Chemical Space Exploration of Oxetanes

International Journal of Molecular Sciences

Article Chemical Space Exploration of Oxetanes

Fernando Rodrigues de Sá Alves 1, Rafael M. Couñago 2 and Stefan Laufer 1,3,* 1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Faculty of Science, Eberhard Karls University of Tübingen, 72074 Tübingen, Germany; [email protected] 2 Centro de Química Medicinal (CQMED), Centro de Biologia Molecular e Engenharia Genética (CBMEG), Structural Genomics Consortium, Departamento de Genética e Evolução, Instituto de Biologia Universidade Estadual de Campinas (UNICAMP), Campinas 13083-875, SP, Brazil; [email protected] 3 Tübingen Center for Academic Drug Discovery, 72076 Tübingen, Germany * Correspondence: [email protected]

Received: 7 October 2020; Accepted: 28 October 2020; Published: 2 November 2020

Abstract: This paper focuses on new derivatives bearing an oxetane group to extend accessible chemical space for further identiﬁcation of kinase inhibitors. The ability to modulate kinase activity represents an important therapeutic strategy for the treatment of human illnesses. Known as a nonclassical isoster of the carbonyl group, due to its high polarity and great ability to function as an acceptor of hydrogen bond, oxetane seems to be an attractive and underexplored structural motif in medicinal chemistry.

Keywords: oxetane; chemical space; nonclassical isosterism; Buchwald–Hartwig reaction; kinases

1. Introduction Oxetane is a four-membered ring having an oxygen atom with an intrinsic ring strain of 1 106 kJ.mol− , which adopts a planar structure with a puckering angle of only 8.7◦ at 140 K (10.7◦ at 90 K). The addition of substituents into the oxetane ring can increment unfavorable eclipsing interactions, resulting in a more puckered conformation [1]. The strained C O C bond angle exposes the oxygen lone pair of electrons, allowing the oxetane − − to act as a good hydrogen-bond acceptor as well as donating electron density as a Lewis base. It was also observed experimentally that oxetanes form more effective H-bonds than other cyclic ethers [2]. Similarly, oxetanes compete as H-bond acceptors with the majority of carbonyl functional groups like aliphatic ketones, aldehydes, and esters [3]. These structural features are important for many of the beneficial properties of substituted oxetanes. Some examples of compounds bearing the oxetane are oxetanocin A, thromboxane A2, mitrephorone A, oxetine and laureatin, as well as the marketed chemotherapy drugs Taxol and Taxotere and a new kinase inhibitor GS-9876 (Figure1). In medicinal chemistry, an oxetane group is employed not only to change the conformational preference of the main scaffold but also to influence physicochemical and biochemical properties such as lipophilicity, aqueous solubility, and metabolic stability [4]. Carreira and co-workers from ETH Zurich have been using oxetanes as surrogates of a carbonyl group in order to generate building blocks providing new opportunities of molecular diversity [5–7]. The group of Professor Bull from Imperial College London has also been working extensively on the synthesis and new methodologies for obtaining compounds bearing an oxetane [8–10]. Recently, Blomgren and co-workers have reported the discovery of a second-generation spleen tyrosine kinase inhibitor, GS-9876 (lanraplenib), which has human pharmacokinetic properties suitable for once-daily administration and it is used against autoimmune diseases [11]. Known as a nonclassical isoster of the carbonyl group [12], due to its high

Int. J. Mol. Sci. 2020, 21, 8199; doi:10.3390/ijms21218199 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 18

Int. J. Mol. Sci. 2020, 21, 8199 2 of 18 polarity and great ability to function as an acceptor of hydrogen bond, the oxetane still remains an underexplored structural motif in drug discovery, especially in the kinase field, in which the oxetane could bind the “hinge” region (Figure2). Nevertheless, the conformational variations or the directional hydrogen bonding differences between sp2- and sp3-hybridized oxygen atoms could be a benefit or a drawbackInt. J. Mol. Sci. for 2020 that, 21 type, x FOR of PEER interaction. REVIEW 2 of 18

Figure 1. Natural products and marketed drugs containing an oxetane group.

In medicinal chemistry, an oxetane group is employed not only to change the conformational preference of the main scaffold but also to influence physicochemical and biochemical properties such as lipophilicity, aqueous solubility, and metabolic stability [4]. Carreira and co-workers from ETH Zurich have been using oxetanes as surrogates of a carbonyl group in order to generate building blocks providing new opportunities of molecular diversity [5–7]. The group of Professor Bull from Imperial College London has also been working extensively on the synthesis and new methodologies for obtaining compounds bearing an oxetane [8–10]. Recently, Blomgren and co-workers have reported the discovery of a second-generation spleen tyrosine kinase inhibitor, GS-9876 (lanraplenib), which has human pharmacokinetic properties suitable for once-daily administration and it is used against autoimmune diseases [11]. Known as a nonclassical isoster of the carbonyl group [12], due to its high polarity and great FigureabilityFigure 2. to2.Schematic Schematicfunction ofasof theanthe acceptorinteractionsinteractions of ofhydrog benzosuberoneen bond, and the oxetane oxetane analogs analogsstill remains in in the the hingean hinge underexplored region. region. structural motif in drug discovery, especially in the kinase field, in which the oxetane could bind the 2. Results and Discussion 2.“hinge” Results region and Discussion (Figure 2). Nevertheless, the conformational variations or the directional hydrogen bondingOur maindifferences goal wasbetween to synthesize sp2- and sp new3-hybridized oxetane derivativesoxygen atoms to furthercould be extend a benefit the or therapeutical a drawback Our main goal was to synthesize new oxetane derivatives to further extend the therapeutical for that type of interaction. kinasekinase space. space. WeWe proposed proposed thethe nonclassicalnonclassical isostericisosteric replacement of the carbonyl carbonyl group group of of some some kinase kinase inhibitorsinhibitors bearingbearing dibenzosuberonedibenzosuberone and benzophenone scaffolds scaﬀolds by by an an oxetane oxetane moiety moiety (Figure (Figure 3).3). ThisThis substitution substitution could could lead lead toto anan interactioninteraction ofof thethe oxetaneoxetane group with the “hinge” region. All All protein protein kinases contain a sequence of amino acids comprising the “hinge” between the two lobes of the catalytic domain. The backbone atoms of the hinge region contain the critical hydrogen bond donor and acceptor atoms anchoring ATP-binding and allowing phosphorylation of the substrate [13].

Figure 2. Schematic of the interactions of benzosuberone and oxetane analogs in the hinge region.

2. Results and Discussion Our main goal was to synthesize new oxetane derivatives to further extend the therapeutical kinase space. We proposed the nonclassical isosteric replacement of the carbonyl group of some kinase inhibitors bearing dibenzosuberone and benzophenone scaffolds by an oxetane moiety (Figure 3). This substitution could lead to an interaction of the oxetane group with the “hinge” region. All protein

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kinaseskinases contain contain a a sequence sequence ofof aminoamino acidsacids comprising the “hinge”“hinge” betweenbetween thethe twotwo lobes lobes of of the the catalytic catalytic Int.domain.domain. J. Mol. The Sci.The2020 backbone backbone, 21, 8199 atoms atoms ofof thethe hingehinge region contain the critical hydrogenhydrogen bondbond donor donor and and acceptor acceptor3 of 18 atomsatoms anchoring anchoring ATP-binding ATP-binding andand allowingallowing phosphorylation ofof thethe substratesubstrate [13].[13].

FigureFigure 3.3. Design of new oxetane derivatives. Figure 3. Design of new oxetane derivatives. 3,3-disubstituted oxetanes can be readily synthesized from 1,3-diols by conversion of the latter 3,3-disubstituted oxetanes can be readily synthesized from 1,3-diols by conversion of the latter into into a3,3-disubstituted halohydrin (or oxetanes another can good be leavingreadily synthesized group) and from then 1,3-diols by addition by conversion of a strong of basethe latter to close into a halohydrin (or another good leaving group) and then by addition of a strong base to close the ring a halohydrin (or another good leaving group) and then by addition of a strong base to close the ring thethrough ring through an intramolecular an intramolecular Williamson Williamson etherification etheriﬁcation reaction [14,15] reaction. However, [14,15]. in However, the case of in 1,3-diols the case through an intramolecular Williamson etherification reaction [14,15]. However, in the case of 1,3-diols ofbearing 1,3-diols two bearing aryl groups, two aryl an groups,1,4-elimination an 1,4-elimination process namely process Grob namely fragmentation Grob fragmentation occurs instead occurs of a bearing two aryl groups, an 1,4-elimination process namely Grob fragmentation occurs instead of a insteadcyclization, of a cyclization,leading to an leading alkene [16–18]. to an alkene Thus, [a16 diff–18erent]. Thus, approach a diﬀ througherent approach a catalytic through functionalization a catalytic cyclization, leading to an alkene [16–18]. Thus, a different approach through a catalytic functionalization functionalizationof tertiary alcohols of was tertiary used alcoholsin order wasto ob usedtain 3,3-diphenyloxetane in order to obtain 3,3-diphenyloxetanederivatives (Figure 4). derivatives (Figureof tertiary4). alcohols was used in order to obtain 3,3-diphenyloxetane derivatives (Figure 4).

Figure 4. Strategies used to synthesize the oxetane scaffolds. Figure 4. Strategies used to synthesize the oxetane scascaffolds.ﬀolds.

Int. J. Mol. Sci. 2020, 21, 8199 4 of 18

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 18 The ketones 1a–c reacted with dimethylsulfonium methylide in a Corey–Chaykovsky epoxidation The ketones 1a–c reacted with dimethylsulfonium methylide in a Corey–Chaykovsky epoxidation reaction [19], leading to the epoxide derivatives 2a–c. In the next step, the aldehydes 3a–c were reaction [19], leading to the epoxide derivatives 2a–c. In the next step, the aldehydes 3a–c were obtained obtained through a Lewis acid catalyzed epoxide rearrangement by a 1,2-hydride shift, namely Meinwald through a Lewis acid catalyzed epoxide rearrangement by a 1,2-hydride shift, namely Meinwald rearrangementrearrangement [20 [20].]. Then Then a a crossed crossed Cannizzaro Cannizzaro reactionreaction was was performe performedd [21], [21], reacting reacting the the aldehydes aldehydes with with formaldehydeformaldehyde in in concentrated concentrated basic basic conditions, conditions, obtainingobtaining the 1,3-diol 1,3-diol derivatives derivatives 4a–c4a–c. For. For the the synthesis synthesis of theof the oxetanes oxetanes applying applying strategy strategy 1, 1, we we usedused thethe conceptconcept of hydroxyl hydroxyl replacement replacement via via oxyphosphonium oxyphosphonium salts,salts, in in which which phosphonium phosphonium salts salts cancan alsoalso bebe form formeded by by reaction reaction of of different different trivalent trivalent phosphorus phosphorus compoundscompounds with with oxidizing oxidizing electrophiles, electrophiles, likelike thethe tris(dimethylamino)phosphine-carbontris(dimethylamino)phosphine-carbon tetrachloride tetrachloride componentcomponent system. system. The The produced produced phosphonium phosphonium intermediates intermediates can can be be trapped trapped with with an an alcohol alcohol to formto alkoxyphosphoniumform alkoxyphosphonium salts, which salts, canwhich then can undergo then unde nucleophilicrgo nucleophilic displacement displacement [22]. Based[22]. Based on the on works the of Castroworks of and Castro Selve and [23 Selve], the [23], dialkyloxetanes the dialkyloxetanes5a–c were 5a–c obtained were obtained in good in yields good yields (Scheme (Scheme1). 1).

SchemeScheme 1. 1.Reagents Reagents and and Conditions: Conditions: (i)(i) (CH(CH33)33SI,SI, NaH, NaH, DMSO, DMSO, r.t. r.t. (ii) (ii) ZnI ZnI2,2 benzene,, benzene, reflux. reflux. (iii) (iii) CH CH2O,2 O, KOH,KOH, ethylene ethylene glycol, glycol, reflux. reflux. (iv) (iv) 1. 1. TDAP,TDAP, CClCCl4, THF, −4040 °C;C; 2. 2. NaOMe, NaOMe, MeOH, MeOH, reflux. reflux. KOH, ethylene glycol, reflux. (iv) 1. TDAP, CCl44, THF, −40 °C;◦ 2. NaOMe, MeOH, reflux.

InIn the the case case of of (5-chloro-2,3-dihydro-1H-indene-1,1-diyl)dimethanol, (5-chloro-2,3-dihydro-1H-indene-1,1-diyl)dimethanol, as as the the Corey–Chaykovsky Corey–Chaykovsky epoxidationepoxidation reaction reaction did did not not work, work, the the 5-chloro-indanone5-chloro-indanone 66 waswas converted converted in in compound compound 7 through7 through a a WittigWittig reaction reaction [24 [24].]. Then Then a a Brown Brown hydroboration hydroboration reactionreaction was was carried carried out out followed followed by by oxidation oxidation of of thethe borate, borate, in in which which the the alkene alkene was was converted converted intointo the primary alcohol alcohol 88, ,using using hydrogen hydrogen peroxide peroxide inin basic basic conditions conditions [25 [25,26].,26]. TheThe alcoholalcohol 8 waswas then oxidized to to the the aldehyde aldehyde 9 9usingusing Dess–Martin Dess–Martin periodinaneperiodinane [27 [27]] and and subsequently subsequently convertedconverted into the diol diol 1010 andand oxetane oxetane 1111 employingemploying the the same same reactionsreactions mentioned mentioned before before (Scheme (Scheme2 ).2).

