Enkephalin analogues with N-phenyl-N-(piperidin-2-ylmethyl) propionamide derivatives: Synthesis and biological evaluations

Abstract

N-Phenyl-N-(piperidin-2-ylmethyl)propionamide based bivalent ligands are unexplored for the design of opioid based ligands. Two series of hybrid molecules bearing N-phenyl-N-(piperidin-2-ylmethyl)propionamide derived small molecules conjugated with an enkephalin analogues with and without a linker (β-alanine) were designed and synthesized. Both bivalent ligand series exhibited remarkable binding affinities from nanomolar to subnanomolar range at both μ and δ opioid receptors and displayed potent agonist activities as well. The replacement of Tyr with Dmt and introduction of a linker between the small molecule and enkephalin analogue resulted in highly potent ligands. Both series of ligands showed excellent binding affinities at both μ (0.6–0.9 nM) and δ (0.2–1.2 nM) opioid receptors respectively. Similarly, these bivalent ligands exhibited potent agonist activities in both MVD and GPI assays. Ligand 17 was evaluated for in vivo antinociceptive activity in non-injured rats following spinal administration. Ligand 17 was not significantly effective in alleviating acute pain. The most likely explanations for this low intrinsic efficacy in vivo despite high in vitro binding affinity, moderate in vitro activity are (i) low potency suggesting that higher doses are needed; (ii) differences in experimental design (i.e. non-neuronal, high receptor density for in vitro preparations versus CNS site of action in vitro); (iii) pharmacodynamics (i.e. engaging signalling pathways); (iv) pharmacokinetics (i.e. metabolic stability). In summary, our data suggest that further optimisation of this compound 17 is required to enhance intrinsic antinociceptive efficacy.

The opioid receptors belong to the superfamily of G-protein coupled receptors (GPCRs) and play important roles in pain perception and regulation. They are classified into three different subtype’s μ, δ and κ opioid receptors. Opioids have been used for the treatment of moderate to severe pain and for their psychotropic effects since ancient times. Indeed, exogenous agonists of opioid receptors elicit marked analgesic effects, along with serious side effects such as respiratory depression, sedation, constipation, nausea, tolerance and dependence. Most of the present opioid analgesics exert their analgesic and adverse effects primarily through the μ opioid receptors. For the past two decades there has been considerable interest in the synthesis of nonselective μ/δ ligands with different combinations of agonist and (or) antagonist activities at each of the opioid receptors, which have been shown to exhibit valuable analgesic properties.^1–8 The new promising approach in the opioid drug development is ligand designed with mixed μ agonist/δ agonist profile or μ agonist/δ antagonist profile.^9–11 Several reports indicate that the dimerization or oligomerization of GPCRs,¹² and the existence of dimers for opioid receptors particularly between μ and δ opioid receptors have been reported.¹³ Recent pharmacological studies have demonstrated that the development of tolerance to μ-opioids can be suppressed by co-administration of δ opioid receptor agonists or antagonists, and that δ agonists can increase the potency and efficacy of μ agonists.^14–17 It appears that this is based on the beneficial modulatory interactions existing between μ and δ opioid receptors.

