Synthesis and anti-influenza a virus activity of 6′-amino-6′-deoxy-glucoglycerolipids analogs

Article abstract of DOI:10.3390/md14060116

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 func

Full text of DOI:10.3390/md14060116

marine drugs
Article
Synthesis and Anti-Influenza A Virus Activity of
61-amino-61-deoxy-glucoglycerolipids Analogs
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; renli@vip.163.com (L.R.); junerjasmine@163.com (J.Z.); zhangxtxm@163.com (H.M.);
sunlinlinenjoylife@163.com (L.S.); xiaoshuangzhang89@126.com (X.Z.); glyu@ouc.edu.cn (G.Y.);
hsguan@ouc.edu.cn (H.G.)
* Correspondence: wwwakin@ouc.edu.cn (W.W.); lchunxia@ouc.edu.cn (C.L.);
Tel.: +86-532-8203-1980 (W.W.); +86-532-8203-2030 (C.L.); Fax: +86-532-8203-3054 (W.W. & C.L.)
† These authors contributed equally to this work.
Academic Editor: Khalid A. El Sayed
Received: 1 April 2016; Accepted: 3 June 2016; Published: 18 June 2016
Abstract: A series of aminoglucoglycerolipids derivatives had been synthesized, including
61-acylamido-glucoglycerolipids 1a
bearing different fatty acids, glucosyl diglycerides 3a
and ether-linked glucoglycerolipids 4a
–
1f and corresponding 21-acylamido-glucoglycerolipids 2a
–
2c
3e bearing different functional groups at C-61
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.
Keywords: synthesis; aminoglucoglycerolipids; anti-influenza A virus
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 [
the 2009 outbreak of swine-origin influenza A/H1N1 [ ] continued to affect many countries, and
caused over 18,000 deaths worldwide [ ]. Considering the frequency of influenza pandemics, it is
urgent to develop novel anti-IAV drugs with high efficiency.
Glycoglycerolipids occur widely in marine algae [ ], cyanobacteria [
The natural glycoglycerolipids possess various biological activities, such as anti-tumor [12
1]. Moreover,
2,3
4
5–
8
9–
11] and higher plants.
13],
,
anti-viral [14–17], and anti-inflammatory activities [18], which make them valuable molecular targets for
further investigation [19]. In our previous work [20–22] synthetic aminoglycoglycerolipids derived from
marine natural product 1,2-dipalmitoyl-3-(N-palmitoyl-61-amino-61-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.
Mar. Drugs 2016, 14, 116; doi:10.3390/md14060116
www.mdpi.com/journal/marinedrugs

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Figure 1. Structures of the AGGL and synthesized compounds 1–4.
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-61position of the sugar ring and the glyceride
a1ttached to anomeric carbon, including 61-acylamido-glucoglycerolipids 1a
–
1f and corresponding
3e bearing
–4c with double-tailed alkyl
2 -acylamido-glucoglycerolipids 2a–2c bearing different fatty acids, glucosyl diglycerides 3a–
1
different functional groups at C-6 and ether-linked glucoglycerolipids 4a
alcohol. The synthetic routes were described as follows.
2.1.1. Synthesis of Compounds 1a–1f
61-amide-61-deoxy-
were synthesized as illustrated in Scheme 1. Activation of thioglycoside
chloride, followed by treatment with sodium azide and benzyl bromide in one-pot method gave
compound . The p-tolylthio group of 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 in 89% as the glycosyl donor. Glycosylation of (S)-isopropylideneglycerol with
donor under trimethylsilyl trifluoromethanesulfonate (TMSOTf) activation in absolute ether afforded
as an inseparable anomeric mixture ( = 12:1) in 97% yield. The small amount of -anomer can
be removed by silica gel column chromatography in the following step of hydrolysis with TsOH
in methanol (89%). Treatment the -anomer diol with excess palmitoyl chloride in the presence of
α
-D-glucoglycerolipids 1a
–
1f bearing different fatty acids at amino group
24] with p-toluene sulfonyl
5
[
6
7
7
8
8
9
α
/
β
β
α
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 C61–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 61-amides 1a 1f, we also synthesized the related 21-amide glycoglycerolipids
2c in order to compare the influence on biological activity of the position of amide group. As shown
–
2a
–
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
After hydrolyzation, esterification and reduction according to the above procedures of 11
20 (54%) was producecd and isolated by silica gel column chromatography. Acylation of the resulting
(α
/
β
= 2:1).
,
α-anomer
1
C2 -NH2 followed by hydrogenation with H2/Pd(OH)2 generated the target compounds 2a–2c.

