Structure guided design of a series of sphingosine kinase (SphK) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.06.030

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each other's loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.

Full text of DOI:10.1016/j.bmcl.2013.06.030

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4608–4616
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure guided design of a series of sphingosine kinase (SphK)
inhibitors
a
b
a
Darin J. Gustin a, Yihong Li a, , Matthew L. Brown , Xiaoshan Min , Mike J. Schmitt ,
Malgorzata Wanska a, Xiaodong Wang a, Richard Connors a, Sheere Johnstone b, Mario Cardozo b,
Alan C. Cheng b, Shawn Jeffries c, Brendon Franks c, Shyun Li c, Shanling Shen c, Mariwil Wong c,
⇑
Holger Wesche c, , Guifen Xu , Timothy J. Carlson , Matthew Plant , Kurt Morgenstern , Karen Rex ,
d
d
e
e
f
⇑
Joanna Schmitt f, Angela Coxon f, Nigel Walker b, Frank Kayser a, Zhulun Wang b,
⇑
a Department of Chemistry, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, United States
b Department of Molecular Structure and Characterization, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, United States
c Department of Oncology Research, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, United States
d Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, United States
e Department of Lead Discovery, Amgen Inc, 360 Binney Street, Cambridge, MA 02041, United States
f Department of Oncology Research, One Amgen Center Drive, Thousand Oaks, CA 91320, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogen-
esis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the pro-
duction of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the
two isoforms can compensate for each other’s loss. Thus, inhibition of both isoforms is likely required to
block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a ser-
ies of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of
human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent
inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate
the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (ꢀ/ꢀ) mice.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 8 April 2013
Revised 4 June 2013
Accepted 11 June 2013
Available online 20 June 2013
Keywords:
Sphingosine kinase 1 inhibitor
Sphingosine kinase 2 inhibitor
Murine SphK
Structure based
Drug design
The sphingolipids are a group of amino or amido di-hydroxyl-
ated lipids.1–3 In addition to their structural roles in the cell mem-
brane, it has become increasingly clear that certain sphingolipids
can act as signaling molecules. Among the sphingolipids, ceramide,
sphingosine (Sph) and sphingosine 1-phosphate (S1P) have re-
ceived considerable attention due to the roles they are reputed
to play in a broad array of biological processes including apoptosis,
mitogenesis, lymphocyte migration, radio and chemo-sensitization
and angiogenesis.4–8
SphK1 expression, severity of disease, drug resistance and reduced
patient survival.9–13 SphK1 activity has also been implicated to
play a role in immunological responses including sepsis, rheuma-
toid arthritis and asthma.4,14–16 SphK2 is less well understood
and its function remains to be clearly defined. In some instances,
SphK2 may act as a tumor suppressor, but in other cases SphK2
expression is correlated with tumor growth and injury repair. It
has been suggested that the role of SphK2 is influenced by its sub-
cellular location.5,9,17–20
Sphingosine kinase (SphK) catalyzes the ATP dependent phos-
phorylation of Sph and carries out the last committed step in the
production of S1P. There are two isoforms of sphingosine kinase;
SphK1 and SphK2. SphK1 has been shown to be up-regulated in a
number of human cancers and there is a correlation between
While SphK1 and SphK2 may have distinct and in some cases
opposing roles, observations made with SphK deficient mice sug-
gest that the two isoforms can compensate for each other, at least
during development. SphK1 knockout mice showed a 50–70%
reduction in circulating S1P levels but are otherwise comparable
to wild-type animals.21,22 Similarly, the whole embryos of SphK2
knockout mice revealed a 60% reduction in S1P, without obvious
defects.22,23 Further reduction in circulating S1P (80%) has been
noted with the ‘SphK triple allele knockout’ mouse in which both
⇑
Corresponding authors. Tel.: +1 650 244 2506.
E-mail addresses: yihongl@amgen.com (Y. Li), hwesche@amgen.com
(H. Wesche), zwang@amgen.com (Z. Wang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.030

D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4608–4616
4609
alleles of SphK1 and one copy of SphK2 have been ablated,24 sug-
gesting that circulating levels of S1P are correlated to SphK activity.
The SphK1/SphK2 double knockout genotype results in embry-
onic death on approximately day 12 of gestation. SphK1/SphK2
deficient embryos have impaired blood vessel maturation owing
to defects in the surrounding endothelium.22 Similar observations
have been made with S1P1 receptor knockout mice, suggesting that
a significant portion of these effects are due to S1P signaling
through the S1P1 receptor.25 Taken together, these results suggest
that interfering with S1P production and signaling may find use in
the treatment of diseases characterized by aberrant angiogenesis.
