Introduction

Immunostimulators, as the adjuvant therapeutic agents for the treatment of many tumors and infectious diseases such as the severe acute respiratory syndrome (SARS) epidemic, have aroused great interest because of their potential value to enhance the ability of the human immune system (Hadden JW, 1993). Many compounds, such as the biological macromolecules including glycoproteins (Billiau and Matthys, 2009), polynucleotides (Anada et al., 2006), and polysaccharides (Chan et al., 2009), and the small molecules including heterocycles (Failli and Caggiano, 1992), nucleosides (Lee et al., 2003), glycosylceramides (glycolipids) (Yang et al., 2004), and iminosugars (Vyavahare et al., 2007), have been demonstrated to possess immunostimulatory properties. Among them, the nucleoside-based small molecule immunostimulants that affect the proliferation and potentiation of the cytokine-producing humoral cells have offered more considerable promise in drug development (Lee et al., 2003). For example, the guanosine derivatives (e.g., Loxoribine, Fig. 1a) have been extensively studied and proved to hold the potential as immunostimulating agent (Reitz et al., 1994). However, as a special kind of nucleoside analogs, nonclassic base-modified nucleosides (Noll et al., 2009) are rarely explored for their immunomodulating activity. It is of great interest and challenge to explore such pseudonucleosides as immunostimulants.

Fig. 1
figure 1

Some chemical structures of small molecule immunostimulating agents (a) Loxoribine, (b) Pidotimod, (c) CGP52608, and (d) C-pseudodisaccharide containing thiazolidin-4-one

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Thiazolidin-4-one ring is a core substructure found in various synthetic pharmaceuticals which associate with diverse biological activities. A few of thiazolidine derivatives, for instance, pidotimod (Du et al., 2008) and CGP52608 (Herrera et al., 2004) (Fig. 1b, c), exhibited strong immunostimulating activity. Recently, we have found that several C-pseudonucleosides containing thiazolidin-4-ones (Fig. 1d) show significant effects on T cell proliferation at low concentration (Chen et al., 2011). The C-pseudonucleosides with phenyl at N-3 position on thiazolidin-4-one ring were observed to have immuno-potentiating efficiency with a bias via TH2-mediated cellular immunity by increasing IL-4 and IL-2 secretions, inhibiting IFN-γ, and promoting CD3+, CD4+ T, and B cell proliferation. In order to find new immnostimulating agents, and to shed more light on the structure–activity relationship (SAR) of this class of C-pseudonucleosides containing thiazolidin-4-one, in the present article, we report the synthesis of the C-pseudonucleosides containing lipophilicity substituents at position 3 or 4 of the phenyl ring at N-3 (Scheme 1), and their effects on T cell proliferation.

Scheme 1
scheme 1

The synthesis of C-pseudonucleosides 4 and 5. Reagents and conditions: (i) 1 (1 mmol), 2 (1 equiv.), dry EtOH (3 mL), r.t., 15–30 min; (ii) 3 (2 equiv.), DCC (1.2 equiv.), DMAP (0.2 equiv.), r.t., 30 min

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Results and discussion

Chemistry

The requisite unprotected sugar aldehyde 1 has been readily prepared in one step from d-glucosamine hydrochloride with the high yield of 85 % (Abdel-Rahman et al., 1999). Following our reported procedure (Chen et al., 2008), the target C-pseudonucleosides 4 and 5 were synthesized by the one-pot three-component condensation from the unprotected sugar aldehyde 1, substituted anilines 2(a–g), and mercaptoacetic acid 3 at room temperature (r.t.) in the presence of the condensation reagent N,N′-dicyclohexyl-carbodiimide (DCC) and the promoter 4-dimethylamino-pyridine (DMAP). The one-pot synthesis afforded the diastereomeric products 4 (less polar) and 5 (more polar), respectively, with the overall yields of 33–42 % (Table 1), after simple working up and purification by reverse silica (C18) gel column chromatography. It was found that the reaction proceeded with low stereoselectivity, and only in the cases of 2e and 2g, a moderate stereoselectivity was observed (Table 1, Entries 5 and 7).

