Alicyclic Ring Size Variation of 4-Phenyl-2-naphthoic Acid Derivatives as P2Y14 Receptor Antagonists.

P2Y14 receptor (P2Y14R) is activated by extracellular UDP-glucose, a damage-associated molecular pattern that promotes inflammation in the kidney, lung, fat tissue, and elsewhere. Thus, selective P2Y14R antagonists are potentially useful for inflammatory and metabolic diseases. The piperidine ring size of potent, competitive P2Y14R antagonist (4-phenyl-2-naphthoic acid derivative) PPTN 1 was varied from 4- to 8-membered rings, with bridging/functional substitution. Conformationally and sterically modified isosteres included N-containing spirocyclic (6-9), fused (11-13), and bridged (14, 15) or large (16-20) ring systems, either saturated or containing alkene or hydroxy/methoxy groups. The alicyclic amines displayed structural preference. An α-hydroxyl group increased the affinity of 4-(4-((1R,5S,6r)-6-hydroxy-3-azabicyclo[3.1.1]heptan-6-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic acid 15 (MRS4833) compared to 14 by 89-fold. 15 but not its double prodrug 50 reduced airway eosinophilia in a protease-mediated asthma model, and orally administered 15 and prodrugs reversed chronic neuropathic pain (mouse CCI model). Thus, we identified novel drug leads having in vivo efficacy.

Effects of Purinergic Receptor Deletion or Pharmacologic Modulation on Pulmonary Inflammation in Mice.

COVID-19 disease is associated with progressive accumulation of SARS-CoV-2-specific mRNA, which is recognized by innate immune receptors, such as TLR3. This in turn leads to dysregulated production of multiple cytokines, including IL-6, IFN-γ, CXCL1, and TNF-α. Excessive production of these cytokines leads to acute lung injury (ALI), which consequently compromises alveolar exchange of O2 and CO2. It is therefore of considerable interest to develop novel therapies that reduce pulmonary inflammation and stem production of pro-inflammatory cytokines, potentially for COVID-19 patients that are at high risk of developing severe disease. Purinergic signaling has a central role in fine-tuning the innate immune system, with P2 (nucleotide) receptor antagonists and adenosine receptor agonists having anti-inflammatory effects. Accordingly, we focused here on the potential role of purinergic receptors in driving neutrophilic inflammation and cytokine production in a mouse model of pulmonary inflammation. To mimic the effects of SARS-CoV-2-specific RNA accumulation in mice, we administered progressively increasing daily doses of a viral mimetic, polyinosinic:polycytidylic acid [poly(I:C)] into the airways of mice over the course of 1 week. Some mice also received increasing daily doses of ovalbumin to mimic virus-encoded protein accumulation. Animals receiving both poly(I:C) and ovalbumin displayed particularly high cytokine levels and neutrophilia, suggestive of both innate and antigen-specific, adaptive immune responses. The extent of these responses was diminished by genetic deletion (P2Y14R, P2X7R) or pharmacologic modulation (P2Y14R antagonists, A3AR agonists) of purinergic receptors. These results suggest that pharmacologic modulation of select purinergic receptors might be therapeutically useful in treating COVID-19 and other pulmonary infections.

Synthesis and pharmacological characterization of multiply substituted 2H-chromene derivatives as P2Y6 receptor antagonists.

P2Y6 receptor (P2Y6R) antagonists represent potential drugs for treating cancer, pain, neurodegeneration, asthma, diabetes, colitis and other disorders. However, there are few chemical classes of known competitive antagonists. We recently explored the structure activity relationship (SAR) of 2H-chromene derivatives as P2Y6R antagonists of moderate affinity. New analogues in this series modified at five positions were synthesized and shown to antagonize Ca2+ transients induced by the native agonist UDP in human (h) P2Y6R-expressing (but not turkey P2Y1R-, hP2Y2R- or hP2Y4R-expressing) astrocytoma cells. Alternatives to the reported 2-(trifluoromethyl)- and 3-nitro- substitutions of this scaffold were not identified. However, 6­fluoro 11 and 6­chloro 12 analogues displayed enhanced potency compared to other halogens, although still in the 1 - 2 µM range. Similar halogen substitution at 5, 7 or 8 positions reduced affinity. 5- or 8­Triethylsilylethynyl extension maintained hP2Y6R affinity, with IC50 0.46 µM for 26 (MRS4853). The 6,8­difluoro analogue 27 (IC50 2.99 µM) lacked off-target activities among 45 sites examined, unlike earlier analogues that bound to biogenic amine receptors. 11 displayed only one weak off-target activity (σ2). Mouse P2Y6R IC50s of 5, 25, 26 and 27 were 4.94, 17.6, 6.15 and 17.8 µM, respectively, but most other analogues had reduced affinity (>20 µM) compared to the hP2Y6R. These analogues are suitable for evaluation in in vivo inflammation and cancer models, which will be performed in the future studies.

