Identification of potent cholecystokinin-B receptor antagonists: Synthesis, molecular modeling and anti-cancer activity against pancreatic cancer cells

Article abstract of DOI:10.1039/c7md00171a

Advanced malignant stages of pancreatic cancer have poor prognosis and very few treatment strategies are available. Pancreatic cancer is known to possess unique growth-related receptors that when activated, stimulate tumour proliferation. Gastrin and its related peptide cholecystokinin (CCK) are also significantly involved in the growth of this cancer type as well as other malignancies through activation of the cholecystokinin-B receptor (CCK-BR). New treatment strategies with CCK-BR antagonists are being suggested that suppress the growth promoting effects of gastrin. In this paper, we report the development of two series of quinazolinone derivatives incorporating hydrazinecarbothioamide (compounds 3a-g) and the hydrazino group (compounds 4a-e) as linkers for developing CCK-BR antagonists. The affinities of the compounds were determined using docking into the CCK-BR homology modeled structure. The compounds were tested for in vitro CCK-BR binding and gastric acid secretion in an isolated lumen-perfused mouse stomach assay. The compounds exhibited CCK-BR binding activity (IC₅₀) in the range of 0.2-975 nM and showed good gastric acid secretion inhibitory activity. Molecular modeling of the compounds was done and pharmacophore mapping results showed good prediction of in vitro activity which correlated well with the experimental antagonistic activity. The compounds were further tested for their cytotoxicity on CCK-BR expressing pancreatic cancer cells. The results of the study provided two potent CCK-BR antagonists which also possess good to moderate growth inhibitory activities against pancreatic cancer cells.

Full text of DOI:10.1039/c7md00171a

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Chowdhary, M. Sikka, P. Verma, P. Jha, A. K. Mishra, D. Saluja and M. Chopra, Med. Chem. Commun.,
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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Identification of Potent Cholecystokinin-B Receptor Antagonists: Synthesis, Molecular Modeling and anti-cancer activity
against Pancreatic Cancer Cells.
Saroj Kumari, †, Joyita Chowdhurya,†, Manisha Sikkaa, Priyanka Vermaa, Prakash Jhaa, Anil K. Mishrab, Daman Salujaa and
Madhu Chopra*a
* Corresponding Author
[†] These authors contributed equally to the work
a. Laboratory of Anticancer Drug Development, Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi,
Delhi 110007, India
E-mail:mchopradu@gmail.com, mchopra@acbr.du.ac.in
b. Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Majumdar Road, Delhi 110054, India
Abstract: Advanced malignant stage of pancreatic cancer has poor prognosis and very few treatment strategies are
available. Pancreatic cancer is known to possess unique growth-related receptors that when activated stimulate tumour
proliferation. Gastrin and its related peptide cholecystokinin (CCK) are also significantly involved with growth of this
cancer type as well as other malignancies through activation of the cholecystokinin-B receptor (CCK-BR). New treatment
strategies with CCK-BR antagonists are being suggested that suppress the growth promoting effects of gastrin. In this
paper, we report development of two series of quinazolinonederivatives incorporating hydrazinecarbothioamide
(compounds 3a-g) and hydrazino group (Compounds 4a-e) as linkers for developing CCK-BR antagonists. The affinities of
the compounds were determined using docking into CCK-BR homology modeled structure. The compounds were tested for
in vitro CCK-BR binding and gastric acid secretion in an isolated lumen-perfused mouse stomach assay. The compounds
exhibited CCK-BR binding activity (IC50) in the range of 0.2 – 975 nM and showed good gastric acid secretion inhibitory
activity. Molecular modeling of the compounds was done and pharmacophore mapping results showed good prediction of
in vitro activity which correlated well with the experimental antagonistic activity. The compounds were further tested for
their cytotoxicity on CCK-BR expressing pancreatic cancer cells. The results of the study provided two potent CCK-BR
antagonists which also possess good to moderate growth inhibitory activities against pancreatic cancer cells.
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Introduction
Pancreatic cancer in advanced malignant stages has a poor prognosis1,2 and currently available therapeutic
modalities have not markedly improved survival rate3. Pancreatic cancer is the twelfth most common cancer in
the world. It is suggested that this malignancy will surpass colon and breast cancer in the next decade with
current rate of rise in incidence and therefore will become second leading cause of cancer related deaths in the
USA.4
Gastrointestinal peptides such as gastrin and cholecystokinin (CCK) have been reported to have growth
stimulatory effects on pancreatic cancer and the receptor for these peptides is markedly over-expressed in
pancreatic cancer5,6. These peptide hormones activate their receptors (CCK in this case) in pancreatic cancer
through induction of AKT pathway7 resulting in cell proliferation. There is sufficient evidence that CCK-BR pathway
is a key driver of pancreatic carcinogenesis, pancreatic cancer and other malignancies8.
Several studies have reported presence of a mutated form of CCK-BR containing the translated 4th intron, termed
as CCK-C or CCK- cancer receptor (CCK-CR) in pancreatic cancer9. Studies suggest that these additional 69 amino
acids of the CCK-C receptor render the receptor constitutively active where it induces proliferation even in the
absence of gastrin10. Thus blocking this receptor in pancreatic cancer becomes even more important to slow down
the proliferation. Moreover, it is also interested to know that both CCK-BR and its splice variant CCK-CR bind to
CCK and gastrin11.
Scheme 1: Design of CCK-BR antagonists
Several treatment strategies are currently being exploited to interrupt the CCK/gastrin: CCK-receptor (CCK-R)
pathway in cancer. In the last decade, several highly selective CCK-A and CCK-B receptor antagonists have been
developed12. This includes use of oligonucleotides to gastrin13, gastrin specific antibodies14, as well as using gastrin
vaccine, “Gastrimmune”, or G17DT. A randomized placebo controlled study using vaccine therapy demonstrated a
significant survival benefit in pancreatic cancer patients that elicited neutralizing antibodies towards gastrin
response to vaccination with G17DT.
In addition to above strategies; studies have been reported to stop the CCK-R activation through use of specific
antagonists though an initial clinical trial done with MK-329, a selective CCK-A receptor antagonist failed15,16. Since
it is well known fact that the CCK-BR subtype is the primary type of receptor that mediates the cancer growth in
pancreatic cancer in humans, the research focus has thus been shifted to use of CCK-BR specific antagonists. E.g. a
highly selective CCK-BR antagonist, netazepide (YF476) was used to treat patients with type 1 gastrin carcinoid
tumour17 and further studies are needed using more potent and selective antagonists in pancreatic cancer. A
phase Ib/IIa clinical trial is underway to evaluate CCKBR antagonist Z-360 in combination with gemcitabine (a
cytosine analogue and antineoplastic agent, used as an anti-cancer chemotherapy drug) in patients with advanced
pancreatic cancer18. In recent years also, there is a study on Pancreatic ductal adenocarcinomas (PDACs) which
are known to constitutively express the CCKBR, using DNA aptamer AP115319, and it is anticipated that
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new CCKBR AP-targeted nanocarriers will have a broad capability to deliver imaging agents or therapeutic
cargos specifically to PDAC cells with minimal off-target effects.
There is dire need for novel therapeutic agents which can target the gastrin: CCK-B–receptor pathways20 which
may help improve survival of advanced gastric cancer. All above studies provide sufficient evidence that new
treatment regimens with CCK-BR antagonists could suppress the growth promoting effects of gastrin. In the
present work, we are demonstrating synthesis, docking and in vitro evaluation of quinazolinone derivatives as
potent CCK-BR antagonists and their Cytotoxicity evaluation against pancreatic cancer cells (MiaPaca-2).
There are several distinct chemical classes of CCK-BR antagonists that have previously been identified through
drug discovery programs. These include benzodiazepine-based compounds (L-365,260), (YM022), and (YF476);
peptoids (PD-134,308)] and indole (JB93182)21. However, despite the variety of CCK-BR targeted ligands, very few
compounds are currently available for clinical use. This may be because many compounds in this class have been
associated with poor or variable pharmacokinetics, both with respect to gastrointestinal absorption and blood-
brain permeability21. Since our compounds are targeted against pancreatic cancer, poor blood–brain permeability
could be a good tool to distinguish between peripheral and central effects of CCKBR antagonism as already
reported in case of S-050922, which is currently at Phase I clinical trials for gastric secretion disorders. Hence, there
is a need to design new CCK-BR antagonists.
