Nascent structure–activity relationship study of a diastereomeric series of kappa opioid receptor antagonists derived from CJ-15,208

Cyclic tetrapeptide c[Phe-pro-Phe-trp] 2, a diastereomer of CJ-15,208 (1), was identified as a potent dual κ/μ opioid receptor antagonist devoid of δ opioid receptor affinity against cloned human receptors: K_i (2) = 3.8 nM (κ), 30 nM (μ); IC₅₀ ([³⁵S]GTPγS binding) = 140 nM (κ), 21 nM (μ). The d-tryptophan residue rendered 2 ca. eightfold and fourfold more potent at κ and μ, respectively, than the corresponding l-configured tryptophan in the natural product 1. Phe analogs 3–10, designed to probe the effect of substituents on receptor affinity and selectivity, possessed K_i values ranging from 14 to 220 nM against the κ opioid receptor with μ/κ ratios of 0.45–3.0. An alanine scan of 2 yielded c[Ala-pro-Phe-trp] 12, an analog equipotent to 2. Agents 2 and 12 were pure antagonists in vitro devoid of agonist activity. Ac-pro-Phe-trp-Phe-NH₂ 16 and Ac-Phe-trp-Phe-pro-NH₂ 17 two of the eight possible acyclic peptides derived from 1 and 2, were selective, modestly potent μ ligands: K_i (16) = 340 nM (μ); K_i (17) = 360 nM (μ).     

Aerobic oxidation of indolomorphinan without the 4,5-epoxy bridge and subsequent rearrangement of the oxidation product to spiroindolinonyl-C-normorphinan derivative

Aerobic oxidation of indolomorphinan 1 without a 4,5-epoxy bridge proceeded in the presence of platinum catalyst to give indoleninomorphinan 2 or quinolono-C-normorphinan 5. The 4-hydroxy group would play an important role in deciding the course of the reaction. Treatment of 2a with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave spiroindolinonyl-C-normorphinan 3a whose structure resembles that of δ opioid receptor agonist spiroindanyloxymorphone (SIOM). Boron trichloride was effective for the reverse reaction from 3a to 2a without side reaction. This practical interconversion method between hydroxyindolenine and spiroindolinone would be useful for the design and construction of drug-like compound libraries. Although the compound 3b was expected to show the selectivity for δ opioid receptor because of the structural resemblance to SIOM, it was rather selective for μ opioid receptor (μ: K_i = 0.75 nM; δ: K_i = 2.90 nM; κ: K_i = 13.4 nM). The result suggests that the slight difference of the spatial location of the benzene rings in these compounds may definitively affect the binding affinity for δ opioid receptor.     

Synthesis and biological evaluation of C-2 halogenated analogs of salvinorin A

Salvinorin A (1), the main active ingredient of Salvia divinorum, is a potent and selective κ opioid receptor (KOPR) agonist. Based on the SAR, its C-2 position is one of the key binding sites and has very little space tolerance (3–4 carbons atoms) and limited to only lipophilic groups. In our attempt to prepare PET brain imaging agent for mapping KOPR, a series of C-2 halogenated analogs have been synthesized and screened for binding affinity at κ (KOPR), μ (MOPR), and δ (DOPR). These C-2 halogenated analogs with sequential changes of atomic radius and electron density serve as excellent molecular probes for further investigating the binding pocket at C-2, particularly on the effects of α verses β configuration at C-2 position. The results of KOPR binding and functional studies reveal β isomer in general binds better than α isomer with the exception of iodinated analogs and none of the C-2 halogenated analogs shows any improvement of KOPR binding affinity. Interestingly, functional assay has characterized that 6b is a partial agonist with E_max of 46% of the kappa receptor full agonist U50,488H at 250 nM (K_i). We have also observed that the affinity to the kappa receptor increases with atomic radius (I > Br > Cl > F) which is in good agreement with halogen bonding interactions reported in the literature.     

Synthesis and opioid receptor activity of indolopropellanes

A series of skeletal rearranged indolomorphinans 7a–d were obtained by N-demethylation of 3-methoxy-N-methyl-14-hydroxymorphinan-6-one 12 followed by N-realkylation, reduction and Fischer indole cyclization. The structure of the novel skeleton was confirmed by X-ray analysis. These new indoles displayed moderate binding affinity and selectivity at the μ receptor, with compound 7b showing the highest affinity at this receptor with a K_i value of 40 nM, and 6- and 25-fold selectivity against δ and κ receptors, respectively. Function assays showed that indolopropellanes 7b and 7c possessed full agonistic activity at all the opioid receptors indicating a different interaction model existed.     

