molecules

Article Enantioenriched Positive Allosteric Modulators Display Distinct Pharmacology at the Dopamine D1 Receptor

Tim J. Fyfe 1, Peter J. Scammells 1, J. Robert Lane 2,3,* and Ben Capuano 1,*

1 Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia; tim.fyfe@griffithhack.com (T.J.F.); [email protected] (P.J.S.) 2 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia 3 School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK * Correspondence: [email protected] (J.R.L.); [email protected] (B.C.)

Abstract: (1) Background: Two first-in-class racemic dopamine D1 receptor (D1R) positive allosteric modulator (PAM) chemotypes (1 and 2) were identified from a high-throughput screen. In particular,

due to its selectivity for the D1R and reported lack of intrinsic activity, compound 2 shows promise as a starting point toward the development of small molecule allosteric modulators to ameliorate the cognitive deficits associated with some neuropsychiatric disease states; (2) Methods: Herein, we describe the enantioenrichment of optical isomers of 2 using chiral auxiliaries derived from (R)- and (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (D- and L-pantolactone, respectively); (3) Results:

We confirm both the racemate and enantiomers of 2 are active and selective for the D1R, but that



the respective stereoisomers show a significant difference in their affinity and magnitude of positive
 allosteric cooperativity with dopamine; (4) Conclusions: These data warrant further investigation

Citation: Fyfe, T.J.; Scammells, P.J.; of asymmetric syntheses of optically pure analogues of 2 for the development of D1R PAMs with Lane, J.R.; Capuano, B. superior allosteric properties. Enantioenriched Positive Allosteric Modulators Display Distinct Keywords: dopamine D1 receptor; positive allosteric modulator; PAM; G protein-coupled recep-

Pharmacology at the Dopamine D1 tors; synthetic medicinal chemistry; pharmacological evaluation; enantioenriched; chiral auxiliary; Receptor. Molecules 2021, 26, 3799. cAMP BRET https://doi.org/10.3390/ molecules26133799

Academic Editor: Jianguo Fang 1. Introduction Schizophrenia (SCZ) is a debilitating neuropsychiatric illness characterised by three Received: 26 May 2021 distinct symptoms domains [1,2]. Positive symptoms describe manifestations of psychosis Accepted: 17 June 2021 such as delusions, whereas negative symptoms are defined as alterations in drive and Published: 22 June 2021 volition [3]. Cognitive deficits are a central feature of SCZ, and include deficits in working memory, attention, learning, and executive functioning [4]. Numerous studies have demon- Publisher’s Note: MDPI stays neutral strated an association between the severity of cognitive impairment and functional, social, with regard to jurisdictional claims in and occupational outcomes in SCZ. Thus, the development of therapeutics that address published maps and institutional affil- this symptom domain are desirable [5]. Unfortunately, current clinical antipsychotic drugs iations. (APDs) fail to address these cognitive symptoms [6,7]. Hypodopaminergic function in the dorsolateral prefrontal cortex (dlPFC), an area associated with cognitive control and executive functions including working memory and selective attention [8], is thought to be related to negative symptoms and cognitive Copyright: © 2021 by the authors. deficits [9,10]. Indeed, both dopamine receptor (DR) antagonists and dopamine (DA) Licensee MDPI, Basel, Switzerland. depletion in the dlPFC impair cognitive function [11–13]. There is mounting evidence This article is an open access article to suggest that D R agonists can reverse these deficits [14,15], although excessive D R distributed under the terms and 1 1 stimulation may impede cognitive function [16,17]. Many orthosteric D R agonists that conditions of the Creative Commons 1 Attribution (CC BY) license (https:// compete with DA for the orthosteric binding site display a lack of subtype selectivity creativecommons.org/licenses/by/ (relative to other DRs) as well as poor pharmacokinetic properties. The benzazepine class 4.0/). of D1R agonists have poor bioavailability [18] and has the propensity to lower seizure

Molecules 2021, 26, 3799. https://doi.org/10.3390/molecules26133799 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 19

Molecules 2021, 26, 3799 2 of 19 poor bioavailability [18] and has the propensity to lower seizure thresholds [19]. Similarly, the D1R agonist dihydrexidine displays poor oral bioavailability and is rapidly metabo- lised in vivo [20]. Orthosteric D1R agonists have also been shown to increase incidence of thresholds [19]. Similarly, the D1R agonist dihydrexidine displays poor oral bioavailability drug-induced hypotension, potentially mediated by D1Rs expressed in the periphery [21], and is rapidly metabolised in vivo [20]. Orthosteric D1R agonists have also been shown to as well as a rapid acquisition of tolerance [22,23]. There is clearly an unmet need for the increase incidence of drug-induced hypotension, potentially mediated by D1Rs expressed indevelopment the periphery of [selective,21], as well bioavailable as a rapid acquisitionsmall molecule of tolerance D1R ligands [22,23 to]. further There is interrogate clearly an their potential utility to treat cognitive deficits associated with SCZ. unmet need for the development of selective, bioavailable small molecule D1R ligands to furtherThere interrogate has been their tremendous potential recent utility progress to treat cognitive in the development deficits associated of novel withD1R positive SCZ. allosteric modulators (PAMs) [24–29] in concert with X-ray crystal [30] and cryo-EM struc- There has been tremendous recent progress in the development of novel D1R positive allosterictures [31,32] modulators that have (PAMs) revealed [24 fascinating–29] in concert structural with X-ray insights crystal into [30 the] and therapeutic cryo-EM poten- struc- turestial of [31 the,32 ]D that1R. haveThis revealedhas culminated fascinating in structuralthe clinical insights evaluation into the of therapeutic the first D potential1R PAM ofLY3154207 the D1R. This(Mevidalen) has culminated [33] which in theis currently clinical evaluation in phase 2 offor the the first amelioration D1R PAM of LY3154207 cognition (Mevidalen)associated with [33] Lewy which body is currently dementias. in phase PAMs 2 forrepresent the amelioration an alternative of cognition approach associated to target- withing the Lewy D1R body and act dementias. to modulate PAMs the represent affinity and/or an alternative efficacy approach of DA from to targetinga topographically the D1R anddistinct act tobut modulate conformationally the affinity linked and/or binding efficacy site. of DAThe fromengagement a topographically of a less conserved distinct but al- conformationallylosteric binding site linked may binding confer site.greater The subtype engagement selectivity of a less than conserved orthosteric allosteric D1R agonists. binding siteA D may1R PAM confer that greater displays subtype positive selectivity allosteric than orthostericcooperativity D1R but agonists. lacks Aintrinsic D1R PAM efficacy that displayswhich, in positive its own allosteric right, might cooperativity maintain but the lacks temporal intrinsic and efficacy spatial which,patterns in of its DA own neuro- right, mighttransmission maintain [34,35]. the temporal and spatial patterns of DA neurotransmission [34,35]. LewisLewis etet al.al. recentlyrecently identifiedidentified twotwo racemicracemic DD11R PAM chemotypeschemotypes (Compound(Compound A,A, ((11)) andand CompoundCompound B,B, ((racrac--22),), FigureFigure1 1))[ [36].36]. 11 waswas shownshown toto bebe aa DD11RR PAMPAM butbut alsoalso actedacted asas anan agonistagonist atat thethe DD22RR andand thusthus waswas notnot investigatedinvestigated further.further. AsAs DD22RR agonismagonism isis knownknown toto exacerbateexacerbate the the positive positive symptoms symptoms of of SCZ, SCZ, achieving achieving selectivity selectivity for for the the D1 DR1 versusR versus the the D2 RD is2R paramountis paramount for for the the development development of efficacious of efficacious cognitive-enhancing cognitive-enhancing therapeutics. therapeutics.rac-2 racwas-2 shownwas shown to have to have superior superior potency potency compared compared to 1 whilst to 1 whilst being being selective selective for the for human the human D1R. ToD1 ourR. To knowledge our knowledge there is there no reported is no reported chemical ch synthesisemical synthesis and biological and biological characterisation characteri- of opticalsation of isomers optical of isomers2. Herein, of 2 we. Herein, report thewe synthesisreport the and synthesis pharmacological and pharmacological characterisation char- ofacterisation (rac)-2, and of enantioenriched(rac)-2, and enantioenriched optical isomers optical of 2 isomers(herein of denoted 2 (herein as (denotedS)-2 and as (R ()-S2)-).2 Theseand (R enantioenriched)-2). These enantioenriched samples were samples accessed were by employingaccessed by various employing chiral various auxiliaries chiral in anauxiliaries asymmetric in an Diels asymmetric Alder (ADA) Diels cycloadditionAlder (ADA) cycloaddition reaction. reaction.

FigureFigure 1.1. High-throughputHigh-throughput screening screening hits hits racemic racemic Compound A [1-(([1-((relrel-1-1SS,3,3RR,6,6RR)-6-)-6- (benzo[(benzo[dd][1,3]dioxol-5-yl)bicyclo][1,3]dioxol-5-yl)bicyclo [4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine][4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] (1)(1) andand racemicracemic CompoundCompound BB [ rel[rel-(9-(9RR,10,10RR,12,12S)-S)-NN-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro--(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro- 9,10-ethanoanthracene-12-carboxamide]9,10-ethanoanthracene-12-carboxamide] ((racrac--22)) reportedreported by by Lewis Lewis et et al. al. [ 36[36].].

ToTo characterise characterise their their pharmacology, pharmacology, we we applied applied analytical analytical pharmacological pharmacological methods methods to determineto determine allosteric allosteric ligand ligand affinity affinity for thefor unoccupiedthe unoccupied receptor, receptor, the strength the strength of modulatory of modu- effectslatory uponeffects DA upon activity DA activity as well as thewell magnitude as the magnitude of allosteric of allosteric agonism agonism [37]. We [37]. demon- We stratedemonstrate that enantiomers that enantiomers of 2 can of be 2 can accessed be accessed in moderate in moderate enantiopurity enantiopurity in four in synthetic four syn- steps.thetic steps. Importantly Importantly we reveal we reveal thatthese that thes enantiomerse enantiomers display display different different affinities affinities for the for Dthe1R D as1R well as well as different as different levels levels of positive of positive cooperativity cooperativity with with DA. TheDA. degreeThe degree of allosteric of allo- cooperativitysteric cooperativity required required for a ‘clinical’ for a ‘clinical’ D1R PAM D1 isR currentlyPAM is currently unknown. unknown. These data These illustrate data theillustrate importance the importance of characterising of characterising optically optically pure analogues pure analogues for future for structure-activity future structure- relationshipactivity relationship studies of studies2. of 2.

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2. Results and2. Discussions Results and Discussions 2.1. Chemistry 2.1. Chemistry Compound racCompound-2 was resynthesisedrac-2 was resynthesisedin three steps according in three steps to Scheme according 1 [38]. to SchemeFirstly, 1[ 38]. Firstly, AlCl3-catalysedAlCl Diels-Alder3-catalysed [4 Diels-Alder+ 2]cycloaddition [4 + 2]cycloaddition of methyl methacrylate of methyl 3 methacrylate with anthracene3 with anthracene 4 afforded the 49,10afforded bridged the ester 9,10 5 bridged as a racemic ester mixture.5 as a racemic Ester mixture.saponification Ester saponificationwas achieved was achieved under forcing underconditions forcing with conditions NaOH in with THF/H NaOH2O at in reflux, THF/H affording2O at reflux, acid affording6. Subsequent acid 6 . Subsequent treatment withtreatment thionyl chloride with thionyl and DMF, chloride followed and by DMF, DMAP-catalysed followed by nucleophilic DMAP-catalysed sub- nucleophilic stitution withsubstitution commercially with available commercially 2,6-dichloro-3-methylaniline available 2,6-dichloro-3-methylaniline (7) afforded rac (7-2). afforded rac-2. High-performanceHigh-performance liquid chromatography liquid chromatography (HPLC) using (HPLC)an amylose using chiral an amylose stationary chiral stationary phase (CSP) verifiedphase (CSP)the presence verified of the two presence enantiomers of two (see enantiomers Supplementary (see Supplementary Materials). Materials).

Scheme 1. ChemicalScheme 1. synthesis Chemical of synthesisrac-2. Reagents of rac and-2. Reagents conditions and:(a )conditions AlCl3, CH: (a2Cl) AlCl2, r.t.3, 72CH h,2Cl 63%;2, r.t. (b 72) NaOH, h, 63%; 1:1 (b THF/H) 2O, NaOH, 1:1 THF/H2O, reflux 7 days, 98%; (c) SOCl2, DMF(cat.), reflux, 24 h; (d) DMAP(cat.) DIPEA, reflux 7 days, 98%; (c) SOCl2, DMF(cat.), reflux, 24 h; (d) DMAP(cat.) DIPEA, MeCN, reflux, 24 h, 25%. MeCN, reflux, 24 h, 25%.

It is not clear from the current literature regarding the newly identified D1R PAM It is not clearscaffold from whether the current a single literature enantiomer regarding or racemic the newly mixture identified is responsible D1R PAM for the quoted scaffold whetherin vitro a singleactivity. enantiomer This led or us race to investigatemic mixture methods is responsible to generate for opticallythe quoted pure in stereoisomers vitro activity. Thisof compound led us to 2investigate. Numerous methods attempts to at generate chiral resolution optically of pure5 (semi-preparative stereoisomers chiral HPLC) of compound and2. Numerous the chromatographic attempts at resolutionchiral resolution of derivatives of 5 (semi-preparative of 5 (diastereomeric chiral amides/esters, HPLC) and thediastereomeric chromatographic salts) resolution using various of derivatives resolving of 5 ligands, (diastereomeric e.g., (R)-1-phenylethan-1-amine, amides/es- ters, diastereomeric(S)-2-amino-2-phenylethan-1-ol, salts) using various resolving and (1R ligands,,2S,5R)-2-isopropyl-5-methylcyclohexan-1-ol e.g., (R)-1-phenylethan-1- ((−)- amine, (S)-2-amino-2-phenylethan-1-ol,menthol)), all failed. Therefore, and (1 toR access,2S,5R enantiopure)-2-isopropyl-5-methylcyclohexan- synthetic precursors, it was necessary 1-ol ((−)-menthol)),to develop all failed. and Therefore, explore the to use acce ofss chiral enantiopure auxiliaries synthetic in an ADAprecursors, reaction. it was The Diels-Alder necessary to developreaction and arises explore from highthe use regio- of andchiral stereoselectivity auxiliaries in an which ADA also reaction. entails theThe use of various Diels-Alder reactionfunctionalised arises from dienes high and regio- dienophiles, and stereoselectivity and much work which has also been entails done the in the last three use of variousdecades functionalised to develop dienes ADA and reactions, dienophiles, based and on much both work enantiopure has been dienes done and in enantiopure the last three decadesdienophiles to develop [39]. Accordingly, ADA reactions, the area based has on been both extensively enantiopure reviewed dienes [ 40and–42 ]. enantiopure dienophiles(−)-Menthol [39]. Accordingly,11, a chiral moleculethe area has with been three extensively stereocentres, reviewed was initially [40– selected as 42]. a practical chiral auxiliary to probe the diastereoselectivity of the Diels-Alder cycload- (−)-Mentholdition. 11, a Achiral derivative molecule of 11with,(− three)-8-phenylmenthol, stereocentres, was has initially previously selected been as shown a to induce practical chiralADA auxiliary reactions to probe with the high diastereoselectivity distereoselectivity of the under Diels-Alder AlCl3 catalysis cycloaddition. [43]. As outlined in A derivative ofScheme 11, (−)-8-phenylmenthol,2, DMAP-catalysed, has EDC-mediated previously been Steglich-type shown to [induce44] esterification ADA reac- of methacrylic tions with highacid distereoselectivity12 with 11 furnished under the AlCl corresponding3 catalysis [43]. auxiliary As outlined13. This in dienophile Scheme 2, was subjected 1 DMAP-catalysed,to a TiClEDC-mediated4-catalysed Steglich-type [4 + 2]cycloaddition, [44] esterification affording cycloadduct(s)of methacrylic 14acid. H12 NMR analysis

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with 11 furnished the corresponding auxiliary 13. This dienophile was subjected to a TiCl4- catalysed [4 + 2]cycloaddition, affording cycloadduct(s) 14. 1H NMR analysis confirmed confirmedthat the (1 thatR,2S the,5R) (1-menthol-acrylateR,2S,5R)-menthol-acrylate chiral auxiliary chiral conferred auxiliary moderate conferred diastereoselec- moderate di- astereoselectivitytivity in the cycloaddition in the cycloaddition as one diastereom as one diastereomerer could be couldidentified be identified as the major as the product major product(>70% d.e.). (>70% Unfortunately, d.e.). Unfortunately, hydrolytic hydrolytic ester cleavage ester cleavage of 14 using of 14 LiOHusing or LiOHNaOH or in NaOH reflux- ining refluxing THF/H THF/H2O failed2O to failed furnish to the furnish acid the(6) acidas no ( 6reaction) as no reaction was evident was evidentvia LC/MS via LC/MSanalysis. analysis.Alternative Alternative conditions conditions for hydrolysis for hydrolysis were sourced were from sourced work fromcompleted work completedby Myers et by al., Myerswho reported et al., who the reported use of various the use synthetic of various exam syntheticples and examples reagents andand reagentsto hydrolyse and amides to hy- drolyseand esters amides [45]. and Such esters additional [45]. Such meth additionalods were methods explored, were including explored, stirring including 14 at stirringreflux in 14theat presence reflux in H the2SO presence4 and 1,4-dioxane, H2SO4 and stirring 1,4-dioxane, 14 reflux stirring in 2:1:114 solutionreflux in of 2:1:1 H2O, solution MeOH and of Htert2O,-butanol MeOH andin thetert presence-butanol inof the NaOH, presence as well of NaOH, as at reflux as well in as a at 4:1 reflux solution in a 4:1 of solutionH2O/1,4- ofdioxane H2O/1,4-dioxane in the presence in the of presence the Lewis of acid the Lewisiron(III) acid chloride iron(III) hexahydrate. chloride hexahydrate. These chemistries These chemistriesall failed to all provide failed tothe provide target acid. the target acid.

