Design, synthesis and biological activity of sphingosine kinase 2 selective inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.11.011

Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in cell survival. SphK phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has been implicated in cancer growth and survival. SphK ex

Full text of DOI:10.1016/j.bmc.2011.11.011

Bioorganic & Medicinal Chemistry 20 (2012) 183–194
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological activity of sphingosine kinase 2
selective inhibitors
Mithun R. Raje a, Kenneth Knott a, Yugesh Kharel b, Philippe Bissel a, Kevin R. Lynch b, Webster L. Santos a,
⇑
a Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, United States
b Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in
cell survival. SphK phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has been
implicated in cancer growth and survival. SphK exists as two different isotypes, namely SphK1 and
SphK2, which play different roles inside the cell. In this report, we describe SphK inhibitors based on
the immunomodulatory drug, FTY720, which is phosphorylated by SphK2 to generate a S1P mimic. Struc-
tural modification of FTY720 provided a template for synthesizing new inhibitors. A diversity-oriented
synthesis generated a library of SphK inhibitors with a novel scaffold and headgroup. We have discovered
subtype selective inhibitors with Ki’s in the low micromolar range. This is the first report describing qua-
ternary ammonium salts as SphK inhibitors.
Received 24 September 2011
Revised 1 November 2011
Accepted 7 November 2011
Available online 15 November 2011
Keywords:
Sphingosine kinase
Structure–activity relationships
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Sphingosine
Lipid
FTY720
Kinase inhibitor
Sphingosine-1-phosphate
Reductive amination
1. Introduction
downregulation.14 The resultant absence of an S1P signal modu-
lates immune function by influencing lymphocyte trafficking, spe-
Sphingosine 1-phosphate (S1P), an intermediate in the
sphingomyelin metabolism pathway, is emerging as a potential
target in cancer therapeutics.1 S1P regulates important cellular
and physiological processes, including cell motility,2 invasion,3
and angiogenesis4 by functioning as a specific ligand for a family of
five G-protein coupled receptors of the rhodopsin family (S1P1–5).5
Recently, histone deacetylase (HDAC) has been identified as a
direct intracellular target of nuclear S1P.6 S1P inhibits HDACs 1
and 2 in repressor complexes, increases histone acetylation and en-
hances gene transcription of p21 and c-fos. Studies linking S1P to
cell proliferation7 and suppression of apoptosis8 have generated
substantial interest about its role in many diseases.9 The approval
of fingolimod (FTY720, Gilenya™) by the Food and Drug Adminis-
tration in 2010 for use in treating relapsing-remitting multiple
sclerosis10,11 underscores the potential of regulating the S1P
pathway for treating disease.12 FTY720 is phosphorylated in vivo
by SphK2 to FTY720-P,13 which induces internalization and
degradation of the S1P1 receptor resulting in prolonged receptor
cifically by decreasing the egress of lymphocytes from secondary
lymphoid tissues.
S1P biosynthetic precursors, in particular ceramide, have been
identified as inducers of apoptosis.15 Since these metabolites are
interconvertible by the actions of various enzymes, a ceramide/
S1P rheostat has been proposed as a determinant of cell fate
(Fig. 1).8 As a result, enzymes in the cell that regulate this pathway
are potential targets for cancer therapeutics. Since S1P is the prox-
imal effector, sphingosine kinase (SphK) plays a crucial role in the
control of this balance. Two isoforms of SphK, SphK1 and SphK2,
have been isolated and characterized.16 It has been hypothesized
that SphK1 and SphK2 might be involved in different cellular func-
tions17 due to differences in their localization within the cell18 (i.e.,
SphK1 is located mainly in the cytoplasm, SphK2 is located in the
nucleus and endoplasmic reticulum), differences in selectivity19
and opposing functions in sphingolipid metabolism.20 Studies have
shown that SphK1 is up-regulated in a variety of solid tumors21
and promotes cell survival while SphK2 is pro-apoptotic because
of its BH3 domain that interacts with BCLXL.22 However, a recent
study suggests an important role for SphK2 in the tumor progres-
sion in MCF-7 breast cancer xenografts.23 These contrasting results
demonstrate the need for a deeper understanding of the role of
⇑
Corresponding author. Tel.: +1 540 231 5742; fax: +1 540 231 3255.
E-mail address: santosw@vt.edu (W.L. Santos).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.011

184
M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
OH
OH
NH
O
Ceramide
Promotes cell death
Ceramide
synthase
Anti-mitogenic
Ceramidase
OH
OH
NH2
Sphingosine (Sph)
Sphingosine-1-
phosphate
Sphingosine kinase
OH
phosphatase
O
P
O-
O-
Promotes cell growth
Mitogenic
O
NH2
Sphingosine-1-phosphate (S1P)
Figure 1. The ceramide/sphingosine-1-phosphate rheostat.
SphK2 in tumor cell models. The widely used inhibitors of SphK,
L
-
were obtained either via screening of commercial small molecule
libraries (ABC294640)33 or by synthesizing analogues of sphingo-
sine (SG-12).34 Recently, the methyl ether of FTY720
((R)-FTY720-OMe) was reported as an inhibitor of SphK2 with Ki
threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine
(DMS),24 are not selective as they also inhibit other enzymes such
as protein kinase C25 and sphingosine-dependent protein kinase
(Fig. 2).26 Indeed, the need for subtype-selective inhibitors has
not gone unnoticed. Recently, a water-soluble, isoenzyme-specific
inhibitor of SphK1, SK1-I, was shown to induce apoptosis in human
leukemia cells and reduced growth of AML xenograft tumors.27 In
another study, SK1-I markedly reduced the tumor growth rate of
glioblastoma xenografts and induced apoptosis.28 SphK1 inhibitors
based on replacing the aminodiol in sphingosine by a serine amide
were reported29 (12aa) and their structure was optimized to in-
crease water solubility and oral bioavailability.30 The most potent
and selective SphK1-selective inhibitors reported to date featured
amidine-based groups (28).31,32 While several studies generated
SphK1 selective inhibitors, reports of SphK2-selective compounds
are scarce. SphK2-selective inhibitors reported in the literature
of 16.5 l
M.35 Unfortunately, rational design of sphingosine kinase
inhibitors has been hampered by the lack of crystal structure of
either protein. While it has been suggested that the C4 domain is
involved in specific recognition of Sph via the interaction of the ba-
sic amine in sphingosine with the Asp177 36
rational design of SphK
,
inhibitors remains a challenge.
Inhibitors of SphKs with the combination of drug-like proper-
ties, potency, and selectivity will be valuable in evaluating these
enzymes as therapeutic targets. The lack of potent SphK2-selective
inhibitors retards detailed scientific studies exploring the role of
SphK2 in many diseases. Herein, we report the discovery of a novel
scaffold and structure-activity studies that reveal SphK2-selective
inhibitors.
OH
OH
OH
OH
C13H27
C13H27
OH
OH
HN
N
NH2
NH2
linker
C5H11
DHS
DMS
SK1-I
OH
head
OH
C8H17
lipophilic
group
HN
H
Cl
N
N
O
O
FTY720
tail
OH
HN
C8H17
12aa
ABC294640
MeO
NH2
NH2
OH
O
Cl
OH
NH2
N
H
OH
NH2
C8H17
C8H17
C12H25
SG-12
(R)-FTY720-OMe
28
Figure 2. Reported sphingosine kinase inhibitors.

M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
185
was obtained after coupling with hydroxylamine. Sodium borohy-
dride reduction of 3 yielded the trans secondary alcohol 9 as the
major product. The synthesis of amine/ammonium functionalized
compounds is shown in Scheme 2. Keeping in mind the stereose-
lective kinase recognition,39 we hypothesized that cis and trans iso-
mers would have different activities. We were thus confronted
with the stereoselective synthesis of a series of N-alkyl-4-(4-octyl-
phenyl)cyclohexanamines. Reductive amination of 3 with various
primary amines generated secondary amines 10a–i predominantly
in either cis or trans form depending on the nature of the reducing
agent. Sodium triacetoxyborohydride provided predominantly cis
isomer whereas sodium cyanoborohydride and lithium borohy-
dride41 gave predominantly trans isomer (Table 1). The stereose-
lectivity of the reductive amination can be explained based on
the steric approach control and torsional strain control
2. Results and discussion
2.1. Design of inhibitors
Fingolimod (FTY720, Fig. 2) has been used as a starting point for
the design of S1P receptor agonists prodrugs37 as well as SphK dual
inhibitors.31 These FTY720 analogues have provided valuable
insight into the structural requirements necessary for phosphory-
lation since FTY720 is a SphK substrate. Synthesis of conformation-
ally biased analogues38 indicates that SphK shows a high degree of
stereoselectivity in substrate recognition.39 Careful structural anal-
ysis of these inhibitors suggests that the highly lipophilic (p-octyl)-
phenyl backbone is a key characteristic feature for SphK inhibitors
and it is noted that inhibitors described in the literature possess a
basic amine functionality as the headgroup (primary, secondary,
tertiary amine, or an amidine) (Fig. 2). The lipophilic tail and the
head group were then connected by a linker (alkyl chain, alkene,
amide or adamantane group). In this study, we chose to incorpo-
rate the lipophilic (p-octyl)phenyl chain, a cyclohexyl linker and
various surrogate head groups in our inhibitor design, which pro-
vided us a template for the synthesis of inhibitors.
