Novel 15-Lipoxygenase-1 Inhibitor Protects Macrophages from Lipopolysaccharide-Induced Cytotoxicity.

Various mechanisms for regulated cell death include the formation of oxidative mediators such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1 (15-LOX-1) is a key enzyme that catalyzes the formation of lipid peroxides. The actions of these peroxides are interconnected with nuclear factor-κB signaling and NO production. Inhibition of 15-LOX-1 holds promise to interfere with regulated cell death in inflammatory conditions. In this study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure-activity relationships were explored. In vitro, this inhibitor protected cells from lipopolysaccharide-induced cell death, inhibiting NO formation and lipid peroxidation. Thus, we provide a novel 15-LOX-1 inhibitor that inhibits cellular NO production and lipid peroxidation, which set the stage for further exploration of these mechanisms.

Introduction

Over recent years, an increasing number of mechanisms for regulated cell death have been identified and versatile roles in numerous diseases were proposed.[1] Cell death via a mechanism other than apoptosis leads to plasma membrane rupture and release of the cellular content, thus providing damage-associated molecular patterns that can induce an autoamplification loop of regulated cell death and inflammation. Such amplification loops are expected to play key roles in diseases such as acute lung injury and acute respiratory distress syndrome.[2] Understanding the underlying mechanisms to develop small-molecule inhibitors to interfere with cell death holds promise for therapeutic control of these disorders.

The discovery of multiple types of cell death provides new challenges to identify the molecular mechanisms involved. One mechanism of nonapoptotic cell death is pyroptosis in which macrophages die by excessive stimulation of Toll-like receptors and activation of the nuclear factor-κB (NF-κB) pathway by, for example, lipopolysaccharides (LPS).[2−6] Normally, pyroptosis is a mechanism to protect multicellular organisms from invading pathogens, such as microbial infections. However, under pathogenic conditions, pyroptosis can be involved in the onset of chronic inflammation. Another mechanism for nonapoptotic cell death is ferroptosis, which is a process in which excessive levels of lipid peroxides cause cell death. It is anticipated that lipoxygenases (LOXs) play key roles in ferroptosis by catalyzing lipid peroxidation.[2,7] The identification of pyroptosis, ferroptosis, and other mechanisms for regulated cell death raises the question how these mechanisms can be exploited for drug discovery.

Although distinct mechanisms for regulated cell death were described, the mechanisms involved are often closely related and crosstalk exists. In this study, we aim to address the crosstalk between macrophage cell death upon LPS stimulation and the enzymatic activity of 15-lipoxygenase-1 (15-LOX-1) as a regulator of cellular lipid peroxidation (Figure ).[8] Activation of the NF-κB pathway results in transcription of downstream genes, such as inducible nitric oxide synthase (iNOS), that plays a critical role in inflammatory responses.[9] iNOS catalyzes the formation of NO radicals that play key roles in many physiological processes.[10] On the other hand, excessive NO production can lead to the formation of reactive nitrogen species (RNOS), which induces cell death and tissue damage.[11]

Figure 1

Several mechanisms of lipopolysaccharide (LPS) signaling in macrophages are connected to cell death. LPS-mediated activation of the NF-κB pathway results in the overexpression of inducible nitric oxide synthase (iNOS). This leads to the production of nitric oxide (NO) and reactive nitrogen species (RNOS), which are involved in cell death. In the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (13-HpODE), the metabolite of 15-LOX-1 activity, can also induce cell death. Both mechanisms act in concert, and crosstalk exists.

Reactive oxygen species (ROS) such as lipid peroxides have been shown to augment LPS-mediated NF-κB activation and thus increase expression of NF-κB target genes,[8,12] which represents a mechanism of crosstalk between lipid peroxidation and NF-κB activation. 15-LOX-1 is a nonheme iron-containing enzyme producing lipid peroxides from polyunsaturated fatty acids, such as arachidonic acid (AA) and linoleic acid (LA).[13−15] 15-LOX-1 oxidizes either AA, to form the corresponding 15-hydroxyeicosatetraenoic acid, or LA, to form the corresponding 13-hydroperoxyoctadecadienoic acid (13-HpODE).[16,17] Apart from these hydroperoxy fatty acids, lipoxins are also derived from the 15-LOXs pathway and play a role as anti-inflammatory mediators.[18] On the other hand, the 15-LOX metabolites eoxins are proposed to be a family of proinflammatory eicosanoids.[19] Altogether, lipid peroxides can be converted further into distinct lipid signaling molecules that have key regulatory roles in immune responses[20−22] and numerous diseases.[23] Importantly, if the production of lipid peroxides is not balanced by the cellular antioxidant system, this can result in ferroptotic cell death and in enhanced activation of the NF-κB pathway, thus providing synergistic crosstalk between two mechanisms of regulated cell death.[24] Thus, 15-LOX-1 is a key enzyme in oxidative stress and regulated cell death in numerous diseases.[13,25,26]

For 15-LOX-1, roles have been described in diseases such as asthma,[14] stroke,[15] atherogenesis,[2] diabetes,[16,17] cancer,[20,21] Alzheimer’s disease,[22,23] and Parkinson’s disease.[25] This triggered the interest in the development of 15-LOX-1 inhibitors for drug discovery. In an early phase, indole-based inhibitors, PD-146176, were identified as r-12/15-LOX inhibitors with a half-maximal inhibitory concentration (IC50) value of 3.81 μM (Figure ).[27] This stimulated efforts to develop inhibitors with an indolyl core (Figure ). More researchers reported the discovery of indole-based or indole-like 15-LOX-1 inhibitors, 371 and Haydi-4b (with IC50 values of 0.006 and 3.84 μM, respectively).[28,29] Our group previously discovered 15-LOX-1 inhibitor Eleftheriadis-14d, which also contains an indole core and demonstrates good potency (IC50 = 90 nM).[30] Furthermore, a 1,3-oxazole-based compound (ML351),[31] a purine-based compound (Anders-6b),[32] and pyrrole-based compound (21B10)[33] were identified as 15-LOX-1 inhibitors as well (Figure ). These inhibitors proved to be effective in various disease models, thus indicating the potential of 15-LOX-1 inhibitors for drug discovery.

Figure 2

Examples of previously reported 15-LOX-1 inhibitors and chemical tools to study lipoxygenase activity. (A) Indole-based 15-LOX-1 inhibitor and inhibitors based on other nitrogen-containing heterocycles. (B) Substrate-based chemical tools to study lipoxygenase activity in cell-based systems.

Complementary to development of inhibitors, efforts were made to engineer 15-LOX-1 substrates for detection of enzyme activity. We developed activity-based probe N144 as a chemical reporter for lipoxygenase activity in cell lysates and tissue samples.[34] Another study employed the omega-alkynyl fatty acid (aAA) to identify the intracellular targets of 12/15-LOX-generated lipid-derived electrophiles.[35] This sets the stage for the development of potent 15-LOX-1 inhibitors and to study their cellular activity.

In this study, we investigated novel substitutions of the indole core and investigated the structure–activity relationships (SARs) for 15-LOX-1 inhibition. For the most potent inhibitor, the effects on cellular 15-LOX-1 inhibition, the effects on formation of reactive oxygen species (ROS), and regulated cell death were investigated on RAW 264.7 macrophages to provide insight into the cellular potency of this type of inhibitors.

Results and Discussion

Chemistry

Scheme presents the general methodology for the synthesis of compounds 5a and 5b. The synthesis started with the assembly of ethyl 6-chloro-1H-indole-2-carboxylate (1) and the corresponding aldehyde (2) using known literature procedures.[30,36] Subsequently, the 2-formyl functionality of the aldehyde 2 was oxidized into its corresponding carboxylic acid (3) via Pinnick oxidation using sodium chlorite (NaClO2), giving a yield of 78%. Attempts to use KMnO4 or Tollens’ reagent did not provide the desired product. Finally, the amide bonds in products 5a and 5b were generated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and N-hydroxybenzotriazole (HOBt) as coupling reagents, giving yields of about 85%.

Scheme 1

Synthetic Route to Compounds 5a and 5b

Reagents and conditions: (a) POCl3, dimethylformamide (DMF), 60 °C, 48 h; (b) NaClO2, t-BuOH, 50 °C, 4 h; (c) amine (4a and 4b), EDCI, HOBt, Et3N, dichloromethane (DCM), room temperature (r.t.), 4 h.

