Synthesis, anticancer activities and molecular modeling studies of novel indole retinoid derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.10.013

In this study, novel (E)-3-(5-substituted-1H-indol-3-yl)-1-(5,5,8,8- tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-en-1-one (5(a-e)) derivatives were synthesized and their anticancer effects were determined in vitro. Novel indole retinoid compounds except 5e have anti-proliferative capacity in liver, breast and colon cancer cell lines. This anti-proliferative effect was further analyzed in breast cancer cell line panel by using the most potent compound 5a. It was determined that 5a can inhibit proliferation at very low IC₅₀ concentrations in all of the breast cancer cell lines. Here, we present some evidence on apoptotic termination of cancer cell proliferation which may be primarily driven by the inhibition of RXRα and, to a lesser extent, RXRγ.

Full text of DOI:10.1016/j.ejmech.2012.10.013

European Journal of Medicinal Chemistry 58 (2012) 346e354
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anticancer activities and molecular modeling studies of novel indole
retinoid derivatives
,
b
c
b
b
A. Selen Gurkan-Alp a , Mine Mumcuoglu , Cenk A. Andac , Emre Dayanc , Rengul Cetin-Atalay ,
*
Erdem Buyukbingol a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara 06100 Turkey
b Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Bilkent, Ankara 06100 Turkey
c Department of Pharmacology, School of Medicine, Dicle University, Diyarbakir 21280, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, novel (E)-3-(5-substituted-1H-indol-3-yl)-1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-
2-yl)prop-2-en-1-one (5(aee)) derivatives were synthesized and their anticancer effects were determined
in vitro. Novel indole retinoid compounds except 5e have anti-proliferative capacity in liver, breast and colon
cancer cell lines. This anti-proliferative effect was further analyzed in breast cancer cell line panel by using the
most potent compound 5a. It was determined that 5a can inhibit proliferation at very low IC50 concentrations
in all of the breast cancer cell lines. Here, we present some evidence on apoptotic termination of cancer cell
Received 14 June 2012
Received in revised form
5 October 2012
Accepted 9 October 2012
Available online 22 October 2012
proliferation which may be primarily driven by the inhibition of RXRa and, to a lesser extent, RXRg.
Keywords:
Indole
Ó 2012 Elsevier Masson SAS. All rights reserved.
MDA-MB-231
Molecular dynamics
Retinoid
T47D
1. Introduction
relied on the magnitude of their influences on these receptors. The
properties of retinoids confer a significant therapeutic potential for
the treatment of dermatological diseases [7] and cancer, including
chemotherapeutic and chemo-preventive applications [8,9]. There
is important evidence that these agents have potent growth
inhibiting activities on cancer cell lines in vitro and in vivo [4].
In vitro studies and animal models show that retinoids have ability
to inhibit carcinogenesis in different tissues [10]. Retinoids have
been evaluated as chemo-preventive agents in cancer treatment
and prevention. Retinoid compounds have been used efficiently in
the treatment of pre-neoplastic diseases such as cervical dysplasia,
leukoplakia and xeroderma pigmentosum. Malignant diseases,
especially acute promyelocytic leukemia (APL), a subtype of acute
myelogenous leukemia (AML) has been successfully treated with
retinoids [11]. Other than leukemia, retinoids have anti-
proliferative action in solid tumors such as breast, liver, lung,
ovarian, prostate and colon cancer [12,13]. Nevertheless, due to the
observation of numerous undesirable side effects i.e. teratogenic
activity [14,15], liver and bone toxicity [16], hypervitaminosis A
syndrome [17], the short and long term applications of retinoids are
limited for the treatment of above-mentioned diseases [18].
Synthesized new retinoid derivatives are required that have
increased beneficial properties and reduced adverse effects.
The indole ring has been deemed as an important moiety found
in many pharmacologically active compounds possessing certain
biological activities in which some studies have been attributed to
its anticancer effectiveness as described in the literature [1e3]. On
the other hand, retinoids, natural and synthetic derivatives of
vitamin A and its most active metabolite all-trans-retinoic acid have
important functions in cell growth, differentiation, modulation of
apoptosis and many physiological processes such as vision and
embryonic development in vertebrates [4,5]. There are two classes
of retinoid nuclear receptors, retinoic acid receptors (RARs) and
retinoid X receptors (RXRs), both having three subtypes (a, b, g) [6].
Retinoid compounds have shown their biological activities via
these receptors in which several mechanistic studies have been
Abbreviations: CPT, camptothecin; DMEM, Dulbecco’s modified Eagle’s medium;
ER, estrogen receptor; FCS, fetal calf serum; MD, molecular dynamics; PBS, phos-
phate buffered saline; RXR, retinoid X receptor; SRB, sulforhodamine B; TCA,
trichloroacetic acid.
* Corresponding author. Tel.: þ90 312 2033080; fax: þ90 312 2131081.
