Synthesis of 4-phenyl-5,6-dihydrobenzo[h]quinazolines and their evaluation as growth inhibitors of carcinoma cells

Article abstract of DOI:10.1039/c5ra24429c

The synthesis of various benzo[h]quinazoline analogs (4a-f, 6a-d, 8a and 8b) was accomplished through the reaction of chalcone with guanidine. The synthesized compounds (4a-f, 6a-d, 8a and 8b) were screened for their anticancer potential against different cancer cells viz MCF-7, DLD1, A549, DU145 & FaDu cell lines. Compounds 4a, 6a-d & 8b showed significant anticancer activity in these cancer cell lines with a range of IC₅₀ values of 1.5-12.99 μM. A functional study of promising molecule 6d at 7 μM (at the IC₅₀ value) over 24 and 48 h showed that it possesses anticancer activity through triggering apoptosis. In a tubulin polymerization assay, 6d effectively inhibited tubulin polymerization with an IC₅₀ of 2.27 μM. In silico docking studies of 6d revealed that 6d has good affinity with an estrogen receptor as well as a tubulin protein on its β-sheet of the colchicines binding site.

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PAPER
Synthesis of 4ꢀphenylꢀ5,6ꢀdihydrobenzo[h]quinazolines and their
evaluation as growth inhibitors of carcinoma cells
Hardesh K Mauryaa, Mohammad Hasanaind, Sarita Singha, Jayanta Sarkard, Vijaya Dubeyb, Aparna
Shuklac, Suaib Luqmanb, Feroz Khan,c Atul Guptaa,#*
5
Synthesis of various benzo[h]quinazoline analogs (4aꢀf, 6aꢀ6d and 8aꢀb) was accomplished through the
reaction of chalcone with guanidine. The synthesized compounds (4aꢀf, 6aꢀ6d & 8aꢀb) were screened for
their anticancer potential against different cancer cells viz MCFꢀ7, DLD1, A549, Du145 & FaDu cell
lines. Compounds 4a, 6aꢀd & 8b showed significant anticancer activity in these cancer cell lines within
10 the range of IC50 values of 1.5ꢀ12.99 µM. Functional study of a promising molecule, 6d, at 7 µM (at IC50
value) for 24 and 48 h showed that it possessed anticancer activity through triggering apoptosis. In tubulin
polymerization assay, 6d effectively inhibited tubulin polymerization at IC50 2.27 µM. In-silico docking
study of 6d revealed that 6d had good affinity with estrogen receptor as well as tubulin protein on its βꢀ
sheet of colchicines binding site.
15
pyridylethylamino)undecanyl]ꢀestradiol
dichloroplatium
(II)
Introduction
45 complexes (IX) have been discovered with potent antiꢀbreast
cancer potential, Figure 1.4ꢀ11 It is reported that estrogen receptorꢀ
α (ERꢀα) dominates in breast cancer tissue and is the main culprit
for excess cell proliferation.
Any defect in programmed cell growth of a normal healthy cell
leads to accumulation of undesired defective cells and ultimately
develop Cancer. Cancer is a leading cause of death worldwide.
20 The present statistics revealed that it accounts for one in every
seven deaths globally which is more than any other infectious
disease.1 By 2030, it is expected that about 21.7 million new
cancer cases and 13.0 million cancer deaths will be reported.2
Lung, prostate, colorectum, stomach, and liver cancers are very
25 common in males while in females stomach, breast, uterine and
cervix cancers are frequent and the common cause of cancers is
imbalance in hormone.3 Among several other estrogenꢀdependent
cancers, breast cancer is a leading pandemic that affects women
over a wide age group.3 Considering the necessity of the
30 anticancer chemotherapeutics for treatment of estrogen dependent
cancers such as breast cancer, different estrane based molecules
such as fulvestrant (ICIꢀ182,780, I), ICIꢀ164,384 (II), SRꢀ16157
(IV), SRꢀ16234 (V), 3ꢀhydroxyꢀestraꢀ1,3,5(10)trienꢀ17ꢀoxoꢀ15βꢀ
morpholinobutanꢀ1ꢀone (VI), estradiolꢀchlorambucil conjugates
50
55
60
65
70
35 (VI),
estraꢀ13,5(10)ꢀtrienꢀ16,17
[3,4]ꢀ
pyrazoloꢀNꢀ(3ꢀ
ehtylpyridino)ꢀ5ꢀcaboxamide (VII), VPꢀ128 (VIII), 16α,βꢀ[11ꢀ(2ꢀ
aMedicinal Chemistry Department, bMolecular Bioprospection
c
Department and Metabolic Structural Biology Departent, CSIR-Central
Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Kukrail Road,
Lucknow 226015, India CSIR-Central Institute of Medicinal and Aromatic
Plants, P.O. CIMAP, Kukrail Road, Lucknow 226015, India
dDivision of Biochemistry, CSIR-Central Drug Research Institute, Sector-
10, Jankipuram Extension, Lucknow 226 031, India, *To whom
40 correspondence should be addressed: Tel: +915222718556, E-mail:
Fig. 1 Some potential 17βꢀestradiol based anticancer drug molecules (Iꢀ
IX) and 17βꢀestradiol.
atisky2001@yahoo.co.in,
No.CIMAP/PUB/2015/02
#CIMAP
communication
75
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using BF3ꢀOEt2 in dioxane in 72 h at room temperature under dry
reaction conditions as reported by us.16 Attempts were made to
find out an alternate method for preparation of 3 which could
60 yield 3 in short reaction time and do not require dry reaction
conditions. For this purpose, we used N/10 HCl in aqueous
medium at room temperature which offered simple reaction
conditions, comparatively short reaction time (50h vs 72h) and
formation of chalcone as sole product. However, yields of the
65 product in this method were moderate. Further, attempts were
made to improve the yields and the best conversion was observed
at 50°C in 50h and used for chalcone synthesis (Scheme 1).
Since 17βꢀestradiol is a native ligand of estrogen receptor (ER),
the possible hypothesis of using estrane in these molecules could
be targeted delivery of therapeutics selectively to the target site. It
is interesting to note that all these compounds possess estradiol as
basic core which has been modified by incorporation of either by
amino, amido, pentafluoroalkyl group or platinum complex at
position 7, 15, 16 or 17 of steroid nucleus. These modification
were made in such a way that modified groups could be
accommodated spatially somewhere in the ring B and/or D region
10 within LBD of ER. However, in this approach, incorporation of
estrane nucleus sometimes creates secondary complications due
its intrinsic estrogen agonistic action. Therefore, search for
effective nonꢀsteroidal ligands for estrogen receptor such as
tamoxifen and other nonꢀsteroidal antiestrogens warranted to
15 address the issue.
5
70
Furthermore, quinazoline nucleus which is eventually present in
lapatinib (X), erlotinib (XI), gefitinib (XII) and vandetanib (XIII)
applied for the treatment of cancer, have been well explored for
development of anticancer drugs Figure 2.12ꢀ15 In our approach to
20 device potential nonꢀsteroidal anticancer agents especially for
estrogen receptor positive breast cancer treatment, we synthesized
75
80
Scheme 1 Synthesis of chalcones (3).
and
evaluated
4ꢀphenylꢀ5,6ꢀdihydrobenzo[h]quinazolin
derivatives for their anticancer potential. The designed molecules
will have a quinazoline embedded tricyclic ring system analogus
25 to rings A, B and C of estradiol and a phenyl ring bearing
alkylamino or ester group flanked more towards ring D of
estradiol core as presented in compounds 1ꢀ9, Figures 1 and 2.
Subsequently, targeted quinazoline analogs (4) were synthesized
though the reaction of chalcone (3) with guanidine hydrochloride.
85 For this purpose, we explored use of green solvents for synthesis
of target compounds in short reaction time. In our attempts, we
found choline chloride (ChCl) as an appropriate green, non toxic,
environment benign solvent for synthesis of highly substituted
5,6ꢀdihydroꢀ8ꢀmethoxybenzo[h]quinazolinꢀamine (4) Scheme 2.
