Discovery of novel spirocyclic inhibitors of fatty acid amide hydrolase (FAAH). Part 2. Discovery of 7-azaspiro[3.5]nonane urea PF-04862853, an orally efficacious inhibitor of fatty acid amide hydrolase (FAAH) for pain

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.     

Synthesis of 2-azaspiro[3.3]heptane-derived amino acids: ornitine and GABA analogues

Synthesis of 6-amino-2-azaspiro[3.3]heptane-6-carboxylic acid and 2-azaspiro[3.3]heptane-6-carboxylic acid was performed. Both four-membered rings in the spirocyclic scaffold were constructed by subsequent ring closure of corresponding 1,3-bis-electrophiles at 1,1-C- or 1,1-N-bis-nucleophiles. The two novel amino acids were added to the family of the sterically constrained amino acids for the use in chemistry, biochemistry, and drug design.     

Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.     

Microwave assisted synthesis and anti-influenza virus activity of 1-adamantyl substituted N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide derivatives

A microwave-assisted three-component one-pot cyclocondensation method was applied for the synthesis of novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide compounds carrying an adamantyl moiety. The structures of the compounds were confirmed by spectral and elemental analysis. All compounds were evaluated for antiviral activity against influenza A (H1N1 and H3N2) and influenza B virus in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. The N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)adamantane-1-carboxamide 3b was the most potent inhibitor [antiviral EC₅₀: 1.4 μM against influenza A/H3N2 virus]. Its strong inhibitory effect in a virus hemolysis assay supports that 3b acts as an influenza virus fusion inhibitor by preventing the conformational change of the influenza virus hemagglutinin at low pH.     

Discovery,synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.     

Synthesis and evaluation of new 1-oxa-8-azaspiro[4.5]decane derivatives as candidate radioligands for sigma-1 receptors

We report the design, synthesis, and evaluation of a series of 1-oxa-8-azaspiro[4.5]decane and 1,5-dioxa-9-azaspiro[5.5]undecane derivatives as selective σ₁ receptor ligands. All seven ligands exhibited nanomolar affinity for σ₁ receptors (K_i(σ₁) = 0.47 – 12.1 nM) and moderate selectivity over σ₂ receptors (K_i(σ₂)/ K_i(σ₁) = 2 – 44). Compound 8, with the best selectivity among these ligands, was selected for radiolabeling and further evaluation. Radioligand [¹⁸F]8 was prepared via nucleophilic ¹⁸F-substitution of the corresponding tosylate precursor, with an overall isolated radiochemical yield of 12–35%, a radiochemical purity of greater than 99%, and molar activity of 94 – 121 GBq/μmol. Biodistribution studies of [¹⁸F]8 in mice demonstrated high initial brain uptake at 2 min. Pretreatment with SA4503 resulted in significantly reduced brain-to-blood ratio (70% − 75% at 30 min). Ex vivo autoradiography in ICR mice demonstrated high accumulation of the radiotracer in σ₁ receptor-rich brain areas. These findings suggest that [¹⁸F]8 could be a lead compound for further structural modifications to develop potential brain imaging agents for σ₁ receptors.     

Novel FFA1 (GPR40) agonists containing spirocyclic periphery: polar azine periphery as a driver of potency

A series of nine compounds based on 3-[4-(benzyloxy)phenyl]propanoic acid core containing a 1-oxa-9-azaspiro[5.5]undecane periphery was designed, synthesized and evaluated as free fatty acid 1 (FFA1 or GPR40) agonists. The spirocyclic appendages included in these compounds were inspired by LY2881835, Eli Lilly’s advanced drug candidate for type II diabetes mellitus that was in phase I clinical trials. These polar spirocyclic, fully saturated appendages (that are themselves uncharacteristic of the known FFA1 ligand space) were further decorated with diverse polar groups (such as basic heterocycles or secondary amides). To our surprise, while seven of nine compounds were found to be inactive (likely due to the decrease in lipophilicity, which is known to be detrimental to FFA1 ligand affinity), two compounds containing 2-pyridyloxy and 2-pyrimidinyloxy groups were found to have EC₅₀ of 1.621 and 0.904?µM, respectively. This result is significant in the context of the worldwide quest for more polar FFA1 agonists, which would be devoid of liver toxicity effects earlier observed for a FFA1 agonist fasiglifam (TAk-875) in clinical studies.     