SchemeScheme 2. 2.Chemical Chemical synthesis synthesis of of menthol-derived menthol-derived diastereomers diastereomers using using ADA ADA chemistry. chemistry. Reagents Reagents and conditions: (a) EDCI, DMAP (cat.), anhydrous CH2Cl2, r.t. 48 h, 55%; (b) TiCl4, dry CH2Cl2, r.t. 15 and conditions: (a) EDCI, DMAP (cat.), anhydrous CH2Cl2, r.t. 48 h, 55%; (b) TiCl4, dry CH2Cl2, r.t. h, 25%; (ci) NaOH, THF/H2O, reflux 7 d; (cii) H2SO4 (conc.), 1,4-dioxane, reflux, 3 h; (ciii) NaOH, 15 h, 25%; (ci) NaOH, THF/H2O, reflux 7 d; (cii)H2SO4 (conc.), 1,4-dioxane, reflux, 3 h; (ciii) NaOH, 2:1:1 H2O, MeOH, t-butanol, reflux 72 h; (civ) FeCl3·6H2O, 1:4 1,4-dioxane/H2O, reflux. 2:1:1 H2O, MeOH, t-butanol, reflux 72 h; (civ) FeCl3·6H2O, 1:4 1,4-dioxane/H2O, reflux.

AnAn additional additional auxiliary, auxiliary, ( R(R)-2-amino-2-phenylethan-1-ol)-2-amino-2-phenylethan-1-ol15 15, was, was investigated investigated for for its its potentialpotential in in ADA ADA chemistry.chemistry. AccordingAccording to Scheme 33,, methacrylicmethacrylic acid 1212 waswas subjected subjected to toa apotassium potassium terttert-butoxide-butoxide mediated mediated direct direct amidation amidation [46] [46] using 15,, yieldingyielding thethe corre-corre- spondingsponding acrylamide acrylamide16 16.. Again, Again, the the newly newly formed formed acrylamide acrylamide was was directly directly utilised utilised in in a a TiClTiCl4-cataysed4-cataysed [4 [4 + + 2]cycloaddition 2]cycloaddition to to furnish furnish the the desired desired ethanoanthracene ethanoanthracene derivative(s) derivative(s) 1717as as a a beige beige solid. solid. HPLC HPLC analysis analysis of of the the purified purified material material indicated indicated the the presence presence of of two two diastereomersdiastereomers in in an an approximate approximate diastereomic diastereomic ratio ratio (3:1) (3:1) and and this this was was further further confirmed confirmed withwith1 H1H NMR NMR spectroscopy spectroscopy (~37% (~37% d.e.). d.e.).

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SchemeScheme 3. 3.Chemical Chemical synthesis synthesis of of 2-phenylglycinol 2-phenylglycinol ethanoanthracene ethanoanthracene diastereomers. diastereomers. Reagents Reagents and and conditions: (a) dry THF, r.t., 2.5 h, 91%; (b) TiCl4, dry DCM, r.t. 7 days, 75%. conditions: (a) dry THF, r.t., 2.5 h, 91%; (b) TiCl4, dry DCM, r.t. 7 days, 75%.

TheThe study study of of ADA ADA induction induction involving involving esters esters derived derived from from achiral achiral acrylic acrylic and methacrylic and meth- acids,acrylic and acids, the chiral and auxiliariesthe chiral (auxiliariesR)- and (S)-3-hydroxy-4,4-dimethyl-1-phenyl-2-pyrrolidinone (R)- and (S)-3-hydroxy-4,4-dimethyl-1-phenyl-2- withpyrrolidinone different dienes with different including dienes anthracene including (which anthracene was of particular (which was interest), of particular catalysed inter- by TiClest),4 hascatalysed been described by TiCl4 [has39]. been These described studies reported [39]. These adducts studies derived reported from adducts anthracene derived and thefrom (R )-pantolactam-acrylateanthracene and the (R)-pantolactam-acrylate ester could be obtained ester with could high be facial-diasteroselectivity obtained with high fa- wherecial-diasteroselectivity subsequent ester where saponification subsequent afforded ester saponification the corresponding afforded enantiopure the corresponding acid in highenantiopure optical purity. acid in Inhigh addition, optical purity. the acrylates In addition, of (R)-pantolactone the acrylates of have (R)-pantolactone also been exten- have sivelyalso been studied extensively for their studied asymmetric for their capacity asymmetr to directic capacity cycloadditions to direct cycloadditions with high facial- with diastereoselectivityhigh facial-diastereoselectivity for a number for of a dienenumber scaffolds of diene under scaffolds TiCl4 undercatalysis, TiCl including4 catalysis, an- in- thracenecluding anthracene [47–49]. Pantolactones [47–49]. Pantolactones and their use and as their chiral use auxiliaries as chiral auxiliaries in ADA chemistry in ADA chem- has beenistry extensivelyhas been extensively reviewed reviewed [50], as well [50], as as mechanistic well as mechanistic models proposed models proposed to explain to the ex- senseplain ofthe asymmetric sense of asymmetric induction induction in TiCl4-catalysed in TiCl4-catalysed ADA reactions ADA reactions of pantolactone-acrylate of pantolactone- derivativesacrylate derivatives [51]. [51]. FollowingFollowing an an adapted adapted procedure procedure from from Camps Camps et et al. al. [39 [39],], and and as as outlined outlined in Schemein Scheme4, commercially4, commercially available availableD-pantolactone D-pantolactone ((R)- 18((R) was)-18) reacted was reacted with methacrylic with methacrylic acid 12 acidunder 12 modifiedunder modified Steglich Steglich esterification esterification [44] conditions [44] conditions to give theto give corresponding the corresponding auxiliary auxiliary (R)-19 in(R moderate)-19 in moderate yield. Subsequent yield. Subsequent [4 + 2] cycloaddition[4 + 2] cycloaddition with anthracene with anthracene4 as the 4 diene as the in diene the presencein the presence of TiCl 4ofas TiCl catalyst,4 as catalyst, followed followed by chromatographic by chromatographic purification purification and recrystalliza- and recrys- 1 tiontallization from EtOH, from affordedEtOH, afforded (S)-20 as (S a)-20 pale as whitea pale crystalline white crystalline solid. solid.H NMR 1H spectroscopicNMR spectro- analysisscopic analysis confirmed confirmed high diasteroselectivity high diasteroselectivity (>90% d.e.) (>90% for the d.e.) asymmetric for the asymmetric induction. induc- Basic ester saponification using a large excess of NaOH in refluxing THF/H O was achieved to tion. Basic ester saponification using a large excess of NaOH in refluxing2 THF/H2O was eventuallyachieved to yield eventually carboxylic yield acid carboxylic (S)-21. Although acid (S)- the21. synthesisAlthough of the ((S synthesis)-21) has been of ((S reported)-21) has inbeen the literaturereported usingin the ( Rliterature)-3-hydroxy-4,4-dimethyl-1-phenyl-2-pyrrolidinone using (R)-3-hydroxy-4,4-dimethyl-1-phenyl-2-pyrroli- as a chiral auxiliarydinone as [39 a] chiral our synthesis auxiliary was [39] performed our synthesis to examine was performed the asymmetric to examine induction the asymmetric potential S 21 ofinduction pantolactone potential chiral auxiliaries.of pantolactone Moreover, chiral (( auxiliaries.)- ) was subsequently Moreover, required((S)-21) towas form subse- the corresponding amide (S)-2, so as it could be evaluated for its in vitro allosteric ligand pa- quently required to form the corresponding amide (S)-2, so as it could be evaluated for its rameters. The enantiopurity of (S)-21 was characterised using polarimetry [α] 25 = −24.9◦ in vitro allosteric ligand parameters. The enantiopurity of (S)-21 was characterisedD using (c 1.0, CHCl3) which was in accordance with literature values [39]. Based on approxima- polarimetry [α]D25 = −24.9° (c 1.0, CHCl3) which was in accordance with literature values tions from the preceding 1H NMR spectrum of (S)-20, an enantiomeric excess (e.e.) of [39]. Based on approximations from the preceding 1H NMR spectrum of (S)-20, an enanti- >90% was likely. Indeed, chiral HPLC (cHPLC) confirmed the presence of predominantly a omeric excess (e.e.) of >90% was likely. Indeed, chiral HPLC (cHPLC) confirmed the pres- single enantiomer, supporting the high facial-diastereoselectivity of the pantolactone chiral ence of predominantly a single enantiomer, supporting the high facial-diastereoselectivity auxiliary in [4 + 2] cycloadditions, with e.e. calculated to be ~88%. The enantioenriched of the pantolactone chiral auxiliary in [4 + 2] cycloadditions, with e.e. calculated to be mixture was successively activated with thionyl chloride and subjected to nucleophilic ~88%. The enantioenriched mixture was successively activated with thionyl chloride and substitution employing conditions outlined previously, yielding enantioenriched (S)-2. subjected to nucleophilic substitution employing conditions outlined previously, yielding CHPLC analysis of (S)-2 determined the e.e. to be ~90%. enantioenriched (S)-2. CHPLC analysis of (S)-2 determined the e.e. to be ~90%.

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Scheme 4. EnantioenrichmentScheme 4. Enantioenrichment of optical isomers of optical of 2 using isomers chiral of 2 auxiliaries using chiral derived auxiliaries from derived pantolactone. from pantolac- Reagents and tone. Reagents and conditions: (a) EDCI, DMAP, dry CH2Cl2, r.t. 10 h, 69–87% ((R)-19, (S)-19); (b) conditions: (a) EDCI, DMAP, dry CH2Cl2, r.t. 10 h, 69–87% ((R)-19,(S)-19); (b) TiCl4, dry CH2Cl2, r.t. 15 h, 80–85% ((S)-20, TiCl4, dry CH2Cl2, r.t. 15 h, 80–85% ((S)-20, (R)-20); (c) NaOH, THF/H2O, reflux, 72 h, 85–98% ((S)- (R)-20); (c) NaOH, THF/H2O, reflux, 72 h, 85–98% ((S)-21,(R)-21); (d) SOCl2 (neat), DMF(cat.) reflux, 24 h, then acyl halide, 21, (R)-21); (d) SOCl2 (neat), DMF(cat.) reflux, 24 h, then acyl halide, DMAP(cat.) DIPEA, MeCN, reflux, 24 S 2 R 2 DMAP(cat.) DIPEA,h, 19–25% MeCN, (( reflux,S)-2, (R 24)-2 h,). 19–25% (( )- ,( )- ). Interestingly, Camps et al. reported that a racemic N-phenyl pantolactam chiral auxil- Interestingly, Camps et al. reported that a racemic N-phenyl pantolactam chiral aux- iary was unable to react with anthracene under AlCl3 catalysis [39]. In addition, the authors iliary was unable to react with anthracene under AlCl3 catalysis [39]. In addition, the au- did not report on the effectiveness of TiCl4 as catalyst for the cycloaddition reaction between thors did not report on the effectiveness of TiCl4 as catalyst for the cycloaddition reaction anthracene and their (S)-N-phenyl pantolactam chiral auxiliary, nor its racemic counterpart. between anthracene and their (S)-N-phenyl pantolactam chiral auxiliary, nor its racemic Thus, the asymmetric induction potential of using the structurally related (S)-pantolactone counterpart.acrylate Thus, esterthe asymmetric ((S)-19) as an induction alternative potential chiral auxiliary of using to the access structurally the previously related unreported (S)-pantolactone(R)-20 wasacrylate unknown. ester (( ThisS)-19 chemistry) as an alternative is illustrated chiral in auxiliary Scheme4 to commencing access the pre- with Steglich viously unreported (R)-20 was unknown. This chemistry is illustrated in Scheme 4 com- esterification [44] of 12 with commercially available L-pantolactone (S)-18) using conditions mencing withoutlined Steglich previously esterification to afford [44] (ofS)- 1219 .with Subsequent commercially cycloaddition available of L-pantolactone the auxiliary ( S)-19 with (S)-18) usingdiene conditions4 gave cycloadductoutlined previously (R)-20 in to good afford yield, (S)-19 with. Subsequent1H NMR cycloaddition indicating a slightly of lower 1 the auxiliarydiastereoselectivity (S)-19 with diene (d.e. 4 gave ~83%). cycloadduct This apparent (R)-20 reduced in good asymmetry yield, with may H haveNMR arisen from indicating a failureslightly for lower (R)- 20diastereoselectivityto recrystallize, or (d.e. is mechanistically ~83%). This apparent inherent reduced in the cycloadditionasym- of metry may( Shave)-19. arisen Hydrolytic from cleavagea failure offor the (R auxiliary)-20 to recrystallize, was achieved or usingis mechanistically forcing alkaline in- conditions, herent in theeventually cycloaddition affording of ( theS)-19 free. Hydrolytic carboxylic acidcleavage (R)-21 of. An the assessment auxiliary was of the achieved enantiopurity was 25 ◦ using forcingagain alkaline made conditions, using polarimetry eventually ([α] Daffording= +24.1 the(c free1.0, carboxylic CHCl3)), whereby acid (R)- subsequent21. An chiral assessmentHPLC of the analysisenantiopurity determined was again the e.e.made to using be ~82%. polarimetry Acyl halide ([α] formationD25 = + 24.1° and (c 1.0, nucleophilic CHCl3)), wherebydisplacement subsequent with 7chiralusing HPLC conditions analysis outlined determined previously the e.e. furnished to be ~82%. the Acyl corresponding halide formationenantioenriched and nucleophilic (R)-2. CHPLC displacement analysis with of (R 7)- using2 determined conditions the outlined e.e. to be previ- ~84%. ously furnished the corresponding enantioenriched (R)-2. CHPLC analysis of (R)-2 deter- mined the 2.2.e.e. to Pharmacology be ~84%.

Previous work by Lewis et al. [36] reported rac-2 as a PAM at the D1R with superior 2.2. Pharmacologypotency (EC50 = 43 nM) and no agonist activity. We tested rac-2 in an assay measuring Previousaccumulation work by Lewis of cyclic et al. adenosine [36] reported monophosphate rac-2 as a PAM (cAMP) at the through D1R with activation superior of the hD1R potency (ECstably50 = 43 expressed nM) and in no FlpIn agonist CHO activity. cells using We a tested BRET rac biosensor-2 in an [ 52assay]. Concentration-response measuring accumulationcurves of cyclic of DA adenosine were generated monophos in thephate presence (cAMP) of increasing through activation concentrations of the of hD test1R compound. stably expressedAn operational in FlpIn CHO model cells of using allostery a BRET was biosensor applied to [52]. these Concentration-response data, allowing us to determine curves of DAestimates were generated of functional in the affinity presence for the of unoccupied increasing concentrations receptor (KB), a of composite test com- measure of pound. Anallosteric operational cooperativity model of allostery (αβ) that was combines applied cooperativityto these data, allowing with DA us affinity to deter- (α) and mod- mine estimatesulatory of functional effect upon affinity DA efficacy for the ( βunoccupied), as well as receptor the intrinsic (KB), a efficacy composite (τB) meas- of the allosteric ure of allostericligand. cooperativity Values of αβ (αβ> 1) that indicate combines a positive cooperativity modulatory with effect. DA affinity Functional (α) and assessment of rac-2 in our hands showed it exhibited modest affinity for the D R(K = 1.6 µM, Figure2A, modulatory effect upon DA efficacy (β), as well as the intrinsic efficacy (τB1) of theB allosteric ligand. ValuesTable of1), αβ acting > 1 indicate to potentiate a positive the potencymodulatory of DA effect. 100-fold Functional ( αβ = assessment 100) as well of as display

Molecules 2021, 26, x FOR PEER REVIEW 7 of 19

Molecules 2021, 26, 3799 7 of 19

rac-2 in our hands showed it exhibited modest affinity for the D1R (KB = 1.6 µM, Figure 2A, Table 1), acting to potentiate the potency of DA 100-fold (αβ = 100) as well as display allostericallosteric agonism agonism ( (ττBB == 2.1, 2.1, Table Table 1).1). Lewis Lewis and and colleagues observed that racrac--22 diddid not not displaydisplay agonism. agonism. This This difference difference likely likely reflects reflects a a difference difference in in the the cell cell background background (CHO (CHO versusversus HEK293) HEK293) and/or and/or a ahigher higher level level of of D D11RR expression expression in in our our cell cell line and/or a a greater greater sensitivitysensitivity of of our our cAMP cAMP assay assay [36]. [36 ].Indeed, Indeed, su suchch effects effects have have been been observed observed for fora PAM a PAM of theof themuscarinic muscarinic M1 acetylcholine M1 acetylcholine receptor receptor and such and observations such observations are consistent are consistent with a two- with statea two-state model of model allostery of allostery [53]. Consistent [53]. Consistent with the withabove the differences, above differences, the potency the of potency DA in ourof DAassay in is our also assay greater is also than greater that observed than that by observedLewis and by coworkers Lewis and in coworkerstheir experiments. in their Noteexperiments. that Lewis Note et al. that also Lewis determined et al. also the determined value of potency the value for of rac potency-2 as 40 for nM.rac -This2 as 40value nM. wasThis determined value was determinedby the measuring by the the measuring potency the with potency which withrac-2which causesrac a -shift2 causes in DA a shiftpo- tencyin DA and potency cannot and be cannotdirectly be compared directly compared with the value with theof K valueB determined of KB determined in our analysis in our thatanalysis reflects that the reflects affinity the of affinity the PAM of thefor PAMthe unoccupied for the unoccupied receptor. receptor.

