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

molecules

Article

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

Kinga Judit Fodor ¹, Dániel Hutai ¹, Tamás Jernei ², Angéla Takács ³, Zsóﬁa Szász ³,

Máté Sulyok-Eiler ⁴, Veronika Harmat ⁴, Rita Oláh Szabó ², Gitta Schlosser ^2,5

,

Ferenc Hudecz ^1,2, László Ko˝hidai ³and Antal Csámpai ^1,

*

1

Department of Organic Chemistry, Eötvös Loránd University (ELTE) Budapest Pázmány P. sétány 1/A,

H-1117 Budapest, Hungary; fodorkinga90@gmail.com (K.J.F.); hutaidani@gmail.com (D.H.);

fhudecz@caesar.elte.hu (F.H.)

MTA-ELTE Research Group of Peptide Chemistry, Budapest Pázmány P. sétány 1/A, H-1117 Budapest,

Hungary; jernei91@gmail.com (T.J.); rita.olah.szabo@gmail.com (R.O.S.); sch@chem.elte.hu (G.S.)

Department of Genetics, Cell- and Immunobiology, Semmelweis University, Nagyvárad tér 4,

H-1089 Budapest, Hungary; angela.takacs1@gmail.com (A.T.); szaszzsoccii@gmail.com (Z.S.);

kohlasz2@gmail.com (L.K.)

Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University,

Budapest Pázmány P. sétány 1/A, H-1117 Budapest, Hungary; etammate95@gmail.com (M.S.-E.);

veronika@chem.elte.hu (V.H.)

Department of Analytical Chemistry, Eötvös Loránd University (ELTE), Budapest, Pázmány P. sétány 1/A,

H-1117 Budapest, Hungary

2

3

4

5

*

Correspondence: csampai@caesar.elte.hu; Tel.: +36-01-372-2500 (ext. 6591)

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

Academic Editor: Maura Pellei

Received: 17 March 2020; Accepted: 30 March 2020; Published: 31 March 2020

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: 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[2⁰,3⁰:3,4]pyrido[1-c]-quinazolines

and 5,5b,17,18-tetrahydroindolo[2⁰,3⁰:3,4]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

1

conformations of the novel polycyclic indole derivatives were determined by H- 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.

Keywords: β-carboline; diastereoselectivity; conformation; ferrocene; organic synthesis; NMR

spectroscopy; X-ray diﬀraction; DFT calculations; cytotoxic activity; structure-activity relationships

Molecules 2020, 25, 1599; doi:10.3390 /molecules25071599

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1. 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 [ ], cardioprotective [ ], trypanocidal and neurotoxic [

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 [ ]. 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- -carbolynes

proved to be inhibitors of microtubule-crosslinking mitotic kinesin Eg5 [ ], cell division-inducing

cyclic dependent kinase (CDKs) [ ] as well as topoisomerase I and II [ 10]. Pointing to an additional

mode of antiproliferative action of -carbolines, a few representative 9-substituted harmine alkaloids

were identiﬁed as DNA-intercalators [10]. -Carboline hybrids with aryl linkers were also shown to

3]

β

8,

8,

β

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 [12–14]

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 [17–21].

2. Results and Discussion

Prompted by the aforementioned highly promising precedents, in the frame of our ongoing

research aimed at identiﬁcation of novel leads including ferrocene hybrids [22–29] we envisaged

atryptamine- and tryptophan-based synthesis, detailed structural analysis and preliminary in vitro

evaluation of 6-aryl-substituted 5,6,8,9,14,14b-hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazolines

(I

), 5,5b,17,18-tetrahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]isoindolo[2,1-a]quinazolin-11(15bH)-ones (II),

and (S_p)-2-formyl-1-ferrocenecarboxylate-derived 5,5b,11,14b,16,17-hexahydroindolo[2⁰,3⁰:3,4]py-

rido[1,2-c]ferroceno[c]pyrrolo[1,2-a]quinazoline-11(14bH)-ones

(III), featuring alkaloid-like

frameworks with diverse stereostructures (Figure 1).

Figure 1. Three types of the targeted polycyclic

β-carbolines with diverse streostructures derived from

tryptamin (R = H) and l-tryptophan (R = CO₂Me).

2.1. Synthesis and Structural Analysis of the 6-Aryl-Substituted

5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]-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

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2-nitrobenzaldehyde followed by nitro group reduction and subsequent aldehyde-mediated cyclisation

of the resulting 2-aminophenyl-substituted -carboline framework to construct the targeted pentacyclic

products (Scheme 1). Accordingly, tryptamine ( ) was ﬁrst converted into the nitrophenyl derivative

β

1

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.

Scheme1. Synthetic routes to novel diastereomeric 6-aryl-5,6,8,9,14,14b-hexahydroindolo-[2⁰,3⁰:3,4]pyrido-

[1,2-c]quinazolines (the relative/absolute conﬁguration is presented for compounds with R = H/R =

CO₂Me.).

The Pd-catalysed hydrogenation of

-carbolines (racemic and enantiopure 6/T in 82% and 75%, resp.) which were then reacted with

aromatic aldehydes 7a in a 5/1 mixture of MeOH and AcOH at reﬂux temperature aﬀording

tetracycles of type and . The facile cyclisations of 6/T could be completed within 2 h providing most

of the targeted esters 9a h/T1 as single enantiomers that were isolated in 62–73% yield (Table 1)

3 and 4/T gave the expected 1-(2-aminophenyl)-substituted

β

5

–

k

8

9

–d,f–

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

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chirality of ring C, is clearly reﬂected from the highly similar (practically almost identical) ¹H- and

¹³C-NMR parameters obtained for the skeletal atoms of this series of esters (cf. Experimental).

Table 1. Isolated yields of pentacyclic compounds types 8 and 9 presented in Scheme 1.

Product

Yield (Reaction Time)

Product

Yield (Reaction Time)

8a/T1

8b/T1

8c/T1

8d/T1

8e/T1

8f/C1

8f/C1/T1

8g/C1

8g/C1/T1

8h/C1/T1

37% (0.5 h)/77% (12 h)

75% (1 h)/72% (12 h)

79% (1 h)/76% (12 h)

74% (1 h)/78% (12 h)

80% (1 h)/75% (12 h)

63% (0.5 h)

8i/C1/T1

8j/T1

8k/T1

9a/T1

9b/T1

9c/T1

9d/T1

9f/T1

9g/T1

9h/T1

b

80% (0.5 h) ^e/35% (12 h)^e

80% (1 h)/87% (12 h)

69% (1 h)/71% (12 h)

64% (2 h)

70% (2 h)

62% (2 h)

73% (2 h)

71% (2 h)

52% (2 h)

56% (2 h)

80% (4 h) ^a/75% (8 h) ^b

39% (0.5 h)

52% (4 h)^c

72% (0.5 h) ^d/25% (12 h) ^d

a

For a ca. 1:3 mixture of cis (C1) and trans (T1) diastereomers. For a ca. 1:4 mixture of cis (C1) and trans (T1

)

c

d

diastereomers. For a ca. 3:7 mixture of cis (C1) and trans (T1) diastereomers. For a ca. 4:3 mixture of cis (C1) and

e

trans (T1) diastereomers. For a ca. 1:1 mixture of cis (C1) and trans (T1) diastereomers.

Figure 2. Stereostructure of trans pentacyclic esther 9f/T1 obtained by single crystal X-ray diﬀraction

(left) and DFT modelling (right).

Of note is the fact that diagnostic interactions detected between proton pairs H-6/H-8 and

H-14b/H-2⁰,6⁰in the NOESY spectra of 9a

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 h/T1 the axial position of H-8 thus, the equatorial

–

d

–d,f–

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_aand H-9_bfeaturing 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 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 reactions of precursor

using MeOH-AcOH (5:1) as solvent at reﬂux temperature. Indicating the acceleration of ring

closures 7b k/T1 enabled by the electron-withdrawing substituents on the aldehyde

5 with benzaldehydes 7a–k were carried out under the same conditions

5+

–e

,j,k→8b–e,j,

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

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

5

+

7a→8a/T1 needed a signiﬁcant extension of reaction time to

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

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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 ¹H/¹³C 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,

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 T1)

5 got converted into pentacycles 8f/C1 (63%) and

/

,

g/

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

allowing the isolation of the mixture of diastereomers 8g

5

, thus 4 h was found to be an optimal choice

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

converted into approximately 4/3 and 1/1 mixtures of isomer pairs 8h

respectively, in high yields (72% for 8h C1 8h T1 and 80% for 8i C1 8i T1). Employing substantially

5

with formylpyridines 7h

,i

got readily

/

C1 8h T1 and 8i

_

/

C1 8i T1,

_

/

_

/

_ /

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

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

/C1_8h/T1 and 35% for 8i/C1_8i/T1) from the darkened reaction mixtures

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].

Table 2. Relative stability of diastereomer pairs 8/T1-8/C1 calculated by DFT modelling.

∆G(T1-C1) [kcal/mol]

8a

8b

8c

−1.13

−1.65

−1.58

−1.48

8e

8f

8g

8h

−3.06

−1.52

−0.47

+0.34

8i

8j

8k

+0.19

−0.75

−1.73

8d

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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).

Scheme 2. Schematic representations and optimized structures of the possible diastereomers of

pentacycle 8a/T1 characterised by relative free energy values and barriers of their interconversion

proceeding via the presented TS structures as disclosed by a series of DFT modelling studies.

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 ¹H- and ¹³C-NMR studies that disclosed highly similar chemical sifts and H-H coupling

patterns for the skeletal CH- and CH₂groups, it can be stated that all the isolated pentacyclic products

types

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)

8 and 9 with trans relative conﬁguration preferably adopt conformation T1. Supporting this view

/

,

/

,

/

∆

/

-

/

∆

/

-

/

= +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

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 ¹H-

–i/C1 diagnostic NOE could be detected between protons H-14b and H-6. Conformation C1 was

/

∆

/

- /

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and ¹³C-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]: H-14b =

5.70/5.95/5.61; H-6 = 5.80/6.20/5.62; H-1 = 7.50/8.22/7.21; C-14b = 60.5/63.3/57.1; C-6 = 79.1/84.6/73.3;

C-8 = 39.6/50.7/38.0) suggesting that 8f C1 is present in the DMSO-d₆solution 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

/

δ

/

]

/

(Scheme 3). These considerations about the skeletal conformation can also be extended to the further

detected cis products 8g–i.

Scheme 3. Structures, relative Gibbs free energy values of 8f/C1 and 8f/C2 and the activation barrier of

the isomerisation of 8f/C1 into 8f/C2 taking place by N7-inversion with simultaneous ﬂip of ring C.

2.2. 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_pplanar 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

the absolute conﬁguration of l-tryptophan-derived 11

/

C1 (5.49). The skeletal structure and the relative- and consequently,

/

C1 were disclosed by ¹H- and ¹³C-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_aand 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 OCH₃singlet (2.83 ppm) can be regarded as conclusive evidences for the equatorial position

of the CO₂CH₃group on ring C being in the anisotropic shielding region of ring D. This proximal

position of the CO₂CH₃group 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

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¹H- and ¹³C-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.

Scheme 4. Cyclization reactions of 1-(2-aminophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indoles 5 and

6/T eﬀected by 2-formylbenzoicacid and (S_p).2-formylferrocenecarboxylic acid 7l and 7m, respectively.

A selection of optimized structures with absolute conﬁguration obtained by DFT modelling.

The reaction of

6

/

T

and 7m, the two components used as single enantiomers, proved to be sluggish

T1 in low yield (ca. 10–15% calculated for

resulting in the formation of trans ferrocenecarboxylate 9m

/

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 ¹H and ¹³C chemical

shifts and H-H coupling constants characteristic for esters type

consequently, absolute conﬁguration.

9/T1 with unambiguous relative- and

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

Molecules 2020, 25, 1599

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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).

2.3. Proposed Mechanism for the Rationalization of the Substituent- and Time-Dependent Interconversion of cis-

and 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 re- or si-face attack on

the O-protonated aldehyde component to construct interconverting rotamer adduct pairs 12⁺13⁺/R

/R_

and their diastereomer counterparts 12⁺

containing N-H and C-OH fragments in approximate anti position preformed for trans elimination, get

converted into iminium cations 14⁺/E and 14⁺

, the key intermediates capable of undergoing either

/S_ ,

13⁺/S. In the subsequent step the rotamers 13⁺/R and 13⁺/S

/

Z

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⁺.

