Novel 3,3′-diindolylmethane derivatives: Synthesis and cytotoxicity, structural characterization in solid state

Article abstract of DOI:10.1016/j.ejmech.2009.05.011

A series of 3,3′-dindolylmethane derivatives have been synthesized and their structures were characterized in solid state by ¹³C CP/MAS NMR and two of them by X-ray diffraction measurements. They exhibited well expressed cytotoxicity against hu

Full text of DOI:10.1016/j.ejmech.2009.05.011

European Journal of Medicinal Chemistry 44 (2009) 4136–4147
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 3,30-diindolylmethane derivatives: Synthesis
and cytotoxicity, structural characterization in solid state
,
a
b
Dorota Maciejewska a , Magdalena Rasztawicka , Irena Wolska ,
*
c
,
c
**
_
Elzbieta Anuszewska
, Beata Gruber
a Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Str, 02 097 Warsaw, Poland
b Department of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, 6 Grunwaldzka Str, 60-780 Poznan´, Poland
c Department of Biochemistry and Biopharmaceuticals, National Medicines Institute, 30/34 Chelmska Str, 00-725 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
A series of 3,30-dindolylmethane derivatives have been synthesized and their structures were charac-
terized in solid state by 13C CP/MAS NMR and two of them by X-ray diffraction measurements. They
exhibited well expressed cytotoxicity against human melanoma cell lines. Derivatives bearing fluoro,
bromo, iodo, and nitro substituents in indole or benzene rings caused 50% inhibition of the viability of
Article history:
Received 6 March 2009
Received in revised form
4 May 2009
Accepted 7 May 2009
Available online 22 May 2009
ME18 and ME18/R cell lines at concentration ranging 9.7–17.3 mM.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
3,30-Dindolylmethane derivatives
13C CP/MAS NMR
X-ray diffraction
Cytotoxicity
1. Introduction
a result, it was established that an enhancement of mitochondrial
release of ROS is responsible for p21 up-regulation. 3,30-Diindo-
lylmethane was found to have low bioavailability, what have
limited its possible widespread use. The discovery of an absorbable
form of 3,30-diindolylmethane [11] allowed the use of a potency of
this compound. Recent reports show that the highly absorbable
microencapsulated formulation of 3,30-diindolylmethane is in I/III
phases of clinical trials for treatment of cervical displasia and
prostate cancer. This formulation is also used for promotion of
healthy estrogen metabolism. These make the bis-indoles an
important class of anticancer chemotherapeutics and potential
source of medicinal lead compounds. Methods of their synthesis
are still developed [12–19]. Taking into account all the facts we
synthesized ten new 3,30-diindolylmethanes (Fig. 1) and studied
their antiproliferative potential against normal cells and two
human melanoma cells.
In the present work we report also the single crystal X-ray
diffraction measurements as well as the analysis of 13C CP/MAS
NMR spectra in solid state. We are hopeful that structural analysis
of the title compounds in solid state can provide useful information
for further development of our work. Examinations of the struc-
tures of the designed compounds could be essential for obtaining
experimental parameters, which can become descriptors in the
planned structure–activity studies.
3,30-Diindolylmethane is considered to be a promising anti-
tumor agent derived from Brassica food plants (cabbage, broccoli,
Brussels sprouts, cauliflower), which could protect against tumor-
igenesis in multiple organs (forestomach, mammary gland, uterus,
liver). However, such phytonutrients may exhibit adverse
promoting activity in certain test protocol or in other organs [1,2].
3,30-Diindolylmethane has reached an important position in the
research works aimed at understanding the mechanisms of anti-
proliferative processes in tumor cell lines, especially in human
breast cancer cells [3–10]. 3,30-Diindolylmethane induced a G1 cell
cycle arrest in human breast cancer, regardless of their estrogen
receptor or p53 tumor suppressor status, by an increase in
expression of the CDK inhibitor, p21Cip1/Waf1. Further investigations
showed that 3,30-diindolylmethane is a strong mitochondrial Hþ-
ATPase inhibitor, and stimulates mitochondrial reactive oxygen
species (ROS) production, which leads to processes involving the
stress-activated protein kinases, c-Jun NH2-terminal and p38. As
* Corresponding author. Tel./fax: þ48 022 5720 643.
** Corresponding author.
E-mail addresses: domac@farm.amwaw.edu.pl (D. Maciejewska), anuszewska@
il.waw.pl (E. Anuszewska).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.011

