Syntheses of L‐rhamnose‐linked amino glycerolipids and their cytotoxic activities against human cancer cells

Article abstract of DOI:10.3390/molecules25030566

A major impediment to successful cancer treatment is the inability of clinically available drugs to kill drug‐resistant cancer cells. We recently identified metabolically stable L‐glucosaminebased glycosylated antitumor ether lipids (GAELs) that were cytotoxic to chemotherapy‐resistant cancer cells. In the absence of commercially available L‐glucosamine, many steps were needed to synthesize the compound and the overall yield was poor. To overcome this limitation, a facile synthetic procedure using commercially available L‐sugars including L‐rhamnose and L‐glucose were developed and the L‐GAELs tested for anticancer activity. The most potent analog synthesized, 3‐amino‐1‐O‐hexadecyloxy‐2R‐(O–α‐L‐rhamnopyranosyl)‐sn‐ glycerol 3, demonstrated a potent antitumor effect against human cancer cell lines derived from breast, prostate, and pancreas. The activity observed was superior to that observed with clinical anticancer agents including cisplatin and chlorambucil. Moreover, like other GAELs, 3 induced cell death by a non‐membranolytic caspase‐independent pathway.

Full text of DOI:10.3390/molecules25030566

Article
Syntheses of ‐Rhamnose‐Linked Amino
L
Glycerolipids and Their Cytotoxic Activities against
Human Cancer Cells
1
2
1
2,3,4
Makanjuola Ogunsina , Pranati Samadder , Temilolu Idowu , Mark Nachtigal
,
1,
2,
Frank Schweizer * and Gilbert Arthur *
1
Department of Chemistry and Biochemistry, Faculty of Science, University of Manitoba,
Winnipeg, MB R3T 2N2, Canada; mogunsina@gmail.com (M.O.); idowut@myumanitoba.ca (T.I.)
Department of Biochemistry and Medical Genetics, Rady Faculty of Health Sciences, University of
Manitoba, Winnipeg, MB R3E 0W9, Canada; sohagdalkutir@gmail.com (P.S.);
Mark.Nachtigal@umanitoba.ca (M.N.)
Department of Obstetrics, Gynecology and Reproductive Sciences, Rady Faculty of Health Sciences,
University of Manitoba, Winnipeg, MB R3E 0W9, Canada
2
3
4
Research Institute in Oncology & Hematology, CancerCare Manitoba, Winnipeg, MB R3E 0V9, Canada
* Correspondence: Frank.Schweizer@umanitoba.ca (F.S.); Gilbert.Arthur@umanitoba.ca (G.A.)
Received: 4 December 2019; Accepted: 26 January 2020; Published: 28 January 2020
Abstract: A major impediment to successful cancer treatment is the inability of clinically available
drugs to kill drug‐resistant cancer cells. We recently identified metabolically stable
based glycosylated antitumor ether lipids (GAELs) that were cytotoxic to chemotherapy‐resistant
cancer cells. In the absence of commercially available ‐glucosamine, many steps were needed to
synthesize the compound and the overall yield was poor. To overcome this limitation, a facile
synthetic procedure using commercially available ‐sugars including ‐rhamnose and ‐glucose
were developed and the ‐GAELs tested for anticancer activity. The most potent analog synthesized,
3‐amino‐1‐O‐hexadecyloxy‐2R‐(O–α‐ ‐rhamnopyranosyl)‐sn‐ glycerol 3, demonstrated a potent
L‐glucosamine‐
L
L
L
L
L
L
antitumor effect against human cancer cell lines derived from breast, prostate, and pancreas. The
activity observed was superior to that observed with clinical anticancer agents including cisplatin
and chlorambucil. Moreover, like other GAELs, 3 induced cell death by a non‐membranolytic
caspase‐independent pathway.
Keywords: glycosylated antitumor ether lipids;
L‐rhamnose‐based glycolipids; chemotherapy
resistant; caspase independent
1. Introduction
Despite increasing investment in cancer research and treatment, the number of new cases and
cancer deaths are increasing in both the developed and the developing world, affecting both males
and females in nearly the same proportion [1]. Glycosylated antitumor ether lipids (GAELs), a
subclass of antitumor ether lipids (AELs) [2,3], are cytotoxic to a wide range of epithelial cancer cell
lines grown as adherent and spheroidal cultures as well as cancer stem cells (CSCs) [4–10]. While
most anticancer drugs kill cells by inducing apoptosis, GAELs kill cells by an apoptosis independent
mechanism [2,8,9], possibly by methuosis [11].
Recently, we reported the synthesis of GAELs with
showed that in spite of the replacement of ‐glucosamine in GAELs with the
compounds formed were equally cytotoxic to cancer cells [7]. The rationale for synthesizing the
L
‐glucosamine as the sugar moiety [7]. We
D
L
‐enantiomer, the
L‐
Molecules 2020, 25, 566; doi:10.3390/molecules25030566
www.mdpi.com/journal/molecules

Molecules 2020, 25, 566
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glucose analogues of the D‐GAELs was to develop compounds that would be resistant to metabolic
degradation (especially by glycosidases) as mammals do not have enzymes that metabolise L‐
glycosidic bonds. In the absence of commercially available L‐glucosamine, synthesis of the L‐
glucosamine derived GAELs was initiated with L‐mannose and required many synthetic steps. By
contrast, L‐sugars such as L‐rhamnose or L‐mannose that are commercially available are relatively
simple to attach to a glycerol moiety. Our previous studies have shown that the activity of GAELs
depends on the maintenance of a cationic amino group in the molecule [3]. The present study was
undertaken to determine whether coupling the L‐sugars to the glycerol moiety and maintaining the
cationic amino moiety on the glycerol would generate biologically active L‐GAELs with activity
similar to the previously described L‐glucosamine GAEL [7]. The advantage would be facile, cost
effective synthetic procedures for potential anticancer compounds. Herein we report the synthesis of
a L‐rhamnose‐GAEL compound and demonstrate its activity against human epithelial cancer cell
lines.
2. Results
We have recently demonstrated that compound 1, L‐glucosamine GAEL (L‐Gln) was cytotoxic
against human epithelial cancer cells [7]. GAELs with L‐sugars other than L‐glucosamine (1) were
synthesized to assess whether we could produce biologically active GAELs and generate insight on
structural features required for activity. Novel L‐GAEL analogues, compounds 2A6 (Figure 1), were
synthesized and assessed for their cytotoxic activity. The neutral glycoglycerolipid 2 was synthesized
to evaluate the effect of replacing L‐glucosamine of compound 1 and D‐glucosamine of 7 with the
neutral sugar L‐rhamnose. Compound 3 was synthesized to evaluate the effect of introducing a
cationic moiety into L‐rhamnose‐based glycoglycerolipid. The primary position of the amine was
selected for ease of synthesis. Compound 4 was synthesized to evaluate the effect of amide linkage
with p‐hydroxyphenyl propionic acid. The L‐glucose‐based cationic compound 5 was synthesized to
evaluate the effect of different types of sugars relative to 3. Compound 6 was synthesized to
investigate the effect of the two amino moieties as prior studies showed that additional amino
substituent at C‐6 position of D‐glucosamine‐derived GAELs enhanced potency.
OC16H33
OC16H33
OC16H33
NH2
O
O
O
O
O
HO
HO
O
O
O
NH2
HO
HO
HO
HO
HO
OH
OH
OH
2
3
1
OC16H33
O
OC16H33
NH2
HO
HO
NH
O
O
O
O
OH
HO
HO
HO
OH
5
4
OC16H33
NH2
OH
O
H2N
O
OC16H33
O
HO
HO
HO
O
O
H2N
HO
OH
7
6
Figure 1. Reference and newly synthesized compounds used in this study. Compound 1 is reference
L‐GAEL, Compounds 2–6 are newly synthesized L‐sugar‐derived GAELs, and compound 7 is
reference D‐GAEL.
2.1. Synthesis of L‐Sugar Derived Glycolipids 2–6
The hydroxyl groups of L‐rhamnose 8 was protected with acetyl groups using acetic anhydride
in pyridine and dimethylaminopyridine as catalyst to give the tetraacetate 9 (Scheme 1). The phenyl
thioglycoside 10 was synthesized from 9 by a BF3∙OEt2 promoted glycosylation with thiophenol.
Silver triflate promoted glycosylation of 10 to the commercially available glycerolipid alcohol 11

