Amphiphilic Modulation of Glycosylated Antitumor Ether Lipids Results in a Potent Triamino Scaffold against Epithelial Cancer Cell Lines and BT474 Cancer Stem Cells

Article abstract of DOI:10.1021/acs.jmedchem.7b01198

The problems of resistance to apoptosis-inducing drugs, recurrence, and metastases that have bedeviled cancer treatment have been attributed to the presence of cancer stem cells (CSCs) in tumors, and there is currently no clinically indicated drug for their eradication. We previously reported that glycosylated antitumor ether lipids (GAELs) display potent activity against CSCs. Here, we show that by carefully modulating the amphiphilic nature of a monoamine-based GAEL, we can generate a potent triamino scaffold that is active against a panel of hard-to-kill epithelial cancer cell lines (including triple-negative breast) and BT474 CSCs. The most active compound of this set, which acts via a nonmembranolytic, nonapoptotic caspase-independent mechanism, is more effective than cisplatin and doxorubicin against these cell lines and more potent than salinomycin against BT474 CSCs. Understanding the combination of factors crucial for the enhanced cytotoxicity of GAELs opens new avenues to develop potent compounds against drug-resistant cancer cells and CSCs.

Full text of DOI:10.1021/acs.jmedchem.7b01198

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Article
Amphiphilic-Modulation of Glycosylated Antitumor Ether
Lipids Results in a potent Triamino Scaffold against
Epithelial Cancer Cell Lines and BT474 Cancer Stem Cells
Temilolu Idowu, Pranati Samadder, Gilbert Arthur, and Frank M. Schweizer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01198 • Publication Date (Web): 18 Oct 2017
Downloaded from http://pubs.acs.org on October 19, 2017
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AmphiphilicꢀModulation of Glycosylated Antitumor
Ether Lipids Results in a Potent Triamino Scaffold
against Epithelial Cancer Cell Lines and BT474 Cancer
Stem Cells
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Temilolu Idowu, Pranati Samadder, Gilbert Arthur,* and Frank Schweizer*
†
Department of Chemistry, Faculty of Science, University of Manitoba, 144 Dysart Road, Winnipeg,
Manitoba R3T 2N2, Canada
‡
Department of Biochemistry and Medical Genetics, Faculty of Health Sciences, University of
Manitoba, 745 Bannatyne Avenue, Winnipeg, Manitoba R3E 0J9, Canada
KEYWORDS: amphiphile, antitumor, cancer stem cell, glycolipid, GAEL, hybrid, nonꢀapoptotic,
tripleꢀnegative breast cancer
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ABSTRACT
The problems of resistance to apoptosisꢀinducing drugs, recurrence, and metastases that have bedeviled
cancer treatment have been attributed to the presence of cancer stem cells (CSCs) in tumors, and there is
currently no clinically indicated drug for their eradication. We previously reported that glycosylated
antitumor ether lipids (GAELs) display potent activity against CSCs. Here, we show that by carefully
modulating the amphiphilic nature of a monoamineꢀbased GAEL, we can generate a potent triamino
scaffold that is active against a panel of hardꢀtoꢀkill epithelial cancer cell lines (including tripleꢀ
negative) and BT474 CSCs. The most active compound of this set, which acts via a nonꢀmembranolytic,
nonꢀapoptotic caspaseꢀindependent mechanism, is more effective than cisplatin and doxorubicin against
these cell lines, and more potent than salinomycin against BT474 CSCs. Understanding the combination
of factors crucial for the enhanced cytotoxicity of GAELs opens new avenues to develop potent
compounds against drugꢀresistant cancer cells and CSCs.
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INTRODUCTION
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The development of resistance to currently used chemotherapeutic drugs, which are mostly proꢀ
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apoptotic, is a major impediment to the treatment of cancer. Also, the presence of cancer stem cells
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(CSCs) in the bulk tumor has been implicated in tumor relapse that usually occurs after the initial
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treatment with existing chemotherapeutic agents. These two factors account for the major reason a
cure for cancer has remained elusive. Glycosylated antitumor ether lipids (GAELs), a subclass of
antitumor ether lipids typified by glycolipid 1 (Figure 1), kill cancer cells via a nonꢀapoptotic cell death
7,8
9,10
mechanism, which bears close resemblance to methuosis.
Methuosis is a nonꢀapoptotic cell death
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that is characterized by vacuolization of macropinosomes which leads to cell rupture.
GAELs have
1
2
also demonstrated the ability to kill CSCs, a characteristic possessed by very few compounds. The
nonꢀapoptotic mechanism of action of this class of drugs provides new opportunities to manipulate cell
death in a therapeutic context and to eliminate apoptosisꢀresistant cancer cells.
Structureꢀactivity studies of GAELs have revealed the importance of the Cꢀ2 amino group as it affects
1
3,14
cytotoxicity.
Other cationic amphiphilic drugs (CADs) have also been shown to induce nonꢀ
1
5
apoptotic cell death pathways implicating lysosomes in the cellꢀdeath processes. Most CADs are easily
protonated at normal body pH, leaving them with a net positive charge. Meanwhile, cancer cell
membranes typically carry a net negative charge relative to normal cell membranes due to an elevated
1
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18,19
expression of anionic molecules such as phosphatidylserine,
Oꢀglycosylated mucins,
sialilated
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gangliosides, and heparan sulfates. The role of cationicity in anticancer agents may be to enhance the
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affinity of CADs for the cancer cells. Upon binding to cell membrane, hydrophobicity of molecules
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becomes crucial in determining how well they permeate such membrane.
Membrane fluidity is
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typically increased in cancer cells relative to normal cells,
which may facilitate cancer cell
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membrane destabilization by membraneꢀbound CADs. Consequently, the intrinsic properties of classical
cationic amphiphiles may allow strong binding to cancer cells and the ability to cross the negatively
charged hydrophobic membrane of cancer cells.
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Previous studies showed that GAELs without the amino group were up to 10ꢀfold less active relative to
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the monoamineꢀbased compound 1. On the other hand, there was a significant loss of activity in
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studies with diglycosylated GAEL 2 (Figure 1) relative to 1. This diglycosylated analog was prepared
to impart two amino functionalities (on different sugars) into a single molecule. In contrast, GAELs with
two amino substituents on a single sugar molecule 3 (αꢀ and βꢀ) exhibited better cytotoxic properties
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than 1 and 2 (Figure 1). The fact that analogs with two amino groups on a single sugar molecule
display better activities than analogs with two amino groups on separate sugars suggests that the
presence of a polar head group with a compensating long lipid (hydrophobic) tail are structural
requirements for active analogs. Hence, we were curious about how the activity would be affected by
three amino groups with an appropriately compensating hydrophobic moiety. We have previously
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demonstrated that fusing other domains with 1 did not significantly alter cytotoxicity. This study
provided insights on possible modifications that could be made to GAELs. More recently, it was
demonstrated that the
Lꢀenantiomers of this class of experimental drugs retained the nonꢀapoptotic
mechanism of action and antiꢀCSC activity, and that introduction of a second amine group enhanced
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potency. Amphiphilicity and cationicity have often been employed to overcome the problems of
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electrostatic interactions and transmembrane navigation in cancer cells, and bacteria. We therefore
envisioned that conjoining myristylamineꢀlike amphiphiles to GAELs would modulate the overall
amphiphilic nature of these agents, and amplify their antitumor activities due to the presence of an
additional amino group. In addition, these compounds would traverse the negatively charged
hydrophobic membrane of cancer cells more easily. Myristylamine (4) (Figure 1) is an amphiphilic
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cationic lipid that exhibits little activity against epithelial cancer cells relative to GAELs. We decided
to investigate the triamino concept by imparting a third amino functionality to βꢀdiamino 3 using
classical amphiphiles with different hydrophobic lengths and a polar (amino) group. Whereas the αꢀ
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anomer of 3 was observed to be slightly more potent than its βꢀanomer, further studies with the
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benzylated αꢀ, and βꢀ analogs showed similar potency between both configurations. We therefore
synthesized six βꢀtriamino GAELs 5 ꢀ 10 (Figure 2) because βꢀconfigurations could be easily
established by chemical methods. Herein, we investigated the effects of a third amino group, the
hydrophobic aliphatic carbon chain lengths, and the point of covalent attachment on the cytotoxicity of
GAEL using a variety of cancer cell lines, including drugꢀresistant human epithelial cancer cells. We
also probed the role of the glycerol backbone on the cytotoxicity of the triamino GAELs. The effect on
the viability of BT474 CSCs was investigated, as well as mechanistic studies were performed to gain
insights into the mode of action of the triamino GAEL derivatives.
