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An efficient regioselective approach to novel functionalized bis(isoxazoles) with a variety of aromatic and aliphatic linkers was elaborated, based on the heterocyclization reaction of electrophilic alkenes under the treatment with tetranitromethane-triethylamine complex affording 3-EWG-5-nitroisoxazoles. The subsequent SNAr reactions of 5-nitroisoxazoles with various O,O-, N,N- and S,S-bis(nucleophiles) provide a wide range of bis(isoxazole) derivatives in good isolated yields. Employing an elaborated method, a series of novel bis(3-EWG-isoxazoles) as the promising allosteric modulators of AMPA receptors were designed and synthesized. The effect of the compounds on the kainate-induced currents was studied in the patch clamp experiments, revealing modulator properties for several of them. The best positive modulator potency was found for dimethyl 5,5′-(ethane-1,2-diylbis(sulfanediyl))bis(isoxazole-3-carboxylate), which potentiated the kainate-induced currents in a wide concentration range (10−12–10−6 M) with maximum potentiation of 77% at 10−10 M. The results were rationalized using molecular docking and molecular dynamics simulations of modulator complexes with the dimeric ligand-binding domain of the GluA2 AMPA receptor. The predicted physicochemical, ADMET, and PAINS properties confirmed that the AMPA receptor modulators based on the bis(isoxazole) scaffold may serve as potential lead compounds for the development of neuroprotective drugs. [ABSTRACT FROM AUTHOR]

Though efficacious in managing chronic, severe pain, opioid analgesics are accompanied by significant adverse effects including constipation, tolerance, dependence, and respiratory depression. The life-threatening risks associated with µ opioid receptor agonist-based analgesics challenges their use in clinic. A rational approach to combatting these adverse effects is to develop agents that incorporate activity at a second pharmacologic target in addition to µ opioid receptor activation. The promise of such bivalent or bifunctional ligands is the development of an analgesic with an improved side effect profile. In this review, we highlight ongoing efforts in the development of bivalent and bifunctional analgesics that combine µ agonism with efficacy at κ and δ opioid receptors, the nociceptin opioid peptide (NOP) receptor, σ receptors, and cannabinoid receptors. Several examples of bifunctional analgesics in preclinical and clinical development are highlighted, as are strategies being employed toward the rational design of novel agents.

Positive allosteric modulators (PAMs) of AMPA receptors represent attractive candidates for the development of drugs for the treatment of cognitive and neurodegenerative disorders. Dimeric molecules have been reported to have an especially potent modulating effect, due to the U-shaped form of the AMPA receptor's allosteric binding site. In the present work, novel bis(pyrimidines) were studied as AMPA receptor modulators. A convenient and flexible preparative approach to bis(pyrimidines) containing a hydroquinone linker was elaborated, and a series of derivatives with varied substituents was obtained. The compounds were examined in the patch clamp experiments for their influence on the kainate-induced currents, and 10 of them were found to have potentiating properties. The best potency was found for 2-methyl-4-(4-((2-methyl-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)phenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[d]pyrimidine, which potentiated the kainate-induced currents by up to 77% in all tested concentrations (10−12–10−6 M). The results were rationalized via the modeling of modulator complexes with the dimeric ligand binding domain of the GluA2 AMPA receptor, using molecular docking and molecular dynamics simulation. The prediction of ADMET, physicochemical, and PAINS properties of the studied bis(pyrimidines) confirmed that PAMs of this type may act as the potential lead compounds for the development of neuroprotective drugs. [ABSTRACT FROM AUTHOR]

An efficient regioselective approach to novel functionalized bis(isoxazoles) with a variety of aromatic and aliphatic linkers was elaborated, based on the heterocyclization reaction of electrophilic alkenes under the treatment with tetranitromethane-triethylamine complex affording 3-EWG-5-nitroisoxazoles. The subsequent SNAr reactions of 5-nitroisoxazoles with various O,O-, N,N- and S,S-bis(nucleophiles) provide a wide range of bis(isoxazole) derivatives in good isolated yields. Employing an elaborated method, a series of novel bis(3-EWG-isoxazoles) as the promising allosteric modulators of AMPA receptors were designed and synthesized. The effect of the compounds on the kainate-induced currents was studied in the patch clamp experiments, revealing modulator properties for several of them. The best positive modulator potency was found for dimethyl 5,5′-(ethane-1,2-diylbis(sulfanediyl))bis(isoxazole-3-carboxylate), which potentiated the kainate-induced currents in a wide concentration range (10−12–10−6 M) with maximum potentiation of 77% at 10−10 M. The results were rationalized using molecular docking and molecular dynamics simulations of modulator complexes with the dimeric ligand-binding domain of the GluA2 AMPA receptor. The predicted physicochemical, ADMET, and PAINS properties confirmed that the AMPA receptor modulators based on the bis(isoxazole) scaffold may serve as potential lead compounds for the development of neuroprotective drugs.

Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A 1 R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A 1 R of an A 1 R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A 1 R antagonists and showed evidence of covalent binding and simultaneous binding across an A 1 R homodimer. Greater selectivity for A 1 R over the adenosine A 3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Purpose: Fibroblast activation protein (FAP) has become a promising cancer-related target for diagnosis and therapy. The aim of this study was to develop a bivalent FAP ligand for both diagnostic PET imaging and endoradiotherapy. Methods: We synthesized a bivalent FAP ligand (ND-bisFAP) and labeled it with 18F or 177Lu. FAP-positive A549-FAP cells were used to study competitive binding to FAP, cellular internalization, and efflux properties in vitro. Micro-PET imaging with [18F]AlF-ND-bisFAPI was conducted in mice bearing A549-FAP or U87MG tumors. Biodistribution and therapeutic efficacy of [177Lu]Lu-ND-bisFAPI were conducted in mice bearing A549-FAP tumors. Results: The FAP binding affinity of ND-bisFAPI is 0.25 ± 0.05 nM, eightfold higher in potency than the monomeric DOTA-FAPI-04 (IC50 = 2.0 ± 0.18 nM). In A549-FAP cells, ND-bisFAPI showed specific uptake, a high internalized fraction, and slow cellular efflux. Compared to the monomeric [18F]AlF-FAPI-42, micro-PET imaging with [18F]AlF-ND-bisFAPI showed higher specific tumor uptake and retention for at least 6 h. Biodistribution studies showed that [177Lu]Lu-ND-bisFAPI had higher tumor uptake than [177Lu]Lu-FAPI-04 at the 24, 72, 120, and 168 h time points (all P < 0.01). [177Lu]Lu-ND-bisFAPI delivered fourfold higher radiation than [177Lu]Lu-FAPI-04 to A549-FAP tumors. For the endoradiotherapy study, 37 MBq of [177Lu]Lu-ND-bisFAPI significantly reduced tumor growth compared to the same dose of [177Lu]Lu-FAPI-04. Half of the dose of [177Lu]Lu-ND-bisFAPI (18.5 MBq) has comparable median survival as 37 MBq of [177Lu]Lu-FAPI-04 (37 vs 36 days). Conclusion: The novel bivalent FAP ligand was developed as a theranostic radiopharmaceutical and showed promising properties including higher tumor uptake and retention compared to the established radioligands [18F]AlF-FAPI-42 and [177Lu]Lu-FAPI-04. Preliminary experiments with 18F- or 177Lu-labeled ND-bisFAPI showed promising imaging properties and favorable anti-tumor responses. [ABSTRACT FROM AUTHOR]

Positive allosteric modulators (PAMs) of AMPA receptors represent attractive candidates for the development of drugs for the treatment of cognitive and neurodegenerative disorders. Dimeric molecules have been reported to have an especially potent modulating effect, due to the U-shaped form of the AMPA receptor’s allosteric binding site. In the present work, novel bis(pyrimidines) were studied as AMPA receptor modulators. A convenient and flexible preparative approach to bis(pyrimidines) containing a hydroquinone linker was elaborated, and a series of derivatives with varied substituents was obtained. The compounds were examined in the patch clamp experiments for their influence on the kainate-induced currents, and 10 of them were found to have potentiating properties. The best potency was found for 2-methyl-4-(4-((2-methyl-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)phenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[d]pyrimidine, which potentiated the kainate-induced currents by up to 77% in all tested concentrations (10−12–10−6 M). The results were rationalized via the modeling of modulator complexes with the dimeric ligand binding domain of the GluA2 AMPA receptor, using molecular docking and molecular dynamics simulation. The prediction of ADMET, physicochemical, and PAINS properties of the studied bis(pyrimidines) confirmed that PAMs of this type may act as the potential lead compounds for the development of neuroprotective drugs.

Glycosyl monomers for the assembly of multivalent ligands are typically synthesized using carbohydrates with biological functions and polymerizable functional groups such as acrylamide or styrene introduced into the carbohydrate aglycon, and monomers polymerized using a radical initiator. Herein, we report the acryloylation of 6-aminohexyl α-d-mannoside and its conversion into the glycosyl monomer bearing an acrylamide group. The general acryloylation procedure afforded the desired N-hexyl α-d-acetylmannosyl acrylamide monomer as well as an unexpected compound with a close Rf value. The compounds were separated and analyzed by nuclear magnetic resonance spectroscopy and mass spectrometry, which revealed the unknown compound to be the bivalent N,N-bis(hexyl α-d-acetylmannosyl) acrylamide monomer, which contains two hexyl mannose units and one acrylamide group. To the best of our knowledge, this side reaction has not previously been disclosed, and may be useful for the construction of multivalent sugar ligands.

Opioid receptors, including the mu and delta opioid receptors (MOR and DOR) are important targets for the treatment of pain. Although there is mounting evidence that these receptors form heteromers, the functional role of the MOR/DOR heteromer remains unresolved. We have designed and synthesized bivalent ligands as tools to elucidate the functional role of the MOR/DOR heteromer. Our ligands (L2 and L4) are comprised of a compound with low affinity at the DOR tethered to a compound with high affinity at the MOR, with the goal of producing ligands with "tuned affinity" at MOR/DOR heteromers compared to DOR homomers. Here we show that both L2 and L4 demonstrate enhanced affinity at MOR/DOR heteromers compared to DOR homomers, thereby providing unique pharmacological tools to dissect the role of the MOR/DOR heteromer in pain.

A convenient synthetic approach to novel functionalized bis(isoxazoles), the promising bivalent ligands of the AMPA receptor, was elaborated. It was based on the heterocyclization reactions of readily available electrophilic alkenes with the tetranitromethane-triethylamine complex. The structural diversity of the synthesized compounds was demonstrated. In the electrophysiological experiments using the patch clamp technique on Purkinje neurons, the compound 1,4-phenylenedi(methylene)bis(5-aminoisoxazole-3-carboxylate) was shown to be highly potent positive modulator of the AMPA receptor, potentiating kainate-induced currents up to 70% at 10−11 M.

