Synthesis, biological evaluation and molecular modeling of substituted indeno[1,2-b]indoles as inhibitors of human protein kinase CK2

Article abstract of DOI:10.3390/ph8020279

Due to their system of annulated 6-5-5-6-membered rings, indenoindoles have sparked great interest for the design of ATP-competitive inhibitors of human CK2. In the present study, we prepared twenty-one indeno[1,2-b]indole derivatives, all of which were tested in vitro on human CK2. The indenoindolones 5a and 5b inhibited human CK2 with an IC₅₀ of 0.17 and 0.61 μM, respectively. The indeno[1,2-b]indoloquinone 7a also showed inhibitory activity on CK2 at a submicromolar range (IC₅₀ = 0.43 μM). Additionally, a large number of indenoindole derivatives was evaluated for their cytotoxic activities against the cell lines 3T3, WI-38, HEK293T and MEF.

Full text of DOI:10.3390/ph8020279

Pharmaceuticals 2015, 8, 279-302; doi:10.3390/ph8020279
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Article
Synthesis, Biological Evaluation and Molecular Modeling of
Substituted Indeno[1,2-b]indoles as Inhibitors of Human Protein
Kinase CK2
Faten Alchab 1,†, Laurent Ettouati 1,†, Zouhair Bouaziz 1, Andre Bollacke 2, Jean-Guy Delcros 3,
Christoph G.W. Gertzen 4, Holger Gohlke 4, Noël Pinaud 5, Mathieu Marchivie 6, Jean Guillon 7,
Bernard Fenet 8,9, Joachim Jose 2 and Marc Le Borgne 1,*
1
EA 4446 Biomolécules Cancer et Chimiorésistances, SFR Santé Lyon-Est CNRS UMS3453 -
INSERM US7, Faculté de Pharmacie - ISPB, Université Lyon 1, 8 avenue Rockefeller, F-69373,
Lyon Cedex 8, France; E-Mails: fatenalchab@yahoo.com (F.A.); laurent.ettouati@univ-lyon1.fr
(L.E.); zouhair.bouaziz@univ-lyon1.fr (Z.B.)
2
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-
Universität Münster, Corrensstr. 48, 48149 Münster, Germany;
E-Mails: andre.bo@uni-muenster.de (A.B.); joachim.jose@uni-muenster.de (J.J.)
3
Laboratoire Récepteurs à dépendance, UMR INSERM U1052/CNRS 5286, Centre de Recherche en
Cancérologie de Lyon, Centre Léon Bérard, Cheney A, 28 rue Laënnec, F-69008, Lyon, France; E-
Mail: jean-guy.delcros@lyon.unicancer.fr
4
Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf, Germany; E-Mails: christoph.gertzen@uni-duesseldorf.de
(C.G.W.G.); gohlke@uni-duesseldorf.de (H.G.)
5
ISM - CNRS UMR 5255, Université de Bordeaux, 351 cours de la Libération, F-33405 Talence
cedex, France; E-Mail: noel.pinaud@u-bordeaux.fr
6
ICMCB, UPR 9048, CNRS, Université de Bordeaux, 87, avenue du Docteur Schweitzer, F-33600
Pessac, France; E-Mail: mathieu.marchivie@u-bordeaux.fr
7
Laboratoire ARNA, INSERM U869, UFR des Sciences Pharmaceutiques, Université de Bordeaux,
146 rue Léo Saignat, F-33076 Bordeaux cedex, France; E-Mail: jean.guillon@u-bordeaux.fr
8
Centre Commun de RMN, Université de Lyon, F-69003 Lyon, France;
E-Mail: bernard.fenet@univ-lyon1.fr
9
ESCPE Lyon, Université Lyon 1, 43 Bd du 11 Novembre 1918, F-69616 Villeurbanne Cedex, France
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: marc.le-borgne@univ-lyon1.fr;
Tel.: +33-478-777-542; Fax: +33-478-777-082.

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Received: 18 March 2015 / Accepted: 26 May 2015 / Published: 8 June 2015
Abstract: Due to their system of annulated 6-5-5-6-membered rings, indenoindoles have
sparked great interest for the design of ATP-competitive inhibitors of human CK2. In the
present study, we prepared twenty-one indeno[1,2-b]indole derivatives, all of which were
tested in vitro on human CK2. The indenoindolones 5a and 5b inhibited human CK2 with
an IC50 of 0.17 and 0.61 µM, respectively. The indeno[1,2-b]indoloquinone 7a also showed
inhibitory activity on CK2 at a submicromolar range (IC50 = 0.43 µM). Additionally, a large
number of indenoindole derivatives was evaluated for their cytotoxic activities against the
cell lines 3T3, WI-38, HEK293T and MEF.
Keywords: indeno[1,2-b]indoles; synthesis; protein kinase CK2; inhibitory activity;
molecular modeling; cytotoxicity
1. Introduction
Protein kinase CK2 is a ubiquitous serine/threonine kinase found in all eukaryotic cells. Known since
1954 [1], CK2 is a newly validated therapeutic target (e.g., pancreatic adenocarcinoma, breast and
prostate carcinoma, hematopoietic tumors) [2–5], is ideally suited for drug design [6,7], and will be a
major target for inhibition for the next decades [8]. Actually, numerous ATP-competitive inhibitors have
been identified [9–11], which consist of small and planar heterocyclic scaffolds, able to fit into the
nucleotide-binding pocket of CK2α and to displace the ATP. Among these inhibitors, are (i)
polyhalogenated benzimidazole and benzotriazole derivatives, (ii) flavonoids, ellagic acid and
coumarins, (iii) anthraquinone, xanthenone and fluorenone, (iv) pyrazolo-triazine derivatives, (v)
carboxyl acid derivatives and, (vi) benzofuran derivatives (Figure 1).
Recently, some new CK2 inhibitors were designed using tricyclic and tetracyclic scaffolds [12,13].
Benzo[g]pyridoindole (BgPI) and indeno[1,2-b]indole scaffolds have sparked great interest for the
design of ATP-competitive inhibitors of CK2 (Figure 2). For example, 5-isopropyl-5H-indeno[1,2-
b]indole-6,9,10-trione 7h inhibits CK2 with an IC50 value of 5.05 µM [14]. Furthermore, this N-
isopropyl derivative 7h inhibited the growth of a series of different tumor cell lines (5637, SISO, KYSE-
70, MCF-7, LCLC, A427) [14]. Indenoindoles represent a wide class of synthetic compounds through
the system of annulated 6-5-5-6-membered rings. They are of great interest due to their potential for
wide biological applications, especially for drug development in oncology (e.g., DNA and
topoisomerase-II, CK2) [15,16].
Structure-Activity Relationship (SAR) studies were ongoing to evaluate the influence of the nature
and position of substituents on the four rings A-D. The first modifications on the C-ring showed that
introduction of a non-bulky group (e.g., ethyl, isopropyl) had a favorable inhibitory effect on human
CK2 [17]. Thus, we continued to explore further structural modifications on the D-ring of the indeno[1,2-
b]indole scaffold, as described in this study (Figure 2).

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Figure 1. Examples of known CK2 inhibitors.
Figure 2. Scaffolds for designing CK2 inhibitors and D-ring modulation on indeno[1,2-b]indoledione.
2. Results and Discussion
Substituted cyclohexane-1,3-diones 1 were mainly commercially available except for
4-methylcyclohexane-1,3-dione 1f and 4-isopropylcyclohexane-1,3-dione 1g, which were synthetized in
good yields by a regioselective and consecutive Michael-Claisen process starting from substituted
acetone and α,β-unsaturated esters in the presence of sodium hydride at 0 °C [18]. Enaminones 2a–g
were prepared in good yields by nucleophilic addition of isopropylamine on substituted cyclohexane-
1,3-diones 1a–g followed by dehydration in toluene at reflux with a Dean-Stark water trap for 6 h

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(Scheme 1). Condensation of ninhydrin 3 with 3-isopropylamino-cyclohexane-2-enones 2a–g at room
temperature gave dihydroxyindeno[1,2-b]indoles 4a–g (Scheme 1) in good to quantitative yields [19].
For example, dihydroxyindeno[1,2-b]indole 4a (R1 = CH3, R2 = H) was isolated as a yellow powder in a
95% yield. In the case of compound 4d (R1 = 4-fluorophenyl, R2 = H), two products were isolated during
the work-up. The main product was identified as dihydroxyindeno[1,2-b]indole 4d. The by-product was
isolated after the first time round and found to be an ammonium salt of 2-[4-(4-fluorophenyl)-2,6-dioxo-
cyclohexyl]-2-hydroxy-indane-1,3-dione 4d’ (Figure 3). Because formation of 4d’ was unexpected, its
structure was consolidated by single-crystal X-ray diffraction analysis (Figure 4). This crystallographic
study confirmed the structure in the solid state as anticipated on the basis of IR and NMR data.
Scheme 1. Synthesis of dihydroxyindeno[1,2-b]indole derivatives 4a–g.
Reagents and conditions: (i). i-PrNH2 (1 eq.), toluene, reflux, 6 h, 74%–100%. (ii). MeOH, room temp., 22 h, 46%–100%.
The key bond lengths and angles of the 2-hydroxyindane-1,3-dione moiety of 4d’ are very similar to
those given in the literature for similarly substituted 2-hydroxyindane-1,3-dione derivatives [20]. The
hydroxyindane-1,3-dione system (C13-C14-C15-C16-C17-C18-C19-C20-C21) of compound 4d’ is
nearly planar with a mean out-of-plane deviation of 0.076 Å with the largest deviation of 0.173(2) Å for
atom C13. In this compound, the cyclohexane moiety (C7-C8-C9-C10-C11-C12) is almost planar with
a maximum deviation from planarity of 0.278(2) Å found for C7. 4d’ contains a cation and an anion in
the solid state. The cation is formed by the ammonium function on the isopropylamine molecule. The
anion is formed after hydrolysis of the intermediate imine; according to the C-O and C-C bond lengths
(ranging from 1.252(3) to 1.255(3) Å, and from 1.399(3) to 1.408(3) Å, respectively) the negative charge
is delocalized over C10 and the two adjacent carbonyl functions. In the crystal, the molecules are linked
together by intermolecular N—H···O hydrogen bonding between the carbonyl groups of the
cyclohexane-dione system and the ammonium group of the isopropylamine. An intramolecular O—
H···O hydrogen bond between the hydroxyl group and the carbonyl oxygen of the cyclohexane-dione
was also noticed in the solid-state conformation.
To optimize the reaction conditions, 4d was only isolated from the mother liquor and used without
further purification for the next step.

