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Article

Synthesis and Anti-Influenza A Virus Activity of 6′-amino-6′-deoxy-glucoglycerolipids Analogs

by
Li Ren
†,
Jun Zhang
†,
Haizhen Ma
,
Linlin Sun
,
Xiaoshuang Zhang
,
Guangli Yu
,
Huashi Guan
,
Wei Wang
* and
Chunxia Li
*
Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering, Key Laboratory of Marine Drugs of Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Mar. Drugs 2016, 14(6), 116; https://doi.org/10.3390/md14060116
Submission received: 1 April 2016 / Revised: 2 June 2016 / Accepted: 3 June 2016 / Published: 18 June 2016
(This article belongs to the Special Issue Drug Design Based on Marine Natural Product Scaffolds)
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Abstract

:
A series of aminoglucoglycerolipids derivatives had been synthesized, including 6′-acylamido-glucoglycerolipids 1a–1f and corresponding 2′-acylamido-glucoglycerolipids 2a–2c bearing different fatty acids, glucosyl diglycerides 3a–3e bearing different functional groups at C-6′ and ether-linked glucoglycerolipids 4a–4c with double-tailed alkyl alcohol. The anti-influenza A virus (IAV) activity was evaluated by the cytopathic effects (CPE) inhibition assay. The results indicated that the integral structure of the aminoglycoglycerolipid was essential for the inhibition of IAV in MDCK cells. Furthermore, oral administration of compound 1d was able to significantly improve survival and decrease pulmonary viral titers in IAV-infected mice, which suggested that compound 1d merited further investigation as a novel anti-IAV candidate in the future.

Graphical Abstract

1. Introduction

Type A influenza, as an infectious disease caused by influenza A virus (IAV), once inflicted more casualties than any other infectious diseases in Europe. It had been the cause of at least three pandemics in the last century, the most severe leading to more than 40 million fatalities in 1918–1919 [1]. Moreover, the 2009 outbreak of swine-origin influenza A/H1N1 [2,3] continued to affect many countries, and caused over 18,000 deaths worldwide [4]. Considering the frequency of influenza pandemics, it is urgent to develop novel anti-IAV drugs with high efficiency.
Glycoglycerolipids occur widely in marine algae [5,6,7,8], cyanobacteria [9,10,11] and higher plants. The natural glycoglycerolipids possess various biological activities, such as anti-tumor [12,13], anti-viral [14,15,16,17], and anti-inflammatory activities [18], which make them valuable molecular targets for further investigation [19]. In our previous work [20,21,22] synthetic aminoglycoglycerolipids derived from marine natural product 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol (AGGL, Figure 1) [23] were found to possess notable anti-IAV activity. Moreover, preliminary results indicated that the type and the length of the acids in the aminomannosylglycerols could influence the inhibitory effect [20]. To explore more potent anti-IAV leading drug candidates, herein, we synthesized new series of aminoglycoglycerolipid derivatives and evaluated their anti-IAV activity in MDCK cells. Some compounds displayed potent antiviral effects, and the primary structure-activity relationship was summarized. Compound 1d was further found to be able to improve survival and decrease pulmonary viral titers in IAV-infected mice significantly.

2. Results and Discussion

2.1. Chemistry

New series of AGGL derivatives 1–4 were designed as Figure 1. The structural modification of AGGL focused on the fatty acid amide linked at C-6′position of the sugar ring and the glyceride attached to anomeric carbon, including 6′-acylamido-glucoglycerolipids 1a–1f and corresponding 2′-acylamido-glucoglycerolipids 2a–2c bearing different fatty acids, glucosyl diglycerides 3a–3e bearing different functional groups at C-6′ and ether-linked glucoglycerolipids 4a–4c with double-tailed alkyl alcohol. The synthetic routes were described as follows.

2.1.1. Synthesis of Compounds 1a–1f

6′-amide-6′-deoxy-α-d-glucoglycerolipids 1a–1f bearing different fatty acids at amino group were synthesized as illustrated in Scheme 1. Activation of thioglycoside 5 [24] with p-toluene sulfonyl chloride, followed by treatment 6 with sodium azide and benzyl bromide in one-pot method gave compound 7. The p-tolylthio group of 7 was removed by N-bromosuccinimide (NBS) in acetone-water, then treatment with trichloroacetonitrile (CCl3CN) and 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) afforded trichloroacetimidate 8 in 89% as the glycosyl donor. Glycosylation of (S)-isopropylideneglycerol with donor 8 under trimethylsilyl trifluoromethanesulfonate (TMSOTf) activation in absolute ether afforded 9 as an inseparable anomeric mixture (α/β = 12:1) in 97% yield. The small amount of β-anomer can be removed by silica gel column chromatography in the following step of hydrolysis with TsOH in methanol (89%). Treatment the α-anomer diol with excess palmitoyl chloride in the presence of N,N-dimethylaminopyridine (DMAP) produced 10 in 96% yield. Reduction of the azido group by Pd/H2 yielded the amino derivative 11 in 95% yield. Introduction of acetyl group on C6′–NH2 with Ac2O and C6-C16 fatty acids under the condition of EDCI/HOBt in CH2Cl2 afforded 12a–12f. Then final compounds 1a–1f were obtained by removal of the benzyl with H2/Pd(OH)2 in 83%–96% yields.

2.1.2. Synthesis of Compounds 2a–2c

In addition to the 6′-amides 1a–1f, we also synthesized the related 2′-amide glycoglycerolipids 2a–2c in order to compare the influence on biological activity of the position of amide group. As shown in Scheme 2, the synthesis started with the diazotransfer reaction [25] to introduce an azide group into 2-C of the glucosamine (13), followed by acetylation in one-pot method to give 14 in 81% yield. The acetate glycoside 14 reacted with p-toluene thiophenol to afford thioglycoside 15 in 85% yield. The acetyl protective group of 15 was changed by benzyl groups to give 16 in 98% yield, and then the p-tolylthio group was transformed to trichloroacetimidate to yield donor 17 (82%). Glycosylation of 17 with (S)-isopropylideneglycerol under TMSOTf condition afforded glycolipid anomers 18 (α/β = 2:1). After hydrolyzation, esterification and reduction according to the above procedures of 11, α-anomer 20 (54%) was producecd and isolated by silica gel column chromatography. Acylation of the resulting C2′-NH2 followed by hydrogenation with H2/Pd(OH)2 generated the target compounds 2a–2c.

2.1.3. Synthesis of Compounds 3a–3e

Glucosyl diglycerides 3a–3e bearing different functional groups at C-6′ were synthesized as illustrated in Scheme 3. Starting from the synthetic compound 22 [26], the trichloroacetimidate donor 23 was prepared, followed by glycosylation with (S)-isopropylideneglycerol to afford the key intermediate 24. TBDPS was chosen to selectively protect the primary hydroxyl of the glucose, which might also provide a remote participating effect in the latter glycosylation reaction to afford absolute α-linked glycoside 24 [27,28]. Under the condition of p-TsOH, acetonide and the TBDPS groups in 24 were removed simultaneously (25, 82%). After esterification and deprotection, 3a was obtained in 62% yield. Besides, in the catalyst of camphorsulfonic acid (CSA), the acetonide of 24 could be selectively hydrolyzed, then esterification of the diols and removal of the silyl ether group to give 27 in 84% yield. Compound 27 was first converted to the corresponding iodo analog 29 upon treatment with triphenylphosphine, imidazole and iodine [29]. The glucuronic acid derivative 30 was prepared upon oxidation of the 6′-OH of 27 into the corresponding carboxylic acid via a TEMPO/BAIB oxidation [30]. 6′-O-sulfated derivative 31 was prepared under the treatment with Py·SO3 complex in DMF at room temperature [31]. In addition, coupling of 27 with galacosyl trichloroacetimidate donor 28 [32] using a catalytic amount of TMSOTf in Et2O gave the desired α-(1→6)-linked disaccharide 32 in 75% yield. Deprotection of 29–32 by Pd-catalyzed hydrogenation afforded the desired compounds 3b–3e.

