Enantioselective Total Syntheses of Plectosphaeroic Acids B and C
Evolution of the synthetic strategy that culminated in the first total syntheses of the structurally unique plectosphaeroic acids B (2) and C (3) is described. The successful enantioselective route to (+)-2 and (+)-3 proceeds in 6 and 11 steps from the known hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione 39, which in turn are available in enantiomerically pure form by chemical synthesis. The central challenge in this synthesis endeavor was uniting the hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione and cinnabarinic acid fragments of these marine alkaloids. Critical for achieving this successful C\xe2\x80\x93N bond formation was the use of an iodocinnabarinic acid diester in which the amino group was masked with two Boc substituents, a Cu(I) carboxylate complex, and the weak base KOAc. The highly congested C\xe2\x80\x93N bond generated in this coupling, in conjunction with the delicate nature of the densely functionalized coupling partners, provide striking testament to the power of modern copper-mediated amination methods. Two approaches, one stereoselective, for introducing the methylthio substituents of (+)-plectosphaeroic acid B were developed. The epitrisulfide ring of (+)-plectosphaeroic acid C was formed by ring expansion of an epidisulfide precursor.
INTRODUCTION<!>Synthesis Plan<!>Initial Development of the C\xe2\x80\x93N Cross-Coupling in Model Systems<!>C\xe2\x80\x93N Cross-Coupling with Cinnabarinic Acid Derivatives and Initial Attempts to Elaborate the Coupled Products<!>Total Synthesis of (+)-Plectosphaeroic Acid B (2)<!>Total Synthesis of (+)-Plectosphaeroic Acid C (3)<!>Preliminary Biological Evaluation<!>Conclusion<!>N6-(tert-Butoxycarbonyl) derivative of gliocladin C (24)<!>Method A<!>Method B<!>tert-Butyl 3-((3aS,8aR)-2-(methylcarbamoyl)-8,8a-dihydropyrrolo[2,3-b]indol-3a(3H)-yl)-1H-indole-1-carboxylate (27)<!>tert-Butyl 3-((5aR,10bS)-6-(2-(methoxycarbonyl)phenyl)-2-methyl-1,3,4-trioxo-1,2,3,4,5a,6-hexahydro-10bH-pyrazino[1\xe2\x80\xb2,2\xe2\x80\xb2:1,5]pyrrolo[2,3-b]indol-10b-yl)-1H-indole-1-carboxylate ((+)-28b)<!>6-Iodo-3-methoxy-2-nitrobenzoic acid (S2)<!>3-Hydroxy-6-iodo-2-nitrobenzoic acid (S3)<!>3-Hydroxy-6-iodoanthranilic acid (29)<!>2-Amino-8-iodo-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid (S5)<!>Dimethyl 2-amino-8-iodo-3-oxo-3H-phenoxazine-1,9-dicarboxylate (31)<!>Dimethyl 2-((tert-butoxycarbonyl)amino)-8-iodo-3-oxo-3H-phenoxazine-1,9-dicarboxylate (32)<!>Dimethyl 2-(di-(tert-butoxycarbonyl)amino)-8-iodo-3-oxo-3H-phenoxazine-1,9-dicarboxylate (33)<!>Cross-coupled product ((\xe2\x88\x92)-35)<!>tert-Butyl 3-((5aR,10bS)-3-hydroxy-2,3-dimethyl-1,4-dioxo-1,2,3,4,5a,6-hexahydro-10bH-pyrazino[1\xe2\x80\xb2,2\xe2\x80\xb2:1,5]pyrrolo[2,3-b]indol-10b-yl)-1H-indole-1-carboxylate (S6)<!>tert-Butyl 3-((5aR,10bS)-3-((tert-butyldimethylsilyl)oxy)-2,3-dimethyl-1,4-dioxo-1,2,3,4,5a,6-hexahydro-10bH-pyrazino[1\xe2\x80\xb2,2\xe2\x80\xb2:1,5]pyrrolo[2,3-b]indol-10b-yl)-1H-indole-1-carboxylate (36)<!>Cross-coupled product (\xe2\x88\x92)-37b (C3\xce\xb2-OTBS epimer)<!>Sharpless dihydroxylation product (\xe2\x88\x92)-38b (C3\xce\xb2-OTBS epimer)<!>Sharpless dihydroxylation product (+)-38a (C3\xce\xb1-OTBS epimer)<!>Method A<!>Method B<!>(3S,5aR,10bR,11S,11aS)-10b-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)-3-((tertbutyldimethylsilyl) oxy)-2,3-dimethyl-1,4-dioxo-1,2,3,4,5a,6,10b,11-octahydro-11aHpyrazino[1\xe2\x80\xb2,2\xe2\x80\xb2:1,5]pyrrolo[2,3-b]indole-11,11a-diyl diacetate ((+)-40b)<!>(5aR,10bR,11S,11aS)-10b-(1-(tert-Butoxycarbonyl)-1H-indol-3-yl)-2-methyl-3-methylene-1,4-dioxo-1,2,3,4,5a,6,10b,11-octahydro-11aH-pyrazino[1\xe2\x80\xb2,2\xe2\x80\xb2:1,5]pyrrolo[2,3-b]indole-11,11a-diyl diacetate ((+)-41)<!>Cross-coupled product ((+)-42a)<!>Cross-coupled product ((+)-42b)<!>Method A<!>Method B<!>(+)-Plectosphaeroic acid B (2)4<!>C3-epi-plectosphaeroic acid B ((+)-S9)<!>Epidisulfidedioxopiperazine ((\xe2\x88\x92)-46)<!>Method A<!>Method B<!>C13-Deoxyplectosphaeroic Acid C ((+)-45)<!>tert-Butyl 3-((3R,5aR,10bR,11S,11aS)-11-acetoxy-3-(acetoxymethyl)-3,11adihydroxy-2-methyl-1,4-dioxo-1,2,3,4,5a,6,11,11a-octahydro-10bHpyrazino[1\xe2\x80\xb2,2\xe2\x80\xb2:1,5]pyrrolo[2,3-b]indol-10b-yl)-1H-indole-1-carboxylate ((+)-50)<!>tert-Butyl 3-((5aR,10bR,11S,11aS)-11-acetoxy-3-(acetoxymethyl)-3,11a-bis((tertbutyldimethylsilyl) oxy)-2-methyl-1,4-dioxo-1,2,3,4,5a,6,11,11a-octahydro-10bHpyrazino[1\xe2\x80\xb2,2\xe2\x80\xb2:1,5]pyrrolo[2,3-b]indol-10b-yl)-1H-indole-1-carboxylate (51)<!>Cross-coupled Product ((+)-52-major)<!>Cross-coupled Product (52-minor)<!>C3,C11a-Dihydroxydioxopiperazine ((\xe2\x88\x92)-53-major)<!>Acetylated Intermediate (54)<!>Method A<!>Method B<!>Plectosphaeroic acid C dimethyl ester ((+)-58)<!>(+)-Plectosphaeroic acid C (3)<!>Cinnabarinic Acid (4)11,32b,c<!>Dimethyl 2-amino-9-iodophenoxazin-3-one 1,10-dicarboxylate (60)81
<p>Epipolythiodioxopiperazines (ETPs) and their methylthio congeners constitute a group of structurally complex and biologically active fungal alkaloids that are characterized structurally by a transannular polysulfide unit joining carbons 3 and 6 of the 2,5-dioxopiperazine ring or thioethers appended at these carbons (Figure 1).1 A spectrum of biological properties has been reported for this family of natural products, including antimicrobial, antiviral, antifungal, and anticancer activities.2 Recent attention has focused on the chemotherapeutic potential of ETP molecules, as several studies indicate in vivo selectivity and cytotoxicity towards several cancer variants.3 These potent activities are generally attributed to the labile S–S σ-bonds of the epidior epipolysulfide functionalities.1</p><p>The plectosphaeroic acids A–C (1–3) were reported in 2009 by Mauk, Andersen and co-workers from the marine fungus Plectosphaerella cucumerina, which was collected in Barkley Sound, British Columbia, Canada. 4 The constitution and relative configuration of these structurally unique alkaloids were elucidated largely by NMR analysis, whereas the absolute configuration of 1–3 was assigned by comparison of circular dichroism (CD) data with values reported for a related group of ETP toxins, the leptosins (e.g., 9).5 The plectosphaeroic acids A–C (1–3) represent a new family of fungal alkaloids that are comprised of a hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione moiety 5 containing sulfur functionality and a cinnabarinic acid portion 4. As both fragments likely arise biosynthetically from tryptophan, the plectosphaeroic acids contain a linear arrangement of four highly modified tryptophan units, a structural feature shared by other, less modified, tryptophan alkaloids such as quadrigemine C6 and psychotetramine.7 A number of ETP alkaloids and their methylthio congeners (e.g., 6–14) share a hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione fragment 5 that is homologous to the northern fragment of 1–3. 8 The southern cinnabarinic acid (4) fragment, particularly its 2-aminophenoxazin-3-one core, is also familiar in natural products some of which show potential cancer chemotherapeutic applications.9</p><p>The plectosphaeroic acids (1–3) were isolated from a program to discover inhibitors of the enzyme indoleamine 2,3-dioxygenase (IDO) from marine sources.4 Inhibition of IDO has been identified as a potential new approach to cancer therapy in which the immune system would be activated to combat solid tumors.10 The plectosphaeroic acids (1–3) and cinnabarinic acid (4) were reported to be equipotent (IC50 = 2 μM) against human recombinant IDO, whereas T988 A (10), which was co-isolated with 1–3, was found to be inactive.11 Considering that some portion of the southern fragment was responsible for IDO inhibition, we became intrigued by the further biological evaluation and the considerable synthetic challenges12–14 associated with the synthesis of these molecules possessing two antitumor pharmacophores.15</p><p>Herein, we describe the development of the first total syntheses of (+)-plectosphaeroic acids B (2) and C (3).16 The successful approach features a late-stage copper-mediated C–N cross-coupling reaction in addition to the stereoselective incorporation of the methylthio or bridging trisulfide substituents into the dioxopiperazine subunits of 2 and 3. In vitro cytotoxicity of plectosphaeroic acids B (2) and C (3) and their dimethyl esters against human prostate and melanoma cancer cell lines is described and compared to that of related structures.</p><!><p>Our synthetic planning was guided by several considerations (Scheme 1). The well-recognized sensitivity of epipolythiodioxopiperazines to strong bases and nucleophiles,1,14 and the similar sensitivity of hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-diones that contain a hydroxyl substituent adjacent to the quaternary carbon stereocenter, 17 places limitations on the reactions that could be employed. Certainly we wanted to defer the sulfidation step until a late stage of the synthesis. Precedent from the Movassaghi,13 Sodeoka18 and our19 groups suggested that the installation of methylthio or epidithio substituents by thiol trapping of N-acyliminium ions derived from intermediate 15 should be possible and likely stereoselective. However, forming the bridging trisulfide functionality of plectosphaeroic acid C (3) by a short sequence was less certain.20 Attractive to us was the potential of ring-expanding the likely more accessible epidisulfide congener 16, a transformation first described by Taylor and co-workers in 1968 for elaborating naturally obtained sporisdesmin A (21) to sporidesmin E (22) (eq 1).21</p><p>Foreseen as a central challenge in these syntheses would be uniting the hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione and cinnabarinic acid fragments. To maximize convergency we hoped to forge the key C–N bond as late as possible, for example between the indoline nitrogen of the pyrazinopyrroloindole fragment 17 and a halogenated congener 18 of cinnabarinic acid. In spite of the remarkable progress recorded in recent years in forging aryl-nitrogen bonds,22 the hindered nature of the required union and the structural complexity of the coupling partners 17 and 18 were without precedent.23,24 Hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione intermediate 17 was envisaged originating from the di-(tert-butoxycarbonyl) derivative of the natural product (+)-gliocladin C (19),8c,12,25 following a sequence our group described recently for preparing gliocladine C (6), (+)-T988 C (7), and (+)-gliocladin A (10).2c,12 We anticipated that biomimetic, oxidative dimerization of 6-halo-3-hydroxyanthranilic acids 20 would provide access to potential electrophilic coupling partners 18.26</p><!><p>As an opening phase of this inquiry, we explored the feasibility of joining the two polycyclic fragments of the plectosphaeroic acids. At the time, there were no reports of N-arylating structurally complex cyclotryptamines. Thus, we decided to examine initially such conversions with simplified aryl fragments in which a 2-halobenzoic acid would substitute for the cinnabarinic acid electrophile. However, the steric environment and nucleophilicity of the indoline nucleophile of the hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione would not be easily modeled. As a result, we chose to access an appropriate amine for our preliminary cross-coupling studies from the di-(tert-butoxycarbonyl) derivative 23 of (+)-gliocladin C, an intermediate that is available on multigram scale by chemical synthesis.2c,12</p><p>Salient results from our efforts to deprotect the indoline nitrogen of 23 are summarized in Table 1. Under thermal conditions, the Boc substituent on the indole nitrogen atom was more labile (entry 1, 2). Lewis acids were more successful, with catalytic amounts of Sc(OTf)3 selectively removing the indoline Boc group to give 25 in 76% yield (entry 3). However, this reaction required careful monitoring to prevent cleavage of the second Boc substituent and proved somewhat unreliable as the reaction was scaled. Thus, an alternative two-step (one-pot) process involving thermal deprotection of both nitrogen atoms of 23, followed by selective reprotection of the indole nitrogen, was utilized to generate large quantities of the cross-coupling precursor 25 (entry 4).</p><p>With indoline 25 in hand, we commenced C–N cross-coupling studies by investigating palladium catalysts (Scheme 2).27 Attempting to join 25 with ethyl 2-bromobenzoate (26a) using 10 mol % of Buchwald's palladacyclic pre-catalyst ligated with RuPhos22f,g and excess Cs2CO3 in t-BuOH at 85 °C resulted in rapid consumption of 25. However, N-arylation products were not detected from this reaction, which returned ring-fragmentation product 27 in low yield. As the α-oxoimide carbonyl group of trioxopiperazines is quite electrophilic—a property we exploit later in our studies—we reasoned that the tert-butoxide anion generated in situ was incompatible with the trioxopiperazine fragment of 25. Consistent with this result, NaOt-Bu in either THF or toluene decomposed 25, forming 27, within hours at 50 °C in the absence of the palladium catalyst.28 Other strong bases such as LHMDS also consumed 25 at 50 °C; whereas 25 was stable to Cs2CO3 in toluene at 85 °C. As a result, our further studies of palladium-catalyzed coupling focused on the use of weak inorganic bases (e.g., Cs2CO3, K2CO3 and K3PO4) in aprotic solvents such as toluene, THF, and 1,4-dioxane. Nevertheless, all attempts to forge the desired C–N bond between 25 and ethyl 2-bromobenzoate (26a) or methyl 2-iodobenzoate (26b) using catalytic or stoichiometric amounts of palladium(0) and various Buchwald biarylmonophosphine ligands such as RuPhos, XPhos, and SPhos,22f,g as well as rac-BINAP and tri-o-tolylphosphine were unproductive.</p><p>Copper(I) salts proved more successful.22a,b,d,e After an extensive examination of reaction parameters, it was found that coupling of pyrazinopyrroloindole 25 with 2 equiv of methyl 2-iodobenzoate in the presence of 4 equiv of either CuI, CuBr or CuCl and 5 equiv of Cs2CO3 in toluene at 110 °C for 24 h promoted C–N bond formation to give 28b as the major product (Scheme 2). Using CuCl, the coupled product 28b was isolated in 85% yield. Reducing the amount of the copper salt lowered the reaction efficiency, as did replacing Cs2CO3 with K2CO3 or K3PO4. In a brief attempt to develop a catalytic transformation, it was found that the combination of 0.5 equiv of CuI and 1,3-bis(diphenylphosphino)propane (dppp) also furnished 28b in 85% yield. Having successfully formed the C–N bond in this model system with copper(I), we shifted to the next phase of our studies, introduction of more complex and delicate cinnabarinic acid electrophiles.</p><!><p>Our cross-coupling approach required an efficient route for the preparation of cinnabarinic acid derivatives having a halide (or pseudohalide) incorporated at C8 (e.g., 31) (Scheme 3). As noted earlier, such intermediates should be available by oxidative dimerization of 6-halo-3-hydroxyanthranilic acids and derivatives (e.g., 29).26, 29, 30 This transformation could be performed with 3-hydroxy-6-iodoanthranilic acid (29);31 however, we found it more convenient to esterify 29, then immediately expose the intermediate ester 30 to oxidation with benzoquinone. 