Alternative Synthesis and Antibacterial Evaluation of 1,5-Dideoxy-1,5-imino-L-rhamnitol

Abstract

A convenient synthesis is described of 5-azido-5-deoxy-2,3-O-isopropylidene-L-rhamnofuranose from L-rhamnose in seven steps and 17% overall yield. A key feature of the synthesis is the selective oxidation of the secondary alcohol in 2,3-O-isopropylidene-L-rhamnofuranose in the presence of the hemiacetal to give the corresponding ketone in good yield using the Parikh-Doering reagent. 5-Azido-5-deoxy-2,3-O-isopropylidene-L-rhamnofuranose is then converted by a literature protocol to 1,5-dideoxy-1,5-imino-L-rhamnitol, which was found to have no significant antimicrobial activity against Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Escherichia coli.

Graphical Abstract

1.1. Introduction

The recent discovery that in Pseudomonas aeruginosa rhamnosylation of a conserved arginine residue in Elongation Factor-P (EF-P) circumvents ribosome stalling at X-PP-X sequences and is essential for bacterial fitness and pathogenicity of this nosocomial pathogen suggests the responsible glycosyl transferase (EarP) and dTDP-L-rhamnose synthesizing enzymes as possible new targets for antibiotic development.¹ Iminosugars have a long and fruitful history as glycosidase and glycosyl transferase inhibitors.^2–3 Pertinently, 1,5-dideoxy-1,5-imino-L-rhamnitol 1 and its N-alkyl and aryl derivatives were prepared by Davis and coworkers and studied as inhibitors of RhamT, a rhamnosyl transferase involved in the assembly of the bridging region linking arabinogalactan units to the peptidoglycan of Mycobacterium tuberculosis cell wall.⁴ As EarP and RhamT employ the same glycosyl donor dTDP-L-Rha, 1 is a possible inhibitor of EarP and therefore of P. aeruginosa. Following earlier work by Fleet and coworkers,⁵ Davis and coworkers prepared 1 and its N-substituted analogs by two routes,⁴ both employing 5-azido-5-deoxy-2,3-O-isopropylidene-L-rhamnofuranose 2 as a key intermediate assembled in seven steps by a literature protocol^5–6 from D-gulonolactone via its 2,3-acetonide⁷ (Scheme 1). 1,5-Dideoxy-1,5-imino-L-rhamnitol 1, alternatively called L-deoxyrhamnonojirimycin, also has been prepared by chemoenzymatic synthesis,⁸ and has been shown to have only weak inhibitory properties for various glycosidase enzymes.^5,8 We describe an alternative route in 17% overall yield and seven steps to the key intermediate 2 from commercial L-rhamnose (Scheme 1), its conversion to 1 by the Davis protocol, and the finding that 1 displays no significant activity against clinical isolates of P. aeruginosa.

Scheme 1.

Existing and New Routes to 1,5-Dideoxy-1,5-imino-L-rhamnitol

1.2. Results and Discussion

Following Khan et al,⁹ L-rhamnose was treated with tetrabutylammonium tribromide in acetone at room temperature to give acetonide 3, in 89% yield after work up. Parikh-Doering oxidation¹⁰ of 3 gave the ketone 4a in 58% yield together with 3% of the ketolactone 4b and 16% of starting material 3. This selective oxidation of the secondary alcohol in the presence of a hemiacetal is noteworthy and contributes significantly to the success of this synthesis. Subsequent reaction with tert-butyldiphenylsilyl chloride, DMAP and imidazole then afforded the silyl glycoside 5 in 96% yield with excellent selectivity in favor of the 1,2-trans-configuration. The anomeric configuration of compounds 3–7, all of which are essentially single isomers, is assigned on the basis of the minimal value of ³J_1,2 in the ¹H-NMR spectra, which is indicative of the 1,2-trans-configuration.¹¹ Reduction of 5 with sodium borohydride in methanol at 0 °C gave 77% of the desired D-gulofuranoside 6a and 11% of the L-rhamno-isomer 6b. The selectivity of this reduction is consistent with that reported previously in the reaction of sodium borohydride with the corresponding methyl glycoside.¹² Reaction of 6a with triflic anhydride and pyridine in dichloromethane at −20 °C afforded an intermediate triflate that on treatment with sodium azide in DMF gave the 5-deoxy-5-azido-L-rhamnose derivative 7 in 64% yield for the two steps. Finally, cleavage of the silyl glycosides with tetrabutylammonium fluoride gave 2 in 71% (Scheme 2), whose spectral data were in full agreement with the literature.⁴ Overall, the synthesis of key intermediate 2 is achieved in six steps and 17% overall yield from the readily available L-rhamnose, thereby providing a useful alternative to the existing synthesis that proceeds in a total of seven steps and 37% yield from the more costly and less available D-gulonolactone. Finally, following the method of Davis and coworkers 2 was then converted to the target 1 in 82% yield (over 2 steps) by reductive cyclization with H₂ over Pd/C and deprotection of acetonide group with aqueous trifluoroacetic acid.