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.

In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic strategy, the CB 1 and CB 2 cannabinoid receptor agonist JWH-018 was conjugated with the opiate analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid pharmacophores were coupled via spacers of different length and chemical structure. In vitro radioligand binding experiments confirmed that the resulting bivalent compounds bound both to the opioid and to the cannabinoid receptors with moderate to high affinity. The highest affinity bivalent derivatives 11 and 19 exhibited agonist properties in [35S]GTPγS binding assays. These compounds activated MOR and CB (11 mainly CB 2 , whereas 19 mainly CB 1) receptor-mediated signaling, as it was revealed by experiments using receptor specific antagonists. In rats both 11 and 19 exhibited antiallodynic effect similar to the parent drugs in 20 μg dose at spinal level. These results support the strategy of multitargeting G-protein coupled receptors to develop lead compounds with antinociceptive properties. Image 1 • Mu opioid (oxycodone or Tyr- d -Ala-Gly-Phe-NH 2) and cannabinoid (JWH-018) receptor agonists were coupled via short spacers. • JWH-018 was labeled with tritium and [3H]JWH-018 was validated as a novel radioligand for cannabinoid receptors. • In vitro studies revealed that the compounds 11 and 19 could bind both to the mu opioid and to the cannabinoid receptors. • The bivalent compounds 11 and 19 were found to be agonists both for the mu opioid and for the cannabinoid receptors. • At spinal level 11 , 19 were equieffective with the parent drugs at 20 μg dose in a chronic osteoarthritis pain model in rats. [ABSTRACT FROM AUTHOR]

Though efficacious in managing chronic, severe pain, opioid analgesics are accompanied by significant adverse effects including constipation, tolerance, dependence, and respiratory depression. The life-threatening risks associated with µ opioid receptor agonist-based analgesics challenges their use in clinic. A rational approach to combatting these adverse effects is to develop agents that incorporate activity at a second pharmacologic target in addition to µ opioid receptor activation. The promise of such bivalent or bifunctional ligands is the development of an analgesic with an improved side effect profile. In this review, we highlight ongoing efforts in the development of bivalent and bifunctional analgesics that combine µ agonism with efficacy at κ and δ opioid receptors, the nociceptin opioid peptide (NOP) receptor, σ receptors, and cannabinoid receptors. Several examples of bifunctional analgesics in preclinical and clinical development are highlighted, as are strategies being employed toward the rational design of novel agents. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: The chemokine receptor CXCR4 (C‐X‐C chemokine receptor type 4 also known as fusin or CD184 (cluster of differentiation 184)) is implicated in various biological and pathological processes of the hematopoietic and immune systems. CXCR4 is also one of the major coreceptors for HIV‐1 entry into target cells and is overexpressed in many cancers, supporting cell survival, proliferation, and migration. CXCR4 is thus an extremely relevant drug target. Among the different strategies to block CXCR4, chemokine‐derived peptide inhibitors hold great therapeutic potential. In this study, we used the N‐terminus of vCCL2/vMIPII, a viral CXCR4 antagonist chemokine, as a scaffold motif to engineer and select CXCR4 peptide inhibitors, called Mimokines, which imitate the chemokine‐binding mode but display an enhanced receptor affinity, antiviral properties, and receptor selectivity. We first engineered a Mimokine phage displayed library based on the first 21 residues of vCCL2, in which cysteine 11 and 12 were fully randomized and screened it against purified CXCR4 stabilized in liposomes. We identified Mimokines displaying up to 4‐fold higher affinity for CXCR4 when compared to the reference peptide and fully protected MT‐4 cells against HIV‐1 infection. These selected Mimokines were then subjected to dimerization, D‐amino acid, and aza‐β3‐amino acid substitution to further enhance their potency and selectivity. Optimized Mimokines exhibited up to 120‐fold enhanced CXCR4 binding (range of 20 nM) and more than 200‐fold improved antiviral properties (≤ 1 μM) compared to the parental Mimokines. Interestingly, these optimized Mimokines also showed up to 25‐fold weaker affinity for ACKR3/CXCR7 and may therefore serve as lead compounds for further development of more selective CXCR4 peptide inhibitors and probes. [ABSTRACT FROM AUTHOR]

Themu opioid receptor (MOR) and metabotropic glutamate receptor = (mGluR5) are well-established pharmacological targets in the management of chronic pain. Both receptors are expressed in the spinal cord. MMG22, a bivalent ligand containing 2 pharmacophores separated by 22 atoms, which simultaneously activates MOR and antagonizes mGluR5, has been shown to produce potent reversal of tactile hypersensitivity in rodent models of lipopolysaccharide (LPS)-and bone cancer-induced chronic pain. This study assessed whether intrathecal MMG22 also is effective in reducing pain of neuropathic origin. Furthermore, we theorized that MMG22 should reduce hyperalgesia in nerve-injured mice in a manner consistent with a synergistic interaction between MOR and mGluR5. Several weeks after spared nerve injury, tactile hypersensitivity was reversed in mice by the intrathecal injection of MMG22 (0.01-10 nmol) but also by its shorter spacer analog, MMG10, with similar potency. The potencies of the bivalent ligands were 10- to 14-fold higher than those of the compounds upon which the bivalent structure was based, the MOR agonist oxymorphone and the mGluR5 antagonist MPEP. Coadministration of oxymorphone and MPEP demonstrated analgesic synergism, an interaction confirmed by isobolographic analysis. This study indicates that in the spared nerve injury-induced model of neuropathic pain, the 2 pharmacophores of the bivalent ligands MMG22 and MMG10 target MOR and mGluR5 as separate receptor monomers. The observed increase in the potency of MMG22 and MMG10, compared with oxymorphone and MPEP, may reflect the synergistic interaction of the 2 pharmacophores of the bivalent ligand acting at their respective separate receptor monomers. [ABSTRACT FROM AUTHOR]

