Synthesis of both enantiomers of 1,2,3,4-tetrahydroisoquinoline derivative IPPAM-1 and enantio-dependency of its positive allosteric modulation of prostacyclin receptor

We present a practical synthesis of both enantiomers of 1,2,3,4-tetrahydroisoquinoline derivative IPPAM-1 (1), which is a positive allosteric modulator (PAM) of prostacyclin receptor (IP) and a candidate for treatment of pulmonary arterial hypertension without the side effects caused by IP agonists. Assay of cAMP production by CHO-K1 cells stably expressing human IP clearly demonstrated that the IPPAM activity resides exclusively on the R-form of 1.     

Synthesis and evaluation of prodrugs of corticotropin-releasing factor-1 (CRF1) receptor antagonist BMS-665053 leading to improved oral bioavailability

A series of phosphate and ester-based prodrugs of anilinopyrazinone 1 (BMS-665053) containing either a methylene or an (acyloxy)alkoxy linker was prepared and evaluated in rat pharmacokinetic studies with the goal of improving the oral bioavailability of the parent (1). The prodrugs, in general, had improved aqueous solubility and oral bioavailability compared to 1. Prodrug 12, which contains an (acyloxy)alkoxy linker, showed the greatest improvement in the oral bioavailability relative to the parent (1), with a seven-fold increase (from 5% to 36%) in rat pharmacokinetic studies.     

A model for a glutamate receptor agonist antibody‐binding site

A combination of mutagenesis, computer modeling and immunoreactivity has been used to develop a structural model of a segment of the glutamate receptor (GluR), termed GluR3B, which is bound by receptor‐activating autoantibodies. In this model, the GluR3B epitope is located in a reverse hairpin loop that places key residues important for antibody recognition and receptor activation in a linear arrangement on the solvent‐exposed surface. The conformation of the loop is stabilized by a hydrophobic core which is critical for functional integrity of the epitope. The proximity of the amino‐ and carboxy‐terminal residues suggested that the GluR3B peptide could be cyclized without diminishing immunoreactivity through replacement of these residues with cysteines and formation of a disulfide bond. This prediction was confirmed experimentally since the cyclized peptide retained full immunoreactivity. The model provides insight into GluR subunit‐specific functional diversity and the role of autoantibodies to this region in neurological disease. Copyright © 1999 John Wiley & Sons, Ltd.     

Synthesis and pharmacological evaluation of nucleoside prodrugs designed to target siderophore biosynthesis in Mycobacterium tuberculosis

The nucleoside antibiotic, 5′-O-[N-(salicyl)sulfamoyl]adenosine (1), possesses potent whole-cell activity against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). This compound is also active in vivo, but suffers from poor drug disposition properties that result in poor bioavailability and rapid clearance. The synthesis and evaluation of a systematic series of lipophilic ester prodrugs containing linear and α-branched alkanoyl groups from two to twelve carbons at the 3′-position of a 2′-fluorinated analog of 1 is reported with the goal to improve oral bioavailability. The prodrugs were stable in simulated gastric fluid (pH 1.2) and under physiological conditions (pH 7.4). The prodrugs were also remarkably stable in mouse, rat, and human serum (relative serum stability: human ∼ rat ≫ mouse) displaying a parabolic trend in the SAR with hydrolysis rates increasing with chain length up to eight carbons (t_1/2 = 1.6 h for octanoyl prodrug 7 in mouse serum) and then decreasing again with higher chain lengths. The permeability of the prodrugs was also assessed in a Caco-2 cell transwell model. All of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction and enhanced permeation in the basolateral-to-apical direction relative to the parent compound 2, resulting in efflux ratios 5–28 times greater than 2. Additionally, Caco-2 cells were found to hydrolyze the prodrugs with SAR mirroring the serum stability results and a preference for hydrolysis on the apical side. Taken together, these results suggest that the described prodrug strategy will lead to lower than expected oral bioavailability of 2 and highlight the contribution of intestinal esterases for prodrug hydrolysis.     