10/C1

Ar

H

Ar

H

OH

H

HO

H

Ar

si-face

attack

-H₂O

8l/C1

from

R

N

H

N

H

-H₂O

N

-H⁺

H

8a-m/C1

NH₂

+H₂O

NH₂

NH

+H⁺

NH

14⁺a-m/E

13⁺a-m/R

12⁺a-m/R

+H⁺

-H⁺

H

7⁺a-m

O

Ar

H

si-face

Ar

H

N

attack

Ar

NH₂

N

H

N

H

N H

H

N

Q

5

H

N

re-face

attack

H

16⁺a-m

17h

17i:

: Q=H

Q=F

15⁺a-m

H

7⁺a-m

O

+H⁺

-H⁺

Ar

H

OH

H

HO

Ar

S

reface

N

H

attack

N

H

N

H

-H₂O

H

-H⁺

+H⁺

NH₂

8a-m/T1

NH₂

NH

+H₂O

NH

14⁺a-m/Z

13⁺a-m/S

12⁺a-m/S

Although all intermediates are racemic mixtures, for simplicity - focusing on the relative configurations - one enantiomer is

presented for each structure formulated in this Scheme. For designation of Ar substituent by a-m: see Schemes 1 and 2.

Scheme 5. Possible pathways with elementary steps suitable for the interpretation of the experimentally

observed substituent-controlled aldehyde-mediated diastereoselective transformations of into

C1 as suggested by comparative

5

pentacyclic products (8/C1 and 8/T1) and hexacyclic lactam 10/

DFT modelling.

Molecules 2020, 25, 1599

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In order to check the plausibility of this complex mechanism, comparative DFT studies

12⁺a

S,

, respectively) and isomer iminium cations

, a set of reasonably selected models containing substituents of markedly diﬀerent electronic

were carried out on diastereomer pairs of adducts and iminium ions (12⁺a

,d,

f,

g/

R

_

,d,g/

13⁺a

,

d

,f,g/R_ ,d,f,g/S ,d,g/E_ ,d,f,g/Z

13⁺a and 14⁺a 14⁺a

15⁺a

,f,g

properties and steric bulk (Table 3).

Table 3. Relative stability of selected isomer pairs to rationalize of the substituent- and time-dependent

interconversion of cis- and trans-pentacycles 8/C1 and 8/T1, as resulted from DFT modeling studies.

Relative Stability of Intermediate Pairs Expressed in ∆G [kcal/mol]

Diastereoselectivity of primary

coupling to 12⁺

12⁺a/R−12⁺a/S

12⁺d/R−12⁺d/S

12⁺f/R−12⁺f/S

12⁺g/R−12⁺g/S

−0.68

+0.17

−0.70

−0.97

Diastereoselectivity of primary

13⁺a/R−13⁺a/S

−0.21

13⁺d/R−13⁺d/S

+2.83

13⁺f/R−13⁺f/S

−3.41

13⁺g/R−13⁺g/S

−0.92

coupling to 13⁺

13⁺a/R−12⁺a/R

−1.48

13⁺d/R−12⁺d/R

+0.79

13⁺f/R−12⁺f/R

−2.21

13⁺g/R−12⁺g/R

−1.77

Readiness of rotation 12⁺/R→13⁺/R ^a

Readiness of rotation 12⁺/S→13⁺/S ^a

13⁺a/S−12⁺a/S

−1.95

13⁺d/S−12⁺d/S

−1.87

13⁺f/S−12⁺f/S

+0.50

13⁺g/S−12⁺g/S

−1.82

Readiness of isomerisation

15⁺a−14⁺a/E

+2.42

15⁺d−14⁺d/E

+1.04

15⁺f−14⁺f/E

+10.24

15⁺g−14⁺g/E

+13.19

14⁺/E→15⁺

Readiness of isomerisation

15⁺→14⁺/Z

14⁺a/Z−15⁺a

−1.61

14⁺d/Z−15⁺d

+0.86

14⁺f/Z−15⁺f

−8.06

14⁺g/Z−15⁺g

−12.04

Preference of isomerization pathway

via 15⁺over that via 16⁺

a

16⁺a−14⁺a

+46.10 ^b/+48.02 ^c

16⁺d−14⁺d

16⁺f−14⁺f

+50.63 ^b/+52.24 ^c

16⁺g−14⁺g

+51.93 ^b/+53.35 ^c

+35.54 ^b/+36.76 ^c

The value can also be considered as a selectivity descriptor for the alternative primary coupling modes

5

+

7⁺→12⁺

b

c

and

5+

7⁺→13⁺disclosing the latter as the preferred one (highlighted by italics). Refers to 16⁺−

1

4⁺/Z

.

Refers to

16⁺−14⁺/E.

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

while the reactions eﬀected by benzaldehyde 7a, and aldehydes 7f

group seem to proceed via intermediates 12⁺and 13⁺

leading to 8a,f,g/C1. On the other hand,

/

T1, the more stable diastereomer that can thus be regarded as the thermodynamic product,

,g

carrying electron-donating

/

R

/

R

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-1⁰atom 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

Molecules 2020, 25, 1599

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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).

13⁺d/S

HOMO-8 (13⁺d/S)

HOMO-11 (13⁺d/S)

Figure 3. Optimized structure 13⁺d/S and its selected MO’s involved in intramolecular interactions

contributing to the stability of this cationic intermediate.

2.4. In vitro Cytotoxic Activity of the Novel Polycyclic β-Carbolines 8/C1, 8/T1, 9/T1, 10/C1 and 11/C1

The in vitro cytotoxicity of the polycondensed β-carboline products expressed in IC₅₀values 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).

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 IC₅₀values in the low micromolar range. It is of note that while trans

diastereomers carrying phenyl- and 3-triﬂuoromethylphenyl substituents (8a

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

/T1 and 8b/T1) display

/

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 IC₅₀values measured for 8c

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 (IC₅₀= 2.1–5.2 M on the investigated cell

lines) and its inactive trans counterpart 8f T1 both carrying 3,4,5-trimethoxyphenyl group on ring D.

/T1 and by the marked diﬀerence in

/

µ

/

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

Molecules 2020, 25, 1599

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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.

Table 4. In vitro cytotoxic eﬀect of novel polycondensed β

-carbolines types 8–11 ^a, on four human

malignant cell lines characterised by half-maximal inhibitory concentration (IC₅₀) values.

IC₅₀[M]

PANC-1

COLO-205

A2058

EBC-1

7.0 × 10⁻⁶± 1.9 × 10⁻⁶

5.6 × 10⁻⁶± 5.6 × 10⁻⁷

1.4 × 10⁻⁵± 3.2 × 10⁻⁶

6.4 × 10⁻⁶± 1.1 × 10⁻⁶

8a/T1

9a/T1

8b/T1

9b/T1

8c/T1

9c/T1

8d/T1

9d/T1

8e/T1

> 10⁻⁴

2.8 × 10⁻⁶± 1.0 × 10⁻⁶

> 10⁻⁴

1.5 × 10⁻⁶± 5.9 × 10⁻⁷

> 10⁻⁴

2.5 × 10⁻⁶± 4.0 × 10⁻⁷

> 10⁻⁴

4.7 × 10⁻⁶± 4.1 × 10⁻⁷

> 10⁻⁴

2.3 × 10⁻⁵± 1.1 × 10⁻⁶

2.5 × 10⁻⁵± 1.2 × 10⁻⁶

> 5.0 × 10⁻⁵

5.0 × 10⁻⁵± 1.9 × 10⁻⁶

2.5 × 10⁻⁵± 9.5 × 10⁻⁷

2.5 × 10⁻⁵± 5.8 × 10⁻⁷

> 5.0 × 10⁻⁵

2.6 × 10⁻⁵± 1.2 × 10⁻⁶

5.2 × 10⁻⁶± 2.3 × 10⁻⁶

> 5.0 × 10⁻⁵

2.5 × 10⁻⁶± 9.4 × 10⁻⁸

2.1 × 10⁻⁶± 1.9 × 10⁻⁷

3.6 × 10⁻⁵± 1.2510⁻⁶

2.6 × 10⁻⁵± 1.6 × 10⁻⁶

> 10⁻⁴

4.1 × 10⁻⁶± 7.8 × 10⁻⁷

1.6 × 10⁻⁵± 3.0 × 10⁻⁶

1.1 × 10⁻⁵± 5.2 × 10⁻⁶

> 10⁻⁴

8f/C1

8f/C1/T1 ^b

9f/T1

> 5.0 × 10⁻⁵

1.3 × 10⁻⁵± 1.2 × 10⁻⁵

> 10⁻⁴

2.0 × 10⁻⁵± 7.6 × 10⁻⁷

> 10⁻⁴

8g/C1

8g/C1/T1 ^c

9g/T1

> 5.0 × 10⁻⁵

3.9 × 10⁻⁵± 1.1 × 10⁻⁶

5.0 × 10⁻⁵± 5.4 × 10⁻⁶

2.3 × 10⁻⁵± 1.1 × 10⁻⁶

> 5.0 × 10⁻⁵

8h/C1/T1 ^d

9h/T1

> 5.0 × 10⁻⁵

4.7 × 10⁻⁵± 2.1 × 10⁻⁶

> 5.0 × 10⁻⁵

8i/C1/T1 ^e

8j/T1

> 5.0 × 10⁻⁵

1.4 × 10⁻⁵± 3.2 × 10⁻⁶

5.5 × 10⁻⁶± 1.9 × 10⁻⁷

> 5.0 × 10⁻⁵

8k/T1

8m*/C1

10/C1

> 5.0 × 10⁻⁵

3.8 × 10⁻⁵± 8.8 × 10⁻⁷

> 10⁻⁴

3.2 × 10⁻⁵± 2.5 × 10⁻⁵

> 10⁻⁵

> 10⁻⁴

2.9 × 10⁻⁵± 2.4 × 10⁻⁶

> 5.0 × 10⁻⁵

5.6 × 10⁻⁶± 1.3 × 10⁻⁷

2.1 × 10⁻⁵± 1.5 × 10⁻⁶

11/C1

^aCompounds 8a

and 11

–g,j,k/T1, 8f,g/C1 and 10/C1 were measured in racemic form, while compounds 8m*/C1, 9a–d,f–h/T1

b

/

C1 were tested as single enantiomers. Approximately 1:4 mixture of cis (C1) and trans (T1) diastereomers.

c

d

Approximately 3:7 mixture of cis (C1) and trans (T1) diastereomers. Approximately 4:3 mixture of cis (C1) and

trans (T1) diastereomers. Approximately 1:1 mixture of cis (C1) and trans (T1) diastereomers.

e

3. 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 ¹H- and ¹³C-NMR spectra were recorded in DMSO-d₆solution in 5 mm

tubes at room temperature (RT), on a Bruker DRX-500 spectrometer (Bruker Biospin, Karlsruhe, Baden

Württemberg, Germany) at 500 (¹H) and 125 (¹³C) MHz, with the deuterium signal of the solvent as the

lock and TMS as internal standard (¹H, ¹³C). 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 ¹H- and ¹³C-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,

Molecules 2020, 25, 1599

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Germany) using Cu -K

α

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.

3.1. (±)-1-(2-Nitrophenyl)-2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]indole (3)

Tryptamine (3.20 g, 20 mmol), 2-nitrobenzaldehyde (3,03 g, 20 mmol) and boric acid (1,24g,

20 mmol) were dissolved in acetic acid (20 mL). The resulting solution was stirred at reﬂux under

argon for 12 h and cooled down to rt. To this cooled reaction mixture stirred intensively, saturated

aqueous Na₂CO₃solution (200 mL) was added in small portions. The precipitated solid was separated

by ﬁltration and recrystallized from EtOH to obtain the product as deep yellow powder. Orange solid.

