N-Substituted 4-sulfamoylbenzoic acid derivatives as inhibitors of cytosolic phospholipase A2α

The N,N-disubstituted 4-sulfamoylbenzoic acid derivative 3, which was found to be an inhibitor of cytosolic phospholipase A2α (cPLA2α) with micromolar activity in a ligand-based virtual screening approach, was structurally modified to increase its enzyme inhibitory potency. Replacing the substituents on the sulfonamide nitrogen with other residues such as naphthyl, naphthylmethyl, indolylalkyl and differently substituted phenyl moieties did not lead to a significant increase in activity. Only strong structural convergence to the potent known benzhydrylindole-substituted benzoic acid derivatives that had served as templates in the virtual screening resulted in compounds with considerable potency. Thus, the sulfamoyl benzoic acid derivatives 85 and 88 showed submicromolar IC50 values against cPLA2α. Graphical abstract Graphical abstract


Introduction
The arachidonic acid cascade is a biochemical pathway that leads to the formation of oxidized arachidonic acid products such as prostaglandins and leukotrienes, which exert a variety of physiological effects [1]. The excessive stimulation of this metabolic route by inflammatory cytokines, however, results in high concentrations of these compounds, known as eicosanoids, being involved in the pathogenesis of inflammatory diseases. The prostaglandin E 2 (PGE 2 ) formed via the cyclooxygenase (COX) pathway and the leukotrienes produced via the 5-lipoxygenase (5-LOX) pathway are particularly responsible for this. Thus, PGE 2 mediates the cardinal symptoms of acute inflammation: rubor (redness), calor (heat), tumor (swelling) and dolor (pain) [2]. Certain leukotrienes are potent chemotractants leading to leukocyte recruitment and activation enhancing the inflammatory process [3]. The therapeutically used COX inhibitors like diclofenac and ibuprofen can amorilate the effect of PGE 2 , but they do not have an impact on leukotriene formation. The inflammatory mediators produced via both pathways could be inhibited simultaneously by blocking the enzyme responsible for releasing the common precursor arachidonic acid from phospholipids, namely cytosolic phospholipase A 2 α (cPLA 2 α). Thus, inhibitors of this enzyme are expected to provide new treatment options for inflammatory conditions [4,5].
Although several highly active inhibitors of cPLA 2 α have been developed, no such compound is available for clinical use today. Among the most effective of the published substances are thiazolidinediones from Shionogi [6], phenylpropanoic acid derivatives from Asahi Kasei Pharma [7,8], benzhydrylindoles from Wyeth (now Pfizer) [9][10][11] and propan-2-ones from AstraZeneca [12]. We have synthesized a series of heteroaryl propan-2-ones structurally related to the latter substance class, which also possess a high enzyme inhibitory potency [13,14]. Unfortunately, these compounds are not suitable for peroral in vivo application, especially due to their rapid metabolic inactivation and their property of being substrates of gastrointestinal efflux transporters. In search of new molecular entities for cPLA 2 α inhibitors, we recently have performed a molecular-modeling approach [15] inspired by the works of Gianella-Borradori et al. and Naylor et al. [16,17]. From a series of six structurally related cPLA 2 α inhibitors of Wyeth with high potency [10,11], such as WAY-196025 (1) (Fig. 1), energetically favorable conformers were calculated and used for a ligand-based virtual screening method including both three-dimensional shape and electrostatic comparison applying the tools OMEGA (conformer generation), ROCS (screening), EON (ranking) and VIDA (visualization) from OpenEye Scientific (Santa Fee, New Mexico, USA). As the database source, the Screening Compound Database (SCD) from MolPort (Riga, Latvia) with around 6 million compounds in 2017 was chosen. Of the compounds with the closest match in terms of shape and electrostatic similarity expressed in terms of the electrostatic shape-Tanimoto (ET) combo score [18], 47 were purchased from MolPort and tested for inhibition of isolated porcine cPLA 2 α. Eight of these substances showed an IC 50 value between 10 µM and 33 µM, among them the 4-sulfamoylbenzoic acid derivative 3 [19] (IC 50 : 19 µM) and the indole-substituted 4-(methylenehydrazineyl)benzoic acid 4 (IC 50 : 22 µM) (Fig. 1). For the commercially available benzhydrylindole-substituted cPLA 2 α inhibitor 2 from Wyeth [9], which is marketed under the name Axon-1609, an IC 50 of 0.21 µM was determined with the same assay. Thus, the hits found with the molecular modeling were about 100-fold less active than 2. Taken into account that the IC 50 values of the six cPLA 2 α inhibitors used as queries in our ligand-based virtual screening had been reported to be even lower than that of 2 [9][10][11], we had to assume that the found hits were substantially less effective than these substances. Nevertheless, we started some structural variations of 3 in order to increase its potency.