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Scheme 1. Reagents and conditions: (
DMF, 86% for 2 steps; ( ) (i) NBS, CO(CH3)2/H2O; (ii) CNCCl3, DBU, CH2Cl2, 89% for 2 steps;
) (S)-isopropylideneglycerol, TMSOTf, Et2O, 97%; ( ) (i) p-TsOH, MeOH, 89%; (ii) CH3(CH2)14COCl,
DMAP, py, 96%; ( ) 10% Pd/C, H2, AcOEt/MeOH, 95%; ( ) (i) EDCI, HOBt, ROH, CH2Cl2 or Ac2O,
CH2Cl2, 41%–89%; (h) 20% Pd(OH)2/C, H2, THF/i-PrOH, 83%–96%.
a) TsCl, DMAP, py, 79%; (b) (i) NaN3, DMF; (ii) BnBr, NaH,
c
(
d
e
f
g
Scheme 2. Reagents and conditions: (
K2CO3, MeOH; (ii) Ac2O, py, 81% for 2 steps; (
CH3OH; (ii) BnBr, NaH, DMF, 98% for 2 steps; (
CH2Cl2, 82% for 2 steps; ( ) (S)-isopropylideneglycerol, TMSOTf, Et2O, 93%; (
(ii) CH3(CH2)14COCl, DMAP, py, 82% for 2 steps; ( ) 10% Pd/C, H2, AcOEt/MeOH, 54%; (
a
) (i) Imidazole-1-sulfonyl azide hydrochloride, CuSO4,
) TolSH, BF3 Et2O, CH2Cl2, 85%; ( ) (i) CH3ONa,
) (i) NBS, (CH3)2CO/H2O; (ii) CNCCl3, DBU,
) (i) p-TsOH, MeOH;
) (i) Ac2O,
b
¨
c
d
e
f
g
h
CH2Cl2 or ROH, EDCI, HOBt, CH2Cl2, 64%–96%; (ii) 20% Pd(OH)2/C, H2, THF/MeOH, 84%–96%. (i).
2.1.3. Synthesis of Compounds 3a–3e
Glucosyl diglycerides 3a
illustrated in Scheme 3. Starting from the synthetic compound 22
–
3e bearing different functional groups at C-61 were synthesized as
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

Mar. Drugs 2016, 14, 116
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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
1
oxidation of the 6 -OH of 27 into the corresponding carboxylic acid via a TEMPO/BAIB oxidation [30].
1
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
Deprotection of 29–32 by Pd-catalyzed hydrogenation afforded the desired compounds 3b–3e.
α
-(1 6)-linked disaccharide 32 in 75% yield.
Ñ
Scheme 3. Reagents and conditions: (
CH2Cl2, 92%; ( ) (S)-isopropylideneglycerol, TMSOTf, Et2O, 83%; (
) (i) CH3(CH2)14COCl, DMAP, py; (ii) 20% Pd(OH)2/C, H2, AcOEt/MeOH, 62% for 2 steps;
) (i) CSA, MeOH, 93%; (ii) CH3(CH2)14COCl, DMAP, py, 88%; ( ) TBAF, THF, 84%; ( ) PPh3, I2,
) TEMPO, BAIB, CH2Cl2/H2O, 92%; ( ) SO3 py, DMF, 86%; ( 28, TMSOTf,
a) (i) NBS, (CH3O)2CO/H2O, 90%; (ii) CNCCl3, DBU,
b
c) p-TsOH, MeOH, 82%;
(
(
d
e
f
g
imidazole, toluene, 70%; (
Et2O, 75%; (k) 20% Pd(OH)2/C, H2, AcOEt/MeOH.
h
i
¨
j)
2.1.4. Synthesis of Compounds 4a–4c
Diacylglycerol residue was substituted by double-tailed alcohol linked at the anomeric carbon
of the 61-amide-61-deoxy-
-D-glucose to afford the derivatives 4a 4c As shown in Scheme 4,
trichloroacetimidate donor reacted with bis (tetradecyl) carbinol 32 33] under TMSOTf activation
in Et2O to afford the absolute -linked intermediate 33 in 49% yield. After reduction, acylation and
deprotection, target analogs 4a–4c were obtained.
α
– .
8
[
α
Scheme 4. Reagents and conditions: (
) Ac2O, CH2Cl2 or ROH, EDCI, HOBt, CH2Cl2, 58%–87%; (
89%–96%.
a
) TMSOTf, Et2O, 49%; (
b
) 10% Pd/C, H2, AcOEt/MeOH, 88%;
(
c
d) 20% Pd(OH)2/C, H2, THF/MeOH,

