Understanding Chemistry and Unique NMR Characters of Novel Amide

magnetochemistry

Article Understanding Chemistry andand UniqueUnique NMRNMR Characters of Novel Amide andand EsterEster Leflunomide LeﬂunomideAnalogues Analogues

Morkos A. Henen 1,2,*, Abdelrahman Hamdi 1, Abdelbasset A. Farahat 1,3 and Morkos A. Henen 1,2,*, Abdelrahman Hamdi 1, Abdelbasset A. Farahat 1,3 ID and Mohammed A. M. Massoud 1 Mohammed A. M. Massoud 1 1 Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Mansoura University, 1 Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt; [email protected] (A.H.); Mansoura 35516, Egypt; [email protected] (A.H.); [email protected] (A.A.F.); [email protected] (A.A.F.); [email protected] (M.A.M.M.) [email protected] (M.A.M.M.) 2 Department of Molecular Genetics Denver, University of Colorado, Aurora, CO 80045, USA 2 Department of Molecular Genetics, Denver, University of Colorado, Aurora, CO 80045, USA 3 Department of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA 3 Department of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA * Correspondence: [email protected] * Correspondence: [email protected] Received: 17 November 2017; Accepted: 29 November 2017; Published: date Received: 17 November 2017; Accepted: 29 November 2017; Published: 5 December 2017

Abstract: A series ofof diversediverse substitutedsubstituted 5-methyl-isoxazole-4-carboxylic5-methyl-isoxazole-4-carboxylic acid amides, imide and esters in whichwhich thethe benzenebenzene ringring isis monomono oror disubstituteddisubstituted waswas prepared.prepared. Spectroscopic and conformational examination was investigated and a new insight involving steric interference and interesting downﬁelddownfield deviation due to additional diamagnetic anisotropic effect of the amidic carbonyl group and the methine protons in 2,6-diisopropyl-aryl derivative (2) as conformationaly carbonyl group and the methine protons in 2,6-diisopropyl-aryl derivative (2) as conformationaly restricted analogues Leflunomide was discussed. Individual substituent electronic effects through restricted analogues Leﬂunomide was discussed. Individual substituent electronic effects through π π resonance of p-substituents and most stable conformation of compound (2) are discussed. resonance of p-substituents and most stable conformation of compound (2) are discussed.

Keywords: leflunomide derivatives; 2,6-diisopropylphenyl anilide chemical shift abnormalities; Keywords: leﬂunomide derivatives; 2,6-diisopropylphenyl anilide chemical shift abnormalities; 5-methyl-4-isoxazole5-methyl-4-isoxazole derivatives

1. Introduction It is known thatthat isoxazoleisoxazole derivatives show diverse biological activity and are known for their potential use against a broad arrayarray ofof diseasesdiseases includingincluding infectiousinfectious diseases,diseases, parasitic infection and for the area of oncology therapeutic therapeuticss [ 1]. For For example example,, Leflunomide Leﬂunomide (Avara), (Avara), is an immunomodulator immunomodulator which is used to treattreat thethe symptomssymptoms associatedassociated with rheumatoid arthritis RA and psoriatic arthritis (Figure1 1))[ [2].]. Leﬂunomide,Leflunomide, as a small,small, low molecular-weightmolecular-weight isoxazole derivative, is one of the most potent but associated with serious sideside effectseffects [[33].].

Figure 1. LeflunomideLeﬂunomide ( (Avara).Avara).

The importance of the amide group for living organisms can be correlated to some of its chemical properties such as planarity [4], relatively high barrier of rotation around the C–NC–N bond, and its hydrogen bonding donor and acceptor properties. These are the key factors related to determiningdetermining the conformations of protein-proteinprotein-protein complexes, enzymes and other biopolymers like DNA and RNA. Studies on amide derivatives have led to many speculations [5]. As NMR provide providess one of the most sensitive biophysical techniques, NMR studies and utilization of chemical shift parameters are

Magnetochemistry 20172017,, 3,, 41;41; doi:doi:10.3390/magnetochemistry3040041 www.mdpi.com/journal/magnetochemistrywww.mdpi.com/journal/magnetochemistry Magnetochemistry 20172017,, 33,, 4141 2 of 11

increasingly being used to tackle more challenging biological problems. It is well known that the increasingly being used to tackle more challenging biological problems. It is well known that the chemical shift depends on electronic and molecular environments [6]. chemical shift depends on electronic and molecular environments [6]. A high rotational barrier due to the partial double-bond character of tertiary amides leads to the A high rotational barrier due to the partial double-bond character of tertiary amides leads to the geometric and magnetic nonequivalence of the nitrogen-attached groups even when both are the geometric and magnetic nonequivalence of the nitrogen-attached groups even when both are the same. same. Amide and related functional groups are planar and exhibit E/Z (Rotational) isomerism [7]. Amide and related functional groups are planar and exhibit E/Z (Rotational) isomerism [7]. There is a known preference of N-aryl amides to exist in an E (Ar and C=O anti) geometry. The There is a known preference of N-aryl amides to exist in an E (Ar and C=O anti) geometry. The N–Ar rotation barrier of a 2-phenylacetamide analog was reduced from 31 kcal mol−1 in the precursor N–Ar rotation barrier of a 2-phenylacetamide analog was reduced from 31 kcal mol−1 in the precursor to 17 kcal mol−1 in the enolate. This dramatic barrier reduction has implications of both N–Ar and to 17 kcal mol−1 in the enolate. This dramatic barrier reduction has implications of both N–Ar and amide C–N rotations [8]. amide C–N rotations [8]. Functional groups with ‘Nsp2–Ar’ as N-aryl amides often prefer twisted geometries. Both the Functional groups with ‘Nsp2–Ar’ as N-aryl amides often prefer twisted geometries. Both geometry of the N–Ar bond and its rotation barrier are crucial features in areas as diverse as the geometry of the N–Ar bond and its rotation barrier are crucial features in areas as diverse as enzyme/substrate binding [9–11]. enzyme/substrate binding [9–11]. Selectively deshielded aromatic protons in some ortho-substituted acetanilides [12] exhibit Selectively deshielded aromatic protons in some ortho-substituted acetanilides [12] exhibit signals signals at unusually low fields for the aromatic proton adjacent to the acetamido group and for the at unusually low fields for the aromatic proton adjacent to the acetamido group and for the amido amido proton itself. proton itself. Based on these facts, in this study, we synthesized novel Leflunomides, which are based on Based on these facts, in this study, we synthesized novel Leflunomides, which are based bioisosterism [13], by changing the substitution pattern at the 4-position of isoxazole ring of on bioisosterism [13], by changing the substitution pattern at the 4-position of isoxazole ring of Leflunomide to: confer different conformations and electronic environment at the amide group that Leflunomide to: confer different conformations and electronic environment at the amide group would exert some effect on the lipophilicity and enhance the activity of the target molecules. New that would exert some effect on the lipophilicity and enhance the activity of the target molecules. substituents are applied, like for example replacing the lipophilic p-CF3 group with the other electron New substituents are applied, like for example replacing the lipophilic p-CF group with the other withdrawing group, adding the electron donating group at either ortho or para3 position, replacing electron withdrawing group, adding the electron donating group at either ortho or para position, the entire ring with phenylethyl ring, or adopting hybrid pharmacophore like isatin and replacing the entire ring with phenylethyl ring, or adopting hybrid pharmacophore like isatin and benzimidazole nucleus or isosteric replacement of amide by ester (Scheme 1). benzimidazole nucleus or isosteric replacement of amide by ester (Scheme1).

