Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives on prostate cancer in vitro. Potential effect against MMP-9

Article abstract of DOI:10.1016/j.ejmech.2015.04.023

A highly regiospecific synthesis of a series of indenoindoles is reported, together with X-ray studies and their activity against human prostate cancer cells PC-3 and LNCaP in vitro. The most effective compound 7,7-dimethyl-5-[(3,4-dichlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9bhexahydro-indeno[1,2-b]indole-9,10-dione 7q reduced the viability in both cell lines in a time and dose-dependent manner. Inhibitory effects were also observed on the adhesion, migration, and invasion of the prostate cancer cells as well as on clonogenic possibly by inhibition of MMP-9 activity. Molecular docking of 7q and 6k into MMP-9 human active site was also performed to determine the probable binding mode.

Full text of DOI:10.1016/j.ejmech.2015.04.023

Accepted Manuscript
Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives on prostate
cancer in vitro. Potential effect against MMP-9
Gricela Lobo, Melina Monasterios, Juan Rodrigues, Neira Gamboa, Mario V.
Capparelli, Javier Martínez-Cuevas, Michael Lein, Klaus Jung, Claudia Abramjuk,
Jaime Charris
PII:
S0223-5234(15)00272-X
10.1016/j.ejmech.2015.04.023
EJMECH 7838
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2014
Revised Date: 8 April 2015
Accepted Date: 9 April 2015
Please cite this article as: G. Lobo, M. Monasterios, J. Rodrigues, N. Gamboa, M.V. Capparelli, J.
Martínez-Cuevas, M. Lein, K. Jung, C. Abramjuk, J. Charris, Synthesis, crystal structure and effect of
indeno[1,2-b]indole derivatives on prostate cancer in vitro. Potential effect against MMP-9, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.04.023.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives
on prostate cancer in vitro. Potential effect against MMP-9.
Gricela Lobo, Melina Monasterios, Juan Rodrigues, Neira Gamboa, Mario V. Capparelli,
Javier Martínez-Cuevas, Michael Lein, Klaus Jung Claudia Abramjuk, Jaime Charris.
,
O
O
HO
MMP-9
R
1
N
OH
R
2
MW Control
7q
R
3
R
4

ACCEPTED MANUSCRIPT
Synthesis, crystal structure and effect of indeno[1,2-b]indole derivatives on prostate
cancer in vitro. Potential effect against MMP-9.
a
a
b
b
Gricela Lobo , Melina Monasterios , Juan Rodrigues , Neira Gamboa , Mario V.
c
d
e,f
e,f
Capparelli , Javier Martínez-Cuevas , Michael Lein , Klaus Jung
Abramjuk , Jaime Charris *
,
Claudia
e,g
a,
a,b
Laboratorio de Síntesis Orgánica, Unidad de Bioquímica, Facultad de Farmacia,
Universidad Central de Venezuela, Apartado 47206, Los Chaguaramos, 1041-A Caracas,
c
Venezuela. Unidad de Estructura Molecular, Fundación Instituto de Estudios Avanzados
d
(
IDEA), Apartado 17606, Caracas 1015-A, Venezuela. Servicio de Difracción de Rayos X,
e
Universidad Autónoma de Barcelona, 08193 Barcelona, Spain. Department of Urology,
University Hospital Charité, Campus Mitte, Schumannstrasse 20/21, 10117 Berlin,
Germany. Berlin Institute for Urologic Research, Schumannstrasse 22/21, 10117 Berlin,
f
g
Germany. Department of Experimental Medicine (FEM), University Hospital Charité,
Campus Mitte, Charitéplatz 1, 10117 Berlin, Germany.
1
. Introduction
Prostate cancer (PCa) represents the second most frequently diagnosed malignancy in men
and the sixth leading cause of cancer death worldwide [1]. Many antitumor compounds
have been developed, but treatment options in patients with advanced stage of PCa have
limited efficacy and are associated with significant side effects and reduced quality of life.
Therefore, developing new alternatives for this malignancy is of great importance. The
ability of indenoindoles as potential lipid peroxidation inhibitors [2], potassium channel
openers [3], DNS intercalators and topoisomerase II inhibitors [4], estrogenic agents [5], or
inhibitors of proteins kinase CK2 [6,7], have been reported. They are considered as a novel
class of potent inhibitors of the human proteins kinase CK2, which is a second-messenger
and phosphorylation independent constitutively active S/T protein kinase. Up to date the
literature reports more than 400 potential physiological targets, showing their important
roles in a variety of non cancer-related diseases (such as neurodegenerative disorders,

ACCEPTED MANUSCRIPT
inflammatory processes, angiogenesis-related diseases and viral infections), as well as in
different types of cancer ( prostate, colon, breast and lung) [8-11].
However, there are no data available about the effect of the indenoindoles structures on the
extracellular matrix (ECM). ECM proteins play important roles during proliferation,
adhesion, migration and invasion of cancer cells as well as in angiogenesis in developing
tumors [12-14]. Each of these events is regulated by the proteolysis of the ECM
components and, thus, by matrix metalloproteinases (MMPs). MMP-9, which activity is
correlated with the progression and the degree of malignancy, contributes to the invasion
and metastatic spread of tumor cells, being recognized as a potential target for the
development of new anti-cancer drugs [15,16]. On the other hand , special attention has
been focused on the indole structure as responsible of the biological properties of some
heterocyclic compounds. Thus, isatin derivatives which contain two carbonyl groups on
the indole core have been tested as potential MMPs inhibitors; however, theses analogs did
not show important effects on the metalloproteinase activities with no significant
inhibitions reported [17].
Despite recent advances in molecular biology and the progress in combinatorial synthetic
methodology, the rate of introduction of new pharmaceutical products has decreased
markedly over the past two decades. Structural diversity in a focused collection of potential
therapeutics is believed to increase the positive hit rate. Most pharmaceutical products in
use are still small synthetic organic molecules that often contain a heterocyclic ring.
However, the range of easily accessible and suitably functionalized heterocyclic building
blocks for the synthesis of structurally diverse libraries is rather limited. The development
of new, rapid, and clean synthetic routes toward focused libraries of such compounds is
therefore of great importance to both medicinal and synthetic chemists. As part of our

ACCEPTED MANUSCRIPT
general interest in the preparation of heterocyclic compounds with potential anticancer
activities [18-23], herein we report the synthesis of indeno[1,2-b]indole derivatives, and
their characterization by X-ray diffraction analysis. Docking simulation was performed
using X-ray crystallographic structure of MMP-9 in complex with the most active inhibitor
to explore the binding mode of the compound at the active site.
2
2
. Results and discussion
.1. Synthesis
Among the existing procedures for the preparation of indenoindoles, the Fischer
indolization starting with an indanone, via the respective phenylhydrazones, serves as the
most common method [24,25]. Recently, two new syntheses by transformation and
reduction of 2-nitrobenzylidenephtalide, generated either by intramolecular cyclization of
2
-(2-nitrophenylethyl)benzoic acid [26], or by reaction of a phthalidyl-phosphonium
bromide with 2-nitrobenzaldehyde [27] and cyclization of the resulting amino compounds,
have been published. The formation of vic-dihydroxy-indenoindolones by the reaction
of ninhydrine 1 with aliphatic, and aromatic amines, or alicyclic, and cyclic enaminones has
been reported elsewhere [28-33].
The intermediates 4 and 5 were achieved according to published procedures by refluxing
cyclohexane-1,3-dione with aromatic amine and a catalytic amount of p-toluensulfonic acid
in toluene and removal of water as an azeotrope with a Dean-Stark water trap [34]. To
prepare the vic-dihydroxy-indenoindiole 6a-q and 7a-q derivatives, a solution of equimolar
amounts of the corresponding enaminone 4 and 5 and ninhydrin 1 in chloroform, stirred at
room temperature for 24 h. TLC (EtAc:Hx 1:1) showed, that only one compound was
1
13
produced (cf. Scheme). Spectroscopic data ( H and C NMR) revealed that this was a

ACCEPTED MANUSCRIPT
1
cyclization product with two H resonances due to OH functionalities at 5 and 7 ppm and
1
3
two C resonances at 83 and 96 ppm approximately.
.2. Biological
2
The effect of compounds 6p-q and 7p-q, on cell viability showed a dose-dependent
response in all the cancer cell lines tested with inhibition in viability from 5µg/ml onwards.
Compound 7q showed the maximal cytotoxic effects. The structure-activity relationship
studies on these compounds revealed that the presence of a dimethyl at position 7 and
dichloro phenyl at position 5 are determinant for the cell viability since compounds which
lack these substitutions were less toxic to the tumor cell lines. In addition, the anti-tumor
activity is also affected by the presence of the H group at position 7 and methoxy and
methyl phenyl groups at position 5. In this context, compound 7q was selected for further
evaluations. This derivative was also active in other human and non-human tumor prostate
cell lines, such as LNCaP and MatLyLu (Table I, Figure 1).
Insert Table I here
Insert Figure 1 here
To examine the effect of this compound on the migration/motility properties of the tumor
cells, the wound-healing assay was used [35]. Compound 7q-free cultures of PC-3 cells
(control vehicle) largely displayed wound recovery within 24 h and cells migrated to the
wound (Figure 2a, 2c). The ability to close the scrape wound was significantly reduced by
compound 7q treatment at its cytotoxic IC50 (Figure 2b, 2d). On the other hand, the results
of cell invasion assay using the Boyden chamber coated with Matrigel revealed that this
compound also decreased the invasion in LNCaP cells after 18 h of incubation (Figure 3).
Insert Figure 2 here