Scheme 2. Reagents and Conditions: (i) 1. Ph3PMeI, t-BuOK, THF, 0 ◦C; 2. 5-chloro-indanone, 0 ◦C to r.t. (ii) 1. BH3, THF, 0 ◦C; 2. NaOH, H2O2, 0 ◦C—r.t. (iii) Dess–Martin periodinane, CH2Cl2, r.t. (iv) CH O, KOH, ethylene glycol, reﬂux. (v) 1. TDAP, CCl , THF, 40 C; 2. NaOMe, MeOH, reﬂux. 2 4 − ◦ Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 18

Scheme 2. Reagents and Conditions: (i) 1. Ph3PMeI, t-BuOK, THF, 0 °C; 2. 5-chloro-indanone, 0 °C to r.t.

Int. J. Mol.(ii) 1. Sci. BH20203, THF,, 21, 81990 °C; 2. NaOH, H2O2, 0 °C—r.t. (iii) Dess–Martin periodinane, CH2Cl2, r.t. (iv) CH2O,5 of 18 KOH, ethylene glycol, reflux. (v) 1. TDAP, CCl4, THF, −40 °C; 2. NaOMe, MeOH, reflux.

The 3,3-diaryloxetane scascaffoldffold 14 was synthesized based on the works of Croft and co-workers [[28]28] through the the catalytic catalytic functionalization functionalization of a of tertiary a tertiary alcohol alcohol to construct to construct a fully a substituted fully substituted carbon carboncenter, center,using Li using as a catalyst Li as a catalystin a Friedel–Crafts in a Friedel–Crafts alkylation alkylation reaction [29]. reaction A halogen-me [29]. A halogen-metaltal exchange reaction exchange of reactionBuLi and ofcompound BuLi and 12 compound, followed 12by, a followed nucleophilic by a addition nucleophilic of the addition organolithium of the organolithiumto the carbonyl group to the carbonylof the oxetan-3-one, group of the gave oxetan-3-one, rise to compound gave rise 13. toThe compound 1H NMR spectrum13. The 1 Hshows NMR two spectrum doublets shows related two to Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 18 doubletstheInt. methyleneInt. J. J.Mol. Mol. related Sci. Sci. 2020 2020groups to, 21, 21 the, x, xFOR ofFOR methylene oxetane, PEER PEER REVIEW REVIEW groupsat 4.88 and of oxetane, 4.91 ppm. at 4.88Additionally, and 4.91 ppm. the OH Additionally, group could the 5be 5ofOH of 18detected 18 group couldas a broad be detected singlet at as 2.92 a broad ppm. singlet Afterwards, at 2.92 the ppm. tertiary Afterwards, alcohol the13 was tertiary reacted alcohol with13 phenol,was reacted leading with to Scheme 2. Reagents and Conditions: (i) 1. Ph3PMeI, t-BuOK, THF, 0 °C; 2. 5-chloro-indanone, 0 °C to r.t. the diaryloxetaneSchemeScheme 2. 2. Reagents Reagents 14 in anda and 60% Conditions: Conditions: yield. Compound(i) 14(i) 1. 1. Ph Ph3PMeI,3PMeI, t14 -BuOK,t-BuOK, was THF, thenTHF, 0 0converted°C; °C; 2. 2. 5-chloro-indanone, 5-chloro-indanone,14 into the triflate 0 0°C °C to to r.t.derivative r.t. 15 phenol,(ii) leading 1. BH3, THF, to the 0 °C; diaryloxetane 2. NaOH, H2O2, 0 °C—r.t.in a 60% (iii) yield.Dess–Martin Compound periodinane, CHwas2Cl2 then, r.t. (iv) converted CH2O, into the (ii)(ii) 1. 1. BH BH3,3 ,THF, THF, 0 0°C; °C; 2. 2. NaOH, NaOH, H H2O2O2,2 ,0 0°C—r.t. °C—r.t. (iii) (iii) Dess–Martin Dess–Martin periodinane, periodinane, CH CH2Cl2Cl2,2 ,r.t. r.t. (iv) (iv) CH CH2O,2O, triflatein a 62% derivativeKOH, yield ethylene [30]15 (Scheme glycol,in a 62% reflux. 3). yield (v) 1. [ 30TDAP,] (Scheme CCl4, THF,3). −40 °C; 2. NaOMe, MeOH, reflux. KOH,KOH, ethylene ethylene glycol, glycol, reflux. reflux. (v) (v) 1. 1. TDAP, TDAP, CCl CCl4,4 THF,, THF, − 40−40 °C; °C; 2. 2. NaOMe, NaOMe, MeOH, MeOH, reflux. reflux. The 3,3-diaryloxetane scaffold 14 was synthesized based on the works of Croft and co-workers [28] TheThe 3,3-diaryloxetane 3,3-diaryloxetane scaffold scaffold 14 14 was was synthesized synthesized based based on on the the works works of of Croft Croft and and co-workers co-workers [28] [28] through the catalytic functionalization of a tertiary alcohol to construct a fully substituted carbon center, throughthrough the the catalytic catalytic functionalization functionalization of of a a tertiary tertiary alcohol alcohol to to construct construct a a fully fully substituted substituted carbon carbon center, center, using Li as a catalyst in a Friedel–Crafts alkylation reaction [29]. A halogen-metal exchange reaction of usingusing Li Li as as a a catalyst catalyst in in a a Friedel–Crafts Friedel–Crafts alkylation alkylation reaction reaction [29]. [29]. A A halogen-me halogen-metaltal exchange exchange reaction reaction of of BuLi and compound 12, followed by a nucleophilic addition of the organolithium to the carbonyl group BuLiBuLi and and compound compound 12 12, followed, followed by by a a nucleophilic nucleophilic addition addition of of the the organolithium organolithium to to the the carbonyl carbonyl group group of the oxetan-3-one, gave rise to compound 13. The 1H NMR spectrum shows two doublets related to ofof the the oxetan-3-one, oxetan-3-one, gave gave rise rise to to compound compound 13 13. .The The 1 H1H NMR NMR spectrum spectrum shows shows two two doublets doublets related related to to the methylene groups of oxetane, at 4.88 and 4.91 ppm. Additionally, the OH group could be detected thethe methylene methylene groups groups of of oxetane, oxetane, at at 4.88 4.88 and and 4.91 4.91 ppm. ppm. Additionally, Additionally, the the OH OH group group could could be be detected detected asScheme a broad 3.singletReagents at 2.92 and ppm. Conditions: Afterwards, (i) then-BuLi, tertiary oxetan-3-one, alcohol 13 was THF, reacted78 C.with (ii) phenol, Li(NTf leading), Bu NPF to , asasScheme a a broad broad singlet3. singlet Reagents at at 2.92 2.92 and ppm. ppm. Conditions: Af Afterwards,terwards, (i) the nthe-BuLi, tertiary tertiary oxetan-3-one, alcohol alcohol 13 13 was wasTHF, reacted reacted −−78 ◦°C. with with (ii) phenol, phenol,Li(NTf 2leading2 leading), Bu44NPF to to 6, theCHCl diaryloxetane, 40 C. (iii) 14 triflicin a 60% anhydride, yield. Compound pyridine, CH 14 wasCl , then r.t. converted into the triflate derivative 15 thetheCHCl diaryloxetane diaryloxetane3, 40 °C.◦ (iii) 14 14 triflic in in a a 60% anhydride,60% yield. yield. Compound Compoundpyridine, CH 14 14 22wasCl was22, r.t.then then converted converted into into the the triflate triflate derivative derivative 15 15 in a 62% yield [30] (Scheme 3). inin a a 62% 62% yield yield [30] [30] (Scheme (Scheme 3). 3). For the last step, the optimal experimental conditions were established for the Buchwald–Hartwig For the last step, the optimal experimental conditions were established for the Buchwald–Hartwig reaction of the halide scaffolds with arylamines, using a catalyst system composed of Pd(OAc)2, reaction of the halide scaffolds with arylamines, using a catalyst system composed of Pd(OAc)2, XPhos XPhos and Cs2CO3, in a methodology adapted from our research group for dibenzosuberone and Cs2CO3, in a methodology adapted from our research group for dibenzosuberone derivatives [31]. derivatives [31]. For the coupling of the aryl triflate with arylamines, a modified method from For the coupling of the aryl triflate with arylamines, a modified method from Buchwald’s group was Buchwald’s group was employed [32,33], through a catalyst system composed of Pd(OAc)2, BINAP and employed [32,33], through a catalyst system composed of Pd(OAc)2, BINAP and Cs2CO3. No formation Cs2CO3. No formation of phenol was detected. The deprotection of the benzyloxyl group was carried of phenol was detected. The deprotection of the benzyloxyl group was carried out with Pd/C under H2 out withScheme Pd/C 3. under Reagents H andatmosphere Conditions: at(i) r.t.n-BuLi, ina oxetan-3-one, mixture of THF, ethanol −78 /°C.acetonitrile, (ii) Li(NTf2), leadingBu4NPF6, to the final atmosphereSchemeScheme at 3.r.t. 3. Reagents Reagentsin a mixture2 and and Conditions: Conditions: of ethanol/acetonit (i) (i) n n-BuLi,-BuLi, oxetan-3-one, oxetan-3-one,rile, leading THF, THF, to − the78−78 °C. final°C. (ii) (ii) compoundsLi(NTf Li(NTf2),2), Bu Bu4NPF4 NPFin 6quantitative,6 , CHCl3, 40 °C. (iii) triflic anhydride, pyridine, CH2Cl2, r.t. compounds3 in3 quantitative yields. The oxetane2 2 derivatives2 2 were reacted with different arylamines. yields. TheCHClCHCl oxetane,3 40, 40 °C. °C. (iii) (iii)derivatives triflic triflic anhydride, anhydride, were pyridine, pyridine,reacted CH CHwithCl2Cl,2 r.t.,different r.t. arylamines. The 2,4-difluoroaniline was The 2,4-difluoroaniline was initially selected, based on previous works of our laboratory, where it had initiallyFor selected, the last based step, the on optimal previous experimental works of ourcondit laboratory,ions were establishedwhere it had for demonstratedthe Buchwald–Hartwig good potency demonstratedForFor the the last good last step, step, potency the the optimal optimal for some experimental experimental kinase inhibitors condit conditionsions [were34 were]. Thenestablished established other fo modificationsfor rthe the Buchwald–Hartwig Buchwald–Hartwig were carried out for reactionsome kinase of the halideinhibitors scaffolds [34]. with Then arylamines other modifi, usingcations a catalyst were system carried composed out in of the Pd(OAc) arylamine2, XPhos subunit, reactionreaction of of the the halide halide scaffolds scaffolds with with arylamines arylamines, ,using using a a catalyst catalyst system system composed composed of of Pd(OAc) Pd(OAc)2,2 ,XPhos XPhos in theand arylamineCs2CO3, in a subunit, methodology selecting adapted both from electron-withdrawing our research group for dibenzosuberone and donating groups. derivatives Additionally, [31]. selectingandand Cs Cs 2bothCO2CO3,3 electron-withdrawing,in in a a methodology methodology adapted adapted and from from donating our our research research groups. group group Additionally, for for dibenzosuberone dibenzosuberone aniline derivatives wasderivatives chosen [31]. [31]. as a non- anilineFor the was coupling chosen of as the a non-substitutedaryl triflate with arylam groupines, to complete a modified the method design from of newBuchwald’s derivatives group(Table was 1). substitutedForFor the the coupling coupling group ofto of thecomplete the aryl aryl triflate triflate the withde withsign arylam arylam of newines,ines, derivatives a a modified modified method (Tablemethod from1). from Buchwald’s Buchwald’s group group was was employedIn order [32,33], to identify through novel a catalyst sca systemffolds composed for molecular of Pd(OAc) recognition2, BINAP inand kinases, Cs2CO3. No we formation selected some employedemployed [32,33], [32,33], through through a a catalyst catalyst system system composed composed of of Pd(OAc) Pd(OAc)2,2 BINAP, BINAP and and Cs Cs2CO2CO3.3 No. No formation formation compoundsof phenol was to bedetected. screened The indeprot a panelection of of kinasesthe benzyloxyl using group the di wasfferential carried out scanning with Pd/C fluorimetry under H2 (DSF) ofof phenol phenol was was detected. Tabledetected. 1. TheN The-arylamine deprot deprotectionection oxetanes of of the the obtainedbenzyloxyl benzyloxyl via group groupBuchwald–Hartwig was was carried carried out out Reaction. with with Pd/C Pd/C under under H H2 2 atmosphere at r.t. in a mixture of ethanol/acetonitrile, leading to the final compounds in quantitative methodatmosphereatmosphere [35,36 at] at atr.t. r.t. SGC-Unicamp. in in a a mixture mixture of of ethanol/acetonit ethanol/acetonit This techniquerile,rile, allows leading leading selectivity to to the the final final profilingcompounds compounds of in anyin quantitative quantitative protein kinase yields. The oxetane derivatives were reacted with different arylamines. The 2,4-difluoroaniline wasYield withoutyields.Entryyields. prior The The oxetane knowledgeoxetaneHalide/Triflate derivatives derivatives of either were were substrate reacted reacted with orwith activity different different of arylamines. Productarylamines. the kinase The The under 2,4-difluoroaniline 2,4-difluoroaniline investigation. was was The most initially selected, based on previous works of our laboratory, where it had demonstrated good potency(%) a promisinginitiallyinitially selected, resultsselected, based were based on obtained on previous previous for works works the of CAMK1G of our our laboratory, laboratory, calcium where where/calmodulin it it had had demonstrated demonstrated dependent good good protein potency potency kinase I for some kinase inhibitors [34]. Then other modifications were carried out in the arylamine subunit, gammaforfor some some and kinase kinase for AAK1 inhibitors inhibitors AP2-associated [34]. [34]. Then Then other other protein modifi modifi kinasecationscations 1,were were as depictedcarried carried out out in 16a:in in Figure the the W arylamine =arylamine 5H, showing subunit, subunit,86 Tm shifts selecting both electron-withdrawing and donating groups. Additionally, aniline was chosen as a non- selectingselecting both both electron-withdrawing electron-withdrawing and and donating donating groups. groups. Additionally, Additionally, aniline aniline16b: Wwas was = 2,4-di-Fchosen chosen as as a a non- 73non- greatersubstituted than 2group◦C. Theto complete pankinase the de inhibitorsign of new staurosporine derivatives (Table (stauro) 1). served as a positive control in substitutedsubstituted1 group group to to complete complete the the de designsign of of new new derivatives derivatives (Table (Table 1). 1). the assay.substituted group to complete the design of new derivatives (Table 1). 16c: W = 2-CF3 77 Table 1. N-arylamine oxetanes obtained via Buchwald–Hartwig16d: Reaction. W = 4-OCH3 86 TableTable 1. 1. N N-arylamine-arylamine oxetanes oxetanes obtained obtained via via Buchwald–Hartwig Buchwald–Hartwig Reaction. Reaction. Table 1. N-arylamine oxetanes obtained via Buchwald–Hartwig Reaction. Yield Entry Halide/Triflate Product YieldYield EntryEntry Halide/TriflateHalide/Triflate Product Product 17a: W = H (%) a 92 a Entry Halide/Triflate Product(%)(%) Yield a a (%) 17b: W = 2,4-di-F (%) 51 2 16a: W = H 86 16a:16a:16a: W17c: W = =H WH = 2-CF3 8686 86 82 16b: W = 2,4-di-F 73 1 16b:16b:16b: WW17d: W = =2,4-di-F W2,4-di-F = 2-OCH 73373 73 82 11 1 16c: W = 2-CF3 77 16c:16c:16c: WW W = =2-CF 2-CF3 3 7777 77 16d: W = 4-OCH3 86 16d:16d:16d: W W = =4-OCH4-OCH 4-OCH33 3 8686 86 18a: W = H 71 17a: W18b: = H W = 2,4-di-F92 76 3 17a:17a:17a: W W = =H H 9292 92 17b:17b: WW18c: == 2,4-di-FW = 2-CF 3 51 51 95 22 17b:17b: W W = =2,4-di-F 2,4-di-F 5151 2 2 17c:17c: WW18d: == 2-CF W =3 2-OCH823 82 80 17c:17c: W W = =2-CF 2-CF3 3 8282 17d:17d: W = 2-OCH3 82 82 17d:17d: W W = =2-OCH 2-OCH3 3 8282