32 Simple filtration delivered 2-amino-8-iodophenoxazin-3-one diester 31 in 56% yield from acid 29. By carefully controlling the amount of Boc2O used—slow addition of 1.2 equiv or adding 2–3 equiv—the 2-amino group of 31 was mono- or diprotected to give 32 or 33 in useful yields.33</p><p>Our efforts to join the two fragments of the plectosphaeroic acids began by attempting to form a C–N bond between the tert-butoxycarbonyl derivative 25 of gliocladin C and iodide 32 (Table 2). However, C–N bond formation was not observed using any of the copper(I) conditions that we had identified previously (e.g., excess CuCl, Cs2CO3, PhMe, 110 °C). In addition, minor modifications of these conditions also were not fruitful.34</p><p>A search for an alternate copper reagent,22a,b,d,e identified copper(I) thiophene-2-carboxylate (CuTC)35,36 as particularly effective (Table 2). When 25 was allowed to react with 2 equiv of 32, 4 equiv of CuTC and excess Cs2CO3 in toluene at 110 °C, coupled product 34 was formed, albeit in low yield (entry 1). The conversion to 34 was improved upon switching to K2CO3 as base (entry 2), whereas a change from toluene to dioxane proved detrimental (data not shown). In contrast to our earlier observations, substantial hydrodehalogenation and minor amounts of homocoupling of iodide 32 were observed when using CuTC; a result suggesting that this copper complex, unlike other complexes investigated to this point, was more efficient in oxidative addition to the C–I σ-bond of 32. A potential explanation for the efficacy of CuTC is its increased solubility in nonpolar solvents.37,38 A breakthrough was made when the electrophile was switched to iodide 33 in which the 2-amino group of the iodophenoxazine was doubly protected. Although the reaction of 25 with 1.5 equiv of 33 in the presence of 3 equiv of CuTC and excess K2CO3 in toluene at 110 °C was inefficient (entry 3),39 carrying out this reaction at 90 °C gave 35 in 48% yield (entry 3). Further reduction of the reaction temperature proved detrimental (entry 5). However, further optimization (entries 6–9) improved the conversion to 35 to 67% yield.40</p><p>We were encouraged by the successful union of hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione 25 and iodide 33 to increase the complexity of the amine nucleophile. We chose a measured approach, first by examining whether a potentially delicate N,O-acetal functionality at C3 would prove problematic. To arrive at a coupling partner of this type, methylmagnesium chloride was added chemoselectively to the α-oxoimide carbonyl group of 25 followed by silylation of the resulting alcohol products to give epimers 36a and 36b (1.2:1.0 dr) in 70% yield over two steps (Scheme 4). Submitting these C3-N,O-acetals to the C–N cross-coupling conditions identified in Table 2 showed that N-arylation was efficient with both epimers to give the coupled products 37 in 68–72% yields.</p><p>Buoyed by this success, we turned to advance toward the plectosphaeroic acids by dihydroxylation of the alkene double bond of coupled products 37a and 37b. In our earlier studies with congeners of 37 bearing a Boc protecting group on the indoline nitrogen, we had observed high α-facial selectivity in dihydroxylation of the C11–C11a π bond.2c,12 To our surprise, oxidation of both 37a and 37b with OsO4/4-methylmorpholine N-oxide (NMO), AD-mix α or AD-mix β41 afforded the undesired, β diols 38a and 38b with high stereoselectivity (see Table, Scheme 4).42 The relative configuration of the syn diol products were assigned from the diagnostic 1H NOE's observed for their C11 methine hydrogens;12 additionally, the minor isomer obtained from dihydroxylation of 37b was transformed into di(methylthio)ether 45b (vide infra). We were forced to turn to investigate the crucial fragment coupling with more complex hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione intermediates already containing oxygen functionality properly installed at C11 and C11a, a strategy that proved ultimately to be successful.</p><!><p>From the outset of this project, we envisaged the pyrazinopyrroloindole fragment 5 of (+)-plectosphaeroic acid B (2) originating from intermediates prepared by our group en route to (+)-gliocladine C (5) and congeners.2c,12 During the synthesis of 5, the di-(tert-butoxycarbonyl) derivative 23 of (+)-gliocladin C was transformed in four efficient steps to products 39a and 39b shown in Scheme 5. For the synthesis of 2, we needed to remove the Boc group from the indoline nitrogen atom of these intermediates, a task anticipated to be complicated by the various acid-labile functionalities that were present.</p><p>Salient results from our efforts to unmask the indoline nitrogen atom of 39a and 39b are summarized in Scheme 5. As we were concerned about the sensitivity of these molecules to acid, we examined initially the two-step (single-pot) procedure developed earlier in our studies (entry 1). This sequence provided products 40a and 40b, whose relative and absolute configuration was confirmed by single-crystal X-ray analysis, in 60–80% yield. 43 In both products, the configuration of the C11a acetate was inverted during the thermolytic Boc-removal step, consistent with higher thermodynamic stability of a β-C11a-epimer in this ring system. Although this sequence was best conducted on small scale (multiple vessels containing 40 mg of 39a or 39b were run in parallel), it allowed us to secure sufficient quantities of 40a and 40b to move forward in the synthesis. Success was registered also in the chemoselective deprotection of 39a under acidic conditions. Exposure of this epimer to 1.5 equiv of trifluoromethanesulfonic (triflic) acid in CH2Cl2 at −40 °C for 2–3 h furnished 40a in 70–80% yield, also with concomitant inversion of the C11a stereocenter. 44 This transformation required careful monitoring, as prolonged reaction times or an increase in temperature resulted in expulsion of the silyloxy group at C3 to give substantial amounts of α-methylene intermediate 41. Under identical conditions, epimer 39b was converted exclusively to elimination product 41 (82% yield). Attempts to prevent the formation of 41 from siloxy epimer 39b by lowering the reaction temperature, reducing the amount of acid or using silyl triflates (e.g., TMSOTf and TBSOTf) were uniformly unsuccessful.</p><p>With convenient access to intermediates 40a and 40b in hand, the essential C–N cross-coupling reaction of these more intricate substrates was examined (Table 3). We began by exploring the union of stereoisomer 40a and cinnabarinic iodide 33. The CuTC/K2CO3 conditions that we had previously found to be optimal with less-elaborate intermediates containing a C11,C11a double bond resulted in inefficient conversion to coupled product 42a (entry 1). A major byproduct formed in this reaction was the C11a thiophene-2-carboxylate analog 43 of the starting diacetate 40a. As activation of the angular N,O-acetal appeared unavoidable, to minimize the formation of 43, KOAc was substituted for K2CO3. This change decreased the formation of 43, giving coupled product 42a in 40% yield (entry 2). It was eventually found that exposure of 40a or 40b to 3 equiv of iodide 33, 6 equiv of CuOAc and excess KOAc in toluene at 90 °C delivered 42a and 42b in 51–58% yield (entry 4).45</p><p>Two methods were developed for introducing the methylthio substituents of (+)-plectosphaeroic acid B (2) (Scheme 6). In the most direct approach, reaction of either N,O-acetal epimer of dioxopiperazines 42a or 42b with a large excess of both BF3•OEt2 and methanethiol at −78 °C with slow warming to room temperature gave an identical 1.3:1.0 separable mixture of di(methylthio)ethers 44b and 44a in high yield (79% from 42a, 92% from 42b). The relative configuration of these products was secured from 1H NOE experiments.46 An identical mixture of epimers was formed when a pure sample of either stereoisomereric product was resubmitted to the reaction conditions, establishing that the thermodynamic ratio of C3 methylthio epimers is produced under these conditions. In addition, this equilibration allowed the undesired C3 α-methylthio epimer 44a to be recycled. Alternatively, in the sequence we currently prefer, reaction of N,O-acetals 42 with excess H2S and BF3•OEt2, followed by alkylation of the resulting thiols with MeI afforded 44b as a single stereoisomer in 80–88% yield.47 Di(methylthio)ether 44b was prepared on useful scales by both of the procedures summarized in Scheme 6.</p><p>The difference in stereochemical outcome of these two sulfidation procedures is striking. Because di(methylthio)ethers 44b and 44a were established to equilibrate under the acidic conditions of the sulfidation step, a similar thermodynamic equilibration would be expected to occur during the BF3•OEt2-promoted reaction of 42 with H2S to form the dithiol congeners of 44a and 44b. Thus, one potential explanation is a considerably higher thermodynamic stability of the cis-dithiol intermediates. Such a trend was seen in a computational study of cis-dithiol than the cis-di(methylthio) isomers in closely related model compounds.48 Alternatively, and in our view more likely, a mixture of dithiol epimers equilibrates49 during the methylation step, with the observed cis stereoselectivity potentially resulting from intramolecular thio coordination of the intermediate potassium thiolate nucleophile.</p><p>All that remained to finish the total synthesis of plectosphaeroic acid B (2) was cleavage of the three ester groups of di(thiomethyl) intermediate 44b. However, the sensitivity of the cinnabarinic acid fragment was worrisome. 50 Methanolysis of the acetyl group at C11 was achieved by exposure of 44b to excess La(OTf)3 and 1 equiv of DMAP in methanol at 50 °C.51 In the absence of DMAP, the deprotection step was sluggish and the resulting product was not stable to prolonged exposure to the reaction conditions. Attempts to promote concomitant hydrolysis of the methyl esters of the phenoxazinone fragment by addition of small amounts of water to this reaction led to decomposition. As a result, the methyl esters were cleaved by classical demethylation with LiI in pyridine at 90 °C.52 In this way, plectosphaeroic acid B (2) was obtained in 65% yield over two steps after purification by HPLC. The optical rotation of synthetic 2, [α]D
23 +228 (c 0.08, MeOH), was higher than the value reported for the natural sample, [α]D
23 +69.8 (c 0.27, MeOH); however, all other spectroscopic data, including CD spectra, compared well.</p><!><p>As a second total synthesis objective in this area, we selected (+)-plectosphaeroic acid C (3) whose synthesis would present two additional challenges: introduction of the bridging trisulfide functionality and incorporation of oxygenation on the one-carbon C3 substituent (Figure 1). We chose to initially address the first obstacle by developing a method to elaborate intermediates prepared en route to (+)-plectosphaeroic acid B to an analogous product containing a trisulfide fragment, 13-deoxyplectosphaeroic acid C (45).</p><p>
</p><p>The ring-expanding sequence that we eventually implemented to incorporate the epitrisulfide functionality originated from several observations made during our efforts to introduce the methylthio groups of plectosphaeroic acid B (2). Specifically, we had observed the formation of epidisulfide 46 (isolated in up to 80% yield) upon exposing N,O-acetals 42 to excess H2S and BF3•OEt2 and subsequently to excess K2CO3 in acetone in the absence of MeI (Scheme 7A). In addition, minor quantities of 47, the trisulfide congener of 46, were also formed from this sequence (10–20% by 1H NMR analysis). When no attempt was made to remove the last traces of H2S from the crude dithiol intermediate, these products were formed within 30 minutes in the second step.53 Consistent with our expectation that trisulfide 47 was formed by ring-expansion of epidisulfide precursor 46,21,54 exposure of a pure sample of 46 to 1 equiv of Na2S in acetone at room temperature provided a 4–5:1 mixture of epitrisulfide 47 and epitetrasulfide 48 products in combined yields of up to 75% (Scheme 7B).55,56</p><p>A streamlined process for installing the epitrisulfide functionality and confirmation that the alcohol and carboxylic acid protecting groups could be removed in the presence of an epitrisulfide was achieved in our successful elaboration of N,O-acetals 42 to 13-deoxyplectosphaeroic acid C (45) (Scheme 8). After BF3•OEt2-mediated reaction of 42 with H2S, the crude dithiol products were dissolved in acetone, followed by sequential additions of K2CO3 and Na2S. In this way, epitrisulfide 47 was isolated in 64% yield from the epimer mixture 42. To our delight, the two-step sequence for cleaving the ester groups developed en route to (+)-plectosphaeroic acid B (2) was not adversely effected by the presence of the epitrisulfide functionality, allowing 13-deoxyplectosphaeroic acid C (45) to be prepared in 55% yield from epitrisulfide precursor 47.</p><p>We turned to the synthesis of (+)-plectosphaeroic acid C (3) itself. In order to install a hydroxyl group at C13, we envisaged oxidizing the exo-methylene double bond of intermediate 41, a product that could be formed in high yield by the reaction of intermediates 39a and 39b with triflic acid (Scheme 5).57 Oxidation of 41 by reaction with catalytic amounts of OsO4 in the presence of NMO provided one major product, diol diacetate 50 (Scheme 9). That the acetyl group had been transferred from the angular C11a hydroxyl group of intermediate 49 was apparent from the NMR signals of the methylene group (1H NMR δ 4.42 (d) and 4.29 (d); 13C NMR δ 65.4). Although minor amounts of 49, in which the C11a acetyl group had not been transferred could be obtained, it was more convenient to promote complete transfer of the acetyl group by stirring the crude reaction product with SiO2 in ethyl acetate prior to chromatographic purification to yield 50. Silylation of the tertiary alcohols of 50 furnished pyrazinopyrroloindole 51, an 8:1 mixture of separable epimers, in 75% overall yield from 41; these isomers undoubtedly result from inconsequential partial epimerization at C3 during the silylation step.49</p><p>Joining of the two fragments of (+)-plectosphaeroic acid C (3) was accomplished by individually treating epimers 51 with 3 equiv of iodide 33 and 6 equiv of CuOAc in the presence of KOAc in PhMe at 90 °C to give coupled products 52-major and 52-minor in 72% and 74% yields, respectively. The increase in efficiency in this C–N coupling step relative to the corresponding N-arylation steps in the synthesis of 2 (50–58% yield) is attributable to the enhanced stability of the C3- and C11a-N,O-acetals of 51 and 52. Specifically, N-acyliminium ion formation at C3 and C11a would be much less favorable in these intermediates wherein the leaving group is a silyl ether rather than an acetoxy substituent.</p><p>To no surprise, this diminished reactivity of the C3- and C11a-N,O-acetals complicated the sulfidation step. For example, treating 52-major with (i) H2S and BF3•OEt2 in CH2Cl2 or MeNO2 at −78 °C to rt, (ii) H2S and Sc(OTf)3 in MeCN at −78 °C to rt, or (iii) Na2S in a 1:1 mixture of TFA/MeNO2 at 0 °C to rt were not successful. In these reactions, either sulfur was not incorporated, or after prolonged exposure (i.e. >12 h) decomposition was observed. For this reason, it was decided to convert the oxygen substituents at C3 and C11a to alternate leaving groups. Removal of both silyl groups of 52-major and 52-minor upon reaction with TBAF and AcOH in THF gave diols 53-major and 53-minor in >90% yields.58</p><p>By incorporating better leaving groups at C3 and C11a, the total synthesis of (+)-plectosphaeroic acid C (3) was successfully completed (Scheme 10).12,18 After standard acetylation of a mixture of epimeric diols 53 arising from the fragment-coupling and desilylation steps, the 5:1 mixture of tetraacetates 54 was exposed to H2S and BF3•OEt2 in CH2Cl2 at −78 °C to room temperature. After removing residual H2S and stirring the crude dithiol products with SiO2 in ethyl acetate, epidisulfide 55 and epitrisulfide 56 were produced in an 85:15 ratio (by 1H NMR analysis). Subsequent ring-expansion of this mixture by reaction with Na2S gave a 2.5:1 mixture of epitrisulfide 56 and epitetrasulfide 57, respectively. On larger scales (30–60 mg), it was more convenient to process the crude dithiol intermediates without rigorous removal of residual H2S to yield a 1:6:2 mixture of epidi-, epitri-, and epitetrasulfide products 55–57.59 As the central S–S σ-bond of tetrasulfides is considerably weaker than the corresponding bonds of disulfides or trisulfides,60 we were able to selectively reduce the epitetrasulfide component of these mixtures by exposing the reaction product to 1 equiv of triphenylphosphine (relative to the amount of 57) in CH2Cl2 at room temperature.61 Ensuing cleavage of the remaining ester groups then provided (+)-plectosphaeroic acid C (3) in 33% overall yield from tetraacetate 54. In the final, dealkylative transformation to liberate the carboxylic acids of 3, switching to the non-coordinating solvent toluene significantly increased the reaction rate,62 which proved necessary as 3—unlike its C3 methyl congener 48—was not stable to prolonged exposure to the reaction conditions. The optical rotation of synthetic 3, [α]D
