Synthesis, cytostatic evaluation and structure activity relationships of novel bis-indolylmethanes and their corresponding tetrahydroindolocarbazoles

Article abstract of DOI:10.1016/j.ejmech.2014.12.008

BIMs (bis-indolylmethanes) (1_a-n) were synthesized using glacial acetic acid as a protic acid for promotion of the condensation reaction of indoles with aldehydes in high yields (86-98 %). Corresponding tetrahydroindolo[2,3-b]carbazoles (2

Full text of DOI:10.1016/j.ejmech.2014.12.008

Accepted Manuscript
Synthesis, Cytostatic Evaluation and Structure Activity Relationships of Novel Bis-
indolylmethanes and their Corresponding Tetrahydroindolocarbazoles
Mardia T. El Sayed , Khadiga M. Ahmed , Kazem Mahmoud , Andreas Hilgeroth
PII:
S0223-5234(14)01117-9
10.1016/j.ejmech.2014.12.008
EJMECH 7568
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 September 2014
Revised Date: 2 November 2014
Accepted Date: 5 December 2014
Please cite this article as: M.T. El Sayed, K.M. Ahmed, K. Mahmoud, A. Hilgeroth, Synthesis,
Cytostatic Evaluation and Structure Activity Relationships of Novel Bis-indolylmethanes and their
Corresponding Tetrahydroindolocarbazoles, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2014.12.008.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis, Cytostatic Evaluation and Structure Activity Relationships of Novel
Bisindolylmethanes and their Corresponding Tetrahydroindolocarbazoles.
Mardia T. El Sayeda,b,*, Khadiga M. Ahmedc, Kazem Mahmouda, and Andreas Hilgerotha.
aInstitute of Pharmacy, Martin-Luther University, Research Group of Drug Development and Analysis, Wolfgang-
Langenbeck-Straße 4, 06120 Halle, Saale, Germany. bApplied Organic Chemistry Department, National Research Centre,
Cairo, Egypt., cNatural Compounds Laboratory, National Research Centre, Cairo Egypt.
Corresponding author: Mardia El Sayed
Mardia_elsayed2009@yahoo.com
Dear Sir,
BIMs (bis-indolylmethanes) (1a-n) were synthesized via using glacial acetic acid as a protic acid for
promotion of the condensation reaction of indoles with aldehydes in high yields (86-98 %).
Corresponding tetrahydroindolo[2,3-b]carbazoles (2a-m) were synthesized via condensation of BIMs
with aldehydes. Ten synthesized compounds have been submitted to the national cancer institute in
the USA where all the submitted samples have been selected for one dose screening. As a result of the
one dose screening of BIMs (1e,f,h,i,n) and of the indolocarbazoles (2e,f,h,i,j) the average highest
cytostatic effects was recorded here for the BIM 1h and the indolocarbazole (2e) that showed the
lowest mean values of “47.39 %” and of “21.63 %” respectively. Both compounds (1h and 2e) were
further tested in five dose screening with the tested substance (1h) being significantly more sensitive
for several cancers cell line as corresponding to their GI50 values. Furthermore, the basically
substituted derivative 2e showed the highest antipoliferative activity in a nanomolar scale towards the
three selected cancers cell lines Non small lung cell NCI-H460 with GI50 “616 nM”, Ovarian Cancer
cell line OVCAR-4 with GI50 “562 nM” and Breast Cancer cell line MCF7 with GI50 “930 nM”.
The present research proved that, all synthesized BIMs (1e,f,h,i,n) showed best activities in the
same cell lines MOLT-4 in leukaemia cell line and in IGROV1 in an ovarian cancer cell line. Also the
basically substituted derivative demonstrates good activity in the renal cancer cell lines CAKI-1 and
UO-31. The TGI and LC50 values were higher than 100 µM so the compound 1h showed noncritical
cytotoxic properties. The basically substituted derivative 2e gave the highest antipoliferative activity
in a nanomolar ranges in selected cell lines with noncritical cytotoxic properties (17 fold higher LC50
“34.6 µM” than GI50 “2 µM” values). Further SAR modifications in compounds 1h and 2e that are
under investigation in our lab to discover more potent antitumor agents.

ACCEPTED MANUSCRIPT
R1
R2
N
H
RCHO
(1:2)mr
gl. AcOH
stirr. , rt.
R1
R1
R1
O
O
R1
R
R
R
R2
R2
R2
R2
RCHO
MeOH, conc. H2SO4
N
H
N
H
N
H
N
H
1:1)mr.
x, 1h
reflu
(
N
N
H
MeO
H,
nc. H2SO4
reflux, 1h
H
N
H
N
H
co
1
2
1a: R1=R2=H, R=Ph
1b: R1=R2=H, R=p-Cl- Ph
c: R1=R2=H, R=p-Br- Ph
1d: R1=R2=H, R=m-Br
1e: R1=R2=H, R=p-N(Me)2- Ph
2a: R1=R2=H, R=Ph
2
b: R1=R2=H, R=p-Cl-Ph
2c: R1=R2=H, R=p-Br-Ph
d: R1=R2=H, R=m-Br-Ph
2e: R1=R2=H, R=p-N(Me)2-Ph
f: R1=R2=H, R=m-OCH2Ph-Ph
2g: R1=R2=H, R=p,m-OH-Ph
h: R1=R2=H, R=p-MeO-m-OCH2Ph-Ph
2i: R1=R2=H, R=m-MeO-p-OCH2Ph-Ph
1
2
4
1
f: R1=R2=H, R=m-OCH2Ph- Ph
1g:R1=R2=H, R=p,m-OH- Ph
1h:R1=R2=H, R=p-MeO-m-OCH2Ph- Ph
i: R1=R2=H, R=m-MeO-p-OCH2Ph- Ph
1j: R1=Cl, R2=H, R=p-MeO-m-OCH2Ph- Ph
1k:R1=H, R2=Cl, R=p-MeO-m-OCH2Ph- Ph
2
2
1
2
j: R1=Cl, R2=H, R=p-MeO-m-OCH2Ph-Ph
2k:R1=H, R2=Cl, R=p-MeO-m-OCH2Ph-Ph
1
l : R1=R2=H, R=3-pridyl
2
1m:R1=R2=H, R=3-indolyl
1n: R1=R2=H, R=1-naphthyl
l: R1=R2=H, R=3-pridyl
2m:R1=R2=H, R=3-indolyl
1h
CHO
MeOH,
conc. H2SO4
reflux, 1h
N
O
O
Scheme (1)
N
H
N
H
N
yield 18 %

1
ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
Synthesis, Cytostatic Evaluation and Structure Activity Relationships of Novel Bis-
indolylmethanes and their Corresponding Tetrahydroindolocarbazoles
Mardia T. El Sayeda,b,*, Khadiga M. Ahmedc, Kazem Mahmouda, and Andreas Hilgerotha.
aInstitute of Pharmacy, Martin-Luther University, Research Group of Drug Development and Analysis, Wolfgang-
Langenbeck-Straße 4, 06120 Halle, Saale, Germany. bApplied Organic Chemistry Department, National Research Centre,
Cairo, Egypt., cNatural Compounds Laboratory, National Research Centre, Cairo Egypt.
Corresponding author: Mardia El Sayed
Email: mardia_elsayed200@yahoo.com
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest to disclose.
Statement of significance
The present research proved that, all synthesized BIMs (1e,f,h,i,n) showed
best activities in the same cell lines MOLT-4 in leukaemia cell line and in
IGROV1 in an ovarian cancer cell line. Also the basically substituted
derivative demonstrates good activity in the renal cancer cell lines CAKI-1
and UO-31. The TGI and LC50 values were higher than 100 µM so the
compound 1h showed noncritical cytotoxic properties. The basically
substituted derivative 2e gave the highest antiproliferative activity in a
nanomolar ranges in selected cell lines with noncritical cytotoxic
properties (17 fold higher LC50 “34.6 µM” than GI50 “2 µM” values).
Further SAR modifications in compounds 1h and 2e that are under
investigation in our lab wishing to discover more potent antitumor agents.

2
ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
Synthesis, Cytostatic Evaluation and Structure Activity Relationships of Novel Bis-
indolylmethanes and their Corresponding Tetrahydroindolocarbazoles
Mardia T. El Sayeda,b,*, Khadiga M. Ahmedc, Kazem Mahmouda, and Andreas Hilgerotha.
aInstitute of Pharmacy, Martin-Luther University, Research Group of Drug Development and Analysis, Wolfgang-
Langenbeck-Straße 4, 06120 Halle, Saale, Germany. bApplied Organic Chemistry Department, National Research Centre,
Cairo, Egypt., cNatural Compounds Laboratory, National Research Centre, Cairo Egypt.
-------------------------------------------------------------------------------------------------------------------------------------------
Article history:
Received:
Accepted:
Abstract
BIMs (bis-indolylmethanes) (1a-n) were synthesized using
glacial acetic acid as a protic acid for promotion of the
condensation reaction of indoles with aldehydes in high yields
(86-98 %). Corresponding tetrahydroindolo[2,3-b]carbazoles (2a-
m) were synthesized via condensation of BIMs with aldehydes.
Ten synthesized compounds have been submitted to the national
cancer institute in the USA where all the submitted samples have
been selected for one dose screening. As a result of the one dose
DOI:
---------------------------------------
Key words:
Bis-indolylmethanes;
tetrahydroindolo[2,3-
b]carbazoles; one dose
screening;
screening; antipoliferative
activity; nanomolar scale.
five
dose
screening of BIMs (1e,f,h,i,n) and of the indolocarbazoles (2e,f,h,i,j
)
the average highest cytostatic effects was recorded here for the
BIM 1h and the indolocarbazole (2e) that showed the lowest mean
values of “47.39 %” and of “21.63 %” respectively. Both
compounds (1h and 2e) were further tested in five dose screening
with the tested substance (1h) being significantly more sensitive
for several cancers cell line as corresponding to their GI50 values.
Furthermore, the basically substituted derivative 2e showed the
highest antipoliferative activity in a nanomolar scale towards the
three selected cancers cell lines Non small lung cell NCI-H460
with GI50 “616 nM”, Ovarian Cancer cell line OVCAR-4 with
GI50 “562 nM” and Breast Cancer cell line MCF7 with GI50 “930
nM”.
---------------------------------------------------------------------------------------------------------------------------
Introduction
In recent years a considerable attention
has been paid on the synthetic ways leading to
indole derivatives because of their biological
activities. Various indole derivatives, such as
3-substituted indoles, are common components
of drugs and are generally found to be of
pharmaceutical interest in a variety of
therapeutic areas [1]. In addition, 3-substituted
indole derivatives are also versatile
intermediates in organic synthesis [2], due to
the feasibility of their 3-position for an
electrophilic substitution. The electrophilic
substitution reactions of indoles with aromatic
aldehydes afford corresponding BIMs. Several
catalysts such as protic acids [3-6], Lewis acids
[7-10], ionic liquids [11], and others are used
to promote these reactions. The 3-position of
indole is the preferred site for the electrophilic
substitution reactions. A simple and direct
method for the synthesis of 3-alkylated indole
derivatives involves the condensation of
indoles or its substituted derivatives with
electrophilies (aldehydes or ketones or
imines). Aldehydes either aliphatic or aromatic

