Cholecystokinin-2/gastrin antagonists: 5-hydroxy-5-aryl-pyrrol-2-ones as anti-inflammatory analgesics for the treatment of inflammatory bowel disease

Article abstract of DOI:10.1039/c6md00707d

Arylated 5-hydroxy-pyrrol-2-ones were prepared in 2 synthetic steps from mucochloric acid and optimised as CCK₂-selective ligands using radiolabelled binding assays. CCK antagonism was confirmed for the ligands in isolated tissue preparations.

Full text of DOI:10.1039/c6md00707d

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Cholecystokinin-2/gastrin antagonists: 5-Hydroxy-5-aryl-pyrrol-2-ones as anti-inflammatory analgesics for
the treatment of inflammatory bowel disease†
E. Lattmann,a J. Sattayasai,b R. Narayaran,c N. Ngoc,a D. Burrell,a P. N. Balaram,d T. Palizdara and P. Lattmannd
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Arylated 5-hydroxy–pyrrol-2-ones were prepared in 2 synthetic Aim of the drug discovery programme was to target the underlying
steps from muco- chloric acid and optimised as CCK2 selective mechanism of the disease, thus targeting Cholecystokinin (CCK)
ligands using radiolabelled binding assays. CCK antagonism was pathways. CCK, a peptide hormone, which is extensively found in
confirmed for the ligands in isolated tissue preparations. DSS the gastrointestinal tract (GIT) is widely distributed through the
(dextran sulfate sodium) induced inflammation was analysed for nervous system2. Cholecystokinin acts as a neuromodulator / gut
derivative 7 and PNB-001 with L-365,260 as standard. The IC50 of hormone and CCK-ligands, agonists as well as antagonists3, have
PNB-001 was determined as 10 nM. Subsequent in vivo evaluation been extensively investigated as potential drug targets4. CCK-
confirmed anti-inflammatory activity with respect to IBD assays. antagonists were studied as growth inhibitors in certain forms of
The best molecule, PNB-001, showed analgesic activity in the cancer5, as anxiolytics6, in the treatment of schizophrenia7, satiety8
formalin test and in the hotplate assay the analgesic effect of 1.5 and as anti-panic agents9. An agonist, the shortened CCK
mg/kg PNB-001 was equivalent to 40 mg/kg tramadol. The CCK2 tetrapeptide, was found to induce panic in patients10
.
selective antagonist PNB-001 protected rats against indomethacin
Despite the progress of several CCK receptor antagonists11 to
different phases of clinical trials, only proglumide was marketed as
Milid for the treatment of gastric ulcer.
induced ulceration at similar doses. The GI protection activity was
found more potent than the 10 mg/kg dose of prednisolone,
which served as standard.
Introduction
O
N
O
O
Inflammatory bowel syndrome (IBS) is a complex symptom of
unknown ethology, characterized by abdominal pain or discomfort
associated with disturbed defecation and often bloating.
Inflammatory bowel disease (IBD) encompasses at least two forms
of intestinal inflammation being expressed as ulcerative colitis and
Crohn's disease. Although many other inflammatory disorders
affect the gastrointestinal tract, most can be distinguished by a
specific underlying process from IBD. Stress may exacerbate IBS
symptoms and the CCK2 receptor is known to mediate anxiety1 and
panic attacks. Though several drugs such as anti-cholinergic-, anti-
spasmodic- agents and tricyclic anti-depressants are of limited use
for IBS and steroids for IBD, inflammatory bowel disease is still
classified as unmet medical need and hundreds of studies are
ongoing worldwide.
O
NH
O
N
N
HN
Ca2+
NH
N
NH
COO-
2
L-365,260
Z-360
Figure 1. CCK2 / gastrin antagonists, Merck standard and most
recent addition.
Merck’s standard CCK2 antagonist12 is outlined in Figure 1. Z-360 is
the most recent derivative derived from this original lead structure,
in which the N was moved to the 5-position. The N-1 was alkylated,
the 5 phenyl group was replaced by a cyclohexyl group and the
water solubility was enhanced by converting the Me group on the
side chain into a carboxylic acid. All structural optimisations did not
address the main underlying problem with respect to poor
pharmacokinetic properties, such as a low solubility and very low
membrane penetration resulting from the large polar surface area
of the molecule.