D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
4137
O
3,30-diindolyl-(3,5-difluorophenyl)methane (1c), the 5,50-diiodo-3,30-
diindolyl-(3,5-difluorophenyl)methane (2c), the 5,50-dimethoxy-3,30-
diindolyl-(3,5-difluorophenyl)methane (3c), and the 5,50-dibro-mo-
3,30-diindolyl-(3,5-difluorophenyl)methane (7c). All examined mole-
cules have two identically substituted indolyl moieties and for the
chemicallyequivalent pairs of atoms from both indole systems we have
observed a different pattern of NMR resonances in the solid state: for
compounds 2a, 2b, 8b and 1c, 2c, 7c, the simple solution-like spectra
but for compounds 4a,1b, 3b and 3c more complicated, double signals’
pattern for majority of atoms.
There could be several reasons for these differences. The solu-
tion-like spectrum is favored in case, when the intermolecular
interaction does not dominate. The double resonances could be
caused by i) the presence of two energetically similar conformers in
solid state (polymorphism), ii) the co-crystallization with solvent
(pseudopolymorphism) or iii) different packing mode of both
indole rings in one conformer. Certainly, spin–spin coupling of 19F
and 14N atoms with 13C atoms also could be observed.
X
X
X
R
C
5
4
R
4'
8'
5'
H
6
8
6'
B
9
3
3'
H3O,
ΔT
A
9'
N
H
10
7
7'
N
H
N
2
2'
1
1'
H
F
12 13
13
12
11
14
11
R =
14
F
R = -H
CH3
12"
R =
16
15
16
15
X = 5-H
X = 5-I
1b
1c
2c
X = 5-H
X = 5-I
X = 5-H
X = 5-I
1a
2b
3b
2a
3a
11"
11"
X = 5-OCH3
X = 5-OCH3
3c
X = 5-OCH3
11"
X = 4-OCH3
4a
The experimental chemical shifts
the theoretical shielding constants
d
[ppm] were compared with
s
[ppm] computed at DFT level
7c
7b
8b
X = 5-Br
X = 5-Br
X = 5-F
employing B3LYP/6-311G (d, p) hybrid functional and CPHF-GIAO
approach for low energy conformers (obtained at PM3 level of
theory) for all compounds apart from these with I-atoms (I atom is
not well parameterized within Gaussian 03). These calculations
helped us to assign properly the 13C NMR resonances and provided
insight into the structural features of the examined compounds in
solid state. Moreover, we determined the geometries of 2a and 8b
by X-ray analysis and compared the obtained conformation of 8b
with its spectroscopic NMR data in solid state, computing the 13C
X = 5-OCH2CH
X = 5-NO2
5a
6a
3
Fig. 1. General synthetic pathway together with compounds and atom numbering.
Structural data for compounds marked with asterisk (*) were already published by us.
2. Results and discussion
shielding constants
Simultaneously, using the same method we computed the shield-
s [ppm] for X-ray derived atomic coordinates.
2.1. Chemistry
ing constants for PM3 geometry of 8a. We have found linear
The principal synthetic route to the bis-indolylmethanes is the
condensation of corresponding indole derivatives with aliphatic or
aromatic aldehydes in the acidic medium (Fig. 1). We made some
modifications of the standard procedure, which are reported in the
Experimental part. Standard 1D and 2D hetero- and homomolecular
1H and 13C NMR experiments in CDCl3 or DMSO-d6 were sufficient to
characterize the studied compounds in solution. Complete assign-
ments of the solution resonances are given in Section 4.
correlations between theoretical
s values and the experimental
chemical shifts [ppm] for both atomic coordinates. The above
d
finding is good evidence that linear correlations between the
computed shielding constants for PM3 geometries and the exper-
imental chemical shifts could be used to analyze the molecular
structure of the studied bis-indoles.
In the spectra of compounds of 1b, 8b, 2c, 7c the strong signal in
the vicinity of 129 ppm was measured. It arises from the inclusion
of benzene molecule into the crystallographic net. The interactions
between benzene and indole rings seem to be responsible for
splitting the resonances of 13C atoms of indole systems in the
spectra of 1b and 8b. This is not revealed in the spectra of 2c and 7c.
For the methoxy derivatives (4a, 3b, 3c) spectral analysis disclosed
double signals of 13C atoms of both indole rings, as was evidenced
previously by us for resemble compounds [24,25]. The methoxy
substituents at C5, C50 atoms of indole rings in compounds 3b, 3c
are situated in aromatic rings’ plane, and are oriented in such a way
that methyl groups point at C4 or C40 atoms. This is confirmed by
upfield shifts of these atoms’ resonances in solid state as compared
to solution one. Similarly, methoxy groups at C4, C40 atoms indole
rings are located in the plane of the aromatic rings, and upfield
shifts of C5 (C50) resonances are observed. The dihedral angles
formed by methoxy groups and indole rings (C1100–O–C5–C4, C1100–
O–C40–C50, C1100–O–C4–C6, C1100–O–C40–C60) are slightly different,
resulting in various values of shielding constants of 13C atoms in
indole rings, and as a consequence the 13C resonances are doubled.
The effect of halogen substituents at indole ring is not so significant
on the geometry of examined compounds (see exemplary spectra
of 2a, 8b, 2c, 7c). The packing of these molecules is stabilized by
different type of symmetric weak intermolecular interactions
which slightly shift downfield the 13C NMR resonances as refer-
enced to solution one. The peak multiplicities and intensity of the
resonances are in agreement with the solution spectra. Only the
2.2. 13C CP/MAS NMR spectra in solid state
From the 1960s the interest in polymorphism of drugs (bound
with the bioavailability of their form) developed among the phar-
maceutical community [20–22], and continues to be, particularly in
the development of new drug entities or new active substances
obtained in laboratory. 13CP/MAS NMR spectra of powdered samples
are very useful for characterization of the solid state structure of
organic compounds [23]. The 13C NMR experimental resonances
could be combined with the theoretical values in an attempt to
predict stable conformations present in the solid state together with
the intramolecular interactions. Some chemical species, which are
employed for such analyses, may soon be used for generating new
pharmaceutical solids with desirable properties.
Exemplary solid state 13C CP/MAS NMR spectra are shown in
Figs. 2 and 3 and the most probable assignments of resonances are
given in Table 1. In Fig. 2 we have presented two spectra of type-
a derivatives (without an additional aromatic ring): 5,50-diiodo-3,30-
diindolylmethane (2a) and the 4,40-dimethoxy-3,30-diindolylmethane
(4a). Below these two, the spectra of type-b derivatives are shown
(type-b derivatives have 4-methylphenyl ring): 5,50-dimethoxy-3,30-
diindolyl-(4-methylphenyl)methane (3b), and 5,50-difluoro-3,30-diin-
dolyl-(4-methylphenyl)methane (8b). In Fig. 3 we have shown the
spectra of four derivatives of type c, having 3,5-difluorophenyl ring: the

Fig. 2. The 13C CP/MAS NMR spectra of 2a, 4a, 3b and 8b in the solid state. Sidebands are marked with an asterisk.

Fig. 3. The 13C CP/MAS NMR spectra of 1c, 2c, 3c and 7c in the solid state. Sidebands are marked with an asterisk.

4140
D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
Table 1
Assignments of resonances in 13C CP/MAS NMR spectra of studied compounds.
No Chemical shifts of 13C in solid state,
13C atoms numbering
d [ppm]
C2;C20 C3;C30 C4;C40
C5;C50 C6;C60 C7;C70 C8;C80
C9;C90 The rest of signals
2a 126.9
4a 123.4
1b 123.9
113.1
123.6
85.0
129.5
116.8 132.1
135.7
23.5-C10
(broad)
101.9
99.1
123.4 157.0
154.7
116.9 118.4
119.9
118.1
120.4
106.4 116.7
115.5
110.9 127.1
139.3
23.9-C10; 55.8, 55.4-C-11*
112.2
136.0
38.1-C-10; 19.5-C12*; 139.7-C11, 129.2-C12, 16; 130.2-C13, 15; 137.2-C14
(broad)
123.4
2b 123.3
119.1
128.5
121.5
131.5
127.6
112.6 130.3
117.4
88
(broad)
135.6
131.9
39.7-C-10; 22.0-C12*; 140.6-C11, 128.7-C12, 16; C13, 15; 136.3-C14
125.2
3b 125.0
118.0 98.6
122.8 99.4
152.1
113.3
112.4 127.2
40.4-C-10; 21.4-C12*; 51.9, 54,5-C11*, 142.9-C11, 130.0-C12, 16; 131.4-C13, 15;
136.0-C14
126.0
7b 124.7
152.5
112.8
–
159.0
156.6
118.9
89.7
113.9
124.7
113.3
128.1
112.8 129.5
133.0
135.1
117.2
122.5
39.1-C-10; 20.9-C12*; 140.0-C11, 127.6-C12, 16; 129.5-C13, 15; 134.9-C14
38.3-C-10; 20.5-C12*; 138.5-C11, 126.9-C12, 16; 128.8-C13, 15; 137.4-C14
8b 123.8
119.6 105.2
118.9 104.2
114.6 117.2
122.8 133.2
110.9
113.0 132.8
134.1
1c 122.7
2c 123.7
121.6
135.2
110.9 125.1
120.3 133.2
135.6
141.2
38.5-C10; 147.5-C11; 110.9-C12, 16; 162.1-C13, 15; 101.9-C14
45.4-C10; 151.9-C11; 116.6-C12, 16; 168.4-C13, 15; 106.5-C14
(with benzene) (broad)
3c 124.0
116.9 97.3
151.3
116.7
115.4
112.4 125.1
130.8
136.2
37.3-C10; 56.1, 51.3-C1100; 146.5-C11; 109.3-C12, 16; 161.5-C13, 15 99.9-C14
40.8-C10; 147.4-C11; 92.3-C12, 16; 163.8-C13, 15 101.7-C14
99.8
7c 124.5
119.3 124.5
124.5
113.1
128.7
(broad)
(broad)
(broad)
(with benzene)
spectrum of fluoroindole derivative 8b (with F atoms at C5, C50
positions, third aromatic ring and benzene molecule included in
the crystallographic net) consists of double peaks due to the two
distinct indole moieties. The spectra of compounds having F atoms
show a number of splitted resonances of 13C atoms proximal to F
atoms due to spin–spin coupling between 13C and 19F atoms. On the
other hand, the effect of phenyl ring (derivatives of types b and c)
on the spatial orientation of two indole systems is not significant.
The angles formed by the bonds at the central C10 atom are close to
110ꢀ, which means that they are similar to the standard tetrahedral
109ꢀ. The solid state forms of derivatives’ types a and b can change
according to the medium used for crystallization. The solvent may
interact with the solute leading to the formation of mixed crystals.
In this work, such phenomenon was observed for compounds 1b,
8b, 2c, 7c, which were solvated by benzene used in synthetic
procedure. The exemplary geometries in solid state determined on
the basis of 13C CP/MAS NMR and molecular modeling are shown in
Fig. 4: for compound 4a (as an example of derivatives of type a), for
compound 7b (as an example of derivatives of type b) and for
compound 1c (as an example of derivatives of type c).
Selected bond lengths, bond angles and torsion angles are listed in
Table 2.
The conformations of the molecules are very similar. The indole
fragments are essentially planar to within 0.020(3) Å for 2a and
0.013(3) Å for 8b with the dihedral angle between their mean
planes of 89.67(6)ꢀ and 86.38(7)ꢀ for 2a and 8b, respectively. The
arrangement of these fragments can be described by the torsion
angle C8–C3–C10–C30 (see Table 2). The halogen atoms are nearly
coplanar with the two-ring frameworks. In 8b, the disposition of
the planar 4-methylphenyl ring at C10 with respect to the indole
fragments can be described by the torsion angles C2–C3–C10–C11
of ꢁ108.3(3)ꢀ and C20;–C30;–C10–C11 of 31.9(4)ꢀ.
In the crystals, the packing of the molecules is stabilized by
dissimilar weak intermolecular interactions. The geometric
parameters of all these bonds are given in Table 3.
The crystal structure of 2a is built of layers of the molecules
parallel to (10-1) plane. Within the layer the molecules are con-
nected via N1–H1/
p
and N10–H10/C7 hydrogen bonds (Fig. 6).
The consecutive inversion centers connect the molecules from
adjacent layers. Short contacts C10/C10 and C2/I0 between the
layers result from the crystal packing of the molecules. The packing
arrangement is shown in Fig. 7.
In the crystal of 8b, there is a three dimensional network of
hydrogen-bonding interactions. The molecules are linked by C6–
H6/F0, C1B–H1B/F and N10ꢁH10/C6 hydrogen bonds forming
layers parallel to (001) plane (Fig. 8). Cohesion between the layers
2.3. Crystal structures of 5,50-diiodo-3,30-diindolylmethane 2a and
5,50-difluoro-3,30-diindolyl(4-methylphenylo)methane 8b
The molecular and crystal structures of 2a and 8b in solid state
were analyzed by single crystal X-ray diffraction. The displacement
ellipsoid representation of the molecules, together with the atomic
numbering scheme, is shown in Fig. 5 (the drawings were per-
formed with Mercury program [26]).
results from the N1ꢁH1/
p contacts (Fig. 9). The packing
arrangement along the b axis (Fig.10) shows the benzene molecules
inside the canals parallel to [101] direction.
Both compounds crystallize in the monoclinic space group P21/n
with a single molecule in the asymmetric unit. Moreover the
crystals of 8b include the disordered solvent molecule in such unit.
The structures consist of two indole systems connected by
a common C atom (C10), different halogen atoms at C5, and
compound 8b has additional aromatic 4-methylphenyl ring at C10.
2.4. Cytotoxic activity of bis-indoles
Compounds 3a, 6a and 1c, 3c were submitted to the National
Cancer Institute for testing against panel of 60 cancer lines. Details of
3a, 6a tests were published [24,27]. Compounds 1c and 3c were
evaluated at concentration of 10 mM against a panel of nine cancer

