Design and syntheses of some iminosugar derivatives as potential immunosuppressants

Guo-Liang Zhang; Xiu-Jing Zheng; Li-He Zhang; Xin-Shan Ye

doi:10.1039/C1MD00098E

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DOI: 10.1039/C1MD00098E (Concise Article) Med. Chem. Commun., 2011, 2, 909-917

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Design and syntheses of some iminosugar derivatives as potential immunosuppressants†

Guo-Liang Zhang , Xiu-Jing Zheng , Li-He Zhang and Xin-Shan Ye *
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, and School of Pharmaceutical Sciences, Peking University, Xue Yuan Road No. 38, Beijing, 100191, China. E-mail: xinshan@bjmu.edu.cn; Fax: +86 10 82802724; Tel: +86 10 82805736

Received 8th April 2011 , Accepted 10th June 2011

First published on 8th July 2011

Abstract

Eleven new iminosugar derivatives were synthesized and the effects of these synthetic iminosugars on proliferation of the mouse splenocytes were evaluated by the cell counting kit-8 (CCK-8) assay. The preliminary structure–activity relationships were deduced from the experimental data. It was found that iminosugar 14 (IC₅₀ = 22 μM) and its C-5 epimer 15 (IC₅₀ = 45 μM) displayed the strongest inhibitory effects, and might hold potential as immunosuppressive agents.

Introduction

Iminosugars, in which the ring oxygen has been replaced by nitrogen,^1,2 were firstly isolated from Streptomyces roseochromogenes R-468 in 1965,³ and since then a large number of iminosugars have been isolated or synthesized.⁴ It was found that iminosugars hold great potential as therapeutic agents in many diseases such as cancer,^5,6 diabetes,^7,8 viral infections⁹ (including HIV,¹⁰ hepatitis B,¹¹ and C virus¹²), and lysosomal storage disorders^13,14 (including Gaucher's disease¹⁵ and Fabry disease¹⁶). Therefore, iminosugars have attracted much attention from both synthetic and biological chemists.¹⁷ However, the inhibition effects of iminosugars on immune system responses and these compounds as immunosuppressive agents have been less explored. Our previous results disclosed that some synthetic iminosugars (i.e. compounds 1–5 in Fig. 1) exhibited immunosuppressive activities.^18–20 Encouraged by these works, and to further understand the structure–activity relationships of this type of compound and to find more potent immunosuppressants, we report herein the syntheses of iminosugar derivatives 6–16 (Fig. 2) and the evaluation of their immunosuppressive activities by the CCK-8 assay.


	Fig. 1 Structures of iminosugars 1–5.


	Fig. 2 Structures of designed iminosugar derivatives 6–16.

Since it was shown that extending the N-side chain may improve the immunosuppressive activity,²⁰ compounds 6–9 were designed to check the inhibitory effect resulting from the carbon chain extension at the C-1 position. Our previous results disclosed that the configuration of the C-1 substituents had little effect on the secretion of IL-4.¹⁸ In addition, β-C-glycosides were easier to prepare,²¹ so compound 10 was designed to investigate whether the 2-OH group in the ring is necessary for the inhibition. On the other hand, it is well-known that one of the current clinically used small-molecule immunosuppressive agents, cyclosporin A (CSA), is made up of eleven hydrophobic amino acids.²² To increase the hydrophobic property of the target molecules, one chlorine atom or cyano group was introduced, thus compounds 11–12 were designed. Considering that the existence of an N-side chain can enhance both the hydrophobic property and the immunosuppressive activity, compounds 13–15 were designed. Compound 16, as the isomer of 4,¹⁸ was designed to evaluate how the configuration of the C-1 substituents can influence the biological activity.

Results and discussion

Chemistry

The preparation of iminosugar derivatives 6 and 8 is shown in Scheme 1. Starting from the aldehyde 17,^18,23 a Wittig reaction²⁴ was performed to provide compound 19 as a chromatographically inseparable mixture of Z- and E- isomers in excellent yield (92%). The double bond in 19 was selectively reduced by 4% NiCl₂·6H₂O solution in methanol in the presence of NaBH₄,²⁵ affording the saturated ester 21. The subsequent reduction of compound 21 by LiAlH₄ produced the alcohol 23 in high yield (86%). In a similar way, alcohol 24 was prepared from the corresponding 5-epimer 18.^18,23 It is noteworthy that the resolution of ¹H NMR spectra of compounds 20–24 was rather low at room temperature, however this problem was successfully solved by increasing the temperature of NMR experiments to 60 °C. Finally, the full deprotection of 23 under hydrogen atmosphere over Pd/C provided the target compound 6 in 94% yield, whereas compound 8 was obtained in 84% yield using BCl₃ solution.²⁶


	Scheme 1 Reagents and conditions: a) Ph₃PCHCOOMe, THF; b) NiCl₂·6H₂O, NaBH₄, CH₂Cl₂, MeOH, 0 °C; c) LiAlH₄, THF; d) Pd/C, H₂, MeOH, 1.25 M HCl; e) BCl₃, CH₂Cl₂, 0 °C.

The preparation of iminosugars 7 and 9 is shown in Scheme 2. The methyl ester 21 was treated with NaOH aqueous solution at 60 °C, which was followed by catalytic hydrogenolysis to yield the target compound 7. In the same way, iminosugar 9 was prepared smoothly from the corresponding 5-epimer 22.


	Scheme 2 Reagents and conditions: a) 1 N NaOH, MeOH, 60 °C; b) Pd/C, H₂, MeOH, 1.25 M HCl.

The preparation of iminosugar 10 also started from the aldehyde 17. As shown in Scheme 3, the base-promoted elimination reaction of compound 17 was carried out. Sodium hydride (NaH) was used as the base. It was found that the amount of NaH was very important to the reaction (Table 1). The amount of NaH should be no more than 0.5 equivalent, otherwise no desired product 25 is obtained. When t-BuOK was used as the base, the yield was low (<50%). The structure of 25 was unambiguously identified by its 1D (¹H, ¹³C, DEPT)- and 2D (COSY, HMBC)- NMR analyses.


	Scheme 3 Reagents and conditions: a) NaH, THF, 30 °C; b) NaBH₄, MeOH, 0 °C; c) Pd/C, MeOH, 1.0 M HCl.

Table 1 The formation of 25via the elimination reaction of 17

Entry	Base	Product	Yield
1	NaH (4.0 eq)	25	0
2	NaH (2.0 eq)	25	0
3	NaH (1.5 eq)	25	0
4	NaH (0.5 eq)	25	83%
5	t-BuOK (1.0 eq)	25	<50%

With compound 25 in hand, the next step was the reduction reaction. Various reducing reagents or solvents were screened (Table 2). The desired product 26 was obtained in 60% isolated yield when NaBH₄ was used as the reductant (entry 5). When NaBH₄ (1.5 equivalents), which was firstly dissolved in MeOH, was added into the flask dropwise, the yield was further improved (74%, entry 6). Interestingly, when LiAlH₄ was used as the reductant, by-product 27 (Fig. 3) was collected (entry 1). Again, the final deprotection of 26viacatalytic hydrogenolysis provided the target compound 10. The structure of 10 was confirmed by its 1D (¹H, ¹³C)- and 2D (COSY, HSQC, NOESY)- NMR analyses.

