Total Synthesis of Phorbazole B

molecules

Article Total Synthesis of Phorbazole B

Yngve Guttormsen 1 , Magnus E. Fairhurst 2, Sunil K. Pandey 2, Johan Isaksson 1 , Article Bengt Erik Haug 2,* and Annette Bayer 1,* Total Synthesis of Phorbazole B 1 Department of Chemistry, UiT The Arctic University of Norway, Hansine Hansens veg 54, 9037 Tromsø, Norway; [email protected] (Y.G.); [email protected] (J.I.) Yngve Guttormsen 1, Magnus E. Fairhurst 2, Sunil K. Pandey 2, Johan Isaksson 1, 2 DepartmentBengt Erik of Haug Chemistry 2,* and andAnnette Centre Bayer for Pharmacy,1,* University of Bergen, Allégaten 41, 5007 Bergen, Norway; [email protected] (M.E.F.); [email protected] (S.K.P.) 1 * Correspondence: Department of [email protected] Chemistry, UiT The Arctic (B.E.H.);University [email protected] of Norway, Hansine Hansens (A.B.); veg 54, Tel.: +47-55-58-34-689037 Tromsø, Norway; (B.E.H.); [email protected]+47-77-64-40-69 (A.B.) (Y.G.); [email protected] (J.I.) 2 Department of Chemistry and Centre for Pharmacy, University of Bergen, Allégaten 41, Academic Editors:5007 Bergen, Magne Norway; Olav [email protected] Sydnes and Joanne Harvey(M.E.F.); [email protected] (S.K.P.) Received:* 19Correspondence: September 2020; bengt Accepted:[email protected] 18 October (B.E.H.); 2020; [email protected] Published: 21 October(A.B.); 2020 Tel.:+47-55-58-34-68 (B.E.H.); +47-77-64-40-69 (A.B.)

Abstract:AcademicPhorbazoles Editors: Magne are polychlorinated Olav Sydnes and Joanne heterocyclic Harvey secondary metabolites isolated from a marine spongeReceived: and several 19 September of these 2020 natural; Accepted: products 18 October have2020; Published: shown inhibitory21 October 2020 activity against cancer cells. In this work, a synthesis of the trichlorinated phorbazole B using late stage electrophilic chlorination Abstract: Phorbazoles are polychlorinated heterocyclic secondary metabolites isolated from a was developed. The synthesis relied on the use of an oxazole precursor, which was protected with an marine sponge and several of these natural products have shown inhibitory activity against cancer iodine incells. the In reactive this work, 4-position, a synthesis followed of the by trichlorinated complete chlorinationphorbazole B ofusing all pyrrole late stage positions. electrophilic Attempts to preparechlorination phorbazole was developed. A and C, The which synthesis contain relied a 3,4-dichlorinatedon the use of an oxazole pyrrole, precursor, were which unsuccessful was as the desiredprotected chlorination with an iodine pattern in onthe thereactive pyrrole 4-position could, notfollowed be obtained. by complete The chlorination identities of of theall pyrrole dichlorinated intermediatespositions. and Attempts products to prepare were phorbazole determined A and using C, which NMR contain techniques a 3,4-dichlorinated including pyrrole, NOESY were/ROESY, 1,1-ADEQUATEunsuccessful and as the high-resolution desired chlorination CLIP-HSQMBC. pattern on the pyrrole could not be obtained. The identities of the dichlorinated intermediates and products were determined using NMR techniques including NOESY/ROESY, 1,1-ADEQUATE and high-resolution CLIP-HSQMBC. Keywords: phorbazole; oxazole; pyrrole; late-stage chlorination Keywords: phorbazole; oxazole; pyrrole; late-stage chlorination

1. Introduction 1. Introduction Phorbazoles A–D (1–4, Figure1) are polychlorinated heterocyclic secondary metabolites isolated Phorbazoles A–D (1–4, Figure 1) are polychlorinated heterocyclic secondary metabolites isolated from thefrom marine the marine sponge spongePhorbas Phorbas aﬀ. aff. Clathrata Clathratao offﬀ the the coast of of South South Africa Africa in 1994 in 1994 [1]. [1].

FigureFigure 1. 1.Phorbazoles Phorbazoles A A–D–D (1 (–14–).4 ).

The phorbazoles were the first natural compounds isolated with the 2-(2′-pyrrolyl)oxazole The phorbazoles were the first natural compounds isolated with the 2-(20-pyrrolyl)oxazole fragment.fragment. They They were were also also the the first first examplesexamples of ofchlorinated chlorinated pyrroles pyrroles found in found nature. in Later nature. on, a N Later- on, methylated analogue of 1 and the 9-chlorinated analogue of 4 were isolated from the marine mollusk N 1 4 a -methylatedAldisa andersoni analogue [2]. No of bioactivityand the has 9-chlorinated so far been reported analogue for 2– of4, whilewere 1 and isolated its N-methylated from the marine molluskanalogueAldisa andersoni and the 9[-2chlorinated]. No bioactivity analogue has of so4 have far been been reported found to forhave2 –a4 feeding, while 1deterrenceand its N effect,-methylated analogue and the 9-chlorinated analogue of 4 have been found to have a feeding deterrence effect, Molecules 2020, 25, x; www.mdpi.com/journal/molecules which suggests that they are involved in chemical defense [2]. The two latter compounds have also been found to inhibit growth of cancer cells in vitro [2]. While the phorbazoles are biosynthetically

Molecules 2020, 25, 4848; doi:10.3390/molecules25204848 www.mdpi.com/journal/molecules Molecules 2020, 25, x 2 of 11 Molecules 2020, 25, x 2 of 11 which suggests that they are involved in chemical defense [2]. The two latter compounds have also Moleculesbeenwhich2020 found suggests, 25, 4848 to inhibit that they growth are involvedof cancer in cells chemical in vitro defense [2]. While [2]. Thethe phorbazolestwo latter compounds are biosynthetically have also2 of 10 producedbeen found by to cyclization inhibit growth of dipeptides of cancer of cells tyrosine in vitro and [2].proline, While a synthesisthe phorbazoles of 3 reported are biosynthetically in 2001 relied onproduced a Robinson by cyclization–Gabriel of cyclodeh dipeptidesydration of tyrosine of a precursorand proline, carrying a synthesis the of appropriately 3 reported in chlorinated 2001 relied produced by cyclization of dipeptides of tyrosine and proline, a synthesis of 3 reported in 2001 relied on pyrroleon a Robinson [3]. –Gabriel cyclodehydration of a precursor carrying the appropriately chlorinated a Robinson–GabrielpyrroleBreitfussin [3]. cyclodehydration A–H (5–12, Figure of 2) a precursor constitute carrying the second the class appropriately of marine chlorinated natural products pyrrole [3]. BreitfussincontainingBreitfussin A–Hthe pyrrole A (5––H12 (,5-oxazole Figure–12, Figure2 ) motif constitute 2) [4,5 constitute]. the Like second the the second phorbazoles, class of class marine of the marine natural breitfussins natural products are products containing highly the pyrrole-oxazolehalogenatedcontaining thenatural motif pyrrole-oxazole products [4,5]. Like derived motif the phorbazoles,from [4,5]. dipeptide Like the the precursors, phorbazoles, breitfussins but are thewith highly breitfussins tryptophan halogenated areinstead highl naturalofy productstyrosine.halogenated derived natural from dipeptide products derived precursors, from but dipeptide with tryptophan precursors, insteadbut with of tryptophan tyrosine. instead of tyrosine. R2 R5