D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
4141
biological activities of bis-indoles, we examined their cytotoxicities
on two human melanoma cell lines ME18 and ME18/R (resistant to
doxorubicin) and compared them with that of the human embryonic
skin fibroblasts WS1. Sensitivity of cells to bis-indole derivatives was
defined on the basis of IC50 values obtained in MTT assay in the
concentrations ranging from 1.25 to 30 mg/ml. The IC50 values were
determined as the concentration of tested compounds decreasing
cell density to 50% as compared to that in the untreated culture after
incubation time. Table 5 summarizes the cytotoxicity data. The
tested bis-indoles demonstrated cytotoxic effects for both human
melanoma cell lines ME18 and ME18/R. 5,50-Diiodo-3,30-diindolyl-
methane (2a), 5,50-dinitro-3,30-diindolylmethane (6a) and 5,50-
dibromo-3,30-diindolyl(4-methylphenylo)methane (7b) proved to
be the most active cytotoxic agents amongst the studied compounds.
3,30-Diindolyl(3,5-difluorophenyl)methane (1c) was characterized
by higher activity against ME18/R cell lines, resistant to doxorubicin.
Compounds 2b, 2c, 3c, and 7c were found to be practically equi-
potent. Derivative 1b displayed the least pronounced cytotoxic
effect.
The cytotoxicity determination was extended to human embry-
onic skin fibroblasts cell line WS1, chosen as representative for
normal, non-malignant cellular population. As was evidenced most
of the bis-indoles (4a, 5a, 6a, 1b, 3b, 7b, 8b, 1c, 2c and 7c) were less
cytotoxic against WS1 cells as compared to the human melanoma
cell lines. Comparable sensitivity of normal fibroblasts and
neoplastic cells – ME18 was noted in the case of 3a which occurred as
less cytotoxic to ME18/R cells. The reference agent 3,30-diindolyl-
methane 1a exhibited the similar effectiveness towards WS1,
comparable to its effects towards ME18 and ME18/R cells.
The following derivatives: 5,50-dinitro-3,30-diindolylmethane
(6a), 5,50-diethoxy-3,30-diindolylmethane (5a) and 4,40-dimethoxy-
3,30-diindolylmethane (4a) occurred to be the most interesting
compounds. They were cytotoxic to ME18/R as well as to ME18 but
not cytotoxic to normal human fibroblasts WS1. Among the tricyclic
derivatives of types b and c we chose three of them which deserve
further testing, i.e., 3,30-diindolyl(3,5-difluorophenyl)methane
1c > 5,50-dibromo-3,30-diindolyl(4-methylphenylo)methane (7b) >
5,50-diiodo-3,30-diindolyl(4-methylphenylo)methane (2b).
3. Conclusion
The most interesting compounds, i.e. cytotoxic to human
melanoma ME18/R and ME18 cell lines but not cytotoxic to normal
human fibroblasts WS1 appeared to be the following derivatives:
5,50-dinitro-3,30-diindolylmethane (6a), 5,50-diethoxy-3,30-diindo-
lylmethane (5a) and 4,40-dimethoxy-3,30-diindolylmethane (4a).
All studied derivatives of 3,30-diindolylmethane existed in solid
state as single molecules in an independent unit, and splitting of
resonances in 13C CP/MAS NMR spectra is due to different packing
mode of both indole rings in one conformer. The conformations of
the alkoxy substituents are crucial for the observed spectral
pattern. As concluded from X-ray diffraction measurement within
Fig. 4. Hypothetical conformations in solid state shown for 4a, 7b and 1c as the
representatives of three types of analyzed compounds.
the layer the molecules are connected via N1ꢁH1/
p
and N10–
H10/C–indole interactions. The halogens located at indole and
phenyl rings are engaged in several intermolecular interactions for
example between C2/I0, C6ꢁH6/F0 and C1B–H1B/F.
cell lines: leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast. Both inhibited strongly growth of
ovarian cancel line IGROV1 (ꢁ6.52 and ꢁ7.10%, respectively),
moderately inhibited growth of renal cancer line UO-31 (52.86 and
43.23%, respectively), melanoma line SK-MEL-2 (58.84 and 62.49%,
respectively), and prostate cancer line PC-3 (67.07 and 50.16%,
respectively). Simultaneously, it was noted that compounds 1c and
3c increased growth of some cancer lines, particularly melanoma
MALME-3M cancer line. To obtain more detailed information about
4. Experimental
All chemicals were purchased from major chemical suppliers as
high or highest purity grade and used without further purification.
Melting points (mp) were determined with a Digital Melting
Point Apparatus 9001 and are uncorrected. Elemental analyses