Table 2 The reduction reaction of unsaturated aldehyde 25

Entry	Reductant	Solvent	product (yield)
a NaBH₄ dissolved in MeOH was added into the reaction mixture dropwise.
1	LiAlH₄ (2.0 eq)	THF	27 (70%)
2	NaBH₄ (4.0 or 2.0 eq)	MeOH	26 (45%), 27 (13%)
3	CaCl₂ (2.0 eq), NaBH₄ (4.0 eq)	MeOH	26 (42%), 27 (13%)
4	CaCl₂ (2.0 eq), NaBH₄ (4.0 eq)	THF-EtOH	26 (62%)
5	NaBH₄ (1.1 eq)	MeOH	26 (60%)
6	NaBH₄ (1.5 eq)^a	MeOH	26 (74%)


	Fig. 3 The structure of by-product 27.

The preparation of iminosugar derivative 11 is shown in Scheme 4. The starting material 28 was obtained according to the procedure described previously.²⁰Alcohol 28 was converted to tosylate 29 in satisfactory yield (90%). Treatment of 29 with TBACN afforded compound 30 in moderate yield (63%), and at the same time by-product 31 (Fig. 4) was detected. The structure of 31 was identified based on the analysis of its 1D (¹H, ¹³C)- and 2D (COSY, HSQC, HMBC)- NMR spectra. As displayed in Fig. 4, the nitrogen atom and the cyano group would competitively attack the carbon to undergo substitution reaction. When the reaction was performed at lower than 60 °C, compound 30 was the major product (path I). Otherwise, compound 31 became the dominant product (path II). Finally, compound 30 was deprotected using BCl₃ thus affording the target compound 11.


	Scheme 4 Reagents and conditions: a) TsCl, Et₃N, DMAP; b) TBACN, toluene, 50 °C; c) BCl₃, CH₂Cl₂, 0 °C. DMAP = N,N-4-dimethylaminopyridine, TBACN = tetra-n-butylammonium cyanide.


	Fig. 4 The formation of compounds 30 and 31.

The preparation of iminosugar derivatives 12–15 is shown in Scheme 5. After treatment of 29 with DBU, aziridine 31 was obtained in 82% isolated yield. Compound 31 was subjected to de-benzylation and aziridine ring opening in the presence of BCl₃ affording the target molecule 12 in high yield. The N-alkyl substituted iminosugar derivatives 32, 33, and 36 were obtained according to the procedure described previously.²⁰ Compound 32 was reacted with MsCl in the presence of Et₃N and DMAP, providing 34 in good yield (94%). Full deprotection of 34 under hydrogen atmosphere over Pd–C provided the target compound 13 in 78% isolated yield. In the same manner, iminosugars 14 and 15 were prepared starting from 33 and 36, respectively.


	Scheme 5 Reagents and conditions: a) DBU, toluene, 80 °C; b) BCl₃, CH₂Cl₂, 0 °C; c) MsCl, Et₃N, DMAP; d) Pd/C, H₂, MeOH, 4 N HCl. DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene.

The preparation of iminosugar derivative 16 is shown in Scheme 6. The alcohol 38¹⁸ was treated with TEMPO/BAIB to give the crude aldehyde 39. Oxidation of 39 with sodium chlorite provided the acid 40 in 75% isolated yield. Full deprotection of 40 under hydrogen atmosphere over Pd/C led to the target compound 16 in quantitative yield.


	Scheme 6 Reagents and conditions: a) TEMPO, BAIB, CH₂Cl₂, 40 °C; b) NaH₂PO₄, 30% H₂O₂, NaClO₂, CH₃CN; c) Pd/C, THF:H₂O:HOAc = 4:2:1. TEMPO = 2,2,6,6-tetramethyl-1-piperidinyloxy free radical, BAIB = (diacetoxyiodo)benzene

Biological assay

The synthetic compounds 6–16 and 1–5 were evaluated in vitro for the effects on concanavalin A (Con A)-induced proliferation of splenocytes in mouse by the cell counting kit-8 (CCK-8) assay.²⁰ The splenocytes were induced by 2.5 μg mL⁻¹ of concanavalin A with 30 μM concentration of the iminosugars at 37 °C, 5% CO₂ for 48 h, using the Con A-treated splenocytes as the experimental control and cyclosporin A (CSA) as the positive control. Compared with the control, the levels of cell proliferation were reduced by 7.8%, 7.2%, 9.7%, no inhibition, 25.5%, 14.6%, 4.9%, 19.3%, 6.4%, 22.3%, 27.9%, 20.8%, 38.9%, 54.1%, 43.6%, and 21.2% when including 30 μM of compounds 1–16, respectively (Fig. 5). Among these compounds, iminosugar 14 (IC₅₀ = 22 μM) and its C-5 epimer 15 (IC₅₀ = 45 μM) displayed the strongest inhibition effects (Fig. 6). It is noteworthy that the immunosuppressive effects of 14 and 15 were increased by 6.9 and 4.5 folds (comparing compounds 14 with 1, 15 with 3). Comparing 6 (14.6%) with 1 (7.8%) and 8 (19.3%) with 3 (9.7%), it seemed that extending the carbon atom number of the terminal group can increase the inhibitory activity. Based on the fact that compound 10 (22.3%) exhibited better inhibition than compound 1 (7.8%), it is clear that a 2-OH in the ring may be not necessary for the inhibition. Comparing 11 (27.9%) with 1 (7.8%), it can be suggested that introducing the cyano group can improve the activity because of the polarity decrease of the compound. Comparing 12 (20.8%) with 1 (7.8%) and 13 (38.9%) with 5 (25.5%), it seemed that bringing one chlorine atom into the structure can increase the inhibition. This suggests that hydrophobic character may be important to the immunosuppressive activity. In addition, the configuration of the C-1 terminal group may have some effects on biological activity based on the inhibition data of compound 16 (21.2%) and 4 (no inhibition at 30 μM). Comparing 14 (54.1%) and 13 (38.9%) with 12 (20.8%), it was found that the N-alkylation is important for the improvement of the inhibitory activity, which agrees with the result previously reported by our group.²⁰ The detailed immunosuppressive mechanisms of these synthetic iminosugars are not well-known.


	Fig. 5 Effects of compounds 1–16 on concanavalin A induced mouse splenocytes proliferation were assessed by the CCK-8 assay. Concentration of CSA was 1μM and concentration of 1–16 was 30 μM. Values are mean ± SEM.


	Fig. 6 Inhibition of 14 and 15 for the cell proliferation. I = inhibition, C = concentration of inhibitor.

The cytotoxicity of compounds 14 and 15 was also assayed by the CCK-8 method using the cells without the Con A-induced proliferation. The experimental data showed that compounds 14 and 15 were low toxic when their concentration was below 110 μM (Fig. 7).


