Structure activity relationships of N-linked and diglycosylated glucosamine-based antitumor glycerolipids

Article abstract of DOI:10.3390/molecules181215288

1-O-Hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-D- glucopyranosyl)-snglycerol (1) was previously reported to show potent in vitro antitumor activity on a range of cancer cell lines derived from breast, pancreas and prostate cancer. This compound was not toxic to mice and was inactive against breast tumor xenografts in mice. This inactivity was attributed to hydrolysis of the glycosidic linkage by glycosidases. Here three N-linked (glycosylamide) analogs 2-4, one triazole-linked analog 5 of 1 as well as two diglycosylated analogs 6 and 7 with different stereochemistry at the C2-position of the glycerol moiety were synthesized and their antitumor activity against breast (JIMT-1, BT-474, MDA-MB-231), pancreas (MiaPaCa2) and prostrate (DU145, PC3) cancer cell lines was determined. The diglycosylated analogs 1-O-hexadecyl-2(R)-, 3-O-di-(2′-amino-2′-deoxy- β-D- glucopyranosyl)-sn-glycerol (7) and the 1:1 diastereomeric mixture of 1-O-hexadecyl- 2(R/S), 3-O-di-(2′-amino-2′-deoxy-β-D- glucopyranosyl)-sn-glycerol (6) showed the most potent cytotoxic activity at CC50 values of 17.5 μM against PC3 cell lines. The replacement of the O-glycosidic linkage by a glycosylamide or a glycosyltriazole linkage showed little or no activity at highest concentration tested (30 μM), whereas the replacement of the glycerol moiety by triazole resulted in CC50 values in the range of 20 to 30 μM. In conclusion, the replacement of the O-glycosidic linkage by an N-glycosidic linkage or triazole-linkage resulted in about a two to three fold loss in activity, whereas the replacement of the methoxy group on the glycerol backbone by a second glucosamine moiety did not improve the activity. The stereochemistry at the C2-position of the glycero backbone has minimal effect on the anticancer activities of these diglycosylated analogs.

Full text of DOI:10.3390/molecules181215288

Molecules 2013, 18, 15288-15304; doi:10.3390/molecules181215288
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
Structure Activity Relationships of N-linked and Diglycosylated
Glucosamine-Based Antitumor Glycerolipids
Makanjuola Ogunsina 1, Hangyi Pan 1, Pranati Samadder 2, Gilbert Arthur 2 and
Frank Schweizer 1,*
1
Department of Chemistry, University of Manitoba, 144 Dysart Rd, Winnipeg, MB R3T 2N2,
Canada
2
Department of Biochemistry and Medical Genetics, University of Manitoba, 745 Bannatyne
Avenue, Winnipeg, MB R3E 0J9, Canada
* Author to whom correspondence should be addressed; E-Mail: schweize@cc.umanitoba.ca;
Tel.: +1-204-474-7012; Fax: +1-204-474-7608.
Received: 15 November 2013; in revised form: 3 December 2013 / Accepted: 4 December 2013 /
Published: 10 December 2013
Abstract: 1-O-Hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-β-D-glucopyranosyl)-sn-
glycerol (1) was previously reported to show potent in vitro antitumor activity on a range
of cancer cell lines derived from breast, pancreas and prostate cancer. This compound was
not toxic to mice and was inactive against breast tumor xenografts in mice. This inactivity
was attributed to hydrolysis of the glycosidic linkage by glycosidases. Here three N-linked
(glycosylamide) analogs 2–4, one triazole-linked analog 5 of 1 as well as two
diglycosylated analogs 6 and 7 with different stereochemistry at the C2-position of the
glycerol moiety were synthesized and their antitumor activity against breast (JIMT-1, BT-474,
MDA-MB-231), pancreas (MiaPaCa2) and prostrate (DU145, PC3) cancer cell lines was
determined. The diglycosylated analogs 1-O-hexadecyl-2(R)-, 3-O-di-(2'-amino-2'-deoxy-
β-D-glucopyranosyl)-sn-glycerol (7) and the 1:1 diastereomeric mixture of 1-O-hexadecyl-
2(R/S), 3-O-di-(2'-amino-2'-deoxy-β-D-glucopyranosyl)-sn-glycerol (6) showed the most
potent cytotoxic activity at CC50 values of 17.5 µM against PC3 cell lines. The replacement
of the O-glycosidic linkage by a glycosylamide or a glycosyltriazole linkage showed little
or no activity at highest concentration tested (30 µM), whereas the replacement of the
glycerol moiety by triazole resulted in CC50 values in the range of 20 to 30 µM. In
conclusion, the replacement of the O-glycosidic linkage by an N-glycosidic linkage or
triazole-linkage resulted in about a two to three fold loss in activity, whereas the
replacement of the methoxy group on the glycerol backbone by a second glucosamine