Indeed, the S1P1 receptor antagonist FTY720 inhibited tumor
growth and angiogenesis in a murine tumor allograft model,26
and an anti-S1P antibody has been shown to inhibit neo-vascular-
ization in both tumor models and a model of age-related macular
degeneration.27,28
Our primary interest in the SphKs was due to their role in angi-
ogenesis. Observations with SphK deficient mice suggested that
complete inhibition of both isoforms of SphK may be required in
order to block SphK driven angiogenesis. Figure 1 shows the struc-
tures and inhibition constants for both SphKs for a number SphK
inhibitors including SK1-I,29 SKI-II,30 ABC 294640,31 and inhibitors
1–3.32–34 To date, few examples of selective SphK2 inhibitors or
dual SphK1/SphK2 inhibitors have been reported.29,35,36
Herein, we describe the structure-based design of a series of
SphK inhibitors that includes examples of both potent dual
SphK1/SphK2 inhibitors and SphK1-specific inhibitors. The series
is highly selective for SphKs, has favorable physicochemical and
pharmacokinetic properties and includes the only known SphK
inhibitors that are also potent against rodent SphK1.
After screening efforts failed to identify tractable leads, we em-
barked on a structure-based design approach to create novel SphK
inhibitors. In doing so, we started with a number of design consid-
erations. First, we decided to target the Sph binding site rather
than the ATP binding site. Calculations based on the X-ray co-crys-
tal structure (see Fig. 2) of SKI-II and ADP with human SphK1 were
used to evaluate the druggability of the two binding sites.37 The
calculations showed that while the pockets are similar in enclosure
and volume, the Sph binding site is over three times as hydropho-
bic as the ATP binding site. Based on binding site surface area; the
ratios of nonpolar to total surface areas are 0.7 and 0.2, respec-
tively. Calculating druggability scores based on published models
that combine these physicochemical properties into a predictive
score yielded a score of 1.9 for the Sph site and 1.0 for the ATP
site.38,39 In this model, scores greater than 1.3 indicate druggable
pockets. Secondly, we proposed that inhibitor designs should be
Sph-like (but not SphK substrates), rather than S1P-like to avoid
building in potentially confounding activity at S1P receptors. Fur-
ther inspiration and a more concrete means to accomplish our
goals were suggested by the X-ray co-crystal structures of SphK1
in complex with SKI-II and Sph.
Figure 2A shows an overlay of the X-ray co-crystal structures of
human SphK1 with bound SKI-II and ADP (2.3 Å) and human SphK1
complexed with Sph (2.0 Å).37 The structural data indicate that
both the SKI-II and the hydrophobic tail of Sph bind to SphK1 in
a hydrophobic pocket that is formed between the strands b10-
b12 of the b-sandwich in the C-terminal domain and helices
a7–
a9. The para-chlorophenyl portion of SKI-II is positioned in the
back of the pocket occupying largely the same location as carbons
15–18 of Sph. In this binding mode, the phenol of SKI-II is located
near Asp178. Removal or methylation of the phenol oxygen of SKI-
II resulted in a complete loss of SphK inhibition. This, in combina-
tion with the structural data, suggested that SKI-II interacts with
SphK in part through donation of a hydrogen bond to Asp178.
The crystallographic data also suggested that Sph interacted with
SphK through donation of a hydrogen bond to Asp178, in this case,
from the secondary hydroxyl on C(3) (Fig. 2A).
Moreover, this hydroxyl group on C(3) appeared to make water
mediated hydrogen bonding interactions with the side chain of
Ser168, the main chain carbonyl of Ala339 and the backbone amide
of Gly342. Carbons 1 and 2 of Sph extend past Asp178 into a rela-
tively polar pocket where the C(1) hydroxyl group appeared to do-
nate a hydrogen bond to Asp81. Interestingly, the C(2) amine of
Sph is located nearly equidistantly between aspartates 81 and 178.
We envisioned that it might be possible to generate SphK inhib-
itors meeting our criteria by simply merging the structures of Sph
and SKI-II. As illustrated in Figure 2B, our plan called for the syn-
thesis of chemical libraries of general formula 4. We initially in-
tended to evaluate the SAR around the para-chlorophenyl
subunit, while at the same time preparing libraries that presented
both hydrogen bond donors and acceptors with the aim of mimick-
ing the hydrogen bonding interactions observed in the crystal
structures.
Cl
OH
H
N
Cl
N
O
N
H
S
After preparing a number of libraries, compound 6 (Table 1) was
identified as a lead. In 6, the para-chlorophenyl substituent on the
thiazole of SKI-II was replaced with a 2-napthyl group that was
identified in an earlier library. Additionally, the phenol of SKI-II
was replaced with an ethylene linker with an (S)-pyrrolidin-2-
ylmethanol group at its terminus. As shown, 6 had an IC50 of
N
SKI-II
ABC 294640
Ki (µM), SphK1
0.5
---
---
9.8
SphK2
NH2
H
N
OH
Me
Me
0.99 and 2.5 lM against SphK1 and SphK2 respectively. X-ray crys-
OH
NHMe
O
OH
tallography showed 6 bound to SphK in the Sph binding site as ex-
pected. Furthermore, 6 was not a substrate for either SphK1 or
SphK2 and showed no binding to S1P receptors 1 or 3 at concentra-
tions below 25 lM. Given that 6 met our initial design criteria, we
investigated the SAR of 6 with the goal of optimizing it for SphK
1
SK1-I
10
>100
1.4
31
Ki (µM), SphK1
H2N
NH HCl
SphK2
O
O
N
inhibition and physico-chemical properties (Table 1).