90 The use of choline chloride (ChCl) containing eutectic solvents
have been reported in various reactions in e.g. ChCl/urea
supported conversion of aromatic boronic acid in phenol,
ChCl/urea supported chemical fixation of carbon dioxide to cyclic
carbonates, ChCl/glycine assisted biotransformations of ethyl
95 valerate to butyl valerate, and bromination of substituted 1ꢀ
aminoanthraꢀ9,10ꢀquinone, ChCl/L(ꢀ)ꢀproline supported aldol
reaction, Knoevenagel condensation in ChCl/amino acid, Dielsꢀ
Alder cycloadditions and Fischer indole annulations in
ChCl/ZnCl2, ring opening of epoxides in ChCl/SnCl2.17ꢀ24
30
35
40
45
100
105
Fig. 2 Quinazoline fragment containing anticancer drugs, 17βꢀestradiol
and proposed prototype.
110 Scheme 2. Synthesis of benzo[h]quinazolinꢀ2ꢀamines (4).
50
Different reaction conditions were attempted in ChCl at different
temperatures and molar ratio of reactants. The desired compounds
were finally synthesized in good yields by the use of 1:1.2 molar
115 ratios of reactants with NaH in ChCl at 140°C within 5h (Table
1). Interestingly, as reported by us, the use of conventional solvent
such as dimethylformamide (DMF) under the reaction conditions
takes longer reaction time viz 36h.16
Results and discussion
Chemistry
The synthesis of designed prototype was started with the
preparation of different chalcones (3), an important intermediate
55 for target compounds. Compound 3 was initially synthesized from
the reaction of 1ꢀtetralones (1) and substituted benzaldehydes (2)
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cells (MCFꢀ7 cells) using PARP assay. The in silico docking
experiments for 4a and 6d on estrogen receptorꢀα and tubulin
45 protein were performed to verify the designing of these molecules.
The results of these studies are elaborated in the following text.
Table1. Optimization of the reaction conditions
In vitro growth inhibition of cancer cells
The compounds 4(a–f), 6(a–d) and 8(a–b) were explored for in
vitro anticancer activity in various human carcinoma cell lines
50 such as DLD1 (colorectal adenocarcinoma), A549 (Lung
carcinoma), FaDu (hypopharyngeal carcinoma), MCFꢀ7 (ER+
Breast adenocarcinoma), DU145 (prostate carcinoma) using
sulphorhodamin B assay. Tamoxifen was used as positive control
in this study. The anticancer activity of the tested compounds is
55 described by half maximal inhibitory concentrations (IC50) value
in Table 2.
Among hydroxyl derivatives 4aꢀf, compounds 4b and 4c which
possessed methoxy and hydroxy groups in its ring D at positions 3
and 4 respectively presented anticancer activity in colon cancer
60 cells at 1.87 and 6.79 µM concentrations whereas 4e and 4f which
had two methoxy groups in ring A and methoxy and hydroxy
groups in its ring D were devoid of anticancer activity. Compound
4d which had two methoxy groups in ring A and a hydroxy group
in its ring D showed significant anticancer activity in colon as
65 well as prostate cancer cells at IC50 value 2.96 and 5.25 µM.
Among others, compound 4a which had a methoxy groups in ring
A and a hydroxy group in its ring D presented potent anticancer
activity in MCFꢀ7, DLDꢀ1, A549 and DU145 cancer cells at 5.89,
1.50, 1.86 and 1.50 µM concentrations respectively compared to
70 tamoxifen, a positive control.
Entry
Base
Solvent
DMF
Temp time purification Yields
(oC)
(h)
1
2
3
4
5
NaH (2 eq)*
NaH(2 eq)
120
36
required
53
0
THF
100
100
140
140
36
5
no reaction
NaH (2 eq) THFꢀChCl
(1:2)
crystallization 85
required 11
crystallization 90
KOH (2 eq) H2OꢀChCl
(1:2)
10
5
NaH (2 eq)
ChCl
*Ref 16.
5
Furthermore, quinazoline (4a) was modified to alkylamine
substituted quinazolines (6) though the reaction of substituted
alkylamine hydrochlorides (5) with 4 in the presence of anhydrous
K2CO3 in chloroformꢀacetone (1:1).16 Similarly, alkylation of 4a
was done using bromo alkylesters the presence of potassium
10 carbonate K2CO3 in chloroformꢀacetone (1:1) which yielded long
chain ester substituted quinazoline (8) in 90ꢀ94% yields.16
In order to improve the biological activity, compound 4a was
chosen for further modification. Interestingly, alkylation of 4a
with different tertiary aminoalkyl groups usually present in
antiestrogens such as tamoxifen, yielded compounds which
75 showed significant anticancer activity invariably in all cancer cell
lines within the range of IC50 values 3.63ꢀ12.13 µM. Further,
compound 8b which had five carbon long chain ester group
showed anticancer activity in MCFꢀ7, DLD1, and A549 cells at
8.03, 15.10 and 12.99 µM concentrations respectively whereas
80 another ester analog 8a was devoid of any activity upto 20 µM
concentration.
15
20
25
Tubulin Polymerization Inhibition
Microtubule/tubulin dynamics interrupting molecules bind to the
85 tubulin protein which perturb mitosis and arrest growth of cells
during interphase.25 Having growth inhibitory activity data in our
hands, we next wished to evaluate whether or not these molecules
could inhibit the growth of cancer cells through inhibition of
tubulin polymerization. For this purpose, compounds 4a and 6d
90 were evaluated for tubulin polymerization inhibitory activity using
tubulin protein in in-vitro model. In this experiment,
podophyllotoxin and nocadazole, standard tubulin polymerization
inhibitors, were used as positive control and DMSO as negative
control. Compounds 4a and 6d effectively inhibited tubulin
95 polymerization at IC50 value 2.92 and 5.97 µM respectively.
Compound 6d presented antitubulin activity very close to the
podophyllotoxin.
Scheme 3. Synthesis of benzo[h]quinazolinꢀ2ꢀamines derivatives (6 and
8).
30
The synthesized compounds were characterized by the use of
different spectroscopic techniques viz NMR, IR, mass
spectrometry.
35 Biology
The synthesized compounds were investigated for in-vitro growth
inhibitory (anticancer) activity of cancer cells in a panel of cancer
cell lines viz MCFꢀ7 A549, FaDu and DU145 using SRB assay.
Some promising molecules (4a and 6d) were evaluated for their
40 antitbulin activity using in vitro tubulin polymerization assay.
Further, compound 6d was evaluated for cell division cycle study
using MCFꢀ7 cells and its capability to induce apoptosis in cancer
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Table 2 In vitro anticancer activity of compounds 4 (aꢀf), 6(aꢀd) and 8 (aꢀb) using SRB assay (IC50 in µM)#,*
Compounds
Carcinoma cell lines (IC50 in µM, mean±SE)
A549
MCFꢀ7
DLD1
DU145
FaDu
4a
4b
4c
4d
4e
4f
5.89±1.22
>20
>20
>20
>20
<1.50
<1.87
6.79±1.14
2.96
>20
>20
1.86±0.18
>20
>20
>20
>20
<1.50
[c]
>20
5.25
>20
[a]
[a]
[a]
[a]
[a]
>20
>20
>20
[a]
7.09±0.66
3.90±0.82
7.97±0.19
7.82±1.17
7.59±1.01
6a
6b
12.13±0.54
7.84±0.91
6.64±1.22
4.22±0.94
11.36±0.54
8.05±0.18
10.41±1.48
7.76±1.41
9.92±0.94
7.51±1.28
6c
6d
6.38±0.37
>20
3.63±0.71
>20
7.35±0.11
>20
6.96±1.27
>20
6.44±0.55
>20
8a
8b
8.03±0.62
8.74±1.17
15.10 [b]
14.65
12.99±0.72
10.37±0.84
>20
>20
[a]
12.14±0.62
Tamoxifen
#values are represented as mean IC50 value±SE of three independent experiments. The concentration of compounds used for IC50 determination was 5 serial
dilutions (2 fold) of the 20µM starting concentration .The Iincubation period of drug treated cells was 48h. *Cell lines used: DLD1 (colorectal
adenocarcinoma), A549 (Lung carcinoma), FaDu (hypopharyngeal carcinoma), MCFꢀ7 (ER+ Breast adenocarcinoma), DU145 (prostate carcinoma), [a] =
not done.