23 +494 (c 0.06, MeOH), was substantially higher than the value reported for the natural sample, [α]D
23 +136 (c 0.17, MeOH); however, the congruence of all other spectroscopic data, including CD spectra, left little doubt that natural (+)-plectosphaeroic acid C (3) had been synthesized.</p><!><p>Having synthesized (+)-plectosphaeroic acids B (2) and C (3) their activities and those of selected related molecules against two invasive cancer cell lines, DU145 (human prostate) and A2058 (melanoma), were determined (Figure 2).63 It was found that 2, 3, and cinnabarinic acid (4)64 were inactive within the detection limits of the assay (>5 μM) (entry 1). However, the dimethyl ester analog 58 of plectosphaeroic acid C exhibited comparable potency to that of T988 C (7) against the two cancer cell lines (entries 2 and 3). 65 Of note, the epidisulfide analog 46 of plectosphaeroic acid B and the structurally simpler ETP 59 having an identical hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione moiety show similar activities (entries 4 and 5). Cinnabarinic acid dimethyl ester (60), a moderately active inhibitor of IDO,11 was inactive (entry 6). In no instance were the corresponding di(methylthio) congeners active against these cell lines (data not shown). Taken together, these data indicate that (i) only the presence of the epipolysulfide functionality is required for in vitro cytotoxicity activity against the DU145 and A2058 cell lines, and (ii) the presence of the carboxylic acid substituents in plectosphaeroic acid C (3) is detrimental to its cell-based cytotoxicity.</p><!><p>The first total syntheses of plectosphaeroic acids B (2) and C (3) are reported, syntheses that confirm the unique structure and absolute configuration of the plectosphaeroic acids.4 The synthetic sequence to (+)-2 and (+)-3 proceeded in 6 and 11 steps from known hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-diones 39a and 39b, which in turn are available in enantiomerically pure form in 14 steps from indole and isatin.12 Our approach to these natural products featured the late-stage merger of the cinnabarinic acid and hexahydro-2H-pyrazinopyrrolo[2,3-b]indole-1,4-dione fragments by a copper-mediated process. The highly congested C–N bond generated in this coupling, in conjunction with the delicate nature of the densely functionalized precursors, provide striking testament to the power of modern copper-mediated amination methods.22a,b,d,e The sequence developed in this investigation for stereoselectively introducing the methylthio substituents of 2 and the ring-expanding procedure for fashioning the bridging trisulfide of 3 were essential to simplifying the synthesis of these polyfunctional natural products. We anticipate that the late-stage ring-expansion procedure employed in these syntheses should be of utility for synthesizing tri- and tetrasulfide congeners of other epipolythiodiketopiperazines.</p><!><p>The di-(tert-butoxycarbonyl) derivative 23 of gliocladin C12 (3 mg, 5.1 μmol) was heated in a sealed vial (neat) at 140 °C (preheated oil bath) for 11 min. The reaction mixture was allowed to cool to rt, then the crude product was directly purified by preparatory thin layer chromatography (1:1 EtOAc/hexanes) to afford the title compound 24 as a yellow solid (2 mg, 83%; formation of minor quantities of gliocladin C (19) also observed, 3:1 24/19): 1H NMR (500 MHz, acetone-d6) δ 10.42 (br s, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 8.1 Hz), 7.40 (d, J = 7.4 Hz), 7.36 (t, J = 7.6 Hz), 7.33 (d, J = 8.2 Hz), 7.15–7.11 (comp, 3H), 7.02 (s, 1H), 6.94 (t, J = 7.4 Hz), 6.83 (s, 1H), 3.23 (s, 3H), 1.54 (s, 9H); 13C NMR (125 MHz, acetone-d6) δ 158.6 (C), 157.9 (C), 152.7 (C), 150.8 (C), 142.8 (C), 138.6 (C), 135.0 (C), 133.9 (C), 129.7 (CH), 126.3 (C), 125.64 (CH), 125.57 (CH), 125.1 (CH), 124.5 (CH), 123.1 (CH), 120.4 (CH), 119.9 (CH), 118.3 (CH), 115.3 (C), 112.9 (CH), 85.4 (CH), 82.9 (C), 59.2 (C), 28.2 (CH3), 27.2 (CH3); IR (film) 3331, 3105, 3058, 2977, 2930, 1714, 1683, 1599, 1478; TLC Rf 0.17 (1:1 EtOAc/hexanes); HRMS (ESI) m/z calcd for C27H24N4O5Na (M+Na)+ 507.1644, found 507.1623.</p><!><p>A solution of scandium triflate (1.6 mg, 3.3 μmol) and MeCN (50 μL) was added dropwise to a solution of the di-(tert-butoxycarbonyl) derivative 23 of gliocladin C (19 mg, 33 μmol) and MeCN (0.33 mL) cooled to 0 °C. The reaction mixture was allowed to warm to rt. After 3 h, the reaction mixture was poured into a mixture of ethyl acetate (EtOAc) (10 mL) and saturated aqueous NaHCO3 (5 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (2 x 5 mL). The combined organic layers were washed sequentially with H2O (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (1:9 EtOAc/hexanes → 3:7 EtOAc/hexanes) to afford the title compound (+)-25 as a yellow solid (12 mg, 76%).</p><!><p>The di-(tert-butoxycarbonyl) derivative 23 of gliocladin C (150 mg, 260 μmol, concentrated in vacuo from a THF solution into a vial) was heated (neat) under a slight vacuum (160 mmHg) at 180 °C (preheated oil bath) for 20 min (reaction times can vary slightly; removal of both Boc groups of 23 were monitored by TLC). The reaction mixture was allowed to cool to rt, then THF (4 mL), di-tert-butyl dicarbonate (Boc2O) (56 mg, 260 μmol) and N,N-dimethyl-4-aminopyridine (DMAP) (6.0 mg, 4.9 μmol) were added to the vial sequentially (under a N2 atmosphere). After 1 h (in some cases, additional Boc2O was added to drive the reaction to completion), the reaction mixture was poured into a mixture of EtOAc (20 mL) and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (1:3 EtOAc/hexanes) to afford the title compound (+)-25 (118 mg, 95%) as a tan foam: [α]D
24 +148, [α]577
24 +144, [α]546
24 +139 (c = 0.2, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.15 (d, J = 8.3 Hz, 1H), 7.60 (s, 1H), 7.33 (d, J = 6.9 Hz, 1H), 7.30 (dt, J = 8.3, 1.0 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 7.15 (dt, J = 7.7, 1.2 Hz, 1H), 7.09 (dt, J = 8.0, 0.9 Hz, 1H), 7.01 (s, 1H), 6.88 (d, J = 7.9 Hz, 1H), 6.75–6.70 (comp, 2H), 6.28 (d, J = 2.6 Hz, 1H), 3.25 (s, 3H), 1.67 (s, 9H); 13C NMR (125 MHz, acetone-d6) δ 158.6 (C), 158.0 (C), 150.7 (C), 150.3 (C), 150.1 (C), 137.9 (C), 133.9 (C), 130.2 (CH), 130.1 (C), 129.0 (C), 125.7 (CH), 125.4 (CH), 125.3 (CH), 124.3 (CH), 123.7 (CH), 122.0 (C), 120.9 (CH), 120.0 (CH), 116.3 (CH), 110.9 (CH), 85.0 (C), 84.0 (CH), 60.6 (C), 28.3 (CH3), 27.2 (CH3); IR (film) 3365, 3102, 3064, 2979, 1736, 1686, 1606, 1452; TLC Rf 0.32 (1:1 EtOAc/hexanes), 0.41 (2:98 MeOH/CH2Cl2, plate was eluted twice); HRMS (ESI) m/z calcd for C27H24N4O5Na (M+Na)+ 507.1644, found 507.1643.</p><!><p>Indoline 25 (12 mg, 25 μmol), Buchwald's palladacyclic pre-catalyst ligated with RuPhos66 (1.0 mg, 1.3 μmol), RuPhos (0.5 mg, 1.0 μmol) and Cs2CO3 (16 mg, 50 μmol) were added to a 1-dram vial (in a nitrogen-filled glovebox). After t-BuOH (100 μL) was added to this mixture, the vial was sealed (with a top, which contained a Teflon®-lined septum) and brought outside of the glovebox. Ethyl 2-bromobenzoate (26a) (3.3 μL, 21 μmol) was injected into the reaction vessel, then the reaction mixture was heated at 85 °C (preheated oil bath) for 12 h, at which point the reaction mixture was cooled to rt and EtOAc (400 μL) was added. The mixture was filtered through a short pad of diatomaceous earth and the filtrate was concentrated in vacuo. The crude residue was purified by preparatory thin layer chromatography (1:4 EtOAc/hexanes) to afford the title compound 27 (2 mg, 19%, with no detectable quantity of the desired coupled product 28a): 1H NMR (500 MHz, CDCl3) δ 8.13 (br d, J = 6.3 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.30 (s, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 7.3, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.15–7.11 (comp, 2H), 6.82 (t, J = 7.4, 1H), 6.76 (d, J = 7.8, 1H), 6.02 (s, 1H), 4.67 (s, 1H), 3.85 (d, J = 18.6 Hz, 1H), 3.70 (d, J = 18.6 Hz, 1H), 2.90 (d, J = 5.1 Hz, 1H), 1.63 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 170.5 (C), 162.7 (C), 149.8 (C), 147.5 (C), 150.1 (C), 132.7 (C), 129.3 (CH), 128.6 (C), 125.3 (CH), 124.8 (CH), 123.4 (CH), 123.0 (C), 122.9 (CH), 120.5 (CH), 119.9 (CH), 115.9 (CH), 111.0 (CH), 96.6 (CH), 84.1 (C), 54.9 (C), 48.4 (CH2), 28.4 (CH3), 26.2 (CH3); HRMS (ESI) m/z calcd for C25H26N4O3Na (M+Na)+ 453.1903, found 453.1908.</p><!><p>Indoline 25 (10 mg, 21 μmol), CuCl (8.0 mg, 81 μmol) [or 0.5 equiv of CuI/1,3-bis(diphenylphosphino)propane (dppp)] and Cs2CO3 (34 mg, 105 μmol) were added to a 1-dram vial (in a nitrogen-filled glovebox). After PhMe (400 μL, degassed via freeze–pump–thaw cycles prior to use) and methyl 2-iodobenzoate (26b) (64 μL, 42 μmol, 10% v/v solution in toluene) were added to this mixture, the vial was sealed (with a Teflon®-lined cap) and brought outside of the glovebox. The heterogenous mixture was stirred at 110 °C (preheated oil bath) for 24 h, at which point the reaction mixture was cooled to rt and EtOAc (400 μL) was added. The mixture was filtered through a short pad of diatomaceous earth, then the filtrate was concentrated in vacuo. The crude residue was purified sequentially by chromatography on silica gel (1:9 EtOAc/hexanes → 3:2 EtOAc/hexanes) then preparatory thin layer chromatography (to separate the product from residual, co-polar 25) (1.5% MeOH/CH2Cl2) to afford the title compound (+)-28b (11 mg, 85%) as an orange solid: [α]D
24 +168, [α]577
24 +170, [α]546
24 +174 (c = 0.9, CH2Cl2); 1H NMR (500 MHz, 353K, DMSO-d6) δ 8.11 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 7.0 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.56 (s, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.36 (t, J = 8.2 Hz, 1H), 7.32 (d, J = 7.4 Hz, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.08 (dt, J = 7.9, 1.1 Hz, 1H), 7.03 (s, 1H), 6.74 (t, J = 7.4 Hz, 1H), 6.72 (s, 1H), 6.15 (d, J = 7.9 Hz, 1H), 3.44 (s, 3H), 3.17 (s, 3H), 1.66 (s, 9H); 13C NMR (125 MHz, 353K, DMSO-d6) δ 165.8 (C), 156.71 (C), 156.66 (C), 149.8 (C), 148.7 (C), 148.6 (C), 139.2 (C), 135.3 (C), 132.9 (CH), 132.0 (C), 130.3 (2 x CH), 128.7 (CH), 127.7 (CH), 127.4 (C), 127.2 (CH), 124.34 (CH), 124.32 (2 x CH), 124.1 (CH), 122.5 (CH), 119.9 (CH), 119.5 (C), 118.5 (CH), 114.7 (CH), 106.7 (CH), 86.6 (CH), 84.1 (C), 58.4 (C), 51.22 (CH3), 27.4 (CH3), 26.3 (CH3); IR (film) 3101, 3054, 2979, 2950, 1731, 1715, 1687, 1598, 1452; TLC Rf 0.35 (1:1 EtOAc/hexanes), 0.50 (2:98 MeOH/CH2Cl2, plate was eluted twice); HRMS (ESI) m/z calcd for C35H30N4O7Na (M+Na)+ 641.2012, found 641.1995.</p><!><p>This procedure is a modification of a procedure reported by Fairfax and Yang.29 To a suspension of 2-methoxy-3-nitrobenzoic acid (S1) (10.0 g, 50.7 mmol) in concentrated H2SO4 (150 mL) was added Ag2SO4 (20.6 g, 65.9 mmol) and iodine (13.5 g, 53.5 mmol). The heterogeneous mixture was shielded from light (by covering the reaction vessel with aluminum foil) and stirred vigorously at rt for 48 h. At which point, the reaction mixture was cooled in an ice-water bath and water (200 mL, ice-water mixture) was slowly added to the reaction mixture. Caution: exotherm or rapid increase in temperature observed over the course of the addition. The resulting precipitate was separated from the mother liquor by filtration and then dissolved in acetone (200–300 mL). The insoluble material was removed by filtration. The mother liquor was dried over Na2SO4 and concentrated under reduced pressure to afford the title compound S2 (12 g, 73%) as a yellow solid, in sufficient purity to use in the next step: 1H NMR (500 MHz, DMSO-d6) δ 8.07 (d, J = 8.9 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 3.91 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 165.8 (C), 150.6 (C), 143.0 (CH), 138.7 (C), 134.1 (C), 117.1 (CH), 81.6 (C), 57.2 (CH3); IR (film) 3074, ~3000 (broad), 2947, 1713; HRMS (ESI) m/z calcd for C8H6INO5Na (M+Na)+ 345.9189, found 345.9188; mp: 178–181 °C.</p><!><p>This procedure is a modification of a procedure reported by Fuji and co-workers.67 To a solution of 6-iodo-3-methoxy-2-nitrobenzoic acid (S2) (14.5 g, 44.9 mmol), Et2S (40 mL) and CH2Cl2 (400 mL) was added AlCl3 (21.0 g, 157 mmol) in a portion-wise fashion. The reaction mixture was stirred at 40 °C for 48 h (the reaction progress was monitored by 1H NMR of aliquots sampled during the course of the reaction), then allowed to cool to rt and concentrated under reduced pressure (to approximately 1/3 volume). The crude residue was poured into a mixture of EtOAc (600 mL) and aqueous solution of HCl (0.125 M, 450 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was crystallized (in some cases, precipitated) from CH2Cl2 (100 mL) in order to remove minor amounts of the hydrodehalogenated by-product, 3-hydroxy-2-nitrobenzoic acid (S4) [602-00-6] (1:10, S4/S3 by 1H NMR). After removal of the mother liquor by filtration, the product was redissolved in Et2O to remove residual, purple-colored insoluble material, which was filtered away. The filtrate was concentrated under reduced pressure to afford the title compound S3 (6.10 g, second crop: 2.10 g; combined 59%) as a tan solid: 1H NMR (500 MHz, CD3OD) δ 7.89 (d, J = 8.8 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 168.7 (C), 152.5 (C), 145.0 (CH), 138.6 (C), 137.2 (C), 122.4 (CH), 79.7 (C); IR (KBr) 3411, 3088, ~3000 (broad), 2898, 2654, 2590, 2526, 1701, 1600, 1442; HRMS (ESI) m/z calcd for C7H3INO5 (M–H)− 307.9056, found 307.9051; mp: 174–176 °C (dec.).</p><!><p>This procedure is a modification of a procedure reported by Fairfax and Yang.29 To a solution of 3-hydroxy-6-iodo-2-nitrobenzoic acid (S3) (7.3 g, 24 mmol) and THF (200 mL) was added a solution of Na2S2O4 (41 g, 240 mmol) and water (100 mL). The reaction mixture was stirred vigorously at 50 °C for 3–4 h, allowed to cool to rt, and EtOAc (300 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated under reduced pressure to afford the title compound 29 (4.6 g, 70%) in sufficient purity to use in the next step: 1H NMR (500 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.80 (br s, 2H), 6.93 (d, J = 8.2 Hz, 1H), 6.48 (d, J = 8.2 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 169.0 (C), 144.8 (C), 137.5 (C), 126.8 (CH), 121.4 (C), 116.4 (CH), 80.7 (C); IR (KBr) 3423, 3221, 2921, 1603; HRMS (ESI) m/z calcd for C7H7INO3 (M+H)+ 279.9471, found 279.9479; mp: 180–182 °C (dec.).</p><!