Design, synthesis and evaluation of cytotoxic properties of bisamino glucosylated antitumor ether lipids against cancer cells and cancer stem cells

Article abstract of DOI:10.1039/c6md00328a

Glycosylated antitumor ether lipids (GAELs) are a class of amphiphilic antitumor agents that kill cancer cells by a non-apoptotic pathway. Previous studies have shown that 2-amino-2-deoxy-d-gluco-based GAELs such as α-GLN and β-GLN show greatly improved antitumor activity against epithelial cancer cells and stem cells. To further optimize the bioactivity, we prepared a series of diamino-d-gluco-based GAELs and their analogs, and screened them against a panel of human epithelial cancer cell lines and cancer stem cells. Most of the new GAEL analogs are more potent than chlorambucil, cisplatin and salinomycin. The most potent bisamine-based GAEL analogs 1, 2, 4 and 8 showed 2- to 3-fold enhanced cytotoxicity against various cancer cell lines when compared to β-GLN, indicating that the addition of a second amino group enhances the cytotoxic effect. The effect of the most active dicationic GAELs 1 and 4 on cancer stem cells isolated from breast (BT-474) and prostate (DU-145) cell lines revealed that the two GAELs inhibited the formation of tumor spheres and resulted in >95% loss of viability of the cancer stem cells at 5 μM. Activity of GAEL 1 against BT-474 cancer stem cells is superior to that of salinomycin.

Full text of DOI:10.1039/c6md00328a

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Ogunsina, P.
Samadder, T. Idowu, G. Arthur and F. Schweizer, Med. Chem. Commun., 2016, DOI:
10.1039/C6MD00328A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Please do not adjust margins
MedChemComm
Page 1 of 12
View Article Online
DOI: 10.1039/C6MD00328A
MedChemComm
ARTICLE
Design, Synthesis and Evaluation of Cytotoxic Properties of
Bisamino Glucosylated Antitumor Ether Lipids against Cancer Cells
and Cancer Stem Cells†
Received 00th January 20xx,
Accepted 00th January 20xx
Makanjuola Ogunsinaa, Pranati Samadderb, Temilolu Idowua, Gilbert Arthurb* and Frank
Schweizera**
DOI: 10.1039/x0xx00000x
www.rsc.org/
Glycosylated Antitumor Ether Lipids (GAELs) are a class of amphiphilic antitumor agents that kill cancer cells by a non-
apoptotic pathway. Previous studies have shown that 2-amino-2-deoxy-D-gluco-based GAELs such as α-GLN and β-GLN
show greatly improved antitumor activity against epithelial cancer cells and stem cells. To further optimize the bioactivity,
we prepared a series of diamino-D-gluco-based GAELs and their analogs, and screened them against a panel of human
epithelial cancer cell lines and cancer stem cells. Most of the new GAEL analogs are more potent than chlorambucil,
cisplatin and salinomycin. The most potent bisamine-based GAEL analogs 1, 2, 4 and 8 analogs showed 2- to 3-fold
enhanced cytotoxicity against various cancer cell lines when compared to β-GLN, indicating that the addition of a second
amino group enhances the cytotoxic effect. The effect of the most active dicationic GAELs 1 and 4 on cancer stem cell
isolated from breast (BT-474) and prostate (DU-145) revealed that the two GAELs inhibited the formation of tumor
spheres and resulted in > 95 % loss of viability of the cancer stem cells at 5 µM. Activity of GAEL 1 against BT-474 cancer
stem cells is superior to that of salinomycin.
There is increasing evidence that the relapse of tumors and
development of drug and radiation resistant tumors as well as
progression to metastases may be due to the presence of a
Introduction
Cancer remains a major health problem worldwide despite the
huge investment to find effective treatments for the disease. A
recent UN report projects that global cancer cases will rise
from 14 million to 22 million per year within the next two
decades, with annual cancer deaths rising from 8.2 million to
13 million.1 The major problems impeding the development of
cures for cancer are resistance to drugs, resistance to
radiotherapy, and metastasis. Many of the existing anticancer
drugs act by disrupting cell DNA, preventing DNA synthesis,
and/or targeting microtubules, while radiotherapy kills cells by
damaging DNA. The cellular perturbation caused by these
treatments induces apoptosis which kills the cancer cells.
While many drugs are initially successful in killing cancer cells
resulting in tumor shrinkage, there is invariably a relapse and
the tumor reappears with cells that resist existing
chemotherapeutic agents2,3,4,5 so the tumors are refractory to
treatment leading to metastases and death. There are also
very few drugs, if any, for treatment of metastasized cancer.
small population of cells in the tumor called cancer stem cells
(CSCs) or tumor initiating cells. CSCs are distinct from the cells
of the bulk tumor in having the capacity for self-renewal,
asymmetric division, and differentiation. CSCs have been
identified and isolated from virtually all solid and
hematological tumors.6,7 These cells resist apoptotic cell death
induced by chemotherapy and radiotherapy, and may
ultimately generate drug-resistant differentiated cells of the
recurrent bulk tumor.6,7 In light of the potential role these cells
play in tumor relapse, drug resistance, and metastases,
effective cancer treatment will require targeting the CSCs
along with the bulk differentiated cells of the tumor.
Only few compounds that kill CSCs have been identified.
They include parthenolide, salinomycin, metformin, lapatinib,
and mitoVES.7 The mechanisms via which they achieve this are
not known but salinomycin, parthenolide and the biguanide,
metformin, induce apoptosis in various human cancer cells.8-12
The potential of the ionophore antibiotic salinomycin is
reinforced by its ability to kill highly multidrug- and apoptosis-
resistant cancer cells11-13 and CSCs.11,15-17 Antitumor ether lipids
(AELs) are synthetic lipids that possess anticancer activity and
comprise three subclasses; the alkyllysophospholipid (ALP),
alkylphosphocholine (APC) and the non-phosphorylated
glycosylated antitumor ether lipids (GAELs). The cytotoxic
properties of GAELs, prototypified by α- GLN and β-GLN, have
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 2 of 12
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
Journal Name
been established to be superior to the most studied AEL,
edelfosine (Figure 1).