Results and discussion
Chemistry For the synthesis of the 4-sulfamoylbenzoic acid derivative 6, bearing a phenyl and a benzyl substituent at the sulfonamide nitrogen, methyl 4-(chlorosulfonyl)benzoate was Fig. 1 Structures of cPLA 2 α inhibitors of Wyeth (1 and 2) and cPLA 2 α inhibitor hits (3 and 4) found in a ligand-based virtual screening approach reacted with N-benzylaniline and the methyl ester group of obtained compound 5 was saponified with aqueous KOH in methanol (Scheme 1). The target compound 11 with a naphthalen-2-yl group instead of the phenyl residue was synthesized analogously using the secondary amine N-benzylnaphthalen-2-amine in the first step. All other N-aryl-Narylmethyl substituted 4-sulfamoylbenzoic acids were prepared in a similar way as outlined for the synthesis of compound 14 in Scheme 2. First, methyl 4-(chlorosulfonyl) benzoate was reacted with aniline or a substituted aniline derivate in dichloromethane and/or THF in the presence of pyridine or triethylamine. Then the benzyl or naphthylmethyl substituent was introduced at the nitrogen by treatment with benzyl chloride or the appropriate (chloromethyl) naphthalene in DMF using K 2 CO 3 as base. Finally, the methylester group was hydrolyzed with aqueous KOH in methanol. Compound 53, substituted at the sulfonamide nitrogen with an indol-3-ylethyl and a naphthalen-2-ylmethyl group, was similarly synthesized using tryptamine instead of an aniline derivative in the first step (Scheme 3). Substitution of the indole nitrogen of 53 with 2,4-dichlorobenzyl or benzyhydryl residues was carried out by reacting the ester intermediate 52 with 2,4-dichlorobenzyl bromide or benzyhydryl bromide in DMF with sodium hydride as base. Under the selected conditions, saponification of the methyl ester groups occurred simultaneously with the formation of the target compounds 54 and 55, respectively (Scheme 3).
The derivatives of 54, in which the N-naphthalen-2-ylmethyl group was replaced by (phenylsulfonylamino)-or (benzylsulfonylamino)alkyl residues were synthesized as shown for compound 63 in Scheme 4. Boc-protected tryptamine was substituted at the indole nitrogen with a 2,4dichlorobenzyl residue by treatment with the corresponding benzyl bromide in DMF using sodium hydride to deprotonate the indole nitrogen. Deprotection of the alkylamine group of the obtained intermediate 56 with trifluoroacetic acid in dichloromethane followed by its reaction with methyl 4-(chlorosulfonyl)benzoate led to the intermediate 58, which was alkylated at the sulfonamide nitrogen with 2-(Boc-amino)ethyl bromide to yield 60. After cleavage of the Boc-protecting group of the alkyl chain amine, a sulfonamide functionality was formed by reaction with benzenesulfonyl chloride in dichloromethane in the presence of triethylamine. Finally, saponification with aqueous KOH in methanol yielded the target compound 63. The corresponding derivative without sulfonylaminoalkyl residue at the sulfamoyl group of the benzoic acid (59) was obtained by alkaline hydrolysis of the intermediate 58.
The derivative of Axon-1609 (2) (Fig. 1), in which the ether oxygen at the benzoic acid residue is replaced by a sulfamoyl group (79), and the corresponding compound, which is additionally methylated at the sulfonamide group (81), were synthesized from the appropriately substituted tryptamine derivative as shown in Scheme 5.
For the synthesis of the two derivatives with 2-hydroxyethyl-and 5-hydroxypentyl-side chain, respectively, at the sulfonamide nitrogen (83 and 85), the N-monosubstituted sulfonamide 78 was reacted with the corresponding ω-bromoalkyl acetate in the presence of K 2 CO 3 (Scheme 6). The ester groups of the intermediates 82 and 84 obtained were saponified with aqueous KOH in methanol to yield the desired target compounds. The preparation of 4-sulfamoyl benzoic acid 88 bearing a more polar hydroxyethylethoxy substituent at the sulfonamide nitrogen is also outlined in Scheme 6. In this case, the sulfonamide 78 was treated with 2-(2-chlororethoxy)ethanol, with the hydroxy group protected as a silyl ether. The alcohol functionality of the substitution product 86 was deprotected with tetrabutyammonium fluoride in THF, and the ester moiety was again cleaved under alkaline conditions to give the test compound 88.