	Fig. 7 Influence on cell viability of 14 and 15 at different concentrations.

Conclusion

In summary, to gather more information on the structure–activity relationships and to search for more potent immunosuppressants, we designed and synthesized 11 new iminosugar derivatives in high yield using concise synthetic routes. All synthetic compounds were evaluated in vitro for the immunosuppressive activities. The experimental data demonstrated that iminosugar 14 (IC₅₀ = 22 μM) and its C-5 epimer 15 (IC₅₀ = 45 μM) displayed the strongest inhibition effects. The preliminary structure–activity relationships were discussed. It was found that the immunosuppressive activities of iminosugars can be improved by decreasing the polarity or increasing the hydrophobicity. In addition, it was disclosed that the 2-OH group in the iminosugar ring may be not necessary for the inhibition. These results may benefit the discovery of new iminosugar derivatives as potent immunosuppressive agents.

Experimental

General

All chemicals were purchased and used without further purification. Tetrahydrofuran (THF) was distilled over sodium/benzophenone, methylene chloride (CH₂Cl₂) over calcium hydride. Reactions were monitored with analytical TLC on silica gel 60-F₂₅₄ precoated aluminium plates and visualized under UV (254 nm) and/or by staining with acidic ceric ammonium molybdate. Column chromatography was performed on silica gel (35–75 μm). NMR spectra were recorded on a Varian VXR-300M or Varian INOVA-500M spectrometer. Mass spectra were recorded using a PE SCLEX QSTAR spectrometer. Elemental analysis data were recorded on PE-2400C elemental analyzer. Concanavalin A and cyclosporin A were purchased from Sigma. Other reagents were from commercial sources.

(2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitolyl)methyl acrylate (19)

To a solution of 17 (170 mg, 0.25 mmol) in dry THF (5.0 mL) was added Ph₃P = CHCOOMe (165 mg, 0.50 mmol). The reaction mixture was stirred overnight at room temperature. The reaction was quenched by NH₄Cl aqueous solution, and the mixture was extracted with EtOAc (3 × 50 mL) and washed with saturated NaHCO₃ aqueous solution (20 mL) and brine (20 mL). The organic phases were combined, dried (Na₂SO₄), filtered, and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 15 [thin space (1/6-em)]

1 to 13

1) to provide 19 (170 mg, 92%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃): δ 3.47 (dd, J = 3.0, 9.0 Hz, 1H), 3.73 (s, 3H), 3.80–3.88 (m, 1H), 4.00–4.07 (m, 3H), 4.19–4.57 (m, 4H), 4.59–4.65 (m, 1H), 4.68 (d, J = 11.5 Hz, 2H), 4.72 (d, J = 12.0 Hz, 2H), 4.78 (d, J = 11.5 Hz, 1H), 4.89 (br.s, 1H), 5.08 (d, J = 7.0 Hz, 1H), 5.20 (d, J = 2.0 Hz, 1H), 5.98–6.03 (m, 1H), 7.18–7.34 (m, 25H). MS-ESI: 742 [M + H⁺]. Anal. Calcd for C₄₆H₄₇NO₈: C, 74.47; H, 6.39; N, 1.89; Found: C, 74.52; H, 6.35; N, 1.88%

2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-L-galacto-heptitol-1-methyl acrylate (20)

Compound 20 was prepared from 18 (84 mg, 0.12 mmol) as described in the preparation of 19, providing 20 (88 mg, 96%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃): δ 3.30 (t, J = 9.0 Hz, 1H), 3.37 (br.s, 1H), 3.70–3.73 (m, 4H), 3.92 (br.s, 1H), 4.26 (dd, J = 6.5, 10.0 Hz, 2H), 4.31–4.37 (m, 2H), 4.52 (d, J = 12.0 Hz, 1H), 4.57–4.67 (m, 4H), 4.73 (d, J = 11.5 Hz, 2H), 5.09 (d, J = 12.0 Hz, 1H), 5.16 (d, J = 12.5 Hz, 1H), 5.36 (br.s, 1H), 6.04 (br.s, 1H), 7.15–7.45 (m, 25H). MS-ESI: 742 [M + H⁺]. Anal. Calcd for C₄₆H₄₇NO₈: C, 74.47; H, 6.39; N, 1.89; Found: C, 74.70; H, 6.56; N, 1.64%

(2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-D- galacto-heptitolyl)methyl propionate (21)

To a solution of 19 (170 mg, 0.23 mmol) in dry CH₂Cl₂ (1.3 mL) and MeOH (8.0 mL) was added 4% solution of NiCl₂·6H₂O in MeOH (2.3 mL, 0.39 mmol) at 0 °C. After stirring for 20 min, a portion of NaBH₄ (35 mg, 0.92 mmol) was added. The reaction mixture was stirred for another five hours at room temperature. The reaction was quenched by HOAc, then the solvent was removed and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 18 [thin space (1/6-em)]

1 to 16

1) to provide 21 (145 mg, 85%) as a yellow oil. ¹H NMR (400 MHz, d₆-DMSO, 70 °C): δ 1.97–2.00 (m, 2H), 2.24–2.27 (m, 2H), 3.52 (s, 3H), 3.63 (br.s, 1H), 3.73–3.82 (m, 2H), 3.91 (br.s, 2H), 4.01 (br.s, 1H), 4.36 (d, J = 12.0 Hz, 2H), 4.42 (d, J = 12.4 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 12.4 Hz, 1H), 4.61 (d, J = 12.0 Hz, 1H), 4.65 (d, J = 12.0 Hz, 2H), 4.69 (d, J = 8.3 Hz, 1H), 4.86 (d, J = 12.8 Hz, 1H), 5.01 (d, J = 12.4 Hz, 1H), 7.20–7.35 (m, 25H). ¹³C NMR (125 MHz, d₆-DMSO): δ 18.99, 30.19, 51.17, 53.85, 54.44, 66.48, 67.70, 71.67, 71.88, 71.95, 73.42, 74.29, 76.20, 79.68, 127.28, 127.36, 127.43, 127.69, 128.02, 128.17, 128.24, 129.12, 129.45, 134.54, 136.68, 138.26, 138.48, 138.87, 154.96, 157.00, 173.03. MS-ESI: 766 [M + Na⁺]. Anal. Calcd for C₄₆H₄₉NO₈: C, 74.27; H, 6.64; N, 1.88; Found: C, 74.53; H, 6.87; N, 1.79%

2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-L-galacto-heptitol-1-methyl propionate (22)