Yield: 4.98 g (85%). M.p. 95–98 ^◦C. ¹H-NMR (DMSO-d₆): 10.61 (s, 1H, (N)H-9); 7.94 (dd, J = 7.7 Hz

and 1.3 Hz 1H, H-3⁰); 7.54 (td, J = 7.8 Hz and 1.3 Hz, 1H, H-5⁰); 7.50 (td, J = 7.7 Hz and 1.3 Hz, 1H,

H-4⁰); 7.43 (br~d, J~8 Hz, 1H, H-5); 7.26 (br~d, J~8 Hz, 1H, H-8); 7.08 (dd, J = 7.7 Hz and 1.3 Hz, 1H,

H-6⁰); 7.04 (t, J = 7.8 Hz, 1H, H-7); 6.97 (t, J = 7.8 Hz, 1H, H-6); 5.66 (s, 1H, H-1); 2.93 (dt, J = 12.7 Hz

and 5.0 Hz, 1H, H-3_b); 2.80 (ddd, J = 12.7 H, 7.6 Hz and 5.0 Hz, 1H, H-3_a); 2.73–2.62 (m, 2H, H-4_a,4_b).

¹³C-NMR (DMSO-d₆): 149.9 (C-2⁰); 137.5 (C-1⁰); 136.4 (C-8a); 133.8 (C-9a); 133.0 (C-5⁰); 131.1 (C-4⁰);

129.0 (C-6⁰); 127.2 (C-4b); 124.8 (C-3⁰); 121.4 (C-7); 118.8 (C-6); 118.3 (C-5); 111.7 (C-8), 109.8 (C-4a); 51.4

(C-1); 40.5 (C-3); 22.5 (C-4). HRMS exact mass calcd. for C₁₇H₁₆N₃O₂[MH]⁺, requires m/z: 294.12370

found m/z: 294.12312.

3.2. Methyl (1R,3S)-1-(2-Nitrophenyl)-2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]indole-3-Carboxylate (4/T)

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 CH₂Cl₂(4

×

15 mL). The organic phase was dried over

Na₂SO₄and 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 Na₂CO₃solution

(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 CH₂Cl₂-MeOH (80:1) as eluent (R_f= 0.20 for

4/T

and 0.23 for as detected by TLC). (No detectable separation could be observed when more polar

4/C

solvents, e.g., CH₂Cl₂-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: 2.95 g (42%). M.p. 105–107 ^◦C. ¹H-NMR (DMSO-d₆):

10.66 (s, 1H, (N)H-9); 7.93 (dd, J = 7.7 Hz and 1.3 Hz 1H, H-3⁰); 7.52 (br~t, J~8 Hz, 1H, H-5⁰); 7.48 (td,

J = 7.7 Hz and 1.3 Hz, 1H, H-4⁰); 7.44 (br~d, J~8 Hz, 1H, H-5); 7.22 (br~d, J~8 Hz, 1H, H-8); 7.02 (t,

J = 7.8 Hz, 1H, H-7); 6.97–6.93 (m, 2H, H-6,6⁰); 5.81 (s, 1H, H-1); 3.63 (ddd, J = 8.4 Hz, 6.8 Hz and 5.0 Hz,

1H, H-3_a); 3.57 (s, 3H, 3-CO₂CH₃); 3.03 (dd, J = 15.0 Hz and 5.0 Hz, 1H, H-4_a); 2.82 (ddd, J = 15.0 Hz,

8.4 Hz and 1.2 Hz, 1H, H-4_b). ¹³C-NMR (DMSO-d₆): 172.8 (3-

C

O₂CH₃);149.9 (C-2⁰); 136.4 (C-8a); 135.2

(C-9a); 133.3 (C-5⁰); 131.1 (C-4⁰); 129.3 (C-6⁰); 128.0 (C-1⁰); 127.1 (C-4b); 125.1 (C-3⁰); 121.6 (C-7); 119.1

(C-6); 118.2 (C-5); 111.7 (C-8), 107.8 (C-4a); 52.2 (3-CO₂CH₃); 52.1 (C-3); 49.8 (C-1); 25.1 (C-4). HRMS

exact mass calcd. for C₁₉H₁₈N₃O₄[MH]⁺, requires m/z: 352.12918, found m/z: 352.12851.

3.3. Hydrogenation of 1-(2-Nitrophenyl)-2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]indoles 3 and 4/T

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 H₂(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.

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3.3.1. (±)-1-(2-Aminophenyl)-2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]indole (5)

Colourless solid. Yield: 2.16 g (82%). M.p. 123–126 ^◦C.¹H-NMR (DMSO-d₆): 10.61 (s, 1H, (N)H-9);

7.41 (d, J = 7.8 Hz, 1H, H-5); 7.25 (d, J = 7.8 Hz, 1H, H-8); 7.04–6.98 (m, 2H, H-3,7); 6.96 (t, J = 7.8 Hz,

1H, H-6); 6.68–6.65 (m, 2H, H-1,4); 6.49 (t, J = 7.7 Hz, 1H, H-2⁰); 5.25 (br s, 2H, 2⁰-NH₂); 5.21 (s, 1H,

H-1); 3.29 (br~s, 1H, (N)H-2); 2.95 (br s, 2H, H-3_a3_b); 2.69 (br s, 2H, H-4_a4_b); ¹³C-NMR (DMSO-d₆):

147.8 (C-2⁰); 136.3 (C-8a); 135.3 (C-9a); 129.9 (C-6⁰); 128.4 (C-4⁰); 127.4 (C-4b); 125.9 (C-1⁰); 120.8 (C-7);

118.6 (C-6); 117.9 (C-5); 116.2 (C-5⁰); 115.8 (C-3⁰); 111.5 (C-8), 108.7 (C-4a); 51.7 (C-1); 41.5 (C-3); 22.8

(C-4). HRMS exact mass calcd. for C₁₇H₁₈N₃[MH]⁺, requires m/z: 264.14952 found m/z: 264.14896.

3.3.2. Methyl (1R,3S)-1-(2-Aminophenyl)-2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]indole-3-Carboxylate

(6/T)

Colourless solid. Yield: 2.41 g (75%). M.p 110–113 ^◦C. ¹H-NMR (DMSO-d₆): 10.69 (s, 1H, (N)H-9);

7.41 (d, J = 7.8 Hz, 1H, H-5); 7.21 (d, J = 7.8 Hz, 1H, H-8); 7.00 (t, J = 7.8 Hz, 1H, H-7); 6.95 (t, J = 7.5 Hz,

1H, H-4⁰); 6.93 (t, J = 7.88 Hz, 1H, H-6); 6.65 (d, J = 7.6 Hz, 1H, H-3⁰); 6.43–6.38 (m, 2H, H-5⁰,6⁰); 5.28 (s,

1H, H-1); 5.22 (br s, 2H, 2⁰-NH₂); 3.60 (s, 3H, 3-CO₂CH₃); 3.57 (br ~s, 1H, H-3); 3.09 (br s, 1H, (N)H-2);

2.98 (dd, J = 15.1 Hz and 5.0 Hz, 1H, H-4_a); 2.79 (dd, J = 15.1 Hz and 9.1 Hz, 1H, H-4_b). ¹³C-NMR

(DMSO-d₆):174.2 (3-C

O₂CH₃); 147.7 (C-2⁰); 136.4 (C-8a); 134.5 (C-9a); 129.4 (C-6⁰); 128.5 (C-4⁰); 127.1

(C-4b); 125.9 (C-1⁰); 121.3 (C-7), 118.8 (C-6); 118.0 (C-5); 116.2 (C-5⁰); 115.9 (C-3⁰); 111.5 (C-8); 107.6

(C-4a); 52.3 (C-3); 52.1 (3-CO₂CH₃); 51.7 (C-1); 25.7 (C-4). HRMS exact mass calcd. for C₁₉H₂₀N₃O₂

[MH]⁺, requires m/z: 322.15500 found m/z: 322.15441.

3.4. Cyclization Reactions of 1-(2-Aminophenyl)-2,3,4,9-Tetrahydro-1H-Pyrido[3,4-b]indoles 5 and 6/T

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 Na₂CO₃solution (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

NaHCO₃in 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 CH₂Cl₂and 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.

3.4.1. (6R*,14bS*)-6-Phenyl-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazoline (8a/T1)

Colourless solid. Yield: 0.260 g (37%) with 0.5 h reaction time; 0.556 g (77%) with 12 h reaction

time. M.p. 153–156 ^◦C. ¹H-NMR (DMSO-d₆): 10.99 (s, 1H, (N)H-14); 7.46 (d, J = 7.4 Hz, 2H, H-2⁰,6⁰);

7.34 (t, J = 7.4 Hz, 2H, H-3⁰,5⁰); 7.26 (t, J = 7.4 Hz, 1H, H-4⁰); 7.39 (d, J = 7.8 Hz, 1H, H-10); 7.30 (d,

J = 7.8 Hz, 1H, H-13); 7.03 (t, J = 7.8 Hz, 1H, H-12); 7.00 (d, J = 7.5 Hz, 1H, H-1); 6.98 (t, J = 7.5 Hz, 1H,

H-3); 6.95 (t, J = 7.8 Hz, 1H, H-11); 6.78 (d, J = 3.4 Hz, 1H, H-5); 6.71 (br~d, J~8 Hz, 1H, H-4); 6.45 (t,

J = 7.6 Hz, 1H, H-2); 5.28 (d, J = 3.4 Hz, 1H, H-6); 4.66 (br s, 1H, H-14b); 3.19 (dd, J = 10.9 Hz and 5.9

Hz, 1H, H-8_b); 2.97 (td, J = 10.9 Hz and 3.6 Hz, 1H, H-8_a); 2.89 (ddd, J = 15.1 Hz, 10.9 Hz and 5.9 Hz,

1H, H-9_b); 2.68 (dd, J = 14.1 Hz and 3.6 Hz, 1H, H-9_a). ¹³C-NMR (DMSO-d₆): 144.0 (C-1⁰); 142.0 (C-4a);

136.3 (C-13a); 134.9 (C-14a); 128.7 (C-3⁰,5⁰); 128.0 (C-1); 127.9 (C-3); 127.8 (C-4⁰); 127.5 (C-2⁰,6⁰); 127.0

(C-9b); 121.1 (C-12), 119.3 (C-14c); 118.8 (C-11); 118.2 (C-10); 115.9 (C-2); 113.6 (C-4); 111.5 (C-13); 106.5

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(C-9a); 72.6 (C-6); 49.9 (C-14b); 46.6 (C-8); 23.2 (C-9). HRMS exact mass calcd. for C₂₄H₂₂N₃[MH]⁺,

requires m/z: 352.18082, found m/z: 352.18022.

3.4.2.

(6R*,14bS*)-6-(3-Triﬂuoromethylphenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]

quinazoline (8b/T1)

Colourless solid. Yield: 0.630 g (75%) with 1 h reaction time. 0.604 g (72%) with 12 h reaction

time. M.p. 253–255 ^◦C. ¹H-NMR (DMSO-d₆): 10.96 (s, 1H, (N)H-14); 7.75–7.71 (overlapping m’s, 2H,

H-2⁰,6⁰); 7.61 (d, J = 7.6 Hz, 1H, H-4⁰); 7.56 (t, J = 7.6 Hz, 1H, H-5⁰); 7.36 (d, J = 7.8 Hz, 1H, H-10); 7.26

(d, J = 7.8 Hz, 1H, H-13); 7.01 (t, J = 7.8 Hz, 1H, H-12); 6.98–6.94 (overlapping m’s, 2H, H-1,3); 6.92 (t,

J = 7.8 Hz, 1H, H-11); 6.86 (d, J = 3.7 Hz, 1H, H-5); 6.70 (d, J = 7.6 Hz, 1H, H-4); 6.43 (td, J = 7.6 Hz

and 1.2 Hz, 1H, H-2); 5.35 (d, J = 3.7 Hz, 1H, H-6); 4.56 (br s, 1H, H-14b); 3.18 (m, 1H, H-8_b); 2.96–2.87

(m, 3H H-8_a,9_a,9_b). ¹³C-NMR (DMSO-d₆): 145.6 (C-1⁰); 141.4 (C-4a); 136.3 (C-13a); 134.6 (C-14a); 131.9

(C-6⁰); 130.0 (C-5⁰); 129.5 (qa, J = 30.8 Hz, C-3⁰); 128.1 (C-1); 128.0 (C-3); 127.1 (qa, J = 272.5 Hz,

CF₃);

126.9 (C-9b); 124.7 (C-4⁰); 124.0 (C-2⁰); 121.2 (C-12), 119.3 (C-14c); 118.9 (C-11); 118.3 (C-10); 116.3 (C-2);

113.8 (C-4); 111.5 (C-13); 106.6 (C-9a); 72.2 (C-6); 49.9 (C-14b); 46.5 (C-8); 22.1 (C-9 HRMS exact mass

calcd. for C₂₅H₂₁F₃N₃[MH]⁺, requires m/z: 420.16766 found m/z: 420.16755.