E-mail address: sgurkan@pharmacy.ankara.edu.tr (A.S. Gurkan-Alp).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.013

A.S. Gurkan-Alp et al. / European Journal of Medicinal Chemistry 58 (2012) 346e354
347
In the present study,
a
series of novel indole retinoid
1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-
compounds 5(aee) (Scheme 1) consisting of both indole and tet-
rahydronaphthalene ring system has been synthesized due to the
requirements of finding new compounds in cancer treatments. The
indole moiety has been reported to exhibit diverse biological
activities including anticancer effects [19]. Therefore, indole ring
comprise beneficial features of some of the existing anticancer
compounds, such as Panobinostat [20,21], Cediranib [22], indole-3-
carbinol [23]. On the other hand, natural and/or synthetic retinoids
have been known influences to regulate inner cell functions to
interfere for the supression of cancer initiation as well as treatment
of the certain cancer occurrences [24]. Therefore, we aimed to
combine the structural features of tetrahydronaphthalene ring
system (retinoid head) and indole moiety with a linker. The anti-
tumoral profiles of the synthesized compounds were investigated.
en-1-one derivatives 5(aee) were prepared by the condensation of
compound 3 with appropriate indole-3-carbaldehyde 4(aee) under
basic conditions [32]. Details are stated in the Experimental section.
3. Results and discussion
In this study, we aimed to synthesize novel indole retinoid
compounds which are expected to have anticancer properties. To
achieve this, five novel indole retinoid derivatives with the
substituents at 5th position of the indole ring expected to possess
anticancer activity were designed and synthesized. The substitu-
tion pattern on the indole ring is thought to have a deterministic
factor over the biological effectiveness of the compounds. Due to
the distinctive properties of the substituents regarding to their
physicochemical behaviors, it might be the way of finding of what
relativeness are able to attract activity-inquires leading to exert the
desired biological activities. In spite of the fact that the absence of
any kind of substitution (hydrogen only) gave the most effective-
ness, both electron-donating and electron-withdrawal substitu-
tions had lesser effects in terms of possessing the activity. Actually,
this could be a very interesting point of view to support the indole
ring system to avoid substituent-inclusion with the enormously
activating and/or deactivating substituents rather than using no
substitution (like hydrogen only) or with substituents having mild
activating/deactivating properties for the future progressions. Thus,
more efficiently activating/deactivating substituents could be
unfavorable for the biological activity studied.
2. Chemistry
The synthetic procedures for the preparation of the compounds
5(aee) are shown in Scheme 1. Commercially available 2,5-
dimethyl-2,5-hexandiole and appropriate 5-substituted indole
derivatives served as starting materials. 2,5-Dichloro-2,5-dimethyl
hexane (1), was prepared in 55% yield by passing dry hydrogen
chloride gas over 2,5-dimethyl-2,5-hexandiole [18,25]. Benzene was
alkylated by compound 1 in dichloromethane catalyzed with
aluminum chloride to produce 1,1,4,4-tetramethyl-1,2,3,4-
tetrahydronaphthalene (2), in 48% yield [26]. Then, 1-(5,5,8,8-
tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethanone (3) was
obtained by acetylation of intermediate 2 with acetyl chloride using
AlCl3 as a catalyst [26,27]. On the other hand, the corresponding
aldehydes 4(aee) were obtained by treating indole derivatives
bearing substituent at position 5 with dimethylformamide, using
phosphorus oxychloride as a catalyst according to literature method
[28e31]. The final compounds (E)-3-(5-substituted-1H-indol-3-yl)-
(E)-3-(5-Substituted-1H-indol-3-yl)-1-(5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)prop-2-en-1-one derivatives 5(aee) were
synthesized in four steps (Scheme 1). Synthesized compounds were
purified by column chromatography using appropriate solvent
systems. Expected chemical structures of the compounds have been
deduced by mass, NMR spectral findings and elemental analyses
O
dry HCl (g)
AcCl
A
lCl3
OH
Cl
Cl
OH abs. Et-OH
AlCl3
1
3
2
CHO
R
R
POCl3 / DMF
N
N
H
H
4a R=H
4b R=OCH3
4c
R=Cl
4d
R=Br
4e R=COOH
R
O
O
CHO
R
KOH / EtOH
+
NH
N
H
3
4(a-e)
5a R=H
5b R=OCH3
5c
R=Cl
5d
R=Br
5e R=COOH
Scheme 1. Synthesis of novel indole retinoid compounds 5(aee).

348
A.S. Gurkan-Alp et al. / European Journal of Medicinal Chemistry 58 (2012) 346e354
Table 1
Table 2
Anti-proliferative capacity of novel indole retinoid derivatives (5(aee)) evaluated by
Anti-proliferative capacity of 5a analyzed in breast cancer cell line panel. IC50 levels
SRB assay in different types of cancer cell lines. IC50 values show
noid concentrations.
m
M levels of reti-
of the compound were shown in
experiment.
mM levels. CPT was used as a positive control in the
Compound
IC50 values
Huh-7
CPT
0.07
5a
Cama1
T47D
MCF7
BT474
MDA-MB-453
BT20
SK-BR-3
MDA-MB-361
MDA-MB-157
MDA-MB-231
ZR-75-1
<0.01
1.16
1.71
1.91
<0.01
1.31
1.29
0.06
1.46
1.83
3.88
3.92
T47D
HCT116
<0.01
<0.01
12.75
<0.01
<0.01
<0.01
0.17
5a
5b
5c
5d
5e
CPT
<0.01
15.50
15.36
13.97
no inh.
0.06
0.10
0.02
11.37
13.14
11.54
no inh.
<0.01
13.88
10.69
10.82
no inh.
<0.01
0.02
<0.01
<0.01
<0.01
indicated in the Experimental section. All spectral data were in accor-
dance with assumed structures. The trans conformation of these
derivatives were confirmed by a reference 3JHH value of 16 Hz obtained
for the HC]CH group of (E)-3-(6-fluoro-1H-indol-3-yl)acrylic acid
[33].