Scheme 1. Synthetic route for novel Leﬂunomide derivatives. Scheme 1. Synthetic route for novel Leflunomide derivatives.

As partpart ofof ourour researchresearch aimingaiming atat thethe synthesissynthesis andand pharmacologicalpharmacological evaluationevaluation ofof diversediverse functionalizedfunctionalized aryl amide and isostericisosteric analoguesanalogues ofof leflunomide,leflunomide, new compoundscompounds have beenbeen investigatedinvestigated forfor further further structure–activity structure–activity relationship relationship (SAR) (SAR) studies. studies. Our Our first publicationfirst publication in this in series this indicatedseries indicated that many that Leflunomide many Leflunomide analogues showedanalogues better showed antifibrotic better activity antifibrotic than Leflunomide activity than [14]. Leflunomide [14].

Magnetochemistry 2017, 3, 41 3 of 11 MagnetochemistryMagnetochemistryMagnetochemistry 2017 20172017,, ,3, 3,,3 ,41, 41 41 33 3of ofof 11 1111

2.2.2. Results ResultsResults and andand Discussion DiscussionDiscussion 2. Results and Discussion 22.2.1..1.1. .Synthetic. SyntheticSynthetic Chemistry ChemistryChemistry 2.1. Synthetic Chemistry InInIn thisthisthis study,study,study, thethethe startingstartingstarting compoundscompoundscompounds assembledassembledassembled bybyby couplingcouplingcoupling thethethe keykeykey intermediate:intermediate:intermediate:intermediate: 55-5-- methylisoxazolemethylisoxazolemethylisoxazoleIn this study,-4--4-4carbonyl--carbonylcarbonyl the starting chloridechloridechloride compounds 77 7 andandand anilines,anilines, assembledanilines, arylarylaryl by ethylamine,ethylamine, couplingethylamine, the isatinisatinisatinisatin key or intermediate:oror phenols,phenols,phenols, inininin 5- dichloromethanedichloromethanedichloromethanemethylisoxazole-4-carbonyl (DCM) (DCM)(DCM) using usingusing chloride trimethylamine trimethylaminetrimethylamine7 and anilines, (TEA) (TEA)(TEA) aryl as ethylamine,asas base basebase [15] [15][15] to isatin toto afford affordafford orphenols, the thethe final finalfinal in products productsproducts dichloromethane 2 2–2–13–1313 inin inin moderatemoderate(DCM)moderate using to toto high highhigh trimethylamine yields yieldsyields (40 (40(40––91%)–91%) (TEA)91%) (Scheme (Scheme(Scheme as base [1, 151,1, Table] TableTable to afford 1). 1).1). The The theThe desiredfinal desireddesired products benzimidazole benzimidazolebenzimidazole2–13 in moderate derivative derivativederivative to high for forfor preparationpreparationyieldspreparation (40–91%) of ofof 4 4 4 (Schemeand andand 13 1313 was 1waswas, Table obtained obtainedobtained1). The inin inindesired a a a good goodgoodbenzimidazole yield yieldyield starting startingstarting derivative from fromfrom heating heatingheating for preparationo oo-phenylenediamine--phenylenediaminephenylenediamine of 4 and (OPDA)(OPDA)13(OPDA)was obtained withwithwith ppp-amino--amino inamino a good ethylbenzoateethylbenzoateethylbenzoate yield starting inininin from thethethe presencepresence heatingpresence o-phenylenediamine ofofof aa a strongstrongstrong dehydratingdehydratingdehydrating (OPDA) agentagentagent with suchsuch p-aminosuch asasas polyphosphoricpolyphosphoricpolyphosphoricethylbenzoate in acid acid theacid presence (PPA) (PPA)(PPA) [16] [16][16] of a or or strongor with withwith dehydrating 4 4-4hydroxybenzaldehyde--hydroxybenzaldehydehydroxybenzaldehyde agent such as polyphosphoricinin inin dimethyl dimethyldimethyl formamide formamideformamide acid (PPA) (DMF) (DMF)(DMF) [16] or usingusingwithusing sodium4-hydroxybenzaldehyde sodiumsodium metabisulfite metabisulfitemetabisulfite as asas oxidizing in oxidizingoxidizing dimethyl agent agentagent formamide [ 17[[1717],],], respectively. respectively.respectively. (DMF) using The TheThe sodium structures structuresstructures metabisulfite of ofof compounds compoundscompounds as oxidizing 2 2–2–13–1313 werewerewereagent approved approvedapproved [17], respectively. on onon the thethe basis basisbasis The of ofof spectralstructures spectralspectral data datadata of (IR, compounds (IR,(IR, mass massmass and andand2 –NMR) NMR)13NMR)were and andand approved elemental elementalelemental on analysis. analysis.analysis. the basis All AllAll of spectral spectralspectral datadatadata were werewere (IR, massinin inin good goodgood and agreement agreementagreement NMR) and with withwith elemental the thethe proposed proposedproposed analysis. structures. structures.structures. All spectral data were in good agreement with the proposed structures. TableTableTable 1. 1.1. Synthesis SynthesisSynthesis of ofof 5 5-5methyl--methylmethyl-4--4-4isoxazoleisoxazole--isoxazoleisoxazole derivatives derivativesderivatives ( 2 ((2–2–13–1313)..) ) .. Table 1. Synthesis of 5-methyl-4-isoxazole derivatives (2–13).

Products -X-Ar * Yield (%) Melting Points (◦C) Colour ProductProductProductss s -X--XX-Ar--ArAr * * * YieldYieldYield (%) (%)(%) MeltingMeltingMelting Points PointsPoints ( ° ((C)°°C)C) ColourColourColour

2 22 2 58585858 145145145 145–146––146–146146 YellowYellowYellow Yellow

3 33 3 68686868 130130130 130–131––131–131131 redredred red

4 44 4 71717171 185185185 185–186––186–186186 graygraygray gray

5 55 5 91919191 148148148 148–149––149–149149 whitewhitewhitewhite

6 66 6 55555555 190190190 190–191––191–191191 redredred red

7 77 7 41414141 898989 89–90––90–9090 yellowyellowyellow yellow

8 88 8 45454545 100100100 100–101––101–101101 yellowyellowyellow yellow

9 99 9 40404040 959595 95–96––96–9696 yellowyellowyellow yellow

101010 484848 808080––81–8181 whitewhitewhite

Magnetochemistry 2017, 3, 41 3 of 11

2. Results and Discussion

2.1. Synthetic Chemistry In this study, the starting compounds assembled by coupling the key intermediate: 5- methylisoxazole-4-carbonyl chloride 7 and anilines, aryl ethylamine, isatin or phenols, in dichloromethane (DCM) using trimethylamine (TEA) as base [15] to afford the final products 2–13 in moderate to high yields (40–91%) (Scheme 1, Table 1). The desired benzimidazole derivative for preparation of 4 and 13 was obtained in a good yield starting from heating o-phenylenediamine (OPDA) with p-amino ethylbenzoate in the presence of a strong dehydrating agent such as polyphosphoric acid (PPA) [16] or with 4-hydroxybenzaldehyde in dimethyl formamide (DMF) using sodium metabisulfite as oxidizing agent [17], respectively. The structures of compounds 2–13 were approved on the basis of spectral data (IR, mass and NMR) and elemental analysis. All spectral data were in good agreement with the proposed structures.