Figure 2. All compounds display allosteric pharmacology at the D1R in an assay measuring cAMP production using a Figure 2. All compounds display allosteric pharmacology at the D1R in an assay measuring cAMP production using a BRET BRETbiosensor. biosensor.rac-2 ( Arac),- the2 (A enantioenriched), the enantioenrichedS-isomer S-isomer (S)-2,(B (),S and)-2, ( theB), enantioenrichedand the enantioenrichedR-isomer R (R-isomer)-2,(C) act(R)- as2, an(C) ago-PAMs, act as an ago-PAMs, potentiating DA potency and exerting allosteric agonism. These data were fitted to an operational model of potentiating DA potency and exerting allosteric agonism. These data were fitted to an operational model of agonism to agonism to derive values of affinity, cooperativity with DA and intrinsic efficacy (see Table 1). Data are presented as mean derive values of affinity, cooperativity with DA and intrinsic efficacy (see Table1). Data are presented as mean ± S.D. from ± S.D. from at least four separate experiments. (D) All compounds were assessed for their activity at non-hD1R/hD2R expressingat least four FlpIn separate CHO-cells experiments. transfected (D) All wi compoundsth the BRET were biosensor, assessed demonstrating for their activity that at non-hD compound1R/hD activity2R expressing is mediated FlpIn throughCHO-cells the transfected D1/D2Rs. Data with are the presented BRET biosensor, as mean demonstrating ± S.D. from at thatleast compound four separate activity experiments. is mediated through the D1/D2Rs. Data are presented as mean ± S.D. from at least four separate experiments. Intriguingly, the optical isomers of rac-2 ((S)-2,(R)-2) were shown to display signif- icant differences both in their affinity for the D1R and their degree of positive allosteric cooperativity with DA. The enantioenriched (S)-2 was determined to have comparable functional affinity, efficacy and cooperativity as compared to rac-2 (KB = 1 µM, αβ = 125, τB = 2.5) (Figure2B, Table1). Conversely, the enantioenriched ( R)-2 displayed 4.8-fold and 7.4-fold lower functional affinity (KB = 7.4 µM αβ = 31) and 3-fold and 4-fold lower cooperativity with DA, relative to rac-2 and (S)-2 respectively (Figure2C, Table1). It is interesting to note that the pharmacology of rac-2 is not significantly different from (S)- 2. This can be reconciled by the lower levels of affinity and cooperativity with DA that (R)-2 displays. These two factors combined mean that (S)-2 would effectively display a 30-fold greater affinity for the DA occupied receptor as compared to (R)-2 even though

Molecules 2021, 26, 3799 8 of 19

their affinities for the unoccupied are a more modest 7-fold different. In the racemate, therefore, (S)-2 would be expected to dominate meaning that the pharmacology of the racemate reflects this enantiomer. We extended our functional characterisation of these compounds to an assay measuring inhibition of forskolin-stimulated cAMP accumulation in hD2LR-expressing FlpIN CHO cells. rac-2 and its respective enantiomers ((R)-2,(S)-2) displayed no activity at the D2R up to a measured concentration of 30 µM. None of the above compounds displayed any off-target response in FlpIn CHO cells transfected with the BRET biosensor, but not expressing either the D1R or the D2R (Figure2D). Together our data indicate that the distinct structural configuration of the two enantiomers of 2 confer differences in their ability to potentiate DA at the D1R.

Table 1. Functional parameters for PAMs derived from cAMP BRET assay at the hD1R.

1 2 3 CPD e.e. (%) pKB (KB, µM) LogτB (τB) Logαβ (αβ) rac-2 0 5.80 ± 0.10 (1.6) 0.32 ± 0.06 (2.1) 2.00 ± 0.12 (100) (S)-2 90 5.99 ± 0.09 (1.0) 0.40 ± 0.06 (2.5) 2.10 ± 0.25 (125) (R)-2 84 5.12 ± 0.06 (7.6) *,ˆ 0.46 ± 0.08 (2.9) 1.51 ± 0.14 (31) *,ˆ 1 Estimate of the negative logarithm of the equilibrium dissociation constant determined in an cAMP functional assay. 2 Estimate of the intrinsic efficacy of the modulator. 3 Estimate of the logarithm of the net cooperativity factor between the modulator and DA. Values represent mean ± S.D. from at least four independent experiments performed in duplicate. Significant differences parameter between (R)-2 and rac-2 * or (S)-2 ˆ, p < 0.05, one-way ANOVA with Tukey’s Post-hoc test (GraphPad Prism Version 7).

3. Experimental 3.1. General Solvents and fine chemicals were commercially available from standard suppliers and used where described without further purification. Davisil silica gel (40–63 µm) for Flash column chromatography used Davisil silica gel (40–63 µm) and was supplied by Grace Davison Discovery Sciences (Victoria, Australia). Deuterated solvents for NMR spectroscopy were sourced by Novachem Pty. Ltd., Victoria, Australia (a distributor for Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA). Reactions were monitored by thin layer chromatography on commercially available precoated aluminum-backed plates (Merck Kieselgel 60 F254). Visualization was by inspection under UV light (254 nm/366 nm). Ninhydrin (in EtOH) was used to visualize primary and secondary amines. Anhydrous Na2SO4 was used to dry all organic extracts collected after aqueous workup procedures before gravity/vacuum filtration and evaporation to dryness. Organic solvents were removed under reduced pressure at a water bath temperature of ≤40 ◦C. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance Nanobay III 400 MHz Ultrashield Plus spectrometer at 400.13 and 100.62 MHz, respectively. Chemical shifts (δ) are recorded in parts per million (ppm) with reference to the chemical shift of the deuterated solvent. Coupling constants (J) are recorded in Hz, and multiplicities described by singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), multiplet (m), doublet of doublets (dd), and doublet of triplets (dt). All NMR experiments were performed in CDCl3 for comparison of spectra of various analogues. For analogues that displayed reduced solubility in CDCl3 then experiments were performed in CD3OD, DMSO-d6, or acetone-d6. LCMS experiments -System A (default): an Agilent 6100 series single quad coupled to an Agilent 1200 series HPLC instrument using the following buffers: buffer A, 0.1% HCOOH in H2O; buffer B, 0.1% HCOOH in CH3CN. The following gradient was used with a Phenomenex Luna 3 µm C8(2) 15 × 4.6 mm column, flow rate of 0.5 mL/min, total run time of 12 min: 0–4 min 95% buffer A/5% buffer B, 4–7 min 0% buffer A/100% buffer B, 7–12 min 95% buffer A/5% buffer B. Mass spectra were acquired in both negative & positive ion modes with a scan range of 0–1000 m/z at 5 V. UV detection was monitored at 254 nm. System B: an Agilent 6120 series single quad coupled to an Agilent 1260 series HPLC instrument using the following buffers; buffer A, 0.1% HCOOH in H2O; buffer B, 0.1% HCOOH in CH3CN. The following gradient was used with a Poroshell 120 EC-C18 50 × 3.0 mm, 2.7 µm column, flow rate of 0.5 mL/min, total run time of 5 min: 0–1 min 95% buffer Molecules 2021, 26, 3799 9 of 19

A/5% buffer B, 1 to 2.5 min 0% buffer A/100% buffer B, held until 3.8 min, 3.8–4 min 95% buffer A/5% buffer B, held until 5 min. Mass spectra were acquired in both negative & positive ion modes with a scan range of 100–1000 m/z. UV detection was monitored at 214 and 254 nm. All retention times (tR) were quoted in minutes. System C: analytical reverse-phase HPLC was performed, to verify purity, on a Waters HPLC system coupled directly to a photodiode array detector and fitted with a Phenomenex Luna C8 (2) 100 Å column (150 × 4.6 mm, 5 µm). A binary solvent system of buffer A, 0.1% TFA/H2O; buffer B, 0.1% TFA/80% CH3CN/H2O was used. Gradient elution was attained using 100% buffer A to 100% buffer B over 12 min at a flow rate of 1 mL/min. All target compounds subjected to biological testing were >95% pure by HPLC at two wavelengths (214 and 254 nm). Analytical chiral-HPLC were conducted on an Agilent Infinity 1260 system fitted with either one of (a) Lux 5 µm Amylose-2 150 × 4.60 mm, or (b) Lux 5 µm Cellulose-1 150 × 4.60 mm. A binary solvent system was used (solvent A: ethanol; solvent B: petroleum spirits), with UV detection at 254 nm. The method used isocratic elution of 1–20% solvent A and 99–80% solvent B, with a flow rate of 1 mL/min. Mass spectra were acquired in in both negative & positive ion mode with a scan range of 100–1000 m/z. UV detection was monitored at 214 and 254 nm with retention times (tR) expressed in minutes. All screening compounds displayed >95% purity unless otherwise specified in the individual monologue.

3.2. Chemistry 3.2.1. General Procedure A for Steglich Esterification A mixture of methacrylic acid (1.0 equiv.) alcohol (1.0 equiv.), EDC (1.1 equiv.) and DMAP (5 mol %) in dry DCM was stirred at rt until complete consumption of the starting acid or alcohol. The reaction mixture was washed with sat. aqueous citric acid (3 × 50 mL) and sat. aqueous NaHCO3 (3 × 50 mL), dried (Na2SO4) and concentrated in vacuo. The residue was purified by flash column chromatography (FCC) with an appropriate eluent as indicated.

3.2.2. General Procedure B for TiCl4-Catalysed Diels-Alder Cycloaddition

A solution of TiCl4 (2.0 equiv.) in anhydrous DCM (20 mL) was added to a solution of the appropriate chiral auxiliary/dienophile (1.0 equiv.) in anhydrous DCM (30 mL), and the mixture stirred at rt for 15 min. Then, a solution of the anthracene (1.0 equiv.) in dry CH2Cl2 (20 mL) was added and the mixture stirred at r.t. for 15 h. A small amount of H2O was added to destroy the TiCl4 complexes, the mixture was filtered and the filtrate was dried with anhydrous Na2SO4. The filtrate was concentrated in vacuo and the residue was purified by FCC using an appropriate eluent as indicated.

3.2.3. General Procedure C for Alkaline Ester Hydrolysis

To a solution of ester (1.0 equiv.) in a 1:1 mixture of THF/H2O was added NaOH (3 equiv.), and the mixture stirred at reflux temperature until complete consumption of the starting material was evident. The THF was removed in vacuo and the remaining aqueous phase was washed with Et2O. The aqueous phase was then acidified (pH = 1), and any precipitated carboxylic acid was collected via vacuum filtration and recrystallised from EtOAc/PE. Similarly, the aqueous phase could be extracted with DCM and the organic extracts dried (Na2SO4) to afford the desired carboxylic acid to maximise the yield.

3.2.4. General Procedure D for Acyl Halide Formation and Nucleophilic Substitution The carboxylic acid (1 equiv.) was taken up in thionyl chloride (5 mL) followed by three drops of DMF and the reaction was stirred at reflux temperature until complete consumption of the starting material. The solvent was removed in vacuo and the residue was taken up in dry MeCN (25 mL). To this solution was added 2,6-dichloro-3-methylaniline (1.1 equiv.), DMAP (0.5 equiv.), DIPEA (2.0 equiv.), and stirred at reflux temperature until complete consumption of the acyl halide was evident. The solvent was evaporated under Molecules 2021, 26, x FOR PEER REVIEW 10 of 19 Molecules 2021, 26, x FOR PEER REVIEW 10 of 19

starting material was evident. The THF was removed in vacuo and the remaining aqueous startingphase was material washed was with evident. Et2O. The The THF aqueous was removed phase was in vacuo then acidifiedand the remaining (pH = 1), aqueousand any phaseprecipitated was washed carboxylic with acid Et2O. was The collected aqueous vi phasea vacuum was filtrationthen acidified and recrystallised (pH = 1), and from any precipitatedEtOAc/PE. Similarly, carboxylic the acid aqueous was collected phase coul viad vacuum be extracted filtration with and DCM recrystallised and the organic from EtOAc/PE.extracts dried Similarly, (Na2SO 4the) to aqueousafford the phase desired coul carboxylicd be extracted acid to with maximise DCM andthe yield.the organic extracts dried (Na2SO4) to afford the desired carboxylic acid to maximise the yield. 3.2.4. General Procedure D for Acyl Halide Formation and Nucleophilic Substitution 3.2.4. General Procedure D for Acyl Halide Formation and Nucleophilic Substitution The carboxylic acid (1 equiv.) was taken up in thionyl chloride (5 mL) followed by threeThe drops carboxylic of DMF andacid the(1 equiv.)reaction was was taken stirre dup at in reflux thionyl temperature chloride (5 until mL) complete followed con- by threesumption drops of of the DMF starting and thematerial. reaction The was solv stirreent wasd at refluxremoved temperature in vacuo and until the complete residue con-was Molecules 2021 26 , , 3799 sumptiontaken up in of drythe startingMeCN (25 material. mL). To The this solv solutionent was was removed added in2,6-dichloro-3-methylaniline vacuo and the residue10 was of 19 taken(1.1 equiv.), up in dryDMAP MeCN (0.5 (25equiv.), mL). DIPEA To this (2.0 solution equiv.), was and added stirred 2,6-dichloro-3-methylaniline at reflux temperature until (1.1complete equiv.), consumption DMAP (0.5 equiv.),of the acyl DIPEA halide (2.0 was equiv.), evident. and The stirred solvent at reflux was evaporatedtemperature under until complete consumption of the acyl halide was evident. The solvent was evaporated under reduced pressure and thethe residueresidue waswas takentaken upup inin EtOAc,EtOAc, washedwashed withwith 11M M aqueousaqueous reduced pressure and the residue was taken up in EtOAc, washed with 1M aqueous NaOH, H2O, 1M 1 M aqueous aqueous HCl, HCl, brine, and the organicorganic layerlayer drieddried (Na(Na22SOSO44). The solventsolvent NaOH,was concentrated H2O, 1M aqueous in vacuo HCl, and thebrine, residue and purifiedpurithe organicfied by FCClayer with dried an (Na appropriate2SO4). The eluent solvent as wasindicated. concentrated in vacuo and the residue purified by FCC with an appropriate eluent as indicated.Methyl (rel-9S,10S,12R)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate [38] (5) Methyl (rel-9S,10S,12R)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate [38] (5) Methyl (rel-9S,10S,12R)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate [38] (5)

Methyl methacrylate (3.29 mL, 30.9 mmol, 1.1 eq.) was added to a suspension of AlCl3 Methyl methacrylate (3.29 mL, 30.9 mmol, 1.1 eq.) was added to a suspension of AlCl3 Methyl(3.74 g, 28.1 methacrylate mmol, 1.0 (3.29 eq.) mL,in dry 30.9 DCM mmol, (100 1.1 mL), eq.) in was a three-necked added to a suspension round-bottom of AlCl flask3 (3.74 g, 28.1 mmol, 1.0 eq.) in dry DCM (100 mL), in a three-necked round-bottom flask (3.74under g, a 28.1positive mmol, pressure 1.0 eq.) of in N dry2. After DCM 1 (100h of mL),stirring, in a anthra three-neckedcene (5.00 round-bottom g, 28.1 mmol, flask 1.0 under a positive pressure of N2. After 1 h of stirring, anthracene (5.00 g, 28.1 mmol, 1.0 undereq.) was a positiveadded portion-wise. pressure of N 2The. After resulting 1 h of stirring,mixture anthracenewas stirred (5.00 at room g, 28.1 temperature mmol, 1.0 eq.)for eq.)was72 h wasaddedor until added portion-wise. complete portion-wise. consumption The resultingThe resulting of mixturethe an mixturethracene was stirred was was stirred atevident. room at temperature roomThe mixture temperature for was 72 then h for or 72 h or until complete consumption of the anthracene was evident. The mixture was then untilpoured complete over ice, consumption the organic oflayer the separate anthracened, washed was evident. with water The mixture (40 mL), was dried then (Na poured2SO4), poured over ice, the organic layer separated, washed with water (40 mL), dried (Na2SO4), overand the ice, solvent the organic evaporated. layer separated, The resulting washed resi withdue waterwas dissolved (40 mL), driedin DCM, (Na 2absorbedSO4), and onto the andsolventsilica the gel evaporated.solvent and purified evaporated. The by resultingFCC The (eluent, resulting residue 9:1 PE/Et wasresidue dissolved2O) wasto afford dissolved in DCM, the title in absorbed compoundDCM, absorbed onto as silica a trans- onto gel silica gel and purified by FCC (eluent, 9:1 PE/Et2O) to afford the title compound as a trans- andparent purified oil which by FCC solidified (eluent, under 9:1 PE/Et high2 vacuumO) to afford to give the titlea white compound solid (5.67 as a g, transparent 72%). LCMS oil parentwhich(m/z): 301.1 solidifiedoil which [M + under Na]solidified+. System high under vacuum C HPLC: high to givevacuumtR 8.389 a white min, to give solid>95% a (5.67white purity g, solid 72%).(214 (5.67& LCMS 254 g, nm). 72%). (m/ 1zH): LCMS NMR 301.1 (m/z): 301.1+ [M + Na]+. System C HPLC: tR 8.389 min, >95% purity (214 & 2541 nm). 1H NMR [M(CDCl + Na]3) δ 7.32–7.28. System C(m, HPLC: 1H), 7.26–7.22tR 8.389 min, (m, >95%2H), 7.19 purity (dd, (214 J = 6.9, & 254 1.6nm). Hz, 1H),H NMR 7.13–7.08 (CDCl (m,3) (CDClδ2H),7.32–7.28 7.08–7.013) δ 7.32–7.28 (m, (m, 1H), 2H), (m, 7.26–7.22 4.401H), (s, 7.26–7.22 1H), (m, 2H),4.25 (m, (t, 7.19 2H),J = (dd,2.7 7.19 Hz,J =(dd, 1H), 6.9, J 1.63.53= 6.9, Hz, (s, 1.6 3H) 1H), Hz, 2.71 7.13–7.081H), (dd, 7.13–7.08 J = (m,12.7, 2H), (m, 3.0 2H),7.08–7.01Hz, 1H), 7.08–7.01 1.39 (m, 2H),(dd, (m, 2H), 4.40J = 12.7, (s,4.40 1H), 2.5 (s, Hz, 1H), 4.25 1H), (t,4.25J 1.05= (t, 2.7 J (s,= Hz, 2.7 3H). 1H),Hz, 13 C1H), 3.53 NMR 3.53 (s, 3H)(CDCl (s, 3H) 2.713) 2.71δ (dd, 177.2, (dd,J = 143.8, 12.7, J = 12.7, 3.0 143.2, Hz,3.0 1313 Hz,1H),141.6, 1H), 1.39 140.5, 1.39 (dd, 126.4, (dd,J = 12.7,J126.2, = 12.7, 2.5 126.2, 2.5 Hz, Hz, 125.7, 1H), 1H), 1.05 125.6, 1.05 (s, (s,124.7, 3H). 3H). 123.6, CC NMR NMR 123.2, (CDCl (CDCl 52.93, 3)52.1,) δδ 177.2,177.2, 44.5, 143.8, 38.9, 26.7.143.2, 141.6, 140.5, 126.4, 126.2, 126.2, 125.7, 125.6, 124.7, 123.6, 123.2, 52.9,52.9, 52.1,52.1, 44.5,44.5, 38.9,38.9, 26.7.26.7. rel-(9S,10S,12R)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid (6) rel-(9S,10S,12R)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acidacid ((66))