In order to analyze anti-cancer properties of the indole retinoid
derivatives, we performed cytotoxicity assay with these derivatives
on different types of cancer cell lines. For cytotoxicity studies, we
performed sulforhodamine B (SRB) assay [34] determining the IC50
values. In this initial screening liver Huh7, breast T47D and colon
HCT116, cancer cell lines were treated with these compounds and
subjected to SRB assay (Table 1).
MCF-12A
of the 11 breast cancer cell lines and one immortalized normal
breast cell line were considered to be screened for the activities of
compound 5a because of their differential ER and RXR expression.
Cells were treated with compound 5a for three days and then
subjected to SRB assay to determine cytotoxicity effect of this
compound (Table 2). Cytotoxicity results showed that 5a can inhibit
cancer cell growth at very low drug concentrations in all of the
breast cancer cell lines. IC50 concentration of 5a for MCF-12A,
which is an immortalized normal epithelial breast cancer cell
SRB assay showed that all of the compounds except compound
5e, were effective in all three of the cancer cell lines. The compound
5a had the lowest IC50 concentration (nanomolar level) similar to
Camptothecin (CPT) among other compounds. In this study, an
anti-cancer agent, CPT was included in to the study as an experi-
mental positive control. Therefore, this preliminary result simply
implies that the compound 5a could be a good candidate as an anti-
proliferative agent for breast, liver and colon cancer cells.
Retinoids have been known and used for their inhibitory effects
on breast cancer in chemoprevention and therapy. Moreover,
selective estrogen receptor modulators (SERMs) (tamoxifen,
raloxifene etc.) or aromatase inhibitors (AIs) (anastrozole, letrozole
etc.) have been used successfully in chemoprevention of estrogen
receptor positive (ER-positive) breast cancers [35]. But these drugs
cannot prevent the progression of ER-negative breast cancer cases.
Herein, the need for new retinoid molecules having the ability of
prevention of both ER-positive breast cancers has emerged as one
of our concerns. Therefore, we wanted to analyze anti-proliferative
effects of compound 5a on other breast cancer cell lines. For this
line, was 3.92 mM. This concentration is four fold higher than IC50
concentration of other breast cancer cell lines. This result might
provide evidence that if 5a is utilized as a drug in cancer prevention
or treatment in breast cancer, it will be less toxic to normal breast
tissue. Breast cancer is a very heterogeneous disease and gene
expression profiling analysis revealed presence of different
molecular subtypes of breast cancer. These subtypes are luminal A,
luminal B, ERBB2-positive, basal-like and normal-like [36]. Every
subtype exhibits specific therapeutic response to therapy and
prognosis [37]. Breast cancer cell lines used in this study represent
all these different subtypes [38]. Compound 5a effectively blocked
proliferation of all these distinct subtypes. Other important aspects
of the results were that compound 5a showed effective anti-
proliferative action at very low concentrations even in MDA-MB-
231 cell line which is an example of triple-negative breast
cancers. Characteristics of triple-negative breast cancer are no
expression of estrogen receptor (ER), progesterone receptor (PR)
and HER-2 genes. Therefore, they present clinical challenge because
they do not respond to endocrine therapy or other available ther-
apies. Furthermore, this subtype of breast cancers shows overall
worst or disease free survival [39]. Therefore, compound 5a could
purpose, we initially analyzed RXR
by RT-PCR (Fig. 1). The cell lines analyzed had varied ER expression
in parallel with RXR , but not RXR . Based on this observation, all
a, RXRg and ER expression levels
g
a
Fig. 1. RXRa, RXRg and ER expressions were determined in breast cancer cell lines by RT-PCR method.

A.S. Gurkan-Alp et al. / European Journal of Medicinal Chemistry 58 (2012) 346e354
349
be a promising drug candidate for the treatment of triple-negative
breast cancers.
Retinoids have been known to regulate cell growth, differenti-
ation and apoptosis [40]. Therefore we investigated whether
compound 5a could generate its anti-proliferative effect in breast
cancer cells through induction of apoptosis. It was then decided to
perform Hoechst staining which is one the apoptosis detection
methods for 5a treated cells. We conducted this assay on T47D and
MDA-MB-231 cell lines as representatives of ER-positive and ER-
negative groups of breast cancer cells. These cells were treated
with 5a for three days starting from 24 h after seeding. Then treated
cells were stained with Hoechst and visualized under florescent
microscope for 5a induced apoptosis. Apoptotic cells were detected
after treatment with compound 5a on both T47D and MDA-MB-231
cell lines compared to DMSO controls (Fig. 2).
To measure the percentage of apoptotic cells, we used flow
cytometry analysis. MDA-MB-231 cells were treated with two
different concentrations of 5a, IC50 (1.8 mM) and IC100 (3.6 mM),
and treatment was stopped on day 2 and day 4 for Annexin V and
Propidium iodide staining by flow cytometry (Fig. 3). We
observed %7.10 and %10.53 of apoptotic cells among the 5a
Fig. 3. Flow cytometry analysis. MDA-MB-231 breast cancer cell line cells were seeded,
and after 24 h, cells were treated with two different concentrations of 5a (1.8
mM and
3.6 M). Measurements were made on two days and four days post-treatment. The rate
m
of apoptosis was determined by Annexin V and Propidium iodide staining, and mean
percentage of Annexin Vþ and Propidium Iodideþ cells were plotted against treatment
modalities. Gray bars represent Day 2, and black bars represent Day 4 of treatment.