Scheme 2.

Synthesis of 2 and 1 from L-Rhamnose

Iminoglycoside 1 was screened for activity against several strains of Pseudomonas aeruginosa. No activity was observed at concentrations of 128 μg/mL or less. Similarly, no activity was observed against the Gram-positive bacterium methicillin resistant Staphylococcus aureus, and the Gram-negative bacterium, Escherichia coli.

1.3. Experimental Section

General Experimental

All experiments were performed in oven dried glassware under an argon atmosphere. The visualization of spots on TLC plates was effected by exposure to iodine or spraying with 10% H₂SO₄ and charring. ¹H NMR spectra were recorded in CDCl₃ solution unless otherwise stated at 400 MHz. ¹³C NMR spectra were recorded in CDCl₃ solution unless otherwise stated at 100 MHz. Mass spectra were recorded in the +ve ion mode using electrospray ionization (ESI-TOF). Specific rotations were recorded in dichloromethane solution at room temperature.

6-Deoxy-2,3-O-isopropylidene-α-L-lyxo-5-hexulofuranose (4a) and L-lyxo-6-deoxy-5-hexulosono-1,4-lactone (4b)

To a stirred solution of 3^9,13 (5.0 g, 24.4 mmol) in dimethyl sulfoxide (55.0 mL) and dichloromethane (82.5 mL) were added triethylamine (8.5 mL, 61.2 mmol) and sulfur trioxide pyridine complex (7.8 g, 48.9 mmol) at room temperature. After stirring for 1.5 h the reaction mixture was washed with water, and extracted with ethyl acetate. The ethyl acetate layer was dried over Na₂SO₄ and concentrated under high vacuum. Column chromatography over silica gel (eluent: 30% ethyl acetate in hexane) afforded 4a (2.4 g, 58%), 4b (0.12 g, 3%), and recovered substrate (0.8 g, 16%).

4a

Mp = 147–148 °C; ${[\alpha ]}_D^{22}=+45.5$ (c 0.55, CH₂Cl₂); IR (neat) ν_max 3346 (–O–H), 1696 (–C=O) cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 5.53 (d, J = 2.1 Hz, 1H, H-1), 5.06 (dd, J = 5.7, 4.2 Hz, 1H, H-3), 4.66–4.62 (m, 2H, H-2, H-4), 2.75 (s, 1H, O-H), 2.23 (s, 3H, H-6), 1.42 (s, 3H, -C(CH₃)₂), 1.28 (s, 3H, -C(CH₃)₂); ¹³C NMR (100 MHz, CDCl₃) δ 204.4, 113.1, 101.4, 85.2, 84.7, 80.8, 27.8, 25.8, 24.5; HRMS (ESI) m/z calcd for C₉H₁₄O₅Na [M+Na]⁺, 225.0739; found, 225.0732.

4b

Mp = 124 – 125 °C; ${[\alpha ]}_D^{22}=-5.7$ (c 2.60, CH₂Cl₂); IR (neat) ν_max 1792 (O–C=O), 1736 (–C=O) cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 5.07 (t, J = 4.6 Hz, 1H, H-3), 4.88 – 4.81 (m, 2H, H-2, H-4), 2.28 (s, 3H, H-6), 1.44 (s, 3H, -C(CH₃)₂), 1.36 (s, 3H, - C(CH₃)₂; ¹³C NMR (100 MHz, CDCl₃) δ 202.3, 172.9, 114.7, 81.4, 76.5, 75.5, 27.6, 26.6, 25.6; HRMS (ESI) m/z calcd for C₉H₁₂O₅Na [M+Na]⁺, 223.0582; found, 223.0575.

tert-Butyldiphenylsilyl 6-deoxy-2,3-O-isopropylidene-α-L-lyxo-5-hexulofuranoside (5)

