Article ArticleOn the Importance of the Thiazole Nitrogen in OnEpothilones: the Importance Semisynthesis of the Thiazole and Microtubule- Nitrogen in Epothilones: Semisynthesis and Microtubule-Binding Binding Affinity of Deaza-Epothilone C Affinity of Deaza-Epothilone C Adriana Edenharter 1, Lucie Ryckewaert 1, Daniela Cintulová 1, Juan Estévez-Gallego 2, 1 1 1 2 AdrianaJosé Fernando Edenharter Díaz 2 ,and Lucie Karl-Heinz Ryckewaert Altmann, Daniela 1,* Cintulová , Juan Estévez-Gallego , José Fernando Díaz 2 and Karl-Heinz Altmann 1,* 1 ETH Zürich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, CH- 1 8093ETH Zürich, Zürich, Switzerland; Department adriana.edenharter@gmx. of Chemistry and Appliedcom Biosciences, (A.E.); [email protected] Institute of Pharmaceutical (L.R.); Sciences, [email protected] Zürich, Switzerland; [email protected](D.C.) (A.E.); [email protected] (L.R.); [email protected] (D.C.) 2 Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, 2 Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain; [email protected] (J.E.-G.); [email protected] (J.F.D.) 28040 Madrid, Spain; [email protected] (J.E.-G.); [email protected] (J.F.D.) * Correspondence: [email protected] * Correspondence: [email protected]

Received: 18 April 2020; Accepted: 21 May 2020; Published: 23 May 2020


Received: 18 April 2020; Accepted: 21 May 2020; Published: 23 May 2020
 Abstract: Deaza-epothilone C, which incorporates a thiophene moiety in place of the thiazole Abstract:heterocycleDeaza-epothilone in the natural epothilone C, which incorporatesside chain, ahas thiophene been prepared moiety inby placesemisynthesis of the thiazole from heterocycleepothilone A, in thein order natural to epothilone assess the sidecontribution chain, has of been the preparedthiazole nitrogen by semisynthesis to microtubule from epothilone binding. A,The in synthesis order to assess was based the contribution on the esterification of the thiazole of anitrogen known toepothilo microtubulene A-derived binding. carboxylic The synthesis acid wasfragment based and on a the fully esterification synthetic alcohol of a known building epothilone block incorporating A-derived the carboxylic modified acid side fragment chain segment and a fullyand syntheticsubsequent alcohol ring-closure building blockby ring-closing incorporating olefin the modified metathesis. side chainThe segmentlatter proceeded and subsequent with ring-closureunfavorable byselectivity ring-closing and olefinin low metathesis. yield. Distinct The latterdifferences proceeded in chemical with unfavorable behavior were selectivity unveiled and inbetween low yield. the Distinctthiophene-derived differences in advanced chemical behaviorintermediates were unveiledand what between has been the thiophene-derivedreported for the advancedcorresponding intermediates thiazole-based and what congeners. has been Compared reported for to the natural corresponding epothilone thiazole-based C, the free congeners.energy of Comparedbinding of deaza-epothilone to natural epothilone C to C, mi thecrotubules free energy was of reduced binding by of ca. deaza-epothilone 1 kcal/mol or less, C tothus microtubules indicating wasa distinct reduced but by non-decisive ca. 1 kcal/mol role or of less, the thus thiazole indicating nitrogen a distinct in the butinteraction non-decisive of epothilones role of the with thiazole the nitrogentubulin/microtubule in the interaction system. of epothilonesIn contrast to with natural the tubulin epothilone/microtubule C, deaza-epothilone system. In contrast C was todevoid natural of epothiloneantiproliferative C, deaza-epothilone activity in vitro C wasup to devoid a concentration of antiproliferative of 10 μM, activity presumablyin vitro dueup toto aan concentration insufficient ofstability 10 µM, in presumably the cell culture due medium. to an insu fficient stability in the cell culture medium.

Keywords: binding aaffinity;ffinity; epothilones;epothilones; deaza-epothilone;deaza-epothilone; microtubules;microtubules; structure-activitystructure-activity relationship; thiophenethiophene.

1. Introduction Epothilones A and B (Epo A and B) (Figure 1 1)) areare 16-membered,16-membered, polyketide-derivedpolyketide-derived macrolidesmacrolides thatthat werewere discovereddiscovered inin 19871987 byby ReichenbachReichenbach etet al.al. in the contextcontext ofof aa screeningscreening forfor newnew antifungalantifungal agents from the soil-dwelling myxobacterium SorangiumSorangium cellulosumcellulosum SoceSoce 9090 [[1,2].1,2]. A number of closely related, butbut lessless prevalent polyketides,polyketides, likelike epothilonesepothilones CC ((EpoEpo CC)) andand DD (Epo D) were later identified identified inin largerlarger scalescale fermentationsfermentations ofof S.S. cellulosumcellulosum [[3].3].

Figure 1. Molecular structures of natural epothilones A–D (Epo A–D).). Chemistry 2020, 2, Firstpage-Lastpage; doi: www.mdpi.com/journal/chemistry

Chemistry 2020, 2, 30; doi:10.3390/chemistry2020030 www.mdpi.com/journal/chemistry ChemistryChemistry 20202020,, 22,, x 30 500 of 5092

