Scaffold-hopping and hybridization based design and building block strategic synthesis of pyridine-annulated purines: Discovery of novel apoptotic anticancer agents

Article abstract of DOI:10.1039/c5ra00052a

A set of novel pyridine-annulated analogs of purinones, adenines and their oxo/thio congeners, xanthines, guanines, and purine-2,4-diamines as potential anticancer agents was considered based on the scaffold-hopping and hybridization of known anticancer a

Full text of DOI:10.1039/c5ra00052a

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Scaffold-hopping and hybridization based design
and building block strategic synthesis of pyridine-
annulated purines: discovery of novel apoptotic
anticancer agents†
Cite this: RSC Adv., 2015, 5, 26051
a
b
b
a
*
Vikas Chaudhary, Sarita Das, Anmada Nayak, Sankar K. Guchhait
and Chanakya N. Kundub
A set of novel pyridine-annulated analogs of purinones, adenines and their oxo/thio congeners, xanthines,
guanines, and purine-2,4-diamines as potential anticancer agents was considered based on the scaffold-
hopping and hybridization of known anticancer agents/drugs, purine derivatives and our recently
developed imidazo-pyridine derivatives. Towards the synthesis of these compounds, a new approach
involving a convenient preparation of 3-amino-2-carboxyethyl substituted imidazo[1,2-a]-pyridine and its
use as a building block for the construction of fused rings was developed. The approach enabled the
preparation of a number of compounds with relevant substitutions for each class. Several of pyridine-
annulated adenine and its oxo/thio analogs, xanthine and purine-2,4-diamine were found to possess
significant anticancer activities in kidney cancer cells and relatively less cytotoxicity to normal cells. They
were relatively more active than the anticancer drugs etoposide and doxorubicin. A representative
pyridine-annulated adenine derivative compound(22) was found to exert significant apoptosis.
Received 2nd January 2015
Accepted 3rd March 2015
DOI: 10.1039/c5ra00052a
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important approach is the molecular hybridization of chemo-
types of known drugs and therapeutic agents.4 A well-known
Introduction
example is azatoxin, which was derived by the structural
hybridization of the topoisomerase II-targeting drugs etoposide
and ellipticine.5 Azatoxin with relevant substitutions showed
better topoisomerase II inhibitory activity than etoposide or
ellipticine. Therefore, an amalgamation of template-hopping
and molecular hybridization on drugs and known bioactive
agents can be an important design approach towards the
discovery of new chemical entities (NCEs).
The purine class of compounds is known for its wide range
of anticancer activities. These compounds interfere with various
biochemical pathways such as the inhibition of CDK,6 PDE7 and
Hsp90,8 and disruption9 of microtubule dynamics. Various
marketed drugs10 also possess a purine nucleus (Fig. 1). QAP 1,
an adenine derivative that shows ATP-competitive catalytic
inhibition of topoisomerase II, is on clinical trial.11 In 2014, the
US FDA approved a drug called idelalisib, an adenine derivative,
for the treatment of various leukemia and lymphoma.12 For
some specic pharmacological activities, purine derivatives
have been found to be less potent, while their analogs with an
annulated ring have been found to possess enhanced pharma-
cological activities and better pharmacokinetic proles.13 These
features incited medicinal and organic chemists to generate a
biologically important class of compounds: heterocyclic-
condensed purines. These compounds have been shown to
possess various bioactivities14 such as, antihypertension,15 anti-
Cancer is the second major cause of death worldwide aer
cardiovascular diseases.1 Towards the development of new
chemical entities (NCEs), various approaches are followed in
medicinal chemistry research. Among them, scaffold/template
hopping2 has been recognized as
a valuable approach.
Scaffold-hopping offers an opportunity to explore new chemo-
types that can possess potentially similar biological activities
and are outside the coverage of existing patents. Several mar-
keted drugs and clinical trial agents have been discovered using
this strategy. For example, the drug vardenal, a PDE5-
inhibitor, was discovered from the heterocyclic scaffold-
hopping of another drug called sildenal, likewise, the
cyclooxygenase-inhibiting anti-inammatory drugs etoricoxib
and valdecoxib were discovered from celecoxib. A selective DPP-
4 inhibitor called imigliptin was developed by a scaffold
hopping-based design on alogliptin and a structure–activity
guided optimization is currently under clinical trial.3 Another
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education
and Research, S. A. S. Nagar, Sector-67, Mohali, Punjab, 160062, India. E-mail:
skguchhait@niper.ac.in; Fax: +91 172 2214692
bSchool of Biotechnology, Kalinga Institute of Industrial Technology (KIIT) University,
Campus-11, Patia, Bhubaneswar, Odisha 751024, India
† Electronic supplementary information (ESI) available: General information,
spectral data, and NMR spectra (1H and 13C) of compounds. See DOI:
10.1039/c5ra00052a
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Several of these new compounds were found to show signicant
anticancer activities, lower cytotoxicities to normal cells, and
apoptosis in cancer cells.