Molecules 2020, 25, 566
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yielded the fully protected glycolipid 12. Sodium methoxide‐catalysed deblocking of 12 in methanol
gave the desired glycolipid 2 (Scheme 1).
OC16H33
OC16H33
O
O
H
O
HO
H
SPh
OH
OAc
11
a
O
b
O
O
O
HO
b
AcO
AcO
93 %
AcO
c, 58 %
HO
AcO
AcO
AcO
OH
OAc
OAc
OAc
12
8
10
9
OC16H33
H
OC16H33
H
OC16H33
H
f
91 %
e
d
75 %
c, 55 %
HO
HO
HO
TsO
HO
N3
60 %
14
15
13
OC16H33
H
OC16H33
H
N3
OC16H33
H
N3
O
O
O
O
O
d
O
O
HO
79 %
AcO
HO
HO
AcO
HO
OH
OAc
OH
2
17
16
O
OH
g
51 %
O
OC16H33
OC16H33
H
NH2
18
O
OH
NH
O
O
O
h, 98 %
HO
HO
HO
OH
HO
OH
OH
3
4
Scheme 1. Synthesis of compounds 2–4. Reagents and conditions: (a) Ac2O, DMAP, Pyridine, 18 h, rt;
(b) PhSH, BF3∙Et2O, DCM,18 h, rt; (c) AgOTf, NIS, DCM, 3 h, rt; (d) MeONa, MeOH, 1 h; (e) TsCl,
Pyridine, DMAP, 0 °C to rt, 18 h; (f) DMF, NaN3, 70 °C; (g) P(CH3)3, THF, H2O, 2 h, rt; (h) TBTU, DMF,
5
h. Abbreviations: 4‐dimethyl amino pyridine (DMAP), dichloromethane (DCM), N,N‐
dimethylformamide (DMF), room temperature (rt), N‐iodosuccinimide (NIS).
Synthesis of compound 3 was achieved by glycosylation of the glycoside donor 10 with the
acceptor azido lipid 15, as described above, to give the fully protected azidoglycolipid 16. The
azidoglycerolipid 15 was made from the commercially available glycerol‐analogue 13 as previously
described [6]. Compound 16 was deacetylated to give the azide 17. Reduction of the azido substituent
of 17 was accomplished by using trimethyl phosphine in THF/water mixture to give monoamino
compound 3 (Scheme 1). The amide 4 was synthesized by coupling 3 to a pre‐activated p‐
hydroxyphenylproprionic acid 18 using TBTU [6] (Scheme 1).
The amino L‐glucose based compound 5 was synthesized from L‐glucose following the same
procedure described above for the synthesis of compound 3 (Scheme 2).

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OH
OH
OAc
OAc
HO
HO
AcO
AcO
a
b
AcO
AcO
SPh
OAc
O
O
O
88 %
88 %
HO
AcO
AcO
20
c, 50 %
19
L-glucose
OC16H33
H
HO
N3
15
OC16H33
H
OC16H33
H
HO
HO
AcO
AcO
O
O
d
O
O
OH
OAc
84 %
N3
N3
HO
AcO
22
21
e
55 %
OC16H33
H
HO
HO
O
O
OH
NH2
HO
5
Scheme 2. Synthesis of compound 5. Reagents and conditions: (a) Ac2O, DMAP, Pyridine, 18 h, rt; (b)
PhSH, BF3∙Et2O, DCM,18, h, rt; (c) AgOTf, NIS, DCM, 3 h, rt; (d) MeONa, MeOH, 1 h; (e) P(CH3)3,
THF, H2O, 2 h, rt; (h) TBTU, DMF, argon, 5 h. Abbreviations: 4‐dimethyl amino pyridine (DMAP),
dichloromethane (DCM), N,N‐dimethylformamide (DMF), room temperature (rt), N‐iodosuccinimide
(NIS).
Compound 6 was synthesized by coupling of glycoside donor 28 to lipid alcohol 15 to afford α‐
glycolipid 29 (Scheme 3). The glycoside donor 28 was synthesized from L‐mannose in six steps. At
first, the hydroxyl functional groups of L‐mannose were acylated to afford pentaacetate 23 as
described above.Compound 23 was subsequently converted into thiophenyl glycoside 24 by BF3∙Et2O
promoted glycosylation with thiophenol. Deacetylation of 24 gave compound 25. The azido
functional group at the C‐6‐position was installed by selective activation of the C‐6 hydroxyl group
in 25 as sulphonate ester to give 26, followed by nucleophilic displacement of the sulphonate group
by sodium azide in DMF to afford the 6‐azido analog 27. Protection of the remaining hydroxyl groups
using acetic anhydride in pyridine produced the glycoside donor 28. Donor 28 was used in
glycosylation reaction with azido lipid alcohol 15 to produce glycolipid 29. The ester groups were
then deprotected to afford the diazido compound 30 which was subsequently subjected to reduced
using trimethyl‐phosphine in THF/water to produce the desired α‐anomeric glycolipid 6 (Scheme 3).

Molecules 2020, 25, 566
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SPh
OH
OAc
HO
HO
AcO
AcO
a
b
AcO
AcO
O
O
O
85 %
70 %
HO
AcO
AcO
24
OH
OAc
OAc
23
L
-mannose
c
91 %
O
SPh
SPh
SPh
N3
HO
TsO
HO
HO
HO
d
e
O
O
95 %
88 %
HO
HO
HO
OH
OH
OH
25
27
26
a
76 %
OC16H33
H
HO
N3
OC16H33
H
OC16H33
H
O
SPh
O
15
c
N3
HO
N3
AcO
N3
AcO
O
O
O
N3
N3
68 %
f, 71 %
HO
AcO
AcO
OH
OAc
OAc
29
30
28
g
62 %
O
OC16H33
H
H2N
HO
O
NH2
HO
OH
6
Scheme 3. Synthesis of compound 6. Reagents and conditions: (a) Ac2O, DMAP, Pyridine, 18 h, rt; (b)
PhSH, BF3∙Et2O, DCM,18, h, rt; (c) MeONa, MeOH, 1 h; (d) TsCl, Pyridine, DMAP, 0 °C to rt, 18 h; (e)
DMF, NaN3, 70 °C; (f) AgOTf, NIS, DCM, 3 h, rt; (g) P(CH3)3, THF, H2O, 2 h, rt. Abbreviations: 4‐
dimethyl amino pyridine (DMAP), dichloromethane (DCM), N,N‐dimethylformamide (DMF), room
temperature (rt), N‐iodosuccinimide (NIS).
2.2. In Vitro Screening of L‐Sugar Derived Glycolipids’ Cytotoxic Activity Against Human Epithelial Cancer
Cell Lines Derived from Breast, Prostate and Pancreas
The cytotoxic properties of compounds 2A6 were initially determined against exponentially
growing human epithelial cancer cell lines including breast (MDA‐MB‐231, JIMT‐1), pancreas
(MiaPaCa2) and prostate (DU‐145, PC‐3) to identify the most active compound. The cells were
incubated with increasing concentrations of the compounds (0–30 μM) for 48 h followed by
assessment of cell viability using the MTS assay. The most potent of the newly synthesized
compounds was the L‐rhamno configured glycoglycerolipid 3. This compound carries a primary
amino group at the sn‐3 position of the glycerolipid and L‐rhamno sugar at the sn‐2 position via an
α‐rhamnosidic linkage. Compound 3 was active with CC50 values of 4.8–14 μM (Figure 2 and Table
1). Greater than 90% loss of cell viability was observed at a concentration range of 7.5–15.0 μM for all
the cell lines. Out of these five cell lines, MDA‐MB‐231, a cisplatin resistant and triple negative breast
cancer cell line, was most sensitive to compound 3.

Molecules 2020, 25, 566
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Figure 2. Effects of compounds 2–6 on the viability of MDA‐MB‐231, JIMT‐1, DU‐145, and MiaPaCa2
cells lines. Twenty four hours after plating, cells were incubated with compounds 2–6 (0–30 μM) for
48 h. Cell viability relative to vehicle control was measured by MTS assay. The results represent the
mean ± standard deviation of 6 independent determinations.
Table 1. CC50 values (μM) of chlorambucil, cisplatin, and compounds 1–7 on a panel of human
epithelial cancer cell lines: breast (MDA‐MB‐231, JIMT1), pancreas (MiaPaCa2) and prostrate (DU145,
PC3).
MDA‐MB‐231
JIMT‐1
>150
NT
6.5
28
5.5
MiaPaCa2
>150
>20
DU‐145
>150
9.2
12.5
25
8.2
30
>30
17.5
9.0
PC‐3
>150
NT
12.5
25.0
11.0
21.5
>30
Chlorambucil
Cisplatin
>150
>20
11.0
26
4.8
23
NT
NT
NT
1*
2
3
4
5
6
7*
7.5
>30
8.5
>30
>30
19
9.0
25
>30
12.5
9.0
25
13.5
The CC50 value (μM) is defined as the concentration required to decrease cell viability by 50% relative
to the untreated control. NT = not tested. * Results from Ref [7].
The second most potent compound was the L‐manno configured bisamine 6 which in contrast
to 3 bears a primary amino substituent at the position C‐6 of the sugar. This compound was typically
2‐ to 4‐times less active than glycolipid 3 with CC50 values in the range of 12.5–25 μM (Table 1). This
is in contrast to previous observation with L‐glucosamine derived GAELs where a primary amino
substituent at the C‐6 of the L‐glucose scaffold enhanced activity [7]. But it is noteworthy that in the
D‐sugar GAEL series, there was significant loss of activity with mannose analogs [10], CC50 was not
reached at 30 μM.
The L‐rhamnose based analog 2 that is devoid of any amino group and amide 4 were
significantly less active than 3 and 6 with CC50 values ≥ 21.5 μM across all the five cell lines. The
reduced cytotoxicity of compounds 2 and 4 when compared to that of compound 3 demonstrated
that the free amine is an important structural feature required for cytotoxicity of L‐sugar‐derived
GAELs. This is consistent with earlier results on D‐glucosamine‐derived GAEL series [10].
Surprisingly, compound 5, an analog of 3 where the L‐rhamnose is replaced with L‐glucose is was
less active. Depending on the cell line, loss of viability caused by 5 ranged from 0–35%. We were
therefore unable to achieve CC50 at the highest concentration tested (30 μM) for any of the cell lines.