5
Table 3 Tubulin polymerization inhibition of 4a and 6d using tubulin polymerization assay (half maximal inhibitory concentrations, IC50 in ꢁM)#
S.No.
Compound
IC 50 (ꢁM)
5.97±0.7
1
2
3
4
4a
6d
2.92±0.16
2.27±0.6
Podophyllotoxin (PDT)
Nocadazole
2.06±0.07
#values are represented as mean IC50 value±SE of three independent experiments
10
trigger apoptosis in MCFꢀ7 cells by Western blot assay after
Cell division cycle study of 6d
25 probing with antiꢀPARP antibody. Poly (ADPꢀribose)
polymerase (PARP) is a protein which is involved in a number
of cellular processes including DNA repair and programmed cell
death through the production of PAR, which stimulates
mitochondria to release apoptosisꢀinducing factor (AIF).26 In the
30 present study, we monitored cleavage of Poly (ADPꢀribose)
polymerase (PARP) which is considered as a marker of apoptosis.
Consistent with flowcytometry data, Western blot analyses of
MCFꢀ7 cell lysates revealed proteolysis of PARP in 6d treated
cells confirming induction of apoptosis (Figure 4). In this
35 experiment, doxorubicin (doxo), a potent anticancer drug was
used as positive control.
Further, compound 6d was selected for Cell division cycle study
using estrogen receptor (ER) positive human breast cancer cell
line (MCFꢀ7). After 24 and 48 hrs incubation with 6d at 7 µM
15 concentration (near IC50 value), substantial accumulation of cells
population at subꢀG0 (apoptotic) and G0/G1 phase compared to the
vehicle treated controls was observed in a time dependent manner
(Figure 3aꢀb). This was associated with concomitant decrease in
cells at S phase while there was no obvious change in G2/M
20 population.
Cell apoptosis study
Following anticancer, tubulin polymerization inhibitory activity
and cell cycle study of 6d, we further investigated ability of 6d to
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Fig. 3a
5
Fig. 3b
Fig. 3 Effect of 6d on cell division cycle (a) MCFꢀ7 cells were treated with 6d at IC50 concentrations for 24 h and 48 h, stained with propidium iodide(PI)
and were subjected to flow cytometry. (b) Histogram showing average population cells in various phases (G1, G2, S) of cell cycle (mean ± S.E. of three
10 independent assays, each performed in duplicate). #P <0.05, *P <0.001 compared with vehicle treated controls.
Fig. 4 6d induces apoptosis in MCFꢀ7 cells. Lysates from treated and untreated cells were subjected to immunoblotting after probing with antiꢀPARP
antibody. Marked cleavage of PARP was observed in 6d treated cells in time dependent manner.
15
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Docking Study
To investigate the mechanism of action of studied molecules, a
Similarly, the interaction study of molecules with tubulin protein
molecular docking study was performed for 4a and 6d against
indicate that control Podophyllotoxin, 4a and 6d showed good
targets ERꢀα and tubulin using Autodock MGL tool v1.5.6. The 20 binding affinity within the colchicine binding site of tubulin
5
docking study with estrogen receptorꢀα (ERꢀα) revealed that
compounds 4a and 6d exhibit promising binding affinity within
the tamoxifen binding site with docking binding energy ꢀ7.12 kcal
molꢀ1 (Ki value 6.01 µM) and ꢀ5.55 kcal molꢀ1 (Ki value 84.85
protein. 4a exhibit highest docking binding energy of ꢀ5.59 kcal
molꢀ1 (Ki value 79.52 µM) and making two Hꢀbonds with amino
acid residues ALA 317 and LYS 352 having bond distance 2.198
and 2.229 Å. Whereas compound 6d showed moderate docking
µM) respectively. The identified key residues for ligandꢀreceptor 25 binding energy of ꢀ5.06 kcal molꢀ1 (Ki value 194.67 µM).
10 interaction are ARG 394, THR 347 and GLY 420. The –NH2
group of amino acid arginine interact with oxygen atom of
hydroxyl group of molecules 4a and 6d with bond distance 2.79
and 2.80 Å respectively. The compound 4a is making two
additional Hꢀbonds with amino acid residues THR 347 and GLY
15 420 having bond distance 2.07 and 1.7 Å. The results are
summarized in table 4 and 5.
30
Fig.5a
Fig.5b
35
Fig.5c
40 Fig. 5. (a) Docking pose of tamoxifen within LBD of Estrogen Receptorꢀα, (b) Docking pose of 4a within LBD of Estrogen Receptorꢀα (c) Docking pose
of 6d within LBD of Estrogen Receptorꢀα. The residues are highlighted with pink stick, ligand is represented with blue ball and stick form and Hꢀbonding
is represented with dashed lines.
45
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Table 4 Docking interactions of tamoxifen, 4a and 6d within ligand binding pocket of estrogen receptorꢀα (ERα).
Compounds
Binding
energy (kcal
molꢀ1)
Ki value
(inhibitory
constant)
Binding pocket residues within 4Ǻ region
Interacting residues
and Hꢀbond length
(Ǻ)
Number
rotatable
bonds
Tamoxifen
ꢀ8.89
ꢀ7.12
ꢀ5.55
304.35 nM
6.01 uM
MET343, LEU346, THR347, ALA350,
ASP351, GLU353, TRP383, LEU384,
LEU387, MET388, LEU391, ARG394,
PHE404, GLU419, GLY420, MET421,
ILE424, LEU428, GLY521, HIS524, LEU525
ARG394 :NH2ꢀ
Tamoxifen: O4
(2.8) &
Tamoxifen:O4ꢀ
GLU353:OE2 (2.4)
8
2
4a
6d
MET343, LEU346, THR347, LEU349,
ALA350, GLU353, LEU384, LEU387,
ARG394, GLU419, GLY420, MET 421,
ILE424GLY521, HIS 524, LEU525
ARG394:NH2ꢀ4a:
O (2.79572), 4a: Hꢀ
THR347:OG1
(2.0738) & 4a: Hꢀ
GLY420: O
(1.73547)
84.85 uM
MET343, LEU346, THR347, ALA350,
ASP351, GLU353, TRP383, LEU384,
LEU387, MET388, LEU391, ARG394,
ILE424, GLY521, LEU525, MET528
6
ARG394:NH2ꢀ6d:
O (2.80547)
5
Fig. 6 Binding affinity of tamoxifen, 4a and 6d with estrogen receptorꢀα (ERα).
10
15
Fig.7a
Fig.7b
20
Fig.7c
Fig. 7 (a) Docking pose of podophyllotoxin within the binding site of tubulin, (b) Docking pose of 4a within the binding site of tubulin, (c) Docking pose
of 6d within the binding site of tubulin. Binding pocket residues are highlighted with cyan color stick form, ligand is represented with blue ball and stick
form and Hꢀbonding is represented with dashed lines.
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Table 5 Docking interactions of podophyllotoxin, 4a and 6d with binding site residues of βꢀtubulin and comparison of binding affinity of in terms of
docking energy.
Compounds
Binding energy
(kcal molꢀ1)
Ki value (inhibitory
constant)
Binding pocket residues within
4Ǻ region
Interacting residues
and Hꢀbond length
(Ǻ)
Number
rotatable
bonds
Podophyllotoxin
ꢀ5.16
ꢀ5.59
165.71 uM
79.52 uM
CYS241, LEU248, ALA250,
ASP251, LYS254, LEU255,
ASN258, VAL315, ALA316,
LYS352, ALA354
ꢀ
5
4a
6d
VAL238, THR239, CYS241,
LEU242, LEU248, ALA250,
LEU255, ASN258, ALA316,
ALA317, ILE318, LYS352,
THR353, ALA354
4a: HꢀALA317:O
(2.19842) & 4a:Hꢀ
LYS352:O
2
(2.22885)
ꢀ5.06
194.67 uM
VAL238, CYS241, LEU242,
LEU248, ALA250, LYS254,
LEU255, ASN258, MET259,
THR314, ALA316, ILE347,
PRO348, ASN349, ASN350,
LYS352
ꢀ
6
5
Fig. 8 Binding affinity of tamoxifen, 4a and 6d with binding site residues of βꢀtubulin.
dihydrobenzo[h]quinazolins may lead to effective and ER
25 selective anticancer lead molecules.
