Cholecystokinin-1 receptor antagonists: 5-hydroxy-5-aryl-pyrrol-2-ones as anticancer agents

Article abstract of DOI:10.1039/c6md00052e

A new class of 5-arylated 5-hydroxypyrrolones was derived from mucochloric acid in 2 synthetic steps and the chemical structure was confirmed additionally by X-ray analysis. Using a radiolabelled binding assay, potent CCK₁ selective ligands were identified (CCK₁: 12 nM) and the antagonism was confirmed by using isolated tissue preparations. A series of isobutyl derivatives displayed unsurmountable CCK antagonistic properties and in vitro excellent inhibition of proliferation was obtained in cholecystokinin related cancer cell lines in the nanomolar range. Finally, using xenograft studies in nude mice, two selected pyrrolone derivatives, X = H and X = F a fluorinated analogue (PNB-028), showed a strong inhibition of tumour growth in a chemo-resistant colon cancer-(MAC 16) and a human pancreatic cell line (MIAPACA) at 50 mg kg^-1 by oral administration.

Full text of DOI:10.1039/c6md00052e

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Graphical abstract
4000
3000
2000
1000
0
Vehicle
Pyrrolone 10
Pyrrolone 9
5
10
15
20
25
Time (days)
-1000
Cholecystokinin-1 receptor antagonists:
5-hydroxy-5-aryl-pyrrol-2-ones as anticancer agents
Hydroxyꢀpyrrolones, which were potent CCK1R antagonists, showed nanomolar in vitro activity and anticancer activity in
vivo for colon and pancreatic cancer.
1

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Cholecystokinin-1 receptor antagonists: 5-hydroxy-5-aryl-pyrrol-2-ones as anticancer
†
agents
a
a
a
a
E. Lattmann, S. T. Russell, C. H. Schwalbe, A. Shortt,
b
a
a
c
P. N. Balaram, E. Theochari, M. Alharbi, R. Narayanan,
b
and P. Lattmann
a.
School of Life and Health Sciences, Aston University, Aston Triangle,
Birmingham B4 7ET, England.
b.
c.
PNB Vesper Life Science PVT, Cochin, Kerala, India.
Department of Medicine, University of Tennessee Health Science Center,
Memphis, TN, USA.
†
The authors declare no competing interests.
Abstract
A new class of 5ꢀarylated 5ꢀhydroxypyrrolones was derived from mucochloric acid in 2
synthetic steps and the chemical structure was confirmed additionally by xꢀray analysis.
Using a radiolabelled binding assay, potent CCK selective ligands were identified (CCK :
1
1
12nM) and the antagonism was confirmed by using isolated tissue preparations. A series of
isobutyl derivatives displayed unsurmountable CCK antagonistic properties and in vitro
excellent inhibition of proliferation was obtained in cholecystokinin related cancer cell lines
in the nanomolar range. Finally, using xenograft studies in nude mice, two selected pyrrolone
derivatives, X=H and X= F a fluorinated analogue ( PNBꢀ028 ), showed a strong inhibition of
tumour growth in a chemoꢀresistant colon cancerꢀ (MAC 16) and a human pancreatic cell
line (MIAPACA) at 50 mg/kg by oral administration.
Introduction
Cholecystokinin (CCK) acts as a neuromodulator in the brain and as a gut hormone in
1
the gastrointestinal tract. CCKꢀligands, agonists as well as antagonists, have been
2

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2
extensively investigated as potential drug molecules. Originally, cholecystokinin was
discovered to cause contractions of the gallbladder, it was then rediscovered as
pancreozymin, triggering the release of pancreatic enzymes and then, it was confirmed
3
that both peptides are identical.
Gastin, closely related to cholecystokinin, triggers the release of gastric acid and in
4
vitro in cell lines, gastric cancer is gastrin dependant. Cholecystokinin does cause
proliferation in colonꢀ and pancreatic cancer cell lines and therefore, CCKꢀantagonists
5
were studied as growth factor inhibitors in certain forms of cancer. They were
6
7
8
evaluated as anxiolytics, in the treatment of schizophrenia, satiety and reviewed as
9
anxiolytics. These properties may be beneficial in cancer patients in addition to their
antiꢀneoplastic activity.
3

