A C–H Activation Approach to the Tricyclic Core of Glionitrin A and B

Abstract

Synthesis of diketopiperazines has been of long-standing interest in both natural product synthesis and medicinal chemistry. Here, we present an operationally convenient and efficient approach to the fused indoline-diketopiperazine tricyclic core of glionitrin A/B and related structures using a Pd-catalyzed C–H activation reaction to form the indoline five-membered ring. Exploratory work aimed at elaborating the tricyclic structures into the corresponding natural products is discussed.

Introduction

2,5-Diketopiperazines (DKPs) are ubiquitously found in natural products, materials, and drugs.^1,2 Consequently, this motif has been the subject of extensive synthetic efforts.²⁻⁴ Natural products containing dithiodiketopiperazines (DTDKPs) have drawn particular interest due to their challenging syntheses and broad spectra of useful biological properties.^5,6 A subclass of this natural product family comprises compounds wherein the DTDKP is fused to an indoline at the 2,3-positions. Such structures have received less synthetic attention than, for instance, the related non-aromatic gliotoxin (5)^7,8 (Figure 1A). Examples include (−)-glionitrin A (1)⁹ and B (2),¹⁰ but the motif is also shared in aromatic gliotoxin homologues like (+)-deoxydehydrogliotoxin (3)¹¹ and (+)-dehydrogliotoxin (4).¹²

Figure 1.

(A) Selected natural products; (B) selected previous C–H activation approaches to indoline-fused scaffolds; and (C) retrosynthesis of the tricyclic core of glionitrin A and B.

The first syntheses of (−)-glionitrin A and B were recently reported by our group.¹³ In this study, the DTDKP motif was constructed in a stepwise manner outgoing from a triketopiperazine precursor. Still, a more direct approach would be to form the disulfide bridge on a DKP scaffold.¹⁴ Practical access to a suitably functionalized substrate of this type is thus of considerable interest for exploring new synthetic pathways to indoline-fused DTDKPs. Moreover, the glionitrins were originally isolated from a co-culture of two microbes, the bacterial strain Sphingomonas KMK-001 and the fungal strain Aspergillus fumigatus KMC-901, and details of their biosyntheses remain unclear.^9,10 It seems plausible that DKP derivatives like 11 (Figure 1C) would lie on the biosynthetic pathway,¹⁵ and access to such compounds may aid in shedding light on the biosynthetic machineries that produce these fascinating structures.

Here, we describe a C–H activation approach leading to concise and practical syntheses of the complete tricyclic cores of glionitrin A/B and dehydrodeoxygliotoxin with defined stereochemistry. Exploratory studies toward elaborating these structures to the corresponding natural products by direct S–S bridge construction or via α-oxidation reactions are also discussed.

Indoline-fused DKPs have been synthesized through several approaches.¹⁶⁻²⁷ Variations drawing on metal-catalyzed bond activation for forming the indoline five-membered ring are particularly attractive as these allow for simple starting materials.²²⁻²⁶ An elegant example is the palladium-catalyzed sp³-C–H activation approach by Baudoin and co-workers to form indoline 8 from DKP 7 (Figure 1B),²⁵ which has since been applied to total syntheses of several DTDKP natural products.^28,29 More recently, Xuan and co-workers described a palladium-catalyzed sp²-C–H activation to produce 10, wherein the amide serving as a directing group for the C–H activation step is also incorporated in the formed ring.²⁶

For the purpose of developing practical syntheses of the tricyclic cores of the glionitrins and homologous structures, we envisioned expanding on Xuan’s approach²⁶ by elaborating the more functionalized dipeptide 13 to indoline 12 (Figure 1C). From this structure, tricyclic 11, carrying the hydroxymethyl side chain of the glionitrins, would be completed by cyclization to a DKP followed by N-methylation and deprotection.

Results and Discussion

We first targeted protected dipeptide 16 as a suitable cyclization precursor. Synthetically, N-boc-protected serine 14 was coupled with nitro-phenylalanine 15 using HATU (Scheme 1A). Protection of the free alcohol was needed to avoid side reactions such as C–O bond cleavage³⁰ or oxidation³¹ during the subsequent C–H activation reaction. A tert-butyldiphenyl silyl (TBDPS) group was found suitable for this purpose and also proved beneficial by improving the solubility of intermediates. We initially explored cyclization of a dipeptide substrate with the requisite N-methyl group already installed. However, in agreement with observations by Xuan,²⁶ this derivative was found incompatible with the C–H activation step due to the inability to form a bidentate coordination with the metal.

Scheme 1. (A) Synthesis of the Tricyclic Core of Gliontrin A and B; (B) scXRD Structure of DKP 11; (C) Synthesis of Tricyclic Structure 21; and (D) Synthesis of Tricyclic Structure 24^,

Reagents and conditions: (1) 15^.HCl (1.05 equiv) or H-Phe-OMe^.HCl (1.1 equiv), HATU (1.0–1.2 equiv), HOBt (1.0–1.2 equiv), Et₃N (4.0 equiv) or DIPEA (4.0 equiv), CH₂Cl₂, 0 °C to rt, 15–21 h; (2) TBDPSCl (1.1–1.2 equiv), Im (1.2–1.4 equiv), DMAP (10 mol %), CH₂Cl₂, 0 °C to rt, 90–105 min; (3) Pd(OAc)₂ (5 mol %), PhI(OAc)₂ (2.0 equiv), toluene, reflux, 16–24 h; (4) TFA (excess), CH₂Cl₂, 0 °C to rt, 0.75–3 h; (5) K₂CO₃ (25 equiv), MeI (excess), acetone, rt or reflux, 6–7 d; (6) HF (aq, 48% w/w, excess), pyridine, rt, 1–20 h. (7) acetic anhydride, 100 °C, microwave irradiation, 2 h. boc = tert-butyl carbamate, DMAP = 4-(dimethylamino)pyridine, Im = imidazole, TBDPSCl = tert-butyldiphenylsilyl chloride, DIPEA = N,N-diisopropylethylamine, HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, and HOBt = 1-hydroxybenzotriazole;

thermal ellipsoids are shown at 50% probability.

With 16 in hand, the stage was set for investigating the C–H activation step to form the indoline five-membered ring of the targeted tricyclic system. To this end, we employed Xuan’s optimized conditions:²⁶ Pd(OAc)₂ (5 mol %) and PhI(OAc)₂ (2.0 equiv) in refluxing toluene. Pleasingly, indoline 12 could be isolated along with recovered starting material in a 2:1 ratio. A brief optimization of the reaction conditions showed that increasing the amount of PhI(OAc)₂ to 4.0 equiv improved the yield to 50% (see Supporting Information, Table S1), but for gram-scale synthesis, we settled on the original protocol.

Due to the difficult separation of the mixture of 16 and 12 obtained in the C–H activation step, the products were directly subjected to trifluoroacetic acid (TFA) to give DKP 17 in good overall yield (49% over four steps). It is worth noting that the sequence of reactions starting from commercially available 14 could be telescoped without chromatographic purification of intermediates to produce >9 g of 17 in a single pass.

Next, we turned our attention to N-methylation of amide 17. This transformation proved challenging due to competing β-siloxy elimination in presence of strong bases like NaH or NaHMDS. Ultimately, we found that amide 17 was smoothly methylated using an excess of MeI with K₂CO₃ as the base³² to give N-methyl DKP 30 in 89% yield. Synthesis of the tricyclic core of glionitrin A and B (11) was then completed by liberation of the primary alcohol with HF (>95% yield). The structure of alcohol 11 was confirmed by single crystal X-ray diffraction (scXRD) analysis (Scheme 1B). The conversion of 11 into the corresponding acetate 18 was also straightforwardly accomplished in 94% yield by reaction with acetic anhydride under microwave irradiation for 2 h at 100 °C (Scheme 1A)

We then applied the same approach to diastereomeric des-nitro derivative 21 (Scheme 1C). This structure was obtained in six steps from N-boc-protected serine 14 in 17% overall yield. In a similar fashion, the corresponding glycine derivative 24 was also produced in 21% yield over four steps (Scheme 1D).

With a practical method to access to the tricyclic cores of the glionitrins and two related structures in place, we explored methods aimed at elaborating these to the corresponding epi-DTDKPs.^{8,22,28,29,33−35} To this end, we first investigated direct installation of the disulfide bridge by reacting DKP enolates with electrophilic sulphur.^8,14 The reactions of unprotected 11, 21, and 24 or the protected 30 and 35 using various bases (LiHMDS, NaHMDS, or KHMDS) and elemental sulfur (S₈) as the electrophile precursor^8,14 following literature procedures⁸ gave slightly varying results, but in no case was formation of the corresponding epi-DTDKP products observed as evident by comparison of the crude ¹H NMR spectra with those of known structures (Scheme 2).

Scheme 2. Attempted Direct Sulfenylation of DKPs 11, 21, and 24^,

Reagents and conditions: (1) NaHMDS, LiHMDS, or KHMDS (5.0–16 equiv), S₈ (1.0–2.0 equiv), THF, −78 °C or room temperature, 1.5–3 h;

using LiHMDS.

In a brief summary, reacting alcohol 11 with LiHMDS (8.0 equiv) and S₈ (1.0 equiv) with the intent of forming glionitrin A gave a mixture of indole 25 along with recovered starting material. Switching the base to NaHMDS or KHMDS gave more complex mixtures of unidentified side products together with indole 25 and unreacted 11. Similar results were obtained when switching the order of addition [LiHMDS (4.0 equiv) first and a mixture of LiHMDS (4.0 equiv)/S₈ (1.0 equiv) second], decreasing the reaction temperature (−78 °C), or doubling the amounts of reagents [LiHMDS (16.0 equiv) and S₈ (2.0 equiv)]. Indole formation was also observed when exposing des-nitro indoline 21 or indoline 24 to the same conditions. In the reaction of 21 with LiHMDS (8.0 equiv) and S₈ (1.0 equiv), we also observed olefin 27 as a minor side product. Reactions of the corresponding O-silylated substrates under the same conditions followed a similar pattern: using LiHMDS, NaHMDS, or KHMDS as the base, silyl ethers 30 and 35 were both converted into complex mixtures.

