Synthesis, Pharmacological Evaluation, and Docking Studies of Novel Pyridazinone-Based Cannabinoid Receptor Type 2 Ligands

Article abstract of DOI:10.1002/cmdc.201800152

In recent years, cannabinoid type 2 receptors (CB₂R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB₂R are devoid of the psychoactive effects typically observed for CB₁R ligands. Based on our recent studies on a class of pyridazinone 4-carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure–activity relationships for this promising scaffold with the aim to develop potent CB₂R ligands. In binding assays, two of the new synthesized compounds [6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (2) and 6-(4-chloro-3-methylphenyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamide (22)] showed high CB₂R affinity, with K_i values of 2.1 and 1.6 nm, respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB₂R inverse agonists. Compound 22 displayed the highest CB₂R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle-switch residue, W6.48.

Full text of DOI:10.1002/cmdc.201800152

Accepted Article
Title: Synthesis, pharmacological evaluation and docking studies of
novel pyridazinone-based cannabinoid receptor type-2 ligands
Authors: Gabriele Murineddu, Patricia Reggio, and Stefania Gessi
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To be cited as: ChemMedChem 10.1002/cmdc.201800152
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Synthesis, pharmacological evaluation and docking studies of
novel pyridazinone-based cannabinoid receptor type-2 ligands
Giulio Ragusa,[a] Serena Bencivenni,[b] Paula Morales,[c] Tyra Callaway,[c] Dow P. Hurst,[c]
Battistina Asproni,[a] Stefania Merighi,[b] Giovanni Loriga,[d] Gerard A. Pinna,[a] Patricia H.
Reggio,[c] Stefania Gessi,*[b] Gabriele Murineddu*[a]
[a] Dr. G. Ragusa, Dr. B. Asproni, Prof. G. A. Pinna, Prof. G Murineddu
Department of Chemistry and Pharmacy
University of Sassari
via F. Muroni 23/A, 07100 Sassari, Italy
E-mail: muri@uniss.it
[b] Dr. S. Bencivenni, Dr. S. Merighi, Prof. S. Gessi
Dipartimento di Scienze Mediche, Sezione di Farmacologia
Università di Ferrara
Via Fossato di Mortara, 17-19, 44121 Ferrara
E-mail: gss@unife.it
[c]
Dr. P. Morales, Dr. T. Callaway, Dr. D. P. Hurst, Prof. P. H. Reggio
Department of Chemistry and Biochemistry
University of North Carolina at Greensboro
Greensboro, NC 27402, USA
[d] Dr. Giovanni Loriga
Institute of Translational Pharmacology
National Research Council
09010 Pula, Cagliari, Italy
Supporting information for this article is given via a link at the end of the document.
Abstract: In the last years cannabinoid type 2 receptors (CB2R)
In the last few years, significant progress have been made in
have emerged as promising therapeutic targets in a wide variety of
diseases. Selective ligands of CB2R are devoid of psychoactive
effect typically observed for CB1R ligands. Taking advantage of our
recent studies on a class of pyridazinone 4-carboxamides, further
structural modifications on pyridazinone core were explored to better
investigate the structure–activity relationships for this promising
scaffold with the aim to develop potent CB2R ligands. In binding
assays two of the new synthetized compounds (2 and 22) showed
high CB2R affinity, with Ki values of 2.1 and 1.6 nM, respectively. In
addition, functional assays of these compounds and other new
active related derivatives showed a pharmacological profile of
inverse agonist for CB2R. Compound 22 displayed the highest CB2R
order to understand the role played by cannabinoid receptors in
many physiological and pathophysiological conditions.
CB1Rs and CB2Rs show certain common features and they
share similar signaling mechanism, nevertheless they differ in
anatomic distribution. The former are expressed in several cell
types and tissues, including muscle and liver cells as well as
spleen, pituitary gland, reproductive organs and adipose
tissues.[4] Nevertheless, they are predominantly distributed in
central nervous system (CNS) in which they are implicated in the
control of many neurobiological processes, including
nociception, control of movement, emesis, emotional response,
motivated behavior, cognitive function and homeostasis.[5,6]
CB2Rs are mostly expressed in immune cells as monocytes,
macrophages, natural killer cells, in which they play an important
role in mediating the immune-modulatory function.[7,8] However,
nowadays it has been reported that CB2Rs are also localized in
other type of cells and tissues such as vascular endothelial cells,
cardiomyocytes, bone cells, tissues of the gastrointestinal
tract.[4] Moreover, CB2Rs have been found in activated microglia
or astroglia, and may be also expressed by central neurons,
especially under pathological conditions.[9,10]
selectivity and potency presenting
a
favorable in silico
pharmacokinetic profile. Furthermore, a molecular modeling study
reveals how 22 produces inverse agonism through blocking the
movement of the toggle switch residue, W6.48.
Introduction
Cannabinoid subtype 2 receptor (CB2R) belongs to the
rhodopsin-like G-protein coupled receptor (GPCR) superfamily
and constitutes, along with cannabinoid subtype 1 receptor
(CB1R), the family of cannabinoid receptors (CBRs).[1,2] CBRs
are mainly activated by three types of ligands: endogenous
In these years, CBRs have been proposed as potential
therapeutic targets for the treatment of several diseases[4,11] and
some non-selective CB1R/CB2R agonists are already in use in
clinic as approved drugs (i. e. Sativex®, Cesamet®, Marinol ®).
However, the psychotropic side effects are to date the major
limitation related to the therapeutic use of non-selective
cannabinoid derivatives. It is well known that the undesirable
effects of cannabinoids on CNS are mediated by CB1R,[12]
cannabinoids,
as
N-arachidonoylethanolamine
and
2-
arachidonoyl glycerol, phytocannabinoids, as (−)-trans-Δ9-
tetrahydrocannabinol, and synthetic cannabinoids.[3]
1
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consequently the new goal for medicinal chemists has become
the discovery of CB2R selective ligands. These ligands are
devoid of the central side-effects due to their limited distribution
in the CNS, although a number of drawbacks could appear in
the immune system (e.g. immunosuppression). Furthermore,
increasing evidences have demonstrated the therapeutic
potential of the pharmacological modulation of CB2R. Preclinical
evidences support that CB2R selective ligands could be used for
the treatment of various CNS-related neurodegenerative
disorders including Alzheimer’s, Huntington’s and Parkinson’s
diseases, multiple sclerosis and amyotrophic lateral
sclerosis.[10,13-15] In addition to be a promising target for the
treatment of pain and inflammation[15-17] CB2R is also indicated
for the treatment of irritable bowel syndrome, myocardial
infarction, bone disorders, and different types of cancer.[4,15,18,19]
However, none of the CB2 ligands tested in clinical phases has
reached the market yet, probably due to their lack of efficacy.[20]
Recently, we synthesized a new series of pyridazinone-based
derivatives[21] endowed with CB2-affinity in the nM range and a
good CB2R-selectivity. Among these novel compounds, the 6-(4-
chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(4-cis-methylcyclo-
hexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide 1 (Figure 1)
showed high CB2-affinity (KiCB2 = 2.0 ± 0.8 nM) and a notable
selectivity (KiCB1R/KiCB2R > 2000). To further explore the
structure-activity relationship (SAR) studies on this new class of
CB2R ligands, we designed the modulation of compound 1,
assumed as lead compound, to obtain derivatives 2-24 (Figure
1). Therefore, we planned structural modifications on three
positions, Ar, R1 and R2, of the dihydropyridazinone core, to
evaluate the effect of their lipophilicity and steric hindarance on
CBR affinity of this series of pyridazinone-based derivatives
acyl halides by the enolate of diethylmalonate in anhydrous THF
furnished diesters 31-33, which were converted into cyclic
derivatives 34-36 by reaction with hydrazine hydrate in refluxing
ethanol. Bromine-assisted oxidation of dihydropyridazinones 34
and 36 in acetic acid provided pyridazinones 37 and 39.
Reaction of 35 with bromine in acetic acid afforded pyridazinone
38 in which bromine led at the same time to ring oxidation and to
bromination of thiopene moiety on C5 position. N-alkylation of
37-39 in anhydrous DMF with the suitable alkyl halide or benzyl
halide in presence of K2CO3, under ultrasound irradiation,
afforded compounds 40-44 which were hydrolyzed with NaOH in
ethanol to the corresponding carboxylic acid 45-49 in almost
quantitative yield.
EDC-HOBt mediated coupling reaction between acids 45-
47,49 and the appropriate amines led to the desired
carboxamide derivatives 2-16 and 22-24. Due to lower yields of
coupling reaction for acid 48 in these conditions, it was
previously activated with thionyl chloride, then, treated with the
suitable amines to furnish the desired derivatives 17-21.
Biological evaluation
The CB1R and CB2R binding affinities of the novel 3-oxo-2,3-
dihydropyridazine-4-carboxamides 2-24 were evaluated by
radioligand binding assays performed by using transfected
human CB1R (hCB1R) and CB2R (hCB2R) Chinese hamster
ovary (CHO) cells. [3H]CP-55,940 was employed as radiolabeled
ligand. The experimental data (IC50 values) were converted into
Ki values.[22] The receptor affinities are shown in Table 1. For
comparison, the Ki value of the mixed CB1R/CB2R ligand WIN
55,212-2 is reported.
The substitution of 3-CH3 group on the benzene ring of the
lead compound 1 with a second chlorine atom led to derivative
2, retaining a good CB2R affinity (Ki = 2.1 ± 0.2 nM) with
moderate selectivity (KiCB1R/KiCB2R = 101), whereas its trans
isomer 3 showed a similar affinity for CB2R and CB1Rs (Ki = 90 ±
7 nM and Ki = 85 ± 7 nM, respectively). The elimination of the
methyl group on cyclohexyl ring (4) and the homologation of the
aliphatic ring to cycloheptyl (5) restored CB2R affinity (Ki = 6.3 ±
0.5 nM and Ki = 7.2 ± 0.9 nM, respectively) and a moderate
selectivity (KiCB1R/KiCB2R = 55 and 31, respectively). Finally,
the introduction of a bulky group in the carboxylic portion, such
the 1-adamantyl amine, gave compound 6 which showed a 10-
fold decrease in CB2R affinity (Ki = 21 ± 2 nM) with respect to
derivative 2.
