Open Journal of Medicinal Chemistry, 2013, 3, 55-59 Published Online September 2013 (
-Galactosyl Phytosphingosine 2,6’-Diamide as an Inducer
of Invariant Natural Killer T Cell
Ying-Cheng Huang1, Wei-Ting Chen2, Shu-Fan Tien2, Ho-Lien Huang2,
Chun-Nan Yeh3, Kun-I Lin2,4*, Chung-Shan Yu2,5*
1Department of Neurosurgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University, Taoyuan, Taiwan
2Department of Biomedical Engineering and Environmental Sciences, National Tsing-Hua University, Hsinchu, Taiwan
3Department of Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University, Taoyuan, Taiwan
4Department of Obsterics and Gynecology, Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan
5Institute of Nuclear Engineering and Science, National Tsing-Hua University, Hsinchu, Taiwan
Email: *
Received April 15, 2013; revised May 14, 2013; accepted May 28, 2013
Copyright © 2013 Ying-Cheng Huang et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
-galactosyl phytosphingosine 2,6’-diamide analogs were prepared from 2,6’-diamino
sine and the aromatic-bearing carboxylic acids. After purification with High Performance Liquid Chromatography, a
flowcytometry for the four compounds for stimulation of human V
11+ NKT cell populations was carried out.
Additional keto groups on the acyl chains of the 2,6’-diamide compound were associated with the enhanced stimulating
Keywords: Phytosphingosine; HPLC; Keto; Flow Cytometry; Simulation
1. Introduction
-galactosyl ceramide (
-GalCer) [1,2] has been associ-
ated with the treatment of immunological disorders such
as certain tumors, infections and autoimmune diseases
(Figure 1) [3-6]. Its immune-stimulation potency is im-
plicated in the activation of the invariant natural killer T
cells (iNKT cells) to trigger the release of cytokines. The
semi-invariant T cell receptor (TCR) which is encoded
by an invariant V
18 chain in humans recognizes
the complex formed from glycolipid antigen and MHC
class I-like protein CD1d. After binding, iNKT cells
could be induced, thereby leading to a rapid release of
the relevant proinflammatory cytokines such as IFN-
and IL-4 which are correlated with Th1 and Th2 pathway,
respectively. Because of the opposing activities affected
by the simultaneously secreted Th1 and Th2 cytokines
that may counteract the therapeutic applications of
GalCer, [7] endeavor has been continuously devoted to
the development of potent
-GalCer analogues with a
biased Th1 and Th2 profile [8-12].
We have recently reported a chemical preparation of
-galactosyl phytosphingosine (
analogue that could be coupled with carboxylic acids to
generate a library consisting of 40 members of 2,6’-dia-
-GalSph analogues. [13] Among them, the com-
pound that exerted minimal cytotoxicity could induce a
moderate proliferation of iNKT cells (Figure 2). Because
the potential compound bears two aromatic groups, a
further study based on this structural feature is therefore
pursued. The present work is aimed to prepare the four
diamide compounds 1-4 with structural variation on the
linker or the aromatic ring (Figure 3). The bioactivities
of the four compounds were also addressed to correlate
with their structures (SAR).
2. Results and Discussion
Preparation of 6-amino
-galactosyl phytosphingosine 5
could be found in the relevant work as described before.
[13] Conjugation of 6-amino
-galactosyl phytosphin-
gosine 5 with four carboxylic acids furnished the diamide
products 1-4 with a fair yield (27% - 46%). Whereas a
number of syntheses of ceramide analogs have been re-
ported, the concern about purity has been rarely addres-
sed. [14] In our case, the four products were still insuffi-
ciently pure after flash chromatography. Further purifica-
tion with High Performance Liquid Chromatography
*Corresponding authors.
opyright © 2013 SciRes. OJMC
Figure 1. Structure of
Figure 2. 2,6’-di-4-butylphenyl
-GalSph amide discovered
from parallel solution phase synthesis exhibited moderate
iNKT cell proliferation.
(HPLC) was needed. Above all, compound 4 after HPLC
purification was additionally purified by recrystalliza-
tion. Whereas the spectra for 1H- and 13C-NMR of the
four compounds were satisfactory, some unknown peaks
related to compound 4 were marked. For example, two
peak clusters were found in 19F-NMR, i.e. cluster 1:δ =
210.26 and 210.28 ppm and cluster 2:δ = 220.17 and
221.67 ppm. The peak cluster 1 was unlikely due to the
residual carboxylic acids because both the HPLC chro-
matograms of the crude and purified compounds showed
solely one peak. Furthermore, the underestimated inte-
grals from 7.6 to 8.0 ppm in 1H-NMR that corresponded
to aromatic protons precluded the presence of an unre-
acted aromatic-containing carboxylic acid. Hence, the
presence of a conformer was speculated to account for
the present observation.