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RESULTS AND DISCUSSION
Chemistry
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In designing the triamino molecules, we wanted to preserve all the functional groups and key features of
the lead monoꢀ (1) and diamino (3) analogs. To ensure physiological protonation of 3, the Cꢀ2 and Cꢀ6
amino groups were alkylated with laurylamine via reductive amination to afford compounds 5 and 7
respectively (Figure 2). The difference in the points of covalent attachment between these compounds
was meant to probe the position of the primary amine of GAELs. The length of the laurylamine domain
was also investigated by keeping the polar head constant while varying the length of carbon chain to
give 6 and 8, which were prepared similarly as 5 and 7. A C12 aliphatic hydrocarbon chain is often
regarded as a safe limit for positive charge and hydrophobic compromise, beyond which hemolytic
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activity dramatically increases with no significant change in efficacy. To determine the role of the
third amino group in 7 we prepared 9 by coupling chlorambucil to the amino moiety of laurylamine
(Figure 2). This transformation converts the terminal primary amine into an amide bond that cannot be
protonated at physiological pH, while retaining the C12 tether and diamino nature of the parent
compound 3. The chlorambucil moiety was used to neutralize the third cationic charge because it has
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1
been shown to be less cytotoxic to the epithelial cancer cells used in this study, and it imparts
additional lipophilicity to the molecule. To contextualize the exact role of the amines, the triamino
scaffold was explored without its glycerol backbone (compound 10) to determine whether cytotoxicity
was solely based on the triamino groups or on the entire molecule. The glycerolipid backbone of GAELs
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has been reported to be essential for cytotoxicity.
Chemical Synthesis
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Synthesis of the triamino GAELs (5 – 10) commenced with the preparation of thioglycoside donor 12
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from commercial glucosamine hydrochloride (11) as earlier reported (Scheme 1).
Donor 12 was then
glycosylated with 12ꢀazidododecanol under Nꢀiodosuccinimide/silver triflateꢀpromoted conditions to
give βꢀglucopyranoside 13, which was subsequently deprotected in a single step with ethylenediamine,
followed by a palladiumꢀcatalyzed hydrogenation to afford 1ꢀ(12ꢀaminododecyl)ꢀ2,6ꢀdiaminoꢀ2,6ꢀ
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dideoxy βꢀglucopyranoside (10) (Scheme 1). nꢀAzidoalkanals 16aꢀb were prepared by S 2 displacement
N
of bromine from commercially available nꢀbromoalcohols 14aꢀb with sodium azide to give nꢀ
azidoalcohols 15aꢀb, and a successive oxidation of the primary alcohols to aldehydes by pyridinium
chlorochromate (PCC) (Scheme 2). Thus, prepared compounds 16aꢀb were immediately reacted with the
glycerolipids 19 and 21, via reductive amination to give protected derivatives 20aꢀb and 22aꢀb
respectively (Scheme 3). A palladiumꢀcatalyzed hydrogenation of 20aꢀb afforded the desired
compounds 5 and 6 in good yields. In a similar manner, 7 and 8 were prepared by deprotecting 22aꢀb
with ethylenediamine, followed by palladiumꢀcatalyzed hydrogenation (Scheme 3).
Chlorambucilꢀlinked glucopyranoside 9 was prepared by deprotecting 22a with ethylenediamine and
reprotecting the primary and secondary amines with diꢀtertꢀbutyl dicarbonate to give a Bocꢀprotected
glucopyranosideꢀsnꢀglycerol 23 (Scheme 4). Catalytic hydrogenation and amide coupling with a preꢀ
activated chlorambucil gave chlorambucilꢀlinked Bocꢀprotected βꢀglucopyranosideꢀsnꢀglycerol 24 in
good yield, and was finally deprotected with trifluoroacetic acid to afford the chlorambucilꢀlinked βꢀ
glucopyranosideꢀsnꢀglycerol compound 9.
In vitro Screening against Epithelial Cancer Cell Lines
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To determine the cytotoxic effects of the newly synthesized triamino compounds 5 – 10 on the viability
of human epithelial cancer cell lines, exponentiallyꢀgrowing cancer cell lines were incubated with
varying concentrations of each compound (0 – 20 µM) for 48 h followed by viability assay using the
MTS reagent. The parent compounds 1, 3 and 4, as well as the clinicallyꢀused drugs cisplatin and
chlorambucil served as reference compounds for the studies.
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The results of the study revealed that of the six triamino compounds synthesized (Figure 2), compound 7
displayed the most potent activity against breast (JIMT1, MDAꢀMBꢀ231, BT474), pancreas
(
MiaPaCa2), and prostate (DU145, PC3) cancer cell lines (Figures 3AꢀC). The CC values ranged from
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.5 ꢀM – 4.0 ꢀM, a consistent 3ꢀ to 7ꢀfold increase in activity (depending on the cell line) when
compared to reference 1 (Table 1). Cisplatin and chlorambucil were also less potent than 7 against these
cell lines (Figure 3G). Although doxorubicin was more active than 7 based on CC values, it could not
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0
achieve a 90 % reduction in cell viability at the highest concentration tested (Figure S1) whereas 7
reduced cell viability to zero across the panel at concentrations between of 4.0 – 10.0 ꢀM (Figure 4).
Compound 5 was the next active triamino GAEL after 7, and its activity was similar to or slightly less
than the activity of derivative 1. Compound 5 was less potent than reference 1 against BT474 cells, and
was only slightly better against prostate cancer cells (DU145, PC3) with a barely 2ꢀfold increase (Table
1
). The shorter chain triamino compounds 6 and 8, as well as the chlorambucilꢀlinked analog 9 displayed
comparably low activities relative to 5 and 7 across all cell lines (CC values ranged from 10.5 ꢀM to >
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0 ꢀM, Table 1 and Figure 3AꢀC). Compound 10 was not active at the highest concentration tested (20
ꢀM). Thus, while increasing the number of amino moieties does lead to enhanced potency, it is clear that
other structural features play a role in influencing the overall activity of the compounds.
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To investigate SAR of GAELs, we compared the activity of compounds designed to explore the position
of the covalent linkage, hydrophobicity, and the role of the glycerol moiety using compound 1 as the
reference compound. Compounds 5 and 7 were designed to explore the effect of fusing a laurylamine
moiety at different positions on the sugar of the GDG on the activity of the compounds. In 5, the
substitution was at the Cꢀ2 position while in 7 it was at the Cꢀ6 position. As mentioned above, 7 was
extremely active and is in fact the most active GAEL synthesized to date. The activity of 5 was 2ꢀ to 7ꢀ
fold less than 7 depending on the cell line, thus the positioning of the moiety has a significant impact on
activity. This view is confirmed by comparing the results of the activities obtained with 6 and 8, which
have shorter hydrophobic moieties that are linked at the Cꢀ2 and Cꢀ6 positions respectively in a manner
analogous to 5 and 7. Compound 8 with the substituent at Cꢀ6 position of the sugar displayed slightly
better activity than 6 that was substituted at Cꢀ2 position with the majority of the cell lines, except for
MiaPaCa2 cells. Although the differences in potency between 6 and 8 were not as pronounced as
between 5 and 7, the phenomenon seems to suggest that the point of covalent attachment (or position of
primary amine) plays a crucial role in the potency of the triamino scaffold. This observation suggests a
requirement for primary amines at Cꢀ2 position (substitutions at Cꢀ6) of the glucosamine moiety that
preserves the exact form of the lead monoamino analog 1 (Figure 1).
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As hydrophobicity is expected to play a major role in amphiphilicꢀmodulation of the compounds, the
effect of the carbon chain length of the hydrophobic moiety on activity was probed by comparing the
activities of 5 with 6 and 7 with 8. The results revealed that 5 and 7 were more active than 6 and 8
respectively. The longer carbon chain length, as presented by 5 and 7, is expected to impart a
compensating hydrophobicity to the terminal amino group while the shorter length in 6 and 8 will result
in molecules that are less hydrophobic (Figure 2). Hydrophobicity plays a vital role in the movement of
amphiphilic molecules across cell membranes or insertion of molecules into membranes. While the
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positively charged amino groups on the compounds (protonated at physiologic pH) electrostatically
localize them to the abundantly expressed anionic charges on cancer cell membranes, their hydrophobic
nature will facilitate their insertion or uptake across the richly lipophilic membranes. Compounds 5 and
0
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probably are inserted or transverse the cell membrane more easily than 6 and 8 and could thus explain
the distinct difference in activity.