4142
D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
Fig. 5. The molecular structures of 2a (a) and 8b with the benzene molecule (b).
were performed on GmbH – Vario EL III C,H,N,S Element Analyzer,
and were averaged from two independent determinations.
Table 2
Selected bond lengths [Å] and angles [ꢀ] and selected torsional angles [deg] for 2a
and 8b.
1H NMR and 13C NMR spectra in solution were recorded at 25 ꢀC
with a Bruker Avance DMX 400 spectrometer. Several (as indicated)
1H NMR and 13C NMR spectra in solutions were recorded with
a Varian Unity plus-500 or Varian VNMRS-300 spectrometers. The
13C CP/MAS NMR spectra in solid state were recorded with a Bruker
Avance DMX 400 spectrometer. Samples were contained in 4 mm
ZrO2 rotors and mounted in standard 4 mm MAS probe, and were
spun at 8 kHz. A contact time of 4 ms, a repetition time of 20 s, and
spectral width of 24 kHz were used for accumulation of 2000–4000
scans. Nonprotonated carbons were selectively observed by
2a
1.360(5)
8b
1.395(4)
N1–C9
N10–C90
N1–C2
N10–C20
C8–C9
C80–C90
C3–C10
C30–C10
C9–N1–C2
C90–N10–C20
C3–C10–C30
C8–C3–C10–C30
C80–C30–C10–C3
N1–C2–C3–C10
N10–C20–C30–C10
1.369(4)
1.372(4)
1.369(5)
1.408(4)
1.412(4)
1.503(5)
1.505(5)
108.8(2)
109.3(3)
115.9(3)
177.7(3)
ꢁ89.5(4)
ꢁ178.5(3)
176.7(3)
1.368(4)
1.389(4)
1.396(4)
1.435(4)
1.421(4)
1.533(4)
1.533(4)
108.4(2)
110.0(2)
111.4(2)
ꢁ162.4(2)
83.6(3)
dipolar-dephasing experiment with delay time 50 ms. Chemical
shifts [ppm] were referenced to TMS. The notation used in the NMR
assignments is given in Fig. 1.
Both crystallographic and optimized atom coordinates for 8b
and optimized for remaining compounds were used for
177.0(2)
179.1(2)

D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
4143
syntheses of 5,50-diiodo-3,30-diindolylmethane (2a) and 3,30-diin-
dolyl(4-methylphenyl)methane (1b) have been already described
[16,18], but our procedure was more facile (it allowed us to avoid
nitrogen and reduce the time of reaction). The structure of
compound 2a was additionally proved by X-ray diffraction
measurement (melting points of 2a in [16] and ours are very
different). The synthesis of 4,40-dimethoxy-3,30-diindolylmethy-
lium tetrafluoroborate was also given [30], but pure compound (4a)
was not obtained until now.
Table 3
Hydrogen-bonding geometry [Å and ꢀ] for 2a and 8b. Intermolecular interactions in
crystals (Å, ꢀ).
D–H/A
d(D–H)
d(H/A)
d(D/A)
<(DHA)
2a
N10–H10/C7i
N1–H1/Cgii
C10/C10iii
0.86
0.86
2.82
2.76
3.579(4)
3.538(9)
3.367(7)
3.620(3)
148
151
iv
C2/I0
8b
i
C6–H6/F0
0.93
0.93
0.86
0.86
2.33
2.56
2.84
2.70
3.194(4)
3.381(8)
3.622(4)
3.507(9)
154
148
153
157
4.1.1.1. 5,50-Diiodo-3,30-diindolylmethane (2a) [16]. According to the
general procedure pinkish solid was obtained. The compound was
recrystallized from benzene to give 80% yield; mp ¼ 148.5–
C1B–H1BA/Fii
N10–H10/C6iii
N1–H1/Cgiv
148.8 ꢀC. 1H NMR (500 MHz, DMSO-d6),
d in ppm: 4.08 (s, 2H,10-H);
Cg in upper part of the table represents the centroid of six-membered ring C40/C50/
C60/C70/C80/C90 of the indole. Symmetry codes (upper part of the table): (i) 1/2 þ x, 1/
2 ꢁ y, 1/2 þ z; (ii) x, ꢁ1 þ y, z; (iii) 1 ꢁ x, 1 ꢁ y, 1 ꢁ z; (iv) –x, 1 ꢁ y, 1 ꢁ z. Cg in lower
part of the table represents the centroid of five-membered ring N10/C20/C30/C80/C90
of the indole. Symmetry codes (lower part of the table): (i) 3/2 ꢁ x, ꢁ1/2 þ y, 3/2 ꢁ z;
(ii) 1 ꢁ x, ꢁy, 1 ꢁ z; (iii) 1/2 ꢁ x, 1/2 þ y, 3/2 ꢁ z; (iv) 1/2 þ x, 1/2 ꢁ y, 1/2 þ z.
7.19 (s, 2H, 4-H, 40-H); 7.21 (d, 2H, J ¼ 8 Hz, 7-H, 70-H); 7.29 (d, 2H,
J ¼ 8 Hz, 6-H, 60-H); 7.85 (s, 2H, 2-H, 20-H); 10.97 (s (broad), 2H,
2NH). 13C NMR (125 MHz, DMSO-d6),
d in ppm: 20.54 (C-10); 81.94
(C-5, C-50); 113.39 (C-3, C-30); 113.93 (C-7, C-70); 124.07 (C-4, C-40);
127.12 (C-2, C-20); 128.71 (C-6, C-60); 129.86 (C-8, C-80); 135.45 (C-9,
C-90). Anal. Calcd for C17H12I2N2 (498.10): C, 40.99%; H, 2.43%; I,
50.95%; N, 5.02%; found: C, 40.89%; H, 2.40%; N, 5.00%.
computation of shielding constants
s
[ppm] of 13C atoms to assign
the resonances in solid state NMR spectra. We have employed the
PM3 semiempirical method for structures’ optimization [28] and
the DFT method with B3LYP/6-311G (d, p) hybrid functional and
CPHF-GIAO approach for the NMR shielding constant computation
using the Gaussian 03 program [29].
4.1.1.2. 4,40-Dimethoxy-3,30-diindolylmethane (4a). After general
procedure and crystallization from benzene colourless solid of 4a
was obtained with 95% yield, mp ¼ 161.3–162.5 ꢀC. 1H NMR
(500 MHz, DMSO-d6), d in ppm: 3.82 (s, 6H, 2OCH3); 4.39 (s, 2H, 10-
H); 6.42 (dd, 2H, J1 ¼1.5 Hz, J2 ¼ 7.0 Hz, 5-H, 50-H); 6.75 (d, 2H,
J ¼ 2 Hz, 2-H, 20-H); 6.90 (dd, 2H, J1 ¼1.5 Hz, J2 ¼ 7.0 Hz, 7-H, 70-H);
6.92 (t, 2H, J ¼ 7.0 Hz, 6-H, 60-H); 10.63 (s (broad), 2H, 2NH); 13C
4.1. Syntheses
4.1.1. General procedure for the preparation of 3,30-diindolylmethanes
and 3,30-diindolyl(aryl)methanes
NMR (125 MHz, DMSO-d6),
d in ppm: 22.22 (C-10); 54.55 (2OCH3);
98.29 (C-5, C-50); 104.46 (C-7, C-70); 115.61 (C-8, C-80); 116.58 (C-3,
C-30); 121.05 (C-2, C-20); 121.07 (C-6, C-60); 137.46 (C-9, C-90);
154.09 (C-4, C-40). Anal. Calcd for C19H18N2O2 (303.36): C, 74.49%; H,
5.92%; N, 9.14%; found: C, 74.29%; H, 5.91%; N, 8.99%.
Indole derivatives (5 mmol) were dissolved in water (25 ml)
with an appropriate derivative of benzaldehyde (2.5 mmol) (for
compounds of types b and c) or with formalin (0.20 g, 2.5 mmol) for
compounds of type a. Next, 2 or 3 drops of concentrated H2SO4
were added. The mixture was kept out of light and stirred at 85–
100 ꢀC for 2.5–5 h. The reaction was monitored by TLC. After
reaching room temperature, the water phase was extracted three
times with diethyl ether (10 ml) and the combined organic phases
were dried with MgSO4 anh. The solvent was evaporated. The oily
residue was recrystallized from benzene, hexane or alcohol. The
4.1.1.3. 3,30-Diindolyl(4-methylphenyl)methane (1b) [18]. According
to the general procedure colourless solid was obtained with 49%
yield. Compound 1b was recrystallized from benzene, mp ¼ 87–
87.3 ꢀC. 1H NMR (400 MHz, CDCl3), in ppm: 2.35 (3H, 1200-H); 5.87
d
(s, 1H, 10-H); 6.63 (s, 2H, 2-H, 20-H); 7.03 (t, 2H, 5-H, 50-H, J ¼ 8 Hz);
7.11 (d, 2H, J ¼ 8 Hz,13-H,15-H); 7.19 (t, 2H, J ¼ 8 Hz, 6-H, 60-H); 7.25
Fig. 6. The interconnections within a layer of 2a.