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2.2. Biological Evaluation
2.2.1. Inhibition of Influenza A Virus Multiplication in Vitro
61-amino-61-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
saturated fatty acyl chains of C6 and C14 at 61-NH2, displayed notable virus inhibition among the
aminoglucoglycerolipids bearing linear fatty acids with C2–C18 length (1a ), suggesting that the
optimum length of saturated fatty acyl chains may be important for their anti-IAV effects in vitro
µM. Compounds 1b and 1d, which contained
–
e
.
1
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
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
µM, which was more than that of AGGL (25.5%) at the same concentration,
–
–
–
–
that the acylamino and glycerol groups of the glycolipids were essential for the 1inhibition of IAV
multiplication. Surprisingly, glucosyl diglyceride 3e bearing another galactose at C-6 exerted excellent
1
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.
Table 1. The inhibitory effect of aminoglycoglycerolipids and analogs on IAV replication in vitro a.
Sample
Virus Inhibition (%)
Sample
Virus Inhibition (%)
Sample
Virus Inhibition (%)
1a
1b
1c
1d
1e
1f
-
27.1
46.1
29.3
46.1
9.4
2a
2b
2c
4a
4b
4c
-
6.1
50.1
19.0
4.0
2.9
0
3a
3b
3c
3d
3e
12.0
6.4
12.5
3.7
51.5
25.5
50.0
10.4
-
AGGL
Ribavirin
-
a
Performed at a concentration of 50 µM.
2.2.2. IAV Infection in Vivo
Considering the structure characteristic and bioactivity in vitro, potent compound 1d
(IC50 = 60.8 M, close to that of Ribavirin (IC50 = 49.7 M)) in the preliminary anti-viral screening was
µ
µ
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].

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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
normal control group; * p < 0.05 vs. virus control group (placebo); (
˘
S.D. (n = 3). Significance: # p < 0.05 vs.
) Survival rate. IAV infected mice
B
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).
Table 2. The anti-IAV activities of compound 1d in vivo.
Groups
Dose (mg/kg/d)
Lung Index a (X ˘ SD)
Inhibitory Rate (%)
Virus control
Normal control
Oseltamivir
1d
-
-
20
5
1.24 ˘ 0.31 #
0.79 ˘ 0.10
0.87 ˘ 0.41 *
1.05 ˘ 0.31
1.15 ˘ 0.49
0
-
29.7
15.3
7.3
1d
10
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)] ˆ 100.
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.

