Design, synthesis, and antileukemic activity of stereochemically defined constrained analogues of FTY720 (Gilenya)

Article abstract of DOI:10.1021/ml4002425

FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has antineoplastic actions. Our published work suggests that at least some of FTY720's anticancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720's S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective antitumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for antileukemic activity.

Full text of DOI:10.1021/ml4002425

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Antileukemic Activity of Stereochemically
Defined Constrained Analogues of FTY720 (Gilenya)
Rebecca Fransson,†,§,∥ Alison N. McCracken,‡,∥ Bin Chen,† Ryan J. McMonigle,‡ Aimee L. Edinger,*,‡
and Stephen Hanessian*,†
†Department of Chemistry, Universite
́ ́ ́
de Montreal, Station Centre-Ville, Montreal, Quebec H3C 3J7, Canada
‡Department of Developmental and Cell Biology, University of California, Irvine, 2128 Natural Sciences 1, California 92697, United
States
S
* Supporting Information
ABSTRACT: FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-
phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has
antineoplastic actions. Our published work suggests that at least some of FTY720’s anticancer
activity is independent of its effects on S1P receptors and due instead to its ability to induce
nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack
FTY720’s S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective
antitumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-
substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human
leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of
compound activity. Moreover, phosphorylation of this group was not required for antileukemic activity.
KEYWORDS: FTY720, leukemia, pyrrolidine, arylmethyl ether
Furthermore, the C8H17 lipophilic appendage on the phenyl
ince its discovery as a synthetic immunomodulatory agent,
SFTY720 (1, also known as Fingolimod), has been the ring of FTY720 was found to be optimal for activity at S1P
receptors.
subject of intensive research efforts on many fronts (Figure 1).
Several research groups have demonstrated that, at higher
doses than are required for its immunosuppressive effect,
FTY720 is an effective and selective anticancer agent.12−16
Although FTY720 is very effective in diverse animal cancer
models, it cannot be used in human patients due to dose-
limiting bradycardia that occurs secondary to the activation of
S1P1 and S1P3.17,18 Until recently, it was thought that the
anticancer activities of FTY720 were intimately associated with
its effects on S1P receptors. Indeed, FTY720’s antiangiogenic
properties depend on S1P1 receptor effects.19 However, the
dose of FTY720 required for anticancer activity is well above
that required for immunosuppression due to S1P receptor
effects suggesting an alternate mode of action in cancer. Recent
studies from our group demonstrated that the anticancer and
S1P receptor effects of FTY720 are clearly separable and
provided a novel mechanism of action for the drug at the
elevated anticancer dose: nutrient transporter down-regula-
tion.20 These studies suggest that an FTY720 analogue that
does not activate S1P receptors has the potential to be an
effective and selective anticancer agent that could be safely used
in humans.
Figure 1. Structures of FTY720 and myriocin.
It is presently marketed as a drug under the trade name
Gilenya for the treatment of relapsing−remitting multiple
sclerosis. The conceptual basis for its structural derivation and
deployment of an aminodiol functionality on an aromatic
moiety bearing a hydrophobic aliphatic chain was inspired from
the natural product myriocin (2). When employed as an
immunosuppressant, FTY720 is a pro-drug as a result of an in
vivo phosphorylation, leading specifically to the pro-S-
phosphate ester isomer. Once phosphorylated, it acts as a
functional antagonist for four of the five sphingosine-1-
phosphate (S1P) receptors, reducing inflammation by prevent-
ing lymphocyte egress from secondary lymphoid organs.1,2
Significant research efforts have been directed toward
developing S1P receptor selective agonists and antagonists. In
an effort to study the stereochemistry of phosphorylation, a
number of synthetic analogues of FTY720 that focus mainly on
the polar subunit have been generated and the biological
activities of the resulting phosphate esters reported.3−11
In a previous study, we had prepared two enantiomeric 2,3,5-
trisubstituted pyrrolidines, compounds 3 and 4 as constrained
Received: June 26, 2013
Accepted: August 3, 2013
© XXXX American Chemical Society
A
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azacyclic analogues of FTY720 (Figure 2).4 Remarkable
selectivity was observed for S1P4 and S1P5 over S1P1 and
Figure 2. Structures of enantiomeric pyrrolidine analogues 3 and 4.
