Identification and characterization of novel indole based small molecules as anticancer agents through SIRT1 inhibition

Article abstract of DOI:10.1016/j.ejmech.2013.08.018

In our pursuit to develop new potential anticancer leads, we designed a combination of structural units of indole and substituted triazole; and a library of 1-{1-methyl-2-[4-phenyl-5-(propan-2-ylsulfanyl)-4H-1,2,4-triazol-3- yl]-1H-indol-3-yl}methanamine derivatives was synthesized and characterized. Cytotoxic evaluations of these molecules over a panel of three human cancer cell lines were carried out. Few molecules exhibited potent growth inhibitory action against the treated cancer cell lines at lower micro molar concentration. An in vitro assay investigation of these active compounds using recombinant human SIRT1 enzyme showed that one of the compounds (IT-14) inhibited the deacetylation activity of the enzyme. The in vivo study of IT-14 exemplified its promising action by reducing the prostate weight to the body weight ratio in prostate hyperplasia animal models. A remarkable decrease in the disruption of histoarchitecture of the prostate tissues isolated from IT-14 treated animal compared to that of the positive control was observed. The molecular interactions with SIRT1 enzyme were also supported by molecular docking simulations. Hence this compound can act as a lead molecule to treat prostatic hyperplasia.

Full text of DOI:10.1016/j.ejmech.2013.08.018

Accepted Manuscript
Identification and characterization of novel indole based small molecules as
anticancer agents through SIRT1 inhibition
Naveen Panathur, Udayakumar Dalimba, Pulla Venkat Koushik, Mallika Alvala,
Perumal Yogeeswari, Dharmarajan Sriram, Vijith Kumar
PII:
S0223-5234(13)00524-2
10.1016/j.ejmech.2013.08.018
EJMECH 6363
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 May 2013
Revised Date: 6 August 2013
Accepted Date: 8 August 2013
Please cite this article as: N. Panathur, U. Dalimba, P.V. Koushik, M. Alvala, P. Yogeeswari, D. Sriram,
V. Kumar, Identification and characterization of novel indole based small molecules as anticancer
agents through SIRT1 inhibition, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.08.018.
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ACCEPTED MANUSCRIPT
Identification and characterization of novel indole based small molecules as
anticancer agents through SIRT1 inhibition
a
a*
b
b
Naveen Panathur , Udayakumar Dalimba , Pulla Venkat Koushik , Mallika Alvala , Perumal
b
b
c
Yogeeswari , Dharmarajan Sriram , Vijith Kumar
This protocol depicts the cytotoxicity and growth inhibitory activity of some indole derivatives,
acting against benign prostatic hyperplasia through SIRT1 inhibition, in terms of in vitro and in
vivo models.

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Highlights
‹
‹
‹
‹
29 Molecules were screened for cytotoxicity evaluation.
In vivo investigations were carried out to examine the activity of the molecule.
Histopathology images and PCR proved the effectiveness of the molecule.
Simple synthetic protocols were used to get a promising lead molecule.

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Identification and characterization of novel indole based small molecules as
anticancer agents through SIRT1 inhibition
a
a,*
b
b
Naveen Panathur , Udayakumar Dalimba , Pulla Venkat Koushik , Mallika Alvala , Perumal
b
b
c
Yogeeswari , Dharmarajan Sriram , Vijith Kumar
a
Organic Chemistry Laboratory, Department of Chemistry, National Institute of Technology
Karnataka, Surathkal, Srinivasanagar, Mangalore-575025, India.
b
Medicinal Chemistry and Drug Discovery Research Laboratory, Pharmacy Group, Birla
Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar, Andhra Pradesh
–
500078, India.
c
Solid state and Structural Chemistry Unit, Indian Institute of Science, Bangalore - 560012,
India.
*
Corresponding author email: udayaravi80@gmail.com, udayakumar@nitk.ac.in.
Abstract
In our pursuit to develop new potential anticancer leads, we designed a combination of
structural units of indole and substituted triazole; and a library of 1-{1-methyl-2-[4-phenyl-5-
(propan-2-ylsulfanyl)-4H-1,2,4-triazol-3-yl]-1H-indol-3-yl}methanamine
derivatives
were
synthesized and characterized. Cytotoxic evaluations of these molecules over a panel of three
human cancer cell lines were carried out. Few molecules exhibited potent growth inhibitory
action against the treated cancer cell lines at lower micro molar concentration. An in vitro assay
investigation of these active compounds using recombinant human SIRT1 enzyme showed that
one of the compounds (IT-14) inhibited the deacetylation activity of the enzyme. The in vivo
study of IT-14 exemplified its promising action by reducing the prostate weight to the body
weight ratio in prostate hyperplasia animal models. A remarkable decrease in the disruption of
histoarchitecture of the prostate tissues isolated from IT-14 treated animal compared to that of
the positive control was observed. The molecular interactions with SIRT1 enzyme were also
supported by molecular docking simulations. Hence this compound can act as a lead molecule to
treat prostatic hyperplasia.
Key words: Indole, leukemia, breast cancer, prostate hyperplasia, human SIRT1.

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1
. Introduction
Cancer continues to be a major health problem all over the world. Apart from heart
disease, cancer turns out to be one of the major killing diseases due to various worldwide factors.
Numerous anticancer agents including taxol [1-3], vinblastine, vincristine, etoposide [4],
camptothecin and its derivatives [5], mitoxantrone [6], 5-fluorouracil [7], indomethacin [8],
cisplatin etc. are in clinical use throughout the world. However, these drugs resulted in adverse
side effects like low blood pressure, bone marrow suppression, gastrointestinal toxicity,
constipation, hair loss etc. Therefore, there is an urgent need for the scientific community to
explore new chemical entities for an effective and safe cure of cancer.
Indole nucleus is constantly drawing interest for the development of newer drug moiety
due to its wide range of pharmacological activities like antibacterial, antifungal [9], anti-malarial
[
[
10], anticonvulsant [11], anti-inflammatory [12], antivascular [13], ischemia/reperfusion injury
14], chronic diabetes [15], HIV inhibitors [16] and anticancer in particular. Since indole
derivatives have shown quite good response as anticancer agents, structural modifications of the
indole based pharmacophores, to scrutinize the pharmacological potency have been a core
interesting strategy among researchers. The 3-substituted indole forms to be a main structural
unit of many natural and pharmacologically active compounds, possessing various biological
activities [17]. For instance, the naturally occurring molecule indole-3-carbinol (I3C) has been
reported to exhibit promising anticancer activities against a number of human cancers acting
through diverse mechanisms [18]. Go et al. have demonstrated the greater antiproliferative
activity by structurally modifying cysmethynil by replacing the acetamide side chain with
tertiary amino groups to give analogues, which are potent in inhibiting isoprenylcysteine
carboxyl methyltransferase (Icmt) [17c]. In this direction, we were interested in the
amalgamation of substituted 1,2,4-triazole-3-thioether unit to the indole nucleus pertaining to the
anticancer properties of the former moiety through assorted mechanisms [19]. In view this, we
were motivated to study the cytotoxicity of compounds based on indole and substituted 1,2,4-
triazole units with the incorporation of different substituted methanamines at position-3 of the
indole nucleus.
+
Silent mating type information regulation 2 homolog 1 (SIRT1), a NAD -dependent
deacetylase enzyme, belonging to the family of class III histone deacetylase is involved in
diverse cellular processes and has recently been emerged as a novel therapeutic target for

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metabolic diseases [20]. Among the seven sirtuins, SIRT1 is the most extensively studied, and
numerous groups have shown that it is involved in gene silencing, genomic stability, stress
resistance, cell division and apoptosis. Over the past several years, many potent small molecules
have been reported as SIRT1 inhibitors viz. Cambinol [21], Sirtinol [20], Splitomicin [22],
,
Salermide [23] suramin [24] etc (Figure 1). Few indole based compounds (for example A and B
in Figure 1) have also been reported to exhibit superior selectivity towards SIRT-1 over other
+
deacetylases and NAD possessing enzymes [25]. Since SIRT1 enzymes are involved in various
physiological functions both in normal and diseased conditions, a strategy of designing small
molecules to act on SIRT1 has been emerging out as a promising practice among medicinal
chemists. Therefore in the present protocol, we present the synthesis of a library of novel indole
derivatives and exploration of their SIRT1 inhibitory activity with respect to in vitro and in vivo
models. The structural considerations of the designed molecules were further supported by the
homology modeling and the docking study, with SIRT1 being the target enzyme.
Figure 1. Potent SIRT1 inhibitors.
2
2
. Results and discussion
.1. Chemistry
Most of the target compounds were designed based on the assumptions of Lipinski rule to
fulfill the drug likeness properties of the molecules. The basic indole moiety was constructed by
straightforward and efficient three-step synthesis based on Fischer indole chemistry protocol
[
26a, 26b] as given in Scheme 1. The synthesis involved the polyphosphoric acid (PPA)
mediated cyclisation of the hydrazones (3), obtained by the condensation of substituted phenyl
hydrazine (2) and ethyl pyruvate. The triazole constituent was introduced by converting indole-
2
-carboxylate (4) into the hydrazide (6) followed by treating it with phenylisothiocyanate in
alcoholic medium to afford the corresponding thiosemicarbazide (7) and then refluxing with
aqueous KOH solution. The indole-triazole compound (9) was then formylated by the
conventional Vilsmeier-Haack method to obtain the pre final intermediate (10). Finally the target
compounds were synthesized by executing the reductive amination protocol between
intermediate (10) and different primary/secondary amines using sodium triacetoxyborohydride as
the reducing agent [27] (Scheme 2). Our preliminary interest was focused in carrying out a study
on the variation in cytotoxicity of the compounds by the assimilation of variety of