Results and discussion
Chemistry
The known methods for the preparation of heterocyclic-
annulated purine derivatives involve mostly the construction
of new annulated rings on purines.13a,c,d,15,19 Following these
methods, the preparation of pyridine-condensed purinones,
adenines, xanthines, and purine-2,4-diamines with a variety of
relevant substitutions would require, for each compound, a
separate reaction route involving the construction of the
annulated pyridine ring on the purine motif. In addition, the
construction of the annulated pyridine ring is difficult. We
considered a building block synthetic strategy via a suitably
functionalized compound that could enable the construction of
a fused pyrimidine ring and follow up derivatization towards
the preparation of the whole series of designed compounds
(Scheme 1a and b and 2). In this direction, we recently devel-
oped a method for the preparation of 3-amino-2-carboxyethyl
substituted imidazo[1,2-a]-pyridine (I) in one step and
explored its use as a building block in the synthesis of pyridine-
fused purinones and adenines (Scheme 1a and b).20 In the
present work, the synthesis of pyridine-annulated xanthines,
guanines and purine-2,4-diamines was explored using the
building block strategy. Some relevant pyridine-fused adenines
(ten compounds) were prepared.
The development of a method towards the preparation of
pyridine-annulated xanthines was initially investigated. The
preparation of a fused pyrimidine-dione ring on 2-amino-
benzenecarboxamide by its reaction with urea at 190 ꢀC is
known.21 Following this method, the reaction of the amide
derivative (II) of the building block (I) with urea was performed.
However, the reaction did not proceed. Variation of the condi-
tions including the addition of acid/base promoters also did not
help. These attempts reveal the poor amine-nucleophilicity of
the C2-carboxyethyl substituted imidazopyridinyl-3-amine
towards urea. A set of reactions on the amide derivative (II)
was then performed using various reactants such as di-ethyl/
methyl carbonate, ethyl chloroformate, and Boc-anhydride,
which could promote the domino conversions of the C3-
amine to carbamate and the follow up trans-amidation.
However, none of these reactions provided the construction of
the fused pyrimidine-dione. In each case, either the reaction did
not proceed or a mixture of non-isolable products was obtained
on prolonging the reaction or enhancing the reaction temper-
ature. The mass spectroscopic studies of the crude mixture
obtained under higher temperature indicated the formation of
the desired product as well as the unwanted product generated
from the intermolecular trans-amidation of the derivative II.
Therefore, this domino reaction is associated with a chemo-
selectivity issue also. The construction of the fused pyrimidine-
dione ring directly from the building block (I) by reaction with
urea, ethyl carbamate or isocyanate was then investigated.
Gratifyingly, the reaction of the building block with isocyanate
Fig. 1 Pharmacological activities of purine-based compounds and
drugs.
inammatory,13a human A3 adenosine receptor antagonism,13c
and inhibition of PDE1/5 (ref. 16) and tyrosine kinase EphB4.17
Recently, we developed imidazo-pyridines, designed based
on the scaffold-hopping of existing drugs and known agents, as
novel anticancer agents that induced apoptosis in the G1/S
phase.18a As part of our program aimed at anticancer drug
discovery,18 employing the amalgamation of scaffold-hopping
and molecular hybridization design approaches on these
structural classes of anticancer agents/drugs, purines (Fig. 1),
annulated purines and imidazo-pyridines, we considered a
series of novel pyridine-annulated purine compounds as
potential anticancer agents (Fig. 2).
Accordingly, studies for the establishment of a diversity-
feasible building block approach for the synthesis of pyridine-
annulated analogs of purinone, adenine and its oxo/thio
congener, xanthine, purine-2,4-diamine and guanine deriva-
tives, preparation of relevant substituted compounds, and
evaluation of their anticancer activities were undertaken.
Fig. 2 Design of target compounds for potential anticancer activity.
