Pyridone-Based Derivatives as Cannabinoid Receptor Type 2 Agonists

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1		Carlos David Pessoa-Mahana	+ 1855 word(s)	1855	2021-10-20 08:56:23	\|
2	format correct	Vivi Li	Meta information modification	1855	2021-11-01 02:35:38	\|

This entry is adapted from the peer-reviewed paper 10.3390/ijms222011212

The activation of the human cannabinoid receptor type II (CB2R) is known to mediate analgesic and anti-inflammatory processes without the central adverse effects related to cannabinoid receptor type I (CB1R). In this work we describe the synthesis and evaluation of a novel series of N-aryl-2-pyridone-3-carboxamide derivatives tested as human cannabinoid receptor type II (CB2R) agonists.

cannabinoids 2-pyridone synthesis CB2R agonists

1. Introduction

The endocannabinoid system comprises a complex network of lipid signaling mediators in which different proteins participate in the modulation of numerous physiological and pathophysiological processes ^[1]^[2]. Cannabinoid receptor type II (CB2R) belongs to the family of heptameric receptors coupled to G proteins (GPCRs). This receptor was identified and cloned from HL60 cells ^[3] and was initially considered as ‘peripheral cannabinoid receptor’ due to its wide distribution in peripheral cells and tissues, particularly in those of the immune system ^[4]^[5]. However, later studies showed its expression also within the Central Nervous System (CNS) especially under states of inflammation ^[4]^[6]. Various studies have shown that the activation of CB2R can block activation of microglia cells but has little effect on the normal functioning of neurons within the CNS ^[7]^[8]. Several reports indicate that the activation of CB2R is analgesic and CB2R agonists have been shown to suppress responses in animal models of both acute and neuropathic pain ^[5]^[9]^[10]. Additionally, cannabinoids have well-established anti-inflammatory properties and recently, effects in the gut-lung-skin barrier epithelia have been reported showing promising results in in vitro and in vivo animal studies ^[11]. Furthermore, the endocannabinoid system is intimately related to neurological function and neurodegenerative diseases with animal models studies showing beneficial effects for the treatment of brain injuries and multiple sclerosis ^[12]. Therefore, CB2R agonists represent potential alternatives for the treatment of pain and inflammation both in the peripheral and CNS ^[13].

The discovery of the CB2R directed research efforts towards the understanding of its role and action. Several reports on the structural requirements for ligand binding to the receptor led to the discovery of many different families of cannabinoid ligands including classical cannabinoids structurally related to THC, eicosanoids analogous to endocannabinoids and synthetic cannabinoids, most of the latter being heterocycles, aminoalkylindoles (represented by WIN55212-2), arylpyrazoles, quinolones and pyridone carboxamide derivatives.

Heterocyclic compounds represent an important source of pharmacologically active molecules and more than 85% of all biologically active compounds contain heterocyclic scaffolds ^[14]. They are frequently used to alter physicochemical properties of molecules such as lipophilicity, polarity and hydrogen bonding capacity which can improve the pharmacodynamic and pharmacokinetic profile ^[15]. The pyridone heterocycle is a 6-membered aromatic ring with a carbonyl group and a nitrogen heteroatom which has found great use in drug discovery strategies ^[16]. Relevant characteristics associated to this structure have been described by Y. Zhan and A. Pike, such as its ability to act as both a hydrogen bond acceptor and donor; act as a bioisostere of amides, phenyls and other nitrogen and oxygen-containing heterocycles, and the capacity to modulate the lipophilicity, solubility, and metabolic stability ^[16].

Previous reports have explored the 2-pyridone scaffold in the cannabinoid system particularly in the CB2R with promising results (Figure 1) ^[17]^[18]^[19]^[20]^[21]^[22]. Kusakabe et al., reported a 2-pyridone-based compound displaying high CB2R affinity and selectivity. They proposed that the pyridone scaffold could provide optimal lipophilicity for the design of CB2R ligands and predicted possible hydrophobic interactions with W194 and F117 ^[19].

Figure 1. Chemical structures of reported pyridone/quinolone based CB2R ligands and target compound.

