Levoglucosenone and Its Pseudoenantiomer iso-Levoglucosenone as Scaffolds for Drug Discovery and Development

Abstract

The bioderived platform molecule levoglucosenone (LGO, 1) and its readily prepared pseudoenantiomer (iso-LGO, 2) have each been subjected to α-iodination reactions with the product halides then being engaged in palladium-catalyzed Ullmann cross-coupling reactions with various bromonitropyridines. The corresponding α-pyridinylated derivatives such as 11 and 24, respectively, are produced as a result. Biological screening of such products reveals that certain of them display potent and selective antimicrobial and/or cytotoxic properties. In contrast, the azaindoles obtained by reductive cyclization of compounds such as 11 and 12 are essentially inactive in these respects. Preliminary mode-of-action studies are reported.

Introduction

The azaindoles represent privileged structures in medicinal chemistry and many derivatives thereof have been explored as drug development candidates for the treatment of a range of diseases.¹ Of particular note has been the exploitation of such compounds as kinase inhibitors,² as antiviral agents,³ as antitumor agents,^2,4 as cannabinoid (CB) receptor agonists,⁵ and as potential treatments for diabetes.⁶ These same heterocycles are also encountered, albeit rather infrequently, in biologically active natural products such as the variolins,⁷ the marinoquinolines,⁸ and the guitarrins.⁹

Recently, we reported the use of a palladium-catalyzed Ullmann cross-coupling/reductive cyclization protocol¹⁰ for the construction of 2,3-annulated-azaindoles from halonitropyridines and α-iodinated cyclohex-2-en-1-ones.¹¹ Simultaneously, we have been exploring the chemistry of the homochiral and α,β-unsaturated ketone levoglucosenone (LGO, 1) (Figure 1),¹² a compound that is now produced commercially in tonne quantities through the high-temperature pyrolysis of acid-treated cellulose.¹³ In addition, the pseudoenantiomeric compound iso-levoglucosenone (iso-LGO, 2) can be obtained in just three steps and on a multigram scale^12c from congener 1.¹⁴ While compounds 1 and 2 are now abundant materials, they remain little explored as scaffolds for drug discovery.¹⁵ Accordingly, we report herein on the deployment of their α-iodinated derivatives as reaction partners in the assembly, via the abovementioned, two-step protocol, of a suite of novel and homochiral azaindoles together with the evaluation of these and their precursors in a range of antimicrobial and anticancer screens. As revealed below, while the targeted heterocycles show little if any biological effects, certain of their nitropyridine-containing precursors are notably active.

Figure 1.

Structures of LGO (1) and iso-LGO (2) and their scalable modes of production.

Results and Discussion

The simple reaction sequence allowing for the elaboration of LGO into a series of α-nitropyridinylated derivatives and thence, through reductive cyclization of these, into the corresponding azaindoles is shown in Scheme 1. Thus, α-iodination of LGO (1) under conditions defined by Isobe and co-workers gave derivative 3(16) (50%) that was then engaged in palladium-catalyzed cross-couplings with the bromonitropyridines 4–8¹¹ and thereby affording the products 9–13, respectively, in yields ranging from 35 to 82%. The structures of two of these (10 and 13) were confirmed by single-crystal X-ray analysis (see the Supporting Information (SI) for details). In an operationally simple process, when methanolic solutions of each of the compounds 9–13 were exposed to hydrogen in the presence of palladium on carbon at 22–50 °C, reductive cyclization reactions took place and, in one case (13), solvent incorporation also occurred. As a result the azaindole derivatives 14–18, respectively, were obtained. The lowest yielding process was the one leading to the 7-azaindole 14, while incomplete reductive cyclization was observed in the case of substrate 13, and such that the N-hydroxyazaindole 18 was obtained. Once again, the structures of certain of these compounds (14, 16, and 17) were confirmed by single-crystal X-ray analysis (see the SI for details).

Scheme 1. Synthetic Sequence Leading from LGO (1) to Test Compounds 9–18.

The iso-LGO-containing azaindoles and their precursors were prepared by the analogous means shown in Scheme 2. Thus, iso-LGO (2) was α-iodinated under Isobe’s conditions to give the previously unreported halide 19 (66%), which was cross-coupled with the bromonitropyridines 4–8 and 20, thereby affording the corresponding products 21–26 (46–84%) with the structures of the second and third of these being confirmed by X-ray analyses (see SI). The outcomes of the reductive cyclizations of compounds 21–26 were quite varied with the reactions of the first four of these substrates under the same conditions as used earlier, leading to identifiable products, viz. 27–30 (48–89%) although single-crystal X-ray analyses of the third and fourth of these revealed (see SI) that the former (viz. 29) was a 1-hydroxy-6-azaindole and not the corresponding, fully reduced system. For reasons that remain unclear, attempts to reduce substrates 25 and 26 under the same or related conditions failed to provide the anticipated azaindoles. In the case of the former compound, complex product mixtures were observed, while in the case of the latter this proved inert to all of the reaction conditions examined.

Scheme 2. Synthetic Sequence Leading from iso-LGO (2) to Test Compounds 21–30.

In our earlier work, we disclosed^11b methods for the reductive cyclization of various α-arylated, α,β-unsaturated carbonyl-containing systems using iron or TiCl₃ under aqueous acidic conditions and thereby affording heterocyclic systems regioisomeric with those obtained using the H₂/Pd on C combination. On applying the first of these alternative conditions to substrate 9 and using aqueous acetic acid, ethanol, or aqueous hydrochloric acid as the reaction medium, compounds 31, 32, and 33(11b) (Figure 2), respectively, were obtained. Once again, the structures of the last two of these were confirmed by single-crystal X-ray analyses (see SI). The precise modes of formation of these distinct products from the common starting material 9 are the subject of ongoing investigations.

Figure 2.

Structures of the products 31–33 arising from the reductive cyclization of cross-coupling product 9 using iron in various acidic media.

All of the LGO-derived cross-coupling products 9–13, the derived azaindoles 14–18 and 31–33, as well as the corresponding collection of iso-LGO-derived systems, viz. compounds 21–30, were subject to biological evaluation against a panel of six antibiotic-sensitive and two antibiotic-resistant Gram-positive and -negative bacteria together with the fungus Candida albicans ATCC90028. In essentially every instance (see SI), the azaindoles proved inactive while, in marked contrast, certain of the precursor α-arylated LGO and iso-LGO derivatives proved to have significant antibacterial effects (Table 1). Notably, the LGO-derived compound 11 showed an IC₅₀ value of 0.6 μM against Bacillus subtilis (ATCC6633).

Table 1. Outcomes of the Antibacterial Screening of Compounds 11,13, 21, 22, and 24–26^a.

		Staphylococcus aureus (ATCC25923)		methicillin-resistant Staphylococcus aureus (MRSA)		Bacillus subtilis (ATCC6633)		Streptococcus pyogenes (D3840)
entry	compound	MIC (μM)	IC50 (μM)	MIC (μM)	IC50 (μM)	MIC (μM)	IC50 (μM)	MIC (μM)	IC50 (μM)
1	11	30	8.1	30	10.0	3.3	0.6	10	5.5
2	13	30	6.0	ntb	nt	30	13.5	30	10.0
3	21	30	10.7	nt	nt	30	10.4	30	10.3
4	22	30	6.8	nt	nt	30	6.8	30	15.9
5	24	30	14.8	nt	nt	30	11.5	30	10.0
6	25	30	27.8	nt	nt	30	10.0	30	10.0
7	26	30	20.0	30	12.3	10	2.9	30	10.7

^aSee the SI for further details.

^bnt, not tested.

Compounds 9–18 and 21–33 were also evaluated as cytotoxic agents against the HepG2, NCIH-460, and SW620 cell lines (which are human hepatocellular, lung, and colorectal cancer cell lines, respectively). Again, the activity of the azaindoles in these assays were of little note (see SI) but the same subset of α-nitropyridinylated LGO and iso-LGO precursors displayed significant effects (Table 2). Specifically, compound 11 and the iso-LGO-derived system 24 display IC₅₀ values of 0.8 and 0.6 μM, respectively, against the SW620 cell line. Compound 21 displays (entry 3, Table 2) similarly notable effects against the NCIH-460 cell line. As such, these compounds appear to possess cytotoxic properties that compare particularly favorably to other LGO-derived systems.¹⁵

Table 2. Outcomes of the Cytotoxicity Screening of Compounds 11,13, 21, 22, and 24–26^a.

entry	compound	HepG2 IC50 (μM)	SW620 IC50 (μM)	NCIH-460 IC50 (μM)
1	11	2.7	0.8	8.0
2	13	5.6	5.6	5.1
3	21	6.5	8.0	1.0
4	22	7.0	3.2	5.1
5	24	8.0	0.6	7.0
6	25	14.6	2.1	12.0
7	26	5.0	1.0	2.9

^aSee SI for further details.