Table 1 The synthesis of C-pseudonucleosides 4 and 5 and their NMR signals of H-2
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The structures of C-pseudonucleosides 4 and 5 were assigned based on the analyses of their spectral data of NMR and HRMS. Each diastereoisomers of 4 and 5 exhibited the similar NMR signals in H-2 as shown in Table 1, i.e., H-2 of the less polar 4 was in more downfield (0.21–0.24 ppm) than that of the more polar 5. Comparing the 1H NMR signals of H-2 in 4 and 5 to those of the known compound D (Fig. 1, R = phenyl) (Chen et al., 2011), the configurations of the new generated chiral carbon (C-2) in 4 and 5 could be tentatively determined to be of (S) and (R), respectively.

Biological assay

The effects of C-pseuodonucleosides 4 and 5 on the concanavalin A (Con A)-induced proliferation of the mice splenocytes were assessed by the MTT method. Our initial studies revealed that the treatment of the mice splenocytes with Con A (20 μM) enhanced cell proliferation by 48 % (p < 0.001) compared with the untreated cells, and all subsequent experiments were carried out using the same concentration of Con A. The assays were conducted 72 h at 37 °C, 5 % CO2 after the administration of 4 and 5 at five different concentrations (5, 10, 25, 50, and 100 μM), using the Con A-treated splenocytes as the experimental control.

As shown in Table 2, compounds 4d, 5d, 4e, 5e, 4f, 5f, 4g, and 5g promoted Con A-induced T cell proliferation to some degree at certain concentrations. Compared with the Con A-treated sample, the treatment with the combination of Con A and compounds 4(d–g) and 5(d–g) (5, 10, 25, 50, and 100 μM) increased the proliferation in the range of 21–106 %. The concentration independent phenomena were found in the all test compounds, i.e., the compounds enhanced the Con A-induced cell proliferation irregularly. Among them, compound 5g showed most pronounced effect on T cell proliferation at 50 μM with the proliferation rate of 106 %. Some compounds, such as 5d, 5e, 5f, and 4g, could significantly increase the proliferation by 62, 63, 70, and 53 %, respectively at low concentration of 5 μM. The other compounds 4(a–c) and 5(a–c) did not exhibit any promotion on T cell proliferation at the tested concentrations. From the view of the SAR, this result suggested that the immunostimulating activity decreased markedly with the introduction of the lipophilicity substituents like chloro, methyl, and methoxyl on the para position of N-3 phenyl at the thiazolidin-4-one ring. However, it was unclear whether compounds 4g and 5g in which the substituent was COOC2H5 on the para position at N-3 phenyl showed good activities. In addition, the similar activities of compounds 4–5(d) and 4–5(e–f) suggested that neither the electron-withdrawing group (chloro) nor the electron-donating group (methyl or methoxyl) had the significant impacts on their immunostimulatory activities. The C-2 configuration of the diastereomeric compounds 4 and 5 had also no significant effects on the T cell proliferation. The above SAR analysis may be helpful to guide us further structural modification of such C-pseudonucleosides as immunostimulating agents. It was necessary to choose the proper concentration for the future pharmacological study.

Table 2 The effects of compounds 4 and 5 on Con A-induced splenocyte proliferation
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Conclusion

Some C-pseudonucleosides containing thiazolidin-4-one were designed and synthesized from the unprotected sugar aldehyde, substituted aniline, and mercaptoacetic acid at r.t. Compounds 5d, 5e, 5f, and 4g could significantly increase the Con A-induced T cell proliferation at low concentration of 5 μM. The SAR analysis indicated that the immunostimulatory efficacy of such C-pseudonucleosides is related to the presence of lipophilic groups (chloro atom, methyl, and methoxyl) in proper position of N-3 phenyl at thiazolidin-4-one ring. Moreover, the C-2 configuration and the electronic property of the lipophilicity substituents likely had slight effects on the immunostimulatory activity.

