Design, synthesis, and pharmacological evaluation of N-(3-carbamoyl-1H-pyrazol-4-yl)-1,3-oxazole-4-carboxamide derivatives as interleukin-1 receptor-associated kinase 4 inhibitors with reduced potential for cytochrome P450 1A2 induction.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical molecule in Toll-like receptor/interleukin-1 receptor signaling and an attractive therapeutic target for a wide range of inflammatory and autoimmune diseases as well as cancers. In our search for novel IRAK4 inhibitors, we conducted structural modification of a thiazolecarboxamide derivative 1, a lead compound derived from high-throughput screening hits, to elucidate structure-activity relationship and improve drug metabolism and pharmacokinetic (DMPK) properties. First, conversion of the thiazole ring of 1 to an oxazole ring along with introduction of a methyl group at the 2-position of the pyridine ring aimed at reducing cytochrome P450 (CYP) inhibition were conducted to afford 16. Next, modification of the alkyl substituent at the 1-position of the pyrazole ring of 16 aimed at improving CYP1A2 induction properties revealed that branched alkyl and analogous substituents such as isobutyl (18) and (oxolan-3-yl)methyl (21), as well as six-membered saturated heterocyclic groups such as oxan-4-yl (2), piperidin-4-yl (24, 25), and dioxothian-4-y (26), are effective for reducing induction potential. Representative compound AS2444697 (2) exhibited potent IRAK4 inhibitory activity with an IC50 value of 20 nM and favorable DMPK properties such as low risk of drug-drug interactions mediated by CYPs as well as excellent metabolic stability and oral bioavailability.

Incidence of spinal instability among patients with discogenic low back pain with different backgrounds.

[Purpose] To investigate the incidence of spinal instability among patients with discogenic low back pain and its various effects with respect to the patients' age, gender, sports activity, and occupation. [Participants and Methods] We assessed 65 patients diagnosed with discogenic low back pain at our clinic between May 2016 and May 2020. After measuring segmental angulation using plain radiographs, we divided these patients into two groups: (1) instability group with >10° of segmental angulation or >3 mm of sagittal translation and (2) stability group with <10° of segmental angulation and <3 mm of sagittal translation. Patient data such as age, gender, sports activity, and occupation were collected using their medical records. [Results] The incidence of spinal instability was 57% (37 patients) among the patients with discogenic low back pain. No significant differences in age, gender, sports activity, and occupation were identified between the groups. [Conclusion] The incidence of spinal instability accounted for more than half of the total number of patients. Additionally, age, gender, sports activity, and occupation did not affect the incidence of spinal instability. Our results support the importance of rehabilitation, such as stability training for spinal instability, for patients with discogenic low back pain.

Therapeutic effects of interleukin-1 receptor-associated kinase 4 inhibitor AS2444697 on diabetic nephropathy in type 2 diabetic mice.

Renal inflammation is a final common pathway of chronic kidney disease including diabetic nephropathy, which is the leading cause of end-stage renal disease and is associated with high cardiovascular risk and significant morbidity and mortality. Interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK-4) is a pivotal molecule for IL-1 receptor- and Toll-like receptor-induced activation of proinflammatory mediators. In this study, we investigated the renoprotective properties of IRAK-4 inhibitor AS2444697 in KK/Ay type 2 diabetic mice. Four-week repeated administration of AS2444697 dose-dependently and significantly improved albuminuria; hyperfiltration, as measured by creatinine clearance; renal injury, including glomerulosclerosis; tubular injury markers, including urinary N-acetyl-ß-D-glucosaminidase activity; and glomerular podocyte injury markers, including urinary nephrin excretion. In addition, AS2444697 attenuated plasma levels of proinflammatory cytokines, including IL-6; plasma levels of endothelial dysfunction markers, including intercellular adhesion molecule-1; and plasma levels and renal contents of oxidative stress markers. In contrast, AS2444697 did not significantly affect food intake or blood glucose levels. These results suggest that AS2444697 attenuates the progression of diabetic nephropathy mainly via anti-inflammatory mechanisms through inhibition of IRAK-4 activity under diabetic conditions and may represent a promising therapeutic option for the treatment of type 2 diabetic nephropathy.