ACCEPTED MANUSCRIPT
Insert Figure 3 here
As shown in Figure 4, compound 7q decreased MMP-9 activity in PC-3 cells at its
cytotoxic IC . The inhibition on this metalloprotease would suggest a consequent
5
0
inhibition on the ability of cells to migrate and invade surrounding areas. No MMP2
activity was reported for these cell lines, as previously shown [36].
Insert Figure 4 here
The anchorage-independent growth is considered an in vitro test, which correlates with
tumorigenesis in vivo [37]. Thus, we examined the ability of PC-3, LNCaP and MatLyLu
cells to grow in a semisoft agar medium after 7q treatment. Cell colonies significantly grew
in vehicle-treated agar on day 14 as previously reported [38]. On the other hand, colonies
were either reduced in size and number or completely absent in cells treated with
compound 7q (Figure 5, Table II).
Insert Table II here
Insert Figure 5 here
New tissue formation, invasion and tumor cell metastasis all depend on cell motility and
migration [39]. Cell migration constitutes an attractive target for the development of
potential antitumor compounds and our results showed that the analog 7q could be
considered as an inhibitor of cell migration after 24 hour-incubation, which could lead to
further decreases in cell invasion and metastasis prevention. Indeed, the ability of this
structure to inhibit cell invasion was also confirmed.
The degree of tumor malignancy is related to the ability of neoplastic cells to invade other
tissues and spreading to other organs. An important role is made by different
metalloproteinases such as MMP-9 which degrades the extracellular matrix (ECM) that is

ACCEPTED MANUSCRIPT
required for migration, invasion and metastasis [40]. This secreted gelatinase is highly
expressed in cancer cells, making it valuable in finding patients who are at high risk of
tumor development [41], thus, compounds that inhibit this protease could represent possible
structures against cancer. Our results showed that compound 7q decreased the activity of
this gelatinase, suggesting that this enzyme could be a molecular target of this structure and
proposing a possible mechanism for its antitumor action.
On the other hand, a specific feature of tumor cells is that they are able to grow in soft agar.
This phenotypic transformation has been positively correlated with the in vivo tumor
growth and metastasis [42]. Our results indicated that a suspension of PC-3, LNCaP or
MatLyLu cells with vehicle successfully developed into relatively large colonies, whereas
those cultured with the compound 7q resulted in fewer and smaller-sized colonies or even a
complete abolishment in the development of colonies, indicating a loss of the transformed
phenotype and providing possible insights about the behavior of this compound on
metastasis in vivo.
2
.3. X-ray crystallography
To confirm the proposed molecular structures, X-ray single-crystal structure analyses of
one compound of each series, viz. 6k and 7k, were carried out. The molecular structures are
shown in Figure 6 and the relevant bond lengths and angles are given in Table III.
Insert Figure 6 here
Insert Table III here
The two stereocenters (C1 and C2) have the same configurations in 6k and 7k. Since both
compounds crystallize in centrosymmetric space groups (cf. Table V), both crystals consist
of equimolar mixtures of the RR and SS diastereomers (Figure 6 shows the RR
configurations).

ACCEPTED MANUSCRIPT
As expected, both compounds display very similar molecular geometries, which are also
closely related the methoxy derivative previously reported by us [19]. Actually, 7k is
isostructural with the OMe derivative. Within experimental error (i.e. three e.s.d.’s) bond
distances are equal in both compounds; the only exception being C5-C6 and C6-C7, which
are significantly longer in 7k due to the C6 methyl substituents (cf. Table IV). The
tetracyclic systems are V-shaped, with the two 5-membered rings making dihedral angles
of 113.75(7)° (6k) and 114.91(7)° (7k). The N-bonded phenyl rings are approximately
perpendicular to the heterocyclic rings [dihedral angles: 74.85(7)° (6k) and 78.52(7)° (7k)].
The phenyl rings are quite planar (r.m.s. deviations: 0.0037- 0.0089 Å) and the two 5-
membered rings are approximately planar (r.m.s. deviations: 0.022-0.029 Å). The C3-C4-
C5-C6-C7-C8 rings display a semi-sofa conformation (i.e. intermediate between boat and
sofa), with distances from the C4, C5, C7, C8 mean plane of 0.166(2) Å and 0.142(2) Å for
the C3 atoms, and of 0.614(2) Å and 0.599(2) Å for the C6 atoms. The puckering
parameters [43] are: q = 0.4522(16) Å, q = -0.1927(16) Å, φ = 3.3(2)°, Q = 0.4915(17) Å
2
3
2
for 6k, and q
k.
2
= 0.4310(146) Å, q
3
= 0.-1911(14) Å, φ
2
= 6.15(18)°, Q = 0.4715(15) Å) for
7
The molecules form O-H···O(keto) intramolecular hydrogen bonds (cf Figure 6). In
addition, in the crystal structure there are intermolecular hydrogen bonds of the types O-
H···O(keto) and (possible) weaker C-H···O(hydroxyl) and C-H···O(keto) (cf. Table IV),
which link the molecules to form a three dimensional network.
Insert Table IV here
2
.4. Molecular docking simulations

ACCEPTED MANUSCRIPT
In order to seek the structure-activity relationships of these synthetic derivative, molecular
docking of the most potent inhibitor 7q and the inactive compound 6k into the catalytic
domain of the MMP-9 enzyme PDB ID: 1GKC [44] (Figure 7) was performed. The
catalytic centre of the active-site includes a zinc ion coordinated by three histidine residues
(
401, 405 and 411) and a glutamic acid residue (402). The main differences between the
catalytic domains of various MMPs occur in the S1’subsite or selectivity pocket (residues
25- 431 in MMP-9). It has been found that chain A is participating in the interactions.
4
Insert Figure 7 here
The highly active compound 7q binds in the catalytic domain through three hydrogen bond.
Carbonyl group of indene rings acts as receptor of the hydrogen bond formed with the NH
group of His 401 (distance 1.88 Å). This interaction is the most important. Furthermore the
dichlorophenyl group is placed into the hinge region (residues 420-431) (Figure 8). Also it
has a very small overall interaction energy (-12.88 kcal/mol). These indicate that this
compound binds to MMP-9 S1’ subsite.
Insert Figure 8 here
The binding energy of compound 6k, which has lower activity, is also smaller (-8.93
kcal/mol). This compound forms several hydrogen bonds but places the phenyl group
outside S1’ pocket.
Insert Figure 9 here
The docking results revealed that His 401 residue located in the catalytic centre of MMP-9
is important to your interaction with 7q, which were stabilized by two hydrogen bonds two
other residues and hydrophobic interactions to the Pro 430 residue within the S1’ pocket.
The compound 7q that binds in the S1’subsite can be a specific inhibitor of MMP-9 mainly
through an indirect mechanism by interacting far from the coordinating Zinc atom.

ACCEPTED MANUSCRIPT
3
. Conclusions
In conclusion, compounds were easily synthesized and with highly regiospecificity. The X-
ray diffraction studies clearly confirmed the structure of the compounds. All tested
compounds proved to be moderately active and, except 7q, showed cytotoxic effects in
different human and non-human prostate cancer cell lines. The mechanism of the antitumor
activity of this structure seems to be related to the decrease of migration, invasion and
clonogenicity possibly by inhibition of MMP-9. Of notice is the inhibition of the
clonogenic potential which might lead to a possible in vivo anticancer effect of this
compound. The structure-activity relationship studies on these compounds revealed that the
presence of a dimethyl at 7 positions and dichloro phenyl at 5 positions are determinant for
the cell viability since compounds which lack these substitutions were less toxic to the
tumor cell lines. Docking studies shows a set of interactions in specific sites that are import
for an inhibitory activity. Further studies will be needed to confirm this hypothesis.
4
. Experimental
4
.1. Chemistry
Melting points were determined on a Thomas micro hot stage apparatus and are
uncorrected. The IR spectra (in KBr pellets) were recorded on a Shimadzu model 470
1
13
spectrophotometer. The H NMR, C NMR spectra were recorded using a Jeol Eclipse 270
(
270 MHz/67.9 MHz) spectrometer using CDCl or DMSO , and are reported in ppm
3
d6
downfield from the residual CHCl or DMSO. Elemental analyses were obtained using a
3
Perkin Elmer 2400 CHN elemental analyzer, the results were within ± 0.4% of the
predicted values. Chemical reagents were obtained from Aldrich Chemical Co, USA. All
solvents were distilled and dried in the usual manner. The intermediates 4 and 5 were
achieved according to published procedures [34].

