Analgesic Effects of 5-Alkyloxy-4-amino-2(5H)-furanones as Cholecystokinin-2 Antagonists

Article abstract of DOI:10.1002/ardp.201600036

4-Amino-2(5H)-furanones were synthesized in high yields over two synthetic steps from readily available mucochloric acid. These 5-alkyloxy-4-amino-2(5H)-furanones were screened in a ^[125]I-CCK-8 radioligand receptor binding assay for CCK₂ affinity and novel active ligands in the nanomolar range were identified. SAR was optimized leading to the cyclohexyl derivative 25 with an IC₅₀ of 27 nM. Furanone 18 was obtained as a stable crystalline material with an IC₅₀ of 85 nM, but had a higher CCK₂ selectivity. It was subsequently tested in the isolated Guinea pig ileum assay with sulfated CCK₈, and the CCK antagonizing properties of the ligand were confirmed. The CCK₂ selective antagonist 18 was found to potentiate analgesia in the tail flick assay in mice, for the strong opiate morphine, the partial opiate agonist tramadol and the tricyclic antidepressant desimipramine. 4-Amino-2(5H)-furanones were synthesized and screened in a ^[125]I-CCK-8 radioligand receptor binding assay for CCK₂ affinity. SAR optimization led to furanone 18 (IC₅₀ = 85 nM). It was confirmed as antagonist in the isolated Guinea pig ileum assay and it was found to potentiate analgesia in the tail flick assay in mice, with morphine, tramadol, and desimipramine at 0.5 mg/kg.

Full text of DOI:10.1002/ardp.201600036

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
Archiv der Pharmazie
Full Paper
Analgesic Effects of 5-Alkyloxy-4-amino-2(5H)-furanones as
Cholecystokinin-2 Antagonists
Eric Lattmann1, Jintana Sattayasai2, Carl H. Schwalbe1, Yodchai Boonprakob2, Simon Dunn1,
Feyisayo Fajana1, and Pornthip Lattmann1
1
The School of Pharmacy, Aston University, Birmingham, UK
Faculty of Medicine, Department of Pharmacology, Khon Kaen University, Khon Kaen, Thailand
2
4-Amino-2(5H)-furanones were synthesized in high yields over two synthetic steps from readily
available mucochloric acid. These 5-alkyloxy-4-amino-2(5H)-furanones were screened in a [125]I-CCK-8
radioligand receptor binding assay for CCK2 affinity and novel active ligands in the nanomolar range
were identified. SAR was optimized leading to the cyclohexyl derivative 25 with an IC50 of 27 nM.
Furanone 18 was obtained as a stable crystalline material with an IC50 of 85 nM, but had a higher
CCK2 selectivity. It was subsequently tested in the isolated guinea pig ileum assay with sulfated CCK8,
and the CCK antagonizing properties of the ligand were confirmed. The CCK2 selective antagonist 18
was found to potentiate analgesia in the tail flick assay in mice, for the strong opiate morphine, the
partial opiate agonist tramadol and the tricyclic antidepressant desimipramine.
Keywords: 2(5H)-Furanones / Analgesia potentiation / CCK antagonists / Isolated tissue preparations /
Radioligand binding
Received: February 11, 2016; Revised: April 5, 2016; Accepted: April 11, 2016
DOI 10.1002/ardp.201600036
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Asperlicin (Fig. 1) was the first non-peptidal CCK antagonist
lead structure from nature and analogs thereof were studied
as CCK ligands [11]. Simplification of the lead structure by
Introduction
Cholecystokinin acts as a neuromodulator as well as gut
hormone and CCK-ligands, agonists as well as antagonists [1],
have been extensively investigated as potential drug mole-
cules [2]. CCK-antagonists were studied as growth inhibitors
in certain forms of cancer [3], as anxiolytics [4], in the
treatment of schizophrenia [5], satiety [6], and as anti-panic
agents [7]. The most simplified agonist, the shortened CCK
tetrapeptide, was found to induce panic in patients and these
effects were blocked by CCK antagonists [8]. In addition to
anxiety [9] and depression [10], the clinically most relevant
therapeutic area for CCK antagonists is pain management.
Merck led to devazepide [12], a potent CCK1 selective
cholecystokinin antagonist, containing a 1,4-benzodiazepine
template and an indole moiety. A phase II trial of devazepide,
which was only published in form of a conference report [13]
showed a significant enhancement of the effect of opiates in
the treatment of chronic and severe pain and the CCK
neuropeptide pathway was reviewed and a featured clinical
application for pain [14].
The indolyl amide of devazepide was replaced by a urea
linkage and this resulted in Merck’s L-365,260, a CCK2 selective
antagonist. Further structure–activity relationship (SAR)
optimization led to Zeria’s Z-360, in which the N-alkyl side
chain and the 5-position (cyclohexyl-) was optimized for
potency (Fig. 1). Additionally, a meta-carboxylic acid on the
aryl urea linkage was introduced to enhance water solubility
of this most recent CCK2 antagonist [15] (Fig. 1).
Correspondence: Dr. Eric Lattmann, Medicinal Chemistry, The
School of Pharmacy, Aston University, Aston Triangle,
Birmingham B4 7ET, UK.
E-mail: e.lattmann@aston.ac.uk
Fax: þ44-121-3590733
In our search for new CCK ligands, the 1,4-benzodiazepine
template was varied by a combinatorial solid phase synthe-
sis [16] and optimized in terms of CCK binding affinity [17].
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
456

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
Analgesia: Synthesis and Evaluation of CCK-2 Antagonists
Archiv der Pharmazie
under reflux using a Dean-Stark trap (Method A). For the iso-
propyloxy-3,4-dichloro-2(5H)furanone excess of the alcohol
was applied (Method B) and the intermediate was obtained in
large crystals once crystalline (Scheme 1).
The 5-alkoxy (5-propargyl-, allyl-, isopropyloxy-, benzyloxy,
and cyclohexyl-methyloxy-) 2(5H)-furanone intermediates
were reacted at slightly elevated temperatures with an excess
of the parent amine into the desired 4-amino-5-alkoxy-2(5H)-
furanones 1–26.
The target molecules were obtained in a low yield for the
propargyl series and in good yields for the allyloxy,
isopropyloxy, and benzyloxy series. Iso-propyloxy derivatives
were generally inactive, but crystalline solids, while the
benzyloxy series provided active and crystalline target
molecules. Chemical stability is linked with crystalline
properties via the melting point, explaining the desire of
the medicinal chemist for white crystalline materials.
Figure 1. Drug design from asperlicin, an isolated natural
product, via devazepide, a CCK1 selective antagonist, to a CCK2
selective urea-based antagonist, L-365,260. Drug optimization
from L-365,260 toward Z-360, a CCK2 antagonist with improved
potency and water solubility.
SAR optimization
The propargyl series contained the original lead structure 1,
which was identified, when the combinatorial library of
antibacterial 4-amino-furanones was screened in a CCK
radioligand binding assay. The 4-isobutyl lead structure 1,
which was identified by chance, was obtained in a low yield
and occurred as sticky brown oil and the optimization of SARs
are outlined in Table 1.