42
(Supplementary Figs. S1 and S2).
To diversify our inhibitors further, secondary amines 10a–c
were transformed into tertiary methyl amines 11a–c by an Esc-
hweiler–Clarke reaction. These tertiary amines were further con-
verted into quaternary ammonium salts 12a–c. The remainder of
the active secondary amines were converted directly into the qua-
ternary ammonium salts 12e, 12h, and 12i by reaction with methyl
iodide in the presence of potassium carbonate.
2.2. Synthesis of inhibitors
Scheme 3 illustrates the synthesis of head group analogs linked
to basic aromatic moieties. Pyrazine 13 was achieved by boration
of 1 followed by Suzuki-Miyaura cross coupling reaction with
5-bromo-2-pyrazineamine and subsequent acidic deprotection.
Similarly, piperazine 14 and pyridinone 15 were synthesized using
the requisite boronic esters and deprotection procedure.
We examined head groups bearing different functionalities to
determine possible lead structures that can be improved. To gener-
ate a library with diverse chemical structures, we envisioned a
divergent synthesis using substituted cyclohexanone 3 as a key
intermediate (Scheme 1). Thus, lithium-halogen exchange of aryl
bromide
1 with n-butyllithium followed by reaction with
2.3. Biological evaluation
1,4-cyclohexanedione monoethylene ketal afforded tertiary alco-
hol 2. Dehydration, hydrogenation of the resulting alkene and
deprotection of the spiroketal40 smoothly provided ketone 3 in
76% yield over three steps. Ketone 3 was the key intermediate that
was converted into compounds 4–9, each consisting of different
functionalities on the cyclohexane ring (Scheme 1). A Strecker
reaction converted the ketone into the corresponding amino-nitrile
4. A Horner–Wadsworth–Emmons olefination of 3 generated the
Table 2 lists the results of the in vitro inhibition assay43 against
SphK1 and SphK2 with 5-10
initially screened at a concentration of 100
structures that could potentially be improved. Possible inhibitors
were defined as those able to inhibit at least 50% at 100
concentration.
l
M sphingosine. The compounds were
lM to identify lead
lM
At the outset, we wanted to test our hypothesis that amine
groups are crucial for compounds to function as inhibitors. We
note that the amino group in sphingosine is hypothesized to
a
,b-unsaturated ester 5 which was subsequently reduced to allylic
alcohol 6 using DIBAL-H. Treatment of 3 with nitromethane and
sodium ethoxide afforded the addition product 7 while oxime 8
a
b, c, d
6
OH
6
6
Br
O
O
O
1
2
3
NH2
CN
OH
h
e
NO2
4
7
6
6
O
O
OH
N
i
f
3
8
5
6
6
g
j
OH
OH
6
9
6
6
Scheme 1. Reagents and conditions: (a) n-BuLi, ꢀ78 °C, THF, then 1,4-cyclohexanedione monoketal, 2 h, 75%; (b) TsOH, toluene, 65 °C, 1 h; (c) H2, Pd/C, EtOH, 20 h; (d) AcOH/
H2O (3:1), 65 °C, 2 h, 76% over 3 steps; (e) KCN, NH4Cl, MeOH, reflux, 23%; (f) NaOtBu, methyl diethylphosphonoacetate, CH2Cl2, ꢀ78 °C to rt, 82%; (g) DIBAL-H (1 M in
toluene), CH2Cl2, 0 °C to rt, 79%; (h) CH3NO2, NaOEt, EtOH, 0 °C to rt, 62%; (i) hydroxylamine hydrochloride, pyridine, 80 °C, 83%; (j) NaBH4, MeOH, rt, 12 h, 72%.

186
M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
Table 2
C8H17
C8H17
Inhibitory effects of various analogs on SphK1 and SphK2
a, b
or c
d
3
Entry
Compound
SphK activity levela (%)
SphK2
R
R
SphK1
100.6
N
H
N
11a-c
10a-i
1
2
3
4
5
6
7
8
9
4
5
6
7
8
9
3
61.8
4
100.8 1.2
100.2 6.1
100.9 2.3
66.7 2.9
52.7 4.3
56.5
70.9 2.3
5.4 0.2
e
f
10a: R = methyl
10b: R = n-propyl
10c: R = isopropyl
10d: R = n-butyl
10e: R = benzyl
10f: R = cyclopropyl
100.4
130.3 21
113.1
9
6
h
C8H17
8
10k: R = H
cis-10a
trans-10a
cis-10b
trans-10b
cis-10c
trans-10c
cis-10d
trans-10d
cis-10e
trans-10e
cis-10f
trans-10f
cis-10g
trans-10g
cis-10h
trans-10h
cis-10i
trans-10i
trans-10j
trans-10k
cis-11a
trans-11a
cis-11b
trans-11b
cis-11c
cis-12a
trans-12a
cis-12b
trans-12b
cis-12c
trans-12c
trans-12e
trans-12h
cis-12i
trans-12i
13b
69.8 6.1
15.7 8.1
7.7 3.6
10g
: R = propargyl
75.6
15.8 2.9
90.0
24.6 7.1
100.2 6.3
20.5 4.4
98.4 11.2
1
18.3 0.3
5.5 0.1
10h: R = allyl
R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
12a-c,e,h,i
N
10i: R = 2-hydroxyethyl
10j: R = 4-picolyl
3
16.5 0.9
23.9 3.9
60.6 3.1
20.1 3.6
67.2 6.3
53.3 3.2
116.1 14.1
77.0 2.3
100.7 5.3
100.0 2.3
100.1 3.6
5.7 0.3
I
Scheme 2. (a) R-NH2, NaBH(OAc)3, CH2Cl2, rt, 2 h, 63–77%; (b) R-NH2, NaBH3CN,
MeOH, 0 °C to rt, 12 h, 64–82%; (c) R-NH2, LiBH4, ꢀ78 °C to rt, 20 h, 73–82%; (d)
(CH2O)n, HCOOH, MeOH, reflux, 6 h, 66–93%; (e) MeI, CH3CN, reflux, 2 h, 68–72%; (f)
MeI, K2CO3, CH3CN, reflux, 2 h, 39–73%; (h) H2, 10% Pd/C, MeOH, 84%.
83.9
4
113.2 12.3
65.0 3.3
100.1 5.1
108.8 1.2
Table 1
91.8
1
Reductive amination of 3 with various reducing agents
19.0 0.2
22.4 3.1
8.9
8.5 2.1
23
2
21.6
33 2.7
47
6
R
4
HN
reducing
agent
R
3
25 3.7
NH2
3
N
H
R
R
83.9 1.1
46.3 2.6
90.7 4.5
77.0 1.1
112.9 13.1
65.8 3.1
10.2 1.1
25.1 1.1
50.8 2.2
9
R
0.6
cis-product
trans-product
65.5 6.2
45.9 6.1
100.6 11.3
8.3 2.2
4.0 0.3
Entry
Amine
cis:trans ratioa (yield, %)
3.2 0.2
b
b
c
NaBH(OAc)3
NaCNBH3
LiBH4
5.3
1
7.2 1.5
10.4 1.1
11.5 1.3
8.3 1.1
2.1 0.1
32.6 4.4
3.0 0.2
99 2.0
100.6 3.2
49.1 6.6
25.7 2.4
30.4 1.1
80.9 5.3
10.5 2.3
89.3 2.1
58 2.1
10a
10b
10c
10d
10e
10f
CH3NH2
nPrNH2
iPrNH2
nBuNH2
BnNH2
Cyclopropyl amine
Propargyl amine
Allylamine
71:29 (77)
66:34 (66)
69:31 (64)
64:36 (68)
69:31 (74)
70:30 (66)
70:30 (75)
78:22 (63)
34:66 (71)
36:64 (69)
41:59 (70)
39:61 (69)
21:79 (82)
38:62 (70)
32:68 (67)
37:63 (64)
4:96 (99)
8:92 (95)
6:94 (97)
9:91 (99)
4:96 (88)
5:95 (97)
4:96 (98)
5:95 (94)
10g
10h
14b
87.3 3.8
93.7 3.8
15b
93.7 3.1
a
b
c
Ratios determined by GC–MS analysis of crude reaction mixture.
Combined isolated yield of cis and trans products.
GC yield.
a
Values are percent activity of SphK1 or SphK2 with 10 and 5
lM Sph, respec-
tively, in the presence of 100 lM inhibitor. Each value is an average of three
experiments. Lower SphK activity level indicates better inhibition.
b
These compounds were assayed at 10 lM.
N
a, b, d
NH2
HCl
electrostatically interact with Asp177 36
Inhibition studies of vari-
ous head groups rapidly established that nitrogen-containing com-
pounds are more potent than other functional groups. For example,
.
C8H17
N
13
a,b-unsaturated ester 5, alcohols 6 and 9, nitromethyl derivative 7
O
and oxime 8 were inactive against SphK1 and did not cross the 50%
threshold with SphK2 (Table 2). In contrast, cyclohexylamine
derivative trans-10k surpassed the 50% inhibition threshold; fur-
ther elaborated amine groups were then synthesized. Biological
testing and analysis of secondary, tertiary and quaternary ammo-
nium salts revealed interesting structure-activity relationships.