(1.8
Whereas 5a (3.6
apoptotic on the same days. Therefore we concluded that four
days of 5a treatment at 3.6 M concentration led to fifty percent
m
M) treated cell population on days 2 and 4, respectively.
m
M) treated cells were found %6.83 and %15.62
the pharmacophore tetramethyltetrahydro naphthalene group in
the original PDB files were referenced to select the lowest r.m.s.d.
m
docked coordinates of compound 5a in the binding site of RXR
a and
increase in apoptotic cell percentage when compared to two days
of 5a treatment.
RXR . The preference for this binding mode of compound 5a relies
g
greatly on the fact that the indole group of compound 5a, which
superimposed with the carboxylic acid side of all-trans-retinoic
acid in another set of docking studies implemented using the
It has been shown that all-trans-retinoic acid and other retinoids
can induce apoptosis in breast cancer cell lines [41,42]. This study
provided evidences that our novel retinoid compound, 5a, exerts
anti-proliferative effects through induction of apoptosis. Addi-
tionally flow cytometry analysis results showed that apoptotic
effect of 5a was gradually increased from day 2 to day 4. This
implies that treatment duration of 5a was an important factor on
apoptotic response which was started from day 4. But this proap-
optotic property needs further analysis to better understand the
precise mechanism of action of compound 5a.
crystal structure of all-trans-retinoic acid in complex with RXR
g
(PDB ID: 2LBD [45]), is more polar than the tetramethyltetrahydro
naphthalene group of compound 5a, which superimposed with the
trimethyl-cyclohexen group of all-trans-retinoic acid in the binding
site of RXR
dynamics (MD) computations were applied for docked coordinates
of compound 5a in the binding site of RXR and RXR . Although
g [45]. After docking studies, 10 ns of molecular
a
g
MD computations equilibrated beyond 4 ns, the MD solution
structures of the complex species were sampled and monitored
between 9 and 10 ns of trajectories in order to assure stability
during sustained equilibration. The MD computations revealed that
To gain more insight into the binding mechanism and estimate
binding affinity at the molecular level, compound 5a was initially
docked into the binding site of RXR-alpha (RXR
a) (PDB ID: 2ZXZ
[43]) and RXR-gamma (RXR ) (PDB ID: 4LBD [44]). Coordinates for
g
Fig. 2. Hoechst staining. T47D and MDA-MB-231 cells were treated with compound 5a for three days and stained with Hoechst. Arrows show apoptotic cells. Pictures were taken at
40ꢀ objective magnification. Camptothecin (CPT) was used as a positive control in the experiment.

350
A.S. Gurkan-Alp et al. / European Journal of Medicinal Chemistry 58 (2012) 346e354
Since RXR
the anti-proliferative effect of compound 5a could be more likely
related to this receptor rather than RXR
Definitions of the energy terms in Table 3 are given in Section
a possesses the highest binding affinity, we suggest that
g.
5.3.3. KD was determined according to
a
generic equation,
D
Gꢁ ¼ ꢂR$T ln (1/KD), where
D
Gꢁ is the binding free energy in
kcal/mol, T is the temperature at 300 K and R is the ideal gas
constant, 1.987 cal/mol K. MMePBSA computations revealed that
a total of non-electrostatic contributions [
(solution)] to the enthalpy of binding are favorable for the
RXR $ compound 5a
DEVDW (gas) þ
DGnonel
a
$ compound 5a (ꢂ63.03 kcal/mol) and RXR
g
(ꢂ58.22 kcal/mol) complex systems, strongly suggesting that
compound 5a possesses a hydrophobic nature and thus it prefers to
interact with hydrophobic binding sites. However, electrostatic
contributions [
binding were found to be unfavorable for compound 5a in complex
with RXR (23.16 kcal/mol) and RXR (27.06 kcal/mol), in which
case an energy difference of 3.90 kcal/mol disfavoring the
RXR $ compound 5a complex species suggest that the binding site
of RXR possesses slightly more hydrophobic amino acid residues
DEel (gas) þ
DGel (solution)] to the enthalpy of
a
g
g
a
(VAL242, GLU243, PRO244, LEU246, PRO264, ILE268, ALA272,
GLN275, LEU279, LEU309, SER312, PHE313, ARG316, LEU325,
LEU326, ALA327, PHE338, VAL349, ILE428, GLY429, CYS432),
Fig. 4A, than that of RXR
g (PHE201, TRP227, PHE230, SER231,
ALA234, CYS237, ILE238, LEU271, MET272, ARG274, ILE275,
ARG278, MET286, PHE288, PHE304, GLY393, ALA397, LEU400,
MET415, LEU416), Fig. 4B.