R2 R5 R3 R6 N N N N 3 6 R1 H R H R O N O N N O N 1 R H O H O O O R4 O Br N Br N H R4 H Br 5 R1 = I, R2N = R3 = R4 = H Br 7 R5 = R6 N= H H H 6 R1 = H, R2 = R4 = H, R3 = Br 11 R5 = H, R6 = Br 5 R1 = I, R2 = R3 = R4 = H 7 R5 = R6 = H 8 R1 = R2 = R3 = R4 = H 12 R5 = Br, R6 = H 6 R1 = H, R2 = R4 = H, R3 = Br 11 R5 = H, R6 = Br 9 R1 = R3 = R4 = H, R2 = H 8 R1 = R2 = R3 = R4 = H 12 R5 = Br, R6 = H 10 R1 = R2 = R3 = H, R4 = I 9 R1 = R3 = R4 = H, R2 = H 1 2 3 4 10 R = RFigure=Figure R = H, 2.R 2.=Breitfussins BreitfussinI s A A–H–H (5 (–512–12). ). Figure 2. Breitfussins A–H (5–12). We haveWe have earlier earlier reported reported on theon the total total syntheses syntheses of of5– 58–,8 which, which were were based based onon thethe utilizationutilization of of two palladium-catalyzedtwo palladiumWe have earlier-cat crossalyzed reported couplings cross on couplings the to total install tosyntheses the install indole theof 5 indole and–8, which pyrrole and were pyrrole onto based the onto on oxazole thethe oxazoleutilization core, core, followed of by halogenationfollowedtwo palladium-catalyzed by ofhalogenation the pyrrole of cross at the a latecouplingspyrrole stage at inato late the install stage case the in of indolethe6 [5 case,6 ]. and Anof 6 pyrrole alternative[5,6]. An onto alternative synthesis the oxazole synthesis of core,6 (and by coincidenceoffollowed 6 (and also byby 8halogenationcoincidence) based on applicationalso of the 8) basedpyrrole of on at the application a Robinson–Gabriellate stage ofin thethe Robinsoncase cyclodehydration of 6 [5,6].–Gabriel An alternativecyclodehydration to form synthesis the oxazoleto form the oxazole ring combined with a late stage bromination strategy [7], has also been reported. ring combinedof 6 (and by with coincidence a late stage also bromination8) based on application strategy [ 7of], the has Robinson also been–Gabriel reported. cyclodehydration to formIn the this oxazole work, ring we combined report the with total a late synthesis stage bromination of 2 via late strategy-stage chlorination[7], has also been and attemptsreported. at In this work, we report the total synthesis of 2 via late-stage chlorination and attempts at synthesizingIn this work,1 and 3 we using report the thesame total strategy. synthesis of 2 via late-stage chlorination and attempts at synthesizing 1 and 3 using the same strategy. synthesizing 1 and 3 using the same strategy. 2. Results and Discussion 2. Results and Discussion 2. Results and Discussion 2.1. Retrosynthetic Analysis of 2 2.1. Retrosynthetic Analysis of 2 2.1. RetrosyntheticWe envisioned Analysis that the of phorbazoles2 should be accessible via chlorination at a late stage, and a Weretrosynthetic envisionedWe envisioned analysis that that the of the2 phorbazoles, which phorbazoles has not should shouldbeen prepared bebe accessible before, viavia is chlorinationshown chlorination in Scheme at a at late 1. a latestage, stage, and a and a retrosyntheticretrosynthetic analysis analysis of 2 of, which2, which has has not not been been prepared prepared before, before, is is shown shown in inScheme Scheme 1. 1. Cl I I N N 3 N Cl 4 Cl I 2 I I N N 3 N O Cl O 1 5 O N 2 N 4 B(OH)2 H Cl H N I 13 Boc O14 HO O2 N TsO O 1 N 5 B(OH) TsO Cl H N 2 H 13 Boc 14 HO 2 TsO TsO CHO 14 C CHO Ts N TsO 15 C 14 Ts N TsOScheme 151. Retrosynthetic analysis for 2. SchemeScheme 1. 1.RetrosyntheticRetrosynthetic analysis analysis for for 2. 2. Due to the intrinsic propensity of both the oxazole and the pyrrole to undergo electrophilic DuearomaticDue to the substitution, to intrinsicthe intrinsic the propensity regioselectivity propensity of of both both is difficult the the oxazole oxazole to control. and and The the the Chen pyrrole pyrrole group to to undergo nicely undergo illustrated electrophilic electrophilic the subtle inherent reactivity differences in the synthesis of 6, where changing the solvent from acetone aromaticaromatic substitution, substitution, the the regioselectivity regioselectivity is difficult diﬃcult to to control. control. The The Chen Chen group group nicely illustrated nicely illustrated the tosubtle tetrahydrofuran inherent reactivity (THF) /pyridinedifferences altered in the the synthesis regioselectivity of 6, where of thechanging halogenation the solvent from from the oxazoleacetone the subtle inherent reactivity diﬀerences in the synthesis of 6, where changing the solvent from 4to-position tetrahydrofuran to the pyrrole (THF)/pyridine 5-position altered[7]. Our the strategy regioselectivity is based on of ou ther realizationhalogenation from from the thebreitfussin oxazole acetone to tetrahydrofuran (THF)/pyridine altered the regioselectivity of the halogenation from syntheses4-position thatto the the pyrrole iodine 5-positionin the oxazole [7]. Our 4-position strategy is isacid based-labile on ourand realizationmay act as froma protecting the breitfussin group the oxazole 4-position to the pyrrole 5-position [7]. Our strategy is based on our realization from syntheses that the iodine in the oxazole 4-position is acid-labile and may act as a protecting group the breitfussin syntheses that the iodine in the oxazole 4-position is acid-labile and may act as a protecting group also toward synthesizing phorbazoles. We thus envisioned that 2 could be obtained by chlorination of compound 13, where the oxazole 4-position is protected. Access to 13 was planned through Suzuki Miyaura coupling on diiodide 14, which we planned to obtain from the Van Leusen methyl isocyanide synthon [8] after cyclisation to the oxazole and subsequent deiodination as we have reported for the breitfussin syntheses [5,6]. Molecules 2020, 25, x 3 of 11

also toward synthesizing phorbazoles. We thus envisioned that 2 could be obtained by chlorination of compound 13, where the oxazole 4-position is protected. Access to 13 was planned through Suzuki MoleculesMiyaura2020, 25, 4848 coupling on diiodide 14, which we planned to obtain from the Van Leusen methyl 3 of 10 isocyanide synthon [8] after cyclisation to the oxazole and subsequent deiodination as we have reported for the breitfussin syntheses [5,6]. 2.2. Synthesis of 2 2.2. Synthesis of 2 The synthesisThe synthesis of the of commonthe common intermediate intermediate 13 commencedcommenced with with a modified a modiﬁed Van Leusen Van Leusen oxazole oxazole synthesissynthesis on the on previously the previously reported reported aldehyde aldehyde15 15 (Scheme(Scheme 2)2 )[[9].9 ].

SchemeScheme 2. 2.Synthesis Synthesis ofof 2 by late late-stage-stage chlorination. chlorination.