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Figure 3. Structure of the ammonium salt 4d’.
Figure 4. Crystal structure of 4d’ with our numbering scheme; displacement ellipsoids are
drawn at the 30% probability level. Drawing was performed using the OLEX2 graphical
interface [21].
Dihydroxyindeno[1,2-b]indole derivatives 4a–g were then converted in good yield (75% to
quantitative) into 5,6,7,8-tetrahydro-indeno[1,2-b]indole-9,10-diones 5a–g (Scheme 2) by means of
N,N,N′,N′-tetraisopropylthionylamide (TIPTA) or N,N,N′,N′-tetraethylthionylamide (TETA) [19]. For
example, the title compound 5a was synthesized in a quantitative yield using TIPTA as reagent.
9-Hydroxy-5H-indeno[1,2-b]indol-10-ones 6a–g were then prepared by oxidation of 5,6,7,8-
tetrahydroindeno[1,2-b]indole-9,10-diones 5a–g with Pd/C 10% at reflux in diphenyl ether. Subsequent
oxidation with molecular dioxygen in the presence of salcomine, also known as
ethylenebis(salicylimine) cobalt(II) salt, at room temperature afforded finally 5H-indeno[1,2-b]indole-
6,9,10-triones 7a–g. Yields were generally good, except for the paraquinone derivative 7g (R1 = H,
R2 = i-C3H7) (yield = 14%). Structural proofs for compounds 5a–g, 6a–g and 7a–g were obtained from
spectroscopic data (1H-NMR, 13C-NMR, IR, HRMS). However, owing to poor nuclear relaxation,
several carbons were not assigned in the 13C-NMR spectra of 7b and 7e,g. Nevertheless, a previous work
1
[22] performed to study H and 13C-NMR assignments of indeno[1,2-b]indole-10-one derivatives
allowed us to achieve a complete spectral assignment for this scaffold.
In summary, three sub-series of compounds, namely D-ring mono-keto derivatives 5a–g, D-ring
hydroxyl derivatives 6a–g and D-ring paraquinone derivatives 7a–g were synthesized, purified and
characterized by spectral techniques.

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Scheme 2. Synthesis of indeno[1,2-b]indole derivatives 5a–g, 6a–g and 7a–g.
Reagents and conditions: (i). TIPTA or TETA (1.5 eq.), DMF, AcOH, room temp., 23 h, 75%–100%. (ii).
Pd/C, diphenyl ether, reflux, 3 h, 20%–89%. (iii). salcomine (1.8 eq.), DMF, O2, room temp., 27 h, 14%–95%.
For testing the inhibitors, the recently established capillary electrophoresis CK2 activity assay was
used [23]. Eleven compounds exhibited percent inhibition higher than 50% at 10 µM; corresponding
IC50 values were calculated from the resulting dose-response curves. In the first sub-series, three
compounds 5a (R1 = CH3, R2 = H), 5b (R1 = i-C3H7, R2 = H) and 5f (R1 = H, R2 = CH3) showed a percent
inhibition superior to 50%. Compound 5a has an IC50 value of 0.17 µM, lower than that obtained with
emodin (IC50 value of 0.58 µM). Still, ellagic acid and TBB remain better CK2 inhibitors than compound
5a. After the aromatization of the D-ring, a second sub-series of compounds was studied. Compounds
6a and 6b, analogues of 5a and 5b, respectively, are active against CK2 with IC50 values of 1.27 and
1.45 µM, respectively. Compounds 6d and 6e, possessing 4-fluorophenyl or 2′-furanyl groups at position
7, displayed IC50 values in the micromolar range (2.77 and 3.63 µM, respectively). In the last sub-series
of D-ring paraquinone derivatives 7a–g, compounds with substituents CH3 and 2′-furanyl on position 7,
are linked to CK2 inhibition, with IC50 values of 0.43 and 1.65 µM, respectively. Among the six
regioisomers of 5a,b, 6a,b and 7a,b (R2 = CH3, i-C3H7), only compound 7f (R2 = CH3) shows a weak
inhibitory activity against CK2 (IC50 = 4.90 µM).
After taking into account the results in our pharmacomodulation works, the introduction of a CH3
group at position 7 (D-ring) allowed us to identify compounds 5a, 6a, and 7a as potent CK2 inhibitors.
Furthermore, a second substituent, the i-C3H7 group at position 7, showed a favorable effect on CK2
inhibition (e.g., compounds 5b and 6b).
The assumption that indeno[1,2-b]indoles as small and planar molecules exhibit their inhibitory effect
towards CK2 by interacting with the ATP binding pocket had already been verified in a previous
investigation [13]. In order to validate the ATP competitive mode of inhibition for the CK2 inhibitors

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described here, the IC50 values of the two most potent inhibitors, 5a and 7a (Figure 5), were determined
at six different ATP concentrations ranging from 6 to 600 µM. IC50 values recorded for the two inhibitors
at different ATP concentrations (0.044, 0.097, 0.17, 0.44, 0.76 and 1.17 µM for 5a and 0.13, 0.34, 0.43,
1.27, 3.32 and 5.16 µM for 7a) were observed to linearly increase with the concentration of ATP, thus
indicating the ATP competitive mode of CK2 inhibition.
Table 1. Synthesized indeno[1,2-b]indole derivatives and inhibition of human CK2 holoenzyme.
Compound
R1
CH3
R2
Inhibition (%) 1) IC50 ± SD (µM)
5a
H
94
94
7
0.17 ± 0.03
0.61 ± 0.03
n.d. 2)
5b
i-C3H7
C6H5
4-F-C6H4
2′-furanyl
H
H
5c
H
5d
H
12
35
52
21
64
60
42
59
65
45
35
87
60
41
42
82
72
45
95
99
99
n.d.
5e
H
n.d.
5f
CH3
9.18 ± 0.92
n.d.
5g
H
i-C3H7
6a
CH3
H
1.27 ± 0.27
1.45 ± 0.32
n.d.
6b
i-C3H7
C6H5
4-F-C6H4
2′-furanyl
H
H
6c
H
6d
H
2.77 ± 0.35
3.63 ± 0.34
n.d.
6e
H
6f
CH3
6g
H
i-C3H7
n.d.
7a
CH3
H
0.43 ± 0.12
4.76 ± 0.29
n.d.
7b
7c
i-C3H7
C6H5
4-F-C6H4
2′-furanyl
H
H
H
7d
H
n.d.
7e
H
1.65 ± 0.14
4.90 ± 0.54
n.d.
7f
CH3
7g
H
i-C3H7
ellagic acid
emodin
TBB
-
-
-
-
0.040 ± 0.007
0.58 ± 0.05
0.060 ± 0.005
-
-
1) Average percent inhibition at 10 µM. 2) n.d.: not determined.
The obtained IC50 value at the highest ATP concentration of 600 µM was 27-fold higher for 5a and
40-fold higher for 7a when compared to the IC50 values obtained at the lowest ATP concentration of
6 µM. The IC50 values obtained at the different ATP concentrations were further used to determine the
Ki values of 5a and 7a. For this purpose, reaction rates at different inhibitor concentrations were plotted
in a Lineweaver-Burk diagram against the varying ATP concentration, resulting in ꢀ1 ꢁꢃꢂꢄꢄ as given
⁄
by the intercepts with the abscissa in both graphs. For determining the Ki values, ꢁꢃꢄꢄ were plotted
ꢂ
against the different inhibitor concentrations as shown for 7a in Figure 6. The Ki value with negative
sign was obtained by the intercept with the abscissa and was found to be 144 ± 22 nM with a regression
coefficient of R2 = 0.9941. For 5a, IC50 values obtained with the highest ATP concentrations at 300 and
600 µM appeared to be not in the linear range in the plot, what precluded the determination of the
corresponding Ki value. These IC50 values were therefore excluded for this compound. Although the

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resulting plot exhibited a poor regression coefficient of R2 = 0.8253, the Ki value for 5a could be obtained
by this plot and was found to be 27 ± 12 nM.
Figure 5. The two most potent CK2 inhibitors 5a and 7a of the novel indeno[1,2-b]indole derivatives.
50
40
30
20
Ki = 148 nM
10
-200
0
200
400
7a [nM]
Figure 6. Determination of the inhibition constants for the indeno[1,2-b]indoloquinone 7a.
ꢁꢂꢃꢄꢄwere plotted against the corresponding inhibitor concentrations.
Four cell lines of various origins were used to evaluate the cytotoxicity of our target compounds
5–7. The selected cell lines were (i) NIH-3T3, a cell line originally established from the primary mouse
embryonic fibroblast cells, (ii) MEF, primary mouse embryonic fibroblasts (used at their early passages)
prepared as previously described [24], (iii) WI-38, a human diploid cell line derived from normal
embryonic lung tissue, and (iv) HEK293T, a transformed variant of the human embryonic kidney cell
line HEK293. After 96 h, cytotoxicity was evaluated using the WST-1 assay. For each compound tested,
the EC50 (effective concentration of drug needed to inhibit cell growth/viability by 50%) was generated
from the dose-response curves for each cell line. Seventeen compounds were evaluated on the cell lines
and then compared to TBB used as a standard. Among the three best CK2 inhibitors (5a, 5b, 7a),
compounds 5a and 5b displayed no marked cytotoxic activity, except for compound 5b on the MEF cell
line (EC50 = 4.4 µM). After 96 h their EC50 values were superior to 10 µM, similar to TBB. No data were
obtained with compound 7a due to the lack of solubility. Four compounds, belonging to the sub-series
of D-ring paraquinone derivatives (7c, 7d, 7f, 7g), displayed cytotoxic activity (0.4 < EC50 µM < 8.8),
especially on HEK293T.

