Novel 1-(1-Arylimiazolin-2-Yl)-3-Arylalkilurea Derivatives with Modulatory Activity on Opioid MOP Receptors

μ-opioid receptor ligands such as morphine and fentanyl are the most known and potent painkillers. However, the severe side effects seen with their use significantly limit their widespread use. The continuous broadening of knowledge about the properties of the interactions of the MOP receptor (human mu opioid receptor, OP3) with ligands and specific intracellular signaling pathways allows for the designation of new directions of research with respect to compounds with analgesic effects in a mechanism different from classical ligands. Allosteric modulation is an extremely promising line of research. Compounds with modulator properties may provide a safer alternative to the currently used opioids. The aim of our research was to obtain a series of urea derivatives of 1-aryl-2-aminoimidazoline and to determine their activity, mechanism of biological action and selectivity toward the MOP receptor. The obtained compounds were subjected to functional tests (cAMP accumulation and β-arrestin recruitment) in vitro. One of the obtained compounds, when administered alone, did not show any biological activity, while when co-administered with DAMGO, it inhibited β-arrestin recruitment. These results indicate that this compound is a negative allosteric modulator (NAM) of the human MOP receptor.


Introduction
Opioid receptors are rhodopsin-like G protein-coupled receptors (GPCRs).They play an important role in the neuromodulation of the nociception process at all levels of neuronal pathways, both at the central and peripheral levels [1].The main molecular target of opioid drugs, such as morphine, fentanyl or oxycodone used in pain therapy, is the µ-opioid receptor.Its activation triggers a cascade of intracellular signaling pathways related to G proteins, arrestin and scaffolding proteins.The key to inducing analgesia is, among others, the inhibition of adenylate cyclase, VGCCs and the opening of GIRK channels [2].Despite the high effectiveness of opiates in pain control, their widespread use is limited by the incidence of side effects, including nausea, vomiting and constipation, as well as the life-threatening depression of the respiratory center [3,4].Additionally, the use of opioids leads to an imbalance and the structural remodeling of the reward system and, consequently, addiction [5].For these reasons, there are ongoing studies on the discovery and development of analgesic drugs, the safety profile of which will be more favorable than that of the opioids currently used.
One of the most promising strategies developed in the search for novel opioid analgesics is allosteric modulation.Positive allosteric modulators (PAMs) stimulate the orthosteric pocket of the MOP receptor by interacting with the allosteric site.Allosteric modulators have characteristic properties that distinguish them from other types of ligands.Particularly important in the pharmacological context is their high selectivity.In contrast to the orthosteric pocket, which is highly conserved in many related receptors, the allosteric site can be located in various areas and exhibits high structural variability, which allows for selectivity toward particular types and even subtypes of GPCRs.In addition, the allosteric modulator, stabilizing the particular conformational state of the receptor, has a different effect on the affinity and/or efficacy of individual orthosteric ligands.What is more, the modulator can induce bias receptor signaling, triggering only the selected signaling pathway [6].Thus, allosteric modulators are an extremely attractive target for the search of new compounds with specific biological activity.They make it possible to obtain drugs with high receptor selectivity and limited side effects, especially compared to some that are already present or have just been introduced to the market, like PAM ligands for the calcium-sensing receptor (cinacalcet), GABA A (benzodiazepines), NAM ligands for chemokine receptor CCR5 (maraviroc), mGluR5 (fenobam, raseglurant, dipraglurant and VU-29) or muscarinic receptor (M1 PAM -MK7622), or are in advanced stages of clinical trials (M2/M4 PAM, CB2 PAM, 5-HT 2C PAM, CCR5 NAM, CXCR3 NAM and β 2 A NAM).
Currently, only a few (Figure 1) exogenous PAMs for the MOP receptor are known: BMS986121, BMS986122 [7], ignavine [8], MS1 and some of its analogs [9]; and NAMs (THC and CBD [10], cannabis secondary metabolites) and two dualsteric ligands, INTA and NCQ [11,12].Developing biased agonists of the MOP receptor could be very promising due to their activation of only one pathway-the G protein pathway, without β-arrestin-2 involvement.Oliceridine was first mentioned in the literature [13,14] and accepted by FDA [15].Unfortunately, after prolonged administration, it produced side effects similar to those of typical MOP receptor agonists, not distinguishing between both pathways and building up to drug tolerance and abuse [16].A new, very promising compound from that group is PZM-21 [17], which is currently undergoing clinical