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The concept of target-specific covalent enzyme inhibitors appears attractive from both an efficacy and a selectivity viewpoint considering the potential for enhanced biochemical efficiency associated with an irreversible mechanism. Aside from potential safety concerns, clearance prediction of covalent inhibitors represents a unique challenge due to the inclusion of nontraditional metabolic pathways of direct conjugation with glutathione (GSH) or via GSH S-transferase-mediated processes. In this article, a novel pharmacokinetic algorithm was developed using a series of Pfizer kinase selective acrylamide covalent inhibitors based on their in vitro-in vivo extrapolation of systemic clearance in rats. The algorithm encompasses the use of hepatocytes as an in vitro model for hepatic clearance due to oxidative metabolism and GSH conjugation, and the use of whole blood as an in vitro surrogate for GSH conjugation in extrahepatic tissues. Initial evaluations with clinical covalent inhibitors suggested that the scaling algorithm developed from rats may also be useful for human clearance prediction when species-specific parameters, such as hepatocyte and blood stability and blood binding, were considered. With careful consideration of clearance mechanisms, the described in vitro-in vivo extrapolation approach may be useful to facilitate candidate optimization, selection, and prediction of human pharmacokinetic clearance during the discovery and development of targeted covalent inhibitors.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.

Spleen tyrosine kinase is a non-receptor tyrosine kinase, overactivation of which is thought to contribute to autoimmune diseases as well as allergy and asthma. Protein kinases have a highly conserved ATP binding site, thus making challenging the design of selective small molecule inhibitors. It has been well documented that some protein kinases can be stabilized in their inactive conformations (Type-II inhibitors). Herein, we describe a protein structure/ligand-based approach to successfully identify ligands that bind to novel conformations of spleen tyrosine kinase. By utilizing kinase protein crystal structures both in the public domain (RCSB) and within Pfizer's protein crystal database, we report the discovery of the first spleen tyrosine kinase Type-II ligands. Compounds 1 and 3 were found to bind to the DFG-out conformation of spleen tyrosine kinase, while compound 2 binds to a DFG-in, C-Helix-out conformation. In this instance, the C-helix moved significantly to create a large hydrophobic pocket rarely seen in kinase protein crystal structures.

Hematopoietic prostaglandin D synthase (HPGDS) is primarly expressed in mast cells, antigen-presenting cells, and Th-2 cells. HPGDS converts PGH2 into PGD2, a mediator thought to play a pivotal role in airway allergy and inflammatory processes. In this letter, we report the discovery of an orally potent and selective inhibitor of HPGDS that reduces the antigen-induced response in allergic sheep.

The immobilized 1,3-regiospecific Rhizomucor miehei lipase (Lipozyme™) was employed to catalyze the transesterification reaction (acidolysis) of 1,2-diacyl-sn-glycero-3-phosphatidylcholine with n-3 polyunsaturated fatty acids under nonaqueous solvent-free conditions. With a concentrate of 55% eicosapentaenoic acid (EPA) and 30% docosahexaenoic acid (DHA) and pure phosphatidylcholine from egg yolk, phospholipids of 32% EPA and 16% DHA content were obtained, presumably as a mixture of phosphatidylcholine and lysophosphatidylcholine. 31P nuclear magnetic resonance (NMR) analysis turned out to be a valuable technique to study the details of the reactions involved. It revealed that when 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine was transesterified with 98% pure EPA, a substantial amount of hydrolysis side reaction took place (39%), leading to a product mixture of 39% phosphatidylcholine, 44% lysophosphatidylcholine, and 17% sn-glycerol-3-phosphatidylcholine. The lysophosphatidylcholine constituent comprised 70% EPA, whereas the phosphatidylcholine component contained 58% EPA. The 31P NMR technique provided valid information about the mechanism of the reaction. It became evident that a high dosage of lipase containing 5% water afforded optimal conditions for the optimal extent of EPA incorporation into the phospholipids, under which the extent of hydrolysis side reaction remained relatively high.

The structure of the title compound, C31H39NO7Si, was determined and found to be a fused tricyclic nitroso acetal. Remarkable features include a twist-boat conformation of the tetrahydro-1,2-oxazine ring and a highly pyramidalized N atom [Sigma (angles) = 310.6(6) degrees]. Three of the contiguous stereocenters in the nitroso acetal are of the same correct relative and absolute configuration as is found in (+)-crotanecine.

A short and efficient asymmetric synthesis of the pyrrolizidine necine base (-)-hastanecine is described. The key reaction in the synthesis is a sequential inter [4+2]/inter [3+2] cycloaddition. The Lewis acid promoted [4+2] cycloaddition between 2-acyloxy nitroalkene 16 and chiral vinyl ether 17 afforded a nitronate which underwent facile [3+2] cycloaddition with dimethyl maleate. The resulting nitroso acetal 19 had all the required stereocenters for (-)-hastanecine. The critical unmasking of the nitroso acetal 19 employed a hydrogenolytic cleavage to give the 1-azabicyclo[3.3.0]octane skeleton of (-)-hastanecine

Bbstract; Nitroalkenes have proven to be extremely versatile 4x-components in heterodiene [4+2]-cycloadditions. Our efforts in this area have been intensively methodological with focus on the reaction scope, mechanism, stereoselectivity and transformations of the cycloadducts. The three modes of tandem inter [4+2]/ intra [3+2] cycloaddition, fused, spiro and bridged have been documented. An efficient and highly selective synthesis of (-)-hastanwine has been completed.

