Conformational restriction leading to a selective CB2 cannabinoid receptor agonist orally active against colitis

Article abstract of DOI:10.1021/ml500439x

The CB₂ cannabinoid receptor has been implicated in the regulation of intestinal inflammation. Following on from the promising activity of a series of 4-oxo-1,4-dihydroquinoline-3-carboxamide, we developed constrained analogues based on a 2H-pyrazolo[4,3-c]quinolin-3(5H)-one scaffold, with improved affinity for the hCB₂ receptor and had very high selectivity over the hCB₁ receptor. Importantly, the lead of this series (26, hCB₂: K_i = 0.39 nM, hCB₁: K_i > 3000 nM) was found to protect mice against experimental colitis after oral administration.

Full text of DOI:10.1021/ml500439x

Letter
pubs.acs.org/acsmedchemlett
Conformational Restriction Leading to a Selective CB Cannabinoid
2
Receptor Agonist Orally Active Against Colitis
,†,∥
‡,⊥
§
§
Jamal El Bakali,* Giulio G. Muccioli, Mathilde Body-Malapel, Madjid Djouina,
†
§
†
†
†
†
Fred
́ ́ ́
erique Klupsch, Alina Ghinet, Amelie Barczyk, Nicolas Renault, Philippe Chavatte,
‡
,†
Pierre Desreumaux, Didier M. Lambert, and Reg
́
is Millet*
†Institut de Chimie Pharmaceutique Albert Lespagnol, Universite
B.P. 83, F-59006 Lille Cedex, France
́
de Lille Nord de France, E.A 4481, IFR 114, 3 rue du Pr. Laguesse,
‡Unite
́
de Chimie Pharmaceutique et de Radiopharmacie, Louvain Drug Research Institute, Universite
avenue E. Mounier UCL-CMFA (7340), B-1200 Bruxelles, Belgium
́
catholique de Louvain, 73
§
Digestive inflammatory diseases: pathophysiology and development of therapeutic targets. U995 INSERM, Universite
France, IFR 114, Amphis J & K, Boulevard du Professeur J. Leclercq, 59045 Lille Cedex, France
́
Lille Nord de
*
S Supporting Information
ABSTRACT: The CB cannabinoid receptor has been implicated in the regulation of intestinal inflammation. Following on
2
from the promising activity of a series of 4-oxo-1,4-dihydroquinoline-3-carboxamide, we developed constrained analogues based
on a 2H-pyrazolo[4,3-c]quinolin-3(5H)-one scaffold, with improved affinity for the hCB receptor and had very high selectivity
2
over the hCB receptor. Importantly, the lead of this series (26, hCB : K = 0.39 nM, hCB : K > 3000 nM) was found to protect
1
2
i
1
i
mice against experimental colitis after oral administration.
KEYWORDS: Cannabinoid receptor, endocannabinoid, colitis, inflammatory bowel disease, quinolone, conformational restriction
nflammatory bowel disease (IBD) represents a group of
(TNBS) and in the interleukin 10-deficient mouse model of
4
−6
I
chronic inflammatory lesions of unknown etiology that affect
colitis. Similarly, the CB receptor selective agonist AM1241
2
1
the digestive tract. The CB cannabinoid receptor emerged as
was shown to significantly reduce inflammation in the TNBS-
2
5
a promising therapeutic target in the treatment of these
pathologies. Indeed, this GPCR has been identified within the
gastrointestinal tract and plays a key role in the regulation of
induced colitis model. The involvement of the CB
2
receptor
was demonstrated by counteracting the effect with the CB
antagonist/inverse agonist AM630 or by evaluating the
compounds in CB
2
2
intestinal inflammation. Additionally, the CB2 receptor
2
receptor deficient mice. Moreover5,
agonists have been shown to exert an anti-inflammatory effect
both in intestinal epithelial cells and in experimental models of
colitis in mice.
AM630 was shown to exacerbate colitis in wild-type mice.
Consistent with these data, we described a series of 4-oxo-1,4-
dihydropyridines (CB selective agonists) in which the lead
2
In vitro experiments highlighted that CB receptor activation
compound, ALICB573 (48 in ref 7), was shown to protect mice
2
results in suppression of the release of pro-inflammatory
against experimental colitis when administered at 10 mg/kg
3
7
cytokines. Data from in vivo studies emphasized the
(i.p. injection). More recently, two isoxazole-based CB
2
importance of this receptor in mediating protection against
agonists were found to be active in the DSS-induced colitis
experimental colitis. Indeed, the CB receptor selective agonist
2
JWH133 proved to be efficient in reducing colonic inflamma-
tion in models of colitis induced by oil of mustard, dextran
sulfate sodium (DSS), or 2,4,6-trinitrobenzenesulfonic acid
Received: October 27, 2014
Accepted: December 4, 2014
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ml500439x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
8
model in mice. Taken together, these different studies provide
bond. A similar strategy was successfully applied to generate
potent topoisomerase II inhibitors.