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moiety did not improve the activity. The stereochemistry at the C2-position of the glycero
backbone has minimal effect on the anticancer activities of these diglycosylated analogs.
Keywords: antitumor ether lipids; glycolipids; anticancer agents; carbohydrates
1. Introduction
Cancer is a devastating disease with significant mortality and morbidity in both developed and
developing countries [1,2]. Acquired and intrinsic resistance to major classes of anticancer agents:
antimetabolites, anthracyclines, taxanes and alkylating agents which are mostly pro-apoptotic present a
serious challenge to management of cancer [3]. Even targeted antibody based therapies such as
trastuzumab (Herceptin) are also affected by the problem of drug resistance [3,4]. So there is need for
new anticancer agents with new mechanisms of action that are apoptosis independent.
Glycosylated antitumor ether lipids (GAELs), represent a subclass of antitumor ether lipids (AELs)
in which the phosphocholine moiety of the prototypic compound, edelfosine is replaced by a sugar
moiety [5]. The antitumor activity of these compounds has been well established [6]. Edelfosine is an
investigational drug that has undergone phase II clinical trials. The best studied GAEL analog,
glucosamine-based glycerolipid 1 (Figure 1) has potent antitumor effects against a range of cancer cell
lines derived from breast, pancreas and prostate cancer with CC50 values in the range of 9–15 µM. This
compound has been reported to kill cancer cells via an apoptosis independent pathway and is more
active than the prototypic AELs, edelfosine, which kills cell via a apoptosis dependent pathway [6–8].
A major drawback for compound 1 is the lack of antitumor activity in in vivo studies using mice.
A maximum tolerability dose could not be attained at concentrations of 500 mg/kg oral dosage or
4 mg/kg given intraperitoneally in Rag2M mice and there was no effect on the body weight of the
animals [9]. Furthermore, at a dose of 100 mg/kg given orally and 4 mg/kg given intraperitoneally, it
had no effect on the growth of JIMT-1 or MDA-MB231 xenografts growing in female Rag2M mice [9].
These observations are consistent with the hypothesis that the likely cause of the in vivo inactivity is
due to cleavage of the -O-glycosidic linkage by glucosidases. A C-glycosidic analogue of 1 has been
synthesized to enhance the metabolic stability of the compound [10]. However the synthesis is very
involved and alternative facile approaches have to be considered to generate metabolically stable
analogues. Based on the rationale that both glycosylamide- and glycosyltriazole linkages will be
resistant towards glycosidic cleavage [11–13], we synthesized compounds 2–4 where the -O-glycosidic
linkage was replaced by a glycosylamide- or a triazole-linkage as in compound 5. In addition, we
synthesized compounds 6 and 7 to study the effects of: (a) diglycosylation and (b) stereochemistry of
the glycerol moiety on antitumor activity in a cell based assay. The rationale behind the synthesis of
compounds 6 and 7 is to test our hypothesis that an additional glucosamine moiety on the molecule
will increase the anticancer activity because glucosamine is cytotoxic against human epidermoid
carcinoma cells in tissue culture [14] and YD-8 human oral cancer cells [15]. Here we report on the
in vitro antitumor activity of compounds 2–7.

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Figure 1. Structures of the synthesized glycolipids used in the study. Compound 1 is the
reference GAEL while compounds 2–5 are glycolipid analogs differing in the nature of the
glycosidic linkage and glycerol moiety. Compounds 6 and 7 are glycolipid analogs where
the glycerolipid moiety contains two glycosidic linkages. Edelfosine is shown as a
prototypic AEL analog.
2. Results and Discussion
2.1. Chemistry
In order to prepare easily accessible analogs with improved metabolic stability towards
glycosidases we initially were interested in analogs that contain a glycosylamide linkage.
Glycosylamides 2–4 were synthesized to evaluate the effect of amide linkage and nature of the lipid
moiety on the antitumor properties. Compound 3 which lacks the methoxy substituent was prepared to
explore how the methoxy group affects the antitumor properties. Compound 4 that contains a
lipophobic polyfluorinated lipid tail instead of a lipophilic carbon chain was prepared to explore how
modifications in the lipid tail affect the antitumor activity. Compound 5 was synthesized to evaluate
the effect of a triazole linkage at the anomeric position. Both linkages, the glycosylamide and
glycosyltriazole linkages are expected to be metabolically stable to hydrolysis by glycosidases in
in vivo studies [11–13]. In addition, the glycosyltriazole linkage will be inert towards peptidases and
proteases which may provide additional benefits for future in vivo studies [16].
GAEL mimetics 2–4 were prepared by coupling of glycosylamine 11 to carboxylic acids 13, 15 and
16 (Scheme 1). Glycosylamine 11 was synthesized in three steps from glucosamine hydrochloride (8)
in 49% yield [17]. The amino substituent at C2 position of glucosamine hydrochloride group was
protected with phthalic anhydride followed by protection of the hydroxyl groups as acetate esters by

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reaction with acetic anhydride in pyridine to afford compound 9. The anomeric amino group in 11 was
installed through conversion of 9 into the corresponding anomeric azide 10. Originally, we planned to
introduce the anomeric azido group by nucleophilic displacement of the α-anomeric chloride. However
conversion of the anomeric acetate into the chloride by reaction with PCl5 did not afford the
corresponding glucosylhalides [18]. However, the anomeric azide 10 was prepared by Fe(III) chloride-
promoted reaction of 9 with trimethylsilyl azide to afford azide 10 in 75% yield. The anomeric azide
was reduced to the amine by catalytic hydrogenation to provide glucosylamine 11 in 96% yield.
Synthesis of the fatty acid compounds 13 and 15 was achieved by Jones oxidation of the corresponding
commercially available alcohol 12 or known alcohol 14 [19]. The polyfluorinated fatty acid 16 was
purchased from commercial source. Coupling of fatty acids 13, 15 and 16 to the glucosylamine 11 was
achieved by using TBTU [20] as coupling agent to afford the protected glycolipids 17, 18 and 19,
respectively. The acetate and phthalimido protecting groups were removed using an ethylenediamine-
butanol mixture (1:1) at 90 °C for 3 h to afford desired target compounds 2, 3 and 4 respectively. The
triazole analog 5 was synthesized by Cu(I)-promoted click chemistry [21] using azide 10 and
3-hexadecyloxyprop-1-yne (22) to produce glycosyltriazole 23. Deblocking using the same method as
described above gave compound 5.
Scheme 1. Synthesis of compounds 2–5.
Reagents and conditions: (a) (i) phthalic anhydride, NaOH, H2O, 16 h, rt; (ii) pyridine, DMAP, Ac2O, 16 h,
rt; (b) TMSN3, FeCl3 as catalyst, DCM, rt; (c) Pd/C, MeOH, H2, 2 h, rt; (d) TBTU, DIPEA. DMF 3 h, rt; (e)
ethylenediamine/butanol (1:1), 90 °C, 2 h; (f) Jones reagent, acetone, 0 °C, 20 min; (g) NaH, DMF, 80 °C;
(h) CuI, acetic acid, DIPEA.

