Discovery of a Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol

Abstract

In a previous communication, our efforts leading from 1 to the identification of spiro[cyclohexane-dihydropyrano[3,4-b]indole]-amine 2a as analgesic NOP and opioid receptor agonist were disclosed and their favorable in vitro and in vivo pharmacological properties revealed. We herein report our efforts to further optimize lead 2a, toward trans-6′-fluoro-4′,9′-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1′(3′H)-pyrano[3,4-b]indol]-4-amine (cebranopadol, 3a), which is currently in clinical development for the treatment of severe chronic nociceptive and neuropathic pain.

Recent publications indicate that small molecules activating both nociceptin/orphanin FQ peptide (NOP) and mu opioid peptide (MOP) receptors may potentiate opiate analgesia and at the same time display an improved side effects profile.^1,2 We have recently reported the discovery of a series of small molecules, characterized by their high NOP and opioid receptor agonistic activity.³ This series included uncyclized (e.g., 1) as well as spirocyclic examples (e.g., 2a). The discovery of spirocyclic 2a originated from the respective uncyclized analogues, which were potent NOP and MOP receptor binders but sometimes hampered by only partial agonistic NOP and MOP receptor activity. In particular, the spiroindole derivates sparked our interest due to their structural novelty and favorable in vitro and in vivo properties. The leading spiroether 2a exhibited strong efficacy in preclinical models of acute (ED₅₀ rat tail-flick: 3.63 nmol/kg i.v.) and neuropathic pain (ED₅₀ rat spinal nerve ligation: 1.05 nmol/kg i.v.) but was hampered by poor pharmacokinetic (PK) properties in rats with high clearance, large volume of distribution, moderate half-life (Cl = 4.0 L/h·kg; V_ss = 7.52 L/kg; t_1/2 = 1.6 h), and a critically very low oral bioavailability (F = 4%).

We herein report our efforts to further optimize the spiroindole lead 2a, which eventually led to the discovery of trans-6′-fluoro-4′,9′-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1′(3′H)-pyrano[3,4-b]indol]-4-amine (3a, cebranopadol), a novel potent analgesic NOP and opioid receptor agonist, currently in clinical development for the treatment of severe chronic nociceptive and neuropathic pain.

The structure–activity relationship (SAR) established around the uncyclized scaffolds (e.g., 1) suggested that a broad variety of linkers such as alcohols, ethers, and amines are tolerated, showing high NOP and MOP receptor binding affinities.³ As a result, we applied this knowledge through analogous structural variations to lead structure 2a (region A, Figure 1). These changes were also combined with a targeted approach to improve the poor PK profile, in particular by addressing metabolically liable regions B and C.

Figure 1.

Sites of modification.

With this in mind, the transformation of the oxacyclic spiro moiety into a carba-, aza-, or thio-cyclic moiety was investigated. Advancing from the oxacyclic spiro 2a to the azacyclic moiety 5a led to equally potent NOP and MOP receptor binders, as well as the introduction of the N-methyl subunit 6a. Similarly, potency was retained when moving from the oxa-cycle 2a to the thio-cycle 7a and carbacycle 8a (Table 1).

Table 1. Modification of Spirocyclic Oxygen, Region A.

			% inhibition @ 1 μM or Ki (nM)
compd	X	config	hNOPr	hMOPr
2ab	O	trans	0.1	0.26
2bb	O	cis	30%	310
5ac	NH	trans	0.19	0.39
6ac	NMe	trans	0.55	0.24
7aa	S	trans	3.7	3.1
8a	CH2	trans	1.26	1.95

^aHemicitrate.

^bHydrochloride.

^cDihydrochloride.

The wide range of tolerated moieties suggests that, upon cyclization, the molecule has been brought into a preferred conformation at both receptors (Figure 2). As a consequence of these results and employing the most favored core from a lipophilicity point of view, we focused our efforts for further optimization on the oxacyclic moiety.

Figure 2.

Three-dimensional structural overlay of the binding modes of 3a in the NOP and MOP receptors. NOP: Binding modes of 3a (orange) in the NOP receptor: NOP ribbons, blue; residues, cyan. MOP: Binding modes of 3a (green) in the MOP receptor: MOP ribbons, red; residues, yellow. MOP ribbons of helix 7, His319/Arg302, removed for clarity.

The synthesis of chemotypes described in this letter is shown in Scheme 1. Tryptophol 9 or its TMS ether 10, thiotryptophol 13, and commercially available tryptamines 14 and 15 were cyclized with 4-dimethylamino-4-phenyl-cyclohexanone 19 to give the corresponding spirocycles 2–7 under similar conditions to the ones reported previously.³

Scheme 1. Synthesis of Investigated Spirocycles.

Reagents and conditions: (a) (i) HN(TMS)₂, TMSCl, THF; (ii) NaHCO₃, Et₂O, 88–100%; (b) (i) Na₂S₂O₃, H₂O, EtOH reflux; (ii) i-PrOH reflux, 75%; (c) 50 v/v H₃PO₄/H₂O, Et₂O, reflux, 96%; (d) (i) HY or 40 w/w HNMe₂/H₂O, MeOH, 4 N HCl, KCN, 0 °C to rt; (ii) H₂O, Et₂O, DCM, 84–97%; (e) (i) PhMgCl, THF, 0 °C to rt; (ii) NH₄Cl, H₂O, 0 °C to rt, Et₂O; (f) 2-butanone, TMSCl, 0 °C to rt, 35–58% over steps (e) and (f); (g) (i) 7.5 N HCl, Et₂O; (ii) NaOH, 78–97%; (h) 5-(TES)-4-pentin-1-ol, MeCN, Ph₃PI₂, imidazole, 70%; (j) (i) t-BuLi/pentane, Et₂O, −75 to −70 °C; (ii) 13, Et₂O, 28%; (k) (i) iodoaniline, [1,3-bis(2,6-i-Pr₂Ph)-imidazol-2-ylidene]-(3-Cl-Py)Pd(II)-chloride, Na₂CO₃, DMF, 100 °C; (ii) Na₂S₂O₃, 66%; (l) TBAF, THF, reflux, 50 °C, 36%; (m) TMSOTf, DCM, rt, 33–99%; (n) (i) H₃PO₄, conc. AcOH; (ii) NaOH, 96%; (o) TfOH, DCM, rt; (ii) NaOH; (iii) MeOH, 65–67%; (p) TFA, DCE, 3–31%; (q) (i) TMSOMs, DCM, rt; (ii) filtration; (iii) evaporation filtrate, recryst EtOH; (iii) citric acid, EtOH, 65 °C to rt, 11% 7a; (r) (i) MsOH, DCM; (ii) filtration, 63–99%; (s) citric acid, hot ethanol, 5 °C 12 h, 53–99%.

To achieve carbacycle 8, another route was necessary: 5-triethylsilyl-4-pentyn-1-ol 20 was converted into the corresponding iodide 21, which after metal–halogen exchange with tBuLi was added to previously described³19 to give 22. Larock cyclization⁴ of the triethylsilyl alkyne moiety with ortho-iodoaniline gave the indole-3-yl-propyl moiety in 23.

After silyl deprotection with TBAF, spirocyclization of 24 was achieved with TMSOTf as promoter to give 8a after separation from the accompanying cis diastereomer by HPLC.⁵

The spirocyclization of 4-methylamino-cyclohexanone 27 gave the corresponding N-monomethyl analogue 28a.

Also cyclic amino heads, such as 4-phenyl-4-pyrrolidin-1-yl-cyclohexanone, were successfully synthesized and spirocyclized in analogous manner to give 38b–40b.

As illustrated in our previous publication, compound 2a, following intravenous administration, showed excellent efficacy in the rat tail-flick and spinal nerve ligation model while having a promising side effects profile. Nevertheless, it suffered from poor oral bioavailability and high clearance. To understand this further we subjected 2a to metabolite ID. These investigations identified N-demethyl compound 28a (Table 2) and 6′-hydroxy compound 48a (Table 3) as metabolites in humans and rodents. Our optimization efforts therefore aimed at targeting both sites of metabolism to improve the PK profile.

Table 2. Variation of the Amine Head, Region C.

			% inhibition @ 1 μM or Ki (nM)
compd	Y	config	hNOPr	hMOPr
2aa	NMe2	trans	0.1	0.26
28ab	NHMe	trans	1.1	0.36
38bc	piperidin-1-yl	cis	<10%	43%
39bd	4-Me-piperazin-1-yl	cis	<10%	<10%
40bc	morpholin-4-yl	cis	<10%	<10%

^aHydrochloride.

^bHemicitrate.

^cMethanesulfonate.

^dDimethanesulfonate.

Table 3. Variation of Substituents at Major Metabolic Positions, Region B.

				% inhibition @ 1 μM or Ki (nM)
compd	R1	R2	config	hNOPr	hMOPr
3aa	F	Me	trans	0.9	0.7
3ba	F	Me	cis	97.4	46
4aa	F	H	trans	11	2
47aa	Me	Me	trans	4.5	2.7
48aa	OH	Me	trans	1	0.4
49ab	OMe	Me	trans	9.4	1.1

^aHemicitrate.

^bHydrochloride.

The dimethyl amine head was tackled first. Synthesis of the N-demethyl metabolite, analogue 28a, resulted in a compound with potent NOP and MOP receptor binding activity. For the cyclic amine heads piperidine 38b, N-methyl piperazine 39b, and morpholine 40b, we only obtained the generally less active cis stereoisomer. However, because the introduction of cyclic amine heads resulted in complete loss of binding affinities, we concluded that there is little room for optimization here and retained the dimethylamino moiety for further optimization. We therefore turned our interest to the 6′-position of the indole spiro scaffold with the aim to block this point of metabolic instability.

As previously reported, the relative stereochemistry remained crucial for high NOP and MOP receptor binding affinities³ with the trans stereoisomer being the favored stereoisomer with respect to binding affinity (2a vs 2b; 3a vs 3b). This has been rationalized, through a bidentate, chelating binding mode to Asp130, which is only feasible from the trans configuration.³

Pleasingly, a variety of small substituents such as Me (47a), F (3a, 3b, and 4a), OH (48a), and OMe (49a) could be obtained by use of trytophols 41–43 and TMS ether 45 and were well tolerated by both receptors. Indeed, as described in Table 3, all trans stereoisomers demonstrated binding affinities below 20 nM at both the NOP and MOP receptor.

Despite the good receptor binding properties, we decided only to progress compounds with a NOP/MOP selectivity factor of <5, as our focus was for equipotent NOP and MOP receptor binders. On this basis, compounds 3a, 4a, 5a, and 48a were advanced into in vitro functional testing.

Equipotent binding at the MOP and NOP receptor was rationalized by overlaying the recently published crystal structures of the MOP and NOP receptors.^6,7 Major differences of the MOP and NOP receptor binding sites are Lys233 (MOP)/Ala216 (NOP), Asn127 (MOP)/Asp110 (NOP), His319 (MOP)/Arg302 (NOP), and Tyr128 (MOP)/Ile111 (NOP) switches (Figure 2).

This should allow for similar pi–pi stacking interactions of aromatic moieties at the NOP and MOP receptors. Hydrophobic cavities are conserved in both receptors. We have subsequently docked compound 3a in both receptors and observed only slightly different binding modes;⁸ therefore, similar binding affinities at the NOP and MOP receptors are to be expected.

More detailed characterization of 3a hemicitrate revealed binding affinities of 2.6 nM (N = 7, ± SD = 1.4) and 18.0 nM (N = 11, ± SD = 20.0 nM) at the hKOP and hDOP receptors.⁹

Functional investigations on 3a, 4a, 5a, and 48a revealed that all compounds acted as full agonists at both the MOP and NOP receptor and could therefore be progressed (Table 4). We nevertheless decided to drop 5a because of concerns with regards to hERG (data not shown).

Table 4. Functional Assay Results for Selected Compounds.

	EC50 GTPγS (nM)		rel. eff.a GTPγS
compd	hNOPr	hMOPr	hNOPr	hMOPr
3ab	13	1.2	89%	104%
4ab	60	2.6	101%	110%
5ac	1.3	0.2	108%	101%
48ab	120	1.2	75%	96%

^aEfficacy of 100% is defined as maximum [³⁵S]GTPγS binding induced by stimulation with nociceptin (NOP receptor) and DAMGO (MOP receptor).

^bHemicitrate.

^cDihydrochloride.

After these promising in vitro results we progressed 3a, 4a, and 48a into in vivo PK profiling. Compound 3a showed good mouse PK properties with low clearance (Cl = 0.96 L/h·kg), moderate half-life (t_1/2 = 2.57 h), medium volume of distribution (V_c = 2.96 L/kg), and good oral bioavailability (F = 44%). In rats, 3a achieved a significant improvement in oral bioavailability (F = 23%) as compared to 2a (F = 4%).³ The mouse PK of 4a is characterized by a very high clearance (Cl = 15.3 L/h·kg), very high volume of distribution (V_z = 72.5 L/kg), longer half-life (t_1/2 = 3.50 h), and good oral bioavailability (F = 44%).

Compounds 3a, 4a, and 48a were initially compared with regards to their activity in a mouse tail-flick model of acute nociceptive pain using a method modified from D’Amour and Smith.¹⁰ Antinociceptive activity of the compounds was quantified after i.v. and p.o. dosing by measuring the increase in the latency for tail withdrawal from a radiant heat source in male NMRI mice¹¹ (Table 5). Compound 3a induced dose-dependent inhibition of heat nociception with ED₅₀ values of 40.1 nmol/kg i.v. and 77.0 nmol/kg p.o. (Figure 3 and Table 5). With both routes of administration, the maximum attainable antinociceptive response was obtained, indicating full antinociceptive efficacy.

Table 5. In Vivo Results for Selected Compounds in a Mouse Tail-Flick Model of Acute Nociceptive Pain.

	tail-flick mouse
compd	ED50 (95% CI) [nmol/kg]	Emax (%MPE)	duration of action (min)e
3ad	i.v.: 40.1 (30.4–48.5)	>95	180
	p.o.: 77.0 (57.8–98.1)	100	>480
4ad	i.v.: 434 (347–499)	100	300
	p.o.: 1432 (1172–1758)	>95	>600
48ac	i.v.: 22.2 (18.0–27.6)	>95	n.d.
	p.o.: 3062 (2622–3538)	100	n.d.
fentanyl citrate	i.v.: 11.2 (8.5–15.1)	100	40
	p.o.: 1956 (1378–2797)	100	90
hydromorphone HCl	i.v.: 622 (529–778)	100	n.d.
	p.o.: 18753 (13466–25284)	>95	n.d.

^aHydrochloride.

^bDihydrochloride.

^cCitrate.

^dHemicitrate.

^eDuration of action was determined for a dose that produces approximately 90% MPE; it describes the duration for which the antinociceptive efficacy is >20% MPE. n.d. = not determined.

Figure 3.

Mouse tail-flick test: Dose–response curves of cebranopadol (3a) hemicitrate (A) and 4a hemicitrate (B) obtained after oral administration. Each point of the graph represents the mean ± SEM of the maximum possible effect (MPE); n = 10 animals per group. *p < 0.05 versus vehicle.

In order to characterize the duration of analgesic action, equi-effective dosages of the high dose range (>90% of the maximum possible effect (MPE)) were chosen. After oral administration, significant antinociceptive activity was observed for more than 8 h (last test point measured), where 85% MPE was still reached. The duration of action after intravenous administration lasted up to 3 h, where 38% MPE was measured. These investigations demonstrate that 3a is highly effective in an animal model of acute nociceptive pain. It is about as equi-potent to the strong opioid fentanyl citrate but shows a significantly longer duration of action and, in contrast to fentanyl, displays high potency and long lasting efficacy as well after oral administration (see Table 5).

Compound 4a also exerted dose-dependent activity in the mouse tail-flick test (Figure 3), with ED₅₀ values of 434 nmol/kg i.v. and 1432 nmol/kg p.o. This is about 10-fold less potent than 3a after both i.v. and p.o. dosing, which can be clearly explained by the observed higher volume of distribution and clearance. The potency of 48a after oral dosing was surprisingly low as compared to cebranopadol 3a and could only be partially explained by its lower oral bioavailability (F = 14%).

A model of streptozotocin (STZ)-induced diabetic polyneuropathy was used to explore the efficacy of 3a in a neuropathic pain state (see Supporting Information for details). A dose of 2.1 nmol/kg i.v. of 3a was tested in STZ-treated diabetic CD-1 mice and, for comparison, in nondiabetic control animals. While 3a induced a significant reduction of thermal hyperalgesia assessed in a hot plate test, it was without an antinociceptive effect in the control animals. The antihyperalgesic efficacy of 2.1 nmol/kg i.v. of 3a was calculated to be 54.1% MPE (n = 10, SEM ± 4.6% MPE; p < 0.001 vs vehicle control) (Figure 4). Thus, in the neuropathic pain state, 3a showed about half-maximum antihyperalgesic activity at a dose being 20 times lower than the ED₅₀ value in the tail-flick model of acute nociception.

Figure 4.

Dose-dependent effects of 3a (cebranopadol) hemicitrate on heat nociception (tail-flick and hot plate in STZ control animals), on thermal hyperalgesia (hot plate in STZ diabetic animals), and on motor coordination (rota rod) after i.v. administration in mice. ^#, Relative effect refers to percentage of maximum possible effect (% MPE) for tail-flick and STZ models, or relative decrease in performance time on the rotating rod (rota rod). Error bars indicate SEM (n = 10). *, p < 0.05 versus vehicle control.

We decided to progress 3a, due to its high efficacy and potency after oral and intravenous administration, for in-depth profiling. The cytochrom-P450 inhibition potential of 3a was estimated after incubation with human CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4/5 and probe substrates selectively metabolized by these enzymes. None of the tested cytochrome P450 enzymes were inhibited in vitro by 3a up to 250 nM.

The interaction of 3a with safety-relevant cardiovascular targets was characterized: L-type Ca²⁺-channel (benzothiazepine site) rat brain cortex K_i (μM) = 0.8 (N = 2); L-type Ca²⁺-channel (phenylalkylamine site) rat brain cortex K_i (μM) = 0.48 (N = 2); L-type Ca²⁺-channel (dihydropyridine site) rat whole brain K_i (μM) > 100 (N = 2); and hERG channel human recombinant K_i (μM) > 100 (N = 2). Taking into account the high affinity of 3a to interact with the primary targets (K_i < 1 nM), the affinities toward these safety relevant targets are expected to be without biological relevance. More detailed electrophysiological investigations using the whole-cell patch-clamp technique¹² revealed that 3a did not affect hERG-mediated potassium currents in transfected HEK293 cells up to 10 μM.

Having ensured a low potential to induce cardiac side effects by 3a we embarked on assessing its side effects profile. Treatment with opioid drugs can be associated with severe side effects on the central nervous system (CNS) and gastrointestinal functions.¹³

In order to investigate its potential to affect the CNS, 3a was investigated in a rota-rod test.¹⁴ In male CD-1 mice, intravenous administration of 3a at doses up to 376 nmol/kg did not significantly influence motor coordination in the rota-rod test. At a dose of 454 nmol/kg, 3a induced a significant impairment of the rota-rod performance. The time that animals were able to remain on the rotating rod was shortened transiently for about 30–60 min after administration. The maximum effect was observed 15 min after administration, when the time on the rod was shortened from the maximum performance time of 120 s to 72 ± 17 s (mean ± SEM). Concurrently, the number of failures increased significantly up to 2.6 ± 0.9 (mean ± SEM). The no observed effect level (NOEL) for 3a was therefore considered to be 376 nmol/kg. An ED₅₀ value could not be calculated but is estimated to be >454 nmol/kg. Compound 3a is, thus, characterized by a safety margin ≥10-fold between the ED₅₀ value in the tail-flick test and the NOEL for effects on motor coordination (Figure 4). This is in clear contrast to standard opioids such as morphine or oxycodone that impair motor coordination within the antinociceptive dose range.¹⁵

Effects of 3a on the gastrointestinal system were investigated in the charcoal transit test in male NMRI mice.¹⁶ In this assay, effects on gut motility were measured as changes in transit rate of an orally administered charcoal suspension. Intravenous administration of 3a at doses between 10 and 454 nmol/kg induced a dose-dependent inhibition of intestinal charcoal transit. NOEL was 21 nmol/kg i.v., and the ED₅₀ was 87 nmol/kg i.v. A comparison of this ED₅₀ value to the ED₅₀ value in the mouse tail-flick assay points toward a separation of the analgesic dose range from doses inhibiting gastrointestinal motility. In contrast to standard opiates like morphine, which inhibits intestinal transit in mice already at doses below the ED₅₀ in the tail-flick assay,^16,173a clearly showed effects on gut motility at doses higher than those required to induce antinociception.

In summary, 3a (cebranopadol) exhibited high efficacy in a mouse model of acute pain with a potency comparable to the strong opioid fentanyl. In a model of polyneuropathic pain, 3a (cebranopadol) showed an about 20 times higher potency as compared to the acute pain model. In contrast to standard opioids, which induce marked CNS side effects and inhibition of gastrointestinal transit at doses below or within the half-maximum effective analgesic dose range,^17,183a (cebranopadol) exhibits distinctly improved margins between the analgesic dose range and doses inducing significant gastrointestinal or CNS side effects, which point towards a broader therapeutic window (Figure 4).

A more detailed investigation of cebranopadol’s pharmacological profile has been published elsewhere.⁹ Currently, cebranopadol is in clinical development for the treatment of severe chronic nociceptive and neuropathic pain.

Glossary

Abbreviations

CHO

Chinese hamster ovary cells

CI

confidence interval

CNS

central nervous system

DOP

delta opioid peptide

EC₅₀

concentration with half-maximum inducible [³⁵S]GTPγS binding

ED₅₀

half-maximum effective dose

E_max

maximum possible effect for the agonist

GTPγS

guanosine-5′-[γ-thio]triphosphate

hNOPr

Human nociceptin/orphanin FQ receptor

hMOPr

Human μ opioid receptor

HCl

hydrochloride

IC₅₀

half-maximum inhibitory concentration

i.v.

intravenous

K_i

dissociation constant for inhibitor binding

KOP

kappa opioid peptide

MeCN

acetonitrile

MOP

mu opioid peptide

MPE

maximum possible effect

NOEL

no observed effect level

NOP

nociceptin/orphanin FQ peptide

SNL

spinal nerve ligation

TES

triethylsilyl

Supporting Information Available

Assay description and experimental procedures for the synthesis and characterization of selected compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.