PNB Vesper Life Science systematically investigated the hydroxyl-
pyrrolone scaffold and the isobutyl derivative13 served as lead
structure on this programme towards novel anti-inflammatory
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analgesics targeting the underlying mechanism of inflammatory
ulceration.
A full biological evaluation of lactame 14, now PNB-001, will be
reported here, in this publication from a CCK-5 pentagastrin
antagonism to initial anti-inflammatory analgesic activity.
Results and discussions
Synthesis
Mucochloric acid was reacted in presence of a Lewis acid via SE
reactions into a series of arylated dichlorinated furanones. In
addition to halogenated X=Cl,
F 5-arylated 2(5H)-furanones
containing further donor substituents were prepared. The chemical
yields were generally lower and binding affinity was also decreased
in the resulting final molecules. Arylated 2(5H)-furanones,
containing nitro- groups or trifluoro-methyl groups could not be
prepared.
Scheme 2. Chemical mechanism for the formation of 5-hydroxy-5-
aryl-pyrrol-2-ones with amines.
Subsequent reaction of the 5-arylated 3,4-dichloro-2(5H)-furanones
with amines, in particular with aryl alkyl amines, furnished 15
arylated N-alkylated hydroxyl-pyrrolones and the 2nd step of the
synthetic sequence is outlined in Scheme 1.
SAR optimisation
Initially SAR were fully explored and optimised for the CCK2/gastrin
receptor, using a radiolabelled binding assay with iodinated CCK8.
The results, expressed in IC50, are outlined in table 1.
Lorglumide served as CCK1 standard and L-365,260 was used as
CCK2 standard. For a better comparison the initial starting point, the
CCK1 antagonist PNB-028, is also included in Table 1.
Cyclic substituents on the N-position, such as cyclopropyl-,
cyclopentyl- and cyclohexyl provided activity in the micromolar
range (Lactame 1-5). Anilines were inactive and the introduction of
the benzyl group furnished a dual ligand in nanomolar range
(lactame 7). Halogen atoms on the 5 phenyl group marginally
enhanced the binding affinity and the best derivative was the
fluorinated N-benzyl lactame 9.
Scheme 1. Synthesis of lactames 1-15 from 3,4-dichloro- 5-arylated
2(5H) furanones. For the synthesis of building blocks from
mucochloric acid see Lattmann et al, MedChemComm 2016.
Donor substituents on the 5-phenyl group, such as para-methoxy
and methyl groups resulted in ligands with manifold lower activity
(lactame 10, 11). The introduction of a chiral amine, such as methyl
benzyl amine provided diastereoisomers (12, 13), which were
separated by column chromatography, but both isomers occurred a
lower affinity than the parent benzyl derivative 7. Further
optimisation on the N-benzyl group was performed, but the
introduction of a para F- and methoxy- group, using p-fluorobenzyl
amine and p-methoxy benzylamine, did not enhance the binding
affinity for the CCK receptor.
The 5-arylated 2(5H)-furanones reacted selectively in the ester
position and no reaction in the 4-position was observed. Previously
the substitution in the 4-position was described for pseudo-
esters14, and here a ring -opening ring -closure mechanism for the
formation of hydroxyl-pyrrolones is proposed in scheme 2.
The first step in the reaction sequence of the dichlorinated 2(5H)-
furanone is the ring opening and amide formation from the
corresponding lactone. The keto form of the acyclic amide was then
in situ converted into a lactame under the elimination of hydrogen
chloride.
The introduction of a spacer, a single CH2 group, resulted in a
phenyl-ethyl derivative 14, which represented a highly CCK2
selective ligand.
Lactame 14 is 450 times selective for the CCK2 / gastrin receptor.
Halogenation, the introduction of a para- chlorine atom on the
phenyl–position, lactame 15, did not enhance binding affinity any
further; possibly due to drug receptor interaction of the phenyl
group with a lipophilic cavity within the CCK receptor.
Analysis by chiral HPLC showed a 50:50 racemic mixture of the
novel template in solution in methanol.
2 | J. Name., 2012, 00, 1-3
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Table 1. CCK binding affinity using radioligands with cortex and the metabolism of the 5-phenyl derivative 14, may result in a p-
pancreatic membranes. IC50 is presented in micromolar; N=3.
hydroxy-phenyl metabolite, which may interact additionally with
His-122 via a strong hydrogen bond and the role of metabolites is
currently under investigation.