Compound 7 was linked to chlorambucil to generate compound 9 because chlorambucil has been
previously reported to exhibit very low activity (CC > 150 ꢀM) against the epithelial cancer cell lines
50
3
1
31
used in this study. However, it modulates the activities of GAELs by imparting lipophilicity. The
chlorambucilꢀlinked analog 9 was synthesized to neutralize the cationic effect of the terminal amino
group through the formation of an amide bond that removes the possibility of protonation. The use of a
Nꢀacetyl derivative to neutralize this cationic charge was undesirable as NHAc groups alter the activities
28
of GAELs against prostate cancer cell lines, whereas chlorambucil does not. Thus, compound 9 is less
cationic than 7, but is more lipophilic. Compound 9 displayed low activity similar to 6 and 8. The loss of
activity of 9 relative to 7 confirms that a certain threshold of hydrophobicityꢀcharge ratio must be
maintained for optimal activity. The TFA salt of compound 9 was stable at 30 ꢀM concentration in a pH
2ꢀ3 in aqueous DMSO solution for at least one week. The results of studies with compound 10 supports
this hydrophobicityꢀcharge balance postulate. Compound 10 that has all the three amino groups but lacks
the hydrophobic domain (glycerol backbone and the long lipid tail) of GAELs (Figure 2) was the least
active of all the analogs tested against all cell lines (Figures 3A–C and Table 1). These results leave us
with two conclusions: 1) a glycerol backbone and long lipid tail is essential for the activity of all GAEL
1
4
analogs, as previously reported. 2) the cationic charges as presented by amino groups are necessary,
but insufficient for the activity of this scaffold. Rather, a balance of charge(s) and an appropriately
compensating hydrophobicity is required.
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The BT474 cell line appeared to be unusually resistant to being killed by the triamino GAELs (Figure
A), the only exception being compound 7, but even this compound did not achieve 100 % cell kill at 20
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µM after 48 h incubation. Compound 6 enhances the growth of BT474 cells up to a concentration of 15
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µM. BT474 cells are characterized by the overꢀexpression of human epidermal growth receptor 2 (HERꢀ
3
5
2) and estrogen receptor (ER), and are resistant to ER antagonists such as tamoxifen. The
overexpression of these receptors could be a reason why the cells resist these compounds. Triple
negative breast cancer (TNBC) cell lines lack HERꢀ2 and ER, as well as the progesterone receptor (PR).
They are very resistant to conventional chemotherapeutic agents and prognosis for patients with TNBC
36
is worse than patients with the receptors. We therefore assessed the effect of 7, the most potent
triamino GAEL, against a panel of TNBC cell lines: MDAꢀMBꢀ453, MDAꢀMBꢀ468, MDAꢀMBꢀ231,
BT549 and Hs578t. The results displayed in Figure 3F show that 7 was able to achieve > 95 % reduction
of cell viability at about 6.0 µM, a > 3ꢀfold increase in activity compared to 1.
We also investigated the effect of 7 on cells derived from other hardꢀtoꢀtreat cancers; brain (U87, U251)
and ovarian (A2780s, A2780cp). A2780cp is a cisplatinꢀresistant isogenic cell line of the drug sensitive
A2780s line. Compound 7 displayed CC values of < 4.0 µM against the brain and ovarian cancer cell
5
0
lines (Figures 3D and E, Table 2) and reduced viability to almost zero at 4.0 ꢀ 8.0 µM. Thus, 7 was
effective against epithelial cancer cell lines representing hardꢀtoꢀtreat cancers including TNBC,
androgenꢀresistant prostate cancer, drug resistant ovarian cancer and brain cancer.
Effect of GAELs on BT474 Cancer Stem Cell Spheroid Viability and Integrity
6
The inability to eliminate CSCs is one of the major impediments to finding a cure for cancer. The
propensity of CSCs to evade apoptosis leads to recurrence subsequent to treatment, and CSCs are
6
thought to be also responsible for metastases. There are only a few compounds that are currently known
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37
to kill CSCs, and it was previously reported that mono and diamino GAELs have the ability to kill
1
2,30,32
these cells.
We therefore evaluated whether the newly synthesized triamino molecules are also
able to kill CSCs. CSCs were isolated from BT474 cells by sorting for cells with high levels of aldehyde
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38–40
dehydrogenase 1 (ALDH 1), a biomarker for breast CSCs.
The isolated cells were grown in low
adhesion plates to form tumorspheres and were subsequently incubated for 3 days with the GAELs. The
viability of the CSCs was determined with the MTS assay.
The results of these studies showed that the triamino GAELs were effective in inhibiting the viability of
the CSCs. The order of potency of these compounds against the BT474 CSCs was similar to what was
previously observed with the unsorted cells; 7 > > 5 > 8, 6 > > > 10 (Figure 5A). Compound 10 and
myristylamine 4 had no effect on the viability of the CSC spheroids at the concentrations examined. It is
worth noting that in spite of differences in the potency of triamino compounds 5 – 8, they all ultimately
reduced the viability of the CSCs to near zero at the highest concentration examined (20 µM).
The activity of the most potent analog 7 was then compared with doxorubicin, salinomycin, and
cisplatin. The results showed that while doxorubicin affected the viability of the CSCs, this compound
was unable to reduce the viability below 20 % despite increase in concentration, whereas the viability of
spheroids incubated with 7 reached almost 2 % viability at about 10 µM (Figure 5B). Cisplatin had very
little effect on the viability of the CSCs and viability was reduced by only 20 % at the highest
concentration tested (20 µM). Its inability to substantially reduce the viability of BT474 CSCs is
4
1
consistent with the characteristic resistance of CSCs to apoptotic cell death, since cisplatin induces
42
apoptosis in cells. Compound 7 also displayed significantly better activity than salinomycin (Figure
43
5
B), a wellꢀdocumented antiꢀCSC experimental agent. Salinomycin has been reported to reduce the
proportion of CSCs by >100ꢀfold relative to paclitaxel, a commonly used breast cancer
4
4
chemotherapeutic drug. The effects of the lead monoamino GAEL 1, diamino 3, and triamino 7 against
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BT474 CSCs show an increasingly better and enhanced cytotoxicity, relative to salinomycin (Figure
C).
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5
The effect of these compounds on the integrity of BT474 CSCs spheroids was also examined. The
results show that after 3 days, the tumor spheres incubated with 7.5 µM of 7 had completely
disintegrated into small amorphous structures while partial disintegration was observed for 5, 6, and 8 at
the same concentrations (Figure 5D). However, at a concentration of 20 µM, where all the compounds
reduced the viability of the CSCs to near zero (Figure 5A), disintegration was complete with 5 and 7
with no difference between the two, while 6 and 8 still showed fairly large amorphous structures (Figure
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D). At this high concentration, 5 and 7 have a similar effect on the integrity of the spheroids. The
presence of the laurylamine in 5 and 7 may explain their superior disintegrative properties compared to 6
and 8 with shorter hydrophobic chains. In contrast, tumor spheres incubated with 7.5 µM of doxorubicin
and salinomycin were still intact, while a higher concentration (20 µM) of salinomycin resulted in
jagged edges, suggesting the onset of disintegration. Spheroids incubated with 4 and 10 were as intact as
the control, although tumor spheres with 20 µM of 4 was not as compact. These results show that the
triamino GAELs retain the intrinsic ability to disintegrate BT474 tumor spheres while compounds 4, 10,
and doxorubicin could not achieve this feat at the highest concentration tested.
Mechanism of Action Studies
Our previous studies showed that mono and diamino GAELs do not induce the activation of caspases
1
4,30
and kill cells by an apoptosisꢀindependent pathway.
act via an apoptosisꢀindependent pathway by assessing the role of caspases in 7ꢀinduced cell death using
the cell permeable panꢀcaspase inhibitor Nꢀ(2ꢀquinolyl)ꢀ ꢀvalylꢀ ꢀaspartylꢀ(2,6ꢀdifluorophenoxy)
We investigated whether triamino GAELs also
L
L
4
5
methylketone 25 (QꢀVDꢀOPh) (Figure S2). When DU145 and JIMT1 cell lines were incubated with
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varying concentrations of doxorubicin or 7, with or without 40 µM of 25, the cytotoxicity of 7 was not
significantly altered (Figure 6A). In contrast, the cytotoxicity of doxorubicin, which induces caspasesꢀ
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dependent apoptosis,
was attenuated in the presence of 25. These results suggest the mechanism of
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cell death initiated by 7 is caspaseꢀindependent and likely similar to that of the mono or diamino GAELs
in being apoptosisꢀindependent.
The membranolytic effect of 7 was also investigated to exclude the possibility that cell death was due to
the effects of 7 on the integrity of the cell membrane. The ability of 7 to perturb the membrane integrity
of DU145 and JIMT1 cells was therefore investigated using membraneꢀimpermeable ethidium
4
9
homodimerꢀ1 dye. The dye emits red fluorescence when it binds to nuclei acids. Incubation of cells
with 6 µM of 7 for 4 h showed noticeable changes in the morphology of the cells under bright field.