Conclusion
In conclusions, we have established a greener approach for
synthesis of substituted benzo[h]quinazoline analogs from
Experimental Section
10 chalcones and guanidine in good yields. The synthesized
compounds presented significant anticancer activity in a panel of
cancer cell lines. The cell apoptosis and tubulin polymerization
inhibitory activities of promising molecule 6d, revealed that the
anticancer activity of compound is due to apoptosis of cancer cell
15 which is very likely due to its tubulin polymerization inhibitory
activity. The in-silico docking experiments of 4a and 6d showed
that these molecules have ability to interact with estrogen
receptorꢀα and colchicine binding site of tubulin protein. The
biological activity and in-silico study results indicate that
20 structural arrangement of 6d allows it to interact with βꢀtubulin
for its anticancer activity in general and has estrogen receptor
mediated activity in ER positive cancer cells (MCFꢀ7 cells) in
particular. Further, structural modifications of these 4ꢀphenylꢀ5,6ꢀ
General
The reagents and the solvents used in this study were of analytical
grade and used without further purification. All the reactions were
30 monitored on Merck aluminium thin layer chromatography (TLC,
UV254nm) plates. Column chromatography was carried out on
silica gel (60ꢀ120 mesh). The melting points were determined on
Buchi melting point M560 apparatus in open capillaries and are
uncorrected. Commercial reagents were used without purification.
35 1H and 13C NMR spectra were recorded on a Bruker WMꢀ300
(300 MHz) using CDCl3 and DMSOꢀd6 as the solvent. Chemical
shift are reported in parts per million shift (δꢀvalue) based on the
This journal is © The Royal Society of Chemistry [2015]
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middle peak of the solvent (CDCl3/DMSOꢀd6) (δ 75.00 ppm for
UV(CH3CN, λmax/nm (ε/dm3 molꢀ1 cmꢀ1): 238 nm (300), 355 nm
(400); H NMR (CDCl3+DMSOꢀD6, 300 MHz, δ ppm): 2.90 (t,
2H, J = 6.0 Hz, CH2), 3.11 (t, 2H, J = 6.0 Hz, CH2), 3.89 (s, 3H,
OCH3), 3.92 (s, 3H, OCH3), 6.74 (s, 1H, ArH), 6.87 (d, 2H, J =
1
13C NMR) as the internal standard. Signal patterns are indicated as
s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t,
triplet; m, multiplet; brm, broad multiplet. Coupling constants (J)
5
are given in Hertz. Infrared (IR) spectra were recorded on a 60 8.4 Hz, ArH), 7.32 (d, 2H, J = 8.4 Hz, ArH), 7.53 (s, 1H, CH),
PerkinꢀElmer AXꢀ1 spectrophotometer in KBr disc and reported
in wave number (cmꢀ1). ESI mass spectra were recorded on
Shimadzu LCꢀMS after dissolving the compounds in acetonitrile
and methanol.
7.67 (s, 1H, ArH), 9.50 (s, 1H, OH); 13C NMR (CDCl3+DMSOꢀ
D6, 75 MHz, δ ppm): 32.56, 33.49, 61.12, 61.22, 114.79, 115.47,
120.87 (2xC), 131.77, 131.99, 136.96 (2xC), 137.77, 141.33,
143.29, 153.29, 158.58, 163.47, 191.41; ESIMS (C19H18O4): m/z
65 = 349 [M+K]+.
10
General procedure for the synthesis of compounds (3aꢀf)
(E)ꢀ2ꢀ(4ꢀhydroxyꢀ3ꢀmethoxybenzylidene)ꢀ6,7ꢀdimethoxyꢀ3,4ꢀ
dihydronaphthalenꢀ1(2H)ꢀone (3e)
(E)ꢀ2ꢀ(4ꢀhydroxybenzylidene)ꢀ6ꢀmethoxyꢀ3,4ꢀ
dihydronaphthalenꢀ1(2H)ꢀone (3a)
Yellow solid, Rf: 0.33 (CHCl3); mp 157ꢀ158oC; 1H NMR (CDCl3,
300 MHz, δ ppm): 2.87 (t, 2H, J = 6.0 Hz, CH2), 3.11 (t, 2H, J =
70 6.0 Hz, CH2), 3.89 (s, 3H, OCH3), 3.92 (s, 6H, 2xOCH3), 5.92 (s,
1H, OH), 6.65 (s, 1H, ArH), 6.92ꢀ7.02 (m, 3H, ArH), 7.60 (s, 1H,
CH), 7.75 (s, 1H, ArH); 13C NMR (CDCl3, 75 MHz, δ ppm):
27.92, 28.95, 56.39, 56.47 (2xC), 110.14, 110.29, 113.20, 114.85,
124.03, 127.13, 128.76, 133.98, 136.73, 138.42, 146.67, 146.83,
75 148.67, 153.88, 187.12; ESIMS (C20H20O5) : m/z = 341 [M+H]+.
In a round bottomed flask, substituted tetralone (1, R1= H, 3.52 g,
15 2.00 mmol) was stirred in N/10 ml aqueous HCl (10 mL) and
substituted benzaldehyde (2, R2 = H, R3 = H, 2.44 g, 2.00 mmol)
was added in the reaction mixture. The reaction mixture was
o
allowed to stir at 50 C for 50 h. The progress of reaction was
monitored using TLC. After completion of reaction, the reaction
20 content was filtered and crude material was subjected to column
chromatography on silica gel using ethyl acetateꢀhexane as eluent
get desired compound (3a) as yellow solid.
(E)ꢀ2ꢀ(3ꢀhydroxyꢀ4ꢀmethoxybenzylidene)ꢀ6,7ꢀdimethoxyꢀ3,4ꢀ
dihydronaphthalenꢀ1(2H)ꢀone (3f)
Yellow solid, Rf: 0.3 (30% EtOAc in hexane); mp 172ꢀ173oC; IR
(KBr, νmax/cmꢀ1): 3357 (NH2), 3489 (OH); H NMR (acetoneꢀd6,
Yellow solid, Rf: 0.32 (CHCl3); mp 169ꢀ170oC; 1H NMR (CDCl3,
300 MHz, δ ppm): 2.86 (t, 2H, J = 6.3 Hz, CH2), 3.11 (t, 2H, J =
80 6.3 Hz, CH2), 3.90 (s, 3H, OCH3), 3.92 (s, 6H, 2xOCH3), 5.75 (s,
1H, OH), 6.65 (s, 1H, ArH), 6.87 (d, 1H, J = 8.1 Hz, ArH), 6.96
(d, 1H, J = 8.1 Hz, ArH), 7.05 (d, 1H, J = 1.5 Hz, ArH), 7.60 (s,
1H, CH), 7.72 (s, 1H, ArH); 13C NMR (CDCl3, 75 MHz, δ ppm):
27.86, 28.95, 56.39, 56.48, 110.11, 110.29, 110.89, 116.14,
85 116.38, 123.43, 127.12, 129.88, 134.38, 136.22, 136.38, 138.52,
145.76, 147.32, 148.64, 153.86, 187.17; ESIMS (C20H20O5) : m/z
= 339 [MꢀH]+.
1
25 300 MHz, δ ppm): 2.92 (t, 2H, J = 6.0 Hz, CH2), 3.11 (t, 2H, J =
6.0 Hz, CH2), 3.88 (s, 3H, OCH3), 6.90 (m, 4H, ArH), 7.39 (d, 2H,
J = 8.4 Hz, ArH), 7.68 (s, 1H, CH), 7.97 (d, 1H, J = 8.4 Hz, ArH),
9.35 (s, 1H, OH); 13C NMR (acetoneꢀd6, 75 MHz, δ ppm): 27.60,
29.30, 55.58, 112.66, 113.87, 116.05 (2xC), 127.50, 127.64,
30 130.49, 132.36 (2xC), 133.54, 136.02, 146.27, 159.04, 164.03,
185.96; ESIMS (C18H16O3): m/z : 281 [M+H]+.