Me
NH HCl
NH2
N
H
Comparison of 6 to its enantiomer (7) revealed a slight prefer-
ence for the (R)-stereoisomer, while methylation of the hydroxy-
methyl group resulted in a loss of potency (cf. compounds 6, 7
and 8). Comparison of 2-hydroxymethyl substituted azetidine, pyr-
rolidine, piperidine and azepane rings indicated that the piperidine
ring was optimal (cf. compounds 5, 7, 9 and 10). Relocation of the
hydroxyl group to either the 3 or 4 position of the piperidine ring
or extending the hydroxymethyl group by one carbon resulted in a
N
O
2
3
Ki (µM), SphK1
0.2
0.5
0.05
4.2
SphK2
Figure 1. Representative SphK inhibitors.

4610
D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4608–4616
B
A
Cl
NH2
HO
OH
HO
N
C12H25
N
S
H
SKI-II
Sph
optimize
aryl-group
R1
R2
N
R
amine
libraries
X
N
S
N
H
4
Figure 2. (A) Overlay of the 2.3 Å X-ray co-crystal structure of SphK1 with bound SKI-II (violet) and ADP (gray) (PDB code: 3VZD) and the 2.0 ÅX-ray co-crystal structure of
SphK1 with bound Sph (green) (PDB code: 3VZB). (B) Initial chemical library design for SphK inhibitors.
complete loss of SphK inhibition (cf. compounds 9, 11, 12, and 13).
Examination of the individual stereoisomers of 9 again revealed
that the (R) absolute configuration was preferred and in this case
a 14-fold separation in activity between the enantiomers was ob-
served (cf. compounds 9, 17 and 18).
Having identified the (R)-2-hydroxymethylpiperidine as a key
fragment, substitutions at the 4-position of the piperidine were
evaluated. As shown in Table 1, the (4-methylpiperazin-2-yl)meth-
anol 14, morpholin-3-ylmethanol 15, and 3-(hydroxymethyl)thi-
omorpholine-1,1-dioxide 16 analogs were poorly tolerated. In
contrast, addition of a single hydroxyl group of (S)-stereochemistry
The synthesis of these compounds is shown in Schemes 1 and 2,
and begins with the preparation of the 2-(hydroxymethyl)
piperidin-4-ol fragment.42 Activation of allyl alcohol as its p-tolu-
enesulfonate, followed by SN2 displacement with (R)-1-phenyleth-
ylamine and iminium cyclization with gloxylic acid provided a
mixture of diastereomeric bicyclic lactones 31 and 33.43,44 An anal-
ogous set of transformations starting with 2-methylprop-2-en-1-ol
provided a mixture of lactones 32 and 34. In both cases, the diaste-
reomers obtained were easily separated. Reduction of 31 or 33
using LiBH4 in tetrahydrofuran provided the amino-diols 35 and
36 respectively in high yield. Cleavage of the phenyethyl-group
of 35 and 36 provided 38 or 39 after ion-exchange chromatogra-
phy. Coupling of 38 with 2-(4-nitrophenyl)acetaldehyde under
reductive amination45 conditions provided intermediate 41.
Reduction of 41 followed by treatment of the aniline obtained with
101-thiocarbonyldiimidazole and excess NH3(g) resulted in thio-
urea 43. Finally, thiazole synthesis was accomplished by heating
a mixture of 43 with 2-bromoacetophenone to afford 70.41 On
some occasions thio-acylation of the hydroxyl groups occurred
during the preparation of 43 leading to variable yields. Addition-
ally, handling of 43 proved difficult due to its polarity. To stream-
line the synthesis, we protected both hydroxyls. Thus, treatment of
diol 35 with TBDPS-Cl, Et3N and Verkade’s super-base provided
bis-silyl ether 37.46 Reductive amination with 2-(4-nitrophenyl)
acetaldehyde provided 42. Thiourea synthesis as described above
provided 44 which proved to be much easier to handle and gave
consistently better results compared to 43. Thiazole synthesis
using 44 proceeded without incident and removal of the TBDPS
protecting groups was easily accomplished by simply adding 70%
HF in pyridine to the reaction mixture once thiazole formation
was complete.
resulted in
a 4-fold improvement in SphK1 inhibition (cf.
compounds 17 and 19), whereas a C(4)-hydroxyl group of (R)-con-
figuration was detrimental (cf. compounds 17, 19 and 20). The
(2R,4S)-2-(hydroxymethyl) piperidin-4-ol analog (19) was an
approximately 23-fold more potent than its enantiomer 21. Lastly,
compound 22 which bears an additional methyl group in the
4-position of the piperidine was 10-fold less potent than 19.
