (4) inhibiting the CK2α/CK2β interaction, analogous pyrrolocarbazoles were prepared and tested for their protein–protein interaction inhibition (PPII). The key step of the synthesis was a multicomponent Levy reaction of 2-(indolyl)acetate 6, benzaldehydes 7, and N-substituted maleimides 8. Targeted modifications were performed by the saponification of the tetracyclic ester 9a, followed by the coupling of the resulting acid 10 with diverse amines. The replacement of the O-atom of the lead furocarbazole 4 by an N-atom in pyrrolocarbazoles retained or even increased the inhibition of the CK2α/CK2β interaction. The large benzyloxazolidinyl moiety of 4 could be replaced by smaller N-substituents without the loss of the PPII. The introduction of larger substituents at the 2-position and/or at p-position of the phenyl moiety at the 10-position to increase the surface for the inhibition of the PPI did not enhance the inhibition of the CK2α/CK2β association. The strong inhibition of the CK2α/CK2β association by the histidine derivative (+)-20a (K_i = 6.1 µM) translated into a high inhibition of the kinase activity of the CK2 holoenzyme (CK2α₂β₂, IC₅₀ = 2.5 µM). Thus, 20a represents a novel lead compound inhibiting CK2 via the inhibition of the association of the CK2α and Ck2β subunits.

1. Introduction

Human serine/threonine kinase CK2 (formerly known as casein kinase II) was identified 70 years ago as the first enzyme able to phosphorylate proteins [1]. The constitutively active CK2 can transfer phosphate moieties from ATP or GTP to more than 500 target proteins [2,3,4]. Due to its broad substrate scope, it is involved in several physiological and pathophysiological processes, including the regulation of the cell cycle, cell movement, cell proliferation, apoptosis, and CNS activity [3,4,5,6,7,8].

The heterotetrameric CK2 holoenzyme is formed by the recruitment of two CK2α and/or CK2α′ subunits to a CK2β dimer [9]. The catalytically active CK2α (CK2α′) subunits contain an ATP binding site each and are responsible for phosphate transfer to the target proteins [9]. The regulatory CK2β subunits stabilize the heterotetrameric holoenzyme and modulate its reactivity and substrate selectivity [9,10,11]. However, the CK2α (CK2α′) subunits alone also show kinase activity. Within the cell, the heterotetrameric holoenzyme and its monomeric CK2α (CK2α′) and CK2β subunits are in a dynamic equilibrium, which contributes to the control of the enzymatic activity in the cell [12].

The kinase CK2 is involved in various central nervous system (CNS) disorders. Recently, it was shown that Okur–Chung neurodevelopmental syndrome (OCNDS) [13] and Poirier–Bienvenu neurodevelopmental syndrome (POBINDS) [14] are related to mutations in the genes for the CK2α and CK2β subunits. Furthermore, increased levels of CK2 have been detected in neurons with pathological neurofibrillary tangles (Alzheimer’s disease) [15,16], and CK2 was identified to contribute to the formation of Levy bodies during the development of Parkinson’s disease [17,18]. In addition to CNS disorders, increased levels of CK2 inducing cell proliferation and inhibiting apoptosis were observed in several types of cancer [19,20,21,22]. Although widely expressed, CK2 represents a promising target for the development of novel therapies to combat cancer, since non-cancer cells appear to be less sensitive to CK2 inhibition with respect to cell proliferation and apoptosis [23,24].

Several CK2 inhibitors differing in efficiency and selectivity have been developed as potential new antitumor drugs [25,26,27]. The tricyclic carboxylic acid silmitasertib (1, CX-4945, Figure 1) targeting the ATP binding site of the CK2α (CK2α′) subunit [28,29] has been clinically evaluated for the treatment of cholangiocarcinoma (bile duct cancer) [30]. Dibenzofurans 2 were shown to strongly inhibit CK2 in an ATP competitive manner. Whereas the dichloro derivative 2a revealed an IC₅₀ value of 29 nM [31], the regioisomeric dibromo derivative 2b was 5-fold more potent (IC₅₀ = 5.8 nM), highly selective for CK2, and showed antiproliferative activity against a human prostate tumor cell line [32]. However, ligands interacting with the ATP binding site usually suffer from lower selectivity, since all other protein kinases contain a similarly structured ATP binding site as well [33,34].

In another approach, the association of CK2α and CK2β subunits is suppressed by protein–protein interaction inhibitors (PPIIs). Thus, peptide-based PPIIs such as the cyclic tridecapeptide Pc (13 amino acids) and its derivatives prevent the formation of the hetero-tetrameric CK2 holoenzyme by the inhibition of the CK2α/CK2β association [35,36,37]. In contrast to peptides, the 4-phenylindole CAM187 (3, Figure 1) is hydrolytically stable and blocks the CK2α/CK2β interaction (IC₅₀ = 44 µM) by binding at the CK2α subunit, but not ATP competitively [38]. Although 3 could inhibit the CK2α/CK2β association, the kinase activity of CK2 was not significantly affected [38].

In addition to peptides and CAM187 (3), W16 (4, Figure 1) with a tetracyclic furocarbazole scaffold was reported to inhibit the CK2α/CK2β interaction, with an IC₅₀ value of 30–40 µM, and the kinase activity of the CK2α subunit, with an IC₅₀ value of 20 µM [39]. We recently confirmed these inhibitory activities of W16 by the determination of a K_i value of 31 µM and an IC₅₀ value of 1.9 µM, respectively (see below). In this context, we also reported on the replacement of the furan ring of 4 by a pyrrole ring, leading to an increased inhibition of the CK2α/CK2β association. As an example, the (3aS,4S,10S,10aS)-configured pyrrolocarbazole 5 exhibited a K_i value of 4.9 µM [40]. Since the lead compounds 4 and 5 inhibit protein–protein interactions, we planned to extend their surface to increase their interactions with the rather flat protein surface. In particular, substituents at the imide N-atom in the 2-position, at the carboxamide in the 4-position, and at the phenyl moiety in the 10-position should be modified. The designed extended pyrrolocarbazoles do not fulfill the rule of five defined by Lipinski et al. [41], as their molecular weight exceeds the upper limit of 500 Da. However, in contrast to drugs addressing the structured binding pockets of enzymes or receptors, protein–protein interaction inhibitors have to interact with a large and rather flat surface of one protein. Therefore, we planned to extend the pyrrolocarbazole system in three directions to enlarge the contacts with the protein surface.

2. Results and Discussion

2.1. Synthesis of Tetracyclic Pyrrolocarbazoles

The tetracyclic framework was prepared by a three-component Levy reaction [42,43]. In a domino reaction, 2-(indolyl)acetate 6 reacted first with trimethoxybenzaldehyde (7a) to form a diene, which underwent a Diels–Alder reaction with maleimides 8, diastereoselectively providing the cis,cis,trans-configured pyrrolocarbazoles 9a and 9b in 79% and 83% yields, respectively. The Diels–Alder reaction with maleimides led to the kinetically favored cis,cis,cis-configured diastereomers, which epimerized at C-4 in the presence of CuSO₄ in refluxing o-xylene to give the thermodynamically favored cis,cis,trans-configured diastereomers 9a and 9b [40,43] (Scheme 1).

In order to obtain diverse ligands, the ester 9a was hydrolyzed with NaOH and the resulting acid 10 [40] was coupled with various primary and secondary amines in the presence of the coupling agent COMU^® [44]. Thus, a diverse set of secondary and tertiary amides 11–17 was obtained. The low yields of 15 and 17 were due to purification issues (Scheme 1).

COMU^® coupling of the acid 10 with enantiomerically pure primary amines derived from amino acids led to diastereomeric secondary amides 18a,b–20a,b. After the separation of diastereomers, the enantiomerically pure secondary amides 18a,b, 19b, and 20a,b were isolated (Scheme 2).

In order to increase potential protein–protein interactions, the tetracyclic pyrrolocarbazole framework was expanded at the 2-position (imide N-atom) and at the phenyl moiety in the 10-position. For this purpose, the Levy reaction was performed with 2-(indolyl)acetate 6, 4-nitrobenzaldehyde (7b) and maleimides 8a-c, bearing various substituents at the N-atom, to obtain the cis,cis,trans-configured pyrrolocarbazoles 21a–c in 25–40% yields. After the reduction of the NO₂ moiety with Zn/NH₄Cl, the primary amine 22c was acylated with 4-methoxybenzoyl chloride to afford the amide 23 in an 82% yield. In the benzamide 23, the tetracyclic pyrrolocarbazole framework was extended at the 2- and 10-positions (Scheme 3).

2.2. Pharmacological Evaluation

2.2.1. Inhibition of the CK2α/CK2β Interaction

A microscale thermophoresis (MST) assay was used to determine the interaction of the CK2α and CK2β subunits. In this assay, a constant concentration of the fluorescently labeled CK2β subunit was added to increasing concentrations of the CK2α subunit and the thermophoretic shift was recorded, respectively. These experiments resulted in a dissociation constant (K_D value) of 11 nM for the CK2α/CK2β interaction, which is in agreement with reported values [40,45]. The same experiment was performed in the presence of 20, 50, or 100 µM of the test compounds (see

Table 1. Inhibition of the CK2α/CK2β association by the tetracyclic pyrrolocarbazoles 9–23 determined in the MST assay compared with the activity of the lead compound W16 (4).

Compd.	R	R′	Conc. Test Compd. (µM)	CK2α/CK2β Interaction KD′ [nM] a	Inhibition of CK2α/CK2β Interaction Ki [µM]
(±)-9a [40]	OEt	H	100	59 ± 25	32 d
(±)-9b	OEt	CH3	20	14 ± 6 b	n.s.
(±)-10 [40]	OH	H	100	758 ± 338	1.9 d
(±)-11	NHCH3	H	50	132 ± 43	6.0
(±)-12	NHCH2C6H5	H	50	62 ± 19	14
(±)-13	NHCH2-4-ClC6H4	H	50	17 ± 3 c	n.s.
(±)-14	NH(CH2)2-indol-3-yl	H	50	158 ± 17	3.8
(±)-15		H	50	89 ± 13	7.4
(±)-16		H	50	72 ± 12	9.7
(±)-17		H	50	93 ± 13	7.1
(+)-18a	CH2OH	H	50	72 ± 2	9.1
(–)-19b	CH2CH2SCH3 (S-RSRR-config.)	H	50	94 ± 24	7.7
(+)-20a		H	50	101 ± 4	6.1
(–)-20b	(S-RSRR-config.)	H	20	29 ± 7 b	n.s.
(±)-21a	NO2	H	100	20 ± 10 c	n.s.
(±)-22a	NH2	H	100	31 ± 11 c	n.s.
(±)-22c	NH2	Bn	100	25 ± 15 c	n.s.
(±)-23	NHC(=O)aryl	Bn	100	24 ± 9 c	n.s.
(+)-4 (W16) [40]			100	61 ± 17	31 d

^a Mean ± SEM values of 2–4 separate experiments resulting from a global analysis of all included data sets in Table 1 and in ref. [40] where K_D′ values were significantly different (unpaired Student’s t-test, p ≤ 0.05) from the K_D value of 12 ± 1 nM (n = 4) for the uninhibited CK2α/CK2β interaction. A global value of K_D = 11 ± 7 nM (n = 4) was calculated in this global analysis. ^b,c K_D′ values were not included in the global analysis. An unpaired Student’s t-test showed each K_D′ value to be non-significantly different (p > 0.05) from a K_D value of 20 ± 9 nM (n = 3) ^b or 12 ± 1 nM (n = 4) ^c for the uninhibited CK2α/CK2β interaction. ^d K_i values in ref. [40] were 32 µM ((±)-9a), 1.8 µM ((±)-10), and 31 µM ((+)-4). n.s.: not significant.

). An increased K_D′ value in the presence of the test compounds was evaluated as the inhibition of the association of the CK2α and CK2β subunits. The increased K_D′ value was then transformed together with the employed concentration of the test compound into the K_i value for the inhibition of the CK2α/CK2β association (formula see Supporting Information). The recorded K_D and K_D′ values, as well as the calculated K_i values for the tetracyclic pyrrolocarbazoles 9–23 and the lead compound W16 (4), are displayed in Table 1.