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aliphatic/aromatic, primary and secondary amines at position-3 and presence of fluoro at
position-5 of indole nucleus. Hence a library of 29 compounds were synthesized following the
above mentioned multi step synthetic protocol. The structural details of the synthesized
compounds are described in Table 1.
Scheme 1. Synthesis of the indole-triazole based scaffolds.
Scheme 2. Synthesis of the target molecules.
Table 1. Structural details of the compounds (the symbol ‘*’ denotes the point of attachment to
the position 3 of indole nucleus)
All the intermediate and final compounds were characterized using various spectroscopic
techniques. One of the final compounds (IT-17) was subjected to single crystal X-Ray diffraction
studies and the 3-dimentional structure of the molecule was confirmed. The ORTEP diagram of
IT-17 is shown in Figure 2.
Figure 2. The ORTEP diagram of the compound IT-17 with the ellipsoids drawn at 50 %
probability.
Crystal data: C25H28FN5OS, M =465.59, triclinic, a = 6.3811 (3), b = 13.0833 (9), c =
3
1
4.7067 (10) Å, α = 101.713 (6), β = 91.147 (5), γ = 93.891 (5), U = 1198.75 (13) Å , T
=
293 K, space group P-1, Z = 2, reflections measured = 4225, unique (Rint = 0.083)
which were used in all calculations.
2
.2. In vitro anticancer activity
The synthesized 29 compounds were tested for cytotoxicity in three different cell lines:
human chronic myeloid leukemia (K562), human metastatic breast cancer (MDA-MB 231) and
human prostate (LNCaP) cancer cells using MTT assay method. All the compounds were
screened at a concentration of 10 µM. Cytotoxic evaluation of the compounds demonstrated that
the molecules IT-16 and IT-17, which consists of 4-methyl piperidine and morpholine entities
respectively, to be potent enough to inhibit the cell growth to about 88 % against K562 cells.
However, the compounds IT-03, IT-04, IT-05 and IT-09 were found to be active against the
same cell lines (K562) with a cell growth inhibition to about more than 50 %. Compounds IT-04,
IT-05, IT-14, IT-15, IT-22, IT-23, IT-26, IT-30 and IT-31 were found to inhibit the cell growth

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to more than 50 % against MDA-MB 231 cells and IT-13, IT-14 and IT-31 showed their
inhibitory activity against LNCaP cell growth to more than 50% at the tested concentration.
Further study on non cancerous HEK293 cells, none of the compounds showed any significant
inhibition suggesting the specificity towards cancer cells. The proliferation assay study results
are presented in Figure 3. Among different amines introduced, 3,4,5-trimethoxy aniline,
cyclopropyl amine, cyclopropylmethyl amine, 4-methyl piperidine, 3-methyl piperidine, 4-
methoxy benzylamine, 3-methoxy benzylamine, morpholine and 2-methoxyethanamine were
found to have an impact in enhancing the activity of the molecules against the tested cell lines. It
is clear from the structural features and activity relation that presence of fluoro at position-5 of
indole moiety also plays a key role in enhancing the activity.
Figure 3. Cell proliferation assay results of the tested compounds at a concentration of 10 µM
against a) cancerous cells; K562, MDA-MB231 and LNCaP b) non cancerous HEK 293 cells.
It has been reported that SIRT1 inhibitors are best studied and used to control the benign
hyperplasia of prostate cancer [28]. Therefore compounds (IT-13, IT-14, IT-25 and IT-31)
showing good inhibitory activity on prostate cancer LNCaP cells were further investigated for in
vitro SIRT1 inhibition using SIRT1 Fluorimetric Drug Discovery Kit. SIRT1 fluorimetric
enzyme assay is based on unique SIRT1 substrate/developer combination. The substrate consists
of 4 amino acids from 379-382 [(Arg-His-Lys-Lys (Ac)] of human p53, which was tagged with
aminomethylcoumarin (AMC). The fluorescence signal is generated in proportional to the
amount of deacetylation of lysine in the substrate. Among the four tested compounds at a
concentration of 40 µM, IT-14 was found to exhibit close to 70 % enzyme inhibitory activity
(Figure 4). Hence we took forward this compound to study its in vivo effects on prostate
hyperplasia animal model.
Figure 4. SIRT1 inhibitory activity of the tested compounds at a concentration of 40 µM.
2
.3. In vivo studies
Male wistar rats weighing 180–220 g were used for in vivo investigations, and the
animals were divided into four groups. Group A served as negative control, Group B served as a
positive control (received only testosterone propionate at 3 mg/kg of body weight dose). Group
C and Group D received standard drug Finasteride (at a dosage of 5 mg/kg) and IT-14 (at a

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dosage of 10 mg/kg) respectively along with testosterone propionate (at a dosage of 3 mg/kg)
daily for 14 days to induce prostatic hyperplasia. Compound IT-14 was suspended in distilled
water by using 5% methyl cellulose and administered intraperitoneally. Testosterone propionate
was diluted with distilled water using Tween 80 as emulgent and injected subcutaneously.
th
Animals were weighed before and after completion of dosage. After dosage on 15 day, animals
were sacrificed by light ether anesthesia, later prostate tissue and seminal vesicles were removed
carefully and weighed immediately. Prostate weights to the body weight ratios were calculated.
Percentage of inhibition was calculated as per the equation given below.
%
I = 100 − [(treated group − negative control)/ (positive control − negative control) × 100]
The results and the percentage inhibition details of the tested molecule (IT- 14) on the rats are
tabulated in Table 2. A graphical representation of prostate gland weight and seminal vesicle
weight of the control and treated rats is shown in Figure 5.
Table 2. Prostate weight to body weight ratio of the rats after treatments.
It is quite clear from the data (Table 2) that there is a marked deviation in the
prostate weight to body weight ratio of Finasteride treated rat (group C) and the
compound IT-14 treated rat (group D) when compared with that of the testosterone treated
rat (group B). It is quite interesting to note that the ratio associated with group D is pretty
approaching that of the control (group A) and is quite comparable with that of Finasteride
treated group. These facts are also supported by the percentage inhibition calculation
where in a growth inhibition of about 56 % and 70 % were observed with respect to an
injection dose of Finasteride and IT-14 respectively. It can be seen that, the growth
inhibitory activity of IT-14 is comparable with that of the known drug Finasteride, which
implies IT-14 can be a lead molecule to act on prostatic hyperplasia.
Figure 5. Prostate gland weight and seminal vesicle weight of the control and treated rats.
2
.3.1. Histopathology of prostate
The isolated prostate tissues were then fixed in bouins solution with haematoxylin and
eosin stain and observed under light microscope. The histopathology images of the prostate
tissues associated with the untreated rat, testosterone treated rat, Finasteride treated rat and IT-14
treated rat were recorded and are shown in Figure 6. The histoarchitecture of the isolated prostate

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gland given in figure 6 (i) showed that the epithelium was cuboidal and regular in size. In the
prostate tissues isolated from positive control rat (group B), there was some disruption in the
histoarchitecture of the prostate tissue. Glandular hyperplasias with epithelial proliferation and
nuclear stratification have been observed, whereas the drug and the compound treated ones
(group C and group D respectively) showed significant reduction in histoarchitecture disruption
when compared to that of the positive control. It can be seen that the cell morphology of the
prostate tissues isolated from group C and group D is convincingly comparable with that of the
prostate tissues isolated from the negative control (group A). The retention in the cell
morphology confirms the inhibitory action of compound IT-14 on prostate hyperplasia.
Figure 6. Histopathological images of prostate tissues isolated from (i) group A, (ii) and (iii)
group B, (iv) group C and (v) group D.
2
.4. Reverse transcriptase PCR
In order to check the SIRT1 mRNA levels in treated and untreated prostate tissues with
respect to β-actin as equal loading control, semi quantitative RT-PCR was performed. Prostate
glands (50 mg) from treated and untreated animals were used for isolation of total mRNA with
TRI reagent (Sigma) according to manufacturer’s protocol. Reverse transcription of 5 µg of total
RNA was conducted with AMV reverse transcriptase enzyme (Sigma). PCR amplification was
o
carried out for 45 cycles, with each cycle consisting of denaturation for 15 sec at 95 C,
o
o
annealing for 1 min at 52-55 C, and an extension for 1 min at 72 C. The PCR products were
analyzed by 1.5 % agarose gel electrophoresis and relative quantification was done by using
Image lab analysis software. Primers used for PCR were: rat SIRT1 primer: sense 5′-
CAGAGCAT CACACGCAAGC-3’, antisense 5′-CAGGAAACAG AAACCCCAG C-3′; rat β-
actin
primer:
sense
5′-GAGAGGGAAATCGTGCGTGAC-3',
rat SIRT2 primer:
antisense
sense
5′-
5′-
TAGAGCCACCAATCCACACAGAG-3′;
AGCAAGGCACCACTAGCCACC-3′, antisense g5′-TGTTCCTCTTTCTCTTTGGTC-3′.
As shown in Figure 7, levels of SIRT1 are significantly decreasing in compound IT-14
treated animals when compared to testosterone induced animals. The mRNA levels of IT-14
treated group were almost similar to that of the negative control, which infers the activity of IT-
1
4 best suits in treating benign prostate hyperplasia conditions.

