Developing pyridazine-3-carboxamides to be CB2 agonists: The design, synthesis, structure-activity relationships and docking studies

Article abstract of DOI:10.1016/j.ejmech.2017.05.060

Herein, we described the design and synthesis of a series of pyridazine-3-carboxamides to be CB2-selective agonists via a combination of scaffold hopping and bioisosterism strategies. The compounds were subjected to assessment of their potential activities through calcium mobilization assays. Among the tested derivatives, more than half of these compounds exhibited moderate to potent CB2 agonist activity. Six compounds showed EC₅₀ values below 35 nM, and several derivatives also exhibited significantly enhanced potency and high selectivity at the CB2 receptor over the CB1 receptor. Specifically, compound 26 showed the highest CB2 agonist activity (EC₅₀ = 3.665 ± 0.553 nM) and remarkable selectivity (Selectivity Index > 2729) against CB1. In addition, logPs of some representative compounds were measured to display significantly decreased values in comparison with GW842166X. Furthermore, docking simulations were conducted to explain the interaction mode of this series.

Full text of DOI:10.1016/j.ejmech.2017.05.060

Accepted Manuscript
Developing pyridazine-3-carboxamides to be CB2 agonists: The design, synthesis,
structure-activity relationships and docking studies
Hai-Yan Qian, Zhi-Long Wang, Xiao-Yu Xie, You-Lu Pan, Gang-Jian Li, Xin Xie, Jian-
Zhong Chen
PII:
S0223-5234(17)30431-2
10.1016/j.ejmech.2017.05.060
EJMECH 9490
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 March 2017
Revised Date: 26 May 2017
Accepted Date: 29 May 2017
Please cite this article as: H.-Y. Qian, Z.-L. Wang, X.-Y. Xie, Y.-L. Pan, G.-J. Li, X. Xie, J.-Z. Chen,
Developing pyridazine-3-carboxamides to be CB2 agonists: The design, synthesis, structure-activity
relationships and docking studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.060.
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ACCEPTED MANUSCRIPT
Novel pyridazine-3-carboxamide derivatives have been synthesized and biologically
evaluated. The results show that the compound 26 has a good agonist activity and high
selectivity for CB2. Docking simulations were performed to predict the receptor-agonist
interactions.

ACCEPTED MANUSCRIPT
Developing pyridazine-3-carboxamides to be CB2 agonists: The design, synthesis,
structure-activity relationships and docking studies
a, §
b, §
a
a
a
b,
Hai-Yan Qian , Zhi-Long Wang , Xiao-Yu Xie , You-Lu Pan , Gang-Jian Li , Xin Xie *,
a,
Jian-Zhong Chen *
a
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058,
China
b
CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
§
These authors contributed equally.
Corresponding Authors:
*
E-mail: xxie@simm.ac.cn, Tel.(Fax): 86-21-50801313 (X. Xie)
E-mail: chjz@zju.edu.cn, Tel.(Fax): 86-571-88208659 (J.-Z. Chen)
1

ACCEPTED MANUSCRIPT
ABSTRACT
Herein, we described the design and synthesis of a series of pyridazine-3-carboxamides
to be CB2-selective agonists via a combination of scaffold hopping and bioisosterism
strategies. The compounds were subjected to assessment of their potential activities through
calcium mobilization assays. Among the tested derivatives, more than half of these
compounds exhibited moderate to potent CB2 agonist activity. Six compounds showed EC50
values below 35 nM, and several derivatives also exhibited significantly enhanced potency
and high selectivity at the CB2 receptor over the CB1 receptor. Specifically, compound 26
showed the highest CB2 agonist activity (EC50 = 3.665 ± 0.553 nM) and remarkable
selectivity (Selectivity Index > 2729) against CB1. In addition, logPs of some representative
compounds were measured to display significantly decreased values in comparison with
GW842166X. Furthermore, docking simulations were conducted to explain the interaction
mode of this series.
Keywords:
Pyridazine-3-carboxamides,CB2 agonist, structure-activity relationships, molecular docking,
logP
2

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1
. Introduction
The endocannabinoid system is composed of a group of lipid-based mediators, relative
targets involved in a variety of synthesizing and metabolizing enzymes, and transporter
receptors [1]. There are two G-protein coupled receptors (GPCRs), cannabinoid receptor 1
(CB1) [2] and cannabinoid receptor 2 (CB2) [3], in the endocannabinoid system. These two
GPCRs share sequence homology of 44% identity at the protein level and 68% similarity in
the seven transmembrane domains [4]. CB1, expressed mainly in the central nervous system
(CNS) [5], has been reported to mitigate numerous pathologies [4, 6], and its ligands have
been proposed for therapeutic use [7]. However, psychotropic side effects induced by the
CB1 ligands limit their pharmaceutical applications. For example, CB1 antagonist
SR141716A (1, Figure 1) was approved to be an anti-obesity agent in 2006, but withdrawn
from European market two years later for its serious psychopathic side effects related to the
inhibition of CB1 in the brain. After that, other CB1 antagonists, like MK-0364 (Taranabant)
and CP-945598 (Otenabant), were stopped in the clinical phase studies for the same reason
[
8]. On the other hand, CB2 is found predominantly in the periphery [9], particularly in the
immune and skeletal systems [10, 11]. Pharmacological studies have demonstrated that CB2
ligands exhibit marvelously therapeutic potentials in the treatment of different pathologies,
including immune-related disorders [12], pain [13], inflammation [14], osteoporosis [15],
neuro-degeneration [16], and cancer [17]. Thus, CB2 selective ligands catch more attention in
the recent years since they avoid the CNS influence by taking effects in the peripheral
system.
3

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Up to now, there were a large number of CB2 ligands reported by pharmaceutical
companies or academic research laboratories [18-20]. As illustrated in Figure 1, some
representatives of CB2 agonists were developed based on traditional cannabinoids, such as
compounds 2 (HU-308) [21], 3 (GW405833) [22], 4 (JWH-015) [23], and 5 (AM1241) [24],
while the pyrazole derivative 6 (SR144528) is a prototypical CB2 antagonist. Besides,
structurally diverse compounds were also discovered to be CB2 ligands, like the CB2 inverse
agonist 7 (JTE-907) [25] displaying to be effective in suppressing itch-associated responses
in NC mice of atopic dermatitis without the adverse effect on the losing weight and
exacerbation of dermatitis [26]. Encouragingly, there were some CB2 ligands getting into
clinic trail studies [17]. For example, the CB2 agonist 8 (GW842166X, Figure 1) [27] has
completed Phase II clinical trials for evaluating its analgesic efficacy in the treatment of
osteoarthritic and dental pain [28]. Unfortunately, clinical trials of GW842166X failed to
achieve satisfactory e� ects. In addition, CB2 agonist 9 (S-777469, Figure 1) was studied to
exhibit moderate bioactivity and excellent selectivity for the CB2 receptor [29].
Pharmacological studies suggested that S-777469 had a significant antipruritic efficiency in
the pruritic animal model, and displayed standout metabolism and pharmacokinetic properties
[
30]. It has also been reported on completing its phase II clinical trials for allergic contact
dermatitis [31]. However, there is no relevant CB2 ligand approved for market. One of
reasons is probably their relatively high lipophilicity to induce inadequate drug-likeness.. The
high lipophilicity of CB2 ligands may be a key factor for CB2 ligands not showing optimal
pharmacokinetic profile, like high binding to plasma proteins, long half-life, low
bioavailability and off-target effects [32]. As we measured, logP value of GW842166X was
4