The endogenous neuropeptides enkephalins, β-endorphins, endomorphins and dynorphins which interact with the δ, μ and κ opioid receptors respectively.¹⁸ These endogenous peptides acts as both neuromodulators and hormones, and are responsible for a broad spectrum of physiological effects. The subsequent elongation or truncation modifications of these endogenous peptides led to highly promising opioid agonists.^6,19 Many interesting and useful selective peptide ligands for the opioid receptors have been developed and used extensively. The peptide fragments for our bivalent ligands (Tyr-DAla-Gly-Phe and Dmt-DAla-Gly-Phe) are potent well established opioid ligands. Portoghese et al.²⁰ reported a conjugation of Tyr-(Gly)_n peptide analogues to the N-phenyl-N-piperidin-4-yl)propionamide moiety and the resulting conjugate compounds showed very weak or no opioid activity. During the last decade our group has shown promising progress in synthesis of bivalent μ/δ ligands based on enkephalin analogues conjugated to the different positions of the 4-anilidopiperidine core and fentanyl molecule. ^4,21–23 Our group reported a Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-yl)propionamide moiety. These ligands exhibited good biological activities in vitro and demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay.⁴ Based on these results recently we reported a series of hybrid molecules containing the C-terminal of enkephalin analogues attached to the amino group of 4-anilidopiperidine core small molecules which were synthesized and pharmacologically evaluated.²⁴ All these bivalent ligands showed good binding affinity as well as potent agonist activities in MVD and GPI assays at both μ and δ opioid receptors. The scientists at Neurosearch²⁵ and Sepracor Inc.²⁶ explored the N-phenyl-N-(piperidin-4-ylmethyl) propionamide, N-phenyl-N-(piperidin-3-ylmethyl)propionamide derivatives with substituted phenethyl groups as potent opioid agonists. They substituted a variety of heterocyclic compounds in place of the phenyl group on the ‘N’ piperidine. The above literature precedence and earlier promising results on opioid bivalent ligands from our group inspired us to explore the bivalent ligands based on the conjugation of enkephalin analogues to 5-amino substituted tetrahydronaphthalen-2-yl) methyl moiety with N-phenyl-N-(piperidin-2-ylmethyl)propionamide based small molecules. Similarly, the second series of molecules explored by enkephalin analogues directly conjugated to the N-phenyl-N-(piperidin-2-ylmethyl)propionamide moiety. The small molecules 1 and 2 are structurally relevant to the 4-anilidopiperidine small molecules, the advantages of these molecules having additional hydrophobic cyclohexyl group and amine substitution on 5th position of tetrahydronaphthalen-2yl-methyl moiety. We expected the lipophilic nature of the molecules 1 and 2 will increase cell permeability of attached peptides, and consequently their bioavailability.²⁷ Another interesting feature of these molecules amino group on tetrahydronaphthalen-2yl-methyl moiety was easily accessible to attach to the C-terminus enkephalin analogues with and without a linker. N-phenyl-N-(piperidin-2-ylmethyl) propionamide 11 analogues remain unexplored in the literature as potential opioid ligands for their structure activity relationship and represent a promising new approach in the design of hybrid nonpeptide-peptide analgesics. The rationale behind the design of second series of bivalent ligands (16–18), Lee et al.⁴ reported a Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-yl)propionamide moiety. The resulted ligands exhibited good biological activities in vitro and demonstrated potent in vivo antihyperalgesic and antiallodynic effects. In our designs we replaced the N-phenyl-N-piperidin-4-yl)propionamide moiety with N-phenyl-N-(piperidin-2-ylmethyl)propionamide (11) and attached the enkephalin peptide analogues in similar fashion. The structural difference between N-phenyl-N-piperidin-4-yl)propionamide moiety and N-phenyl-N-(piperidin-2-ylmethyl)propionamide (11) is connectivity of piperidine ring to N-phenylpropionamide group, one is at 4^th position and the other one is 2nd position. Our interest here is to study the effect of piperdine ring connectivity to N-phenylpropionamide group on opioid receptors (μ and δ) binding affinity and activity. The present study constitutes ongoing developments in our laboratory towards the design and synthesis of novel bivalent ligands based on enkephalin analogues and 4-anilidopiperidine small molecules.

We take the N-phenyl-N-(piperidin-2-ylmethyl)propionamide 11 as the core template, which can be expanded towards designing novel ligands. The first series of bivalent ligands were designed based on the small molecule 1 (N-((1-(((R)-5-amino-5,6,7,8-tetrahydronaphthalen-2-yl)methyl)piperidin-2-yl)methyl)-N-phenylpropionamide) and fluoro analogue 2 conjugated to the C-terminal of the enkephalin peptide derivatives with and without a linker (Fig. 1). The rationale for choosing fluoro analogue 2 into bivalent ligands, the fluorine imparts a variety of properties to the molecules, including enhanced binding interactions, metabolic stability, changes in physical properties, and selective reactivities. ²⁸ The Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-(piperidin-4-yl)propionamide moiety gave a promising results (in vitro and in vivo) at the μ and δ opioid receptors,⁴ and similar approach we followed here in the design of second series ligands. The C-terminal enkephalin peptide derivatives conjugated to the N-phenyl-N-(piperidin-2-ylmethyl)propionamide 11 with and without a linker (Fig. 1).