The recent crystal structure of the κ-opioid receptor (κ-OR) revealed, unexpectedly, that the antagonist JDTic is a bivalent ligand: in addition to the orthosteric pocket occupied by morphinans, JDTic also occupies a distinct (allotopic) pocket. Mutagenesis data suggest that salvinorin A (1) also binds to this allotopic pocket, adjacent to the aspartate residue that anchors the basic nitrogen atom of classical opiates (Asp138). It has been suggested that an H-bond donor appended to 1 might interact with Asp138, increasing affinity. Such a bivalent ligand might also possess altered functional selectivity. Based on modeling and known N-furanylmethyl opioid antagonists, we appended H-bond donors to the furan ring of 1. (Dimethylamino)methyl groups at C-15 or C-16 abolished affinity for κ-OR. Hydroxymethylation at C-16 was tolerated, but 15,16-bis-hydroxymethylation was not. Since allosteric modulators may go undetected in binding assays, we also tested these and other low-affinity derivatives of 1 for allosteric modulation of dynorphin A in the [35S]GTPγS assay. No modulation was detected. As an alternative attachment point for bivalent derivatives, we prepared the 2-(hydroxyethoxy)methyl ether, which retained high affinity for κ-OR. We discuss alternative design strategies for linked, fused or merged bivalent derivatives of 1.

Activation of β 2 -adrenergic receptor (β 2 AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. ( R , R ′)-4′-methoxy-1-naphthylfenoterol [( R , R ′)-MNF] is a bivalent compound that agonizes β 2 AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent β 2 AR activation and GPR55 blockade in C6 glioma cells using ( R , R ′)-MNF as a marker ligand. Our data show that ( R , R ′)-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of ( R , R ′)-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of β 2 AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in ( R , R ′)-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by ( R , R ′)-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of β-catenin occurred with ( R , R ′)-MNF, and we provided new evidence of ( R , R ′)-MNF-mediated inhibition of oncogenic β-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of β 2 AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer. [ABSTRACT FROM AUTHOR]

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used forin vitrobinding assays, forin vivoantinociception models andin vitrohypothalamic perfusion test, to evaluate the neurotransmitters release. [ABSTRACT FROM AUTHOR]

Opioids are widely prescribed pain relievers with multiple side effects and potential complications. They produce analgesia via G-protein-protein coupled receptors: μ-, δ-, κ-opioid and opioid receptor-like 1 receptors. Bivalent ligands targeted to the oligomerized opioid receptors might be the key to developing analgesics without undesired side effects and obtaining effective treatment for opioid addicts. In this review we will update the biological effects of μ-opioids on homo- or hetero-oligomerized μ-opioid receptor and discuss potential mechanisms through which bivalent ligands exert beneficial effects, including adenylate cyclase regulation and receptor-mediated signaling pathways.

FimH is a mannose-specific bacterial lectin found on type 1 fimbriae with a monovalent carbohydrate recognition domain (CRD) that is known from X-ray studies. However, binding studies with multivalent ligands have suggested an additional carbohydrate-binding site on this protein. In order to prove this hypothesis, a bivalent glycopeptide ligand with the capacity to bridge two putative carbohydrate binding sites on FimH was designed and synthesized. Anti-adhesion assays with the new bivalent ligand and type 1-fimbriated bacteria have revealed, that verification of the number of carbohydrate binding sites on FimH with a tailor-made bivalent glycopeptide requires further investigation to be conclusive.

A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM 1–5 ) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM 1 , hM 2 and hM 3 on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM 1 and hM 2 receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain. [ABSTRACT FROM AUTHOR]

We have previously used poly- l -proline linkers for the development of bivalent-type ligands for the chemokine receptor, CXCR4. The bivalent ligands with optimum linkers showed specific binding to CXCR4, suggesting the existence of CXCR4 possibly as a dimer on the cell membrane, and enabled definition of the amount of CXCR4 expressed. This paper reports the synthesis by a copper-catalyzed azide–alkyne cycloaddition reaction as the key reaction, of bivalent CXCR4 ligands with near infrared (NIR) dyes at the terminus or the center of the poly- l -proline linker. Some of the NIR-labeled ligands, which would be valuable probes useful in studies of the behavior of cells expressing CXCR4, have been obtained. The information concerning the effects of the labeling positions of NIR dyes on their binding properties is useful for the design of modified bivalent-type CXCR4 ligands. [ABSTRACT FROM AUTHOR]

