4.1.3. [(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH<sup>2</sup> (UFP-112)

The chemical modifications of the phenyl ring in Phe4 residue that is essential for NOP receptor activation by inserting pF along with the replacement of Ala at position 7 by α-aminoisobutyric acid (Aib) in N/OFQ sequence resulted in generation of more potent lig-

ands [74–76]. By applying these two chemical modifications to [Arg14, Lys15]N/OFQ-NH2, [(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2, also known as (UFP-112), was synthesized [67]. This ligand acts as a potent (100-fold higher than N/OFQ) and a selective NOP receptor agonist. A long-lasting dose dependent antinociceptive effect was observed after the i.t. administration of UFP-112 (1–100 pmol) in the mouse tail withdrawal assay. In contrast, the same dose of UFP-112 produced a pronociceptive effect and a long-lasting reduction in the locomotor activity when it was administered intracerebroventricularly. Subsequent to intravenous (i.v.) administration of UFP-112 in rats, diuresis as well as reduction in heart rate, blood pressure, and urinary sodium excretion were significantly observed. Consistent with the mouse tail withdrawal assay finding, a long-lasting dose dependent antinociceptive effect was also observed after the i.t. administration of UFP-112 (1–10 nmol) in monkeys without inducing itching by using acute and chronic primate pain modalities (acute noxious stimulus and capsaicin-induced thermal hyperalgesia, respectively) [68]. Notably, the spinal administration of a subthreshold dose of UFP-112 (1 nmol) synergized a morphine analgesic effect without increasing pruritus.

#### 4.1.4. [Phe1Ψ(CH2-NH)Gly<sup>2</sup> (pF)Phe4Aib7Arg14Lys15]N/OFQ-NH<sup>2</sup> (UFP-113)

The combination of [Phe1Ψ(CH2-NH)Gly<sup>2</sup> ](N/OFQ-NH<sup>2</sup> that was synthesized to further avoid the protease degradation [63] and the mentioned above [(pF)Phe4Aib7Arg14Lys15] N/OFQ-NH2, UFP-112, led to the generation of [Phe1Ψ(CH2-NH)Gly<sup>2</sup> (pF)Phe4Aib7Arg<sup>14</sup> Lys15]N/OFQ-NH2, also referred to as UFP-113 [77]. In vitro pharmacological characterization studies that include the functional [35S]-GTPγS binding in CHO cells that express the human NOP receptor and electrically stimulated mouse and rat vas deferens and guinea pig ileum tissues, reveals that UFP-113 acts as a selective partial agonist for NOP receptor [77]. The spinal catheterization of UFP-113 induced an analgesic response in rats at doses that range between (0.001 and 1 nmol); however, in the knockout of rats for the NOP receptor gene the analgesic effect no longer persisted, implying that the antinociceptive effect of UFP-113 is mediated through the NOP receptor stimulation [69].

#### 4.1.5. PWT2-N/OFQ

By employing a novel chemical strategy using peptide wilding approach (PWT), three tetrabranched derivatives of N/OFQ that include PWT1-N/OFQ, PWT2-N/OFQ, and PWT3-N/OFQ were generated [78]. Both in vitro ([35S]-GTPγS binding, calcium mobilization, and electrically stimulated mouse vas deferens assays) and in vivo studies using NOP receptor gene knocked out [NOP receptor (−/−)], revealing that these PWT derivatives act as full NOP receptor agonists that have high potency and a long duration of action of, particularly in PWT2-N/OFQ (40-fold more potent than N/OFQ) [70]. Additionally, analgesic effects were reported after the spinal administration of PWT2-N/OFQ using the nociceptive pain model (tail withdrawal assay) and the neuropathic pain model (chronic constriction injury) in mice and monkeys [71]. PWT2-N/OFQ exhibited higher potency (40-fold more potent) and longer duration (10-fold longer duration of action) in comparison to N/OFQ.