4144
D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
Fig. 7. Projection of the crystal structure of 2a along the b axis with layers (black and red colours) and contacts (blue dashed lines) between them. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of the article.)
(m, 2H, 12-H, 16-H); 7.34 (d, 2H, 7-H, 70-H, J ¼ 8 Hz); 7.42 (d, 2H, 4-
H, 14-H, J ¼ 8 Hz); 7.80 (s (broad), 2H, 2NH); 13C NMR (100 MHz,
(C-4, C-40); 129.33 (C-13, C-15); 129.69 (C-8, C-80); 130.62 (C-6, C-
60); 135.98 (C-9, C-90); 136.15 (C-14); 140.24 (C-11). Anal. Calcd for
C24H18I2N2 (588.23): C, 49.01; H, 3.08; I, 143.15; N, 4.76; found: C,
48.96%; H, 3.11%; N, 4.75%.
CDCl3),
d
in ppm: 21.28 (C-1200); 39.95 (C-10), 111.20 (C-7, C-70);
119.38 (C-5, C-50); 120.07 (C-3, C-30); 120.16 (C-4, C-40); 122.06 (C-6,
C-60); 123.74 (C-2, C-20); 127.29 (C-8, C-80); 128.76 (C-12, C-16);
129.11 (C-13, C-15); 135.68 (C-14); 136.86 (C-9, C-90); 141.18 (C-11).
4.1.1.5. 5,50-Dimethoxy-3,30-diindolyl(4-methylphenyl)methane (3b).
After general procedure, colourless solid was recrystallized from
methanol to give 60% yield of compound 3b, mp ¼ 215.4–215.7 ꢀC.
4.1.1.4. 5,50-Diiodo-3,30-diindolyl(4-methylphenylo)methane (2b).
According to the general procedure colourless solid of 2b was
obtained with 18% yield. Compound 2b was recrystallized from
1H NMR (400 MHz, CDCl3),
d
in ppm: 2.51 (s, 3H, 1200-CH3); 3.47 (s,
6H, 2OCH3); 5.67 (s, 1H, 10-H); 6.66 (dd, 2H, J1 ¼8 Hz, J2 ¼ 1.2 Hz, 6-
H, 60-H); 6.70 (d, 2H, J ¼ 1.2 Hz, 4-H, 40-H); 6.78 (s, 2H, 2-H, 20-H);
7.04 (m, 2H, 13-H, 15-H); 7.20 (m, 2H, 12-H, 16-H); 7.22 (d, 2H,
J ¼ 8 Hz, 7-H, 70-H); 10.60 (s, broad, 2H, 2NH). 13C NMR (100 MHz,
benzene, mp ¼ 224.7–225.0 ꢀC. 1H NMR (400 MHz, CDCl3),
d in
ppm: 2.26 (s, 3H, 1200-H); 5.63 (s, 1H, 10-H); 6.52 (s, 2H, 2-H, 20-H);
7.04 (m, 2H, 15-H, 13-H); 7.07 (d, 2H, J ¼ 8 Hz, 7-H, 70-H); 7.34 (dd,
2H, J1 ¼8 Hz, J2 ¼1.2 Hz, 6-H, 60-H); 7.61 (d, 2H, J2 ¼ 1.2 Hz, 4-H, 40-
CDCl3),
d in ppm: 20.74 (C-11); 39.36 (C-10); 55.39 (2OCH3), 101.69
H); 7.87 (s (broad), 2H, 2NH). 13C NMR (100 MHz, CDCl3),
d
in ppm:
(C-4, C-40); 110.62 (C-6, C-60); 112.08 (C-7, C-70); 118.00 (C-3, C-30);
124.20 (C-2, C-20); 127.15 (C-8, C-80); 128.33 (C-12, C-16); 128.71
(C-13, C-15); 131.94 (C-9, C-90); 134.70 (C-14); 142.08 (C-11); 152.76
21.30 (C-1200); 39.56 (C-10); 83.14 (C-5, C-50); 113.30 (C-7, C-70);
119.24 (C-3, C-30); 124.51 (C-2, C-20); 128.55 (C-12, C-16); 128.75
Fig. 8. The interconnections within the layer of 8b.