S1P3, demonstrating that chemical modification of the
conformationally flexible aminodiol portion of FTY720 could
lead to selective affinities toward the S1P receptors.
Furthermore, one of the diastereomers, compound 4, was not
susceptible to phosphorylation by sphingosine kinase 1 or 2,
and when chemically rather than enzymatically phosphorylated,
the individual (2R) and (5S) phosphate esters of 4 had no
activity at S1P1 or S1P3. In an effort to simplify the synthetic
protocol and incorporate a conformationally more flexible aryl
appendage, we synthesized a series of enantiomerically pure
and stereochemically diverse O-substituted benzyl ethers of
pyrrolidines starting with appropriately substituted 4-hydroxy
D- or L-prolines (Figure 3). Herein, we describe the anticancer
effects of these analogues in multiple human cancer cell lines
using FTY720 as a control.
Figure 4. Cytotoxic action of diastereomeric 2-hydroxymethyl
pyrrolidine 3- or 4-arylmethyl ethers on Sup-B15 leukemia cells. Cell
viability was measured by vital dye exclusion and flow cytometry at 72
h.
the hydroxymethyl group in this series has a strong influence
over the ability of these compounds to kill these leukemia cells.
Furthermore, it is clear that the novel, constrained diaster-
eoisomers represented by compounds 5−8 trigger cell death
with similar potency to the more flexible parent FTY720, with a
distinct preference for compound 6.
Given the observed influence of stereochemistry over
activity, we next evaluated the antileukemic activity of
compounds 9−12 in which the position of the ether appendage
was changed to be spatially proximal to the hydroxymethyl
group (Figure 3).
Compound 10 was 6-fold less active than compound 6,
similar to the other members of this series of analogues (Figure
4). Thus, it appears that the stereochemistry and particular
orientation of the ether appendage in compound 6 is unique in
conferring enhanced anticancer activity relative to its
diastereomeric congeners and to FTY720 in this leukemic
cell line.
While the ability to interfere with S1P1 receptor signaling is
critical for its immunosuppressive activity at low nanomolar
doses, activation of S1P1 and S1P3 by FTY720 prevents it from
being used in cancer therapy. Since the constrained analogues
might also be subject to phosphorylation in the cells, we
synthesized a series of compounds in which the hydroxymethyl
group was modified or entirely removed. For example, in
compounds 13 and 14, the hydroxymethyl group present in 5
and 6, respectively, was replaced with a methyl group (Figure
5). Loss of this potential phosphorylation site had no detectable
effect on the potency of 13 relative to 5 but decreased the
activity of 14 relative to 6 (Figure 6). Similarly, protecting the
hydroxyl group as the O-methyl ether as in compound 15
reduced the potency relative to 6. Removing the hydroxymethyl
group from 5 to give the pyrrolidine 16 had only a marginal
effect on activity in cell viability assays. However, the
enantiomeric pyrrolidine analogue 17 exhibited a 6-fold
reduction in activity relative to 6. As compounds 13−17
cannot be phosphorylated but retain the ability to kill leukemia
cells, the results are consistent with our model where
phosphorylation is not important for the ability of FTY720
to kill cancer cells.20 Although phosphorylation is not required
Figure 3. Structures of diastereomeric 2-hydroxymethyl pyrrolidine 3-
and 4-ether analogues, 5−12.
We first compared the activity of a diastereomeric series of
compounds comprising two enantiomeric pairs (5, 6 and 7, 8)
in cell viability assays using the BCR-ABL positive human acute
lymphoblastic leukemia (ALL) cell line, Sup-B15. All
compounds in this series killed leukemia cells as efficiently as
FTY720 (Figure 4 and Supporting Information). Interestingly,
the (2R,4S)-isomer 6 showed a 4-fold increase in activity
compared to its enantiomer 5. Moreover, compound 8, the syn-
diastereomer in this series, exhibited a dramatic 8-fold decrease
in activity compared to compound 6 and was 2-fold weaker
than its enantiomer 7. Together, these results demonstrate that
the 3-dimensional orientation of the ether appendage relative to
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introduction of the BCR-ABL fusion protein p190. Nalm-6,
Blin-1, and CCRF-CEM are also ALL cell lines but do not
express the oncogenic BCR-ABL fusion protein. In these three
human leukemia cell lines, 6 no longer exhibited increased
potency relative to other compounds in the series. The effect of
compound 6 and its distereoisomeric congeners on the prostate
cancer cell lines PC3 and DU145 was also determined.