Synthesis and evaluation of a conditionally-silent agonist for the α7 nicotinic acetylcholine receptor

We introduce the term ‘silent agonists’ to describe ligands that can place the α7 nicotinic acetylcholine receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming a complex that can subsequently generate currents when treated with an allosteric modulator. KC-1 (5′-phenylanabaseine) was synthesized and identified as a new silent agonist for the α7 nAChR; it binds to the receptor but does not activate α7 nAChR channel opening when applied alone, and its agonism is revealed by co-application with the type II positive allosteric modulator PNU-120596 in the Xenopus oocyte system. The concise synthesis was accomplished in three steps with the C–C bonds formed via Pd-catalyzed mono-arylation and organolithium coupling with N-Boc piperidinone. Comparative structural analyses indicate that a positive charge, an H-bond acceptor, and an aryl ring in a proper arrangement are needed to constitute one class of silent agonist for the α7 nAChR. Because silent agonists may act on signaling pathways not involving ion channel opening, this class of α7 nAChR ligands may constitute a new alternative for the development of α7 nAChR therapeutics.     

Expression profile and role of prostacyclin receptor (PTGIR) in peri-implantation porcine conceptuses

Prostacyclin (prostaglandin I₂ [PGI₂]) signaling system not only plays a pivotal role in vascular function in many species but is also important during early pregnancy in rodents and ruminants. Recently, abundant concentrations of PGI₂ were found in the endometrium and uterine lumen of gilts at the time of implantation. In the present study, conceptuses collected on Days 10, 12, 14, 16, and 18 of pregnancy were examined for the expression of PGI₂ receptors, PTGIR. Moreover, the effect of iloprost (a PGI₂ analogue) on attachment, proliferation, and apoptosis in conceptus trophoblast (Tr) cells was investigated in vitro. Increased PTGIR mRNA expression was observed in Day 16 trophoblasts compared with Days 10, 12, and 14 conceptuses (P < 0.001) and Day 18 trophoblast tissue (P < 0.01). Embryos from Day 18 of gestation revealed greater PTGIR mRNA expression compared with Day 16 embryos (P < 0.01). In contrast to mRNA, PTGIR protein level in conceptus and trophoblast tissue was high on Days 12 and 14, followed by a decrease observed on Day 16. On Day 18 of pregnancy, PTGIR protein was detected in both trophoblast and embryonic tissue. Iloprost stimulated attachment and proliferation of Tr cells, but this effect was abolished by the addition of the PTGIR-specific antagonist, CAY10441, into culture medium. Addition of iloprost neither did affect the ratio of BAX/BCL-2 gene expression in cultured Tr cells nor did protect these cells from staurosporine-induced apoptosis. In summary, PTGIR is expressed in porcine conceptuses, and PGI₂ acting through this receptor may promote the attachment and proliferation of Tr cells, thereby facilitating conceptus implantation.     

Discovery of evocalcet,a next-generation calcium-sensing receptor agonist for the treatment of hyperparathyroidism

The calcium-sensing receptor (CaSR) plays an important role in sensing extracellular calcium ions and regulating parathyroid hormone secretion by parathyroid gland cells, and the receptor is a suitable target for the treatment of hyperparathyroidism. Cinacalcet hydrochloride is a representative CaSR agonist which widely used for the hyperparathyroidism. However, it has several issues to clinical use, such as nausea/vomiting and strong inhibition of CYP2D6. We tried to improve these issues of cinacalcet for a new pharmaceutical agent as a preferable CaSR agonist. Optimization from cinacalcet resulted in the identification of pyrrolidine compounds and successfully led to the discovery of evocalcet as an oral allosteric CaSR agonist. Evocalcet, which exhibited highly favorable profiles such as CaSR agonistic activity and good DMPK profiles, will provide a novel therapeutic option for secondary hyperparathyroidism.     