Table 1. Synthesis of 5-methyl-4-isoxazole derivatives (2–13).

Products -X-Ar * Yield (%) Melting Points (°C) Colour

2 58 145–146 Yellow

3 68 130–131 red

4 71 185–186 gray

5 91 148–149 white

6 55 190–191 red

7 41 89–90 yellow

8 45 100–101 yellow Magnetochemistry 2017, 3, 41 4 of 11

9 Table 1. Cont.40 95–96 yellow

Magnetochemistry 2017, 3, 41 4 of 11 MagnetochemistryMagnetochemistry10 102017 2017 ,, 3, 3,, 41, 41 4848 80 80–81–81 whitewhite 44 of of 11 11

111111 11 424242 42 124124 124–125124––125125–125 whitewhitewhitewhite

1212 12 5151 51 188188188––189189–189 BuffBuffBuff Magnetochemistry1212 2017 , 3, 41 5151 188 188–189–189 4 of 11 Buff Buff

13131311 13 42 404040 40 124 –125 150150 150–151150white––151151–151 WhiteWhiteWhite White

12 ** General General* General* General structure structure structure structure is is provided51 isprovided is provided provided on on on 188 ontop top top– top189 of of theof the the table tabletable tableBuff

22.2.22.2.. ..Spectroscopic2 Spectroscopic. Spectroscopic Spectroscopic Examination Examination Examination 2.2. Spectroscopic13 Examination 40 150–151 White

6 DimethylDimethylDimethyl sulfoxide sulfoxide sulfoxide sulfoxide--d-d6- 6d(DMSO) d(DMSO)6 (DMSO)(DMSO) was was was was the the the the solvent solvent solvent solvent of of of ofchoice,choice, choice, choice, not not not not only only only for for for for its its its excellent excellent excellent solvation solvation solvation properties,properties,properties, but but but also also also for for for the the *the Generalfact fact fact that thatstructure that amide amide amide is provided proton proton proton on topchemical chemical chemicalof the table shifts shifts shifts in in in DMSO DMSO DMSO were were were clearly clearly clearly sepa sepa sepa separablerablerablerable fromfrom the the aromatic aromatic region. region. The The downfield downfield chemical chemical shifts shifts in inDMSO DMSO (about (about 12.8 12.8 ppm ppm in case in case of 2– of5)2 are–5) fromfrom2. 2the .the Spectroscopic aromatic aromatic Examination region. region. The The downfield downfield chemical chemical shifts shifts in in DMSO DMSO (about (about 12.8 12.8 ppm ppm in in case case of of 2 2––55) )are are undoubtedlyundoubtedlyundoubtedlyare undoubtedly due due due to to due tohydrogen hydrogen hydrogen to hydrogen bonding bonding bonding bonding of of ofthe the ofthe amide theamide amide amide proton proton proton proton with with with with solvent. solvent. solvent. solvent. The The The The substituents substituents substituents substituents exert exert exert Dimethyl sulfoxide-d6 (DMSO) was the solvent of choice, not only for its excellent solvation relatively small influences influences on the δ of the N N–H–H proton as the anisotropy effect depends on the spatial relativelyrelativelyproperties, small small but influences alsoinfluences for the facton on thethat the δamide δ of of the theproton N N––H chemicalH proton proton shifts as as the thein DMSOanisotropy anisotropy were clearly effect effect sepa depends dependsrable on on the the spatial spatial arrangement,arrangement,arrangement,from the aromatic but but but it region.it is it is independentis independent independentThe downfield of of chemical of the the the nuclei nuclei nucleishifts being inbeing beingDMSO observed observed (about observed 12.8 [18]. [18]. ppm [[18].18 ]. in case of 2–5) are 1 13 undoubtedly due to hydrogen bonding5 3 of the amide3 1 1proton1 with solvent. 4The substituents1313 13 exert TheTheThe chemical chemical chemical shift shift shift of of ofC C5 C C5CH 5CH CH3 3and and3 and C C3 C3H H3 Hin in in1 HH -HH-NMRNMR-NMR-NMR and and and C C4 C4C=O C=O4 C=O in in in 13 C C-NMRC-C-NMRNMR-NMR having having having having nearly nearly nearly nearly the the the samerelatively chemical chemical small influences shift shift value value on the meaning meaning δ of the N a a– similarH similar proton special specialas the anisotropy arrangement arrangement effect depends like like 5 5-Methylisoxazole-4-carboxylic- Methylisoxazoleon the spatial -4-carboxylic samesamearrangement, chemical chemical shiftbut shift it valueis value independent meaning meaning of the a a similar nucleisimilar being special special observed arrangement arrangement [18]. like like 5 5-Methylisoxazole-Methylisoxazole-4-4-carboxylic-carboxylic arrangement, but it is independent of the nuclei13 being observed [18]. acid [ 19]. Due Due to to planar planar delocalization, delocalization,1313 1CC-NMR-NMR chemical chemical shifts shifts13 for for the the amidic amidic CON CON indicates indicates (amidic (amidic acidacid [ 19[19].The]. Due Due chemical to to planar planar shift ofdelocalization, delocalization, C5 CH3 and C3 H inCC -1NMR-HNMR-NMR chemical chemicaland C4 C=O shifts shifts in 13 Cfor for-NMR the the havingamidic amidic nearly CON CON the indicates indicates (amidic (amidic sp2sp2sp2 samecarbon carbon carbon chemical near near near shift188 188 188 valueHz) Hz) Hz) meaningdue due due to to to electronic aelectronic similar electronic special interactions interactions interactions arrangement and and like and steric 5steric- Methylisoxazole steric effects effects effects over over - over4-carboxylic these these thesethese atoms. atoms. atoms.atoms. 13 acidAmidesAmidesAmides [19]. Due 2 2– to–424 –planarand 4and4 and 6 delocalization,6 exert 6exert exert the the the same same 13 same C-NMR sign sign sign chemical for for for angle angle angleshifts Ө Ө for betweenӨ between the between amidic carbonyl carbonylCON carbonyl indicates and and and (amidic isoxazole isoxazole isoxazole or or or aromatic aromatic aromatic sp2 carbon near 188 Hz) due to electronic interactions and steric effects over these atoms. ringringring like like like leflunomide leflunomide leflunomide leflunomide (cis (cis (cis relation relation relation between between between isoxazole isoxazole isoxazole and and and aromatic aromatic aromatic rings). rings). rings). While, While, While, 5 5 and 5and and 7 7– –13713– 13exert exert exert one one one Amides 2–4 and 6 exert the same sign for angle Ө between carbonyl and isoxazole or aromatic opposite signssigns (trans (trans relation relation between between isoxazole isoxazole and aromaticand aromatic rings) asrings) shown as inshown (Table 2in). In(Table compound 2). In oppositeoppositering like signs signsleflunomide (trans(trans (cis relationrelation relation between betweenbetween isoxazole isoxazoleisoxazole and aromatic andand aromaticrings).aromatic While, rings)rings) 5 and 7 as–as13 shown exertshown one inin (Table(Table 2).2). InIn compoundcompoundcompound6,opposite N is imidic 6signs 6, ,N6 N, isN so (transis imidicis theimidic imidic relation lone so so pairsothe thebetween the lone oflone lone electrons pair isoxazolepair pair of of ofelectrons are electrons and electrons delocalized aromatic are are are delocalized rings)delocalized overdelocalized as 2 shown C=O over over groupsoverin 2(Table 2 C=O 2C=O C=O and 2). groups groups thusIngroups theand and and aryl thus thus thus protons the the the arylarylarylarecompound protons protonsmore protons deshielded. are6 are, Nare moreis more imidicmore deshielded. deshielded. so deshielded. the lone pair of electrons are delocalized over 2 C=O groups and thus the aryl protons are more deshielded. Table 2.2. DihedralDihedral angle angle between between C=O C=O and and phenyl phenyl ring, ring, and and Dihedral Dihedral angle angle between C C–O–O and TableTableTable 2.2. 2. Dihedral DihedralDihedral angle angleangle between betweenbetween C=O C=OandC=O phenyl andand phenylring,phenyl and ring, ring,Dihedral andand angle DihedralDihedral between angle angleC–O and betweenbetween CC––OO andand isoxazoleTable 2. Dihedral ring. angle between C=O and phenyl ring, and Dihedral angle between C–O and isoxazoleisoxazoleisoxazoleisoxazole ring ringring ring.. . .

