Anti-allodynic effect of 2-(aminomethyl)adamantane-1-carboxylic acid in a rat model of neuropathic pain: A mechanism dependent on CaV2.2 channel inhibition

Neuropathic pain is a serious physical disabling condition resulting from lesion or dysfunction of the peripheral sensory nervous system. Despite the fact that the mechanisms underlying neuropathic pain are poorly understood, the involvement of voltage-gated calcium (Ca_V) channels in its pathophysiology has justified the use of drugs that bind the Ca_V channel α₂δ auxiliary subunit, such as gabapentin (GBP), to attain analgesic and anti-allodynic effects in models involving neuronal sensitization and nerve injury. GBP binding to α₂δ inhibits nerve injury-induced trafficking of the α₁ pore forming subunits of Ca_V channels, particularly of the N-type, from the cytoplasm to the plasma membrane of pre-synaptic terminals in dorsal root ganglion neurons and dorsal horn spinal neurons. In the search for alternative forms of treatment, in this study we describe the synthesis and pharmacological profile of a GABA derivative, 2-aminoadamantane-1-carboxylic acid (GZ4), which displays a close structure–activity relationship with GBP. Behavioral assessment using von Frey filament stimuli showed that GZ4 treatment reverted mechanical allodynia/hyperalgesia in an animal model of spinal nerve ligation-induced neuropathic pain. In addition, using the patch clamp technique we show that GZ4 treatment significantly decreased whole-cell currents through N-type Ca_V channels heterologously expressed in HEK-293 cells. Interestingly, the behavioral and electrophysiological time course of GZ4 actions reflects that its mechanism of action is similar but not identical to that of GBP. While GBP actions require at least 24 h and imply uptake of the drug, which suggests that the drug acts mainly intracellularly affecting channels trafficking to the plasma membrane, the faster time course (1–3 h) of GZ4 effects suggests also a direct inhibition of Ca²⁺ currents acting on cell surface channels.     

Effect of prolonged administration of bovine lactoferrin in neuropathic pain: Involvement of opioid receptors,nitric oxide and TNF-α

AimsThis study aimed to investigate the effect of prolonged administration of bovine milk lactoferrin (bLF) on hyperalgesia and allodynia in a rat model of neuropathic pain and to determine the involvement of c-Fos, TNF-α, nitric oxide and opioidergic systems in this effect.Main methodsNeuropathic pain was induced in rats by loose ligation of the right sciatic nerve and evaluated by tests measuring the mechanical and thermal hyperalgesia and allodynia. bLF (50, 100, and 200 mg/kg) alone or in combination with opioidergic antagonists were administered intraperitoneally to the rats with neuropathic pain. c-Fos and NADPH-d immunocytochemistry and Western blotting for TNF-α, iNOS and nNOS were performed in the lumbar spinal cord of rats. Plasma TNF-α levels were determined with ELISA.Key findingsProlonged, but not single, administration of bLF produced antihyperalgesic and antiallodynic effects in neuropathic rats. Pretreatment with opioidergic antagonists significantly decreased this effect. Prolonged administration of bLF decreased c-Fos and NADPH-d immunoreactivity and TNF-α and iNOS expressions at 50 and 100 mg/kg and nNOS expression at 100 mg/kg in the lumbar spinal cord of neuropathic rats. Plasma TNF-α levels remained unchanged after bLF treatment.SignificanceProlonged administration of bLF exerts antihyperalgesic and antiallodynic effect in neuropathic rats; down-regulation of both TNF-α and iNOS expressions and potentiation of opioidergic system in the lumbar spinal cord can contribute to this effect.     

The discovery of potent glycine transporter type-2 inhibitors: Design and synthesis of phenoxymethylbenzamide derivatives

We describe the discovery of phenoxymethylbenzamide derivatives as a novel class of glycine transporter type-2 (GlyT-2) inhibitors. We found hit compound 1 (human GlyT-2, IC₅₀ = 4040 nM) in our library and converted its 1-(1-(naphthalen-2-ylmethyl)piperidin-4-yl)pyrrolidin-3-yl group to an 1-(N,N-dimethylaminopropyl)piperidyl group and its tert-butyl group to a trifluoromethyl group to obtain N-(1-(3-(dimethylamino)propyl)piperidin-4-yl)-4-((4-(trifluoromethyl)phenoxy)methyl)benzamide (20). Compound 20 showed good inhibitory activity against human GlyT-2 (IC₅₀ = 15.3 nM) and exhibited anti-allodynia effects in a mouse neuropathic pain model.     