The 1,4-benzodiazepine structure [18] was replaced by an
achiral diphenyl pyrazolone template, giving novel CCK
antagonists with an indole carboxylic acid [19] and a phenyl
urea moiety [20] and they displayed excellent animal data on
anxiety and depression [21].
The change of the butyl group in this series resulted
generally in lower binding affinity and the benzyl derivative 2
was found of twofold lower and the N-methyl benzyl analog
of a similar activity.
Again, having realized the poor pharmacokinetic proper-
ties of these agents, a search for a completely novel, smaller
template with a molecular weight <350, a log p about 3 and a
polar surface area for membrane penetration of less that
100A, with no urea linkage was initiated.
This resulted in the discovery and SAR-optimization of 4-
amino-2(5H)-furanones, which are reported here in this
publication. For the first time now, a totally novel, non-urea,
and non-benzodiazepine template [22] was available as
potent CCK2 receptor antagonist. In vivo evaluation in mice
using the tail flick test [23] was initiated with tramadol,
morphine, and finally desimipramine and resulted in a
relevant potentiation of analgesia.
Results and discussion
Chemistry
Alkoxy-2(5H)-furanones were generally prepared from
mucochloric acid using previously developed methods [24].
The overall starting material of this short chemical sequence is
furfural, which is converted into mucochloric acid on an
industrial scale, mainly for the use of this intermediate in
plant protection and pharmaceuticals.
Scheme 1. See experimental section for furanone building
blocks. Synthetic route for the formation of 5-alkoxy-4-amino-
furan-2(5H)-ones 1–26.
Propargylalcohol, allylalcohol, benzylalcohol, and cyclo-
hexyl methanol were reacted in the presence of toluene
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
457

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
E. Lattmann et al.
Archiv der Pharmazie
Table 1. SAR optimization with respect to receptor binding affinity for 4-amino-5-alkoxy-furan-2(5H)-ones 1–26.
Entry
R1
R2
CCK-B
CCK-A
1
2
3
4
5
6
7
8
Propargyl-
Propargyl-
Propargyl-
Propargyl-
Propargyl-
Allyl-
Isobutylamino-
Benzylamino-
N-Methyl-benzylamino-
Benzylpiperazinyl-
3,5-Dimethylmorpholino-
Isobutylamino-
220 ꢀ 29
470 ꢀ 34
280 ꢀ 32
>10000
>10000
1239 ꢀ 103
>10000
4312 ꢀ 242
1200
>10000
–
–
–
–
–
Allyl-
Allyl-
Sec butylamino-
n-Butylamino-
–
–
9
Isopropyl-
Isopropyl-
Isopropyl-
Isopropyl-
Isopropyl-
Isopropyl-
Isopropyl-
Isopropyl-
Isopropyl-
Benzyl-
Benzyl-
Benzyl-
Benzyl-
Benzyl-
Isopropylamino-
Cyclopropylamino-
Cyclopentylamino-
Cyclohexylamino-
Phenylethylamino-
Benzylamino-
N-Methyl-benzylamino-
Dimethylanilino-
Indolino-
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4300
5700
2300
6700
1300
2700
–
–
–
–
–
–
2600 ꢀ 120
–
5400
–
Isobutylamino-
n-Butylamino-
85 ꢀ 11
548 ꢀ 43
627 ꢀ 43
162 ꢀ 13
263 ꢀ 18
180 ꢀ 33
503 ꢀ 48
27 ꢀ 6
2566 ꢀ 343
–
Sec butylamino-
3-Methylpyrazolo-
Cyclopropylamino-
Cyclopentylamino-
Cyclohexylamino-
Isobutylamino-
–
2344 ꢀ 321
>10000
2598 ꢀ 436
–
Benzyl-
Benzyl-
Cyclohexylmethyl-
Cyclohexylmethyl-
260 ꢀ 21
–
Methylamino-
>10000
IC50 in nM. N ¼ 3.
The introduction of piperazinyl-, morpholinyl-, and other
heterocyclic groups was found to result in the inactive
molecules 4 and 5.
The isobutyl analog 6 containing an allyloxy-group, was
five times less potent and the closely related n-butyl
derivative 8 was found 20 times less potent in the receptor
binding assay.
Subsequently, the 5-alkoxy C3 unit was varied and a series
of isopropyloxy furanones 9–17 was prepared and tested and
an unspecific micromolar activity was determined.
The benzyl group is a classical bioisostere of the propargyl
group, and the benzyl analog of the isobutyl furanone 18 was
three times more potent than lead structure 1. It was also
obtained in a good chemical yield as a white, crystalline
material.
For n-butyl and for the secondary butyl 5-benzyloxy-
furanones 19 and 20, the bioactivity was decreased.
The pyrazol derivative 21, as well as the cyclopropyl
furanone 22 and the cyclopentyl derivative 23 were found
of a high nanomolar binding affinity, while the cyclohexyl
analog 24 had a significantly lower activity.
resulted in enhanced binding affinity for the privileged
isobutyl amino structure 25. A similar enhancement phenyl
versus cyclohexyl was previously observed in Z-360, a recent
CCK2 antagonist [25].
Thus, overall four potent CCK ligands in the nanomolar
range were identified. The furanone 18 showed an IC50 of
85 nM and displayed 30 times selectivity toward the CCK2
receptor. Furanones 21 and 23 displayed slightly lower
binding affinities and are outlined in Fig. 2.
The cyclohexyl derivative 25 showed an enhanced IC50 of
about 27 nM, but was less selective, only 10 times for the
CCK2R. It was obtained as an oily solid, which makes
purification under GMP conditions by recrystallization
impossible.
The isobutyl-amino-benzyloxy-furanone 18 was recrystal-
lized from methanol and the crystal structure was
determined.
The crystal structure of the high yield – high purity –
compound, furanone 18, is outlined in Fig. 3, supporting
excellent manufacturing properties due to appropriate
physical chemical properties. In the crystal structure, the 5-
benzyloxy moiety is orientated below the furanone plain and
the 4-amino substituent is located above.
In order to rationalize drug ligand interactions of furano-
none 18 with the CCK2/gastrin receptor, molecular modeling
studies were performed.
For the 5-cyclohexyl methyl aminofuranones series, the
methyl analog 26 showed no binding affinity up to 10
micromolar and the isobutyl derivative 25 was identified as
the most potent derivative of the entire series. The replace-
ment of the aromatic phenyl group by a cyclohexyl group
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
458

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
Analgesia: Synthesis and Evaluation of CCK-2 Antagonists
Archiv der Pharmazie
Figure 4. Docking of the furanone 18 into the active site of the
CCK2 receptor.
Functional assay – isolated guinea pig ileum
preparation
Figure 2. Overview of nanomolar CCK2 ligands.
In a radio-labeled binding assay with native brain and
pancreatic membranes, potent and selective ligands were
identified. The CCK ligands may act as antagonists, and this
was investigated using isolated tissue preparations.