The origins of the abovementioned and notable antimicrobial and/or cytotoxic properties of compounds 11,13, 21, 22, and 24–26 are most likely connected with their capacities to act as Michael acceptors¹⁵ and thereby engage in covalent binding with nucleophilic amino acid residues at the target receptor(s).¹⁷ In support of such notions, we have observed that when equimolar mixtures of compounds 9, 11, or 21 and l-cysteine (or the corresponding methyl ester) are subject to electrospray ionization (ESI) mass spectrometric analysis (in either positive or negative mode), then prominent molecular-associated ions arising from their mutual conjugation are observed in each instance (this was not the case with the associated azaindoles). Furthermore, a solution-phase reaction of congener 10 (Scheme 3) with a half-molar equivalent¹⁸ of l-cysteine in methanol/water under ambient conditions for 48 h afforded, after chromatographic purification, the adducts 34 (18%) and 35 (38%). Spectroscopic analysis of each of these clearly suggested that they are diastereoisomers arising from conjugate addition of the sulfhydryl group of the amino acid to the less-congested α-face of the substrate and therefore differ in the configuration (R vs S) of the nitropyridyl residue at the α-position of the LGO framework. The illustrated configurations of these diastereoisomers are tentatively assigned on the basis that the resonances due to the diastereotopic methylene protons of the cysteine side chain in the cis-isomer 34 are much more strongly differentiated than their counterparts in trans-isomer 35 because of the greater impact of the proximal α-nitropyridinyl residue in the former case (Δδ_H = 0.35 vs < 0.01). The latter conjugate, viz. compound 35 (which predominates), is presumed to be the thermodynamically more stable one^15d (the fragile nature of compounds 34 and 35 prevented the conduct of relevant equilibration studies so as to determine if this is indeed the case).

Scheme 3. Reaction of the α-Arylated and LGO-Derived Enone 10 with l-Cysteine Leading to the Isolable Adducts 34 and 35.

These conjugation experiments clearly indicate that the α-pyridinylated LGO and iso-LGO systems reported here are effective Michael acceptors. The predominance of the later systems among the active compounds (see Tables 1 and 2) could be taken as suggesting that these are more disposed toward such addition reactions than their LGO-based counterparts (and perhaps because the inductive effects of the internal and γ-related acetal enhancing the electrophilic character of the adjacent β-carbon). Regardless of such details, the seeming capacity to engage both frameworks in conjugation reactions suggests that peptide-incorporated derivatives (viz. peptide–drug conjugates or PDCs)¹⁹ could serve as targeted prodrug forms of the active compounds and thus allow for the assembly of highly selective antibacterial and/or antitumor compounds. Of course, such adducts or related (simpler) ones might simply serve as a means for stabilizing otherwise highly reactive Michael acceptors and thereby enhancing bioavailability and improving therapeutic potential.²⁰

Conclusions

The results detailed above clearly demonstrate that the bioderived platform molecule LGO (1) and its ready accessible pseudoenantiomer iso-LGO (2) represent new and useful molecular scaffolds for drug development. As such they warrant further study in this regard, not least because of the significant array of derivatives likely to be available through manipulation of the associated α,β-unsaturated ketone residues. Of course, the internal acetal units within these compounds also provide capacities for their manipulation in distinct ways and so offer further potential for diversifying these frameworks.

Experimental Section

Chemical Synthesis Studies

General Experimental Protocols

Unless otherwise specified, proton (¹H) and proton-decoupled carbon [¹³C{¹H}] NMR spectra were recorded at room temperature in base-filtered CDCl₃ on a Bruker spectrometer operating at 400 MHz for proton and 100 MHz for carbon nuclei. For ¹H NMR spectra, signals arising from the residual protio-forms of the solvent were used as the internal standards. ¹H NMR data are recorded as follows: chemical shift (δ) [multiplicity, coupling constant(s) J (Hz), relative integral] where multiplicity is defined as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or combinations of the above. The signal due to residual CHCl₃ appearing at δ_H 7.26 and the central resonance of the CDCl₃ “triplet” appearing at δ_C 77.16 were used to reference ¹H and ¹³C{¹H} NMR spectra, respectively. Attenuated total reflectance (ATR) IR spectra were recorded on a Bruker Alpha-P instrument with samples being prepared by allowing a CDCl₃ or CH2Cl₂ solution of these to evaporate on the sampling plate before the spectrum was acquired. In some instances, solids were applied directly to the sampling plate. Optical rotations were measured on a Perkin-Elmer 241 polarimeter at the sodium-D line (λ = 589 nm) between 17 and 20 °C at the concentration (c) (g/100 mL) indicated using, unless otherwise specified, spectroscopic grade chloroform as solvent. Measurements were carried out in a cell with a path length (l) of 1 dm. Low-resolution ESI mass spectra were recorded on a single quadrupole liquid chromatograph–mass spectrometer, while high-resolution measurements were conducted on a time-of-flight instrument. Low- and high-resolution EI mass spectra were recorded on a magnetic-sector machine. Melting points were measured on a Reichert melting point microscope and are uncorrected. Analytical thin-layer chromatography (TLC) was performed on aluminum-backed 0.2 mm thick silica gel 60 F₂₅₄ plates as supplied by Merck. Eluted plates were visualized using a 254 nm UV lamp and/or by treatment with a suitable dip followed by heating. These dips included phosphomolybdic acid/ceric sulfate/sulfuric acid (concn)/water (37.5 g: 7.5 g: 37.5 g: 720 mL) or potassium permanganate/potassium carbonate/5% sodium hydroxide aqueous solution/water (3 g: 20 g: 5 mL: 300 mL). Flash chromatographic separations were carried out following protocols defined by Still et al.²¹ with silica gel 60 (40–63 μm) as the stationary phase and using the AR- or HPLC-grade solvents indicated. Starting materials and reagents were generally available from the Sigma-Aldrich, Merck, TCI, Strem, or Lancaster Chemical Companies and were used as supplied. Drying agents and other inorganic salts were purchased from the AJAX, BDH, or Unilab Chemical Companies. Tetrahydrofuran (THF), methanol, and dichloromethane were dried using a Glass Contour solvent purification system that is based upon a technology originally described by Grubbs et al.²² Petroleum ether refers to the fraction boiling between 40 and 60 °C. Where necessary, reactions were performed under a nitrogen atmosphere.

Specific Chemical Transformations

(1S,5R)-3-(2-Nitropyridin-3-yl)-6,8-dioxabicyclo- [3.2.1]oct-2-en-4-one (9)

A magnetically stirred solution of compound 3 (2.77 g, 11.0 mmol), pyridine 4 (1.0 g, 5.0 mmol), and copper(I) iodide (1.43 g, 7.5 mmol) containing copper powder (1.28 g, 20.0 mmol) and maintained at 50 °C was treated with Pd(dppf)Cl₂·CH₂Cl₂ (204 mg, 0.25 mmol). After 5 h, the reaction mixture was cooled to 22 °C, diluted with ethyl acetate (20 mL), and then filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (1 × 40 mL) and the combined filtrates themselves washed with ammonia (2 × 25 mL of a 5% v/v aqueous solution), water (2 × 25 mL), then brine (1 × 25 mL) before being dried (Na₂SO₄) and filtered and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 9 (1.133 g, 91%) as a yellow, crystalline solid, mp = 170 °C, [α]_D = −140.0 (c 0.07, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.59 (dd, J = 4.7 and 1.7 Hz, 1H), 7.79 (dd, J = 7.6 and 1.7 Hz, 1H), 7.65 (dd, J = 7.6 and 4.7 Hz, 1H), 7.32 (d, J = 4.7 Hz, 1H), 5.51 (s, 1H), 5.20 (m, 1H), 4.03 (m, 1H), 3.97 (d, J = 7.0 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 185.2, 148.7, 144.3, 141.4, 134.6, 127.9, 123.3, 101.2, 72.1, 66.7 (one signal obscured or overlapping); IR (ATR) ν_max 2971, 1702, 1541, 1406, 1364, 1101, 984, 930, 890, 819, 647 cm^–1; MS (ESI, +ve) m/z 271 [(M + Na)⁺, 100%], 249 [(M + H)⁺, 29%]; HRMS (ESI, +ve) 249.0509 (M + H)⁺ (calcd for C₁₁H₈N₂O₅ 249.0506).

(1S,5R)-3-(3-Nitropyridin-2-yl)-6,8-dioxabicyclo[3.2.1]oct-2-en-4-one (10)

A magnetically stirred mixture of compound 3 (1.50 g, 6.0 mmol), copper(I) iodide (857 mg, 4.5 mmol), copper powder (763 mg, 12.0 mmol), and Pd(dppf)Cl₂·CH₂Cl₂ (122 mg, 0.15 mmol) in dry DMSO (10 mL) maintained at 50 °C was treated, dropwise over 2 h, with a solution of pyridine 5 (609 mg, 3.0 mmol) in dimethyl sulfoxide (DMSO) (30 mL). After 4 h, the reaction mixture was cooled to 22 °C then diluted with ethyl acetate (20 mL) and water (20 mL) before being filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (3 × 20 mL) and the combined filtrates washed with water (2 × 30 mL) then brine (1 × 30 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 10 (589 mg, 79%) as a yellow, crystalline solid, no mp, decomposition above 120 °C, [α]_D = −231.7 (c 0.06, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.84 (dd, J = 4.8 and 1.5 Hz, 1H), 8.34 (dd, J = 8.3 and 1.5 Hz, 1H), 7.72 (d, J = 4.8 Hz, 1H), 7.52 (m, 1H), 5.51 (s, 1H), 5.29 (m, 1H), 4.02 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 185.3, 153.1, 146.4, 146.3, 146.1, 136.7, 132.3, 124.0, 101.2, 72.3, 66.6; IR (ATR) ν_max 3074, 2965, 2899, 1703, 1527, 1357, 1109, 1101, 984, 890, 574 cm^–1; MS (ESI, +ve) m/z 271 [(M + Na)⁺, 100%]; HRMS (ESI, +ve) 249.0506 (M + H)⁺ (calcd for C₁₁H₈N₂O₅ 249.0506).