Potency optimization of Huwentoxin-IV on hNav1.7: A neurotoxin TTX-S sodium-channel antagonist from the venom of the Chinese bird-eating spider Selenocosmia huwena

The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNa_v1.7 (IC₅₀ determined herein as 17 ± 2 nM). Na_v1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure–function study into Na_v1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu¹, Glu⁴, Phe⁶ and Tyr³³ were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNa_v1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNa_v1.5. Peptide 47 (Gly¹, Gly⁴, Trp³³-HwTx) demonstrated the largest potency increase on hNa_v1.7 (IC₅₀ 0.4 ± 0.1 nM).     

Discovery of novel analgesic and anti-inflammatory 3-arylamine-imidazo[1,2-a]pyridine symbiotic prototypes

We describe herein the design, synthesis and pharmacological evaluation of novel 3-arylamine-imidazo[1,2-a]pyridine derivatives structurally designed as novel symbiotic prototypes presenting analgesic and anti-inflammatory properties. The derivatives obtained were submitted to in vivo assays of nociception, hyperalgesia and inflammation, and to in vitro assays of human PGHS-2 inhibition. These assays allowed the identification of compound LASSBio-1135 (3a) as an anti-inflammatory and analgesic symbiotic prototype. This compound inhibited moderately the human PGHS-2 enzyme activity (IC₅₀ = 18.5 μM) and reverted the capsaicin-induced thermal hyperalgesia (100 μmol/kg, po) similarly to p38 MAPK inhibitor SB-203580 (2). Additionally, LASSBio-1135 (3a) presented activity similar to celecoxib (1) regarding the reduction of the carrageenan-induced rat paw edema (33% of inhibition at 100 μmol/kg, po). We also discovered derivatives LASSBio-1140 (3c) and LASSBio-1141 (3e) as analgesic and anti-inflammatory prototypes, which were able to attenuate the capsaicin-induced thermal hyperalgesia (100 μmol/kg, po) and reduce the carrageenan-induced paw edema (ED₅₀ = 11.5 μmol/kg (3.3 mg/kg) and 14.5 μmol/kg (4.1 mg/kg), respectively), being both more active than celecoxib (1), despite the fact that their effects involve a different mechanism of action. Additionally, derivative LASSBio-1145 (3j) showed remarkable analgesic (ED₅₀ = 22.7 μmol/kg (8.9 mg/kg)) and anti-inflammatory (ED₅₀ = 8.7 μmol/kg (3.4 mg/kg)) profile in vivo (100 μmol/kg; po), in AcOH-induced abdominal constrictions in mice and carrageenan-induced rat paw edema models, respectively, being a novel orally-active anti-inflammatory drug candidate that acts as a selective PGHS-2 inhibitor (IC₅₀ = 2.8 μM).     

Bioavailable pyrrolo-benzo-1,4-diazines as Nav1.7 sodium channel blockers for the treatment of pain

A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized to improve the profile of the previous lead compound 1. The syntheses, structure–activity relationships, and selected pharmacokinetic data of these analogs are described. The optimization efforts allowed the identification of 33, a quinoline amide exhibiting potent Na_v1.7 inhibitory activity and moderate selectivity over Na_v1.5. Compound 33 displayed anti-nociceptive oral efficacy in a rat CFA inflammatory pain model at 100 mpk and in a rat spinal nerve ligation neuropathic pain model with an EC₅₀ 75 μM.     