The asymmetric variant of the tandem [4 + 2]/[3 + 2] cycloaddition of nitroalkenes with chiral vinyl ethers has been extensively explored in recent years.1 To date, the chiral vinyl ethers that have been utilized in the tandem sequence are derived from bornane-2,3-diol, 1, trans-2phenylcyclohexanol, 2, and 2,2-diphenylcyclopentanol, 3, Figure 1. These auxiliaries have served admirably in many aspects of the cycloaddition process, but some limitations still exist, such as the following: (1) length of synthesis, (2) low selectivity in exo mode cycloadditions or (3) unavailability in both enantiomeric series. In the course of a number of total synthesis projects that employ the tandem sequence, we were forced to consider alternatives that addressed some of the limitations noted above.2 In particular, material through-put became an important issue since the critical unmasking of the nitroso acetals to R-hydroxy lactams involves up to a 50%mass loss due to the size of the chiral auxiliaries employed. Thus, it is of critical importance that the auxiliaries be easily prepared and inexpensive. Comins has recently disclosed the use of an easily prepared auxiliary, trans-2-cumylcyclohexanol, 4 (TCC), for the diastereoselective addition of Grignard reagents to alkoxypyridines.3 The preparation of TCC by Comins is considerably more simple than that previously reported by Whitesell, making this auxiliary an interesting alternative for use in the nitroalkene cycloaddition chemistry.4 By analogy with the preparation of the vinyl ethers derived from phenylcyclohexanol, 2, and 2,2-diphenylcyclopentanol, 3, the transetherification of 4 with a vinyl ether was explored.5 TCC is a very sterically crowded auxiliary, since the 1-methyl-1-phenylethane presents a larger steric bulk than the phenyl group in 2-phenylcyclohexanol. Therefore, a higher boiling vinyl ether was required for the trans-etherification. n-Butyl vinyl ether was found to be the vinyl ether of choice as was also the case for 2,2-diphenylcyclopentanol.6 Optimized reaction conditions employed two portions of 0.6 equiv of mercury(II) acetate in a dilute solution of n-butyl vinyl ether as solvent, Scheme 1. The vinyl ether, (-)-5, was isolated in 62% yield along with 33% of recovered alcohol, (-)-4. To evaluate the utility of this new auxiliary in the tandem [4 + 2]/[3 + 2] cycloaddition, the nitroalkene 6 was selected as the model substrate since it had been used previously with all the other chiral vinyl ethers.6 Furthermore, it was imperative to deduce the sense of asymmetric induction exercised by this new auxiliary. We, therefore, needed to establish to which enantiomeric series the final R-hydroxy lactam belonged. Accordingly, slow addition of the nitroalkene 6 to a cold solution of methylaluminum bis(2,6-diphenylphenoxide) (MAPh)7 and vinyl ether (+)-5 afforded the nitroso acetal 7 in 85% yield, Scheme 2. By 1H NMR analysis of HC(6) the product composition was estimated to be a 145/1.8/1.0 (exo/exo/endo) mixture of diastereomeric nitroso acetals. The major diastereomer was assumed to have a trans relationship between HC(4a) and HC(6) on the basis of the documented preference of MAPh to promote exo-mode [4 + 2] cycloadditions.8 The nitroso acetal 7 was then unmasked under the standard hydrogenolysis conditions (Raney nickel/1 atm H2/rt/MeOH). The R-hydroxy lactam, (-)-8, isolated in 76% yield (along with an (1) Denmark, S. E.; Thorarensen, A. Chem. Rev. 1996, 96, 137. (2) Denmark, S. E.; Thorarensen, A.; Middleton, D. S. J. Org. Chem. 1995, 60, 3574. (3) (a) Comins, D. L.; Salvador, J. M. J. Org. Chem. 1993, 58, 4656. (b) Comins, D. L.; Salvador, J. M. Tetrahedron Lett. 1993, 34, 801. (c) Comins, D. L.; LaMunyon, D. H. Tetrahedron Lett. 1994, 35, 7343. (4) Whitesell, J. K.; Lawrence, R. M. Chimia 1986, 40, 318. (5) The vinyl ether of phenmenthol has been prepared earlier in a two-step procedure, by the preparation of the acetylenic ether followed by a partial reduction. Denmark, S. E.; Senanayake, C. B. W.; Ho, G. D. Tetrahedron 1990, 46, 4857. (6) (a) Denmark, S. E.; Schnute, M. E.; Senanayake, C. B. W. J. Org. Chem. 1993, 58, 1859. (b) Denmark, S. E.; Schnute, M. E.; Marcin, L. R.; Thorarensen, A. J. Org. Chem. 1995, 60, 3205. (7) (a) Maruoka, K.; Itoh, T.; Yamamoto, H. J. Am. Chem. Soc. 1985, 107, 4573. (b) Nonoshita, K.; Banno, H.; Maruoka, K.; Yamamoto, H. J. Am. Chem. Soc. 1990, 112, 316. (c) Maruoka, K.; Ooi, T.; Yamamoto, H. J. Am. Chem. Soc. 1990, 112, 9011. (d) Maruoka, K.; Itoh, T.; Shirasaka, T.; Yamamoto, H. J. Am. Chem. Soc. 1988, 110, 310. (8) Denmark, S. E.; Schnute, M. E.; Senanayake, C. B. W. J. Org. Chem. 1993, 58, 1859. Figure 1. Chiral auxiliaries employed in the tandem [4 + 2]/[3 + 2] cycloadditions of nitroalkenes.