GeneralGeneral StructureStructure Structure GeneralGeneral S Structuretructure Dihedral Angle Dihedral Angle Dihedral AngleDihedralDihedralDihedral between A Angle C=O Anglengle DihedralDihedralDihedralDihedral Angle between Angle Angle Angle C–O between C=ODihedral and Anglebetween C–O andDihedral Angle and Phenylbetween Ring C=O and andbetween Isoxazolering C–O and Phenylbetweenbetween Ring C=O C=O and Isoxazoleringand betweenbetween C C––OO and and PhenylPhenylPhenyl Ring Ring Ring IsoxazoleringIsoxazoleringIsoxazolering Leflunomide Leﬂunomide Leflunomide LeflunomideLeflunomide −30.03−30.03° ◦ −173.3° −173.3◦ −−30.0330.03−30.03° ° ° −173−173−173.3.3° °.3 ° 2 0.09°0.09 ◦ 179.6° 179.6◦ 3 24.5°24.5 ◦ 7.17° 7.17◦ ◦ ◦ 4 19.2119.21° 160.2° 160.2 22 2 ◦0.090.090.09° ° ° 179.6179.6179.6◦ ° ° ° 5 2 −63.3−°63.3 0.09° 8.84° 179.68.84 ° 3 ◦ 24.5° 7.17◦ ° 6 33 19.2119.21° 24.524.5° ° 160.2° 160.27.177.17° ° 7 129.3◦ −164.3◦ 7 44 4 129.3° 19.2119.2119.21° ° ° −164.3° 160.2160.2160.2° ° ° 8 4 −97.8819.21◦ ° 160.2176◦ ° 8 −97.88° 176° 9 55 5 −74◦−63−63−63.3.3° °.3 ° 8.84155.68.848.84° ° ° 9 −74° ◦ 155.6 ◦ 10 66 6 −47 19.2119.2119.21° ° ° 160.216.5160.2160.2° ° ° 10 6 −47° ◦19.21° 16.5° 160.2◦° 11 7 −64 129.3° 177.6−164.3° 1211 77 −64°− 3.08129.3◦ 129.3° ° 177.6° −164−−16420.9.3◦.3° ° 1312 88 8 −3.0833.9° ◦−97−97−97.88.88.88° ° ° −20.9° 4.93176176176◦° ° ° 13 99 9 33.9° −74°−74°−74° 4.93° 155.6155.6155.6

1010 10 −47°−47°−47° 16.516.516.5° ° ° 1111 11 −64°−64°−64° 177.6177.6177.6° ° ° 1212 12 −3−3.08−3.08.08° ° ° −−2020−.920.9° °.9 °

1313 13 33.933.933.9° ° ° 4.934.934.93° ° °

Magnetochemistry 2017, 3, 41 5 of 11 Magnetochemistry 2017, 3, 41 5 of 11

The contributions for for different different anilide anilide groups groups in in the the surroundings surroundings of ofour our system system is relative is relative to correspondingto corresponding chemical chemical shift shift-- for for Hbase Hbase of of acetanilide. acetanilide. For For example, example, compound compound 33 whichwhich hashas substitution in p p-position-position creates a push and pull effect which leads leads to to more more relevant relevant long long-range-range effect on the chemical shift and makes extra stability of the negative charge due to extended resonance (highlighted by by arrows, arrows, as as O O atom atom stabilize stabilize –ve –ve charge charge more more than than N atom N atom in indicated in indicated R groups). R groups). As Asthe thepresence presence of conjugation of conjugation normally normally leads leads to upfield to upfield shift shift of o of-protonso-protons (Figure (Figure 1).1 While). While in in4 the4 the o- protonso-protons is ismore more deshielded deshielded due due to to the the – –II effect effect of of the the positively positively charged charged nitrogen. nitrogen. The The added added parapara-- group should notnot significantlysignificantlyaffect affect either either barriers barriers or or rotamer rotamer populations, populations, and and it isit is present present simply simply as asan an analogue analogue of of leflunomide leflunomide (Figure (Figure2). 2). It isIt is reported reported that that the the1 H1H chemical chemical shiftshift isis notnot asas sensitive as 15NN or or 1313CC to to conjugation, conjugation, and and presence presence of of the the amide amide group group at at the the end end of the conjugation in this case can have the higher hand in effect on the 11H chemical shift value.

Figure 2. EffectEffect of conjugation and para para-substitution-substitution on the barrier or rotamer population.population.