Compound 22 was prepared from 20 (125 mg, 0.17 mmol) as described in the preparation of 21, providing 22 (100 mg, 80%) as a yellow oil. ¹H NMR (500 MHz, d₆-DMSO, 70 °C): δ 1.55–1.63 (m, 1H), 2.12–2.20 (m, 1H), 2.23–2.34 (m, 2H), 3.50–3.53 (m, 4H), 3.57–3.63 (m, 1H), 3.89 (dd, J = 3.0, 10.0 Hz, 1H), 3.94 (t, J = 2.5 Hz, 1H), 4.02 (dd, J = 6.5, 10.0 Hz, 1H), 4.44 (s, 2H), 4.55 (s, 3H), 4.58 (d, J = 12.0 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.64 (d, J = 11.5 Hz, 1H), 4.68 (d, J = 12.0 Hz, 1H), 4.73 (br.s, 1H), 5.07 (ABq, J = 12.5 Hz, 2H), 7.21–7.34 (m, 25H). ¹³C NMR (125 MHz, DMSO): δ 23.60, 23.95, 30.12, 51.13, 52.34,54.05, 54.49, 66.76, 69.27, 70.96, 71.29, 71.51, 71.76, 71.93, 73.62, 73.86, 75.02, 127.23, 127.33, 127.36, 127.42, 127.53, 127.76, 128.09, 128.13, 128.15, 128.21, 128.32, 136.68, 137.97, 138.32, 138.56, 138.85, 156.06, 173.05. MS-ESI: 766 [M + Na⁺]. Anal. Calcd for C₄₆H₄₉NO₈: C, 74.27; H, 6.64; N, 1.88; Found: C, 74.02; H, 6.64; N, 1.86%

2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-propanol (23)

To a solution of 21 (46 mg, 0.06 mmol) in dry THF (3.5 mL) was added LiAlH₄ (4.70 mg, 0.12 mmol) at −15 °C. The reaction mixture was stirred overnight at room temperature. Ethyl acetate (1.0 mL) was added dropwise to decompose the excess LiAlH₄, followed by addition of 2 N NaOH (1.5 mL). The suspension was filtered through a bed of Celite. The filtrate was extracted with EtOAc (3 × 30 mL) and washed with saturated NaHCO₃ aqueous solution (10 mL) and brine (10 mL). The organic phases were combined, dried (Na₂SO₄), filtered, and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 6 [thin space (1/6-em)]

1 to 4

1) to provide 23 (38 mg, 86%) as a yellow oil. ¹H NMR (500 MHz, d₆-DMSO, 70 °C): δ 1.29–1.37 (m, 1H), 1.38–1.46 (m, 1H), 1.72–1.75 (m, 2H), 3.38 (dd, J = 6.0, 11.5 Hz, 2H), 3.60 (br.s, 1H), 3.72 (dd, J = 3.0, 8.5Hz, 1H), 3.80 (dd, J = 5.0, 8.5 Hz, 1H), 3.92–3.96 (m, 2H), 4.02 (br.s, 1H), 4.12 (t, J = 5.0 Hz, 1H), 4.36 (d, J = 12.0 Hz, 1H), 4.42 (d, J = 12.0 Hz, 1H), 4.47 (d, J = 12.0 Hz, 2H), 4.58 (s, 2H), 4.64–4.71 (m, 3H), 4.87 (d, J = 13.0 Hz, 1H), 5.01 (d, J = 12.5 Hz, 1H), 7.20–7.33 (m, 25H). ¹³C NMR (125 MHz, DMSO): δ 19.82, 29.10, 54.15, 60.28, 65.82, 66.64, 67.89, 71.72, 71.88, 71.96, 73.46, 74.41, 76.41, 79.79, 127.29, 127.33, 127.47, 127.76, 128.01, 128.12, 128.17, 128.21, 128.25, 136.91, 138.18, 138.58, 138.89, 156.85. MS-ESI: 738 [M + Na⁺]. Anal. Calcd for C₄₅H₄₉NO₇: C, 75.50; H, 6.90; N, 1.96; Found: C, 75.46; H, 6.93; N, 1.85%

2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-L-galacto-heptitol-1-propanol (24)

Compound 24 was prepared from 22 (54 mg, 0.07 mmol) as described in the preparation of 23, providing 24 (25 mg, 79% based on the recovery of starting material) as a yellow oil. The material 22 (21 mg) was recovered. ¹H NMR (500 MHz, d₆-DMSO, 60 °C): δ 1.24–1.33 (m, 1H), 1.39–1.41 (m, 2H), 1.84–1.91 (m, 1H), 3.34 (br.s, 2H), 3.50 (dd, J = 5.5, 9.5 Hz, 1H), 3.61 (t, J = 8.5 Hz, 1H), 3.86 (dd, J = 3.0, 10.0 Hz, 1H), 3.94–3.95 (m, 1H), 3.99 (dd, J = 6.5, 10.5 Hz, 1H), 4.14–4.16 (m, 1H), 4.41 (d, J = 12.0 Hz, 1H), 4.46 (d, J = 12.5 Hz, 1H), 4.52–4.70 (m, 8H), 5.08 (ABq, J = 13.0 Hz, 2H), 7.25–7.33 (m, 25H). ¹³C NMR (125 MHz, DMSO): δ 25.35, 29.51, 53.08, 53.44, 54.60, 60.63, 66.69, 69.24, 70.95, 71.22, 71.49, 71.90, 73.54, 73.88, 75.12, 75.23, 127.22, 127.26, 127.33, 127.45, 127.56, 127.77, 127.95, 128.10, 128.13, 128.22, 128.34, 136.76, 138.02, 138.32, 138.66, 138.82, 155.80. MS-ESI: 738 [M + Na⁺]. Anal. Calcd for C₄₅H₄₉NO₇: C, 75.50; H, 6.90; N, 1.96; Found: C, 75.50; H, 6.97; N, 1.83%

1,5-Dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-propanol (6)

A mixture of 23 (80 mg, 0.11 mmol) and 10% Pd/C (10.0 mg) in MeOH (2.0 mL) and 1.25 M HCl solution was stirred for 72 h under H₂ atmosphere. The solid was removed by filtration through Celite and the filtrate was concentrated. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH 3 [thin space (1/6-em)]

1 to 1

3) and C-18 reversed-phase column chromatography (H₂O as eluent), giving 6 (23 mg, 94%) as a white solid in the form of hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 1.66–1.79 (m, 3H), 1.96–2.09 (m, 1H), 3.57–3.59 (m, 1H), 3.63–3.72 (m, 3H), 3.90 (dd, J = 2.5, 8.5Hz, 1H), 3.93 (d, J = 6.5 Hz, 2H), 4.14 (dd, J = 4.5, 8.0 Hz, 1H), 4.21(s, 1H). ¹³C NMR (75 MHz, D₂O): δ 24.21, 31.04, 56.98, 58.08, 60.87, 63.73, 68.57, 69.70, 71.65. HRMS (ESI, positive) Calcd for [(M + H) ⁺] C₉H₂₀NO₅ 222.1336; Found: 222.1331

1,5-Dideoxy-1,5-imino-D-glycero-L-galacto-heptitol-1-propanol (8)

To a solution of 24 (21 mg, 0.03 mmol) in dry CH₂Cl₂ (3.0 mL) was added BCl₃ (1 M solution in hexane, 0.45 mL, 0.45 mmol). The reaction mixture was stirred for 5 h at 0 °C, and then the solids formed were dissolved by addition of MeOH (1.0 mL) and water (1.0 mL). The solvent was removed and the residue was purified by flash chromatography on C18-reversed phase silica gel (H₂O as eluent), thus providing 8 (6.2 mg, 82%) as a yellow solid in the form of hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 1.55–1.62 (m, 1H), 1.64–1.79 (m, 2H), 1.81–1.92 (m, 1H), 3.37–3.41 (m, 1H), 3.51 (dd, J = 5.5, 9.5 Hz, 1H), 3.59–3.68 (m, 2H), 3.85 (dd, J = 6.5, 13.0 Hz, 1H), 3.96 (dd, J = 2.5, 12.5 Hz, 1H), 4.04 (dd, J = 2.5, 11.0 Hz, 1H), 4.07–4.10 (m, 2H). ¹³C NMR (125 MHz, D₂O): δ 24.89, 27.82, 55.19, 56.79, 58.99, 61.74, 63.90, 67.88, 69.72. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₉H₂₀NO₅ 222.1336; Found: 222.1333.