The trans isomers (10a–d, 10f–h) of secondary amines were signif-
icantly more potent inhibitors of SphK1 than the corresponding cis
isomers. In the case of trans-10e, both isomers were ineffective
while for trans-10i, both isomers were equally effective. Against
SphK2, however, different trends were observed. For compounds
10b, 10d, and 10h, the trans isomer was significantly more active
than the cis isomer. However, for compounds 10a, 10c, 10e, and
10i, both the cis and trans isomers were equally effective. More-
over, both isomers of compounds 10f–g were largely inactive;
N
N
B
O
N
Boc
N
N
1
c, d
NH
14
C8H17
HCl
O
O
O
B
N
O
N
C8H17
N
N
c
15
Scheme 3. Reagents and conditions: (a) n-BuLi, ꢀ78 °C, THF, 15 min, then B(OMe)3,
rt, 1 h, then 10% aq. HCl, 58%; (b) 5-Bromo-2-pyrazineamine, Pd(OAc)2, K2CO3,
SPhos, CH3CN/H2O (1:1.5), reflux, 6 h, 72%; (c) Pd(OAc)2, K2CO3, SPhos, CH3CN/H2O
(1:1.5 equiv), reflux, 12 h, 81–92%; (d) HCl (g), THF, 1 min., 92–95%.

M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
187
the origin of the unfavorable interaction with the cyclopropyl and
propargyl groups is currently not clear. Further, we discovered that
both cis and trans isomers of tertiary amines (11a–c) were gener-
ally not effective as SphK1/2 inhibitors; hence these structures
were not pursued. The exception to this is the activity of trans-
11a towards SphK2 (vide supra).
During the course of our studies, quaternary ammonium salts
were discovered to be effective inhibitors of SphK1/2 comparable
to secondary amines. Because quaternary ammonium salts have
the desired water solubility and potential cell permeability, a series
of compounds (12) were synthesized. Consistent with the results
with secondary amines, trans isomers were more potent than cis
isomers specifically with SphK1 (compare trans- vs cis-12a–c &
12i) (Table 2). The data indicate that the cis isomers (12a–c &
specific inhibition, we added exogenous FTY720 in these cells
and monitored the phosphorylation of FTY720 with or without
compounds in cell extracts using LC/MS. Because FTY720 is a spe-
cific substrate of SphK2, a decrease in FTY720-P concentration
would suggest SphK2-selective inhibition. Gratifyingly, both com-
pounds trans-12a and trans-12b significantly suppressed the pro-
duction of FTY720-P (Fig. 3A) and exaggerated the accumulation
of FTY720 in cells (Fig. 3B), which suggests that trans-12a/12b in-
hibit SphK2.
In addition, we assayed for S1P-dependent Akt/ERK phosphory-
lation status. It was reported recently that treatment with SphK
inhibitors resulted in decreased cell survival as a consequence of
S1P biosynthesis blockade, a process monitored by the decreased
phosphorylation of ERK and Akt.47,48 When U937 cells were treated
with different concentrations of trans-12a and 12b for 16 h, a dose-
dependent decrease in Akt and ERK phosphorylation was observed
(Fig. 3C). Specifically, both trans-12a/12b quantitatively inhibited
ERK phosphorylation; however, trans-12b appeared to be more po-
tent than trans-12a as evidenced by the complete disappearance of
p-Akt band in the Western blot. Collectively, our data suggest that
our inhibitors are cell permeable compounds that may interfere
with S1P signaling.
12i) are selective towards SphK2, but follow-up assays at 10 lM
inhibitor concentration revealed moderate inhibition against
SphK2 (data not shown).
Additional head group analogs containing pyrazyl or pyridyl
rings (13–15) were assayed for inhibition but unfortunately the de-
sired activity was not observed. Although the pyridyl group is ex-
pected to be protonated at physiological pH, the lack of activity
may be attributed to the replacement of the cyclohexyl ring with
a flat aromatic ring that displays the important functional group
in unfavorable orientation.
To confirm the potencies of select compounds identified as hits
in the initial screen (vide supra), their Ki values were determined.
The Ki values as well as SphK2 selectivity are listed in Table 3. Be-
3. Conclusion
The large number of reports that implicate SphK in various dis-
ease states highlights the importance of this enzyme as a key reg-
ulator of sphingolipid homeostasis. Although SphK1 and SphK2
share many features, they have been reported to possess different
functions. SphK1 promotes cell growth and survival; it is overex-
pressed in many tumor types such as brain, breast, colon, prostate,
skin and others. As a result, selective targeting of SphK1 for the
treatment of cancer has caught the attention of the scientific com-
munity. SphK2 has been reported to play an important role in tu-
mor progression such as in MCF-7 breast cancer xenografts. On
the other hand, some studies suggest SphK2 has the opposite ef-
fect—it inhibits cell growth and induces apoptosis which is attrib-
uted to the release of its BH3 domain upon proteasomal
degradation. These contradictory functions for SphK2 need to be
further understood. Clearly, it is still unknown whether selective
inhibition of SphK1, SphK2 or both is beneficial. Indeed, selective
small molecule inhibitors are key to answering these questions.
Unfortunately, these inhibitors are currently lacking—and even
more so with SphK2.
cause SphK1/2 have different Km values (10 and 5 lM, respec-
tively),44,45 the selectivity ratios were normalized with the
respective Km values. These data suggest that quaternary ammo-
nium salts are more potent and selective towards SphK2 relative
to secondary and tertiary amines (entries 1–4 vs 5–8). As the size
of substituents on the amine increase, we found that the potency
decreases, presumably as a result of increased steric interaction
in the enzyme binding pocket. Among the quaternary ammonium
salts tested, trans-12a and trans-12b are the most potent
(Ki = 8
l
M) and selective (ꢁfourfold for trans-12a and ꢁthreefold
for trans-12b) (Table 3). To the best of our knowledge, this is the
first demonstration of a SphK inhibitor scaffold containing a qua-
ternary amine and is consistent with the observation that a posi-
tive charge is essential in SphK inhibitors.
2.4. Inhibition of SphK2 and Akt/ERK phosphorylation in intact
cells
In this report, we documented our efforts in developing
SphK2-selective inhibitors. We discovered a novel scaffold that
afforded compounds with low micromolar inhibitory activities that
are moderately SphK2 selective. In general, trans isomers bearing
small quaternary ammonium salts are good SphK2 inhibitors. Fi-
nally, we demonstrated that trans-12a/12b inhibited Akt/ERK
phosphorylation suggesting that these compounds inhibit the
SphK-dependent phosphorylation cascade. Current efforts are
aimed at modifying several regions of lead compounds to arrive
at more potent and selective SphK2 inhibitors.
We next decided to investigate the effect of our inhibitors in in-
tact cells. We first measured S1P levels in the presence of trans-
12a/12b U937 cells (human histiocytic leukemia cells) using LC/
MS46 and found no change (data not shown). To confirm SphK2
Table 3
Ki Values for select compounds
a
b
Entry
Compound
Ki (
SphK1
lM)
SphK2 selectivity
SphK2
4. Experimental section
4.1. Chemistry
1
2
3
4
5
6
7
8
cis-10a
>100
22
32
40
60
47
>100
70
40
6
5
5
5
2
1
7
2
—
trans-10i
trans-10k
trans-11a
trans-12a
trans-12b
trans-12c
trans-12i
2
4
7
6
4
38
29
27
8
8
33
14
1.23
0.55
0.74
3.75
2.94
—
4.1.1. General procedures
Melting points were recorded using a Büchi B-540 melting point
instrument and are uncorrected. 1H NMR spectra were recorded on a
JEOL EclipsePlus-500 (500 MHz) or a Varian Inova-400 (400 MHz)
spectrometer. Chemical shifts are reported in ppm from tetrameth-
ylsilane with the solvent resonance as an internal standard
8
2.5
a
Ki = [I]/(K0m/Km ꢀ 1); Km of sphingosine at SphK1 = 10
lM; Km of sphingosine at
SphK2 = 5 lM.
b
SphK2
Selectivity = (Ki/Km
)
SphK1/(Ki/Km
)
.

188
M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
13C NMR spectra were recorded on an EclipsePlus-500 (126 MHz)
1.5×10-10
1.0×10-10
5.0×10-11
0.0
A
spectrometer or a Varian Inova-400 (101 MHz) spectrometer with
complete proton decoupling. Chemical shifts are reported in ppm
with the solvent resonance as the internal standard (CDCl3:
77.16 ppm). Low resolution mass spectrometry (ESI-MS) was per-
formed on a Thermo Instrument TSQ triple quadrupole mass spec-
trometer (Thermo Finnigan, San Jose, CA, USA), equipped with an
ESI source, which was used in the positive ion mode. High resolution
mass spectroscopy (HRMS) was performed on an Agilent 6220 LC/
MS time-of-flight mass spectrometer using either electrospray ion-
ization (ESI) or fast atom bombardment (FAB). Column chromatog-
raphy was performed either on a CombiFlashÒ Rf automated
chromatography or by either using flash grade silica gel (SiO2, 32–
63 lm) or neutral, activated, Brockmann I aluminum oxide (Al2O3,
μ
μ
μ
ꢁ150 mesh, 58 Å). Thin layer chromatography (TLC) was performed
either on EMD silica gel 60 F254 plates or EMD aluminum oxide 60
F254 neutral plates. All reactions were conducted in oven or flame
dried glassware under an inert atmosphere of nitrogen using mag-
netic stirring. Solvents were dried using the PureSolv™ solvent puri-
fication system. All other chemical reagents were purchased from
commercial sources and were used without further purification.