Structures 33,, 44, ,1010, 12, 12 andand 13 13havehave magnetically magnetically equivalents equivalents p-substituted p-substituted phenyl phenyl with high with ortho high couplingortho coupling (J value (J valuebetween between 8 and 88.4 and Hz) 8.4 like Hz) leflunomide. like leflunomide. While While the others the others have havemagnetically magnetically non- equivalent phenyl or substituted phenyl. Structure 11 contains NO2 group which may have a small non-equivalent phenyl or substituted phenyl. Structure 11 contains NO2 group which may have a anisotropicsmall anisotropic effect effectsimilar similar to that to of that C=O of group C=O groupin Structure in Structure 12, with12 ,a with deshielding a deshielding region region in the inplane the planeof aromatic of aromatic ring. The ring. ortho The proton(s) ortho proton(s) relative relative to nitro to or nitro acetyl or group acetyl is group strongly is strongly downfield, downfield, in part duein part to this due interaction. to this interaction. Structure 2 has symmetrically ortho disubstitited but contain containss magnetically non non-equivalent-equivalent meta aryl protons (aromatic protons is away from the carbonyl, and is shifted downfield downfield by 0.3 ppm which is generallygenerally proposed proposed by by dispersion dispersion interactions. interactions. In addition, In addition, the 2-ortho the 2- di-isopropylortho di-isopropyl methine methine protons protonsare non-equivalent are non-equivalent (no plane (no of plane symmetry); of symmetry) chemical; chemical shift is indicated shift is indicated at two multiplets at two multiplets at 4.84 and at 4.845.15. and The 5.15. spectrum The spectrum shows howshows dramatic how dramatic the effect thecan effect be, can indicating be, indicating a quite a quite large large downfield downfield shift shiftrelative relative to the to corresponding the corresponding known known practical practical range or range calculated or calculated values. Thevalues. prediction The prediction of chemical of chemicalshift of the shift isopropyl of the CHisopropyl group wasCH calculatedgroup was using calculated the Curphy-Morrison using the Curphy Additivity-Morrison Constants Additivity for CoProtonnstants NMR for [Proton20]. NMR [20]. 1 The predicted predicted H1H-NMR-NMR chemical chemical shift shift = 1.55 = + 1.55 1.45 += 3 1.45 ppm. = The 3 ppm. actual value The actual was 4.84 value–5.15. was So in4.84–5.15. case of the So inupfield case ofvalue the, upfield ∆δ = 4.84 value, − 3 =∆ 1.84,δ = 4.84 while− 3in = case 1.84 ,of while the downfield in case of value, the downfield ∆δ = 5.15 value, − 3 = 2.15.∆δ = To 5.15 the− 3best = 2.15 of our. To knowledge, the best of our this knowledge, is the first this spectacular is the first report spectacular of such report deviation of such and deviation further studyand further in this study areain is thisneeded. area isIn needed. addition, In addition,examination examination of anticancer of anticancer activity activityof compound of compound 2 using2 Swissusing SwissTarget Target Prediction Prediction software software [21] indicated [21] indicated a very a high very susceptibility high susceptibility to cytochrome to cytochrome P450. P450. The secondarysecondary amide amide is adopting is adopting trans conformation,trans conformation, this means this more means rigidity more and conformational rigidity and conformationalstability [22] Conformational stability [22] Conformational analysis of compound analysis2 using of compound MarvenSuit 2 using software Marven (Marvin Suit 16.7.18.0,software (Marvin2016, ChemAxon, 16.7.18.0, 2016, http://www.chemaxon.com ChemAxon, http://www.chemaxon.com) showed dihedral) showed angle dihedral between angle the planebetween of the planearomatic of the ring aromatic and C=O ring is −and93.64 C=O◦. is −93.64°. 휋 휋 휋 The compoundcompound (2(2)) has has three three different differentπ systems. systems. The Theπ 2 can 2 becan conjugated be conjugated with πwith1 as anilide 1 as (anilide2a), loss (2a of), conjugationloss of conjugation between between nitrogen nitrogen and aryl leadsand aryl to the leads fact to that the the fact compound that the behavescompound as behavesamide rather as amide than aniliderather duethan toanilide the bulky due orthoto the 2,6-diisopropyl bulky ortho 2,6 groups,-diisopropyl or π 3 asgroups, carbonyl or moiety휋 3 as (carbonyl2b), very moiety unstable, (2b has), very possibility unstable, of h diverseas possibility dipolar of interactions diverse dipolar (Figure interactions3). (Figure 3).

Magnetochemistry 2017, 3, 41 6 of 11 MagnetochemistryMagnetochemistryMagnetochemistry 2017, 3, 41 20172017 ,, 33,, 4141 6 of 11 66 ofof 1111

Figure FigureFigure3. Diverse 3.3. DiverseDiverse possible possible possiblepossible dipolar dipolar dipolar dipolarinteraction interaction interactioninteraction (2a and (2a (( 2aand2a2b and)and 2band ) 2b and2b the)) andand the most most thethe stable mostmost stable conformation stablestable conformation conformationconformation with with planar withwith ┴ ┴ planar carbonylplanarcarbonylplanar carbonylcarbonyl group group groupgroup thethe oo┴-diisopropyl-diisopropyl thethe oo--diisopropyldiisopropyl phenylphenyl ringphenylringphenyl ((2c2c ).ringring). ((2c2c).).

Magnetochemistry 2017, 3, 41 1 1 4 of 11 In additionInIn addition addition,addition, the relatively,, the the relatively relatively H-NMR 1HHH-NMR -deshielded-NMRNMR deshieldeddeshielded methine methinemethine proton proton protonon 3° isopropyl on on 3° 33°◦ isopropylisopropylisopropyl carbon carbon carbon(indicated (indicated(indicated → → by )byby in →2 )) cannotin in 22 cannotcannot be explained be be explained explained by co -byplanarity by co co-planarityco--planarityplanarity or private oror privateprivate dipolar dipolardipolar structure structurestructure 2a & 222aaba &or& 222bbybb oror by by hyperconjugationhyperconjugationhyperconjugation of methine ofof methinemethine proton proton protonas indicated asas indicatedindicated by curved byby curvedcurved arrow arrowinarrow 2a. The inin 2a2a diamagnetic.. TheThe diamagneticdiamagnetic anisotropy anisotropyanisotropy 11 hyperconjugation of methine proton42 as indicated by124 curved–125 arrowwhite in 2a . The diamagnetic anisotropy of of the oftheofconjugated thethe conjugated conjugatedconjugated plannar plannar plannarplannar carbonyl carbonyl carbonylcarbonyl group group groupgroupwhich which whichwhichis is nearly nearly isis nearlynearly perpendicularperpendicular perpendicularperpendicular toto the the aromatic totoaromatic thethe aromaticaromatic ring, ring, as indicated as ring,ring, asas indicatedindicatedbyindicated 2cbywhich 2c bywhichby creates 2c2c whichwhichcreates two createscreates additivetwo additive twotwo environments additiveadditive environments environmentsenvironments of diamagnetic of diamagnetic ofof anisotropy,diamagneticdiamagnetic anisotropy, is anisotropy, inanisotropy, close is contactin close isis inin to closeclose the 2 contactcontact to the 2to methine the 2 methine protons. protons. Conformational Conformational analysis analysis of compound of compound 2 using 2 3Dusing molecular 3D molecular model model 12 methinecontact to protons. the 2 methine Conformational protons. Conformational51 analysis of compound188 analysis–189 2ofusing compoundBuff 3D molecular 2 using 3D model molecular examination model examinationexamination resulted resulted in a better in a insightbetter insight (Figure (Figure 4). 4). resultedexamination in a betterresulted insight in a better (Figure insight4). (Figure 4).