2.1.4. Synthesis of Compounds 4a–4c

Diacylglycerol residue was substituted by double-tailed alcohol linked at the anomeric carbon of the 6′-amide-6′-deoxy-α-d-glucose to afford the derivatives 4a–4c. As shown in Scheme 4, trichloroacetimidate donor 8 reacted with bis (tetradecyl) carbinol 32 [33] under TMSOTf activation in Et2O to afford the absolute α-linked intermediate 33 in 49% yield. After reduction, acylation and deprotection, target analogs 4a–4c were obtained.

2.2. Biological Evaluation

2.2.1. Inhibition of Influenza A Virus Multiplication in Vitro

6′-amino-6′-deoxy-glycoglycerolipid (AGGL) and its derivatives were tested for their antiviral activity by the cytopathic effects (CPE) inhibition assay [34]. As shown in Table 1, natural AGGL displayed moderate virus inhibition at 50 μM. Compounds 1b and 1d, which contained saturated fatty acyl chains of C6 and C14 at 6′-NH2, displayed notable virus inhibition among the aminoglucoglycerolipids bearing linear fatty acids with C2–C18 length (1a–e), suggesting that the optimum length of saturated fatty acyl chains may be important for their anti-IAV effects in vitro. Compound 2b bearing hexanoic acid (C6) at 2′-NH2 also exhibited similar inhibitory activity with the control of ribavirin. Whereas, compound 1b which also had a hexanoyl group at a different position, showed comparably good anti-IAV activity (46.1%) in vitro, suggesting that the hexanoyl group may be indispensable for rational design of novel anti-IAV drugs based on AGGL structure. In addition, compound 1f with an aromatic ring at the acylamino group displayed a decreased effect on inhibition of IAV multiplication in MDCK cells, which was consistent with our previous results [20]. Moreover, our previous result [20] indicated that the tripalmitoyl derivative of the 6-aminomannoglycerolipid had 48% IAV inhibition at 50 μM, which was more than that of AGGL (25.5%) at the same concentration, suggesting that mannose may be able to increase the activity of these lipids. In addition, compounds 3a–3d and 4a–4c, which replaced the fatty acyl amide with other groups (3a–3d), or substituted the glycerolipid with a dialkyl alcohol (4a–4c), had much lower inhibition effect. These results indicated that the acylamino and glycerol groups of the glycolipids were essential for the inhibition of IAV multiplication. Surprisingly, glucosyl diglyceride 3e bearing another galactose at C-6′ exerted excellent inhibitory effect (51.5% at 50 μM), and hence the introduction of galactose at 6′-position of the glucosyl moiety may be able to enhance its activity.

2.2.2. IAV Infection in Vivo

Considering the structure characteristic and bioactivity in vitro, potent compound 1d (IC50 = 60.8 μM, close to that of Ribavirin (IC50 = 49.7 μM)) in the preliminary anti-viral screening was selected for further study of its inhibitory effect on IAV infection in vivo. In brief, IAV-infected mice received oral administration of Oseltamivir (20 mg/kg/d), compound 1d (5, 10 mg/kg/d) or placebo (PBS) once daily for the entire experiment, and then sacrificed at 4 d p.i. Subsequently, the pulmonary viral titers were determined by performing neuraminidase activity assay [35,36]. As shown in Table 2, compound 1d could reduce the lung index in IAV-infected mice with an inhibition rate of 15.3% at a dose of 5 mg/kg/d. Moreover, neuraminidase (NA) assay exhibited a significant reduction of virus titers in the lungs of 1d-treated mice (10 mg/kg/d) 4 d post infection, as compared to the control group (p < 0.05) (Figure 2A). The data suggested that oral therapy of infected mice with 1d resulted in a reduction of viral titers in the lung [37].
Furthermore, influenza A virus can induce lethal infections in certain mouse strains usually within 2 weeks [38]. Therefore, the survival experiments were also performed to evaluate the effect of compound 1d on the survival of IAV-infected mice. As shown in Figure 2B, the survival rate significantly increased in the 1d and oseltamivir-treated groups, as compared to the placebo-treated control group (p < 0.5). By d 14 after infection, only 30% of the individuals in the placebo group survived whereas 90% of those in the 1d (5 mg/kg/d) treated group survived, superior to that in the Oseltamivir treated group (Figure 2B).
Moreover, some researchers reported that small molecules such as flavonoids had no inhibitory effect against inflammatory related diseases at excessive concentrations, and the potential toxicity of flavonoids at high doses was possibly due to the generation of reactive oxygen species [39]. Herein, the anti-IAV effect of compound 1d also declined when elevating the dose from 5 mg/kg/d to 10 mg/kg/d, thus we supposed that treatment of compound 1d at high dose (10 mg/kg/d) may cause toxicity to mice due to the generation of reactive oxygen species or excessive pro-inflammatory cytokines in IAV infected mice. In addition, Sugawara et al. reported that oral administration of glycoglycerolipids may cause hydrolytic degradation and poor adsorption of these compounds [40]. However, Maeda et al. showed that the oral administration of cyclodextrin-galactosyldiacylglycerol complex could inhibit the tumor growth in mice despite the probable digestive degradation of these compounds [41]. Thus, the oral administration of compound 1d may also cause hydrolytic degradation but it may be able to inhibit IAV replication in vivo through other mechanisms.

3. Experimental Section

3.1. Chemical Procedures

3.1.1. General Information

Solvents were purified in a conventional manner. Thin layer chromatography (TLC) was performed on precoated HSGF254 plates (Yantai Chemical Industry Institute, Yantai, China). Flash column chromatography was performed on silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). 1H-NMR and 13C-NMR spectra were taken on a JEOL JNM-ECP 600 MHz (JEOL Ltd., Tokyo, Japan) and an Agilent 500 MHz DD2 spectrometer (Agilent Technologies, Santa Clara, CA, USA) with tetramethylsilane (Me4Si) as the internal standard, and chemical shifts were recorded as δ values. Mass spectra were recorded on a Global Q-TOF mass spectrometer (Waters Ltd., Wilmslow, UK) and IonSpec 4.7 Tesla FTMS (Varian Inc., Palo Alto, CA, USA) (MALDI/DHB).

3.1.2. Chemistry: General Methods

General Procedure for Compounds 1a–1f

A solution of 12a–12f in THF/i-PrOH (9:1) was treated with 20% palladium hydroxide and stirred at ambient temperature under hydrogen atmosphere for 3.5 h. After filtration, the solvent was evaporated and the residue was purified by silica column chromatography (CH2Cl2-MeOH 30:1) to afford 1a–1f (83%–96%) as white solids.

1,2-Dipalmitoyl-3-O-(N-acetyl-6′-amino-6′-deoxy-α-d-glucopyranosyl)-sn-glycerol (1a)

30 mg, 91% yield; 1H-NMR (600 MHz, CDCl3): δ 6.26–6.18 (m, 1H, NH-CO), 5.24–5.20 (m, 1H, Hsn-2), 4.80 (d, J = 3.5 Hz, 1H, H-1), 4.38 (dd, J = 12.0, 3.6 Hz, 1H, Hsn-1a), 4.14 (dd, J = 12.0, 6.1 Hz, 1H, Hsn-1b), 3.93–3.87 (m, 1H, H-5), 3.77 (dd, J = 10.8, 5.1 Hz, 1H, Hsn-3a), 3.73 (t, J = 9.0 Hz, 1H, H-3), 3.62 (dd, J = 10.9, 5.8Hz, 1H, Hsn-3b), 3.58–3.55 (m, 1H, H-6a), 3.52–3.48 (m, 1H, H-2), 3.18–3.04 (m, 2H, H-4, H-6b), 2.32–2.28 (m, 4H, 2 × CO–CH2), 2.04 (s, 3H, NH–CO–CH3), 1.63–1.58 (m, 4H, 2 × CO–CH2–CH2), 1.29–1.24 (m, 48H, 2 × CH2–CH2–(CH2)12–CH3), 0.87 (t, J = 7.0 Hz, 6H, 2 × CH3); 13C-NMR (150 MHz, CDCl3): δ 172.8, 172.5, 171.7, 98.7, 72.4, 71.5, 70.3, 69.5, 69.1, 65.9, 61.6, 39.2, 33.5, 33.4, 31.2, 29.0–28.4, 24.2, 22.3, 22.0, 13.4; HR-ESI-MS m/z calcd. for C43H82NO10 [M + H]+ 772.5933, found 772.5944.