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The effects of L‐mannose, L‐glucose, rhamnose (8) were also tested to determine if these L‐sugars
displayed any cytotoxicity against the cancer cells. The results revealed that rhamnose, L‐mannose
and L‐glucose were not cytotoxic at the highest dose (30 μM) tested (data not shown).
The activity of the most active analogue, L‐rhamnose linked glycerolipid 3, was compared with
results published on the activity of L‐ and D‐glucosamine based analogs 1 and 7 [7]. Compound 3 is
as active or more active (CC50 = 4.8–11.0 μM) than 1 (6.5–12 μM) or 7 (8.0–13.5 μM) across all the cell
lines (Table 1).
The activities of compounds 2–6 against the cancer cell lines were compared with those of
clinically‐used anticancer agents: chlorambucil and cisplatin. As shown in Table 1, compounds 2–6
were more potent across the five cell lines than chlorambucil. A CC50 concentration for chlorambucil
was not attained even at a concentration as high as 150 μM. Compounds 3 and 6 also displayed
superior cytotoxicity than cisplatin against all the cell lines. Cisplatin did not achieve 50% cell death
at 20 μM against MDA‐MB‐231 and MiaPaCa‐2 cell lines. Thus, the activity of 3 in vitro was better
than that of anticancer drugs like chlorambucil and cisplatin. The activity of 3 was similar or better
than that of L‐glucosamine derived compound 1.
Having identified 3 as the most active of the synthesised L‐sugar analogues, its effects on the
viability of additional cancer cell lines including BT549, MDA‐MB‐453, MDA‐MB‐468, and Hs578t
(triple negative breast cancer lines), and BT474 (hormone receptor positive breast cancer cells) (Figure
3) was investigated. Concentrations of compound 3 between 6–15 μM were sufficient to kill all the
triple negative cell lines with the exception of MDA‐MB‐453 where viability was inhibited by 60% at
the highest concentration tested. 30 μM of 3 was required for complete loss of viability of the hormone
sensitive breast cancer BT474 cell line.
BT-474
BT-549
MDA-MB-453
120
100
80
60
40
20
0
Hs 578T
MDA-MB-468
0.0
2.5
5.0
7.5
M
10.0 12.5 15.0
Figure 3. Dose‐dependent alteration of cell viability. Human breast adenocarcinoma cell lines were
treated with increasing concentrations of 3. Twenty four hours after plating, cells were incubated with
0–15 μM 3 for 48 h. Cell viability relative to vehicle control was measured by MTS assay. The results
represent the mean ± standard deviation of 6 independent determinations.
2.3. Determination of the Effect of Pan‐caspases Inhibition on Cytotoxicity of Compound 3 in JIMT‐1 and
DU‐145 Cell Lines
One of the hallmark features of cytotoxic GAELs is their ability to kill cancer cells via an
apoptosis‐independent mechanism [2,4,5,7–9]. We evaluated whether the mechanism of action of 3
was similar to other GAELs. The ability of 3 to kill JIMT‐1 and DU‐145 cell lines in the presence or
absence of QVD‐OPh (QVD), an effective pan‐caspase inhibitor [12], was investigated. There were no
statistically significant differences in cytotoxicity in the absence or presence of 40 μM QVD (Figure
4). In contrast, QVD significantly reduced cytotoxicity of Adriamycin, an apoptosis inducing
anticancer agent, in JIMT‐1 and DU‐145 cell lines at all concentration tested. The results of the studies
show that compound 3 kills cancer cells via a caspase‐independent pathway(s).

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Figure 4. Demonstration of caspase/apoptosis independent cell death. DU‐145 or JIMT‐1 cell lines
were treated with increasing concentrations of 3 or Adriamycin in the presence or absence of 40 μM
QVD. Four hours after plating, cells were treated with QVD. After 20 h, the cells were incubated with
0–15 μM 3 or 0–4 μM Adriamycin for 48 h. Cell viability relative to vehicle control was measured by
MTS assay. The results represent the mean ± standard deviation of 6 independent determinations.
2.4. Evaluation of Effect of L‐Rhamnose Based GAEL 3 on Integrity of DU145 or JIMT‐1 Cell Membranes
After determining that the mechanism of cell death of compound 3 is independent of caspases‐
mediated apoptosis, we evaluated if cell death was due to membrane disruption or lysis using the
cell impermeant ethidium homodimer‐1 (EthD‐1) dye that emits red fluorescence upon binding to
DNA [13,14]. DU‐145 and JIMT‐1 were treated with compound 3 at 10 and 15 μM, respectively. After
4–6 h, the cells were treated with EthD‐1 dye and monitored under fluorescence microscopy.
Comparison of untreated control cells and cells treated with compound 3 in both DU‐145 and JIMT‐
1 cell lines demonstrated that there was no significant cell membrane lysis. In contrast, cells incubated
with 0.01% Triton X‐100 for 10 min stained bright red (Figure 5).
Figure 5. Evaluation of effect of compound 3 on cell membrane of DU‐145 and JIMT‐1 cell lines using
cell impermeant ethidium homodimer‐1 (EthD‐1) dye that emits red fluorescence upon binding to
DNA.

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2.5. Hemolytic Properties of L‐Rhamno Configured GAEL 3
To further evaluate the membrane lytic properties of compound 3, we investigated its capacity
to lyse ovine erythrocytes [13]. The highest hemolysis observed with compound 3 was about 6.6% at
100 μM compared to the 1% ammonium hydroxide positive control (Figure 6). These results indicated
that compound 3 is not lytic and may be compatible with intravenous administration without concern
for hemolysis.
Figure 6. Hemolytic properties of GAEL 3 using freshly isolated ovine erythrocytes. 1% ammonium
hydroxide was used for 100% hemolysis, the control. The values were determined as % of control.
3. Discussion
GAELs are a promising class of investigational anticancer agents with potent antitumor activity
against a range of cancer cell lines and CSCs [2,3,5,6]. The biologically active GAELs that we have
developed thus far typically have a glycerol backbone with a methoxy group at the C‐2 position, a D‐
or L‐glucose at the C‐3 position, and an amino group attached to the sugar [3,4,6,7]. Due to the relative
expense and complexity in synthesizing L‐glucosamine GAEL analogues, the objective of the present
study was to develop biologically active GAELs with L‐sugars via a facile approach that would
facilitate large scale synthesis of compounds for in vivo studies. Our previous studies identified that
the presence of a primary amine in the molecule is essential for good cytotoxic activity. To simplify
synthesis, the amino group was placed at the C‐3 position of the glycerol backbone while the L‐sugar
was placed at the C‐2 position. It was reasoned that this would satisfy the requirement for a free
amine needed for activity. This was tested with the synthesis of L‐rhamnose GAEL (3) and L‐glucose‐
GAEL (5). The results showed that this GAEL scaffold did in fact result in bioactive compounds.
Comparison of the activity of the novel compounds synthesised (2–6) against epithelial cancer cell
lines derived from breast, pancreas and prostate tumors, revealed that the most potent analog was
the L‐rhamnose configured analog 3. The L‐rhamnose based analog 2, which does not have a primary
amine, or compound 4 with an amide linkage, were significantly less potent than 3 with a primary
amino substituent and L‐rhamnose at position sn‐3 and sn‐2 of the glycerolipid, respectively. This
observation shows that the primary amino substituent is essential for cytotoxicity of this series of
GAELs and is akin to what was observed with glucose‐based GAELs which required an amino group
on the sugar [2,6,10]. However, compound 5, an analog in which the L‐rhamnose of 3 was replaced
with L‐glucose showed minimal cytotoxicity. Indeed, CC50 values were not attained at the highest
tested concentration for all the cell lines. Thus, the nature and type of sugar moiety in GAELs play a
crucial role in their cytotoxicity. We have previously demonstrated with the GAEL series that the
nature of the sugar also affects activity [10].
Compound 3 was active against a wide range of breast cancer cells differing in hormone receptor
status suggesting that the presence or absence of the receptors was not a determinant in the
susceptibility of the cells to compound 3. Thus, BT‐474 hormone receptor (HER/2, ER, PR) positive
cells and the MDA‐MB‐453, a TNBC cell line, had similar CC50’s of 14 μM vs. 13.8 μM. Also, JIMT‐1
cell line (overexpression of Her2) was more sensitive to compound 3 than some TNBC cell lines.
Studies conducted to provide insight on the mode of cell death induced by 3 revealed that death
occurs via an apoptosis‐independent pathway. Thus, the mechanism of action of 3 may be similar to