Epo A and B were subsequently found to be highly cytotoxic in the 60-cell line panel of the NationalEpo Cancer A and Institute,B were subsequently although the foundmechanisti to bec underpinnings highly cytotoxic of inthis the effect 60-cell remained line panel unknown of the atNational the time Cancer [4]. Interest Institute, in epothilones although the then mechanistic surged in underpinnings 1995, when Bollag of this et e al.ffect demonstrated remained unknown that Epo at Athe and time B [were4]. Interest new microtubule-stabilizing in epothilones then surged agents in 1995,and, thus, when inhibited Bollag et cell al. demonstratedproliferation by that a Epotaxol- A likeand mechanismB were new [5]. microtubule-stabilizing At the time of discovery agents of their and, mode thus, of inhibited action, Epo cell A proliferation and B were bythe a only taxol-like non- taxanemechanism compound [5]. At class the time known of discovery to stabilize of theirmicrotubules, mode of action, but in Epocontrast A and to Btaxolwere they the retained only non-taxane almost fullcompound activity (i.e., class IC known50 values to stabilizein the nM microtubules, range) against but multidrug-resistant in contrast to taxol cancer they retainedcells expressing almost the full P-glycoproteinactivity (i.e., IC efflux50 values pump in or the harboring nM range) tubulin against mutations multidrug-resistant [6,7]. cancer cells expressing the P-glycoproteinNumerous etotalfflux syntheses pump or harboringof natural epothilones tubulin mutations have been [6,7]. reported in the literature, and these effortsNumerous have been total reviewed syntheses extensively of natural [8–13]. epothilones Based on have the chemistry been reported developed in the in literature, the context and of these the totalefforts synthesis have been work, reviewed hundreds extensively of synthetic [8–13 ].anal Basedogues on of the epothilones chemistry developedhave been inprepared the context for structure-activityof the total synthesis relationship work, hundreds (SAR) studies of synthetic and with analogues the objective of epothilones to deliver compounds have been preparedwith an improvedfor structure-activity overall pharmacological relationship (SAR)profile studies [14,15]. and In addition, with the objectivesemisynthetic to deliver approaches compounds have been with pursuedan improved to explore overall the pharmacological epothilone-like structural profile [14 sp,15ace,]. Inin particular addition, semisyntheticby Höfle and co-workers. approaches (This have workbeen pursuedis summarized to explore in ref. the [16]). epothilone-like The most important structural semisynthetic space, in particular epothilone by Höfle derivative and co-workers. is the Epo B(This lactam work ixabepilone, is summarized which in is ref. approved [16]). The by mostthe FDA important (under semisynthetic the tradename epothilone Ixempra® derivative) for the ® treatmentis the Epo of B metastaticlactam ixabepilone, or locally advanced which is approved breast cancer by the [17]. FDA At least (under eight the additional tradename epothilone- Ixempra ) typefor theagents treatment have been of metastatic advanced orto locallyclinical advancedtrials in oncology, breast cancer including [17]. the At natural least eight product additional Epo A [14,15],epothilone-type and Epo agentsD has havealso been advancedinvestigated to clinicalin Phase trials I cl ininical oncology, trials for including Alzheimer’s the natural disease product [18]. TheEpo development A [14,15], and ofEpo most D has of these also beencompounds investigated has be inen Phase discontinued I clinical trials (including for Alzheimer’s the development disease [18 of]. EpoThe D development for Alzheimer’sof most disease), of these but compounds an analog termed has been utidelone discontinued (or UTD1) (including is currently the development being studied of inEpo Phase D for III Alzheimer’s clinical trials disease), against butbreast an ca analogncer (in termed combination utidelone with (or UTD1)capecitabine) is currently [19]. being studied in PhaseWhile III numerous clinical trials side against chain-modified breast cancer epothilone (in combination analogs with have capecitabine) been investigated [19]. as part of comprehensiveWhile numerous SAR studies side [15,16], chain-modified a specific epothilonequestion that analogs has been have addressed been investigated only indirectly as partrelates of tocomprehensive the importance SAR of studiesthe N-atom [15,16 in], the a specific thiazole question ring for that microtubule has been addressed binding and only cellular indirectly potency. relates Thus,to the Nicolaou importance and of co-workers the N-atom have in the shown thiazole that ringamong for the microtubule three possible binding pyridyl-Epo and cellular B variants potency. (FigureThus, Nicolaou 2), the isomer and co-workers with the N-atom have shownin the position that among ortho the to threethe vinyl possible linkerpyridyl-Epo between the B pyridinevariants ring(Figure and2), the the macrolactone isomer with the coreN -atom(i.e., o in-Pyr-Epo the position B) isortho the tomost the vinylpotent linker with between regard theto both pyridine the promotionring and the of macrolactone tubulin polyme corerization (i.e., o-Pyr-Epo and the Binhibition) is the most of potentcancer withcell growth regard toin both vitro the [20]. promotion At the sameof tubulin time, polymerizationthe meta and para and isomers the inhibition (m-Pyr-Epo of cancer B and cell p-Pyr-Epo growth Bin, vitrorespectively)[20]. At still the sameretained time, a significantthe meta and tubulin-polymerizingpara isomers (m-Pyr-Epo capacity, B and whichp-Pyr-Epo reflects B ,the respectively) ability of stilla compound retained ato significant stabilize microtubules.tubulin-polymerizing capacity, which reflects the ability of a compound to stabilize microtubules.

FigureFigure 2. 2. MolecularMolecular structures structures of of the the three three possible possible pyridyl-Epopyridyl-Epo B B variantsvariants and and of of phenyl-Epophenyl-Epo D D..

Nicolaou’sNicolaou’s data data were were in in line line with with the the results results of of an an earlier earlier study study by by Danishefsky Danishefsky and and co-workers co-workers whowho had had reported reported the the phenyl-based phenyl-based EpoEpo D D analoganalog Ph-EpoPh-Epo D D (Figure(Figure 22)) toto bebe aa potentpotent inducerinducer ofof tubulintubulin polymerization, polymerization, albeit albeit to to a a lo lowerwer extent extent than than the the natural natural product product Epo D D itselfitself [21]. [21]. These These data data suggestedsuggested that that the the presence presence of of a a heterocyclic heterocyclic NN-atom-atom next next to to the the vinyl vinyl linker linker moiety moiety in in epothilone epothilone analogsanalogs is is required required for maximum inductioninduction ofof tubulintubulin assembly assembly and, and, consequently, consequently, for for the the inhibition inhibition of ofcancer cancer cell cell proliferation. proliferation. However, However, neither neither of of the the above above studies studies included included a quantitativea quantitative assessment assessment of ofthe the microtubule-binding microtubule-binding affi nityaffinity of the of analogs the analogs investigated. investigated. In this context,In this itcontext, needs toit beneeds recognized to be recognizedthat the assessment that the ofassessment tubulin polymerization of tubulin polymerization induction is useful induction for the is unequivocaluseful for the identification unequivocal of identificationcompounds with of compoundspoor tubulin with assembly poor properties,tubulin assembly but is less properties, suited for but the high-resolutionis less suited for quantitative the high- resolutiondifferentiation quantitative between differentiation potent assembly between inducers. pote Innt ourassembly own work, inducers. we have In our demonstrated own work, we that have the demonstrated that the microtubule-binding affinities of quinoline-based epothilone analogs (Figure

Chemistry 2020, 2, 30 501 of 509 Chemistry 2020, 2, x 3 microtubule-binding affinities of quinoline-based epothilone analogs (Figure3)[ 22] that incorporate the 3) [22] that incorporate the side chain N-atom in the “natural” position (i.e., m-Quin-Epo B and m- side chain N-atom in the “natural” position (i.e., m-Quin-Epo B and m-Quin-Epo D) are ca. one order Quin-Epo D) are ca. one order of magnitude higher than those of the corresponding regioisomers p- of magnitude higher than those of the corresponding regioisomers p-Quin-Epo B and p-Quin-Epo D, Quin-Epo B and p-Quin-Epo D, respectively (Kb’s of 92 × 107 and 88 × 107 for m-Quin-Epo B and m- respectively (K ’s of 92 107 and 88 107 for m-Quin-Epo B and m-Quin-Epo D, respectively, Quin-Epo D, respectively,b × vs. 6.9 × 107 ×and 6.1 × 107 for p-Quin-Epo B and p-Quin-Epo D). Quite vs. 6.9 107 and 6.1 107 for p-Quin-Epo B and p-Quin-Epo D). Quite intriguingly, and for reasons intriguingly,× and for× reasons not understood, the difference in the microtubule-binding affinity not understood, the difference in the microtubule-binding affinity between m-Quin-Epo D and between m-Quin-Epo D and p-Quin-Epo D translates into a corresponding difference in the cellular p-Quin-Epo D translates into a corresponding difference in the cellular activity, while the epoxides activity, while the epoxides m-Quin-Epo B and p-Quin-Epo B are virtually equipotent (and highly m-Quin-Epo B and p-Quin-Epo B are virtually equipotent (and highly active). active).