1,2-Dipalmitoyl-3-O-(N-hexanoyl-6′-amino-6′-deoxy-α-d-glucopyranosyl)-sn-glycerol (1b)

44 mg, 90% yield; 1H-NMR (600 MHz, CDCl3): δ 5.96 (dd, J = 8.0, 5.1 Hz, 1H, NH–CO), 5.25–5.21 (m, 1H, Hsn-2), 4.80 (d, J = 4.0 Hz, 1H, H-1), 4.38 (dd, J = 12.0, 4.0 Hz, 1H, Hsn-1a), 4.14 (dd, J = 12.0, 6.0 Hz, 1H, Hsn-1b), 4.01–3.97 (m, 1H, H-5), 3.78 (dd, J = 11.0, 4.8 Hz, 1H, Hsn-3a), 3.73 (t, J = 9.3 Hz, 1H, H-3), 3.62 (dd, J = 11.0, 6.0 Hz, 1H, Hsn-3b), 3.57 (dt, J = 9.7, 2.3 Hz, 1H, H-6a), 3.54–3.46 (m, 1H, H-2), 3.12–3.04 (m, 2H, H-4, H-6b), 2.32–2.28 (m, 4H, 2 × CO–CH2), 2.24 (dt, J = 7.4, 3.1 Hz, 2H, NH–CO–CH2), 1.63–1.58 (m, 6H, 3 × CO–CH2–CH2), 1.29–1.24 (m, 52H, 2 × CH2–CH2–(CH2)12–CH3, CH2–CH2–(CH2)2–CH3), 0.91–0.84 (m, 9H, 3 × CH3); 13C-NMR (150 MHz, CDCl3): δ 175.0, 172.7, 172.5, 98.7, 72.3, 71.6, 70.4, 69.2, 69.1, 66.1, 61.4, 39.0, 35.7, 33.5, 33.3, 31.2, 30.7, 29.0–28.4, 24.6, 24.1, 21.9, 21.6, 13.4, 13.2; HR-ESI-MS m/z calcd. for C47H90NO10 [M + H]+ 828.6559, found 828.6557.

1,2-Dipalmitoyl-3-O-(N-lauroyl-6′-amino-6′-deoxy-α-d-glucopyranosyl)-sn-glycerol (1c)

48 mg, 89% yield; 1H-NMR (600 MHz, CDCl3): δ 5.91 (dd, J = 8.1, 4.7 Hz, 1H, NH–CO), 5.24–5.20 (m, 1H, Hsn-2), 4.80 (d, J = 3.9 Hz, 1H, H-1), 4.38 (dd, J = 11.9, 4.0 Hz, 1H, Hsn-1a), 4.12 (dd, J = 11.9, 5.9 Hz, 1H, Hsn-1b), 4.01 (ddd, J = 15.0, 8.3, 2.4 Hz, 1H, H-5), 3.78 (dd, J = 11.0, 4.8 Hz, 1H, Hsn-3a), 3.73 (t, J = 9.3 Hz, 1H, H-3), 3.62 (dd, J = 11.0, 6.0 Hz, 1H, Hsn-3b), 3.57 (dt, J = 9.6, 2.5 Hz, 1H, H-6a), 3.52–3.48 (m, 1H, H-2), 3.09 (t, J = 9.6 Hz, 1H, H-4), 3.04 (ddd, J = 14.7, 4.5, 2.8 Hz, 1H, H-6b), 2.32–2.28 (m, 4H, 2 × CO–CH2), 2.25–2.22 (m, 2H, NH–CO–CH2), 1.63–1.58 (m, 6H, 3 × CO–CH2–CH2), 1.29–1.24 (m, 64H, 2 × CH2–CH2–(CH2)12–CH3, CH2–CH2–(CH2)8–CH3), 0.87 (t, J = 7.0 Hz, 9H, 3 × CH3); 13C-NMR (150 MHz, CDCl3): δ 175.0, 172.7, 172.5, 98.7, 72.3, 71.6, 70.4, 69.2, 69.1, 66.1, 61.3, 38.9, 35.7, 33.5, 33.3, 31.2, 29.0–28.4, 24.9, 24.1, 21.9, 13.3; HR-ESI-MS m/z calcd. for C53H102NO10 [M + H]+ 912.7498, found 912.7493.

1,2-Dipalmitoyl-3-O-(N-myristoyl-6′-amino-6′-deoxy-α-d-glucopyranosyl)-sn-glycerol (1d)

45 mg, 83% yield; 1H-NMR (600 MHz, CDCl3): δ 5.95 (dd, J = 7.9, 4.9 Hz, 1H, NH-CO), 5.24–5.20 (m, 1H, Hsn-2), 4.80 (d, J = 3.9 Hz, 1H, H-1), 4.38 (dd, J = 11.9, 4.0 Hz, 1H, Hsn-1a), 4.12 (dd, J = 12.0, 6.0 Hz, 1H, Hsn-1b), 3.99 (ddd, J = 14.9, 8.3, 2.7 Hz, 1H, H-5), 3.77 (dd, J = 11.0, 4.8 Hz, 1H, Hsn-3a), 3.73 (t, J = 9.3 Hz, 1H, H-3), 3.62 (dd, J = 11.0, 5.9 Hz, 1H, Hsn-3b), 3.57 (dt, J = 9.8, 2.7 Hz, 1H, H-6a), 3.48 (dd, J = 9.6, 3.9 Hz, 1H, H-2), 3.10 (t, J = 9.5 Hz, 1H, H-4), 3.06–3.03 (m, 1H, H-6b), 2.33–2.28 (m, 4H, 2 × CO–CH2), 2.26–2.17 (m, 2H, NH–CO–CH2), 1.62–1.58 (m, 6H, 3 × CO–CH2–CH2), 1.29–1.24 (m, 68H, 2 × CH2–CH2–(CH2)12–CH3, CH2–CH2–(CH2)10–CH3), 0.87 (t, J = 7.0 Hz, 9H, 3 × CH3); 13C-NMR (150 MHz, CDCl3): δ 175.0, 172.8, 172.5, 98.7, 72.4, 71.6, 70.4, 69.4, 69.2, 66.1, 61.5, 39.0, 35.8, 33.6, 33.4, 31.2, 29.1–28.4, 25.0, 24.2, 22.0, 13.4; HR-ESI-MS m/z calcd. for C55H106NO10 [M + H]+ 940.7811, found 940.7801.

1,2-Dipalmitoyl-3-O-(N-stearoyl-6′-amino-6′-deoxy-α-d-glucopyranosyl)-sn-glycerol (1e)

68 mg, 96% yield; 1H-NMR (600 MHz, CDCl3): δ 6.03–5.99 (m, 1H, NH-CO), 5.25–5.20 (m, 1H, Hsn-2), 4.80 (d, J = 4.0 Hz, 1H, H-1), 4.38 (dd, J = 11.9, 3.9 Hz, 1H, Hsn-1a), 4.13 (dd, J = 11.9, 6.0 Hz, 1H, Hsn-1b), 3.98–3.95 (m, 1H, H-5), 3.77 (dd, J = 10.9, 4.9 Hz, 1H, Hsn-3a), 3.73 (t, J = 9.0 Hz, 1H, H-3), 3.62(dd, J = 11.0, 5.9 Hz, 1H, Hsn-3b), 3.57 (dt, 1H, J = 9.2, 2.8 Hz, H-6a), 3.47 (dd, J = 9.2, 2.9 Hz, 1H, H-2), 3.13–3.05 (m, 2H, H-4, H-6b), 2.32–2.27 (m, 4H, 2 × CO–CH2), 2.26–2.19 (m, 2H, NH–CO–CH2), 1.63–1.56 (m, 6H, 3 × CO–CH2–CH2), 1.33–1.22 (m, 76H, 2 × CH2–CH2–(CH2)12–CH3, CH2–CH2–(CH2)14–CH3), 0.87 (t, J = 7.0 Hz, 9H, 3 × CH3); 13C-NMR (150 MHz, CDCl3): δ 174.9, 172.8, 172.6, 98.7, 72.4, 71.6, 70.4, 69.5, 69.2, 66.1, 61.6, 39.1, 35.8, 33.6, 33.4, 31.2, 29.0–28.4, 25.0, 24.2, 22.0, 13.4; HR-ESI-MS m/z calcd. for C59H114NO10 [M + H]+ 996.8437, found 996.8425.