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4
.27 [33]. The logP was recognized to be the important drug-likeness property for a bioactive
compound to be developed as therapeutic agent. Reducing logP could be a strategy for CB2
ligands to have desired pharmacokinetic properties [34].
In the previous reports, GW842166X (Figure 1) was demonstrated to possess high potency,
efficacy, and selectivity due to low molecular weight and polar surface area, despite its low
aqueous solubility [35]. The aforementioned facts motivated us to investigate new aromatic
core with improved aqueous solubility. In this context, bioisosterism strategy was applied for
designing pyridazine-3-carboxamides as CB2 agonists by replacing the central pyrimidine
core of GW842166X with a pyridazine nucleus, since the basic pK of pyridazine (2.33) is
a
higher than pyrimidine (1.3) and pyridazine showed measurable aqueous solubility, which
could be improved through salt formation [36]. Based on critical pharmacophore features
predicted by our docking-simulated interaction mode of GW842166X binding to the CB2
receptor (Figure S1 in the Supporting Information), we designed and synthesized a series of
pyridazine-3-carboxamides (general structure A, Figure 1). The structural pieces suggested
good alignment with pharmacophoric features of GW842166X. The cell-based calcium
mobilization assays [37] were used to evaluate in vitro functional activities of synthesized
pyridazine-3-carboxamide derivatives. Structure-activity relationships (SARs) studies were
mainly performed on the substituent (R ) on the pyridazine nucleus, the C-3 amide groups
1
(
R ), and the C-6 substituent group (R ). The bioactive tests indicated that the synthesized
2
3
compounds displayed good agonist activity towards CB2 and high selectivity against CB1,
and some compounds showed much higher potency in relative to GW842166X. Finally,
molecular docking simulations were carried out with the aim of understanding the structural
5

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requirements for new derivatives binding to the CB2 receptor.
2
. Chemistry
The synthetic route for the target pyridazine-3-carboxamide derivatives 21-55 was depicted
1
in Scheme 1. The structures of pyridazine-3-carboxamide derivatives were confirmed by H
1
3
NMR, C NMR spectrum, and mass spectrometry.
As shown in Scheme 1, the commercially available material α-ketoglutaric acid 10 was
converted into the 6-oxo-1,4,5,6-tetrahydropyridazine-3-carboxylic acid 11 upon treatment
with hydrazine sulfate in the presence of sodium hydroxide in water. In the next step,
acid-catalyzed enol formation, bromination, and elimination of HBr, provided
6
-oxo-1,6-dihydropyridazine-3-carboxylic acid 12 [38]. Analogously to the reported
procedures [39, 40], 12 was subsequently submitted to chlorination using thionyl chloride to
give the corresponding acid chloride 13, and then reacted with diverse primary amines to
afford the amide analogs 14a-14c. Subsequent nucleophilic substitution of the key
intermediates 14a-14c with morpholine provided the final pyridazine-3-carboxamide
derivatives 21-23.
On the other hand, treatment of the intermediate 12 by refluxing in ethanol of hydrogen
chloride led to the ethyl 6-oxo-1,6-dihydropyridazine-3-carboxylate 15, and which was
subsequently reacted with phosphorus oxychloride to yield the ethyl 6-chloropyridazine-
3
-carboxylate 16 [41]. Successively, compound 16 was then subjected to Minisci Reaction
with homolytic alkylation by free isopropyl radical, generated by silver-catalyzed oxidative
decarboxylation of isobutyric acid, and led to a mixture (ca 1:10 regioisomeric ratio) of
regioisomers 17a and 17b that were separated by flash chromatography on silica gel column
6

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[
42]. The two regioisomers 17a and 17b were identified on the basis of the chemical shifts of
the aromatic protons (shown in the Supporting Information). Furthermore, it has been
reported that similar pyridazines with a carbonitrile group instead of the ethyl ester function
reacted with pivalic acid under the same conditions to obtained a 3:7 mixture of two
regioisomers, and the major component confirmed by X-ray analysis, having the same
regiochemistry of our synthesized intermediate 17b [43]. Compounds 17a and 17b afforded
the corresponding carboxylic acids 18a and 18b, respectively, through the alkaline hydrolysis
with lithium hydroxide in good overall yields. The key intermediates 20a-20f were obtained
according to the above procedures that were utilized for preparing the amide analogs 14a-14c,
by refluxing the newly formed 18a-18b with thionyl chloride, and then distilling off the
excess reagent to afford the crude acid chlorides 19a-19b. Afterwards, treatment of 19a-19b
with the suitable primary amines in the presence of triethylamine eventually gave the amide
derivatives 20a-20f in moderate to good yield. Finally, the targeted compounds 24-53 were
produced via the reaction between the compounds 20a-20f and the aromatic amines in
acetonitrile heated to 160 ºC [44] or the aliphatic amines in 1,4-dioxane at reflux temperature
[
39].
3
. Results and discussion
3
.1. In vitro biological evaluations
All the synthesized compounds were evaluated in vitro for their functional activities by
using our developed cell-based calcium mobilization assays [37]. Both GW842166X and
JTE-907 were included in the experiments as the reference standards of CB2 agonist and
7

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antagonist, respectively. Two commercially available ligands, CP55940 and SR141716A
were used as positive controls for agonist mode and antagonist mode, respectively, in the
bioactive assays. At first, all compounds were screened for their functional effects towards
CB1 and CB2 at 10 µM concentration, respectively, using the method described in the
Supporting Information, and the results are summarized in Table S1. The initial screening
results indicated that most compounds demonstrated activating bioactivity to CB2. Those
compounds displaying more than 50% activation activity were further evaluated in
dose-response studies to determine their EC50 values. As summarized in Tables 1-3, more
than half of the tested compounds exhibited moderate to potent CB2 full agonist activities.
Six compounds showed EC values below 35 nM for the CB2 receptor with high selectivity
50
against CB1. Typically, compound 26 demonstrates a full agonist activity with the EC value
5
0
of 3.665 ± 0.553 nM, which is 94-fold and 8-fold more potent than GW842166X and
CP55940, respectively. Besides, other four compounds 36 (EC50 = 5.701 ± 1.993 nM), 38
(
EC = 4.955 ± 0.633 nM), 39 (EC = 18.08 ± 2.89 nM), and 42 (EC = 17.13 ± 1.59 nM)
50 50 50
display higher CB2 agonist activities and have much higher selectivity over the CB1 receptor
than both GW842166X and CP55940. Moreover, it was noticed that all these potent CB2
agonists have clogD values, calculated by using ACD/ADME Suite software [45] less than 5
and exhibit remarkably improved solubility in comparison with GW842166X.
The logS for pyridazine-3-carboxamide derivatives 21-55 along with GW842166X and
CP55940 were calculated using ACD/ADME Suite software [45] to predict their aqueous
solubility. As listed in Tables 1-3, the predicted results reveal that all our synthesized
compounds possess higher calculated logSPH7.4 (-1.95 ~ -4.95) compared with GW842166X
8

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(clogSPH7.4 = -5.65) and CP55940 (clogSPH7.4 = -5.27), suggesting that these newly
pyridazine-3-carboxamide derivatives displayed substantially improved aqueous solubility.
The logP is an important drug-likeness property, and appropriate logP for a drug
candidate was proposed to be 1 ~ 3 [46]. In order to position drug-like physicochemical
properties of the synthesized compounds, four representative compounds 26, 38, 39, and 42
were selected to measure their logP values using the methods described in the previous
manuscript [33]. As results, these four compounds display their logP values to be 2.41, 2.01,
3
.40, and 3.16, respectively, decreasing significantly in comparison with GW842166X with
the measured logP value of 4.27 [33]. By structural optimization, we developed pyridazine-3-
carboxamides to be CB2-selective agonists, not only showing high potency on CB2 and
selectivity against CB1 but also having appropriate logP.
3
.2. Structure-activity relationships
We started structure-activity relationships studies by the synthesis of a series of derivatives
with various substitutions on pyridazine-3-carboxamide scaffold to investigate the impact of
this variable on their activities. Among three compounds 21-23 without a substituent (R ) on
1
the C4- or C5-position, 21 and 22 were detected to have no bioactivity on neither CB1 nor
CB2, while compound 23 showed a low CB2 agonist activity (EC = 8425 ± 4183 nM). By
5
0
comparing the bioactivities of compounds without a substituent (21, 22, 23) and compounds
with a R 4-isopropyl (24, 25, 26) or 5-isopropyl (27, 28, 29), it was found that a substituent
1
at the C-4 or C-5 site of pyridazine-3-carboxamide scaffold would be a desirable feature for
improving the CB2 agonist activity. In the meantime, it was noticed that 24-26 bearing a R1
9