Compared to compound 1 and 4, which exhibited limi-
ted activity, compound 2 and compound 3 both induced
meaningful cellular populations (Figure 4). This could
be rationalized by the contribution of the keto group. In
contrast, other structural alterations did not significantly
affect the bioactivity. For example, the bioactivity of
compound 2 was tolerated by the introduction of the bi-
phenyl group. Furthermore, the presence of the germinal
dimethyl groups or a double bond did not enhance the
activity. The role played by the keto group was therefore
elucidated by molecular docking (Figure 5). The binding
site for docking was defined by employing the crystallo0
graphic data for a complex of
-GalCer with V
chain of iNKT cells (PDB:4EN3). [15] Interestingly, all
compounds (1-4) failed to well dock to the site. Only
when adopting a site defined by a complex of
(PDB code: 3SDX) [16] and using a truncated compound
3, in which the aryl group on the amide chain of the
sugar moiety was removed deliberately while the rest
Figure 3. Structures of the four diamide compounds 1-4.
groups including the keto group and the other amide resi-
due on backbone were retained, an acceptable docking
result arose with a score of 65 points. The two hydroxy
groups of the sugar moiety i.e. 2-OH and 3-OH could act
as hydrogen bond acceptors with Arg94. In addition,
3-OH could act as hydrogen bond donor with ASP93.
The 6-amido group of the sugar moiety did not form in-
termolecular hydrogen bond with the neighboring amino
acids but form intramolecular hydrogen bonding with
4-OH. In spite of these stabilizations gained by hydrogen
bondings between the sugar moiety and the surrounding
amino acid residues, an inherent difference in stereo-
chemistry between
-GalPhy and
-GalCer could not be
overlooked. In brief, the present
-GalSph analogs 2 and
3 with an additional keto group on the amide linkage of
sugar moiety and backbone warrants a further study.
3. Experimental
3.1. General
All reagents and solvents were purchased from Sig-
ma-Aldrich, Malingkrodt, Acros, Alfa, Tedia, or Fluka.
All preparations of compounds were routinely conducted
in dried glassware under a positive pressure of nitrogen
at room temperature unless otherwise noted. CH2Cl2 was
dried over CaH2. DMF and NEt3 were distilled under
reduced pressure prior use. Reagents and solvents were
of reagent grade. The eluents for chromatography in-
cluding MeOH and CHCl3 were reagent grade and used
without further purification. NMR spectroscopy includ-
ing 1H-NMR (500 MHz) and 13C-NMR (125 MHz,
DEPT-135) was measured on Varian UnityInova 500
MHz. Low-resolution mass spectrometry (LRMS) was
performed on an ESI-MS spectrometry employing
VARIAN 901-MS Liquid Chromatography Tandem Mass
Q-Tof Spectrometer at the Department of Chemistry of
National Tsing-Hua University (NTHU). High-Resolu-
tion Mass Spectrometry (HRMS) was performed using a
Copyright © 2013 SciRes. OJMC
Figure 4. Potencies of analogs 1-4 for stimulation of human
11+ NKT cell populations. Peripheral blood mono-
nuclear cells (PBMC) from a normal healthy donor were
incubated with each individual compound at a final con-
centration of 100 nM. After 14 days of culture, NKT cell
frequencies were determined by flow cytometry. NKT cell
frequencies were defined as the percentage of V
NKT cells among gated lymphocytes in the upper right (UR)
corner (Q2) for each case. Shown here are the profiles of
PBMC harvested from 14-day cultures containing (control)
vehicle alone (DMSO, UN) and the compounds 1-4 tested.
varian HPLC (prostar series ESI/APCI) coupled mass
detector of Varian 901-MS (FT-ICR Mass) and triple
quadrapole. Flash chromatography was performed using
Geduran Si 60 silica gel (230 - 400 mesh). The final
conjugation products were further purified and analyzed
by HPLC, consisting of an Angilent 1100 pump and a
linear UVIS detector (254 nm). A ZORBAX SILcolumn
(250 mm × 9.4 mm, 5 m, Si-100) was used as the sta-
tionary phase and the eluents of a combination of MeOH
and CH2Cl2 with a flow rate of 3 mL/min were used as
Figure 5. Molecular docking of truncated compound 3 onto
the complex of CD1d and V
24+ chain. Software: Discovery
Studio 2.1 with Ligandfit. Active site was defined by using
Auto-Vα24-CD1d-β-GalCer complex (PDB entry of 3SDX).
the mobile phase.
