Novel Polycondensed Partly Saturated β-Carbolines Including Ferrocene Derivatives: Synthesis, DFT-Supported Structural Analysis, Mechanism of Some Diastereoselective Transformations and a Preliminary Study of Their in vitro Antiproliferative Effects.

Use of a Pictet-Spengler reaction of tryptamine and l-tryptophan methyl ester and subsequent reduction of the nitro group followed by further cyclocondensation with aryl aldehydes and formyl-substituted carboxylic acids, including ferrocene-based components, furnished a series of diastereomeric 6-aryl-substituted 5,6,8,9,14,14b-hexahydroindolo[2',3':3,4]pyrido[1-c]-quinazolines and 5,5b,17,18-tetrahydroindolo[2',3':3,4]pyrido[1,2-c]isoindolo[2,1-a]quinazolin-11-(15bH)-ones with the elements of central-, planar and conformational chirality. The relative configuration and the conformations of the novel polycyclic indole derivatives were determined by 1H- and 13C-NMR methods supplemented by comparative DFT analysis of the possible diastereomers. The structure of one of the pentacyclic methyl esters with defined absolute configuration "S" was also confirmed by single crystal X-ray diffraction measurement. Accounting for the characteristic substituent-dependent diastereoselective formation of the products multistep mechanisms were proposed on the basis of the results of DFT modeling. Preliminary in vitro cytotoxic assays of the products revealed moderate-to-significant antiproliferative effects against PANC-1-, COLO-205-, A-2058 and EBC-1 cell lines that proved to be highly dependent on the stereostructure and on the substitution pattern of the pending aryl substituent.


Introduction
Aromatic or partly saturated β-carbolines can be found as key structural motifs in a variety of biologically active compounds, including both synthetic and natural products that display remarkable pharmacological activities, e.g., antimalarial [1], cardioprotective [2], trypanocidal and neurotoxic [3] effects. A number of compounds belonging to this group of heterocycles have also been identified as potent anticancer agents with marked cytotoxicity against malignant human cell lines [4,5]. Since the last years have witnessed a growing interest in novel chemotherapeutic agents with a multitarget mechanisms of action, it is of pronounced importance that representative tetrahydro-β-carbolynes proved to be inhibitors of microtubule-crosslinking mitotic kinesin Eg5 [6,7], cell division-inducing cyclic dependent kinase (CDKs) [8,9] as well as topoisomerase I and II [8,10]. Pointing to an additional mode of antiproliferative action of β-carbolines, a few representative 9-substituted harmine alkaloids were identified as DNA-intercalators [10]. β-Carboline hybrids with aryl linkers were also shown to exhibit substantial antiproliferative activity on a wide range of malignant human cells acting mainly by CT-DNA intercalation [11]. On the other hand, synergistically strengthening the antiproliferative effects associated with selective binding-based classical targeted mechanisms of action, electron transfer processes via the Fenton pathway induced by redox active fragments, e.g., by suitably positioned ferrocenyl group(s) in the molecular scaffold, may play a key role in mitochondrial generation of reactive oxygen species (ROS), e.g., nitric oxide, superoxide anion and other forms of free radicals [12][13][14] that can be involved in biological regulatory processes finally leading to programmed cell death (apoptosis) [15]. Supporting the relevance of this view, convincing preclinical evidence about the interplay between particular binding-induced-and redox signalling pathways implicated in cancer has been disclosed [16]. Accordingly, due to their marked effects on malignant cell lines, a plethora of ferrocene derivatives with diverse molecular architectures have been emerged as potent antiproliferative agents in the last decades [17][18][19][20][21].

Introduction
Aromatic or partly saturated β-carbolines can be found as key structural motifs in a variety of biologically active compounds, including both synthetic and natural products that display remarkable pharmacological activities, e.g. antimalarial [1], cardioprotective [2], trypanocidal and neurotoxic [3] effects. A number of compounds belonging to this group of heterocycles have also been identified as potent anticancer agents with marked cytotoxicity against malignant human cell lines [4,5]. Since the last years have witnessed a growing interest in novel chemotherapeutic agents with a multitarget mechanisms of action, it is of pronounced importance that representative tetrahydro-β-carbolynes proved to be inhibitors of microtubule-crosslinking mitotic kinesin Eg5 [6,7], cell division-inducing cyclic dependent kinase (CDKs) [8,9] as well as topoisomerase I and II [8,10]. Pointing to an additional mode of antiproliferative action of β-carbolines, a few representative 9-substituted harmine alkaloids were identified as DNA-intercalators [10]. β-Carboline hybrids with aryl linkers were also shown to exhibit substantial antiproliferative activity on a wide range of malignant human cells acting mainly by CT-DNA intercalation [11]. On the other hand, synergistically strengthening the antiproliferative effects associated with selective binding-based classical targeted mechanisms of action, electron transfer processes via the Fenton pathway induced by redox active fragments, e.g. by suitably positioned ferrocenyl group(s) in the molecular scaffold, may play a key role in mitochondrial generation of reactive oxygen species (ROS), e.g. nitric oxide, superoxide anion and other forms of free radicals [12][13][14] that can be involved in biological regulatory processes finally leading to programmed cell death (apoptosis) [15]. Supporting the relevance of this view, convincing preclinical evidence about the interplay between particular binding-induced-and redox signalling pathways implicated in cancer has been disclosed [16]. Accordingly, due to their marked effects on malignant cell lines, a plethora of ferrocene derivatives with diverse molecular architectures have been emerged as potent antiproliferative agents in the last decades [17][18][19][20][21].