Development of Dibenzothiazepine Derivatives as Multifunctional Compounds for Neuropathic Pain.

Neuropathic pain is a chronic and sometimes intractable condition caused by lesions or diseases of the somatosensory nervous system. Many drugs are available but unfortunately do not provide satisfactory effects in patients, producing limited analgesia and undesirable side effects. Thus, there is an urgent need to develop new pharmaceutical agents to treat neuropathic pain. To date, highly specific agents that modulate a single target, such as receptors or ion channels, never progress to the clinic, which may reflect the diverse etiologies of neuropathic pain seen in the human patient population. Therefore, the development of multifunctional compounds exhibiting two or more pharmacological activities is an attractive strategy for addressing unmet medical needs for the treatment of neuropathic pain. To develop novel multifunctional compounds, key pharmacophores of currently used clinical pain drugs, including pregabalin, fluoxetine and serotonin analogs, were hybridized to the side chain of tianeptine, which has been used as an antidepressant. The biological activities of the hybrid analogs were evaluated at the human transporters of neurotransmitters, including serotonin (hSERT), norepinephrine (hNET) and dopamine (hDAT), as well as mu (µ) and kappa (κ) opioid receptors. The most advanced hybrid of these multifunctional compounds, 17, exhibited multiple transporter inhibitory activities for the uptake of neurotransmitters with IC50 values of 70 nM, 154 nM and 2.01 µM at hSERT, hNET and hDAT, respectively. Additionally, compound 17 showed partial agonism (EC50 = 384 nM) at the µ-opioid receptor with no influence at the κ-opioid receptor. In in vivo pain animal experiments, the multifunctional compound 17 showed significantly reduced allodynia in a spinal nerve ligation (SNL) model by intrathecal administration, indicating that multitargeted strategies in single therapy could considerably benefit patients with multifactorial diseases, such as pain.

Bridged Piperidine Analogues of a High Affinity Naphthalene-Based P2Y14R Antagonist.

High affinity phenyl-piperidine P2Y14R antagonist 1 (PPTN) was modified with piperidine bridging moieties to probe receptor affinity and hydrophobicity. Various 2-azanorbornane, nortropane, isonortropane, isoquinuclidine, and ring-opened cyclopentylamino derivatives preserved human P2Y14R affinity (fluorescence binding assay), and their pharmacophoric overlay was compared. Enantiomeric 2-azabicyclo[2.2.1]hept-5-en-3-one precursors assured stereochemically unambiguous, diverse products. Pure (S,S,S) 2-azanorbornane enantiomer 15 (MRS4738) displayed higher affinity than 1 (3-fold higher affinity than enantiomer 16) and in vivo antihyperallodynic and antiasthmatic activity. Its double prodrug 143 (MRS4815) dramatically reduced lung inflammation in a mouse asthma model. Related lactams 21-24 and dicarboxylate 42 displayed intermediate affinity and enhanced aqueous solubility. Isoquinuclidine 34 (IC50 15.6 nM) and isonortropanol 30 (IC50 21.3 nM) had lower lipophilicity than 1. In general, rigidified piperidine derivatives did not lower lipophilicity dramatically, except those rings with multiple polar groups. P2Y14R molecular modeling based on a P2Y12R structure showed stable and persistent key interactions for compound 15.

Adipocyte P2Y14 receptors play a key role in regulating whole-body glucose and lipid homeostasis.

Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated Gi-type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y14R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y14R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y14R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y14R antagonist. In conclusion, we demonstrate that adipose P2Y14Rs were critical regulators of whole-body glucose and lipid homeostasis, suggesting that P2Y14R antagonists might be beneficial for the therapy of obesity and T2D.

Structure activity relationship of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives as P2Y6 receptor antagonists.