Compounds 9a–j were synthesized using procedures as shown in Scheme . As a first step, the 2-formyl functionality of aldehyde 2 was employed for the Wittig reaction with (tert-butoxycarbonylmethylene)triphenylphosphorane to provide the α,β-unsaturated ester 6. Initially, attempts were made to obtain compound 7 by refluxing aldehyde 2 with the Wittig reagent in toluene overnight. However, this provided compound 6 as a mixture of E- and Z-isomers (E/Z = approximately 9/1).[37] Changing the solvent from toluene to ethanol at 80 °C enabled the reaction to finish in 2 h with the E-alkene as the major product that could be isolated in a yield of 70% after purification. The E- and Z-isomer could be distinguished by their J values of 16.0 and 7.0 Hz, respectively. Finally, intermediate 7 was converted into the amides 9a–j by removal of the tert-butyl protecting group using trifluoroacetic acid (TFA) treatment and subsequent coupling of the corresponding amines using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and N-hydroxybenzotriazole (HOBt) as coupling reagents in yields between 80 and 90% over two steps.

Scheme 2

Synthetic Route to Compounds 9a–j

Reagents and conditions: (d) (tert-butoxycarbonylmethylene)triphenylphosphorane, EtOH, reflux, 2 h; (e) TFA, DCM, r.t., overnight; (f) amine (8a–j), EDCI, HOBt, Et3N, DCM, r.t., 4 h.

Products 12a–g were produced starting from compound 10 as shown in Scheme . The carboxylic acid 10 was coupled to amines 11a–g using EDCI and HOBt as coupling reagents, which gives the desired products in 80–90% yield. Afterward, compound 13 was obtained from compound 12c using a Mannich reaction with dimethylamine, formaldehyde, and acetic acid to provide the desired product in a yield of 93%. As shown in Scheme , compounds 14–17 were obtained from the corresponding 2-carboxy ethyl indoles (9c, Eleftheriadis-14d and Eleftheriadis-14e) over two steps in a similar way as for 12a–g in a high yield (80–90%).

Scheme 3

Synthetic Route to Compounds 12a–g and 13

Reagents and conditions: (g) amine (11a–g), EDCI, HOBt, Et3N, DCM, r.t., 4 h; (h) dimethylamine, formaldehyde, acetic acid, MeOH, reflux, 4 h.

Scheme 4

Synthetic Route to Compounds 14–17

Reagents and conditions: (i) LiOH, THF, H2O, 50 °C, 2 h; (j) EDCI, HOBt, Et3N, DCM, r.t., 4 h.

Structure–Activity Relationships

Inhibition of 15-LOX-1 enzyme activity was performed using an activity assay as described previously by us.[30,33,38] The activity of 15-LOX-1 was monitored by measuring the conversion of LA into the UV-active 13-HpODE (λmax 234 nm). This assay was used to determine IC50 of each compound. SARs for binding to 15-LOX-1 were investigated starting from ethyl 6-chloro-1H-indole-2-carboxylate (1). We aimed to introduce structural modifications to replace the lipid chain at the 3-position and the ethylcarboxylate at the 2-position. Key structural modifications are shown in Figure starting from previously identified inhibitors 1 and Eleftheriadis-14d.[30]

Figure 3

Systematic modifications of 15-LOX-1 inhibitor 1 as a core scaffold and the previously described inhibitor Eleftheriadis-14d[30] to provide the new inhibitors 5b, 9c, 15–17 and their IC50 values for 15-LOX-1 inhibition.

The SAR of the previously identified inhibitor Eleftheriadis-14d was explored with respect to aliphatic acyl substitutions at the indole 3-position. To expand the SAR in novel directions, the carboxyl ethyl ester at the indole 3-position was replaced by an amide to gain metabolic stability. Both compounds 5a and 5b (Scheme and Table ) provided IC50 values above 1 μM, which is much higher compared to that of the previously reported series of inhibitors with the carboxyl ethyl ester.[30] Apparently, replacement of the carbonyl at the indole 3-position for an amide is unfavorable for 15-LOX-1 inhibition.

Table 1

IC50 Values for 15-LOX-1 Inhibition by Amide-Substituted Indoles at the 3-Position (Analogues 5a and 5b)

The SAR at the indole 3-position was further explored by replacement of the carbonyl at the 3-position for a double bond using Wittig chemistry. Using this chemistry, we aimed to replace the aliphatic lipid chain in Eleftheriadis-14d for less flexible substituents. Thus, we investigated a series of E-alkenes as shown in Table . Clear SARs were observed for this series of compounds. Compound 9c (i472) proved to be the most potent 15-LOX-1 inhibitor with an IC50 value of 0.19 μM. Comparison of inhibitor 9c (i472) to inhibitor 9f indicates that the ortho-methoxy substitution on benzyl provides a 10-fold gain in potency compared to that of a nonsubstituted benzyl. Importantly, the IC50 values decrease if the methoxy group is moved from the ortho to the meta or para position on benzyl 9d and 9e. Extending the benzyl to an ethylphenyl group in inhibitors 9g–j did not improve their potencies either. Taken together, the 2-methoxybenzyl group in 9c (i472) provides the most potent inhibitor in this series.

Table 2

IC50 Values against 15-LOX-1 with Different Variations in R (Analogues 9a–j)

To further explore the SAR of previously identified inhibitor Eleftheriadis-14d, variations were made at the indolyl 2-position. Toward this aim, the ester group was replaced with various amides to provide inhibitors 12a–g, as shown in Table . The results indicate that amide substitution provides inhibitors with potencies in the micromolar range. However, the SAR for modifications with methyl, ethyl, n-propyl, n-butyl, n-pentyl, and branched alkyl groups proved to be relatively flat with potency differences of no more than 2–3-fold. Remarkably, cyclopropyl substitution in 12g turns out to be inactive (IC50 > 20 μM). Taken together, the investigated series of amide-substituted indoles did not provide improved potency and the most potent inhibitor in this series is compound 12c with a propyl substitution.

Table 3

IC50 Values against 15-LOX-1 with Different Variations in the Amide Tail (Analogues 12a–g)

As a next step, we combined ethyl- or propyl-substituted amides at the indole 2-position with substitutions at the 3-position to provide inhibitors 14–17, as shown in Table . Unfortunately, the combination of both modifications provided inhibitors with low potency. Apparently, the more polar amide bond is not well tolerated for enzyme inhibition and combination of modifications at the 2- and 3-position caused a greatly reduced potency. Inhibitor 13 also showed a complete loss in potency against 15-LOX-1. Taken together, we concluded that inhibitor 9c (i472) has the highest potency of this series and that the IC50 value is in the same range as for the previously identified inhibitor Eleftheriadis-14d.

Table 4

IC50 Values against 15-LOX-1 with Different Variations in R1 and R2 (Analogues 13–18)

Docking 15-LOX-1

To understand the observed SAR key inhibitors were docked in the 15-LOX-1 active site. In this study, docking was performed using Discovery Studio (Dassault Systèmes) version 2018. Moreover, the rabbit reticulocyte 15-LOX-1 crystal structure (Protein Data Bank (PDB) ID: 1LOX) was used for docking because of its high sequence similarity in the active site.[39] In this crystal structure, the ligand in the crystal structure was removed and the center of the binding sphere was set at the same position. Based on this position, CDocker, a CHARMm-based method, was used and resulted in ten highest-ranked poses for all selected inhibitors.

Based on the observed SAR, the most potent inhibitor 9c (i472) was docked and compared to 9f (lacking the methoxy group) and 16 (in which the ester is replaced for an amide). In both cases, the potency decreased by at least 10-fold. The docking model suggested several interactions between the active site of 15-LOX-1 and 9c (i472), as shown in Figure A. Upon comparison of the docking of 9c (i472) and 9f, the 2-methoxy group on the benzyl functionality provides two hydrogen bonds with GLN 548 and ILE 593, respectively (Figure A). This may provide an explanation for the 10-fold potency difference between 9c (i472) and 9f. In addition, because of the hydrophobic character of 15-LOX-1, except from the edge of the active site, the majority of the pocket is hydrophobic, as shown in brown in Figure B,C. The hydrophilic sites are shown in blue. Docking of compound 16, in which an amide replaces the carboxy ethyl ester at the indole 2-position, shows a positionally inverted orientation compared to 9c (i472) and 9f (Figures C and S1C). Apparently, the amide with an additional hydrogen bond donor does not fit at the same position as the ester in 9c (i472). This change in orientation upon docking is also in line with the observed drop in potency for 16 compared to 9c (i472) and 9f.