18a: W = H 71 18a:18a: W W = =H H 7171 18b: W = 2,4-di-F 76 3 18b:18b: W W = =2,4-di-F 2,4-di-F 7676 3 3 18c: W = 2-CF3 95 18c:18c: W W = =2-CF 2-CF3 3 9595 18d: W = 2-OCH3 80 18d:18d: W W = =2-OCH 2-OCH3 3 8080

Int.Int. J. J.Mol. Mol. Sci. Sci. 2020 2020, 21, 21, x, xFOR FOR PEER PEER REVIEW REVIEW 5 5of of 18 18

SchemeScheme 2. 2. Reagents Reagents and and Conditions: Conditions: (i) (i) 1. 1. Ph Ph3PMeI,3PMeI, t-BuOK, t-BuOK, THF, THF, 0 0°C; °C; 2. 2. 5-chloro-indanone, 5-chloro-indanone, 0 0°C °C to to r.t. r.t.

(ii)(ii) 1. 1. BH BH3, 3THF,, THF, 0 0°C; °C; 2. 2. NaOH, NaOH, H H2O2O2, 20, 0°C—r.t. °C—r.t. (iii) (iii) Dess–Martin Dess–Martin periodinane, periodinane, CH CH2Cl2Cl2, 2r.t., r.t. (iv) (iv) CH CH2O,2O, KOH,KOH, ethylene ethylene glycol, glycol, reflux. reflux. (v) (v) 1. 1. TDAP, TDAP, CCl CCl4, 4THF,, THF, − 40−40 °C; °C; 2. 2. NaOMe, NaOMe, MeOH, MeOH, reflux. reflux.

TheThe 3,3-diaryloxetane 3,3-diaryloxetane scaffold scaffold 14 14 was was synthesized synthesized based based on on the the works works of of Croft Croft and and co-workers co-workers [28] [28] throughthrough the the catalytic catalytic functionalization functionalization of of a atertiary tertiary alcohol alcohol to to construct construct a afully fully substituted substituted carbon carbon center, center, usingusing Li Li as as a acatalyst catalyst in in a aFriedel–Crafts Friedel–Crafts alkylation alkylation reaction reaction [29]. [29]. A A halogen-me halogen-metaltal exchange exchange reaction reaction of of BuLiBuLi and and compound compound 12 12, followed, followed by by a anucleophilic nucleophilic addition addition of of the the organolithium organolithium to to the the carbonyl carbonyl group group ofof the the oxetan-3-one, oxetan-3-one, gave gave rise rise to to compound compound 13 13. The. The 1 H1H NMR NMR spectrum spectrum shows shows two two doublets doublets related related to to thethe methylene methylene groups groups of of oxetane, oxetane, at at 4.88 4.88 and and 4.91 4.91 ppm. ppm. Additionally, Additionally, the the OH OH group group could could be be detected detected asas a abroad broad singlet singlet at at 2.92 2.92 ppm. ppm. Af Afterwards,terwards, the the tertiary tertiary alcohol alcohol 13 13 was was reacted reacted with with phenol, phenol, leading leading to to thethe diaryloxetane diaryloxetane 14 14 in in a a60% 60% yield. yield. Compound Compound 14 14 was was then then converted converted into into the the triflate triflate derivative derivative 15 15 inin a a62% 62% yield yield [30] [30] (Scheme (Scheme 3). 3).

SchemeScheme 3. 3. Reagents Reagents and and Conditions: Conditions: (i) (i) n -BuLi,n-BuLi, oxetan-3-one, oxetan-3-one, THF, THF, − 78−78 °C. °C. (ii) (ii) Li(NTf Li(NTf2),2 ),Bu Bu4NPF4NPF6, 6,

CHClCHCl3, 340, 40 °C. °C. (iii) (iii) triflic triflic anhydride, anhydride, pyridine, pyridine, CH CH2Cl2Cl2, 2r.t., r.t.

ForFor the the last last step, step, the the optimal optimal experimental experimental condit conditionsions were were established established fo for rthe the Buchwald–Hartwig Buchwald–Hartwig reactionreaction of of the the halide halide scaffolds scaffolds with with arylamines arylamines, using, using a acatalyst catalyst system system composed composed of of Pd(OAc) Pd(OAc)2,2 XPhos, XPhos andand Cs Cs2CO2CO3,3 in, in a amethodology methodology adapted adapted from from our our research research group group for for dibenzosuberone dibenzosuberone derivatives derivatives [31]. [31]. ForFor the the coupling coupling of of the the aryl aryl triflate triflate with with arylam arylamines,ines, a amodified modified method method from from Buchwald’s Buchwald’s group group was was employedemployed [32,33], [32,33], through through a acatalyst catalyst system system composed composed of of Pd(OAc) Pd(OAc)2,2 BINAP, BINAP and and Cs Cs2CO2CO3.3 No. No formation formation ofof phenol phenol was was detected. detected. The The deprot deprotectionection of of the the benzyloxyl benzyloxyl group group was was carried carried out out with with Pd/C Pd/C under under H H2 2 atmosphereatmosphere at at r.t. r.t. in in a amixture mixture of of ethanol/acetonit ethanol/acetonitrile,rile, leading leading to to the the final final compounds compounds in in quantitative quantitative yields.yields. The The oxetane oxetane derivatives derivatives were were reacted reacted with with different different arylamines. arylamines. The The 2,4-difluoroaniline 2,4-difluoroaniline was was initiallyinitially selected, selected, based based on on previous previous works works of of our our laboratory, laboratory, where where it it had had demonstrated demonstrated good good potency potency forfor some some kinase kinase inhibitors inhibitors [34]. [34]. Then Then other other modifi modificationscations were were carried carried out out in in the the arylamine arylamine subunit, subunit, selectingselecting both both electron-withdrawing electron-withdrawing and and donating donating groups. groups. Additionally, Additionally, aniline aniline was was chosen chosen as as a anon- non- substitutedsubstituted group group to to complete complete the the de designsign of of new new derivatives derivatives (Table (Table 1). 1).

TableTable 1. 1. N N-arylamine-arylamine oxetanes oxetanes obtained obtained via via Buchwald–Hartwig Buchwald–Hartwig Reaction. Reaction.

YieldYield EntryEntry Halide/TriflateHalide/Triflate Product Product (%)(%) a a

16a:16a: W W = =H H 8686 16b:16b: W W = =2,4-di-F 2,4-di-F 7373 1 1 16c:16c: W W = =2-CF 2-CF3 3 7777 16d:16d: W W = =4-OCH 4-OCH3 3 8686

Int. J. Mol. Sci. 2020, 21, 8199 6 of 18 17a:17a: W W = =H H 9292 17b:17b: W W = =2,4-di-F 2,4-di-F 5151 2 2 Table 1. Cont. 17c:17c: W W = =2-CF 2-CF3 3 8282 17d:17d: W W = =2-OCH 2-OCH3 3 8282 a Entry Halide/Triﬂate Product Yield (%) 18a:18a:18a: W W = =HH H 7171 71 18b:18b:18b: W W = =2,4-di-F 2,4-di-F 7676 76 33 3 18c:18c:18c: W W = =2-CF 2-CF33 3 9595 95 Int.Int.Int.Int. J. J. J. Mol. J.Mol. Mol. Mol. Sci. Sci. Sci. Sci. 2020 2020 2020 2020, ,21 ,21 ,21 ,21 ,x ,x ,FORx xFOR FOR FOR PEER PEER PEER PEER REVIEW REVIEW REVIEW REVIEW 66 of6 6of of of18 18 18 18 18d:18d: W W = =2-OCH2-OCH 2-OCH33 3 8080 80

19a:19a:19a:19a:19a: W WW WW = = =H =H H H 83838383 83 19b:19b:19b:19b:19b: W WW WW = == =2,4-di-F =2,4-di-F 2,4-di-F2,4-di-F 71717171 71 444 4 4 19c:19c:19c:19c:19c: W WW WW = == =2-CF =2-CF 2-CF2-CF3 3 33 88888888 88 19d:19d:19d:19d:19d: W W WW = = =2-OCH =2-OCH 2-OCH2-OCH3 3 33 81818181 81