><p>To a solution of 3-hydroxy-4-iodoanthranilic acid 29 (50 mg, 0.18 mmol) in MeOH (4 mL) was added 1,4-benzoquinone (33 mg, 0.30 mmol, freshly recrystallized from petroleum ether).11,32 The reaction mixture was maintained at 50 °C for 3 h (the reaction was shielded from light by covering the reaction vessel with aluminum foil), followed by placement in a freezer (−20 °C) for 12 h. The resulting suspension was concentrated under reduced pressure (to approximately 1/2 volume), then filtered to separate the mother liquor from the precipitate, which was subsequently washed with EtOAc/hexane (10–20 mL, 1:1) to afford the title compound S5 (25 mg, 66%) as an orange-red solid with no further purification: 1H NMR (500 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.92 (s, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 6.62 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 178.0 (C), 168.7 (C), 167.9 (C), 152.7 (C), 150.9 (C), 148.1 (C), 142.1 (C), 138.6 (CH), 137.7 (C), 127.7 (C), 118.3 (CH), 105.5 (CH), 92.4 (C), 87.0 (C); IR (KBr) 3402, 3282, 3082, 2917, 1721, 1645, 1578; HRMS (ESI) m/z calcd for C14H6IN2O6 (M–H)− 424.9271, found 424.9274.</p><!><p>To a suspension of 3-hydroxy-4-iodoanthranilic acid 29 (4.4 g, 16 mmol) in a co-solvent mixture of PhMe and MeOH (160, 50 mL, respectively) was slowly added trimethylsilyl diazomethane (TMSCHN2) (8.8 mL, 18 mmol, 2.0 M solution in hexane) while stirring. Caution: evolution of N2(g) over the course of the addition. After 30 min, the resulting solution was poured into a mixture of EtOAc (50 mL) and water (25 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure to afford methyl 3-hydroxy-4-iodoanthranilate (30), which was used immediately in the following transformation: TLC Rf 0.45 (3:7 EtOAc/hexanes). To a solution of 2-aminophenol 30 (16 mmol) in MeOH (160 mL) was added 1,4-benzoquinone (3.0 g, 27 mmol, freshly recrystallized from petroleum ether).11,32 The reaction mixture was maintained at 50 °C for 3 h (the reaction was shielded from light by covering the reaction vessel with aluminum foil), followed by placement in a freezer (−20 °C) for 12h. The resulting suspension was concentrated under reduced pressure (to approximately 1/2 volume), then filtered to separate the mother liquor from the precipitate, which was subsequently washed with EtOAc/hexane (100 mL, 1:1) to afford the title compound 31 (2.0 g, 56%) as an orange-red solid of sufficient purity to use in the next step: 1H NMR (500 MHz, DMSO-d6) δ 8.00 (br s, 2H), 7.87 (d, J = 7.4 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 6.49 (s, 1H), 3.92 (s, 3H), 3.80 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 178.4 (C), 167.2 (C), 167.1 (C), 148.9 (C), 148.4 (C), 146.2 (C), 141.3 (C), 138.6 (C), 138.0 (CH), 131.6 (C), 118.4 (CH), 104.3 (CH), 99.0 (C), 86.6 (C), 52.7 (CH3), 51.6 (CH3); IR (film) 3446, 3326, 3075, 2948, 2922, 2854, 1730, 1672, 1641, 1575; TLC Rf 0.36 (3:7 EtOAc/hexanes), 0.52 (3:7 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C16H11IN2O6Na (M+Na)+ 476.9560, found 476.9573; mp: 233–235 °C.</p><!><p>To a solution of 2-aminophenoxazinone 31 (300 mg, 0.66 mmol) in THF (13 mL) were added sequentially Boc2O (170 mg, 0.79 mmol) and DMAP (16 mg, 0.13 mmol). After 2 h, the reaction mixture was poured into a mixture of EtOAc (60 mL) and saturated aqueous NH4Cl (30 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 30 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (1:3 EtOAc/hexanes) to afford the title compound 32 (200 mg, 54%; the remainder of mass balance consisted of a co-polar mixture of starting material 31 and the di-tert-butoxycarbonyl derivative 33) as an orange/red solid: 1H NMR (500 MHz, CDCl3) δ 8.01 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 6.48 (s, 1H), 4.03 (s, 3H), 3.92 (s, 3H), 1.50 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 179.0 (C), 166.8 (C), 164.4 (C), 151.1 (C), 148.1 (C), 147.4 (C), 142.6 (C), 141.3 (CH), 141.0 (C), 135.0 (C), 131.9 (C), 118.4 (CH), 116.9 (C), 105.2 (CH), 86.4 (C), 83.0 (C), 53.3 (CH3), 52.5 (CH3), 28.2 (CH3); IR (film) 3330, 2980, 2950, 2917, 2849, 1737, 1631, 1509, 1492; TLC Rf 0.64 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C21H19IN2O8Na (M+Na)+ 577.0084, found 577.0086.</p><!><p>To a THF (88 mL) solution of 2-aminophenoxazinone 31 (2.0 g, 4.4 mmol) was sequentially added Boc2O (2.3 g, 11 mmol) and DMAP (50 mg, 0.10 mmol). After 2 h (in some cases, additional Boc2O was added to drive the reaction to completion), the reaction mixture was poured into a mixture of EtOAc (300 mL) and saturated aqueous NH4Cl (200 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (1:3 EtOAc/hexanes) to afford the title compound 33 (2.2 g, 76%) as a red solid: 1H NMR (500 MHz, CDCl3) δ 8.01 (s, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 6.42 (s, 1H), 3.99 (s, 3H), 3.91 (s, 3H), 1.41 (s, 18H); 13C NMR (125 MHz, CDCl3) δ 179.9 (C), 166.5 (C), 162.6 (C), 149.2 (C), 148.2 (C), 147.5 (C), 143.5 (C), 142.8 (CH), 141.4 (C), 138.5 (C), 133.8 (C), 131.3 (C), 118.7 (CH), 107.2 (CH), 86.2 (C), 84.2 (C), 53.3 (CH3), 53.0 (CH3), 27.9 (CH3); IR (film) 2980, 2953, 1801, 1745, 1640; TLC Rf 0.48 (3:7 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C26H27IN2O10Na (M+Na)+677.0608, found 677.0611.</p><!><p>The components of this reaction were combined in a screw-top vial inside a N2-filled glovebox. The reaction vial was sealed with a Teflon®-lined cap, then brought outside the glovebox and heated in an aluminum block for the period of time indicated. The reaction vessel was charged with indoline 25 (10 mg, 21 μmol), phenoxazinone iodide 33 (30 mg, 46 μmol), copper(I) thiophene-2-carboxylate (CuTC)36 (12 mg, 62 μmol) and K2CO3 (11 mg, 83 μmol), then PhMe (200 μL) was added by syringe. The heterogeneous mixture was stirred at 90 °C for 36 h. After being allowed to cool to rt, the reaction mixture was passed directly through a short plug of silica gel (eluting with EtOAc) to remove the metal salts, then concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:19 EtOAc/hexanes → 1:3 EtOAc/hexanes) to afford the title compound (−)-35 (14 mg, 67%) as a red solid: [α]D
24 −68.7, [α]577
24 −72.2 (c = 0.075, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.18 (d, J = 7.0 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.6–7.5 (comp, 2H), 7.35 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 7.3 Hz, 1H), 7.05 (d, J = 4.1 Hz, 1H), 6.96 (d, J = 7.6, 1H), 6.83 (s, 1H), 6.78 (m, 1H), 6.75 (s, 1H), 6.45 (s, 1H), 6.37 (d, J = 7.0 Hz, 1H), 3.84 (s, 3H), 3.63 (s, 3H), 3.41 (s, 3H), 1.70 (s, 9H), 1.43 (s, 18H); 13C NMR (125 MHz, CDCl3) δ 180.1 (C), 165.2 (C), 162.6 (C), 157.2 (C), 157.0 (C), 150.4 (C), 149.6 (C), 149.1 (C), 148.7 (C), 148.5 (CH), 147.3 (C), 143.0 (CH), 138.3 (C), 136.5 (C), 136.3 (C), 135.8 (C), 133.8 (C), 133.5 (CH), 131.6 (C), 131.0 (C), 129.8 (CH), 127.8 (C), 126.9 (C), 126.3 (CH), 125.4 (CH), 124.9 (CH), 124.1 (CH), 123.4 (CH), 120.8 (CH), 119.5 (2 x CH), 119.1 (C), 116.2 (CH), 108.2 (CH), 106.9 (CH), 88.0 (CH), 85.0 (C), 84.2 (C), 59.3 (C), 52.9 (2 x CH3), 28.4 (CH3), 27.9 (CH3), 27.7 (CH3); IR (film) 3056, 2980, 2952, 2933, 1804, 1741, 1714, 1692, 1642; TLC Rf 0.41 (4:6 EtOAc/hexanes); HRMS (ESI) m/z calcd for C53H50N6O15Na (M+Na)+ 1033.3232, found 1033.3240.</p><!><p>This procedure is a modification of a procedure reported by Overman and coworkers.12 A THF (11 mL) solution of indoline 25 (260 mg, 0.54 mmol) was cooled to −78 °C and methylmagnesium chloride (540 μL, 1.6 mmol, 2.9 M solution in THF) was added slowly. The reaction mixture was maintained at −78 °C for 3 h and AcOH (120 μL, 2.0 mmol) was added. The cooling bath was removed and the reaction mixture was allowed to warm to rt. The solvent was removed under reduced pressure and H2O (15 mL) was added to the concentrate. The aqueous phase was extracted with EtOAc (3 x 15 mL) and the combined organic layers were washed with brine (15 mL), dried over Na2SO4, then concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:3 EtOAc/hexanes → 2:3 EtOAc/hexanes) to afford the title compound S6 (190 mg, 71%, 3:2 mixture of alcohol epimers) as an amorphous colorless solid: 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 6.4 Hz, 0.6H), 8.08 (d, J = 6.4 Hz, 0.4H), 7.49 (s, 0.6H), 7.46 (s, 0.4H), 7.27 (m, 0.4H), 7.21 (t, J = 7.7 Hz, 0.4H), 7.14 (t, J = 7.5 Hz, 0.4H), 7.08–7.00 comp, 3.8H), 6.94 (d, J = 7.5 Hz, 1H), 6.77–6.72 (comp, 1H), 6.67 (d, J = 7.9 Hz, 0.6H), 6.67 (d, J = 7.8 Hz, 0.4H), 6.59 (s, 1H), 6.57 (s, 1H), 6.20 (d, J = 2.6 Hz, 0.6H), 6.12 (app s, 0.4H), 5.42 (app s, 0.4H), 5.38 (app s, 0.6H), 4.29 (s, 0.6H), 4.25 (s, 0.4H), 3.10 (s, 1.8H), 3.09 (s, 1.2H), 1.73 (s, 3H), 1.68–1.66 (comp, 12H); 13C NMR (125 MHz, CDCl3) δ 165.0 (C), 164.5 (C), 155.7 (2 x C), 149.8 (2 x C), 148.0 (C), 147.9 (C), 136.4 (2 x C), 132.5 (C), 132.2 (C), 129.7 (C), 129.42 (CH), 129.38 (CH), 129.2 (C), 128.1 (2 x C), 125.1 (CH), 124.9 (CH), 124.8 (CH), 124.7 (CH), 123.3 (2 x CH), 123.1 (CH), 123.0 (CH), 121.8 (CH), 121.5 (C), 121.2 (CH), 121.1 (C), 120.2 (CH), 119.9 (CH), 119.8 (CH), 115.8 (CH), 115.7 (CH), 110.0 (CH), 109.8 (CH), 85.3 (C), 84.9 (C) 84.6 (C), 84.5 (C), 83.2 (CH), 83.1 (CH), 59.8 (C), 59.5 (C), 28.44 (CH3), 28.42 (CH3), 27.5 (CH3), 27.3 (CH3), 26.5 (CH3), 26.1 (CH3); IR (film) 3339, 2979, 1734, 1675, 1642, 1453; TLC Rf 0.15 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C28H28N4O5Na (M+Na)+ 523.1957, found 523.1953.</p><!><p>This procedure is a modification of a procedure reported by Overman and coworkers.12 To a solution of alcohols S6 (190 mg, 0.38 mmol), DMAP (46 mg, 0.38 mmol), and triethylamine (0.53 mL, 3.8 mmol) in DMF (4.0 mL) maintained at 0 °C was added TBSOTf (0.52 mL, 2.3 mmol) slowly. The cold bath was removed and the reaction mixture was maintained at rt for 12 h, whereupon EtOAc (20 mL) was added followed by saturated aqueous NH4Cl (20 mL). The mixture was transferred to a separatory funnel, the layers were separated, and the aqueous phase was further extracted with EtOAc (2 x 15 mL). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:3 EtOAc/hexanes → 2:3 EtOAc/hexanes) to afford the title compounds 36 as colorless solids (125 mg of the major epimer 36a, 105 mg of the minor epimer 36b; 98% overall yield, 1.2:1.0 ratio of C3-epimers). Data for the major, C3α-OTBS epimer, (+)-36a: [α]D
24 +187, [α]577
24 +195, [α]546
24 +220, [α]435
24 +352 (c = 0.54, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.14 (d, J = 7.2 Hz, 1H), 7.46 (s, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.15–7.05 (comp, 3H), 7.02 (d, J = 7.4 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 6.74 (t, J = 7.4 Hz, 1H), 6.57 (s, 1H), 6.16 (s, 1H), 5.35 (s, 1H), 3.07 (s, 3H), 1.74 (s, 3H), 1.67 (s, 9H), 0.86 (s, 9H), 0.10 (s 3H), 0.01 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 163.0 (C), 156.0 (C), 149.7 (C), 148.1 (C), 136.4 (C), 132.6 (C), 129.6 (C), 129.3 (CH), 128.2 (C), 125.0 (CH), 124.7 (CH), 123.5 (CH), 123.0 (CH), 121.3 (C), 120.9 (CH), 120.1 (2 x CH), 115.7 (CH), 109.9 (CH), 87.1 (C), 84.3 (C) 83.4 (CH), 59.5 (C), 28.4 (CH3), 27.8 (CH3), 26.5 (CH3), 25.8 (CH3), 18.4 (C), −3.6 (CH3), −3.9 (CH3); IR (film) 3356, 3054, 2953, 2931, 1736, 1682, 1649, 1607, 1453; TLC Rf 0.38 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C34H42N4O5SiNa (M+Na)+ 637.2822, found 637.2820. Data for minor, C3β-OTBS epimer, (+)-36b: [α]D
24 +118, [α]577
24 +121, [α]546
24 +134, [α]435
24 +155 (c = 0.54, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.13 (d, J = 6.5 Hz, 1H), 7.46 (s, 1H), 7.27 (t, J = 7.1 Hz, 1H), 7.15–7.05 comp, 3H), 6.74 (d, J = 7.4, 1H), 6.74 (d, J = 7.7 Hz, 1H), 6.73 (t, J = 7.6 Hz, 1H), 6.59 (s, 1H), 6.21 (s, 1H), 5.38 (s, 1H), 3.07 (s, 3H), 1.73 (s, 3H), 1.67 (s, 9H), 0.86 (s, 9H), 0.12 (s 3H), 0.01 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 163.1 (C), 155.6 (C), 149.7 (C), 148.0 (C), 136.4 (C), 132.5 (C), 129.7 (C), 129.3 (CH), 128.2 (C), 125.0 (CH), 124.7 (CH), 123.4 (CH), 123.0 (CH), 121.3 (C), 121.0 (CH), 120.0 (CH), 119.9 (CH), 115.7 (CH), 109.7 (CH), 87.1 (C), 84.4 (C) 83.4 (CH), 59.3 (C), 28.4 (CH3), 27.9 (CH3), 27.1 (CH3), 25.8 (CH3), 18.5 (C), −3.5 (CH3), −3.8 (CH3); IR (film) 3365, 3064, 2979, 1736, 1686, 1606, 1452; TLC Rf 0.44 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C34H42N4O5SiNa (M+Na)+ 637.2822, found 637.2825.</p><!><p>The components of this reaction were combined in a screw-top vial inside a N2-filled glovebox. The reaction vial was sealed with a Teflon®-lined cap, then brought outside the glovebox and heated in an aluminum block for the period of time indicated. The reaction vessel was charged with indoline 36b (40 mg, 65 μmol), phenoxazinone iodide 33 (130 mg, 200 μmol), copper(I) thiophene-2-carboxylate (CuTC)36 (63 mg, 330 μmol) and K2CO3 (50 mg, 360 μmol), then PhMe (2.6 mL) was added by syringe. The heterogeneous mixture was stirred at 90 °C for 48 h. After being allowed to cool to rt, the reaction mixture was passed directly through a short plug of silica gel (eluting with EtOAc) to remove the metal salts, then concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:19 EtOAc/hexanes → 1:3 EtOAc/hexanes) to afford the title compound 37b (51 mg, 68%) as a red solid: [α]D
24 −150 (c = 0.05, MeOH); 37b was observed as a 9:1 mixture of atropisomers by NMR at 298 K (subsequent data is provided for the major isomer only); 1H NMR (500 MHz, acetone-d6) δ 8.20 (d, J = 8.3 Hz, 1H), 7.98 (br d, 1H), 7.80–7.70 (comp, 2H), 7.36 (m, 1H), 7.20–7.10 (comp, 4H), 6.77 (t, J = 6.9 Hz, 1H), 6.67 (s, 1H), 6.56 (s, 1H), 6.52 (s, 1H), 6.38 (d, J = 7.9 Hz, 1H), 3.89 (s, 1H), 3.58 (br s, 3H), 3.09 (s, 1H), 1.68 (s, 9H), 1.55 (s, 3H), 1.39 (s, 18H), 0.83 (s, 9H), −0.08 (s, 3H), −0.35 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 180.6 (C), 165.7 (C), 165.4 (C), 163.6 (2 x C), 155.9 (C), 150.3 (C), 150.0 (C), 149.8 (C), 149.6 (C), 147.6 (C), 143.8 (C), 138.9 (C), 137.3 (C), 136.5 (C), 134.8 (CH), 133.4 (C), 131.7 (C), 129.7 (CH), 129.0 (C), 125.66 (CH), 125.59 (CH), 125.4 (C), 123.9 (CH), 121.1 (C), 121.0 (CH), 120.9 (CH), 120.8 (CH), 119.9 (CH), 116.5 (CH), 109.0 (CH), 106.9 (CH), 88.7 (CH), 88.0 (C), 85.1 (C), 83.7 (C), 59.9 (C), 53.2 (CH3), 52.7 (CH3), 28.3 (CH3), 28.0 (CH3), 27.6 (CH3), 27.4 (CH3), 26.3 (CH3), 19.3 (C), −2.7 (CH3), −3.4 (CH3);68 IR (film) 2977, 2953, 2917, 2850, 1806, 1743, 1701, 1643; TLC Rf 0.35 (3:7 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C60H68N6O15SiNa (M+Na)+ 1163.4409, found 1163.4402.</p><!><p>These procedures are modifications of procedures reported by Overman and coworkers.12 A flask was charged with AD-mix-α (150 mg),41 (−)-37b (15 mg, 13 μmol), methane sulfonamide (6.3 mg, 66 μmol), and (DHQ)2PHAL (1.3 mg, 1.6 μmol), then t-BuOH/H2O/acetone (3:2:1, 3 mL) was added and additional K2OsO4•H2O (1.2 mg, 3.3 μmol).69 The resulting heterogeneous mixture was stirred vigorously at rt for 4 h, the reaction was cooled to 0 °C and solid Na2SO3 (450 mg) was added. The cold bath was removed and the mixture was stirred for 1 h at rt. Water (5 mL) was added to the reaction mixture, which was transferred to a separatory funnel then the aqueous phase was extracted with EtOAc (3 x 8 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:10 EtOAc/hexanes → 2:3 EtOAc/hexanes) to afford the title compound (−)-38b (10 mg, 65%, >10:1 dr) as a red solid. The corresponding reaction of (−)-37b (10 mg, 8.8 μmol) using AD-mix-β (with additional K2OsO4•H2O and (DHQD)2PHAL) provided isomers (−)-38b and S7 (8 mg, 77%, 7:1 dr). The analogous reaction of (−)-37b (10 mg, 8.8 μmol) using OsO4 (22 mg, 2.1 μmol, 2.5 wt % in t-BuOH) and NMO (2 mg, 17 μmol) in acetone/H2O (4:1, 2 mL) provided isomers (−)-38b and S7 (5 mg, 49%, reaction was quenched prior to full consumption of starting material, 5:1 dr) as red solids. Data for the minor isomer S7:70 1H NMR (500 MHz, acetone-d6) δ 8.55 (s 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 7.10 (s, 1H), 7.09 (t, 1H), 6.94 (d, J = 7.3 Hz, 1H), 6.77 (s, 1H), 6.70 (t, J = 7.4 Hz, 1H), 6.53 (s, 1H), 6.30 (d, J = 7.9 Hz, 1H), 6.00 (s, 1H), 5.52 (d, J = 5.7 Hz, 1H), 4.74 (s, 1H), 3.90 (s, 3H), 3.64 (s, 3H), 3.02 (s, 3H), 1.70 (s, 12H), 1.40 (s, 18H), 0.78 (s, 9H), 0.07 (s, 3H), −0.47 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 180.6 (C), 171.2 (C), 166.5 (C), 165.9 (C), 163.7 (C), 150.6 (C), 149.9 (C), 149.5 (C), 149.1 (C), 147.5 (C), 143.5 (C), 138.83 (C), 138.75 (C), 138.0 (C), 136.0 (C), 134.8 (C), 134.0 (C), 133.1 (CH), 131.6 (C), 131.2 (C), 129.5 (CH), 129.1 (CH), 124.7 (CH), 124.4 (CH), 123.4 (CH), 121.1 (CH), 120.7 (CH), 119.8 (CH), 116.9 (C), 116.4 (CH), 109.9 (CH), 106.9 (CH), 88.3 (C), 86.5 (CH), 85.5 (C), 84.6 (C), 83.7 (C), 82.4 (CH), 57.7 (C), 53.2 (CH3), 52.7 (CH3), 28.4 (CH3), 28.0 (CH3), 27.4 (CH3), 26.3 (2 x CH3), 19.4 (C), −2.46 (CH3), −3.39 (CH3); TLC Rf 0.07 (2:3 EtOAc/hexanes); LRMS (ESI) m/z calcd for C60H70N6O17SiNa (M+Na)+ 1197.4, found 1197.5. Data for the major isomer (−)-38b: [α]D