Synthesis and Pharmacological Evaluation of 3-[(4-Oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)(phenyl)amino]propanenitrile Derivatives as Orally Active AMPA Receptor Antagonists.

In our search for novel orally active α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, we found that conversion of an allyl group in the lead compound 2-[allyl(4-methylphenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-one (4) to a 2-cyanoethyl group significantly increased inhibitory activity against AMPA receptor-mediated kainate-induced toxicity in rat hippocampal cultures. Here, we synthesized 10 analogs bearing a 2-cyanoethyl group and administered them to mice to evaluate their anticonvulsant activity in maximal electroshock (MES)- and pentylenetetrazol (PTZ)-induced seizure tests, and their effects on motor coordination in a rotarod test. 3-{(4-Oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)[4-(trifluoromethoxy)phenyl]amino}propanenitrile (25) and 3-[(2,2-difluoro-2H-1,3-benzodioxol-5-yl)(4-oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)amino]propanenitrile (27) exhibited potent anticonvulsant activity in both seizure tests and induced minor motor disturbances as indicated in the rotarod test. The protective index values of 25 and 27 for MES-induced seizures (10.7 and 12.0, respectively) and PTZ-induced seizures (6.0 and 5.6, respectively) were considerably higher compared with those of YM928 (5) and talampanel (1).

Synthesis, structure-activity relationships, and anticonvulsant activities of 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one derivatives as orally active AMPA receptor antagonists.

As part of a program aimed at discovering orally active 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonists, we screened our compound library and identified 2-[allyl(4-methylphenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-one (7) as a lead compound that inhibited kainate-induced neurotoxicity mediated by AMPA receptors in rat hippocampal cultures. Structure-activity relationship studies of a series of 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one derivatives revealed that substituents on the phenyl ring attached to the 2-amino group and the 4H-pyrido[3,2-e][1,3]thiazin-4-one ring system play an important role in inhibitory activity against kainate-induced neurotoxicity. Several analogs bearing a phenyl group with a 4-substituent or five- or six-membered ring fused at the 3,4-positions exhibited potent inhibitory activity against kainate-induced neurotoxicity. Further, some of these compounds exhibited significant suppression of maximal electroshock seizure in mice following oral administration. Of these compounds, 2-[(4-chlorophenyl)(methyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-one (16i) (YM928) demonstrated the most potent inhibitory effect with an ED50 value of 7.4mg/kg.

Renoprotective effects of novel interleukin-1 receptor-associated kinase 4 inhibitor AS2444697 through anti-inflammatory action in 5/6 nephrectomized rats.

Renal inflammation is a final common pathway of chronic kidney disease (CKD), and its progression can be used to effectively gauge the degree of renal dysfunction. Interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK-4) has been reported to be a pivotal molecule for IL-1 receptor- and Toll-like receptor-induced signaling and activation of proinflammatory mediators. In this study, we hypothesized that if inflammation plays a key role in renal failure, then the anti-inflammatory effect of IRAK-4 inhibitor should be effective in improving CKD. To determine its pharmacological potency, we investigated the renoprotective properties of the novel IRAK-4 inhibitor AS2444697 (N-[3-carbamoyl-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl]-2-(2-methylpyridin-4-yl)-1,3-oxazole-4-carboxamide hydrochloride (1:1)) in 5/6 nephrectomized (Nx) rats, a model of CKD. Six weeks' repeated administration of AS2444697 (0.3-3 mg/kg, twice daily) dose-dependently and significantly reduced urinary protein excretion and prevented the development of glomerulosclerosis and interstitial fibrosis without affecting the blood pressure. In addition, AS2444697 showed beneficial effects on renal function as demonstrated by the decrease in levels of plasma creatinine and blood urea nitrogen and attenuation of decline in creatinine clearance. 5/6 Nx rats exhibited low-grade inflammation as evidenced by increased renal mRNA expression and plasma levels of proinflammatory cytokines (IL-1ß, IL-6, TNF-α, and MCP-1) and C-reactive protein as a marker of systemic inflammation. AS2444697 significantly reduced or showed a decreasing trend in expression and levels of these inflammatory parameters. These results suggest that AS2444697 suppresses the progression of chronic renal failure via anti-inflammatory action and may therefore be potentially useful in treating CKD patients.