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Table 2. EC50 values of selected compounds 5–7 and TBB against four cell lines.
EC50 in µM ± SD
Compound
Normal cell lines 1)
NIH-3T3
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
WI-38
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
HEK293T
> 10
MEF
> 10
4.4 ± 0.3
> 10
5a
5b
5c
5f
5g
6a
6b
6c
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
6d
6e
6f
> 10
> 10
7a
7b
7c
7d
7f
not soluble not soluble not soluble not soluble
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
1.6 ± 0.2
4.9 ± 2.4
4.4 ± 2.2
> 10
> 10
8.5 ± 0.4
> 10
7g
1.5 ± 0.8
> 10
2.5 ± 0.4 0.40 ± 0.02
> 10
> 10
> 10
> 10
ellagic acid
emodin
TBB
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
1) Cells were cultured in presence of various concentrations of the respective conjugate. After 96 h, cytotoxicity
was evaluated using the WST-1 assay; see Experimental Section 3.3.4.
In order to understand observed differences in the inhibitory activity of compounds 5a, 7a, 5h
(R1 = R2 = H) [13] and 7h (R1 = R2 = H) [14] (Table S1 in the supplementary data), the compounds were
structurally aligned with the program vROCS [24] onto an inhibitor with pyridocarbazolone scaffold co-
crystallized with CK2 (PDB code: 3OWJ [12]). Subsequently, the compounds were energy minimized in
the presence of the enzyme with the program Moloc [26], keeping all protein atoms fixed. For this the
co-crystallized water molecule 429 was removed, as it clashed with the ligands (distance ≤ 2.2 Å); in
contrast, water molecule 382 was kept as its distance to the ligands was  4 Å. In addition, the
minimization was performed with all binding site residues within 5 Å of the ligands (L45-R47, S51,
V53, V66, K68, I95, F113-V116, H160-N161, M163, and I174-W176) allowed to move, in order to
investigate if the ligand binding would cause conformational changes within the binding site. The
resulting protein structures showed only minor differences (Figure S1 in the supplementary data) that
are within the experimental uncertainty of the structure determination (root mean-square deviation of
the heavy atoms of the binding pocket < 0.4 Å); these differences are mainly caused by optimized
intramolecular hydrogen-bonding of the CK2 residues. Thus, we focused on the binding modes of 5a,
7a, 5h and 7h obtained with fixed protein atoms for a better comparison to the crystal structure. The
obtained binding modes of 5a, 7a, 5h and 7h agree very well with the one of the co-crystallized inhibitor
(Figure S2 in the supplementary data). These binding modes provide explanations as to the observed

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differences in the inhibitory activity (Figure 7): (I) A carbonyl oxygen at position 6 points towards the
negatively charged side chain of Asp175 resulting in the vicinity of two hydrogen bond acceptor groups.
This unfavorable interaction increases the IC50 value of 7h [14] by a factor of 15 compared to 5h [13].
(II) Methyl substitution in position 7, first, decreases the polarity of the carbonyl group and, second,
partially shields the group from solvation in the unbound state, making desolvation effects upon binding
less costly. This results in an increase in inhibitory activity for 7-methyl derivatives of the paraquinone
series as observed for 7h compared to its methylated analogue 7a (IC50 values 5.55 and 0.43 µM,
respectively). (III) The methyl group also makes contacts with the Cα atom of Gly46 (Figure 7). A
complete burial of a methyl group can increase a binding constant by up to 10-fold [27]. Here, the methyl
group becomes only partially buried, which can explain the only two-fold increase of the inhibitory
activity of 5a compared to 5h.
Figure 7. (A) Predicted binding mode of 7a in the binding pocket of CK2. Green:
hydrophobic surface patches, yellow: hydrophilic surface patches, blue (red): positively
(negatively) polarized surface patches. Val53 and Ile174 shown in panel B are hidden behind
Gly46. (B) Schematic view of the interactions between 7a and neighboring amino acids
(modified from a PoseView drawing [28]).
3. Experimental Section
3.1. General
Melting points were measured in capillary tubes using a BUCHI 510 apparatus and were uncorrected.
IR spectra were recorded on a Perkin Elmer 1310 spectrometer and a Spectrum One spectrometer using
1
KBr pellets ( cm−1). H-NMR and 13C-NMR spectra (broadband decoupling and DEPT-135) were
recorded on a Brucker Avance 400 (400 MHz for 1H and 100 MHz for 13C) or a Brucker Avance 500
1
13
spectrometer (500 MHz for H and 125 MHz for C) using CDCl3 or DMSO-d6 as solvents. NMR
analysis of compounds 4–7 was performed with the same experiments described in [22]. Chemical shifts
(δ) are referred to that of the solvent. Low resolution mass spectra were recorded on an Agilent 1290

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Infinity system equipped with an Agilent 1260 DAD detector and an Agilent 6120 Quadrupole mass
detector with an ESI source in positive mode. HRMS spectra were performed on a Q-Tof Micro Water
with an ESI source in positive mode. Flash chromatography was performed on 230-400-mesh silica.
3.2. Synthesis
3.2.1. General Procedure: Synthesis of Enaminones 2a–g
Isopropylamine and the corresponding substituted cyclohexane-1,3-dione were dissolved in toluene
in equimolar amounts. The reaction mixture was refluxed with a Dean-Stark trap until separation of H2O
ceased (ca. 6 h). Solvent was removed in vacuo and the residual solid or viscous oil was treated with
ethyl acetate to give after filtration and drying the product as a powder.
5-Methyl-3-isopropylaminocyclohex-2-enone (2a). Pale yellow powder; 75% yield; mp 106.7 °C; IR
1
(KBr  cm−1) 3250 (N-H), 1552 (C=O); H-NMR (DMSO-d6,  ppm, J Hz) 6.82 (d, 1H, J = 6.04,
N-H), 4.78 (s, 1H, H-2), 3.46 (m, 1H, NCHMe2), 2.28 (m, 1H, H-6), 2.03–2.11 (m, 3H, H-4 and H-6),
1.82 (m, 1H, H-5), 1.10 (d, J = 4.3, 6H, NCHMe2), 0.95 (d, 3H, J = 5.79, CH3-5); 13C-NMR (DMSO-d6,
 ppm) 194.1 (C-1), 162.4 (C-3), 94.2 (C-2), 44.7 (C-6), 43.0 (C-5), 36.6 (C-4), 28.7 (NCHMe2), 21.6
(CH3), 21.5 (CH3), 20.8 (CH3); MS- ESI (m/z) 168.2 [M+H]+.
5-Isopropyl-3-isopropylaminocyclohex-2-enone (2b). Pale yellow powder; 74% yield; mp 125.2 °C; IR
(KBr,  cm−1) 3259 (N-H) ,1538 (C=O); 1H-NMR (DMSO-d6,  ppm, J Hz) 6.86 (d, 1H, J = 6.5, N-H),
4.78 (s, 1H, H-2), 3.46 (m, 1H, NCHMe2), 2.30 (d, 1H, J = 13.8, H-6), 2.08 (m, 2H, H-4 and H-6), 1,87
(dd, 1H, J = 12.3 and 15.6, H-4), 1.65 (m, 1H, CHMe2-5 or H-5), 1.49 (m, 1H, H-5 or CHMe2-5), 1.11
(d, 3H, J = 6.2 NCHMe2), 1.09 (d, J = 6.3, 3H, NCHMe2), 0.87 (d, J = 6.5, 6H, CHMe2-5);
13C-NMR (DMSO-d6,  ppm) 195.1 (C-1), 163.7 (C-3), 95.7 (C-2), 43.9 (NCHMe2), 40.9 (C-6), 40.9
(C-5), 33.2 (C-4), 32.1 (CHMe2), 22.5 (CH3), 22.3 (CH3), 20.3 (2 CH3); MS-ESI (m/z) 391.5
[2 M+H]+, 196.3 [M+H]+.
3-Isopropylamino-5-phenylcyclohex-2-enone (2c). Pale yellow powder; quantitative yield; mp 145 °C;
1
IR (KBr,  cm−1) 3225 (N-H), 1537 (C=O); H-NMR (DMSO-d6,  ppm, J Hz) 7.28–7.40 (m, 5H,
Ar-H), 7.02 (d, 1H, J = 6.8, N-H), 4.98 (s, 1H, H-2), 3.60 (m, 1H, NCHMe2), 3.28 (m, 1H, H-5), 2.69
(m, 1H, H-6), 2,52 (m, 2H, H- 4), 2.31 (dd, 1H, J = 3.5 and 15.9, H-6 ), 1.21 (d, 3H, J = 6.5, CH3 ), 1.19
13
(d, 3H, J = 6.3, CH3 ); C-NMR (DMSO-d6,  ppm) 194.2 (C-1), 163.1 (C-3), 144.9 (C-1′), 129.2
(C-2′ and C-6′), 127.6 (C-3′ and C-5′), 126.1 (C-4′), 95.2 (C-2), 44.6 (C-6), 44.0 (NCHMe2), 40.2 (C-5),
36,7 (C-4), 22.5 (CH3), 22,3 (CH3); MS-ESI (m/z) 481.3 [2 M+Na]+, 230.1 [M+H]+.
5-(4-Fluorophenyl)-3-isopropylaminocyclohex-2-enone (2d). Pale yellow powder; quantitative yield;
mp 155 °C; IR (KBr,  cm−1) 3674 (N-H), 1562 (C=O); 1H-NMR (DMSO-d6,  ppm, J Hz) 7.32 (m, 2H,
Ar-H), 7.13–7.06 (m, 3H, Ar-H and N-H), 4.77 (s,1H, H-2 ), 3.30–3.15 (m, 2H, NCHMe2 and H-5),
2.32–2.39 (dd, 2H, J = 11.6 and 15.8, H- 4 and H-6 ), 2.17–2.22 (dd, 2H, J = 4.3 and 15.8, H-4 and H-
6), 1.17 (d, 6H, J = 6.3, 2 CH3 ); 13C-NMR (DMSO-d6,  ppm) 190.9 (C-1), 161.2 (d, 1JC-F = 241, C-4′),
142.1 (d, 4JC-F = 2.2, C-1′), 142.1 (C-3), 129.2 (d, 3JC-F = 7.3, C-2′ and C-6′), 115.4 (d, 2JC-F = 21.3, C-3′