The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H 2 receptor (H 2 R) agonists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activities, but considerably improved stability against hydrolytic cleavage. These compounds achieved up to 2500 times the potency of histamine when studied in [ 35 S]GTPγS assays on recombinant human and guinea pig H 2 R. Unlike 3-(imidazol-4-yl)propyl substituted carbamoylguanidines, the corresponding 2-amino-4-methylthiazoles revealed selectivity over histamine receptor subtypes H 1 R, H 3 R and H 4 R in radioligand competition binding studies. H 2 R binding studies with three fluorescent compounds and one tritium-labeled ligand, synthesized from a chain-branched precursor, failed due to pronounced cellular accumulation and high non-specific binding. However, the dimeric H 2 R agonists proved to be useful pharmacological tools for functional studies on native cells, as demonstrated for selected compounds by cAMP accumulation and inhibition of fMLP-stimulated generation of reactive oxygen species in human monocytes. [ABSTRACT FROM AUTHOR]

The design of bivalent ligands targeting G protein-coupled receptors (GPCRs) often leads to the development of new, highly selective and potent compounds. To date, no bivalent ligands for the human cannabinoid receptor type 2 (hCB2R) of the endocannabinoid system (ECS) are described. Therefore, two sets of homobivalent ligands containing as parent structure the hCB2R selective agonist 13a and coupled at different attachment positions were synthesized. Changes of the parent structure at these positions have a crucial effect on the potency and efficacy of the ligands. However, we discovered that bivalency has an influence on the effect at both cannabinoid receptors. Moreover, we found out that the spacer length and the attachment position altered the efficacy of the bivalent ligands at the receptors by turning agonists into antagonists and inverse agonists. [ABSTRACT FROM AUTHOR]

Opioids are widely prescribed pain relievers with multiple side effects and potential complications. They produce analgesia via G-protein-protein coupled receptors: µ-, d-, ?-opioid and opioid receptor-like 1 receptors. Bivalent ligands targeted to the oligomerized opioid receptors might be the key to developing analgesics without undesired side effects and obtaining effective treatment for opioid addicts. In this review we will update the biological effects of µ-opioids on homo- or hetero-oligomerized µ-opioid receptor and discuss potential mechanisms through which bivalent ligands exert beneficial effects, including adenylate cyclase regulation and receptor-mediated signaling pathways. [ABSTRACT FROM AUTHOR]

[Display omitted] • The crosstalk between opioid and chemokine receptors play a role in chronic pain. • The putative MOR-CXCR4 heterodimers may be involved in pain modulation. • Bivalent ligands are powerful to investigate GPCR dimerization and relevant diseases. • Bivalent ligand 1a showed reasonable recognition to both the MOR and CXCR4. • Bivalent ligand 1a showed potential antinociception in lactic acid-induced pain model. The functional interactions between opioid and chemokine receptors have been implicated in the pathological process of chronic pain. Mounting studies have indicated the possibility that a MOR-CXCR4 heterodimer may be involved in nociception and related pharmacologic effects. Herein we have synthesized a series of bivalent ligands containing both MOR agonist and CXCR4 antagonist pharmacophores with an aim to investigate the functional interactions between these two receptors. In vitro studies demonstrated reasonable recognition of designed ligands at both respective receptors. Further antinociceptive testing in mice revealed compound 1a to be the most promising member of this series. Additional molecular modeling studies corroborated the findings observed. Taken together, we identified the first bivalent ligand 1a showing promising antinociceptive effect by targeting putative MOR-CXCR4 heterodimers, which may serve as a novel chemical probe to further develop more potent bivalent ligands with potential application in analgesic therapies for chronic pain management. [ABSTRACT FROM AUTHOR]

Abstract: The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y1R, Y2R, Y4R and Y5R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y1R, Y2R and Y5R, but such compounds are still lacking for the Y4R. In this work, the structures of the high affinity selective (R)-argininamide-type Y1R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y1R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y1R versus Y4R subtype, resulting in comparable affinities of bivalent ligands for Y1R and Y4R (e.g. UR-MK177 (( R , R )-49): K i =230nM (Y1R) and 290nM (Y4R)). With a K i value of 130nM and a K b value of 20nM, UR-MK188 (( R , R )-51) was superior to all Y4R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 (( S , S )-49) and UR-MEK388 (( S , S )-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y1R, stereochemical discrimination by the Y4R was not observed. This may pave the way to selective Y4R antagonists. [Copyright &y& Elsevier]

Bivalent ligands often display high affinity/avidity for and long residence time at their target. Thereto responsible is the synergy that emanates from the simultaneous binding of their two pharmacophores to their respective target sites. Thermodynamic cycle models permit the most complete description of the binding process, and thereto, corresponding differential equation-based simulations link the 'microscopic' rate constants that govern the individual binding steps to the 'macroscopic' bivalent ligand's binding properties. Present simulations of heterobivalent ligand binding led to an appreciably simpler description thereof. The thermodynamic cycle model can be split into two pathways/lanes that the bivalent ligand can solicit to reach fully bound state. Since the first binding event prompts the still free pharmacophore to stay into 'forced proximity' of its target site, such lanes can be looked into by the equations that also apply to induced fit binding mechanisms. Interestingly, the simplest equations apply when bivalency goes along with a large gain in avidity. The overall bivalent ligand association and dissociation will be swifter than via each lane apart, but it is the lane that allows the fastest bidirectional 'transit' between the free and the fully bound target that is chiefly solicited. The bivalent ligand's residence time is governed not only by the stability of the fully bound complex but also by the ability of freshly dissociated pharmacophores to successfully rebind. Hence, the presence of a slow-associating pharmacophore could be counterproductive. Yet, a long residence time is unfortunately also responsible for the slow attainment of binding equilibrium. [ABSTRACT FROM AUTHOR]