The reactions of 6/T with aldehyde components 7e,i-k carrying substituents adjacent to the formyl group gave highly complex mixtures of undefined components. The failure of these experiments can probably be attributed to an interference of the CO2Me group with the negatively polarized carbonyl oxygen and the aromatic ring, the two rotating molecular fragments that avoid to get in proximal position destabilized by steric crowding (in case of bulky 7e,j) or electrostatic repulsion (in case of 7i,k with negative fluorine-centre).
The Pd-catalysed hydrogenation of 3 and 4/T gave the expected 1-(2-aminophenyl)-substituted β-carbolines (racemic 5 and enantiopure 6/T in 82% and 75%, resp.) which were then reacted with aromatic aldehydes 7a-k in a 5/1 mixture of MeOH and AcOH at reflux temperature affording tetracycles of type 8 and 9. The facile cyclisations of 6/T could be completed within 2 h providing most of the targeted esters 9a-d,f-h/T1 as single enantiomers that were isolated in 62-73% yield (Table 1) with a well-defined stereostructure, that was confirmed for 9f/T1 by single-crystal X-ray diffraction ( Figure 2).
Employing prolonged reaction times the isolated yields could not be increased as the ring closures were accompanied by decomposition. (T1 is an arbitrary designation of the relative configuration as will be discussed later along with T2 and T3 representing two further types of possible diastereomers.) The unanimous and rigid conformation of 9a-d,f-h/T1, determined by the carbon-and nitrogen stereogenic centres (C-6, C-8, C-14b and the non-inverting N-7) as well as by the conformational chirality of ring C, is clearly reflected from the highly similar (practically almost identical) 1 H-and 13 C-NMR parameters obtained for the skeletal atoms of this series of esters (cf. Experimental).  Substituted 5,6,8,9,14,3':3,4

]-pyrido[1,2-c]quinazolines
A straightforward retrosynthetic analysis of pentacycles type I set up an obvious synthetic pathway starting with a Pictet-Spengler (PS) annelation involving tryptophan-based precursors and 2-nitrobenzaldehyde followed by nitro group reduction and subsequent aldehyde-mediated cyclisation of the resulting 2-aminophenyl-substituted β-carboline framework to construct the targeted pentacyclic products (Scheme 1). Accordingly, tryptamine (1) was first converted into the nitrophenyl derivative 3 by an efficient PS protocol using an acetic acid/boric acid system at reflux temperature to promote the reaction [30] which practically went to completion within 4 h and allowed the isolation of the product as a racemic mixture in 85% yield. The analogous reaction of the methyl ester of L-tryptophan (2), conducted under the same conditions, gave a hardly separable 1/2 mixture of cis-and trans-diastereomer esters 4/C and 4/T. By means of flash column chromatography on silica using CH2Cl2-MeOH (80:1) as eluent, 4/T could be isolated in pure form in acceptable yield (42%), but the complete separation of 4/C from 4/T could not be achieved.
The Pd-catalysed hydrogenation of 3 and 4/T gave the expected 1-(2-aminophenyl)-substituted β-carbolines (racemic 5 and enantiopure 6/T in 82% and 75%, resp.) which were then reacted with aromatic aldehydes 7a-k in a 5/1 mixture of MeOH and AcOH at reflux temperature affording tetracycles of type 8 and 9. The facile cyclisations of 6/T could be completed within 2 h providing most of the targeted esters 9a-d,f-h/T1 as single enantiomers that were isolated in 62-73% yield (Table 1) with a well-defined stereostructure, that was confirmed for 9f/T1 by single-crystal X-ray diffraction ( Figure 2). Employing prolonged reaction times the isolated yields could not be increased as the ring closures were accompanied by decomposition. (T1 is an arbitrary designation of the relative configuration as will be discussed later along with T2 and T3 representing two further types of possible diastereomers.) The unanimous and rigid conformation of 9a-d,f-h/T1, determined by the carbonand nitrogen stereogenic centres (C-6, C-8, C-14b and the non-inverting N-7) as well as by the conformational chirality of ring C, is clearly reflected from the highly similar (practically almost identical) 1 H-and 13 C-NMR parameters obtained for the skeletal atoms of this series of esters (cf. Experimental).  Of note is the fact that diagnostic interactions detected between proton pairs H-6/H-8 and H-14b/H-2 ,6 in the NOESY spectra of 9a-d also confirm the trans arrangement of H-6 and H-14b as well as the spatial proximity of H-6 and H-8. In 9a-d,f-h/T1 the axial position of H-8 thus, the equatorial position of the methoxycarbonyl group (R), can also be regarded as evidenced by the characteristic coupling pattern of the signals originated from the skeletal protons H-8, H-9 a and H-9 b featuring a dd split of H-8 signal with coupling constants at around 8 Hz and 4 Hz.
The reactions of 6/T with aldehyde components 7e,i-k carrying substituents adjacent to the formyl group gave highly complex mixtures of undefined components. The failure of these experiments can probably be attributed to an interference of the CO 2 Me group with the negatively polarized carbonyl oxygen and the aromatic ring, the two rotating molecular fragments that avoid to get in proximal position destabilized by steric crowding (in case of bulky 7e,j) or electrostatic repulsion (in case of 7i,k with negative fluorine-centre).
