Structural Requirements and Docking Analysis of Amidine-Based Sphingosine Kinase 1 Inhibitors Containing Oxadiazoles

Article abstract of DOI:10.1021/acsmedchemlett.6b00002

Sphingosine 1-phosphate (S1P) is a potent growth-signaling lipid that has been implicated in cancer progression, inflammation, sickle cell disease, and fibrosis. Two sphingosine kinases (SphK1 and 2) are the source of S1P; thus, inhibitors of the SphKs have potential as targeted cancer therapies and will help to clarify the roles of S1P and the SphKs in other hyperproliferative diseases. Recently, we reported a series of amidine-based inhibitors with high selectivity for SphK1 and potency in the nanomolar range. However, these inhibitors display a short half-life. With the goal of increasing metabolic stability and maintaining efficacy, we designed an analogous series of molecules containing oxadiazole moieties. Generation of a library of molecules resulted in the identification of the most selective inhibitor of SphK1 reported to date (705-fold selectivity over SphK2), and we found that potency and selectivity vary significantly depending on the particular oxadiazole isomer employed. The best inhibitors were subjected to in silico molecular dynamics docking analysis, which revealed key insights into the binding of amidine-based inhibitors by SphK1. Herein, the design, synthesis, biological evaluation, and docking analysis of these molecules are described.

Full text of DOI:10.1021/acsmedchemlett.6b00002

Letter
pubs.acs.org/acsmedchemlett
Structural Requirements and Docking Analysis of Amidine-Based
Sphingosine Kinase 1 Inhibitors Containing Oxadiazoles
Joseph D. Houck,*,†,§ Thomas K. Dawson,*,† Andrew J. Kennedy,†,∥ Yugesh Kharel,‡ Niels D. Naimon,†
Saundra D. Field,† Kevin R. Lynch,‡ and Timothy L. Macdonald†,‡
†Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, Virginia 22904, United States
‡Department of Pharmacology, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, Virginia 22904, United States
S
* Supporting Information
ABSTRACT: Sphingosine 1-phosphate (S1P) is a potent growth-signaling lipid that has
been implicated in cancer progression, inflammation, sickle cell disease, and fibrosis. Two
sphingosine kinases (SphK1 and 2) are the source of S1P; thus, inhibitors of the SphKs
have potential as targeted cancer therapies and will help to clarify the roles of S1P and
the SphKs in other hyperproliferative diseases. Recently, we reported a series of amidine-
based inhibitors with high selectivity for SphK1 and potency in the nanomolar range.
However, these inhibitors display a short half-life. With the goal of increasing metabolic
stability and maintaining efficacy, we designed an analogous series of molecules
containing oxadiazole moieties. Generation of a library of molecules resulted in the
identification of the most selective inhibitor of SphK1 reported to date (705-fold
selectivity over SphK2), and we found that potency and selectivity vary significantly
depending on the particular oxadiazole isomer employed. The best inhibitors were subjected to in silico molecular dynamics
docking analysis, which revealed key insights into the binding of amidine-based inhibitors by SphK1. Herein, the design,
synthesis, biological evaluation, and docking analysis of these molecules are described.