Figure 2. Structures of diamine-based and N-substituted
Figure 1 Structures of various glycosylated antitumor ether
lipids (GAELs) and edelfosine, an antitumor ether lipid (AEL) of
the ALP subclass
glucosylated antitumor ether lipids (GAELs) used in this study.
that the anomeric configuration can influence the antitumor
Unlike ALPs and APCs which kill cells by apoptosis, GAELs kill
cells by an apoptotic-independent mechanism.18-21 Though the
mechanism of action of GAELs is yet to be fully understood, it
appears to involve the perturbation of endocytosis pathway to
generate large acidic vacuoles that ultimately leads to the
release of acid hydrolases to induce cell death.19-21 This ability
to kill cells by an apoptosis-independent pathway led to the
postulation that GAELs could kill CSCs because their mode of
action will not be impacted by the strategies cells use to
prevent death by apoptosis. Recently this hypothesis was
validated by the demonstration that a number of GAELs were
cytotoxic against CSCs isolated from BT-474 cell lines.22
potency in monocationic GAELs.18,21,23 Compound
3 with a
phthalimido group at the C2-position of glucose was
synthesized to study how the absence of a cationic charge at
that position affects cytotoxicity. In addition, the phthalimido
group was selected as this group exhibits significant
cytotoxicity against both human and murine cancer cell
lines.25,26 Compounds
4 and 5 were synthesized to explore the
effect of the methoxy substituent at the sn-2 position of the
glycero moiety. Previous studies on gluco-based GAELs had
shown that the nature of the methoxy substituent is not
critical for antitumor activity and can be replaced by
hydrogen.23 Another reason for synthesizing tetraamine-
Our ongoing structure activity studies on GAELs have led to the
observation that the anticancer potency of GAELs is intimately
linked to their cationic nature. Our previously identified leads,
containing bisglycolipid
replacement of the methoxy substituent by an additional 2,6-
diamino- -D-glucose moiety may lead to enhanced antitumor
activity as seen for other polycationic structures.27 Dicationic
glycolipids and were synthesized to explore how the
positioning of a second amino substituent on the glycerol
moiety affects the antitumor properties. Azido compound
was also prepared to study how the absence of a cationic
charge in affects cytotoxicity. Compound was synthesized
5 was based on the hypothesis that
α
-GLN and β-GLN, contain a positively charged 2-amino group
β
in the gluco-portion of the glycolipid (Figure 1). Replacement
of the 2-amino substituent by a neutral hydroxyl or azido
group, as well as replacement by a cationic guanidino and
secondary amine-based substituents including benzyl amine-
substituted groups, resulted in more than 3-fold reduced
activity against various cancer cell lines.23,24 The observation
that primary amine-based GAELs are significantly more potent
than their non-amine-based counterparts raised the question
whether introduction of a second primary amino group in
GAELs could further enhance the antitumor properties. We
disclose here our structure activity study on diamine-based as
7
8
6
7
9
to explore how the presence of two lipid tails and modification
of the free amino substituent at the sn-2-position affect the
biological properties of compound
8. We also synthesized
carbamate 10 to explore whether the presence of a methoxy
carbamate substituent at the sn-2-position of the glycerolipid
will induce prostate cancer selectivity based on previous
findings.28
well as N-substituted gluco-based GAEL analogs 1-10 (Figure
2).
Compounds 5, 8, 9, and 10 were studied as diastereomeric
1:1 mixture based on the stereochemistry at sn-2 position of
the glycerolipid. We have previously reported that the
stereochemistry at this position has little or no effect on
anticancer activity.24 Moreover, edelfosine (Figure 1) the most
studied AEL is used as a racemic mixture.29
Results
Chemistry
To study the effects of diamino and N-substituted GAELs, a
second amino substituent was introduced at the C6-position of
Compound
1 was synthesized by coupling of glycoside
glucose as shown in compounds
the presence of a second amino substituent, glycolipids
were also selected to study the nature of the glycosidic
1
and
2
(Figure. 2). Besides
donor 17 to the commercially available lipid alcohol 18 to
afford a mixture of α- and β-glycolipid 19 and 20 in a 4:1 ratio,
respectively (Scheme 1). The glycoside donor 17 was
synthesized from glucosamine hydrochloride 11 in seven steps.
1
and
2
linkage on antitumor activity as previous reports had indicated
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 3 of 12
View Article Online
DOI: 10.1039/C6MD00328A
Journal Name
ARTICLE
OH
OAc
OAc
O
O
11
O
AcO
a
AcO
b
c
HO
HO
AcO
OAc
NPhth
SPh
AcO
SPh
NPhth
NPhth
22 (49%)
23 (77%)
24 (80%)
d
OTs
N3
N3
O
O
HO
AcO
O
f
HO
SPh
SPh
NPhth
HO
HO
AcO
SPh
NPhth
NPhth
e
26 (90%)
27(87%)
25 (74%)
g
18
N3
OC16H33
N3
OC16H33
H
O
O
O
H
O
O
O
AcO
AcO
28 (47%)
c
HO
HO
NPhth
NPhth
30 (62%)
h
i
N3
OC16H33
H
NH2
OC16H33
H
O
O
O
O
O
O
HO
HO
HO
HO
NH2
29 (55%)
NPhth
3 (67%)
i
NH2
OC16H33
H
O
O
HO
HO
O
NH2
2 (78%)
Scheme 2. Synthesis of compounds
2 and 3. Reagents and
Scheme 1. Synthesis of compound 1. Reagents and conditions:
(a) 1. TfN3, CuSO4, Et3N, H2O, rt; 2. Ac2O, DMAP, pyridine, 18 h,
rt (b) PhSH, BF3.Et2O, DMAP, DCM, 18 h, rt (c) MeONa, MeOH,
conditions: (a) 1. Phthalic anhydride, NaOH, H2O, 18 h, rt; 2.
Ac2O, DMAP, pyridine, 18 h, rt (b) PhSH, BF3.Et2O, DMAP, DCM,
18 h, rt (c) MeONa, MeOH, 20 mins (d) TsCl, pyridine, DMAP, 0
1 hr (d) TsCl, pyridine, DMAP, 0
C, 24 h (f) Ac2O, DMAP, pyridine, 18 h, rt (g) AgOTf, NIS, DCM,
3 h, rt (h) MeONa, MeOH, 30 mins (i) P(CH3)3, THF, H2O, 2 h, rt.