Molecules 2020, 25, 566
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that described for glucose‐based GAELs [4,7–9,11]. This non‐apoptotic mechanism of cell death may
explain why GAELs are effective against resistant cancer lines as cells escape cell death through many
mechanisms including over expression of proteins that inhibit caspases and apoptosis [15–17]. Also,
the result of membrane disruption experiment using cell impermeant EthD‐1 dye demonstrated that
3 does not kill cells by membrane disruption. The fact that the cells were rounding up as they were
dying may imply possible interference with the cell cytoskeleton. Our hemolysis assay on ovine
erythrocytes further confirmed that L‐sugar GAELs induced cell death is not due to interference with
cell membrane integrity. The results also indicate that L‐rhamnose GAEL 3 may be safely delivered
by intravenous administration.
In conclusion, this study identified a novel scaffold for active L‐GAELs. The most active
compound was L‐rhamnose GAEL 3 that has potent cytotoxicity against a spectrum of cancer cell
lines. The ease of synthesis makes 3 a suitable compound for large scale synthesis for preclinical
studies in rodent models of human cancer.
4. Materials and Methods
4.1. Synthesis of GAELs
1
13
Solvents were dried over CaH2. H‐, C‐NMR spectra were recorded on an Avance 300 NMR
spectrometer (Bruker, Rheinstetten, Germany), and chemical shifts reported (in ppm) relative to
1
13
internal Me4Si (δ = 0.0) and at 75 MHz, respectively. NMR spectra ( H‐NMR, C‐NMR) of compounds
2–6 are provided in the Supplementary Materials. Thin‐layer chromatography (TLC) was carried out
on aluminum or glass‐backed silica gel GF plates (250 μm thickness) and plates were visualized by
charring with 5% H2SO4 in MeOH and or short wavelength UV light. Compounds were purified by
flash chromatography on silica gel 60 (230–400 ASTM mesh). ESI‐MS analyses were performed on a
500 MS Ion Trap Mass Spectrometer (Varian, Walnut Creek, CA, USA). MALDI‐TOF‐MS were
performed on a Bruker Daltonics Ultraflex MALDI TOF/TOF Mass Spectrometer (Bremen, Germany).
1
Purity of compound 2–6 was assessed by H‐NMR spectroscopy after reverse phase chromatography.
1,2,3,4‐L‐Rhamnopyranosyl Tetraacetate (9)
L‐Rhamnose 8 (0.99 g, 5.49 mmol) was dissolved in 20.0 mL pyridine at room temperature, then
acetic anhydride (5.20 mL, 54.90 mmol) and dimethyl amino pyridine (0.10 g) were sequentially
added and the reaction was vigorously stirred for 18 h after which it was stopped by addition of
methanol (10.0 mL) and then stirred for 15 min. The solvents were removed under high vac. The
resulting residue was then dissolved in ethyl acetate (50.0 mL) and washed with 3% HCl solution (1
time), saturated sodium bicarbonate (2 times), distilled water (1 time) and brine (1 time). The resulting
organic layer was dried over Na2SO4 and concentrated to dryness and purified by flash
chromatography using ethyl acetate and hexane (1:1) to give 9 (1.71 g, 5.12 mmol) as α, β mixture
1
(9:1). Yield was 93%. NMR data for α‐ anomer of compound 9: H‐NMR (300 MHz, chloroform‐d) δ
6.01 (d, J = 1.8 Hz, 1H, H‐1), 5.31 (dd, J = 10.1, 3.5 Hz, 1H, H‐3), 5.25 (dd, J = 3.5, 1.8 Hz, 1H, H‐2), 5.12
(dd, J = 10.1, 9.9 Hz, 1H, H‐4), 3.93 (m, 1H), 2.17 (s, 3H), 2.16 (s, 3H), 2.06 (s, 3H), 2.00 (s, 3H), 1.24 (d,
13
J = 6.2 Hz, 3H, H‐6). C‐NMR (75 MHz, CDCl3) δ 170.05, 169.81, 169.79, 168.35, 90.65, 70.48, 68.77,
+
68.72, 68.65, 20.89, 20.77, 20.74, 20.67, 17.44. ES‐MS: calculated (calcd): C14H20O9Na m/z: 355.1, found
+
[M + Na] m/z: 355.5.
Phenyl‐2,3,4‐triacetyl‐1‐thio‐α‐L‐rhamnopyranoside (10)
The tetraacetate 9 (1.71 g, 5.12 mmol) was dissolved in in 20.0 mL DCM, then thiophenol (0. 68
g, 6.14 mmol) and BF3∙Et2O (0.87 g, 6.14 mmol) were sequentially added. The reaction was stirred
vigorously for 18 h after which it was stopped with 20 mL saturated sodium bicarbonate at 0 °C. The
organic layer was separated using separatory funnel and subsequently washed with saturated 20.0
mL of sodium bicarbonate (2 times), 20.0 mL of water (1 time) and 20.0 mL of brine (1 time). The
organic layer was dried over anhydrous sodium bicarbonate and then concentrated under vacuum.

Molecules 2020, 25, 566
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The residue was then purified by flash chromatography using ethyl acetate and hexane (4:6) to give
mainly α‐anomer of 10 (1.72 g, 4.5 mmol). Yield was 88%. NMR data for the anomer of compound 10:
1
H‐NMR (300 MHz, chloroform‐d) δ 7.59–7.22 (m, 5H, aromatic protons), 5.51 (dd, J = 3.4, 1.1 Hz, 1H,
H‐2), 5.42 (d, J = 1.1 Hz, 1H, H‐1), 5.30 (dd, J = 10.0, 3.4 Hz 1H, H‐3), 5.19 (dd, J = 9.6, 10.0 Hz, 1H, H‐
13
4), 4.42–4.34 (m, 1H, H‐5), 2.15 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 1.26 (d, J = 6.2 Hz, 3H, H‐6). C‐ NMR
(75 MHz, CDCl3) δ 169.99, 169.98, 169.91, 132.08, 131.85, 129.19, 127.89, 85.71, 71.34, 71.17, 69.40, 67.79,
+
+
20.91, 20.82, 20.69, 17.35. ES‐MS: calcd: C18H22O7SNa m/z: 405.1, found [M + Na] m/z: 405.3.
1‐Hexadecyloxyl‐2R‐methoxyl‐3‐(2’3’4’‐triacetyl‐α‐L‐rhamnopyranosyl)‐sn‐glycerol (12)
The fully protected glycoside donor 10 (153 mg, 0.4 mmol) and the glycoside acceptor 11 (140
mg, 0.4 mmol) were dissolved in 15 mL of DCM under argon atmosphere, then AgOTf (0.02 g, 0.08
mmol) and N‐iodosuccinimide (0.14 g, 0.60 mmol) were simultaneously added. The reaction was
vigorously stirred for 2 h after which it was stopped with saturated solution of sodium thiosulphate
(5.0 mL) and then washed with 25.0 mL of saturated sodium thiosulphate solution (1 time), saturated
sodium bicarbonate (3 times), water (1 time) and brine (1 time). The organic layer was then dried over
anhydrous sodium sulphate and then concentrated under vac. The residue, was purified by flash
chromatography using ethyl acetate/hexane mixture (4:6) to give the α‐anomer, 12 (0.19 g, 0.31 mmol)
1
as a white solid. Yield was 58%. NMR data of 12: H‐NMR (300 MHz, chloroform‐d) δ 5.36–5.19 (m,
2H, H‐2, H‐3), 5.04 (dd, J = 9.7, 9.7 Hz, 1H, H‐4), 4.73 (d, J = 1.5 Hz, 1H, H‐1), 3.93–3.84 (m, 1H, H‐5),
3.79–3.63 (m, 1H), 3.61–3.34 (m, 9H), 2.12 (s, 3H), 2.02 (s, 3H), 1.96 (s, 3H), 1.50–1.48 (m, 2H), 1.23
13
(broad s, 26H, lipid tail), 1.20 (d, J = 6.2 Hz, 3H, H‐6), 0.85 (t, J = 6.6 Hz, 3H, terminal lipid CH3). C‐
NMR (75 MHz, CDCl3) δ 170.01, 169.92, 169.87, 97.58, 78.94, 71.78, 71.12, 69.80, 69.67, 69.12, 67.35,
66.34, 58.20, 31.89, 29.66, 29.62, 29.47, 29.33, 26.09, 22.65, 20.85, 20.74, 20.67, 17.39, 14.08. ES‐MS: calcd:
+
+
C32H58O10Na m/z: 625.4, found [M + Na] m/z: 624.8.
1‐Hexadecyl‐2R‐methoxyl‐3‐O‐α‐L‐rhamnopyranosyl‐sn‐glycerol (2)
Compound 12 (0.19 g, 0.31 mmol) was dissolved in 15.0 mL of methanol, then catalytic amount
of sodium methoxide was added and the reaction was vigorously stirred for 3 h. The reaction was
stopped by acidic ion exchange resin. The resin was filtered and the filtrate was concentrated under
vacuum and the residue was purified by flash chromatography using ethyl acetate/hexane mixture
1
(9:1) to give 2 (0.11 g, 0.23 mmol) as a white solid. Yield was 75%. NMR data of 2: H‐NMR (300 MHz,
chloroform‐d) δ 4.78 (d, J = 1.1, 1H, H‐1), 4.12 (s, 3H, OH, rhamnose‐OH), 3.97 (dd, J = 1.1, 3.3 Hz, 1H,
H‐2), 3.83–3.63 (m, 3H), 3.63–3.53 (m, 1H), 3.53–3.36 (m, 9H), 1.56 (m, 2H), 1.31 (d, J = 6.0 Hz, 3H, H‐
13
6), 1.28 (broad s, 26H, lipid tail), 0.89 (t, J = 6.5 Hz, 3H, terminal lipid CH3). C‐NMR (75 MHz, CDCl3)
δ 99.92, 79.03, 72.80, 71.84, 71.68, 70.89, 69.99, 68.24, 66.71, 58.04, 31.94, 29.72, 29.69, 29.37, 26.13, 22.70,
+
+
17.55, 14.12. MALDI‐HRMS: calcd: C26H52O7Na m/z: 499.3611, found [M + Na] m/z: 499.3615.
3‐Hexadecyloxy‐2R‐hydroxyl propyl‐1‐p‐toluene sulphonate (14)
The lipid diol 13 (2.00 g, 6.32 mmol) was dissolved in dissolved in 20 mL DCM, cooled to 0 C,
then Et3N (1.28 g, 1.80 mL,) was added followed by 4‐toluenesulphonyl chloride (1.33 g, 6.95 mmol)
and DMAP (0.04 g, 0.32 mmol). The temperature was allowed to increase to room temperature (23
°C) and the mixture was stirred for 4 h. At the end of reaction, the mixture was diluted with ethyl
(60.0 mL) acetate, washed with saturated aqueous ammonium chloride (3 times), brine (3 times). The
organic layer was then dried over sodium sulphate, concentrated under vacuum and the residue was
purified using flash chromatography using hexane/ethyl acetate (8:2) to give 14 (1.80 g, 3.80 mmol)
1
as a white flaky solid. Yield was 60%. NMR data of 14: H NMR (300 MHz, chloroform‐d) δ 7.78 (d, J
= 8.2 Hz, 2H, aromatic protons), 7.33 (d, J = 8.1 Hz, 2H, aromatic protons), 4.11–4.00 (m, 2H, TsO‐CH2),
3.99–3.89 (m, 1H, HO‐CH), 3.46–3.31 (m, 4H), 2.80 (d, J = 5.4 Hz, 1H, OH), 2.42 (s, 3H, toluene‐CH3),
13
1.55–1.41 (m, 2H), 1.25 (s, 26H, Lipid tail), 0.87 (t, J = 6.4 Hz, 3H, lipid terminal‐CH3). C‐NMR (75
MHz, CDCl3) δ 144.90, 132.77, 129.88, 127.99, 71.73, 70.77, 70.56, 68.25, 31.93, 29.71, 29.68, 29.64, 29.61,