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Compounds 6 and 7 were synthesized to study the effects of diglycosylation and stereochemistry of
the glycerol moiety on the antitumor properties. Glycolipids 6 and 7 were prepared from known
thioglycoside donor 24 [22] by glycosylation with commercially lipid alcohol 25 or racemic alcohol 26
using silver triflate as catalyst and N-iodosuccinimde as promoter to afford protected glycolipids 27 or
28, respectively (Scheme 2). The acetate and phthalimido protective groups were removed using
ethylenediamine:butanol mixture (1:1) at 90 °C for 3 h to provide compounds 6 and 7, respectively.
Scheme 2. Synthesis of compounds 6 and 7.
Reactions and conditions: (a) BF3·Et2O, DCM, reflux 16 h; (b) AgOTf, N-iodosuccinimide,
DCM, rt, 5 h; (c) ethylenediamine/butanol (1:1), 90 °C, 4 h.
2.2. Cytotoxicity
The cytotoxicity of compounds 1–7 was evaluated against a number of epithelial cancer cell lines
using the MTS assay [22]. The cells were derived from cancers of the breast (JIMT-1, BT-474,
MDA-MB-231), pancreas (MiaPaCa2), and prostrate (DU145, PC3). The cytotoxic effect of compounds
2–6 were compared with that of 1 the most studied glycosylated antitumor ether lipid [5–7,9] (see
Table 1). Exponentially growing cells were treated with test compound and then incubated for 48 h.
The result for compounds 2–4 are shown in Figure 2, while that of compounds 5–7 are shown in
Figure 3. The CC50 values that lead to a reduction of cell viability by 50% relative to untreated control
are reported in Table 1. At the highest concentration tested none of the N-linked glycolipids 2–4 was

Molecules 2013, 18
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able to achieve 50% reduction in viability against all the four cell lines tested (see Figure 2). It is
noteworthy that compound 2 with similar glycerolipid moiety as reference compound 1, is consistently
more active than compounds 3 and 4, leading to a 19%–29% reduction in viability relative to untreated
control at the highest dose tested (30 µM).
Table 1. Cytotoxicity of compounds 1–7 on a panel of human epithelial cancer cell lines:
breast (BT474, JIMT1, MDA-MB-231), prostrate (DU145, PC3), pancreas (MiaPaCa2).
The CC50 value is defined as the concentration required to decrease cell viability by 50%
relative to the untreated control. Values were determined by MTS assay. The CC50 values
were obtained by estimating the drug concentration at 50% viability on the y axis using
line plots (graph not shown); NT = not tested. Please note that CC50 values for compound 1
were obtained from our previously published work [21].
CC50 (µM)
Compd
JIMT1 MiaPaCa2 DU145 PC3 BT474 MDA-MB-231
1
2
3
4
5
6
7
9
9
10
>30
>30
>30
>30
20
13.5
NT
6.5
>30
>30
>30
28
NT
NT
NT
NT
>30
>30
>30
>30
>30
>30
23
>30
>30
>30
>30
>30
>30
NT
NT
>30
17.5
17.5
27
>30
>30
29
25
Figure 2. Effects of compounds 2–4 on the viability of epithelial cancer cell lines: breast
(BT474, JIMT1), prostrate (DU 145), pancreas (MiaPaCa2). The results represent the
means ± standard deviations of six independent determinations (compound 1 is not
included in this chart because we have previously published the data [22]).
DU 145
BT 474
120
100
80
60
40
20
0
140
120
100
80
2
3
4
2
3
4
60
40
20
0
0
5
10 20 30
0
5
10 20 30
Doses (µM)
Doses (µM)

Molecules 2013, 18
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Figure 2. Cont.
Mia Paca 2
JIMT‐1
120
100
80
60
40
20
0
120
100
80
60
40
20
0
2
3
4
2
3
4
0
5
10 20 30
0
5
10 20 30
Doses (µM)
Doses (µM)
At this dose and lower concentration, compound 4 with polyfluorinated lipid moiety is consistently
the least active analogue among these N-linked (amide) glycolipids. This relative difference in activity
is an indication that the nature of the lipids of this class of compounds plays an important role on their
cytotoxicity against human cancer cell lines. The triazole-linked glycolipid compound 5 at
concentration of 30 µM or below was unable to achieve CC50 values against prostrate and pancreas
cell lines used in this experiment, indicating that both the nature of the glycosidic linkage and the
glycero moiety influences the antitumor properties. It is noteworthy that compound 5 was able to
achieve more than 80% reduction in viability of JIMT1, a trastuzumab resistant cell line [4] at the dose
of 30 µM, (CC50: 23µM) and more than 60% reduction in viability of BT474, a trastuzumab sensitive
cell lines (see Figure 3). In vivo, triazole-linked glycolipid 5 is expected to be metabolically stable
relative to lead compound 1, however the loss of activity does not make it a worthwhile compound for
further development.
The diglycosylated analogues, compounds 6 and 7 with different stereochemistry at position 2 of
the glycerol backbone have very similar activity but are significantly less active than GAEL-based
reference compound 1 (Figure 2 and Table 1) indicating that additional glucosamine moieties on the
glycerol backbone reduce the cytotoxic effect of the compounds. This reduction in activity may be due
to increased polarity of the compound which in turn can affect the absorption of the drug across cell
membrane, and subsequent decreased drug concentration in the cell. The fact that both compounds
despite the difference in stereochemistry at the C-2 position of the glycerol backbone have similar
activity especially in the more sensitive prostrate cell lines (Figure 2) is an indication that
stereochemistry at this position has minimal to no effect on the anticancer activity.

Molecules 2013, 18
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Figure 3. Effects of compounds 5–7 on the viability of epithelial cancer cell lines: breast
(BT474, JIMT1, MDA-MB-231), prostrate (DU145, PC3), pancreas (MiaPaCa2). The
results represent the means ± standard deviations of six independent determinations.
PC‐3
120
100
80
5
6
7
60
40
20
0
0
10
20
30
Doses (µM)
Mia Paca 2
JIMT‐1
120
100
80
60
40
20
0
120
100
80
60
40
20
0
5
6
7
5
6
7
0 2.5 5 7.5 10 15 20 30
0
10
20
30
Doses (µM)
Doses (µM)
BT 474
MDA‐MB‐231`
120
100
80
60
40
20
0
120
100
80
60
40
20
0
5
5
6
7
6
7
0 2.5 5 7.5 10 15 20 30
0 2.5 5 7.5 10 15 20 30
Doses (µM)
Doses (µM)