Sulfated CCK8 resulted in dose-dependent contractions of
the GPI, which are outlined in Fig. 5. From 1 nM onwards, a
small response was obtained, which reached a plateau at
about 100 nM concentrations. A total of 500 nM final bath
concentration of the test molecule 18 shifted the curve to the
right and 1 mM concentration of the furanone 18 resulted in
nearly a complete inhibition of CCK8S-induced contractions,
so that it is now confirmed, that the ligand furanone 18 acted
as CCK antagonist.
The small alkyl amino-benzyloxyfuranone molecule 18
showed a cLogP of about 2.6 compared with a cLogP of
diazepam of 2.7. Therefore, a sufficient brain penetration
should be anticipated, essential for CNS activity. A high
percentage of preclinical candidates drop out due to a poor
pharmacokinetic profile and this is in particular complex for a
neuromodulator.
In Fig. 4, the docking of the furanone 18 into the CCK2
receptor is outlined and key drug ligand interactions are
analyzed as follows:
Van der Waals interactions of the isobutyl group with
Ala106 explain the importance of the two methyl groups of
isobutyl, which both interact with the methyl side chain of
alanin. Therefore, even minor modifications of the alkyl
group resulted in less binding affinity.
The carbonyl oxygen of the ligand binds to Arg 106, while
the O1 showed hydrogen binding with the alpha H of Trp 105.
The aromatic benzyl group interacts with a lipophilic
pocket of the CCK2 receptor and in particular interactions
with Ile122 explained an enhanced binding affinity for the
cyclohexyl ring system of ligand furanone 25.
CCK antagonists potentiate the analgesic effects of opiates
and for this potent CCK2 antagonist 18, the scope of analgesia
was investigated.
Tramadol is a partial opiate agonist and also NA/SE
reuptake inhibitor. Here, for the first time the tricyclic
antidepressant desipramine was included in the evaluation
of one selected CCK antagonist, furanone 18.
In vivo analgesic tests
In vivo assay – analgesia potentiation assay [26] in mice for
furanone 18
The effects of CCK on the modulation of pain transmission
and the opioid effects are well established [27, 28], and
typically a low dose of morphine or a high dose of a weak
opiate agonist will result in a good potentiation of analgesia
[29] in the tail immersion test [30].
Figure 3. Crystal structure of 5-benzyloxy-3-chloro-4-isobutyl-
amino-5H-furan-2-one 18, sample recrystallized from methanol.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
459

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
E. Lattmann et al.
Archiv der Pharmazie
For morphine, the potentiation of analgesia was also
confirmed. Morphine analgesia is potentiated by a CCK
antagonist and the 2 mg/kg morphine dose is, in the presence
of furanone 18, equivalent to 8 mg/kg morphine, which is
therapeutically relevant to reduce side effects of opiates.
Most interestingly, for desimipramine at 0.5 mg/kg, a
maximum possible effect of 6% was determined. Tricyclic
antidepressants (TCAs), such as desimipramine, show clinically
a useful analgesic effect in man and in mice, this effect is very
small (0.3% MPE). Here, in the presence of 0.5 mg/kg CCK
antagonist 18, the 20 mg/kg desimipramine dose is equivalent
to 20–40 mg tramadol. Thus, 0.5 mg/kg of furanone 18
potentiated the analgesic effect of a classical TCA by factor 20.
The effects of CCK on the modulation of pain transmission
and the opioid effects are well established [27, 28] and we
have shown here the useful adjunct therapy of these agents in
pain management. Previously, it was shown that the CCK2
antagonist CI-988 potentiated the analgesia of morphine and
clomipramine [31] and therefore, it may be concluded that
this represents a general therapeutic application of CCK2
antagonists. Trimipramine is in use as second line treatment
for persistent neuropathic pain, which still represents an
unmet medical need. CCK antagonists also block the
development of morphine tolerance [32, 33], another further
possible therapeutic feature in pain management.
Figure 5. Isolated GPI. Cummulative log concentration response
curves to CCK8S only (*) and CCK8S in the presence of 18: 0.5 mM
(~), 1.0 mM (!) on the guinea pig ileum, expressed as change of
tension in gram. Each point represents the mean and the
standard errors obtained from three different experiments.
The furanone 18 was intraperitoneally injected as a first
injection to build up a plasma concentration of the CCK
antagonist and then, tramadol/morphine/desimipramine was
subcutaneously injected and subsequently, the analgesic
effect was evaluated in the tail flick assay (Fig. 6).
In all treated groups, no effect on nociception [29] for doses
of up to 5 mg/kg was observed for the furanone 18 in the tail
immersion test [30] as a single agent. Administered in
conjunction with tramadol/morphine and desimipramine at
0.05 mg/kg showed no potentiation of analgesia and a
clinically relevant significant effect was observed at 0.5 mg/kg.
For tramadol (20 mg/kg) and concomitant administration of
furanone 18 (0.5 mg), the analgesic effect in the tail flick test is
equivalent to a 2 mg/kg dose of morphine.
Conclusions
Chemically, the bis-substituted amino-furanones are not
related to previously known CCK antagonizing small organic
molecules and they do not contain the widely used urea
linkage. Our amino-furanones represent a totally novel “new
chemical entity” for a CCK antagonist, a CNS drug-like
molecule and most preferred an adjunct in pain management.
The tail immersion test is a robust assay to evaluate
analgesia of selected CCK ligands in combination with
approved analgesics. A classical tissue preparation confirmed
reliably the antagonism of the ligands. Overall, a good
in vitro/in vivo correlation was found.
During scale up, the yield for the solid isobutyl derivative 18
was increased to >70% over two steps, and this molecule is
available in >99.7% purity and was selected for preclinical
development. The greater CCK2 selectivity of 18, compared
with 25 may result in less toxicity due to less interference with
the physiological role of cholecystokinin, mediated by CCK1
receptors.
Experimental
General synthesis
The chemicals were obtained from Aldrich (Gillingham, UK)
and Lancaster (Lancaster, UK). Atmospheric pressure chemical
ionization mass spectroscopy (APCI), negative or positive
mode, was carried out using a Hewlett-Packard 5989b
Figure 6. Maximum possible effect (MPE) of three doses of
furanone 18 in the tail flick test in mice in % MPE for tramadol-,
morphine-, and desimipramine potentiation.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
460

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
Analgesia: Synthesis and Evaluation of CCK-2 Antagonists
Archiv der Pharmazie
quadrupole instrument (Vienna, Austria). Proton and carbon
NMR spectra were obtained on a Bruker AC 250 instrument
(Follanden, Switzerland), operating at 250 MHz, calibrated
with the solvent reference peak or TMS. IR spectra were
plotted from KBr discs on a Mattson 300 FTIR spectrometer.
resulting in a light–dark brown-colored oil, which was
distilled under vacuum giving a colorless liquid. Yield ¼ 73%;
1H NMR (CDCl3) 250 MHz: d ¼ 5.90 (s, 1H), 4.25 (m, 1H),
1.29 þ 1.42 (d, 6H) ppm. 13C NMR (CDCl3) 250 MHz: d ¼ 163.7,
148.0, 123.9, 100.3, 75.1, 22.6, 22.0 ppm; IR (KBr-disc) y max:
3392, 2975, 2927, 1797, 1645, 1460, 1380, 1326, 1234, 1160,
Melting points were recorded from
a Stuart Scientific
(Coventry, UK) melting point analyzer and are uncorrected.
