Discovery of 7-Azanorbornane-Based Dual Agonists for the Delta and Kappa Opioid Receptors through an In Situ Screening Protocol

In medicinal chemistry, the copper-catalyzed click reaction is used to prepare ligand candidates. This reaction is so clean that the bioactivities of the products can be determined without purification. Despite the advantages of this in situ screening protocol, the applicability of this method for transmembrane proteins has not been validated due to the incompatibility with copper catalysts. To address this point, we performed ligand screening for the µ, δ, and κ opioid receptors using this protocol. As we had previously reported the 7-azanorbornane skeleton as a privileged scaffold for the G protein-coupled receptors, we performed the click reactions between various 7-substituted 2-ethynyl-7-azanorbornanes and azides. Screening assays were performed without purification using the CellKeyTM system, and the putative hit compounds were re-synthesized and re-evaluated. Although the “hit” compounds for the µ and the δ receptors were totally inactive after purifications, three of the four “hits” for the κ receptor were true agonists for this receptor and also showed activities for the δ receptor. Although false positive/negative results exist as in other screening projects for soluble proteins, this in situ method is effective in identifying novel ligands for transmembrane proteins.


Introduction
Click reaction is a term used to indicate a group of reactions that connect two components with excellent functional selectivity, extremely high yields, and inoffensive byproducts, if they exist [1].A representative example is the copper-catalyzed 1,3-dipolar cycloaddition reaction between azides and alkynes (copper-catalyzed azide-alkyne cycloaddition, CuAAC) [2,3].This reaction is used in many fields, including material sciences [4], chemical biology [5], and medicinal chemistry [6,7].In drug discovery projects, the CuAAC reaction is used, by the power of combinatory processes, to prepare a large library of compounds.There are many examples in which appendage diversity of the library components is achieved through this reaction [8,9].In addition, the robustness of this reaction enables biological evaluation of the resultant 1,4-disubstituted triazoles without purification [10].
Thus, a library of compounds can be prepared by reacting azides with alkynes in a microplate and directly used for screening assays.This in situ screening protocol has enabled the effortless preparations of numerous ligand candidates and has led to the discovery of novel enzyme inhibitors [11][12][13][14].Despite these successful examples, applying this method to transmembrane proteins has been difficult.In screening ligands for transmembrane proteins, biological evaluations are usually performed by using whole cells due to the difficulty in the purification and isolation of these transmembrane proteins.However, the cell-based assay systems are incompatible with the copper catalysts used in the CuAAC reactions due to the toxicity of the catalysts [15,16].The cytotoxicity of the catalysts is problematic not only in in situ screening projects, but also in other biological and medicinal applications.To address this concern, copper-free click reactions were developed [17,18].One example is the 1,3-dipolar cycloaddition reaction between strained cyclooctynes and azides (strain-promoted azide-alkyne cycloaddition, SPAAC) [19].This click reaction is also used in in situ ligand screening [15].However, the bulkiness of the cyclooctyne moiety hampers application of this method with broader protein targets.Therefore, few reports have applied the in situ screening method for transmembrane proteins [16,20,21].
G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors, are important drug targets, and nearly one-third of the approved drugs bind to and act on these proteins [22].Previously, we identified some agonists for GPCRs including the κ opioid receptor (KOR) from our original 7-azanorbornane-based compound library [23].Since sp 3 -rich compounds are advantageous in drug development [24], we constructed a small-sized library by modifying the sp 3 -rich 7-azanorbornane scaffold with click reactions (Scheme 1).Screening assays were performed after purification of all the product 1,4-disubstituted triazoles.Although an in situ screening protocol would enhance the efficacy of our screening project, the scarcity of reports in which this method had been applied for GPCRs had prevented us from choosing this protocol.
this reaction enables biological evaluation of the resultant 1,4-disubstituted triazoles without purification [10].Thus, a library of compounds can be prepared by reacting azides with alkynes in a microplate and directly used for screening assays.This in situ screening protocol has enabled the effortless preparations of numerous ligand candidates and has led to the discovery of novel enzyme inhibitors [11][12][13][14].Despite these successful examples, applying this method to transmembrane proteins has been difficult.In screening ligands for transmembrane proteins, biological evaluations are usually performed by using whole cells due to the difficulty in the purification and isolation of these transmembrane proteins.However, the cell-based assay systems are incompatible with the copper catalysts used in the CuAAC reactions due to the toxicity of the catalysts [15,16].The cytotoxicity of the catalysts is problematic not only in in situ screening projects, but also in other biological and medicinal applications.To address this concern, copper-free click reactions were developed [17,18].One example is the 1,3-dipolar cycloaddition reaction between strained cyclooctynes and azides (strain-promoted azide-alkyne cycloaddition, SPAAC) [19].This click reaction is also used in in situ ligand screening [15].However, the bulkiness of the cyclooctyne moiety hampers application of this method with broader protein targets.Therefore, few reports have applied the in situ screening method for transmembrane proteins [16,20,21].
G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors, are important drug targets, and nearly one-third of the approved drugs bind to and act on these proteins [22].Previously, we identified some agonists for GPCRs including the κ opioid receptor (KOR) from our original 7-azanorbornane-based compound library [23].Since sp 3 -rich compounds are advantageous in drug development [24], we constructed a small-sized library by modifying the sp 3 -rich 7-azanorbornane scaffold with click reactions (Scheme 1).Screening assays were performed after purification of all the product 1,4-disubstituted triazoles.Although an in situ screening protocol would enhance the efficacy of our screening project, the scarcity of reports in which this method had been applied for GPCRs had prevented us from choosing this protocol.GPCRs are undoubtedly a most important drug target class, and the applicability of the in situ screening protocol should be explored to encourage future drug discovery.To address this point, here, we constructed the 7-azanorbornane-based 1,4-disubstituted triazole library on microplates and performed screening assays without purification of the compounds.We selected as the targets the KOR, for which we had identified the ligands in a previous report, and the µ and the δ opioid receptors (MOR and DOR, respectively), which are closely related to the KOR.We were able to apply this protocol to GPCRs, but the reliability of the screening results may depend on the sensitivity of the assay systems.GPCRs are undoubtedly a most important drug target class, and the applicability of the in situ screening protocol should be explored to encourage future drug discovery.To address this point, here, we constructed the 7-azanorbornane-based 1,4-disubstituted triazole library on microplates and performed screening assays without purification of the compounds.We selected as the targets the KOR, for which we had identified the ligands in a previous report, and the µ and the δ opioid receptors (MOR and DOR, respectively), which are closely related to the KOR.We were able to apply this protocol to GPCRs, but the reliability of the screening results may depend on the sensitivity of the assay systems.