1,2-Dipalmitoyl-3-O-(N-hydrocinnamoyl-6′-amino-6′-deoxy-α-d-glucopyranosyl)-sn-glycerol (1f)

30 mg, 88% yield; 1H-NMR (600 MHz, CDCl3): δ 7.30 (t, J = 7.5 Hz, 2H, ArH), 7.23–7.18 (m, 3H, ArH), 5.86 (dd, J = 8.1, 4.6 Hz, 1H, NH–CO), 5.22–5.18 (m, 1H, Hsn-2), 4.73 (d, J = 3.9 Hz, 1H, H-1), 4.36 (dd, J = 12.0, 4.0 Hz, 1H, Hsn-1a), 4.10 (dd, J = 12.0, 6.0 Hz, 1H, Hsn-1b), 3.99–3.96 (m, 1H, H-5), 3.74 (dd, J = 11.0, 4.7 Hz, 1H, Hsn-3a), 3.69 (t, J = 9.4 Hz, 1H, H-3), 3.59 (dd, J = 12.0, 6.0 Hz, 1H, Hsn-3b), 3.51 (dt, J = 9.9, 2.5 Hz, 1H, H-6a), 3.02–2.91 (m, 4H, H-2, H-6b, NHCO-CH2), 2.90 (t, J = 9.4 Hz, 1H, H-4), 2.58–2.54 (m, 2H, CH2Ph), 2.29–2.25 (m, 4H, 2 × CO–CH2), 1.60–1.57 (m, 4H, 2 × CO–CH2–CH2), 1.29–1.24 (m, 48H, 2 × CH2–CH2–(CH2)12–CH3), 0.87 (t, J = 7.0 Hz, 6H, 2 × CH3); 13C-NMR (150 MHz, CDCl3): δ 173.8, 172.9, 172.6, 139.7, 128.1, 127.7, 125.8, 98.7, 72.4, 71.6, 70.3, 69.2, 69.1, 66.1, 61.6, 39.1, 37.5, 33.6, 33.5, 31.3, 30.8, 29.4–28.5, 24.2, 22.1, 13.5; HR-ESI-MS m/z calcd. for C50H88NO10 [M + H]+ 862.6403, found 862.6394.

General Procedure for 2a–2c

Pd(OH)2/C (20%) was added to a mixture of compounds 21a–21c in THF/MeOH (1:1). Upon stirring for 4 h under H2, the mixture was filtered off and the solvent was evaporated. After purification by column chromatography (CH2Cl2/MeOH 20:1), compounds 2a–2c (84%–96%) were afforded as white solids.

1,2-Dipalmitoyl-3-O-(N-acetyl-2′-amino-2′-deoxy-α-d-glucopyranosyl)-sn-glycerol (2a)

40 mg, 92% yield; 1H-NMR (600 MHz, CDCl3): δ 6.57 (d, J = 8.9 Hz, 1H, NHCO), 5.20–5.18 (m, 1H, Hsn-2), 4.77 (d, J = 3.3 Hz, 1H, H-1), 4.46 (dd, J = 11.9, 4.1 Hz, 1H, Hsn-1a), 4.08–4.06 (m, 1H, H-2), 4.05 (dd, J = 11.9, 6.0 Hz, 1H, Hsn-1b), 3.91–3.88 (m, 1H, H-6a), 3.79–3.76 (m, 1H, H-6b), 3.75 (dd, J = 11.2, 4.9 Hz, 1H, Hsn-3a), 3.69–3.67 (m, 2H, H-5, H-3), 3.57–3.55 (m, 1H, H-4), 3.52 (dd, J = 11.0, 5.7 Hz, 1H, Hsn-3b), 2.32–2.28 (m, 4H, 2 × CO–CH2), 2.05 (s, 3H, NH–CO–CH3), 1.61–1.58 (m, 4H, 2 × CO–CH2–CH2), 1.29–1.24 (m, 48H, 2 × CH2–CH2–(CH2)12–CH3), 0.87 (t, J = 7.0 Hz, 6H, 2 × CH3); 13C-NMR (126 MHz, CDCl3): δ 173.56, 173.18, 172.40, 97.93, 73.14, 72.01, 70.33, 69.83, 65.66, 61.94, 61.31, 53.52, 34.28, 34.10, 31.92, 29.71–29.13, 24.92, 24.87, 23.09, 22.69, 14.12; HR-ESI-MS m/z calcd. for C43H82NO10 [M + H]+ 772.5933, found 772.5925.

1,2-Dipalmitoyl-3-O-(N-hexanoyl-2′-amino-2′-deoxy-α-d-glucopyranosyl)-sn-glycerol (2b)

32 mg, 84% yield; 1H-NMR (600 MHz, CDCl3): δ 6.36 (d, J = 8.6 Hz, 1H, NHCO), 5.20–5.17 (m, 1H, Hsn-2), 4.77 (d, J = 3.5 Hz, 1H, H-1), 4.45 (dd, J = 11.7, 4.2 Hz, 1H, Hsn-1a), 4.08 (m, 1H, H-2), 4.04 (dd, J = 11.9, 6.0 Hz, 1H, Hsn-1b), 3.89 (dd, J = 11.8, 2.2 Hz, 1H, H-6a), 3.79–3.76 (m, 1H, H-6b), 3.75 (dd, J = 11.0, 4.9 Hz, 1H, Hsn-3a), 3.70–3.64 (m, 2H, H-3, H-5), 3.56–3.54 (m, 1H, H-4), 3.52 (dd, J = 11.0, 5.6 Hz, 1H, Hsn-3b), 2.31–2.21 (m, 6H, 3 × CO–CH2), 1.60–1.57 (m, 6H, 3 × CO–CH2–CH2), 1.33–1.23 (m, 52H, CH2–(CH2)2–CH3, 2 × CH2–CH2–(CH2)12–CH3), 0.92–0.84 (m, 9H, 3 × CH3); 13C-NMR (126 MHz, CDCl3): δ 175.40, 173.48, 173.10, 97.91, 73.27, 72.00, 70.50, 69.81, 65.66, 61.94, 61.34, 53.42, 36.35, 34.27, 34.08, 31.92, 31.44, 30.54, 29.70–29.14, 25.26, 24.92, 24.87, 22.68, 22.38, 14.12, 13.98; HR-ESI-MS m/z calcd. for C47H90NO10 [M + H]+ 828.6559, found 828.6565.

1,2-Dipalmitoyl-3-O-(N-palmitoyl-2′-amino-2′-deoxy-α-d-glucopyranosyl)-sn-glycerol (2c)

65 mg, 96% yield; 1H-NMR (500 MHz, CDCl3): δ 6.35 (d, J = 8.1 Hz, 1H, NHCO), 5.20–5.17 (m, 1H, Hsn-2), 4.76 (d, J = 3.7 Hz, 1H, H-1), 4.53 (dd, J = 11.5, 4.7 Hz, 1H, Hsn-1a), 4.09–4.05 (m, 1H, H-2), 4.02 (dd, J = 11.5, 5.6 Hz, 1H, Hsn-1b), 3.88–3.82 (m, 2H, H-6 × 2), 3.80 (dd, J = 11.1, 4.5 Hz, 1H, Hsn-3a), 3.69 (t, J = 9.3 Hz, 1H, H-3), 3.64–3.56 (m, 2H, H-5, H-4), 3.52 (dd, J = 11.3, 5.4 Hz, 1H, Hsn-3b), 2.36–2.27 (m, 6H, 3 × CO–CH2), 1.61–1.58 (m, 6H, 3 × CO–CH2–CH2), 1.30–1.25 (m, 72H, 3 × CH2–CH2–(CH2)12–CH3), 0.88 (t, J = 6.9 Hz, 9H, 3 × CH3); 13C-NMR (126 MHz, CDCl3): δ 178.46, 173.54, 173.16, 97.77, 74.06, 71.72, 71.42, 69.83, 65.63, 62.01, 61.72, 53.58, 34.29, 34.09, 33.87, 31.91, 29.70–29.08, 25.62, 24.92, 24.88, 24.72, 22.68, 14.10; HR-MALDI-MS calcd. for C57H109NO10 Na [M + Na]+ 990.7949, found 990.7952.