Figure 4

Molecular modeling of selected compounds in the active site of 15-LOX-1 (PDB ID: 1LOX). The surface in the pocket is colored based on the relative hydrophobicity: brown for hydrophobic and blue for hydrophilic areas. (A) Interactions of compound 9c (i472) with the active site of the enzyme. (B) Preferred orientation of compound 9c (i472) in the active site of the enzyme. (C) Preferred orientation of compound 16 in the active site of the enzyme that is inverted compared to 9c (i472).

Physicochemical Properties of Inhibitor 9c (i472)

The α,β-unsaturated amide functionality in 9c (i472) is, as a Michael acceptor, reactive toward conjugate addition by nucleophiles, such as thiols. To monitor this reactivity, the UV spectrum of 9c (i472) was recorded upon incubation with 2-mercaptoethanol at pH 7.4. No changes in the UV spectrum were observed, which indicates that the chromophore, including the α,β-unsaturated system, did not change (Figure S2), thus indicating a reasonable stability of compound 9c (i472) toward nucleophilic substitution. This stability might be attributed to the conjugation of the α,β-unsaturated double bond with the aromatic indole core.

Inhibitor 9c (i472) has a calculated Log P (ChemDraw Professional version 12.0) of 4.7, which is more than 2 orders of magnitude lower compared to the previously identified inhibitor Eleftheriadis-14d (cLog P = 6.9), whereas both inhibitors have molecular weights around 400 g/mol. Considering the physicochemical properties, the newly identified inhibitor 9c (i472) has fewer rotatable bonds and a cLog P that is more favorable for cellular permeability compared to the previously identified inhibitor Eleftheridis-14d.

LOX Inhibitory Potency of 9c (i472) in Cells by Activity-Based Labeling

As a next step in the characterization of inhibitor 9c (i472), the inhibition of cellular LOX activity was investigated. Toward this aim, we employed a method for activity-based labeling of LOX activity in cell-based systems that we developed recently.[34] In this method, a covalent inhibitor of lipoxygenase activity is equipped with a terminal alkene for bioorthogonal labeling with biotin using the oxidative Heck reaction.[40] Here, we employed this method to estimate the inhibition of cellular lipoxygenase activity by inhibitor 9c (i472) in RAW 264.7 macrophages. Inhibitor-treated and nontreated cell lysates were exposed to covalent inhibitor N144 (Figure ) for 2 min, and subsequently, the samples were subjected to the oxidative Heck reaction to link a biotinylated phenylboronic acid for detection. In parallel to the labeling, the same samples were subjected to staining for β-actin as a loading control and antibody-based detection of the amount of 15-LOX-1. Representative blots are shown in Figure . We observed a decreased intensity for the band for activity-based lipoxygenase labeling as detected by streptavidin–horseradish peroxidase (HRP). For comparison, the bands normalized with the β-actin antibody and the 15-LOX antibody were included as well, which show comparable intensities. Quantifications of the bands from three independent experiments are shown in Figure . From these results, we conclude that 15-LOX-1 inhibitor 9c (i472) is able to inhibit the activity of cellular lipoxygenases.

Figure 5

Detection of the effect of inhibitor 9c (i472) on the activity of 15-LOX-1 by an activity-based probe. Labeling was performed on cell lysis of RAW 264.7 cells. Positive control (with probe and without inhibitor), negative control (without probe or inhibitor), and incubation of 9c (i472) were performed with the 15-LOX antibody, β-actin antibody, and streptavidin–HRP (n = 3).

Figure 6

Quantification of Western blots for detection and analysis of activity-based labeling: The values are measured by integrating the gray values by ImageJ 1.44. The integrated gray values of streptavidin–HRP and 15-LOX are normalized to β-actin, respectively. All of the values were expressed as mean ± standard error of the mean (SEM). The results were normalized by three independent experiments (n = 3). *p < 0.05, **p < 0.005, and ***p < 0.001 compared to control by the two-tailed test.

Protection of RAW 264.7 Macrophages from LPS-Induced Cytotoxicity

After identifying compound 9c (i472) as a potent inhibitor for recombinant expressed 15-LOX-1 and cellular LOX activity, we moved on to investigate the potency of this compound in cell-based studies. As the insight into the mechanism, we presume that 15-LOX-1 inhibitors inhibit the formation of lipid peroxides in cells, thereby preventing ferroptotic cell death. Additionally, we expect this mechanism to have crosstalk with the NF-κB pathway. Activation of this pathway can also lead to cell death. To test this hypothesis, we employed a model in which we stimulated RAW 264.7 macrophages with LPS to cause cell death, as reported previously.[3] This study reported an LD50 of 89.5 μg/mL for LPS-induced cell death in macrophages. In our experiments, 40% inhibition of cell viability was obtained at 100 μg/mL (Figure S3). Subsequently, as shown in Figure , we employed an LPS concentration of 100 μg/mL and investigated the protection from cell death by treatment with lipoxygenase inhibitors. The 5-LOX inhibitor Zileuton and the previously identified 15-LOX-1 inhibitor Eleftheriadis-14d comparably improved the viability of LPS-treated RAW 264.7 macrophages. In addition, inhibitor 9c (i472) showed stronger, dose-dependent effects with a 20% viability increase at 5 μM. Thus, these data demonstrated that inhibiting 15-LOX-1 by compound 9c (i472) can protect RAW 264.7 macrophages from LPS-induced cell death.

Figure 7

Inhibitor 9c (i472) protects RAW 264.7 macrophages from LPS-induced cytotoxicity. RAW 264.7 macrophages were treated with lipopolysaccharides (LPSs) (100 μg/mL) and 9c (i472) together for 24 h. Then, the cell viability was determined by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (n = 3–13). All of the values were expressed as mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001 compared to positive control by the two-tailed test that is only with the treatment of LPS (100 μg/mL) for 48 h.

NF-κB Activity Determination

To gain further insight into the mechanism of protection for LPS-induced cell death, we investigated the effect of inhibitor 9c (i472) on NF-κB activity using an NF-κB reporter assay on RAW-Blue macrophages (modified RAW 264.7 macrophages). These RAW-Blue macrophages can stably express a secreted embryonic alkaline phosphatase (SEAP) gene that is inducible by NF-κB and AP-1 transcription factors. Previous evidence demonstrated that the product of 15-LOX-1 and 13-HpODE can increase NF-κB activation but has no effect on AP-1.[8] The cells were stimulated with LPS, interferon γ (IFNγ), and inhibitor 9c (i472).[41] This provided significant but not complete inhibition of NF-κB transcriptional activation upon LPS/INFγ stimulation (Figure ). These results are in line with the anticipated crosstalk between 15-LOX-1 inhibition and NF-κB signaling.

Figure 8

Inhibitor 9c (i472) reduces NF-κB activity. RAW-Blue macrophages were pretreated with inhibitor 9c (i472) at 0.2, 1, and 5 μM for 20 h, after which inflammatory lipopolysaccharide (LPS) and interferon γ (IFNγ) stimuli (10 ng/mL of each) were given for another 4 h in continued presence of inhibitor 9c. All of the values were expressed as mean ± SEM (n = 8–16). *p < 0.05, **p < 0.005, and ***p < 0.001 compared to positive control that is treated with LPS and IFNγ by the two-tailed test.

Gene Expression

Subsequently, we turned our attention to the influence of 15-LOX-1 inhibition by 9c (i472) on the gene expression of NF-κB-related gene iNOS (Figure ). As a model, we used RAW 264.7 macrophages that were activated by LPS and IFNγ (10 ng/mL of each).[42] The gene expression of iNOS was downregulated by approximately 50% at 5 μM. This finding is in line with but more pronounced than the observed decrease in NF-κB transcriptional activity (Figure ).