Table 1 shows that the PPII of several prepared pyrrolocarbazoles exceeded the activity of the lead compound 4, and some compounds even revealed K_i values below 10 µM. Particularly high activity with K_i values below 7 µM was observed for the racemic acid (±)-10 (K_i = 1.9 µM), N-methylcarboxamide (±)-11 (K_i = 6.0 µM), the 2-(indolyl)ethyl derivative (±)-14 (K_i = 3.8 µM), and the enantiomerically pure histidine derivative (+)-20a (K_i = 6.1 µM). These results indicate that the large benzyloxazolidinyl substituent of the lead compound 4 is not essential to achieve a high inhibition of the CK2α/CK2β association. Smaller substituents at the carboxamide in the 4-position, such as the small methyl moiety of 11, also lead to a high PPII. However, an ester moiety instead of the carboxamide in the 4-position (e.g., 9, 21–23) is not appropriate to strongly inhibit the CK2α/CK2β association. An extension at the 2-position with an additional benzyl moiety, as in 22c, or an extension of the phenyl moiety at the 10-postion, as in 23, did not result in a significant inhibition of the CK2α/CK2β association.

2.2.2. Inhibition of the Enzymatic Activity

The inhibition of the catalytic activity of the CK2 holoenzyme (CK2α₂β₂) was determined in an enzyme assay. In brief, the CK2 holoenzyme was incubated with the decapeptide RRRDDDSDDD and ATP. The amount of phosphorylated peptide (Ser7) was recorded by capillary electrophoresis (CE) [40,46]. In this assay, the lead compound W16 (4) inhibited the CK2 activity with an IC₅₀ value of 1.9 µM [40].

In a first screening with a test compound concentration of 10 µM, only the histidine derivative (+)-20a exhibited more than 50% enzyme inhibition, indicating an IC₅₀ value below 10 µM. Therefore, concentration-dependent CK2 inhibition was recorded only for (+)-20a, resulting in an IC₅₀ value of 2.5 µM, which is close to the IC₅₀ value of 4. Thus, the strong inhibition of the CK2α/CK2β association (K_i = 6.1 µM) by the histidine derivative (+)-20a was transferred into a strong inhibition of the enzymatic activity of the CK2 holoenzyme (IC₅₀ = 2.5 µM).

3. Conclusions

In order to study the relationships between the structural features and the inhibition of the interactions between the CK2α and CK2β subunits of the protein kinase CK2, a series of pyrrolocarbazoles was prepared. The thermodynamically favored cis,cis,trans-configured pyrrolocarbazoles were obtained by a multicomponent Levy reaction as the key step allowing for modifications at the 2-, 4-, and 10-positions of the tetracyclic framework. The replacement of the O-atom of the furocarbazole lead compound W16 (4) by an N-atom retained or even increased the inhibition of the CK2α/CK2β association. Smaller N-substituents of the carboxamide in the 4-position instead of the large benzyloxazolidinyl substituent of the lead compound 4 were well tolerated and increased the PPII, as shown for the N-methylcarboxamide 11 (K_i = 6.0 µM), the N-indolylethylcarboxamide 14 (K_i = 3.8 µM), and the histidine derivative (+)-20a (K_i = 6.1 µM). Additional substituents at the imide N-atom in the 2-position or in the p-position at the phenyl moiety in the 10-position to increase the interactions with the protein surface did not lead to an increased inhibition of the CK2α/CK2β association. The inhibition of the CK2α/CK2β association of the histidine derivative (+)-20a translated into an inhibition of the kinase activity of the CK2 holoenzyme (IC₅₀ = 2.5 µM), which is in the same range as the CK2 inhibition of the furocarbazole lead compound 4.

4. Experimental Part

4.1. Chemistry, General Methods

Oxygen- and moisture-sensitive reactions were carried out under nitrogen dried with silica gel with a moisture indicator (orange gel, VWR, Darmstadt, Germany) and in dry glassware (Schlenk flask or Schlenk tube). The temperature was controlled with dry ice/acetone (−78 °C), ice/water (0 °C), a Cryostat (Julabo TC100E-F, Seelbach, Germany), a magnetic stirrer MR 3001 K (Heidolph, Schwalbach, Germany), or am RCT CL (IKA, Staufen, Germany), together with the temperature controller EKT HeiCon (Heidolph) or VT-5 (VWR) and PEG or a silicone bath. All solvents were of analytical- or technical-grade quality. o-Xylene and toluene were dried with molecular sieves (3 Å). Demineralized water was used. Thin-layer chromatography (tlc) was used with tlc silica gel 60 F₂₅₄ on aluminum sheets (VWR). Flash chromatography (fc) was used with silica gel 60, 40–63 µm (VWR) with parentheses including the diameter of the column (Ø), length of the stationary phase (l), fraction size (v), and eluent. Automated flash chromatography used Isolera^TM Spektra One (Biotage^®, Global Go-to Separations Company, Stockholm, Sweeden), with parentheses including the cartridge size, flow rate, eluent, and a fractions size always of 20 mL. The melting point system MP50 (Mettler Toledo, Gießen, Germany) was open-capillary and uncorrected. A MicroTOFQII mass spectrometer (Bruker Daltonics, Bremen, Germany) was used, and deviations of the exact masses found from the calculated exact masses were 5 mDa or less; the data were analyzed with Data-Analysis^® (Bruker Daltonics). NMR spectra were recorded in deuterated solvents on Agilent DD2 400 MHz and 600 MHz spectrometers (Agilent, Santa Clara, CA, USA); chemical shifts (δ) are reported in parts per million (ppm) against the reference substance tetramethylsilane and were calculated using the solvent residual peak of the undeuterated solvent. Coupling constants are given with a 0.5 Hz resolution. The assignment of ¹H and ¹³C NMR signals was supported by 2-D NMR techniques where necessary. Am FT/IR Affinity^®-1 spectrometer (Shimadzu, Düsseldorf, Germany) using ATR technique was utilized.

4.2. HPLC Method for the Determination of the Purity

Equipment 1, as follows: Pump: L-7100, degasser: L-7614, autosampler: L-7200, UV detector: L-7400, interface: D-7000, data transfer: D-line, and data acquisition: HSM-Software (all from Merck Hitachi, Darmstadt, Germany). Equipment 2, as follows: Pump: LPG-3400SD, degasser: DG-1210, autosampler: ACC-3000T, UV-detector: VWD-3400RS, interface: DIONEX UltiMate 3000, and data acquisition: Chromeleon 7 (equipment and software from Thermo Fisher Scientific, Lauenstadt, Germany). The column was as follows: LiChrospher^® 60 RP-select B (5 µm), LiChroCART^® 250–4 mm cartridge. The flow rate was 1.0 mL/min, the injection volume was 5.0 µL, and detection was performed at λ = 210 nm. The solvents were as follows: A: demineralized water with 0.05% (V/V) trifluoroacetic acid, B: CH₃CN with 0.05% (V/V) trifluoroacetic acid; gradient elution (% A): 0–4 min: 90%; 4–29 min: gradient from 90% to 0%; 29–31 min: 0%; 31–31.5 min: gradient from 0% to 90%; 31.5–40 min: 90%.

4.3. Synthetic Procedures

• Ethyl (3aRS,4SR,10RS,10aRS)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl9a)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate () [40]
• Under N₂, indolylacetate 6 (203 mg, 1.00 mmol), maleimide (8a, 292 mg, 3.01 mmol) and 3,4,5-trimethoxybenzaldehyde (7a, 296 mg, 1.51 mmol) were dissolved in dry o-xylene (10 mL) in a pressure-resistant Schlenk tube. Crushed CuSO₄ ∙ 5 H₂O (25.2 mg, 0.10 mmol) was added to the solution and the mixture was heated to reflux for 16 h (oil bath temperature of 185 °C). After cooling to room temperature, the mixture was filtered and the filter was washed with CH₂Cl₂ (3 × 10 mL). The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (ethyl acetate/cyclohexane = 45:55, Ø 4 cm, h = 16 cm, v = 30 mL). Yellow solid, mp 220 °C, yield of 379 mg (79%). C₂₆H₂₆N₂O₇ (478.5). TLC (ethyl acetate/CH₂Cl₂ = 3:7): R_f = 0.38.
• Purity (HPLC): 97.4%, (t_R = 19.3 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 1.31 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 3.53–3.57 (m, 1H, 10a-H), 3.57 (s, 3H, 4-OCH₃), 3.61 (s, 6H, 3-OCH₃, 5-OCH₃), 4.05 (dd, J = 9.0/3.4 Hz, 1H, 3a-H), 4.20–4.33 (m, 2H, OCH₂CH₃), 4.49 (d, J = 3.5 Hz, 1H, 4-H), 4.74 (d, J = 8.3 Hz, 1H, 10-H), 6.21 (s, 2H, 2-H_TMP, 6-H_TMP), 6.88 (ddd, J = 8.0/7.0/1.0 Hz, 1H, 8-H), 7.05 (ddd, J = 8.1/6.9/1.2 Hz, 1H, 7-H), 7.20 (dd, J = 8.0/1.1 Hz, 1H, 9-H), 7.37 (dt, J = 8.2/0.9 Hz, 1H, 6-H), 10.92 (s, 2H, 2-H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 14.0 (1C, OCH₂CH₃), 36.9 (1C, C-4), 38.0 (1C, C-10), 42.5 (1C, C-3a), 45.5 (1C, C-10a), 55.6 (2C, 3-OCH₃ 5-OCH₃), 59.9 (1C, 4-OCH₃), 61.7 (1C, OCH₂CH₃), 106.0 (2C, C-2_TMP, 6-C_TMP), 111.3 (1C, C-9b), 111.5 (1C, C-6), 118.4 (1C, C-9), 118.7 (1C, C-8), 121.6 (1C, C-7), 125.4 (1C, C-9a), 128.5 (1C, C-4a), 136.2 (1C, C-4_TMP), 136.4 (1C, C-1_TMP), 136.6 (1C, C-5a), 152.1 (2C, C-3_TMP, C-5_TMP), 171.1 (1C, C-4-C=O), 177.7 (1C, C-1), 179.5 (1C, C-3). Exact mass (APCI): m/z = 479.1785 (calcd. 479.1813 for C₂₆H₂₇N₂O₇ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2987 (w, C-H_aliph.), 1775 (w, C=O), 1709 (s, C=O), 1119 (s, C-O), 752 (m, C-H_arom.).

• Ethyl (3aRS,4SR,10RS,10aRS)-2-methyl-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate (9b)
• Under N₂, indolylacetate 6 (3.01 g, 14.8 mmol), N-methylmaleimide (8b, 5.00 g, 45.0 mmol), and 3,4,5-trimethoxybenzaldehyde (7a, 4.42 g, 22.5 mmol) were dissolved in dry o-xylene (100 mL). Crushed CuSO₄ ∙ 5 H₂O (351 mg, 1.41 mmol) was added to the solution and the mixture was heated to reflux for 16 h. After cooling to -20 °C, the mixture was filtered and the obtained solid was washed with water (3 × 25 mL) and n-pentane (5 × 25 mL) to give the product without further purification. Colorless solid, mp 215 °C, yield of 6.32 g (83%). C₂₇H₂₈N₂O₇ (492.5). TLC (ethyl acetate/cyclohexane = 1:1): R_f = 0.41. Purity (HPLC): 97.3%, (t_R = 20.5 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 1.30 (t, J = 7.1 Hz, 3H; OCH₂CH₃), 2.35 (s, 3H, NCH₃), 3.55 (s, 3H, 4-OCH₃), 3.61 (s, 6H, 3-OCH₃, 5-OCH₃), 3.63 (t, J = 8.2 Hz, 1H, 10a-H), 4.06 (dd, J = 8.7/3.3 Hz, 1H, 3a-H), 4.21–4.33 (m, 2H, OCH₂CH₃), 4.57 (d, J = 3.0 Hz, 1H, 4-H), 4.76 (d, J = 8.0 Hz, 1H, 10-H), 6.13 (s, 2H, 2-H_TMP, 6-H_TMP), 6.88 (ddd, J = 7.9/7.0/1.0 Hz, 1H, 8-H), 7.05 (ddd, J = 8.2/7.0/1.2 Hz, 1H, 7-H), 7.19 (d, J = 7.9 Hz, 1H, 9-H), 7.38 (dt, J = 8.0/0.9 Hz, 1H, 6-H), 10.96 (s, 1H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 14.0 (1C, OCH₂CH₃), 23.7 (1C, NCH₃), 36.6 (1C, C-4), 38.0 (1C, C-10), 41.4 (1C, C-3a), 44.7 (1C, C-10a), 55.7 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 61.8 (1C, OCH₂CH₃), 106.0 (2C, C-2_TMP, C-6_TMP), 110.8 (1C, C-9b), 111.5 (1C, C-6), 118.4 (1C, C-9), 118.7 (1C, C-7), 121.7 (1C, C-8), 125.4 (1C, C-9a), 128.4 (1C, C-4a), 136.1 (1C, C-1_TMP), 136.3 (1C, C-4_TMP), 136.7 (1C, C-5a), 152.1 (2C, C-3_TMP, C-5_TMP), 171.0 (1C, C-4-C=O), 176.4 (1C, C-1), 178.0 (1C, C-3). Exact mass (ESI): m/z = 493.1958 (calcd. 493.1969 for C₂₇H₂₉N₂O₇ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2970 (w, C-H_aliph.), 1743,1701 (s, C=O), 1180, 1115 (s, C-O).