Molecules 2020, 25, 566
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+
+
29.48, 29.37, 26.01, 22.68, 21.58, 14.11. ES‐MS: calcd: C26H46NO5Na m/z: 493.3, found [M + Na] m/z:
493.7
3‐Hexadecyloxy‐2R‐hdroxyl propyl‐1‐azide (15)
Compound 14 (1.30 g, 2.76 mmol) and sodium azide (1.81 g, 27.60 mmol) were suspended in
anhydrous DMF and the mixture was stirred at 90 °C for 18 h. at the end of the reaction the mixture
was concentrated then diluted with ethyl acetate and filtered to remove excess sodium azide. The
filtrate was then concentrated and purified with flash chromatography using hexane/ethyl acetate
1
(9:1) to give 15 (0.85 g, 2.50 mmol) as a white wax like solid. Yield was 91%. H‐NMR (300 MHz,
Chloroform‐d) δ 3.92–3.86 (m, 1H, HO‐CH), 3.48–3.34 (m, 4H), 3.31 (dd, J = 5.5, 2.9 Hz, 2H, ‐CH2N3),
3.17 (s, 1H, OH), 1.55–1.41 (m, 2H, 1.25 (s, 26H, Lipid tail)), 0.85 (t, J = 6.6 Hz, 3H, terminal lipid‐
13
CH3). C‐NMR (75 MHz, CDCl3) δ 71.92, 71.71, 69.59, 53.54, 31.93, 29.71, 29.67, 29.61, 29.52, 29.47,
+
+
29.37, 26.05, 22.67, 14.03.ES‐MS: calcd: C19H39N3O2Na m/z: 364.3, found [M + Na] m/z: 364.5
3‐Azido‐1‐hexadecyloxyl‐2R‐(2’3’4’‐tri‐O‐acetyl‐O‐α‐L‐rhamnopyranosyl)‐sn‐glycerol (16)
The fully protected glycoside donor 10 (0.15 g, 0.41 mmol) and the glycoside acceptor 15 (0.12 g,
0.36 mmol) were dissolved in 15.0 mL of DCM under argon atmosphere, then AgOTf (0.02 g, 0.08
mmol) and N‐iodosuccinimide (0.14 g, 0.60 mmol) were simultaneously added. The reaction was
vigorously stirred for 2 h after which it was stopped with saturated solution of sodium thiosulphate
(5.0 mL) and then washed with 25.0 mL of saturated sodium thiosulphate solution (1 time), saturated
sodium bicarbonate (3 times), water (1 time) and brine (1 time). The organic layer was then dried over
anhydrous sodium sulphate and then concentrated under vac. The residue was purified by flash
chromatography using ethyl acetate/hexane mixture (4:6) to give α‐anomers, 16 (0.12 g, 0.20 mmol)
1
as a white solid. Yield was 55%. NMR data of 16: H‐NMR (300 MHz, chloroform‐d) δ 5.30 (dd, J =
10.0, 3.6 Hz, 1H, H‐3), 5.25 (dd, J = 3.6, 1.7 Hz, 1H, H‐2), 5.06 (dd, J = 9.8, 9.9 Hz, 1H, H‐4), 4.93 (d, J =
1.7 Hz, 1H, H‐1), 4.18–3.99 (m, 1H, H‐5), 3.95–3.83 (m, 1H), 3.58–3.29 (m, 6H), 2.14 (s, 3H), 2.03 (s, 3H),
1.98 (s, 3H), 1.57–1.52 (m, 2H), 1.25 (broad s, 26H, lipid tail), 1.20 (d, J = 6.3 Hz, 3H, H‐6), 0.87 (t, J =
13
6.6 Hz, 3H). C‐NMR (75 MHz, CDCl3) δ 170.01, 169.95, 169.84, 97.22, 76.46, 71.77, 71.09, 70.48, 70.01,
68.92, 66.68, 51.68, 31.91, 29.68, 29.49, 29.34, 26.13, 20.87, 20.75, 20.67, 17.34, 14.09. ES‐MS: calcd:
+
+
C31H55N3O9Na m/z: 636.4, found [M + Na] m/z: 636.5.
3‐Azido‐1‐hexadecyloxyl‐2R‐O‐α‐L‐rhamnopyranosyl‐sn‐glycerol (17)
Compound 16 (0.12 g, 0.20 mmol) was dissolved in 15.0 mL of methanol, then catalytic amount
of sodium methoxide was added and the reaction was vigorously stirred for 3 h. The reaction was
stopped by acidic ion exchange resin. The resin was filtered and the filtrate was concentrated under
vacuum and the residue was purified by flash chromatography using ethyl acetate/hexane mixture
1
(9:1) to give 17 (0.08 g, 0.16 mmol) as a white solid. Yield was 79%. NMR data of 17: H‐NMR (300
MHz, chloroform‐d) δ 4.95 (d, J = 1.1, 1H, H‐1), 4.19–3.95 (m, 1H, H‐5), 4.03–3.85 (m, 2H), 3.77 (d, J =
8.3, 3.5 Hz, 1H, H‐3), 3.62–3.27 (m, 10H), 1.58–1.54 (m, 2H), 1.32 (d, J = 6.4 Hz, 3H, H‐6), 1.27 (broad s,
13
26H), 0.88 (d, J = 7.1 Hz, 3H). C‐NMR (75 MHz, CDCl3) δ 100.04, 76.26, 72.70, 71.83, 71.60, 71.09,
+
70.33, 68.67, 51.71, 31.94, 29.73, 29.52, 29.38, 26.11, 22.70, 17.48, 14.12. ES‐MS: calcd: C25H49N3O6Na
m/z: 500.4, found [M + Na] m/z: 500.4.
+
3‐Amino‐1‐O‐hexadecyloxy‐2R‐(O–α‐L‐rhamnopyranosyl)‐sn‐glycerol (3)
To a solution compound 17 (0.10 g, 0.21 mmol) in THF (7.0 mL) was added 1.5 mL of water and
2.6 mL of 1 M trimethylphosphine in THF. The reaction was vigorously stirred for 2 h at room
temperature after which it was concentrated under vac. The residue was purified by C‐18 column
using gradient elution with water/methanol to give 3 (0.06 g, 0.13 mmol) as a white solid. Yield was
1
61%. NMR data for 3: H‐NMR (300 MHz, methanol‐d4) δ 4.65 (d, J = 1.3 1H, H‐1) 3.65 (dd, J = 1.3, 3.4
Hz, 1H, H‐2), 3.48–3.56 (m, 2H), 3.45 (dd, J = 9.5, 3.4 Hz, 1H, H‐3), 3.37–3.29 (m, 1H, H‐5), 3.29–3.11
(m, 4H), 2.59–2.42 (m, 2H), 1.40–1.34 (m, 2H), 1.08 (broad s, 29H, H‐6, lipid tail), 0.69 (t, J = 6.4 Hz, 3H,