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and C-5′), 101.4 (C-2), 43.43 (C-4 and C-6), 43.1 (NCHMe2), 40.1 (C-5), 22.0 (2 CH3); MS-ESI (m/z)
270.1 [M+Na]+, 248.1 [M+H]+.
5-Furan-2-yl-3-isopropylaminocyclohex-2-enone (2e). Pale yellow powder; 99% yield; mp 120 °C; IR
1
(KBr,  cm−1) 3258 (N-H), 1537 (C=O); H-NMR (DMSO-d6,  ppm, J Hz) 7.55 (d, 1H, J = 1.76,
H-5′), 7.00 (d, 1H, J = 7.0, N-H), 6.37 (dd, 1H, J = 2.0 and 3.3, H-4′), 6.11 (d, 1H, J = 3.3, H-3′), 4.85
(s, 1H, H-2 ), 3.5 (m, 1H, H-5), 3.31 (m, 1H, NCHMe2), 2.65 (dd, 1H, J = 4.0 and 15.9, H-4 or H-6),
2.51 (m, 1H, H- 4 or H 6), 2.40 (dd, 1H, J = 4.5 and 16.1, H-4 or H-6), 2.30 (dd, 1H, J = 10.3 and 16.1,
H-4 or H-6), 1.12 (d, 3H, J = 6.3, CH3 ), 1.10 (d, 3H, J = 6.3, CH3); 13C-NMR (DMSO-d6,  ppm) 193.0
(C-1), 162.1 (C-3), 157.6 (C-2′) 142.1 (C-5′), 110.9 (C-3′ or C-4′), 104.9 (C-4′ or C-3′), 95.1 (C-2), 43.8
(CHMe2), 41.3 (C-4 or C-6), 33.8 (C-6 or C-4), 33.3 (C-5), 22.2 (CH3), 22.3 (CH3); MS-ESI (m/z) 461.3
[2 M+Na]+, 220.1 [M+H]+.
3-Isopropylamino-6-methyl-cyclohex-2-enone (2f). Pale yellow powder; 82% yield; mp 106.5 °C; IR
(KBr  cm−1) 3261 (N-H), 1540 (C=O); 1H-NMR (DMSO-d6,  ppm, J Hz) 6.76 (d, 1H, J = 6.4, N-H ),
4.76 (s, 1H, H-2), 3.46 (m, 1H, NCHMe2), 2.26–2.39 (m, 2H, H-4 and H-6), 2.07 (m, 1H, H-4), 1.2 (m,
1H, H-5), 1.49 (m, 1H, H-5), 1.10 (d, 3H, J = 6.4, NCHMe2), 1.09 (d, 3H, J = 6.4, NCHMe2), 0.98 (d,
3H, J = 6.95, CH3-6); 13C-NMR (DMSO-d6,  ppm) 196.5 (C-1), 162.5 (C-3), 94.0 (C-2), 43.0
(NCHMe2), 38.8 (C-6), 29.6 (C-5), 21.65 (NCHMe2), 21.7 (NCHMe2), 16.0 (CH3-6); MS-ESI (m/z)
168.2 [M+H]+.
3-Isopropylamino-6-isopropylcyclohex-2-enone (2g). Pale yellow powder; 78% yield; mp 121 °C; IR
(KBr,  cm−1) 3268 (N-H), 1572 (C=O); RMN 1H (DMSO-d6,  ppm, J Hz) 6.74 (d, 1H, J = 6.7, N-H),
4.78 (s, 1H, H-2), 3.44 (m, 1H, NCHMe2), 2.4–2.2 (m, 3H, CHMe2 and H-6 and H-4), 1.75–1.86 (m,
2H, H-5 and H-4), 1.57 (m, 1H, H-5), 1.10 (d, 3H, J = 4.7, NCH(Me)2), 1.07 (d, 3H, J = 4.7, NCHMe2),
0.87 (d, 3H, J = 7.1, CHMe2-6), 0.70 (d, 3H, J = 6.8, CHMe2-6); 13C-NMR (DMSO-d6,  ppm) 195.33
(C-1), 162.25 (C-3), 95.06 (C-2), 49.92 (NCHMe2), 42.96 (C-6), 27.98 (C-4), 25.70 (CHMe2-6), 21.65
(CH3), 21.53 (CH3), 21.31 (C-5), 20.54 (CH3), 18.03 (CH3); MS-ESI (m/z) 196.2 [M+H]+.
3.2.2. General Procedure: Synthesis of Substituted 4b,9b-Dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-diones 4a–g
Equimolar amounts of the corresponding enaminone 2a–g and ninhydrin 3 were dissolved in
methanol and stirred at room temperature for 22 h. Solvent was removed in vacuo and the residual solid
or viscous oil was treated with diethyl ether to give the product as a powder after filtration and drying.
A further amount was recovered from the mother liquor.
4b,9b-Dihydroxy-5-isopropyl-7-methyl-4b,5,6,7,8,9b-hexahydroindeno[1,2-b]indole-9,10-dione (4a).
Yellow powder; 95% yield; mp 198.9 °C; IR (KBr  cm−1) 3320 (OH), 3189 (OH), 1742 (C=O);
1H-NMR (CHCl3,  ppm, J Hz) 7.83 and 7.80 (m, 2H, H-1 and H-4), 7.71 and 7.69 (m, 1H, H-3), 7.51
and 7.49 (m, 1H, H-2), 5.28 (br s, 2H, 2 OH), 4.63 (m, 1H, NCHMe2), 2.78 (dd, 1H, J = 4.1 and 16.5,
H-6), 2.57 (dd, 1H, J = 3.3 and 17, H-6), 2.33 (m, 1H, H-7), 2.29 (m, 1H, H-6), 2.27 (m, 1H, H-8), 2.04
(m, 3H, H-6 and H-7 and H-8), 1.93 (m, 1H, H-8), 1.57 (d, 1H, J = 7, NCHMe2), 1.53 (d, 1H, J = 7.1,

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NCHMe2), 1.35 (d, 3H, J = 7.3, NCHMe2), 1.32 (d, 3H, J = 7.2, NCHMe2), 1.03 (d, J = 6.2, 3H, CH3),
1.00 (d, J = 6.5, 3H, CH3); 13C-NMR (CHCl3,  ppm) 197.7 and 197.1 (C-10), 191.7 and 191.3 (C-9),
165.9 and 165.7 (C-5a), 148.5 and 148.0 (C-4a), 135.7 (C-3), 135.4 and 135.3 (C-10a), 130.4 and 130.4
(C-2), 124.9 and 124.7 (C-1 or C-4), 124.1 and 123.7 (C-1 or C-4), 105.6 and 105.4 (C-9a), 96.3 and
96.0 (C-4b), 82.7 and 82.3 (C-9b), 46.4 and 46.1 (CHMe2), 44.6 and 45.1 (C-8), 33.2 and 33.1 (C-6),
30.2 and 29.8 (C-7), 23.6 and 23.3 (CH3), 22.8 and 22.7 (CH3), 21.4 and 21.1 (CH3); MS-ESI (m/z) 677.3
[2 M+Na]+, 350.1 [M+Na]+, 328.2 [M+H]+.
4b,9b-Dihydroxy-5,7-diisopropyl-4b,5,6,7,8,9b-hexahydroindeno[1,2,b]indole-9,10-dione (4b). Yellow
powder; quantitative yield; mp 200.8 °C; IR (KBr,  cm−1) 3252 (OH), 3180 (OH), 1728 (C=O); for
complete NMR data see [22]; MS-ESI (m/z) 733.7 [2 M+Na]+, 378.4 [M+Na]+, 356.3 [M+H]+.
4b,9b-Dihydroxy-5-isopropyl-7-phenyl-4b,5,6,7,8,9b-hexahydroindeno[1,2-b]indole-9,10-dione (4c).
Yellow powder; quantitative yield; mp 181.1 °C; IR (KBr, cm−1) 1719 (C=O); 1H-NMR (DMSO-d6, 
ppm, J Hz) 7.99 (m, 1H, Ar-H), 7.83 (m, 1H, Ar-H), 7.75 (m, 1H, Ar-H), 7.63 (m, 1H, Ar-H), 7.39–7.2
(m, 5H, Ar-H), 6.88 and 6.78 (2 s, 1H, OH), 5.81 and 5.72 (2 s, 1H, OH), 4.64 (m, 1H, NCHMe2), 3.22
and 3.13 (2 m, 1H, H-7), 2.92 (m, 1H, H-6 or H-8), 2.80 (m, 1H, H-8 or H-6), 2.47 (m, 1H, H-6 or
H-8), 2.21 (m, 1H, H-8 or H-6), 1.50 and 1.43 (2 d, J = 6.8, 3H, CH3), 1.28 and 1.26 (2 d, 3H, J = 7.3
13
and 8.3, CH3); C-NMR (DMSO-d6,  ppm) 198.7 and 198.1 (C-10), 188.7 and 188.2 (C-9), 164.7
(C-5a), 149.4 and 148.7 (C-4a), 144.6 and 144.5 (C-1′), 136.5 and 136.4 (C-3), 135.9 and 135.7 (C-10a),
131.2 and 131.1 (C-2), 129.3 (C-3′ and C-5′), 128.0 and 128.0 (C-2′and C-6′), 127.5 and 127.5 (C-4′),
125.7 and 125.3 (C-4), 124.3 and 124.0 (C-1), 105.8 (C-9a), 97.1 and 96.9 (C-4b), 84.0 and 83.7 (C-9b),
46.0 and 45.8 (NCHMe2), 45.5 and 45.1 (C-8), 41.3 and 40.5 (C-7), 32.3 and 31.6 (C-6), 23.8 and 23.3
(CH3), 23.5 and 23.4 (CH3); MS-ESI (m/z) 801.4 [2 M+Na]+, 412.1 [M+Na]+, 390.2 [M+H]+.
4b,9b-Dihydroxy-7-(4-Fluoro-phenyl)-5-isopropyl-4b,5,6,7,8,9b-hexahydroindeno[1,2-b]indole-9,10-
dione (4d). Yellow powder; 75% yield; mp 161.1 °C; IR (KBr,  cm−1) 3150 (OH), 3186 (OH), 1741
(C=O); for complete NMR data see [22]; MS-ESI (m/z) 408.2 [M+H]+; HRMS-ESI (m/z) [M+H]+ calcd
for C24H23FNO4 408.1606, found 408.1587.
Isopropylammonium
4-(4-fluorophenyl)-1-(2-hydroxy-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)-2,6-
dioxocyclohexan-1-ide (4d’). Pale yellow powder; 10% yield; mp 161.1 °C; IR (KBr  cm−1) 3150 (OH),
1741 (C=O), 1706 (C=O); 1H-NMR (DMSO-d6,  ppm, J Hz) 7.87 (m, 2H, 2 H-Phe), 7.82 (m, 2H, 2 H-
Phe), 7.66 (br s, 2H, OH), 7.35 (m, 2H, 2 H-Phe), 7.10 (m, 2H, 2 H-Ar), 3.23 (m, 1H, NCHMe2), 3.22
(m; 1H, CH-Ar), 2.38 (dd, 2H, J = 11.9 and 16.1, CH2), 2.13 (dd, 2H, J = 4.3 and 16.1, CH2), 1.15 (d,
13
4
6H, J = 6.4, 2 CH3); C-NMR (DMSO-d6,  ppm) 202.4 (2 C=O), 190.1 (2 C=O), 160.6 (d, JC-F
=
240.2, C-4′), 141.0 (d, J = 2.2, C-1′), 140.0 (C-OH), 134.8 (C-2 and C-3), 128.6 (d, 3JC-F = 8.0, C-2′ and
C-6′), 122.4 (C-1 and C-4), 114.8 (d, 2JC-F = 20.8, C-3′ and C-5′), 105.1 (Cq), 80.0 (C-OH), 42.9 (2 CH2
andCHPhe), 38.7 (NCHMe2), 20.2 (NCHMe2); MS-ESI (m/z) 755.2 [2 M+Na]+, 367.1 [M+H]+; HRMS-
ESI (m/z) [M+Na]+ calcd for C21H15NFNaO5 389.0792, found 389.0796.
7-Furan-2-yl-4b,9b-dihydroxy-5-isopropyl-4b,5,6,7,8,9b-hexahydroindeno[1,2-b]indole-9,10-dione
(4e). Yellow powder; quantitative yield; mp 135 °C; IR (KBr,  cm−1) 3159 (OH), 3150 (OH), 1719