3
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
are the most important and widely used
electrophiles in such reactions.
screening. The tumor growth inhibition
properties of the ten compounds 1e, 1f, 1h, 1i,
1n, 2e, 2f, 2h, 2i and 2j with the NCI codes NSC
D-755521/1, D-755518/1, D-755517/1, D-
755519/1, D-755520/1, D-758513/1, D-
758511/1, D-758510/1, D-758512/1 and D-
758514/1 were evaluated. The selected
compounds were screened on human tumor
cell lines at 10-5 M at the 60-Cell-Line
Screenings of the Developmental Therapeutics
Program (DTP) of the National Cancer
Institute (NCI, Bethesda, Maryland, USA)
under the drug discovery program of the NCI.
Among the selected 10 compounds the two
compounds 1h (NSC D- 755517/1) and 2e (D-
758513/1) were further screened for five-log
dose molar range as they have shown
Bisindolylalkane derivatives are found in
bioactive metabolites of terrestrial and marine
origin. Recently, Maciejewska et al. [12] used
DNA-based electrochemical biosensors to
prove that bis(5-methoxyindol-3-yl)methane
[13] considerably reduces the growth of cancer
cell lines such as HOP-92 (lung), A498 (renal)
and MDAMB-231/1TCC (breast) Their results
also indicate that BIMs could potentially be
applied as chemotherapeutic agents against
tumors [14]. It has been reported that, DIM-C-
p-PhC6H5 substituted in the phenyl ring with a
para-t-butyl,
trifluoromethyl
(DIM-C-p-
PhCF3) substituent and indole ring-substituted
analogs are selective PPARγ modulators [15]
in several cancer cell lines with high
antiproliferative activity [16 - 25]. Other study
investigated the antileukaemic activity and
molecular mechanisms of action of a newly
synthesized ring-substituted diindolylmethane
derivative, 1,1-bis[3/-(5-methoxyindolyl)]-1-
(p-t-butylph- enyl) methane in acute
myelogenous leukemia (AML) cells [26].
Inaddition, Indolocarbazoles have been
reported as a primary compound for the
synthesis of various drugs with important
biological, pharmacological, and medicinal
activities [27-32]. Indolocarbazoles are
associated with anticancer, antimicrobial, and
antifungal activities. In most cases biological
activity is correlated with indolocarbazoles
containing heteroatoms. The biological
activity depends on the interaction potential
with DNA [33,34]. Furthermore, many
experimental studies have indicated that the
size, shape and planarity of this structure are
important criteria in such DNA interaction
[35].
prominent cell growth inhibition at 10-5
M
concentration against variety of cancers cell
lines. All the one dose mean graphs, the
superposition curves and the dose response
curves are included in the electronic
supplementary file. The 60-cell-line-screening
of the NCI includes 60 different tumor cell
lines, the nine various organs and tumor types
derived (leukaemia, non-small-cell lung
cancer, colon cancer, CNS cancer, melanoma,
ovarian cancer, renal cancer, prostate cancer
and breast cancer).
Chemistry
Many procedures for the synthesis of BIMs
have been published by varying the nature of
the catalyst used. Generally, BIMs are
synthesized by a reaction analogous to the
Ehrlich test, where indoles react with aliphatic
or aromatic aldehydes or ketones in presence
of acid catalyst to give azafulven [36,37,38],
which undergoes further addition with the
second indole molecule to afford BIMs. In
view of our previous work performed in our
lab using glacial acetic acid as a protic acid for
efficient promotion of the condensation
reaction of indoles with different types of
aldehydes, we have synthesized the BIMs via
using glacial acetic acid with indoles and
aromatic aldehydes. In brief, glacial acetic acid
Result and Discussion
All the submitted compounds [BIMs
(1e,f,h,i,n) and the indolocarbazoles (2e,f,h,i,j)] to
National Cancer Institute (NCI), USA, have
been selected by the NCI for anticancer

4
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
R1
R2
N
H
RCHO
(1:2)mr
gl. AcOH
stirr. , rt.
R1
R1
R1
O
O
R1
R
R
R
R2
R2
R2
R2
RCHO
MeOH, conc. H2SO4
1a
N
H
N
H
N
H
N
H
1:1)mr.
x, 1h
reflu
(
N
N
H
MeO
H,
conc. H2SO4
reflux,
H
N
H
N
H
1
1
h
2
1a: R1=R2=H, R=Ph
1b: R1=R2=H, R=p-Cl- Ph
c: R1=R2=H, R=p-Br- Ph
1d: R1=R2=H, R=m-Br
1e: R1=R2=H, R=p-N(Me)2- Ph
2a: R1=R2=H, R=Ph
2
b: R1=R2=H, R=p-Cl-Ph
2c: R1=R2=H, R=p-Br-Ph
d: R1=R2=H, R=m-Br-Ph
2e: R1=R2=H, R=p-N(Me)2-Ph
f: R1=R2=H, R=m-OCH2Ph-Ph
2g: R1=R2=H, R=p,m-OH-Ph
h: R1=R2=H, R=p-MeO-m-OCH2Ph-Ph
2i: R1=R2=H, R=m-MeO-p-OCH2Ph-Ph
1
2
4
1
f: R1=R2=H, R=m-OCH2Ph- Ph
1g:R1=R2=H, R=p,m-OH- Ph
1h:R1=R2=H, R=p-MeO-m-OCH2Ph- Ph
i: R1=R2=H, R=m-MeO-p-OCH2Ph- Ph
1j: R1=Cl, R2=H, R=p-MeO-m-OCH2Ph- Ph
1k:R1=H, R2=Cl, R=p-MeO-m-OCH2Ph- Ph
2
2
1
2
j: R1=Cl, R2=H, R=p-MeO-m-OCH2Ph-Ph
2k:R1=H, R2=Cl, R=p-MeO-m-OCH2Ph-Ph
1
l : R1=R2=H, R=3-pridyl
2
1m:R1=R2=H, R=3-indolyl
1n: R1=R2=H, R=1-naphthyl
l: R1=R2=H, R=3-pridyl
2m:R1=R2=H, R=3-indolyl
1h
CHO
MeOH,
conc. H
2SO4
reflux,
1h
N
O
O
Scheme (1)
N
H
N
H
N
yield 18 %
without solvent was used to catalyze the
reaction of indoles (two equivalent moles) and
aryl or heteroaryl aldehydes (one equivalent
mole). With our new method via glacial acetic
acid as a solvent the corresponding BIMs
were formed in a high yields (86-98 %) and
after a few hours (4-6) hours of stirring at
room temperature. Some BIMs (1a,b,c,d,e,k,n)
are known [39-45], their identities were
proven by means of MS, NMR, IR spectra,
and the other BIMs, (1f,g,h,i,j,l), are novel and
could not be found in the literature. The short
reaction time and the simplicity of the reaction
procedure makes this method one of the most
efficient methods for the synthesis of this class
of compounds.
according to the few reported cases of
condensation of BIMs with aldehydes or
ketones [46] in which the BIM and the
aromatic aldehyde (the same aldehyde which
condensed with indoles in the synthesis of the
used BIM) in molar ratios (1:1) were dissolved
in methanol and few drops of conc. H2SO4
were added dropwisly. The mixture was
refluxed under stirring for about 1 h. The
product precipitated and was isolated from the
reaction mixture while the solution is hot
yielding few amounts of the pure
tetrahydroindolo[2,3-b]carbazoles of type 2a-m
.
The rest of the compounds 2a-m could be
extracted and purified from the reaction
mixture affording the second crop in good to
better yields, scheme (1). The formation of the
tetrahydroindolo[2,3-b]carbazoles (2a-m) was
due to the fact that a cyclizative condensation
can occur by an acid catalyzed nucleophilic
attack of an indole nucleus at the 2-position,
when the 2-position is free. The unsubstituted
As an extending study of our present lab work,
we used the prepared BIMs 1a-n as a starting
materials for the synthesis of biologically
active tetrahydroindolo[2,3-b]carbazoles of
type 2a-m, 3 and the extended spirocyclic
biscarbazoles 4. The reaction has been done

5
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
2-position of the two indole nucleus in BIMs
can react with a carbonyl group of either
aldehydes or ketones, affording the
corresponding tetrahydroindolo[2,3-b]carba-
-zoles. The 1H-NMR of compounds 2a-m
showed the two aliphatic CH protons as a
single signal at δ between 5.50 to 5.90 ppm.
The acid catalyzed condensation of indoles
with aldehydes has been reported as a method
for the preparation of substituted isomers of
R
R
H
N
H
N
N
N
H
N
H
N
H
H
R
R
tetrahydroindolo[2,3-b]carbazole (2a) tetrahydroindolo[3,2-b]carbazole
dihydroindolo[3,2-b]carbazole
Trans
- isomer
dehydrated form
Cis- isomer
Figure (1): Cis and Trans isomers of indolocarbazoles.
In this context and as a continuation of our
work
concerning
the
synthesis
of
tetrahydroindolo[2,3-b]carbazoles, the reaction
of BIMs (1h) (1 mole equivalent) and p-
tetrahydroindolo[3,2-b]carbazole
(trans
isomer) and tetrahydroindolo[2,3-b]carbazoles
(cis-isomer), figure (1) in the presence of
phosphoryl chloride as the acid catalyst [47].
However the products are not stable under this
reaction condition where they readily
converted via oxidation with air to the
dihydroindolocarbazoles. The formation of the
trans isomer has also recently confirmed and
published by Rong Gu and et al. [47a].
However the reaction was accomplished using
indoles with aromatic aldehydes in (1:1) molar
ratio in presence of 2 mol % of iodine as a
catalyst in acetonitrile under reflux affording
6,12-trans–isomer which was confirmed by x-
ray crystallography, figure (1). This behaviour
is due to the presence of the free 2- and 3-
positions of the indole ring which both of them
can undergo a neucleophilic attack at a
carbonyl group leading to the expected
formation of a mixture of tetrahydroindolo-
[2,3-b]carbazoles and tetrahydroindolo[3,2-
b]carbazoles, figure (1). However the reaction
of indoles with aromatic aldehydes using
iodine as a catalyst is a selective reaction for
the preparation of tetrahydroindolo [3,2-
b]carbazoles and none of the other isomer
dimethylaminobenzaldehyde
(1
mole
equivalent) has been done by the method of
methanol sulphuric solution as a possible route
for the synthesis of 4-(8-(3-(benzyloxy)-4-
methoxyphenyl)1,1a,2,2a,3,7b,8,8a-octahydro-
indolo[2,3-b]carbazol-2-yl)-N,N-dimethylani-
lin (3). TLC of the reaction mixture showed
the formation of four products were identified
after purification by column chromatography
as BIM (1e) as a main product with a 30 %
yield, compound 2e with a 15 % yield,
compound 1h with a yield of 10 % and the
formation of desired compound 3 with a 18 %
yield. The reaction products were identified by
ESI-MS and compared by TLC with all
products which have been prepared separately.
Our desired compound 3 was confirmed by the
1
means of H-NMR, ESI-MS and IR spectra,
1
where the H-NMR of 3, indicated the singlet
signal for 2-protons at 5.79 ppm for two
aliphatic CH protons. The extended spirocyclic
structure (4) was synthesized in a better yield
of 52 %, by the way of MeOH and conc.H2SO4
using BIM (1a) 2 moles equivalent and 1,4-
cyclohexanedione, 1 mole equivalent. The
reaction solution turned from pink colour to
dark violet by leaving it stirring for one hour
under reflux. The product detected, purified
and confirmed by means of ESI-MS (m/z:
719.29[M+-H], EI-MS (720[M+] 32 %). Its 1H-
NMR spectrum showed singlet signal at δ =
5.91 ppm for 2 protons (2CH), and 2 triplet
signals every one for 4 protons (2CH2) at δ =
2.03 ppm and 2.27 ppm, also the four NH
indole protons appeared at 9.94 ppm as a
broad signal. These data proved the similarity
tetrahydroindolo[2,3-b]carbazoles
were
observed in the reaction mixture. In our
reaction using BIMs and aldehydes in
methanolic sulfuric acid solution, the cis
isomer of tetrahydroindolo[2,3-b]carbazoles
(2a-m) were obtained. The products 2a-m which
were prepared by this method were found to be
more stable than the same products using
POCl3 as a catalyst as they were rapidly
converted into the oxidized form, figure (1).

6
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
of the structure which confirmed additionally
by its 13C-NMR spectrum.
of the percent growth of the treated cells when
compared to the untreated control cells. After
obtaining the results for one dose assay,
In vitro cancer screen
analysis
of
historical
Development
(DTP) was
Therapeutics
Programme
All the submitted compounds BIMs
(1e,f,h,i,n) and the indolocarbazoles (2e,f,h,i,j)) to
National Cancer Institute (NCI), USA, have
been selected by the NCI for anticancer
screening. The tumour growth inhibition
properties of the ten compounds 1e, 1f, 1h, 1i,
1n, 2e, 2f, 2h, 2i and 2j with the NCI codes NSC
D-755521/1, D-755518/1, D-755517/1, D-
755519/1, D-755520/1, D-758513/1, D-
758511/1, D-758510/1, D-758512/1 and D-
758514/1. The selected compounds were
screened on 60-human tumour cell lines at 10-5
M at the 60-Cell-Line Screenings of the
Developmental Therapeutics Program (DTP)
of the National Cancer Institute (NCI,
Bethesda, Maryland, USA) under the drug
discovery program of the NCI. Among the
selected 10 compounds the two compounds 1h
(NSC D- 755517/1) and 2e (D-758513/1) were
further screened for five-log dose molar range
as they have shown prominent cell growth
inhibition at 10-5 M concentration against
variety of cancers cell lines. The 60-cell-line-
screening of the NCI includes 60 different
tumour cell lines, the nine various organs and
tumour types derived (leukaemia, non-small-
cell lung cancer, colon cancer, CNS cancer,
melanoma, ovarian cancer, renal cancer,
prostate cancer and breast cancer).
performed and compound 1h (NSC D-
755517/1) which satisfied predetermined as
effective inhibition criteria was selected for
NCI full panel 5 dose assays. The tested BIMs
showed a distinctive pattern of selectivity with
regard to sensitivity against individual cell
lines all the percent growth inhibition and the
mean growth percent has been collected in
table (1). Compound 1h (NSC D-755517/1)
exhibited broad spectrum cell growth
inhibition against leukaemia cancer cell
MOLT-4 (growth inhibition 20.53 %), non
small lung cancer cell NCI-H460 (growth
inhibition 9.25 %), colon cancer cells HCT-
116 and HT29 with recorded growth inhibition
values 19.91 % and 20.89% respectively,
melanoma cancer cell M14 (growth inhibition
19.50 %), ovarian cancer cell IGROV1
(growth inhibition 23.79 %), and renal cancer
cells (CAKI-1 and UO-31) with growth
inhibition 15.65 % and 18.10 % respectively.
This data confirmed that as a result of a Single
dose assay concentration of 10-5 M the average
highest cytostatic effects were recorded for the
compound 1h (NSC D-755517/1) that showed
the lowest over all mean value (47.39 %), see
maen graph figures in the supporting
information file. The two substituted
derivatives 1g and 1i were observed as
moderate cytostatic properties with over all
All the five selected BIMs (1e,f,h,i,n), by the
NCI for in vitro anticancer assay were
evaluated for their anticancer activity. Primary
in vitro One dose anticancer assay was
performed in full NCI 60 cell panel
representing leukaemia, melanoma and
cancers of lung, colon, brain breast, ovary,
kidney and prostate in accordance with the
protocol of the NCI, USA. The compounds
were added at a single concentration (10-5 M)
and the culture was incubated for 48 h. End
point determinations were made with a protein
binding dye, Sulforhodamine B. Results for
each compound were reported as a mean graph
mean values 75.51
%
and 86.38
%
respectively, especially for the cancer cell
lines “leukaemia MOLT-4, non small lung
cancer NCI-H460, ovarian cancer cell lines
IGROVI and OVCAR-3 and renal cancer cell
lines CAKI-1 and UO-31” with growth percent
values in a range from 58.65 % to 34.07 %.
Compounds 1e and 1l were shown as inactive
cytostatics against all selected cell lines with a
mean values 101.60 % and 92.63 %
respectively. Table (1) showed the sixty
human tumour cell line anticancer screening
data at single dose assay (10-5 M) as percent