However, the cells did not stain red indicating that the membrane was intact. In contrast, cells incubated
with 0.01 % triton Xꢀ100 for 10 min were all rounded up, and stained red (Figure 6B). Compound 7 also
displayed less than 20 % hemolysis of ovine erythrocytes at a concentration that was about fifteen times
the CC value against BT474 cells in vitro (Figure S3). Taken together, these results indicate that 7 is
50
not lytic and cell death is not due to necrosis.
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CONCLUSIONS
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Despite the huge investment in cancer research, several cancers such as brain, ovarian, pancreatic, and
triple negative breast cancer do not have any effective treatments. In addition, the problem of drug
resistance which leads to recurrence of tumors after initial treatments has limited the effectiveness of
current anticancer therapies, leading to growing morbidity and mortality. CSCs, which are refractory to
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51
most current chemotherapeutic agents, are believed to drive tumor growth and generate drugꢀresistant
variants. The elimination of CSCs is key in the fight to find a cure for cancer. GAELs are novel agents
1
2,30,32
that kill cells by a nonꢀapoptotic mechanism, and are also able to kill CSCs.
Our previous studies
that showed a correspondence between amphiphilicity and cytotoxicity of GAELs led to the postulation
that manipulating the balance between these two parameters could enhance the potency of this class of
drugs without changing the mechanism of action.
We conducted SAR studies to explore the effect of an extra amino group with hydrophobic moiety of
different chain lengths attached at different positions of glucosamineꢀderived GAEL 3. The results of
this study show that changes to the amino groups (cationic charge) alone or hydrophobicity alone is
insufficient for optimum activity, but a combination of both, maintained at a certain threshold is needed.
Compound 7 with an additional amino group attached to a C12 moiety at position 6 of the sugar was
identified as the most active GAEL synthesized to date. GAEL 7 showed significant activity against a
range of drugꢀresistant cancer cell lines derived from several tissues, as well as BT474 breast CSCs. The
activity against TNBC lines (≥ 95 % cell kill at 6 ꢀM) is noteworthy as there are no drugs currently
available to treat TNBC. With respect to CSCs, compound 7 was more potent than doxorubicin, cisplatin
and the wellꢀstudied antiꢀCSC agent salinomycin. The mode of action of GAEL 7 was independent of
caspase activation, and it was not due to necrosis.
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The validation of GAELs as a potent agent against TNBC cells and one of the very few agents capable
of killing CSCs, perhaps due to their apoptosisꢀindependent mechanism of action, provides a promising
avenue to develop novel therapeutics that can prevent tumor relapse as well as circumvent resistance to
proꢀapoptotic drugs. The potential of these compounds to overcome the twinꢀproblem of resistance and
tumor relapse represent a viable alternative to expand the chemotherapeutic space in cancer
management.
0
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EXPERIMENTAL SECTION
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Chemistry. All chemicals and reagents were purchased from SigmaꢀAldrich (Oakville, ON, Canada),
except 1ꢀOꢀhexadecylꢀ2ꢀOꢀmethylꢀsnꢀglycerol (17) that was purchased from ChemꢀImplex Inc. (Wood
Dale, IL, USA). The chemicals were all used without further purification. Air and moistureꢀsensitive
reactions were performed under a nitrogen atmosphere with dry solvents. Thinꢀlayer chromatography
0
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(TLC) was carried out on aluminumꢀbacked silica gel 60 F254 GF plates (Merck KGaA, Germany) with
the indicated solvents, and visualized under ultraviolet light and/or charring with 10 % H SO in
2
4
methanol. Compounds were purified by flash chromatography on silica gel 60 (230ꢀ400 ASTM mesh) or
reverseꢀphase C18 silica gel (Silicyle, USA). Yields refer to chromatographyꢀpurified homogenous
1
13
materials, except otherwise stated. H and C NMR spectra were recorded on Bruker AMXꢀ300 and
AMXꢀ500 spectrometers (Germany) as solutions, and reported in the order of chemical shifts (δ) in ppm
relative to the indicated solvent, multiplicity (s, singlet; d, doublet; t, triplet and m, multiplet), number of
1
13
protons, and coupling constants (J) in hertz (Hz). H and C of compounds were assigned based on
Proton, COSY, Carbonꢀ13, DEPTꢀ135 and HSQC experiments. Electrospray ionization mass
spectrometry (ESIꢀMS) on a Varian 500ꢀMS Ion Trap spectrometer (USA) was obtained for all
intermediates while matrix assisted laser desorption ionization (MALDI) coupled with time of flight
(ToF) mass analyzer was performed on a Bruker Daltonics Ultraflex MALDI TOF/TOF mass
spectrometer to characterize the molecular weight of final compounds. Optical rotations of final
®
compounds were measured on an Autopol IV automatic digital polarimeter (Rudolph Research
Analytical, USA). The purity of final compounds as determined by elemental analysis was > 95 %.
Representative Procedure A: Catalytic Hydrogenation for Preparation of Compounds 5 and 6.
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1
-O-Hexadecyl-2-O-methyl-3-O-[2’-N-(12-aminododecyl)-6’-amino-2’,6’-dideoxy-β-D-
glucopyranoside]-sn-glycerol (5): A solution of 20a (0.20 g, 0.28 mmol) in methanol (5.0 ml) was
treated with a catalytic amount of Pd/C catalyst (10 % wt.) and stirred under H gas atmosphere for 1 h.
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0
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The resulting solution was filtered, concentrated in vacuo and purified by reverseꢀphase C18 silica gel
25
using a gradient elution of water and methanol to give 5 (0.14 g, 76 %). [α]D = ꢀ5.2° (c = 0.1,
1
methanol); H NMR (500 MHz, CD OD) δ 4.30 (d, J = 8.1 Hz, 1H, Hꢀ1), 3.91 (dd, J = 10.5, 4.7 Hz,
3
1H), 3.63 (dd, J = 10.5, 4.5 Hz, 1H), 3.70 – 3.50 (m, 2H), 3.50 – 3.40 (m, 6H), 3.34 – 3.32 (m, 1H), 3.26
– 3.23 (m, 1H, Hꢀ3), 3.20 – 3.12 (m, 1H), 3.03 (dd, J = 13.5, 2.7 Hz, 1H), 2.96 – 2.88 (m, 1H), 2.76 –
2.65 (m, 3H), 2.34 (dd, J = 9.8, 8.1 Hz, 1H, Hꢀ2) 1.60 – 1.45 (m, 6H), 1.38 – 1.22 (m, 43H), 0.88 (t, J =
13
6.9 Hz, 3H, terminal CH ); C NMR (126 MHz, CD OD) δ 104.0 (Cꢀ1), 79.2, 76.3, 75.0, 72.2 (Cꢀ3),
3
3
7
2
1.2, 70.2, 68.1, 63.6 (Cꢀ2), 56.7, 49.0, 42.5, 40.5, 31.7, 30.6, 29.6, 29.4, 29.38, 29.36, 29.34, 29.33,
9.31, 29.28, 29.18, 29.12, 29.06, 27.0, 26.5, 25.8, 22.3, 13.1 (terminal CH ). MALDI TOFꢀMS m/e
3
+
calc’d for C H N O K: 712.5606, measured m/e: 712.5598 [M + K]
3
8
79
3
6
1
-O-Hexadecyl-2-O-methyl-3-O-[2’-N-(3-aminopropyl)-6’-amino-2’,6’-dideoxy-
β-D-glucopyranoside]-
sn-glycerol (6): A solution of 20b (0.11 g, 0.18 mmol) was reduced via catalytic hydrogenation and
2
5
1
purified by reverseꢀphase C18 silica gel to give 6 (0.072 g, 72 %). [α]D = ꢀ3.6° (c = 0.1, methanol); H
NMR (300 MHz, CD OD) δ 4.43 (d, J = 8.1 Hz, 1H, Hꢀ1), 3.99 (dd, J = 10.6, 4.2 Hz, 1H), 3.89 (dd, J =
3
1
2
0
1.9, 2.0 Hz, 1H), 3.77 – 3.65 (m, 2H), 3.65 – 3.23 (m, 12H), 3.22 – 3.06 (m, 1H), 3.02 – 2.89 (m, 1H),
.55 (dd, J = 10.4, 8.1 Hz, 1H, Hꢀ2) 1.90 – 1.77 (m, 2H), 1.65 – 1.52 (m, 2H), 1.45 – 1.23 (m, 27H),
13
.91 (t, J = 6.9 Hz, 3H); C NMR (75 MHz, CD OD) δ 104.1 (Cꢀ1), 80.6, 78.2, 75.7, 72.7, 72.0, 71.5,
3
70.0, 64.4, 62.7, 58.2, 33.1, 30.80, 30.77, 30.6, 30.5, 29.4, 27.3, 23.8, 14.5. MALDI TOFꢀMS m/e calc’d
+
for C H N O Na: 570.4458, measured m/e: 570.4461 [M + Na]
2
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Representative Procedure B: Deprotection of Amine (removal of phthalimido group) for Preparation of
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1
1
1
1
1
1
2
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6
Compounds 7 and 8.