Abbreviations: 4-dimethylaminopyridine (DMAP),
dichloromethane (DCM), 2,2,N,N-dimethylformamide (DMF),
room temperature (rt) 23 C, N-iodosuccinimide (NIS),
°C to rt, 18 h (e) DMF, NaN3, 70
°
C to rt , 18 h (e) DMF, NaN3, 70
pyridine, 18 h, rt (g) AgOTf, NIS, DCM,
ethylenediamine/butanol (1:1), 90 C, 2 h, rt (i) P(CH3)3, THF,
°
C, 24 h (f) Ac2O, DMAP,
°
3
h, rt (h)
°
H2O, 2 h, rt. Abbreviations: 4-dimethylaminopyridine (DMAP),
dichloromethane (DCM), 2,2,N,N-dimethylformamide (DMF),
°
room temperature (rt) 23 °C, N-iodosuccinimide (NIS).
triisopropyl benzyl sulphonate ester (OTIBs).
achieved by exposure to ethylenediamine in butanol (1:1 v/v)
At first, glucosamine 11 was converted to azide 14 as
previously reported.23 The azido function at the C-6-position
was installed by selective activation of the C-6 hydroxyl group
in 14 as sulphonate ester 15, followed by nucleophilic
displacement of the sulphonate group by sodium azide in DMF
at high temperature to yield azide 29 which was subsequently
reduced to the desired dicationic glycolipid
2. To synthesize
the target compound 3, the acetate protecting groups of
compound 28 were selectively removed using catalytic amount
of sodium methoxide in methanol to give 6-azido-2-
phthalimido analog 30. Reduction of the azido group via
to afford the 2,6-diazido analog 16. Protection of the
remaining hydroxyl groups using acetic anhydride in pyridine
produced the glycoside donor 17. Donor 17 was used in
glycosylation reaction with commercially available lipid alcohol
18 to produce a mixture of the glycolipids 19 and 20 in ratio
7:3. The α-glycolipid 19 was isolated in pure form and the ester
groups were deprotected to afford the 2,6-diazido compound
21 which was subsequently subjected to azide reduction using
trimethylphosphine in THF to produce the desired α-anomeric
Staudinger reaction produced glycolipid
3 (Scheme 2).
To synthesize compound , the glycoside donor 27 was
4
glycosylated to lipid alcohol 31,23 to produce glycolipid 32
(Scheme 3). Deblocking of the acetate and phthalimido
protecting groups produced azide 33 which was reduced to the
desired diamine-based glycolipid
4. Target molecule 5 was
synthesized by glycosylating commercially available lipophilic
diol 34 to thioglycoside donor 27 to afford protected
diglycosylated lipid 35. Deblocking of diglycosylated lipid
produced bisazido compound 36 which was subsequently
glycolipid
1
(Scheme 1).
and 3 were prepared by coupling of lipid
Glycolipids
2
alcohol 18 to thioglycoside donor 27 (Scheme 2). Donor 27 was
prepared from glucosamine hydrochloride 11 in seven steps.
11 was converted into the unprotected thioglycoside 24 using
established procedures.23 The azido group at C-6 was installed
by selective activation of the 6-hydroxy group as sulphonate
ester 25 followed by nucleophilic displacement with sodium
azide in DMF to produce azido analog 26 which was acylated
to afford the desired thioglycoside donor 27. Donor 27 was
coupled to lipid alcohol 18 to produce glycolipid 28. Removal
of the acetate- and phthalimido-based protecting groups was
reduced to produce tetraamine-based glycolipid
5 (Scheme 3).
Compounds 6 and 7 were synthesized as outlined in Scheme 4.
Azido lipid alcohol 39 was synthesized from commercially
available lipophilic diol 37 in two steps. First, the primary
hydroxyl group in diol 37 was selectively activated as
sulphonate ester 38 followed by a SN2-type displacement with
sodium azide to produce azido acceptor 39. The glycoside
donor 23 was glycosylated with acceptor 39 to afford the
protected glycolipid 40
.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 4 of 12
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
Journal Name
Scheme 5. Synthesis of compounds
conditions: (a) DIAD, Ph3P, Me3SiN3, DCM, 8 h (b) AgOTf, NIS,
DCM, h, rt (c) P(CH3)3, THF, H2O, h, rt (d)
ethylenediamine/butanol (1:1), 90 C, 2 h, rt. (e) Et3N,
ClCO2Me, DCM, 15 (f) TBTU, DIPEA, DMF, h, rt.
Abbreviations: dichloromethane (DCM), 2,2,N,N-
dimethylformamide (DMF), room temperature (rt) 23 C, N-
iodosuccinimide (NIS), diisopropylethylamine (DIPEA),
diisopropyl azodicarboxylate (DIAD).
8-10. Reagents and
Scheme 3. Synthesis of compounds
4
and
5
. Reagents and
conditions: (a) AgOTf, NIS, DCM,
ethylenediamine/butanol (1:1), 90
H2O, 2 h, rt. Abbreviations: dichloromethane (DCM), 2,2,N,N-
3
h, rt (b)
3
2
°C, 2 h, rt (c) P(CH3)3, THF,
°
h
3
dimethylformamide (DMF), room temperature (rt) 23
iodosuccinimide (NIS).
°C, N-
°
The acetate and phthalimido protective groups of 40 were
removed with ethylenediamine in butanol (1:1) at 90 C for 2 h
to give the desired compound . The azido substituent of
was reduced as described above to afford the diamino analog
(Scheme 4).
°
6
6
Bisamino glycolipid 8, was synthesized according to Scheme 5.
The lipid alcohol 41, was synthesized from commercially
available lipid alcohol as previously reported,16 and coupled to
the glycoside donor 23 to afford protected glycolipid 42
(Scheme 5). Reduction of the azido substituent in 42 gave
compound 43 which was subsequently deprotected using
ethylenediamine in butanol (1:1) at elevated temperature to
7
yield dicationic glycolipid
8. To synthesize glycolipid 9, the
amine 43 was coupled to palmitic acid 44 by condensation
with TBTU,30 to give compound 45 which was deprotected to
produce target glycolipid
9 (Scheme 5). Finally, compound 10
was synthesized by reaction of amine 43 with methyl
chloroformate 46 to produce carbamate 47 which was
subsequently deblocked to afford the desired carbamate-
based glycolipid 10 (Scheme 5).18
Biological Studies
Scheme 4 Synthesis of compounds
conditions: (a) TsCl, Pyridine, DMAP, 0
NaN3, 70 24 (c) AgOTf, NIS, DCM,
ethylenediamine/butanol (1:1), 90 C, 2 h, rt (e) P(CH3)3, THF,
6
and
C to rt, 18 h (b) DMF,
h, rt (d)
7. Reagents and
°
In vitro screening of GAELs’ activity against epithelial cancer
cell lines
°C
h
3
°
The cytotoxicity of compounds
1 – 10 was evaluated
H2O, 2 h, rt. Abbreviations: 4-dimethylaminopyridine (DMAP),
against exponentially growing epithelial cancer cell lines
including BT-474, JIMT-1 and MDA-MB-231 (breast), DU-145
and PC3 (prostate), and MiaPaCa2 (pancreas). Compounds
dichloromethane (DCM), 2,2,N,N-dimethylformamide (DMF),
room temperature (rt) 23 °C, N-iodosuccinimide (NIS).
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 5 of 12
Journal Name
were incubated with varying concentrations of 1 – 10 (0 – 30 position of glycerol. GAEL
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
8
was also more active than β-GLN
µM) for 48 h, followed by determination of cell viability using against all the cell lines with the exception of the BT-474 cell
MTS assay.22 The results of the viability studies are shown in line. This result indicates that activity of this class of
Figure 2. Lead compound β-GLN, the most studied GAEL to compounds is better when the sugar moiety is on sn-3 position
date, was selected as the reference compound for comparison. of the glycerolipid.