ACCEPTED MANUSCRIPT
4
4
.1.2. General procedure to obtain 5-(substitutedphenyl)-(4bRS,9bRS)-dihydroxy-
b,5,6,7,8,9b-hexahydroindeno[1,2-b]indole-9,10-dione derivatives, 6a-q, 7a-q.
Enaminones (1.35 mmol) and 1 (1.12 mmol) were dissolved in chloroform 5 mL and stirred
at rt. (24 h). The solvent was evaporated to dryness under reduced pressure, the solid thus
obtained was collected by filtration, washed with diethyl ether and recrystallized from
ethanol to afford the title compound.
4
.1.2.1. 5-(2-methoxyphenyl)-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydro-indeno[1,2-
b]-indole-9,10-dione, 6a
Yield: 68% , mp: 234-236 C, IR cm : 3504, 3120 (OH), 1718 (C=O); NMR- H, ppm δ:
.92 (m, 2H, H ), 2.12 (m, 2H, H ), 2.35 (m, 2H, H ), 3.95 (s, 3H, OCH ), 5.74 (s, 1H,
OH), 6.64 (dd, 1H, H , J: 7.9, 2.3Hz), 6.81(dd, 1H, H3´, J:7.8, 2.1Hz), 6.90(t, 1H, H4´, J:7.6,
.1Hz), 7.12(t, 1H, H , J:7.8, 2.2Hz), 7.8 (m, 3H, OH, Ar), 7.51 (t, 1H, H , J:7.6, 1.8Hz),
o
-1
1
1
7
6
8
3
4
2
7
1
1
3
4
5
´
2
1
3
.83 (dd, 1H, H , J: 8.2, 2.1Hz). NMR- C, ppm δ: 21.8, 24.1, 37.3, 55.9, 82.7, 96.8, 106.3,
1
14.7, 115.1, 124.7, 125.2, 127.0, 127.7, 129.8, 131.3, 135.7, 135.4, 149.9, 150.0, 167.7,
93.1, 197.5. Anal. C H NO : C, 70.02; H, 5.07; N, 3.71. Found: C, 69.92; H, 5.10; N,
2
2
19
5
.80 %.
.1.2.2. 5-(3-methoxyphenyl)-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydro-indeno[1,2-
b]-indole-9,10-dione, 6b
Yield: 80%, mp: 128-130 C, IR cm : 3504, 3152 (OH), 1712 (C=O); NMR- H, ppm δ:
.90 (m, 2H, H ), 2.22 (m, 4H, H ), 3.83 (s, 3H, OCH ), 5.52 (brs, 1H, OH), 6.79(d, 1H,
o
-1
1
1
7
6, 8
3
H , J: 7.9Hz), 6.90 (m, 2H, Ar), 7.03(d, 1H, H , J:7.9Hz), 7.13(m, 1H, H5´,2´), 7.45(m, 2H,
4
´
6´
1
3
H ), 7.81 (d, 1H, H , J: 7.6Hz). NMR- C, ppm δ: 21.8, 23.8, 36.7, 55.6, 82.9, 96.3,
2
´,3
1
1
06.3, 114.5, 114.6, 124.6, 125.2, 126.4, 127.9, 130.3, 131.0, 135.0, 135.3, 147.8, 159.8,

ACCEPTED MANUSCRIPT
1
5
4
67.2, 193.1, 197.9. Anal. C H NO : C, 70.02; H, 5.07; N, 3.71. Found: C, 70.11; H,
22
19
5
.18; N, 3.97 %.
.1.2.3. 5-(4-methoxyphenyl)-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydro-indeno[1,2-
b]-indole-9,10-dione, 6c
o
-1
1
Yield: 59% , mp: 204 C, IR cm : 3632, 3232 (OH),1708 (C=O); NMR- H, ppm δ: 1.92
(
m, 2H, H ), 2.17 (m, 4H, H ), 3.86 (s, 3H, OCH ), 5.60 (brs, 1H, OH), 6.90(dd, 1H, H ,
7 6, 8 3 4
J:8.2, 2.1Hz), 6.94 (d, 2H, H3´,5´, J:8.2Hz), 7.16(d, 2H, H2´,6´, J:78.2Hz), 7.48(t, 1H, H3,
J:7.6Hz), 7.49(s, 1H, OH), 7.50 (t, 1H, H , J: 7.6Hz), 7.83(dd, 1H, H , J:8.4, 2.0Hz). NMR-
2
1
1
3
C, ppm δ: 21.8, 23.9, 36.8, 55.6, 82.9, 96.4, 106.3, 114.5, 124.6, 125.3, 127.9, 130.0,
1
3
4
35.0, 135.3, 147.8, 159.8, 167.3, 193.1, 197.2. Anal. C22
.71. Found: C, 70.04; H, 5.08; N, 3.75 %.
5
H19NO : C, 70.02; H, 5.07; N,
.1.2.4.
5-(2,4-dimethoxyphenyl)-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydro-
indeno[1,2-b]-indole-9,10-dione, 6d
o
-1
1
Yield: 93%, mp: 188-190 C, IR cm : 3568, 2896 (OH), 1712 (C=O), NMR- H, ppm δ:
1
1
.94 (m, 2H, H ), 2.17 (m, 4H, H ), 3.85 (s, 3H, OCH ), 3.98 (s, 3H, OCH ), 5.27 (brs,
7 6, 8 3 3
H, OH), 6.36(d, 1H, H , J:1.9Hz), 6.61 (dd, 1H, H , J:7.9, 1.9Hz), 6.74 (m, 2H, Ar), 7.42
3
´
5´
1
3
(
s, 2H, OH, Ar), 7.58 (m, 1H, Ar), 7.81(dd, 1H, H
3.5, 36.8, 55.2, 55.7, 81.5, 95.5, 99.3, 104.6, 105.9, 116.9, 124.4, 124.6, 129.9, 132.0,
34.8, 135.0, 148.2, 157.0, 161.4, 193.4, 198.0. Anal. C H NO : C, 67.80; H, 5.20; N,
1
, J:8.0, 2.1Hz). NMR- C, ppm δ: 21.7,
2
1
3
4
2
3
21
6
.44. Found: C, 67.83; H, 5.26; N, 3.63 %.
.1.2.5. 5-(3,4-dimethoxyphenyl)-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydro-
indeno[1,2-b]-indole-9,10-dione, 6e
o
-1
1
Yield: 87%, mp: 220-222 C, IR cm : 3536, 3105 (OH), 1715 (C=O); NMR- H, ppm δ:
1
7 3 3
.96 (m, 2H, H ), 2.28 (m, 4H, H6, 8), 3.86 (s, 3H, OCH ), 3.94 (s, 3H, OCH ), 6.03 (brs,

ACCEPTED MANUSCRIPT
1
H, OH), 6.74(dd, 1H, H , J:8.4, 2.1Hz), 6.90 (d, 1H, H , J:8.6Hz), 7.01(s, 1H, H ), 7.03
4
5´
2´
(
dd, 1H, H6´, J:8.4, 1.5Hz), 7.46 (t, 1H, H
J:7.5Hz), 7.83(dd, 1H, H , J:8.3, 1.8Hz). NMR- C, ppm δ: 21.8, 23.9, 36.7, 56.1, 56.3,
3.1, 96.8, 106.2, 110.8, 113.5, 122.2, 124.4, 125.5, 128.1, 130.3, 135.1, 147.9, 149.2,
49.3, 167.3, 193.0, 197.8. Anal. C H NO : C, 67.80; H, 5.20; N, 3.44. Found: C, 68.04;
4 2
, J:7.5Hz), 7.48 (s, 1H, OH), 7.51 (t, 1H, H ,
1
3
1
8
1
23
21
6
H, 5.33; N, 3.57 %.
4
.1.2.6.
5-(2,5-dimethoxyphenyl)-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydro-
indeno[1,2-b]-indole-9,10-dione, 6f
o
-1
1
Yield: 87%, mp: 142-144 C, IR cm : 3504, 3104 (OH), 1712 (C=O); NMR- H, ppm δ:
1
3
.93 (m, 2H, H
7
), 2.15 (t, 2H, H6, J: 6.8Hz), 2.36 (t, 2H, H8, J: 6.7Hz), 3.10 (s, 3H, OCH
3
),
.86 (s, 3H, OCH
3
), 4.32 (brs, 1H, OH), 6.72(d, 1H, H , J:7.9Hz), 6.75 (d, 1H, H3´,
4
J:8.9Hz), 6.96 (dd, 1H, H , J: 8.9, 3.0Hz), 7.00(s, 1H, OH), 7.3 (d, 1H, H , J: 3.0Hz), 7.45
4
´
6´
1
3
(
m, 2H, H ), 7.83(d, 1H, H , J:7.9Hz). NMR- C, ppm δ: 21.7, 23.6, 36.3, 55.6, 56.0, 82.8,
2,3 1
9
1
5
4
6.3, 106.5, 112.4, 116.0, 116.7, 124.6, 125.0, 130.0, 134.9, 135.0, 148.1, 150.0, 154.0,
69.3, 192.9, 198.0. Anal. C H NO : C, 67.80; H, 5.20; N, 3.44. Found: C, 67.81; H,
23
21
6
.35; N, 3.59 %.
.1.2.7. 5-(3,4,5-trimethoxyphenyl)-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydro-
indeno[1,2-b]-indole-9,10-dione, 6g
o
-1
1
Yield: 46%, mp: 140-142 C, IR cm : 3525, 2986 (OH), 1723 (C=O); NMR- H, ppm δ:
1
.78 (m, 2H, H ), 2.10 (m, 4H, H ), 3.73 (s, 9H, OCH ), 5.95 (s, 1H, OH), 6.67(s, 2H,
7 6, 8 3
H2´,6´), 6.93 (d, 1H, H , J:7.7Hz), 7.13(s, 1H, OH), 7.55 (t, 1H, H , J:7.7Hz), 7.66 (t, 1H, H ,
4
3
2
1
3
J:7.7Hz), 7.73(d, 1H, H , J:7.6Hz). NMR- C, ppm δ: 22.1, 24.0, 37.6, 56.6, 60.7, 84.0,
1
9
7.0, 106.6, 108.2, 123.6, 125.8, 130.7, 131.7, 135.3, 135.4, 137.7, 147.8, 153.1, 165.5,