KEYWORDS: Sphingosine 1-phosphate, sphingosine kinase, cancer, inhibitor, oxadiazoles
phingosine 1-phosphate (S1P) is a potent signaling lipid
kinases, SphK1 is activated and translocated to the inner leaflet
of the plasma membrane.16 Then, in an inside-out signaling
mechanism, S1P is shuttled outside the cell by the membrane-
spanning spinster 2 protein (or by members of the ABC
transport family), where it may bind with any of the G protein-
coupled S1P1−5. Agonism at these receptors initiates a cascade
of signaling events affecting growth, motility, immune cell
trafficking, metastasis, and angiogenesis.12
Because of the complexity of S1P signaling, a large and
diverse set of pharmacological tools is necessary to probe the
underlying physiological pathways and conclusively validate
both SphK1 and 2 in different disease models. Small-molecule
SphK inhibitors are an important example of such a tool, and
ideally should have the following characteristics: single-digit
nanomolar potency, at least 100-fold selectivity for either of the
SphK isoforms, and high metabolic stability.10 Currently, no
single molecule fulfills each criterion simultaneously, but a new
generation of inhibitors is nearing this mark. In particular,
recent efforts to improve our highly potent (but unstable)
amidine-based inhibitors have yielded a class of very effective
guanidine-based, oxadiazole-containing molecules.17−19 These
compounds are the most potent and selective inhibitors that
elicit prolonged shifts in blood S1P concentrations; the ability
S
that acts on five membrane-bound receptors (S1P1−5)1
and various intracellular targets.2 S1P has been shown to
regulate a host of processes that affect the growth and fate of
cells,3 and aberrant S1P signaling has been linked to numerous
diseases including cancer,2,4 asthma,5 fibrosis,6 and sickle cell
disease.7 Cellular synthesis of S1P is exclusively dependent on
the action of two sphingosine kinases (SphK1 and 2) that
directly phosphorylate sphingosine (Sph).3 As the sole sources
of cellular S1P, the SphKs influence cell survival8,9 and have
been identified as targets of interest in pharmacology and the
pharmaceutical industry.4,10 Therefore, SphK inhibitors, by
affecting S1P levels, have therapeutic potential and are
necessary to clarify the many roles of S1P and each SphK in
physiology and disease.10−12
S1P signaling is complex, and the roles of SphK1 and 2 in
various pathways differ significantly. These differences are due,
in large part, to subcellular compartmentalization; SphK2
possesses an N-terminal nuclear localization sequence not
present in SphK1.13 Indeed, SphK2 is located primarily in the
nucleus and other organelles and may influence gene expression
through S1P-dependent inhibition of histone deacetylase 1 and
2 (HDAC1 and 2).14 Additionally, S1P produced by SphK2 in
the endoplasmic reticulum and mitochondria has the effect of
stimulating apoptosis.15 In contrast, SphK1 is located primarily
in the cytosol, and SphK1-derived S1P generally elicits
prosurvival and proliferative effects through interactions with
S1P1−5. Upon phosphorylation by the extracellular-regulated
Received: January 4, 2016
Accepted: March 1, 2016
© XXXX American Chemical Society
A
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to lower or raise blood S1P levels is an important biomarker for
inhibition of SphK1 or 2, respectively.20
Further improvements were the result of in silico screening of a
library of molecules with varied, rigidified tails. This analysis
was carried out with a SphK1 homology model based on
diacylglycerol kinase beta, and yielded 6, a SphK1 inhibitor with
a KI of 47 nM and 180-fold selectivity over SphK2 (Table 1).22
In the work described presently, we followed a similar
approach by optimizing a lead molecule with a library of
synthetic variants and then following with in silico modeling. In
this new series, the amide moiety was replaced with three
distinct oxadiazoles; the reasoning for this is 3-fold: (1) our
previous inhibitors have displayed poor half-lives in vivo and
oxadiazoles are well-documented amide surrogates,23−27 (2)
oxadiazoles have been effective structural subunits in our
previous work with SphK2 substrates,28 and (3) oxadiazole
linkers improved the efficacy of guanidine-based SphK1
inhibitors.17
To further improve SphK inhibitors, particularly regarding
potency, it is necessary to continue to probe the Sph-binding
pockets of SphK1 and 2 with structurally diverse sets of
molecules. To this end, we have developed and characterized a
small library of molecules combining an amidine headgroup
with various oxadiazole linkers, elaborating on our earlier
studies.21,22 Herein, we report an inhibitor with double-digit
nanomolar potency and 705-fold selectivity for SphK1 over
SphK2. Additionally, we reveal two key insights into the
structure−activity relationships governing SphK1 inhibition,
namely, that potency and selectivity are greatly affected by
oxadiazole subtypes and alkyl tail positioning.
Our previous work yielded a series of amidine-based
molecules displaying high potency and selectivity for SphK1.