The CC50 values for all compounds and CC90 values for the most The increased potency of compound
8 compared to β-GLN
active analogs are summarized in Tables 1 and 2. The most against most of the cell lines also shows that replacement of
potent GAEL amongst the compounds tested against six cell the methoxy group at sn-2 position of the glycerolipid with
lines is dicationic glycolipid 1, with CC50 values of 3.0 to 7.5 μM, amino substituent enhanced cytotoxicity.
while 90 % loss of cell viability was observed at a concentration
range of 4.5 to 9.5 µM, depending on cell lines. This compound of
Comparison of the activity of compounds
7 and 8 with that
1
,
2, and 4 demonstrates that better activities can be
bears two primary amino groups at positions 2 and 6 of the achieved when both amino groups are placed on the sugar as
glucose moiety and has α-glucosidic linkage to the compared to a combination where one amino group is present
glycerolipid. The next most potent analog was compound at the sugar and the other one is attached to the glycerol
which differs from merely in its β-glucosidic linkage, with a portion of the lipid.
typical 1.5- to 3-fold less activity relative to (Table 1). This is Compound with a second hydrophobic C-16 moiety
a
2
1
1
9
consistent with our previous results in the monocationic GAEL attached to the sn-2 position of the glycerolipid via an amide
series, which showed that α-GLN was more active than its β- linkage was not active at the highest concentration tested, 30
anomer.22,23 Comparison of the β-dicationic analog
2
with β- µM. At this concentration, there was no statistically significant
monocationic analog β-GLN indicates that the dicationic GAEL difference in viability of cells incubated with compared to
showed better cytotoxicity than its monocationic analog vehicle-treated cells (controls). The lack of activity might be
except against BT-474 cell lines. Compound containing an caused by increased lipophilicity which exceeds the
9
3
uncharged phthalimido group at the C-2 position showed weak lipophilicity-hydrophilicity balance required for optimal cellular
antitumor activity against all six cell lines (CC50 > 15 µM), absorption of this compound.
indicating that the primary amino group at the C-2 position is
crucial for antitumor activity. GAEL , an analog of without a sn-2 position of the glycerolipid, was synthesized to potentially
methoxy substituent at the glycero moiety, displayed promote selectivity for prostate cancer cell lines. This is
comparable activity to . Statistical analysis indicated that consequent upon previous report that describes edelfosine
there was no significant difference between the activity of analog bearing carbamate at sn-2 position of glycerolipid as
and
across all cell lines except for BT-474 cells. This suggests being more selective towards prostate cancer cell lines.28 The
Compound 10 with a methyl-carbamate substituent at the
4
2
2
2
4
that the methoxy substituent is not essential for manifestation CC50 values of compound 10 which is in the range of 14 – 23
of antitumor activity in the β-anomeric dicationic glycolipids. µM is much less active than that of β-GLN indicating that the
This is consistent with previous SAR studies on monocationic, carbamate substituent neither increases activity nor promoted
2-amino-D-gluco-based GAEL that demonstrated the methoxy selectivity for prostrate cell line in GAEL series.
group as not being crucial for antitumor activity.22 However,
tetracationic glycolipid which was generated by replacement were 2- to 3-fold more toxic toward the drug-resistant cancer
It is noteworthy that the most active analogs 1, 2, 4 and 8
5
of the methoxy substituent at the sn-2 position by a second cell lines, JIMT-1, MB-MDA-231, DU-145, and PC-3, relative to
2,6-diamino-β-D-gluco residue had a greatly reduced activity β-GLN (Table 1). The reason(s) for the indistinguishable
and was unable to achieve 50 % loss of cell viability at the activities of
highest concentration (30 µM) tested. This may be due to its To compare the cytotoxic activity of our synthesized
increased hydrophilicity leading to reduced cellular absorption. compounds with that of drugs in clinical use or development,
Compounds were synthesized primarily to determine we tested chlorambucil, cisplatin and salinomycin against all
the effect of the position of the sugar on the glycerolipid. The the six cell lines used in this study (Table 1). Chlorambucil, a
azido compound has its CC50 values in the range of 6 - 22 µM. DNA alkylating agent, did not kill up to 50 % of all cell lines at
1 and β-GLN against BT-474 cells is unclear.
6
– 8
6
It was more cytotoxic against PC-3 cell lines than β-GLN; CC50 the highest concentration (150 µM) tested. Similarly, we were
of 6.0 µM compared to 13.5 µM, but less against BT-474, (22 unable to reach CC50 values for cisplatin against BT-474, MDA-
µM and 8 µM respectively). For other cell lines, their activities MB-231 and MiaPaCa2 cell lines at the highest dose (20 µM)
are comparable. The dicationic GAEL
group at the sn-3 position of the glycerolipid is less potent CSCs killing properties, we were able to demonstrate that the
than mono-cationic across all cell lines. This indicates that most potent GAEL displayed > 6-fold activity against MDA-
the positioning of the second amino group on the glycerol MB-231, > 4-fold activity against DU-145, 2-fold higher against
moiety is not optimal. Compound with the sugar moiety on MiaPaCa2 and BT-474. Improved activity over salinomycin was
sn-3 position of the glycerolipid, was significantly more active also observed with GAELs and
compared to compound with the aminosugar on sn-2
7 with a primary amino tested. In the case of salinomycin, an experimental drug with
6
1
8
2,
4
8.
7
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 6 of 12
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
Journal Name
Figure 3. Effects of compounds
1 - 10, and β-GLN, on the viability of MDA-MB-231 (A), JIMT-1 (B), BT-474 (C), DU-145 (D), PC-3
(E) and MiaPaCa2 (F) cell lines. Effect of cisplatin (G) and salinomycin (H) on viability of DU-145, BT-474, MDA-MB-231 and
MiaPaCa2 were included for comparison. MDA-MB-231, JIMT-1, DU-145 and MiaPaCa2 cells were cultured in DMEM medium
supplemented with 10 % FBS, BT-474 cells were cultured in DMEM/F12 medium supplemented with 10 % FBS and PC-3 cells
were cultured in F12K medium supplemented with 10 % FBS. Equal numbers were dispersed into 96-well plates. After 24 h, the
cells were incubated with compounds
1 – 11 (0 – 30 µM) for 48 h. At the end of the incubation, MTS reagent (20 % v/v) was
added and the plates were incubated for 1 – 4 h. The OD490 was read with a plate reader. 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. Compounds 5, 8, 9, and 10 were studied as diastereomeric 1:1 mixture based on the stereochemistry at sn-2
position of the glycerolipid.