Molecules 2020, 25, 566
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13
lipid terminal‐CH3). C‐NMR (75 MHz, MeOD) δ 101.91, 79.55, 73.98, 72.69, 72.66, 72.45, 72.39, 70.10,
+
43.50, 33.10, 30.81, 30.78, 30.66, 30.50, 27.33, 23.76, 18.08, 14.47. MALDI‐HRMS: calcd: C25H51NO6Na
m/z: 484.3614, found [M + Na] m/z: 484.3611.
+
3‐(‐3‐(p‐Hydroxyphenylpropyl)‐amido‐1‐O‐Hexadecyloxy‐2R‐(O‐α‐L‐rhamnopyranosyl)‐sn‐glycerol
(4)
To a solution of p‐hydroxyphenyl propionic acid 18 (0.02 g, 0.11 mmol), TBTU (0.05 g, 0.14 mmol)
and diisopropyl ethyl amine (0.02 g, 0.14 mmol) in 5.0 mL of DMF which has been stirring for 20 min
was added 3 (0.05 g, 0.11 mmol). The reaction was vigorously stirred for 8 h after which it was diluted
with methanol. The solvents were removed in vacuo and the residue was purified by flash
chromatography using ethylacetate to give 4 (0.07 g, 0.11 mmmol) as an off white solid. Yield was
1
98%. NMR data of 4: H‐NMR (300 MHz, methanol‐d4) δ 6.94 (d, J = 8.3 Hz, 2H, aromatic proton), 6.62
(d, J = 8.3 Hz, 2H, aromatic proton), 4.72 (d, J = 1.9 Hz, 1H, H‐1), 3.80–3.62 (m, 2H), 3.55 (dd, J = 9.8,
5.7 Hz, 1H), 3.41–3.21 (m, 7H), 3.16 (dd, J = 13.8, 5.6 Hz, 1H), 2.73 (t, J = 7.5 Hz, 2H, propionamide
CH2), 2.36 (t, J = 7.7 Hz, 2H, propionamide CH2), 1.49–1.41 (m, 2H), 1.21 (broad s, 26H, lipid tail), 1.16
13
(d, J = 6.2 Hz, 3H, H‐6), 0.82 (t, J = 6.4 Hz, 3H, lipid terminal CH3). C‐NMR (75 MHz, MeOD) δ 175.67,
156.96, 132.79, 130.30, 116.30, 101.26, 76.32, 74.02, 72.67, 72.53, 72.34, 70.00, 49.89, 48.18, 40.92, 39.32,
+
33.10, 32.20, 30.82, 30.70, 30.50, 27.32, 23.76, 18.09, 14.48. MALDI‐HRMS: calcd: C34H59NO7Na m/z:
+
632.4138, found [M + Na] m/z: 632.4590.
α/β‐L‐Glucopyranosyl‐1,2,3,4,5‐pentaacetate (19)
L‐Glucose (0.90 g, 5.00 mmol) was dissolved in 20.0 mL pyridine at room temperature, then acetic
anhydride (5.20 mL, 54.90 mmol) and dimethyl amino pyridine (0.10 g,) were sequentially added and
the reaction was vigorously stirred for 18 h after which it was stopped by addition of methanol (10.0
mL) and then stirred for 15 min. The solvents were removed under high vac. The resulting residue
was then dissolved in ethyl acetate (50.0 mL) and washed with 3% HCl solution (1 time), saturated
sodium bicarbonate (2 times), distilled water (1 time) and brine (1 time). The resulting organic layer
was dried over Na2SO4 and concentrated to dryness and purified by flash chromatography using
ethyl acetate and hexane (1:1) to give 19 (1.70 g, 4.40 mmol) as α, β mixture (3:2). Yield was 88%.
1
Characteristic proton NMR data for 19: H‐NMR (300 MHz, chloroform‐d) δ 6.35 (d, J = 3.7 Hz, 3H, α‐
+
+
H‐1), 5.73 (d, J = 8.2 Hz, 2H, β‐H‐1). ES‐MS: calcd: C16H22O11Na m/z: 413.1, found [M + Na] m/z: 413.4.
Phenyl‐2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐β‐L‐glucopyranoside (20)
The pentaaacetate 19 (1.70 g, 4.40 mmol) was dissolved in in 20.0 mL DCM, then thiophenol (0.
68 g, 6.14 mmol) and BF3∙Et2O (0.87 g, 6.14 mmol) were sequentially added. The reaction was stirred
o
vigorously for 18 h after which it was stopped with 20.0 mL saturated sodium bicarbonate at 0 C. The
organic layer was separated using separatory funnel and subsequently washed with saturated 20.0
mL of sodium bicarbonate (2 times), 20.0 mL of water (1 time) and 20.0 mL of brine (1 time). The
organic layer was dried over anhydrous sodium bicarbonate and then concentrated in vacuo. The
residue was then partially purified by flash chromatography using ethyl acetate and hexane (4:6) to
give mainly β‐anomer of 20 (1.72 g, 4.50 mmol). Yield was 88%. Compound 20 was not characterised.
3‐Azido‐1‐hexadecyloxyl‐2R‐(2’3’4’6’‐tetra‐O‐acetyl‐O‐β‐L‐glucopyranosyl)‐sn‐glycerol (21)
The fully protected glycoside donor 20 (0.18 g, 0.40 mmol) and the glycoside acceptor 15 (0.15 g,
0.44 mmol) were dissolved in 15.0 mL of DCM under argon atmosphere, then AgOTf (0.02 g, 0.08
mmol) and N‐iodosuccinimide (0.18 g, 0.80 mmol) were simultaneously added. The reaction was
vigorously stirred for 2 h after which it was stopped with saturated solution of sodium thiosulphate
(5.0 mL) and then washed with 25.0 mL of saturated sodium thiosulphate solution (1 time), saturated
sodium bicarbonate (3 times), water (1 time) and brine (1 time). The organic layer was then dried over
anhydrous sodium sulphate and then concentrated under vac. The residue was purified by flash
chromatography using ethyl acetate/hexane mixture (4:6) to give 21 (0.13g, 0.20 mmol) as a white

Molecules 2020, 25, 566
14 of 19
1
solid. Yield was 50%. H‐NMR (300 MHz, chloroform‐d) δ 5.22 (dd, J = 9.4, 9.4 Hz, 1H, H‐4), 5.10 (dd,
J = 9.6, 9.6 Hz, 1H, H‐3), 4.97 (dd, J = 9.6, 7.9 Hz 1H, H‐2), 4.75 (d, J = 7.9 Hz, 1H, H‐1), 4.25–4.18 (m,
2H), 4.02–3.90 (m, 1H), 3.78–3.69 (m, 1H), 3.53–3.23 (m, 6H), 2.10 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.02
(s, 3H), 1.59–1.52 (m, 2H), 1.27 (broad singlet, 26H, Lipid tail), 0.89 (h, J = 6.1 Hz, 3H, Lipid terminal‐
13
CH3). C‐NMR (75 MHz, CDCl3) δ 170.65, 170.29, 169.37, 169.17, 100.44, 77.64, 72.83, 71.93, 71.85,
71.47, 70.58, 68.47, 61.90, 52.06, 31.93, 29.70, 29.66, 29.61, 29.49, 29.36, 26.12, 22.70, 20.72, 20.62, 14.12.
+
+
ES‐MS: calcd: C33H57N3O11Na m/z: 694.4, found [M + Na] m/z: 694.8.
3‐Azido‐1‐hexadecyloxyl‐2R‐O‐β‐L‐glucopyranosyl‐sn‐glycerol (22)
Compound 21 (0.13 g, 0.20 mmol) was dissolved in 15.0 mL of methanol, then the catalytic
amount of sodium methoxide was added and the reaction was vigorously stirred for 3 h. The reaction
was stopped by acidic ion exchange resin. The resin was filtered and the filtrate was concentrated
under vacuum and the residue was dissolved in ethyl acetate and filtered through a pad of silica gel
to give 22 (0.09 g, 0.17 mmol) as a white solid. Yield was 84%. Compound 22 was not characterised.
+
+
ES‐MS: calcd: C25H49N3O7Na m/z: 526.4, found [M + Na] m/z: 526.5.
3‐Amino‐1‐hexadecyloxyl‐2R‐O‐β‐L‐glucopyranosyl‐sn‐glycerol (5)
To a solution of compound 22 (0.09 g, 0.17 mmol) in THF (7.0 mL) was added 1.5.0 mL of water
and 2.6 mL of 1 M trimethylphosphine in THF. The reaction was vigorously stirred for 2 h at room
temperature after which it was concentrated under vac. The residue was purified by C‐18 column
using gradient elution with water/methanol to give 5 (0.06 g, 0.09 mmol) as a white solid. Yield was
1
55%. NMR data for 5: H‐NMR (300 MHz, methanol‐d4) δ 4.35 (d, J = 7.7, Hz, 1H, H‐1), 3.81 (dd, J =
13.7, 3.6 Hz, 2H, ‐CH‐CH2O‐), 3.69–3.36 (m, 6H), 3.34–3.07 (m, 4H), 1.59–1.38 (m, 3H), 1.22 (broad s,
13
26H, Lipid tail), 0.85 (t, J = 7.3, 3H, Lipid terminal‐CH3). C‐NMR (75 MHz, MeOD) δ 103.87, 79.65,
78.17, 77.96, 75.13, 72.76, 72.35, 71.66, 62.80, 43.87, 33.09, 30.81, 30.78, 30.49, 27.26, 23.75, 14.46. MALDI‐
+
+
HRMS: calcd: C25H51NO7Na m/z: 500.3563, found [M + Na] m/z: 500.3740.
1,2,3,4,6‐Pentaacetyl α/β‐L‐mannopyranoside (23)
L‐Mannose (2.00 g, 11.10 mmol), was dissolved in pyridine (40.0 mL), then acetic anhydride
(11.00 mL, 111.00 mmol) was added followed by dimethyl amino pyridine (DMAP, 0.27 g 2.20 mmol).
The mixture was stirred vigorously for 18 h at room temperature and it was stopped by addition of
methanol (10.0 mL) and then stirred for 15 min. The solvents were removed under high vac. The
resulting residue was then dissolved in ethyl acetate (50.0 mL) and washed with 3% HCl solution (1
time), saturated sodium bicarbonate (2 times), distilled water (1 time) and brine (1 time). The resulting
organic layer was dried over Na2SO4 and concentrated to dryness and purified by flash
chromatography using ethyl acetate and hexane (1:1) to give 23 (3.70 g, 9.48 mmol) as α, β mixture
1
(4:1). Yield was 85%. NMR data for α‐ anomer of compound 23: H‐NMR (300 MHz, chloroform‐d) δ
5.94 (d, J = 1.9 Hz, 1H, H‐1), 5.24–5.06 (m, 3H, H‐2), 4.14 (dd, J = 12.7, 4.9 Hz, 1H, H‐6a), 4.05–3.85 (m,
13
2H, H‐5, H‐6b), 2.09 (s, 3H), 2.01 (s, 3H), 1.96 (s, 3H), 1.92 (s, 3H), 1.82 (s, 3H). C‐NMR (75 MHz,
CDCl3) δ 170.34, 169.73, 169.50, 169.34, 167.88, 90.44, 70.45, 68.63, 68.20, 65.39, 61.94, 20.62, 20.53, 20.48,
+
+
20.44, 20.41. ES‐MS: calcd: C16H22O11Na m/z: 414.1, found [M + Na] m/z: 414.5
Phenyl‐2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐α‐L‐mannopyranoside (24)
The pentaaacetate 23 (1.50 g, 3.35 mmol) was dissolved in in 30.0 mL DCM, then thiophenol (1.30
g, 11.50 mmol) and BF3∙Et2O (1.60 g, 11.50 mmol) were sequentially added. The reaction was stirred
o
vigorously for 18 h after which it was stopped with 30 mL saturated sodium bicarbonate at 0 C. The
organic layer was separated using separatory funnel and subsequently washed with saturated 35.0
mL of sodium bicarbonate (2 times), 30.0 mL of water (1 time) and 30.0 mL of brine (1 time). The
organic layer was dried over anhydrous sodium bicarbonate and then concentrated in vacuo. The
residue was then purified by flash chromatography using ethyl acetate and hexane (4:6) to give