(E)ꢀ2ꢀ(4ꢀhydroxyꢀ3ꢀmethoxybenzylidene)ꢀ6ꢀmethoxyꢀ3,4ꢀ
dihydronaphthalenꢀ1(2H)ꢀone (3b)
1
Yellow solid, Rf: 0.4 (CHCl3); mp 129ꢀ130oC; H NMR (CDCl3,
General procedure for the synthesis of compounds (4aꢀf)
35 300 MHz, δ ppm): 2.90 (t, 2H, J = 6.3 Hz, CH2), 3.12 (t, 2H, J =
6.3 Hz, CH2), 3.85 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 5.91 (s,
1H, OH), 6.68 (s, 1H, ArH), 6.69 (d, 1H, J = 2.4 Hz, ArH), 6.84ꢀ
7.03 (m, 3H, ArH), 7.77 (s, 1H, CH), 8.10 (d, 1H, J = 8.7 Hz,
ArH); 13C NMR (CDCl3, 75 MHz, δ ppm): 27.71, 29.62, 55.83,
40 56.39, 112.66, 113.19, 113.64, 114.84, 124.08, 127.59, 128.76,
131.09, 134.15, 136.76, 145.95, 146.69, 146.81, 163.90, 187.14;
ESIMS (C19H18O4): m/z = 311 [M+H]+.
90 8ꢀMethoxyꢀ4ꢀphenylꢀ5,6ꢀdihydrobenzo[h]quinazolinꢀ2ꢀamine
(4a)
In a 100 mL round bottom flask, NaH (50 mg, 2.00 mmol) and
guanidine hydrochloride (1.43 g, 1.50 mmol) in choline chloride
(ChCl) (10 mL) was stirred on ice bath at 0°C for 10 minutes.
95 Afterwards, 3a (2.80 g, 1.00 mmol)) dissolved in choline chloride
(ChCl, 2.5 mL) was added to the reaction mixture and reaction
mixture was allowed to stirred at 140°C for 5 h. The progress of
reaction was monitored by thin layer chromatography. After the
completion of reaction, the mixture was diluted with water and
(E)ꢀ2ꢀ(3ꢀhydroxyꢀ4ꢀmethoxybenzylidene)ꢀ6ꢀmethoxyꢀ3,4ꢀ
dihydronaphthalenꢀ1(2H)ꢀone (3c)
45 Yellow solid, Rf: 0.26 (22% EtOAc in hexane); mp 173ꢀ174oC; 100 extracted with ethyl acetate. The organic phase was separated,
1H NMR (DMSOꢀD6, 300 MHz, δ ppm): 2.87 (t, 2H, J = 6.3 Hz,
CH2), 3.01 (t, 2H, J = 6.3 Hz, CH2), 3.77 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 6.85ꢀ6.94 (m, 5H, ArH), 7.87 (s, 1H, CH), 9.18 (s,
1H, OH); 13C NMR (DMSOꢀD6, 75 MHz, δ ppm): 27.58, 29.10,
dried over anhydrous sodium sulphate and concentrated to give
crude material which on column chromatography on basic
alumina using methanol in chloroform as eluent gave desired
compound 4a.
50 56.36, 56.49, 112.94, 113.12, 114.44, 117.69, 123.10, 127.34, 105 Offꢀwhite solid, Rf: 0.17 (10% methanol in chloroform); mp 263ꢀ
1
128.99, 130.67, 134.21, 136.23, 146.64, 147.18, 149.31, 164.01,
186.30; ESIMS (C19H18O4): m/z = 349 [M+K]+.
265oC; IR (KBr, νmax/cmꢀ1): 3357 (NH2), 3489 (OH); H NMR
(DMSOꢀD6, 300 MHz, δ ppm): 2.74 (bs, 2xCH2), 3.80 (s, 3H,
OCH3), 5.74 (s, 1H, OH), 6.31 (s, 2H, NH2), 6.83ꢀ6.92 (m, 4H,
ArH), 7.42 (d, 2H, J = 8.4 Hz, ArH), 8.09 (d, 1H, J = 8.4 Hz,
110 ArH); 13C NMR (DMSOꢀD6, 75 MHz, δ ppm): 24.71, 28.94,
56.09, 113.38, 113.50, 113.77, 115.52 (2xC), 126.86, 127.63,
(E)ꢀ2ꢀ(4ꢀhydroxybenzylidene)ꢀ6,7ꢀdimethoxyꢀ3,4ꢀ
dihydronaphthalenꢀ1(2H)ꢀone (3d)
55 Yellow solid, Rf: 0.53 (50% EtOAc in hexane); mp 247ꢀ248oC;
This journal is © The Royal Society of Chemistry [2015]
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DOI: 10.1039/C5RA24429C
129.97, 131.14 (2xC), 142.19, 158.86, 160.50, 161.78, 162.81, 55 Offꢀwhite solid, Rf: 0.66 (EtOAc); mp 223ꢀ224oC; UV(CH3CN,
165.07; ESIMS (C19H17N3O2): m/z = 320.4 [M+H]+.
λmax/nm (ε/dm3 molꢀ1 cmꢀ1): 208 nm (230), 282 nm (70), 313 nm
(60), 357 nm (90); H NMR (DMSOꢀd6, 300 MHz, δ ppm): 2.62
1
4ꢀ(2ꢀaminoꢀ8ꢀmethoxyꢀ5,6ꢀdihydrobenzo[h]quinazolinꢀ4ꢀyl)ꢀ2ꢀ
methoxyphenol (4b)
(t, 2H, J = 4.5 Hz, CH2), 2.70 (t, 2H, J = 4.5 Hz, CH2), 3.75 (s,
3H, OCH3), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 6.25 (s, 2H,
60 NH2), 6.74 (d, 1H, J = 1.8 Hz, ArH), 6.84 (s, 1H, ArH), 6.86 (s,
1H, ArH), 6.96 (d, 1H, J = 1.2 Hz, ArH), 7.70 (s, 1H, ArH); 7.70
(s, 1H, OH); 13C NMR (DMSOꢀd6, 75 MHz, δ ppm): 24.92,
28.13, 56.40, 56.47 (2xC), 109.05, 111.88, 112.23, 113.94,
117.52, 118.39, 126.31, 132.04, 133.74, 148.36, 149.88, 150.06,
65 151.52, 160.37, 162.65, 165.66; ESIMS (C21H21N3O4): m/z = 380
[M+H]+.
5
Offꢀwhite solid, Rf: 0.57 (10% methanol in chloroform); mp 271ꢀ
o
272 C; UV (CH3CN, λmax/nm (ε/dm3 molꢀ1 cmꢀ1): 203 nm (280),
1
224 nm (150), 280 nm (90), 348 nm (110); H NMR (DMSOꢀD6,
300 MHz, δ ppm): 2.69ꢀ2.76 (m, 2xCH2), 3.73 (s, 3H, OCH3),
3.76 (s, 3H, OCH3), 6.10 (bs, 2H, NH2), 6.73 (d, 1H, J = 8.1 Hz,
10 ArH), 6.80 (d, 1H, J = 2.1 Hz, ArH), 6.85 (d, 1H, J = 2.4 Hz,
ArH), 6.88 (m, 1H, ArH), 7.04 (s, 1H, ArH), 8.03 (d, 1H, J = 8.7
Hz, ArH); 13C NMR (DMSOꢀD6, 75 MHz, δ ppm): 24.74, 28.91,
56.10, 56.57, 113.49, 113.78, 114.03, 115.69, 122.69, 126.71,
127.67, 142.21, 148.00, 148.40, 161.82, 162.63; ESIMS
15 (C20H19N3O3): m/z = 350 [M+H]+.