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1
(C=O); H-NMR (DMSO-d6,  ppm, J Hz) 7.99 (m, 1H, H-1), 7.82 (m, 1H, H-4), 7.74 (m, 1H, H-3),
7.65–7.5 (m, 2H, Ar-H and H-5′), 6.90 and 6.81 (2 s, 1H, OH), 6.40 and 6.32 (2 dd, 1H, J = 1.8, and 3,
H-4′), 6.23 and 6.08 (2 d, 1H, J = 3.2, H-3′), 5.79 and 5.70 (2 s, 1H OH), 4.65 (m, 1H, NCHMe2), 3.43
and 3.33 (2 m, 1H, H-7), 3.09 (dd, 1H, J = 4.3 and 16.9, H-6), 2.68 (m, 1H, H-8), 2.47–2.27 (m, 2H,
H-6 and H-8), 1.52 and 1.47 (2 d, 3H, J = 7, CH3), 1.32 and 1.30 (2 d, 3H, J = 4 and 4.3, CH3);
13C-NMR (DMSO-d6,  ppm) 198.1 and 197.7 (C-10), 187.3 and 186.9 (C-9), 163.5 and 163.2 (C-5a),
156.9 (C-2′), 148.8 and 148.3 (C-4a), 142.1 and 142.1 (C-5′), 136.1 and 136.0 (C-3), 135.5 and 135.3
(C-10a), 130.8 and 130.7 (C-2), 125.0 and 125.2 (C-1), 123.9 and 123.6 (C-4), 110.9 and 110.8 (C-3′ or
C-4′), 105.5 and 105.2 (C-4′ or C-3′), 96.5 and 96.4 (C-4b), 83.5 and 83.3 (C-9b), 45.6 and 45.4
(NCHMe2), 42.1 and 41.5 (C-8), 34.0 and 33.4 (C-7), 29.4 and 29.2 (C-6), 23.3 and 23.1 (CH3), 23.0
and 23.0 (CH3); MS-ESI (m/z) 781.3 [2 M+Na]+, 402.1 [M+Na]+, 380.2 [M+H]+.
4b,9b-Dihydroxy-5-isopropyl-8-methyl-4b,5,6,7,8,9b-hexahydro-indeno[1,2-b]indole-9,10-dione (4f).
Yellow pale powder; 46% yield; mp 198.9 °C; IR (KBr  cm−1) 3190 (OH), 3160 (OH), 1725 (C=O);
1H-NMR (DMSO-d6,  ppm, J Hz) 7.94 and 7.92 ( 2 d, 1H, J = 7.8, H-1), 7.78 (m, 1H, H-4), 7.70 and
7.67 (2 d, 1H, J = 7.5, H-3), 7.57 (m, 1H, H-2), 6.71 (s, 1H, OH minor - 45%), 6.64 (s, 1H, OH major -
55%), 5.60 (s, 1H, OH major), 5.59 (s, 1H, OH minor), 4.56 (m, 1H, CHMe2), 2.76 (m, 1H, H-8 major),
2.66 (m, 1H, H-6 or H-7 minor), 2.58 (m, 1H, H-8 minor), 2.44 (m, 1H, H-6 or H-7 major), 2.1 (m, 1H,
H-6 or H-7 minor), 2.04 (m, 1H, H-6 or H-7 major), 1.92 (m, 1H, H-6 or H-7 major), 1.57 (m, 1H, H-7
major), 1.45 (d, 3H, J = 7.0, CH3 major), 1.42 (d, 3H, J = 7.0, CH3 minor), 1.41 (m, 1H, H-7 minor),
1.24 (d, 3H, J = 7.0, CH3 major), 1.21 (d, 3H, J = 7.2, CH3 minor), 0.97 (d, 3H, J = 6.8, CH3 major), 0.93
(d, 3H, J = 6.9, CH3 minor); 13C-NMR (DMSO-d6,  ppm) 197.1 and 196.6 (C-10), 190.7 and 190.4 (C-
9), 163.4 and 163.3 (C-5a), 147.7 and 147.3 (C-4a), 134.7 and 134.7 (C-3), 134.2 and 134.0 (C-10a),
129.5 and 129.4 (C-2), 124.0 and 123.6 (C-4), 122.6 and 122.3 (C-1), 103.8 and 103.5 (C-9a), 95.1 and
95.0 (C-4b), 82.5 and 82.4 (C-9b), 44.2 and 44.0 (CHMe2), 39.6 and 39.1 (C-8), 30.2 and 29.7 (C-6),
23.7 and 23.4 (C-7), 22.7 and 22.5 (CH3), 22.3 and 22.2 (CH3), 15.7 and 15.4 (CH3-8); MS-ESI+ (m/z)
677.3 [2 M+Na]+, 350.2 [M+Na]+, 328.2 [M+H]+.
4b,9b-Dihydroxy-5,8-diisopropyl-4b,5,6,7,8,9b-hexahydroindeno[1,2-b]indole-9,10-dione (4g). Yellow
1
powder; 55% yield; mp 220 °C; IR (KBr,  cm−1) 3675 (OH), 3158 (OH), 1719 (C=O); H-NMR
(DMSO-d6,  ppm, J Hz) 7.93 (m, 1H, H-1), 7.80 (m, 1H, H-4), 7.70 (m, 1H, H-3), 7.58 (m, 1H, H-2),
6.75 (2 s, 1H, OH), 5.64 (br s, 1H, OH), 4.55 (m, 1H, NCHMe2), 2.84 (m, 1H, H-8), 2.80 (m, 1H, H-8),
2.60 (m, 2H, H-6 and H-8), 2.37 (m, 1H, CHMe2-8), 2.17 (m, 1H, CHMe2-8), 1.82 (m, 2H, H-7 and H-
6), 1.64 (m, 1H, H-7), 1.51 (m, 1H, H-7), 1.45 (d, 3H, J = 7.0, CH3), 1.41 (d, 3H, J = 7.0, CH3), 1.23 (d,
3H, J = 7.0, CH3), 1.20 (d, 3H, J = 7.0, CH3), 0.86 (d, 3H, J =7.0, CH3), 0.80 (d, 3H, J = 7.0, CH3),
0.74 (d, 3H, J = 6.8, CH3), 0.64 (d, 3H, J = 6.8, CH3); 13C-NMR (DMSO-d6,  ppm) 197.9 and 197.3
(C-10), 191.0 and 189.7 (C-9), 163.8 and 163.6 (C-5a), 148.4 and 147.9 (C-4a), 135.4 and 135.3 (C-3),
134.9 and 134.6 (C-10a), 130.1 and 130.0 (C-2), 124.6 and 124.3 (C-1), 123.2 and 123.0 (C-4), 105.3
and 105.0 (C-9a), 95.8 and 95.6 (C-4b), 83.3 and 82.9 (C-9b), 50.5 and 49.9 (NCHMe2), 44.8 and 44.6
(C-8), 25.9 and 25.2 (CHMe2-8), 23.8 and 23.1 (C-7), 22.7 and 22.4 (CH3), 22.2 and 22.1 (CH3), 21.9
and 21.8 (C-6), 20.6 and 20.5 (CH3), 18.4 and 18.0 (CH3); MS-ESI (m/z) 356.2 [M+H]+.

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3.2.3. Synthesis of N,N,N′,N′-Tetraisopropylthionylamide (TIPTA) and N,N,N′,N′-
Tetraethylthionylamide (TETA)
Diisopropylamine (161.01 mmol) was introduced into 150 mL of distilled diethyl ether at −40 °C. A
thionyl chloride solution (4.25 mL) in diethyl ether (10 mL) was introduced dropwise during one hour
with release of a white smoke. Temperature increased to −20 °C at the end of the addition. The viscous
mixture was stirred for 3 h with the temperature reaching 0 °C. The mixture was then filtered under
vacuum to remove the diisopropylammonium chloride precipitate and washed with diethyl ether. The
filtrate was concentrated and evaporated to dryness to afford a white precipitate of TIPTA (85% yield;
mp 57.1–58.2 °C).
N,N,N′,N′-tetraethylthionylamide (TETA) was prepared by the same method unless diisopropylamine
was replaced by diethylamine (93% yield).
3.2.4. General Procedure: Synthesis of Substituted 5,6,7,8-Tetrahydroindeno[1,2-b]indole-9,10-diones
5a–g
The corresponding dihydroxy derivative 4a–g was dissolved in 25 mL DMF and 5 mL acetic acid.
After addition of TIPTA or TETA (1.5 eq.) the mixture was stirred for 23 h at room temperature and
then the mixture was poured into ice water (200 mL). After 1.50 h the precipitate 5a–g was recovered
by filtration.
5-Isopropyl-7-methyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (5a). Orange powder;
quantitative yield; mp 205.9 °C; IR (KBr,  cm−1) 1710 (C=O), 1667 (C=O); 1H-NMR (CHCl3,  ppm,
J Hz) 7.74 (d, 1H, J = 7.1, H-1), 7.23 (m, 1H, H-3), 7,11 (m, 2H, H-2 and H-4), 4.62 (sept, 1H, J = 7.1,
NCH(Me)2), 2.92 (dd, 1H, J = 4.3 and 16.2, H-6), 2.54 (dd, 1H, J = 3.6 and 16.2, H-8), 2.50 (m, 1H,
H-6), 2.39 (m, 1H, H-7), 2.20 (dd, 1H, J = 11.8 and 16.2, H-8), 1.65 (d, 3H, J = 7.3, NCH(Me)2), 1.64
(d, 3H, J = 7.0, NCH(Me)2), 1.17 (d, 3H, J = 6.5, CH3); 13C-NMR (CHCl3,  ppm) 191.9 (C-9), 184.2
(C-10), 151.8 (C-4b), 148.7 (C-5a), 138.9 (C-10a), 135.4 (C-4a), 132.2 (C-3), 128.1 (C-2), 123,8 (C-1),
118.8 (C-4), 120.8 (C-9b), 117.4 (C-9a), 49.4 (N-CH(Me)2), 46.1 (C-8), 31.7 (C-6), 31.1 (C-7), 22.0
(CH3), 21.9 (CH3), 21.3 (CH3-7); MS-ESI (m/z) 609.3 [2 M+Na]+, 316.1 [M+Na]+, 294.1 [M+H]+;
HRMS-ESI (m/z) [M+Na]+ calcd for C19H19NO2Na 316.1308, found 316.1312.
5,7-Diisopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (5b). Orange powder; quantitative
yield; mp 191 °C; IR (KBr,  cm−1) 1710 (C=O), 1667 (C=O); for complete NMR data see [22];
MS-ESI (m/z) 665.8 [2 M+Na]+, 344.4 [M+Na]+, 322.4 [M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for
C21H23NO2Na 344.1621, found 344.1616.
5-Isopropyl-7-phenyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (5c). Orange powder; 76%
yield; mp 278 °C; IR (KBr,  cm−1) 1703 (C=O), 1651 (C=O); 1H-NMR (CDCl3,  ppm, J Hz) 7.40 (d,
1H, J = 6.8, H-1), 7.31 (m, 2H, Ar-H), 7.12 (m, 4H, Ar-H), 7.07 (m, 2H, Ar-H), 4.56 (sept, J = 6.7, 1H,
NCHMe2), 3.46 (m, 1H, H-7), 3.08 (dd, 1H, J = 4.8 and 16.4, H-6), 2.93 (dd, 1H, J = 11.1 and 16.4, H-
8), 2.69 (m, 2H, H-8 and H-6), 1.59 (d, 3H, J = 6.8, CH3), 1.58 (d, 3H, J = 7.0, CH3); 13C-NMR (CDCl3,
 ppm) 191.1 (C-9), 184.4 (C-10), 152.2 (C-4b), 148.3 (C-5a), 142.9 (C-1′), 139.0 (C-4a), 135.5 (C-
10a), 132.4 (C-3), 129.0 (C-3′ and C-5′), 128.4 (C-2), 127.4 (C-4′), 127.0 (C-2′ and C-6′), 124.0 (C-1),