3.4.3.

(6R*,14bS*)-6-[3,5-Bis(triﬂuoromethyl)phenyl]-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido-

[1,2-c]quinazoline (8c/T1)

Colourless solid. Yield: 0.770 g (79%) with 1 h reaction time. 0.741 g (76%) with 12 h reaction

time. M.p. 247–250 ^◦C. ¹H-NMR (DMSO-d₆): 10.96 (s, 1H, (N)H-14); 8.04 (br s, 2H, H-2⁰,6⁰); 8.02 (br s,

1H, H-4⁰); 7.36 (d, J = 7.8 Hz, 1H, H-10); 7.28 (d, J = 7.8 Hz, 1H, H-13); 7.02 (br~t, J~8 Hz, 1H, H-12);

6.99–6.95 (overlapping m’s, 3H, H-1,3,5); 6.93 (br~t, J~8 Hz, 1H, H-11); 6.74 (br~d, J~8 Hz, 1H, H-4);

6.46 (td, J = 7.8 Hz and 1.2 Hz, 1H, H-2); 5.48 (d, J = 3.2 Hz, 1H, H-6); 4.54 (br s, 1H, H-14b); 3.21 (m,

1H, H-8_b); 2.96–2.87 (m, 3H H-8_a,9_a,9_b). ¹³C-NMR (DMSO-d₆): 147.9 (C-1⁰); 141.0 (C-4a); 136.3 (C-13a);

134.2 (C-14a); 130.8 (qa, J = 32.6 Hz, C-3⁰,5⁰); 128.4 (br, C-2⁰,6⁰); 128.3 (C-3); 128.1 (C-1); 126.9 (C-9b); 123.

8 (qa, J = 273.6 Hz, 3⁰,5⁰- F₃); 121.9 (br, C-4⁰); 121.2 (C-12), 119.3 (C-14c); 118.9 (C-11); 118.3 (C-10);

C

116.7 (C-2); 114.0 (C-4); 111.5 (C-13); 106.6 (C-9a); 71.7 (C-6); 49.8 (C-14b); 46.5 (C-8); 22.0 (C-9). HRMS

exact mass calcd. for C₂₆H₂₀F₆N₃[MH]⁺, requires m/z: 488.15559, found m/z: 488.15512.

3.4.4. (6R*,14bS*)-6-(4-Nitromethylphenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-

quinazoline (8d/T1)

Light orange solid. Yield: 0.587 g (74%) with 1 h reaction time. 0.618 g (78%) with 12 h reaction

time. M.p. 292–295 ^◦C. ¹H-NMR (DMSO-d₆): 10.94 (s, 1H, (N)H-14); 8.19 (d, J = 8.8 Hz, 2H, H-3⁰,5⁰);

7.69 (d, J = 8.8 Hz, 2H, H-2⁰,6⁰); 7.36 (d, J = 7.8 Hz, 1H, H-10); 7.26 (d, J = 7.8 Hz, 1H, H-13); 7.01 (t,

J = 7.8 Hz, 1H, H-12); 6.98–6.94 (overlapping m’s, 2H, H-1,3); 6.92 (t, J = 7.8 Hz, 1H, H-11); 6.88 (d,

J = 3.9 Hz, 1H, H-5); 6.71 (dd, J = 7.6 Hz and 1.2 Hz, 1H, H-4); 6.43 (td, J = 7.6 Hz and 1.2 Hz, 1H, H-2);

5.38 (d, J = 3.7 Hz, 1H, H-6); 4.54 (br s, 1H, H-14b); 3.20 (dd, J = 10.2 Hz and 4.6 Hz, 1H, H-8_b); 2.93

(td, J = 10.2 Hz and 4.0 Hz, 1H, H-8_a); 2.97–2.84 (m, 2H H-9_a,9_b). ¹³C-NMR (DMSO-d₆): 151.7 (C-1⁰);

147.4 (C-4⁰); 141.3 (C-4a); 136.5 (C-13a); 134.5 (C-14a); 129.0 (C-2⁰,6⁰); 128.1 (C-3); 128.0 (C-1); 127.0

(C-9b); 124.1 (C-3⁰,5⁰); 121.2 (C-12), 119.3 (C-14c); 119.0 (C-11); 118.3 (C-10); 116.4 (C-2); 113.9 (C-4);

111.5 (C-13); 106.6 (C-9a); 72.4 (C-6); 60.2 (C-14b); 46.7 (C-8); 22.3 (C-9). HRMS exact mass calcd. for

C₂₄H₂₁N₄O₂[MH]⁺, requires m/z: 397.16600, found m/z: 397.16542.

3.4.5. (6R*,14bS*)-6-(2-Nitrophenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-

quinazoline (8e/T1)

Yellow solid. Yield: 0.634 g (80%) with 1 h reaction time. 0.595 g (75%) with 12 h reaction time.

M.p. 275–279 ^◦C. ¹H-NMR (DMSO-d₆): 10.97 (s, 1H, (N)H-14); 7.75 (br~d, J~8 Hz, 1H, H-3⁰); 7.62 (t,

J = 7.7 Hz, 1H, H-5⁰); 7.57 (dd, J = 7.7 Hz and 1.7 Hz, 1H, H-6⁰); 7.48 (dt, J = 7.7 Hz and 1.7 Hz, 1H,

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H-4⁰); 7.33 (d, J = 7.8 Hz, 1H, H-10); 7.25 (d, J = 7.8 Hz, 1H, H-13); 7.01 (t, J = 7.8 Hz, 1H, H-12); 6.98 (t,

J = 7.5 Hz, 1H, H-3); 6.94 (d, J = 7.5 Hz, 1H, H-1); 6.91 (t, J = 7.8 Hz,1H, H-11); 6.85 (d, J = 4.1 Hz, 1H,

H-5); 6.73 (d, J = 7.5 Hz, 1H, H-4); 6.43 (t, J = 7.5 Hz, 1H, H-2); 5.84 (d, J = 4.1 Hz, 1H, H-6), 4.31 (br s,

1H, H-14b); 3.21 (m, 1H, H-8_b); 2.96–2.87 (m, 3H H-8_a,9_a,9_b). ¹³C-NMR (DMSO-d₆): 149.4 (C-2⁰); 141.4

(C-4a); 136.9 (C-1⁰); 136.3 (C-13a); 134.0 (C-14a); 132.5 (C-5⁰); 129.4 (C-4⁰); 129.3 (C-6⁰); 128.2 (C-3); 127.8

(C-1); 126.9 (C-9b); 125.2 (C-3⁰); 121.2 (C-12); 119.1 (C-14c); 118.9 (C-11); 118.2 (C-10); 116.6 (C-2); 113.9

(C-4); 111.5 (C-13); 106.6 (C-9a); 69.5 (C-6); 49.7 (C-14b); 46.9 (C-8); 21.9 (C-9). HRMS exact mass calcd.

for C₂₄H₂₁N₄O₂[MH]⁺, requires m/z: 397.16600, found m/z: 397.16590.

3.4.6. (6R*,14bR*)-6-(3,4,5-Trimethoxyphenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]

quinazoline (8f/C1)

◦

1

Colourless solid. Yield: 0.556 g (63%) with 0.5 h reaction time. M.p. 235–238 C. H-NMR

(DMSO-d₆): 11.27 (s, 1H, (N)H-14); 7.35 (d, J = 7.8 Hz, 1H, H-13); 7.31 (d, J = 7.8 Hz, 1H, H-10); 7.20 (d,

J = 7.9 Hz, 1H, H-1); 7.02 (t, J = 7.8 Hz, 1H, H-12); 6.95 (t, J = 7.9 Hz, 1H, H-3); 6.93 (t, J = 7.8 Hz, 1H,

H-11); 6.91 (s, 2H, H-2⁰,6⁰); 6.73 (d, J = 7.9 Hz, 1H, H-4); 6.54 (t, J = 7.9 Hz, 1H, H-2); 6.28 (br s, 1H, H-5);

5.60 (two coalesced lines, 2H, H-6,14b); 3.66 (s, 6H, 3⁰,5⁰-OCH₃); 361 (s, 3H, 4⁰-OCH₃); 2.73 (m, 1H,

H-8_b); 2.60–2.47 (overlapping m’s, 3H, H-8_a9_a,9_b). ¹³C-NMR (DMSO-d₆): 153.2 (C-3⁰,5⁰); 144.0 (C-4a);

139.4 (C-1⁰); 137.2 (C-4⁰); 136.6 (C-13a); 134.9 (C-14a); 127.9 (C-1); 127.5 (C-3); 126.8 (C-9b); 121.4 (C-14c);

121.1 (C-12), 118.8 (C-11); 118.2 (C-10); 117.6 (C-2); 115.6 (C-4); 111.5 (C-13); 107.3 (C-9a); 105.0 (C-2⁰,6⁰);

73.2 (C-6); 60.5 (4⁰-O H₃); 56.4 (3⁰,5⁰-O

C CH₃); 57.5 (C-14b); 38.0 (C-8); 21.9 (C-9). HRMS exact mass

calcd. for C₂₇H₂₈N₃O₃[MH]⁺, requires m/z: 442.212520, found m/z: 442.21261.

3.4.7. (6R*,14bS*)-6-(3,4,5-Trimethoxyphenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]

quinazoline (8f/T1)

Colourless solid. Yield: present in ca. 80% of the isomer mixture 8f

/

T1 and 8f

/

C1 isolated after 8

1

h reaction time with weight of 0.662 g (75%). H-NMR (DMSO-d₆): 10.91 (s, 1H, (N)H-14), 7.35 (d,

J = 7.8 Hz, 1H, H-10); 7.27 (d, J = 7.8 Hz, 1H, H-13); 7.03–6.98 (overlapping m’s, 2H, H-1,12); 6.94 (t,

J = 7.9 Hz, 1H, H-3); 6.91 (t, J = 7.8 Hz, 1H, H-11); 6.74 (s, 2H, H-2⁰,6⁰); 6.68 (d, J = 3.1 Hz, 1H, H-5); 6.65

(d, J = 7.9 Hz, 1H, H-4); 6.42 (t, J = 7.9 Hz, 1H, H-2); 5.14 (d, J = 3.1 Hz, 1H, H-6), 4.74 (br s, 1H, H-14b);

3.63 (s, 6H, 3⁰,5⁰-OCH₃); 3.59 (s, 3H, 4⁰-OCH₃); 3.12 (ddd, J = 10.6 Hz, 5.6 Hz and 2.0 Hz, 1H, H-8_b);

2.92 (td, J = 10.6 Hz, 3.9 Hz, 1H, H-8_a); 2.82 (m, 1H, H-9_b); 2.64 (br~d, J~15 Hz, 1H, H-9_a). ¹³C-NMR

(DMSO-d₆): 153.3 (C-3⁰,5⁰); 141.9 (C-4a); 139.7 (C-1⁰); 137.3 (C-4⁰); 136.5 (C-13a); 135.0 (C-14a); 128.1

(C-3); 128.0 (C-1); 127.2 (C-9b); 121.6 (C-12), 119.4 (C-14c); 119.0 (C-11); 118.4 (C-10); 116.0 (C-2); 113.8

C CH₃); 50.5 (C-14b); 46.7 (C-8);

(C-4); 111.4 (C-13); 106.5 (C-9a); 72.4 (C-6); 60.7 (4⁰-O H₃); 56.5 (3⁰,5⁰-O

21.8 (C-9).