ProtectionProtection of the of phenolthe phenol is is required required both for for the the lithiation lithiation step and step to andavoid to chlorination avoid chlorination on the on the phenolphenol later later in in the the synthesis synthesis [[10].10]. In In order order to to avoid avoid deprotectio deprotectionn of the of tosyl the tosylgroup, group, 1,8-diazabicyclo 1,8-diazabicyclo [5.4.0] undec-7-en (DBU) was used as base instead of K2CO3. Heating the aldehyde 15 and the TosMIC [5.4.0] undec-7-en (DBU) was used as base instead of K CO . Heating the aldehyde 15 and the TosMIC reagent with DBU in 1,2-dimethoxyethane (DME) (702 °C, 35 h) gave oxazole 16 in 70% yield. This reagentmaterial with DBU was in dilithiated 1,2-dimethoxyethane by treatment with (DME) lithium (70 bis(◦C,hexamethyl 5 h) gavesilyl oxazole)amide16 (inLiHMDS 70% yield.) in THF This at - material was dilithiated78 °C for 1 by h treatmentbefore addition with of lithiumiodine to bis(hexamethylsilyl)amidegive the diiodinated oxazole 14 (LiHMDS)in 79% yield. in While THF this at 78 C − ◦ for 1 h beforeprotocol addition also resulted of iodinein the formation to give the of the diiodinated monoiodinated oxazole 2-iodo14 oxazolein 79% 17 yield. in 12% Whileyield, it this does protocol also resultednot require in theprolonged formation reaction of times the monoiodinatedas has been reported 2-iodo for other oxazole methods17 [11].in The 12% diiodo yield, oxazole it does not require14 prolonged was subsequently reaction coupled times toas Boc has-protected been reported (1H-pyrrol for-2 other-yl)boronic methods acid in [11 a ]. Suzuki The- diiodoMiyauraoxazole reaction. The coupling reaction proceeded with excellent regioselectivity favoring the 2-substituted 14 wasoxazolyl subsequently-pyrrole 18 coupled in 75% yield to Boc-protected after 68 h at rt. (1WeH were-pyrrol-2-yl)boronic not able to detect any acid product in a Suzuki-Miyaurastemming reaction.from The coupling coupling at the reaction oxazole proceeded4-position, although with excellent this result regioselectivity cannot be completely favoring ruled the out. 2-substituted Next, oxazolyl-pyrrolethe Boc group18 in was 75% removed yield afterusing 68 trimethylsilyl h at rt. We trif wereluoromethanesulfonate not able to detect any(TMSOTf product) under stemming basic from couplingconditions at the oxazole to give 4-position,the common althoughintermediate this 13 result in high cannot yield (89%). be completely Chlorination ruled of the out. common Next, the Boc group wasintermediate removed 13 using using trimethylsilylPalau’chlor [12], trifluoromethanesulfonategave a mixture of di- and trichlorinated (TMSOTf) products, under basicwhile conditionsthe recently developed procedure of Gustafson [13] using N-chlorosuccinimide (NCS)/Ph3PS resulted in to give the common intermediate 13 in high yield (89%). Chlorination of the common intermediate 13 complete conversion. At first 3.07 equiv. of NCS and 20 mol% of Ph3PS was added; however, when using Palau’chlorHRMS analysis [12 ],revealed gave a uncomplete mixture of reaction di- and (see trichlorinated experimental products,part for details), while a thefurther recently 0.2 equiv developed. procedureof NCS of Gustafson was added. [Thus,13] using usingN a-chlorosuccinimide total of 3.27 equiv. of NCS, (NCS) we/Ph obtained3PS resulted the desired in complete trichlorinated conversion. At firstpyrrole 3.07 equiv. 19 in of 73% NCS yield. and No 20 mol% chlorination of Ph 3 onPS the was phenyl added; ring however, was observed. when Next,HRMS we analysis opted to revealed uncompletedeiodinate reaction 19 using (see the experimental conditions identified part for through details), our total a further syntheses 0.2 of equiv. breifussins of NCSA–D ( was5–9, added. Thus, usingFigure a 2 total); however, of 3.27 treatment equiv. of of 19 NCS, under we acidic obtained conditions the did desired not give trichlorinated any traces of deiodinated pyrrole 19 in 73% product. On the other hand, simultaneous deprotection of the tosyl and iodine protecting groups of yield. No chlorination on the phenyl ring was observed. Next, we opted to deiodinate 19 using 19 was achieved using zinc in refluxing ethanolic sodium hydroxide, to give 2 in 87% yield. The NMR the conditionsspectra of identified synthetic material through in (CD our3)2 totalSO (see syntheses Figures S1 ofand breifussins S2) matched A–Dthe reported (5–9, Figurespectra2 of); the however, treatmentnatural of 19 phorbazoleunder acidic B [1] (see conditions Table S1 didfor a notcomparison give any). traces of deiodinated product. On the other hand, simultaneous deprotection of the tosyl and iodine protecting groups of 19 was achieved using zinc in refluxing ethanolic sodium hydroxide, to give 2 in 87% yield. The NMR spectra of synthetic material in (CD3)2SO (see Figures S1 and S2) matched the reported spectra of the natural phorbazole B[1] (see Table S1 for a comparison).

2.3. Attempted Synthesis of 1 and 3 We envisioned that it might be possible to access natural products 1 and 3 by shielding the pyrrole 5-position with a bulky triisopropylsilyl (TIPS) group on the pyrrole N-atom, giving 20a and 20b as intermediates for the synthesis of 1 and 3, respectively (Scheme3). The literature suggests that the extent of shielding that TIPS protection of the pyrrole can infer on C-5 (our numbering, see Scheme3) of the pyrrole, varies depending on the chlorination reagent and solvent that is employed; however, MoleculesMolecules 20202020,, 2255,, xx 44 ofof 1111

2.3.2.3. AttemptedAttempted SynthesisSynthesis ofof 11 andand 33 WeWe envisioned envisioned that that it it might might be be possible possible to to access access natural natural products products 11 andand 33 byby shielding shielding the the pyrrolepyrrole 55--positionposition withwith aa bulkybulky triisopropylsilyltriisopropylsilyl ((TIPSTIPS)) groupgroup onon thethe pyrrolepyrrole NN--atom,atom, givinggiving 20a20a andand Molecules 2020, 25, 4848 4 of 10 20b20b asas intermediatesintermediates forfor thethe synthesissynthesis ofof 11 andand 33,, respectivelyrespectively (Scheme(Scheme 3).3). TheThe literatureliterature suggestssuggests thatthat thethe extent extent of of shielding shielding that that TIP TIPSS protectionprotection of of the the pyrrole pyrrole can can infer infer on on C C--55 (our (our numbering, numbering, see see conditionsSchemeScheme that 3)3) ofof result thethe pyrrole,pyrrole, in full variesvaries shielding dependingdepending of the onon 2-position thethe chlorinationchlorination of pyrrole reagentreagent itself andand have solventsolvent not thatthat been isis employedemployed achieved;; [14]. however, conditions that result in full shielding of the 2-position of pyrrole itself have not been On thehowever other, conditions hand, for thatbromination result in full and shielding iodination, of the the 2-position TIPS group of pyrrole is reported itself have to o notﬀer been excellent achievedachieved [14].[14]. OnOn thethe otherother hand,hand, forfor brominationbromination andand iodination,iodination, thethe TIPSTIPS groupgroup isis reportedreported toto offeroffer shielding as both exposure of N-TIPS-protected pyrrole to NBS or NIS resulted in clean formation of excellentexcellent shieldingshielding asas bothboth exposureexposure ofof NN--TIPSTIPS--protectedprotected pyrrolepyrrole toto NBSNBS oror NISNIS resultedresulted inin cleanclean the 3-bromo or 3-iodo derivative [14–17]. formationformation ofof thethe 33--bromobromo oror 33--iodoiodo derivativederivative [[1414––1717].].