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121.0 (C-9b), 119.0 (C-4), 117.6 (C-9a), 49.3 (N-CHMe2), 44.9 (C-8), 41.9 (C-7), 31.8 (C-6), 22.2
(CH3), 22.0 (CH3); MS-ESI (m/z) 733.3 [2 M+Na]+, 378.1 [M+Na]+, 356.2 [M+H]+; HRMS-ESI (m/z)
[M+Na]+ calcd for C24H21NNaO2 378.1465, found 378.1461.
7-(4-Fluorophenyl)-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione
(5d).
Orange
powder; 78% yield; mp 264 °C; IR (KBr,  cm−1) 1701 (C=O), 1670 (C=O); for complete NMR data
see [22]; HRMS-ESI (m/z) [M+Na]+ calcd for C24H20FNNaO2 396.1370, found 396.1362.
7-Furan-2-yl-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (5e). Orange powder; 78%
yield; mp 233.1 °C; IR (KBr,  cm−1) 1705 (C=O), 1661 (C=O); 1H-NMR (CDCl3,  ppm, J Hz) 7.38
(d, 1H, J = 7.0, H-1), 7.17 (m, 2H, 2 H-Ar), 7.05 (2H, H-Ar and H-5′), 6.31 (m, 1H, H-3′), 6.02 (d, J =
2.9, 1H, H-4′), 4.55 (m, 1H, NCHMe2), 3.54 (m, 1H, H-7), 3.17 (dd, 1H, J = 4.6 and 16.4, H-6 or H-8),
2.96 (dd, 1H, J = 9.7 and 16.3, H-8 or H-6), 2.77 (dd, 1H, J = 5.4 and 17.9, H-6 or H-8), 2.36 (dd, 1H, J
= 10.9 and 16.4, H-6 or H-8), 1.57 (d, 3H, J = 6.8, CH3), 1.56 (d, 3H, J = 6.8, CH3); 13C-NMR (CDCl3,
 ppm) 190.2 (C-9), 184,2 (C-10), 155.7 (C-2′), 152.1 (C-4b), 147.2 (C-5a), 141.6 (C-5′), 138.9 (C-10a),
135.3 (C-4a), 132.2 (C-3), 128.3 (C-2), 124.0 (C-1), 118.8 (C-4), , 117.5 (C-9), 110.3 (C-3′ or C-4′),
105.2 (C-4′ or C-3′), 41.9 (C-8), 49.6 (NCHMe2), 34.9 (C-7), 28.6 (C-6), 22.05 (CH3), 21.9 (CH3); MS-
ESI (m/z) 713.3 [2 M+Na]+, 368.1 [M+Na]+, 346.1 [M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for
C22H19NNaO3 368.1257, found 368.1258.
5-Isopropyl-8-methyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione
(5f).
Orange
powder;
quantitative yield; mp 200 °C; 1H-NMR (DMSO-d6,  ppm, J Hz) 7.37–7.33 (m, 2H, Ar-H), 7.30 (d, 1H,
J = 7.0, Ar-H), 7.17 (m, 1H, Ar-H), 4.72 (m, 1H, NCHMe2), 2.99 (m, 1H, H-6 or H-7), 2.91 (m, 1H, H-
6 or H-7), 2.41 (m, 1H, H-8), 2.14 (m, 1H, H-6 or H-7), 1.88 (m, 1H, H-6 or H-7), 1.55 (d, 3H,
J = 6.8, NCHMe2), 1.54 (d, 3H, J = 6.8, NCHMe2), 1.07 (d, 3H, J = 6.9, CH3); 13C-NMR (DMSO-d6, 
ppm) 193.6 (C-9), 183.3 (C-10), 151.2 (C-4b), 150.02 (C-5a), 138.0 (C-4a), 134.65 (C-10a), 132.8
(C-3), 128.2 (C-2), 122.9 (C-1), 120.4 (C-9b), 119.4 (C-4), 116.0 (C-9a), 46.1 (N-CH(Me)2), 40.6
(C-8), 30.3 (C-6), 22.2 (C-7), 21.4 (CH3), 21.2 (CH3), 14.8 (CH3-8); MS-ESI (m/z) 609.3 [2 M+Na]+
316.2 [M+Na]+, 294.2 [M+H]+; HRMS-ESI (m/z) [M+H]+ calcd for C19H19 NO2Na 316.1308, found
316.1315.
5,8-Diisopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (5g). Orange powder; 75% yield; mp
1
215 °C; IR (KBr,  cm−1) 1738 (C=O), 1712 (C=O); H-NMR (CDCl3,  ppm, J Hz) 7.45 (m, 1H,
Ar-H), 7.23 (m, 1H, Ar-H), 7.12–7.06 (m, 2H, Ar-H), 4.59 (m, 1H, NCHMe2), 2.94 (m, 1H, H-6 or
H-7), 2.78 (m, 1H, H-6 or H-7), 2.47 (m, 1H, H-8), 2.17 (m, 2H, H-6 and H-7), 2.01 (m, 1H, H-6 or
H-7), 1.63 (d, 3H, J = 7.0, NCHMe2), 1.62 (d, 3H, J = 7.0, NCHMe2), 0.98 (d, 3H, J = 6.9, CHMe2-8),
13
0.90 (d, 3H, J = 6.8, CHMe2-8); C-NMR (CDCl3,,  ppm) 193.6 (C-9), 184.2 (C-10), 151.6 (C-4b),
148.1 (C-5a), 139.0 (C-4a), 135.4 (C-10a), 132.0 (C-3), 128.0 (C-2), 123.7 (C-1), 121.0 (C-9b), 118.6
(C-4), 118.6 (C-9a), 52.3 (N-CHMe2), 49.2 (C-8), 25.81 (CHMe2-8), 23.7 (C-6 or C-7), 22.9 (C-7 or
C-6), 22.0 (CH3), 21.9 (CH3), 21.6 (CH3), 20.8 (CH3); MS-ESI (m/z) 665.4 [2 M+Na]+, 322.2
[M+H]+; HRMS-ESI (m/z) [M+H]+ calcd for C21H24NO2 322.1802, found 322.1810.

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3.2.5. General Procedure: Synthesis of Substituted 9-Hydroxy-5H-indeno[1,2-b]indol-10-ones 6a–g
The corresponding mono-keto derivative 5a–g (4 mmol) was introduced into 15 mL diphenyl ether
then Pd/C (150% m/m) was added. The reaction mixture was refluxed for 3 h. After cooling, methanol
(50 mL) was added. The mixture was then filtrated on Celite and the solution concentrated. The obtained
dark red oil was purified by column chromatography on silica gel (cyclohexane then ethyl
acetate/cyclohexane: 1/2).
9-Hydroxy-5-isopropyl-7-methyl-5H-indeno[1,2-b]indol-10-one (6a). Dark red powder; 70% yield; IR
(KBr,  cm−1) 3181 (OH), 1701 (C=O); 1H-NMR (CDCl3,  ppm, J Hz) 7.35 (d, 1H, J = 6.9, H-1), 7.22
(t, J = 7.5, 1H, H-3), 7.16 (d, J = 7.2, 1H, H-4), 7.11 (t, J = 7.3, 1H, H-2), 6.72 (s, 1H, H-6), 6.58 (s, 1H,
OH), 6.49 (s, 1H, H-8), 4.81 (sept, J = 6.9, 1H, NCHMe2), 2.39 (s, 3H, CH3-7), 1.70 (d, 6H, J= 7.0,
NCHMe2); 13C-NMR (CDCl3,  ppm) 186.1 (C-10), 155.3 (C-4b), 149.7 (C-9), 142.9 (C-5a), 140.6 (C-
10a), 136.5 (C-4a), 135.7 (C-7), 132.2 (C-3), 129.1 (C-2), 123.3 (C-1), 119.8 (C-4), 111.4 (C-9a), 109.2
(C-8), 104.9 (C-6), 49.5 (NCHMe2), 22.2 (CH3), 21.8 (2 CH3); MS-ESI (m/z) 605.3 [2 M+Na]+, 314.1
[M+Na]+, 292.1 [M+H]+; HRMS-ESI (m/z) calcd for C19H17NNaO2 314.1151, found 314.1153.
9-Hydroxy-5,7-diisopropyl-5H-indeno[1,2-b]indol-10-one (6b). Dark red powder; 89% yield; mp
188.1 °C; IR (KBr,  cm−1) 3411 (OH), 1653 (C=O); for complete NMR data see [22]; MS-ESI (m/z)
661.7 [2 M+Na]+, 342.4 [M+Na]+, 320.4 [M+H]+; HRMS-ESI (m/z) [M+H]+ calcd for C21H22NO2
320.1645, found 320.1649.
9-Hydroxy-5-isopropyl-7-phenyl-5H-indeno[1,2-b]indol-10-one (6c). Dark red powder; 56% yield; mp
198 °C; IR (KBr,  cm−1) 3396 (OH), 2924 (CH), 1659 (C=O); 1H-NMR (CDCl3,  ppm, J Hz) 7.61 (m,
2H, Ar-H), 7.47–7.41 (m, 3H, Ar-H), 7.35 (m, 1H, Ar-H), 7.29–7.22 (m, 2H, Ar-H), 7.17 (m, 1H, Ar-
H), 7.19 (s, 1H, H-6), 6.93 (s, 1H, H-8), 6.73 (s, 1H, OH), 4.90 (sept, J = 7, 1H, NCHMe2), 1.76 (d, 6H,
13
J = 7.0, 2 CH3); C-NMR (CDCl3,  ppm) 186.0 (C-10), 156.0 (C-4b), 150.1 (C-9), 143.10 (C-5a),
141.8 (C-10a), 140.7 (C-4a), 139.3 (C-1′), 136.4 (C-7), 132.4 (C-3), 129.4 (C-2), 128.8 (C-3′ and C-5′),
127.3 (C-2′ and C-6′), 127.2 (C-4′), 123.6 (C-1), 120.1 (C-4), 115.7 (9b), 112.8 (C-9a), 107.6 (C-8),
103.7 (C-6), 49.8 (NCHMe2), 21.9 (2 CH3); MS-ESI (m/z) 730.3 [2 M+Na]+, 376.2 [M+Na]+, 354.1
[M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for C24H19NNaO2 376.1308, found 376.1300.
7-(4-Fluorophenyl)-9-hydroxy-5-isopropyl-5H-indeno[1,2-b]indol-10-one (6d). Dark red powder; 52%
yield; mp 208.1 °C; IR (KBr,  cm−1) 3391 (OH), 1659 (C=O); for complete NMR data see [22]; 394
[M+Na]+, 372 [M+H]+; HRMS-ESI (m/z) [M+H]+ calcd for C24H19FNO2 372.1394, found 372.1377.
7-Furan-2-yl-9-hydroxy-5-isopropyl-5H-indeno[1,2-b]indol-10-one (6e). Dark red powder; 50% yield;
mp 191 °C; IR (KBr,  cm−1) 3391 (OH), 1672 (C=O); 1H-NMR (CDCl3,  ppm, J Hz) 7.43 (s, 1H, OH),
7.37 (d, 1H, J=7.1, H-1), 7.25–7.18 (m, 3H, 2 Ar-H and H-5′), 7.13 (t, J = 7.3, 1H, Ar-H), 6.94 (s, 1H,
H-6), 6.67 (s, 1H, H-8), 6.59 (d, 1H, J = 3.4, H-3′), 6.45 (m, 1H, H-4′), 4.86 (sept, J = 7.0, 1H, NCHMe2),
1.72 (d, 6H, J = 7.0, CH3); 13C-NMR (CDCl3,  ppm) 185.9 (C-10), 155.9 (C-4b), 154.2 (C-2′), 150.0
(C-9), 142.9 (C-5a), 141.7 (C-5′), 140.5 (C-10a), 136.3 (C-7), 132.3 (C-3), 129.3 (C-2), 128.4 (C-4a),
123.5 (C-1), 120.1 (C-4), 116.0 (C-9b), 113.0 (C-8), 104.9 (C-4′), 104.1 (C-3′), 100.3 (C-6), 49.6