It was found that there was a preferred distance between the
secondary alcohol on the piperidine (or pyrrolidine) and the anili-
no-thiazole fragment. Compound 23 (Table 2, which contains a
3,4-dichlorophenyl substitution for the 2-napthyl group found in
compound 19) inhibited SphK1 and Sphk2 with IC50s of 0.02 and
1.6 lM respectively. Extending the linker by a single methylene
group resulted in a 100-fold loss in potency against SphK1 and a
complete loss of SphK2 inhibition (cf. compounds 23 and 24). In
contrast, combination of tethers consisting of either 2 or 3 methy-
lene groups with a (3R,5R)-5-(hydroxymethyl)pyrrolidin-3-ol sub-
unit indicated that the 3-carbon tether was preferred with the
smaller pyrrolidine ring (cf. compounds 25 and 26). The same trend
was observed with the diastereomeric (3S,5R)-5-(hydroxy-
methyl)pyrrolidin-3-ol moiety (cf. compounds 27 and 28). Compar-
ison of compounds 25–28 demonstrated that the stereochemistry
of the pyrrolidine secondary alcohol was less critical compared to
the piperidines. In fact, in the case of the pyrrolidines, the trans-diol
configuation was more potent against both SphK isoforms. Com-
pound 28 inhibited SphK1 with potency comparable to that of com-
The synthesis of compounds 26 and 28 (Scheme 2) begins with
Cbz-protected D-cis-hydroxyproline, 45. Reduction of 45 with bor-
ane in THF, followed by cleavage of the Cbz group and treatment
with HCl in MeOH provided the hydrochloride salt 47. The prepa-
ration of 28 began with esterification of 45 with (trimethyl-
silyl)diazomethane followed by Mitsunobu inversion47 of the
C(3) hydroxyl with DIAD and para-nitrobenzoic acid to give 49.
Reduction of 49 with LiBH4 gave 50. Lastly, hydrogenolysis and
acidic work-up gave the hydrochloride salt 51. The synthesis of
the thiazole containing fragment of 26 and 28 started with 1-
(4-(3-hydroxypropyl)phenyl)thiourea (53) which was prepared
pound 23 (IC50s = 0.03 and 0.02
significantly improved inhibition of SphK2 (IC50 = 0.09
l
M
respectively) and had
M), dem-
l
onstrating dual SphK1/2 inhibitors existed within this series of
compounds. Unfortunately, compound 28 and related pyrrolidine
based compounds had poor pharmacokinetic properties and thus
were not developed further.41 By comparison, the piperidine-based
inhibitors had reasonable pharmacokinetic properties in both rats
and mice (vide infra) and were developed further.
from 3-(4-aminophenyl)propan-1-ol,
ammonia and
101-
thiocarbonyldiimidazole as described above. Reaction of 53 with
2-bromo-1-(3,4-dichlorophenyl)ethanone in ethanol provided 57,

D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4608–4616
4611
Table 1
Human SphK1 and SphK2 biochemical assay data for compounds 5-22a
R
S
N
H
N
Compound
R
SphK1
SphK2
Compound
R
SphK1
SphK2
IC50
(l
M)
IC50
(
lM)
IC50
(lM)
IC50
(lM)
Me
N
N
OH
5
1.6
2.5
14
1.0
1.7
N
O
N
OH
OH
N
N
6
7
0.99
0.58
2.5
2.7
15
3.1
6.1
>10
3.0
OH
O
O
S
16b
OH
N
N
OH
OH
OH
N
N
8
9
2.3
5.7
2.9
17
18
0.08
1.1
1.6
7.2
OMe
OH
0.14
N
OH
N
10
0.92
>10
19
0.02
0.76
N
OH
OH
OH
OH
OH
OH
N
11
12
13
2.1
1.8
>10
3.3
3.3
>10
20
21
22
0.22
0.45
0.23
1.7
2.2
3.8
N
OH
N
OH
N
Me OH
N
OH
N
OH
a
All data reported are the average of at least two individual tests. See the bibliography for a description of the assays.40
In this case, the data reported were obtained with the 3,4-dichlorophenyl replacement of the 2-naphthyl group compare to compound 23, Table 2.
b
which was converted to its p-toluenesulfonate (60) by treatment
with TsCl and triethylamine in CH2Cl2. Finally, heating a mixture
of hydrochloride salts 46 or 51 with 60 in methanol and in the
presence of potassium carbonate provided SphK inhibitors 26 or
28. Starting from the thiourea 52, an analogous set of synthetic
steps was used to prepare tosylates 58 and 59 which were further
elaborated to the compounds given in Tables 1 and 2 under similar
conditions.
Table 3 summarizes the in vitro data for a series of thiazoles
with substitutions in the 4-positon. Comparison of compounds
possessing a hydroxy group in the 4-position of the piperidine frag-
ment to those lacking this functional group, again indicated that
significant improvement in SphK1 inhibition was provided by this
single hydroxy group as noted above (cf. compounds 61 and 70; 63
and 72; 68 and 79; and 69 and 80). The contribution of this hydro-
xy group was particularly apparent within the context of com-
pounds with small substituents in the 4-position of the thiazole.
For example, compounds 61 and 70 in which an un-substituted
phenyl ring is appended to the thiazole, showed that the hydroxyl
group in question provided a 100-fold boost in potency against
SphK1. However, the hydroxyl group did not similarly affect po-
tency against SphK2 and thus compound 70 was selective for SphK
isoform 1. The differentiation between SphK isoforms noted with
compounds 19 and 70 was typical of compounds bearing aliphatic
groups or 3,4 di-substituted aryl rings on C(4) of the thiazole. An
example is compound 83 (Table 3, R2 = n-butyl) which inhibited
SphK1with an IC50 of 0.07 lM and was over 70-fold selective for
SphK1 versus SphK2. Likewise, cyclopentyl, cyclohexyl and 1-ada-
mantyl groups (compounds 86–88) showed at least a 40-fold sep-
aration between SphK isoforms.
