Fentanyl analog structure-activity relationships demonstrate determinants of diverging potencies for antinociception and respiratory depression.

Opioid overdoses, particularly those involving fentanyl-related substances (FRS), present a significant public health challenge in the United States. This structure-activity relationship (SAR) study evaluated the relationship between the chemical structure of seventeen FRS and their in vivo mu-opioid-receptor (MOR) mediated effects. SAR evaluations included fluorine substitutions on the aniline or phenethyl ring and variations in N-acyl chain length. Adult male Swiss Webster mice were administered fluorinated regioisomers of fentanyl, butyrylfentantyl and valerylfentanyl, and compared to MOR standards including morphine, buprenorphine, and fentanyl to determine if they would elicit prototypical opioid-like effects including hyperlocomotion (open-field test), antinociception (warm-water tail-withdrawal test), and hypoventilation (whole-body plethysmography test). To determine if the MOR was the pharmacological mechanism responsible for these effects, naltrexone or naloxone pretreatments were administered to evaluate their actions on FRS-induced antinociception and hypoventilation. There were three main findings. First, FRS elicited hyperlocomotion, antinociception, and hypoventilation in mice to varying degrees, similar to prototypical MOR standards. Second, the rank order of potencies for hypoventilatory effects of FRS were different for each series including FRS with increasing N-acyl chain length (i.e., acetylfentanyl, fentanyl, butyrylfentanyl, valerylfentanyl, hexanoylfentanyl), phenethyl-fluorinated regioisomers (e.g., 2'-fluorofentanyl, 3'-fluorofentanyl, 4'-fluorofentanyl), and aniline-fluorinated regioisomers (e.g., ortho-fluorofentanyl, meta-fluorofentanyl, para-fluorofentanyl). Third, the degree of separation in potencies observed for the antinociceptive and hypoventilatory effects of these drugs did not always follow that which was observed for their antinociceptive and hyperlocomotor effects. This study clarifies the in vivo activities for these FRS and elucidates a SAR for MOR-mediated effects among structural isomers.

Respiratory depressant effects of fentanyl analogs are opioid receptor-mediated.

Opioid-related fatalities involving synthetic opioids have reached unprecedented levels. This study evaluated the respiratory depressant effects of seven fentanyl analogs that have either emerged in the illicit drug supply or been identified in toxicological analyses following fatal or non-fatal intoxications. Adult male Swiss Webster mice were administered fentanyl analogs (isobutyrylfentanyl, crotonylfentanyl, para-methoxyfentanyl, para-methoxybutyrylfentanyl, 3-furanylfentanyl, thiophenefentanyl, and benzodioxolefentanyl) and their effects on minute volume as compared to mu-opioid receptor (MOR) agonist standards (fentanyl, morphine, and buprenorphine) were measured using whole body plethysmography (WBP). All drugs elicited significant (p ≤ 0.05) hypoventilation relative to vehicle for at least one dose tested: morphine (1, 3.2, 10, 32 mg/kg), buprenorphine, (0.032, 0.1, 0.32, 1, 3.2 mg/kg), fentanyl (0.0032, 0.01, 0.032, 0.1, 1, 32 mg/kg), isobutyrylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), crotonylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxyfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxybutyrylfentanyl (0.32, 1, 3.2, 10 mg/kg), 3-furanylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), thiophenefentanyl (1, 3.2, 10, 32, 100 mg/kg), and benzodioxolefentanyl (3.2, 10, 32, 100 mg/kg). The ED50 values for hypoventilation showed a rank order of potency as follows: fentanyl (ED50 = 0.96 mg/kg) > 3-furanylfentanyl (ED50 = 2.60 mg/kg) > crotonylfentanyl (ED50 = 2.72 mg/kg) > para-methoxyfentanyl (ED50 = 3.31 mg/kg) > buprenorphine (ED50 = 10.8 mg/kg) > isobutyrylfentanyl (ED50 = 13.5 mg/kg) > para-methoxybutyrylfentanyl (ED50 = 16.1 mg/kg) > thiophenefentanyl (ED50 = 18.0 mg/kg) > morphine (ED50 = 55.3 mg/kg) > benzodioxolefentanyl (ED50 = 10,168 mg/kg). A naloxone pretreatment (10 mg/kg) attenuated the hypoventilatory effects of all drugs. These results establish that the respiratory depressant effects of these fentanyl analogs are at least in part mediated by the MOR.

Novel amide and imidazole compounds as potent hematopoietic prostaglandin D2 synthase inhibitors.