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1
Fig.1 Drug design from Asperlicin, via Devazepide, a CCK selective antagonist, a
CCK
2
selective antagonist, Lꢀ365,260 towards Zꢀ360, a potent and water soluble
CCK antagonist.
2
4

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Asperlicin (Fig. 1) was the first nonꢀpeptidal CCK antagonist lead structure from
1
0
nature and analogues thereof, were studies as CCK ligands. Simplification of the
11
lead structure by Merck led to Devazepide, a potent CCK
1
selective cholecystokinin
antagonist, containing a 1,4ꢀbenzodiazepine template and an indole moiety.
1
2
Devazepide was tested clinically in 18 patients with advanced cancer without
escalating doses and failed to show significant effects due to its poor pharmacokinetic
properties.
The indolyl amide of devazepide was replaced by a urea linkage and Merck’s Lꢀ
3
65,260 resulted in a CCK
led to Zeria’s improved Zꢀ360, in which the Nꢀalkyl side chain, the 5ꢀ position
cyclohexyl) was optimised for potency and a metaꢀcarboxylic acid on the aryl urea
linkage was introduced to enhance water solubility of the CCK antagonists (Fig. 1).
Zꢀ360 is a CCK –gastrin receptor antagonist and progressed into phase 2 trial with
2
selective antagonist. Further subsequent SAR optimisation
13
(
2
2
14
pancreatic cancer.
Lorglumide is 25 times more potent in blocking the cholecystokinin induced
contractions of the gallbladder than proglumide, but the activity to block pancreatic
1
5
amylase secretion is only better by factor 2, indicating a heterogeneity of the CCK
receptor.
1
Proglumide with mM activity is too nonꢀpotent, requiring multi gramme quantities for
potential cancer treatment.
1
6
In our search for new CCK ligands, in which the 1,4ꢀbenzodiazepine structure was
replaced by an achiral diphenyl pyrazolone template, novel CCK antagonists with an
1
7
18
indole carboxylic acid and a phenyl urea moiety were found and optimised with
1
9
excellent animal data on anxiety and depression. The 1,4ꢀbenzodiazepine template of
2
0
devazepide was varied by a combinatorial solid phase synthesis and was optimised in
21
terms of CCK binding affinity.
Again, having realized the poor pharmacokinetic properties of these agents, a search
2
2
for a completely novel, smaller template with a molecular weight <350, a log p about
and a polar surface area for membrane penetration of less that 100A, with no urea
3
linkage was initiated (Fig. 2).
Here, we identified within a novel chemical class, potent irreversible cholecystokinin
antagonists, which translated in vitro and in vivo efficiency into new chemical entities
5

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(
NCE) for the treatment of colon and pancreatic cancer. The pyrrolone 10 entered
preclinical development (PNBꢀ028) and showed excellent metabolic stability, a very
high bioavailability (98%) and a halfꢀlife of 3.5 h in rats.
CCK
2
CCK
1
antagonists
Cl
HO
HN
Cl
O
N
Cl
O
O
O
5ꢀ(benzyloxy)ꢀ3ꢀchloroꢀ4ꢀ(isobutylamino)furanꢀ
2(5H)ꢀone
4
ꢀchloroꢀ5ꢀ(4ꢀchlorophenyl)ꢀ5ꢀhydroxyꢀ1ꢀisobutylꢀ
Hꢀpyrrolꢀ2(5H)ꢀone
1
Lactone
Lactame ꢀ template
Fig. 2 From 4ꢀaminoꢀ(2H)ꢀ furanones to novel hydroxylꢀpyrrolones, a conversion of
gastrin antagonists into cholecystokinin antagonists.
Results and discussion
Chemistry
The arylated dichloroꢀ2(5H)ꢀfuranones (Lactone A, B, C) were synthesised from
mucochloric acid (Scheme 1), which is available from furfural under oxidising
conditions with hydrochloric acid. Theses arylated intermediates were chemically
2
3
optimised by us previously and evaluated as anticancer agents.
Under mild conditions, using ether as solvent, the arylated furanones A, B, C were
reacted with a range of amines, required for SAR optimisation towards the CCK1
6