13 40 150–151 White

* General structure is provided on top of the table

2.2. Spectroscopic Examination

Dimethyl sulfoxide-d6 (DMSO) was the solvent of choice, not only for its excellent solvation properties, but also for the fact that amide proton chemical shifts in DMSO were clearly separable from the aromatic region. The downfield chemical shifts in DMSO (about 12.8 ppm in case of 2–5) are undoubtedly due to hydrogen bonding of the amide proton with solvent. The substituents exert relatively small influences on the δ of the N–H proton as the anisotropy effect depends on the spatial arrangement, but it is independent of the nuclei being observed [18]. The chemical shift of C5 CH3 and C3 H in 1H-NMR and C4 C=O in 13C-NMR having nearly the same chemical shift valueFigure meaning 4. 3D a similar structure special of compound arrangement2, showing like 5- theMethylisoxazole dihedral angle-4 between-carboxylic C=O group and the Figure Figure4. 3D structure4. 3D structure of compound of compound 2, showing 2, showing the dihedral the dihedral angle betweenangle between C=O group C=O andgroup the and the acid [19]. Due to planar delocalization,benzeneFigure 4. ring. 3D structure 13C-NMR of chemical compound shifts 2, showing for the amidicthe dihedral CON angleindicates between (amidic C=O group and the benzenebenzene ring. ring. sp2 carbon near 188 Hz) duebenzene to electronic ring. interactions and steric effects over these atoms. Amides 2–4 and 6 exert the same sign for angle Ө between carbonyl◦ and isoxazole or aromatic The veryTheThe small very very differencesmall small difference difference of Ө angle of of Ө 3.46°angle is 3.46° 3.46 responsible isis responsible responsible for the for fornon the the-equivalence non non-equivalence-equivalence and difference and and difference difference ring like leflunomide (cisThe relation very between small difference isoxazole of and Ө angle aromatic 3.46° rings). is responsible While,ortho 5 forand the 7– 13non exert-equivalence one and difference in chemicalinin chemical chemical shift of shift the oftwo thethe methine twotwo methinemethine protons. protons. protons. The added The The added addedortho-diisopropyl ortho-diisopropyl-diisopropyl groups groups groups serves serves servesas the as aslock the the lock lock by opposite signs (transin chemicalrelation betweenshift of the isoxazole two methine and aromaticprotons. Therings) added as shown ortho-diisopropyl in (Table 2).groups In serves as the lock by raisingbyraising raising the the N – N–ArtheAr Nrotation rotation–Ar rotation barrier; barrier; barrier; and and isis and proposedproposed is proposed [ 23[23,24],24]. [23,24] Therefore,. Therefore,. Therefore, individual individual individual substituent substituent substituent electronic compound 6, N is imidicby raising so the lonethe N pair–Ar of rotation electronsπ barrier; are delocalized and is proposed over 2 C=O [23,24] groups. Therefore, and thus theindividual substituent electronicelectroniceffects effects through effectsthrough well-deﬁned through well-defined well–resonance-defined π–resonance π units–resonance indicateunits indicateunits that indicate these that units these that behave unitsthese bothbehaveunits as behave isolatedboth as both and as as aryl protons are moreelectronic deshielded. effects through well-defined π–resonance units indicate that these units behave both as isolatedisolatedconjugated and as and conjugated fragments, as conjugated fragments, depending fragments, depending onthe depending substituents. on the onsubstituents. the substituents. isolated and as conjugated fragments, depending on the substituents.1 Linear free energy relationships (LFER) were applied to the H-NMR1 1 spectral data of compounds LinearLinear free energyfree energy relationships relationships (LFER) (LFER) were appliedwere applied to the to Hthe-NMR 1H -NMRspectral spectral data ofdata of Table 2. Dihedral angleLinear between free C=Oenergy and relationshipsphenyl ring, and (LFER) Dihedral were angle applied between to Cthe–O andH -NMR spectral data of compoundscompounds7–13 and7–13 IR and 7 spectral.–13 IR and spectral. IR A spectral. variety A variety of A substituentsvariety of substituents of substituents were were employed employed were employed for phenylfor phenyl for substitution phenyl substitution substitution and fairly isoxazole ring. compounds 7–13 and IR spectral. A variety of substituents were employed for phenyl substitution and fairlyandgood goodfairly correlations correlationsgood correlations were were obtained obtained were using obtained using the simple theusing simple the Hammett simple Hammett andHammett theand Hammett–Taft the and Hammett the Hammett– dualTaft dual substituent–Taft dual and fairly good correlations were obtained using the simple Hammett and the Hammett1 –Taft dual parameter equations [25,26]. The correlation results of the substituent induced H-NMR1 1 chemical substituentGeneralsubstituent parameter Structure parameter equations equations [25,26] [25,26]. The correlation. The correlation results results of the substituentof the substituent induced induced H-NMR 1H -NMR substituent parameter equationsDihedral [25,26] A.ngle The correlationDihedral results Angle of the substituent induced H-NMR chemicalchemical shifts (SCS)shifts of(SCS) the ofCH the3 at CH C5 3isoxazole at C5 isoxazole spins indicatedspins indicated different different sensitivity sensitivity with respect with respect to to chemical shifts (SCS) of the CHbetween3 at C C=O5 isoxazole and spinsbetween indicated C–O anddifferent sensitivity with respect to electronicelectronicelectronic substituent substituentsubstituent effects. effects.effects. The following TheThePhenyl followingfollowing equation Ring equation equation was applied wasIsoxazoleringwas appliedapplied. ..

Leflunomide −30.03° −173.3°

2 0.09° 179.6° 3 24.5° 7.17° 4 19.21° 160.2° 5 −63.3° 8.84° 6 19.21° 160.2° 7 129.3° −164.3° 8 −97.88° 176° 9 −74° 155.6 10 −47° 16.5° 11 −64° 177.6° 12 −3.08° −20.9° 13 33.9° 4.93°

Magnetochemistry 2017, 3, 41 7 of 11

Magnetochemistry 2017, 3, 41 7 of 11 shifts (SCS) of the CH3 at C5 isoxazole spins indicated different sensitivity with respect to electronic substituent effects. The following equation was applied. S = ρσ + h S = ρσ + h S is substituent dependent value (absorption frequency in cm−1, or chemical shift), ρ is the proportionalityS is substituent constant, dependent σ is Hammett value (absorptionconstant, h is frequency the intercept in cm(Table−1, or3 and chemical Figure shift), 5). ρ is the proportionality constant, σ is Hammett constant, h is the intercept (Table3 and Figure5). Table 3. Summary of results of simple Hammett equation fit. Table 3. Summary of results of simple Hammett equation ﬁt. Parameter Value ρ −12.24 ± 1.419 Parameter Value R 0.9370 ρ −12.24 ± 1.419 RSD 1.419 0.9370 SDh 0.169 1.419 h 0.169 Sy.x 0.266 Sy.x 0.266

Simple Hammett equation 4

2 of isoxazole 5 1 at C at 3 CH

δ 0 -0.25 -0.20 -0.15 -0.10 -0.05 0.00 σ

Figure 5. Hammett equation ﬁtfit of the chemical shift of CH33 at C 5 ofof isoxazole isoxazole of of compounds compounds 77––1212..

Detailed studies of this new observation with other o-substituted anilines and conformational determination is essential for biological correlations.