Lactame
X=
R=
CCK-A
CCK-B
[µM]
[µM]
Lorglumide
-
-
>10
0.17±0.01
L-356,260
-
-
0.25±0.01
0.012±0.001
7.5±0.3
0.003±0.001
0.75±0.21
>10
PNB-028
F
Isobutyl-
Cyclopropyl-
Cyclopropyl-
Cyclopentyl-
Cyclopentyl-
Cyclohexyl-
Ph-
1
H
Cl
H
Cl
H
H
H
Cl
F
2
>10
4.0±0.3
3
0.36±0.30
2.50±0.20
>10
0.84±0.40
>10
4
5
>10
6
>10
>10
7
Bz-
0.80±0.04
0.51±0.04
0.32±0.01
0.21±0.02
1.5±0.04
0.60±0.04
0.022±0.002
0.020±0.004
Figure 2. Docking of CCK antagonist PNB-001 into the CCK2
receptor.
8
Bz-
9
Bz-
0.017±0.002
4.5±0.4
The molecular structure of lactame 14, (Table 1) which is now PNB-
001 was designed from an iso-propyl lead structure via the benzyl
derivative 7 (Figure 3). PNB-001 contains the N-phenyl-ethyl
substituent, which is required for high CCK2 / gastrin selectivity15.
10
11
12
MeO Bz-
Me
H
Bz-
4.1±0.3
Methylbenzyl-
0.21±0.01
13
14
15
H
H
Cl
Methylbenzyl-
Phenylethyl-
Phenylethyl-
0.42±0.03
>10
0.21±0.15
Cl
Cl
7
0.022±0.002
0.030±0.001
HO
HO
O
O
>10
N
N
Molecular modelling - Ligand docking
Lead structure
Cl
The docking of the phenylethyl pyrrolone 14 into the CCK2 receptor
is outlined in figure 2 for one final pose of minimal energy and some
key drug receptor interactions are highlighted.
HO
N
The 2-carbonyl group of the central pyrrolone template interacts via
hydrogen binding with the N group of Trp-114. The phenyl group of
the N-phenylethyl- side chain binds towards the aromatic indole
system of Trp-114 and electron withdrawing groups should enhance
these aromatic interactions. The lipophilic pocket in the CCK2
receptor comprising of Ile-184 and Leu-133, allows principally a
wide range of substituents, but only phenyl and not cyclo-hexyl can
be realised synthetically. The 5-phenyl group of the pyrrolone
template binds via Ile-184 and Leu-133, based on van der Waals
interactions and not aromatic interactions. Therefore, the
introduction of electron withdrawing groups on the 5- phenyl
group, such as a chlorine atom, do not enhance binding affinity and
optimisation on this side is overall with limited effect. Interestingly,
O
PNB-001
14
Figure 3. Lead design. From a CCK1 antagonist to a CCK2 selective
molecule.
The mixed dual acting N- benzylated pyrrolone 7 (Table 1) is
showing the expected pharmacological profile of a mixed CCK
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antagonist16 and its role in brain cancer is currently being Inflammatory bowel disease, IBD, is classified an unmet medicinal
elucidated.
need by the FDA and allows fast track approval. The indomethacin
induced ulceration is a standard in vivo assay for IBD21.
The phenyl ethyl derivative PNB-001 completed preclinical
development as anti-inflammatory analgesic and progressed into In vitro inflammation
Phase 1 clinical trials for the treatment of IBD.
In vitro, spontaneous contractions correlate with inflammation and
anti-inflammatory steroids such as dexamethasone reduced
spontaneous contractions of the duodenum22.
Isolated tissue preparations
DSS, dextran sulfonic acid sodium, is a standard agent to induce
inflammation for IBD in vivo and in vitro the effect is slower and
milder than for Trinitro-benzene sulfonic acid, TNBSA23.
CCK antagonism
The CCK2 selective ligand PNB-001, occurred a potent binding
affinity and the CCK-antagonism was studied17 using pentagastrin,
CCK-5 induced contractions of the rat duodenum.
DSS was used here in vitro using isolated organ preparations and
preparations of the duodenum worked best in this assay24.