Figure 1. Structural design of pyridazinone compounds 2-24.
The same trend was observed in the small series of
derivatives bearing a methylenecyclohexyl chain on the N2 of
the pyridazinone ring, derivatives 7-11. All compounds showed a
good CB2R affinity (Ki = 4.2 - 25 nM), resulting the N-(4-cis-
methylcyclohexyl)-carboxamide 7 having the best CB2R binding
profile within this series (Ki = 4.2 ± 0.3 nM), comparable to that
of lead compound 1 and analogue 2.
Then, we wanted to evaluate the effect of the insertion of a
chloropentyl substituent on N2 of pyridazinone, synthesizing
compounds 12-16. Derivatives of this series maintained a good
CB2R affinity (Ki, 9 - 85 nM) and carboxamide 12 showed the
high CB2R selectivity (KiCB1R/KiCB2R > 1111) even if its KiCB2R
affinity was 4.5-fold less than lead compound 1.
In this study, we report the synthesis, the preliminary
pharmacological data and docking studies on the new
pyridazinones 2-24 reported in Table 1.
Results and Discussion
Chemistry
Target 3-oxo-6-aryl-2,3-dihydropyridazines 2 24 were
synthesized as outlined in Scheme 1.
Alpha-halogenation of commercial ketones 25-27 with bromine
gave acyl halides 28-30. The nucleophilic substitution on the
2
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Scheme 1. Scheme 1. Reagents and conditions: a) Br2, AcOH (for 25 and 27) or DCM (for 26), rt, 8 h; b) diethylmalonate, NaH, THF, 0 °C - rt, 20-24 h; c)
NH2NH2 x H2O, EtOH., reflux, 24 h; d) Br2, AcOH, rt, 6 h; e) K2CO3, DMF, R1X, ultrasonic irradiation, rt, 2 h; f) NaOH 2 M, EtOH, reflux, 8 h; g) HOBt, EDC,
R2NH2, DCM, 0 °C - rt, 12-24 h (for 2-16,22-24); h) SOCl2, Toluene, reflux, 3 h, then Et3N, R2NH2, DCM, 0 °C - rt, 12 h (for 17-21).
Therefore, the double chlorine atom substitution on the
CB1R/CB2R-stimulated cAMP binding
aromatic ring in C6 both keeping the N2-4-fluorobenzyl group (2)
and introducing N2-methylcyclohexyl group (7) led to
a
Selected compounds characterized by high affinity and
selectivity towards CB2Rs was studied in functional assays to
assess their behavior as agonists, antagonists or inverse
agonists at CB2Rs. Therefore, their modulation of forskolin-
stimulated cAMP levels in human CB2Rs CHO cells has been
investigated in comparison to the reference agonist compound
WIN 55,212-2. This ligand (0.1 μM) reduced forskolin-stimulated
cAMP levels of 78% and the compounds 2, 7 and 22
antagonized this effect in the range of concentration 0.01-1 μM,
with the 22 and 2 being the most potent molecules, according to
binding data. However compounds 12, 13, 17 and 21, even
though with high affinity values towards CB2Rs, did not display
an antagonistic effect on adenylyl cyclase activity inhibition
induced by WIN 55,212-2 (Figure 2).
In addition, all the ligands tested in the functional assay were
also evaluated for their ability to increase forskolin-stimulated
cAMP production in the range of concentration 1-100 nM. As
shown in Figure 3 the molecules under examination induced an
increase in forskolin-stimulated cAMP levels suggesting their
role as inverse agonists at CB2R.
derivatives with similar KiCB2R values to lead 1, whereas the
cyclopentyl N2 substitution gave the compound 9 with a lower
KiCB2R affinity.
Once evaluated the effects of the substituent in the N2
position of pyridazinone, we wanted to investigate the
replacement of the disubstituted phenyl in C6 of the
pyridazinone ring with a thiophene, synthesizing compounds 17-
21. Within this series, both the derivative 17, bioisoster of the
lead 1, and the N-(adamantan-1-yl)-carboxamide 21 showed an
interesting CB2R affinity (Ki = 5.5 nM and 3.1 nM, respectively),
the latter resulting endowed with good selectivity
(KiCB1R/KiCB2R > 1976).
Finally, we synthesized derivatives 22-24, three analogues
of lead 1 bearing a pentyl chain on N2 position, to confirm that
the N-4-cis-methylcyclohexyl group was the preferred
substituent in the carboxamide moiety, as resulted from data
obtained for compound 22 (KiCB2R = 1.6 ± 0.17 nM), which
displayed the highest CB2R selectivity (KiCB1R/KiCB2R > 6250).
3
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Table 1. Structures and binding data[a] for compounds 2-24
Ki CB1R
(nM)
Ki CB2R
(nM)
Selectivity
(KiCB1R/KiCB2R)
Compound
Ar
R1
R2
4-cis-Methyl-
cyclohexyl
4-trans-Methyl-
cyclohexyl
2
3
3,4-dichlorophenyl
3,4-dichlorophenyl
4-fluorobenzyl
4-fluorobenzyl
213±18
85±7
2.1±0.2
90±7
101
0.94
4
5
6
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
Cyclohexyl
Cycloheptyl
Adamantly
350±31
226±25
6.3±0.5
7.2±0.9
21±2
55
31
57
1200±123
4-cis-Methyl-
cyclohexyl
4-trans-Methyl-
cyclohexyl
7
8
3,4-dichlorophenyl
3,4-dichlorophenyl
cyclohexylmethyl
cyclohexylmethyl
3300±315
> 10000
4.2±0.3
10±1
786
> 1000
9
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
cyclohexylmethyl
cyclohexylmethyl
cyclohexylmethyl
Cyclohexyl
Cycloheptyl
Adamantly
6000±578
> 10000
93±9
11±1
10±1
25±3
545
> 1000
4
10
11
4-cis-Methyl-
cyclohexyl
4-trans-Methyl-
cyclohexyl
12
13
3,4-dichlorophenyl
3,4-dichlorophenyl
chloropentyl
chloropentyl
> 10000
9.0±0.8
16±2
> 1111
250
4000±395
14
15
16
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
chloropentyl
chloropentyl
chloropentyl
Cyclohexyl
Cycloheptyl
Adamantly
5300±542
147±16
85±8
55±6
16±2
62
3
> 10000
> 625
4-cis-Methyl-
cyclohexyl
4-trans-Methyl-
cyclohexyl
17
18
5-bromothiophen
5-bromothiophen
4-fluorobenzyl
4-fluorobenzyl
1600±146
200±23
5.5±0.7
291
19
10.5±0.9
19
20
21
5-bromothiophen
5-bromothiophen
5-bromothiophen
4-fluorobenzyl
4-fluorobenzyl
4-fluorobenzyl
Cyclohexyl
Cycloheptyl
Adamantly
5600±559
800±78
8.4±0.9
110±13
3.1±0.3
667
7
6125±583
1976
4-cis-Methyl-
cyclohexyl
22
4-chloro-3-methylphenyl
pentyl
> 10000
1.6±0.2
> 6250
23
24
4-chloro-3-methylphenyl
4-chloro-3-methylphenyl
pentyl
pentyl
Cyclohexyl
2500±271
666±68
13±2
7.5±0.9
6.1±0.8
2.5±0.3
333
109
5.2
Cycloheptyl
WIN 55212
[a] Ki values were determined by competitive displacement of radioligand [3H]CP55,940, for CB2Rs and CB1Rs. The Ki was calculated from the IC50 values
determined from the binding curves, using the Cheng-Prusoff equation.[22] Values are the mean ± SEM of at least three independent experiments run in triplicate.
Molecular modeling
the 1,8-naphthyridin-2(1H)-one-3-carboxamides. The most
potent and selective CB2R inverse agonist, 22, was docked in a
previously published CB2R R state model.[23-26] This homology
In previous work, it was identified the binding site for the
CB2R antagonist, SR144528, using our CB2R inactive state (R)
homology model.[23] These studies suggested that the
SR144528 amide functional group is important for interaction at
CB2R. In addition, aromatic stacking interactions in the TMH5/6
aromatic cluster were revealed to be crucial in the
SR144528/CB2R complex. Further exploration of the 1,8-
naphthyridin-2(1H)-one-3-carboxamide scaffold in the CB2R R
model, showed that antagonists from this series bind in the
TMH2-3-6-7 region with the C-6 naphthyridine substituent
directly blocking the W6.48(258) toggle switch.[24]
model
was
pre-equilibrated
in
a
stearoyl-
docosahexaenoylphosphatidylcholine (SDPC) bilayer for 300 ns
to allow it to adjust in a lipidic environment[23] (see experimental
section).
A complete conformational analysis of 22 was
performed to identify the global minimum energy conformer, as
well as, other minimum energy conformations that 22 can adopt.
This global minimum energy conformer has an intramolecular
hydrogen bond between the pyridazin-3(2H)-one carbonyl
oxygen and the amide hydrogen. Docking studies used this
global minimum energy conformer. As depicted in Figure 4(A),
pyridazinone 22 occupies the CB2R TMH2-3-6-7 region.
In the current work, Glide docking studies revealed that the
novel pyridazinones reported here bind in a similar orientation as
4
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Figure 2. Inhibition of forskolin-stimulated cAMP levels by WIN 55,2122 (100 nM) and antagonism by 22, 7, 2, 21, 17, 12 and 13 (10-1000 nM) in hCB2R-CHO
cells. Forskolin (5 μM) effect was set to 100%. Values are the mean±SEM of four independent experiments (N=4). *P<0.05, compared with WIN 55,212-2;
analysis was by ANOVA followed by Dunnett’s test.