1
2,13
the proof of concept that CB agonists are effective in reducing
2
colitis and can therefore be considered as promising agents for
Thus, we designed, synthesized, and evaluated against both
the treatment of IBD.
CB and CB receptors a series of 2H-pyrazolo[4,3-c]quinolin-
1
2
In light of these findings, we aimed to develop new CB2
receptor agonists effective in the treatment of IBD. In terms of
ligand profile, the new ligands should (i) display nanomolar
3(5H)-one based on the already established pharmacophore in
the series of 4-oxo-1,4-dihydroquinoline-3-carboxamide.
Therefore, the n-pentyl chain was retained at the N-5 position
of the rigid heterocycle and we only varied the moieties at the
N-2 position and to a lesser extent the C-4 position.
9,10
affinity for the CB receptor, (ii) behave as agonists, (iii) be
2
selective for the CB receptor over the CB receptor, to limit
2
1
CNS side effects, and (iv) be active in an experimental model of
colitis in rodents.
Key 2H-pyrazolo[4,3-c]quinolin-3(5H)-one compounds 8−
27 were prepared by a linear step-by-step synthesis that include
the construction of the 4-oxo-1,4-dihydroquinoline scaffold
Our groups previously described a series of 4-oxo-1,4-
dihydroquinoline-3-carboxamides as selective ligands of the
14
(Scheme 1). The latter was obtained using two different
synthetic routes depending on the nature of the R1 substituent
(at C-4). The first one consisted of a condensation of aniline
and diethyl ethoxymethylenemalonate followed by cyclization
9
,10
CB receptor. One of the commonly used strategies in drug
2
design to increase affinity and selectivity of a given “flexible”
lead for its pharmacological target is to conformationally
constrain it to mimic the so-called bioactive conformation.
Therefore, we designed constrained analogues of the 4-oxo-1,4-
dihydroquinoline-3-carboxamide series based on a 2H-
pyrazolo[4,3-c]quinolin-3(5H)-one scaffold (Figure 1).
9
into 4-oxo-1,4-dihydroquinoline 2. Subsequent N-alkylation
with 1-bromopentane yielded compound 4. The alternative
route, allowing the introduction of a phenyl moiety at C-4,
starts with the N-alkylation of isatoic anhydride. The resulting
product was then reacted with ethyl benzoylacetate in the
presence of sodium hydride to afford the desired 4-oxo-1,4-
15
dihydroquinoline 5. The latter as well as its analogue 4 were
converted into the more reactive thioxo-derivatives 6 and 7.
Finally, the 2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (10−27)
were obtained either by reacting 6 and 7 with the
corresponding substituted hydrazine or via initial cyclization
with monohydrate hydrazine (leading to 8 and 9) and
14
subsequent N-2 alkylation.
The affinities of target compounds 8−27 (Table 1) for the
Figure 1. Design of conformationally constrained quinolone
analogues.
human cannabinoid receptors (hCB1 and hCB ) were
2
determined by a competitive radioligand displacement assay
3
using [ H]-CP-55,940 as the radioligand for both the hCB and
2
7
hCB receptors. We also investigated the functional activity on
1
Our strategy relies on the fact that the amide at the C-3
position of the 4-oxo-1,4-dihydroquinoline ring may adopt
different conformations, of which one is the result of a
hydrogen bond between the amide (C-3) and the heterocyclic
carbonyl (Figure 1). We hypothesized that this hydrogen-
bonded conformation may also be the biologically active
conformation of this series of ligands. Accordingly, to accurately
orientate the aliphatic or aromatic moiety borne by the amide,
we built an additional ring that acts as a permanent
conformational lock in place of the intramolecular hydrogen
the CB receptor of four representative compounds of this
series using a [ S]-GTPγS binding assay (Table 2).