The reactions of precursor 5 with benzaldehydes 7a-k were carried out under the same conditions using MeOH-AcOH (5:1) as solvent at reflux temperature. Indicating the acceleration of ring closures 5+7b-e,j,k→8b-e,j,k/T1 enabled by the electron-withdrawing substituents on the aldehyde components, no significant change could be detected in the yields of the products when the reaction was conducted for a substantially longer time (1 h: 65-80%, 12 h: 63-87%). In keeping with this observation, in the lack of activation effect induced by electron-withdrawing group on the aryl ring, the diastereoselective transformation 5+7a→8a/T1 needed a significant extension of reaction time to produce a substantially increased isolated yield (1 h: 37%, 12 h: 77%). Except for the non-precipitated fraction of the products no component with well-defined structure could be unequivocally identified by NMR in the highly complex mixtures recovered from the solutions. The stereostructure of 8a-e,k/T1 determined by the helicity of ring C and the relative configuration of the stereogenic centres C-6, N-7 Molecules 2020, 25, 1599 5 of 25 and C-14b was evidenced by the NOESY interactions detected between H-14b and the protons of the 6-Ar group as well as by the highly similar chemical shifts of 1 H/ 13 C signal pairs H-6/C-6 and H-14b/C-14b discernible in narrow ranges of the appropriate spectra of compounds type 8/T1 verifying to their unanimous skeletal structure. Pointing to the spectacular substituent-dependence of the diastereoselectivity of the studied cyclocondensations, on short treatment (0.5 h) with aldehydes 7f and 7g containing electron-donating aryl group, 5 got converted into pentacycles 8f/C1 (63%) and 8g/C1 (39%), respectively, in which H-6 and H-14b are in cis position on ring D as proved by NOESY measurements. Besides small portions of products type 8/C1, traces of their trans counterparts (8f,g/T1) could also be identified by NMR in the complex mixtures recovered from the solution. When aldehyde 7f was used as reactant, the extension of reaction time from 0.5 h to 4 h gave rise to a spectacular change in the product ratio resulting in the isolation of the epimeric mixture of 8f/C1 and 8f/T1 in 80% yield markedly enriched in the trans component (C1/T1~1/3). Further prolongation of the reaction time to 8 h led to the isolation of an approximately 1/4 mixture of these diastereomers with slightly decreased isolated yield of 75%, pointing to a slow development of an equilibrium system. On the other hand, undefined decomposition processes prevented a substantial extension of the reaction time of the formylferrocene-mediated cyclisation of 5, thus 4 h was found to be an optimal choice allowing the isolation of the mixture of diastereomers 8g/C1 and 8g/T1 in 52% yield enriched in the trans component (C1/T1~3/7). A series of attempts for the chromatographic separation of isomer pairs 8f/C1_8f/T1 and 8g/C1_8g/T1 failed.
It is of interest that even with only 0.5 h of treatment of 5 with formylpyridines 7h,i got readily converted into approximately 4/3 and 1/1 mixtures of isomer pairs 8h/C1_8h/T1 and 8i/C1_8i/T1, respectively, in high yields (72% for 8h/C1_8h/T1 and 80% for 8i/C1_8i/T1). Employing substantially prolonged reaction time (12 h) the ratio of the diastereomers were not changed within experimental error pointing to the fast formation of the rapidly interconverting isomers with slightly different or nearly identical thermodynamic stability, while both diastereomer pairs could be isolated in dramatically decreased yields (25% for 8h/C1_8h/T1 and 35% for 8i/C1_8i/T1) from the darkened reaction mixtures indicating uncontrolled decompositions of these pyridine derivatives. On the other hand, using 3-bromopicolinaldehyde 7j, a sterically more demanding ring-closing component, the reaction of 5 practically was completed within 1 h to afford trans diastereomer 8j/T1 isolated as single product in high yield (80%). Since after an extended reaction time (12 h) this trans diastereomer could again be isolated in excellent yield (87%), it seems reasonable that the abovementioned decomposition of the equilibrating isomer pars of 8h,i might take place via the appropriate cis isomer.
The experimental product distributions corroborate with the relative free energy [∆G(8/T1-8/C1)] values (Table 2) obtained by DFT comparative analysis of the isomeric pairs carried out by B3PW91 functional [31] employing DGTZVP basis set [32] together with IEFPCM solvent model [33]. For all calculations, a dielectric constant (ε = 28.2) was used to represent the approximate polarity of the medium composed of the solvent mixture MeOH-AcOH (5:1). It must be noted here that the comparative DFT modeling studies were performed on models also including ferrocene derivatives, so we selected B3PW91 for this purpose as this functional has been demonstrated to provide an improvement over B3LYP regarding the description of bonding parameters in metal-containing fragments with metal-carbon bond(s) [34]. Although the relative trans position of protons H-6 and H-14b in compounds types 8 and 9 was unambiguously evidenced by NOESY experiments disclosing interactions between H-14b and the protons of the proximal Ar groups, the possible N-7-inversion with simultaneous or separated flip of ring C might allow the formation of equilibrium mixtures of three diastereomers designated as T1, T2 and T3 (Scheme 2). Their interconversions, in principle enabled by the abovementioned processes, were modelled by the DFT analysis of 8a. The computations identified these types of conformers as three local minima on the potential energy surface (PES) connected by saddle points representing the corresponding transition states TS(8a/T1-T2), TS(8a/T2-T3) and TS(8a/T1-T3) (Scheme 2). Although the relative trans position of protons H-6 and H-14b in compounds types 8 and 9 was unambiguously evidenced by NOESY experiments disclosing interactions between H-14b and the protons of the proximal Ar groups, the possible N-7-inversion with simultaneous or separated flip of ring C might allow the formation of equilibrium mixtures of three diastereomers designated as T1, T2 and T3 (Scheme 2). Their interconversions, in principle enabled by the abovementioned processes, were modelled by the DFT analysis of 8a. The computations identified these types of conformers as three local minima on the potential energy surface (PES) connected by saddle points representing the corresponding transition states TS(8a/T1-T2), TS(8a/T2-T3) and TS(8a/T1-T3) (Scheme 2).