The InChI codes of the synthesized compounds are provided
in the Supporting Information.
1118, 951, 892, 746 cmꢂ1
.
Preparation of 5-benzyloxy-3,4-dichloro-5H-furan-2-one D
Method A: To a solution of (10 g, 0.06 mol) mucochloric acid in
toluene (50 mL), benzyl alcohol (2 eq., 13.8 g, 0.12 mol) and
concentrated sulfuric acid (0.2 mL) were added and reacted
according to Method A. The crude furanone, which was
obtained as a light brown-colored oil, was distilled under
vacuum giving a yellow liquid. Yield ¼ 77%; 1H NMR (CDCl3)
250 MHz: d ¼ 7.48 (m, 5H), 5.94 (s, 1H), 4.88 (m, 2H) ppm; 13C
NMR (CDCl3) 250 MHz: d ¼ 163.3, 147.7, 135.2, 128.9, 128.5,
126.2, 124.5, 99.7, 71.4 ppm; IR (KBr-disc) y max: 3432, 3034,
2928, 2371, 2344, 1794, 1642, 1452, 1330, 1231, 1148, 1022,
Preparation of 3,4-dichloro-5-alkoxy-furan-2(5H)-
one building blocks
Preparation of 3,4-dichloro-5-propargyloxy-furan-2(5H)-
one A
Method A (toluene-Dean–Stark): To a solution of (10 g,
0.06 mol) mucochloric acid in toluene (50 mL), propargyl
alcohol (2 eq., 6.5 g, 0.12 mol) and concentrated sulfuric acid
(0.2 mL) were added. The resulting solution was left for 2 days
to reflux using Dean and Stark apparatus. The brown oily tar-
like crude product was analyzed by TLC and then quenched
with saturated sodium bicarbonate solution (50 mL). After
leaving the crude mixture to settle for 10 min, the product was
extracted with ether (3 ꢁ 50 mL). The organic layers were
combined and dried using magnesium sulfate. The solvent
was removed in vacuum, resulting in a dark brown-colored oil.
Yield ¼ 61%; 1H NMR (CDCl3) 250 MHz: d ¼ 6.11 (s, 1H), 4.58
(m, 2H), 2.96 (t, J ¼ 2.4 Hz, 1H); 13C NMR (CDCl3) d ¼ 163.1,
147.5, 128.2, 98.6, 78.1, 77.7, 57.4 ppm; IR (KBr-disc) y max:
3578, 3293, 2938, 2881, 2122, 1799, 1634, 1450, 1355, 1232,
909, 746, 697 cmꢂ1
.
Preparation of 3,4-dichloro-5-cyclohexylmethoxy-5H-
furan-2-one E
Method A: To a solution of (10 g, 0.06 mol) mucochloric acid in
toluene (50 mL), cyclohexyl methanol (2 eq. 13.5 g, 0.12 mol)
and concentrated sulfuric acid (0.2 mL) were added and the
reaction mixture was reacted according to Method A. The
crude furanone was distilled under vacuum to give an orange
liquid. Yield ¼ 57%; 1H NMR (CDCl3) 250 MHz: d ¼ 5.73 (s, 1H),
3.90 (m, 2H), 1.39–1.76 (m, 11H) ppm; 13C NMR (CDCl3)
250 MHz: d ¼ 161.4, 147.6, 125.3, 101.2, 68.6, 40.3, 29.5,
26.7, 25.7 ppm; IR (KBr-disc) y max: 3334, 2926, 2851, 2669,
1143, 1017, 903, 748, 687 cmꢂ1
.
Preparation of 5-allyloxy-3,4-dichloro-5H-furan-2-one B
Method A: To a solution of (10 g, 0.06 mol) mucochloric acid in
toluene (50 mL), allyl alcohol (2 eq., 7.1 g, 0.12 mol) and
concentrated sulfuric acid (0.2 mL) were added. The resulting
solution was left for 2 days according to Method A. The crude
furanone, which was obtained as dark brown-colored oil, was
distilled under vacuum to give an orange liquid. Yield ¼ 69%;
1H NMR (CDCl3) 250 MHz: d ¼ 5.99 (s, 1H), 5.78 (m, 1H), 5.27 (m,
1H), 5.48 (m, 1H), 4.32 (m, 2H) ppm. 13C NMR (CDCl3) d ¼ 163.3,
147.9, 131.9, 124.2, 119.5, 99.7, 71.1 ppm; IR (KBr-disc) y max:
3413, 3092, 2935, 2870, 2365, 2339, 1796, 1642, 1334, 1236,
1794, 1732, 1634, 1444, 1177, 1018, 729, 697 cmꢂ1
.
Preparation of 3-chloro-4-substituted amino-5-
alkyloxyloxy-5H-furan-2-ones
General method: Building block (2 mmol A–E/0.41, 0.42, 0.42,
0.52, 0.53 g) was dissolved in DMF (2 mL) and placed into 30 mL
glass vials. Amines (5 mmol) were added carefully dropwise to
each vial. The vials were placed in a heating block and heated
to 45°C for 48 h. The compound was extracted with ether
(10 mL) and washed twice with water (10 mL). The organic
layer was dried using anhydrous magnesium sulfate and
removed in vacuum to give a dark brown oil, which was
purified using column chromatography (solvent system: 50:50
ether/petrol ether) to give the desired product.
1157, 1020, 899, 778, 748 cmꢂ1
.
Preparation of 3,4-dichloro-5-isopropoxy-5H-furan-2-
one C
Method B (excess alcohol): To a solution of (10 g, 0.06 mol)
mucochloric acid in excess isopropanol (60 mL), concentrated
sulfuric acid (0.2 mL) was added. The resulting solution was
left for 2 days to reflux. The brown oily crude product was
analyzed by TLC and then quenched with saturated sodium
bicarbonate solution (50 mL). After leaving the crude mixture
to settle for 10 min, the product was extracted with ether
(3 ꢁ 50 mL). The organic layers were combined and dried using
magnesium sulfate. The solvent was removed in vacuum,
3-Chloro-4-isobutylamino-5-prop-2-ynyloxy-5H-furan-2-
one 1
Yield ¼ 12% brown oil; MW: 243.7; MS (APCI(þ)): 244/246
(Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 5.92 (s, 1H), 5.19 (bs,
NH), 4.41 (s, 2H), 3.28 (m, 2H), 2.55 (m, 1H), 1.84 (m, 1H), 1.08
13
–
(m, 6H) ppm; C NMR (CDCl ) d ¼ 166.2 (C O), 155.3, 104.5,
–
3
94.5, 76.7, 78.1, 56.1, 51.1, 29.6, 19.7 ppm; IR (KBr-disc) y max:
3306, 3103, 2967, 2937, 2881, 2370, 1748, 1646, 1542, 1458,
1434, 1335, 1126, 969, 747, 701 cmꢂ1
.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
461