Our lab is developing CCK-BR specific antagonists which have been designed by taking lead from naturally
occurring compound, Asperlicin, a weak and non-selective CCK ligand. In a previous communication, we have
shown design and synthesis of one such potent antagonist linked to fluorescein moiety and used it as imaging
agent to study receptor trafficking in CCK-BR positive cells23. In another communication we have shown synthesis
and in vivo biological activity of another quinazolinone derivative24. We have shown that the two key moieties, a
quinazolinone ring and an aryl ring linked with a proper linker are crucial for CCK-BR binding. Continuing our
efforts further, in the present paper, we report synthesis, biological evaluation of several derivatives of these two
series of compounds. We decided to explore the effect of two types of linkers. One series of compounds (3a-g)
were prepared by incorporating hydrazine carbothioamide –NHNH-CS-NH- as linker between the quinazolinone
and the aryl moiety and the other (4a-e) were synthesized by keeping hydrazino group (=N-NH-) as linker
between the quinazolinone and the indanone. (Scheme 1) and the effect of substitutions in aryl ring was studied.
Results and Discussion
Chemistry
The compounds described in this paper were prepared according to Scheme 2 incorporating either
hydrazinecarbothioamide (Compounds 3a-g) or a hydrazine linker (Compounds 4a-e). In the former series,
quinazolinone ring was prepared by one pot reaction of anthranilic acid with phenylisothiocyanates in glacial
acetic
Scheme 2:
Scheme for the synthesis of Quinazolinone
Derivatives (i) ACOH, reflux (ii) anhy.hydrazine, abs. EtOH (iii)
isothiocyanates, anhy. DMF, 110 oC (iv) isatin, abs. EtOH, reflux.
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acid under reflux conditions. The hydrazone derivatives (compound 2a-g) were obtained by treatment of the
thioxoquinazolinones (compounds 1a-f) with excess of anhydrous hydrazine in refluxing absolute ethanol. The
isothiocyanates which were not commercially available were prepared by the standard methodology reported in
the literature.
The hydrazinoquinazolinones (compounds 2a-g) were treated either with isothiocyanates or isatin to provide
compounds (3a-g) and (4a-e), respectively. One such compound 3a was attached with fluorescein in our previous
communication and has been shown to have affinity in nM range and we successfully used it as fluorescence
probe to image CCK-BR positive cells23. In order to improve pharmacokinetic properties we are reporting synthesis
of derivative of these series of compounds and their antagonistic activities. The cytotoxic activities of these
compounds have also been evaluated against CCK-BR expressing human pancraetic cancer cells (MiaPaca-2).
Molecular Modeling
Pharmacophore mapping and prediction of activity
In a previous paper, we developed pharmacophore model based on six chemically diverse series of compounds
taken from literature25. The pharmacophore model was validated on a test series of compounds taken from
literature and resulted in good prediction of antagonistic activities. The basic features essential for CCK-BR
antagonistic activities identified in the paper were used here for the synthesis of quinazolinone derivatives. The
pharmacophore25 was used in present work to predict the antagonistic activities of the synthesized compounds
which correlated very well with the in-vitro results. All the newly synthesized compounds gave a pharmacophore
fit value in the range of 7.83 – 5.33 with predicted activities in the range of 0.82 nM – 257 nM upon mapping to
the features of the pharmacophore model. (Table1).
Figure1. Mapping of the CCKBR antagonists onto the
pharmacophore model (hypothesis 1). The blue contour
represents the HY-ALI features, the cyan contour represents HY-
AR features, and the dark pink contour represents the HBD
features.
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Comp.
no.
Substitution
Potential Fit
Features Estimated Actual Activity
Activity
scale
CDOCKER
ENERGY
kcalmol-1
CDOCKER
INTERACTION acid
Gastric
Cytotoxicity Evaluation
IC50 (µM)
energy
value
mapped activity
Activity Scale
kcalmol-1
(Estimated) (Actual)
ENERGY
secretion
inhibition
(%)
kcalmol-1
IC50
(nM)
IC50 in
nM
at
100 nM
ND
YF476
-
-
-41.44
0.1167
9.864
7.946
1111
1011
0.0076
0.0636
0.10a
5.2a
+++
+++
+++
+++
-22.52
-19.09
-37.96
-33.84
ND
L365,260
-
-
ND
0.0018
R
R1
3a
3b
3c
3d
3e
3f
p-
OC2H5
m-Br
m-
-
7.75
7.49
7.17
5.33
7.72
7.81
7.83
6.78
5.48
1011
1011
1011
1001
1011
1011
1011
1011
1011
0.985
1.74
3.79
257
0.66
+++
+++
+++
+
+++
+++
+++
+
-19.33
-18.38
-17.73
-20.87
-17.91
-21.37
-16.09
4.78
-36.16
-37.46
-37.85
-35.01
-37.05
-34.33
-37.12
-31
58
62
39
280
150
200
180
80
COOH 67.816
m-
COOH
m-
-67.18
8.64
p-Br
H
-
0.2001
975.4
9.715
2.005
11.48
17.36
56.49
COOH 66.109
m-
COOH
p-
OC2H5
p-Br
-65.37
48 at
µM
1
p-Cl
-44.5
1.05
0.863
0.82
9.37
185.5
+++
+++
+++
+++
++
+++
+++
+++
+++
++
53
p-
OC2H5
p-
OC2H5
p-
OC2H5
p-CH3
-
39
58
48
10
43.528
-44.29
3g
4a
4b
P-Cl
250
80
-
-
48.58
32.48
2.26
-28.26
67 at 300
nM
10
4c
4d
4e
p-Cl
-
-
-
33.82
35.85
34.84
5.55
5.74
6.38
1011
1011
1011
158.2
100.8
23.1
ND
++
++
++
ND
+
5.3
4.5
1.9
-33.43
-28.41
-35.36
58
100
92
m-Cl
m-Br
384.2
19.46
58
62
+++
200
Table 1: CCKBR Antagonists 3a-g and 4a-e produced via Scheme 1.
aactivities were taken from literature determined through similar assay (J. Med. Chem., 1993, 36 (26), pp 4276–4292&G. S. Baldwin, PNAS, 1994, 91, 7593–7597)
bActivity scale: +++ (0-20nM, highly active), ++ (30-200 nM, moderately active), + (>200 nM, poorly active) (J. Chem. Inf. Model., 2005, 45, 1934–1942))
We also compared the fit values and estimated activities with known CCK-BR antagonists YF476 and L365, 260
.
The compound YF476, a highly potent and orally active CCK-BR antagonist showed a fit value of 9.864 with an
estimated activity of 0.0076 nM and mapped to all the four features and has a reported IC50 of 0.10 nM using a
similar assay26. Another compound, L-365,260, showed a fit value of 7.946 and an estimated activity of 0.06367
nM (Actual activity, IC50 = 3.8nM) mapped to three features of thepharmacophore27. As we have shown earlier25; a
fit value from 6 – 8 bits suggests that a compound could be highly active and those with fit value less than 6 bits
might be moderately active or weakly active compounds. The compounds developed in this study showed high
(Fit value 7.83, 0.82 nM as estimated activity) to moderate activity (Fit value 5.33, 257 nM as estimated activity)
and
correlated
well
with
the
corresponding
actual
activities
active compounds mapped similar to the potent compound L-365,260 (Figure. 1).
(Table
1).
The
highly
Homology Modeling of CCK-BR
The sequence alignment of human CCK-BR (ID: P32239) and the template human A2A adenosine receptor was
generated using Clustal W and manually adjusted to avoid the insertions and deletions in the conserved
transmembrane regions. The sequence similarity and sequence identity of the aligned sequence are 19.2 and
36.3% respectively. The best modeled structure with the least DOPE score28 (38262.156250) and PDF energy
score28 (-8161.8936 kcalmol-1) was selected for molecular dynamics simulations
Molecular dynamics simulation and validation
Molecular dynamics (MD) simulations were performed to improve the structure of CCK-BR and to study its bio-
physical properties. The MD simulation results show that the model clearly diverged and the conserved disulphide
bridge remained stable throughout the simulations. The root mean square deviation (RMSD) of less than 1 nm
was obtained in the plot of backbone atoms.
The potential energy analysed during the 4ns molecular dynamics simulation showed that the molecular system is
stabilized and remained stable throughout the molecular dynamics simulations with the average of potential
energy stabilized around -3.47 × 104 kJ/mol.