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Figure 7. Semi quantitative RT-PCR analysis of SIRT1, equal loading was confirmed by β-actin.
Relative levels of SIRT1 were calculated by Image analysis software.
3
. Molecular docking
The three dimensional model of hSIRT1 (uniprot code: Q96EB6, 244-498 amino acid
residues) was developed by threading method using PRIME homology modeling program
Schrödinger L.L.C., USA). The multi-step Schrödinger's Protein preparation tool (PPrep) has
(
been used for final preparation of receptor model. Hydrogen’s were added to the model
automatically via the Maestro interface. PPrep neutralizes side chains and residues which are not
involving in salt bridges. This step is then followed by restrained minimization using the OPLS
2
005 force field to RMSD of 0.3 Ǻ. This model has 92.9 % residues in most favored regions and
Prosa-Web Z score of -6.38. Active site pocket was identified by using SITEMAP, module of
Schrödinger. The synthesized compound IT-14 was sketched by using chemdraw and prepared
for docking using Ligprep, module of Schrödinger. GLIDE program was used for docking with
grid coordinates of X:-12.9111; Y:-27.7633; Z:-33.29672.
Molecular docking simulation study to understand the interaction of IT-14 with the
protein i.e. homology model of hSIRT1 (244-498 amino acid residues), indicated that Lys134
and Glu 167 involve in hydrogen bonding and strong π- π stacking with Phe 179 residues played
0
key role with the docking score of -4.334. Docking pose and interacting amino acids within 5 A
distances are shown in Figure 8.
Figure 8. a) Ligand interaction diagram of IT-14. b) Docking pose of IT-14 in the catalytic core
of hSIRT1. Pink dots denotes hydrogen bond and green dots denote hydrophobic
interactions.
4
. Conclusions
We have designed a set of indole based molecules and synthesized a library of indole
derivatives that showed potent growth inhibitory activity against three cancer cell lines. Six
molecules were found to be active against K562 cell lines with a cell growth inhibition to about
more than 50 %, among which two molecules (IT-16 and IT-17) were found to inhibit the cell
growth to about 88 %. Among the synthesized indole derivatives, nine molecules were found to
inhibit the cell growth to more than 50% against MDA-MB-231 cells and three molecules
showed inhibitory activity against LNCaP cell growth to more than 50 % at the tested

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concentration. Further none of the compounds were found to be toxic against noncancerous
HEK-293 cell line, signifying the specificity of the compounds towards cancer cells. In the
present study we screened a sequence of molecules for SIRT1 inhibitory activity. We found that
IT-14 prevents prostatic hyperplasia by inhibiting SIRT1. Compound IT-14 treatment
significantly reduced the development of testosterone induced hyperplasia in rats. This was
clearly evident with reduction of prostate weight, seminal vesicle weight and prostate weight to
body weight ratio. Treatment of compound IT-14 did not show significant change in the body
weight during the treatment, but the effect was significant on the prostate weight to body weight
ratio. Moreover semi quantitative RT-PCR revealed decreased transcript levels of SIRT1 in
prostate tissues treated with IT-14, indicating the activity of the molecule on SIRT1. Further the
experimental studies were well supported by the computer simulation study where the electronic
environment of the molecule was found to be quite favorable for the molecular interactions with
the amino acids constituting the receptor enzyme. Hence from these studies, we conclude that
IT-14 can be a good lead candidate to treat prostate hyperplasia.
5
5
. Experimental
.1. Materials and instruments
All chemicals and solvents were procured from Sigma Aldrich (Germany), Merck (India)
and Spectrochem Chemicals Pvt.Ltd. All the solvents were distilled and dried before usage. The
progress of the reactions was monitored by TLC using pre coated aluminum sheets with 60 F254
silica gel (Merck KGaA). Melting point of the synthesized compounds was recorded by a Stuart
1
SMP3 melting point apparatus. H NMR spectra of the intermediates and final compounds were
recorded using Bruker 300 MHz, 400 MHz and 500 MHz NMR spectrometers using TMS as
1
3
internal standard. C NMR spectra of the compounds were recorded using Bruker 400 MHz and
00 MHz NMR spectrometers. Elemental analysis was done using a Thermo electron
5
corporation EA-112 series C,H,N,S analyzer. LC-MS was recorded using an Agilent 1200 series
mass spectrometer and HPLC purity of the compound was recorded using a Schimadzu
Prominence HPLC instrument. Mass spectra were recorded using a Waters micro mass Q-Tof
micro spectrometer with an ESI source.

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5
.2. Animal studies
Male Wistar albino rats weighing (180–220 g) were procured from the Institutional
Animal Facility Centre. They were housed individually in clean and transparent polypropylene
cages maintained at room temperature with 12-h light/dark cycle and had free access to food and
water. After 3 days of acclimatization, they were randomly distributed into experimental groups.
All the experimental procedures were carried out in accordance with CPCSEA (Committee for
the purpose of control and supervision of experiments on animals) guidelines. The study was
reviewed and approved by the Institutional Animal Ethics Committee.
5
5
6
.3. Synthesis
.3.1. General procedure for the synthesis of indole-2-carbohydrazide intermediates (6a and
b):
The ethyl 1-methyl-1H-indole-2-carboxylate intermediate (5) was suspended in ethanol, to which
excess (about 2 equivalents) hydrazine hydrate was added portion wise. The reaction mass was
then refluxed for about 45 min. After the completion of reaction, the reaction mass was cooled
and the solid obtained was filtered, washed with cold ethanol and dried under vacuum to obtain
the hydrazide intermediates (6).
5
.3.1.1. 1-Methyl-1H-indole-2-carbohydrazide (6a): Intermediate 6a was prepared by following
the above mentioned procedure for compound 5a (9 g, 44.28 mmol) in ethanol (45 mL), to
1
obtain the corresponding hydrazide (6a) as white solid (7.8 g, 94.3 %); mp 160-161 °C; H
NMR (300 MHz, [D ]DMSO): δ = 9.75 (s, 1H, NHCO), 7.00-7.62 (m, 5Haromatic), 4.49 (s, 2H,
6
13
NH ), 3.98 (s, 3H, N-CH ); C NMR (100 MHz, [D ]DMSO): δ = 160.25, 148.67, 145.20,
2
3
6
1
37.99, 124.22, 123.12, 120.39, 119.65, 112.86, 38.66.
5
.3.1.2. 5-Fluoro-1-methyl-1H-indole-2-carbohydrazide (6b): Intermediate compound 6b was
prepared by following the above mentioned procedure for compound 5b (8 g, 44.28 mmol) in
1
ethanol (45 mL), to obtain the hydrazide (6b) as white solid (6.9 g, 92.1 %); mp 180-181 °C; H
NMR (300 MHz, [D ]DMSO): δ = 9.79 (s, 1H, NHCO), 6.92-7.65 (m, 4Haromatic), 4.48 (s, 2H,
6
13
NH ), 3.99 (s, 3H, N-CH ); C NMR (100 MHz, [D ]DMSO): δ = 162.56, 159.72, 144.11,
2
3
6
1
32.25, 122.79, 121.65, 120.09, 119.63, 118.63, 38.52.