In general, compounds having lipophilic and electron-with-
drawing groups on the aryl ring bound to the thiazole had im-

4612
D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4608–4616
Table 2
R
R
Human SphK1 and SphK2 biochemical assay data for compounds 23–28a
O
O
O
O
d, e
R
a, b, c
R
Cl
Cl
S
N
N
OH
N
H
N
Ph
Me
Ph
Me
29, R = H
30, R = Me
31, R = H
32, R = Me
33, R = H
34, R = Me
Compound
R
SphK1
SphK2
IC50
(l
M)
IC50
(lM)
OR1
R
OR1
N
R
23
24
0.02
1.6
f , g
h
HO
OH
OR1
OR1
N
N
H
N
N
N
2.5
>10
HO
Ph
Me
OH
OH
35, R = H, R1 = H
38, R = H, R1 = H
N
36, R = Me, R1 = H
39
, R = Me, R1 = H
25
26
27
28
0.13
0.03
0.41
0.03
0.09
0.27
0.25
0.09
, R = H, R1 = TBDPS
40, R = H, R1 = TBDPS
HO
37
OH
OR1
i, j
OR1
HO
N
N
S
N
OH
OR1
OR1
H2N
O2N
N
H
HO
, R1 = H
, R1 = H
43
41
OH
42, R1 = TBDPS
44, R1 = TBDPS
HO
a
OH
All data reported are the average of at least two individual tests. See the bib-
liography for a description of the assays.38
k, l
N
S
N
N
OH
H
proved inhibition of both SphK1 and SphK2. For example, installing
a chlorine atom in the ortho, meta or para position provided a 7-, 4-
and 9-fold potency improvement against SphK1, respectively and a
3-, 9-, and 14-fold improvement (respectively) in SphK2 inhibition
(cf. compounds 70, 72, 74 and 79). In contrast, while methoxy-
substituted phenyl groups were tolerated by SphK1; modest po-
tency losses were seen with the same compounds against SphK2
(cf. compounds 70, 76 and 81). Some of the most interesting results
were obtained with a series of compounds in which the terminal
aryl ring carried either a fluorine or trifluoromethyl substituent.
A single fluorine atom in either the meta or para position resulted
in a 3- to 4-fold loss in both SphK1 and SphK2 inhibition (cf. com-
pounds 70, 73 and 78). By comparison, the ortho-fluorophenyl ana-
log 71, showed a 4-fold improvement in SphK1 inhibition and a 6-
fold improvement against SphK2. Similar to the results seen with
chlorine, addition of a trifluoromethyl group to either the meta
(77) or para (82) position resulted in a significant improvement
in both SphK1 (4- to 14-fold) and SphK2 (8- to 13-fold) inhibition.
Lastly, combination of the SAR seen with the fluorine and trifluoro-
methyl groups proved to be additive and resulted in the identifica-
tion of compounds 89-91 which displayed inhibitory activities of
70
Scheme 1. Synthesis of compound 70 (Table 3). Reagents and conditions: (a) p-
TsCl, Et3N, THF; (b) (S) or (R)-1-phenylethylamine, Et3N, THF 23 °C 40 h; (c)
glyoxylic acid, water 60 °C, 60% overall. (d) Diastereomer separation. (e) LiBH4, THF,
23 °C 90–96%. (f) Verkade superbase, TBDPS-Cl, Et3N, DMF, 60 °C, 71%. (g) H2,
Pd(OH)2, with either EtOAc or EtOAc/MeOH TFA as solvents. (h) 2-(4-Nitro-
phenyl)acetaldehyde, AcOH, NaHB(OAc)3, 51–85%. (i) H2, 10% Pd/C, EtOH. (j) 101-
Thiocarbonyldiimidazole, THF, 23 °C, 30 min; NH3. (g) Sparge 10 min; 75–90%. (k)
2-Bromoacetophenone, EtOH, 75 °C, 66%. (l) HF, pyridine, EtOH.
unexpected as qPCR experiments indicated that SphK2 expression
was significantly lower than SphK1 expression in these cells (data
not shown). The data obtained in WM266-4 cells was used to dif-
ferentiate compounds based on physical-chemical properties. For
example, while compounds 77 and 85 exhibited comparable pro-
files in the biochemical assays, compound 77 was over 10-fold
more potent in these tumor cells. Similarly, compounds 88–91
were significantly more potent in the biochemical assays com-
pared to compounds 72 and 82. However, there was no associated
improvement in potency in WM266-4 cells. These results may be
due to the high concentration of Sph used in the assay. To illus-
less than 0.01 lM against SphK1 and as low as a 4-fold separation
between the two isoforms of SphK. The data provided in Table 3
along with compounds 19 and 23, showed that SphK1 tolerated a
much wider set of thiazole substituents compared to SphK2 within
this series of inhibitors and provides a means of targeting both iso-
forms of SphK or of blocking SphK1 selectively by the judicious
choice of the thiazole substituent.