Synthesised according to general procedure C using LiOH.H2O (2.15 g, 89.8 mmol). After acidification,Synthesised according the precipitated to gene ralsolids procedure were washedC using LiOH.Hwith water2O (2.15 and g,recrystallised 89.8 mmol). Afterfrom acidification,SynthesisedEtOAc/PE, affording according the precipitated the to generalcompound solids procedure aswere white washed C needles using LiOH.Hwith (4.75 water g,2O quantitative). (2.15and g,recrystallised 89.8 mmol).LCMS ( Afterfromm/z): EtOAc/PE,acidification,263.1 [M − affordingH] the+. System precipitated the Ccompound HPLC: solids tR as7.181 were white min, washed needles >95% with (4.75 purity water g, quantitative).(214 and & recrystallised254 nm). LCMS 1H (NMR fromm/z): + R 1 263.1EtOAc/PE,(CDCl [M3) δ −7.31–7.28 H] affording. System (m, the 1H), C compound HPLC: 7.27–7.23 t as 7.181(m, white 3H min,), needles7.23–7.19 >95% (4.75purity (m, g,1H), quantitative).(214 7.12 & (ddd,254 nm). JLCMS = 6.1, H 5.6, (NMRm/ 3.7z): + 1 (CDCl263.1Hz, 2H), [M3) δ 7.08 −7.31–7.28H] (dd,. SystemJ =(m, 7.2, 1H), 1.5 C HPLC:Hz,7.27–7.23 1H),tR 7.06–7.01 (m,7.181 3H min,), 7.23–7.19(m, >95% 1H), 4.37purity (m, (s,1H), (2141H), 7.12 4.26 & (ddd, 254 (t, nm).J J= = 2.7 6.1, Hz,H 5.6, NMR 1H), 3.7 δ Hz,(CDCl2.62 2H),(dd,3) 7.08J =7.31–7.28 12.7, (dd, 3.0 J = (m,Hz,7.2, 1H),1.51H), Hz, 7.27–7.231.40 1H), (dd, 7.06–7.01 J (m, = 12.7, 3H), (m,2.5 7.23–7.19 1H),Hz, 1H), 4.37 (m, 1.07(s, 1H), (s, 3H). 7.124.26 13(t, (ddd,C J NMR= 2.7J = Hz, (CDCl 6.1, 1H), 5.6,3) 13 2.623.7δ 181.7, Hz, (dd, 2H),143.6, J = 12.7, 7.08 143.3, (dd,3.0 Hz,141.3,J = 1H), 7.2, 140.5, 1.5 1.40 Hz, 126.5,(dd, 1H), J 126.3,= 7.06–7.0112.7, 126.2,2.5 Hz, (m, 125.8, 1H), 1H), 125.7,1.07 4.37 (s, (s, 125.2, 3H). 1H), 4.26123.5,C NMR (t, 123.3,J = (CDCl 2.7 52.6, Hz,3) 13 δ1H),48.5, 181.7, 2.6244.5, 143.6, (dd, 38.7, J143.3, =26.9. 12.7, 141.3,Analytical 3.0 Hz,140.5, 1H), data 126.5, 1.40 including 126.3, (dd, J 126.2,= 1H 12.7, NMR 125.8, 2.5 and Hz, 125.7, 1H),13C 125.2,NMR 1.07 (s,123.5,spectra 3H). 123.3, areC NMRin 52.6, ac- Molecules 2021, 26, x FOR PEER REVIEW(CDCl ) δ 181.7, 143.6, 143.3, 141.3, 140.5, 126.5, 126.3,1 126.2, 125.8,13 125.7, 125.2, 123.5,11 123.3, of 19 48.5,cordance 44.5,3 with 38.7, those 26.9. publishedAnalytical [26,39].data including H NMR and C NMR spectra are in ac- cordance52.6, 48.5, with 44.5, those 38.7, 26.9.published Analytical [26,39]. data including 1H NMR and 13C NMR spectra are in accordance with those published [26,39]. rel-(9R,10R,12S)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanth rel-(9R,10R,12S)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthra- cene-12-carboxamideracene-12-carboxamide (rac(rac-2)- 2)

Synthesised according to general procedure D using oxalyl chloride (243 µL, 2.84 mmol), relSynthesised-(9S,10S,12 accordingR)-12-methyl-9,10-dihydro-9,10-ethano to general procedure D using oxalylanthracene-12-carboxylic chloride (243 µL, 2.84 mmol),acid rel(6)- (375(9S,10 mg,S,12 1.42R)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic mmol), DMAP (4.32 mg, 35.4 mmol), DIPEA (120 µL, 707 acidµmol) (6) and (375 2,6- mg, dichloro-3-methylaniline (65.4 mg, 371 µmol). Purification by FCC (eluent, 10:1 PE/EtOAc) afforded the racemic compound as a white solid (75 mg, 50%). LCMS (m/z): 446.1 [M + Na]+, 423.1 [M + H]+. System C HPLC: tR 8.496 min, >95% purity (214 & 254 nm). HRMS (m/z): C25H21Cl2NO: requires 422.1083 [M + H]+; found 422.1073. 1H NMR (CDCl3) δ 7.40–7.37 (m, 1H), 7.34–7.31 (m, 1H), 7.30–7.25 (m, 2H), 7.18–7.11 (m, 3H), 7.08–7.01 (m, 3H), 6.89 (s, 1H), 4.52 (s, 1H), 4.34 (t, J = 2.6 Hz, 1H), 2.64 (dd, J = 12.5, 2.8 Hz, 1H), 2.29 (s, 3H), 1.66 (dd, J = 12.4, 2.7 Hz, 1H), 1.18 (s, 3H). 13C NMR (CDCl3) δ 174.7, 143.3, 143.2, 141.5, 141.4, 136.1, 133.8, 132.2, 130.8, 129.6, 127.4, 126.5, 126.4, 126.2, 125.9, 125.9, 125.7, 123.5, 123.2, 52.7, 49.2, 44.7, 40.3, 28.3, 20.5. Analytical data including 1H NMR and 13C NMR spectra are in accordance with those published [26]. (1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl methacrylate (13)

Synthesised according to general procedure A using methacrylic acid (1.62 mL, 19.2 mmol), (1R,2S,5R)-2-isopropyl-5-methylcyclohexan-1-ol (3.00 g, 19.2 mmol), EDC (4.05 g, 21.2 mmol) and DMAP (117 mg, 960 µmol). Purification by FCC (eluent, DCM) gave the title compound as a transparent oil (3.20 g, 74%). LCMS (m/z): 225.2 [M + H]+. System C HPLC: tR 9.042 min, >95% purity (214 & 254 nm). 1H NMR (CDCl3) δ 6.23–6.20 (m, 1H), 5.81 (dd, J = 2.0, 1.2 Hz, 1H), 3.39 (td, J = 10.4, 4.3 Hz, 1H), 2.16 (dtd, J = 14.0, 7.0, 2.7 Hz, 1H), 1.99 (dt, J = 1.5, 0.9 Hz, 3H), 1.95–1.91 (m, 1H), 1.61 (ddd, J = 16.1, 14.6, 7.9 Hz, 2H), 1.45–1.37 (m, 2H), 1.14–1.05 (m, 1H), 0.96 (dd, J = 12.1, 4.1 Hz, 1H), 0.94–0.88 (m, 6H), 0.80 (dd, J = 7.0, 0.9 Hz, 3H). 13C NMR (CDCl3) δ 163.2, 135.9, 129.1, 71.7, 50.3, 45.2, 34.7, 31.8, 25.9, 23.3, 22.3, 21.1, 16.2. Analytical data in accordance with those published [54]. (1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12- carboxylate (14)

Synthesised according to general procedure B using TiCl4 (4.38 µL, 3.93 mmol), (1R,2S,5R)- 2-isopropyl-5-methylcyclohexyl methacrylate (13) (440 mg, 1.96 mmol) and anthracene (350 mg, 1.96 mmol). Purification by FCC (eluent, 10:1 PE/EtOAc] gave the anthracene ester derivative as a light-yellow oil (400 mg, 51%, d.e. >70%). LCMS (m/z): 401.1 [M − H]‒ . System C HPLC: tR 2.204 min, >95% purity (214 & 254 nm). 1H NMR (CDCl3) δ 7.30–7.27 (m, 1H), 7.25–7.18 (m, 3H), 7.11–7.07 (m, 2H), 7.07–6.98 (m, 2H, H3), 4.48 (td, J = 10.8, 4.3 Hz, 1H), 4.38 (s, 1H), 4.23 (t, J = 2.6 Hz, 1H), 2.70 (dd, J = 12.7, 3.0 Hz, 1H), 1.86 (tt, J = 11.3, 3.5 Hz, 1H), 1.67–1.57 (m, 3H), 1.41–1.36 (m, 2H), 1.36–1.32 (m, 1H), 1.02 (s, 3H), 0.98 (dd,

Molecules 2021, 26, x FOR PEER REVIEW 11 of 19

Molecules 2021, 26, x FOR PEER REVIEW 11 of 19

rel-(9R,10R,12S)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthra- rel-(9R,10R,12S)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthra-cene-12-carboxamide (rac-2) cene-12-carboxamide (rac-2)

Molecules 2021, 26, 3799 11 of 19

Synthesised according to general procedure D using oxalyl chloride (243 µL, 2.84 mmol), Synthesisedrel-(9S,10S,12 accordingR)-12-methyl-9,10-dihydro-9,10-ethano to general procedure D usinganthracene-12-carboxylic oxalyl chloride (243 µL, 2.84 acid mmol), (6) 1.42rel(375-(9 mmol),mg,S,10 S1.42,12 DMAPR mmol),)-12-methyl-9,10-dihydro-9,10-ethano (4.32 DMAP mg, (4.32 35.4 mmol),mg, 35.4 DIPEA mmol), (120 anthracene-12-carboxylicDIPEAµL, 707 (120µmol) µL, 707 and µmol) 2,6-dichloro-3- acid and 2,6-(6) (375dichloro-3-methylanilinemethylaniline mg, 1.42 mmol), (65.4 mg, DMAP 371 (65.4µ mol).(4.32 mg, Purificationmg, 371 35.4 µmol). mmol), by FCCPurification DIPEA (eluent, (120 10:1 byµL, PE/EtOAc) FCC707 µmol) (eluent, affordedand 10:12,6- dichloro-3-methylanilinePE/EtOAc)the racemic afforded compound the asracemic (65.4 a white mg,compound solid 371 (75µmol). as mg, a white 50%).Purification solid LCMS (75 ( mbymg,/ z): FCC50%). 446.1 (eluent,LCMS [M + ( Na]m/z10:1+):, PE/EtOAc) afforded++ the racemic+ compound as a white solid (75 mg, 50%). LCMS (m/z): 446.1423.1 [M + Na] H] ., System423.1 [M C + HPLC: H] . SystemtR 8.496 C HPLC: min, >95% tR 8.496 purity min, (214 >95% & 254purity nm). (214 HRMS & 254 (m nm)./z): 446.1 [M + Na]+, 423.1 [M + H]+. System C HPLC:+ tR 8.496+ min,1 >95% purity1 (214 & 254 nm). HRMSC25H21 (Cm/zl2NO:): C25 requiresH21Cl2NO: 422.1083 requires [M 422.1083 + H] ; found [M + H] 422.1073.; found 422.1073.H NMR (CDCl H NMR3) δ (CDCl7.40–7.373) δ + 1 HRMS7.40–7.37(m, 1H), (m/z 7.34–7.31 (m,): C1H),25H21 (m,7.34–7.31Cl2NO: 1H), requires 7.30–7.25 (m, 1H), 422.1083 (m, 7.30–7.25 2H), [M 7.18–7.11 (m,+ H] 2H),; found (m, 7.18–7.11 3H), 422.1073. 7.08–7.01 (m, 3H),H NMR (m, 7.08–7.01 3H), (CDCl 6.89 (m,3) (s, δ 1H),7.40–7.373H), 6.89 4.52 (s, (m, 1H), 1H), 1H), 4.52 4.34 7.34–7.31 (s, (t, 1H),J = 2.6(m, 4.34 Hz,1H), (t, 1H),J 7.30–7.25= 2.6 2.64 Hz, (dd, 1H),(m,J 2H),=2.64 12.5, (dd,7.18–7.11 2.8 J = Hz, 12.5, (m, 1H), 2.8 3H), 2.29 Hz, 7.08–7.01 (s,1H), 3H), 2.29 1.66(m, (s, 3H), 6.89 (s, 1H), 4.52 (s, 1H), 4.34 (t, J 13= 2.6 Hz,13 1H), 2.64 (dd, J = 12.5, 2.8 Hz, 1H), 2.29 (s, 3H),(dd, J1.66= 12.4, (dd, 2.7J = Hz,12.4, 1H), 2.7 Hz, 1.18 1H), (s,3H). 1.18 (s,C 3H). NMR C (CDCl NMR3 (CDCl) δ 174.7,3) δ 143.3,174.7, 143.2,143.3, 143.2, 141.5, 141.5, 141.4, 136.1,3H),141.4, 1.66 133.8,136.1, (dd, 132.2,133.8, J = 12.4, 130.8,132.2, 2.7 Hz, 130.8, 129.6, 1H), 129.6, 127.4, 1.18 (s,127.4, 126.5, 3H). 126.5, 126.4, 13C NMR 126.4, 126.2, (CDCl 126.2, 125.9,3) δ 125.9, 174.7, 125.9, 125.7,143.3, 125.7, 123.5,143.2, 123.5,141.5, 123.2, 141.4,123.2,52.7, 49.2, 136.1,52.7, 44.7, 49.2, 133.8, 40.3, 44.7, 132.2, 28.3, 40.3, 130.8, 20.5. 28.3, Analytical129.6, 20.5. 127.4,Analytical data 126.5, including data 126.4, including 1126.2,H NMR 125.9, 1H and NMR 125.9,13C and NMR 125.7, 13C spectra 123.5,NMR 123.2,spectraare in accordance52.7, are in49.2, accordance 44.7, with 40.3, those with 28.3, published those 20.5. published Analytical [26]. [26]. data including 1H NMR and 13C NMR (1R,2S,5R)-2-Isopropyl-5-methylcyclohexylspectra are in accordance with those published methacrylate [26]. (13) (1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl methacrylate (13) (1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl methacrylate (13)