The lead molecule, 1, was modified to optimize tail length,
amide orientation, and headgroup identity (2−4, Table 1).21
As a starting point, 11c, an analogue of 2 and 3, was
synthesized as follows (Scheme 1). First, 1-dodecene was
reduced to the alkyl borane with 9-BBN and coupled to 4-
bromobenzonitrile using Suzuki conditions to p-alkylbenzoni-
trile 7c. Next, conversion to aryl amidoxime 8c was achieved
using hydroxylamine hydrochloride and triethylamine in
ethanol. PyBop-mediated coupling of the amidoxime to 1-
cyano-1-cyclopropanecarboxylic acid yielded 9c. Cyclization of
the coupled amidoxime with TBAF29 gave oxadiazole 10c and
was subjected to base-catalyzed Pinner conditions30 to yield
amidine 11c.
Table 1. Previously Described Amidine-Based SphK
Inhibitors20,21
KI values were determined by a [γ-32P]ATP in vitro assay of
SphK enzymatic activity (Table 2).31 Compound 11c maintains
potency and SphK1 selectivity, validating the oxadiazole as a
suitable replacement for the amide group.
The incorporation of an oxadiazole into the molecular
scaffold significantly alters the angle between the tail and head
groups. With this in mind, a variant of 11c was synthesized with
meta-substitution about the phenyl ring. Interestingly, upon
attaching the tail at the meta position, potency and selectivity
increased dramatically, with 11g displaying a KI of 40 nm and
705-fold selectivity for SphK1 over SphK2. As a control, a meta-
substituted analogue of our amide compounds, 13, was
synthesized (Scheme SI-1). This compound lost potency
proportionately at both kinases (Table 3). meta-Substituted
analogues present the amidine to the γ-phosphate of ATP such
that the amidine is no longer antiperiplanar with the aromatic
ring. Deletion of the cyclopropane ring alpha to the amidine
resulted in a loss of activity in oxadiazole 15 (Scheme SI-2,
Table SI-1). The unique torsional angle of the cyclopropane
a
KI = [I]/(KM/KM − 1); KM of sphingosine at SphK1 = 10 μM; KM of
b
sphingosine at SphK2 = 5 μM. Selectivity = (KI/KM)SphK2/(KI/
KM)SphK1
.
Scheme 1. Synthesis of Oxadiazole Amidine 11c
B
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structure, PDB 4L02), one can see that meta amide (13) and
meta oxadiazole (11g) adopt very different configurations
(Figure 1). The oxadizaole inhibitor fills out the tail-binding
region of the pocket to a greater extent than the amide. Further
analysis reveals three cationic attractions of the amidine
headgroup: to the γ-phosphate of ATP (as previously
reported), and to Asp 81 and Asp178. Additionally, π-stacking
between the oxadiazole and Phe192, which forms the roof of
the pocket above the linker region, contributes to binding, as
well as a favorable arene−H interaction between the phenyl
ring and Ile-174.
Table 2. KI Values of Oxadiazole 11c Compared to Amides 2
and 3
Following the evaluation of 11c and 11g, four additional
compounds were synthesized to determine the relative efficacy
of the three oxadiazole subtypes, as it has been reported in the
literature that each individual regioisomer exhibits unique
physical and pharmacological properties.33 For each oxadiazole
subtype, both meta- and para-substituted variants were
synthesized (Schemes SI-3 and SI-4).
a
KI = [I]/(KM/KM − 1); KM of sphingosine at SphK1 = 10 μM; KM of
b
sphingosine at SphK2 = 5 μM. Selectivity = (KI/KM)SphK2/(KI/
KM)SphK1
.
Biological evaluation revealed substantial differences in the
inhibitory qualities of molecules containing isomeric oxadia-
zoles (Table 4). Surprisingly, the largest distinction occurred
between compounds containing the 3-aryl (11c and 11g) and
5-aryl (21a and 21b) 1,2,4-oxadiazole isomers. Inhibitors 11c
and 11g display significantly higher potency and selectivity for
SphK1 than do 21a and 21b. Compounds containing 1,3,4-
oxadiazoles (26a and 26b) display intermediate potency.
