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The quest for novel agents to regulate the generation of prostaglandin E2 (PGE2) is of high importance because this eicosanoid is a key player in inflammatory diseases. We synthesized a series of N-acylated and N-alkylated 2-aminobenzothiazoles and related heterocycles (benzoxazoles and benzimidazoles) and evaluated their ability to suppress the cytokine-stimulated generation of PGE2 in rat mesangial cells. 2-Aminobenzothiazoles, either acylated by the 3-(naphthalen-2-yl)propanoyl moiety (GK510) or N-alkylated by a chain carrying a naphthalene (GK543) or a phenyl moiety (GK562) at a distance of three carbon atoms, stand out in inhibiting PGE2 generation, with EC50 values ranging from 118 nM to 177 nM. Both GK510 and GK543 exhibit in vivo anti-inflammatory activity greater than that of indomethacin. Thus, N-acylated or N-alkylated 2-aminobenzothiazoles are novel leads for the regulation of PGE2 formation.

2-Oxoesters constitute an important class of potent and selective inhibitors of human cytosolic phospholipase A2 (GIVA cPLA2) combining an aromatic scaffold or a long aliphatic chain with a short aliphatic chain containing a free carboxylic acid. Although highly potent 2-oxoester inhibitors of GIVA cPLA2 have been developed, their rapid degradation in human plasma limits their pharmaceutical utility. In an effort to address this problem, we designed and synthesized two new 2-oxoesters introducing a methyl group either on the α-carbon to the oxoester functionality or on the carbon carrying the ester oxygen. We studied the in vitro plasma stability of both derivatives and their in vitro inhibitory activity on GIVA cPLA2. Both derivatives exhibited higher plasma stability in comparison with the unsubstituted compound and both derivatives inhibited GIVA cPLA2, however to different degrees. The 2-oxoester containing a methyl group on the α-carbon atom to the oxoester functionality exhibits enhancement of the metabolic stability and retains considerable inhibitory potency.

[This corrects the article DOI: 10.1021/acsomega.8b01214.].

Ca2+-independent phospholipase A2 (GVIA iPLA2) has gained increasing interest recently as it has been recognized as a participant in biological processes underlying diabetes development and autoimmune-based neurological disorders. The development of potent GVIA iPLA2 inhibitors is of great importance because only a few have been reported so far. We present a novel class of GVIA iPLA2 inhibitors based on the β-lactone ring. This functionality in combination with a four-carbon chain carrying a phenyl group at position-3 and a linear propyl group at position-4 of the lactone ring confers excellent potency. trans-3-(4-Phenylbutyl)-4-propyloxetan-2-one (GK563) was identified as being the most potent GVIA iPLA2 inhibitor ever reported ( XI(50) 0.0000021, IC50 1 nM) and also one that is 22 000 times more active against GVIA iPLA2 than GIVA cPLA2. It was found to reduce β-cell apoptosis induced by proinflammatory cytokines, raising the possibility that it can be beneficial in countering autoimmune diseases, such as type 1 diabetes.

In recent years, halogen‐bonded complexes (XBCs), in solution, have played a pivotal role in inducing photochemical organic reactions. In this work, we explore the ability of various tertiary amines to act as XB acceptors in the presence of the XB donor CBr4 by computational and spectroscopic studies. DFT studies clearly showcase the formation of XBCs between the studied tertiary amines and CBr4. Simultaneously, computational and experimental UV‐Vis studies display intense red shifts that are consistent with charge transfer observed from tertiary amines to CBr4. A detailed NMR study revealed a clear chemical shift of the carbon carrying the bromine atoms upon mixing the XB acceptor with the donor, suggesting that this spectroscopic technique is indeed an experimental tool to identify the generation of XBCs. An application of the ability of such XBCs to activate a carboxylic acid under UVA irradiation or sunlight is presented for amino acid coupling. Among the various tertiary amines studied, the pair DABCO‐CBr4 was found to work well for the photochemical amide bond formation. Direct infusion‐HRMS studies allowed us to propose a general mechanism for the photochemical amino acid coupling in the presence of a tertiary amine and CBr4, initiated by the photoactivation of an XBC. [ABSTRACT FROM AUTHOR]

Cytosolic phospholipase A2 (GIVA cPLA2) has attracted great interest as a medicinal target because it initiates the eicosanoid cascade and is involved in a number of inflammatory diseases. As a consequence, the development of potent synthetic inhibitors is of great importance. We have developed highly potent 2-oxoester inhibitors of GIVA cPLA2 presenting XI(50) values between 0.000019 and 0.000066. We demonstrate that the 2-oxoester functionality is essential for in vitro inhibitory activity, making these inhibitors useful research reagents. However, their high reactivity results in rapid degradation of the inhibitors in human plasma, limiting their pharmaceutical utility without further modification.