Although examples of successful formation of epi-DTDKPs on indoline-fused DKP scaffolds have been reported using the here-evaluated procedures in moderate to good yields,^8,14,36 our results suggest that substrates like 11 are not amenable to these conditions and that the encountered difficulties are not easily circumvented by variations in reaction conditions and operational procedures.

An alternative approach to construct disulfide bridges on DKP scaffolds is the biomimetic α-oxidation with permanganate reagents pioneered by Movassaghi.^32,37−42 The hemi-aminal DKPs formed with this procedure have been successfully transformed into highly complex natural product targets via acid-promoted sulfenylation.^32,37,38 Inspired by this work, we explored oxidation of DKPs 18, 24, 30, and 35 with silver(I)bispyridine permanganate (Scheme 3).⁴³

Scheme 3. α-Oxidation of DKPs 18, 24, 30, and 35.

Reagents and conditions: (1) silver(I)bispyridine permanganate (4.0–8.0 equiv), CH₂Cl₂, rt, 15–24 h; (2) HF^.py, pyridine, rt, 2 h.

Thus, oxidation of silyl ether 30 with silver(I)bispyridine permanganate (8.0 equiv) in CH₂Cl₂ at room temperature gave a difficult to separate 40:60 mixture of unreacted starting material and C10a mono-oxidized 31 as a single diastereomer (35% yield). Trace amounts of the C3a/C10a doubly oxidized product were detected by HRMS analysis of the crude reaction mixture. Treatment of the mixture of 30 and 31 with HF provided diol 32.

Oxidation of acetate 18 with silver(I)bispyridine permanganate (8.0 equiv) was also evaluated and gave a mixture of C10a mono-oxidized 33 (22% yield) and C3a/C10a doubly oxidized 34 (8% yield). In contrast, des-nitro silyl ether 35, which is less deactivated at the C3a position, reacted smoothly with silver(I)bispyridine permanganate (4.0 equiv) to produce diol 36 in 46% yield as a single observed diastereomer. Similarly, oxidation of indoline 24 produced diol 37, albeit in only 12% isolated yield.

Assignment of the relative configuration for the oxidized products was attempted using 2D-NMR spectroscopy experiments; however, the results were inconclusive. For 31–34 and 36/37, the differences in distance between the C10a hydroxyl group proton and protons at C3/C3a position are too small to unambiguously distinguish between the cis and trans configurations by NOESY correlations. For 32, 34, and 36/37, a further complication was magnetization transferred between the alcohol protons due to chemical exchange.

Sulfenylation of the obtained hemi-aminals 31, 34, and 36 was investigated next (Scheme 4). Substrates with hemi-aminals at C10a cleanly eliminated into the corresponding indoles, whereas Lewis acid-promoted thio-aminal formation proved fruitful at the C3a position. The reaction of alcohol 31 with BF₃·Et₂O in the presence of 4-methoxy-α-toluenethiol thus gave indole 38 (>95% yield). Under the same reaction conditions, diol 34 was converted into C3a sulfenylated indole 39 (60% yield) and diol 36 into C3a sulfenylated indole 40 (74% yield).

Scheme 4. Sulfenylation of Hemi-Aminals 31, 34, and 36 and Attempted Sulfenylation of Indole 38.

Reagents and conditions: (1) BF₃·Et₂O (20.0 equiv), 4-methoxy-α-toluenethiol (10.0 equiv), CH₂Cl₂, rt, 1 h; (2) rac-camphorsulfonic acid (10.0 equiv) or HBr (33% w/w in AcOH, excess) or BF₃·Et₂O (10.0 equiv), 4-methoxy-α-toluenethiol (4.0 equiv), CH₂Cl₂, reflux, 1 h.

Finally, we evaluated conversion of indole 38 to the corresponding C10a sulfenylated product with 4-methoxy-α-toluenethiol using various acids⁴⁴ (camphorsulfonic acid, HBr, or BF₃·Et₂O). In all cases, 38 was recovered unreacted.

Thus, while the tricyclic substrates were successfully oxidized at C10a, and in case of 18, 24, and 35, also at C3/C3a, the propensity to eliminate to the corresponding indoles was found prohibitive for the purpose of conversion to the corresponding epi-DTDKP natural products under the investigated conditions.

Conclusions

In conclusion, a concise, simple, and scalable route to the complete indoline-fused DKP core of the glionitrins and related natural products has been developed. Xuan’s palladium-catalyzed C–H activation²⁶ was applied to form the indoline five-membered ring from simple dipeptides which, in turn, were readily prepared from abundant and enantiomerically pure amino acids. The approach connects with putative intermediates in the biosyntheses of several natural products. Investigations aimed to elaborate the obtained tricyclic structures into their corresponding DTDKPs, either by direct S–S bridge construction or sulfenylation via oxidative conversion to the corresponding bis-hemi-aminals, were not met with success due to competing indole formation. The development of milder conditions for direct S–S bridge construction and for sulfenylation of C10a hemi-aminals thus remains an outstanding challenge with sensitive indoline-fused DKP scaffolds. An additional interesting question is that of absolute stereocontrol in such reactions. Studies aimed at addressing these problems are under way in our laboratory and will be reported in due course.

Experimental Section

General Procedures

All reactions were conducted in air unless otherwise stated. Reactions involving hydrogen fluoride were carried out in plastic vessels. The experiment involving microwave irradiation was performed using a Biotage Initiator+. All reagents and solvents were bought from commercial suppliers and used as received unless otherwise stated. Dichloromethane and toluene were obtained from a MBraun MB-SPS 800 solvent purification system. THF was distilled over sodium benzophenone ketyl. S₈ was recrystallized from benzene. ¹H and ¹³C (¹H decoupled) NMR spectroscopy data were collected on a Bruker AVANCE II 400 MHz (¹H 400 MHz; ¹³C 101 MHz) equipped with a 5 mm BBOF Z-gradient probe. Multiplicities are denoted by singlet (s), doublet (d), doublet of doublets (dd), doublet of doublet of doublets (ddd), triplet of doublets (td), triplet (t), apparent triplet (app. t), doublet of triplets (dt), and multiplet (m). Broad peaks are denoted by (br). IR spectra were recorded on a Bruker ALPHA II spectrometer and peaks denoted as strong (s), medium (m), weak (w), and broad (br). Optical rotations [α]_D^T were recorded at room temperature (∼20 °C) using a PerkinElmer model 341 polarimeter. D represents the sodium D line (589 nm), and concentrations (c) are reported in g/100 mL. HRMS data were obtained using an ESI-QTOF mass spectrometer (Waters Xevo-G2) in the positive mode between m/z 50–1200, employing lockmass correction according to the manufacturer’s instructions. Thin layer chromatography (TLC) was performed using Merck 60 F₂₅₄ silica gel bound to aluminum plates. Purification by column chromatography was performed using Merck 60 Å (40–63 μm particle size) silica or using the Biotage Isolera One system.

Methyl (R)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-hydroxypropanamido)-3-(4-nitrophenyl)propanoate (13)

To a stirred suspension of serine 14 (7.61 g, 37.1 mmol) and nitrophenyl alanine 15^.HCl⁴⁵ (10.2 g, 38.9 mmol) in CH₂Cl₂ (500 mL) under a N₂ atmosphere was added N,N-diisopropylethylamine (27.1 mL, 157 mmol) in one portion. The resulting clear yellow solution was cooled to 0 °C using an ice-water bath, and 1-hydroxybenzotriazole hydrate (5.26 g, 38.9 mmol) was added in one portion, followed by portionwise addition of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (14.8 g, 38.9 mmol). After 21 h, the resulting reaction mixture was washed with HCl (2 × 200 mL, aq, 1 M), NaHCO₃ (2 × 200 mL, sat. aq), and brine (200 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was used without further purification in the next reaction. Analytically pure dipeptide 13 was obtained by purification by column chromatography (50–100% EtOAc/n-heptane). Yield: 14.4 g (crude). Purified 13 was isolated as faint yellow crystals. >95% pure by NMR and a single spot by TLC. R_f: 0.33 in 75% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −54.1 (c = 1.06, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.19–8.13 (m, 2H), 7.36–7.30 (m, 2H), 7.24–7.15 (m, 1H), 5.51 (br, d, J = 7.6 Hz, 1H), 4.94–4.85 (m, 1H), 4.20–4.00 (m, 2H), 3.74 (s, 3H), 3.68–3.58 (m, 1H), 3.30 (dd, J = 14.0, 5.6 Hz, 1H), 3.17 (dd, J = 14.0, 6.8 Hz, 1H), 2.58 (dd, J = 8.8, 4.8 Hz, 1H), 1.42 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 171.5, 171.2, 156.3, 147.4, 143.7, 130.3, 123.9, 81.0, 62.8, 55.2, 53.0, 52.9, 37.7, 28.4 ppm. FTIR (neat): 3535 (br), 3486 (br), 3357 (br), 3258 (br), 1733 (m), 1682 (m), 1664 (s), 1542 (m), 1524 (s), 1346 (s), 1279 (m), 1245 (m), 1161 (s), 1051 (m), 859 (m), 756 (m), 687 (m), 518 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₂₅N₃O₈, 412.1720; found, 412.1716. mp 92–94 °C (obtained by crystallization from EtOAc/n-heptane).