1
-O-Hexadecyl-2-O-methyl-3-O-[2’-amino-6’-N-(12-aminododecyl)-2’,6’-dideoxy-β-D-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
glucopyranoside]-sn-glycerol (7): A solution of 22a (0.45 g, 0.53 mmol) in 1ꢀbutanol (5.0 ml) was
treated with ethylenediamine (5.0 ml) and stirred for 3 h at 90 °C. The mixture was concentrated under
high vacuo and purified by flash chromatography (dichloromethane/methanol, 3:1, v/v). The resulting
compound was then reduced by catalytic hydrogenation (following representative procedure A) and
25
1
purified by reverseꢀphase C18 silica gel to afford 7 (0.198g, 55 %). [α]D = ꢀ5.9° (c = 0.1, methanol); H
NMR (500 MHz, CD OD) δ 4.48 (d, J = 8.1 Hz, 1H, Hꢀ1), 3.94 (dd, J = 10.5, 6.7 Hz, 1H), 3.68 (dd, J =
3
10.5, 6.7 Hz, 1H), 3.57 – 3.52 (m, 2H), 3.51 (m, 1H), 3.49 – 3.40 (m, 6H), 3.37 (dd, J = 10.2, 8.8 Hz,
1
H, Hꢀ3), 3.31 – 3.28 (m, 2H), 3.28 – 3.24 (m, 2H), 3.08 – 3.00 (m, 1H), 2.88 – 2.82 (m, 1H), 2.58 (dd,
J = 10.2, 8.1 Hz, 1H, Hꢀ2), 1.62 – 1.52 (m, 6H), 1.41 – 1.24 (m, 43H), 0.89 (t, J = 6.8 Hz, 3H, terminal
1
3
CH ); C NMR (126 MHz, CD OD) δ 101.9 (Cꢀ1), 79.0, 75.9, 73.5 (Cꢀ3), 71.4, 71.3, 70.1, 67.8, 62.7
3
3
(Cꢀ2), 56.7, 51.2, 51.0, 31.69, 31.67, 29.44, 29.39, 29.36, 29.35, 29.33, 29.29, 29.28, 29.25, 29.20,
29.14, 29.08, 28.89, 28.5, 28.3, 26.7, 26.4, 25.8, 22.3, 13.1 (terminal CH ). MALDI TOFꢀMS m/e calc’d
3
+
for C H N O Na: 696.5867, measured m/e: 696.5871 [M + Na]
3
8
79
3
6
1
-O-Hexadecyl-2-O-methyl-3-O-[2’-amino-6’-N-(3-aminopropyl)-2’,6’-dideoxy-β-D-glucopyranoside]-
sn-glycerol (8): Compound 22b (0.17 g, 0.24 mmol) was treated with ethylenediamine and purified by
flash chromatography (dichloromethane/methanol, 3:1, v/v). The resulting compound was subsequently
reduced by catalytic hydrogenation and purified by reverseꢀphase C18 silica gel to give 8 (0.064g, 45.5
2
5
1
%). [α]D = ꢀ2.8° (c = 0.1, methanol); H NMR (500 MHz, CD OD) δ 4.23 (d, J = 8.0 Hz, 1H, Hꢀ1),
3
3
.90 (dd, J = 10.5, 4.9 Hz, 1H), 3.66 (dd, J = 10.5, 3.9 Hz, 1H), 3.61 – 3.50 (m, 2H), 3.50 – 3.42 (m,
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9
1
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1
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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6
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H), 3.40 – 3.38 (m, 4H), 3.26 (dd, J = 10.0, 8.7 Hz, 1H), 3.15 – 3.09 (m, 1H), 2.94 (dd, J = 14.2, 2.4
Hz, 1H), 2.68 – 2.55 (m, 4H), 1.83 – 1.68 (m, 3H), 1.61 – 1.51 (m, 2H), 1.39 – 1.27 (m, 26H), 0.89 (t, J
13
=
6.8 Hz, 3H); C NMR (126 MHz, CD OD) δ 102.5 (Cꢀ1), 79.0, 75.7, 74.5, 72.6, 71.2, 69.8, 68.4,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6.7, 54.7, 51.4, 49.1, 31.7, 29.4, 29.34, 29.26, 29.19, 29.06, 26.3, 25.8, 22.3, 13.0. MALDI TOFꢀMS
+
m/e calc’d for C H N O : 548.4697, measured m/e: 548.4701 [M + H]
2
9
62
3
6
1
-O-Hexadecyl-2-O-methyl-3-O-[6’-N-(12-N-chlorambucil dodecyl)-2’,6’-diamino-2’,6’-dideoxy-β-D-
glucopyranoside]-sn-glycerol.2TFA (9): A solution of 24 (0.10 g, 0.086 mmol) in DCM (5.0 ml) was
treated with trifluoroacetic acid (2.0 ml) and stirred for 1 h. The reaction was then concentrated in vacuo
and purified by flash chromatography (dichloromethane/methanol, 5:1, v/v) to give compound 9 (0.07g,
25
1
85 %). [α]D = 7.3° (c = 0.1, methanol); H NMR (500 MHz, CD OD) δ 7.09 – 7.01 (m, 2H), 6.71 –
3
6
.64 (m, 2H), 4.66 (d, J = 8.4 Hz, 1H, Hꢀ1), 4.01 (dd, J = 10.6, 4.9 Hz, 1H), 3.77 (dd, J = 10.7, 3.0 Hz,
1
H), 3.74 – 3.44 (m, 18H), 3.27 – 3.11 (m, 4H), 3.09 – 3.04 (m, 2H), 2.99 – 2.85 (m, 1H), 2.51 (m, 2H),
2.16 (t, J = 7.6 Hz, 2H), 1.90 – 1.80 (m, 2H), 1.74 – 1.65 (m, 2H), 1.56 (m, 2H), 1.48 (t, J = 6.8 Hz, 2H),
13
1
1
.29 (m, 42H), 0.89 (t, J = 6.9 Hz, 3H); C NMR (126 MHz, CD OD) δ 174.5 (Cꢀamide), 144.6, 130.4,
3
29.2, 112.1, 99.0 (Cꢀ1), 78.8, 72.2, 72.1, 71.9, 71.4, 69.4, 69.3, 57.0, 55.8, 53.2, 40.3, 38.9, 35.1, 33.8,
31.6, 29.35, 29.34, 29.31, 29.24, 29.22, 29.19, 29.17, 29.06, 29.04, 28.99, 28.95, 28.8, 27.6, 26.6, 26.2,
2
9
5.8, 25.7, 22.3, 13.0. MALDI TOFꢀMS m/e calc’d for C H Cl N O Na: 981.6554, measured m/e:
52 96 2 4 7
+
81.6559 [M + Na]
1
-(12-aminododecyl)-2,6-diamino-2,6-dideoxy β-D-glucopyranoside (10): Compound 13 (0.11 g, 0.18
mmol) was deprotected following representative procedure B, and the crude was purified by flash
chromatography (dichloromethane/ methanol, 7:1, v/v) to afford 1ꢀ(12ꢀazidododecyl)ꢀ2,6ꢀdiaminoꢀ2,6ꢀ
dideoxy glucopyranoside (0.049 g, 67 %). This compound was then treated with catalytic amount of
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Pd/C, following representative procedure A, and filtered from the Pd catalyst. The crude was
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9
1
1
1
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1
1
1
1
1
1
2
2
2
2
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5
6
concentrated in vacuo and purified by reverseꢀphase C18 silica gel to give compound 10 (0.032 g, 80
2
5
1
%
). [α]D = 36.6° (c = 0.1, methanol); H NMR (300 MHz, CD OD) δ 4.28 (d, J = 7.9 Hz, 1H, Hꢀ1),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
.96 – 3.85 (m, 1H), 3.61 – 3.50 (m, 1H), 3.50 – 3.40 (m, 3H), 3.36 ꢀ3.20 (m, 4H), 2.70 – 2.55 (m, 1H,
13
Hꢀ2), 1.73 – 1.52 (m, 4H), 1.48 – 1.28 (m, 16H); C NMR (75 MHz, CD OD) δ 104.4 (Cꢀ1), 77.4, 77.2,
3
7
2.8, 70.8, 58.4, 52.8, 52.5, 30.8, 30.7, 30.66, 30.65, 30.5, 30.3, 30.0, 27.9, 27.2. MALDI TOFꢀMS m/e
+
calc’d for C H N O Na: 400.2578, measured m/e: 400.2581 [M + Na]
1
8
39
3
4
Phenyl 3,4-di-O-acetoxy-6-deoxy-6-azido-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (12):
Compound 12 was prepared from a commercially available glucosamine hydrochloride (11) as
3
1,32
previously reported and NMR data were consistent with earlier reports.