Although the calculated activation barriers (Scheme 2) might point to a relative flexibility of the skeletal structure, in agreement with the relative energetics expressed in Gibbs free energy values, the NMR data discussed above clearly indicate that 8a/T1 can be regarded as the most stable and practically exclusively detectable diastereomer in the analysed sample dissolved in DMSO-d6. It is of importance that on the basis of the well-established solid-state structure of 9f supplemented by the results of 1 H-and 13 C-NMR studies that disclosed highly similar chemical sifts and H-H coupling patterns for the skeletal CH-and CH2 groups, it can be stated that all the isolated pentacyclic products types 8 and 9 with trans relative configuration preferably adopt conformation T1. Supporting this view about the conformational space, the same series of DFT analyses carried out for esther 9a identified the expected diastereomers 9a/T1, 9a/T2 and 9a/T3 along with their relative energetics and activation barriers of their interconversions proceeding through transition states TS(9a/T1-T2), TS(9a/T2-T3) and TS(9a/T1-T3). Albeit in the minor components 9a/T2 and 9a/T3 the methoxycarbonyl group is in axial position, the calculated relative energetics [ΔG(9a/T1-9a/T2) = +4.53 kcal/mol and ΔG(9a/T1-9a/T3) = +0.87 kcal/mol] refer to a population Although the calculated activation barriers (Scheme 2) might point to a relative flexibility of the skeletal structure, in agreement with the relative energetics expressed in Gibbs free energy values, the NMR data discussed above clearly indicate that 8a/T1 can be regarded as the most stable and practically exclusively detectable diastereomer in the analysed sample dissolved in DMSO-d 6 .
It is of importance that on the basis of the well-established solid-state structure of 9f supplemented by the results of 1 H-and 13 C-NMR studies that disclosed highly similar chemical sifts and H-H coupling patterns for the skeletal CH-and CH 2 groups, it can be stated that all the isolated pentacyclic products types 8 and 9 with trans relative configuration preferably adopt conformation T1. Supporting this view about the conformational space, the same series of DFT analyses carried out for esther 9a identified the expected diastereomers 9a/T1, 9a/T2 and 9a/T3 along with their relative energetics and activation barriers of their interconversions proceeding through transition states TS(9a/T1-T2), TS(9a/T2-T3) and TS(9a/T1-T3). Albeit in the minor components 9a/T2 and 9a/T3 the methoxycarbonyl group is in axial position, the calculated relative energetics [∆G(9a/T1-9a/T2) = +4.53 kcal/mol and ∆G(9a/T1-9a/T3) = +0.87 kcal/mol] refer to a population distribution similar to that of the conformers of 8a. We also undertook the conformational analysis of the detectable or purely isolated cis pentacyclic products. In 8f-i/C1 diagnostic NOE could be detected between protons H-14b and H-6. Conformation C1 was identified by further comparative DFT studies carried out on representative model diastereomers 8f/C1 and 8f/C2. Besides the relative energetics [∆G(8f/C1-8f/C2) = +0.89 kcal/mol] diagnostic 1 H-and 13 C-chemical shifts were also calculated for the optimized structures of these isomers and compared to a selection of diagnostic experimental data (calcd. for C1/C2/measured [ppm]: δH-14b = 5.70/5.95/5.61; δH-6 = 5.80/6.20/5.62; δH-1 = 7.50/8.22/7.21; δC-14b = 60.5/63.3/57.1; δC-6 = 79.1/84.6/73.3; δC-8 = 39.6/50.7/38.0) suggesting that 8f/C1 is present in the DMSO-d 6 solution subjected to NMR measurements. These calculations were performed by GIAO method [35] using B3LYP functional [36] and 6-311++G(2d,p) basis set [37]. Due to the significant steric crowdance in the endo region of the bent ring system, 8f/C3 was not taken into account as a realistic component of the conformational space (Scheme 3). These considerations about the skeletal conformation can also be extended to the further detected cis products 8g-i. distribution similar to that of the conformers of 8a. We also undertook the conformational analysis of the detectable or purely isolated cis pentacyclic products. In 8f-i/C1 diagnostic NOE could be detected between protons H-14b and H-6. Conformation C1 was identified by further comparative DFT studies carried out on representative model diastereomers 8f/C1 and 8f/C2.  [37]. Due to the significant steric crowdance in the endo region of the bent ring system, 8f/C3 was not taken into account as a realistic component of the conformational space (Scheme 3). These considerations about the skeletal conformation can also be extended to the further detected cis products 8g-i.

Preparative-, Spectral and Theoretical Studies on the Mechanism and Products of the Substituent-Controlled Annulations of 5 and 6/T Effected by Formyl-Substituted Carboxylic Acids
In order to obtain targeted polycyclic lactams types II and III (Figure 1), using our standard conditions with slightly prolonged reaction time (5 h), precursors 5 and 6/T were also reacted with 2-formylbenzoicacid (7l) and the enantiomerically pure planar chiral (Sp)-2-formyl-1-carboxyferrocene (7m), respectively (Scheme 4).
The reactions of 5 and 6/T with 7l afforded polycondensed lactams 10/C1 and 11/C1 in good yields, (88% and 71%, respectively,) pointing to fast sequential annelations that certainly proceed via the appropriate cis pentacyclic diastereomer (8l/C1 and 9l/C1). However, on treatment with 7m the racemic precursor 5 got converted into cis pentacyclic product 8m*/C1 in mediocre yield (47%) with well-defined relative and, consequently, absolute configuration ultimately controlled by the planar chiral ferrocene unit embodied in the heterocyclic skeleton. This relatively rigid molecular architecture is stabilized by an intramolecular H-bond involving the carboxylic group and the N-7 atom of which basic character is probably enhanced relative to that of N-5 atom. Taking this seven-membered chelate ring and the Sp planar chirality of the ferrocene moiety as structural constrains into account, the absolute configuration of 8m*/C1 was deduced from the NOESY interactions of the unsubstituted Cp ring with the proximal (C)H-14b and (N)H-5 protons.