Compounds 5 and 6 induced cell death to a similar extent,
and the potency of the constrained analogues was slightly
reduced relative to FTY720. From these findings, we conclude
that the enhanced potency of 6 over its diastereoisomers is a
characteristic associated with hematologic but not prostate
cancers and may be linked to expression of the BCR-ABL
fusion protein. BCR-ABL-dependent signaling drives the
survival and proliferation of chronic myelogenous leukemias
and a subset of ALLs. The discovery of the Abl kinase inhibitor
imatinib (Gleevec) and development of more potent second
generation drugs has allowed for the long-term survival of
patients with chronic myelogenous leukemia. However,
imatinib and its analogues do not lead to a cure, and drug
resistance often develops over time. Thus, novel agents with
activity against BCR-ABL positive leukemias could have clinical
utility.
Figure 5. Structures of 2-substituted pyrrolidine 4-ethers (13−17).
Finally, to test whether the constrained analogues were
working through the antineoplastic mechanism we previously
identified for FTY720, we compared the effect of FTY720 and
compounds 5, 6, 13, and 14 on surface expression of the amino
acid transporter-associated protein, 4F2hc. All five compounds
triggered the rapid down-regulation of 4F2hc suggesting that
FTY720 and the conformationally constrained analogues
described herein kill cancer cells through the same mechanism
(Figure 7).20
Figure 6. Cytotoxic action of 2-substituted pyrrolidine 4-arylmethyl
ethers and 3-pyrrolidine arylmethyl ethers on Sup-B15 leukemia cells.
for activity, compound 6 appears to dominate the stereo-
chemical spectrum in these enantiomerically pure 2-hydrox-
ymethyl pyrrolidines given its enhanced activity relative to the
other stereoisomers and to FTY720. Finally, we also noted that
the nature and length of the aliphatic chain was critical to the
activity. Analogues of compound 5 with shorter and longer
chains or with a simple heptyl chain were less active, as was a
MOM ether (see Supporting Information).
Having observed an increased potency of 6 relative to its
diastereoisomers 5, 7, and 8, we wished to determine whether
this differential activity was also seen in other cancer cell lines.
Cell viability assays were used to compare the activity of the
constrained analogues and FTY720 in an additional BCR-ABL
positive ALL cell line, BV173. In this cell line, compound 6 was
again more active than its stereoisomers 5, 7, and 8 (Table 1).
Interestingly, compound 6 was also 10-fold more active than
compound 5 in murine bone marrow transformed by
Figure 7. Diastereomeric 2-hydroxymethyl pyrrolidine 4-arylmethyl
ethers (10 μM) trigger nutrient transporter loss in Sup-B15 leukemia
cells.
In conclusion, we have reported on the anticancer activity of
a series of novel constrained analogues of FTY720, including a
stereochemically distinct isomer (6) with enhanced activity. A
Table 1. Mean IC50 (in μM SEM) of Analogues in Cell Viability Assays in a Range of Human Cancer Cell Lines and BCR-Abl-
Expressing Murine Bone Marrow (BM); Viability Was Measured by Vital Dye Exclusion and Flow Cytometry at 72 h; When
Compared to FTY720 Using a t-test (two-tailed): * p < 0.05; ** p < 0.01; *** p < 0.001
SupB15
BV173
CCRF-CEM
Nalm-6
Blin-1
PC3
DU145
prostate
Ph + ALL
BM-p190
Ph + ALL
Ph − ALL
Ph − ALL
Ph − ALL
prostate
1
5
6
7
8
6.8 0.7
7.7 0.8
3.3 0.2
6.3 0.4
6.8 0.3
9.6 1.9
15.0 2.1
13.5 2.4
5.5 0.1
9.8 0.9
14.3 1.2*
13.5 2.6
6.5 0.9
5.7 1.1*
0.5 0.1***
4.0 0.4
10.4 0.7***
3.8 0.4**
9.7 1.0**
13.8 0.8***
11.0 1.3***
8.2 0.7
7.5 0.1***
6.9 0.3**
10.8 0.4*
15.1 1.0**
2.0 0.2***
8.3 0.8
8.1 1.4
16.7 2.4***
8.4 1.5**
11.6 1.1***
C
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ASSOCIATED CONTENT
■
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* Supporting Information
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1
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AUTHOR INFORMATION
Corresponding Authors
*(A.L.E.) aedinger@uci.edu
■
*(S.H.) E-mail: stephen.hanessian@umontreal.ca
Present Address
§(R.F.) Department of Medicinal (Organic Pharmaceutical)
Chemistry, Uppsala University, Uppsala, Sweden.