Initially CCK4 was used18, but CCK4 has a low solubility and low
potency in the micro-molar range. The best CCK2 selective agonist
is CCK-5 and this was used to analyse the agonist or antagonist
properties of the ligand.
L-365,260 was applied as CCK2 standard and the mixed CCK
antagonist HPhBz, lactame 7, together with the highly CCK2
selective antagonist PNB-001 were all tested under the same
conditions. The concentration response curves, showing how these
agents inhibit contractions a very low concentrations, are outlined
in Figure 5.
100
PNB001
L365260
HPhBz
80
60
40
20
0
0.01
0.1
1
10
100 1000 10000
log C (nM)
Figure 5.The inhibitory concentration-response curves of PNB-001,
L-365,260 and HPhBz (lactame 7) for DSS stimulated contractions.
Figure 4. Responses to CCK-5 in the absence and presence of PNB-
001; CCK-5, CCK-5 +10 nM PNB-001, CCK-5 +30 nM PNB-001, CCK-5 +
100 nM PNB-001; N = 2 for each data point. Values are ±SEM.
An IC50 of 1 nM was determined for CCK2 antagonist PNB-001. The
Merck CCK2 standard occurred a ten times lower IC50 and the
benzylated derivative HPhBz, lactame 7, had the lowest anti-
inflammatory effect. Considering the chemical similarity between
the lactame 7 and PNB-001, the addition of an extra CH2 group
resulted in a remarkable effect. The CCK1 antagonism may oppose
the anti-inflammatory effect, but the possible mechanistic pathway
is outside the scope of this publication.
The concentration response curve of pentagastrin, CCK-5, was
recorded and shifted to the right by a nanomolar concentration of
PNB-001, thus confirming the antagonistic properties19 of this
ligand.
100 nM of PNB-001 with 30 min of incubation time fully blocked the
CCK4 and CCK5 induced contractions. For the 5 min incubation cycle,
10 nM of PNB-001 shifted the CCK-5 concentration response curve
to the right and from 30 nM onwards, the antagonist PNB-001
showed a reduced maximum response. At high nano molar
Based on the in vitro results with respect to anti-inflammatory
activity, PNB-001 was selected for further in vivo assays. This
programme was concerned with inflammation of the GI tract and
the indomethacin induced ulceration is the best model for IBD,
inflammatory bowel disease and ulcerative colitis, which is
associated with the release of gastric acid. Here, the anti-
inflammatory action of the molecule, supported by anti-gastric
activity, was supposed to create a synergistic therapeutic effect.
concentrations
PNB-001
acted
as
unsurmountable
gastrin/cholecystokinin antagonist.
As the clinical trial outcome of selective CCK/gastrin antagonists
was found questionable in the treatment of anxiety and
depression20, the aim of the programme was, to focus on selectively
designing CCK2 antagonists for the gastrointestinal system and to
focus on inflammation thereof.
4 | J. Name., 2012, 00, 1-3
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In vivo evaluation
Inflammation scores
Inflammatory/ulceration protection
Proximal Duodenum
Distal Jejunum
Proximal ileum
Proximal colon
Distal colon
Gastrin triggers the release of gastric acid in the stomach and the
content of the GI system moves from the duodenum, jejunum via
ileum to the colon. The inflammation was assessed in this order by
organ weight and inflammation scores. The measurement of weight
as inflammation parameter is reliable and the induction of
inflammation / ulceration resulted in an increased organ weight.
10
8
Whole caecum
6
4
2
0
Weights of different parts of GI tract
Vehicle
1.5
1.0
0.5
0.0
Indomethacin
Indo+5 mpk PNB-001
Indo+20 mpk PNB-001
Indo+10 mpk Predni
Figure 7. Inflammation score of the GI tract treated by PNB-001.
Treatment is presented for each individual organ and scores of
inflammation were determined. Most interestingly the gaps in the
treatment groups with zero inflammation are present in the
duodenum for both the 5 and the 20 mg /kg dose of PNB-001 via
oral administration. Indomethacin induced an inflammation score
of 5 in the duodenum, which was fully protected by both doses of
the CCK B antagonist and reduced to 2 by prednisolone at 10 mg/kg.
Figure 6. Indomethacin induced ulceration. Weights of the GI tract
treated by PNB-001.