ACCEPTED MANUSCRIPT
1
3
4
90.2, 198.1. Anal. C H NO : C, 65.90; H, 5.30; N, 3.20. Found: C, 66.08; H, 5.37; N,
2
4
23
7
.47 %.
.1.2.8. 5-[(2-methylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno[1,2-
b]indole-9,10-dione, 6h
Yield: 55%, mp: 219-222 C, IR cm : 3560, 3184 (OH), 1718 (C=O); NMR- H, ppm δ:
.15 (s, 3H, CH ), 1.78 (m, 2H, H ), 1.98 (m, 2H, H ), 2.16 (m, 2H, H ), 5.91 (s, 1H, OH),
o
-1
1
1
3
7
6
8
6
.62 (d, 1H, H , J: 5.4), 7.27 (m, 2H, OH, Ar), 7.40 (m, 2H, Ar), 7.56 (m, 2H, H ), 7.65
4 2,3
1
3
(
m, 2H, Ar), 7.76 (d, 1H, H , J: 8.9). NMR- C, ppm δ: 17.0, 21.6, 23.8, 36.0, 83.1, 97.2,
1
1
1
5
4
06.4, 124.9, 126.0, 126.2, 127.4, 129.5, 129.7, 130.4, 131.0, 132.0, 135.2, 135.6, 148.0,
68.1, 192.4, 198.0. Anal. C22
.31; N, 4.07 %.
4
H19NO : C, 73.12; H, 5.30; N, 3.88. Found: C, 73.17; H,
.1.2.9. 5-[(3-methylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno[1,2-
b]indole-9,10-dione, 6i
Yield: 75%, mp: 211-212 °C, IR cm : 3424, 2992 (OH), 1708 (C=O); NMR- H, ppm δ:
-1
1
1
6
1
.79 (m, 2H, H ), 2.01 (m, 2H, H ), 2.11 (m, 2H, H ), 2.34 (s, 3H, CH ), 5.95 (s, 1H, OH),
7 6 8 3
.65 (d, 1H, H , J: 8.2), 7.08 (d, 1H, Ar, J: 7.7), 7.15 (s, 1H, Ar ), 7.17 (s, 1H, OH), 7.27 (d,
4
2
H, Ar, J: 7.7), 7.36 (t, 1H, Ar
5
1
, J: 7.7), 7.55 (m, 2H, H2,3), 7.71 (dd, 1H, H , J: 8.2, 1.7). ).
1
3
NMR- C, ppm δ: 18.5, 21.4, 24.0, 35.8, 83.5, 97.2, 107.5, 124.7, 125.3, 126.4, 129.1,
29.8, 130.0, 130.5, 134.9, 135.2, 135.4, 139.5, 147.7, 167.6, 191.7, 197.6. Anal.
C H NO : C, 73.12; H, 5.30; N, 3.88. Found: C, 73.15; H, 5.38; N, 4.12 %.
1
22
19
4
4
.1.2.10. 5-[(4-methylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno[1,2-
b]indole-9,10-dione, 6j
Yield: 73%, mp: 228-231 °C, IR cm : 3513, 3184 (OH), 1721 (C=O); NMR- H, ppm δ:
.78 (m, 2H, H ), 1.93 (m, 2H, H ), 2.0 (m, 2H, H ), 2.38 (s, 3H, CH ), 5.92 (s, 1H, OH),
-1
1
1
7
6
8
3

ACCEPTED MANUSCRIPT
6
8
3
1
5
4
.66 (d, 1H, H , J: 6.9), 7.14 (s, 1H, OH), 7.18 (d, 2H, Ar , J: 8.2), 7.29 (d, 2H, Ar , J:
4
3,5
2,6
1
3
1
.2), 7.55 (m, 2H, H2,3), 7.71 (dd, 1H, H , J: 7.6, 1.2); NMR- C, ppm δ: 21.3, 22.3, 24.2,
7.7, 84.1, 96.9, 107.0, 123.8, 125.5, 129.8, 130.0, 130.7, 133.8, 135.3, 135.4, 138.0, 147.9,
65.4, 190.2, 198.2. Anal. C H NO : C, 73.12; H, 5.30; N, 3.88. Found: C, 73.19; H,
22
19
4
.32; N, 4.17 %.
.1.2.11. 5-[(2,4-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno-
1,2-b]indole- 9,10-dione, 6k
Yield: 88%, mp: 230-233 °C, IR cm : 3567, 3184 (OH), 1715 (C=O); NMR- H, ppm δ:
[
-1
1
1
.11 (s, 3H, CH
.88 (s, 1H, OH), 6.67 (d, 1H, H
H, Ar, J: 7.9), 7.58 (m, 2H, H2,3), 7.75 (dd, 1H, H
3
), 1.76 (m, 2H, H
, J: 8.4), 7.05 (s, 1H, Ar
, J: 7.2). NMR- C, ppm δ: 17.0, 21.2,
7
), 1.98 (m, 2H, H
6
), 2.13 (m, 2H, H
8 3
), 2.34 (s, 3H, CH ),
5
4
3
), 7.21 (m, 2H, OH, Ar), 7.52 (d,
1
3
1
1
2
1.6, 23.8, 36.0, 83.0, 97.1, 106.4, 124.9, 125.0, 128.1, 130.4, 130.7, 131.0, 135.1, 135.6,
36.5, 139.6, 148.0, 165.3, 191.8, 198.0. Anal. C H NO : C, 73.58; H, 5.64; N, 3.73.
1
23
21
4
Found: C, 73.63; H, 5.71; N, 3.97 %.
4
.1.2.12. 5-[(2,5-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno-
1,2-b]indole- 9,10-dione, 6l
Yield: 79%, mp: 227-229 °C, IR cm : 3497, 3168 (OH), 1715 (C=O); NMR- H, ppm δ:
[
-1
1
1
3 7 6 8 3
.09 (s, 3H, CH ), 1.76 (m, 2H, H ), 1.97 (m, 2H, H ), 2.13 (m, 2H, H ), 2.36 (s, 3H, CH ),
5
.89 (s, 1H, OH), 6.66 (m, 1H, H ), 7.12 (d, 1H, Ar , J: 7.9), 7.21 (m, 2H, OH, Ar), 7.47 (s,
4
3
1
3
1
H, Ar ), 7.55 (m, 2H, H ), 7.75 (dd, 1H, H , J: 8.6, 2.7). NMR- C, ppm δ: 16.5, 21.0,
6 2,3 1
2
1.6, 23.8, 35.6, 83.1, 97.2, 100.2, 124.9, 125.0, 130.3, 130.4, 130.7, 131.3, 133.3, 135.0,
35.6, 137.2, 147.9, 165.4, 191.7, 198.0. Anal. C H NO : C, 73.58; H, 5.64; N, 3.73.
1
23
21
4
Found: C, 73.59; H, 5.67; N, 3.87 %.

ACCEPTED MANUSCRIPT
4
.1.2.13. 5-[(3,4-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno-
[
1,2-b]indole-9,10-dione, 6m
o
-1
1
Yield: 79%, mp: 233-236 C, IR cm : 3562, 3184 (OH),1718 (C=O); NMR- H, ppm δ:
1
.77 (m, 2H, H ), 2.01 (m, 2H, H ), 2.10 (m, 2H, H ), 2.23 (s, 3H, CH ), 2.28 (s, 3H CH ),
7 6 8 3 3
5
.92 (s, 1H, OH), 6.69 (d, 1H, H , J: 6.7), 7.01 (m, 1H, Ar ), 7.10 (s, 2H, OH, Ar), 7.23 (d,
4 6
1
3
1
H, Ar , J: 8.2), 7.55 (m, 2H, H ), 7.71 (dd, 1H, H , J: 7.4, 1.2); NMR- C, ppm δ: 19.6,
5 2,3 1
1
9.9, 22.2, 24.1, 37.7, 84.0, 96.8, 106.8, 123.7, 125.5, 127.2, 130.3, 130.6, 130.7, 134.0,
35.2, 135.3, 136.7, 137.3, 147.9, 165.4, 190.1, 198.2. Anal. C H NO : C, 73.58; H, 5.64;
1
2
3
21
4
N, 3.73. Found: C, 73.72; H, 5.69; N, 4.01 %.
4
.1.2.14. 5-[(3,5-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno-
1,2-b] indole- 9,10-dione, 6n
Yield: 60%, mp: 215-217 °C, IR cm : 3543, 3200 (OH), 1718 (C=O); NMR- H, ppm δ:
[
-1
1
1
6
.78 (m, 2H, H ), 1.95 (m, 2H, H ), 2.01 (m, 2H, H ), 2.29 (s, 6H, CH ), 5.91 (s, 1H, OH),
7 6 8 3
.69 (d, 1H, H , J: 6.9), 6.92 (s, 2H, Ar ), 7.09 (s, 1H, Ar ), 7.10 (s, 1H, OH), 7.55 (m, 2H,
4
4
2,6
1
3
H ), 7.71 (d, 1H, H , J: 6.4); NMR- C, ppm δ: 21.4, 22.3, 24.3, 37.7, 84.1, 96.9, 107.1,
2
,3
1
1
23.7, 125.6, 127.4, 129.9, 130.7, 135.3, 136.4, 138.5, 147.9, 165.3, 190.3, 198.2. Anal.
: C, 73.58; H, 5.64; N, 3.73. Found: C, 73.71; H, 5.67; N, 3.91 %.
.1.2.15. 5-[(4-bromophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno[1,2-
b]indole-9,10-dione, 6o
Yield: 73%, mp: 200 °C, IR cm : 3549, 3184 (OH),1721 (C=O); NMR- H, ppm δ: 1.75 (m,
23 4
C H21NO
4
-1
1
2
7
1
H, H ), 1.93 (m, 2H, H ), 2.11 (m, 2H, H ), 6.00 (s, 1H, OH), 6.67 (d, 1H, H , J: 7.2Hz),
7 6 8 4
.27 (m, 3H, H3´,5´, OH), 7.53 (t, 1H, H , J: 7.2Hz), 7.68 (d, 2H, H2´,6´, J: 8.7Hz), 7.72 (d,
3,
1
3
H, H J: 7.2Hz); NMR- C, ppm δ: 22.2, 24.0, 37.7, 84.0, 97.0, 107.6, 121.6, 123.8, 125.3,
1
,