Figure 9

Effect of inhibition of 15-LOX-1 by 9c (i472) on iNOS in RAW 264.7 cells: Lipopolysaccharide (LPS)/interferon γ (IFNγ) (10 ng/mL of each)-stimulated cells were normalized to the positive control. All experimental groups were treated with compound 9c (i472) at 0.2, 1, and 5 μM for 20 h and stimulated with LPS/IFNγ for another 4 h (n = 3–4). All of the values were expressed as mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001 compared to the LPS/IFNγ-treated positive control group by the two-tailed test.

Quantification of Nitric Oxide (NO) Production

Gene transcription of iNOS is connected to NO production, which plays an important role in the regulation of immune response and apoptosis. In our study, we compared the inhibitory effects of 9c (i472) on the ratio of total nitrate to nitrite in RAW 264.7 macrophages (Figure ). We demonstrated that 9c (i472) as a 15-LOX-1 inhibitor provided dose-dependent inhibition of NO production, which is consistent with the results of reduced activity of NF-κB and the gene expression of iNOS. The observations that 15-LOX inhibition inhibits NF-κB reporter gene activity, iNOS expression, and NO levels are in line with the idea that there is crosstalk between 15-LOX-1 activity and cell death via activity of the NF-κB pathway and NO production.

Figure 10

Dose-dependent effect of 9c (i472) on the expression of total nitrate/nitrite in RAW 264.7 cells: Lipopolysaccharide (LPS)/interferon γ (IFNγ) (10 ng/mL of each)-stimulated cells were corrected to 100% as positive control. All experimental groups were treated with compound 9c (i472) at 0.2, 1, and 5 μM for 20 h and stimulated with LPS/IFNγ for another 4 h (n = 3). All of the values were expressed as mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001 compared to the LPS/IFNγ-treated control group by the two-tailed test.

Lipid Peroxidation

Oxidative stress can cause a series of toxic effects through the production of lipid peroxides that play a role in cell death.[43] The effect of lipoxygenase inhibitor 9c (i472) on lipid peroxidation in RAW 264.7 cells was investigated using the fluorescent dye dipyrrometheneboron difluoride (BODIPY) 581/591 C11 and fluorescence-activated cell sorting (FACS).[44] As shown in Figure , 15-LOX inhibitor PD-146176 and 9c (i472) revealed comparable effects that both of them significantly attenuated the boost of lipid peroxides at 5 μM after the treatment of LPS/IFNγ (10 ng/mL of each). This result could be attributed to loss of 15-LOX products, such as 13-HpODE. Furthermore, compared to the 5-LOX inhibitor, Zileuton, both 15-LOX-1 inhibitors showed a more pronounced effect on lipid peroxidation, fitting the result of LPS-induced cell death assay that the 15-LOX-1 inhibitor has a better rescue effect. Although 15-LOX-1 is not the only pathway that can trigger lipid peroxide formation, these results indicate that inhibition of 15-LOX-1 has a strong influence on lipid peroxidation in this model system.

Figure 11

Analysis of lipid peroxidation using BODIPY 581/591 C11 staining and FACS analysis. Cells were treated with lipopolysaccharide (LPS)/interferon γ (IFNγ) (10 ng/mL of each) and PD-146176 (5 μM), Zileuton (5 μM), or 9c (i472) (5 μM). Results are represented as mean ± SEM (n = 3). *p < 0.05, **p < 0.005, and ***p < 0.001 compared to the LPS/IFNγ-treated control group by the two-tailed test.

Conclusions

In this study, compound 9c (i472) was developed as a potent 15-LOX-1 inhibitor with a novel substituent pattern (IC50 = 0.19 μM) and its SARs were explored. Using activity-based labeling, we demonstrated that inhibitor 9c (i472) was able to inhibit cellular lipoxygenases. Further characterization of this compound demonstrated that it was able to protect RAW 264.7 macrophages from LPS-induced cell death. We explored the influence of inhibitor 9c (i472) on different pathways of cell death. We investigated NF-κB activation, iNOS expression, and NO formation as a line of events that can trigger cell death. Treatment with inhibitor 9c (i472) enabled downregulation of the NF-κB transcriptional activity in a reporter gene assay. Furthermore, we demonstrated that iNOS gene expression and the levels of NO in RAW 264.7 macrophages decreased significantly upon 9c (i472) treatment. As a direct effect of inhibiting lipoxygenase activity, we investigated inhibition of cellular lipid peroxidation upon 9c (i472) treatment, for which we observed a clear reduction back to baseline levels. Having explored both mechanisms, we can conclude that inhibitor 9c (i472) influences both NO production and lipid peroxidation, potentially via a crosstalk mechanism. Thus, we conclude that we provide a novel 15-LOX-1 inhibitor 9c (i472) with cellular activity that inhibits the formation of oxidative mediators, such as NO and lipid peroxides, that are connected to different mechanisms for cell death.

Experimental Section

General

All reagents, solvents, and catalysts were purchased from commercial sources (Acros Organics, Sigma-Aldrich, and abcr GmbH, Netherlands) and used without purification. Reactions that required exclusion of oxygen or water were performed in oven-dried flasks under nitrogen atmosphere. Reactions were monitored by thin-layer chromatography (TLC) on TLC precoated (250 μm) silica gel 60 F254 aluminum foil (EMD Chemicals Inc.). Visualization was achieved using UV light. Alternatively, non-UV-active compounds were detected after staining with potassium permanganate. Flash column chromatography was performed on silica gel (32–63 μm, 60 Å pore size). 1H NMR (500 MHz) and 13C NMR (126 MHz) spectra were recorded with a Bruker Avance four-channel NMR spectrometer with a TXI probe. Chemical shifts (δ) are reported in ppm. Abbreviations are as follows: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). Fourier transform mass spectrometry and electrospray ionization were performed on an Applied Biosystems/SCIEX API 3000-triple quadrupole mass spectrometer. High-performance liquid chromatography (HPLC) analysis was performed for confirming purity with a Shimadzu LC-10AT HPLC, with a Shimadzu SP-M10A ELSD detector, and with a Shimadzu SPD-M10A photodiode array detector. Analytical HPLC was performed using a Kinetex C18 column (150 mm × 4.6 mm, 5 μm) with 5–95% MeCN gradient in H2O as a mobile phase, confirming purity ≥95%. Retention time (RT) of HPLC was also reported.

Synthesis and Characterization

Ethyl 6-Chloro-3-formyl-1H-indole-2-carboxylate (2)

To a solution of POCl3 (0.30 mL, 3.2 mmol, 1.2 equiv) in DMF (6.0 mL) stirred at 0 °C for 0.5 h, 1 (0.60 g, 2.7 mmol) was added, and the reaction mixture was heated to 50 °C for 46 h. After completion, the reaction mixture was slowly poured into a mixture of crushed ice and H2O (300 mL). The product was obtained by filtration of the resulting suspension. The residue was washed with acetonitrile and dried at r.t., giving the title intermediate 2 as a yellow solid in a yield of 83% (0.55 g, 2.2 mmol). The NMR spectra were the same as reported previously.[36]

6-Chloro-2-(ethoxycarbonyl)-1H-indole-3-carboxylic Acid (3)

To a solution of 2 (0.10 g, 0.39 mmol) and NaClO2 (71 mg, 0.78 mmol) was added 5.0 mL of t-BuOH at 50 °C for 4 h. After reaction completion, the mixture was concentrated and H2O (50 mL) was added, followed by extraction with EtOAc (3 × 15 mL). The organic phases were collected and evaporated, giving a white crude product without further purification in 82% yield.

General Synthetic Procedure 1: Amide Bond Formation

The respective carboxylic acid (1.0 equiv) was added to a mixture of HOBt (0.40 equiv), EDCI (2.0 equiv), and Et3N (1.0 equiv) in CH2Cl2 (20 mL) at room temperature for 30 min. After stirring, the respective amine (1.5 equiv) was added to this reaction mixture, which was subsequently stirred at room temperature for 4 h. Then, the reaction mixture was washed with 1.0 M aqueous HCl (5.0 mL), sat. aqueous NaHCO3 (5.0 mL), and brine (5.0 mL); dried over MgSO4; filtered; and concentrated under reduced pressure. The crude product was purified by column chromatography and eluted with 20% ethyl acetate in DCM as a solvent to obtain a white solid product with a general yield from 70 to 80%.