Heteroarylureas with spirocyclic diamine cores as inhibitors of fatty acid amide hydrolase

A series of mechanism based heteroaryl urea fatty acid amide hydrolase (FAAH) inhibitors with spirocyclic diamine cores is described. A potent member of this class, (37), was found to inhibit FAAH centrally, elevate the brain levels of three fatty acid ethanolamides [FAAs: anandamide (AEA), oleoyl ethanolamide (OEA) and palmitoyl ethanolamide (PEA)], and was moderately efficacious in a rat model of neuropathic pain.     

Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.     

Identification of potent, noncovalent fatty acid amide hydrolase (FAAH) inhibitors

Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel, noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors. The mechanism of action for these compounds was determined through a combination of SAR and co-crystallography with rat FAAH.     

Design and synthesis of a novel series of (1′S,2R,4′S)-3H-4′-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2′-bicyclo[2.2.2]octanes] with high affinity for the α7 neuronal nicotinic receptor

We describe an efficient and convergent synthesis of a series of (1′S,2R,4′S)-3H-4′-azaspiro[benzo[4,5]imidazo[2,1-b]oxazole-2,2′-bicyclo[2.2.2]octanes] displaying potency for the α7 nicotinic acetylcholine receptor (nAChR) and good selectivity vs. the related 5-HT_3A receptor.     

Design,synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists

The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described. In this series, optimization of the right piperidine N-capping group (R²) and the left aryl group (R³) led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic rats.     

Synthesis and biological evaluation of nonpeptide integrin antagonists containing spirocyclic scaffolds

Analogues of isoxazoline alpha(v)beta(3) antagonist 1 designed to further restrict the four carbon alkyl tether were prepared by incorporating two spirocyclic scaffolds, 1-oxa-2-azaspiro[4,5]dec-2-ene and 1-oxa-2,7-diazaspiro[4,4]non-2-ene. Additional optimization provided potent antagonists of both alpha(v)beta(3) and alpha(5)beta(1) which are selective over GPIIb/IIIa.     

Sulphonamide 1,4-dithia-7-azaspiro[4,4]nonane derivatives as gelatinase A inhibitors

Gelatinase A, a zinc-containing endopeptidase, has been shown to be an essential therapeutic target for tumor intervention owing to its participation in almost all types of solid tumors. Based on our previous work with respect to quinoxalinone peptidomimetics, a novel series of sulphonamide-containing 1,4-dithia-7-azaspiro[4,4]nonane (DAN) derivatives have been synthesized and evaluated as potential gelatinase A inhibitors hereby. The results revealed that the majority of tested compounds displayed satisfactory inhibition activity against gelatinase A. Among the tested compounds, 2b, 3a, 4a–d, 6a, 6d, 7a–d exhibited more potent gelatinase A inhibition than the positive control LY52. Furthermore, two test compounds 2b and 6a demonstrated moderate anti-proliferative in vitro and anti-metastatic activities in vivo, which might be utilized as potential leads in future chemical optimization.     

Structure-activity relationship of [Nphe1]-NC-(1-13)-NH2, a pure and selective nociceptin/orphanin FQ receptor antagonist.

A series of analogs of the ORL1 receptor antagonist [Nphe1]-NC(1-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe1]-NC(1-13)-NH2 is high (pKB 6.43) compared with those of all other compounds except [(S)(betaMe)Nphe1]NC(1-13)-NH2 (pK(B) 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK(B)) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.     