Design synthesis and structure–activity relationship of 5-substituted (tetrahydronaphthalen-2yl)methyl with N-phenyl-N-(piperidin-2-yl)propionamide derivatives as opioid ligands

Here, we report the design, synthesis and structure activity relationship of novel small molecule opioid ligands based on 5-amino substituted (tetrahydronaphthalen-2-yl)methyl moiety with N-phenyl-N-(piperidin-2-yl)propionamide derivatives. We synthesized various molecules including amino, amide and hydroxy substitution on the 5th position of the (tetrahydronaphthalen-2-yl)methyl moiety. In our further designs we replaced the (tetrahydronaphthalen-2-yl)methyl moiety with benzyl and phenethyl moiety. These N-phenyl-N-(piperidin-2-yl)propionamide analogues showed moderate to good binding affinities (850–4 nM) and were selective towards the μ opioid receptor over the δ opioid receptors. From the structure activity relationship studies, we found that a hydroxyl substitution at the 5th position of (tetrahydronapthalen-2yl)methyl group, ligands 19 and 20, showed excellent binding affinities 4 and 5 nM, respectively, and 1000 fold selectivity towards the μ opioid relative to the delta opioid receptor. The ligand 19 showed potent agonist activities 75 ± 21 nM, and 190 ± 42 nM in the GPI and MVD assays. Surprisingly the fluoro analogue 20 showed good agonist activities in MVD assays 170 ± 42 nM, in contrast to its binding affinity results.     

Redefining the structure–activity relationships of 2,6-methano-3-benzazocines. Part 8. High affinity ligands for opioid receptors in the picomolar Ki range: Oxygenated N-(2-[1,1′-biphenyl]-4-ylethyl) analogues of 8-CAC

N-[2-(4′-methoxy[1,1′-biphenyl]-4-yl)ethyl]-8-CAC (1) is a high affinity (K_i = 0.084 nM) ligand for the μ opioid receptor and served as the lead compound for this study. Analogues of 1 were made in hopes of identifying an SAR within a series of oxygenated (distal) phenyl derivatives. A number of new analogues were made having single-digit pM affinity for the μ receptor. The most potent was the 3′,4′-methylenedioxy analogue 18 (K_i = 1.6 pM).     

Evaluation of N-substituent structural variations in opioid receptor profile of LP1

The benzomorphan scaffold has great potential as lead structure and the nature of the N-substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N-substituent (9–15). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity (K_i^MOR = 38 ± 4 nM) displaying good selectivity versus DOR and KOR. In the electrically stimulated GPI, compound 9 was inactive as agonist but produced an antagonist potency value (pA₂) of 8.6 in presence of MOR agonist DAMGO. Moreover, subcutaneously administered it antagonized the antinociceptive effects of morphine with an AD₅₀ = 2.0 mg/kg in mouse-tail flick test. Modeling studies on MOR revealed that compound 9 fit very well in the binding pocket but in a different way in respect to the agonist LP1. Probably the replacement of its N-substituent on the III, IV and V TM domains reflects an antagonist behavior. Therefore, compound 9 could represent a potential lead to further develop antagonists as valid therapeutic agents and useful pharmacological tools to study opioid receptor function.     

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC₅₀ = 150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC₅₀ = 25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC₅₀ = 7.5 nM) and TNF-α production in mouse splenocytes (IC₅₀ = 9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID₅₀ = 18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand–enzyme complex.     

Novel highly potent serotonin 5-HT7 receptor ligands: Structural modifications to improve pharmacokinetic properties

Here we report the synthesis, pharmacological and pharmacokinetic evaluation of a pilot set of compounds structurally related to the potent and selective 5-HT₇ ligand LP-211. Among the studied compounds, N-pyridin-3-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (4b) showed high affinity for 5-HT₇ receptors (K_i = 23.8 nM), selectivity over 5-HT_1A receptors (>50-fold), in vitro metabolic stability (82%) and weak interaction with P-glycoprotein (BA/AB = 3.3). Compound 4b was injected ip in mice to preliminarily evaluate its distribution between blood and brain.     

An LP1 analogue,selective MOR agonist with a peculiar pharmacological profile,used to scrutiny the ligand binding domain

The hypothesis that central analgesia with reduced side effects is obtainable by occupying an ‘allosteric’ site in the MOR ligand binding domain requires the development of new ligands with peculiar pharmacological profile to be used as tools. New benzomorphan derivatives, analogues of LP1, a multitarget MOR agonist/DOR antagonist, were designed to examine in depth MOR ligand binding domain. Compound 5, bearing a diphenylic N-substituent on the benzomorphan nucleus, showed an affinity (K_i^μ = 0.5 ± 0.2 nM) comparable to that of LP1 and a better selectivity versus DOR and KOR. It elicits antinociceptive effects in ex vivo (GPI) and in vivo. This new compound engages receptor amino acidic residues not reached by LP1 and by other established MOR ligands. Molecular modeling studies, conducted on 5 and on several reference compounds, allowed us to propose possible residues in the MOR ligand binding domain essential for their interactions with ‘orthosteric’ and ‘allosteric’ binding sites.     

Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC₅₀ = 77 nM; μ:κ and δ:κ IC₅₀ ratios >400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC₅₀ = 20 nM; μ:κ = 36, δ:κ = 415) was also shown to reverse κ-agonist induced rat diuresis in vivo.     

Discovery of μ-opioid selective ligands derived from 1-aminotetralin scaffolds made via metal-catalyzed ring-opening reactions

A series of 1-aminotetralin scaffolds was synthesized via metal-catalyzed ring-opening reactions of heterobicyclic alkenes. Small libraries of amides and amines were made using the amino group of each scaffold as a handle. Screening of these libraries against human opioid receptors led to the identification of (S)–(S)-5.2a as a high-affinity selective μ ligand (IC₅₀ μ = 5 nM, κ = 707 nM, δ = 3,795 nM) displaying μ-agonist/antagonist properties due to its partial agonism (EC₅₀ = 2.6 μM; E_max = 18%).     

3-(Arylsulfonyl)-1-(azacyclyl)-1H-indoles are 5-HT6 receptor modulators

Novel 3-(arylsulfonyl)-1-(azacyclyl)-1H-indoles 6 were synthesized as potential 5-HT₆ receptor ligands, based on constraining a basic side chain as either a piperidine or a pyrrolidine. Many of these compounds had good 5-HT₆ binding affinity with K_i values <10 nM. Depending on substitution, both agonists (e.g., 6o: EC₅₀ = 60 nM, E_max = 70%) and antagonists (6y: IC₅₀ = 17 nM, I_max = 86%) were identified in a 5-HT₆ adenylyl cyclase assay.     

A new Suzuki synthesis of triphenylethylenes that inhibit aromatase and bind to estrogen receptors α and β

The design and synthesis of dual aromatase inhibitors/selective estrogen receptor modulators (AI/SERMs) is an attractive strategy for the discovery of new breast cancer therapeutic agents. Previous efforts led to the preparation of norendoxifen (4) derivatives with dual aromatase inhibitory activity and estrogen receptor binding activity. In the present study, some of the structural features of the potent AI letrozole were incorporated into the lead compound (norendoxifen) to afford a series of new dual AI/SERM agents based on a symmetrical diphenylmethylene substructure that eliminates the problem of E,Z isomerization encountered with norendoxifen-based AI/SERMs. Compound 12d had good aromatase inhibitory activity (IC₅₀ = 62.2 nM) while also exhibiting good binding activity to both ER-α (EC₅₀ = 72.1 nM) and ER-β (EC₅₀ = 70.8 nM). In addition, a new synthesis was devised for the preparation of norendoxifen and its analogues through a bis-Suzuki coupling strategy.     

Discovery of a potent,selective, and orally bioavailable histamine H3 receptor antagonist SAR110068 for the treatment of sleep–wake disorders

Previous studies have shown that compound 1 displayed high affinity towards histamine H₃ receptor (H₃R), (human (h-H₃R), K_i = 8.6 nM, rhesus monkey (rh-H₃R), K_i = 1.2 nM, and rat (r-H₃R), K_i = 16.5 nM), but exhibited high affinity for hERG channel. Herein, we report the discovery of a novel, potent, and highly selective H₃R antagonist/inverse agonist 5a(SS) (SAR110068) with acceptable hERG channel selectivity and desirable pharmacological and pharmacokinetic properties through lead optimization sequence. The significant awakening effects of 5a(SS) on sleep–wake cycles studied by using EEG recording in rats during their light phase support its potential therapeutic utility in human sleep–wake disorders.     

Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin A ligands

Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand–receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ-, δ- and μ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (K_i = 2 nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (K_i = 21, 36 and 39 nM).     