2. Chemistry

All compounds were synthesized as shown in Scheme 1. Firstly, three N-aryl-4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carboxylic acids were synthesized from compound 3 using different substituted anilines (step c, Scheme 1) to obtain the corresponding substituted amides 4a, 4b and 4c. These amides were cyclized using acetylacetone and piperidine as a catalyst to yield compounds 5a, 5b and 5c. The pyridone derivatives were then hydrolyzed using potassium hydroxide in ethanol 80% under reflux heating obtaining the carboxylic acids derivatives 6a, 6b and 6c.

Scheme 1. Synthetic route for obtaining pyridone-derived carboxylic acids. Reagents and conditions: (a) hydrazine hydrate, ethanol, 0 °C; rt, 2 h; (b) water, HCl, acetylacetone, rt, 5 h; (c) amine, toluene, reflux, 4 h; (d) acetylacetone, N-substituted cyanoacetamide, water:ethanol, piperidine, reflux, 4 h; (e) water, KOH, reflux, 24 h; HCl, rt.

It is noteworthy to mention that hydrolysis of the nitrile derivatives proved to be harder than expected and both acid and basic conditions were studied. As described in Scheme 2, different side products were obtained depending on the reaction conditions. Only decarboxylated product 9a was obtained under acidic medium while under basic medium, the obtained product depended upon the reaction temperature. Heating the reaction below 100 °C stopped the reaction at the amide intermediate 10a whereas heating the reaction above 100 °C completely hydrolyzed the precursors to product 6a (95% relative yield).

Scheme 2. Preparation of the carboxylic acid derivative from 2-cyanopyridone and decarboxylation of the pyridone ring.

The carboxylic acid derivatives 6a, 6b and 6c were finally reacted with different cycloalkyl amines (cyclohexylamine, cycloheptylamine and 1-adamantylamine), according to Scheme 3. The products 7 and 8 were obtained using the same synthetic procedure whereby the respective amines were coupled to the carboxylic acid in the presence of BOP as coupling reagent. The relative yield for compounds 7 varied between 50% and 70% and for compounds 8 varied between 70 and 80%.

Scheme 3. Synthesis of a series N-aryl-pyridone-2-carboxamides. Reagents and conditions: (a) DMF, DIPEA, BOP, rt, 10 min; amine reagent, rt, 2 h.

3. Human CB2R cAMP Assay (Agonism Effect)

Functional activity of the synthesized compounds was evaluated through their ability to decrease the accumulation of intracellular cAMP levels (Eurofins Cerep services), ^[23] and the results are displayed in Table 1. The results show a dependence of activity on the nature of the group present in position X. Heteroaryl derivatives presented little to no activity in contrast to cycloalkyl derivatives with three of the compounds (8b, 8c and 8d) showing activity above 30%. No significant activity was observed in the 2-benzothiazole derivative compounds (7a and 7g) while 8d showed the highest agonist response and the EC50 was determined to be 112 nM (Figure 2).

Figure 2. Concentration-response curve of compound 8d in the CB2R. The result showed an EC50 = 0.11 μM. Each point represents mean values with standard error (n = 3).

Table 1. Synthesized target compounds and human CB2R agonist effect for derivatives 7 and 8 at a concentration 10 µM.


Compound	X	Y	% CB2 Agonist Response ¹	EC50 (μM)
7a	S	H	2	ND
7b	NH	H	8	ND
7c	O	H	13	ND
7d	O	CH₃	0	ND
7e	NH	CH₃	0	ND
7f	NH	OH	13	ND
7g	S	OH	0	ND

Compound	X	Y	% CB2R Agonist Response ¹	EC50 (μM)
8a	Cyclohexyl	H	12	ND
8b	Cycloheptyl	H	31	ND
8c	Adamantyl	H	51	ND
8d	Adamantyl	CH₃	95	0.11
8e	Cycloheptyl	CH₃	0	ND
8b	Cycloheptyl	H	31	ND
8f	Cycloheptyl	OH	0	ND
8g	Cyclohexyl	OH	4	ND

¹ CB2R agonist response expressed as percentage relative to control. Activity was defined as the rounded mean of the two assays. ND = no data.

Regarding the cycloalkyl-substituted derivatives, the presence of an adamantyl group seemed to favor activity over the cycloheptyl ring (8c and 8d vs. 8b). Compound (8a) showed the lowest percentage of agonist response and followed by the cycloheptyl derivative compound (8b). Replacement with a bulkier adamantyl group, the percentage of human CB2R agonist response increased over 50% at concentration 10 µM (8c).