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(NCHMe2), 21.9 (2 CH3); MS-ESI (m/z) 709.3 [2 M+Na]+, 366.2 [M+Na]+, 344.2 [M+H]+;
[M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for C22H17NNaO3 366.1094, found 366.1101.
9-Hydroxy-5,8-diisopropyl-5H-indeno[1,2-b]indol-10-one (6g). Dark red powder; 20% yield; mp
235 °C; IR (KBr,  cm−1) 3422 (OH), 1665 (C=O); 1H-NMR (CDCl3 + TFA,  ppm, J Hz) 7.32 (d, 1H,
J = 7.2, Ar-H), 7.23 (m, 1H, Ar-H), 7.16 (m, 2H, Ar-H), 7.05 (m, 1H, Ar-H), 4.81 (m, 1H, NCHMe2),
3.35 (m, 1H, CHMe2-8), 1.71 (d, 6H, J = 7, 2 CH3), 1.26 (d, 6H, J = 6.9, 2 CH3); 13C-NMR (CDCl3 +
TFA,  ppm) 187.63 (C-10), 158.38 (C-4b), 146.14 (C-5a), 141.24 (C-4a), 140.75 (C-10a), 135.45
(C-9), 132.93 (C-3), 130.36 (C-2), 129.47 (C-8), 124.30 (C-1), 123.49 (C-7 or C-6), 123.38 (C-6 or
C-7), 120.85 (C-4), 114.68 (C-9a or C-9b), 113.81 (C-9b or C-9a), 50.41 (NCHMe2), 26.42 (CHMe2-8),
22.73 (2 CH3), 21.60 (2 CH3); MS-ESI (m/z) 661 [2 M+Na]+, 342 [M+Na]+, 320 [M+H]+;
HRMS-ESI (m/z) [M+Na]+ calcd for C21H21NNaO2 342.1465, found 342.1460.
3.2.6. General Procedure: Synthesis of Substituted 5H-Indeno[1,2-b]indole-6,9,10-triones 7a–g
The corresponding 9-hydroxyindeno[1,2-b]indolone 6a–g and salcomine (1.8 eq.) were introduced
into 5 mL DMF under oxygen and the solution was stirred at room temperature for 27 h. The reaction
mixture was then poured into ice water to afford the crude product as a red powder after filtration. The
filtrate was extracted with diethyl ether and combined with the precipitate. The crude product 7a–g was
finally purified by column chromatography on silica (ethyl acetate/cyclohexane: 1/2).
5-Isopropyl-7-methyl-5H-indeno[1,2-b]indole-6,9,10-trione (7a). Red powder; 67% yield; mp 235 °C;
IR (KBr,  cm−1) 1721 (C=O), 1660 (C=O), 1647 (C=O); 1H-NMR (CDCl3,  ppm, J Hz) 7.62 (d, 1H,
J = 7.2, H-1), 7.43 (dt, J = 0.9 and 7.5 1H, H-4), 7.42 (m, 1H, H-3), 6.50 (q, J = 1.5, 1H, H-8), 5.83 (br
13
s, 1H, NCHMe2), 2.10 (d, 3H, J = 1.5, CH3-7), 1.69 (d, 6H, J = 7.1, NCHMe2); C RMN (CDCl3,, 
ppm) 183.6 (C-10), 181.8 (C-9), 178.6 (C-6), 154.8 (C-4b), 146.7 (C-5a), 140.2 (C-4a), 134.4 (C-10a),
134.1 (C-9b), 133.3 (C-4), 132.7 (C-8), 130.0 (C-2), 124.7 (C-1), 123.3 (C-9a), 121.6 (C-7), 121.2
(C-3), 50.6 (NCHMe2), 21.1 (2CH3), 16.4 (CH3); MS-ESI (m/z) 633.2 [2 M+Na]+, 328.1 [M+Na]+, 306.1
[M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for C19H15NNaO3 328.0944, found 328.0945.
5,7-Diisopropyl-5H-indeno[1,2-b]indole-6,9,10-trione (7b). Red powder; 74% yield; mp 230.4 °C; IR
(KBr,  cm−1) 1716 (C=O), 1656 (C=O), 1641 (C=O); for complete NMR data see [22]; MS-ESI (m/z)
689.7 [2 M+Na]+, 356.3 [M+Na]+, 334.4 [M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for C21H19 NNaO3
356.1257, found 356.1254.
5-Isopropyl-7-phenyl-5H-indeno[1,2-b]indole-6,9,10-trione (7c). Red powder; yield 44%; mp 230.4 °C;
IR (KBr, cm−1) 1715 (C=O), 1658 (C=O), 1591 (C=O); 1H-NMR (CDCl3,  ppm, J Hz) 7.65 (m, 1H,
H-1), 7.50–7.45 (m, 7H, Ar-H), 7.35 (m, 1H, Ar-H), 6.75 (s, 1H, H-8), 5.86 (br s, 1H, NCHMe2), 1.72
(d, 6H, J = 7.0, 2CH3); 13C-NMR (CDCl3,,  ppm) 183.8 (C-10), 181.9 (C-9), 177.9 (C-6), 155.5 (C-4b),
147.6 (C-5a), 140.4 (C-4a), 134.6 (C-10a), 134,4 (C-9b), 133.9 (C-1′), 133.6 (C-8), 133.2 (C-4), 130.3
(C-2), 130.1 (C-4′), 129.9 (C-3′ et C-5′), 128.7 (C-2′ et C-6′), 125.0 (C-1), 121.9 (C-3, C-9a and C-7),
51.0 (NCHMe2), 21.3 (2 CH3); MS-ESI (m/z) 757.3 [2 M+Na]+, 390.2 [M+Na]+, 368.1 [M+H]+; HRMS-
ESI (m/z) [M+Na]+ calcd for C24H17NNaO3 390.1101, found 390.1094.

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7-(4-Fluorophenyl)-5-isopropyl-5H-indeno[1,2-b]indole-6,9,10-trione (7d). Red powder; 58% yield;
mp 258.1 °C; IR (KBr,  cm−1) 1716 (C=O), 1650 (C=O), 1599 (C=O); for complete NMR data see [22];
MS-ESI (m/z) 793.3 [2 M+Na]+, 408.1 [M+Na]+, 386.2 [M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for
C24H16FNNaO3 408.1006, found 408.1003.
7-Furan-2-yl-5-isopropyl-5H-indeno[1,2-b]indole-6,9,10-trione (7e). Red orange powder; 58% yield;
mp 216.1 °C; IR (KBr,  cm−1) 1713 (C=O), 1655 (C=O), 1586 (C=O); 1H-NMR (CDCl3,  ppm, J Hz)
7.62 (d, 1H, J = 7.2, H-Ar), 7.55 (d, 1H, J = 1.7, H-5′), 7.50 (d, 1H, J = 3.4, H-3′), 7.43 (m, 2H, H-Ar),
7.30 (m, 1H, H-Ar), 6.98 (s, 1H, H-8), 6.57 (dd, 1H, J = 1.8 and 3.5, H-4′), 5.85 (br s, 1H, NCHMe2),
1.72 (d, 6H, J = 7.1, 2 CH3); 13C-NMR (CDCl3,  ppm) 183.4 (C-10), 181.4 (C-9), 176.2 (C-6), 155.0
(C-4b), 147.1 (C-5a), 145.0 (C-5′), 140.1 (C-4a), 134.7 (C-10a), 134.2 (C-2′), 133.9 (C-9b), 133.3
(C-4), 129.9 (C-2), 126.1 (C-8), 124.6 (C-1), 123.5 (C-9a), 121.7 (C-3 and C-7), 117.9 (C-4′ or C-3′),
113.2 (C-3′ or C-4′), 50.4 (NCHMe2), 21.0 (2 CH3); MS-ESI (m/z) 737.2 [2 M+Na]+, 380.1 [M+Na]+,
358.1 [M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for C22H15NNaO4 380.0893, found 380.0891.
5-Isopropyl-8-methyl-5H-indeno[1,2-b]indole-6,9,10-trione (7f). Red powder; 95% yield; IR (KBr, 
1
cm−1) 1717 (C=O), 1642 (C=O), 1656 (C=O); H-NMR (CDCl3 + TFA,  ppm, J Hz) 7.55 (d, 1H,
J = 7.1, Ar-H), 7.47–7.40 (m, 2H, 2 Ar-H), 7.30 (m, 1H, Ar-H), 6.51 (s, 1H, H-7), 5.71 (m, 1H,
NCHMe2), 2.1 (s, 3H, CH3-8), 1.68 (d, 6H, J = 7.0, NCHMe2); 13C-NMR (CDCl3 + TFA,  ppm) 185.8
(C-10), 182.4 (C-9), 179.0 (C-6), 155.8 (C-4b), 145.6 (C-5a), 139.6 (C-4a), 134.3 (C-7), 134.1 (C-4),
133.9 (C-9b), 130.4 (C-2), 125.1 (C-1), 122.1 (C-3), 121.6 (C-9a), 50.8 (NCHMe2), 20.8 (2CH3), 15.2
(CH3); MS-ESI (m/z) 633.3 [2 M+Na]+, 328.1 [M+Na]+, 306.2 [M+H]+; HRMS-ESI (m/z) [M+Na]+
calcd for C21H19NNaO3 328.0944, found 328.0955.
5,8-Diisopropyl-5H-indeno[1,2-b]indole-6,9,10-trione (7g). Red powder; 14% yield; mp 226 °C; IR
1
(KBr,  cm−1) 1710 (C=O), 1643 (C=O), 1604 (C=O); H-NMR (CDCl3,  ppm, J Hz) 7.47 (d, 1H,
J = 7.0, H-1), 7.30–7.24 (m, 2H, Ar-H), 7.15 (m, 1H, Ar-H), 6.22 (s, 1H, H-7), 5.64 (br s, 1H, NCHMe2),
2.99 (sept, 1H, J = 6.7, CHMe2-8), 1.53 (d, 6H, J = 7.0, 2 CH3), 1.01 (d, 6H, J = 6.8, 2 CH3); 13C-NMR
(CDCl3,  ppm) 183.6 (C-10), 181.3 (C-9), 179.0 (C-6), 154.5 (C-4b), 154.1 (C-5a), 139.9 (C-4a), 134.1
(C-10a), 133.5 (C-9b), 133.2 (C-4), 131.7 (C-7), 129.8 (C-2), 124.5 (C-1), 123.5 (C-9a), 121.3 (C-3),
50.1 (NCHMe2), 26.5 (CHMe2-8), 21.7 (2 CH3), 20.9 (2 CH3); MS-ESI (m/z) 689.3 [2 M+Na]+, 356.2
[M+Na]+, 334.2 [M+H]+; HRMS-ESI (m/z) [M+Na]+ calcd for C21H19NNaO3 356.1257, found 356.1253.
3.3. Biological Activities
3.3.1. Preparation of Recombinant Human CK2 Holoenzyme
The preparation of the human recombinant CK2 holoenzyme was performed according to a protocol
described previously [29]. For the expression of the α-subunit (CSNK2A1) and β-subunit (CSNK2B) of
the human protein kinase CK2 a pT7-7 expression system in Escherichia coli BL21(DE3) was used.
Newly transformed starter cultures were grown overnight at 37 °C in LB-medium to the stationary phase.
New medium was inoculated with the separate starter cultures for both subunits and cultivated until an
OD500 of 0.6 was reached. Expression was induced by addition of IPTG (1 mM final concentration) and