3.2. General Procedure for Preparation of the
Four Amide Products
mido)-3,4- dihydrox yoctadecyl)ox y)-3,4,5 -trihydr oxytetra
The conjugating procedure adopted the protocol as
reported before [9].
To a mixture of Gemfibrozil (4 eq., 42 mg, 0.167
mmol), HBTU (4.0 equiv, 64 mg, 0.167 mmol) and DMF
(1 mL) was added diisopropylethylamine (8.0 equiv, 60
μL, 0.334 mmol) under N2. After stirring for 30 min,
TLC (EtOAc:n-hexane = 2.5:7.5) indicated the forma-
tion of the ester intermediate (Rf = 0.73) and consump-
tion of the starting Gemfibrozil (Rf = 0.12). To this mix-
ture was added the solution of compound 5 ((1 equiv, 20
mg, 41.8 μmol) in DMF (1 mL). After stirring for 24 h,
TLC (MeOH/CH2Cl2 = 1:8) indicated the formation of
the product 1 (Rf = 0.80) and consumption of the active
ester (Rf = 0.90). The mixture was concentrated under
reduced pressure. The residue obtained was purified with
HPLC using column chromatography (MeOH:CH2Cl2 =
1:10 MeOH:CH2Cl2 = 1:9) to afford the crude product
1 in 38% yield (15 mg). The sample was further purified
using MeOH/CH2Cl2 = 1:9 as eluent at a flow rate of 3
mL/min to afford an oil (7 mg) which was recorded as
retention time at tR = 5.79 min.
Anal. C54H90N2O11, M (calcd.) = 942.6 (m/z), ESI +
Q-TOF:M = 798.6 (m/z), [M + H]+ = 943.9 (100%), [M +
Na]+ = 965.9 (50%); HRMS (ESI) calcd. [M + H]+ =
943.66229 (m/z), found [M + H]+ = 943.66552 (m/z), =
Copyright © 2013 SciRes. OJMC
3.42 ppm; 1H-NMR (500 MHz, CD3OD):
0.88 (t, 3H,
Haliphatic), 1.18 - 1.20 (m, 12H, Haliphatic), 1.25 - 1.39 (m,
24H, Haliphatic), 1.54 - 1.72 (m, 10H, Haliphatic), 2.12 (s, 6H,
2 × OPhMe), 2.26 (s, 6H, 2 × OPhMe), 3.24 - 3.29 (m,
1H), 3.50 - 3.57 (m, 2H), 3.60 - 3.71 (m, 3H), 3.74 - 3.84
(m, 4H), 3.88 - 3.89 (m, 4H, PhO-CH2), 4.18 - 4.22 (m,
1H), 4.88 (d, 1H, J1,2 = 3.5 Hz, H-1), 6.59 (d, 2H, J = 7.5
Hz, ArH), 6.64 (s, 2H, ArH), 6.93 (d, 2H, J = 7.5 Hz,
ArH); 13C-NMR (125 MHz, CD3OD):δ 14.44 (CH3,
Caliphatic), 16.08 (CH3), 16.12 (CH3), 21.50 (CH3), 23.74
(CH2), 25.68 (CH3), 25.86 (CH3), 25.95 (CH3), 26.02
(CH3), 26.28 (CH2), 26.34 (CH2), 27.04 (CH2), 30.48
(CH2), 30.77 (CH2), 30.81 (CH2), 30.85 (CH2), 30.93
(CH2), 33.08 (CH2), 38.64 (CH2), 41.39 (CH2), 43.02 (C),
43.10 (C), 51.97 (CH), 68.09 (CH2), 69.13 (CH2), 69.23
(CH2), 70.03 (CH), 70.43 (CH), 71.25 (CH), 71.37 (CH),
72.93 (CH), 75.55 (CH), 101.29 (CH, C-1), 113.09 (CH,
aromatic), 121.78 (CH, aromatic), 121.83 (CH, aromatic),
124.42 (C, aromatic), 131.20 (CH, aromatic), 131.23
(CH, aromatic), 137.61 (C, aromatic), 158.33 (C, aro-
matic), 179.65 (C, amide), 180.91 (C, amide).