ACCEPTED MANUSCRIPT
4
-isopropyl group displayed higher bioactivities than 5-isopropyl analogs 27-29. These
results revealed the crucial role of R 4-isopropyl group for pyridazine-3-carboxamides to
1
interact with CB2. By comparing the binding modes of our synthesized pyridazine-3-
carboxamides with GW842166X, it could be proposed that the pyridazine core may be
embraced by hydrophobic residues V113, F117, F177, W258, and V261 of the CB2 receptor.
In addition, structural alignments indicated that the R1 4-isopropyl group of
pyridazine-3-carboxamides may superimpose with the 4-CF of GW842166X. Therefore, a
3
proper hydrophobic substituent on the 4-position of pyridazine core would be benefit to have
hydrophobic interactions with the CB2 receptor. Typically, 26 with R 4-isopropyl, R2
1
2
0
-adamantyl, and R morpholino ring displays the highest CB2 bioactivity (EC = 3.665 ±
3 50
.553 nM) and selectivity against CB1 among all of the synthesized pyridazine-3-
carboxamides.
Moreover, it was predicted that the amide moiety of GW842166X might lie deeply in a
hydrophobic sub-pocket embraced by the residues F91, F94, F106, K109, and I110 (Figure
S1 in the Supporting Information), so a modest steric hindrance adjacent to the amide would
be a preferred building block for the CB2 agonist activity of pyridazine-3-carboxamides. This
hypothesis was confirmed by the replacement of the R cyclohexyl subunit with an adamantyl
2
group, which had a significant impact on the CB2 agonist activity. Either compound 25/26
with a R 1-adamantyl group or 28/29 with a R 2-adamantyl group possess higher CB2
2
2
agonist activity in comparison with the corresponding compound 24 and 27 with a R2
cyclohexyl group. Therefore, a R adamantyl group was demonstrated to have more positive
2
impact on the CB2 agonist activity of pyridazine-3-carboxamides than a R cyclohexyl.
2
1
0

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After preliminary identification of the preference for the R 4-isopropyl substitution,
1
4
-isopropylpyridazine-3-carboxamides 30-38 with diverse R groups were synthesized to
3
characterize the effect of R 1-adamantyl or 2-adamantyl on the CB2 bioactivity. It was
2
observed that EC50 values increased for 4-isopropylpyridazine-3-carboxamide derivatives
with the corresponding R subunit in the order of cyclohexyl (30), 1-adamantyl (33), and
2
2
-adamantyl (36). Similarity, this trend is also seen in other different R substituted
3
compounds, whose EC50 values are decreased in the order of 2-adamantyl (37 or 38),
-adamantyl (34 or 35), and cyclohexyl (31 or 32), respectively. These results further
confirmed that the R2 2-adamantyl group would be the best choice to
-isopropylpyridazine-3-carboxamides as CB2 agonists. Among these compounds, 36 or 38
1
4
shows the CB2 agonist activity similar to 26. Actually, docking simulations discussed later
will suggest that R 2-adamantyl of 26 would have maximal hydrophobic space-filling in the
2
pocket surrounded by the key residues F91, F94, F106, K109, and I110 (Figure 2A).
SAR studies were continued to explore the influence of R units on the CB2 agonist
3
activity of 4-isopropylpyridazine-3-carboxamides with a constant R 2-adamantyl group. As
2
shown in Table 2, compound 39 with a R piperidinyl group shows more than 4-fold lower
3
CB2 agonist activity than 26 with a R morpholino, and N-methylpiperazin derivative 40
3
results in complete loss of activity. By introducing a cyclohexylamino group in place of the
morpholino group of 26, the obtained compound 41 shows more than 108-fold drop in CB2
agonist activity. However, compound 36 with a cyclopentylamino group or 42 with a
cyclopropylamino group maintains CB2 agonist activity similar as 26. In addition, either 43
with a cyclopropylmethylenamino group or 37 with an n-butylamino group was also
11

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examined to be a CB2 agonist, showing 13-times lower bioactivity than 26. Moreover, it
would not be helpful to introduce a branched pentylamino chain (44) at the R position.
3
Furthermore, replacement of the R n-butylamino of 37 with 2-hydroxyethylamino led to
3
analog 38, which has the CB2 agonist activity similar to 26. As discussed later, docking
simulations will indicate the flexible R 2-hydroxyethylamino of 38 would have a similar
3
H-bond interaction with T114 like the R morpholino ring of 26 (Figure 2D). On the other
3
hand, compound 45 with a R aromatic moiety do not show potency on neither CB1 nor CB2
3
receptors, suggesting the R aromatic substitution unfavorable for CB2 agonist activity.
3
Herein, a modest steric hindrance or more flexible substituent at the R position would be
3
beneficial for improving CB2 agonist activity of pyridazine-3- carboxamides. Furthermore,
by detecting the CB2 agonist activity of compounds 36 (EC = 5.701 ± 1.993 nM) with a
50
hydrophobic R cyclopentylamino group or 38 (EC = 4.955 ± 0.633 nM) with a hydrophilic
3
50
R 2-hydroxyethylamino group, it was found that both hydrophobicity and hydrophilicity are
3
tolerated for R substituent.
3
Furthermore, SAR analyses were performed to investigate the influence of R substituent
3
of 5-isopropylpyridazine-3-carboxamides on the CB2 bioactivity. As summarized in Table 3,
some 5-isopropylpyridazine-3-carboxamides also display potent CB2 agonist activity, such as
4
6
3
8 (EC = 34.42 ± 2.97 nM, E = 114 ± 4%), 51 (EC = 96.24 ± 17.98 nM, E = 93 ±
50 max 50 max
%), and 52 (EC = 99.86 ± 21.14 nM, E = 127 ± 10%). As like 4-isopropylpyridazine-
5
0
max
-carboxamides, replacement of the R morpholino group with a piperidinyl resulted in the
3
reduced EC value by more than 8-times, as illustrated by the activity of 46 vs 29. Whereas,
5
0
both the R cyclohexylamino (47) and cyclopropylamino (48) substitutions may enhance the
3
1
2

ACCEPTED MANUSCRIPT
CB2 agonist activity in comparison with the R morpholino group (29). In particular, 48
3
displays an improved bioactivity by more than 10-fold compared with GW842166X. The R3
cyclopropylmethylenamino derivative 49 shows a decrease in the CB2 agonist activity than
4
8. Moreover, the R n-butylamino substituted analog 50 displays 3-times lower bioactivity
3
than 48. In addition, there is an apparent decrease in CB2 selectivity on compounds 49 and 50
owing to their CB1 agonist activity. Compound 51 with a R branched pentylamino chain are
3
inactive on both CB1 and CB2. Furthermore, the polar 2-hydroxyethylamino compound 52
was found to have more than 4-fold higher CB2 agonist activity than 29. However,
compound 55 with a R 3-chlorophenylamino group is deprived of the CB2 activity. Taken
3
together, our SAR results suggest that a R group with appropriate steric hindrance is required
3
for pyridazine-3-carboxamides to be a potent CB2 agonist. In the meantime, a substituent at
C-4 position was confirmed to be a preferred for the CB2 receptor bioactivity in comparison
with the C5-substitution by comparing the CB2 agonist activities of 26 vs 29, 36 vs 48, or 38
vs 52, which could pave the way to develop high potency and selectivity ligands for CB2
receptor in the future.
3
.3. Docking-predicted interaction modes of pyridazine-3-carboxamides binding to the
CB2 receptor
In an attempt to explain how our designed compounds interact with active site of the CB2
receptor and how this relates to their CB2 agonist activities, flexible docking simulations
were carried out to predict the receptor-ligand interactions by using the FlexiDock module of
Sybyl-X1.3 [47]. As shown in Figure 2, Several representative compounds with different
1
3