(1S,5R)-3-(3-Nitropyridin-4-yl)-6,8-dioxabicyclo[3.2.1]oct-2-en-4-one (11)

A magnetically stirred mixture of compound 3 (2.2 g, 8.8 mmol), pyridine 6 (812 mg, 4.0 mmol), copper(I) iodide (1.14 g, 6.0 mmol), and copper powder (1.27 g, 20.0 mmol) in dry DMSO (40 mL) maintained at 50 °C was treated with Pd(dppf)Cl₂·CH₂Cl₂ (34 mg, 0.04 mmol). After 5 h, the reaction mixture was cooled to 22 °C before being diluted with ethyl acetate (20 mL) then filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (1 × 40 mL) and the combined filtrates washed with ammonia (2 × 25 mL of a 5% v/v aqueous solution), water (2 × 25 mL), and then brine (1 × 25 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 11 (348 mg, 35%) as a yellow, crystalline solid, no mp, decomposition above 90 °C, [α]_D = −268.3 (c 0.06, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 9.28 (s, 1H), 8.87 (d, J = 4.7 Hz, 1H), 7.36 (d, J = 4.7 Hz, 1H), 7.25 (s, 1H), 5.52 (s, 1H), 5.22 (s, 1H), 4.25-3.82 (complex m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 184.6, 154.1, 145.6, 144.4(2), 144.3(7), 136.6, 135.2, 125.2, 101.1, 72.1, 66.8; IR (ATR) ν_max 2983, 2901, 1712, 1544, 1524, 1355, 1107, 984, 935, 891, 579 cm^–1; MS (ESI, +ve) m/z 271 [(M + Na)⁺, 100%]; HRMS (ESI, +ve) 249.0506 (M + H)⁺ (calcd for C₁₁H₈N₂O₅ 249.0506).

(1S,5R)-3-(6-Methyl-3-nitropyridin-2-yl)-6,8-dioxabicyclo[3.2.1]oct-2-en-4-one (12)

A magnetically stirred mixture of compound 3 (717 mg, 2.8 mmol), pyridine 7 (308 mg, 1.4 mmol), copper(I) iodide (407 mg, 2.1 mmol), and copper powder (362 mg, 5.7 mmol) in dry DMSO (40 mL) maintained at 50 °C was treated with Pd(dppf)Cl₂·CH₂Cl₂ (23 mg, 0.03 mmol). After 5 h, the reaction mixture was cooled to 22 °C, diluted with ethyl acetate (20 mL), and then filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (1 × 40 mL) and the combined filtrates washed with ammonia (2 × 25 mL of a 5% v/v aqueous solution), water (2 × 25 mL), and then brine (1 × 25 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 12 (207 mg, 56%) as a yellow, crystalline solid, no mp, decomposition above 105 °C, [α]_D = +18.3 (c 0.06, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 4.8 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 5.50 (s, 1H), 5.25 (m, 1H), 4.02 (broad s, 2H), 2.66 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 185.5, 163.6, 146.0, 145.8, 144.0, 137.0, 132.5, 123.6, 101.3, 72.3, 66.6, 24.7; IR (ATR) ν_max 3074, 2977, 2892, 1702, 1574, 1521, 1351, 1110, 985, 890, 834, 548 cm^–1; MS (ESI, +ve) m/z 285 [(M + Na)⁺, 100%], 263 [(M + H)⁺, 15]; HRMS (ESI, +ve) 263.0661 (M + H)⁺ (calcd for C₁₂H₁₀N₂O₅ 263.0662).

(1S,5R)-3-(5-Chloro-3-nitropyridin-2-yl)-6,8-dioxabicyclo[3.2.1]oct-2-en-4-one (13)

A magnetically stirred mixture of compound 3 (1.5 g, 6.0 mmol), copper(I) iodide (857 mg, 4.5 mmol), copper powder (763 mg, 12.0 mmol), and Pd(dppf)Cl₂·CH₂Cl₂ (122 mg, 0.15 mmol) in dry DMSO (10 mL) maintained at 50 °C was treated, dropwise over 2 h, with a solution of pyridine 8 (742 mg, 3.0 mmol) in DMSO (30 mL). After 4 h, the reaction mixture was cooled to 22 °C then diluted with ethyl acetate (20 mL) and water (20 mL) before being filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (3 × 20 mL) and the combined filtrates washed with water (2 × 30 mL) then brine (1 × 30 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 13 (699 mg, 82%) as a yellow, crystalline solid, no mp, decomposition above 150 °C, [α]_D = −416.0 (c 0.05, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.79 (d, J = 2.1 Hz, 1H), 8.34 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 4.8 Hz, 1H), 5.50 (s, 1H), 5.27 (m, 1H), 4.06–3.98 (complex m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 185.0, 152.1, 146.9, 145.6, 144.2, 135.8, 132.3, 131.9, 101.1, 72.3, 66.6; IR (ATR) ν_max 3070, 2963, 2900, 1705, 1551, 1527, 1447, 1348, 1108, 984, 895, 440 cm^–1; MS (ESI, +ve) m/z 307 and 305 [(M + Na)⁺, 35 and 100%]; HRMS (ESI, +ve) 283.0118 (M + H)⁺ (calcd for C₁₁H₇ClN₂O₅ 283.0116).

(6S,9R)-6,7,9,10-Tetrahydro-5H-6,9-epoxyoxepino[4′,3′:4,5]pyrrolo[2,3-b]pyridine (14)

A magnetically stirred mixture of compound 9 (100 mg, 0.4 mmol) and 10% palladium on carbon (10 mg) in methanol (8 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 16 h, the reaction mixture was flushed with nitrogen then filtered through a pad of diatomaceous earth and the filtrate concentrated under reduced pressure. The residue thus obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 14 (19 mg, 23%) as a yellow, crystalline solid, no mp, decomposition above 130 °C, [α]_D = +11.1 (c 0.09, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 11.75 (broad s, 1H), 8.31 (broad s, 1H), 7.83 (dd, J = 7.8 and 1.5 Hz, 1H), 7.09 (dd, J = 7.8 and 4.8 Hz, 1H), 6.28 (s, 1H), 5.02 (m, 1H), 4.12 (m, 1H), 3.71 (dd, J = 7.5 and 2.2 Hz, 1H), 3.43 (ddd, J = 15.8, 4.1 and 1.4 Hz, 1H), 2.60 (dd, J = 15.8 and 1.1 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 148.4, 142.1, 134.6, 127.3, 119.8, 115.8, 102.7, 95.6, 72.8, 68.1, 28.8; IR (ATR) ν_max 3140, 3050, 2976, 2919, 2847, 2735, 1419, 1139, 1077, 946, 906, 767 cm^–1; MS (ESI, +ve) m/z 203 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 203.0812 (M + H)⁺ (calcd for C₁₁H₁₀N₂O₂ 203.0815).

(6R,9S)-6,8,9,10-Tetrahydro-5H-6,9-epoxyoxepino[4′,3′:4,5]pyrrolo[3,2-b]pyridine (15)

A magnetically stirred mixture of compound 10 (124 mg, 0.5 mmol) and 10% palladium on carbon (13 mg) in methanol (10 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 2 h, the reaction mixture was flushed with nitrogen then stirred at 50 °C for 16 h before being cooled to 22 °C then filtered through a pad of diatomaceous earth. The filtrate was concentrated under reduced pressure and the residue thus obtained subjected to flash column chromatography (silica, 1:4 v/v acetone/dichloromethane elution). Concentration of the appropriate fractions (R_f = 0.5 in 1:1 v/v acetone/dichloromethane) gave compound 15 (104 mg, 95%) as a yellow, crystalline solid, no mp, decomposition above 150 °C, [α]_D = +52.0 (c 0.05, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 10.89 (broad s, 1H), 8.43 (dd, J = 4.7 and 1.4 Hz, 1H), 7.62 (dd, J = 8.2 and 1.4 Hz, 1H), 7.09 (m, 1H), 6.14 (s, 1H), 4.96 (broadened s, 1H), 4.04 (m, 1H), 3.68 (dd, J = 7.5 and 2.3 Hz, 1H), 3.46 (m, 1H), 2.69 (dd, J = 16.2 and 2.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 144.1, 142.7, 138.4, 129.2, 119.6, 117.0, 104.4, 95.5, 72.8, 68.2, 28.2; IR (ATR) ν_max 3051, 2971, 2898, 2843, 1359, 1136, 1080, 982, 882, 769, 579, 488 cm^–1; MS (ESI, +ve) m/z 203 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 203.0812 (M + H)⁺ (calcd for C₁₁H₁₀N₂O₂ 203.0815).

(1R,4S)-3,4,5,10-Tetrahydro-1H-1,4-epoxyoxepino[4′,3′:4,5]pyrrolo[2,3-c]pyridine (16)

A magnetically stirred mixture of compound 11 (124 mg, 0.5 mmol) and 10% palladium on carbon (13 mg) in methanol (10 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 2 h, the reaction mixture was flushed with nitrogen then stirred at 50 °C for 16 h before being cooled to 22 °C then filtered through a pad of diatomaceous earth and the filtrate concentrated under reduced pressure. The residue thus obtained was subjected to flash column chromatography (silica, 5:95 v/v methanol/dichloromethane elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.5 in 10:90 v/v methanol/dichloromethane), compound 16 (94 mg, 93%) as a yellow, crystalline solid, mp = 173 °C, [α]_D = +8.0 (c 0.05, CHCl₃). ¹H NMR [400 MHz, (CD₃)₂CO] δ 8.81 (broad s, 1H), 8.20 (broad s, 1H), 7.46 (broad s, 1H), 6.25 (s, 1H), 5.02 (broadened s, 1H), 4.03 (m, 1H), 3.66 (dd, J = 7.6 and 2.2 Hz, 1H), 3.31 (ddd, J = 15.8, 4.2 and 1.5 Hz, 1H), 2.69 (d, J = 15.8 Hz, 1H) (signal due to N–H group proton not observed); ¹³C NMR (150 MHz, (CD₃)₂SO) δ 138.1, 137.5, 134.6, 130.0, 114.5, 103.1, 94.5, 71.9, 67.6, 28.0; IR (ATR) ν_max 3045, 2966, 2906, 2846, 2725, 1704, 1449, 1365, 1136, 1082, 945, 810, 590 cm^–1; MS (ESI, +ve) m/z 203 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 203.0816 (M + H)⁺ (calcd for C₁₁H₁₀N₂O₂ 203.0815).