Molecules 2013, 18
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3. Experimental
3.1. General Methods
All fine chemicals such as glucosamine hydrochloride, phthalic anhydride, pyridine, 1-hexadecanol,
4-toluenesulfonyl chloride, 60% sodium hydride, dimethylformamide (DMF), 1,3-dihydroxypropane,
sodium methoxide, concentrated sulfuric acid, palladium on carbon, trimethylsilyl azide, toluene,
phosphorus pentachloride, boron trifluoride, N,N-diisopropyl-ethylamine, ethylenediamine and iron
(III) chloride. All solvents such as dichloromethane (DCM), hexane, acetone, methanol, ethyl acetate,
butanol were purchased from Sigma-Aldrich (Oakville, ON, Canada). Carboxylic acid 16
(2H,2H,3H,3H-perfluoroundecanoic acid) were purchased from Fluorous Technologies now available
through Sigma-Aldrich. 1-O-hexadecyl-2-O-methyl-sn-glycerol was purchased from Chem-Implex
(Wood Dale, IL, USA). 5 ¾ and 9 inch pipets used in lab were obtained from Fisher Scientific
(Ottawa, ON, Canada). TLC plates (CCM Gel silica 60 F254) were visualized by ultraviolet light or by
charring (9:1 methanol and sulfuric acid). All chromatography solvents were prepared by mixing
hexane and ethyl acetate in various ratios based on the polarity of the compound. Column
chromatography was performed by using silica gel p60 (40–63 µm) or reverse-phase C18 silica gel.
Solutions were concentrated on reduced pressure rotary evaporator (IKA RV 10 Basic, that was
13
connected to building vacuum and high vacuum pump (Welch 8907). 1H-NMR and C-NMR spectra
were recorded on a 300 MHz (Bruker AMX-300 spectrometer). Low-resolution mass spectrometry
(MS) data were obtained on a Varian 500-MS IT Mass spectrometer using electrospray ionization (ESI).
3.2. General Procedure for the Synthesis of N-Linked Compounds 2–5
1,3,4,6-Tetra-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (9). Glucosamine hydro-
chloride 8 (3.016 g, 14 mmol) and NaOH (28 mmol) were dissolved in water (50 mL). The resulting
mixture was stirred at room temperature for 30 min. Phthalic anhydride (2.34 g, 0.0157 mol) was added
to the solution. The mixture was stirred vigorously at room temperature for 16 h. The mixture was
concentrated and dried using rotary evaporator. The residue was dissolved in pyridine (30 mL), and
then Ac2O (19.8 mL) was added to the solution. The resulting solution was allowed to stir vigorously
overnight. The reaction was checked by the TLC. Methanol (6 mL) was used to quench the excess of
Ac2O, and then excess pyridine was removed under high vacuum. The remaining solid was dissolved
in CH2Cl2 (40 mL), and then the solution was washed with 10% HCl (40 mL×1), Saturated NaHCO3
solution (40 mL×3), H2O (40 mL×1) and brine (40 mL×1) and dried over anhydrous MgSO4. The final
solution was concentrated under reduced pressure, and the obtained product 8 (3.3g, 49.4%) was dried
overnight. NMR data were consistent with data in the literature [23].
3,4,6-tri-O-Acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl azide (10). Compound 9 (1.8 g, 8 mmol)
and trimethylsilyl azide (1.7 g, 148 mmol) were both dissolved in CH2Cl2 (20 mL) in a 100 mL-round
bottom flask with vigorous stirring. Then FeCl3 (1.77 g) was added to the reacting mixture. The
reaction was allowed to stir for 24 h and then progress was checked by TLC. The dispersion solution
was made by mixing hexane and ethyl acetate in 1:1 ratio. The solution was concentrated under
reduced pressure by rotary evaporator. The product 10 was isolated and purified by column

Molecules 2013, 18
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chromatography (1:2 ethyl acetate/hexane). The obtained product 10 (1.3 g, 76.53%) was a light
yellow solid. NMR data were consistent with previously published data [16].
3,4,6-tri-O-Acetyl-1-amino-2-deoxy-2-N-phthalimido-β-D-glucopyranose (11). Compound 10 (0.11 g,
0.24 mmol) was dissolved in methanol (2 mL) in a 25 mL-round bottom flask with vigorous stirring,
and then Pd/C (0.29 g) was added. After that, round bottom flask was connected to a hydrogen
balloon. The reaction was allowed to take place for half hour, and checked by TLC. The reaction was
stopped when all starting material has disappeared, and the solution was concentrated to provide
1
compound 11 (100 mg, 96%). This compound was not characterised by H-NMR, because it is
chemically unstable. Therefore it was directly used for the next step, i.e., coupling of carboxylic acids
using TBTU as coupling reagent.
3-(Hexadecyloxy)-2-methoxypropanoic acid (13). A 2.7 M solution of Jones reagent (0.5 mL) was
added dropwise into a stirred solution of commercially available 3-O-hexadecyloxy-2-O-methyl-sn-
glycerol 12 (23 mg) in acetone (5 mL) at 0 °C. The reaction was monitored by TLC and was
completed after 1 h. Isopropyl alcohol was added dropwise until a green color remained, to remove
excess of CrO3 totally. The organic solvent was removed under reduced pressure. The solid was
dissolved in water, and then 3-(hexadecyloxy)-2-methoxypropanoic acid was extracted using ethyl
acetate. The combined organic layers were washed with brine, dried using MgSO4 and concentrated
under vacuum. The 3-(hexadecyloxy)-2-methoxypropanoic acid (13) was purified by silica gel column
flash chromatography (4:1 hexane/ethyl acetate) to yield white solid (16 mg, 70%). 1H-NMR (CDCl3):
δ = 4.00–3.75 (m, 3H), 3.55 (s, 3H, –OCH3), 2.66 (t, J = 6.4 Hz, 2H, –OCH2–CH2–(CH2)13), 1.59 (m,
2H, –CH2–(CH2)13), 1.23–1.34 (brs, 26H, –(CH2)13), 0.90 (t, J = 6.9 Hz, 3H, –CH3). ESMS: calcd for
C20H40O4Na+ m/z 367.3; found: m/z [M+Na]+ 367.3.
3-(Hexadecyloxy)-propanoic acid (15). A 2.7 M solution of Jones reagent (2.0 mL) was added
dropwise into a stirred solution of compound 14 (0.124 g, 0.4 mmol) in acetone (20 mL) at 0 °C. The
reaction was monitored by TLC and was completed after 1 h. Isopropyl alcohol was added dropwise
until a stable green color appeared, to remove the excess of CrO3. The organic solvent was removed
under reduced pressure. The solid was dissolved in water, and then compound 15 was extracted using
ethyl acetate. The combined organic layers were washed by brine, dried using MgSO4 and concentrated
under reduced pressure. The compound 15 was purified by silica gel column flash chromatography
(4:1 hexane/ethyl acetate) to yield white solid (63 mg, 50.1%). 1H-NMR (CDCl3): δ = 3.72 (t, J = 6.3 Hz,
2H, –CH2–COOH), 3.48 (t, J = 6.7 Hz, 2H, –OCH2–CH2–COOH), 2.65 (t, J = 6.3 Hz, 2H, –OCH2–
CH2–(CH2)13), 1.59 (m, 2H, –CH2–(CH2)13), 1.23–1.34 (brs, 26H, –(CH2)13 0.90 (t, J = 6.9 Hz, 3H, CH3).
ESMS: calcd for C19H38O3Na+ m/z 337.3; found: m/z [M+Na]+ 337.4
3-Hexadecyloxy-2-methoxyl-1-N-(3,4,6-tri-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-
propanamide (17). Glucosylamine 11 (110 mg, 0.25 mmol), carboxylic acid 13 (110 mg, 0.32 mmol),
DIPEA (0.40 mL) and TBTU (0.245 g) were dissolved in DMF (10 mL) in a round bottom flask with
vigorous stirring overnight and was monitored by TLC. At the completion of the reaction DMF was
removed under reduced pressure to obtain a solid residue. The solid residue was dissolved in water,
and the product was extracted by ethyl acetate. The combined organic layers were concentrated under