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
E. Lattmann et al.
Archiv der Pharmazie
4-(Benzylamino)-3-chloro-5-(prop-2-ynyloxy)furan-2(5H)-
one 2
21.4, 10.3 ppm. IR (KBr-disc)
y
max: 3296, 3088, 2978,
2978, 2939, 2881, 1753, 1649, 1546, 1467, 1338, 1150, 972,
709 cmꢂ1
Yield ¼ 36% brown oil; MW: 277.7; MS (APCI(þ)): 278/280
(Mþ1) m/z; 1H NMR (CDCl3) 300 K d: 7.36 (m, 5H); 5.98 (s, 1H),
5.20 (s, NH), 4.66 (s, 2H), 4.45 (m, 1H), 4.41 (m, 2H), 2.55 (m, 1H)
ppm. 13C NMR (CDCl3) d ¼ 168.1, 155.8, 135.6, 129.0, 128.6,
126.3, 108.3, 96.6, 78.2, 76.6, 54.9 ppm. IR (KBr-disc) y max:
3380, 3283, 2358, 2338, 1752, 1646, 1455, 1326, 1123, 971,
.
5-Allyloxy-4-butylamino-3-chloro-5H-furan-2-one 8
Yield ¼ 57% oily solid; MW: 245.7 (APCI(þ)): 188/190 (Mþ),
246/248 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 5.90 (m, 1H),
5.76 (s, CH), 5.32 (m, 2H), 4.29 (m, 2H), 3.43 (bs, 2H), 1.63 (m,
2H), 1.38 (m, 2H), 0.98 (t, J ¼ 8.2 Hz, 3H) ppm. 13C NMR (CDCl3)
d ¼ 165.9, 151.3, 132.4, 119.4, 105.6, 96.2, 69.6, 43.6, 32.7, 19.7,
13.7 ppm. IR (KBr-disc) y max: 3332, 3095, 2974, 2941, 2892,
1754, 1655, 1540, 1457, 1333, 1227, 1138, 1087, 969, 748,
695 cmꢂ1
.
4-(Benzyl-methyl-amino)-3-chloro-5-prop-2-ynyloxy-5H-
furan-2-one 3
Yield ¼ 49% oily solid; MW: 291.7; MS (APCI(þ)): 292/294
(Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 7.16–7.41 (m, 5H),
5.96 (s, 1H), 4.70 (m, 2H), 4.40 (m, 2H), 3.04 (s, 3H), 2.48 (m,
1H) ppm; 13C NMR (CDCl3) d ¼ 168.2, 155.6, 135.7, 129.0, 128.6,
127.3, 107.3, 94.6, 78.1, 76.9, 55.9, 38.1 ppm. IR (KBr-disc) y
max: 3441, 3296, 3037, 2930, 2374, 2343, 2128, 1766, 1643,
700 cmꢂ1
.
5-Benzyloxy-3-chloro-4-isobutylamino-5H-furan-2-one 18
Yield ¼ 76%; mp: 105–107°C; MW: 295.8; MS (APCI(þ)): 188/
190 (Mþ), 296/298 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz:
d ¼ 7.01–7.49 (m, 5H), 5.79 (s, CH), 5.10 (bs, NH), 4.71 (bs, 2H),
3.22 (m, 2H), 1.79 (m, 1H), 0.97 (m, 6H) ppm; 13C NMR (CDCl3):
d ¼ 170.4, 170.4, 135.4, 128.8, 106.3, 95.6, 70.4, 51.0, 29.5,
19.7 ppm. IR (KBr-disc) y max: 3460, 3252, 2977, 2937, 2877,
2368, 2341, 1741, 1631, 1433, 1353, 1329, 1255, 1128, 1028,
1460, 1419, 1353, 1270, 1229, 1116, 983, 747, 703 cmꢂ1
.
4-(4-Benzyl-piperazin-1-yl)-3-chloro-5-prop-2-ynyloxy-5H-
furan-2-one 4
Yield ¼ 50%; mp: 120–123°C; MW: 346.8; MS (APCI(þ)): 346/
348 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 7.18–7.38 (m,
5H), 5.87 (s, 1H), 4.36 (s, 2H), 3.76 (m, 4H), 3.51 (s, 2H), 2.42–
2.39 (m, 5H) ppm. 13C NMR (CDCl3) 250 MHz: d ¼ 168.1, 154.0,
137.2, 129.2, 128.4, 127.5, 103.0, 94.3, 76.9, 76.8, 62.7, 55.7,
52.7, 47.5 ppm. IR (KBr-disc) y max: 3253, 2935, 2815, 2126,
961, 750, 703 cmꢂ1
15H18ClNO3, Mr ¼ 295.75, T ¼ 293(2) K, tabular, 0.20 ꢁ 0.15
.
C
̊
ꢁ 0.05 mm, colorless, MoKa radiation: l ¼ 0.71073 A, mono-
̊
̊
̊
clinic, P21/c, a ¼ 11.420(5) A, b ¼ 10.736(2) A, c ¼ 12.798(5) A,
3
b ¼ 102.83(4), V ¼ 1529.9(9) A , Z ¼ 4, Dx ¼ 1.284 Mg/mꢂ3, Dm
̊
not measured, R [F2 > 2s(F2)] ¼ 0.0791, wR(F2) ¼ 0.1845, 3207
1758, 1446, 1347, 1277, 1111, 985, 849, 740, 693 cmꢂ1
.
reflections, 187 parameters.
Selected geometric parameters are listed in Table 2.
3-Chloro-4-(2,6-dimethyl-morpholin-4-yl)-5-prop-2-
ynyloxy-5H-furan-2-one 5
5-Benzyloxy-4-butylamino-3-chloro-5H-furan-2-one 19
Yield ¼ 68%; mp: 109–112°C; MW: 295.8; MS (APCI(þ)): 188/
190 (Mþ), 296/298 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz:
d ¼ 7.39 (m, 5H), 5.74 (s, 1H), 4.64 (m, 3H), 3.31 (bs, 2H), 1.569
(m, 2H), 1.39 (m, 2H), 0.89 (t, J ¼ 6.6 Hz, 3H) ppm. 13C NMR
(CDCl3) d ¼ 171.4, 153.4, 135.5, 128.8, 128.1, 124.5, 107.5, 95.5,
70.3, 43.7, 29.5, 19.7, 13.7 ppm; IR (KBr-disc) y max: 3419, 2067,
3028, 2928, 2371, 2338, 1794, 1642, 1502, 1456, 1327, 1234,
Yield ¼ 47% oily solid; MW: 285.7; MS (APCI(þ)): 286/288
1
(Mþ1) m/z; H NMR (CDCl3) 250 MHz: d ¼ 5.94 (s, 1H), 4.36 (s,
2H), 3.89–4.25 (m, 2H), 3.58–3.85 (m, 2H), 2.51–2.98 (m, 3H),
1.46 (m, 6H) ppm. 13C NMR (CDCl3): d ¼ 170.4, 153.7, 104.2,
94.3, 77.8, 77.6, 66.0, 55.8, 52.6, 18.5, 18.4 ppm. IR (KBr-disc) y
max: 3484, 3255, 2981, 2928, 2883, 2366, 2108, 1743, 1639,
1267, 1083, 981, 751, 697 cmꢂ1
.