Synthesis of a novel universal opioid receptor agonist with the 1,3,5-trioxazatriquinane skeleton and its pharmacologies

We designed and synthesized of 1,3,5-trioxazatriquinanes with o- or p-hydroxyphenyl rings as analogs of the κ opioid receptor agonist SYK-146 with m-hydroxyphenyl groups. Although almost all tested compounds did not bind to the opioid receptors, only 17b (SYK-524) with two o-hydroxyphenyl rings showed moderate or potent binding affinities and exhibited agonistic activities for the three opioid receptor types. Because the basicity of the nitrogen atom in the 1,3,5-trioxazatriquinane structure was predicted to be very low due to the electron withdrawing effect of the three oxygen atoms, SYK-524 was a novel non-morphinan and nonpeptidic opioid universal agonist lacking a basic nitrogen atom.     

Design,synthesis, and biological evaluation of pyrrolobenzodiazepine-containing hypoxia-activated prodrugs

The ability of various pyrrolobenzodiazepine(PBD)-containing cytotoxic compounds to function as hypoxia-activated prodrugs was assessed. These molecules incorporated a 1-methyl-2-nitro-1H-imidazole hypoxia-activated trigger (present in the clinically evaluated compound TH-302) in a manner that masked a reactive imine moiety required for cytotoxic activity. Incubation of the prodrugs with cytochrome P450-reductase under normoxic and hypoxic conditions revealed that some, but not all, were efficient substrates for the enzyme. In these experiments, prodrugs derived from PBD-monomers underwent rapid conversion to the parent cytotoxic compounds under low-oxygen conditions while related PBD-dimers did not. The ability of a given prodrug to function as an efficient cytochrome P450-reductase substrate correlated with the ratio of cytotoxic potencies measured for the compound against NCI460 cells under normoxic and hypoxic conditions.     

Synthesis and SAR study of a novel series of dopamine receptor agonists

The synthesis of a novel series of dopamine receptor agonists are described as well as their in vitro potency and efficacy on dopamine D₁ and D₂ receptors. This series was designed from pergolide and (4aR,10aR)-1-propyl-1,2,3,4,4a,5,10,10a-octahydro-benzo[g]quinolin-6-ol (PHBQ) and resulted in the synthesis of (2R,4aR,10aR)-2-methylsulfanylmethyl-4-propyl-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazin-9-ol (compound 27), which has a D₁ and D₂ receptor profile similar to that of the most recently approved drug for Parkinson’s disease, rotigotine.     

Design,synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor

An absolute or relative deficiency of pancreatic β-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect β-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces β-cell replication and attenuate β-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic β-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional β-cell mass.     

Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.     

Role of endogenous prostacyclin in gastric ulcerogenic and healing responses—a study using IP-receptor knockout mice

Endogenous prostaglandins (PGs) play an important role in the cytoprotective and healing responses in the stomach, by altering various functions, i.e., an increase of the mucosal blood flow, yet the role of prostacyclin (PGI₂) and its receptor (IP-receptor) in these responses remains unclarified. In the present study, we used IP-receptor knockout mice [IP (−/−)] and examined the importance of IP-receptors in gastric ulcerogenic, cytoprotective and healing responses in these animals. The studies included the ulcerogenic response to cold-restraint stress, the cytoprotective response to a mild irritant (20 mM taurocholate: TC) and capsaicin, and the healing response of chronic gastric ulcers induced by thermo-cauterization. We first checked the absence of IP-receptors by examining the effect of cicaprost (a PGI₂ agonist, topical mucosal application) on gastric mucosal blood flow and found that this agent increased the mucosal blood flow in wild-type [WT (+/+)] mice but not in IP (+/−) mice. Cold-restraint stress (4 h) induced gastric lesions in both groups of mice, but the severity of damage was significantly greater in IP (−/−) mice. Prior p.o. administration of both TC and capsaicin exhibited a marked cytoprotection against HCl/ethanol-induced gastric damage in WT (+/+) mice, both responses being significantly mitigated in the presence of indomethacin. The adaptive cytoprotection induced by TC was similarly observed in IP (−/−) mice, while the capsaicin protection was totally attenuated in the animals lacking IP receptors. On the other hand, the healing of gastric ulcers was significantly delayed by daily administration of indomethacin in WT (+/+) mice. However, this process was not altered in IP (−/−) mice. These results suggest that endogenous PGI₂ is involved in the gastric ulcerogenic response to stress, but not in the healing of pre-existing gastric ulcers. In addition, PGI₂ and its receptors may play a crucial role in capsaicin-induced gastric protection but not in the adaptive cytoprotection-induced by mild irritants.     