Figure 3. Molecular structures of quinoline-derived epothilone analogs.

While ourour data data for for quinoline-based quinoline-based epothilone epothilone analogs analogs seem seem to re-enforce to re-enforce the conclusions the conclusions derived fromderived the from earlier the studies earlier by studies Nicolaou by andNicolaou Danishefsky and Danishefsky on pyridyl- on and pyridyl- phenyl-based and phenyl-based epothilone analogs,epothilone respectively, analogs, respectively, they need tothey be need interpreted to be interpreted with some with care, some in light care, of in thelight significant of the significant overall modificationoverall modification of the side of chainthe side vs. naturalchain vs. epothilones. natural epothilones. Finally, the recentFinally, X-ray the crystalrecent structureX-ray crystal of a tubulin-structureEpo of Aa tubulin-complexEpo [23 ]A invokes complex a hydrogen [23] invokes bond a betweenhydrogen the bond thiazole between nitrogen the andthiazole the side nitrogen chain hydroxyand the side group chain of βhydroxyThr297. group Overall, of theβThr297. available Overall, experimental the available data ex suggestperimental that data the presence suggest that of a properlythe presence positioned of a properly heterocyclic positionedN-atom heterocyclic in side chain-modified N-atom in side epothilone chain-modified analogs isepothilone required, inanalogs order tois required, maximize in interactions order to maximize with the interactions tubulin/microtubule with the tubulin/microtubule system. At the same system. time, At the the magnitudesame time, ofthe the magnitude effect associated of the effect with associated the simple with removal the simple of this removal nitrogen of fromthis nitrogen the natural fromside the chain,natural quite side surprisingly,chain, quite surprisingly, has never been has assessed. never been We assessed. have thus We been have interested thus been for interest some timeed for in thesome synthesis time in ofthe a thiophene-containingsynthesis of a thiophene-containi analog of a naturalng analog epothilone of a natural and the epothilone determination and ofthe its determination microtubule-binding of its amicrotubule-bindingffinity in comparison affinity with the in naturalcomparison parent with structure. the natural In this parent paper, structure. we describe In this the paper, results we of thesedescribe efforts. the results of these efforts.

2. Materials and Methods Detailed protocols for the synthesis of new compoundscompounds and the associated analytical data can be found in the Supplementary Materials.Material.

3. Results and Discussion

3.1. Synthesis of Deaza-Epo C (5) The initial initial synthetic synthetic target target of of our our work work was was deaza-Epodeaza-Epo A (1 A), (which1), which we planned we planned to access to access from fromketone ketone 2 by 2meansby means of the of theWittig Wittig or orHorner Horner–Woodsworth–Emmons–Woodsworth–Emmons (HWE) (HWE) reaction reaction (Scheme (Scheme 11).). Ketone 2 can be accessed from Epo A by TMS-protection and ozonolysis [[24];24]; and its conversion into TMS-protected 3 3via via the the transformation transformation of of the the ketone ketone moiety moiety into into the the corresponding corresponding vinyl vinyl boronic boronic acid, followedacid, followed by iodination by iodination and Stilleand Stille coupling, coupling, had beenhad been described described earlier earlier by Höfle by Höfle and co-workersand co-workers [25]. [25]. However, no experimental protocols for this reaction sequence are provided in ref. [25], and no yields are reported for the iodination and Stille coupling steps. On the other hand, we had been

Chemistry 2020, 2, 30 502 of 509

Chemistry 2020, 2, x 4 However,Chemistry 2020 no, 2,experimental x protocols for this reaction sequence are provided in ref. [25], and no4 yields are reported for the iodination and Stille coupling steps. On the other hand, we had been successful ourselves in the elaboration of ketone 2 into epothilone A analogs bearing modified successful ourselves in the elaboration of ketone 2 into epothilone A analogs bearing modified thiazole, thiazole,successful pyrimidine ourselves orin pyridinethe elaboration moieties of in ketone place of2 intothe naturalepothilone thiazole A analogs heterocycle bearing by meansmodified of pyrimidine or pyridine moieties in place of the natural thiazole heterocycle by means of HWE chemistry HWEthiazole, chemistry pyrimidine (Hauenstein or pyridine & Altmamoietiesnn, inunpublished place of the experiments; natural thiazole see alsoheterocycle refs. [26] by and means [27]). of (Hauenstein & Altmann, unpublished experiments; see also refs. [26,27]). Unfortunately, the attempted Unfortunately,HWE chemistry the (Hauenstein attempted HWE & Altma couplingnn, unpublished of 2 with phosphonate experiments; 4 did see not also yield refs. any [26] of the and desired [27]). HWE coupling of 2 with phosphonate 4 did not yield any of the desired olefin (Scheme1). olefinUnfortunately, (Scheme 1).the attempted HWE coupling of 2 with phosphonate 4 did not yield any of the desired olefin (Scheme 1).