Experimental

Chemistry

Melting points were measured on an SGW® X-4 micro melting point apparatus and were uncorrected. Optical rotations were determined on an SGW®-1 automatic polarimeter. IR spectra were determined on a WQF-510 as KBr tablets for solid samples and were expressed in cm−1 scale. 1H NMR and 13C NMR spectra were measured on a RT-NMR Bruker AVANCE 600 MHz, NMR spectrometer using tetramethylsilane (Me4Si) as an internal standard. Mass spectra (MS) and high-resolution mass spectra (HRMS) were carried out on a FTICR-MS (Ionspec 7.0T) mass spectrometer with electrospray ionization (ESI). The optical densities for examining the activities of immunological activities were measured on a BioRad Model 3550 microplate spectrophotometer. Thin-layer chromatography (TLC) was performed on precoated plates (Qingdao GF254) with detection by UV light or with phosphomolybdic acid in EtOH/H2O followed by heating. Column chromatography was performed using reverse silica gel (C18, 50 μM). Concanavalin A (type IV) was purchased from Sigma. Cell culture was carried out under sterile condition.

General procedure for the synthesis of compounds 4 and 5

The unprotected aldehyde 1 (0.16 g, 1 mmol) was dissolved in 3 mL anhydrous EtOH. To the solution, substituted aniline 2 (1 mmol) was added and the mixture was stirred at r.t. for 15–30 min. Then, mercaptoacetic acid 3 (0.14 mL, 2 mmol) was added, followed by DMAP (0.2 equiv.) and DCC (1.2 equiv.). After continued stirring for 30 min at r.t., the mixture was neutralized with solid K2CO3. Solvent was evaporated under reduced pressure to get a crude product, which was purified using reverse silica (C18) gel column chromatography (H2O–MeOH V/V = 90:10 for 4, 5(a, b, f, g), H2O–MeOH V/V = 95:5 for 4, 5(c–e) to get the mixture of two diastereomers 4 and 5.

(S)-3-(4-Chlorophenyl)-2-((2S,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)thiazolidin-4-one (4a)

Light yellow solid, yield 24.9 %, m.p. 65–66 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−12.0 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3421, 2925, 1671, 1491, 1394, 1012. 1H NMR (600 MHz, CD3OD) δ H (ppm): 3.51 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5), 3.61 (1H, dd, J = 12.0 Hz, 3.6 Hz, H-5′), 3.65 (1H, d, J = 15.6 Hz, H-5), 3.71–3.75 (1H, m, H-4′), 3.94–3.97 (2H, m, H-1′, H-5′), 4.04 (1H, t, J = 6.0 Hz, H-2′), 4.24 (1H, t, J = 6.0 Hz, H-3′), 5.54 (1H, dd, J = 4.8 Hz, 1.2 Hz, H-2), 7.51 (4H, q, J = 8.4 Hz, 4CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 17.0, 31.8, 57.0, 61.4, 64.7, 77.7, 83.9, 127.8, 128.8, 132.5,137.0, 172.0; HRESIMS: calcd for C14H16ClNO5SNa ([M+Na]+) 368.0335, found: 368.0326.

(R)-3-(4-Chlorophenyl)-2-((2S,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)thiazolidin-4-one (5a)

Light yellow solid, yield 13.1 %, m.p. 136–137 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−93.1 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3436, 2951, 1682, 1495, 1401, 1023. 1H NMR (600 MHz, CD3OD) δ H (ppm): 3.55 (1H, d, J = 15.6 Hz, H-5), 3.60 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5′), 3.78 (1H, dd, J = 10.2 Hz, 1.8 Hz, H-5), 3.89 (1H, d, J = 7.8 Hz, H-5′), 3. 93 (1H, d, J = 4.8 Hz, H-1′), 3.95–3.97 (2H, m, H-2′, H-4′), 4.07 (1H, t, J = 7.2 Hz, H-3′), 5.32 (1H, s, H-2), 7.43 (2H, d, J = 8.4 Hz, 2CH), 7.49 (2H, d, J = 8.4 Hz, 2CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 32.6, 61.6, 66.9, 76.2, 77.7, 80.7, 83.9, 128.3, 129.2, 133.2, 135.9, 172.6; HRESIMS: calcd for C14H16ClNO5SNa ([M+Na]+) 368.0335, found: 368.0338.