Figure 1.

Design principle of bivalent ligands.

The synthetic procedures for small molecules 1 and 2, and their structure activity relationship studies are going to communicate as a different account. The enkephalin analogues 3 (Boc-Tyr-DAla-Gly-Phe-OH), 4 (Boc-Dmt-DAla-Gly-Phe-βAla-OH) and 12 (Boc-Dmt-DAla-Gly-Phe-OH) were prepared by solution phase peptide synthesis following the N^α-Boc strategy by previously reported.²¹ The hybrid molecules 8–10 were prepared starting from the small molecules 1 and 2 according to Scheme 1. The enkephalin peptides 3 and 4 are conjugated to the small molecules 1 and 2 in presence of HATU/DIPEA in acetonitrile to afford the Boc-protected bivalent ligands 5, 6 and 7. Surprisingly, during the coupling of enkephalin peptide 4 to the small molecule 2, we obtained two products wherein the major product 6 has a single peptide unit attached to the small molecule, and the other minor product 7 has two peptide units conjugated to the small molecule. Without further purification the Boc-protected analogues 5 (mixture of 6 and 7) were treated with 50% trifluoroacetic acid in dichloromethane to yield the crude final bivalent ligands 8, 9 and 10. The second series of bivalent ligands were prepared according to Scheme 2. The enkephalin peptides 3, 4 and 12 were conjugated to the N-phenyl-N-(piperidin-2-ylmethyl)propionamide moiety 11 in the presence of HATU/DIPEA in acetonitrile to afford the Boc-protected analogues 13, 14 and 15 which were followed by removal of the Boc group with 50% trifluoroacetic acid in dichloromethane thus obtaining the crude bivalent ligands 16, 17 and 18. The Boc-protected analogues 5–7 and 13–15 were triturated with ether, filtered and washed with excess ether, and without further purification the Boc group was deprotected to obtain the final bivalent ligands. The final crude ligands were purified by RP-HPLC to give pure ligands as white powders in overall yields of 35–40%. The purity of the final ligands was determined as >95% by analytical HPLC. The final bivalent ligands were characterised and their purity confirmed by analytical HPLC, ¹H NMR, HRMS. For all the data, see Supporting information.

Scheme 1.

Preparation of enkephalin analogues with 5-amino substituted tetrahydronaphthalen-2-yl)methyl bivalent ligands.

Scheme 2.

Preparation of enkephalin analogues with N-phenyl-N-(piperidin-2-ylmethyl)propionamide bivalent ligands.

The opioid receptor binding affinities of the synthesized bivalent ligands were evaluated using human δ opioid receptors (hDOR) and rat μ-opioid receptors (rMOR) with cells that stably express these receptors as previously described.²⁹ [³H]DPDPE and [³H]DAMGO were used as competitive radio ligands, respectively. The tissue bioassays (mouse vas deferens: MVD, guinea pig isolated ileum: GPI) also were performed to characterise the agonist function at δ and μ opioid receptors as described previously.³⁰ The affinities of the two series of bivalent ligands were evaluated at the μ and δ opioid receptor. In addition, the functional activity of these hybrid molecules was also investigated through MVD and GPI assay (Tables 2 and 3). The two series of new opioid bivalent ligands showed very promising binding affinities and functional bioactivities at the δ and μ opioid receptors.

Table 2.

Functional assay result for bivalent ligands at MOR and DOR

Compd	a MVD (δ)	a GPI (μ)
	IC50 (nM)
1	18% 1 μM	1005 ± 58
2	22% 1 μM	10% 1 μM
8	540 ± 230	95 ± 16
9	57 ± 9.4	5.7 ± 2.5
10	6.1 ± 1.5	2.1 ± 0.45

^aConcentration at 50% inhibition of muscle contraction at electrically stimulated isolated tissues.

Table 3.