Representative Procedure C. Glycosylation Reaction.
1
-(12-azidododecyl)-6-azido-6-deoxy-3,4-di-O-acetoxy-2-deoxy-2-N-phthalimido
13): A solution of the thioglycoside donor 12 (0.15 g, 0.29 mmol), 12ꢀazidododecanol (glycoside
acceptor 15a) (0.08 g, 0.35 mmol) and Nꢀiodosuccinimide (NIS) (0.10g, 0.44 mmol) in dry CH Cl (15.0
β-D-glucopyranoside
(
2
2
ml) were treated with AgOTf (0.011 g, 0.044 mmol) and stirred for 3 h under nitrogen gas. The
insoluble NIS was filtered using celite and the filtrate washed with Na S O (×2), NaHCO (×3), H O
2
2
3
3
2
(×1) and saturated brine (×1) successively. The organic layer was dried over anhydrous Na SO ,
2
4
concentrated in vacuo and purified by flash chromatography (hexanes/ ethyl acetate, 3:1, v/v) to give
1
(
13) (0.12 g, 66 %). H NMR (300 MHz, CDCl ) δ 7.90 – 7.65 (m, 4H), 5.77 (dd, J = 10.8, 9.0 Hz, 1H,
3
Hꢀ3), 5.37 (d, J = 8.5 Hz, 1H, Hꢀ1), 5.02 (dd, J = 10.1, 9.0 Hz, 1H, Hꢀ4), 4.29 (dd, J = 10.8, 8.5 Hz, 1H,
Hꢀ2), 4.15 – 3.96 (m, 2H), 3.90 – 3.77 (m, 2H), 3.50 – 3.38 (m, 1H), 3.26 – 3.15 (m, 2H), 2.01 (s, 6H),
1
3
1
.63 – 1.50 (m, 4H), 1.48 – 1.28 (m, 16H); C NMR (75 MHz, CDCl ) δ 171.1, 169.6, 134.3, 131.4,
3
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23.5, 97.9 (Cꢀ1), 73.6, 70.6, 70.4, 70.0, 64.6, 54.7, 51.4, 51.2, 29.5, 29.4, 29.3, 29.1, 28.8, 26.7, 25.9,
+
+
2
5.7, 21.0, 20.4, 14.2. ESIꢀMS: m/z [M + Na] calc’d for C H N O Na : 650.80, found: 650.8
30 41
7
8
NꢀAzidoalkanals (16a and 16b): Commercially available 12ꢀbromoꢀ1ꢀdodecanol 14a (0.20 g, 0.75
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
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8
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0
1
2
3
4
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6
7
8
9
0
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2
3
4
5
6
7
8
9
0
mmol) was dissolved in dry DMF (5.0 ml) and treated with NaN (0.49 g, 7.54 mmol) at 70 °C for 3 h.
3
The crude product was concentrated under vacuo, worked up with H O (×2) and brine (×1) successively,
2
and reꢀconcentrated to give 12ꢀazidoꢀ1ꢀdodecanol 15a in excellent yield. This compound was
subsequently dissolved in dry DCM (5.0 ml) and treated with pyridinium chlorochromate PCC (0.48 g,
2
.25 mmol) for 2 h. The reaction was monitored with TLC using KMnO stain. The mixture was filtered
4
through a pad of silica and concentrated under low vacuo to give 12ꢀazidododecanal 16a. Compound
16b was also prepared from 3ꢀbromoꢀ1ꢀpropanol 14b following the same procedure. The resulting
compounds were used immediately without further purification.
1
-O-Hexadecyl-2-O-methyl-3-O-(6’-azido-6’-deoxy-3’,4’-di-O-acetoxy-2’-deoxy-2’-N-phthalimido-
glucopyranoside)-sn-glycerol (18): Thioglycoside donor 12 (0.58 g, 1.14 mmol) was glycosylated with
ꢀOꢀhexadecylꢀ2ꢀOꢀmethylꢀsnꢀglycerol (17) (0.45 g, 1.36 mmol) as described in representative
β-D-
1
procedure C, and purified by flash chromatography (hexanes/ethyl acetate, 3:2, v/v) to afford 18 (0.72 g,
1
7
0 %). H NMR (300 MHz, CDCl ) δ 7.90 – 7.70 (m, 4H), 5.85 (dd, J = 10.8, 8.9 Hz, 1H, Hꢀ3), 5.39 (d,
3
J = 8.4 Hz, 1H, Hꢀ1), 5.06 (dd, J = 9.5 Hz, 1H, Hꢀ4), 4.33 (dd, J = 10.8, 8.4 Hz, 1H, Hꢀ2), 3.96 – 3.82
(m, 1H), 3.62 (dd, J = 10.7, 5.0 Hz, 1H), 3.57 – 3.40 (m, 4H), 3.37 – 3.05 (m, 7H), 2.04 (s, 3H, ꢀ
13
COCH ), 1.87 (s, 3H, ꢀCOCH ), 1.39 (m, 2H), 1.26 (s, 26H), 0.88 (t, J = 6.9 Hz, 3H, CH CH ); C
3
3
2
3
NMR (75 MHz, CDCl ) δ 171.2, 170.1, 134.2, 130.9, 123.5, 98.5 (Cꢀ1), 79.9, 78.6, 73.6, 71.9, 71.6,
3
70.6, 70.4, 69.8, 68.7, 62.7, 57.6, 54.6, 51.2, 31.9, 29.7, 29.65, 29.63, 29.60, 29.5, 29.4, 26.1, 26.0, 22.7,
+
+
2
0.6, 20.5, 14.1. ESIꢀMS: m/z [M + Na] calc’d for C H N O Na : 753.41, found: 753.4.
38 58 4 10
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-O-Hexadecyl-2-O-methyl-3-O-(2’-amino-6’-azido-2’,6’-dideoxy-β-D-glucopyranoside)-sn-glycerol
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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3
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4
4
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4
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4
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5
5
5
5
5
5
5
5
6
(19): Compound 18 (0.070 g, 0.096 mmol) was deprotected in a single step following representative
procedure B, and purified by flash chromatography (dichloromethane/methanol, 4:1, v/v) to give 19
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(
0.036 g, 72.8 %). H NMR (300 MHz, CD OD) δ 4.26 (d, J = 8.0 Hz, 1H, Hꢀ1), 3.91 (dd, J = 9.1, 4.5
3
Hz, 1H), 3.68 (dd, J = 10.6, 4.5 Hz, 1H), 3.61 – 3.51 (m, 3H), 3.51 – 3.37 (m, 8H), 3.30 – 3.21 (m, 2H),
.60 (dd, J = 8.0, 3.8 Hz, 1H, Hꢀ2), 1.60 1.55 (m, 2H), 1.30 – 1.27 (m, 26H), 0.89 (t, J = 6.7 Hz, 3H, ꢀ
2
1
3
CH CH ); C NMR (75 MHz, CD OD) δ 103.2 (Cꢀ1), 79.0, 76.0, 75.8, 71.2, 70.0, 68.0, 56.9, 56.6,
2
3
3
+
+
5
5
1.3, 31.7, 29.4, 29.3, 29.2, 29.1, 25.8, 22.3, 13.0. ESIꢀMS: m/z [M + H] calc’d for C H N O :
26 53 4 6
16.39, found: 516.4.
Representative Procedure D. Reductive Amination.