24 −47.6 (c = 0.03, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.17 (d, J = 8.5 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 7.3 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.7 Hz, 1H), 6.76 (d, J = 7.5 Hz, 1H), 6.50 (s, 1H), 6.41 (s, 1H), 6.34 (d, J = 7.5 Hz, 1H), 5.18 (br s, 1H), 5.11 (app s, 1H), 4.72 (br s, 1H), 3.88 (s, 3H), 3.73 (s, 3H), 2.98 (s, 3H), 1.67 (s, 9H), 1.65 (s, 3H), 1.39 (s, 18H), 0.89 (s, 9H), 0.15 (s, 3H), −0.01 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 180.6 (C), 168.5 (C), 166.7 (C), 166.2 (C), 163.7 (C), 150.3 (C), 150.2 (C), 149.8 (C), 149.4 (C), 147.6 (C), 143.6 (C), 138.9 (C), 137.2 (C), 136.6 (C), 136.5 (CH), 135.7 (C), 134.9 (C), 131.8 (C), 129.5 (CH), 129.4 (C), 129.3 (C), 128.7 (CH), 125.4 (CH), 125.1 (CH), 123.8 (CH), 123.0 (C), 121.3 (CH), 119.7 (CH), 119.0 (CH), 116.2 (CH), 108.3 (CH), 106.8 (CH), 86.9 (C), 84.9 (C), 84.5 (C), 83.7 (C), 79.9 (2 x CH), 63.3 (CH3), 55.6 (C), 52.5 (CH3), 28.3 (CH3), 28.0 (CH3), 27.9 (CH3), 26.1 (CH3), 25.0 (CH3), 18.9 (C), −2.12 (CH3), −2.96 (CH3); IR (film) 3457, 2978, 2953, 2932, 2857, 1806, 1740, 1699, 1643, 1371; TLC Rf 0.20 (2:3 EtOAc/hexanes); HRMS (ESI) m/z calcd for C60H70N6O17SiNa (M+Na)+ 1197.4464, found 1197.4486. Note, for comparison, the 1H NMR of (−)-38b was also obtained in CDCl3: 1H NMR (500 MHz, CDCl3) δ 8.14 (br d, J = 5.7 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.60 (s, 1H), 7.51 (br d, 1H), 7.36 (br d, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.18–7.12 (comp, 2H), 6.83 (t, J = 7.4 Hz, 1H), 6.43 (s, 1H), 6.40 (br s, 1H), 4.96 (s, 1H), 3.87 (s, 3H), 3.81 (br s, 1H), 3.67 (br s, 1H), 3.17 (s, 3H), 3.01 (s, 3H), 1.67 (s, 9H), 1.65 (s, 3H), 1.41 (s, 18H), 0.88 (s, 9H), 0.14 (s, 3H), 0.04 (s, 3H).</p><!><p>Following a similar procedure as described for the formation of (−)-37b, indoline (+)-36a (40 mg, 65 μmol) and phenoxazinone iodide 33 (130 mg, 200 μmol) were converted to intermediate 37a (53 mg, 72%) as a red solid: TLC Rf 0.25 (3:7 EtOAc/hexanes, plate was eluted twice), 37a is co-polar with the hydrodehalogenated byproduct of 33 and the yield is based on the corresponding ratio obtained upon 1H NMR analysis. Analytically pure samples of 37a were not obtained for characterization purposes. Following a similar procedure as described for formation of 38b, dihydroxylation of 37a (15 mg, 13 μmol) using AD-mix-α provided the title compound (+)-38a (15 mg, 97%, >10:1 dr) as a red solid. The corresponding reaction of 37a (10 mg, 8.8 μmol) using AD-mix-β (with additional K2OsO4•H2O and (DHQD)2PHAL) provided (+)-38a (8 mg, 77%, >10:1 dr). The analogous reaction of 37a (10 mg, 8.8 μmol) using OsO4 (22 mg, 2.1 μmol, 2.5 wt % in t-BuOH) and NMO (2 mg, 17 μmol) in acetone/H2O (4:1, 2 mL) provided (+)-38a (8 mg, 77%, >10:1 dr): Subsequent data is provided for the major isomer only; [α]D
24 +61.8 (c = 0.05, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.14 (br d, J = 7.2 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.69 (s, 1H), 7.42 (d, J = 8.7 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.34–7.32 (comp, 2H), 7.17 (app t, J = 7.1 Hz, 1H), 7.15 (app t, J = 6.9 Hz, 1H), 6.81 (app t, J = 7.4 Hz, 1H), 6.45 (comp, 1H), 6.44 (s, 1H), 6.43 (s, 1H), 5.01 (d, J = 2.5 Hz, 1H), 3.98 (br s, 1H), 3.88 (s, 3H), 3.73 (s, 1H), 3.46 (s, 3H), 2.96 (s, 3H), 1.68 (s, 9H), 1.53 (s, 3H), 1.41 (s, 18H), 0.80 (s, 9H), −0.06 (s, 3H), −0.19 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 180.0 (C), 167.4 (C), 166.2 (C), 165.4 (C), 162.8 (C), 149.8 (C), 149.4 (C), 149.2 (C), 148.5 (C), 146.7 (C), 142.7 (C), 138.3 (C), 137.0 (C), 136.3 (C), 136.0 (C), 134.8 (CH), 134.0 (C), 131.3 (C), 130.2 (CH), 128.2 (C), 127.9 (CH), 127.8 (C), 125.0 (CH), 123.7 (CH), 123.4 (CH), 121.1 (C), 120.9 (CH), 120.4 (CH), 117.3 (CH), 115.6 (CH), 109.3 (CH), 106.8 (CH), 85.9 (C), 84.4 (C), 84.0 (C), 83.5 (C), 82.7 (CH), 78.7 (CH), 54.4 (C), 53.0 (CH3), 52.7 (CH3), 28.5 (CH3), 28.4 (CH3), 27.9 (CH3), 27.0 (CH3), 26.1 (CH3), 19.0 (C), −2.91 (CH3), −3.24 (CH3); IR (film) 3443, 2979, 2951, 2931, 2855, 1805, 1741, 1643; TLC Rf 0.16 (2:3 EtOAc/hexanes); HRMS (ESI) m/z calcd for C60H70N6O17SiNa (M+Na)+ 1197.4464, found 1197.4436.</p><!><p>To a solution of intermediate 39a12 (40 mg, 48 μmol) in CH2Cl2 maintained at −78 °Cwas added TfOH (84 μL, 48 μmol, 5% solution in CH2Cl2). The reaction mixture was warmed to −40 °C then maintained at −40 °C for 4 h (note: reaction times may vary, close monitoring was required to prevent further reaction), whereupon it was quenched by the addition of pyridine (10 μL). After 5 min, the mixture was poured into a mixture of EtOAc (20 mL) and saturated aqueous NaHCO3 (10 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (10 mL). The combined organic layers were washed sequentially with H2O (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (1:9 EtOAc/hexanes → 1:3 EtOAc/hexanes) to afford the title compound (+)-40a (28 mg, 80%, in some of these reactions enamide 41 was also isolated as a minor by-product).</p><!><p>In multiple, sealed vials, intermediate 39a (5 × 40 mg, 0.048 mmol) was maintained (neat) at 175 °C in a pre-heated oil bath for 40–50 min. The reaction vessels were allowed to cool to rt, then THF (0.48 mL), DMAP (1 mg) and Boc2O (10 mg, 0.048 mmol, 0.1 mL of a pre-made 0.5 M THF solution) were added sequentially to the reaction mixtures. After 1 h, the reaction mixtures were combined and poured into a mixture of EtOAc (20 mL) and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (10 mL). The combined organic layers were washed successively with H2O (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (1:9 EtOAc/hexanes → 1:3 EtOAc/hexanes) to afford the title compound (+)-40a (125 mg, 71%; in some of these reactions the di-tert-butoxycarbonyl derivative (−)-S8a was also isolated as a minor by-product also as a colorless foam)71 as a colorless foam: [α]D
24 +93.8, [α]577
24 +95.0, [α]546
24 +100, [α]435
24 +178 (c 0.14, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.10 (br d, J = 5.5 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 6.92 (t, J = 7.4 Hz, 1H), 6.64 (d, J = 7.8 Hz, 1H), 6.52 (s, 1H), 6.26 (s, 1H), 4.86 (s, 1H), 3.02 (s, 3H), 1.90 (s, 3H), 1.61 (s, 9H), 1.45 (s, 3H), 1.37 (s, 3H), 0.98 (s, 9H), 0.37 (s 3H), 0.33 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 169.3 (C), 168.13 (C), 168.08 (C), 159.1 (C), 149.5 (C), 147.2 (C), 136.4 (C), 129.5 (C), 129.3 (CH), 128.4 (C), 126.7 (CH), 125.6 (CH), 124.6 (CH), 122.6 (CH), 121.4 (CH), 119.9 (CH), 117.4 (C), 115.6 (CH), 109.7 (CH), 90.6 (C), 87.2 (C) 84.3 (C), 82.4 (CH), 80.9 (CH), 58.5 (C), 28.3 (CH3), 28.0 (CH3), 25.9 (CH3), 24.0 (CH3), 20.5 (CH3), 20.4 (CH3), 18.8 (C), −2.4 (CH3), −3.1 (CH3); IR (film) 3372, 2954, 2930, 2856, 1741, 1682, 1609, 1370, 1216; TLC Rf 0.28 (1:3 EtOAc/hexanes); HRMS (ESI) m/z calcd for C38H48N4O9SiNa (M+Na)+ 755.3088, found 755.3083. The relative and absolute configuration of (+)-40a was confirmed by single crystal X-ray diffraction of an isopropanol solvate, mp 150–153°C, formed by slow evaporation of the title compound from isopropanol.43 Data for di-tert-butoxycarbonyl derivative (−)-S8a: [α]D
24 − 3.2, [α]577
24 −3.5, [α]546
24 −2.8, [α]435
24 −16.0 (c 0.16, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.11 (br d, J = 6.6 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.40 (s, 1H), 7.34 (s, 1H), 7.32–7.28 (comp, 3H), 7.25 (t, J = 7.6 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 6.11 (s, 1H), 3.03 (s, 3H), 1.90 (s, 3H), 1.61 (s, 9H), 1.49 (s, 9H), 1.43 (2 × s, 6H), 0.98 (s, 9H), 0.43 (s 3H), 0.40 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 168.3 (C), 168.0 (C), 166.9 (C), 158.9 (C), 151.9 (C), 149.4 (C), 141.9 (C), 136.4 (C), 131.7 (C), 129.3 (CH), 128.0 (C), 127.5 (CH), 125.6 (CH), 124.7 (CH), 123.4 (CH), 122.9 (CH), 121.5 (CH), 116.8 (C), 115.8 (CH), 115.5 (CH), 92.0 (C), 87.1 (C), 84.4 (C), 83.4 (CH), 82.8 (C), 80.1 (CH), 56.9 (C), 28.5 (CH3), 28.2 (CH3), 27.9 (CH3), 26.1 (CH3), 24.8 (CH3), 20.5 (CH3), 20.3 (CH3), 18.9 (C), −2.38 (CH3), −2.42 (CH3); IR (film) 2953, 2929, 2853, 1766, 1740, 1714, 1682, 1484, 1455, 1371; TLC Rf 0.35 (1:3 EtOAc/hexanes); HRMS (ESI) m/z calcd for C43H56N4O11SiNa (M+Na)+ 855.3613, found 855.3622.</p><!><p>Following a similar procedure as described in Method B for formation of (+)-40a, intermediate 39b12 (200 mg, 0.26 mmol) was converted to the title compound (+)-40b (110 mg, 63%; in some of these reactions the di-tert-butoxycarbonyl derivative (−)-S8b was also isolated as a minor by-product also as a colorless foam), which was isolated as a colorless foam: [α]D
24 +50.7, [α]577
24 +52.4, [α]546
24 +60.5, [α]435
24 +112, [α]405
24 +144 (c 0.10, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.16 (d, J = 8.3 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.52 (s, 1H), 6.49 (d, J = 2.8 Hz, 1H), 6.11 (s, 1H), 3.03 (s, 3H), 1.97 (s, 3H), 1.63 (s, 9H), 1.44 (s, 3H), 1.41 (s, 3H), 0.93 (s, 9H), 0.34 (s 3H), 0.16 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 168.7 (C), 168.5 (C), 167.7 (C), 160.2 (C), 150.0 (C), 149.9 (C), 137.1 (C), 130.2 (C), 130.0 (CH), 129.2 (C), 126.8 (CH), 125.7 (CH), 125.4 (CH), 123.3 (CH), 121.9 (CH), 119.6 (CH), 119.2 (C), 116.4 (CH), 110.6 (CH), 91.2 (C), 86.9 (C) 85.0 (C), 84.1 (CH), 82.2 (CH), 59.6 (C), 29.4 (CH3), 28.2 (CH3), 28.0 (CH3), 26.3 (CH3), 20.5 (CH3), 20.2 (CH3), 19.2 (C), −2.9 (CH3), −3.6 (CH3); IR (film) 3376, 2954, 2917, 2850, 1766, 1740, 1682, 1609, 1371, 1215; TLC Rf 0.25 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C38H48N4O9SiNa (M+Na)+ 755.3088, found 755.3098. Data for di-tert-Butoxycarbonyl derivative (−)-S8b: [α]D
24 −20.5, [α]577
24 − 31.4, [α]546
24 −37.8, [α]435
24 −82.9 (c 0.18, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.12 (br d, J = 6.4 Hz, 1H), 7.72 (br d, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.40 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.27 (s, 1H), 7.22–7.18 (comp, 2H), 6.11 (s, 1H), 3.04 (s, 3H), 1.94 (s, 3H), 1.61 (s, 9H), 1.55 (s, 9H), 1.45 (s, 3H), 1.33 (s, 3H), 0.92 (s, 9H), 0.33 (s 3H), 0.14 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 167.8 (C), 167.3 (C), 165.4 (C), 159.6 (C), 152.0 (C), 149.4 (C), 142.1 (C), 136.3 (C), 132.0 (C), 129.3 (CH), 128.0 (C), 127.1 (CH), 125.7 (CH), 124.7 (CH), 123.7 (CH), 122.7 (CH), 120.9 (CH), 116.7 (C), 116.4 (CH), 115.7 (CH), 91.1 (C), 85.9 (C), 84.5 (C), 83.9 (CH), 83.1 (C), 81.8 (CH), 56.9 (C), 28.5 (CH3), 28.4 (CH3), 28.3 (CH3), 28.1 (CH3), 25.9 (CH3), 20.5 (CH3), 20.3 (CH3), 18.6 (C), −3.2 (CH3), −4.1 (CH3); IR (film) 2954, 2917, 2850, 1768, 1737, 1716, 1685, 1476, 1455, 1370; TLC Rf 0.35 (1:3 EtOAc/hexanes); HRMS (ESI) m/z calcd for C43H56N4O11SiNa (M+Na)+ 855.3613, found 855.3600.</p><!><p>To a solution of intermediate 39b (100 mg, 0.12 mmol) in CH2Cl2 (2.5 mL) maintained at −78 °C was added TfOH (16 μL, 0.18 mmol, 5% solution in CH2Cl2). The reaction mixture was warmed to −40 °C, maintained at −40 °C for 4 h (note: reaction times may vary, monitoring required to prevent further cleavage of the remaining Boc group), then quenched by the addition of pyridine (20 μL). After 5 min, the mixture was poured into a mixture of EtOAc (20 mL) and saturated aqueous NaHCO3 (10 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (10 mL). The combined organic layers were washed successively with H2O (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. A second batch of 39b (220 mg, 0.26 mmol) was processed in a similar fashion. The crude product from both reactions were combined and purified by silica gel chromatography (1:9 EtOAc/hexanes → 1:3 EtOAc/hexanes) to afford the title compound (+)-41 (185 mg, 82%) as a colorless solid: [α]D
24 +36.0, [α]577
24 +46.1, [α]546
24 +44.9, [α]435
24 +87.9 (c 0.13, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.15 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.33 (t, J = 8.2 Hz, 1H), 7.24 (t, J = 7.3 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.51 (s, 1H), 6.38 (s, 1H), 6.19 (s, 1H), 5.83 (s, 1H), 5.21 (s 1H), 3.23 (s, 3H), 1.64 (s, 9H), 1.43 (s, 6H); 13C NMR (125 MHz, acetone-d6) ™169.9 (C), 168.7 (C), 160.6 (C), 159.0 (C), 150.0 (C), 149.8 (C), 139.1 (C), 137.1 (C), 130.1 (CH), 129.3 (C), 126.8 (CH), 125.9 (CH), 125.5 (CH), 123.5 (CH), 122.2 (CH), 119.7 (CH), 119.0 (C), 116.3 (CH), 110.8 (CH), 103.8 (CH2), 91.9 (C), 85.0 (C), 83.9 (CH), 81.7 (CH), 58.9 (C), 28.2 (CH3), 20.30 (CH3), 20.28 (CH3);72 IR (film) 3374, 3055, 2979, 2917, 2849, 1740, 1695, 1615, 1371; TLC Rf 0.28 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C32H32N4O8Na (M+Na)+ 623.2118, found 623.2111.</p><!><p>The components of this reaction were combined in a screw-top vial inside a nitrogen-filled glovebox. The reaction vial was sealed with a Teflon®-lined cap, then brought outside the glovebox and heated in an aluminum block for the period of time indicated. The reaction vessel was charged with indoline 40a (10 mg, 14 μmol), phenoxazinone iodide 33 (27 mg, 42 μmol), copper(I) acetate (CuOAc) (10 mg, 82 μmol) and KOAc (3.0 mg, 31 μmol), then PhMe (200 μL) was added by syringe. The reaction mixture was maintained at 90 °C for 8 h. After being allowed to cool to rt, the reaction mixture was passed directly through a short plug of silica gel (eluting with EtOAc) to remove the metal salts, then concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:19 EtOAc/hexanes → 1:3 EtOAc/hexanes) to afford the title compound (+)-42a (10 mg, 58%) as a red solid: [α]D