Despite having high potency and selectivity of the previously mentioned NOP receptor peptides in targeting NOP receptor, their pharmacokinetic properties, specifically their poor penetration across the blood-brain barrier have limited their therapeutic indications. However, these peptides have substantially contributed to the detailed understanding of the various responses of the peripheral (respiratory, gastrointestinal, genitourinary, immune, and cardiovascular systems) and central (pain transmission, anxiety, food intake, locomotion, and drug addiction) systems that are related to the N/OFQ–NOP receptor system.

#### *4.2. Non-Peptide NOP Receptor Ligands Targeting Pain*

To overcome the poor metabolic stability of peptide ligands related to N/OFQ and require to be administered either intrathecally or intracerebroventricularly, several studies were conducted to identify new selective non-peptide ligands that are suitable for intraperitoneal or oral administration. High-throughput screening and medicinal chemistry research have led to the discovery of multiple classes of chemical compounds including piperidines, spiropiperidines, nortropanes, 4-amino-quinolines, and quinazolines that act as NOP receptor ligands with enhanced metabolic stability. The non-peptides that have antinociceptive activity are summarized in Table 2 and described below.


**Table 2.** Non-peptide NOP receptor ligands targeting pain.

#### 4.2.1. Ro 65-6570

The high-throughput screening of 8-acenaphthene-l-yl-l-phenyl-l,3,8-triaza-spiro[4.5]decan-4-one was performed to develop Ro 65-6570, 8-(1,2-dihydroacenaphthylen-1-yl)-1-phenyl-1,3,8 triazaspiro[4,5]decan-4-one, by a group of scientists at Roche laboratories [86]. In vitro studies that include radioligand binding and cAMP inhibition assays in (CHO) cells expressing the recombinant human NOP receptor indicated that Ro 65-6570 acts as a NOP receptor full agonist with poor selectivity in comparison to other opioid receptors [87]. In mice, i.v. administration of Ro 65-6570 resulted in dose-dependent antinociceptive effects without modifying motor coordination using formalin paw and orofacial formalin (OFF) tests [79,80]. Further in vitro functional selectivity studies such as the BRET-based assay revealed that Ro 65-6570 is a G protein-biased agonist which exhibited antinociceptive effects in β-arrestin 2 knockout mice as compared to the wild-type [88,89].

#### 4.2.2. Ro 64-6198

In an effort to develop a new NOP receptor agonist with high selectivity (greater than 100-fold over canonical opioid receptors) and potency, [(1*S*,3a*S*)-8-(2,3,3a,4,5, 6- hexahydro-1*H*-phenalen-1-yl)-1-phenyl-1,3,8-triaza- spiro[4.5] decan-4-one], also known as Ro 64-6198, was identified by a group of scientists at Hoffman La Roche in Switzerland [81,90,91]. Using Ro 64-6198 as a valuable pharmacological tool highlighted therapeutic applications for NOP receptor agonist such as anxiety, neuropathic pain, addiction, cough, and anorexia, in addition to the undesirable effects it has on learning, memory, motor activity, and body temperature (hypothermia) [92]. Similar to morphine, analgesic effects in the hot plate and shock threshold assays were observed after the systemic administration of Ro 64-6198 (3 mg/kg, intraperitoneal (i.p)) in wild-type mice but not in NOP receptor

knockout mice [82,83]. Conversely, increased pain sensitivity was observed as an opposite effect in the tail flick assay, implying the complex role of NOP receptor in pain processing. Furthermore, coadministration of low doses (1 mg/kg) of Ro 64-6198 and morphine resulted in an additive analgesic effect [83]. Consistent with these findings, analgesic effects without causing depression, itching, and reinforcing responses were observed after the subcutaneous (s.c.) administration of Ro 64-6198 (0.001–0.06 mg/kg) in both acute (acute noxious stimulus) and chronic (capsaicin-induced neuropathic pain) pain modalities in monkeys [93]. Pretreatment with J-113397 (0.1 mg/kg), a selective nonpeptidic NOP receptor antagonist, blocked Ro 64-6198-induced antinociception, emphasizing that the antinociceptive actions of Ro 64-6198 is mediated via NOP receptor. Despite the robust analgesic effects of systemically administered Ro 64-6198 in non-human primates, several in vivo studies using tail flick and immersion, tactile or cold water stimulation and foot shock assays revealed that Ro 64-6198 does not modulate pain processing in rodents, except mouse hot plate assay [81,83,93–95].