Figure 3. Effect of 22, 7, 2, 21, 17, 12 and 13 (1-100 nM) expressed as % of increase of forskolin-stimulated cAMP accumulation in hCB2R-CHO cells. Forskolin
(5 μM) effect was set to 100%. Values are the mean±SEM of four independent experiments (N=4). *P<0.05, compared with Forskolin (5 μM); analysis was by
ANOVA followed by Dunnett’s test.
The energy-minimized 22/CB2R state complex (Figure 4(A))
displays four main binding site interactions. The carbonyl oxygen
of the pyridazinone core is engaged in a hydrogen bond with
S7.39(285) [hydrogen bond (O−O) distance = 2.90 Å and (O-
H−O) angle = 166°], and the carbonyl oxygen of the 4-cis-
methylcyclohexyl amide substituent establishes a hydrogen
bond with K3.28(109) [hydrogen bond (O−O) distance = 2.90 Å
and (O-H−O) angle = 150°]. In addition, the central pyridazin-
3(2H)-one ring forms a tilted-T aromatic stack with F2.57(87)
(ring centroid to ring centroid distance = 5.60 Å, and the angle
Å of 22 were also assessed. The ligand conformational cost for
22 was 0.89 kcal/mol, bringing the total Interaction Energy to -
65.70 kcal/mol. (see Supplementary, Table S1). The Glide score
for this dock was -9.50. The conformational change of W6.48
(χ1 g+→trans) is associated with receptor activation in Class A
GPCRs such as CB2R. This change can modulate the Pro kink
in TMH6 (P6.50), causing an opening at the IC end of the
receptor to form for G-protein interaction.[24] W6.48 is therefore
considered to be part of the “toggle switch” for activation. As
illustrated in Figure 4(B), the 4-chloro-3-methylphenyl
pyridazinone substituent of 22 points toward TMH5 and extends
deep enough in the binding pocket to block the movement of
W6.48(258) and therefore keep its χ1 at g+. This is consistent
with 22 acting as an inverse agonist at CB2R.
between the ring planes
= 63°). Moreover, M6.55(265)
establishes a Met−aromatic interaction with the 4-chloro-3-
methylphenyl moiety [distances to the central ring centroid from
M6.55 side chain atoms CG (3.92Å), SD (3.60 Å), and CE (3.89
Å)]. The ligand/receptor interaction energies for residues within 5
5
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B
A
Figure 4. (A) This figure presents an extracellular view of Compound 22 (lavender) docked in the TMH2-3-6-7 region of CB2R. Residues with which 22 has direct
interactions are shown in purple carbons. (B) This figure presents a side view of 22 (lavendar, atoms contoured at their Van der Waals radii) docked at CB2R. It is
clear here that 22 is adjacent to the Toggle Switch residue, W6.48 (Green), and blocks the W6.48 χ1 g+→trans associated with actvation.
Therefore, this docking study provides a rational structural
explanation for the ability of ligands like 22 to prevent G-protein
dependent CB2R activation.
abundant CYP450 isoenzymes in human liver and intestine are
CYP3A4, CYP2C9 and CYP2D6.[36] The pharmacokinetic
behavior of drugs can be determined by their interactions with
these enzymes, since their inhibition can affect drug clearance,
consequently enhancing toxicity. Therefore, it is important to
predict whether a new compound is a substrate, an inhibitor or
an inducer of these CYP isoforms in early drug discovery
phases. As depicted in Table S2, compounds 22 24 present the
best metabolic profiles since they are substrates but non-
inhibitors of the most abundant isoform CYP3A4. These
compounds will not hamper the biotransformation of drugs
metabolized by this cytochrome P450 enzyme, whereas certain
inhibitors could trigger adverse side effects. This difference in
the metabolism among pyridazinones may be due to the
presence of halogens in the molecule (derivatives 22 24 have
only one halogen, whereas the other derivatives have at least
two halogens per structure).[37,38]
In terms of toxicity, as shown in Table S3, the safety profile
of all ligands reported here are favorable, since they present low
cardiotoxicity, being nonmutagenic and noncarcinogenic. Low
oral toxicity values were found also for the reference compounds
SR144528 and HU308. In addition, the predicted median lethal
dose (LD50 in rat model) for these compounds is in the range of
most FDA approved small molecule drugs.[39,40] Therefore, taking
the pharmacological and ADMET prediction data into
consideration, the highly selective CB2R inverse agonist 22 may
serve as a lead structure for further optimizing this novel class of
CB2R ligands, not only with regard to potency, but also
concerning its pharmacokinetic properties.
In silico ADMET parameters
In silico prediction of properties related to absorption,
distribution, metabolism, excretion and toxicity (ADMET) can
help to efficiently prioritize the most promising molecules to be
further developed from the early stages of the drug discovery
process. The high predictive potential and the reduced time and
cost associated with these in silico approaches have increased
its use in the search of novel drug candidates.[27-29]
Pharmacokinetic properties of the newly synthesized
pyridazinones and of well-known CB2R ligands such as
SR144528 and HU308 were calculated in silico using QikProp
from Maestro (Schrödinger package). This program calculates a
set of 34 physicochemical descriptors based on the global
minimum energy conformer of each molecule.
According to our drug-likeness prediction studies, the
pyridazinones described herein follow the Lipinski and
Jorgensen pharmacokinetics rules (Table 2).[30,31] As shown in
Table 2, the prediction of blood-brain barrier permeability,
human oral absorption, bioavaibility, human intestinal
permeability or binding to human serum albumin suggest that
pyridazinones 2 24 have a suitable druggability profile. The
novel compounds presented here have solubility values that fall
outside the range predicted by QikProp for 95% of known oral
drugs. Solubility issues are widely known in cannabinoid
pharmacology because of their lipophilic nature.[32,33] Within all
the structural subsets of pyridazinones, the derivatives bearing
an adamantyl group at the R1 position display higher in silico
lipophilicity values (6, 11, 16, 21). 6-(4-Chloro-3-methylphenyl)-
3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamides (22 24)
display the best solubility values among this series of
compounds being better than the widely used CB2R reference
ligands SR144528 and HU308.
Conclusions
The modulation of the 6-aryl-3-oxo-2,3-dihydropyridazine-4-
carboxamide scaffold in three different positions, the N2 chain,
C4 carboxamide moiety and C6-aryl ring, led us to determine the
key features of this novel class of CB2R ligands. In general, the
N-4-cis-methylcyclohexyl group was the preferred substituent in
the C4 carboxamide moiety, resulting derivatives bearing this
group those with high CB2R affinity (KiCB2R ranging from 1.6 nM
and 9.0 nM) and selectivity (KiCB1R/KiCB2R from 101 to >6250).
To further explore in silico metabolism and toxicity for these
novel compounds, we calculated the parameters summarized in
Tables S2 and S3 in Supplementary Material by using the
admetSAR server.[34] Over 70% of human drug metabolism
occurs via cytochrome P450 oxidases (CYP450).[35] The most
6
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10.1002/cmdc.201800152
ChemMedChem
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Concerning the N2 position, all inserted chains showed a good
CB2R binding profile, especially the 5-chloropentyl and 4-
fluorobenzyl groups; at the same time, the presence of a 3,4-
disubstituted aromatic system in C6 position provides a better
binding profile for CB2Rs.
profile. Compound 22, and its congeners, display the best
solubility values from this series, and additionally have improved
solubility over the reference compounds, SR144528 and HU308.
Finally, calculations to predict toxicity and metabolism showed
compound 22 acts as a substrate, but not an inhibitor, for the
most abundant CYP450 isoenzymes, and presents the best
metabolic profile in this novel class of pyridazinones.
To sum up, 22, a very potent and selective CB2R inverse
agonist has been identified with favorable properties that
indicate this compound could be a lead for drug development.
Docking studies on compound 22 provides
a rational
structural explanation for the ability of these novel derivatives to
act as inverse agonists at CB2R, preventing G-protein
dependent CB2R activation.
The in silico prediction of the pharmacokinetic properties
suggest that novel pyridazinones have a suitable druggability
Table 2. Physicochemical descriptors of compounds 2 24, SR144528 and HU308 calculated by QikProp integrated in
Maestro (Schrödinger, LLC, New York, USA).
%Human oral
absorption GI[f]
Compound
QPlogP[a]
QlogBB[b]
QPPMDCK[c]
QPlogKhsa[d]
QPPCaco[e]
2
7.52
7.56
7.21
7.45
7.96
7.50
7.49
7.12
7.33
7.86
7.15
7.26
7.40
7.70
8.12
7.09
7.12
6.76
7.02
7.54
6.69
6.65
6.88
7.01
7.61
-0.01
-0.07
-0.10
-0.04
-0.13
-0.22
-0.22
-0.04
-0.03
-0.14
-0.11
-0.24
-0.23
-0.21
-0.13
0.05
10000
10000
10000
10000
9996
1.64
1.69
1.53
1.62
1.87
1.81
1.80
1.60
1.69
1.96
1.50
1.52
1.57
1.69
1.87
1.52
1.52
1.36
1.50
1.72
1.30
1.48
1.57
1.62
1.91
2524
2248
2111
2328
2053
2149
2165
2905
2869
2412
1966
1840
1964
2054
2445
2629
2893
2802
2375
2631
2494
1963
2076
3738
4606
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
3
4
5
6
7
5819
8
5819
9
10
8058
7953
11
6590
12
10000
10000
10000
10000
10000
10000
10000
10000
9935
13
14
15
16
17
18
0.11
19
0.10
20
0.01
21
0.05
10000
2874
22
-0.37
-0.56
-0.52
-0.52
0.11
23
2219
24
2357
HU308
SR144528
2057
5587
[a] Predicted octanol/water partition coefficient [-2.0-6.5]. [b] Predicted log of the brain/blood partition coefficient [-3.0-1.2].