SchemeScheme 1.1. Reagents and conditions:conditions: a) nBuLi, −7878 °C◦C to to RT, RT, 0%. Scheme 1. Reagents and conditions: a) nBuLi, −−78 °C to RT, 0%. In lightlight ofof aa previousprevious reportreport byby HöfleHöfle andand co-workers,co-workers, who had been unable of convert 2 intointo phenyl-EpoIn light AAof oror a topreviousto reconstructreconstruct report EpoEpo by AAHöfle fromfrom and 22 byby co-workers, aa varietyvariety of ofwho olefinationolefination had been methodsmethods unable of [[24],24 convert], wewe diddid 2 into notnot furtherphenyl-Epo pursuepursue A the theor directtodirect reconstruct elaboration elaboration Epo of of A2 2 intofrom into1 .12. Instead, byInstead, a variety we we turned turned of olefination our our attention attention methods to to a dia [24],ffdifferenterent we aspect did aspect not of theoffurther the chemistry chemistry pursue of theEpo of direct AEpothat elaborationA had that been had unveiled ofbeen 2 into unveiled by1. Instead, Höfle’s by work weHöfle’s turned on semisyntheticwork our onattention semisynthetic epothilone to a different derivativesepothilone aspect andderivativesof the that chemistry involves and that of the Epoinvolves targeted A that the removal had targeted been of the unveiledremoval C13–C15 ofby thesegment Höfle’s C13– C15 ofwork the segment on macrocycle semisynthetic of the to macrocycle generate epothilone acid to 8generatederivatives[28] (Scheme acid and 82 ).[28] that The (Scheme latterinvolves was 2). the thenThe targeted latter to be esterifiedwas removal then withto of be thealcohol esterified C13–7C15, andwith segment the alcohol resulting of 7 ,the and diene macrocycle the wouldresulting beto cyclizeddienegenerate would by acid ring-closing be 8 cyclized[28] (Scheme olefin by ring-closing 2). metathesis The latter olefin (RCM) was meta then in analogythesis to be (RCM)esterified to previous in analogywith work alcohol to on previous the 7, and total thework synthesis resulting on the of Epototaldiene Asynthesis would[29–31 ].be of Deprotection cyclized Epo A [29–31]. by ring-closing would Deprotecti then olefin yieldon woulddeaza-Epometathesis then C (RCM)yield(5). deaza-Epo in analogy C to (5 previous). work on the total synthesis of Epo A [29–31]. Deprotection would then yield deaza-Epo C (5).

Scheme 2. Retrosynthesis of deaza-Epo C (5). SchemeScheme 2.2. RetrosynthesisRetrosynthesis ofof deaza-deaza-EpoEpo CC ((55).). When elaborating this strategy, we were cognizant of the fact that the conversion of the Epo C Epo C analogWhenWhen 5 into elaboratingelaborating 1 might be thisthis impaired strategy,strategy, by wethewe werecompetingwere cognizantcogniz epoxidationant ofof thethe factfact of thethatthat C16 thethe– conversionC17conversion double ofbond,of thethe whichEpo C analogisanalog more5 5nucleophilicinto into1 1might might than be be impaired impaired in Epo C by ,by and/or the the competing competing oxidation epoxidation epoxidationat sulfur [32]. of of the At the C16–C17the C16 same–C17 doubletime, double we bond, alsobond, whichfelt which that is Epo C moreouris more question nucleophilic nucleophilic about than thanthe in effects in Epo, andofC, and/orthe/or oxidationremoval oxidation atof sulfur theat sulfur thiazole [32]. [32]. At thenitrogen At samethe same time,could time, we be alsowe addressed also felt thatfelt that ourby questioncomparingour question about the about themicrotubule-binding eff ectsthe ofeffects the removal of the affinity ofremoval the of thiazole 5 ofwith the nitrogen thatthiazole of couldEpo nitrogen C be, addressedas thecould latter bybe had comparingaddressed also been theby 5 Epo C microtubule-bindingreportedcomparing to bethe a potentmicrotubule-binding affi inducernity of ofwith tubulin thataffinity polyme of of rization5 ,with as the that[33] latter (althoughof hadEpo also C, no beenas microtubule the reported latter had to binding be also a potent beendata inducerforreported the compound of to tubulin be a potent polymerizationexist inducer in the literature). of tubulin [33] (although polyme norization microtubule [33] (although binding no data microtubule for the compound binding existdata infor the theIn literature). compoundthe forward exist direction, in the literature).the synthesis of acid 8 commenced with the deoxygenation of Epo A with InIn3-methylbenzo[d]thiazole-2(3 thethe forwardforward direction,direction, thethe synthesissynthesisH)-selenone ofof acidacid(10), 8 8prepared commencedcommenced according withwith thethe to deoxygenationdeoxygenationCalo and co-workers of EpoEpo by AA withrefluxingwith 3-methylbenzo[d]thiazole-2(33-methylbenzo[d]thiazole-2(3 methylbenzothiazolium iodideH)-selenone and elemental ( 10), prepared selenium accordingaccording in pyridine toto CaloCalo [34] andand(Scheme co-workersco-workers 3). byby refluxingrefluxingThe deoxygenation methylbenzothiazoliummethylbenzothiazolium reaction gave iodideiodide Epo andand C in elementalelemental highly variable seleniumselenium yields, inin pyridinepyridine due to [ incomplete34[34]] (Scheme (Scheme conversion3 ).3). and theThe formation deoxygenation of an unknown reaction gave side productEpo C in that highly was variable difficult yields, to remove. due to It incompletewas found eventually,conversion however,and the formation that the ofsubsequent an unknown ri ng-openingside product reaction that was proceeded difficult to remove.equally Itwell was without found eventually, the prior removalhowever, of thatthis impurity,the subsequent thus obviating ring-opening the need reaction for the proceededtedious purification equally wellof the without intermediate the prior Epo Cremoval. The ring-opening of this impurity, of the thus ma obviatingcrocycle thewith need ethylene for the in tedious the presence purification of the of theGrubbs intermediate–Hoveyda Epo II C. The ring-opening of the macrocycle with ethylene in the presence of the Grubbs–Hoveyda II

Chemistry 2020, 2, x 5 catalystChemistry furnished2020, 2, 30 diene 9 (as described [28]), which was converted into acid 8 by sequential treatment503 of 509 with TBSOTf and LiOH at elevated temperature under microwave conditions in 61% overall yield.

Chemistry 2020, 2, x 5

catalyst furnished diene 9 (as described [28]), which was converted into acid 8 by sequential treatment with TBSOTf and LiOH at elevated temperature under microwave conditions in 61% overall yield.

SchemeScheme 3. 3. ReagentsReagents and and conditions: conditions: a) a) 1010, ,TFA, TFA, DCM, DCM, 6 6 h, h, RT, RT, 43%; 43%; b) b) Hoveyda Hoveyda–Grubbs-II–Grubbs-II cat., cat., DCM, DCM,

2020 h, RT, 61%;61%; c)c) i) i) 2,6-lutidine, 2,6-lutidine, DCM, DCM, 10 10 min, min, RT, RT, ii) TBSOTf,ii) TBSOTf, 5 min, 5 min, 110 ◦110C, microwave;°C, microwave; d) LiOH, d) LiOH, H2O, HiPrOH,2O, iPrOH, 5 min, 5 150min,◦ C,150 microwave, °C, microwav 61%e, over61% twooversteps. two steps.