4-([1,1’-b iphenyl]-4- yl)-N-(((2 R,3R,4S,5 R,6S)-6-(((2 S
H-pyran-2-yl)methyl)-4-oxobutanamide (2).
The same procedure as that for amide coupling of
compound 1 was used. Fenbufen (42 mg, 0.167 mmol)
was used as the carboxylic acid for the conjugation. The
required reagents were the same as that described above.
Column chromatography was performed by using eluents
of MeOH:CH2Cl2 = 1:11 to provide the white crude
product 2 in 40% yield (16 mg). After further purifica-
tion with HPLC using eluents of MeOH:CH2Cl2 = 1:11, a
white solid was obtained in an overall yield of 15% (6
mg). tR = 9.04 min. Anal. C56H74N2O11, HRMS (ESI) M
(calcd.) = 950.5293 (m/z), [M + H]+ = 951.5371 m/z, [M
H] = 949.5214 m/z; found [M + H]+ = 951.5289 (m/z),
= 8.62 ppm; [M H] = 949.5216 (m/z), = 0.21 ppm;
1H-NMR (600 MHz, CD3OD):δ 0.79 (t, 3H, J = 7.2 Hz,
Haliphatic), 1.15 - 1.34 (m, 24 H, Haliphatic), 1.44 - 1.46 (m,
1H, Haliphatic), 1.57 - 1.60 (m, 1H, Haliphatic), 2.54 - 2.62 (m,
4H aliphatic), 3.18 - 3.36 (m, 6H), 3.50 - 3.65 (m, 4H),
3.72-3.78 (m, 3H), 3.85 (dd, 1H, J2,3 = 10.5, J2,1 = 3.5 Hz,
H-2), 4.15 - 4.16 (m, 1H), 4.82 (d, 1H, J1,2 = 3.5 Hz, H-1),
7.38 - 7.97 (m, 18H, Phamide); 13C-NMR (150 MHz,
CD3OD):δ 20.50 (C), 29.17 (CH2), 32.35 (CH2), 35.86
(CH2), 36.16 (CH2), 36.22 (CH2), 36.26 (CH2), 36.31
(CH2), 38.42 (CH2), 39.27 (CH2), 40.26 (CH2), 40.32
(CH2), 46.15 (CH2), 55.81 (CH2), 73.55 (CH2), 75.55
(CH), 76.44 (CH), 78.64 (CH), 81.04 (CH), 105.94 (CH,
C-1), 133.72 (CH, aromatic), 134.82 (CH, aromatic),
135.25 (CH, aromatic), 135.48 (CH, aromatic), 141.61
(C, aromatic), 146.17 (C, aromatic), 146.22 (C, aromatic),
152.59 (C, aromatic), 152.67 (C, aromatic), 179.20 (C,
CO), 180.63 (C, CO), 205.60 (C, CO), 206.17 (C, CO)
)-2-(4-(4-ch lorophen yl)-4-oxobu tanamido)-3 ,4-dih ydrox
yl)methyl)-4-oxobutanamide (3).
3-(4-Chlorobenzoyl)propionic acid (53 mg, 0.251
mmol) was used as the carboxylic acid for the conjuga-
tion. Column chromatography was performed by using
eluents of MeOH:CH2Cl2 = 1:9 to provide the white
crude product 3 in 46% yield (25 mg). After further puri-
fication with HPLC using eluents of MeOH:CH2Cl2 =
1:11, a white solid was obtained in an overall yield of
28% (15 mg). tR = 10.94 min. Anal. C44H64Cl2N2O11, M
(calcd.) = 866.4 (m/z); found: ESI + Q-TOF:[M + Na]+ =
889.5 (47.1%); HRMS (ESI) M (calcd.) = 866.38872
(m/z), [M + H]+ = 867.39654 amu; found [M + H]+ =
867.39501, = 1.76 ppm; 1H-NMR (600 MHz, CD3OD):
δ 0.89 (t, 3H, J = 7.2 Hz, Haliphatic), 1.23-1.38 (m, 24H,
Haliphatic), 1.51 (m, 1H, Haliphatic), 1.60 - 1.62 (m, 1H,
Haliphatic), 2.58 - 2.64 (m, 4H aliphatic), 3.26 - 3.29 (m, 2H),
3.30 - 3.34 (m,2H), 3.47 - 3.50 (m, 1H), 3.58 - 3.63 (m,
2H), 3.68 - 3.70 (m, 1H), 3.77 - 3.80 (m, 3H), 3.84 - 3.90
(m, 2H), 4.21 - 4.22 (m, 1H), 7.46 - 7.48 (m, 4H, arom.,
7.96 - 7.98 (m, 4H, arom.); 13C-NMR (150 MHz,
CD3OD):δ14.45 (CH3), 23.75 (CH2), 27.00 (CH2), 30.49
(CH2), 30.72 (CH2), 30.78 (CH2), 30.83 (CH2), 30.87
(CH2), 30.90 (CH2), 33.08 (CH2), 34.82 (CH2), 41.33
(CH2), 51.94 (CH), 68.03 (CH2), 70.21 (CH), 70.55 (CH),
71.28 (CH), 71.30 (CH), 72.98 (CH), 75.49 (CH), 101.01
(CH, C-1), 129.95 (CH, arom.), 129.96 (CH, arom.),
130.84 (CH, arom.), 136.56 (C, arom.), 136.58 (C,
arom.), 140.49 (C, arom.), 140.53 (C, arom.), 174.42 (C,
CO), 175.32 (C, CO), 199.39 (C, CO), 199.52 (C, CO).