Structure–activity relationships of diphenylpiperazine N-type calcium channel inhibitors

A novel series of compounds derived from the previously reported N-type calcium channel blocker NP118809 (1-(4-benzhydrylpiperazin-1-yl)-3,3-diphenylpropan-1-one) is described. Extensive SAR studies resulted in compounds with IC₅₀ values in the range of 10–150 nM and selectivity over the L-type channels up to nearly 1200-fold. Orally administered compounds 5 and 21 exhibited both anti-allodynic and anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain.     

Spinal activation of the NPY Y1 receptor reduces mechanical and cold allodynia in rats with chronic constriction injury

Neuropeptide tyrosine (NPY) and its associated receptors Y1R and Y2R have been previously implicated in the spinal modulation of neuropathic pain induced by total or partial sectioning of the sciatic nerve. However, their role in chronic constrictive injuries of the sciatic nerve has not yet been described. In the present study, we analyzed the consequences of pharmacological activation of spinal Y1R, by using the specific Y1R agonist Leu³¹Pro³⁴-NPY, in rats with chronic constriction injury (CCI). CCI and sham-injury rats were implanted with a permanent intrathecal catheter (at day 7 after injury), and their response to the administration of different doses (2.5, 5, 7, 10 or 20 μg) of Leu³¹Pro³⁴-NPY (at a volume of 10 μl) through the implanted catheter, recorded 14 days after injury. Mechanical allodynia was tested by means of the up-and-down method, using von Frey filaments. Cold allodynia was tested by application of an acetone drop to the affected hindpaw. Intrathecal Leu³¹Pro³⁴-NPY induced an increase of mechanical thresholds in rats with CCI, starting at doses of 5 μg and becoming stronger with higher doses. Intrathecal Leu³¹Pro³⁴ also resulted in reductions in the frequency of withdrawal to cold stimuli, although the effect was somewhat more moderate and mostly observed for doses of 7 μg and higher. We thus show that spinal activation of the Y1R is able to reduce neuropathic pain due to a chronic constrictive injury and, together with other studies, support the use of a spinal Y1R agonist as a therapeutic agent against chronic pain induced by peripheral neuropathy.     

Peripherally acting novel lipo-endomorphin-1 peptides in neuropathic pain without producing constipation

We previously described two novel analogues of endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂, 1), modified with an 8-carbon lipoamino acid (C8LAA) with or without replacement of Tyr¹ with 2,6-dimethyltyrosine (Dmt) at the N-terminus of the peptide (compounds 3 and 4, respectively). They were shown to be more stable and permeable, and acted as potent μ-opioid receptor agonists. In this study we report that the C8LAA modification resulted in successful systemic delivery of both analogues. They produced potent dose-dependent pain relief in a chronic constriction injury-rat model of neuropathic pain after intravenous administration with ED₅₀ values obtained at 6.58 (±1.22) μmol/kg for 3 and 6.18 (±1.17) μmol/kg for 4. Using two different rat models of constipation that assess the effects of μ-opioid receptor agonists on stool hydration and gastro-intestinal motility, compound 3 produced insignificant constipation at 16 μmol/kg, whereas morphine elicited significant constipation at 2 μmol/kg. Compound 3 in contrast to morphine, did not attenuate the hypercapnic ventilatory response at 5 μmol/kg, a dose that fully alleviated hindpaw sensitivity at the time of peak effect in CCI-rats. This finding revealed the lack of respiratory depression effect at antinociceptive dose.     

Synthesis and in vitro evaluation of 2,4-diamino-1,3,5-triazine derivatives as neuronal voltage-gated sodium channel blockers

Neuronal sodium channels blockers interfere with ion flux and have been used for managing neuropathic pain, epilepsy, and cerebral ischemic disorders. In the current study, four groups of 2,4-diamino-1,3,5-triazine derivatives were synthesized and investigated for their neuronal sodium channels binding activity. 5-Aryl-1,3,5-triazaspiro[5.5]undeca-1,3-diene-2,4-diamines (4a–4j) were found to have the best neuronal sodium binding activity among the four groups of triazines evaluated. Derivatives 4a–4j blocked the sodium channels with IC₅₀ values ranged from 4.0 to 14.7 μM. The result from this study showed that analogues of 2,4-diamino-1,3,5-triazines could be used as leads for the discovery of neuronal sodium channels blockers for managing central nervous system related disorders.     