7
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growth inhibition of BIMs (1e,f,h,i,n), see all the
figures of the one dose mean graph for all the
tested compounds in the supporting
information file.
Delta and the Range. The MG-MID or the
average is the calculated logarithmic of a mean
panel of GI50, TGI or LC50. The Delta is the
differences of concentrations with parameters
between the most sensitive cell line and the
mean. Similarly, the Range is the number of
log10 units by which the delta of the most
sensitive line(s) of the panel differs from the
delta of the least sensitive lines. On the other
hand the given Delta and Range values quite
accurately reflect a true range of differential
sensitivity among the full panel of cell lines to
the compound under investigation. Likewise,
the given MG-MID (Average) value quite
accurately reflects a true overall panel-average
sensitivity of the cell lines to this agent, and
therefore is a useful basis for comparison of
overall potency of the given agent with related
or unrelated compounds. Compound 1h has
average GI50 responses at micromolar
concentrations (11 µM), cytostatic effects at
micromolar concentrations (95.5 µM) and the
average cytotoxic effects on cancer cell lines
at micromolar concentrations ”LC50 (100 µM)
value which is 10 fold higher than GI50 (11
µM) value”. Based on all these data compound
1h showed a high degree of variability in its
response. Compound 1h with GI50 values in the
micromolar range is more effective than the
cytostatic drugs Etoposid, Melphalan and
Irinotecan (GI50 values of 38.9 µM, 14.5 µM
and 14.1 µM respectively) [49h].
Compound under investigation 1h (NSC D-
755517/1) exhibited remarkable anticancer
activity against most of the tested cell lines
representing nine different subpanels with GI50
values between ‘‘1.20 – 9.56 µM’’ as shown
in table (2). Whereas three cell lines of non
small lung cancer cell subpanel namely HOP-
62, melanoma cancer cell line MALME-3M
and breast cancer cell line HS 578T were
found to be insensitive at the highest tested
concentration 100 µM therefore a sign of ‘‘>’’
is used as prefix to the concentration. With
regard to the sensitivity against some
individual cell lines, compound 1h (NSC D-
755517/1) showed obvious activity toward
CNS cancer cell lines SNB-7 and U251,
Melanoma cell lines MDA-MB-43 and
UACC-62, renal cancer cell lines A498 and
RXF 393 and breast cancer cell line MDA-
MB-468, (GI50 value ranging from 1.20 to 1.87
µM). The criterion for selectivity of a
compound depends upon the ratio obtained by
dividing the full panel MID (the average
sensitivity of all cell lines toward the test
agent) by their individual subpanel MID (the
average sensitivity of all cell lines of a
particular subpanel toward the test agent).
Ratios between 3 and 6 refer to moderate
selectivity, ratios greater than 6 indicate high
selectivity toward the corresponding cell line,
while compounds not meeting either of these
criteria rated non-selective. As per this
criterion, compound under investigation was
found to be non selective toward all the cell
panels, table (2). The five dose screening for
1h gave the parameters log GI50, log TGI and
log LC50, see mean graph of the five dose in
the supporting information file, which are
summarized in table (2). In the full NCI
screening data report, three additional numbers
are printed at the base of each of the three
respective mean-graphs provided. These
numbers are the MG-MID (Average), the
We further developed the series of the
substituted
(BIMs) with the synthesis of new structures as
aryl substituted tetrahydroindolo[2,3-
bis(indolyl)phenylmethanes
b]carbazoles to constrain the flexibility of the
molecule. The NCI selected five derivatives of
these substituted indolocarbazoles for the one-
dose screening program at a concentration of
10-5 µM. The selected substances (2e,f,h,i,j
)
showed a distinctive pattern of selectivity with
regard to sensitivity against individual cell
lines all the percent growth inhibition and the
mean growth percent has been collected in
table (3). Compound 2e exhibited broad
spectrum cell growth inhibition against non

8
European Journal of Medicinal Chemistry
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small lung cancer cell NCI-H23 (growth
inhibition 5.55 %), colon cancer cell lines
HCT-116 and SW-620 (growth inhibition 7.08
% and 6.72 %), renal cancer cell line ACHN,
CAKI-1 and UO-31 (growth inhibition 9.61 %,
13.72 % and 12.35 % respectively) and breast
cancer cell line BT-549 (growth inhibition
8.92 %) at single dose assay concentration of
10-5 M. The average highest anticancer activity
were scored for the compound 2e that showed
the lowest mean value (21.63 %). The two
substituted derivatives 2h and 2i were observed
as moderate cytostatic properties with mean
values of 84.67 % and of 76.84 % respectively
and the other two derivatives 2f and 2j were
shown as inactive cytostatics.
N
N
N
H
H
N
2e
Figure (2): Superposition of all growth curves for compound 2e (nanomolar active).
Compound under investigation 2e (D-
758513/1) exhibited remarkable anticancer
activity against most of the tested cell lines
representing nine different subpanels with GI50
values between 1.07 and 5.65 µM except the
two cancer cell lines non small cell lung
cancer NCI-H460 and breast cancer cell line
MCF7 with GI50 values of 6.22 and 9.29 µM
respectively, table (4). From the five- dose
screening for 2e and similar to compound 1h
the criterion for selectivity of a compound 2e
indicated that it also non selective toward the
cancer subpanels with selectivity ratio in range
of 0.62 - 1.46, table (4). Compound 2e has
average GI50 responses at a micromolar
concentrations (2 µM), cytostatic effects at
micromolar concentrations (48.9 µM) and the
average cytotoxic effects on cancer cell lines
at micromolar concentrations (34.6 µM) which
is 17 fold higher than GI50. Furthermore, the
basically substituted derivative 2e gave the
highest antiproliferative activity being
“nanomolar active” towards the selected
cancer cell lines which is non small lung
cancer cell line NCI-H460 with GI50 = 616 nM
and the ovarian cancer cell line OVCAR-4
with GI50 = 562 nM with non critical cytotoxic
properties. Based on these data we observed
that compound 2e is more effective than the
cytostatic drugs etoposid, melphalan and
irinotecan (GI50 values of 38.9 µM, 14.5 µM
and 14.1 µM respectively).
Figure(3): Theonedose maengraphof compound2e
comparison of 1i and 1h the position of the
functional groups is of great importance of
being either meta or para. Where the para-
methoxy is much more favourable than meta
substituent. Moreover the comparison of 1h
with 1f indicated that the methoxy function in
addition to a benzyloxy group ensures mainly
increased activity. If the methoxy function is
positioned in meta position the effect is similar
concerning no favour of a meta methoxy
function as indicated by a comparison of 1i and
1f. The lipophilic fixed substituent in the
naphthyl derivative 1n is not favourable
compared to the rotable benzyloxy substituent
in compound 1f. BIM 1e containing the basic
substituent (NMe2) which was unfavourable
Structure activity relationship (SAR)
Structure-activity
synthesized BIMs revealed that, by
correlation
of
the
a

9
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
concerning the all over anticancer activities
whereas, its activities in a renal cancer cells
indicated different anticancer activities
comparable to compound 1f. In conclusion, all
BIMs (1e,f,h,i,n) showed best activities in the
same cell lines MOLT-4 as a leukaemia cell
line, IGROV1 and as an ovarian cell line and
the cell lines CKAI-1 and UO-31 renal cancer
cell lines. Also the basically substituted
derivative demonstrates good activity for
leukaemia cell line MOLT-4, non small cell
lung cancer NCI-H460, colon cancer cell lines
HCT-116 and HT29, melanoma cell line M14,
ovarian cancer cell line IGROV1, the renal
cancer cell lines CAKI-1 and UO-31, and the
breast cancer cell line MCF7. Moreover
compound 1h showed non critical cytotoxic
properties.
Experimental
The melting points were measured on a
Boetius-Mikroheiztisch the company "VEB
weighing, Rapido Radebeul/VEB NAGEMA
"measured and are uncorrected. The carbon,
hydrogen and nitrogen content of the
substances was performed on a "CHNS-932"
automatic analyzer of the company "LECO
Corporation" in the automatic Micro chemical
determined. The halogen content was
determined by titration in semi micro method
determined. For the analyzes TLC were with
aluminium foil fluorescent indicator from
Merck KGaA (silica gel 60 F254, layer
thickness 0.2 mm) used. Rf -values (run level
relative to the solvent front), The separations
were with column chromatography at
atmospheric pressure on silica gel 60 (Grain
size from 0.063 to 0.200 mm) from Merck
KGaA. The NMR spectra were recorded on a
"Gemini 2000" (400/100 MHz). The ATR
spectra were recorded on a FT-IR spectrometer
"IFS 28" by "Bruker", the KBr spectra on a
FT-IR Spectrometer "Spectrum BX" "the
Company "Perkin-Elmer" measured. The ESI
mass spectra were recorded on a "Finnigan
LCQ Classic" by "thermal Electron measured"
the sample was injected directly. The 60-Cell-
Line Screenings of the Developmental
Therapeutics Program (DTP) were examined
in the National Cancer Institute (USA) on a
The structure activity relationship our
synthesized indolocarbazoles indicated that,
the basicly substituted derivative has the
highest activity which recorded the very potent
and broad spectrum of activity against several
cancers cell lines indicated with the negative
values of percent growth (-7.03 % to -52.72
%) promoted at one dose with GI50 value of
(1.07 µM to 5.65 µM) against almost all the
selected cell lines at five dose assay. The
chloro-substitution on the indole phenyl ring is
unfavorable with a main loss of activity in the
selected cancer cell lines. Comparing
compound 2h with 2i a para-benzyloxy
substituent increases the activity in some novel
sensitive cell line (NCI-H522 as non small
lung cancer cell lines and CAKI-1 and UO-31
as renal cancer cell. By comparison of
possible
human
tumor
cell
lines.
General procedure for the preparation of
compounds 1a-n :
compound 2h and 2f
a
para-methoxy
In a flask containing 5 ml of glacial acetic
acid and 2 mmol of indole (0.234 gm) or 5-
chloroindole 0.303 gm or 6-chloroindole
0.303 gm was added under stirring until all the
indole was dissolved. Then 1 mmol of the
appropriate aromatic or heterocyclic aldehyde
was added under vigorous stirring. The
reaction mixture was allowed to stir over 4 to
6 h, where the reaction solution turned from
light yellow to light pink to dark red colour.
The product was detected by TLC (100 %
CH2Cl2), and when the reaction was finished
10 ml of water were added and the solution
substituent ensures the activity, especially in
selected cancer cell lines. The para-benzyloxy
compound 2i is more active than the meta-
benzyloxy compound 2f. The basically
substituted derivative 2e gave the highest
antipoliferative activity in a nanomolar ranges
in selected cell lines (non small lung cancer
cell NCI-H460 and ovarian cancer cell
OVCAR-4) with non critical cytotoxic
properties because LC50 value (34.6 µM) is 17
fold higher than GI50 (2 µM) value. However,
this compound was found to be non selective
toward the cancer subpanels. See figure (2)
and (3) illustrating the superposition of all
growth curves for compound 2e (nanomolar
active) and its one dose mean graph.