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receptor, into the desired arylated pyrrolones 1ꢀ16 in a 1 step synthesis in high yields
(
Scheme 1).
Cl
Cl
Cl
Cl
O
O
HO
O
O
HO
Mucochloric acid
Arene, RT,
solvent and reagent
AlCl3
X
X=H
X=Cl
X=F
Cl
Cl
Cl
Amine, ether,
RT, 30 min
HO
O
N
O
O
X
R
X
Lactone A, B,C
Lactame 1ꢀ16
Scheme 1 Synthesis of arylated lactams 1ꢀ16.
The experimental details are described in ESI under the section “materials and
methods” and no acyclic ketone, the ring opened form of the hydroxylꢀpyrrolone, was
observed. On a small scale purification was performed by column chromatography and
on a multigramme scale, the isobytyl series 9ꢀ11 was purified by recrystallisation with
methanol.
As the gold standard of structure characterisation, the xꢀray structure of pyrrolone 9
was determined. Both stereoisomers are displayed in Fig. 3. The molecule is not
present in a keto form and fully occurred in the 5ꢀmembered ring form, as a hydroxyl
–
pyrrolone.
7

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Fig. 3 Xꢀray structure of 4ꢀChloroꢀ5ꢀhydroxyꢀ1ꢀisobutylꢀ5ꢀphenylꢀ1,5ꢀdihydroꢀpyrrolꢀ
ꢀone, the lactame 9 recrystallised from methanol.
2
SAR optimisation
The initial step of evaluation was to screen for potent binding affinity and to identify a
1
CCK selective ligand for the treatment of cholecystokinin related conditions.
Using radiolabel iodinated cholecystokinin, inhibition of binding was determined for
all test molecules and the IC50 are outlined in Table 1.
Nꢀmethyl group, present in lactame 1, showed no binding affinity and an increase in
the length of the side chain resulted in micromolar binding for the isoꢀpropyl group, as
seen in lactame 3 and 4.
Ring closure on the Nꢀsubstituent, from isopropyl to cycloꢀpropyl resulted in a loss of
activity (lactame 5, 6) and ring enlargement from a 3ꢀ membered to a 5ꢀ membered
ring system resulted in no significant increase of binding affinity.
The change from a propyl into a butyl group resulted in a manifold increase of activity
and the best substituent was found isobutyl, as shown in Figure 4 for the isoꢀbutyl
series 9ꢀ11. The introduction of a halogen atom resulted in an increase of binding
8

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affinity, possibly due to enhanced lipophilicity and the best overall molecule is
pyrrolone 11 with an IC about 8 nM. The tꢀbutyl analogue 12 was formed in very
50
low yields, same as a pentyl analogue (not included).
Table 1 CCK binding affinity using radioligands with cortex and pancreatic
membranes. IC50 is presented in micromolar; N=3.
Pyrrolone
X= R=
CCKꢀA
M]
CCKꢀB
[µM]
[
µ
1
2
3
4
5
6
7
8
9
H
Me
Cl Me
IꢀPr
Cl IꢀPr
2.5
2.0
0.2
0.3
±
±
±
±
±
0.2
>10
>10
0.2
H
0.02
0.04
0.3
0.9±
0.03
0.4
3.7±
H
Cyclopropyl 7.5
>10
>10
0.84
10
Cl Cyclopropyl 4
±
0.3
0.3
0.2
H
Cyclopentyl 0.36
±
±0.4
Cl Cyclopentyl 2.5
±
H
F
Isobutyl
Isobutyl
0.020
0.012
0.008
±
±
±
0.01 1.2
±
0.3
0.2
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
0.01 0.75
±
Cl Isobutyl
0.01 0.4
±
0.2
H
H
tꢀbutyl
Hexyl
0.12
±
0.22 0.9
±
0.03
4.5
±
±
0.3
0.3
>10
Cl Hexyl
3.6
>10
>10
>10
H
H
H
Cyclohexyl >10
Ph
Bz
ꢀ
>10
0.80
0.17
±
±
0.04 0.022
±0.002
Lorglumide ꢀ
0.01 >10
The nꢀhexyl lactame 13, 14 clearly lost activity and the same was observed for the
cyclohexylderivative 15. Lactames, such as pyrrolone 16, containing an aromatic ring
directly connected to the Nꢀposition showed a micromolar activity > 10
µ
M. Most
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interestingly, the introduction of a Nꢀbenzyl substituent to the pyrrolone template,
instead of an alkyl group, resulted in the mixed CCKꢀligand, pyrrolone 17 (Fig. 4).
This is a new lead structure towards CCK
optimisation is in high progress.
2
selective antagonists and a full lead
Cl
Cl
HO
HO
O
O
N
N
X
X
X=H, Cl, F
Pyrrolone 17
Pyrrolone 9, 10, 11
Fig. 4 From a CCK
1
antagonist towards a CCK
2
lead structure. Overview of selected
pyrrolones (lactames).
Generally, within the isobutyl series the halogen atom on the 5ꢀarylated ring enhanced
the binding affinity. The IC50 was enhanced from 20 nM for pyrrolone 9, towards the
1
0nM range for the halogenated entities. Most importantly the metabolism, such as an
expected hydroxylation in the paraꢀphenyl position, would be blocked, resulting in
agents with improved pharmacokinetic properties.
Molecular modelling studies were performed to rationalise drug receptor interactions
of the pyrrolone 11 with the CCK receptor.
1
The isobutyl group of the ligand interacted with a hydrophobic cave of the receptor,
centred at Alaꢀ14. The carbonyl group in the 2ꢀ position bond via hydrogen binding
towards the CCK receptor with Argꢀ9 and the Nꢀ atom of the lactame interacted with
Gluꢀ17. The 5ꢀhydroxyl group of the lactame ligand displayed interactions with of
Asnꢀ6, which are outlined in Fig. 5.
1
0