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5
.3.2. General procedure for the synthesis of indole-2-thiosemicarbazide intermediate (7a and
b): A suspension of hydrazide (6) and phenylisothiocyanate in ethanol was heated under reflux
7
in an oil bath for 3 h. After the completion of the reaction, the solvent was removed under
reduced pressure and the residue thus obtained was taken in minimum quantity of ethanol, stirred
for 10 min and filtered. The compound was recrystallized from ethanol to afford the
phenylisothiosemicarbazide (7).
5
.3.2.1. 1-(1-Methyl-1H-indole-2-carbonyl)-4-phenylthiosemicarbazide (7a): Compound 7a was
prepared by following the above procedure for the intermediate 6a (7 g, 36.99 mmol) with
phenylisothiocyanate (5.50 g, 40.69 mmol) in ethanol (70 mL) to afford the
1
phenylisothiosemicarbazide (7a) as white crystalline solid (10.2 g, 85 %); mp 212-213 °C; H
NMR (400 MHz, [D ]DMSO) δ = 11.73 (s, 1H, CONH), 10.54 (s, 1H, NH-NH-CS), 9.87 (s, 1H,
6
1
3
CS-NH), 7.03-7.66 (m, 10Haromatic), 3.99 (s, 3H, N-CH ); C NMR (100 MHz, [D ]DMSO): δ =
3
6
1
1
62.92, 158.12, 139.76, 137.09, 130.18, 128.45, 127.41, 126.37, 125.50, 124.18, 122.17, 120.38,
12.79, 104.51, 31.33.
5
.3.2.2.
1-(5-Fluoro-1-methyl-1H-indole-2-carbonyl)-4-phenylthiosemicarbazide
(7b):
Compound 7b was prepared by following the above procedure for the intermediate 6b (6.5 g,
3
1.40 mmol) with phenylisothiocyanate (4.70 g, 34.54 mmol) in ethanol (65 mL) to afford the
1
phenylisothiosemicarbazide (7b) as white crystalline solid (9.2 g, 85.58 %); mp 180-181 °C; H
NMR (400 MHz, [D ]DMSO) δ = 10.56 (s, 1H, CONH), 9.87 (s, 1H, NH-NH-CS), 9.78 (s, 1H,
6
13
CS-NH), 7.14-7.62 (m, 9Haromatic), 4.00 (s, 3H, N-CH ); C NMR (100 MHz, [D ]DMSO): δ =
3
6
1
1
61.88, 159.04, 156.72, 139.69, 135.82, 132.09, 128.42, 126.40, 126.02, 125.49, 113.17, 112.47,
12.37, 106.58, 106.35, 105.89, 32.22.
5
.3.3. General procedure for the synthesis of 5-(1-methyl-1H-indol-2-yl)-4-phenyl-4H-1,2,4-
triazole-3-thiol derivatives (8a and 8b): A suspension of thiosemicarbazide (7) in aqueous
potassium hydroxide was refluxed for 2 h. Then the reaction mixture was cooled to room
temperature and was mixed with of water. The aqueous layer was washed with diethyl ether
(
2x100mL). Then the aqueous layer was cooled to 0 ºC, acidified to pH ≈ 4. The precipitate thus
obtained was filtered and washed thoroughly with water and dried to obtain the desired product
8).
(

ACCEPTED MANUSCRIPT
5
.3.3.1.
5-(1-Methyl-1H-indol-2-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol
(8a):
The
thiosemicarbazide compound 7a (11 g, 33.91 mmol) was treated with aqueous potassium
hydroxide (50 mL, 4 mmol) as per the above mentioned procedure to afford the intermediate
1
product 8a as white fluffy solid (8.4 g, 80.85 %); mp 238-239 °C; H NMR (400 MHz,
[
D ]DMSO) δ = 7.00-7.68 (m, 9Haromatic), 6.06 (s, 1H, indole-CH), 4.02 (s, 1H, SH), 3.87 (s, 3H,
6
1
3
-
NCH ); C NMR (100 MHz, [D ]DMSO): δ = 159.12, 157.26, 145.33, 134.96, 132.12, 130.11,
3 6
1
29.99, 129.14, 126.62, 126.44, 122.12, 119.86, 113.22, 113.18, 107.55, 105.86, 32.41.
5
.3.3.2. 5-(5-Fluoro-1-methyl-1H-indol-2-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol (8b): The
intermediate 8b was prepared by treating the thiosemicarbazide intermediate 7b (9 g, 27.74
mmol ) with aqueous potassium hydroxide (50 mL, 4 mmol) as per the above mentioned
1
procedure to obtain 8b as white solid (7.1 g, 83.53 %); mp 287-288 °C; H NMR (400 MHz,
[
D ]DMSO) δ = 7.08-7.56 (m, 8Haromatic), 6.07 (s, 1H, indole-CH), 4.03 (s, 1H, SH), 3.88 (s, 3H,
6
1
3
-
NCH ); C NMR (100 MHz, [D ]DMSO): δ = 168.88, 158.98, 156.66, 144.67, 134.93, 130.10,
3 6
1
29.81, 129.15, 126.50, 126.15, 118.26, 112.63, 112.40, 112.30, 106.08, 105.85, 32.41.
5
.3.4. General procedure for the synthesis of 1-methyl-2-[4-phenyl-5-(propan-2-ylsulfanyl)-4H-
1
,2,4-triazol-3-yl]-1H-indole intermediates (9a and 9b): To a solution of intermediate 8 in DMF,
calculated amount of K CO was added and stirred at RT for about 30 min. Excess
2
3
isopropylbromide (5 equivalents) was then charged in to the reaction mass and allowed to stir
overnight at RT. After the completion of the reaction, the reaction mass was quenched with ice
cold water and kept stirring for about an hour to precipitate the crude product. The solid mass
was then filtered, washed with water, dried and recrystalised from ethanol/water mixture (60 %)
to afford pure product (9).
5
.3.4.1. 2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indole (9a): The
intermediate 9a was prepared by carrying out the reaction for the compound 8a (8 g, 26.11
mmol) in DMF with K CO (9 g, 65.28 mmol) and isopropylbromide (16.06 g, 130.57 mmol)
2
3
according to the procedure mentioned above to obtain 9a as white crystalline solid (8.2 g, 90.11
1
%
); mp 177-178 °C; H NMR (400 MHz, CDCl ) δ = 7.03-7.55 (m, 9Haromatic), 5.94 (s, 1H,
3
1
3
indole-CH), 4.13 (s, 3H, N-CH ), 4.04 (m, 1H, S-CH), 1.47 (d, 6H, J = 6.8 Hz); C NMR (100
3
MHz, CDCl ): δ = 152.71, 148.88, 138.24, 134.29, 130.09, 129.93, 127.68, 126.75, 125.25,
3
1
23.33, 121.27, 120.06, 109.87, 104.81, 38.56, 32.25, 23.52.

ACCEPTED MANUSCRIPT
5
.3.4.2. 5-Fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indole (9b):
The intermediate 9b was prepared by carrying out the reaction for the compound 8b (7 g, 21.58
mmol) in DMF with K CO (7.45 g, 53.95 mmol) and isopropylbromide (13.27 g, 107.9 mmol)
2
3
according to the procedure mentioned above to obtain 9b as white crystalline solid (7.1 g, 89.76
1
%
); mp 192-193 °C; H NMR (400 MHz, CDCl ) δ = 6.97-7.55 (m, 8Haromatic), 5.88 (s, 1H,
3
1
3
indole-CH), 4.11 (s, 3H, N-CH ), 4.00-4.07 (m, 1H, S-CH), 1.47 (d, 6H, J = 6.4 Hz); C NMR
3
(
100 MHz, CDCl ): δ = 159.13, 156.78, 152.91, 148.57, 134.91, 134.14, 130.22, 120.00, 127.61,
3
1
26.82, 126.67, 112.11, 111.85, 110.72, 105.80, 104.46, 38.58, 32.50, 23.51.
5
.3.5. General procedure for the synthesis of 2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-
yl]-1-methyl-1H-indole-3-carbaldehydes (10a and 10b): The iminium cation was generated by
adding POCl to a round bottomed flask (RBF) containing DMF, at very slow rate maintaining
3
the internal temperature below 0 °C. After the formation of iminium cation, a solution of
intermediate (9) in DMF was introduced into the RBF and the mass was heated to 55 °C for 1 h.
Reaction was monitored using TLC and after the starting material was completely consumed, the
temperature was brought back to RT and 50 mL of ice cold water was added and kept stirring for
3
0 min. The precipitated solid was filtered, washed and dried to obtain the formylated product
(10).
5
.3.5.1.
2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indole-3-
carbaldehyde (10a): The intermediate compound 10a was synthesized by carrying out above
reaction for 9a (7 g, 20.08 mmol) with DMF (24.11 mmol) and POCl (24.11 mmol) to afford
3
1
the product as white solid (6.2 g, 82.01 %); mp 179-180 °C; H NMR (400 MHz, CDCl ) δ =
3
9
.81 (s, 1H, CHO), 8.27 (d, 1Haromatic), 7.11-7.39 (m, 8Haromatic), 4.09-4.16 (m, 1H, S-CH), 3.68
1
3
(
s, 3H, N-CH ), 1.51 (d, 6H, J = 6.8 Hz); C NMR (100 MHz, CDCl ): δ = 184.55, 154.07,
3
3
1
3
45.69, 137.80, 133.46, 132.81, 130.00, 126.29, 125.08, 124.85, 123.65, 122.39, 118.10, 110.14,
8.94, 31.48, 23.54.
5
.3.5.2.
5-Fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indole-3-
carbaldehyde (10b): The intermediate compound 10b was synthesized by carrying out above
reaction for 9b (7 g, 19.10 mmol) with DMF (22.92 mmol) and POCl (22.92 mmol) to afford
3
1
the product as white solid (6.6 g, 83.44 %); mp 165-166 °C; H NMR (400 MHz, CDCl ) δ =
3
9
.76 (s, 1H, CHO), 7.96 (d, 1Haromatic), 7.09-7.44 (m, 7Haromatic), 4.12-4.15 (m, 1H, S-CH), 3.68