Biochemical activity
The cPLA 2 α used in the biological assay was isolated from porcine platelets. The cPLA 2 α inhibitory activity of the target compounds was evaluated by determining the arachidonic acid release by the enzyme from the substrate 1stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine in the presence of the vesicle forming lipid 1,2-dioleoyl-sn-glycerol by HPLC and MS detection [20]. Deuterated arachidonic acid was used as an internal standard.
The structure of the hit 3 ( Fig. 1) was first simplified by replacing the benzothiophene residue by a phenyl ring and omitting the two substituents of the benzyl residue. In contrast to 3, for which an IC 50 of 19 µM was measured, the derivative thus obtained (6) showed no cPLA 2 α inhibition at the highest test concentration of 33 µM (Table 1). Fusing a benzene ring to the phenyl or benzyl moiety led to the compounds 9, 11, 14 and 16, respectively, which exhibited some activity against the enzyme at 33 µM (35% -48% inhibition).
Next, in the two derivatives with naphthalen-1-ylmethyl and naphthalen-2-ylmethyl moieties (14 and 16), various substituents such as chlorine, methyl, methoxy and phenyl were introduced into the phenyl group at position 4 or the phenyl group was replaced by a benzyl residue. For the compound with the naphthalen-1-ylmethyl substituent (14), these structural changes did not significantly improve the potency ( Table 2). In contrast, more pronounced effects occurred in case of the analogous naphthalen-2-ylmethyl derivative 16. Thus, the cPLA 2 α inhibitory potency increased by a factor of 3 when a chlorine atom was introduced in position 4 of the phenyl residue (IC 50 of 33: 12 µM). A phenyl and a benzyl radical at this position had an analogous effect, as shown by the IC 50 values of the compounds 42 and 45.
Since Wyeth's potent cPLA 2 α inhibitors, which were the starting point for our ligand-based virtual screening for new cPLA 2 α inhibitors, contain a 3-alkyl-substituted indole ring, a derivative of 16 was prepared that beared a 2-(indol-2-yl) ethyl residue in place of the phenyl ring. However, with an IC 50 value of 27 µM this compound (53) was not significantly more potent than 16. The introduction of a 2,4dichlorophenyl moiety, which was present in one of our other screening hits (4) (Fig. 1), at the indole-1 position of 53 resulted in a negligible increase in potency by a factor of about 1.5 (IC 50 of 54: 18 µM). In contrast, compound 55, which like the Wyeth compounds carries a benzhydryl residue at position 1 of the indole, showed a more pronounced inhibitory effect. With an IC 50 value of 5.8 µM, it was the most active inhibitor in this series.
In Wyeth's benzhydrylindoles, a significant increase in cPLA 2 α inhibition was achieved by introducing benzylsulfonylaminoethyl residues in position 2 of the indole [9][10][11]. Therefore, we next attempted to address the binding site of this residue in the enzyme also with our compounds. We began these studies not with the benzyhydryl-substituted derivative 55, but with the slightly less active derivative 54, since we did not want to get too close structurally to the Wyeth compounds. Moreover, we wanted to introduce this residue not at the indole 2-position but at the sulfonamide nitrogen instead of the naphthalen-2-ylmethyl unit (Table 3). This latter residue apparently does not have a positive effect on the cPLA 2 α inhibition of compound 54, since its replacement by hydrogen did not significantly change the activity as shown by the inhibition data of 59. Unfortunately, this structural variation also did not lead to more effective compounds despite the variation in the length of the alkyl spacer between the two sulfonylamino groups and the replacement of the terminal benzyl by a phenyl residue, respectively (Table 3). Thus, the IC 50 s of the investigated compounds 69, 71 and 75 as well as those of the corresponding phenylsulfonylaminoalkyl derivatives 63 and 67 were between 14 µM and 18 µM.
Therefore, we finally decided to align the structure of our 4-sulfamoylbenzoic acid derivatives with that of the Wyeth substances more closely. For this purpose, the benzhydrylsubstituted 5-chloro-2-methylindole derivative 79 and its Nmethylated analogue 81 were synthesized. With an IC 50 of 1.8 µM, compound 79 was significantly more active than the 4-sulfamoylbenzoic acid derivatives synthesized so far ( Table 4). The corresponding compound 81 with additional methyl group on the sulfonamide nitrogen had approximately the same activity (IC 50 : 1.4 µM).
For the 4-sulfamoylbenzoic acid derivatives with N-(dichlorobenzyl)indolylethyl residues, it was shown that the introduction of a second large substituent on the sulfonamide nitrogen, such as naphthylmethyl or benzylsulfonylaminoalkyl, did not significantly affect the cPLA 2 α inhibitory activity of the molecules (Table 3). Therefore, it could be assued that in this area of the molecules, without loss of activity functional groups could be attached with which the physicochemical or pharmacokinetic properties of the inhibitors can be controlled. A disadvantage of the investigated compounds is their extremely high lipophilicity. For example, compounds 79 and 81 have log P values of 5.0 and 5.8, and that of the reference substance Axon-1609 is even 6.2 (Table 4). Based on these observations, the polarity of the compounds should be increased by adding a polar residue to the sulfonamide nitrogen. By introducing a hydroxyethyl moiety to the sulfonamide nitrogen, the log P decreased to 4.6, while the enzyme- inhibitory effect of the obtained compound 83 was maintained (IC 50 : 1.5 µM). With a slightly more polar hydroxyethylethoxy radical (88), the inhibitory effect could even be improved by a factor of 2 (IC 50 : 0.66 µM). The replacement of the ether oxygen atom of this compound by a carbon led, as expected, to a more lipophilic substance (log P of 85: 5.0). At the same time, however, surprisingly, the inhibitory potency of this compound against cPLA 2 α increased markedly. With an IC 50 of 0.25 µM, compound 85 was about as effective as the reference substance Axon-1609. Accordingly, in the case of the N-indolylethyl-substituted 4-sulfamoylbenzoic acid derivatives, it is nevertheless possible to increase the inhibitory effect on cPLA 2 α by adding a second substituent at the sulfonamide nitrogen.