Molecules 2013, 18
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reduced pressure to yield a light yellow compound 17 (130 mg, 68%). 1H-NMR (CDCl3): δ = 8.84 (s,
br. 1H), 7.93–7.67 (m, 4H, phthalimido), 7.11(d, J = 9.0 Hz, 1H, H–1), 6.14–5.96 (m, 2H, H–2, H–3),
5.19 (dd, J1 = J2 = 12 Hz, 1H, H–4), 4.44–3.54 (m, 8H), 3.26 (s, 3H, –OCH3), 2.12(s, 3H,
acetate–CH3), 2.06 (s, 3H, acetate–CH3), 1.89 (s, 3H, acetate–CH3), 1.53 (m, 2H–CH2–(CH2)13),
1.39–1.15 (brs, 26H, (CH2)13), 0.90 (t, J = 6.0 Hz, 3H, CH3). ES-MS: calcd for C40H60N2O12Na+ m/z
783.4: found: m/z [M+Na]+ 783.6.
3-Hexadecyloxy-1-N-(3,4,6-tri-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-propanamide
(18). Glucosylamine 11 (100 mg, 0.23 mmol) and carboxylic acid 15 (43.7 mg, 0.15 mmol) and
DIPEA (0.04 g) and TBTU (0.04 g) and DMF (4 mL) were added into a 25-mL round bottom flask
with vigorous stirring. The reaction allowed to stir for overnight and was monitored by TLC. DMF
was removed under reduced pressure to obtain solid. The solid was dissolved in water, and the product
was extracted by ethyl acetate. The combined organic layers were concentrated by reduced pressure
rotary evaporator to yield a light yellow compound 18 (50 mg, 45.5%). 1H-NMR (CDCl3): δ = 8.84 (s,
br. 1H), 7.89–7.76 (m, 4H, phthalimido), 7.03 (d, J = 9.0 Hz, 1H, H–1), 6.08 (dd, J1 = J2 = 9.8 Hz, 1H,
H-3), 6.02 (dd, J1 = J2 = 9.6 Hz, 1H, H–2), 5.18 (dd, J1 = J2 = 9.8 Hz, 1H, H–4), 4.36 (m, 1H, H–5),
4.28 (dd, J1 = J2 = 10.2 Hz, 2H, ), 4.09 (dd, J1 = J2 = 10.0 Hz, 2H, H–6), 3.51 (m, 1H), 3.34 (m, 2H),
2.26 (m, 2H,), 2.12 (s, 3H, acetate–CH3), 2.05 (s, 3H, acetate–CH3), 1.88 (s, 3H, acetate–CH3), 1.53
(m, 2H, CH2–(CH2)13), 1.31–1.28 (brs, 26H, –(CH2)13), 0.90 (t, J = 6.9 Hz, 3H, –CH3). ES-MS: calcd
for C39H58N2O11Na+ m/z 753.4: found: m/z [M+Na]+ 753.8.
Heptadecylfluoro-1-N-(3,4,6-tri-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-undecanamide
(19). Glucosylamine 11 (110 mg, 0.25 mmol), fluorinated carboxylic acid (250 mg, 0.51 mmol),
DIPEA (0.04 g), TBTU (0.245 g) and DMF (10 mL) were added into a 25-mL round bottom flask with
vigorous stirring. The reaction allowed to stir for overnight and was monitored by TLC. DMF was
removed under reduced pressure to obtain solid. The solid was dissolved in water, and the product was
extracted by ethyl acetate. The combined organic layers were concentrated by reduced pressure rotary
1
evaporator to yield a light yellow compound 19 (105 mg, 45%). H-NMR (CDCl3): δ = 8.93 (s, br.
1H), 7.93–7.69 (m, 4H, phthalimido), 7.05 (d, J = 9.0 Hz, 1H, H–1), 6.15–5.99 (m, 2H, H–2, H–3),
5.17 (dd, J1 = J2 = 9.0 Hz, 1H, H–4), 4.45–3.98 (m, 5H), 2.38 (m, 2H), 2.13 (s, 3H, acetate–CH3), 2.06
(s, 3H, acetate–CH3), 1.89 (s, 3H, acetate–CH3). ES-MS: calcd for C31H25F17N2O10Na+ m/z 931.1:
found: m/z [M+Na]+ 931.5.
3-Hexadecyloxy-2-methoxy-1-N-(-2-deoxy-2-amino-β-D-glucopyranosyl)-propanamide (2). Compound
17 (130 mg, 0.17 mmol) was dissolved in mixture of butanol (2 mL) and ethylenediamine (2 mL). The
resulting mixture was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC plate.
The solution was concentrated to dryness. The resulting reside was purified by flash chromatography
(reverse-phase C18 silica gel). The collected product was 20 mg (yield 23%). The compound was
acidified with TFA to convert it into the TFA salt. 1H-NMR (CD3OD): δ = 5.14 (d, J = 9.6 Hz, 1H, H–1),
3.87–3.52 (m, 6H), 3.50–3.24 (m, 7H, H–3), 3.08 (dd, J = 9.6 Hz, 10 Hz, 1H, H–2), 1.56–1.36 (m, 2H,
13
CH2–(CH2)13), 1.25–1.13 (brs, 26H, –(CH2)13), 0.80 (t, J = 6.9 Hz, 3H, –CH3). C-NMR (75 MHz,
CD3OD) characteristic data: 174.03 (C–1), 82.50, 80.41, 78.00, 74.87, 72.88, 71.49, 71.25, 62.11,