1,2-O-Dipalmitoyl-3-O-(6′-O-palmitoyl-α-d-glucopyranosyl)-sn-glycerol (3a)

To a solution of 25 (58 mg, 0.11 mmol) in dry pyridine (5 mL), DMAP (3 mg, 0.02 mmol) and palmitoyl chloride (0.3 mL, 0.67 mmol) were added at 80 °C. The reaction mixture was stirred for 4 h, and then was concentrated and diluted with CH2Cl2 (30 mL) and washed sequentially with 1 M HCl and saturated NaHCO3. The organic phase was dried over Na2SO4, filtrated, concentrated. Purification by flash chromatography (AcOEt-petroleum ether 1:8) gave a crude. To a solution of the crude in 5 mL AcOEt/MeOH (1:1), 20% palladium hydroxide (80 mg) was added and stirred at r.t. under hydrogen atmosphere for 1 h. After filtration the solvent was evaporated and the residue was purified by silica column chromatography (AcOEt-petroleum ether 1:1) to afford 3a (66 mg, 62% for 2 steps) as a white solid. 1H-NMR (600 MHz, CDCl3): 5.26–5.23 (m, 1H, Hsn-2), 4.86 (d, J = 3.9 Hz, 1H, H-1), 4.55 (dd, J = 12.2, 4.0 Hz, 1H, H-6a), 4.40 (dd, J = 11.8, 4.3 Hz, 1H, Hsn-1a), 4.19 (dd, J = 12.3, 2.1 Hz, 1H, H-6b), 4.12 (dd, J = 11.9, 5.9 Hz, 1H, Hsn-1b), 3.84 (dd, J = 11.0, 4.6 Hz, 1H, Hsn-3a), 3.74–3.71 (m, 2H, H-3, H-5), 3.61 (dd, J = 11.0, 6.0 Hz, 1H, Hsn-3b), 3.50 (dd, J = 9.3, 3.7 Hz, 1H, H-2), 3.34 (t, J = 9.5 Hz, 1H, H-4), 2.37–2.29 (m, 6H, CH2 × 3), 1.63–1.60 (m, 6H, CH2 × 3), 1.29–1.24 (m, 72H, (CH2)12 × 3), 0.88 (t, J = 7.0 Hz, 9H, CH3 × 3); HR-MALDI-MS m/z calcd. for C57H108O11 Na [M + Na]+ 991.7784, found 991.7783.

General Procedure for Compounds 3b–3e

Pd(OH)2/C (20%) was added to a mixture of compounds 30–32 in AcOEt/MeOH (1:1). Upon stirring for 4 h under H2, the mixture was filtered off and the solvent was evaporated. After purification by column chromatography (CH2Cl2/MeOH 10:1), compounds 3c–3e were afforded as white solids.
Under the hydrogenation-reduction method mentioned above, only deiodinated product was found from the starting material 29. After a quick purification by column chromatography (petroleum ether/AcOEt 12:1), the deiodinated product converted to the target compound 3b as white solid by hydrogenation-reduction once more.

1,2-Dipalmitoyl-3-O-(6-deoxy-α-d-glucopyranosyl)-sn-glycerol (3b)

78 mg, 81% yield; 1H-NMR (600 MHz, CDCl3): δ5.27–5.22 (m, 1H, Hsn-2), 4.77 (d, J = 3.6 Hz, 1H, H-1), 4.38 (dd, J = 12.0, 3.7 Hz, 1H, Hsn-1a), 4.16 (dd, J = 12.0, 6.1 Hz, 1H, Hsn-1b), 3.80 (dd, J = 10.7, 5.4 Hz, 1H, Hsn-3a), 3.67 (t, J = 9.3, 1H, H-3), 3.65–3.60 (m, 1H, H-5), 3.58 (dd, J = 10.7, 5.8 Hz, 1H, Hsn-3b), 3.48 (dd, J = 9.1, 3.3 Hz, 1H, H-2), 3.13 (t, J = 9.3 Hz, 1H, H-4), 2.32–2.28 (m, 4H, 2 × CO–CH2), 1.62–1.58 (m, 4H, 2 × CO–CH2–CH2), 1.31–1.24 (m, 51H, 2 × CH2–CH2–(CH2)12–CH3, CH3), 0.87 (t, J = 7.0 Hz, 6H, 2 × CH3); 13C-NMR (150 MHz, CDCl3): δ 173.9, 173.4, 98.8, 75.4, 74.1, 72.3, 69.8, 67.7, 65.9, 62.4, 34.4, 34.2, 32.0, 29.6–29.2, 25.0, 24.9, 22.8, 17.7, 14.2; HR-ESI-MS m/z calcd. for C41H79O9 [M + H]+ 715.5719, found 715.5709.

1,2-Dipalmitoyl-3-O-α-d-glucosyluronate-sn-glycerol (3c)

84 mg, 76% yield; 1H-NMR (600 MHz, DMSO-D6): δ 5.12–5.10 (m, 1H, Hsn-2), 4.70 (d, J = 3.1 Hz, 1H, H-1), 4.33 (dd, J = 11.7, 1.5 Hz, 1H, Hsn-1a), 4.22 (dd, J = 12.1, 6.6 Hz, 1H, Hsn-1b), 3.81 (d, J = 9.9 Hz, 1H, H-5), 3.70 (dd, J = 11.0, 4.9 Hz, 1H, Hsn-3a), 3.55 (dd, J = 11.4, 5.4 Hz, 1H, Hsn-3b), 3.39 (t, J = 9.1, 1H, H-3), 3.31 (t, J = 9.4 Hz, 1H, H-4), 3.23 (dd, J = 9.2, 3.1 Hz, 1H, H-2), 2.30–2.22 (m, 4H, 2 × CO–CH2), 1.51–1.48 (m, 4H, 2 × CO–CH2–CH2), 1.34–1.20 (m, 48H, 2 × CH2–CH2–(CH2)12–CH3), 0.84 (t, J = 6.8 Hz, 6H, 2 × CH3); 13C-NMR (150 MHz, CDCl3): δ 172.3, 172.0, 170.7, 99.2, 72.3, 71.7, 71.4, 71.1, 69.3, 65.3, 61.9, 33.3, 33.2, 31.1, 28.9–28.2, 24.2, 24.1, 21.9, 13.6; HR-ESI-MS m/z calcd. for C41H76O11 Na [M + Na]+ 767.5280, found 767.5274.

1,2-Dipalmitoyl-3-O-(6′-O-sulfonato-α-d-glucopyranoside)-sn-glycerol (3d)

46 mg, 88% yield; 1H-NMR (600 MHz, DMSO-D6): δ 5.14–5.10 (m, 1H, Hsn-2), 4.63 (d, J = 2.8 Hz, 1H, H-1), 4.33–4.28 (m, 1H, Hsn-1a), 4.14–4.10 (m, 1H, Hsn-1b), 3.95–3.92 (m, 1H, Hsn-3a), 3.78–3.75 (m, 1H, Hsn-3b), 3.69–3.66 (m, 1H, H-6a), 3.48–3.35 (m, 3H, H-2, H-5, H-6b), 3.19–3.16 (m, 1H, H-3), 3.08–3.03 (m, 1H, H-4), 2.29–2.23 (m, 4H, 2 × CO–CH2), 1.53–1.49 (m, 4H, 2 × –CH2), 1.26–1.18 (m, 48H, (CH2)12 × 2), 0.85–0.82 (m, 6H, 2 × CH3); 13C-NMR (126 MHz, DMSO-D6): δ 172.88, 172.61, 99.42, 73.20, 72.13, 71.18, 70.45, 69.93, 66.10, 65.53, 62.73, 34.05, 33.89, 31.79, 29.57–28.95, 24.89, 22.58, 14.35; LR-ESI-MS m/z calcd. for C41H77O13S [M − H]− 809.5, found 809.7.