Conclusion
In conclusion, in the case of the hit 3 ( Fig. 1), which was discovered by ligand-based virtual screening, both replacement of the benzothiophene residue with substituted phenyl moieties and substitution of the benzyl residue by naphthylmethyl groups did not significantly improve the compound's cPLA 2 α inhibitory activity. Only a clear structural approximation to the benzhydrylindole-Scheme 6 Reagents and conditions: a 2-Bromoethyl acetate or 5-bromopentyl acetate, K 2 CO 3 , DMF, room temperature, 4 h or 16 h; b aqueous KOH (10%), methanol, THF, room temperature, overnight; c tert-butyl[2-(2-chloroethoxy)ethoxy]diphenylsilane, K 2 CO 3 , DMF, 100 o C, 2 h and room temperature 6 h; d tetrabutylammonium fluoride, THF, room temperature, overnight; e aqueous KOH (10%), methanol, THF, room temperature, overnight substituted benzoic acids such as WAY-196025 [9,10], on which the virtual screening was based, brought about some increase in activity. The most potent derivatives were approximately as active as the reference inhibitor Axon-1609 (2). In this context, however, it should be noted that this compound is reported to be significantly less active than its highly potent derivatives such as WAY-196025 [8,10]. Future studies will attempt to further improve the potency and especially the physicochemical properties of the 4-sulfamoylbenzoic acid derivatives investigated, e.g., by introducing acidic or basic residues on the sulfonamide nitrogen.

