Synthetic Sphingolipids with 1,2-Pyridazine Appendages Improve Antiproliferative Activity in Human Cancer Cell Lines

Article abstract of DOI:10.1021/acsmedchemlett.9b00553

A synthetic sphingolipid related to a ring-constrained hydroxymethyl pyrrolidine analog of FTY720 that was known to starve cancer cells to death was chemically modified to include a series of alkoxy-tethered 3,6-substituted 1,2-pyridazines. These derivati

Full text of DOI:10.1021/acsmedchemlett.9b00553

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Letter
Synthetic sphingolipids with 1,2-pyridazine appendages
improve anti-proliferative activity in human cancer cell lines
Sylvestre P. J. T. Bachollet, Vito Vece, Alison N. McCracken, Brendan T. Finicle, Elizabeth Selwan,
Nadine Ben Romdhane, Amogha Dahal, Cuauhtemoc Ramirez, Aimee L. Edinger, and Stephen Hanessian
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00553 • Publication Date (Web): 12 Feb 2020
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Synthetic sphingolipids with 1,2-pyridazine appendages improve
anti-proliferative activity in human cancer cell lines
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Sylvestre P. J. T. Bachollet,†,# Vito Vece,†,# Alison N. McCracken,‡,# Brendan T. Finicle,‡ Elizabeth
Selwan,‡ Nadine Ben Romdhane,‡ Amogha Dahal,‡ Cuauhtemoc Ramirez,‡ Aimee L. Edinger*,‡ and
Stephen Hanessian*,†
† Department of Chemistry, Université de Montréal, P.O. Box 6128, Station Centre-Ville, Montréal, Quebec H3C 3J7,
Canada
‡ Department of Developmental and Cell Biology, University of California, Irvine, 2128 Natural Sciences 1, California
92697-2300,United States
KEYWORDS Sphingolipid, anti-cancer, KRAS mutant cancer, nutrient transporter, vacuolation.
ABSTRACT. A synthetic sphingolipid related to a ring-constrained hydroxymethyl pyrrolidine analog of FTY720 that was known
to starve cancer cells to death was chemically modified to include a series of alkoxy tethered 3,6- substituted 1,2-pyridazines. These
derivatives exhibited excellent anti-proliferative activity against 8 human cancer cell lines from 4 different cancer types. A 2.5-9
fold reduction in IC50 in these cell lines was observed relative to the lead compound which lacked the appended heterocycle.
Figure 1. Structures of anti-proliferative synthetic compounds
related to sphingosine (1 – 3); 3-pyridyl appended analog of 3 (4).
IC50 in FL5.12 cells shown.
Sphingolipids are a family of evolutionarily-conserved natural
compounds that play essential roles in the life cycle of cells.1–3
A subset of sphingolipids, such as phytosphingosine, slow cell
growth, induce differentiation, and trigger cell death (Figure 1,
compound 1). As part of the adaptive response to stress,
compound 1 down-regulates nutrient transporters in yeast,
reducing access to amino acids and uracil and inducing a
stress-resistant quiescent state.4 Although mammalian cells do
not produce phytosphingosine, the sphingolipid ceramide, a
4,5-unsaturated congener, has a similar effect on mammalian
nutrient transporters for amino acids and glucose.5,6 While
limiting nutrient access produces adaptive quiescence in
normal cells, the oncogenic mutations in cancer cells
constitutively drive growth, rendering transformed cells
hypersensitive to interruptions in the nutrient supply.7,8 Indeed,
many standard of care chemotherapies target biosynthetic
pathways and cancer therapies that disrupt anabolism are
under development.9
Our group has developed synthetic sphingolipid analogs that
recapitulate the ability of natural sphingolipids to starve cancer
cells to death.8,10,11 Being cognizant of the challenges
associated with using a natural molecule like ceramide as a
drug (e.g. poor solubility and rapid metabolism), we turned to
the synthetic sphingolipid FTY720 (Fingolimod). At higher