Substitution about the phenyl ring again proved to be a very
important factor, and all compounds with meta-substituted alkyl
tails are much more potent and selective than para-substituted
analogues.
These marked differences in in vitro activity between
oxadiazole subtypes can be rationalized, in part, with additional
in silico analyses. Compounds 11g, 21b, and 26b were docked
into the substrate-binding domain of SphK1, where the
respective heterocycles appear to adopt different binding
orientations (Figure 2). Most notably, electrostatic repulsion
between the nonbonding electrons of the oxadiazole isomers
and the backbone carbonyl of Leu268 destabilizes binding of
compounds 21b and 26b. This interaction alters the position-
ing of the amidine headgroup, limiting binding interactions
with the γ-phosphate of kinase-bound ATP. Furthermore,
ring provides improved presentation of the amidine in the
active site.
Because the incorporation of oxadiazoles increases the overall
molecular length, we altered the length of the alkyl tail to
accommodate this change. Compounds with tails ranging from
10 to 13 carbons in length, both para- and meta-substituted,
were synthesized through the same methods illustrated in
Scheme 1 (KI values summarized in Table 3). The tail
derivatives follow the same trend as our previous amidine-based
inhibitors.21,22 Potency increases with tail length before
dropping off at a length of 13 carbons. Tail substitution at
the para position reaches a maximum potency and selectivity
for SphK1 at a length of 11 carbons, with a KI of 0.26 μM
(11b). Because an oxadiazole is one atom longer than an amide,
it is expected that the 11-carbon tail fills out the sphingosine-
binding pocket similarly to a 12-carbon tail on an amide
analogue.
By integrating our previous work in the development of a
SphK1 homology model22 with the recently determined SphK1
crystal structure,32 we have identified several key binding
interactions between 11g and SphK1. On docking compounds
11g and 13 into a SphK1 model (generated with the crystal
Table 3. KI Values of para- and meta-Substituted Oxadiazoles 11a−h Compared to Amide Analogues 2 and 13
a
b
KI = [I]/(KM/KM − 1); KM of sphingosine at SphK1 = 10 μM; KM of sphingosine at SphK2 = 5 μM. Selectivity = (KI/KM)SphK2/(KI/KM)SphK1
.
C
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Figure 1. Docking studies with SphK1. (left) meta-Substituted oxadiazole (11g, black) and amide (13, yellow) derivatives docked into the crystal
structure of SphK1. (right) Two-dimensional representation of 11g and its side chain interactions.
Table 4. KI Values of Heterocyclic Isomers with meta- and para-Substitution about the Phenyl Ring
a
b
KI = [I]/(KM/KM − 1); KM of sphingosine at SphK1 = 10 μM; KM of sphingosine at SphK2 = 5 μM. Selectivity = (KI/KM)SphK2/(KI/KM)SphK1
.
Figure 2. In silico docking of 11g (A), 21b (B), and 26b (C). White sphere = Mg2+.
nitrogen atoms on the oxadiazoles of 21b and 26b likely
undergo a repulsive interaction with the Asp81 residue.
These simulations suggest that, relative to isoforms present
in other compounds, the particular 1,2,4-oxadiazole of 11g is
subjected to minimal repulsive interactions in the SphK1 active
site. This favorable binding orientation, coupled with meta-
substitution about the phenyl ring, results in a significantly
enhanced inhibitory profile. Compound 11g is not only highly
potent but, with 705-fold selectivity for SphK1, is the most
selective SphK1 inhibitor reported to date.
also, meta-substitution about the phenyl ring increased efficacy
for all isomers. Docking analysis revealed important interactions
between 11g and specific amino acid residues in the SphK1
active site, and provided justification for the favorable binding
of this compound. This study contributes to the growing
understanding of the structural requirements for SphK1
inhibition, and compounds such as 11g provide the ideal
scaffold for future inhibitors. Simultaneous optimization of
potency, selectivity, and pharmacokinetic properties will be
necessary to further interrogate both SphKs in pathophysiology
and to identify suitable therapeutic candidates in the fight
against cancer and hyperproliferative diseases.