TheThe deoxygenationsynthesis of alcohol reaction 7 gavestartedEpo from C in 3-thienylmethanol highly variable yields, (11), due which to incomplete was TBS-protected; conversion deprotonationand the formation of TBS-ether of an unknown 12 with side nBuLi product (1.1 equiv) that was and direactionfficult to with remove. a two-fold It was excess found of eventually, MeI then providedhowever, thatTBS-ether the subsequent 13 in 62% ring-opening yield (Scheme reaction 4). The proceeded deprotection equally of well 13 withoutwith TBAF, the priorfollowed removal by oxidationof thisScheme impurity, of the3. Reagents ensuing thus obviatingand free conditions: alcohol the needundera) 10, TFA, for Swer theDCM,n tediousconditions, 6 h, RT, purification 43%; afforded b) Hoveyda ofthe the –desiredGrubbs-II intermediate aldehyde cat., DCM,Epo 15 in C . 71%The ring-openingoverall20 h, RT, yield. 61%; of Thec) thei) Wittig2,6-lutidine, macrocycle reaction DCM, with of 10 ethylene15 min, with RT, 16 in ii)then the TBSOTf, presence delivered 5 min, of homologated the 110 Grubbs–Hoveyda °C, microwave; aldehyde d) LiOH, II 17 catalyst (71% yield).furnishedH 2O, i dienePrOH,9 5 (asmin, described 150 °C, microwav [28]), whiche, 61% was over converted two steps. into acid 8 by sequential treatment with TBSOTf and LiOH at elevated temperature under microwave conditions in 61% overall yield. TheThe synthesissynthesis ofof alcoholalcohol 77 startedstarted fromfrom 3-thienylmethanol3-thienylmethanol ((1111),), whichwhich waswas TBS-protected;TBS-protected; deprotonationdeprotonation of TBS-ether 1212 withwith nnBuLiBuLi (1.1 (1.1 equiv) equiv) and and reaction reaction with with a two-fold a two-fold excess excess of MeI of MeIthen thenprovided provided TBS-ether TBS-ether 13 in13 62%in 62% yield yield (Scheme (Scheme 4).4 ).The The deprotection deprotection of of 1313 withwith TBAF, TBAF, followed byby oxidationoxidation ofof the the ensuing ensuing free free alcohol alcohol under under Swern Swer conditions,n conditions, afforded afforded the desiredthe desired aldehyde aldehyde15 in 15 71% in overall71% overall yield. yield. The Wittig The Wittig reaction reaction of 15 with of 1516 withthen 16 delivered then delivered homologated homologated aldehyde aldehyde17 (71% 17 yield). (71% yield).

Scheme 4. Reagents and conditions: a) TBSCl, imidazole, DMF, 5 h, 45 °C, quant.; b) nBuLi, MeI, THF,

18 h, −35 °C to RT, 62%; c) TBAF, THF, 3 h, RT; d) oxalyl chloride, DMSO, DCM, NEt3, 2 h, −78 °C to RT, 71% (2 steps); e) 16, benzene, 23 h, 103 °C, 71%; f) 18, dibutylboron triflate, DIPEA, DCM, 4 h, −78 °C to 0 °C, 51%; g) TBSCl, imidazole, DMF, 4.5 h, 45 °C, 79%; h) DIBAL-H, DCM, 2 h, −78 °C, 80%; i) 22, NaHMDS, THF, 2 h, −78 °C, 88%; j) TBAF, THF, 5 h, 0 °C to RT, 94%.

The reaction of 17 with the dibutylboron enolate of acetylsultam 18, obtained by the successive treatmentSchemeScheme of 4.the4. ReagentsReagents latter with andand conditions:conditions:dibutylboron a) TBSCl, triflate imidazole,im andidazole, DIPEA DMF, [22,35], 55 h,h, 4545 ◦ °C,proceededC, quant.; b) withnBuLi, only MeI, moderate THF, selectivity,1818 h,h, − 3535to °CCafford toto RT,RT, 62%;a 62%; ca.c) 2:1c) TBAF, TBAF, mixture THF, THF, 3 of h,3 RT;h,aldol RT; d) oxalyld)products, oxalyl chloride, chloride, in DMSO,favor DMSO, of DCM, the DCM, NEtdesired NEt, 2 h,3, 2isomer 78h, −78C to°C 19 RT, to [36]. − ◦ 3 − ◦ Preparing71%RT, 71% (2 the steps); (2 dibutylboron steps); e) 16 ,e) benzene, 16, benzene, triflate 23 h, in 10323 situh, C,103 from 71%; °C, f)71%;tr18iethylborane, dibutylboronf) 18, dibutylboron and triflate, trifluorom triflate, DIPEA, DIPEA,ethanesulfonic DCM, DCM, 4 h, 78 4 h, Cacid −to78 did ◦ − ◦ not lead0°CC, to 51%;to0 °C, an g)51%; TBSCl,improved g) TBSCl, imidazole, drimidazole,. Although DMF, DMF, 4.5 h,tedious, 4. 455 h,C, 45 79%; °C,isomer 79%; h) DIBAL-H, h)separation DIBAL-H, DCM, DCM,was 2 h, possible2 78h, −C,78 80%;°C, by 80%; i)column22 i), ◦ ◦ − ◦ chromatography,NaHMDS,22, NaHMDS, THF, and THF, 2 h, 19 2 78 h,was −◦C,78 finally 88%;°C, 88%; j) TBAF,obtained j) TBAF, THF, THF,as 5 h,a 05single ◦h,C 0 to °C RT,isomer to 94%.RT, 94%.in 51% yield; the latter was then − protected as its TBS-ether 20. The reductive cleavage of the auxiliary with DIBAL-H, followed by The reaction of 17 with the dibutylboron enolate of acetylsultam 18, obtained by the successive treatment of the latter with dibutylboron triflate and DIPEA [22,35], proceeded with only moderate selectivity, to afford a ca. 2:1 mixture of aldol products, in favor of the desired isomer 19 [36]. Preparing the dibutylboron triflate in situ from triethylborane and trifluoromethanesulfonic acid did not lead to an improved dr. Although tedious, isomer separation was possible by column chromatography, and 19 was finally obtained as a single isomer in 51% yield; the latter was then protected as its TBS-ether 20. The reductive cleavage of the auxiliary with DIBAL-H, followed by

Chemistry 2020, 2, 30 504 of 509

The reaction of 17 with the dibutylboron enolate of acetylsultam 18, obtained by the successive treatment of the latter with dibutylboron triflate and DIPEA [22,35], proceeded with only moderate selectivity, to afford a ca. 2:1 mixture of aldol products, in favor of the desired isomer 19 [36]. Preparing the dibutylboron triflate in situ from triethylborane and trifluoromethanesulfonic acid did not lead to an improved dr. Although tedious, isomer separation was possible by column chromatography, and 19 was finally obtained as a single isomer in 51% yield; the latter was then protected as its TBS-ether 20. Chemistry 2020, 2, x 6 The reductive cleavage of the auxiliary with DIBAL-H, followed by Julia Kocienski olefination of − theJulia ensuing−Kocienski aldehyde olefination21 with of sulfone the ensuing22 and subsequentaldehyde 21 TBS-deprotection, with sulfone 22 gave and homoallylic subsequent alcohol TBS- 7deprotection,in ca. 8.5% gave overall homoallylic yield for thealcohol 10-step 7 in sequence ca. 8.5% overall from 3-thienylmethanol yield for the 10-step (11 ).sequence The attempted from 3- Wittigthienylmethanol olefination of(11 aldehyde). The21 withattempted methyltriphenylphosphonium Wittig olefination bromideof aldehyde in combination 21 withwith variousmethyltriphenylphosphonium bases had been found previously bromide toin induce comb theination elimination with various of TBSOH bases (Cintulov had ábeen& Altmann, found unpublished).previously to induce the elimination of TBSOH (Cintulová & Altmann, unpublished). The esterificationesterification ofof alcohol 7 with acid 8 under Yamaguchi conditions [[37]37] at RT furnished diene 6 in moderate but acceptable yields of 44% 65% (Scheme5). 6 in moderate but acceptable yields of 44%−−65% (Scheme 5).