1,2-Dipalmitoyl-3-O-[α-d-galactopyranosyl-(1′′→6′)-α-d-glucopyranosyl]-sn-glycerol (3e)

109 mg, 89% yield; 1H-NMR (500 MHz, CDCl3/CD3OD = 1:1): δ 5.20–5.18 (m, 1H, Hsn-2), 4.86 (d, J = 2.7 Hz, 1H, H-1′′), 4.78 (d, J = 3.4 Hz, 1H, H-1′), 4.37 (dd, J = 12.0, 3.1 Hz, 1H, Hsn-1a), 4.11 (dd, J = 12.0, 6.6 Hz, 1H, Hsn-1b), 4.00 (dd, J = 10.9, 2.8 Hz, 1H, Hsn-3a), 3.94 (ds, 1H, H-4′′), 3.81 (m, 1H, H-3′′), 3.77 (dd, J = 10.9, 5.3 Hz, 1H, H-6a′), 3.73–3.69 (m, 3H, H-2′′, H-5′′, H-6a′′), 3.64–3.55 (m, 4H, H-3′, H-5′, H-6b′, Hsn-3b), 3.53–3.45 (m, 2H, H-4′, H-6b′′), 3.42 (dd, J = 9.5, 3.4 Hz, 1H, H-2′), 2.28–2.24 (m, 4H, O=CCH2 × 2), 1.56–1.52 (m, 4H, O=CCH2CH2 × 2), 1.28–1.20 (m, 48H, (CH2)12 × 2), 0.83 (t, J = 6.9 Hz, 6H, CH3 × 2); 13C-NMR (126 MHz, CDCl3): δ 173.87, 173.58 (O=C × 2), 99.42 (C-1′), 98.64 (C-1′′), 73.73 (C-5′), 71.81 (C-2′), 70.96 (C-3′), 70.51 (C-3′′), 70.32 (C-4′), 69.97 (Csn-2), 69.57 (C-5′′), 69.38 (C-2′′), 69.20 (C-4′′), 66.30 (Csn-3), 65.56 (C-6′), 62.50 (Csn-1), 61.39 (C-6′′), 34.18, 34.02 (COCH2 × 2), 31.83 (COCH2CH2 × 2), 29.61–29.03 (CH2 × 20), 24.82, 24.80 (CH2 × 2), 22.58 (CH2 × 2), 13.93 (CH3 × 2); HR-ESI-MS m/z calcd. for C47H88O15 Na [M + Na]+ 915.6015, found 915.6012.

General procedures for 4a–4c

Pd(OH)2/C (20%) was added to a mixture of compounds 35a–35c in THF/MeOH (1:1). Upon stirring for 4 h under H2, the mixture was filtered off and the solvent was evaporated. After purification by column chromatography (CH2Cl2/MeOH 20:1), compounds 4a–4c were afforded as white solids.

Bis(tetradecyl)methyl-N-acetyl-6-amino-6-deoxy-α-d-glucopyranoside (4a)

25 mg, 89% yield; 1H-NMR (500 MHz, CDCl3): δ 5.87 (dd, J = 7.5, 4.8 Hz, 1H, -NH), 4.89 (d, J = 4.2 Hz, 1H, H-1), 4.07–4.02 (m, 1H, H-6a), 3.73 (t, J = 9.3 Hz, 1H, H-3), 3.69–3.66 (m, 1H, H-5), 3.58–3.53 (m, 1H, OCH), 3.48 (dd, J = 9.2, 3.9 Hz, 1H, H-2), 3.13 (t, J = 9.4 Hz, 1H, H-4), 3.01–2.99 (m, 1H, H-6b), 2.06 (s, 3H, COCH3), 1.53–1.48 (m, 4H, OCH-(CH2)2), 1.34–1.25 (m, 48H, (CH2)12 × 2), 0.88 (t, J = 6.7 Hz, 6H, CH3 × 2); 13C-NMR (126 MHz, CDCl3): δ 172.65 (C=O), 97.49 (C-1), 78.96 (O–C), 73.71 (C-3), 72.69 (C-2), 71.15 (C-5), 70.07 (C-4), 40.01 (C-6), 34.57, 33.44, 32.08 (CH2 × 2), 29.97, 29.92, 29.85–29.81, 29.78, 29.77, 29.72 29.52 (CH2 × 2), 25.62, 25.22, 23.18 (CH3), 22.85 (CH2 × 2), 14.27 (CH3 × 2); HR-ESI-MS m/z calcd. for C37H74NO6 [M + H]+ 628.5511, found 628.5500.

Bis(tetradecyl)methyl-N-hexanoyl-6-amino-6-deoxy-α-d-glucopyranoside (4b)

34 mg, 94% yield; 1H-NMR (500 MHz, CDCl3): δ 5.88–5.86 (m, 1H, -NH), 4.89 (d, J = 4.2 Hz, 1H, H-1), 4.07–4.02 (m, 1H, H-6a), 3.72 (t, J = 9.1 Hz, 1H, H-3), 3.69–3.66 (m, 1H, H-5), 3.58–3.53 (m, 1H, OCH), 3.47 (dd, J = 9.1, 3.7 Hz, 1H, H-2), 3.09 (t, J = 9.4 Hz, 1H, H-4), 3.01–2.98 (m, 1H, H-6b), 2.26–2.22 (m, 2H, COCH2), 1.67–1.62 (m, 2H, COCH2CH2), 1.51–1.46 (m, 4H, OCH-(CH2)2), 1.33–1.25 (m, 52H, (CH2)2, (CH2)12 × 2), 0.96–0.86 (m, 9H, CH3 × 3). 13C-NMR (126 MHz, CDCl3): δ 175.80 (C=O), 97.48 (C-1), 78.91 (O–C), 73.66 (C-3), 72.70 (C-2), 71.20 (C-5), 70.13 (C-4), 39.84 (C-6), 36.60, 34.57, 33.43, 32.08 (CH2 × 2), 31.57, 29.97, 29.92, 29.85–29.72, 29.51 (CH2 × 2), 25.62, 25.48, 25.22, 22.84 (CH2 × 2), 22.50, 14.27 (CH3 × 2), 14.07 (CH3); HR-ESI-MS m/z calcd. for C41H82NO6 [M + H]+ 684.6137, found 684.6130.

Bis(tetradecyl)methyl-N-palmitoyl-6-amino-6-deoxy-α-d-glucopyranoside (4c)

43 mg, 96% yield; 1H-NMR (500 MHz, CDCl3): δ 5.83 (dd, J = 8.3, 4.7 Hz, 1H, -NH), 4.89 (d, J = 4.2 Hz, 1H, H-1), 4.07 (ddd, J = 14.4, 8.6, 2.0 Hz, 1H, H-6a), 3.72 (t, J = 9.3 Hz, 1H, H-3), 3.68–3.66 (m, 1H, H-5), 3.59–3.53 (m, 1H –OCH–), 3.46 (dd, J = 9.5, 3.9 Hz, 1H, H-2), 3.08 (t, J = 9.5 Hz, 1H, H-4), 3.37 (dt, J = 14.1, 4.4 Hz, 1H, H-6b), 2.25 (dt, J = 7.0, 1.3 Hz, 2H, –COCH2–), 1.65–1.62 (m, 2H, COCH2CH2), 1.52–1.48 (m, 4H, OCH(CH2)2), 1.33–1.22 (m, 72H, COCH2CH2(CH2)12, (CH2)12 × 2), 0.88 (t, J = 6.9 Hz, 9H, CH3 × 3); 13C-NMR (126 MHz, CDCl3): δ 175.86 (C=O), 97.48 (C-1), 78.92 (O–C), 73.68 (C-3), 72.73 (C-2), 71.22 (C-5), 70.09 (C-4), 39.85 (C-6), 36.65, 34.58, 33.43, 32.08 (CH2 × 3), 29.98, 29.92, 29.85–29.77, 29.72, 29.63, 29.52 (CH2 × 2), 29.46, 29.44, 25.82, 25.61, 25.22, 22.85 (CH2 × 3), 14.28 (CH3 × 3), HR-MALDI-MS m/z calcd. for C51H101O6N Na [M + Na]+ 846.7527, found 846.7520.