ACCEPTED MANUSCRIPT
CB2 agonist activities (26, 29, 37, and 38) were selected to investigate the different
interaction modes with the CB2 receptor on the basis of our previously generated homology
model [48].
Evidently, these four typical analogs display similar binding poses but a little bit different
interaction patterns (Figure 2). In each of the CB2-ligand complexes, the carbonyl group of
the C-3 amide moiety conferred a critical H-bond interaction with the side chain hydroxyl
group of S285. Similarly, the corresponding H-bond interaction was also observed in the
binding mode of GW842166X (Figure S1 in the Supporting Information). As formerly
hypothesized, the nitrogen atoms of pyridazine nucleus engaged in three H-bonds with the
side chain of residue R177, suggesting that the pyridazine core would be a better choice than
pyrimidine template. S285 and R177 were also proposed to play important roles in the
binding of CP55940 to the CB2 receptor according to the docking and molecular dynamics
simulations [49]. Docking of the most active ligand 26 showed a slightly different binding
mode with other compounds (Figure 2A). In the simulated structural model of complex
CB2-26, the polar morpholino group points to a hydrophobic pocket surrounded by the side
chains of W172, Y190, W194, and F197, which had been identified as crucial residues for
ligand recognition in the CB2 receptor [50-52]. Meanwhile, the oxygen atom of morpholino
may form an H-bond interaction with the side chain hydroxyl group of residue T114. The
bond length and angle are 2.34 Å and 160.58°, respectively. The R 2-adamantyl moiety may
2
fully occupy the hydrophobic sub-pocket composed of the side chains of the residues F91,
F94, F106, K109, and I110, which indicating that a moderate bulky group is favorable at this
position. For comparison, analogs 24 with a R cyclohexyl and 25 with a R 1-adamantyl
2
2
1
4

ACCEPTED MANUSCRIPT
group were selected to study the different binding modes of agonists with CB2 receptor. A
three-dimensional superposition of these three ligands in the CB2 receptor binding cavity is
shown in Figure S2 in the Supporting Information. It was noted that a smaller hydrophobic
cyclohexyl group of 24 would be minimally space-filling in the binding pocket. Whereas, the
introduced 1-adamantyl group of 25 squeezes into the hydrophobic region adjacent to the side
chains of residue F91, making 25 undergo movements to fit the binding cavity, thus reducing
the interactions with R177. The tighter contact may be the major reason for the considerably
decreased activity of 25. These observations led us to think that the occupancy of this region
is fundamental for a ligand.
As for docking-simulated structural model of CB2-29 complex (Figure 2B), because of the
restriction of the R 5-isopropyl group, compound 29 has its R morpholino group move
2
3
away to miss the H-bond interaction with the residue T114. This is probably the reason for
4
5
2
-isopropylpyridazine-3-carboxamides showing higher CB2 agonist activities than
-isopropylpyridazine-3-carboxamides. For the docking-simulated CB2-37 complex (Figure
C), the R n-butyl chain tightly stretches into the hydrophobic sub-pocket composed of
3
W172, Y190, W194, and F197. the cyclopentyl group, the n-butyl group would be at a
disadvantage in van der Waals interaction with the hydrophobic residues, inducing lower
bioactivity of 37 than 36. In the simulated structural model of complex CB2-38 (Figure 2D),
3
8 with a polar 2-hydroxyethylamino as the R group displays similar receptor-ligand
3
interaction as that of the complex CB2-26. The hydroxyl oxygen atom of 38 may also serve
as an H-bond receptor for residue T114. As for bioactivity assays data, both 26 and 38
compounds showed potent CB2 agonist activities. Consequently, the H-bond interaction with
1
5

ACCEPTED MANUSCRIPT
the side chain hydroxyl group of T114 would be beneficial for the CB2 bioactivity of
pyridazine-3-carboxamides.
Furthermore, the core ring of these compounds contacts with the side chains of residues
V113, F117, W258, V261, and C288. In the docking-simulated model of CB2-GW842166X,
the aromatic side chains of residues F117 (3.36) and W258 (6.48) of CB2 may form a parallel
π-π interaction with a distance of 5.07 Å. Previous studies have revealed that the
F3.36/W6.48 interaction may be the rotamer toggle switch for the protein activation [49, 53,
5
4]. In the docking-simulated structural models of four complexes, our docking results are
congruent with the predicted interaction mode between CB2 and the GW842166X, which has
the W258’s side chain indole ring to maintain the parallel π-π interactions with the side chain
aromatic ring of F117. Overall, these effects are consistent with their activity data.
4
. Conclusion
In summary, we have described the synthesis and biological evaluation of
pyridazine-3-carboxamide derivatives as novel CB2-selective agonists. Most of these
compounds exhibit moderate to potent CB2 agonist activity and high selectivity against the
CB1 receptor. Among these derivatives, six compounds were confirmed as potent CB2
agonists with EC values below 35 nM. Especially, compound 26 showed the highest CB2
5
0
agonist activity with an EC value of 3.665 ± 0.553 nM and remarkable selectivity index of
50
more than 2729 over the CB1 receptor. The SAR analyses revealed that a R group at C-4
1
position, a hydrophobic R moiety with 2-adamantyl substituent, and a R steric moderate
2
3
aliphatic group would be favorable to the CB2 agonist activity of pyridazine-3-carboxamides.
1
6

ACCEPTED MANUSCRIPT
In addition, molecular docking simulations were carried out to predict their plausible binding
modes and corresponding interaction patterns with the key residues around the active site of
the CB2 receptor. The docking results suggested that pyridazine-3-carboxamides share
similar receptor-ligand interactions like that of GW842166X. Taken together, novel series of
pyridazine-3-carboxamide derivatives and molecular docking simulations could provide a
viable starting point for the development of potent CB2 agonists and give a guideline for
further structural optimization.
5
5
5
. Experimental section
.1. Compound syntheses
.1.1. Materials and instrumentation
All reactions involving air or moisture-sensitive compounds were performed under
nitrogen atmosphere and reaction progress was monitored by thin-layer chromatography
TLC) on silica gel plates (GF254). Column chromatography was performed using silica gel
00-300 mesh. Melting points were measured with a Büchi Melting Point B-540 apparatus
(
2
1
13
(
Büchi Labortechnik, Flawil, Switzerland). H NMR and C NMR spectra were recorded on
5
00 MHz and 125 MHz instruments (Bruker Bioscience, Billerica, MA, USA), respectively,
with TMS as an internal standard. The values of chemical shifts (δ) were given in ppm and
coupling constants (J) in Hz. The abbreviations used to characterize the signals are as follows:
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), etc. Mass
spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA).
Dichloromethane (DCM), 1,2-dichloroethane and N,N-dimethylformamide (DMF) were dried
1
7