Methyl (R)-2-((S)-2-((tert-Butoxycarbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)propanamido)-3-(4-nitrophenyl)propanoate (16)

To a stirred solution of crude dipeptide 13 (14.4 g) in CH₂Cl₂ (250 mL) at 0 °C under a N₂ atmosphere was added imidazole (2.86 g, 42.0 mmol) in one portion. tert-Butyl(chloro)diphenylsilane (10.0 mL, 38.5 mmol) was then added dropwise over 5 min, after which 4-(dimethylamino)pyridine (429 mg, 3.50 mmol) was added in one portion. The reaction mixture turned white and cloudy. After 45 min, the cooling bath was removed, and after a further 45 min, the resulting reaction mixture was washed with HCl (3 × 100 mL, aq, 1 M) and brine (100 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was used in the next reaction without further purification. Analytically pure silyl ether 16 was obtained by purification by column chromatography (33–75% EtOAc/n-heptane). Yield: 26.6 g (crude). Purified 16 was isolated as a white amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.25 in 33% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −20.8 (c = 0.53, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.13–8.05 (m, 2H), 7.67–7.57 (m, 4H), 7.50–7.36 (m, 6H), 7.31–7.23 (m, 2H), 7.01 (br, d, J = 8.0 Hz, 1H), 5.20 (br, d, J = 7.6 Hz, 1H), 4.94 (dt, J = 7.6, 6.0 Hz, 1H), 4.32–4.20 (m, 1H), 4.12 (dd, J = 10.4, 4.4 Hz, 1H), 3.79 (dd, J = 10.4, 4.8 Hz, 1H), 3.74 (s, 3H), 3.29 (dd, J = 13.6, 6.0 Hz, 1H), 3.17 (dd, J = 13.6, 6.0 Hz, 1H), 1.45 (s, 9H), 1.05 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 171.1, 170.2, 155.7, 147.3, 143.6, 135.7, 135.6, 132.9, 132.5, 130.4, 130.21, 130.19, 128.1, 128.0, 123.8, 80.7, 64.1, 56.4, 53.0, 52.7, 38.0, 28.4, 26.9, 19.4 ppm. FTIR (neat): 3339 (w), 3322 (w), 2952 (w), 2855 (w), 1735 (m), 1652 (m), 1515 (s), 1344 (m), 1109 (m), 700 (m), 613 (m), 508 (m), 487 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₄H₄₃N₃O₈Si, 650.2898; found, 650.2897. mp 130–131 °C (obtained by concentration from EtOAc/n-heptane).

Methyl (R)-1-[N-(tert-Butoxycarbonyl)-O-(tert-butyldiphenylsilyl)-l-seryl]-6-nitroindoline-2-carboxylate (12)

A stirred suspension of crude silyl ether 16 (26.5 g), PhI(OAc)₂ (22.5 g, 70.0 mmol), and Pd(OAc)₂ (393 mg, 1.75 mmol) in toluene (250 mL) was heated to reflux. After 16 h, the resulting reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was taken up in CH₂Cl₂, filtered through a plug of silica, and concentrated under reduced pressure. The resulting crude residue was used in the next reaction without further purification. Analytically pure indoline 12 was obtained by purification by column chromatography (25% EtOAc/n-heptane). Yield: 37.8 g (crude). Purified 12 was isolated as yellow oil. >95% pure by NMR and a single spot by TLC. R_f: 0.27 in 33% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: +27.4 (c = 0.50, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.93 (br, s, 1H), 7.95 (dd, J = 8.0, 2.0 Hz, 1H), 7.75–7.56 (m, 4H), 7.54–7.19 (m, 7H), 5.79 (br, d, J = 8.8 Hz, 1H), 5.09 (br, d, J = 8.8 Hz, 1H), 4.59–4.45 (m, 1H), 4.06 (dd, J = 10.4, 4.8 Hz, 1H), 3.88 (dd, J = 10.4, 8.4 Hz, 1H), 3.80–3.52 (m, 4H), 3.41 (br, d, J = 16.0 Hz, 1H), 1.43 (s, 9H), 1.08 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 171.3, 170.2, 155.9, 148.2, 143.2, 137.0, 135.74, 135.70, 133.2, 133.1, 130.1, 130.0, 128.0, 127.9, 124.6, 120.2, 113.0, 80.4, 63.6, 61.1, 54.8, 53.2, 33.3, 28.4, 26.9, 19.4 ppm. FTIR (film): 3342 (br), 2955 (w), 2931 (w), 2858 (w), 1748 (m), 1704 (m), 1668 (m), 1525 (m), 1481 (m), 1428 (m), 1344 (m), 1161 (m), 1111 (s), 1007 (m), 909 (m), 819 (m), 732 (m), 701 (s), 613 (m), 503 (m), 486 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₄H₄₁N₃O₈Si, 648.2741; found, 648.2753.

(3S,10aR)-3-{[(tert-Butyldiphenylsilyl)oxy]methyl}-7-nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (17)

To a stirred solution of crude indoline 12 (37.8 g) in CH₂Cl₂ (250 mL) at 0 °C under a N₂ atmosphere was added TFA (10 mL) dropwise over 5 min. Additional TFA (20 mL) was then added in one portion. After 30 min, the cooling bath was removed, and after a further 115 min, TFA (10 mL) was added in one portion. After a further 1 h, water (200 mL) was added, and the resulting mixture was neutralized with NaHCO₃ (s). The organic phase was separated, and the aqueous phase was extracted with CH₂Cl₂ (3 × 100 mL). The combined organic extracts were concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (50–75% EtOAc/n-heptane) to give diketopiperazine 17. Yield: 9.37 g (49% over four steps from 14). Isolated as a beige amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.19 in 50% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −8.5 (c = 0.53, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.94 (d, J = 2.2 Hz, 1H), 8.04 (dd, J = 8.2, 2.2 Hz, 1H), 7.62–7.54 (m, 4H), 7.46–7.30 (m, 7H), 6.39 (d, J = 4.0 Hz, 1H), 5.09 (app. t, J = 10.8, 10.0 Hz, 1H), 4.26 (dd, J = 10.4, 4.0 Hz, 1H), 4.21–4.16 (m, 1H), 3.93 (dd, J = 10.4, 2.8 Hz, 1H), 3.49 (ddd, J = 17.4, 10.8, 1.6 Hz, 1H), 3.37 (dd, J = 17.4, 10.0 Hz, 1H), 1.03 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 168.5, 163.8, 148.2, 142.2, 136.8, 135.57, 135.56, 132.3, 132.1, 130.4, 128.20, 128.16, 125.3, 120.9, 111.5, 66.7, 60.6, 60.3, 31.8, 27.0, 19.3 ppm. FTIR (film): 3242 (br), 2931 (w), 2858 (w), 1684 (s), 1601 (w), 1526 (m), 1479 (m), 1429 (m), 1343 (m), 1107 (m), 738 (m), 702 (m), 505 (w) cm^–1. HRMS-ESI (m/z): [M + NH₄]⁺ calcd for C₂₈H₂₉N₃O₅Si, 533.2220; found, 533.2219. mp 209–210 °C (obtained by concentration from EtOAc/n-heptane).

(3S,10aR)-3-{[(tert-Butyldiphenylsilyl)oxy]methyl}-2-methyl-7-nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (30)

To a stirred suspension of diketopiperazine 17 (5.87 g, 11.4 mmol) and K₂CO₃ (39.3 g, 284 mmol) in acetone (100 mL) was added iodomethane (60.0 mL, 964 mmol) in one portion. The flask was wrapped in aluminum foil. After 7 d, the resulting reaction mixture was filtered and concentrated under reduced pressure. Safety note: Iodomethane is toxic. Handling of this reagent and decontamination of equipment require appropriate protocols to avoid exposure. The resulting crude residue was purified by column chromatography (33–75% EtOAc/n-heptane) to give N-methyl diketopiperazine 30. Yield: 5.35 g (89%). Isolated as a beige amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.40 in 50% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −2.4 (c = 0.21, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.93 (d, J = 2.2 Hz, 1H), 8.04 (dd, J = 8.2, 2.2 Hz, 1H), 7.61–7.52 (m, 4H), 7.46–7.30 (m, 7H), 5.15 (app. t, J = 10.8, 10.0 Hz, 1H), 4.26 (dd, J = 11.8, 3.6 Hz, 1H), 4.04 (dd, J = 11.8, 2.8 Hz, 1H), 4.04–4.00 (m, 1H), 3.50 (ddd, J = 17.4, 10.8, 1.2 Hz, 1H), 3.39 (dd, J = 17.4, 10.0 Hz, 1H), 2.99 (s, 3H), 1.01 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 166.8, 163.8, 148.2, 142.0, 137.5, 135.6, 135.5, 132.3, 132.0, 130.4, 128.23, 128.17, 125.3, 120.9, 111.5, 67.3, 63.6, 60.5, 32.7, 32.2, 27.0, 19.3 ppm. FTIR (film): 2931 (w), 2858 (w), 1677 (s), 1527 (m), 1483 (m), 1430 (m), 1346 (m), 1110 (m), 740 (m), 704 (m) cm^–1. HRMS-ESI (m/z): [M + NH₄]⁺ calcd for C₂₉H₃₁N₃O₅Si, 547.2377; found, 547.2371. mp 154–156 °C (obtained by concentration from EtOAc/n-heptane).