(S)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(p-tolyl)thiazolidin-4-one (4b)

Light yellow solid, yield 24.6 %, m.p. 65–71 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−56.0 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3436, 2898, 1662, 1496, 1397, 1024. 1H NMR (600 MHz, CD3OD) δ H (ppm): 2.37 (3H, s, CH3), 3.51 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5), 3.61–3.65 (2H, m, H-5, H-5′), 3.74–3.77 (1H, m, H-4′), 3.93–3.96 (2H, m, H-5′, H-1′), 4.03 (1H, dd, J = 6.6 Hz, 4.2 Hz, H-2′), 4.30 (1H, t, J = 6.0 Hz, H-3′), 5.49 (1H, dd, J = 4.2 Hz, 1.8 Hz, H-2), 7.25 (2H, d, J = 7.8 Hz, 2CH), 7.43 (2H, d, J = 8.4 Hz, 2CH); 13C NMR (150 MHz, CD3OD) δ C (ppm):19.8, 32.8, 61.7, 67.4, 76.2, 77.7, 80.7, 83.8, 126.7, 129.7, 134.4, 138.0, 172.6; HRESIMS: calcd for C15H19NO5SNa (M+Na)+: 348.0881, found: 348.0872.

(R)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(p-tolyl)thiazolidin-4-one (5b)

White solid, yield 16.4 %, m. p. 168–170 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−184.5 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3384, 2912, 1654, 1401,1398, 1060. 1H NMR (600 MHz, CD3OD) δ H (ppm): 2.38 (3H, s, CH3), 3.54 (1H, d, J = 15.6 Hz, H-5), 3.60 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5′), 3.78 (1H, dd, J = 12.0 Hz, 2.4 Hz, H-5), 3.91–3.95 (3H, m, H-5′, H-1′, H-2′), 3.96–4.00 (1H, m, H-4′), 4. 06 (1H, t, J = 7.2 Hz, H-3′), 5.26 (1H, s, H-2), 7.29 (4H, m, 4CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 19.8, 32.8, 61.7, 67.3, 76.2, 77.7, 80.7, 83.8, 126.7, 129.7, 134.4, 138.0, 172.6; HRESIMS: calcd for C15H20NO5S (M+H)+: 326.1062, found: 326.1074.

(S)-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(4-methoxyphenyl)thiazolidin-4-one (4c)

Light yellow solid, yield 22.8 %, m.p. 127–129 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−72.1 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3347, 2923, 1663, 1511, 1239, 1072. 1H NMR (600 MHz, CD3OD) δ H (ppm): 3.51 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5), 3.63–3.65 (2H, m, H-5′, H-5), 3.76–3.78 (1H, m, H-4′), 3.83 (3H, s, CH3), 3.92–3.97 (2H, m, H-5′, H-1′), 4.05 (1H, dd, J = 4.2 Hz, 1.2 Hz, H-2′), 4.31 (1H, t, J = 6.6 Hz, H-3′), 5.43 (1H, dd, J = 4.2 Hz, 1.8 Hz, H-2), 6.98 (2H, d, J = 8.4 Hz, 2CH), 7.36 (2H, d, J = 9.0 Hz, 2CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 31.8, 54.6, 61.5, 65.6, 77.1, 77.5, 82.6, 83.5, 114.0, 127.9, 130.7, 159.0, 172.2; HRESIMS: calcd for C15H20NO6S (M+H)+: 342.1011, found: 342.1025.

(R)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(4-methoxyphenyl)thiazolidin-4-one (5c)

Light yellow solid, yield 16.3 %, m.p. 168–169 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−102.5 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3358, 2923, 1649, 1519, 1246, 1028. 1H NMR (600 MHz, CD3OD) δH (ppm): 3.53 (1H, d, J = 15.0 Hz, H-5), 3.61 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5′), 3.79 (1H, dd, J = 12.0 Hz, 1.8 Hz, H-5), 3.83 (3H, s, CH3), 3.90–3.94 (3H, m, H-1′, H-5′, H-2′), 3.96–3.99 (1H, m, H-4′), 4. 06 (1H, t, J = 7.2 Hz, H-3′), 5.20 (1H, s, H-2), 7.02 (2H, d, J = 9.0 Hz, 2CH), 7.36 (2H, d, J = 9.0 Hz, 2CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 32.6, 64.4, 61.7, 76.3, 77.7, 80.6, 83.8, 114.4, 128.3, 129.6, 159.6, 172.8; HRESIMS: calcd for C15H20NO6S (M+H)+: 342.1011, found: 342.1008.