24 +43.9 (c 0.063, MeOH); 42a was observed as a 3:1 mixture of atropisomers by NMR at 298 K; 1H NMR (500 MHz, acetone-d6) δ 8.21 (d, J = 8.3 Hz, 1.33H), 7.76 (d, J = 7.5 Hz, 1.33H), 7.67 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.56 (s, 1H), 7.53 (s, 0.33H), 7.51 (d, J = 8.1 Hz, 0.33H), 7.41–7.38 (comp, 1.66H), 7.31 (d, J = 8.8 Hz, 1H), 7.30–7.19 (comp, 3H), 7.08 (t, J = 7.4 Hz, 1H), 6.92 (d, J = 8.8 Hz, 0.33H), 6.89 (s, 1H), 6.72 (s, 0.33H), 6.69 (d, J = 8.0 Hz, 0.33H), 6.48 (s, 1H), 6.44 (s, 0.33H), 6.31 (d, J = 7.9 Hz, 1H), 6.20 (s, 1H), 6.11 (s, 0.33H), 3.94 (s, 1H), 3.89 (s, 1H), 3.79 (s, 3H), 3.04 (s, 1H), 3.02 (s, 3H), 2.88 (s, 3H), 2.00 (s, 1H), 1.89 (s, 3H), 1.65 (s, 12H), 1.52 (s, 4H), 1.39 (s, 3H), 1.39 (s, 6H), 1.37 (2 × s, 19H), 1.01 (s, 1H), 0.98 (s, 9H), 0.40 (s, 1H), 0.35 (s, 1H), 0.32 (s 3H), 0.30 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 180.6 (C, major), 170.0 (C, major), 169.6 (C, minor), 168.5 (C, major), 168.3 (C, minor), 168.2 (C, major), 167.4 (C, minor), 166.4 (C, minor), 165.4 (C, major), 163.73 (C, minor), 163.65 (C, major), 159.3 (C, minor), 159.2 (C, major), 151.0 (C, minor), 150.3 (C, minor), 150.2 (C, major), 150.0 (C, major), 149.8 (C, major), 148.8 (C, major), 148.4 (C, minor), 148.2 (C, major), 144.2 (C, major), 144.0 (C, minor), 140.3 (C, minor), 139.0 (C, major), 137.9 (C, minor), 137.33 (C, minor), 137.27 (C, major), 137.0 (C, major), 136.1 (CH, major), 134.83 (CH, minor), 134.75 (C, major), 134.71 (C, major), 131.84 (C, major), 131.79 (C, minor), 131.3 (C, minor), 130.22 (C, major), 130.19 (CH, minor), 130.1 (CH, major), 129.1 (C, major), 128.8 (C, minor), 128.6 (CH, minor), 128.1 (CH, major), 126.1 (CH, major), 125.8 (CH, minor), 125.6 (C, minor), 125.5 (CH, major), 123.6 (CH, major), 123.5 (CH, minor), 122.5 (CH, minor), 122.3 (CH, major), 122.1 (CH, minor), 120.5 (CH, major), 120.4 (CH, minor), 119.4 (CH, major), 118.5 (C, minor), 118.1 (C, major), 116.5 (CH, minor), 116.4 (CH, major), 114.3 (CH, minor), 108.8 (CH, major), 106.8 (CH, major), 106.7 (CH, minor), 92.0 (C, major), 91.5 (C, minor), 88.5 (CH, minor), 88.2 (C, minor), 87.9 (C, major), 86.7 (CH, major), 85.34 (C, minor), 85.25 (C, major), 83.74 (CH, minor), 83.70 (C, major), 83.4 (CH, major), 59.6 (C, minor), 58.5 (C, major), 53.3 (CH3, minor), 53.2 (CH3, minor), 53.1 (CH3, major), 51.8 (CH3, major), 28.3 (CH3, major), 28.2 (CH3, major), 27.93 (CH3, minor), 27.92 (CH3, major), 26.35 (CH3, minor), 26.32 (CH3, major), 24.83 (CH3, minor), 24.80 (CH3, major), 20.5 (CH3, major), 20.3 (CH3, major), 20.1 (CH3, minor), 19.30 (C, minor), 19.28 (C, major), −2.0 (CH3, minor), − 2.3 (CH3, major), −2.4 (CH3, minor), −2.7 (CH3, major);73 IR (film) 2954, 2918, 2850, 1807, 1744, 1683, 1643, 1578, 1369; TLC Rf 0.10 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C64H74N6O19SiNa (M+Na)+ 1281.4675, found 1281.4697. During preliminary cross-coupling experiments, allowing 40a to react with varying amounts of 33, CuTC, and K2CO3 in PhMe at 90 °C resulted in the formation of 43 as a tan solid, in minor quantities. Data for thiophene-2-carboxylate adduct (+)-43: [α]D
24 +29.8, [α]577
24 +38.3, [α]546
24 +44.0, [α]435
24 +93.8 (c 0.10, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.16 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 4.8 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.71 (d, J = 6.8 Hz, 1H), 7.49 (s, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.3 Hz, 1H), 6.98 (t, J = 4.3 Hz, 1H), 6.62 (d, J =1.5 Hz, 1H), 6.56 (s, 1H), 6.49 (s, 1H), 6.48 (d, J = 7.8 Hz, 1H), 6.37 (s, 1H), 3.05 (s, 3H), 1.99 (s, 3H), 1.64 (s, 9H), 1.50 (s, 3H), 1.01 (s, 9H), 0.42 (s 3H), 0.32 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 167.9 (C), 167.8 (C), 160.3 (C), 159.0 (C), 149.3 (C), 149.0 (C), 136.5 (C), 135.4 (CH), 135.0 (CH), 131.8 (C), 129.7 (CH), 129.6 (C), 128.5 (C), 128.2 (CH), 126.0 (CH), 125.1 (CH), 124.8 (CH), 122.8 (CH), 121.6 (CH), 119.03 (C), 119.00 (CH), 115.7 (CH), 110.3 (CH), 91.8 (C), 87.3 (C) 84.4 (C), 82.9 (CH), 81.6 (CH), 59.0 (C), 27.65 (CH3), 27.57 (CH3), 25.7 (CH3), 23.9 (CH3), 19.8 (CH3), 18.7 (C), −2.9 (CH3), −3.4 (CH3); IR (film) 3376, 2954, 2918, 2851, 1765, 1736, 1710, 1683, 1608, 1371, 1218; TLC Rf 0.56 (3:7 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C41H48N4O9SSiNa (M+Na)+ 823.2809, found 823.2798.</p><!><p>Following a similar procedure as described for formation of 42a, intermediate (+)-40b (17 mg, 23 μmol) was converted to the title compound (+)-42b (15 mg, 51%), which was isolated as a red solid: [α]D
24 +28.0 (c 0.053, MeOH); 1H NMR (500 MHz, acetone-d6) δ 8.22 (d, J = 8.3 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.56 (s, 1H), 7.41–7.38 (comp, 3H), 7.29 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.09 (t, J = 7.7 Hz, 1H), 6.79 (s, 1H), 6.48 (s, 1H), 6.34 (d, J = 7.6 Hz, 1H), 6.11 (s, 1H), 3.78 (s, 3H), 3.04 (s, 3H), 2.84 (s, 3H), 1.96 (s, 3H), 1.65 (s, 9H), 1.59 (s, 3H), 1.40 (2 × s, 21H), 0.98 (s, 9H), 0.24 (s 3H), 0.09 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 180.6 (C), 169.4 (C), 168.3 (C), 167.8 (C), 165.3 (C), 163.6 (C), 159.8 (C), 150.1 (C), 150.0 (C), 149.7 (C), 149.0 (C), 148.1 (C), 144.2 (C), 139.0 (C), 137.22 (C), 137.17 (CH), 136.7 (CH), 134.79 (C), 134.76 (C), 131.9 (C), 130.1 (CH), 129.8 (C), 129.1 (C), 127.9 (CH), 126.1 (CH), 125.6 (CH), 123.3 (CH), 121.8 (CH), 120.6 (CH), 119.4 (CH), 118.3 (C), 116.5 (CH), 108.9 (CH), 106.9 (CH), 91.6 (C), 88.0 (CH), 87.1 (C), 85.3 (C), 84.3 (CH), 83.4 (C), 59.0 (C), 53.1 (CH3), 51.7 (CH3), 29.4 (CH3),74 28.2 (CH3), 28.1 (CH3), 27.9 (CH3), 26.2 (CH3), 20.6 (CH3), 20.1 (CH3), 19.2 (C), −2.8 (CH3), − 3.1 (CH3); IR (film) 2979, 2952, 2929, 2851, 1807, 1768, 1747, 1687, 1577, 1370; TLC Rf 0.24 (4:6 EtOAc/hexanes); HRMS (ESI) m/z calcd for C64H74N6O19SiNa (M+Na)+ 1281.4675, found 1281.4680.</p><!><p>Hydrogen sulfide (bp −60 °C, ca. 200 [L) was condensed at −78 °C in a thick-walled, glass pressure tube fitted with a rubber septum. A solution of (+)-42a (5.0 mg, 4.0 μmol) in CH2Cl2 (400 [L) and BF3•OEt2 (10 μL, 80 μmol) were injected sequentially into the reaction vessel maintained at −78 °C. The rubber septum was replaced by a Teflon®screw cap, which was used to seal the vessel. The cold bath was removed and the reaction mixture was allowed to warm to rt behind a blast shield. After 1 h, the reaction mixture was cooled to −78 °C, the Teflon®cap was replaced by the rubber septum, which was equipped with a needle vented to base (KOH/isopropanol) and bleach traps (attached in series). The cooling bath was removed and the resulting brown suspension was allowed to warm up to rt. Upon evolution of the majority of hydrogen sulfide gas, an argon-filled balloon was attached (by needle) to fully purge the reaction mixture, which was subsequently poured into a mixture of EtOAc (2 mL) and saturated aqueous NH4Cl (2 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 mL). The combined organic layers were washed with brine (4 mL), dried over Na2SO4 and filtered. To this solution was immediately added MeI (20 [L, 200 [mol) and K2CO3 (14 mg, 100 [mol). Performing the S-alkylation step prior to any further manipulations prevented competitive oxidation of the dithiol intermediate to the undesired epidisulfide congener. The reaction mixture was stirred for 12 h at rt, then poured into a mixture of EtOAc (10 mL) and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by preparatory thin layer chromatography (6/4 EtOAc/hexanes) to afford the title compound (−)-44b (3.0 mg, 88%) as a single diastereomer (by 1H NMR analysis). In corresponding experiments with N,O-acetal intermediate (+)-42b (10 mg, 8.0 μmol), the title compound (−)-44b (5.5 mg, 80%) was obtained in similar efficiency and diastereoselectivity as a red solid.</p><!><p>Methanethiol (bp 6 °C, ca. 200 [L) was condensed at −78 °C in a thick-walled, glass pressure tube fitted with a rubber septum. A solution of (+)-42a (23 mg, 18 μmol) in CH2Cl2 (400 [L) and BF3•OEt2 (30 μL, 240 μmol) were injected sequentially into the reaction vessel maintained at −78 °C. The rubber septum was replaced by a Teflon®screw cap, which was used to seal the vessel. The cold bath was removed and the reaction mixture was allowed to warm to rt behind a blast shield. After 1 h, the reaction mixture was cooled back down to −78 °C, the Teflon®cap was replaced by the rubber septum, which was equipped with a needle vented to base (KOH/isopropanol) and bleach traps (attached in series). The cooling bath was removed and the resulting brown suspension was allowed to warm up to rt. Upon evolution of the majority of methanethiol gas, an argon-filled balloon was attached (by needle) to fully purge the reaction mixture, which was subsequently poured into a mixture of EtOAc (10 mL) and saturated aqueous NaHCO3 (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and then concentrated under reduced pressure. This reaction was conducted three times consecutively, and then the crude residues were combined for purification by preparatory thin layer chromatography (6/4 EtOAc/hexanes) to afford the title compound (−)-44b (21 mg, 45%) and (−)-44a (16 mg, 34%) as red solids. In corresponding experiments with (+)-42b (30 mg, 24 μmol), the title compound (−)-44b and (−)-44a were obtained in similar efficiency (19 mg, 92% overall) and diastereoselectivity (1.3:1 dr, respectively) as a red solid. Data for cis-di(thiomethyl)ether (−)-44b: [α]D
24 −142, [α]577
24 −168, [α]546
24 −217 (c 0.050, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.21 (s, 1H), 7.71–7.67 (comp, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.26 (s, 1H), 7.16–7.13 (comp, 1H), 7.10 (s, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.02 (d, J = 2.5 Hz, 1H), 6.89 (t, J = 7.4 Hz, 1H), 6.45 (s, 1H), 6.29 (s, 1H), 6.22 (d, J = 7.8 Hz, 1H), 3.75 (s, 3H), 3.10 (s, 3H), 2.65 (s, 3H), 2.16 (s, 3H), 2.00 (s, 3H), 1.87 (s, 3H), 1.32 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 178.5 (C), 169.1 (C), 168.9 (C), 166.1 (C), 163.8 (C), 163.5 (C), 150.8 (C), 150.2 (C), 149.4 (C), 145.9 (C), 141.1 (C), 137.3 (C), 135.6 (C), 134.7 (C), 132.0 (C), 131.4 (CH), 129.7 (C), 129.6 (CH), 126.1 (C), 126.0 (CH), 124.2 (CH), 122.3 (CH), 121.1 (CH), 120.1 (CH), 118.8 (CH), 117.4 (CH), 112.9 (C), 111.8 (CH), 107.7 (CH), 105.0 (CH), 98.2 (C), 86.1 (CH), 81.2 (CH), 72.6 (C), 67.2 (C), 57.5 (C), 51.6 (CH3), 51.3 (CH3), 29.2 (CH3), 23.7 (CH3), 20.5 (CH3), 16.4 (CH3), 14.6 (CH3); IR (film) 3412, 3308, 2947, 2918, 2849, 1745, 1668, 1581, 1374; TLC Rf 0.38 (1:1 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C43H38N6O10S2Na (M+Na)+ 885.1989, found 885.1968. Note, for comparison, the 1H NMR of (−)-44b was also obtained in acetone-d6: 1H NMR (500 MHz, acetone-d6) δ 10.37 (s, 1H), 7.73 (d, J = 7.3 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.2–7.1 (comp, 3H), 7.07 (s, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.90 (t, J = 7.4 Hz, 1H), 6.44 (s, 1H), 6.30 (s, 1H), 6.16 (d, J = 7.8 Hz, 1H), 3.73 (s, 3H), 3.08 (s, 3H), 2.69 (s, 3H), 2.13 (s, 3H), 2.05 (s, 3H), 1.85 (s, 3H), 1.29 (s, 3H). Data for trans-di(thiomethyl)ether (−)-44a: [α]D
24 −103, [α]577
24 −110, [α]546
24 −136 (c 0.11, CH2Cl2); 44a was observed as a 4:1 mixture of atropisomers by NMR at 298 K (subsequent data is provided for the major isomer only); 1H NMR (500 MHz, acetone-d6) δ 10.39 (s, 1H), 7.73 (d, J = 7.3 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.2–7.1 (comp, 3H), 7.05 (t, J = 7.8 Hz, 1H), 7.04 (s, 1H), 6.92 (t, J = 7.5 Hz, 1H), 6.45 (s, 1H), 6.23 (s, 1H), 6.19 (d, J = 7.9 Hz, 1H), 3.73 (s, 3H), 3.11 (s, 3H), 2.62 (s, 3H), 2.16 (s, 3H), 2.12 (s, 3H), 1.74 (s, 3H), 1.29 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 179.0 (C), 169.3 (C), 168.6 (C), 166.2 (C), 165.1 (C), 163.6 (C), 151.3 (C), 151.2 (C), 150.4 (C), 146.8 (C), 142.1 (C), 138.7 (C), 136.4 (C), 135.1 (C), 132.6 (C), 132.1 (CH), 130.9 (C), 130.1 (CH), 127.2 (CH), 127.1 (C), 125.1 (CH), 122.5 (CH), 121.9 (CH), 119.8 (CH), 119.3 (CH), 118.2 (CH), 112.98 (CH), 112.96 (C), 108.1 (CH), 105.3 (CH), 87.3 (CH), 81.6 (CH), 74.5 (C), 73.8 (C), 58.6 (C), 51.7 (CH3), 51.2 (CH3), 30.2 (CH3), 25.2 (CH3), 20.3 (CH3), 16.1 (CH3), 13.2 (CH3);75 IR (film) 3389, 3307, 2947, 2917, 2849, 1747, 1667, 1581, 1377; TLC Rf 0.28 (1:1 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C43H38N6O10S2Na (M+Na)+ 885.1989, found 885.1960.</p><!><p>To a solution of cis-di(thiomethyl)ether (−)-44b (8.0 mg, 9.3 μmol) in MeOH (0.10 mL) was added lanthanum(III) trifluoromethanesulfonate (56 mg, 93 μmol) and DMAP (1.4 mg, 11 μmol). The reaction mixture was maintained at 45 °C. After 5 h (the consumption of the starting material was monitored by TLC, heating times varied in some instances), the reaction mixture was allowed to cool to rt, then poured into a mixture of EtOAc (10 mL) and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by preparatory thin layer chromatography (7:3 EtOAc/hexanes) to afford the deacetylated, C11-secondary alcohol intermediate (6.0 mg, contaminated with a closely eluting byproduct arising from the in-situ hydrolysis of a methyl ester group) as a red solid. This solid was dissolved in pyridine (0.10 mL) and LiI (50 mg, 370 μmol) was added to the solution. The reaction mixture was stirred for 12 h at 90 °C (formation of product was observed by RP-18 TLC), allowed to cool to rt, and poured into a mixture of EtOAc (5 mL) and 1N HCl (2 mL). The layers were separated, the organic layers was washed with 1 N HCl (2 × 2 mL). The combined aqueous layers were extracted with EtOAc (4 mL), the combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by reverse-phase HPLC (step-gradient eluting 20%, 40%, 60%, 65%, 70%, 75%, 90% MeOH/H2O + 0.1% TFA) to afford plectosphaeroic acid B (2) (4.8 mg, 65% over two steps, eluting at 75% MeOH/H2O + 0.1% TFA) as a red solid: [α]D
24 +228 (c 0.08, MeOH), compare with reported value [α]D
24 +69.8 (c 0.27, MeOH) of the natural sample; 1H NMR (500 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.72 (s, 1H), 8.91 (s, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.36 (comp, 2H), 7.09 (m, 1H), 7.06 (m, 1H), 6.90 (t, J = 7.6 Hz, 1H), 6.74 (s, 1H), 6.68 (s, 1H), 6.56 (t, J = 7.4 Hz, 1H), 5.98 (d, J = 7.9 Hz, 1H), 5.73 (br s, 1H), 5.44 (br s, 1H), 2.93 (s, 3H), 2.14 (s, 3H), 1.88 (s, 3H), 1.59 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 178.1 (C), 168.9 (C), 167.2 (C), 163.6 (C), 163.5 (C), 152.6 (C), 151.0 (C), 149.1 (C), 147.6 (C), 141.2 (C), 137.1 (C), 135.0 (C), 133.4 (C), 133.3 (C), 130.7 (CH), 127.9 (CH), 127.4 (C), 125.3 (C), 122.5 (CH), 122.4 (CH), 121.5 (CH), 120.9 (CH), 118.6 (CH), 118.0 (CH), 117.8 (CH), 115.9 (C), 111.4 (CH), 106.7 (CH), 105.5 (CH), 92.4 (C), 86.1 (C), 78.6 (CH), 74.1 (C), 66.3 (C), 58.6 (C), 28.5 (CH3), 22.6 (CH3), 15.7 (CH3), 13.9 (CH3); RP-18 TLC Rf 0.57 (4:1 MeOH/H2O); HRMS (ESI) m/z calcd for C39H31N6O9S2 (M–H)− 791.1594, found 791.1610.</p><!><p>Following a similar procedure as described for formation of plectosphaeroic acid B (2), (−)-44a (18 mg, 19 μmol) was converted to the title compound (+)-S9 (8.0 mg, 48%), which was isolated as a red solid: [α]D
24 +236 (c 0.062, MeOH); S9 was observed as an 8:1 mixture of atropisomers by NMR at 298 K (subsequent data is provided for the major isomer only); 1H NMR (500 MHz, DMSO-d6) δ 10.87 (s, 1H), 9.72 (s, 1H), 8.91 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.6–7.5 (comp, 2H), 7.35 (d, J = 8.1 Hz, 1H), 7.31 (s, 1H), 7.1–7.0 (comp, 2H), 6.93 (t, J = 7.7 Hz, 1H), 6.69 (s, 1H), 6.68 (s, 1H), 6.60 (t, J = 7.3 Hz, 1H), 6.10 (br s, 1H), 6.01 (d, J = 7.9 Hz, 1H), 5.41 (s, 1H), 2.97 (s, 3H), 2.10 (s, 3H), 1.71 (s, 3H), 1.53 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 178.1 (C), 168.9 (C), 167.1 (C), 164.7 (C), 162.8 (C), 152.6 (C), 150.9 (C), 149.3 (C), 147.6 (C), 141.2 (C), 137.1 (C), 135.2 (C), 133.2 (2 × C), 130.6 (CH), 128.7 (CH), 127.6 (C), 125.3 (C), 122.9 (CH), 122.4 (CH), 121.4 (CH), 120.9 (CH), 118.6 (CH), 118.3 (CH), 117.7 (CH), 115.8 (C), 111.4 (CH), 106.9 (CH), 105.5 (CH), 92.4 (C), 86.7 (CH), 78.7 (CH), 75.4 (C), 73.3 (C), 58.7 (C), 29.4 (CH3), 25.1 (CH3), 15.3 (CH3), 12.3 (CH3); IR (film) 3374, 3270, 3062, 2919, 2850, 1681, 1588, 1487, 1385; RP-18 TLC Rf 0.53 (4:1 MeOH/H2O); HRMS (ESI) m/z calcd for C39H31N6O9S2 (M–H)− 791.1594, found 791.1595.</p><!><p>A similar procedure for sulfidation as described for formation of 44 using H2S and BF3•OEt2 was followed, except the organic layer upon quenching of this step was concentrated under reduced pressure, and the resulting crude mixture was redissolved in acetone (0.5 mL) and K2CO3 (14 mg, 100 [mol) was added. The mixture was subsequently processed in a similar fashion. Using this procedure, intermediate 42a and 42b (5–10 mg/run) were converted to epidisulfide (−)-46 containing minor amounts of trisulfide (+)-47 (3–6 mg, 70–80%, 5–10:1 ratio of products). An alternative procedure for the S–S bond-forming step involved adding SiO2 (100 mg; instead of K2CO3) to the crude mixture obtained from sulfidating 26 mg of 42 dissolved in acetone (0.5 mL). The resulting heterogeneous mixture was vigorously stirred for 24 h (reaction times varied), filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparatory thin layer chromatography (7:3 EtOAc/hexanes) to afford (−)-46 and (+)-47 (12 mg, 70%, 7:1 ratio) as a red solid: [α]D