To a stirred solution of 4a (0.230 g, 1.14 mmol) in DMF (3.0 mL) were added tert-butylchlorodiphenylsilane (0.44 mL, 1.71 mmol), imidazole (0.155 g, 2.3 mmol), and dimethylaminopyridine (0.014 g, 0.11 mmol). The reaction mixture was stirred for 5 h at room temperature before TLC (40% ethyl acetate in hexane) showed reaction completion. The reaction mixture was washed with water, and extracted with ethyl acetate. The ethyl acetate layer was dried over Na₂SO₄ and concentrated. Column chromatography over silica gel (eluent: 10% ethyl acetate in hexane) afforded 5 (0.480 g, 96%) as a colorless oil. ${[\alpha ]}_D^{22}=-2.5$ (c 1.00, CH₂Cl₂); IR (neat) ν_max 1722 (–C=O) cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 7.67–7.61 (m, 4H, ArH), 7.50–7.32 (m, 6H, ArH), 5.51 (s, 1H, H-1), 5.13–5.05 (m, 1H, H-3), 4.67 (d, J = 5.7 Hz, 1H, H-4), 4.60 (d, J = 4.1 Hz, 1H, H-2), 2.19 (s, 3H, H-6), 1.36 (s, 3H, -C(CH₃)₂), 1.27 (s, 3H, -C(CH₃)₂), 1.07 (s, 9H, ^tBuH); ¹³C NMR (100 MHz, CDCl₃) δ 205.5, 135.6, 135.5, 132.91, 132.6, 129.9, 127.8, 127.7, 112.9, 101.8, 86.1, 85.1, 80.9, 28.0, 26.7, 26.5, 25.7, 24.4, 19.2; (ESI) m/z calcd for C₂₅H₃₂O₅SiNa [M+Na]⁺, 463.1917; found, 463.1907.

tert-Butyldiphenylsilyl 6-deoxy-2,3-O-isopropylidene-β-D-gulofuranoside (6a) and tert-Butyldiphenylsilyl 2,3-O-isopropylidene-α-L-rhamnofuranoside (6b)

To a stirred solution of 5 (0.14 g, 0.32 mmol) in methanol (2 mL) was added sodium borohydride (0.018 g, 0.48 mmol) at 0 °C. The reaction mixture was stirred for 1 h at 0 °C before TLC (30% ethyl acetate in hexane) showed reaction completion. The mixture was neutralized with sat. NH₄Cl solution, washed with water, and extracted with ethyl acetate. The ethyl acetate layer was dried over Na₂SO₄ and concentrated. Column chromatography on silica gel (eluent: 10% ethyl acetate in hexane) afforded 6a (0.11 g, 77%) and 6b (0.16 g, 11%).

6a

${[\alpha ]}_D^{22}=-38.8$ (c 1.35, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 7.69 – 7.62 (m, 4H, ArH), 7.46–7.32 (m, 6H, ArH), 5.40 (s, 1H, H-1), 4.82 (dd, J = 5.8, 3.6 Hz, 1H, H-3), 4.73 (d, J = 5.9 Hz, 1H, H-2), 4.13–4.07 (m, 1H, H-5), 3.98 (dd, J = 6.0, 3.7 Hz, 1H, H-4), 2.55 (s, 1H, O-H), 1.40 (s, 3H, -C(CH₃)₂), 1.30 (s, 3H, -C(CH₃)₂), 1.27 (d, J = 6.4 Hz, 3H, H-6), 1.07 (s, 9H, ^tBuH); ¹³C NMR (100 MHz, CDCl₃) δ 135.7, 135.5, 133.0, 132.8, 129.9, 129.8, 127.7, 127.6, 112.5, 101.1, 87.5, 83.6, 80.2, 66.45, 26.7, 25.8, 24.5, 19.2, 18.6. HRMS (ESI) m/z calcd for C₂₅H₃₄O₅SiNa [M+Na]⁺, 465.2073; found, 465.2081.

6b

${[\alpha ]}_D^{22}=-29.3$ (c 1.95, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 7.67 – 7.61 (m, 4H, ArH), 7.44 – 7.35 (m, 6H, ArH), 5.39 (s, 1H, H-1), 4.95 (dd, J = 5.8, 3.5 Hz, 1H, H-3), 4.71 (d, J = 5.9 Hz, 1H, H-2), 4.01–3.95 (m, 2H, H-4, H-5), 2.40 (s, 1H, O-H), 1.41 (s, 3H, -C(CH₃)₂), 1.32 (s, 3H, -C(CH₃)₂), 1.27 (d, J = 5.9 Hz, 3H, H-6), 1.06 (s, 9H, ^tBuH); ¹³C NMR (100 MHz, CDCl₃) δ 135.7, 135.5, 133.1, 132.9, 129.8, 127.7, 127.6, 112.5, 101.2, 86.8, 83.6, 79.9, 66.5, 26.7, 25.8, 24.6, 20.4, 19.2; HRMS (ESI) m/z calcd for C₂₅H₃₄O₅SiNa [M+Na]⁺, 465.2073; found, 465.2075. To obtain better resolution in the NMR spectrum and assist with configurational assignment 6b to the acetate 6c.