• (3aRS,4SR,10RS,10aRS)-1,3-Dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylic acid (10) [40]
• A solution of NaOH (2.60 g, 6.50 mmol) in water (50 mL) was added to a solution of ester 9a (5.06g, 10.56 mmol) in THF (50 mL). The mixture was stirred for 30 min at room temperature. Ethyl acetate (100 mL) was added and the phases were separated. The aqueous layer was washed with ethyl acetate (3 × 50 mL) to remove non-acidic impurities. HCl solution (2 M in water, 30 mL) was added to the aqueous layer and the resulting suspension was extracted with ethyl acetate (3 × 50 mL). The combined organic layers of the last extraction step were dried (Na₂SO₄), filtered, and concentrated in vacuo to obtain the product without further purification. Yellow solid, mp 162 °C, yield of 4.58 g (96%). C₂₄H₂₂N₂O₇ (450.4). TLC (MeOH/CH₂Cl₂ = 15:85 + 0.1% acetic acid): R_f = 0.33. Purity (HPLC): 94.7%, (t_R = 16.5 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 3.54 (t, J = 8.4 Hz, 1H, 10a-H), 3.56 (s, 3H, 4-OCH₃), 3.62 (s, 6H, 3-OCH₃, 5-OCH₃), 4.09 (dd, J = 9.0/3.6 Hz, 1H, 3a-H), 4.41 (d, J = 3.6 Hz, 1H, 4-H), 4.72 (d, J = 8.2 Hz, 1H, 10-H), 6.22 (s, 2H, 2-H_TMP, 6-H_TMP), 6.87 (ddd, J = 7.8/6.8/0.9 Hz, 1H, 8-H), 7.02 (ddd, J = 8.2/7.0/1.2 Hz, 1H, 7-H), 7.21 (d, J = 7.9 Hz, 1H, 9-H), 7.39 (d, J = 8.1 Hz, 1H, 6-H), 10.89 (s, 1H, 2-H), 10.92 (s, 1H, 5-H), 13.40 (s, 1H, COOH). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 37.0 (1C, C-4), 38.1 (1C, C-10), 42.3 (1C, C-3a), 45.5 (1C, C-10a), 55.6 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 106.0 (2C, C-2_TMP, C-6_TMP), 111.1 (1C, C-9b), 111.6 (1C, C-6), 118.2 (1C, C-9), 118.6 (1C, C-8), 121.4 (1C, C-7), 125.4 (1C, C-9a), 128.9 (1C, C-4a), 136.1 (1C, C-4_TMP), 136.5 (1C, C-1_TMP), 136.6 (1C, C-5a), 152.1 (2C, C-3_TMP, C-5_TMP), 172.4 (1C, COOH), 177.8 (1C, C-1), 179.9 (1C, C-3). Exact mass (ESI): m/z = 451.1477 (calcd. 451.1500 for C₂₄H₂₃N₂O₇ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2939, 2835 (m, C-H_aliph.), 1709 (s, C=O), 1119 (m, C-O), 745 (m, C-H_arom.).

• (3aRS,4SR,10RS,10aRS)-N-Methyl-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxamide (11)
• Carboxylic acid 10 (212 mg, 0.47 mmol), COMU^® (242 mg, 0.56 mmol), and DIPEA (140 mg, 1.08 mmol) were dissolved in dry THF (15 mL) at 0 °C. Then, a solution of methylamine (17.6 mg, 0.56 mmol) in THF (2 mL) was added dropwise. The mixture was stirred for 2 h at 0 °C. After the completion of the transformation, the solution was allowed to warm to ambient temperature and ethyl acetate (30 mL) was added. The mixture was extracted with HCl solution (1 M in water, 2 × 30 mL) and saturated NaCl solution. The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by automatic flash column chromatography (cartridge: SNAP 50 g, flow rate of 50 mL/min, ethyl acetate) to yield a yellow solid. For further purification, the obtained solid was dissolved in ethyl acetate (50 mL) and extracted with NaHCO₃ solution (0.1 M in water, 3 × 50 mL). The organic layer was dried (Na₂SO₄), filtered, and the solvent was removed in vacuo. Yellow solid, mp 215 °C, yield of 106 mg (49%). C₂₅H₂₅N₃O₆ (463.5). TLC (ethyl acetate): R_f = 0.31. Purity (HPLC): 95.6%, (t_R = 16.0 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 2.81 (d, J = 4.6 Hz, 3H, NHCH₃), 3.52 (dd, J = 9.6/7.9 Hz, 1H, 10a-H), 3.55 (s, 3H, 4-OCH₃), 3.65 (s, 6H, 3-OCH₃, 5-OCH₃), 4.00 (dd, J = 9.5/5.0 Hz, 1H, 3a-H), 4.36 (d, J = 5.0 Hz, 1H, 4-H), 4.74 (d, J = 7.9 Hz, 1H, 10-H), 6.27 (s, 2H, 2-H_TMP, 6-H_TMP), 6.90 (ddd, J = 8.0/7.0/1.0 Hz, 1H, 8-H), 7.03 (ddd, J = 8.1/7.0/1.1 Hz, 1H, 7-H) 7.34–7.36 (m, 1H, 9-H), 7.36–7.37 (m, 1H, 6-H), 8.61 (q, J = 4.6 Hz, 1H, NH_amide), 10.67 (s, 1H, 2-H), 10.88 (s, 1H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 26.3 (1C, NHCH₃), 38.1 (1C, C-10), 38.6 (1C, C-4), 42.6 (1C, C-3a), 46.2 (1C, C-10a), 55.7 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.7 (2C, C-2_TMP, C-6_TMP), 111.4 (1C, C-9b), 111.6 (1C, C-6), 117.8 (1C, C-9), 118.6 1C, C-8), 121.2 (1C, C-7), 125.5 (1C, C-9a), 131.3 (1C, C-4a), 136.1 (1C, C-4_TMP), 136.3 (1C, C-1_TMP), 137.0 (1C, C-5a), 152.2 (1C, C-3_TMP, C-5_TMP), 170.5 (1C, CO_amide), 178.1 (1C, C-1), 180.4 (1C, C-3). Exact mass (ESI): m/z = 464.1818 (calcd. 451.1816 for C₂₅H₂₅N₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2889 (m, C-H_aliph.), 1697, 1678 (s, C=O), 1119 (m, C-O), 741 (m, C-H_arom.).

• (3aRS,4SR,10RS,10aRS)-N-Benzyl-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxamide (12)
• Carboxylic acid 10 (135 mg, 0.30 mmol), COMU^® (168 mg, 0.39 mmol), and DIPEA (90.6 mg, 0.70 mmol) were dissolved in dry THF (5 mL) at room temperature. Then, a solution of benzylamine (42.1 mg, 0.39 mmol) in THF (2 mL) was added dropwise. The mixture was stirred for 2 h at room temperature. After the completion of the transformation, ethyl acetate (50 mL) was added. The mixture was extracted successively with NaHCO₃ solution (0.1 M in water, 3 × 25 mL), HCl solution (1 M in water, 3 × 25 mL), and saturated NaCl solution (3 × 25 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (ethyl acetate/cyclohexane = 6:4, Ø 2 cm, h = 18 cm, v = 10 mL). Yellow solid, mp 157 °C, yield of 136 mg (84%). C₃₁H₂₉N₃O₆ (539.6). TLC (ethyl acetate/cyclohexane = 6:4): R_f = 0.41. Purity (HPLC): 96.9%, (t_R = 19.3 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 3.56 (s, 3H, 4-OCH₃), 3.57 (dd, J = 9.5/8.0 Hz, 1H, 10a-H), 3.64 (s, 6H, 3-OCH₃, 5-OCH₃), 4.03 (dd, J = 9.0/4.9 Hz, 1H, 3a-H), 4.26 (dd, J = 15.2/4.2 Hz, 1H, PhCH₂NH), 4.48 (d, J = 5.0 Hz, 1H, 4-H), 4.75 (dd, J = 15.3/7.3 Hz, 1H, PhCH₂NH), 4.76 (d, J = 8.1 Hz, 1H, 10-H), 6.29 (s, 2H, 2-H_TMP, 6-H_TMP), 6.91 (ddd, J = 7.9/7.0/1.0 Hz, 1H, 8-H), 7.04 (ddd, J = 8.2/7.0/1.2 Hz, 1H, 7-H), 7.28 (tt, J = 7.3/1.7 Hz, 1H, 4-H_benzyl), 7.33–7.39 (m, 4H, 6-H, 9-H, 3-H_benzyl, 5-H_benzyl), 7.40–7.42 (m, 2H, 2-H_benzyl, 6-H_benzyl), 9.09 (dd, J = 7.3/4.3 Hz, 1H, NH_amide), 10.73 (s, 1H, 2-H), 10.91 (s, 1H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 38.1 (1C, C-10), 38.7 (1C, C-4), 42.9 (1C, C-3a), 43.1 (1C, PhCH₂NH), 46.1 (1C, C-10a), 55.7 (2C, 3-OCH_3, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.8 (2C, C-2_TMP, C-6_TMP), 111.3 (1C, C-9b) 111.7 (1C, C-6), 117.9 (1C, C-9), 118.7 (1C, C-8), 121.3 (1C, C-7), 125.5 (1C, C-9a), 126.9 (1C, C-4_benzyl), 127.5 (2C, C-2_benzyl, C-6_benzyl), 128.3 (2C, C-3_benzyl, C-5_benzyl), 131.2 (1C, C-4a), 136.1 (1C, C-4_TMP), 136.3 (1C, C-1_TMP), 137.0 (1C, C-5a), 139.0 (1C, C-1_benzyl), 152.2 (2C, C-3_TMP, C-5_TMP), 170.4 (1C, CO_amide), 178.1 (1C, C-1), 180.3 (1C, C-3). Exact mass (ESI): m/z = 540.2125 (calcd. 540.2129 for C₃₁H₃₀N₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2889 (s, C-H_aliph.), 1712 (s, C=O), 1153,1122 (s, C-O), 741 (m, C-H_arom.).

