Design, synthesis, and validation of a β-turn mimetic library targeting protein-protein and peptide-receptor interactions

The design and synthesis of a β-turn mimetic library as a key component of a small-molecule library targeting the major recognition motifs involved in protein-protein interactions is described. Analysis of a geometric characterization of 10,245 β-turns in the protein data bank (PDB) suggested that trans-pyrrolidine-3,4-dicarboxamide could serve as an effective and synthetically accessible library template. This was confirmed by initially screening select compounds against a series of peptide-activated GPCRs that recognize a β-turn structure in their endogenous ligands. This validation study was highlighted by identification of both nonbasic and basic small molecules with high affinities (K(i) = 390 and 23 nM, respectively) for the κ-opioid receptor (KOR). Consistent with the screening capabilities of collaborators and following the design validation, the complete library was assembled as 210 mixtures of 20 compounds, providing a total of 4200 compounds designed to mimic all possible permutations of 3 of the 4 residues in a naturally occurring β-turn. Unique to the design and because of the C(2) symmetry of the template, a typical 20 × 20 × 20-mix (8000 compounds prepared as 400 mixtures of 20 compounds) needed to represent 20 variations in the side chains of three amino acid residues reduces to a 210 × 20-mix, thereby simplifying the library synthesis and subsequent screening. The library was prepared using a solution-phase synthetic protocol with liquid-liquid or liquid-solid extractions for purification and conducted on a scale that insures its long-term availability for screening campaigns. Screening the library against the human opioid receptors (KOR, MOR, and DOR) identified not only the activity of library members expected to mimic the opioid receptor peptide ligands but also additional side-chain combinations that provided enhanced receptor binding selectivities (>100-fold) and affinities (as low as K(i) = 80 nM for KOR). A key insight to emerge from the studies is that the phenol of Tyr in endogenous ligands bearing the H-Tyr-Pro-Trp/Phe-Phe-NH(2) β-turn is important for MOR binding but may not be important for KOR (accommodated, but not preferred) and that the resulting selectivity for KOR observed with its removal can be increased by replacing the phenol OH with a chlorine substituent, further enhancing KOR affinity.     

New mu-opioid receptor agonists with phenoxyacetic acid moiety

New muq-opioid receptor (MOR) agonists containing 4-hydroxypiperidine, piperidine and piperazine moieties were synthesized and evaluated to find a peripheral opioid analgesic. Among the synthesized compounds, 12-[1-[3-(N,N-dimethylcarbamoyl)-3,3-diphenylpropyl]-4-hydroxypiperidin-4-yl]phenoxy]acetic acid (8: SS620) having phenoxyacetic acid and 4-hydroxypiperidine moieties showed the highest agonist potency on the MOR in an isolated guinea-pig ileum preparation, and it also had selectivity to the human MOR expressed in Chinese hamster ovary (CHO)-K1 cells compared with the same types of delta- and kappa-opioid receptors (DOR and KOR). In addition, compound 8 showed a 10 times more potent MOR agonist activity than loperamide. Furthermore, compound 8 showed a peripheral analgesic activity in vivo screening on rat.     

Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists

The delta opioid receptor (DOR) is a crucial receptor system that regulates pain, mood, anxiety, and similar mental states. DOR agonists, such as SNC80, and DOR-neutral antagonists, such as naltrindole, were developed to investigate the DOR in vivo and as potential therapeutics for pain and depression. However, few inverse agonists and non-competitive/irreversible antagonists have been developed, and none are widely available. This leaves a gap in our pharmacological toolbox and limits our ability to investigate the biology of this receptor. Thus, we designed and synthesized the novel compounds SRI-9342 as an irreversible antagonist and SRI-45128 as an inverse agonist. These compounds were then evaluated in vitro for their binding affinity by radioligand binding, their functional activity by ³⁵S-GTPγS coupling, and their cAMP accumulation in cells expressing the human DOR. Both compounds demonstrated high binding affinity and selectivity at the DOR, and both displayed their hypothesized molecular pharmacology of irreversible antagonism (SRI-9342) or inverse agonism (SRI-45128). Together, these results demonstrate that we have successfully designed new inverse agonists and irreversible antagonists of the DOR based on a novel chemical scaffold. These new compounds will provide new tools to investigate the biology of the DOR or even new potential therapeutics.     