In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole "linker" between the pyrimidine or pyridine "core" ring and the "tail" ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes.

Improved homology modeling of the human & rat EP4 prostanoid receptors.

BACKGROUND: The EP4 prostanoid receptor is one of four GPCRs that mediate the diverse actions of prostaglandin E2 (PGE2). Novel selective EP4 receptor agonists would assist to further elucidate receptor sub-type function and promote development of therapeutics for bone healing, heart failure, and other receptor associated conditions. The rat EP4 (rEP4) receptor has been used as a surrogate for the human EP4 (hEP4) receptor in multiple SAR studies. To better understand the validity of this traditional approach, homology models were generated by threading for both receptors using the RaptorX server. These models were fit to an implicit membrane using the PPM server and OPM database with refinement of intra and extracellular loops by Prime (Schrödinger). To understand the interaction between the receptors and known agonists, induced-fit docking experiments were performed using Glide and Prime (Schrödinger), with both endogenous agonists and receptor sub-type selective, small-molecule agonists. The docking scores and observed interactions were compared with radioligand displacement experiments and receptor (rat & human) activation assays monitoring cAMP. RESULTS: Rank-ordering of in silico compound docking scores aligned well with in vitro activity assay EC50 and radioligand binding Ki. We observed variations between rat and human EP4 binding pockets that have implications in future small-molecule receptor-modulator design and SAR, specifically a S103G mutation within the rEP4 receptor. Additionally, these models helped identify key interactions between the EP4 receptor and ligands including PGE2 and several known sub-type selective agonists while serving as a marked improvement over the previously reported models. CONCLUSIONS: This work has generated a set of novel homology models of the rEP4 and hEP4 receptors. The homology models provide an improvement upon the previously reported model, largely due to improved solvation. The hEP4 docking scores correlates best with the cAMP activation data, where both data sets rank order Rivenprost>CAY10684 > PGE1 ≈ PGE2 > 11-deoxy-PGE1 ≈ 11-dexoy-PGE2 > 8-aza-11-deoxy-PGE1. This rank-ordering matches closely with the rEP4 receptor as well. Species-specific differences were noted for the weak agonists Sulprostone and Misoprostol, which appear to dock more readily within human receptor versus rat receptor.

Optimization of LpxC Inhibitors for Antibacterial Activity and Cardiovascular Safety.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.

Difluoromethylene at the γ-Lactam α-Position Improves 11-Deoxy-8-aza-PGE1 Series EP4 Receptor Binding and Activity: 11-Deoxy-10,10-difluoro-8-aza-PGE1 Analog (KMN-159) as a Potent EP4 Agonist.

A series of small-molecule full agonists of the prostaglandin E2 type 4 (EP4) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

Biochemical Characterization and Structure-Based Mutational Analysis Provide Insight into the Binding and Mechanism of Action of Novel Aspartate Aminotransferase Inhibitors.

Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize a metabolic pathway through the enzyme aspartate aminotransferase 1 [also known as glutamate oxaloacetate transaminase 1 (GOT1)] to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor-GOT1 interactions with iGOT1-01 analogues that possess improved solubility and potency properties. These results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and the thermal stability of GOT1 while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.

A novel glucose 6-phosphate isomerase from Listeria monocytogenes.

D-Arabinose 5-phosphate isomerases (APIs) catalyze the interconversion of D-ribulose 5-phosphate and D-arabinose 5-phosphate (A5P). A5P is an intermediate in the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo), an essential component of lipopolysaccharide, the lipopolysaccharide found in the outer membrane of Gram-negative bacteria. The genome of the Gram-positive pathogen Listeria monocytogenes contains a gene encoding a putative sugar isomerase domain API, Q723E8, with significant similarity to c3406, the only one of four APIs from Escherichia coli CFT073 that lacks a cystathionine-ß-synthase domain. However, L. monocytogenes lacks genes encoding any of the other enzymes of the Kdo biosynthesis pathway. Realizing that the discovery of an API in a Gram-positive bacterium could provide insight into an alternate physiological role of A5P in the cell, we prepared and purified recombinant Q723E8. We found that Q723E8 does not possess API activity, but instead is a novel GPI (D-glucose 6-phosphate isomerase). However, the GPI activity of Q723E8 is weak compared with previously described GPIS. L. monocytogenes contains an ortholog of the well-studied two-domain bacterial GPI, so this maybe redundant. Based on this evidence glucose utilization is likely not the primary physiological role of Q723E8.