• (3aRS,4SR,10RS,10aRS)-N-(4-Chlorobenzyl)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxamide (13)
• Carboxylic acid 10 (135 mg, 0.30 mmol), COMU^® (155 mg, 0.36 mmol), and DIPEA (86.1 mg, 0.67 mmol) were dissolved in dry THF (10 mL) at 0 °C. Then, a solution of 4-chlorobenzylamine (51.2 mg, 0.36 mmol) in THF (2 mL) was added dropwise. The mixture was stirred for 30 min at 0 °C and 1 h at room temperature. After the completion of the transformation, ethyl acetate (30 mL) was added. The mixture was extracted with HCl solution (1 M in water, 5 × 30 mL) and saturated NaCl solution (2 × 25 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was adsorbed onto silica and purified by automatic flash column chromatography (cartridge: SNAP 50g, flow rate of 50 mL/min, ethyl acetate/cyclohexane = 30:70 → 0:100). Yellow solid, mp 204 °C, yield of 123 mg (71%). C₃₁H₂₈ClN₃O₆ (574.0). TLC (ethyl acetate/cyclohexane = 6:4): R_f = 0.38. Purity (HPLC): 96.9%, (t_R = 20.3 min). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = 3.56 (s, 3H, 4-OCH₃), 3.56 (dd, J = 9.6/8.0 Hz, 1H, 10a-H), 3.64 (s, 6H, 3-OCH₃, 5-OCH₃), 4.00 (dd, J = 9.5/4.9 Hz, 1H, 3a-H), 4.25 (dd, J = 15.4/4.4 Hz, 1H, PhCH₂NH), 4.46 (d, J = 4.9 Hz, 1H, 4-H), 4.71 (dd, J = 15.4/7.1 Hz, 1H, PhCH₂NH), 4.76 (d, J = 8.0 Hz, 1H, 10-H), 6.28 (s, 2H, 2-H_TMP, 6-H_TMP), 6.91 (ddd, J = 8.0/7.0/1.0 Hz, 1H, 8-H), 7.04 (ddd, J = 8.2/7.0/1.2 Hz, 1H, 7-H), 7.34 (d, J = 7.8 Hz, 1H, 9-H), 7.37 (d, J = 8.0 Hz, 1H, 6-H), 7.40–7.45 (m, 4H, 2-H_benzyl, 3-H_benzyl, 5-H_benzyl, 6-H_benzyl), 9.11 (dd, J = 7.1/4.5 Hz, 1H, NH_amide), 10.75 (s, 1H, 2-H), 10.92 (s, 1H, 5-H). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 38.0 (1C, C-10), 38.7 (1C, C-4), 42.4 (1C, PhCH₂NH), 42.9 (1C, C-3a), 46.1 (1C, C-10a), 55.7 (2C, 3-OCH_3, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.8 (2C, C-2_TMP, C-6_TMP), 111.3 (1C, C-9b) 111.7 (1C, C-6), 118.0 (1C, C-9), 118.7 (1C, C-8), 121.3 (1C, C-7), 125.5 (1C, C-9a), 128.2 (2C, C-2_benzyl, C-6_benzyl), 129.3 (2C, C-3_benzyl, C-5_benzyl), 131.1 (1C, C-4a), 131.4 (1C, C-4_benzyl), 136.1 (1C, C-4_TMP), 136.3 (1C, C-1_TMP), 137.0 (1C, C-5a), 138.1 (1C, C-1_benzyl), 152.2 (2C, C-3_TMP, C-5_TMP), 170.5 (1C, CO_amide), 178.1 (1C, C-1), 180.3 (1C, C-3). Exact mass (ESI): m/z = 574.1730 (calcd. 574.1739 for C₃₁H₂₉ClN₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2970, 2940 (s, C-H_aliph.), 1717, 1682 (s, C=O), 1173, 1126 (s, C-O), 745 (m, C-H_arom.).

• (3aRS,4SR,10RS,10aRS)-N-[2-(Indol-3-yl)ethyl]-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxamide (14)
• Carboxylic acid 10 (136 mg, 0.30 mmol), COMU^® (154 mg, 0.36 mmol), and DIPEA (117 mg, 0.90 mmol) were dissolved in ethyl acetate (10 mL) at room temperature. Then, 2-(indol-3-yl)ethanamine (58.4 mg, 0.36 mmol) was added to the solution. The mixture was stirred for 4 h at room temperature. After the completion of the transformation, ethyl acetate (30 mL) was added. The mixture was extracted with HCl solution (1 M in water, 3 × 20 mL) and saturated NaCl solution (2 × 25 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by automatic flash column chromatography (cartridge: SNAP 50 g, flow rate of 50 mL/min, ethyl acetate/cyclohexane = 35:65 → 0:100) to obtain a yellow solid. For further purification, the solid was recrystallized (ethyl acetate/cyclohexane), filtered, and washed with cold ethanol (2 × 10 mL). Colorless solid, mp 182 °C, yield of 93 mg (52%). C₃₄H₃₂N₄O₆ (592.7). TLC (ethyl acetate/cyclohexane = 7:3): R_f = 0.33. Purity (HPLC): 99.4%, (t_R = 19.5 min). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = 3.01 (t, J = 7.6 Hz, 2H, NHCH₂CH₂), 3.40–3.47 (m, 1H, NHCH₂CH₂), 3.53 (dd, J = 9.5/8.0 Hz, 1H, 10a-H), 3.56 (s, 3H, 4-OCH₃), 3.65 (s, 6H, 3-OCH₃, 5-OCH₃), 3.62–3.68 (m, 1H, NHCH₂CH₂), 3.99 (dd, J = 9.4/4.6 Hz, 1H, 3a-H), 4.42 (d, J = 4.6 Hz, 1H, 4-H), 4.74 (d, J = 8.0 Hz, 1H, 10-H), 6.27 (s, 2H, 2-H_TMP, 6-H_TMP) 6.89 (ddd, J = 7.9/7.0/1.0 Hz, 1H, 8-H), 7.00 (ddd, J = 7.9/7.0/1.1 Hz, 1H, 5-H_indolyl), 7.02 (ddd, J = 8.2/7.0/1.2 Hz, 1H, 7-H), 7.09 (ddd, J = 8.1/6.9/1.2 Hz, 1H, 6-H_indolyl), 7.24 (d, J = 2.2 Hz, 1H, 2-H_indolyl), 7.31 (dt, J = 8.2/0.8 Hz, 1H, 6-H), 7.32 (d, J = 8.0 Hz, 1H, 9-H), 7.36 (dt, J = 8.1/0.9 Hz, 1H, 7-H_indolyl), 7.62 (d, J = 7.9 Hz, 1H, 4-H_indolyl), 8.73 (t, J = 5.6 Hz, 1H, NH_amide), 10.42 (s, 1H, 5-H), 10.87 (d, J = 2.4 Hz, 1H, 1-H_indolyl), 10.89 (s, 1H, 2-H). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 25.0 (1C, NHCH₂CH₂), 38.1 (1C, C-10), 38.5 (1C, C-4), 40.3 (1C, NHCH₂CH₂), 42.7 (1C, C-3a), 46.1 (1C, C-10a), 55.7 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.9 (2C, C-2_TMP, C-6_TMP), 111.4 (1C, C-6), 111.4 (1C, C-7_indolyl), 111.6 (1C, C-9b), 111.7 (1C, C-3_indolyl), 117.9 (1C, C-9), 118.3 (1C, C-5_indolyl), 118.3 (1C, C-4_indolyl), 118.7 (1C, C-8), 121.0 (1C, C-6_indolyl), 121.3 (1C, C-7), 122.7 (1C, C-2_indolyl), 125.6 (1C, C-9a), 127.2 (1C, C-3a_indolyl), 131.2 (1C, C-4a), 136.1 (1C, C-4_TMP), 136.2 (1C, C-5a), 136.3 (1C, C-7a_indolyl), 136.9 (1C, C-1_TMP), 152.2 (2C, C-3_TMP, C-5_TMP), 170.1 (1C, CO_amide), 178.1 (1C, C-1), 180.3 (1C, C-3). Exact mass (ESI): m/z = 593.2370 (calcd. 593.2395 for C₃₄H₃₃N₄O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2940 (s, C-H_aliph.), 1701 (s, C=O), 1177, 1123 (s, C-O), 733 (s, C-H_arom.).

• (3aRS,4SR,10RS,10aRS)-1,3-Dioxo-N-(pyridin-3-yl)-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxamide (15)
• Carboxylic acid 10 (135 mg, 0.30 mmol), COMU^® (155 mg, 0.36 mmol), and DIPEA (85.5 mg, 0.66 mmol) were dissolved in dry THF (10 mL) at room temperature. Then, pyridin-3-amine (37.7 mg, 0.40 mmol) was added to the solution. The mixture was stirred for 12 h at room temperature. After the completion of the transformation, ethyl acetate (50 mL) was added. The mixture was extracted with HCl solution (1 M in water, 5 × 20 mL) and saturated NaCl solution (2 × 20 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by automatic flash column chromatography (cartridge: SNAP 50g, flow rate of 50 mL/min, MeOH/CH₂Cl₂ = 0:100 → 10:90). Colorless solid, mp 198 °C, yield of 21 mg (12%). C₂₉H₂₆N₄O₆ (526.5). TLC (MeOH/CH₂Cl₂ = 5:95): R_f = 0.14. Purity (HPLC): 97.6%, (t_R = 15.7 min). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = 3.57 (s, 3H, 4-OCH₃), 3.60 (dd, J = 9.6/8.0 Hz, 1H, 10a-H), 3.68 (s, 6H, 3-OCH₃, 5-OCH₃), 4.14 (dd, J = 9.5/4.9 Hz, 1H, 3a-H), 4.67 (d, J = 5.0 Hz, 1H, 4-H), 4.81 (d, J = 7.9 Hz, 1H, 10-H), 6.31 (s, 2H, 2-H_TMP, 6-H_TMP), 6.92 (ddd, J = 7.9/7.1/1.0 Hz, 1H, 8-H), 7.04 (ddd, J = 8.2/7.0/1.2 Hz, 1H, 7-H), 7.34 (d, J = 8.1 Hz, 1H, 6-H), 7.40 (d, J = 7.9 Hz, 1H, 9-H), 7.45 (dd, J = 8.3/4.7 Hz, 1H, 5-H_pyridinyl), 8.22 (ddd, J = 8.3/2.6/1.5 Hz, 1H, 6-H_pyridinyl), 8.36 (dd, J = 4.7/1.5 Hz, 1H, 4-H_pyridinyl), 8.93 (d, J = 2.5 Hz, 1H, 2-H_pyridinyl), 10.95 (s, 1H, 5-H), 10.97 (s, 1H, 2-H), 11.02 (s, 1H, NH_amide). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 38.0 (1C, C-10), 42.8 (1C, C-3a), 46.0 (1C, C-10a), 55.8 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.9 (2C, C-2_TMP, C-6_TMP), 111.3 (1C, C-9b), 112.0 (1C, C-6), 118.0 (1C, C-9), 118.8 (1C, C-8), 121.5 (1C, C-7), 123.7 (1C, C-5_pyridinyl), 125.4 (1C, C-9a), 126.7 (1C, C-6_pyridinyl), 130.4 (1C, C-4a), 135.9 (1C, C-3_pyridinyl), 136.2 (1C, C-4_TMP), 136.4 (1C, C-5a), 137.0 (1C, C-1_TMP), 141.2 (1C, C-2_pyridinyl), 144.5 (1C, C-4_pyridinyl), 152.3 (2C, C-3_TMP, C-5_TMP), 169.7 (1C, CO_amide), 178.0 (1C, C-1), 180.3 (1C, C-3). The signal for C-4 C-atom is overlaid by the DMSO signals at 39.5 ppm. Exact mass (ESI): m/z = 527.1919 (calcd. 527.1925 for C₂₉H₂₇N₄O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2932 (s, C-H_aliph.), 1709 (s, C=O), 1173, 1123 (s, C-O), 745 (s, C-H_arom.).