ClCl RR RR II 3 NN Cl NN NN 3 Cl 4 22 4

O O O 1 O NN O NN O 1 NN 55 H TIPS H 11:: RR == CCll H TIPS 1313 H HO TsO 2200aa:: RR == HH TsO HO 33:: RR == HH TsO TsO 20b20b:: RR == II SchemeSchemeScheme 3. 3.3.Retrosynthetic RetrosyntheticRetrosynthetic analysisanalysis analysis forfor for 11 and1andand 33.. 3.

TheThe TIPSThe TIPSTIPS protection protectionprotection of of13of 1313(Scheme (Scheme(Scheme4 ) 4)4) turned turnedturned out outout toto be be aa a delicatedelicate delicate step,step, step, asas asaa reactionreaction a reaction usingusing using THFTHF THF asas as solventsolventsolvent was waswas successful successfulsuccessful with withwith 96% 96%96% yield yieldyield the thethe firstfirstfirst time time itit it waswas was testedtested tested;;; howeverhowever however,,, allall alllaterlater later attemptsattempts attempts usingusing using this solvent only gave back the starting material. The use of DMF as a solvent and NaH as a base [18], this solventthis solvent only only gave gave back back the the starting starting material. material. The u usese of of DMF DMF as asa solvent a solvent and and NaH NaH as a base as a [18] base, [18], andand quenchingquenching thethe reactionreaction byby additionaddition ofof crushedcrushed iceice consistentlyconsistently gavegave thethe crudecrude productproduct ofof 20b20b inin and quenching the reaction by addition of crushed ice consistently gave the crude product of 20b aroundaround 94%94% yieldyield andand withwith hhighigh purity.purity. Unfortunately,Unfortunately, 20b20b provedproved toto bebe somewhatsomewhat unstableunstable uponupon in around 94% yield and with high purity. Unfortunately, 20b proved to be somewhat unstable upon flashflash chromatographychromatography purificationpurification andand waswas thereforetherefore,, forfor thethe mostmost partpart,, usedused inin thethe nextnext stepstep withoutwithout flashpurification.purification. chromatography Upon Upon purification prolonged prolonged storage, storage, and was the the therefore, TIPS TIPS group group for was was the mostcleaved cleaved part, off off used to to give give in theback back next the the step s startingtarting without purification.material.material. Upon prolonged storage, the TIPS group was cleaved off to give back the starting material.

SchemeSchemeScheme 4. 4.4. Attempted AttemptedAttempted synthesissynthesis synthesis ofof of 33.. 3.

ChlorinationChlorinationChlorination of 20bofof 20b20bwas waswas performed performedperformed analogously analogouslyanalogously toto to thethe the chlorinationchlorination chlorination ofof the ofthe the unprotectedunprotected unprotected pyrrolepyrrole pyrrole in 13inin(see 1313 (( above).seesee aboveabove By).). ByBy using usingusing 2.05 2.052.05 equivalents equivalentsequivalents ofof of NCS,NCS, NCS, oneone one majormajor major dichlorinateddichlorinated dichlorinated productproduct product waswas formedformed was formedasas evident by 11H-NMR analysis of the crude product. The clean conversion of 20b to one major as evidentevidentby by1 H-NMRH-NMR analysis of of the the crude crude product. product. The The clean clean conversion conversion of 20b of 20bto oneto majorone major dichlorinateddichlorinated productproduct waswas veryvery encouragingencouraging;; however,however, uponupon globalglobal deprotectiondeprotection ((videvide infrainfra),), thethe dichlorinated product was very encouraging; however, upon global deprotection (vide infra), the NMR NMRNMR spectraspectra ofof thethe materialmaterial obtainedobtained diddid notnot matchmatch thosethose reportedreported forfor naturalnatural productproduct 33 [1].[1]. ItIt turnedturned spectra of the material obtained did not match those reported for natural product 3 [1]. It turned out outout thatthat,, insteadinstead ofof thethe desireddesired chlorinationchlorination ofof thethe pyrrolepyrrole 33-- andand 44--positions,positions, selectiveselective dichlorinationdichlorination that,onon instead thethe pyrrolepyrrole of the 44 desired-- andand 55--positionspositions chlorination hadhad occurredoccurred of the pyrrole toto givegive 3- regioisomerregioisomer and 4-positions, 2121 inin anan selective 84%84% isolatedisolated dichlorination yieldyield (see(see on the pyrroleNMRNMR discussiondiscussion 4- and 5-positions belowbelow forfor thethe had structurestructure occurred assignmentassignment to give regioisomer ofof 2121).). 21 in an 84% isolated yield (see NMR discussionUponUpon below flashflash for columncolumn the structure purificationpurification assignment ofof thethe crudecrude of 21 product).product followingfollowing thethe chlorinationchlorination reaction,reaction, wewe experiencedUponexperienced flash thatthat column thethe materialmaterial purification decomposeddecomposed of the whenwhen crude ethylethyl product acetateacetate ((0 following0––2020%)%) inin hexaneshexanes the chlorination waswas usedused asas reaction,thethe we experiencedeluent,eluent, toto givegive that TIPSTIPS the--deprotecteddeprotected material 22 decomposed22 asas thethe majormajor when product.product. ethyl WeWe acetatespeculatespeculate (0–20%) thatthat thethe inpresencepresence hexanes ofof aceticacetic was used as the eluent, to give TIPS-deprotected 22 as the major product. We speculate that the presence of acetic acid in the ethyl acetate might provide an explanation for the observed decomposition. By using CH2Cl2 (0–100%) in heptane as the eluent, decomposition was avoided completely. Complete deprotection—i.e., removal of the iodine, the tosyl and the TIPS protecting groups—could be facilitated using Zn in refluxing ethanolic NaOH (vide supra) to give 23 in high yield (88%). Under acidic conditions, using Zn in refluxing ethanolic HCl, the tosyl group was retained providing compound 24—the tosyl-protected analogue of 23. In the face of the undesired regioselectivity for the dichlorination step and the lability of the TIPS group of 20b, further attempts to synthesize 1 and 3 were suspended. Molecules 2020, 25, 4848 5 of 10