Effect of dicationic GAELs 1, 2, 4, or 8 on the integrity and Figures 4A and 5A show the results of the effects of GAELs on
viability of BT-474 and DU-145 cancer stem cells (CSCs)
the integrity and viability of BT-474 CSC spheroids after 6 days
of treatment. Incubations with the vehicle revealed the
To assess the effect of the most potent GAELs on cancer stem spheroids grew larger and more compact. In contrast,
cells (CSCs), high expressing aldehyde dehydrogenase incubations with the GAELs resulted in disintegration of the
1
(ALDH1) cells were sorted from BT-474 and DU-145 cells, and spheroids. Similar results were obtained with CSCs derived
cultured in low-adhesion culture plates in stem cell growth from DU-145 prostate cancer cell lines (Figures 4B and 5B). The
media. The spheroids were isolated by cell sieving, trypsinised GAELs were able to completely inhibit the viability of the CSCs
and counted. Equal numbers were dispersed in wells and and disintegrate the CSCs spheroids at concentrations ranging
allowed to form spheroids. The active dicationic GAELs,
or were added to the spheroids.
1,
2,
4,
from 5 – 10 µM depending on the compound.
8
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 7 of 12
View Article Online
DOI: 10.1039/C6MD00328A
Journal Name
ARTICLE
Figure 4. Effect of GAEL compounds
1
,
2
,
4
, and
8
on the integrity of (A) BT-474 breast cancer stem cell spheroids, (B) DU-145
, salinomycin, myristylamine and cisplatin on BT-474
prostate cancer stem cells spheroids, and (C) comparison of effects of
1
CSCs. Equal numbers of BT-474 or DU-145 cancer stem cells were seeded into ultra-low adhesion 48-well plates and grown for 5
days to allow for spheroid formation. The spheroids were incubated with or without 10 µM GAELs for up to 6 days (A and B) or 3
days (C). The images were taken after 4 days (A and B) or 3 days (C) of incubation with an Olympus I
(magnification 10).
×70 microscope
×
The most active compounds were
and and α-GLN for DU-145 CSCs. When compared to 474 CSCs was superior (Figure 4 and 5).
salinomycin, an investigational anti-CSCs drug; cisplatin, a
clinical anticancer agent, and myristylamine, known
1, 2 and 4 for BT474 CSCs surfactant, the activity of the most active analog 1 against BT-
1, 4
a
Figure 5. Effects of compounds
1,
2,
4,
8,
α-GLN on the viability of cancer stems cells isolated from BT-474 breast cancer (
A), DU-
145 cell lines ( ) and comparison of
B
1
with salinomycin, cisplatin, myristylamine ( ). α-GLN has been previously reported to be
C
more active than our reference β-GLN against cancer cell lines22, 23 and CSCs obtained from BT-474 cell line.22 Also it is being
tested on CSCs from DU-145 for the first time. BT-474 and DU-145 cancer stem cells were obtained by staining for ALDH1 and
sorting the cells by flow cytometry. The spheroids from BT-474 stems cells were grown in ultra-low adhesion plates in
mammocult medium for 6 days, while the spheroids from DU-145 stem cells were grown in ultra-low adhesion plates in
prostatosphere growth medium for 6 days. The spheroids formed were harvested, trypsinised and equal numbers were seeded
in 48-well low adhesion plates for 5 – 6 days to allow formation of spheroids. The spheroids were incubated with varying
concentrations of compounds 1, 2, 4, 8 (0 - 30 µM) for 6 days (
reagent was added to each well and the plates were incubated in a 5 % CO2 incubator for 4 h. The absorbance was read at 470
nm in a plate reader. The results are the means ± standard deviation for 4 independent determinations ( and ) and 6
).
A and B) and 3 days (C). At the end of the incubation, the MTS
A
B
independent
determination
(C
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 8 of 12
View Article Online
DOI: 10.1039/C6MD00328A
MedChemComm
ARTICLE
Table 1. CC50 (A) and CC90 (B) values of compounds 1-10, cisplatin, salinomycin, chlorambucil and β-GLN on a panel of human
epithelial cancer cell lines: breast (BT474, JIMT1, MDA-MB-231), pancreas (MiaPaCa2) and prostrate (DU145, PC3). The CC50 value
is defined as the concentration required to decrease cell viability by 50% relative to the untreated control, while the CC90 value is
defined as the concentration required to decrease cell viability by 90% relative to untreated control. The values were obtained
by estimating the drug concentration at 50% and 10% viability on the y-axis using line plots. Compounds 5, 8,9, and 10 were
studied as diastereomeric mixtures (1:1) based on the stereochemistry at sn-2 position of the glycerolipid. NT – Not tested, ND –
Not determined.
A
CC50 values (µM)
B
CC90 values (µM)
MDA-MB-
231
DU-
145
JIMT-1
MiaPaC
PC-3
BT-474
MDA-
DU-
145
JIMT-1
MiaPaC
PC-3
BT-474
Compd
1
a2
3.5
MB- 231
4.5
a2
6.5
3.0
5.2
3.5
3.5
7.5
7.4
4.9
6.0
9.5
± 0.1
± 0.3
± 0.2
± 0.1
± 0.1
± 0.3
± 0.1
± 0.1
± 0.1
± 0.1
± 0.2
± 0.1
2
3
5.5
6.0
4.2
± 0.1
>30
7.0
11.0
± 0.3
15.0
± 0.5
11.5
± 0.5
NT
7.0
8.5
6.5
12.0
14.0
14.0
± 0.2
20.0
± 2.1
± 0.2
± 0.3
± 0.1
± 0.1
± 0.2
± 0.3
± 0.1
± 0.1
ND
4
4.5
6.0
4.0
6.5
8.0
8.5
7.0
8.5
6.0
13.0
14.0
17.5
± 0.1
± 0.1
± 0.3
± 0.5
± 0.9
± 0.2
± 0.1
± 0.1
± 0.1
± 0.1
± 0.2
± 0.1
5
6
>30
ND
ND
ND
11.0
± 0.1
12.0
± 0.4
6.0 ±
0.2
12.5±
0.6
9.5
± 0.2
14.0
± 0.1
5.5
11.5
± 0.1
15.0
± 0.7
8.5
6.0
± 0.1
9.5
22.0
±1.1
25.0
± 0.4
15.5
± 0.4
15.0
± 0.3
17.5
± 0.2
9.0
18.0±
0.5
13.5
± 0.1
19.0
± 0.5
7.5
18.5
± 0.3
28.0
± 1.3
14.0
± 0.1
15.0
± 0.1
17.0
± 0.4
14.0
± 0.1
28.0
± 0.5
>30
7
8
17.5±
0.4
25.0±
1.0
± 0.3
9.0
6.0
9.0
28.0
± 0.1
± 0.1
± 0.3
± 0.1
± 0.1
± 0.1
± 0.1
± 0.8
9
>30
10
16.0
± 0.3
NT
14.0±
0.1
12.5
± 0.1
9.0
18.0
± 0.5
9.0
20.0
± 0.3
13.5
± 0.2
NT
23.0
± 0.8
8.0
19.0
± 0.4
NT
19.0
± 0.5
15.0±
0.4
15.0
± 0.2
16.0
± 0.2
29.0
± 0.9
18.0
± 0.6
29.0
±1.0
28.0
± 0.9
29.0
± 1.5
13.0
± 0.5
β-GLN
10.0±
0.5
± 0.1
NT
± 0.1
>20
± 0.3
>20
Cisplatin
>20
>20
14.8±
0.3
>20
NT
6.5 ±
0.1
NT
14.0 ±
0.3
Salinomycin
Chlorambucil
>150
Compound 1 demonstrated a better anti-CSC activity than salinomycin. Cisplatin did not show any significant reduction in
viability at highest dose tested (20 µM). Myristylamine, a classical amphiphile did not show any activity at the highest dose
tested (20 µM) indicating that the disruption of mammosphere formed by BT-474 CSCs is not due to detergent effect.