Radiosynthesis and evaluation of new PET ligands for peripheral cannabinoid receptor type 1 imaging

Cannabinoid receptor type 1 (CB1) is mainly expressed in the brain, as well as being expressed in functional relevant concentrations in various peripheral tissues. 1-(4-Chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 1) was developed as a potent allosteric antagonist for CB1 and its oral administration led to reductions in the appetite and body weight of rats. Several analogs of 1 (compounds 2 and 3) were recently identified through a series of structure-activity relationship studies. Herein, we report the synthesis of radiolabeled analogs of these compounds using [¹¹C]COCl₂ and an evaluation of their potential as PET ligands for CB1 imaging using in vitro and in vivo techniques. [¹¹C]2 and [¹¹C]3 were successfully synthesized in two steps using [¹¹C]COCl₂. The radiochemical yields of [¹¹C]2 and [¹¹C]3 were 17 ± 8% and 20 ± 9% (decay-corrected to the end of bombardment, based on [¹¹C]CO₂). The specific activities of [¹¹C]2 and [¹¹C]3 were 42 ± 36 and 37 ± 13 GBq/μmol, respectively. The results of an in vitro binding assay using brown adipose tissue (BAT) homogenate showed that the binding affinity of 2 for CB1 (K_D = 15.3 µM) was much higher than that of 3 (K_D = 26.0 µM). PET studies with [¹¹C]2 showed a high uptake of radioactivity in BAT, which decreased in animals pretreated with AM281 (a selective antagonist for CB1). In conclusion, [¹¹C]2 may be a useful PET ligand for imaging peripheral CB1 in BAT.     

Pyrazole-hydrazone derivatives as anti-inflammatory agents: Design,synthesis, biological evaluation,COX-1,2/5-LOX inhibition and docking study

A new series of pyrazole-hydrazone derivatives 4a-i were designed and synthesized, their chemical structures were confirmed by IR, ¹H NMR, ¹³C NMR, MS spectral data and elemental analysis. IC₅₀ values for all prepared compounds to inhibit COX-1, COX-2 and 5-LOX enzymes were determined in vitro. Compounds 4a (IC₅₀ = 0.67 μM) and 4b (IC₅₀ = 0.58 μM) showed better COX-2 inhibitory activity than celecoxib (IC₅₀ = 0.87 μM) with selectivity index (SI = 8.41, 10.55 in sequent) relative to celecoxib (SI = 8.85). Also, compound 4a and 4b exhibited superior inhibitory activity against 5-LOX (IC₅₀ = 1.92, 2.31 μM) higher than zileuton (IC₅₀ = 2.43 μM). All target pyrazoles were screened for their ability to reduce nitric oxide production in LPS stimulated peritoneal macrophages. Compounds 4a, 4b, 4f and 4i displayed concentration dependent reduction and were screened for in vivo anti-inflammatory activity using carrageenan-induced rat paw edema assay. Compound 4f showed the highest anti-inflammatory activity (% edema inhibition = 15–20%) at all doses when compared to reference drug celecoxib (% edema inhibition = 15.7–17.5%). Docking studies were carried out to investigate the interaction of target compounds with COX-2 enzyme active site.     

Rational design and synthesis of dihydropyrimidine based dual binding site acetylcholinesterase inhibitors

Based on the pharmacological importance of dihydropyrimidine (DHPM) scaffold, substituted DHPMs linked with acetamide linker to substituted aromatic anilines were synthesized and evaluated for their potency as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The good AChE inhibitory activity of 4-dihydropyrimidine-2-thione (4a–h) and 2-amino-1,4-dihyropyrimidines (5a–h) series was observed with compound 4a and 4d identified as the most potent compounds with IC₅₀ values of 0.17 ± 0.01 and 0.39 ± 0.04 μM respectively. The inhibition of BChE was found in a broader range of concentrations (2.37–56.32 μM). To explore the binding insights into the enzyme, molecular docking study was carried out using GOLD software. The binding mode analysis indicated that all of these inhibitors are well accommodated in the active site and interact with the key amino acid residues of Catalytic anionic site (CAS) and peripheral anionic site (PAS). Furthermore, in silico ADMET predictions suggest that these compounds are non-AMES toxic with good blood brain barrier (BBB) penetration, human intestinal absorption.     