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carried out at 30 °C for 5–6 h for CSNK2A1 and at 37 °C for 3 h for CSNK2B. Cells were harvested by
centrifugation (6000× g for 10 minutes at 4 °C) and disrupted by sonication (three times 30 seconds on
ice). Preparations were then centrifuged to remove the cells debris and the bacterial extracts for both
subunits were combined and purified by a three-column procedure. Fractions exhibiting CK2 activity
were combined and analyzed by SDS-PAGE and Western Blot.
3.3.2. Capillary Electrophoresis Based Assay for the Testing of Inhibitors of the Human CK2
Testing of the inhibitors of the human CK2 was performed by the recently established capillary
electrophoresis based CK2 activity assay of Gratz et al. [23]. Therefore, 2 µL of the dissolved inhibitors
(stock solution in DMSO) were mixed with 78 µL of CK2 supplemented kinase buffer which was
composed of 1 µg CK2 holoenzyme, 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2 and 1
mM DTT. The reaction was initiated by the addition of 120 µL assay buffer, which was composed of 25
mM Tris/HCl (pH 8.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µM ATP and 0.19 mM of the
substrate peptide RRRDDDSDDD. The reaction was carried out for 15 minutes at 37 °C and stopped by
the addition of 4 µL EDTA (0.5 M). Subsequently the reaction mixture was analyzed by a PA800
capillary electrophoresis from Beckman Coulter (Krefeld, Germany). Acetic acid (2 M, adjusted with
conc. HCl to a pH of 2.0) was used as the electrolyte for electrophoretic separation. The separated
substrate and product peptide were detected at 214 nm using a DAD-detector. Pure solvent was used as
negative control (0% inhibition), assays devoid of CK2 were used as positive control (100% inhibition).
For primary testing an inhibitor concentration of 10 µM was used. Compounds that revealed at least 50%
inhibition at 10 µM were used for IC50 determinations. For the determination of IC50, inhibition was
determined using nine inhibitor concentrations ranging from 0.001 µM to 100 µM. IC50 were calculated
from the resulting dose-response curves.
3.3.3. Mode of Inhibition and Determination of Ki
The CE based CK2 activity assay as described was used to investigate whether 5a and 7a inhibit CK2
by an ATP competitive mode. For this purpose, enzymatic reactions and IC50 determinations were
performed as described in Section 3.3.2., but varying compositions of the assay buffer with respect to
the ATP concentration were used. IC50 values were measured at six different final ATP concentrations
ranging from 6 to 600 µM. The obtained IC50 values were subsequently used to determine the Ki values
of 5a and 7a. For this purpose, reaction velocities at different inhibitor concentrations were plotted in a
Lineweaver-Burk diagram against the varied ATP concentrations and the corresponding ꢀ1 ꢁꢃꢂꢄꢄ were
⁄
obtained by the intercepts with the abscissa. Finally the ꢁꢂꢃꢄꢄ values as obtained were plotted against the
different inhibitor concentrations. By this method, the Ki values with a negative sign were given by the
intercepts of the linear slope with the abscissa.
3.3.4. Cytotoxicity Assay on Cell Lines
All cells were grown in DMEM medium supplemented with 10% fetal calf serum, glutamine (2 mM),
penicillin (100 U/mL), and streptomycin (50 μg/mL) at 37 °C under a humidified 5% CO2 atmosphere
(Gibco). The assays were performed in sterile 96-wells microtiter plates (Falcon). Cells were plated at

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500 (NIH-3T3), 750 (WI-38) or 1000 (MEF; HEK293T) cells/100 µL/well. Drug solutions (10 μL per
well) of appropriate concentration (prepared by dilution in the culture medium from 2 mM stock
acetonitrile solution) were added 24 h after cell seeding. For ellagic acid and emodin, DMSO was used
to prepare the stock solution. After 96 h, the cytotoxicity is evaluated using the Cell Proliferation Reagent
WST-1 following the procedure described by the manufacturer (Roche Applied Science). The drugs
were assayed at 10, 5, 1, 0.2 and 0.04 µM in triplicate. The vehicle was used as control. The experiments
were repeated three times.
For each compound tested, the EC50 (concentration of drug needed to inhibit cell growth/viability by
50%) was generated from the dose-response curves for each cell line.
3.4. Crystallography
The structure of compound 4d’ has been established by X-ray crystallography (Figure 4). Yellow
single crystal (0.76 × 0.13 × 0.09 mm3) of 4d’ was obtained after 10 days at 17 °C by slow evaporation
from a methanol/chloroform (80/20) solution. It crystallized in the monoclinic space group P21/c,
a = 13.103(2) Å, b = 10.0799(11) Å, c = 16.411(2) Å, α= 90°, β= 95.439(13)°, = 90°, V = 2157.8(5)
Å3, Z = 4, (calcd) = 1.310 Mg.m−3, FW = 425.44 for C24H24FNO5, F(000) = 896, final R indexes were
R1 = 0.0519 (I > 2σ(I)) and wR2 = 0.1437 (all data). Crystallographic data were acquired on a
Bruker-Nonius Kappa CCD. Full crystallographic results have been deposited at the Cambridge
Crystallographic Data Centre (CCDC-977015), UK, as supplementary Material [30]. The data were
corrected for Lorentz and polarization effects and for empirical absorption correction [31]. The structure
was solved by direct methods with ShelxS 2013 [31] and refined using ShelxL 2013programs [32], both
pieces of software were found within the OLEX2 package [21].
3.5. Molecular Modeling
All four inhibitors 5a, 7a, 5h [13], and 7h [14] were structurally aligned using the program
vROCS [25]. For this a pharmacophore match based on shape and H-bond donor and acceptor groups
with the co-crystallized inhibitor (from PDB code: 3OWJ) was performed. Subsequently, the inhibitors
were energy minimized in the binding region of CK2 (from PDB code: 3OWJ) using the program
Moloc [26].
4. Conclusions
A library of twenty-one new indeno[1,2-b]indole derivatives was synthesized and evaluated as
inhibitors of human protein kinase CK2. This preliminary SAR study confirmed that compounds with
substituents at position 5 (C-ring) and position 7 (D-ring) of the indeno[1,2-b]indole scaffold showed
CK2 inhibitory activity at micromolar and submicromolar range of concentrations (e.g., compounds 5a,
5b and 7a have IC50 values of 0.17, 0.61 and 0.43 µM, respectively). In comparison with three standards,
we showed that compound 5b and 7a are as active as emodin but less potent than ellagic acid and TBB.
However compound 5a is a better CK2 inhibitor than emodin (0.17 versus 0.58 µM). Furthermore, the
low cytotoxicity of compound 5a against four cell lines encourages us to continue our medicinal
chemistry investigations. For example 5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-diones 5, 9-hydroxy-

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5H-indeno[1,2-b]indol-10-ones 6 and 5H-indeno[1,2-b]indole-6,9,10-triones 7 were included in
building a 3D-QSAR model. The third generation of indeno[1,2-b]indoles as CK2 inhibitors are actually
synthesized and evaluated.
Acknowledgments
We acknowledge financial support to FAC by the “Bonus Qualité Recherche” (BQR) of the
University Claude Bernard Lyon 1, by the “Cluster 5 Chimie Durable et Chimie pour la Santé” of the
Region Rhône-Alpes and by the “ARC 1 Santé” of the Region Rhône-Alpes. The “Institut des Sciences
Pharmaceutiques et Biologiques” (ISPB) is also gratefully acknowledged for the funding of an
uHPLC/DAD/MS system.
M. Le Borgne thanks “Cancéropôle Lyon Auvergne Rhône-Alpes” (CLARA) and “Université France
Allemagne” (UFA) for their funding of ChemBioInteract network.
M. Le Borgne and Dr. Laurent Ettouati are grateful to Christine Ranquet for her assistance in the
stock management of the small molecule library of EA 4446 B2C. We also thank ChemAxon for
providing us a license to their chemoinformatics software.
Author Contribution
M.L.B. and J.J. planned and organized the whole study; F.A., L.E. and Z.B. carried out syntheses;
A.B. performed the CK2 assay; J.G.D. performed the cytotoxicity assay; C.G.W.G. and H.G. performed
the molecular modeling studies; N.P., M.M. and J.G. performed the crystallography part; L.E. and B.F.
performed NMR experiments and analyzed the data. All authors contributed to the writing of the paper
and approval of the versions.
Conflict of Interest
The authors declare no conflict of interest.
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