Various 6-alkynyl analogues of a known 3-nitro-2-(trifluoromethyl)-2H-chromene antagonist 3 of the Gq-coupled P2Y6 receptor (P2Y6R) were synthesized using a Sonogashira reaction to replace a 6-iodo group. The analogues were tested in a functional assay consisting of inhibition of calcium mobilization in P2Y6R-expressing astrocytoma cells elicited by native P2Y6R agonist UDP. 6-Ethynyl and 6-cyano groups were installed, and the alkynes were extended through both alkyl and aryl spacers. The most potent antagonists, with IC50 of ~1 µM, were found to be trialkylsilyl-ethynyl 7 and 8 (3-5 fold greater affinity than reference 3), t-butyl prop-2-yn-1-ylcarbamate 14 and p-carboxyphenyl-ethynyl 16 derivatives, and 3 and 8 displayed surmountable antagonism of UDP-induced production of inositol phosphates. Other chain-extended terminal carboxylate derivatives were less potent than the corresponding methyl ester derivatives. Thus, the 6 position in this chromene series is suitable for derivatization with flexibility of substitution, even with sterically extended chains, without losing P2Y6R affinity. However, a 3-carboxylic acid or 3-ester substitution did not serve as a nitro bioisostere, as the affinity was eliminated. These compounds provide additional ligand tools for the underexplored P2Y6R, which is a target for inflammatory, neurodegenerative and metabolic diseases.

Structure-Activity Relationship of Heterocyclic P2Y14 Receptor Antagonists: Removal of the Zwitterionic Character with Piperidine Bioisosteres.

A known zwitterionic, heterocyclic P2Y14R antagonist 3a was substituted with diverse groups on the central phenyl and terminal piperidine moieties, following a computational selection process. The most potent analogues contained an uncharged piperidine bioisostere, prescreened in silico, while an aza-scan (central phenyl ring) reduced P2Y14R affinity. Piperidine amide 11, 3-aminopropynyl 19, and 5-(hydroxymethyl)isoxazol-3-yl) 29 congeners in the triazole series maintained moderate receptor affinity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave the most potent naphthalene-containing (32; MRS4654; IC50, 15 nM) and less active phenylamide-containing (33) scaffolds. Thus, a zwitterion was nonessential for receptor binding, and molecular docking and dynamics probed the hydroxymethylisoxazole interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate group to provide neutral analogues, which were cleavable by liver esterase, and in vivo efficacy demonstrated. We have, in stages, converted zwitterionic antagonists into neutral molecules designed to produce potent P2Y14R antagonists for in vivo application.

Biological Evaluation of 5'-(N-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors.

The heat shock protein 90 kDa (Hsp90) family of chaperones is highly sought-after for the treatment of cancer and neurodegenerative diseases. Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum localized isoform that is responsible for the maturation of proteins involved in cell adhesion and the immune response, including Toll-like receptors, immunoglobulins, and integrins. Consequently, Grp94 has been implicated in many different diseases including cancer metastasis, glaucoma, and viral infection. 5'-(N-Ethylcarboxamido)adenosine (NECA) was identified from a high-throughput screen as one of the first molecules to exhibit isoform selectivity toward Grp94, with the ethyl group projecting into a unique pocket within the ATP binding site of Grp94. This pocket has since been exploited by several groups to develop Grp94 selective inhibitors. Despite success in the development of other classes of inhibitors, relatively little work has been done to further develop inhibitors with the NECA scaffold. Unfortunately, NECA is also a potent adenosine receptor agonist, which is likely to confound any biological activity. Therefore, structure-activity relationship studies were performed on the NECA scaffold leading to the discovery of several molecules that displayed similar selectivity and affinity as the parent compound.

Exploration of Alternative Scaffolds for P2Y14 Receptor Antagonists Containing a Biaryl Core.

Various heteroaryl and bicyclo-aliphatic analogues of zwitterionic biaryl P2Y14 receptor (P2Y14R) antagonists were synthesized, and affinity was measured in P2Y14R-expressing Chinese hamster ovary cells by flow cytometry. Given this series' low water solubility, various polyethylene glycol derivatives of the distally binding piperidin-4-yl moiety of moderate affinity were synthesized. Rotation of previously identified 1,2,3-triazole attached to the central m-benzoic acid core (25) provided moderate affinity but not indole and benzimidazole substitution of the aryl-triazole. The corresponding P2Y14R region is predicted by homology modeling as a deep, sterically limited hydrophobic pocket, with the outward pointing piperidine moiety being the most flexible. Bicyclic-substituted piperidine ring derivatives of naphthalene antagonist 1, e.g., quinuclidine 17 (MRS4608, IC50 ≈ 20 nM at hP2Y14R/mP2Y14R), or of triazole 2, preserved affinity. Potent antagonists 1, 7a, 17, and 23 (10 mg/kg) protected in an ovalbumin/Aspergillus mouse asthma model, and PEG conjugate 12 reduced chronic pain. Thus, we expanded P2Y14R antagonist structure-activity relationship, introducing diverse physical-chemical properties.