1144, 1022, 972, 902, 750, 707 cmꢂ1
.
5-Allyloxy-3-chloro-4-isobutylamino-5H-furan-2-one 6
Yield ¼ 62% oily solid; MW: 245.7; MS (APCI(þ)): 188/190
(Mþ), 246/248 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 5.89
(m, 1H), 5.73 (s, 1H), 5.34 (m, 2H), 4.79 (bs, NH), 4.27 (m, 2H),
3.22 (bs, 2H), 1.83 (m, 1H), 1.16–0.98 (m, 6H) ppm; 13C NMR
(CDCl3): d ¼ 172.1, 156.7, 132.2, 119.6, 108.0, 95.9, 69.3,
51.0, 29.6, 19.7 ppm; IR (KBr-disc) y max: 3296, 3088, 2978,
2978, 2939, 2881, 1753, 1649, 1546, 1467, 1338, 1150, 972,
5-Benzyloxy-4-sec-butylamino-3-chloro-5H-furan-
2-one 20
Yield ¼ 64%; mp: 93–96°C; MW: 295.8; MS (APCI(þ)): 188/190
(Mþ), 296/298 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 7.30
(m, 5H), 5.73 (s, 1H), 5.11 (bs, NH), 4.68 (m, 2H), 4.40 (bs, NH),
3.49 (bs, 1H), 1.46 (m, 2H), 1.16 (m, 3H), 0.84 (m, 3H) ppm; 13
C
NMR (CDCl3) d ¼ 169.3, 155.5, 135.6, 128.9, 128.7, 124.3, 105.9,
95.7, 70.2, 51.1, 30.5, 21.6, 21.6 ppm; IR (KBr-disc) y max: 3272,
3085, 2977, 2929, 2880, 2368, 1732, 1632, 1560, 1454, 1348,
709 cmꢂ1
.
5-Allyloxy-4-sec-butylamino-3-chloro-5H-furan-2-one 7
Yield ¼ 49% oily solid; MW: 245.7; MS (APCI(þ)): 190/192
(Mþ), 246/248 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 5.89
(m, 1H), 5.74 (s, 1H), 5.28 (m, 2H), 4.27 (m, 2H), 1.68 (m, 2H),
1.82 (m, 3H), 0.99 (t, J ¼ 4.6 Hz, 3H) ppm; 13C NMR (CDCl3):
d ¼ 168.3, 153.5, 132.5, 119.0, 106.3, 96.5, 69.6, 49.6, 30.5,
1231, 1123, 966, 749, 700 cmꢂ1
.
5-Benzyloxy-3-chloro-4-(3-methyl-pyrazol-1-yl)-5H-furan-
2-one 21
Yield ¼ 76%; mp: 89–91°C; MW: 304.7; MS (APCI(þ)): 305/307
(Mþ1) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 8.25 (m, 1H), 7.31
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
462

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
Analgesia: Synthesis and Evaluation of CCK-2 Antagonists
Archiv der Pharmazie
̊
Table 2. Selected geometric parameters (A, °).
Cl(7)-C(2)
O(5)-C(1)
O(5)-C(4)
O(6)-C(1)
1.707(7)
1.365(8)
1.432(7)
1.202(7)
O(12)-C(4)
C(3)-N(8)
C(2)-C(3)
1.386(7)
1.331(7)
1.354(9)
C(4)-O(12)-C(13)
O(12)-C(13)-C(14)
N(8)-C(3)-C(4)
113.7(5)
108.2(6)
124.5(6)
111.5(5)
O(6)-C(1)-O(5)
C(1)-C(2)-Cl(7)
C(3)-C(2)-Cl(7)
C(3)-N(8)-C(9)
120.3(5)
122.9(5)
127.0(5)
124.5(6)
O(12)-C(4)-O(5)
(m, 5H), 6.49 (s, 1H) 6.40 (m, 1H), 4.94 (m, 2H), 2.41 (s, 3H) ppm.
13C NMR (CDCl3): d ¼ 165.7, 153.7, 147.3, 135.5, 130.9, 128.7,
128.6, 128.55, 111.3, 102.7, 99.1, 72.6, 13.7 ppm; IR (KBr-disc) y
max: 3440, 3176, 2950, 2941, 2885, 2362, 2336, 1790, 1674,
51.1, 37.7, 29.8, 29.7, 27.0, 25.7, 19.74, 19.71 ppm; IR (KBr-disc)
y max: 3285, 2971, 2933, 2863, 2370, 1745, 1678, 1630, 1470,
1333, 1256, 1147, 1025, 948, 750, 718 cmꢂ1
.
1551, 1443, 1324, 1264, 1122, 1016, 983, 735, 695 cmꢂ1
.
3-Chloro-5-cyclohexylmethoxy-4-methylamino-5H-furan-
2-one 26
5-Benzyloxy-3-chloro-4-cyclopropylamino-5H-furan-2-
one 22
Yield ¼ 55%; mp: 117–120°C; MW: 259.7; MS (APCI(þ)): 164/
166 (Mþ1), 260/262 (Mþ) m/z; 1H NMR (CDCl3) 250 MHz:
d ¼ 5.72 (s, 1H), 4.89 (bs, NH), 3.49 (m, 1H), 3.37 (m, 1H), 3.23
Yield ¼ 81%; mp: 104–107°C; MW: 279.7; MS (APCI(þ)): 278/
280 (Mþ) m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 7.80 (m, 5H), 5.95
(s, 1H), 4.84 (m, 3H), 2.99 (m, 1H), 0.79 (m, 7H) ppm; 13C NMR
(CDCl3) d ¼ 161.4, 153.4, 133.6, 128.8, 128.7, 126.4, 106.1, 95.7,
70.8, 25.5, 18.3 ppm; IR (KBr-disc) y max: 3453, 3265, 3071,
2935, 2864, 2366, 1788, 1742, 1636, 1442, 1348, 1245, 983, 753,
(m, 3H), 2.76 (bs, 1H), 1.72 (m, 5H), 0.77–1.39 (m, 6H) ppm; 13
C
NMR (CDCl3) d ¼ 164.1, 151.3, 102.6, 97.0, 74.2, 37.7, 30.6, 29.7,
27.0, 25.7 ppm. IR (KBr-disc) y max: 3267, 2925, 2848, 2370,
2338, 1742, 1684, 1632, 1451, 1329, 1255, 1159, 1016, 949, 755,
716 cmꢂ1
.
701 cmꢂ1
.