ACCEPTED MANUSCRIPT
by distillation from calcium hydride. Triethylamine (Et N) was dried by distillation from
3
potassium hydroxide. All other solvents and reagents were purchased from commercial
sources and used as received without further purification.
5
.1.2. Preparation of 6-oxo-1,4,5,6-tetrahydropyridazine-3-carboxylic acid (11)
A well-stirred solution of α-ketoglutaric acid (1.14 g, 7.81 mmol) in hot water (1.8 mL)
was added slowly, with stirring to a solution of sodium hydroxide (0.69 g, 17.25 mmol) and
hydrazinium sulfate (1.02 g, 7.85 mmol) in hot water (4.5 mL). The mixture was heated to
moderate boiling overnight, and then refrigerated in ice bath for several hours. The precipitate
was collected by filtration and recrystallized with 2 N HCl to provide 11 as a colorless needle
crystal (691.1 mg, yield 62.3%). Mp: 195.4-195.9 ºC (Ref. melting point 195-196 ºC [55]).
5
.1.3. Preparation of 6-oxo-1,6-dihydropyridazine-3-carboxylic acid (12)
To a solution of 11 (568 mg, 4.00 mmol) in glacial acetic acid (2.0 mL) was vigorously
stirred at slight boiling temperature, then bromine (230 µL, 4.50 mmol) was added via
dropping funnel. When all of the bromine had been introduced, water (2.0 mL) was added
and the reaction mixture was heated under reflux temperature overnight. After cooling to
room temperature, the precipitate was filtrated, and then dried to give crude product 12
without further purification. The average yield of acid which precipitated was 334.1 mg
(60.0%). Mp: 258.4-258.6 ºC (Ref. melting point 257 ºC [55]).
5
.1.4. Preparation of 6-chloropyridazine-3-carbonyl chloride (13)
Dry DMF (18 µL), thionyl chloride (250 µL, 3.52 mmol), and dry 1,2-dichloroethane (2.5
mL) was added to 6-oxo-1,6-dihydropyridazine-3-carboxylic acid 12 (100 mg, 0.72 mmol),
and the mixture was heated at 68 ºC overnight. After completion, the excess solvent was
1
8

ACCEPTED MANUSCRIPT
concentrated in vacuo, and the crude product of acyl chloride 13 was used immediately
without further purification.
5
.1.5. General procedures for the preparation of amide analogues 14a-14c
Amine (1.08 mmol) and triethylamine (1.44 mmol) were added to dry DCM (5.4 mL) at
ice bath in another flask, and the newly synthesized acyl chloride 13 was dissolved in dry
DCM (2.5 mL) and added dropwise via dropping funnel. The mixture was stirred at room
temperature overnight. The resultant was poured into cooled water, and the organic layer was
washed with 1 N HCl, saturated NaHCO aqueous solution, and saturated NaCl aqueous
3
solution, dried over anhydrous MgSO , and evaporated in vacuo. The residue was purified by
4
flash chromatography on silica gel (200-300 mesh, hexane: ethyl acetate = 8:1 to 5:1) to
obtain the compounds 14a-14c.
5
.1.5.1. 6-chloro-N-cyclohexylpyridazine-3-carboxamide (14a)
1
Yellow solid. Yield: 55.2%. Mp: 157.1-158.2 ºC. H NMR (CDCl , 500 MHz): δ 8.29 (d, J
3
=
9 Hz, 1H), 7.94-7.96 (m, 1H), 7.68 (d, J = 8.5 Hz, 1H), 4.03-3.98 (m, 1H), 2.05-2.02 (m,
2
H), 1.82-1.77 (m, 2H), 1.68-1.65 (m, 1H), 1.49-1.40 (m, 2H), 1.39-1.31 (m, 2H), 1.30-1.24
+
(
m, 1H). MS (ESI), m/z: 240.12 [M+H] .
5
.1.5.2. N-(adamantan-1-yl)-6-chloropyridazine-3-carboxamide (14b)
1
Yellow solid. Yield: 50.4%. Mp: 200.8-202.1 ºC. H NMR (CDCl , 500 MHz): δ 8.25 (d, J
3
=
8.5 Hz, 1H), 7.86 (s, 1H), 7.66 (d, J = 8.5 Hz, 1H), 2.16 (s, 9H), 1.78-1.72 (m, 6H). MS
+
(
ESI), m/z: 292.13 [M+H] .
5
.1.5.3. N-(adamantan-2-yl)-6-chloropyridazine-3-carboxamide (14c)
1
Yellow solid. Yield: 51.6%. Mp: 171.2-172.5 ºC. H NMR (CDCl , 500 MHz): δ 8.46 (d, J
3
1
9

ACCEPTED MANUSCRIPT
=
5 Hz, 1H), 8.29 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 4.30-4.28 (m, 1H), 2.08 (s,
2
H), 1.97 (d, J = 13.5 Hz, 2H), 1.92 (s, 6H), 1.80 (s, 2H), 1.72 (d, J = 13 Hz, 2H). MS (ESI),
+
m/z: 292.13 [M+H] .
5
.1.6. Preparation of ethyl 6-oxo-1,6-dihydropyridazine-3-carboxylate (15)
A solution of 12 (1.11 g, 7.92 mmol) in 8 mL of hydrogen chloride in ethanol (hydrogen
chloride gas generated by reacting sodium chloride with concentrated sulfuric acid was
passed into ethanol in an ice bath) was reflux for 14 h. After cooling to room temperature, the
precipitate was filtered off and dried to give 15 as a white solid (1.17 g, yield 87.5%). Mp:
1
25.0-125.8 ºC (Ref. melting point 127-128 ºC [56]).
5
.1.7. Preparation of ethyl 6-chloropyridazine-3-carboxylate (16)
A solution of 15 (1.17 g, 6.96 mmol) in phosphorus oxychloride (12 mL) was heated at 100
ºC for 2 h. After cooling, the solution was concentrated in vacuo to 3 mL, and the residue was
carefully poured into ice water, slowly neutralized with 10% NaOH aqueous solution in ice
bath, and then extracted with ethyl acetate. The organic layer was washed with saturated
NaCl aqueous solution, dried over anhydrous MgSO , and evaporated in vacuo. The residue
4
was purified by flash chromatography on silica gel (200-300 mesh, hexane: ethyl acetate =
3
:1) to obtain the compound 16 as a white solid (892 mg, yield 68.7%). Mp: 145.6-146.7 ºC
(Ref. melting point 154-155 ºC [56]).
5
.1.8. General procedures for the preparation of intermediates 17a-17b
To a suspension of ethyl 6-chloropyridazine-3-carboxylate 16 (534 mg, 2.87 mmol) in
distilled water (9 mL), isobutyric acid (60 µL, 0.65 mmol), conc. H SO (230 µL, 4.31 mmol)
2
4
and AgNO (48.75 mg, 0.29 mmol) were added at room temperature. The mixture was heated
3
2
0

ACCEPTED MANUSCRIPT
at 65-75 ºC and a solution of NH S O (982.40 mg, 4.31 mmol) in distilled water (5 mL) was
4
2
8
added dropwise in 10-15 minutes. The reaction was stirred for an additional 30 minutes at
0-75 ºC, then poured over ice, then neutralized with a 30% aqueous solution of NH OH and
7
4
immediately extracted twice with dichloromethane. The collected organic layers were dried
over anhydrous MgSO , and evaporated in vacuo. The residue was purified by flash
4
chromatography on silica gel (200-300 mesh, hexane: ethyl acetate = 5:1) to obtain the
regioisomer 17a (29.4 mg, yield 4.5%) and regioisomer 17b (294.0 mg, 44.9%).
Ethyl 6-chloro-4-isopropylpyridazine-3-carboxylate (17a)
1
Yellow liquid. H NMR (CDCl , 500 MHz): δ 7.51 (s, 1H), .4.54-4.49 (q, 2H), 3.47-3.41
3
+
(
m, 1H), 1.46 (t, J = 7 Hz, 3H), 1.31 (s, 3H), 1.29 (s, 3H). MS (ESI), m/z: 229.11 [M+H] .
Ethyl 6-chloro-5-isopropylpyridazine-3-carboxylate (17b)
1
Yellow liquid. H NMR (CDCl , 500 MHz): δ 8.06 (s, 1H), 4.57-4.53 (q, 2H), 3.38-3.32 (m,
3
+
1
H), 1.48 (t, J = 7 Hz, 3H), 1.35 (s, 3H), 1.33 (s, 3H). MS (ESI), m/z: 229.10 [M+H] .
.1.9. General procedures for the preparation of intermediates 18a-18b
5
To a solution of ethyl ester 17 (1 equiv.) in a 4:1 mixture of THF/H2O (0.05 M solution)
LiOH hydrate (1.2 equiv.) was added at 0 ºC. After the hydrolysis is completed (TLC
monitoring), the solution was acidified carefully with a 1 N HCl, heated to room temperature
and extracted with ethyl acetate. The collected organic layers were dried over anhydrous
MgSO , filtrated and the solvent removed under reduced pressure leading to the free
4
carboxylic acid 18 that was used without further purification.
5
.1.10. General procedures for the preparation of acyl chlorides 19a-19b
The similar procedures for the preparation of 13 were used to prepare the compounds 19a
2
1