10
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ACCEPTED MANUSCRIPT
was extracted with ethylacetate, washed with
water and brine, dried over anhydrous sodium
sulfate and concentrated in vacuum. The
product was purified by passing over a column
and eluted with dichloromethane.
123.48, 124.99, 127.89, 129.99, 136.50,
144.02, Rf 0.65(CH2Cl2).
3,3'-((3-Bromophenyl)methylene)bis(1H-
indole) (1d) [39-45]: red crystals, Mp. 93-95
0C, yield 98%, C23H17BrN2, 401.30 g/mol,
ESI-MS: 401.26[M++H], 399.31 [M+-H], IR
3,3'-(Phenylmethylene)bis(1H-indole) (1a)
[39-45]: pink powder, 90 % yield, C23H18N2,
322.40 g/mol, mp:126-1270C, ESI-MS:
(ATR,cm-1):
3405(NH),
1H-NMR(400
MHz,DMSO-d6): δ (ppm): 5.86(s, 1H, CH),
6.85-6.86(m, 3H), 7.03(t, 2H, J=7.6Hz), 7.22(t,
1H, J=7.8Hz), 7.28(d, 2H, J=7.9Hz), 7.34-
7.37(m, 5H), 7.49(s, 1H), 10.84(s, 2H, 2NH),
13C-NMR (100 MHz,DMSO-d6)δ (ppm):
39.16(CH), 111.38, 117.20, 118.16, 118.80,
120.84, 121.25, 123.51, 126.32, 127.23,
128.54, 130.08, 130.69, 136.42, 147.78, EA.
Calcd. C, 68.84, H, 4.27, Br, 19.91, N, 6.98,
found C, 68.90, H, 4.30, Br, 19.95, N, 7.00, Rf
0.74(CH2Cl2).
1
321.32[M+-H], IR (ATR,cm-1)3141(NH), H-
NMR(400 MHz,acetone-d6) δ (ppm): 5.90(s,
1H, CH), 6.79(d, 2H, J=1.5Hz), 6.87(t, 2H,
J=7.2Hz), 7.04(t, 2H, J=7.6Hz), 7.16(d, 1H,
J=7.3Hz), 7.25(t, 2H, J=7.5Hz), 7.32-7.39(m,
6H), 9.99(s, 2H, 2NH), 13C-NMR(100
MHz,CDCl3) δ (ppm): 40.26(CH), 110.94,
119.68, 120.59, 121.79, 121.85, 123.49,
123.99, 125.99, 126.98, 128.08, 128.59,
136.55, 143.88, EA: Calcd. C, 85.68, H, 5.63,
N, 8.69, Found C, 85.72, H, 5.58, N, 8.66, Rf
0.76(CH2Cl2).
4-Di(1H-indol-3-yl)methyl)-N,N-dimeth-
ylaniline (1e) [39-45]: pink powder, Mp.
3,3'-((4-Chlorophenyl)methylene)bis(1H-
0
225-226 C, yield 91, C25H23N3, 365.47 g/mol,
indole) (1b) [39-45]: pink powder, Mp:104-
ESI-MS:
IR(ATR,cm-1):
366.25[M++H], 364.38[M+-H],
0
106 C, in 99% yield, C23H17ClN2, 356.85
3314(NH),
1H-NMR(400
g/mol, ESI-MS: 355.11[M+-H], IR(ATR,cm-1):
3410(NH), 1H-NMR(400 MHz,DMSO-d6)δ
(ppm): 5.85(s, 1H, CH), 6.83(d, 2H, J=7.2Hz),
6.86(t, 2H, J=7.4Hz), 7.04(t, 2H, J=7.6Hz),
7.28(d, 2H, J=7.9Hz), 7.29-7.36(m, 6H),
MHz,DMSO-d6)δ (ppm):
4.60(s,br., 6H,
2CH3), 5.89(s, 1H, CH), 6.84-6.88(m, 4H),
7.03(t, 2H, J=7.99Hz), 7.28(d, 2H, J=7.9Hz),
7.34(d, 2H, J=8.1Hz), 7.49(t, 4H, J=10.6Hz),
10.84(s, 2H, 2NH), 13C-NMR(100 MHz,
DMSO-d6) δ(ppm): 40.13(CH3), 43.62(CH3),
45.07(CH), 111.39, 114.52, 117.43, 118.13,
10.83(s,
2H,
2NH),
13C-NMR(100
MHz,DMSO-d6): 59.65(CH), 111.38, 117.48,
118.14, 118.89, 119.85, 123.48, 124.99,
127.84, 129.97, 130.16, 136.49, 143.87. EA.
Calcd. C, 77.41; H, 4.80; Cl, 9.94; N, 7.85,
found C, 77.50, H, 5.01, Cl, 10.00, N, 7.89. Rf.
0.87(CH2Cl2)
118.85, 119.08, 120.83,
121.40, 123.47,
124.23, 126.37, 129.47, 136.46, 141.84, EA
calcd. C, 82.16; H, 6.34; N, 11.50, found C,
82.20, H, 6.37, N, 11.53, Rf 0.29(CH2Cl2).
3,3/(3-Benzyloxy)phenyl)methylene)bis(1H-
indole (1f): white powder, Mp. 190-1920C,
yield 87%, C30H24N2O, 428.52g/mol, ESI-MS.
3,3'-((4-bromophenyl)methylene)bis(1H-
indole) (1c) [39-45]: yellow crystals, Mp 100-
0
103 C, in yield 76%, C23H17BrN2, 401.30
1
428.24[M+-H], IR(ATR,cm-1): 3425(NH), H-
g/mol, ESI-MS: 402 [M++H], IR(ATR,cm-1):
4356(NH),
1H-NMR(400
MHz,acetone-
NMR(400 MHz,acetone-d6)δ(ppm): 5.01(s,
2H, CH2), 5.90(s, 1H, CH), 6.82(d, 2H,
J=7.5Hz), 6.85(d, 2H, J=7.2Hz), 6.90(t, 2H,
J=7.5Hz), 7.00-7.11(m, 4H), 7.18(t, 1H,
J=7.9Hz), 7.26-7.33(m, 2H), 7.37-7.39(m,
6H), 9.95(s, br., 2H, 2NH), 13C-NMR(100
d6)δ(ppm): 5.91(s, 1H, CH), 6.79(d, 2H,
J=7.2Hz), 6.87(t, 2H, J=7.5Hz), 7.07(t, 2H,
J=7.4Hz), 7.28(d, 2H, J=8Hz), 7.36-7.40(m,
13
6H), 10.93(s, 2H, 2NH), C-NMR (100MHz,
acetone-d6): 57.50(CH), 111.40, 117.48,
118.14, 118.99, 119.89, 120.80, 120.99,
MHz,
acetone-d6)δ(ppm):
41.18(CH),

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70.31(CH2-O), 112.06, 112.91, 116.41,
119.26, 119.63, 120.21, 121.98, 122.13,
123.51, 124.45, 128.04, 128.32, 128.37,
129.07, 129.23, 129.69, 137.98, 138.38,
147.55, 159.66, EA calcd. C, 84.08; H, 5.65;
N, 6.54, found C, 84.12, H, 5.55, N, 6.58, Rf
0.79(CH2Cl2).
7.09(s, 1H), 7.29(d, 1H, J=7.5Hz), 7.33-
7.37(m, 6H), 7.47(d, 2H, J=7.7Hz), 9.95(s, 2H,
2NH), 13C-NMR(100 MHz,acetone-d6)δ(ppm):
40.75(CH),
56.19(OMe),
71.61(OCH2),
112.05, 112.10, 114.33, 114.93, 119.23,
120.09, 120.32, 121.47, 121.98, 124.32,
124.47, 128.12, 128.45, 128.49, 129.12,
138.08, 138.83, 139.31, 147.72, 150.64, EA
calcd. C, 81.20; H, 5.72; N, 6.11, found C,
81.02, H, 5.90, N, 6.22, Rf 0.71 (CH2Cl2).
4-(Di(1H-indol-3-yl)methyl)benzene-1,2-diol
(1g): light brown powder, Mp 105-1070C,
yield 73%, C23H18N2O2, 354.40 g/mol, ESI-
MS: 392.89[M++K], 354.25[M+], 353.24[M+-
H], IR(ATR,cm-1): broad 3400(NH and OH),
1H-NMR(400 MHz,acetone-d6)δ(ppm): 5.77(s,
1H, CH), 6.45(s, 1H), 6.76(d, 2H, J=8.9Hz),
6.86-6.89(m, 2H), 7.04(s, 2H), 7.29(s, 1H),
7.35(s, 4H), 7.55(s, 1H), 9.89(s, 2H, 2NH), EA
calcd. C, 77.95; H, 5.12; N, 7.90, found C,
78.01, H, 5.20, N, 7.96, Rf 0.62(CH2Cl2).
3,3/-((3-Benzyloxy)-4-methoxyphenyl)meth-
ylene)bis(5-chloro-1H-indole (1j): yellow
powder,
Mp
82-850C,
yield
91%,
C31H24Cl2N2O2, 527.44 g/mol, ESI-MS:
1
528.18[M++H], IR(ATR,cm-1): 3369(NH), H-
NMR(400 MHz, CDCl3)δ (ppm): 3.77(s, 3H,
OMe), 4.93(s, 2H, CH2), 5.52(s, 2H, CH),
6.41(d, 2H, J=7.6Hz), 6.73(t, 4H, J=7.3Hz),
7.02(d, 2H, J=7Hz), 7.13(d, 2H, J=8.6Hz),
7.18(dd, 6H, J=3.1, 7.1Hz), 7.88(s, 2H, 2NH),
3,3/-(3-Benzyloxy)-4-methoxyphenyl)meth-
13C-NMR(100
39.39(CH),
MHz,
55.99(OMe),
acetone-d6)δ(ppm):
71.03(OCH2),
ylene)bis(1H-indole (1h): orange crystals, Mp
75-780C,
yield
89%,
C31H26N2O2,
458.55g/mol, ESI-MS 481.16 [M++Na],
457.24[M+-H], IR (ATR,cm-1): 3398 (NH), 1H-
NMR (400 MHz, DMSO-d6)δ(ppm): 3.71(s,
3H, OMe), 4.95(s, 2H, CH2), 5.71(s, 1H, CH),
6.74-6.76(m, 2H), 6.81-6.86(m, 4H), 7.02(t,
2H, J=7.5Hz), 7.06(s, 1H), 7.23(d, 2H,
J=7.9Hz), 7.29-7.31(m, 6H), 7.34(s, 1H),
10.73(s, 2H, 2NH), 13C-NMR (100 MH,
111.51, 111.76, 112.12, 115.37, 119.15,
121.20, 122.31, 124.77, 124.99, 126.91,
127.46, 127.50, 127.66, 127.96, 128.64,
135.04, 135.74, 137.10, 147.63, 148.36, EA
calcd. C, 70.59; H, 4.59; Cl, 13.44; N, 5.31,
found C, 70.62, H, 4.55, Cl, 13.55, N, 5.51,
Rf 0.68 (CH2Cl2).
3,3/-((3-(Benzyloxy)-4-methoxyphenyl)me-
thylene)bis(6-chloro-1H-indole (1k): light
orange crystals, Mp 85-870C, yield 93%,
C31H24Cl2N2O2, 527.44 g/mol, ESI-MS
526.14[M+-H], IR(ATR,cm-1): 1253(C-O),
2866, 2928(CH), 3420(NH), 1H-NMR(400
MHz,DMSO-d6):δ (ppm): 3.70(s, 3H, OMe),
4.94(s, 2H, OCH2), 5.69(s, 1H, CH), 6.77(d,
2H, J=2Hz), 6.79(d, 1H, J=1.9Hz), 6.84(t, 2H,
J=7.9Hz), 7.00(d, 1H, J=2Hz), 7.17(d, 2H,
J=8.6Hz), 7.30(t, H, J=5.7Hz), 7.37(d, 2H,
J=1.6Hz), 10.91(s, 2H, 2NH), 13C-NMR(100
DMSO-d6):
55.59(CH),
59.70(OMe),
70.08(OCH2), 111.29, 111.98, 114.94, 118.00,
118.24, 119.03, 120.71, 123.29, 126.24,
126.56, 127.63, 127.75, 127.86, 128.18,
128.35, 136.49, 137.09, 137.39, 147.14,
147.38, EA calcd.C, 81.20; H, 5.72; N, 6.11,
found C, 81.22, H, 5.75, N, 6.14, Rf 0.79
(CH2Cl2).
3,3/-((4-Benzyloxy)-3-methoxyphenyl)meth-
ylene)bis(1H-indole (1i): light orange crystals,
Mp 215-2190C, yield 92%, C31H26N2O2,
458.55 g/mol, ESI-MS: 457.20[M+-H],
IR(ATR,cm-1):
MHz,acetone-d6)δ(ppm): 3.70(s, 3H, OMe),
5.04(s, 2H, CH2), 5.85(s, 1H, CH), 6.81(s,
2H), 6.85-6.92(m, 4H), 7.04(t, 2H, J=7.6Hz),
MHz,DMSO-d6) δ(ppm):
26.78(CH),
3416(NH),
1H-NMR(400
55.99(OMe), 70.41(OCH2), 111.47, 112.41,
115.11, 118.79, 118.95, 120.83, 121.08,
125.02, 125.79, 126.10, 127.02, 128.17,
128.29, 128.71, 128.89, 137.22, 137.40,