Pharmacology
5-Benzyloxy-3-chloro-4-cyclopentylamino-5H-furan-2-
one 23
Cholecystokinin binding assay, [125]I-CCK-8 receptor
binding assay
Yield ¼ 78%; mp: 101–103°C; MW: 307.8; MS (APCI(þ)): 284/
286 (Mþ), 306/308 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz:
d ¼ 7.49 (m, 5H), 5.78 (s, 1H), 4.78 (m, 2H), 4.01 (bs, NH), 1.33–
2.15 (m, 8H) ppm; 13C NMR (CDCl3) d ¼ 165.2, 151.3, 135.5,
128.8, 128.2, 124.6, 104.2, 95.6, 70.2, 55.3, 34.6, 23.7 ppm; IR
(KBr-disc) y max: 3459, 3306, 3070, 2961, 2859, 2367, 2342,
CCK1 and CCK2 receptor binding assays were performed by
using guinea pig cerebral cortex (CCK2) or rat pancreas (CCK1).
Male guinea pig brain tissues were prepared according to the
modified method described by Saita et al. [34]. Pancreatic
membranes were prepared as described by Charpentier et al.
[35].
Tissues were homogenized in ice cold sucrose (0.32 M,
25 mL) for 15 strokes at 500 rpm and centrifuged at 13000 rpm
for 10 min. The supernatant was re-centrifuged at 13000 rpm
for 20 min. The resulting pellet was re-dispersed to the
required volume of buffer at 500 rpm and stored in aliquots at
70°C.
1735, 1626, 1547, 1352, 1231, 1144, 1007, 959, 758, 707 cmꢂ1
.
5-Benzyloxy-3-chloro-4-cyclohexylamino-5H-furan-2-
one 24
Yield ¼ 49% oily solid; MW: 321.8; MS (APCI(þ)): 322/324 (Mþ)
m/z; 1H NMR (CDCl3) 250 MHz: d ¼ 7.30 (m, 5H), 5.78 (s, 1H),
4.96 (bs, NH), 4.76 (m, 2H), 1.04–2.18 (m, 10H) ppm; 13C NMR
(CDCl3) d ¼ 167.5, 155.5, 135.5, 128.7, 128.4, 124.2, 104.5, 95.6,
67.0, 52.7, 34.5, 25.5, 25.1 ppm; IR (KBr-disc) y max: 3293, 3067,
2941, 2861, 2375, 2355, 1754, 1648, 1550, 1446, 1337, 1231,
Binding was achieved using radioligand 125I-Bolton-
Hunter-labeled CCK, NEN at 25 pM. The samples were
incubated with membranes (0.1 mg/mL) in 20 mM Hepes,
1 mM EGTA, 5 mM MgCl2, 150 mM NaCl, at pH 6.5 for 2 h at
RT and then centrifuged at 11000 rpm for 5 min. The
membrane pellets were washed twice with water and the
bound radioactivity was measured in a Packard Cobra
counter.
1134, 1091, 955, 746, 700 cmꢂ1
.
3-Chloro-5-cyclohexylmethoxy-4-isobutylamino-5H-furan-
2-one 25
Yield ¼ 59%; mp: 119–122°C; MW: 301.8; MS (APCI(þ)): 206/
208 (Mþ), 302/304 (Mþ1) m/z; 1H NMR (CDCl3) 250 MHz:
d ¼ 5.75 (s, 1H), 4.89 (bs, NH), 3.53 (m, 1H), 3.49 (m, 1H), 3.37
(bs, 2H), 2.76 (bs, 1H), 1.53–1.79 (m, 5H), 0.81–1.42 (m,
12H) ppm; 13C NMR (CDCl3) d ¼ 165.5, 153.6, 104.2, 97.0, 74.0,
Isolated tissue preparation
Adult male guinea pigs, weighing 200–250 g, were used and
from the abdomen of the animals, the ileum was carefully
excised at a site 15 cm away from the ileocaecal junction and
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
463

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
E. Lattmann et al.
Archiv der Pharmazie
washed with physiological solution. The mesentery of the
ileum was removed and the ileal lumen was gently flushed
with Tyrode’s solution to clear luminal contents. The prepared
isolated tissue was rapidly incubated in Tyrode’s solution
maintained at 37°C and gassed with 95% O2/5% CO2.
Tyrode’s solution of the following quantity was freshly
prepared daily (g/L): NaCl, 8.0; KCl, 0.2; CaCl2, 0.2; MgSO4, 0.1;
NaH2PO4, 0.05; NaHCO3, 1.0; glucose, 1.0. Stock solutions of
test compounds were prepared weekly, diluted to required
concentration as required, and stored at a temperature of 4°C.
From the isolated tissue preparation, ileal strips of
appropriate length were mounted vertically in organ bath
containing Tyrode’s solution, under a tension of 1 g and
allowed to equilibrate for 30 min. One end of the strip was
attached to the hook at the bottom of the organ bath and the
other end connected by a thread to the external isometric
force transducer. During equilibration, tension was continu-
ously adjusted to 1 g when required and the Tyrode’s solution
in the organ bath was changed every 20 min. All spontaneous
contractions of longitudinal muscles were recorded with the
aid of an isometric transducer linked to a power lab chart
computer unit.
were expressed as a percentage of Maximal Possible Effect
(% MPE) using the equation:
% MPE ¼ fðTT ꢂ BTÞ=ð45 ꢂ BTÞg ꢁ 100
DMSO (5%), furanone (in 5% DMSO) was intraperitoneally
injected as the first injection. Twenty minutes after the first,
the second injection was done by subcutaneously injecting
with tramadol at either 10, 20, or 40 mg/kg body weight.
Molecular modeling
For target preparation, the protein structures, pdb identifier
1HZN for the CCK1 and 1L4T for the CCK2-gastrin receptor
were downloaded from the protein data bank (www.rcs.org)
and docking was performed using Autodock Vina and Hex.
After several docking trials for the CCK2 receptor, the results
were analyzed and visualized using Chimera and Designer
Studio 4.5. After visual inspection and scores, results were
presented to understand drug–ligand interaction with the
CCK2 receptor.
Statistical methods
The data were expressed as mean ꢀ SD and one-way analysis
of variance (ANOVA) and supplementary Tukey test for
pairwise comparison was tested to determine any significant
difference at p < 0.05.
Effects on CCK-8 stimulated isolated guinea pig ileum
To study the effect of furanone 18 on strips prepared from
guinea pig ileum, CCK8S was dissolved in distilled water to
prepare a stock solution of 500 mM solution, from which
cumulative additions of increasing concentrations (0.1, 1, 5,
10, 20, 30, and 40 nM) were tested to plot a dose–response
curve. Test compounds and Lorglumide/L365260 as standard
were added to the organ bath 10 min before exposure to the
next CCK8S serial concentrations.
The authors deeply appreciate assistance of Wanchai Airarat
in the animal experiments and are grateful for funding from
PNB Vesper life Sciences PVT.
The authors have declared no conflicts of interest.
Animal studies
References
Experiments were conducted in male standard IRC mice
obtained from the animal house, Faculty of Medicine, Khon
Kaen University. Each experimental group consisted of six
animals and the treatment procedures were approved by the
ethical committee, Faculty of Medicine, Khon Kaen University
(BEA030699).
[1] I. M. McDonald, Exp. Opin. Ther. Patents 2001, 11,
445–462.
[2] M. G. Bock, R. M. DiPardo, E. C. Mellin, N. C. Newto,
J. Med. Chem. 1994, 37, 722–724.
[3] E. Lattmann, P. Arayarat, KKU. Sci. J. 2003, 31, 178–193.