(3S,10aR)-3-(Hydroxymethyl)-2-methyl-7-nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (11)

To a stirred solution of N-methyl diketopiperazine 30 (670 mg, 1.26 mmol) in pyridine (6 mL) was added hydrofluoric acid (2 mL, 48% w/w) in one portion. After 1 h, the resulting reaction mixture was neutralized with NaHCO₃ (s). The resulting mixture was diluted with water and extracted with EtOAc (2 × 20 mL). The combined organic extracts were washed with HCl (20 mL, aq, 1 M) and brine (50 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (EtOAc) to give alcohol 11. Yield: 365 mg (>95%). Isolated as a yellow amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.19 in EtOAc. Optical rotation: [α]_D²⁰: +79.4 (c = 0.64, DMSO). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.65 (d, J = 2.2 Hz, 1H), 8.00 (dd, J = 8.2, 2.2 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 5.54 (app. t, J = 5.6, 5.2 Hz, 1H), 5.20 (app. t, J = 10.8, 10.0 Hz, 1H), 4.17 (app. t, J = 2.8, 2.4 Hz, 1H), 3.92 (ddd, J = 11.7, 5.2, 2.4 Hz, 1H), 3.88 (ddd, J = 11.7, 5.6, 2.8 Hz, 1H), 3.46 (dd, J = 17.6, 10.0 Hz, 1H), 3.32 (ddd, J = 17.6, 10.8, 1.2 Hz, 1H), 2.96 (s, 3H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 166.4, 165.1, 146.9, 141.9, 139.0, 125.9, 120.2, 109.3, 66.6, 61.0, 59.9, 31.7, 31.6 ppm. FTIR (neat): 3368 (br), 1680 (m), 1650 (m), 1519 (m), 1482 (m), 1402 (m), 1331 (m), 1074 (m), 1060 (m), 892 (m), 815 (m), 742 (m), 522 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₃N₃O₅, 292.0933; found, 292.0934. mp 224–225 °C (obtained by concentration from EtOAc).

[(3S,10aR)-2-Methyl-7-nitro-1,4-dioxo-1,2,3,4,10,10a-hexahydropyrazino(1,2-a)indol-3-yl]methyl Acetate (18)

A stirred suspension of alcohol 11 (291 mg, 1.00 mmol) in acetic anhydride (3.0 mL) was heated with microwave irradiation to 100 °C. After 2 h, the resulting reaction mixture was cooled to room temperature and diluted with EtOAc (25 mL). The resulting solution was washed with NaHCO₃ (3 × 10 mL) and brine (10 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (50–75% EtOAc/n-heptane) to give acetate 18. Yield: 313 mg (94%). Isolated as a yellow amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.22 in 75% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: +103 (c = 0.1, CHCl₃). ¹H NMR (CDCl₃, 400 MHz): δ 8.87 (d, J = 2.4 Hz, 1H), 8.03 (dd, J = 8.4, 2.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 5.03 (app. t, J = 10.8, 10.0 Hz, 1H), 4.68 (dd, J = 12.0, 4.0 Hz, 1H), 4.48 (dd, J = 12.0, 3.6 Hz, 1H), 4.25 (app. t, J = 4.0, 3.6 Hz, 1H), 3.58 (ddd, J = 17.2, 10.8, 1.6 Hz, 1H), 3.52 (dd, J = 17.2, 10.0 Hz, 1H), 3.10 (s, 3H), 2.08 (s, 3H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 170.0, 166.4, 162.6, 148.2, 141.9, 137.2, 125.3, 121.1, 111.7, 64.3, 62.6, 60.1, 32.9, 32.4, 20.9 ppm. FTIR (film): 2938 (w), 1746 (m), 1675 (s), 1601 (w), 1525 (m), 1484 (m), 1435 (m), 1403 (m), 1346 (m), 1226 (m), 1076 (w), 1058 (w), 1035 (w), 892 (w), 831 (w), 739 (w) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₅H₁₅N₃O₆, 334.1039; found, 334.1036. mp 164–166 °C (obtained by concentration from EtOAc/n-heptane).

Methyl (tert-Butoxycarbonyl)-l-seryl-l-phenylalaninate (41)⁴⁶

To a stirred solution of serine 14 (2.05 g, 10.0 mmol) and l-phenylalanine methyl ester hydrochloride (2.37 g, 11.0 mmol) in CH₂Cl₂ (100 mL) under a N₂ atmosphere was added N,N-diisopropylethylamine (6.97 mL, 40.0 mmol) in one portion. The resulting clear colorless solution was cooled to 0 °C with an ice-water bath, and 1-hydroxybenzotriazole hydrate (1.62 g, 12.0 mmol) was added in one portion, followed by portionwise addition of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (4.56 g, 12.0 mmol). The reaction mixture turned bright yellow. After 19 h, the resulting reaction mixture was washed with NaHCO₃ (2 × 40 mL, sat. aq), HCl (2 × 40 mL, aq, 1 M), and brine (40 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was used in the next reaction without further purification. Analytically pure dipeptide 41 was obtained by purification by column chromatography (50–75% EtOAc/n-heptane). Yield: 9.1 g (crude). Purified 41 was isolated as colorless oil that partially solidified upon standing. >95% pure by NMR and a single spot by TLC. ¹H NMR (CDCl₃, 400 MHz): δ 7.35–7.17 (m, 3H), 7.17–7.08 (m, 2H), 7.07–6.90 (m, 1H), 5.55–5.39 (m, 1H), 4.91–4.76 (m, 1H), 4.16 (br, s, 1H), 4.00 (br, d, J = 11.2 Hz, 1H), 3.73 (s, 3H), 3.60 (dd, J = 11.2, 5.6 Hz, 1H), 3.18 (dd, J = 14.0, 5.6 Hz, 1H), 3.05 (dd, J = 14.0, 6.8 Hz, 1H), 2.77 (br, s, 1H), 1.44 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 171.9, 171.2, 156.0, 135.8, 129.3, 128.8, 127.3, 80.6, 63.0, 55.1, 53.5, 52.6, 37.8, 28.4 ppm. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₂₆N₂O₆, 367.1869; found, 367.1867.

Methyl N-(tert-Butoxycarbonyl)-O-(tert-butyldiphenylsilyl)-l-seryl-l-phenylalaninate (19)

To a stirred solution of crude dipeptide 41 (9.1 g) in CH₂Cl₂ (100 mL) under a N₂ atmosphere were added imidazole (953 mg, 14.0 mmol) and 4-(dimethylamino)pyridine (122 mg, 1.00 mmol) in one portion. The reaction mixture was cooled to 0 °C with an ice-water bath, and tert-butyl(chloro)diphenylsilane (3.11 mL, 12.0 mmol) was added dropwise over 2 min. After 105 min, the resulting reaction mixture was washed with HCl (3 × 50 mL, aq, 1 M) and brine (50 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (12.5–50% EtOAc/n-heptane) to give silyl ether 19. Yield: 5.40 g (89% over two steps from 14). Isolated as a white amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.44 in 33% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: +24.2 (c = 0.53, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.72–7.57 (m, 4H), 7.48–7.33 (m, 6H), 7.31–7.17 (m, 3H), 7.16–7.03 (m, 2H), 6.91 (br, d, J = 7.2 Hz, 1H), 5.19 (br, s, 1H), 4.93–4.83 (m, 1H), 4.25 (br, s, 1H), 4.01 (dd, J = 10.0, 4.4 Hz, 1H), 3.77 (dd, J = 10.0, 5.8 Hz, 1H), 3.65 (s, 3H), 3.20–3.05 (m, 2H), 1.42 (s, 9H), 1.04 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 171.6, 170.1, 155.6, 135.9, 135.7, 135.6, 133.0, 132.6, 130.0, 130.0, 129.4, 128.7, 128.00, 127.98, 127.3, 80.3, 64.1, 55.9, 53.6, 52.4, 38.3, 28.4, 26.9, 19.4 ppm. FTIR (film): 3342 (br), 3070 (w), 2931 (w), 2858 (w), 1745 (m), 1677 (s), 1496 (m), 1366 (m), 1168 (m), 1112 (s), 742 (w), 702 (s), 505 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₄H₄₄N₂O₆Si, 605.3056; found, 605.3060. mp 116–117 °C (obtained by concentration from EtOAc/n-heptane).

Methyl (S)-1-[N-(tert-Butoxycarbonyl)-O-(tert-butyldiphenylsilyl)-l-seryl]indoline-2-carboxylate (42)

A stirred suspension of silyl ether 19 (5.00 g, 8.27 mmol), PhI(OAc)₂ (5.33 g, 16.5 mmol), and Pd(OAc)₂ (92.8 mg, 413 μmol) in toluene (83 mL) was heated to reflux. After 24 h, the resulting reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (12.5% EtOAc/n-heptane) to give indoline 42. Yield: 2.16 g (43%). Isolated as golden oil/foam. >95% pure by NMR and a single spot by TLC. R_f: 0.29 in 25% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −50.6 (c = 0.69, CHCl₃). ¹H NMR (CDCl₃, 400 MHz): δ 8.33 (d, J = 8.0 Hz, 1H), 7.61–7.51 (m, 4H), 7.47–7.34 (m, 3H), 7.33–7.23 (m, 4H), 7.18–7.12 (m, 1H), 7.12–7.04 (m, 1H), 5.39 (d, J = 9.2 Hz, 1H), 5.28 (dd, J = 9.2, 4.0 Hz, 1H), 4.75 (td, J = 9.2, 5.6 Hz, 1H), 3.88 (dd, J = 9.2, 5.6 Hz, 1H), 3.83–3.73 (m, 4H), 3.38–3.21 (m, 2H), 1.43 (s, 9H), 0.99 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 171.8, 170.6, 154.8, 142.2, 135.6, 135.5, 132.7, 132.7, 130.0, 129.2, 128.0, 127.9, 124.7, 124.5, 117.6, 79.8, 66.1, 60.6, 53.8, 53.3, 33.2, 28.4, 26.9, 19.3 ppm. FTIR (film): 3433 (w), 2955 (w), 2931 (w), 2858 (w), 1743 (m), 1712 (m), 1658 (m), 1480 (m), 1414 (m), 1366 (m), 1163 (m), 1106 (m), 1014 (m), 909 (m), 823 (w), 756 (m), 731 (s), 701 (s), 613 (m), 504 (m), 489 (w) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₄H₄₂N₂O₆Si, 603.2890; found, 603.2894.