doses than are required to affect sphingosine-1-phosphate
receptors, FTY720 limits nutrient access and starves cancer
cells to death.10,11 In addition to reducing the levels of glucose
and amino acid transporters on the cell surface, FTY720 also
disrupts lysosomal fusion reactions.6,11 This disruption,
visualized as cellular vacuolation, additionally limits access to
nutrients acquired from the digestion of LDL particles,
autophagosomes, and macropinosomes.8,11,12 Despite its
activity in multiple pre-clinical cancer models, FTY720 cannot
be repurposed as a cancer therapy because the phosphorylated
form triggers severe bradycardia at the doses required to kill
neoplastic cells.10,13–16
OH
C14H29
OH
OH
OH NH2
NH2
HO
We have reported that the synthetic sphingolipid compound 3
(SH-BC-893), a constrained variant of FTY720, exhibits
favorable activity as an anti-cancer agent without the negative
cardiovascular effects associated with FTY720.11,16,17 Like
FTY720, compound 3 triggers the internalization of nutrient
transporters and blocks lysosome-dependent nutrient
generation pathways.6,11 Compound 3 significantly inhibits the
growth of colon cancer xenografts and autochthonous prostate
tumors in a genetically modified mouse model.11 Importantly,
compound 3 affects both normal and transformed cells, but is
only toxic to tumor cells. Compound 3 did not disrupt normal
organ function, cause bone marrow suppression, or negatively
1
2
Phytosphingosine
IC50 = 2.9 µM ± 0.5
FTY720
IC50 = 2.4 µM ± 0.5
OH
N
N
H
H
O
N
4
3
3-Pyridyltetryl ether derivative
IC50 = 2.1 µM ± 0.5
SH-BC-893
IC50 = 1.7 µM ± 0.3
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affect the rapidly proliferating cells of the gut even upon
With this premise in mind we developed a strategy that
engaged the terminal olefinic ether appendages of 3 with 3,6-
disubstituted 1,2,4,5-tetrazines in an inverse electron demand
Diels–Alder reaction to give the corresponding 3,6
disubstituted 1,2-pyridazines28 as 1:1 mixtures of 4,5-
regioisomers (Scheme 1). Cytotoxicity, nutrient transporter
down-regulation, and vacuolation induced by the products
were then measured in FL5.12 murine hematopoietic cells, a
cell line that grows in suspension and is therefore well-suited
to flow cytometry-based assays for transporter loss.
Cytotoxicity was also evaluated in a panel of human cancer
cell lines for compounds of interest (see Figure 3 and
chronic administration of the anti-cancer dose.11 Thus,
compound 3 is both efficacious and well-tolerated, at least in
mice.
1
2
3
4
5
6
7
8
These results showed the benefits and feasibility of
concurrently blocking parallel nutrient access pathways that
are essential for proliferating cancer cells. Because compound
3 simultaneously reduces access to glucose, amino acids, and
cholesterol,6,11 it is expected to be effective even against
heterogeneous tumors and may be less susceptible to the rapid
development of resistance observed with many targeted
therapies. While it has many activities desirable in an anti-
cancer agent, the low micromolar potency exhibited by
compound 3 might be improved by additional derivatization.
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Supplementary Table 3).
R1
C8H17
N
N
N
N
C8H17
In previous studies, we delineated the structural and functional
features in compound 3 that contributed to cytotoxicity.18 The
phenyloctyl appendage was found to be susceptible to changes
in the length of the octyl chain and the presence of polar
groups, except at the benzylic position. Extension of the
hydroxymethyl group to include alkyl ethers containing
heterocyclic moieties such as a 3-pyridyl appended variant
(Figure 1, compound 4) was tolerated.19 We were intrigued
that the inclusion of a 3-pyridyl unit as an appendage did not
negatively affect the cytotoxicity of the original compound 3.