12
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
137.59, 147.70, 147.99, EA calcd. C, 70.59; H,
4.59; Cl, 13.44; N, 5.31, found C, 70.63, H,
4.72, Cl, 13.53, N, 5.34, Rf 0.68 (CH2Cl2).
124.13, 125.15, 125.19, 125.42, 125.68,
126.43, 126.54, 128.42, 131.23, 133.49,
136.56, 140.18, EA calcd. C, 87.07; H, 5.41;
N, 7.52, Rf 0.87 (CH2Cl2).
3,3/-(Pyridin-3-ylmethylene)bis(1H-indole
(1l): light pink powder, Mp 98-1010C, yield
95%, C22H17N3, 323.39 g/mol, ESI-MS:
General procedure for the preparation of
compounds 2a-m
1
324.16[M++H], IR(ATR,cm-1): 3403(NH), H-
In a round bottom flask containing 1mmol of
BIMs derivatives 1a-n was stirred with 50ml
MeOH under heating until it completely
dissolved. The aromatic or heterocyclic
aldehyde 1mmol which has been used for the
synthesis of the BIMs was added and the
reaction mixture was stirred under heating
until the reaction solution became clear. Then
a few drops of conc. H2SO4 were added. The
reaction solution became pink turned to dark
red by refluxing for about 1h. Upon the
reaction completion, as monitored by TLC
(100%CH2Cl2) the reaction was worked up by
adding 50ml water, which was neutralized by
NH4OH addition, extracted with ethylacetate
100ml for two times, washed with water and
then brine, dried over anhydrous Na2SO4,
filtered and concentrated in vacuum. The
crude reaction mixture was purified via
column chromatography on silica gel using
(30%EtAc/hexane) as a solvent to afford the
alternative carbazole derivatives 2a-n [4].
NMR(400 MHz,DMSO-d6)δ(ppm): 5.70(s,
1H, CH), 5.88(s, 1H, CH), 6.84(t, 4H,
J=7.1Hz), 7.01(t, 2H, J=7.6Hz), 7.22-7.29(m,
3H), 7.32(d, 2H, J=8.1Hz), 7.65(d, 1H,
J=7.9Hz), 8.34-8.37(m, 1H), 8.58(d, 1H,
J=7.9Hz), 10.84(s, 2H, 2NH), 13C-NMR (100
MHz, DMSO-d6)δ (ppm): 54.79(CH),
54.78(CH), 111.45, 117.07, 118.24, 118.83,
120.94, 123.15, 123.56, 126.29, 135.47,
136.51, 140.15, 146.99, 149.50, EA C, 81.71;
H, 5.30; N, 2.99, found C, 81.90, H, 5.35, N,
13.02, Rf 0.46 (7%MeOH/CH2Cl2).
Tri(1H-indol-3-yl)methane (1m): light yellow
0
powder, Mp 235-240 C, C25H19N3, 361.44
g/mol, yield 98%, ESI-MS: 360.32[M+-H],
IR(ATR,cm-1 ): 3424(NH), 1H-NMR(400
MHz,acetone-d6): δ (ppm): 6.19(s, 1H, CH),
6.85-6.93(m, 6H), 7.03(t, 4H, J=7.6Hz), 7.37(t,
3H, J=7.8Hz), 7.48(t, 2H, J=7.4Hz), 9.88(s,
13
3H, 3NH), C-NMR(100 MHz,acetone-d6): δ
(ppm): 31.33(CH), 111.13, 118.95, 119.08,
120.12, 121.09, 123.17, 124.60, 127.35,
128.17, 137.19, EA calcd. C, 83.08; H, 5.30;
N, 11.63, found C, 83.09, H, 5.33, N,11.71,
Rf 0.73(CH2Cl2).
2,8-Diphenyl-1,2,3,8-tetrahydroindolo[2,3-
b]carbazole (2a) [46]: Color and shape: light
brown powder, Mp 352-3550C, yield 81%,
C30H22N2, 410.51g/mol, ESI-MS: 409.35[M+-
1
H], IR(ATR,cm-1): 3389(NH), H-NMR(400
3,3/-(Naphthalen-1-ylmethylene)bis(1H-
indole (1n): white powder, Mp 252-255 C,
MHz, DMSO-d6)δ(ppm): 5.66 (s, 2H, 2CH),
6.74(t, 2H, J=7.5Hz), 6.74(t, 2H, J=7.5Hz),
6.91(t, 2H, J=7.6Hz), 7.05(d, 2H, J=7.9Hz),
7.15-7.26(m, 8H), 7.30(d, 4H, J=7.1Hz),
0
yield 97%, C27H20N2, 372.46 g/mol, ESI-MS
1
371.30[M+-H], IR(ATR,cm-1): 3407(NH), H-
NMR(400 MHz,DMSO-d6)δ(ppm): 5.71(s,
1H, CH), 6.59(s, 1H), 6.68(d, 2H, J=7Hz),
6.81(t, 2H, J=7.5Hz), 6.99(t, 2H, J=7.6Hz),
7.23(d, 4H, J=8.1Hz), 7.32(t, 2H, J=9Hz),
7.41(t, 2H, J=7.7Hz), 7.73(d, 1H, J=8Hz),
7.88(d, 1H, J=7.5Hz), 8.22(d, 1H, J=8Hz),
10.63(s,
MHz,DMSO-d6)δ
2H,
2NH),
(ppm):
13C-NMR(100
30.57(CH),
39.40(CH), 109.67, 110.85, 117.93, 118.24,
120.31, 125.46, 126.14, 128.01, 128.23,
128.98, 129.79, 136.38, 136.88, 143.84, EA
calcd. C, 87.77; H, 5.40; N, 6.82, found C,
87.79, H, 5.36, N, 6.86, Rf 0.89 (CH2Cl2)
10.74(s,
2H,
2NH),
13C-NMR(100
MHz,DMSO-d6)δ(ppm): 35.33(CH), 111.41,
117.62, 118.15, 118.84, 120.77, 123.84,

13
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
44.58(Me), 52.81(CH), 109.59, 111.06,
118.27, 118.34, 120.66, 125.39, 127.80,
129.61, 133.32, 136.24, 137.01, EA calcd. C,
82.22, H, 6.49, N, 11.28, found C, 82.25, H,
6.51, N, 11.38, Rf 0.66 (CH2Cl2).
2,8-Bis(4-Chlorophenyl)-1,2,3,8-tetrahydro-
indolo[2,3-b]carbazole (2b) [46]: light green
powder, Mp 339-342 0C, yield 56%,
C30H20Cl2N2,
479.40
g/mol,
ESI-MS:
1
478.27[M+-H], IR(ATR,cm-1): 3414(NH), H-
NMR (400 MHz, DMSO-d6)δ(ppm): 5.69(s,
2H, 2CH), 6.78(t, 2H, J=7.5Hz), 6.84(t, 2H,
J=7.6Hz), 6.93(t, 2H, J=7.4Hz), 7.06(dd, 2H,
J=8, 15.2Hz), 7.21(d, 2H, J=8Hz), 7.24-
7.32(m, 3H), 7.40(d, 1H, J=8Hz), 7.67(d, 1H,
J=8.3Hz), 7.75(d, 1H, J=8.3Hz), 10.57(s, 1H,
NH), 10.72(s, 1H, NH), 13C-NMR(100 MHz,
DMSO-d6)δ (ppm): 38.66(CH), 40.17(CH),
109.54, 111.10, 118.32, 120.73, 125.40,
128.18, 129.26, 130.23, 130.88, 131.93,
132.68, 136.19, 137.09, 142.89, EA calcd. C,
75.16; H, 4.21; Cl, 14.79; N, 5.84, found C,
75.18, H, 4.24, Cl, 14.82, N, 5.79, Rf 0.96
(CH2Cl2).
2,8-Bis(3-bromophenyl)-1,2,3,8-tetrahydro-
indolo[2,3-b]carbazole (2e): light green
powder, Mp 255-257 0C, yield 54 %,
C30H20Br2N2,
568.30
g/mol,
ESI-MS:
569.16[M++H], 567.01[M+-H], IRATR,cm-1):
1
3390(NH), H-NMR(400 MHz,DMSO-d6): δ
(ppm): 5.75(s, 2H, 2CH), 6.82(t, 4H,
J=7.4Hz), 6.97(t, 2H, J=7.3Hz), 7.09(d, 2H,
J=7.9Hz), 7.26(d, 2H, J=7.5Hz), 7.27-7.34(m,
4H), 7.47(s, 2H), 10.81(s, 2H, 2NH), 13C-
NMR(100
MHz,DMSO-d6):
40.12(CH),
109.39, 111.15, 118.31, 120.82, 121.53,
125.32, 127.55, 128.07, 129.32, 130.88,
136.03, 137.06, 146.41, 146.68, EA calcd. C,
63.40; H, 3.55; Br, 28.12; N, 4.93, found C,
63.40, H, 3.58, Br, 28.18, N, 5.00, Rf 0.92
(CH2Cl2)
2,8-Bis(4-bromophenyl)-1,2,3,8-tetrahydro-
indolo[2,3-b]carbazole (2c): yellow powder,
Mp >350 0C, yield 87%, C30H20Br2N2, 568.30
g/mol, ESI-MS: 569.19[M++H], 567.12[M+-
1
H], IR(ATR,cm-1): 3436(NH), H-NMR(400
2,8-Bis(3-(benzyloxy)phenyl)-1,2,3,8-tetra-
MHz, DMSO-d6)δ(ppm): 5.66(s, 2H, 2CH),
6.78(t, 2H, J=7.5Hz), 6.94(t, 2H, J=7.5 Hz),
7.05(d, 2H, J=7.9 Hz), 7.12-7.29(m, 6H),
7.43(d, 4H, J=8Hz), 10.71(s, 2H, 2NH), 13C-
hydroindolo[2,3-b]carbazole (2f): white
powder, Mp 275-279 0C,
yield 72 %,
g/mol, ESI-MS:
C44H34N2O2,
622.75
623.26[M++H], 621.31[M+-H], IR(ATR,cm-1):
1
NMR(100
MHz,
DMSO-d6
)δ(ppm):
3390(NH), H-NMR(400 MHz,DMSO-d6): δ
39.02(CH), 39.99(CH), 110.22, 112.34,
119.05, 120.22, 126.40, 128.80, 130.55,
136.40, 137.62, 139.00, 143.01, EA calcd. C,
63.40; H, 3.55; Br, 28.12; N, 4.93, found C,
63.42, H, 3.58, Br, 28.16, N, 4.98, Rf 0.87
(CH2Cl2).
(ppm): 5.00(s, 4H, 2CH2), 5.62(s, 2H, 2CH),
6.76(t, 4H, J=7.3Hz), 6.82(d, 2H, J=7.9Hz),
6.93(t, 2H, J=7.2Hz), 7.03(d, 2H, J=7.7Hz),
7.14(t, 2H, J=8Hz), 7.23(d, 2H, J=7.9Hz),
7.24-7.32(m, 6H), 7.37(d, 4H, J=6.7Hz),
10.62(s, 2H, 2NH), EA calcd. C, 84.86; H,
5.50; N, 4.50, found C, 84.89, H, 5.54, N,
4.53, Rf 0.85(CH2Cl2).
4,4/-(8,3,2,1-Tetrahydroindolo[2,3-b]carbaz-
ole-2,8-diyl)bis(N,N-dimethyl aniline) (2d)
[46]: dark gray powder, Mp 324-3250C, yield
91 %, C34H32N4, 496.64g/mol, ESI-MS
497.21[M++H], IR: (ATR, cm-1):3304(NH),1H-
NMR(400 MHz,DMSO-d6)δ(ppm): 3.05(s,
12H, 4Me), 5.73(s, 2H, 2CH), 6.78(t, 2H,
J=7.5Hz), 6.94(t, 2H, J=7.5Hz), 7.09(d, 2H,
J=7.9Hz), 7.23(d, 2H, J=8.1Hz), 7.35-7.41(m,
8H), 10.73(s, 2H, 2NH), 13C-NMR(100
4,4-(1,2,3,8-Tetrahydroindolo[2,3-b]carbaz-
ole-2,8-diyl)dibenzene-1,2-diol (2g):
0
dark brown powder, Mp 273-275 C, yield 45
%, C30H22N2O4, 474.51 g/mol, ESI-MS:
475.10[M++H], 473.09[M+-H], IR(ATR,cm-1):
3250(OH), 3430(NH), 1H-NMR(400 MHz,
acetone-d6): δ(ppm): 5.53(s, 2H, 2CH), 6.65(d,
2H, J=2Hz), 6.74(d, 7H, J=7.9Hz), 6.78-
6.92(m, 4H), 6.94(t, 2H, J=7Hz), 7.18(d, 2H,
MHz,DMSO-d6)δ
(ppm):
38.59(Me),