Molecules 2013, 18
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58.94 56.46, + multiple methylene carbons, 14.40 (CH3). ES-HRMS: calcd for C26H52N2O7Na+ m/z
527.3667, found: m/z [M+Na]+ 527.3677.
3-Hexadecyloxy-1-N-(-2-deoxy-2-amino-β-D-glucopyranosyl)-propanamide (3). Compound 8 (50 mg,
0.07 mmol) was dissolved in mixture of butanol (2 mL) and ethylenediamine (2 mL). The resulting
mixture was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC. The solution was
concentrated to dryness. The resulting reside was purified by flash chromatography (reverse-phase
C18 silica gel). The collected product was 18 mg (yield 54%). The compound 3 was acidified with
TFA to enhance solubility in methanol. 1H-NMR (CD3OD): δ = 5.14 (d, J = 10 Hz, 1H, H–1), 3.74–3.56
(m, 4H), 3.48–3.42 (m, 1H), 3.40–3.32 (m, 2H), 3.30–3.25 (m, 2H), 2.89 (dd, J1 = J2 = 10.0 Hz, 1H,
H–2), 2.44 (t, J = 6 Hz, 2H,), 1.51–1.42 (m, 2H, –CH2–(CH2)13), 1.25–1.18 (brs, 26 H, –(CH2)13), 0.81
13
(t, J = 7 Hz, 3H, –CH3). C-NMR (CD3OD) characteristic data: 174.50 (amide), 80.30 77.83, 74.87,
72.43, 71.43, 67.11, 62.18, 56.90, 37.54, (multiple methylene carbons), 14.43 (CH3). ES-HRMS: calcd
for C25H50N2O6Na+ m/z 497.3567: found: m/z [M+Na]+ 497.3554.
Heptadecylfluoro-1-N-(-2-deoxy-2-amino-β-D-glucopyranosyl)-dodecanamide (4). Compound 19 (105 mg,
0.11 mmol) was dissolved in mixture of butanol (2 mL) and ethylenediamine (2 mL). The resulting
mixture was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC plate. The
solution was concentrated to dryness. The resulting residue was purified by flash chromatography
(reverse-phase C18 silica gel) to yield product 4 (50 mg, 64% yield). The compound 3 was acidified
1
with TFA to enhance solubility in methanol. H-NMR (CD3OD): δ = 5.16 (d, J = 9.9 Hz, 1H, H–1),
3.80–3.73 (m, 1H, H–6a), 3.65–3.55 (m, 1H, H–6b), 3.52–3.43 (m, 1H, H–5), 3.34–3.17 (m, 4H), 2.91
13
(dd, J1 = 9.9 Hz, J2 = 9.9 Hz, 1H, H–2), 2.59–2.33 (m, 2H). C-NMR (CD3OD) for sugar carbons
173.32 (amide), 80.33, 77.96, 74.88, 71.45, 62.21, 56.87. ES-HRMS: calcd for C17H17F17N2O5Na+m/z
675.0758: found: m/z [M+Na]+ 675.0786.
3-Hexadecyloxyl-propy-1-ne (22). Compound 20 (8.25 mmol, 2.0 g) and 21 (9.9 mmol, 0.884 mL)
were dissolved in dry DMF (20 mL) under nitrogen atmosphere, then NaH (9.9 mmol, 237 mg) was
added and the reaction mixture was heated to 90 °C overnight. The reaction was stopped by addition of
water (8 mL). The mixture was concentrated under high vacuum and the residue was purified by flash
chromatography using hexane/ethyl acetate mixture (9.5:0.5) to give compound 22 as a white sticky
solid yield 60%. The NMR data correspond to the previously reported data [24].
4-(Hexadecyloxy)methyl)-1-(3,4,6-tri-O-acetyl-2'-deoxy-2'-N-phthalimido-β-D-glucopyranosyl)-1,2,3-
triazole (23). Compound 10 (0.09 mmol, 40 mg) and compound 22 (0.1 mmol, 29 mg) were dissolved
in DCM (5 mL), copper-I-iodide (0.009 mmol, 1.65 mg), acetic acid (0.17 mmol, 12.88 mg), and
diisopropylethylamine (0.17 mmol, 22.48 mg) were added at the same time and the reaction mixture
were left stirring for 6 h. At the end of the reaction (TLC monitoring), the reaction mixture was
concentrated under vacuum and purified by flash chromatography using a hexane/ethyl acetate mixture
(6:4) to give compound 23 as a white solid. Yield 58 mg (90%). 1H-NMR (CDCl3): δ = 7.81–7.71 (m,
5H, phthalimido and triazole), 6.79 (d, J = 9.9 Hz, 1H, H–1), 6.03 (dd, J1 = 10.2 Hz, J2 = 9.6 Hz, 1H,
H–3), 5.34 (dd, J1 = J2 = 9.6Hz, 1H, H–4), 4.85 (dd, J1 = 9.9 Hz, J2 = 10.2 Hz, 1H, H–2), 4.55 (s, 2H,
–CH2–O–CH2), 4.37 (m, 1H, H–5), 4.26–4.05 (m, 2H, H–6), 3.42 (t, J = 6.7 Hz, 2H), 2.14 (s, 3H,