• (3aRS,4SR,10RS,10aRS)-4-[(Piperidin-1-yl)carbonyl]-10-(3,4,5-trimethoxyphenyl)-4,5,10,10a-tetrahydropyrrolo[3,4-b]carbazole-1,3(2H,3aH)-dione (16)
• Carboxylic acid 10 (136 mg, 0.30 mmol), COMU^® (168 mg, 0.39 mmol), and DIPEA (89.6 mg, 0.69 mmol) were dissolved in dry THF (10 mL) at 0 °C. Then, a solution of piperidine (32.2 mg, 0.38 mmol) in THF (3 mL) was added dropwise. The mixture was stirred for 2 h at 0 °C. After the completion of the transformation, the solution was allowed to warm to ambient temperature and ethyl acetate (30 mL) was added. The mixture was extracted with NaHCO₃ solution (0.1 M in water, 3 × 25 mL), HCl solution (1 M in water, 3 × 25 mL), and saturated NaCl solution (1 × 25 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (ethyl acetate/cyclohexane = 6:4 → 7:3, Ø 2 cm, h = 25 cm, v = 10 mL). Colorless solid, mp 293 °C, yield of 103 mg (66%). C₂₉H₃₁N₃O₆ (517.6). TLC (ethyl acetate/cyclohexane = 6:4): R_f = 0.29. Purity (HPLC): 96.7%, (t_R = 18.4 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 1.43–1.74 (m, 6H, N(CH₂CH₂)₂CH₂), 3.22 (ddd, J = 12.8/9.7/3.0 Hz, 1H, N(CH₂CH₂)₂CH₂), 3.51 (dd, J = 9.3/8.0 Hz, 1H, 10a-H), 3.57 (s, 3H, 4-OCH₃), 3.59–3.62 (m, 1H, N(CH₂CH₂)₂CH₂), 3.63 (s, 6H, 3-OCH₃, 5-OCH₃), 3.92 (dt, J = 13.7/4.2 Hz, 1H, N(CH₂CH₂)₂CH₂), 3.97 (dd, J = 9.1/4.3 Hz, 1H, 3a-H), 3.95–4.02 (m, 1H, N(CH₂CH₂)₂CH₂), 4.76 (d, J = 8.3 Hz, 1H, 10-H), 4.77 (d, J = 4.0 Hz, 1H, 4-H), 6.28 (s, 2H, 2-H_TMP, 6-H_TMP), 6.89 (ddd, J = 7.9/7.0/1.0 Hz, 1H, 8-H), 7.03 (ddd, J = 8.1/7.0/1.2 Hz, 1H, 7-H), 7.27 (dd, J = 8.0/1.0 Hz, 1H, 9-H), 7.33 (dd, J = 8.1/0.9 Hz, 1H, 6-H), 10.78 (s, 1H, 5-H), 10.91 (s, 1H, 2-H). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 24.2 (1C, N(CH₂CH₂)₂CH₂) 25.3 (1C, N(CH₂CH₂)₂CH₂), 26.0 (1C, N(CH₂CH₂)₂CH₂), 34.3 (1C, C-4), 38.3 (1C, C-10), 43.3 (1C, N(CH₂CH₂)₂CH₂), 44.0 (1C, C-3a), 46.3 (1C, C-10a), 47.0 (1C, N(CH₂CH₂)₂CH₂), 55.5 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.9 (2C, C-2_TMP, C-6_TMP), 111.3 (1C, C-6), 111.5 (1C, C-9b), 118.0 (1C, C-9), 118.6 (1C, C-8), 121.2 (1C, C-7), 125.5 (1C, C-9a), 131.1 (1C, C-4a), 136.1 (1C, C-4_TMP), 136.4 (1C, C-5a), 136.6 (1C, C-1_TMP), 152.2 (2C, C-3_TMP, C-5_TMP), 168.5 (1C, CO_amide), 178.0 (1C, C-1), 180.2 (1C, C-3). Exact mass (ESI): m/z = 518.2280 (calcd. 518.2286 for C₂₉H₃₂N₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2886 (s, C-H_aliph.), 1709 (s, C=O), 1150, 1119 (s, C-O), 799, 745 (s, C-H_arom.).

• (3aRS,4SR,10RS,10aRS)-N-(1H-Benzimidazol-2-yl)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxamide (17)
• Carboxylic acid 10 (136 mg, 0.30 mmol), COMU^® (167 mg, 0.39 mmol), and DIPEA (128 mg, 0.99 mmol) were dissolved in dry THF (10 mL) at room temperature. Then, benzimidazol-2-amine (52.2 mg, 0.39 mmol) was added. The mixture was stirred for 2 h at room temperature. After the completion of the transformation, ethyl acetate (50 mL) was added. The mixture was extracted with saturated NH₄Cl solution (3 × 25 mL), NaHCO₃ solution (0.1 M in water, 3 × 25 mL), and saturated NaCl solution (1 × 25 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was dissolved in ethyl acetate (10 mL), and n-hexane (30 mL) was added. The resulting mixture was filtered, and the precipitate was washed with cold Et₂O. Colorless solid, mp 233 °C, yield of 39 mg (23%). C₃₁H₂₇N₅O₆ (565.6). TLC (ethyl acetate/cyclohexane = 6:4): R_f = 0.16. Purity (HPLC): 97.3%, (t_R = 17.7 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 3.57 (s, 3H, 4-OCH₃), 3.65 (dd, J = 9.8/7.9 Hz, 1H, 10a-H), 3.70 (s, 6H, 3-OCH₃, 5-OCH₃), 4.19 (dd, J = 9.7/5.8 Hz, 1H, 3a-H), 4.79 (d, J = 5.8 Hz, 1H, 4-H), 4.83 (d, J = 8.0 Hz, 1H, 10-H), 6.34 (s, 2H, 2-H_TMP, 6-H_TMP), 6.93 (ddd, J = 7.9/6.9/1.0 Hz, 1H, 8-H), 7.03 (ddd, J = 8.1/6.9/1.2 Hz, 1H, 7-H), 7.11–7.16 (m, 2H, 5-H_BI, 6-H_BI), 7.30–7.34 (m, 1H, 6-H), 7.44 (d, J = 7.9 Hz, 1H, 9-H), 7.50–7.54 (m, 2H, 4-H_BI, 7-H_BI), 11.03 (s, 1H, 5-H), 11.03 (s, 1H, 2-H), 12.20 (s, 2H, NH_amide, 1-H_BI). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 38.1 (1C, C-10), 42.9 (1C, C-3a), 46.1 (1C, C-10a), 55.7 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.5 (2C, C-2_TMP, C-6_TMP), 111.4 (1C, C-6), 112.1 (1C, C-9b), 117.9 (1C, C-9), 118.8 (1C, C-8), 121.3 (2C, C-5_BI, C-6_BI), 121.4 (1C, C-7), 125.4 (1C, C-9a), 130.4 (1C, C-4a), 136.1 (1C, C-4_TMP), 136.3 (1C, C-5a), 137.1 (1C, C-1_TMP), 152.3 (2C, C-3_TMP, C-5_TMP), 172.0 (1C, CO_amide), 178.1 (1C, C-1), 180.3 (1C, C-3). Signals for C-3a_BI, C-4_BI, C-7_BI, and C-7a_BI C-atoms were not observed in the spectrum. The signal for C-4 C-atom was overlaid by the DMSO signal at 39.5 ppm. Exact mass (ESI): m/z = 566.2037 (calcd. 566.2034 for C₃₁H₂₈N₅O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2909, 2835 (w, C-H_aliph.), 1701 (s, C=O), 1177, 1126 (s, C-O), 798, 745 (s, C-H_arom.).

• Methyl N-{[(3aS,4R,10S,10aS)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazol-4-yl]carbonyl}-(S)-serinate ((+)-18a) and methyl N-{[(3aR,4S,10R,10aR)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazol-4-yl]carbonyl}-(S)-serinate ((-)-18b)
• Carboxylic acid 10 (136 mg, 0.30 mmol), COMU^® (168 mg, 0.39 mmol), and DIPEA (128 mg, 0.99 mmol) were dissolved in dry DMF (10 mL) at 0 °C. After the addition of (S)-serine methyl ester HCl (38.9 mg, 0.25 mmol), the mixture was stirred for 2 h at 0 °C. After the completion of the transformation, the solution was allowed to warm to ambient temperature, and ethyl acetate (30 mL) was added. The mixture was washed with HCl solution (1 M in water, 3 × 25 mL) and saturated NaCl solution (1 × 25 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (ethyl acetate/cyclohexane = 8:2, Ø 2 cm, h = 15 cm, v = 10 mL) to give both diastereomers.
• (+)-18a [R_f = 0.41 (ethyl acetate/cyclohexane = 8:2)]: Yellow solid, mp 174 °C, yield of 37 mg (27%). C₂₈H₂₉N₃O₉ (551.6). Purity (HPLC): 93.7%, (t_R = 16.5 min). Specific rotation: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}=$ +100 (c = 1.7, THF). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 3.51 (dd, J = 9.6/7.8 Hz, 1H, 10a-H), 3.56 (s, 3H, 4-OCH₃), 3.68 (s, 6H, 3-OCH₃, 5-OCH₃), 3.78 (s, 3H, CO₂CH₃), 3.78–3.84 (m, 1 H, CHCH₂OH), 3.97 (dt, J = 11.0/5.1 Hz, 1H, CHCH₂OH), 4.08 (dd, J = 9.6/5.2 Hz, 1H, 3a-H), 4.60–4.69 (m, 1H, CHCH₂OH), 4.67 (d, J = 5.3 Hz, 1H, 4-H), 4.76 (d, J = 7.8 Hz, 1H, 10-H), 5.41 (t, J = 5.2 Hz, 1H, CHCH₂OH), 6.33 (s, 2H, 2-H_TMP, 6-H_TMP), 6.95 (ddd, J = 7.9/7.0/1.0 Hz, 1H, 8-H), 7.08 (ddd, J = 8.1/7.0/1.2 Hz, 1H, 7-H), 7.34 (dt, J = 8.0/0.9 Hz, 1H, 6-H), 7.42 (dd, J = 7.9/1.0 Hz, 1H, 9-H), 9.35 (d, J = 7.9 Hz, 1H), NH_amide), 10.38 (s, 1H, 5-H), 10.93 (s, 1H, 2-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 38.1 (1C, C-10), 38.2 (1C, C-4), 41.7 (1C, C-3a), 46.3 (1C, C-10a), 52.7 (1C, CO₂CH₃), 55.3 (1C, CHCH₂OH), 55.8 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 61.0 (1C, CHCH₂OH₎, 105.8 (2C, C-2_TMP, C-5_TMP), 111.0 (1C, C-6), 112.0 (1C, C-9b), 118.1 (1C, C-9), 119.0 (1C, C-8), 121.6 (1C, C-7), 125.6 (1C, C-9a), 131.3 (1C, C-4a), 135.7 (1C, C-5a), 136.2 (1C, C-4_TMP), 136.9 (1C, C-1_TMP), 152.3 (2C, C-3_TMP, C-5_TMP), 170.3 (1C, CO₂CH₃), 172.7 (C-4-C=O), 178.1 (1C, C-1), 180.5 (1C, C-3). Exact mass (ESI): m/z = 552.1972 (calcd. 552.1977 for C₂₈H₃₀N₃O₉ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 3314 (m, OH), 2931, 2847 (w, C-H_aliph.), 1708, 1674 (s, C=O), 1123 (s, C-O), 745 (s, C-H_arom.).
• (−)-18b [R_f = 0.35 (ethyl acetate/cyclohexane = 8:2)]: Yellow solid, mp 183 °C, yield of 37 mg (27%). C₂₈H₂₉N₃O₉ (551.6). Purity (HPLC): 93.2%, (t_R = 16.0 min). Specific rotation: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}=$ −115 (c = 1.3, THF). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 3.50 (dd, J = 9.5/7.8 Hz, 1H, 10a-H), 3.56 (s, 3H, 4-OCH₃), 3.66 (s, 6H, 3-OCH₃, 5-OCH₃), 3.71 (s, 3H, CO₂CH₃), 3.82–3.89 (m, 1H, CHCH₂OH), 3.94 (dt, J = 10.8/4.7 Hz, 1H, CHCH₂OH), 4.03 (dd, J = 9.7/4.4 Hz, 1H, 3a-H), 4.62 (d, J = 4.6 Hz, 1H, 4-H), 4.62–4.65 (m, 1H, CHCH₂OH), 4.75 (d, J = 7.8 Hz, 1H, 10-H), 5.83 (t, J = 5.3 Hz, 1H, CHCH₂OH), 6.29 (s, 2H, C-2_TMP, C-6_TMP), 6.92 (ddd, J = 8.0/7.0/1.0 Hz, 1H, 8-H), 7.05 (ddd, J = 8.2/6.9/1.2 Hz, 1H, 7-H), 7.30 (dt, J = 8.1/0.9 Hz, 1H, 6-H), 7.36 (d, J = 7.9 Hz, 1H, 9-H), 9.15 (d, J = 8.1 Hz, 1H, NH_amide), 10.58 (s, 1H, 5-H), 10.90 (s, 1H, 2-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 38.0 (1C, C-10), 38.2 (1C, C-4), 41.9 (1C, C-3a), 46.3 (1C, C-10a), 52.1 (1C, CO₂CH₃), 55.1 (1C, CHCH₂OH), 55.7 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 60.8 (1C, CHCH₂OH₎, 105.8 (2C, C-2_TMP, C-6_TMP), 111.0 (1C, C-6), 111.7 (1C, C-9b), 118.0 (1C, C-9), 118.8 (1C, C-8), 121.4 (1C, C-7), 125.6 (1C, C-9a), 130.9 (1C, C-4a), 136.0 (1C, C-5a), 136.1 (1C, C-4_TMP), 136.8 (1C, C-1_TMP), 152.2 (2C, C-3_TMP, C-5_TMP), 170.5 (1C, C-4-C=O), 170.6 (CO₂CH₃), 178.1 (1C, C-1), 180.3 (1C, C-3). Exact mass (ESI): m/z = 552.1972 (calcd. 552.1977 for C₂₈H₃₀N₃O₉ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 3309 (m, OH), 2927, 2850 (w, C-H_aliph.), 1708, 1674 (s, C=O), 1123 (s, C-O), 745 (s, C-H_arom.).

