Synthesis and evaluation of cyclopentane-based muraymycin analogs targeting MraY

Abstract

Antibiotic resistance is one of the most challenging global health issues and presents an urgent need for the development of new antibiotics. In this regard, phosphor-MurNAc-pentapeptide translocase (MraY), an essential enzyme in the early stages of peptidoglycan biosynthesis, has emerged as a promising new antibiotic target. We recently reported the crystal structures of MraY in complex with representative members of naturally occurring nucleoside antibiotics, including muraymycin D2. However, these nucleoside antibiotics are synthetically challenging targets, which limits the scope of medicinal chemistry efforts on this class of compounds. To gain access to active muraymycin analogs with reduced structural complexity and improved synthetic tractability, we prepared and evaluated cyclopentane-based muraymycin analogs for targeting MraY. For the installation of the 1,2-synamino alcohol group of analogs, the diastereoselective isocyanoacetate aldol reaction was explored. The structure–activity relationship analysis of the synthesized analogs suggested that a lipophilic side chain is essential for MraY inhibition. Importantly, the analog 20 (JH-MR-23) showed antibacterial efficacy against Staphylococcus aureus. These findings provide insights into designing new muraymycin-based MraY inhibitors with improved chemical tractability.

Graphical Abstract

1. Introduction

The global burden of multidrug-resistant infections is a major threat to global public health [1]. A decline in chemical and pharmaceutical research to develop new antibiotics has resulted in few truly new antibiotics in the pipeline. At the same time, widespread antibiotic resistance is steadily eroding the effectiveness of existing treatment options. Therefore, an urgent need exists for new antibiotics with novel modes of action.

Peptidoglycan is a cross-linked polymer of carbohydrates and amino acids that comprises the cell wall of both Gram-negative and Gram-positive bacteria [2]. It is essential for bacterial survival as it is responsible for maintaining cell shape by stabilizing the membrane against osmotic pressure. Hence, biosynthesis of peptidoglycan is a well-established target for antibiotic development. The late stages of peptidoglycan biosynthesis (i.e., cross-linking) have been extensively explored, which has resulted in the development of the penicillin and vancomycin classes of antibiotics. However, the early stages have been underexplored despite the fact that there are many natural product inhibitors targeting these stages. Therefore, these early steps offer excellent opportunities for new antibiotic development.

Among the enzymes involved in the early stages of peptidoglycan biosynthesis, phospho-MurNAc-pentapeptide translocase (MraY) is a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate-transferase (PNPT) superfamily. MraY is an integral membrane protein that catalyzes the first membrane step of bacterial cell wall biosynthesis, the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate (C₅₅-P) [3]. This is an essential membrane step of peptidoglycan biosynthesis, and as a result, the inhibition of MraY leads to cell lysis. Therefore, MraY has long been considered a promising target for the development of new antibiotics [4–6]. However, despite many years of effort, the development of antibiotics targeting MraY has been stagnant largely due to insufficient understanding of MraY structure, function, and inhibition. Recently, we reported the crystal structures of Aquifex aeolicus MraY (MraY_AA) alone and in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors [7–9]. The analysis of these crystal structures enhanced our understanding of the mechanisms of MraY catalysis and inhibition by natural products and paved the way to the development of new MraY-targeting antibiotics.

Among the naturally occurring nucleoside antibiotics, the muraymycins (Figure 1a) were isolated from a broth of a Streptomyces sp. and represent a promising class of new nucleoside antibiotics targeting MraY [10]. They have a glycyl-uridine motif connected via an aminopropyl linker to a urea peptide moiety consisting of L-leucine or L-hydroxyleucine, L-epicapreomycidine, and L-valine. The promising MraY inhibitory activity of muraymycins makes them attractive candidates for future antibacterial agent development.

Figure 1.

(a) The structure of muraymycins. (b) Conformation of muraymycin D2 (1d, green color) in the 1d-bound MraY structure (PDB: 5CKR). The hydrogen bond, water–hydrogen bond, π–π stacking interaction, and salt bridge interaction between 1d and MraY are shown in green, cyan, magenta, and orange dashed lines, respectively. Water molecules are presented as red spheres.

Since the first total synthesis of muraymycin D2 reported by Ichikawa, Matsuda, and co-workers [11], there have been a number of reports on the synthesis and biological evaluation of muraymycins and muraymycin analogs [12–27]. However, despite great efforts in the synthesis of muraymycins, they are still challenging synthetic targets. Moreover, the structure–activity relationship (SAR) of muraymycins reported to date has primarily focused on the peptide motif and the 5’-position with the amino ribose [16–18]; little is known about the role of the ribose core of muraymycins in MraY inhibition [28]. Therefore, development of muraymycin analogs with modifications on the ribose moiety would help to elucidate the role of the ribose unit of muraymycins in MraY inhibition.

Towards this goal, we embarked on the synthesis of cyclopentane core analogs of muraymycins. As the ribose of muraymycins has no specific interaction with the amino acid residues within the active site of MraY (Figure 1b) [8], we reasoned that a cyclopentane with a similar ring conformation as the tetrahydrofuran ring of the ribose would be an excellent substitute for the ribose moiety of muryamycins. More importantly, it is more amenable to modifications with various substituents than the ribose ring of muraymycins. Indeed, it has been reported that the replacement of a ribose with a cyclopentane ring improved the biological activity of the cyclopentane analog over the original compound [29]. By substituting the ribose of muraymycins with a cyclopentane ring, we anticipated to gain access to active muraymycin analogs with reduced structural complexity and/or improved synthetic tractability. Here we report the synthesis and evaluation of cyclopentane core-based muraymycin analogs.

2. Results and discussion

2.1. Chemistry

The synthesis of the key intermediate 5 started with the commercially available (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one (2) (Scheme 1). Catalytic hydrogenation of 2 followed by N-Boc protection gave the lactam 3 [30] (81% over two steps). Next, the reductive ring cleavage of 3 with NaBH₄ and boiling water catalyzed neutral N-Boc deprotection [31] gave the known amine 4 [30] in good yield (64% over two steps). To introduce a uracil group, we coupled 4 with 3-ethoxyacryloyl isocyanate following the previously reported procedure [32]. The final acidmediated cyclization of the urea intermediate completed the synthesis of the key intermediate 5.

Scheme 1.