Synthesis, biological evaluation, and metabolic stability of acrylamide derivatives as novel CCR3 antagonists.

Our laboratory has identified several acrylamide derivatives with potent CCR3 inhibitory activity. In the present study, we evaluated the in vitro metabolic stability (CL(int); mL/min/kg) of these compounds in human liver microsomes (HLMs), and assessed the relationship between their structures and CL(int) values. Among the compounds identified, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-[1-(2-hydroxybenzoyl)piperidin-4-ylidene]acetamide (30j) was found to be a potent inhibitor (IC(50)=8.4nM) with a high metabolic stability against HLMs.

Design and synthesis of 6-fluoro-2-naphthyl derivatives as novel CCR3 antagonists with reduced CYP2D6 inhibition.

In our previous study on discovering novel types of CCR3 antagonists, we found a fluoronaphthalene derivative (1) that exhibited potent CCR3 inhibitory activity with an IC(50) value of 20 nM. However, compound 1 also inhibited human cytochrome P450 2D6 (CYP2D6) with an IC(50) value of 400 nM. In order to reduce its CYP2D6 inhibitory activity, we performed further systematic structural modifications on 1. In particular, we focused on reducing the number of lipophilic moieties in the biphenyl part of 1, using ClogD(7.4) values as the reference index of lipophilicity. This research led to the identification of N-{(3-exo)-8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3-yl}-3-(piperidin-1-ylcarbonyl)isonicotinamide 1-oxide (30) which showed comparable CCR3 inhibitory activity (IC(50)=23 nM) with much reduced CYP2D6 inhibitory activity (IC(50)=29,000 nM) compared with 1.

Synthesis and structure-activity relationships of N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide derivatives as novel CCR3 antagonists.

A novel class of potent CCR3 receptor antagonists were designed and synthesized starting from N-{1-[(6-fluoro-2-naphthyl)methyl]piperidin-4-yl}benzamide (1),which was found by subjecting our chemical library to high throughput screening (HTS). The CCR3 inhibitory activity of the synthesized compounds against eotaxin-induced Ca(2+) influx was evaluated using CCR3-expressing preB cells. Systematic chemical modifications of 1 revealed that the 6-fluoro-2-naphthylmethyl moiety was essential for CCR3 inhibitory activity in this new series of CCR3 antagonists. Further structural modifications of the benzamide and piperidine moieties of 1 led to the identification of exo-N-{8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3- yl}biphenyl-2-carboxamide [corrected] (31) as a potent CCR3 antagonist with an IC(50) value of 0.020 microM.

Effect of YM928, a novel AMPA receptor antagonist, on seizures in EL mice and kainate-induced seizures in rats.

Kainate-induced seizures and seizures induced by tossing stimulation in epilepsy-prone EL mice are considered as models of complex partial seizures. We used these models to evaluate the anticonvulsive effects of 2-[ N-(4-chlorophenyl)- N-methylamino]-4 H-pyrido[3.2-e]-1,3-thiazin-4-one (YM928), a novel alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist. In the kainate-induced seizure test in rats, wet-dog shakes (WDS) were reduced by oral administration of YM928 at doses of 7.5 mg/kg and 30 mg/kg. YM928 (15 mg/kg) reduced the number of WDS within the first 80 min, but then prolonged the time of occurrence compared with the other groups. Significant reduction in kainate-induced motor seizure was observed with 4-30 mg/kg. YM928 did not induce apparently abnormal behaviour at doses of 2-15 mg/kg but did induce sedation at 30 mg/kg. Carbamazepine (40 or 80 mg/kg), valproate (600 mg/kg), diazepam (2.5 mg/kg), and phenobarbital (20 or 40 mg/kg) exerted anticonvulsant effects against motor seizures, but only valproate, at a dose that also caused sedation, suppressed WDS. Phenytoin and ethosuximide did not show significant anti-kainate effects. In the tossing stimulation test in EL mice, i.p. injection of YM928 at 5 mg/kg or 10 mg/kg significantly increased the number of stimulations required to elicit generalized seizure. Carbamazepine (4 or 8 mg/kg), phenytoin (8 or 16 mg/kg), valproate (100-400 mg/kg), diazepam (0.5 mg/kg), phenobarbital (1.3 or 2.5 mg/kg) and ethosuximide (75-300 mg/kg) exerted significant anticonvulsant effects against these seizures. These results indicate that YM928 has anticonvulsant effects on seizure models that are characteristic of partial onset seizures in humans. YM928 is expected to have beneficial effects against human complex partial seizure with secondary generalization or temporal lobe epilepsy.