[c] Predicted apparent MDCK (Madin-Darby canine kidney) cell permeability in nm/sec [<25 poor, >500 great]. [d]
Prediction of binding to human serum albumin [-1.5-1.5]. [e] Apparent Caco-2 cell permeability in nm/s (intestinal drug
permeability) [<25 poor, >500 excellent]. QikProp predictions are for non-active transport. [f] Human Oral Absorption in GI
[<25% is poor]; [range of 95% of drugs].
Chemistry
Experimental Section
7
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All reactions involving air or moisture-sensitive compounds
were performed under nitrogen atmosphere. Solvents and
reagents were obtained from commercial suppliers and were
used without further purification. The starting ketones 25-27
and amines for the synthesis of final compounds were
purchased from Sigma-Aldrich®. Ultrasonic irradiation
experiments were carried out in Bandelin® Sonorex ultrasonic
bath RK-100H. Flash column chromatography was performed
automatically on Flash-master (Biotage®) with pre-packed
Biotage® SNAP silica gel cartridges or manually on silica gel
(Kieselgel 60, 0.040−0.063 mm, Merck®). Thin layer
chromatography (TLC) was performed with Polygram SIL N-
HR/HV254 pre-coated plastic sheets (0.2 mm) on aluminum
sheets (Kieselgel 60 F254, Merck®). Melting points were
The resulting residue was purified by column flash
chromatography using the appropriate eluents.
Bromo-1-(3,4-dichlorophenyl)ethan-1-one (28): Following
method A, ketone 25 led to obtain a yellow solid which was
purified by flash chromatography (petroleum ether/EtOAc
95:5) to afford 28 as a white solid (14.9 g, 94%), Rf =0.45
(petroleum ether/EtOAc 95:5); mp 44-45 °C; 1H NMR (400
MHz, Chloroform-d) δ 8.07 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H),
7.58 (d, J = 8.3 Hz, 1H), 4.38 (s, 2H).
Bromo-1-(thiophen-2-yl)ethan-1-one
(29):
Following
method B, ketone 26 led to obtain a dark oil which was
purified by flash chromatography (petroleum ether/EtOAc
95:5) to afford 29 as a yellow oil (14.9 g, 92%). Rf =0.33
(petroleum ether/EtOAc 95:5); 1H NMR (400 MHz,
Chloroform-d) δ 7.81 (t, J = 2.9 Hz, 1H), 7.72 (d, J = 4.5 Hz,
1H), 7.17 (d, J = 2.4 Hz, 1H), 4.36 (s, 2H).
obtained on
a Köfler melting point apparatus and are
uncorrected. IR spectra were recorded as nujol mulls on NaCl
plates with a Jasco FT/IR 460 plus spectrophotometer and
are expressed in  (cm-1). NMR experiments were run on a
Bruker Avance III Nanoboy 400 system (400.13 MHz for 1H,
and 100.62 MHz for 13C). Spectra were acquired using
deuterated chloroform (chloroform-d) or deuterated
dimethylsulfoxide (DMSO-d6) as solvents. Chemical shifts ()
See the Supporting Information for compound 30.
General Procedure for the preparation of ketodiester
derivatives (31-33). To
a suspension of NaH (60%
1
for H- and 13C-NMR spectra are reported in parts per million
dispersion in mineral oil, 0.480 g, 20 mmol, 2 eq) in
anhydrous THF (26 mL) was added dropwiswe a solution of
diethylmalonate (1.83 mL, 12 mmol, 1.2 eq) in anhydrous
THF (6.5 mL) under N2 flow at 0 °C, and the mixture was
stirred for 1 h. Then a solution of bromoketone (10 mmol,
1eq) in anhydrous THF (4 mL) was added dropwise and the
resulting mixture of reaction was stirred at room temperature
until complete conversion of the starting materials (TLC). The
reaction was quenched with saturated NH4Cl aqueous
solution, and the aqueous layer extracted with EtOAc. The
combined organic layers were washed with H2O and brine,
then dried over Na2SO4. The solvent was removed under
reduced pressure and the resulting residue was purified by
column flash chromatography with the appropriate eluents.
(ppm) using the residual non-deuterated solvent resonance
as the internal standard (for chloroform-d: 7.26 ppm, H and
77.16 ppm, 13C; for DMSO-d6: 2.50 ppm, 1H, 39.52 ppm, 13C).
Data are reported as follows: chemical shift (sorted in
descending order), multiplicity (s for singlet, br s for broad
singlet, d for doublet, dd for double doublet, t for triplet, q for
1
quadruplet,
constants (J) in Hertz (Hz). LC/MS analyses were run on an
Agilent 1100 LC/MSD system consisting of single
m for multiplet), integration and coupling
a
quadrupole detector (SQD) mass spectrometer (MS)
equipped with an electrospray ionization (ESI) interface and a
photodiode array (PDA) detector; PDA range was 120−550
nm. ESI in positive mode was applied; mobile phases: (A)
ACN in H2O (8:2). Analyses were performed with a flow rate
of 0.9 mL/min and at room temperature. All final compounds
displayed ≥ 95% purity as determined by elemental analysis
on a Perkin-Elmer 240-B analyser, for C, H, and N.
Diethyl
2-[2-(3,4-dichlorophenyl)-2-oxoethyl]malonate
(31): A mixture of diethylmalonate, NaH and 28 was stirred at
room temperature for 20 h. The crude product of reaction was
purified by flash chromatography (petroleum ether/EtOAc 9:1)
to afford 31 as a yellow oil (2.7 g, 77%), Rf = 0.30 (petroleum
ether/EtOAc 9:1); 1H NMR (400 MHz, Chloroform-d) δ 8.05 (s,
1H), 7.79 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 4.11 (q,
J = 7.1 Hz, 4H), 4.03 (t, J = 6.9 Hz, 1H), 3.55 (d, J = 7.0 Hz,
2H), 1.28 (t, J = 7.1 Hz, 6H).
Synthetic procedures for representative compounds
General Procedure for the preparation of acyl bromides
(28-30)
Method A: Bromine (3.02 mL, 59,3 mmol, 1 eq) was
dropwise added to a stirred solution of appropriate ketone
(59.3 mmol, 1 eq) in glacial AcOH (65 mL), the resulting
solution was stirred at room temperature for 8 h. Then ice-
water was poured into reaction flask and the resulting
precipitate was filtered off, washed with H2O, and air-dried.
The resulting crude product was purified by column flash
chromatography using the appropriate eluents.
See the Supporting Information for compounds 32 and 33.
General
procedure
for
the
preparation
of
dihydropyridazinone derivatives (34-36). To an ice-bath
cooled solution of malonate derivatives 31-33 (15 mmol, 1 eq)
in absolute ethanol (75 mL), hydrazine monohydrate 64-65%
(0.79 mL, 16.5 mmol, 1.1 eq) was added dropwise. The
resulting solution was warmed to room temperature, then
refluxed for 24 h. The solution was allowed to stand at room
temperature and the resulting precipitate was isolated by
filtration in vacuum, washed with cold water, and dried to air.
The obtained solid was purified by column flash
chromatography using the appropriate eluents.
Method B: A solution of bromine (4.0 mL, 79.2 mmol, 1 eq) in
DCM (40 mL) was dropwise added to a stirred solution of
ketone (79.2 mmol, 1 eq) in DCM (48 mL). The resulting
mixture was stirred at room temperature for 1 h, then
neutralized with saturated NaHCO3 aqueous solution. The
organic layer was separated in a separatory funnel and
washed with brine. Therefore the organic solution was dried
(Na2SO4) and the solvent removed under reduced pressure.
8
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Ethyl 5-(3,4-dichlorophenyl)-2-oxo-1,2,3,4-tetrahydropyri-
dine-3-carboxylate (34): From 31 was obtained a solid which
was purified by flash chromatography (petroleum ether/EtOAc
6:4) to give 34 as a pale yellow solid (2.79 g, 59%). Rf = 0.51
(petroleum ether/EtOAc 6:4); mp 166-168 °C; 1H NMR (400
MHz, Chloroform-d) δ 8.96 (s, 1H, NH, exch. with D2O), 7.84
(s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 4.25
(q, J = 7.2 Hz, 2H), 3.61 (t, J = 7.5 Hz, 1H), 3.39 (dd, J = 17.0,
7.9 Hz, 1H), 3.05 (dd, J = 17.0, 6.9 Hz, 1H), 1.28 (t, J = 7.2
Hz, 3H).
(petroleum ether/EtOAc 7:3), mp 139-140 °C; 1H NMR (400
MHz, Chloroform-d) δ 8.15 (s, 1H), 7.90 (s, 1H), 7.62 (d, J =
8.3 Hz, 1H), 7.57 – 7.48 (m, 3H), 7.02 (t, J = 8.0 Hz, 2H), 5.40
(s, 2H), 4.43 (q, J = 6.9 Hz, 2H), 1.41 (t, J = 7.0 Hz, 3H); 13C
NMR (101 MHz, CDCl3) δ 163.93 (C), 163.30 (CF), 161.28
(CF), 156.24 (C), 141.39 (C), 134.10 (C), 133.94 (C), 133.59
(C), 131.41 (CH), 131.18 (C), 131.13 (CH), 131.04 (CH x2),
130.60 (C), 127.63 (CH), 124.84 (CH), 115.71 (CH), 115.50
(CH), 62.49 (CH2), 55.82 (CH2), 14.21 (CH3).
See the Supporting Information for compounds 41-44.
See the Supporting Information for compounds 35 and 36.