1.5×10-08
1.0×10-08
5.0×10-09
0.0
B
4.1.2. Synthesis and characterization of compounds
4.1.2.1. 8-(4-Octylphenyl)-1,4-dioxaspiro[4.5]decan-8-ol (2).
To a solution of 1-bromo-4-octylbenzene (1 g, 3.60 mmol) in
25 mL THF at ꢀ78 °C, n-butyllithium (2.5 M in hexanes, 1.965 mL,
4.32 mmol) was added and the solution stirred for 10 min. Then,
a solution of 1,4-dioxaspiro[4.5]decan-8-one (0.696 g, 4.32 mmol)
in 10 mL THF was added dropwise at ꢀ78 °C. The reaction was stir-
red for 2 h at ꢀ78 °C, warmed to 0 °C and quenched with dropwise
addition of a saturated solution of NH4Cl. The reaction mixture was
partitioned between water and EtOAc. The aqueous phase was ex-
tracted with EtOAc and the combined organic phases were washed
with brine, dried over anhydrous sodium sulfate and concentrated
on a rotary evaporator. The resulting residue was purified by col-
umn chromatography over silica gel (95/5 dichloromethane/ace-
tone) to give the title compound (0.96 g, 75%) as a white solid,
mp 52 °C; 1H NMR (400 MHz, CDCl3) d 7.44–7.40 (m, 2H), 7.18–
7.13 (m, 2H), 4.03–3.93 (m, 4H), 2.67–2.54 (m, 2H), 2.22–2.04
(m, 4H), 1.85–1.77 (m, 2H), 1.72–1.55 (m, 5H), 1.40–1.20 (m,
10H), 0.87 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 145.7,
141.6, 128.2, 124.4, 108.5, 72.2, 64.3, 64.2, 36.6, 35.5, 31.9, 31.5,
30.8, 29.5, 29.4, 29.3, 22.7, 14.1; HRMS (FAB+) m/z calcd for
μ
μ
μ
C
C
22H33O2 [MꢀOH]+ 329.2481, found 329.24603.
β
4.1.2.2. 4-(4-Octylphenyl)cyclohexanone (3).
p-Toluenesul-
fonic acid monohydrate (0.106 g, 0.556 mmol) was added to a solu-
tion of 2 (1.96 g, 5.66 mmol) in toluene (40 mL) and the reaction
mixture was heated at 65 °C for 1 h. Toluene was evaporated under
reduced pressure to give a pink oil which was dissolved in EtOAc. It
was washed with NaHCO3, brine, dried under sodium sulfate, and
the solution concentrated on a rotary evaporator. The resulting
oil was dissolved in ethanol and transferred to a 2-neck flask
equipped with a magnetic stirrer. 10% Pd on activated carbon
(10 mol %) was added and the reaction run under H2 gas for 20 h.
Pd/C was filtered through a plug of celite and the filtrate concen-
trated on a rotary evaporator. Acetic acid (45 mL) and water
(15 mL) was added to the resulting oil and the solution heated at
65 °C for 2 h. The reaction mixture was cooled to rt and partitioned
between hexanes and water. The aqueous layer was extracted with
hexanes and the combined organic extracts washed with NaHCO3,
brine, and dried with sodium sulfate. The organic solvents were
evaporated under reduced pressure and the resulting oil purified
by column chromatography over silica gel (90/10 hexanes/EtOAc)
to give the title compound (1.24 g, 76%) as a white solid, mp
36.0–37.0 °C; 1H NMR (500 MHz, CDCl3) d 7.18–7.10 (m, 4H),
μ
Figure 3. SphK2 inhibition in U937 cells. (A) and (B) Cultured U937 cells were
exposed to FTY720 and 10 M inhibitors as indicated in the figure. After 2 h, cells
l
were harvested by centrifugation and the amounts of FTY720-P and FTY720
associated with the cell pellet were measured by LC/MS. (A): FTY720-P; (B): FTY720.
Amounts are expressed as the number of moles per million cells. Data are
means SD of three independent experiments. ⁄p <0.05; ⁄⁄p <0.005 (Unpaired two-
tailed T-test). (C) trans-12b and trans-12a inhibit Akt and ERK phosphorylation.
U937 cells were pretreated with indicated concentration of inhibitor for 24 h. Cells
were lysed and equal amounts of proteins were analyzed by Western blotting with
the indicated antibodies. PD098059 (10
inhibition of ERK phosphorylation.
lM) was used as a positive control for
(CDCl3: 7.26 ppm). Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
br = broad, m = multiplet), coupling constants (Hz), and integration.

M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
189
2.99 (tt, J = 3.0 Hz, 12.0 Hz, 1H), 2.62–2.43 (m, 6H), 2.26–2.16 (m,
2H), 1.99–1.86 (m, 2H), 1.65–1.53 (m, 2H), 1.36–1.20 (m, 10H),
0.87 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 211.3, 141.9,
141.2, 128.6, 126.5, 42.4, 41.4, 35.6, 34.1, 31.9, 31.5, 29.5, 29.4,
29.3, 22.7, 14.1; HRMS (FAB+) m/z calcd for C20H30O [M+H]+
287.2375, found 287.23669.
31.5, 29.5, 29.4, 29.3, 28.6, 22.7, 14.1; HRMS (FAB+) m/z calcd for
C
22H33 [MꢀOH]+ 297.25823, found 297.25844.
4.1.2.6.
1-(Nitromethyl)-4-(4-octylphenyl)cyclohexanol (7).
Nitromethane (0.37 mL, 6.98 mmol) was added to a solution of 3
(0.4 g, 1.396 mmol) in ethanol (8 mL). The mixture cooled to 0 °C
and sodium ethoxide (0.114 g, 1.676 mmol) dissolved in ethanol
(4 mL) was added dropwise. The mixture was warmed to rt and
stirred for 4 h. It was quenched by the addition of saturated NH4Cl.
It was then partitioned between EtOAc and water. The aqueous
layer was extracted with EtOAc and the combined organic extracts
were washed with brine and dried with sodium sulfate. The organ-
ic solvents were evaporated under reduced pressure and the
resulting residue purified by column chromatography over silica
gel (75/25 hexanes/EtOAc, Rf = 0.37) to give the title compound
(0.3 g, 62%) as a white solid, mp 45.8–46.5 °C; 1H NMR (500 MHz,
CDCl3) d 7.12 (s, 4H), 4.68 (s, 2H), 3.09 (s, 1H), 2.62 (tt, J = 3.0 Hz,
12.5 Hz, 1H), 2.57 (t, J = 7.5 Hz, 2H), 2.01–1.90 (m, 4H), 1.77 (td,
4.1.2.3. 1-Amino-4-(4-octylphenyl)cyclohexanecarbonitrile (4).
A solution of potassium cyanide (0.091 g, 1.396 mmol) and ammo-
nium chloride (0.021 g, 0.394 mmol) in water (2.5 mL) was added
to a solution of 3 (0.1 g, 0.349 mmol) in methanol (2.5 ml). The
mixture was stirred overnight at 60 °C. After cooling to rt, the mix-
ture was diluted with water and extracted with EtOAc. The organic
layer was washed with brine, dried with sodium sulfate and evap-
orated under reduced pressure. The residue obtained was purified
by column chromatography on silica gel (90/10 EtOAc/hexanes) to
give the title compound as a colorless oil; 1H NMR (500 MHz,
CDCl3) d 7.16–7.09 (m, 4H), 2.58–2.54 (m, 2H), 2.48 (tt, J = 3.6 Hz,
12.3 Hz, 1H), 2.18–2.12 (m, 2H), 1.98–1.78 (m, 6H), 1.67–1.61 (m,
2H), 1.61–1.56 (m, 2H), 1.35–1.21 (m, 10H), 0.87 (t, J = 7.0 Hz,
3H); 13C NMR (101 MHz, CDCl3) d 142.5, 141.1, 128.4, 126.6,
123.7, 51.5, 42.6, 38.3, 35.5, 31.9, 31.5, 30.7, 29.4, 29.4, 29.2,
22.6, 13.9.
J = 3.7 Hz,
13.4 Hz,
2H),
1.63–1.49
(m,
4H),
1.37–1.21 (m, 10H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz,
CDCl3) d 142.2, 141.1, 128.5, 126.5, 81.3, 71.2, 42.2, 35.9, 35.5,
31.9, 31.5, 30.5, 29.4, 29.4, 29.2, 22.6, 14.1.
4.1.2.7. 4-(4-Octylphenyl)cyclohexanone oxime (8).