R1
R2
N
N
O
(
)
n
O
(
)
DMSO
80 °C, 24 h
n
N
N
R2
Boc
Boc
n = 1, 2
C8H17
C8H17
HCl (4M in dioxane)
rt, 2 h
R1
R1
N
+
O
O
(
)
n
(
)
n
N
N
N
Cl
N
N
Cl
R2
H
H
H
H
R2
In an effort to further explore the effects of heteroaromatic
appendages, we turned our attention to 1,2-pyridazine as a
heterocycle that has found many applications as a versatile
biological probe.20–26
Scheme 1. General scheme for the synthesis of the 3,6-
disubstituted 1,2-pyridazine derivatives as 1:1 mixtures of 4,5-
regioisomers.
Table 1 lists the activity of a variety of analogs. We were
pleased that a 1:1 mixture of regioisomers of compound 5
having methyl and 1,3-pyrimidine substituents on the 1,2-
pyridazine core surpassed the activity of the parent 3, reaching
the high nanomolar threshold for the first time in this series. In
fact, the two regioisomers 5a and 5b could be separated by
chromatography and exhibited IC50 values of 0.5 and 0.7 µM
respectively. Despite its increased potency in cytotoxicity
assays, compound 5 did not down-regulate amino acid
transporters or vacuolate FL5.12 cells at the concentrations
that killed cells; 10 µM was required to observe these effects
(See Supplementary Table 1). However, it is of particular
interest that compound 5 efficiently triggered transporter loss
and vacuolation in MDA-MB-468 breast cancer cells (See
Supplementary Figure 1). These discrepancies may arise from
differences in the subunit composition of PP2A heterotrimers,
post-translational modification of PP2A subunits, and/or the
subcellular localization of PP2A in these various cell lines. In
sum, given its ability to fully phenocopy the PP2A-dependent
effects of compound 3 in cancer cells, it is likely that
compound 5 shares the PP2A-related target of compound 3.
On the other hand, the ability of compound 5 to efficiently kill
FL5.12 cells without inducing transporter loss or vacuolation
suggests for the first time that there may be another target of
this chemical series that makes an important, and partially
redundant, contribution to its anti-cancer effects. On-going
target deconvolution studies are expected to provide insight on
these points.
hydrophobic
pocket
A
R1
possible
pi-stacking;
hydrophobic
interaction
N
O
N
charge
interaction
O
H
H
O
(
)
n
N
R2
R1, R2 = aryl, heteroaryl, alkyl, alkyne combinations
Figure 2. Hypothetical interactions of compound 3/pyridazine
ether-appended constructs (A). Only one regioisomer is shown in
the 1,2-pyridazine ring.
In view of the presence of an extended (or folded)
hydrophobic chain, and an appended aromatic heterocycle
anchored on a basic pyrrolidine core, it is possible to envisage
certain hypothetical interactions with a biological target as
exemplified by structure A in Figure 2. The synthetic protocol
we chose allowed for the generation of a series of 3,6-
disubstituted 1,2-pyridazines appended to the lead compound
3 via a 2-3 carbon chain ether linker. We further surmised that
the relatively flexible single bonds of the 3,6-substituents
would adopt conformationally favorable spatial orientations.
Although we have previously established that compound 3
activates PP2A6,11,27, we do not as yet know the mechanism by
which this occurs.
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Importantly, compounds consisting of only the pyridazine
1
2
3
4
5
6
7
8
moiety (15), and compound 6 devoid of the phenyloctyl chain
(16), were inactive (Figure 3, Table 2, Supplementary Table
2). However, compound 17 with the ether linker but without
the pyridazine minimally affected the cytotoxicity of 3.
Clearly, adding the pyridazine moiety to the linker in
compound 17, particularly exemplified by compounds 5a and
5b (or the 1:1 mixture of regioisomers), improves the activity
of the current lead compound 3.
C8H17
R1
N
N
O
C8H17
9
N
Cl
Me
Me
R2
10
11
12
13
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H
H
N
HO
N
N
N
O
O
Comp.
5a
n
R1
R2
IC50
N
N
N
N
H
H
Cl
N
N
H
H
Cl
N
N
16
15
17
2
CH3
0.5 µM ± 0.2
Figure 3. 1,2-Pyridazine derivatives and a 1-butenyl ether of 3 for
comparisons of biological activities.