ACCEPTED MANUSCRIPT
1
3
(
s, 3H, N-CH ), 1.52 (d, 6H, J = 6.4 Hz); C NMR (100 MHz, CDCl ): δ = 184.24, 161.31,
3
3
1
1
58.92, 154.23, 145.39, 134.46, 132.72, 130.17, 130.05, 126.22, 125.43, 117.93, 113.94, 111.20,
11.11, 107.83, 107.59, 38.96, 31.74, 23.52.
5
.3.6. General procedure for the synthesis of final molecules: The prefinal aldehyde compound
(10a – 10b) (100 mg) was dissolved in 1,2-dichloroethane (for reaction with primary amines) or
THF (for reaction with secondary amines), to which calculated quantity of amine was added
followed by a drop of glacial acetic acid. After stirring for about an hour, sodium
triacetoxyborohydride (1.5 equivalents) was added and the reaction mass was further stirred at
RT for 4 hr. After the completion of reaction, the reaction mass was washed with 10 % sodium
bicarbonate solution, the organic layer was then washed with water three times, dried over
Na SO and evaporated under vacuum. The crude product was purified using column
2
4
chromatography with dichloromethane/methanol as eluent to obtain the pure compounds.
.4. Characterization of the final molecules
The characterization data of the final compounds are listed below.
.4.1. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
5
5
yl}methyl}-3,4,5-trimethoxybenzenamine (IT-03).
1
H NMR (400 MHz, CDCl ) δ = 6.99-7.06 (m, 3H), 7.19-7.38 (m, 5H), 5.74 (s, 2H), 4.12 (s,
3
13
2
H), 4.05 (m, 1H), 3.72-3.80 (m, 9H), 3.55 (s, 3H), 1.48 (d, 6H, J = 6.8 Hz); C NMR (100
MHz, CDCl ): δ = 158.13, 155.78, 152.86, 146.34, 143.76, 132.21, 129.24, 128.79, 128.45,
3
1
5
6
25.76, 125.01, 123.84, 114.58, 111.45, 111.18, 109.82, 103.76, 103.53, 97.17, 89.63, 60.08,
+
5.22, 38.57, 37.76, 22.50; ESI-MS (m/z) 562.2 (M+H) ; Anal. calculated for C H FN O S; C,
3
0
32
5
3
4.15; H, 5.74; N, 12.47; O, 8.55; S, 5.71. Found: C, 64.28; H, 5.68; N, 12.42; O, 8.55; S, 5.68.
5
.4.2. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}cyclopropanamine (IT-04).
1
H NMR (300 MHz, CDCl ) δ = 6.99-7.43 (m, 8H), 4.06-4.15 (m, 1H), 3.400 (s, 3H), 2.12-2.16
3
1
3
(
m, 1H), 1.52 (d, 6H, J = 6.6 Hz), 0.60 (m, 2H), 0.51 (m, 2H); C NMR (100 MHz, CDCl ) δ =
3
1
1
6
58.99, 157.10, 152.95, 147.54, 134.29, 133.31, 129.78, 127.51, 126.07, 125.08, 116.41, 112.36,
10.61, 104.86, 43.16, 38.89, 31.33, 30.04, 23.51, 5.98; Anal. calculated for C H FN S; C,
2
4
26
5
6.18; H, 6.02; N, 16.08; S, 7.36. Found C, 66.16; H, 5.92; N, 15.97; S, 7.29.

ACCEPTED MANUSCRIPT
5
.4.3. Cyclopropyl-N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-
1
H-indol-3-yl}methyl}methanamine (IT-05).
1
H NMR (300 MHz, CDCl ) δ = 7.06-7.28 (m, 4H), 7.40-7.51 (m, 4H), 4.29 (s, 2H), 4.08-4.15
3
(m, 1H), 3.13 (s, 3H), 2.80 (d, 2H, J = 7.5 Hz), 1.54 (d, 6H, J = 6.6 Hz), 1.43-1.53 (m, 1H), 0.65-
1
3
0
1
4
.72 (m, 2H), 0.34-0.38 (m, 2H); C NMR (100 MHz, CDCl ) δ = 159.37, 157.48, 153.66,
3
47.38, 134.40, 133.12, 129.95, 127.51, 127.43, 126.26, 113.00, 110.99, 104.70, 58.53, 52.28,
+
+
3.50, 42.21, 38.99, 38.90, 31.67, 23.56; ESI-MS (m/z) 450.2 (M+H) , 472.2 (M+Na) ; Anal.
calculated for C H FN S; C, 66.79; H, 6.28; N, 15.58; S, 7.13. Found: C, 66.70; H, 6.21; N,
2
5
28
5
1
5.49; S, 7.18.
5
.4.4. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}-2-morpholinoethan-1-amine (IT-06).
1
H NMR (500 MHz, CDCl ) δ = 7.04-7.17 (m, 4H), 7.40-7.56 (m, 4H), 4.21 (s, 2H), 4.07 (m,
3
1
H), 3.70 (t, 4H, J = 4.5 Hz), 3.17 (s, 3H), 3.03 (t, 2H, J = 5.5 Hz), 2.74 (t, 2H, J = 5.5 Hz), 2.44
13
(
broad peak, 4H), 1.56 (d, 6H, J = 7 Hz); C NMR (100 MHz, CDCl ): δ = 158.72, 154.62,
3
1
6
6
47.11, 130.71, 129.02, 126.75, 126.29, 124.60, 122.18, 120.84, 115.12, 113.22, 111.31, 110.01,
7.13, 54.8, 54.4, 47.22, 42.50, 36.50, 23.82; Anal. calculated for C H FN OS; C, 63.75; H,
2
7
33
6
.54; N, 16.52; S, 6.30. Found: C, 63.65; H, 6.45; N, 16.43; S, 6.21.
5
.4.5. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}-3-morpholinopropan-1-amine (IT-07).
1
H NMR (500 MHz, CDCl ) δ = 7.06-7.18 (m, 4H), 7.42-7.63 (m, 4H), 4.25 (s, 2H), 4.07-4.11
3
(m, 1H), 3.54-3.57 (m, 4H), 3.14-3.19 (m, 5H), 2.53-2.57 (m, 6H), 2.03-2.07 (m, 2H), 1.52 (d,
1
3
6
1
5
6
H, J = 6.6 Hz); C NMR (125 MHz, CDCl ) δ 159.62, 157.72, 154.04, 146.72, 134.11, 132.97,
3
30.09, 127.28, 127.11, 125.81, 113.50, 113.28, 111.21, 110.37, 104.77, 104.57, 66.63, 57.45,
3.43, 47.32, 41.67, 39.00, 31.79, 23.50, 22.15; Anal. calculated for C H FN OS; C, 64.34; H,
2
8
35
6
.75; N, 16.08; O, 3.06; S, 6.13. Found: C, 64.45; H, 6.86; N, 16.20; O, 3.09; S, 6.18.
5
.4.6. N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}cyclopropanamine (IT-08).
1
H NMR (500 MHz, CDCl ) δ = 7.20-7.80 (m, 9H), 4.70 (s, 2H), 3.10-3.21 (m, 1H), 3.35 (s,
3
1
3
3
1
H), 2.35-2.55 (m, 1H), 1.80 (d, 4H), 1.55 (d, 6H); C NMR (125 MHz, CDCl ) δ = 158.21,
3
56.54, 152.99, 148.52, 136.31, 133.81, 130.09, 128.91, 125.27, 124.97, 115.71, 113.79, 110.52,