Scheme 5. a) Reagents and conditions: NEt NEt33, ,2,4,6-trichlorobenzoyl 2,4,6-trichlorobenzoyl chloride chloride (TCBC), (TCBC), DMAP, DMAP, 3 3 h, h, RT, RT, 44%–65%; b)b) GrubbsGrubbs IIII cat.,cat., toluene,toluene, 4040 ◦°C,C, 17%.

A series of screening experiments was then performedperformed on an analytical scale to identify the best conditions for the crucial ring-closure reaction, includingincluding a range of solvents (DCM, benzene, toluene, THF)THF) and different different metathesis metathesis ca catalyststalysts (Grubbs (Grubbs I and I and II, II,Hoveyda Hoveyda−GrubbsGrubbs II, Grubbs II, Grubbs III) [38]. III) [Not38]. − Notunexpectedly, unexpectedly the, reaction the reaction under under all conditions all conditions investigated investigated suffered suff eredfromfrom low lowselectivity; selectivity; low lowselectivity selectivity has also has been also reported been reported for the for RCM the of RCM bis-TBS-protected of bis-TBS-protected Epo C withEpo the C withGrubbs the I Grubbscatalyst I[29–31]).catalyst However, [29–31]). However,in contrast in to contrastthe latter, to which the latter, delivered which the delivered E/Z isomeric the E mixture/Z isomeric of macrocycles mixture of macrocyclesin high yield, in diene high yield, 6 appeared diene 6 toappeared be of limited to be of stability limited stabilityunder the under reaction the reaction conditions conditions and/or and to /beor toconsumed be consumed by alternative by alternative reaction reaction pathways. pathways. Overall, Overall, the screening the screening experiments, experiments, unfortunately, unfortunately, did didnot notprovide provide consistent consistent guidance guidance on onhow how to toma maximizeximize the the yield yield of of the the desired desired macrocycle macrocycle 23.. Ultimately, thethe reactionreaction waswas conductedconducted with 0.1 equiv. of GrubbsGrubbs II catalystcatalyst in toluenetoluene at 4040 ◦°CC and quenchedquenched before completecomplete conversionconversion of thethe startingstarting material.material. On a 20 mg scale,scale, thesethese conditionsconditions providedprovided 3.53.5 mgmg ofof slightlyslightly impureimpure23 23(17%) (17%) together together with with 6.9 6.9 mg mg (33%) (33%) of of the the corresponding corresponding 12,13- 12,13-E isomer.E isomer. When When the the reaction reaction was was carried carried out out for for 3 3 h h at at reflux, reflux, the theE E isomerisomer ofof 2323 waswas obtainedobtained in 51% isolatedisolated yield.yield. The didifficultiesfficulties with with the the RCM RCM of diene of diene6 were 6 thenwere further then further aggravated aggravated by the fact by that the no fact conditions that no couldconditions be identified could be that identified would that have would allowed have for allowed the clean for deprotection the clean deprotection of 23, including of 23, conditions including thatconditions have been that employedhave been successfullyemployed successful in the deprotectionly in the deprotection of bis-TBS-protected of bis-TBS-protectedEpo C [29– Epo31]. C These [29– findings,31]. These which findings, point which to a distinctpoint to instability a distinct ofinstability23 (or 5) of compared 23 (or 5) to compared (protected) to Epo(protected) C, called Epo for C a, changecalled for to a change protecting to a group protecting more group easily removablemore easily than removable TBS. An than obvious TBS. candidateAn obvious that candidate would meet that thiswould requirement meet this requirement was the TMS was group the andTMS we grou feltp thatand thewe correspondingfelt that the corresponding acid 8a (Figure acid4) 8a could (Figure be readily4) could available be readily from available9 in analogy from 9 to in the analogy preparation to the preparation of 8, by simply of 8 substituting, by simply substituting TMSOTf for TMSOTf TBSOTf. for TBSOTf.

Figure 4. Molecular structures of acid 8a.

Chemistry 2020, 2, x 6

Julia−Kocienski olefination of the ensuing aldehyde 21 with sulfone 22 and subsequent TBS- deprotection, gave homoallylic alcohol 7 in ca. 8.5% overall yield for the 10-step sequence from 3- thienylmethanol (11). The attempted Wittig olefination of aldehyde 21 with methyltriphenylphosphonium bromide in combination with various bases had been found previously to induce the elimination of TBSOH (Cintulová & Altmann, unpublished). The esterification of alcohol 7 with acid 8 under Yamaguchi conditions [37] at RT furnished diene 6 in moderate but acceptable yields of 44%−65% (Scheme 5).

Scheme 5. a) Reagents and conditions: NEt3, 2,4,6-trichlorobenzoyl chloride (TCBC), DMAP, 3 h, RT, 44%–65%; b) Grubbs II cat., toluene, 40 °C, 17%.

A series of screening experiments was then performed on an analytical scale to identify the best conditions for the crucial ring-closure reaction, including a range of solvents (DCM, benzene, toluene, THF) and different metathesis catalysts (Grubbs I and II, Hoveyda−Grubbs II, Grubbs III) [38]. Not unexpectedly, the reaction under all conditions investigated suffered from low selectivity; low selectivity has also been reported for the RCM of bis-TBS-protected Epo C with the Grubbs I catalyst [29–31]). However, in contrast to the latter, which delivered the E/Z isomeric mixture of macrocycles in high yield, diene 6 appeared to be of limited stability under the reaction conditions and/or to be consumed by alternative reaction pathways. Overall, the screening experiments, unfortunately, did not provide consistent guidance on how to maximize the yield of the desired macrocycle 23. Ultimately, the reaction was conducted with 0.1 equiv. of Grubbs II catalyst in toluene at 40 °C and quenched before complete conversion of the starting material. On a 20 mg scale, these conditions provided 3.5 mg of slightly impure 23 (17%) together with 6.9 mg (33%) of the corresponding 12,13- E isomer. When the reaction was carried out for 3 h at reflux, the E isomer of 23 was obtained in 51% isolated yield. The difficulties with the RCM of diene 6 were then further aggravated by the fact that no conditions could be identified that would have allowed for the clean deprotection of 23, including conditions that have been employed successfully in the deprotection of bis-TBS-protected Epo C [29– 31]. These findings, which point to a distinct instability of 23 (or 5) compared to (protected) Epo C, called for a change to a protecting group more easily removable than TBS. An obvious candidate that would meet this requirement was the TMS group and we felt that the corresponding acid 8a (Figure Chemistry4) could2020 be readily, 2, 30 available from 9 in analogy to the preparation of 8, by simply substituting TMSOTf505 of 509 for TBSOTf.