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Scheme 1 (a) Synthesis of pyridine-condensed purinones (1–7).20 (b) Synthesis of pyridine-condensed adenines and their oxo/thio analogs (9–
32).20
was found to be promising. In the reaction, bases were found to also suitable for the construction of thioxanthine (42) by reac-
be more efficient as promoters than acids. In the evaluation of tion of the building block with isothiocyanate.
various bases, to our delight, the NaOEt-mediated reaction
Towards the synthesis of pyridine-condensed guanines
under microwave irradiation was found to be the most effective starting from the building block, condensed thioxanthine (42)
and afforded the pyridine-annulated xanthine in good yield was considered as a suitable substrate. The thio-methylation
(Scheme 2). This developed method enabled the preparation of and SNAr with amines produced pyridine-enlarged guanines
various pyridine-annulated xanthines (33–41). The protocol was (47–49).
Scheme 2 Synthesis of pyridine-condensed xanthines (33–41), thioxanthine (42), purin-2,4-diamines (43–46) and guanines (47–49).
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Purine-2,6-diamines are well known for their inhibitory apoptotic cells (Q3) were observed for cells treated with 2.5 mM
activities against various kinases.22 This incited us to also of the compound and more than 54 percent necrotic cells (Q4)
investigate the use of the building block in the synthesis of were observed for cells treated with 4 mM of the compound.
pyridine-enlarged purines with 2,4-differential amine substitu- Thus, the result indicated that cells were moving towards the
tions. In this aspect, we were required to obtain the chemo- necrotic phase followed by a true apoptotic phase with
selective differential amination of the dichloro-derivative of the increasing concentration of compound 22 (Fig. 6).
purine-annulated xanthine (VI), prepared by deoxychlorination
Expressions of CASPASE 3. Increased expression of CASPASE
of the pyridine-condensed xanthine (V). Compared to the 2-Cl 3, an important marker, is an indication of apoptosis. To check
group, the 4-Cl functionality was found to be more reactive for whether exposure to compound 22 caused the apoptosis in the
SNAr and underwent rapid reaction at room temperature, HEK 293T cells, we measured the CASPASE 3 expression by
whereas the reaction of the 2-Cl with amine required microwave immunostaining CASPASE 3 aer exposure to compound 22.
irradiation at a higher temperature and neat conditions. This HEK 293T cells were treated with compound 22 of increasing
differential reactivity of the 4-Cl vs. 2-Cl group enabled the concentrations and probed with the CASPASE-3 antibody (cat
preparation of the desired annulated purine-2,6-diamines (43– #9662 from Cell Signaling, MA, USA). They were re-probed with
46) in moderate to good yields (Scheme 2). Using the developed secondary anti-rabbit conjugated to TRITC (tetramethylrhod-
building block synthetic strategy, we prepared new pyridine amine, a red uorescent dye) followed by counterstaining with
ring-condensed analogs of all known classes of purine scaffolds, DAPI and visualized under
a uorescence microscope.
purinone, adenine and its oxo/thio-congeners, xanthine, Increasing expression of CASPASE-3 (red uorescence) was
guanine, and purine-2,6-diamine. This approach is convenient, noted with an increasing dose of the compound (Fig. 7).
concise, and exible for the incorporation of various
Measurement of apoptosis using DAPI stain. To further
substituents/functionalities, including those that are thera- conrm the apoptotic effect of compound 22, a nuclear staining
peutically important, into the products (Fig. 3). This approach experiment using HEK 293T cells was performed. The cells were
bears potential application in the synthesis of various annu- treated with compound 22 of increasing concentrations and the
lated purines.
uorescence was observed aer staining with DAPI. Signi-
cantly higher chromatin condensation and nuclear fragmenta-
tion in treated cells compared to untreated cells were observed
and these were found to enhance with increasing concentra-
Biological studies
Cytotoxicity assay. The cytotoxic activities of the synthesized tions of the compound (Fig. 8A and B). Approximately 4, 13 and
compounds (1–49, Fig. 3) were measured by a well-known 15 fold increases in apoptotic nuclei in cells treated with the
colorimetric-based MTT assay. A representative cancer cell, compound at 1, 2.5 and 4 mM concentrations compared to
kidney cancer cell line (HEK 293T) and its corresponding untreated cells were observed.