3.2. Biological Methods

3.2.1. Cell Culture and Virus Infection

Madin-Darby canine kidney (MDCK) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in RPM1640 medium (Hangzhou genome biomedical Ltd., Hangzhou, China) supplemented with 10% FBS, 100 units/mL of penicillin and 100 μg/mL of streptomycin. Influenza virus (A/Puerto Rico/8/34 [H1N1]; PR/8) was propagated in 10-d-old embryonated eggs for 3 d at 36.5 °C.
The virus infection experiments in vitro were performed as described previously [36]. In brief, virus propagation solution was diluted in PBS containing 0.2% bovine serum albumin and was added to cells at the indicated multiplicity of infection (MOI). Virus was allowed to adsorb for 60 min at 4 °C. After removing the virus inoculum, cells were maintained in infecting media (RPM1640, 4 μg/mL trypsin, Hangzhou genome biomedical Ltd.) at 37 °C in 5% CO2.

3.2.2. Infectivity Antiviral Assays

The antiviral activity was evaluated by the cytopathic effects (CPE) inhibition assay. MDCK cell cultures in 96-well plates were firstly infected with IAV (MOI = 1.0), and then treated with different compounds in triplicate after removal of the virus inoculum. After 48 h incubation, the cells were fixed with 4% formaldehyde for 20 min at room temperature. After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for 30 min. The plates were washed and dried, and the intensity of crystal violet staining for each well was measured at 570 nm. The virus inhibition (%) was calculated by the equation:
Virus inhibition (%) = [(Asample570 − Avirus570)/(Amock570 − Avirus570)] × 100
where, Amock570 was the absorbance without virus infection, Asample570 was absorbance with virus infection and drug treatment, Avirus570 was absorbance with virus infection but without drugs.

3.2.3. In Vivo Experiments

Four-week-old female Kunming mice (average weight, 13.0 ± 1.0 g) were housed and studied under protocols approved by the Animal Ethics Committee of Ocean University of China. Briefly, mice were randomly divided into different experimental groups (10 mice/group). The drugs administration started 1 d prior to virus inoculation. Virus-infected control group (virus control) and uninfected control group (normal control) received 1 × PBS as a placebo. On the day of virus inoculation, mice were lightly anaesthetized and each was inoculated intranasally with PR8 virus (4 HAU/mouse) diluted in 40 μL of 1 × PBS. Two hours after inoculation, mice received oral administration of 1d (5 or 10 mg/kg/d) or oseltamivir (20 mg/kg/d), and the treatments were repeated once daily for the entire experiment. Mice were weighed and killed on Day 4 after inoculation, and lungs were removed and weighed. The lung index was calculated by the following equation using the obtained values:
Lung index = [Lung weight (g)/Mice weight (g)] × 100
By this index, the severity of lung injury in pneumonia mice was evaluated. Subsequently, lung specimens of animals from each experimental group were homogenized in 1 × PBS (pH 7.4) for determination of viral titers by neuraminidase activity assay.
In the survival experiments, 10 mice per group were intranasally infected with PR/8 virus (Wuhan Institute of Virology, Wuhan, China) (6 HAU/mouse) at Day 0. IAV infected mice received oral administration of compound 1d (5 or 10 mg/kg/d) or oseltamivir (20 mg/kg/d), and the virus control group and normal control group received PBS as a placebo. The drugs administration started 1 d prior to virus infection and was repeated once daily during the course of the experiment, and survival was assessed in all groups for 14 d after infection.

4. Conclusions

In this study, four series of glycoglycerolipids (1–4), seventeen analogs of AGGL, were designed and prepared. The successful total synthesis afforded enough samples for anti-IAV screening, and the results indicated that the acylamino and glycerol groups of the glycolipids were essential for the inhibitory effect on IAV multiplication. Furthermore, the potent derivative 1d was able to significantly improve survival and decrease pulmonary viral titers in IAV-infected mice, which could provide novel insights into deeper exploration of the unique aminoglycoglycerolipids in drug discovery of pneumonia diseases caused by viruses.

Supplementary Materials

The following are available online at www.mdpi.com/1660-3397/14/6/116/s1, The Chemical synthesis of compounds 7, 9–11, 12a–12f, 13, 14, 16, 18–20, 21a–21c, 24–27, 29–34, 35a–35c and their 1H-NMR and MS Data.

Acknowledgments

This work was supported by NSFC-Shandong Joint Fund for Marine Science Research Centers (Grant No. U1406402), National Natural Science Foundation of China (81302811 and 31500646), Taishan Scholar Project Special Funds, Major Science and Technology Project in Shandong Province (2015ZDJS04002), Special Fund for Marine Scientific Research in the Public Interest (201405038).