• Methyl N-{[(3aR,4S,10R,10aR)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazol-4-yl]carbonyl}-(S)-methioninate ((-)-19b)
• Under N₂, carboxylic acid 10 (136 mg, 0.30 mmol) and CDI (53.5 mg, 0.33 mmol) were dissolved in dry CH₂Cl₂ (10 mL) at room temperature. The mixture was stirred for 2 h. Then, (S)-methionine methyl ester HCl (120 mg, 0.60 mmol) was added. The mixture was stirred for 48 h at room temperature. After the completion of the transformation, the resulting suspension was filtered through Celite^®, followed by rinsing with CH₂Cl₂ (40 mL). The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (ethyl acetate/cyclohexane = 6:4, Ø 2 cm, h = 20 cm, v = 10 mL). Yellow solid, mp 127 °C, yield of 39 mg (22%). C₃₀H₃₃N₃O₈S (595.7). TLC (ethyl acetate/cyclohexane = 6:4): R_f = 0.24. Purity (HPLC): 98.1%, (t_R = 19.6 min). Specific rotation: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}=$ −113 (c = 1.2, DMSO). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 2.02–2.07 (m, 1H, CH₃SCH₂CH₂CH), 2.09 (s, 3H, SCH₃), 2.11–2.17 (m, 1H, CH₃SCH₂CH₂CH), 2.58–2.69 (m, 2H, CH₃SCH₂CH₂CH), 3.55 (dd, J = 9.7/8.0 Hz, 1H, 10a-H), 3.57 (s, 3H, 4-OCH₃), 3.67 (s, 6H, 3-OCH₃, 5-OCH₃), 3.76 (s, 3H, CO₂CH₃), 4.04 (dd, J = 9.6/5.3 Hz, 1H, 3a-H), 4.52 (d, J = 5.3 Hz, 1H, 4-H), 4.69 (ddd, J = 9.0/7.6/4.7 Hz, 1H, CH₃SCH₂CH₂CH), 4.78 (d, J = 8.0 Hz, 1H, 10-H), 6.32 (s, 2H, 2-H_TMP,6-H_TMP), 6.95 (ddd, J = 8.0/7.0/1.0 Hz, 1H, 8-H), 7.07 (ddd, J = 8.2/7.0/1.2 Hz, 1H, 7-H), 7.36 (dt, J = 8.1/0.9 Hz, 1H, 6-H), 7.42 (d, J = 8.0 Hz, 1H, 9-H), 9.18 (d, J = 7.7 Hz, 1H, NH_amide), 10.34 (s, 1H, 5-H), 10.97 (s, 1H, 2-H). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 14.6 (1C, SCH₃), 29.5 (1C, CH₃SCH₂CH₂CH), 30.7 (1C, CH₃SCH₂CH₂CH), 38.0 (1C, C-10), 38.4 (1C, C-4), 42.1 (1C, C-3a), 46.0 (1C, C-10a), 51.6 (1C, CH₃SCH₂CH₂CH), 52.7 (1C, CO₂CH₃), 55.8 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.8 (2C, C-2_TMP, C-6_TMP), 111.1 (1C, C-6), 112.1 (1C, C-9b), 118.1 (1C, C-9), 119.0 (1C, C-8), 121.6 (1C, C-7), 125.6 (1C, C-9a), 130.8 (1C, C-4a), 135.9 (1C, C-5a), 136.2 (1C, C-4_TMP), 137.0 (1C, C-1_TMP), 152.3 (2C, C-3_TMP, C-5_TMP), 170.3 (1C, CO_amide), 173.5 (1C, CO₂CH₃), 178.1 (1C, C-1), 180.3 (1C, C-3). Exact mass (ESI): m/z = 596.2062 (calcd. 566.2061 for C₃₀H₃₄N₃O₈S [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2889 (w, C-H_aliph.), 1774, (w, C=O), 1713 (s, C=O), 1643 (w, C=O), 1173, 1123 (s, C-O), 745 (s, C-H_arom.).

• Methyl N-{[(3aS,4R,10S,10aS)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazol-4-yl]carbonyl}-(S)-histidinate∙HCl ((+(-20a HCl) and methyl N-{[(3aR,4S,10R,10aR)-1,3-dioxo-10-(3,4,5-trimethoxyphenyl)-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazol-4-yl]carbonyl}-(S)-histidinate ((-)-20b)
• Carboxylic acid 10 (450 mg, 1.00 mmol), COMU^® (471 mg, 1.10 mmol), and (S)-histidine methyl ester HCl (267 mg, 1.10 mmol) were dissolved in dry THF (20 mL) at room temperature. Then, DIPEA (426 mg, 3.30 mmol) was added dropwise. The mixture was stirred for 16 h at room temperature. After the completion of the transformation, ethyl acetate (100 mL) was added. The mixture was extracted with NaHCO₃ solution (0.1 M in water, 4 × 50 mL) and saturated NaCl solution (1 × 50 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by automatic flash column chromatography (cartridge: SNAP 100 g, flow rate of 50 mL/min, MeOH/CH₂Cl₂ = 2:98 → 10:90) to obtain the diastereomers 20a and 20b.
• Then, 20a was dissolved in CH₂Cl₂ (10 mL) and HCl solution (2 M in Et₂O, 5 mL) was added. The resulting suspension was filtered and the precipitate was washed with cold CH₂Cl₂ (2 × 10 mL) to give 20a∙HCl.
• (+)-20a∙HCl [R_f = 0.43 (MeOH/CH₂Cl₂ = 10:90, free base)]: yellow solid, mp 253 °C, yield of 125 mg (20%). C₃₁H₃₂ClN₅O₈ (638.1). Purity (HPLC): 95.0%, (t_R = 15.3 min). Specific rotation: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}=$ +121 (c = 1.7, DMSO). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 3.27 (dd, J = 15.2/6.4 Hz, 1H, NHCHCH₂), 3.34 (dd, J = 15.1/8.3 Hz, 1H, NHCHCH₂), 3.53 (t, J = 8.9 Hz, 1H, 10a-H), 3.57 (s, 3H, 4-OCH₃), 3.63 (s, 6H, 3-OCH₃, 5-OCH₃), 3.65 (s, 3H, CO₂CH₃), 3.85 (dd, J = 9.0/3.7 Hz, 1H, 3a-H), 4.52 (d, J = 3.8 Hz, 1H, 4-H), 4.67–4.75 (m, 1H, NHCHCH₂), 4.72 (d, J = 8.2 Hz, 1H, 10-H), 6.25 (s, 2H, 2-H_TMP, 6-H_TMP), 6.88 (ddd, J = 8.0/7.1/1.0 Hz, 1H, 8-H), 7.04 (ddd, J = 8.1/7.0/1.2 Hz, 1H, 7-H), 7.20 (d, J = 7.9 Hz, 1H, 9-H), 7.39 (dt, J = 8.1/0.9 Hz, 1H, 6-H), 7.51 (s, 1H, 5-H_imidaz.), 8.99 (s, 1H, 2-H_imidaz.), 9.22 (d, J = 7.4 Hz, 1H, NH_amide), 10.52 (s, 1H, 5-H), 10.91 (s, 1H, 2-H). A signal for the NH_imidaz. proton is not observed in the spectrum. ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 26.0 (1C, NHCHCH₂), 37.7 (1C, C-4), 38.1 (1C, C-10), 42.8 (1C, C-3a), 45.9 (1C, C-10-a), 52.2 (1C, NHCHCH₂), 52.3 (1C, CO₂CH₃), 55.7 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 106.1 (2C, C-2_TMP, C-6_TMP), 111.4 (1C, C-6), 111.4 (1C, C-9b), 117.2 (1C, C-5_imidaz.) 118.2 (1C, C-9), 118.7 (1C, C-8), 121.5 (1C, C-7), 125.6 (1C, C-9a), 130.4 (1C, C-4a), 133.9 (1C, C-2_imidaz.), 136.2 (1C, C-4_TMP), 136.3 (1C, C-5a), 136.6 (1C, C-1_TMP), 152.1 (2C, C-3_TMP, C-5_TMP), 170.8 (1C, CO_amide), 171.0 (1C, CO₂CH₃), 178.0 (1C, C-1), 179.8 (1C, C-3). A signal for the C-4_imidaz. C-atom was not observed in the spectrum. Exact mass (ESI): m/z = 602.2251 (calcd. 602.2245 for C₃₁H₃₂N₅O₈ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2886 (s, C-H_aliph.), 1713 (s, C=O), 1686 (m, C=O), 1154, 1119 (s, C-O), 745 (s, C-H_arom.).
• (−)-20b [R_f = 0.32 (MeOH/CH₂Cl₂ = 10:90)]: Yellow solid, mp 233 °C, yield of 172 mg (29%). C₃₁H₃₁N₅O₈ (601.6). Purity (HPLC): 94.3%, (t_R = 15.9 min). Specific rotation: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}=$ −169 (c 1.5, DMSO). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 3.10 (dd, J = 14.8/9.3 Hz, 1H, NHCHCH₂), 3.16 (dd, J = 14.9/4.2 Hz, 1H, NHCHCH₂), 3.50 (dd, J = 9.5/7.8 Hz, 1H, 10a-H), 3.56 (s, 3H, 4-OCH₃), 3.66 (s, 9H, 3-OCH₃, 5-OCH₃, CO₂CH₃), 4.03 (dd, J = 9.6/4.9 Hz, 1H, 3a-H), 4.29 (s broad, 1H, NHCHCH₂), 4.53 (d, J = 5.0 Hz, 1H, 4-H), 4.75 (d, J = 7.8 Hz, 1H, 10-H), 6.31 (s, 2H, 2-H_TMP, 6-H_TMP), 6.93 (td, J = 7.5/7.1/0.9 Hz, 1H, 8-H), 7.03 (s, 1H, 5-H_imidaz.), 7.06 (ddd, J = 8.2/7.0/1.1 Hz, 1H, 7-H), 7.37–7.43 (m, 2H, 6-H, 9-H), 7.80 (s, 1H, 2-H_imidaz.), 9.36 (s, 1H, NH_amide), 10.90 (s, 1H, 2-H), 11.95 (s, 1H, 5-H), 12.07 (s, 1H, τ-NH_imidaz.). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 27.8 (1C, NHCHCH₂), 38.1 (1C, C-10), 38.3 (1C, C-4), 42.0 (1C, C-3a), 46.2 (1C, C-10-a), 52.1 (1C, CO₂CH₃), 52.3 (1C, NHCHCH₂), 55.8 (2C, 3-OCH₃, 5-OCH₃), 59.9 (1C, 4-OCH₃), 105.8 (2C, C-2_TMP, C-6_TMP), 111.2 (1C, C-6), 111.8 (1C, C-9b), 113.7 (1C, C-5_imidaz.), 117.9 (1C, C-9), 118.7 (1C, C-8), 121.5 (1C, C-7), 125.5 (1C, C-9a), 130.4 (1C, C-4a), 135.1 (1C, C-2_imidaz.), 136.1 (1C, C-4_TMP), 136.1 (1C, C-5a), 137.0 (1C, C-1_TMP), 152.3 (2C, C-3_TMP, C-5_TMP), 170.1 (1C, CO_amide), 172.1 (1C, CO₂CH₃), 178.1 (1C, C-1), 180.2 (1C, C-3). A signal for the C-4_imidaz. C-atom was not observed in the spectrum. Exact mass (ESI): m/z = 602.2254 (calcd. 602.2245 for C₃₁H₃₂N₅O₈ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2886 (s, C-H_aliph.), 1705, 1674 (s, C=O), 1153, 1125 (s, C-O), 745 (s, C-H_arom.).