Synthesis of cyclopentane-based MraY inhibitors 10 (JH-MR-21) and 11 (JH-MR-22).^a

^a Reagents and conditions: (a) Pd/C, H₂, MeOH, 25 °C, 5 h; (b) Boc₂O, DMAP, MeCN, 25 °C, 1 h, 81% for two steps; (c) NaBH₄, MeOH, 25 °C, 3 h, 80%; (d) H₂O, reflux, 20 h, 80%; (e) 3-ethoxyacryloyl isocyanate, 4 Å MS, DMF, −20 to 25 °C, 15 h, 49%; (f) 1 N H₂SO₄, reflux, 30 min, 68%; (g) IBX, MeCN, 80 °C, 1.5 h; (h) Ph₃P=CHCO₂t-Bu, THF, 0 °C, 12 h, 45% for two steps; (i) CbzNH₂, K₂OsO₂(OH)₄, (DHQD)2AQN, t-BuOCl, n-PrOH, 0.6 N NaOH, 5 to 25 °C, 2 h, 46% (dr >20:1); (j) 8, NIS, TESOTf, CH2Cl2, 4 Å MS, 25 °C, 30 min, 53%; (k) PPh₃, THF/toluene (1/1), H₂O, 25 °C, 12 h, 63%; (l) 80% TFA in THF/H₂O (1/1), 25 °C, 14 h; For 10: 41%; For 11: 91%.

After the preparation of 5, the installation of the 1,2-syn-amino alcohol moiety of muraymycins began with the oxidation of 5 to the corresponding aldehyde. Initial attempts for the oxidation of 5 such as Swern or PCC oxidation did not provide the desired aldehyde, which led us to use of the IBX oxidation conditions reported by Matsuda and co-workers [33]. The IBX oxidation of 5 smoothly proceeded to provide the corresponding aldehyde, which was converted to the α,β-unsaturated ester 6 by treating with (tert-butoxycarbonylmethylene)triphenylphosphorane. The Sharpless aminohydroxylation reaction [33] of 6 was carried out with (DHQD)₂AQN as a chiral ligand to afford the 1,2-syn-amino alcohol 7 (46%) as a single diastereomer. Interestingly, the aminohydroxylation reaction of 6 did not give other diastereomeric 2,3-amino alcohols as previously reported by others [34]. However, the stereoselective aminohydroxylation reaction of 6 suffered from the reproducibility and low-yield issues, which prompted us to explore other methods for the installation of the 1,2-syn-amino alcohol moiety (vide infra). Next, the glycosylation reaction of 7 with various glycosyl donors and activators was explored. After an extensive search for reaction conditions (see the Supplementary Information for details), treatment of 7 with the n-pentenyl glycoside 8 [35], TESOTf, and NIS provided the desired glycosylation product in 53% as a single diastereomer. The azide group of the glycosylation product was reduced under Staudinger’s conditions to give the amine 9 (63%). The final global deprotection under acidic conditions gave either the partially deprotected t-Bu ester 10 (JH-MR-21) or the fully deprotected carboxylic acid 11 (JH-MR-22) as the final product depending on purity of 9 and reaction time.

Since the lipophilic peptide chains of muraymycins play an important role in MraY inhibition [11, 16, 18], we embarked on the synthesis of a cyclopentane analog with a lipophilic side chain (analog 20) starting from the common intermediate 5. As mentioned above, when we prepared the cyclopentane-based analogs 10 and 11, the Sharpless aminohydroxylation gave low yield and inconsistent diastereoselectivity. To address these issues, we turned our attention to the diastereoselective isocyanoacetate aldol reaction reported by Dixon and co-workers [36] (Scheme 2). The IBX oxidation of 5 followed by the coupling of the resulting aldehyde with methyl isocyanoacetate in the presence of chiral aminophosphine ligand 12 and Ag₂O proceeded to give a 2:1 mixture of the aldol products 14a and 14b. The yield and stereoselectivity of the isocyanoacetate aldol reaction were sensitive to catalyst activation time and temperature (see the Supplementary Information for details). When we adopted the procedure reported by Shibasaki and co-workers (CuCl, PPh₃ and DIPEA) [37], the isocyanoacetate aldol reaction gave a 1:5 (14a:14b) mixture. To unambiguously established the configuration of the major aldol reaction product to be (2S,3R), we subjected 13 to acid-catalyzed hydrolysis and subsequent Cbz protection. The NMR spectral data of the Cbz-protected major aldol product 14a was identical with the major diastereomer of the Sharpless aminohydroxylation reaction (Scheme 1), confirming that the major isocyanoacetate aldol product was the desired 1,2-syn-amino alcohol (see the Supplementary Information for details).

Scheme 2.

Synthesis of a cyclopentane-based MraY inhibitor with a lipophilic side chain 20 (JH-MR-23).^a

^a Reagents and conditions: (a) IBX, MeCN, 80 °C, 1 h, 70%; (b) methyl isocyanoacetate, Ag₂O, 12, EtOAc, 4 Å MS, −78 to 0 °C, 1.5 days; (c) 4 N HCl, THF, 25 °C, 1 h; (d) CbzCl, NaHCO₃, THF/H₂O (2/1), 0 °C, 15 h, 20% for three steps; (e) 8, NIS, TESOTf, CHCl, 4 Å MS, 25 °C, 30 min, 60%; (f) Zn powder, NH₄Cl, EtOH/H₂O (3/1), 25 °C, 1.5 h; (g) Boc₂O, NaHCO₃, CH₂Cl₂, 25 °C, 2 h, 52% for two steps; (h) Pd/C, H₂, MeOH, 25 °C, 3 h; (i) 16, NaBH₃CN, HOAc, MeOH, 25 °C, 15 h, 59% for two steps; (j) Zn powder, NH₄Cl, MeOH, 25 °C, 25 h; (k) 18, EDCl, HOBt, CH₂Cl₂, 25 °C, 20 h, 12% for two steps; (l) Ba(OH)₂·8H2O, THF/H₂O (4/1), 0 to 25 °C, 20 h, 40%; (m) 80% TFA in H₂O, 25 °C, 18 h, quantitative.

Following the isocyanoacetate aldol reaction, the 1,2-syn-amino alcohol 14 was treated with the glycosyl donor 8 [35], TESOTf, and NIS to afford the glycosylation product in 60%. The azide reduction of the glycosylation product by Zn powder and NH₄Cl followed by a subsequent Boc protection of the resulting amine gave the Boc-protected amine 15 in 52% for two steps. The Cbz deprotection of 15 by Pd/C followed by reductive alkylation of the resulting amine with 16 [38] and NaBH₃CN/HOAc gave the carbamate 17 in 59% for two steps. The Troc group of 17 was removed by treatment with Zn powder in MeOH, and the resulting amine was acylated with the carboxylic acid 18 [16] in the presence of EDCI and HOBt to afford the amide 19. Treatment of 19 with Ba(OH)₂‧8H₂O provided the carboxylic acid (40%). Finally, the global deprotection by aqueous TFA successfully completed the synthesis of the cyclopentane-based analog 20 (JH-MR-23) with a lipophilic side chain.

2.2. Biological characterization

After the completion of analog synthesis, we performed the UMP-Glo^™ glycosyltransferase assay [39] to assess the effect of the cyclopentane core on MraY inhibition. The analogs (10, 11 and 20) we prepared inhibited MraY activity in a dose-dependent manner (Figure 2a–c). Among the analogs tested, the analog 20 (JH-MR-23) showed the most potent inhibitory activity (IC₅₀: 75 ± 9 μM) against MraY from Aquifex aeolicus (MraY_AA). The analogs 10 (JH-MR-21) and 11 (JH-MR-22) without a lipophilic side chain exhibited significantly lower inhibitory activity (IC₅₀: 340 ± 42 μM for 10 and 500 ± 69 μM for 11) than 20. This data indicated that a lipophilic side chain is crucial for MraY inhibition. However, none of them was as potent as muraymycin D2 [8]. We then tested the biological activity of our most potent analog 20 (JH-MR-23) against Staphylococcus aureus (strain SA113) and determined the minimal inhibitory concentration (MIC) is 54 ± 6.8 μg/mL (Figure 2d).

Figure 2.

Dose-response curves and IC₅₀ values of (a) 10 (JH-MR-21), (b) 11 (JH-MR-22), and (c) 20 (JH-MR-23) with MraY_AA solubilized in the detergent CHAPS. Each IC50 measurement was made by using the UMP-Glo^™ assay. Data are shown as the mean ± standard deviation of three technical replicates. (d) MIC of 20 (JH-MR-23) against S. aureus (strain SA113) is 54 ± 6.8 μg/mL. Representative images of the MIC determination for 20 (JH-MR-23) with growth and sterility controls (4 wells each) as indicated in row B and C (n=8, MIC value is reported as mean ± standard error).

3. Conclusion

MraY is an essential enzyme in the peptidoglycan biosynthesis and a promising target for new antibiotic development. We recently reported the crystal structures of MraY in complex with representative members of the natural nucleoside inhibitors, including muraymycin D2. To harness our recent findings and to improve the chemical tractability of muraymycin analogs, we prepared cyclopentane-based muraymycin analogs by replacing the ribose and lipophilic peptide chain groups of muraymycins with a cyclopentane ring and a modified lipophilic side chain. We also explored the diastereoselective isocyanoacetate aldol reaction for the installation of the 1,2-syn-amino alcohol group of muraymycins. We found that our cyclopentane analogs are less potent than muraymycin D2 (1d) in inhibiting MraY activity. Future structural and functional studies are necessary to elucidate the reason for the low efficacy of cyclopentane analogs in MraY function. However, among the cyclopentane analogs, analog 20 (JH-MR-23) has the most potent MraY inhibition and exhibits antibacterial activity against the Gram-positive bacteria S. aureus to levels comparable to some of the reported muramycin analogs [18]. Our SAR analysis of the analogs suggested that a lipophilic side chain is important for MraY inhibition and antibacterial efficacy. Our recent structural and functional analysis revealed that MraY contains six druggable hot spots, all of which can be exploited in a combinatorial manner to improve existing MraY inhibitors or develop new types of MraY inhibitors [6]. Because our cyclopentane-based muraymycin analogs are more amenable to modification with various substituents, our synthetic route will be a valuable foundation for the future development of new muraymycin-derived antibiotics targeting MraY.

4. Materials and methods

4.1. Synthesis of cyclopentane-based muraymycin analogs

General chemistry procedures.

All reactions were conducted in oven-dried glassware under nitrogen or argon. Unless otherwise stated all reagents were purchased from commercial suppliers and used without further purification. All solvents were American Chemical Society (ACS) grade or better and used without further purification except tetrahydrofuran (THF), which was freshly distilled from sodium/benzophenone each time before use. Analytical thin layer chromatography (TLC) was performed with glass backed silica gel (60 Å) plates with fluorescent indication (Whatman). Visualization was accomplished by UV irradiation at 254 nm and/or by staining with p-anisaldehyde solution. Flash column chromatography was performed by using silica gel (particle size 230–400 mesh, 60 Å). All ¹H spectra were recorded with a Varian 400 (400 MHz) and a Bruker 500 (500 MHz) spectrometer. All NMR δ values are given in parts per million (ppm) and are referenced to the residual solvent signals (CDCl₃: δ = 7.26 ppm, CD₃OD: δ = 3.31 ppm, (CD₃)₂SO: δ = 2.50 ppm) for ¹H NMR spectra, or the solvent signals for ¹³C spectra. Coupling constants (J) are given in Hertz (Hz) and multiplicities are indicated using the conventional abbreviation (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or overlap of non-equivalent resonances, br = broad). Electrospray ionization (ESI) mass spectrometry (MS) was recorded with an Agilent 1100 series (LC/MSD trap) spectrometer in order to obtain the molecular masses of compounds.

tert-Butyl (1S,4R)-3-oxo-2-azabicyclo[2.2.1]heptane-2-carboxylate (3). [Reduction] To a solution of the commercially available (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one (2) (10 g, 91.70 mmol) in anhydrous MeOH (180 mL) was added 10% palladium on activated carbon (3 g). After stirring at 25 °C for 5 h under H₂ atmosphere, the reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give the corresponding lactam [30] (9.17 g). The crude lactam was used in the following step without further purification: ¹H NMR (400 MHz, CDCl₃) δ 5.75 (br s, 1H), 3.89 (s, 1H), 2.74 (s, 1H), 1.94–1.91 (m, 1H), 1.88–1.80 (m, 2H), 1.65–1.57 (m, 2H), 1.42–1.36 (m, 1H); [Boc Protection] A mixture of the lactam (9.17 g, 82.51 mmol), Boc₂O (28.60 g, 131.04 mmol), and DMAP (5.04 g, 41.25 mmol) in anhydrous MeCN (300 mL) was stirred at 25 °C for 1 h. The solvents were removed in vacuo and the residue was purified by column chromatography (silica gel, hexanes/EtOAc, 5/2) to afford the known Boc-protected lactam 3 [30] (15.68 g, 81% for two steps) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 4.53 (s, 1H), 2.85 (s, 1H), 1.95–1.90 (m, 2H), 1.80–1.73 (m, 2H), 1.51 (s, 9H), 1.43–1.39 (m, 2H).

((1R,3S)-3-Aminocyclopentyl)methanol (4). A mixture of 3 (15.68 g, 74.25 mmol) and NaBH₄ (5.62 g, 14.85 mmol) in MeOH (300 mL) was stirred at 25 °C for 3 h. The solvents were removed in vacuo, and the residue was partitioned between EtOAc and H₂O. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 2/1) to afford the Boc-protected amino alcohol (12.79 g, 80%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 3.90 (br s, 1H), 3.56 (d, J = 5.0 Hz, 2H), 2.19–2.10 (m, 2H), 1.92–1.83 (m, 1H), 1.74–1.71 (m, 1H), 1.47–1.42 (m, 2H), 1.40 (s, 9H), 1.14–1.10 (m, 1H); [Boc Deprotection] A solution of the Boc-protected amino alcohol (12.79 g, 59.44 mmol) in H₂O (700 mL) was stirred at 100 °C for 20 h. The solvents were removed to give the amino alcohol 4 [30] (5.47 g, 80%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 3.58–3.46 (m, 3H), 2.75 (br s, 2H), 2.40–2.30 (m, 1H), 1.99–1.92 (m, 1H), 1.80–1.69 (m, 3H), 1.50–1.45 (m, 1H), 1.38–1.28 (m, 1H); HRMS (ESI) m/z 116.1073 [(M+H)⁺ calcd for C₆H₁₃NO 116.1069].

1-((1S,3R)-3-(Hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,3H)-dione (5). [Coupling] To a solution of 4 (4.50 g, 39.1 mmol) in anhydrous DMF (135 mL) were slowly added 4 Å molecular sieves and 3-ethoxyacryloyl isocyanate [40] (84.5 mL, 50.8 mmol) in anhydrous benzene at −20 °C. After stirring at 25 °C for 15 h, the molecular sieves were filtered off and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 20/1) to afford the urea intermediate (4.89 g, 49%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.63 (d, J = 7.3 Hz, 1H), 8.41 (s, 1H), 7.62 (d, J = 12.1 Hz, 1H), 5.24 (d, J = 12.2 Hz, 1H), 4.21–4.14 (m, 1H), 3.96 (q, J = 7.1 Hz, 2H), 3.58 (d, J = 5.8 Hz, 2H), 2.28–2.12 (m, 2H), 2.09–1.90 (m, 1H), 1.85–1.75 (m, 1H), 1.65–1.55 (m, 2H), 1.52–1.41 (m, 1H), 1.35 (t, J = 7.0 Hz, 3H); [Uracil Formation] The urea (4.50 g, 17.56 mmol) was dissolved in 1 N H₂SO₄ (150 mL) and the resulting reaction mixture was refluxed under N₂ atmosphere. After stirring for 30 min, the reaction was quenched by an addition of saturated aqueous 2 N NaOH, and the resulting mixture was diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 40/1) to afford the uracil alcohol 5 (2.50 g, 68%) as a colorless oil: ¹H NMR (400 MHz, (CD₃)₂SO) δ 11.20 (br s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 5.56 (d, J = 8.0 Hz, 1H), 4.77–4.64 (m, 1H), 4.56 (br s, 1H), 3.36 (d, J = 6.2 Hz, 2H), 2.08–1.92 (m, 2H), 1.90–1.78 (m, 1H), 1.70–1.60 (m, 2H), 1.55–1.43 (m, 1H) 1.38–1.27 (m, 1H); HRMS (ESI) m/z 211.1082 [(M+H)⁺ calcd for C₁₀H₁₄N₂O₃ 211.1077].

tert-Butyl (E)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)acrylate (6). [Oxidation] To a solution of 5 (2.50 g, 11.89 mmol) in anhydrous MeCN (400 mL) was added IBX (8.32 g, 29.72 mmol). After stirring at 80 °C for 1.5 h, the insoluble was filtered off and the filtrate was concentrated in vacuo to afford the corresponding aldehyde (2.50 g). The crude aldehyde was used in the following step without further purification: ¹H NMR (400 MHz, (CD₃)₂CO) δ 9.98 (br s, 1H), 9.71 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 5.60 (d, J = 8.0 Hz, 1H), 5.08–4.90 (m, 1H), 3.05–2.95 (m, 1H), 2.29–2.21 (m, 2H), 2.18–2.08 (m, 2H), 1.95–1.89 (m, 1H), 1.79–1.69 (m, 1H); [Wittig Reaction] To a cooled (0 °C) solution of the aldehyde (300 mg, 1.44 mmol) in anhydrous THF (15 mL) was added (tert-butoxycarbonylmethylene)triphenylphosphorane (2.10 g, 5.76 mmol). After stirring at 0 °C for 12 h, the solvents were removed in vacuo and the residue was purified by column chromatography (silica gel, hexanes/i-PrOH, 6/1) to afford the α,β-unsaturated ester 6 (198.33 mg, 45% for two steps) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 7.23 (d, J = 8.1 Hz, 1H), 6.84 (dd, J = 7.3, 1.4 Hz, 1H), 5.76 (s, 1H), 5.74 (d, J = 6.6 Hz, 1H), 5.04–4.90 (m, 1H), 2.82–2.62 (m, 1H), 2.28–2.23 (m, 1H), 2.20–2.13 (m, 1H), 2.04–1.91 (m, 1H), 1.80–1.64 (m, 2H), 1.55–1.50 (m, 1H), 1.46 (s, 9H); ¹³C NMR (125 MHz, CDCl₃) δ 165.79, 163.09, 151.00, 148.78, 140.65, 122.65, 102.89, 80.50, 56.06, 40.38, 37.74, 30.11, 28.13.

tert-Butyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)-3-hydroxypropanoate (7). tert-Butyl hypochlorite (446 μL, 3.95 mmol) was added to a solution of benzyl carbamate (92.40 mg, 3.91 mmol) in 0.6 N NaOH/n-PrOH (1/1, 15 mL) at 15 °C. After stirring at 15 °C for 15 min, the reaction mixture was warmed to 25 °C and sequentially treated with (DHQD)₂AQN (167.10 mg, 0.19 mmol) in nPrOH (2.50 mL), 6 (200 mg, 0.65 mmol) in n-PrOH (2.5 mL), and K₂OsO₂(OH)₄ (71.80 mg, 0.19 mmol) in n-PrOH (2.50 mL). After stirring at 25 °C for 2 h, the reaction was quenched by an addition of H₂O and the resulting mixture was diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 50/1) to afford the amino alcohol 7 (140 mg, 46%) as a yellow solid: ¹H NMR (400 MHz, CDCl₃) δ 8.17 (s, 1H), 7.38–7.26 (m, 6H), 5.72 (d, J = 8.4 Hz, 1H), 5.52–5.44 (m, 1H), 5.13 (s, 2H), 4.87–4.78 (m, 1H), 4.34–4.27 (m, 1H), 3.94–3.87 (m, 1H), 2.32–2.19 (m, 2H), 2.18–2.08 (m, 2H), 1.75–1.68 (m, 1H), 1.47 (s, 10H), 1.28–1.19 (m, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 170.39, 163.83, 157.16, 156.76, 151.16, 141.51, 136.26, 136.18, 102.40, 82.72, 67.11, 66.96, 60.45, 57.90, 56.83, 40.88, 34.61, 29.90, 26.95, 21.06, 14.19; HRMS (ESI) m/z 474.2236 [(M+H)⁺ calcd for C₂₄H₃₁N₃O₇ 474.2235].

tert-Butyl (2S,3R)-3-(((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-diethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)oxy)-2-(((benzyloxy)carbonyl)amino)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)propanoate (9). [Glycosylation] To a solution of 7 (90 mg, 0.19 mmol) in CH₂Cl₂ (8 mL) were added 8 [35] (78.70 mg, 0.25 mmol), 4 Å molecular sieves, and NIS (74.80 mg, 0.33 mmol). After stirring at 25 °C for 10 min, TESOTf (20 μL, 0.09 mmol) was added and the resulting mixture was stirred for 30 min. The reaction was quenched by an addition of saturated aqueous NaHCO₃, and the resulting mixture was diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 40/1) to afford the corresponding azide (60 mg, 53%) as a yellow solid: ¹H NMR (400 MHz, CDCl₃) δ 9.20 (br s, 1H), 7.41–7.26 (m, 5H), 7.22 (d, J = 7.7 Hz, 1H), 6.06 (d, J = 9.6 Hz, 1H), 5.72 (d, J = 8.2 Hz, 1H), 5.11 (s, 1H), 5.09 (d, J = 13.9 Hz, 2H), 4.91–4.77 (m, 1H), 4.58–4.52 (m, 2H), 4.30 (d, J = 9.8 Hz, 1H), 4.27–4.21 (m, 1H), 4.09 (q, J = 7.2 Hz, 2H), 4.00 (d, J = 7.2 Hz, 1H), 3.50–3.32 (m, 2H), 2.30–2.17 (m, 5H), 2.00–1.89 (m, 2H), 1.70–1.60 (m, 2H), 1.58–1.48 (m, 2H), 1.45 (s, 9H), 0.90–0.78 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 178.07, 171.12, 169.29, 156.88, 151.07, 141.02, 136.50, 117.42, 111.13, 102.54, 86.28, 84.66, 84.22, 82.54, 81.94, 66.97, 60.37, 56.52, 56.21, 53.19, 40.94, 35.55, 29.58, 29.45, 28.87, 27.94, 21.01, 14.17, 8.35, 7.35, 6.77, 6.39; HRMS (ESI) m/z 721.3161 [(M+Na)⁺ calcd for C₃₄H₄₆N₆O₁₀ 721.3168]; [Azide Reduction] To a solution of the azide (5 mg, 0.007 mmol) in H₂O (0.05 mL) and THF/toluene (1/1, 1 mL) was added PPh₃ (3.70 mg, 0.01 mmol). After stirring at 25 °C for 12 h, the solvents were removed in vacuo and the residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 10/1) to afford the amine 9 (3 mg, 63%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.24 (m, 6H), 5.71 (d, J = 8.2 Hz, 1H), 5.14–5.09 (m, 3H), 4.84–4.78 (m, 1H), 4.62–4.49 (m 3H), 4.35 (d, J = 9.4 Hz, 1H), 4.24–4.17 (m, 1H), 4.03 (d, J = 8.4 Hz, 1H), 2.90–2.84 (m, 1H), 2.72–2.68 (m, 1H), 2.58–2.50 (m, 1H), 1.69–1.62 (m, 6H), 1.57–1.51 (m, 5H), 1.45 (s, 9H), 0.90–0.79 (m, 6H); HRMS (ESI) m/z 673.3444 [(M+H)⁺ calcd for C₃₄H₄₈N₄O₁₀ 673.3443].

tert-Butyl (2S,3R)-3-(((2R,3R,4S,5R)-5-(aminomethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-2-(((benzyloxy)carbonyl)amino)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)propanoate (10). Compound 9 (3 mg, 0.004 mmol) was treated with 80% TFA in THF/H₂O (1/1, 1 mL) and stirred at 25 °C for 14 h. The reaction mixture was concentrated in vacuo and triturated from CH₂Cl₂ to afford the partially deprotected t-Bu ester 10 (JH-MR-21) and the fully deprotected carboxylic acid 11 (JH-MR-22). The global deprotection reaction 10 or 11 as the final product depending on purity of 9 and reaction time. Compounds 10 and 11 were purified by HPLC (YMC J′sphere ODS M80, 10 mm ×150 mm, 0.1% formic acid, a linear gradient from 60 to 99% of MeCN-H₂O for 30 min, flow injection: 1 mL/min) to afford 10 (1 mg, 41%) and 11 (2 mg, 91%): For 10, t_R 3.263 min; HRMS (ESI) m/z 605.2816 [(M+H)⁺ calcd for C₂₉H₄₀N₄O₁₀ 605.2711]; For 11, t_R 3.329 min; ¹H NMR (500 MHz, CD₃OD) δ 7.70 (d, J = 7.9 Hz, 1H), 7.40–7.25 (m, 5H), 5.69 (d, J = 7.9 Hz, 1H), 5.50 (s, 2H), 5.12 (d, J = 9.8 Hz, 1H), 5.03 (d, J = 9.8 Hz, 1H), 4.96 (s, 1H), 4.18 (s, 1H), 4.11–4.03 (m, 2H), 4.02–3.95 (m, 2H), 3.96–3.90 (m, 1H), 3.14–3.08 (m, 2H), 2.18–2.11 (m, 2H), 2.10–2.00 (m, 1H), 1.90–1.80 (m, 1H), 1.78–1.70 (m, 1H), 1.68–1.58 (m, 2H); ¹³C NMR (125 MHz, CD₃OD) δ 164.85, 157.75, 157.52, 151.56, 151.40, 142.87, 128.11, 127.80, 127.72, 127.68, 110.36, 101.25, 85.71, 80.86, 78.42, 75.13, 72.05, 66.61, 56.91, 56.74, 56.40, 42.71, 41.07, 34.87, 28.73, 26.22, 22.80; HRMS (ESI) m/z 549.2188 [(M+H)⁺ calcd for C₂₅H₃₂N₄O₁₀ 549.2191].

Methyl 5-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)-4,5-dihydrooxazole-4-carboxylate (13). [Oxidation] To a solution of 5 (2.50 g, 11.89 mmol) in anhydrous MeCN (400 mL) was added IBX (8.32 g, 29.72 mmol). After stirring at 80 °C for 1 h, the insoluble was filtered off and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 30/1) to afford the corresponding aldehyde (1.72 g, 70%); [Aldol Reaction] The pre-catalyst 12 [36] (28.10 mg, 0.04 mmol) was dissolved in EtOAc (0.60 mL) and Ag₂O (5.57 mg, 0.02 mmol) was added at –78 °C. The resulting mixture was stirred for approximately 1 min before sequentially treated with methyl isocyanoacetate (51.50 μL, 0.56 mmol) and powdered 4 Å molecular sieves at −78 °C. The aldehyde (200 mg, 0.96 mmol) in EtOAc (1 mL) was added to the reaction mixture. The resulting mixture was slowly warmed to 0 °C and stirred at 0 °C for 1.5 d. Ag₂O was removed by filtering through a pad of Celite with MeOH to afford the trans-oxazoline 13. The crude 13 was used in next step without further purification.

Methyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)-3-hydroxypropanoate (14a). [Hydrolysis] To a solution of 13 (200 mg, 0.65 mmol) in THF (1.40 mL) was added 4 N HCl in dioxane (0.70 mL) at 0 °C. After stirring under N₂ at 25 °C for 1 h, the reaction mixture was concentrated in vacuo to afford the crude amino alcohol, which was used in the following step without further purification; [Cbz Protection] To a solution of the crude amino alcohol (200 mg, 0.67 mmol) in THF/H₂O (2/1, 6 mL) were added NaHCO₃ (113 mg, 1.35 mmol) and CbzCl (0.14 mL, 1.01 mmol). After stirring at 0 °C for 15 h, the reaction was quenched by an addition of H₂O, and the resulting mixture was diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 30/1) to afford the syn-amino alcohol 14a (84.9 mg, 20.5% for three steps) and the diastereomeric syn-amino alcohol 14b (42.7 mg, 10.3% for three steps) each as a white solid: Data for the (2S,3R)-syn-amino alcohol: ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.28 (m, 5H), 7.23 (d, J = 7.8 Hz, 1H), 5.76 (d, J = 9.5 Hz, 1H, -NH), 5.70 (d, J = 8.0 Hz, 1H), 5.12 (s, 2H), 4.82–4.74 (m, 1H), 4.47 (d, J = 9.7 Hz, 1H), 4.01 (d, J = 8.4 Hz, 1H), 3.76 (s, 3H), 2.29–2.20 (m, 1H), 2.18–2.05 (m, 2H), 1.94–1.85 (m, 1H), 1.78–1.70 (m, 1H), 1.68–1.57 (m, 2H); HRMS (ESI) m/z 432.1771 [(M+H)⁺ calcd for C₂₁H₂₅N₃O₇ 432.1765]; Data for the diastereomeric (2R,3S)-synamino alcohol: ¹H NMR (400 MHz, CDCl₃) δ 8.80 (br s, 1H), 7.42–7.28 (m, 5H), 7.20 (d, J = 8.0 Hz, 1H), 5.74 (d, J = 7.5 Hz, 1H), 5.46 (dd, J = 11.8, 9.8 Hz, 2H), 5.14 (s, 2H), 4.89–4.86 (m, 1H), 4.66 (d, J = 9.8 Hz, 1H), 3.74 (s, 3H), 3.67–3.60 (m, 1H), 2.41–2.30 (m, 1H), 2.21–2.07 (m, 2H), 1.74–1.70 (m, 1H), 1.68–1.64 (m, 1H), 1.50–1.46 (m, 2H).

Methyl (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-(((3aR,4R,6R,6aR)-6-(((tert-butoxycarbonyl)amino)methyl)-2,2-diethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)propanoate (15). [Glycosylation] To a solution of 14 (20 mg, 0.04 mmol) in CH₂Cl₂ (1.50 mL) were added 8 [35] (20 mg, 0.06 mmol), 4 Å molecular sieves, and NIS (18 mg, 0.08 mmol). After stirring at 25 °C for 10 min, TESOTf (5 μL, 0.02 mmol) was added and the resulting solution was stirred for 30 min. The reaction was quenched by an addition of saturated aqueous NaHCO₃, and the resulting mixture was diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 30/1) to afford the corresponding azide (15.70 mg, 60%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.80 (br s, 1H), 7.42–7.29 (m, 5H), 7.22 (d, J = 8.1 Hz, 1H), 6.05 (d, J = 9.9 Hz, 1H), 5.74 (dd, J = 8.0, 2.4 Hz, 1H), 5.14 (d, J = 3.4 Hz, 2H), 5.08 (s, 1H), 4.88–4.85 (m, 1H), 4.60 (dd, J = 5.9, 1.6 Hz, 1H), 4.54 (d, J = 6.1 Hz, 1H), 4.47 (d, J = 9.7 Hz, 1H), 4.28–4.23 (m, 1H), 4.07 (d, J = 8.3 Hz, 1H), 3.75 (s, 3H), 3.48–3.40 (m, 2H), 2.33–2.25 (m, 2H), 2.20–2.14 (m, 1H), 2.01–1.91 (m, 2H), 1.80–1.70 (m, 2H), 1.69–1.63 (m, 2H), 1.60–1.50 (m, 2H), 0.98–0.78 (m, 6H); HRMS (ESI) m/z 657.2879 [(M+H)⁺ calcd for C₃₁H₄₀N₆O₁₀ 657.2883]; [Azide Reduction] To a solution of the azide (19 mg, 0.02 mmol) in EtOH/H₂O (3/1, 0.80 mL) were added activated Zn powder (2.60 mg, 0.03 mmol) and NH₄Cl (3.70 mg, 0.07 mmol). After stirring at 25 °C for 1.5 h, the reaction was quenched by an addition of saturated aqueous NaHCO₃, and the resulting mixture was diluted with CH₂Cl₂. The layers were separated, and the aqueous layer was extracted with CH₂Cl₂. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo to afford the corresponding amine. The crude amine was used in the following step without further purification: ¹H NMR (400 MHz, CDCl₃) δ 7.51 (br s, 1H), 7.40–7.29 (m, 5H), 7.25–7.21 (d, J = 8.2 Hz, 1H), 5.75 (d, J = 8.0 Hz, 1H), 5.12 (s, 2H), 5.09 (s, 1H), 4.91–4.82 (m, 1H), 4.60–4.50 (m, 3H), 4.25 (br s, 1H), 4.08 (d, J = 7.6 Hz, 1H), 3.77 (s, 3H), 2.97–2.92 (m, 1H), 2.87–2.82 (m, 1H), 2.28–2.21 (m, 3H), 2.15–2.10 (m, 1H), 2.05–1.90 (m, 1H), 1.75–1.69 (m, 2H), 1.68–1.64 (m, 2H), 1.60–1.50 (m, 2H), 0.92–0.82 (m, 6H); [Boc Protection] To a solution of the crude amine (8 mg, 0.01 mmol) in anhydrous CH₂Cl₂ (1 mL) were added NaHCO₃ (2 mg, 0.02 mmol) and Boc₂O (11 mg, 0.04 mmol). After stirring at 25 °C for 2 h, the reaction was quenched by an addition of H₂O, and the resulting mixture was diluted with CH₂Cl₂. The layers were separated, and the aqueous layer was extracted with CH₂Cl₂. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 20/1) to afford the Boc-protected glycosylation product 15 (9.20 mg, 52% for two steps) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 7.40–7.29 (m, 5H), 7.23 (d, J = 8.9 Hz, 1H), 5.75 (dd, J = 5.8, 2.2 Hz, 1H), 5.58–5.45 (m, 1H), 5.40 (br s, 1H), 5.12 (d, J = 7.5 Hz, 2H), 5.07 (s, 1H), 4.91–4.82 (m, 1H), 4.60 (d, J = 6.2 Hz, 1H), 4.54 (d, J = 9.8 Hz, 1H), 4.49 (d, J = 6.1 Hz, 1H), 4.25 (t, J = 5.1 Hz, 1H), 4.04 (d, J = 8.0 Hz, 1H), 3.79 (s, 3H), 3.25–3.18 (m, 2H), 2.28–2.21 (m, 2H), 2.00–1.88 (m, 1H), 1.75–1.62 (m, 6H), 1.60–1.50 (m, 11H), 0.92–0.82 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 171.43, 163.96, 156.52, 156.01, 150.82, 141.03, 136.00, 128.31, 128.25, 117.17, 112.51, 106.68, 87.00, 86.31, 85.24, 82.21, 79.52, 67.41, 56.69, 56.39, 52.94, 43.06, 41.00, 35.28, 29.64, 29.29, 29.00, 26.87, 8.40, 7.55; HRMS (ESI) m/z 753.3324 [(M+Na)⁺ calcd for C₃₆H₅₀N₄O₁₂ 753.3317].

Methyl (2S,3R)-3-(((3aR,4R,6R,6aR)-6-(((tert-butoxycarbonyl)amino)methyl)-2,2-diethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)cyclopentyl)-2-((3-(((2,2,2-trichloroethoxy)carbonyl)amino)propyl)amino)propanoate (17). [Cbz Deprotection] To a solution of 15 (45 mg, 0.06 mmol) in anhydrous MeOH (2.25 mL) was added 10% palladium on activated carbon (27 mg). After stirring under H₂ atmosphere at 25 °C for 3 h, the reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give the corresponding amine. The crude amine was used in the following step without further purification; HRMS (ESI) m/z 597.3138 [(M+H)⁺ calcd for C₂₈H₄₄N₄O₁₀ 597.3130]; [Reductive amination] To a solution of the crude amine (45 mg, 0.05 mmol) in anhydrous MeOH (3 mL) were treated with 16 [38] (22.60 mg, 0.09 mmol) in HOAc (30 μL, 0.54 mmol) and NaBH₃CN (15 mg, 0.24 mmol). After stirring at 25 °C for 15 h, the reaction was quenched by an addition of saturated aqueous NaHCO₃, and the resulting mixture was diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 30/1) to afford the carbamate 17 (27 mg, 59% for two steps) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.89 (br s, 1H), 7.25–7.22 (m, 1H), 5.87–5.76 (m, 1H), 5.73 (d, J = 8.1 Hz, 1H), 5.06 (s, 1H), 4.88–4.79 (m, 1H), 4.72–4.70 (m, 2H), 4.60 (d, J = 6.0 Hz, 1H), 4.50–4.46 (m, 1H), 4.29–4.24 (m, 1H), 3.75 (s, 3H), 3.36–3.29 (m, 3H), 3.26–3.16 (m, 4H), 2.93–2.82 (m, 1H), 2.49–2.32 (m, 2H), 2.31–2.23 (m, 1H), 2.21–2.12 (m, 1H), 1.84–1.80 (m, 2H), 1.66–1.49 (m, 4H), 1.48–1.43 (m, 3H), 1.42 (s, 12H), 0.86–0.84 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 178.02, 173.79, 163.00, 154.58, 150.81, 141.21, 116.90, 112.71, 103.69, 102.56, 86.96, 85.51, 82.32, 79.22, 74.46, 74.39, 64.08, 56.73, 53.43, 52.36, 46.54, 43.17, 41.04, 39.80, 37.22, 35.01, 32.21, 29.90, 29.82, 29.42, 29.04, 28.99, 27.08, 8.41, 7.52; HRMS (ESI) m/z 828.2740 [(M+H)⁺ calcd for C₃₄H₅₂Cl₃N₅O₁₂ 828.2751].

Methyl (2S,3R)-3-(((3aR,4R,6R,6aR)-6-(((tert-butoxycarbonyl)amino)methyl)-2,2-diethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)-2-((3-((S)-2-heptadecanamido-5-(3-((2,2,5,6,8-pentamethylchroman-7-yl)sulfonyl)guanidino)pentanamido)propyl)amino)propanoate (19). [Troc Deprotection] A solution of 17 (27 mg, 0.03 mmol) in anhydrous MeOH (1.50 mL) were treated with NH₄Cl (49 mg, 0.92 mmol) and Zn powder (97% purity, 31.80 mg, 0.48 mmol). After stirring at 25 °C for 25 h, the insoluble was filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give the corresponding amine. The crude amine was used in the following step without further purification; HRMS (ESI) m/z 654.3713 [(M+H)⁺ calcd for C₃₁H₅₁N₅O₁₀ 654.3709]; [Coupling] To a solution of the crude amine (27 mg, 0.04) in CH₂Cl₂ (1 mL) were added EDCI (3.20 mg, 0.01 mmol), HOBt (1.9 mg, 0.01 mmol) and 18 [16] (8.40 mg, 0.01 mmol). After stirring at 25 °C for 20 h, the reaction was quenched by an addition of 1 N HCl solution, and the reaction mixture was diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The combined organic layers were purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 30/1) to afford the amide 19 (5 mg, 12% for two steps) as a white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.67 (d, J = 8.0 Hz, 1H), 5.67 (d, J = 7.9 Hz, 1H), 5.08 (s, 1H), 4.64–4.54 (m, 2H), 4.33–4.22 (m, 1H), 4.18–4.10 (m, 1H), 3.92–3.87 (m, 1H), 3.73 (s, 3H), 3.40–3.20 (m, 3H), 3.18–3.09 (m, 2H), 2.75–2.72 (m, 1H), 2.68–2.62 (m, 2H), 2.55–2.54 (m, 6H), 2.49–2.39 (m, 2H), 2.23–2.18 (m, 3H), 2.11–2.06 (m, 4H), 1.99 (s, 1H), 1.90–1.47 (m, 25H), 1.42 (s, 3H), 1.29 (s, 3H), 1.27 (s, 9H), 1.24–1.12 (m, 21H), 0.88–0.86 (m, 9H); HRMS (ESI) m/z 1328.8127 [(M+H)⁺ calcd for C₆₈H₁₁₃N₉O₁₅S 1328.8150].

Methyl (2S,3R)-3-(((2R,3R,4S,5R)-5-(aminomethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-((1R,3S)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)-2-((3-((S)-5-guanidino-2-heptadecanamidopentanamido)propyl)amino)propanoate formate (20). [Hydrolysis] Ba(OH)₂‧8H₂O (3.20 mg, 0.01 mmol) was added to a solution of 19 (5 mg, 0.003 mmol) in THF/H₂O (4/1, 0.10 mL) at 0 °C and the resulting mixture was stirred at 0 °C for 10 min. The mixture was warmed to 25 °C and stirred for 20 h. The reaction was quenched by an addition of 1 N HCl and diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The organic layers were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH, 5/1) to afford the corresponding carboxylic acid (2 mg, 40%) as a white solid: ¹H NMR (500 MHz, CD₃OD) δ 7.70 (d, J = 8.0 Hz, 1H), 5.69 (d, J = 7.9 Hz, 1H), 5.18 (s, 1H), 4.70–4.58 (m, 2H), 4.30–4.25 (m, 1H), 4.22–4.18 (m, 1H), 3.82 (br s, 1H), 3.22–3.18 (m, 3H), 3.13–3.08 (m, 2H), 2.71–2.68 (m, 3H), 2.60–2.57 (m, 6H), 2.42–2.37 (m, 2H), 2.23–2.18 (m, 3H), 2.10 (s, 2H), 2.01 (s, 1H), 1.88–1.53 (m, 25H), 1.42 (s, 3H), 1.31 (s, 3H), 1.28 (s, 9H), 1.26–1.20 (m, 21H), 0.88–0.86 (m, 9H); HRMS (ESI) m/z 1314.7990 [(M+H)⁺ calcd for C₆₇H₁₁₁N₉O₁₅S 1314.7993]; [Global Deprotection] After the carboxylic acid (2 mg, 0.001 mmol) was treated with 80% TFA in H₂O (0.30 mL), the resulting mixture was stirred at 25 °C for 18 h. The reaction mixture was concentrated in vacuo and triturated from CH₂Cl₂ to afford 20 (2 mg, quantitative) as a white solid: HRMS (ESI) m/z 880.5862 [(M+H)⁺ calcd for C₄₃H₇₇N₉O₁₀ 880.5866].

4.2. UMP-Glo assay

The UMP-Glo^™ glycosyltransferase assay [39] was performed according to the manufacturer’s specifications (Promega Corporation). Reaction mixtures containing 250 μM undecaprenyl phosphate (C₅₅-P) and 150 μM UDP-MurNAc-pentapeptide (UM5A) were initiated with the addition of 50 nM MraY_AA. The reaction buffer consisted of 100 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl₂, and 20 mM (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) (CHAPS, Anatrace). To determine the IC₅₀ for 10 (JH-MR-21), the following concentrations were used: 0, 0.5, 5, 25, 100, 250, 750, 1000 μM. Reactions conducted in the presence of 10 had a final concentration of 2% DMSO. To determine the IC₅₀ for 11 (JH-MR-22), the following concentrations were used: 0, 10, 100, 750, 1000, 1500, 2000, and 2500 μM. Reactions conducted in the presence of 11 had a final concentration of 5% DMSO. To determine the IC₅₀ for 20 (JH-MR-23), the following concentrations were used: 0, 0.1, 5, 50, 200, 500, 1500, 2000 μM. Reactions conducted in the presence of 20 had a final concentration of 4% DMSO. All reactions were carried out for 5 min at 45 °C. A SpectraMax M3 multi-mode microplate reader in luminescence mode was used to make measurements, which were normalized to a negative control reaction without enzyme. Data were fit by using GraphPad Prism 7 software and IC₅₀ values were calculated using the log(inhibitor) versus response–variable slope model. Hill slope used for 10 (JH-MR-21), 11 (JH-MR-22), and 20 (JH-MR-23) is −1.2, −1, −0.9 respectively.

4.3. MIC

MIC was determined using the established protocol [41]. Briefly, S. aureus SA113 (ATCC) were grown overnight on TB agar plate at 37 °C. Direct colony resuspension method was used to resuspend S. aureus colonies in 2× YT media to match with 0.5 MacFarland standard and then diluted 100-fold. 50 μL of the inoculum was mixed with 50 μL of 2× YT media with different concentrations of 20 (JH-MR-23) diluted in. The MIC was performed in 96-well plate (Corning 3596); the plate was incubated overnight at 37 °C. Eight biological replicates (n=8) were performed, and the average was reported as the MIC value.

Highlights.

• Synthesis and biological evaluation of cyclopentane-based MraY inhibitors

• Exploration of the diastereoselective isocyanoacetate aldol reaction

• Analysis of the SAR of cyclopentane-based MraY inhibitors