(6R,9S)-2-Methyl-6,8,9,10-tetrahydro-5H-6,9-epoxyoxepino[4′,3′:4,5]pyrrolo[3,2-b]pyridine (17)

A magnetically stirred mixture of compound 12 (103 mg, 0.4 mmol) and 10% palladium on carbon (10 mg) in methanol (8 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 2 h, the reaction mixture was flushed with nitrogen then stirred at 50 °C for 16 h before being cooled to 22 °C then filtered through a pad of diatomaceous earth. The filtrate was concentrated under reduced pressure and the residue thus obtained subjected to flash column chromatography (silica, 1:4 v/v acetone/dichloromethane elution). Concentration of the appropriate fractions (R_f = 0.5 in 1:1 v/v acetone/dichloromethane) then gave compound 17 (71 mg, 85%) as a white, crystalline solid, no mp, decomposition at ca. 180 °C, [α]_D = 20.0 (c 0.05, CHCl₃). ¹H NMR (400 MHz, CD₃OD) δ 7.67 (dd, J = 8.4 and 2.3 Hz, 1H), 7.03 (dd, J = 8.4 and 2.3 Hz, 1H), 6.17 (broad s, 1H), 5.00 (m, 1H), 4.04 (m, 1H), 3.65 (d, J = 7.5 Hz, 1H), 3.41-3.24 (complex m, 1H), 2.75 (broadened d, J = 16.0 Hz, 1H), 2.60 (s, 3H) (signal due to N–H group proton not observed); ¹³C NMR (100 MHz, CD₃OD) δ 150.5, 143.0, 138.1, 127.5, 120.0, 116.6, 102.6, 95.4, 72.7, 67.7, 27.7, 22.2; IR (ATR) ν_max 3016, 2901, 2843, 2715, 1565, 1425, 1386, 1288, 1119, 1083, 888, 806 cm^–1; MS (ESI, +ve) m/z 217 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 217.0979 (M + H)⁺ (calcd for C₁₂H₁₂N₂O₂ 217.0977).

(6R,9R,10S)-3-Chloro-10-methoxy-6,8,9,10-tetrahydro-5H-6,9-epoxyoxepino[4′,3′:4,5]-pyrrolo-[3,2-b]pyridin-5-ol (18)

A magnetically stirred mixture of compound 13 (141 mg, 0.5 mmol) and 10% palladium on carbon (10 mg) in methanol (8 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 2 h, the reaction mixture was flushed with nitrogen then stirred at 50 °C for 16 h before being cooled to 22 °C and filtered through a pad of diatomaceous earth. The filtrate was concentrated under reduced pressure and the residue thus obtained subjected to flash column chromatography (silica, 1:4 v/v acetone/dichloromethane elution). Concentration of the appropriate fractions (R_f = 0.5 in 1:1 v/v acetone/dichloromethane) then gave compound 18 (56 mg, 40%) as a white, crystalline solid, mp = 150 °C, [α]_D = +42.9 (c 0.07, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 10.09 (broad s, 1H), 8.09 (d, J = 2.1 Hz, 1H), 7.16 (d, J = 2.1 Hz, 1H), 6.50 (s, 1H), 5.08 (m, 1H), 4.33 (m, 1H), 4.05 (m, 1H), 3.93 (s, 3H), 3.34 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 142.5, 136.7, 136.3, 125.8, 125.3, 116.2, 101.2, 98.9, 93.4, 74.2, 63.5, 58.2; IR (ATR) ν_max 3050, 2897, 1478, 1272, 1170, 1099, 1080, 1034, 983, 954, 892, 737 cm^–1; MS (ESI, +ve) m/z 589 and 587 [(2M + Na)⁺, 100%], 285 and 283 [(M + H)⁺, 30 and 80]; HRMS (ESI, +ve) 283.0475 (M + H)⁺ (calcd for C₁₂H₁₁ClN₂O₄ 283.0480).

(1R,5R)-3-Iodo-6,8-dioxabicyclo[3.2.1]oct-3-en-2-one (19)

A magnetically stirred mixture of iso-LGO (2, 700 mg, 5.56 mmol) and pyridine (530 mg, 6.67 mmol) in dichloromethane (28 mL) maintained at 22 °C was treated with molecular iodine (1.83 g, 7.78 mmol). The ensuing mixture was stirred for 18 h then diluted with ethyl acetate (100 mL) and the resulting mixture washed with Na₂S₂O₃ (1 × 50 mL of a 1 M aqueous solution), water (1 × 100 mL), and brine (1 × 100 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 7:93 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 19 (911 mg, 66%) as a yellow, crystalline solid, mp = 112 °C, [α]_D = +158.8 (c 0.07, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J = 3.5 Hz, 1H), 5.63 (d, J = 3.5 Hz, 1H), 5.01 (dd, J = 6.3 and 1.5 Hz, 1H), 4.13 (dd, J = 8.4 and 6.3 Hz, 1H), 3.66 (dd, J = 8.4 and 1.5 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 188.4, 155.6, 102.3, 97.5, 78.2, 62.9; IR (ATR) ν_max 3065, 2964, 2898, 2890, 1701, 1686, 1580, 1336, 1300, 1101, 979, 907, 524 cm^–1; MS (ESI, +ve) m/z 252 (M^+•, 100%), 209 (17), 53 (22); HRMS (EI, 70 eV) 251.9289 (M^+•) (calcd for C₆H₅IO₃ 251.9283).

(1R,5R)-3-(2-Nitropyridin-3-yl)-6,8-dioxabicyclo[3.2.1]oct-3-en-2-one (21)

A magnetically stirred mixture of compound 19 (126 mg, 0.5 mmol), pyridine 4 (121 mg, 0.6 mmol), copper(I) iodide (143 mg, 0.75 mmol), and copper powder (159 mg, 2.5 mmol) in dry DMSO (5 mL) maintained at 50 °C was treated with Pd(dppf)Cl₂·CH₂Cl₂ (8 mg, 0.01 mmol). After 5 h, the reaction mixture was cooled to 22 °C, diluted with ethyl acetate (10 mL), then filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (1 × 20 mL) and the combined filtrates washed with ammonia (2 × 15 mL of a 5% v/v aqueous solution), water (2 × 15 mL), and then brine (1 × 15 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 1:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 21 (102 mg, 82%) as yellow, crystalline solid, no mp, decomposition above 120 °C, [α]_D = −20.0 (c 0.02, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.58 (dd, J = 4.6 and 1.7 Hz, 1H), 7.78 (dd, J = 7.6 and 1.7 Hz, 1H), 7.67 (dd, J = 7.6 and 4.6 Hz, 1H), 7.17 (d, J = 3.4 Hz, 1H), 5.96 (d, J = 3.4 Hz, 1H), 4.89 (dd, J = 6.3 and 1.5 Hz, 1H), 4.17 (dd, J = 8.4 and 6.3 Hz, 1H), 3.90 (dd, J = 8.4 and 1.5 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 190.9, 156.4, 148.8, 143.7, 141.5, 135.1, 128.5, 123.2, 96.5, 79.1, 62.5; IR (ATR) ν_max 3063, 2964, 2898, 1709, 1538, 1405, 1359, 1116, 986, 915, 730, 645, 533 cm^–1; MS (ESI, +ve) m/z 271 [(M + Na)⁺, 100%]; HRMS (ESI, +ve) 249.0506 (M + H)⁺ (calcd for C₁₁H₈N₂O₅ 249.0506).

(1R,5R)-3-(3-Nitropyridin-2-yl)-6,8-dioxabicyclo[3.2.1]oct-3-en-2-one (22)

A magnetically stirred mixture of compound 19 (126 mg, 0.5 mmol), copper(I) iodide (143 mg, 0.75 mmol), copper powder (159 mg, 2.5 mmol), and Pd(dppf)Cl₂·CH₂Cl₂ (8 mg, 0.01 mmol) in dry DMSO (2 mL) maintained at 50 °C was treated, dropwise over 2 h, with the solution of pyridine 5 (121 mg, 0.6 mmol) in DMSO (3 mL). After 4 h, the reaction mixture was cooled to 22 °C, diluted with ethyl acetate (10 mL) and water (10 mL), and then filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (3 × 10 mL) and the combined filtrates washed with water (2 × 10 mL) then brine (1 × 10 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 1:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 22 (105 mg, 84%) as a yellow, crystalline solid, no mp, decomposition above 120 °C, [α]_D = +52.5 (c 0.04, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.85 (dd, J = 4.7 and 1.5 Hz, 1H), 8.36 (dd, J = 8.3 and 1.5 Hz, 1H), 7.78-7.40 (complex m, 2H), 6.03 (d, J = 3.4 Hz, 1H), 4.91 (dd, J = 6.3 and 1.5 Hz, 1H), 4.19 (dd, J = 8.4 and 6.3 Hz, 1H), 3.93 (dd, J = 8.4 and 1.5 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 191.1, 153.3, 146.1, 145.8, 136.9, 132.3, 124.2, 96.7, 79.2, 62.5 (one signal obscured or overlapping); IR (ATR) ν_max 2967, 1702, 1593, 1526, 1356, 1114, 986 cm^–1; MS (ESI, +ve) m/z 271 [(M + Na)⁺, 100%]; HRMS (ESI, +ve) 249.0506 (M + H)⁺ (calcd for C₁₁H₈N₂O₅ 249.0506).

(1R,5R)-3-(3-Nitropyridin-4-yl)-6,8-dioxabicyclo[3.2.1]oct-3-en-2-one (23)

A magnetically stirred mixture of compound 19 (166 mg, 0.66 mmol), pyridine 7 (160 mg, 0.79 mmol), copper(I) iodide (190 mg, 1 mmol), and copper powder (209 mg, 3.3 mmol) in dry DMSO (7 mL) maintained at 22 °C was treated with Pd(dppf)Cl₂·CH₂Cl₂ (27 mg, 0.03 mmol). After 16 h, the reaction mixture was diluted with ethyl acetate (10 mL) then filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (1 × 20 mL) and the combined filtrates washed with ammonia (2 × 15 mL of a 5% v/v aqueous solution), water (2 × 15 mL), and then brine (1 × 15 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 23 (84 mg, 51%) as a yellow, crystalline solid, no mp, decomposition above ca. 90 °C, [α]_D = −15.0 (c 0.04, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 9.29 (s, 1H), 8.87 (broad s, 1H), 7.25 (d, J = 4.9 Hz, 1H), 7.20 (d, J = 3.4 Hz, 1H), 5.99 (d, J = 3.4 Hz, 1H), 4.92 (dd, J = 6.3 and 1.5 Hz, 1H), 4.20 (dd, J = 8.4 and 6.3 Hz, 1H), 3.95 (dd, J = 8.4 and 1.5 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 190.3, 154.4, 145.7, 143.9, 136.2, 135.5, 125.1, 96.4, 79.2, 62.6 (one signal obscured or overlapping); IR (ATR) ν_max 3058, 2965, 1708, 1598, 1544, 1524, 1359, 1110, 1033, 987, 918, 856, 583, 533 cm^–1; MS (ESI, +ve) m/z 303 [(M + Na + MeOH)⁺, 100%]; HRMS (ESI, +ve) 249.0505 (M + H)⁺ (calcd for C₁₁H₈N₂O₅ 249.0506).

(1R,5R)-3-(6-Methyl-3-nitropyridin-2-yl)-6,8-dioxabicyclo[3.2.1]oct-3-en-2-one (24)

A magnetically stirred mixture of compound 19 (252 mg, 1.0 mmol), copper(I) iodide (286 mg, 1.5 mmol), copper powder (312 mg, 5.0 mmol), and Pd(dppf)Cl₂·CH₂Cl₂ (40 mg, 0.05 mmol) in dry DMSO (4 mL) maintained at 50 °C was treated, dropwise over 2 h, with a solution of pyridine 7 (260 mg, 1.2 mmol) in DMSO (6 mL). After 4 h, the reaction mixture was cooled to 22 °C, diluted with ethyl acetate (10 mL) then filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (1 × 20 mL) and the combined filtrates washed with ammonia (2 × 15 mL of a 5% v/v aqueous solution), water (2 × 15 mL), and then brine (1 × 15 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 24 (191 mg, 73%) as a white, crystalline solid, no mp, decomposition above ca. 115 °C, [α]_D = +6.3 (c 0.08, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 3.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 6.01 (d, J = 3.4 Hz, 1H), 4.88 (dd, J = 6.3 and 1.5 Hz, 1H), 4.17 (dd, J = 8.3 and 6.3 Hz, 1H), 3.94 (dd, J = 8.3 and 1.5 Hz, 1H), 2.66 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 191.2, 163.8, 145.5, 145.4, 144.1, 137.2, 132.5, 123.8, 96.7, 79.2, 62.5, 24.7; IR (ATR) ν_max 3079, 2996, 2898, 1702, 1573, 1519, 1349, 1118, 986, 922, 736, 535 cm^–1; MS (ESI, +ve) m/z 285 [(M + Na)⁺, 100%]; HRMS (ESI, +ve) 263.0664 (M + H)⁺ (calcd for C₁₂H₁₀N₂O₅ 263.0662).

(1R,5R)-3-(5-Chloro-3-nitropyridin-2-yl)-6,8-dioxabicyclo[3.2.1]oct-3-en-2-one (25)

A magnetically stirred mixture of compound 19 (252 mg, 1.0 mmol), copper(I) iodide (286 mg, 1.5 mmol), copper powder (312 mg, 5.0 mmol), and Pd(dppf)Cl₂·CH₂Cl₂ (40 mg, 0.05 mmol) in dry DMSO (4 mL) maintained at 50 °C was treated, dropwise over 2 h, with a solution of pyridine 8 (298 mg, 1.25 mmol) in DMSO (6 mL). After 4 h, the reaction mixture was cooled to 22 °C then diluted with ethyl acetate (10 mL) and water (10 mL) before being filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (3 × 15 mL) and the combined filtrates washed with water (2 × 15 mL) then brine (1 × 15 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 1:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 25 (156 mg, 55%) as a yellow, crystalline solid, no mp, decomposition above ca. 110 °C, [α]_D = +46.0 (c 0.05, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.80 (d, J = 2.2 Hz, 1H), 8.35 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 3.4 Hz, 1H), 6.03 (d, J = 3.4 Hz, 1H), 4.91 (dd, J = 6.3 and 1.5 Hz, 1H), 4.19 (dd, J = 8.4 and 6.3 Hz, 1H), 3.90 (dd, J = 8.4 and 1.5 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 190.9, 152.3, 146.3, 145.7, 143.9, 136.0, 132.5, 131.9, 96.6, 79.2, 62.5; IR (ATR) ν_max 3070, 2899, 1705, 1551, 1528, 1448, 1350, 1114, 986, 899, 776, 563 cm^–1; MS (ESI, +ve) m/z 307 and 305 [(M + Na)⁺, 35 and 100%]; HRMS (ESI, +ve) 283.0118 (M + H)⁺ (calcd for C₁₁H₇ClN₂O₅ 283.0116).

(1R,5R)-3-(4-Nitropyridin-3-yl)-6,8-dioxabicyclo[3.2.1]oct-3-en-2-one (26)

A magnetically stirred mixture of compound 19 (170 mg, 0.67 mmol), pyridine 20 (137 mg, 0.67 mmol), copper(I) iodide (190 mg, 1.0 mmol), and copper powder (214 mg, 3.37 mmol) in dry DMSO (7 mL) maintained at 50 °C was treated with Pd(dppf)Cl₂·CH₂Cl₂ (5 mg, 0.06 mmol). After 5 h, the reaction mixture was cooled to 22 °C then diluted with ethyl acetate (10 mL) before being filtered through a plug of TLC-grade silica topped with diatomaceous earth. The solids so retained were washed with ethyl acetate (1 × 20 mL) and the combined filtrates washed with ammonia (2 × 15 mL of a 5% v/v aqueous solution), water (2 × 15 mL), and then brine (1 × 15 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 26 (78 mg, 46%) as a yellow, crystalline solid, no mp, decomposition above 105 °C, [α]_D = −52.0 (c 0.05, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 8.96 (broad s, 1H), 8.69 (broad s, 1H), 7.89 (s, 1H), 7.22 (d, J = 3.3 Hz, 1H), 5.98 (d, J = 3.3 Hz, 1H), 4.89 (m, 1H), 4.17 (dd, J = 8.5 and 6.3 Hz, 1H), 3.90 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 191.0, 153.8, 152.5, 152.2, 143.8, 134.0, 122.1, 117.1, 96.5, 79.1, 62.4; IR (ATR) ν_max 3098, 3059, 2965, 1709, 1529, 1355, 1108, 986, 913, 858, 720, 535 cm^–1; MS (ESI, +ve) m/z 303 [(M + Na + MeOH)⁺, 100%], 271 [(M + Na)⁺, 60]; HRMS (ESI, +ve) 249.0506 (M + H)⁺ (calcd for C₁₁H₈N₂O₅ 249.0506).

(6R,9S)-5,8,9,10-Tetrahydro-6H-6,9-epoxyoxepino[4′,5′:4,5]pyrrolo[2,3-b]pyridine (27)

A magnetically stirred mixture of compound 21 (194 mg, 0.78 mmol) and 10% palladium on carbon (20 mg) in methanol (16 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 2 h, acetic acid (47 μL, 0.78 mmol) was added to the reaction mixture that was then flushed with nitrogen before being stirred at 50 °C for 16 h. After this time, the mixture was cooled to 22 °C then filtered through a pad of diatomaceous earth and the filtrate concentrated under reduced pressure. The residue thus obtained was subjected to flash column chromatography (silica, ethyl acetate elution) and so afforded, after concentration of the appropriate fractions (R_f = 0.5 in 1:99 v/v methanol/ethyl acetate), compound 27 (75 mg, 48%) as a white, crystalline solid, no mp, decomposition above ca. 160 °C, [α]_D = −21.3 (c 0.08, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 12.41 (broadened s, 1H), 8.23 (dd, J = 4.8 and 1.5 Hz, 1H), 7.78 (dd, J = 7.8 and 1.5 Hz, 1H), 7.07 (dd, J = 7.8 and 4.8 Hz, 1H), 6.04 (d, J = 2.4 Hz, 1H), 5.43 (d, J = 4.1 Hz, 1H), 4.13 (d, J = 6.7 Hz, 1H), 4.02 (dd, J = 6.7 and 4.1 Hz, 1H), 3.16 (ddd, J = 15.7, 2.4 and 1.1 Hz, 1H), 2.79 (d, J = 15.6 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 148.5, 141.1, 135.4, 127.0, 120.5, 115.7, 102.3, 101.2, 73.7, 70.5, 30.7; IR (ATR) ν_max 3143, 3057, 2940, 2886, 1587, 1417, 1288, 1144, 1001, 996, 768, 514 cm^–1; MS (ESI, +ve) m/z 203 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 203.0822 (M + H)⁺ (calcd for C₁₁H₁₀N₂O₂ 203.0821).

(6S,9R)-6,7,9,10-Tetrahydro-5H-6,9-epoxyoxepino[4′,5′:4,5]pyrrolo[3,2-b]pyridine (28)

A magnetically stirred mixture of compound 22 (170 mg, 0.68 mmol) and 10% palladium on carbon (17 mg) in methanol (14 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 2 h, acetic acid (40 μL, 0.68 mmol) was added to the reaction mixture that was flushed with nitrogen before being stirred at 50 °C for 16 h. After this time, the reaction mixture was cooled to 22 °C then filtered through a pad of diatomaceous earth and the filtrate concentrated under reduced pressure. The residue thus obtained was subjected to flash column chromatography (silica, ethyl acetate elution) and so afforded, after concentration of the appropriate fractions (R_f = 0.5 in 5:95 v/v methanol/ ethyl acetate), compound 28 (123 mg, 89%) as a yellow oil, [α]_D = −30.2 (c 0.04, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 10.87 (broadened s, 1H), 8.33 (d, J = 4.8 Hz, 1H), 7.67 (dd, J = 8.2 and 1.4 Hz, 1H), 7.07 (ddd, J = 8.2, 4.8 and 1.3 Hz, 1H), 5.94 (d, J = 2.3 Hz, 1H), 5.28 (d, J = 4.2 Hz, 1H), 4.02 (d, J = 6.6 Hz, 1H), 3.88 (dd, J = 6.6 and 4.2 Hz, 1H), 3.13 (dd, J = 16.0 and 2.3 Hz, 1H), 2.85 (d, J = 16.0 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 143.9, 141.3, 139.9, 129.5, 120.0, 116.5, 103.4, 101.0, 73.4, 70.4, 30.3; IR (ATR) ν_max 3138, 3051, 2954, 2888, 1556, 1412, 1359, 1106, 999, 845, 729, 517 cm^–1; MS (ESI, +ve) m/z 203 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 203.0822 (M + H)⁺ (calcd for C₁₁H₁₀N₂O₂ 203.0821).

(6R,9S)-5,6,8,9-Tetrahydro-10H-6,9-epoxyoxepino[4′,5′:4,5]pyrrolo[2,3-c]pyridin-10-ol (29)

A magnetically stirred mixture of compound 23 (87 mg, 0.35 mmol) and 10% palladium on carbon (9 mg) in methanol (7 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 2 h, acetic acid (20 μL, 0.35 mmol) was added to the reaction mixture that was flushed with nitrogen then stirred at 50 °C for 16 h. After this time, the reaction mixture was cooled to 22 °C then filtered through a pad of diatomaceous earth and the filtrate concentrated under reduced pressure. The residue thus obtained was subjected to flash column chromatography (silica, ethyl acetate elution) and so afforded, after concentration of the appropriate fractions (R_f = 0.1 in 1:4 v/v methanol/dichloromethane), compound 29 (42 mg, 55%) as a yellow, crystalline solid, no mp, decomposition above ca. 150 °C, [α]_D = +31.4 (c 0.07, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 13.91 (broadened s, 1H), 8.58 (m, 1H), 7.75 (s, 1H), 7.34 (m, 1H), 6.00 (d, J = 2.3 Hz, 1H), 5.72 (d, J = 3.9 Hz, 1H), 4.10 (m, 2H), 3.10 (dd, J = 15.7 and 2.5 Hz, 1H), 2.78 (d, J = 15.7 Hz, 1H); ¹³C NMR [100 MHz, (CD₃)₂CO] δ 139.9, 134.6, 130.0, 127.4, 113.4, 104.3, 100.7, 99.0, 72.9, 68.2, 30.1; IR (ATR) ν_max 3062, 2955, 2896, 1637, 1611, 1440, 1111, 1026, 954, 858, 825, 737 cm^–1; MS (ESI, +ve) m/z 219 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 219.0761 (M + H)⁺ (calcd for C₁₁H₁₀N₂O₃ 219.0764).

(6S,9R)-2-Methyl-6,7,9,10-tetrahydro-5H-6,9-epoxyoxepino[4′,5′:4,5]pyrrolo[3,2-b]pyridine (30)

A magnetically stirred mixture of compound 24 (194 mg, 0.74 mmol) and 10% palladium on carbon (19 mg) in methanol (15 mL) maintained at 22 °C was placed under a balloon of hydrogen. After 24 h, the reaction mixture was filtered through a pad of diatomaceous earth and the filtrate concentrated under reduced pressure. The residue thus obtained was subjected to flash column chromatography (silica, 1:99 v/v methanol/ethyl acetate elution) and so afforded, after concentration of the appropriate fractions (R_f = 0.5 in 1:9 v/v methanol/ethyl acetate), compound 30 (136 mg, 85%) as a yellow, crystalline solid, no mp, decomposition above ca. 128 °C, [α]_D = −52.0 (c 0.05, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 11.05 (broad s, 1H), 7.51 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 5.90 (d, J = 2.4 Hz, 1H), 5.19 (s, 1H), 3.98 (d, J = 6.6 Hz, 1H), 3.84 (m, 1H), 3.08 (dd, J = 16.0 and 2.4 Hz, 1H), 2.83 (d, J = 16.0 Hz, 1H), 2.60 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 151.2, 144.4, 138.3, 127.1, 119.2, 116.8, 103.8, 101.2, 73.5, 70.4, 30.3, 24.1; IR (ATR) ν_max 2954, 2920, 1567, 1423, 1291, 1107, 1055, 1002, 845, 734, 525 cm^–1; MS (ESI, +ve) m/z 217 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 217.0979 (M + H)⁺ (calcd for C₁₂H₁₂N₂O₂ 217.0977).

(5S,6R,9R)-6,7,9,10-Tetrahydro-5H-6,9-epoxyoxepino[4′,3′:4,5]pyrrolo[2,3-b]pyridin-5-ol (31)

A magnetically stirred mixture of compound 9 (50 mg, 0.2 mmol) and iron powder (56 mg, 1 mmol) in dimethyl ether (DME) (1 mL) was treated with acetic acid (1 mL of a 1 M aqueous solution) then heated at 50 °C for 8 h before being cooled and filtered through a pad of diatomaceous earth. The filtrate was diluted was ethyl acetate (50 mL) then washed with water (1 × 20 mL) and brine (1 × 20 mL) before being dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 3:1 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 31 (29 mg, 68%) as white, crystalline solid, no mp, decomposition above ca. 130 °C, [α]_D = −124.3 (c 0.07, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 12.26 (broad s, 1H), 8.10 (dd, J = 7.9 and 1.5 Hz, 1H), 8.02 (dd, J = 4.8 and 1.5 Hz, 1H), 7.15 (dd, J = 7.9 and 4.8 Hz, 1H), 5.65 (s, 1H), 4.96 (ddd, J = 6.7, 2.3 and 1.5 Hz, 1H), 4.53 (d, J = 1.5 Hz, 1H), 4.01 (dd, J = 8.0 and 6.7 Hz, 1H), 3.39 (dd, J = 8.0 and 2.3 Hz, 1H) (signal due to OH group proton not observed); ¹³C NMR (100 MHz, CDCl₃) δ 148.1, 142.3, 134.9, 128.2, 119.0, 116.3, 105.1, 94.9, 79.1, 66.4, 63.8; IR (ATR) ν_max 3153, 3054, 2977, 1557, 1418, 1284, 1145, 1080, 983, 942, 894, 773, 535 cm^–1; MS (ESI, +ve) m/z 219 [(M + H)⁺, 100%]; HRMS (ESI, +ve) 219.0771 (M + H)⁺ (calcd for C₁₁H₁₀N₂O₃ 219.0770).

(5S,6R,9R)-5-Ethoxy-6,7,9,10-tetrahydro-5H-6,9-epoxyoxepino[4′,3′:4,5]pyrrolo[2,3-b]pyridine (32)

A magnetically stirred mixture of compound 9 (124 mg, 0.5 mmol), CaCl₂ (175 mg, 1.5 mmol), and iron powder (84 mg, 1.5 mmol) in water/ethanol (5 mL of a 1:4 v/v mixture) was heated under reflux for 2 h. The cooled reaction mixture was filtered through a pad of diatomaceous earth and the filtrate diluted was ethyl acetate (50 mL) before being washed with water (1 × 20 mL) and brine (1 × 20 mL) then dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 1:3 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 32 (67 mg, 55%) as a white, crystalline solid, no mp, decomposition above ca. 125 °C, [α]_D = −140.0 (c 0.02, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ 12.01 (s, 1H), 8.34 (broad s, 1H), 7.98 (dd, J = 7.9 and 1.4 Hz, 1H), 7.15 (m, 1H), 6.31 (s, 1H), 5.07 (m, 1H), 4.40 (s, 1H), 4.08 (dd, J = 7.8 and 6.7 Hz, 1H), 3.93-3.60 (complex m, 2H), 3.47 (dd, J = 7.8 and 2.3 Hz, 1H), 1.29 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 148.5, 142.5, 136.1, 128.1, 119.8, 116.6, 103.4, 95.5, 76.3, 73.1, 64.1(0), 64.0(6), 15.7; IR (ATR) ν_max 3150, 3051, 2974, 2893, 1555, 1416, 1280, 1147, 1078, 984, 889, 773 cm^–1; MS (ESI, +ve) m/z 269 [(M + Na)⁺, 100%], 247 [(M + H)⁺, 11]; HRMS (ESI, +ve) 247.1083 (M + H)⁺ (calcd for C₁₃H₁₄N₂O₃ 247.1083).

(6R,9S)-9,10-Dihydro-6H-6,9-epoxyoxepino[4′,5′:4,5]pyrrolo[2,3-b]pyridin-5(8H)-one (33)

A magnetically stirred mixture of compound 9 (50 mg, 0.2 mmol) and iron powder (56 mg, 1 mmol) in DME (1 mL) was treated with HCl (1 mL of a 1 M of an aqueous solution) then heated at 50 °C for 18 h before being cooled then filtered through a pad of diatomaceous earth. The filtrate was diluted was ethyl acetate (50 mL) before being washed with water (1 × 20 mL) and brine (1 × 20 mL) then dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue so obtained was subjected to flash column chromatography (silica, 2:1 v/v ethyl acetate/40–60 petroleum ether elution) and thus afforded, after concentration of the appropriate fractions (R_f = 0.2), compound 33(11b) (26 mg, 58%) as white, crystalline solid, mp = 275 °C, [α]_D = −221.7 (c 0.06, CHCl₃). ¹H NMR [400 MHz, (CD₃)₂CO] δ 11.59 (broad s, 1H), 8.37 (broadened s, 1H), 8.25 (dd, J = 7.8 and 1.6 Hz, 1H), 7.29 (dd, J = 7.8 and 4.6 Hz, 1H), 5.95 (d, J = 4.8 Hz, 1H), 5.44 (s, 1H), 4.11 (dd, J = 7.2 and 4.6 Hz, 1H), 3.96 (d, J = 7.2 Hz, 1H) (signal due to N–H group proton not observed); ¹³C NMR [100, MHz, (CD₃)₂CO] δ 185.6, 151.3, 148.9, 144.8, 128.7, 118.6, 116.5, 106.2, 102.3, 71.2, 67.1; IR (ATR) ν_max 2981, 2888, 1679, 1592, 1480, 1416, 1109, 1075, 889, 805 cm^–1; MS (ESI, +ve) m/z 239 [(M + Na)⁺, 100%], 217 [(M + H)⁺, 10]; HRMS (ESI, +ve) 217.0609 (M + H)⁺ (calcd for C₁₁H₈N₂O₃ 217.0608).

Formation of l-Cysteine Adducts with Compounds 9–12, 14–17 and 21–30 for Mass Spectral Analysis

A magnetically stirred solution of the relevant compound (0.01 mmol) in methanol (0.2 mL) maintained at 22 °C was treated with l-cysteine (1.4 mg, 0.01 mmol) or the equivalent amount of the corresponding methyl ester and after 24 h the reaction mixture was diluted with methanol (2 mL) then analyzed using both +ve and −ve ionization modes of ESI mass spectrometry.

S-((1R,2S,3R,5R)-3-(3-Nitropyridin-2-yl)-4-oxo-6,8-dioxabicyclo[3.2.1]octan-2-yl)-l-cysteine (34) and S-((1R,2S,3S,5R)-3-(3-Nitropyridin-2-yl)-4-oxo-6,8-dioxabicyclo[3.2.1]octan-2-yl)-l-cysteine (35)

A magnetically stirred solution of compound 10 (100 mg, 0.4 mmol) in methanol (1.4 mL) maintained at 22 °C was treated with a solution of l-cysteine (24 mg, 0.2 mmol) in water (700 μL). After 48 h the resulting mixture was diluted with methanol (10 mL) then filtered through a pad of diatomaceous earth and the filtrate concentrated under reduced pressure. The residue thus obtained was subjected to flash column chromatography (silica, 1:10:10 v/v/v acetic acid/methanol/ethyl acetate elution) and thus afforded, two fractions, A and B.

Concentration of fraction A (R_f = 0.3 in 1:10:10 v/v/v acetic acid/methanol/ethyl acetate) afforded compound 34 (13 mg, 18%) as a yellow oil, [α]_D = −93.3 (c 0.03, CH₃OH). ¹H NMR (400 MHz, CD₃OD) δ 8.74 (dd, J = 4.7 and 1.4 Hz, 1H), 8.30 (dd, J = 8.3 and 1.4 Hz, 1H), 7.46 (dd, J = 8.3 and 4.7 Hz, 1H), 5.28 (s, 1H), 4.31 (m, 1H), 4.20 (m, 1H), 3.84-3.70 (complex m, 2H), 3.56 (m, 1H), 3.30 (m, 1H), 3.09 (dd, J = 14.7 and 3.7 Hz, 1H), 2.74 (dd, J = 14.7 and 9.8 Hz, 1H) (signals due to NH₂ and CO₂H group protons not observed); ¹³C NMR (100 MHz, CD₃OD) δ 168.7, 152.4, 152.1, 146.9, 132.9, 122.8, 98.6, 80.6(4), 80.6(1), 68.3, 54.4, 51.8, 36.4, 33.7; IR (ATR) ν_max 3384, 2925, 1704, 1595, 1565, 1525, 1407, 1347, 1279, 1107, 1013, 818, 617, 455 cm^–1; MS (ESI, +ve) m/z 424 [(M+CH₃OH+Na)⁺, 100%]; HRMS (ESI, +ve) 402.0956 (M+CH₃OH+H)⁺ (calcd for C₁₄H₁₅N₃O₇S 402.0966).

Concentration of fraction B (R_f = 0.2 in 1:10:10 v/v/v acetic acid/methanol/ethyl acetate) afforded compound 35 (28 mg, 38%) as a yellow oil, [α]_D = −60.0 (c 0.02, CH₃OH). ¹H NMR (400 MHz, D₂O) δ 8.80 (dd, J = 4.8 and 1.5 Hz, 1H), 8.52 (dd, J = 8.4 and 1.5 Hz, 1H), 7.64 (dd, J = 8.4 and 4.8 Hz, 1H), 5.18 (s, 1H), 4.40 (q, J = 6.3 Hz, 1H), 4.18 (dd, J = 8.5 and 6.8 Hz, 1H), 4.00 (dd, J = 8.5 and 6.4 Hz, 1H), 3.88 (m, 1H), 3.75–3.39 (complex m, 2H), 3.01–2.89 (complex m, 2H) (signals due to NH₂ and CO₂H group protons not observed); ¹³C NMR (100 MHz, D₂O) δ 173.9, 172.3, 152.6, 152.2, 146.5, 134.4, 123.7, 100.1, 79.2, 68.0, 54.0, 48.9, 37.1, 32.8; IR (ATR) ν_max 3408, 2927, 1704, 1595, 1564, 1525, 1407, 1347, 1279, 1107, 1013, 807, 617, 454 cm^–1. Satisfactory MS and HRMS data could not be obtained on this compound.

Crystallographic Studies

Crystallographic Data

Compound 10

C₁₁H₈N₂O₅, M = 248.19, T = 150 K, orthorhombic, space group P2₁2₁2₁, Z = 4, a = 9.6892(4) Å, b = 9.8837(4) Å, c = 10.8874(4) Å; V = 1042.63(7) ų, D_x = 1.581 Mg m^–3, 2443 unique data (2θ_max = 58.33°), R = 0.0370 [for 2263 reflections with I > 2.0σ(I)]; R_w = 0.0831 (all data), S = 1.053.

Compound 13

C₁₁H₇ClN₂O₅, M = 282.64, T = 150 K, orthorhombic, space group P2₁2₁2₁, Z = 4, a = 7.9009(2) Å, b = 10.9614(3) Å, c = 13.2772(4) Å; V = 1149.87(6) ų, D_x = 1.633 Mg m^–3, 2681 unique data (2θ_max = 58.444°), R = 0.0360 [for 2483 reflections with I > 2.0σ(I)]; R_w = 0.0764 (all data), S = 1.037.

Compound 14

C₁₁H₁₀N₂O₂, M = 202.21, T = 150 K, monoclinic, space group P2₁, Z = 8, a = 9.90829(8) Å, b = 9.74229(7) Å, c = 19.67523(17) Å; α = 90°, β = 102.5485(8)°, γ = 90°; V = 1853.87(3) ų, D_x = 1.449 Mg m^–3, 7371 unique data (2θ_max = 147.8°), R = 0.036 [for 7251 reflections with I > 2.0σ(I)]; R_w = 0.097 (all data), S = 1.04.

Compound 16

C₁₁H₁₀N₂O₆, M = 202.21, T = 150 K, orthorhombic, space group P2₁2₁2₁, Z = 4, a = 5.12226(4) Å, b = 9.87199(79) Å, c = 18.24358(17) Å; α = 90°, β = 90°, γ = 90°; V = 922.52(2) ų, D_x = 1.456 Mg m^–3, 1875 unique data (2θ_max = 147.8°), R = 0.023 [for 1860 reflections with I > 2.0σ(I)]; R_w = 0.063 (all data), S = 1.06.