ACCEPTED MANUSCRIPT
1
1
04.25, 39.82, 36.52, 28.68, 22.50, 6.20 ; Anal. calculated for C H N S; C, 69.03; H, 6.52; N,
2
4
27
5
6.77; S, 7.68. Found: C, 69.11; H, 6.48; N, 16.69; S, 7.72.
5
.4.7. Cyclopropyl-N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-
3
-yl}methyl}methanamine (IT-09).
1
H NMR (500 MHz, CDCl ) δ = 7.16-7.79 (m, 9H), 4.43 (s, 2H), 4.07-4.16 (m, 1H), 3.08 (s,
3
3
H), 2.82 (d, 2H, J = 7.2 Hz), 1.53 (d, 6H, J = 6.9 Hz), 1.33-1.41 (m, 1H), 0.66-0.70 (m, 2H),
1
3
0
1
.34-0.39 (m, 2H); C NMR (125 MHz, CDCl ) δ = 154.34, 147.48, 137.52, 132.99, 130.14,
3
27.28, 125.92, 124.64, 121.84, 119.25, 110.25, 109.04, 50.52, 41.01, 39.06, 31.77, 23.53, 8.02;
+
+
ESI-MS (m/z) 432.2 (M+H) , 454.2 (M+Na) ; Anal. calculated for C H N S; C, 69.57; H,
2
5
29
5
6
.77; N, 16.23; S, 7.43. Found: C, 69.51; H, 6.70; N, 16.14; S, 7.65.
5
.4.8. N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-yl}methyl}-
2
-morpholinoethan-1-amine (IT-10).
1
H NMR (500 MHz, CDCl ) δ = 7.10-7.8 (m, 9H), 4.35 (s, 2H), 4.06-4.09 (m, 1H), 3.66 (t, 4H),
3
1
3
3
.14 (s, 3H), 3.10 (t, 2H, J = 5.5 Hz), 2.75 (t, 2H, J = 5.5 Hz), 2.40 (t, 4H), 1.55 (d, 6H); C
NMR (100 MHz, CDCl ) δ = 153.10, 146.03, 136.56, 132.10, 129.15, 129.02, 128.83, 126.02,
3
1
25.40, 124.93, 123.72, 122.76, 120.91, 118.53, 109.19, 108.09, 65.56, 52.47, 51.89, 44.51,
1.15, 40.30, 38.09, 30.75, 22.53; Anal. calculated for C H N OS;C, 66.09; H, 6.98; N, 17.13;
4
2
7
34
6
S, 6.54. Found: C, 66.01; H, 6.89; N, 17.18; S, 6.49.
5
.4.9. N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-yl}methyl}-
-morpholinopropan-1-amine (IT-11).
3
1
H NMR (500 MHz, CDCl ) δ = 7.13-7.45 (m, 8H), 7.92 (m, 1H), 4.28 (s, 2H), 4.06-4.09 (m,
3
1
H), 3.48-3.49 (m, 4H), 3.21 (s, 3H), 3.16 (t, 2H, J = 7 Hz), 2.48-2.53 (m, 6H), 1.99-2.01 (m,
1
3
2
H), 1.51 (d, 6H, J = 8.5 Hz); C NMR (100 MHz, CDCl ) δ 159.86, 156.98, 154.12, 146.77,
3
1
35.97, 131.82, 130.11, 127.35, 127.56, 124.22, 112.10, 112.99, 110.11, 110.84, 103.45, 102.87,
6.55, 53.06, 52.11, 46.32, 43.59, 36.58, 35.14, 28.29, 22.55; Anal. calculated for C H N OS;
6
2
8
36
6
C, 66.63; H, 7.19; N, 16.65; S, 6.35. Found: C, 66.59; H, 7.24; N, 16.71; S, 6.32.

ACCEPTED MANUSCRIPT
5
.4.10. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}-2-(pyrrolidin-1-yl)ethan-1-amine (IT-12).
1
H NMR (500 MHz, CDCl ) δ = 6.98-7.41 (m, 8H), 4.21 (s, 2H), 4.02-4.07 (m, 1H), 3.43 (s,
3
1
3
3
H), 2.98 (broad peak, 4H), 2.72-2.77 (m, 4H), 1.84-1.88 (m, 4H), 1.49 (d, 6H, J = 6.5 Hz); C
NMR (125 MHz, CDCl ) δ = 159.12, 156.77, 152.88, 147.49, 134.27, 133.21, 129.69, 127.12,
3
1
26.09, 125.04, 116.09, 112.33, 110.58, 104.94, 54.88, 53.99, 46.05, 43.39, 38.81, 31.22, 23.48;
Anal. calculated for C H FN S; C, 65.82; H, 6.75; N, 17.06; S, 6.51. Found: C, 65.79; H, 6.68;
2
7
33
6
N, 17.12; S, 6.48.
.4.11. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
5
yl}methyl}-2-methoxyethan-1-amine (IT-13).
1
H NMR (500 MHz, CDCl ) δ = 6.91-7.31 (m, 8H), 3.98-4.01 (m, 1H), 3.71 (s, 1H), 3.39 (t, 2H,
3
13
J = 6.5 Hz), 3.35 (s, 3H), 3.25 (s, 3H), 2.68 (t, 2H, J = 6.5 Hz), 1.41 (d, 6H, J = 8.5 Hz); C
NMR (100 MHz, CDCl ) δ = 158.44, 156.08, 152.27, 146.37, 133.24, 132.22, 128.84, 128.74,
3
1
25.03, 111.72, 111.46, 109.83, 109.73, 103.79, 103.55, 69.15, 57.93, 46.29, 41.90, 37.92, 30.54,
2.51; Anal. calculated for C H FN OS; C, 63.55; H, 6.22; N, 15.44; S, 7.07. Found: C, 63.45;
2
24
28
5
H, 6.18; N, 15.42; S, 7.15.
5
.4.12. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}(4-methoxyphenyl)methanamine (IT-14).
1
H NMR (300 MHz, CDCl ) δ = 6.80-7.55 (m, 12H), 4.35 (s, 2H), 4.21 (s, 2H), 4.01-4.10 (m,
3
1
3
1
1
1
H), 3.86 (s, 3H), 3.72 (s,3 H), 1.50 (d, 6H, J = 6.6 Hz); C NMR (100 MHz, CDCl ) δ =
3
59.90, 157.52, 153.86, 146.75, 134.16, 133.02, 131.69, 130.72, 129.98, 127.67, 125.81, 120.56,
19.14, 113.45, 111.11, 110.24, 104.93, 104.69, 67.08, 55.36, 46.55, 41.32, 39.12, 31.85, 23.00;
Anal. calculated for C H FN OS; C, 67.55; H, 5.86; F, 3.68; N, 13.58; O, 3.10; S, 6.22. Found:
2
9
30
5
C, 67.52; H, 5.80; N, 13.62; S, 6.18; LC-MS: Compound molecular weight = 515.64, Obtained
+
mass = 516.6 (M+H) ; HPLC Purity: > 98 %.
5
.4.13. N-{{5-fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}(3-methoxyphenyl)methanamine (IT-15).
1
H NMR (500 MHz, CDCl ) δ = 6.78-7.36 (m, 12H), 4.40 (s, 2H), 4.02-4.09 (m, 1H), 3.81 (s,
3
1
3
3
1
H), 3.79 (s, 2H), 3.46 (s, 3H), 1.48 (d, 6H, J = 8.5 Hz); C NMR (100 MHz, CDCl ) δ =
3
61.21, 158.57, 148.56, 142.97, 138.91, 133.38, 131.55, 130.62, 130.78, 129.11, 127.27, 126.94,

ACCEPTED MANUSCRIPT
1
23.36, 121.57, 120.44, 120.09, 117.55, 114.61, 114.08, 113.46, 110.69, 55.36, 54.84, 42.82,
8.22, 31.80, 23.51; Anal. calculated for C H FN OS; C, 67.55; H, 5.86; N, 13.58; S, 6.22.
3
2
9
30
5
Found: C, 67.57; H, 5.81; N, 13.51; S, 6.29.
5
.4.14. 2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-[(4-methylpiperidin-1-
yl)methyl]-1H-indole (IT-16).
1
H NMR (400 MHz, CDCl ) δ = 7.09-7.39 (m, 8H), 7.72-7.74 (m, 1H), 4.01-4.05 (m, 1H), 3.54
3
(s, 3H), 2.76-2.79 (m, 2H). 2.02-2.16 (m, 4H), 1.48-1.54 (m, 5H), 1.46 (d, 6H, J = 6.8 Hz), 0.86
1
3
(
d, 3H, J = 6.4 Hz); C NMR (100 MHz; CDCl ) δ =151.99, 148.06, 137.44, 133.47, 129.73,
3
1
29.56, 129.34, 127.40, 126.38, 123.79, 123.12, 120.46, 119.83, 109.71, 55.52, 53.57, 52.68,
8.78, 34.55, 31.06, 23.45; Anal. calculated for C H N S; C, 70.55; H, 7.24; N, 15.24; S, 6.98.
3
2
7
33
5
Found: C, 70.51; H, 7.28; N, 15.19; S, 7.02.
5
.4.15.
5-Fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-
(morpholinomethyl)-1H-indole (IT-17).
1
H NMR (500 MHz, [D ]DMSO) δ = 7.06-7.10 (m, 1H), 7.30-7033 (m, 2H), 7.42-7.49 (m, 5H),
6
3
.76-3.82 (m, 1H), 3.54 (s, 3H), 3043 (broad peak, 4H), 2.18 (broad peak, 4H), 1.36 (d, 6H, 5
1
3
Hz); C NMR (100 MHz; CDCl ) δ = 158.92, 156.53, 152.25, 147.70, 134.27, 133.43, 129.43,
3
1
27.38, 126.18, 125.07, 112.07, 111.80, 110.42, 110.33, 105.43, 67.04, 53.58, 53.37, 38.84,
+
+
3
1.14, 23.52; ESI-MS (m/z) 466.2 (M+H) , 488.2 (M+Na) ; Anal. calculated for C H FN OS;
25 28 5
C, 64.49; H, 6.06; N, 15.04; S, 6.89. Found: C, 64.46; H, 6.09; N, 15.08; S, 6.81.
5
.4.16. 2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-(morpholinomethyl)-1H-
indole (IT-18).
1
H NMR (500 MHz, CDCl ) δ = 7.09-7.35 (m, 8H), 7.72-7.74 (m, 1H), 4.02-4.09 (m, 1H), 3.51-
3
1
3
3
.62 (m, 7H), 3.38 (broad peak, 2H), 2.31 (broad peak, 4H), 1.47 (d, 6H, J = 6.8 Hz); C NMR
(
125 MHz, CDCl ) δ = 152.03, 148.01, 137.56, 133.51, 129.37, 127.21, 126.21, 123.55, 123.19,
3
1
20.42, 119.68, 114.79, 109.58, 66.99, 53.46, 53.14, 38.78, 31.87, 29.64, 23.46; Anal. calculated
for C H N OS; C, 67.08; H, 6.53; N, 15.65; S, 7.16. Found: C, 67.15; H, 6.49; N, 15.58; S,
2
5
29
5
7
.11.