A Journey through Diastereomeric Space: The Design,Synthesis, In Vitro and In Vivo Pharmacological Activity,and Molecular Modeling of Novel Potent Diastereomeric MOR Agonists and Antagonists

Four sets of diastereomeric C9-alkenyl 5-phenylmorphans, varying in the length of the C9-alkenyl chain, were designed to examine the effect of these spatially distinct ligands on opioid receptors. Functional activity was obtained by forskolin-induced cAMP accumulation assays and several compounds were examined in the [³⁵S]GTPgS assay and in an assay for respiratory depression. In each of the four sets, similarities and differences were observed dependent on the length of their C9-alkenyl chain and, most importantly, their stereochemistry. Three MOR antagonists were found to be as or more potent than naltrexone and, unlike naltrexone, none had MOR, KOR, or DOR agonist activity. Several potent MOR full agonists were obtained, and, of particular interest partial agonists were found that exhibited less respiratory depression than that caused by morphine. The effect of stereochemistry and the length of the C9-alkenyl chain was also explored using molecular modeling. The MOR antagonists were found to interact with the inactive (4DKL) MOR crystal structures and agonists were found to interact with the active (6DDF) MOR crystal structures. The comparison of their binding modes at the mouse MOR was used to gain insight into the structural basis for their stereochemically induced pharmacological differences.     

Unpredictable stereochemical preferences for mu opioid receptor activity in an exhaustively stereodiversified library of 1,4-enediols

Using olefin cross-metathesis, we synthesized a novel stereodiversified library of compounds 3 containing a trans-1,4-enediol. Screening this library for mu opioid receptor (MOR) affinity identified multiple high-affinity ligands and revealed that the stereochemical configuration varied widely among those ligands having the highest affinity. It was not possible to predict the configurations of the most active compounds 3 on the basis of the configuration of endomorphin-2, a known MOR peptide ligand, validating the diversity-based approach to ligand discovery.     

Design, synthesis and biological evaluation of a novel series of potent, orally active adenosine A1 receptor antagonists with high blood-brain barrier permeability

A novel series of 3-(2-substituted-3-oxo-2,3-dihydropyridazin-6-yl)-2-phenylpyrazolo[1,5-a]pyridines (5-38) were synthesized and evaluated for their in vitro adenosine A1 and A(2A) receptor binding activities, and in vitro metabolism by rat liver in order to search for orally active compounds. Most of the test compounds were potent adenosine A1 receptor antagonists with high A1 selectivity and the A1 affinity and A1 selectivity of carbonyl derivatives (5-11) was particularly high. In particular, compound 7 was an extremely potent and selective adenosine A1 antagonist with high A1 selectivity (Ki=0.026 nM, A(2A)/A1=5400). In terms of metabolic stability, 2-oxopropyl (5), 2-hydroxypropyl (12), N-methylacetamide (16), 2-(piperidin-1-yl)ethyl (28) and 1-methylpiperidin-4-yl (32, FR194921) were the most stable compounds in this series of analogues. Further in vivo evaluation indicated that compounds 5, 13, 17, 28 and 32 were detected in both plasma and brain after oral administration in rats. In particular, 32 displayed good plasma and brain concentrations (dose: 32 mg/kg (n=3); after 30 min, plasma conc.=3390+/-651nM, brain conc.=3670+/-496nM; after 60min, plasma conc.=1580+/-348nM, brain conc.=2143+/-434nM), and a good brain/plasma ratio (1.11+/-0.060 (30min), 1.39+/-0.172 (60min)). As a result, we could show that 32 is a good candidate for an orally active adenosine A1 receptor antagonist with high blood-brain barrier permeability and good bioavailability (Ki=6.6nM, A(2A)/A1=820, BA=60.6+/-4.9% (32 mg/kg)).     

Short synthesis of a novel class of salvinorin A analogs with hemiacetalic structure

Novel semisynthetic analogs of salvinorin A, a full agonist having extraordinary affinity as well as selectivity for the κ-opioid receptor (KOR), were obtained in good yields. The derivatives are remarkable for their unusual and unique hemiacetal structure in the salvinorin series of compounds. The formation of the hemiacetal occurs with epimerization at C-12, thus preserving the original configuration of salvinorin A. The dimethyl ester derivative of the hemiacetal was found to have an affinity for both KOR and MOR (μ-opioid receptor).     

Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional μ/δ-Opioid Agonist–Neurokinin Antagonist Peptidomimetics

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH₂ (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.     

Synthesis,Pharmacological Evaluation,and Computational Studies of Cyclic Opioid Peptidomimetics Containing β3-Lysine

Our formerly described pentapeptide opioid analog Tyr-c[D-Lys-Phe-Phe-Asp]NH₂ (designated RP-170), showing high affinity for the mu (MOR) and kappa (KOR) opioid receptors, was much more stable than endomorphine-2 (EM-2) in the rat brain homogenate and displayed remarkable antinociceptive activity after central (intracerebroventricular) and peripheral (intravenous ) administration. In this report, we describe the further modification of this analog, which includes the incorporation of a β³-amino acid, (R)- and (S)-β³-Lys, instead of D-Lys in position 2. The influence of such replacement on the biological properties of the obtained analogs, Tyr-c[(R)-β³-Lys-Phe-Phe-Asp]NH₂ (RP-171) and Tyr-c[(S)-β³-Lys-Phe-Phe-Asp]NH₂, (RP-172), was investigated in vitro. Receptor radiolabeled displacement and functional calcium mobilization assays were performed to measure binding affinity and receptor activation of the new analogs. The obtained data revealed that only one of the diastereoisomeric peptides, RP-171, was able to selectively bind and activate MOR. Molecular modeling (docking and molecular dynamics (MD) simulations) suggests that both compounds should be accommodated in the MOR binding site. However, in the case of the inactive isomer RP-172, fewer hydrogen bonds, as well as instability of the canonical ionic interaction to Asp¹⁴⁷, could explain its very low MOR affinity.     

Fenchone Derivatives as a Novel Class of CB2 Selective Ligands: Design,Synthesis, X-ray Structure and Therapeutic Potential

A series of novel cannabinoid-type derivatives were synthesized by the coupling of (1S,4R)-(+) and (1R,4S)-(−)-fenchones with various resorcinols/phenols. The fenchone-resorcinol derivatives were fluorinated using Selectfluor and demethylated using sodium ethanethiolate in dimethylformamide (DMF). The absolute configurations of four compounds were determined by X-ray single crystal diffraction. The fenchone-resorcinol analogs possessed high affinity and selectivity for the CB2 cannabinoid receptor. One of the analogues synthesized, 2-(2′,6′-dimethoxy-4′-(2″-methyloctan-2″-yl)phenyl)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (1d), had a high affinity (K_i = 3.51 nM) and selectivity for the human CB2 receptor (hCB2). In the [³⁵S]GTPγS binding assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC₅₀ = 2.59 nM, E_(max) = 89.6%). Two of the fenchone derivatives were found to possess anti-inflammatory and analgesic properties. Molecular-modeling studies elucidated the binding interactions of 1d within the CB2 binding site.     

HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variants

The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14-16-fold), moderate binders (35-80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance.     

Novel Delta‐Opioid‐Receptor‐Selective Ligands in the 14‐Alkoxy‐Substituted Indolo‐ and Benzofuromorphinan Series

Several new indolo‐ and benzofuromorphinans substituted at the positions 5 and 14 were prepared and tested in vitro by means of opioid‐receptor binding and functional ([³⁵S]GTPγS binding) assays. All compounds 1 – 11 displayed high affinity for δ opioid‐binding sites (Table 1). Compound 4 proved to be an agonist, and all other compounds were antagonists. The presence of a Me group at position 5 induced no change in δ affinity (see 1 vs. 3 ), but decreased the μ and κ affinities. An EtO group at position 14 conferred a very high affinity and also high selectivity to δ opioid receptors (see 2 and 10 ). Chain elongation of the 14‐alkoxy group resulted in compounds with reduced δ affinity and selectivity (see 4 and 11 and also 5 – 9 ). The results of the present study indicate that the 5‐ and 14‐positions of indolo‐ and benzofuromorphinans represent critical sites that could be a trigger to develop new compounds with increased δ affinity and/or selectivity.     

Synthesis and characterization of orally active nonpeptide vasopressin V2 receptor antagonists.