Table 3 also displays data for the inhibition of SphK activity in
the context of the human melanoma cell line WM266-4. In this as-
say, WM266-4 cells were incubated with 5 lM C17-Sph resulting
in the intracellular, SphK dependent production of C17-S1P which
was quantified by Rapid-fire™ LC–MS analysis.48,49 As the data
show, this series of compounds were potent inhibitors of SphK
activity in WM266-4 cells and there was a correlation between
potency in our cell-based assay and in our SphK1 biochemical
assay. However, there was no obvious relationship between SphK2
activity and potency in WM266-4 cells. This was not entirely
trate, compound 82 had an IC50 of less than 0.004
the endogenous levels of S1P in WM266-4 cells compared to an
IC50 of 0.09 M in the presence of exogenously added Sph under
lM in reducing
l
the same conditions. Thus, the free fraction of individual com-
pounds under the assay conditions may account for the results.
The structure activity relationship of these SphK inhibitors can
be rationalized in part through the X-ray co-crystal structure of
compound 23 with SphK1 shown in Figure 3. Compound 23 bound
to SphK1 in the same pocket as Sph and occupied space along the
entire length of Sph. As expected, the tri-aryl fragment of 23 was
oriented like SKI-II (Fig. 3A). In this position the two chlorine atoms
are located in a hydrophobic pocket composed of residues on heli-
ces a9 and 310, strands b10–b12, and the loop b10–b11. The chlo-
rine in the meta-position made van der Waals contacts with
Met306, Leu261 and Leu268. At the same time, the chlorine in
the para-position appeared to make van der Waals contacts with

D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4608–4616
4613
O
NO2
HO
HO
a, b
O
HO
e, f
c, d
CO2R
CO2Me
N
N
Cbz
N
Cbz
N
OH
R2
OH
R2
50, R2 = Cbz
46, R2 = Cbz
45, R = H
48
49
, R2 = H HCl
51
, R2 = H HCl
, R = Me
R3
47
n
g
n
h
OH
OH
S
N
H2N
N
H
S
N
H
R
3 = 2-napthyl
52,
53, n = 2
n = 1
54.
,
OH
n = 1
55, n = 1, R3 = 3,4-diCl-Ph
56, n = 2, R2 = 3,4-diCl-Ph
N
HO
R3
n
OTs
i
N
S
NH
S
N
H
N
Cl
3 = 2-napthyl
58.
,
26.
3R
28, 3S
n = 1
R
59, n = 1, R3 = 3,4-diCl-Ph
60, n = 2, R2 = 3,4-diCl-Ph
Cl
Scheme 2. Synthesis of compounds 26 and 28 (Table 2). Reagents and conditions: (a) BH3, THF 65 °C, 2 h 98%. (b) H2, (1 atm), 20% Pd(OH)2/C, EtOH, 17 h, then HCl in MeOH
83%. (c) TMSCHN2, MeOH (quantitative); (d) p-nitrobenzoic acid, Ph3P, DIAD, THF, 3 min 98%. (e) LiBH4, THF 23 °C, 58%. (f) H2, (1 ATM), 20% Pd(OH)2/C EtOH, then HCl in
MeOH 90%. (g) 2-bromo-30,40-dichloroacetophenone or 2-bromo-1-(napthalen-2-yl)ethanone, EtOH, 60 °C, 10 min-1 h, 52–76%. (h) p-toluenesulfonyl chloride, Et3N, CH2Cl2,
60–90%. (i) 46 or 51, K2CO3, MeOH/THF 80 °C, 17 h.
Table 3
Phe288, His311 and Leu319. In SphK2, amino acid 288 is cysteine
Human SphK1 and SphK2 biochemical assay data and WM266-4 cell assay data for
compounds 61–91a
rather than the phenylalanine of isoform 1. A deeper pocket, due
to the smaller size of cysteine relative to phenylalanine, may ex-
plain why a substituent in the meta or para position of the aryl-thi-
azole unit was required for potent inhibition of SphK2.
R1
N
S
R2
One of the most satisfying discoveries made from the X-ray co-
crystal structure of compound 23 and SphK1 was that the hydroxyl
groups of 23 appeared to mimic the locations and interactions
noted in the crystal structure of Sph with SphK1. The overlay of
the co-crystal structures of Sph and compound 23 with SphK1
showed that the oxygen atom of the hydroxyl in the piperidine-
4-position lay in a position like the C(1) hydroxyl of Sph, where
the angle (120°) and distance (2.8 Å) to the carboxyl group of
Asp81 suggested hydrogen bonding. Likewise, the oxygen atom
of the piperidine hydroxymethyl-group was located at an angle
just over 120° and distance of 2.5 Å from the carboxyl of Asp178
and again suggested the presence of a hydrogen bond. The oxygen
of the piperidine-hydroxymethyl group also appeared to partici-
N
N
H
OH
SphK2
Compound
R1
R2
SphK1
(IC50
WM
266-4
(EC50
lM)
(IC50
lM)
l
M)
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
H
H
H
H
H
H
H
H
Phenyl
2-F-phenyl
2.0
3.7
2.1
1.9
5.5
5.4
1.7
1.2
0.37
—
0.12
0.15
1.1
2-Cl-phenyl
2-Me-phenyl
2-OMe-phenyl
2-CF3-phenyl
3-Cl-phenyl
4-Cl-phenyl
4-CN-phenyl
Phenyl
2.1
0.17
0.27
0.08
>10
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
0.07
0.02
0.01
0.32
0.02
0.01
0.05
0.005
0.36
0.008
0.007
0.03
0.02
0.07
>5
0.005
0.01
0.13
0.003
0.005
0.004
0.003
1.3
0.31
0.13
0.08
1.2
pate in
a water mediated hydrogen bonding network with
2-F-phenyl
0.22
0.43
>5
0.15
0.70
3.0
Ala339, Gly342, Asp341 and Ser168 like that seen with Sph. Aspar-
tate 178 appeared to interact with compound 23 in a bi-dentate
manner in which one oxygen atom participated in the hydrogen
bonding network with the hydroxymethyl group, while the other
engaged in a salt-bridge with the piperidine nitrogen. It is interest-
ing to note that Asp178 is rotated 90° in complex with compound
23 compared to its complex with SKI-II. In addition to these polar
interactions, the piperidine fragment completely filled the polar
pocket containing Asp81 and Asp178. Recently, investigators at
Pfizer disclosed the structure of PF-543, a SphK inhibitor that
incorporates a (R)-2-hydroxymethylpyrrolidine subunit.50 While
to the best of our knowledge, no X-ray co-crystal data exist for
PF-543, one may infer from our results that the pyrrolidine of PF-
543 binds SphK in a manner similar to that of the SphK inhibitors
described here.
2-Cl-phenyl
3-F-phenyl;
3-Cl-phenyl
3-Me-phenyl
3-OMe-phenyl
3-CF3-phenyl
4-F-phenyl
4-Cl-phenyl
4-Me-phenyl
4-OMe-phenyl
4-CF3-phenyl
n-butyl
t-butyl
1-Adamantyl
Cyclohexyl
Cyclopentyl
2,4-Dichloroph
2F,3CF3phenyl
2F,4CF3phenyl
2F,5CF3phenyl
0.11
0.87
0.12
1.2
0.17
4.6
0.09
0.11
0.94
0.10
>5
>10
0.20
0.39
>5
0.04
0.04
0.02
0.05
0.13
0.10
0.09
0.37
1.9
0.28
0.85
0.36
0.25
0.12
0.39
In moving forward, we chose to profile compounds 82 and 88 in
greater detail.51 Table 4 summarizes the pharmacokinetic parame-
ters of these compounds in rat. Each of these compounds showed
moderate clearance in rat. Compound 88, while more potent in
a
All data reported are the average of at least two individual tests. See the bib-
liography for a description of the assays.38

4614
D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4608–4616
Figure 3. (A) Overlay of the co-crystal structures of SphK1 with compound 23 (cyan) and Sph (green). (B) Co-crystal structure of SphK1 with compound 23 (PDB code: 4L02)
highlighting the piperidine moiety.
on either S1P receptors 1 or 3, no significant activity at concentra-
tions below 10 M against an internal panel of GPCRs, kinases and
ion channels. Table 4 also shows the potencies of these compounds
against murine SphK1 (mSphK1) and murine SphK2 (mSphK2). As
indicated, compounds 82 and 88 inhibited mSphK1 with IC50s of
Table 4
l
Pharmacokinetic parameters in rats, murine SphK1 and SphK2 biochemical data and
WM266-4 cell assay data for compounds 82 and 88a
Compound
82
88
CL (L/kg/h)b
MRT (h)b
1.4
12
17
0.04/0.03
0.07
>10
1.3
14
18
0.02/0.01
0.02
>10
0.07 and 0.02 lM, respectively. However, neither showed any
Vdss (L/kg)b
Fub (h/r)c
appreciable inhibition of mSphK2 at concentrations below 10
lM.
These results were typical for this series of compounds.
mSphK1 (IC50
mSphK2 (IC50
WM266-4 (IC50
,
,
l
l
l
M)
M)
M)
The in vivo activity of compound 82 was evaluated in a pharma-
codynamic assay in which circulating levels of S1P and plasma lev-
els of 82 were examined.51 Compound 82 was administered by oral
gavage daily to athymic nude mice at a dose of 100 mg/kg. On days
1, 4 and 7, plasma was collected at trough levels and S1P and plas-
ma drug concentration were determined (Fig. 4A and B). Treatment
with compound 82 led to a significant (p <0.0001) sustained de-
crease of S1P that correlated with drug exposure through day 7.