ACCEPTED MANUSCRIPT
-19b by replacing the reactant 12 with intermediate compound 18. The crude products of acyl
chlorides 19a-19b were used for next step.
5
.1.11. General procedures for the preparation of amide analogues 20a-20f
The similar procedures for the preparation of intermediate compound 14 were used to
prepare the compounds 20a-20f by replacing the reactant 13 with intermediate compound 19.
6
-chloro-N-cyclohexyl-4-isopropylpyridazine-3-carboxamide (20a)
1
Yellow solid. Yield: 49.4%. Mp: 116.4-118.4 ºC. H NMR (CDCl , 500 MHz): δ 7.80 (d, J
3
=
6.5 Hz, 1H), 7.54 (s, 1H), 4.33-4.27 (m, 1H), 3.98-3.91 (m, 1H), 2.05-2.01 (m, 2H),
1
1
.80-1.76 (m, 2H), 1.68-1.64 (m, 1H), 1.47-1.38 (m, 3H), 1.36-1.31 (m, 2H), 1.29 (s, 3H),
+
.28 (s, 3H). MS (ESI), m/z: 282.17 [M+H] .
N-(adamantan-1-yl)-6-chloro-4-isopropylpyridazine-3-carboxamide (20b)
1
Yellow solid. Yield: 32.2%. Mp: 148.5-149.5 ºC. H NMR (CDCl , 500 MHz): δ 7.60 (s,
3
1
H), 7.52 (s, 1H), 4.27-4.22 (m, 1H), 2.15 (s, 9H), 1.76-1.70 (m, 6H), 1.29 (s, 3H), 1.27 (s,
+
3
H). MS (ESI), m/z: 334.20 [M+H] .
N-(adamantan-2-yl)-6-chloro-4-isopropylpyridazine-3-carboxamide (20c)
1
Yellow solid. Yield: 28.2%. Mp: 118.5-120.0 ºC. H NMR (CDCl , 500 MHz): δ 8.31 (d, J
3
=
7 Hz, 1H), 7.55 (s, 1H), 4.34-4.28 (m, 1H), 4.25-4.23 (m, 1H), 2.07 (s, 2H), 1.94 (d, J = 14
Hz, 2H), 1.91 (s, 6H), 1.78 (s, 2H), 1.70 (d, J = 12.5 Hz, 2H), 1.30 (s, 3H), 1.29 (s, 3H). MS
+
(
ESI), m/z: 334.19 [M+H] .
6
-chloro-N-cyclohexyl-5-isopropylpyridazine-3-carboxamide (20d)
1
White solid. Yield: 97.2%. Mp: 129.4-130.5 ºC. H NMR (CDCl , 500 MHz): δ 8.20 (s,
3
1
H), 7.97 (d, J = 7.5 Hz, 1H), 4.04-3.97 (m, 1H), 3.37-3.31 (m, 1H), 2.04-2.01 (m, 2H),
2
2

ACCEPTED MANUSCRIPT
1
1
.81-1.77 (m, 2H), 1.69-1.65 (m, 1H), 1.49-1.41 (m, 2H), 1.39-1.36 (m, 2H), 1.34 (s, 3H),
+
.33 (s, 3H), 1.29-1.24 (m, 1H). MS (ESI), m/z: 282.16 [M+H] .
N-(adamantan-1-yl)-6-chloro-5-isopropylpyridazine-3-carboxamide (20e)
1
White solid. Yield: 68.6%. Mp: 97.5-98.1 ºC. H NMR (CDCl , 500 MHz): δ 8.18 (s, 1H),
3
7
.88 (s, 1H), 3.35-3.29 (m, 1H), 2.16 (s, 9H), 1.77-1.69 (m, 6H), 1.32 (s, 3H), 1.31 (s, 3H).
+
MS (ESI), m/z: 334.19 [M+H] .
N-(adamantan-2-yl)-6-chloro-5-isopropylpyridazine-3-carboxamide (20f)
1
White solid. Yield: 75.0%. Mp: 132.6-134 ºC. H NMR (CDCl , 500 MHz): δ 8.48 (d, J =
3
7
.5 Hz, 1H), 8.21 (s, 1H), 4.29-4.27 (m, 1H), 3.37-3.32 (m, 1H), 2.07 (s, 2H), 1.97 (d, J =
1
3.5 Hz, 2H), 1.92 (s, 6H), 1.79 (s, 2H), 1.70 (d, J = 12.5 Hz, 2H), 1.34 (s, 3H), 1.33 (s, 3H).
+
MS (ESI), m/z: 334.20 [M+H] .
5
.1.12. General procedures for the preparation of pyridazine-3-carboxamide derivatives
1-44 and 46-52
Aliphatic amine (0.18 mmol) and N,N-diisopropylethylamine (61 µL) were added into a
2
solution of intermediate compound 14 or 20 (0.12 mmol) in 1,4-dioxane (2 mL), and the
reaction was heated under reflux overnight. After cooling to room temperature, the mixture
was poured into water and extracted with ethyl acetate. The organic layer was washed with
saturated NaCl aqueous solution, dried over anhydrous MgSO , and evaporated in vacuo. The
4
residue was purified by flash chromatography on silica gel (200-300 mesh, hexane: ethyl
acetate = 5:1 to 1:1) to obtain the compounds 21-44 and 46-52.
N-cyclohexyl-6-morpholinopyridazine-3-carboxamide (21)
1
White solid. Yield: 89.7%. Mp: 146.4-147.0 ºC. H NMR (CDCl , 500 MHz): δ 8.04 (d, J
3
2
3

ACCEPTED MANUSCRIPT
=
=
9.5 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 6.96 (d, J = 9.5 Hz, 1H), 4.00-3.94 (m, 1H), 3.86 (t, J
4.5 Hz, 4H), 3.72 (t, J = 5.0 Hz, 4H), 2.02-1.99 (m, 2H), 1.79-1.75 (m, 2H), 1.65-1.62 (m,
1
3
1
1
H), 1.47-1.39 (m, 2H), 1.34-1.22 (m, 3H). C NMR (CDCl , 125 MHz): δ 162.16, 160.37,
3
45.37, 126.94, 112.10, 66.44, 48.16, 45.01, 33.09, 25.55, 24.79. MS (ESI), m/z: 291.18
+
[
M+H] .
N-(adamantan-1-yl)-6-morpholinopyridazine-3-carboxamide (22)
1
White solid. Yield: 92.5%. Mp: 191.6-192.0 ºC. H NMR (CDCl , 500 MHz): δ 8.02 (d, J
3
=
9.5 Hz, 1H), 7.77 (s, 1H), 6.96 (d, J = 9.5 Hz, 1H), 3.86 (t, J = 4.5 Hz, 4H), 3.72 (t, J = 5.0
1
3
Hz, 4H), 2.14 (s, 9H), 1.76-1.70 (m, 6H). C NMR (CDCl , 125 MHz): δ 162.00, 160.33,
3
1
46.01, 126.60, 112.22, 66.43, 51.82, 45.08, 41.65, 36.39, 29.49. MS (ESI), m/z: 343.23
+
[
M+H] .
N-(adamantan-2-yl)-6-morpholinopyridazine-3-carboxamide (23)
1
White solid. Yield: 85.2%. Mp: 219.4-219.8 ºC. H NMR (CDCl , 500 MHz): δ 8.34 (d, J
3
=
8.0 Hz, 1H), 8.05 (d, J = 9.5 Hz, 1H), 6.97 (d, J = 9.5 Hz, 1H), 4.27-4.25 (m, J = 8.5 Hz,
1
1
1
2
H), 3.86 (t, J = 4.5 Hz, 4H), 3.73 (t, J = 5.0 Hz, 4H), 2.05 (s, 2H), 1.99 (d, J = 13.0 Hz, 2H),
1
3
.90-1.88 (m, 6H), 1.77 (s, 2H), 1.67 (d, J = 13.0 Hz, 2H). C NMR (CDCl , 125 MHz): δ
3
62.20, 160.42, 145.62, 126.95, 112.13, 66.44, 53.40, 45.09, 37.59, 37.19, 32.16, 31.90,
+
7.28, 27.21. MS (ESI), m/z: 343.24 [M+H] .
N-cyclohexyl-4-isopropyl-6-morpholinopyridazine-3-carboxamide (24)
1
White solid. Yield: 35.4%. Mp: 109.4-110.3 ºC. H NMR (CDCl , 500 MHz): δ 7.89 (d, J
3
=
8.0 Hz, 1H), 6.80 (s, 1H), 4.34-4.29 (m, 1H), 3.94-3.90 (m, 1H), 3.86 (t, J = 4.5 Hz, 4H),
3
.70 (t, J = 5.0 Hz, 4H), 2.02-1.99 (m, 2H), 1.78-1.71 (m, 2H), 1.66-1.62 (m, 1H), 1.45-1.37
2
4