The unambiguous determination of the chlorination pattern of fully protected 21 and partially deprotected 24 was not trivial because of the absence of protons in the area. The spectral assignments were made by a combination of 1H-1D, 13C-1D, HSQC, HMBC, COSY, NOESY/ROESY, 1,1-ADEQUATE and high-resolution CLIP-HSQMBC (see Figures S36–S48). In order to establish the chlorination pattern, it was necessary to unambiguously determine the position of the pyrrole CH-proton. From the spectral data obtained of 24, we could exclude that this compound contained a proton on the pyrrole 5-position due to the absence of any NOESY or ROESY correlation to the pyrrole NH proton (Figure S48). The experimental chemical shift for the pyrrole CH was determined to be 111.3 ppm, which is not consistent with this carbon atom being next to a nitrogen, for which a chemical shift of around 130 ppm would have been expected. Thus, in order to differentiate between positions 3 and 4 on the pyrrole, we had to establish which carbon atoms carried chlorine and differentiate these from C-2 of the pyrrole, which experimentally resonates at 117.3 ppm—inconveniently near the two chlorinated carbon atoms C-5 (117.1 ppm) and C-4 (111.9 ppm). The pyrrole C-4 signal was sharp and a ~0.6 Hz 35,37Cl isotope shift could be directly observed at 214 MHz field for 13C. The pyrrole C-5 resonance was however broad and the isotope shift could not be observed directly in 13C-NMR 1 n nor indirectly in a high-resolution selective CLIP-HSQMBC. A weak long-range JCH was observed between the proton and the carbon in the 2-position of the oxazole, and supports the proton sitting in position 3 of the pyrrole, but since the coupling was very weak it could not be used alone to rule out a potential four-bond coupling from the pyrrole 4 position. In intermediate 21, the 35,37Cl isotope shift could however be observed for both the C-4 (113.8 ppm) and the C-5 (123.4 ppm) of the pyrrole, in both 13C-NMR and selective CLIP-HSQMBC (Figure S41), presumably because the absence of the exchangeable pyrrole NH no longer causes line broadening of the neighboring carbon resonances. The presence of the N-TIPS group in 21 did, however, affect the chemical shifts significantly. On the other hand, the long range coupling constants between the oxazole C-2 and the pyrrole H-3 (2.0 Hz), the pyrrole C-2 and the pyrrole H-3 (5.4 Hz) and the pyrrole C-5 and H-3 (9.5 Hz) were not significantly changed by the TIPS group, and the measurements of these couplings for all intermediates were used to ensure that the chemical shift assignment of the chlorinated pyrrole carbons could be transferred from 21 to 24.2 Knowing the unambiguous assignments of pyrrole carbons C-2 and C-5, 1,1-ADEQUATE correlations from pyrrole H-3 to C-2 and C-4 could be used to conclusively determine the chlorination pattern.

3. Materials and Methods

3.1. General Information Chemicals and solvents were purchased from Sigma-Aldrich and used as delivered unless otherwise stated. All moisture sensitive reactions were carried out under argon atmosphere in oven-dried (130 ◦C) equipment that has been cooled down under vacuum. Anhydrous THF was either obtained from a sodium/benzophenone still or an anhydrous solvent delivery system (SPS-800 system from M. Braun GmbH, Garching, Germany). Flash column chromatography was performed using silica gel from Merck (Silica gel 60, 0.040–0.063 mm). Thin layer chromatography (TLC) analyses were performed on aluminum sheets coated with Merck TLC silica gel 60 F254 and visualization was achieved by using ultraviolet light (254 nm) or a solution of sodium permanganate. The NMR experiments were recorded on a Bruker 400 MHz, a Bruker BioSpin AV500 or a Bruker BioSpin Ascend spectrometer operating at 850 MHz for 1H equipped with an inverse-detected triple resonance (TCI) cryoprobe. 1 13 H and C chemical shifts (δ) are reported in ppm with reference to the solvent residual peak (CDCl3: δH 7.26 and δC 77.16; (CD3)2SO: δH 2.50 and δC 39.98). All coupling constants are given in Hz. Positive and negative ion electrospray ionization mass spectrometry was conducted on a Thermo electron LTQ Orbitrap XL spectrometer (Thermos, Bremen, Germany) with methanol as solvent. Flash chromatography was performed on a Biotage SP1 HPFC system (Biotage, Uppsala, Sweden) or a PuriFlash XS 420 system (Interchim, Montlucon Cedex, France). Molecules 2020, 25, 4848 6 of 10

3.2. Methods

3.2.1. Synthesis of Phorbazole B (2) The trichlorinated pyrrole 19 (7.65 mg, 12.5 µmol) was dissolved in ethanol (0.2 mL) and 6 M aqueous NaOH (50 µL) was added. The reaction mixture was heated to reﬂux, zinc (12 mg, 190 µmol) was added and heating was continued for 1 h. To the reaction mixture 1 mL 10% NaHCO3 was added and extracted with 3 1 mL CHCl on a Biotage phase separator with a Na SO ﬁlter plug. × 3 2 4 The solution was evaporated to give the title compound. 1 Colorless solid; yield 3.58 mg (87%); H-NMR (400 MHz, (CD3)2SO) δ = 13.57 (s, 1H), 9.86 (s, 1H), 13 7.63 (d, J = 7.7, 2H), 7.60 (s, 1H),6.89 (d, J = 7.7, 2H); C-NMR (101 MHz, (CD3)2SO) δ = 158.0, 151.6, 35 150.3, 125.7, 121.1, 118.3, 115.9, 115.8, 115.0, 110.0, 108.5; HRMS (ESI) m/z calcd. for C13H6O2N2 Cl3 [M H] : 326.9500; found: 326.9496. The NMR spectra in (CD ) SO (Table S1) matched the original − − 3 2 spectra [1].

3.2.2. Synthesis of 4-(4-Iodo-2-(1H-pyrrol-2-yl)oxazol-5-yl)phenyl 4-methyl-benzenesulfonate (13)

To a solution of Boc-protected pyrrole 18 (530 mg, 0.87 mmol) in anhydrous CH2Cl2 (5 mL) at 0 ◦C was slowly added Et3N (1.20 mL, 8.74 mmol) and TMSOTf (1.58 mL, 8.74 mmol). The reaction mixture was stirred at room temperature for 16 h, after which no starting material could be observed by TLC analysis. The reaction mixture was diluted with ethyl acetate, quenched with cold water and extracted with ethyl acetate (3 20 mL). The organic phase was concentrated and dissolved in a × small amount of CH2Cl2 before adsorption on a Biotage snaplet precolumn and puriﬁed by ﬂash chromatography on a Biotage SNAP Ultra column using an eluent with 0–80% ethyl acetate in heptane to give the title compound. 1 Orange solid; yield 395 mg (89%); mp 53.4–54.7 ◦C; H-NMR (400 MHz, CDCl3) δ = 9.58 (bs, 1H), 7.93 (d, J = 8.8, 2H), 7.74 (d, J = 8.2, 2H), 7.34 (d, J = 8.0, 2H), 7.08 (d, J = 8.8, 2H), 7.00–6.96 (m, 1H), 13 6.91–6.87 (m, 1H), 6.34–6.30 (m, 1H), 2.46 (s, 3H); C-NMR (101 MHz, CDCl3) δ = 157.5, 149.6, 147.4, 145.7, 132.3, 130.0, 128.7, 127.0, 126.4, 122.9, 122.6, 119.1, 111.7, 110.8, 79.7, 29.9 (grease), 21.9: HRMS + (ESI) m/z calcd. for C20H16O4N2IS [M + H] : 506.9870; found: 506.9865.

3.2.3. Synthesis of 4-(2,4-diiodooxazol-5-yl)phenyl 4-methylbenzenesulfonate (14) Oxazole 16 (5.00 g, 15.9 mmol) was dissolved in anhydrous THF (75 mL) and cooled to 78 C. − ◦ Freshly prepared LiHMDS (1M in THF/hexanes, 47.6 mL, 47.6 mmol. Prepared by adding a 2.5 M n-BuLi solution in hexanes into HMDS in THF) was added dropwise. The resulting mixture was stirred at 78 C for 1 h, during which the solution turned very viscous. A solution of iodine (12.10 g, 47.6 mmol) − ◦ in anhydrous THF (10 mL) was added very slowly under thorough stirring. The resulting mixture was heated to room temperature for 1 h before 10% aqueous Na2S2O3 (100 mL) was added to quench the reaction and the resulting two layers were separated. The aqueous layer was extracted with ethyl acetate (2 50 mL) and the combined organic layers were washed with saturated NaCl (50 mL), dried over × Na2SO4 and evaporated. The resulting residue was purified by flash chromatography on a Biotage SNAP Ultra column using an eluent with 5–40% ethyl acetate in heptane to give the title compound. 1 Yellow solid; yield 6.99 g (79%); mp 57.3–58.7 ◦C; H-NMR (400 MHz, CDCl3): δ = 7.86 (d, J = 8.9, 2H), 7.73 (d, J = 8.4, 2H), 7.33 (d, J = 8.0, 2H), 7.09 (d, J = 8.9, 2H), 2.46 (s, 3H); 13C-NMR (101 MHz, CDCl3): δ = 156.0, 150.3, 145.8, 132.3, 130.0, 128.7, 127.4, 125.3, 123.0, 101.4, 80.00, 21.9; HRMS (ESI) m/z + calcd for C16H11O4KNI2S [M + K] : 605.8130; found: 605.8129.