normal cell (Vero) were considered for testing the cytotoxic
activities. Doxorubicin (DOX) and etoposide (clinically used
anticancer drugs) were used as positive controls. The cells were
treated with doxorubicin, etoposide and the test compounds for
Experimental section
Chemistry
48 h, each with increasing concentrations, following a protocol
General considerations. The starting materials and solvents
described in the experimental section. The percent viability was were used as received from commercial sources without further
measured (Table 1). The IC50 (concentration of compound to purication. The 1H and 13C NMR spectra were recorded in
effect 50% cell growth inhibition in culture) values of 22, 23, 30, CDCl3/DMSO-d6/CD3OD solvents on 400 MHz spectrometer
32, 34, 37 and 46 were found to be relatively more active and using TMS as the internal standard. HRMS was measured using
showed remarkably low IC50 values (2.5, 3, 2.5, 3, 4, 3 and 2.5 a TOF analyzer. The melting points determined are uncorrected.
mM, respectively) in HEK 293T as compared to doxorubicin (20 Microwave assisted reactions were carried out in a sealed
mM) or etoposide (22 mM). For the Vero cell line these reaction vessel using a Biotage Initiator.
compounds exhibited relatively higher IC50 (58, 54, 50, 60, 56.5,
Ethyl 3-aminoimidazo[1,2-a]pyridine-2-carboxylate (I), 3-
45 and 40 mM, respectively) (Fig. 4). To further conrm the aminoimidazo[1,2-a]pyridine-2-carboxamide (II), N-benzoyli-
cytotoxicity, a clonogenic assay was performed according to the midazo[1,2-a]pyridine-3-amine-2-carboxamide (III), pyrido[1,2-
protocol described in the experimental section. Table 2 e]purin-4(3H)-one (1–7), pyrido[1,2-e]purin-4(3H)-one (8), 4-
demonstrates the LC50 (concentration of compound to cause chloropyrido[1,2-e]purine (IV), pyrido[1,2-e]purin-4-amines (9,
50% death of cells) values. For compounds 22, 23, 30, 32, 34, 37 11, 12, 14, 17, 19, 20, 22, 25, 27, 28, 29, 31 and 32) were prepared
and 46, the LC50 values were found to be 1.4, 1.1, 1.7, 1.2, 3.4, 1.4 by our reported procedures.20 Some more relevant pyridine-
and 2.2 mM respectively. A dose dependent decrease in colony fused adenines (10, 13, 15, 16, 18, 21, 23, 24, 26 and 30) were
formation was observed (Fig. 5).
prepared by following our reported procedures.20
Measurement of apoptosis in HEK 293T cells by Annexin V/
Representative experimental procedure for the synthesis of
FITC assay. The apoptosis measurement was performed by 3-benzylpyrido[1,2-e]purine-2,4(1H,3H)-dione (33). To a solu-
Annexin-V/FITC staining followed by FACS aer treatment with tion of ethyl 3-aminoimidazo[1,2-a]pyridine-2-carboxylate (I,
compound 22 for 48 h. Fig. 6 demonstrates the distribution of 205 mg, 1 mmol) in anhyd. ethanol (2 mL) in a vial, were added
cells in various phases of apoptosis. Approximately 46.8 percent subsequently benzyl isocyanate (200 mg, 1.5 mmol) and sodium
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Fig. 3 Synthesized pyridine-condensed purinones, adenines and their oxo/thio analogs, xanthines, guanines, and purine-2,4-diamines as
potential anticancer agents.
ethoxide (68 mg, 1 mmol) under a ow of N2. The reaction provided 3-benzylpyrido[1,2-e]purine-2,4(1H,3H)-dione (33, 181
mixture was heated at 120 ꢀC under microwave irradiation. Aer mg, 62%).
completion of the reaction (monitored by TLC, 20 min), the
The other compounds (34–42, Scheme 2) were prepared
solvent was evaporated under a vacuum. The column chro- following this procedure.