• Ethyl (3aRS,4SR,10RS,10aRS)-10-(4-nitrophenyl)-1,3-dioxo-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate (21a)
• Under N₂, indolylacetate 6 (205 mg, 1.01 mmol), maleimide (8a, 292 mg, 3.00 mmol), and 4-nitrobenzaldehyde (7b, 228 mg, 1.51 mmol) were dissolved in dry o-xylene (15 mL) in a pressure-resistant Schlenk tube. Crushed CuSO₄ ∙ 5 H₂O (26.4 mg, 0.11 mmol) was added to the solution and the mixture was heated to reflux for 40 h (oil bath temperature 180 °C). After cooling to room temperature, the mixture was diluted with CH₂Cl₂ until a clear solution formed. After filtration, the filter was washed with CH₂Cl₂ (3 × 10 mL). The filtrate was concentrated in vacuo. The residue was again dissolved in CH₂Cl₂ (2 mL) and adsorbed onto silica gel, followed by purification by automatic flash column chromatography (cartridge: SNAP 100g, flow rate of 50 mL/min, ethyl acetate/cyclohexane = 10:90 → 0:100). For further purification, the resulting solid was left stirring in Et₂O (5 mL) for 30 min. The suspension was filtrated, washed with cold Et₂O (3 × 10 mL), and dried under vacuum. Yellow solid, mp 262 °C, yield of 107 mg (25%). C₂₃H₁₉N₃O₆ (433.4). TLC (ethyl acetate/cyclohexane = 1:1): R_f = 0.26. Purity (HPLC): 94.9%, (t_R = 20.1 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 1.31 (t, J = 7.1 Hz, 3H, COCH₂CH₃), 3.72 (t, J = 9.0 Hz, 1H, 10a-H), 4.09 (dd, J = 9.0/3.8 Hz, 1H, 3a-H), 4.22–4.33 (m, 2H, COCH₂CH₃), 4.56 (d, J = 3.7 Hz, 1H, 4-H), 4.99 (d, J = 8.9 Hz, 1H, 10-H), 6.83 (ddd, J = 7.9/6.8/1.0 Hz, 1H, 8-H), 6.98 (d, J = 7.8 Hz, 9H), 7.04 (ddd, J = 8.1/6.9/1.1 Hz, 1H, 7H), 7.25–7.30 (m, 2H, 2-H_NO2Ph, 6-H_NO2Ph), 7.35–7.39 (dt, J = 8.1/0.8 Hz, 1H, 6-H), 8.03–8.07 (m, 2H, 3-H_NO2Ph, 5-H_NO2Ph), 10.97 (s, 1H, 2-H), 11.10 (s, 1H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 14.0 (1C, COCH₂CH₃), 37.1 (1C, C-4), 37,4 (1C, C-10), 42.6 (1C, C-3a), 45.0 (1C, C-10a), 61.7 (1C, COCH₂CH), 109.9 (1C, C-9b), 111.6 (1C, C-6), 118.1 (1C, C-9), 118.9 (1C, C-8), 121.7 (1C, C-7), 123.0 (2C, C-3_NO2Ph, C-3_NO2Ph), 125.2 (1C, C-9a), 129.2 (1C, C-4a), 130.2 (2C, C-2_NO2Ph, C-6_NO2Ph), 136.6 (1C, C-5a), 146.3 (1C, C-4_NO2Ph), 149.1 (1C, C-1_NO2Ph), 170.9 (1C, COCH₂CH₃), 177.5 (1C, C-1), 178.2 (1C, C-3). Exact mass (ESI): m/z = 434.1342 (calcd. 434.1347 for C₂₃H₂₀N₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2893 (w, C-H_aliph.), 1720 (s, C=O), 1508, 1342 (s, N-O) 1141 (s, C-O), 745 (m, C-H_arom.).

• Ethyl (3aRS,4SR,10RS,10aRS)-2-methyl-10-(4-nitrophenyl)-1,3-dioxo-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate (21b)
• Under N₂, indolylacetate 6 (101 mg, 0.50 mmol), N-methylmaleimide (8b, 281 mg, 2.53 mmol), and 4-nitrobenzaldehyde (7b, 132 mg, 0.87 mmol) were dissolved in dry o-xylene (15 mL) in a pressure-resistant Schlenk tube. Crushed CuSO₄ ∙ 5 H₂O (12.4 mg, 0.05 mmol) was added to the solution and the mixture was heated to reflux for 40 h (oil bath temperature of 180 °C). After cooling to room temperature, the mixture was filtered, and the filter was washed with CH₂Cl₂ (3 × 10 mL). The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (ethyl acetate/cyclohexane = 4:7, Ø 4 cm, h = 12 cm, v = 20 mL). Yellow solid, mp 237 °C, yield of 82 mg (40%). C₂₄H₂₁N₃O₆ (447.4). TLC (ethyl acetate/cyclohexane = 4:6): R_f = 0.21. Purity (HPLC): 95.3%, (t_R = 21.3 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 1.30 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 2.36 (s, 3H, NHCH₃), 3.80 (t, J = 8.8 Hz, 1H, 10a-H), 4.10 (dd, J = 8.6/3.4 Hz, 1H, 3a-H), 4.16–4.36 (m, 2H, OCH₂CH₃), 4.62 (d, J = 3.4 Hz, 1H, 4-H), 5.01 (d, J = 8.9 Hz, 1H, 10-H), 6.82 (ddd, J = 8.0/7.0/1.0 Hz, 1H, 8-H), 6.93 (d, J = 8.2 Hz, 1H, 9-H), 7.03 (ddd, J = 8.2/7.0/1.3 Hz, 1H, 7-H), 7.17–7.25 (m, 2H, 2-H_NO2Ph 6-H_NO2Ph), 7.37 (dt, J = 8.1/0.9 Hz, 1H, 6-H), 7.98–8.10 (m, 3-H_NO2Ph, 5-H_NO2Ph), 11.14 (s, 1H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 14.0 (1C, OCH₂CH₃), 23.8 (1C, NHCH₃), 37.0 (1C, C-4), 37.6 (1C, C-10), 41.6 (1C, C-3a), 44.0 (1C C-10a), 61.7 (1C, OCH₂CH₃), 109.6 (1C, C-9b), 111.5 (1C, C-6), 118.2 (1C, C-9), 118.9 (1C, C-8), 121.7 (1C, C-7), 122.9 (2C, C-3_NO2Ph, C-5_NO2Ph) 125.1 (1C, C-9a), 129.1 (1C, C-4a), 130.0 (2C, C-2_NO2Ph, C-6_NO2Ph), 136.6 (1C, C-5a), 146.3 (1C, C-4_NO2Ph), 148.8 (1C, C-1_NO2Ph), 170.8 (1C, CO_ester) 176.2 (1C, C-1), 177.4 (1C, C-3). Exact mass (ESI): m/z = 446.1506 (calcd. 448.1503 for C₂₄H₂₂N₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978 (w, C-H_aliph.), 1782, (w, C=O), 1717 (s, C=O), 1196,1150 (s, C-O), 745 (m, C-H_arom.).

• Ethyl (3aRS,4SR,10RS,10aRS)-2-benzyl-10-(4-nitrophenyl)-1,3-dioxo-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate (21c)
• Under N₂, indolylacetate 6 (104 mg, 0.52 mmol), N-benzylmaleimide (8c, 282 mg, 1.50 mmol), and 4-nitrobenzaldehyde (7b, 113 mg, 0.75 mmol) were dissolved in dry o-xylene (15 mL) in a pressure-resistant Schlenk tube. Crushed CuSO₄ ∙ 5 H₂O (12.9 mg, 0.05 mmol) was added to the solution and the mixture was heated to reflux for 24 h (oil bath temperature of 180 °C). The mixture was allowed to cool down to ambient temperature and CH₂Cl₂ (20 mL) was added until a clear solution formed. After filtration, the filter was washed with CH₂Cl₂ (3 × 10 mL). The filtrate was concentrated in vacuo. The residue was purified by flash column chromatography (ethyl acetate/CH₂Cl₂ = 2:98, Ø 3 cm, h = 22 cm, v = 20 mL). Yellow solid, mp 199 °C, yield of 109 mg (40%). C₃₀H₂₅N₃O₆ (523.5). TLC (ethyl acetate/CH₂Cl₂ = 2:98): R_f = 0.33. Purity (HPLC): 98.7%, (t_R = 23.2 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) 1.33 (t, J = 7.1 Hz, 3H, OCH₂CH₃), 3.86 (t, J = 9.0 Hz, 1H, 10a-H), 4.15 (d, J = 14.8 Hz, 1H, NCH₂Ph), 4.20 (d, J = 14.8 Hz, 1H, NCH₂Ph), 4.24 (dd, J = 9.3/4.6 Hz, 1H, 3a-H), 4.26–4.40 (m, 2H, OCH₂CH₃), 4.62 (d, J = 4.5 Hz, 1H, 4-H), 5.04 (d, J = 8.7 Hz, 1H, 10-H), 6.83 (ddd, J = 8.1/7.1/1.4 Hz, 1H, 8-H), 6.88–6.95 (m, 2H, C-2_benzyl, C-6_benzyl), 7.02–7.08 (m, 5H, 7-H, 9-H, 3-H_benzyl, 4-H_benzyl, 5-H_benzyl), 7.10–7.13 (m, 2H, 2-H_NO2Ph, 6-H_NO2Ph), 7.35–7.40 (m, 1H, 6-H), 7.75–7.79 (m, 2H, 3-H_NO2Ph, 5-H_NO2Ph), 11.07 (s, 1H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 14.0 (1C, OCH₂CH₃), 37.3 (1C, C-10), 37.6 (1C, C-4), 41.5 (1C, NCH₂Ph), 41.6 (1C, C-3a), 43.8 (1C, C-10a), 61.9 (1C, OCH₂CH₃), 110.3 (1C, C-9b), 111.6 (1C, C-6), 118.0 (1C, C-9), 118.9 (1C, C-8), 121.7 (1C, C-7), 123.0 (2C, C-3_NO2Ph, C-5_NO2Ph), 125.1 (1C, C-9a), 127.1 (1C, C-4_benzyl), 127.9 (2C, C-3_benzyl, C-5_benzyl), 128.0 (2C, C-2_benzyl, C-6_benzyl), 129.1 (1C, C-4a), 129.8 (2C, C-2_NO2Ph, C-6_NO2Ph), 135.3 (1C C-1_benzyl), 136.5 (1C, C-5a), 146.1 (1C, C-4_NO2Ph), 148.2 (1C, C-1_NO2Ph), 170.9 (1C, C-4-C=O), 175.9 (1C, C-1), 177.7 (1C, C-3). Exact mass (ESI): m/z = 524.1794 (calcd. 524.1816 for C₃₀H₂₆N₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2928 (w, C-H_aliph.), 1732, 1701 (s, C=O), 1342 (s, N-O) 1141 (s, C-O_ester), 745, 689 (m, C-H_arom.).

• Ethyl (3aRS,4SR,10RS,10aRS)-10-(4-aminophenyl)-1,3-dioxo-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate∙HCl (22a.HCl)
• Aromatic nitro compound 21a (119 mg, 0.28 mmol) was dissolved in THF (20 mL), and saturated NH₄Cl solution (20 mL) was added. Under vigorous stirring, a large excess of Zn dust (1.01 g, 15.4 mmol) was poured into the mixture, which was then stirred for 2 h at room temperature. After the completion of the transformation, saturated NaHCO₃ solution (30 mL) was added, and the suspension was stirred for an additional 30 min at room temperature. The reaction mixture was filtered through Celite^®, followed by rinsing with ethyl acetate (150 mL). The filtrate was poured into a separating funnel and the layers were separated. The aqueous layer was then extracted with ethyl acetate (2 × 50 mL). The combined organic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (ethyl acetate/cyclohexane = 8:2, Ø 2 cm, h = 12 cm, v = 10 mL). For conversion into the HCl salt, the resulting solid (74 mg) was dissolved in CH₂Cl₂ (5 mL). HCl solution (1 M in Et₂O, 3 mL) was added, and the suspension was concentrated in vacuo. Orange solid, mp 244 °C, yield of 75 mg (62%). C₂₃H₂₂ClN₃O₄ (439.9). TLC (ethyl acetate/cyclohexane = 8:2): R_f = 0.21 (free base). Purity (HPLC): 95.9%, (t_R = 15.1 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 1.31 (t, J = 7.1 Hz, 3H, COCH₂CH₃), 3.62 (t, J = 8.9 Hz, 1H, 10a-H), 4.06 (dd, J = 9.1/3.9 Hz, 1H, 3a-H), 4.21–4.35 (m, 2H, COCH₂CH₃), 4.51 (d, J = 3.9 Hz, 1H, 4-H), 4.82 (d, J = 8.7 Hz, 1H, 10-H), 6.82 (ddd, J = 7.9/6.9/1.0 Hz, 1H, 8-H), 6.99 (d, J = 7.9 Hz, 1H, 9-H), 7.01–7.07 (m, 3H, 7-H, 3-H_NH2Ph, 5-H_NH2Ph), 7.10 (d, J = 8.1 Hz, 2H, 2-H_NH2Ph, 6-H_NH2Ph), 7.32–7.40 (m, 1H, 6-H), 9.79 (s, 3H, NH₃⁺), 10.95 (s, 1H, 2-H), 11.01 (s, 1H, 5-H). ¹³C NMR (151 MHz, DMSO-d₆): δ (ppm) = 14.0 (1C, COCH₂CH₃), 37.1 (1C, C-4), 37.2 (1C, C-10), 42.6 (1C, C-3a), 45.2 (1C, C-10a), 61.7 (1C, COCH₂CH), 110.9 (1C, C-9b), 111.5 (1C, C-6), 118.1 (1C, C-9), 118.7 (1C, C-8), 121.6 (1C, C-7), 121.8 (2C, C-2_NH2Ph, C-6_NH2Ph), 125.2 (1C, C-9a), 129.0 (1C, C-4a), 130.0 (2C, C-3_NH2Ph, C-5_NH2Ph), 136.5 (1C, C-5a), 171.0 (1C, COCH₂CH₃), 177.6 (1C, C-1), 179.1 (1C, C-3). Signals for C-1_NH2Ph and C-4_NH2Ph C-atoms were not observed in the spectrum. Exact mass (ESI): m/z = 404.1606 (calcd. 404.1605 for C₂₃H₂₂N₃O₄ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978 (w, N-H_{amine salt}), 2983, 2862 (w, C-H_aliph.), 1782 (w, C=O), 1701 (s, C=O), 1180 (s, C-O), 1153 (m, C-O), 741 (m, C-H_arom.).