2
35
16
The first step of this study was to evaluate the impact of the
phenyl substituent at the C-4 position. While in the 4-oxo-1,4-
dihydroquinoline series this modification induced a switch in
11
10
functional activity (from agonist to inverse agonist), in the
present case, the substitution was found to be deleterious for
the affinity, since none of the C-4 substituted analogues (9−12
and 17−18) displayed activity against CB receptor. As a
2
consequence, no functional activity was measured. Conversely,
a
Scheme 1
a
Reagents and conditions: (i) 100 °C, 1 h ; (ii) Ph O, 240 °C, 2 h ; (iii) NaH, C H Br, DMF, 90 °C, 16 h ; (iv) ethyl benzoylacetate, NaH, DMF,
2
5
11
1
20 °C, 1 h ; (v) P S , pyridine, reflux, 16 h ; (vi) NH NH ,H O, EtOH, reflux, 16 h ; (vii) NaH, R -Br, DMF, 90 °C, 16 h ; (viii) R -NHNH ,
4 10 2 2 2 2 2 2
EtOH, reflux, 16 h.
B
dx.doi.org/10.1021/ml500439x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 1. Binding Affinity of Compounds 8−27 and
a
Reference Compounds for hCB Cannabinoid Receptor
2
Table 2. Functional Activity of Selected 2H-Pyrazolo[4,3-
c]quinolin-3(5H)-ones and Reference Compounds for hCB2
Cannabinoid Receptor
[35
S]-GTPγS (hCB2)
Cpd
EC50 (nM)
Emax (%)
1
2
2
2
6
4
5
6
204 ± 41
14.3 ± 4.0
5.4 ± 1.1
64 ± 10
24.5 ± 1.6
1.8 ± 0.9
160 ± 3
149 ± 4
141 ± 3
163 ± 4
207 ± 10
21.6 ± 2.7
(
R)-(+) WIN 55,212-2
SR144528
the absence of a substituent at C-4 provides compounds with
very high affinity toward the CB receptor. Therefore, for the
2
rest of our investigations, we decided to leave this position
unsubstituted and vary only the moieties at the N-2 position,
which were selected on the basis of the previously established
pharmacophore (aromatic or aliphatic groups).
The importance of a substituent at the N-2 position was
illustrated by the poor affinity of compound 8 (R = H) for
2
both receptor subtypes. Similarly, aromatic groups are generally
not well tolerated at this position. Indeed, with the notable
exception of 15 (hCB : K = 29.2 nM), none of the compounds
2
i
presenting an aromatic group at this position (spaced by an
alkyl linker or not) displayed significant affinity for the CB2
receptor.
In contrast, the introduction of aliphatic substituents at N-2
is crucial for the affinity, since all evaluated compounds
presenting an alkyl or cycloalkyl group at this position were
found to bind the CB receptor with affinities in the nanomolar
2
range (16 and 21−27). When comparing 16, 24, and 25, it is
evident that the distance of the cycloalkyl group from the
heterocycle is important, with the methyl linker being optimal.
This observation was confirmed when the cycloalkyl was an
adamantyl, with 26 (methyl linker) displaying a 67-fold higher
affinity than 27 (ethyl linker). In addition, the size of the
cycloalkyl group also impacts on the affinity. For instance, when
comparing 23, 24, and 26, the compound affinity improves as
the cycloalkyl size increases (adamantyl > cyclohexyl >
cyclopropyl), with 26 displaying a K for hCB of 0.39 nM.
i
2
The drastic loss of affinity when the cycloalkyl is replaced by a
phenyl group is noteworthy (24 or 26 vs 19). Lastly, linear
alkyl chains are also well tolerated, as evidenced by the high
affinity of compounds 21 and 22.
Interestingly, none of the evaluated compounds displayed
affinity for the hCB receptor (hCB : K > 3000 nM), pointing
1
1
i
at this series as highly selective for the hCB receptor.
2
From a functional activity perspective, compounds 16 and
2
4−26 showed a profile of CB partial agonist with EC values
2
50
ranging from 5.4 nM to 204 nM and E values on the order of
max
1
50% (Table 2). In this assay CB receptor full agonist (R)-(+)
2
WIN 55,212-2 displayed an EC value of 24.5 nM and an E
50
max
value of 207%, while SR144528 (inverse agonist) had an EC50
of 1.8 nM and an Emax value of 21.6%. The rigidification
approach has no effect on the functional activity as the parent
4
-oxo-1,4-dihydroquinoline derivatives were also described as
7
a
agonists.