Construction of the Compound Library by Click Reactions and a Screening Assay without Compound Purification
In this study, as in our previous one, we used the CellKey TM system which uses impedance biosensors to detect the changes of the behavior of the cells upon treatment with test compounds to determine the agonistic activities of the compounds [25].Prior to the screening assay, we estimated the effects of the copper catalyst, copper sulfate on the assay system.We compared the concentration-response curves of DAMGO, a MOR agonist, on MOR-expressing cells in the presence and absence of copper sulfate (Figure S1).The curves were almost identical, and we concluded that the copper catalyst has little effect on the assay system.
Encouraged by this result, we next prepared the library by the click reactions in microplates to be screened by the CellKey TM system without purification (Figure 1).In targeting opioid receptors, we presumed that the presence of basic nitrogens and phenolic groups in the candidates would enhance the hit rates.This idea is based on the observation in the crystal structures of the complexes between morphinan derivatives and the opioid receptors, the presence of ionic interactions between the basic nitrogens of the compounds and Asp 3.32  of the receptors and the hydrogen bonds between the phenolic groups of the compounds and His 6.52 of the receptors through water molecules are preserved [26][27][28] (Figure 2, the superscript denotes Ballesteros-Weinstein numbering [29]).Hence, we prepared the exo-and endo-alkynes 1 with their basic nitrogens on the 7-position of the bicyclic scaffolds and the alkynes 2-4 with the basic nitrogens on the pyridine rings on the 7-substituents (Figure 1).For comparison, we also prepared alkynes 5-7 without basic nitrogens (See Scheme 3 in Section 4 for the preparation methods of the alkynes).By adding alkynes 8 in our previous report [23], we obtained 16 alkynes to be used in the click reactions.In addition to phenolic group-bearing aromatic azides a-c, we also prepared azides d-h with heteroaryl groups whose heteroatoms would function as hydrogen bond acceptors.Again, for comparison, azides i and j without heteroatoms and azide k without the aromatic moiety were also prepared.These 11 azides and the above eight alkynes were conjugated by the click reactions in microplates according to the reported methods [10].

