Biological Effects on μ-Receptors Affinity and Selectivity of Arylpropenyl Chain Structural Modification on Diazatricyclodecane Derivatives

Opioid analgesics are clinically used to relieve severe pain in acute postoperative and cancer pain, and also in the long term in chronic pain. The analgesic action is mediated by μ-, δ-, and κ-receptors, but currently, with few exceptions for k-agonists, μ-agonists are the only ones used in therapy. Previously synthesized compounds with diazotricyclodecane cores (DTDs) have shown their effectiveness in binding opioid receptors. Fourteen novel diazatricyclodecanes belonging to the 9-propionyl-10-substituted-9,10-diazatricyclo[4.2.1.12,5]decane (compounds 20–23, 53, 57 and 59) and 2-propionyl-7-substituted-2,7-diazatricyclo[4.4.0.03,8]decane (compounds 24–27, 54, 58 and 60) series, respectively, have been synthesized and their ability to bind to the opioid μ-, δ- and κ-receptors was evaluated. Five of these derivatives, compounds 20, 21, 24, 26 and 53, showed μ-affinity in the nanomolar range with a negligible affinity towards δ- and κ-receptors and high μ-receptor selectivity. The synthesized compounds showed μ-receptor selectivity higher than those of previously reported methylarylcinnamyl analogs.


Introduction
Pain is an essential defense the human body activates as a result of noxious stimuli. It can be defined as a sensorial and emotional experience correlated to tissue damage. The perception of pain as such is subjective when considering severity and tolerance. Pain is classified into acute and chronic. Acute pain is mediated by nociceptors activation in the tissue damage site, it is transient, and intensity decreases with the healing of the injury that caused it. Chronic pain (CNP), on the other hand, lasts longer than six months and may continue after the injury or illness has been treated. Therefore, CNP is considered a pathologic process involving the somato-sensory system, caused by abnormal processing of stimuli arriving from cellular damage location in the central nervous system (CNS) or peripheral nervous system (PNS) [1]. Chronic neuropathic pain (CNP) can result from surgical treatment, diabetes, spinal injury, multiple sclerosis and several other conditions. It affects a high percentage of adults globally and the treatment of chronic pain is a crucial issue worldwide. Opioid analgesics are the oldest and still the most potent drugs widely Structure-activity relationship (SAR) studies on these templates have emphasized that the cinnamyl portion played a pivotal role in µ-affinity. To further evaluate the influence of the endoethanic bridge of 3,8-diazatricyclo[3.2.1]octane (DBO) structure on the receptor-drug complex stability, it was considered useful to evaluate its homologation to an endopropanic bridge to give the 3,9-diazabicyclo[3.2.1]nonane (DBN) scaffold (compounds 3 and 4, Figure 1). The in vitro data of diazabicycloctane and diazabicyclononane analogs showed that the dimensions of the loop induced different effects on the corresponding ligands [22,23]. Nevertheless, the endoethanic and endopropanic bridges played a pivotal role in the interaction with the µ-receptor.
Therefore, assuming that the introduction of a second endoethanic bridge on the piperazine portion of the DBO could be a powerful feature for the interaction with the µ-receptor, two novel cores were synthesized: the 9,10-diazatricyclo[4.2.1.1 2,5 ]decane (DTD) moiety 5, containing two bridges between atoms 1,6 and 2,5 of DTD and its isomer 2,7diazatricyclo  The appropriate replacement on the nitrogen atoms of the two DTDs, both with the propionyl group and the cinnamyl chain, led to the identification of compounds typified by general structures 7 and 8 [24] (Figure 3), whose in vitro binding studies indicated a significant selectivity towards µ receptors, concerning to κ and δ, for both series of DTDs, some of which, for R 1 = CH 3 , with µ-affinity values (K i = 1.29 − 4.07 nM) comparable to morphine (K i = 1.07 nM) [24]. In order to better define the influence of the cinnamyl side chain for the interaction of 7 and 8 with the receptor site, two different modifications were designed: the incorporation of a CH 3 group on the cinnamyl chain both into a rigid benzocondensed structure and into a bicyclic heteroaromatic system. Therefore, we started with the substitution of the methylcinnamyl chain with an indenylidenic group to afford derivatives of general structures 9 and 10 ( Figure 4) by reacting the appropriate aldehydes with the DTD. Unexpectedly, and only for the condensation between the aldehydes and the 2-propionyl-2,7-diazatricylo[4.4.0.0 3,8 ]decane bicyclic system, the endo derivative 11 was also obtained ( Figure 4).
As the second step of our project, we planned the introduction of a heteroaromatic system that mimics the cinnamyl chain, synthesizing derivatives of general structures 12 and 13 ( Figure 4). In this paper we report the synthesis and the binding data against µ-, δand κ-receptors of novel DTD derivatives, compounds 20-27, 53-54 and 57-60, reported in Table 1, in which the side cinnamyl chain is forced into a limited number of conformations.