(S)-3-(3-Chlorophenyl)-2-((2S,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)thiazolidin-4-one (4d)

White solid, yield 28.6 %, m.p. 172–173 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−81.4 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3340, 2923, 1654, 1412, 1118, 1032. 1H NMR (600 MHz, CD3OD) δ H (ppm): 3.51 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5), 3.66 (1H, d, J = 15.6 Hz, H-5), 3.73–3.75 (1H, m, H-4′), 3.95–3.98 (2H, m, H-1′, H-5′), 4.04 (1H, t, J = 6.0 Hz, H-2′), 4.27 (1H, t, J = 6.0 Hz, H-3′), 5.59 (1H, dd, J = 4.8 Hz, 1.2 Hz, H-2), 7.33–7.34 (1H, m, CH), 7.41–7.42 (2H, m, 2CH), 7.57 (1H, s, CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 31.8, 61.4, 64.7, 77.0, 77.6, 83.2, 83.8, 124.2, 126.5, 127.0, 129.9, 134.0, 139.5, 171.9; HRESIMS: C14H16ClNO5SNa ([M+Na]+) 368.0335, found: 368.0341.

(R)-3-(3-Chlorophenyl)-2-((2S,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)thiazolidin-4-one (5d)

White solid, yield 11.4 %, m. p. 186–188 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−92.4 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3379, 2887, 1647, 1058, 1415, 971. 1H NMR (600 MHz, CD3OD) δ H (ppm): 3.56 (1H, d, J = 15.6 Hz, H-5), 3.91 (1H, dd, J = 7.2 Hz, 5.4 Hz, H-5′), 3.93–3.98 (3H, m, H-1′, H-4′, H-2′), 4. 07 (1H, t, J = 7.2 Hz, H-3′), 5.37 (1H, s, H-2), 7.38–7.40 (2H, m, 2CH), 7.55 (1H, s, CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 32.7, 61.7, 66.8, 76.2, 77.7, 80.9, 83.9, 124.8, 126.7, 127.6, 130.3, 134.5, 138.5, 172.5; HRESIMS: calcd for C14H16ClNO5SNa ([M+Na]+) 368.0335, found: 368.0324.

(S)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(m-tolyl)thiazolidin-4-one (4e)

White solid, yield 7.0 %, m.p. 204–205 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−71.2 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3348, 2913, 1656, 1407, 1120, 1031. 1H NMR (600 MHz, CD3OD) δ H (ppm): 2.38 (3H, s, CH3), 3.51 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5), 3.62–3.66 (2H, m, H-5, H-5′), 3.74–3.78 (1H, m, H-4′), 3.95–3.97 (2H, m, H-1′, H-5′), 4.03 (1H, dd, J = 6.0 Hz, 4.8 Hz, H-2′), 4.31 (1H, t, J = 6.0 Hz, H-3′), 4.39 (2H, dd, J = 14.4 Hz, 7.2 Hz, CH2), 5.52 (1H, dd, J = 4.2 Hz, 1.8 Hz, H-2), 7.16 (1H, d, J = 7.8 Hz, CH), 7.24 (1H, d, J = 7.8 Hz, CH), 7.29 (1H, s, CH), 7.32 (1H, d, J = 7.8 Hz, CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 19.9, 32.0, 61.4, 65.4, 77.0, 77.4, 82.5, 83.4, 123.3, 127.0, 128.0, 128.6, 138.0, 139.0, 172.0; HRESIMS: calcd for C15H19NO5SNa (M+Na)+: 348.0881, found: 348.0879.