Cytosolic phospholipase A2 (GIVA cPLA2) is the only PLA2 that exhibits a marked preference for hydrolysis of arachidonic acid containing phospholipid substrates releasing free arachidonic acid and lysophospholipids and giving rise to the generation of diverse lipid mediators involved in inflammatory conditions. Thus, the development of potent and selective GIVA cPLA2 inhibitors is of great importance. We have developed a novel class of such inhibitors based on the 2-oxoester functionality. This functionality in combination with a long aliphatic chain or a chain carrying an appropriate aromatic system, such as the biphenyl system, and a free carboxyl group leads to highly potent and selective GIVA cPLA2 inhibitors (X I(50) values 0.00007-0.00008) and docking studies aid in understanding this selectivity. A methyl 2-oxoester, with a short chain carrying a naphthalene ring, was found to preferentially inhibit the other major intracellular PLA2, the calcium-independent PLA2. In RAW264.7 macrophages, treatment with the most potent 2-oxoester GIVA cPLA2 inhibitor resulted in over 50% decrease in KLA-elicited prostaglandin D2 production. The novel, highly potent and selective GIVA cPLA2 inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of inflammatory diseases.

Calcium-independent phospholipase A2 (GVIA iPLA2) has recently attracted interest as a medicinal target. The number of known GVIA iPLA2 inhibitors is limited to a handful of synthetic compounds (bromoenol lactone and polyfluoroketones). To expand the chemical diversity, a variety of 2-oxoamides based on dipeptides and ether dipeptides were synthesized and studied for their in vitro inhibitory activity on human GVIA iPLA2 and their selectivity over the other major intracellular GIVA cPLA2 and the secreted GV sPLA2. Structure-activity relationship studies revealed the first 2-oxoamide derivative (GK317), which presents potent inhibition of GVIA iPLA2 (XI(50) value of 0.007) and at the same time significant selectivity over GIVA cPLA2 and GV sPLA2.

Cytosolic GIVA phospholipase A2 (GIVA cPLA2) initiates the eicosanoid pathway of inflammation and thus inhibitors of this enzyme constitute novel potential agents for the treatment of inflammatory diseases. Traditionally, GIVA cPLA2 inhibitors have suffered systemically from high lipophilicity. We have developed a variety of long chain 2-oxoamides as inhibitors of GIVA PLA2. Among them, AX048 was found to produce a potent analgesic effect. We have now reduced the lipophilicity of AX048 by replacing the long aliphatic chain with a chain containing an ether linked aromatic ring with in vitro inhibitory activities similar to AX048.

The development of inhibitors for phospholipase A2 (PLA2) is important in elucidating the enzymes implication in various biological pathways. PLA2 enzymes are an important pharmacological target implicated in various inflammatory diseases. Computational chemistry, organic synthesis, and in vitro assays were employed to develop potent and selective inhibitors for group VIA calcium-independent PLA2. A set of fluoroketone inhibitors was studied for their binding mode with two human cytosolic PLA2 enzymes: group IVA cPLA2 and group VIA iPLA2. New compounds were synthesized and assayed toward three major PLA2s. This study led to the development of four potent and selective thioether fluoroketone inhibitors as well as a thioether keto-1,2,4-oxadiazole inhibitor for GVIA iPLA2, which will serve as lead compounds for future development and studies. The keto-1,2,4-oxadiazole functionality with a thioether is a novel structure, and it will be used as a lead to develop inhibitors with higher potency and selectivity toward GVIA iPLA2.

Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is the rate-limiting provider of pro-inflammatory mediators in many tissues and is thus an attractive target for the development of novel anti-inflammatory agents. In this work, we present the synthesis of new thiazolyl ketones and the study of their activities in vitro, in cells, and in vivo. Within this series of compounds, methyl 2-(2-(4-octylphenoxy)acetyl)thiazole-4-carboxylate (GK470) was found to be the most potent inhibitor of GIVA cPLA2, exhibiting an XI(50) value of 0.011 mole fraction in a mixed micelle assay and an IC50 of 300 nM in a vesicle assay. In a cellular assay using SW982 fibroblast-like synoviocytes, it suppressed the release of arachidonic acid with an IC50 value of 0.6 μM. In a prophylactic collagen-induced arthritis model, it exhibited an anti-inflammatory effect comparable to the reference drug methotrexate, whereas in a therapeutic model, it showed results comparable to those of the reference drug Enbrel. In both models, it significantly reduced plasma PGE2 levels.

Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various neurological disorders. In the current work, we explore the significance of the introduction of a substituent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhibition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2.