3.4.8. (6R*,14bR*)-6-Ferrocenyl-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazoline

(8g/C1)

1

Yellow solid. Yield: 0.358 g (39%) with 0.5 h reaction time. M.p. 211–215 ^◦C (decomp.). H-NMR

(DMSO-d₆): 11.23 (s, 1H, (N)H-14); 7.33 (d, J = 7.8 Hz, 1H, H-13); 7.27 (d, J = 7.8 Hz, 1H, H-10); 7.15 (d,

J = 7.6 Hz, 1H, H-1); 7.00 (t, J = 7.8 Hz, 1H, H-12); 6.94 (t, J = 7.6 Hz, 1H, H-3); 6.89 (t, J = 7.8 Hz, 1H,

H-1,11); 6.81 (d, J = 7.6 Hz, 1H, H-4); 6.49 (t, J = 7.6 Hz, 1H, H-2); 5.60 (br s, 1H, H-6); 5.53 (br s, 1H, H-5);

5.49 (br s, 1H, H-14b); 4.55 (br s, 1H, H-2⁰); 4.34 (br s, 1H, H-5⁰); 4.22 (s, 5H, ⁵-C₅H₅); 4.19 (br s, 2H,

η

H-3⁰,4⁰); 2.80 (m, 1H, H-8_b); 2.48-2.43 (overlapping m’s, 3H, H-8_b,9_a,9_b). ¹³C-NMR (DMSO-d₆): 143.8

(C-4a); 136.5 (C-13a); 134.9 (C-14a); 127.9 (C-1); 127.4 (C-3); 126.7 (C-9b); 120.9 (C-12); 120.8 (C-14c);

118.6 (C-11); 118.3 (C-10); 115.0 (C-2); 113.5 (C-4); 111.4 (C-13); 107.0 (C-9a); 86.7 (C-1⁰); 71.0 (C-6); 69.2

(

η⁵C₅H₅); 68.4 (C-5⁰); 67.8 (two coalesced lines, C-3⁰,4⁰); 67.0 (C-2⁰); 57.2 (C-14b); 37.4 (C-8); 21.3 (C-9).

-

HRMS exact mass calcd. for C₂₈H₂₆FeN₃[MH]⁺, requires m/z: 460.14706, found m/z: 460.14641.

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3.4.9. (6R*,14bS*)-6-Ferrocenyl-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazoline

(8g/T1)

Yield: present in ca. 70% of the isomer mixture 8g

/

T1 and 8g

/

C1 isolated after 8 h reaction time

1

with weight of 0.477 g (52%). H-NMR (DMSO-d₆): 11.02 (s, 1H, (N)H-14); 7.31 (d, J = 7.8 Hz, 1H,

H-10); 7.24 (d, J = 7.8 Hz, 1H, H-13); 6.99 (t, J = 7.8 Hz, 1H, H-12); 6.95 (d, J = 7.6 Hz, 1H, H-1); 6.91 (d,

J = 7.6 Hz, 1H, H-3); 6.88 (t, J = 7.8 Hz, 1H, H-1,11); 6.67 (d, J = 7.6 Hz, 1H, H-4); 6.45 (d, J = 3.4 Hz, 1H,

H-5); 6.36 (t, J = 7.6 Hz, 1H, H-2); 5.18 (d, J = 3.4 Hz, 1H, H-6); 4.75 (br s, 1H, H-14b); 4.63 (t, J = 2.2 Hz,

1H, H-2⁰); 4.08 (t, J = 2.2 Hz, 1H, H-5⁰); 4.00 (br s, 2H, H-3⁰,4⁰); 4.21 (s, 5H, ⁵-C₅

η H₅); 3.01 (m, 1H, H-8_b);

2.80–2.77 (overlapping m’s, 2H, H-8_b,9_a); 2.56 (m, 1H, H-9_b). ¹³C-NMR (DMSO-d₆): 142.2 (C-4a); 136.3

(C-13a); 135.0 (C-14a); 127.7 (C-1); 127.5 (C-3); 126.9 (C-9b); 120.9 (C-12); 118.9 (C-14c); 118.6 (C-11);

118.3 (C-10); 115.6 (C-2); 113.3 (C-4); 111.2 (C-13); 106.5 (C-9a); 91.5 (C-1⁰); 73.5 (C-2⁰); 70.9 (C-6); 70.1

(C-5⁰); 69.3 (η⁵-C₅H₅); 67.7 (two coalesced lines, C-3⁰,4⁰);49.5 (C-14b); 46.2 (C-8); 21.6 (C-9).

3.4.10.

(6R*,14bR*)-6-(Pyridine-2-yl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazoline

(8h/C1)

Light yellow solid. Yield: present in ca. 60% of the isomer mixture 8h

/

T1 and 8h

/

C1 isolated after

1

0.5 reaction time with weight of 0.508 g (72%). H-NMR (DMSO-d₆): 11.36 (s, 1H, (N)H-14); 8.67 (dd,

J = 4.5 Hz and 1.2 Hz, 1H, H-6⁰); 7.92 (td, J = 8.2 Hz and 1.2 Hz, 1H, H-4⁰); 7.73 (d, J = 8.2 Hz, 1H,

H-3⁰); 7.43 (dd, J = 8.2 Hz and 4.5 Hz, 1H, H-5⁰); 7.41 (d, J = 7.8 Hz, 1H, H-13); 7.36 (d, J = 7.8 Hz, 1H,

H-10); 7.25 (d, J = 7.6 Hz, 1H, H-1); 7.08 (t, J = 7.8 Hz, 1H, H-12); 7.03-6.94 (m, 2H, H-3,11); 6.86 (d,

J = 7.8 Hz, 1H, H-4); 6.57 (t, J = 7.5 Hz, 1H, H-2); 6.38 (br s, 1H, H-5); 5.77 (br s, 1H, H-6), 5.71(br s, 1H,

H-14b); 2.63 (br ~d, J~10 Hz, 1H, H-8_b); 2.56–2.51 (m, 3H, H-8_a,9_a,9_b). ¹³C-NMR (DMSO-d₆): 157.1

(C-2⁰); 146.9 (C-6⁰); 143.3 (C-4a); 137.5 (C-4⁰); 136.3 (C-13a); 135.2 (C-14a); 127.9 (C-1); 127.7 (C-3); 126.9

(C-9b); 123.6 (C-5⁰); 122.4 (C-3⁰); 121.2 (C-12), 120.9 (C-14c); 118.8 (C-11); 118.2 (C-10); 117.3 (C-2); 113.6

(C-4); 111.6 (C-13); 107.0 (C-9a); 73.1 (C-6); 57.7 (C-14b); 38.7 (C-8); 21.9 (C-9). HRMS exact mass calcd.

for C₂₃H₂₁N₄[MH]⁺, requires m/z: 353.17571, found m/z: 353.17607.

3.4.11.

(6R*,14bS*)-6-(Pyridine-2-yl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-Quinazoline

(8h/T1)

Yield: present in ca. 40% of the isomer mixture 8h

/

T1 and 8h

/

C1 isolated after 0.5 reaction time

1

with weight of 0.508 g (72%). H-NMR (DMSO-d₆): 11.00 (s, 1H, (N)H-14); 8.54 (dd, J = 4.6 Hz and

1.2 Hz, 1H, H-6⁰); 7.79 (dd, J = 8.3 Hz and 1.2 Hz, 1H, H-4⁰); 7.49 (d, J = 8.2 Hz, 1H, H-4⁰); 7.36 (d,

J = 7.8 Hz, 1H, H-10); 7.32 (d, J = 7.8 Hz, 1H, H-13); 7.29 (dd, J = 8.3 Hz and 4.6 Hz, 1H, H-5⁰); 7.05 (dd,

J = 7.6 Hz and 1.2 Hz, 1H, H-1); 7.03 (t, J = 7.8 Hz, 1H, H-12); 7.01-6.92 (m, 2H, H-3,11); 6.71 (d, J = 3.6

Hz, 1H, H-5); 6.68 (d, J = 7.9 Hz, 1H, H-4); 6.47 (t, J = 7.5 Hz, 1H, H-2); 5.28 (d, J = 3.6 Hz, 1H, H-6), 4.79

(br s, 1H, H-14b); 3.20 (m, 1H, H-8_b); 2.99 (td, J = 12.4 Hz and 4.0 Hz, 1H, H-8_a); 2.63 (m, 1H, H-9_b); 2.70

(dd, J = 15.5 Hz and 4.0 Hz, 1H, H-9_a). ¹³C-NMR (DMSO-d₆): 162.0 (C-2⁰); 147.2 (C-6⁰); 142.2 (C-4a);

137.1 (C-4⁰); 135.8 (C-13a); 135.2 (C-14a); 128.0 (C-3); 127.8 (C-1); 126.9 (C-9b); 123.2 (C-5⁰); 122.0 (C-3⁰);

121.2 (C-12), 119.3 (C-14c); 118.8 (C-11); 118.3 (C-10); 115.9 (C-2); 113.7 (C-4); 111.6 (C-13); 106.9 (C-9a);

74.3 (C-6); 50.3 (C-14b); 47.2 (C-8); 22.0 (C-9).

3.4.12.

(6R*,14bR*)-6-(3-Fluoropyridine-2-yl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazoline

(8i/C1)

Colourless solid. Yield: present in ca. 50% of the isomer mixture 8i/T1 and 8i/C1 isolated after

0.5 reaction time with weight of 0.593 g (80%). ¹H-NMR (DMSO-d₆): 11.37 (s, 1H, (N)H-14); 8.56

(br~d, J~5 Hz, 1H, H-6⁰); 7.83 (dd, J = 10.2 Hz and 8.2 Hz, 1H, H-4⁰); 7.60 (m, 1H, H-5⁰); 7.40 (d,

J = 7.8 Hz, 1H, H-13); 7.36 (d, J = 7.8 Hz, 1H, H-10); 7.24 (d, J = 7.6 Hz, 1H, H-1); 7.04–6.94 (m, 4H,

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H-1,3,11,12); 6.85 (d, J = 7.9 Hz, 1H, H-4); 6.59 (t, J = 7.6 Hz, 1H, H-2); 6.21 (d, J = 3.4 Hz, 1H, H-5); 6.00

(d, J = 3.4 Hz, 1H, H-6), 5.23 (br s, 1H, H-14b); ~2.5 (overlapped by the CD₂H solvent signal, H-8_b,9_a,9_b);

3.01 (td, J = 11.8 Hz and 4.2 Hz, 1H, H-8_a); 2.84 (ddd, J = 15.5 Hz 11.8 Hz and 6.3 Hz, 1H, H-9_b); 2.66

(dd, J = 15.5 Hz and 4.2 Hz, 1H, H-9_a). ¹³C-NMR (DMSO-d₆): 153.5 (d, J = 258.2 Hz, C-3⁰); 151.2 (d,

J = 11.6 Hz, C-2⁰); 144.2 (d, J = 5.0 Hz, C-6⁰); 142.9 (C-4a); 136.5 (C-13a); 134.7 (C-14a); 128.1 and 128.0

(C-1 and C-3); 126.8 (C-9b); 126.1 (d, J = 4.5 Hz, C-5⁰); 124.8 (d, J = 20.8 Hz, C-5⁰); 121.3 (C-12); 120.5

(C-14c); 118.8 (C-11); 118.2 (C-10); 117.8 (C-2); 116.1 (C-4); 111.5 (C-13); 106.8 (C-9a); 69.0 (C-6); 57.5

(C-14b); 38.3 (C-8); 21.7 (C-9). HRMS exact mass calcd. for C₂₃H₂₀FN₄[MH]⁺, requires m/z: 371.16665,

found m/z: 371.16614.

3.4.13.

(6R*,14bS*)-6-(3-Fluoropyridine-2-yl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-quinazoline

(8i/T1)

Yield: present in ca. 50% of the isomer mixture 8i

/

T1 and 8i

/

C1 isolated after 0.5 reaction time

1

with weight of 0.593 g (80%). H-NMR (DMSO-d₆): 11.13 (s, 1H, (N)H-14); 8.36 (dd, J = 4.6 Hz and

1.2 Hz, 1H, H-6⁰); 7.69 (dd, J = 10.2 Hz and 8.2 Hz, 1H, H-4⁰); 7.42 (m, 1H, H-5⁰); 7.37 (d, J = 7.8 Hz, 1H,

H-10); 7.30 (d, J = 7.8 Hz, 1H, H-13); 7.04 (t, J = 7.8 Hz, 1H, H-12); 7.00–6.93 (m, 3H, H-1,3,11); 6.59 (d,

J = 7.9 Hz, 1H, H-4); 6.51 (d, J=3.0 Hz, 1H, H-5); 6.45 (t, J = 7.5 Hz, 1H, H-2); 5.61 (d, J = 3.0 Hz, 1H,

H-6), 4.97 (br s, 1H, H-14b); 3.20 (dd, J = 11.8 Hz and 6.3 Hz 1H, H-8_b); 3.01 (td, J=11.8 Hz and 4.2

Hz, 1H, H-8_a); 2.84 (ddd, J = 15.5 Hz 11.8 Hz and 6.3 Hz, 1H, H-9_b); 2.66 (dd, J = 15.5 Hz and 4.2 Hz,

1H, H-9_a). ¹³C-NMR (DMSO-d₆): 156.7 (d, J = 258.6 Hz, C-3⁰); 149.5 (d, J = 11.6 Hz, C-2⁰); 144.8 (d,

J = 5.0 Hz, C-6⁰); 142.6 (C-4a); 136.3 (C-13a); 134.6 (C-14a); 127.70 and 127.67 (C-1 and C-3); 126.9 (C-9b);

125.2 (d, J = 4.0 Hz, C-5⁰); 124.2 (d, J = 19.1 Hz, C-5⁰); 121.0 (C-12); 119.3 (C-14c); 118.8 (C-11); 118.2

(C-10); 115.5 (C-2); 113.4 (C-4); 111.4 (C-13); 106.5 (C-9a); 69.1 (C-6); 50.0 (C-14b); 47.7 (C-8); 22.3 (C-9).