• Ethyl (3aRS,4SR,10RS,10aRS)-10-(4-aminophenyl)-2-benzyl-1,3-dioxo-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate∙HCl (22c.HCl)
• Aromatic nitro compound 21c (263 mg, 0.50 mmol) was dissolved in THF (30 mL) and saturated NH₄Cl solution (30 mL) was added. Under vigorous stirring, a large excess of Zn dust (5.00 g, 76.5 mmol) was poured into the mixture, which was then stirred for 2 h at room temperature. After the completion of the transformation, saturated NaHCO₃ solution (30 mL) was added, and the suspension was stirred for an additional 30 min at room temperature. The reaction mixture was filtered through Celite^®, followed by rinsing with ethyl acetate (80 mL). The filtrate was poured into a separating funnel and the layers were separated. The aqueous layer was then extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo. For conversion into the HCl salt, the resulting solid was dissolved in CH₂Cl₂ (5 mL). HCl solution (1 M in Et₂O, 3 mL) was added, and the suspension was concentrated in vacuo. Orange solid, mp 206 °C, yield of 126 mg (48%). C₃₀H₂₈ClN₃O₄ (530.0). TLC (ethyl acetate/cyclohexane = 7:3): R_f = 0.38 (free base). Purity (HPLC): 95.9%, (t_R = 18.7 min). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) = 1.30 (t, J = 7.1 Hz, 3H, COCH₂CH₃), 3.81 (t, J = 8.8 Hz, 1H, 10a-H), 3.98 (d, J = 15.1 Hz, 1H, NCH₂Ph), 4.12 (d, J = 15.1 Hz, 1H, NCH₂Ph), 4.20 (dd, J = 8.9/4.0 Hz, 1H, 3a-H), 4.22–4.34 (m, 2H, COCH₂CH₃), 4.57 (d, J = 4.0 Hz, 1H, 4-H), 4.88 (d, J = 8.5 Hz, 1H, 10-H), 6.82 (ddd, J = 8.0/7.0/1.0 Hz, 1H, 8-H), 6.93–7.09 (m, 8H, 7-H, 9-H, 2-H_NH2Ph, 3-H_NH2Ph, 5-H_NH2Ph, 6-H_NH2Ph, 3-H_benzyl, 5-H_benzyl), 7.18–7.23 (m, 3H, 2-H_benzyl, 4-H_benzyl, 6-H_benzyl), 7.38 (dt, J = 8.1/1.1/0.9 Hz, 1H, 6-H), 9.77 (s, 3H, NH₃⁺), 11.08 (s, 1H, 5-H). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) = 14.1 (1C, COCH₂CH₃), 37.51 (1C, C-4), 37.53 (1C, C-10), 41.1 (1C, NCH₂Ph), 41.8 (1C, C-3a), 44.4 (1C, C-10a), 61.8 (1C, COCH₂CH), 110.7 (1C, C-9b), 111.6 (1C, C-6, 118.2 (1C, C-9), 118.8 (1C, C-8), 121.6 (1C, C-7), 122.1 (2C, C-2_NH2Ph, C-6_NH2Ph), 125.2 (1C, C-9a), 127.2 (2C, C-3_benzyl, C-5_benzyl), 127.3 (1C, C-4_benzyl), 128.3 (2C, C-2_benzyl, C-6_benzyl), 129.1 (1C, C-4a), 130.0 (2C, C-3_NH2Ph, C-5_NH2Ph), 135.5 (1C, C-1_benzyl), 136.5 (1C, C-5a), 170.8 (1C, COCH₂CH₃), 176.2 (1C, C-1), 177.6 (1C, C-3). Signals for C-1_NH2Ph and C-4_NH2Ph C-atoms are not observed in the spectrum. Exact mass (APCI): m/z = 494.2064 (calcd. 494.2074 for C₃₀H₂₈N₃O₄ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978 (w, N-H_{amine salt}), 2978, 2808 (w, C-H_aliph.), 1697 (s, C=O), 1184, 1146 (m, C-O), 745 (m, C-H_arom.).

• Ethyl (3aRS,4SR,10RS,10aRS)-2-benzyl-10-[4-(4-methoxybenzamido)phenyl]-1,3-dioxo-1,2,3,3a,4,5,10,10a-octahydropyrrolo[3,4-b]carbazole-4-carboxylate (23)
• Ammonium chloride 22c∙HCl (77.5 mg, 0.14 mmol) was suspended in dry CH₂Cl₂ (20 mL) followed by the addition of DIPEA (80 µL, 0.47 mmol) to obtain the free base. 4-methoxybenzoyl chloride (43.1 mg, 0.28 mmol) was added and the mixture was stirred for 2 h at room temperature. After the completion of the transformation, the solution was washed with HCl solution (0.1 M, 2 × 10 mL) and saturated NaCl solution (1x 10 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (THF/CH₂Cl₂ = 3:97, Ø 3 cm, h = 14 cm, v = 10 mL). Yellow solid, mp 249 °C, yield of 76 mg (82%). C₃₈H₃₃N₃O₆ (627.7). TLC (THF/CH₂Cl₂ = 3:97): R_f = 0.19. Purity (HPLC): 94.5%, (t_R = 22.8 min). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) = 1.32 (t, J = 7.1 Hz, 3H, COCH₂CH₃), 3.78 (t, J = 8.9 Hz, 1H, 10a-H), 3.83 (s, 3H, OCH₃), 4.04 (d, J = 15.2 Hz, 1H, NCH₂Ph), 4.19 (dd, J = 9.2/4.3 Hz, 1H, 3a-H), 4.20 (d, J = 15.1 Hz, 1H, NCH₂Ph), 4.24–4.37 (m, 2H, COCH₂CH₃), 4.57 (d, J = 4.3 Hz, 1H, 4-H), 4.83 (d, J = 8.4 Hz, 1H, 10-H), 6.83–6.88 (m, 3H, 8-H, 2-H_NH2Ph, 6-H_NH2Ph), 6.89–6.93 (m, 2H, 2-H_benzyl, 6-H_benzyl), 7.01–7.09 (m, 4H, 7-H, 9-H, 3-H_MeObz, 5-H_MeObz), 7.09–7.18 (m, 3H, 3-H_benzyl, 4-H_benzyl, 5-H_benzyl), 7.38 (dt, J = 8.3/0.9 Hz, 1H, 6-H), 7.48–7.54 (m, 2H, 3-H_NH2Ph, 5-H_NH2Ph), 7.90–7.96 (m, 2H, 2-H_MeObz, 6-H_MeObz), 9.98 (s, 1H, NH_amide), 11.00 (s, 1H, 5-H). ¹³C NMR: (151 MHz, DMSO-d₆) δ (ppm) = 14.0 (1C, COCH₂CH₃), 37.5 (2 × 1C, C-4_, C-10), 41.2 (1C, NCH₂Ph), 41.8 (1C, C-3a), 44.7 (1C, C-10a), 55.4 (1C, OCH₃), 61.7 (1C, COCH₂CH), 111.3 (1C, C-9b), 111.5 (1C, C-6), 113.6 (2C, C-3_MeObz, C-5_MeObz), 118.2 (1C, C-9), 118.8 (1C, C-8), 119.5 (2C, C-3_NH2Ph, C-5_NH2Ph), 121.5 (1C, C-7), 125.3 (1C, C-9a), 127.0 (1C, C-4_benzyl), 127.0 (1C, C-1_MeObz), 127.2 (2C, C-2_benzyl, C-6_benzyl) 128.1 (2C, C-3_benzyl, C-5_benzyl), 128.8 (2C, C-2_NH2Ph, C-6_NH2Ph), 128.9 (1C, C-4a), 129.5 (2C, C-2 _MeObz, C-6 _MeObz), 134.9 (1C, C-1_NH2Ph), 135.5 (1C, C-1_benzyl), 136.5 (1C, C-5a), 138.1 (1C, C-4_NH2Ph), 161.8 (1C, C-4 _MeObz), 164.7 (1C, CO_amide), 170.9 (1C, COCH₂CH₃), 176.2 (1C, C-1), 177.8 (1C, C-3). Exact mass (APCI): m/z = 628.2400 (calcd. 628.2442 for C₃₈H₃₄N₃O₆ [MH]⁺). IR (neat): ṽ [cm⁻¹] = 2978, 2889 (m, C-H_aliph.), 1775 (w, C=O), 1694 (s, C=O), 1643, 1605 (m, C=C_aryl) 1184 (s, C-O), 1153 (m, C-O).

4.4. Microscale Thermophoresis Assay

The recombinant expression and purification [37,47] of human CK2α^1–335 and human CK2β¹⁻¹⁹³, as well as the microscale thermophoresis (MST) assay to quantify the interaction of the two CK2 subunits, were performed as described previously [40,45]. Briefly, MST experiments followed the interaction between 20 nM of fluorescently labelled CK2β^1–193 and 16 serial dilutions of CK2α^1–335 (0.1526–5000 nM) in 25 mM Tris-HCl, 500 mM NaCl, pH 8.5, 1% (v/v) DMSO, and 0.05% (v/v) Tween 20. The compounds were investigated at a final concentration of 20, 50, or 100 µM. These experiments resulted in dissociation constants of K_D and K_D′ for the uninhibited and inhibited CK2 subunit interactions, respectively, which were then subjected to an unpaired Student’s t test to analyze for the statistical significance of the difference in the values. Data in the absence of inhibitors and in the presence of those compounds leading to a statistically significant increase in the dissociation constant (p ≤ 0.05) were analyzed again by setting K_D to a global value and, in addition, using the equation K_D′ = K_D [1 + (c_compound/K_i)]. The global analysis gave a K_D of 11 nM, which was in agreement with reported values [40,45] and provided the K_i values listed in Table 1. Data analysis and statistical evaluation were conducted with GraphPad Prism v.6 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com).

4.5. Capillary Electrophoresis Assay

The capillary electrophoresis assay to determine the inhibition of the CK2 holoenzyme (CK2α₂β₂) was performed as described in refs. [40,46]. The CK2 holoenzyme was recombinantly expressed in E. coli BL21(DE3) and purified according to the protocol by Grankowski et al. [48]. Enzymatic activity was determined in the presence of 1 µg of CK2α₂β₂, 60 µM of ATP, and 114 µM of the substrate peptide RRRDDDSDDD. The assay buffer contained 60 mM NaCl and 10 mM MgCl₂. Inhibition was determined in three independent experiments at a compound concentration of 10 µM, and the mean value and the standard deviation (SD) were calculated. When the holoenzyme inhibition exceeded 50% in comparison to the enzymatic activity without inhibitors, obtained with the same amount of DMSO, an IC₅₀ value was determined three times independently.