The vehicle treated groups had plasma S1P levels of approximately
0.09
0.36
a
Values are means of at least two individual test subjects. See the bibliography
for a description of the assays.
b
Following 0.5 mg/kg dose iv.
c
Determined by ultra-centrifugation as described previously.52
our biochemical assays, had a free fraction half of that seen with
compound 82 in human and rat plasma. While circumstantial, this
observation is consistent with our assertion that the correlation
between the performance of these inhibitors in our biochemical
and whole cell assays may be related to the free fraction of individ-
ual inhibitors under the assay conditions. In mice, compound 82
was cleared at a rate of 0.7 L/kg/h, had a MRT of 13 h and reason-
able bioavailability (32% at 5 mg/kg in methylcellulose). In addition
to having favorable PK parameters, compound 82 was very selec-
tive for SphKs and showed no binding or receptor internalization
320 lg/L, which was comparable to S1P levels in wild type mice re-
ported by Hla.24
In conclusion, using a combination of structure-based design
and focused chemical library synthesis; we identified a novel series
of SphK inhibitors. A critical structural feature of this series of SphK
inhibitors is the (2R,4S)-2-(hydroxymethyl)piperidin-4-ol moiety
which appeared to make key hydrogen bonding interactions with
Asp81 and Asp178 of SphK1. Overall, the series contains highly po-
tent and selective SphK inhibitors with favorable physicochemical
properties and reasonable PK parameters. The identification of
B
A
0.20
500
100 mg/kg
400
0.15
0.10
Vehicle
300
200
*
*
*
100 mg/kg
IC50 = 0.076 µM
0.05
100
0
* = p<0.0001
0.00
0
2
4
6
8
0
2
4
6
8
Time [Day]
Time [Day]
Figure 4. (A) S1P levels and (B) unbound plasma concentration of compound 82 following oral dosing in athymic nude mice. Data are mean SE (n = 5). P values correspond
to statistical difference between groups treated with vehicle and compound 82 by Oneway Anova followed by Dunnett’s post hoc test. S1P and drug concentration determined
by LC–MS/MS.

D. J. Gustin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4608–4616
4615
33. 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. J. Med. Chem. 2011, 54, 3524.
34. 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. J. Med. Chem.
2010, 53, 2766.
35. Raje, M. R.; Knott, K.; Kharel, Y.; Bissel, P.; Lynch, K. R.; Santos, W. L. Bioorg.
Med. Chem. 2012, 20, 183.
36. Beljanski, V.; Knaak, C.; Zhuang, Y.; Smith, C. D. Invest. New Drugs 2011, 29,
1132.
37. 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. Structure 2013, 21, 798.
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these SphK inhibitors represents advancement in several areas.
First, this series contains the only potent inhibitors of both human
isoforms of SphK yet described. Second, these SphK inhibitors are,
to the best of our knowledge, the most potent inhibitors of rodent
SphK1 and the only series of SphK inhibitors with a combination of
potency and drug-like properties to enable the effective in vivo
inhibition of SphK1 in mice.53 We fully expect that these com-
pounds will complement existing SphK inhibitors and find use in
elucidating SphKs biological role(s) more precisely.
39. Halgren, T. A. J. Chem. Inf. Model 2009, 49, 377.
Acknowledgement
40. The ability of a test compound to inhibit SPHK was determined in the following
manner: A solution of test compound in DMSO (at various concentrations) was
added to a mixture containing purified SPHK1 enzyme, 1.11 mM sphingosine
(Enzo Life Sciences, cat# EI155-0025—resuspended in DMSO), 1 mM ATP,
0.375 mCi [g-33P] ATP, 0.005% Triton X-100, and 2 mM DTT in 50 mM HEPES at
pH 7.5 and containing 3 mM MgCl2, and 10 mM NaF. The assay mixtures were
incubated in 96-well Lumitrac 600 high-binding plates (E&K Scientific, cat#
EK-25074) at 30 °C for 50 mins before being stopped by the addition of 2.5 M
guanidine HCl and incubated for an additional 30 min. Assay plates were then
washed three times with 200 mL of PBS per well and allowed to air dry for
20 mins. Under these conditions the 33P-S1P produced coats the interior of the
assay plate well. Finally, 50 mL of MicroScint-20 (PerkinElmer, cat# 6013621)
was dispensed into each well and the 33P-S1P produced was quantitated using
a TopCount detector (PerkinElmer). The determination of a test compounds
ability to inhibit SPHK2 was determined using the conditions described for
SPHK1 using an assay buffer in which 0.005% Triton X-100 was replaced with
0.0025% NP-40. All other conditions were the same. The ability of a test
compound to inhibit SPHK activity in whole cells was determined in the
following manner: One million cells and a test compound were incubated in
serum-free culture media (Cellgro MEM-10-010-CV) for 20 min. at 37 °C in 5%
CO2. C17-Sphingosine (Avanti Polar Lipids) was added and the mixture
incubated for 15 mins. The cells were pelleted by centrifugation. The
supernatant was decanted before sealing the plates and freezing at ꢀ80 °C.
Frozen cell pellets were thawed and 75 mL of methanol was added to each
well. The samples were then transferred to a 96 well PCR plate sealed and
pulsed for 6 s per well (E200-S0%250 mV500 setting) using a Covaris AFA cell
disruptor. Dichloromethane was added and the plate incubated at room
temperature for 2 h. 52 mL of H2O was added and the plates were centrifuged.
70 mL of the upper phase was transferred to a new PCR plate and mixed with
70 mL of (80:20) MeOH:H2O, 2% formic acid and 150 mM ammonium acetate.
We thank Bill Romanow and Steve Thibault for providing pro-
tein support for the structural work.
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a RapidfireTM
chromatography system interfaced with TSQ Vantage triple stage
a
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