D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
4145
Fig. 9. Projection of the crystal structure of 8b along the a axis with layers (black and red colours) and N1–H1/p contacts (blue dashed lines) between them. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of the article.)
(C-5, C-50). Anal. Calcd for C26H24N2O2 (396.49): C, 78.76%; H, 6.10%;
N, 7.07%; found: C, 78.37%; H, 6.06%; N, 7.00%.
benzene to give 52% yield of compound 8b, mp ¼ 90.8–91.2 ꢀC. 1H
NMR (400 MHz, CDCl3), d
in ppm: 2.30 (s, 3H, 1200-CH3); 5.65 (s, 1H,
10-H); 6.63 (d, 2H, J ¼ 1 Hz, 2-H, 20-H); 6.87 (td, 2H, J1 ¼8.8 Hz,
J2 ¼ 2.0 Hz, 6-H, 60-H); 6.97 (dd, 2H, J1 ¼9.6 Hz, J2 ¼ 2.0 Hz, 4-H, 40-
H); 7.06 (m, 2H, 13-H, 15-H); 7.16 (m, 2H, 12-H, 16-H); 7.18 (d,
J ¼ 8.8 Hz, 2H, 7-H, 70-H); 7.76 (s (broad), 2H, 2NH). 13C NMR
4.1.1.6. 5,50-Dibromo-3,30-diindolyl(4-methylphenylo)methane (7b).
After general procedure, colourless solid was recrystallized from
benzene/hexane (1:2 v/v) to give 28% yield of compound 7b,
mp ¼ 214.5–214.7 ꢀC. 1H NMR (400 MHz, CDCl3),
d
in ppm: 2.20 (s,
(100 MHz, CDCl3), d
in ppm: 21.24 (C-12000); 40.00 (C-10); 104.96 (d,
3H, 1200-CH3); 5.56 (s, 1H, 10-H); 6.47 (s, 2H, 2-H, 20-H); 6.95 (m, 2H,
13-H,15-H); 7.03 (m, 2H,12-H,16-H); 7.05 (d, 2H, J ¼ 8.5 Hz, 7-H, 70-
H); 7.11 (dd, 2H, J1 ¼8.5 Hz, J2 ¼ 1 Hz, 6-H, 60-H); 7.40 (s (broad), 2H,
JC-F ¼ 23.3 Hz, C-4, C-40); 110.51 (d, JC-F ¼ 26.2 Hz, C-6, C-60); 111.50
(d, JC-F ¼ 6.8 Hz, C-7, C-70); 119.79 (d, JC-F ¼ 4.7 Hz, C-3, C-30); 125.38
(C-2, C-20); 127.53 (d, JC-F ¼ 9.7 Hz, C-8, C-80); 128.62 (C-12, C-16);
129.28 (C-13, C-15); 133.36 (C-9, C-90); 136.04 (C-14); 140.45 (C-
11); 157.69 (d, JC-F ¼ 233 Hz, C-5, C-50). Anal. Calcd for
C24H18F2N2$½C6H6 (411.47) C, 78.81%; H, 5.14%; N, 6.781%; found: C,
78.42%; H, 5.27%; N, 6.49%.
4-H, 40-H); 7.74 (s (broad), 2H, 2NH). 13C NMR (100 MHz, CDCl3),
d in
ppm: 21.27 (C-1200); 39.68 (C-10); 112.77 (C-7, C-70); 112.85 (C-5, C-
50); 119.46 (C-3, C-30); 122.50 (C-4, C-40); 124.90 (C-2, C-20); 125.13
(C-6, C-60); 128.57 (C-12, C-16); 128.89 (C-8, C-80); 129.34 (C-13, C-
15); 135.54 (C-14); 136.16 (C9, C90); 140.23 (C-11). Anal. Calcd for
C24H18Br2N2$½C6H6 (533.29): C, 60.81%; H, 3.97%; Br, 29.397%; N,
5.25%; found: C, 59.87%; H, 3.90%; N, 5.30%.
4.1.1.8. 3,30-Diindolyl(3,5-difluorophenyl)methane (1c). After general
procedure, colourless solid was dissolved in ethanol and drops of
water were slowly added at boiling point to give 54% yield of
4.1.1.7. 5,50-Difluoro-3,30-diindolyl(4-methylphenylo)methane (8b).
After general procedure, colourless solid was recrystallized from
compound 1c, mp ¼ 159–160 ꢀC. 1H NMR (400 MHz, CDCl3),
d in
ppm: 5.83 (s, 1H, 10-H); 6.59 (s, 2H, 2-H, 20-H); 6.64 (tt, 1H,
J1 ¼9 Hz, J2 ¼ 2 Hz, 14-H); 6.84 (m, 2H, 12-H, 16-H); 7.02 (t, 2H,
J ¼ 8 Hz, H-5, H-50); 7.18 (t, 2H, J ¼ 8 Hz, H-6, H-60); 7.32 (d, 2H,
J ¼ 8 Hz, H-7, H-7); 7.36 (d, 2H, J ¼ 8 Hz, H-4, H-40); 7.80 (s (broad),
2H, 2NH). 13C NMR (100 MHz, CDCl3),
d in ppm: 40.23 (C-10), 101.97
(t, C-14, JC-F ¼ 25.4 Hz); 111.45 (C-7, C-70), 111.78 (tt, C-12, C-16, JC-
¼ 24.6 Hz), 118.56 (C-3, C-30), 119.73 (C-5, C-50), 119.86 (C-4, C-40),
F
122.44 (C-6, C-60), 123.81 (C-2, C-20), 126.97 (C-8, C-80), 136.88 (C-9,
C-90), 148.62 (t, J ¼ 8.1 Hz, C-11); 163.23 (dd, C-13, C-15, JC-
¼ 246 Hz, JC-F ¼ 12.7 Hz). Anal. Calcd for C23H16F2N2 (347.35) C,
F
77.08%; H, 4.50%; N, 7.82%; found: C, 77.07%; H, 4.45%; N, 7.88%.
4.1.1.9. 5,50-Diiodo-3,30-diindolyl(3,5-difluorophenyl)methane (2c).
After general procedure, colourless solid was dissolved in benzene
and drops of hexane were slowly added at boiling point to give 40%
yield of compound 2c, mp ¼ 109.9–110.5 ꢀC. 1H NMR (300 MHz,
CDCl3),
d
in ppm: 5.70 (s, 10-H); 6.59 (d, 2H, 2-H, 20-H, J ¼ 1.5 Hz);
6.69 (tt, 1H, J1 ¼9 Hz, J2 ¼ 2 Hz, 14-H); 6.79 (m, 2H, 12-H, 16-H); 7.14
(d, 2H, J ¼ 9 Hz, 7-H, 70-H); 7.44 (dd, 2H, J1 ¼9 Hz, J2 ¼1.5 Hz, 6-H, 60-
H); 7.65 (d, 2H, J ¼ 1.5 Hz, 4-H, 40-H); 7.97 (s (broad), 2NH). 13C NMR
(75 MHz, CDCl3), d
in ppm: 39.73 (C-10); 83.43 (C-5, C-50); 102.42 (t,
JC-F ¼ 25.2 Hz, C-14); 111.64 (m, C-12, C-16); 113.50 (C-70, C-70);
117.67 (C-3, C-30); 124.57 (C-2, C-20); 128.44 (C-4, C-40); 129.34 (C-8,
C-80); 131.00 (C-6, C-60); 135.97 (C-9, C-90); 147.60 (t, JC-F ¼ 7.5 Hz, C-
11); 163.29 (dd, JC-F ¼ 246.9 Hz, JC-F ¼ 12.6 Hz, C-13, C-15). Anal.
Calcd for C23H14F2I2N2$C6H6 (610.19) C, 50.61%; H, 2.93%; N, 4.07%;
found: C, 50.81%; H, 2.99%; N, 4.09%.
Fig. 10. The packing arrangement of 8b along the b axis.