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Model Evaluation
The model was evaluated using verify 3D score, ERRAT score and favourable residues in Ramachandran plot (See
Supplementary material). The verify 3D score of simulated protein, was 77.83, which is greater than expected low
score as well as the verify score of the modelled protein before simulations. The phi and psi angles of the receptor
were verified by Ramachandran plot which showed 81.8 % residues falling in the favoured region, 99% within
additionally allowed region and none of the binding site residue in the disallowed region. The ERRAT score was
95.68 suggesting ‘very good quality’ of the modelled protein structure.
Active Site Prediction
After obtaining the final model, active sites of the protein were predicted using discovery studio based on
receptor cavity method and further modified manually using the active site residues namely Asn353A6.55
,
Arg356A6.58(TM6), His207(EL2), and Tyr189A4.60(TM4) which are know to be involved in CCK-BR antagonism for
non-peptide antagonists through site directed mutagenesis and docking Studies reported earlier29,30
.
Molecular Docking
The energy minimised structures of the compounds in the study were docked into the active site of the modeled
CCK-BR structure. L365,260 and YF476 both, on being docked intothe modelled CCK-BR active site revealed H-
bond interactions with Arg356A6.58 L365,260), Asn353A6.55and Arg356A6.58
( (YF476). Both the compounds showed
π – positive charge interaction with Arg356A6.58 through their respective aromatic rings (Figure 2). YF476 showed
hydrophobic alkyl interaction with Arg356A6.58 in addition to π–Sulphur bond with Met117 (EL1) and π–Alkyl
hydrophobic bond with Ile375A7.38, Leu113A2.63 and Pro114A2.64. In the case of L365, 260, the benzodiazepine core
also showed π–Alkyl hydrophobic bond with His364 (EL3) and Ala366 (EL3) and Hydrophobic – Alkyl interaction
with Ile375A7.38. Both the ligands were further stabilized by common van der Waals interactions to residues
His376A7.39, Pro114A2.64, Ile375A7.38 and Ala352A6.54
.
Figure 2: Docked structures of L365,260 and YF476, into the homology modelled CCK-B structure.
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Similarly, the common binding pocket for all our synthesized ligands is composed of residues Arg356A6.58
,
Asn353A6.55, Leu113A2.63, Pro114A2.64, His376A7.39, Ser379A7.42, Thr111A2.61, Ile375A7.38and Val349A6.51, which are
in the distance interval of 1.5 to 10 Å from the closest ligand atom. In addition to many hydrophobic contacts that
contributed the most to the binding, ionic and hydrogen bonds could also be identified. The most active
compound 3c showed π– bonding of quinazolinone core with Arg356A6.58, similar to that shown by L365, 260 and
YF476 (discussed above). 3c also interacted through hydrophobic π– π T-shaped bond with His376A7.39and two H
– bonds with Asn353A6.55. The residues, namely Leu113A2.63, Pro114A2.64 and Arg356A6.58 were also found to form
π– alkyl hydrophobic bonds with 3c. Similar to YF476 and L365, 260, it also showed van der Waals interactions
with residues Leu113A2.63, Pro114A2.64, and Ile375A7.38 (Figure3). The compound 3f also showed π– bonding of
quinozolinone core with Arg356A6.58 as seen in L365, 260 and YF476 along with H-bond with Asn353A6.55. It also
showed π– alkyl hydrophobic bonds with His376A7.39 and Pro114A2.64 were further stabilised by hydrophobic-alkyl
bonds with Ile372 and Met134.
Figure 3 :Docked structures of 3a, 3f, 4a and 4b into the homology modelled CCK-B structure.
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In the case of 4a, there was one close contact between the carbonyl group of the quinozolinone core and
Asn353A6.55 (TM6). Compound 4a and 4b, both showed π– bonding of quinozolinone core and Arg356A6.58as seen
in L365, 260 and YF476. Compound 4a also exhibited H-bond with residue Arg213 (EL2) and Vanderwaals
interactions with Pro114A2.64, Ala352A6.54, Ile375A7.38 and Thr111A2.61 (Figure 3). The ligand 4aalso showed
hydrophobic π– alkyl interactions with Arg356A6.58, Val349A6.51and Ala352A6.54, whereas Leu113A2.63, Pro114A2.64
and Arg356A6.58 were found to form π– alkyl hydrophobic bonds with 4b, as also reported in the case of 3c. This is
in agreement with reported interacting residues in earlier studies29,30. Similar to YF476 and L365,260, both 4a and
4b were further stabilized by common van der Waals interactions to residues Pro114A2.64, Ile375A7.38and
Val349A6.51
.
Therefore, the two amino acids Asn353A6.55, Arg356A6.58 (TM6), appear to be much more involved in antagonistic
activity of all ligands through H-Bonds and several residues with hydrophobic interactions. The pharmacophore
mapping results of L365, 260 and YF476 also exhibited four features namely two hydrogen bond donors and two
hydrophobic features. These features/groups were found to be interacting within the binding site of the CCK-BR
receptor through the similar groups in the ligands. Hence the ligand based approach used in our previous study25
could be utilized here to optimize the compounds and docking of these compounds showed important binding
interactions.
Biological Evaluation
Receptor Binding Assay and SAR
The affinity of the compounds for the CCK-BR was determined by a competitive binding assay with a Bolton-
Hunter labelled CCK-8 radioligand, 125I-BH-CCK-8 (Perkin Elmer). Compounds in both the series showed good
binding activities in the range of 0.2 – 975 nM (Table 1). Compound 3c with p-bromo substitution in ring A and m-
COOH in ring B showed the highest affinity (IC500.200nM)followed by 3a23(p-OC2H5,ring A; m-COOH, ring B; IC50
0.66 nM). Compound 3g (p-OC2H5, ring A; p-Cl, ring B) showed IC50 11.48 nM, whereas3e (p-Cl, ring A; p-OC2H5)
,
and 3b (m-Br, ring A; m-COOH, ring B) showed IC50 9.715 and 8.64 nM respectively. Compound 3f (p-OC2H5 ring A;
p-Br, ring B) also showed good affinity (2.005 nM) whereas the unsubstituted ring A in compound 3d resulted in
IC50 975 nM. Therefore, the substitution in Ring A is important for improving the activity and a polar substituent in
ring B improves the activity.
The other series of compounds with hydrazino linker and substitution in only ring A and where ring B being
indanone resulted in overall less active compounds. In this series the most active compound 4a with p-OC2H5
substitution in ring A (IC50 17.36 nM) favoured over p-CH3 (4b, IC50 56.49 nM). m-Br substitution in compound 4e
(IC50 19.46 nM) in ring A resulted in better affinity over m-Cl (IC50 384 nM),. Overall electron donating group
improved the activity in this series of compounds.
To summarize the SAR, we suggest that overall the substitution in ring A is important for CCK-BR antagonistic
activity with p-OC2H5 substitution (3a) and p-Br (3c) in Ring A along with m-COOH group in Ring Bbeing favourable
in the series with hydrazinocarbothioamide linker. Both these compounds were also predicted highly active
through pharmacophore mapping exercise and their actual activities correlated well to the estimated activities
with fit values greater than 7 (designated as +++ in Table 1). The most favoured substitution in the compounds
with hydrazino group as linker and indanone as Ring B was p-OC2H5 (4a) which was found to be active in in vitro
binding experiment. Compounds 3c and 4a also exhibited favourable interactions in the CCK-BR binding site as
discussed in docking results above and shown in Table 1.
Gastric acid secretion assay
To further establish the ability of the compounds in two series, these were tested to antagonize pentagastrin-
induced gastric acid secretion in an isolated lumen perfused mouse stomach assay. All the compounds produced
concentration dependent, non-parallel shifts to the right of the concentration response curves to Boc-
pentagastrin12,31 and decreased the maximum response. Therefore, pA2 (negative algorithm of the molar
concentration of a dose of agonist to that of half the dose) could not be calculated. The compounds were capable
of inhibiting the gastric acid secretion (Table 1) at 100 nM. The compounds showed almost 50-60 % inhibition and
one compound with no substitution in ring A showed an inhibition of 39% at much higher concentration (1µM)
again suggesting that the substitution in ring A is important for specific CCK-BR antagonistic activity in both the
series of compounds.