Accordingly, the proximity of the iron centre can also be considered as evidenced by the substantial downfield shift of the H-14b signal (6.23 ppm) relative to that measured for the racemic ferrocenyl-substituted derivative 8g/C1 (5.49). The skeletal structure and the relative-and consequently, the absolute configuration of L-tryptophan-derived 11/C1 were disclosed by 1 H-and 13 C-NMR methods. The presence of the fused isoindolone fragment was evidenced by the HMBC correlation between the signals of H-15b and C-11.
The characteristic coupling pattern of the signals of the skeletal protons H-17, H-18a and H-18b featuring a dd split for H-17 resonance (J = 7.9 Hz and 3.8 Hz) and the highly spectacular upfield shift of the OCH3 singlet (2.83 ppm) can be regarded as conclusive evidences for the equatorial

Preparative-, Spectral and Theoretical Studies on the Mechanism and Products of the Substituent-Controlled Annulations of 5 and 6/T Effected by Formyl-Substituted Carboxylic Acids
In order to obtain targeted polycyclic lactams types II and III (Figure 1), using our standard conditions with slightly prolonged reaction time (5 h), precursors 5 and 6/T were also reacted with 2-formylbenzoicacid (7l) and the enantiomerically pure planar chiral (S p )-2-formyl-1-carboxyferrocene (7m), respectively (Scheme 4).
The reactions of 5 and 6/T with 7l afforded polycondensed lactams 10/C1 and 11/C1 in good yields, (88% and 71%, respectively,) pointing to fast sequential annelations that certainly proceed via the appropriate cis pentacyclic diastereomer (8l/C1 and 9l/C1). However, on treatment with 7m the racemic precursor 5 got converted into cis pentacyclic product 8m*/C1 in mediocre yield (47%) with well-defined relative and, consequently, absolute configuration ultimately controlled by the planar chiral ferrocene unit embodied in the heterocyclic skeleton. This relatively rigid molecular architecture is stabilized by an intramolecular H-bond involving the carboxylic group and the N-7 atom of which basic character is probably enhanced relative to that of N-5 atom. Taking this seven-membered chelate ring and the S p planar chirality of the ferrocene moiety as structural constrains into account, the absolute configuration of 8m*/C1 was deduced from the NOESY interactions of the unsubstituted Cp ring with the proximal (C)H-14b and (N)H-5 protons.
Accordingly, the proximity of the iron centre can also be considered as evidenced by the substantial downfield shift of the H-14b signal (6.23 ppm) relative to that measured for the racemic ferrocenyl-substituted derivative 8g/C1 (5.49). The skeletal structure and the relative-and consequently, the absolute configuration of l-tryptophan-derived 11/C1 were disclosed by 1 H-and 13 C-NMR methods. The presence of the fused isoindolone fragment was evidenced by the HMBC correlation between the signals of H-15b and C-11.
The characteristic coupling pattern of the signals of the skeletal protons H-17, H-18 a and H-18 b featuring a dd split for H-17 resonance (J = 7.9 Hz and 3.8 Hz) and the highly spectacular upfield shift of the OCH 3 singlet (2.83 ppm) can be regarded as conclusive evidences for the equatorial position of the CO 2 CH 3 group on ring C being in the anisotropic shielding region of ring D. This proximal position of the CO 2 CH 3 group and ring D was also confirmed by the diagnostic NOE's detected between signal pairs H-5b/H-15b and H-15/OCH 3 . Accordingly, the HMBC correlation and the NOE's involving signal pairs H-15b/C-11 and H-5b/H-15b, respectively, complemented with the highly similar Molecules 2019, 24, x; doi: www.mdpi.com/journal/molecules position of the CO2CH3 group on ring C being in the anisotropic shielding region of ring D. This proximal position of the CO2CH3 group and ring D was also confirmed by the diagnostic NOE's detected between signal pairs H-5b/H-15b and H-15/OCH3. Accordingly, the HMBC correlation and the NOE's involving signal pairs H-15b/C-11 and H-5b/H-15b, respectively, complemented with the highly similar 1 H-and 13 C-NMR data, clearly indicate that the racemic lactam 10/C1 and its ester analogue 11/C1 with well-defined absolute configuration have identical skeletal constitution and conformation. The reaction of 6/T and 7m, the two components used as single enantiomers, proved to be sluggish resulting in the formation of trans ferrocenecarboxylate 9m/T1 in low yield (ca. 10-15% calculated for its approximate 60% ratio in the isolated highly contaminated crude product). Due to its decreased stability and a hardly separable array of undefined contaminations, the attempted purifications of this ester by column chromatography and crystallisation have failed so far. It is also of note that on prolongation of the reaction time this compound underwent a complete decomposition. On the other hand, its skeletal structure could be identified by NMR measurements producing 1 H and 13 C chemical shifts and H-H coupling constants characteristic for esters type 9/T1 with unambiguous relative-and consequently, absolute configuration. The reaction of 6/T and 7m, the two components used as single enantiomers, proved to be sluggish resulting in the formation of trans ferrocenecarboxylate 9m/T1 in low yield (ca. 10-15% calculated for its approximate 60% ratio in the isolated highly contaminated crude product). Due to its decreased stability and a hardly separable array of undefined contaminations, the attempted purifications of this ester by column chromatography and crystallisation have failed so far. It is also of note that on prolongation of the reaction time this compound underwent a complete decomposition. On the other hand, its skeletal structure could be identified by NMR measurements producing 1 H and 13 C chemical shifts and H-H coupling constants characteristic for esters type 9/T1 with unambiguous relative-and consequently, absolute configuration.