14
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
J=7.9Hz), 7.25(d, 2H, J=8Hz), 7.59(s,br., 4H,
4OH), 9.75(s, 2H, 2NH), C-NMR(100 MHz,
calcd. C, 80.92; H, 5.61; N, 4.10, found C,
80.95, H, 5.64, N, 4.17, Rf 0.65(CH2Cl2).
13
acetone-d6):
δ(ppm):40.67(CH),
111.42,
2,8-Di(pyridin-3-yl)-1,2,3,8-tetrahydroin-
111.79, 115.93, 116.39, 119.32, 119.97,
121.05, 121.69, 127.63, 136.81, 138.28,
138.55, 144.82, 145.94, EA calcd. C, 75.94; H,
4.67; N, 5.90, found C, 75.99, H, 4.69, N,
5.93, Rf 0.54 (10%MeOH/CH2Cl2).
dolo[2,3-b]carbazole (2j): light pink powder,
0
Mp 129-132 C, yield 58 %, C28H20N4, 412.49
g/mol, ESI-MS
413[M++H], EI-MS
412[M+]10%, 334[M+-pyridine]5%, 323[M+-
pyridine.CH]100%,
45[indolyl.CH.indolyl]80%,
1338(C=N), 3398(NH),
IR(ATR,cm-1):
1H-NMR(400
2,8-Bis(3-(benzyloxy)-4-methoxyphenyl)-
1,2,3,8-tetrahydroindolo[2,3-b]carbazole
(2h): white powder, Mp 310-3130C, yield 84
%, C46H38N2O4, 682.80g/mol, Mp 310-3130C,
ESI-MS: 705.19[M++Na], 681.41[M+-H], IR
MHz,DMSO-d6): δ (ppm): 5.43(s, 1H, CH),
5.89(s, 1H, CH), 6.86(d, 4H, J=7.3Hz), 7.02(t,
2H, J=7.5Hz), 7.22-7.25(m, 1H), 7.28(d, 2H,
J=8Hz), 7.35(d, 2H, J=7.5Hz), 7.66-7.69(m,
2H), 8.35(d, 1H, J=7.6Hz), 8.51(d, 1H,
J=7.6Hz), 8.57(dd, 1H, J=1.6, 11.6Hz),
(ATR,cm-1):
3389(NH),
1H-NMR(400
MHz,DMSO-d6): δ (ppm): 3.66(s, 6H, 2OMe),
5.53(s, 4H, 2CH2), 5.69(s, 2H, 2CH), 6.69-
6.78(m, 4H), 6.82(d, 2H, J=8.3Hz), 6.92(t, 2H,
J=7.3Hz), 6.98(d, 2H, J=7.9Hz), 7.06(d, 2H,
J=7.7Hz), 7.19(d, 2H, J=10.4Hz), 7.22-7.26(m,
4H), 7.32(dd, 4H, J=3, 6.6Hz), 7.41(d, 4H,
J=7.9Hz), 10.51(s, 2H, 2NH), 13C-NMR(100
10.86(s,
2H,
2NH),
13C-NMR(100
MHz,DMSO-d6)δ(ppm):37.72(CH),
53.34(CH), 101.78, 112.04, 117.67, 118.85,
119.42, 121.55,
123.85, 124.15, 126.89,
134.13, 134.75, 136.09, 137.12, 140.75,
147.56, 148.39, 150.04, 150.06, EA calcd. C,
81.53; H, 4.89; N, 13.58, found C, 81.50, H,
4.95, N, 13.62, Rf 0.49 (7% MeOH /CH2Cl2).
MHz,DMSO-d6):
δ
(ppm):38.97(CH),
55.63(OMe), 70.09(OCH2), 109.68, 110.00,
111.55, 112.44, 114.04, 118.35, 119.21,
119.44, 120.55, 121.00, 123.50, 127.20,
127.55, 127.61, 128.24, 137.19, 137.96,
138.40, 146.83, 149.75, EA calcd.C, 80.92; H,
5.61; N, 4.10, found C, 80.95, H, 5.62, N,
4.16, Rf 0.71(CH2Cl2).
2,8-Di(1H-indol-3-yl)-1,2,3,8-tetrahydroin-
dolo[2,3-b]carbazole (2k): light yellow
powder, Mp 190-193 0C, yield 47 %,
C34H24N4,
488.58
g/mol,
ESI-MS
489.18[M++H], IR(ATR,cm-1): 3406(NH), H-
NMR(400 MHz,acetone-d6): δ (ppm): 5.85(s,
2H, 2CH), 6.81-6.89(m, 2H), 7.00-7.07(m,
2H), 7.14(t, 2H, J=7Hz), 7.31-7.37(m, 4H),
7.46(d, 2H, J=7.2Hz), 7.48-7.55(m, 4H),
7.74(s, 1H), 8.16(s, 1H), 9.95(s, 2H, 2NH),
10.13(s, 2H, 2NH), 13C-NMR(100 MHz,
1
2,8-Bis(4-(benzyloxy)-3-methoxyphenyl)-
1,2,3,8-tetrahydroindolo[2,3-b]carbazole
(2i): Color and shape: dark green powder, Mp
0
289-291 c,yield 88%, C46H38N2O4, 682.80
g/mol, ESI-MS 683.20[M++H], IR(ATR,cm-1):
1
3301(NH), H-NMR(400 MHz,acetone-d6): δ
acetone-d6):
δ
(ppm):27.42(CH), 111.22,
(ppm): 3.70(s, 6H, 2OMe), 5.04(s, 4H, 2CH2),
5.84(s, 2H, 2CH), 6.81(d, 4H, J=1.7Hz), 6.84-
6.92(m, 4H), 7.04(t, 4H, J=8Hz), 7.09(d, 2H,
J=1.9Hz), 7.28(d, 2H, J=7.3Hz), 7.33-7.37(m,
4H), 7.46(d, 4H, J=7Hz), 9.95(s, 2H, 2NH),
13C-NMR(100 MHz,acetone-d6): δ (ppm):
112.00, 115.23, 118.55, 119.38, 120.28,
120.55, 121.89, 122.10, 124.52, 124.61,
129.00, 130.32, 138.00, 138.26, 142.55, EA
calcd. C, 83.58; H, 4.95; N, 11.47, found C,
83.55, H, 5.01, N, 11.49, Rf 0.65 (CH2Cl2).
39.87(CH),
55.31(OMe),
70.72(OCH2),
2,8-Bis(3-(benzyloxy)-4-methoxyphenyl)-
6,10-dichloro-1,2,3,8-tetra-hydroindolo[2,3-
b]carbazole (2l): brown powder, Mp 320-322
0C, yield 89 %, C46H36Cl2N2O4, 751.70 g/mol,
110.98, 111.22, 113.44, 113.98, 114.04,
118.35, 119.20, 119.44, 120.58, 121.10,
123.59, 127.23, 127.56, 127.61, 128.24,
137.19, 137.94, 138.42, 146.83, 149.75, EA

15
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
ESI-MS:
IR(ATR,cm-1):
752.10[M++H],
3304(NH),
749.17[M+-H],
1H-NMR(400
dissolved. After that few drops of conc. H2SO4
were dropwisly added. Then the reaction was
allowed to stirring under reflux for about one
hour. TLC of the reaction mixture showed the
formation of four products. The reaction was
worked up by adding 10 ml of water,
neutralization with a solution of NH4OH,
extracted by CH2Cl2, dried over anhydrous
Na2SO4, evaporated and purified by column
chromatography eluted with CH2Cl2 to
separate the four products that were identified
as compound 1e as a main product with a 30 %
yield, compound 1d in a 15 % yield,
compound 1h with a 10 % yield and our
desired compound 3 in a 18 % yield, as light
pink powder, C40H35N3O2, 589.72 g/mol, Mp
MHz,DMSO-d6): δ (ppm): 3.70(s, 6H, 2OMe),
4.97(s, 4H, 2OCH2), 5.60(s, 2H, 2CH),
6.82(dd, 2H, J=1.9, 8.2Hz), 6.90(d, 2H,
J=8.2Hz), 6.97(dd, 2H, J=2, 8.6Hz), 7.05(dd,
4H, J=1.9, 8.6Hz), 7.23-7.27(m, 8H), 7.33(dd,
4H, J=2.6, 6.7Hz), 10.81(s, 2H, 2NH), 13C-
NMR
(100
MHz,
DMSO-d6)δ(ppm):
26.29(CH),
55.53(OMe),
70.13(OCH2),
109.45, 112.19, 112.54, 114.48, 117.67,
120.44, 120.85, 122.64, 126.86, 127.72,
127.89, 128.19, 128.34, 133.00, 135.56,
135.81, 136.89, 138.57, 147.56, 148.00, EA
calcd. C, 73.50; H, 4.83; Cl, 9.43; N, 3.73,
found C, 73.52, H, 4.85, Cl, 9.45, N, 3.75, Rf
0.85(CH2Cl2).
299-3010C,
ESI-MS:
590.26[M++H],
1H-NMR(100
IR(ATR,cm-1):
3416(NH),
MHz,acetone-d6): δ (ppm): 2.03(s, 6H, 2Me),
3.78(s, 3H, OMe), 4.98(s, 2H, CH2), 5.79(s,
2H, 2CH), 6.73(s, 2H), 6.85-6.91(m, 4H),
7.03(t, 2H, J=7.2Hz), 7.09(d, 2H, J=7.9Hz),
7.27-7.37(m, 8H), 9.93(s, br., 2H, 2NH), Rf
0.55 (CH2Cl2).
2,8-Bis(3-(benzyloxy)-4-methoxyphenyl)-
5,11-dichloro-1,2,3,8-tetra hydroindolo[2,3-
b]carbazole (2m): white powder, Mp 322-
3240C, yield 90%, C46H36Cl2N2O4, 751.70
g/mol, ESI-MS: 751.27[M+], 752.30[M++H],
1
750.26[M+-H], IR(ATR,cm-1): 3348(NH), H-
NMR(400 MHz,acetone-d6): δ (ppm): 3.69(s,
6H, 2OMe), 4.94(s, 4H, 2OCH2), 5.52(s, 2H,
2CH), 6.71(dd, 2H, J=1.9, 8.6Hz), 6.81(s, 4H),
6.96(d, 4H, J=7.2Hz), 7.17-7.21(m, 8H),
7.30(dd, 4H, J=2, 7.5Hz), 10.31(s, 2H, 2NH),
Procedure of the preparation of the
Spirocyclic structure (4):
In a round bottom flask containing 50ml of
MeOH 2 mmol (0.65 mg) of BIMs derivatives
1a was added under stirring until it completely
dissolved. Cyclohexane-1,4-dione (1mmol,
0.112 mg) was added to the reaction mixture.
When the reaction solution became clear, few
drops of conc. H2SO4 were added slowly. The
reaction solution became pink and the colour
turned to dark violet by leaving it stirring
under reflux for one hour. Upon the reaction
completion as monitored by TLC (100 %
CH2Cl2) the reaction was worked up by added
of 50 ml of water, neutralized by NH4OH,
extracted with ethylacetate 200 ml two times
washed with water and brine, dried over
anhydrous Na2SO4, filtered and concentrated
in vacuum. The crude reaction mixture was
purified via column chromatography on silica
gel eluted with (30 % EtAc/hexane) to afford
compound 4 in a moderate yield 52%, as light
13C-NMR(100
30.55(CH),
MHz,acetone-d6)
δ(ppm):
56.00(OMe), 74.05(OCH2),
108.99, 112.89, 112.99, 114.50, 115.20,
117.68, 120.00, 120.95, 122.90, 126.58,
127.72, 127.89, 128.18, 128.34, 135.56,
135.81, 136.89, 138.55, 147.56, 148.05, EA
calcd. C,73.50; H, 4.83; Cl, 9.43; N, 3.73,
found C,73.49, H, 4.88, Cl, 9.39, N, 3.80, Rf
0.79 (CH2Cl2).
Procedure for the preparation of 4-(8-(3-
(Benzyloxy)-4-methoxyphenyl)-1,2,3,8-tetra
hydroindolo[2,3-b]carbazol-2-yl)-N,N-dime-
thylaniline (3): BIM (1i) 1mmol, 0.5 gm was
dissolved in 25 ml of MeOH, and 1mmol
0.149
mg
of
p-N,N-
dimethylaminobenzaldehyde was added to the
reaction mixture. The reaction was allowed to
stir under reflux until all the reactants had