Author Contributions
The manuscript was written by A.L.E. and S.H. and edited by
A.N.M. All authors have given approval to the final version of
the manuscript.
Author Contributions
∥R.F. and A.N.M. contributed equally to this work.
Funding
A.N.M. is supported by Grant Number T32CA009054 from the
National Cancer Institute (NCI). A.L.E. is supported by grants
from the NIH (R01 GM089919), the American Cancer Society
(RSG-11-111-01-CDD), the Department of Defense
(W81XWH-11-1-0535), University of California, Irvine Coun-
cil on Research, Computing, and Libraries (MIIG-2011-12-24
with S.H.), and the William Lawrence and Blanche Hughes
Foundation. S.H. is supported by grants from Isis Pharmaceut-
icals, Sigma Tau USA, NSERC Canada, Servier Canada, and
Servier France. S.H. is the Isis Pharmaceuticals Chairholder at
́ ́
the Universite de Montreal.
Notes
The authors declare no competing financial interest.
(13) Azuma, H.; Takahara, S.; Ichimaru, N.; Wang, J. D.; Itoh, Y.;
Otsuki, Y.; Morimoto, J.; Fukui, R.; Hoshiga, M.; Ishihara, T.;
Nonomura, N.; Suzuki, S.; Okuyama, A.; Katsuoka, Y. Marked
prevention of tumor growth and metastasis by a novel immunosup-
pressive agent, FTY720, in mouse breast cancer models. Cancer Res.
2002, 62, 1410−1419.
(14) Chua, C. W.; Lee, D. T.; Ling, M. T.; Zhou, C.; Man, K.; Ho, J.;
Chan, F. L.; Wang, X.; Wong, Y. C. FTY720, a fungus metabolite,
inhibits in vivo growth of androgen-independent prostate cancer. Int. J.
Cancer 2005, 117, 1039−1048.
(15) Azuma, H.; Takahara, S.; Horie, S.; Muto, S.; Otsuki, Y.;
Katsuoka, Y. Induction of apoptosis in human bladder cancer cells in
vitro and in vivo caused by FTY720 treatment. J. Urol. 2003, 169,
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ACKNOWLEDGMENTS
■
The authors would like to thank Jordan Thompson, Saurabh
Ghosh Roy, Gang Liu, Tiffany Nguyen, and Jessica Kemp for
their contributions to IC50 assays.
ABBREVIATIONS
■
ALL, acute lymphoblastic leukemia; BCR-ABL, breakpoint
cluster region-Abelson tyrosine kinase fusion protein; Ph,
Philadelphia chromosomal translocation t(9;22)(q34;q11);
S1P, sphingosine-1-phosphate; S1P1−5, sphingosine-1-phos-
phate receptor 1−5; 4F2hc, 4F2 heavy chain amino acid
transporter-associated protein
(16) Neviani, P.; Santhanam, R.; Oaks, J. J.; Eiring, A. M.; Notari, M.;
Blaser, B. W.; Liu, S.; Trotta, R.; Muthusamy, N.; Gambacorti-
Passerini, C.; Druker, B. J.; Cortes, J.; Marcucci, G.; Chen, C. S.;
Verrills, N. M.; Roy, D. C.; Caligiuri, M. A.; Bloomfield, C. D.; Byrd, J.
C.; Perrotti, D. FTY720, a new alternative for treating blast crisis
chronic myelogenous leukemia and Philadelphia chromosome−
positive acute lymphocytic leukemia. J. Clin. Invest. 2007, 117,
2408−2421.
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NOTE ADDED AFTER ASAP PUBLICATION
■
This manuscript was published on the Web on August 26,
2013. A citation number was corrected in the text and the
revised version was reposted on August 28, 2013.
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