Contrary to intermediate carboxylates 8l/C1 and 9l/C1, the ferrocene analogues 8m*/C1 and 9m/T1 could not be forced to undergo intramolecular acylation even after using a prolonged reaction time (12 h), instead, uncontrolled processes were promoted resulting in the formation of substantial amounts of tarry materials that allowed the isolation of unchanged pentacycle 8m*/C1 with significantly decreased yield (8%), while the trans isomer 9m/T1 completely decomposed. The resistance of these carboxyferrocene derivatives to cyclization can probably be ascribed to the substantial overall strain and steric crowdance in the hypothetic polycyclic ferrocene lactams (12m*/C1, 13m/T1 or 13m/T3, a more stable conformer) incorporating two diectly fused five-membered rings. This view is strongly supported by the results of DFT modelling studies revealing dramatic reactivity difference between the carboxyphenyl-substituted intermediates (8l/C1 and 9l/C1) and the carboxyferrocenyl analogues (8m*/C1 and 9m/T1) in terms of their propensity to undergo cyclisation, as spectacularly shown by the changes in the free energy values calculated for the corresponding transformations (Scheme 4).

Proposed Mechanism for the Rationalization of the Substituent-and Time-Dependent Interconversion of cisand trans-Pentacycles 8/C1 and 8/T1 Based on the Results of DFT Modelling Studies
Finally, we put forward a rationalization of the characteristic substituent-and time-dependent diastereoselectivity observed in the aldehyde-mediated cyclization reactions of precursor 5 assumed to proceed along the multistep reaction sequences outlined in Scheme 5. It was hypothesized that preferably the nitrogen atom in ring C of enhanced nucleophilicity might initiate a reor si-face attack on the O-protonated aldehyde component to construct interconverting rotamer adduct pairs 12 + /R_13 + /R and their diastereomer counterparts 12 + /S_13 + /S. In the subsequent step the rotamers 13 + /R and 13 + /S, containing N-H and C-OH fragments in approximate anti position preformed for trans elimination, get converted into iminium cations 14 + /E and 14 + /Z, the key intermediates capable of undergoing either ring closure constructing diastereomeric pentacycles 8/C1 and 8/T1, or interconversion by reversible 1,3-proton-migration proceeding via the same iminium cation 15 + . In order to check the plausibility of this complex mechanism, comparative DFT studies were carried out on diastereomer pairs of adducts and iminium ions (12 + a,d,f,g/R_12 + a,d,g/S,  13 + a,d,f,g/R_13 + a,d,f,g/S and 14 + a,d,g/E_14 + a,d,f,g/Z, respectively) and isomer iminium cations  15 + a,d,f,g, a set of reasonably selected models containing substituents of markedly different In order to check the plausibility of this complex mechanism, comparative DFT studies were carried out on diastereomer pairs of adducts and iminium ions (12 + a,d,f,g/R_12 + a,d,g/S,  13 + a,d,f,g/R_13 + a,d,f,g/S and 14 + a,d,g/E_14 + a,d,f,g/Z, respectively) and isomer iminium cations 15 + a,d,f,g, a set of reasonably selected models containing substituents of markedly different electronic properties and steric bulk (Table 3). Table 3. Relative stability of selected isomer pairs to rationalize of the substituent-and time-dependent interconversion of cisand trans-pentacycles 8/C1 and 8/T1, as resulted from DFT modeling studies.

Relative Stability of Intermediate Pairs Expressed in
The value can also be considered as a selectivity descriptor for the alternative primary coupling modes 5+7 + →12 + and 5+7 + →13 + disclosing the latter as the preferred one (highlighted by italics). b Refers to 16 + −14 + /Z. c Refers to The calculations were carried out again at B3PW91/DGTZVP level of theory supported by the IEFPCM solvent model using a dielectric constant (ε = 28.2) to represent the polarity of the reaction mixture MeOH-AcOH (5:1). The calculated ∆G(12 + /R−12 + /S) and ∆G(13 + /R−13 + /S) suggest that the transformation with electron-deficient 4-nitrobenzaldehyde 7d directly results in the formation of 8d/T1, the more stable diastereomer that can thus be regarded as the thermodynamic product, while the reactions effected by benzaldehyde 7a, and aldehydes 7f,g carrying electron-donating group seem to proceed via intermediates 12 + /R and 13 + /R leading to 8a,f,g/C1. On the other hand, regardless to the nature of the aryl group, the ∆G(13 + −12 + ) values point to the preference of the primary addition 5+7 + →13 + over the alternative step 5+7 + →12 + constructing the appropriate rotamer and, accordingly, to the readiness of rotation 12 + →13 + . It is also of pronounced importance that the experimentally observed substantial decrease in the propensity of 8f,g/C1 to undergo epimerization to 8f,g/T1 can be adequately interpreted in terms of the reluctance of iminium cations 14 + f,g/E to undergo 1,3-proton migration switching off the resonance-stabilization contributed by the electron-donating aryl substituent [cf. ∆G(15 + −14 + /E) values in Table 3].
Finally, the data calculated for the reaction sequence involving O-protonated 4-nitro-benzaldehyde 7 + d, suggest that the multistep annulation of 5 with electron-deficient aldehyde components proceeds via the primary formation of adducts type 13 + /S referring to the identical outcome of the kinetic-and thermodynamic controls.
This assumption gains support from the n→π* type donor-acceptor interaction between the amino group and the C-1 atom of the 4-nitrophenyl substituent as clearly seen from the HOMO-8 identified in the optimized structure of 13 + d/S (Figure 3). It must be noted here that besides this interaction the lone pair of the amino group is also incorporated in a chelate-like intramolecular H-bond with the protonated skeletal secunder amine residue (cf. HOMO-11: Figure 3).