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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.,
1
Ltd., Qingdao, China). H-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
1
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):
6.26–6.18 (m, 1H, N -CO), 5.24–5.20 (m, 1H,
sn-2), 4.80 (d, J = 3.5 Hz, 1H, H-1), 4.38 (dd, J = 12.0, 3.6 Hz, 1H, sn-1a), 4.14 (dd, J = 12.0, 6.1 Hz,
1H, sn-1b), 3.93–3.87 (m, 1H, H-5), 3.77 (dd, J = 10.8, 5.1 Hz, 1H, sn-3a), 3.73 (t, J = 9.0 Hz, 1H,
H-3), 3.62 (dd, J = 10.9, 5.8Hz, 1H, sn-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–C 2), 2.04 (s, 3H, NH–CO–C 3), 1.63–1.58 (m, 4H,
CO–CH2–CH2), 1.29–1.24 (m, 48H, 2 CH2–CH2–(C 2)12–CH3), 0.87 (t, J = 7.0 Hz, 6H, 2 3);
δ
H
H
H
H
H
H
,
ˆ
H
H
2
ˆ
ˆ
H
ˆ
CH
13C-NMR (150 MHz, CDCl3):
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,
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,
found 772.5944.
1
1
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):
5.96 (dd, J = 8.0, 5.1 Hz, 1H, N –CO), 5.25–5.21
(m, 1H, sn-2), 4.80 (d, J = 4.0 Hz, 1H, H-1), 4.38 (dd, J = 12.0, 4.0 Hz, 1H, sn-1a), 4.14 (dd, J = 12.0,
6.0 Hz, 1H, sn-1b), 4.01–3.97 (m, 1H, H-5), 3.78 (dd, J = 11.0, 4.8 Hz, 1H, sn-3a), 3.73 (t, J = 9.3 Hz,
1H, H-3), 3.62 (dd, J = 11.0, 6.0 Hz, 1H, sn-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–C 2), 1.63–1.58 (m, 6H, 3 CO–CH2–C 2), 1.29–1.24 (m, 52H, 2 CH2–CH2–(C 2)12–CH3,
CH2–CH2–(C 2)2–CH3), 0.91–0.84 (m, 9H, 3 175.0, 172.7, 172.5,
3); 13C-NMR (150 MHz, CDCl3):
δ
H
H
H
H
H
H
,
ˆ
H
ˆ
H
H
ˆ
H
H
ˆ
C
δ
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
1
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):
5.91 (dd, J = 8.1, 4.7 Hz, 1H, N –CO), 5.24–5.20
(m, 1H, sn-2), 4.80 (d, J = 3.9 Hz, 1H, H-1), 4.38 (dd, J = 11.9, 4.0 Hz, 1H, sn-1a), 4.12 (dd, J = 11.9,
5.9 Hz, 1H, sn-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, sn-3a), 3.73
(t, J = 9.3 Hz, 1H, H-3), 3.62 (dd, J = 11.0, 6.0 Hz, 1H, sn-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),
δ
H
H
H
H
H
H