Suppression of fully kindled seizure and retardation of kindling acquisition by YM928 in the rat kindling model of epilepsy.

We investigated the effects of 2-[N-(4-chlorophenyl)-N-methylamino]-4H-pyrido[3.2-e]-1,3-thiazin-4-one (YM928), a selective alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, in the rat kindling model of complex partial seizures. YM928 (10 and 30 mg/kg p.o.) markedly suppressed the motor seizures and afterdischarge induced by electrical stimulation of the amygdala at generalized seizure-triggering threshold intensity. YM928 (10 mg/kg p.o.) did not induce apparent abnormal behavior, but did induce sedation at a dose of 30 mg/kg p.o. YM928 (30 mg/kg p.o.) showed a similar anticonvulsant effect at twice the threshold intensity as it did at threshold intensity. Diazepam (10 mg/kg p.o.) and phenobarbital (60 mg/kg p.o.) also exerted anticonvulsant activities. Diazepam (10 mg/kg) showed a similar effect at twice the threshold as at threshold, but the anticonvulsant effect of phenobarbital (60 mg/kg p.o.) was reversed when the stimulus was doubled. When YM928 (10 mg/kg p.o.) was administered 60 min before daily stimulation of the amygdala, the development of kindling seizure was significantly retarded. These results indicate that YM928 has anticonvulsant effects and suppresses kindling acquisition without sedative effects, and may be suitable as an antiepileptic drug for the treatment of complex partial seizures in humans.

Effects of 2-[N-(4-chlorophenyl)-N-methylamino]-4H-pyrido[3.2-e]-1,3-thiazin-4-one (YM928), an orally active alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, in models of generalized epileptic seizure in mice and rats.

The anticonvulsant activity of 2-[N-(4-chlorophenyl)-N-methylamino]-4H-pyrido[3.2-e]-1,3-thiazin-4-one (YM928), a novel alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, was studied in animal models of generalized seizure. YM928 exerted significant anticonvulsant effects in the maximal electroshock (MES) seizure test (ED50 = 7.4 mg/kg p.o.), pentylenetetrazol (PTZ)-induced seizure test (ED50 = 9.6 mg/kg p.o.), AMPA-induced seizure test (ED50 = 5.5 mg/kg p.o.), and strychnine-induced seizure test (ED50 = 14.0 mg/kg p.o.) in mice. Effects in rats were detected in the MES seizure test (ED50 = 4.0 mg/kg p.o.) and PTZ-induced seizure test (ED50 = 6.2 mg/kg p.o.). The profile of YM928 was compared with that of established antiepileptics. Valproate showed beneficial effects in all tests used. In contrast, carbamazepine, phenytoin, lamotrigine, phenobarbital, diazepam, ethosuximide, and gabapentin were not active against seizures induced by at least one stimulant. In the rotarod test, YM928 impaired motor coordination (TD50 = 22.5 mg/kg p.o.). The protective index (TD50 value of the rotarod test/ED50 value of MES seizure) was 3.0, suggesting that YM928 can exert antiepileptic effects with only minor motor disturbances. YM928 at doses of 2, 4, and 8 mg/kg p.o. did not significantly affect the threshold of electroshock seizure in rats after 16 days of repeated administration. These data indicate that YM928 does not induce tolerance after subchronic administration. These results indicate that YM928 is a broad-spectrum anticonvulsant that would prove useful for the treatment of generalized seizure in human epileptic patients.