Potent inhibitors of CDK5 derived from roscovitine: Synthesis,biological evaluation and molecular modelling

Cyclin dependent kinase 5 (CDK5) is a serine/threonine kinase belonging to the cyclin dependent kinase (CDK) family. CDK5 is involved in numerous neuronal diseases (including Alzheimer’s or Parkinson’s diseases, stroke, traumatic brain injury), pain signaling and cell migration. In the present Letter, we describe syntheses and biological evaluations of new 2,6,9-trisubstituted purines, structurally related to roscovitine, a promising CDK inhibitor currently in clinical trials (CDK1/Cyclin B, IC₅₀ = 350 nM; CDK5/p25, IC₅₀ = 200 nM). These new molecules were synthesized using an original Buchwald–Hartwig catalytic procedure; several compounds (3j, 3k, 3l, 3e, 4k, 6b, 6c) displayed potent kinase inhibitory potencies against CDK5 (IC₅₀ values ranging from 17 to 50 nM) and showed significant cell death inducing activities (IC₅₀ values ranging from 2 to 9 μM on SH-SY5Y). The docking of the inhibitors into the ATP binding domain of the CDK5 catalytic site highlighted the discriminatory effect of a hydrogen bond involving the CDK5 Lys-89. In addition, the calculated final energy balances for complexation measured for several inhibitors is consistent with the ranking of the IC₅₀ values. Lastly, we observed that several compounds exhibit submicromolar activities against DYRK1A (dual specificity, tyrosine phosphorylation regulated kinase 1A), a kinase involved in Down syndrome and Alzheimer’s disease (3g, 3h, 4m; IC₅₀ values ranging from 300 to 400 nM).     

Design,synthesis and biological evaluation of novel tetrahydroisoquinoline quaternary derivatives as peripheral κ-opioid receptor agonists

A novel series of tetrahydroisoquinoline quaternary derivatives 4 were synthesized as peripheral κ-opioid receptor agonists. All the target compounds were evaluated in κ-opioid receptor binding assays, and compounds 4l, 4m, and 4n exhibited high affinity for κ-opioid receptor. Furthermore, compound 4l (κK_i = 0.94 nM) produced potent antinociceptive activity in the mouse acetic acid-induced writhing assay, with lower sedative side effects than the parent compound MB-1c.     

Synthesis,in vitro evaluation and molecular docking studies of novel amide linked triazolyl glycoconjugates as new inhibitors of α-glucosidase

A series of N-substituted amide linked triazolyl β-d-glucopyranoside derivatives (4a-l) were synthesized and their in vitro inhibitory activity against yeast α-glucosidase enzyme [EC.3.2.1.20] was assessed. Compounds 4e (IC₅₀ = 156.06 μM), 4f (IC₅₀ = 147.94 μM), 4k (IC₅₀ = 127.71 μM) and 4l (IC₅₀ = 121.33 μM) were identified as the most potent inhibitors for α-glucosidase as compared to acarbose (IC₅₀ = 130.98 μM) under the same in vitro experimental conditions. Kinetic study showed that both 4e and 4f inhibit the enzyme in a competitive manner with p-nitrophenyl α-d-glucopyranoside as substrate. Molecular docking studies of 4e, 4f, 4k and 4l were also carried out using homology model of α-glucosidase to find out the binding modes responsible for the inhibitory activity. This study revealed that the binding affinity of compounds 4e, 4f, 4k and 4l for α-glucosidase were −8.2, −8.6, −8.3 and −8.5 kcal/mol respectively, compared to that of acarbose (−8.9 kcal/mol). The results suggest that the N-substituted amide linked triazole glycoconjugates can reasonably mimic the substrates for the yeast α-glucosidase.     