Chemistry
Final derivatives 20-27, were synthesized as reported in Scheme 1, by using aldehydes synthesized as described in Scheme 2. Compounds 53-54 and 57-60 were synthesized as reported in Schemes 3 and 4, respectively. The amine intermediates 9-propionyl-9,10diazatricyclo[4.2.1.1 2,5 ]-decane (14) and 2-propionyl-2,7-diazatricyclo[4.4.0.0 3,8 ]decane (15), synthesized following the literature [25], were used as starting compounds. A first attempt to prepare derivatives 20-27 provided for a synthetic approach similar to that used for both DBO and DBN series, consisting of a simple alkylation of amines 14 and 15 with the required alkyl chlorides, but unexpectedly this condensation failed. Therefore, we planned a sodium cyanoborohydride reductive amination between a slight molar excess of amine 14 and the appropriate aldehyde 16-19 in methanol, in the presence of a catalytic amount of acetic acid. Subsequent purification by flash chromatography of the crude products gave the desired compounds 20-23 (Scheme 1).   The same reaction with the amine 15 led to a mixture of compounds, whose separation by flash chromatography, led us to obtain two fractions in a 2:1 ratio: the former, the expected exo compounds 24-27, whereas the latter corresponded to the the unexpected endo derivatives 28-31 (Scheme 1).
The three-step synthetic route for the preparation of aldehydes 16-19 is depicted in Scheme 2. The commercially available indanones 32-35 underwent a Horner-Wadsworth-Emmons reaction to afford the corresponding esters 36-39, in accordance with literature for 35 [26]. This synthetic pathway, providing the use of triethyl phosphonacetate (TEFA) and sodium hydride in anhydrous toluene, under a N 2 atmosphere, afforded a mixture of the exo-E (36a-39a), exo-Z (36b-39b) and endo (36c-39c) isomer esters and a 25-30% amount of unreacted starting product. The exo-E isomers 36a-39a, separated by flash chromatography, were reduced to the respective alcohols 40-43 with diisobutylaluminium hydride ( [25], were reacted with the indole carbonyl chloride 46, obtained by thionyl chloride treatment of the corresponding commercial acid, to give derivatives 47 and 48, respectively. Their N-debenzylation with hydrogen on 10% palladium on carbon afforded 49 and 50, whose reduction with lithium aluminum hydride in tetrahydrofuran gave compounds 51 and 52. The final acylation with propionic anhydride yielded derivatives 53 and 54. N-propionyldecanes (14, 15) served as starting compounds also for the synthesis of derivatives 57-60. The reaction of intermediates 14 and 15 with the commercial chloromethyl quinoline hydrochloride (55) or bromomethyl quinoxaline (56) [27] in the presence of K 2 CO 3 , furnished the desired derivatives (Scheme 4).

Radioligand Binding Assay
The newly synthesized compounds 20-27, 53-54 and 57-60 were assayed in binding studies on µ-, δ-, and κ-opioid receptors (Table 1), performed on mouse brain homogenates in the presence of 3 H-DAMGO for µ-receptor, 3 H-DELTORPHINE II for δ-receptor and 3 H-U69593 for κ-receptor. By comparison, the K i value of the reference compound morphine is reported. Within the 9,10-diazatricyclo which showed comparable effects on µ-binding affinity. Compound 24 showed a 2-fold lower affinity (K i = 100 nM) if compared to its isomer 20, whereas the introduction of a chlorine atom in the indene ring, compound 26, led to a comparable µ-receptor affinity (K i = 75 nM) with both 20 and 24. The introduction of the indolic chain, compound 54, resulted in a 23-fold lower affinity (K i = 500 nM) with respect to its isomer 53. In general, the introduction of a methylene-quinoline (57, 58) or -quinoxaline (59, 60) substituent on a nitrogen atom of DTD templates led to a decrease of µ-receptor affinity.

General Procedure for the Synthesis of Alcohols 40-43
To a solution of esters 36a-39a (540 mg, 0.228 mmol) in dry toluene (15.60 mL) under nitrogen atmosphere, a 25% solution of diisobutylaluminium hydride (DIBAL-H) in dry toluene (4.32 mL, 6.42 mmol) was added at 0 • C and the mixture stirred for 1 h. Then, a saturated K + /Na + tartrate aqueous solution was added and the mixture was stirred overnight at room temperature. The mixture was taken up with diethyl ether and the organic phase was separated, washed (H 2 O), dried (Na 2 SO 4 ) and evaporated under vacuum to afford the pure alcohols as an oil (40) [29] or a solid (41-43).

General Procedure for the Synthesis of Aldehydes 16-19
A solution of alcohol 40-43 (180 mg, 0.753 mmol) and MnO 2 85% (161 mg, 1.85 mmol) in CH 2 Cl 2 was stirred at room temperature for 3 h. The unreacted MnO 2 was filtered off and the solvent was evaporated under vacuum to give the pure aldehydes 16-19 as yellow solids.