OOOO 20a:20a:20a:20a:20a: W WW WW = = =H =H H H 85858585 85 20b: W = 2,4-di-F 66 b 20b:20b:20b:20b: W W WW = = =2,4-di-F =2,4-di-F 2,4-di-F2,4-di-F 66666666 5 bbb 20c: W = 4-CF3 71 55 5b 5 20c:20c:20c:20c: W W WW = = =4-CF =4-CF 4-CF4-CF3 3 33 71717171 20d: W = 4-OCH 54 N 3 HOHOHOHO N NN 20d:20d:20d: W WW = = =4-OCH 4-OCH4-OCH3 3 33 545454 HHHH WWWW 20d:20e: W W == 4-OCH4-CN 54 70 20e:20e:20e:20e: W W WW = = =4-CN =4-CN 4-CN4-CN 70707070 a Isolated yields after purification by column chromatography. b Products obtained after deprotection of benzyl ethera aIsolated aIsolateda Isolated group.Isolated yields yields yields yields after after after after purification purification purification purification by by by by column column column column chromatography. chromatography. chromatography. chromatography. b b Productsb Productsb Products Products obtained obtained obtained obtained after after after after deprotection deprotection deprotection deprotection ofofof ofbenzyl benzyl benzylbenzyl ether ether etherether group. group. group.group. 4-(3-phenyloxetan-3-yl)phenol derivatives 20c and 20e showed a ∆Tm of 5.8 ◦C and 5.2 ◦C at 10 µM,In respectivelyInInIn order order orderorder to to toto identify identify identifyidentify against novel novel novelnovel CAMK1G scaffolds scaffolds scaffoldsscaffolds (Figure for for forfor molecular molecular molecularmolecular6). These recognition recognition recognitionrecognition results suggest in in inin kinases, kinases, kinases,kinases, that we we electron-withdrawingwewe selected selected selectedselected some some somesome compoundscompounds to to be be screened screened in in a apa panelnel of of kinases kinases using using the the differential differential scanning scanning fluorimetry fluorimetry (DSF) (DSF) groupscompoundscompounds on the arylto to be be ring screened screened are well in in a tolerated, apa panelnel of of kinases inkinases which using using the the 4-CF the differential differential3 and 4-CN scanning scanning analogs fluorimetry fluorimetry showed similar (DSF) (DSF) values. methodmethodmethodmethod [35,36] [35,36] [35,36][35,36] at at at at SGC-Unicamp. SGC-Unicamp. SGC-Unicamp.SGC-Unicamp. This This ThisThis technique technique techniquetechnique allows allows allowsallows selectivity selectivity selectivityselectivity profiling profiling profilingprofiling of of ofof any any anyany protein protein proteinprotein kinase kinase kinasekinase However, both an electron-donating group like 4-OCH3 (20d) and the group 2,4-difluoryl (20b) were without prior knowledge of either substrate or activity of the kinase under investigation. The most detrimentalwithoutwithoutwithout prior priorprior to CAMK1Gknowledge knowledgeknowledge interaction.of ofof either eithereither substrate substratesubstrate Calcium–calmodulin or oror activity activityactivity of ofof the thethe kina kina dependentkinasesese under underunder investigation. protein investigation.investigation. kinase The TheThe I most (CaMKI) mostmost is promisingpromisingpromisingpromising results results resultsresults were were werewere obtained obtained obtainedobtained for for forfor the the thethe CAMK1G CAMK1G CAMK1GCAMK1G calcium/calmodulin calcium/calmodulin calcium/calmodulincalcium/calmodulin dependent dependent dependentdependent protein protein proteinprotein kinase kinase kinasekinase I I I I part of a calmodulin-dependent protein kinase cascade and is implicated in various physiological gammagammagammagamma and and and and for for for for AAK1 AAK1 AAK1 AAK1 AP2-associated AP2-associated AP2-associated AP2-associated protein protein protein protein kinase kinase kinase kinase 1, 1, 1, 1,as as as as depicted depicted depicted depicted in in in inFigure Figure Figure Figure 5, 5, 5, 5,showing showing showing showing Tm Tm Tm Tm shifts shifts shifts shifts greater greater greater greater processes, being expressed in many tissues. CaMKI regulates transcriptional activator activity, the cell thanthanthanthan 2 2 2°C. 2°C. °C.°C. The The TheThe pankinase pankinase pankinasepankinase inhibitor inhibitor inhibitorinhibitor staurosporine staurosporine staurosporinestaurosporine (sta (sta (sta(stauro)uro)uro)uro) served served servedserved as as asas a a apositive apositive positivepositive control control controlcontrol in in in inthe the thethe assay. assay. assay.assay. cycle, cell differentiation, actin filament organization and neurite outgrowth [37]. However, the role of CAMK1G is still mainly unclear. Compounds 20c and 19b showed a ∆Tm of 5.2 ◦C and 4.5 ◦C, respectively against AAK1 (Figure7). Adaptor-associated kinase 1 (AAK1), a member of Ark1/Prk1 family of serine/threonine kinases, is involved in the endocytic pathway. Although AAK1 has been mostly related to the regulation of receptor endocytosis, several studies suggested that AAK1 may be associated to psychiatric and neurological disorders [38]. Recently, Kostich and co-workers have showed that the inhibition of AAK1 kinase could be a novel therapeutic approach for the treatment of neuropathic pain [39]. The inhibition of AAK1 also showed to be important as an antivirus strategy, especially against HCV [40,41]. In addition to these preliminary results, new structural modifications and experiments to measure the Kd and IC50 will be carried out for a better understanding of the structure-activity relationship.

Int. J. Mol. Sci. 2020, 21, 8199 7 of 18 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 18

Kinases Compounds 16b 20b 20d 20c 20e 19b 17b 18b stauro DMSO AAK1 2.78 2.12 0.11 5.20 2.60 4.54 2.65 1.69 8.13 0.00 BMP2K 0.80 0.29 0.01 1.19 0.47 0.39 0.37 0.28 7.34 0.00 BMX -0.23 -0.36 0.00 0.47 0.23 0.13 0.18 0.56 4.26 0.00 BRAF 0.23 0.23 0.22 0.26 0.15 0.15 0.24 -0.02 0.00 CAMK1D -1.49 -1.62 -1.60 -1.41 -1.93 0.41 -0.38 -0.63 3.71 0.00 CAMK1G -0.401.241.245.825.231.191.140.944.980.00 CAMKK1 -1.87 -1.69 -2.07 -1.81 -1.93 -1.56 -0.68 5.35 0.00 CAMKK2 -0.25 -0.09 -0.22 1.82 0.33 0.56 0.54 0.06 15.90 0.00 CDC42BPA 0.29 -0.22 -0.20 -0.13 0.26 0.14 0.02 0.14 0.92 0.00 CDK2 2.20 2.10 2.20 2.53 1.97 1.95 2.21 2.35 6.99 0.00 CDKL1 0.22 0.31 0.26 0.23 0.15 0.62 0.07 0.33 0.93 0.00 CHEK2 -0.06 -0.61 -0.29 0.48 -0.49 -0.26 0.29 0.11 0.87 0.00 CLK1 0.21 -0.36 -0.30 2.00 -0.10 -0.37 0.01 0.19 6.36 0.00 CSNK1G1 0.63 0.60 0.74 0.44 0.46 0.37 0.22 0.62 0.23 0.00 CSNK1G3 -0.38 -0.37 -0.07 -0.07 -0.35 -0.07 0.03 -0.10 0.04 0.00 CSNK2A1 0.26 0.27 0.24 0.22 0.29 0.31 0.08 0.55 0.26 0.00 DYRK1 -1.06 -1.24 -0.78 -1.59 -1.14 -0.50 -0.41 -0.65 2.20 0.00 DYRK2 0.24 0.35 0.45 0.65 0.22 0.19 0.32 0.34 1.23 0.00 EPHA2 -0.21 -0.14 -0.08 0.40 -0.05 -0.42 -0.39 -0.16 1.79 0.00 GAK -0.99 -1.21 -1.47 -1.51 -1.22 -0.82 -0.30 -0.82 3.68 0.00 GSG2 0.08 0.12 -0.38 -13.16 -6.62 0.21 -0.33 0.16 4.57 0.00 MAPK1 -0.51 -0.33 -0.66 -0.33 -0.29 -0.46 -0.30 -0.32 -0.21 0.00 MAPK14B -0.13 -0.68 -1.28 -0.38 -0.82 -0.66 -0.26 -0.50 -0.63 0.00 MAPK3 0.50 0.03 -0.53 0.41 -0.28 -0.10 -0.31 0.23 1.14 0.00 PHKG2A 0.34 0.94 0.84 1.72 1.37 1.16 1.30 0.76 16.68 0.00 PIM1 -0.27 -0.72 -1.41 0.24 -0.41 -0.53 -0.86 -0.60 4.67 0.00 PKMYT1A -2.48 -2.17 -1.63 -1.46 -1.50 -1.28 -1.01 -1.48 -0.01 0.00 PRPF4B -0.97 -0.41 -0.21 0.28 -0.30 -0.57 -0.56 0.00 0.12 0.00 RPS6KA1A -1.20 -1.21 -0.46 -1.53 -0.24 -0.39 0.05 -0.91 -0.12 0.00 RPS6KA5A -0.70 -0.96 -0.85 -2.20 -1.34 0.20 -0.14 -0.70 3.74 0.00 RPS6KA6A -3.26 -3.90 -1.70 -4.42 0.06 -2.54 -2.60 -2.75 8.94 0.00 SLK -1.85 -2.98 -1.85 -0.57 -2.06 0.09 -1.74 15.03 0.00 SRPK1 -0.55 -0.92 -0.66 -0.97 -0.12 -0.60 -0.71 2.31 0.00 SRPK2 -0.77 -1.09 -0.79 -1.42 -1.08 -0.41 -0.63 -0.57 0.10 0.00 STK3 -0.24 -0.66 -0.65 -0.51 0.29 -0.47 0.09 0.67 0.00 STK6 0.06 0.25 -0.71 0.24 -0.24 -0.82 0.01 11.88 0.00 STK10 -0.11 -1.19 -0.51 -0.31 0.53 0.20 22.26 0.00 STK38 -0.75 -0.22 -0.26 0.69 0.25 0.53 -0.01 0.03 5.12 0.00 TRIB2 -0.69 -0.74 -3.36 -1.69 -0.89 -0.49 -1.46 -0.98 -0.41 0.00 TTKA -1.53 -1.12 -1.20 -0.70 -0.76 -0.06 1.68 0.00 WNK3 -0.59 -0.31 -0.25 -0.60 -0.42 -0.28 -0.25 -0.97 -0.46 0.00 VRK1 0.76 -0.20 0.08 0.38 -0.34 0.41 1.43 0.58 0.00 VRK2 -0.36 -1.32 -1.36 -2.29 1.17 -1.28 -0.75 0.05 0.92 0.00 Figure 5. ResultsResults obtained obtained from a kinase panel usin usingg differential diﬀerential scanning ﬂuorimetryfluorimetry (DSF).

4-(3-phenyloxetan-3-yl)phenol derivatives 20c and 20e showed a ΔTm of 5.8 °C and 5.2 °C at 10 µM, respectively against CAMK1G (Figure 6). These results suggest that electron-withdrawing groups on the aryl ring are well tolerated, in which the 4-CF3 and 4-CN analogs showed similar values. However, both an electron-donating group like 4-OCH3 (20d) and the group 2,4-difluoryl (20b) were detrimental to CAMK1G interaction. Calcium–calmodulin dependent protein kinase I (CaMKI) is part of a calmodulin-dependent protein kinase cascade and is implicated in various physiological processes, being expressed in many tissues. CaMKI regulates transcriptional activator activity, the

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 18

cell cycle, cell differentiation, actin filament organization and neurite outgrowth. [37]. However, the role of CAMK1G is still mainly unclear.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 18 cell cycle, cell differentiation, actin filament organization and neurite outgrowth. [37]. However, the Int. J. Mol.Figure Sci. 2020 6. ,∆21Tm, 8199 values obtained for the CAMK1G calcium/calmodulin dependent protein kinase I gamma.8 of 18 role of CAMK1G is still mainly unclear. Compounds 20c and 19b showed a ΔTm of 5.2 °C and 4.5 °C, respectively against AAK1 (Figure 7). Adaptor-associated kinase 1 (AAK1), a member of Ark1/Prk1 family of serine/threonine kinases, is involved in the endocytic pathway. Although AAK1 has been mostly related to the regulation of receptor endocytosis, several studies suggested that AAK1 may be associated to psychiatric and neurological disorders [38]. Recently, Kostich and co-workers have showed that the inhibition of AAK1 kinase could be a novel therapeutic approach for the treatment of neuropathic pain [39]. The inhibition of AAK1 also showed to be important as an antivirus strategy, especially against HCV [40,41]. In addition to these preliminary results, new structural modifications and experiments to measure the Kd and IC50 will be carried out for a better understanding of the structure-activity relationship.Figure 6. ∆∆TmTm values values obtained obtained for for the the CAMK1G CAMK1G calcium/calmodulin calcium/calmodulin dependent dependent protein protein kinase kinase I I gamma. gamma.

Compounds 20c and 19b showed a ΔTm of 5.2 °C and 4.5 °C, respectively against AAK1 (Figure 7). Adaptor-associated kinase 1 (AAK1), a member of Ark1/Prk1 family of serine/threonine kinases, is involved in the endocytic pathway. Although AAK1 has been mostly related to the regulation of receptor endocytosis, several studies suggested that AAK1 may be associated to psychiatric and neurological disorders [38]. Recently, Kostich and co-workers have showed that the inhibition of AAK1 kinase could be a novel therapeutic approach for the treatment of neuropathic pain [39]. The inhibition of AAK1 also showed to be important as an antivirus strategy, especially against HCV