tests.In recent years, Matosiuk et al. have obtained a number of 1-aryl-imidazoline-2-yl urea derivatives with interesting antinociceptive properties [18,19].Some of them have MOP receptor affinity at the micromolar level, which was evaluated using binding assay tests.On the other hand, behavioral tests ('hot plate' and 'writhing test') carried out on mice confirmed their strong analgesic effects, partially reversible after the administration of naloxone.Their administration in under-threshold doses increased significantly the effect of morphine (lower than 5 mg/kg) or DAMGO (lower than 0.5 mg/kg) [20], producing strong antinociception.Importantly, these compounds did not cause depression of the respiratory center in laboratory animals.The obtained results indicate that they can interact with the MOP receptor in a different way than its classical and non-classical ligands [19].
In this paper, we present the results of the synthesis of 1-arylimidazoline urea derivatives (Scheme 1, Table 1.) and in vitro examination of their effect on the MOP receptor, including functional tests (cAMP inhibition and β-arrestin recruitment assays).1-aryl-2aminoimidazoline hydrobromides, which were necessary for synthesis, were obtained from respective N-aryl-1,2-diaminoethanes and cyanogen bromide according to our previous publication [17].1,2-Diaminoethanes were obtained through the Lehmann method [21] with the use of aziridine and anhydrous aluminum trichloride or through the Strecker method with the Takeda modification [22,23] (for methoxy derivatives) with the use of methanal, sodium cyanide and a solution of ammonia.Commercially available arylakylamines were bought.Other branched arylalkylamines were synthesized from the beginning.1-Arylpropyl-2-amine derivatives (6a-r) were obtained from nitroethane and aromatic aldehydes via Henry's method (acetic acid, ammonium acetate, catalytic reduction with hydrogen in the presence of Raney nickel) [24,25] or heteroaromatic aldehydes (methanol in the presence of butyl amine, reduction with lithium aluminum tetrahydride) [26].Benzylideneacetone derivatives were obtained via the Cleisen-Schmidt method [27].Further on, they were transformed into oximes [28].The reduction of oximes with hydrogen and Raney nickel or with lithium aluminum tetrahydride led to respective 1-arylbutyl-3-amines (7a-p) [26,29].The presence of the urea moiety diminished the solubility of obtained compounds, whereas the introduction of longer and branched aliphatic linkers improved it.Therefore, from among 54 synthesized derivatives, only 22 were selected and tested in vitro.The selection was made based on the verification of solubility via spectrophotometric methods.Less soluble derivatives were omitted due to the production of unreliable results.The methodology of that spectral technique was described in our previous publication from this series [19].In this paper, we present the results of the synthesis of 1-arylimidazoline urea derivatives (Scheme 1, Table 1.) and in vitro examination of their effect on the MOP receptor, including functional tests (cAMP inhibition and β-arrestin recruitment assays).1-aryl-2-aminoimidazoline hydrobromides, which were necessary for synthesis, were obtained from respective N-aryl-1,2-diaminoethanes and cyanogen bromide according to our previous publication [17].1,2-Diaminoethanes were obtained through the Lehmann method [21] with the use of aziridine and anhydrous aluminum trichloride or through the Strecker method with the Takeda modification [22,23] (for methoxy derivatives) with the use of methanal, sodium cyanide and a solution of ammonia.Commercially available arylakylamines were bought.Other branched arylalkylamines were synthesized from the beginning.1-Arylpropyl-2-amine derivatives (6a-r) were obtained from nitroethane and aromatic aldehydes via Henry's method (acetic acid, ammonium acetate, catalytic reduction with hydrogen in the presence of Raney nickel) [24,25] or heteroaromatic aldehydes (methanol in the presence of butyl amine, reduction with lithium aluminum tetrahydride) [26].Benzylideneacetone derivatives were obtained via the Cleisen-Schmidt method [27].Further on, they were transformed into oximes [28].The reduction of oximes with hydrogen and Raney nickel or with lithium aluminum tetrahydride led to respective 1-arylbutyl-3-amines (7a-p) [26,29].The presence of the urea moiety diminished the solubility of obtained compounds, whereas the introduction of longer and branched aliphatic linkers improved it.Therefore, from among 54 synthesized derivatives, only 22 were selected and tested in vitro.The selection was made based on the verification of solubility via spectrophotometric methods.Less soluble derivatives were omitted due to the production of unreliable results.The methodology of that spectral technique was described in our previous publication from this series [19].

Results
The tested compounds (in agonist mode, Table 2, Figure 2) exhibited no effect on cAMP accumulation both in 0.1 and 10 µM concentrations or on allosteric properties at both concentrations (Table 3, Figure 3).