Discussion and Conclusion
primary amino substituent at the C-2 position of the sugar
GAELs represent a novel class of potential anticancer moiety shows higher toxicity against cancer cells compared to
agents. The most active compounds reported herein display analogs without amino function,18 we explored the possibility
CC50 values in the range of 3.0 to 7.5 μM. Since GAELs with a
that increasing the number of cationic charges would lead to
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 8
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 9 of 12
Journal Name
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
enhanced cytotoxic activity. Our results demonstrated that this to eradicate them using conventional chemotherapeutic
was indeed the case. Diamino GAELs and but not agents. Thus, compounds that kill cells by non-apoptotic
bisamino GAEL were 2- to 3-fold more active than our mechanisms are attractive potential anti-CSC agents, as they
previous monobasic lead structure β-GLN. The fact that GAEL could by-pass a variety of strategies adapted by CSCs to evade
which has the carbohydrate moiety linked at the sn-2 position apoptotic cell death.
1,
2,
4
8
7
7
shows reduced antitumor activity when compared to other
In summary, the current study has demonstrated the
dibasic GAELs may indicate that the carbohydrate moiety ability of glucose-configured diamine-based GAELs to
should be linked to the sn-3 position of the glycerolipid for disintegrate and kill CSC spheroids. The retention of this ability
optimal antitumor activity. Within the class of diamine-based across board demonstrates that the various structural
GAELs, glycolipid 1, containing an α-glucosidic-2,6-diamino modifications made to generate the diamine-based glycolipids
head group, consistently showed the highest activity against all did not fundamentally alter their mechanism of action. Thus,
6 cancer cell lines, indicating that the α-glucosidic linkage and the ability to disrupt the integrity of CSCs may be a general
positioning of the two cationic charges in the glucose moiety characteristic of GAELs.
are optimal for anticancer activity. The lack of activity of
compound
3 in comparison to β-GLN despite the free amino
Experimental
group at the C-6 position of glucose indicates that the amino
group at C-2 remains critical for anticancer activity.
Materials and methods: Synthesis of GAELs
Solvents were dried over CaH2. 1H, 13C NMR spectra were
recorded with a JMN A500 FT NMR spectrometer at 500 or 300
MHz and at 125 or 75 MHz, respectively, and chemical shifts
were reported in parts per million (ppm) relative to the
indicated solvent Thin-layer chromatography (TLC) was carried
out on an aluminum-backed silica gel GF plates (250 mm
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). Matrix assisted laser desorption ionization-
time of flight (MALDI-TOF)-MS was recorded on a voyager RP
mass spectrometer. High-resolution (HR) mass spectra were
recorded on a JEOL JMS700 under FAB conditions. Purity of
compounds 1 – 10 were assessed by elemental analysis, and
were within ± 0.5 % of the calculated theoretical values.
Comparison of the activity of GAELs
6 and 7 with that of
compound 8 showed that glycosylation of the hydroxyl group
at the sn-3 position of the glycero moiety appears to be
optimal for antitumor activity. The significantly higher activity
of compound
2 when compared to that of compound 8
indicates that the presence of both amino substituents on the
glucose moiety is optimal for anticancer activity. Similar to
previous findings in the monocationic GAEL series,22,23
replacing the methoxy substituent in the glycero portion by a
H-atom had little effect on the cytotoxicity against most cancer
cell lines except the PC-3 and BT-474 cell lines where a slight
improvement was observed. On the other hand, replacement
of the methoxy substituent by a second β-glycosidic 2,6-
amino-D-glucosyl moiety as in
5, or acylation of GAEL 8 by
palmitic acid to afford 9, resulted in a loss of antitumor
activity. While the exact reasons for the loss of activity are not
clear, the substitutions could affect the physical properties of
the compound which may impact absorption and thus the
differential in activity. Unlike edelfosine, replacement of the
methoxy group at sn-2 position of the glycerolipid with methyl
carbamate as in 10 significantly reduced anticancer activity
and it did not enhance selectivity toward prostate cancer.
Chemistry:
General method for acetylation
Acetylation reaction were carried out in pyridine with catalytic
amount of dimethyl amino pyridine (DMAP, 0.2 equiv.) using
acetic anhydride (2 equiv.). After stirring for 18 h at room
Juxtaposing the cytotoxicity of
1
,
cisplastin and
temperature 23 °C, the reaction was stopped by addition of
methanol, and concentrated to dryness. The resulting residue
salinomycin, the most researched ant-CSCs agent,31 against BT-
474, DU-145 and MDA-MB-231 cancer cell lines and CSCs
isolated from BT474 cell lines, it is clear that GAELs exhibited
the best anticancer properties (Figure 3 G, 3H, 4C and 5C).
Interestingly, the classical amphiphile myristylamine did not
decrease the viability and disintegrate CSC spheroids,
indicating that the anti-CSCs properties of GAELs is not the
result of a detergent effect.
was dissolved in ethyl acetate, washed with saturated sodium
bicarbonate (×3) and distilled water (×2). The resulting organic
layer was dried over Na2SO4 and concentrated to dryness. The
residue was purified by flash chromatography, with yields
ranging from 40 – 80 %.