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2.32–2.28 (m, 4H, 2
1.29–1.24 (m, 64H, 2
ˆ
CO–C
H
2), 2.25–2.22 (m, 2H, NH–CO–C
2)12–CH3, CH2–CH2–(C
3); 13C-NMR (150 MHz, CDCl3):
CH δ 175.0, 172.7, 172.5, 98.7, 72.3, 71.6, 70.4, 69.2, 69.1, 66.1, 61.3,
H
2), 1.63–1.58 (m, 6H, 3
ˆ
CO–CH2–CH2),
ˆ
CH2–CH2–(C
H
H
2)8–CH3), 0.87 (t, J = 7.0 Hz, 9H,
3
ˆ
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
1
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):
5.95 (dd, J = 7.9, 4.9 Hz, 1H, N -CO), 5.24–5.20
(m, 1H, sn-2), 4.80 (d, J = 3.9 Hz, 1H, H-1), 4.38 (dd, J = 11.9, 4.0 Hz, 1H, sn-1a), 4.12 (dd, J = 12.0,
6.0 Hz, 1H, sn-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, sn-3a), 3.73
(t, J = 9.3 Hz, 1H, H-3), 3.62 (dd, J = 11.0, 5.9 Hz, 1H, sn-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,
CO–C 2), 2.26–2.17 (m, 2H, NH–CO–C 2), 1.62–1.58 (m, 6H, 3 CO–CH2–C 2), 1.29–1.24
(m, 68H, 2 CH2–CH2–(C 2)12–CH3, CH2–CH2–(C 2)10–CH3), 0.87 (t, J = 7.0 Hz, 9H, 3 3);
13C-NMR (150 MHz, CDCl3):
δ
H
H
H
H
H
H
2
ˆ
H
ˆ
H
ˆ
H
H
H
ˆ
CH
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
1
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): 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, sn-1a), 4.13 (dd, J = 11.9, 6.0 Hz,
1H, sn-1b), 3.98–3.95 (m, 1H, H-5), 3.77 (dd, J = 10.9, 4.9 Hz, 1H, sn-3a), 3.73 (t, J = 9.0 Hz, 1H,
H-3), 3.62(dd, J = 11.0, 5.9 Hz, 1H, sn-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–C 2), 2.26–2.19 (m, 2H,
NH–CO–C 2), 1.63–1.56 (m, 6H, 3 CO–CH2–C 2), 1.33–1.22 (m, 76H, 2 CH2–CH2–(C 2)12–CH3,
CH2–CH2–(C 2)14–CH3), 0.87 (t, J = 7.0 Hz, 9H, 3 174.9, 172.8,
3); 13C-NMR (150 MHz, CDCl3):
δ
H
H
H
H
,
ˆ
H
H
ˆ
H
ˆ
ˆ
H
H
C
H
δ
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
1
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):
7.30 (t, J = 7.5 Hz, 2H, Ar ), 7.23–7.18 (m, 3H,
), 5.86 (dd, J = 8.1, 4.6 Hz, 1H, N –CO), 5.22–5.18 (m, 1H, sn-2), 4.73 (d, J = 3.9 Hz, 1H, H-1), 4.36
sn-1a), 4.10 (dd, J = 12.0, 6.0 Hz, 1H, sn-1b), 3.99–3.96 (m, 1H, H-5), 3.74
sn-3a), 3.69 (t, J = 9.4 Hz, 1H, H-3), 3.59 (dd, J = 12.0, 6.0 Hz, 1H, sn-3b),
H-6b, NHCO-C 2), 2.90 (t, J = 9.4 Hz, 1H,
CO–C 2), 1.60–1.57 (m, 4H, 2 CO–CH2–C 2),
2)12–CH3), 0.87 (t, J = 7.0 Hz, 6H, 2
3); 13C-NMR (150 MHz,
δ
H
ArH
H
H
(dd, J = 12.0, 4.0 Hz, 1H,
(dd, J = 11.0, 4.7 Hz, 1H,
H
H
H
H
3.51 (dt, J = 9.9, 2.5 Hz, 1H, H-6a), 3.02–2.91 (m, 4H, H-2
H-4), 2.58–2.54 (m, 2H, C 2Ph), 2.29–2.25 (m, 4H, 2
1.29–1.24 (m, 48H, 2 CH2–CH2–(C
CDCl3):
,
H
H
ˆ
H
ˆ
H
ˆ
H
ˆ
CH
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.