The present study was undertaken to evaluate whether a novel series of 2,6-diaza-5-oxobicyclo[5.4.0]undeca-1(7),8,10-triene derivatives exhibited antagonistic activity for vasopressin V1 and V2 receptors. Most of these compounds were synthesized and showed a high affinity potential for V2 receptor and low to moderate affinity potential for V1 receptor. The most potent and V2-selective compound, N-[4-[2,6-diaza-6-[2-(4-methylpiperazinyl)-2-oxoethyl] -5- oxobicyclo[5.4.0]undeca-1(7),8,10-trien-2-yl]-carbonyl]pheny l][2-(4- methylphenyl)phenyl]-formamide (11b), exhibited IC50's of 2.9 nM for the V2 receptor and 200 nM for the V1 receptor, respectively. When administered orally to rat, 11b showed an approximately 18-fold increased urine volume in comparison with control rat.     

Structure-activity relationships of 2-(benzothiazolylthio)acetamide class of CCR3 selective antagonist

The structure activity relationships of novel selective CCR3 receptor antagonists, 2-(benzothiazolylthio)acetamimde derivatives were described. A lead structure (1a) was discovered from the screening of the focused library that was based on the structure of our dual antagonists for the human CCR1 and CCR3 receptors. Derivatization of 1a including incorporation of substituent(s) into each benzene ring of the benzothiazole and piperidine side chain resulted in the identification of potent and selective compounds (1b, r, s) exhibiting nano-molar binding affinity (IC(50)s: 1.5-3.0 nM) and greater than 800-fold selectivity for the CCR3 receptor over the CCR1 receptor.     

Synthesis and structure-activity relationships of 4-amino-5-chloro-N-(1,4-dialkylhexahydro-1,4-diazepin-6-yl)-2-methoxybenzamide derivatives,novel and potent serotonin 5-HT3 and dopamine D2 receptors dual antagonist

In search of a dopamine D2 and serotonin 5-HT3 receptors dual antagonist as a potential broad antiemetic agent, a number of benzamides were prepared from 4-amino-5-chloro-2-methoxybenzoic acid derivatives and 6-amino-1,4-dialkylhexahydro-1,4-diazepines and evaluated for their binding affinity for the dopamine D2 and the serotonin 5-HT3 receptors using rat brain synaptic and rat cortical membranes, respectively. From the results of both in vitro receptor binding and in vivo biological assays for the dopamine D2 receptor, 1-ethyl-4-methylhexahydro-1,4-diazepine ring was selected as an optimum amine moiety. Introduction of one methyl group on the nitrogen atom at the 4-position and/or modification of the substituent at the 5-position of the 4-amino-5-chloro-2-methoxybenzoyl moiety caused a marked increase in the dopamine D2 receptor binding affinity along with a potent 5-HT3 receptor binding affinity. Among the compounds, 5-chloro-N-(1-ethyl-4-methylhexahydro-1,4-diazepin-6-yl)-2-methoxy-4-methylaminobenzamide (82), 5-bromo (110), and 5-iodo (112) analogues exhibited a much higher affinity for the dopamine D2 receptor than that of metoclopramide (IC50=17.5-61.0 nM vs. 483 nM). In particular, 82 showed a potent antagonistic activity for both receptors in vivo tests. Optical resolution of the racemate 82 brought about a dramatic change in the pharmacological profile with the (R)-enantiomer exhibiting a strong affinity for both the dopamine D2 and the 5-HT3 receptors, while the corresponding (S)-enantiomer had a potent and selective serotonin 5-HT3 receptor binding affinity.     

Computational Methods for Understanding the Selectivity and Signal Transduction Mechanism of Aminomethyl Tetrahydronaphthalene to Opioid Receptors