In figure 8 selected photos of the gastrointestinal system with
ulceration are depicted. In the duodenum the perforation is clearly
visible and this is fully prevented by a low 5 mg /kg dose of PNB-
001. A similar effect is seen for the jejunum on indomethacin
induced ulcers and this damage of the GI system was prevented by
PNB-001.
Indomethacin increased weight in all observed organ parts and this
increase of weight was reduced dose dependently by PNB-001 for
the duodenum, jejunum, and ileum. The effect of protection is
observed for all parts of the GI system and PNB-001 performed
better than the steroid standard prednisolone.
The second accessed parameter was scores of inflammation and
the results are outlined in Figure 7.
Figure 8. Top: Photos of duodenum; below jejunum. Selected parts
of the GI system, indomethacin induced ulcers and prevented by
CCK2 / gastrin antagonist PNB-001.
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Hot Plate Test
40
30
20
10
0
Analgesic evaluation
CCK antagonists potentiate the analgesia of opiates and usually,
except for proglumide, have no analgesic effect on their own 25. For
Z-360 an interesting weak analgesic effect in the formalin test was
observed26.
Thus, a first evaluation of the analgesic properties of PNB-001 was
performed using the formalin test.
1st phase
200
2nd phase
150
100
50
Figure 10. Hotplate test of PNB-001 in mice by IP – administration.
0
Bar 1 is the baseline with a response time about 12 s. The maximum
analgesic effect in the hotplate is 26 s for 40 mg/kg tramadol, a
more than 100% of change from the control.
The analgesic efficacy of 0.5 mg/kg PNB-001 by IP administration as
a single agent was found equivalent to 40 mg/kg tramadol (SC
administration). Interestingly, there is no significant potentiation of
tramadol analgesia with PNB-001. The selective CCK2 / gastrin
antagonist, worked here in this assay as analgesic on its own. This
analgesic activity may be found useful in the treatment of IBD/IBS
pain, additionally.
Figure 9. Analgesic anti-inflammatory activity of PNB-001 on
formalin induced pain in rats.
Opiates, such as morphine, reduce the response in form of licks and
bites to formalin in the first phase of the assay. Anti-inflammatory
agents are active in this assay in the second phase.
Conclusions
This novel template, which originally occurred CCK1 activity27 was
optimised as CCK2/gastrin selective antagonist.
PNB-001 showed only at the 1.5 mg/kg dose some opiate like
effects (phase 1) and displayed at this dose also the best anti-
inflammatory effect (phase 2). The 5 mg dose by IP administration is
equivalent to the 20 mg/kg dose by oral administration, but had a
lower analgesic effect than the 1.5mg/kg dose. However, the
activity in the second phase of the formalin test, reconfirmed the
The target molecule was synthesised in only 2 steps from readily
available starting materials and will potentially deliver affordable
therapeutic agents.
By designing a potent CCK2 /gastrin antagonist for inflammatory-
diseases and not CNS disorders, a first in class CCK2 antagonist with
analgesic anti-inflammatory activity was developed under the
consideration of membrane penetration, half-life and
bioavailability.
anti-inflammatory activity found in vitro and in vivo
.
In order to evaluate the pain managing properties of the new agent,
the partial opiate agonist tramadol was included in a last study, in
which the hot plate assay was used to evaluate analgesic activity.
By focussing on unmet medical needs such as inflammatory bowel
disease, IND was granted in India and the FDA approval is expected
for late 2019.
The mild analgesic effect of PNB-001 may offer an alternative drug
treatment to Tramadol, which is closing the gap of pain
management between NSAIDs and opiates.
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25 E. Lattmann, J. Sattayasai, C. H. Schwalbe, Y. Boonprakob, S.
Dunn, F. Fajana and P. Lattmann, Arch Pharm (Weinheim), 2016,
349, 456.
Acknowledgements
26 K. Yoshinaga, T. Horii, H. Hamano, R. Eta, T. Ozaki, Y. Orikawa, K.
Yoshii, Y. Kawabata, Y. Hori, K. Seto, M. Takei and Y. Kuraishi,
Biol Pharm Bull., 2010; 33, 244.
The experimental work was partly supported by PNB Vesper Life
Sciences Pvt, India.
27 S. Ponnusamy, E. Lattmann, P. Lattmann, T. Thiyagarajan, B. N.
Padinjarethalakal and R. Narayanan, Oncology Reports, 2016,
35, 2097.
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