Synthesis, transport and antiviral activity of Ala-Ser and Val-Ser prodrugs of cidofovir

We report the synthesis and biological evaluation of Ala-(Val-)l-Ser-CO₂R prodrugs of 1, where a dipeptide promoiety is conjugated to the P(OH)₂ group of cidofovir (1) via esterification by the Ser side chain hydroxyl group and an ethyl group (4 and 5) or alone (6 and 7). In a murine model, oral administration of 4 or 5 did not significantly increase total cidofovir species in the plasma compared to 1 or 2, but 7 resulted in a 15-fold increase in a rat model and had an in vitro EC₅₀ value against human cytomegalovirus comparable to 1. Neither 6 nor 7 exhibited toxicity up to 100 μM in KB or HFF cells.     

反向激动剂：受体研究中的一个新发现

静息时就有一部分受体处于激活性状它们与非激活状态的受体处于动态平衡状态之中。某些原来认为的受体拮抗对Ｒ的亲和性远远高于对Ｒ的亲和性,导致动态平衡中的Ｒ数量减少,因而产生生物效应,对某些受体突变或爱 体过度表达引起的疾病,反向激动剂具有应用前景。     

Synthesis and biological evaluation of pyrimidine derivatives with diverse azabicyclic ether/amine as novel GPR119 agonist

A class of novel pyrimidine derivatives bearing diverse conformationally restricted azabicyclic ether/amine were designed, synthesized and evaluated for their GPR119 agonist activities against type 2 diabetes. Most compounds exhibited superior hEC₅₀ values to endogenous lipid oleoylethanolamide (OEA). Analogs with 2-fluoro substitution in the aryl ring showed more potent GPR119 activation than those without fluorine. Especially compound 27m synthesized from endo-azabicyclic alcohol was observed to have the best EC₅₀ value (1.2 nM) and quite good agonistic activity (112.2% max) as a full agonist.     

The liver X receptor agonist TO901317 protects mice against cisplatin-induced kidney injury

Liver X receptors are in the nuclear receptor superfamily and are contained in the regulation of lipid and cholesterol metabolism. Besides, liver X receptors are considered crucial regulators of the inflammatory response and innate immunity. The current study evaluates the in vivo effects that the synthetic liver X receptor agonist TO901317 protects against cisplatin-induced kidney injury in mice. Mice received cisplatin administration through a single intraperitoneal injection (20 mg/kg in saline). And then the mice were treated with the TO901317 by daily gavage (10 mg/kg/day) 12 h postcisplatin administration, and cisplatin nephrotoxicity was evaluated. At 72 h after cisplatin treatment, elevated plasma urea and creatinine levels (P < 0.05) were evidenced which indicates the renal dysfunction of the vehicle-treated mice, consistent with tubular necrosis, protein cast, dilation of renal tubules, and desquamation of epithelial cells in renal tubules. In contrast, the severity of renal dysfunction and histological damage was reduced in TO901317 treated mice (P < 0.05). In accordance, circulating tumor necrosis factor alpha levels, renal tumor necrosis factor alpha, p47^phox, gp91^phox, and protein expression levels and COX-2 mRNA, renal monocyte chemoattractant protein 1, VACAM-1 mRNA and intercellular adhesion molecule-1 contents, and renal prostaglandin E₂ amounts, were higher in samples from cisplatin-treated mice in comparison with controls (P < 0.05) but attenuated in the TO901317 treatment group (P < 0.05). Taken together, treatment with the liver X receptor agonist TO901317 ameliorated the inflammatory response and oxidative stress in cisplatin-induced kidney injury in mice.     