1,5-Dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-propionic acid (7)

To a solution of 21 (55 mg, 0.07 mmol) in MeOH (2.0 mL) was added 1M NaOH aqueous solution (0.28 mL, 0.28 mmol). After stirring for five hours at 60 °C, a portion of cation exchange resin was added until the pH value remained between 5 and 6. The solvent was removed and the residue was purified by simple column chromatography on silica gel (petroleum ether/EtOAc 3 [thin space (1/6-em)]

1 to 1

1) to provide the crude product (51 mg). The crude product was treated under the conditions as described in the preparation of 6, yielding 7 (16 mg, 80%, two steps) as a white solid in the form of a hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 1.81–1.92 (m, 1H), 1.99–2.03 (m, 1H), 2.33–2.43 (m, 2H), 3.43–3.47 (m, 2H), 3.79–3.84 (m, 3H), 4.05–4.08 (m, 1H), 4.13 (d, J = 3.0 Hz, 1H). ¹³C NMR (125 MHz, D₂O): δ 21.15, 35.92, 55.06, 56.12, 60.07, 67.78, 68.40, 70.20, 182.93. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₉H₁₈NO₆ 236.1129; Found: 236.1120.

1,5-Dideoxy-1,5-imino-D-glycero-L-galacto-heptitol-1-propionic acid (9)

Compound 9 was prepared from 22 (90 mg, 0.12 mmol) as described in the preparation of 7, providing 9 (27 mg, 83%) as a white solid in the form of a hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 1.98 (dd, J = 7.5, 14.5 Hz, 2H), 2.34–2.45 (m, 2H), 3.37–3.41 (m, 1H), 3.48 (t, J = 7.0 Hz, 1H), 3.88 (dd, J = 6.5, 13.0 Hz, 1H), 4.00 (d, J = 3.0 Hz, 1H), 4.03 (dd, J = 3.0, 9.0 Hz, 1H), 4.06 (d, J = 3.5 Hz, 1H), 4.13 (t, J = 3.0 Hz, 1H). ¹³C NMR (125 MHz, D₂O): δ 25.24, 34.35, 55.32, 56.74, 59.61, 64.43, 68.86, 70.07, 182.43. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₉H₁₈NO₆ 236.1129; Found: 236.1122.

3,4,6-Tri-O-benzyl-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-galacto-1-ene-heptitol-1-aldehyde (25)

To a solution of 17 (35 mg, 0.05 mmol) in dry THF (3.0 mL) was added a solution of NaH (1.0 mg, 0.03 mmol) in THF (1.0 mL). The reaction mixture was stirred for 3 h at 30 °C. The reaction was quenched by NH₄Cl aqueous solution and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 15 [thin space (1/6-em)]

1 to 14

1) to provide 25 (24 mg, 83%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 3.83–3.86 (m, 2H), 3.87 (t, J = 4.5 Hz, 1H), 4.02 (t, J = 4.5 Hz, 1H), 4.44 (d, J = 12.5 Hz, 1H), 4.56(d, J = 12.0 Hz, 1H), 4.62 (d, J = 11.5 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.65 (d, J = 12.0 Hz, 1H), 4.72 (d, J = 12.0 Hz, 1H), 4.81–4.85 (m, 1H), 5.18 (d, J = 12.0 Hz, 1H), 5.21 (d, J = 12.5 Hz, 1H), 6.03 (d, J = 4.0 Hz, 1H), 7.25–7.34 (m, 20H), 9.33 (s, 1H). ¹³C NMR (125 MHz, CDCl₃): δ 54.50, 65.68, 68.54, 69.29, 71.90, 72.84, 73.00, 77.12, 120.01, 127.51, 127.59, 127.64, 127.80, 127.89, 128.16, 128.29, 128.41, 128.45, 128.59, 135.31, 136.69, 137.54, 137.91, 138.33, 154.10, 186.75. HRMS (ESI, positive) Calcd for [(M + Na)⁺] C₃₆H₃₅NO₆Na 600.2357; Found: 600.2356.

3,4,6-Tri-O-benzyl-N-benzyloxycarbonyl-1,2,5-trideoxy-1,5-imino-D-galacto-1-ene-heptitol-1-methanol (26)

To a solution of 25 (46 mg, 0.08 mmol) in freshly dry MeOH (4.0 mL) was added a solution of NaBH₄ (4.6 mg, 0.12 mmol) in MeOH (0.8 mL) at 0 °C. The reaction mixture was stirred for 2 h at 0 °C. The solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 8 [thin space (1/6-em)]

1 to 7

1) to provide 26 (34 mg, 74%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 3.78 (t, J = 4.5 Hz, 1H), 3.83 (dd, J = 2.5, 11.0 Hz, 1H), 3.89–3.95 (m, 2H), 4.17 (d, J = 5.5 Hz, 2H), 4.40 (d, J = 12.0 Hz, 1H), 4.50 (d, J = 12.0 Hz, 1H), 4.61 (d, J = 11.5 Hz, 3H), 4.73 (d, J = 12.0 Hz, 1H), 4.92–4.96 (m, 1H), 5.15 (d, J = 12.5 Hz, 1H), 5.18 (d, J = 12.5 Hz, 1H), 5.30 (d, J = 4.5 Hz, 1H), 7.22–7.35 (m, 20H). ¹³C NMR (75 MHz, CDCl₃): δ 54.70, 64.41, 66.10, 68.07, 68.39, 71.24, 72.58, 72.76, 76.57, 112.65, 127.44, 127.60, 127.88, 128.23, 128.32, 128.44, 128.54, 135.71, 137.63, 138.22, 138.57, 154.43. MS-ESI: 580 [M + H⁺]. Anal. Calcd for C₃₆H₃₇NO₆: C, 74.59; H, 6.43; N, 2.42; Found: C, 74.67; H, 6.61; N, 2.35%

3,4,6-Tri-O-benzyl-1,2,5-trideoxy-1,5-imino-D-galacto-1-ene-heptitol-N-cyclic carbamate (27)