Hydrox-
4.1.2.4. Methyl 2-(4-(4-octylphenyl)cyclohexylidene)acetate (5).
ylamine hydrochloride (72.8 mg, 1.047 mmol) was added to a solu-
tion of 3 (100 mg, 0.349 mmol) in pyridine (1 mL) and the resulting
solution was heated at 80 °C for 24 h. Pyridine was evaporated un-
der reduced pressure and the resulting residue was dissolved in
EtOAc. It was washed with NaHCO3, brine, dried over sodium sul-
fate and filtered. The resulting solution was concentrated on a ro-
tary evaporator to give the title compound (87 mg, 83%) as a white
solid, mp 69.2–70.3 °C; 1H NMR (400 MHz, CDCl3) d 8.35 (br s, 1H),
7.04 (s, 4H), 3.44–3.36 (m, 1H), 2.68 (tt, J = 3.3 Hz, 12.1 Hz, 1H),
2.53–2.42 (m, 3H), 2.19 (td, J = 4.7 Hz, 13.6 Hz, 1H), 2.05–1.93 (m,
2H), 1.81 (td, J = 5.2 Hz, 13.9 Hz, 1H), 1.68–1.47 (m, 4H), 1.30–
1.14 (m, 10H), 0.80 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 159.9, 142.8, 140.9, 128.5, 126.5, 43.3, 35.6, 34.1, 33.0, 32.0,
31.9, 31.5, 29.5, 29.4, 29.3, 24.2, 22.7, 14.2; HRMS (FAB+) m/z calcd
for C20H32NO [M+H]+ 302.2484, found 302.2457.
To
a solution of methyl diethylphosphonoacetate (0.329 mL,
1.745 mmol) in CH2Cl2 (5 mL) at ꢀ78 °C was added sodium tert-
butoxide (0.138 g, 1.396 mmol) over a period of 15 min. The reac-
tion mixture was stirred for 1 h at ꢀ78 °C. Then, a solution of 3
(0.2 g, 0.698 mmol) in CH2Cl2 (2 mL) was added dropwise.
The solution was allowed to warm to rt and then stirred overnight.
The reaction was quenched by the addition of saturated NH4Cl. The
reaction mixture was partitioned between CH2Cl2 and water and
the aqueous layer extracted with CH2Cl2. The combined organic
layers were washed with saturated NaHCO3, brine, dried over so-
dium sulfate and filtered. The filtrate was concentrated on a rotary
evaporator and the residue was purified by column chromatogra-
phy over silica gel (95/5 hexanes/EtOAc, Rf = 0.38) to give the title
compound (0.195 g, 82%) as a white solid, mp 39.1–40.0 °C; 1H
NMR (500 MHz, CDCl3) d 7.11 (s, 4H), 5.69 (s, 1H), 4.02–3.93 (m,
1H), 3.71 (s, 3H), 2.77 (tt, J = 3.8 Hz, 11.9 Hz, 1H), 2.57 (t,
J = 7.9 Hz, 2H), 2.44–2.31 (m, 2H), 2.11–2.00 (m, 3H), 1.69–1.55
(m, 4H), 1.37–1.22 (m, 10H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR
(101 MHz, CDCl3) d 167.4, 162.6, 143.3, 141.0, 128.6, 126.8,
113.4, 51.1, 43.9, 38.0, 35.9, 35.8, 35.1, 32.1, 31.8, 29.8, 29.7,
29.6, 29.5, 22.9, 14.3; ESI-MS m/z calcd for C23H34O2 [M+H]+
343.26, found 343.30.
4.1.2.8. (1r,4r)-4-(4-Octylphenyl)cyclohexanol (9).
Sodium
borohydride (0.031 g, 0.826 mmol) was added to a solution of 3
(0.1 g, 0.349 mmol) in methanol (2 mL) at 0 °C. The reaction was
warmed to rt and stirred overnight. The solvent was removed by
evaporation under reduced pressure and water was added to the
resulting residue. The aqueous phase was extracted 3 times with
EtOAc and the combined organic extracts were washed with brine
and dried over sodium sulfate. The solvent was evaporated under
reduced pressure and the resulting residue was purified by column
chromatography on silica gel (50/50 hexanes/EtOAc) to give the ti-
tle compound (0.073 mg, 72% yield) as a white solid; 1H NMR
(500 MHz, CDCl3) d 7.10 (s, 4H), 3.72-3.65 (m, 1H), 2.58–2.53 (m,
2H), 2.46 (tt, J = 3.5 Hz, 12.2 Hz, 1H), 2.12–2.06 (m, 2H),
1.95–1.89 (m, 2H), 1.62–1.22 (m, 16H), 0.87 (t, J = 7.5 Hz, 3H);
13C NMR (126 MHz, CDCl3) d 143.8, 140.8, 128.4, 126.7, 70.8,
43.1, 36.1, 35.6, 32.6, 32.0, 31.6, 29.6, 29.5, 29.4, 22.8, 14.2; HRMS
(FAB+) m/z calcd for C20H32O 288.2453, found 288.2457.
4.1.2.5.2-(4-(4-Octylphenyl)cyclohexylidene)ethanol (6).
Dii-
sobutylaluminum hydride (1 M in toluene, 2.2 mL, 2.2 mmol) was
added dropwise to a solution of 5 (0.247 g, 0.721 mmol) in CH2Cl2
(7.2 mL) at 0 °C. The solution was allowed to warm to rt and stirred
for 1 h. The solution was diluted with careful dropwise addition of
0.09 mL water, 0.14 mL 10% NaOH and then 0.22 mL water and
stirred for 30 min. The resulting precipitate was filtered through
a plug of celite and the filtrate was washed with brine solution
and dried with sodium sulfate. Evaporation of the organic solvent
gave a residue which was purified by column chromatography on
silica gel (75/25 hexanes/EtOAc, Rf = 0.32) to give the title com-
pound (0.179 g, 79%) as a white solid, mp 36.7–37.4 °C; 1H NMR
(500 MHz, CDCl3) d 7.10 (s, 4H), 5.44 (t, J = 7.1 Hz, 1H), 4.18 (d,
J = 7.1 Hz, 2H), 2.79–2.73 (m, 1H), 2.68 (tt, J = 3.5 Hz, 12.2 Hz,
1H), 2.58–2.53 (m, 2H), 2.39–2.29 (m, 1H), 2.27–2.18 (m, 1H),
2.03–1.88 (m, 3H), 1.63–1.41 (m, 4H), 1.36–1.21 (m, 11H), 0.87
(t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 143.8, 143.2,
140.6, 128.3, 126.6, 120.9, 58.7, 44.1, 36.8, 35.6, 35.5, 35.1, 31.9,
4.1.3. General procedure for reductive amination with sodium
triacetoxyborohydride
Primary amine (1.1 equiv) was added to a solution of 3 (1 equiv)
in CH2Cl2 and the solution stirred for 5 min. Sodium triacetoxy-
borohydride (1.4 equiv) was added and the mixture was stirred
for 1 h. The reaction mixture was diluted by the addition of satu-
rated NaHCO3 and stirred for an additional 10 min. It was then
partitioned between water and CH2Cl2. The aqueous layer was

190
M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
extracted 3 times with CH2Cl2 and the combined organic layers
were washed with brine, dried with sodium sulfate and filtered.
The organic solvent was removed by evaporation under reduced
pressure and the crude product was purified by column chroma-
tography on neutral alumina.
d 144.3, 140.5, 128.3, 126.6, 56.8, 49.2, 43.7, 35.6, 33.9, 33.1,
31.9, 31.5, 29.5, 29.4, 29.3, 23.6, 22.7, 14.1, 11.9; HRMS (ESI+) m/
z calcd for C23H39N [M+H]+ 330.3155, found 330.3153.
4.1.5.5. (1s,4s)-N-Isopropyl-4-(4-octylphenyl)cyclohexanamine
(cis-10c).
Colorless oil; 1H NMR (400 MHz, CDCl3) d 7.23–7.04
4.1.4. General procedure for reductive amination with sodium
cyanoborohydride
(m, 4H), 3.01–2.95 (m, 1H), 2.95–2.87 (m, 1H), 2.65–2.50 (m,
3H), 1.86–1.55 (m, 10H), 1.46–1.21 (m, 11H), 1.08 (d, J = 7.5 Hz,
6H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 144.5,
140.6, 128.5, 126.9, 48.6, 45.1, 43.1, 35.8, 32.2, 31.8, 30.7, 29.8,
Primary amine (1.1 equiv) was added to a solution of 3 (1 equiv)
in MeOH at 0 °C. After 15 min., sodium cyanoborohydride
(0.7 equiv) and acetic acid (1.2 equiv) were added and the mixture
was warmed to rt. After 12 h, the solution was evaporated under
reduced pressure, saturated NaHCO3 was added and the mixture
extracted with EtOAc. The organic phase was washed with brine,
dried with sodium sulfate, and concentrated on a rotary evapora-
tor. The crude product was purified by column chromatography
on neutral alumina.
29.7, 29.5, 28.5, 23.6, 22.9, 14.4; ESI-MS m/z calcd for C23H39
N
[M+H]+ 330.31, found 330.30.
4.1.5.6. (1r,4r)-N-Isopropyl-4-(4-octylphenyl)cyclohexanamine
(trans-10c).