8 | J. Name., 2012, 00, 1-3
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Cytotoxicity Evaluation
All the compounds synthesized in this study were then tested for anti-proliferative activity against pancreatic
cancer cells (MiaPaca-2) using MTT assay32. The growth inhibition effect evaluated through dose response graphs
showed that all the quinazolinone derivatives exhibited growth inhibitory effect and IC50 in the range of 10 – 280
µM was obtained. The positive control for the experiment L365, 260 showed IC50 of 0.0018 µM. The compound
3fwith hydrazine linker and substitution p-OC2H5 (Ring A) and p-Br (ring B) exhibited best IC50 (10 µM) whereas
compound 4b showed similar activity (IC50 10 µM) in indole series. Therefore, our results provided two potent
CCK-BR antagonists exhibiting good CCKBR antagonistic activity as well as cytotoxicity against CCK-BR expressing
pancreatic cancer cells (MiaPaca-2) (Table 1).
Figure 4: Time and concentration dependent effect of antagonist 3f and 4b on the cell viability of MIAPaCa 2 cells. The
lines depict various concentrations of antagonists (alone), Boc-pentagastrin (alone) and/or antagonist and Boc-
pentagastrin together. DMSO control at all the time points is taken as 100%.
Inhibition of proliferation of MiaPaca-2 cells induced by Boc-Pentagastrin
33
Smith et al reported that the growth responses of several human pancreatic cancer cell lines are stimulated by
CCK in serum-free medium, and its tropic effect can be blocked by a specific antagonist 5. They also demonstrated
that gastrin exerts a growth-stimulating effect on pancreatic cancer in vivo and in vitro 34. Therefore, we checked
whether the cytotoxicity shown by compounds 3f and 4b in MiaPaca-2 cells is specific towards gastrin induced
proliferation or not. The cellular proliferation induced by one of the potent and specific CCK-BR agonist, Boc-
Pentagastrin was undertaken. The Boc-Pentagastrin showed proliferative effect in the MiaPaca-2 cells from a
concentration of 1 pM to 10µM tested for a time period of 48 h to 120 h. No cellular growth was observed in first
24 h. Maximum cellular growth was observed at 10nM at 96 h (also reported earlier)33. Both the compounds 3f
and 4b were tested at 10µM (IC50 concentration as determined from MTT assay) and at 1 µM for evaluating their
inhibitory effect on specific growth induced by Boc-Pentagastrin. The compounds exhibited anti-proliferative
action against Miapaca-2 cells at both the tested concentrations (Figure 4a & 4b), exhibiting maximum inhibition
at 72 h when given alone. The compounds also inhibited the proliferative effects of Boc-Pentagastrin when used
in combination. Several studies have been performed to show presence of CCK-C receptor subtype in pancreatic
cancers but not in normal tissues35. Our results suggest compounds 3f and 4b might show some kind of selectivity
towards CCK-C receptors in MiaPaCa-2 cell line which has been shown to express this splice variant9.
ADMET Prediction
The intestinal absorption and blood brain barrier penetration were predicted by developing an ADME model using
descriptors 2D PSA and AlogP98 that include 95% and 99% confidence ellipses. These ellipses define regions
where well-absorbed compounds are expected to be found. The results of DS 4.0-ADME model screening showed
that both our active compounds 3f and 4b possess 99% confidence levels for human intestinal absorption, and
low blood brain barrier (BBB) penetration. Since we are targeting pancreatic cancer, the low BBB penetration of
our active compounds is advantageous.Also; TOPKAT results predicted that both 3f and 4b are non-toxic, non-
mutagen and non-irritant to the skin, which makes our active compounds competitive to be new developmental
compounds for the Pancreatic Cancer. Overall the compounds 3f and 4b are being suggested as the best
compounds which showed good CCK-BR antagonistic activity, showed lower IC50 (cytotoxicity), specifically
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inhibited the growth stimulatory effects of gastrin in MiaPaca-2 cells as well as exhibited best computed ADMET
properties.
Experimental Section
Analytical grade materials were purchased from commercial suppliers and were used without purification, unless
otherwise noted. Melting points were determined on a Thomas Hoover apparatus and are uncorrected. The 1HNMR
spectra were determined on a Bruker 300 MHz spectrometer at ambient temperature. IR spectroscopy was performed
with a FTIR apparatus (Model 1600, Perkin Elmer). Mass spectra were recorded on AP1, PE SCEIX system. Fast atom
bombardment (FAB) mass spectra were recorded with an SX 102/DA-6000 mass spectrometer (JEOL) with m-
nitrobenzylalcohol as matrix, elemental analysis was done using a Carlo Erba 1108 (Heraus, Hanau, Germany). All
experiments involving animals were approved by the Institutional Animal Ethics Committee under the Committee for the
purpose of control and Supervision and Experimentation on Animals (CPCSEA), Ministry of Environment, Government of
India.
General method for synthesis of 3-aryl-2-thioxo-2, 3-dihyroquinazolinone-4-(1H)-one (1a-f)
Anthralinic acid (3.0 g, 0.021 M) and appropriate substituted aryl isothiocynate(0.022 M) were refluxed in glacial
acetic acid (75 mL) for 16 h. The white lustrous crystals was separated by filtration and washed with water until
the washings showed neutral pH. The crystalline product was dried and checked for purity using TLC (ethyl
acetate - petroleum ether in 50%).
3-(4-Methylphenyl)-2-thiooxo-2,3-dihydroquinazoline -4-(1H)-one (1a): Yield, 4.5 g (85 %);mp>300 oC; IR
1
(KBr)
ν
max/cm-1; 3245.02 (NH), 1662.43 (C=O), 1200.05 (C=S); H NMR (DMSO–d6);
exchangeable), 7.953(d, 1H, J = 7.8 Hz), 7.797 (t, 1H, J = 7.65 Hz), 7.450 (d, 1H, J = 8.1 Hz), 7.359 (t, 1H, J = 6.0 Hz),
7.276 (d, 2H, J = 6.6 Hz), 7.140 – 7.120 (m, 1H), 2.364 (S, 3H).
δ
12.965 (Brs, 1H, D2O
3-(4-ethoxyphenyl)-2-thiooxo-2,3-dihydroquinazoline-4-(1H)-one (1b): Yield, 4.914g (80%);mp 285 oC;IR
(KBr)ν
max/cm-1; 3223.54(NH), 1662.59 (C=O), 1205.10 (C=S). 1H NMR (DMSO–d6);δ 11.987 (Brs., 1H, D2O
exchangeable), 7.942 (d,1H, J = 8.7 Hz), 7.688–7.637 (m, 2H), 7.339 (t, 1H, J = 5 Hz), 7.122 (d, 2H, J = 8.7 Hz), 6.809
(d, 2H, J = 9.0 Hz), 4.223 (q, 2H, J = 6.8 Hz), 1.392 (t, 3H, J = 6.9 Hz).
3-phenyl-2-thiooxo-2,3-dihydroquinazoline-4-(1H)-one (1c): Yield, 1.930 g (90 %);mp>300
3219 (NH), 1662.5 (C=O), 1197.7 (C=S), 1H NMR (DMSO–d6);
7.8 Hz), 7.798 (t, 2H, J = 5.0 Hz), 7.472–7.253 (m, 5H).
°
C, IR (KBr)
ν
max/cm-1;
δ13.037 (Brs, 1H, D2O exchangeable), 7.958 (d, 2H, J =
3-(4-bromophenyl)-2-thiooxo-2,3-dihydroquinazoline-4-(1H)-one (1d): Yield, 1.608 g (99%);mp>320
ν
°C; IR
(KBr) 13.097 (Brs, 1H, D2O exchangeable),
max/cm-1;3235.35 (NH),1663 (C=O),1199.05 (C=S), 1H NMR (DMSO–d6);
7.973 (d, 1H, J = 7.8 Hz), 7.839 (t, 1H, J = 5.1 Hz), 7.695 (d, 2H, J = 7.2 Hz), 7.461 (d, 1H, J = 8.1 Hz), 7.383 (t, 1H, J =
5.0 Hz) 7.290 (d, 2H, J = 7.2 Hz).
δ
3-(4-chlorophenyl)-2-thiooxo-2,3-dihydroquinazoline-4-(1H)-one (1e): Yield, 1.783 g (100%);mp>320
(KBr)ν δ 11.971 (Brs, 1H, D2O
°
C; IR
1
max/cm-1;3246.0 (NH), 1662.5 (C=O), 1199.6 (C=S), 759.9 (C-Cl), H NMR (DMSO–d6);
exchangeable), 7.964 (d, 1H, J = 6.3 Hz) 7.815 (t, 1H, J = 7.6 Hz), 7.557 (d, 1H, J = 5.0 Hz), 7.445–7.376 (m, 4H),
7.350–7.267 (m, 1H).