Synthesised according to general procedure A using methacrylic acid (1.62 mL, 19.2 Synthesisedmmol), (1R,2 Saccording ,5R)-2-isopropyl-5-methylcyclohexan-1-ol to general procedure A using methacrylic(3.00 g, 19.2 mmol), acid (1.62 EDC mL, (4.05 19.2 g, mmol),21.2 mmol) (1 R,2 ,2andS,5,5 RDMAP)-2-isopropyl-5-methylcyclohexan-1-ol (117 mg, 960 µmol). Purification (3.00 (3.00by FCC g, g, 19.2 19.2 (eluent, mmol), mmol), DCM) EDC gave (4.05 the g, 21.2title compoundmmol) and asDMAP a transparent (117 mg, oil 960 (3.20 µµmol).mol). g, 74%). PurificationPurification LCMS by(m/z FCC): 225.2 (eluent, [M + DCM) H]+. System gave the C ++ titleHPLC: compound tR 9.042 min, as aa transparenttransparent>95% purity oiloil (214 (3.20(3.20 & g,254g, 74%). 74%). nm). LCMS LCMS1H NMR ( m(m/z/ z(CDCl):): 225.2225.23) δ [M[M 6.23–6.20 ++ H]H] . System (m, 1H), C 1 HPLC:5.81 (dd, tR J9.042 9.042= 2.0, min, 1.2 Hz, >95% 1H), purity purity 3.39 (214(td, (214 J & = 25410.4, nm). 4.3 Hz, HH NMR1H), 2.16 (CDCl (dtd,33)) δδJ 6.23–6.20= 14.0, 7.0, (m, 2.7 1H), Hz, 5.811H), (dd, 1.99 J(dt, = 2.0, J = 1.2 1.5, Hz, 0.9 1H), Hz, 3H),3.39 1.95–1.91(td, J = 10.4, (m, 4.3 1H), Hz, 1.61 1H), (ddd, 2.16 J(dtd, = 16.1, J = 14.6, 14.0, 7.9 7.0, Hz, 2.7 2H), Hz, 1H),1.45–1.37 1.99 (dt,(m, 2H),J = 1.5, 1.14–1.05 0.9 Hz, (m, 3H), 1H), 1.95–1.91 0.96 (dd, (m, J =1H), 12.1, 1.61 4.1 (ddd,Hz, 1H), J = 16.1,0.94–0.88 14.6, (m, 7.9 6H),Hz, 2H), 0.80 1.45–1.37(dd, J = 7.0, (m, 0.9 2H), Hz, 1.14–1.05 3H). 13C (m,NMR 1H), (CDCl 0.96 3 (dd,) δ 163.2, JJ = 12.1, 135.9, 4.1 129.1,Hz, 1H), 71.7 0.94–0.88 , 50.3, 45.2, (m, 34.7, 6H), 31.8, 0.80 13 (dd,25.9, J23.3, = 7.0, 22.3, 0.9 21.1,Hz, 3H).16.2. AnCC NMRalytical NMR (CDCl (CDCl data 33in)) δ δaccordance 163.2,163.2, 135.9, 135.9, with 129.1, 129.1, those 71.7 71.7, published, 50.3, 50.3, 45.2, 45.2, [54]. 34.7, 31.8, 25.9, 23.3, 22.3, 21.1, 16.2. An Analyticalalytical data in accordance with those published [[54].54]. (1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-car(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12- (1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12- carboxylateboxylate (14 ()14) carboxylate (14)

Synthesised according to general procedure B using TiCl 4 (4.38 µL, 3.93 mmol), (1R,2S,5R)- Synthesised2-isopropyl-5-methylcyclohexyl according to general methacrylateprocedure B using (13) (440TiCl 4mg, (4.38 1.96 µL, mmol)3.93 mmol), and (1anthraceneR,2S,5R)- 2-isopropyl-5-methylcyclohexyl(350Synthesised mg, 1.96 according mmol). Purification to general methacrylate procedure by FCC (e Bluent, using(13) (440 TiCl10:1 4 mg,PE/EtOAc](4.38 1.96µL, mmol) 3.93 gave mmol), andthe anthracene (1R,2S,5R)- (350ester2-isopropyl-5-methylcyclohexyl mg,derivative 1.96 mmol). as a light-yellow Purification methacrylateoil by (400 FCC mg, (e 51%,luent, (13) d.e. (44010:1 >70%). mg,PE/EtOAc] 1.96 LCMS mmol) gave(m/z): andthe 401.1 anthraceneanthracene [M − H]‒ ‒ ester.(350 System mg,derivative C 1.96 HPLC: mmol). as taR light-yellow2.204 Purification min, >95% oil by (400 purity FCC mg, (eluent, (214 51%, & d.e. 254 10:1 >70%).nm). PE/EtOAc] 1H LCMS NMR gave( m/z(CDCl): the 401.13) anthraceneδ 7.30–7.27 [M − H] .(m,ester System 1H), derivative 7.25–7.18C HPLC: as t(m,R a 2.204 light-yellow 3H), min, 7.11–7.07 >95% oil (m,purity (400 2H), mg, (214 7.07–6.98 51%, & 254 d.e. nm).(m, >70%). 2H, 1H NMRH3), LCMS 4.48 (CDCl ( m(td,/3z) ):Jδ = 7.30–7.27 401.1 10.8, [M4.3 - 1 (m,Hz,− H] 1H),. System 7.25–7.184.38 (s, C 1H), HPLC: (m, 4.23 3H),t (t,R 7.11–7.072.204 J = 2.6 min, Hz, (m, >95%1H), 2H), 2.70 purity 7.07–6.98 (dd, (214 J = (m,12.7, & 2542H, 3.0 nm). H3),Hz, 1H),4.48H NMR 1.86(td, J(tt, (CDCl= 10.8,J = 11.3,3 4.3) δ Hz,3.57.30–7.27 Hz, 1H), 1H), 4.38 (m, 1.67–1.57 (s, 1H), 1H), 7.25–7.18 4.23 (m, (t,3H), J (m,= 1.41–1.362.6 3H), Hz, 7.11–7.071H), (m, 2.70 2H), (m,(dd, 1.36–1.322H), J = 12.7, 7.07–6.98 (m, 3.0 1H), Hz, (m,1.02 1H), 2H, (s, 1.86 3H), H3), (tt, 0.98 4.48J = 11.3,(dd, (td, 3.5J = 10.8,Hz, 1H),4.3 Hz,1.67–1.57 1H), 4.38(m, 3H), (s, 1H), 1.41–1.36 4.23 (t, (m,J = 2H), 2.6 Hz,1.36–1.32 1H),2.70 (m, 1H), (dd, J1.02= 12.7, (s, 3H), 3.0 0.98 Hz, 1H),(dd, 1.86 (tt, J = 11.3, 3.5 Hz, 1H), 1.67–1.57 (m, 3H), 1.41–1.36 (m, 2H), 1.36–1.32 (m, 1H), 1.02 (s, 3H), 0.98 (dd, J = 13.1, 3.2 Hz, 1H), 0.91 (d, J = 7.0 Hz, 3H), 0.88–0.85 (m, 2H), 0.82 (d, 13 J = 6.5 Hz, 3H), 0.68 (d, J = 6.9 Hz, 3H). C NMR (CDCl3) δ 176.3, 143.9, 143.4, 141.5, 141.0, 126.3, 126.1, 126.0, 125.5, 125.5, 125.2, 123.4, 123.2, 74.7, 52.9, 47.1, 44.6, 40.6, 38.8, 34.3, 31.4, 27.3, 26.0, 23.2, 22.1, 21.1, 16.1. (R)-N-(2-Hydroxy-1-phenylethyl)methacrylamide [55] (16) Molecules 2021,, 26,, xx FORFOR PEERPEER REVIEWREVIEW 12 of 19 Molecules 2021, 26, x FOR PEER REVIEW 12 of 19

JJ == 13.1,13.1, 3.23.2 Hz,Hz, 1H),1H), 0.910.91 (d,(d, JJ == 7.07.0 Hz,Hz, 3H),3H), 0.88–0.850.88–0.85 (m,(m, 2H),2H), 0.820.82 (d,(d, JJ == 6.56.5 Hz,Hz, 3H),3H), 0.680.68 J = 13.1, 3.2 Hz, 1H),13 0.91 (d, J = 7.0 Hz, 3H), 0.88–0.85 (m, 2H), 0.82 (d, J = 6.5 Hz, 3H), 0.68 (d,(d, JJ == 6.96.9 Hz,Hz, 3H).3H). 13C NMR (CDCl33)) δ 176.3,176.3, 143.9,143.9, 143.4,143.4, 141.5,141.5, 141.0,141.0, 126.3,126.3, 126.1,126.1, 126.0,126.0, 13 (d,125.5, J = 125.5,6.9 Hz, 125.2, 3H). 123.4,C NMR 123.2, (CDCl 74.7,3) 52.9,δ 176.3, 47.1, 143.9, 44.6, 143.4, 40.6, 141.5,38.8, 34.3, 141.0, 31.4, 126.3, 27.3, 126.1, 26.0, 126.0, 23.2, 125.5, 125.5, 125.2, 123.4, 123.2, 74.7, 52.9, 47.1, 44.6, 40.6, 38.8, 34.3, 31.4, 27.3, 26.0, 23.2, Molecules 2021, 26, 3799 22.1, 21.1, 16.1. 12 of 19 22.1, 21.1, 16.1. (R)-N-(2-Hydroxy-1-phenylethyl)methacrylamide(R)-N-(2-Hydroxy-1-phenylethyl)methacrylamide [55][55] ((16)) (R)-N-(2-Hydroxy-1-phenylethyl)methacrylamide [55] (16)

KOtt-Bu-Bu (3.27(3.27 g,g, 29.229.2 mmol)mmol) waswas dissolveddissolved inin THTHF (100 mL; technical grade, containing ca. KOt-Bu (3.27 g, 29.2 mmol) was dissolved in THF (100 mL; technical grade, containing ca. 0.2% H22O) with stirring in air at rt for 1 min. Methyl methacrylate (1.55 mL, 14.6 mmol) KOt-Bu (3.27 g, 29.2 mmol) was dissolved in THF (100 mL; technical grade, containing ca. 0.2%and ( RH)-2-amino-2-phenylethan-1-ol)-2-amino-2-phenylethan-1-ol2O) with stirring in air at rt for (2.00(2.00 1 min. g,g, 14.614.6 Methyl mmol)mmol) methacrylate werewere addedadded (1.55 immediatelyimmediately mL, 14.6 and andmmol) thethe 0.2% H2O) with stirring in air at rt for 1 min. Methyl methacrylate (1.55 mL, 14.6 mmol) andmixture (R)-2-amino-2-phenylethan-1-ol was stirred at rt for 1 h. After (2.00 evaporating g, 14.6 mmol) the THF were under added reduced immediately pressure, and H the2O andmixture (R)-2-amino-2-phenylethan-1-ol was stirred at rt for 1 h. After (2.00 evaporating g, 14.6 mmol) the THF were under added reduced immediately pressure, and H the2O mixture(75(75 mL)mL) wasandand stirredDCMDCM (75(75at rt mL)mL) for 1werewere h. After addedadded evaporating andand thethe organicorganic the THF layerlayer under waswas reduced separatedseparated pressure, andand drieddried H2O mixture was stirred at rt for 1 h. After evaporating the THF under reduced pressure, H2O (75(Na mL)2SO4 ).and The DCM solvent (75 was mL) evaporated were added under and reducedthe organic pressure layer wasand residueseparated was and purified dried (75(Na mL)2SO4). and The DCM solvent (75 was mL) evaporated were added under and thereduced organic pressure layer wasand separatedresidue was and purified dried (Naby FCC2SO4 ).(eluent, The solvent 1:10 DCM/MeOH) was evaporated to giveunder the reduced corresponding pressure amide and residue as a yellow/orange was purified (Na2SO4). The solvent was evaporated under reduced pressure and residue was purified bysolid FCC (2.45 (eluent, g, 89%). 1:10 LCMS DCM/MeOH) (m/z): 206.1 to [M give + H] the++. Systemcorresponding C HPLC: amide tR 4.465 as min, a yellow/orange >95% purity bysolid FCC (2.45 (eluent, g, 89%). 1:10 LCMS DCM/MeOH) (m/z): 206.1 to [M give + H] the. System corresponding C HPLC: amide tR 4.465 as amin, yellow/orange >95% purity solid (2.45 g, 89%).1 LCMS (m/z): 206.1 [M + H]+. System C HPLC: tR 4.465 min, >95% purity (214(214 && 254254 nm).nm). 1H NMR (CDCl33)) δ 7.38–7.337.38–7.33 (m,(m, 2H),2H),+ 7.32–7.287.32–7.28 (m,(m, 3H),3H), 6.576.57 (d,(d, JJ == 5.65.6 Hz,Hz, solid (2.45 g, 89%).1 LCMS (m/z): 206.1 [M + H] . System C HPLC: tR 4.465 min, >95% (2141H), &5.75 254 (s, nm). 1H), H 5.37–5.36 NMR1 (CDCl (m, 31H),) δ 7.38–7.33 5.10 (dt, (m, JJ == 2H),7.0,7.0, 5.0 5.07.32–7.28 Hz,Hz, 1H),1H), (m, 3.893.89 3H), (d,(d, 6.57 JJ == (d, 4.94.9 J =Hz,Hz, 5.6 2H), 2H),Hz, purity (214 & 254 nm). H NMR (CDCl3) δ 7.38–7.33 (m, 2H), 7.32–7.28 (m, 3H), 6.57 (d, 1H), 5.75 (s, 1H), 5.37–5.36 (m, 1H),13 5.10 (dt, J = 7.0, 5.0 Hz, 1H), 3.89 (d, J = 4.9 Hz, 2H), 2.96J = 5.6 (s, Hz, 1H),1H), 1.98–1.97 5.75 (s, (m, 1H), 3H). 5.37–5.36 13C NMR (m, (CDCl 1H), 5.1033)) δ (dt, 168.9,168.9,J = 139.8,139.8, 7.0, 5.0 139.2,139.2, Hz,1H), 129.0,129.0, 3.89 128.0,128.0, (d, J 126.8,126.8,= 4.9 2.96 (s, 1H), 1.98–1.97 (m, 3H). 13C NMR (CDCl3) δ 168.9, 139.8, 139.2, 129.0, 128.0, 126.8, 120.3,Hz, 2H), 66.6, 2.96 56.1, (s, 1H),18.8. 1.98–1.97Analytical (m, data 3H). in13 accordanceC NMR (CDCl with) thoseδ 168.9, published 139.8, 139.2, [55]. 129.0, 128.0, 120.3, 66.6, 56.1, 18.8. Analytical data in accordance with3 those published [55]. 126.8,N-((R)-2-Hydroxy-1-phenylethyl)-11-methyl-9,10-dihydro-9,10-ethanoanthracene-11-carbox- 120.3, 66.6, 56.1, 18.8. Analytical data in accordance with those published [55]. N-((R)-2-Hydroxy-1-phenylethyl)-11-methyl-9,10-dihydro-9,10-ethanoanthracene-11-carbox-amideN-((R)-2-Hydroxy-1-phenylethyl)-11-methyl-9,10-dihydro-9,10-ethanoanthracene-11-carboxamide ((17)) (17) amide (17)

Synthesised according to general procedure B using TiCl 44 (1.45(1.45 mL,mL, 13.013.0 mmol),mmol), ((R)-)-N-(2--(2- Synthesisedhydroxy-1-phenylethyl)methacryla according to general procedure mide (1.34 B using g, 6.51 TiCl mmol)4 (1.45(1.45 (16 mL, mL,)) andand 13.0 13.0 anthraceneanthracene mmol), mmol), ( (RR (1.16(1.16)-)-N-(2--(2- g,g, hydroxy-1-phenylethyl)methacrylamide (1.34 g, 6.51 mmol) (16) and anthracene (1.16 g, hydroxy-1-phenylethyl)methacryla6.51 mmol). Residue chromatographedmide with(1.34 5:1g, 6.51 PE/EtOAc mmol) to(16 afford) and anthracenea pair of diastere- (1.16 g, 6.51omers mmol).mmol). (2:1 ratio) ResidueResidue as a chromatographed transparentchromatographed oil (2.00 with with g, 5:1 80%). 5:1 PE/EtOAc PE/EtOAc LCMS to(m/z affordto): afford384.2 a pair [Ma pair of+ H] diastereomers of++. Systemdiastere- C omers (2:1 ratio) as a transparent oil (2.00 g, 80%). LCMS (m/z): 384.2 [M+ + H] . System C (2:1 ratio) as a transparent oil (2.00 g, 80%). LCMS (m/z): 384.2 [M + H] . System+ C HPLC: omersHPLC: (2:1 tR 7.254min, ratio) as a>95% transparent purity (214 oil (2.00 & 254 g, nm).80%). 11 HLCMS NMR ( m/z(401): 384.2MHz, [M CDCl + H]3) δ. System7.36–7.25 C HPLC: tR 7.254min, >95% purity (214 & 2541 nm). H NMR (401 MHz, CDCl3) δ 7.36–7.25 t 7.254min, >95% purity (214 & 254 nm). H NMR1 (401 MHz, CDCl ) δ 7.36–7.25 (m, 12H), HPLC:(m,(m,R 12H),12H), tR 7.254min,7.20–7.117.20–7.11 (m,(m,>95% 6H),6H), purity 7.08–6.997.08–6.99 (214 &(m,(m, 254 4H),4H), nm). 6.046.04 H (s,(s, NMR 0.5H),0.5H), (401 5.755.75 MHz, (s,(s,3 1H),1H), CDCl 4.864.863) (dt,(dt,δ 7.36–7.25 JJ == 12.5,12.5, 7.20–7.11 (m, 6H), 7.08–6.99 (m, 4H), 6.04 (s, 0.5H), 5.75 (s, 1H), 4.86 (dt, J = 12.5, 5.8 Hz, (m,5.8 Hz,12H), 1.7H), 7.20–7.11 4.34 (d, (m, JJ ==6H), 2.22.2 Hz,Hz,7.08–6.99 3H),3H), 3.803.80 (m, (dd,(dd,4H), J J6.04 == 4.6,4.6, (s, 2.42.4 0.5H), Hz,Hz, 1H),1H),5.75 3.68(s,3.68 1H), (dd,(dd, 4.86 JJ == 11.3,11.3,(dt, J 5.6 5.6= 12.5, Hz,Hz, 1.7H), 4.34 (d, J = 2.2 Hz, 3H), 3.80 (dd, J = 4.6, 2.4 Hz, 1H), 3.68 (dd, J = 11.3, 5.6 Hz, 1H), 5.81H), Hz, 3.59 1.7H), (dd, 4.34JJ == 11.3,11.3, (d, J3.93.9 = 2.2 Hz,Hz, Hz, 1H),1H), 3H), 2.582.58 3.80 (dd,(dd, (dd, JJ == J12.8,12.8, = 4.6, 3.03.0 2.4 Hz,Hz, Hz, 0.7H),0.7H), 1H), 3.682.382.38 (dd,(dd,(dd, JJ === 11.3,13.1,13.1, 5.63.03.0 Hz,Hz, 3.59 (dd, J = 11.3, 3.9 Hz, 1H), 2.58 (dd, J = 12.8, 3.0 Hz, 0.7H), 2.38 (dd, J = 13.1, 3.0 Hz, 1H), 1H),1H), 3.591.59 (dd,(dd, JJ === 11.3,13.2,13.2, 3.92.52.5 Hz,Hz,Hz, 1H),1H),1H), 2.581.551.55 (dd,(dd,(dd, JJJ === 12.8,13.4,13.4, 3.03.03.0 Hz,Hz,Hz, 0.7H),0.6H),0.6H), 2.381.101.10 (dd,(s,(s, JJ ==J =2.62.6 13.1, Hz,Hz, 3.0 1.4H),1.4H), Hz, 1.59 (dd, J = 13.2, 2.5 Hz, 1H), 1.55 (dd, J = 13.4, 3.0 Hz, 0.6H), 1.10 (s, J = 2.6 Hz, 1.4H), 1.09 1H),1.09 (s,1.59 3H). (dd, J = 13.2, 2.5 Hz, 1H), 1.55 (dd, J = 13.4, 3.0 Hz, 0.6H), 1.10 (s, J = 2.6 Hz, 1.4H), 1.09(s, 3H). (s, 3H). (R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl(R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl methacrylatemethacrylatemethacrylate ((((((RR)-)-)-1919))) (R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl methacrylate ((R)-19)