Comparing compounds with an adamantyl group in position X, activity was also influenced by the substituent in position Y. A p-tolyl group presented maximum response (8d) whereas a phenyl group (8c) showed half the maximal response at the same concentration (10 μM), suggesting that polar groups in this region are not favorable for activity.

4. Molecular Docking Studies

Molecular docking studies on the CB2R predicted that the designed ligands bind in the transmembrane (TM) region defined by TM2-TM3 and TM5-TM7 in a similar disposition as that shown by WIN55212-2 in the Cryo-EM structure of human CB2R ^[24].

Based on reported 3D structure data, three distinct cavities in the binding pocket can be identified which accommodate the three-group scaffold of the agonist WIN55212-2 and antagonist AM10257, in what has been described as a “three-arm pose” ^[24]^[25]. According to the interactions established by WIN55212-2 and AM10257, respectively, we define as cavity 1 by residues F87, F91 and F94 which form a hydrophobic pocket that binds the naphthyl or adamantly moiety, referred to as arm 1; cavity 2 by residues I186, W194 and M265 which participate in hydrophobic interactions with the morpholine moiety or hydroxypentyl chain, referred to as arm 2; and cavity 3 defined by residues F117, V113, F183, F281, W258 and V261 that establish π-π stacking and hydrophobic interactions with the oxazinoindole or pyrazole core. Here a downward extension of a longer group towards W258 in the antagonist structure defines arm 3 (Figure 3A).

Figure 3. (a) Compound 8d interacting with residues of the binding pocket in the CB2R. The three arm-structure and residues from the three cavities are shown; (b) CB2R binding pocket with the three main cavities indicated.

Our results showed that the most active compound 8d stabilized in the orthosteric site forming an “L-shape” pose (Figure 3B). The pyridone core acted as a central scaffold within the center of the binding site and cavity 3 engaging in hydrophobic interactions with V113, V261 and M265 and π-π interactions with F117 and F183. As we expected, the 2-pyridone moiety acted as a pivot directing the substituent groups to the binding cavities. The adamantyl group (arm 1) extended towards cavity 1 interacting with F94, H95 and I110, while the p-tolyl group (arm 2) was oriented towards cavity 2 were aromatic and hydrophobic interactions with residues Y190, L191 and W194 were possible (Figure 3B).

Furthermore, Hua et al., have also reported the crystal structure of CB2R bound to the selective agonist AM12033 with a resolution of 3.2 Å (PDB:6KPC) ^[26]. Even though AM12033 is structurally different to WIN-55212-2 and 8d, all of them share a common “L-shape” conformation in the orthosteric site ^[24]^[26]. In all cases, a hydrophobic central core occupies part of cavity 3 and connects an arm 2 side chain in cavity 2 with a voluminous and hydrophobic arm 1 that extends towards cavity 1 (Figure 4B).

Figure 4. (a) Structurally different CB2R agonists; (b) Superposition of the CryoEM structure of CB2R with bound agonist WIN-55212-2 (PDB:6PT0, both cyan), the crystal structure of CB2R with the selective agonist AM12033 (PDB:6KPC, both orange) and the predicted binding pose of 8d (green). Residues F94 (red, cavity 1) W194 (blue, cavity 2), F183 (yellow, cavity 3) and I110 (magenta, common residue) are shown for spatial references.

Within the same general scaffold, the steric effect of an additional substituent group in cavity 3 was found to be critical in distinguishing agonists from antagonists. Inverse agonism activity was observed when deep insertion of longer sidechains in cavity 3 allowed strong π-π interaction (3.0 Å) with W258 which plays a key role in receptor activation/inactivation (Figure 5b) ^[24]^[26]. Superposition of the predicted binding pose of 8d with agonist WIN 55,212-2 and antagonist AM10257 showed that 8d does not reach residue W258 (>5 Å) consistent with the observed agonist profile in functional assays (Figure 5 and Figure 6).

Figure 5. (a) Predicted binding mode of compound 8d in CB2R. Hydrophobic and π-π interactions are shown in dashed lines; (b) Distance between W258 residue and 8d.

Figure 6. (a) Superposition of docked 8d (cyan) with bound agonist WIN 55,212-2, (green) in the activated CB2R complex (residues in yellow); (b) Superposition of docked 8d (cyan) with bound antagonist AM10257, (gray) in the activated CB2R complex (residues in yellow).

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Carlos David Pessoa-Mahana

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