[40,41]. In additionFigure to 7. these∆Tm valuespreliminary obtained results, for the new AAK1 structural AP2-associated modifications protein kinase and 1. experiments to measure the Kd andFigure IC 507. will∆Tm bevalues carried obtained out forfor the a AAK1better AP2-associatedunderstanding protein of the kinase structure-activity 1. relationship.3. Materials and Methods 3. Materials and Methods All commercially available reagents and solvents were used without further purification. 1 13 H and AllC commercially nuclear magnetic available resonance reagents (NMR) and solven spectrats were used determined without in further DMSO-d purification.6, CDCl3 and1H and 1 13 Acetone-d13C nuclear6 solutions magnetic using resonance a Bruker (NMR) Avance spectra 200 were ( H determined= 200 MHz; in DMSO-dC = 506, CDCl MHz)3 and or a Acetone-d Bruker 6 Avancesolutions 400 (using1H = 400a Bruker MHz ;Avance13C = 100200 MHz)(1H = 200 spectrometer. MHz; 13C = 50 The MHz) chemical or a Bruker shifts areAvance given 400 in (1 partsH = 400 perMHz; million 13C (ppm)= 100 MHz) from solventspectrometer. residual The peaks chemical and theshifts coupling are given constant in parts values per million (J) are (ppm) given infrom Hz.solvent Signal residual multiplicities peaks areand represented the coupling by: constant s (singlet), values d (doublet), (J) are given dd (doublein Hz. Signal doublet), multiplicities t (triplet), are m (multiplet)represented and by: br s (broad(singlet), signal). d (doublet), The evaluation dd (double of spectradoublet), was t (triplet), performed m using(multiplet) the MestReNova and br (broad program.signal). Infrared The evaluation spectra of were spectra obtained was performed using a Thermo using the Scientific MestReNova Nicolet’s program. Avatar iS10Infrared spectrometer spectra were equippedobtained with using a smart a Thermo endurance Scientific diamond Nicolet’s ATR Avatar unit iS10 for directspectrometer measurements. equipped ESI with mass a smart spectra endurance were obtaineddiamond from ATRFigure a TLC-MS unit 7. for ∆Tm direct interface values measur obtained CAMAGements. for in the ESI negative AAK1 mass AP2-associated spectra mode [Mwere Hobtained protein+]. EI-MS kinase from mass 1. a TLC-MS spectra interface were − obtainedCAMAG from in thenegative Mass Spectrometrymode [M − H Department+]. EI-MS mass of thespectra Institute were of obtained Organic from Chemistry, the Mass University Spectrometry of 3. Materials and Methods Tübingen:Department Finnigan of the MAT, Institute TSQ of 70 Organic Tripel Chemistry, quadrupol; Un ioniversity source of temperature Tübingen: Finnigan at 200 ◦C; MAT, temperature TSQ 70 Tripel of evaporation:quadrupol;All commercially 30–300ion source◦ availableC; temperature ionization reagents energy: at 200 and °C; 70solven temper eV.ts The wereature compounds usedof evaporation: without were further 30–300 purified purification. °C; through ionizationcolumn 1H energy:and ® 13chromatography,C 70nuclear eV. The magnetic compounds using resonance silica were gel (NMR)purified as stationary spectra through phase were column (Gedurandetermined chromatography,Si in 60, DMSO-d 0.063–0.200 using6, CDCl silica mm).3 and gel The Acetone-d as purity stationary of6 solutionscompoundsphase (Geduranusing was a determined Bruker® Si 60, Avance 0.063–0.200 by HPLC 200 ( 1 (Merckmm).H = 200 The Hitachi MHz; purity 13 L-6200C of = compounds50 intelligentMHz) or wasa pump, Bruker determined Merck Avance Hitachi by 400 HPLC (1 AS-2000H = (Merck 400 MHz;autoHitachi sampler, 13C = L-6200 100 Merck MHz) intelligent Hitachi spectrometer. L-4250pump, UV Merck The vis chemical detector)Hitachi AS-2shifts using000 are a ZORBAXauto given sampler, in Eclipse parts Merck per XDB million Hitachi C8 column (ppm) L-4250 (5 from mm), UV vis solventemploying residual a gradient peaks of and 0.01 the M coupling KH2PO4 constant(pH 2.3) andvalues methanol (J) are given as solvent in Hz. system Signal with multiplicities a flow rate are of represented1.5 mL/min andby: s detection (singlet), at d 254(doublet), nm. dd (double doublet), t (triplet), m (multiplet) and br (broad signal).Kinase The evaluation selectivity of assay: spectra Starting was performed from 100 µusMing protein the MestReNova stocks, all the program. kinases Infrared present spectra in the panelwere obtainedwere diluted using to a 1ThermoµM in Scientific buffer 100 Nicolet’s mM K2 AvatarHPO4 pHiS10 7.5 spectrometer containing equipped 150 mM NaCl,with a 10%smart glycerol endurance and diamond1X dye (Applied ATR unit Biosystems for direct measur catalogements. 4461806). ESI The mass protein spectra/dye were mixture obtained was from transferred a TLC-MS column-wise interface CAMAGto a 384-well in negative PCR microplate mode [M with− H+]. 20 EI-MSµL per mass well. spectra Compounds were obtained at 10 mM from concentration the Mass Spectrometry in DMSO Department(from a separate of the plate Institute arranged of Organic in rows) Chemistry, were added University next, of in Tübingen: 20 nL volume, Finnigan using MAT, a liquid TSQ handling70 Tripel quadrupol;device setup ion with source a pin temperature head to make at 200 a °C; final temper 10 µMature compound of evaporation: in the assay30–300 plate. °C; ionization The final energy: DMSO 70concentration eV. The compounds in all wells were is 0.2%,purified including through the column reference chromatography, with DMSO only.using Thermal silica gel shift as stationary data was phasemeasured (Geduran in a qPCR® Si 60, instrument 0.063–0.200 (Applied mm). The Biosystems purity of QuantStudiocompounds was 6) programmed determined by to equilibrateHPLC (Merck the Hitachiplate at L-6200 25 ◦C for intelligent 5 min followed pump, Merck by ramping Hitachi the AS-2 temperature000 auto sampler, to 95 ◦C atMerck a rate Hitachi of 0.05 L-4250◦C per second.UV vis Data was processed on Protein Thermal shift software (Applied Biosystems) fitting experimental curves to a Boltzmann function to calculate differential thermal shifts (dTm) referenced to protein/dye

Int. J. Mol. Sci. 2020, 21, 8199 9 of 18 in 0.2% DMSO. The kinases were cloned, expressed (bacteria or insect cell expression) and puriﬁed at the laboratory of SGC.

3.1. General Procedure A—Corey-Chaykovsky Epoxidation 4.0 eq. of NaH (60% dispersion in mineral oil) was placed in a three-necked ﬂask under argon and then DMSO (0.6 M) and the ketone were added, after which 2.25 eq. of trimethylsulfonium iodide was also added, all at once. The reaction mixture was stirred at ambient temperature. After controlling through TLC, the mixture was poured over ice and extracted with diethyl ether and ethyl acetate (1:1). The extracts were combined, washed with brine and afterwards dried over Na2SO4. The solvent was removed under vacuum, and the residue was used without puriﬁcation for the next step. Adapted from Winter [19].

1 2-bromo-6,7,8,9-tetrahydrospiro[benzo[7]annulene-5,20-oxirane](2a): Yellow oil. 95% yield. H-NMR (200 MHz, CDCl3) δ in ppm: 1.62–1.80 (m, 2H), 1.80–2.15 (m, 2H), 2.74 (d, 1H, J = 6.0 Hz), 2.82–2.87 (m, 2H), 13 2.98 (d, 1H, J = 6.0 Hz), 7.30 (m, 3H); C-NMR (50 MHz, CDCl3) δ in ppm: 27.0, 28.6, 35.9, 36.0, 53.9, 60.8, 121.2, 126.9, 129.1, 131.8, 140.1, 143.3.

1 6-bromo-3,4-dihydro-2H-spiro[naphthalene-1,20-oxirane] (2b): Yellow solid. 90% yield. H-NMR (200 MHz, CDCl3) δ in ppm: 1.75–1.85 (m, 2H), 2.06–2.11 (m, 2H), 2.83–2.87 (m, 2H), 2.97 (s, 2H), 6.92–6.96 (d, 2H), 13 7.27–7.29 (s, 1H); C-NMR (50 MHz, CDCl3) δ in ppm: 21.9, 29.6, 31.7, 56.4, 58.9, 121.5, 125.4, 129.6, 131.4, 134.9, 141.7.

1 2-(4-bromophenyl)-2-methyloxirane (2c): Yellow solid. 90% yield. H-NMR (200 MHz, CDCl3) δ in ppm: 1.70 (s, 3H), 2.74–2.77 (d, 1H), 2.97–2.99 (d, 1H), 7.22–7.26 (d, J = 8.0 Hz, 2H), 7.44–7.48 (d, J = 8.0 Hz, 13 2H); C-NMR (50 MHz, CDCl3) δ in ppm: 21.7, 56.5, 57.1, 121.6, 127.2, 131.6, 140.4.

3.2. General Procedure B—Meinwald Rearrangement

The crude epoxide was dissolved in benzene (0.3 M) and 0.3 eq. of ZnI2 was added; the reaction mixture was heated at reflux under argon. After controlling through TLC, it was then diluted with ethyl acetate and washed with water. The organic layer was dried with Na2SO4, and the solvent was evaporated to afford the pure aldehyde, which was used without purification for the next step. Adapted from Snyder et al. [20]. 2-bromo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-5-carbaldehyde (3a): Brown oil. 98% yield. 1H-NMR (200 MHz, CDCl3) δ in ppm: 1.59–2.25 (m, 6H, CH2), 2.68 (t, 2H, CH2), 3.71 (d, 1H, CH), 6.95 (d, 1H, J = 8.0 Hz), 7.31 (m, 2H), 9.89 (s, 1H). 6-bromo-1,2,3,4-tetrahydronaphthalene-1-carbaldehyde (3b): Brown oil. 95% yield. 1H-NMR (200 MHz, CDCl3) δ in ppm: 1.92 (m, 2H), 2.24 (m, 2H), 2.72–2.78 (t, 2H), 3.58 (t, 1H), 6.99–7.03 (d, 1H), 13 7.30–7.34 (m, 2H), 9.64 (s, 1H); C-NMR (50 MHz, CDCl3) δ in ppm: 20.2, 22.9, 29.1, 51.3, 121.2, 129.4, 129.9, 131.3, 132.6, 140.3, 201.4.

1 2-(4-bromophenyl)propanal (3c): Brown oil. 98% yield. H-NMR (200 MHz, CDCl3) δ in ppm: 1.41–1.45 (d, 3H), 13 3.55–3.66 (q, 1H), 7.06–7.11 (d, 2H), 7.48–7.52 (d, 2H), 9.65 (s, 1H); C-NMR (50 MHz, CDCl3) δ in ppm: 14.7, 52.5, 121.7, 130.1, 132.3, 136.8, 200.5.

3.3. General Procedure C—Crossed Cannizzaro Reaction A mixture of 35% formalin, KOH 50% aqueous solution and the aldehyde in ethylene glycol (0.4 M) was heated at reﬂux overnight. Water was added to the resulting mixture, and the organic layer was extracted with CH2Cl2, washed with brine, and dried over Na2SO4. After concentration of the solution, hexane was added immediately with vigorous stirring, and then the mixture was cooled to 0 ◦C. White precipitate was collected, washed with hexane, and dried under air to obtain the diol. Adapted from Sato et al. [21]. Int. J. Mol. Sci. 2020, 21, 8199 10 of 18

(2-bromo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-5,5-diyl)dimethanol (4a): White solid. 61% yield. 1 HPLC: 6.948 min (83%) at 254 nm. IR (ATR): 3276, 2931, 2882, 2855, 1556, 1445 cm− . ESI-MS: Calculated: 284.0 for C H BrO . Found: 283 [M H] . 1H-NMR (200 MHz, CDCl ) δ in ppm: 1.26 (s, 2H, CH ), 13 17 2 − − 3 2 1.79 (m, 4H, CH2), 2.31 (s, 2H, CH2), 2.90 (br, 2H), 3.88 (d, 2H, J = 10 Hz), 4.04 (d, 2H, J = 12 Hz), 13 7.27–7.30 (m, 3H); C-NMR (50 MHz, CDCl3) δ in ppm: 23.9, 27.3, 30.9, 35.9, 48.6, 68.5, 120.7, 129.3, 129.9, 134.4, 140.2, 144.8. (6-bromo-1,2,3,4-tetrahydronaphthalene-1,1-diyl)dimethanol (4b): White solid. 45% yield. HPLC: 6.950 min 1 (96%). IR (ATR): 3269, 2928, 2868, 2360, 1479 cm− . ESI-MS: Calculated: 270.03 for C12H15BrO2. 1 Found: 269 [M H]−. H-NMR (200 MHz, CDCl3) δ in ppm: 1.77–1.92 (m, 4H), 1.93 (s, 2H), 2.72–2.78 − 13 (t, 2H), 3.71–3.76 (d, 2H, J = 10 Hz), 3.87–3.92 (d, 2H, J = 10 Hz), 7.27 (s, 3H); C-NMR (50 MHz, CDCl3) δ in ppm: 18.9, 27.7, 30.5, 43.3, 69.7, 120.6, 128.9, 129.2, 132.4, 136.9, 141.2. (5-chloro-2,3-dihydro-1H-indene-1,1-diyl)dimethanol (10): White solid. 30% yield. HPLC: 5.580 min (98%). 1 IR (ATR): 3280, 2933, 2877, 2843, 1451, 1200, 824 cm− . ESI-MS: Calculated: 212.0 for C11H13ClO2. 1 Found: 211 [M H]−. H-NMR (200 MHz, CDCl3) δ in ppm: 2.04–2.12 (m, 4H), 2.88–2.96 (t, 2H), − 13 3.72–3.87 (q, 4H), 7.12–7.25 (m, 3H); C-NMR (50 MHz, CDCl3) δ in ppm: 30.2, 31.4, 54.2, 68.2, 125.2, 125.4, 126.8, 133.6, 143.2, 146.8. 2-(4-bromophenyl)-2-methylpropane-1,3-diol (4c): White solid. 67% yield. HPLC: 5.280 min (100%). ESI-MS: Calculated: 244.01/246.01 for C H BrO . Found: 243/245 [M H] . 1H-NMR (200 MHz, 10 13 2 − − CDCl3) δ in ppm: 1.25 (s, 3H), 2.22 (s, 2H), 3.78–3.83 (d, 2H), 3.91–3.97 (d, 2H), 7.29–7.33 (d, 2H), 13 7.46–7.51 (d, 2H); C-NMR (50 MHz, CDCl3) δ in ppm: 20.9, 44.5, 70.0, 120.8, 128.7, 131.8, 142.3.