24 −23.8, [α]577
24 −33.2 (c 0.12, MeOH); 1H NMR (500 MHz, acetone-d6) δ 10.42 (br s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.3 Hz, 1H), 7.64 (m, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 7.3 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 7.01 (s, 1H), 6.44 (s, 1H), 6.42 (br d, J = 7.2 Hz, 1H), 6.36 (s, 1H), 3.74 (s, 3H), 3.01 (s, 3H), 2.60 (br s, 3H), 1.91 (s, 3H), 1.47 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 178.9 (C), 169.2 (C), 169.0 (C), 166.3 (C), 164.1 (C), 162.8 (C), 151.1 (C), 150.4 (C), 149.4 (C), 146.7 (C), 141.7 (C), 138.8 (C), 134.4 (C), 132.8 (CH), 132.3 (C), 130.7 (C), 130.5 (CH), 127.5 (CH), 127.4 (C), 126.1 (CH), 125.9 (CH), 122.7 (CH), 122.3 (CH), 120.6 (CH), 120.1 (CH), 117.6 (CH), 113.0 (CH), 111.7 (C), 109.1 (CH), 105.2 (CH), 99.1 (C), 86.8 (CH), 80.6 (CH), 77.2 (C), 76.2 (C), 60.6 (C), 51.8 (CH3), 51.4 (CH3), 27.6 (CH3), 20.4 (CH3), 18.0 (CH3); IR (film) 3411, 3323, 3055, 2950, 2917, 2849, 1737, 1703, 1578, 1489; TLC Rf 0.33 (4:6 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C41H32N6O10S2Na (M+Na)+ 855.1519, found 855.1542.</p><!><p>Hydrogen sulfide (bp −60 °C, ca. 400 [L) was condensed at −78 °C in a thick-walled, glass pressure tube fitted with a rubber septum. A solution of 42 (12 mg, 9.5 μmol) in CH2Cl2 (400 [L) and BF3•OEt2 (20 μL, 160 μmol) were injected sequentially into the reaction vessel maintained at −78 °C. The rubber septum was replaced by the corresponding Teflon®screw cap, which was used to seal the vessel. The cold bath was removed and the reaction mixture was allowed to warm to rt behind a blast shield. After 1 h, the reaction mixture was cooled to −78 °C, the Teflon®cap was replaced by the rubber septum, which was equipped with a needle vented to base (KOH/isopropanol) and bleach traps (attached in series). The cooling bath was removed and the resulting brown suspension was allowed to warm up to rt. Upon evolution of the majority of hydrogen sulfide gas, an argon-filled balloon was attached (by needle) to fully purge the reaction mixture, which was subsequently poured into a mixture of EtOAc (2 mL) and saturated aqueous NH4Cl (2 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 mL). The combined organic layers were washed with brine (4 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was dissolved in acetone (1 mL) and K2CO3 (10 mg, 72 [mol) was added. The resulting heterogenous mixture was stirred for 1 h, at which point Na2S (0.7 mg, 9.0 μmol) was added. The reaction mixture was stirred for 12 h at rt, then poured into a mixture of EtOAc (10 mL) and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by preparatory thin layer chromatography (6:4 EtOAc/hexanes) to afford the title compound (+)-47 (5.0 mg, 64%) as a red solid.76</p><!><p>To a solution of disulfide (−)-46 (5.0 mg, 6.0 μmol) was added Na2S (0.50 mg, 6.4 μmol). The reaction mixture was stirred for 1 h at rt, then poured into a mixture of EtOAc (20 mL) and water (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 20 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. An identical reaction was conducted on 9 mg of (−)-46. The crude residue from both reactions was combined for purification by preparatory thin layer chromatography (3:7 EtOAc/hexanes) to afford the title compound (+)-47 and 48 (11 mg, 75%; 5:1 ratio). Data for epitrisulfide (+)-47: [α]D
24 +61.3, [α]577
24 +64.9, [α]546
24 +72.4 (c 0.12, MeOH); 1H NMR (500 MHz, acetone-d6) δ 10.38 (br s, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.27 (t, J = 7.7 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.04 (s, 1H), 6.99 (t, J = 7.5 Hz, 1H), 6.95 (s, 1H), 6.47 (s, 1H), 6.43 (d, J = 8.1 Hz, 1H), 6.33 (s, 1H), 3.74 (s, 3H), 3.21 (s, 3H), 2.66 (s, 3H), 1.74 (s, 3H), 1.47 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 179.0 (C), 169.2 (C), 168.5 (C), 166.8 (C), 166.4 (C), 164.1 (C), 152.4 (C), 151.1 (C), 150.4 (C), 146.8 (C), 142.1 (C), 138.8 (C), 135.4 (C), 135.2 (C), 132.7 (CH), 132.4 (C), 131.3 (CH), 127.7 (C), 127.2 (2 × CH), 127.0 (C), 122.6 (CH), 122.0 (CH), 120.3 (CH), 120.1 (CH), 117.9 (CH), 113.0 (CH), 111.3 (C), 109.5 (CH), 105.3 (CH), 99.1 (C), 85.9 (CH), 85.7 (C), 84.1 (CH), 73.2 (C), 58.4 (C), 51.8 (CH3), 51.4 (CH3), 29.6 (CH3), 21.4 (CH3), 20.7 (CH3);77 IR (film) 3410, 3313, 3056, 2997, 2948, 2917, 2849, 1738, 1686, 1579, 1488; TLC Rf 0.40 (4:6 EtOAc/hexanes, plate was eluted twice; compare with Rf 0.37 for 48); HRMS (ESI) m/z calcd for C41H32N6O10S3Na (M+Na)+ 887.1240, found 887.1238. Only minor quantities of tetrasulfide 48 suitable for mass spectrometric analysis could be obtained in pure form: HRMS (ESI) m/z calcd for C41H32N6O10S4Na (M+Na)+ 919.0961, found 919.0952).</p><!><p>Following a similar procedure as described for formation of 2, intermediate (+)-47 (5 mg, 5.8 μmol) was converted to acid (+)-45 (2.6 mg, 55%) as a red solid: [α]D
24 +416, (c 0.06, MeOH); 1H NMR (500 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.72 (s,1H), 8.90 (s, 1H), 8.01 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.38–7.35 (comp, 2H), 7.11–7.04 (comp, 3H), 6.71 (t, J = 7.5 Hz, 1H), 6.68 (s, 1H), 6.64 (s, 1H), 6.33 (d, J = 7.0 Hz, 1H), 6.28 (d, J = 8.0 Hz, 1H), 5.53 (d, J = 6.9 Hz, 1H), 3.06 (s, 3H), 1.55 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 178.1 (C), 168.9 (C), 167.5 (C), 166.3 (C), 163.3 (C), 152.6 (C), 150.9 (C), 150.5 (C), 147.6 (C), 141.2 (C), 137.1 (C), 135.3 (C), 132.1 (C), 131.3 (CH), 129.8 (C), 129.3 (CH), 127.3 (C), 125.1 (C), 125.0 (CH), 122.6 (C), 121.1 (CH), 121.0 (CH), 119.5 (CH), 118.8 (CH), 117.3 (CH), 113.7 (C), 111.5 (CH), 108.7 (CH), 105.5 (CH), 92.5 (C), 86.8 (C), 85.7 (CH), 81.8 (CH), 71.9 (C), 58.4 (C), 27.8 (CH3), 20.7 (CH3); IR (film) 3363, 3243, 2954, 2916, 2849, 1681, 1584, 1481; RP-18 TLC Rf 0.36 (7:3 MeOH/H2O); HRMS (ESI) m/z calcd for C37H25N6O9S3Na2 (M–H+2Na)+ 839.0641, found 839.0637.</p><!><p>To a solution of enamide (+)-41 (70 mg, 0.12 mmol), NMO (28 mg, 0.24 mmol) and a co-solvent mixture of acetone and H2O (1.2 mL and 0.3 mL, respectively) was added OsO4 (60 μL, 6 μmol, 2.5 wt % solution in t-BuOH). The reaction mixture was stirred for 2 h at rt, then poured into a mixture of EtOAc (10 mL) and 10% aqueous sodium thiosulfate. The layers were separated and the aqueous layer was extracted with EtOAc (2 × 5 mL). The combined organic layers were washed sequentially with H2O (10 mL) and brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was redissolved in EtOAc (1 mL) and SiO2 (100 mg) was added. The resulting suspension was stirred for 1 h at rt (reaction times can vary; the reaction was monitored for consumption of the minor product, 49 by TLC: Rf 0.12, 7:3 EtOAc/Hexanes), filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (3:7 → 7:3 EtOAc/hexanes) to afford the title compound (+)-50 (58 mg, 78%) as a colorless solid: [α]D
24 +140, [α]577
24 +145, [α]546
24 +162, [α]435
24 +330 (c 0.48, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.13 (d, J = 8.2 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.46 (s, 1H), 7.40 (d, 1H), 7.32 (t, J = 7.4 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.80 (t, J = 7.4 Hz, 1H), 6.72 (d, J = 7.7 Hz, 1H), 6.47 (s, 1H), 6.25 (s, 1H), 6.20 (s, 1H), 5.50 (s, 1H), 4.42 (d, J = 11.3 Hz, 1H), 4.29 (d, J = 11.3 Hz, 1H), 2.99 (s, 3H), 1.90 (s, 3H), 1.63 (s, 9H), 1.45 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 170.6 (C), 168.8 (C), 166.7 (C), 165.3 (C), 150.0 (C), 149.9 (C), 137.0 (C), 130.1 (C), 129.8 (CH), 129.5 (C),126.1 (CH), 125.6 (CH), 125.3 (CH), 123.3 (CH), 122.6 (CH), 119.9 (C), 119.2 (CH), 116.1 (CH), 110.6 (CH), 90.6 (C), 84.8 (C), 84.7 (CH), 83.8 (CH), 80.8 (CH), 65.4 (CH2), 58.5 (C), 28.2 (CH3), 27.6 (CH3), 20.9 (CH3), 20.7 (CH3); IR (film) 3365, 2979, 2917, 2849, 1740, 1650, 1453, 1370; TLC Rf 0.27 (7:3 EtOAc/hexanes); HRMS (ESI) m/z calcd for C32H34N4O10Na (M+Na)+ 657.2173, found 657.2175.</p><!><p>To a solution of 50 (200 mg, 0.32 mmol), DMAP (38 mg, 0.31 mmol), and triethylamine (0.41 mL, 3.2 mmol) in DMF (3.2 mL) maintained at 0 °C was added TBSOTf (0.360 mL, 1.6 mmol) slowly. The cold bath was removed and the reaction mixture was maintained at rt for 12 h, whereupon EtOAc (30 mL) was added followed by saturated aqueous NH4Cl (20 mL). The mixture was transferred to a separatory funnel, the layers were separated, and the aqueous phase was further extracted with EtOAc (2 × 15 mL). The combined organic extracts were washed sequentially with 10% aqueous LiCl (2 × 15 mL) and brine (15 mL), dried over Na2SO4, then concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:10 EtOAc/hexanes → 1:3 EtOAc/hexanes) to afford the title compounds 51 as colorless solids (230 mg of the major epimer 51-major, 30 mg of the minor epimer 51-minor; 96% overall yield, 8:1 ratio of C3-epimers). Data for the major epimer, (+)-51-major: [α]D
24 +126.8, [α]577
24 +131, [α]546
24 +152, [α]435
24 +293, [α]405
24 +396 (c 0.34, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.13 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.41 (s, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 6.51 (d, J = 2.2 Hz, 1H), 6.48 (br s, 1H), 6.25 (s, 1H), 4.45 (d, J = 11.5 Hz, 1H), 4.33 (d, J = 11.5 Hz, 1H), 3.14 (s, 3H), 2.11 (s, 3H), 1.62 (s, 9H), 1.41 (s, 3H), 1.00 (s, 9H), 0.44 (s, 9H), 0.40 (s, 3H), 0.28 (s, 3H), 0.20 (s, 3H), 0.01 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 170.3 (C), 168.7 (C), 165.5 (C), 163.5 (C), 150.0 (2 × C), 137.0 (C), 130.5 (CH), 129.6 (C), 129.4 (C), 126.1 (CH), 125.7 (CH), 125.3 (CH), 123.1 (CH), 122.5 (CH), 120.8 (C), 119.9 (CH), 116.2 (CH), 111.2 (CH), 92.5 (C), 86.7 (C), 84.9 (C), 83.6 (CH), 80.9 (CH), 69.0 (CH2), 59.0 (C), 30.1 (CH3), 28.2 (CH3), 26.7 (CH3), 26.2 (CH3), 20.9 (CH3), 20.7 (CH3), 19.9 (C), 18.6 (C), −2.4 (CH3), −2.8 (CH3), −3.7 (CH3), −4.2 (CH3); IR (film) 3380, 2954, 2929, 2856, 1756, 1739, 1694, 1678, 1454, 1371; TLC Rf 0.61 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C44H62N4O10Si2Na (M+Na)+ 885.3902, found 885.3907. Data for the minor epimer, (+)-51-minor: [α]D
24 +57.7, [α]577
24 +58.3, [α]546
24 +69.1, [α]435
24 +140.0, [α]405
24 +190 (c 0.34, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.14 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.40 (s, 1H), 7.33 (t, J = 7.7 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.86 (t, J = 7.4 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 6.55 (br s, 1H), 6.53 (d, J = 3.2 Hz, 1H), 6.16 (s, 1H), 4.72 (d, J = 10.9 Hz, 1H), 4.50 (d, J = 10.9 Hz, 1H), 3.02 (s, 3H), 2.15 (s, 3H), 1.62 (s, 9H), 1.38 (s, 3H), 0.94 (s, 9H), 0.48 (s, 9H), 0.43 (s, 3H), 0.21 (s, 3H), 0.14 (s, 3H), 0.01 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ170.2 (C), 168.8 (C), 164.2 (C), 160.1 (C), 150.2 (C), 149.9 (C), 137.0 (C), 130.3 (CH), 129.9 (C), 129.6 (C), 127.0 (CH), 125.8 (CH), 125.3 (CH), 123.4 (CH), 122.3 (CH), 120.7 (C), 119.4 (CH), 116.2 (CH), 110.9 (CH), 91.8 (C), 86.8 (C), 84.9 (C, CH), 81.5 (CH), 65.7 (CH2), 59.1 (C), 28.2 (CH3), 28.1 (CH3), 26.40 (CH3), 26.36 (CH3), 21.1 (CH3), 20.7 (CH3), 19.2 (C), 18.7 (C), −2.4 (CH3), −3.1 (CH3), −3.6 (CH3), − 3.7 (CH3); IR (film) 3380, 2954, 2928, 2856, 1754, 1740, 1681, 1454, 1372; TLC Rf 0.47 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C44H62N4O10Si2Na (M+Na)+ 885.3902, found 885.3905.</p><!><p>Following a similar procedure as described for formation of 42a, intermediate (+)-51-major (3 runs in parallel: 50 mg, 58 μmol per run) and phenoxazinone iodide 33 (114 mg, 170 μmol per run) were converted to (+)-52-major (168 mg combined, 72%) as an orange solid: [α]D
24 +26.2 (c 0.052, MeOH); 1H NMR (500 MHz, acetone-d6) δ 8.19 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.66–7.62 (comp, 3H), 7.50 (s, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.00 (t, J = 7.3 Hz, 1H), 6.99 (s, 1H), 6.49 (s, 1H), 6.31 (s, 1H), 6.30 (d, 1H), 4.42 (d, J = 11.4 Hz, 1H), 4.34 (d, J = 11.3 Hz, 1H), 3.83 (s, 3H), 3.12 (s, 3H), 3.09 (s, 3H), 2.15 (s, 3H), 1.65 (s, 9H), 1.38 (s, 18H), 1.35 (s, 3H), 0.93 (s, 3H), 0.55 (s, 3H), 0.25 (s, 3H), 0.21 (s, 3H), 0.18 (s, 3H), 0.07 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ180.6 (C), 170.2 (C), 168.4 (C), 165.60 (C), 165.55 (C), 163.6 (C), 163.5 (C), 150.0 (C), 149.7 (C), 149.3 (C), 147.9 (C), 144.1 (C), 139.0 (C), 137.2 (C), 137.0 (C), 136.2 (CH), 135.8 (C), 134.8 (C), 131.9 (C), 130.7 (C), 129.6 (CH), 129.4 (C), 127.2 (CH), 126.0 (CH), 125.4 (CH), 123.1 (CH), 122.7 (CH), 120.9 (CH) 119.8 (C), 118.9 (CH), 116.3 (CH), 109.9 (CH), 106.9 (CH), 93.9 (C), 86.5 (CH), 86.2 (C), 85.1 (C), 83.9 (CH), 83.7 (C), 65.4 (CH2), 58.1 (C), 53.1 (CH3), 52.1 (CH3), 28.2 (CH3), 27.9 (CH3), 26.6 (CH3), 26.4 (CH3), 21.1 (CH3), 20.5 (CH3), 19.9 (C), 18.8 (C), −2.4 (CH3), −2.8 (CH3), −3.7 (CH3), −3.9 (CH3);78 IR (film) 2956, 2929, 2856, 1807, 1746, 1712, 1679, 1461, 1371; TLC Rf 0.45 (3:7 EtOAc/hexanes); HRMS (ESI) m/z calcd for C70H88N6O20Si2Na (M+Na)+ 1411.5490, found 1411.5460.</p><!><p>Following a similar procedure as described for formation of 42a, intermediate (+)-51-minor (30 mg, 35 μmol) and phenoxazinone iodide 33 (68 mg, 100 μmol per run) were converted to 52-minor (36 mg, 74%) as an orange solid: 1H NMR (500 MHz, acetone-d6) δ 8.20 (d, J = 8.2 Hz, 1H), 7.70–7.64 (comp, 3H), 7.53 (d, J = 7.8 Hz, 1H), 7.43 (s, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.27 (t, J = 7.3 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.3 Hz, 1H), 6.89 (s, 1H), 6.48 (s, 1H), 6.29 (d, J = 7.9 Hz, 1H), 6.21 (s, 1H), 4.64 (d, J = 10.8 Hz, 1H), 4.55 (d, J = 10.8 Hz, 1H), 3.78 (s, 3H), 3.03 (s, 3H), 2.78 (s, 3H), 2.19 (s, 3H), 1.64 (s, 9H), 1.36 (s, 18H), 1.35 (s, 3H), 0.96 (s, 3H), 0.57 (s, 3H), 0.28 (s, 3H), 0.15 (s, 3H), 0.15 (s, 3H), 0.07 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ180.6 (C), 170.1 (C), 168.6 (C), 165.3 (C), 164.32 (C), 164.25 (C), 163.6 (C), 150.0 (C), 149.7 (C), 149.5 (C), 147.9 (C), 143.9 (C), 139.0 (C), 137.3 (C), 136.4 (C), 135.7 (CH), 134.8 (C), 133.5 (C), 132.1 (C), 132.0 (C), 130.5 (CH), 129.8 (C), 129.7 (C), 128.1 (CH), 126.1 (CH), 125.4 (CH), 123.5 (CH), 122.3 (CH), 120.6 (CH) 119.6 (C), 119.1 (CH), 116.3 (CH), 109.3 (CH), 106.8 (CH), 92.6 (C), 87.31 (C), 87.29 (CH), 85.12 (C), 85.05 (CH), 83.7 (C), 65.4 (CH2), 58.3 (C), 53.1 (CH3), 51.6 (CH3), 28.2 (CH3), 27.9 (CH3), 26.6 (CH3), 26.3 (CH3), 21.2 (CH3), 20.6 (CH3), 19.3 (C), 18.8 (C), −2.0 (CH3), −2.2 (CH3), −3.3 (CH3), −3.6 (CH3); IR (film) 2952, 2929, 2856, 1806, 1747, 1712; TLC Rf 0.20 (3:7 EtOAc/hexanes, copolar with iodide 32); HRMS (ESI) m/z calcd for C70H88N6O20Si2Na (M+Na)+ 1411.5490, found 1411.5490.</p><!><p>Tetra-n-butylammonium fluoride (TBAF) (85 μL, 85 μmol, 1.0 M in THF) was added to a solution of intermediate (+)-52-major (56 mg, 40 μmol), acetic acid (AcOH) (7.0 μL, 12 μmol) and THF (0.40 mL) at 0 °C. The reaction mixture was maintained at 0 °C for 30 min then poured into a mixture of EtOAc (10 mL) and saturated aqueous NH4Cl (5 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (2 × 5 mL). The combined organic layers were washed successively with saturated aqueous NH4Cl (2 × 5 mL) and brine (5 mL), dried over Na2SO4, then concentrated under reduced pressure. The crude product was purified by silica gel chromatography (7:3 EtOAc/hexanes) to afford the title compound (−)-53-major as a red solid (45 mg, 96%): [α]D
24 −94.4 (c 0.050, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.19 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.64–7.60 (comp, 2H), 7.57 (s, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.27 (t, J = 7.4 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 6.93 (t, J = 7.3 Hz, 1H), 6.49 (s, 1H), 6.32 (s, 1H), 6.29 (d, J = 7.8 Hz, 1H), 6.17 (s, 1H), 6.01 (s, 1H), 4.48 (d, J = 11.4 Hz, 1H), 3.87 (s, 3H), 3.86 (d, 1H), 3.24 (s, 3H), 2.94 (s, 3H), 2.08 (s, 3H), 1.67 (s, 9H), 1.40 (s, 3H), 1.39 (s, 18H); 13C NMR (125 MHz, acetone-d6) δ180.6 (C), 170.3 (C), 168.5 (C), 166.8 (C), 166.0 (C), 165.3 (C), 163.8 (C), 150.2 (C), 150.1 (C), 150.0 (C), 149.7 (C), 147.7 (C), 144.0 (C), 138.9 (C), 137.4 (C), 137.2 (C), 136.4 (CH), 136.1 (C), 134.8 (C), 131.6 (C), 130.9 (C), 130.0 (CH), 129.3 (C), 126.7 (CH), 125.9 (CH), 125.4 (CH), 123.4 (CH), 122.7 (CH), 120.3 (CH) 119.1 (C), 119.0 (CH), 116.2 (CH), 109.1 (CH), 106.8 (CH), 91.6 (C), 86.1 (CH), 85.1 (C), 84.6 (C), 84.3 (CH), 83.7 (C), 64.7 (CH2), 57.7 (C), 53.1 (CH3), 52.3 (CH3), 28.2 (CH3), 27.9 (CH3), 27.5 (CH3), 21.0 (CH3), 20.5 (CH3); IR (film) 3360, 2981, 2952, 2933, 2918, 2849, 1806, 1747, 1644, 1371; TLC Rf 0.17 (7:3 EtOAc/hexanes); HRMS (ESI) m/z calcd for C58H60N6O20Na (M+Na)+ 1183.3760, found 1183.3765.</p><!><p>Tetra-n-butylammonium fluoride (TBAF) (310 μL, 0.31 mmol, 1.0 M in THF) was added to a solution of intermediate epimers 52 (227 mg, 5:1 ratio of epimers, 0.16 mmol), acetic acid (AcOH) (25 μL, 0.40 mmol) and THF (0.40 mL) at 0 °C. The reaction mixture was maintained at 0 °C for 30 min then poured into a mixture of EtOAc (10 mL) and saturated aqueous NH4Cl (5 mL). The layers were separated and the aqueous layer was further extracted with EtOAc (2 × 5 mL). The combined organic layers were washed successively with saturated aqueous NH4Cl (2 × 5 mL) and brine (5 mL), dried over Na2SO4 and concentrated under reduced pressure. The diol intermediates 53 were of sufficient purity to be taken forward without further purification. To a solution of this intermediate in CH2Cl2 (7.3 mL) was added DMAP (142 mg, 1.2 mmol) and acetic anhydride (50 μL, 0.52 mmol). The resulting mixture was maintained for 12 h at rt, then poured into a mixture of EtOAc (30 mL) and saturated aqueous NH4Cl (20 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 15 mL). The combined organic layers were washed successively with H2O (20 mL) and brine (20 mL), dried over Na2SO4, then concentrated under reduced pressure. The crude residue was purified by chromatography on silica gel (1:5 EtOAc/hexanes → 6:4 EtOAc/hexanes) to afford the title compounds 54 (175 mg, 86% over two steps, 5:1 ratio of epimers) as a deep red solid: Analytical samples for both epimers were obtained. Data for (−)-54-major: [α]D
24 −45.8, [α]577
24 −48.6 (c 0.058, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.17 (d, J = 8.3 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.48 (s, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.28–7.22 (comp, 2H), 7.11 (t, J = 7.4 Hz, 1H), 6.98 (s, 1H), 6.49 (s, 1H), 6.43 (s, 1H), 6.94 (d, J = 7.9 Hz, 1H), 4.86 (d, J = 11.9 Hz, 1H), 4.55 (d, J = 11.9 Hz, 1H), 3.81 (s, 3H), 3.00 (s, 3H), 2.80 (s, 3H), 2.23 (s, 3H), 1.65 (s, 9H), 1.50 (s, 3H), 1.47 (s, 3H), 1.39 (s, 3H), 1.37 (s, 18H); 13C NMR (125 MHz, acetone-d6) δ180.6 (C), 170.2 (C), 169.5 (C), 169.4 (C), 168.8 (C), 165.5 (C), 163.7 (C), 163.3 (C), 160.6 (C), 150.1 (C), 150.0 (C), 149.7 (C), 148.7 (C), 148.0 (C), 144.1 (C), 139.0 (C), 137.2 (2 × C), 135.4 (CH), 134.83 (C), 134.77 (C), 131.8 (C), 130.8 (C), 130.3 (CH), 129.4 (C), 128.0 (CH), 126.3 (CH), 125.5 (CH), 124.0 (CH), 123.4 (CH), 120.8 (CH) 119.3 (CH), 117.7 (C), 115.9 (CH), 109.1 (CH), 106.9 (CH), 92.8 (C), 87.6 (CH), 86.7 (C), 85.2 (C), 83.7 (C), 83.0 (CH), 64.6 (CH2), 57.7 (C), 53.1 (CH3), 51.8 (CH3), 29.4 (CH3), 28.2 (CH3), 27.9 (CH3), 20.9 (CH3), 20.7 (CH3), 20.4 (CH3), 20.3 (CH3); IR (film) 2979, 2917, 2849, 1806, 1747, 1694, 1644, 1369; TLC Rf 0.18 (1:1 EtOAc/hexanes); HRMS (ESI) m/z calcd for C62H64N6O22Na (M+Na)+ 1267.3971, found 1267.3987. Data for (+)-54-minor: [α]D
24 +50.5 (c 0.043, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 8.23 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 7.4 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.55 (s, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.24 (t, J = 7.7 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.98 (s, 1H), 6.59 (s, 1H), 6.46 (s, 1H), 6.39 (d, J = 7.9 Hz, 1H), 4.75 (d, J = 11.4 Hz, 1H), 4.59 (d, J = 11.4 Hz, 1H), 3.80 (s, 3H), 3.05 (s, 3H), 2.87 (s, 3H), 2.20 (s, 3H), 2.12 (s, 3H), 1.65 (s, 9H), 1.47 (s, 3H), 1.46 (s, 3H), 1.37 (s, 18H); 13C NMR (125 MHz, acetone-d6) δ 180.6 (C), 170.2 (C), 168.7 (C), 168.5 (C), 167.4 (C), 165.6 (C), 163.7 (C), 162.1 (C), 162.0 (C), 150.1 (C), 150.0 (C), 149.7 (C), 149.2 (C), 147.7 (C), 144.0 (C), 138.9 (C), 137.5 (CH), 137.3 (C), 136.6 (C), 135.1 (C), 134.8 (C), 131.6 (C), 130.3 (CH), 130.0 (C), 129.4 (C), 128.0 (CH), 126.3 (CH), 125.6 (CH), 123.6 (CH), 122.1 (CH), 120.5 (CH) 118.9 (CH), 117.9 (C), 116.5 (CH), 108.8 (CH), 106.8 (CH), 91.7 (C), 87.20 (CH), 87.17 (C), 85.3 (C), 83.7 (C), 83.2 (CH), 64.2 (CH2), 57.8 (C), 53.1 (CH3), 51.8 (CH3), 29.2 (CH3), 28.2 (CH3), 27.9 (CH3), 21.1 (CH3), 21.0 (CH3), 20.6 (CH3), 20.6 (CH3); IR (film) 2979, 2917, 2848, 1806, 1747, 1703, 1644, 1370; TLC Rf 0.11 (1:1 EtOAc/hexanes); HRMS (ESI) m/z calcd for C62H64N6O22Na (M+Na)+ 1267.3971, found 1267.3956.</p><!><p>Hydrogen sulfide (bp −60 °C, ca. 0.20 mL) was condensed at −78 °C in a thick-walled, glass pressure tube fitted with a rubber septum. A solution of 54 (3 runs in series: 30 mg, 50 mg, 60 mg; 5:1 mixture of epimers) in CH2Cl2 (0.60 mL) and BF3•OEt2 (30 μL, 0.24 mmol) were injected sequentially into the reaction vessel maintained at −78 °C. The rubber septum was replaced by a Teflon®screw cap, which was used to seal the vessel. The cold bath was removed and the reaction mixture was allowed to warm to rt behind a blast shield. After 1 h, the reaction mixture was cooled to −78 °C, the Teflon®cap was replaced by the rubber septum, which was equipped with a needle vented to base (KOH/isopropanol) and bleach traps (attached in series). The cooling bath was removed and the resulting brown suspension was allowed to warm up to rt. Upon evolution of the majority of hydrogen sulfide gas, an argon-filled balloon was attached (by needle) to fully purge the reaction mixture, which was subsequently poured into a mixture of EtOAc (2 mL) and saturated aqueous NH4Cl (2 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 mL). The combined organic layers were washed with brine (4 mL), dried over Na2SO4, and concentrated under reduced pressure (residual volatile components were not rigorously removed). The crude residue was immediately dissolved in acetone (1 mL) and 400 wt % of SiO2 (120, 150, 180 mg, respectively) was added. The resulting heterogeneous mixture was stirred vigorously for 12–48 h (reaction times were found to be variable, and required monitoring for completion, see Table S4 for more details) then filtered, and the filtrate was concentrated under reduced pressure. The residue from all three reactions was purified by silica gel chromatography (3:7 EtOAc/hexanes) to afford a mixture of ETP products, disulfide 55, trisulfide 56 and tetrasulfide 57 (50 mg, 48%, 1:6:2 ratio of products, determined by 1H NMR) as an orange solid.</p><!><p>The procedure described above was followed for processing 54 (10 mg, 8.0 μmol), except upon quenching the sulfidation step, the crude residue was dissolved in EtOAc (2 mL) and concentrated under reduced pressure three additional cycles prior to the addition of EtOAc (1 mL) and SiO2 (40 mg). Upon purification, an inseparable mixture of ETP products, disulfide 55 and trisulfide 56 (7 mg, 7:1 ratio of products, determined by 1H NMR) as an orange solid was obtained. To a solution of this mixture of ETPs 55 and 56 (7 mg, 7.8 μmol) in acetone (1 mL) was added Na2S (0.60 mg, 7.6 μmol). The resulting heterogeneous mixture was stirred for 0.5 h at rt, then poured into a mixture of EtOAc (2 mL) and saturated aqueous NH4Cl (2 mL). The combined organic layers were washed with brine (4 mL), dried over Na2SO4 then concentrated under reduced pressure to afford a mixture of ETP products, trisulfide 56 and tetrasulfide 57 (2.5:1 ratio of products, determined by 1H NMR).79 The mixture was dissolved in CH2Cl2 (0.5 mL) then Ph3P (40 μL, 0.05 M solution in CH2Cl2) was added. After 0.5 h, the mixture was eluted through a short column of silica gel (1:2 EtOAc/hexanes, then 2:1 EtOAc/hexanes) to afford the title compound (+)-56 (5.0 mg, 67%) as a red solid: [α]D
24 +47.3, [α]577
24 +49.5, [α]546
24 +53.9 (c 0.06, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 10.41 (br s, 1H), 7.96 (d, J = 8.7 Hz, 1H), 7.70–7.67 (comp, 2H), 7.63 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.27 (t, J = 7.7 Hz, 1H), 7.18 (t, J = 7.3 Hz, 1H), 7.11 (t, J = 7.2 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.00 (d, J = 7.4 Hz, 1H), 6.94 (s, 1H), 6.46 (s, 1H), 6.43 (d, J = 8.0 Hz, 1H), 6.36 (s, 1H), 4.65 (d, J = 12.3 Hz, 1H), 4.36 (d, J = 12.3 Hz, 1H), 3.73 (s, 3H), 3.27 (s, 3H), 2.68 (s, 3H), 1.96 (s, 3H), 1.47 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 179.0 (C), 169.9 (C), 169.2 (C), 168.5 (C), 166.7 (C), 166.4 (C), 162.1 (C), 152.3 (C), 151.1 (C), 150.4 (C), 146.8 (C), 142.1 (C), 138.8 (C), 135.3 (C), 135.0 (C), 132.8 (CH), 132.4 (C), 131.4 (CH), 127.5 (C), 127.2 (CH), 127.04 (CH), 126.95 (C), 122.7 (CH), 121.8 (CH), 120.4 (CH), 120.2 (CH), 118.0 (CH), 113.0 (CH), 111.3 (C), 109.4 (CH), 105.3 (CH), 99.1 (C), 86.0 (CH), 85.1 (C), 84.0 (CH), 74.9 (C), 61.6 (CH2), 58.6 (C), 51.8 (CH3), 51.4 (CH3), 28.5 (CH3), 20.6 (CH3), 20.6 (CH3); IR (film) 3394, 3322, 2917, 2849, 1746, 1694, 1576, 1486; TLC Rf 0.25 (4:6 EtOAc/hexanes); HRMS (ESI) m/z calcd for C43H34N6O12S3Na (M+Na)+ 945.1295, found 945.1281. Data for epidisulfidedioxopiperazine 55: 1H NMR (500 MHz, acetone-d6) δ 10.40 (br s, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.71 (d, J = 7.4 Hz, 1H), 7.64 (m, 1H), 7.69–7.62 (comp, 2H), 7.24 (t, J = 7.8 Hz, 1H), 7.21–7.16 (comp, 2H), 7.08–7.04 (comp, 2H), 7.02 (s, 1H), 6.44 (d, 1H), 6.43 (s, 2H), 6.39 (s, 1H), 4.82 (d, J = 12.7 Hz, 1H), 4.77 (d, J = 12.7 Hz, 1H), 3.71 (s, 3H), 3.10 (s, 3H), 2.60 (br s, 3H), 2.11 (s, 3H), 1.50 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 178.9 (C), 170.1 (C), 169.1 (C), 169.0 (C), 166.2 (C), 163.8 (C), 161.1 (C), 151.0 (C), 150.4 (C), 149.3 (C), 146.7 (C), 141.7 (C), 138.8 (C), 138.7 (C), 132.8 (CH), 132.3 (C), 131.4 (C), 130.6 (CH), 127.6 (CH), 127.42 (C), 127.38 (C), 126.0 (CH), 122.7 (CH), 122.3 (CH), 120.7 (CH), 120.2 (CH), 117.6 (CH), 113.0 (CH), 111.5 (C), 109.4 (CH), 105.2 (CH), 99.0 (C), 86.9 (CH), 80.3 (CH), 77.3 (C), 77.1 (C), 60.9 (CH2), 60.8 (C), 51.8 (CH3), 51.5 (CH3), 28.5 (CH3), 20.6 (CH3), 20.5 (CH3); TLC Rf 0.25 (4:6 EtOAc/hexanes); HRMS (ESI) m/z calcd for C43H34N6O12S2Na (M+Na)+ 913.1574, found 913.1587.</p><!><p>To a solution of ETP (+)-56 (11 mg, 11 μmol) in MeOH (0.30 mL) was added lanthanum(III) trifluoromethanesulfonate (66 mg, 110 μmol) and DMAP (1.6 mg, 12 μmol). The reaction mixture was maintained at 45 °C. After 5 h (the consumption of the starting material was monitored by TLC), the reaction mixture was allowed to cool to rt, then poured into a mixture of EtOAc (10 mL) and saturated aqueous NH4Cl (10 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by preparatory thin layer chromatography (7:3 EtOAc/hexanes) to afford intermediate (+)-58 (7.0 mg, 76%) as a red solid:80 [α]D
24 +181.4, [α]577
24 +190.0, [α]546
24 +203.6 (c 0.07, CH2Cl2); 1H NMR (500 MHz, acetone-d6) δ 10.25 (br s, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.18–7.11 (comp, 2H), 7.07 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 7.3 Hz, 1H), 6.78 (s, 1H), 6.47 (s, 1H), 6.45 (d, J = 7.7 Hz, 1H), 5.52 (d, J = 4.8 Hz, 1H), 5.17 (d, J = 4.8 Hz, 1H), 4.25 (app t, J = 7.0 Hz, 1H), 3.99 (dd, J = 12.5, 6.4 Hz, 1H), 3.86 (s, 3H), 3.82 (dd, J = 12.5, 7.6 Hz, 1H), 3.37 (s, 3H), 3.24 (s, 3H), 1.96 (s, 3H), 1.47 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 179.0 (C), 169.2 (C), 168.4 (C), 167.5 (C), 164.1 (C), 151.9 (C), 151.1 (C), 150.3 (C), 146.7 (C), 141.9 (C), 138.6 (C), 136.3 (C), 135.0 (C), 132.5 (CH), 132.4 (C), 130.6 (CH), 130.2 (C), 127.4 (C), 126.7 (CH), 125.0 (CH), 122.5 (CH), 122.4 (CH), 120.5 (CH), 120.0 (CH), 117.9 (CH), 114.3 (C), 112.5 (CH), 109.9 (CH), 105.3 (CH), 99.2 (C), 87.6 (C), 87.1 (CH), 84.4 (CH), 76.6 (C), 61.9 (CH2), 59.5 (C), 52.4 (CH3), 51.9 (CH3), 27.8 (CH3); IR (film) 3390, 3323, 2951, 2918, 2849, 1738, 1681, 1582; TLC Rf 0.41 (7:3 EtOAc/hexanes, plate was eluted twice); HRMS (ESI) m/z calcd for C39H30N6O10S3Na (M+Na)+ 861.1083, found 861.1087.</p><!><p>To a solution of ETP (+)-58 (5.0 mg, 6.0 μmol) in PhMe (0.10 mL) was added LiI (3 mg, 22 μmol). The reaction mixture was stirred for 1 h at 90 °C (formation of product was observed by RP-18 TLC), then a second portion of LiI (3 mg, 22 μmol) was added, and the reaction mixture was heated for 1 h at 90 °C. This sequence was repeated 2 more times (total: 12 mg of LiI, 4 h at 90 °C) before the reaction was allowed to cool to rt and poured into a mixture of EtOAc (5 mL) and 0.1% aqueous TFA (2 mL). The layers were separated, the aqueous layers were extracted with EtOAc (4 mL), and then the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. This reaction was conducted in series on 1, 2, 5, 5 mg scales (processed identically), then the crude residue from all four reactions was purified by reverse-phase HPLC (step-gradient eluting 20%, 40%, 50%, 60%, 70%, 95% MeCN/H2O + 0.1% TFA) to afford plectosphaeroic acid C (3) (9 mg, 64%, eluting at 60% MeCN/H2O + 0.1% TFA) as a red solid: [α]D
24 +494 (c 0.06, MeOH), compare with reported value [α]D
24 +136 (c 0.17, MeOH) of the natural sample; 1H NMR (500 MHz, DMSO-d6) δ 10.86 (br s, 1H), 9.72 (br s, 1H), 8.92 (br s, 1H), 8.02 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.37 (m, 1H), 7.36 (m, 1H), 7.10 (t, J = 7.0 Hz, 1H), 7.06 (comp, 2H), 6.72 (t, J = 7.4 Hz, 1H), 6.70 (s, 1H), 6.68 (s, 1H), 6.45 (br s, 1H), 6.32 (d, J = 7.8 Hz, 1H), 5.54 (s, 1H), 5.48 (br s, 1H), 3.78 (m, 1H), 3.61 (m, 1H), 3.11 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 178.1 (C), 168.9 (C), 167.5 (C), 166.8 (C), 162.1 (C), 152.7 (C), 150.8 (C), 150.3 (C), 141.1 (C), 137.1 (C), 135.5 (C), 132.0 (C), 131.2 (CH), 129.8 (C), 129.3 (CH), 127.4 (C), 125.1 (C, CH), 122.6 (CH), 121.1 (2 × CH), 119.5 (CH), 118.8 (CH), 117.3 (CH), 113.8 (C), 111.5 (CH), 108.7 (CH), 105.6 (CH), 92.5 (C), 86.3 (C), 85.7 (C), 81.7 (CH), 75.6 (C), 59.5 (CH2), 58.5 (C), 27.3 (CH3); RP-18 TLC Rf 0.50 (1:1 MeCN/H2O); HRMS (ESI) m/z calcd for C37H25N6O10S3 (M–H)− 809.0794, found 809.0779.</p><!><p>This procedure is a modification of a procedure reported by Manthey and coworkers.32b To a solution of 3-hydroxyanthranilic acid (S10) (350 mg, 2.3 mmol) and EtOH (200 mL) was added 1,4-benzoquinone (420 mg, 3.3 mmol, freshly recrystallized). The reaction mixture was maintained at 40 °C for 12 h, then cooled to rt and the desired product (250 mg, 72%) was filtered away from the mother liquor as an orange/red solid in sufficient purity. The spectroscopic data obtained for the product matched previously reported data;32c 1H NMR (500 MHz, DMSO-d6) δ 9.72 (br s 1H), 8.78 (br s, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 6.60 (s, 1H).</p><!><p>To a solution of 3-hydroxyanthranilic acid methyl ester (S11)82 (280 mg, 1.7 mmol) and EtOH (56 mL) was added 1,4-benzoquinone (310 mg, 2.8 mmol, freshly recrystallized). The reaction mixture was maintained at 40 °C for 24 h, then cooled to rt and concentrated in vacuo. The crude residue was suspended in ethyl acetate/hexanes (1:1, 60 mL), and the desired product (160 mg, 56%) was filtered away from the mother liquor as an orange/red solid in sufficient purity. The spectroscopic data obtained for the product matched previously reported data:11 1H NMR (500 MHz, DMSO-d6) δ 7.8 (br s, 2H), 7.65 (d, J = 6.1 Hz, 1H), 7.59–7.54 (m, 2H), 6.49 (s, 1H), 3.89 (s, 3H), 3.83 (s, 3H).</p>