General procedure for the preparation of pyridazinone
carboxilic acid derivatives 45-49. To a stirred solution of
pyridazinone ethyl ester (3 mmol) in EtOH (50 mL), a solution
of NaOH 2 M (50 mL) was added. The resulting mixture was
heated to reflux and stirred for overnight. Then the solution
was allowed to cool at room temperature and acidified with
HCl 6M. The resulting precipitate was filtered under vacuum,
washed with H2O and air-dried to yield the analytically pure
acid.
General procedure for the preparation of pyridazinone
derivatives (37-39). To
a stirred solution of dihydro-
pyridazinone 34-36 (5 mmol, 1 eq) in glacial AcOH (30 mL)
was added dropwise (30 min) a solution of bromine (0.51 mL,
10 mmol, 2 eq) in glacial AcOH (8 mL). The resulting mixture
was stirred at room temperature for 6-8 h. Then the solution
was poured into ice-water, and aqueous solution was
extracted with a separatory funnel with EtOAc. The collected
organic phases were washed with H2O, saturated NaHCO3
solution and brine. Then, dried (Na2SO4) and concentrated
under reduced pressure to yield a residue which was purified
by column flash chromatography using the appropriate
eluents.
6-(3,4-Dichlorophenyl)-2-(4-fluorobenzyl)-3-oxo-2,3-dihy-
dropyridazine-4-carboxylic
acid
(45):
alkaline
saponification in hydroalcoholic NaOH of pyridazinone ethyl
ester 40 furnished acid 45 as a pink solid (1.1 g, 92%), Rf =
1
0.46 (CHCl3/MeOH 9:1); mp 200-201 °C; H NMR (400 MHz,
Ethyl 6-(3,4-dichlorophenyl)-3-oxo-2,3-dihydropyridazine-
4-carboxylate (37): General procedure for the preparation of
pyridazinones was used to oxidize 34. The reaction mixture
was stirred for 8 h and the obtained crude product purified by
flash chromatography (petroleum ether/EtOAc 1:1) to afford
37 as a pale yellow solid (0.85 g, 54%). Rf = 0.47 (petroleum
ether/EtOAc 1:1); mp 188-190 °C; 1H NMR (400 MHz,
Chloroform-d) δ 8.26 (s, 1H), 7.94 (s, 1H), 7.66 (d, J = 8.3 Hz,
1H), 7.55 (d, J = 8.3 Hz, 1H), 4.47 (q, J = 7.1 Hz, 2H), 1.44 (t,
J = 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 162.79 (C),
158.25 (C), 143.09 (C), 134.31 (C), 133.65 (C), 133.63 (CH),
132.71 (CH), 131.08 (C), 130.75 (C), 127.76 (CH), 124.93
(CH), 62.60 (CH2), 14.22 (CH3).
Chloroform-d) δ 8.60 (s, 1H), 7.97 (s, 1H), 7.67 (d, J = 8.5 Hz,
1H), 7.60 (d, J = 8.4 Hz, 1H), 7.54 – 7.48 (m, 2H), 7.07 (t, J =
8.1 Hz, 2H), 5.49 (s, 2H); 13C NMR (101 MHz, CDCl3) δ
164.24 (CF), 162.72 (C), 161.78 (CF), 160.41 (C), 144.63 (C),
135.19 (C), 133.98 (C), 132.99 (CH), 132.96 (C), 131.32 (CH),
131.21 (CH), 131.13 (CH), 129.93 (C), 127.99 (CH), 126.72
(C), 125.14 (CH), 116.10 (CH), 115.88 (CH), 56.32 (CH2).
See the Supporting Information for compounds 46 - 49.
General procedures for the synthesis of carboxamide
derivatives (2-24).
Procedure A: To a stirred solution of acids 45-47, 49 (1 mmol,
1 eq) in DCM (10 mL), HOBt (0.20 g, 1.5 mmol, 1.5 eq) and
EDC (0.38 g, 2 mmol, 2 eq) were added. The resulting
mixture was stirred at room temperature for 30 min. Afterward
a solution of suitable amine in DCM (10 mL) was added
dropwise at 0 °C. The mixture of reaction was allowed to
stand at room temperature, and then stirred for 12-24 h. The
solution was then poured into a separatory funnel and H2O
was added. The aqueous phase was separated, and
extracted with DCM. The combined organic phases were
washed with H2O and brine, dried (Na2SO4) and concentrated
under reduced pressure. The analytically pure product was
isolated by column flash chromatography purification using
the appropriate eluents.
See the Supporting Information for compounds 38 and 39.
General procedure for the preparation of N-alkyl
pyridazinone derivatives (40-44). To
a
solution of
pyridazinones 37-39 (4 mmol, 1 eq) in anhydrous DMF (18
mL) was added K2CO3 (1.65 g, 12 mmol, 3 eq) and then the
suitable alkyl/benzyl halide (12 mmol, 3 eq). The resulting
suspension was underwent to ultrasound irradiation for 2 h at
room temperature. Then the mixture of reaction was poured
into ice-water, filtered off, and extracted with EtOAc in a
separatory funnel. The collected organic phases were
washed with H2O, LiCl 5% solution and brine, then dried
(Na2SO4) and concentrated under reduced pressure.
Resulting residue was purified by column flash
chromatography using the appropriate eluents.
Procedure B: A solution of acid 48 (410 mg, 1.0 mmol, 1 eq)
and thionyl chloride (0.22 mL, 3 mmol, 3 eq) in toluene (9 mL)
was refluxed for 3 h. Then, the excess of thionyl chloride was
removed under reduced pressure and the resulting dark solid
was dissolved in DCM (15 mL). To a resulting solution was
added dropwise at 0 °C a solution of appropriate amine (1.5
eq) and Et3N (1.5 eq) in DCM (15 mL). The mixture of
reaction was allowed to stand at room temperature and
Ethyl 6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-3-oxo-2,3-
dihydropyridazine-4-carboxylate (40): From pyridazinone
37 and 4-fluorobenzylbromide was obtained a residue which
was purified by flash chromatography (petroleum ether/EtOAc
8:2) to obtain 40 as a pale yellow solid (1.3 g, 78%), Rf = 0.47
9
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10.1002/cmdc.201800152
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FULL PAPER
stirred for 12 h. Then the mixture was poured into a
separatory funnel and H2O was added. The aqueous phase
was separated and extracted with DCM. The combined
organic phases were washed with H2O, dried (Na2SO4) and
concentrated under reduced pressure. The analytically pure
product was isolated by column flash chromatography
purification using the appropriate eluents.
95:5). 7 (90 mg, 19%), white solid, Rf = 0.38 (petroleum
ether/EtOAc 9:1); mp 131-132 °C; IR 1688 (C=O), 3273 (NH);
1H NMR (400 MHz, Chloroform-d) δ 9.88 (d, J = 7.8 Hz, 1H,
NH, exch. with D2O), 8.63 (s, 1H), 7.98 (s, 1H), 7.69 (d, J =
8.3 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 4.25 – 4.21 (m, 1H),
4.19 (d, J = 7.5 Hz, 2H), 2.10 – 2.00 (m, 1H), 1.89 – 1.57 (m,
11H), 1.34 – 1.04 (m, 8H), 0.97 (d, J = 6.4 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 159.73 (C), 159.17 (C), 141.36 (C),
133.27 (C), 132.95 (C), 132.51 (C), 129.96 (CH), 129.36 (CH),
128.70 (C), 126.79 (CH), 124.00 (CH), 57.72 (CH2), 44.95
(CH), 35.91 (CH), 29.99 (CH), 29.52 (CH2 x2), 29.09 (CH2 x2),
28.44 (CH2 x2), 25.24 (CH2), 24.62 (CH2 x2), 20.44 (CH3); MS
(ESI): C25H31Cl2N3O2 requires m/z 475.2, found 476.2 [M +
1]+; Anal. calcd for C25H31Cl2N3O2: C, 63.02; H, 6.56; N, 8.82;
Found: C, 62.88; H, 6.55; N, 8.80. 8 (47 mg, 10%), white solid,
Rf = 0.55 (petroleum ether/EtOAc 9:1); mp 133-135 °C; IR
6-(3,4-diChlorophenyl)-2-(4-fluorobenzyl)-cis-N-(-4-me-
thylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxa-
mide (2) and 6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-
trans-N-(-4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazi-
ne-4-carboxamide (3): General procedure
A for the
synthesis of carboxamides was used to convert acid 45 and 4
-methylcyclohexylamine into the title products. The mixture of
reaction was stirred for 18 h, then the cis/trans isomers were
separated by flash chromatography (petroleum ether/EtOAc
9:1). 2 (111 mg, 23%), white solid, Rf = 0.41 (petroleum
ether/EtOAc 85:15); mp 130-132 °C; IR 1681 (C=O), 3274
(NH); 1H NMR (400 MHz, Chloroform-d) δ 9.80 (d, J = 7.9 Hz,
1H, NH, exch. with D2O), 8.63 (s, 1H), 7.98 (d, J = 2.1 Hz,
1H), 7.68 (dd, J = 8.5, 2.2 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H),
7.49 (dd, J = 8.5, 5.5 Hz, 2H), 7.05 (t, J = 8.6 Hz, 2H), 5.45 (s,
2H), 4.26 – 4.18 (m, 1H), 1.86 – 1.77 (m, 2H), 1.71 – 1.56 (m,
5H), 1.33 – 1.20 (m, 2H), 0.98 (d, J = 6.5 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 163.96 (CF), 161.50 (CF), 160.43 (C),
159.74 (C), 143.14 (C), 134.22 (C), 134.00 (C), 133.59 (C),
131.09 (C), 131.03 (CH), 130.91 (CH), 130.87 (CH), 130.79
(CH), 130.39 (C) 127.86 (CH), 125.06 (CH), 115.80 (CH),
115.59 (CH), 55.54 (CH2), 49.95 (CH), 30.98 (CH), 30.06
(CH2 x2), 29.45 (CH2 x2), 21.49 (CH3);; MS (ESI):
C25H24FCl2N3O2 requires m/z 487.1, found 488.1 [M + 1]+;
Anal. calcd for C25H24FCl2N3O2: C, 61.48; H, 4.95; N, 8.60;
Found: C, 61.59; H, 4.94; N, 8.61. 3 (58 mg, 12%), white solid,
Rf = 0.50 (petroleum ether/EtOAc 85:15); mp 136-138 °C; IR
1
1690 (C=O), 3275 (NH); H NMR (400 MHz, Chloroform-d) δ
9.52 (d, J = 8.0 Hz, 1H, NH, exch. with D2O), 8.63 (s, 1H),
7.98 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H),
4.42 (d, J = 7.3 Hz, 1H), 4.17 (d, J = 7.4 Hz, 2H), 3.93 – 3.83
(m, 1H), 2.12 – 2.02 (m, 3H), 1.82 – 1.55 (m, 9H), 1.40 – 1.03
(m, 7H), 0.92 (d, J = 6.5 Hz, 3H); 13C NMR (101 MHz, CDCl3)
δ 160.12 (C), 142.49 (C), 134.23 (C), 134.00 (C), 133.53 (C),
132.97 (C), 130.98 (CH), 130.54 (CH), 129.69 (C), 127.82
(CH), 125.04 (CH), 58.99 (CH2), 49.06 (CH), 36.95 (CH),
33.80 (CH2 x2), 32.81 (CH2 x2), 31.94 (CH), 30.59 (CH2 x2),
26.23 (CH2), 25.62 (CH2 x2), 22.18 (CH3); MS (ESI):
C25H31Cl2N3O2 requires m/z 475.2, found 476.2 [M + 1]+; Anal.