Synthesis and biological activity of novel tert-amylphenoxyalkyl (homo)piperidine derivatives as histamine H3R ligands

As a continuation of our search for novel histamine H₃ receptor ligands a series of twenty new tert-amyl phenoxyalkylamine derivatives (2–21) was synthesized. Compounds of four to eight carbon atoms spacer alkyl chain were evaluated on their binding properties at human histamine H₃ receptor (hH₃R). The highest affinities were observed for pentyl derivatives 6–8 (K_i = 8.8–23.4 nM range) and among them piperidine derivative 6 with K_i = 8.8 nM. Structures 6, 7 were also classified as antagonists in cAMP accumulation assay (with EC₅₀ = 157 and 164 nM, respectively). Moreover, new compounds were also evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA). Seven compounds (2–4, 9, 11, 12 and 20) showed anticonvulsant activity at maximal electroshock (MES) test in the dose of 30 mg/kg at 0.5 h. In the subcutaneous pentetrazole (scMET) test compound 4 showed protection at 100 and 300 mg/kg dose at mice, however compounds showed high neurotoxicity in rotarod test at used doses. Also, molecular modeling studies were undertaken, to explain affinity of compounds at hH₃R (taking into the consideration X-ray analysis of compound 18). In order to estimate “drug-likeness” of selected compounds in silico and experimental evaluation of lipophilicity, metabolic stability and cytotoxicity was performed.     

Synthesis,biological activity and structure–activity relationship of endomorphin-1/substance P derivatives

Endomorphins have been shown to produce potent analgesia in various rodent models of pain. However, their central administration led to the development of tolerance and physical dependence. Conjugation of C-terminal substance P (SP) fragments to opioids and opioid peptides was previously shown to produce hybrid peptides with strong analgesic activity, with low or no propensity to develop tolerance. In this study, four peptides (2–5) comprised of endomorphin-1 (1) and C-terminal fragments of SP (four or five amino acids, SP_8–11 (2) or SP_7–11 (4), respectively), with an overlapping Phe residue, were synthesized. To overcome low metabolic stability and poor membrane permeability of the peptide, the N-terminus of 2 and 4 was further modified with a C10-carbon lipoamino acid (C10LAA) achieving 3 and 5, respectively. LAA-modification of the hybrid peptides resulted in a significant increase in metabolic stability and membrane permeability compared to peptides 1, 2 and 4. Compound 5 showed potent μ-opioid receptor binding affinity (K_iμ = 3.87 ± 0.51 nM) with dose-dependent agonist activity in the nanomolar range (IC₅₀ = 45 ± 13 nM). In silico modeling was used to investigate the binding modes and affinities of compounds 1–5 in the active site of μ-opioid receptors. The docking scores were in agreement with the K_iμ values obtained in the receptor binding affinity studies. The more active LAA-modified hybrid peptide showed a lower total interaction energy and higher negative value of MolDock score.     

Synthesis and pharmacological evaluation of novel N-aryl-3,4-dihydro-1′H-spiro[chromene-2,4′-piperidine]-1′-carboxamides as TRPM8 antagonists

A novel series of N-aryl-3,4-dihydro-1′H-spiro[chromene-2,4′-piperidine]-1′-carboxamides was identified as transient receptor potential melastatin 8 (TRPM8) channel blockers through analogue-based rational design, synthesis and screening. Details of the synthesis, effect of aryl groups and their substituents on in-vitro potency were studied. The effects of selected functional groups on the 4-position of the chromene ring were also studied, which showed interesting results. The 4-hydroxy derivatives showed excellent potency and selectivity. Optical resolution and screening of alcohols revealed that (R)-(–)-isomers were in general more potent than the corresponding (S)-(+)-isomers. The isomer (R)-(–)-10e (IC₅₀: 8.9 nM) showed a good pharmacokinetic profile upon oral dosing at 10 mg/kg in Sprague–Dawley (SD) rats. The compound (R)-(–)-10e also showed excellent efficacy in relevant rodent models of neuropathic pain.     

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC₅₀ = 150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC₅₀ = 25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC₅₀ = 7.5 nM) and TNF-α production in mouse splenocytes (IC₅₀ = 9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID₅₀ = 18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand–enzyme complex.