Synthesis of a Pseudodisaccharide Suitable for Synthesis of Ring I Modified 4,5–2-Deoxystreptamine Type Aminoglycoside Antibiotics.

Abstract

To facilitate the synthesis of paromomycin and/or neomycin analogs we describe a cleavage of ring I from paromomycin that proceeds in the presence of azides and affords a glycosyl acceptor for the installation of a modified ring I. A paromomycin 4’,6’-diol is oxidized by the Dess-Martin periodinane followed by m-chloroperoxybenzoic acid. Base treatment then affords a protected pseudodisaccharide, which functions as a glycosyl acceptor. The method should also apply to the cleavage of pyranosyl 4,6-diols from oligosaccharides and glycoconjugates.

Graphical Abstract

Owing to the ever-increasing spread of multidrug-resistant infections there is much interest in the development of new and improved antibacterial agents.¹ Next generation aminoglycoside antibiotics in particular are attracting attention because of their many attractive features^2–4 including high efficacy and broad spectrum potency, rapid bactericidal potency, minimal protein binding and metabolic stability, lack of drug-related allergy, and absence of interaction with the hosts intestinal microbiome and with other pharmaceutical agents. Indeed, modification of ring I of the 2-deoxystreptamine aminoglycosides paromomycin and/or neomycin, which makes an essential contact with A1408 within the drug binding pocket on the small ribosomal subunit,⁵ has proven useful in increasing antibacterial activity while decreasing susceptibility to resistance and, in some cases, increasing selectivity for bacterial over eukaryotic ribosomes.^6–13 Many modifications to ring I can be made by taking advantage of the extensive reported chemistry of these pseudotrisaccharides (Scheme 1),^{14, 15} but others are best addressed by a deglycosylation-reglycosylation strategy in which ring I is removed to give a pseudodisaccharide followed by reinstallation of a preformed modified ring I.^{10, 16} More drastic modifications involving the replacement of ring I by a heteroaromatic ring have to proceed via cleavage of ring I,¹⁷ or be prepared by synthesis from 2-deoxystreptamine or its derivatives (Scheme 1).¹⁸

Scheme 1.

Strategies for the Synthesis of Ring I Modified Derivatives of Paromomycin and Neomycin.

Several routes have been devised for the cleavage of ring I from paromomycin and/or neomycin analogs but all have limitations. The Farmitalia protocol involving diazotization of the 2’-amino group in ring I is effective but is preceded by a low yielding step involving selective protection of all other amino groups in the molecule.^{10, 16} Removal of ring I initiated by sodium metaperiodate cleavage of the 3’,4’-diol is effective but is typically conducted without protection of the various other hydroxyl groups, thereby necessitating subsequent selective protection steps before reinstallation of ring I.^{10, 19–21} Finally, reducing metal initiated cleavage of 6’-deoxy-6’-iodo derivatives of paromomycin require installation of the iodide, and are not compatible with azide protection of the multiple amino groups present.^{17, 22} We describe a protocol for the cleavage of ring I from paromomycin that takes advantage of the selective installation of a 4’,6’-O-benzylidene acetal in ring I and that functions in the presence of multiple azide groups.

Paromomycin was converted through three steps of azide installation, benzylidene acetal formation, and benzylation as described previously to give the fully protected derivative 1.²³ Also as described previously,²³ exposure of 1 to toluenesulfonic acid in methanol then gave the 4’,6’-diol 2 in excellent yield (Scheme 2). Stirring of 2 with the Dess Martin periodinane (DMP)²⁴ in dichloromethane at room temperature was followed by addition of m-chloroperoxybenzoic acid and stirring for a further 24 h. Finally, the reaction mixture was worked-up by stirring with aqueous sodium hydroxide and sodium thiosulfate and purification by chromatography over silica gel. In this manner the selectively protected pseudodisaccharide 3 was isolated in 47% overall yield from 2 in the form of a white gum (Scheme 2).

Scheme 2.

Synthesis of pseudodisaccharide 3 from paromomycin.

