Neuropeptide FF (NPFF) is an octapeptide with the sequence Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2. It interacts with two G-protein coupled receptors (GPCRs), NPFF1-R and NPFF2-R, which are mainly present in the central nervous system (CNS) [[1], [2], [3]]. NPFF represents a neurotransmitter system which acts as a modulator of opioid action [4]. NPFF has been found to elicit both anti-opioid and pro-opioid actions in animal models of pain [2]. Whereas the intracerebroventricular (i.c.v.) administration of NPFF reverses morphine-induced analgesia in rats, intrathecal (i.t.) administration of NPFF analogues results in a long-lasting, opioid-induced analgesia and potentiates morphine-induced analgesia [3,5,6]. These opposing actions have led some to speculate that these effects could be arising from interaction with the different receptor subtypes [2,7,8]. However, to this day, such theory has not been substantially corroborated. To have a better understanding of this elusive receptor system, many studies have been undertaken to identify high affinity and selective NPFF receptor ligands. While this field initially started employing peptidomimetic molecules, some small molecule campaigns have been successful [[9], [10], [11], [12], [13], [14], [15], [16]]. One of the main goals of these previous studies was to delineate a precise role for NPFF1-R and NPFF2-R as either anti- or pronociceptive systems in in vivo models.

A major structural feature of most NPFF-R ligands, both peptide-based and small molecule, is the presence of a guanidine group. This guanidine group is thought to anchor the NPFF ligands in the receptor binding site via salt bridge formation with a conserved aspartate residue at position 6.59 [10]. Despite the success of guanidine based NPFF-R ligands, the guanidine group could be considered the “Achilles heel” of these molecules. The guanidine group suffers from an elevated pKa, large polar surface area, high plasma protein binding, all of which lead to limited blood-brain barrier (BBB) penetration [17]. This could negatively affect the drug-like properties of guanidine-based NPFF-R ligands and hamper their development into viable CNS-directed molecules. Thus, it would be beneficial, from a drug development point of view, to steer away from this precarious functional group. There are literature reports of NPFF-R ligands that are devoid of guanidine or guanidino-mimetic groups. However, most of them lack the desired pharmacological profile in terms of selectivity and high receptor binding affinity [9,18]. Taken together, these findings underscore the importance of developing novel, non-guanidine based small molecule NPFF-R ligands with improved biological profiles.

For a long time, the focus of our group had been directed towards design and synthesis of guanidine-based small molecule NPFF ligands. Through our studies, we were able to identify several NPFF-R ligand scaffolds, both agonists and antagonists [14]. One of our leading molecules was a piperidino-guanidine derivative (Fig. 1) which displayed sub-micromolar binding affinity at NPFF1-R and NPFF2-R. MES304 is non-subtype selective and binds to NPFF1-R with Ki = 112 ± 19 nM and to NPFF2-R with Ki = 30 ± 5 nM. Moreover, the compound behaved as an antagonist in our in vitro and in vivo assays [14]. Accordingly, MES304 presented a promising starting point for further development. Given the potential disadvantages presented with the guanidine group, our primary aim for this study was to explore new chemical entities beyond the guanidine group, which always remained the cornerstone of high affinity peptidomimetics, as well as small molecule NPFF-R ligands. Our end goal revolved around two objectives: 1) identify novel, non-guanidine based NPFF small molecule scaffolds that could have better subtype selectivity profiles than the parent/lead molecule and; 2) removal of the guanidine group to improve the overall physicochemical properties of the NPFF ligands in anticipation for their use as in vivo pharmacological tools.

We initially started searching for literature examples of functional groups that served as bioisosteric replacements of the guanidine functionality. First, we came across a study whereby the unsubstituted guanidine group of an anti-HIV peptidomimetic compound was bioisosterically replaced with a squaryldiamide moiety [19]. Additionally, we found reports of acylguanidines and carbamoylguanidines successfully serving as bioisosteres of guanidine groups on peptidomimetic NPY and NPFF receptor ligands [20,21]. Soll et al., showed that an aminohydrazone motif was an effective functional group mimic of the highly basic guanidine group in a series of small molecule thrombin inhibitors and resulted in reduced basicity, as well as enhanced oral bioavailability of these compounds [22]. Although these bioisosteric replacements may prove successful in terms of biological activity, synthetic feasibility may hamper access to such chemical entities. Besides, many of the bioisosteres were merely guanidine derivatives and not replacements.

On the other hand, Bihel et al., were successfully able to completely replace the guanidine group of their high affinity peptide-based NPFF-R ligand 4a (Fig. 2) with various piperidine and piperazine derivatives [9], with the resultant analogues retaining the nanomolar NPFF-R binding affinity of the parent molecule. This is of particular importance since it was believed that a guanidine group or a guanidine mimetic is essential for high affinity NPFF receptor binding. Additionally, some of the synthesized analogues demonstrated preference in binding affinity for one NPFF-R subtype over the other. One likely explanation for this phenomenon could be that the basic nitrogen atom of these cyclic tertiary amines is engaging in an ionic interaction with the same biding site residues as the guanidine group.

The same strategy was adopted by la Rochelle et al., in the design of their chimeric, peptidomimetic mu-opioid receptor (MOR) agonist/NPFF-R antagonist KGFF09 [23] (Fig. 3). In this study, the authors described combining the pharmacophores of ligands of both MOR and NPFF receptors to form a series of bifunctional ligands. By substituting the guanidine of the NPFF core on one of their analogues, KGFF03, with piperidine and 4-benzyl piperidine, they obtained KGFF08 and KGFF09, respectively. Despite some minor loss in binding affinity at NPFF receptors, the Ki values of both analogues remained in the low nanomolar range. Moreover, the preference in binding affinity for the NPFF2-R subtype exhibited by the parent molecule, KGFF03, was preserved. In the in vitro functional assay, KGFF09 behaved as a MOR agonist/NPFF-R antagonist and demonstrated anti-hyperalgesic effects in mice in vivo.

Taken together, these two studies demonstrate that the guanidine of the sequence RF-amide can be successfully replaced with piperidine and/or piperazine derivatives, leading to viable peptidomimetic NPFF-R ligands.

Considering this work and based on the promising results presented in the latter two studies, we decided to adopt this concept and apply it to our small molecule NPFF-R ligands. Since the guanidine moiety is a common feature of both our small molecules and the peptidomimetic NPFF-R ligands, we wanted to explore the prospect of substituting the guanidine group of our small molecules with various cyclic amines. Our plan was to combine our leading NPFF-R antagonist MES304 (Fig. 1) with the cyclic amines used in Fig. 2, Fig. 3. We believed that these cyclic amines serve as an excellent substitute for the guanidine group since they are basic in nature as guanidines, albeit with lower pKa and lower polar surface area. Accordingly, we envisioned that they could improve the overall pharmacokinetic profile of our NPFF-R probe candidates.

We started by replacing the guanidine group of our lead molecule, MES304 with various cyclic amines. Afterwards, optimization efforts on different parts of the molecule lead to the development of a library of piperidine and piperazine derivatives, for which the binding affinity at the NPFF receptors was determined.