Compound 27 was prepared from 25 (21 mg, 0.04 mmol) as described in the preparation of 23, providing 27 (12 mg, 70%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 3.85 (br.s, 1H), 4.00 (dd, J = 3.5, 11.0 Hz, 1H), 4.06 (t, J = 4.5 Hz, 1H), 4.11–4.14 (m, 1H), 4.45 (d, J = 12.0 Hz, 1H), 4.55 (br.s, 1H), 4.62 (d, J = 12.0 Hz, 1H), 4.65 (d, J = 12.0 Hz, 1H), 4.67 (d, J = 12.0 Hz, 1H), 4.69–4.82 (m, 5H), 7.24–7.37 (m, 15H). ¹³C NMR (125 MHz, CDCl₃): δ 51.31, 65.84, 68.41, 71.47, 72.18, 72.68, 75.49, 92.10, 127.40, 127.58, 127.71, 127.94, 128.28, 128.35, 128.52, 136.05, 137.52, 138.37, 138.50, 155.64. HRMS (ESI, positive) Calcd for [(M + Na)⁺] C₂₉H₂₉NO₅Na 494.1938; Found: 494.1934.

1,2,5-Trideoxy-1,5-imino-L-glycero-D-galacto-heptitol-1-methanol (10)

A mixture of 26 (24 mg, 0.04 mmol) and 10% Pd/C (5.0 mg) in THF (2.0 mL), H₂O (1.0 mL) and 1.0 M HCl aqueous solution (cat.) was stirred for 72 h under H₂ atmosphere. The solid was removed by filtration through Celite and the filtrate was concentrated. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH 3 [thin space (1/6-em)]

1 to 1

3) and C–18 reversed-phase column chromatography (H₂O as eluent), giving 10 (7.7 mg, 80%) as a white solid in the form of a hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 1.86–1.97 (m, 2H), 3.37–3.42 (m, 2H), 3.72 (dd, J = 6.5, 12.5 Hz, 1H), 3.83 (dd, J = 3.5, 12.5 Hz, 1H), 3.87–3.91 (m, 2H), 3.93–3.97 (m, 1H), 4.10 (s, 1H). ¹³C NMR (75 MHz, D₂O): δ 27.44, 57.41, 59.81, 60.54, 61.78, 66.30, 68.18. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₇H₁₆NO₄ 178.1074; Found: 178.1079.

1-Tosyl-2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol (29)

To a solution of 28 (40 mg, 0.07 mmol) in CH₂Cl₂ (3.0 mL) was added TsCl (21 mg, 0.11 mmol), Et₃N (20 μL, 0.14 mmol) and DMAP (cat.). The reaction mixture was stirred for 12 h at room temperature. The solvent was removed and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 5 [thin space (1/6-em)]

1 to 4

1) to provide 29 (46 mg, 90%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 2.40 (s, 3H), 3.22 (br.s, 1H), 3.41–3.47 (m, 2H), 3.54 (dd, J = 2.5, 7.0 Hz, 1H), 3.69 (br.s, 1H), 3.77 (br.s, 1H), 3.88 (t, J = 3.5 Hz, 1H), 4.03–4.10 (m, 2H), 4.40 (d, J = 12.0 Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H), 4.48 (d, J = 12.0 Hz, 1H), 4.49 (d, J = 11.5 Hz, 1H), 4.53 (d, J = 12.0 Hz, 1H), 4.56 (d, J = 11.0 Hz, 1H), 4.63 (d, J = 12.0 Hz, 2H), 7.15–7.17 (m, 2H), 7.24–7.36 (m, 20H), 7.75 (d, J = 8.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 21.61, 50.38, 53.22, 68.06, 68.43, 72.80, 73.00, 73.14, 73.20, 74.65, 75.68, 127.44, 127.54, 127.64, 127.83, 127.97, 128.35, 129.03, 129.83, 132.68, 137.82, 138.22, 138.38, 138.44, 144.77. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₄₂H₄₆NO₇S 708.2989; Found: 708.2987.

2,3,4,6-Tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-acetonitrile (30)

To a solution of 29 (48 mg, 0.07 mmol) in dry toluene (2.0 mL) was added a solution of TBACN (91 mg, 0.35 mmol) in toluene (2.0 mL). The reaction mixture was stirred for 8 h at 50 °C. The solvent was removed and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 5 [thin space (1/6-em)]

1 to 2

1) to provide 30 (24 mg, 63%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 1.85 (br.s, 1H), 2.43 (dd, J = 9.0, 17.0Hz, 1H), 2.61 (dd, J = 6.0, 17.0 Hz, 1H), 3.18 (br.s, 1H), 3.44–3.48 (m, 1H), 3.52 (dd, J = 5.5, 9.5 Hz, 1H), 3.60 (dd, J = 2.5, 7.5 Hz, 1H), 3.72–3.73 (m, 1H), 3.83 (br.s, 1H), 3.95 (t, J = 3.5 Hz, 1H), 4.50 (s, 2H), 4.55 (d, J = 11.5 Hz, 1H), 4.56 (d, J = 11.5 Hz, 1H), 4.61 (d, J = 11.5 Hz, 1H), 4.63 (d, J = 11.5 Hz, 1H), 4.71 (d, J = 12.0 Hz, 2H), 7.25–7.37 (m, 20H). ¹³C NMR (125 MHz, CDCl₃): δ 18.00, 49.55, 53.29, 67.94, 73.06, 73.29, 73.36, 74.62, 76.48, 77.00, 77.26, 118.47, 127.49, 127.62, 127.68, 127.72, 127.92, 128.00, 128.09, 128.31, 128.37, 128.41, 128.48, 137.74, 138.12, 138.33, 138.38. MS-ESI: 585 [M + Na⁺]. Anal. Calcd for C₃₆H₃₈N₂O₄: C, 76.84; H, 6.81; N, 4.98; Found: C, 76.85; H, 6.84; N, 4.95%

1,5-Dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-acetonitrile (11)

Compound 11 was prepared from 30 (24 mg, 0.04 mmol) as described in the preparation of 8, providing 11 (7.8 mg, 78%) as a yellow semi-solid in the form of hydrochloride salt. ¹H NMR (300 MHz, D₂O): δ 2.89 (dd, J = 6.0, 17.4 Hz, 1H), 3.05 (dd, J = 7.2, 17.4 Hz, 1H), 3.40–3.55 (m, 1H), 3.72–3.80 (m, 3H), 3.99–4.06 (m, 3H). ¹³C NMR (75 MHz, D₂O): δ 17.63, 52.89, 58.99, 60.26, 67.87, 69.02, 71.28, 119.86. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₈H₁₅N₂O₄ 203.1026; Found: 203.1032.