3.4.14.

(6R*,14bS*)-6-(3-Bromopyridine-2-yl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazoline

(8j/T1)

Light yellow solid. Yield: 0.736 g (80%) with 1 h reaction time. 0.800 g (87%) with 12 h reaction

time. M.p. 219–223 ^◦C. ¹H-NMR (DMSO-d₆): 11.07 (s, 1H, (N)H-14); 8.45 (dd, J = 4.6 Hz and 1.2 Hz,

1H, H-6⁰); 8.00 (dd, J = 8.3 Hz and 1.2 Hz, 1H, H-4⁰); 7.34 (d, J = 7.8 Hz, 1H, H-10); 7.26 (d, J = 7.8 Hz,

1H, H-13); 7.22 (dd, J = 8.3 Hz and 4.6 Hz, 1H, H-5⁰); 7.00 (t, J = 7.8 Hz, 1H, H-12); 6.95–6.89 (m, 3H,

H-1,3,11); 6.54 (d, J = 7.9 Hz, 1H, H-4); 6.48 (d, J = 3.2 Hz, 1H, H-5); 6.39 (t, J = 7.5 Hz, 1H, H-2); 5.54 (d,

J = 3.2 Hz, 1H, H-6), 4.76 (br s, 1H, H-14b); 3.13 (m, 1H, H-8_b); 2.92 (td, J = 12.2 Hz and 4.0 Hz, 1H,

H-8_a); 2.84 (ddd, J = 15.5 Hz 12.2 Hz and 6.4 Hz, 1H, H-9_b); 2.62 (dd, J = 15.5 Hz and 4.0 Hz, 1H, H-9_a).

¹³C-NMR (DMSO-d₆): 158.5 (C-2⁰); 147.6 (C-6⁰); 142.7 (C-4a); 141.7 (C-4⁰); 136.3 (C-13a); 134.5 (C-14a);

127.8 (C-3); 127.6 (C-1); 126.9 (C-9b); 124.9 (C-5⁰); 121.1 (C-12), 120.5 (C-3⁰); 119.2 (C-14c); 118.9 (C-11);

118.2 (C-10); 115.2 (C-2); 113.3 (C-4); 111.4 (C-13); 106.7 (C-9a); 73.0 (C-6); 49.6 (C-14b); 47.8 (C-8); 22.4

(C-9). HRMS exact mass calcd. for C₂₃H₂₀BrN₄[MH]⁺, requires m/z: 431.08658, found m/z: 431.08596.

3.4.15.

(6R*,14bS*)-6-[3,5-Diﬂuorophenyl]-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazoline

(8k/T1)

Colourless solid. Yield: 0.535 g (69%) with 1 h reaction time. 0.550 g (71%) with 12 h reaction

time. M.p. 208–211 ^◦C. ¹H-NMR (DMSO-d₆): 11.03 (s, 1H, (N)H-14); 7.34–7.30 (overlapping m’s, 2H,

H-6⁰,10); 7.27 (d, J = 7.8 Hz, 1H, H-13); 7.17 (ddd, J = 11.1 Hz, 10.6 Hz and 2.3 Hz, 1H, H-3⁰); 7.04 (td,

J = 8.2 Hz, 2.3 Hz, 1H, H-5⁰); 7.01-6.96 (overlapping m’s, 2H, H-1,12); 6.91 (t, J = 7.8 Hz, 1H, H-11); 6.65

(br~d, J~4 Hz, 1H, H-5); 6.63 (d, J = 7.7 Hz, 1H, H-4); 6.48 (t, J = 7.7 Hz, 1H, H-2); 5.45 (br~d, J~4 Hz,

1H, H-6); 4.68 (br s, 1H, H-14b); 3.12 (dd, J = 11.4 Hz,5.6 Hz, 1H, H-8_b); 2.89 (dd, J = 11.4 Hz, 3.9 Hz,

1H, H-8_a); 2.77 (m, 1H, H-9_b); 2.60 (dd, J = 15.4 Hz and 3.9 Hz, 1H, H-9_a). ¹³C-NMR (DMSO-d₆): 162.3

(dd, J = 243.3 Hz, 14.3 Hz, C-4⁰); 159.9 (dd, J = 250.4 Hz, 12.2 Hz, C-2⁰); 141.9 (C-4a); 136.3 (C-13a);

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134.5 (C-14a); 130.0 (dd, J = 9.8 Hz, 5.6 Hz, C-6⁰); 128.2 (C-3); 128.0 (C-1); 127.3 (dd, J = 12.4 Hz, 3.3 Hz,

C-1⁰); 126.9 (C-9b); 121.2 (C-12); 118.9 (two coalesced lines, C-11,14c); 118.2 (C-10); 116.3 (C-2); 113.5

(C-4); 111.5 (C-13); 111.1 (dd, J = 20.8 Hz, 3.0 Hz, C-5⁰); 106.5 (C-9a); 104.7 (t, J = 26.6 Hz, C-3⁰); 67.8

(C-6); 50.0 (C-14b); 47.1 (C-8); 22.2 (C-9). HRMS exact mass calcd. for C₂₄H₂₀F₂N₃[MH]⁺, requires m/z:

388.16198, found m/z: 388.16146.

3.4.16.

2-(6S,14bS,S_p)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]quinazolin-6-yl)-ferrocene-1-

carboxylic acid (8m*/C1)

Light orange solid. Yield: 0.473 g (47%) with 5 h reaction time. 0.080 g (8%) with 12 h reaction

time. M.p. 215–218 ^◦C. ¹H-NMR (DMSO-d₆): 11.44 (s, 1H, (N)H-14); 7.38 (d, J = 7.8 Hz, 1H, H-13); 7.31

(d, J = 7.8 Hz, 1H, H-10); 7.24 (d, J = 7.5 Hz, 1H, H-1); 7.06 (t, J = 7.8 Hz, 1H, H-12); 7.03 (t, J = 7.5 Hz,

1H, H-3); 6.94–6.92 (overlapping m’s, 2H, H-4,11); 6.62 (t, J = 7.5 Hz, 1H, H-2); 6.25 (br s, 1H, H-5); 6.23

(br s, 1H, H-6); 5.78 (br s, 1H, H-14b); 4.89 (br s, 1H, H-5⁰); 4.83 (br s, 1H, H-3⁰); 4.48 (~t, J~2 Hz, 1H,

H-4⁰4.27 (s, 5H,

η

⁵-C₅H₅); 2.72 (m, 1H, H-8_b); 2.60–2.51 (overlapping m’s 3H, H-8_a,9_a,9_b); ¹³C-NMR

O₂H); 142.6 (C-4a); 136.8 (C-13a); 132.8 (C-14a); 128.0 (two coalesced lines,

(DMSO-d₆): 171.8 (2⁰-

C

C-1,3); 126.5 (C-9b); 121.6 (C-12), 119.8 (C-14c); 119.1 (C-11); 118.4 (C-10); 118.2 (C-2); 115.9 (C-4); 111.7

-C

(C-13); 106.6 (C-9a); 83.4 (C-1⁰); 74.2 (C-3⁰); 73.6 (C-2⁰); 71.4 (η⁵₅H₅); 70.5 (C-5⁰); 70.3 (C-6); 69.3 (C-4⁰);

55.9 (C-14b); 20.6 (C-9).

3.4.17.

Methyl-(6S,8S,14bR)-6-Phenyl-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-Quinazoline-8-

Carboxylate (9a/T1)

◦

1

Colourless solid. Yield: 0.525 g (64%) with 2 h reaction time. M.p. 180–183 C. H-NMR

(DMSO-d₆): 10.98 (s, 1H, (N)H-14); 7.43 (d, J = 7.4 Hz, 2H, H-2⁰,6⁰); 7.32 (t, J = 7.4 Hz, 2H, H-3⁰,5⁰); 7.24

(t, J = 7.4 Hz, 1H, H-4⁰); 7.36 (d, J = 7.8 Hz, 1H, H-10); 7.27 (d, J = 7.8 Hz, 1H, H-13); 7.03 (t, J = 7.8 Hz,

1H, H-12); 7.01 (d, J = 7.5 Hz, 1H, H-1); 6.97 (t, J = 7.5 Hz, 1H, H-3); 6.93 (t, J = 7.8 Hz, 1H, H-11); 6.72

(d, J = 3.6 Hz, 1H, H-5); 6.68 (br~d, J~8 Hz, 1H, H-4); 6.43 (t, J = 7.6 Hz, 1H, H-2); 5.27 (d, J = 3.6 Hz,

1H, H-6); 4.81 (br s, 1H, H-14b); 3.73 (s, 3H, 8-CO₂CH₃); 2.98 (br~d, J~8 Hz 2H, H-9_a,9_b). ¹³C-NMR

(DMSO-d₆): 173.8 (8-C

O₂CH₃); 143.4 (C-1⁰); 141.9 (C-4a); 136.3 (C-13a); 134.6 (C-14a); 128.9 (C-3⁰,5⁰);

128.3 (C-3); 128.1 (C-4⁰); 127.9 (C-1); 127.4 (C-2⁰,6⁰); 126.6 (C-9b); 121.4 (C-12), 119.1 (C-11); 118.9 (C-14c);

118.3 (C-10); 116.3 (C-2); 113.6 (C-4); 111.5 (C-13); 104.1 (C-9a); 69.0 (C-6); 57.4 (C-8); 52.6 (8-CO₂CH₃);

49.9 (C-14b); 26.0 (C-9). HRMS exact mass calcd. for C₂₆H₂₄N₃O₂[MH]⁺, requires m/z: 410.18630,

found m/z: 410.18595.

3.4.18.

Methyl-(6S,8S,14bR)-6-(3-Triﬂuoromethylphenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]-Pyrido

[1,2-c]quinazoline-8-Carboxylate (9b/T1)

◦

1

Colourless solid. Yield: 0.669 g (70%) with 2 h reaction time. M. p. 231–234 C. H-NMR

(DMSO-d₆): 10.97 (s, 1H, (N)H-14); 7.75 (d, J = 7.6 Hz, 1H, H-6⁰); 7.71 (br s, 1H, H-2⁰); 7.63 (d, J = 7.6 Hz,

1H, H-4⁰); 7.63 (d, J = 7.6 Hz, 1H, H-4⁰); 7.59 (t, J = 7.6 Hz, 1H, H-5⁰); 7.37 (d, J = 7.8 Hz, 1H, H-10); 7.28

(d, J = 7.8 Hz, 1H, H-13); 7.03 (t, J = 7.8 Hz, 1H, H-12); 7.00 (dd, J = 8.0 Hz and 7.6 Hz, 1H, H-3); 6.94 (t,

J = 7.8 Hz, 1H, H-11); 6.90 (d, J = 7.6 Hz, 1H, H-1); 6.79 (d, J = 3.7 Hz, 1H, H-5); 6.73 (dd, J = 8.0 Hz

and 1.2 Hz, 1H, H-4); 6.46 (td, J = 7.6 Hz and 1.2 Hz, 1H, H-2); 5.38 (d, J = 3.7 Hz, 1H, H-6); 4.75 (br

s, 1H, H-14b); 3.76 (s, 3H, 8-CO₂CH₃); 3.73 (dd, J = 11.2 Hz and 7.6 Hz, 1H, H-8); 2.97 (~d, J~8 Hz,

2H H-9_a,9_b). ¹³C-NMR (DMSO-d₆): 173.9 (8-

(C-14a); 131.7 (C-6⁰); 130.2 (C-5⁰); 129.7 (qa, J = 31.0 Hz, C-3⁰); 128.4 (C-3); 127.9 (C-1); 126.7 (C-9b);

124.9 (qa, J = 3.5 Hz, C-4⁰); 124.7 (qa, J = 273.0 Hz, F₃); 123.8 (qa, J = 3.8 Hz, C-2⁰); 121.6 (C-12), 119.2

C

O₂CH₃); 145.1 (C-1⁰); 141.3 (C-4a); 136.3 (C-13a); 133.5

C

(C-11); 119.0 (C-14c); 118.3 (C-10); 116.8 (C-2); 113.8 (C-4); 111.6 (C-13); 104.2 (C-9a); 68.6 (C-6); 57.3

(C-8); 52.6 (8-CO₂CH₃); 49.8 (C-14b); 26.3 (C-9). HRMS exact mass calcd. for C₂₇H₂₃F₃N₃O₂[MH]⁺,

requires m/z: 478.17369, found m/z: 478.17327.