Figure 2.
Interaction between MP1104, a KOR agonist with the morphinan scaffold (black), and the residues in KOR (red) [28].The hydrogen-bonding water molecules are depicted in blue.The A-ring of MP1104 is indicated in the structure.

Validation of the Activities after Purification of the Compounds
The triazoles selected above were synthesized by the standard click reactions using copper (II) sulfate pentahydrate and sodium L-ascorbate as the catalysts [30] (Scheme 2).Then, their agonistic activities were determined (Figure 3).Despite our expectations, exo-6f and exo-7e did not show significant activity for the MOR.As for the DOR, endo-1f, exo-5a, and exo-6d were not active, either.In contrast, three of the four hit triazoles, endo-1b, endo-1i, and endo-7c, showed significant activity for the KOR.These compounds also showed activity for the DOR.In addition, exo-7e, one of the false hits for the MOR, was  Interaction between MP1104, a KOR agonist with the morphinan scaffold (black), and the residues in KOR (red) [28].The hydrogen-bonding water molecules are depicted in blue.The A-ring of MP1104 is indicated in the structure.
The results of the first screening assays are shown in Figure S2.For the MOR and KOR, some compounds exhibited impedance changes three times greater than those of vehicle treatments (MOR: exo-6f and exo-7e, KOR: endo-1b, exo-1b, endo-1i, and endo-7c).Therefore, we re-synthesized these triazoles to validate the activities.As for DOR, no compound met this criterion.Hence, the three most potent compounds, endo-1f, exo-5a, and exo-6d, were selected, although the selection criteria were rather arbitrary.

Validation of the Activities after Purification of the Compounds
The triazoles selected above were synthesized by the standard click reactions using copper (II) sulfate pentahydrate and sodium L-ascorbate as the catalysts [30] (Scheme 2).Then, their agonistic activities were determined (Figure 3).Despite our expectations, exo-6f and exo-7e did not show significant activity for the MOR.As for the DOR, endo-1f, exo-5a, and exo-6d were not active, either.In contrast, three of the four hit triazoles, endo-1b, endo-1i, and endo-7c, showed significant activity for the KOR.These compounds also showed activity for the DOR.In addition, exo-7e, one of the false hits for the MOR, was revealed to be a dual agonist for the DOR and the KOR.At this stage, we evaluated the activities of the parental azides and alkynes of the selected triazoles.Although the azides were completely inactive, to our surprise, four of the six alkynes (endo-1, exo-1, exo-5, and endo-7) showed significant activity for the KOR.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 2 Scheme 2. Syntheses of the selected triazoles to validate the activities.
To confirm that these effects were truly mediated by binding to the DOR or the KOR we evaluated the compounds in the presence and absence of selective antagonists for the receptors [31][32][33][34] (Figure 4).Indeed, the agonistic activities of the four DOR agonists and the eight KOR agonists were abolished in the presence of naltrindole and norBNI, respec tively.revealed to be a dual agonist for the DOR and the KOR.At this stage, we evaluated the activities of the parental azides and alkynes of the selected triazoles.Although the azides were completely inactive, to our surprise, four of the six alkynes (endo-1, exo-1, exo-5, and endo-7) showed significant activity for the KOR.To confirm that these effects were truly mediated by binding to the DOR or the KOR, we evaluated the compounds in the presence and absence of selective antagonists for the receptors [31][32][33][34] (Figure 4).Indeed, the agonistic activities of the four DOR agonists and the eight KOR agonists were abolished in the presence of naltrindole and norBNI, respectively.
Although it was difficult to detect the concentration dependence of the weak agonists such as exo-1, exo-5, and endo-7 for the KOR, most of the agonists showed concentrationdependent effects on the receptors (Figure 5).Even at the highest concentrations (10 −5 M), most of the concentration-response curves did not reach plateaus.Despite this outcome, we determined the preliminary E max and logEC 50 values from these curves (Table S1).Two of the hit triazoles for the KOR, endo-1b and endo-7c, seemed to be full agonists for the KOR and partial agonists for the DOR.In addition, exo-7e, which was not regarded as the hit compound for the DOR nor the KOR at the first screening, was revealed to be a full dual agonist for these receptors.Even more surprisingly, one of the parental alkynes endo-1, with the very low molecular weight of 211.3 Da, was the most potent KOR agonist of all.Although it was difficult to detect the concentration dependence of the weak agonists such as exo-1, exo-5, and endo-7 for the KOR, most of the agonists showed concentrationdependent effects on the receptors (Figure 5).Even at the highest concentrations (10 −5 M), most of the concentration-response curves did not reach plateaus.Despite this outcome, we determined the preliminary Emax and logEC50 values from these curves (Table S1).Two of the hit triazoles for the KOR, endo-1b and endo-7c, seemed to be full agonists for the KOR and partial agonists for the DOR.In addition, exo-7e, which was not regarded as the hit compound for the DOR nor the KOR at the first screening, was revealed to be a full dual agonist for these receptors.Even more surprisingly, one of the parental alkynes endo-1, with the very low molecular weight of 211.3 Da, was the most potent KOR agonist of all.