N
N
5b
6
2
1
CH3
0.7 µM ± 0.4
1.7 µM ± 0.4
Comp.
3
IC50
1.7 ± 0.3
> 40
N
N
CH3
15
N
7
8
9
2
2
2
CH3
CH3
CH3
1.6 µM ± 0.5
1.3 µM ± 0.4
2.8 µM ± 0.4
16
> 40
N
17
2.9 ± 0.2
Table 2. Cytotoxicity of pyridazine control compounds in FL5.12
cells. Values represent mean ± SD.
Encouraged by the improved cytotoxicity of compound 5 in
FL5.12 cells, we conducted additional tests to evaluate its anti-
neoplastic potential compared to the parent compound 3 in 8
human cancer cell lines from 4 different cancer types. We
were pleased to find that the superior activity of compound 5
was indeed reproduced. In fact, as a 1:1 mixture of isomers,
compound 5 was consistently more potent than the parent
compound 3, with IC50 values from ~2.5 to 9-fold lower than 3
(Figure 4), substantiating the role of the pyridazine core unit
as a preferred heterocyclic appendage. We further confirmed
that, as in FL5.12, the presence of a pyridine (compound 4) is
not sufficient for increased potency, and that the nature of the
substituents on the pyridazine plays a role in activity
(compound 5 vs 9) in HCT116 colon cancer cells (see
Supplementary Table 3). Finally, it is worth noting that in 3
KRAS mutant cancer cell lines, compound 5 is between 7 and
>20 times more potent than the published small molecule
activator of PP2A (SMAP), which is reported to inhibit
proliferation of KRAS mutant lung cancers29 (see
Supplementary Figure 2). Even the parent compound 3 is at
least 2-fold more potent than SMAP in these cell lines.
F
10
11
12
2
2
2
CH3
CH3
3.3 µM ± 0.3
1.3 µM ± 0.3
1.8 µM ± 0.6
CH3
F
F
13
14
2
2
1.4 µM ± 0.3
0.9 µM ± 0.1
N
N
Table 1. Cytotoxicity of the synthetic pyridazine derivatives in
FL5.12 cells. Values represent mean ± SD. Compounds 6-10 are
1:1 mixtures of 4- and 5- substituted isomers. The IC50 value for
a 1:1 mixture of 5a and 5b was 0.9 µM ± 0.1
Although there was a clear preference for a methyl and a
pyrimidine group attached to the 3- and 6- positions of the
pyridazine in analogs 5a and 5b compared to all the others in
Table 1, the differences in IC50 values only varied by a factor
of 5 for the least active compound (10). Decreasing the
number of carbons in the linker from 3 to 2 as in compound 6
resulted in a 3-fold increase in IC50 compared to 5a or 5b
without affecting its ability to down-regulate nutrient
transporters or to vacuolate (Supplemental Table 1). The
relatively non-remarkable SAR within this limited series of
derivatives suggests that the central pyridazine core with a 3,6-
substitution pattern of small or large groups such as methyl
and aryl or heteroaryl respectively is well-tolerated.
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Author Contributions
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#These authors contributed equally.
3
4
5
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ACKNOWLEDGMENT
The Montreal group thanks NSERC for financial assistance.
Funding for A.L.E. was provided through a grant from the Chao
Family Comprehensive Cancer Center Anti-Cancer Challenge.
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Figure 4. Compound 5 has a lower IC50 than compound 3 across
human cancer cell lines.
In conclusion, we have established a novel series of synthetic
sphingolipids related to a lead compound 3, with appended
1,2-pyridazine units bearing substituents at the 3,6-positions
via a 3-carbon ether linker. While a combination of
substituents were tolerated in maintaining cytotoxicity against
FL5.12 cells, inclusion of a methyl and a pyrimidinyl group
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Extension of these activities to selected human cancer cell
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ASSOCIATED CONTENT
The Supporting Information contains the biological and chemical
methods and chemical experimental procedures and is available
free of charge via the internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: stephen.hanessian@umontreal.ca.
*E-mail: aedinger@uci.edu.
prevention of tumor growth and metastasis by
a
novel
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