3-(3-chlorophenyl)-2-thiooxo-2,3-dihydroquinazoline-4-(1H)-one (1f): Yield, 1.67 g (98%);mp298 °C; IR (KBr)νmax/cm-
1
1;3205 (NH), 1664.78 (C=O), 1199.21(C=S), 761 (C-Cl), H NMR (DMSO–d6);
δ13.105 (Brs, 1H, D2O exchangeable), 7.974 (d,
1H,J = 7.2 Hz), 7.824 (t, 1H, J = 7.6 Hz), 7.548 – 7 .468 (m, 3H), 7.439 (s, 1H), 7.388 (d, 1H, J = 7.5 Hz)7.338 (t, 1H, J = 7.0 Hz).
3-(3-bromophenyl)-2-thiooxo-2,3-dihydroquinazoline-4-(1H)-one (1g): Yield: 0.50 g (38%); mp295 °C, IR (KBr)νmax/cm-
1
1;3210 (NH), 1672.42 (C=O), 1197.74 (C=S), H NMR(DMSO–d6);
δ13.105 (Brs, 1H, D2O exchangeable), 7.973 (d, 1H, J = 8.1
Hz), 7.824 (t, 1H, J = 7.6 Hz), 7.636–7.612 (m, 2H), 7.462 (d, 2H, J = 7.5 Hz), 7.386–7.334 (m, 2H).
General procedure for synthesis of 2-hydrazino-3-arylquinazoline-4-(3H)-one (2a-g)
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A mixture of 1(0.016 M) and anhydrous hydrazine (5.87 mL, 0.16 M) in anhydrous methanol was refluxed for 18 h. The solid
was separated by filtration, washed with cold methanol and was dried.
2-hydrazino-3-(4-methylphenyl)quinazoline -4-(3H)-one (2a): Yield, 2.2 g (71%);mp 193–194 °C; IR (KBr)νmax/cm-1; 3497.67,
1
3311.65, 3209.05 (NH’s), 1673.02 (C=O); H NMR (DMSO–d6);
δ7.925 (d, 1H, J = 7.8 Hz), 7.787–7.761 (d, 1H, J = 7.8 Hz),
7.670 (t, 1H, J = 7.0 Hz), 7.389(m, 1H), 7.361 (d, 2H, J = 7.5 Hz), 7.187 (d, 2H, J = 7.8 Hz), 6.787 (Brs, 1H, D2O exchangeable),
4.391 (Brs, 2H, D2O exchangeable), 2.393 (s, 3H).
2-hydrazino-3-(4-ethoxyphenyl)quinazoline-4-(3H)-one (2b): Yield, 3.4 g (79 %);mp 222 °C;IR (KBr)νmax/cm-1; 3423.4 (NH-
NH2), 1706.9 (C=O), 1H NMR (DMSO–d6);
δ7.924 (d, 1H, J = 7.8 Hz), 7.669 (t, 1H, J = 7.6 Hz), 7.359 (d, 1H, J = 7.2 Hz), 7.211 (d,
2H, J = 8.7 Hz), 7.158 (t, 1H, J = 5.0 Hz), 7.070 (d, 2H, J = 9.0 Hz), 6.906 (Brs, 1H, D2O, exchangeable), 4.458 (Brs,2H, D2O
exchangeable), 4.122–4.053 (q, 2H, J = 6.9 Hz), 1.392–1.346 (t, 3H, J = 6.9 Hz).
2-hydrazino-3-phenylquinazoline-4-(3H)-one (2c): Yield, 2.5 g (80 %);mp 205 °C;IR (KBr)νmax/cm-1; 3487.67, 3229.05 (NH’s),
1
1673.02 (C=O), H NMR (DMSO–d6);
δ
8.220 (Brs, 1H, D2O exchangeable),7.910 (m, 2H), 7.655 (d, 1H, J = 7.2 Hz), 7.557 (m,
2H, J = 8.4 Hz), 7.399 (d, 1H, J = 8.1 Hz), 7.372–7.327 (m, 2H), 7.161 (d, 1H, J = 7.8 Hz), 4.419 (Brs, 2H, D2O exchangeable).
2-hydrazino-3-(4-bromophenyl)quinazoline-4-(3H)-one (2d): Yield, 0.185 g (37%); mp 210 °C; IR (KBr)νmax/cm-1;3291.67,
3330.86 (NHs), 1676.23 (C=O), 1H NMR (DMSO–d6);
δ8.225 (Brs, 1H, D2O exchangeable), 8.011 (d, 1H, J = 7.8 Hz), 7.962 (d,
2H, J = 8.1 Hz), 7.694 (t, 1H, J = 7.5 Hz), 7.543 (d, 2H, J = 8.4 Hz), 7.413 (d, 1H, J = 8.1 Hz), 7.278 (t, 1H, J = 7.5 Hz), 4.386 (Brs,
2H, D2O exchangeable).
2-hydrazino-3-(4-chlorophenyl)quinazoline-4-(3H)-one (2e): Yield, 0.58 g (58%);mp 190 °C; IR (KBr)νmax/cm-1;3210.33,
3306.27 and 3328.41 (NHs), 1675.57 (C=O), 1H NMR (DMSO–d6);
δ8.352 (Brs, 1H, D2O exchangeable), 8.022 (d, 1H, J = 9.9
Hz), 7.940 (d, 1H, J = 9.0 Hz), 7.693 (t, 1H, J = 7.5 Hz), 7.422 (d, 2H, J = 9.0 Hz), 7.331 (d, 2H, J = 8.4 Hz), 7.251 (t, 1H, J = 7.5
Hz), 4.41 (Brs, 2H, D2O exchangeable).
2-hydrazino-3-(3-chlorophenyl)quinazoline-4-(3H)-one (2f): Yield, 0.267 g (26%), mp 170 °C; IR (KBr)νmax/cm-1;3312.35
(NHs) 1674.79 (C=O), 759.19 (C-Cl), 1H NMR (DMSO–d6);
δ8.823 (Brs, 1H, D2O exchangeable), 8.025 (d, 1H, J = 7.8 Hz), 7.290
(d, 1H, , J = 5.0 Hz), 7.131 (d, 1H, J = 7.8 Hz), 7.404 (d, 1H, J = 8.1 Hz), 7.686 (t, 1H, J = 4.0 Hz), 7.574 - 7.493 (m, 2H), 4.370
(Brs, 2H, D2O exchangeable).
2-hydrazino-3-(3-bromophenyl)quinazoline-4-(3H)-one (2g): Yield, 0.289 g (28%);mp 190°C; IR (KBr)νmax/cm-1;3306.60
(NHs), 1672.42 (C=O), 1H NMR(DMSO–d6);
δ8.248 (Brs, 1H, D2O exchangeable), 4.382 (Brs, 2H, D2O exchangeable), 8.025(d,
1H, J= 7.8 Hz), 7.971 (d, 1H, J = 7.8 Hz), 7.822 (t, 1H, J = 7.6 Hz), 7.711–7.661 (t, 1H,, J = 7.5 Hz), 7.61 (s, 1H), 7.461–7.266
(m, 3H).
General method for synthesis of {N-aryl-2-[3,4-dihydro-3 or 4-substituted aryl)-4-oxo-2 quinazonyl]}hydrazine
carbothioamide (3a-g)
An equimolar mixture of appropriate compound 2 (0.878 mM) and 3- or 4-substituted arylisothiocynate (0.878 mM) was
stirred in DMF under inert atmosphere at 110 ºC for 12 h. DMF was evaporated by using rotavapour. The residue obtained
was washed with cold methanol, filtered and dried to give 3a-g.