[4] C. T. Dourish, S. Ravard, Trends Pharmacol. Sci. 1990, 11,
271–273.
[5] K. Rasmussen, J. F. Czachura, M. E. Stockton, J. J. Howbert,
J. Pharmacol. Exp. Ther. 1993, 264, 480–488.
[6] C. T. Dourish, W. Rycroft, S. D. Iversen, Science 1989, 245,
1509–1511.
Mice were intraperitoneally injected with either test
compound dissolved in 5% DMSO at the volume not more
than 0.2 mL/animal. At 30 min after treatment, animals were
tested as described in the following sections.
Nociception test
The tail immersion test: The thermal response latency was
measured by the tail immersion test. The animals were
placed into individual restraining cages leaving the tail
hanging freely. The tail was immersed into water preset at
50°C. The response time, at which the animal reacted by
withdrawing its tail from water, was recorded and the cut-
off time was 10 s in order to avoid damaging the animal’s
tissue. The base line withdrawal thresholds (BT) were
recorded prior to the first injection. Test thresholds (TT)
were measured 60 min after the second injection. The TT
[7] B. K. Trivedi, Curr. Med. Chem. 1994, 1, 313–327.
[8] J. Bradwejn, D. Koszycki, G. Meterissian, Can. J. Psychia-
try 1990, 35, 83–85.
[9] E. Lattmann, J. Sattayasai, P. Lattmann, D. C. Billington,
C. H. Schwalbe, J. Boonprakob, W. Airarat, H. Singh,
M. Offel, Drug Discov. Ther. 2007, 1, 45–56.
[10] E. Lattmann, Y. Boonprakob, J. Sattayasai, Drug Discov.
Ther. 2008, 2, 344–352.
[11] E. Lattmann, D. C. Billington, D. R. Poyner, S. B. Howitt,
M. Offel, Drug Des. Discov. 2001, 17, 219–230.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
464

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 456–465
Analgesia: Synthesis and Evaluation of CCK-2 Antagonists
Archiv der Pharmazie
[12] B. 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, D. J. Cerino, J. Med.
Chem. 1988, 31, 2235–2246.
[13] K. Simpson, M. Serpell, T. D. McCubbins, N. L. Padfield,
N. Edwards, K. Markam, D. Eastwood, R. Block,
D. J. Rowbotham, Management of neuropathic pain
inpatients using a CCK antagonist devazepide as an
adjunct to strong opioids, 4th International Conference
on the Mechanisms and Treatment of Neuropathic Pain,
Sep. 2001, San Francisco, USA.
[23] Y. Yu, A. Jawa, W. Pan, A. J. Kastin, Peptides 2004, 25,
2257–2289.
[24] E. Lattmann, N. Sattayasai, C. H. Schwalbe, S. Niamsanit,
D. C. Billington, P. Lattmann, C. A. Langley, H. Singh,
S. Dunn, Curr. Drug Discov. Technol. 2006, 3, 125–134.
[25] K. Yoshinaga, T. Horii, H. Hamano, R. Eta, T. Ozaki,
Y. Orikawa, K. Yoshii, Y. Kawabata, Y. Hori, K. Seto,
M. Takei, Y. Kuraishi, Biol. Pharm. Bull. 2010, 33,
244–248.
[26] H. G. Vogel, W. H. Vogel (Eds.), Drug Discovery and
Evaluation: Pharmacological Assays, Springer, Berlin
1997, p. 232.
[27] M. F. O’Neill, C. T. Dourish, S. D. Iversen, NeuroPharma-
cology 1989, 28, 243–247.
[28] M. F. O’Neill, C. T. Dourish, S. D. Iversen, Eur. J.
Pharmacol. 1990, 193, 203–208.
[29] J. M. Walker, W. C. Dixon, Physiol. Behav. 1983, 30,
481–483.
[14] J. Hughes, G. N. Woodruff, Arzneim. ꢂ Forsch./Drug Res.
1992, 42, 250–255.
[15] A. M. Grabowska, T. M. Morris, A. J. McKenzie,
R. Kumari, H. Hamano, Y. Emori, K. Yoshinaga,
S. A. Watson, Regul. Peptides 2008, 146, 46–57.
[16] E. Lattmann, D. C. Billington, D. R. Poyner, P. Arayarat,
S. B. Howitt, S. Lawrence, M. Offel, Drug Des. Discov.
2002, 18, 9–21.
[17] E. Lattmann, J. Sattayasai, D. C. Billington, D. R. Poyner,
P. Puapairoj, S. Tiamkao, W. Airarat, H. Singh, M. Offel,
J. Pharm. Pharm. 2002, 54, 827–834.
[30] A. Cowan, in Modern Methods in Pharmacology Volume
6: Testing and Evaluation of Drugs of Abuse (Eds.
M. W. Adler, A. Cowan), Wiley-Liss Inc., New York 1990,
Recent approaches in the testing of analgesics in
animals, p. 33.
[18] M. Offel, P. Lattmann, H. Singh, D. C. Billington,
Y. Bunprakob, J. Sattayasai, E. Lattmann, Archiv der
Pharmazie/Chem. Life Sci. 2006, 339, 163–173.
ꢀ
[31] M. A. Coudore-Civiale, C. Courteix, M. Boucher,
ꢀ
M. Meen, J. Fialip, A. Eschalier, D. Ardid, Neurosci. Lett.
[19] E. Lattmann, H. Singh, J. Boonprakob, P. Lattmann,
J. Sattayasai, J. Pharm. Pharm. 2006, 58, 1–9.
[20] E. Lattmann, J. Sattayasai, J. Boonprakob, P. Lattmann,
2000, 286, 37–40.
[32] C. T. Dourish, M. F. O’Neill, J. Coughlan, S. J. Kitchener,
D. Hawley, S. D. Iverson, Eur. J. Pharmacol. 1990, 176,
35–44.
[33] C. T. Dourish, M. F. O’Neill, L. W. Schaffer, P. K. Siegi,
S. D. Iversen, J. Pharmacol. Exp. Ther. 1990, 255, 1158–1165.
[34] Y. Saita, H. Yazawa, Y. Honma, A. Nishida, K. Miyata,
K. Honda, Eur. J. Pharmacol. 1994, 269, 249–281.
[35] B.Charpentier,D.Pelaprat,C.Durieux,A.Dor,M.Reibaud,
J. C. Blanchard, B. P. Roques, Proc. Natl. Acad. Sci. U. S. A.
1988, 85, 1968–1973.
H. Singh, Arzneim.
251–258.
ꢂ
Forsch./Drug Res. 2005, 55,
[21] E. Lattmann, J. Sattayasai, Y. Boonprakob, H. Singh,
P. Lattmann, S. Dunn, Drug Discov. Ther. 2008, 2,
156–167.
[22] E. Lattmann, P. Lattmann, J. Boonprakob, W. Airarat,
H. Singh, M. Offel, J. Sattayasai, Arzneim. ꢂ Forsch./Drug
Res. 2009, 59, 61–71.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
465