General
Column chromatography was performed on silica gel 60, particle size 0.040-0.063 mm, from Macherey & Nagel (Düren, Germany). Melting points were determined on a Büchi B-540 apparatus (Essen, Germany) and are uncorrected. 1 H NMR spectra were recorded on a DD2 spectrometer (400 MHz) or a DD2 spectrometer (600 MHz) from Agilent (Santa Clara, USA). Electron ionization (EI) mass spectra were obtained on a Finnigan (Bremen, Germany) GCQ apparatus. The high resolution mass spectra (HR-MS) were recorded on a Bruker (Bremen, Germany) micrOTOF-Q II spectrometer applying atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). Purity of the target compounds was determined by reversed phase HPLC with UV-detection. The samples were prepared by mixing 20 μL of a 5 mM solution of the respective compound in DMSO with 180 μL of acetonitrile. 5 μL of the solutions was injected into the HPLC-system. Separation was performed using a Nucleosil 100 RP18 3 µm column (3 mm (I.D.) x 125 mm) at a flow rate of 0.4 mL/min with a gradient consisting of acetonitrile/water/trifluoroacetic acid (18:82:0.1 to 86:14:0.1, v/v/v). UV-absorbance was measured at 254 nm. Under these conditions, most compounds had a purity of 95 % or more, with the exception of compounds 86, 88 and 91, whose purity levels were between 90% and 94%. For the syntheses of the target compounds not described below: see Supplementary Material.

Methyl 4-(N-benzyl-N-phenylsulfamoyl)benzoate (5)
A solution of benzylaniline (187 mg, 1.02 mmol) was treated with a mixture of dry dichloromethane/dry THF (4:1, 5 mL) followed by pyridine (1 mL). Then methyl 4-(chlorosulfonyl)benzoate (200 mg, 0.85 mmol) was added portionwise and the obtained mixture was stirred overnight at room temperature. After dilution with aqueous HCl (1 M), the organic phase was separated. The aqueous phase was extracted exhaustively with ethyl acetate. The combined organic phases were washed with water and brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo to yield 5 as a solid (180 mg, 55%). C 21