ACCEPTED MANUSCRIPT
5
.4.17. 3-[(2,6-Dimethylmorpholino)methyl]-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-
yl]-1-methyl-1H-indole (IT-19).
1
H NMR (500 MHz, CDCl ) δ = 7.09-7.28 (m, 8H), 7.67-7.69 (m, 1H), 3.94-3.97 (m, 1H), 3.79-
3
3
.83 (m, 2H), 3.5 (s, 3H), 3.43 (s, 2H), 2.49-2.52 (m, 4H), 1.39 (d, 6H, J = 6.8 Hz), 1.04 (d, 3H,
1
3
J = 6.4 Hz), 0.99 (d, 3H, 6 Hz); C NMR (125 MHz, CDCl ) δ = 153.07, 148.22, 136.76,
3
1
7
6
35.24, 132.87, 130.99, 129.47, 128.98, 125.96, 123.21, 123.01, 121.47, 120.03, 115.19, 110.52,
0.22, 60.31, 49.19, 37.56, 32.01, 23.45, 20.85; Anal. calculated for C H N OS; C, 68.18; H,
2
7
33
5
.99; N, 14.72; S, 6.74. Found: C, 68.27; H, 6.91; N, 14.63; S, 6.76.
5
.4.18. N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}-2-(pyrrolidin-1-yl)ethan-1-amine (IT-20).
1
H NMR (500 MHz, CDCl ) δ = 7.05-7.31 (m, 8H), 7.63-7.65 (m, 1H), 3.96-3.99 (m, 1H), 3.75
3
(s, 2H), 3.35 (s, 3H), 2.73 (t, 2H, J = 6.8 Hz), 2.61 (t, 2H, J = 6.4 Hz), 2.52-2.55 (m, 4H), 1.69-
1
3
1
1
4
.73 (m, 4H), 1.41 (d, 6H, J = 6.8 Hz); C NMR (125 MHz, CDCl ) δ = 152.72, 147.74, 137.63,
3
33.31, 129.64, 129.48, 126.89, 126.14, 123.59, 120.12, 119.89, 116.24, 109.69, 54.87, 53.93,
+
6.44, 43.30, 38.78, 30.98, 29.63, 23.47; ESI-MS (m/z) 475.2 (M+H) ; Anal. calculated for
C H N S; C, 68.32; H, 7.22; N, 17.71; S, 6.76. Found: C, 68.38; H, 7.29; N, 17.69; S, 6.70.
27
34
6
5
.4.19. 2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-[(2-methylpiperidin-1-
yl)methyl]-1H-indole (IT-21).
1
H NMR (300 MHz, CDCl ) δ = 7.13-7.93 (m, 9H), 4.48 (s, 2H), 4.04-4.18 (m, 1H), 3.47 (s,
3
3
H), 3.18 (broad peak, 2H), 2.76 (broad peak, 1H), 1.52 (d, 6H, J = 6.6 Hz), 1.27-1.35 (m, 9H);
1
3
C NMR (100 MHz, CDCl ) δ = 151.92, 148.16, 137.38, 133.59, 129.46, 129.32, 127.78,
3
1
2
7
26.44, 123.67, 122.90, 120.76, 119.46, 117.23, 109.49, 56.87, 51.46, 47.70, 38.81, 34.74, 30.87,
5.99, 23.52; Anal. calculated for C H N S; C, 70.55; H, 7.24; N, 15.24; S, 6.98. Found C,
2
7
33
5
0.51; H, 7.29; N, 15.14; S, 7.06.
5
.4.20. 2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-[(3-methylpiperidin-1-
yl)methyl]-1H-indole (IT-22).
1
H NMR (400 MHz, CDCl ) δ = 7.14-7.36 (m, 8H), 7.75-7.77 (m, 1H), 4.04-4.07 (m, 1H), 3.55
3
(s, 3H), 2.86-2.94 (m, 2H), 2.03-2.17 (m, 4H), 1.61-1.83 (m, 5H), 1.48 (d, 6H, J = 6.8 Hz), 0.82
1
3
(
d, 3H, J = 8Hz); C NMR (100 MHz, CDCl ) δ = 149.25, 147.23, 138.58, 133.89, 130.17,
3
1
29.98, 129.01, 128.08, 127.86, 125.98, 123.94, 123.11, 121.09, 199.52, 118.03, 110.08, 56.28,

ACCEPTED MANUSCRIPT
5
7
4.64, 48.12, 37.51, 35.86, 34.09, 30.16, 23.55, 23.20, 15.91; Anal. calculated for C H N S; C,
2
7
33
5
0.55; H, 7.24; N, 15.24; S, 6.98. Found: C, 70.51; H, 7.28; N, 15.32; S, 6.89.
5
.4.21. N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}(3-methoxyphenyl)methanamine (IT-23).
1
H NMR (300 MHz, CDCl ) δ = 6.82-7.38 (m, 12H), 7.69-7.72 (m, 1H), 4.06-4.13 (m, 1H), 3.91
3
1
3
(
s, 2H), 3.85 (s, 2H), 3.84 (s, 3H), 3.43 (s, 3H), 1.51 (d, 6H, J = 6.6 Hz); C NMR (100 MHz,
CDCl ) δ = 159.69, 152.57, 147.84, 140.95, 137.72, 133.38, 129.71, 129.56, 129.39, 129.26,
3
1
5
27.02, 126.13, 123.45, 120.57, 120.48, 120.02, 116.55, 113.43, 113.38, 113.00, 109.69, 55.194,
2.73, 43.68, 38.79, 31.88, 29.65, 23.50.
5
.4.22.
2-[5-(Isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-{[4-(pyrrolidin-1-
yl)piperidin-1-yl]methyl}-1H-indole (IT-24).
1
H NMR (400 MHz, CDCl ) δ = 7.01-7.71 (m, 9H), 3.91-3.94 (m, 1H), 3.44 (s, 3H), 2.69 (broad
3
peak, 2H), 2.49 (broad peak, 4H), 2.09 (m, 1H), 1.61-1.82 (m, 4H), 1.44 (d, 6H, J = 6.4
1
3
Hz).1.01-1.39 (m, 8H); C NMR (100 MHz, CDCl ) δ = 153.02, 144.65, 136.76, 132.41,
3
1
3
7
31.77, 129.03, 128.96, 125.26, 124.04, 123.81, 122.61, 121.36, 117.06, 109.09, 37.91, 37.70,
0.46, 28.71, 28.68, 28.64, 22.51, 21.67, 13.10; Anal. calculated for C H N S; C, 70.00; H,
3
0
38
6
.44; N, 16.33; S, 6.23. Found: C, 70.11; H, 7.39; N, 16.29; S, 6.21.
5
.4.23. N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}-2-methoxyethan-1-amine (IT-25).
1
H NMR (300 MHz, CDCl ) δ = 7.16-7.77 (0M, 9H), 4.23 (s, 2H), 4.05-4.16 (m, 1H), 3.64 (t,
3
1
3
2
H, J = 4.5 Hz), 3.38 (s, 3H), 3.23 (s, 3H), 3.01 (t, 2H, J = 4.5 Hz), 1.51 (d, 6H, J = 6.9 Hz); C
NMR (100 MHz, CDCl ) δ = 153.15, 152.29, 147.79, 137.59, 133.34, 129.79, 127.35, 127.15,
3
1
26.26, 126.06, 124.54, 123.66, 121.27, 120.91, 119.59, 109.85, 70.96, 58.87, 47.07, 42.68,
8.88, 31.27, 23.49; Anal. calculated for C H N OS; C, 66.18; H, 6.71; N, 16.08; S, 7.36.
3
2
4
29
5
Found: C, 66.22; H, 6.78; N, 16.18; S, 7.45.
5
.4.24.
N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}(4-methoxyphenyl)methanamine (IT-26).
1
H NMR (300 MHz, CDCl ) δ = 6.3-7.35 (m, 12H), 7.84-7.87 (m, 1H), 3.98-4.08 (m, 1H), 3.83
3
1
3
(
s, 2H), 3.68 (s, 3H), 3.32 (s, 3H), 3.09 (s, 2H), 1.49 (d, 6H, J = 6.6 Hz); C NMR (100 MHz,