3.2.4. Synthesis of 4-Formylphenyl 4-methylbenzenesulfonate (15) 4-Hydroxy-benzaldehyde (20.33 g, 166 mmol) and DMAP (68 mg, 0.61 mmol) were dissolved in a mixture of CH2Cl2 (200 mL) and Et3N (75 mL) and cooled to 0 ◦C. Tosyl chloride (31.7 g, 166 mmol) was dissolved in CH2Cl2 (60 mL) and added via an addition funnel for 20 min. The resulting mixture was left to heat to room temperature overnight and was then poured into water (200 mL). The phases Molecules 2020, 25, 4848 7 of 10 were separated and the organic phase was washed with 1M aqueous HCl (2x100 mL), water (100 mL) and saturated NaCl (100 mL) before drying over MgSO4 and evaporation. The residue was dissolved in a small amount of ethyl acetate and heated to reflux before cooling to -20 ◦C overnight. The precipitated crystals were filtered off and dried to afford 25.90 g of the title compound. The mother liquid was evaporated and the crystallization was repeated to give an additional 2.81 g of the title compound. The remaining mother liquid was evaporated and crystallized from 10–15% water in ethanol to give an additional 9.10 g of the title compound. 1 Colorless solid; yield 37.81 g (82%); mp 72.5–73.5 ◦C; H-NMR (400 MHz, CDCl3): δ = 9.96 (s, 1H), 7.82 (d, J = 8.2, 2H), 7.71 (d, J = 7.9, 2H), 7.32 (d, J = 8.0, 2H), 7.16 (d, J = 8.2, 2H), 2.44 (s, 3H); 13C-NMR (101 MHz, CDCl3) δ = 190.7, 154.0, 146.0, 134.9, 132.1, 131.4, 130.1, 128.6, 123.2, 21.8; HRMS (ESI) m/z calcd for C H O S [M H] : 375.0384; found: 375.0384. 14 11 4 − − 3.2.5. Synthesis of 4-(Oxazol-5-yl)phenyl 4-methylbenzenesulfonate (16) Aldehyde 15 (2.0 g, 7.24 mmol) and TosMIC (1.48 g, 7.60 mmol) were dissolved in DME (10 mL). DBU (1.14 mL, 7.60 mmol) was added using a syringe and the resulting mixture was heated to reflux until TLC analysis showed that the starting material had been consumed (approx. 5 h). The reaction mixture was evaporated, re-dissolved in a small amount of acetone, adsorbed onto a Biotage snaplet precolumn and purified by flash chromatography on a Biotage SNAP Ultra column using an eluent with 15–100% ethyl acetate in heptane to give the title compound. 1 Pale yellow solid; yield 1.59 g (70%); mp 112.2–114.1 ◦C; H-NMR (400 MHz, CDCl3) δ = 7.90 (s, 1H), 7.72 (d, J = 8.3, 2H), 7.57 (d, J = 8.8, 2H), 7.32 (m, 3H), 7.05 (d, J = 8.8, 2H), 2.45 (s, 3H); 13C-NMR (101 MHz, CDCl3) δ = 150.9, 150.5, 149.6, 145.7, 132.3, 130.0, 128.7, 126.8, 125.8, 123.2, 122.2, 21.9; + + HRMS (ESI) m/z calcd for C16H14NO4S [M + H] : 354.0197, found 354.0201.

3.2.6. Synthesis of 4-(2-Iodooxazol-5-yl)phenyl 4-methylbenzenesulfonate (17) The title compound was isolated as a side-product during the synthesis of 14. 1 Yellow solid; yield 837 mg (12%); mp 39.1–41.4 ◦C; H-NMR (400 MHz, CDCl3): δ = 7.71 (d, J = 8.4, 2H), 7.51 (d, J = 8.8, 2H), 7.31 (d, J = 8.0, 2H), 7.24 (s, 1H), 7.04 (d, J = 8.8, 2H), 2.46 (s, 3H); 13C-NMR (101 MHz, CDCl3): δ = 152.7, 149.9, 146.8, 145.8, 132.3, 130.0, 128.7, 126.0, 125.5, 124.0, 123.4, 29.8 + (grease), 21.9; HRMS (ESI) m/z calcd for C16H13O4NIS [M + H] : 441.9604; found: 441.9604.

3.2.7. Synthesis of t-Butyl 2-(4-iodo-5-(4-(tosyloxy)phenyl)oxazol-2-yl)-1H-pyrrole-1-carboxylate (18)

Diiodooxazol 16 (3.22 g, 5.77 mmol), Cs2CO3 (5.64 g, 17.3 mmol), (N-Boc-pyrrol-2-yl)boronic acid (1.46 g, 6.93 mmol) and PdCl (dppf) CH Cl (235 mg, 0.289 mmol) were dissolved in a mixture of 2 · 2 2 degassed dioxane (18 mL) and degassed water (6 mL). The solution was stirred at room temperature for 6 h under argon upon which TLC analysis showed some remaining starting material. Additional boronic acid (0.5 equiv.) and catalyst (5 mol%) were added and the resulting mixture was stirred for 42 h after which TLC still showed starting material left. Additional boronic acid (0.5 equiv.) and catalyst (5 mol%) were added and the mixture was stirred for 18 h, after which TLC showed no sign of starting material. The mixture was filtered through a plug of celite, diluted with ethyl acetate, washed with water (20 mL) and saturated NaCl (20 mL), dried over Na2SO4 and evaporated. The residue was purified by flash chromatography on a Biotage SNAP Ultra column using an eluent with 0–80% ethyl acetate in heptane to give the title compound. 1 Orange solid; yield 2.64 g (75%); mp 73.1–73.3 ◦C; H-NMR (400 MHz, CDCl3): δ = 7.94 (d, J = 8.8, 2H), 7.73 (d, J = 7.9, 2H), 7.44 (s, 1H), 7.32 (d, J = 8.1, 2H), 7.26 (s, 1H), 7.15–7.02 (m, 2H), 6.75 (s, 1H), 13 6.29 (d, J = 3.5, 1H), 2.45 (s, 3H), 1.44 (s, 9H); C-NMR (101 MHz, CDCl3): δ = 156.6, 149.8, 149.2, 148.2, 145.7, 132.4, 130.0, 128.7, 127.2, 126.4, 125.3, 122.9, 119.7, 119.7, 111.3, 85.1, 79.3, 27.8, 21.9; HRMS (ESI) + m/z calcd for C25H24O6N2IS [M + H] : 607.0394; found: 607.0394. Molecules 2020, 25, 4848 8 of 10