White solid, mp 41.5–42.5 °C; 1H NMR (500 MHz,
CDCl3) d 7.13–7.07 (m, 4H), 3.14–3.04 (m, 1H), 2.69 (tt, J = 3.9 Hz,
11.3 Hz, 1H), 2.55 (t, J = 8.0 Hz, 2H), 2.48 (tt, J = 3.5 Hz, 12.5 Hz,
1H), 2.12–2.03 (m, 2H), 1.98 (br s, 1H), 1.96–1.88 (m, 2H), 1.62–
1.55 (m, 2H), 1.49 (qd, J = 3.0 Hz, 12.5 Hz, 2H), 1.37–1.20 (m,
12H), 1.14 (d, J = 6.3 Hz, 6H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR
(126 MHz, CDCl3) d 144.1, 140.7, 128.4, 126.7, 53.5, 45.3, 43.5,
35.6, 33.5, 33.2, 32.0, 31.6, 29.6, 29.5, 29.3, 22.8, 14.2; ESI-MS m/
z calcd for C23H39N [M+H]+ 330.31, found 330.28; HRMS (ESI+)
m/z calcd for C23H39N [M+H]+ 330.3155, found 330.3168.
4.1.5. General procedure for reductive amination with lithium
borohydride
Primary amine (3 equiv) was added to a solution of 3 (1 equiv)
in methanol. The mixture was stirred for 1 h at rt, cooled to ꢀ78 °C,
and treated with a 2 M solution of lithium borohydride in THF
(1.1 equiv). After stirring at ꢀ78 °C for 1 h, the mixture was slowly
warmed to rt and stirred for 16 h. It was quenched by slow addi-
tion of a saturated solution of NaHCO3. The resulting mixture
was partitioned between EtOAc and water. The aqueous layer
was extracted with EtOAc and the combined organic phases
washed with brine, dried over sodium sulfate, filtered, and concen-
trated. The crude product was purified by column chromatography
on neutral alumina.
4.1.5.7. (1s,4s)-N-Butyl-4-(4-octylphenyl)cyclohexanamine (cis-
10d).
Colorless oil; 1H NMR (500 MHz, CDCl3) d 7.22–7.05 (m,
4H), 2.91–2.85 (m, 1H), 2.69–2.47 (m, 5H), 1.90–1.73 (m, 4H),
1.73–1.57 (m, 6H), 1.56–1.21 (m, 15H), 1.04–0.81 (m, 6H); 13C
NMR (126 MHz, CDCl3) d 144.7, 140.5, 128.5, 127.0, 52.1, 47.3,
43.3, 35.8, 32.8, 32.2, 31.8, 30.7, 29.8, 29.7, 29.5, 28.5, 22.9, 20.9,
14.4, 14.3; ESI-MS m/z calcd for C24H41N [M+H]+ 344.32, found
344.30.
4.1.5.1.
(cis-10a).
(1s,4s)-N-Methyl-4-(4-octylphenyl)cyclohexanamine
Colorless oil; 1H NMR (400 MHz, CDCl3) d 7.18–7.13
(m, 2H), 7.13–7.07 (m, 2H), 2.79–2.74 (m, 1H), 2.59–2.48 (m,
3H), 2.43 (s, 3H), 1.89–1.71 (m, 4H), 1.68–1.55 (m, 6H), 1.37–
1.20 (m, 11H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 144.5, 140.3, 128.2, 126.7, 53.8, 43.4, 35.6, 34.2, 31.9, 31.6,
30.1, 29.5, 29.4, 29.3, 28.3, 22.7, 14.1; HRMS (FAB+) m/z calcd for
4.1.5.8.
(trans-10d).
(1r,4r)-N-Butyl-4-(4-octylphenyl)cyclohexanamine
Yellow oil; 1H NMR (400 MHz, CDCl3) d 7.18–
7.05 (m, 4H), 2.69–2.63 (m, 2H), 2.60–2.43 (m, 4H), 2.09–2.00
(m, 2H), 1.95–1.88 (m, 2H), 1.64–1.44 (m, 6H), 1.42–1.18 (m,
14H), 0.93 (t, J = 7.3 Hz, 3H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR
(101 MHz, CDCl3) d 144.3, 140.5, 128.3, 126.6, 56.9, 47.0, 43.7,
35.6, 34.0, 33.1, 32.7, 31.9, 31.6, 29.5, 29.4, 29.3, 22.7, 20.6, 14.1,
14.0; ESI-MS m/z calcd for C24H41N [M+H]+ 344.32, found 344.30.
C
21H36N [M+H]+ 302.2848, found 302.2838.
4.1.5.2.
(trans-10a).
(1r,4r)-N-Methyl-4-(4-octylphenyl)cyclohexanamine
Yellow oil; 1H NMR (500 MHz, CDCl3) d 7.16–
7.05 (m, 4H), 2.58–2.52 (m, 2H), 2.51–2.36 (m, 5H), 2.10–2.02
(m, 2H), 1.96–1.88 (m, 2H), 1.65–1.54 (m, 3H), 1.54–1.44 (m,
2H), 1.34–1.17 (m, 12H), 0.87 (t, J = 7.0, 3H); 13C NMR (126 MHz,
CDCl3) d 144.4, 140.6, 128.4, 126.7, 58.6, 43.8, 35.7, 33.8, 33.5,
33.1, 32.0, 31.6, 29.6, 29.5, 29.4, 22.8, 14.2; HRMS (FAB+) m/z calcd
for C21H36N [M+H]+ 302.2848, found 302.2838.
4.1.5.9.
(cis-10e).
(1s,4s)-N-Benzyl-4-(4-octylphenyl)cyclohexanamine
Yellow oil; 1H NMR (500 MHz, CDCl3) d 7.39–7.31
(m, 4H), 7.27–7.23 (m, 1H), 7.17–7.14 (m, 2H), 7.12–7.08 (m,
2H), 3.80 (s, 2H), 2.96–2.91 (m, 1H), 2.59–2.50 (m, 3H), 1.92–1.81
(m, 4H), 1.68–1.56 (m, 6H), 1.48 (br s, 1H), 1.37–1.22 (m, 10H),
0.88 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) d 144.7, 141.3,
140.4, 128.5, 128.3, 128.2, 126.9, 126.8, 51.4, 51.2, 43.4, 35.7,
32.0, 31.7, 30.6, 29.6, 29.5, 29.4, 28.3, 22.8, 14.2; ESI-MS m/z calcd
for C27H39N [M+H]+ 378.31, found 378.25; HRMS (ESI+) m/z calcd
for C27H40N [M+H]+ 378.3155, found 378.3168.
4.1.5.3.
(cis-10b).
(1s,4s)-4-(4-Octylphenyl)-N-propylcyclohexanamine
Colorless oil; 1H NMR (400 MHz, CDCl3) d 7.17–7.12
(m, 2H), 7.12–7.07 (m, 2H), 2.88–2.82 (m, 1H), 2.59–2.49 (m,
5H), 1.85–1.72 (m, 4H), 1.69–1.46 (m, 8H), 1.37–1.19 (m, 10H),
0.94 (t, J = 7.3 Hz, 3H), 0.88 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz,
CDCl3) d 144.5, 140.3, 128.2, 126.7, 51.8, 49.3, 43.1, 35.6, 31. 9,
31.6, 30.5, 29.5, 29.4, 29.3, 23.6, 22.7, 14.1, 11.9; HRMS (ESI+) m/
z calcd for C23H39N [M+H]+ 330.3155, found 330.3126.
4.1.5.10.
(trans-10e).
(1r,4r)-N-Benzyl-4-(4-octylphenyl)cyclohexanamine
White solid, mp 41.5–42.5 °C; 1H NMR (400 MHz,
CDCl3) d 7.36–7.31 (m, 4H), 7.27–7.23 (m, 1H), 7.13–7.06 (m,
4H), 3.85 (s, 2H), 2.60–2.52 (m, 3H), 2.48 (tt, J = 3.0, 12.0, 1H),
2.11–2.04 (m, 2H), 1.94–1.87 (m, 2H), 1.62–1.41 (m, 6H), 1.33–
1.23 (m, 11H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 144.3, 140.9, 140.5, 128.4, 128.3, 128.1, 126.6, 56.1, 51.2, 43.7,
35.6, 33.8, 33.1, 31.9, 31.5, 29.5, 29.4, 29.3, 22.7, 14.1; ESI-MS m/
z calcd for C27H39N [M+H]+ 378.31, found 378.25; HRMS (ESI+)
m/z calcd for C27H40N [M+H]+ 378.3155, found 378.3169.
4.1.5.4.
(trans-10b).
(1r,4r)-4-(4-Octylphenyl)-N-propylcyclohexanamine
Colorless oil; 1H NMR (400 MHz, CDCl3) d 7.14–
7.07 (m, 4H), 2.67–2.41 (m, 6H), 2.09–2.00 (m, 2H), 1.96–1.87
(m, 2H), 1.65–1.42 (m, 6H), 1.38–1.17 (m, 13H), 0.93 (t,
J = 7.4 Hz, 3H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3)

M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
191
4.1.5.11.
amine (cis-10f).