General method for glycosylation reaction
Despite the large number of chemotherapeutic agents in
clinical use for the management of cancer, the overall
treatment outcomes have at best been disappointing. There
are compelling evidences that tie tumor relapse to the inability
to eradicate CSCs.6,7, 22 A major goal of current cancer research
is to identify mechanisms and/or means of killing or
inactivating CSCs.7 Several approaches to curtail the activity of
CSCs in tumors have been suggested7,32-35 but because of their
capacity to resist apoptotic cell death, it has been challenging
The glycoside donor and 1.1 molar equivalent of the lipid
alcohol, the glycoside acceptor, were dissolved in anhydrous
dichloromethane (DCM) under argon atmosphere. N-
Iodosuccinimide (NIS, 1.5 equiv) and silver triflate (AgOTf, 0.2
equiv) were simultaneously added. The reaction mixture was
stirred for 3 h. Upon completion, the reaction mixture was
diluted with DCM and filtered over celite. The resulting organic
layer was washed with saturated sodium thiosulphate solution
(×2), saturated sodium bicarbonate (×3), and water (×2). The
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 10 of 12
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
Journal Name
organic layer was then dried over anhydrous Na2SO4 and flash chromatography (hexanes/ethylacetate (9:1, v/v) and
concentrated under vacuum to give a residue which was 100% ethyl acetate for protected glycolipid).
purified by flash chromatography (hexanes/ethyl acetate, 2:3)
Synthetic procedures for the synthesis of intermediates and
General method for conversion of primary hydroxyl group to final compounds
azide
See supplementary information
Triisopropylbenzylsulphonylchloride (TIBS) (1.5 equiv) or p-
toluenesulphonylchloride (1.1 equiv) were used to activate the
primary hydroxyl group at the C-6 position of the sugar or Biological methods
primary hydroxyl group of the glycerolipid-based diol, using
DMAP (0.2 equi) as catalyst in anhydrous pyridine under argon Effect of GAELs on viability of epithelial cancer cell lines
or nitrogen atmosphere. The reaction was stirred at rt for 12 The cell lines were cultured from frozen stocks originally
to 24 h, after which it was stopped by addition of methanol. obtained from ATCC. MDA-MB-231, JIMT-1 and DU-145 were
The methanol and pyridine were removed under high vacuum. grown in DMEM medium supplemented with 10 % FBS. BT-474
The crude mixture was dissolved in EtOAc and washed with 5 cells were grown in DMEM/F12 medium supplemented with
% HCl (
×
2), saturated sodium bicarbonate solution (
×2), and 10 % FBS. MiaPaCa-2 was cultured in DMEM supplemented
1), to give a dark brown organic layer. The organic with 10 % FBS and 2.5 % horse serum. PC-3 cells were cultured
water (×
solvent was concentrated and purified by flash in F12K medium supplemented with 10 % FBS. All the media
chromatography (hexanes/ethyl acetate, 2:3) or 100 % ethyl contained penicillin/streptomycin.
acetate. The sulphonate ester was displaced with azide in a The effects of the GAELs on the viability of the various
nucleophilic substitution reaction using sodium azide (10 epithelial cancer cell lines was determined as previously
equiv) in anhydrous DMF at 70 – 90
atmosphere for 12 to 24 h. Upon completion, the mixture was dispersed into 96-well plates. After 24 h, the cells were
concentrated, resuspended in ethyl acetate, filtered, re- incubated with the compounds (0 – 30 M) for 48 h. At the
°
C under argon or nitrogen described.12,13,16 Briefly, equal numbers of the cells were
ꢀ
concentrated, and purified using flash chromatography.
end of the incubation, MTS reagent (20 % v/v) was added and
the plates were incubated for 1 – 4 h in a CO2 incubator. The
OD490 was read with a plate reader (Molecular Devices). Wells
with media but no cells were treated in similar fashion and the
General method for deprotection of acetate group
The acetate protected compounds were dispersed in methanol values utilized as blank. The results represent the mean ±
followed by the addition of a catalytic quantity of NaOMe (0.5 standard deviation of 6 independent determinations.
equiv). The mixture was stirred for 1 h and stopped with ion
exchange resin (H+). When the reaction mixture was clear, the Isolation of breast cancer stem cells from BT-474 and prostate
resin was filtered, concentrated under vacuum, and purified by cancer stem cells from DU145 cell lines and determination of
flash chromatography (100% ethyl acetate).
the effect of GAELs on the viability of the cancer stem cells.
For selective deprotection of acetate in the presence of
phthalimido protective group, the reaction was stirred for A population enriched in BT474 breast cancer stem cells or DU-
lesser time (20 to 25 mins).
145 prostate cancer stem cells was obtained by staining the
cells for aldehyde dehydrogenase using the Aldefluor assay kit
General method for simultaneous deprotection of acetate from Stem Cell Technologies (Vancouver, BC, Canada)
and phthalimido group according to the manufacturer’s instructions, with appropriate
To simultaneously remove acetate and phthalimido protecting controls. The stained cells were sorted from the bulk
group, the protected compound is dissolved in a mixture of population by flow cytometry on a 4 laserMoFloXPP high
ethylenediamine/n-butanol (1:1, v/v) and stirred at 90 °C for 2 speed/pressure cell sorter. The cells were pelleted by
h. Then the reaction mixture was concentrated under high centrifugation. BT-474 cells were resuspended into ultra-low
vacuum and purified using reverse phase C-18 silica gel column adhesion plates in mammocult medium (Stem cell
by gradient elution of water and methanol.
Technologies), and DU-145 stem cells in their growth medium
(DMEM/F12 medium supplemented with 20 ng/ml EGF, and 10
General method for reduction of azide
ng/ml basic FGF,
5
µg/ml insulin, 0.4% BSA, with 1%
dishes were incubated at 37 C in a CO2
compounds were suspended in a mixture of THF/water (9:1), incubator for 4 days for spheroid formation.
The
To reduce the azido protecting group to free amine, the azido antibiotics).36
°
then trimethyl phosphine in THF (1 M, 5 – 10 equiv) was The spheres are separated from single cells with a 40 µm nylon
added. The reaction was stirred at rt for 2 h and concentrated cell strainer. The spheres retained in the strainer were washed
under vacuum. Residues of final compounds were purified with PBS and trypsinised to obtain single cells. The cell
using C-18 coated silica gel column chromatography by numbers were counted with a Coulter ZM counter and the
gradient elution of water and methanol, while the lipid cells were dispersed into 48-well low adhesion plates (Grenier)
molecule with azido group was purified using normal phase in a volume of 500 µl. The cells were incubated for 4 days to
allow for formation of spheroids. Subsequently, the stock
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 11 of 12
Journal Name
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
GAELs in ethanol were diluted to twice the final concentration
in the media and a volume of 500 µl was added to the wells.
Wells with growth medium but no cells were treated similarly
as wells with cells. After 5 days incubation, MTS reagent (2 %
v/v) was added to each well and the plates were incubated for
1 – 4 h for formation of colour. The OD490 were read in a
Molecular Device absorbance plate reader using the
SpectroMax software.