In summary, we have generated a library of amidine-based
SphK1 inhibitors with in vitro potencies in the nanomolar
range. Surprisingly, different oxadiazole subtypes elicited
dramatically different effects regarding potency and selectivity;
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Brocklyn, J. R.; Wang, F. Sphingosine-1-phosphate in cell growth and
cell death. Ann. N. Y. Acad. Sci. 1998, 845, 11−18.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00002.
■
S
(9) Pitson, S. M. Regulation of sphingosine kinase and sphingolipid
signaling. Trends Biochem. Sci. 2011, 36, 97−107.
(10) Santos, W. L.; Lynch, K. R. Drugging sphingosine kinases. ACS
Chem. Biol. 2015, 10, 225−233.
(11) Plano, D.; Amin, S.; Sharma, A. K. Importance of sphingosine
kinase (SphK) as a target in developing cancer therapeutics and recent
developments in the synthesis of novel SphK inhibitors. J. Med. Chem.
2014, 57, 5509−5524.
Experimental details for the synthesis of all compounds,
compound characterization, and description of the SphK
assay (PDF)
(12) Gandy, K. A. O.; Obeid, L. M. Targeting the sphingosine
kinase/sphingosine 1-phosphate pathway in disease: review of
sphingosine kinase inhibitors. Biochim. Biophys. Acta, Mol. Cell Biol.
Lipids 2013, 1831, 157−166.
(13) Igarashi, N.; Okada, T.; Hayashi, S.; Fujita, T.; Jahangeer, S.;
Nakamura, S. Sphingosine kinase 2 is a nuclear protein and inhibits
DNA synthesis. J. Biol. Chem. 2003, 278, 46832−46839.
(14) Hait, N. C.; Allegood, J.; Maceyka, M.; Strub, G. M.; Harikumar,
K. B.; Singh, S. K.; Luo, C.; Marmorstein, R.; Kordula, T.; Milstien, S.;
Spiegel, S. Regulation of histone acetylation in the nucleus by
sphingosine-1-phosphate. Science 2009, 325, 1254−1257.
(15) Neubauer, H. a; Pitson, S. M. Roles, regulation and inhibitors of
sphingosine kinase 2. FEBS J. 2013, 280, 5317−36.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: jdhouck@umd.edu.
*E-mail: tkd5gx@virginia.edu.
■
Present Addresses
§Department of Chemistry and Biochemistry, University of
Maryland, 0107 Chemistry Building, College Park, Maryland
20742, United States.
∥University of Alabama at Birmingham, 1825 University
Boulevard, Shelby Building, Birmingham, Alabama 35294,
United States.
(16) Pitson, S. M.; Moretti, P. A. B.; Zebol, J. R.; Lynn, H. E.; Xia, P.;
Vadas, M. A.; Wattenberg, B. W. Activation of sphingosine kinase 1 by
ERK1/2-mediated phosphorylation. EMBO J. 2003, 22, 5491−5500.
(17) Patwardhan, N. N.; Morris, E. A.; Kharel, Y.; Raje, M. R.; Gao,
M.; Tomsig, J. L.; Lynch, K. R.; Santos, W. L. Structure−activity
relationship studies and in vivo activity of guanidine-based sphingosine
kinase inhibitors: discovery of sphK1- and sphK2-selective inhibitors. J.
Med. Chem. 2015, 58, 1879−1899.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding
This work was supported by grants from the National Institutes
of Health (R01 GM067958 to K.R.L. and T.L.M.; R01
GM104366 to K.R.L.).
(18) Knott, K.; Kharel, Y.; Raje, M. R.; Lynch, K. R.; Santos, W. L.
Effect of alkyl chain length on sphingosine kinase 2 selectivity. Bioorg.
Med. Chem. Lett. 2012, 22, 6817−20.
Notes
The authors declare no competing financial interest.
(19) Kharel, Y.; Morris, E. A.; Thorpe, S. B.; Tomsig, J. L.; Santos, W.
L.; Lynch, K. R. Sphingosine kinase type 2 and blood sphingosine 1-
phosphate. J. Pharmacol. Exp. Ther. 2015, 355, 23−31.
(20) Kharel, Y.; Raje, M.; Gao, M.; Gellett, A. M.; Tomsig, J. L.;
Lynch, K. R.; Santos, W. L. Sphingosine kinase type 2 inhibition
elevates circulating sphingosine 1-phosphate. Biochem. J. 2012, 447,
149−157.
(21) Mathews, T. P.; Kennedy, A. J.; Kharel, Y.; Kennedy, P. C.;
Nicoara, O.; Sunkara, M.; Morris, A. J.; Wamhoff, B. R.; Lynch, K. R.;
Macdonald, T. L. Discovery, biological evaluation, and structure-
activity relationship of amidine based sphingosine kinase inhibitors. J.
Med. Chem. 2010, 53, 2766−2778.
(22) Kennedy, A. J.; Mathews, T. P.; Kharel, Y.; Field, S. D.; Moyer,
M. L.; East, J. E.; Houck, J. D.; Lynch, K. R.; Macdonald, T. L.
Development of amidine-based sphingosine kinase 1 nanomolar
inhibitors and reduction of sphingosine 1-phosphate in human
leukemia cells. J. Med. Chem. 2011, 54, 3524−3548.
(23) Warmus, J. S.; Flamme, C.; Zhang, L. Y.; Barrett, S.; Bridges, A.;
Chen, H.; Gowan, R.; Kaufman, M.; Sebolt-Leopold, J.; Leopold, W.;
Merriman, R.; Ohren, J.; Pavlovsky, A.; Przybranowski, S.; Tecle, H.;
Valik, H.; Whitehead, C.; Zhang, E. 2-Alkylamino- and alkoxy-
substituted 2-amino-1,3,4-oxadiazoles-O-alkyl benzohydroxamate es-
ters replacements retain the desired inhibition and selectivity against
MEK (MAP ERK Kinase). Bioorg. Med. Chem. Lett. 2008, 18, 6171−
6174.
ACKNOWLEDGMENTS
Support for Thomas Dawson was provided by the ARCS
Foundation MWC and Mr. Gary Hoffman.
■
ABBREVIATIONS
■
HDAC, histone deacetylace; S1P, sphingosine 1-phosphate;
Sph, sphingosine; SphK1, sphingosine kinase 1; SphK2,
sphingosine kinase 2; SphKs, sphingosine kinases
REFERENCES
■
(1) Blaho, V. a; Hla, T. An update on the biology of sphingosine 1-
phosphate receptors. J. Lipid Res. 2014, 55, 1596.
(2) Pyne, N. J.; Pyne, S. Sphingosine 1-phosphate and cancer. Nat.
Rev. Cancer 2010, 10, 489−503.
(3) Hannun, Y. A.; Obeid, L. M. Principles of bioactive lipid
signalling: lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 2008, 9,
139−150.
(4) Pitman, M. R.; Pitson, S. M. Inhibitors of the sphingosine kinase
pathway as potential therapeutics. Curr. Cancer Drug Targets 2010, 10,
354−367.
(5) Lai, W.-Q.; Wong, W. S. F.; Leung, B. P. Sphingosine kinase and
sphingosine 1-phosphate in asthma. Biosci. Rep. 2011, 31, 145−150.
(6) Long, D. A.; Price, K. L. Sphingosine kinase-1: a potential
mediator of renal fibrosis. Kidney Int. 2009, 76, 815−817.
(7) Zhang, Y.; Berka, V.; Song, A.; Sun, K.; Wang, W.; Zhang, W.;
Ning, C.; Li, C.; Zhang, Q.; Bogdanov, M.; Alexander, D. C.; Milburn,
M. V.; Ahmed, M. H.; Lin, H.; Idowu, M.; Zhang, J.; Kato, G. J.;
Abdulmalik, O. Y.; Zhang, W.; Dowhan, W.; Kellems, R. E.; Zhang, P.;
Jin, J.; Safo, M.; Tsai, A. L.; Juneja, H. S.; Xia, Y. J. Elevated
sphingosine-1-phosphate promotes sickling and sickle cell disease
progression. J. Clin. Invest. 2014, 124, 2750−2761.