#### 4.2.3. SCH221510

SCH221510 is a potent and selective non-peptide NOP receptor agonist that was reported to induce analgesia in neuropathic pain when administered orally and intrathecally in mice and rat models, respectively [96–98]. It is also reported to attenuate the respiratory depression and itch response that were observed after the systemic administration of buprenorphine to a non-human primate, as well as reinforcing MOP receptor agonists induced responses in rats [97,99]. Conversely, a s.c. administration of SCH221510 (3 and 10 mg/kg) in hot-plate test did not produce analgesia, while SCH221510 administration (3 mg/kg) reduced morphine-induced analgesia. The co-administration of SCH221510 (3 mg/kg) and morphine (10 mg/kg) accelerated the tolerance development to the antinociceptive effect of morphine in female mice [100].

#### *4.3. Bifunctional and Mixed NOP Receptor Compounds*

Considering the potential ability of intracerebroventricularly administered N/OFQ to attenuate morphine tolerance and suppress drug reinforcing response, the development of new synthetic agonists may constitute an innovative pharmacological approach for analgesics that target both MOP receptor and NOP receptor to enhance their analgesic effect and minimize their side effects as depicted in Figure 2 [99,101–105]. Additionally, multiple pathophysiological pathways are involved in the pain process, so developing analgesic agents with multiple mechanisms of actions could be an innovative strategy for developing new effective and safe analgesics [106]. Accordingly, several compounds including AT-121 (a partial agonist of NOP receptor and MOP receptor), buprenorphine (semisynthetic multifunctional opioid), and its analogue BU08028 were synthesized (reviewed in [107–109]).

#### 4.3.1. SR 16435

SR 16435 (Figure 3), also referred to as [1-(1-(bicyclo[3.3.1]nonan-9-yl)piperidin-4 yl)indolin-2-one] behaved as a bi-functional NOP receptor /MOP receptor partial agonist with high binding affinity was synthesized by Toll group [110]. In mice, SR 16435 administration produced an analgesic effect (s.c. and i.t.) which was effective and potent in attenuating both neuropathic and inflammatory pain (i.t) with diminished tolerance development to the antinociceptive effect of SR 16435 [96,110]. Nonetheless, the conditioned place preference (CPP) that was primarily mediated by MOP receptor activation was induced after the administration of SR 16435. This finding emphasizes that full agonistic activity at NOP receptor could be required to reduce the rewarding properties associated with MOP receptor [110].

**Figure 2.** Rational design of new safer analgesics. (**A**) Beneficiary and side effects produced by MOP receptor activation. (**B**) Beneficiary (synergizing analgesic effect) and protective (ameliorating typical-opioid side effect profile) effects produced by developing a new compound with simultaneous agonistic activity at NOP receptor and MOP receptor.

**Figure 3.** Chemical structures and in vitro pharmacological profiles of bifunctional and mixed NOP receptor ligands that target pain [99,110–119].

#### 4.3.2. AT-121

AT-121 (Figure 3) is a non- morphinan compound which acts as a bifunctional NOP receptor /MOP receptor partial agonist with high binding affinity [114]. It was synthesized to optimize the pharmacological profile of MOP receptor agonists by synergizing their therapeutic effects (analgesia and treatment of substance abuse) and minimizing their side effects (respiratory depression, tolerance dependent, and abuse liability) via targeting NOP receptor. In monkeys, s.c. administration of AT-121 produced morphine-like analgesic and antiallodynic effects using the warm water tail-withdrawal assay and capsaicin-induced allodynia, respectively, without trigging itch, physical dependence, respiratory depression, and hyperalgesia mediated by opioid. These effects were confirmed to be mediated by MOP receptor and NOP receptor activation by using selective dose of MOP receptor and NOP receptor antagonists, J-113397 (0.1 mg/kg) and naltrexone (0.03 mg/kg), respectively. Additionally, AT-121 could be therapeutically implicated for opioid addiction as it lacks the abuse potential (reinforcing effects) and diminished oxycodone reinforcing response.