Applicability of the In Situ Screening Protocol for the Opioid Receptors
In this study, we applied the in situ screening method for the opioid receptors, which are members of the GPCR family.Three of the four putative hit compounds for KOR were proven to be true agonists for this receptor after re-synthesis and re-evaluation.Hence, it is safe to say that this in situ screening method is reliable enough for the KOR.Contrary to this, the putative hit compounds for the MOR and the DOR were revealed to be inactive toward these receptors.What can account for these results?Four of these false hits for the MOR and the DOR have pyridine moieties that can coordinate with the copper catalyst.Hence, one possibility is that these complexes interfered with the screening system to enumerate the false hits.As another possibility, the difference in the dynamic ranges of the assay systems may explain the results.In this study, we used the change of impedance of the cells as the indicator of the agonistic activities.In the case of the KOR, the "change of impedance" value of the cells treated with the positive control was 13 fold greater than that of vehicle treatment (Figure 2).In the case of the MOR and DOR, the differences between the vehicle and the positive controls were only five and ten-fold, respectively.Thus, the higher noise ratio of the assay system may have concealed the activities of the agonists.Indeed, the agonistic activities of exo-7e, endo-1b, endo-1i, and endo-7c for the DOR were not detected at the first in situ screening stage.As for the KOR, two weak agonists found in our previous study, exo-8b and exo-8d, whose efficacies at the concentration of 10 −5 M were almost 30% of that of U-50,488H [23], were not detected as hit compounds in this in situ screening.Such discrepancies between the in situ screening results and validation assays have been frequently reported in other reports and the inactivity of a compound in an in situ screening does not rule out the possibility that the compound is indeed an agonist [16,20,35].Hence, although we have not obtained an agonist for the MOR from our 7azanorbornane-based library so far, there remains the possibility that such compounds may be identified from this library by using screening systems with higher dynamic ranges and lower noise ratios.

Applicability of the In Situ Screening Protocol for the Opioid Receptors
In this study, we applied the in situ screening method for the opioid receptors, which are members of the GPCR family.Three of the four putative hit compounds for KOR were proven to be true agonists for this receptor after re-synthesis and re-evaluation.Hence, it is safe to say that this in situ screening method is reliable enough for the KOR.Contrary to this, the putative hit compounds for the MOR and the DOR were revealed to be inactive toward these receptors.What can account for these results?Four of these false hits for the MOR and the DOR have pyridine moieties that can coordinate with the copper catalyst.Hence, one possibility is that these complexes interfered with the screening system to enumerate the false hits.As another possibility, the difference in the dynamic ranges of the assay systems may explain the results.In this study, we used the change of impedance of the cells as the indicator of the agonistic activities.In the case of the KOR, the "change of impedance" value of the cells treated with the positive control was 13 fold greater than that of vehicle treatment (Figure 2).In the case of the MOR and DOR, the differences between the vehicle and the positive controls were only five and ten-fold, respectively.Thus, the higher noise ratio of the assay system may have concealed the activities of the agonists.Indeed, the agonistic activities of exo-7e, endo-1b, endo-1i, and endo-7c for the DOR were not detected at the first in situ screening stage.As for the KOR, two weak agonists found in our previous study, exo-8b and exo-8d, whose efficacies at the concentration of 10 −5 M were almost 30% of that of U-50,488H [23], were not detected as hit compounds in this in situ screening.Such discrepancies between the in situ screening results and validation assays have been frequently reported in other reports and the inactivity of a compound in an in situ screening does not rule out the possibility that the compound is indeed an agonist [16,20,35].Hence, although we have not obtained an agonist for the MOR from our 7-azanorbornane-based library so far, there remains the possibility that such compounds may be identified from this library by using screening systems with higher dynamic ranges and lower noise ratios.