Results
The tested compounds (in agonist mode, Table 2, Figure 2) exhibited no effect on cAMP accumulation both in 0.1 and 10 µM concentrations or on allosteric properties at both concentrations (Table 3, Figure 3).

Results
The tested compounds (in agonist mode, Table 2, Figure 2) exhibited no effect on cAMP accumulation both in 0.1 and 10 µM concentrations or on allosteric properties at both concentrations (Table 3, Figure 3).

Results
The tested compounds (in agonist mode, Table 2, Figure 2) exhibited no effect on cAMP accumulation both in 0.1 and 10 µM concentrations or on allosteric properties at both concentrations (Table 3, Figure 3).

Results
The tested compounds (in agonist mode, Table 2, Figure 2) exhibited no effect on cAMP accumulation both in 0.1 and 10 µM concentrations or on allosteric properties at both concentrations (Table 3, Figure 3).

Results
The tested compounds (in agonist mode, Table 2, Figure 2) exhibited no effect on cAMP accumulation both in 0.1 and 10 µM concentrations or on allosteric properties at both concentrations (Table 3, Figure 3).

% of maximalresponse
% of maximalresponse The tested compounds (in agonist and antagonist mode, Table 4, Figures 4 and 5) had no effect on β-arrestin recruitment, except for compound 7i, for which its Emax% at a concentration of 10 µM in the antagonistic mode was 48%.In the structure of the investigated compounds, two distinct fragments can be re nized.One is based on an imidazoline ring with an aromatic substituent in position 1. imidazoline system makes a rigid element for the structure and, even though it cont

Structure-Activity Relationship
In the structure of the investigated compounds, two distinct fragments can be re nized.One is based on an imidazoline ring with an aromatic substituent in position 1 imidazoline system makes a rigid element for the structure and, even though it con nitrogen atoms, shows no basicity due to its incorporation into the urea moiety and

Structure-Activity Relationship
In the structure of the investigated compounds, two distinct fragments can be recognized.One is based on an imidazoline ring with an aromatic substituent in position 1.The imidazoline system makes a rigid element for the structure and, even though it contains nitrogen atoms, shows no basicity due to its incorporation into the urea moiety and the very strong polarization effect of the carbonyl group.Therefore, activity is expected to be affected only by substituents in the aromatic ring.Pi-Pi interactions or hydrophobic interactions produced by the aromatic ring and its substituents can be the main effects taken into account.The second fragment is an aromatic-aliphatic moiety with different lengths of the aliphatic linker and its branching.The linker, especially the branched one, was introduced to enhance solubility and bioavailability.The aromatic ring at the end of the linker plays a similar role to the imidazoline substituent-it is responsible for generating hydrophobic interactions.It was confirmed that compounds with a short linker (one or two carbon atoms) (3a-g, 4a-g, 5a-g) exhibited very weak activity and no differences between agonist and antagonist modes of action.For three-(6a-r) and especially four-carbon (7a-p) branched linkers, more profound activity was confirmed for derivatives containing 1-(4-chlorophenyl) (three carbon atom-6d, 6f, 6r) or 1-(4-methylphenyl)imidazoline (four carbon atoms-7c, 7i, 7m) moiety and 4-chloro-(6d) or 4-fluorosubstituted (6f, 7i) phenyl rings.Significant activity was also confirmed for derivatives containing thiophene-2-yl (7m) or 5-methyl-thiophene-2-yl substituents (6r).It is interesting that similar patterns, namely the presence of the urea moiety, branched aliphatic linker and thiophen-2-yl aromatic substituent, can be observed in the structure of PMZ-21 [17].

Chemistry
Chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA) or Merck KGaA (Damstadt, Germany) and used without further purification.Melting points were measured using a Boetius apparatus and are given uncorrected. 1H (600 MHz) and 13 C (151 MHz) NMR spectra were recorded on a Bruker Avance 600 apparatus (Bruker BioSpin GmbH, Rheinstetten, Germany) in DMSO-d 6 with TMS as the internal standard.Mass analyses were carried out using the atmospheric pressure chemical ionization (APCI) mass spectrometer microTOF-Q II (Bruker Daltonics, Bremen, Germany).TLC was performed on commercial Merck silica gel F254 plates with hexane/ethyl acetate (7:3) eluent system; visualization was performed under UV light with λ = 254 nm.Flash chromatography was performed using a puriFlash 430 apparatus using commercial column PF-15SIHP puriFlash (Interchim, Montluçon, France).The purity of the compounds based on spectral data was over 95%.