P2Y14 Receptor Antagonists Reverse Chronic Neuropathic Pain in a Mouse Model.

Eight P2Y14R antagonists, including three newly synthesized analogues, containing a naphthalene or phenyl-triazolyl scaffold were compared in a mouse model of chronic neuropathic pain (sciatic constriction). P2Y14R antagonists rapidly (≤30 min) reversed mechano-allodynia, with maximal effects typically within 1 h after injection. Two analogues (4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid 1 and N-acetyl analogue 4, 10 µmol/kg, i.p.) achieved complete pain reversal (100%) at 1 to 2 h, with relief evident up to 5 h for 4 (41%). A reversed triazole analogue 7 reached 87% maximal protection. Receptor affinity was determined using a fluorescent antagonist binding assay, indicating similar mouse and human P2Y14R affinity. The mP2Y14R affinity was only partially predictive of in vivo efficacy, suggesting the influence of pharmacokinetic factors. Thus P2Y14R is a potential therapeutic target for treating chronic pain.

Structure activity relationship of 2-arylalkynyl-adenine derivatives as human A3 adenosine receptor antagonists.

Recognition of nucleosides at adenosine receptors (ARs) is supported by multiple X-ray structures, but the structure of an adenine complex is unknown. We examined the selectivity of predicted A1AR and A3AR adenine antagonists that incorporated known agonist affinity-enhancing N 6 and C2 substituents. Adenines with A1AR-favoring N 6-alkyl, cycloalkyl and arylalkyl substitutions combined with an A3AR-favoring 2-((5-chlorothiophen-2-yl)ethynyl) group were human (h) A3AR-selective, e.g. MRS7497 17 (∼1000-fold over A1AR). In addition, binding selectivity over hA2AAR and hA2BAR and functional A3AR antagonism were demonstrated. 17 was subjected to computational docking and molecular dynamics simulation in a hA3AR homology model to predict interactions. The SAR of nucleoside AR agonists was not recapitulated in adenine AR antagonists, and modeling suggested an alternative, inverted binding mode with the key N2506.55 H-bonding to the adenine N 3 and N 9, instead of N 6 and N 7 as in adenosine agonists.

Isobolographic Analysis of Drug Combinations With Intrathecal BRL52537 (κ-Opioid Agonist), Pregabalin (Calcium Channel Modulator), AF 353 (P2X3 Receptor Antagonist), and A804598 (P2X7 Receptor Antagonist) in Neuropathic Rats.

BACKGROUND: Neuropathic pain should be treated with drug combinations exhibiting multiple analgesic mechanisms of action because the mechanism of neuropathic pain involves multiple physiological causes and is mediated by multiple pathways. In this study, we defined the pharmacological interaction of BRL52537 (κ-opioid agonist), pregabalin (calcium channel modulator), AF 353 (P2X3 receptor antagonist), and A804598 (P2X7 receptor antagonist). METHODS: Animal models of neuropathic pain were established by spinal nerve ligation (SNL) in male Sprague-Dawley rats, and responses to the mechanical stimulation using von Frey filaments were measured. Drugs were administered by intrathecal route and were examined for antiallodynic effects, and drug interactions were evaluated using isobolographic analysis. The mRNA expression levels of pain-related receptors in each spinal cord or dorsal root ganglion of naïve, SNL, and drug-treated SNL rats were evaluated using real-time polymerase chain reaction. RESULTS: Intrathecal BRL52537, pregabalin, AF 353, and A804598 produced antiallodynic effects in SNL rats. In the drug combination studies, intrathecal coadministration of BRL52537 with pregabalin or A804598 exhibited synergistic interactions, and other drugs combinations showed additivity. The rank order of potency was observed as follows: BRL52537 + pregabalin > BRL52537 + A804598 > pregabalin + AF 353 > A804598 + pregabalin > BRL52537 + AF 353 > AF 353 + A804598. Real-time polymerase chain reaction indicated that alterations of P2X3 receptor and calcium channel mRNA expression levels were observed, while P2X7 receptor and κ-opioid receptor expression levels were not altered. CONCLUSIONS: These results demonstrated that intrathecal combination of BRL52537, pregabalin, AF 353, and A804598 synergistically or additively attenuated allodynia evoked by SNL, which suggests the possibility to improve the efficacy of single-drug administration.

Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors.

Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

Prevalence rate and clinical characteristics of esophageal ectopic sebaceous glands in asymptomatic health screen examinees.

Ectopic sebaceous glands in the esophagus have rarely been reported and, thus, represent an obscure medical condition. The aim of this study is to identify the prevalence rate and clinical characteristics of this lesion in an asymptomatic population. We prospectively enrolled health screen examinees who underwent esophagogastroduodenoscopy for gastric cancer screening. An esophageal biopsy was performed in the cases in which esophageal ectopic sebaceous glands were suspected. The general characteristics of the examinees were analyzed based on their medical records. A total of 9989 examinees were enrolled, and five examinees were diagnosed with esophageal ectopic sebaceous glands between December 2012 and June 2014. The endoscopic findings of the esophageal ectopic sebaceous glands indicated multiple yellowish patches or papules, which varied in size. The histopathological findings indicated several lobulated sebaceous glands in the squamous epithelium with inflammatory infiltration. The follow-up endoscopic findings indicated that there was no grossly discernible change. In conclusion, esophageal ectopic sebaceous glands are present in 0.05% of asymptomatic subjects. This lesion is thought to be benign and is not related to clinical symptoms. Therefore, esophageal ectopic sebaceous glands do not require further treatment or follow-up, which makes endoscopists free from active efforts for differential diagnosis with other malignant diseases.

Design and synthesis of potent and selective P2X3 receptor antagonists derived from PPADS as potential pain modulators.

Pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (7a, PPADS), a nonselective P2X receptor antagonist, was extensively modified to develop more stable, potent, and selective P2X3 receptor antagonists as potential antinociceptive agents. Based on the results of our previous report, all strong anionic groups in PPADS including phosphate and sulfonate groups were changed to carboxylic acids or deleted. The unstable azo (-NN-) linkage of 7a was transformed to more stable carbon-carbon, ether or amide linkages through the synthesis of the 5-hydroxyl-pyridine moieties with substituents at 2 position via a Diels-Alder reaction. This resulted in the retention of antagonistic activity (IC50 = 400 ∼ 700 nM) at the hP2X3 receptor in the two-electrode voltage clamp (TEVC) assay system on the Xenopus oocytes. Introduction of bulky aromatic groups at the carbon linker, as in compounds 13 h-n, dramatically improved the selectivity profiles of hP2X3 when compared with mP2X1 and hP2X7 receptors. Among the substituents tested at the 2-position, the m-phenoxybenzyl group showed optimum selectivity and potency at the hP2X3 receptor. In searching for effective substituents at the 4- and 3-positions, we found that compound 36j, with 4-carboxaldehyde, 3-propenoic acid and 2-(m-phenoxy)benzyl groups, was the most potent and selective hP2X3 receptor antagonist with an IC50 of 60 nM at hP2X3 and marginal antagonistic activities of 10 µM at mP2X1 and hP2X7. Furthermore, using an ex-vivo assay system, we found that compound 36j potently inhibited pain signaling in the rat dorsal horn with 20 µM 36j displaying 65% inhibition while 20 µM pregabalin, a clinically available drug, showed only 31% inhibition.

Structure-activity relationships and optimization of 3,5-dichloropyridine derivatives as novel P2X(7) receptor antagonists.

Screening of a library of chemical compounds showed that the dichloropyridine-based analogue 9 was a novel P2X(7) receptor antagonist. To optimize its activity, we assessed the structure-activity relationships (SAR) of 9, focusing on the hydrazide linker, the dichloropyridine skeleton, and the hydrophobic acyl (R(2)) group. We found that the hydrazide linker and the 3,5-disubstituted chlorides in the pyridine skeleton were critical for P2X(7) antagonistic activity and that the presence of hydrophobic polycycloalkyl groups at the R(2) position optimized antagonistic activity. In the EtBr uptake assay in hP2X(7)-expressing HEK293 cells, the optimized antagonists, 51 and 52, had IC(50) values of 4.9 and 13 nM, respectively. The antagonistic effects of 51 and 52 were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1ß, by LPS/IFN-γ/BzATP stimulation of THP-1 cells (IC(50) = 1.3 and 9.2 nM, respectively). In addition, 52 strongly inhibited iNOS/COX-2 expression and NO production in THP-1 cells, further indicating that this compound blocks inflammatory signaling and suggesting that the dichloropyridine analogues may be useful in developing P2X(7) receptor targeted anti-inflammatory agents.