16
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
pink powder, Mp 149-152 0C, C52H40N4,
720.90 g/mol, ESI-MS 719.29[M+-H], EI-MS:
drug addition (Tz). Experimental drugs are
solubilized in dimethylsulfoxide at 400-fold
the desired final maximum test concentration
and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate
is dissolved and diluted to twice the desired
final maximum test concentration with
complete medium containing 50 mg/ml
gentamicin. Additional four, 10-fold or ½ log
serial dilutions are made to provide a total of
five drug concentrations plus control. Aliquots
of 100 ml of these different drug dilutions are
added to the appropriate microtiter wells
already containing 100 ml of medium,
resulting in the required final drug
concentrations. Following drug addition, the
plates are incubated for an additional 48 h at
720
[M+]
32
%,
322
[3,3'-
(phenylmethylene)bis(1H-indole)] 100 %, 245
[indolyl.CH.indolyl] 75 %, 117 [indolyl] 75%,
90 [Ph.CH] 31 %, IR(ATR,cm-1): 3409 (NH),
1H-NMR(400 MHz,acetone-d6):
δ
(ppm):
2.03(t, 4H, 2CH2, J=7Hz), 2.27(t, 4H, 2CH2,
J=11.4Hz), 5.91(s, 2H, 2CH), 6.79(s, 2H),
6.88(t, 2H, J=7.5Hz), 7.04(t, 4H, J=7.6Hz),
7.11-7.20(m, 4H), 7.25(t, 4H, J=7.5Hz),
7.35(dd, 4H, J=7.9, 15.8Hz), 7.38(d, 4H,
J=8Hz), 7.47(t, 2H, J=8.8Hz), 9.94(s, br., 2H,
2NH),
d6)δ(ppm):26.69(CH2),
13C-NMR(100
MHz,acetone-
26.96(CH2),
29.66(CH), 29.69(C), 110.22, 111.73, 117.21,
118.48, 120.39, 122.24, 123.27, 125.58,
125.65, 126.72, 127.82, 128.22, 128.50,
128.82, 128.84, 129.46, 130.09, 130.86,
130.89, 134.11, 137.03, 140.96, Rf 0.97
(CH2Cl2).
0
37 C, 5 % CO, 95 % air, and 100 % relative
humidity. For adherent cells, the assay is
terminated by the addition of cold TCA. Cells
are fixed in situ by the gentle addition of 50 ml
of cold 50 % TCA (final concentration, 10 %
TCA) and incubated for 60 min at 4 C. The
supernatant is discarded, and the plates are
washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 ml) at
0.4% in 1 % acetic acid is added to each well,
and plates are incubated for 10 min at room
temperature. After staining, unbound dye is
removed by washing five times with 1 %
acetic acid and the plates are air dried. Bound
stain is subsequently solubilized with 10 µM
trizma base, and the absorbance is read on an
automated plate reader at a wavelength of 515
nM. For suspension cells, the methodology is
the same except that the assay is terminated by
fixing settled cells at the bottom of the wells
by gently adding 50 ml of 80 % TCA (final
concentration, 16 % TCA). Using the seven
absorbance measurements [time zero, (Tz),
control growth, (C), and test growth in the
presence of drug at the five concentration
levels (Ti)], the percentage growth is
In-vitro Cancer Screen
The screening is
a
two-stage process,
beginning with the evaluation of all
compounds against the 60 cell lines at a single
dose of 10-5 M. The output from the single
dose screen is reported as a mean graph and is
available for analysis by the COMPARE
program. Compounds which exhibit significant
growth inhibition are evaluated against the 60
cell panel at five concentration levels. The
human tumor cell lines of the cancer-screening
panel are grown in RPMI 1640 medium
containing 5 % fetal bovine serum and 2 µM
L-glutamine. For
a
typical screening
experiment, cells are inoculated into 96 well
microtiter plates in 100 mL at plating densities
ranging from 5000 to 40,000 cells/well
depending on the doubling time of individual
cell lines. After cell inoculation, the microtiter
plates are incubated at 37 C, 5 % CO, 95 % air
and 100 % relative humidity for 24 h prior to
addition of experimental drugs. After 24 h,
two plates of each cell line are fixed in situ
with TCA, to represent a measurement of the
cell population for each cell line at the time of
calculated
at
each
of
the
drug
concentrations levels. Percentage growth
inhibition is calculated as: [(Ti-Tz)/(C-Tz)] x
100 for concentrations for which Ti > / = Tz ,
[(Ti-Tz)/Tz] x 100 for concentrations for

17
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
which Ti < Tz. Three dose response
parameters are calculated for each
[2] (a): Moore, R. E.; Cheuk, C.; Yang, X. Q.;
Patterson, G.M. L.; Bonjouklian, R.; Smita, T.
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experimental agent. Growth inhibition of 50%
(GI50) is calculated from [(Ti-Tz)/(C-Tz)] x
100 = 50, which is the drug concentration
resulting in a 50 % reduction in the net protein
increase (as measured by SRB staining) in
control cells during the drug incubation. The
drug concentration resulting in total growth
inhibition (TGI) is calculated from (Ti = Tz).
The LC50 (concentration of drug resulting in a
50 % reduction in the measured protein at the
end of the drug treatment as compared to that
at the beginning) indicating a net loss of cells
following treatment is calculated from [(Ti-
Tz)/Tz] x 100 = -50. Values are calculated for
each of these three parameters if the level of
activity is reached; however, if the effect is not
reached or is exceeded, the value for that
parameter is expressed as greater or less than
the maximum or minimum concentration
tested [49].
[4] a) Ramesh, C.; Ravindranath, N.; Das, B.
J. Chem. Res. (S) 2003, 72., b) Nagawade, R.
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Conclusion:
poor-Baltork,
I.;
Memarian,
H.
R.;
The present research proved that, all
Khosropour, A. R.; Nikoofar, K. Lett. Org.
Chem. 2006, 3, 768.,
synthesized BIMs (1e,f,h,i,n
)
showed best
activities in the same cell lines MOLT-4 in
leukaemia cell line and in IGROV1 in an
ovarian cancer cell line. Also the basically
substituted derivative demonstrates good
activity in the renal cancer cell lines CAKI-1
and UO-31. The TGI and LC50 values were
higher than 100 µM so the compound 1h
showed noncritical cytotoxic properties. The
basically substituted derivative 2e gave the
[5] a) Thirupathi Reddy, Y.; Narsimha Reddy,
P.; Sunil Kumar, B.; Rajitha, B.; Indian, J.
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highest antiproliferative activity in
a
nanomolar ranges in selected cell lines with
noncritical cytotoxic properties (17 fold higher
LC50 “34.6 µM” than GI50 “2 µM” values).
Further SAR modifications in compounds 1h
and 2e that are under investigation in our lab
wishing to discover more potent antitumor
agents.
[8] Fertuck, K. C.; Kumar, S.; Sikka, H. C.;
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Tables:
Table (1): 60 human tumour cell line anticancer screening data at single dose assay (10-5 M) as percent growth inhibition of BIMs.
Panel/Cell Line ------------------------------------------------- Growth Percent-----------------------------------------------------------
BIM (1h)
BIM (1e)
BIM (1g)
BIM (1i)
BIM (1l)

21
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
Leukaemia ..................................................
......................
94.29
..........................
65.84
......................
82.70
..................
90.92
100.22
76.69
75.09
93.10
CCRF-CEM
HL-60(TB)
K-562
41.24
47.74
37.52
20.53
42.97
38.00
128.59
91.47
96.36
94.00
85.73
63.92
52.47
75.01
97.61
71.05
58.65
94.76
MOLT-4
RPMI-8226
SR
100.85
67.99
77.82
89.79
Non-Small Cell Lung Cancer .......................
.........................
98.14
........................
70.37
....................
80.66
....................
78.08
A549/ATCC
EKVX
HOP-62
44.24
33.06
85.39
37.86
75.60
48.28
53.46
9.25
94.88
103.12
97.98
103.98
105.48
89.81
111.93
103.14
70.13
117.07
77.26
96.26
83.58
76.91
40.92
81.51
75.34
89.05
74.21
86.74
83.17
72.67
87.36
90.05
92.09
96.56
65.77
95.12
90.04
94.19
90.80
88.29
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
44.03
Colon Cancer ..............................................
............................
104.13
.......................
98.17
.....................
102.68
102.35
63.15
.....................
103.48
101.88
78.21
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
28.69
52.61
19.91
43.97
20.89
28.51
37.08
107.97
89.91
100.56
100.05
102.97
93.36
89.58
49.68
71.42
67.05
61.54
66.72
80.96
80.75
88.20
84.23
93.30
92.07
83.31
87.23
KM12
SW-620
CNS Cancer . ..............................................
.........................
116.79
.......................
67.22
......................
89.60
.....................
99.12
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
52.76
41.84
75.91
56.58
64.21
39.48
98.27
91.54
109.25
92.69
90.26
90.70
88.91
69.63
86.27
97.53
101.55
80.78
94.01
75.79
104.56
113.06
76.20
111.50
71.87
91.64
Melanoma ..................................................
........................
96.58
......................
70.34
.....................
84.68
.....................
91.70
104.86
91.67
104.96
117.60
110.32
104.38
97.73
LOX IMVI
MALME-3M
M14
35.11
76.44
19.50
39.23
62.89
75.02
53.71
64.60
67.03
108.26
102.85
100.64
108.23
115.84
102.49
113.36
92.49
86.23
60.84
66.55
87.10
94.00
69.90
88.90
65.78
95.57
94.58
97.07
106.14
107.74
89.22
97.97
75.87
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
90.79
Ovarian Cancer ..........................................
.....................
99.14
......................
51.22
....................
53.76
.....................
92.57
IGROV1
23.79
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer ................................................
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
UO-31
42.79
68.99
52.85
54.18
43.84
82.74
108.57
108.38
102.74
100.41
105.48
105.24
57.39
85.86
84.10
84.14
83.51
99.10
95.78
93.36
77.30
99.85
95.00
92.84
89.75
105.91
92.79
98.97
95.63
100.06
......................
105.42
95.52
......................
101.34
.......................
93.63
......................
96.46
66.10
63.36
37.06
15.65
71.08
45.51
18.10
106.51
93.95
74.45
115.46
103.75
64.28
104.66
63.05
34.27
90.57
71.34
89.10
74.33
57.43
110.45
87.36
86.38
53.07
94.67
103.59
66.86
42.30
51.55
Prostate Cancer ..........................................
........................
88.06
120.00
........................
105.91
............................
58.42
74.41
...........................
52.85
.....................
67.79
109.45
......................
76.47
....................
69.02
105.82
...................
104.09
79.28
102.05
99.38
101.32
PC-3
DU-145
31.31
63.31
Breast Cancer .............................................
MCF7
21.93
MDA-MB-231/ATCC
BT-549
T-47D
50.81
80.58
43.17
56.56
104.98
109.82
89.79
110.54
86.20
102.59
65.05
68.39
99.13
72.49
119.11
MDA-MB-468
97.31
Mean
47.39 %
Selected for 5-dose
101.60 %
Non selected
75.51 %
Non selected
86.38 %
Non selected
92.63 %
Non selected
Table (2): NCI in vitro testing result of compound 1h (NSC D-755517/1) at five dose level in µM.
Panel/Cell Line
GI50
TGI
LC50
LogGI50
Log TGI
Log LC50
Concentration
per cell line
MIDb
selectivity ratio
(MIDa:MIDb)

22
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
Leukemia .............................
.......
6.91
8.67
6.49
5.51
3.83
7.20
........
7.18
5.83
> 1.00
3.03
3.49
8.10
7.51
4.54
3.15
........
9.47
9.31
5.05
4.53
4.74
5.12
7.71
.......
2.79
9.07
4.12
2.35
1.87
1.28
........
5.76
> 1.00
3.91
1.50
3.78
6.12
3.81
6.40
1.42
........
6.98
6.95
6.22
5.04
6.81
9.56
........
1.46
3.01
4.78
1.20
5.22
5.47
3.52
........
4.47
3.81
........
5.67
2.84
>1.00
9.32
1.27
6.44
0.79
............
> 1.00
> 1.00
> 1.00
4.83
......
> 1.00
.......
-5.16
........... ...
...............
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer ....
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer ..........................
COLO 20
HCC-2998
HCT-116
HCT-1
>
>
>
-4.00
-4.00
-4.00
-4.32
-4.00
-4.00
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
.........
-5.06
-5.19
-5.26
-5.42
-5.14
..........
-5.14
-5.23
> -4.00
-5.52
-4.46
-5.09
-4.12
-5.34
-5.50
...........
-5.02
-5.03
-5.30
-5.34
-5.32
-5.29
-5.11
.........
-4.55
-5.04
-4.39
-4.63
-4.73
-4.89
.........
-5.24
>-4.00
-5.41
-4.82
-5.42
-4.21
-4.42
-4.19
-4.85
............
-4.16
-5.16
-5.21
-5.30
-4.17
-5.02
.........
-4.83
-5.52
-5.32
-4.92
-5.28
-5.26
-5.45
.........
-5.35
-4.42
........
-5.25
-5.55
> - 4.00
-5.03
-4.90
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
.............
> 1.00
> 1.00
5.28
>
>
5.35
0.96
...............
...............
>
>
>
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
>
>
>
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
6.56
0.78
..............
..............
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
..........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
.........
>
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
>
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
HT29
KM12
SW-620
CNS Cancer ............................
SF-268
SF-29
SF-539
SNB-19
SNB-7
U251
Melanoma........................
LOX IMVI
MALME-3M
M14
MDA-MB-43
SK-MEL-2
SK-MEL-28
SK-MEL-
UACC-257
UACC-62
Ovarian Cancer .......................
IGROV1
OVCAR-3
OVCAR-4
OVCAR-
OVCAR-8
NCI/ADR-RES
Renal Cancer ............................
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer ........................
PC-3
DU-14
Breast Cancer ...........................
MCF7
MDA-MB-231/ATCC 0.594
HS 578T
3.58
4.09
1.42
..............
................
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
1.24
.............
................
>
>
-4.00
-4.00
-4.28
-4.00
-4.10
-4.00
-4.00
-4.00
-4.00
>
>
>
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
> 1.00
7.87
>
> 1.00
> 1.00
> 1.00
> 1.00
............
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
...........
> 1.00
> 1.00
.........
>
>
>
>
6.93
3.5
0.73
1.45
...............
...............
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
..............
...............
>
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
>
>
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
-4.00
2.1
2.42
............
..............
> 1.00
> 1.00
.........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
>
>
-4.00
-4.00
>
>
-4.00
-4.00
4.78
1.07
.............
.............
> 1.00
5.57
> 1.00
> 1.00
> 1.00
>
-4.00
-4.25
-4.00
-4.00
-4.00
>
>
>
>
>
-4.00
-4.00
-4.00
-4.00
-4.00
>
>
>
T-47D
MDA-MB-468
MIDa
5.09
Average:
-4.96
-4.02
-4.0
(11 µM)
(95.5 µM)
(˃100µM)
Delta:
Range:
0.59
1.55
0.30
0.32
0.00
0.00
MIDa: Average sensitivity of all cell line in µM. MIDb: Average sensitivity of all cell line of a particular subpanel in µM.
Table (3): 60 cell line anticancer screening data at single dose assay (10-5 M) as percent growth inhibition of indolo-carbazoles.
Panel/Cell Line................................................................................. Growth Percent (%).................................................................................
2e
2f
2h
2i
2l