Mar. Drugs 2016, 14, 116
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1
1
1,2-Dipalmitoyl-3-O-(N-acetyl-2 -amino-2 -deoxy-α-D-glucopyranosyl)-sn-glycerol (2a)
1
40 mg, 92% yield; H-NMR (600 MHz, CDCl3):
δ
6.57 (d, J = 8.9 Hz, 1H, N
sn-1a), 4.08–4.06 (m, 1H,
sn-1b), 3.91–3.88 (m, 1H, H-6a), 3.79–3.76 (m, 1H, H-6b), 3.75
sn-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,
2), 2.05 (s, 3H, NH–CO–C 3), 1.61–1.58 (m, 4H,
CH2–CH2–(C 2)12–CH3), 0.87 (t, J = 7.0 Hz, 6H, 2 3);
HCO), 5.20–5.18
(m, 1H,
H-2), 4.05 (dd, J = 11.9, 6.0 Hz, 1H,
(dd, J = 11.2, 4.9 Hz, 1H,
5.7 Hz, 1H, sn-3b), 2.32–2.28 (m, 4H, 2
CO–CH2–C 2), 1.29–1.24 (m, 48H, 2
13C-NMR (126 MHz, CDCl3):
Hsn-2), 4.77 (d, J = 3.3 Hz, 1H, H-1), 4.46 (dd, J = 11.9, 4.1 Hz, 1H, H
H
H
,
H
ˆ
ˆ
CO–C
H
H
2
ˆ
H
H
ˆ
CH
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
1
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):
6.36 (d, J = 8.6 Hz, 1H, N CO), 5.20–5.17 (m, 1H,
sn-2), 4.77 (d, J = 3.5 Hz, 1H, H-1), 4.45 (dd, J = 11.7, 4.2 Hz, 1H, sn-1a), 4.08 (m, 1H, H-2), 4.04
(dd, J = 11.9, 6.0 Hz, 1H, sn-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, sn-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, sn-3b), 2.31–2.21 (m, 6H, 3 CO–C 2), 1.60–1.57 (m, 6H, 3 CO–CH2–C 2), 1.33–1.23 (m, 52H,
CH2–CH2–(C 2)12–CH3), 0.92–0.84 (m, 9H, 3
3); 13C-NMR (126 MHz,
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,
δ
H
H
H
H
H
,
H
ˆ
H
ˆ
H
CH2–(C
CDCl3):
H
δ
2)2–CH3, 2
ˆ
H
ˆ
CH
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
1
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):
6.35 (d, J = 8.1 Hz, 1H, N CO), 5.20–5.17 (m, 1H,
sn-2), 4.76 (d, J = 3.7 Hz, 1H, H-1), 4.53 (dd, J = 11.5, 4.7 Hz, 1H, sn-1a), 4.09–4.05 (m, 1H, H-2), 4.02
(dd, J = 11.5, 5.6 Hz, 1H, sn-3a), 3.69
sn-1b), 3.88–3.82 (m, 2H, H-6 ˆ 2), 3.80 (dd, J = 11.1, 4.5 Hz, 1H,
(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, sn-3b),
2.36–2.27 (m, 6H, 3 CO–C 2), 1.61–1.58 (m, 6H, 3 CO–CH2–C 2), 1.30–1.25 (m, 72H,
CH2–CH2–(C 2)12–CH3), 0.88 (t, J = 6.9 Hz, 9H, 3 178.46,
3); 13C-NMR (126 MHz, CDCl3):
δ
H
H
H
H
H
,
H
ˆ
H
ˆ
H
3
ˆ
H
ˆ
CH
δ
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
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
1
2 steps) as a white solid. H-NMR (600 MHz, CDCl3): 5.26–5.23 (m, 1H,
H
H
sn-2), 4.86 (d, J = 3.9 Hz, 1H,
sn-1a), 4.19 (dd, J = 12.3, 2.1
sn-3a), 3.74–3.71
Hsn-3b), 3.50 (dd, J = 9.3, 3.7 Hz, 1H, H-2), 3.34
H-1), 4.55 (dd, J = 12.2, 4.0 Hz, 1H, H-6a), 4.40 (dd, J = 11.8, 4.3 Hz, 1H,
Hz, 1H, H-6b), 4.12 (dd, J = 11.9, 5.9 Hz, 1H,
(m, 2H, H-3 H-5), 3.61 (dd, J = 11.0, 6.0 Hz, 1H,
Hsn-1b), 3.84 (dd, J = 11.0, 4.6 Hz, 1H, H
,
(t, J = 9.5 Hz, 1H, H-4), 2.37–2.29 (m, 6H, CH2 ˆ 3), 1.63–1.60 (m, 6H, CH2 ˆ
3), 1.29–1.24 (m, 72H,
(CH2
)
12 ˆ 3), 0.88 (t, J = 7.0 Hz, 9H, CH3 ˆ 3); HR-MALDI-MS m/z calcd. for C57H108O11 Na [M + Na]+
991.7784, found 991.7783.

