Synthesis and biological evaluation of fentanyl analogues modified at

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Synthesis and biological evaluation of fentanyl analogues modified at phenyl groups with alkyls Yajuan Qin, Luofan Ni, Jiawei Shi, Zhiying Zhu, Saijian Shi, Ai Leen Lam, Julia Magiera, Sunderajhan Sekar, Andy Kuo, Maree T. Smith, and Tingyou Li ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00363 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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ACS Chemical Neuroscience

Synthesis and biological evaluation of fentanyl analogues modified at phenyl groups with alkyls

Yajuan Qin†, Luofan Ni†, Jiawei Shi†, Zhiying Zhu†, Saijian Shi†, Ai-leen Lam‡, Julia Magiera‡, Sunderajhan Sekar‡, Andy Kuo‡, Maree T. Smith*,‡, Tingyou Li*,†,§

†

School of Pharmacy, Nanjing Medical University, Nanjing 211166, China

‡

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland,

Brisbane, QLD 4072, Australia §

Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative

Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China

*Corresponding

authors:

T

Li,

Tel&

Fax:

+86-25-8686-8467;

e-mail:

[email protected]; MT Smith, Tel: +61-7-3365-2554; Fax: +61-3346-7391; e-mail: [email protected]

Abbreviations: cAMP, Cyclic Adenosine monophosphate; MOP, µ opioid; KOP, κ opioid; DOP, δ opioid; HEK, human embryonic kidney; Dmt, 2,6-dimetyl-L-tyrosine; Tmp, 2,3,6-trimethyl-L-phenylalanine; Dmp, 2,6-dimethyl-L-phenylalanine；DAMGO, [D-Ala2, N-MePhe4, Gly-ol] enkephalin; DPDPE, [D-Pen2, D-Pen5] enkephalin; NTB, naltriben; NX, naloxone; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid.

1

ACS Paragon Plus Environment

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Abstract: A series of fentanyl analogues modified at the phenyl group of the phenethyl with alkyl and/or hydroxyl and alkoxy, and the phenyl group in the anilido moiety replaced with benzyl or substituted benzyl, were synthesized. The in vitro opioid receptor functional activity of these compounds was evaluated by assessment of their ability to modulate forskolin-stimulated cAMP accumulation and by their ability to induce β-arrestin2 recruitment. Compound 12 is a potent µ-opioid (MOP) receptor agonist, a potent κ-opioid (KOP) receptor antagonist with weak β-arrestin2 recruitment activity. Compounds 10 and 11 are potent MOP receptor agonists with weak δ-opioid (DOP) receptor antagonist activity and moderate KOP receptor antagonist activity as well as weak β-arrestin2 recruitment activity at the MOP receptor. These compounds are promising leads for discovery of potent opioid analgesics with reduced side effects relative to clinically available strong opioid analgesics.

Keywords: fentanyl, analgesics, opioid, β-arrestin2

Introduction Fentanyl is one of the most commonly used strong opioid analgesics for the relief of acute and chronic nociceptive pain. After its discovery, scientists have synthesized a huge number of fentanyl analogues,1,

2

which led to discovery of

sufentanil, alfentanil, lofentanil and remifentanil. However, fentanyl formulated as transdermal patches is the most prescribed analgesic of this group for the relief of chronic nociceptive pain.1 Fentanyl has a rapid onset of analgesia and it shows more potent activity than morphine. Long-term administration of fentanyl, like morphine, for relief of 2


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cancer-related pain, produces a series of undesired side effects with analgesic tolerance and constipation being particularly troublesome.1, 2 Therefore, the synthesis of new fentanyl analogs with high potency but reduced opioid-related side effects is desirable.3-8 Recent advances in understanding opioid receptor signal transduction have suggested new possibilities regarding opioid receptor activation to evoke analgesia, while reducing or eliminating unwanted side effects.9-11 Pharmacological studies on opioid receptors have found that δ-opioid (DOP) receptors modulate the function of µ-opioid (MOP) receptors.12,13 Administration of a MOP agonist in conjunction with a DOP antagonist14 or a MOP agonist mixed with DOP antagonism exhibit analgesia but with reduced tolerance and physical dependence liabilities.15-19 Within this context, fentanyl was used to synthesize bivalent ligands comprising fentanyl conjugated to a DΟΡ receptor antagonist, Dmt-Tic. However, this bivalent ligand showed a substantial loss in MOP receptor functional activity.20 Opioid receptors are G-protein-coupled receptors. Apart from activating Gi/o-G-proteins to inhibit downstream cAMP formation, they also activate the β-arrestin2-coupled downstream signaling system. Recent research by others suggests that MOP receptor agonists biased to signal primarily via Gi/o proteins with minimal to no β-arrestin2 signaling, induce analgesic activity but with less constipation and respiratory depression relative to morphine.9-11 TRV130, a novel MOP receptor G protein-biased ligand, is reportedly a potent analgesic. It induces reduced gastrointestinal dysfunction and respiratory depression than morphine at equi-analgesic doses both in rats and mice.21-22 Recently, TRV130 completed Phase 3 clinical trials for the relief of moderate to severe acute pain, and the New Drug Application has been filed with FDA.23-26 Very recently, several novel G protein-biased ligands, such as PZM2127 and the SR-series compounds,28 have been reported as potent MOP-receptor mediated analgesic agents that show no respiratory depression and no hyperlocomotive response in mice at analgesic doses.27,28 These 3



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advances in the opioid pharmacology field have provided renewed impetus for the discovery of novel opioid analgesics with a reduced propensity to evoke respiratory depression, gastrointestinal discomfort and/or analgesic tolerance and dependence. In our previous work, we found that endomorphin analogues modified at the 1,3-position

of

their

aromatic

Dmt-Pro-Tmp/Dmp/Trp-Phe-NH229 2,6-dimetyl-L-tyrosine,

Tmp:

side

chains

with

alkyls,

and

Dmt-Pro-Tmp-Tmp-NH230

2,3,6-trimethyl-L-phenylalanine,

such

as

(Dmt: Dmp:

2,6-dimethyl-L-phenyl alanine)， are potent MOP agonists with DOP antagonism. Furthermore, Dmt-Pro-Tmp-Tmp-NH2 exhibited no β-arrestin2 recruitment activity.30 Opioids with such a pharmacological profile are expected to have fewer gastrointestinal side effects, and less respiratory depression, with a low propensity to evoke tolerance and physical dependence compared with an equi-analgesic dose of morphine. The in vivo biological activities of these compounds are currently under investigation. Opioid peptides generally have low oral bioavailability, primarily due to their inability to be absorbed across the gastrointestinal mucosa, their poor capacity to go through the blood-brain barrier and their low stability towards peptidases. In previous work on the structure-activity relationships of endomorphins, it was found that the side chains of the 1,3-position aromatic amino acids in these opioid peptides play a critical role in binding to and/or activating opioid receptors.30 Interestingly, fentanyl also has two phenyl groups, although they may have different binding modes with the MOP receptor compared with Tyr containing opioid peptides.31 Nevertheless, they provide two possible modifying moieties that have not been extensively studied.1 We applied the modification strategy used successfully for the endomorphin analogues to fentanyl to investigate if we could induce DOP-receptor antagonistic activity in fentanyl derivatives and inhibit the recruitment of β-arrestin2. We now report on a series of fentanyl analogues derived by modification of fentanyl’s two phenyl groups with alkyl groups, as well as their MOP, DOP and KOP functional activities in the forskolin-stimulated cAMP assay, and β-arrestin2 recruitment activity. 4


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Results and discussion Chemical synthesis.

Three series of fentanyl analogues modified at the two

phenyl groups were synthesized (Scheme 1). 2-Arylbromoethanes (3) were prepared from the corresponding arylbromides (1) through Grignard reaction subsequent to a bromination reaction (Scheme 2). 3,5-Dimethylbenzylamine was synthesized from 3,5-dimethylbenzoic

acid

as

reported.32

The

preparation

of

N-3,5-dimethylbenzyl-N-(4-piperidinyl)-propanamide (7b) was achieved following the procedure reported for the N-phenyl-N-(4-piperidinyl) propanamide (7a)7 starting from N-benzypiperidine-4-one (4) (Scheme 3). Briefly, N-benzypiperidine-4-one (4) reacted with aniline or 3,5-dimethylbenzylamine to give the corresponding Schiff base which was then reduced with NaBH4 to yield the corresponding N-benzyl-4-anilinopiperidine

(5a)

or

N-benzyl-4-(3,5-dimetnylbenzylamino)-piperidine (5b). Compound 5 was acylated using propionyl chloride. The benzyl group of the resulting compound 6 was removed by hydrogenation to give N-phenyl-N-(4-piperidinyl) propanamide (7a) or N-(3, 5-dimethylbenzyl)-N-(4-piperidinyl)-propanamide (7b). N-Benzyl-N-(4-piperidinyl) propanamide (19) (Scheme 4) was synthesized starting from N-Boc-piperidine-4-one (16) with a series of reactions analogous to those used in the preparation of compound 5. The synthesized propanamide derivatives (7a, 7b and 19) were then N-alkylated with 2-arylbromoethane to yield the desired fentanyl analogues (Scheme 3 and 4). The structures of the analogues were verified by MS and NMR. The purities were determined by HPLC, all the compounds showed purities greater than 98%.

Scheme 1. Structural formulae of fentanyl analogues. 5



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Scheme 2. Synthesis of 2-arylbromoethane. Reagents and conditions: (i) Mg, CH3CH2Br, THF, 30℃, 6.5 h, then ethylene oxide, -15℃ to rt, 12 h; (ii) PBr3, CCl4, 60℃, 1 h; (iii) BBr3, CH2Cl2, -15℃ to rt, 2 h.

Scheme 3. Synthesis of fentanyl analogues. Reagents and conditions: (i) RNH2, p-TsOH, 4Å MS, toluene, reflux 12 h; (ii) NaBH4, MeOH, reflux 3 h; (iii) propionyl chloride, CHCl3, -15℃ to rt, 12 h; (iv) Pd/C, 1 atm H2, MeOH, 50℃, 3d; (v) ArCH2CH2Br, Et3N, toluene, reflux 24 h.

6


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Scheme 4. Synthesis of fentanyl analogues. Reagents and conditions: (i) PhCH2NH2, p-TsOH, 4Å MS, toluene, reflux 12 h; (ii) NaBH4, MeOH, reflux 3 h; (iii) propionyl chloride, CHCl3, -15℃ to rt, 12 h; (iv) 6.4 mol/L HCl/dioxane, rt, 1 h; (v) ArCH2CH2Br, Et3N, toluene, reflux 48 h.

Inhibition of forskolin-stimulated cAMP formation in HEK-293 cells stably transfected with MOP, DOP or KOP receptors. Following opioid agonist binding at HEK-MOP, HEK-DOP and HEK-KOP cells, forskolin-stimulated cAMP formation is inhibited. Hence, the agonist and antagonist potencies of the new fentanyl analogues were evaluated using this assay as previously described.30 (Figures 1-6). For these cell based assays undertaken in 384-well plates, the final concentration of DMSO in each well was

Synthesis and biological evaluation of fentanyl analogues modified at

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