3.2.8. Synthesis of 4-(4-Iodo-2-(3,4,5-trichloro-1H-pyrrol-2-yl)oxazol-5-yl)phenyl 4-methylbenzenesulfonate (19) 2-(4-Iodooxazole)pyrrole 13 (101 mg, 0.20 mmol) and triphenylphosphine sulfide (12 mg, 40 µmol) were dissolved in CH Cl (0.8 mL) and cooled to 20 C. NCS (82 mg, 0.61 mmol) was added in one 2 2 − ◦ portion and the reaction mixture was allowed to heat to room temperature for 10 min. A small aliquot was subjected to HRMS analysis, which revealed that the trichlorination was not complete. The reaction mixture was again cooled to 20 C and additional NCS (5 mg, 37 µmol) was added. After heating − ◦ to room temperature no traces of dichlorinated compound(s) could be observed, and the reaction mixture was directly applied onto a Biotage SNAP Ultra column and purified by flash chromatography using 40–100% CH2Cl2 in heptane to give the title compound. This material was used in the next step without further purification; however, 1H-NMR analysis revealed the presence of minor amounts of residual solvent/and or grease. A small sample was purified further by normal and C18 flash chromatography for characterization by 1H- and 13C-NMR analysis. 1 Pale orange solid; yield 88 mg (73%); mp 97.7–99.4 ◦C; H-NMR (400 MHz, (CD3)2SO) δ = 8.02 (d, J = 8.9, 2H), 7.78 (d, J = 8.2, 2H), 7.50 (d, J = 8.2, 2H), 7.24 (d, J = 8.9, 2H), 2.44 (s, 3H); 13C-NMR (101 MHz, CDCl3) δ = 154.7, 149.4, 147.4, 146.5, 131.7, 130.8, 128.8, 127.3, 126.4, 123.3, 117.1, 115.0, 111.8, 109.2, 83.6, 21.7; HRMS (ESI) m/z calcd. for C H O N S35Cl 37ClI [M H] : 608.8526; found: 608.8514. 20 11 4 2 2 − − 3.2.9. Synthesis of 4-(4-Iodo-2-(1-(triisopropylsilyl)-1H-pyrrol-2-yl)oxazol-5-yl)-phenyl 4-methylbenzenesulfonate (20b) 2-(4-Iodo-oxazole)pyrrole 13 (479 mg, 0.95 mmol) was dissolved in anhydrous DMF (20 mL) and the solution was cooled at 0 ◦C in an ice/water bath. NaH (60% in mineral oil, 92 mg, 2.28 mmol) was added slowly and the color of the solution changed from green to orange. The flask was flushed with argon and stirring was continued for 15 min before TIPS-Cl (0.25 mL, 1.14 mmol) was added dropwise upon which the reaction mixture changed to a peachy color. Stirring was continued for 15 min at 0 ◦C before the cooling bath was removed and stirring continued at room temperature for 2 h when TLC analysis showed complete conversion of the starting material (TLC sample quenched with ice and extracted with ethyl acetate before spotting). The reaction mixture was cooled on ice, diluted with ethyl acetate (20 mL) and quenched by slow addition of crushed ice. Phases were separated, and the aqueous phase was extracted with ethyl acetate (2 25 mL). The combined organic phases were × washed with water (25 mL) and saturated NaCl (25 mL), dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by flash chromatography using a Biotage SNAP Ultra column using 0–20% ethyl acetate in heptane to give the title compound. For the most part, the crude product from this step was used in the next step without further purification. 1 Colorless oil; 558 mg (89%); H-NMR (400 MHz, CDCl3) δ = 7.94 (d, J = 8.8, 2H), 7.74 (d, J = 8.4, 2H), 7.33 (d, J = 8.1, 2H), 7.12–7.02 (m, 4H), 6.35 (t, J = 3.1, 1H), 2.46 (s, 3H), 1.86 (hept, J = 7.6, 3H), 13 1.13 (d, J = 7.6, 18H); C-NMR (101 MHz, CDCl3) δ = 158.0, 149.4, 147.0, 145.7, 132.4, 131.1, 130.0, 128.7, 126.9, 126.7, 124.4, 122.8, 117.6, 111.1, 79.3, 29.84 (impurity), 21.9, 18.4, 13.5; HRMS (ESI) m/z calcd. + for C29H35O4N2SiSINa [M + Na] : 685.1024; found: 685.1022.

3.2.10. Synthesis of 4-(2-(4,5-Dichloro-1-(triisopropylsilyl)-1H-pyrrol-2-yl)-4-iodo-oxazol-5-yl)phenyl 4-methylbenzenesulfonate (21) TIPS-protected pyrrole 20b (100 mg, 0.161 mmol) and triphenylphosphine sulfide (10 mg, 32 µmol) were dissolved in CH Cl (0.8 mL) and cooled to 20 C. NCS (44 mg, 0.330 mmol) was added in one 2 2 − ◦ batch, and the reaction mixture was allowed to warm to room temperature for 10 min. A small aliquot was subjected to HRMS analysis, which showed that complete dichlorination had occurred. The reaction mixture was directly applied onto a Biotage SNAP Ultra column and purified using flash chromatography using 0–100% CH2Cl2 in heptane to give the title compound. 1 Colorless oil; yield 99 mg (84%); H-NMR (400 MHz, CDCl3) δ = 7.91 (d, J = 8.8, 2H), 7.74 (d, J = 8.1, 2H), 7.34 (d, J = 8.1, 2H), 7.09 (d, J = 8.8, 2H), 6.75 (s, 1H), 2.46 (s, 3H), 1.72 (hept, J = 7.5, Molecules 2020, 25, 4848 9 of 10

13 3H), 1.10 (d, J = 7.5, 18H); C-NMR (101 MHz, CDCl3) δ = 157.1, 149.9, 148.7, 145.8, 132.4, 130.0, 128.7, 127.2, 126.1, 123.8, 123.4, 123.0, 118.5, 113.8, 79.1, 29.9 (imp.), 21.9, 18.5, 13.7; HRMS (ESI) m/z calcd. for 35 + C29H33N2O4N2SiS Cl2IK [M + K] : 768.9984; found: 768.9987.

3.2.11. Synthesis of 4-(2-(4,5-Dichloro-1H-pyrrol-2-yl)oxazol-5-yl)phenol (23) TIPS-protected dichlorinated pyrrole 21 (48 mg, 66 µmol) was dissolved in ethanol (0.7 mL) at 70 ◦C and 20% aqueous NaOH (0.3 mL) was added, upon which the color changed from clear to orange. The mixture was heated to reflux and, after 5 min, zinc (64 mg, 0.984 mmol) was added and heating was continued for 45 min. The reaction mixture was filtered through a pad of Celite and partitioned between ethyl acetate (10 mL) and water (10 mL). The aqueous phase was extracted with ethyl acetate (2 10 mL) and the combined organic phases were washed with saturated NaCl, × dried over Na2SO4 and evaporated. The residue was adsorbed onto a Biotage snaplet precolumn using CH2Cl2 and purified by flash chromatography using a Biotage SNAP Ultra column using 0–100% ethyl acetate in heptane to give the title compound. 1 Colorless solid; yield 17 mg (88%); mp 246 ◦C (dec.); H-NMR (400 MHz, (CD3)2SO) δ = 13.12 (s, 1H), 9.82 (s, 1H), 7.62 (d, J = 8.4, 2H), 7.51 (s, 1H), 6.94–6.75 (m, 3H); 13C-NMR (101 MHz, (CD3)2SO) δ = 157.8, 153.0, 150.1, 125.6, 121.1, 118.9, 118.5, 115.8, 114.7, 109.12, 109.11; HRMS (ESI) m/z calcd. 35 + for C13H9O2N2 Cl2 [M + H] : 295.0036; found: 295.0037.