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3.4.19.

Methyl-(6S,8S,14bR)-6-[3,5-Bis(triﬂuoromethyl)phenyl]-5,6,8,9,14,14b-Hexahydroindolo-[2⁰,3⁰:3,4]

pyrido[1,2-c] quinazoline-8-Carboxylate (9c/T1)

◦

1

Colourless solid. Yield: 0.677 g (62%) with 2 h reaction time. M.p. 281–284 C. H-NMR

(DMSO-d₆): 10.98 (s, 1H, (N)H-14); 8.05 (br s, 2H, H-2⁰,6⁰); 8.02 (br s, 1H, H-4⁰); 7.37 (d, J = 7.8 Hz, 1H,

H-10); 7.29 (d, J = 7.8 Hz, 1H, H-13); 7.02 (br~t, J~8 Hz, 1H, H-12); 7.01–6.97 (overlapping m’s, 3H,

H-1,3,5); 6.93 (t, J = 7.8 Hz, 1H, H-11); 6.77 (br~d, J~8 Hz, 1H, H-4); 6.46 (td, J = 7.8 Hz and 1.2 Hz, 1H,

H-2); 5.50 (d, J = 3.3 Hz, 1H, H-6); 4.75 (br s, 1H, H-14b); 3.78 (s, 3H, 8-CO₂CH₃); 3.76 (dd, J = 10.9 Hz

and 7.5 Hz, 1H, H-8); 2.97 (~d, J~8 Hz, 2H H-9_a,9_b). ¹³C-NMR (DMSO-d₆): 173.9 (8-

(C-1⁰); 141.0 (C-4a); 136.3 (C-13a); 134.1 (C-14a); 130.7 (qa, J = 32.6 Hz, C-3⁰,5⁰); 128.2 (br, C-2⁰,6⁰); 128.3

(C-3); 128.1 (C-1); 126.8 (C-9b); 123. 7 (qa, J = 273.8 Hz, 3⁰,5⁰- F₃); 121.9 (br, C-4⁰); 121.4 (C-12), 119.2

(C-14c); 119.0 (C-11); 118.3 (C-10); 116.8 (C-2); 113.8 (C-4); 111.6 (C-13); 104.3 (C-9a); 68.5 (C-6); 57.1

(C-8); 52.6 (8-CO₂

H₃); 49.7 (C-14b); 26.3 (C-9). HRMS exact mass calcd. for C₂₈H₂₂F₆N₃O₂[MH]⁺,

CO₂CH₃); 147.5

C

requires m/z: 546.16107, found m/z: 546.16123.

3.4.20.

Methyl-(6S,8S,14bR)-6-(4-Nitrophenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-

Quinazoline-8-Carboxylate (9d/T1)

◦

1

Light oange solid. Yield: 0.709 g (73%) with 2 h reaction time. M.p. 235–237 C. H-NMR

(DMSO-d₆): 11.00 (s, 1H, (N)H-14); 8.22 (d, J = 8.7 Hz, 2H, H-3⁰,5⁰); 7.69 (d, J = 8.7 Hz, 2H, H-2⁰,6⁰); 7.36

(d, J = 7.8 Hz, 1H, H-10); 7.27 (d, J = 7.8 Hz, 1H, H-13); 7.02 (t, J = 7.8 Hz, 1H, H-12); 6.99 (t, J = 7.5

Hz, 1H, H-3); 6.93 (t, J = 7.8 Hz,1H, H-11); 6.88 (d, J = 7.5 Hz, 1H, H-1); 6.87 (d, J= 3.9 Hz, 1H, H-5);

6.73 (d, J = 7.5 Hz, 1H, H-4); 6.45 (t, J = 7.5 Hz, 1H, H-2); 5.42 (d, J = 3.9 Hz, 1H, H-6), 4.71 (br s, 1H,

H-14b); 3.76 (s, 3H, 8-CO₂CH₃); 3.71 (dd, J = 9.3 Hz, 6.1 Hz, 1H, H-8_a); 2.94 (dd, J = 14.9 Hz 9.3 Hz, 1H,

H-9_b); 2.88 (dd, J = 14.9 Hz 6.1 Hz, 1H, H-9_a). ¹³C-NMR (DMSO-d₆): 173.8 (8- O₂CH₃); 151.3 (C-1⁰);

C

147.5 (C-4⁰); 141.1 (C-4a); 136.3 (C-13a); 133.3 (C-14a); 128.8 (C-2⁰,6⁰); 128.4 (C-3); 127.8 (C-1); 126.5

(C-9b); 124.3 (C-3⁰,5⁰); 121.5 (C-12), 119.2 (C-11); 118.9 (C-14c); 118.3 (C-10); 116.8 (C-2); 113.9 (C-4);

111.6 (C-13); 104.2 (C-9a); 68.6 (C-6); 57.3 (C-8); 52.7 (8-CO₂CH₃); 50.0 (C-14b); 26.5 (C-9). HRMS exact

mass calcd. for C₂₆H₂₃N₄O₄[MH]⁺, requires m/z: 455.17138, found m/z: 455.17071.

3.4.21.

Methyl-(6S,8S,14bR)-6-(3,4,5-Trimethoxyphenyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]-pyrido

[1,2-c]quinazoline-8-Carboxylate (9f/T1)

◦

1

Colourless solid. Yield: 0.709 g (71%) with 2 h reaction time. M.p. 240–243 C. H-NMR

(DMSO-d₆): 10.86 (s, 1H, (N)H-14); 7.36 (d, J = 7.8 Hz, 1H, H-10); 7.29 (d, J = 7.8 Hz, 1H, H-13); 7.02

(t, J = 7.8 Hz, 1H, H-12); 6.00-6.95 (overlapping m’s, H-1,3); 6.93 (t, J = 7.8 Hz, 1H, H-11); 6.76 (s,

2H, H-2⁰,6⁰); 6.67 (d, J = 7.6 Hz, 1H, H-4); 6.63 (d, J = 3.0 Hz, H-5); 6.48 (t, J=7.7 Hz, 1H, H-2); 5.14

(d, J = 3.0 Hz, 1H, H-6), 4.95 (br s, 1H, H-14b); 3.71 (dd, J = 8.4 Hz, 5.3 Hz, 1H, H-8_a); 3.70 (s, 3H,

8-CO₂CH₃); 3.65 (s, 6H, 3⁰,5⁰-OCH₃); 3.61 (s, 3H, 4⁰-OCH₃); 2.95 (dd, J = 15.4 Hz 8.4 Hz, 1H, H-9_b);

2.92 (dd, J = 15.4 Hz 5.3 Hz, 1H, H-9_a). ¹³C-NMR (DMSO-d₆): 173.8 (8- O₂CH₃); 153.5 (C-3⁰,5⁰); 137.7

C

(C-4⁰); 142.0 (C-4a); 137.7 (C-4⁰); 136.3 (C-13a); 133.9 (C-14a); 128.2 (C-3); 128.0 (C-1); 126.7 (C-9b); 121.4

(C-12), 119.0 (C-11); 118.8 (C-14c); 118.3 (C-10); 116.5 (C-2); 113.7 (C-4); 111.7 (C-13); 105.1 (C-2⁰,6⁰);

104.8 (C-1⁰); 104.1 (C-9a); 69.1 (C-6); 60.5 (4⁰-O H₃); 56.9 (C-8); 56.5 (3⁰,5⁰-O

C CH₃); 52.7 (8-CO₂CH₃);

50.3 (C-14b); 24.8 (C-9). HRMS exact mass calcd. for C₂₉H₃₀N₃O₅[MH]⁺, requires m/z: 500.21800

found m/z: 500.21791.

3.4.22. Methyl-(6S,8S,14bR)-6-Ferrocenyl-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-

Quinazoline-8-Carboxylate (9g/T1)

◦

1

Light orange solid. Yield: 0.538 g (52%) with 2 h reaction time. M.p. 201–205 C. H-NMR

(DMSO-d₆): 11.12 (s, 1H, (N)H-14); 7.33 (d, J = 7.8 Hz, 1H, H-10); 7.26 (d, J = 7.8 Hz, 1H, H-13); 7.00 (t,

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J = 7.8 Hz, 1H, H-12); 6.95 (t, J = 7.5 Hz, 1H, H-3); 6.90 (br~t, J~8 Hz, 1H, H-11); 6.87 (d, J = 7.5 Hz, 1H,

H-1); 6.78 (d, J = 7.9 Hz, 1H, H-4); 6.58 (d, J = 3.7 Hz, 1H, H-5); 6.39 (t, J = 7.5 Hz, 1H, H-2); 5.30 (d,

J = 3.7 Hz, 1H, H-6), 4.98 (br s, 1H, H-14b); 4.22 (dt, J = 2.3 Hz, 1.5 Hz, 1H, H-2⁰); 4.18 (s, 5H, ⁵-C₅H₅);

η

4.11 (qa, J = 2.3 Hz, 1H, H-5⁰); 4.03 (~t, J~2 Hz, 2H, H-3⁰,4⁰); 3.77 (s, 3H, 8-CO₂CH₃); 3.59 (dd, J = 10.3

Hz, 5.3 Hz, 1H, H-8_a); 2.88 (dd, J = 14.9 Hz 5.3 Hz, 1H, H-9_a); 2.83 (dd, J = 14.9 Hz 10.3 Hz, 1H, H-9_b).

¹³C-NMR (DMSO-d₆): 174.7 (8-

C

O₂CH₃); 142.2 (C-4a); 136.3 (C-13a); 134.3 (C-14a); 128.1 (C-3); 127.7

(C-1); 126.5 (C-9b); 121.4 (C-12), 118.9 (C-11); 118.5 (C-14c); 118.3 (C-10); 116.0 (C-2); 113.4 (C-4); 111.5

(C-13); 104.3 (C-9a); 91.2 (C-1⁰); 69.3 (η⁵₅H₅); 68.9 (C-5⁰); 68.1 and 68.2 (C-3⁰,4⁰); 67.1 (C-6); 66.9 (C-2⁰);

-C

57.2 (C-8); 52.5 (8-CO₂CH₃); 49.6 (C-14b); 26.8 (C-9). HRMS exact mass calcd. for C₃₀H₂₈FeN₃O₂

[MH]⁺, requires m/z: 518.15254, found m/z: 518.15078.