Procedure for the Synthesis of Compounds 47 and 48
A mixture of 44 or 45 (3 mmol) in toluene (7 mL), acyl chloride (46) (3 mmol) and triethylamine (3 mmol), was stirred at room temperature for 3 h. Then, 4 mL of water was added and continued to stir for another 10 min. The reaction mothers were extracted with CH 2 Cl 2 , which by in vacuum evaporation supplied crude products such as light-yellow solids.

Procedure for the Synthesis of Compounds 49 and 50
To an ethanol solution (7 mL) of the appropriate derivatives 47 or 48 (1.9 mmol), 0.20 g of 10% Pd/C was added. The mixture was hydrogenated at 60 • C for 7 h, then the catalyst was removed by Celite ® filtration and the solvent evaporated to obtain the pure compounds 49 and 50 as white solids.

Procedure for the Synthesis of Compounds 51 and 52
A solution of the derivative 49 or 50 (1.5 mmol) in tetrahydrofuran (THF) (23 mL) was dripped into a solution in THF of LiAlH 4 (6 mmol) previously cooled to 0 • C with ice. The mixture was stirred overnight at room temperature and then it was cooled to 0 • C and taken up with ethyl ether (10 mL) and water (1 mL). The white solid that formed was removed and the solution was concentrated, the oily residue was taken up with a 1:1 mixture of water and dichloromethane; the separated organic phase was dried (Na 2 SO 4 ) and evaporated under vacuum to give compounds 51 and 52 as light-yellow solids.  (15) (0.96 mmol) respectively, in 9 mL of acetone and in the presence of K 2 CO 3 (0.96 mmol). The mixture was stirred overnight at 60 • C. In the end, the salt was filtered off and the liquor mothers were evaporated to obtain derivatives 57-60 as crude products that were purified by flash chromatography.

Molecular Docking
Docking simulations were conducted with GOLD version 5.2 (The Cambridge Crystallographic Data Centre, Cambridge, UK). This program uses a genetic algorithm to calculate up to ten docking poses per input-ligand. The resulting poses were evaluated with the scoring function GoldScore that takes into account hydrogen bonding, ligand internal strains, and steric aspects of the receptor-ligand complex. The crystal structure of the β-FNA-MOR complex (PDB-entry 4DKL1) [33] was prepared for docking by adding hydrogens and deleting all water molecules except 718 and 719. The remaining two water molecules were set to "toggle and spin". This allowed the program to automatically decide to include the water molecule in a simulation run and to optimize the orientation of the water molecule. The area of 6 Å around the co-crystallized ligand was defined as the binding site.

Conclusions
In summary, the purpose of this study was to better describe the impact of the cinnamyl side chain to enhance the binding with the opioid receptor site. This was achieved by the inclusion of a methyl group on the cinnamyl chain both into a rigid benzocondensed structure and into a bicyclic heteroaromatic system. Herein we have reported the synthesis of a small series of compounds containing 9-propionyl-10-substituted-9,10-diazatricyclo Derivatives 20-27, 53, 54 and 57-60 were evaluated in µ-, δand κ-opioid receptor binding assays and, in general, both series showed higher µ-receptor selectivity than that of previously reported methylarylcinnamyl analogs. On the other hand, these novel ligands showed a reduced µ-affinity compared to the previous series.
From these studies, it is possible deduce that the incorporation of the methyl group on cinnamic chain into a rigid benzo-condensed structure led to templates endowed with 10-fold less affinity towards µ-receptors and negligible for δand κ-receptors with >1 µM K i affinity values, but at the same time the resulting indenylidenic group is responsible of increased µ-receptor selectivity. Compound 20 turned out as the most promising derivative from the indenylidenic-9,10-diazatricyclo[4.2.1.12,5]decane series with a µ-opioid receptor affinity of 50 nM. The substitution with a fluorine (21), chlorine (22) or bromine atom (23) on C5 of 2,3-dihydro-1H-indene system uncovers a different effect on µ-receptor affinity. The sole compound 21, bearing a fluorine atom, conserved a similar receptor affinity (K i = 65 nM) when compared to parental compound 20. While derivatives 22 and 23 showed K i values higher than 1 µM.
Concerning the virtual structural stiffening of cinnamyl chain, by introducing three different heterocyclic systems on DTD templates, only the indolic ring seems to be positive for this class of compounds, resulting in derivative 53 endowed with the high µ-receptor affinity, being the best among all compounds herein reported. By molecular docking mechanism assessment, arises a stronger interaction with the target binding site due to the nitrogen atom of the indole moiety that acts as hydrogen bond donor for Asp147.
In conclusion, this work evidenced as the flexibility of the cinnamyl chain is a prerequisite for the µ-receptor affinity of these derivatives, whereas its constriction in benzocondensed or hetero-bicyclic system seems responsible for its selectivity for the same receptors. This work also showed a series of new derivatives endowed with an increased selectivity towards µOR associated with a lower µOR affinity. Further investigations will be carried out to synthesize new benzocondensed derivatives with a higher µOR affinity.

Informed Consent Statement: Not applicable.
Data Availability Statement: All the data are reported in this paper.