3-(2-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)hydrazinecarbothioamido)benzoic acid(3a): Yield, 1.90
(60 %); mp 239 °C;IR (KBr)νmax/cm-1; 3442.5 (NH), 1662.5 (C=O), 1249.8 (C=S), 2924.52 (-OC2H5); 1H NMR (DMSO–d6);
g
δ
12.995 (s, 1H, D2O exchangeable), 11.500 (Brs, 1H, D2O exchangeable), 9.520 (Brs, 1H, D2O exchangeable), 8.234 (Brs, 1H,
D2O exchangeable), 8.207 (s, 1H), 7.955 (d,J= 7.56, 1H), 7.796 (t, 1H,J = 7.6 Hz), 7.686 (t, 1H, J = 7.5 Hz), 7.446 (d, 1H, J =
8.34 Hz), 7.381 (t, 1H, J = 7.652 Hz), 7.204 – 7.314 (m, 1H), 7.204(d, 2H, , J = 8.1 Hz), 7.094 (d, 1H, J = 7.8 Hz), 6.994 (d, 2H,
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J= 7.9 Hz), 4.101(q, 2H, , J = 6.4 Hz), 1.380 (t, 3H, , J = 6.6 Hz); MASS (m/z) = 474 (C24H21N5SO4[M – 1]+).Anal.Calcd.for
C24H21N5SO4: C, 60.62; H, 4.45; N, 14.73.Found:C, 60.76; H, 4.52; N, 14.89.
3-(2-(3-(3-bromophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)hydrazinecarbothioamido)benzoic acid(3b): Yield: 0.658
(65 %);mp 195 °C;IR (KBr)νmax/cm-1; 3327.92 (NH), 1670.16 (C=O), 1197.92 (C=S), 759.96 (C-Br); 1H NMR (DMSO–
d6); 12.564 (s, 1H, D2O exchangeable), 11.545 (Brs, 1H, D2O exchangeable), 9.296 (Brs, 1H, D2O exchangeable), 8.503 (Brs,
g
δ
1H, D2O exchangeable), 8.384 (d, 1H, J = 7.6 Hz), 7.976 (d, 1H, J = 8.3 Hz), 7.984 (t, 1H, J = 5.0 Hz), 7.892 (d, 1H, J = 7.2 Hz),
7.836 - 7.829 (m, 3H), 7.908 (d, 2H, J = 7.8 Hz), 7.743 - 7.712 (m, 1H), 6.923 (d, 2H, J = 5.0 Hz); MASS (m/z) M+= 509
(C22H16BrN5O3S).Anal.Calcd.forC22H16BrN5O3S:C, 51.77; H, 3.16; N, 13.72. Found: C, 51.89; H, 3.28; N, 13.83.
3-(2-(3-(4-bromophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)hydrazinecarbothioamido)benzoic acid (3c): Yield, 0.352 g
(50 %);mp220 °C;IR (KBr)νmax/cm-1; 3330.73 (NH), 1676.02 (C=O), 1223.82 (C=S), 762.19 (C-Br), 1H NMR (DMSO–
d6);δ12.539 (s, 1H, D2O exchangeable), 11.691 (Brs, 1H, D2O exchangeable), 9.295 (Brs, 1H, D2O exchangeable), 8.399 (Brs,
1H, D2O exchangeable), 8.359 (d, 1H, J = 6.3 Hz), 7.975 (d, 1H, J = 9.4 Hz), 7.940 (t, 1H, J = 7.2 Hz), 7.892 (d, 1H, J = 7.3 Hz),
7.836 – 7.819 (m, 3H), 7.808 (d, 2H, J = 7.8 Hz), 7.753 – 7.712 (m, 1H), 6.823 (d, 2H, J = 5.1 Hz);MASS(m/z) M+ =
509(C22H16BrN5O3S).Anal.Calcd.forC22H16BrN5O3S: C, 51.77; H, 3.16; N, 13.72. Found: C, 51.88; H, 3.29; N, 13.85.
3-(2-(4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)hydrazinecarbothioamido)benzoicacid(3d):Yield, 1.354 g (50 %);mp263
°C; IR (KBr)νmax/cm-1; 3435.92 (NH), 1675.93 (C=O), 1298.66 (C=O); 1H NMR (DMSO–d6);
δ12.784 (s, 1H, D2O exchangeable),
11.504 (Brs, 1H, D2O exchangeable), 9.534 (Brs, 1H, D2O exchangeable), 8.292 (Brs, 1H, D2O exchangeable), 8.334 (d, 1H, J
= 7.8 Hz), 7.956 (d, 1H, J = 9.4 Hz), 7.924 -7.909 (t, 1H, J = 5.0 Hz), 7.892 (d, 1H, J = 7.2 Hz), 7.836 - 7.829 (m, 3H), 7.908 (d,
2H,
J
=
9.2 Hz), 7.743
-
7.712 (m, 2H), 6.723 (d, 2H,
J
= = 431
5.0 Hz);MASS (m/z) M+
(C22H17N5O3S).Anal.Calcd.forC22H17N5O3S:C, 61.24; H, 3.97; N, 16.23. Found: C, 61.39; H, 3.90; N, 16.10.
N-(4-chlorophenyl)-2-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)hydrazinecarbothioamide (3e):Yield, 0.798 g
1
(65 %);mp242 – 244 °C; IR (KBr)νmax/cm-1; 3330 (NH), 1698.94 (C=O), 1296.55 (C=S), 2967.2 (C-H); H NMR (DMSO–d6);
δ10.775 (Brs, 1H, D2O exchangeable), 9.932 (Brs, 1H, D2O exchangeable), 8.659 (Brs, 1H, D2O exchangeable), 7.955 (d, 1H, J
= 7.2 Hz), 7.797 (t, 1H, J = 7.6 Hz), 7.544(t, 1H, J = 9.3 Hz), 7.450 (d, 1H, J = 8.7 Hz), 7.395 (d, 2H, J = 9.0 Hz), 7.164 (d, 2H, J =
8.7 Hz), 7.078 (d, 2H, J = 8.7 Hz), 6.994 (d, 2H, J = 8.7 Hz), 4.131 (q, 2H, J = 7.0 Hz), 1.391 (t, 3H, J = 6.8 Hz).MASS (m/z) M+=
465 (C23H20ClN5O2S).Anal.calcd. forC23H20ClN5O2S: C, 59.29; H, 4.33; N, 15.03. Found: C, 59.10; H, 4.25; N, 15.21.
N-(4-bromophenyl)-2-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)hydrazinecarbothioamide (3f): Yield, 0.285 g
(57 %); mp>250°C;I.R.(KBr) νmax/cm-1; 3400 (NH), 1662.83 (C=O), 1250.86(C=S), 2967.06 (C-H); 1H NMR, (DMSO–d6);
δ
10.945 (Brs, 1H, D2O exchangeable), 10.235 (Brs, 1H, D2O exchangeable), 9.198 (Brs, 1H, D2O exchangeable), 8.246 (d, 1H, J
= 6.6 Hz), 7.918 (d, 2H, J = 7.8 Hz), 7.699 (d, 2H, J = 7.5 Hz), 7.363 - 7.298 (m, 4H), 7.198 (d, 1H, J = 7.2 Hz), 6.860 (d, 2H, J=
7.5 Hz), 3.992 (q, 2H, J = 6.2 Hz), 1.342 (t, 3H, J = 6.1 Hz); MASS (m/z) M+= 509 (C23H20BrN5O2S).Anal.calcd.
forC23H20BrN5O2S: C, 54.12; H, 3.95; N, 13.72. Found: C, 54.28; H, 3.86; N, 13.91.
2-(3-(4-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(4-ethoxyphenyl)hydrazinecarbothioamide (3g): Yield, 0.82 g
(72 %); mp 260 °C; IR (KBr)νmax/cm-1; 3435.26 (NH), 1616.47 (C=O), 1225.79 (C=S), 2973.73 (C-H); 1H NMR (DMSO–d6);
δ13.355 (Brs, 1H, D2O exchangeable), 10.450 (Brs, 1H, D2O exchangeable), 10.303 Brs, 1H, D2O exchangeable), 8.123 (d, 1H,
J = 8.1 Hz), 7.990–7.921 (m, 1H), 7.831 (d, 1H, J = 7.8 Hz), 7.588 (d, 2H, J = 7.6 Hz), 7.399–7.324 (m, 4H), 7.270 (t, 1H, J =
7.5 Hz), 7.106 (d, 2H,J = 8.4 Hz), 4.116 (q, 2H, , J = 6.6 Hz), 1.388 (t, 3H, J = 6.4 Hz); MASS (m/e) M+= 465 (C23H20ClN5O2S).