4. Conclusions In conclusion, we have achieved the total synthesis of phorbazole B in six steps with an overall yield of 23%, showing the utility of a simple catalyzed chlorination and demonstrating the use of iodine as a protection group for oxazoles. Furthermore, we have concluded that phorbazoles A and C were not accessible through our synthetic approach based on previous literature reports and our own observation of predominantly 4,5-dichlorination of the pyrrole instead of the desirable 3,4-dichlorination pattern of phorbazole A and C.

Supplementary Materials: The following are available online, Figures S1–S22: 1D NMR spectra for 2 and 13–24, Table S1: Comparison of spectral data for synthetic and isolated 2, synthesis protocol for 24, structural assignment of 21 and 24, Figures S25–S37: 1D and 2D NMR spectra for assignment of chlorination pattern for 21 and 24. Author Contributions: Conceptualization, Y.G., J.I., B.E.H. and A.B.; funding acquisition, B.E.H. and A.B.; methodology, Y.G., M.E.F., S.K.P. and J.I.; writing—original draft, Y.G., J.I., B.E.H. and A.B.; writing—review and editing, Y.G., M.E.F., S.K.P., J.I., B.E.H. and A.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Research Council of Norway (grants 224790/O30 and 244264/O30) and the University of Bergen (PhD funding M.E.F.). The Research Council of Norway is further acknowledged for its support through the Norwegian NMR Platform (NNP) 226244/F50. The publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway Acknowledgments: The authors would like to thank Jarl Underhaug for the assistance with running NMR experiments. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Rudi, A.; Stein, Z.; Green, S.; Goldberg, I.; Kashman, Y.; Benayahu, Y.; Schleyer, M. Phorbazoles A-D, novel chlorinated phenylpyrrolyloxazoles from the marine sponge phorbas aﬀ. clathrata. Tetrahedron Lett. 1994, 35, 2589–2592. [CrossRef] 2. Nuzzo, G.; Ciavatta, M.L.; Kiss, R.; Mathieu, V.; Leclercqz, H.; Manzo, E.; Villani, G.; Mollo, E.; Lefranc, F.; D’Souza, L.; et al. Chemistry of the Nudibranch Aldisa andersoni: Structure and Biological Activity of Phorbazole Metabolites. Mar. Drugs 2012, 10, 1799–1811. [CrossRef][PubMed] 3. Radspieler, A.; Liebscher, J. Total synthesis of phorbazole C. Tetrahedron 2001, 57, 4867–4871. [CrossRef] Molecules 2020, 25, 4848 10 of 10

4. Hanssen, K.Ø.; Schuler, B.; Williams, A.J.; Demissie, T.B.; Hansen, E.; Andersen, J.H.; Svenson, J.; Blinov, K.; Repisky, M.; Mohn, F.; et al. A Combined Atomic Force Microscopy and Computational Approach for the Structural Elucidation of Breitfussin A and B: Highly Modiﬁed Halogenated Dipeptides from Thuiaria breitfussi. Angew. Chem. Int. Ed. 2012, 51, 12238–12241. [CrossRef][PubMed] 5. Hansen, K.O.; Andersen, J.H.; Bayer, A.; Pandey, S.K.; Lorentzen, M.; Jorgensen, K.B.; Sydnes, M.O.; Guttormsen, Y.; Baumann, M.; Koch, U.; et al. Kinase Chemodiversity from the Arctic: The Breitfussins. J. Med. Chem. 2019, 62, 10167–10181. [CrossRef][PubMed] 6. Pandey, S.K.; Guttormsen, Y.; Haug, B.E.; Hedberg, C.; Bayer, A. A Concise Total Synthesis of Breitfussin A and B. Org. Lett. 2015, 17, 122–125. [CrossRef][PubMed] 7. Khan, A.H.; Chen, J.S. Synthesis of Breitfussin B by Late-Stage Bromination. Org. Lett. 2015, 17, 3718–3721. [CrossRef][PubMed] 8. van Leusen, A.M.; Hoogenboom, B.E.; Siderius, H. A novel and eﬃcient synthesis of oxazoles from tosylmethylisocyanide and carbonyl compounds. Tetrahedron Lett. 1972, 13, 2369–2372. [CrossRef] 9. Min, G.K.; Bjerglund, K.; Kramer, S.; Gogsig, T.M.; Lindhardt, A.T.; Skrydstrup, T. Generation of stoichiometric ethylene and isotopic derivatives and application in transition-metal-catalyzed vinylation and enyne metathesis. Chem. Eur. J. 2013, 19, 17603–17607. [PubMed] 10. Maddox, S.M.; Dinh, A.N.; Armenta, F.; Um, J.; Gustafson, J.L. The Catalyst-Controlled Regiodivergent Chlorination of Phenols. Org. Lett. 2016, 18, 5476–5479. [CrossRef] 11. Vedejs, E.; Luchetta, L.M. A Method for Iodination of Oxazoles at C-4 via 2-Lithiooxazoles. J. Org. Chem. 1999, 64, 1011–1014. [CrossRef][PubMed] 12. Rodriguez, R.A.; Pan, C.M.; Yabe, Y.; Kawamata, Y.; Eastgate, M.D.; Baran, P.S. Palau’chlor: A practical and reactive chlorinating reagent. J. Am. Chem. Soc. 2014, 136, 6908–6911. [CrossRef][PubMed] 13. Maddox, S.M.; Nalbandian, C.J.; Smith, D.E.; Gustafson, J.L. A practical Lewis base catalyzed electrophilic chlorination of arenes and heterocycles. Org. Lett. 2015, 17, 1042–1045. [CrossRef][PubMed] 14. Morrison, M.D.; Hanthorn, J.J.; Pratt, D.A. Synthesis of Pyrrolnitrin and Related Halogenated Phenylpyrroles. Org. Lett. 2009, 11, 1051–1054. [CrossRef][PubMed] 15. Muchowski, J.M.; Naef, R. 3-Lithiopyrroles by Halogen-Metal Interchange of 3-Bromo-1- (triissopropylsilyl)pyrroles. Synthesis of Verucarin E and Other 3-Substituted Pyrroles. Preliminary Communication. Helv. Chim. Acta 1984, 67, 1168–1172. [CrossRef] 16. Bray, B.L.; Mathies, P.H.; Naef, R.; Solas, D.R.; Tidwell, T.T.; Artis, D.R.; Muchowski, J.M. N-(Triisopropylsilyl)pyrrole. A progenitor “par excellence” of 3-substituted pyrroles. J. Org. Chem. 1990, 55, 6317–6328. [CrossRef] 17. Higashino, T.; Osuka, A. 2,3,17,18-Tetrahalohexaphyrins and the First Phlorin-type Hexaphyrins. Chem. Asian J. 2013, 8, 1994–2002. [CrossRef][PubMed] 18. Yang, Q.; Sheng, M.; Henkelis, J.J.; Tu, S.; Wiensch, E.; Zhang, H.; Zhang, Y.; Tucker, C.; Ejeh, D.E. Explosion Hazards of Sodium Hydride in Dimethyl Sulfoxide, N,N-Dimethylformamide, and N,N-Dimethylacetamide. Org. Process Res. Dev. 2019, 23, 2210–2217. [CrossRef]

Sample Availability: No samples are available from the authors.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aﬃliations.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).