7
R. Zobalova, M. Stantic, M. Stapelberg, K. Prokopova,
J. Dong and J. Truksa, J. In: Shostak S, editor. Cancer
Stem Cells Theories and Practice, ISBN: 978-953-307-
225-8, 2011.
8
9
C. Naujokat, D. Fuchs and G. Opelz, Mol. Med. Rep.,
2010, 3, 555-559.
M. L. Guzman, L. Karnischky, D. R. Peterson, D. S.
Howard and C. T. Jordan, Blood, 2005, 105, 4163-
4169.
Statistical analysis
10 Z. Xiao, B. Sperl, A. Ullrich and P. Knyazev,
Oncotarget, 2014, , 12877-12890.
The results represent the mean ± standard deviation of 6
independent determinations. Statistical significant difference
tests were carried out using GraphPadInstat software. The
mean values were subjected to one way analysis of variance
(ANOVA) followed by Tukey-Kramer multiple comparison tests
as 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 1 – 10 tested and the lead compound β-GLN was
also compared using ANOVA, followed by Turkey-Kramer
multiple comparison tests at the following concentrations: 5,
7.5 and 10 µM to determine statistical significance in potency.
A p-value > 0.05 indicates no statistical differences while a p-
value of < 0.001 indicated statistical significant differences.
The statistical analysis data are not included in this report.
5
11 D. Fuchs, V. Daniel, M. Sadeghi, G. Opelz, C. Naujokat,
Biochem. Biophys. Res. Comm., 2010, 394, 1098-
1104.
12 D. Fuchs, A. Heinold, G. Opelz, V. Daniel, C. Naujokat,
Biochem. Biophys. Res. Commun., 2009, 390, 743-
749.
13 C. Gong, H. Yao, Q. Liu, J. Chen, J. Shi, F. Su and E.
Song, Plos ONE, 2010, 5, Article ID e15630.
14 K. Y. Kim, S. N. Yu and S. Y. Lee, Biochem. Biophys.
Res. Comm., 2011, 413, 80-86.
15 M. R. Bardsley, V. J. Horvth and D. T. Azuzu,
Gastroenterology, 2010, 139, 942-952.
16 G. N. Zhang, Y. Liang, J. Zhou, S. Chen, G. Chen, T.
Zhanga, T. Kang and Y. Zhao, Cancer lett., 2011, 313
,
137-144.
17 Y. Zhang, H. Zhang, J. Wang, X. Wang and Q. Zhang,
Biomaterials, 2012, 33, 679-691
18 R.V. Erukulla, X. Zhou, P. Samadder, G. Arthur and R.
Bittman, J. Med. Chem., 1996, 39, 1545–1548
19 P. Samadder, R. Bittman, H.-S. Byun and G. Arthur,
Cell Biol., 2009, 87, 401–414
Acknowledgements
20 P. Samadder, R. Bittman, H.-S. Byun and G. Arthur,
Anticancer Res., 2011, 31, 3809–3818.
This study was supported by research grants from the
Canadian Breast Cancer Foundation Prairie/NWT to GA and
Natural Science and Engineering Council of Canada (NSERC)
grant to FS. Makanjuola Ogunsina is a recipient of the
University of Manitoba and Manitoba provincial government
graduate fellowships (UMGF and MGS).
21 P. Samadder, R. Bittman, H.-S. Byun and G. Arthur,
Anticancer Res.,1998, 18, 465–470.
22 P. Samadder, Y. Xu, F. Schweizer and G. Arthur, Eur. J.
Med. Chem., 2014, 78, 225-235.
23 Y. Xu, M. Ogunsina, P. Samadder, G. Arthur and F.
Schweizer, ChemMedChem, 2013, 8, 511–520.
24 M. Ogunsina, H. Pan, P. Samadder, G. Arthur and F.
Schweizer, Molecules, 2013, 18, 15288-15304.
References
1
World Health Organization: Cancer Fact Sheet #297
http://www.who.int/mediacentre/factsheets/fs297/e
n/ (Viewed June 20,2016).
25 H. Miyachi, A. Azuma, A. Ogasawara, E. Uchimura, N.
Watanabe, Y. Kobayashi, F. Kato, M. Kato and Y.
Hashimoto, J. Med. Chem., 1997, 40, 2858-2865.
26 M. A. H. Zahran, Y. G. Abdin, A. M. A Osman, A. M
Gamal-Eldeen, R. M. Talaat and E. B Pedersen, Arch.
Pharm., 2014, 347, 642–649.
2
3
D. S. Tan, M. B. Gerlinger and C. Swanton, Eur. J.
Cancer, 2010, 46, 2166 – 2177.
X. Li, M. T. Lewis, J. Huang, C. Gutierrez, C. K. Osborne
and M. F. Wu, J. Natl. Cancer Inst., 2008, 100, 672 –
679.
27 F. Schweizer, Eur. J. Pharmacol., 2009, 625, 190-194.
28 H.-S. Byun, R. Bittman, P. Samadder, and G. Arthur,
4
M. Tanner, A. I. Kapanen, T. Junttila, O. Raheem, S.
Grenman, J. Elo, K. Elenius, J. Isola, Mol. Cancer Ther.,
ChemMedChem, 2010,
29 L. Jahreiss, M. Renna, R. Bittman, G. Arthur and D.
Rubinsztein, Autophagy, 2009, , 835–846.
5, 1045-1052.
2004, 3, 1585 – 1592.
5
5
6
J. A. Ajani, J. G. Izzo and J. -S. Lee, J. Clin. Oncol., 2009,
30 S. Bera, R. Dhondikubeer, B. Findlay, G. G. Zhanel and
F. Schweizer, Molecules, 2012, 17, 9129–9141.
31 M. Antoszczak, and A. Huczyński, Anticancer Agents
Med. Chem., 2015, 15, 575-591.
27, 162 - 163.
B. K. Garvalov and T. Acker, J. Mol. Med., 2011, 89, 95
– 107.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 12 of 12
View Article Online
DOI: 10.1039/C6MD00328A
ARTICLE
32 G. Arthur and R. Bittman, Anticancer Agents Med.
Chem., 2014, 14, 592-596.
Journal Name
36 L. Salvatori F. Caporuscio A. Verdina G. Starace S.
Crispi and M. R. Nicotra, PLoS ONE, 2012, 7(2),
33 X. Ning, J. Shu, Y. Du, Q. Ben and Z. Li, Cancer Biol.
Ther., 2013, 14, 295 – 303.
e31467. doi: 10.1371/journal.pone.0031467.
34 K. Chen, Y. Huang and J. Chen, Acta Pharmacol. Sin.,
2013, 34, 732-740.
35 N. Oishi and X. W. Wang, Int. J. Biol. Sci., 2011, 7, 517
– 535.
12 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins