A Convenient Protecting Group for Uridine Ureido Nitrogen: (4,4'-Bisfluorophenyl)methoxymethyl group

Abstract

(4,4'-Bisfluorophenyl)methoxymethyl (BFPM) group of uridine ureido nitrogen shows good relative stability in a variety of chemical transformation reactions for uridine. The BFPM group can be cleaved via 2% of TFA in CH₂Cl₂ without affecting the Boc group.

Graphical Abstract

Uridine is a building block of a large number of biologically important natural products.¹ Uridine-containing antibiotics, muraymycins and capuramycin, have been of our interest in developing novel antibacterial and anticancer drug leads.^2a-k Muraymycin analogues, APPB and APPU, display unique antiproliferative activity with strong anti-metastatic activity against certain solid cancers (e.g., pancreatic and cervical cancers) by targeting N-acetylglucosaminephosphotransferase1 (DPAGT1).^2a,b Protection of the uridine ureido nitrogen is essential to achieve the synthesis of uridine-containing complex natural products. Benzyloxymethyl (BOM) group has widely been utilized as a convenient protecting group for the uridine ureido nitrogen.³ However, BOM deprotection of uridine derivatives under hydrogenation conditions often resulted in unpredictable results with over-reduction followed by cleavage of the N-glycoside bond.⁴ Alternatively, strong Lewis acids are applied, however, acid-sensitive glyosyl bonds are often cleaved during the BOM-deproection steps.^4a Thus, we determined that the BOM protecting group is not suitable for systematic synthesis of DPAGT1 inhibitors.^2b To this end, we have introduced (2,6-dichloro-4-methoxyphenyl)(2,4,6-trichlorophenyl)methoxymethyl chloride [1, monomethoxydiphenylmethoxymethyl chlroide (MPPM-Cl) and (2,6-dichloro-4-methoxyphenyl) (2,4-dichlorophenyl) methoxymethyl chloride [2, monomethoxytetrachlorodiphenylmethoxymethyl (MTPM-Cl) for the protection of the uridine ureido nitrogen for synthesis of complex uridine-containing natural products (A in Figure 1).⁵ MPPM and MTPM groups exhibited excellent stability against a variety of Brønsted and Lewis acids such as 20% TFA, 10% TMSOTf, 10% HCl, 30% HF, 90% AcOH, 30% TsOH, La(OTf)₃, BF₃•OEt₂, and TiCl₄ at room temperature, however, these groups could be deprotected with 30% TFA in CH₂Cl₂.^5a The precursors of MPPM and MTPM groups require synthesizing in two chemical steps including Friedel–Crafts and carbonyl reduction reactions. Thus, we have sought a convenient diphenyl methoxymethyl group that can 1) readily be synthesized with commercially available building blocks, and 2) tolerate to a wide range of chemical transformations applied for modifications of uridine under neutral and basic conditions. The other consideration is that deprotections of MPPM and MTPM groups require 30% or higher concentrations of TFA in CH₂Cl₂. Under these conditions, Boc, silyl, and ketal groups are removed. To synthesize the versatile intermediate E (Figure 1b) for systematic generation of DPAGT1 inhibitors, it is required that the uridine ureido nitrogen protecting group is stable against a series of Lewis and Brønsted acids (e.g., BF₃•OEt₂, CF₃CO₂H/H₂O, AcOH, Ti(OⁱPr)₄), and can be deprotected without the cleavage of the Boc group. To fulfil these criteria, we rescreened 4,4’-disubstituted diphenylmethoxymethyl groups. Relative stability of 3-((bisphenyl)methoxy)methyl)-1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione derivatives (1-4) against BF₃•OEt₂ in CH₂Cl₂, TFA in CH₂Cl₂, 4N HCl in dioxane, 80% AcOH at 50 °C, and a hydrogenation condition (H₂, 10% Pd-C in ⁱPrOH-H₂O) is summarized in Table 1.

Figure 1. Acid-cleavable diphenylmethoxylmethyl groups for uridine ureido nitrogen.

a: MPPM and MTPM groups introduced for the syntheses of muraymycin D1 and its analogues (previous works)

b: Reinvestigation of a convenient diphenylmethoxymethyl group to synthesize the intermediate E (this work)

Table 1.

Relative reactivity of differently substituted diphenylmethymethyl protecting groups.

reactivityb╲compound	1: X, Y = H	2: X = OMe, Y = F	3: X = F, Y = F	4: X = Cl, Y = Cl
BF3•OEt2 (10 equiv.) / CH2Cl2, RT	H	H	L	L
1% TFA / CH2Cl2	H	H	M	L
2% TFA / CH2Cl2	H	H	H	L
5% TFA / CH2Cl2	H	H	H	H
10% TFA / CH2Cl2	H	H	H	H
4N HCl / dioxane	Lc	Hc	Lc	Lc
80% AcOH / H2O 50 °C	M c	M c	Lc	Lc
H2, Pd-C / iPrOH-H2O	L	L	L	L

^aH (High) indicates the protecting group is readily cleaved.; M (Marginal) indicates that the protecting group may be stable or may be cleaved slowly.; L (Low) indicates that the protecting group is stable.

^bRecation time: 3 h.

^cThe acetonide group was cleaved.

The (bisphenyl)methoxymethyl and (fluorophenyl)(methoxyphenyl)methoxymethyl groups of uridine acetonide (1 and 2) were readily cleaved under BF₃•OEt₂ (10 equiv.) in CH₂Cl₂ and showed acid instability against 1-10%

TFA in CH₂Cl₂. On the other hand, the (4,4'-bisfluorophenyl)methoxymethyl (BFPM) and (4,4'-bischlorophenyl)methoxymethyl (BCPM) groups were intact under BF₃•OEt₂ (10 equiv.) in CH₂Cl₂. These preliminary studies may indicate that BFPM is stable under the chemical transformations (steps 1~7 and 9) and can be deprotected selectively without affecting the Boc group (step 8) in Figure 1b. In contrast, the BCPM group is difficult to remove selectively in the presence of the Boc group(s).

In order to demonstrate utility of the BFPM group as a protecting group for the uridine ureido nitrogen, the BFPM-protected uridine 5 was transformed to a wide range of advanced building blocks that are utilized for the syntheses of bioactive uridine derivatives. Selected examples are summarized in Table 2. Ketal formations of 5 under TsOH•H₂O in acetone provided the corresponding 2’,3’-protected derivative 3 in 89% yield (entry 1). The primary TBS group of 6 could be deprotected selectively with 10% HF in CH₃CN to furnish 7 without deprotection of the BFPM group (entry 2). The trityl group of 8 was selectively removed by using BF₃•OEt₂ in the presence of TolSH without deprotection of the BFPM group (entry 3). The secondary alcohol 6 could be oxidized under CrO₃•Pyridine•Ac₂O in CH₂Cl₂ to afford 9 in >81% yield. Reduction of the C3’-ketone of 9 with LiBH₄ in THF furnished a 1 : 1 mixture of 10α and 10β (6) in 90% yield; in this reduction condition, no carbonyl reductions of the uridine base and cleavage of the BFPM group were observed (entry 5). Selective hydrogenolytic deprotection of the benzyl group of 11 was carried out via 10% Pd-C in ⁱPrOH-water to furnish 3 without over-reduction of the uracil double bond (entry 6). Olefination of the carbonodithioate 12 was achieved by using ⁿBu₃SnH and AIBN in toluene at 100 °C to provide 2',3'-didehydro-2',3'-dideoxy derivative 13 in 85% yield, during which cleavage of the BFPM group was not observed (entry 7). Hydrogenation of the 2’,3’-double bond of 13 was also achieved under a kinetically-controlled hydrogenation condition with 10% Pd-C in MeOH to provide the BFPM-protected uridine-2',3'-dideoxy derivative 14 in 93% yield (entry 8). Thus, it was experimentally proved that the BFPM group is a durable protecting group for the uridine ureido nitrogen to synthesize a wide variety of uridine derivatives.

Table 2.

Functionalizations of BFPM-protected uridines.

Entry	Starting material	Conditions	Product	Yield (%)a
1		Acetone/TsOH•H2O, rt, 3h		89
2		10% HF / CH3CN, rt, 10 min.		83
3		BF3•OEt2 (0.5 equiv), TolSH / CH2Cl2, rt, 1h		84
4		CrO3•Pyridine•Ac2O / CH2Cl2, rt, 0.5h		>81
5		LiBH4 / THF, 0 °c, 1h		90
6		H2 (1 atm), 10% Pd-C / iPrOH-watert, 2h		88
7		nBuSnH, AIBN / toluene, 100 °C, 3h		85
8		H2 (1 atm), 10% Pd-C MeOH, 2h		93b

^aProduct was isolated by SiO₂ chromatography.

^bno over-reduction was observed.

We applied the BFPM group for the synthesis of the muraymycin analogue intermediate 16 (E in Figure 1b). The advanced intermediate 15 was synthesized according to the synthetic scheme established previously (Figure 1b); the synthetic procedures and physical data for the new molecules are summarized in Supporting Information.^2c The acetals of 15 were deported with 50% TFA in H₂O, under which the Boc and BFPM groups were intact. The BFPM group could be deprotected with 2% TFA in CH₂Cl₂ within 2h at room temperature without deprotection of the Boc group. Finally, hydrogenation with 10% Pd-C in ⁱPrOH-H₂O furnished 16 with 76% overall yield for 3 chemical steps (Scheme 1).

Scheme 1.

Deprotections of the protecting groups of 15, leading to the advanced intermediate 16.

In conclusion, we have reinvestigated diphenyl methoxymethyl protecting groups for the uridine ureido nitrogen. The (4,4'-bisfluorophenyl)methoxymethyl (BFPM) group displays excellent relative stability in general chemical transformations for advancing uridine. Uniquely, the BFPM group is tolerated in aqueous acidic and hydrogenation conditions, but can be cleaved with 2% TFA in CH₂Cl₂. Thus, the BFPM group can be distinguished from the Boc group. BFPM-Cl can readily be synthesized from commercially available bis(4-fluorophenyl)methanol in two chemical steps with high yield using inexpensive chemicals. Application of the BFPM group is not be limited to uridine; all reported synthetic intermediates possessing the benzyloxymethyl (BOM) or its related groups can be replaced with BFPM group.⁶ In this regard, the BFPM group has been applied to primary and secondary alcohol protections in our laboratory.

General experimental details

All chemicals were purchased from commercial sources and used without further purification unless otherwise noted. THF, CH₂Cl₂, and DMF were purified via Innovative Technology's Pure-Solve System. All reactions were performed under an Argon atmosphere. All stirring was performed with an internal magnetic stirrer. Reactions were monitored by TLC using 0.25 mm coated commercial silica gel plates (EMD, Silica Gel 60F₂₅₄). TLC spots were visualized by UV light at 254 nm, or developed with ceric ammonium molybdate or anisaldehyde or copper sulfate or ninhydrin solutions by heating on a hot plate. Reactions were also monitored by using SHIMADZU LCMS-2020 with solvents: A: 0.1% formic acid in water, B: acetonitrile. Flash chromatography was performed with SiliCycle silica gel (Purasil 60 Å, 230-400 Mesh). Proton magnetic resonance (¹H-NMR) spectral data were recorded on 400, and 500 MHz instruments. Carbon magnetic resonance (¹³C-NMR) spectral data were recorded on 100 and 125 MHz instruments. For all NMR spectra, chemical shifts (δH, δC) were quoted in parts per million (ppm), and J values were quoted in Hz. ¹H and ¹³C NMR spectra were calibrated with residual undeuterated solvent (CDCl₃: δH = 7.26 ppm, δC = 77.16 ppm; CD₃CN: δH = 1.94 ppm, δC = 1.32ppm; CD₃OD: δH =3.31 ppm, δC =49.00 ppm; DMSO-d₆: δH = 2.50 ppm, δC = 39.52 ppm; D₂O: δH = 4.79 ppm) as an internal reference. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, dd = double doublets, t = triplet, q = quartet, quin = quintet, hept = heptet, m = multiplet, br = broad. Infrared (IR) spectra were recorded on a Perkin-Elmer FT1600 spectrometer. HPLC analyses were performed with a Shimadzu LC-20AD HPLC system. HR-MS data were obtained from a Waters Synapt G2-Si (ion mobility mass spectrometer with nanoelectrospray ionization).

Procedure for synthesis of 4,4'-((chloromethoxy)methylene)bis(fluorobenzene) (BFPM-Cl)

To a stirred suspension of NaH (1.4 g, 60% in oil, 35 mmol) in THF (17 mL), bis(4-fluorophenyl)methanol (3.8 g, 17 mmol) was added at 0 °C. After being stirred for 0.5 h, chloromethyl methyl sulfide (2.9 mL, 39 mmol) was added. After 3 h at 0 °C, the reaction mixture was quenched with sat. aq. NH₄Cl, and extracted with EtOAc. The combined extract was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 99/1) to afford (bis(4-fluorophenyl))methoxymethyl methyl sulfide as pale yellow oil (4.0 g, 82%). IR (thin film) ν_max = 2922, 1603, 1507, 1433, 1297, 1223, 1181, 1155, 1098, 1048, 1014, 964, 877, 860, 832, 793, 781, 731, 684 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.29 (dd, J = 8.5, 5.5 Hz, 4H), 7.02 (t, J = 8.6 Hz, 4H), 5.85 (s, 1H), 4.61 (s, 2H), 2.19 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.44, 160.99, 136.86, 136.83, 129.06 (2C), 128.98 (2C), 115.49 (2C), 115.28 (2C), 72.53, 13.95; HRMS (ESI+) m/z calcd for C₁₅H₁₄F₂NaOS [M + Na] 303.0631, found: 303.0637.

To a stirred solution of (bis(4-fluorophenyl))methoxymethyl methyl sulfide (4.0 g, 14 mmol) in CH₂Cl₂ (28 mL) was added sulfuryl chloride (3.5 mL, 43 mmol) at 0 °C. After being stirred for 3 h at 0 °C, the reaction mixture was concentrated in vacuo. The crude mixture (BFPM-Cl) was used for next reaction without purification. IR (thin film) ν_max = 2930, 1602, 1507, 1409, 1316, 1225, 1157, 1139, 1101, 1057, 1015, 966, 928, 859, 836, 767 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.29 (dd, J = 8.5, 5.4 Hz, 4H), 7.05 (t, J = 8.6 Hz, 4H), 5.88 (s, 1H), 5.44 (s, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 133.66, 133.63, 129.51, 129.42, 129.27, 129.19, 115.68, 115.63, 115.46, 115.42, 79.56, 62.71; HRMS (ESI+) m/z calcd for C₁₄H₁₁ClF₂NaO [M + Na] 291.0364, found: 291.0370.

3-((Bis(4-fluorophenyl)methoxy)methyl)-1-((3R,4S)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (5)

To a stirred solution of BFPM-Cl (14 mmol) in DMF (14 mL), uridine (3.5 g, 14 mmol) and DBU (4.3 mL, 29 mmol) were added. After being stirred for 8 h at r.t., the reaction mixture was quenched with 1.0 M HCl, extracted with EtOAc. The combined extract was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 2/1 - CHCl₃/MeOH = 88/12) to afford 5 as yellow oil (5.2 g, 77% for 2 steps). [a]$[\mathrm{a}{]}_{\mathrm{D}}^{22}$ + 0.027 (c = 0.31, CHCl₃); IR (thin film) ν_max = 3453 (br), 2959, 2929, 2874, 2861, 1723, 1664, 1603, 1508, 1460, 1379, 1274, 1224, 1155, 1122, 1074, 1015, 834, 744 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.55 (d, J = 8.2 Hz, 1H), 7.29 (dd, J = 8.6, 5.5 Hz, 4H), 6.98 (t, J = 8.7 Hz, 4H), 5.70 (s, 1H), 5.68 (d, J = 8.2 Hz, 1H), 5.60 (d, J = 4.2 Hz, 1H), 5.51 (s, 2H), 4.34 – 4.28 (m, 2H), 4.23 (q, J = 2.8 Hz, 1H), 3.96 (dd, J = 11.9, 2.6 Hz, 1H), 3.82 (dd, J = 11.8, 2.7 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 163.43, 162.34, 160.98, 151.83, 139.23, 137.41, 137.38, 128.64, 128.57, 128.56, 128.49, 115.38 (2C), 115.17 (2C), 101.72, 93.52, 86.00, 82.05, 75.40, 71.14, 69.80, 62.07; HRMS (ESI+) m/z calcd for C₂₃H₂₂F₂N₂NaO₇ [M + Na] 499.1293, found: 499.1302.

3-((Bis(4-fluorophenyl)methoxy)methyl)-1-((3aR,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (3)

To a stirred solution of 5 (5.2 g, 11 mmol) and 2,2-dimethoxypropane (6.8 mL, 55 mmol) in acetone (11 mL), TsOH^•H₂O (0.21 g, 1.1 mmol) was added. After being stirred for 3 h at r.t., the reaction mixture was quenched with sat. aq. NaHCO₃, extracted with EtOAc. The combined extract was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 1/1 to 1/2) to afford 3 as yellow foam (5.1 g, 89%). [α]$[\alpha {]}_{\mathrm{D}}^{21}$ - 0.129 (c = 0.61, CHCl₃); IR (thin film) ν_max = 3474 (br), 2935, 1716, 1667, 1604, 1508, 1457, 1275, 1221, 1156, 1078, 835 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.29 (ddd, J = 8.4, 5.4, 2.3 Hz, 4H), 7.24 (d, J = 8.1 Hz, 1H), 6.98 (td, J = 8.7, 2.0 Hz, 4H), 5.70 (s, 1H), 5.66 (d, J = 8.1 Hz, 1H), 5.51 (s, 3H), 4.96 (dd, J = 6.5, 3.3 Hz, 1H), 4.91 (dd, J = 6.5, 2.9 Hz, 1H), 4.29 (q, J = 3.0 Hz, 1H), 3.93 (dd, J = 12.0, 2.5 Hz, 1H), 3.81 (dd, J = 12.0, 3.3 Hz, 1H), 1.58 (s, 3H), 1.37 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.38, 162.26, 160.93, 150.88, 141.15, 137.48, 128.55, 128.54, 128.47, 128.46, 115.35, 115.33, 115.14, 115.12, 114.39, 102.09, 96.51, 86.72, 83.59, 82.06, 80.17, 69.87, 62.70, 27.24, 25.23; HRMS (ESI+) m/z calcd for C₂₆H₂₆F₂N₂NaO₇ [M + Na] 539.1606, found: 539.1620.

Selective desilylation of 3-((Bis(4-fluorophenyl)methoxy)methyl)-1-((2R,3R,4R)-3-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (6)

To a stirred solution of 6 (13 mg, 0.018 mmol) in CH₃CN (0.16 mL), 50% HF in H₂O (0.04 mL) was added. After being stirred for 10 min at r.t., the reaction mixture was diluted with H₂O, extracted with EtOAc. The combined extract was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 1/1 - 1/2) to afford 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R,3R,4R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione as colorless oil (7, 9.0 mg, 83%). $[\alpha {]}_{\mathrm{D}}^{21}$ - 0.058 (c = 0.29, CHCl₃); IR (thin film) ν_max = 3480 (br), 2954, 2930, 2858, 1714, 1665, 1604, 1508, 1459, 1273, 1259, 1224, 1155, 1114, 1076, 1014, 862, 836, 812, 782 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.49 (d, J = 8.1 Hz, 1H), 7.29 (ddd, J = 8.5, 5.4, 2.6 Hz, 4H), 6.99 (t, J = 8.6 Hz, 4H), 5.73 (d, J = 8.1 Hz, 1H), 5.69 (s, 1H), 5.55 (d, J = 5.0 Hz, 1H), 5.46 (s, 2H), 4.55 (t, J = 5.0 Hz, 1H), 4.20 – 4.16 (m, 1H), 4.15 – 4.12 (m, 1H), 3.98 (dd, J = 12.2, 2.1 Hz, 1H), 3.82 (dd, J = 12.1, 2.1 Hz, 1H), 0.87 (s, 9H), 0.05 (s, 3H), 0.02 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.40, 162.32, 160.96, 150.98, 140.76, 137.42, 137.38, 128.71, 128.64, 128.63, 128.56, 115.34, 115.32, 115.13, 115.10, 102.22, 93.42, 85.22, 81.50, 74.05, 70.70, 69.27, 62.18, 25.59, 17.91, −4.86, −5.21; HRMS (ESI+) m/z calcd for C₂₉H₃₆F₂N₂NaO₇Si [M + Na] 613.2158, found: 613.2162.

Detritylation of 3-((Bis(4-fluorophenyl)methoxy)methyl)-1-((3aR,4R,6aR)-2,2-dimethyl-6-((trityloxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (8)

To a stirred solution of 8 (65 mg, 0.086 mmol) and TolSH (16 mg, 0.13 mmol) in CH₂Cl₂ (0.43 mL), BF₃^•OEt₂ (5.3 μL, 0.043 mmol) was added at r.t. After being stirred for 1 h, the reaction mixture was quenched with sat. aq. NaHCO₃, extracted with EtOAc. The combined extract was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 1/1 - 1/2) to afford 3 as yellow foam (45 mg, 84%).

Oxidation of 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R,3R,4R)-3-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (6)

To a stirred suspension of CrO₃ (35 mg, 0.35 mmol) in CH₂Cl₂ (0.30 mL), Ac₂O (99 mL, 1.04 mmol) and pyridine (0.17 mL, 2.09 mmol) were added at 0 °C. After being stirred for 0.5 h at 0 °C, 0.5 h at r.t., 6 (61 mg, 0.087 mmol) was added. After 0.5 h, the reaction mixture was diluted with EtOAc, filtered through celite and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 9/1 to 8/2) to afford 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R,3S)-3-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-oxotetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione as colorless oil (9, 49 mg, 81%). [α]$[\alpha {]}_{\mathrm{D}}^{21}$ + 0.525 (c = 0.46, CHCl₃); IR (thin film) ν_max = 2955, 2930, 2858, 1786, 1723, 1672, 1604, 1508, 1454, 1272, 1255, 1222, 1070, 834, 780 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.77 (d, J = 8.2 Hz, 1H), 7.30 (dd, J = 8.4, 5.4 Hz, 4H), 6.98 (t, J = 8.4 Hz, 4H), 6.27 (d, J = 7.9 Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H), 5.69 (s, 1H), 5.49 (d, J = 9.8 Hz, 1H), 5.44 (d, J = 9.8 Hz, 1H), 4.24 (s, 1H), 4.12 (d, J = 7.9 Hz, 1H), 3.93 (d, J = 11.7 Hz, 1H), 3.89 (d, J = 11.3 Hz, 1H), 0.88 (s, 9H), 0.79 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H), 0.02 (s, 3H), −0.09 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 208.35, 163.42, 162.17, 160.97, 151.03, 137.75, 137.44, 137.42, 137.39, 128.69, 128.66, 128.61, 128.58, 115.33 (2C), 115.11 (2C), 103.18, 85.51, 81.85, 81.40, 69.36, 62.84, 25.72 (3C), 25.26 (3C), 18.19, 18.04, −4.88, −5.51, −5.64, −5.79; HRMS (ESI+) m/z calcd for C₃₅H₄₈F₂N₂NaO₇Si₂ [M + Na] 725.2866, found: 725.2889.

Reduction of 9

To a stirred solution of 9 (12 mg, 0.018 mmol) in THF (0.35 mL), LiBH₄ (4.0M in THF, 8.8 μL, 0.035 mmol) was added at 0 °C. After being stirred for 1 h, the reaction mixture was quenched with sat. aq. NaHCO₃, extracted with EtOAc. The combined extract was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 85/15 to 80/20) to afford mixture of 10α (6) and 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R,3R,4S)-3-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (10β, 11 mg, 90%) as colorless oil. ¹H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 8.2 Hz, 1H), 7.30 (ddd, J = 7.9, 5.3, 2.3 Hz, 4H), 7.02 – 6.94 (m, 4H), 5.72 (s, 1H), 5.69 (s, 1H), 5.58 (d, J = 8.2 Hz, 1H), 5.49 (d, J = 9.8 Hz, 1H), 5.46 (d, J = 9.8 Hz, 1H), 4.40 (s, 1H), 4.30 (dd, J = 12.3, 3.4 Hz, 1H), 4.22 – 4.18 (m, 2H), 4.15 (s, 1H), 4.10 (s, 1H), 0.91 (s, 9H), 0.90 (s, 9H), 0.19 (s, 3H), 0.15 (s, 3H), 0.14 (s, 3H), 0.13 (s, 3H).

Debenzylation of 1-((3aR,4R,6aR)-6-((benzyloxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-3-((bis(4-fluorophenyl)methoxy)methyl)pyrimidine-2,4(1H,3H)-dione (11)

To a stirred solution of 11 (28 mg, 0.046 mmol) in a 10/1 mixture of ⁱPrOH/H₂O (0.5 mL) was added 10% Pd/C (2.8 mg). H₂ gas was introduced and the reaction mixture was stirred under H₂ atmosphere. After 2 h, the solution was filtered through Celite and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 1/1 to 1/2) to afford 3 as yellow foam (21 mg, 88%).

Deoxygenations of 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R,3R,4S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

To a stirred suspension of NaH (11 mg, 60% in oil, 0.28 mmol) in THF (0.46 mL), 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R,3R,4S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (55 mg, 0.092 mmol) was added at 0 °C. After being stirred for 0.5 h, CS₂ (17 mL, 0.28 mmol) was added. After 2 h, MeI (17 mL, 0.28 mmol) was added. After 4 h, the reaction mixture was quenched with sat. aq. NaHCO₃, and extracted with EtOAc. The combined extract was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc = 90/10 to 85/15) to afford O,O'-((2R,3R,4R)-2-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-3,4-diyl) S,S'-dimethyl bis(carbonodithioate) as pale yellow oil (12, 70 mg, 99%). [α]$[\alpha {]}_{\mathrm{D}}^{21}$ – 0.250 (c = 1.42, CHCl₃); IR (thin film) ν_max = 2955, 2929, 2858, 1721, 1675, 1604, 1508, 1456, 1260, 1222, 1198, 1095, 1075, 833, 810, 780 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.78 (d, J = 8.2 Hz, 1H), 7.31 – 7.27 (m, 4H), 6.98 (t, J = 8.5 Hz, 4H), 6.56 (d, J = 7.2 Hz, 1H), 6.22 (d, J = 5.5 Hz, 1H), 6.03 (dd, J = 7.0, 5.7 Hz, 1H), 5.71 – 5.67 (m, 2H), 5.50 (d, J = 9.9 Hz, 1H), 5.47 (d, J = 9.8 Hz, 1H), 4.45 (s, 1H), 4.02 (dd, J = 11.4, 1.5 Hz, 1H), 3.92 (dd, J = 11.1, 1.5 Hz, 1H), 2.61 (s, 3H), 2.53 (s, 3H), 0.94 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 214.74, 214.41, 163.41, 163.38, 162.18, 160.96, 160.93, 151.06, 138.18, 137.48, 137.45, 137.39, 137.36, 128.74, 128.66 (2C), 128.58, 115.30 (2C), 115.09 (2C), 102.86, 85.95, 84.31, 81.48, 79.91, 78.85, 69.63, 63.25, 25.93 (3C), 18.33, −5.36, −5.65; HRMS (ESI+) m/z calcd for C₃₃H₄₀F₂N₂NaO₇S₄Si [M + Na] 793.1353, found: 793.1361.

To a stirred solution of 12 (46 mg, 0.059 mmol) in toluene (0.60 mL) were added ⁿBu₃SnH (80 mL, 0.30 mmol) and AIBN (29 mg, 0.18 mmol). After being stirred for 3 h at 100 °C, the reaction mixture was concentrated in vacuo. The crude product was purified by silica gel column chromatography (10% w/w anhydrous K₂CO₃-SiO₂, hexanes/EtOAc = 85/15 to 80/20) to afford 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-2,5-dihydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione as colorless oil (13, 28 mg, 85%). [α]$[\alpha {]}_{\mathrm{D}}^{21}$ – 0.177 (c = 0.70, CHCl₃); IR (thin film) ν_max = 2956, 2929, 2858, 1718, 1669, 1604, 1508, 1458, 1258, 1223, 1139, 1075, 836, 808, 781 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 7.74 (d, J = 8.2 Hz, 1H), 7.31 (dt, J = 8.9, 5.3 Hz, 4H), 7.00 (d, J = 2.9 Hz, 1H), 6.97 (dd, J = 8.5, 2.7 Hz, 4H), 6.24 (d, J = 5.9 Hz, 1H), 5.76 (d, J = 5.6 Hz, 1H), 5.72 (s, 1H), 5.61 (d, J = 8.1 Hz, 1H), 5.51 (s, 2H), 4.88 (s, 1H), 3.91 (dd, J = 11.7, 2.8 Hz, 1H), 3.83 (dd, J = 11.7, 2.6 Hz, 1H), 0.88 (s, 9H), 0.06 (d, J = 1.9 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 163.36, 162.73, 160.91, 151.27, 139.71, 137.64, 137.61, 134.30, 128.65, 128.63, 128.57, 128.54, 126.44, 115.28 (2C), 115.07 (2C), 101.80, 90.25, 87.28, 81.66, 69.66, 64.10, 25.86 (3C), 18.48, −5.42, −5.60; HRMS (ESI+) m/z calcd for C₂₉H₃₄F₂N₂NaO₅Si [M + Na] 579.2103, found: 579.2119.

Hydrogenation of 13

To a stirred solution of 13 (14 mg, 0.026 mmol) in MeOH (5.0 mL) was added 10% Pd/C (2.8 mg). H₂ gas was introduced and the reaction mixture was stirred under H₂ atmosphere. After 2 h, the solution was filtered through Celite and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc = 85/15 to 80/20) to afford 3-((bis(4-fluorophenyl)methoxy)methyl)-1-((2R)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione as colorless oil (14, 13 mg, 93%). [α]$[\alpha {]}_{\mathrm{D}}^{22}$ + 0.171 (c = 1.13, CHCl₃); IR (thin film) ν_max = 2955, 2929, 2858, 1713, 1663, 1604, 1507, 1459, 1276, 1260, 1222, 1127, 1073, 1014, 998, 863, 836, 807, 779 cm⁻¹; ¹H NMR (400 MHz, Chloroform-d) δ 8.01 (d, J = 8.1 Hz, 1H), 7.30 (ddd, J = 8.2, 5.4, 2.3 Hz, 4H), 6.97 (td, J = 8.7, 3.1 Hz, 4H), 5.99 (dd, J = 6.5, 2.6 Hz, 1H), 5.72 (s, 1H), 5.59 (d, J = 8.2 Hz, 1H), 5.52 (d, J = 9.9 Hz, 1H), 5.49 (d, J = 9.8 Hz, 1H), 4.14 (td, J = 8.0, 1.8 Hz, 1H), 4.06 (dd, J = 11.6, 2.2 Hz, 1H), 3.69 (dd, J = 11.6, 2.2 Hz, 1H), 2.41 – 2.31 (m, 1H), 2.02 – 1.83 (m, 3H), 0.92 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.34, 162.89, 160.90, 150.78, 139.29, 137.72, 137.69, 137.65, 137.61, 128.65, 128.57 (2C), 128.48, 115.26 (2C), 115.04 (2C), 100.72, 86.84, 82.23, 81.72, 69.58, 63.33, 33.52, 25.86 (3C), 23.99, 18.41, −5.52, −5.61; HRMS (ESI+) m/z calcd for C₂₉H₃₆F₂N₂NaO₅Si [M + Na] 581.2259, found: 581.2267.

Deprotections of tert-butyl (((2R,3S,4R,5S)-5-((1S,2S)-3-amino-2-((3-aminopropyl)amino)-1-((2S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-oxopropoxy)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)carbamate (15)

To a stirred solution of 15 (17 mg, 0.016 mmol) in H₂O (0.5 mL) was added TFA (0.5 mL). After being stirred for 2 h at r.t., all volatile were evaporated in vacuo. To a stirred solution of the crude mixture in CH₂Cl₂ (1.0 mL) was added TFA (0.02 mL). After 2 h, all volatile were evaporated in vacuo. To a stirred solution of the crude mixture in ⁱPrOH-H₂O (10:1, 1.0 mL) was added 10% Pd/C (10 wt % 6.8 mg). H₂ gas was introduced and the reaction mixture was stirred for 6 h under H₂. The solution was filtered through Celite and concentrated in vacuo. The crude mixture was purified by C18 reverse-phase HPLC [column: Luna® (100 Å, 10 μm, 250 x 10 mm), solvents: 25:75 MeOH:0.05 M NH₄HCO₃ in H₂O, flow rate: 3.0 mL/min, UV: 254 nm] to afford tert-butyl (((3aR,4R,6S,6aR)-6-((1S,2S)-3-amino-2-((3-(((benzyloxy)carbonyl)amino)propyl)amino)-1-((4R,6R)-6-(3-((bis(4-fluorophenyl)methoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-3-oxopropoxy)-2,2-diethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)carbamate as colorless oil (16, 7.2 mg, 76% for 3 steps, retention time: 13.0 min): [α]$[\alpha {]}_{\mathrm{D}}^{21}$ + 0.386 (c = 0.35, MeOH); IR (thin film) ν_max = 3329 (br), 3312 (br), 2979, 2929, 1678, 1572, 1508, 1459, 1392, 1367, 1279, 1131, 1115, 1077, 1057, 1018 cm⁻¹; ¹H NMR (400 MHz, Deuterium Oxide) δ 7.76 (d, J = 7.8 Hz, 1H), 5.79 (s, 1H), 5.76 (d, J = 7.8 Hz, 1H), 5.05 (s, 1H), 4.25 – 4.13 (m, 3H), 4.03 (dd, J = 10.5, 6.8 Hz, 3H), 3.52 (d, J = 6.4 Hz, 1H), 3.36 – 3.31 (m, 2H), 3.29 (s, 1H), 2.97 (t, J = 7.3 Hz, 2H), 2.65 (t, J = 7.1 Hz, 2H), 1.82 – 1.73 (m, 2H), 1.34 (s, 9H); ¹³C NMR (101 MHz, D₂O) δ 176.10, 161.92, 161.73, 160.96, 146.67, 140.44, 109.60, 102.04, 90.59, 90.39, 82.37, 81.77, 80.86, 79.92, 75.26, 74.88, 41.07, 38.32, 38.19, 27.69 (3C); HRMS (ESI+) m/z calcd for C₂₄H₄₀N₆NaO₁₂ [M + Na] 627.2602, found: 627.2613.