4146
D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
4.1.1.10. 5,50-Dimethoxy-3,30-diindolyl-(3,5-difluorophenyl)methane
(3c). After general procedure, colourless solid was recrystallized
from methanol to give 60% yield of compound 3c, mp ¼ 195.4–
Table 4
Crystal data, data collection and structure refinement for compounds 2a and 8b.
Compound
2a
8b
196 ꢀC. 1H NMR (500 MHz, CDCl3),
d in ppm: 3.72 (s, 6H, 2OCH3);
Empirical formula
Formula weight
T (K)
Wavelength (Å)
Crystal system, space group
Unit cell dimensions
a (Å)
C17H12N2I2
498.09
293(2)
0.71073
Monoclinic, P21/n
C30H24N2F2
450.51
120(2)
0.71073
Monoclinic, P21/n
5.74 (s, 1H, 10-H); 6.66 (tt (hided), 1H, 14-H); 6.66 (d, 2H, J ¼ 2 Hz,
2-H, 20-H); 6.78 (d, 2H, J ¼ 2.5 Hz, 4-H, 40-H); 6.86 (dd, 2H,
J1 ¼8.5 Hz, J2 ¼ 2.5 Hz, 6-H, 60-H); 6.87 (dd, 2H, J1 ¼8.5 Hz,
J2 ¼ 2.0 Hz, 12-H, 16-H); 7.25 (d, 2H, J ¼ 8.5 Hz, 7-H, 70-H); 7.86 (s
(broad), 2H, 2NH). 13C NMR (125 MHz, CDCl3),
d in ppm: 40.05 (C-
11.6634(5)
9.0383(3)
15.7982(5)
109.600(4)
1568.9(1)
4, 2.109
4.003
936
4.16–25.03
ꢁ10 ꢂ h ꢂ 13
ꢁ10 ꢂ k ꢂ 10
ꢁ18 ꢂ l ꢂ 18
9.8917(6)
19.4883(8)
13.7389(6)
108.628(6)
2509.7(2)
4, 1.192
0.080
944
4.18–25.02
ꢁ11 ꢂ h ꢂ 11
ꢁ22 ꢂ k ꢂ 23
ꢁ16 ꢂ l ꢂ 16
b (Å)
c (Å)
10); 54.89 (2OCH3); 101.71 (t, JC-F ¼ 20.7 Hz, C-14), 101.66 (C-4, C-
40); 111.53 (m, C-12, C-16); 111.86 (C-6, C-60); 112.13 (C-7, C-70);
117.92 (C-3, C-30); 124.40 (C-2, C-20); 127.17 (C-8, C-80); 131.84 (C-9,
C-90); 148.34 (t, JC-F ¼ 8.40 Hz, C-11); 153.89 (C-5, C-50); 163.00 (dd,
JC-F ¼ 196.3 Hz, JC-F ¼ 10.3 Hz, C-13, C-15). Anal. Calcd for
C25H20F2N2O2 (418.42): C, 71.76%; H, 4.82%; N, 6.69%; found: C,
71.69%; H, 4.88%; N, 6.70%.
b
(ꢀ)
Volume (Å3)
Z, Dx (Mg/m3)
m
(mmꢁ1
)
F(000)
range for data collection (ꢀ)
q
hkl range
4.1.1.11. 5,50-Dibromo-3,30-diindolyl(3,5-difluorophenylo)methane
(7c). After general procedure, colourless solid was recrystallized
from benzene/hexane (1:1, v/v) to give 19% yield of compound 7c,
Reflections
Collected
Unique (Rint
9225
2754 (0.014)
2260
2754/0/190
Multi-scan
1.040
0.0218
0.0654
0.557/ꢁ0.719
23 459
4404(0.023)
3253
4404/42/307
Multi-scan
1.078
0.0676
0.2208
0.849/ꢁ0.439
)
mp ¼ 118.7–119.3 ꢀC. 1H NMR (400 MHz, CDCl3),
d in ppm: 5.74 (s,
Observed (I > 2s(I))
1H, H-10); 6.67 (d, 2H, J ¼ 1.5 Hz, 2-H, 20-H); 6.71 (tt, 1H,
J1 ¼9.2 Hz, J2 ¼ 2 Hz, 14-H); 6.81 (m, 2H, 12-H, 16-H); 7.26 (d, 2H,
J ¼ 8.4 Hz, 7-H, 70-H); 7.29 (dd, 2H, J1 ¼8.4 Hz, J2 ¼1.5 Hz, 6-H, 60-
H), 7.47 (d, 2H,J ¼ 1.5 Hz, 4-H, 40-H); 8.02 (s (broad), 2H, 2NH). 13C
Data/restraints/parameters
Absorption correction
Goodness-of-fit on F2
R(F) (I > 2
s(I))
wR(F2) (all data)
Max/min. Dr (e/Å3)
NMR (100 MHz, CDCl3),
d in ppm: 39.78 (C-10); 102.35 (t,
J ¼ 25.2 Hz, C-14); 111.60 (m, C-12, C-16); 112.91 (C-7, C-70); 113.12
(C-5, C-50); 117.87 (C-3, C-30); 122.14 (C-4, C-40); 124.89 (C-2, C-20);
125.47 (C-6, C-60); 128.48 (C-8, C-80); 135.47 (C-9, C-90); 147.52 (t,
JC-F ¼ 8.40 Hz, C-11); 163.25 (dd, JC-F ¼ 246.7 Hz, JC-F ¼ 12.9 Hz, C-
13, C-15). Anal. Calcd for C23H14Br2F2N2$C6H6 (594.30): C, 58.61%;
H, 3.39%; Br, 26.89%; F, 6.39%; N, 4.71%; found: C, 58.21%, H, 3.30%;
N, 4.61%.
by contacting The Cambridge Crystallographic Data Centre, 12,
Union Road, Cambridge CB2 1EZ, UK; fax: þ44 1223 336033.
4.3. Biological assays
4.3.1. In vitro assays: chemicals, solutions and other materials
The cell culture flasks and the 96-well microplates were
provided by Nunc. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide, DMSO (dimethyl sulfoxide) and antimycotic
antibiotics (penicillin and streptomycin) were obtained from Sigma
Co. MEM (Minimum Essential Medium) was purchased from Gibco
Co. PBS (phosphate–Ca2þ and Mg2þ-free buffered saline) was
obtained from IITD, Poland. Fetal bovine serum was bought from
Bioproduct, Hungary. The stock solutions of tested compounds were
freshly prepared in DMSO and diluted in medium just before use. In
the final dilutions obtained the concentrations of DMSO never
exceeded 0.4%.
4.2. X-ray diffraction measurements’ details
Crystals of 2a and 8b, suitable for X-ray analysis, were grown by
slow evaporation from benzene solution. All details of the measure-
ments, crystal data and structure refinement are given inTable 4. The
data were collected on an Oxford Diffraction KM4CCD diffractometer
[31], using graphite-monochromated Mo Ka radiation, at 293 K and
120 K for 2a and 8b, respectively. The unit cells parameters were
determined by least-squares treatment of setting angles of highest-
intensity reflections chosen from the whole experiments. Intensity
data were corrected for the Lorentz and polarization effects [32]. The
structures were solved by direct methods by use of the SHELXS97
program [33] and refined by the full-matrix least-squares method
with the SHELXL97 program [34]. Three reflections for 2a and two for
Table 5
The IC50 values of the tested compounds (in
m
mol/ml, x ꢃ SEM).
8b were excluded from the reflection file due to their large
Compound
WS1-human
embryonic skin
fibroblasts
ME18-human
melanoma
ME18/R-human melanoma/
resistant to doxorubicin
P
2
2
2
2 2
(jFoj ꢁ jFcj ) differences. The function w(jFoj ꢁ jFcj ) was mini-
mized with wꢁ1 ¼ [
s
2(Fo)2 þ (0.0388P)2 þ1.0105P] for 2a and
wꢁ1 ¼ [
s
2(Fo)2 þ (0.1257P)2 þ1.6736P] for 8b, where P ¼ (Fo2 þ 2F2c)/3.
1a
2a
3a
4a
5a
6a
1b
2b
3b
7b
8b
1c
2c
3c
7c
36.6 ꢃ 6.5
13.0 ꢃ 1.0
22.8 ꢃ 4.9
31.0 ꢃ 3.3
32.7 ꢃ 4.3
33.3 ꢃ 3.6
58.4 ꢃ 4.0
16.2 ꢃ 3.6
38.6 ꢃ 6.3
26.3 ꢃ 2.8
45.9 ꢃ 9.9
36.0 ꢃ 2.0
28.5 ꢃ 1.7
26.5 ꢃ 7.9
24.2 ꢃ 2.9
37.0 ꢃ 0.4
9.7 ꢃ 2.7
40.7 ꢃ 2.0
17.3 ꢃ 4.0
30.7 ꢃ 7.8
22.5 ꢃ 4.2
19.7 ꢃ 4.6
14.3 ꢃ 4.0
35.9 ꢃ 4.9
21.1 ꢃ 6.8
33.0 ꢃ 1.5
17.4 ꢃ 5.7
32.5 ꢃ 10.5
17.0 ꢃ 5.2
23.8 ꢃ 2.1
28.2 ꢃ 5.0
22.7 ꢃ 5.4
All non-hydrogen atoms were refined with anisotropic thermal
parameters. The atoms of the benzene ring (of the solvent which is
included in crystals of 8b) appeared disordered, so restrains for the
thermal parameters and the bond lengths for these atoms, according
to SHELXL97, were applied. The coordinates of the hydrogen atoms
were calculated in idealized positions and refined as a riding model
withtheirthermalparameters calculatedas 1.2(1.5for methyl group)
times Ueq of the respective carrier carbon atom.
21.2 ꢃ 6.5
21.2 ꢃ 7.8
23.6 ꢃ 4.8
16.9 ꢃ 5.8
46.7 ꢃ 1.9
22.6 ꢃ 3.1
32.9 ꢃ 6.6
18.0 ꢃ 1.9
43.0 ꢃ 1.2
31.1 ꢃ 4.6
21.3 ꢃ 1.1
28.4 ꢃ 0.9
23.4 ꢃ 2.9
The deposition numbers CCDC 730183 for 2a and 730184 for 8b
contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge via www.ccdc.cam.ac.uk/
data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or