After determination of binding mechanism and affinity of 5a to
RXR
receptors in our breast cancer cell lines. RXR
of the breast cancer cell lines. Nevertheless RXR
absent in SK-BR-3 and MDA-MB-231 cell lines. Since compound 5a
showed high binding affinity pattern toward both RXR and RXRg,
a
and RXR
g
, we checked the expression level of these two
was expressed in all
expression was
a
g
a
in silico, still there could be an ambiguous explanation of the data
obtained from in vitro biological assays and molecular dynamics. In
this case, there seems to be a conflict between in vitro experimental
results and molecular dynamics predictions. The molecular
dynamics predictions showed high binding affinities for both
receptors whereas in vitro experimental results indicated the
existence of RXRa expression, but not RXRg. This observation may
Fig. 4. Compound 5a (green) confined to the binding site of (A) RXRa and (B) RXRg at
point out that in silico prediction approaches are quite far from the
explanation of accurate interaction patterns of in vivo ligande
receptor relationships. Molecular dynamics studies obviously
provide a road-map for such interactions to assume the consider-
ation of possible mechanisms for the biological phenomena.
More likely, different mechanisms could be attributed if there
were being utilized with different cancer cell lines which have an
expression of RXRg. But in our case, RXRa was found to have the
main factor, which is responsible in the occurrence of the biological
activity. The other receptor involvements were not included in this
10 ns of MD computation. Amino acid residues neighboring compound 5a are anno-
tated in black. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
compound 5a is mainly confined to the binding sites of RXR
Fig. 4A, and RXR , Fig. 4B.
a,
g
MMePBSA binding enthalpy energy terms are listed in Table 3.
It was determined that compound 5a possesses very high binding
affinities toward RXR
a
(D
Gꢁ ¼ ꢂ20.19 kcal/mol, KD ¼ 1.9 ꢀ 10ꢂ15 kcal/
mol) and RXR
g
(D
Gꢁ ¼ ꢂ13.13 kcal/mol, KD ¼ 2.7 ꢀ 10ꢂ10 kcal/mol).
study due to the main approach has been focused on the RXRa.
However, studies for finding out though different mechanisms are
under progress.
Table 3
MMPBSA binding energies (kcal/mol) and dissociation constants KD (kcal/mol) for
compound 5a.
4. Conclusions
RXRa
RXRg
Breast cancer is the second leading cause of cancer death in
women in the world. Therefore generation of new agents for the
prevention and the treatment of the breast cancer are very critical.
Our results have determined that most of our novel indole retinoid
compounds have anti-proliferative effects in different cancer cell
lines especially in breast cancer cell lines. Compound 5a showed
the lowest IC50 level in cytotoxicity assays in our breast cancer cell
line panel, which includes ER-positive and ER-negative cell lines.
Furthermore, we observed that 5a was less toxic in MCF-12A, which
D
D
D
D
D
D
D
Eel
EVDW
Eint
ꢂ23.53 ꢃ 3.25
ꢂ56.16 ꢃ 2.70
0.00 ꢃ 0.01
ꢂ10.45 ꢃ 3.24
ꢂ51.25 ꢃ 2.74
ꢂ0.01 ꢃ 0.01
ꢂ61.71 ꢃ 4.19
ꢂ6.97 ꢃ 0.13
37.51 ꢃ 3.07
ꢂ31.18 ꢃ 4.74
ꢂ18.05 ꢃ 7.71
ꢂ13.13
Hgas
Gnonel
Gel
ꢂ79.69 ꢃ 3.69
ꢂ6.87 ꢃ 0.12
45.69 ꢃ 2.86
ꢂ40.87 ꢃ 3.85
ꢂ20.68 ꢃ 10.92
ꢂ20.19
Hꢁ
T$
Gꢁ
KD
D
Sꢁ
D
1.9 ꢀ 10ꢂ15
2.7 ꢀ 10ꢂ10

A.S. Gurkan-Alp et al. / European Journal of Medicinal Chemistry 58 (2012) 346e354
351
is a normal-like breast epithelial cell line. Compound 5a induced
apoptosis was the cause of the anti-proliferative effect. We also
performed molecular binding studies to analyze the involvement of
Jo ¼ 8 Hz), 7.43 (s, 1H, indole eH(2)), 7.33 (d, 1H, Jo ¼ 9.2 Hz), 6.95
(dd, 1H, Jo ¼ 8.8 Hz, Jm ¼ 2 Hz), 3.92 (s, 3H, eOCH3), 1.73 (s, 4H, e
CH2eCH2e), 1.34 (d, 12H, e(CH3)2, e(CH3)2). Elemental analysis,
Calcd for C26H29NO2: C 80.59; H 7.54; N 3.61. Found: C 80.34; H
7.79; N 3.33.
retinoic receptors. We predicted that 5a binds RXR
a and RXRg.
These results implied that these novel indole retinoid compounds,
particularly 5a, could be a promising anti-cancer agent candidate in
prevention or treatment of different cancers. Nevertheless, further
investigation is needed to determine exact mechanisms underlying
this effect.
5.1.4. (E)- 3-(5-Chloro-1H-indol-3-yl)-1-(5,5,8,8-tetramethyl-
5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-en-1-one (5c)
13% Yield, yellow solid, m.p. 195e197 ꢁC. ESIþ-MS (m/z %): 392
(M þ H,100), 394 (M þ H þ 2, 30). 1H NMR
d ppm (CDCl3): 8.79 (br.s,
5. Experimental section
1H, eNH), 8.02 (d, 1H, Jm ¼ 2 Hz), 8.01 (d, 1H, J ¼ 16 Hz), 7.96 (d, 1H,
Jm ¼ 2 Hz), 7.80 (dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2 Hz), 7.61 (d, 1H), 7.52 (d,
1H, J ¼ 15.6 Hz), 7.46 (d, 1H, Jo ¼ 8 Hz), 7.36 (d, 1H, Jo ¼ 8.4 Hz), 7.26
(dd, 1H, Jo ¼ 8.8 Hz, Jm ¼ 2 Hz), 1.73 (s, 4H, eCH2eCH2e), 1.34 (d,
12H, e(CH3)2, e(CH3)2). Elemental analysis, Calcd for C25H26ClNOe
0.55H2O: C 74.72; H 6.79; N 3.48. Found: C 74.88; H 7.16; N 3.08.