tert-Butyldiphenylsilyl 5-O-Acetyl-2,3-O-isopropylidene-α-L-rhamnofuranoside (6c)

Pyridine (0.25 mL) and acetic anhydride (0.25 mL) were added to 6b (0.010 g, 0.023 mmol). The reaction mixture was stirred at room temperature for 24 h before TLC (30% ethyl acetate in hexane) showed completion. The solvents were evaporated, and column chromatography of the residue over silica gel (eluent: 5% ethyl acetate in hexane) afforded 6c (0.010 g, 91%) as a colorless oil; ${[\alpha ]}_D^{22}=-28.8$ (c 0.55, CH₂Cl₂); IR (neat) ν_max 1746 (–C=O) cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 7.65–7.61 (m, 4H, ArH), 7.47–7.30 (m, 6H, ArH), 5.33 (s, 1H, H-1), 5.09 (dq, J = 8.4, 6.3 Hz, 1H, H-5), 4.81 (dd, J = 5.8, 3.7 Hz, 1H, H-3), 4.69 (d, J = 5.8 Hz, 1H, H-2), 4.15 (dd, J = 8.4, 3.6 Hz, 1H, H-4), 2.05 (s, 3H,-COCH₃), 1.36 (s, 3H, -C(CH₃)₂), 1.28 (s, 3H, -C(CH₃)₂), 1.27 (d, J = 4.0 Hz, 3H, H-6), 1.07 (s, 9H, ^tBuH); ¹³C NMR (100 MHz, CDCl₃) δ 170.0, 135.5, 133.2, 129.8, 127.7, 111.8, 101.1, 86.5, 81.8, 79.2, 68.2, 26.0, 25.7, 24.5, 21.5, 18.9, 17.0; HRMS (ESI) m/z calcd for C₂₇H₃₆O₆SiNa [M+Na]⁺, 507.2179; found, 507.2189.

tert-Butyldiphenylsilyl 5-Azido-5-deoxy-2,3-O-isopropylidene-α-L-rhamnofuranoside (7)

To a stirred solution of 6a (0.67 g, 1.51 mmol) in pyridine (1.14 mL) and dichloromethane (4.2 mL) was added triflic anhydride (0.47 g, 1.66 mmol). The reaction mixture was stirred at −20 °C for 1 h before TLC (30% ethyl acetate in hexane) showed completion. The reaction mixture was neutralized with 1N hydrochloric acid, washed with water, and extracted with dichloromethane, dried over Na₂SO₄ and concentrated. The residue was taken up in DMF (10 mL) and sodium azide (0.491 g, 7.55 mmol) added. After stirring for 0.5 h TLC (20% ethyl acetate in hexane) showed completion. The mixture was washed with water, extracted with ethyl acetate, dried over Na₂SO₄ and concentrated. Column chromatography over silica gel (eluent: 10% ethyl acetate in hexane) afforded 7 (0.45 g, 64%) as a colorless oil; ${[\alpha ]}_D^{22}=-27.7$ (c 0.90, CH₂Cl₂); IR (neat) ν_max 2094 (–N₃) cm^{−1; 1}H NMR (400 MHz, CDCl₃) δ 7.67–7.62 (m, 4H), 7.43–7.34 (m, 6H), 5.32 (s, 1H, H-1), 4.87 (dd, J = 5.8, 3.6 Hz, 1H, H-3), 4.72 (d, J = 5.8 Hz, 1H, H-2), 3.91 (dd, J = 9.6, 3.5 Hz, 1H, H-4), 3.80–3.73 (m, 1H, H-5), 1.40 (s, 3H, -C(CH₃)₂), 1.33 (s, 3H, -C(CH₃)₂), 1.30 (d, J = 6.5 Hz, 3H, H-6), 1.07 (s, 9H, ^tBuH); ¹³C NMR (100 MHz, CDCl₃) δ 135.7, 135.5, 134.8, 133.0, 132.9, 129.8, 129.6, 127.6, 127.7, 127.6, 112.5, 101.5, 86.9, 82.4, 79.5, 55.3, 26.7, 26.5, 25.9, 24.7, 19.2, 17.0; HRMS (ESI) m/z calcd for C₂₅H₃₃N₃O₄SiNa [M+Na]⁺, 490.2138; found, 490.2136.