1
-O-Hexadecyl-2-O-methyl-3-O-[2’-N-(12-azidododecyl)-6’-azido-2’6’-dideoxy-β-D-glucopyranoside]-
sn-glycerol (20a): A solution of 19 (0.20 g, 0.39 mmol) in dry DCE was treated with 12ꢀazidododecanal
(16a) (0.088 g, 0.39 mmol) and two drops of acetic acid. The reaction was stirred at RT for 6 h under
nitrogen gas. Sodium borohydride, NaBH (0.045 g, 1.16 mmol) was then added to the mixture and
4
stirred overnight at 0 °C to RT. The resulting mixture was quenched with saturated sodium bicarbonate,
extracted with DCM (×3), concentrated in vacuo, and purified by flash chromatography (hexanes/ethyl
acetate, 2:1 to 100% ethyl acetate, then dichloromethane/methanol, 20:1, v/v) to afford 20a (0.20 g, 69
1
%
). H NMR (300 MHz, CD OD) δ 4.60 (d, J = 8.1 Hz, 1H, Hꢀ1), 3.98 (dd, J = 8.9, 4.2 Hz, 1H), 3.74
3
(dd, J = 10.5, 3.8 Hz, 1H), 3.64 – 3.38 (m, 11H), 3.36 – 3.24 (m, 4H), 3.18 – 3.04 (m, 1H), 3.02 – 2.90
(m, 1H), 2.69 (dd, J = 10.2, 8.1 Hz, 1H, Hꢀ2), 1.72 – 1.50 (m, 6H), 1.48 – 1.28 (m, 42H), 0.88 (t, J = 6.9
1
3
Hz, 3H); C NMR (75 MHz, CD OD) δ 102.7 (Cꢀ1), 80.4, 77.4, 74.6, 72.9, 72.8, 71.5, 69.4, 64.0, 58.2,
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2
2
2
2
2
2
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5
2.7, 52.5, 33.2, 30.9, 30.86, 30.76, 30.71, 30.6, 30.4, 30.0, 29.3, 28.1, 27.9, 27.3, 23.8, 14.6. ESIꢀMS:
+
+
m/z [M + H] calc’d for C H N O H : 726.58, found: 726.7
38 75
7
6
1
-O-Hexadecyl-2-O-methyl-3-O-[2’-N-(3-azidopropyl)-6’-azido-2’,6’-dideoxy-glucopyranoside]-sn-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
glycerol (20b): A solution of 19 (0.15 g, 0.29 mmol) was treated with 3ꢀazidopropanal (16b) (0.03 g,
0.30 mmol) via reductive amination (representative procedure D) and purified by flash chromatography
1
(dichloromethane/methanol, 7:1, v/v) to afford 20b (0.11 g, 64 %). H NMR (300 MHz, CD OD) δ 4.43
3
(d, J = 8.1 Hz, 1H, Hꢀ1), 3.99 (dd, J = 10.6, 4.2 Hz, 1H), 3.89 (dd, J = 11.9, 2.0 Hz, 1H), 3.77 – 3.65 (m,
2H), 3.65 – 3.23 (m, 13H), 3.22 – 3.06 (m, 1H), 3.02 – 2.89 (m, 1H), 2.55 (dd, J = 10.4, 8.1 Hz, 1H, Hꢀ
13
2
) 1.90 – 1.77 (m, 2H), 1.65 – 1.52 (m, 2H), 1.45 – 1.23 (m, 26H), 0.91 (t, J = 6.9 Hz, 3H); C NMR
(75 MHz, CD OD) δ 104.1 (Cꢀ1), 80.6, 78.2, 75.7, 72.7, 72.0, 71.5, 69.7, 64.4, 62.7, 58.2, 33.1, 30.80,
3
+
+
3
0.77, 30.6, 30.5, 29.4, 27.3, 23.8, 14.5. ESIꢀMS: m/z [M + Na] calc’d for C H N O Na : 622.43,
29 57 7 6
found: 622.5
1
-O-hexadecyl-2-O-methyl-3-O-(2’-N-phthalimido-6’-amino-2’,6’-dideoxy-β-D-glucopyranoside)-sn-
glycerol (21): Compound 18 (0.62 g, 0.85 mmol) was dissolved in a solution of sodium methoxide (0.05
g, 0.93 mmol) in methanol (20.0 ml) and stirred for 25 mins. The reaction was monitored with TLC and
quenched with a catalytic amount of Dowex 50WX2 (50ꢀ100 mesh) ion exchange resin. The resulting
mixture was filtered by suction, concentrated in vacuo and purified by flash chromatography
(hexanes/ethyl acetate, 1:1, v/v). This was then reduced by catalytic hydrogenation (following
representative procedure A) and purified by flash chromatography (dichloromethane/methanol, 6:1, v/v)
1
to yield 21 (0.282 g, 55 %). H NMR (500 MHz, CD OD) δ 7.95 – 7.68 (m, 4H), 5.25 (d, J = 8.5 Hz,
3
1
1
H, Hꢀ1), 4.30 (dd, J = 10.4, 9.5 Hz, 1H) 4.04 – 3.83 (m, 3H), 3.59 – 3.29 (m, 4H), 3.28 – 3.09 (m, 8H),
13
.45 – 1.35 (m, 2H), 1.35 – 1.15 (m, 26H), 0.90 (t, J = 6.9 Hz, 3H, ꢀCH CH ); C NMR (126 MHz,
2
3
CD OD) δ 170.1, 134.1, 122.8, 99.0 (Cꢀ1), 78.8, 76.3, 72.8, 71.1, 70.8, 69.4, 67.9, 57.2, 56.4, 42.3, 31.7,
3
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+
+
2
6
9.4, 29.3, 29.12, 29.10, 29.05, 25.7, 22.3, 13.0. ESIꢀMS: m/z [M + Na] calc’d for C H N O Na :
34 56 2 8
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
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5
5
5
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6
43.39, found: 644.4.
1-O-Hexadecyl-2-O-methyl-3-O-[2’-N-pthalimindo-6’-N-(12-azidododecyl)-2’,6’-β-D-dideoxy-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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4
5
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7
8
9
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5
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7
8
9
0
1
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4
5
6
7
8
9
0
glucopyranoside]-sn-glycerol (22a): A solution of compound 21 (0.45 g, 0.73 mmol) in DCM was
treated with 12ꢀazidododecanal (16a) (0.17 g, 0.73 mmol) via reductive amination (representative
procedure D) and purified by flash chromatography (dichloromethane/methanol, 9:1, v/v) to afford 22a
1
(
0.46 g, 73 %). H NMR (500 MHz, CD OD) δ 7.92 – 7.78 (m, 4H), 5.21 (d, J = 8.5 Hz, 1H, Hꢀ1), 4.32
3
(dd, J = 10.8, 8.6 Hz, 1H, Hꢀ3), 4.00 (dd, J = 10.8, 8.5 Hz, 1H, Hꢀ2), 3.84 – 3.72 (m, 1H), 3.63 – 3.53
(
m, 1H), 3.50 – 3.38 (m, 3H), 3.30 – 3.23 (m, 5H), 3.23 – 3.09 (m, 6H), 3.07 – 3.01 (m, 2H), 1.87 – 1.80
1
3
(
m, 2H), 1.73 – 1.66 (m, 2H), 1.50 – 1.17 (m, 44H), 0.89 (t, J = 6.9 Hz, 3H); C NMR (126 MHz,
CD OD) δ 134.1, 134.0, 122.7, 98.8 (Cꢀ1), 78.6, 73.2, 71.4, 71.1, 70.6, 69.0, 68.5, 56.7, 56.5, 54.4, 53.4,
3
3
2
8
3.0, 32.6, 31.7, 29.38, 29.35, 29.33, 29.30, 29.22, 29.20, 29.18, 29.17, 29.15, 29.10, 29.06, 29.05,
+
+
8.98, 28.4, 27.8, 26.5, 25.8, 24.4, 22.3, 13.0. ESIꢀMS: m/z [M + Na] calc’d for C H N O Na :
46 79
5
6
53.09, found: 853.1
1
-O-Hexadecyl-2-O-methyl-3-O-[2’-N-pthalimido-6’-N-(3-azidopropyl)-2’,6’-dideoxy-β-D-
glucopyranoside]-sn-glycerol (22b): A solution of 21 (0.21 g, 0.34 mmol) in DCM was treated with 3ꢀ
azidopropanal (16b) (0.035 g, 0.34 mmol) via reductive amination (representative procedure D) and
purified by flash chromatography (dichloromethane/methanol, 10:1, v/v) to afford 22b (0.17 g, 70 %).
1
H NMR (500 MHz, CD OD) δ 7.92 – 7.78 (m, 4H), 5.17 (d, J = 8.0 Hz, 1H, Hꢀ1), 4.33 (dd, J = 10.5,
3
4
.9 Hz, 1H), 3.90 (dd, J = 10.5, 3.9 Hz, 1H), 3.61 – 3.42 (m, 7H), 3.38 (t, J = 6.7 Hz, 4H), 3.26 (dd, J =
10.0, 8.7 Hz, 1H), 3.15 – 3.09 (m, 1H), 2.94 (dd, J = 14.2, 2.4 Hz, 1H), 2.68 – 2.55 (m, 5H), 1.83 – 1.68
1
3
(
m, 3H), 1.61 – 1.51 (m, 2H), 1.39 – 1.27 (m, 26H), 0.89 (t, J = 6.8 Hz, 3H); C NMR (126 MHz,
CD OD) δ 134.42, 133.93, 122.0, 97.8 (Cꢀ1), 77.0, 73.4, 70.6, 70.4, 70.2, 69.3, 68.0, 55.9, 56.4, 54.4,
3
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3.4, 31.5, 30.4, 29.25, 29.23, 29.19, 29.17, 29.15, 29.10, 29.06, 29.05, 28.98, 28.4, 27.8, 26.5, 25.7,
+
+
4.5, 22.3, 13.0. ESIꢀMS: m/z [M + Na] calc’d for C H N O Na : 726.44, found: 726.5
37 61
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-O-Hexadecyl-2-O-methyl-3-O-[6’-N-Boc-(12-azidododecyl)-2’-N-boc-2’,6’-dideoxy-β-D-
glucopyranoside]-sn-glycerol (23): Compound 22a (0.45 g, 0.53 mmol) was deprotected following
representative procedure B and purified by flash chromatography (dichloromethane/methanol, 3:1, v/v).