Highlights: [•] We designed, synthesized and studied a new bivalent D-peptide antagonist of CXCR4. [•] This peptide shows very high binding affinity for CXCR4. [•] This peptide inhibits calcium influx and cancer cell migration triggered by SDF-1α. [•] This peptide inhibits HIV-1 infection and is not cytotoxic. [•] This peptide is a new probe of CXCR4 function and lead for therapeutic development. [ABSTRACT FROM AUTHOR]

Abstract: We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides. [Copyright &y& Elsevier]

Bivalent ligands are increasingly important therapeutic agents. Although the naturally occurring antibodies are predominant, it is becoming more common to combine different antibody fragments or even low molecular weight compounds to generate heterobivalent ligands. Such ligands exhibit markedly increased affinity (i.e. avidity) and target residence time when both pharmacophores can bind simultaneously to their target sites. This is because binding of one pharmacophore forces the second tethered one to stay close to its corresponding site. This 'forced proximity' favours its binding and rebinding (once dissociated) to that site. However, rebinding will also take place when the diffusion of freshly dissociated ligands is merely slowed down. The present differential equation-based simulations explore the way both situations affect ligand binding. Both delay the attainment of binding equilibrium (resulting in steep saturation curves) and also increase the target residence time. Competitive ligands are able to interfere in a concentration-dependent manner, although much higher concentrations are required in the 'forced proximity' situation. Also, it is only in that situation that the ligand shows increased affinity. These simulations shed light on two practical consequences. Depending on the pharmacokinetic half-life of the bivalent ligand in the body, it may not have sufficient time to achieve equilibrium with the target. This will result in lower potency than expected, although it would have significant advantages in terms of residence time. In in vitro experiments, the manifestation of steep saturation curves and of accelerated dissociation in the presence of competitive ligands could mistakenly be interpreted as evidence for non-competitive, allosteric interactions. [ABSTRACT FROM AUTHOR]

Abstract: Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone–adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton. [Copyright &y& Elsevier]

This paper analyzes the biochemical equilibria between bivalent receptors, homo-bifunctional ligands, monovalent inhibitors, and their complexes. Such reaction schemes arise in the immune response, where immunoglobulins (bivalent receptors) bind to pathogens or allergens. The equilibria may be described by an infinite system of algebraic equations, which accounts for complexes of arbitrary size n ( n being the number of receptors present in the complex). The system can be reduced to just 3 algebraic equations for the concentrations of free (unbound) receptor, free ligand and free inhibitor. Concentrations of all other complexes can be written explicitly in terms of these variables. We analyze how concentrations of key (experimentally-measurable) quantities vary with system parameters. Such measured quantities can furnish important information about dissociation constants in the system, which are difficult to obtain by other means. We provide analytical expressions and suggest specific experiments that could be used to determine the dissociation constants. [ABSTRACT FROM AUTHOR]

It is now acknowledged that G protein-coupled receptors, the largest class of drug targets, adopt multiple active states that can be preferentially stabilized by orthosteric ligands or allosteric modulators, thus giving rise to the phenomenon of pathway-biased signaling. In the past few years, researchers have begun to explore the potential of linking orthosteric and allosteric pharmacophores to yield bitopic hybrid ligands. This approach is an extension of the more traditional bivalent ligand concept and shares some of the same challenges, including the choice and role of the linker between the two pharmacophores and the validation of mechanism of action. Nonetheless, the promise of bitopic ligands is the generation of novel chemical tools that have improved affinity and//or selectivity profiles. Previously identified functionally selective compounds (and medicines) also may act via a bitopic mechanism, suggesting that the phenomenon is more widespread than currently appreciated. [ABSTRACT FROM AUTHOR]

Abstract: Merging two arylamidoalkyl substituted phenylpiperazines as prototypical recognition elements for dopamine D 2-like receptors by oligoethylene glycol linkers led to a series of bivalent ligands. These dimers were investigated in comparison to their monomeric analogues for their dopamine D 2long, D 2short, D 3 and D 4 receptor binding. Radioligand binding experiments revealed strong bivalent effects for some para-substituted benzamide derivatives. For the D3 subtype, the target compounds 32, 34 and 36 showed an up to 70-fold increase of affinity and a substantial enhancement of subtype selectivity when compared to the monovalent analogue 24. Analysis of the binding curves displayed Hill slopes very close to one indicating that the bivalent ligands displace 1equiv of radioligand. Obviously, the two pharmacophores occupy an orthosteric and an allosteric binding site rather than adopting a receptor-bridging binding mode. [Copyright &y& Elsevier]

Abstract: We report the synthesis, binding properties and intrinsic activity at MT1 and MT2 melatonin receptors of new dimeric melatonin receptor ligands in which two units of the monomeric agonist N-{2-[(3-methoxyphenyl)methylamino]ethyl}acetamide (1) are linked together through different anchor points. Dimerization of compound 1 through the methoxy substituent leads to a substantial improvement in selectivity for the MT1 receptor, and to a partial agonist behavior. Compound 3a, with a trimethylene linker, was the most selective for the MT1 subtype (112-fold selectivity) and compound 3d, characterized by a hexamethylene spacer, had the highest MT1 binding affinity (pK iMT1 =8.47) and 54-fold MT1-selectivity. Dimerization through the aniline nitrogen of 1 abolished MT1 selectivity, leading to compounds with either a full agonist or an antagonist behavior depending on the nature of the linker. [Copyright &y& Elsevier]