Mar. Drugs 2016, 14, 116
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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):
H-1), 4.38 (dd, J = 12.0, 3.7 Hz, 1H, sn-1a), 4.16 (dd, J = 12.0, 6.1 Hz, 1H,
5.4 Hz, 1H, sn-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,
sn-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–C 2),
1.62–1.58 (m, 4H, 2 CO–CH2–C 2), 1.31–1.24 (m, 51H, 2 CH2–CH2–(C 2)12–CH3, C 3), 0.87
(t, J = 7.0 Hz, 6H, 2 173.9, 173.4, 98.8, 75.4, 74.1, 72.3, 69.8, 67.7,
3); 13C-NMR (150 MHz, CDCl3):
δ5.27–5.22 (m, 1H,
H
sn-2), 4.77 (d, J = 3.6 Hz, 1H,
sn-1b), 3.80 (dd, J = 10.7,
H
H
H
H
ˆ
H
ˆ
H
ˆ
H
H
ˆ
C
H
δ
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,
H
H
H
sn-2), 4.70
sn-1b), 3.81
sn-3b), 3.39
(d, J = 3.1 Hz, 1H, H-1), 4.33 (dd, J = 11.7, 1.5 Hz, 1H,
(d, J = 9.9 Hz, 1H, H-5), 3.70 (dd, J = 11.0, 4.9 Hz, 1H,
H
H
sn-1a), 4.22 (dd, J = 12.1, 6.6 Hz, 1H,
sn-3a), 3.55 (dd, J = 11.4, 5.4 Hz, 1H,
(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,
CO–C 2), 1.51–1.48 (m, 4H, 2 CO–CH2–C 2), 1.34–1.20 (m, 48H, 2 CH2–CH2–(C 2)12–CH3),
0.84 (t, J = 6.8 Hz, 6H, 2 172.3, 172.0, 170.7, 99.2, 72.3, 71.7, 71.4,
3); 13C-NMR (150 MHz, CDCl3):
2
ˆ
H
ˆ
H
ˆ
H
ˆ
CH
δ
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
1,2-Dipalmitoyl-3-O-(6 -O-sulfonato-α-D-glucopyranoside)-sn-glycerol (3d)
46 mg, 88% yield; 1H-NMR (600 MHz, DMSO-D6):
1H, H-1), 4.33–4.28 (m, 1H, sn-1a), 4.14–4.10 (m, 1H,
(m, 1H,
3.08–3.03 (m, 1H, H-4), 2.29–2.23 (m, 4H, 2
(CH2 2), 0.85–0.82 (m, 6H, 2
3); 13C-NMR (126 MHz, DMSO-D6):
δ
5.14–5.10 (m, 1H,
sn-1b), 3.95–3.92 (m, 1H,
H-5 H-6b), 3.19–3.16 (m, 1H, H-3),
2), 1.53–1.49 (m, 4H, 2 –C 2), 1.26–1.18 (m, 48H,
172.88, 172.61, 99.42,
H
sn-2), 4.63 (d, J = 2.8 Hz,
H
H
H
sn-3a), 3.78–3.75
H
sn-3b), 3.69–3.66 (m, 1H, H-6a), 3.48–3.35 (m, 3H, H-2
CO–C
,
,
ˆ
H
ˆ
H
δ
)
ˆ
ˆ
CH
12
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.
11
1
1,2-Dipalmitoyl-3-O-[α-D-galactopyranosyl-(1 Ñ6 )-α-D-glucopyranosyl]-sn-glycerol (3e)
109 mg, 89% yield; 1H-NMR (500 MHz, CDCl3/CD3OD = 1:1):
5.20–5.18 (m, 1H,
(d, J = 2.7 Hz, 1H, H-111), 4.78 (d, J = 3.4 Hz, 1H, H-11), 4.37 (dd, J = 12.0, 3.1 Hz, 1H,
δ
H
H
sn-2), 4.86
sn-1a), 4.11
Hsn-1b), 4.00 (dd, J = 10.9, 2.8 Hz, 1H, Hsn-3a), 3.94 (ds, 1H, H-4 ), 3.81 (m, 1H,
11
(dd, J = 12.0, 6.6 Hz, 1H,
H-311), 3.77 (dd,1J = 10.9, 5.3 Hz, 1H, H-6a1), 3.73–3.691(1m, 3H, H-2
1 1 1 1
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
,
H-5
11
11
,
H-6a11), 3.64–3.55 (m, 4H,
(m, 4H, O=CCH2 ˆ 2), 1.56–1.52 (m, 4H, O=CCH2CH2 ˆ 2), 1.28–1.20 (m, 48H, (CH2
)
ˆ
2), 0.83
12
1
2), 99.42 (C-1 ), 98.64
(t, J = 6.9 Hz, 6H, CH3 ˆ 2); 13C-NMR (126 MHz, CDCl3):
δ
173.87, 173.58 (O=C
ˆ
11
1
11
1
1
1
11
1
11
11
(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
11
(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
ˆ
2), 31.83