4-(N-{2-[1-(2,4-Dichlorobenzyl)indol-3-yl]ethyl}sulfamoyl) benzoic acid (59)
A suspension of compound 58 (35 mg, 0.068 mmol) in a mixture of methanol (6 mL) and aqueous KOH solution (20%) (3 mL) was stirred at 70°C for 5 h. The cooled reaction mixture was treated with aqueous HCl solution (3 M) until the product precipitated, and then extracted exhaustively with ethyl acetate. The combined organic phases were dried over anhydrous Na 2 SO 4 and concentrated to give 59 as a solid A solution of 58 (390 mg, 0.75 mmol) in dry dimethylacetamide (8 mL) was successively treated with Cs 2 CO 3 (368 mg, 1.13 mmol) and 2-(Boc-amino)ethyl bromide (203 mg, 0.91 mmol). The obtained suspension was stirred at 60°C for 16 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and washed three times with water. The organic phase was dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The residue was chromatographed on silica gel (cylcohexane to cyclohexane/ethyl acetate, 7:3) to yield 60 as a solid ( To a solution of 60 (150 mg, 0.23 mmol) in dry dichloromethane (5 mL) was added trifluoroacetic acid (0.5 mL). The solution was stirred for 4 h at room temperature. A saturated aqueous NaHCO 3 solution was added in portions until the mixture was slightly alkaline. After exhaustive extraction with ethyl acetate, the combined organic phases were dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to give 61 as an oil (127 mg, quantitative). To a solution of 61 (120 mg, 0.21 mmol) in dry dichloromethane (5 mL) was added triethylamine (0.5 mL) followed by benzenesulfonyl chloride (55 mg, 0.31 mmol). After stirring at room temperature for 16 h, the mixture was diluted with ethyl acetate and washed with aqueous HCl solution (1 M) and water. The organic phase was dried over anhydrous Na 2 SO 4 and the solvent was removed in vacuo. The residue was chromatographed on silica gel (cyclohexane to cyclohexane/ethyl acetate, 6:4) to yield 62 as an oil (75 mg, 50%). C 33 H 31 Cl 2 N 3 O 6 S 2 (700.6); 1 H-NMR To a solution of 76 (150 mg, 0.49 mmol) in dry DMF (5 mL) was added sodium hydride (60% dispersion in mineral oil) (39 mg, 0.98 mmol) in portions under ice cooling in a nitrogen atmosphere. The mixture was stirred for 30 min at room temperature and then benzhydryl bromide (192 mg, 0.78 mmol) was added. After stirring for another 10 min, the reaction mixture was carefully quenched with water, stirred for further 15 min, diluted with ethyl acetate, and washed three times with water. The combined organic phases were dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The residue was chromatographed on silica gel (cyclohexane to cyclohexane/ ethyl acetate, 9:1) to yield 77 as an oil (135 mg, 59%). Compound 77 (100 mg, 0.17 mmol) was saponified according to the procedure described for the synthesis of 59. The product was additionally purified by chromatography on silica gel (cyclohexane to cyclohexane/ethyl acetate/ formic acid, 7:3:0.1) to give 79 as a solid (65 mg, 67%  To a solution of 76 (241 mg, 0.78 mmol) in dry THF (5 mL) was added dropwise, under a nitrogen atmosphere and cooling on ice, a LiAlH 4 solution (1 M in dry THF) (3.12 mL). The mixture was stirred under reflux for 2 h. After cooling to room temperature, the reaction mixture was carefully quenched with water and then treated with aqueous NaOH solution (1 M). The resulting precipitate was filtered off. The filtrate was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to afford 2-(5chloro-2-methylindol-3-yl)-N-methylethan-1-amine as an oil (138 mg, 79%). An aliquot of this compound (130 mg, 0.58 mmol) was dissolved in a mixture of dry dichloromethane/dry THF (8 mL, 4:1) and triethylamine (1 mL  To a solution of 82 (50 mg, 0.076 mmol) in a mixture of methanol and THF (3:1, 8 ml) was added aqueous KOH solution (10%) (4 mL). After stirring at room temperature overnight, the mixture was treated with aqueous HCl solution (3 M) until the product precipitated, and then extracted exhaustively with ethyl acetate. The combined organic phases were dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The residue was chromatographed on silica gel (cyclohexane to cyclohexane/ethyl acetate/ formic acid, 6:4:0.1) to yield 83 as a solid (27 mg, 59%).

Biochemical and physicochemical analysis
Inhibition of cytosolic phospholipase A 2 α (cPLA 2 α) Inhibition of cPLA 2 α was determined according to a published procedure [20]. Briefly, cPLA 2 α isolated from porcine platelets was incubated with co-vesicles consisting of the substrate 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (200 µM) and 1,2-dioleoyl-sn-glycerol (100 µM). The enzyme reactions were terminated after 60 min, and cPLA 2 α activity was measured by quantifying the arachidonic acid released by the enzyme in the presence and the absence of a test compound using reversed-phase HPLC and single quad MS-detection. In parallel, blank incubations were performed in the absence of the enzyme. Inhibition of cPLA 2 α was calculated from the amount of arachidonic acid liberated by the enzyme in the presence and the absence of a test compound (corrected for blank). IC 50 values were determined by probit transformation [26].

Determination of log P values
The partition coefficients (log P) were determined by reversed-phase HPLC according to a published OECD method as recently described [27].
Funding Open Access funding enabled and organized by Projekt DEAL.

Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.