#### 4.3.3. Buprenorphine and Its Analog BU08028

Buprenorphine (Figure 3) is a natural derived alkaloid of the opium poppy with a mixed pharmacological activity (MOP receptor /NOP receptor partial agonist and DOP receptor /KOP receptor low partial agonist) clinically approved to treat pain and substance abuse [99,115]. In rodent, full analgesic effects were produced after the administration of buprenorphine in both chronic and acute pain models [120]. After a systemic administration of 0.01–0.1 mg/kg to a non-human primate, an antinociceptive effect was present in a dose-dependent manner. A resultant respiratory depression and itch were observed and subsequently confirmed to be induced by MOP receptor activation. These side effects associated with buprenorphine were found to be attenuated by the co-administration of an NOP receptor selective agonist such as Ro 64-6198 and SCH 221510 [99]. As such, the combination emphasized the therapeutic potential of mixed MOP receptor /NOP receptor agonists as innovative analgesics. A buprenorphine analog that is known as BU08028 (Figure 3) demonstrated a similar binding profile to buprenorphine with improved binding affinity and efficacy to NOP receptor. In mice, an intrathecal administration of BU08028 produced an analgesic effect, which was more potent than morphine in attenuating both neuropathic and inflammatory pain [96]. Consistent with these results, a systemic administration of BU08028 to a non-human primate produced a long-lasting analgesic effect (>24 h) with a reduced reinforcing effect as compared to cocaine, remifentanil, or buprenorphine and without causing respiratory depression and CVS adverse effects [121].

#### 4.3.4. BPR1M97

By applying a high-throughput screening, BPR1M97 (Figure 3) was identified as a dual agonist that produced a significant analgesic effect in a tail-flick assay in mice [122]. Both in vitro assays (radioligand binding, c-AMP production, membrane potential, β-Arrestin-2 recruitment, and internalization assays) and in vivo behavior assays (tail flick and clip, respiratory and cardiovascular functional, acetone drop, von Frey hair, charcoal meal, glass bead, locomotor activity, conditioned place preference (CPP) and naloxone precipitation assays) proved that BPR1M97 behaved as a dual agonist for MOP receptor (full agonist) and NOP receptor (G-protein biased agonist) [118]. Notably, rapid analgesic actions (more potent than morphine in cancer-induced sensory allodynia) were observed after the BPR1M97 s.c. administration with less undesirable side effect as compared to morphine.

#### 4.3.5. BU10038

A naltrexone-derived bifunctional MOP receptor /NOP receptor agonist, also referred to as BU10038 (Figure 3), behaved as a partial MOP receptor and NOP receptor agonist was synthesized by Husbands and Ko groups [117]. In non-human primate, both systemic (0.001–0.01 mg/kg) and intrathecal (3 mg) administrations of BU10038 resulted in a

long-lasting antinociceptive with neither reinforcing effects nor other effects like itching, respiratory depression, and tolerance when administered repeatedly.

#### 4.3.6. JTC-801

JTC-801 (Figure 3), also referred to as [N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide hydrochloride], behaved as a NOP receptor antagonist and was developed by a group of scientists at the Central Pharmaceutical Research Institute [119]. JTC-801 produced antinociceptive effects in a hot plate test and a formalin test using mice and rats, respectively. Although the injectable and oral formulations of JTC-801 entered Phase II of its clinical trials in both Japan and the UK to treat the neuropathic and postoperative pain, it was suspended for unknown reasons [123].