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mainly α‐anomer of 24 (0.99 g, 2.35 mmol). Yield was 70%. NMR data of compound 24 is similar to
+
+
previously reported data [6]. ES‐MS: calcd: C20H24O9Na m/z: 463.1, found [M + Na] m/z: 462.9
Phenyl‐1‐thio‐α‐L‐mannopyranoside (25)
Compound 24 (1.00 g, 2.35 mmol) was dissolved in 15.0 mL of methanol, then catalytic amount
of sodium methoxide was added and the reaction was vigorously stirred for 3 h. The reaction was
stopped by acidic ion exchange resin. The resin was filtered and the filtrate was concentrated under
vacuum and the residue was dissolved in ethyl acetate and filtered again through a pad of silica gel
to give 25 (0.58 g, 2.14 mmol) as a white solid. Yield was 91%. This compound was used without
further purification and characterization for the next step.
Phenyl‐6‐tosyl‐1‐thio‐α‐L‐mannopyranoside (26)
Compound 25 (1.31 g, 4.80 mmol) was dissolved in dissolved in 20.0 mL pyridine, cooled to 0
C, then toluenesulphonychloride (1.06 g, 5.54 mmol) and DMAP (0.05 g, 0.41 mmol) were added.
The temperature was allowed to increase to room temperature and the mixture was stirred for 18 h.
At the end of reaction, the mixture was diluted with methanol after which the solvents were removed
in vacuo. The residue was diluted with 40.0 mL ethyl acetate and the washed sodium bicarbonate
solution (3 times), brine (3 times). The organic layer was then dried over sodium sulphate and then
concentrated under vacuum and the residue was partially purified by flash chromatography using
ethyl acetate (100%) to give 26 (1.81 g, 4.24 mmol) as a white solid. Yield was 88%. 26 was not
+
+
characterized using NMR. ES‐MS: calcd: C19H22O7S2Na m/z: 449.1, found [M + Na] m/z: 449.5.
Phenyl‐6‐azido‐1‐thio‐α‐L‐mannopyranoside (27)
Compound 26 (1.81 g, 4.24 mmol) and sodium azide (1.80 g, 27.60 mmol) were suspended in
anhydrous DMF and the mixture was stirred at 90 C for 18 h. at the end of the reaction the mixture
was concentrated then diluted with ethyl acetate and filtered to remove excess sodium azide. The
filtrate was then concentrated and partially purified with flash chromatography using hexane/ethyl
acetate (1:9) to give 27 (1.20 g, 4.04 mmol) as a white solid. Yield was 95%. ES‐MS: calcd:
+
+
C12H15N3O4SNa m/z: 320.1, found [M + Na] m/z: 320.3
Phenyl‐2,3,4‐tri‐O‐acetyl‐6‐azido‐1‐thio‐α‐L‐mannopyranoside (28)
Compound 27 (1.20 g, 4.04 mmol) was dissolved in pyridine (50.0 mL), then acetic anhydride
(2.00 mL, 20.00 mmol) was added followed by dimethyl amino pyridine (DMAP, 0.05 g, 0.41 mmol).
The mixture was stirred vigorously for 18 h at room temperature and it was stopped by addition of
methanol (10.0 mL) and then stirred for 15 min. The solvents were removed under high vac. The
resulting residue was then dissolved in ethyl acetate (50.0 mL) and washed with 3% HCl solution (1
time), saturated sodium bicarbonate (2 times), distilled water (1 time) and brine (1 time). The resulting
organic layer was dried over Na2SO4 and concentrated to dryness and purified by flash
chromatography using ethyl acetate and hexane (1:1) to give 28 (1.31 g, 3.10 mmol) white solid. Yield
1
was 76%. NMR data of compound 28: H‐NMR (300 MHz, chloroform‐d) δ 7.54–7.10 (m, H, aromatic
protons), 5.54–5.37 (m, 2H, H‐1, H‐3), 5.32–5.19 (m, 2H, H‐2, H‐4), 4.43–4.38 (m, 1H, H‐5), 3.41–3.17
13
(m, 2H, H‐6), 2.07 (s, 3H), 2.03 (s, 3H), 1.90 (s, 3H). C‐NMR (75 MHz, CDCl3) δ 169.72, 169.64, 132.49,
132.01, 129.27, 128.12, 85.64, 71.01, 70.81, 69.18, 67.14, 50.98, 20.69, 20.58, 20.50. ES‐MS: calcd:
+
+
C18H21N3O7SNa m/z: 446.1, found [M + Na] m/z: 446.4