(3S,10aS)-3-{[(tert-Butyldiphenylsilyl)oxy]methyl}-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (20)

To a stirred solution of indoline 42 (2.16 g, 3.58 mmol) in CH₂Cl₂ (30 mL) at 0 °C under a N₂ atmosphere was added TFA (10 mL) dropwise over 5 min. After 50 min, the resulting reaction mixture was poured out onto water (50 mL) and neutralized with NaHCO₃ (s). The organic phase was separated, and the aqueous phase was extracted with CH₂Cl₂ (2 × 50 mL). The combined organic extracts were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (40% EtOAc/n-heptane) to give diketopiperazine 20. Yield: 1.22 g (72%). Isolated as yellow foam. >95% pure by NMR and a single spot by TLC. R_f: 0.40 in 50% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −26.4 (c = 0.72, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.00 (d, J = 7.6 Hz, 1H), 7.70–7.61 (m, 4H), 7.50–7.36 (m, 6H), 7.29–7.18 (m, 2H), 7.10 (dt, J = 7.6, 1.2 Hz, 1H), 6.43 (br, s, 1H), 4.80 (t, J = 10.0 Hz, 1H), 4.37–4.29 (m, 2H), 3.93 (dd, J = 12.0, 11.2 Hz, 1H), 3.58 (dd, J = 16.8, 10.0 Hz, 1H), 3.40 (dd, J = 16.8, 10.0 Hz, 1H), 1.10 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 168.5, 162.8, 140.9, 135.7, 135.6, 132.6, 132.4, 130.34, 130.31, 129.6, 128.2, 128.0, 125.3, 125.1, 116.2, 63.0, 60.1, 56.4, 31.1, 27.0, 19.3 ppm. FTIR (film): 3214 (br), 3071 (w), 2956 (w), 2857 (w), 1680 (s), 1486 (m), 1411 (m), 1112 (m), 739 (m), 702 (m), 505 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₃₀N₂O₃Si, 471.2104; found, 471.2103. mp 77–80 °C (obtained by concentration from EtOAc/n-heptane).

(3S,10aS)-3-{[(tert-Butyldiphenylsilyl)oxy]methyl}-2-methyl-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (35)

To a stirred suspension of diketopiperazine 20 (1.00 g, 2.12 mmol) and K₂CO₃ (7.31 g, 53.1 mmol) in acetone (30 mL) was added iodomethane (20 mL, 321 mmol) in one portion. The flask was wrapped in aluminum foil. After 5 d, the reaction mixture was heated to reflux. After a further 17 h, the resulting reaction mixture was cooled to room temperature and concentrated under reduced pressure. Safety note: iodomethane is toxic. Handling of this reagent and decontamination of equipment require appropriate protocols to avoid exposure. The resulting residue was mixed with water (100 mL) and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic extracts were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (50% EtOAc/n-heptane) to give N-methyl diketopiperazine 35. Yield: 852 mg (83%). Isolated as a white amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.24 in 50% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −48.4 (c = 0.61, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.12–8.06 (m, 1H), 7.60–7.51 (m, 4H), 7.45–7.36 (m, 2H), 7.36–7.23 (m, 6H), 7.14 (dt, J = 7.6, 1.2 Hz, 1H), 4.80 (t, J = 10.0 Hz, 1H), 4.40–4.34 (m, 1H), 4.13–4.09 (m, 1H), 4.09 (dd, J = 15.2, 2.4 Hz, 1H), 3.41 (d, J = 10.0 Hz, 2H), 3.02 (s, 3H), 0.92 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 167.0, 162.3, 141.8, 135.8, 135.7, 133.0, 132.6, 130.2, 130.1, 130.0, 127.9, 127.94, 127.92, 125.3, 125.0, 117.0, 64.3, 62.0, 60.1, 32.8, 31.2, 26.8, 19.3 ppm. FTIR (film): 3071 (w), 3050 (w), 2857 (w), 1670 (s), 1485 (m), 1428 (m), 1113 (m), 756 (m), 736 (m), 703 (m), 505 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₉H₃₂N₂O₃Si, 485.2260; found, 485.2260. mp 142–143 °C (obtained by concentration from EtOAc/n-heptane).

(3S,10aS)-3-(Hydroxymethyl)-2-methyl-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (21)⁴⁷

A solution of diketopiperazine 35 (362 mg, 747 μmol) in pyridine (4 mL) was added dropwise to stirred hydrofluoric acid (2 mL, 48% w/w) over 12 min. A white suspension formed immediately. Pyridine (4 mL) was added, and a clear, colorless solution formed. After 17 h, the resulting reaction mixture was neutralized with NaHCO₃ (s) and then diluted with water (25 mL) and NaHCO₃ (25 mL, sat. aq). The aqueous mixture was extracted with EtOAc (4 × 25 mL). The combined organic extracts were washed with HCl (25 mL, aq, 1 M) and brine (25 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (EtOAc) to give alcohol 21. Yield: 135 mg (73%). Isolated as a white crystalline solid. >95% pure by NMR and a single spot by TLC. R_f: 0.21 in EtOAc. Optical rotation: [α]_D²⁰: −45.0 (c = 0.10, CHCl₃). ¹H NMR (CDCl₃, 400 MHz): δ 8.04 (d, J = 8.0 Hz, 1H), 7.31–7.22 (m, 2H), 7.13 (dt, J = 7.6, 1.2 Hz, 1H), 4.75 (app. t, J = 10.0, 9.6 Hz, 1H), 4.52–4.42 (m, 1H), 4.18–4.07 (m, 2H), 3.64 (dd, J = 16.8, 9.6 Hz 1H), 3.43 (dd, J = 16.8, 10.0 Hz, 1H), 3.18 (s, 3H), 3.04–2.96 (m, 1H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 168.5, 163.8, 140.7, 130.1, 128.0, 125.6, 125.2, 116.4, 61.4, 59.9, 59.3, 31.7, 29.8 ppm. FTIR (neat): 3362 (br), 1659 (m), 1642 (m), 1600 (m), 1484 (m), 1415 (m), 1394 (m), 1238 (m), 1064 (m), 1010 (m), 754 (s), 409 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₄N₂O₃, 247.1083; found, 247.1082. mp 130–133 °C (obtained by concentration from EtOAc/n-heptane).

Methyl (R)-2-{2-[(tert-Butoxycarbonyl)amino]acetamido}-3-(4-nitrophenyl)propanoate (ent-9)²⁶

To a stirred suspension of glycine 22 (7.71 g, 44.0 mmol) and nitrophenyl alanine 15^.HCl⁴⁵ (10.4 g, 40 mmol) in CH₂Cl₂ (500 mL) under a N₂ atmosphere was added triethylamine (22.3 mL, 160 mmol) in one portion. The resulting clear yellow solution was cooled to 0 °C with an ice-water bath, and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (16.7 g, 44.0 mmol) was added portionwise, followed by addition of 1-hydroxybenzotriazole hydrate (5.95 g, 44 mmol) in one portion. After 30 min, the cooling bath was removed. After a further 15 h, the resulting reaction mixture was washed with HCl (2 × 200 mL, aq, 1 M), NaHCO₃ (2 × 200 mL, sat. aq), and brine (100 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (50–75% EtOAc/n-heptane) to give dipeptide ent-9. Yield: 16.6 g (93%). Isolated as yellow oil that crystallized upon standing. >95% pure by NMR. R_f: 0.17 in 50% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −53.2 (c = 0.62, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 8.18–8.11 (m, 2H), 7.33–7.27 (m, 2H), 6.72 (br, d, J = 6.0 Hz, 1H), 5.08 (br, s, 1H), 4.92 (dt, J = 7.6, 6.0 Hz, 1H), 3.83 (dd, J = 17.0, 6.0 Hz, 1H), 3.73 (s, 3H), 3.72 (dd, J = 17.0, 6.0 Hz, 1H), 3.30 (dd, J = 13.8, 6.0 Hz, 1H), 3.17 (dd, J = 13.8, 6.0 Hz, 1H), 1.43 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 171.2, 169.5, 156.2, 147.3, 143.7, 130.4, 123.9, 80.8, 52.9, 52.8, 44.6, 37.9, 28.4 ppm. FTIR (film): 3342 (w), 3307 (w), 1738 (m), 1682 (m), 1667 (s), 1521 (s), 1347 (m), 1282 (m), 1162 (m), 855 (m), 700 (m), 539 (m) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₇H₂₃N₃NaO₇, 404.1434; found, 404.1430. mp 96–98 °C (obtained by concentration from EtOAc/n-heptane).

Methyl (R)-1-[(tert-Butoxycarbonyl)glycyl]-6-nitroindoline-2-carboxylate (ent-10)²⁶

A stirred suspension of dipeptide ent-9 (11.6 g, 30.5 mmol), PhI(OAc)₂ (19.6 g, 61.0 mmol), and Pd(OAc)₂ (342 mg, 1.52 mmol) in toluene (300 mL) was heated to reflux. After 20 h, the resulting reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude residue was used in the next step without further purification. Analytically pure indoline ent-10 was obtained by purification by column chromatography (50% EtOAc/n-heptane). Yield: 9.11 g (crude). Purified ent-10 was isolated as a faint yellow amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.17 in 50% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: +54.1 (c = 0.51 CH₂Cl₂) ¹H NMR (CDCl₃, 400 MHz): δ 8.99 (br, s, 1H), 7.95 (dd, J = 8.0, 2.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 5.44 (br, s, 1H), 5.10 (br, d, J = 9.2 Hz, 1H), 4.16 (br, d, J = 14.4 Hz, 1H), 3.93–3.56 (m, 5H), 3.41 (br, d, J = 17.2 Hz, 1H), 1.46 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 170.7, 168.1, 156.0, 148.4, 143.3, 135.7, 124.7, 120.2, 112.5, 80.3, 60.4, 53.6, 43.7, 33.7, 28.5 ppm. FTIR (film): 3399 (br), 2978 (w), 2929 (w), 1744 (m), 1709 (m), 1682 (s), 1524 (s), 1481 (m), 1345 (s), 1246 (m), 1667 (s), 740 (w) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₇H₂₁N₃O₇, 380.1458; found, 380.1458. mp 111–113 °C (obtained by concentration from EtOAc/n-heptane).

(R)-7-Nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (23)

To a stirred solution of crude indoline ent-10 (9.11 g) in CH₂Cl₂ (100 mL) at 0 °C under a N₂ atmosphere was added TFA (20 mL) in one portion. After 15 min, the cooling bath was removed. After a further 45 min, the resulting reaction mixture was neutralized with NaHCO₃ (s). The resulting mixture was diluted with NaHCO₃ (100 mL, sat. aq) and then extracted with CH₂Cl₂ (2 × 100 mL). The combined organic extracts were washed with brine (100 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (0–20% MeOH/EtOAc) to give a dark brown solid (4.6 g), which was triturated from boiling EtOAc/MeOH (10:7, 170 mL) to give diketopiperazine 23. Yield: 1.84 g (24% over two steps from ent-9). Isolated as a beige amorphous solid. >95% pure by NMR. R_f: 0.16 in EtOAc. Optical rotation: [α]_D²⁰: +90.0 (c = 0.05, acetone). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.62 (d, J = 2.4 Hz, 1H), 8.45 (br, d, J = 5.0 Hz, 1H), 7.99 (dd, J = 8.4, 2.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 5.12 (t, J = 10.0 Hz, 1H), 4.29 (dd, J = 17.0, 0.4 Hz, 1H), 3.76 (dd, J = 17.0, 5.0 Hz, 1H), 3.45 (d, J = 10.0 Hz, 2H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 168.3, 164.7, 147.0, 141.7, 138.5, 126.0, 119.9, 108.7, 59.8, 46.4, 30.2 ppm. FTIR (neat): 3316 (br), 1675 (m), 1657 (m), 1598 (w), 1514 (m), 1474 (m), 1416 (m), 1340 (m), 1090 (m), 1074 (m), 889 (m), 709 (m) cm^–1. HRMS-ESI (m/z): [M + NH₄]⁺ calcd for C₁₁H₉N₃O₄, 265.0937; found, 265.0936. mp 237–239 °C (obtained by trituration from EtOAc/MeOH).

(R)-2-Methyl-7-nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (24)

To a stirred suspension of diketopiperazine 23 (1.79 g, 7.23 mmol) and K₂CO₃ (25.0 g, 181 mmol) in acetone (70 mL) was added iodomethane (40 mL, 642 mmol) in one portion. The flask was wrapped in aluminum foil. After 7 d, the resulting reaction mixture was concentrated under reduced pressure. Safety note: iodomethane is a toxic reagent. Handling of this reagent and decontamination of equipment require appropriate protocols to avoid exposure. The resulting residue was mixed with water (100 mL) and EtOAc (100 mL). The organic phase was separated, and the aqueous phase was extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with brine (50 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give diketopiperazine 24. Yield: 1.9 g (>95%). Isolated as an orange crystalline solid. >95% pure by NMR. R_f: 0.27 in EtOAc. Optical rotation: [α]_D²⁰: +74.1 (c = 0.87, DMSO). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.58 (d, J = 2.4 Hz, 1H), 7.98 (dd, J = 8.4, 2.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 5.13 (t, J = 9.6 Hz, 1H), 4.50 (dd, J = 16.8, 1.2 Hz, 1H), 3.99 (d, J = 16.8 Hz, 1H), 3.53–3.39 (m, 2H), 2.94 (s, 3H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 166.4, 163.7, 147.0, 141.6, 138.8, 126.0, 120.0, 108.8, 59.8, 53.3, 33.2, 30.8 ppm. FTIR (neat): 2905 (w), 1663 (s), 1598 (w), 1517 (m), 1479 (m), 1415 (m), 1396 (m), 1331 (m), 1215 (m), 1061 (m), 881 (m), 740 (m), 417 (m) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₂H₁₁N₃NaO₄, 284.0647; found, 284.0645. mp 205–207 °C (obtained by concentration from EtOAc).

General Procedure for the Attempted Direct Sulfenylation of DKPs

To a stirred suspension of S₈ (1.0 equiv) in THF was added LiHMDS, NaHMDS, or KHMDS (3.0–4.0 equiv, 1 M in THF) dropwise over 2 min at room temperature under a N₂ atmosphere. After 5 min, a solution of the diketopiperazine (1.0 equiv) in THF was added dropwise over 2 min. After 5 min, LiHMDS, NaHMDS, or KHMDS (2.0–4.0 equiv, 1 M in THF) was added dropwise over ∼30 s. After 1.5 h, NaHCO₃ (sat. aq) was added. The resulting mixture was extracted with CH₂Cl₂. The combined organic extracts were passed through a phase separator and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (50–100% EtOAc/n-heptane).

(±)-3-(Hydroxymethyl)-2-methyl-7-nitro-2,3-dihydropyrazino[1,2-a]indole-1,4-dione (rac-25)

Following the general procedure using diketopiperazine 11 (29.1 mg, 100 μmol), S₈ (25.6 mg, 100 μmol), and LiHMDS (800 μL, 1 M in THF). Yield: 2 mg (7%). Isolated as a yellow amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.09 in 75% EtOAc/n-heptane. ¹H NMR (DMSO-d₆, 400 MHz): δ 9.11 (d, J = 2.4 Hz, 1H), 8.27 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.51 (s, 1H), 5.44 (app. t, J = 6.0, 5.6 Hz, 1H), 4.66–4.61 (m, 1H), 4.06–3.97 (m, 1H), 3.92–3.84 (m, 1H), 3.06 (s, 3H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 166.0, 156.1, 146.4, 135.0, 134.4, 133.1, 123.8, 120.3, 111.8, 111.3, 66.7, 61.4, 31.8 ppm. FTIR (film): 3385 (br), 2930 (w), 1720 (m), 1648 (m), 1592 (w), 1580 (w), 1522 (m), 1468 (m), 1433 (m), 1399 (s), 1340 (w), 1244 (w), 1219 (w), 1119 (w), 1065 (w), 1039 (w), 895 (w), 840 (w), 754 (w), 730 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₃H₁₁N₃NaO₅, 312.0596; found, 312.0598. mp 267–269 °C (obtained by concentration from EtOAc/n-heptane).

(±)-3-(Hydroxymethyl)-2-methyl-2,3-dihydropyrazino[1,2-a]indole-1,4-dione (rac-26)

Following the general procedure using diketopiperazine 21 (24.6 mg, 100 μmol), S₈ (25.6 mg, 100 μmol), and LiHMDS (800 μL, 1 M in THF). Yield: 9 mg (37%). Isolated as a white amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.11 in 75% EtOAc/n-heptane. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.35 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.52 (dt, J = 8.0, 1.2 Hz, 1H), 7.41 (dt, J = 8.0 Hz, 1.2 Hz, 1H), 7.35 (s, 1H), 5.37 (app. t, J = 6.0, 5.6 Hz, 1H), 4.56–4.50 (m, 1H), 4.00 (ddd, J = 11.6, 7.6, 2.8 Hz, 1H), 3.88 (ddd, J = 11.6, 7.6, 1.6 Hz, 1H), 3.04 (s, 3H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 165.1, 156.3, 134.0, 130.0, 128.8, 127.1, 124.9, 122.5, 115.7, 111.6, 66.1, 60.7, 31.1 ppm. FTIR (film): 3368 (br), 2933 (w), 1711 (m), 1638 (m), 1589 (m), 1574 (w), 1447 (m), 1396 (s), 1337 (m), 1250 (w), 1067 (w), 843 (w), 752 (w), 738 (w) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₂N₂O₃, 245.0926; found, 245.0923. mp 200–202 °C (obtained by concentration from EtOAc/n-heptane).

2-Methyl-7-nitro-2,3-dihydropyrazino[1,2-a]indole-1,4-dione (28)

Following the general procedure using diketopiperazine 24 (26.1 mg, 100 μmol), S₈ (25.6 mg, 100 μmol), and LiHMDS (500 μL, 1 M in THF). Yield: 4 mg (15%). Isolated as a yellow amorphous solid. >90% pure by NMR and a single spot by TLC. R_f: 0.19 in 75% EtOAc/n-heptane. ¹H NMR (CDCl₃, 400 MHz): δ 9.32 (d, J = 2.0 Hz, 1H), 8.29 (dd, J = 8.8, 2.0 Hz, 1H), 7.83 (dd, J = 8.8, 0.8 Hz, 1H), 7.50 (d, J = 0.8 Hz, 1H), 4.52 (s, 2H), 3.21 (s, 3H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 161.2, 155.3, 147.3, 133.8, 133.7, 133.1, 123.0, 120.7, 113.2, 113.0, 53.8, 33.5 ppm. FTIR (film): 3121 (w), 2926 (w), 2855 (w), 1723 (m), 1656 (m), 1589 (w), 1572 (m), 1521 (m), 1494 (m), 1393 (m), 1365 (s), 1349 (s), 1337 (m), 1292 (w), 1249 (w), 1210 (w), 1067 (w), 1031 (w), 907 (w), 853 (w), 840 (w), 731 (m), 670 (w), 632 (w), 550 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₂H₉N₃NaO₄, 282.0491; found, 282.0483. mp 189–191 °C (obtained by concentration from CDCl₃/n-heptane).

General Procedure for the Oxidation of DKPs

To a stirred solution of the diketopiperazine (1.0 equiv) in CH₂Cl₂ was added silver(I)bispyridine permanganate⁴³ (4.0–8.0 equiv) in one portion. After 15–24 h, sodium bisulfite (1 M, aq) was added. The resulting mixture was passed through a phase separator, and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were washed with CuSO₄ (sat. aq), NH₄Cl (sat. aq), and brine. The organic phase was then passed through a phase separator and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (25–100% EtOAc/n-heptane).

(3S)-3-{[(tert-Butyldiphenylsilyl)oxy]methyl}-10a-hydroxy-2-methyl-7-nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (31)

Following the general procedure using diketopiperazine 30 (265 mg, 500 μmol), CH₂Cl₂ (10 mL), and silver(I)bispyridine permanganate (1.54 g, 4.00 mmol). Yield: 157 mg consisting of a difficult to separate 40:60 mixture of 30/31 (35%). A purified sample of 31 was isolated as a white solid by exhaustive column chromatography. >90% pure by NMR and a single spot by TLC. R_f: 0.09 in 33% EtOAc/n-heptane. ¹H NMR (CDCl₃, 400 MHz): δ 8.98 (d, J = 2.0 Hz, 1H), 8.10 (dd, J = 8.4, 2.0 Hz, 1H), 7.66–7.59 (m, 4H), 7.53–7.40 (m, 7H), 5.70 (br, s, 1H), 4.22 (dd, J = 10.8, 1.6 Hz, 1H), 4.08 (dd, J = 3.2, 1.6 Hz, 1H), 3.94 (dd, J = 11.2, 3.2 Hz, 1H), 3.76 (d, J = 18.0 Hz, 1H), 3.47 (d, J = 18.0 Hz, 1H), 2.93 (s, 3H), 1.02 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 165.4, 163.8, 148.2, 140.7, 136.4, 135.73, 135.65, 130.9, 130.83, 130.81, 130.5, 128.4, 125.6, 121.1, 111.8, 89.1, 66.4, 62.4, 39.8, 32.3, 26.9, 19.2 ppm. FTIR (film): 3321 (br), 2957 (w), 2932 (w), 2859 (w), 1685 (w), 1602 (w), 1528 (m), 1483 (m), 1436 (m), 1404 (m), 1349 (m), 1240 (w), 1184 (w), 1150 (m), 1112 (m), 1089 (w), 1065 (w), 1032 (w), 997 (w), 911 (w), 883 (w), 825 (w), 740 (m), 703 (m), 615 (w), 505 (w), 491 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₂₉H₃₁NaN₃O₆Si, 568.1880; found, 568.1879. mp 96–100 °C (obtained by concentration from CH₂Cl₂/n-heptane).

[(3S)-10a-Hydroxy-2-methyl-7-nitro-1,4-dioxo-1,2,3,4,10,10a-hexahydropyrazino(1,2-a)indol-3-yl]methyl Acetate (33)

Following the general procedure using diketopiperazine 18 (167 mg, 500 μmol), CH₂Cl₂ (10 mL), and silver(I)bispyridine permanganate (1.54 g, 4.00 mmol). Yield: 39 mg (22%). Isolated as a white amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.23 in 75% EtOAc/n-heptane. ¹H NMR (CDCl₃, 400 MHz): δ 8.87 (d, J = 2.4 Hz, 1H), 8.08 (dd, J = 8.0, 2.4 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 4.77 (br, s, 1H), 4.72 (dd, J = 11.6, 4.4 Hz, 1H), 4.64 (dd, J = 11.6, 4.4 Hz, 1H), 4.36 (t, J = 4.4 Hz, 1H), 3.76 (dd, J = 18.4, 1.6 Hz, 1H), 3.46 (d, J = 18.4 Hz, 1H), 3.10 (s, 3H), 2.12 (s, 3H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 169.8, 165.1, 163.0, 148.2, 140.6, 135.5, 125.5, 121.3, 112.0, 89.3, 64.0, 63.2, 40.9, 33.4, 20.8 ppm. FTIR (film): 3291 (br), 2952 (w), 2931 (w), 1747 (m), 1686 (s), 1656 (s), 1603 (w), 1526 (s), 1483 (m), 1438 (m), 1406 (m), 1349 (s), 1222 (s), 1185 (w), 1153 (w), 1059 (m), 912 (w), 885 (w), 831 (w), 740 (m), 638 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₅H₁₅NaN₃O₇, 372.0808; found, 372.0806. mp 102–105 °C (obtained by concentration from EtOAc/n-heptane).

[3,10a-Dihydroxy-2-methyl-7-nitro-1,4-dioxo-1,2,3,4,10,10a-hexahydropyrazino(1,2-a)indol-3-yl]methyl Acetate (34)

Following the general procedure using diketopiperazine 18 (167 mg, 500 μmol), CH₂Cl₂ (10 mL), and silver(I)bispyridine permanganate (1.54 g, 4.00 mmol). Yield: 14 mg (8%). Isolated as a yellow amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.19 in 75% EtOAc/n-heptane. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.65 (d, J = 2.2 Hz, 1H), 8.10 (dd, J = 8.4, 2.2 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.42 (s, 1H), 4.64 (d, J = 11.2 Hz, 1H), 4.32 (d, J = 11.2 Hz, 1H), 3.68 (d, J = 18.4 Hz, 1H), 3.39 (d, J = 18.4 Hz, 1H), 2.97 (s, 3H), 1.89 (s, 3H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 169.4, 166.4, 163.5, 147.1, 140.5, 137.3, 126.3, 120.7, 110.2, 88.2, 84.9, 63.5, 40.6, 27.3, 20.3 ppm. FTIR (film): 3349 (br), 2979 (w), 2924 (w), 1750 (m), 1699 (s), 1605 (w), 1528 (m), 1486 (w), 1437 (m), 1396 (m), 1350 (m), 1257 (m), 1157 (m), 1060 (m), 913 (w), 828 (w), 739 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₅H₁₅NaN₃O₈, 388.0757; found, 388.0758. mp 70–74 °C (obtained by concentration from CH₂Cl₂/n-heptane).

[3,10a-Dihydroxy-2-methyl-1,4-dioxo-1,2,3,4,10,10a-hexahydropyrazino(1,2-a)indol-3-yl]methyl Acetate (36)

Following the general procedure using diketopiperazine 35 (72.7 mg, 150 μmol), CH₂Cl₂ (3 mL), and silver(I)bispyridine permanganate (231 mg, 600 μmol). Yield: 36 mg (46%). Isolated as yellow foam. >95% pure by NMR and a single spot by TLC. R_f: 0.21 in 50% EtOAc/n-heptane. ¹H NMR (CDCl₃, 400 MHz): δ 8.00 (d, J = 7.6 Hz, 1H), 7.63–7.52 (m, 4H), 7.50–7.20 (m, 8H), 7.15 (t, J = 7.2 Hz, 1H), 5.22 (br, s, 1H), 5.00 (br, s, 1H), 4.29 (d, J = 10.8 Hz, 1H), 4.02 (d, J = 10.8 Hz, 1H), 3.69 (d, J = 17.2 Hz, 1H), 3.35 (d, J = 17.2 Hz, 1H), 3.01 (s, 3H), 0.96 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 168.2, 163.4, 139.9, 135.8, 135.7, 132.3, 132.1, 130.3, 130.2, 128.17, 128.10, 128.06, 128.0, 125.7, 125.3, 116.8, 88.6, 86.7, 64.2, 40.3, 28.2, 26.8, 19.3 ppm. FTIR (film): 3339 (br), 3072 (w), 3051 (w), 2956 (w), 2930 (w), 2894 (w), 2858 (w), 1688 (s), 1663 (s), 1606 (w), 1482 (m), 1463 (m), 1427 (s), 1396 (m), 1363 (w), 1320 (w), 1273 (w), 1186 (w), 1113 (s), 1056 (m), 912 (w), 822 (m), 777 (w), 753 (m), 735 (s), 613 (w), 505 (m), 489 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₂₉H₃₂N₂NaO₅Si, 539.1973; found, 539.1978.

(±)-3,10a-Dihydroxy-2-methyl-7-nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (rac-37)

Following the general procedure using diketopiperazine 24 (52.0 mg, 200 μmol), CH₂Cl₂ (4 mL), and silver(I)bispyridine permanganate (385 mg, 1.00 mmol). Yield: 7 mg (12%). Isolated as a yellow amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.10 in 75% EtOAc/n-heptane. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.61 (d, J = 2.4 Hz, 1H), 8.06 (dd, J = 8.4, 2.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.37 (d, J = 6.6 Hz, 1H), 5.21 (d, J = 6.6 Hz, 1H), 3.65 (d, J = 18.4 Hz, 1H), 3.31 (d, J = 18.4 Hz, 1H), 2.96 (s, 3H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 165.9, 164.0, 147.0, 140.6, 137.3, 126.1, 120.3, 109.7, 88.7, 81.5, 40.6, 31.2 ppm. FTIR (film): 3258 (br), 2924 (w), 2854 (w), 1690 (s), 1667 (m), 1602 (w), 1526 (m), 1482 (m), 1439 (w), 1404 (m), 1350 (m), 1242 (w), 1185 (w), 1149 (w), 1068 (w), 1046 (m), 1027 (m), 940 (w), 884 (w), 829 (w), 777 (w), 740 (w) cm^–1. HRMS-ESI (m/z): [M – H]⁺ calcd for C₁₂H₁₁N₃O₆, 292.0570; found, 292.0572. mp 200–202 °C (obtained by concentration from CH₂Cl₂/n-heptane).