(1s,4s)-N-Cyclopropyl-4-(4-octylphenyl)cyclohexan-
Colorless oil; 1H NMR (500 MHz, CDCl3) d
3.59 (m, 2H), 2.93–2.85 (m, 1H), 2.83–2.73 (m, 2H), 2.59–2.48
(m, 3H), 2.12 (br s, 2H), 1.84–1.53 (m, 10H), 1.39–1.18 (m, 10H),
0.87 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 144.3, 140.4,
128.3, 126.6, 61.3, 51.5, 48.5, 43.1, 35.5, 31.9, 31.6, 30.6, 29.5,
29.4, 29.3, 28.2, 22.7, 14.1; ESI-MS m/z calcd for C22H38NO
[M+H]+ 332.29, found 332.30.
7.16–7.07 (m, 4H), 3.02–2.97 (m, 1H), 2.58–2.49 (m, 3H), 2.14–
2.08 (m, 1H), 1.92–1.84 (m, 2H), 1.80–1.70 (m, 2H), 1.67–1.55
(m, 7H), 1.35–1.21 (m, 10H), 0.87 (t, J = 6.8 Hz, 3H), 0.46–0.40 (m,
2H), 0.40–0.32 (m, 2H); 13C NMR (126 MHz, CDCl3) d 144.7,
140.4, 128.3, 126.8, 52.3, 43.3, 35.6, 32.0, 31.6, 30.8, 29.6, 29.5,
29.4, 28.7, 28.5, 22.8, 14.2, 6.4; ESI-MS m/z calcd for C23H37
N
4.1.5.18. 2-((1r,4r)-4-(4-Octylphenyl)cyclohexylamino)ethanol
[M+H]+ 328.29, found 328.32.
(trans-10i).
53% Yield (NaBH3CN), white solid, mp 79.3–
80.0 °C; 1H NMR (500 MHz, CDCl3) d 7.12–7.07 (m, 4H), 3.65 (t,
J = 5.0 Hz, 2H), 2.83 (t, J = 5.0 Hz, 2H), 2.57–2.47 (m, 4H), 2.08–
2.04 (m, 2H), 1.94–1.90 (m, 2H), 1.61–1.43 (m, 4H), 1.35–1.18
(m, 12H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) d
144.2, 140.7, 128.4, 126.7, 61.5, 56.5, 48.3, 43.7, 35.6, 34.2, 33.2,
32.0, 31.6, 29.6, 29.5, 29.3, 22.8, 14.2; HRMS (FAB+) m/z calcd for
4.1.5.12.
amine (trans-10f).
(1r,4r)-N-Cyclopropyl-4-(4-octylphenyl)cyclohexan-
Yellow oil; 1H NMR (500 MHz, CDCl3) d
7.15–7.08 (m, 4H), 2.66 (tt, J = 4.0 Hz, 11.5 Hz, 1H), 2.57 (t,
J = 7.5 Hz, 2H), 2.47 (tt, J = 3.5 Hz, 12.0 Hz, 1H), 2.19–2.10 (m,
3H), 1.95–1.89 (m, 2H), 1.68–1.47 (m, 5H), 1.38–1.19 (m, 12H),
0.89 (t, J = 7.0 Hz, 3H), 0.49–0.42 (m, 2H), 0.41–0.34 (m, 2H); 13C
NMR (126 MHz, CDCl3) d 144.4, 140.6, 128.4, 126.8, 57.6, 43.9,
35.7, 34.3, 33.3, 32.0, 31.6, 29.6, 29.5, 29.4, 28.5, 22.8, 14.2, 6.6;
ESI-MS m/z calcd for C23H37N [M+H]+ 328.29, found 328.33.
C
22H38NO [M+H]+ 332.2953, found 332.2972.
4.1.5.19.
cyclohexanamine (trans-10j).
(1r,4r)-4-(4-Octylphenyl)-N-(pyridin-4-ylmethyl)
20% Yield (LiBH4); white solid;
1H NMR (400 MHz, CDCl3) d 8.58–8.53 (m, 2H), 7.31–7.27 (m,
2H), 7.10 (s, 4H), 3.88 (s, 2H), 2.59–2.43 (m, 4H), 2.12–2.04 (m,
2H), 1.96–1.88 (m, 2H), 1.64–1.55 (m, 2H), 1.55–1.42 (m, 2H),
1.39–1.21 (m, 13H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz,
CDCl3) d 150.1, 149.8, 144.1, 140.6, 128.3, 126.6, 122.9, 56.2,
49.9, 43.6, 35.6, 33.9, 33.0, 31.9, 31.5, 29.5, 29.4, 29.3, 22.7, 14.1.
4.1.5.13. (1s,4s)-N-(Prop-2-ynyl)-4-(4-octylphenyl)cyclohexan-
amine (cis-10g).
Colorless oil; 1H NMR (500 MHz, CDCl3) d
7.19–7.15 (m, 2H), 7.14–7.09 (m, 2H), 3.47 (d, J = 2.4 Hz, 2H),
3.16–3.10 (m, 1H), 2.61–2.50 (m, 3H), 2.22 (t, J = 2.4 Hz, 1H),
1.90–1.77 (m, 4H), 1.70–1.58 (m, 6H), 1.38–1.22 (m, 10H), 1.12
(br s, 1H), 0.90 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) d
144.6, 140.4, 128.3, 126.8, 82.9, 71.0, 50.2, 43.6, 35.8, 35.7, 32.0,
31.7, 30.4, 29.6, 29.5, 29.4, 28.3, 22.7, 14.2; ESI-MS m/z calcd for
4.1.5.20. (1r,4r)-4-(4-Octylphenyl)cyclohexanamine (trans-
10k).
White solid; 1H NMR (500 MHz, CDCl3) d 7.10 (s, 4H),
C
23H35N [M+H]+ 326.28, found 326.27.
2.77–2.69 (m, 1H), 2.55 (t, J = 7.5 Hz, 2H), 2.44 (tt, J = 3.4 Hz,
11.6 Hz, 1H), 2.0–1.86 (m, 4H), 1.75 (br s, 2H), 1.63–1.45 (m, 4H),
1.39–1.20 (m, 12H), 0.88 (t, J = 7.5 Hz, 3H); 13C NMR (126 MHz,
CDCl3) d 143.3, 140.6, 128.7, 128.4, 50.5, 43.3, 37.1, 35.7, 33.3,
32.0, 31.6, 30.8, 29.6, 29.5, 29.4, 22.8, 14.2; HRMS (FAB+) m/z calcd
for C20H34N+ [M+H]+ 288.2691, found 288.2680.
4.1.5.14. (1r,4r)-N-(Prop-2-ynyl)-4-(4-octylphenyl)cyclohexan-
amine (trans-10g).
White solid, mp 48.1–49.1 °C; 1H NMR
(500 MHz, CDCl3) d 7.16–7.09 (m, 4H), 3.50 (d, J = 2.4 Hz, 2H),
2.75 (tt, J = 3.4 Hz, 11.6 Hz, 1H), 2.56 (t, J = 8.0 Hz, 2H), 2.48 (tt,
J = 3.0 Hz, 12.0 Hz, 1H), 2.22 (t, J = 2.4 Hz, 1H), 2.05–1.98 (m, 2H),
1.96–1.89 (m, 2H), 1.63–1.48 (m, 4H), 1.37–1.20 (m, 13H), 0.88
(t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) d 144.3, 140.7,
128.3, 126.7, 82.6, 71.4, 55.0, 43.6, 35.7, 35.4, 33.4, 32.9, 32.0,
4.1.6. General procedure for the Eschweiler–Clarke methylation
of secondary amines
Formic acid (4 equiv) was added to a solution of secondary
amine (1 equiv) and paraformaldehyde (4 equiv) in methanol at
rt. The reaction mixture was refluxed for 6 h. After cooling to rt,
it was partitioned between ether and water. 10% NaOH was added
to the aqueous layer until the pH is ꢁ12. The basic aqueous layer
was then extracted 3 times with ether. The combined organic ex-
tracts were washed with brine, dried with sodium sulfate and
the solvent removed by evaporation under reduced pressure. The
product was purified by column chromatography on neutral
alumina.
31.6, 29.6, 29.5, 29.4, 22.7, 14.1; ESI-MS m/z calcd for C23H35
N
[M+H]+ 326.28, found 326.27.
4.1.5.15. (1s,4s)-N-Allyl-4-(4-octylphenyl)cyclohexanamine (cis-
10h).
Colorless oil; 1H NMR (500 MHz, CDCl3) d 7.17–7.14 (m,
2H), 7.12–7.09 (m, 2H), 6.02–5.90 (m, 1H), 5.20 (dq, J = 1.7 Hz,
17.2 Hz, 1H), 5.09 (dq, J = 1.6 Hz, 10.0 Hz, 1H), 3.27 (dt, J = 1.4 Hz,
6.0 Hz, 2H), 2.95–2.88 (m, 1H), 2.59–2.50 (m, 3H), 1.86–1.75 (m,
4H), 1.70–1.56 (m, 6H), 1.38–1.21 (m, 11H), 0.88 (t, J = 7.0 Hz,
3H); 13C NMR (126 MHz, CDCl3) d 144.5, 140.4, 137.7, 128.4,
126.7, 115.5, 51.3, 50.0, 43.2, 35.5, 32.0, 31.7, 30.5, 29.6, 29.5,
29.4, 28.3, 22.7, 14.2; ESI-MS m/z calcd for C23H37N [M+H]+
328.29, found 328.33.