Anal.calcd. forC23H20BrN5O2S: C, 59.29; H, 4.33; N, 15.03. Found: C, 59.12; H, 4.40; N, 15.18.
General procedure for synthesis of (Z)-3-aryl-2-(2-(2-oxoindolin-3-ylidene)hydrazinyl)quinazolin-4(3H)-one(4a-e)
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An equimolar mixture of 2, 3-dioxoindole (0.499 g, 0.0034 M) and appropriate 2-hydrazino-3-arylquinazoline- 4(3H)-one
(0.0034 M) was refluxed in 50 mL of absolute ethanol for 2 h. The precipitate thus formed was filtered, washed with cold
ethanol, dried and crystallized from ethanol to give the compounds 4a-e.
(Z)-3-(4-ethoxyphenyl)-2-(2-(2-oxoindolin-3-ylidene)hydrazinyl)quinazolin-4(3H)-one (4a): Yield, 2.5 g (90%); mp 275 - 280
°C;IR (KBr)νmax/cm-1; 3064.7, 3184.3 (NH’s), 1708.8 and 1622 (C=O); 1H NMR (DMSO–d6);
δ11.51 (s, 1H, D2O exchangeable),
10.4696 (S, 1H, D2O exchangeable), 8.006 (d, 1H, J = 7.5 Hz), 7.856 (d, 1H, J = 8.4 Hz), 7.767 (t, 1H, J = 7.2 Hz), 7.340 (d, 2H,
J = 8.7 Hz), 7.311 (t, 1H, J =8.6 Hz), 7.176 (t, 1H, J = 7.2 Hz), 7.128 (d, 2H, J = 8.7 Hz), 6.980 (d, 1H, J = 7.5 Hz), 6.755 (d,
1H, J = 7.5 Hz), 6.520 (t, 1H, J = 7.5 Hz), 4.112–4.06 (q, 2H, J = 6.8 Hz), 1.380 (t, 3H, J = 6.6 Hz); MASS (m/z)M+ = 425
(C24H19N5O3). Anal.calcd. forC24H19N5O3: C, 67.76; H, 4.50; N, 16.46. Found: C, 67.51; H, 4.62; N, 16.30.
(3Z)-1H-indole-2,3-dione 3-{[3- (4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2- yl]hydrazone} (4b): Yield, 0.6
g
(68%);mp 280 °C;IR (KBr)νmax/cm-1; 3184.2 (NH), 1708.7 (C=O);1H NMR (DMSO–d6);
δ11.53 (Brs, 1H, D2O exchangeable),
10.43 (s, 1H, D2O exchangeable), 8. (d, 1H, J = 7.5 Hz), 7.879 (d, 1H, J = 9.2 Hz), 7.768 (t, 1H, J=6.6 Hz Hz), 7.407 (d, 2H, J =
8.1 Hz), 7.310 (t, 2H, J = 8.2 Hz), 7.154 (t, 2H, J =6.2 Hz), 6.839 (d, 1H, J = 7.5 Hz), 6.740 (d, 1H, J = 7.5 Hz), 6.501 – 6.451 (t,
1H, J =7.47 Hz), 2.45 (s, 3H). MASS (m/z) M+ = 395 (C23H17N5O2).Anal.calcd. for: C, 69.86; H, 4.33; N, 17.71. Found:C, 69.69;
H, 4.44; N, 17.57.
(3Z)-1H-indole-2,3-dione-3-{[4- (4-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2- yl]hydrazone} (4c): Yield, 0.269 g (70%);
mp 280 °C; IR (KBr)νmax/cm-1; 3313.58 (NH’s), 1691.14 (C=O);1H NMR (DMSO–d6);
δ11.632 (Brs, 1H, D2O exchangeable),
10.622 (Brs, 1H, D2O exchangeable), 8.455 (d, 1H, J = 8.4 Hz), 8.027 (d, 1H, J = 7.5 Hz), 7.818 (d, 2H, J = 8.7 Hz), 7.739 –
7.641 (m, 1H), 7.421 – 7.291 (m, 4H), 7.061 (t, 2H, J = 7.5 Hz), 6.896 (d, 1H, J = 7.8 Hz); MASS (m/z) M+ = 415
(C22H14ClN5O2).Anal.calcd. forC22H14ClN5O2: C, 63.54; H, 3.39; N, 16.84. Found: C, 63.39; H, 3.48; N, 16.70.
(3Z)-1H-indole-2,3-dione 3-{[3- (3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2- yl]hydrazone} (4d): Yield, 0.052 g
(34%);mp 335 °C; IR (KBr)νmax/cm-1; 3434.84 (NH), 1623.32(C=O), 756.94 (C-Cl);1H NMR (DMSO–d6);
δ11.70 (Brs, 1H, D2O
exchangeable), 10.58 (Brs, 1H, D2O exchangeable), 8.018 (d, 1H, J = 8.1 Hz), 6.91 (d, 1H, J = 6.9 Hz), 6.777 (d, 1H, J = 7.8 Hz),
6.600 (t, 1H, J = 6.6 Hz), 7.322 (t, 1H, J = 8.1 Hz), 7.188 (t, 1H, J = 8.2 Hz), 7.810 - 7.762 (m, 3H), 7.677 - 7.641 (m, 3H);
MASS (m/z) M+ = 415 (C22H14ClN5O2).Anal.calcd. forC22H14ClN5O2: C, 57.41; H, 3.07; N, 15.22. Found: C, 57.27; H, 3.22; N,
15.39.
(3Z)-1H-indole-2,3-dione 3-{[3- (3-bromophenyl)-4-oxo-3,4-dihydroquinazolin-2- yl]hydrazone} (4e): Yield, 0.050 g
(38%);mp295 °C; IR (KBr)νmax/cm-1; 3332.73(NH), 1710.51(C=O);1H NMR (DMSO–d6);
δ13.141 (Brs, 1H, D2O exchangeable),
11.644 (Brs, 1H, D2O exchangeable), 8.012 – 7.943 (m, 1H), 7.876 (d, 1H, J = 8.4 Hz), 7.805 – 7.758 (m, 2H), 7.609 –
7.433 (m, 2H), 7.385 – 7.266 (m, 2H), 7.236 (t, 1H, J = 7.5 Hz), 6.907 (d, 1H, J = 7.5), 6.769 (d, 1H, J = 7.5 Hz) 6.612 (t, 1H, J
= 7.5 Hz); MASS (m/e) M+ = 459 (C22H14BrN5O2). Anal.Calcd.forC22H14BrN5O2: C, 63.54; H, 3.39; N, 16.84. Found: C, 63.36; H,
3.29; N, 16.98.
Molecular modeling
All molecular modeling work was performed using Discovery Studio 2.5.5 and Discovery Studio 4.0 (Accelrys Inc.). The
molecules were built using “Builder” module. The energies were minimized using “CHARMm” force field in a step wise
manner by using “steepest descent” followed by “conjugate gradient” till a RMSD derivative of 0.001 was achieved.
Conformational models of the molecules were generated using “best quality” conformational search option in “Catalyst”
using a constraint of 20 kcalmol-1 energy threshold above the global energy minimum and “CHARMm” force field
parameters. A maximum of 250 conformations were generated using “best fit method” to ensure maximum coverage in
the conformational space. The pharmacophore mapping exercise was performed using “Best mapping” protocol keeping
the “flexible method” and “maximum omitted features” at 2. All the activities were predicted as nM and fit values were
calculated by the software.
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Homology Modeling
The sequence of Human CCK-BR (ID: P32239), was retrieved from SwissProt database36.The template structure and
sequence of Human A2A Adenosine Receptor X-ray structure (3EML)37 were downloaded from PDB (www.rcsb.org).The
sequence alignment was generated with Clustal W multiple sequence alignment tool and manually adjusted to avoid
insertions and deletions in the transmembrane regions.
The intracellular loop (IL3) of CCKBR was deleted as the template 3EML lacked this region. Most of the third cytoplasmic
loop (Leu 2095.70 – Ala2216.23 of the template was replaced with lysozyme from T4 bacteriophage and the carboxyl terminal
tail (Ala317-Ser412) was deleted in the template 3EML.pdb to improve the likelihood of crystallization. Moreover, it has
also been reported that most of the ligands binds to the extracellular region and the conserved transmembrane region of
the CCK-BR38. Hence coordinates for this region could not be generated for this study.
Total 10 models of the CCK-BR along with 30 loop models were built using “MODELER” module present in Discovery Studio
4.0 at low optimization level. The loop modeling was performed for the modeling of gaps created during the sequence
alignment. For the specific case of CCK-BR, the conserved disulphide bridge between Cys127 and Cys205 was manually
introduced according to experimental data39. This disulphide bridge has importance in ligand binding interaction and the
structure stability of the protein. All the models were sorted on the basis of their PDF total energy as well as DOPE score. A
model is better optimized against the homology restraint when it has a lower PDF total energy.
For accessing the validity of homology model with sequence of residues it contain, verify score of each model was
calculated by using “Profile-3D” module present in the Discovery studio 4.0. Finally the model having lowest PDF total
energy score as well as highest verify score was selected for molecular dynamics simulations
Molecular Dynamics Simulation
The modelled structure of the CCK-BR generated above was used as starting structure for molecular dynamics simulations.
The stability of the structure was verified through the molecular dynamics (MD) simulations using GROMACS40 in an explicit
membrane aqueous system. The total system consisted of 123 molecules of DPPC lipids, 31444 water molecules, 358
amino acids, Na+ and Cl- ions. Energy minimization was carried out by steepest descent algorithms applying Berger lipids
parameters41 for the lipid component in combination with the GROMOS42 representation of the protein. Energy
minimization was carried out by steepest descent algorithm with strong position restrained force on protein, so that, the
protein position does not change during energy minimization. The steepest descent algorithm converged to Fmax<1000
KJ/mol/nm in 1248 steps. Parinello- Rahman coupling43, Berendson44 and Nose- Hoover thermostat coupling45 were used to
equilibrate the system in stable environment (323K, 1bar). Electrostatic energy was computed by Particle Mesh Edwald
(PME) method46 and linear constraint solver (LINCS) algorithm47 was applied to fix all bonds involving hydrogen. The
structure in the medium was equilibrated for 100ps in NVT followed by 1000 ps in NPT due to membrane simulation.
Finally, a 4 nanosecond molecular dynamics simulation was carried out for the structure.The RMSD (root-mean-square
deviations) from the initial structure was calculated during 4ns simulation run. After MD simulation, the stereochemistry
quality of the structures was validated with PROCHECK48 and Verify 3D49 and the quality factors of the protein models were
calculated
using
ERRAT250,
available
in
SAVES
(Structural
Analysis
and
Verification
Server)
[http://nihserver.mbi.ucla.edu/SAVES/]. The Ramachandran plot51 was created by submitting the model toPDBsum52.
Molecular Docking
All molecular docking work was done using Discovery Studio 4.0 software packages (Accelrys, California, USA). All
the simulations were performed on Intel(R) Pentium(R) IV 2.4GHz, running Windows XP professional version 2006
operating system. The modelled structure was defined as the receptor molecule and was chosen for ‘input
receptor molecule parameter in the C-DOCKER protocol for molecular docking.All the molecules YF476, L365,260,
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the synthesized compounds 3a-g and 4a-e were drawn and energy minimized in separate molecular windows and
saved as .dsvfile. The saved structures were then used as ‘input ligands’ in CDOCKER protocol and docked into the
active site of CCK-BR.
ADME parameters screening for drug likeness
The compounds were subjected to ADMET calculations. Parameters such as aqueous solubility, absorption, plasma protein
binding, cytochrome P450 2D6 inhibition, and hepatotoxicity were determined using the ADMET protocol in DS4.0 (Table
TS1 and Figure S6 in supplementary file). Moreover, the toxicity potential (i.e., carcinogenicity and mutagenicity) of the
compounds was also predicted using the TOPKAT (TOxicity Prediction by Komputer Assisted Technology) protocol in DS4.0
(Table TS2 in supplementary file).
Biological evaluation
Receptor binding assay
The affinity of the test compounds for the CCK-B/gastrin receptor, solubilized in detergent, was determined by a
competitive binding assay with a Bolton–Hunter-labelled CCK-8 radioligandas previously described53. For this plasma
membrane prepared from rat cerebral cortex (100 µg per tube) was mixed with 125I-Bolton Hunter-CCK-8 (Perkin Elmer)
radio-ligand (20 pM) in absence and presence of increasing concentrations of the test compounds. Non-specific binding
was determined in presence of 1 µM unlabeled CCK-8s peptide (Bachem, Bubendorf, Switzerland). The tubes were
incubated at 25 °C for 1 h to achieve steady state binding. Bound radio-ligand was rapidly separated from free radio-ligand
using Millipore receptor binding filtermats. Bound radioactivity was quantified with Microbeta liquid scintillation counter
from Perkin Elmer. Data was analysed using Graphpad software using non-linear least square curve fitting routine in the
prism 5 software of the program.
Gastric acid secretion inhibition assay
For determination of acid secretion, the whole stomach was excised from albino mice (25-30 g) and was placed in tissue
organ bath purchased from local suppliers and perfused by the methods described by Black et. Al54. The effluent from the
perfused stomach was monitored for change in pH using continuous probe (Ecoscan pH meter from Eutech Instruments)
inserted into the organ bath. The basal acidic pH and increase in acid output was produced by a sub-maximal concentration
of Boc-pentagastrin (10 nM) before and 45 min after incubation with various concentrations of the test compound and the
resultant percentage inhibition was calculated.
In vitro cytotoxicity assay
Cell survival was measured by using the MTT[3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide] micro-
culture tetrazolium assay, according to the method described by Mosmann32. A total of 1000 cells/well were seeded in a
96-well plate. After 24 h incubation in a 5% humidified CO2 incubator at 37°C, varying concentrations of compounds were
added to final volume of 200 μL of standard growth medium/well. After 24 h incubation at 37°C, 20 µl of MTT (Invitrogen)
(5 mg/mL in PBS) was added to each well and incubated for 4 h at 37°C. The medium was removed and formazancrystals
thus formed were dissolved in DMSO. The plates were read immediately in a microplate reader (Tecan, GENios-Pro,
Austria) operating at 540 nm.
Exogenous Gastrin Assay
The tropic effects of gastrin to promote tumour growth can be blocked by specific antagonists33. To show whether the
stimulatory effect of gastrin was blocked by 3f and 4b, the MiaPaca-2 cells were incubated with Boc-Pentagastrin (Bachem)
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for 24 h and then treated with 3f, 4b and Boc-pentagastrin either alone or in combination. Cellular proliferation was
studied using the MTT assay as described above. Briefly, the cells were seeded in 96 well plate (4000cells/well) in 10% FCS-
DMEM. Cells were then incubated in incomplete media for 24 h to attain the quiescent stage. The Boc-Pentagastrin and the
antagonists were then added in incomplete media. The MTT assay was carried out after 48-120h of incubation by replacing
fresh media and same concentration of Boc-Pentagastrin and/or the antagonist every day. The absorbance was measured
as described above.
Conclusions
To summarize, the quinazolinone ring system has been employed as a template for the synthesis of two series of CCK-BR
antagonists. The study resulted in development of potent CCK-BR antagonists. All the compounds were predicted for
activities using pharmacophore mapping exercise and a good correlation was found between the experimentally
determined and predicted antagonistic activities. The compounds showed favorable interactions within the binding site of
homology modeled CCK-BR structure. Two best compounds 3c and 4a exhibited remarkable antagonistic activities of 0.2
nM and 17.36 nM respectively. The compounds also showed promising results in a functional gastric acid secretion assay
using lumen-perfused isolated mouse stomach assay and inhibited growth of pancreatic cancer cells (MiaPaca-2) exhibiting
good Cytotoxicity resulting in the most active compounds 3f (hydrazine thiocarboxamide linker) and 4b (hydrazine linker,
indanone series). The compound 3f being the best overall compound exhibiting good receptor binding affinity and
cytotoxicity activity. Thus the optimized compounds in this study can be taken further for pre-clinical evaluation in drug
discovery process to develop anticancer compounds against pancreatic cancer.
Acknowledgements
This work was supported by Department of Science and Technology (DST), Government of India and Department of
Biotechnology (DBT), Government of India for providing Bioinformatics Facility at ACBR.
Conflict of Interest
The authors declare co competing interests.
Keywords:
Cholecystokinin Receptor Antagonist• Quinazolinone• Pancreatic Cancer • Biological activity • Molecular Modeling
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