Abstract: Bivalent ligands constituted by two identical pharmacophores structurally related to the Nociceptin Opioid Receptor (NOPr) antagonist JTC-801 were synthesized and their binding affinities for NOPr were evaluated. The novel ligands are formed by two modified JTC-801 units linked by di-iminic and di-aminic spacers with length ranging from three to ten methylene units. Moreover, the synthesis and the pharmacological characterization were extended to the corresponding univalent ligands. The latter compounds consisted in a single modified JTC-801 unit and an alkyl or alkylamino or alkylimino tail. The purpose of this study is to feature the location and surroundings of the allosteric binding site(s) of pharmacophores containing the 4-aminoquinoline structure. Most important, the bivalent ligands were exploited to reveal the eventual occurrence of a supramolecular receptorial architecture of the NOPr. All the bivalent derivatives 4 and 5 proved to be active in the nanomolar range with no outstanding dependence on the chain length. They showed potencies from three to ten times higher than the corresponding monomers. Consequently, results clearly indicated a positive role of the second pharmacophore in the ligand–protein interaction. The pharmacological profile of the monomers 7 and 8 clarified the contribution of the linker chain to NOP receptor affinity and suggested the presence of a lipophilic acidic site neighbouring the binding site of the JTC-like ligands. Selectivity of saturated compounds 5, 7, and 8 was tested by binding experiments on δ, κ and μ opioid receptors. Results indicated a general loss of selectivity as compared to JTC-801. In the [35S]GTPγS binding assay, all the compounds revealed antagonistic properties at the NOP Receptor. In conclusion the present study set the basis for a systematic investigation on the structural modifications that can be introduced into novel ligands for NOPr and helped to feature the surrounds of the allosteric site of NOPr. [Copyright &y& Elsevier]

Estrogen receptors are known drug targets that have been linked to several kinds of cancer. The structure of the estrogen receptor ligand binding domain is available and reveals a homodimeric layout. In order to improve the binding affinity of known estrogen receptor inhibitors, bivalent compounds have been developed that consist of two individual ligands linked by flexible tethers serving as spacers. So far, binding affinities of the bivalent compounds do not surpass their monovalent counterparts. In this article, we focus our attention on the molecular spacers that are used to connect the individual ligands to form bivalent compounds, and describe their thermodynamic contribution during the ligand binding process. We use computational methods to predict structural and entropic parameters of different spacer structures. We find that flexible spacers introduce a number of effects that may interfere with ligand binding and possibly can be connected to the low binding affinities that have been reported in binding assays. Based on these findings, we try to provide guidelines for the design of novel molecular spacers. [ABSTRACT FROM AUTHOR]

This paper analyzes the equilibria between immunoglobulins ( R), homo-bifunctional ligands ( L), monovalent ligands ( I), and their complexes. We present a mathematical model that can be used to estimate the concentration of each species present in a mixture of R, L, and I, given the initial conditions defining the total concentration of R, L, I, and four dissociation constants ( $$ K_{\rm{d}}^{\rm{inter}} $$, $$ K_{\rm{d}}^{\rm{intra}} $$, $$ K_{\rm{d}}^{\rm{mono}} $$, and α). This model is based on fewer assumptions than previous models and can be used to describe exactly a broad range of experimental conditions. A series of curves illustrates the dependence of the equilibria upon the total concentrations of receptors and ligands, and the dissociation constants. We provide a set of guidelines for the design and analysis of experiments with a focus on estimating the binding constants from experimental binding isotherms. Two analytical equations relate the conditions for maximum aggregation in this system to the binding constants. This model is a tool to quantify the binding of immunoglobulins to antigens and a guide to understanding and predicting the experimental data of assays and techniques that employ immunoglobulins. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: Steroidal bivalent ligands were designed on the basis of the described closer proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of seven progesterone–adenine hybrids were described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. [Copyright &y& Elsevier]

Abstract: Steroidal bivalent ligands for the estrogen receptor (ER) were designed using crystal structures of ERα dimers as a template. The syntheses of several 17α-ethynylestradiol-based bivalent ligands with varying linker compositions and lengths are described. The binding affinities of these bivalent ligands for ERα and ERβ were determined. In the two series of bivalent ligands that we synthesized, there is a clear correlation between linker length and binding affinity, both of which reach a maximum at the same tether length. Further studies are underway to explore aspects of bivalent ligand and control compound binding to the ERs and their effects on ER dimer formation; these results will be reported in a subsequent publication. [Copyright &y& Elsevier]

Abstract: A set of bivalent mannose 6-phosphonate ‘molecular rulers’ has been synthesized to examine ligand binding to the M6P/IGF2R. The set is estimated to span a P–P distance range of 16–26Å (MMFF energy minimization on the hydrated phosphonates). Key synthetic transformations include sugar triflate displacement for phosphonate installation and Grubbs I cross-metathesis to achieve bivalency. Relative binding affinities were tested by radioligand displacement assays versus PMP-BSA (pentamannosyl phosphate-bovine serum albumin). These compounds exhibit slightly higher binding affinities for the receptor (IC50’s=3.7–5μM) than the parent, monomeric mannose 6-phosphonate ligand and M6P itself (IC50 =11.5±2.5μM). These results suggest that the use of an α-configured anomeric alkane tether is acceptable, as no significant thermodynamic penalty is apparently paid with this design. On the other hand, the modest gains in binding affinity observed suggest that this ligand set has not yet found true bivalent interaction with the M6P/IGF2R (i.e., simultaneous binding to two distinct M6P-binding pockets). [Copyright &y& Elsevier]