This compound was then treated with Boc O (0.23 g, 1.05 mmol) and Et N (0.2 ml, 1.44 mmol) in
2
3
methanol, and stirred at 50 °C for 5 h. The resulting mixture was concentrated in vacuo and purified by
1
flash chromatography (hexanes/ethyl acetate, 1:1, v/v) to afford 23 (0.21 g, 51.2 %). H NMR (500
MHz, CD OD) δ 4.28 (d, J = 8.1 Hz, 1H, Hꢀ1), 3.81 (dd, J = 10.5, 4.6 Hz, 1H), 3.64 – 3.52 (m, 3H),
3
3
1
1
5
2
.50 – 3.40 (m, 8H), 3.39 – 3.32 (m, 5H), 3.29 – 3.25 (m, 2H), 3.18 – 3.08 (m, 1H), 1.63 – 1.51 (m, 6H),
13
.45 (s, 18H, boc), 1.41 – 1.24 (m, 42H), 0.90 (t, J = 6.9 Hz, 3H); C NMR (126 MHz, CD OD) δ
3
57.0, 156.8, 128.5, 102.1 (Cꢀ1), 79.8, 79.5, 79.1, 78.57, 78.53, 75.3, 73.8, 72.2, 71.3, 70.1, 68.0, 56.9,
1.1, 36.2, 31.7, 29.41, 29.38, 29.31, 29.29, 29.27, 29.23, 29.1, 28.9, 28.5, 27.5, 27.4, 26.6, 26.5, 25.9,
+
+
4.2, 22.3, 13.1. ESIꢀMS: m/z [M + Na] calc’d for C H N O Na : 922.68, found: 922.8
4
8
93
5
10
1-O-Hexadecyl-2-O-methyl-3-O-[6’-N-Boc-(12-N-chlorambucil dodecyl)-2’-N-boc-2’,6’-dideoxy-β-D-
glucopyranoside]-sn-glycerol (24): A solution of chlorambucil (0.067 g, 0.22 mmol) and
diisopropylethylamine (0.2 ml) in dry DMF (5.0 ml) was preꢀactivated with TBTU (0.07 g, 0.22 mmol).
In a separate flask, compound 23 (0.21 g, 0.23 mmol) was reduced by catalytic hydrogenation following
representative procedure A, concentrated, and added to the solution containing the preꢀactivated
chlorambucil. This was stirred for 5 h and purified by flash chromatography (hexanes/ethyl acetate, 1:1,
1
v/v) to yield 24 (0.15 g, 55.6 %). H NMR (500 MHz, CD OD) δ 7.09 – 7.01 (m, 2H), 6.71 – 6.64 (m,
3
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H), 4.66 (d, J = 8.4 Hz, 1H, Hꢀ1), 4.01 (dd, J = 10.6, 4.9 Hz, 1H), 3.77 (dd, J = 10.7, 3.0 Hz, 1H), 3.74
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
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2
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3.44 (m, 18H), 3.27 – 3.11 (m, 4H), 3.09 – 3.04 (m, 2H), 2.99 – 2.85 (m, 1H), 2.51 (t, J = 7.6 Hz, 1H),
.16 (t, J = 7.6 Hz, 2H), 1.90 – 1.80 (m, 2H), 1.74 – 1.65 (m, 2H), 1.56 (t, J = 6.8 Hz, 2H), 1.48 (t, J =
0
1
2
3
4
5
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8
9
0
1
2
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4
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8
9
0
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9
0
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0
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9
0
13
.8 Hz, 2H), 1.45 (s, 18H, Boc), 1.29 (m, 44H), 0.89 (t, J = 6.9 Hz, 3H); C NMR (126 MHz, CD OD)
3
δ 174.5 (Cꢀamide), 157.0, 156.8, 144.6, 130.4, 129.2, 128.5, 112.1, 98.8 (Cꢀ1), 78.6, 72.1, 72.0, 71.8,
7
2
1.3, 69.4, 69.3, 57.0, 55.8, 53.2, 40.3, 38.9, 35.1, 33.8, 31.6, 29.35, 29.34, 29.31, 29.24, 29.22, 29.19,
+
9.17, 29.06, 29.04, 28.99, 28.95, 28.8, 27.6, 26.6, 26.2, 25.8, 25.7, 22.3, 13.0. ESIꢀMS: m/z [M + H]
+
calc’d for C H Cl N O Na : 1181.76, found: 1181.8
6
2
112
2
4
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Biology. The cytotoxic effects of compounds 1, 5 – 10 against a panel of human epithelial cell lines
3
2
were assessed using the MTS assay. The cell lines were derived from cancers of the breast (JIMT1,
BT474, MDAꢀMBꢀ231, MDAꢀMBꢀ453, MDAꢀMBꢀ468, BT549, Hs578t), pancreas (MiaPaCa2),
prostate (DU145, PC3), brain (U87, U251) and ovaries (A2780s, A2780cp). Exponentially growing cells
were treated with test compounds and then incubated for 48 h. All compounds were tested up to 20 µM
and 7 reꢀtested at much lower concentrations (Figure 4). The results for compounds 1, 5 – 10 are shown
in figure 3 and the CC values are displayed in Tables 1 and 2, along with the values obtained for 1 and
0
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4
4 from previous studies.
Cell Culture
MDAꢀMBꢀ231, BT474, MiaPaCa2, DU145 and PC3 cell lines were grown from frozen stocks of cell
lines that were originally obtained from ATCC (Manassas, VA, USA). JIMT1 cells were grown from
frozen stocks of cells obtained from DSMZ (Braunschweig, Germany). MDAꢀMBꢀ231, JIMT1 and
DU145 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM), PC3 cells grown in F12K
medium while BT474 cells were grown in Hybricare medium (ATCC). MiaPaCa2 was grown in DMEM
supplemented with FBS to a concentration of 10 % and horse serum to a final concentration of 2.5 %.
The cells were grown in media supplemented with 10 % fetal bovine serum (FBS), penicillin (100 U/ml)
and streptomycin (0.1 mg/ml) in a humidified 5 % atmospheric incubator at 37 ºC.
Isolation of ALDH-positive breast cancer cell population
The ALDEFLUOR assay kit (STEMCELL Technologies, Vancouver, BC, Canada) was used to isolate a
CSC enriched cell population from BT474 cells according manufacturer’s instructions, with nonꢀstained
cells and cells stained in the presence of ALDH1 inhibitor (diethylaminobenzaldehyde, DEAB) as
controls for the sorting. The optimum time for staining at 37 °C was determined to be 45 mins for
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BT474 cells. Following the staining, ALDH1ꢀstained cells were sorted from the bulk population by flow
cytometery on a 4ꢀlaser MoFloXPP high speed/pressure cell sorter. The sorted cells were pelleted by
centrifugation, resuspended in supplemented Mammocult medium (STEMCELL Technologies) and
dispersed into ultraꢀlow adhesive 6ꢀwell plates or 35 mm dishes for 7 days to allow the formation of
spheroids (mammospheres). After 7 days, the spheres were separated from the media and single cells by
sieving the contents of the well through a 40 µm nylon cell strainer (BD Falcon). The spheres retained in
the sieve were washed with Hanks buffer and subsequently trypsinised to obtain a single cell suspension
of CSCs. The cell numbers were determined with a Coulter ZM counter and the cells were seeded into
the appropriate low adhesion tissue culture ware for various studies.
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Cytotoxicity Assay
Cell viability was determined with the CellTitre 96 Aqueous One solution (MTS assay kit; Promega).
Equal numbers of cancer cells (7500 – 9000) in media (100 µL) were dispersed into 96ꢀwell plates. As
blanks, media without cells (100 µL) were also placed in some wells and treated similarly to the cellꢀ
containing wells. After an incubation period of 24 h, a solution of test compound (100 µL) in medium at
twice the desired concentration was added to each well. The treated cells were then incubated further for
4
8 h, after which 20 % v/v MTS reagent, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ
(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium was added to each well. The plates were then incubated for 1 – 4 h on a
Nutator mixer in a 5 % CO incubator. The optical density (OD) was read at 490 nm on a SpectraMax
2
M2 plate reader (Molecular Devices Corp., Sunnyvale, CA, USA). The values of blank were subtracted
from each value and the viability values of the treated samples relative to the controls with vehicle were
calculated. The values for the plots are the means ± standard deviation. Values of zero indicate there are
no viable cells in the wells.
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