3.4. General Procedure D—Wittig Reaction Methyltriphenylphosphonium bromide (1.287 g, 3.601 mmol) was suspended in 15 mL of dry THF at 0 ◦C. KOtBu (0.404 g, 3.601 mmol) was added to the suspension and a bright yellow color was observed. The mixture was stirred at 0 ◦C for 30 min. Then 5-chloro-2,3-dihydro-1H-inden-1-one (0.200 g, 1.200 mmol) was added neat and the reaction mixture was stirred at 0 ◦C to r.t. for 4 h (the solution turned green and then dark brown). Water was added to the mixture and the product was extracted with CH2Cl2 and dried with Na2SO4, ﬁltrated and concentrated in vacuum. The residue was puriﬁed by column chromatography using n-hexane. Adapted from Liwosz and Chemler [24]. 5-chloro-1-methylene-2,3-dihydro-1H-indene (7): Incolor oil. 83% yield. HPLC: 9.720 min (99.4%). 1 H-NMR (200 MHz, CDCl3) δ in ppm: 2.77–2.85 (m, 2H), 2.93–3.00 (m, 2H), 5.04–5.07 (t, 1H), 5.42–5.44 (t, 1H), 7.15–7.19 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.38–7.42 (d, J = 8.0 Hz, 1H); 13C-NMR (50 MHz, CDCl3) δ in ppm: 13.1, 37.6, 119.7, 124.1, 126.3, 129.2, 130.7, 139.5, 144.8, 146.1.

3.5. General Procedure E—Brown Hydroboration—Oxidation A solution of 1.6 g (9.537 mmol) of 5-chloro-1-methylene-2,3-dihydro-1H-indene in 10 mL of THF was treated with a solution of 19.0 mL of BH3-THF complex at ice-bath temperature. The reaction mixture was stirred until disappearance of the alkene. It was then cooled in a water-ice bath, and carefully treated with methanol followed by addition of 31.8 mL (95.365 mmol) of NaOH 3.0 M and 25.3 mL (247.959 mmol) of 30% H2O2. The resulting mixture was stirred at room temperature, poured into water, acidified with 10% HCl, and extracted with ether. The organic layer was dried with Na2SO4 and the solvent was evaporated to afford the pure alcohol after purification by column in silica gel (20% EtOAc:n-hexane). Adapted from Zimmerman and Nesterov [26]. (5-chloro-2,3-dihydro-1H-inden-1-yl)methanol (8): Yellow oil. 80% yield. HPLC: 9.720 min (99.4%). 1 1 IR (ATR): 3323, 2940, 2866, 1598, 1471, 1067, 870 cm− . H-NMR (400 MHz, CDCl3) δ in ppm: 1.62 (br s, 1H), 1.91–1.98 (sextet, 1H), 2.23–2.32 (sextet, 1H), 2.82–2.99 (m, 2H), 3.27–3.34 (quintet, 1H), 13 3.76–3.77 (d, 2H), 7.13–7.15 (d, 1H), 7.19–7.21 (m, 2H); C-NMR (50 MHz, CDCl3) δ in ppm: 28.7, 31.4, 47.1, 65.9, 125.1, 125.3, 126.5, 132.8, 142.5, 146.8. Int. J. Mol. Sci. 2020, 21, 8199 11 of 18

3.6. General Procedure F—Dess–Martin Reaction To a solution of 0.470 g (2.573 mmol) of (5-chloro-2,3-dihydro-1H-inden-1-yl)methanol in 20 mL of CH2Cl2 (0.13 M) 1.6 g (3.860 mmol) of Dess–Martin periodate was added. The reaction was allowed to stir at room temperature for 3 h. TLC and HPLC analysis showed formation of the desired product. The solution was diluted with CH2Cl2 and a 10% Na2S2O3 solution to consume the excess Dess–Martin reagent was added. The mixture was stirred until the two layers separated. The CH2Cl2 layer was collected, washed with saturated Na2CO3 solution and dried with Na2SO4, ﬁltered and evaporated to give the crude aldehyde, which was used in the next step without puriﬁcation. Adapted from Thongsornkleeb and Danheiser [27].

1 5-chloro-2,3-dihydro-1H-indene-1-carbaldehyde (9): Yellow oil. 98% yield. H-NMR (200 MHz, CDCl3) δ in ppm: 2.36–2.45 (m, 2H), 2.95–3.03 (t, 2H), 3.88–3.95 (t, 1H), 7.20 (s, 3H), 9.64 (s, 1H); 13C-NMR (50 MHz, CDCl3) δ in ppm: 25.8, 31.7, 57.3, 125.5, 126.0, 127.1, 134.0, 137.0, 146.8, 200.2.

3.7. General Procedure G—Nucleophilic Addition to the Carbonyl Group Compound 9 was synthesized according to General Procedure H: 2,6 mL (6.513 mmol) of n-BuLi was added dropwise over 5 min to a solution of 1.71 g (6.513 mmol) of 1-(benzyloxy)-4-bromobenzene in 20 mL of THF at 78 C. The reaction mixture was stirred at 78 C for a further 30 min. − ◦ − ◦ Then, 0.36 g (5.010 mmol) of oxetan-3-one was added dropwise to the reaction mixture. After a further 45 min at 78 C the reaction mixture was warmed to room temperature then quenched with − ◦ water. The layers were separated and the aqueous portion extracted with diethylether. The organic extracts were combined, washed with brine, dried over Na2SO4, filtered and concentrated in vacuum. Purification by column chromatography (40% EtOAc:n-hexane) afforded the product. Adapted from Croft et al. [28]. 3-(4-(benzyloxy)phenyl)oxetan-3-ol (13): White solid. 70% yield. HPLC: 6.898 min (98%). 1H-NMR (400 MHz, CDCl3) δ in ppm: 2.92 (br, 1H), 4.86–4.91 (m, 4H), 5.09 (s, 2H), 7.00–7.02 (d, 2H, J = 8.0 Hz), 7.33–7.35 13 (d, 2H, J = 8.0 Hz), 7.38–7.45 (m, 4H), 7.47–7.49 (d,2H, J = 8.0 Hz); C-NMR (100 MHz, CDCl3) δ in ppm: 70.2, 75.8, 85.7, 115.2, 126.1, 127.6, 128.2, 128.8, 134.9, 136.9, 158.6.

3.8. General Procedure H—Oxetane Formation via Williamson Etheriﬁcation

The diol was dissolved in THF (0.1 M) and then 4 eq. of CCl4 was added. The solution was cooled to 48 C and then 1.1 eq. of Tris(dimethylamino)phosphine in 1 mL of THF was added slowly to this − ◦ solution during 5 min. The solution turned slightly turbid. The flask was transferred to an ice-bath and then agitated until it reached room temperature. After controlling using HPLC, the solvent was evaporated and then methanol was added followed by 4 eq. of a 5.4 M solution of sodium methoxide. The mixture was refluxed at 90 ◦C during 1 h. The reaction was then diluted with ethyl acetate and washed with water. The organic layer was dried with Na2SO4 and the solvent was evaporated to afford a product after purification by column in silica gel (5% EtOAc: n-hexane). Adapted from Castro and Selve [23].

2-bromo-6,7,8,9-tetrahydrospiro[benzo[7]annulene-5,30-oxetane] (5a): Yellow oil. 47% yield. HPLC: 8.497 min + 1 (93%). EI-MS: Calculated 266.03 for C13H15BrO. Found: 235.9 [M-CH2O] •. H-NMR (200 MHz, CDCl3) δ in ppm: 1.53–1.59 (t, 2H), 1.99–2.01 (t, 2H), 2.12–2.15 (d, 2H), 2.4–2.45 (d, 2H), 4.70–4.73 (d, J = 6.0 Hz, 2H), 4.97–5.00 (d, J = 6.0 Hz, 2H), 6.92–6.96 (d, 1H), 7.21–7.33 (m, 2H). 13C-NMR (50 MHz, CDCl3) δ in ppm: 26.4, 26.9, 34.9, 36.5, 47.9, 80.4, 120.0, 129.2, 132.5, 143.4, 144.4.

6-bromo-3,4-dihydro-2H-spiro[naphthalene-1,30-oxetane] (5b): Yellow oil. 48% yield. HPLC: 8.230 min + 1 (98%). EI-MS: Calculated 252.01 for C12H13BrO. Found: 221.9 [M-CH2O] •. H-NMR (200 MHz, CDCl3) δ in ppm: 1.65–1.77 (m, 2H), 2.14–2.20 (t, 2H), 2.69–2.75 (t, 2H), 4.62–4.65 (d, J = 6.0 Hz, 2H), 4.75–4.78 (d, J = 6.0 Hz, 2H), 7.21–7.22 (s, 1H), 7.38–7.43 (d, 1H), 7.77–7.81 (d, 1H). 13C-NMR (50 MHz, CDCl3) δ in ppm: 20.0, 29.9, 35.3, 42.2, 85.4, 120.4, 128.4, 129.9, 131.7, 138.7, 139.2. Int. J. Mol. Sci. 2020, 21, 8199 12 of 18

5-chloro-2,3-dihydrospiro[indene-1,30-oxetane] (11): Yellow oil. 47% yield. HPLC: 7.664 min (97%). + 1 EI-MS: Calculated 194.0 for C11H11ClO. Found: 163.9 [M-CH2O] •. H-NMR (200 MHz, CDCl3) δ in ppm: 2.43–2.50 (t, J = 6.0 Hz, 2H), 2.83–2.90 (t, J = 6.0 Hz, 2H), 4.80 (s, 4H), 7.17 (s, 1H), 7.18–7.29 (d, 1H), 13 7.56–7.60 (d, 1H). C-NMR (50 MHz, CDCl3) δ in ppm: 30.4, 37.9, 50.9, 84.2, 123.8, 124.8, 127.4, 133.4, 144.7, 145.4. 3-(4-bromophenyl)-3-methyloxetane (5c): Yellow solid. 35% yield. HPLC: 7.390 min (98%). EI-MS: Calculated + 1 226.00 for C10H11BrO. Found: 195.9 [M-CH2O] •. H-NMR (200 MHz, CDCl3) δ in ppm: 1.71 (s, 3H), 4.62–4.64 (d, 2H), 4.90–4.93 (d, 2H), 7.07–7.11 (d, J = 8.0 Hz, 2H), 7.46–7.50 (d, J = 8.0 Hz, 2H). 13C-NMR (50 MHz, CDCl3) δ in ppm: 27.7, 43.3, 83.64, 120.3, 127.0, 131.8, 145.6.

3.9. General Procedure I—Oxetane Formation via Friedel-Crafts Reaction Quantities of 0.008 g (0.028 mmol) of lithium bis(trifluoromethanesulfonimide) and 0.006 g (0.014 mmol) of tetrabutylammonium hexafluorophosphate were added to a solution of 0.066 g (0.258 mmol) of 3-(4-(benzyloxy)phenyl)oxetan-3-ol and 0.12 g (1.288 mmol) of phenol in 0.5 mL of chloroform. The reaction mixture was stirred at 40 ◦C for 1 h then quenched with sat. aq. NaHCO3. CH2Cl2 was added and the layers were separated. The organic extracts were combined, dried over Na2SO4, filtered and concentrated in vacuum. Purification by column chromatography (30% EtOAc:n-hexane) afforded the oxetane. Adapted from Croft et al. [28]. 4-(3-(4-(benzyloxy)phenyl)oxetan-3-yl)phenol (14): White solid. 60% yield. HPLC: 8.286 min (93%). ESI-MS: Calculated: 332.14 for C H O . Found: 331.1 [M H] . 1H-NMR (200 MHz, CDCl ) δ in 22 20 3 − − 3 ppm: 5.06 (s, 2H), 5.21 (s, 4H), 6.78–6.82 (d, J = 8.0 Hz, 2H), 6.94–6.98 (d, J = 8.0 Hz, 2H), 7.05–7.15 13 (m, 4H), 7.36–7.42 (m, 5H); C-NMR (50 MHz, CDCl3) δ in ppm: 50.5, 70.2, 85.2, 114.9, 115.5, 127.6, 127.8, 127.9, 128.2, 128.8, 137.1, 138.3, 138.5, 154.4, 157.6.

3.10. General Procedure J—Triflate Formation First, 0.2 mL (2.708 mmol) of pyridine, then 0.3 mL (2.031 mmol) of triflic anhydride were added to a solution of 4-(3-(4-(benzyloxy)phenyl)oxetan-3-yl)phenol in 3.0 mL of CH2Cl2. The reaction mixture was stirred at room temperature. After controlling through TLC and HPLC, the work-up was done adding water followed by CH2Cl2. The layers were separated and the aqueous portion was extracted with CH2Cl2. The organic extracts were combined, dried over Na2SO4, filtered and concentrated in vacuum. Purification by column chromatography (20% EtOAc:n-hexane) afforded the triflate. Adapted from Thompson et al. [30]. 4-(3-(4-(benzyloxy)phenyl)oxetan-3-yl)phenyl trifluoromethanesulfonate (15): White solid. 90% yield. HPLC: 10.332 min (100%). ESI-MS: Calculated: 464.09 for C23H19F3O5S. Found: 463.0 [M H]−. 1 − H-NMR (200 MHz, CDCl3) δ in ppm: 5.06 (s, 2H), 5.21 (s, 4H), 6.78–6.82 (d, J = 8.0 Hz, 2H), 6.94–6.98 (d, J = 8.0 Hz, 2H),7.05–7.15 (m, 4H), 7.36–7.42 (m, 5H).