Opioid receptors are members of the group of G protein-couple receptors, which have been proven to be effective targets for treating severe pain. The interactions between the opioid receptors and corresponding ligands and the receptor’s activation by different agonists have been among the most important fields in opioid research. In this study, with compound M1, an active metabolite of tramadol, as the clue compound, several aminomethyl tetrahydronaphthalenes were designed, synthesized and assayed upon opioid receptors. With the resultant compounds FW-AII-OH-1 (Ki = 141.2 nM for the κ opioid receptor), FW-AII-OH-2 (Ki = 4.64 nM for the δ opioid receptor), FW-DI-OH-2 (Ki = 8.65 nM for the δ opioid receptor) and FW-DIII-OH-2 (Ki = 228.45 nM for the δ opioid receptor) as probe molecules, the structural determinants responsible for the subtype selectivity and activation mechanisms were further investigated by molecular modeling and molecular dynamics simulations. It was shown that Y^7.43 was a key residue in determining the selectivity of the three opioid receptors, and W^6.58 was essential for the selectivity of the δ opioid receptor. A detailed stepwise discovered agonist-induced signal transduction mechanism of three opioid receptors by aminomethyl tetrahydronaphthalene compounds was proposed: the 3–7 lock between TM3 and TM7, the DRG lock between TM3 and TM6 and rearrangement of I^3.40, P^5.50 and F^6.44, which resulted in the cooperative movement in 7 TMs. Then, the structural relaxation left room for the binding of the G protein at the intracellular site, and finally the opioid receptors were activated.     

Synthesis and evaluation of peptide–fentanyl analogue conjugates as dual µ/δ-opioid receptor agonists for the treatment of pain

Novel peptide-fentanyl analogue conjugates were synthesized by the covalent coupling of carfentanyl derivatives to the C-terminus or N-terminus of the conformationally constrained dermorphin tetrapeptide BVD03 via a chemical linker. The carfentanyl-related analogues displayed distinct binding and functional activities at µ/δ opioid receptors (MOR/DOR) and antinociceptive effects when conjugated to the peptide. The most potent compound, SW-LJ-11, displayed mixed MOR/DOR agonist properties in the low nanomolar range and significant analgesic efficacy in vivo in four classic mouse models of pain. Interestingly, SW-LJ-11 did not exhibit any physical dependence or respiratory depression, in contrast to an equipotent analgesic dose of morphine or BVD03, indicating that the use of opioid peptide–fentanyl analogue conjugates as dual MOR/DOR agonists may be a promising strategy for obtaining safer opioids.     

Fluoromethylated and hydroxymethylated derivatives of N-methyl-D-aspartate receptor antagonist 1-[1-(2-thienyl)cyclohexyl]piperidine.

Derivatives with fluoromethyl and hydroxymethyl groups on the cyclohexyl ring of 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor, were tested in a radioligand binding assay to evaluate their ability to inhibit [3H]TCP binding by rat brain homogenates. The potencies of these compounds as antagonists of NMDA and L-glutamate responses were also compared using a rat cortical slice preparation. One of the analogs, cis-2-hydroxymethyl-r-1-(N-piperidyl)-1-(2-thienyl) cyclohexane (5) was found to show a high affinity (IC50 = 16 nM) for the phencyclidine (PCP) binding sites, very close to that of TCP, and to be 38-fold more potent in binding than its trans isomer. Fluoromethyl and hydroxymethyl substitutions at C4 position of the cyclohexyl ring of TCP clearly reduced the affinity by at least one order of magnitude relative to TCP.     

Biological properties of opioid peptides replacing Tyr at position 1 by 2,6-dimethyl-Tyr.

To understand the effect of the replacement of Tyr residue at position 1 in opioid peptides by 2,6-dimethyl-Tyr (Dmt) on the biological property, chiral (D or L) Dmt1 analogs of Leu-enkephalin (Enk) and Tyr-D-Arg-Phe-beta Ala-NH2 (YRFB) were synthesized and their enzymatic stabilities, in vitro bioactivities and receptor binding affinities compared with those of parent peptides. [L-Dmt1]Enk (1) exhibited 4-fold higher stability against aminopeptidase-M and possessed dramatically increased activities in guinea pig ilium (GPI) (187-fold) and mouse vas deferens (MVD) (131-fold) assays, and in rat brain receptor binding assays (356-fold at mu receptor and 46-fold at delta receptor) as compared to Enk. [L-Dmt1]YRFB (3) also exhibited increased activities in GPI (46-fold) and MVD (177-fold) assays, and in the binding assays (69-fold at mu receptro and 341-fold at delta receptor) as compared to the parent peptide. [D-Dmt1]Enk (2) and [D-Dmt1]YRFB (4) exhibited activities with diminished or lesser potency than the parent peptide in all assays. These results indicate that there is a tendency for mu affinity to be enhanced more than delta affinity with introduction of L-Dmt into delta ligand peptide (Enk), and for delta affinity to be enhanced more than mu affinity in case of mu ligand peptide (YRFB), resulting in reduced receptor selectivities at the receptors.