(3S)-10a-Hydroxy-3-(hydroxymethyl)-2-methyl-7-nitro-2,3,10,10a-tetrahydropyrazino[1,2-a]indole-1,4-dione (32)

To a stirred 40:60 mixture of 30/31 (70.0 mg) in pyridine (1.5 mL) was added pyridine hydrofluoride (500 μL). After 2 h, the resulting reaction mixture was neutralized with NaHCO₃ (s). The resulting mixture was diluted with water and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with CuSO₄ (2 × 10 mL, sat. aq) and brine (10 mL). The organic phase was then dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (50–100% EtOAc/n-heptane) to give diol 32. Yield: 11 mg (28%). Isolated as a white crystalline solid. >95% pure by NMR and a single spot by TLC. R_f: 0.12 in 75% EtOAc/n-heptane. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.67 (d, J = 2.4 Hz, 1H), 8.06 (dd, J = 8.2, 2.4 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.10 (s, 1H), 6.53 (t, J = 4.4 Hz, 1H), 4.37 (app. t, J = 4.0, 3.2 Hz, 1H), 4.04–3.91 (m, 2H), 3.66 (dd, J = 18.4, 1H), 3.29 (d, J = 18.4 Hz, 1H), 2.99 (s, 3H) ppm. ¹³C NMR (DMSO-d₆, 101 MHz): δ 165.3, 164.7, 147.0, 140.5, 137.2, 126.1, 120.3, 109.7, 88.8, 66.1, 60.8, 32.4 ppm. The signal corresponding to the benzylic carbon atom coincides with the residual DMSO signal. FTIR (film): 3259 (br), 2927 (w), 1681 (s), 1603 (w), 1526 (m), 1483 (m), 1437 (m), 1405 (m), 1348 (m), 1348 (m), 1243 (w), 1203 (w), 1185 (w), 1148 (w), 1107 (w), 1085 (w), 1066 (w), 908 (w), 887 (w), 830 (w), 818 (w), 741 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₁₃H₁₃N₃NaO₆, 330.0702; found, 330.0692. mp 239–240 °C (obtained by recrystallization from EtOH).

General Procedure for the Sulfenylation of DKP Hemi-Aminals

To a stirred solution of the hemi-aminal (1.0 equiv) and 4-methoxy-α-toluene thiol (10.0 equiv) in CH₂Cl₂ under a N₂ atmosphere was added dropwise BF₃·Et₂O (20.0 equiv). After 1 h, NaHCO₃ (sat. aq) was added, and the resulting mixture was extracted with CH₂Cl₂. The combined organic extracts were passed through a phase separator and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography (25–50% EtOAc/n-heptane).

(S)-3-{[(tert-Butyldiphenylsilyl)oxy]methyl}-2-methyl-7-nitro-2,3-dihydropyrazino[1,2-a]indole-1,4-dione (38)

Following the general procedure using hemi-aminal 31 (14.0 mg, 25.7 μmol), 4-methoxy-α-toluene thiol (33 μL, 257 μmol), CH₂Cl₂, (1 mL), and BF₃·Et₂O (69 μL, 513 mmol). Yield: 13 mg (>95%). Isolated as a white/yellow amorphous solid. >95% pure by NMR and a single spot by TLC. R_f: 0.13 in 33% EtOAc/n-heptane. Optical rotation: [α]_D²⁰: −181 (c = 1.0 in CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 9.33 (d, J = 2.2 Hz, 1H), 8.28 (dd, J = 8.8, 2.2 Hz, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.50–7.22 (m, 11H), 4.33 (app. t, J = 2.4, 2.0 Hz, 1H), 4.21 (dd, J = 10.8, 2.0 Hz, 1H), 4.05 (dd, J = 10.8, 2.4 Hz, 1H), 3.03 (s, 3H), 0.69 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 164.2, 156.6, 147.1, 135.5, 135.4, 134.1, 133.9, 133.5, 131.9, 131.7, 130.3, 130.2, 128.03, 127.97, 122.8, 120.5, 112.7, 112.4, 66.8, 63.2, 32.0, 26.3, 18.8 ppm. FTIR (film): 3122 (w), 3072 (w), 2953 (w), 2931 (w), 2886 (w), 2858 (w), 1723 (m), 1660 (m), 1591 (w), 1579 (w), 1522 (m), 1470 (m), 1429 (m), 1396 (m), 1360 (s), 1291 (w), 1245 (w), 1210 (w), 1105 (m), 1068 (w), 1045 (w), 931 (w), 908 (w), 854 (w), 730 (s), 702 (m), 616 (w), 504 (w), 489 (m), 429 (w) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₉H₂₉N₃O₅Si, 528.1955; found, 528.1961. mp 69–77 °C (obtained by concentration from CH₂Cl₂/n-heptane).

(±)-(3-((4-Methoxybenzyl)thio)-2-methyl-7-nitro-1,4-dioxo-1,2,3,4-tetrahydropyrazino[1,2-a]indol-3-yl)methyl Acetate (rac-39)

Following the general procedure using bis-hemi-aminal 34 (14.0 mg, 38.3 μmol), 4-methoxy-α-toluene thiol (49 μL, 383 μmol), CH₂Cl₂ (1 mL), and BF₃^·Et₂O (103 μL, 766 mmol). Yield: 11 mg (60%). Isolated as a yellow crystalline solid. >95% pure by NMR and a single spot by TLC. R_f: 0.29 in 50% EtOAc/n-heptane. ¹H NMR (CDCl₃, 400 MHz): δ 9.01 (d, J = 2.4 Hz, 1H), 8.26 (dd, J = 8.8, 2.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.45 (s, 1H), 6.98–6.90 (m, 2H), 6.39–6.28 (m, 2H), 4.76–4.67 (m, 2H), 3.79 (d, J = 14.8 Hz, 1H), 3.57 (d, J = 14.8 Hz, 1H), 3.44 (s, 3H), 3.32 (s, 3H), 1.92 (s, 3H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 169.6, 162.1, 158.9, 156.3, 147.1, 133.6, 133.5, 131.5, 129.2, 126.4, 122.7, 120.6, 113.6, 113.3, 113.2, 74.1, 64.2, 54.9, 34.2, 28.6, 20.6 ppm. FTIR (film): 3124 (w), 2954 (w), 2837 (w), 1751 (m), 1751 (m), 1720 (m), 1663 (m), 1609 (w), 1580 (w), 1522 (m), 1512 (m), 1464 (m), 1422 (m), 1397 (m), 1358 (s), 1341 (s), 1250 (m), 1210 (m), 1177 (m), 1037 (w), 897 (w), 840 (w), 731 (m) cm^–1. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₃H₂₁N₃O₇S, 484.1178; found, 484.1175. mp 176–179 °C (obtained by concentration from EtOAc/n-heptane).

(±)-3-{[(tert-Butyldiphenylsilyl)oxy]methyl}-3-[(4-methoxybenzyl)thio]-2-methyl-2,3-dihydropyrazino[1,2-a]indole-1,4-dione (rac-40)

Following the general procedure using bis-hemi-aminal 36 (36.0 mg, 69.8 μmol), 4-methoxy-α-toluene thiol (97 μL, 698 μmol), CH₂Cl₂ (2 mL), and BF₃·Et₂O (172 μL, 1.40 mmol). Yield: 33 mg (74%). Isolated as colorless oil. >95% pure by NMR and a single spot by TLC. R_f: 0.17 in 25% EtOAc/n-heptane. ¹H NMR (CDCl₃, 400 MHz): δ 8.30–8.25 (m, 1H), 7.71–7.66 (m, 1H), 7.53–7.23 (m, 13H), 6.98–6.91 (m, 2H), 6.38–6.31 (m, 2H), 4.29 (d, J = 10.0 Hz, 1H), 3.84 (d, J = 10.0 Hz, 1H), 3.66 (d, J = 14.0 Hz, 1H), 3.53 (d, J = 14.0 Hz, 1H), 3.38 (s, 3H), 3.19 (s, 3H), 0.74 (s, 9H) ppm. ¹³C NMR (CDCl₃, 101 MHz): δ 163.5, 158.7, 157.7, 135.6, 135.1, 132.4, 132.1, 130.2, 130.1, 129.7, 129.0, 128.04, 128.01, 127.9, 127.8, 127.2, 125.3, 122.3, 117.0, 114.1, 113.6, 77.7, 65.9, 54.9, 34.0, 28.4, 26.4, 19.1 ppm. FTIR (film): 3071 (w), 2953 (w), 2931 (w), 2893 (w), 2857 (w), 1711 (m), 1656 (w), 1609 (w), 1589 (w), 1574 (w), 1449 (m), 1424 (s), 1353 (s), 1303 (w), 1251 (m), 1222 (w), 1204 (w), 1176 (w), 1107 (s), 1035 (w), 908 (w), 865 (w), 822 (m), 735 (s), 702 (s), 611 (w), 504 (m), 488 (w) cm^–1. HRMS-ESI (m/z): [M + Na]⁺ calcd for C₃₇H₃₈N₂NaSSi, 657.2219; found, 657.2224.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c00810.

• Crystallographic information for 11, optimization of the C–H activation reaction to form 12 from 16, and copies of ¹H and ¹³C NMR spectra of 9, 11–13, 16–21, 23–26, 28, and 30–42 (PDF)

• Crystallographic data for DKP-11 (CIF)

The authors declare no competing financial interest.