Unless specified otherwise, compounds in this table display K values
i
b
c
for hCB receptor greater than 3000 nM. K (hCB ) > 1000 nM. K
It is instructive to compare the CB affinity of our lead
2
1
i
1
i
(
hCB ) = 52.4 ± 1.2 nM.
compound 26 with its analogue in the 4-oxo-1,4-dihydroquino-
1
C
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ACS Medicinal Chemistry Letters
Letter
2
0
line series (ALICB353). As can be seen from Table 1, the latter
previously described protocol. Compound 26 was orally
administered (in carboxymethyl cellulose, CMC) once daily at
dosages of 0.1, 1, and 10 mg/kg over 7 days, starting 2 days
before colitis induction. Control mice were treated with vehicle
only (CMC).
compound displayed a K for the hCB receptor of 21.6 nM
i
2
(
reported in ref 10 to have a K of 50.6 nM). Therefore, the
i
conformational constriction of this 4-oxo-1,4-dihydroquinoline
leading to 26 (hCB : K = 0.39 nM) allowed a 55-fold increase
2
i
in binding affinity.
Our results suggest that the rigidification strategy allows an
We examined survival rates during the full length of the study
and evaluated the body weight loss before euthanasia of the
animals (5 days after TNBS administration). We noted that
mice treated with 26 show reduced mortality compared to their
untreated counterparts (Figure 3A). Moreover, daily treatment
with compound 26 attenuated, in a dose-dependent manner,
body weight loss generally observed during the development of
TNBS colitis (Figure 3B).
increase in affinity for the CB receptor while not impacting the
2
functional activity. In addition, very high selectivity for the CB2
receptor over the CB receptor was achieved. This contrasts
1
with a previously reported study on this series that suggested
that the 2H-pyrazolo[4,3-c]quinolin-3(5H)-one was not a good
template for designing CB selective ligands, since all of the
2
described ligands behaved as low micromolar/high nanomolar
17
dual CB /CB ligands. We demonstrate herein that high
1
2
affinity for the CB receptor and selectivity over the CB1
2
receptor could be achieved using this template, when both the
N-2 and N-5 positions are substituted with an alkyl or
cycloalkyl group.
To investigate potential binding modes, compound 26 and
its 4-quinolone analogue (ALICB353) was docked into an
18
agonist-biased state model of the CB receptor. In contrast
2
with ALICB353, which produced diverse docking solutions, 26
is not only well tolerated but selects exclusively docking poses
that fit in a unique binding mode, satisfying interactions with
Figure 3. Survival rates and body weight loss evaluation: compound
2
6 attenuated both mortality and body weight loss (data are the mean
critical amino acids known to bind reference CB agonists
2
± SEM of 10 mice per group; *p < 0.05; vs vehicle).
(
Figure S1 in the Supporting Information). The most
representative docking pose for each molecule shows both
the adamantyl group in a hydrophobic cavity formed by helices
After euthanasia of the different groups, the colon of each
mouse was examined and damages were assessed using a
semiquantitative scoring system. Mice that received the vehicle
only showed macroscopic colitis reflected by thickening of the
bowel and areas of ulceration (macroscopic score of 4.9, Figure
2
(Phe87, Phe91, Phe94) and 3 (Phe106, Ile110, Val 113), as
well as a common hydrogen bond with the hydroxyl group of
Ser285 (Figure 2). The docking poses of the central
4
A). Compound 26 exerts a dose-dependent decrease in
macroscopic score (Figure 4A), with a very strong effect when
administered orally at 10 mg/kg (1.7 vs 4.9). This protective
Figure 2. Preferred docking pose for 26 (cyan) and ALICB353
(
green) in an agonist-biased state model of the CB receptor.
2
heterocycle and n-pentyl, however, differ slightly for the two
compounds. The tricyclic ring of 26 orients orthogonally to the
4
-quinolone scaffold of ALICB353, and the n-pentyl chain of 26
extends toward a hydrophobic cavity formed by helices 5
Phe200, Leu201) and 6 (Val261, Met265) whereas it folds
behind the 4-quinolone ring, toward extracellular loop 2
Leu182, Leu192) in the case of ALICB353.
(
Figure 4. Macroscopic (A) and histological (B) score. TNFα and IL-
(
1
β mRNA levels (C, D) were measured in the colon after TNBS-
19
Our lead compound in this series (26) was evaluated in a
mouse model of acute colitis induced by TNBS using a
induced colitis and treatment with compound 26 (data are the mean ±
SEM of 10 mice per group; *p < 0.05; ***p < 0.001 vs vehicle).