3.4.23. Methyl-(6S,8S,14bR)-6-(pyridine-2-yl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-

Quinazoline-8-Carboxylate (9h/T1)

Yellow solid. Yield: 0.460 g (56%) with 2 h reaction time. M.p. 190–193 ^◦C. ¹H-NMR (DMSO-d₆):

10.91 (s, 1H, (N)H-14); 8.50 (br ~d, J~4 Hz, 1H, H-6⁰); 7.79 (td, J = 7.7 Hz, 1.3 Hz, 1H, H-4⁰); 7.50

(d, J = 7.8 Hz, 1H, H-3⁰) 7.36 (d, J = 7.8 Hz, 1H, H-10); 7.30-7.27 (overlapping m’s, H-5⁰,13); 7.02 (t,

J = 7.8 Hz, 1H, H-12); 6.99-6.95 (overlapping m’s, H-1,3); 6.93 (t, J = 7.8 Hz, 1H, H-11); 6.63 and 6.62

(partly overlapping d’s, J = 7.6 Hz and 3.0 Hz, resp., 2

5.24 (d, J = 3.0 Hz, 1H, H-6), 4.94 (br s, 1H, H-14b); 3.69 (s, 3H, 8-CO₂C

1H, H-8_a); 2.98 (dd, J = 15.8 Hz 5.6 Hz, 1H, H-9_b); 2.91 (dd, J = 15.8 Hz 8.8 Hz, 1H, H-9_a). ¹³C-NMR

×

1H, H-4 and H-5); 6.47 (t, J = 7.6 Hz, 1H, H-2);

H

₃); 3.68 (dd, J = 8.8 Hz, 5.6 Hz,

(DMSO-d₆): 173.6 (8-C

O₂CH₃); 161.3 (C-2⁰); 149.2 (C-6⁰); 142.4 (C-4a); 137.5 (C-4⁰); 136.3 (C-13a); 133.6

(C-14a); 128.2 (C-3); 127.8 (C-1); 126.6 (C-9b); 123.5 (C-5⁰); 122.5 (C-3⁰); 121.4 (C-12), 119.1 (C-11); 119.0

(C-14c); 118.3 (C-10); 116.3 (C-2); 113.9 (C-4); 111.6 (C-13); 104.1 (C-9a); 70.8 (C-6); 57.6 (C-8); 52.5

(8-CO₂C

H₃); 50.6 (C-14b); 25.3 (C-9). HRMS exact mass calcd. for C₂₅H₂₃N₄O₂[MH]⁺, requires m/z:

411.18155, found m/z: 411.18091.

3.4.24.

2-((6S,8S,14bR,S_p)-8-(methoxycarbonyl)-5,6,8,9,14,14b-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]

quinazolin-6-yl)-Ferrocene-1-Carboxylic acid (9m/T1).

Since upon attempted chromatographic puriﬁcations this compound underwent uncontrolled

1

decomposition, this product was identiﬁed as the major component (ca. 60% by H-NMR) in the

isolated sample (0.250 g) contaminated with undeﬁned components. Due to the presence of these

unidentiﬁed components with uncertain composition, 10–15% can be given as an approximate yield

1

of this unstable product. H-NMR (DMSO-d₆): 11.19 (s, 1H, (N)H-14); 7.36 (d, J = 7.8 Hz, 1H, H-10);

7.28 (d, J = 7.8 Hz, 1H, H-13); 7.04 (t, J = 7.8 Hz, 1H, H-12); 7.01 (d, J = 7.5 Hz, 1H, H-3); 6.96-6.94

(overlapping m’s, 2H, H-1,11); 6.75 (d, J = 7.5 Hz, 1H, H-4); 6.48 (t, J = 7.5 Hz, 1H, H-2); 6.35 (br s, 1H,

H-5); 5.93 (d, J = 3.2 Hz, 1H, H-6); 5.08 (br s, 1H, H-14b); 4.73 (br s, 1H, H-3⁰); 4.34 (~t, J~2 Hz, 1H,

H-4⁰); 4.31 (br s, 1H, H-5⁰); 4.24 (s, 5H,

η

⁵-C₅H₅); 3.80 (s, 3H, 8-CO₂CH₃); 3.70 (dd, J = 8.2 Hz, 5.1 Hz,

O₂CH₃); 172.1

O₂H); 141.2 (C-4a); 136.5 (C-13a); 132.5 (C-14a); 128.6 (C-3); 127.9 (C-1); 126.3 (C-9b); 121.6 (C-12);

119.3 (C-11); 118.4 (C-10); 117.7 (C-14c); 116.7 (C-2); 113.9 (C-4); 111.5 (C-13); 104.3 (C-9a); 73.7 (C-3⁰);

73.2 (C-1⁰); 72.5 (C-2⁰); 71.3 (η⁵C₅H₅); 70.7 (C-5⁰); 69.5 (C-4⁰); 69.2 (C-6); 57.3 (C-8); 52.9 (8-CO₂

H₃);

1H, H-8_a); 2.94-2.89 (overlapping m’s 2H, H-9_a,9_b); ¹³C-NMR (DMSO-d₆): 172.5 (8-

(2⁰-

C

-

C

50.9 (C-14b); 26.3 (C-9). HRMS was not measured for this sample.

3.4.25.

(5bR*,15bS*)-5,5b,17,18-Tetrahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]isoindolo[2,1-a]quinazolin-11(15bH)-

One (10/C1)

◦

1

Colourless solid. Yield: 0.667 g (88%) with 5 h reaction time. M.p. 272–274 C. H-NMR

(DMSO-d₆): 11.47 (s, 1H, (N)H-5); 8.54 (dd, J = 8.2 Hz and 1.1 Hz, 1H, H-9); 7.85 (d, J = 7.6 Hz, 1H,

H-12); 7.74 (t, J = 7.6 Hz, 1H, H-14); 7.69 (d, J = 7.6 Hz, 1H, H-15); 7.64 (t, J = 7.6 Hz, 1H, H-13); 7.56 (d,

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J = 7.6 Hz, 1H, H-6); 7.40 (d, J = 7.8 Hz, 1H, H-4); 7.33 (d, J = 7.8 Hz, 1H, H-1); 7.30 (dd, J = 8.2 Hz

and 7.6 Hz, 1H, H-8); 7.09 (t, J = 7.8 Hz, 1H, H-7); 7.07 (t, J = 7.9 Hz, 1H, H-3); 6.93 (ddd, J = 7.9 Hz,

7.2 Hz and 1.0 Hz, 1H, H-2); 6.32 (s, 1H, H-15b); 5.75 (s, 1H, H-5b); 2.89 (dt, J = 14.9 Hz and 2.8 Hz, 1H,

H-17_a); 2.62 (m, 1H, H-18_b); 2.49 (dt, J = 14.9 Hz and 2.8 Hz, 1H, H-17_b); 2.46 (m, 1H, H-18_a); ¹³C-NMR

(DMSO-d₆): 165.5 (C-11); 140.1 (C-15a); 136.6 (C-4a); 135.1 (C-9a); 133.6 (C-14); 133.5 (two coalesced

lines, C-5a, C-11a); 130.5 (C-13); 128.5 (C-6); 128.1 (C-8); 126.6 (C-18b); 124.4 (C-5c); 124.3 (C-5); 124.2

(C-7); 123.9 (C-12); 121.6 (C-3); 119.1 (C-2); 118.6 (C-1); 118.2 (C-9); 111.7 (C-4); 107.5 (C-18a); 77.0

(C-15b); 57.1 (C-5b); 38.4 (C-17); 21.4 (C-18). HRMS exact mass calcd. for C₂₅H₂₀N₃O [MH]⁺, requires

m/z: 378.16009, found m/z: 378.15966.

3.4.26. Methyl

(5bR,15bS,17S)-11-Oxo-5,5b,11,15b,17,18-Hexahydroindolo[2⁰,3⁰:3,4]pyrido[1,2-c]-isoindolo[2,1-a]

quinazoline-17-Carboxylate (11/C1)

◦

1

Colourless solid. Yield: 0.619 g (71%) with 5 h reaction time. M.p. 239–242 C. H-NMR

(DMSO-d₆): 11.54 (s, 1H, (N)H-5); 8.65 (dd, J = 8.2 Hz and 1.1 Hz, 1H, H-9); 7.79 (dd, J = 7.6 Hz and

1.1 Hz, 1H, H-12); 7.65 (td, J = 7.6 Hz and 1.1 Hz 1H, H-14); 7.60 (d, J = 7.6 Hz, 1H, H-15); 7.58 (td,

J = 7.6 Hz and 1.1 Hz, 1H, H-13); 7.42 (d, J = 8.2 Hz, 1H, H-4); 7.35 (two coalesced d’s, J~8 Hz, 2H,

H-1,6); 7.31 (ddd, J = 8.2 Hz, 7.6 Hz and 1.1 Hz, 1H, H-8); 7.09 (ddd, J = 8.2 Hz, 7.9 Hz and 1.0 Hz, 1H,

H-3); 7.05 (td, J = 7.6 Hz and 1.1 Hz, 1H, H-7); 6.95 (t, J=7.9 Hz, 1H, H-2); 6.48 (s, 1H, H-15b); 5.92 (s,

1H, H-5b); 3.48 (dd, J = 8.9 Hz and 5.2 Hz, 1H, H-17_a); 2.98 (dd, J = 15.4 Hz and 8.9 Hz, 1H, H-18_b);

2.83 (s, 3H, 17-CO₂CH₃); 2.70 (dd, J = 15.4 Hz and 5.2 Hz, 1H, H-18_a). ¹³C-NMR (DMSO-d₆): 172.5

(17-CO₂CH₃); 165.2 (C-11); 139.4 (C-15a); 136.5 (C-4a); 135.5 (C-11a); 133.7 (C-9a); 133.2 (C-5a); 132.4

(C-14); 130.4 (C-13); 128.4 (C-8); 128.2 (C-6); 126.25 (C-18b); 126.20 (C-15); 124.6 (C-5c); 124.1 (C-7); 123.3

(C-12); 121.8 (C-3); 119.3 (C-2); 118.5 (C-1); 117.5 (C-9); 105.1 (C-18a);; 77.1 (C-15b); 58.7 (C-5b); 54.5

(C-17); 51.9 (17-CO₂CH₃); 26.5 (C-18). HRMS exact mass calcd. for C₂₇H₂₂N₃O₃[MH]⁺, requires m/z:

436.16557, found m/z: 436.16519.

4. Conclusions

Using a straightforward three-step reaction sequence comprising Pictet-Spengler annelation

followed by nitro-reduction and aldehyde-mediated cyclization, tryptamine and l-tryptophan were

converted into a series of partly saturated novel polycondensed β-carbolines obtained as racemic

mixtures and single enantiomers, respectively, with well-deﬁned conformations identiﬁed by single

crystal X-ray analysis and NMR measurements. On the basis of the results of comparative DFT

studies, a plausible mechanism was proposed for the ﬁnal cyclization that might account for the

observed stereochemical outcome controlled by the electronic properties and steric bulk of the

reactants. The prepared compounds were evaluated for their antiproliferative/cytotoxic activity

on human malignant cell lines PANC-1, COLO-205, A2058 and EBC-1 disclosing characteristic

structure-activity relationships and cell-selectivity that might be utilized in rational structure-reﬁnement

in the development of more potent antiproliferative agents with enhanced activity and selectivity.

Accordingly, besides the comparative tests of the particular tryptamine-derived enantiomers envisaged

to be available by stereoselective cyclization procedures (using e.g., organocatalysis) or chromatographic

separation, the synthesis and evaluation of the d-tryptophan-derived enantiomers and further models,

with an array of substituents in the diﬀerent regions of the heterocyclic skeletons, will be the subjects

of subsequent papers.

Supplementary Materials: Description of cell culture and viability assays; copies of NMR spectra; details of x-ray

analysis of 9f/T1.

Author Contributions: Conceptualization, F.H., L.K. and A.C., formal analysis, M.S.-E., V.H. and G.S.; funding

acquisition, A.C.; investigation, K.J.F., D.H., T.J., A.T., Z.S., R.O.S. and A.C.; methodology, K.J.F., D.H., T.J., A.T.,

Z.S., M.S.-E., V.H. and R.O.S.; supervision, L.K. and A.C.; validation, V.H., G.S. and L.K.; visualization, A.C.;

writing—original draft, K.J.F. and A.C.; writing—review & editing, F.H., L.K. and A.C. All authors have read and

agreed to the published version of the manuscript.

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Funding: This work was funded by the Hungarian Scientiﬁc Research Fund [OTKA K_129037], by the National

Research, Development and Innovation Oﬃce, Hungary [NVKP_16-1-2016-0036] and by the ELTE Thematic

Excellence Programme supported by the Hungarian Ministry for Innovation and Technology. [SzintPlusz_1117].

G.S was supported by the MTA Premium Post-Doctorate Research Program of the Hungarian Academy of Sciences

(HAS, MTA). Purchase of the UHRMS mass spectrometer was supported by grant (VEKOP-2.3.3-15-2017-00020)

from the European Union and the State of Hungary, co-ﬁnanced by the European Regional Development Fund.

The crystallographic study, as part of research within project No. VEKOP-2.3.3-15-2017-00018 was supported by

the European Union and the State of Hungary, co-ﬁnanced by the European Regional Development Fund.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds reported in this contribution are available from the authors.

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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access

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Article Doi

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

DOI: 10.3390/molecules25071599

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Full text of DOI:10.3390/molecules25071599

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