Novel GlyT1 inhibitor chemotypes by scaffold hopping. Part 2: Development of a [3.3.0]-based series and other piperidine bioisosteres

This Letter describes the development and SAR of a novel series of GlyT1 inhibitors derived from a scaffold hopping approach, in lieu of an HTS campaign, which provided intellectual property position. Members within this new [3.3.0]-based series displayed excellent GlyT1 potency, selectivity, free fraction, and modest CNS penetration. Moreover, enantioselective GlyT1 inhibition was observed, within this novel series and a number of other piperidine bioisosteric cores.     

1-Indol-1-yl-propan-2-ones and related heterocyclic compounds as dual inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgetic and anti-inflammatory drugs. We recently reported that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (10) and related compounds are inhibitors of cPLA₂α. Since cPLA₂α and FAAH possess several common structural features, we now screened this substance series together with some new derivatives for FAAH inhibition. Some of the assayed compounds proved to be selective cPLA₂α inhibitors, while others showed high FAAH and moderate cPLA₂α inhibitory potency. Furthermore, several derivatives were favorably active against both enzymes and, therefore, could represent agents, which have improved analgetic and anti-inflammatory qualities in comparison with selective cPLA₂α and FAAH inhibitors.     

N-[1-(2-Phenylethyl)pyrrolidin-3-yl]-1-adamantanecarboxamides as novel 5-HT2 receptor antagonists

A series of 1-adamantanecarboxamides was synthesized and examined for their potency as a selective 5-HT2 receptor antagonist. We found (S)-N-[1-[2-(4-fluorophenyl)ethyl]pyrrolidin-3-yl]-1-adamantane carboxamide hydrochloride hydrate (10-(S), Y-39241) to have a high affinity and selectivity for 5-HT2 receptors, and this potent anti-platelet effect of Y-39241 was confirmed both in vitro and in vivo.     

N-(3,4-Dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide: A promising positron emission tomography ligand for fatty acid amide hydrolase

To visualize fatty acid amide hydrolase (FAAH) in brain in vivo, we developed a novel positron emission tomography (PET) ligand N-(3,4-dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[¹¹C]methylphenyl)thiazol-2-yl]-1-carboxamide ([¹¹C]DFMC, [¹¹C]1). DFMC (1) was shown to have high binding affinity (IC₅₀: 6.1 nM) for FAAH. [¹¹C]1 was synthesized by C–¹¹C coupling reaction of arylboronic ester 2 with [¹¹C]methyl iodide in the presence of Pd catalyst. At the end of synthesis, [¹¹C]1 was obtained with a radiochemical yield of 20 ± 10% (based on [¹¹C]CO₂, decay-corrected, n = 5) and specific activity of 48–166 GBq/μmol. After the injection of [¹¹C]1 in mice, high uptake of radioactivity (>2% ID/g) was distributed in the lung, liver, kidney, and brain, organs with high FAAH expression. PET images of rat brains for [¹¹C]1 revealed high uptakes in the cerebellar nucleus (SUV = 2.4) and frontal cortex (SUV = 2.0), two known brain regions with high FAAH expression. Pretreatment with the FAAH-selective inhibitor URB597 reduced the brain uptake. Higher than 90% of the total radioactivity in the rat brain was irreversible at 30 min after the radioligand injection. The present results indicate that [¹¹C]1 is a promising PET ligand for imaging of FAAH in living brain.     

Spirocyclic nonpeptide glycoprotein IIb-IIIa antagonists. Part 2: design of potent antagonists containing the 3-azaspiro[5.5]undecanes

The synthesis and biological activity of novel glycoprotein IIb-IlIa anatagonists containing 3-azaspiro[5.5]undec-9-yl nucleus are described. The potent activity of these compounds as platelet aggregation inhibitors demonstrates the utility of the monoazaspirocyclic structure as central template for nonpeptide RGD mimics.