Synthesised according to general procedure A using methacrylic acid (1.49 g, 17.3 mmol), Synthesised(Synthesised(R)-3-hydroxy-4,4-dimethyldihydrofuran-2(3)-3-hydroxy-4,4-dimethyldihydrofuran-2(3 according according to to general general procedure procedure A AH using using)-one)-one methacrylic(2.25(2.25 methacrylic g,g, 17.317.3 mmol),acidmmol), acid (1.49 (1.49 EDCEDC g, g, 17.3 17.3(3.31(3.31 mmol), mmol), g,g, 17.317.3 (mmol)R)-3-hydroxy-4,4-dimethyldihydrofuran-2(3 and DMAP (106 mg, 864 µmol). PurificatiHH)-one)-oneon (2.25by (2.25 FCC g, g,17.3 (eluent, 17.3 mmol), mmol), DCM) EDC EDCgave (3.31 the (3.31 g, 17.3title g, µ Molecules 2021, 26, x FOR PEER REVIEWmmol)17.3compound mmol) and asDMAP and a transparent DMAP (106 mg, (106 oil 864 mg, (3.55 µmol). 864 g, 67%). Purificatimol). LCMS Purificationon (m/z by): ):FCC 221.1221.1 by (eluent, [M[M FCC ++ Na]Na] (eluent,DCM)++.. SystemSystem gave DCM) CC the HPLC:HPLC:13 gave oftitle 19 + compoundthe title compound as a transparent as a transparent oil (3.55 oilg, 67%). (3.551 g,LCMS 67%). (m/z LCMS): 221.1 (m/ [Mz): 221.1+ Na] [M+. System + Na] C. SystemHPLC: ttR 6.0106.010 min,min, >95%>95% puritypurity (214(214 && 254254 nm).nm). 1H NMR (CDCl33)) δ 6.26–6.156.26–6.15 (m,(m, 1H),1H), 5.685.68 (dd,(dd, t 1 1 δ tJCJR == 6.010 HPLC: 2.6,2.6, 1.21.2 min, RHz,Hz,6.010 >95% 1H),1H), min, purity 5.415.41 >95% (d,(d, (214 JJ == purity 0.80.8& 254 Hz,Hz, (214 nm). 1H),1H), & 2544.09–4.014.09–4.01H NMR nm). (CDCl (m,(m,H NMR 2H),2H),3) δ 1.98 1.98(CDCl6.26–6.15 (dd,(dd,3) JJ (m,6.26–6.15== 1.7,1.7, 1H), 0.80.8 5.68Hz,Hz, (m, 3H),3H), 1H),(dd, J5.68 = 2.6, (dd, 1.2J Hz,= 2.6, 1H), 1.2 5.41 Hz, (d, 1H), J = 5.410.8 Hz, (d, 1H),J = 0.8 4.09–4.01 Hz, 1H), (m, 4.09–4.01 2H), 1.98 (m, (dd, 2H), J = 1.981.7, 0.8 (dd, Hz,J = 3H), 1.7, 1.21 (s, 3H), 1.13 (s, 3H). 13C NMR (CDCl3) δ 172.4, 165.9, 135.1, 127.5, 76.2, 75.2, 23.1, 19.9, 0.8 Hz, 3H), 1.21 (s, 3H), 1.13 (s, 3H). 13C NMR (CDCl ) δ 172.4, 165.9, 135.1, 127.5, 76.2, 18.2. 3 75.2, 23.1, 19.9, 18.2. (S)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl methacrylatemethacrylate ((((S)-)-19))

Synthesised according to general procedure A using methacrylic acid (2.32 g, 26.9 mmol), (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (3.50 g, 26.9 mmol), EDC (5.16 g, 26.9 mmol), and DMAP (164 mg, 1.34 mmol). Purification by FCC (eluent, DCM) gave the title compound as a transparent oil (1.05 g, 69%). LCMS (m/z): 221.1 [M + Na]+. System C HPLC: tR 6.010 min, >95% purity (214 & 254 nm). 1H NMR (CDCl3) δ 6.26–6.15 (m, 1H), 5.68 (dd, J = 2.6, 1.2 Hz, 1H), 5.41 (d, J = 0.8 Hz, 1H), 4.09–4.01 (m, 2H), 1.98 (dd, J = 1.7, 0.8 Hz, 3H), 1.21 (s, 3H), 1.13 (s, 3H). 13C NMR (CDCl3) δ 172.4, 165.9, 135.1, 127.5, 76.2, 75.2, 23.1, 19.9, 18.2. (R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthra- cene-12-carboxylate ((S)-20)

Synthesised according to general procedure B using TiCl4 (2.39 mL, 21.8 mmol), (R)-4,4- dimethyl-2-oxotetrahydrofuran-3-yl methacrylate ((R)-19) (2.06 g, 10.4 mmol) and anthra- cene (1.85 g, 10.4 mmol). Purification by FCC (eluent, DCM) afforded the title compound as a brown solid. Recrystallization from EtOH gave white crystals (3.25 g, 83%, d.e. ~90%). LCMS (m/z): 399.1 [M+Na]+. System C HPLC: tR 8.320 min, >95% purity (214 & 254 nm). 1H NMR (401 MHz, CDCl3) δ 7.33 (dt, J = 5.4, 3.3 Hz, 1H), 7.30–7.26 (m, 2H), 7.24–7.21 (m, 1H), 7.16–7.10 (m, 2H), 7.10–7.02 (m, 2H), 5.18 (s, 1H), 4.40 (s, 1H), 4.30 (t, J = 2.7 Hz, 1H), 4.04 (d, J = 9.0 Hz, 1H), 3.97 (d, J = 9.0 Hz, 1H), 2.76 (dd, J = 12.8, 3.0 Hz, 1H), 1.50 (dd, J = 12.8, 2.6 Hz, 1H), 1.25 (s, 3H), 1.18 (s, 3H), 1.17 (s, 3H). 13C NMR (CDCl3) δ 175.6, 172.1, 143.7, 143.5, 141.2, 140.4, 126.6, 126.6, 125.7, 125.5, 124.8, 123.9, 123.4, 76.2, 75.5, 52.6, 49.0, 44.4, 39.2, 27.2, 23.0, 20.3. (S)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(R)-12-methyl-9,10-dihydro-9,10-ethanoanthra- cene-12-carboxylate ((R)-20)

Synthesised according to general procedure B using TiCl4 (1.61 mL, 14.7 mmol), (S)-4,4- dimethyl-2-oxotetrahydrofuran-3-yl methacrylate ((S)-19) (1.39 g, 7.01 mmol) and anthra- cene (1.25 g, 7.01 mmol). Purification by FCC (eluent, DCM) afforded the title compound as a beige solid (2.15 g, 82%, d.e. ~83%) LCMS (m/z): 399.1 [M + Na]+. System C HPLC: tR 8.320 min, >95% purity (214 & 254 nm). 1H NMR (CDCl3) δ 7.33 (dt, J = 5.4, 3.3 Hz, 1H), 7.30–7.26 (m, 2H), 7.24–7.21 (m, 1H), 7.16–7.10 (m, 2H), 7.10–7.02 (m, 2H), 5.18 (s, 1H), 4.40 (s, 1H), 4.30 (t, J = 2.7 Hz, 1H), 4.04 (d, J = 9.0 Hz, 1H), 3.97 (d, J = 9.0 Hz, 1H), 2.76 (dd, J = 12.8, 3.0 Hz, 1H), 1.50 (dd, J = 12.8, 2.6 Hz, 1H), 1.25 (s, 3H), 1.18 (s, 3H), 1.17 (s, 3H). 13C NMR (CDCl3) δ 175.6, 172.1, 143.7, 143.5, 141.2, 140.4, 126.6, 126.6, 125.7, 125.5, 124.8, 123.9, 123.4, 76.2, 75.5, 52.6, 49.0, 44.4, 39.2, 27.2, 23.0, 20.3.

Molecules 2021, 26, x FOR PEER REVIEW 13 of 19

Molecules 2021, 26, x FOR PEER REVIEW 13 of 19

1.21 (s, 3H), 1.13 (s, 3H). 13C NMR (CDCl3) δ 172.4, 165.9, 135.1, 127.5, 76.2, 75.2, 23.1, 19.9, 1.2118.2. (s, 3H), 1.13 (s, 3H). 13C NMR (CDCl3) δ 172.4, 165.9, 135.1, 127.5, 76.2, 75.2, 23.1, 19.9, 18.2. (S)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl methacrylate ((S)-19) (S)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl methacrylate ((S)-19) Molecules 2021, 26, 3799 13 of 19

Synthesised according to general procedure A using methacrylic acid (2.32 g, 26.9 mmol), Synthesised(S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3 according to general procedure AHH )-oneusing)-one (3.50methacrylic (3.50 g, g,26.9 26.9 mmol), acid mmol), (2.32 EDC g, EDC 26.9(5.16 (5.16mmol), g, 26.9 g, (mmol),26.9S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3 mmol), and DMAP and DMAP (164 (164mg, 1.34 mg, 1.34mmol). mmol). PurificationH Purification)-one (3.50 by FCC g, by 26.9 FCC(eluent, mmol), (eluent, DCM) EDC DCM) gave (5.16 gave the g, 26.9title the compoundmmol),title compound and as DMAP a transparent as a (164 transparent mg, oil 1.34 (1.05 oil mmol). (1.05g, 69%). g,Purification 69%). LCMS LCMS (m/z by): ( FCCm221.1/z): (eluent, [M 221.1 + Na] [M DCM)+. + System Na] gave+. SystemC the HPLC: title C compound as a transparent oil (1.05 g, 69%).1 LCMS (1m/z): 221.1 [M + Na]+. System C HPLC: tHPLC:R 6.010 tmin,R 6.010 >95% min, purity >95% (214 purity & 254 (214 nm). & 254 H NMR nm). (CDClH NMR3) δ (CDCl 6.26–6.153) δ 6.26–6.15(m, 1H), 5.68 (m, 1H),(dd, Jt5.68 R= 6.010 2.6, (dd, 1.2 min,J Hz,= 2.6,>95% 1H), 1.2 purity5.41 Hz, (d, 1H), (214 J = 5.41 0.8& 254Hz, (d, nm). 1H),J = 0.8 14.09–4.01H Hz,NMR 1H), (CDCl (m, 4.09–4.01 2H),3) δ 1.98 6.26–6.15 (m, (dd, 2H), J =(m, 1.981.7, 1H), 0.8 (dd, 5.68Hz,J = 3H),(dd, 1.7, 13 13 1.21J0.8 = 2.6, Hz, (s, 1.23H), 3H), Hz, 1.13 1.21 1H), (s, (s, 5.413H). 3H), (d, 1.13C J NMR= (s,0.8 3H).Hz, (CDCl 1H),C3) NMRδ4.09–4.01 172.4, (CDCl 165.9, (m,3 )2H), 135.1,δ 172.4, 1.98 127 165.9,(dd,.5, 76.2, J = 135.1, 1.7, 75.2, 0.8 127.5, 23.1, Hz, 19.9, 76.2,3H), 1.2118.2.75.2, (s, 23.1, 3H), 19.9, 1.13 18.2. (s, 3H). 13C NMR (CDCl3) δ 172.4, 165.9, 135.1, 127.5, 76.2, 75.2, 23.1, 19.9, (R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthracene18.2. -12-car (R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthra- boxylate ((S)-20) cene-12-carboxylate(R)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthra- ((S)-20) cene-12-carboxylate ((S)-20)

Synthesised according to general procedure B using TiCl 4 (2.39 mL, 21.8 mmol), (R)-4,4- Synthesised according to general procedure B using TiCl4 (2.39 mL, 21.8 mmol), (R)-4,4- dimethyl-2-oxotetrahydrofuran-3-ylSynthesised according to general procedure methacrylate B using ((R)-19 TiCl) (2.064 (2.39 g, 10.4 mL, mmol) 21.8 mmol),and anthra- (R)- dimethyl-2-oxotetrahydrofuran-3-ylcene4,4-dimethyl-2-oxotetrahydrofuran-3-yl (1.85 g, 10.4 mmol). Purification methacrylate by F methacrylateCC (eluent, ((R)- DCM)19 ((R) (2.06)-19 afforded) g, (2.06 10.4 g,themmol) 10.4 title mmol)andcompound anthra- and asceneanthracene a brown (1.85 g, solid. (1.8510.4 Recrystallizationmmol). g, 10.4 Purification mmol). Purificationfrom by EtOH FCC (eluent,gave by FCC white DCM) (eluent, crystals afforded DCM) (3.25 the g, afforded 83%,title compoundd.e. the ~90%). title LCMSascompound a brown (m/z ):solid. as 399.1 a brown Recrystallization [M+Na] solid.+. System Recrystallization from C HPLC: EtOH t R fromgave 8.320 EtOHwhite min, gavecrystals>95% white purity (3.25 crystals (214g, 83%, & (3.25254 d.e. nm). g,~90%). 83%, 1H + + 1 NMRLCMSd.e. ~90%). (401 (m/z ):MHz, LCMS 399.1 CDCl [M+Na] (m/z3):) δ 399.1 .7.33 System [M+Na] (dt, CJ HPLC:= 5.4,. System 3.3 tR 8.320Hz, C HPLC:1H), min, 7.30–7.26 >95%tR 8.320 purity (m, min, (2142H), >95% &7.24–7.21 254 purity nm).(214 (m, H 1 NMR1H),& 254 7.16–7.10 (401 nm). MHz,H (m, NMR CDCl 2H), (401 37.10–7.02) δ MHz, 7.33 (dt, CDCl(m, J 2H), =3 )5.4,δ 5.187.33 3.3 (s, (dt,Hz, 1H),J 1H),= 4.40 5.4, 7.30–7.26 3.3(s, 1H), Hz, 1H),(m,4.30 2H), 7.30–7.26(t, J =7.24–7.21 2.7 Hz, (m, 1H),2H), (m, 4.041H),7.24–7.21 (d,7.16–7.10 J = (m, 9.0 1H),Hz,(m, 2H),1H), 7.16–7.10 7.10–7.023.97 (d, (m, J = 2H),(m, 9.0 2H), 7.10–7.02Hz, 5.181H), (s,2.76 (m, 1H), (dd, 2H), 4.40 J5.18 = (s,12.8, (s,1H), 1H),3.0 4.30 Hz, 4.40 (t, 1H), J (s, = 2.71.50 1H), Hz, (dd, 4.30 1H), J(t, = 12.8,4.04J = 2.7 (d,2.6 Hz, J Hz,= 1H),9.0 1H), Hz, 4.04 1.251H), (d, (s, 3.97J =3H), 9.0 (d, Hz,1.18J = 9.0 1H),(s, Hz,3H), 3.97 1H), 1.17 (d, 2.76 J(s,= 9.03H).(dd, Hz, J13 =C 1H), 12.8,NMR 2.763.0 (CDCl Hz, (dd, 1H),3J) =δ 12.8,175.6,1.50 (dd, 3.0 172.1, Hz, J = 13 13 1H),12.8,143.7, 1.502.6 143.5, Hz, (dd, 141.2,1H),J = 12.8, 1.25 140.4, 2.6(s, 126.6, Hz,3H), 1H), 1.18126.6, 1.25 (s, 125.7, 3H), (s, 3H), 1.17125.5, 1.18 (s, 124.8, (s,3H). 3H), 123.9,C 1.17 NMR 123.4, (s, 3H).(CDCl 76.2,3C) 75.5, δ NMR 175.6, 52.6, (CDCl 172.1, 49.0,3) 143.7,44.4,δ 175.6, 39.2, 143.5, 172.1, 27.2, 141.2, 143.7,23.0, 140.4, 20.3. 143.5, 126.6, 141.2, 126.6, 140.4, 125.7, 126.6, 125.5, 126.6, 124.8, 125.7, 123.9, 125.5, 123.4, 124.8, 76.2, 123.9, 75.5, 123.4, 52.6, 49.0, 76.2, 44.4,75.5, 39.2, 52.6, 27.2, 49.0, 23.0, 44.4, 20.3. 39.2, 27.2, 23.0, 20.3. (S)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(R)-12-methyl-9,10-dihydro-9,10-ethanoanthracene(S)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(R)-12-methyl-9,10-dihydro-9,10-ethanoanthra--12-car (S)-4,4-Dimethyl-2-oxotetrahydrofuran-3-yl-(R)-12-methyl-9,10-dihydro-9,10-ethanoanthra- cene-12-carboxylateboxylate ((R)-20) ((R)-20) cene-12-carboxylate ((R)-20)