ACCEPTED MANUSCRIPT
1
3
(
m, 3H), 1.31-1.28 (m, 2H), 1.26 (s, 3H), 1.24 (s, 3H). C NMR (CDCl , 125 MHz): δ
3
1
2
63.95, 160.61, 151.34, 144.01, 108.92, 66.49, 48.21, 45.03, 33.09, 27.77, 25.61, 24.95,
+
2.86. MS (ESI), m/z: 333.23 [M+H] .
N-(adamantan-1-yl)-4-isopropyl-6-morpholinopyridazine-3-carboxamide (25)
1
White solid. Yield: 26.0%. Mp: 156.6-158.0 ºC. H NMR (CDCl , 500 MHz): δ 7.74 (s,
3
1
H), 6.79 (s, 1H), 4.31-4.25 ( m, 1H), 3.86 (t, J = 5 Hz, 4H), 3.69 (t, J = 5.0 Hz, 4H),
13
2
.14-2.11 (m, 9H), 1.75-1.69 (m, 6H), 1.25 (s, 3H), 1.23 (s, 3H). C NMR (CDCl , 125
3
MHz): δ 164.09, 160.59, 151.14, 144.87, 109.07, 66.49, 51.84, 45.10, 41.58, 36.46, 29.53,
+
2
7.72, 22.86. MS (ESI), m/z: 385.27 [M+H] .
N-(adamantan-2-yl)-4-isopropyl-6-morpholinopyridazine-3-carboxamide (26)
1
White solid. Yield: 29.7%. Mp: 180.6-181.7 ºC. H NMR (CDCl , 500 MHz): δ 8.36 (d, J
3
=
8.0 Hz, 1H), 6.81 (s, 1H), 4.34-4.29 (m, 1H), 4.22-4.21 (m, 1H), 3.87 (t, J = 4.5 Hz, 4H),
3
2
1
2
.71 (t, J = 5.0 Hz, 4H), 2.05 (s, 2H), 1.98 (d, J = 13.0 Hz, 2H), 1.89-1.86 (m, 6H), 1.77 (s,
1
3
H), 1.66 (d, J = 13.0 Hz, 2H), 1.26 (s, 3H), 1.25 (s, 3H). C NMR (CDCl , 125 MHz): δ
3
64.06, 160.69, 151.35, 144.31, 108.96, 66.50, 53.44, 45.10, 37.62, 37.25, 32.08, 32.00,
+
7.81, 27.31, 27.28, 22.88. MS (ESI), m/z: 385.26 [M+H] .
N-cyclohexyl-5-isopropyl-6-morpholinopyridazine-3-carboxamide (27)
1
White solid. Yield: 44.1%. Mp: 186.3-186.7 ºC. H NMR (CDCl , 500 MHz): δ 8.09 (s,
3
1
4
H), 8.00 (d, J = 8.0 Hz, 1H), 4.02-3.96 (m, 1H), 3.91 (t, J = 4.5 Hz, 4H), 3.35 (t, J = 4.5 Hz,
H), 3.18-3.13 (m, 1H), 2.02-1.99 (m, 2H), 1.79-1.75 (m, 2H), 1.67-1.63 (m, 1H), 1.49-1.40
1
3
(
m, 3H), 1.35-1.32 (m, 1H), 1.30 (s, 3H), 1.29 (s, 3H), 1.27-1.23 (m, 1H). C NMR (CDCl ,
3
1
25 MHz): δ 163.75, 162.01, 149.45, 142.94, 124.60, 66.80, 51.43, 48.23, 33.01, 27.52,
2
5

ACCEPTED MANUSCRIPT
+
2
5.53, 24.74, 23.12. MS (ESI), m/z: 333.23 [M+H] .
N-(adamantan-1-yl)-5-isopropyl-6-morpholinopyridazine-3-carboxamide (28)
1
White solid. Yield: 47.4%. Mp: 149.7-150.0 ºC. H NMR (CDCl , 500 MHz): δ 8.08 (s,
3
1
9
1
H), 7.91 (s, 1H), 3.91 (t, J = 4.5 Hz, 4H), 3.34 (t, J = 4.5 Hz, 4H), 3.17-3.12 (m, 1H), 2.15 (s,
1
3
H), 1.77-1.71 (m, 6H), 1.29 (s, 3H), 1.28 (s, 3H). C NMR (CDCl , 125 MHz): δ 163.66,
3
61.86, 150.02, 142.95, 124.26, 66.80, 51.96, 51.44, 41.55, 36.36, 29.45, 27.49, 23.08. MS
+
(
ESI), m/z: 385.25 [M+H] .
N-(adamantan-2-yl)-5-isopropyl-6-morpholinopyridazine-3-carboxamide (29)
1
White solid. Yield: 58.2%. Mp: 232.2-232.6 ºC. H NMR (CDCl , 500 MHz): δ 8.49 (d, J
3
=
8.5 Hz, 1H), 8.11 (s, 1H), 4.29-4.27 (m, 1H), 3.92 (t, J = 4.5 Hz, 4H), 3.35 (t, J = 5.0 Hz,
4
2
1
2
H), 3.19-3.14 (m, 1H), 2.05 (s, 2H), 2.00 (d, J = 13.5 Hz, 2H), 1.91-1.88 (m, 6H), 1.78 (s,
1
3
H), 1.68 (d, J = 12.0 Hz, 2H), 1.31 (s, 3H), 1.29 (s, 3H). C NMR (CDCl , 125 MHz): δ
3
63.83, 162.07, 149.74, 142.97, 124.60, 66.82, 53.53, 51.50, 37.58, 37.18, 32.15, 31.89,
+
7.54, 27.28, 27.20, 23.07. MS (ESI), m/z: 385.26 [M+H] .
N-cyclohexyl-6-(cyclopentylamino)-4-isopropylpyridazine-3-carboxamide (30)
1
White solid. Yield: 10.0%. Mp: 172.1-173.1 ºC. H NMR (CDCl , 500 MHz): δ 7.89 (d, J
3
=
8.0 Hz, 1H), 6.55 (s, 1H), 4.89 (s, 1H), 4.30-4.23 (m, 2H), 3.94-3.86 (m, 1H), 2.17-2.10 (m,
2
H), 2.01-1.98 (m, 2H), 1.79-1.74 (m, 4H), 1.70-1.67 (m, 1H), 1.65-1.62 (m, 2H), 1.56-1.49
1
3
(
m, 2H), 1.45-1.34 (m, 3H), 1.30-1.26 (m, 2H), 1.23(s, 3H), 1.22 (s, 3H). C NMR (CDCl ,
3
1
2
6
25 MHz): δ 164.20, 159.79, 151.21, 143.77, 110.00, 53.35, 48.17, 33.40, 33.13, 27.54, 25.65,
+
4.99, 23.81, 22.78. MS (ESI), m/z: 331.27 [M+H] .
-(butylamino)-N-cyclohexyl-4-isopropylpyridazine-3-carboxamide (31)
2
6