R)-3,4,5- trihydrox y-6-((( E)-3-(4-(trifluoromethyl)phenyl
trans-4-(Trifluoromethyl) cinnamic acid (54 mg, 0.251
mmol) was used as the carboxylic acid for the conjuga-
tion. Column chromatography was performed using elu-
ents of MeOH:CH2Cl2 = 1:10 MeOH:CHCl2 = 1:8 to
provide the white crude product 4 in 27% yield (15 mg).
After additional purification with HPLC using eluents of
MeOH:CH2Cl2 = 1:7, a white solid was obtained in 22%
yield (12 mg). The sample was further purified by using
recrystallization from MeOH to provide the white solid
in an overall yield of 13% (7 mg). tR = 8.49 min. Anal.
C44H60F6N2O9, M (calcd.) = 874.4 (m/z); found:ESI +
Q-TOF:[M + H]+ = 875.4, [M + Na]+ = 897.4; 1H-NMR
(500 MHz, CD3OD) :δ 0.88 (t, 3H, J = 7.0Hz, Haliphatic),
1.20 - 1.42 (m, 24H, Haliphatic), 1.45 - 1.65 (m, 2H,
Haliphatic), 3.35 - 3.40 (m, 1H), 3.44 - 3.49 (m, 1H), 3.52 -
3.56 (m, 1H), 3.61 - 3.77 (m, 3H), 3.79 - 3.82 (m, 1H),
Copyright © 2013 SciRes. OJMC
Copyright © 2013 SciRes. OJMC
3.85 (m, 1H), 3.92 - 3.98 (m, 2H), 4.34 - 4.37 (m, 1H),
4.89 (d, 1H, J1,2 = 3.5 Hz, H-1), 6.72 (d, 1H, J = 16.0 Hz),
6.76 (d, 1H, J = 16.0 Hz), 7.45 (d, 1H, J = 16.0 Hz), 7.48
(d, 1H, J = 16.0 Hz), 7.63 - 7.66 (m, 8H, Hamide-aromatic);
13C-NMR (125 MHz, CD3OD):δ 14.44 (CH3, Caliphatic),
23.74 (CH2), 26.79 (CH2), 30.47 (CH2), 30.71 (CH2),
30.75 (CH2), 30.78 (CH2), 30.85 (CH2), 33.07(CH2),
33.20 (CH2), 41.75 (CH2), 52.56 (CH), 68.13 (CH2),
70.50 (CH), 71.02 (CH), 71.26 (CH), 71.57 (CH), 72.83
(CH), 75.40 (CH), 100.93 (CH, C-1), 124.50 (CH),
124.74 (CH), 126.77 (CH), 129.35 (CH), 140.02 (CH),
140.08 (CH), 167.41 (C, COamide), 168.26 (C, COamide).
19F-NMR (470 MHz, CD3OD) δ 210.26, 210.28,
220.17, 221.67.
Conformers were speculated to assign for δ 220.17
and 221.67.
4. Acknowledgements
We are grateful to the National Science Council of Tai-
wan, Chang-Bing Show Chwan Memorial Hospital,
CGMH_NTHU Joint Research, and Chang-Gung Medi-
cal Research Project for providing financial support through
grant numbers NSC-101-2113-M-007-010, NSC-97-2314-
B-182A-020-MY3, NSC-97-2314-B-182A-020-MY3,
CGTH96N2342E1, CMRPG3B0531, CMRPG390661,
CMRPG390931, and CMRPG3B0361.
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