Molecules 2013, 18
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acetate–CH3), 2.05 (s, 3H, acetate–CH3), 2.01 (s, 3H, acetate–CH3), 1.45–1.55 (m, 2H, CH2–(CH2)13),
1.25 (brs, 26 H, –(CH2)13), 0.88 (t, J = 6.6 Hz, 3H, –(CH2)13–CH3). 13C-NMR (CDCl3) δ 171.18,
170.59, 169.91, 169.43, 146.16, 134.56, 123.90, 120.75, 83.03, 77.50, 77.28, 77.08, 76.65, 75.05,
73.58, 70.96, 70.47, 68.24, 64.05, 61.69, 60.40, 54.03, 31.92, 29.69, 29.65, 29.59, 29.49, 29.35, 26.07,
+
22.68, 21.03, 20.71, 20.66, 20.58, 20.34, 14.19, 14.11. ESMS: calcd for C39H56N4O10Na m/z: 763.4,
found m/z [M+Na]+ 763.7.
4-(Hexadecyloxy)methyl)-1-(2'-deoxy-2'-amino-β-D-glucopyranosyl)-1,2,3-triazole (5). Compound 23
(58 mg) was dissolved in mixture of butanol (2 mL) and ethylenediamine (2 mL). The resulting
mixture was heated to 90 °C and stirred for 3 h. The reaction was monitored by TLC. The solution was
concentrated to dryness. The resulting residue was purified by flash gradient chromatography (reverse-
phase C18 silica gel, 100 water to 100 methanol) to give compound 5 as a white solid in a yield 22 mg
1
(60%). H-NMR (CD3OD) δ = 8.21 (s, 1H, triazole H), 5.60 (d, J = 9.2 Hz, 1H, H–1), 3.91 (dd,
J = 12.2, 12.1 Hz, 1H H–3), 3.74 (dd, J = 12.2, 5.0 Hz, 1H, H–4), 3.67–3.25 (m, 8H), 1.69–1.53 (m,
2H, –CH2–(CH2)13), 1.32 (brs, 26H, –(CH2)13), 0.93 (t, J = 6.9 Hz, 3H, –CH3). 13C-NMR (CD3OD)
δ = 124.58 (triazole–CH), 81.27 (–C1), 71.85, 64.63, 62.43, 49.89, 49.60, 49.32, 49.04, 48.75, 48.47,
48.19, 33.09, 30.79, 30.75, 30.62, 30.48, 27.24, 23.75, 14.47. ES-HRMS: calcd for C25H48N4O5Na+ m/z
507.3517: found: m/z [M+Na]+ 507.3532.
3.3. General Procedure for the Synthesis of Diglycosylated Compounds 6 and 7
Compounds 6 and 7 were synthesized by diglycosylation of commercially available lipids 25 and
racemic alcohol 26, respectively. The previously reported thioglycoside donor 24 [19] was synthesized
.
from compound 9 and thiophenol using BF3 Et2O as promoter in DCM at 60 °C for 16 h. The
glycosylation reaction was carried out using silver triflate and N-Iodosuccinimide in anhydrous DCM
under argon atmosphere for 5 h. The reaction was stopped by addition of saturated sodium thiosulphate
solution followed by washing with saturated NaHCO3 solution (×3). The organic layer was
concentrated under vacuum to give a brownish residue which was purified with flash chromatography
using hexane/ethyl acetate mixture (6:4) to give protected diglycosylated glycolipids 27 and 28 as a
white foam (yield 45%). Compounds 27 and 28 were subsequently deprotected using a 1:1 mixture of
ethylenediamine and butanol for 4 h, followed by removal of solvent under vacuum and purified with
Ethyl acetate/methanol mixture (7:3) to give compounds 6 and 7, respectively (yield 70%).
3-Hexadecyloxy-1,2R-di(3,4,6-tri-O-acetyl-2'-deoxy-2'-N-phthalimido-β-D-glucopyranosyl)-glycerol
1
(27). H-NMR (CDCl3) δ = 7.93–7.71 (m, 8H, phthalimido), 5.75–5.62 (m, 2H), 5.53 (d, J = 8.5 Hz,
1H), 5.17 (d, J = 8.7 Hz, 1H), 5.14–5.00 (m, 2H), 4.41–4.38 (m, 2H), 4.26–4.09 (m, 3H), 3.91–3.83
(m, 1H), 3.82–3.66 (m, 4H), 3.50–3.33 (m, 2H), 3.09–3.15 (m, 3H), 2.17 (s, 3H), 2.12 (s, 3H), 1.95 (d,
J = 2.8 Hz, 6H, acetate–CH3), 1.91 (d, J = 2.8 Hz, 6H, acetate–CH3), 1.64 (m, 2H), 1.43–1.05 (brs,
26H, –(CH3)13), 0.88 (t, J = 6.6 Hz, 3H). 13C-NMR (CDCl3) δ = 171.22, 170.27, 169.74, 168.66,
165.86, 134.16, 123.51, 123.49, 98.76, 96.97, 77.56, 76.71, 71.64, 71.61, 70.66, 70.47, 69.89, 69.16,
68.91, 62.18, 54.61, 54.40, 37.02, 31.94, 29.72, 29.51, 29.38, 25.98, 22.70, 22.70, 20.80, 14.13.
ESMS: Calcd for C59H78N2O21Na+ m/z 1173.5, found m/z [M+Na]+ 1173.6.