Ethyl 6-Chloro-3-(propylcarbamoyl)-1H-indole-2-carboxylate (5a)

The product was obtained using general procedure 1 starting from carboxylate 3 and propylamine. The product was obtained as a yellow solid in 86% yield. 1H NMR (500 MHz, dimethyl sulfoxide (DMSO)-d6) δ 12.96 (s, 1H), 8.92 (t, J = 5.5 Hz, 1H), 8.57 (d, J = 2.5 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.35 (dd, J = 8.5, 2.5 Hz, 1H), 4.31 (q, J = 7.0 Hz, 2H), 3.26–3.23 (m, 2H), 1.59–1.52 (m, 2H), 1.32 (t, J = 7.0 Hz, 3H), 0.91 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.37, 160.16, 154.97, 139.38, 136.90, 130.43, 130.37, 124.08, 120.02, 119.90, 63.31, 41.49, 22.49, 14.26, 11.88. High-resolution mass spectrometry (HRMS), calcd for C15H18ClO2N3 [M + H]+: 309.1000, found 309.1002. HPLC: purity 96%, retention time 17.7 min.

Ethyl (R)-6-Chloro-3-((2,6-dimethylheptyl)carbamoyl)-1H-indole-2-carboxylate (5b)

The product was obtained using general procedure 1 starting from carboxylate 3. The product was obtained as a yellow solid in 82% yield. 1H NMR (500 MHz, CDCl3) δ 10.75 (s, 1H), 9.40 (s, 1H), 8.43 (d, J = 8.5 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 8.5, 2.0 Hz, 1H), 5.75 (s, 1H), 4.56 (q, J = 7.0 Hz, 2H), 3.25–3.06 (m, 1H), 1.54 (m, 1H), 1.51 (t, J = 5.0 Hz, 3H), 1.36–1.15 (m, 8H), 0.96 (d, J = 6.0 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.71, 160.13, 154.91, 139.28, 136.81, 131.58, 130.40, 124.03, 120.33, 119.92, 63.29, 45.55, 38.70, 36.64, 36.34, 27.81, 24.04, 22.87, 20.86, 14.26, 14.18. HRMS, calcd for C21H30ClN2O3 [M + H]+: 393.1939, found 393.1938. HPLC: purity 99%, retention time 14.3 min.

Ethyl (E)-3-(3-(tert-Butoxy)-3-oxoprop-1-en-1-yl)-6-chloro-1H-indole-2-carboxylate (6)

2 (0.10 g, 0.39 mmol), (tert-butoxycarbonylmethylene)triphenylphosphorane (0.17 g, 0.50 mmol), and EtOH (10 mL) were mixed under an atmosphere of nitrogen in an oven-dried flask. The mixture was heated at reflux for 2 h. Then, the solvent was evaporated under reduced pressure. The product was purified by column chromatography and eluted with 20% ethyl acetate in petroleum as a solvent, and a yellow solid product was obtained in 84% yield. The NMR spectra were the same as reported previously.[45]

(E)-3-(6-Chloro-2-(ethoxycarbonyl)-1H-indol-3-yl)acrylic Acid (7)

6 (0.50 g, 1.7 mmol) was dissolved in DCM (1 mL). Then, trifluoroacetic acid (1.0 mL, 2.0 mmol) was added and the mixture was stirred at 0 °C for 2 h. After evaporation of the solvent, the crude product did not need further purification. The NMR spectra were the same as reported previously.[45]

Ethyl (E)-6-Chloro-3-(3-((2,4-dichlorobenzyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9a)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 89% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.31 (s, 1H), 8.63 (t, J = 6.0 Hz, 1H), 8.34 (d, J = 16.0 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 2.0 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.48–7.43 (m, 2H), 7.28 (dd, J = 8.5, 2.0 Hz, 1H), 7.24 (d, J = 16.0 Hz, 1H), 4.48 (d, J = 6.0 Hz, 2H), 4.41 (q, J = 7.0 Hz, 2H). 1.39 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.16, 161.20, 137.57, 136.10, 133.64, 132.81, 132.02, 131.12, 130.47, 129.09, 127.88, 127.66, 123.97, 123.71, 122.58, 122.37, 117.17, 113.10, 105.90, 61.59, 14.69. HRMS, calcd for C21H18Cl3N2O3 [M + H]+: 451.0378, found 451.0377. HPLC: purity 95%, retention time 19.2 min.

Ethyl (E)-6-Chloro-3-(3-((2-chlorobenzyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9b)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 89% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.32 (s, 1H), 8.59 (t, J = 5.5 Hz, 1H), 8.34 (d, J = 16.0 Hz, 1H), 8.07 (d, J = 6.0 Hz, 1H), 8.55 (d, J = 2.0 Hz, 1H), 7.48 (dd, J = 7.0, 2.0 Hz, 1H), 7.43 (dd, J = 7.0, 2.0 Hz, 1H), 7.35 (m, 2H), 7.28 (dd, J = 7.0, 2.0 Hz, 1H), 7.03 (d, J = 16 Hz, 1H), 4.50 (d, J = 6.0 Hz, 2H), 4.42 (q, J = 6.0 Hz, 2H), 1.39 (t, J = 6.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 171.36, 166.33, 141.59, 140.42, 134.65, 134.42, 133.89, 133.21, 131.51, 130.11, 129.20, 127.64, 126.67, 125.90, 122.91, 122.09, 117.00, 99.98, 65.72, 34.31, 19.24. HRMS, calcd for C21H19O3N2Cl2 [M + H]+: 417.0767, found 417.0767. HPLC: purity 96%, retention time 19.2 min.

Ethyl (E)-6-Chloro-3-(3-((2-methoxybenzyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9c)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 79% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.28 (s, 1H), 8.39 (t, J = 5.5 Hz, 1H), 8.31 (d, J = 16.0, Hz, 1H), 8.08 (d, J = 9.0, Hz, 1H), 7.54 (dd, J = 7.0, 2.0 Hz, 1H), 7.42–7.29 (m, 3H), 7.03 (s, 1H), 7.01 (d, J =7.0 Hz, 1H), 6.94 (td, J = 7.0, 2.0 Hz, 1H), 4.43–4.38 (m, 4H), 3.83 (s, 3H), 1.39 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.97, 161.25, 157.28, 141.43, 137.57, 131.43, 130.44, 128.78, 127.53, 125.63, 124.96, 124.11, 123.96, 122.30, 120.66, 117.37, 113.09, 111.04, 61.57, 55.78, 38.81, 14.73. HRMS, calcd for C22H22ClN2O4 [M + H]+: 413.1263, found 413.1261. HPLC: purity 96%, retention time 18.9 min.

Ethyl (E)-6-Chloro-3-(3-((3-methoxybenzyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9d)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 91% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.30 (s, 1H), 8.55 (t, J = 6.0 Hz, 1H), 8.34 (d, J = 16.0 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.55 (d, J =2.0 Hz, 1H), 7.29–7.25 (m, 2H), 6.94 (d, J = 16.0 Hz, 1H), 6.91 (m, 2H), 6.85 (dd, J = 7.0, 2.0 Hz, 1H), 4.44–4.40 (m, 4H), 3.74 (s, 3H), 1.39 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.92, 161.24, 159.82, 141.43, 137.57, 131.69, 130.45, 129.68, 127.53, 125.63, 124.96, 123.97, 123.05, 122.34, 120.13, 117.30, 113.66, 112.71, 61.58, 55.54, 42.84, 14.75. HRMS, calcd for C22H22ClN2O4 [M + H]+: 413.1263, found 413.1262. HPLC: purity 96%, retention time 14.4 min.

Ethyl (E)-6-Chloro-3-(3-((4-methoxybenzyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9e)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 84% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.29 (s, 1H), 8.49 (t, J = 6.0 Hz, 1H), 8.34 (d, J = 16.0 Hz, 1H), 8.04 (d, J = 6.0 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.26 (m, 3H), 6.97–6.91 (m, 3H), 4.43 (q, J = 6.0 Hz, 2H), 4.35 (d, J = 6.0 Hz, 2H), 3.74 (s, 3H), 1.39 (t, J = 6.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.77, 161.24, 158.78, 137.56, 131.76, 131.52, 130.43 (2×), 129.35, 127.48, 123.95, 123.72, 123.22, 122.32, 117.33, 114.27, 114.21, 113.11, 61.58, 55.52, 42.35, 14.67. HRMS, calcd for C22H22ClN2O4 [M + H]+: 413.1263, found 413.1262. HPLC: purity 95%, retention time 14.4 min.