ACCEPTED MANUSCRIPT
1
5
4
31.8, 132.5, 135.2, 135.6, 136.0, 147.6, 164.9, 190.4, 198.0. Anal. C H BrNO : C,
2
1
16
4
9.17; H, 3.78; N, 3.29. Found: C, 59.23; H, 3.83; N, 3.47 %.
.1.2.16. 5-[(4-chlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno[1,2-
b]indole-9,10-dione, 6p
Yield: 73%, mp: 170-172 °C, IR cm : 3485, 3184 (OH), 1721 (C=O); NMR- H, ppm δ:
.87 (m, 2H, H ), 1.96 (m, 2H, H ), 2.23 (m, 2H, H ), 4.05 (brs, 1H, OH), 6.91 (d, 1H, H ,
-1
1
1
7
6
8
4
J: 7.6Hz), 7.28 (d, 2H, H3´,5´, J: 8.9Hz ), 7.44 (d, 1H, H2´,6´, J: 8.9Hz), 7.49 (t, 1H, H , J:
3
1
3
7
.6Hz), 7.51 (s, 1H, OH), 7.54 (t, 1H, H , J: 7.5Hz), 7.82 (d, 1H, H , J: 7.6Hz) ; NMR- C,
2 1
ppm δ: 21.7, 23.9, 35.3, 82.8, 97.6, 107.7, 119.5, 124.9, 125.1, 129.7, 130.7, 133.8, 134.7,
35.2, 135.7, 147.6, 164.9, 192.7, 197.4. C21 : C, 66.06; H, 4.22; N, 3.67. Found:
C, 66.14; H, 4.29; N, 3.83 %.
.1.2.17. 5-[(3,4-dichlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-hexahydroindeno-
1,2-b]-indole-9,10-dione, 6q
Yield: 53%, mp: 160-162 °C, IR cm : 3561, 3173 (OH), 1727 (C=O); NMR- H, ppm δ:
1
H16ClNO
4
4
[
-1
1
1
6
7
3
1
6
4
.8-2.02 (m, 2H, H ), 2.25-2.33 (m, 2H, H ), 2.38-2.46 (m, 2H, H ), 4.52 (brs, 1H, OH),
7 6 8
.98 (d, 1H, H , J: 7.2Hz), 7.27 (dd, 1H, H , J: 8.9, 2.2Hz ), 7.47 (d, 1H, H , J: 2.2Hz),
4
6´
2´
1
3
1
.51-7.60 (m, 4H, H2,3,5´, OH), 7.84 (d, 1H, H , J: 7.6Hz) ; NMR- C, ppm δ: 21.6, 23.9,
5.9, 82.9, 97.4, 107.8, 124.9, 125.0, 129.0, 130.7, 131.0, 131.3, 133.3, 133.5, 134.9, 135.0,
35.7, 147.4, 167.0, 192.6, 197.1. C H Cl NO : C, 60.59; H, 3.63; N, 3.36. Found: C,
2
1
15
2
4
0.62; H, 3.67; N, 3.61 %.
.1.2.18. 7,7-dimethyl-5-[(2-methoxyphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7a
-1
1
Yield: 81%, mp: 170-172 °C, IR cm : 3600, 3104 (OH), 1712 (C=O); NMR- H, ppm δ: 0.8
(
s, 3H, CH ), 0.94 (s, 3H, CH ), 1.95 (d, 2H, H , J: 17Hz), 2.00 (d, 2H, H , J: 17 Hz), 3.16
3
3
6
8

ACCEPTED MANUSCRIPT
(
s, 3H, OCH ), 5.81 (s, 1H, OH), 6.52 (dd, 1H, H , J: 8.2, 2.1Hz), 7.05 (t, 1H, H , J:
3
4
4´
7
.9Hz), 7.07 (m, 3H, OH, Ar), 7.12 (t, 1H, H
3
, J: 7.6Hz),7.48 (m, 3H, H
2
, Ar), 7.62 (dd, 1H,
1
3
H
1
6´, J: 8.8, 1.8Hz), 7.69(d, 1H, H , J: 8.1Hz); NMR- C, ppm δ: 28.5, 28.6, 33.7, 36.9, 52.0,
5
1
7
4
5.5, 83.8, 96.6, 105.0, 112.5, 121.1, 123.6, 124.7, 125.0, 130.3, 130.8, 131.8, 134.9, 135.1,
48.4, 156.5, 165.6, 189.5, 198.5. Anal. C H NO : C, 71.10; H, 5.72; N, 3.45. Found: C,
24
23
5
1.19; H, 5.75; N, 3.68 %.
.1.2.19. 7,7-dimethyl-5-[(3-methoxyphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7b
-1
1
Yield: 95%, mp: 202-204 °C, IR cm : 3552, 2992 (OH), 1715 (C=O); NMR- H, ppm δ:
0
3
8
.8 (s, 3H, CH
.16 (s, 3H, OCH
.4, 2.1Hz), 7.08 (s, 1H, OH), 7.12 (t, 1H, H , J: 7.6Hz),7.48 (m, 3H, Ar), 7.63 (dd, 1H,
3
), 0.94 (s, 3H, CH
3
), 1.92 (d, 2H, H
6
, J: 17Hz), 2.01 (d, 2H, H
8
, J: 17 Hz),
3
), 5.82 (s, 1H, OH), 6.51 (dd, 1H, H
4
, J: 8.3, 2.2Hz), 7.03 (dd, 1H, H4´, J:
3
1
3
H , J: 8.0, 1.5Hz), 7.69(d, 1H, H , J: 8.2Hz); NMR- C, ppm δ: 28.5, 28.6, 33.7, 36.7, 51.9,
6
´
1
5
5.5, 83.8, 96.5, 104.7, 112.5, 121.1, 123.5, 124.7, 125.0, 130.2, 130.7, 131.8, 134.9, 148.3,
56.4, 165.4, 189.3, 198.4. Anal. C H NO : C, 71.10; H, 5.72; N, 3.45. Found: C, 71.16;
1
24
23
5
H, 5.81; N, 3.59 %.
4
.1.2.20.
7,7-dimethyl-5-[(4-methoxyphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7c
-1
1
Yield: 85%, mp: 120-122 °C, IR cm : 3440, 3100 (OH), 1715 (C=O); NMR- H, ppm δ:
0
1
2
.91 (s, 3H, CH ), 1.02 (s, 3H, CH ), 2.05 (d, 2H, H , J: 17Hz), 2.22 (dd, 2H, H , J: 16.1,
3 3 6 8
8Hz), 3.91 (s, 3H, OCH ), 4.09 (brs, 1H, OH), 6.86 (dd, 1H, H , J: 8.3, 2.2Hz), 6.96 (d,
3
4
H, H3´,5´, J: 8.7Hz), 7.12 (d, 2H, H2´,6´, J: 8.7Hz), 7.48 (m, 3H, OH, Ar), 7.87(d, 1H, H1,
1
3
J: 8.0Hz); NMR- C, ppm δ: 27.7, 29.5, 34.3, 37.6, 50.5, 55.6, 82.7, 96.8, 105.3, 114.6,

ACCEPTED MANUSCRIPT
1
24.7, 125.2, 128.0, 130.3, 130.8, 135.0, 135.3, 147.7, 159.9, 166.1, 191.5, 197.5. Anal.
C
24
H23NO
5
: C, 71.10; H, 5.72; N, 3.45. Found: C, 71.27; H, 5.77; N, 3.70 %.
4
.1.2.21.
7,7-dimethyl-5-[(2,4-dimethoxyphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione 7d [19]
.1.2.22. 7,7-dimethyl-5-[(3,4-dimethoxyphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione 7e
4
-1
1
Yield: 72%, mp: 156-158 °C, IR cm : 3456, 3088 (OH), 1715 (C=O); NMR- H, ppm δ:
0
1
.87 (s, 3H, CH ), 0.95 (s, 3H, CH ), 1.77 (d, 1H, H , J: 17.6Hz), 1.88 (d, 1H, H , J:
3
3
6
8
5.6Hz), 2.10 (d, 1H, H
8
, J: 15.6Hz), 2.34 (d, 1H, H
6
, J: 17.6Hz), 3.71 (s, 3H, OCH
3
), 3.82
(s, 3H, OCH
3
), 5.95 (s, 1H, OH), 6.73 (dd, 1H, H6´, J: 8.3, 2.1Hz), 6.80 (d, 1H, H
4
, J:
7
.2Hz), 6.96 (d, 1H, H2´, J: 2.1Hz), 7.03 (d, 1H, H6´, J:8.4Hz), 7.15 (s, 1H, OH), 7.53 (t,
1
3
1
H, H , J: 7.2Hz), 7.61 (t, 1H, H , J: 7.2Hz), 7.71 (d, 1H, H , J: 7.7Hz); NMR- C, ppm δ:
3 2 1
2
8.4, 33.5, 36.7, 51.9, 55.4, 55.9, 83.7, 96.3, 99.4, 104.6, 105.3, 117.2, 123.4, 125.0, 130.1,
32.3, 134.8, 135.0, 148.4, 157.3, 161.1, 165.7, 189.2, 198.4. Anal. C H NO : C, 68.95;
1
2
5
25
6
H, 5.79; N, 3.22. Found: C, 68.97; H, 5.81; N, 3.41 %.
4
.1.2.23.
7,7-dimethyl-5-[(2,5-dimethoxyphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7f
-1
1
Yield: 80%, mp: 210-212 °C, IR cm : 3488, 3056 (OH), 1712 (C=O); NMR- H, ppm δ:
0
1
8
7
.80 (s, 3H, CH ), 0.94 (s, 3H, CH ), 1.92 (d, 2H, H , J: 17.5Hz), 2.00 (d, 2H, H , J:
3 3 6 8
7,5Hz), 3.12 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 5.83 (s, 1H, OH), 6.60 (dd, 1H, H , J:
3
3
4
.2, 1.5Hz), 6.97 (d, 1H, H , J: 9.2Hz), 7.04 (dd, 1H, H , J: 9.2, 2.9Hz), 7.09 (s, 1H, OH),
3
´
4´
.29 (d, 1H, H , J:2.9Hz), 7.49 (t, 1H, H , J:7.2Hz), 7.54 (t, 1H, H , J: 7.2Hz), 7.69 (d, 1H,
6
´
3
2
1
3
H , J: 8.1Hz); NMR- C, ppm δ: 28.4, 28.6, 36.9, 52.0, 55.7, 56.1, 56.6, 83.4, 96.7, 105.0,
1