Synthesised according to general procedure B using TiCl 4 (1.61 mL, 14.7 mmol), (S)-4,4- Synthesiseddimethyl-2-oxotetrahydrofuran-3-yl according to general procedure methacrylate B using ((S )-TiCl19) 4(1.39 (1.61 g, mL, 7.01 14.7 mmol) mmol), and anthra-(S)-4,4- cenedimethyl-2-oxotetrahydrofuran-3-ylSynthesised (1.25 g, 7.01 according mmol). to Purification general procedure methacrylate by FCC (eluent, B using ((S)- DCM)19 TiCl) (1.394 afforded(1.61 g, 7.01 mL, themmol) 14.7 title and mmol),compound anthra- (S)- ascene4,4-dimethyl-2-oxotetrahydrofuran-3-yl a beige (1.25 g,solid 7.01 (2.15 mmol). g, 82%, Purification d.e. ~83%) by LCMSFCC methacrylate (eluent, (m/z): 399.1 DCM) ((S )-[M19 afforded )+ (1.39Na]+. g,Systemthe 7.01 title mmol) Ccompound HPLC: and tR + 8.320asanthracene a beige min, solid>95% (1.25 (2.15 purity g, 7.01 g, 82%,(214 mmol). &d.e. 254 ~83%) Purification nm). LCMS 1H NMR by(m/z (CDCl FCC): 399.1 (eluent,3) δ[M 7.33 + Na] DCM) (dt,. JSystem = afforded 5.4, 3.3 C HPLC:Hz, the 1H), title tR + 8.3207.30–7.26compound min, (m, >95% as 2H), a beige purity 7.24–7.21 solid (214 (2.15 (m, & 2541H), g, 82%,nm). 7.16–7.10 d.e.1H NMR ~83%) (m, 2H),(CDCl LCMS 7.10–7.023) ( mδ /7.33z): (m, 399.1(dt, 2H), J [M= 5.4,5.18 + Na] 3.3 (s, 1H),Hz,. System 1H),4.40 1 (s,7.30–7.26C HPLC:1H), 4.30 (m,tR (t,8.320 2H), J = 2.7 min,7.24–7.21 Hz, >95% 1H), (m, purity 4.04 1H), (d, 7.16–7.10 (214 J = 9.0 & 254 Hz, (m, nm). 1H), 2H), 3.97 H7.10–7.02 NMR (d, J (CDCl= (m,9.0 2H),Hz,3) δ1H), 5.187.33 2.76(s, (dt, 1H), (dd,J = 4.40 5.4, J = 12.8,(s,3.3 1H), Hz, 3.0 1H),4.30 Hz, (t,7.30–7.261H), J = 1.502.7 Hz, (m,(dd, 1H), 2H), J = 12.8, 7.24–7.214.04 (d,2.6 J Hz,= (m, 9.0 1H), 1H), Hz, 1.25 7.16–7.101H), (s, 3.97 3H), (m,(d, 1.18 J 2H), = 9.0 (s, 7.10–7.02 Hz,3H), 1H), 1.17 (m, 2.76 (s, 2H), 3H). (dd, 5.18 13J C= 13 12.8,NMR(s, 1H), 3.0 (CDCl 4.40 Hz, (s,31H),) δ 1H), 175.6, 1.50 4.30 172.1,(dd, (t, JJ 143.7,= 2.712.8, Hz,143.5, 2.6 1H), Hz, 141.2, 4.041H), 140.4, (d, 1.25J = 126.6,(s, 9.0 3H), Hz, 126.6, 1.18 1H), 125.7,(s, 3.97 3H), (d,125.5, 1.17J = 124.8, 9.0(s, Hz,3H). 123.9, 1H), C Molecules 2021, 26, x FOR PEER REVIEWNMR2.76 (dd, (CDClJ = 12.8,3) δ 175.6, 3.0 Hz, 172.1, 1H), 143.7, 1.50 (dd,143.5,J = 141.2, 12.8, 2.6140.4, Hz, 126.6, 1H), 1.25126.6, (s, 125.7, 3H), 1.18125.5, (s, 124.8, 3H), 1.1714 123.9, of (s,19 123.4, 76.2, 75.5, 52.6, 49.0, 44.4, 39.2, 27.2, 23.0, 20.3. 3H). 13C NMR (CDCl ) δ 175.6, 172.1, 143.7, 143.5, 141.2, 140.4, 126.6, 126.6, 125.7, 125.5, 123.4, 76.2, 75.5, 52.6, 49.0,3 44.4, 39.2, 27.2, 23.0, 20.3. 124.8, 123.9, 123.4, 76.2, 75.5, 52.6, 49.0, 44.4, 39.2, 27.2, 23.0, 20.3. (S)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid S 21 (S)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid ((((S)-)-21))

Synthesised according to general procedure C using (R)-4,4-Dimethyl-2-oxotetrahydrofu- ran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate ((S)-20) (3.00 g, 7.97 mmol) and NaOH (956 mg, 23.9 mmol). The aqueous phase was extracted with DCM, and the organic extracts were dried (Na2SO4) and evaporated to dryness to afford the desired acid as a white foam (2.05 g, 95%, e.e. ~88%). [α]D25 = −24.9° (c 1.0, CHCl3), lit[α]D20 = −26.7° (c 1.08, CHCl3) [39], LCMS (m/z): 263.1 [M − H]‒. System C HPLC: tR 7.181 min, >95% purity (214 & 254 nm). Analytical data including 1H NMR and 13C NMR spectra are in accordance with those reported by Camps et al. [39] (R)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid ((R)-21) O

H3C (R) OH

(S) (S) Synthesised according to general procedure C using (S)-4,4-Dimethyl-2-oxotetrahydrofu- ran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate ((R)-20) (1.40 g, 3.72 mmol) and NaOH (446 mg, 11.2 mmol). The aqueous phase was extracted with DCM, and the organic extracts were dried (Na2SO4) and evaporated to dryness to afford the desired acid as a white solid (956 mg, 98%, e.e. ~83%). [α]D25 = +24.1° (c 1.0, CHCl3), LCMS (m/z): 263.1 [M − H]‒. System C HPLC: tR 7.181 min, >95% purity (214 & 254 nm). 1H NMR (CDCl3) δ 7.31–7.28 (m, 1H), 7.27–7.23 (m, 3H), 7.23–7.19 (m, 1H), 7.12 (ddd, J = 6.1, 5.6, 3.7 Hz, 2H), 7.08 (dd, J = 7.2, 1.5 Hz, 1H) 7.06–7.01 (m, 1H), 4.37 (s, 1H), 4.26 (t, J = 2.7 Hz, 1H), 2.62 (dd, J = 12.7, 3.0 Hz, 1H), 1.40 (dd, J = 12.7, 2.5 Hz, 1H), 1.07 (s, 3H). 13C NMR (CDCl3) δ 181.7, 143.6, 143.3, 141.3, 140.5, 126.5, 126.3, 126.2, 125.8, 125.7, 125.2, 123.5, 123.3, 52.6, 48.5, 44.5, 38.7, 26.9. (9S,10R,12R)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene- 12-carboxamide ((S)-2)

Synthesised according to general procedure D using (S)-12-methyl-9,10-dihydro-9,10-eth- anoanthracene-11-carbonyl chloride ((S)-21) (260 mg, 919 µmol), 2,6-dichloro-3-methyl- aniline (162 mg, 920 µmol), and DMAP (56.2 mg, 460 µmol). Purification by FCC (eluent, 1:10 EtOAc/PE) afforded the title compound as a white solid (75 mg, 19%, e.e. 90%). LCMS (m/z): 446.1 [M + Na]+, 423.1 [M + H]+. System C HPLC: tR 8.496 min, >95% purity (214 & 254 nm). HRMS (m/z): C25H21Cl2NO: requires 422.1083 [M + H]+; found 422.1073. 1H NMR (CDCl3) δ 7.40–7.37 (m, 1H), 7.34–7.31 (m, 1H), 7.30–7.25 (m, 2H), 7.18–7.11 (m, 3H), 7.08– 7.01 (m, 3H), 6.89 (s, 1H), 4.52 (s, 1H), 4.34 (t, J = 2.6 Hz, 1H), 2.64 (dd, J = 12.5, 2.8 Hz, 1H), 2.29 (s, 3H), 1.66 (dd, J = 12.4, 2.7 Hz, 1H), 1.18 (s, 3H). 13C NMR (CDCl3) δ 174.7, 143.3, 143.2, 141.5, 141.4, 136.1, 133.8, 132.2, 130.8, 129.6, 127.4, 126.5, 126.4, 126.2, 125.9, 125.9, 125.7, 123.5, 123.2, 52.7, 49.2, 44.7, 40.3, 28.3, 20.5.

Molecules 2021, 26, x FOR PEER REVIEW 14 of 19 Molecules 2021, 26, x FOR PEER REVIEW 14 of 19

(S)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid ((S)-21) (S)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid ((S)-21)

Molecules 2021, 26, 3799 14 of 19

Synthesised accordingaccording toto general general procedure procedure C C using using (R ( )-4,4-Dimethyl-2-oxotetrahydrofuran-R)-4,4-Dimethyl-2-oxotetrahydrofu- Synthesised according to general procedure C using (R)-4,4-Dimethyl-2-oxotetrahydrofu- ran-3-yl-(3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylateS)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate ((S )-((20S)-)20 (3.00) (3.00 g, ran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate ((S)-20) (3.00 g,7.97 7.97 mmol) mmol) and and NaOH NaOH (956 (956 mg, mg, 23.9 23.9 mmol). mmol). TheThe aqueousaqueous phasephase waswas extractedextracted with g, 7.97 mmol) and NaOH (956 mg, 23.9 mmol). The aqueous phase was extracted with DCM, and the organic extracts werewere drieddried (Na(Na22SO4)) and and evaporated to dryness to afford DCM, and the organic extracts were dried (Na2SO4) and evaporated25 to dryness to afford the desired acidacid asas aa whitewhite foam foam (2.05 (2.05 g, g, 95%, 95%, e.e. e.e. ~88%). ~88%). [ α[α] ]D25 == −−24.9°24.9◦ (c 1.0, CHClCHCl3), D 25 3 thelit desired2020 acid ◦as a white foam (2.05 g, 95%, e.e. ~88%). [α]‒D = -−24.9° (c 1.0, CHCl3), [[αα]]DD = =−26.7°−26.7 (c 1.08,(c 1.08, CHCl CHCl3) [39],3)[39 LCMS], LCMS (m/z (m): /263.1z): 263.1 [M − [M H]−. SystemH] . System C HPLC: C HPLC: tR 7.181tR lit 20 ‒ min,7.181[α]D >95% min, = −26.7° >95%purity (c purity (2141.08, & CHCl (214 254 nm). &3) 254[39], Analytical nm). LCMS Analytical (m/z data): 263.1including data [M including − 1H]H NMR. System1H and NMR C13 CHPLC: and NMR13 C tspectraR NMR7.181 1 13 min,arespectra in >95% accordance are purity in accordance (214with & those 254 with nm). reported those Analytical reported by Camps data by including Campset al. [39] et al.H [NMR39] and C NMR spectra (R)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylicare in accordance with those reported by Camps et al. [39] acid ((R)-21) (R)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid ((R)-21) (R)-12-Methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylic acid ((R)-21) O O H3C (R) OH H3C (R) OH (S) (S)(S) Synthesised according to general procedure(S) C using (S)-4,4-Dimethyl-2-oxotetrahydrofu- Synthesisedran-3-yl-(S)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate according toto general general procedure procedure C C using using (S ()-4,4-Dimethyl-2-oxotetrahydrofuran-S)-4,4-Dimethyl-2-oxotetrahydrofu- ((R)-20) (1.40 ran-3-yl-(g,3-yl-( 3.72S )-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylatemmol)S)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxylate and NaOH (446 mg, 11.2 mmol). The aqueous phase was (( Rextracted (()-R20)-)20 (1.40) (1.40with g, g, 3.72 mmol) and NaOH (446 mg, 11.2 mmol). The aqueous phase was extracted with DCM,3.72 mmol) and the and organic NaOH extracts (446 mg, were 11.2 dried mmol). (Na2SO The4) and aqueous evaporated phase wasto dryness extracted to afford with DCM, and the organic extracts were dried (Na2SO4) and evaporated25 to dryness to afford theDCM, desired andthe acid organic as a white extracts solid were (956 dried mg, (Na98%,2SO e.e.4) ~83%). and evaporated [α]D = +24.1° to dryness (c 1.0, to CHCl afford3), 25 the desired acid as a white‒ solid (956 mg, 98%, e.e. ~83%). [α]D25 = +24.1°◦ (c 1.0, CHCl13), LCMSthe desired (m/z): acid 263.1 as [M a white − H] . solidSystem (956 C mg,HPLC: 98%, tR 7.181 e.e. ~83%). min, >95% [α]D purity= +24.1 (214 (&c 1.0,254 CHClnm). 3H), LCMS (m/z): 263.1 [M − H]‒. System- C HPLC: tR 7.181 min, >95% purity (214 & 254 nm). 1H NMRLCMS (CDCl (m/z):3) δ 263.1 7.31–7.28 [M − (m,H] 1H),. System 7.27–7.23 C HPLC: (m, 3H),tR 7.1817.23–7.19 min, (m, >95% 1H), purity 7.12 (ddd, (214 J & = 2546.1, NMR1 (CDCl3) δ 7.31–7.28 (m, 1H), 7.27–7.23 (m, 3H), 7.23–7.19 (m, 1H), 7.12 (ddd, J = 6.1, 5.6,nm). 3.7H Hz, NMR 2H), (CDCl 7.08 3(dd,) δ 7.31–7.28 J = 7.2, 1.5 (m, Hz, 1H), 1H) 7.27–7.23 7.06–7.01 (m, (m, 3H), 1H), 7.23–7.19 4.37 (s, (m,1H), 1H), 4.26 7.12 (t, J (ddd,= 2.7 5.6,Hz,J = 6.1, 3.71H), 5.6,Hz, 2.62 3.72H), (dd, Hz, 7.08 J 2H),= 12.7,(dd, 7.08 J3.0 = (dd,7.2, Hz, J1.5 1H),= 7.2,Hz, 1.40 1.51H) (dd, Hz, 7.06–7.01 J 1H) = 12.7, 7.06–7.01 (m, 2.5 1H),Hz, (m, 1H),4.37 1H), 1.07(s, 4.371H), (s, (s,3H). 4.26 1H), 13(t,C 4.26 JNMR = 2.7 (t, 13 Hz,(CDClJ = 2.71H),3 Hz,) δ 2.62 181.7, 1H), (dd, 143.6, 2.62 J = (dd,12.7, 143.3, J3.0= 141.3, 12.7,Hz, 1H), 3.0140.5, Hz, 1.40 126.5, 1H), (dd, 126.31.40 J = 12.7, (dd,, 126.2, 2.5J = Hz,125.8, 12.7, 1H), 2.5125.7, 1.07 Hz, 125.2, (s, 1H), 3H). 123.5, 1.07 C (s, 123.3,NMR 3H). 13 (CDCl52.6,C NMR 48.5,3) δ 181.7, (CDCl44.5, 38.7, 143.6,3) δ 26.9.181.7, 143.3, 143.6, 141.3, 143.3, 140.5, 141.3, 126.5, 140.5, 126.3,126.5, 126.2, 126.3,125.8, 126.2,125.7, 125.8,125.2, 123.5, 125.7, 123.3, 125.2, 52.6,123.5, 48.5, 123.3, 44.5, 52.6, 38.7, 48.5, 26.9. 44.5, 38.7, 26.9. (9S,10R,12R)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene- (9S,10R,12R)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-car (9S,10R,12R)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide ((S)-2) 12-carboxamideboxamide ((S)- 2(()S)-2)