Preliminary Structure-Activity Relationship Information of the Obtained Agonists
In this study, the triazoles exo-7e, endo-1b, endo-1i, and endo-7c were revealed to be dual agonists for DOR and KOR.With respect to substituents on the 7-nitrogen atoms, two of the triazoles have cyclopropane carbonyl groups and the other two have benzyl groups.Hence, contrary to our expectations, the 7-nitrogen atoms on the 7-azanorbornanes did not serve as a surrogate for the basic nitrogen atoms of the morphinan derivatives.As for the substituents on the triazole rings, the four triazole agonists have aromatic rings and endo-1i without a hydrogen bond acceptor is slightly less potent than the others.Hence, this moiety may interact with the receptors in a similar manner as the A rings of the morphinan derivatives (Figure 2).We are now undertaking a structure-activity relationship study to elucidate the interaction mode between the opioid receptors and 7-azanorbornane-based triazole agonists.
A few dual agonists for DOR and KOR have been identified, such as MP1104 [36] and a literature compound 7a [37], and these agonists are morphinan derivatives.In this study, we succeeded in identifying a novel class of dual agonists that are structurally discrete from known agonists.Compared to the morphinan derivatives with EC 50 values in nanomolar to subnanomolar ranges [36,37], the 7-azanorbornane derivatives have been much less potent.Yet, two of them, exo-7e (309.37 Da, CLogP = −0.302)and endo-7c (324.38 Da, CLogP = 0.528, calculated using ChemDraw Professional version 22.2 (PerkinElmer Informatics, Waltham, MA, USA)), fell in the "lead-like space" (molecular weight: 200 to 350 Da and CLogP: −1 to 3 [38]) and stand as a reasonable starting point for structural development.These derivatives did not show activity toward the MOR, which is associated with opioid addiction [39,40].Such dual agonists for DOR and KOR have recently been expected to be candidates for antinociceptive agents without drug dependence [36,41].Hence, the triazoles discovered herein will serve as a reasonable starting point for the development of analgesic drugs without abuse liability by the concept of polypharmacology.
In addition to the above-mentioned dual agonists, we found that endo-1 was a KOR agonist which is more potent than the triazoles.Such a small agonist has not been discovered thus far.Future structure-activity relationship studies will determine the minimal structural unit which is indispensable for agonistic activity and may reveal the interaction pattern between the compound and the receptor.
Molecules 2023, 28, x FOR PEER REVIEW Scheme 3. Preparation of the alkynes to be used in the click reactions.

Preparation of Alkynes 1-7 2-endo-Ethynyl-7-benzyl-7-azabicyclo [2.2.1]heptane (endo-1)
To a solution of endo-9 (118 mg, 0.535 mmol) in dichloromethane (4 mL) wa trifluoroacetic acid (0.5 mL) at 0 °C.After stirring at ambient temperature for 1 h, ture was concentrated under reduced pressure, and the excess trifluoroacetic a removed by repeated evaporation with toluene.The crude ammonium salt was the next reaction without further purification.To a suspension of the crude amm salt and potassium carbonate (355 mg, 2.57 mmol) in dry acetone (2 mL) was added bromide (89 µL, 0.75 mmol) at 0 °C.After stirring for 3 h at this temperature, w

Preparation of Alkynes 1-7 2-endo-Ethynyl-7-benzyl-7-azabicyclo [2.2.1]heptane (endo-1)
To a solution of endo-9 (118 mg, 0.535 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (0.5 mL) at 0 • C.After stirring at ambient temperature for 1 h, the mixture was concentrated under reduced pressure, and the excess trifluoroacetic acid was removed by repeated evaporation with toluene.The crude ammonium salt was used for the next reaction without further purification.To a suspension of the crude ammonium salt and potassium carbonate (355 mg, 2.57 mmol) in dry acetone (2 mL) was added benzyl bromide (89 µL, 0.75 mmol) at 0 • C.After stirring for 3 h at this temperature, water (30 mL) was added to the mixture.The mixture was then extracted with dichloromethane (30 mL × 3), and the combined organic layers were washed with brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure.Column chromatography (silica gel 5 g, n-hexane/ethyl acetate, 100:1 to 30:1) gave the title compound (107 mg, 0.506 mmol, 94.6%) as a colorless oil. 1