(24) McBriar, M. D.; Clader, J. W.; Chu, I.; Del Vecchio, R. A.;
Favreau, L.; Greenlee, W. J.; Hyde, L. A.; Nomeir, A. A.; Parker, E. M.;
Pissarnitski, D. A.; Song, L.; Zhang, L.; Zhao, Z. Discovery of amide
and heteroaryl isosteres as carbamate replacements in a series of orally
active gamma-secretase inhibitors. Bioorg. Med. Chem. Lett. 2008, 18,
215−219.
̀
(25) Bostrom, J.; Hogner, A.; Llinas, A.; Wellner, E.; Plowright, A. T.
̈
Oxadiazoles in medicinal chemistry. J. Med. Chem. 2012, 55, 1817−
(8) Spiegel, S.; Cuvillier, O.; Edsall, L. C.; Kohama, T.; Menzeleev,
R.; Olah, Z.; Olivera, A.; Pirianov, G.; Thomas, D. M.; Tu, Z.; Van
1830.
E
DOI: 10.1021/acsmedchemlett.6b00002
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(26) Borg, S.; Estenne-Bouhtou, G.; Luthman, K.; Csoeregh, I.;
Hesselink, W.; Hacksell, U. Synthesis of 1,2,4-oxadiazole-, 1,3,4-
oxadiazole-, and 1,2,4-triazole-derived dipeptidomimetics. J. Org. Chem.
1995, 60, 3112−3120.
(27) Garfunkle, J.; Ezzili, C.; Rayl, T. J.; Hochstatter, D. G.; Hwang,
I.; Boger, D. L. Optimization of the central heterocycle of alpha-
ketoheterocycle inhibitors of fatty acid amide hydrolase. J. Med. Chem.
2008, 51, 4392−4403.
(28) Foss, F.; Mathews, T.; Kharel, Y.; Kennedy, P.; Snyder, A.;
Davis, M.; Lynch, K.; Macdonald, T. Synthesis and biological
evaluation of sphingosine kinase substrates as sphingosine-1-phosphate
receptor prodrugs. Bioorg. Med. Chem. 2009, 17, 6123−6136.
(29) Gangloff, A. R.; Litvak, J.; Shelton, E. J.; Sperandio, D.; Wang, V.
R.; Rice, K. D. Synthesis of 3,5-disubstituted-1,2,4-oxadiazoles using
tetrabutylammonium fluoride as a mild and efficient catalyst.
Tetrahedron Lett. 2001, 42, 1441−1443.
(30) Schaefer, F. C.; Peters, G. A. Base-catalyzed reaction of nitriles
with alcohols: A convenient route to imidates and amidine salts. J. Org.
Chem. 1961, 26, 412−418.
(31) Kharel, Y.; Mathews, T. P.; Kennedy, A. J.; Houck, J. D.;
Macdonald, T. L.; Lynch, K. R. A rapid assay for assessment of
sphingosine kinase inhibitors and substrates. Anal. Biochem. 2011, 411,
230−235.
(32) Wang, Z.; Min, X.; Xiao, S.-H.; Johnstone, S.; Romanow, W.;
Meininger, D.; Xu, H.; Liu, J.; Dai, J.; An, S.; Thibault, S.; Walker, N.
Molecular basis of sphingosine kinase 1 substrate recognition and
catalysis. Structure 2013, 21, 798−809.
(33) Goldberg, K.; Groombridge, S.; Hudson, J.; Leach, A. G.;
MacFaul, P. A.; Pickup, A.; Poultney, R.; Scott, J. S.; Svensson, P. H.;
Sweeney, J. Oxadiazole isomers: All bioisosteres are not created equal.
MedChemComm 2012, 3, 600−604.
F
DOI: 10.1021/acsmedchemlett.6b00002
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