Ethyl (E)-3-(3-(Benzylamino)-3-oxoprop-1-en-1-yl)-6-chloro-1H-indole-2-carboxylate (9f)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 88% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.30 (s, 1H), 8.57 (t, J = 6.0 Hz, 1H), 8.33 (d, J = 16.0 Hz, 1H), 8.05 (d, J = 6.0 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.38–7.25 (m, 6H), 6.67 (d, J = 16.0 Hz, 1H), 4.44–4.40 (m, 4H), 1.38 (t, J = 6.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.91, 161.24, 139.86, 137.56, 131.68, 131.62, 130.44, 128.86 (2×), 127.98, 127.52, 127.38, 123.96, 123.77, 123.08, 122.34, 117.30, 113.02, 61.59, 42.88, 14.76. HRMS, calcd for C21H20ClN2O3 [M + H]+: 383.1157, found 383.1158. HPLC: purity 98%, retention time 7.1 min.

Ethyl (E)-6-Chloro-3-(3-oxo-3-(phenethylamino)prop-1-en-1-yl)-1H-indole-2-carboxylate (9g)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 76% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.28 (s, 1H), 8.29 (d, J = 16.0 Hz, 1H), 8.19 (t, J = 6.0 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.34–7.21 (m, 6H), 6.90 (d, J = 16.0 Hz, 1H), 4.42 (q, J = 6.0 Hz, 2H), 4.45 (q, J = 6.0 Hz, 2H), 2.81 (t, J = 6.0 Hz, 2H), 1.38 (t, J = 6.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.90, 161.24, 144.40, 139.98, 137.58, 137.13, 131.26, 130.41 (2×), 129.09, 128.82, 126.57, 123.97, 123.70, 123.26, 122.28, 117.34, 113.06, 61.55, 39.77, 35.74, 14.71. HRMS, calcd for C22H22ClN2O3 [M + H]+: 397.1313, found 397.1311. HPLC: purity 96%, retention time 14.5 min.

Ethyl (E)-6-Chloro-3-(3-((2-chlorophenethyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9h)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 90% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.28 (s, 1H), 8.28 (d, J = 16.0 Hz, 1H), 8.24 (t, J = 6.0 Hz, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.46 (dd, J = 8.0, 2.0 Hz, 1H), 7.38 (dd, J = 8.0, 2.0 Hz, 1H), 7.33–7.26 (m, 3H), 6.87 (d, J = 16.0 Hz, 1H), 4.42 (q, J = 7.0 Hz, 2H), 3.45 (q, J = 7.0 Hz, 2H), 2.95 (t, J = 7.0 Hz, 2H), 1.40 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.01, 161.53, 137.21, 136.78, 133.50, 131.21, 129.66, 129.44, 128.67, 128.58, 127.78, 127.68, 125.82, 125.02, 123.80, 120.88, 118.46, 112.26, 61.13, 38.63, 33.14, 14.71. HRMS, calcd for C22H21O3N2Cl2 [M + H]+: 431.0924, found 431.0924. HPLC: purity 95%, retention time 14.2 min.

Ethyl (E)-6-Chloro-3-(3-((2,4-dichlorophenethyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9i)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 89% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.29 (s, 1H), 8.28 (d, J = 16.0 Hz, 1H), 8.22 (t, J = 6.0 Hz, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.40 (m, 2H), 7.28 (m, 1H), 6.85 (d, J = 16.0 Hz, 1H), 4.42 (q, J = 7.0 Hz, 2H), 3.48 (q, J = 7.0 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H), 1.36 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.97, 161.22, 137.55, 136.49, 134.57, 132.79, 132.21, 131.38, 130.44, 129.12, 127.86, 127.49, 123.97, 123.65, 123.13, 122.32, 117.29, 113.07, 61.56, 38.69, 32.96, 14.70. HRMS, calcd for C22H20O3N2Cl3 [M + H]+: 465.0534, found 465.0534. HPLC: purity 95%, retention time 18.2 min.

Ethyl (E)-6-Chloro-3-(3-((2-methoxyphenethyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxylate (9j)

The product was obtained using general procedure 1 starting from 7. The product was obtained as a white solid in 81% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.27 (s, 1H), 8.27 (d, J = 16.0 Hz, 1H), 8.17 (t, J = 5.5 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.27 (dd, J = 8.0, 2.0 Hz, 1H), 7.22 (m, 1H), 7.18 (dd, J = 7.0, 2.0 Hz, 1H), 6.99 (d, J = 16.0 Hz, 1H), 6.89 (m, 2H), 4.42 (q, J = 7.0 Hz, 2H), 3.81 (s, 3H), 3.32 (q, J = 7.0 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H), 1.39 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.84, 161.25, 157.71, 137.56, 131.17, 130.43, 128.09, 127.61, 127.41, 123.96, 123.75, 123.68, 123.41, 122.31, 117.38, 113.11, 113.00, 111.12, 61.56, 55.80, 55.71, 30.50, 14.75. HRMS, calcd for C23H24O4N2Cl [M + H]+: 427.1419, found 427.1418. HPLC: purity 96%, retention time 17.2 min.

6-Chloro-N-methyl-1H-indole-2-carboxamide (12a)

The product was obtained using general procedure 1 starting from 10. The product was obtained as a light brown solid in 92% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.72 (s, 1H), 8.53 (d, J = 5.0 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 7.05 (dd, J = 8.5, 2.0 Hz, 1H), 2.82 (d, J = 4.5 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 172.08, 162.03, 143.15, 135.19, 125.34, 125.14, 122.83, 113.47, 99.98, 35.69. HRMS, calcd for C10H10ClN2O [M + H]+: 209.0476, found 209.0475. HPLC: purity 95%, retention time 18.5 min.

6-Chloro-N-ethyl-1H-indole-2-carboxamide (12b)

The product was obtained using general procedure 1 starting from 10. The product was obtained as a light brown solid in 94% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.70 (s, 1H), 8.65 (t, J = 6.0 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 8.5, 2.0 Hz, 1H), 3.31 (m, 2H), 1.15 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 160.95, 137.05, 133.44, 128.15, 126.35, 123.55, 120.62, 112.17, 102.68, 34.13, 15.38. HRMS, calcd for C11H12ON2Cl [M + H]+: 223.0633, found 223.0633. HPLC: purity 98%, retention time 10.8 min.

6-Chloro-N-propyl-1H-indole-2-carboxamide (12c)

The product was obtained using general procedure 1 starting from 10. The product was obtained as a light brown solid in 91% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.70 (s, 1H), 8.56 (t, J = 5.5 Hz, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 7.05 (dd, J = 8.5, 2.0 Hz, 1H), 3.25 (q, J = 7.0 Hz, 2H), 1.55 (q, J = 7.5 Hz, 2H), 0.91 (d, J = 2.0 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 161.14, 137.07, 133.42, 128.18, 126.36, 123.54, 120.57, 112.18, 102.75, 41.06, 22.97, 11.94. HRMS, calcd for C12H14ON2Cl [M + H]+: 237.0789, found 237.0789. HPLC: purity 95%, retention time 14.5 min.

N-Butyl-6-chloro-1H-indole-2-carboxamide (12d)

The product was obtained using general procedure 1 starting from 10. The product was obtained as a light brown solid in 90% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.96 (s, 1H), 7.45 (t, J = 6.0 Hz, 1H), 6.64 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.20 (dd, J = 8.5, 2.0 Hz, 1H), 3.32 (q, J = 7.5 Hz, 2H), 1.57–1.53 (m, 2H), 1.39–1.34 (m, 2H), 0.90 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 161.07, 137.05, 133.42, 128.15, 126.36, 123.57, 120.59, 112.13, 102.70, 38.93, 31.79, 20.10, 14.20. HRMS, calcd for C13H16ClN2O [M + H]+: 251.0946, found 251.0947. HPLC: purity 99%, retention time 14.5 min.