Chemistry 2020, 2, x Figure 4. Molecular structures of acid 8a.. 7

Unfortunately,Unfortunately, the the treatment treatment of of 9 9withwith TMSOTf TMSOTf under under a variety a variety of ofconditions conditions did did not not lead lead to completeto complete conversion conversion to the to bis-TMS the bis-TMS ether ether 8a, even8a, evenif a large if a excess large excess of TMSOTf of TMSOTf was employed was employed (up to ( up to 75-fold); in addition, the retro aldol cleavage of the C3 C4 bond was frequently observed as 75-fold); in addition, the retro aldol cleavage of the C3−C4 bond− was frequently observed as a side reactiona side reaction [39]. The [39 purification]. The purification of acid of8a acidby silica8a by gel silica chromatography gel chromatography was possible was possible to some to extent, some butextent, was butcomplicated was complicated by the limited by the stability limited of stability the compound of the compoundunder the chromatographic under the chromatographic conditions. Inconditions. contrast to In 8a contrast, mono-TMS-ether to 8a, mono-TMS-ether 26 (Scheme26 6)(Scheme could be6) couldobtained be obtainedin 53% yield in 53% after yield column after chromatography,column chromatography, when TMSOTf when TMSOTf and 2,6-lutidine and 2,6-lutidine were premixed were premixed in DCM in DCMbefore before the addition the addition of 9 to of the9 to reaction the reaction mixture mixture (Scheme (Scheme 6). 6While). While the the use use of of26 26presentedpresented its its own own problems problems in in the the subsequent subsequent esterificationesterification step step ( (videvide infra infra),), sufficient sufficient quantities quantities of of this this materi materialal could could be be produced produced to to complete complete the the synthesissynthesis of of the the target target structure. structure.

SchemeScheme 6. 6. ReagentsReagents and and conditions: conditions: a) a) TMSOTf, TMSOTf, 2,6-lu 2,6-lutidine,tidine, DCM, DCM, 1.5 1.5 h, h, 0 0 °C◦C to to RT, RT, 53%; 53%; b) b) 77, ,TCBC, TCBC, NEtNEt33, ,THF, THF, DMAP, DMAP, 2 2 h, h, 0 0 °C◦C to to RT, RT, 39% 39% for for the the 2:1 2:1 mixture mixture (not (not separated) separated) of of 2626 (26%)(26%) and and 2727 (13(13%);%); c)c) GrubbsGrubbs II cat., cat., toluene, toluene, RT, RT, 18 h, 13% ( 28) and 19% (29); d) PPTS, EtOH, 0 ◦°C,C, 90 min, 67%.

Thus,Thus, the the reaction reaction of of 2626 withwith 77 underunder Yamaguchi Yamaguchi conditions conditions gave gave the the desired desired ester ester 2626 andand the the dimericdimeric structure structure 2727 [40] [40] inin a a ca. ca. 2:1 2:1 ratio ratio and and with with an an overall overall yield yield of of 39%. 39%. The The separation separation of of 2626 andand 2727 waswas possible, possible, if if tedious, tedious, but but was was better better performed performed after after the the RCM RCM step. step. Intriguingly, Intriguingly, two two attempts attempts atat the the Yamaguchi Yamaguchi esterification esterification of of doubly doubly TMS-protected TMS-protected acid acid 8a8a onlyonly led led to to slow slow decomposition, decomposition, withwith alcohol alcohol 77 beingbeing recovered in almost quantitative yields. RCMRCM with with 60 60 mg mg of of the the 2:1 2:1 mixture mixture of of 2626 andand 2727 withwith Grubbs Grubbs I catalyst I catalyst gave gave 4.5 4.5mg mg (13%) (13%) of Z of- productZ-product 28 28andand 6.6 6.6mg mg (19%) (19%) of E of-isomerE-isomer 29 after29 after two two preparative preparative RP-HPLC RP-HPLC runs. runs. Furthermore, Furthermore, 6.9 mg6.9 mg(19%) (19%) of diene of diene 26 26werewere recovered; recovered; no no other other products products we werere characterized. characterized. The The configurational configurational assignmentassignment of of the the C12–C13 C12–C13 double double bond bond in in 2828 andand 2929 waswas based based on on JJ couplingcoupling constants constants obtained obtained via via NMRNMR decoupling experiments.experiments. ForForZ Z-product-product28, 28,a couplinga coupling constant constant of 10.9 of 10.9 Hz was Hz determined,was determined, while whileE-isomer E-isomer29 showed 29 showed a coupling a coupling constant constant of 15.3 Hzof 15.3 (see theHz Supporting(see the Supporting Material). Material). With the protectedWith the protectedmacrocycle macrocycle in hand, removal in hand, of theremoval TMS moietyof the wasTMS then moiety attempted was then with citricattempted acid (MeOH, with citric RT, 12acid h), (MeOH, RT, 12 h), as this had proven successful for other TMS-protected epothilone analogs [27]; however, the mass of 5 was not detected in the reaction mixture, and RP-HPLC indicated the formation of multiple products. In contrast, the final deprotection of 28 with PPTS in EtOH gratifyingly afforded deaza-Epo C (5) in 67% yield after HPLC purification (Scheme 6).

3.2. Biochemical and Cellular Assessment With deaza-Epo C (5) in hand, we assessed the binding of the compound to cross-linked microtubules at different temperatures in comparison with natural Epo C. As can be seen from the data presented in Table 1, for temperatures up to 35 °C, the binding constant of deaza-Epo C (5) for microtubules is ca. 4-fold to 6-fold lower than that of Epo C, corresponding to a free energy difference

Chemistry 2020, 2, 30 506 of 509 as this had proven successful for other TMS-protected epothilone analogs [27]; however, the mass of 5 was not detected in the reaction mixture, and RP-HPLC indicated the formation of multiple products. In contrast, the final deprotection of 28 with PPTS in EtOH gratifyingly afforded deaza-Epo C (5) in 67% yield after HPLC purification (Scheme6).