5.1. General synthetic
All starting materials and reagents were high-grade commercial
products purchased from Aldrich, Merck or Fluka. The structures of
all synthesized compounds were assigned on the basis of 1H NMR
and Mass spectral analyses. Analytical thin-layer chromatographies
were run on silica gel 60 F254plates (Merck, Germany). Column
5.1.5. (E)-3-(5-Bromo-1H-indol-3-yl)-1-(5,5,8,8-tetramethyl-
5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-en-1-one (5d)
chromatographies were accomplished on silica gel 60 (40e63
mm
18% Yield, a yellow solid, m.p. 199e200 ꢁC. 1H NMR
d ppm
particle size) (Merck, Germany). Melting points were determined
with an Electrothermal 9100 melting point apparatus (Electro-
thermal Engineering, Essex, UK) and uncorrected. 1H NMR
(400 MHz) spectra were recorded with a Varian Mercury-400
spectrometer (Varian Inc., Palo Alto, CA, USA), in CDCl3 or DMSO-
(CDCl3): 8.84 (br.s, 1H, eNH), 8.12 (d, 1H, Jm ¼ 1.6 Hz), 8.02 (d, 1H,
J ¼ 1.2 Hz), 8.01 (d, 1H, J ¼ 16 Hz), 7.79 (dd, 1H, Jo ¼ 8.4 Hz,
Jm ¼ 2 Hz), 7.58 (d, 1H), 7.51 (d, 1H, J ¼ 16 Hz), 7.46 (d, 1H,
Jo ¼ 8.4 Hz), 7.39 (dd, 1H, Jo ¼ 8.8 Hz, Jm ¼ 2 Hz), 7.31 (d, 1H,
Jo ¼ 8.4 Hz), 1.73 (s, 4H, eCH2eCH2e), 1.34 (d, 12H, e(CH3)2, e
(CH3)2). Elemental analysis, Calcd for C25H26BrNO: C 68.81; H
6.01; N 3.21. Found: C 68.88; H 5.92; N 3.12.
d6,
d scale (ppm) from internal standard TMS. Mass spectra were
recorded on a Waters ZQ micromass LCeMS spectrometer (Waters
Corporation, Milford, MA, USA) by the method of ESIþ. Elemental
analyses were performed on LECO CHNS-932 instrument (Leco, St
Joseph, MI, USA) and satisfactory results ꢃ0.4% of calculated values
(C, H, N) were obtained. 1H NMR, Mass, and elemental analyses
were performed at The Central Instrumentation Laboratory of the
Pharmacy Faculty of Ankara University, Ankara, Turkey.
5.1.6. (E)-3-(3-Oxo-3-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-
2-yl)prop-1-enyl)-1H-indole-5-carboxylic acid (5e)
5% Yield, yellow solid, m.p. 274e275 ꢁC. ESIþ-MS (m/z %): 402
(M þ H, 100). ESIꢂ-MS (m/z %): 400 (M ꢂ H, 80). 1H NMR
d ppm
(DMSO-d6): 12.69 (br.s, 1H, eNH), 12.19 (s, 1H, eCOOH), 8.60 (s, 1H),
8.26 (s, 1H), 8.01 (d, 1H, J ¼ 16 Hz), 7.99 (s, 1H), 7.85 (d, 1H,
Jo ¼ 8.4 Hz), 7.78 (d, 1H, Jo ¼ 8.4 Hz), 7.65 (d, 1H, J ¼ 15.2 Hz), 7.55
(m, 2H), 1.70 (s, 4H, eCH2eCH2e), 1.32 (d, 12H, e(CH3)2, e(CH3)2).
Elemental analysis, Calcd for C26H27NO3e0.7H2O: C 75.41; H 6.91;
N 3.38. Found: C 75.30 H, 6.79; N 3.22.
5.1.1. General methods for the preparation of the (E)-3-(5-
substituted-1H-indol-3-yl)-1-(5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)prop-2-en-1-one derivatives 5(aee)
To
a
solution
of
1-(5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)ethanone (3) (2 mmol) and appro-
priate indole-3-carbaldehyde 4(aee) (2 mmol) in 4 ml of ethanol
and 2 ml of water was added 2 g of solid KOH. The reaction mixture
was refluxed for at least 12 h. The end of the reaction was moni-
tored by TLC. The resulting mixture was cooled in an ice-water bath,
and then acidified with 4 ml of concentrated HCl and diluted with
20 ml of water. The precipitate was then collected by filtration and
dried [32]. The crude product was purified by column chromatog-
raphy eluting with n-hexane/ethyl acetate (3:1) solvent system to
give 5(aee).