6-Chloro-N-pentyl-1H-indole-2-carboxamide (12e)

The product was obtained using general procedure 1 starting from 10. The product was obtained as a light brown solid in 91% yield. 1H NMR (500 MHz, DMSO-d6) δ 1H NMR (500 MHz, DMSO-d6) δ: 11.69 (s, 1H), 7.51 (t, J = 6.0 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H), 7.43 (s, 1H), 7.14 (m, 1H), 7.03 (dd, J = 8.5, 2.0 Hz, 1H), 3.30 (q, J = 7.5 Hz, 2H), 1.57–1.53 (m, 2H), 1.24–1.20 (m, 4H), 0.92 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 161.05, 137.05, 133.42, 128.15, 126.35, 123.54, 120.55, 112.15, 102.72, 39.21, 29.36, 29.15, 22.36, 14.44. HRMS, calcd for C14H18ClN2O [M + H]+: 265.1102, found 265.1103. HPLC: purity 99%, retention time 14.3 min.

6-Chloro-N-isopropyl-1H-indole-2-carboxamide (12f)

The product was obtained using general procedure 1 starting from 10. The product was obtained as a light brown solid in 84% yield. 1H NMR (500 MHz, DMSO-d6) δ 1H NMR (500 MHz, DMSO-d6) δ: 11.67 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H), 7.43 (s, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.03 (dd, J = 8.5, 2.0 Hz, 1H), 4.17–4.09 (m, 1H), 1.20 (d, J = 6.5 Hz, 6H). 13C NMR (126 MHz, DMSO-d6) δ 160.28, 137.04, 133.50, 128.14, 126.33, 123.46, 120.56, 112.10, 102.88, 41.24, 41.14, 22.87. HRMS, calcd for C12H14ON2Cl [M + H]+: 237.0789, found 237.0789. HPLC: purity 99%, retention time 10.8 min.

6-Chloro-N-cyclopropyl-1H-indole-2-carboxamide (12g)

The product was obtained using general procedure 1 starting from 10. The product was obtained as a light brown solid in 91% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.70 (s, 1H), 8.54 (d, J = 2.0 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 2.0 Hz 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.06 (dd, J = 8.5, 2.5 Hz, 1H), 2.90–2.83 (m, 1H). 0.75–0.71 (m, 2H), 0.61–0.55 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 162.36, 137.09, 133.14, 128.24, 126.32, 123.54, 120.63, 112.16, 103.01, 23.12 (2×), 6.30. HRMS, calcd for C12H12ON2Cl [M + H]+: 235.0633, found 235.0632. HPLC: purity 97%, retention time 11.8 min.

6-Chloro-3-((dimethylamino)methyl)-N-propyl-1H-indole-2-carboxamide (13)

6-Chloro-N-propyl-1H-indole-2-carboxamide (12c) (50 mg, 0.20 mmol), dimethylamine (9.5 mg, 0.20 mmol), paraformaldehyde (6.5 mg, 0.20 mmol), and 0.20 mL of acetic acid were dissolved in 10 mL of MeOH. The reaction mixture was refluxed for 4 h. After completion, the product was purified by column chromatography and eluted with 10% ethyl acetate in petroleum ether as a solvent to obtain a white solid product with 91% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.75 (s, 1H), 10.53 (t, J = 2.0 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.5, 2.0 Hz, 1H), 3.69 (s, 2H), 3.29 (q, J = 6.5 Hz, 2H), 2.24 (s, 6H), 1.71–1.66 (m, 2H), 1.04 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 161.68, 135.54, 132.22, 128.27, 127.28, 121.36, 120.42, 112.11, 112.05, 52.57, 44.00, 43.97, 41.30, 22.75, 12.00. HRMS, calcd for C15H21ON3Cl [M + H]+: 294.1368, found 294.1367. HPLC: purity 99%, retention time 13.9 min.

General Synthetic Procedure 2: Hydrolysis Reaction

The ester (1.0 equiv) was dissolved in THF (15 mL) while stirring. Then, a solution of lithium hydroxide trihydrate (3.0 equiv) in demiwater (15 mL) was added and the mixture was stirred at 50 °C for 2 h. Subsequently, the aqueous layer was extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product 11 was used without further purification.

(R)-6-Chloro-3-(3,7-dimethyloctanoyl)-N-propyl-1H-indole-2-carboxamide (14)

The product was obtained using general procedures 1 and 2 starting from compounds Eleftheriaidis-14d and 12c. The product was obtained as a light brown solid in 65% yield over two steps. 1H NMR (500 MHz, CDCl3) δ 11.83 (s, 1H), 11.45 (t, J = 2.0 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.31 (dd, J = 8.5, 2.5 Hz), 3.62 (q, J = 6.5 Hz, 2H), 3.06 (m, 2H), 2.28 (m, 1H), 1.83 (m, 2H), 1.56 (m, 1H), 1.42 (m, 2H), 1.33 (m, 2H), 1.22 (m, 2H), 1.12 (t, J = 7.5 Hz, 3H), 1.04 (d, J = 7.5 Hz, 3H), 0.89 (d, J = 7.5 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 199.61, 159.84, 136.61, 135.02, 130.45, 125.26, 123.66, 123.16, 115.54, 113.22, 51.18, 42.02, 39.08, 37.33, 29.61, 27.92, 24.79, 22.69, 22.62, 22.56, 20.01, 11.66. HRMS, calcd for C22H32O2N2Cl [M + H]+: 391.2147, found 391.2148. HPLC: purity 97%, retention time 21.4 min.

(S)-6-Chloro-3-(3,7-dimethyloctanoyl)-N-propyl-1H-indole-2-carboxamide (15)

The product was obtained using general procedures 1 and 2 starting from compounds Eleftheriaidis-14e and 12c. The product was obtained as a light brown solid in 55% yield over two steps. 1H NMR (500 MHz, CDCl3) δ 11.63 (s, 1H), 11.41 (t, J = 2.0 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.31 (dd, J = 8.5, 2.5 Hz), 3.60 (q, J = 7.0 Hz, 2H), 3.19–2.96 (m, 2H), 2.30–2.25 (m, 1H), 1.85–1.78 (m, 2H), 1.60–1.52 (m, 1H), 1.46–1.16 (m, 6H), 1.12 (t, J = 7.5 Hz, 3H), 1.04 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.5 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 199.60, 159.81, 136.62, 134.97, 130.46, 125.27, 123.66, 123.17, 115.53, 113.18, 51.18, 42.00, 39.08, 37.32, 29.60, 27.92, 24.79, 22.69, 22.62, 22.56, 20.00, 11.66. HRMS, calcd for C22H32O2N2Cl [M + H]+: 391.2147, found 391.2149. HPLC: purity 99%, retention time 22.9 min.

(E)-6-Chloro-N-ethyl-3-(3-((2-methoxybenzyl)amino)-3-oxoprop-1-en-1-yl)-1H-indole-2-carboxamide (16)

The product was obtained using general procedures 1 and 2 starting from compounds 9c and 11b. The product was obtained as a white solid in 62% yield over two steps. 1H NMR (500 MHz, DMSO-d6) δ 12.01 (s, 1H), 8.47 (t, J = 6.0 Hz, 1H), 8.31 (t, J = 6.0 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.50 (s, 1H), 7.25 (m, 3H), 7.04 (d, J = 7.5 Hz, 1H), 6.92 (t, J = 7.5 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 4.38 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H), 3.33 (m, 2H), 1.19 (t, J = 6.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.34, 161.31, 157.29, 136.87, 134.45, 132.09, 129.02, 128.73, 127.32, 124.16, 123.14, 121.79, 120.79, 120.63, 113.16, 112.59, 111.05, 55.85, 37.93, 34.56, 31.15, 15.04. HRMS, calcd for C22H23ClN3O3 [M + H]+: 412.4122, found 412.4123. HPLC: purity 96%, retention time 13.8 min.