22.3..3. Antioxidant Antioxidant Activity The antioxidant activity of the final ﬁnal compounds 2–13 had been tested using L-ascorbic acid as reference assay in triplicate and average values were considered. The ABTS antioxidant assay [[26]26] is applied as follows:

1. 900900 µLµL of of (ABTS/MnO 2 2mix)mix) was was transferred transferred to to cuvette cuvette of of spectrophotometer spectrophotometer (SPEKOL11) (SPEKOL11) and control thethe absorbance absorbance (A (Acontrol) was) was measured measured at at 734 734 nm nm against against blan blankk (methanol/phosphate (methanol/phosphate buffer buffer (1:1); (1:1); readingreading ca. ca. 0.2. 0.2. 2. 900900 µLµL of of mix mix was was transferred to to 100 µLµL standard ascorbic acid in cuvette and the absorbance waswas measured measured against against blank blank (methanol/phosphate (methanol/phosphate buffer buffer (1:1) (1:1) + + 100 100 µLµL of of ascorbic ascorbic acid). acid). 3. 900 µL of mix to 100 µL of sample was transferred in cuvette and the absorbance (Atest) was 3. 900 µL of mix to 100 µL of sample was transferred in cuvette and the absorbance (Atest) was measuredmeasured against against blank blank (methanol/phosphate (methanol/phosphate buffer buffer (1:1) (1:1) + + 100 100 µLµL of of sample). sample). 4. % Inhibition was calculated using the following equation; % Inhibition = ([Acontrol − Atest]/Acontrol) × 4. % Inhibition was calculated using the following equation; % Inhibition = ([Acontrol − Atest]/Acontrol) 100. × 100. The results of the preliminary qualitative antioxidant screening of 21 compounds are listed in The results of the preliminary qualitative antioxidant screening of 21 compounds are listed in (Table 4). (Table4).

Magnetochemistry 2017, 3, 41 8 of 11

Table 4. Results of the preliminary qualitative antioxidant screening.

Compound A % Inhibition ABTS Control 0.480 0.00 Ascorbic acid 0.059 87.71 Leﬂunomide 0.315 34.38 2 0.360 25.00 3 0.331 31.04 4 0.252 47.50 5 0.351 26.88 6 0.355 26.04 7 0.372 22.50 8 0.385 19.79 9 0.397 17.29 10 0.406 15.42 11 0.332 30.83 12 0.355 26.04 13 0.239 50.2

Most of compounds beside Leﬂunomide showed moderate antioxidant activity. In general:

1. Changing the amide linkage with ester linkage decreased the antioxidant activity more than most of the amide Leflunomide analogues. 2. The benzimidazole derivatives of Leflunomide 4 and 13 showed higher % of inhibition of radical production than Leflunomide. Benzimidazole derivatives are considered to be good chelating agents [27,28], therefore, our finding can pave the way for further in vivo studies of compounds 4 and 13. In addition, this also shows the linkage between antifibrotic and antioxidant activity which has been reported in literature [29].

3. Experimental Section

General Melting points were recorded using a Mel-Temp 3.0 melting point apparatus (Barnstead, Dubuque, IA, USA). IR spectra were performed on a Mattson 5000 FT-IR spectrometer (Fremont, CA, USA) in KBr disks at the Faculty of Pharmacy, Mansoura University. 1H and 13C-NMR spectra were obtained using a Bruker 400 MHz spectrometer (Billerica, MA, USA) and DMSO-d6 as solvent. Mass spectra (m/z) were obtained from the Cairo University Mass Spectrometry Laboratory, Cairo, Egypt. High resolution mass (HRMS) were obtained from Georgia State University, Atlanta, GA 30303-3083, USA. Elemental analysis was done at the Microanalysis Centre, Cairo University, Egypt from a CHNS Elemental Analyser. The major chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fluka (St. Louis, MO, USA). 5-Methylisoxazole-4-carbonyl chloride (1). [18,30]. Thionylchloride (3.53 g, 0.0278 mol) was added to a solution of 5-Methylisoxazole-4-carboxylic acid (2.7 g, 0.0185 mol) in anhydrous dichloromethane (50 mL) with catalytic drops of DMF. The reaction was heated under reflux for 12 h then followed by removing the solvent under reduced pressure. DCM (20 mL) was added and evaporated 3 times to produce a brown oil that was used directly in the next step. General procedure for the synthesis of compounds (2–13). To a stirred solution of the amines or phenols (0.0024 mol) and trimethylamine (0.0025 mol) in dichloromethane (40 mL), 5-methylisoxazole-4-carbonyl-chloride (0.003 mol) was added dropwise at 0–5 ◦C. Then, reaction mixture was refluxed at 40 ◦C for 24 h. After completion of the reaction as indicated from the TLC, the solvent was evaporated under vacuum and the residue was purified using preparative TLC. Magnetochemistry 2017, 3, 41 9 of 11