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3‐Azido‐1‐hexadecyloxyl‐2R‐(6′‐azido‐2′3′4′‐tri‐O‐acetyl‐O‐α‐L‐mannopyranosyl)‐sn‐glycerol (29)
The fully protected glycoside donor 28 (0.20 g, 0.47 mmol) and the glycoside acceptor 15 (0.18 g,
0.52 mmol) were dissolved in 15.0 mL of DCM under argon atmosphere, then AgOTf (0.02 g, 0.09
mmol) and N‐iodosuccinimide (0.16g, 0.71 mmol) were simultaneously added. The reaction was
vigorously stirred for 2 h after which it was stopped with saturated solution of sodium thiosulphate
(5.0 mL) and then washed with 25.0 mL of saturated sodium thiosulphate solution (1 time), saturated
sodium bicarbonate (3 times), water (1 time) and brine (1 time). The organic layer was then dried over
anhydrous sodium sulphate and then concentrated under vac. The residue was purified by flash
chromatography using ethyl acetate/hexane mixture (4:6) to give 29 (0.220 g, 0.34 mmol) as a white
1
solid. Yield was 71%. NMR data of 29: H‐NMR (300 MHz, chloroform‐d) δ 5.38 (dd, J = 9.9, 3.3 Hz,
1H, H‐3), 5.32–5.20 (m, 2H, H‐2, H‐4), 5.06 (d, J = 1.8 Hz, 1H, H‐1), 4.20 (ddd, J = 9.5, 5.6, 3.4 Hz, 1H,
H‐5), 4.07–3.89 (m, 1H), 3.64–3.23 (m, 8H, H‐6), 2.18 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H), 1.61–1.54 (m,
13
2H), 1.27 (broad s, 26H, lipid tail), 0.89 (t, J = 6.5 Hz, 3H, lipid terminal‐CH3). C‐NMR (75 MHz,
CDCl3) δ 170.01, 169.81, 97.13, 76.84, 71.80, 70.35, 70.23, 69.69, 68.66, 67.20, 51.76, 51.18, 31.93, 29.70,
+
29.64, 29.52, 29.37, 26.15, 22.70, 20.88, 20.72, 14.12. ES‐MS: calcd: C31H54N6O9Na m/z: 677.4, found [M
+
+ Na] m/z: 677.8
3‐Azido‐1‐hexadecyloxyl‐2R‐(6′‐azido‐O‐α‐L‐mannopyranosyl)‐sn‐glycerol (30)
Compound 29 (0.22 g, 0.34 mmol) was dissolved in 15.0 mL of methanol, then catalytic amount
of sodium methoxide (0.05 g) was added and the reaction was vigorously stirred for 3 h. The reaction
was stopped by acidic ion exchange resin. The resin was filtered and the filtrate was concentrated
under vacuum and the residue was purified by flash chromatography using ethyl acetate/hexane
1
mixture (9:1) to give 30 (0.120 g, 0.23 mmol) as a white solid. Yield was 68%. NMR data of 30: H‐
NMR (300 MHz, methanol‐d4) δ 5.02 (d, J = 1.9 Hz, 1H, H‐1), 4.10–3.89 (m, 2H, H‐3), 3.96–3.81 (m, 2H,
H‐2), 3.78–3.57 (m, 3H), 3.56–3.28 (m, 6H), 1.62–1.58 (m, 2H), 1.33 (broad s, 26H, lipid tail), 0.91 (t, J =
13
6.6 Hz, 3H, lipid terminal‐CH3). C‐NMR (75 MHz, MeOD) δ 101.93, 77.92, 74.29, 72.72, 72.18, 72.15,
+
71.65, 69.45, 53.23, 52.92, 33.16, 30.89, 30.79, 30.57, 27.33, 23.82, 14.59. ES‐MS: calcd: C25H48N6O6Na
m/z: 528.4, found [M + Na] m/z: 528.7
+
3‐Amino‐1‐hexadecyloxyl‐2R‐(6′‐amino‐6′deoxy‐O‐α‐L‐mannopyranosyl)‐sn‐glycerol (6)
To a solution of compound 30 (0.12 g, 0.23 mmol) in THF (7.0 mL) was added 1.5 mL of water
and 2.6 mL of 1 M trimethylphosphine in THF. The reaction was vigorously stirred for 2 h at room
temperature after which it was concentrated under vacuum. The residue was purified by C‐18
column using gradient elution with water/methanol to give 6 (0.07 g, 0.14 mmol) as a white solid.
1
Yield was 62%. NMR data for 6: H‐NMR (300 MHz, methanol‐d4) δ 4.83 (d, J = 2.0 Hz, 1H, H‐1), 3.79
(dd, J = 5.0, 2.0 Hz, 1H, H‐2), 3.71–3.62 (m, 2H), 3.58–3.43 (m, 3H, H‐5), 3.38–3.17 (m, 4H), 2.94–2.82
(m, 1H), 2.83–2.60 (m, 2H, H‐6), 1.59–1.38 (m, 2H), 1.21 (broad s, 26H, lipid tail), 0.83 (t, J = 6.6 Hz, 3H,
13
lipid terminal‐CH3). C‐NMR (75 MHz, MeOD) δ 101.52, 79.00, 74.36, 72.75, 72.70, 72.44, 72.34, 69.51,
+
43.45, 43.26, 33.12, 30.84, 30.70, 30.52, 27.37, 23.78, 14.51. MALDI‐HRMS: calcd: C25H52N2O6Na m/z:
+
499.3723, found [M+Na] m/z: 499.3409.
4.2. Biological Methods
4.2.1. Determination of Cytotoxicity of GAELs on Cancer Cell Lines
The cell lines were cultured from frozen stocks originally obtained from ATCC (Manassas, VA,
USA). MDA‐MB‐231, JIMT‐1, DU145, MDA‐MB‐468, Hs578t and MDA‐MB‐453 were grown in
DMEM medium supplemented with 10% FBS. BT‐474 cells were grown in DMEM/F12 medium
supplemented with 10% FBS. MiaPaCa2 was cultured in DMEM supplemented with 10% FBS and
2.5% horse serum. All the media contained penicillin/streptomycin. U‐251 and U‐87 in Eagle MEM
supplemented by 10% FBS, 1% Non‐essential amino acids, 1 mM sodium pyruvate and 2 mM
glutamine. BT‐549 was cultured in RPMI 1640 medium supplemented with 10% FBS.

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The effects of the GAELs on the viability of the epithelial cancer cell lines were determined as
previously described [4,6,7]. Briefly, equal numbers of the cells were dispersed into 96‐well plates.
After 24 h, the cells were incubated with the compounds (0–30 μM) for 48 h. MTS reagent [3‐(4,5‐
dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt;
Promega, Madison, WI, USA] (20% v/v) was subsequently added and the plates were incubated for
1–4 h in a CO2 incubator followed by measurement of the medium at OD490 with a plate reader
(Molecular Devices). Wells with media but no cells were treated in similar fashion and the values
utilized as blank. The results represent the mean ± standard deviation of 6 independent
determinations.
4.2.2. Determination of Membranolytic Effects of GAELs
Equal numbers of JIMT‐1 and DU145 cells were dispersed into 96‐well plates. After 24 h the cells
were incubated with varying concentration of compounds 3 or 4 (4–6 μM) for 5–6 h. Subsequently, 2
μM of the cell membrane impermeant dye, ethidium homodimer‐1 (EthD‐1 (Molecular Probes,
Eugene, OR, USA) that emits red fluorescence upon binding to DNA was added and the cells
analysed by fluorescence microscopy. EthD‐1 staining was compared to negative controls with no
treatment and positive control treated with 0.01% Triton X‐100 for 10 min.
4.2.3. Demonstration of Caspase‐mediated‐apoptosis Independent Mode of Cell Death
Equal numbers of JIMT‐1, or DU145 cells were dispersed into 96‐well plates, and after 4 h the
cells were treated with pan‐caspase inhibitor QVD‐OPh (40 μM). After 20 h, the cells were incubated
with the varying concentration of the compounds (0–9 μM) for 48 h. At the end of the incubation, the
MTS assay was used to assess cell viability as described above. The results represent the mean ±
standard deviation of 6 independent determinations.
4.2.4. Hemolytic Assay
The hemolytic activity of the GAEL analogs was evaluated using ovine erythrocyte. Sheep whole
blood was collected from a slaughter house into a vessel containing disodium EDTA (1.2 g/mL) in a
buffered saline (10 mM Tris, 150 mM NaCl, pH 7.4). The erythrocytes were prepared and washed
with buffered saline as previously reported [4]. For the assay, the erythrocyte suspension, varying
amounts of the GAEL drugs or vehicle were pipetted into 1.5 mL microcentrifuge tubes to give a final
8
volume of 1500 μL and cell density of 2.5 × 10 cells/mL. The suspensions were incubated with gentle
shaking in Eppendorf thermomixer for 30 min. The 1.5 mL microcentrifuge tubes were cooled in ice
water and centrifuged at 2000× g and 4 °C for 5 min to pellet cell debris. 200 μL of the supernatant
was dissolved in 1800 μL of 0.5% NH4OH and the optical density (OD) was recorded using 1 mL
cuvette at 540 nm in a spectrophotometer. For 0% hemolysis, buffer and vehicle were added instead
of the drug, and 1% NH4OH was used to generate 100% hemolysis. The % hemolysis was calculated
using the optical density (OD) values as shown below in Equation (1) [4]:
% hemolysis = (X − 0%)/(100% − 0%)
(1)
where X is the OD values of the drug(s) at varying concentration.
4.2.5. Statistical Analysis
The results represent the mean ± standard deviation of 6 independent determinations. Statistical
significant difference test was carried using GraphPadInstat software (GraphPad Software, San
Diego, CA, USA). The mean values were subjected to one‐way analysis of variance (ANOVA)
followed by Tukey‐Kramer multiple comparison post hoc test. Comparisons were carried out
between the viability of controls and drug treated cells to determine if statistically significant
differences existed between the two groups. The results of the effects of different concentrations of
the compounds were also compared for statistically significant differences to determine if the
cytotoxic activities of the drugs are dose dependent. The anticancer activities of all the compounds

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2–6 tested were compared using ANOVA followed by Tukey‐Kramer multiple comparison tests at
the following concentrations: 5, 7.5 and 10 μM to determine if the difference in the potency of the
drugs are statistically significant or not. A p value > 0.05 indicates no statistical differences while a p
value <0.001 indicated statistical significant differences. The statistical analysis data are not included
in this report.
Supplementary Materials: The NMR spectra (1H‐NMR, 13C‐NMR) of compounds 2–6 are available online at
www.mdpi.com/xxx/s1.
Author Contributions: Conceptualization M.O., F.S., G.A. Methodology M.O., P.S. F.S., G.A.; Formal analysis
M.O., P.S., T.I., F.S., M.N., G.A.; Investigation, M.O., P.S., G.A.; Writing—original draft preparation, M.O., M.N.,
G.A.,T.I; Writing—review and editing, M.O., P.S., T.I., F.S., M.N., G.A.; Funding acquisition, F.S., M.N., G.A.
Authorship must be limited to those who have contributed substantially to the work reported. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was supported by research grants from Cancer Care Manitoba to M.N./G.A. and Natural
Sciences and Engineering Council of Canada (NSERC‐DG‐06047) grant to F.S. M.O. was the recipient of a
University of Manitoba and Manitoba provincial government graduate fellowships (UMGF and MGS).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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Sample Availability: Samples of the compound 3 are available from the authors.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).