calcd for C25H31Cl2N3O2: C, 63.02; H, 6.56; N, 8.82; Found: C,
63.10; H, 6.58; N, 8.83.
See the Supporting Information for compounds 9 - 11.
2-(5-Chloropentyl)-6-(3,4-dichlorophenyl)-cis-N-(4-methyl-
cyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxa-mide
(12) and 2-(5-chloropentyl)-6-(3,4-dichlorophenyl)-trans-
N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazi-ne-4-
carboxamide (13): General procedure A for the synthesis of
1
1677 (C=O), 3280 (NH); H NMR (400 MHz, Chloroform-d) δ
9.40 (d, J = 8.0 Hz, 1H, NH, exch. with D2O), 8.63 (s, 1H),
7.98 (s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H),
7.47 (t, J = 6.9 Hz, 2H), 7.04 (t, J = 8.5 Hz, 2H), 5.43 (s, 2H),
3.97 – 3.75 (m, 1H), 2.09 – 1.98 (m, 2H), 1.83 – 1.71 (m, 2H),
1.64 (s, 1H), 1.39 – 1.23 (m, 2H), 1.17 – 1.03 (m, 2H), 0.91 (d,
J = 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 162.93 (CF),
160.47 (CF), 159.39 (C), 158.61 (C), 142.18 (C), 133.23 (C),
132.96 (C), 132.58 (C), 130.06 (C), 130.02 (CH), 129.96 (C),
129.74 (CH), 129.66 (CH), 129.29 (CH), 126.86 (CH), 124.06
(CH), 114.82 (CH), 114.60 (CH), 54.79 (CH2), 48.10 (CH),
32.76 (CH2 x2), 31.76 (CH2 x2), 30.90 (CH), 21.16 (CH3); MS
(ESI): C25H24FCl2N3O2 requires m/z 487.1, found 488.1 [M +
1]+; Anal. calcd for C25H24FCl2N3O2: C, 61.48; H, 4.95; N,
8.60; Found: C, 61.55; H, 4.96; N, 8.59.
carboxamides was used to convert acid 47 and
4 -
methylcyclohexylamine into the title products. The mixture of
reaction was stirred for 18 h, then the cis/trans isomers were
separated by flash chromatography (petroleum ether/EtOAc
87:13). 12 (82 mg, 17%), colourless oil, Rf = 0.42 (petroleum
ether/EtOAc 85:15); IR 1678 (C=O), 3301 (NH); 1H NMR (400
MHz, Chloroform-d) δ 9.90 (d, J = 7.8 Hz, 1H, NH, exch with
D2O), 8.64 (s, 1H), 7.99 (s, 1H), 7.69 (dd, J = 8.4, 2.2 Hz, 1H),
7.55 (d, J = 8.4 Hz, 1H), 4.34 (t, J = 7.4 Hz, 2H), 4.28 – 4.21
(m, 1H), 3.47 (t, J = 7.0 Hz, 2H), 1.99 – 1.79 (m, 4H), 1.72 –
1.59 (m, 9H), 1.56 – 1.44 (m, 2H), 0.97 (d, J = 6.5 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 159.64 (C), 158.90 (C), 141.75
(C), 133.20 (C), 133.00 (C), 132.53 (C), 129.97 (CH), 129.51
(CH), 128.79 (C), 126.81 (CH), 124.00 (CH), 69.25 (CH2)
51.94 (CH2), 44.80 (CH), 30.12 (CH), 29.06 (CH2 x2), 28.53
(CH2 x2), 28.39 (CH2), 27.31 (CH2), 22.41 (CH2), 20.56 (CH3);
MS (ESI): C23H28Cl3N3O2 requires m/z 483.1, found 484.1 [M
+ 1]+; Anal. calcd for C23H28Cl3N3O2: C, 56.99; H, 5.80; N,
8.64; Found: C, 56.90; H, 5.82; N, 8.67. 13 (48 mg, 10%),
colourless oil, Rf = 0.53 (petroleum ether/EtOAc 85:15); IR
See the Supporting Information for compounds 4 - 6.
2-(Cyclohexylmethyl)-6-(3,4-dichlorophenyl)-cis-N-(4-me-
thylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxa-
mide (7) and 2-(cyclohexylmethyl)-6-(3,4-dichlorophenyl)-
trans-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazi-
ne-4-carboxamide (8): General procedure
A for the
1
synthesis of carboxamides was used to convert acid 46 and 4
-methylcyclohexylamine into the title products. The mixture of
reaction was stirred for 18 h, then the cis/trans isomers were
separated by flash chromatography (petroleum ether/EtOAc
1673 (C=O), 3298 (NH); H NMR (400 MHz, Chloroform-d) δ
9.52 (d, J = 8.0 Hz, 1H, NH, exch. with D2O), 8.63 (d, J = 5.3
Hz, 1H), 7.99 (s, 1H), 7.69 – 7.67 (m, 1H), 7.46 – 7.40 (m,
1H), 4.33 (dd, J = 13.1, 5.7 Hz, 2H), 3.94 – 3.83 (m, 1H), 3.51
10
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10.1002/cmdc.201800152
ChemMedChem
FULL PAPER
– 3.40 (m, 2H), 2.09 – 1.88 (m, 4H), 1.80 – 1.72 (m, 2H), 1.70
– 1.58 (m, 6H), 1.35 – 1.23 (m, 3H), 0.91 (d, J = 6.5 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 159.44 (C), 158.92 (C), 141.68
(C), 133.11 (C), 133.05 (C), 132.22 (C), 129.79 (CH), 129.22
(CH), 128.80 (C), 126.69 (CH), 124.03 (CH), 69.26 (CH2),
51.90 (CH2), 42.72 (CH), 29.00 (CH2 x2), 28.30 (CH2 x2),
28.25 (CH), 28.09 (CH2), 27.33 (CH2), 22.42 (CH2), 13.52
(CH3); MS (ESI): C23H28Cl3N3O2 requires m/z 483.1, found
484.2 [M + 1]+; Anal. calcd for C23H28Cl3N3O2: C, 56.98; H,
5.82; N, 8.67; Found: C, 57.05; H, 5.83; N, 8.65.
colourless oil. Rf = 0.22 (petroleum ether/EtOAc 95:5) IR
1683 (C=O), 3261 (NH); H NMR (400 MHz, Chloroform-d) δ
1
9.96 (d, J = 7.8 Hz, 1H, NH, exch. with D2O), 8.65 (s, 1H),
7.73 (s, 1H), 7.64 (d, J = 8.3, 2.3 Hz, 1H), 7.43 (d, J = 8.3 Hz,
1H), 4.33 (t, J = 7.6 Hz, 2H), 4.27 – 4.19 (m, 1H), 2.45 (s, 3H),
2.19 – 2.02 (m, 1H), 1.98 – 1.79 (m, 3H), 1.80 – 1.55 (m, 4H),
1.51 – 1.22 (m, 7H), 0.97 (d, J = 6.5 Hz, 3H), 0.92 (t, J = 6.3
Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 159.91 (C), 158.95 (C),
143.16 (C), 135.83 (C), 135.04 (C), 131.81 (C), 129.88 (CH),
128.67 (CH), 128.50 (C), 127.35 (CH), 123.61 (CH), 51.88
(CH2), 44.79 (CH), 30.10 (CH), 29.08 (CH2 x2), 28.51 (CH2
x2), 27.77 (CH2), 27.18 (CH2), 21.31 (CH2), 19.19 (CH3 x2),
12.94 (CH3); MS (ESI): C24H32ClN3O2 requires m/z 429.2,
found 430.1 [M + 1]+; Anal. calcd for C24H32ClN3O2: C, 67.04;
H, 7.50; N, 9.77; Found: C, 66.93; H, 7.48; N, 9.80.
See the Supporting Information for compounds 14 - 16.
6-(5-Bromothiophen-2-yl)-2-(4-fluorobenzyl)-cis-N-(4-me-
thylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxa-
mide (17) and 6-(5-bromothiophen-2-yl)-2-(4-fluoroben-
zyl)-trans-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyri-
dazine-4-carboxamide (18): General procedure B for the
synthesis of carboxamides was used to convert acid 48 and 4
-methylcyclohexylamine into the title products. The cis/trans
isomers were separated by flash chromatography (petroleum
ether/EtOAc 85:15). 17 (90 mg, 19%), yellow solid, Rf = 0.30
(petroleum ether/EtOAc 85:15); mp 158-159 °C; IR 1683
See the Supporting Information for compounds 23 and 24.