(R)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(m-tolyl)thiazolidin-4-one (5e)

Light yellow solid, yield 35.0 %, m.p. 65–66 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−77.5 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3367, 2912, 1658, 1397, 1106, 1027. 1H NMR (600 MHz, CD3OD) δ H (ppm): 2.39 (3H, s, CH3), 3.54 (1H, d, J = 15.0 Hz, H-5), 3.61 (1H, dd, J = 12.0 Hz, 5.4 Hz, H-5′), 3.78 (1H, dd, J = 12.0 Hz, 2.4 Hz, H-5), 3.91–3.96 (3H, m, H-1′, H-5′, H-2′), 3.97–4.00 (1H, m, H-4′), 4. 06 (1H, t, J = 7.2 Hz, H-3′), 5.28 (1H, s, H-2), 7.20 (2H, t, J = 6.0 Hz, 2CH), 7.24 (1H, s, CH), 7.36 (1H, t, J = 7.8 Hz, CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 19.9, 32.8, 61.7, 76.3, 77.7, 80.7, 83.9, 123.8, 127.3, 128.4, 129.0, 137.0, 139.4, 172.6; HRESIMS: calcd for C15H19NO5SNa (M+Na)+: 348.0881, found: 348.0893.

(S)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(3-methoxyphenyl)thiazolidin-4-one (4f)

Light yellow solid, yield 16.4 %, m.p. 48–50 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−106.9 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3349, 2823, 1656, 1404, 1123, 1028. 1H NMR (600 MHz, CD3OD) δ H (ppm): 3.52 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5), 3.62–3.67 (2H, m, H-5′, H-5), 3.76–3.79 (1H, m, H-4′), 3.82 (3H, s, CH3), 3.95–3.97 (2H, m, H-5′, H-1′), 4.05 (1H, dd, J = 6.6 Hz, 4.8 Hz, H-2′), 4.30 (1H, t, J = 6.0 Hz, H-3′), 5.53 (1H, dd, J = 4.8 Hz, 1.2 Hz, H-2), 6.91 (1H, dd, J = 8.4 Hz, 1.8 Hz, CH), 7.01 (1H, d, J = 7.8 Hz, CH), 7.08 (1H, s, CH), 7.34 (1H, t, J = 7.8 Hz, CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 32.0, 54.5, 61.4, 65.3, 77.0, 77.4, 82.6, 83.5, 112.3, 113.1, 118.1, 129.4, 139.1, 160.3, 172.0; HRESIMS: calcd for C15H20NO6S (M+H)+: 342.1011, found: 342.1019.

(R)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(3-methoxyphenyl)thiazolidin-4-one (5f)

White solid, yield 20.6 %, m. p. 112–113 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−181.4 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3405, 2885, 1647, 1493, 1177, 1049. 1H NMR (600 MHz, CD3OD) δ H (ppm): 3.55 (1H, d, J = 15.0 Hz, H-5), 3.61 (1H, dd, J = 12.6 Hz, 5.4 Hz, H-5′), 3.78 (1H, dd, J = 12.0 Hz, 2.4 Hz, H-5), 3.82 (3H, s, CH3), 3.91–3.94 (2H, m, H-1′, H-5′), 3.96 (1H, d, J = 7.2 Hz, H-2′), 3.97–4.00 (1H, m, H-4′), 4. 07 (1H, t, J = 7.2 Hz, H-3′), 5.32 (1H, s, H-2), 6.94–6.99 (2H, m, 2CH), 7.01 (1H, s, CH), 7.368 (1H, t, J = 8.4 Hz, CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 32.9, 54.6, 61.8, 76.3, 77.7, 80.8, 83.9, 112.5, 113.5, 118.6, 129.9, 138.1, 160.6, 172.5; HRESIMS: calcd for C15H19NO6SNa (M+Na)+: 364.0830, found: 364.0843.

(S)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-(3-ethoxycarbonylphenyl)thiazolidin-4-one (4g)

Yellow solid, yield 24.8 %, m. p. 55–60 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−47.6 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3435, 2912, 1727, 1661, 1403, 1277, 1115, 1019. 1H NMR (600 MHz, CD3OD) δ H (ppm): 1.41 (3H, t, J = 7.2 Hz, CH3), 3.49 (1H, dd, J = 12.0 Hz, 4.8 Hz, H-5), 3.58 (1H, dd, J = 12.0 Hz, 3.0 Hz, H-5′), 3.66 (1H, d, J = 15.6 Hz, H-5), 3.70–3.72 (1H, m, H-4′), 3.95 (1H, t, J = 6.0 Hz, H-1′), 4.00 (1H, d, J = 16.2 Hz, H-5′), 4.06 (1H, t, J = 6.0 Hz, H-2′), 4.22 (1H, t, J = 5.4 Hz, H-3′), 4.39 (2H, dd, J = 14.4 Hz, 7.2 Hz, CH2), 5.69 (1H, dd, J = 5.4 Hz, 1.2 Hz, H-2), 7.62 (2H, d, J = 8.4 Hz, 2CH), 8.07 (2H, d, J = 8.4 Hz, 2CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 13.2, 32.0, 60.9, 61.4, 64.2, 77.1, 77.8, 84.1, 126.3, 128.4, 129.8, 142.6, 166.0, 171.8; HRESIMS: calcd for C17H21NO7SNa (M+Na)+: 406.0936, found: 406.0929.