2,3,4,6-Tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-bicyclo[4.1.0] aziridine (31)

To a solution of 29 (29 mg, 0.04 mmol) in dry toluene (3.0 mL) was added DBU (61 μL, 0.40 mmol). The reaction mixture was stirred for 8 h at 80 °C. The solvent was removed and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 4 [thin space (1/6-em)]

1 to 2

1) to provide 31 (18 mg, 82%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 1.54 (d, J = 4.0 Hz, 1H), 2.09 (d, J = 6.0 Hz, 1H), 2.50–2.53 (m, 1H), 2.63–2.66 (m, 1H), 3.33 (dd, J = 1.5, 8.5 Hz, 1H), 3.68 (dd, J = 5.5, 9.0 Hz, 1H), 3.72 (t, J = 8.5 Hz, 1H), 4.00 (t, J = 1.5 Hz, 1H), 4.44 (d, J = 11.5 Hz, 1H), 4.49 (d, J = 11.5 Hz, 1H), 4.49–4.51 (m, 1H), 4.59 (d, J = 12.0 Hz, 1H), 4.68 (s, 2H), 4.74 (d, J = 11.5 Hz, 1H), 4.78 (d, J = 11.5 Hz, 1H), 4.89 (d, J = 11.5 Hz, 1H), 7.23–7.37 (m, 20H). ¹³C NMR (125 MHz, CDCl₃): δ 34.88, 35.78, 64.45, 70.92, 72.29, 72.52, 73.38, 74.17, 74.71, 76.93, 81.44, 127.36, 127.48, 127.57, 127.67, 127.76, 127.96, 128.14, 128.33, 138.07, 138.54, 138.70, 138.88. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₃₆H₃₉N₂O₄ 536.2795; Found:536.2809.

1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-chloromethane (12)

Compound 12 was prepared from 31 (35 mg, 0.07 mmol) as described in the preparation of 8, providing 12 (14 mg, 93%) as a yellow solid in the form of a hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 3.63–3.66 (m, 1H), 3.87 (dd, J = 3.5, 8.5Hz, 1H), 3.94–4.04 (m, 4H), 4.11 (dd, J = 4.0, 12.0 Hz, 1H), 4.22–4.25 (m, 2H). ¹³C NMR (75 MHz, D₂O): δ 39.03, 56.31, 56.53, 58.66, 66.43, 67.20, 69.69. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₇H₁₅NO₄Cl 212.0684; Found: 212.0686.

2,3,4,6-Tetra-O-benzyl-N-hexyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-chloromethane (34)

To a solution of 32 (30 mg, 0.05 mmol) in CH₂Cl₂ (3.0 mL) was added MsCl (7.3 μL, 0.10 mmol), Et₃N (13 μL, 0.10 mmol) and DMAP (cat.). The reaction mixture was stirred for 12 h at room temperature. The solvent was removed and the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 40 [thin space (1/6-em)]

1 to 30

1) to provide 34 (29 mg, 94%) as a colorless oil. ¹H NMR (500 MHz, C₆D₅N): δ 0.82 (t, J = 6.5 Hz, 3H), 1.12–1.62 (m, 8H), 2.79–2.91 (m, 2H), 3.52 (br.s, 1H), 3.67–3.70 (m, 1H), 3.95–4.11 (m, 4H), 4.39 (s, 2H), 4.57 (d, J = 12.0 Hz, 1H), 4.61 (d, J = 11.5 Hz, 1H), 4.73 (d, J = 12.0 Hz, 2H), 4.80 (d, J = 12.0 Hz, 1H), 4.85 (d, J = 12.0 Hz, 2H), 4.92 (s, 1H), 5.03 (br.s, 1H), 7.18–7.55 (m, 20H). ¹³C NMR (75 MHz, CDCl₃): δ 14.13, 22.70, 26.83, 29.74, 31.83, 41.68, 50.27, 57.95, 68.21, 72.70, 73.14, 73.26, 74.60, 75.42, 77.19, 78.18, 127.36, 127.50, 127.63, 127.72, 128.20, 128.30, 138.47, 138.71, 139.09. MS-ESI: 656 [M + H⁺], 658 [M + 2 + H⁺]. Anal. Calcd for C₄₁H₅₀ClNO₄: C, 75.03; H, 7.68; N, 2.13; Found: C, 75.13; H, 7.66; N, 2.07%

2,3,4,6-Tetra-O-benzyl-N-nonyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-chloromethane (35)

Compound 35 was prepared from 33 (38 mg, 0.06 mmol) as described in the preparation of 34, providing 35 (35 mg, 89%) as a colorless oil. ¹H NMR (300 MHz, C₆D₅N): δ 0.74 (t, J = 6.9 Hz, 3H), 1.12–1.35 (m, 14H), 2.68–2.84 (m, 2H), 3.42 (br.s, 1H), 3.54–3.65 (m, 1H), 3.85–4.00 (m, 4H), 4.29 (br.s, 2H), 4.46 (d, J = 11.7 Hz, 1H), 4.52 (d, J = 11.7 Hz, 1H), 4.61–4.77 (m, 5H), 4.82 (s, 1H), 4.91 (br.s, 1H), 7.17–7.44 (m, 20H). ¹³C NMR (75 MHz, C₆D₅N): δ 13.98, 22.63, 27.07, 29.31, 29.66, 29.69, 30.24, 31.80, 42.19, 50.03, 56.96, 60.94, 68.52, 70.94, 72.53, 72.90, 73.16, 75.71, 80.03, 127.60, 127.83, 128.41, 128.46, 139.09, 139.30, 139.62. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₄₄H₅₇ClNO₄ 698.3971; Found: 698.3979.

N-Hexyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-chloromethane (13)

A mixture of 34 (49 mg, 0.07 mmol) and 10% Pd/C (10.0 mg) in MeOH (4.0 mL) and 4.0 M HCl aqueous solution (cat.) was stirred for 72 h under H₂ atmosphere. The solid was removed by filtration through Celite and the filtrate was concentrated. The residue was purified by C-18 reversed-phase column chromatography (H₂O as eluent), giving 13 (20 mg, 78%) as a white solid in the form of a hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 0.88 (t, J = 7.0 Hz, 3H), 1.27–1.42 (m, 6H), 1.82 (br.s, 2H), 3.49–3.52 (m, 2H), 3.83–4.17 (m, 7H), 4.32 (br.s, 2H). ¹³C NMR (100 MHz, D₂O): δ 15.97, 24.46, 27.66, 27.90, 33.15, 37.74, 40.84, 54.11, 59.27, 64.52, 68.31, 71.55. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₁₃H₂₇ClNO₄ 296.1623; Found: 296.1624.

N-Nonyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitol-1-chloromethane (14)

Compound 14 was prepared from 35 (24 mg, 0.03 mmol) as described in the preparation of 13, providing 14 (10 mg, 77%) as a white solid in the form of a hydrochloride salt. ¹H NMR (500 MHz, CD₃OD): δ 0.89 (t, J = 7.0 Hz, 3H), 1.23–1.48 (m, 14H), 2.71–2.78 (m, 2H), 2.89 (s, 1H), 3.31–3.35 (m, 1H), 3.51–3.52 (m, 1H), 3.71 (dd, J = 3.0, 11.0 Hz, 1H), 3.80–3.84 (m, 3H), 3.99–4.06 (m, 2H). ¹³C NMR (75 MHz, CD₃OD): δ 14.44, 23.73, 28.08, 30.31, 30.42, 30.66, 30.70, 33.06, 41.20, 50.89, 60.76, 61.99, 69.36, 71.52, 73.04. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₁₆H₃₃ClNO₄ 338.2093; Found: 338.2097.