ACCEPTED MANUSCRIPT
1
1
4
13.0, 114.9, 118.1, 123.5, 125.2, 130.3, 134.9, 135.1, 148.3, 150.6, 153.4, 165.4, 189.5,
98.3. Anal. C25
6
H25NO : C, 68.95; H, 5.79; N, 3.22. Found: C, 69.12; H, 5.86; N, 3.30 %.
.1.2.24. 7,7-dimethyl-5-[(3,4,5-trimethoxyphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7g
-1
1
Yield: 90%, mp: 198 °C, IR cm : 3600, 3136 (OH), 1715 (C=O); NMR- H, ppm δ: 0.89 (s,
3
1
H, CH ), 0.96 (s, 3H, CH ), 1.84 (d, 2H, H , J: 17Hz), 1.89 (d, 1H, H , J: 15Hz), 2.12(d,
3 3 6 8
H, H8, J: 15Hz), 3.73 (s, 6H, OCH ), 3.74 (s, 3H, OCH ), 6.10 (brs, 1H, OH), 6.90 (d, 1H,
3
3
H , J: 7.7Hz), 6.63 (s, 2H, H2´,6´), 7.17 (s, 1H, OH), 7.54 (t, 1H, H , J:6.7Hz), 7.65 (t, 1H,
4
3
1
3
H
2
1
, J:6.7Hz), 7.71 (d, 1H, H , J: 7.4Hz); NMR- C, ppm δ: 27.0, 30.0, 33.9, 37.4, 51.8,
5
1
6.7, 60.8, 84.0, 97.3, 105.6, 108.2, 123.6, 126.9, 130.8, 131.8, 135.3, 135.4, 137.8, 147.7,
53.2, 164.4, 189.7, 198.2. Anal. C26 : C, 67.09; H, 5.85; N, 3.01. Found: C, 67.12;
H27NO
7
H, 5.91; N, 3.17 %.
.1.2.25. 7,7-dimethyl-5-[(2-methylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno [1,2-b]indole-9,10-dione, 7h
4
-1
1
Yield: 82%, mp: 231-234 °C, IR cm : 3424, 2996 (OH), 1712 (C=O); NMR- H, ppm δ:
0
5
.89 (s, 3H, CH ), 0.95 (s, 3H, CH ), 1.18 (s, 3H, CH ), 1.86 (s, 2H, H ), 2.04 (s, 2H, H ),
3
3
3
6
8
4
.88 (s, 1H, OH), 6.67 (d, 1H, H , J: 6.2), 7.27 (m, 2H, OH, Ar), 7.37 (m, 2H, Ar4,5), 7.57
1
3
(
m, 2H, H2,3), 7.62 (m, 1H, Ar), 7.76 (dd, 1H, H
8.6, 33.8, 36.9, 52.0, 84.1, 97.3, 104.7, 124.2, 125.2, 127.2, 129.4, 130.7, 131.3, 132.0,
34.6, 135.4, 135.7, 137.9, 148.7, 164.3, 189.5, 198.7. C H NO : C, 74.02; H, 5.95; N,
1
, J: 7.4); NMR- C, ppm δ: 17.3, 28.4,
2
1
3
4
2
4
23
4
.60. Found: C, 74.11; H, 6.01; N, 3.92 %.
.1.2.26. 7,7-dimethyl-5-[(3-methylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno [1,2-b]indole-9,10-dione, 7i

ACCEPTED MANUSCRIPT
-1
1
Yield: 56%, mp: 118-120 °C, IR cm : 3524, 3092 (OH), 1718 (C=O); NMR- H, ppm δ:
0
5
.88 (s, 3H, CH
3
), 0.95 (s, 3H, CH
3
), 1.92 (s, 2H, H
6
), 2.10 (s, 2H, H
8
), 2.35 (s, 3H, CH
3
),
.96 (s, 1H, OH), 6.65 (d, 1H, H
4
, J: 7.9), 7.02 (d, 1H, Ar
6
, J: 7.7), 7.15 (s, 1H, Ar
2
), 7.20
(
s, 1H, OH), 7.27 (d, 1H, Ar , J: 7.7), 7.36 (t, 1H, Ar , J: 7.7), 7.54 (m, 2H, H ), 7.71 (dd,
4 5 2,3
1
3
1
1
1
4
H, H , J: 8.1, 1.9); NMR- C, ppm δ: 21.6, 27.1, 30.0, 34.1, 37.7, 51.8, 84.0, 97.3, 106.0,
1
23.8, 125.6, 127.0, 129.2, 129.3, 130.4, 130.8, 135.3, 135.4, 136.6, 140.0, 147.8, 164.1,
89.8, 198.2. C H NO : C, 74.02; H, 5.95; N, 3.60. Found: C, 74.07; H, 5.98; N, 3.73 %.
24
23
4
.1.2.27.
7,7-dimethyl-5-[(4-methylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno [1,2-b]indole-9,10-dione, 7j
-1
1
Yield: 99%, mp: 206-207 °C, IR cm : 3602, 3214 (OH), 1721 (C=O); NMR- H, ppm δ:
0
2
6
3
5
1
4
.95 (s, 3H, CH
3 3 6 6
), 1.03 (s, 3H, CH ), 1.98 (d, 1H, H J: 17Hz), 2.15 (d, 1H, H , J: 17Hz),
.20 (d, 1H, H , J: 16Hz), 2.28 (d, 1H, H8, J: 16Hz), 2.44 (s, 3H, CH ), 5.97 (s, 1H, OH),
8
3
.86 (d, 1H, H , J: 8.7), 7.10 (d, 2H, AH3´,5´, J: 8.2Hz), 7.26 (d, 2H, H2´,6´, J: 8.2), 7.48 (m,
4
1
3
H, H , OH), 7.86 (d, 1H, H , J: 8.6Hz); NMR- C, ppm δ: 21.3, 27.6, 30.5, 34.4, 37.6,
2
,3
1
1.8, 82.7, 96.8, 105.6, 124.7, 125.2, 129.2, 130.0, 132.9, 134.9, 135.3, 138.8, 147.7, 165.7,
91.7, 197.5. C H NO : C, 74.02; H, 5.95; N, 3.60. Found: C, 63.91; H, 6.13; N, 3.85 %.
24
23
4
.1.2.28.
7,7-dimethyl-5-[(2,4-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7k
-1
1
Yield: 59%), mp: 215-218 °C, IR cm : 3583, 3184 (OH), 1718 (C=O); NMR- H, ppm δ:
0
2
.82 (s, 3H, CH ), 0.94 (s, 3H, CH ), 1.13 (s, 3H, CH ), 1.83 (s, 2H, H ), 2.03 (s, 2H, H ),
3 3 3 6 8
.33 (s, 3H, CH ), 5.86 (s, 1H, OH), 6.71 (d, 1H, H , J: 8.2), 7.05 (d, 1H, Ar , J: 1), 7.17
3
4
3
(
dd, 1H, Ar , J: 8.2, 1.2), 7.22 (s, 1H, OH), 7.47 (d, 1H, Ar , J: 7.9), 7.57 (m, 2H, H ),
5 6 2,3
1
3
7
.75 (dd, 1H, H , J: 8.9, 1.2). NMR- C, ppm δ: 17.1, 21.2, 28.7, 34.1, 37.1, 50.9, 82.9,
1