Compound 17

C₁₂H₁₂N₂O₂, M = 216.24, T = 150 K, orthorhombic, space group P2₁2₁2₁, Z = 8, a = 7.82892(6) Å, b = 11.64835(9) Å, c = 23.20164(18) Å; α = 90°, β = 90°, γ = 90°; V = 2115.85(3) ų, D_x = 1.358 Mg m^–3, 4293 unique data (2θ_max = 147.6°), R = 0.026 [for 4199 reflections with I > 2.0σ(I)]; R_w = 0.069 (all data), S = 1.03.

Compound 22

C₁₁H₈N₂O₅, M = 248.19, T = 150 K, monoclinic, space group P2₁, Z = 2, a = 5.6724(2) Å, b = 9.6556(3) Å, c = 9.4591(4) Å; β = 103.910(4)°; V = 502.89(3) ų, D_x = 1.639 Mg m^–3, 2450 unique data (2θ_max = 58.478°), R = 0.0347 [for 2314 reflections with I > 2.0σ(I)]; R_w = 0.0778 (all data), S = 1.072.

Compound 23

C₁₁H₈N₂O₅, M = 248.19, T = 150 K, orthorhombic, space group P2₁2₁2₁, Z = 4, a = 10.0284(3) Å, b = 11.8376(3) Å, c = 18.0484(5) Å; V = 2142.57(10) ų, D_x = 1.539 Mg m^–3, 5260 unique data (2θ_max = 58.734°), R = 0.0390 [for 4752 reflections with I > 2.0σ(I)]; R_w = 0.0823 (all data), S = 1.071.

Compound 29

C₁₁H₁₁N₂O_3.5, M = 227.22, T = 150 K, trigonal, space group P3₂21, Z = 6, a = 14.9944(3) Å, b = 14.9944(3) Å, c = 9.21720(10) Å; α = 90°, β = 90°, γ = 120°; V = 1794.68(7) ų, D_x = 1.261 Mg m^–3, 2443 unique data (2θ_max = 147.48°), R = 0.0311 [for 2394 reflections with I > 2.0σ(I)]; R_w = 0.0962 (all data), S = 1.133.

Compound 30

C₁₃H₁₄Cl₂N₂O₂, M = 301.16, T = 150 K, triclinic, space group P1, Z = 2, a = 8.3394(3) Å, b = 9.2251(3) Å, c = 10.2483(3) Å; α = 112.584(3)°, β = 102.063(3)°, γ = 99.352(3)°; V = 685.67(4) ų, D_x = 1.459 Mg m^–3, 4429 unique data (2θ_max = 147.6°), R = 0.031 [for 4363 reflections with I > 2.0σ(I)]; R_w = 0.087 (all data), S = 1.05.

Compound 32

C₁₃H₁₄N₂O₃, M = 246.26, T = 150 K, orthorhombic, space group P2₁2₁2₁, Z = 4, a = 9.26094(4) Å, b = 9.56903(5) Å, c = 13.38456(6) Å; α = 90°, β = 90°, γ = 90°; V = 1186.12(1) ų, D_x = 1.379 Mg m^–3, 2405 unique data (2θ_max = 147.6°), R = 0.041 [for 2393 reflections with I > 2.0σ(I)]; R_w = 0.120 (all data), S = 1.19.

Compound 33

C₁₁H₈N₂O₆, M = 216.19, T = 150 K, orthorhombic, space group P2₁2₁2₁, Z = 4, a = 4.7466(1) Å, b = 12.197927 Å, c = 16.5531(3) Å; α = 90°, β = 90°, γ = 90°; V = 958.40(3) ų, D_x = 1.498 Mg m^–3, 1931 unique data (2θ_max = 148.182°), R = 0.0313 [for 1846 reflections with I > 2.0σ(I)]; R_w = 0.0831 (all data), S = 1.063.

Structure Determinations

Data for compounds 14, 16, 17, 29, 30, 32, and 33 were measured on a Rigaku SuperNova diffractometer using Cu Kα, graphite monochromator (λ = 1.54184 Å), while those for compounds 10, 13, 22, and 23 were recorded using Mo Kα, graphite monochromator (λ = 0.71073 Å). Data collection, cell refinement, and data reduction employed the CrysAlis PRO program,²³ while SHELXT²⁴ and SHELXL²⁵ were used for structure solution and refinement. Atomic coordinates, bond lengths and angles, and displacement parameters have been deposited at the Cambridge Crystallographic Data Centre (CCDC nos. 1990200–1990210). These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Accession Codes

CCDC depositions 1990200–1990210 contain the supplementary crystallographic data for this article. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by e-mailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.

Biological Studies

Antibacterial Assays

The bacterium to be tested was streaked onto a tryptic soy agar plate then incubated at 37 °C for 24 h. One colony was then transferred to fresh tryptic soy broth (15 mL) and the cell density was adjusted to 10⁴–10⁵ CFU/mL. The compounds to be tested were dissolved in DMSO and diluted with H₂O to give 600 μM stock solution (20% DMSO). For the single data point experiment, an aliquot (10 μL) from 600 μM stock solution was transferred to a 96-well microtiter plate, and freshly prepared microbial broth (190 μL) was added to each well to give final concentrations of 30 μM in 1% DMSO. For the serial dilution experiments, the 600 μM stock solution was serially diluted with 20% DMSO to give concentrations from 600 to 0.2 μM in 20% DMSO. An aliquot (10 μL) of each dilution was transferred to a 96-well microtiter plate, and freshly prepared microbial broth (190 μL) was added to each well to give final concentrations of 30–0.01 μM in 1% DMSO. The plates were incubated at 37 °C for 24 h, and the optical density of each well was measured spectrophotometrically at 600 nm using a POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Each test compound was screened against the Gram-negative bacteria Escherichia coli ATCC11775 and Pseudomonas aeruginosa ATCC10145; the Gram-positive bacteria Staphylococcus aureus ATCC25923, B. subtilis ATCC6633, and Streptococcus pyogenes (ATCC2727); and three clinical isolates S. pyogenes (D3840), vancomycin-resistant Enterococci (VRE), and methicillin-resistant S. aureus (MRSA). Rifampicin/ampicillin and vancomycin/methicillin were used as positive controls (40 μg/mL in 10% DMSO) for susceptible and multidrug-resistant strains, respectively. The IC₅₀ value was calculated as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 (GraphPad Software Inc., La Jolla, CA). The outcomes of the relevant tests are shown in Figure S12.

Antifungal Assays

The fungus C. albicans ATCC10231 was streaked onto a Sabouraud agar plate and was incubated at 37 °C for 48 h. One colony was then transferred to fresh Sabouraud broth (15 mL) and the cell density adjusted to 10⁴–10⁵ CFU/mL. For the single data point experiments, an aliquot (10 μL) from 600 μM stock solution (20% DMSO) was transferred to a 96-well microtiter plate, and freshly prepared fungal broth (190 μL) was added to each well to give final concentrations of 30 μM in 1% DMSO. The plates were incubated at 37 °C for 24 h, and the optical density of each well was measured spectrophotometrically at 600 nm using a POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Ketoconazole was used as a positive control (30 μg/mL in 10% DMSO). Where relevant, IC₅₀ values were calculated as the concentration of the compound or antifungal drug required for 50% inhibition of the fungal cells using Prism 7.0 (GraphPad Software Inc., La Jolla, CA).

Cytotoxicity Assays

Adherent human hepatocellular (HepG2), human colorectal (SW620), and lung carcinoma (NCI-H460) cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640. All cells were cultured as adherent monolayers in flasks supplemented with 10% fetal bovine serum, l-glutamine (2 mM), penicillin (100 unit/mL), and streptomycin (100 μg/mL) in a humidified 37 °C incubator supplied with 5% CO₂. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 3,000 cells/well after which they were incubated for 18 h at 37 °C with 5% CO₂ (to allow cells to attach as adherent monolayers). Test compounds were dissolved in DMSO and diluted with phosphate-buffered saline (PBS) to give a 600 μM stock solution (20% DMSO), which was serially diluted with 20% DMSO to give concentrations from 600 to 0.2 μM in 20% DMSO. An aliquot (10 μL) of each dilution was applied to cells to give final concentrations of 30–0.01 μM in 1% DMSO. After 48 h incubation at 37 °C with 5% CO₂, an aliquot (20 μL) of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS (5 mg/mL) was added to each well (final concentration of 0.5 mg/mL) and microtiter plates were incubated for further 4 h at 37 °C with 5% CO₂. After final incubation, the medium was aspirated and precipitated formazan crystals dissolved in DMSO (100 μL/well). The absorbance of each well was measured at 600 nm with a PowerWave XS Microplate Reader from Bio-Tek Instruments Inc. (Vinooski, VT). IC₅₀ values were calculated using Prism 7.0 (GraphPad Software Inc., La Jolla, CA) as the concentration of the analyte required for 50% inhibition of cancer cell growth (compared to negative controls). The negative control was 1% aqueous DMSO, while the positive control was vinblastine (30 μM). All experiments were performed in duplicate. The outcomes of the relevant tests are shown in Figure S13.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01331.

• Plots from crystallographic studies, additional biological test results, and ¹H and ¹³C NMR spectra of compounds 9–19 and 21–35 (PDF)

• X-ray data for compound 10 (CIF)

• X-ray data for compound 13 (CIF)

• X-ray data for compound 14 (CIF)

• X-ray data for compound 16 (CIF)

• X-ray data for compound 17 (CIF)

• X-ray data for compound 22 (CIF)

• X-ray data for compound 23 (CIF)

• X-ray data for compound 29 (CIF)

• X-ray data for compound 30 (CIF)

• X-ray data for compound 32 (CIF)

• X-ray data for compound 33 (CIF)

Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.