General Procedure
Quantities of 0.0083 mol (2.46 g) of triphosgene and 30 cm 3 of dry toluene were placed in a round-bottom flask equipped with a reflux condenser and a magnetic stirrer.A solution of 0.025 mol of arylalkylamine and 0.025 mol (2.53 g) of triethylamine in 30 cm 3 of dry toluene was slowly added dropwise to the mixture.The mixture was stirred for 2 h at room temperature; then, a solution of 0.025 mol of 1-aryl-2-aminoimidazoline-2 and 0.025 mol (2.53 g) of triethylamine in 30 cm 3 of dry toluene was slowly added dropwise and heated at 100 • C for 5 h.Triethylamine hydrochloride (reaction by-product) was filtered out, the solvent of the filtrate was distilled off in a vacuum evaporator and the yellow-brown oily residue was dissolved in a small amount of propan-2-ol.The obtained white precipitate of 1-(1-arylimidazoline) urea derivative was dried and purified via flash chromatography using a puriFlash 430 apparatus and a PF-15SIHP puriFlash column.Hexane and ethyl acetate were used as the eluent system in a 7:3 volume ratio.

Molecules 2024 , 15 Figure 1 .
Figure 1.Structures of some positive, negative, allosteric and biased ligands of the opioid MOP receptor.

Figure 1 . 15 Scheme 1 .
Figure 1.Structures of some positive, negative, allosteric and biased ligands of the opioid MOP receptor.Molecules 2024, 29, x FOR PEER REVIEW 4 of 15

Figure 2 .Table 3 .
Figure 2. Screening for agonistic properties at MOP receptor performed for tested compounds at 10 and 0.1 µM concentrations.

Figure 3 .
Figure 3. Screening for allosteric properties at opioid mu receptor performed for tested compounds at 10 and 0.1 µM concentration.

Figure 4 .
Figure 4. Screening for agonistic properties at opioid mu receptor performed for tested compo at 10 µM and 0.1 µM concentrations.

Figure 4 .
Figure 4. Screening for agonistic properties at opioid mu receptor performed for tested compounds at 10 µM and 0.1 µM concentrations.

Figure 4 .
Figure 4. Screening for agonistic properties at opioid mu receptor performed for tested compo at 10 µM and 0.1 µM concentrations.

Figure 5 .
Figure 5. Screening for antagonistic properties at opioid mu receptor performed for tested pounds at 10 µM and 0.1 µM concentrations.

Figure 5 .
Figure 5. Screening for antagonistic properties at opioid mu receptor performed for tested compounds at 10 µM and 0.1 µM concentrations.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 1 .
List of synthesized urea derivatives.

Table 2 .
The effect of tested compounds on forskolin-induced cAMP levels in CHO cells stably expressing MOP receptor (OP3).Results are normalized to the maximal response induced by the reference OP3 receptor agonist-DAMGO.(n.d.-not determined)

Table 2 .
The effect of tested compounds on forskolin-induced cAMP levels in CHO cells stably expressing MOP receptor (OP3).Results are normalized to the maximal response induced by the reference OP3 receptor agonist-DAMGO.(n.d.-not determined)

Table 2 .
The effect of tested compounds on forskolin-induced cAMP levels in CHO cells stably expressing MOP receptor (OP3).Results are normalized to the maximal response induced by the reference OP3 receptor agonist-DAMGO.(n.d.-not determined)

Table 2 .
The effect of tested compounds on forskolin-induced cAMP levels in CHO cells stably expressing MOP receptor (OP3).Results are normalized to the maximal response induced by the reference OP3 receptor agonist-DAMGO.(n.d.-not determined)

Table 2 .
The effect of tested compounds on forskolin-induced cAMP levels in CHO cells stably expressing MOP receptor (OP3).Results are normalized to the maximal response induced by the reference OP3 receptor agonist-DAMGO.(n.d.-not determined)

Table 2 .
The effect of tested compounds on forskolin-induced cAMP levels in CHO cells stably expressing MOP receptor (OP3).Results are normalized to the maximal response induced by the reference OP3 receptor agonist-DAMGO.(n.d.-not determined).

Table 4 .
The effect of the tested compounds on β-arrestin recruitment in U2OS cells with stable MOP expression in both agonist and antagonist modes.
Results are normalized to the maximal response induced by the reference OP3 receptor agonist, DAMGO (agonist mode), and to the maximal response induced by the reference OP3 receptor antagonist, ß-Funaltrexamine (antagonist mode).Molecules 2024, 29, x FOR PEER REVIEW 10