In principle C1↔T1 epimerization might also take place by the revesible deprotonation of C-6 proceeding via cations type 16 + (Scheme 5) enabled by the protonation of both adjacent skeletal nitrogen atoms, however this pathway must be regarded as hardly feasible as unequivocally evidenced by the values of relative energetics ∆G(16 + −14 + /Z) and ∆G(16 + −14 + /Z) even though the enhanced anion-stabilizing effect of the 4-nitrophenyl group is also reflected from the calculated data (Table 3). Finally, it seems reasonable that the pronounced readiness of pyridyl derivatives 8h,i to undergo C1↔T1 isomerization, mentioned in Section 2.1 can be attributed to a highly feasible pathway proceeding via enamine type tautomers 17h,i (Scheme 5).
Molecules 2019, 24, x; doi: www.mdpi.com/journal/molecules undergo 1,3-proton migration switching off the resonance-stabilization contributed by the electron-donating aryl substituent [cf. ΔG(15 + -14 + /E) values in Table 3]. Finally, the data calculated for the reaction sequence involving O-protonated 4-nitro-benzaldehyde 7 + d, suggest that the multistep annulation of 5 with electron-deficient aldehyde components proceeds via the primary formation of adducts type 13 + /S referring to the identical outcome of the kinetic-and thermodynamic controls.
This assumption gains support from the n→π* type donor-acceptor interaction between the amino group and the C-1' atom of the 4-nitrophenyl substituent as clearly seen from the HOMO-8 identified in the optimized structure of 13 + d/S (Figure 3). It must be noted here that besides this interaction the lone pair of the amino group is also incorporated in a chelate-like intramolecular H-bond with the protonated skeletal secunder amine residue (cf. HOMO-11: Figure 3). In principle C1↔T1 epimerization might also take place by the revesible deprotonation of C-6 proceeding via cations type 16 + (Scheme 5) enabled by the protonation of both adjacent skeletal nitrogen atoms, however this pathway must be regarded as hardly feasible as unequivocally evidenced by the values of relative energetics ΔG(16 + -14 + /Z) and ΔG(16 + -14 + /Z) even though the enhanced anion-stabilizing effect of the 4-nitrophenyl group is also reflected from the calculated data (Table 3). Finally, it seems reasonable that the pronounced readiness of pyridyl derivatives 8h,i to undergo C1↔T1 isomerization, mentioned in section 2.1 can be attributed to a highly feasible pathway proceeding via enamine type tautomers 17h,i (Scheme 5). The in vitro cytotoxicity of the polycondensed β-carboline products expressed in IC 50 values was determined on four selected human tumour cell cultures (PANC-1: pancreatic carcinoma of ductal origin, COLO-205: colon adenocarcinoma, A-2058: malignant melanoma with high invasiveness and EBC-1: lung squamous cell carcinoma). These cell lines are dedicated models for cancerous diseases with high mortality due to the lack of efficient chemotherapy. Thus, to find new antitumor agents against our investigated tumour models is a principal goal in the field of oncology drug development. The cells were treated with the compounds at 0.1-100 µM concentration range and the cell viability was determined by real-time impedimetry analysis (adherent cells-PANC-1) or colorimetric alamarBlue assay (cultures characterizing with weak/negligible adhesion-A2058, EBC-1, and COLO-205) (see Supplementary Materials).

In vitro Cytotoxic
The data listed in Table 4 show negligible or moderate-to-significant antiproliferative effects against PANC-1-, COLO-205-, A-2058 and EBC-1 cell lines that proved to be strongly dependent on the stereostructure and on the substitution pattern of the pending aryl substituent of aldehyde-origin. Thus, although the majority of the products do not have marked effect on the investigated cell lines, several highly active pentacycles were identified as promising models with significant antiproliferative activity characterized by IC 50 values in the low micromolar range. It is of note that while trans diastereomers carrying phenyl-and 3-trifluoromethylphenyl substituents (8a/T1 and 8b/T1) display outstanding effects on each investigated cell line even in racemic form, their ester derivatives isolated as single enantiomers (9a/T1 and 9b/T1) proved to be inactive in the tests. The strong impact of the substitution pattern on the cytotoxicity is spectacularly indicated by the introduction of the second trifluoromethyl group into the phenyl substituent leading to a decrease of ca. one order of magnitude in the cytotoxicity as reflected from the IC 50 values measured for 8c/T1 and by the marked difference in the anticancer potential of the inactive 4-nitrophenyl derivative 8d/T1 and its 2-nitrophenyl-substituted counterpart 8e/T1, the latter one exerting detectable effect on PANC-1 cell line. The combined effect of the substituents and the stereostructure is exemplified by comparing these results to those obtained in the tests of the highly potent racemic cis-pentacycle 8f/C1 (IC 50 = 2.1-5.2 µM on the investigated cell lines) and its inactive trans counterpart 8f/T1 both carrying 3,4,5-trimethoxyphenyl group on ring D. Interestingly, an opposite order of diastereomer activity was manifested in the tests of the inactive 8g/C1, the racemic ferrocenyl derivative isolated as single diastereomer, and the 1:3 mixture of 8g/C1 and 8g/T1 having substantial cytotoxicity on COLO-205, A-2058 and EBC-1 cell lines. (Obviously, the measured effects must be displayed by the trans isomer.) PANC-1 cells did not present detectable sensitivity towards this mixture. Similar degree and profile of activity were detected for 8*m/C1, the chiral carboxyferrocene-containing cis-diastereomer that can be regarded as a further indication of the combined effect of the substituents and the stereostructure. In accord with this view, contrary to the inactive racemic lactam 10/C1, its ester derivative 11/C1, obtained as a single enantiomer, displayed marked antiproliferative activity on three cell lines including PANC-1. Interestingly this model proved to be inactive against COLO-205 cells.

Materials and Methods
All fine chemicals were obtained from commercially available sources (Merck, Budapest, Hungary), Fluorochem (Headfield, U.K.), Molar Chemicals (Budapest, Hungary), (VWR, Budapest, Hungary) and used without further purification. Merck Kieselgel (230-400 mesh, 60 Å) was used for flash column chromatography. Melting points (uncorrected) were determined with a M-560 instrument (Büchi, Essen, Germany). The 1 H-and 13 C-NMR spectra were recorded in DMSO-d 6 solution in 5 mm tubes at room temperature (RT), on a Bruker DRX-500 spectrometer (Bruker Biospin, Karlsruhe, Baden Württemberg, Germany) at 500 ( 1 H) and 125 ( 13 C) MHz, with the deuterium signal of the solvent as the lock and TMS as internal standard ( 1 H, 13 C). The 2D-COSY, NOESY, HSQC, and HMBC spectra were obtained by using the standard Bruker pulse programs cosygpppqf (2D COSY with gradient pulses for selection and purge pulses before relaxation delay d1) for COSY, noesygpphpp (2D phase sensitive NOESY with gradient pulses in mixing time and purge pulses before relaxation delay d1 for NOESY), hsqcetgp (2D phase sensitive HSQC using Echo/Antiecho-TPPI gradient selection with decoupling during acquisition and using trim pulses in inept transfer) for HSQC and hmbcgpndqf (2D H-1/X HMBC optimized on long range couplings, no decoupling during acquisition using gradient pulses for selection for HMBC). For each compound characterized in this session the numbering of atoms used for assignment of 1 H-and 13 C-NMR signals correspond to IUPAC rules reflected from the given systematic names. The exact mass measurements were performed using a Q Exactive Focus orbitrap instrument (Thermo Scientific Bremen, Bremen, Germany) equipped with heated electrospray ionization source. X-ray diffraction data were collected on an XtaLab Synergy-R diffractometer (Rigaku, Neu-Isenburg, Germany) using Cu -Kα radiation (λ = 1.54184 Å). Data reduction was carried out using the software provided with the diffractometer (CrysAlisPro 1.171.40.14e, Rigaku OD, 2018)). All calculations were carried out by using the Gaussian 09 software (Gaussian Incorporation, Pittsburgh, U.S.) package [38]. The optimized structures are available from the authors. To a cooled and stirred solution of L-tryptophan methyl ester hydrochloride (5.09 g, 20 mmol) in water (50 mL) a solution of KOH (1.12 g, 20 mmol in 10 mL of water) was added in small portions and the resulting mixture was extracted with CH 2 Cl 2 (4 × 15 mL). The organic phase was dried over Na 2 SO 4 and evaporated to dryness. The oily residue, 2-nitrobenzaldehyde (3.03 g, 20 mmol) and boric acid (1.24 g, 20 mmol) were dissolved in acetic acid and (20 mL). The solution was stirred at reflux under argon for 12 h and cooled down to rt. To the stirred reaction mixture saturated Na 2 CO 3 solution (200 mL) was added in small portions. The precipitated solid containing ca. 1:2 mixture of 4/C and 4/T was subjected to flash chromatography on silica using CH 2 Cl 2 -MeOH (80:1) as eluent (R f = 0.20 for 4/T and 0.23 for 4/C as detected by TLC). (No detectable separation could be observed when more polar solvents, e.g., CH 2 Cl 2 -MeOH (from 50:1 to 10:1) were used as eluent.) The complete separation of the eluting fractions could not be achieved, so the non-overlapping upper part of the second band was collected and evaporated to dryness. The oily residue was crystallized with cold MeOH to obtain the product as a yellow powder. Orange solid. Yield The corresponding 2-nitrophenyl derivative (10 mmol) was dissolved in MeOH (20 mL) In the presence of 10% Pd/C (1.21 g) the reaction mixture was stirred under H 2 (1 atm, balloon) for 6 h. The catalyst was removed by filtration over celite and the filtrate was concentrated in vacuo. The reulting solid was triturated with cold MeOH and filtered off to obtain the product as colorless crystals. The corresponding 2-aminophenyl derivative (2 mmol) and the aldehyde component (1.1 mmol) were stirred and heated under argon at reflux temperature in a mixture of MeOH (10 mL) and AcOH (2 mL) for 0.5-16 h and cooled down to rt. (The exact reaction times are given along with the isolated yields of the products as presented in the sections below describing product characterization.) To this reaction mixture, cooled with ice-water, saturated aqueous Na 2 CO 3 solution (15 mL) was added in small portions. (The reaction mixtures obatined by the cyclizations involving 7m as aldehyde component were diluted with 10 mL of water and carefully neutralized by the addition of solid NaHCO 3 in small portions.) The precipitated solid was separated by filtration, triturated with a cold 2:5 mixture of MeOH and water (ca. 15 mL), filtered off again and dried in dessicator over KOH pellets. Analytical samples were recrystallized from MeOH. (In order to remove the traces of tarry contaminations formed in uncontrolled processes, the crude products of the cyclization reactions with ferrocene reagents 7l and 7m were dissolved in 20 mL of CH 2 Cl 2 and passed through a celite pad.) The solution was evaporated to obtain an oily or solid residue which was triturated with a cold 1:5 mixture of MeOH and water (ca. 10 mL). The purified product was filtered off and dried in dessicator over KOH pellets.