Characterization of the molecular mechanisms of the coupling between intracellular loops of prostacyclin receptor with the C-terminal domain of the Galphas protein in human coronary artery smooth muscle cells

The C-terminal domain of the Gs protein alpha subunit (Galphas Ct) and the first intracellular loop (iLP1) of prostacyclin receptor (IP) have been predicted to be involved in the receptor signaling mediated through the IP/Gs protein coupling by our previous NMR studies using synthetic peptides. To test whether the results of the peptide studies can be applied to the protein interaction between the IP receptor and the Gs protein in cells, a minigene technique was used to construct cDNAs that encoded either the amino acid residues of the Galphas or that of the individual intracellular loops of the IP receptor. The effects of the minigene-expressed protein fragments on cAMP production mediated by the IP/Gs coupling were evaluated through experiments that co-expressed peptides either through the Galphas Ct or the IP intracellular loops with the IP receptor in HEK293 cells. The first (iLP1) and third (iLP3) IP intracellular loops, as well as the Galphas Ct, which are important to the IP/Gs coupling-mediated signaling, were identified by the significant reduction of cAMP production when the corresponding peptides were expressed in the cells. Furthermore, the cAMP productions were significantly impaired in Galphas-knockout cells co-expressing the IP receptor with the Galphas C-terminal mutants (E392A, L393A and L394A), compared with the Galphas wild type. Blocking of the endogenous IP/Gs coupling by the minigene-expressed peptides of the Galphas CT, iLP1 and iLP3 was further observed in the human coronary artery smooth muscle cells (SMCs). These results indicate that the three residues (E392-L394) of the Galphas protein predicted from NMR peptide studies, and the IP iLP1 and iLP3 play important roles in the Galphas-mediated IP receptor signaling in the cells, which may be a general binding site for the corresponding regions of the other prostanoid receptors that couple to Gs protein.     

Design,synthesis and ex vivo evaluation of colon-specific azo based prodrugs of anticancer agents

Colon-specific azo based prodrugs of anticancer agents like methotrexate (6), gemcitabine (7) and analogue of oxaliplatin (RTB-4) (8) were synthesized and characterized by modern analytical techniques. The prepared prodrugs were stable in acidic (pH 1.2) and basic (pH 7.4) buffers which showed their stability in upper GIT environment. Further, an assay was performed which demonstrated the presence of azoreductase enzyme in the rat fecal material, rat cecum content and other parts of intestinal content which reduce specifically the azo bond and release the drug. The in vitro cytotoxicity assay was also performed which clearly indicated that these azo based prodrugs are active against human colorectal cancer cell lines (COLO 205, COLO 320 DM and HT-29). The release behavior of prodrugs (10, 11 and 15) was 60–70% after 24 h incubation at 37 °C. Therefore, the synthesized azo linked prodrugs of methotrexate, gemcitabine and RTB-4 are the potential candidates for colon targeted drug delivery system with minimal undesirable side effects.     

Design,synthesis, and evaluation of potential prodrugs of DFMO for reductive activation

A series of potential DFMO prodrugs was designed through the incorporation of 4-nitrobenzyl ester or carbamate groups for potential activation by trypanosomal nitroreductase. It was found that only modification of N^ε-amino group of DFMO by 4-nitro-2-fluorobenzyloxycarbonyl resulted in significant trypanocidal activity and could serve as a lead for further investigation.