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Fig. 5 Drug receptor interactions of lactame 11 with the CCK receptor.
1
The phenyl group has no interaction with tryptophan or phenylalanine and Pi ꢀ alkyl
interactions of the chloroꢀphenyl group with Leuꢀ29 and Ileꢀ28 explain the small
increase in specific binding. The ligand acted as irreversible inhibitor in vitro and the
rd
3
1
extracellular loop of the CCK receptor with the natural ligand CCK8s binding site,
was selected to initiate modelling. It was assumed that irreversible, possibly covalent
interaction with this loop of the receptor may deform the site and explain the
superiority of the antineoplastic activity of this class of agents compared with
competitive inhibitors.
Pharmacology
Opiate agonists, such as morphine and CCK antagonists, such as lorglumide and
devazepide reduced electrically induced contractions on the GPI. From the
radioligand binding assay the isoꢀbutylꢀpyrrolones were identified as the most potent
ligands, and the classical isolated tissue preparation served as initial functional assay,
confirming the antagonistic properties of these ligands.
1
1

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Fig. 6 The inhibitory effect of 0.1, 0.5 and 1.0 µM of pyrrolone 11 on electrically
stimulated rat duodenum tissue contractions.
Using the isolated rat duodenum preparation, a stable amplitude was generated and a
reduction of this amplitude was observed dose dependently for the isobutyl series,
which is outlined in figure 6 for the pyrrolone 11. This assay represents a fast and
efficient way to screen for CCK antagonists using classical isolated tissue
preparations.
Cholecystokinin, CCK8s, induced contractions of the guinea pig gall bladder and this
second tissue based assay was applied to reconfirm with this standard preparation the
antagonistic properties of the potent CCK ligands.
CCKꢀ8s induced contractions of the gall bladder and these contractions were reduced
dose dependently, which is outlined for pyrrolone 11 in fig. 7. The concentration ꢀ
ꢀ5
response curve was calculated based on the percentage response to 10 M ACh for a
better comparison. Increased concentrations of the antagonist, pyrrolone 11 = ClIBA
were added to the bath cumulatively and a shift of the curve to the right was observed.
The function of the halogen atom is to enhance binding affinity/ increase lipophilicity
and most importantly, to block metabolism of the molecules in the para – phenyl
position.
1
2