matographic purication of the crude mass on neutral alumina
Experimental procedure for the synthesis of 3-benzyl-2-
(methylthio)pyrido[1,2-e]purin-4(3H)-one (VIII, Scheme 2). 3-
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Table 1 IC50 (mM) values for the cytotoxicities in HEK 293T and Vero cells
Compound
IC50 (mM) HEK 293T
IC50 (mM) Vero
Compound
IC50 (mM) HEK 293T
IC50 (mM) Vero
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
49 Æ 2.0
60 Æ 1.5
60 Æ 1.8
29 Æ 3.0
25 Æ 2.0
30 Æ 1.5
28 Æ 1.7
12 Æ 1.8
5 Æ 2.0
60 Æ 1.9
50 Æ 1.0
50 Æ 1.1
55 Æ 2.1
49 Æ 2.0
55 Æ 1.8
58 Æ 2.5
48 Æ 1.8
49 Æ 1.1
30 Æ 1.2
28 Æ 1.0
29 Æ 2.1
25.5 Æ 1.0
27 Æ 1.5
29 Æ 2.1
24.2 Æ 0.5
29.5 Æ 0.8
30 Æ 1.1
22 Æ 1.2
60 Æ 1.0
38 Æ 0.8
58 Æ 0.5
54 Æ 1.1
50 Æ 1.2
52 Æ 1.0
25 Æ 0.5
44 Æ 0.2
60 Æ 1.1
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
IV
V
8 Æ 1.7
2.5 Æ 1.1
2.5 Æ 0.5
3 Æ 1.6
3 Æ 1.5
10 Æ 2.0
4 Æ 0.7
4 Æ 1.8
12 Æ 1.3
3 Æ 0.5
4 Æ 2.0
18 Æ 2.5
12 Æ 1.8
4 Æ 2.1
5 Æ 2.0
4 Æ 2.2
4 Æ 1.8
6 Æ 1.0
2.5 Æ 1.5
2.5 Æ 0.5
8.5 Æ 1.1
6 Æ 1.0
8 Æ 1.9
10 Æ 2.1
8 Æ 1.0
6 Æ 1.1
6.5 Æ 1.5
22 Æ 1.5
40 Æ 1.7
44 Æ 2.0
50 Æ 1.5
38 Æ 2.2
60 Æ 1.7
50 Æ 2.5
56.5 Æ 1.0
35 Æ 1.5
42 Æ 2.0
45 Æ 2.2
55 Æ 1.1
60 Æ 2.5
58 Æ 1.2
38 Æ 0.7
36 Æ 0.5
24 Æ 0.2
18 Æ 0.5
24 Æ 1.1
40 Æ 2.0
8 Æ 2.1
8 Æ 1.6
5.2 Æ 3.1
7 Æ 3.0
6 Æ 2.5
6.2 Æ 2.2
3.0 Æ 2.1
6.0 Æ 2.0
1.5 Æ 1.8
4 Æ 2.0
8 Æ 1.9
4 Æ 2.2
5 Æ 2.5
14 Æ 2.5
10 Æ 2.0
44 Æ 2.0
60 Æ 2.0
42 Æ 2.1
50 Æ 1.1
60 Æ 2.0
50 Æ 1.5
2.5 Æ 1.0
3 Æ 1.1
4 Æ 1.7
25
26
27
4 Æ 2.0
VI
5 Æ 2.2
VII
VIII
Etoposide
5 Æ 1.5
Doxorubicin
20 Æ 1.1
Benzyl-2-thiopyrido[1,2-e]purin-4(1H)-one (42) was taken in an neutral alumina provided pyrido[1,2-e]purine-2,4(1H,3H)-dione
ethanol–water mixture (1 : 1, 2 mL) in a round bottom ask. (V, 102 mg, 50%).
Sodium hydroxide (40 mg, 1 mmol) and methyl iodide (142 mg,
Experimental procedure for the synthesis of 2,4-dichlor-
1 mmol) were subsequently added to it. The reaction mixture opyrido[1,2-e]purine (VI). Pyrido[1,2-e]purine-2,4(1H,3H)-dione
was heated at 60 ꢀC in a closed system. Aer completion of the (V, 203 mg, 1 mmol) was taken in a round bottom ask. N,N-
reaction (monitored by TLC, 1 h) the solvent mixture was Diethylaniline (149 mg, 1 mmol) and POCl3 (2 mL) were added
evaporated under a vacuum. The column chromatographic subsequently to it under a ow of N2. The reaction mixture was
purication of the crude mass on neutral alumina provided 3- heated at reux. Aer reaction completion (monitored by TLC, 4
benzyl-2-(methylthio)pyrido[1,2-e]purin-4(3H)-one (VIII, 209 h), the reaction mixture was poured into ice cold water (30 mL).
mg, 65%).