Chlorophenoxy aminoalkyl derivatives as histamine H3R ligands and antiseizure agents

A series of twenty new chlorophenoxyalkylamine derivatives (9–28) was synthesized and evaluated on their binding properties at the human histamine H₃ receptor (hH₃R). The spacer alkyl chain contained five to seven carbon atoms. The highest affinities have shown the 4-chloro substituted derivatives 10 and 25 (K_i = 133 and 128 nM, respectively) classified as antagonists in cAMP accumulation assay (EC₅₀ = 72 and 75 nM, respectively). Synthesized compounds were also evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA). Two compounds (4-chloro substituted derivatives: 20 and 26) were the most promising and showed in the MES seizure model in rats (after ip administration) ED₅₀ values of 14 mg/kg and 13.18 mg/kg, respectively. Protective indexes (PI = TD₅₀/ED₅₀) were 3.2 for 20 and 3.8 for 26. Moreover, molecular modeling and docking studies were undertaken to explain affinity at hH₃R of target compounds, and the experimentally and in silico estimation of properties like lipophilicity and metabolism was performed. Antiproliferative effects have been also investigated in vitro for selected compounds (10 and 25). These compounds neither possessed significant antiproliferative and antitumor activity, nor modulated CYP3A4 activity up to concentration of 10 μM.     

Investigation of various N-heterocyclic substituted piperazine versions of 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol: Effect on affinity and selectivity for dopamine D3 receptor

Here we report on the design and synthesis of several heterocyclic analogues belonging to the 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol series of molecules. Compounds were subjected to [³H]spiperone binding assays, carried out with HEK-293 cells expressing either D2 or D3 dopamine receptors, in order to evaluate their inhibition constant (K_i) at these receptors. Results indicate that N-substitution on the piperazine ring can accommodate various substituted indole rings. The results also show that in order to maintain high affinity and selectivity for the D3 receptor the heterocyclic ring does not need to be connected directly to the piperazine ring as the majority of compounds included here are linked either via an amide or a methylene linker to the heterocyclic moiety. The enantiomers of the most potent racemic compound 10e exhibited differential activity with (?)-10e (K_i; D2 = 47.5 nM, D3 = 0.57 nM) displaying higher affinity at both D2 and D3 receptors compared to its enantiomer (+)-10e (K_i; D2 = 113 nM, D3 = 3.73 nM). Additionally, compound (?)-10e was more potent and selective for the D3 receptor compared to either 7-OH-DPAT or 5-OH-DPAT. Among the bioisosteric derivatives, the indazole derivative 10g and benzo[b]thiophene derivative 10i exhibited the highest affinity for D2 and D3 receptors. In the functional GTPγS binding study, one of the lead molecules, (?)-15, exhibited potent agonist activity at both D2 and D3 receptors with preferential affinity at D3.     

Design,synthesis and anticonvulsant activity of new hybrid compounds derived from N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)-propanamides and -butanamides

The focused library of 21 new N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, 2-(3-methyl-2,5-dioxopyrrolidin-1-yl)propanamide, and 2-(2,5-dioxopyrrolidin-1-yl)butanamide derivatives as potential new hybrid anticonvulsant agents was synthesized. These hybrid molecules were obtained as close analogs of previously described N-benzyl derivatives and fuse the chemical fragments of clinically relevant antiepileptic drugs such as ethosuximide, levetiracetam, and lacosamide. The initial anticonvulsant screening was performed in mice (ip) using the ‘classical’ maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests, as well as in the six-Hertz (6 Hz) model of pharmacoresistant limbic seizures. Applying the rotarod test, the acute neurological toxicity was determined. The broad spectra of activity across the preclinical seizure models in mice (ip) displayed compounds 4, 5, 11, and 19. The most favorable anticonvulsant properties demonstrated 4 (ED₅₀ MES = 96.9 mg/kg, ED₅₀ scPTZ = 75.4 mg/kg, ED₅₀ 6 Hz = 44.3 mg/kg) which showed TD₅₀ = 335.8 mg/kg in the rotarod test that yielded satisfying protective indexes (PI MES = 3.5, PI scPTZ = 4.4, PI 6 Hz = 7.6). Consequently, compound 4 revealed comparable or better safety profile than model antiepileptic drugs (AEDs): ethosuximide, lacosamide, and valproic acid. In the in vitro assays, compound 4 was observed as relatively effective binder to the neuronal voltage-sensitive sodium and diltiazem site of L-type calcium channels.     

Diether derivatives of homo- or substituted piperidines as non-imidazole histamine H3 receptor ligands

Synthesis and biological activities of a series of homo- or substituted piperidine unsymmetrical diethers are described. The novel compounds were evaluated for histamine H₃ receptor binding affinities at recombinant human H₃ receptor stably expressed in HEK-293 cells. All diethers showed in vitro affinities in nanomolar concentration range. The most potent compounds are 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]-3-methylpiperidine 11 (K_i = 3.2 nM) and 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]azepane 13 (K_i = 3.5 nM).