We envisage that this oxidative cleavage reaction takes place by oxidation of the 1,3-diol 2 to the corresponding α-formyl ketone, whose enolized form 4 is oxidized by the peracid to afford an α-hydroxy-α-formyl ketone 5. On treatment with base fragmentation of the hemiacetal ensues resulting overall in the cleavage of ring I and formation of 3 (Scheme 3).

Scheme 3.

Proposed Mechanism for the Formation of 3 from diol 2.

To establish that 3 is suitable for the installation of a highly modified paromomycin ring I, we prepared a 3,4-dideoxy glycopyranosyl donor 11 as outlined in Scheme 4 from the readily available galactenitol derivative 6 and 1-octanol.²⁵ Noteworthy in this preparation is the ability to carry the primary sulfonate ester through the hydrolysis of 10 to 11 in hot aqueous trifluoroacetic acid.

Scheme 4.

Glycosyl donor preparation.

Subsequent coupling of 3 with 11 to give 12 was achieved smoothly in 95% yield as a single α-anomer under dehydrative conditions using diphenyl sulfoxide and triflic anhydride as reagents (Scheme 5).²⁶ Although we have not made a study of the mechanism of this highly selective glycosylation reaction, it is reasonable in the light of current knowledge to suggest that it proceeds by displacement of an intermediate a β-O-glycosyl diphenylsulfonium ion.^27–29

Scheme 5.

Glycosylation of Acceptor 3.

Conclusion

We have provided a straightforward method for the synthesis of the functioning glycosyl acceptor 3 from paromomycin in five simple steps. While the focus here is on controlled removal of a pyranosyl ring from an aminoglycoside antibiotic, the chemistry presented should be readily extended to the cleavage of a pyranoside ring capable of benzylidene acetal formation from the reducing terminus of any readily available oligosaccharide or glycoconjugate.

Experimental

GENERAL EXPERIMENTAL

High resolution mass spectra were collected on a Waters LCT Premier XE ESI-TOF mass spectrometer. Optical rotations were measured using an automated polarimeter. NMR spectra were collected at 400, 500, or 600 MHz as indicated and assigned by 1D and 2D techniques including COSY, HSQC, HMBC, and TOCSY.

1,3‐Diazido‐1,3‐dideamino‐5‐O‐[2’,5’‐dibenzyl‐3’‐O‐(2”,6”‐diazido‐2”,6”‐ dideamino‐3”,4”‐di‐O‐benzyl‐α‐L‐idopyranosyl)‐β‐D‐ribofuranosyl]‐6‐O‐benzyl‐2‐deoxystreptamine (3).

DMP (1.58 g, 3.73 mmol) was added to a stirred solution of 2²³ (2.0 g, 1.55 mmol) in dry DCM (25 mL). The reaction mixture was stirred for 1.5 h at room temperature before mCPBA (402 mg, 2.3 mmol) was added, after which stirring was continued for 24 h. 3 N NaOH (5 mL) and aqueous Na₂S₂O₃ solution (5 mL) were added to the reaction mixture and stirred for additional 1 h. After completion, the reaction mixture was diluted with EtOAc and the organic layer was washed with aqueous NaHCO₃ followed by brine, dried with Na₂SO₄, and concentrated. The crude product was purified via silica gel chromatography eluting with 10% to 20% EtOAc in hexanes to give 3 (739 mg, 47%) as a white gum;[α]_D²⁵= +47.1 (c 5.2, DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.50 – 7.14 (m, 23H, ArH), 7.13 – 7.02 (m, 2H, ArH), 5.28 (s, 1H, H-1’), 5.06 (s, 1H, OH), 4.95 (s, 1H, H-1”), 4.88 (d, J = 11.1 Hz, 1H, OCH₂Ph), 4.84 – 4.75 (m, 2H, H-3’, OCH₂Ph), 4.70 – 4.54 (m, 3H, OCH₂Ph), 4.51 – 4.27 (m, 5H, H-4’, OCH₂Ph), 4.23 (d, J = 11.7 Hz, 1H, OCH₂Ph), 4.02 – 3.89 (m, 2H, H-2 ‘, H-5”), 3.86 – 3.77 (m, 2H, H-5’, H-3”), 3.75 – 3.60 (m, 2H, H-5’, H-6”), 3.49 (s, 1H, H-2”), 3.46 – 3.29 (m, 3H, H-1, H-3, H-5), 3.29 – 3.09 (m, 4H, H-4, H-6, H-4”, H-6”), 2.06 (dt, J = 13.4, 4.3 Hz, 1H, H-2), 1.22 (q, J = 13.0 Hz, 1H, H-2); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 137.9 (ArC), 137.5 (ArC), 137.2 (ArC), 128.7 (ArC), 128.6 (ArC), 128.5 (ArC), 128.4 (ArC), 128.3 (ArC), 127.9 (ArC), 127.8 (ArC), 127.7 (ArC), 127.3 (ArC), 127.2 (ArC), 106.4 (C-1’), 98.2 (C-1”), 85.0 (C-5), 83.4 (C-4), 80.64 (C-2’), 80.57 (C-4’), 75.3 (OCH₂Ph), 74.9 (C-6), 74.34 (C-5”), 74.27 (C-3’), 73.7 (OCH₂Ph), 73.1 (C-3”), 72.6 (OCH₂Ph), 72.1 (OCH₂Ph), 72.0 (OCH₂Ph), 71.6 (C-4”), 67.5 (C-5’), 60.6 (C-3), 59.5 (C-1), 57.8 (C-2”), 51.0 (C-6”), 32.3 (C-2). ESI-HRMS: m/z calcd. for C₅₂H₅₆N₁₂NaO₁₀ [M+Na]⁺ 1031.4140; found, 1031.4126.

Octyl 6-O-acetyl-3,4-dideoxy-α-D-glycero-hex-3-enopyranosid-2-ulose (7).

A solution of 2,3,4,6-tetra-O-acetyl-1,5-anhydro-D-lyxo-hex-1-enitol²⁵ 6 (500 mg, 1.5 mmol) and octanol (0.45 mL, 3.0 mmol) in dry DCM (30 mL) was cooled to −78 °C and SnCl₄ (0.2 mL, 1.8 mmol) was added. The reaction mixture was stirred at 0 °C for 2 h before dilution with DCM. The resulting organic solution was washed twice with saturated aqueous NaHCO₃ solution, and then with brine. The organic extract was dried with sodium sulfate, filtered, and concentrated. The crude product was purified by gradient chromatography over silica gel (eluent: 5% to 8% EtOAc/hexanes) to yield 7 (335 mg, 74%) as a colorless oil; [α]_D²⁵= −3.6 (c 1.1, DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.94 (dd, J = 10.6, 1.6 Hz, 1H), 6.17 (dd, J = 10.6, 2.5 Hz, 1H), 4.86 (s, 1H), 4.74 (tt, J = 4.6, 2.1 Hz, 1H), 4.35 (dd, J = 11.6, 5.5 Hz, 1H), 4.25 (dd, J = 11.6, 4.6 Hz, 1H), 3.80 (dt, J = 9.6, 6.8 Hz, 1H), 3.62 (dt, J = 9.6, 6.6 Hz, 1H), 2.10 (s, 3H), 1.68 – 1.52 (m, 2H), 1.38 – 1.18 (m, 10H), 0.93 – 0.81 (m, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 188.5, 170.6, 147.0, 126.3, 97.7, 69.9, 66.9, 64.6, 31.8, , 29.4, 29.3, 29.2, 25.9, 22.6, 20.7, 14.1. ESI-HRMS: m/z calcd. for C₁₆H₂₆NaO₅ [M+Na]⁺ 321.1678; found, 321.1665.

Octyl 3,4-dideoxy-α-D-erythro-hexopyranoside (8).

Compound 7 (2.4 g, 7.47 mmol) dissolved in EtOAc (100 mL) and 10% Pd/C (200 mg) was subjected to catalytic hydrogenation at 45 psi for 3 h. The reaction mixture was filtered through Celite^®. The crude compound (1.97 g, 83%) was pure enough to be used in the next step. A solution of this crude product (500 mg, 1.66 mmol) in dry MeOH (50 mL) was cooled to −30 °C and NaBH₄ (70 mg, 1.8 mmol) was added. After 2 h, NaOMe (108 mg, 2 mmol) was added, and the reaction was continued for 1 h at room temperature. The solution was neutralized by the addition of glacial acetic acid and concentrated. The crude product was purified by gradient chromatography over silica gel (eluent: 40% to 50% EtOAc/hexanes) to yield 8 (335 mg, 74%, 61% overall from 7) as a white solid; [α]_D²⁵= +70.5 (c 1.0, DCM); ¹H NMR (400 MHz, CDCl₃) δ 4.77 (d, J = 3.6 Hz, 1H), 3.81 – 3.67 (m, 2H), 3.64 – 3.53 (m, 2H), 3.54 – 3.37 (m, 2H), 2.03 (dd, J = 7.4, 5.0 Hz, 1H), 1.94 (d, J = 11.2 Hz, 1H), 1.88 (dq, J = 12.2, 4.0 Hz, 1H), 1.72 (ddd, J = 13.3, 12.0, 4.3 Hz), 1.68 – 1.53 (m, 3H), 1.46 (tdd, J = 13.4, 11.6, 4.0 Hz, 1H), 1.40 – 1.18 (m, 10H), 0.94 – 0.83 (m, 3H); ¹³C NMR{¹H} (101 MHz, CDCl₃) δ 98.1, 68.8, 68.1, 67.8, 65.4, 31.8, 29.6, 29.4, 29.2, 27.1, 26.2, 26.0, 22.6, 14.1. ESI-HRMS: m/z calcd. for C₁₄H₂₈NaO₄ [M+Na]⁺ 283.1885; found, 283.1882.

Octyl 3,4-dideoxy-6-O-trisyl-α-D-erythro-hexopyranoside (9).

Compound 8 (1.3 g, 5.0 mmol) and triethylamine (2.8 mL, 20 mmol) were dissolved in dry DCM (15 mL) and the resulting solution was stirred and ice cooled before trisyl chloride (1.9 g, 6.3 mmol) was added portionwise. The reaction mixture was stirred for 48 h at rt under argon, then diluted with ethyl acetate and the organic layer was washed with aqueous NaHCO₃ followed by brine, dried with Na₂SO₄, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with 5% to 15% EtOAc in hexanes to give 9 (2.0 g, 76%) as a white solid; [α]_D²⁵= +34.7 (c 1.1, DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.18 (s, 2H), 4.71 (d, J = 3.6 Hz, 1H), 4.14 (hept, J = 6.8 Hz, 2H), 4.01 – 3.89 (m, 3H), 3.70 (dt, J = 9.7, 6.7 Hz, 1H), 3.55 (tt, J = 11.2, 4.6 Hz, 1H), 3.39 (dt, J = 9.7, 6.5 Hz, 1H), 2.91 (hept, J = 6.9 Hz, 1H), 1.93 – 1.79 (m, 2H, H-3), 1.76 – 1.65 (m, 2H), 1.57 (q, J = 6.8 Hz, 2H), 1.48 – 1.37 (m, 1H), 1.35 – 1.20 (m, 28H), 0.93 – 0.83 (m, 3H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 153.7, 150.8, 123.7, 97.9, 70.8, 68.0, 67.7, 66.1, 34.2, 31.8, 29.6, 29.5, 29.4, 29.2, 27.0, 26.6, 26.2, 24.7, 24.7, 23.5, 22.6, 14.1. ESI-HRMS: m/z calcd. for C₂₉H₅₀NaO₆S [M+Na]⁺ 549.3229; found, 549.3247.

Octyl 2-O-benzyl-3,4-dideoxy-6-O-trisyl-α-D-erythro-hexopyranoside (10).

Compound 9 (1.3 g, 2.5 mmol) was dissolved in dry THF (15 mL) and the resulting solution was stirred and ice cooled before benzyl bromide (1.5 mL, 12.3 mmol) and NaH (60% dispersion in oil) (200 mg, 5 mmol) was added portionwise. The reaction mixture was stirred for 3 h at 0 °C under argon and 3 h at rt, then diluted with ethyl acetate and the organic layer was washed with aqueous NaHCO₃ followed by brine, dried with Na₂SO₄, and concentrated in vacuo. The crude product was purified via silica gel chromatography eluting with 3% to 15% EtOAc in hexanes to give 10 (1.5 g, quant.) as a white solid; [α]_D²⁵= +35.3 (c 0.5, DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.37 – 7.26 (m, 5H), 7.17 (s, 2H), 4.77 (d, J = 3.2 Hz, 1H), 4.56 (q, J = 12.3 Hz, 2H), 4.13 (hept, J = 6.8 Hz, 2H), 4.04 – 3.89 (m, 3H), 3.64 (dt, J = 9.8, 7.0 Hz, 1H), 3.46 – 3.35 (m, 2H), 2.90 (hept, J = 6.7 Hz, 1H), 1.98 – 1.79 (m, 2H), 1.76 – 1.67 (m, 1H), 1.66 – 1.53 (m, 2H), 1.47 – 1.17 (m, 29H), 0.92 – 0.85 (m, 3H); ¹³C NMR{¹H} (101 MHz, CDCl₃) δ 153.6, 150.8, 128.3, 127.6, 123.7, 96.5, 74.7, 70.8, 70.6, 67.8, 65.9, 34.2, 31.8, 29.6, 29.5, 29.3, 26.8, 26.2, 24.7, 24.7, 23.5, 23.4, 22.7, 14.1. ESI-HRMS: m/z calcd. for C₃₆H₅₆NaO₆S [M+Na]⁺ 639.3695; found, 639.3712.

2-O-Benzyl-3,4-dideoxy-6-O-trisyl-α,β-D-erythro-hexopyranose (11).

Compound 10 (263 mg, 0.43 mmol) was suspended in 50% aqueous trifluoroacetic acid (8 mL) and stirred at 60 °C for 6 h. The reaction mixture was co-evaporated with toluene three times. The crude product was purified via silica gel chromatography eluting with 5% to 15% EtOAc in hexanes to give 11 (160 mg, 75%) as a white solid (α:β = 1:0.5); (Major isomer)¹H NMR (400 MHz, CDCl₃) δ 7.40 – 7.24 (m, 5H), 5.17 (d, J = 2.4 Hz, 1H), 4.62 – 4.52 (m, 2H), 4.21 – 4.09 (m, 4H, H-5), 4.06 – 3.97 (m, 2H), 3.45 (ddd, J = 11.3, 5.1, 3.2 Hz, 1H), 2.91 (hept., J = 6.9 Hz, 1H), 2.85 (d, J = 7.3 Hz, 1H), 1.96 – 1.79 (m, 2H), 1.54 – 1.33 (m, 2H), 1.26 (d, J = 6.9 Hz, 18H); ¹³C{¹H} NMR (101 MHz, CDCl₃) δ 153.7, 150.8, 128.5, 127.9, 127.8, 127.7, 127.6, 123.8, 123.7, 90.9, 74.3, 70.7, 70.6, 65.9, 34.2, 29.6, 26.1, 24.8, 24.7, 22.6. ESI-HRMS: m/z calcd. for C₂₈H₄₀NaO₆S [M+Na]⁺ 527.2443; found, 527.2433.

1,3,2”’,6”’-Tetraazido-6,2”,5”,3”’,4”’-penta-O-benzyl-1,3,2’,2”’,6”’-pentadeamino-2’-benzyloxy-3’,4’-dideoxy-6’-O-trisyl paromomycin (12).

Donor 11 (90 mg, 0.18 mmol), diphenyl sulfoxide (58 mg, 0.23 mmol) and TTBP (88 mg, 0.35 mmol) were charged to a round bottom flask, co-evaporated with toluene three times and dried in vacuo overnight. The flask was purged with argon and the mixture was dissolved in dry DCM (1 mL) and stirred with activated 4 Å M.S. for 1 h. The reaction mixture was cooled to −78 °C and treated with Tf₂O (39 μL, 0.23 mmol) then stirred for 1.5 h at −40 °C. A solution of compound 3 (90 mg, 0.08 mmol) in dry DCM (1 mL) was stirred with activated 4 Å M.S. for 3 h before added to the reaction mixture. The reaction mixture was stirred for 3 h at −40 °C before the reaction was quenched with triethylamine (0.2 mL), diluted with EtOAc and washed with aqueous NaHCO₃ and brine then concentrated. The crude product was purified using silica gel column chromatography (eluent: 5% - 15% EtOAc/hexanes) give 12 (128 mg, 95%) as a white foam; [α]_D²⁵= +28.6 (c 0.8, DCM); ¹H NMR (600 MHz, CDCl₃) δ 7.44 – 7.13 (m, 32H, ArH), 5.87 (d, J = 3.4 Hz, 1H, H-1’), 5.54 (d, J = 3.2 Hz, 1H, H-1”), 4.82 (d, J = 11.2 Hz, 1H, OCH₂Ph), 4.76 (d, J = 2.0 Hz, 1H, H-1”’), 4.72 – 4.55 (m, 4H, CH₂Ph), 4.52 – 4.48 (m, 2H, OCH₂Ph), 4.48 – 4.44 (m, 2H, OCH₂Ph), 4.42 (d, J = 11.9 Hz, 1H, CH₂Ph), 4.38 – 4.34 (m, 2H, OCH₂Ph), 4.34 – 4.24 (m, 5H, H-5’, H-3”, H-4”, OCH₂Ph), 4.20 – 4.07 (m, 2H, o-CH(CH₃)₂), 4.04 – 4.01 (m, 1H, H-2”), 4.00 (t, J = 5.0 Hz, 1H, H-6’), 3.95 (dd, J = 10.0, 4.7 Hz, 1H, H-6’), 3.78 – 3.69 (m, 4H, H-4, H-5, H-3”’, H-5”’), 3.66 (d, J = 9.6 Hz, 1H, H-5”’), 3.62 – 3.53 (m, 2H, H-5”, H-6”’), 3.48 (ddt, J = 12.6, 9.2, 4.6 Hz, 1H, H-3), 3.39 – 3.32 (m, 3H, H-1, H-2’, H-2”’), 3.22 (t, J = 8.8 Hz, 1H, H-6), 3.14 (s, 1H, H-4”’), 2.98 (dd, J = 12.8, 4.5 Hz, 1H, H-6”’), 2.90 (hept, J = 7.0 Hz, 1H, p-CH(CH₃)₂), 2.20 (dt, J = 13.2, 4.8 Hz, 1H, H-2), 1.89 (qd, J = 12.7, 12.0, 4.1 Hz, 1H, H-3’), 1.78 – 1.70 (m, 2H, H-3’, H-4’), 1.43 – 1.33 (m, 2H, H-2, H-4’), 1.26 (dd, J = 6.9, 3.6 Hz, 18H, 3 × CH(CH₃)₂); ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 153.6 (ArC), 150.8 (ArC), 138.5 (ArC), 137.9 (ArC), 137.8 (ArC), 137.0 (ArC), 136.9 (ArC), 128.7 (ArC), 128.5 (ArC), 128.4 (ArC), 128.3 (ArC), 128.2 (ArC), 128.0 (ArC), 127.9 (ArC), 127.7 (ArC), 127.5 (ArC), 127.4 (ArC), 127.3 (ArC), 123.7 (ArC), 107.2 (C-1”), 98.2 (C-1”’), 95.3 (C-1’), 83.0 (C-6), 81.9 (C-4”), 81.5 (C-5), 81.2 (C-2”), 76.0 (C-4), 75.6 (C-3”), 74.8 (C-2’), 74.7 (OCH₂Ph), 74.0 (C-5”), 73.1 (OCH₂Ph), 73.0 (C-3”), 72.7 (OCH₂Ph), 72.5 (OCH₂Ph), 71.9 (OCH₂Ph), 71.6 (C-4”’), 71.2 (OCH₂Ph), 70.9 (C-6’), 70.8 (C-5”), 66.0 (C-5’), 60.4 (C-1), 59.6 (C-3), 57.6 (C-2”’), 51.0 (C-6”’), 34.2 (CH(CH₃)₂), 32.1 (C-2), 29.7 (CH(CH₃)₂), 29.6 (C-4’), 26.7 (CH(CH₃)₂), 24.8 (CH(CH₃)₂), 22.7 (C-3’). ESI-HRMS: m/z calcd. for C₈₀H₉₄N₁₂NaO₁₅S [M+Na]⁺ 1517.6580; found, 1517.6512.