2,3,4,6-Tetra-O-benzyl-N-nonyl-1,5-dideoxy-1,5-imino-D-glycero-L-galacto-heptitol-1-chloromethane (37)

Compound 37 was prepared from 36 (20 mg, 0.03 mmol) as described in the preparation of 34, providing 37 (16 mg, 80%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃): δ 0.88 (t, J = 7.0 Hz, 3H), 1.09–1.40 (m, 14H), 2.63–2.69 (m, 1H), 2.79–2.86 (m, 1H), 3.07 (td, J = 3.5, 9.0 Hz, 1H), 3.15–3.19 (m, 1H), 3.54–3.58 (m, 2H), 3.64 (dd, J = 3.5, 10.5 Hz, 1H), 3.66–3.74 (m, 2H), 3.78 (dd, J = 3.0, 9.0 Hz, 1H), 3.90 (dd, J = 2.5, 5.0 Hz, 1H), 4.31 (d, J = 12.0 Hz, 1H), 4.35 (d, J = 12.0 Hz, 1H), 4.38 (d, J = 12.0 Hz, 1H), 4.43 (d, J = 12.5 Hz, 1H), 4.47 (d, J = 2.5 Hz, 2H), 4.51 (d, J = 12.0 Hz, 1H), 4.53 (d, J = 12.5 Hz, 1H), 7.21–7.32 (m, 20H). ¹³C NMR (75 MHz, CDCl₃): δ 14.12, 22.67, 23.92, 27.21, 29.25, 29.53, 29.66, 31.89, 43.10, 49.52, 58.64, 59.30, 67.73, 71.59, 72.23, 72.38, 73.28, 74.33, 74.40, 77.18, 127.55, 127.77, 128.04, 128.18, 128.27, 128.51, 138.13, 138.39, 138.57. MS-ESI: 698 [M + H⁺]. Anal. Calcd for C₄₄H₅₆ClNO₄: C, 75.67; H, 8.08; N, 2.01; Found: C, 75.94; H, 8.35; N,1.85%

N-Nonyl-1,5-dideoxy-1,5-imino-D-glycero-L-galacto-heptitol-1-chloromethane (15)

Compound 15 was prepared from 37 (25 mg, 0.04 mmol) as described in the preparation of 13, providing 15 (10 mg, 75%) as a white solid in the form of hydrochloride salt. ¹H NMR (500 MHz, D₂O): δ 0.85–0.87 (m, 3H), 1.28–1.41 (m, 12H), 1.75–1.76 (m, 2H), 3.24–3.34 (m, 1H), 3.41–3.50 (m, 2H), 3.91–4.10 (m, 5H), 4.13 (t, J = 4.0 Hz, 1H), 4.20–4.22 (m, 1H), 4.41–4.42 (m, 1H). ¹³C NMR (100 MHz, D₂O): δ 13.36, 19.98, 21.98, 25.34, 28.14, 28.25, 28.39, 31.06, 38.63, 48.28, 54.38, 59.17, 60.86, 62.52, 67.26, 68.37. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₁₆H₃₃ClNO₄ 338.2093; Found: 338.2097.

2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-imino-L-glycero-L-galacto-heptitol-1-acid (40)

To a solution of 38 (71 mg, 0.10 mmol) in CH₂Cl₂ (4.0 mL) was added BAIB (68 mg, 0.20 mmol) and TEMPO (16.5 mg, 0.10 mmol). The reaction mixture was stirred for 30 h at 40 °C. The solvent was removed under reduced pressure, the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 18 [thin space (1/6-em)]

1 to 15

1) to provide yellow crude product. The crude product was dissolved in acetonitrile (1.5 mL), and sodium dihydrogen phosphate in water (1.5 mL, 100 mg mL⁻¹), 30% H₂O₂ (0.38 mL) and sodium chlorite in water (3.0 mL, 130 mg mL⁻¹) were added to the mixture. After stirring for 36 h at room temperature, the aqueous solution was extracted with ethyl acetate. The combined organic layers were dried (Na₂SO₄) and evaporated in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc 3 [thin space (1/6-em)]

1 to 1

1) to yield 40 (54 mg, 75%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 3.44 (s, 1H), 3.69–3.74 (m, 2H), 4.00 (s, 1H), 4.37–4.51 (m, 8H), 4.59 (d, J = 11.5 Hz, 1H), 4.70–4.78 (m, 2H), 4.98–5.08 (m, 2H), 7.22–7.30 (m, 25H). HRMS (ESI, positive) Calcd for [(M + H)⁺] C₄₃H₄₄NO₈ 702.3061; Found: 702.3082.

1,5-Dideoxy-1,5-imino-L-glycero-L-galacto-heptitol-1-formic acid (16)

A mixture of 40 (27 mg, 0.04 mmol) and 10% Pd/C (8.0 mg) in THF (2.0 mL), H₂O (1.0 mL) and HOAc (0.4 mL) was stirred for 72 h under H₂ atmosphere. The solid was removed by filtration through Celite and the filtrate was concentrated. The residue was purified by column chromatography on silica gel (CH₂Cl₂/MeOH 3 [thin space (1/6-em)]

1 to 1

3) and C-18 reversed-phase column chromatography (H₂O as eluent), giving 16 (8.0 mg, 100%) as a white solid. ¹H NMR (500 MHz, D₂O): δ 3.69 (br.s, 2H), 3.86 (dd, J = 7.0, 12.5Hz, 1H), 3.95–3.98 (m, 2H), 4.07 (dd, J = 2.5, 8.5 Hz, 1H), 4.40 (br.s, 1H). ¹³C NMR (75 MHz, D₂O): δ 57.30, 61.53, 62.12, 67.94, 72.26, 72.50. HRMS (ESI, positive) Calcd for [(M + H)⁺] C₇H₁₄NO₆ 208.0816; Found: 208.0819.

Cell proliferation assay

Male BALB/c mouse splenocytes (8 × 10⁵cells per well), retreated with Con A alone (2.5 μg mL⁻¹ concentration) or along with 30 μM concentration of synthetic iminosugar compounds, were incubated at 37 °C for 48 h under 5% CO₂ in a RPMI-1640 medium containing 10% fetal bovine serum (FBS). The proliferation of the mouse splenocytes was assayed using the CCK-8 reduction method. CCK-8 (20 μL) was added to each well and the plates were incubated for 2 h at 37 °C. Optical density was measured using a Microplate Reader at 450 nm. All data were presented as mean ± SEM. The cytotoxicity was assayed by the CCK-8 method using the cells without the Con A-induced proliferation.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China.

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Footnote

† Electronic supplementary information (ESI) available: NMR spectra for new compounds. See DOI: 10.1039/c1md00098e

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