CellKey TM Assay
The CellKey TM assay has been described previously [52].Briefly, CellKey TM buffer was prepared by adding 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 0.1% bovine serum albumin to Hanks's balanced salt solution (1.3 mM CaCl 2 •2H 2 O, 0.81 mM MgSO 4 , 5.4 mM KCl, 0.44 mM KH 2 PO 4 , 4.2 mM NaHCO 3 , 136.9 mM NaCl, 0.34 mM Na 2 HPO 4 and 5.6 mM D-glucose).At day 1, the cells were seeded onto poly-D-lysinecoated CellKey TM 96-well microplates at a density as follows: flag-MOR, 6×10 4 cells per well; T7-DOR and myc-KOR, 7 × 10 4 cells per well.At day 2, the cells were washed with CellKey TM buffer, and incubated in this buffer at ambient temperature for 30 min (for antagonist assay, 10 µM of naltrindole or nor-BNI was added to this CellKey TM buffer).After monitoring the impedance baseline for 5 min, the compounds were added, and impedance currents within 30 min were measured.The values in Figures 3-5 and Figures S1 and S2 were calculated by dividing the changes of impedance by the compounds (maxmin) by the changes of impedance by vehicle treatment (max-min).Statistical significance of the difference was assessed by one-way ANOVA followed by Dunnet's test (Figure 3) or Bonferroni's test (Figure 4).DAMGO, SNC80 and naltrindole were purchased from

Scheme 1 .
Scheme 1. Construction of the 7-azanorbornane-based compound library constructed by the click reactions.Structures of the KOR agonists identified in our previous study are also shown.

Scheme 1 .
Scheme 1. Construction of the 7-azanorbornane-based compound library constructed by the click reactions.Structures of the KOR agonists identified in our previous study are also shown.

20 Figure 1 .
Figure 1.Schematic outline of library construction and first screening assay using the CellKey TM system.

Figure 1 .
Figure 1.Schematic outline of library construction and first screening assay using the CellKey TM system.

Figure 1 .
Figure 1.Schematic outline of library construction and first screening assay using the CellKey TM system.

52 AFigure 2 .
Figure 2.Interaction between MP1104, a KOR agonist with the morphinan scaffold (black), and the residues in KOR (red)[28].The hydrogen-bonding water molecules are depicted in blue.The A-ring of MP1104 is indicated in the structure.

Scheme 2 .
Scheme 2. Syntheses of the selected triazoles to validate the activities.

Figure 3 .
Figure 3. Agonistic activities of the triazoles and the parental alkynes and azides toward the opioid receptors.The positive controls are DAMGO (for MOR), SNC80 (for DOR), and (-)-U-50,488H (for KOR).All the compounds, including the positive controls were treated at the concentration of 10 µM.

Figure 3 .
Figure 3. Agonistic activities of the triazoles and the parental alkynes and azides toward the opioid receptors.The positive controls are DAMGO (for MOR), SNC80 (for DOR), and (-)-U-50,488H (for KOR).All the compounds, including the positive controls were treated at the concentration of 10 µM.

Figure 4 .
Figure 4. Agonistic activities in the presence and absence of selective antagonists for (a) DOR and (b) KOR.The concentrations of both the agonists and the antagonists were 10 −5 M.

Figure 4 . 20 Figure 5 .
Figure 4. Agonistic activities in the presence and absence of selective antagonists for (a) DOR and (b) KOR.The concentrations of both the agonists and the antagonists were 10 −5 M. Molecules 2023, 28, x FOR PEER REVIEW 8 of 20

Figure 5 .
Figure 5.The dose-response curves of the triazoles and the alkynes for (a) DOR and (b) KOR.

Figure 5 .
Figure 5.The dose-response curves of the triazoles and the alkynes for (a) DOR and (b) KOR.

or 3 .
Scheme 3. Preparation of the alkynes to be used in the click reactions.