20% aq. NaOH solution was added dropwise till pH 8 was
Representative experimental procedure for the synthesis of 3- achieved. Then the mixture was extracted with EtOAc (2 Â 40
benzyl-2-(hexylamino)pyrido[1,2-e]purin-4(3H)-one (47, Scheme 2). mL). The combined organic layers were washed with water,
3-Benzyl-2-(methylthio)pyrido[1,2-e]purin-4(3H)-one (VIII, 322 mg, dried with anhyd. Na2SO4, and concentrated under a vacuum.
1 mmol) was taken in a sealed tube and hexylamine (2 mL) was The column chromatographic purication of the crude mass on
added to it and the tube was closed. The reaction mixture was silica gel provided 2,4-dichloropyrido[1,2-e]purine (VI, 127 mg,
heated at 170 ꢀC. Aer maximum conversion in the reaction 53%).
(monitored by TLC, 8 h), the column chromatographic purica-
tion of the crude mass on neutral alumina provided 3-benzyl-2- of 2-morpholino-N-phenethylpyrido[1,2-e]purin-4-amine (43,
(hexylamino)pyrido[1,2-e]purin-4(3H)-one (47, 199 mg, 53%). Scheme 2). To a solution of 2,4-dichloropyrido[1,2-e]purine
The products 48 and 49 were prepared following this (VI) in THF (1 mL) in a round bottom ask, phenethylamine
Representative experimental procedure for the synthesis
procedure.
(121 mg, 1 mmol) and triethylamine (101 mg, 1 mmol) were
Experimental procedure for the synthesis of pyrido[1,2-e]- added. The reaction mixture was stirred at room temperature
ꢀ
purine-2,4(1H,3H)-dione (V). To a solution of ethyl 3-amino- (25–27 C). Aer the completion of the reaction (monitored
imidazo[1,2-a]pyridine-2-carboxylate (I, 205 mg, 1 mmol) in by TLC, 10 min), the solvent was evaporated under a vacuum.
phenol (2 mL) in a round bottom ask, urea (315 mg, 5 mmol) To this crude reaction mixture morpholine (1 mL) was added
was added. The reaction mixture was heated at 150 ꢀC. Aer the and the mixture was further heated at 120 ꢀC under micro-
completion of the reaction (monitored by TLC, 24 h), the wave irradiation. Aer the completion of the reaction
column chromatographic purication of the crude mass on (monitored by TLC, 10 min), the column chromatographic
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Fig. 5 Clonogenic cell survival in HEK 293T cells.
,
,
,
,
,
,
symbols represent compounds 22, 23, 30, 32, 34, 37 and 46,
respectively. Data presented is the mean Æ SD of three individual sets
of experiments.
cells (cat #CCL-81), were grown in a DMEM medium supple-
mented with 10% FBS, 1% antibiotic (100 units of penicillin and
10 mg streptomycin per mL in 0.9% normal saline) and main-
tained in 5% carbon dioxide in humidied conditions at 37 ꢀC.
All cell culture reagents were purchased from Hi Media, Mum-
bai, India.
MTT assay. The cell proliferation ability of the investigated
compounds (1–49) was measured by performing a colorimetric
based assay using MTT reagent. Approximately 10 000–12 000
cells per well were seeded in a 96 well tissue culture plate and
were incubated for 24 h. Aer 24 h of incubation the cells were
exposed to different concentrations of the investigated
compounds for 48 h. Then the cells were washed with 1Â PBS
followed by the addition of 100 mL of 0.05% MTT reagent to each
well. The plate was incubated overnight at 37 ꢀC. Purple for-
mazan crystals were formed which were dissolved using 10%
NP-40 with 4 mM HCl. The plate was incubated for 1 h at 37 ꢀC
and then the intensity of the developed color was measured at
570 nm using a microplate reader (Barthold, Germany). The
data were calculated and a graph representing the percent
viability was plotted in comparison to the control. The data
presented are the mean Æ SD of at least three experiments.
Clonogenic assay. The colony forming abilities of the cells
aer treatment with the investigated compounds were tested by
performing a clonogenic assay. This assay was carried out to
check the anti-proliferating or colony forming ability of the
selected investigated compounds. Approximately 500 cells per
well of HEK 293T, a kidney cancer cell line, were seeded in 12
well plates. The plates were incubated for 24 h. The cells were
treated with various concentrations of the selected compounds
for 48 h. Then the media was aspirated and replaced with fresh
media. The plates were incubated further to allow the cells to
grow for 5–6 doublings by changing the media every 72 h. Then
the media was removed and the cells were washed with 1Â PBS
followed by the addition of 0.2% crystal violet stain to each well
of the plates. It was kept for 1 h and the excess stain was
removed by washing with 1Â PBS. The plates were air dried and
Fig. 4 Cell survivals in the MTT assay for the selected compounds 22,
23, 30, 32, 34, 37 and 46. Cells were treated with the investigated
compounds and doxorubicin of various concentrations for 48 h.