ACCEPTED MANUSCRIPT
CDCl ) δ = 158.58, 150.12, 149.92, 146.71, 135.18, 134.45, 132.99, 131.08, 130.11, 128.17,
3
1
21.68, 119.08, 113.68, 112.89, 110.54, 104.69, 63.13, 56.85, 43.65, 38.14, 32.66, 23.28; Anal.
calculated for C H N OS; C, 69.99; H, 6.28; N, 14.07; S, 6.44. Found: C, 69.91; H, 6.39; N,
2
9
31
5
1
4.14; S, 6.34.
5
.4.25.
N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}-3,4,5-trimethoxybenzenamine (IT-27).
1
H NMR (300 MHz, CDCl ) δ = 7.10-7.42 (m, 9 H), 5.78 (s, 2H), 4.18 (s, 2H), 4.01-4.14 (m,
3
1
3
1
1
1
H), 3.74-3.82 (m, 9H), 3.52 (s, 3H), 1.47 (d, 6H, J = 6.8 Hz); C NMR (100 MHz, CDCl ) δ =
3
53.88, 152.86, 147.66, 144.95, 137.79, 133.32, 130.10, 129.60, 129.39, 126.56, 126.34, 123.65,
23.37, 120.14, 119.84, 115.76, 109.94, 92.70, 90.52, 61.11, 55.96, 39.57, 38.76, 31.07, 23.53;
+
+
ESI-MS (m/z) 544.2 (M+H) , 566.2 (M+Na) ; Anal. calculated for C H N O S; C, 66.27; H,
3
0
33
5
3
6
.12; N, 12.88; S, 5.90. Found: C, 66.15; H, 6.18; N, 12.94; S, 5.81.
5
.4.26. 5-Fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-[(3-
methylpiperidin-1-yl)methyl]-1H-indole (IT-30).
1
H NMR (500 MHz, CDCl ) δ 7.00-7.46 (m, 8H), 4.03-4.09 (m, 1H), 3.56 (s, 3H), 3.32 (s, 2H),
3
1
3
2
.65 (broad peak, 4H), 1.55-1.65 (m, 5H), 1.48 (d, 6H, J = 7 Hz), 0.80 (d, 3H, J = 7 Hz); C
NMR (100 MHz, CDCl ) δ = 161.08, 156.58, 152.16, 147.83, 134.20, 133.49, 129.43, 129.36,
3
1
3
1
27.64, 126.28, 111.93, 111.67, 110.28, 110.19, 105.60, 105.37, 61.66, 53.75, 53.23, 38.86,
2.85, 31.12, 29.71, 25.32, 23.48, 19.66; Anal. calculated for C H FN S; C, 67.89; H, 6.75; N,
2
7
32
5
4.66; S, 6.71. Found: C, 67.92; H, 6.65; N, 14.72; S, 6.63.
.4.27. 5-Fluoro-2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-3-[(4-
5
methylpiperidin-1-yl)methyl]-1H-indole (IT-31).
1
H NMR (400 MHz. CDCl ) δ = 6.97-7.41 (m, 8H), 4.00-4.07 (m, 1H), 3.53 (s, 3H), 3.35 (broad
3
peak, 2H), 2.69-2.72 (m, 2H), 2.17 (s, 2H), 1.46-1.53 (m, 9H), 1.26 (broad peak, 2H), 0.86 (d,
1
3
3
1
3
1
H, J = 8 Hz); C NMR (100 MHz, CDCl ) δ = 158.92, 156.58, 152.13, 147.86, 134.17, 133.48,
3
29.42, 127.62, 127.52, 126.28, 111.93, 111.66, 110.31, 110.21, 105.49, 105.25, 53.73, 53.03,
8.85, 34.23, 31.11, 30.66, 23.52, 21.83; Anal. calculated for C H FN S C, 67.89; H, 6.75;
2
7
32
5
4.66; S, 6.71. Found: C, 67.95; H, 6.71; 14.55; S, 6.76.

ACCEPTED MANUSCRIPT
5
.4.28.
N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-
yl}methyl}-1H-benzo[d]imidazol-2-amine (IT-35).
1
H NMR (500 MHz, CDCl ) δ = 7.18-7.78 (m, 13 H), 4.68 (s, 2H), 4.05-4.14 (m, 1H), 3.37 (s,
3
1
3
3
1
1
1
H), 1.51 (d, 6H, J = 8 Hz); C NMR (100 MHz, CDCl ) δ = 152.12, 147.56, 141.52, 139.68,
3
38.14, 130.35, 129.93, 127.96, 126.06, 124,38, 124.01, 122.22, 121.59, 120.85, 119.09, 115.37,
13.16, 112.55, 38.56, 35.19, 30.46, 23.59; Anal. calculated for C H N S; C, 68.13; H, 5.51; N,
2
8
27
7
9.86; S, 6.50. Found: C, 68.06; H, 5.59; N, 19.78; S, 6.57.
5
.4.29. N-{{2-[5-(isopropylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]-1-methyl-1H-indol-3-yl}methyl}
pyrazin-2-amine (IT-36).
1
H NMR (400 MHz, CDCl ) δ = 7.14-7.38 (m, 9H), 7.72-7.95 (m, 3H), 4.65 (s, 2H), 4.03-4.10
3
1
3
(
m, 1H), 3.36 (s, 3H), 1.48 (d, 6H, J = 6.8 Hz); C NMR (100 MHz, CDCl ) δ = 154.55, 152.84,
3
1
1
47.81, 141.87, 137.74, 134.00, 133.33, 132.63, 129.70, 129.54, 126.36, 126.18, 123.59, 123.57,
+
20.26, 119.84, 119.26, 109.71, 55.51, 38.79, 31.02, 23.46; ESI-MS (m/z) 456.2 (M+H) ; Anal.
calculated for C H N S; C, 65.91; H, 5.53; N, 21.52; S, 7.04. Found: C, 65.98; H, 5.43; N,
2
5
25
7
2
1.63; S, 6.96.
Acknowledgments
N.P. thank NITK, Surathkal for the research fellowship. We thank Prof. T. N. Guru Row
and single crystal X-ray diffraction unit, SSCU, IISc Bangalore for the single crystal X-ray
analysis. We acknowledge the NMR research centre, IISc, Bangalore, IIT Madras and CDRI
Lucknow for providing the NMR characterization facility. We thank SAIF Punjab, Chandigarh
for providing the Mass spectrometer facility.
The CCDC reference for the crystal structure (IT-17) is 913951.
References
[
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[
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List of contents
Table 1. Structural details of the compounds (the symbol ‘*’ denotes the point of attachment to
the position 3 of indole nucleus).
Table 2. Prostate weight to body weight ratio of the rats after treatments.
Figure 1. Potent SIRT1 inhibitors.
Figure 2. The ORTEP diagram of the compound IT-17 with the ellipsoids drawn at 50 %
probability.
Figure 3. Cell proliferation assay results of the tested compounds at a concentration of 10 µM
against a) cancerous cells; K562, MDA-MB231 and LNCaP, b) non cancerous HEK 293 cells.
Figure 4. SIRT1 inhibitory activity of the tested compounds at a concentration of 40 µM.
Figure 5. Prostate gland weight and seminal vesicle weight of the control and treated rats.
Figure 6. Histopathological images of prostate tissues isolated from (i) group A, (ii) and (iii)
group B, (iv) group C and (v) group D.
Figure 7. Semi quantitative RT-PCR analysis of SIRT1, equal loading was confirmed by β-actin.
Relative levels of SIRT1 were calculated by Image analysis software.
Figure 8. a) Ligand interaction diagram of IT-14. b) Docking pose of IT-14 in the catalytic core
of hSIRT1. Pink dots denotes hydrogen bond and green dots denote hydrophobic interactions.
Scheme 1. Synthesis of the indole-triazole based scaffolds.
Scheme 2. Synthesis of the target molecules.

ACCEPTED MANUSCRIPT
Table 1. Structural details of the compounds (the symbol ‘*’ denotes the point of attachment to
the position 3 of indole nucleus)
Entry Compound
code
R1
R2
Entry Compound
code
R1
R2
O
O
O
∗
∗
N
1
3
IT-03
IT-05
F
F
2
4
IT-04
IT-06
F
F
N
H
H
O
H
N
N
∗
HN
∗
O
N
∗
N
5
IT-07
IT-09
F
6
8
IT-08
IT-10
H
H
NH
H
∗
O
H
N
N
∗
7
9
H
HN
∗
O
∗
N
N
H
N
IT-11
H
10
IT-12
F
NH
∗
H
O
N
∗
∗
O
1
1
3
IT-13
IT-15
F
F
12
14
IT-14
IT-16
F
NH
∗
O
N
1
H
H
N
∗