Biological assays
Radioligand binding assays at hCB1Rs and hCB2Rs:
[3H]CP-55,940 (specific activity, 180 Ci/mmol) and CHO cells
transfected with hCB1 and hCB2Rs were derived from Perkin-
Elmer Life and Analytical Sciences (U.S.). All other reagents
were obtained from Sigma (Milan, Italy). CHO cells
expressing CB1 and CB2Rs were cultured in Ham’s F12
additioned with 10% fetal bovine serum, penicillin (100 U/mL),
streptomycin (100 μg/mL), and Geneticin (G418, 0.4 mg/mL)
at 37 °C in 5% CO2/95% air.[49] Membranes from transfected
cells to be used in binding experiments were derived as
previously reported.[49] In brief, the cells were detached in ice-
cold hypotonic buffer (5 mM Tris-HCl, 2 mM EDTA, pH 7.4),
triturated with a Polytron and centrifuged for 10 min at 1000g.
Then the supernatant was centrifuged for 30 min at 100000g
and the membrane pellet was diluted in 50 mM Tris-HCl
buffer, 0.5% BSA (pH 7.4) containing 5 mM MgCl2, 2.5 mM
EDTA or 1 mM EDTA for hCB1R or hCB2R, respectively.
Competition binding experiments were carried out with
[3H]CP-55,940 in the presence of ligands under examination
in the range of concentrations 1 nM to 10 μM or WIN 55,212-
2, the CB1R/CB2R standard agonist. Incubations lasted 90 or
60 min at 30 °C for CB1 or CB2Rs, respectively. Bound and
free radioactivities were separated by filtering the assay
mixture through Whatman GF/C glass fiber filters using a
Brandel cell harvester (Brandel Instruments, Unterfꢀhring,
Germany). The filter bound radioactivity was counted on a
Perkin-Elmer 2810 TR scintillation counter (Perkin-Elmer Life
and Analytical Sciences, U.S.).
1
(C=O), 3308 (NH); H NMR (400 MHz, Chloroform-d) δ 9.81
(d, J = 7.7 Hz, 1H, NH, exch with D2O), 8.50 (s, 1H), 7.52 –
7.44 (m, 2H), 7.23 (d, J = 4.0 Hz, 1H), 7.04 (t, J = 6.5 Hz, 3H),
5.36 (s, 2H), 4.33 – 4.13 (m, 1H), 1.86 – 1.76 (m, 2H), 1.70 –
1.56 (m, 4H), 1.32 – 1.20 (m, 3H), 0.97 (d, J = 6.5 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 162.94 (CF), 160.48 (CF),
159.32 (C), 158.48 (C), 140.00 (C), 138.99 (C), 130.07 (C),
129.98 (CH), 129.91 (CH), 129.90 (CH), 129.21 (C), 128.90
(CH), 125.42 (CH), 115.07 (C), 114.73 (CH), 114.52 (CH),
53.96 (CH2), 44.94 (CH), 29.96 (CH), 29.04 (CH2 x2), 28.42
(CH2 x2), 20.48 (CH3); MS (ESI): C23H23BrFN3O2S requires
m/z 503.1, found 504.2 [M
+
1]+; Anal. calcd for
C23H23BrFN3O2S: C, 54.77; H, 4.60; N, 8.33; Found: C, 54.88;
H, 4.61; N, 8.34. 18 ( 55 mg, 11%), yellow solid, Rf = 0.48
(petroleum ether/EtOAc 85:15); mp 140-412 °C; IR 1681
1
(C=O), 3305 (NH); H NMR (400 MHz, Chloroform-d) δ 9.42
(d, J = 8.1 Hz, 1H, NH, exch with D2O), 8.50 (s, 1H), 7.51 –
7.43 (m, 2H), 7.24 (d, J = 4.0 Hz, 1H), 7.09 – 7.00 (m, 3H),
5.34 (s, 2H), 3.94 – 3.75 (m, 1H), 2.07 – 2.00 (m, 2H), 1.80 –
1.71 (m, 2H), 1.62 – 1.58 (m, 1H), 1.41 – 1.23 (m, 2H), 1.15 –
1.01 (m, 2H), 0.91 (d, J = 6.6 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 162.93 (CF), 160.47 (CF), 159.32 (C), 158.38 (C),
140.06 (C), 138.94 (C), 129.98 (C), 129.92 (CH), 129.85 (CH),
129.77 (CH), 129.15 (C), 129.07 (CH), 125.47 (CH), 115.12
(C), 114.77 (CH), 114.55 (CH), 54.23 (CH2), 48.09 (CH),
32.76 (CH2 x2), 31.74 (CH2 x2), 30.89 (CH), 21.16 (CH3); MS
(ESI): C23H23BrFN3O2S requires m/z 503.1, found 504.2 [M +
1]+; Anal. calcd for C23H23BrFN3O2S: C, 54.77; H, 4.60; N,
8.33; Found: C, 54.85; H, 4.59; N, 8.34.
cAMP assays for hCB2Rs: CHO cells expressing hCB2Rs
were
preincubated
with
0.5
mM
4-(3-butoxy-4-
methoxybenzyl)-2-imidazolidinone (Ro 20-1724), as
a
phosphodiesterase inhibitor. Then the effect of a selection of
compounds characterized by high affinity and selectivity
towards CB2Rs was tested at increasing concentrations
(0.001-1 μM) with or without WIN 55,212-2 (0.1 μM) in the
presence of forskolin (5 μM), by using the fluorimetric cAMP
kit (AlphaScreen cAMP assay kit, from Perkin Elmer)
according to the manufacturer’s instructions.
See the Supporting Information for compounds 19 - 21.
6-(4-Chloro-3-methylphenyl)-cis-N-(4-methylcyclohexyl)-
3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamide (22):
General procedure A for the synthesis of carboxamides was
used to convert acid 44 and 4-methylcycloheylamine into the
title product. The mixture of reaction was stirred for 18 h, then
the crude of reaction was purified by flash chromatography
(petroleum ether/EtOAc 95:5) to afford 22 (98 mg, 23%) as a
11
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10.1002/cmdc.201800152
ChemMedChem
FULL PAPER
Data analysis: The protein concentration was measured by a
Bio-Rad method[50] with bovine albumin as reference standard.
Inhibitory binding constants, Ki, were obtained from the IC50
values following the Cheng and Prusoff equation: Ki = IC50/(1
+ [C]/KD), where [C] is the concentration of the radioligand
of TMH3-4-5. The initial CB2R homology model was refined
by calculating the low free energy conformations for any TMH
with an important sequence divergence from rhodopsin and
replacing the corresponding helix from the initial model with
one that more accurately reflects the sequence dictated TMH
geometries in CB2R. This includes TMH2 (GG helix distorting
motif in Rho vs no Pro or GG in CB2R) and TMH5 (Pro at
5.50 in Rho vs no Pro at 5.50 in CB2R). The resultant CB2R
model has been tested using results from substituted cysteine
accessibility studies to identify binding pocket facing
residues,[43,44] from mutation studies of key ligand interactions
sites,[43-46] and from covalent labeling studies of CB2R that
support a lipid entry pathway for CB2R ligands.[47] To permit
adjustment to a lipid bilayer environment, the resultant model
and KD its dissociation constant (Cheng Prusoff).[22]
A
weighted nonlinear least-squares curve fitting program,
LIGAND, was used for computer analysis of the inhibition
experiments.[51] All the data are presented as the mean ±
SEM of n = 4 independent experiments. Statistical analysis of
the data was done using ANOVA followed by Dunnett’s test.
Computational studies
was
pre-equilibrated
in
a
stearoyl-
Conformational analysis: The Ballesteros−Weinstein
numbering system for GPCR amino acid residues is used. In
this numbering system, the label 0.50 is assigned to the most
docosahexaenoylphosphatidylcholine (SDPC) bilayer for 300
ns.[25] While the toggle switch residue, W6.48(258), remained
in its inactive state g+ χ1 dihedral angle after the equilibration
in SDPC, some notable changes did occur during this
equilibration. The R3.50(131) and D6.30(240) salt bridge at
the intracellular ends of TMHs3/6 (analogous to the
R3.50(135)/ E6.30(247) salt bridge in the dark state of
rhodopsin) rearranged quickly to form a salt bridge between
R3.55(136) and D6.30(240), with Y3.51(132) supporting the
salt bridge by hydrogen bonding to the exposed backbone
carbonyl of L6.29(239). A second notable change was the
development of additional helical turns in the IC-3 (TMH5-
TMH6) loop after the original end of TMH5.[25]
highly conserved residue in Class
A GPCRs in each
transmembrane helix (TMH).[41] This is preceeded by the TMH
number. In this system, for example, the most highly
conserved residue in TMH6 is P6.50. The residue
immediately before this would be labeled 6.49, and the
residue immediately after this would be labeled 6.51. When
referring
to
a
specific
CB2R
residue,
the
Ballesteros−Weinstein name is followed by the absolute
sequence number given in parentheses (e.g., K3.28(109));
however, when referring to a highly conserved residue among
Class A GPCRs (and not a specific residue in CB2R), only the
Ballesteros−Weinstein name is given.