ACCEPTED MANUSCRIPT
9
1
4
7.0, 104.7, 124.9, 128.0, 130.3, 131.0, 131.6, 135.1, 135.2, 135.5, 139.5, 148.2, 166.5,
91.7, 197.8. C25
4
H25NO : C, 74.42; H, 6.25; N, 3.47. Found: C, 74.56; H, 6.30; N, 3.75 %.
.1.2.29. 7,7-dimethyl-5-[(2,5-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7l
-1
1
Yield: 98%, mp: 173-175 °C, IR cm : 3595, 3296 (OH), 1721 (C=O); NMR- H, ppm δ:
0
.82 (s, 3H, CH ), 0.94 (s, 3H, CH ), 1.11 (s, 3H, CH ), 1.94 (m, 4H, H ), 2.35 (s, 3H,
3
3
3
6,8
CH ), 5.86 (s, 1H, OH), 6.69 (m, 1H, H ,), 7.12 (d, 1H, Ar , J: 7.9), 7.20 (m, 3H, OH, 2Ar),
3
4
3
1
3
7
3
1
.42 (s, 1H, Ar ), 7.56 (m, 2H, H ), 7.74 (m, 1H, H ). NMR- C, ppm δ: 16.7, 21.0, 28.7,
6 2,3 1
4.1, 37.2, 50.2, 82.8, 97.1, 105.0, 124.9, 130.4, 130.7, 131.6, 133.4, 133.7, 135.1, 135.5,
37.3, 148.1, 167.0, 191.6, 198.0. C25 : C, 74.42; H, 6.25; N, 3.47. Found: C, 74.61;
H25NO
4
H, 6.39; N, 3.79 %.
.1.2.30. 7,7-dimethyl-5-[(3,4-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7m
4
-1
1
Yield: 58%, mp: 185-188 °C, IR cm : 3520, 3168 (OH), 1718 (C=O); NMR- H, ppm δ:
0
2
.82 (s, 3H, CH ), 0.90 (s, 3H, CH ), 1.93 (s, 2H, H ), 2.06 (s, 2H, H ), 2.21 (s, 3H, CH ),
3 3 6 8 3
.25 (s, 3H, CH ), 5.95 (s, 1H, OH), 6.69 (d, 1H, H , J: 6.7), 6.86 (d, 1H, Ar , J: 8.2), 7.04
3
4
6
(
s, 1H, Ar
2
5 1
), 7.17 (s, 1H, OH), 7.21 (d, 1H, Ar , J: 8.2), 7.53 (m, 2H, H2,3), 7.7 (d, 1H, H ,
1
3
J: 7.2); NMR- C, ppm δ: 19.57, 19.95, 27.16, 29.55, 33.86, 37.54, 51.37, 83.75, 97.15,
1
1
7
4
05.31, 123.81, 125.53, 127.24, 130.43, 130.64, 130.93, 133.65, 134.99, 135.54, 137.11,
37.64, 147.73, 165.08, 190.59, 198.36. C H NO : C, 74.42; H, 6.25; N, 3.47. Found: C,
25
25
4
4.43; H, 6.27; N, 3.59 %.
.1.2.31. 7,7-dimethyl-5-[(3,5-dimethylphenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione, 7n

ACCEPTED MANUSCRIPT
-1
1
Yield: 64%), mp: 143-145 °C, IR cm : 3561, 3168 (OH), 1705 (C=O); NMR- H, ppm δ:
0
5
.84 (s, 3H, CH
3
), 0.91 (s, 3H, CH
3
), 1.94 (s, 2H, H
6
), 2.07 (s, 2H, H
8 3
), 2.26 (s, 6H, CH ),
.95 (s, 1H, OH), 6.67 (d, 1H, H
4
, J: 6.7), 6.81 (s, 1H, Ar
4
), 7.08 (s, 2H, Ar2,6), 7.19 (s, 1H,
1
3
OH), 7.54 (m, 2H, H ), 7.70 (dd, 1H, H , J: 7.4, 2.2); NMR- C, ppm δ: 21.3, 27.5, 29.6,
2
,3
1
3
4.5, 37.7, 50.7, 82.7, 96.7, 105.6, 124.7, 125.3, 126.9, 130.4, 130.5, 134.9, 135.2, 135.4,
39.1, 147.7, 165.6, 191.7, 197.6. C H NO : C, 74.42; H, 6.25; N, 3.47. Found: C, 74.47;
1
25
25
4
H, 6.28; N, 3.62 %.
4
.1.2.32.
7,7-dimethyl-5-[(4-bromophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno-[1,2-b]indole-9,10-dione, 7o
-1
1
Yield: 75%, mp: 140-142 °C, IR cm : 3644, 3008 (OH), 1718 (C=O); NMR- H, ppm δ:
0
2
.93 (s, 3H, CH
3 3 6
), 1.00 (s, 3H, CH ), 1.96 (d, 1H, H , J:17 Hz), 2.05 (d, 1H, H6, J:17 Hz),
.20 (d, 1H, H , J:15 Hz), 2.29 (d, 1H, H , J: 15Hz), 3.60 (brs, 1H, OH), 6.86 (d, 1H, H ,
8
8
4
J:6.7 Hz), 7.16 (d, 2H, H3´,5´, J:8.2 Hz), 7.49 (m, 3H, H2,3, OH), 7.60 (d, 2H, H2´,6´, J: 8.2
1
3
Hz), 7.83 (d, 1H, H , J: 7.6 Hz); NMR- C, ppm δ: 27.7, 29.4, 34.4, 37.7, 50.0, 82.8, 123.0,
1
1
24.9, 125.1, 128.8, 130.6, 131.0, 132.7, 134.5, 134.6, 134.8, 135.7, 147.5, 191.4, 197.3.
C H BrNO : C, 60.81; H, 4.44; N, 3.08. Found: C, 60.93; H, 4.52; N, 3.27 %.
23
20
4
4
.1.2.33.
7,7-dimethyl-5-[(4-chlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno-[1,2-b]indole-9,10-dione 7p
-1
1
Yield: 67%, mp: 147-148 °C, IR cm : 3637, 3018 (OH), 1714 (C=O); NMR- H, ppm δ:
0
2
.90 (s, 3H, CH ), 1.03 (s, 3H, CH ), 1.98 (d, 1H, H , J:15 Hz), 2.05 (d, 1H, H J:15 Hz),
3 3 6 6,
.23 (d, 1H, H , J:17 Hz), 2.31 (d, 1H, H , J: 17Hz), 3.67 (brs, 1H, OH), 6.81 (d, 1H, H ,
8
8
4
J:6.8 Hz), 7.14 (d, 2H, H3´,5´, J:8.0 Hz), 7.47 (m, 3H, H2,3, OH), 7.62 (d, 2H, H2´,6´, J: 8.0
1
3
Hz), 7.83 (d, 1H, H , J: 7.6 Hz); NMR- C, ppm δ: 28.0, 30.1, 34.7, 36.9, 51.0, 81.7, 122.8,
1

ACCEPTED MANUSCRIPT
1
25.3, 125.9, 128.3, 131.1, 131.7, 132.0, 133.9, 134.7, 135.2, 135.9, 147.7, 191.3, 197.2.
C
23
H
20ClNO
4
: C, 67.40; H, 4.92; N, 3.42. Found: C, 67.44; H, 4.99; N, 3.63 %.
4
.1.2.34.
7,7-dimethyl-5-[(3,4-dichlorophenyl)]-(4bRS,9bRS)-dihydroxy-4b,5,6,7,8,9b-
hexahydroindeno[1,2-b]indole-9,10-dione 7q
-1
1
Yield: 47%, mp: 180-182 °C, IR cm : 3568, 2647 (OH), 1724 (C=O); NMR- H, ppm δ:
0
2
7
.96 (s, 3H, CH ), 1.00 (s, 3H, CH ), 2.00 (d, 1H, H J: 17Hz), 2.17 (d, 1H, H , J: 17Hz),
3 3 6, 6
.20 (d, 1H, H , J: 15Hz), 2.26 (d, 1H, H J: 15Hz), 3.7 (brs, 1H, OH), 6.93 (d, 1H, H , J:
8
8,
4
.2Hz), 7.19 (dd, 1H, H , J: 8.9, 2.2Hz ), 7.48-7.56 (m, 5H, H2´,5´,3,2,OH), 7.84 (d, 1H, H1,
6
´
1
3
J: 7.6Hz) ; NMR- C, ppm δ: 27.5, 29.5, 37.7, 50.1, 82.6, 97.4, 106.7, 124.9, 128.8, 130.7,
31.1, 131.2, 133.4, 134.8, 135.1, 135.7, 147.4, 165.6, 168.9, 191.9, 197.1. C23 NO
C, 62.17; H, 4.31; N, 3.15. Found: C, 62.23; H, 4.35; N, 3.41 %.
1
H
19Cl
2
4
:
4
4
.2. Anticancer assays
.2.1. Cell Culture
Human and non-human prostate tumor cell lines PC-3, LNCaP and MatLyLu were obtained
from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig,
Germany). Cells were grown in culture RPMI medium supplemented with fetal bovine
serum (FBS, 10%), penicillin (50 units/ml) and streptomycin (50 ꢀg/ml), in a humidified
2
atmosphere (95% air, 5% CO , 37°C). Culture medium was purchased from Gibco-In
Vitrogen, Karlsruhe, Germany and PAA Laboratories, Pasching, Austria.
4
.2.2. Cell Viability Test
3
4
4
5
×10 PC-3, 1.2×10 LNCaP or 1×10 MatLyLu cells were seeded in a 96-well microtiter
plate containing 100 µl of culture growth RPMI medium/well. After 24 h of culture, cells
were exposed to the compounds previously dissolved in DMSO (72h, 5-100 ꢀg/ml ). The
final concentration of this solvent in the culture media was always lower than 0.2%, a