General procedure for the synthesis of compounds (6aꢀd)
4ꢀ(4ꢀ(2ꢀ(piperidinꢀ1ꢀyl)ethoxy)phenyl)ꢀ5,6ꢀdihydroꢀ8ꢀ
70 methoxybenzo[h] quinazolinꢀ2ꢀamine (6a)
4ꢀ(2ꢀaminoꢀ8ꢀmethoxyꢀ5,6ꢀdihydrobenzo[h]quinazolinꢀ4ꢀyl)ꢀ2ꢀ
methoxyphenol (4c)
In
a
round bottom Flask, 4ꢀ(2ꢀaminoꢀ5,6ꢀdihydroꢀ8ꢀ
methoxybenzo[h]quinazolinꢀ4ꢀyl)phenol (4a, 320 mg, 1.00 mmol)
was stirred in the mixture of acetone (40 mL) and chloroform (25
mL) at 80°C. After stirring for ten minutes, anhydrous K2CO3
75 (210 mg, 1.50 mmol) and 2ꢀpiperidinylethylchloride
hydrochloride 5 (2.40 mg, 1.30 mmol) was added in reaction
mixture. The progress of reaction was monitored by thin layer
chromatography. After the completion of the reaction, solvent was
evaporated and residue was diluted with chloroform and extracted
80 with water. The organic phase was separated, evaporated and
dried. The crude was purified by column chromatography with a
mixture of chloroformꢀhexane (10%) to give the desired product
6a.
Offꢀwhite solid, Rf: 0.56 (10% methanol in chloroform); mp 223ꢀ
224oC; UV (CH3CN, λmax/nm (ε/dm3 molꢀ1 cmꢀ1): 204 nm (760),
20 226 nm (450), 279 nm (250), 347 nm (350); 1H NMR (DMSOꢀD6,
300 MHz, δ ppm): 2.76 (bs, 4H, 2xCH2), 3.81 (s, 6H, 2xOCH3),
6.31 (s, 2H, NH2), 6.86ꢀ7.04 (m, 5H, ArH), 8.10 (d, 1H, J = 8.4
Hz, ArH), 9.15 (s, 1H, OH); 13C NMR (DMSOꢀD6, 75 MHz, δ
ppm): 24.70, 28.99, 56.20, 56.57, 113.52, 113.75, 117.26, 117.37,
25 120.96, 127.01, 127.75, 132.57, 142.20, 146.91, 146.94, 149.32,
161.00, 162.11, 162.79, 165.23; ESIMS (C20H19N3O3): m/z = 350
[M+H]+.
4ꢀ(2ꢀaminoꢀ8ꢀmethoxyꢀ5,6ꢀdihydrobenzo[h]quinazolinꢀ4ꢀyl)ꢀ2ꢀ
methoxyphenol (4d)
Offꢀwhite solid; Rf: 0.23 (10% methanol in chloroform); mp 152ꢀ
85 155oC; IR (KBr, νmax/cmꢀ1): 3396 (NH2); H NMR (CDCl3, 300
1
30 Offꢀwhite solid, Rf: 0.11 (2% methanol in chloroform); mp 248ꢀ
249oC; UV (CH3CN, λmax/nm (ε/dm3 molꢀ1 cmꢀ1): 204 nm (250),
225 nm (160), 279 nm (110), 354 nm (10 0); 1H NMR (DMSOꢀd6,
300 MHz, δ ppm): 2.75 (bs, 2xCH2), 3.81 (s, 6H, 2xOCH3), 6.27
(s, 2H, NH2), 6.83 (s, 1H, ArH), 6.87 (d, 2H, J = 6.9 Hz, ArH),
35 7.42 (d, 2H, J = 8.4 Hz, ArH), 7.70 (s, 1H, ArH), 8.25 (s, 1H,
OH); 13C NMR (DMSOꢀd6, 75 MHz, δ ppm): 24.87, 28.11, 56.42
(2xC), 109.07, 111.91, 113.88, 115.54 (2xC), 126.00, 126.26,
131.12 (2xC), 133.74, 148.39, 151.58, 158.80, 160.55, 162.70,
165.04; ESIMS (C20H19N3O3): m/z = 350 [M+H]+.
MHz, δ ppm): 1.26 (bs, 2H, CH2), 1.46 (bs, 4H, 2xCH2), 2.54 (bs,
4H, 2xNCH2), 2.77ꢀ2.86 (bs, 6H, NCH2 & 2xCH2), 3.85 (s, 3H,
OCH3), 4.17 (t, 2H, OCH2), 5.00 (s, 2H, NH2), 6.73 (d, 1H, J =
2.1 Hz, ArH), 6.86 (d, 1H, J = 2.4 Hz, ArH), 6.89 (d, 2H, J = 2.1
90 Hz, ArH), 7.50 (d, 2H, J = 8.7 Hz, ArH), 8.20 (d, 1H, J = 8.7 Hz,
ArH); 13C NMR (CDCl3, 75 MHz, δ ppm): 24.52, 24.73, 26.24
(2xC), 29.16, 55.43 (2xC), 55.72, 58.20, 66.41, 112.95, 113.24,
114.72 (2xC), 115.43, 126.67, 127.83, 130.54 (2xC), 131.41,
141.92, 159.79, 161.36, 161.94, 161.98, 165.04; ESIMS
95 (C26H30N4O2): m/z = 431.5 [M+H]+.
40 4ꢀ(2ꢀaminoꢀ8ꢀmethoxyꢀ5,6ꢀdihydrobenzo[h]quinazolinꢀ4ꢀyl)ꢀ2ꢀ
methoxyphenol (4e)
4ꢀ(4ꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethoxy)phenyl)ꢀ5,6ꢀdihydroꢀ8ꢀ
methoxybenzo[h]quinazolinꢀ2ꢀamine (6b)
Offꢀwhite solid, Rf: 0.34 (EtOAc); mp 271ꢀ272oC; UV(CH3CN,
λmax/nm (ε/dm3 molꢀ1 cmꢀ1): 209 nm (400), 281 nm (100), 313 nm
Offꢀwhite solid; Rf: 0.23 (10% methanol in chloroform); mp 118ꢀ
1
122oC; IR (KBr, νmax/cmꢀ1): 3398 (NH2); H NMR (DMSOꢀD6,
1
(60), 357 nm (160); H NMR (DMSOꢀd6, 300 MHz, δ ppm): 2.70
100 300 MHz, δ ppm): 1.18 (bs, 1H, CH2), 1.47 (bs, 2H, CH2), 1.68
(bs, 3H, CH2), 2.47ꢀ2.84 (bs, 8H, 3x NCH2 & CH2), 3.77 (s, 3H,
OCH3), 4.11(bs, 2H, OCH2), 6.33 (s, 2H, NH2), 6.82 (s, 1H, ArH),
6.90 (d, 1H, J = 8.7 Hz, ArH), 6.99 (d, 2H, J = 8.1 Hz, ArH), 7.48
(d, 2H, J = 8.1 Hz, ArH), 8.07 (d, 1H, J = 8.4 Hz, ArH); 13C NMR
105 (DMSOꢀD6, 75 MHz, δ ppm): 22.96, 23.96 (2xC), 24.65, 25.73,
28.89, 54.83 (2xC), 55.00, 56.09, 67.38, 113.42, 113.85, 114.70
(2C), 126.79 (2xC), 127.67, 131.09, 131.60, 142.19, 159.67,
160.62, 161.83, 162.85; ESIMS (C25H28N4O2): m/z = 417.5
[M+H]+.
45 (bs, 2H, CH2), 2.83 (bs, 2H, CH2), 3.75 (s, 3H, OCH3), 3.81 (s,
3H, 2xOCH3), 6.18 (s, 2H, NH2), 6.71 (d, 1H, J = 8.1 Hz, ArH),
6.88 (s, 1H, ArH), 6.94 (d, 1H, J = 8.1 Hz), 7.08 (s, 1H, ArH),
7.70 (s, 1H, OH), 13C NMR (DMSOꢀd6, 75 MHz, δ ppm): 25.33,
28.30, 56.44, 56.50 (2xC), 109.14, 111.89, 113.61, 114.30,
50 116.66, 123.72, 126.63, 133.55, 142.00, 148.36, 149.40, 151.39,
154.00, 160.19, 162.59, 165.65; ESIMS (C21H21N3O4): m/z =
380.4 [M+H]+.