3.11. General Procedure K—Buchwald-Hartwig Reaction via Halides

A mixture of 1.0 eq. of aryl halide, 1.0 eq. of amine, 3.0 eq. of Cs2CO3 as the base, 0.5 eq. of X-Phos as the ligand and 0.1 eq. of Pd(OAc)2 as the catalyst in dry 1,4-dioxane and absolute tert-butanol (4:1) (0.05 M) was stirred at reflux under atmosphere of argon. After controlling through TLC and HPLC, the mixture was allowed to cool to room temperature. It was diluted with water and subsequently extracted with ethyl acetate. The extracts were combined, washed with brine and afterwards dried over Na2SO4. The solvent was removed under vacuum, and the residue was purified by column chromatography, affording the desired product. Adapted from Martz et al. [31].

N-phenyl-6,7,8,9-tetrahydrospiro[benzo[7]annulene-5,30-oxetan]-2-amine (16a): Brown solid. 86% yield. HPLC: 9.015 min (96%). ESI-MS: Calculated: 279.1 for C H NO. Found: 278.0 [M H] . 1H-NMR 19 21 − − (400 MHz, CDCl3) δ in ppm: 1.59 (m, 2H, CH2), 2.01 (m, 2H, CH2), 2.18 (t, 2H, J = 8.0 Hz, CH2), Int. J. Mol. Sci. 2020, 21, 8199 13 of 18

2.42 (t, 2H, J = 8.0 Hz, CH2), 4.73 (d, 2H), 5.04 (d, 2H), 5.78 (br, 1H), 6.82 (s, 1H), 6.91–7.00 (m, 3H), 13 7.08 (d, 2H, J = 8.0 Hz),7.27 (t, 2H, J = 8.0 Hz); C-NMR (100 MHz, CDCl3) δ in ppm: 26.5, 27.3, 35.5, 37.1, 47.7, 80.7, 115.1, 117.9, 119.4, 121.0,126.2, 129.5, 137.1, 141.8, 143.4.

N-(2,4-diﬂuorophenyl)-6,7,8,9-tetrahydrospiro[benzo[7]annulene-5,30-oxetan]-2-amine (16b): Brown solid. 73% yield. HPLC: 8.542 min (98%). ESI-MS: Calculated: 315.1 for C19H19F2NO. Found: 314.2 [M H]−. 1 − H-NMR (400 MHz, CDCl3) δ in ppm: 1.49 (m, 2H), 1.91 (m, 2H), 2.08 (t, 2H), 2.31 (t, 2H), 4.63–4.64 (d, 2H, J = 4.0 Hz), 4.93–4.94 (d, 2H, J = 4.0 Hz), 5.46 (br, 1H), 6.66 (s, 1H), 6.70–6.82 (m, 3H), 6.89 (d, 1H, 13 J = 8.0 Hz), 7.18 (s, 1H); C-NMR (100 MHz, CDCl3) δ in ppm: 26.5, 27.3, 37.1, 47.7, 80.7, 104.1 (dd, J1 = 23.6 Hz, J2 = 23.7 Hz), 110.9 (dd, J1 = 3.64 Hz, J2 = 3.71 Hz), 114.9, 119.3, 119.5 (dd, J1 = 2.28 Hz, J2 = 3.17 Hz), 126.4, 127.9 (dd, J1 = 2.35 Hz, J2 = 2.36 Hz), 137.6, 141.4, 143.6.

N-(2-(trifluoromethyl)phenyl)-6,7,8,9-tetrahydrospiro[benzo[7]annulene-5,30-oxetan]-2-amine(16c): Yellow solid. 77% yield. HPLC: 10.008 min (96%). ESI-MS: Calculated: 347.1 for C20H20F3NO. Found: 346.1 [M H]−. 1 − H-NMR (400 MHz, CDCl3) δ in ppm: 1.58 (m, 2H), 1.99 (m, 2H), 2.17 (t, 2H), 2.42 (t, 2H), 4.73 (d, 2H), 5.02 (d, 2H), 6.02 (br, 1H), 6.84 (s, 1H), 6.90–6.96 (m, 2H), 7.01 (d, 1H, J = 8.0 Hz), 7.32–7.39 (m, 2H), 13 7.54 (d, 1H, J = 8.0 Hz); C-NMR (100 MHz, CDCl3) δ in ppm: 26.5, 27.2, 35.4, 36.9, 47.8, 80.7, 117.2, 117.5, 117.7 (d, J = 10.6 Hz), 119.6, 122.0, 123.6, 126.4, 127.0 (dd, J1 = 5.5 Hz, J2 = 5.4 Hz), 132.8, 139.0, 140.1, 142.5, 143.7.

N-(4-methoxyphenyl)-6,7,8,9-tetrahydrospiro[benzo[7]annulene-5,30-oxetan]-2-amine (16d): Yellow solid. 86% yield. HPLC: 7.976 min (98%). ESI-MS: Calculated: 309.1 for C20H23NO2. Found: 308.0 [M H]−. 1 − H-NMR (200 MHz, CDCl3) δ in ppm: 1.58 (m, 2H), 1.98 (m, 2H), 2.17 (t, 2H), 2.43 (t, 2H), 3.80 (s, 3H), 4.67 (d, 2H, J = 6.0 Hz), 4.99 (d, 2H, J = 6.0 Hz), 5.45 (br, 1H), 6.63–6.74 (m, 3H), 6.84–6.93 (m, 3H), 13 7.04 (t, 2H); C-NMR (100 MHz, CDCl3) δ in ppm: 26.6, 27.3, 35.6, 37.2, 47.9, 55.7, 112.8, 114.8, 117.3, 122.2, 126.2, 135.6, 136.0, 143.3, 143.9, 155.4.

N-phenyl-3,4-dihydro-2H-spiro[naphthalene-1,30-oxetan]-6-amine (17a): Brown solid. 92% yield. HPLC: 7.967 min (97%). ESI-MS: Calculated: 265.1 for C H NO. Found: 264.0 [M H] . 1H-NMR (200 MHz, 18 19 − − CDCl3) δ in ppm: 1.75–1.84 (m, 2H), 2.24 (t, 2H, J = 6.0 Hz), 2.75 (t, 2H, J = 6.0 Hz), 4.66 (d, 2H, J = 6.0 Hz), 4.87 (d, 2H, J = 6.0 Hz), 6.84 (s, 1H), 6.94–7.10 (m, 4H), 7.33 (t, 2H), 7.83 (d, 2H, J = 8.0 Hz); 13 C-NMR (50 MHz, CDCl3) δ in ppm: 20.3, 30.4, 35.9, 42.1, 85.8, 116.8, 117.4, 117.9, 121.1, 127.7, 129.5, 132.1, 138.1, 141.7, 143.2.

N-(2,4-difluorophenyl)-3,4-dihydro-2H-spiro[naphthalene-1,30-oxetan]-6-amine(17b): Brown solid. 51% yield. HPLC: 8.586 min (98%). ESI-MS: Calculated: 301.1 for C H F NO. Found: 299.9 [M H] . 1H-NMR 18 17 2 − − (200 MHz, CDCl3) δ in ppm: 1.70–1.79 (m, 2H), 2.19 (t, 2H, J = 6.0 Hz), 2.70 (t, 2H, J = 6.0 Hz), 4.61 (d, 2H, J = 6.0 Hz), 4.80 (d, 2H, J = 6.0 Hz), 6.70–6.99 (m, 4H), 7.19–7.31 (m, 1H), 7.79 (d, 2H, J = 10 Hz); 13C-NMR (50 MHz, CDCl3) δ in ppm: 20.3, 30.4, 35.8, 42.1, 85.8, 103.9 (dd, J1 = 23.5 Hz, J2 = 23.5 Hz), 110.8 (dd, J1 = 3.76 Hz, J2 = 3.79 Hz), 116.5, 117.3, 119.6 (dd, J1 = 3.22 Hz, J2 = 3.30 Hz), 127.9, 132.7, 138.3, 141.3, 151.0 (d, J = 11.8 Hz), 154.5 (d, J = 11.1 Hz), 155.9 (d, J = 11.8 Hz), 159.3 (d, J = 11.3 Hz).

N-(2-(triﬂuoromethyl)phenyl)-3,4-dihydro-2H-spiro[naphthalene-1,30-oxetan]-6-amine (17c): White solid. 82% yield. HPLC: 9.943 min (98%). ESI-MS: Calculated: 333.1 for C19H18F3NO. Found: 332.2 [M H]−. 1 − H-NMR (200 MHz, CDCl3) δ in ppm: 1.71–1.74 (m, 2H), 2.20 (t, 2H, J = 6.0 Hz), 2.72 (t, 2H, J = 6.0 Hz), 4.63 (d, 2H, J = 6.0 Hz), 4.82 (d, 2H, J = 6.0 Hz), 6.03 (br, 1H), 6.83 (s, 1H), 6.94 (t, 1H, J = 8.0 Hz), 7.06 (d, 1H, J = 8.0 Hz), 7.32–7.43 (m, 2H), 7.54 (d, 1H, J = 8.0 Hz), 7.84 (d, 1H, J = 8.0 Hz); 13C-NMR (50 MHz, CDCl3) δ in ppm: 20.3, 30.3, 35.8, 42.2, 85.8, 117.3, 117.9, 118.0, 118.9, 119.8, 120.1, 126.9 (dd, J1 = 5.5 Hz, J2 = 5.5 Hz), 127.9, 132.7, 134.1, 138.3, 140.1, 142.2.

N-(2-methoxyphenyl)-3,4-dihydro-2H-spiro[naphthalene-1,30-oxetan]-6-amine (17d): Yellow solid. 82% yield. HPLC: 9.221 min (95%). ESI-MS: Calculated: 295.1 for C H NO . Found: 294.4 [M H] . 1H-NMR 19 21 2 − − (200 MHz, CDCl3) δ in ppm: 1.72–1.83 (m, 2H), 2.24 (t, 2H, J = 6.0 Hz), 2.76 (t, 2H, J = 6.0 Hz), 3.93 (s, 3H), 4.66 (d, 2H, J = 6.0 Hz), 4.87 (d, 2H, J = 6.0 Hz), 6.92 (m, 4H), 7.13 (d, 1H), 7.32 (m, 1H), 7.84 (d, 1H, Int. J. Mol. Sci. 2020, 21, 8199 14 of 18

13 J = 8.0 Hz); C-NMR (50 MHz, CDCl3) δ in ppm: 20.4, 30.4, 35.9, 42.1, 55.7, 85.8, 110.7, 114.9, 117.5, 118.2, 120.0, 120.9, 127.7, 132.3, 133.0, 138.0, 141.3, 148.4.

N-phenyl-2,3-dihydrospiro[indene-1,30-oxetan]-5-amine (18a): Brown solid. 71% yield. HPLC: 8.231 min (95%). ESI-MS: Calculated: 251.1 for C H NO. Found: 250.1 [M H] . 1H-NMR (200 MHz, CDCl ) 17 17 − − 3 δ in ppm: 2.52 (t, 2H, J = 8.0 Hz), 2.89 (d, 2H, J = 8.0 Hz), 4.83–4.92 (m, 4H), 5.82 (br, 1H), 6.94–7.12 (m, 13 5H), 7.28–7.36 (m, 2H), 7.57 (d, 2H, J = 8.0 Hz); C-NMR (50 MHz, CDCl3) δ in ppm: 30.6, 38.4, 50.9, 84.6, 113.8, 117.4, 177.9, 121.1, 123.6, 129.5, 138.8, 143.0, 143.5, 144.9.

N-(2,4-diﬂuorophenyl)-2,3-dihydrospiro[indene-1,30-oxetan]-5-amine (18b): Brown solid. 76% yield. HPLC: 8.670 min (100%). ESI-MS: Calculated: 287.1 for C H F NO. Found: 286.1 [M H] . 1H-NMR 17 15 2 − − (200 MHz, CDCl3) δ in ppm: 2.47 (t, 2H, J = 6.0 Hz), 2.89 (d, 2H, J = 6.0 Hz), 4.79–4.87 (m, 4H), 6.76–6.98 13 (m, 4H), 7.17–7.29 (m, 1H), 7.53 (d, 2H, J = 8.0 Hz); C-NMR (100 MHz, CDCl3) δ in ppm: 30.6, 38.3, 50.9, 84.6, 103.9, 104.4 (J = 2.87 Hz), 110.8 (J1 = 3.83, J2 = 3.97 Hz), 113.6, 117.3, 119.6 (J1 = 2.86, J2 = 3.34 Hz), 123.7, 139.3, 142.7, 145.1.

N-(2-(triﬂuoromethyl)phenyl)-2,3-dihydrospiro[indene-1,30-oxetan]-5-amine (18c): White solid. 95% yield. HPLC: 9.588 min (100%). ESI-MS: Calculated: 319.1 for C H F NO. Found: 318.1 [M H] . 1H-NMR 18 16 3 − − (200 MHz, CDCl3) δ in ppm: 2.49 (t, 2H, J = 8.0 Hz), 2.86 (d, 2H, J = 8.0 Hz), 4.80–4.88 (m, 4H), 6.06 (br, 1H), 6.90–6.97 (m, 2H), 7.04 (d, 2H, J = 8.0 Hz), 7.29–7.41 (m, 2H), 7.58 (t, 2H, J = 8.0 Hz); 13C-NMR (50 MHz, CDCl3) δ in ppm: 30.6, 38.2, 50.9, 84.5, 116.5, 117.9, 119.8, 123.7, 127.1, 132.8, 140.7, 141.4, 142.5, 145.1.