ACCEPTED MANUSCRIPT
concentration that has neither a cytotoxic effect nor causes any interference with the
colorimetric detection method. The dose-dependent effects of each compound on were
assessed using the XTT test (Roche Applied Science, Mannheim, Germany). After 72 h of
exposure with the compounds, cells were incubated with XTT (4h, 37°C) and the formazan
was recorded at 492 nm (Microplate reader HT II, Anthos, Salzburg, Austria). The IC50
value was defined as the concentration of tested compound resulting in a 50% reduction in
cell viability compared to vehicle-treated cells. Further evaluations of the best compound
were performed using this IC value [36,45].
5
0
4
.2.3. Cell Migration and Motility Evaluation
Cell migration and motility was tested using the scrape wound repair assay. 8×104 PC-3
cells were grown to confluence on 24-well plates in RPMI medium (48 h, 37°C). A
sterilized micropipette tip was used to introduce a wound across the entire cell monolayer,
and the medium was removed. After washing gently with PBS, the most cytotoxic
compound on the XTT assay was added (IC ) in fresh medium and incubated for 24 h in
5
0
the presence of endothelial growth factor (1 pg/ml). Cover slips were mounted onto a light
microscope and images of the wounds were captured on a computer system using a digital
camera immediately following wounding (0 h) and after 24 h of compound or vehicle
incubation. The wound area in each image was measured using the ImageJ program for
Windows (http://rsb.info.nih.gov/ij/) and quantified by following the change in wound area
over time compared with the original wound area. The results were expressed as the
percentage of wound closure [35].
4
.2.4. Invasion Assay.
5
Human tumor LNCaP cells (1×10 cells/ml) were pretreated with the compound (cytotoxic
IC50, 24h). Cells then were seeded into the upper part of a Boyden chamber membrane

ACCEPTED MANUSCRIPT
coated with Matrigel (Becton Dickinson Biosciences, Heidelberg, Germany) in 50 ꢀL of
serum-free medium and incubated (18 h, 37°C). The bottom of the chamber contained 0.5
mL of standard medium (20% FBS). Invaded cells at the lower surface of the chamber
were reacted with calcein (4 ꢀg/ml) in Hank´s Buffered Salt Solution (1 h, 37°C). The
fluorescence of invaded cells was read at 485/530 nm (Fluoroskan Ascent, Thermo
Labsystems Oy, Helsinki, Finland) [22,36].
4
.2.5. Measurement of Clonogenic Potential
The ability of cells to grow in an anchorage-independent manner was tested in cells grown
in agar (0.6%). In short, 1 mL of a mixture of 1.2% Noble agar (Gibco-In Vitrogen,
Karlsruhe, Germany) and RPMI medium (1:1) was added into each well of a six-well plate.
PC3, LNCaP and MatLyLu cells (1×105) suspended in completed RPMI medium (FBS
2
0%) containing Noble agar (0.3%) were overlaid on the semisolid bottom layer. The plates
were kept at room temperature for 15 min and incubated for 24 h (37°C, 95% O , 5% CO ).
2
2
The following day, 1 mL of medium with the compound at its IC50 concentrations was
added to each well. After 2 weeks of incubation cells were stained with crystal violet
(
0.01%) for 18 h at 37°C. Pictures were taken under light microscopy, and the total number
of colonies and the relative colony size were determined [22,46].
.2.6. MMP Zymography
4
To measure MMP-2 and MMP-9 activities, PC-3 and LNCaP cells (80% confluent in six-
well plates) were washed twice with PBS and treated with the compound at its respective
IC concentration in serum-free medium (2.5 mL, 24 h, 37°C) in a humidified atmosphere
5
0
(
95% O , 5% CO ). 22 ꢀl of a mixture composed of the conditioned medium and sample
2 2
buffer (without mercaptoethanol, 0.75:0.25) were subjected to electrophoresis on 10%
SDS-polyacrylamide gels gelatin-copolymerized (1 mg/mL). MMP-2 and MMP-9

ACCEPTED MANUSCRIPT
gelatinolytic activity in the conditioned culture medium was assayed as previously
described [22,47]. Bands quantification was performed by using ImageJ Software for
Windows. Pure human MMP-9 and MMP-2 protein were used as a positive control.
4
.3. X-ray crystallography
Crystals of 6k and 7k suitable for X-ray diffraction were obtained by slow evaporation of a
solution in ethanol. Crystal data, intensity data collection parameters and final refinement
results are summarised in Table V. Diffraction data were measured on a Bruker Smart-
Apex diffractometer using graphite-monochromated Mo-Kα radiation (λ = 0.71070 Å). The
2
structure was solved by direct methods and refined on F by full-matrix least-squares, using
2
2
o
2
-1
2
2
all reflections and weights w = [σ (F ) + (a P) + b P] , with P = (F + 2 F )/3. The C-
o
c
bonded H atoms were placed in calculated positions and refined using a riding atom model
with fixed C-H distances (0.93 Å for CH, 0.97 Å for CH , 0.96 Å for CH ), and with U =
2
3
iso
p U (parent atom) (p = 1.2 for CH and CH , 1.5 for CH ). The O-bonded H atoms were
eq
2
3
placed at idealized tetrahedral geometries, with fixed O-H distances (0.82 Å) and Uiso = 1.5
eq(parent atom).
U
The following computer programs were used: data collection, SMART [48]; cell refinement
and data reduction, SAINT [49]; absorption correction, SADABS [50]; structure solution
and refinement, SHELXL-97 [51]; molecular graphics, ORTEP-3 [52]; geometrical
calculations, PLATON [53]; The structure solution, the refinement and the drawings were
carried out with the aid of the WinGX [54] suite of programs. Comprehensive
crystallographic data (excluding structure factors) for the structural analysis of 6k and 7k
have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data
(CIF files) can be obtained, free of charge, on application to CCDC, 12 Union Road,

ACCEPTED MANUSCRIPT
Cambridge
CB2
1EZ,
UK,
fax:
+44-(0)1223-336033,
or
from
www.ccdc.cam.ac.uk/data_request/cif, quoting deposition No. CCDC 1022641 (6k) and
CCDC 1022819 (7k).
4
.4. Molecular docking methodology
The three dimensional structure of MMP-9 was downloaded from the Protein Data Bank
(http://www.rscb.org/pdb) server with PDB code: 1GKC. Internal ligands and the
crystallographic water molecules were removed from the protein and missing Hydrogens
were added. Ligands were built with the molecular editor of CAChe 6.0 [55] using the
crystallographic data structures 7k and 6k as reference. They were geometrically optimized
by using MM3 [56] force field by CAChe 6.0 and PM3 of the Arguslab 4.0.1 [57] with
RMSD 0.0001. The active site of the enzyme was defined and the ligand was placed in a
box with dimensions of X = 20 Å, Y = 20 Å, Z = 20 Å and a resolution of the grid of
0
.4000 Å. Docking was performed using Arguslab 4.0.1employing genetic algorithm to
generate the poses of the ligands at the binding site, allowing flexibility of the ligand. The
results of docking were quantifies in terms of the score and minimized energy.
Acknowledgments
We thank the IIF and CDCH-UCV (grants IIF.01-2014, PG. 06-8627-2013/1) programs for
financial support, and Professor Milagros Avendaño for her help in the Molecular Docking
Simulations.
References
[1] A. Jemal, F. Bray, M. Center, J. Ferlay, E. Ward, D. Forma, Global cancer statistics,
CA Canc. J. Clin. 61 (2011) 69-90, DOI: 10.3322/caac.20107.

ACCEPTED MANUSCRIPT
[
2] D. Brown, P. Graupner, M. Sainsbury, H. Shertzer, New antioxidants incorporating
indole and indoline chromopheres, Tetrahedron, 47 (1991) 4383-4408,
DOI:10.1016/S0040-4020(01)87108-8.
[
3] J. Butera, S. Antane, B. Hirth, J. Lennox, J. Sheldon, N. Norton, D. Warga, T.
Argentieri, Synthesis and potassium channel opening activity of substituted 10H-
Benzo[4,5]furo[3,2-b]indole- and 5,10-dihydro-indeno[1,2-b]indole-1-carboxilic
acid, Bioorg. Med. Chem. Lett. 11 (2001) 2093-2097, DOI:10.1016/S0960-
8
94X(01)00385-7.
[
4] C. Bal, B. Baldeyrou, F. Moz, A. Lansiaux, L. ColsoKraus-Berthier, S. Léonce, A.
Pierre, M. Boussard, A. Rousseau, M. Wierzbicki, C. Bailly, Novel antitumor
indenoindole derivatives targeting DNA and antitopoisomerase II, Biochem.
Pharmacol. 68 (2004) 1911-1922, DOI: 10.1016/j.bcp.2004.07.008.
[
5] C. Miller, M. Collini, B. Tran, Indenoindoles and benzocarbazoles as estrogenic
agents, US 6107292 A, (2000).
[
6] H. Hemmerling, C. Götz, J. Jose, Substituierte indeno [1,2-b] indoldrivative alse
neue hemmstoffe der protein kinase CK2 und ihre verwendung als tumor
therapeutika, cytostatike und diagnostika, WO 2008040547 A1, (2008).
7] C. Hundsdörfer, H. Hemmerling, C. Götz, F. Totzke, P. Bednarski, M. Borgne, J.
Jose, Indeno[1,2-b]indole derivatives as a novel class of potent human protein
kinase CK2 inhibitors, Bioorg. Med. Chem. 20 (2012) 2282-2289.
DOI:10.1016/j.bmc.2012.02.017.
[
[
8] M. Salvi, S. Sarno, L. Cesaro, H. Nakamura, L. Pinna, Extraordinary pleiotropy of
protein kinase CK2 revealed by weblogo phosphoproteome analysis, Biochim.