#### 4.3.7. Cebranopadol

The rational optimization strategy of spiro[cyclohexanedihydropyrano[3,4-b]indole] amine resulted in the discovery of cebranopadol (Figure 3) that represents the first in its class to be a highly potent and efficacious antinociceptive agent with combined agonistic activity at MOP receptor, NOP receptor (subnanomolar affinity), KOP receptor, and DOP receptor (low nanomolar affinity) [111,112,124]. Behavior in vivo studies including acute and chronic pain models in rodents (tail-flick, formalin test, rheumatoid arthritis, bone cancer, spinal nerve ligation, diabetic neuropathy) further indicated the high potency and extremely long-lasting analgesic effect of cebranopadol in comparison with selective MOP receptor agonist, particularly in the chronic pain model [111,125]. Extensive preclinical safety and tolerability studies have been conducted on rodent models to reveal the possible side effects on the CNS, the respiratory system, and the gastrointestinal system (reviewed in [126]). Limited range of unwanted effects were also observed, as cebranopadol did not decrease respiratory rate, develop a tolerance, or impair the motor coordination, unlike the effects of morphine. The G-protein-biased agonistic activity at NOP receptor could be the reason behind these favorable side effect profiles of cebranopadol [125]. Notably, cebranopadol is equipotent and equi-efficacious toward the G protein activation at both MOP receptor and NOP receptor without inducing phosphorylation or NOP receptor internalization and without recruiting B-arrestin2 at NOP receptor only in BRET assay [125,127]. The noncompartmental analysis in phase I and phase II clinical trials was used to assess the pharmacokinetics profiles of cebranopadol. The maximum plasma concentration [Cmax] (4–6 h) with a long half-value duration (14–15 h) was reached after oral administration of immediate release formulation of cebranopadol. After the administration of multiple once-daily oral doses of cebranopadol in patients, the steady state was reached in nearly 2 weeks. Following single- and multiple-doses administration of cebranopadol in healthy subjects and patients, a two-compartment disposition model with first-order elimination process and a two lagged transition compartments was observed [128]. Several phase II clinical trials were conducted and listed as complete in patients suffering from acute (bunionectomy trial) and chronic (diabetic neuropathy, osteoarthritis, chronic low back pain, and diabetic polyneuropathy) pain to evaluate the efficacy, safety, and tolerability of a single oral dose of cebranopadol [129–135]. While most phase III clinical trials have recently proven the effectiveness, safety, and tolerability of cebranopadol when administered orally (200–1000 µg per day) to cancer patients who suffer from moderate to severe chronic pain [136,137].

#### **5. Future Directions and Conclusions**

In this review, the rational design of NOP receptor ligands with various pharmacological profiles as a promising alternative for conventional opioid analgesic is discussed. The crystal structure, distribution, and signaling pathway of NOP receptor are also highlighted. It is important to note that other therapeutic indications for NOP receptor in the treatment of various neurological disorders and alcohol abuse have not been explored in this review. Notably, NOP receptor-related peptides have substantially attributed in expanding our

knowledge regarding the various peripheral and central responses related to N/OFQ–NOP receptor system, but their poor bioavailability has limited their therapeutic implications. Regardless of the controversial results between the spinal and supraspinal administration of endogenous neuropeptide of NOP receptor that remains poorly understood, the NOP receptor ligands exhibit favorable pharmacological activity and side effects, particularly the mixed which target multiple opioid receptors. So far, cebranopadol represents the most promising NOP receptor ligand to treat acute and chronic pain without reducing respiratory rate, developing a tolerance, or impairing the motor coordination as compared to the clinically approved opioid analgesic. However, further work needs to be done to resolve the high-resolution structure of NOP receptor in its active state to elucidate the distinct residues responsible for NOP receptor agonist binding [138]. Conceivably, a deep understanding of the NOP receptor signaling pathway and structure along with computer-aided molecular docking and behavior studies will facilitate the discovery of polypharmacological ligands that target multiple receptors including NOP receptor as new effective and safe analgesics.

**Author Contributions:** A.E.D. and T.C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by R35GM143061 to T.C.

**Conflicts of Interest:** The authors have no conflict of interests to declare.

#### **References**