Receptor affinity and functional bioactivity of bivalent ligands

Compd	R3	Binding Ki (nM)				Ki μ/δ	c MVD δ	c GPI μ
		b log IC50	a MOR (μ)	b log IC50	a DOR (δ)
16	Tyr-DAla-Gly-Phe–	−8.49 ± 0.06	2	−8.31 ± 0.08	2	1	7.1 ± 1.5	21 ± 7.1
17	Dmt-DAla-Gly-Phe–	−8.76 ± 0.30	0.8	−9.28 ± 0.03	0.2	4/1	0.47 ± 0.06	0.73 ± 0.06
18	Dmt-DAla-Gly-Phe-βAla	−9.56 ± 0.30	0.9	−8.92 ± 0.04	1.2	1	0.45 ± 0.26	4.1 ± 2.1

^aCompetition analyses against radiolabeled ligand ([³H]DPDPE for DOR δ [³H]DAMGO for μ were carried out using rat brain membranes.

^bLogarithmic values determined from the nonlinear regression analysis of data collected from at least two independent experiments.

^cConcentration at 50% inhibition of muscle contraction at electrically stimulated isolated tissues.

The small molecules 1 and 2 are showed very good binding affinity (69 nM and 33 nM) at μ opioid receptors respectively, with high selectivity towards the μ opioid receptor. When the C-terminus of the enkephalin analogue was attached to the amino group of the small molecule 1 the resultant bivalent ligand 8 exhibited 1 nM a 33 fold increase in binding affinity for the μ receptor, and a 56 fold increases for a δ receptor biding affinity of 160 nM comparative to the small molecule 1. The introduction of a linker (β-alanine) in between the enkephalin analogues and the small molecules, and the replacement of Tyr¹ with Dmt¹ attached to the fluoro analogue of small molecule 2 the bivalent ligand 9 which exhibited μ receptor binding affinity retained (0.7 nM) and almost 400 fold increase in the δ receptor binding affinity to 0.4 nM compared to 8. The bivalent ligand 10 contains two enkephalin peptide units with small molecule exhibited almost similar binding affinities (0.6 nM) at both receptors as does ligand 9 which contains one enkephalin unit. In second series of bivalent ligands 16, 17 and 18 enkephalin analogues directly attached to the N-phenyl-N-(piperidin-2-ylmethyl)propionamide moiety with and without a linker. The second series of bivalent ligands 16, 17 and 18 showed excellent binding affinities towards μ and δ opioid receptors (Table 3). The enkephalin peptide 3 directly attached to the small molecule 11 resulting in the bivalent ligand 16 showed good and balanced binding affinities (2 nM) at μ and δ opioid receptors. Ligand 17 retained similar binding affinities like ligand 16 with 0.8 nM affinity at the μ opioid receptor, with a 10 fold increased binding affinity of 0.2 nM at the δ opioid receptor. In ligand 18 the introduction of a linker between enkephalin analogue and small molecule showed almost the same binding affinities as ligand 17. Thus replacement of Tyr with Dmt and introducing a β-alanine as the linker in this series of ligands not significant influence the binding affinities towards the receptors as were observed profoundly in the earlier series.

All analogues showed good opioid agonist activities in the GPI and MVD assays and were observed to correlate to their in vitro binding affinities at μ and δ opioid receptors respectively (Tables 1–3). Among these novel bivalent ligands 17 and 18 are found to have very potent agonist activities at both in MVD (0.47 and 0.45 nM) and GPI (0.73 and 4.1 nM) assays respectively. The bivalent ligands 17 and 18 showed very balanced activities at μ and δ opioid receptors. The lead compound 17 was evaluated in vivo (Fig. 2).

Table 1.

Binding affinities of bivalent ligands at μ/δ opioid receptors

Compd	R	R1	R2	Binding Ki (nM)				Ki μ/δ
				b log IC50	a MOR (μ)	b log IC50	a DOR (δ)
1	H	H	H	−6.83 ± 0.09	69	−4.92 ± 0.09	5200	1/79
2	F	H	H	−7.25 ± 0.09	33	−4.72 ± 0.70	9000	1/270
8	H	Tyr-DAla-Gly-Phe–	H	−8.69 ± 0.17	1	−6.47 ± 0.08	160	1/160
9	F	Dmt-DAla-Gly-Phe-βAla–	H	−9.10 ± 0.23	0.7	−8.79 ± 0.06	0.4	1.75/1
10	F	Dmt-DAla-Gly-Phe-βAla–	Dmt-DAla-Gly-Phe-βAla–	−8.89 ± 0.21	0.6	−8.85 ± 0.04	0.6	1

^aCompetition analyses against radiolabeled ligand ([³H]DPDPE for DOR δ [³H]DAMGO for μ were carried out using rat brain membranes.