4ꢀ(2ꢀaminoꢀ8ꢀmethoxyꢀ5,6ꢀdihydrobenzo[h]quinazolinꢀ4ꢀyl)ꢀ2ꢀ
methoxyphenol (4f)
This journal is © The Royal Society of Chemistry [2015]
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DOI: 10.1039/C5RA24429C
4ꢀ(4ꢀ(2ꢀ(dimethylamino)ethoxy)phenyl)ꢀ5,6ꢀdihydroꢀ8ꢀ
methoxybenzo[h]quinazolinꢀ2ꢀamine (6c)
Off white solid; Rf: 0.40 (30% acetone in hexane); mp 109ꢀ110
1
oC; H NMR (DMSOꢀd6, 300 MHz, δ ppm): 1.19 (t, 3H, J = 7.2
Hz, CH3), 1.40 (bs, 4H, 2xCH2), 2.38 (t, 2H, J = 6.9 Hz, CH2),
60 2.77 (bs, 4H, 2xCH2), 3.82 (s, 3H, OCH3), 4.05 (m, 4H, 2xOCH2),
6.34 (s, 2H, NH2), 6.86 (s, 1H, ArH), 6.92 (d, 1H, J = 8.7 Hz,
ArH), 7.01 (d, 2H, J = 8.4 Hz, ArH), 7.52 (d, 2H, J = 8.4 Hz,
ArH), 8.11 (d, 1H, J = 8.4 Hz, ArH); 13C NMR (CDCl3, 75 MHz,
δ ppm): 14.98, 22.07, 24.65, 28.86, 34.02, 35.00, 56.10, 60.59,
65 68.01, 113.41, 113.53, 113.85, 114.65 (2xC), 126.81, 127.66,
131.08 (2xC), 131.49, 142.20, 159.85, 160.59, 161.83, 162.86,
164.77, 173.65; ESIMS (C26H29N3O4): m/z = 448.2 [M+H]+.
Offꢀwhite solid; Rf: 0.23 (10% methanol in chloroform); mp 114ꢀ
1
116oC; IR (KBr, νmax/cmꢀ1): 3318 (NH2); H NMR (DMSOꢀD6,
5
300 MHz, δ ppm): 2.21 (s, 6H, 2xNCH3), 2.48 (s, 2H, NCH2),
2.62 (bs, 4H, 2xCH2), 3.79 (s, 3H, OCH3), 4.07(d, 1H, J = 4.8 Hz,
OCH2), 6.34 (s, 2H, NH2), 6.83 (s, 1H, ArH), 6.90 (d, 1H, J = 7.8
Hz, ArH), 6.99 (d, 2H, J = 7.5 Hz, ArH), 7.49 (d, 2H, J = 6.9 Hz,
ArH), 8.08 (d, 1H, J = 8.4 Hz, ArH); 13C NMR (DMSOꢀD6, 75
10 MHz, δ ppm): 24.65, 28.90, 46.40 (2xC), 56.10, 58.51, 66.75,
113.40, 113.53, 113.84, 114.68 (2xC), 126.81, 127.66, 131.08
(2xC), 131.55, 142.20, 159.75, 160.60, 161.82, 162.85, 164.75;
ESIMS (C23H26N4O2): m/z = 391.5 [M+H]+.
In-vitro Cancer Cell growth inhibition assay
In vitro anticancer activity of synthesized compounds was studied
70 by sulphorhodamine B (SRB) dye based plate assay. In brief, 104
cells per well were added in 96ꢀwell culture plates and incubated
at 37 oC in 5% carbondioxide concentration. After overnight
incubation of cells, serial dilutions of synthesized compound were
added to the wells. Untreated cells served as control. After 48 h,
75 cells were fixed with iceꢀcold triꢀchloroacetic acid (50% w/v, 100
ml per well), stained with SRB (0.4% w/v in 1% acetic acid, 50
ml per well), washed and airꢀdried. Bound dye was solubilize with
10 mM tris base (150 ml per well) and absorbance was read at 540
nm on a plate reader. The cytotoxic effect of compound was
80 calculated as % inhibition in cell growth as per formula: [1ꢀ
(absorbance of drug treated cells/absorbance of untreated
cells)x100]. Determination of IC50 (50% inhibitory concentration)
was based on doseꢀresponse curves.
4ꢀ(4ꢀ(1ꢀ(dimethylamino)propanꢀ2ꢀyloxy)phenyl)ꢀ5,6ꢀdihydroꢀ
15 8ꢀmethoxybenzo[h]quinazolinꢀ2ꢀamine (6d)
Offꢀwhite solid; Rf: 0.24 (10% methanol in chloroform); mp 104ꢀ
106oC; IR (KBr, νmax/cmꢀ1): 3318 (NH2); 1H NMR (DMSOꢀD6,
300 MHz, δ ppm): 1.04 (d, 3H, J = 4.6 Hz, CH3) (s, 6H, 2xNCH3),
2.48 (s, 1H, NCH2), 2.73 (bs, 4H, 2xCH2), 2.91 (d, 1H, J = 5.1
20 Hz, NCH2), 3.78 (s, 3H, OCH3), 4.04(d, 1H, J = 4.8 Hz, OCH),
6.34 (s, 2H, NH2), 6.83 (s, 1H, ArH), 6.90 (d, 1H, J = 7.8 Hz,
ArH), 6.99 (d, 2H, J = 7.5 Hz, ArH), 7.49 (d, 2H, J = 6.9 Hz,
ArH), 8.08 (d, 1H, J = 8.4 Hz, ArH); 13C NMR (DMSOꢀD6, 75
MHz, δ ppm): 24.66, 25.72, 28.90, 41.84(2xC), 46.75, 56.09,
25 58.40, 70.49, 113.40, 113.52, 113.83, 114.72, 115.60, 126.81,
127.66, 131.09, 131.55, 142.19, 159.85, 160.60, 161.82, 162.86,
164.75; ESIMS (C24H28N4O2): m/z : 405.5 [M+H]+.
Tubulin polymerization assay
General procedure for the synthesis of compounds (8aꢀb)
85 To a pre warmed 96 well plate, compounds were added at a conc
of 10, 2, 0.4 and 0.08 ꢁg/ml. Paclitaxel was used as a standard at a
conc of 10ꢁM.Tubulin protein provided in the kit was added to a
pre prepared ice cold tubulin polymerization buffer [80mM PIPES
pH 6.9, 2mM MgCl2, 0.5mM EGTA] immediately before use and
90 was kept on ice. The ratio at which tubulin protein is mixed with
the tubulin polymerization buffer was as 200 ꢁL of protein with
420ꢁL of tubulin polymerization buffer that gave a final
concentration of 3mg/mL tubulin in 80mM PIPES pH 6.9, 2mM
MgCl2, 0.5mM EGTA.
30 Ethyl
4ꢀ(4ꢀ(2ꢀaminoꢀ8ꢀmethoxyꢀ5,6ꢀ
dihydrobenzo[h]quinazolinꢀ4ꢀyl)phenoxy)butanoate (8a)
In a 100 mL round bottom flask, 4ꢀ(2ꢀaminoꢀ5,6ꢀdihydroꢀ8ꢀ
methoxybenzo[h]quinazolinꢀ4ꢀyl)phenol (4a, 319 mg, 1.00
mmol)) and was stirred in dimethylformamide (DMF, 9 mL) at
35 80°C. After five minutes, anhydrous K2CO3 (210 mg, 1.5 mmol)
and substituted ethyl nꢀbromoalkanoate 7 (172 mg, 1.50 mmol)
was added in reaction mixture and stirred the reaction for 5 h. The
reaction mixture was poured in cold water with vigorous stirring
and neutralized it with N/5 HCl solution. The precipitate was
40 filtered and dried. The crude was purified by column
chromatography with a mixture of chloroformꢀmethanol (0.5 to
1.0%) to give the desired product 8a.
95 50 ꢁL of this diluted tubulin solution was added to each well
containing the treated compound at different concentration and
immediately the plate was read at 37 oC with pre set kinetic
parameters at 340 nm wavelength for a period of 60 min.