5.2. General biological assays
5.2.1. Cell culture
Most of the cell lines were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum (FCS)
and 50 mg/ml penicillin/streptomycin. CAMA-1 and MDA-MB-157
were grown in DMEM containing 10% FCS, 50 mg/ml penicillin/
streptomycin and 1% sodium pyruvate. Glucose rich (4.5 g/l) RPMI
medium for ZR-75-1 and McCoy medium for SK-BR-3 were used
and supplemented with 10% FCS and 50 mg/ml penicillin/strepto-
mycin. Each cell line was maintained in a humidified incubator at
37 ꢁC supplied with 5% CO2.
5.1.2. (E)-3-(1H-Indol-3-yl)-1-(5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)prop-2-en-1-one (5a)
14% Yield, yellow crystalline solid, m.p.196e198 ꢁC. ESIþ-MS (m/z
%): 358 (M þ H, 100). 1H NMR
d
ppm (CDCl3): 8.62 (br.s, 1H, eNH),
5.2.2. Sulforhodamine B (SRB) cytotoxicity assay
8.09 (d, 1H, J ¼ 15.2 Hz), 8.03 (d, 1H, Jm ¼ 2 Hz), 8.01 (m, 1H), 7.81
(dd, 1H, Jo ¼ 8 Hz, Jm ¼ 1.6 Hz), 7.61 (s, 1H, indole eH(2)), 7.59 (d, 1H,
J ¼ 15.6 Hz), 7.45 (m, 2H), 7.31 (m, 2H),1.73 (s, 4H, eCH2eCH2e),1.34
(d,12H, e(CH3)2, e(CH3)2). Elemental analysis, Calcd for C25H27NO: C
83.99; H 7.61; N 3.92. Found: C 83.71; H 7.47; N 3.86.
Cancer cells (104 cells/well) were inoculated into 96 well
plates and after 24 h, cells were treated with retinoid compounds.
After three days of incubation with retinoids, cells were fixed
using 60
4
m
l of ice-cold 10% trichloroacetic acid (TCA) for 60 min at
l 0.4% SRB solution was administered and cells
ꢁC. Next 100
m
were incubated for 10 min at room temperature. To remove
unbound dye, cells were washed with 1% acetic acid five times
and air dried. 10 mM Tris-Base solution was applied to solubilize
SRB dye and absorbance was acquired at 515 nm in micropipette
reader. All the experiments were conducted in triplicate and
DMSO was used as negative control in corresponding
concentrations.
5.1.3. (E)-3-(5-Methoxy-1H-indol-3-yl)-1-(5,5,8,8-tetramethyl-
5,6,7,8-tetrahydronaphthalen-2-yl) prop-2-en-1-oþne (5b)
14% Yield, yellow solid, m.p. 138e139 ꢁC. ESI -MS (m/z %): 388
(M þ H, 40). 1H NMR
d ppm (CDCl3): 8.57 (br.s, 1H, eNH), 8.08 (d,
1H, J ¼ 15.6 Hz), 8.02 (d, 1H, Jm ¼ 1.6 Hz), 7.80 (dd, 1H, Jo ¼ 8.4 Hz,
Jm ¼ 2 Hz), 7.59 (d, 1H), 7.51 (d, 1H, J ¼ 15.6 Hz), 7.44 (d, 1H,

352
A.S. Gurkan-Alp et al. / European Journal of Medicinal Chemistry 58 (2012) 346e354
5.2.3. Hoechst staining
parameter in pmemd) [51]. The coordinates of the starting structure
were initially relaxed to remove bad close contacts over 1000
iterations. The temperature of the relaxed system was then equil-
ibrated at 300 K through 10 ps of MD using 2 fs time steps over
5000 iterations. Langevin dynamics [52] was used to equilibrate the
temperature of the system at 300 K by using a collision frequency of
10 psꢂ1 and a velocity limit of 10 temperature units. The final
coordinates of the temperature equilibration routine (after 10 ps)
were then used to run 10 ns molecular dynamics using 2 fs time
steps over 5 million iterations, during which the temperature was
kept at 300 K using the same Langevin dynamics parameters as
applied before. During the temperature equilibration and MD
routines, a non-bonded cutoff distance of 999 Å was applied by the
Particle Mesh Ewald method [53] to handle electrostatic interac-
tions in implicit solvent media and SHAKE method [54] was applied
to keep the bond lengths of protons attached to heteroatoms
constant. Coordinates and energy outputs for the relaxation and the
molecular dynamics routines were saved every 5000 iterations.
Human cancer cells (50.000 cells/well) were seeded into six-
well plates and 24 h later retinoid compounds were applied.
Hoechst staining was performed after 72 h of incubation with
retinoid compounds (IC50 concentration). Cells were incubated
with 1
mg/ml concentration of Hoechst 33258 (SigmaeAldrich,
861405) in 1ꢀ phosphate buffered saline (PBS) for 5 min at room
temperature at dark. Then, the cells were detained with ddH2O for
10 min and mounted onto slides. Apoptotic cells were visualized
under a fluorescent microscope (Zeiss, Axiovision Rel 4.6) at 40ꢀ
objective magnification.