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400
300
200
100
0
CCK-8
CCK-8 + 0.10 µM ClIBA
CCK-8 + 0.50 µM ClIBA
CCK-8 + 1.0 µM ClIBA
-11
-10
-9
-8
-7
-6
-5
log CCK-8 concentration (M)
Fig. 7 Mean cumulative concentration–response curve for CCKꢀ8s in the absence and
presence of 0.1, 0.5 and 1.0 µM ClIBA/pyrrolone 11 determined for the isolated
guinea pig gallbladder (Internal standard: ACh 10 µM = 100 % contraction).
It appeared that the effect of the antagonist in the gallbladder assay is insurmountable
and an irreversible inhibitor is ideal for the development of antineoplastic agents.
Reactive chlorine in the 4ꢀ position may be suitable for covalent drug receptor
interactions, explaining an irreversible antagonism for the isobutylꢀ pyrrolone series.
The antineoplastic properties of selected NCE and standard CCK antagonists, such as
2 1
Lꢀ365,260 (CCK ), devazepide and lorglumide (CCK ) was subsequently investigated,
using a range of CCK associated cancer cell lines.
In vitro cancer cell based testing
CCK antagonists are associated with an array of therapeutic applications, but the focus
of our research programme was to provide a nonꢀtoxic orally available CCK
antagonist for the treatment of cancers.
In vitro tests - Cytotoxicity assays
In vitro tests were originally performed using a cell count assay, as the aim was to
inhibit proliferation and not to optimise cytotoxicity, but the results were identical in
1
3

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the methylthiazolyltetrazolium (MTT) assay, possible due to induced apoptosis. It was
screened for an inhibitor of viability in certain CCK related cancers cell lines using the
MTT assay.
MIA PACA: Miapaca is a human pancreatic carcinoma cell line and selected data are
outlined in Table 2. Pyrrolone 9 was found 20 times better in terms of IC50
concentrations than lorglumide. Devazepide, which gave an IC50 about 1 µM showed
agonistic activity (unpublished results). Lorglumide, a CCK
1
antagonist, was acting on
the gallbladder CCK receptor and not the pancreatic CCK receptor. None of the
1
known CCK antagonists, devazepide or lorglumide proved to be clinical useful, which
is in line with our results.
AGS: AGS is a human stomach cancer cell line and was selected to screen for antiꢀ
cancer activity within the entire GI system including the stomach. The stomach
contains CCK /gastrin receptors and pyrrolone 17 occurred some CCK activity.
2
2
CCK
CCK
2
1
is gastrin and it is no surprise that a more CCK
2
antagonist, (or the combination
2
/CCK ) is working best on this cell line.
PANC: The PANC cell line is a human pancreatic cell line. Pyrrolone 9 is many times
better than devazepide / lorglumide on this selected pancreatic cell line. Cancer
treatment is combination therapy, even for hormone dependent cancers. For this
pancreatic cell line the synergistic effects of MKꢀ329 = devazepide in conjunction
24
with cisplatin were reported and studies to investigate pyrrolone 10, which is now
PNBꢀ028 with cis ꢀ platin and 5ꢀFU are ongoing.
Overall, pyrrolone 9, 10, 11 were not significantly different from each other and the
parent pyrrol 9 and the halogenate fluorinated pyrrolone 10 were tested subsequently
in vivo in xenograft models. In vitro in cell based assays they were found of equal
selective toxicity towards 2 human pancreatic and 2 colon cancer cell lines. The
cytotoxicity results for CCK related cell lines are outlined in table 2 with lorglumide
1 2
and devazepide as CCK standard and Lꢀ365,260 as CCK standard.
U373MG: The anticancer activity of the agents is not limited to GI cancers. The
cytotoxicity of the glioblastoma cell line U373MG, a brain cancer cell line, was
determined for a small series of test compounds including for Merck’s CCK
2
1
4

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1
antagonist Lꢀ365,260. The CCK antagonist, pyrrolone 9 and Lꢀ365,260 were found of
high nanomolar activity. The mixed CCK antagonist, pyrrolone 16 showed low
nanomolar activity and a programme for the development of mixed CCK antagonists
on this lead structure was initiated and the results will be communicated in a second
publication.
Table 2 Cytotoxicity assay, IC50 of selected examples against a variety of GI and brain
cell lines. IC50 values are based on inhibition of viability in the MTT assay in various
cell lines.
IC50
MIA
AGS
PANC U373MG MAC13 MAC16
[
nM]
PACA
Lꢀ365,260
Devazepide
Lorglumide
Pyrrolone 9
>5000 675
1200 >5000
425
15
±
54
>1000 541
±
87
32 >5000
21 >5000
>5000
1100
>5000
1000
480
±
±
±
23 289
±
±
±
±
14
316
421
536
508
526
±
±
±
±
11 861±22
±
4
4
4
262
222
242
18
16
18
27
18
22
±
9
6
9
±
765
665
475
±
±
±
29
28
25
32 96
35 76
34 86
±
25
20
23
Pyrrolone 10 11
Pyrrolone 11 13
Pyrrolone 17 230
±
±
±
±
±
±
±
11 53
±
4
674
33 23
±
3
>1000
>1000
A mixed antagonist may work best for treating brain cancers, as mengionomas express
2
5
1 2
CCK receptors and astrocytomas CCK receptors. A human glioma cell line, Uꢀ87
26
responded to CCK antagonists, unknown uncharacterised primary tumour cell lines
2
only to mixed CCK antagonists (Lattmann & Buchfelder unpublished).
1
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The antineoplastic effect, mediated by blocking CCK receptors, which are expressed
in parts of the GI system, correlated well in vitro cell culture and isolated tissue
preparations.
MAC 16: MAC 13 and MAC 16 cancers are derived from the colon of the mouse and
MAC 16 is of particular interest, as it is resistant to alkylating agents.
For pyrrolone 9, an IC50 about 100 nM was determined for the MAC 16 cell line and
the fluorinated analogue 10 was slightly more potent. Most interestingly, no other
CCK antagonist, such as Lꢀ365,260, devazepide or lorglumide was found active for
this cell line, which is, when transplanted into mice lethal within weeks. Therefore, it
may be concluded, that the unsurmountable irreversible properties of the antagonists
are key to colon cancer.
Subsequently, this tumour was studied initially in mice to analyse, if the in vitro
results can be translated into relevant antiꢀtumour activity.
Xenograft studies in nude mice
MAC 16 tumour xenograft study
MAC 16 tumours developed quickly within 10 days after implantation and grew
3
robustly. Once the tumours reached 100 mm , (Fig. 8) the animals were divided into
the various treatment groups and treated with vehicle or the fluorinated pyrrolone 10
and the nonꢀhalogenated derivative 9. Though MAC 16 tumours grew robustly from
3
3
1
00 mm to over 1500 mm within 2ꢀ3 weeks, the animals treated with both agents
tumours grew slower. Both pyrrolones inhibited the tumour growth significantly from
the day treatment was initiated and maintained tumour inhibitory activity until
sacrifice. At sacrifice, interestingly in some animals growths was not only inhibited,
but tumours shrinked after reaching a maximum volume. Most interestingly, while
MAC 16 tumours in the vehicleꢀtreated group metastasized to lungs, tumours in drugꢀ
treated groups did not metastasize to lungs.
Cholecystokinin is known to mediate cancer progression and metastasis, which were
2
7
antagonised by sufficient concentrations of devazepide.
1
6

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One animal in vehicleꢀtreated group died during the course of the study, which is
typical for MAC 16 induced cancer, showing the lethality of this selected cancer
model (Figure 8).
4000
Vehicle
3000
Pyrrolone 10
Pyrrolone 9
2000
1000
0
5
10
15
20
25
Time (days)
-1000
Fig. 8 Effect of pyrrolone 9 and 10 on MAC 16 colon cancer tumour growth.
1
Colon cancer is generally linked with the CCK receptor and only under certain
2
8
conditions it is associated with the CCK receptor. Gastric cancer is correlated with
2
the CCK
2
receptor / gastrin receptor and in the human gastric cancer cell line HTꢀ29,
antagonists
gastrinꢀ17 stimulated growths and theses effects were abolished by CCK
2
such as Lꢀ365,260. In detail Colucci tested only one 1 nM concentration of
devazepide, which stimulated growths in the study and by our own observations. Low
nanomolar devazepide concentrations occur in vitro a CCK agonistic effect and this
1
2
may explain why devazepide showed no significant effect in cancer previously.
Overall for the MAC 16 cell line proliferation was blocked in vitro and in vivo by our
pyrrolone based CCK antagonists. Here, the link CCK
confirmed experimentally.
1
receptor and colon cancer was
MIAPACA tumour xenograft study
MIAPACA tumours developed quickly after implantation and grew modestly, though
3
not as robustly as MAC 16 tumours. Once the tumours reached 100 mm , the animals
were divided into various treatment groups and were treated with vehicle or respective
1
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3
drugs. Though vehicleꢀtreated MIAPACA tumours grew from 100 mm to over 750
3
mm within 3 weeks, pyrrolone 9 and 10 ꢀ treated tumours grew slower.
Both agents inhibited tumour growth significantly from the day treatment was initiated
and maintained the growth inhibitory properties until sacrifice (Fig. 9).
5
00
00
00
00
00
Vehicle
4
Pyrrolone 10
Pyrrolone 9
3
2
1
0
2
4
6
8
10
12
14
16
Time (days)
-100
Fig. 9 Effect of pyrrolone 9 and 10 on MIAPACA pancreatic cancer tumour growth.
Overall, it was clearly shown, that colon and pancreatic cancers response to CCK
1
2
9
antagonists and not CCK antagonists. We agree, it is unlikely that, gastrin is more
2
important in pancreatic cancer than cholecystokinin. It may be wise to say, that it is
pancreozymin, via the pancreatic cholecystokinin receptor, that promotes cancer
(
CCK = pancreozymin receptor and not CCK cholecystokinin receptor) or possibly
1b 1a
the CCKꢀC receptor. Smith clearly stated the role of the CCKꢀC receptor in pancreatic
30
cancer and reviewed the cholecystokinin receptor as molecular target in treating
31
pancreatic cancer. If it is the CCKꢀC receptor, then according to recent nomenclature
the CCK receptorꢀ would be the cancer target and molecular modelling based on the
ꢀ
3
whole receptor, generated by homology modelling would be the starting point for
further optimisation towards the CCK receptor. This would be advantageous as the
3
1
CCK receptor is required for the physiological role of cholecystokinin. Blocking of
1
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the contractions of the gall bladder leads to gallstones in the long term and reductions
of bile flow will result generally in liver toxicity.
Conclusions
This novel arylated hydroxylꢀpyrrolone template was synthesised in only 2 synthetic
steps in high overall yields from readily available starting material.
1
For the potent CCK ligands, a full nonꢀcompetitive, possible irreversible CCK
antagonism was confirmed classically, using isolated tissue preparations, which is
ideal for an antineoplastic agent. For this novel template, it was found, that CCK
antagonism translated into antiꢀcancer activity within the GI system and the known
CNS activity of CCK antagonists may be of additional therapeutic benefit in treating
cancer –associated diseases.
The isobutyl series stopped the growths of colonꢀ and pancreatic cancer models and a
full preꢀclinical development programme was launched for the fluorinated pyrrolone
1
0. The new chemical entity PNB-028 progressed into late stage preꢀclinical
development for the treatment of human pancreatic and colon cancer.
All experiments were performed in compliance with the relevant laws and institutional
guidelines.
The xenograft studies (RDꢀ09/102014ꢀ4482403) in mice were performed and approved by
the bioethics committee at the University of Tennessee Health Science Center, Cancer
Research Building 19, Manassas, Memphis, TN 38103 and it was performed from May 2014
ꢀ
July 2014 .
No human subjects were involved in this study.
The study was sponsored by PNB Vesper Life Sciences.
References
1
2
I. M. McDonald, Exp. Opin. Ther. Patents., 2001, 11, 445.
M. G. Bock, R. M. DiPardo, E. C. Mellin and N. C. Newton, J. Med. Chem., 1994, 37
,
722.
3
4
5
E. Jorpes and V. Mutt, Acta Physiologica Scandinavica, 1966, 66, 196.
J. Bradwejn, D. Koszycki and G. Meterissian, Can. J. Psychiatry, 1990, 35, 83.
E. Lattmann and P. Arayarat, KKU. Sci. J., 2003, 31, 178.
1
9