Synthesised according to general procedure D using (S)-12-methyl-9,10-dihydro-9,10-eth- Synthesisedanoanthracene-11-carbonyl according to general chloride procedure ((S)-21 )D (260 using mg, (S )-12-methyl-9,10-dihydro-9,10-eth-919 µmol), 2,6-dichloro-3-methyl- anoanthracene-11-carbonylanilineSynthesised (162 mg, according 920 µmol), to general andchloride DMAP procedure ((S )-(56.221) (260mg D, using460mg, µmol). 919 (S)-12-methyl-9,10-dihydro-9,10- µmol), Purificati 2,6-dichloro-3-methyl-on by FCC (eluent, aniline1:10ethanoanthracene-11-carbonyl EtOAc/PE) (162 mg, afforded 920 µmol), the and title chloride DMAP compound (((56.2S)-21 as mg) a (260 ,white 460 mg, µmol). solid 919 (75 µPurificatimol), mg, 2,6-dichloro-3-methyl19%,on e.e.by FCC90%). (eluent, LCMS 1:10(anilinem/z ):EtOAc/PE) 446.1 (162 [M mg, +afforded 920Na]µ+,mol), 423.1 the and title[M DMAP+compound H]+. System (56.2 as mg, Ca whiteHPLC: 460 µ solidmol). tR 8.496 (75 Purification mg, min, 19%, >95% e.e. by purity FCC90%). (eluent, (214LCMS & + +. R (1:10254m/z nm).): EtOAc/PE) 446.1 HRMS [M + afforded (Na]m/z):, C423.125 theH21 title[MCl2NO: + compound H] requires System as 422.1083 C a whiteHPLC: [M solid t +8.496 H] (75+;mg, foundmin, 19%, >95% 422.1073. e.e. purity 90%). 1H (214 LCMSNMR & + 25 21 l2 + + 1 254((CDClm/ nm).z):3 446.1) δ HRMS 7.40–7.37 [M +(m/z Na] (m,): C ,1H), 423.1H 7.34–7.31C [MNO: + H]requires (m,. System 1H), 422.1083 7.30–7.25 C HPLC: [M (m, +tR H] 2H),8.496; found 7.18–7.11 min, 422.1073. >95% (m, purity 3H), H NMR7.08– (214 + 1 (CDCl&7.01 254 (m,3 nm).) δ3H), 7.40–7.37 HRMS 6.89 (s, ((m,m 1H),/z 1H),): 4.52 C25 7.34–7.31 H(s,21 1H),Cl2NO: 4.34(m, requires 1H),(t, J = 7.30–7.25 2.6 422.1083 Hz, 1H), (m, [M 2.642H), + H](dd, 7.18–7.11; J found = 12.5, (m, 422.1073. 2.8 3H), Hz, 7.08– 1H),H NMR (CDCl ) δ 7.40–7.37 (m, 1H), 7.34–7.31 (m, 1H), 7.30–7.2513 (m, 2H), 7.18–7.11 (m, 3H), 7.012.29 (m,(s, 3H), 3H), 3 1.666.89 (dd,(s, 1H), J = 4.5212.4, (s, 2.7 1H), Hz, 4.34 1H), (t, 1.18 J = 2.6 (s, Hz,3H). 1H), C 2.64NMR (dd, (CDCl J = 12.5,3) δ 174.7,2.8 Hz, 143.3, 1H), 13 2.29143.2,7.08–7.01 (s, 141.5, 3H), (m, 1.66141.4, 3H), (dd, 6.89 136.1, J (s, = 1H),12.4,133.8, 4.522.7 132.2, Hz, (s, 1H), 130.8,1H), 4.34 1.18 129.6, (t, (s,J =127.4,3H). 2.6 Hz, 126.5,C 1H),NMR 126.4, 2.64 (CDCl (dd, 126.2,3)J δ= 174.7,125.9, 12.5, 2.8 143.3,125.9, Hz, Molecules 2021, 26, x FOR PEER REVIEW143.2,1H), 2.29 141.5, (s, 3H),141.4, 1.66 136.1, (dd, J133.8,= 12.4, 132.2, 2.7 Hz, 130.8, 1H), 129.6, 1.18 (s, 127.4, 3H). 13126.5,C NMR 126.4, (CDCl 126.2,) δ 125.9,174.7, 15125.9, 143.3, of 19 125.7, 123.5, 123.2, 52.7, 49.2, 44.7, 40.3, 28.3, 20.5. 3 125.7,143.2, 123.5, 141.5, 123.2, 141.4, 52.7, 136.1, 49.2, 133.8, 44.7, 132.2, 40.3, 130.8,28.3, 20.5. 129.6, 127.4, 126.5, 126.4, 126.2, 125.9, 125.9,

125.7, 123.5, 123.2, 52.7, 49.2, 44.7, 40.3, 28.3, 20.5. (9R,10S,12S)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene(9R,10S,12S)-N-(2,6-Dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene--12-car 12-carboxamideboxamide ((R)- 2(()R)-2)

Synthesised according to general procedure D using (R)-12-methyl-9,10-dihydro-9,10-eth- anoanthracene-11-carbonyl chloride ((R)-21) (135 mg, 477 µmol), 2,6-dichloro-3-methyl- aniline (84. 1 mg, 477 µmol), and DMAP (29.1 mg, 239 µmol). Purification by FCC (eluent, 1:10 EtOAc/PE) afforded the title compound as a white solid (50 mg, 25%, e.e. 84%). LCMS (m/z): 446.1 [M + Na]+, 423.1 [M + H]+. System C HPLC: tR 8.496 min, >95% purity (214 & 254 nm). HRMS (m/z): C25H21Cl2NO: requires 422.1083 [M + H]+; found 422.1073. 1H NMR (CDCl3) δ 7.40–7.37 (m, 1H), 7.34–7.31 (m, 1H), 7.30–7.25 (m, 2H), 7.18–7.11 (m, 3H), 7.08– 7.01 (m, 3H), 6.89 (s, 1H), 4.52 (s, 1H), 4.34 (t, J = 2.6 Hz, 1H), 2.64 (dd, J = 12.5, 2.8 Hz, 1H), 2.29 (s, 3H), 1.66 (dd, J = 12.4, 2.7 Hz, 1H), 1.18 (s, 3H). 13C NMR (CDCl3) δ 174.7, 143.3, 143.2, 141.5, 141.4, 136.1, 133.8, 132.2, 130.8, 129.6, 127.4, 126.5, 126.4, 126.2, 125.9, 125.9, 125.7, 123.5, 123.2, 52.7, 49.2, 44.7, 40.3, 28.3, 20.5.

3.3. Pharmacological Characterisation 3.3.1. Materials Dulbecco’s modified Eagle’s medium, Flp-In CHO cells, and hygromycin B (Invitro- gen, Carlsbad, CA, USA). Fetal bovine serum (FBS) (ThermoTrace, Melbourne, VIC, Aus- tralia). All other reagents (Sigma Aldrich, St. Louis, MO, USA).

3.3.2. General Cell Culture FlpIn Chinese Hamster Ovary (CHO) cells (Invitrogen, Carlsbad, CA, USA) were grown in DMEM supplemented with 10% FBS and maintained at 37 °C in a humidified incubator (5% CO2). The Flp-In-CHO cells were transfected with the pOG44 vector encod- ing Flp recombinase and the pDEST vector encoding the D2LR or the hD1R at a ratio of 9:1 using PEI as transfection reagent. Following transfection (24 h), cells were subcultured and the medium supplemented with 700 mg/mL HygroGold (Invivogen) as selection agent, to obtain cells stably expressing the D1R or D2LR. FlpIn Chinese Hamster Ovary CHO cells stably expressing the hD1R or hD2LR were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, and 200 µg/mL of Hy- gromycin-B, and maintained at 37 °C in a humidified incubator (5% CO2).

Cell Culture and Transfection for cAMP Assay

FlpIn CHO cells stably expressing the hD1R were maintained in DMEM supple- mented with 5% fetal calf serum and 0.2 mg/mL hygromycin at 37 °C in a humidified incubator (5% CO2). For transfection, the cells were grown in 10 cm culture dishes until 60% confluent. A mixture of 4 µg plasmid DNA containing BRET-based cAMP (CAMYEL) biosensor construct [52] and 25 µg 20 kDa linear PEI in 500 µL 150 mM NaCl was added into a dish of cells.

3.3.3. cAMP Assay Measurement Cellular cAMP levels were measured with the CAMYEL BRET-based biosensor for cAMP [52]. One day following transfection, cells were trypsinised and seeded in white 96- well microplates, cultured for an additional day, rinsed (×2) with Hank’s Balanced Salt Solution (HBSS), then incubated in fresh HBSS. For the D1R functional assay, the cells were stimulated with DA together with addition of the modulators. The BRET signals were measured using a BMG Lumistar counter 30 min after stimulation. For the D2R functional

Molecules 2021, 26, 3799 15 of 19

Synthesised according to general procedure D using (R)-12-methyl-9,10-dihydro-9,10- ethanoanthracene-11-carbonyl chloride ((R)-21) (135 mg, 477 µmol), 2,6-dichloro-3-methy laniline (84. 1 mg, 477 µmol), and DMAP (29.1 mg, 239 µmol). Purification by FCC (eluent, 1:10 EtOAc/PE) afforded the title compound as a white solid (50 mg, 25%, e.e. 84%). LCMS + + (m/z): 446.1 [M + Na] , 423.1 [M + H] . System C HPLC: tR 8.496 min, >95% purity (214 + 1 & 254 nm). HRMS (m/z): C25H21Cl2NO: requires 422.1083 [M + H] ; found 422.1073. H NMR (CDCl3) δ 7.40–7.37 (m, 1H), 7.34–7.31 (m, 1H), 7.30–7.25 (m, 2H), 7.18–7.11 (m, 3H), 7.08–7.01 (m, 3H), 6.89 (s, 1H), 4.52 (s, 1H), 4.34 (t, J = 2.6 Hz, 1H), 2.64 (dd, J = 12.5, 2.8 Hz, 13 1H), 2.29 (s, 3H), 1.66 (dd, J = 12.4, 2.7 Hz, 1H), 1.18 (s, 3H). C NMR (CDCl3) δ 174.7, 143.3, 143.2, 141.5, 141.4, 136.1, 133.8, 132.2, 130.8, 129.6, 127.4, 126.5, 126.4, 126.2, 125.9, 125.9, 125.7, 123.5, 123.2, 52.7, 49.2, 44.7, 40.3, 28.3, 20.5.

3.3. Pharmacological Characterisation 3.3.1. Materials Dulbecco’s modified Eagle’s medium, Flp-In CHO cells, and hygromycin B (Invitrogen, Carlsbad, CA, USA). Fetal bovine serum (FBS) (ThermoTrace, Melbourne, VIC, Australia). All other reagents (Sigma Aldrich, St. Louis, MO, USA).

3.3.2. General Cell Culture FlpIn Chinese Hamster Ovary (CHO) cells (Invitrogen, Carlsbad, CA, USA) were grown in DMEM supplemented with 10% FBS and maintained at 37 ◦C in a humidified incubator (5% CO2). The Flp-In-CHO cells were transfected with the pOG44 vector encod- ing Flp recombinase and the pDEST vector encoding the D2LR or the hD1R at a ratio of 9:1 using PEI as transfection reagent. Following transfection (24 h), cells were subcultured and the medium supplemented with 700 mg/mL HygroGold (Invivogen) as selection agent, to obtain cells stably expressing the D1R or D2LR. FlpIn Chinese Hamster Ovary CHO cells stably expressing the hD1R or hD2LR were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, and 200 µg/mL of Hygromycin-B, ◦ and maintained at 37 C in a humidified incubator (5% CO2).

Cell Culture and Transfection for cAMP Assay

FlpIn CHO cells stably expressing the hD1R were maintained in DMEM supplemented with 5% fetal calf serum and 0.2 mg/mL hygromycin at 37 ◦C in a humidified incubator (5% CO2). For transfection, the cells were grown in 10 cm culture dishes until 60% confluent. A mixture of 4 µg plasmid DNA containing BRET-based cAMP (CAMYEL) biosensor construct [52] and 25 µg 20 kDa linear PEI in 500 µL 150 mM NaCl was added into a dish of cells.

3.3.3. cAMP Assay Measurement Cellular cAMP levels were measured with the CAMYEL BRET-based biosensor for cAMP [52]. One day following transfection, cells were trypsinised and seeded in white 96-well microplates, cultured for an additional day, rinsed (×2) with Hank’s Balanced Salt Solution (HBSS), then incubated in fresh HBSS. For the D1R functional assay, the cells were stimulated with DA together with addition of the modulators. The BRET signals were measured using a BMG Lumistar counter 30 min after stimulation. For the D2R functional assay, cells were stimulated with DA in the presence of 10 µM forskolin (final concentration). DA and modulators were added 15 min prior to stimulation and BRET signals were measured using a BMG Lumistar counter 30 min after stimulation. The BRET signal (BRET ratio) was detected at 445–505 nm and 505–565 nm using a LUMIstar Omega instrument (BMG LabTech, Offenburg, Germany), and quantified by calculating the ratio of light emitted at 535 ± 30 nm (YFP) to light emitted at 475 ± 30 nm (RLuc). Molecules 2021, 26, 3799 16 of 19

3.3.4. Data Analysis of Allsotery Simulations, statistical analyses and computerized nonlinear regression were per- formed using Prism 6.0 (GraphPad Prism 6.0b Software, San Diego, CA, USA). Analysis of functional data. All concentration-response data were fitted to the follow- ing modified four-parameter Hill equation in order to derive potency estimates [56].

(E − Basal)·[A]nH E = Basal + max (1) nH nH [A ] + EC50

where E = the effect of the system; nH = Hill slope; EC50 = [A] (concentration of agonist) that provides the midpoint response between basal and maximal effect of DA or other agonists (Emax), which are the lower and upper asymptotes of the response, respectively. To determine the mode of interaction of compounds 2, and optical isomers of 2 at the D1R in relation to the agonist DA, all data were analyzed using a complete operational model of allosterism and agonism according to Equation (2) [34]

E (τ [A](K + αβ[B]) + τ [B]K )nH = m A B B A E nH nH (2) ([A]KB + KAKB + KA[B] + α[A][B]) + (τA[A](KB + αβ[B]) + τB[B]KA)

where Em = maximum possible cellular response, [A]&[B] = concentrations of orthosteric and allosteric ligands, respectively, KA & KB = equilibrium dissociation constants of orthos- teric and allosteric ligands, respectively, τA & τB = operational measures of orthosteric and allosteric ligand efficacy (which incorporate both signal efficiency and receptor density), respectively, α = binding cooperativity parameter between orthosteric and allosteric ligand, and β = magnitude of the allosteric effect of modulator on efficacy of orthosteric agonist. The KA for DA was determined through receptor depletion by phenoxybenzamine alkylation as the proportional relationship of RT to measured τ whilst KA is invariant with receptor depletion. Thus, unique estimates of KA could be measured by direct fitting of the operational model to the family of concentration-response curve for DA [57,58]. A series of cAMP inhibition assays were conducted in cells which were pre-treated with phenoxybenzamine (an alkylating agent which is used to inhibit high affinity orthosteric interactions at the D2R) [59]. The data for DA evaluated in the presence of the alkylating agent were then fit to an operational model of receptor depletion in order to determine values of KA and τA (log KA = −5.78 ± 0.16, log τA = 1.84 ± 0.16). These data were used to constrain values of KA and τA when we fitted the operational model of allostery (Equation (2)) to functional data. The value of the Hill slope (nH) was fixed to unity. The logarithms of affinity and cooperativity values are normally distributed, whereas the absolute values (antilogarithms) are not, and therefore all statistical analyses were performed on the logarithmic values. However, for ease of interpretation, allosteric parameter antilogarithms are highlighted in the main text [60].

4. Conclusions In this study, we describe the first reported enantioenrichment of optical isomers of rac- 2 using chiral auxiliaries derived from enantiomers of 3-hydroxy-4,4-dimethyldihydrofuran- 2(3H)-one and their pharmacological characterisation. Interestingly, (R)-2 was shown to display 4-fold lower positive cooperativity with DA as compared to (S)-2 and a 7-fold lower affinity for the D1R. Our findings illustrate the importance of further investigation into asymmetric syntheses of analogues of 2 and/or isolation of optically pure analogues as part of future SAR efforts aimed at developing enhanced D1R PAMs based on this scaffold.

Supplementary Materials: The following are available online. Figure S1: Chiral HPLC chro- matogram for Compound 5, Figure S2: Chiral HPLC chromatogram for Compound (S)-21, Figure S3: Chiral HPLC chromatogram for Compound (R)-21, Figures S4–S7: HPLC & chiral HPLC chro- matograms, 1H& 13C NMR spectra for 2, Figure S8: HPLC chromatogram (MeOH blank), Figures S9–S12: Molecules 2021, 26, 3799 17 of 19

HPLC & chiral HPLC chromatograms, 1H& 13C NMR spectra for (S)-2, Figures S13–S16: HPLC & chiral HPLC chromatograms, 1H& 13C NMR spectra for (R)-2. Author Contributions: T.J.F. synthesized the target compounds and carried out their spectroscopic identification. P.J.S.and B.C. contributed to the spectroscopic identification. T.J.F. and J.R.L. performed the in vitro pharmacological evaluation of the synthesized target compounds. J.R.L., P.J.S. and B.C. proposed the work and T.J.F. prepared the manuscript for publication. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by a National Health and Medical Research Council (NHMRC) of Australia under project grant no. APP1049564. Data Availability Statement: Data is contained within the article or Supplementary Materials. Acknowledgments: The authors would like to thank the Australian government for their support through an Australian Postgraduate Award to T.J.F. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are available from the authors.

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