Mar. Drugs 2016, 14, 116
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(COCH2CH2
ˆ
2), 29.61–29.03 (CH2
ˆ
20), 24.82, 24.80 (CH2
ˆ
2), 22.58 (CH2
ˆ
2), 13.93 (CH3
ˆ
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)
1
25 mg, 89% yield; H-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, OC ), 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
H
(m, 1H, H-6b), 2.06 (s, 3H, COC
H
3), 1.53–1.48 (m, 4H, OCH-(C
H
2)2), 1.34–1.25 (m, 48H, (CH2
δ 172.65 (C=O), 97.49 (C-1), 78.96
)
ˆ
2),
12
0.88 (t, J = 6.7 Hz, 6H, CH3 ˆ 2); 13C-NMR (126 MHz, CDCl3):
(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
ˆ
ˆ
2), 29.97,
2), 14.27
29.92, 29.85–29.81, 29.78, 29.77, 29.72 29.52 (CH2
ˆ
2), 25.62, 25.22, 23.18 (CH3), 22.85 (CH2
(CH3 ˆ 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):
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,
OC ), 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, COC
(C 2)2, (CH2
δ 5.88–5.86 (m, 1H, -NH), 4.89 (d, J = 4.2 Hz, 1H,
H
H
2), 1.67–1.62 (m, 2H, COCH2C
ˆ
H
2), 1.51–1.46 (m, 4H, OCH-(C
H
2)2), 1.33–1.25 (m, 52H,
175.80 (C=O), 97.48
H
)
2), 0.96–0.86 (m, 9H, CH3 ˆ 3). 13C-NMR (126 MHz, CDCl3):
δ
12
(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
14.27 (CH3
ˆ
2), 31.57, 29.97, 29.92, 29.85–29.72, 29.51 (CH2
ˆ
2), 25.62, 25.48, 25.22, 22.84 (CH2
ˆ
2), 22.50,
ˆ
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)
1
43 mg, 96% yield; H-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 –OC –), 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, –COC 2–), 1.65–1.62 (m, 2H,
2)2), 1.33–1.22 (m, 72H, COCH2CH2(C 2)12, (CH2 2),
175.86 (C=O), 97.48 (C-1), 78.92 (O–C),
H
H
COCH2C
H
2), 1.52–1.48 (m, 4H, OCH(C
H
H
)
ˆ
12
0.88 (t, J = 6.9 Hz, 9H, CH3 ˆ 3); 13C-NMR (126 MHz, CDCl3):
δ
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
ˆ
3), 29.98,
3), 14.28
29.92, 29.85–29.77, 29.72, 29.63, 29.52 (CH2
ˆ
2), 29.46, 29.44, 25.82, 25.61, 25.22, 22.85 (CH2
ˆ
(CH3 ˆ 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

Mar. Drugs 2016, 14, 116
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˝
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
trypsin, Hangzhou genome biomedical Ltd.) at 37 C in 5% CO2.
µg/mL
˝
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 p%q “ rpAsample570 ´ Avirus570q{pAmock570 ´ Avirus570qs ˆ 100
(1)
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
were lightly anaesthetized and each was inoculated intranasally with PR8 virus (4 HAU/mouse)
diluted in 40 L of 1 PBS. Two hours after inoculation, mice received oral administration of 1d
ˆ
PBS as a placebo. On the day of virus inoculation, mice
µ
ˆ
(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 “ rLung weight pgq{Mice weight pgqs ˆ 100
(2)
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
determination of viral titers by neuraminidase activity assay.
ˆ
PBS (pH 7.4) for
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.

Mar. Drugs 2016, 14, 116
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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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