Docking Study in the CB2R Inactive State Model:
Compound 22 was docked into the SR144528/CB2R binding
site previously identified by Glide docking studies.[23,24,26]
Compound 22 was flexibly docked using Glide (version 7.5,
Schrödinger, LLC, New York, NY) and the dock with the best
Glide score, as ranked by Emodel, was chosen. Glide was
used to generate a grid based on the centroid of select
residues in the binding pocket. The box size was set to the
default value of 24 x 24 x 24 Å, with the inner box size set to
10 x 10 x 10 Å. This box size encompasses the entire CB2R
binding pocket both in width and depth. No scaling of VdW
radii was used. S7.39(285) was defined to be a required
interaction during the docking procedure, by analogy to
mutation literature for HU243 at CB2R.[48] Extra precision (XP)
and flexible docking with 50K maximum initial poses to be
retained for the initial phase of docking were selected for the
docking setup. During docking only trans amides were
allowed, aromatic hydrogens were set as donors and
halogens as acceptors. After docking, a 800 step conjugate
gradient minimization was performed on the ligand pose by
Glide using OPLS3 (distance dependent dielectric with a base
constant of 2). The resulting ligand/receptor complex with the
best Glide score was minimized using the OPLS3 all-atom
force field in Macromodel. An 8.0 Å nonbonded cutoff
(updated every 10 steps), a 20.0 Å electrostatic cutoff, and a
4.0 Å hydrogen bond cutoff were used in each stage of the
calculation. The first stage consisted of 4000 steps of
A complete conformational analysis of 22 was performed
using the Mixed Torsional/Low-mode Sampling technique as
encoded in Macromodel (version 11.6, Schrödinger, LLC,
New York, NY). Torsional moves of 30° or greater were set
for all rotatable bonds within 22, including full exploration of
the 4-cis cyclohexyl ring. The search was comprised of 20K
Monte Carlo steps using the OPLS3 force field with a
distance dependent dielectric and a dielectric base constant
of 2. An 8.0 Å non-bonded cutoff (updated every 10 steps), a
20.0 Å electrostatic cutoff, and a 4.0 Å hydrogen bond cutoff
were applied in the minimization of each conformation to a
gradient of 0.01 kcal/mol·Å. Duplicate conformations
identified by an RMSD ≤ 0.7 were removed. To calculate the
energy difference between the global minimal energy
conformer of 22 and its final docked conformation, the single
point energy of each was calculated in OPLS3 and the
difference was calculated.
Model Development: Complete details on the generation of
the CB2R inactive state model used here are available in our
previous publication.[25] Briefly, the crystal structure of the
Class A GPCR, rhodopsin in the dark state, was used as the
template for the creation of our CB2R inactive state model.[42]
This template was chosen because no mutations or
modifications were made to its structure for crystallization and
there are no structural distortions due to crystal packing for
this structure. In addition, the cannabinoid receptors and
rhodopsin share some unusual sequence motifs. For example,
Polak−Ribier conjugate gradient minimization using
a
distance-dependent dielectric function with a base constant of
2. No harmonic constraints were placed on the side chains,
but 100 kJ/mol torsional constraints were applied to hold all
the backbone ϕ/ψ torsion angles. During the second stage of
these receptors share
a TMH4 GWNC motif at their
extracellular ends. Here a Trp residue forms an aromatic
stacking interaction with Y5.39, influencing the EC positions
12
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10.1002/cmdc.201800152
ChemMedChem
FULL PAPER
[3]
[4]
R. G. Pertwee, R. A. Ross, Prostaglandins Leukot. Essent. Fatty
Acids 2002, 66, 101-121.
1000 steps, all torsional constraints were released. To relax
the loops, an additional 1000-step Polak−Ribier conjugate
gradient minimization of the loop regions was performed. The
loop and termini regions were left free, while the
transmembrane regions were not allowed to move during this
final minimization. An 8.0 Å extended nonbonded cutoff
(updated every 10 steps), 20.0 Å electrostatic cutoff, and 4.0
Å hydrogen bond cutoff were used in this calculation, and the
generalized Born/surface area (GB/SA) continuum solvation
model for water available in Macromodel was employed.
M. Maccarrone, I. Bab, T. Bíró, G.A. Cabral, S. K. Dey, V. Di Marzo,
J. C. Konje, G. Kunos, R. Mechoulam, P. Pacher, K. A. Sharkey, A.
Zimmer, Trends Pharmacol. Sci. 2015, 36, 277-296.
R. G. Pertwee, Int. J. Obes. 2006, 30, S13–S18.
D. Piomelli, Nat. Rev. Neurosci. 2003, 4, 873-884.
A. B. Lynn, M. Herkenham, J. Pharmacol. Exp. Ther. 1994, 268,
1612-1623.
[5]
[6]
[7]
[8]
[9]
R. G. Pertwee, Br. J. Pharmacol. 2009, 156, 397-411.
I. Svízenská, P. Dubový, A. Sulcová, Pharmacol. Biochem. Behav.
2008, 90, 501–511.
[10] G. Navarro, P. Morales, C. Rodriguez-Cueto, J. Fernandez-Ruiz, N.
Jagerovic, R. Franco, Front. Neurosci. 2016, 10, 406.
[11] V. Di Marzo, M. Bifulco, L. De Petrocellis, Nat. Rev. Drug Discov.
2004, 3, 771-784.
Assessment of Pairwise Interaction Energies: After the
atoms of 22 had been defined as one group (group 1) and the
atoms corresponding to a residue that lines the binding site in
the final ligand/CB2R complex as another group (group 2),
Macromodel was used to output the pairwise interaction
energy (Coulombic and van der Waals) for a given pair of
atoms. The pairs corresponding to group 1 (ligand) and group
2 (residue of interest) were then summed to yield the energy
of interaction between the ligand and that residue. The
22/CB2R complex was found to have interaction energies
totaling to −66.59 kcal/mol. Taking the conformational energy
cost for 22 (0.89 kcal/mol) in the final complex into account,
we found the final total energy for the 22/CB2R complex to be
−65.70 kcal/mol. A breakdown of interaction energies is
provided in Table S1 in Supplementary Material.
[12] D. R. Compton, K. C. Rice, B. R. De Costa, R. K. Razdan, L. S.
Melvin, M. R. Johnson, B. R. Martin, J. Pharmacol. Exp. Ther. 1993,
265, 218-226.
[13] T. Bisogno, V. Di Marzo, CNS Neurol. Disord. Drug Targets 2010, 9,
564-573.
[14] J. L. Shoemaker, K. A. Seely, R. L. Reed, J. P. Crown, P. L. Prather,
J. Neurochem. 2007, 101, 87-98.
[15] M. A. Tabrizi, P. G. Baraldi, P. A. Borea, K. Varani, Chem. Rev.
2016, 116, 519-560.
[16] F. Vincenzi, M. Targa, C. Corciulo, M. A. Tabrizi, S. Merighi, S.
Gessi, G. Saponaro, P. G. Baraldi, P. A. Borea, K. Varani, Pain
2013, 154, 864-873.
[17] A. Quartilho, H. Mata, M. Ibrahim, T. Vanderah, P. Porreca, A.
Makriyannis, T. Malan, Anesthesiology 2003, 99, 955-960.
[18] C. Blazquez, L. Gonzalez-Feria, L. Alvarez, A. Haro, M. L.
Casanova, M. Guzmán, Cancer Res. 2004, 64, 5617-5623.
[19] S. Pisanti, P. Picardi, A. D'Alessandro, C. Laezza, M. Bifulco,
Trends Pharmacol. Sci. 2013, 34, 273-282.
In Silico ADMET Calculations: A set of 34 physicochemical
descriptors were computed using QikProp version 3.5 integrated in
Maestro (Schrꢀdinger, LLC, NY 2016). The QikProp descriptors
are shown in Table 2. The 3D conformations used in the calculation
of QikProp descriptors were generated using the OPLS3 force field
and the Mixed Torsional Low Mode Sampling Monte Carlo
Conformational Search method in Macromodel 11.3 (Schrꢀdinger,
LLC, NY 2016). Possible tautomerization and ionization states were
calculated using Epik integrated in the Maestro software. No
[20] P. Morales, L. Hernandez-Folgado, P. Goya, N. Jagerovic, Expert
Opin. Ther. Pat. 2016, 26, 843–856
[21] G. Ragusa, M. Gómez-Cañas, P. Morales, C. Rodríguez-Cueto, M.
R. Pazos, B. Asproni, E. Cichero, P. Fossa, G. A. Pinna, N.
Jagerovic, J. Fernández-Ruiz, G. Murineddu, Eur. J. Med. Chem.
2017, 127, 398-412.
[22] Y. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099-
3108.
tautomer was found for pyridazinones
. Toxicity and
metabolism predictions were performed by using the admetSAR
prediction tool (http://lmmd.ecust.edu.cn/admetsar1/).[34] Tables S2
and S3 summarize the parameters calculated, classifying the
compounds according to previously reported methods (see
Supplementary Material).
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Acknowledgements
This work was supported by NIH grants RO1 DA003934 and
KO5 DA021358 (PHR).
[26] G. Ragusa, M. Gómez-Cañas, P. Morales, D. P. Hurst, F. Deligia, R.
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Conflict of interest
[28] J. M. Sanders, D. C. Beshore, J. C. Culberson, J. I. Fells, J. E.
Imbriglio, H. Gunaydin, A. M. Haidle, M. Labroli, B. E. Mattioni, N.
Sciammetta, W. D. Shipe, R. P. Sheridan, L. M. Suen, A. Verras, A.
M. Walji, E. M. Joshi, T. Bueters, J. Med. Chem. 2017, 60, 6771-
6780.
None of the authors have conflict of interest to declare
Keywords: synthesis • cannabinoid receptors • CB2 inverse
agonism • docking studies • ADMET calculations
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Table of Contents
The modulation of the 6-aryl-3-oxo-2,3-dihydropyridazine-4-carboxamide scaffold in three different positions, the N2 chain, C4
carboxamide moiety and C6-aryl ring, led us to determine the key features of this novel class of CB2R ligands. Derivative 22, a very
potent and selective CB2R inverse agonist, has been identified with favorable properties that indicate this compound could be a lead
for drug development.
15
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