3.2. Biochemical and Cellular Assessment With deaza-Epo C (5) in hand, we assessed the binding of the compound to cross-linked microtubules at different temperatures in comparison with natural Epo C. As can be seen from the data presented in Table1, for temperatures up to 35 ◦C, the binding constant of deaza-Epo C (5) for microtubules is ca. 4-fold to 6-fold lower than that of Epo C, corresponding to a free energy difference ∆∆G of ca. 0.7 kcal/mol to 1 kcal/mol. Thus, the loss in binding free energy incurred by the replacement of the thiazole nitrogen in Epo C by a CH group is rather modest and appears to be comparable with the loss in binding energy observed upon removal of the epoxide oxygen from Epo A (to form Epo C) (Table1) or the Epo B Epo D transformation [41]. The difference appears somewhat less pronounced → than for the quinoline-based Epo D analogs o-Quin-Epo D and p-Quin Epo D (Figure3), where the difference in binding constants is >10-fold; however, the differences are small and should not be overinterpreted. At the same time, the data for the quinoline-based epothilones depicted in Figure3 suggest that the difference in binding free energy observed here between Epo C and deaza-Epo C (5) can most likely be extrapolated to epothilones A, B and D and their corresponding deaza analogs. For temperatures above 35 ◦C, a marked drop in the microtubule-binding affinity of deaza-Epo C (5) was observed (K of 5 105 at 42 C); the effect is significantly more pronounced than for Epo C (K of b × ◦ b 88 105 at 42 C). The cause for this behavior is unclear at this point, but may be related to the limited × ◦ chemical stability of the compound 5 at higher temperatures (vide infra).

Table 1. Binding constants of deaza-Epo C (5), Epo C and Epo A for stabilized microtubules. 1

7 1 Compound Kb [10 M− ]

26 ◦C 30 ◦C 35 ◦C 5 0.39 0.03 0.33 0.02 0.37 0.05 ± ± ± Epo C 1.46 0.33 1.19 0.15 1.93 0.27 ± ± ± Epo A 2 7.48 1.00 5.81 1.08 3.63 0.51 ± ± ± 1 Association constant Kb with glutaraldehyde-stabilized microtubules at different temperatures, determined as described in ref. [42]. Numbers are average values from three independent experiments standard deviation. 2 From ref. [41]. ±

Epothilone analogs with similar Kb’s as deaza-Epo C (5) have been reported to exhibit sub-µM antiproliferative activity [26], and it was, therefore, surprising that 5 showed no growth inhibition of the human cancer cell lines MCF7 (breast) or A549 (lung) up to a concentration of 10 µM. In contrast, and as expected from the literature, Epo C inhibited the growth of MCF7 and A549 cells with IC50 values of 9 nM and 103 nM, respectively [33]. These findings triggered experiments on the stability of 5 in cell culture medium, which revealed that the compound was degraded with a half-life of less than 2 h; this is significantly lower than the half-life of Epo C under identical conditions (see the Supporting Material). Due to limitations in the sensitivity of the analytical system, the experiments had to be carried out at a 100 µM concentration, which is 10-fold higher than the highest concentration tested in the growth inhibition experiments. While our stability data, thus, are largely qualitative in nature, they do indicate that the limited stability of deaza-Epo C (5) in cell culture medium may contribute to the lack of cellular potency.

4. Conclusions Deaza-epothilone C (5), which incorporates a thiophene heterocycle in place of the thiazole moiety in natural epothilones, has been prepared by semisynthesis from epothilone A. The synthesis Chemistry 2020, 2, 30 507 of 509

of building blocks 8 (from epothilone A) and 7 (from 3-thienylmethanol) was accomplished with reasonable efficiency; the synthesis of the alternatively protected acids 8a and 23 proved to be more difficult. Likewise, the elaboration of these building blocks into deaza-epothilone C was hampered by significant difficulties, which (partly) reflect distinct differences in the chemical behavior between the thiophene-containing intermediates and their thiazole-derived congeners. As a consequence, deaza-epothilone C (5) was obtained from 7 in only 2.3% overall yield (three steps). Nevertheless, sufficient material could be procured to assess the binding of 5 to microtubules and its cellular activity. While the replacement of the thiazole nitrogen by a simple CH group causes a drop in the the free energy of binding of 5 relative to epothilone C, in agreement with structural data for the tubulin/Epo A complex and also previous (binding) data for quinoline-based epothilone analogs, the magnitude of the decrease (between 0.7 and 1 kcal/mol) is rather modest. Thus, the data indicate that, in principle, thiophene-derived analogs of epothilone A or B (or macrocycle-modified variants thereof) should be high-affinity microtubule binders. However, high-affinity microtubule-binding of such analogs may not translate into potent cellular activity, as indicated by the reduced stability (compared to epothilone C) and lack of cellular activity observed for deaza-epothilone C (5). At the same time, stability may be enhanced by appropriate modification of the thiophene ring (e.g., by the replacement of the methyl group by a quasi-isosteric electron-withdrawing chlorine atom). These questions will have to be clarified in future experiments.

Supplementary Materials: The following are available online at http://www.mdpi.com/2624-8549/2/2/30/s1, synthesis protocols and analytical data for all new compounds, Figure S1: Decoupling experiments with 28, Figure S2: Decoupling experiments with 29, Figure S3: Stability of deaza-Epo C (5) in Dulbecco’s Modified Eagle Medium (DMEM) over a 2 h time period, Figure S4: Stability of deaza-Epo C (5) in DMEM over a 24 h time period, Figure S5: Stability of Epo C in DMEM over a 2 h time period, Figure S6: Stability of Epo C in DMEM over a 24 h time period. Author Contributions: Conceptualization, K.-H.A.; methodology, K.-H.A., A.E., J.F.D.; formal analysis, A.E., K.-H.A., J.F.D.; investigation, A.E., L.R., D.C., J.E.-G.; data curation, A.E., J.E.-G.; writing, K.-H.A.; supervision, K.-H.A.; funding acquisition, J.F.D. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by institutional funding from ETH Zürich (A.E., K.-H.A., L.R., D.C.) and by Ministerio de Economia y Competitividad grants BFU2016-75319-R to J.F.D. (both AEI/FEDER, UE) and H2020-MSCA-ITN-2019 860070 TUBINTRAIN to J.F.D. and a donation from Club deportivo Escuela Hungaresa de Pontevedra. No funds were received to cover the costs for publication in open access. Acknowledgments: The authors acknowledge networking contributions by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”. We are indebted to Bernhard Pfeiffer (ETHZ) for NMR support, and to Xiangyang Zhang, Louis Bertschi, Rolf Häfliger and Oswald Greter (ETHZ) for HRMS spectra. Kurt Hauenstein is acknowledged for excellent technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

References and Notes

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