Light yellow solid; Rf: 0.26 (30% acetone in hexane); mp 148ꢀ149
oC; IR (KBr, νmax/cmꢀ1): 3474 (NH2), 1728 (C=O) ; 1H NMR
45 (DMSOꢀd6, 300 MHz, δ ppm): 1.67 (t, 3H, J = 6.9 Hz, CH3), 1.97
(t, 2H, J = 6.9 Hz, CH2), 2.45 (p, 2H, J = 7.2 Hz, CH2), 2.72 (bs,
4H, 2xCH2), 3.78 (s, 3H, OCH3), 4.00 (m, 4H, 2xOCH2), 6.31 (bs,
2H, NH2), 6.88 (d, 1H, J = 2.1 Hz, ArH), 6.90 (d, 1H, J = 8.7 Hz,
ArH), 6.98 (d, 2H, J = 8.7 Hz, ArH), 7.49 (d, 2H, J = 8.7 Hz,
The increase in absorbance at the pre set Kinetic parameters ( 340
100 nm, 37 C over 60 min period of time) is graphically obtained as
o
the polymerization curve and the Vmax values obtained by the
Skanit software 4.0 are used to interpret the results for analyzing
the effect of the compounds on the tubulin polymerization.
Cell cycle analysis
50 ArH), 8.08 (d, 1H, J = 8.7 Hz, ArH); 13C NMR (DMSOꢀd6, 75 105 Cell cycle distribution was measured in concentration and time
MHz, δ ppm): 14.92, 24.61, 25.05, 28.86, 31.03, 56.08, 60.79,
67.46, 113.41, 113.52, 113.94, 114.67 (2xC) 126.72, 127.68,
131.07 (2xC), 131.53, 142.22, 159.71, 160.64, 161.85, 162.79,
164.75, 173.48; ESIMS (C25H27N3O4): m/z = 434.2 [M+H]+.
dependent manner by flow cytometric analysis of PIꢀstained
cellular DNA, as described earlier. Briefly, MCFꢀ7 cells (8 x 105
per well) were seeded in 60 mm tissue culture dishes and grown
overnight (37 ºC, 5% CO2). Compound treated cells were
110 harvested by trypsinization and fixed (30 min, 4˚C) with iceꢀcold
70% ethanol at indicated time points. The pellets were washed
55 Ethyl
5ꢀ(4ꢀ(2ꢀaminoꢀ8ꢀmethoxyꢀ5,6ꢀ
dihydrobenzo[h]quinazolinꢀ4ꢀyl)phenoxy)pentanoate (8b)
This journal is © The Royal Society of Chemistry [2015]
RSC Adv., 2015, XXX, 01–09
| 11

RSC Advances
Page 12 of 13
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DOI: 10.1039/C5RA24429C
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with PBS and reꢀsuspended in a solution containing PI (20 µg/ml),
Triton X100 (0.1%) and RNase (0.1mg/ml) in PBS. After
incubation (45 min, in the dark, 37˚C), cells were analysed on a
FACS Calibur flow cytometer (BD Biosciences). Distribution of
cells in different phases of cell cycle was calculated using “Cell
Quest” software.
5
Koskimies,
L,
Pirkkala, U.S.
Pat.
Appl.
Publ. 2006, US 20060281710 A1 20061214. (b) J. Messinger,
H. H. Thole, B. Husen, M. Weske, P. Koskimies, L, Pirkkala,
60
Western blot assay
M.
Weske,
PCT
Int.
Cells were grown overnight in 60 mm tissue culture dishes and
Appl. 2006, WO 2006125800 A1 20061130.
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M. Simard, G. Berube, E. Asselin, Endocr. Rel. Can. 2009, 16,
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70 11. A. Gupta, P. Saha, C. Descoteaux, V. Leblanc, E. Asselin,
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T. Pienkowski, A. JagielloꢀGruszfeld, J. Crown, A. Chan, B.
Kaufman, D. Skarlos, M. Campone, N. Davidson, M. Berger,
10 were exposed to vehicle and test molecule for 24 and 48 h at IC50
concentration. Cells were then harvested and lysed with MꢀPER
reagent (Thermo Scientific) supplemented with protease and
phosphatase inhibitors. After centrifugation at 12000 rpm,
supernatant was collected and protein quantity was measured by
15 BCA protein assay kit (Thermo Scientific). Equal amount of
proteins (20 µg) were resolved in 8% SDSꢀPAGE, transferred
onto PVDF membrane and probed overnight with antiꢀPARP
(Cell Signaling Technolgy) and antiꢀactin (SigmaꢀAldrich)
antibodies. After incubation with HRPꢀconjugated secondary
20 antibody for 1 h at room temperature, ECL solution (BioꢀRad)
was added to the membrane and luminescence was detected by
Chemidoc XRS+ system (BioꢀRad).
65
75
C. Oliva, S. D. Rubin, S. Stein, D. Cameron, N. Engl. J. Med.
2006, 355, 2733.
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Ligand and protein preparation for docking experiments
A. Decosterd, AꢀC. GairardꢀDory, G. UbeaudꢀSéquier, N.
Widmer, Ther. Drug Monit., 2015, 37, 2.
The molecular docking of control inhibitors tamoxifen, colchicine
25 and compounds 4aꢀf, 6aꢀd and 8aꢀb were executed by using
software Autodock v 1.5.6 [23]. The 3.5 Å 3D crystallographic
structure of estrogen receptor alpha & tubulin were retrieved
80 14. T. Araki, H. Yashima, K. Shimizu, T. Aomori, T. Hashita, K.
Kaira, T. Nakamura, K. Yamamoto, Clin. Med. Insights: Oncol.
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Drug Metab. Toxicol. 2014, 10, 469. (b) A. D. Luca, A.
through
Brookhaven
Protein
DataBank
(PDB)
(http://www.pdb.org) (PDB ID: 3ERT and 4O2B). The ligand and
30 receptor preparation was done by using Discovery studio v3.5
(Accelrys, Inc., San Diego, CA, USA). The receptor grid was
generated using the ligands tamoxifen and colchicine coꢀ
crystallized in receptor. The standardization of software was done
by redocking study of bound inhibitor. Visualization of the
35 docked conformation was performed in pymol.
85
D’Alessio, M. R. Maiello, M. Gallo, S. Bevilacqua, D.
Frezzetti, A. Morabito, F. Perrone, N. Normanno, Expert.
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17. http://en.wikipedia.org/wiki/Choline.
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Acknowledgements
The authors thank the Director, CSIRꢀCIMAP and CSIRꢀCDRI
for financial support. The work was carried out under project
ChemBio (BSCꢀ203) to AG and GAP 0115 (sponsored by DST,
40 India) to JS. AS and FK acknowledge the Science & Engineering
Research Board (SERB), Department of Science & Technology
(DST), New Delhi, India for financial support through GAPꢀ260
project (Sanction No.: SR/FT/LSꢀ25/2010; 02/05/2012).
95 20. S. B. Phadtare, G. S. Shankarling, Green Chem., 2010, 12,
458.
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Notes and references
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This journal is © The Royal Society of Chemistry [2015]
RSC Adv., 2015, XXX, 01–09
| 12

Page 13 of 13
RSC Advances
View Article Online
DOI: 10.1039/C5RA24429C
RSC Advances
Graphical Abstract:
Synthesis of 4-phenyl-5,6-dihydrobenzo[h]quinazolines and their evaluation as growth inhibitors of
carcinoma cells
Hardesh K Maurya, Mohammad Hasanain, Sarita Singh, Jayanta Sarkar, Vijaya Dubey, Aparna Shukla,
Suaib Luqman, Feroz Khan, Atul Gupta*
Synthesis of various benzo[h]quinazoline analogs (4a-f, 6a-6d and 8a-b) was accomplished through the reaction of chalcone with guanidine. The
synthesized compounds (4a-f, 6a-6d & 8a-b) were screened for their anticancer potential against different cancer cells viz MCFꢀ7, DLD1, A549,
Du145 & FaDu cell lines. Compounds 4a, 6a-d & 8b showed significant anticancer activity in these cancer cell lines within the range of IC50
values of 1.5ꢀ12.99 µM. Functional study of a promising molecule, 6d, at 7 µM (at IC50 value) for 24 and 48 h showed that it possessed
anticancer activity through triggering apoptosis. In tubulin polymerization assay, 6d effectively inhibited tubulin polymerization at IC50 2.27 µM.
In-silico docking study of 6d revealed that 6d had good affinity with estrogen receptor as well as tubulin protein on its βꢀsheet of colchicines
binding site.
NH2
OH
16
N
B
N
C
D
C
R
15
A
B
A
D
7
HO
H3CO
OR
4-phenyl-5,6-dihydrobenzo[hquinazolines]
17 -Estradiol
Orientation of modified groups