N-(2-methoxyphenyl)-2,3-dihydrospiro[indene-1,30-oxetan]-5-amine (18d): Yellow solid. 80% yield. HPLC: 8.718 min (98%). ESI-MS: Calculated: 281.1 for C H NO . Found: 280.1 [M H] . 1H-NMR 18 19 2 − − (200 MHz, CDCl3) δ in ppm: 2.48 (t, 2H, J = 8.0 Hz), 2.86 (d, 2H, J = 8.0 Hz), 3.89 (s, 3H), 4.79–4.89 (m, 4H), 6.15 (br, 1H), 6.85–6.91 (m, 3H), 7.02 (s, 1H), 7.07 (d, 1H), 7.28 (m, 1H), 7.54 (d, 2H, J = 8.0 Hz); 13 C-NMR (50 MHz, CDCl3) δ in ppm: 30.6, 38.4, 50.9, 55.7, 84.6, 110.6, 114.5, 114.8, 118.1, 119.9, 120.9, 123.5, 133.3, 138.9, 142.6, 144.8, 148.3. 4-(3-methyloxetan-3-yl)-N-phenylaniline (19a): Brown solid. 83% yield. HPLC: 7.967 min (96%). ESI-MS: Calculated: 239.1 for C H NO. Found: 238.0 [M H] . 1H-NMR (400 MHz, CDCl ) δ in ppm: 1.75 (s, 16 17 − − 3 3H, CH3), 4.65–4.66 (d, 2H, J = 4.0 Hz, CH2), 4.98–4.99 (d, 2H, J = 4.0 Hz, CH2), 5.76 (br, 1H), 6.94–6.97 (t, 1H, J = 8.0 Hz), 7.08–7.11 (m, 4H), 7.16–7.18 (d, 2H, J = 8.0 Hz), 7.28–7.36 (t, 2H, J = 8.0 Hz); 13C-NMR (100 MHz, CDCl3) δ in ppm: 27.7, 43.0, 84.2, 117.8, 118.2, 121.1, 126.2, 129.5, 139.1, 141.6, 143.4. 2,4-diﬂuoro-N-(4-(3-methyloxetan-3-yl)phenyl)aniline (19b): Brown solid. 71% yield. HPLC: 7.689 min (100%). ESI-MS: Calculated: 275.1 for C H F NO. Found: 274.0 [M H] . 1H-NMR (200 MHz, 16 15 2 − − CDCl3) δ in ppm: 1.73 (s, 3H), 4.62 (d, 2H, J = 6.0 Hz), 4.94 (d, 2H, J = 6.0 Hz), 6.75–6.95 (m, 2H), 13 6.99 (d, 2H, J = 10 Hz), 7.14 (d, 2H, J = 8.0 Hz), 7.23–7.28 (d, 1H); C-NMR (50 MHz, CDCl3) δ in ppm: 27.7, 42.9, 84.1, 103.9 (dd, J1 = 23.5 Hz, J2 = 23.5 Hz), 110.8 (dd, J1 = 3.8 Hz, J2 = 3.8 Hz), 117.9, 119.3 (dd, J1 = 3.4 Hz, J2 = 3.3 Hz), 126.3, 127.8 (dd, J1 = 3.4 Hz, J2 = 3.1 Hz), 139.6, 141.2, 150.9 (d, J = 11.9 Hz), 154.5 (d, J = 11.1 Hz), 155.8 (d, J = 11.7 Hz), 159.3 (d, J = 11.1 Hz). N-(4-(3-methyloxetan-3-yl)phenyl)-2-(triﬂuoromethyl)aniline (19c): White solid. 88% yield. HPLC: 9.306 min (100%). ESI-MS: Calculated: 307.1 for C H F NO. Found: 306.0 [M H] . 1H-NMR 17 16 3 − − (400 MHz, CDCl3) δ in ppm: 1.78 (s, 3H, CH3), 4.67–4.70 (d, 2H, J = 6.0 Hz, CH2), 4.99–5.02 (d, 2H, J = 6.0 Hz, CH2), 6.10 (br, 1H), 6.94–7.02 (t, 1H, J = 8.0 Hz), 7.13–7.25 (q, 4H), 7.30–7.41 (m, 2H), 7.59–7.63 13 (d, 2H, J = 8.0 Hz); C-NMR (100 MHz, CDCl3) δ in ppm: 27.7, 43.1, 84.0, 114.3, 117.7, 119.8, 120.7, 126.4, 127.1, 132.8, 140.0, 140.7, 141.1, 142.4. 2-methoxy-N-(4-(3-methyloxetan-3-yl)phenyl)aniline (19d): Yellow solid. 81% yield. HPLC: 8.251 min 1 (98%). EI-MS: Calculated: 269.1 for C17H19NO2. Found: 269.1. H-NMR (400 MHz, CDCl3) δ in ppm: 1.73 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 4.62–4.64 (d, 2H, J = 8.0 Hz, CH2), 4.96–4.97 (d, 2H, J = 4.0 Hz, Int. J. Mol. Sci. 2020, 21, 8199 15 of 18

13 CH2), 6.85–6.90 (m, 3H), 7.15 (s, 4H), 7.27–7.29 (d, 1H); C-NMR (100 MHz, CDCl3) δ in ppm: 27.7, 43.0, 55.7, 84.2, 110.7, 114.7, 118.9, 120.0, 120.9, 126.1, 133.2, 139.2, 141.2, 148.4.

3.12. General Procedure L—Buchwald–Hartwig Reaction via Triﬂates—Benzyl Ether Deprotection

A mixture of aryl triflate, amine, Cs2CO3 as the base, BINAP as the ligand and Pd(OAc)2 as the catalyst in dry 1,4-dioxane and absolute tert-butanol (4:1) (0.03 M) was stirred at 110 ◦C under an atmosphere of argon. After controlling through TLC and HPLC, the mixture was allowed to cool to room temperature. It was diluted with water and subsequently extracted with ethyl acetate. The extracts were combined, washed with brine and afterwards dried over Na2SO4. The solvent was removed under vacuum, and the residue was purified by column chromatography, affording the product. Adapted from Åhman and Buchwald [33]. Then the benzyl ether deprotection was performed as follows: 0.6 eq. of palladium on carbon (10% w/w) was added to a solution of oxetane in ethanol/acetonitrile. The reaction mixture was stirred under an atmosphere of hydrogen at 25 ◦C until HPLC analysis indicated the end of reaction. The reaction mixture was filtered through celite, washed through with ethanol and then concentrated in vacuum to afford the final oxetane. Adapted from Croft et al. [28]. 4-(3-(4-(phenylamino)phenyl)oxetan-3-yl)phenol (20a): White solid. 85% yield. HPLC: 7.618 min (96%). ESI-MS: Calculated: 317.1 for C H NO . Found: 316.2 [M H] . 1H-NMR (400 MHz, d-acetone) 21 19 2 − − δ in ppm: 5.13 (s, 4H), 6.82–6.85 (t, 3H), 7.10–7.17 (m, 8H), 7.20–7.24 (t, 2H), 7.41 (br, 1H); 13C-NMR (100 MHz, d-acetone) δ in ppm: 51.3, 84.9, 116.1, 117.9, 118.1, 120.9, 128.0, 129.9, 138.5, 139.5, 142.9, 144.7, 156.7. 4-(3-(4-((2,4-difluorophenyl)amino)phenyl)oxetan-3-yl)phenol (20b): Brown solid. 66% yield. HPLC: 7.930 min (98%). ESI-MS: Calculated: 353.1 for C H F NO . Found: 352.0 [M H] . 1H-NMR 21 17 2 2 − − (400 MHz, d-acetone) δ in ppm: 5.12 (s, 4H), 6.81–6.83 (d, 2H, J = 8.0 Hz), 6.90–6.95 (t, 1H), 7.0–7.02 (d, 2H, J = 8.0 Hz), 7.11–7.13 (d, 2H, J = 8.0 Hz), 7.15–7.17 (d, 2H, J = 8.0 Hz), 7.32–7.38 (m, 1H), 8.29 (br, 1H); 13C-NMR (100 MHz, d-acetone) δ in ppm: 51.6, 85.0, 104.9, 105.1 (d, J = 3.0 Hz), 105.4, 111.9 (d, J = 4.0 Hz), 112.1 (d, J = 4.0 Hz), 116.3, 117.9, 122.1 (d, J = 3.0 Hz), 122.2 (d, J = 3.0 Hz), 128.3, 128.5, 138.7, 140.0, 143.3, 156.9. 4-(3-(4-((4-(trifluoromethyl)phenyl)amino)phenyl)oxetan-3-yl)phenol (20c): White solid. 71% yield. HPLC: 7.967 min (96%). ESI-MS: Calculated: 385.1 for C H F NO . Found: 383.9 [M H] . 1H-NMR 22 18 3 2 − − (200 MHz, d-acetone) δ in ppm: 5.15 (s, 4H), 6.82–6.86 (d, 2H, J = 8.0 Hz), 7.11–7.29 (m, 8H), 7.49–7.53 (d, 2H, J = 8.0 Hz), 7.93 (br, 1H), 8.34 (br, 1H); 13C-NMR (50 MHz, d-acetone) δ in ppm: 51.5, 84.8, 115.7, 116.1, 120.5, 121.0, 127.3, 127.4, 128.2, 128.4, 138.3, 140.9, 141.8, 148.8, 156.8. 4-(3-(4-((4-methoxyphenyl)amino)phenyl)oxetan-3-yl)phenol (20d): Yellow solid. 54% yield. HPLC: 7.159 min (99%). ESI-MS: Calculated: 347.1 for C H NO . Found: 345.9 [M H] . 1H-NMR 22 21 3 − − (400 MHz, d-acetone) δ in ppm: 3.75 (s, 3H), 5.11 (s, 4H), 6.80–6.89 (t, 4H), 6.93–6.97 (d, 2H, J = 8.0 Hz), 7.07–7.13 (m, 7H); 13C-NMR (100 MHz, d-acetone) δ in ppm: 27.7, 43.0, 84.2, 117.8, 118.2, 121.1, 126.2, 129.5, 139.1, 141.6, 143.4. 4-((4-(3-(4-hydroxyphenyl)oxetan-3-yl)phenyl)amino)benzonitrile (20e): Yellow solid. 70% yield. HPLC: 6.883 min (96%). ESI-MS: Calculated: 342.1 for C H N O . Found: 341.1 [M H] . 1H-NMR 22 18 2 2 − − (200 MHz, d-acetone) δ in ppm: 5.15 (s, 4H), 6.81–6.86 (d, 2H, J = 10 Hz), 7.11–7.16 (d, 2H, J = 10 Hz), 7.20–7.31 (dd, 4H, J = 10 Hz, J = 8.0 Hz), 7.52–7.56 (d, 2H, J = 8.0 Hz), 8.12 (br, 1H), 8.32 (br, 1H); 13C-NMR (50 MHz, d-acetone) δ in ppm: 51.6, 84.8, 101.5, 115.7, 116.2, 120.4, 121.5, 128.4, 128.5, 134.5, 138.3, 140.1, 142.7, 149.7, 156.9.

4. Conclusions We successfully synthesized the spiro and the methyl-phenyl oxetane scaffolds in good and reproducible yields. The hydroxyl replacement via oxyphosphonium salts proved to be a winning Int. J. Mol. Sci. 2020, 21, 8199 16 of 18 strategy for the synthesis of oxetanes from the aryl-alkyl diols through a cyclization reaction. Additionally, the reaction of phenol with a functionalized oxetan-3-ol was pivotal to obtain the 3,3-diaryloxetanes. Five scaffolds and novel compounds possessing different electronic properties were synthesized using the Buchwald–Hartwig reaction, which worked well for all halides and triflate derivatives. We succeeded in finding three new hits against the CAMK1G and AAK1 through a screening in a kinase panel using the DSF method. The results indicate that it is possible to explore compounds bearing the oxetane motif in medicinal chemistry, aiming at the discovery of novel kinase inhibitor prototypes. However, some results demonstrated that the oxetane group is probably not interacting in the hinge region, which could be associated to conformational variations or to directional hydrogen bonding differences between sp2- and sp3-hybridized oxygen atoms. As a perspective, new experiments could be carried out to determine the Kd and IC50 of the hit compounds. Furthermore, it is also possible to extensively expand the chemical space of oxetanes using other palladium-catalyzed coupling reactions, such as Sonogashira, Suzuki and Heck. In addition to these preliminary results, new structural modifications and experiments to measure the Kd and IC50 will be carried out for a better understanding of the structure-activity relationship.

Author Contributions: Conceptualization, F.R.d.S.A. and S.L.; methodology, F.R.d.S.A. and R.M.C.; writing—original draft preparation, F.R.d.S.A. and S.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Brazilian agencies FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) (2013/50724-5 and 2014/50897-0) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (465651/2014-3) and the University of Tübingen. Acknowledgments: The present work was possible with financial support of the Brazilian National Council for Scientific and Technological Development (CNPq) and of the University of Tübingen. The authors also thank Gerd Helms for some NMR experiments and Paulo Godoi for the Kinase experiments at the Structural Genomics Consortium—SGC (Campinas, Brazil). The authors thank Kristine Schmidt for proof-reading (language) of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

TDAP Tris(dimethylamino)phosphine ATP Adenosine triphosphate BINAP 2,20-bis(diphenylphosphino)-1,10-binaphthyl Bu4NPF6 Tetrabutylammonium hexaﬂuorophosphate (CH3)3SI Trimethylsulfonium iodide Li(NTf2) Lithium bis(triﬂuoromethanesulfonimide) Ph3PMeI Methyltriphenylphosphonium iodide XPhos 2-Dicyclohexylphosphino-20,40,60-triisopropylbiphenyl

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