Molecules 2013, 18
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3-Hexadecyloxy-1,2S/R-di(3,4,6-triacetyl-2'-deoxy-2'-N-phthalimido-β-D-glucopyranosyl)-glycerol
(28). The NMR data of compound 28 shown here are for the 2R and 2S 1:1 diastereomeric mixture and
1
the four anomeric protons were identified and labeled accordingly. H-NMR (CDCl3) characteristic
data: δ = 7.90–7.68 (m, 16H aromatic protons), 5.76–5.63 (m, 2H, H-3 (sugar 1), H-3 (sugar 2), 5.53
(d, J = 8.5 Hz, 1H, H–1 (sugar 1-2R-isomer), 5.39 (d, J = 8.5 Hz, 1H, H–1 (sugar 1 2S isomer), 5.33
(d, J = 8.3 Hz, 1H, H–1 (sugar 2-2S isomer), 5.17 (d, J = 8.7 Hz, 1H, H–1 (sugar 2-2R-isomer),
1.43–1.05 (brs, 52H, –(CH3)13), 0.85 (t, J = 6.6 Hz, 6H). 13C-NMR (CDCl3) characteristic data:
δ = 170.72, 170.05, 169.55, 167.80, 134.25, 131.47, 123.70, 123.41, 98.77, 98.36, 78.65, 71.76, 71.64,
71.37, 70.76, 70.71, 69.90, 69.17, 69.00, 68.91, 62.19, 62.02, 54.63, 54.42, 31.95, 29.63, 29.38, 28.94,
22.71, 20.80, 14.13. ESMS: Calcd for C59H78N2O21Na+ m/z 1173.5, found m/z [M+Na]+ 1173.5.
1
3-Hexadecyloxy-1,2S/R-di(-2'-deoxy-2'-amino-β-D-glucopyranosyl)-glycerol (6). H-NMR (CD3OD)
characteristic data for the diastereomeric mixture: δ 4.83–4.73 (m, 2H, H–1 (sugar 1, 2R/2S),
4.71–4.57 (m, 2H, H–1 (sugar 2, 2R/2S), 1.59 (m, 4H, –CH2–(CH)13), 1.32 (brs, 52H, (CH2)13), 0.93
(2xt, 6H, -CH3). 13C-NMR (MeOD) δ 102.75, 102.01, 101.47, 100.89 (anomeric carbons) 78.82, 78.75,
78.69, 78.53, 78.12, 75.45, 75.16, 74.95, 73.18, 72.18, 72.14, 71.85, 69.97, 62.80, 62.71, 62.12, 61.93,
58.33, 58.16, 50.24, 49.67, 34.98, 34.86, 33.45, 31.16, 31.14, 31.06, 31.02, 30.84, 27.57, 24.65, 24.11,
19.13, 14.82. ES-HRMS: calcd for C31H62N2O11Na+ m/z 661.4246,found: m/z [M+Na]+ 661.4241.
3-Hexadecyloxy-1,2R-di(-2'-deoxy-2'-amino-β-D-glucopyranosyl)-glycerol (7). 1H-NMR (methanol-d4)
δ = 4.78 (dd, J = 8.3, 7.9 Hz, 1H, H–1a), 4.68 (dd, J = 8.3 Hz, 7.8 Hz, 1H, H–1b) 4.27–4.19 (m, 1H),
4.09–3.90 (m, 4H), 3.85–3.62 (m, 4H), 3.62–3.45 (m, 4H), 3.45–3.25 (m, 4H), 2.98–2.79 (m, 2H,
H–2a, H–2b), 1.66–1.53 (m, 2H, –CH2–CH2–(CH2)13), 1.32 (brs, 26H, –(CH2)13–), 0.93 (t, J = 6.8 Hz,
13
3H, –CH3). C-NMR (CD3OD) δ = 101.92, 100.38, 78.17, 77.74, 74.40, 71.83, 71.69, 71.35, 71.24,
69.52, 67.17, 62.37, 57.95, 57.71, 35.99, 33.08, 30.80, 27.19, 23.74, 14.44. ES-HRMS: calcd for
C31H62N2O11Na+ m/z 661.4246, found: m/z [M+Na]+ 661.4222.
3.4. Biological Activity
3.4.1. Cell Culture
Breast (BT474, MDA-MB-231), prostrate (DU 145, PC3), pancreas (MiaPaCa2) cell lines were
grown from frozen stocks of cells that were originally obtained from ATCC (Manassas, VA, USA).
JIMT-1 breast cancer cells were originally obtained from DSMZ (Braunschweig, Germany). JIMT1,
DU145 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), PC3 cells were grown in
F12K medium. The cells were grown in media supplemented with 10% fetal bovine serum (FBS),
penicillin (100 U/mL) and streptomycin (0.1 mg/mL)
3.4.2. Cytotoxicity Assay
The cytotoxicity assay was carried out using a previously reported method [22]. Cell viability was
determined with the cell Titre 96 AQueous One Solution (MTS assay, Promega, Madison, WI, USA.
Equal numbers of cancer cells (7500–9500) in media (100 µL) were dispersed into 96-well plates. As
blanks, media without cells (100 µL) were also placed in some wells and treated similarly to the cell

Molecules 2013, 18
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containing wells. After an incubation period of 24 h, a solution of test compound ((100 µL) in medium
at twice the desired concentration was added to each well. The treated cells were incubated for a
further 48 h, after which time methanethiosulfonate (MTS) reagent (20%V/V) was added to each well.
The plates were incubated for 1–4 h on a Nutating mixer in a 5% CO2 incubator, and then the optical
density (OD) was read at 490 nm by using a SpectraMax M2 plate reader (Molecular Devices Corp.,
Sunnyvale, CA, USA). The blank values were substracted from each value, and the viability values of
the treated samples relative to controls with vehicle were calculated. The values for the plots are the
means ± standard deviation of six different wells.
4. Conclusions
Six novel cationic GAEL analogs were synthesized to explore novel SAR in this class of
compounds. We were especially interested to explore how changes in the nature of the anomeric
linkage, the nature of the hydrophobic lipid tail and the stereochemistry of the glycerol moiety affects
the cytotoxic properties against breast, pancreas and prostate cancer cell lines. During our study, we
discovered that replacement of the O-glycosidic linkage of lead compound 1 with an N-glycosidic
linkage or a triazole linkage resulted in significant loss of anticancer activity against all six cancer cell
lines tested. Moreover, replacement of the hydrophobic lipid tail by a fluorinated tail as well as
replacement of the methoxy substituent by a second glucosamine moiety resulted in decreased
cytotoxic activity.
Acknowledgements
This study was supported by the Natural Science and Engineering Research Council of Canada
(NSERC) to FS and the Canadian Breast Cancer Foundation Prairie/Northwest Territories (NMT) to
GA. Makanjuola Ogunsina is grateful to University of Manitoba and Manitoba Provincial Government
for graduate scholarships, UMGF (University of Manitoba Graduate Fellowship) and MGS (Manitoba
Graduate Scholarship), respectively.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2–7 are available from the authors.
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