Abstract: The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties. [Copyright &y& Elsevier]

Although highly controversial just a few years ago, the idea that G-protein-coupled receptors (GPCRs) may undergo homo-oligomerization or hetero-oligomerization has recently gained considerable attention. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based on a number of different biochemical and biophysical approaches. Although much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains elusive. An additional source of uncertainty stems from the realization that homo-oligomerization and hetero-oligomerization of GPCRs may affect receptor binding and activity in different ways, depending on the type of interacting receptors. In this brief review, the functional and pharmacological effects of the hetero-oligomerization of GPCR on binding and cell signaling are critically analyzed. [ABSTRACT FROM AUTHOR]

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G4-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the β-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67phox SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G4-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains. [ABSTRACT FROM AUTHOR]

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ETA and ETB receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds. 2. In saturation binding studies with increasing concentrations (0.77–200 pM) of 125I-ET-1 (nonselective bivalent radioligand), 125I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a KD of 71 pM and a Bmax of 120 fmol mg−1. When 1.0 μM BQ-123 (ETA antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of KD and 8.0 fmol mg−1 of Bmax, whereas 10 nM sarafotoxin S6c (ETB agonist) exerted little change in these binding parameters ( KD, 72 pM; Bmax, 110 fmol mg−1). 3. Competition binding studies with a fixed amount (3.8 pM) of 125I-ET-1 revealed that when 1.0 μM BQ-123 was present in the incubation buffer, ETB receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ETB agonist), and BQ-788 (ETB antagonist) competitively inhibited 125I-ET-1 binding with Kis of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for 125I-ET-1 binding in the case of absence of BQ-123. 4. In cold-ligand saturation studies with a fixed amount (390 pM) of 125I-IRL 1620 (ETB radioligand), IRL1620 bound to a single population of the ETB receptor, and no change was observed in binding characteristics in the presence of 1.0 μM BQ-123. 125I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with Kis of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 μM BQ-123. 5. Two nonbivalent ETA antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for 125I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c. 6. Taken together with the present finding that mRNAs encoding the rat ETA and the ETB receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ETA and ETB receptors with a functional binding capability for ET receptor-ligands, the ETB receptor does not independently recognize ET-1 without the aid of the ETA receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ETA–ETB receptor heterodimer. [ABSTRACT FROM AUTHOR]

A convenient synthetic approach to novel functionalized bis(isoxazoles), the promising bivalent ligands of the AMPA receptor, was elaborated. It was based on the heterocyclization reactions of readily available electrophilic alkenes with the tetranitromethane-triethylamine complex. The structural diversity of the synthesized compounds was demonstrated. In the electrophysiological experiments using the patch clamp technique on Purkinje neurons, the compound 1,4-phenylenedi(methylene)bis(5-aminoisoxazole-3-carboxylate) was shown to be highly potent positive modulator of the AMPA receptor, potentiating kainate-induced currents up to 70% at 10−11 M. [ABSTRACT FROM AUTHOR]

Prostate-specific membrane antigen (PSMA) has become as an outstanding prostate cancer-related target for diagnostic imaging and targeted radiotherapy. Clinical studies on a few PSMA radiotracers are currently underway to determine their efficacy as imaging agents to detect prostate cancer. To improve tumor retention and tumor-to-normal tissue contrast, we herein report the synthesis and preclinical evaluation of an Al18F-labeled bivalent PSMA ligand (18F-Bi-PSMA). 18F-Bi-PSMA was successful automated preparation and in vitro evaluation showed that 18F-Bi-PSMA was potent binding affinity, high specificity, and rapid internalization in PSMA-expressing cells. Biodistribution studies revealed a high and specific tumor uptake of 20.5 ± 3.5 %ID/g in 22Rv1 tumor-bearing mice. Furthermore, compared to the clinically used monomeric PSMA-targeting tracers, 68Ga-PSMA-11 and 18F-PSMA-1007, 18F-Bi-PSMA exhibited improved pharmacokinetics and higher tumor uptake, as well as better tumor-to-normal tissue contrast, resulting in considerably high imaging quality. Our findings indicated that the bivalent PSMA radioligand, 18F-Bi-PSMA, was successfully synthesized and ideal imaging properties. The bivalent radioligand, 18F-Bi-PSMA, where two targeting groups are tethered to a single Al18F complex, dramatically outperforms the monomeric probes, 68Ga-PSMA-11 and 18F-PSMA-1007, with respect to tumor uptake and imaging quality. [Display omitted] • Automated production of bivalent PSMA radioligand, 18F-Bi-PSMA. • 18F-Bi-PSMA exhibited good pharmacokinetic property, high specificity, and high PSMA-positive tumor uptake. • Compared to 68Ga-PSMA-11 and 18F-PSMA-1007, 18F-Bi-PSMA improve tumor uptake and imaging quality. [ABSTRACT FROM AUTHOR]