23
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
Leukaemia .....................................
HL-60(TB)
K-562
MOLT-4
RPMI-8226
Non-Small Cell Lung Cancer...........
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer ................................
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS Cancer .................................
SF-268
SF-295
SF-539
SNB-19
U251
Melanoma ...... ............................
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer .........................
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer.............................
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer.........................
PC-3
DU-145
Breast Cancer..........................
MCF7
MDA-MB-231/ATCC
HS 578T
.........
85.63
56.77
61.55
103.63
..........
35.07
28.58
-44.87
-20.13
58.36
5.55
54.20
-15.58
-23.83
..........
35.06
78.09
7.08
...........
93.01
93.77
87.83
93.28
...........
101.29
85.39
103.65
117.34
113.20
99.57
92.60
101.36
76.28
...........
102.90
94.79
93.94
91.90
90.98
105.92
91.96
............
104.78
108.54
89.59
104.31
97.54
...........
91.90
89.19
87.83
102.34
96.34
113.79
104.58
93.61
..........
94.67
124.29
92.04
88.96
107.28
106.83
105.46
...........
97.88
113.64
98.38
80.17
112.55
96.88
106.78
57.58
..........
107.22
115.99
..........
89.39
...........
93.83
90.31
89.45
101.84
............
87.05
100.47
75.37
98.69
121.56
85.95
94.27
11.95
69.76
..........
46.32
102.40
72.65
97.68
97.62
70.71
11.44
............
95.54
97.81
87.95
101.34
44.79
............
91.85
70.52
86.36
90.59
87.37
100.59
109.35
90.58
............
94.15
122.18
78.48
94.14
59.24
75.38
94.19
............
84.04
86.53
94.06
84.65
105.50
97.15
95.31
60.84
...........
58.82
39.16
...........
77.15
86.89
91.24
87.17
92.62
108.96
...........
92.43
69.13
57.59
85.35
...........
70.47
60.37
87.43
56.36
95.18
72.69
74.83
68.71
40.64
...........
89.45
89.55
58.49
67.40
57.82
73.25
80.96
...........
93.10
67.24
95.85
89.22
75.14
.............
76.41
95.88
78.68
79.49
95.93
71.78
97.58
77.85
...........
62.03
96.52
73.12
85.87
89.33
79.55
94.58
............
76.33
78.86
70.22
41.36
85.59
81.56
94.84
39.78
...........
48.49
98.24
...........
59.07
63.58
88.18
81.59
59.13
112.86
..........
71.69
91.85
100.00
98.07
...........
102.55
100.02
82.44
89.60
113.72
87.64
106.18
74.67
67.55
...........
104.17
nd
92.35
42.53
35.90
44.00
6.72
101.24
103.79
104.52
75.56
..........
111.10
99.02
100.18
109.29
91.47
...........
95.39
101.60
102.10
100.85
102.29
106.20
106.52
100.27
............
105.11
108.60
101.27
104.54
96.21
108.71
91.66
...........
92.18
118.27
104.77
95.32
104.21
99.00
139.73
76.77
..........
76.27
97.18
.........
..........
37.80
-23.09
38.81
25.73
-51.91
..........
42.67
21.37
-29.41
44.51
17.60
45.69
83.91
39.50
.........
33.23
-52.72
14.28
36.61
20.00
19.48
26.87
..........
-35.79
-7.03
9.61
13.72
30.66
27.74
-18.32
12.35
.........
36.54
46.83
..........
24.23
-30.21
-8.41
94.71
84.64
97.29
94.79
96.62
121.33
89.23
109.72
113.56
85.57
BT-549
T-47D
MDA-MB-468
8.92
28.15
47.27
115.17
Mean
21.63 %
Selected
98.65 %
Non selected
84.67%
Non selected
76.84%
Non selected
98.24%
Non selected
Table (4): NCI in vitro testing result of compound 2e (D-758513/1) at five dose level in µM.
Panel/Cell Line
TGI
LC50
Log GI50
Log TGI
Log LC50
GI50
Concentration

24
European Journal of Medicinal Chemistry
ACCEPTED MANUSCRIPT
per cell line
MIDb
selectivity ratio
(MIDa:MIDb)
1.0
Leukemia ...................................
........
2.41
..........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
.........
nd
..........
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
> 1.00
.........
nd
3.90
nd
2.83
3.42
> 1.00
9.01
4.34
...........
6.77
> 1.00
5.11
..........
nd
-4.62
nd
nd
nd
.........
> -4.00
> -4.00
> -4.00
> -4.00
> -4.00
> -4.00
..........
nd
...........
> -4.00
> -4.00
> -4.00
> -4.00
> -4.00
> -4.00
..........
nd
-4.41
nd
-4.55
-4.47
> -4.00
-5.05
-5.36
...........
-4.17
> -4.00
-5.29
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer ....
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
Colon Cancer ......................
COLO 20
HCC-2998
HCT-116
HCT-1
HT29
KM12
SW-620
CNS Cancer ......................
SF-268
SF-29
SF-539
SNB-19
SNB-7
U251
Melanoma........................
LOX IMVI
MALME-3M
M14
MDA-MB-43
SK-MEL-2
SK-MEL-28
SK-MEL-
UACC-257
UACC-62
Ovarian Cancer .......................
IGROV1
nd
2.41
nd
nd
nd
nd
nd
......
1.51
1.92
1.16
1.07
3.28
1.69
1.46
6.22
.......
4.22
1.69
1.34
4.24
2.36
3.77
1.15
.......
1.79
1.19
3.00
1.52
1.34
1.13
.......
2.56
nd
2.29
1.06
.........
-5.82
-5.72
-5.94
-5.97
-5.48
-5.77
-5.83
-6.21
..........
-5.38
-4.77
-5.87
-5.37
-4.63
-5.42
-4.94
..........
-5.75
-5.92
-5.52
-5.82
-5.87
-5.95
...........
-5.59
nd
5.72
2.38
3.73
1.08
4.59
3.63
1.88
..........
2.05
> 1.00
2.61
2.12
6.72
> 1.00
2.36
...........
5.35
2.69
9.53
3.35
5.24
2.33
..........
1.21
nd
-5.24
-5.62
-5.43
-4.97
-5.34
-5.44
-5.72
...........
-4.69
>-4.00
-5.58
-4.67
-5.17
>-4.00
-5.63
..........
-5.27
-5.57
-5.02
-5.47
-5.28
-5.63
...........
-4.92
nd
2.68
0.9
8.44
-4.07
> 1.00
> 1.00
4.86
..........
1.99
6.06
> 1.00
7.40
> 1.00
nd
...........
> 1.00
> 1.00
5.51
> 1.00
> 1.00
nd
> -4.00
> -4.00
-5.31
..........
-4.70
-5.22
> -4.00
-5.13
> -4.00
nd
..........
> -4.00
> -4.00
-5.26
> -4.00
> -4.00
nd
1.66
2.51
1.46
0.96
1.67
3.10
nd
3.03
> 1.00
nd
-5.78
-5.51
nd
-5.52
>-4.00
nd
1.62
2.57
3.54
2.48
.......
2.06
1.67
5.65
2.39
1.72
1.82
2.19
.......
1.60
1.53
1.96
1.90
3.19
2.00
1.52
1.82
.......
nd.
nd
-5.79
-5.59
-5.45
-5.61
...........
-5.69
-5.78
-6.25
-5.62
-5.76
-5.74
-5.66
..........
-5.80
-5.82
-5.71
-5.72
-5.50
-5.70
-5.82
-5.74
...........
nd
nd
7.81
5.93
1.15
..........
nd
3.03
2.89
6.97
nd
5.77
5.72
..........
2.95
3.28
4.61
6.11
9.24
6.81
2.85
5.41
...........
> 1.00
3.30
...........
nd
2.74
-5.11
-4.23
-4.94
...........
nd
-5.52
-5.54
-5.16
Nd
-5.24
-5.24
...........
-5.53
-5.48
-5.34
-5.21
-5.03
-5.17
-5.54
-5.27
...........
>-4.00
-5.48
...........
nd
-4.56
> 1.00
> 1.00
..........
> 1.00
5.51
1.58
2.60
> 1.00
3.99
1.97
...........
5.43
7.05
1.22
5.51
4.14
> 1.00
5.34
>-4.00
> -4.00
...........
> -4.00
-5.26
-4.80
-4.58
> -4.00
-4.40
-4.70
..........
-5.27
-5.15
-4.91
-4.26
-4.38
> -4.00
-5.27
2.5
0.97
OVCAR-3
OVCAR-4
OVCAR-
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer ............................
786-0
A498
ACHN
1.94
1.25
CAKI-1
RXF 393
SN12C
TK-10
UO-31
2.01
-4.70
Prostate Cancer ........................
PC-3
DU-14
Breast Cancer ...........................
MCF7
MDA-MB-231/ATCC 0.594
HS 578T
1.77
3.86
1.37
0.62
...........
> 1.00
6.13
...........
> 1.00
5.20
1.29
> 1.00
> 1.00
..........
> -4.00
-5.21
...........
> -4.00
-5.28
-4.89
> -4.00
> -4.00
1.77
.......
9.29
1.41
2.40
2.46
3.74
-5.75
...........
-6.03
-5.85
-5.62
-5.61
-5.43
2.70
5.08
nd
-5.57
-5.29
Nd
T-47D
MDA-MB-468
5.23
-4.28
_MIDa
2.42
Avarege:
-5.69
(2 µM)
0.56
-5.01
(10 µM)
0.71
-4.46
(34.6 µM)
0.9
Delta:
Range:
1.63
1.72
1.36
MIDa: Average sensitivity of all cell line in µM. MIDb: Average sensitivity of all cell line of a particular subpanel in µM.

Research Highlights
ACCEPTED MANUSCRIPT
•
•
•
•
We synthesized BIMs and tetrahydroindolocarbazoles,
Ten compounds have selected by the NCI for anticancer screening,
Compound 2e gave the highest antiproliferative activity in a nanomolar ranges.
Compound 2e showed non critical cytotoxcity.

ACCEPTED MANUSCRIPT
Supporting information for
Synthesis, Cytostatic Evaluation and Structure Activity Relationships of Novel Bis-
indolylmethanes and their Corresponding Tetrahydroindolocarbazoles.
Mardia T. El Sayeda,b,*, Khadiga M. Ahmedc, Kazem Mahmouda, and Andreas Hilgerotha.
aInstitute of Pharmacy, Martin-Luther University, Research Group of Drug Development and Analysis, Wolfgang-
Langenbeck-Straße 4, 06120 Halle, Saale, Germany. bApplied Organic Chemistry Department, National Research Centre,
Cairo, Egypt., cNatural Compounds Laboratory, National Research Centre, Cairo Egypt.
Corresponding author: Mardia El Sayed
Mardia_elsayed2009@yahoo.com
N
N
N
H
H
N
2e

ACCEPTED MANUSCRIPT
Mean graphs of One and Five dose anticancer screening
Figure (1): Maen graph one dose screening of 1g