D. Maciejewska et al. / European Journal of Medicinal Chemistry 44 (2009) 4136–4147
4147
[5] X. Chang, J.C. Tou, C. Hong, et al., Carcinogenesis 26 (2005) 771–778.
[6] M. Nachshon-Kedmi, F.A. Fares, S. Yannai, Prostate 61 (2004) 153–160.
[7] C. Hong, H.A. Kim, G.L. Firestone, et al., Carcinogenesis 23 (2002) 1297–1305.
[8] C. Hong, G.L. Firestone, L.F. Bjeldanes, Biochem. Pharmacol. 63 (2002)
1085–1097.
[9] L. Xue, G.L. Firestone, L.F. Bjeldanes, Oncogene 24 (2005) 2343–2353.
[10] Y. Gong, H. Sohn, L. Xue, G.L. Firestone, L.F. Bjendales, Cancer Res. 66 (2006)
4880–4887.
[11] M.A. Zeligs, C. Jacobs, Formulation and Use of Controlled Release Indole
Alkaloids, US Patent 6,416,793; 9.07.2002.
[12] H. Shirani, B. Stensland, J. Bergman, T. Janosik, Synlett 15 (2006) 2459.
[13] H. Shirani, T. Janosik, Synthesis 17 (2007) 2690.
[14] A. McDougal, M.S. Gupta, K. Ramamoorthy, G. Sun, S.H. Safe, Cancer Lett. 151
(2000) 169.
[15] P.K. Pradhan, S. Dey, V.S. Giri, P. Jaisankar, Synthesis (2005) 1779–1782.
[16] S. Fo¨ldeak, J. Czombos, B. Matkowics, Acta Univ. Sjeged. Acta Phys. Chem. 11
(1965) 115–125.
4.3.2. Cell lines and culture conditions
The cell lines WS1 (human embryonic skin fibroblasts) were
supplied by ATCC, ME18 (human melanoma cells) was a gift from
M. Sk1odowska-Curie Memorial Cancer Center and Institute of
Oncology, Warsaw, Poland, ME18/R (the subline resistant to doxo-
rubicin) was obtained experimentally in National Medicines Insti-
tute [35]. Cells are also resistant to vinblastine and methotrexate,
which indicates that they have acquired pattern of pleiotropic
multidrug resistance [36]. Cells were cultured as monolayers in
MEM medium supplemented with 10% fetal bovine serum and
antibiotics in humidified atmosphere at 37 ꢀC and 5% CO2. The cell
line ME18/R was maintained in MEM with doxorubicin, 0.04
cell cultures were mycoplasma-free.
mM. All
[17] C. Pal, S. Dey, S.K. Mahato, J. Vinayagam, P.K. Pradhan, V.S. Giri, P. Jaisankar,
T. Hossain, S. Baruri, D. Ray, S.M. Biswas, Bioorg. Med. Chem. Lett. 17 (2007)
4924–4928.
[18] S.J. Ji, S.Y. Wang, Y. Zhang, T.P. Loh, Tetrahedron 60 (2004) 2051–2055.
[19] R. Nagarajan, P.T. Perumal, Chem. Lett. 33 (2004) 288–289.
[20] J. Haleblian, W. McCrane, J. Pharm. Sci. 58 (1969) 911–929.
[21] D.C. Apperley, R.A. Fletton, R.K. Harris, R.W. Lancaster, S. Tavener, T.L. Threlfall,
J. Pharm. Sci. 88 (1999) 1275.
4.3.3. MTT assay
Cell survival was evaluated by using the MTT-dye reduction
assay, which is based on the ability of viable cells to metabolize
a yellow tetrazolium salt to violet formazan product that can be
detected spectrophotometrically. The assay was carried out by
the method previously described [37] with minor modifications
[38].
The suspension of cells was diluted in MEM to concentration of
105 cells/ml and 0.05 ml of suspension was placed into individual
well on 96-well multiplate. Then, 0.05 ml of the tested compounds
solved in DMSO and diluted in MEM were added at double strength
dilution. After a 48 h continuous exposure the medium was
[22] A. Bauer-Brandl, Int. J. Pharm. 140 (1996) 195.
[23] R.H. Harris, J. Pharm. Pharmacol. 59 (2007) 225–239.
[24] D. Maciejewska, M. Niemyjska, I. Wolska, M. W1ostowski, M. Rasztawicka, Z.
Naturforsch. 59b (2004) 1137–1142.
[25] M. Rasztawicka, I. Wolska, D. Maciejewska, J. Mol. Struct. 831 (2007)
174–179.
[26] C.F. Macrae, P.R. Edgington, P. McCabe, E. Pidcock, G.P. Shields, R. Taylor,
M. Towler, J. van de Streek, Mercury: visualization and analysis of crystal
structures, J. Appl. Crystallogr. 39 (2006) 453.
_
[27] D. Maciejewska, I. Wolska, M. Niemyjska, P. Zero, J. Mol. Struct. 753 (2005)
removed and 50 ml of MTT solution in PBS (5 mg/ml) was added.
53–60.
The plates were incubated for 4 h at 37 ꢀC in a humidified 5% CO2
atmosphere. The formazan crystals yielded were dissolved in
DMSO and the absorbance was measured at 500 nm. The final
[28] HyperChem 7.01, Hypercube Inc., Canada, 2002.
[29] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega,
G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li,
J.E. Knox, H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts,
R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski,
P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg,
V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick,
A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul,
S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,
I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng,
A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen,
M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision B.02, Gaussian, Inc.,
Pittsburgh, PA, 2003.
concentration of bis-indole derivatives ranged from 1.25 to 30 mg/
ml. The cells treated as control were kept in bis-indole-free medium
or in medium with DMSO. In all experiments, three replicate wells
were used at each point. IC50 values were determined graphically
and analyzed statistically by the ANOVA test.
Acknowledgments
The authors thank Interdisciplinary Center for Mathematical
and Computational Modeling (ICM) of Warsaw University for the
computational grant.
[30] U. Pindur, H. Witzel, Monatsh. Chem. 121 (1990) 77–80.
[31] Oxford Diffraction Poland, CrysAlisCCD, CCD Data Collection GUI, Version
1.171.31.8 (2007) and 1.171.32.5 (2007).
[32] Oxford Diffraction Poland, CrysAlisRED, CCD Data Reduction GUI, Version
1.171.31.5 (2006) and 1.171.32.5 (2007).
References
[33] G.M. Sheldrick, Acta Crystallogr. A46 (1990) 467–473.
[34] G.M. Sheldrick, SHELXL97, Program for the Refinement of Crystal Structures,
[1] R.H. Dashwood, A.T. Fong, D.E. Williams, J.D. Hendricks, G.S. Bailey, Cancer Res.
51 (1991) 2362–2365.
University of Go
¨ttingen, Germany, 1997.
[35] E.L. Anuszewska, B.M. Gruber, J.H. Koziorowska, Biochem. Pharmacol. 54
(1997) 597–603.
[2] D.J. Kim, B.S. Han, B. Ahn, R. Hasegawa, T. Shirai, N. Ito, H. Tsuda, Carcino-
genesis 18 (1997) 377–381.
[36] E.L. Anuszewska, J.H. Koziorowska, Mutat. Res. 431 (1999) 25–30.
[37] T. Mosmann, J. Immunol. Methods 65 (1983) 55–60.
[38] B.M. Gruber, E.L. Anuszewska, Toxicol. In Vitro 16 (2002) 663–667.
[3] C.A. De Kruif, J.W. Marsman, J.C. Venekamp, et al., Chem. Biol. Interact. 80
(1991) 305–315.
[4] I. Chen, A. McDougal, F. Wang, S. Safe, Carcinogenesis 19 (1998) 1631–1639.