MedChemComm
Page 20 of 21
View Article Online
DOI: 10.1039/C6MD00052E
6
7
C. T. Dourish and S. Ravard, Trends Pharmacol. Sci., 1990, 11, 271.
K. Rasmussen, J. F. Czachura, M. E. Stockton and J. J. Howbert, J. Pharmacol. Exp.
Ther., 1993, 264, 480.
8
9
1
C. T. Dourish, W. Rycroft and S. D. Iversen, Science, 1989, 245, 1509.
B. K. Trivedi, Curr. Med. Chem., 1994, 1, 313.
0E. Lattmann, D. C. Billington, D. R. Poyner, S. B. Howitt and M. Offel, Drug Des.
Discov., 2001, 17, 219.
1
1B. E. Evans, K. E. Rittle, M. G. Bock, R. M. DiPardo, R. M. Freidinger, W. L.
Whitter, G. F. Lundell, D. F. Veber, P. S. Anderson, R. S. Chang and D. J. Cerino, J.
Med. Chem., 1988, 31, 2235.
1
2 J.L. Abbruzzese, C.F. Gholson, K. Daugherty, E. Larson, R. DuBrow, R. Berlin, B. Levin,
Pancreas, 1992, 7, 165.
13
Y. Orikawa, H. Kato, K. Seto, N. Kobayashi, K. Yoshinaga, H. Hamano, Y. Hori, Mol.
Pain, 2010, 6 , 1744.
14 T. Meyer, M. E. Caplin, D. H. Palmer, J. W. Valle, M. Larvin, J. S. Waters, F. Coxon,
I. Borbath, M. Peeters, E. Nagano and H. Kato, Eur. J. Cancer., 2010, 46, 526.
1
1
1
1
1
2
2
2
5F. Makovec, R. Chistè, M. Bani, M. A. Pacini, I. Setnikar and L. A. Rovati, Arzneim.-
Forsch. / Drug Res., 1985, 35, 1048.
6M. Offel, P. Lattmann, H. Singh, D. C. Billington, Y. Bunprakob, J. Sattayasai and E.
Lattmann, Archiv der Pharmazie / Chemistry in Life Science, 2006, 339, 163.
7E. Lattmann, H. Singh, J. Boonprakob, P. Lattmann and J. Sattayasai, J. Pharm.
Pharm., 2006, 58, 1.
8E. Lattmann, J. Sattayasai, J. Boonprakob, P. Lattmann and H. Singh, Arzneim.-
Forsch.
9E. Lattmann, J. Sattayasai, Y. Boonprakob, H. Singh, P. Lattmann and S. Dunn, Drug
Discov. Ther., 2008, , 156.
/ Drug Research, 2005, 55, 251.
2
0E. Lattmann, D. C. Billington, D. R. Poyner, P. Arayarat, S. B. Howitt, S. Lawrence
and M. Offel, Drug Des. Discov., 2002, 18, 9.
1E. Lattmann, J. Sattayasai, D. C. Billington, D. R. Poyner, P. Puapairoj, S. Tiamkao,
W. Airarat, H. Singh and M. Offel, J. Pharm. Pharm., 2002, 54, 827.
2E. Lattmann, P. Lattmann, H. Singh. (Aston University) WO2004106315A3. 2004.
2
0

Page 21 of 21
MedChemComm
View Article Online
DOI: 10.1039/C6MD00052E
2
3E. Lattmann, W. O. Ayuko, D. Kinchinaton, C. A. Langley, L. Karimi, H. Singh and
M. J. Tisdale, J. Pharm. Pharm., 2003, 55, 1259.
2
4R. Jamshidipour, E. B. Pinho, D. K. Hom and S. B. Howell, Cancer Chemother.
Pharmacol., 1994, 34, 484.
25J. C. Reubi, J. C. Schaer and B. Waser, Cancer Research, 1997, 57, 1377.
26E. Oikonomou, M. Buchfelder and E. F. Adams, Neuropeptides, 2008, 42, 255.
27G. L. Matters, T. K. Cooper, C. O. McGovern, E. L. Gilius, J. Liao, B. M. Barth, M.
Kester and J. P. Smith, Dig. Dis. Sci., 2014, 59, 1180.
2
8 R. Colucci, C. Blandizzi, M. Tanini, C. Vassalle, M. C. Breschi and M. Del Tacca,
Br. J. Pharmacol., 2005, 144, 338.
2
9G. L. Matters, C. McGovern, J. F. Harms, K. Markovic, K. Anson, C. Jayakumar, M.
Martenis, C. Awad and J. P. Smith, Int. J. Oncol., 2011, 38, 593.
30 J. P. Smith, W. B. Stanley, M. F. Verderame and I. S. Zagon, Pancreas, 2004, 29, 271.
3
1J. P. Smith and T. E. Solomon, Am. J. Physiol. Gastrointest. Liver. Physiol., 2014,
06, G91.
3
2
1