N-(2,6-Diisopropylphenyl)-5-methylisoxazole-4-carboxamide (2). Using 2,6-diisopropylaniline. IR (KBr) −1 1 υ/cm : 3268, 1606, 1532; H-NMR (DMSO-d6, 400 MHz): δ 1.31 (br t, 12H), 2.64 (s, 3H), 4.84–5.15 (2q, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 8.77 (s, 1H), 12.8 (br s, 1H, D2O exchangeable); m/z: Calcd. for C17H23N2O2: 287.1754; Found: 287.1756 [M + H]+; Anal. Calcd. For C17H22N2O2 (286.37): C, 71.30; H, 7.74; N, 9.78, Found: C, 71.23; H, 7.79; N, 9.75. Ethyl 4-(5-methylisoxazole-4-carboxamido)benzoate (3). Using ethyl aniline p-carboxylate. IR (KBr) −1 1 υ/cm : 3307, 1713, 1639, 1547; H-NMR (DMSO-d6, 400 MHz): δ 1.25 (t, J = 6.8 Hz, 3H), 2.64 (s, 3H), 4.18–4.12 (m, 2H), 6.58 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H) Compare with previous 13 2a, 8.77 (s, 1H), 12.8 (br s, 1H, D2O exchangeable). C-NMR (DMSO-d6, 100 MHz): δ 24.52, 46.06, 61.27, 110.36, 119.91, 120.23, 130.76, 131.41, 141.99, 166.75, 174.26, 187.71; ESI-HRMS: m/z Calcd. + For C14H15N2O4: 275.1105; Found: 275.1107 [M + H] . Anal. Calcd. For C14H14N2O4 (274.27): C, 61.31; H, 5.14; N, 10.21, Found: C, 61.35; H, 5.24; N, 10.11. N-(4-(1H-Benzo[d]imidazol-2-yl)phenyl)-5-methylisoxazole-4-carboxamide (4). Using 4-(1H-Benzo[d] −1 1 imidazol-2-yl)aniline. IR (KBr) υ/cm : 3420, 3345, 1602, 1548; H-NMR (DMSO-d6, 400 MHz): δ 2.51 (s, 3H), 6.57 (s, 2H), 7.23–7.25 (m, 1H), 7.65 (s, 1H), 8 (d, J = 8 Hz, 2H), 8.34 (d, J = 8 Hz, 2H), 8.76 (s, 1H), 12.8 (brs, 1H, D2O exchangeable).; (MS-EI): m/z 318 [M+, 49.64%]. Anal. Calcd. For C18H14N4O2 (318.33): C, 67.91; H, 4.43; N, 17.60, Found: C, 67.85; H, 4.46; N, 17.68. Methyl-N-phenethylisoxazole-4-carboxamide (5). Using phenylethyl amine. IR (KBr) υ/cm−1: 3345, 1593, 1 1555; H-NMR (DMSO-d6, 400 MHz): δ 2.51 (s, 3H), 3.11 (t, J = 7.2 Hz, 2H), 3.72 (t, J = 7.2 Hz, 2H), 13 7.23–7.31 (m, 5H), 8.77 (s, 1H), 12.8 (br s, 1H, D2O exchangeable); C-NMR (DMSO-d6, 100 MHz): δ 22.26, 35.14, 41.14, 116.76, 126.69, 128.84, 129.08, 138.83, 139.36, 169.29, 188.79; (MS-EI): m/z 230 [M+, + 39.46%]; ESI-HRMS: m/z Calcd. for C13H13N2O2Na2: 275.0772; Found: 275.0769 [M − H + Na2] ; Anal. Calcd. For C13H14N2O2 (230.26): C, 67.81; H, 6.13; N, 12.17, Found: C, 67.88; H, 6.23; N, 12.19. Bromo-1-(5-methylisoxazole-4-carbonyl)indoline-2,3-dione (6). Using isatin. IR (KBr) υ/cm−1: 3307, 1650, 1 1639, 1547; H-NMR (DMSO-d6, 400 MHz): δ 2.64 (s, 3H), 8.11 (s, J = 8.4 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.76 (s, 1H,), 8.83 (s, 1H). Anal. Calcd. For C13H7BrN2O4 (335.11): C, 46.59; H, 2.11; N, 8.36, Found: C, 46.69; H, 2.17; N, 8.3. Phenyl 5-methylisoxazole-4-carboxylate (7). Using phenol. IR (KBr) υ/cm−1: 1688, 1575; 1H-NMR (DMSO-d6, 400 MHz): δ 2.64 (s, 3H), 6.58 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.65 (s, 1H), 7.7 (d, J = 8 Hz, 13 1H), 7.9 (s, 1H), 8.77 (s, 1H). C-NMR (DMSO-d6, 100 MHz): δ 22.84, 116.57, 119.25, 122.45, 126.58, 129.9, 150.73, 163.64, 188.98; (MS-EI): m/z 203 [M+, 5.71%]; Anal. Calcd. For C11H9NO3 (203.19): C, 65.02; H, 4.46; N, 6.89, Found: C, 64.90; H, 4.33; N, 6.84. o-Tolyl 5-methylisoxazole-4-carboxylate (8). Using o-cresol. IR (KBr) υ/cm−1: 1683, 1570; 1H-NMR (DMSO-d6, 400 MHz): δ 2.13 (s, 3H), 2.5 (s, 3H), 7.08 (d, J = 7.6 Hz, 1H), 7.15-7.25 (m, 2H), 7.29 (d, J = 6.8 Hz, 1H), 8.77 (s, 1H); (MS-EI): m/z 217 [M+, 13.88%]; Anal. Calcd. For C12H11NO3 (217.22): C, 66.35; H, 5.10; N, 6.45, Found: C, 66.45; H, 5.19; N, 6.50. m-Tolyl 5-methylisoxazole-4-carboxylate (9). Using m-cresol. IR (KBr) υ/cm−1: 1721, 1602; 1H-NMR (DMSO-d6, 400 MHz): δ 2.32 (s, 3H), 2.5 (s, 3H), 6.92–7.03 (m, 2H), 7.08 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 8.77 (s, 1H); (MS-EI): m/z 217 [M+, 13.3%]; Anal. Calcd. For C12H11NO3 (217.22): C, 66.35; H, 5.10; N, 6.45, Found: C, 66.30; H, 5.08; N, 6.41. p-Tolyl 5-methylisoxazole-4-carboxylate (10). Using p-cresol. IR (KBr) υ/cm−1: 1676, 1596; 1H-NMR (DMSO-d6, 400 MHz): δ 2.3 (s, 3H), 2.51 (s, 3H), 7.01 (d, J = 8 Hz, 2H), 7.2 (d, J = 8 Hz, 2H), 8.77 (s, 1H); (MS-EI): m/z 217 [M+, 12.65%]; Anal. Calcd. For C12H11NO3 (217.22): C, 66.35; H, 5.10; N, 6.45, Found: C, 66.21; H, 5.05; N, 6.48. 2-Nitrophenyl 5-methylisoxazole-4-carboxylate (11). Using 2-nitrophenol. IR (KBr) υ/cm−1: 1703, 1528, 1 1348; H-NMR (DMSO-d6, 400 MHz): δ 2.6 (s, 3H), 7.61 (t, J = 8 Hz, 1H), 8.02 (d, J = 8 Hz, 1H), 8.21 (d, Magnetochemistry 2017, 3, 41 10 of 11

J = 8 Hz, 1H), 8.34 (s, 1H), 8.73 (s, 1H); (MS-EI): m/z 248 [M+, 1.61%]; Anal. Calcd. For C11H8N2O5 (248.19): C, 53.23; H, 3.25; N, 11.29, Found: C, 53.32; H, 3.28; N, 11.34. 4-Acetylphenyl 5-methylisoxazole-4-carboxylate (12). Using p-hydroxyacetophenone. IR (KBr) υ/cm−1: 1 1702, 1679, 1591; H-NMR (DMSO-d6, 400 MHz): δ 2.63 (s, 3H), 2.64 (s, 3H), 8 (d, J = 8 Hz, 2H), 8.09 (d, 13 J = 8 Hz, 2H), 8.77 (s, 1H). C-NMR (DMSO-d6, 100 MHz): δ 22.71, 26.91, 115.97, 119.92, 122.56, 126.58, 132, 154.75, 163.97, 187.3, 197.1; (MS-EI): m/z 245 [M+, 3.88%]; Anal. Calcd. For C13H11NO4 (245.23): C, 63.67; H, 4.52; N, 5.71, Found: C, 63.52; H, 4.44; N, 5.59. 4-(1H-Benzo[d]imidazol-2-yl)phenyl 5-methylisoxazole-4-carboxylate (13). Using 2-(4-hydroxyphenyl) −1 1 benzimidazole (0.5 g, 0.0024 mol) as phenol; IR (KBr) υ/cm : 3307, 1639, 1547; H-NMR (DMSO-d6, 400 MHz): δ 2.64 (s, 3H), 6.92 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 3.2 Hz, 2H), 7.54 (d, J = 3.2 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 8.78 (s, 1H). Anal. Calcd. For C18H13N3O3 (319.31): C, 67.71; H, 4.10; N, 13.16, Found: C, 67.74; H, 4.19; N, 13.13.

Acknowledgments: The authors are grateful for Mohamed M. Abu Habib, Pharmacognosy Department, Mansoura University, Egypt, for his valuable help in performing the antioxidant assay. Author Contributions: All Authors contributed equally to the work mentioned here and to the manuscript writing. Conflicts of Interest: Some of the work mentioned here was extracted from the master thesis supervised by the 2nd author (Faculty of Pharmacy, Mansoura University, Egypt 2016). The authors confirm no conflict of interest.

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