The group IVA cytosolic phospholipase A(2) (GIVA cPLA(2)) plays a central role in inflammation. Long chain 2-oxoamides constitute a class of potent GIVA cPLA(2) inhibitors that exhibit potent in vivo anti-inflammatory and analgesic activity. We have now gained insight into the binding of 2-oxoamide inhibitors in the GIVA cPLA(2) active site through a combination of molecular docking calculations and molecular dynamics simulations. Recently, the location of the 2-oxoamide inhibitor AX007 within the active site of the GIVA cPLA(2) was determined using a combination of deuterium exchange mass spectrometry followed by molecular dynamics simulations. After the optimization of the AX007-GIVA cPLA(2) complex using the docking algorithm Surflex-Dock, a series of additional 2-oxoamide inhibitors have been docked in the enzyme active site. The calculated binding affinity presents a good statistical correlation with the experimental inhibitory activity (r(2) = 0.76, N = 11). A molecular dynamics simulation of the docking complex of the most active compound has revealed persistent interactions of the inhibitor with the enzyme active site and proves the stability of the docking complex and the validity of the binding suggested by the docking calculations. The combination of molecular docking calculations and molecular dynamics simulations is useful in defining the binding of small-molecule inhibitors and provides a valuable tool for the design of new compounds with improved inhibitory activity against GIVA cPLA(2).

Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A(2) (PLA(2)) superfamily plays important roles in SCI. PLA(2) enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA(2) group IVA (cPLA(2) GIVA) and calcium-independent PLA(2) group VIA (iPLA(2) GVIA)], and a secreted form [secreted PLA(2) group IIA (sPLA(2) GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA(2)s play differing roles. cPLA(2) GIVA mediates protection, whereas sPLA(2) GIIA and, to a lesser extent, iPLA(2) GVIA are detrimental. Furthermore, completely blocking all three PLA(2)s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA(2) and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA(2) (sPLA(2) and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.

Phospholipases represent one of the earliest enzyme activities to be identified and studied, and the phospholipase A2 superfamily traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. Both electrostatic and hydrophobic interactions contribute to the interfacial binding of sPLA2 to anionic phospholipid membranes. The interaction between basic residues on the binding surface with anionic vesicles plays an important role in interfacial binding. The major functions will be summarized below and include the ability to kill Gram-positive and Gram-negative bacteria, thereby affecting host defense against bacterial infections. sPLA2 may be involved in the pathogensis of inflammatory bowel disease including Crohn's disease and ulcerative colitis. GIIA sPLA2 protein and mRNA were detected in Paneth cells of the small intestinal mucosa in the intestine in Crohn's disease patients.

Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective.

A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A(2) (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase A(2) (GV sPLA(2)) were evaluated. Derivatives containing a free carboxyl group are selective GIVA cPLA(2) inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-oxoamide, which preferentially inhibits GVIA iPLA(2). The effect of 2-oxoamides on the generation of arachidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA(2) inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC(50) value of 2muM.

An analysis of group IVA (GIVA) phospholipase A(2) (PLA(2)) inhibitor binding was conducted using a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models of the GIVA PLA(2) inhibitors pyrrophenone and the 2-oxoamide AX007 docked into the protein were designed on the basis of deuterium exchange results, and extensive molecular dynamics simulations were run to determine protein-inhibitor contacts. The models show that both inhibitors interact with key residues that also exhibit changes in deuterium exchange upon inhibitor binding. Pyrrophenone is bound to the protein through numerous hydrophobic residues located distal from the active site, while the oxoamide is bound mainly through contacts near the active site. We also show differences in protein dynamics around the active site between the two inhibitor-bound complexes. This combination of computational and experimental methods is useful in defining more accurate inhibitor binding sites and can be used in the generation of better inhibitors against GIVA PLA(2).

The phospholipase A(2) (PLA(2)) superfamily hydrolyzes phospholipids to release free fatty acids and lysophospholipids, some of which can mediate inflammation and demyelination, hallmarks of the CNS autoimmune disease multiple sclerosis. The expression of two of the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) and two of the secreted PLA(2)s (sPLA(2) GIIA and sPLA(2) GV) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We show using small molecule inhibitors, that cPLA(2) GIVA plays a role in the onset, and iPLA(2) GVIA in the onset and progression of EAE. We also show a potential role for sPLA(2) in the later remission phase. These studies demonstrate that selective inhibition of iPLA(2) can ameliorate disease progression when treatment is started before or after the onset of symptoms. The effects of these inhibitors on lesion burden, chemokine and cytokine expression as well as on the lipid profile provide insights into their potential modes of action. iPLA(2) is also expressed by macrophages and other immune cells in multiple sclerosis lesions. Our results therefore suggest that iPLA(2) might be an excellent target to block for the treatment of CNS autoimmune diseases, such as multiple sclerosis.

The development of selective inhibitors for individual PLA(2) enzymes is necessary in order to target PLA(2)-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA(2) inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA(2), a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the alpha' position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA(2) inhibitors.