ACCEPTED MANUSCRIPT
1
Yellow solid. Yield: 16.2%. Mp: 109.5-110.7 ºC. H NMR (CDCl , 500 MHz): δ 7.89 (d, J
3
=
8.0 Hz, 1H), 6.54 (s, 1H), 4.81 (s, 1H), 4.30-4.25 (m, 1H), 3.94-3.86 (m, 1H), 3.48-3.44 (q,
2
H), 2.01-1.98 (m, 2H), 1.78-1.74 (m, 2H), 1.70-1.64 (m, 2H), 1.63-1.61 (m, 1H), 1.49-1.45
(m, 2H), 1.43-1.33 (m, 3H), 1.31-1.26 (m, 2H), 1.23(s, 3H), 1.22 (s, 3H), 0.98 (t, J = 7.5 Hz,
1
3
3
3
H). C NMR (CDCl , 125 MHz): δ 164.18, 160.05, 151.28, 143.87, 109.81, 48.16, 41.70,
3
+
3.12, 31.50, 27.57, 25.64, 24.98, 22.80, 20.15, 13.81. MS (ESI), m/z: 319.25 [M+H] .
N-cyclohexyl-6-((2-hydroxyethyl)amino)-4-isopropylpyridazine-3-carboxamide (32)
1
White solid. Yield: 11.6%. Mp: 169.2-171.2 ºC. H NMR (CDCl , 500 MHz): δ 7.78 (d, J
3
=
7.5 Hz, 1H), 6.63 (s, 1H), 5.46 (s, 1H), 4.26-4.20 (m, 1H), 3.92 (t, J = 5.0 Hz, 2H),
3
1
.94-3.87 (m, 1H), 3.71-3.68 (q, 2H), 2.02-1.98 (m, 2H), 1.78-1.74 (m, 2H), 1.65-1.61 (m,
1
3
H), 1.48-1.36 (m, 3H), 1.31-1.26 (m, 2H), 1.23 (s, 3H), 1.21 (s, 3H). C NMR (CDCl , 125
3
MHz): δ 164.00, 160.00, 151.48, 144.57, 111.26, 62.12, 48.23, 44.48, 33.07, 27.60, 25.63,
+
2
4.94, 27.75. MS (ESI), m/z: 307.23 [M+H] .
N-(adamantan-1-yl)-6-(cyclopentylamino)-4-isopropylpyridazine-3-carboxamide (33)
1
White solid. Yield: 12.4%. Mp: 229.6-230.6 ºC. H NMR (CDCl , 500 MHz): δ 7.73 (s,
3
1
1
1
H), 6.54 (s, 1H), 4.87 (s, 1H), 4.27-4.21 (m, 2H), 2.15-2.11 (m, 11H), 1.78-1.68 (m, 10H),
1
3
.55-1.50 (m, 2H), 1.22 (s, 3H), 1.21 (s, 3H). C NMR (CDCl , 125 MHz): δ 164.33, 159.69,
3
51.04, 144.50, 110.23, 53.31, 51.73, 41.58, 36.48, 33.40, 29.53, 27.51, 23.80, 22.81. MS
+
(
ESI), m/z: 383.30 [M+H] .
N-(adamantan-1-yl)-6-(butylamino)-4-isopropylpyridazine-3-carboxamide (34)
1
White solid. Yield: 11.2%. Mp: 77.2-73.0 ºC. H NMR (CDCl , 500 MHz): δ 7.74 (s, 1H),
3
6
.53 (s, 1H), 4.79 (s, 1H), 4.27-4.21 (m, 1H), 3.47-3.43 (q, 2H), 2.13-2.11 (m, 9H), 1.75-1.69
2
7

ACCEPTED MANUSCRIPT
(m, 6H), 1.68-1.63 (m, 2H), 1.49-1.41 (m, 2H), 1.22 (s, 3H), 1.21 (s, 3H), 0.97 (t, J = 7.5 Hz,
1
3
3
H). C NMR (CDCl , 125 MHz): δ 164.32, 159.99, 151.09, 144.72, 109.91, 51.72, 41.70,
3
+
4
1.60, 36.49, 31.53, 29.55, 27.50, 22.79, 20.13, 13.77. MS (ESI), m/z: 371.29 [M+H] .
N-(adamantan-1-yl)-6-((2-hydroxyethyl)amino)-4-isopropylpyridazine-3-carboxamide (35)
1
Yellow solid. Yield: 12.6%. Mp: 82.4-83.2 ºC. H NMR (CDCl , 500 MHz): δ 7.58 (s, 1H),
3
6
.61 (s, 1H), 5.48 (s, 1H), 4.22-4.17 (m, 1H), 3.92-3.90 (t, J = 5.0 Hz, 2H), 3.70-3.67 (m, 2H),
13
2
.13-2.11 (m, 9H), 1.75-1.68 (m, 6H), 1.22 (s, 3H), 1.21 (s, 3H). C NMR (CDCl , 125
3
MHz): δ 164.17, 160.11, 151.31, 145.31, 111.14, 61.94, 51.88, 44.59, 41.54, 36.45, 29.52,
+
2
7.59, 27.73. MS (ESI), m/z: 359.27 [M+H] .
N-(adamantan-2-yl)-6-(cyclopentylamino)-4-isopropylpyridazine-3-carboxamide (36)
1
Yellow solid. Yield: 10.2%. Mp: 138.8-139.2 ºC. H NMR (CDCl , 500 MHz): δ 8.36 (d, J
3
=
8.0 Hz, 1H), 6.55 (s, 1H), 4.81 (d, J = 6.0 Hz, 1H), 4.31-4.24 (m, 2H), 4.21-4.19 (m, 1H),
2
4
3
3
.17-2.11 (m, 2H), 2.04 (s, 2H), 1.98 (d, J = 13.0 Hz, 2H), 1.89-1.86 (m, 6H), 1.79-1.76 (m,
H), 1.70-1.68 (m, 2H), 1.65 (d, J = 13.0 Hz, 2H), 1.54-1.50 (m, 2H), 1.23 (s, 3H), 1.22 (s,
1
3
H). C NMR (CDCl , 125 MHz): δ 164.28, 159.76, 151.25, 143.88, 110.23, 53.34, 53.32,
3
7.66, 37.26, 33.38, 32.11, 32.00, 27.60, 27.33, 27.29, 23.81, 22.80. MS (ESI), m/z: 383.28
+
[
M+H] .
N-(adamantan-2-yl)-6-(butylamino)-4-isopropylpyridazine-3-carboxamide (37)
1
White solid. Yield: 40.5%. Mp: 116.2-117.7 ºC. H NMR (CDCl , 500 MHz): δ 8.38 (d, J =
3
8
.0 Hz, 1H), 6.56 (s, 1H), 4.89 (s, 1H), 4.30-4.24 (m, 1H), 4.20 (d, J = 8.5 Hz, 1H), 3.50-3.46
(
(
m, 2H), 2.03 (s, 2H), 1.97 (d, J = 13.0 Hz, 2H), 1.88-1.86 (m, 6H), 1.76 (s, 2H), 1.70-1.63
1
3
m, 4H), 1.49-1.42 (m, 2H), 1.23 (s, 3H), 1.22 (s, 3H), 0.97 (t, J = 7.5 Hz, 3H). C NMR
2
8