(R)-2-((2S,3S,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-3-ethoxycarbonylphenyl)thiazolidin-4-one (5g)

Light yellow solid, yield 8.3 %, m.p. 80–82 °C, \( [{{\upalpha}}]_{\text{D}}^{25} \)−61.5 (c 1.0, MeOH); IR (KBr) ν (cm−1): 3472, 2926, 1700, 1662, 1401, 1282, 1105, 1060. 1H NMR (600 MHz, CD3OD) δ H (ppm): 1.41 (3H, t, J = 7.2 Hz, CH3), 3.56–3.60 (2H, m, H-5, H-5′), 3.77 (1H, dd, J = 12.0 Hz, 2.4 Hz, H-5′), 3.90 (1H, d, J = 7.2 Hz, H-5), 3.93 (1H, d, J = 7.2 Hz, H-1′), 3. 95–3.97 (1H, m, H-4′), 3.99 (1H, d, J = 15.6 Hz, H-2′), 4. 08 (1H, t, J = 7.2 Hz, H-3′), 4.39 (2H, dd, J = 13.8 Hz, 7.2 Hz, CH2), 5.48 (1H, s, H-2), 7.61 (2H, d, J = 9.0 Hz, 2CH), 8.11 (2H, d, J = 7.2 Hz, 2CH); 13C NMR (150 MHz, CD3OD) δ C (ppm): 13.2, 32.9, 61.0, 61.6, 66.6, 76.1, 77.7, 81.1, 83.8, 125.8, 129.1, 130.2, 141.4, 165.8, 129.1; HRESIMS: calcd for C17H21NO7SNa (M+Na)+: 406.0936, found: 406.0932.

Immunological activities assay

Preparation and cultivation of splenocytes

The spleens from BALb/c mice were taken out in sterile conditions and soaked in non-serum RPMI-1640 cell culture medium. The spleens were grinded using a wire mesh. Filter the cell suspension with a 200-mesh nylon net. The filtrate of the splenocytes was centrifuged at 2,000 g for 10 min, and then the supernatant was removed. Dissolving the precipitation in 5 mL of pH 7.2 Tris–NH4Cl solution and incubating the cells at 37 °C for 6–10 min to lyse the red cells. Then, the cells were centrifuged at 2,000 g for 7 min, and the cell pellets were dissolved in RPMI-1640 culture medium with 10 % newborn calf serum and 20 μM Con A. Counting cell and adjusting the concentration of cells solution to 5 × 106/mL. Add 4.5 × 105 cells into each well of 96-well plates. Subsequently, adding different concentrations of each compound into each well, and incubating at 37 °C, 5 % CO2 for 72 h. The supernatant was collected and centrifuged at 2,000 g for 5 min. The supernatant was collected and stored at −20 °C until for assay.

Measurement of the proliferation of T cell

Splenocytes from BALb/C mice were aseptically removed and minced, and cell suspensions were incubated at 4.5 × 105 cell/well, 90 μL/well in 96-well microtiter plates using an RPMI 1640 medium with 10 % FCS. Spleen cells were cultured with 20 μM of Con A for 72 h at 37 °C in 5 % CO2 in the presence or absence of the tested compounds. Wells containing Con A without tested compounds were used as blanks. All the tests were performed at least three times in quadruplicate (p < 0.01). Cell proliferation was measured using the MTT assay, testing OD (A) at wavelength 570 nm.

$$ {\text{Cell}}\,{\text{proliferation}}\,\left( \% \right) = \left( {A_{{{\text{treated}}}} - A_{{{\text{control}}}} } \right)/A_{{{\text{control}}}} \times 100$$