Symbols,
,
,
,
represent HEK 293T/compound, Vero/
compound, HEK 293T/DOX and Vero/DOX, respectively. Data pre-
sented is the mean Æ SD of three independent sets of experiments.
Table 2 LC50 (mM) values for the clonogenic assay using HEK 293T
cells
Compound LC50 (mM) HEK 293T Compound LC50 (mM) HEK 293T
22
23
30
32
1.4 Æ 0.5
1.1 Æ 0.8
1.7 Æ 1.1
1.2 Æ 0.2
34
37
46
3.4 Æ 1.5
1.4 Æ 0.5
2.2 Æ 1.0
purication of the crude mass on neutral alumina provided
2-morpholino-N-phenethylpyrido[1,2-e]purin-4-amine
(43,
307 mg, 82%).
The other products (44–46, Scheme 2) were prepared following
this procedure. 2-Chloro-N-phenethylpyrido[1,2-e]purin-4-amine
(VII) was obtained only by 4-amination of compound VI. The
column chromatographic purication of the crude mass on
neutral alumina provided VII in 90% isolated yield.
Biology
Cell line and culture conditions. HEK 293T (cat #CRL-
11268), a kidney cancer cell line, and Vero, normal kidney
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Fig. 6 Measurement of apoptosis by the Annexin-V/FITC method. Cells were treated with compound 22 of various concentrations for 48 h and
the experiment was carried out as described in the experimental section. Data given here is one of three separate experiments.
Fig. 8 (A) DAPI nuclear staining: images were taken under a fluores-
cence microscope at 40Â magnification (scale bar 10 mm). (B) Bar-
diagram presentation is the number of apoptotic and non-apoptotic
nuclei taken from (A).
Annexin-V/FITC staining. Annexin-V/FITC staining is used to
detect the distribution of cells in early, apoptotic and late
apoptotic (necrosis) phases. For this assay, the cells were seeded
in a 6 well cell culture plate at a density of 1 Â 105 cells per well.
70–80 percent conuent grown cells were treated with varying
concentrations of compound 22 for 48 h. Then, the cells were
harvested, washed and stained with Annexin-V/FITC and PI
according to manufacturer’s protocol (Sigma). Finally, the
stained cells were sorted by FACS using the PE-A (to capture the
PI stained cells) and FITC-A (to capture the Annexin-V stained
cells) channels. According to principle, Annexin-V dye stains
living and apoptotic cells, whereas PI stains all cells including
necrotic cells. The percentage of cells population in Q1, Q2, Q3
and Q4 represent the normal, early apoptotic, apoptotic and
necrotic cells, respectively.
Fig. 7 Compound 22 increased the CASPASE 3 expression in HEK
293T cells. The left and middle panels represent the cells stained with
DAPI and CASPASE 3/TRITC (tetramethylrhodamine, a red fluorescent
dye) and the right panel represents the MERGED image of both.
Images were taken under a fluorescence microscope at 40Â magni-
fication. Images presented here are the best of three individual sets of
experiments (scale bar 10 mm).
the colonies were counted under a gel documentation system
(UVP, Germany). The colonies formed in the treated wells were
counted against the untreated well that served as the control
(100% survival) and a graph was plotted representing the
percent survival of the cells. Data presented here are the mean Æ
SD of three different experiments.
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Immunouorescence of CASPASE 3. Immunouorescence
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Acknowledgements
of CASPASE 3 was performed to check the expression of
CASPASE 3 aer the treatment of the cells with compound 22. We gratefully acknowledge nancial support from DST, New
HEK 293T cells (1 Â 104) were seeded on sterile cover slips and Delhi for this investigation. VC is thankful to CSIR, New Delhi
allowed to attain 50–60% conuency. The cells were treated for his fellowship.
with various concentrations of the drug for 48 h. The drug
treated media were removed and the cells were washed with
Notes and references
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Conclusions
A set of novel pyridine annulated purinones, adenines and their
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were considered as potential anticancer agents based on
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