^bLogarithmic values determined from the nonlinear regression analysis of data collected from at least two independent experiments.

Figure 2.

(A) Ligand 17 was evaluated in SD rats using a radiant heat assay (B) Ligand 17 antinociceptive dose-response curve (C) Ligand 17 dose-dependency was assessed by constructing a dose response curve.

Three doses of Ligand 17 or vehicle (0.1, 1.0, and 10 μg in 5 μl; n = 5/treatment) were evaluated in non-injured rats using a radiant heat assay. At no time were paw withdrawal latencies (PWLs) of rats treated with Ligand 17 significantly longer than those vehicle-treated rats and baseline values (p >0.05; Fig. 2a). The area under the curve (AUC) was calculated to determine if the duration of effect depended on dose; no significantly differences in the AUC were observed (Fig. 2c). The % antinociception was calculated and reached a maximal level of 63 ± 19 % (10 μg/5μL) 5 minutes after intrathecal administration; this was not significantly higher than vehicle treatment (p = 0.65; Fig. 2b). Ligand 17 did not significantly alleviate acute thermal pain in non-injured rats despite high affinity and in vitro functional activity at μ and δ opioid receptors. Detailed in vivo experimental procedures are given in the notes section.

The enkephalin analogues conjugated with N-phenyl-N-(piperidin-2-ylmethyl)propionamide derivatives represent an new family of μ and δ hybrid ligands and exhibit very good and balanced binding affinities with good agonist properties at μ and δ opioid receptors. From the structure activity relationship studies the small molecule 2 exhibited excellent binding affinities and selectivity towards the μ opioid receptor. The conjugation of enkephalin analogues to the small molecules, and the resultant bivalent ligands exhibited good binding affinities and agonistic activities as well. The introduction of a linker between the small molecule and peptide, and the replacement of Tyr with Dmt afforded highly potent ligands at both receptors. In the second series of bivalent ligands (16–18) introduction of a linker in between small molecule and peptide does not have a much effect on the bioactivity. Ligand 17 was evaluated for in vivo antinociceptive activity in non-injured rats following spinal administration. This local administration at the site of the first synapse was chosen to optimise delivery to the site of action and thus evaluate maximal intrinsic efficacy. At the doses tested, Ligand 17 was not significantly effective in alleviating acute pain. The most likely explanations for this low intrinsic efficacy in vivo despite high in vitro binding affinity, moderate in vitro activity are (i) low potency suggesting that higher doses are needed; (ii) differences in experimental design (i.e. non-neuronal, high receptor density for in vitro preparations versus CNS site of action in vitro); (iii) pharmacodynamics (i.e. engaging signalling pathways); (iv) pharmacokinetics (i.e. metabolic stability). In summary, our data suggest that further optimisation of this compound 17 is required to enhance intrinsic antinociceptive efficacy.

Abbreviations

ACN

acetonitrile

Boc

tert-butyloxycarbonyl

CHO

Chinese hamster ovary

DAMGO

[D-Ala2, NMePhe4, Gly5-ol]enkephalin

DALEA

[D-Ala2, Leu5] enkephalinamide

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

HBTU

N,N,N′,N′-tetra methyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

hDOR

human δ opioid receptor

Dmt

2,6-dimethyltyrosine

DPDPE

c[D-Pen2, DPen5]enkephalin

GPI

guinea pig isolated ileum

HATU

1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate

HRMS

high resolution mass spectrometry

rMOR

rat μ opioid receptor

MVD

mouse vas deferens

RP-HPLC

reverse phase high performance liquid chromatography

RT

room temperature

SAR

structure–activity relationship

TFA

trifluoroacetic acid

TLC

thin layer chromatography