Author Contributions

C.L., W.W., G.Y. and H.G. initiated the project. C.L. and L.R. designed the experimental work and analyzed the data. J. Z., H.M. and L.S. were in charge of the chemical synthesis. W.W. and X.Z. was in charge of biological evaluation. L.R. and J.Z. were in charge of writing and checking the manuscript. All the authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Marinedrugs 14 00116 g001 1024
Figure 1. Structures of the AGGL and synthesized compounds 1–4.
Figure 1. Structures of the AGGL and synthesized compounds 1–4.
Marinedrugs 14 00116 g001
Marinedrugs 14 00116 sch001 1024
Scheme 1. Reagents and conditions: (a) TsCl, DMAP, py, 79%; (b) (i) NaN3, DMF; (ii) BnBr, NaH, DMF, 86% for 2 steps; (c) (i) NBS, CO(CH3)2/H2O; (ii) CNCCl3, DBU, CH2Cl2, 89% for 2 steps; (d) (S)-isopropylideneglycerol, TMSOTf, Et2O, 97%; (e) (i) p-TsOH, MeOH, 89%; (ii) CH3(CH2)14COCl, DMAP, py, 96%; (f) 10% Pd/C, H2, AcOEt/MeOH, 95%; (g) (i) EDCI, HOBt, ROH, CH2Cl2 or Ac2O, CH2Cl2, 41%–89%; (h) 20% Pd(OH)2/C, H2, THF/i-PrOH, 83%–96%.
Scheme 1. Reagents and conditions: (a) TsCl, DMAP, py, 79%; (b) (i) NaN3, DMF; (ii) BnBr, NaH, DMF, 86% for 2 steps; (c) (i) NBS, CO(CH3)2/H2O; (ii) CNCCl3, DBU, CH2Cl2, 89% for 2 steps; (d) (S)-isopropylideneglycerol, TMSOTf, Et2O, 97%; (e) (i) p-TsOH, MeOH, 89%; (ii) CH3(CH2)14COCl, DMAP, py, 96%; (f) 10% Pd/C, H2, AcOEt/MeOH, 95%; (g) (i) EDCI, HOBt, ROH, CH2Cl2 or Ac2O, CH2Cl2, 41%–89%; (h) 20% Pd(OH)2/C, H2, THF/i-PrOH, 83%–96%.
Marinedrugs 14 00116 sch001
Marinedrugs 14 00116 sch002 1024
Scheme 2. Reagents and conditions: (a) (i) Imidazole-1-sulfonyl azide hydrochloride, CuSO4, K2CO3, MeOH; (ii) Ac2O, py, 81% for 2 steps; (b) TolSH, BF3·Et2O, CH2Cl2, 85%; (c) (i) CH3ONa, CH3OH; (ii) BnBr, NaH, DMF, 98% for 2 steps; (d) (i) NBS, (CH3)2CO/H2O; (ii) CNCCl3, DBU, CH2Cl2, 82% for 2 steps; (e) (S)-isopropylideneglycerol, TMSOTf, Et2O, 93%; (f) (i) p-TsOH, MeOH; (ii) CH3(CH2)14COCl, DMAP, py, 82% for 2 steps; (g) 10% Pd/C, H2, AcOEt/MeOH, 54%; (h) (i) Ac2O, CH2Cl2 or ROH, EDCI, HOBt, CH2Cl2, 64%–96%; (ii) 20% Pd(OH)2/C, H2, THF/MeOH, 84%–96%. (i).
Scheme 2. Reagents and conditions: (a) (i) Imidazole-1-sulfonyl azide hydrochloride, CuSO4, K2CO3, MeOH; (ii) Ac2O, py, 81% for 2 steps; (b) TolSH, BF3·Et2O, CH2Cl2, 85%; (c) (i) CH3ONa, CH3OH; (ii) BnBr, NaH, DMF, 98% for 2 steps; (d) (i) NBS, (CH3)2CO/H2O; (ii) CNCCl3, DBU, CH2Cl2, 82% for 2 steps; (e) (S)-isopropylideneglycerol, TMSOTf, Et2O, 93%; (f) (i) p-TsOH, MeOH; (ii) CH3(CH2)14COCl, DMAP, py, 82% for 2 steps; (g) 10% Pd/C, H2, AcOEt/MeOH, 54%; (h) (i) Ac2O, CH2Cl2 or ROH, EDCI, HOBt, CH2Cl2, 64%–96%; (ii) 20% Pd(OH)2/C, H2, THF/MeOH, 84%–96%. (i).
Marinedrugs 14 00116 sch002
Marinedrugs 14 00116 sch003 1024
Scheme 3. Reagents and conditions: (a) (i) NBS, (CH3O)2CO/H2O, 90%; (ii) CNCCl3, DBU, CH2Cl2, 92%; (b) (S)-isopropylideneglycerol, TMSOTf, Et2O, 83%; (c) p-TsOH, MeOH, 82%; (d) (i) CH3(CH2)14COCl, DMAP, py; (ii) 20% Pd(OH)2/C, H2, AcOEt/MeOH, 62% for 2 steps; (e) (i) CSA, MeOH, 93%; (ii) CH3(CH2)14COCl, DMAP, py, 88%; (f) TBAF, THF, 84%; (g) PPh3, I2, imidazole, toluene, 70%; (h) TEMPO, BAIB, CH2Cl2/H2O, 92%; (i) SO3·py, DMF, 86%; (j) 28, TMSOTf, Et2O, 75%; (k) 20% Pd(OH)2/C, H2, AcOEt/MeOH.
Scheme 3. Reagents and conditions: (a) (i) NBS, (CH3O)2CO/H2O, 90%; (ii) CNCCl3, DBU, CH2Cl2, 92%; (b) (S)-isopropylideneglycerol, TMSOTf, Et2O, 83%; (c) p-TsOH, MeOH, 82%; (d) (i) CH3(CH2)14COCl, DMAP, py; (ii) 20% Pd(OH)2/C, H2, AcOEt/MeOH, 62% for 2 steps; (e) (i) CSA, MeOH, 93%; (ii) CH3(CH2)14COCl, DMAP, py, 88%; (f) TBAF, THF, 84%; (g) PPh3, I2, imidazole, toluene, 70%; (h) TEMPO, BAIB, CH2Cl2/H2O, 92%; (i) SO3·py, DMF, 86%; (j) 28, TMSOTf, Et2O, 75%; (k) 20% Pd(OH)2/C, H2, AcOEt/MeOH.
Marinedrugs 14 00116 sch003
Marinedrugs 14 00116 sch004 1024
Scheme 4. Reagents and conditions: (a) TMSOTf, Et2O, 49%; (b) 10% Pd/C, H2, AcOEt/MeOH, 88%; (c) Ac2O, CH2Cl2 or ROH, EDCI, HOBt, CH2Cl2, 58%–87%; (d) 20% Pd(OH)2/C, H2, THF/MeOH, 89%–96%.
Scheme 4. Reagents and conditions: (a) TMSOTf, Et2O, 49%; (b) 10% Pd/C, H2, AcOEt/MeOH, 88%; (c) Ac2O, CH2Cl2 or ROH, EDCI, HOBt, CH2Cl2, 58%–87%; (d) 20% Pd(OH)2/C, H2, THF/MeOH, 89%–96%.
Marinedrugs 14 00116 sch004
Marinedrugs 14 00116 g002 1024
Figure 2. The therapeutic effect of 1d on influenza A virus-infected mice. (A) Viral titers in lungs evaluated by performing neuraminidase activity assay. The mean fluorescence intensity of each sample was used to evaluate the relative neuraminidase content. The intensity of normal control group was assigned values of 1.0 and the data presented as mean ± S.D. (n = 3). Significance: # p < 0.05 vs. normal control group; * p < 0.05 vs. virus control group (placebo); (B) Survival rate. IAV infected mice received intraperitoneal therapy once daily with drugs or placebo for the entire experiment. Results are expressed as percentage of survival, evaluated daily for 15 d. Significance: * p < 0.05 vs. control group (placebo).
Figure 2. The therapeutic effect of 1d on influenza A virus-infected mice. (A) Viral titers in lungs evaluated by performing neuraminidase activity assay. The mean fluorescence intensity of each sample was used to evaluate the relative neuraminidase content. The intensity of normal control group was assigned values of 1.0 and the data presented as mean ± S.D. (n = 3). Significance: # p < 0.05 vs. normal control group; * p < 0.05 vs. virus control group (placebo); (B) Survival rate. IAV infected mice received intraperitoneal therapy once daily with drugs or placebo for the entire experiment. Results are expressed as percentage of survival, evaluated daily for 15 d. Significance: * p < 0.05 vs. control group (placebo).
Marinedrugs 14 00116 g002
Table
Table 1. The inhibitory effect of aminoglycoglycerolipids and analogs on IAV replication in vitro a.
Table 1. The inhibitory effect of aminoglycoglycerolipids and analogs on IAV replication in vitro a.
SampleVirus Inhibition (%)SampleVirus Inhibition (%)SampleVirus Inhibition (%)
1a27.12a6.13a12.0
1b46.12b50.13b6.4
1c29.32c19.03c12.5
1d46.14a4.03d3.7
1e9.44b2.93e51.5
1f10.44c0AGGL25.5
----Ribavirin50.0
a Performed at a concentration of 50 μM.
Table
Table 2. The anti-IAV activities of compound 1d in vivo.
Table 2. The anti-IAV activities of compound 1d in vivo.
GroupsDose (mg/kg/d)Lung Index a (X ± SD)Inhibitory Rate (%)
Virus control-1.24 ± 0.31 #0
Normal control-0.79 ± 0.10-
Oseltamivir200.87 ± 0.41 *29.7
1d51.05 ± 0.3115.3
1d101.15 ± 0.497.3
Values are means ± S.D. (n = 10); # p < 0.05 vs. normal control group; t-test, * p < 0.05 vs. virus control group; t-test; a Lung index = [lung weight (g)/mice weight (g)] × 100.

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Ren, L.; Zhang, J.; Ma, H.; Sun, L.; Zhang, X.; Yu, G.; Guan, H.; Wang, W.; Li, C. Synthesis and Anti-Influenza A Virus Activity of 6′-amino-6′-deoxy-glucoglycerolipids Analogs. Mar. Drugs 2016, 14, 116. https://doi.org/10.3390/md14060116

AMA Style

Ren L, Zhang J, Ma H, Sun L, Zhang X, Yu G, Guan H, Wang W, Li C. Synthesis and Anti-Influenza A Virus Activity of 6′-amino-6′-deoxy-glucoglycerolipids Analogs. Marine Drugs. 2016; 14(6):116. https://doi.org/10.3390/md14060116

Chicago/Turabian Style

Ren, Li, Jun Zhang, Haizhen Ma, Linlin Sun, Xiaoshuang Zhang, Guangli Yu, Huashi Guan, Wei Wang, and Chunxia Li. 2016. "Synthesis and Anti-Influenza A Virus Activity of 6′-amino-6′-deoxy-glucoglycerolipids Analogs" Marine Drugs 14, no. 6: 116. https://doi.org/10.3390/md14060116

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