5-Azido-5-deoxy-2,3-O-isopropylidene-α,β-L-rhamnofuranose (2)

A stirred solution of 7 (0.20 g, 0.42 mmol) in THF (2.0 mL) was treated with TBAF (0.85 mL, 0.85 mmol, 1M solution in THF) and stirred for 1 h before TLC (20% ethyl acetate in hexane) showed completion. The reaction mixture was neutralized with Et₃N (0.2 mL), washed with water, and extracted with dichloromethane. The extracts were dried over Na₂SO₄ and concentrated. Column chromatography over silica gel (eluent: 30% ethyl acetate in hexane) afforded 2 (0.07 g, 71%) as a mixture of anomers (α;β = 5.5:1), whose spectra data were consistent with the literature values.⁴ IR (neat) ν_max 3429 (–O–H), 2092 (–N₃) cm^{−1; 1}H NMR (400 MHz, CDCl₃, 5.5:1 ratio of α:β anomers) δ 5.36 (s, 5.5H, H-1α), 4.99 (br s, 1H, H-1β), 4.80 (dd, J = 5.7, 3.2 Hz, 5.5H, H-3α), 4.72 (dd, J = 6.0, 3.2 Hz, 1H, H-3β), 4.60 (d, J = 6.1 Hz, 5.5H, H-2α), 4.52 (dd, J = 6.1, 3.6 Hz, 1H, H-2β), 3.87 (dd, J = 9.3, 3.2 Hz, 5.5H, H-4α), 3.85–3.77 (m, 6.5H, H-5α, H-5β), 3.22 (dd, J = 9.3, 3.2 Hz, 1H, H-4β), 1.53 (s, 3H, -C(CH₃)₂β), 1.46 (s, 16.5H, -C(CH₃)₂α), 1.40 (d, J = 6.9 Hz, 3H, H-6β), 1.39 (s, 3H, -C(CH₃)₂β), 1.38 (d, J = 6.5 Hz, 16.5H, H-6α), 1.34 (s, 16.5H, -C(CH₃)₂α); ¹³C NMR (100 MHz, CDCl₃) δ 113.2, 112.6, 101.1, 96.9, 85.3, 82.4, 79.5, 79.1, 78.4, 78.2, 55.3, 55.2, 25.9, 25.7, 24.8, 24.6, 17.2, 17.0; HRMS (ESI) m/z calcd for C₉H₁₅N₃O₄Na [M+Na]⁺, 252.0960; found, 252.0965.

1,5-Dideoxy-1,5-imino-L-rhamnitol hydrochloride (1)

A stirred solution of 2 (0.07 g, 0.30 mmol) in EtOH (2.0 mL) was treated with Pd/C (25.0 mg) and stirred under 1 atm of H₂ for 24 h before TLC (60% ethyl acetate in hexane) showed completion. The reaction mixture was filtered through a Celite pad, washing with MeOH, and the filtrate was concentrated and dried under high vacuum. The residue was dissolved in THF (1.0 mL), treated with TFA/H₂O (0.25 mL/0.25 mL), and stirred at room temperature for 3 h. The reaction mixture was concentrated and the crude product was purified through a Sephadex C-25 column (eluent: 10–20% NH₄OH) to give 1 (36 mg, 82%) as a free amine, which was converted to the hydrochloride salt 1.HCl by dissolving in 1N HCl and lyophilization. The spectra data of 1.HCl were consistent with the literature values.^5,8 ${[\alpha ]}_D^{22}=+32.0$ (c 1.00, H₂O); Lit.⁵ ${[\alpha ]}_D^{20}=+37.5$ (c 0.67, H₂O); Lit.⁸ ${[\alpha ]}_D^{25}=+39.3$ (c 1.09, H₂O); ¹H NMR (400 MHz, D₂O) δ 4.11 (br s, 1H, H-2), 3.55 (t, J = 9.3 Hz, H-4) 3.52 (dd, J = 2.9, 9.7 Hz, H-3), 3.24 (dd, J = 13.6, 2.9 Hz, 1H, H-1), 3.08 (d, J = 13.6 Hz, 1H, H-1′), 3.03–2.92 (m, 1H, H-5), 1.28 (d, J = 6.5 Hz, 3H, H-6); ¹³C NMR (100 MHz, D₂O) δ 72.1 (C-3), 70.0 (C-4), 65.9 (C-2), 55.4 (C-5), 47.5 (C-1), 14.3 (C-6); HRMS (ESI) m/z calcd for C₆H₁₄NO₃ [M+H]⁺, 148.0974; found, 148.0970.

Antibacterial assays

Clinical isolates of P. aeruginosa, E. coli and S. aureus were obtained from the Diagnostic Department, Institute of Medical Microbiology, University of Zurich. MIC values were determined by broth microdilution assays as described.¹⁴

Highlights.

• Selective oxidation

• Stereoselective reduction

• Antibacterial assay

• Efficient synthesis