D
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ACS Medicinal Chemistry Letters
Letter
effect was confirmed by the results of the histological
evaluation. Indeed, 26 was able to attenuate the histological
score by 65% (Figure 4B). We also quantified colon levels of
TNFα and Il-1β, two cytokines involved in the inflammatory
response leading to epithelial injury. As can be seen from Figure
(35.5% inhibition of tail current at 1 μM) and so may have
some toxicity at a high dose. Compound 26 did however show
good in vitro metabolic stability, with 64% of compound
remaining after 60 min of incubation with human liver
microsomes. The DMPK-tox profile of compound 26 needs
to be improved, and future optimization regarding this series
will have to focus on this particular point.
4C and D, compound 26 completely abolished Il-1β mRNA
expression but failed to significantly decrease TNFα mRNA
expression.
All together, the above data clearly support that compound
In summary, the present study shows that 2H-pyrazolo[4,3-
c]quinolin-3(5H)-ones, constrained analogues of 4-oxo-1,4-
2
6 protects mice against experimental colitis after oral
dihydroquinolines, are potent and highly selective CB
agonists. The rigidification approach applied in this paper
resulted in increased affinity for the CB receptor while not
2
receptor
administration in a dose-dependent manner, an effect attributed
in part to the strong anti-inflammatory property of this
compound. Although different studies have already emphasized
2
altering the functional activity. Despite displaying a nonideal in
vitro DMPK-Tox profile, compound 26 (ALICB459), the lead
of this series, exerts a strong protective effect in the
experimental model of TNBS-induced colitis. This effect was
achieved after oral administration and was shown to be dose-
dependent.
4
−8
the efficacy of CB agonists in colitis, all were active after i.p.
2
injection, which from a drug development perspective is not
desirable. Favorably, compound 26 is active in a mouse model
of colitis after oral administration.
Compound 26 has also been profiled for in vitro metabolic
stability, plasma protein binding, intestinal absorption, and
hERG toxicity (Table 3).
21
ASSOCIATED CONTENT
Supporting Information
Experimental procedures and spectroscopic data for all
synthesized compounds, detailed pharmacology and all docking
poses of 26 and ALICB353 in an agonist-biased state model of
■
*
S
Table 3. Determination and Evaluation of Selected
Physicochemical and in Vitro DMPK-Tox Parameters for
Compound 26
the CB receptor. This material is available free of charge via
2
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*
Dr. Jamal El Bakali, e-mail: je331@cam.ac.uk.
Prof. Regis Millet, e-mail: regis.millet@univ-lille2.fr. Phone:
33320964374.
Present Addresses
*
+
́
a
Parameters
∥
(
J.E.B.) University of Cambridge, Department of Chemistry,
MW (g/mol)
cLogP
403.6
5.6
Lensfield Road, Cambridge CB2 1EW, United Kingdom.
⊥
(
G.G.M.) Bioanalysis and Pharmacology of Bioactive Lipids
tPSA
35.9
Lab, Louvain Drug Research Institute, Universite Catholique de
́
In vitro ADME-tox
Louvain, 72 Avenue E. Mounier, B-1200 Bruxelles, Belgium.
b
Human PPB (% free)
<1
A-B Caco-2 permeability (×10 cm·s−1
−
6
)
c
0.8
30.6
64
Funding
d
́
We are grateful for financial support from the Conseil regional
du Nord-Pas de Calais and University of Lille 2.
P-gp inhibition (% at 10 μM)
Metabolic stability (human liver microsomes, % parent remaining
e
after 1 h)
Notes
f
hERG (% inhibition of tail current at 1 μM)
35.5
The authors declare no competing financial interest.
a
b
Determined with ChemDraw Ultra 10.0. Assessed by equilibrium
dialysis (18 h) at 37 °C. Compound 26 (10 μM) was incubated (0
and 60 min) at 37 °C with Caco-2 cell line (pH 6.5/7.4). Performed
c
ABBREVIATIONS
d
■
hCB1&2, human cannabinoid receptor 1 and 2; DSS, dextran
sulfate sodium; TNBS, 2,4,6-trinitrobenzenesulfonic acid; i.p.,
intraperitoneal; Il-1β, interleukin-1β; CMC, carboxymethyl
cellulose
on MDR1-MDCKII cell line, using cellular uptake of calcein AM (30
e
min incubation/37 °C). Compound 26 (1 μM) was incubated (0 and
6
0 min) at 37 °C with human liver microsomes (0.3 mg/mL).
f
Compound 26 (1 μM) was incubated (5−10 min, cumulatively) at
room temperature with hERG HEK 293 cells (conventional whole-cell
patch clamp).
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■
(
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