(E)-6-Chloro-3-(3-((2-methoxybenzyl)amino)-3-oxoprop-1-en-1-yl)-N-propyl-1H-indole-2-carboxamide (17)

The product was obtained using general procedures 1 and 2 starting from compounds 9c and 11c. The product was obtained as a white solid in 62% yield over two steps. 1H NMR (500 MHz, DMSO-d6) δ 12.03 (s, 1H), 8.47 (t, J = 5.5 Hz, 1H), 8.29 (t, J = 5.5 Hz, 1H), 8.07 (d, J = 16.0 Hz, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.23 (m, 3H), 7.20 (dd, J = 7.5, 2.0 Hz, 1H), 6.94 (td, J = 6.5, 2.0 Hz, 1H), 6.90 (d, J = 16.0 Hz, 1H), 4.38 (d, J = 6.0 Hz, 2H), 3.86 (s, 3H), 3.28 (q, J = 6.0 Hz, 2H), 1.68 (m, 2H), 0.95 (t, J = 6.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 166.32, 161.45, 157.26, 136.87, 134.57, 132.07, 128.98, 128.69, 128.66, 127.30, 124.13, 123.12, 121.77, 120.73, 120.62, 113.06, 112.58, 111.03, 55.84, 41.44, 37.92, 22.72, 12.01. HRMS, calcd for C23H25ClN3O3 [M + H]+: 426.1579, found 426.1580. HPLC: purity 96%, retention time 13.8 min.

Human 15-LOX-1 Enzyme Inhibition Studies

The 15-LOX-1 enzyme was expressed and purified as described before.[46] Furthermore, the 15-LOX-1 enzyme activity studies were done using procedures previously described by our group as well.[30] 15-LOX-1 activity was determined by the conversion of linoleic acid to hydroperoxy-(9Z,11E)-octadecadienoic acid (λmax of 234 nm) in a 96-well plate. The conversion rate was followed by UV absorbance at 234 nm. The conversion rate was evaluated at the linear part of the plot, and the substrate depletion covers the first 16 min. The optimum concentration of 15-LOX-1 was determined by an enzyme activity assay and proved to be a 40-fold dilution. The assay buffer consists of 25 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid titrated to pH 7.4. The substrate, linoleic acid (LA) (Sigma-Aldrich, L1376), was dissolved in ethanol to a concentration of 500 nM. The absorbance increased at 234 nm over time for the conversion of linoleic acid in the presence (positive control) of the enzyme or remained constant in the absence (blank control) of the enzyme.

To determine IC50 values, 140 μL of the inhibitors (0–71 μM, 2× dilution series) was incubated with 50 μL of 1:40 enzyme solution for 10 min at room temperature in a 96-well plate. After 10 min incubation, 10 μL of 500 nM LA was added to the mixture, which resulted in desired concentrations of the inhibitors (0–50 μM, 2× dilution series), a final dilution of the enzyme of 1:160, and 25 nM LA. The linear absorbance increased in the absence of the inhibitor was set to 100%, whereas the absorbance increased in the absence of the enzyme was set to 0%. All experiments were performed at least in triplicate. The average values and their standard deviations were plotted. Data analysis was performed using Microsoft Excel professional plus 2013 and GraphPad Prism 5.01.

Cell Culture and MTS and RAW-Blue NF-κB Reporter Gene Assays

RAW 264.7 murine macrophages were obtained from ATCC (Wesel, Germany) and cultured in Dulbecco’s modified Eagle’s medium + GlutaMAX (Gibco by Life Technologies, The Netherlands) supplemented with 10% (v/v) fetal bovine serum and 100 U/mL 1% penicillin/streptomycin (Gibco, The Netherlands) in a humidified 5% CO2 atmosphere at 37 °C. RAW-Blue macrophages were obtained from InvivoGen (Toulouse, France) and cultured in the same conditions, with the addition of 200 μg/mL Zeocin to the culture medium as reported by the manufacturer.

RAW 264.7 cells were seeded at 5000 cells per well in a 96-well plate 1 day prior to the experiment. Cells were treated with 9c (i472) at 0.1, 1, 5, 10, and 50 μM for 48 h. The cell viability of the treated cells was determined by adding 20 μL of the CellTiter reagent to each well. After 2 h incubation with the CelTiter reagent at 37 °C, the absorbance at 490 nm was measured using a Synergy H1 plate reader.

RAW-Blue cells were seeded at 10 × 104 cells per well in a 96-well plate 1 day before the start of the experiment. Cells were treated with 9c (i472) at 0.2, 1, and 5 μM and stimulated with 10 ng/mL LPS (Sigma-Aldrich, The Netherlands) and 10 ng/mL IFNγ (Sigma-Aldrich, The Netherlands) for 24 h. The secreted embryonic alkaline phosphatase (SEAP) release was measured to monitor the NF-κB levels using the QuantiBlue reagent (InvivoGen, Toulouse, France). After 2 h incubation at 37 °C, the absorbance at 635–655 nm was measured using a Synergy H1 plate reader according to the manufacturer’s instructions.

LPS-Induced Cell Death

RAW 264.7 cells were seeded at 5000 cells per well in a 96-well plate. The ability of rescue was tested with the treatment of 9c (i472) at 0.2, 1, and 5 μM or Zileuton at 5 μM with 100 μg/mL LPS for 48 h. The cell viability was determined by the MTS assay as described above.[3]

Gene Expression Analysis by Quantitative Reverse Transcription Polymerase Chain Reaction (PCR)

Total RNA was isolated from RAW 264.7 cells using the SV total RNA isolation system (Promega, Leiden, The Netherlands) according to the protocol of the manufacturer. RNA integrity was determined by 28S/18S ratio detection on an agarose gel, which was consistently found to be intact. For gene expression analysis, RNA was reverse-transcribed using a reverse-transcription kit (Promega). Subsequently, 10 ng of cDNA was applied for each real-time PCR, which was performed on an ABI Prism 7900HT sequence detection system (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). The primers for NF-κB1 (Fw, 5′-GAAATTCCTGATCCAGACAAAAAC-3′, Rv, 5′-ATCACTTCAATGGCCTCTGTGTAG-3′), NF-κB2 (Fw, 5′-CTGGTGGACACATACAGGAAGAC-3′, Rv, 5′- ATAGGCACTGTCTTCTTTCACCTC-3′), RelA (Fw, 5′-CTTCCTCAGCCATGGTACCTCT-3′, Rv, 5′- CAAGTCTTCATCAGCATCAAACTG-3′), RelB (Fw, 5′-CTTTGCCTATGATCCTTCTGC-3′, Rv, 5′- GAGTCCAGTGATAGGGGCTCT-3′) and iNOS (Fw, 5′-TATCAGGAAGAAATGCAGGAGAT-3′, Rv, 5′- GAGCACGCTGAGTACCTCATT-3′) were purchased from Sigma. For each sample, the real-time PCR reactions were performed in triplicate, and the averages of the obtained Ct values were used for further calculations. Gene expression levels were normalized to the expression of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was not influenced by the experimental conditions, resulting in the ΔCt value. Gene expression levels were calculated by the comparative Ct method (2–ΔΔ).

Nitric Oxide (NO) Assay

The level of nitric oxide was measured in RAW 264.7 cells. Macrophage cells (2 × 106 per well) were seeded in six-well plates and incubated 24 h with or without 10 ng/mL LPS (Sigma, The Netherlands) and 10 ng/mL IFNγ (Sigma-Aldrich, The Netherlands) in the presence or absence of 5 μM 9c (i472). The nitric oxide level in each sample was quantified using the commercially available colorimetric nitric oxide assay kit (abcam, ab 65328, U.K.) following the manufacturer’s instructions.

Lipid Peroxidation

RAW 264.7 cells were seeded into a six-well plate containing 10 × 106 cells per well. After overnight incubation, cells were treated with 10 ng/mL LPS (Sigma, The Netherlands) and 10 ng/mL IFNγ (Sigma, The Netherlands) for 24 h in the presence or absence of 5 μM PD146176, Zileuton, or 9c (i472), respectively. Cells without LPS/IFNγ treatment were taken as a control. Lipid peroxidation was detected by staining with BODIPY 581/591 C11 (Invitrogen, Karlsruhe, Germany) at a final concentration of 2 mM for 30 min at 37 °C. The shift in fluorescence from red to green was analyzed by fluorescence-activated cell sorting (FACS) using the Guava Easy Cite 6-2L system (Merck Millipore, Darmstadt, Germany) by excitation at 488 nm. At least three independent experiments were performed per condition.