5.2.4. Flow cytometry analysis
Cells were seeded at 5.105 cells per well onto 75 mm2 tissue
culture plates and incubated in humidified incubators at 37 ꢁC, with
5% CO2. The next day, cells were treated with two different
concentrations of 5a (1.8 mM and 3.6 mM). On day 2 and day 4,
1.106 cells were sampled and stained with FITC Annexin V
Apoptosis detection Kit (BD Pharmingen, Cat: 556570) according to
the manufacturer’s instructions. Control groups include corre-
sponding DMSO concentrations as negative controls and CPT
5.3.3. MMePBSA computations
MMePBSA (molecular mechanicsePoisson Boltzmann/surface
area) binding energy computations were conducted by the mme
pbsa [55] module of AMBER v11. For the formation of the
complex, as generically shown in EQ. (1), MMePBSA
(5 mM) and 1% v/v hydrogen peroxide, as positive controls. Stained
cells were kept from light on ice and analyzed immediately using
Becton Dickinson FACScalibur Flow Cytometer. Flow cytometry
results were analyzed using WinMDI 2.9 software (http://facs.
scripps.edu/software.html) for differentially stained percentage of
cells over controls and results were plotted and analyzed using
GraphPad Prism version 5.00 (GraphPad Software, San Diego Cal-
ifornia USA).
Receptor þ Ligand)/Complex
(1)
free energy computations were implemented for the complex
(RXR compound 5a), receptor (RXR and the ligand
a/g
.
a/g)
(compound 5a). The absolute free energy (G) of the complex
systems, their receptors, and the ligand were computed in a clas-
sical manner as in EQ. (2), in which T is the temperature of the
system at 300 K.
5.3. Computational studies
5.3.1. System set-up and initial structures
X-ray coordinates for human retinoid X receptor alpha (RXRa) in
complex with 4-[2-(1,1,3,3-tetramethyl-2,3-dihydro-1H-inden-5-
yl)-1,3-dioxolan-2-yl] benzoic acid and human retinoid X receptor
gamma (RXRg) in complex with 3-fluoro-4-[2-hydroxy-2-(5,5,8,8-
tetramethyl-5,6,7,8-tetrahydro-naphtalen-2-yl)-acetylamino] ben-
zoic acid were obtained from Protein Data Bank (PDB ID: 2ZXZ [43]
and 4LBD [44], respectively). All ligands and water molecules were
initially removed from the X-ray structures. Amino acid residues
G ¼ H ꢂ T$S
(2)
The binding free energies (
computed as in EQ. (3) where Gcomp is the absolute free energy of
the complex, Grec is the absolute free
D
G) of the complex systems were
h
i
D
G ¼ Gcomp ꢂ Grec þ Glig
(3)
245e261 in the X-Ray structure of RXR
a are not resolved. In
energy of the receptor, and Glig is the absolute free energy of the
ligand. 50 Snapshots were extracted for the coordinates of the
solute species (complex, receptor and ligand) at 20 ps time intervals
between 9 ns and 10 ns of the trajectories. MMePBSA energies
were computed for each snapshot and averaged out to constitute
mean binding free energies (
The enthalpy term in EQ. (2) is dissected into subenergy terms as
in EQ. (4).
general, the RXR and RXR proteins in the PDB files share 34.35%
a
g
amino acid sequence identity with very similar three dimensional
motifs. Therefore, amino acid backbone (NHeCHeCOe) coordi-
nates for the missing residues of RXR
acid residues 203e219 in the X-ray structure of RXR
acid residue names were mutated to comply with those of RXR
Compound 5a, shown in Scheme 1 was docked by Auto Dock
v4.2 [46] into the binding site of RXR and RXR using a flexible
a
were obtained from amino
g, whose amino
DG).
a
.
a
g
binding site and flexible ligand strategy applied by Auto Dock.
Molecular dynamics (MD) and molecular mechanics-Poisson
Boltzmann/surface area (MMePBSA) computations were imple-
mented by AMBER v11 (2010) suite of programs [47] running under
64 bit Scientific Linux at the TR-Grid e-Infrastructure of Turkey.
AMBER1999Sbildn [48] and general AMBER force fields (GAFF) [49]
Htot ¼ Hgas þ Gsolv
(4)
where Hgas is the potential energy of the solute in gas phase which
is determined as a sum of van der Waals (EVDW), electrostatic (Eel)
and internal (Eint) energies as in Cornell et al. (1995) force field [54].
Gsolv is the solvation free energy for transferring the solute from
vacuum into solvent and is a sum of electrostatic (Gel) and non-
electrostatic (hydrophobic) contributions (Gnonel) as seen in EQ. (5).
were used together to parameterize the RXR
a $ compound 5a and
RXR $ compound 5a complexes for implicit solvent simulations by
g
the LeaP [50] module of AMBER v11.
Gsolv ¼ Gel þ Gnonel
(5)
5.3.2. Molecular dynamics
Temperature equilibration and MD routines were conducted in
implicit solvent environment by the parallel pmemd (Particle Mesh
Ewald Molecular Dynamics) module of AMBER v10 [47] using
a generalized Born solvent model of Onufriev et al. (igb ¼ 5
Gel was computed at 0.15 M salt concentration by the pbsa
module of AMBER v11. [47] using Poisson Boltzmann equations
[55,56]. Gnonel was computed by the molsurf module of AMBER v11
[55]. The entropy energy term, S in EQ. (2), was computed for each

A.S. Gurkan-Alp et al. / European Journal of Medicinal Chemistry 58 (2012) 346e354
353
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(2007).
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nmode module of AMBER v11.
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Acknowledgment
We would like to thank Professor Hakan Goker and Dr. Mehmet
Alp from Ankara University, Central Instrumentation Laboratory of
Faculty of Pharmacy, for their support for the acquisition of the
instrumental analyses of this study. This study is partially sup-
ported by DPT KANILTEK Project.
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