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase (GV sPLA(2)) was evaluated. We show that an amide based on (R)-gamma-norleucine is a highly selective inhibitor of GV sPLA(2).

We provide evidence that two members of the intracellular phospholipase A(2) family, namely calcium-dependent group IVA (cPLA(2) GIVA) and calcium-independent group VIA (iPLA(2) GVIA) may play important roles in Wallerian degeneration in the mouse sciatic nerve. We assessed the roles of these PLA(2)s in cPLA(2) GIVA(-/-) mice, and mice treated with a selective inhibitor of iPLA(2) GVIA (FKGK11). Additionally, the effects of both these PLA(2)s were assessed by treating cPLA(2) GIVA(-/-) mice with the iPLA(2) inhibitor. Our data suggest that iPLA(2) GVIA may play more of a role in the early stages of myelin breakdown, while cPLA(2) GIVA may play a greater role in myelin clearance by macrophages. Our results also show that the delayed myelin clearance and Wallerian degeneration after sciatic nerve crush injury in mice lacking cPLA(2) and iPLA(2) activities is accompanied by a delay in axon regeneration, target re-innervation and functional recovery. These results indicate that the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) contribute significantly to various aspects of Wallerian degeneration in injured peripheral nerves, which is then essential for successful axon regeneration. This work has implications for injury responses and recovery after peripheral nerve injuries in humans, as well as for understanding the slow clearance of myelin after CNS injury and its potential consequences for axon regeneration.

A variety of lipophilic 2-oxoamides containing sulfonamide analogs of gamma-amino acids as well as acyl sulfonamides of gamma-aminobutyric acid were synthesized. Their ability to inhibit intracellular GIVA cPLA2 and GVIA iPLA2 as well as secreted GV sPLA2 was evaluated. The sulfonamide group seems a bioisosteric group suitable to replace the carboxyl group in 2-oxoamide inhibitors of GVIA cPLA2.

A variety of lipophilic 2-oxoamides containing sulfonamide analogs of gamma-amino acids as well as acyl sulfonamides of gamma-aminobutyric acid were synthesized. Their ability to inhibit intracellular GIVA cPLA2 and GVIA iPLA2 as well as secreted GV sPLA2 was evaluated. The sulfonamide group seems a bioisosteric group suitable to replace the carboxyl group in 2-oxoamide inhibitors of GVIA cPLA2.

A variety of lipophilic 2-oxoamides based on gamma-aminobutyric and delta-aminovaleric analogues were synthesized. 2-oxoamides containing a tetrazole, a thioethyl or a thioacetyl group are weak inhibitors of GIVA cPLA(2), while derivatives containing a methyl tetrazole, a diethyl phosphonate or a thioethyl group are weak inhibitors of GV sPLA(2).

A variety of lipophilic 2-oxoamides based on gamma-aminobutyric and delta-aminovaleric analogues were synthesized. 2-oxoamides containing a tetrazole, a thioethyl or a thioacetyl group are weak inhibitors of GIVA cPLA(2), while derivatives containing a methyl tetrazole, a diethyl phosphonate or a thioethyl group are weak inhibitors of GV sPLA(2).

Inhibitors of the Group IVA phospholipase A(2) (GIVA cPLA(2)) and GVIA iPLA(2) are useful tools for defining the roles of these enzymes in cellular signaling and inflammation. We have developed inhibitors of GVIA iPLA(2) building upon the 2-oxoamide backbone that are uncharged, containing ester groups. Although the most potent inhibitors of GVIA iPLA(2) also inhibited GIVA cPLA(2), there were three 2-oxoamide compounds that selectively and weakly inhibited GVIA iPLA(2). We further show that several potent 2-oxoamide inhibitors of GIVA cPLA(2) containing free carboxylic groups (Kokotos et al. J. Med. Chem. 2002, 45, 2891-2893) do not inhibit GVIA iPLA(2) and are, therefore, selective GIVA cPLA(2) inhibitors.

The direct amide bond formation between a carboxylic acid and an amine still constitutes a challenging reaction for both academia and industry. We demonstrate herein that several pairs of amines (halogen bond acceptors) and organohalogen sources may be used for the photochemical amidation reaction under either UVA or sunlight irradiation. Our studies led to the identification of pyridine‐CBr4 as an efficient agent to perform amide synthesis under LED 370 nm irradiation, avoiding super‐stoichiometric quantities. An extended substrate scope was demonstrated, showing that the widely used amino and carboxyl protecting groups are compatible with this photochemical protocol, while a number of industrially interesting products and bioactive compounds were synthesized. Direct infusion‐high resolution mass spectrometry studies suggest an unprecedented type of carboxylic acid activation mode upon irradiation, involving the generation of a symmetric anhydride, an active ester with pyridine N‐oxide and a mixed anhydride with hypobromous acid. [ABSTRACT FROM AUTHOR]