4.1.6.1.
amine (cis-11a).
(1s,4s)-N,N-Dimethyl-4-(4-octylphenyl)cyclohexan-
91% Yield, colorless oil; 1H NMR (500 MHz,
CDCl3) d 7.19–7.16 (m, 2H), 7.10–7.07 (m, 2H), 2.65–2.51 (m,
3H), 2.24 (s, 6H), 2.10–2.06 (m, 1H), 1.99–1.86 (m, 4H), 1.65–1.56
(m, 4H), 1.56–1.47 (m, 2H), 1.35–1.22 (m, 10H), 0.87 (t,
J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) d 144.4, 140.3, 128.2,
127.0, 61.1, 43.6, 43.0, 35.6, 32.0, 31.7, 29.6, 29.5, 29.4, 28.9,
28.6, 22.8, 14.2; ESI-MS m/z calcd for C22H37N [M+H]+ 316.29,
found 316.29.
4.1.5.16.
(trans-10h).
(1r,4r)-N-Allyl-4-(4-octylphenyl)cyclohexanamine
Colorless oil; 1H NMR (400 MHz, CDCl3)
d
7.17–7.05 (m, 4H), 6.01–5.87 (m, 1H), 5.19 (dq, J = 1.6 Hz,
17.2 Hz, 1H), 5.12–5.07 (m, 1H), 3.32 (dt, J = 1.3 Hz, 6.0 Hz, 2H),
2.61–2.42 (m, 4H), 2.10–2.00 (m, 2H), 1.96–1.86 (m, 2H), 1.63–
1.44 (m, 4H), 1.38–1.17 (m, 13H), 0.87 (t, J = 6.9 Hz, 3H); 13C
NMR (126 MHz, CDCl3)
d
144.4, 140.6, 137.4, 128.4, 126.7,
4.1.6.2.
amine (trans-11a).
(1r,4r)-N,N-Dimethyl-4-(4-octylphenyl)cyclohexan-
85% Yield, colorless oil; 1H NMR
115.7, 56.2, 49.8, 43.8, 35.7, 34.0, 33.2, 32.0, 31.6, 29.6, 29.5,
29.4, 22.8, 14.2; HRMS (FAB+) m/z calcd for C23H37N [M+H]+
328.2999, found 328.3001.
(500 MHz, CDCl3) d 7.13–7.09 (m, 4H), 2.58–2.54 (t, J = 7.5 Hz,
2H), 2.44 (tt, J = 3.0 Hz, 11.5 Hz, 1H), 2.32 (s, 6H), 2.25 (tt,
J = 3.0 Hz, 11.5 Hz, 1H), 2.05–1.94 (m, 4H), 1.63–1.56 (m, 2H),
1.57–1.44 (m, 2H), 1.41–1.22 (m, 12H), 0.88 (t, J = 7.5 Hz, 3H);
13C NMR (126 MHz, CDCl3) d 144.3, 140.6, 128.4, 126.7, 63.5,
43.8, 41.8, 35.7, 33.6, 32.0, 31.6, 29.6, 29.5, 29.4, 29.1, 22.8, 14.2;
4.1.5.17. 2-(((1s,4s)-4-(4-Octylphenyl)cyclohexyl)amino)ethanol
(cis-10i).
(400 MHz, CDCl3) d 7.16–7.12 (m, 2H), 7.12–7.08 (m, 2H), 3.69–
76% Yield (NaBH(OAc)3), colorless oil; 1H NMR

192
M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
HRMS (FAB+) m/z calcd for C22H37N [M+H]+ 316.3004, found
316.3009.
1.77 (m, 2H), 1.68–1.56 (m, 4H), 1.38–1.19 (m, 10H), 1.04 (t,
J = 7.3 Hz, 3H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (126 MHz, CDCl3)
d 140.9, 138.6, 128.9, 127.0, 72.7, 64.1, 49.0, 35.5, 33.9, 32.0,
31.5, 29.6, 29.5, 29.4, 28.8, 22.8, 21.9, 16.5, 14.2, 10.9; ESI-MS m/
z calcd for C25H44N+ 358.35, found 358.34.
4.1.6.3. (1s,4s)-N-Methyl-4-(4-octylphenyl)-N-propylcyclohex-
anamine (cis-11b).
93% Yield, colorless oil; 1H NMR
(400 MHz, CDCl3) d 7.22–7.16 (m, 2H), 7.13–7.07 (m, 2H), 2.72–
2.62 (m, 1H), 2.60–2.52 (m, 2H), 2.45–2.34 (m, 3H), 2.22 (s, 3H),
2.05–1.93 (m, 2H), 1.92–1.81 (m, 2H), 1.66–1.40 (m, 8H), 1.36–
1.21 (m, 10H), 0.88 (t, J = 7.3 Hz, 6H); 13C NMR (126 MHz, CDCl3)
d 144.2, 140.2, 128.3, 127.1, 58.7, 56.0, 41.9, 38.9, 35.6, 32.0,
31.7, 29.6, 29.5, 29.4, 28.8, 28.1, 22.8, 19.4, 14.2, 12.1; ESI-MS m/
z calcd for C24H41N [M+H]+ 344.32, found 344.35.
4.1.7.4. (1r,4r)-N,N-Dimethyl-4-(4-octylphenyl)-N-propylcyclo-
hexanaminium iodide (trans-12b).
71% Yield, white solid,
mp 162.6–163.7 °C; 1H NMR (400 MHz, CDCl3) d 7.12–7.05 (m,
4H), 3.77–3.68 (m, 1H), 3.60–3.51 (m, 2H), 3.34 (s, 6H), 2.60–2.50
(m, 3H), 2.37–2.29 (m, 2H), 2.17–2.10 (m, 2H), 1.92–1.80 (m,
2H), 1.78–1.65 (m, 4H), 1.61–1.52 (m, 2H), 1.35–1.20 (m, 10H),
1.07 (t, J = 7.3 Hz, 3H), 0.85 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz,
CDCl3) d 141.7, 141.4, 128.6, 126.6, 72.0, 64.5, 49.5, 42.4, 35.6,
32.6, 32.0, 31.6, 29.6, 29.5, 29.3, 26.6, 22.7, 16.5, 14.2, 10.9; ESI-
MS m/z calcd for C25H44N+ 358.35, found 358.34; HRMS (FAB+)
m/z calcd for C25H44N+ 358.3463, found 358.3470.
4.1.6.4. (1r,4r)-N-Methyl-4-(4-octylphenyl)-N-propylcyclohex-
anamine (trans-11b).
66% Yield, colorless oil; 1H NMR
(500 MHz, CDCl3) d 7.13–7.07 (m, 4H), 2.58–2.53 (m, 2H), 2.50–
2.38 (m, 4H), 2.28 (s, 3H), 1.99–1.90 (m, 4H), 1.62–1.54 (m, 2H),
1.54–1.36 (m, 6H), 1.35–1.21 (m, 10H), 0.92–0.84 (m, 6H); 13C
NMR (126 MHz, CDCl3) d 144.4, 140.6, 128.4, 126.7, 62.3, 56.0,
44.0, 38.1, 35.7, 33.9, 32.0, 31.6, 29.6, 29.5, 29.4, 28.7, 22.8, 21.2,
14.2, 12.1; HRMS (FAB+) m/z calcd for C24H41N [M+H]+ 344.3312,
found 344.3314.
4.1.7.5.
(1s,4s)-N-Isopropyl-N,N-dimethyl-4-(4-octylphe-
70% Yield, white so-
nyl)cyclohexanaminium iodide (cis-12c).
lid; 1H NMR (500 MHz, CDCl3) d 7.20-7.07 (m, 4H), 4.06–3.98 (m,
1H), 3.84 (tt, J = 3.2 Hz, 11.8 Hz, 1H), 3.14–3.05 (m, 1H), 2.96 (s,
6H), 2.54 (t, J = 7.9 Hz, 2H), 2.49–2.42 (m, 2H), 2.17–2.00 (m, 4H),
1.68–1.52 (m, 4H), 1.48 (d, J = 6.5 Hz, 6H), 1.34–1.20 (m, 10H),
0.85 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 140.9, 139.0,
129.0, 127.2, 71.7, 63.9, 44.7, 35.6, 34.2, 32.1, 31.7, 29.7, 29.6,
29.5, 29.1, 22.9, 22.4, 17.8, 14.3; ESI-MS m/z calcd for C25H44N+
358.35, found 358.32.
4.1.6.5.
hexanamine (cis-11c).
(1s,4s)-N-Isopropyl-N-methyl-4-(4-octylphenyl)cyclo-
86% Yield, colorless oil; 1H NMR
(400 MHz, CDCl3) d 7.21–7.15 (m, 2H), 7.14–7.07 (m, 2H), 3.27–
3.10 (m, 1H), 2.73–2.52 (m, 4H), 2.14 (s, 3H), 2.05–1.86 (m, 4H),
1.67–1.48 (m, 6H), 1.39–1.19 (m, 10H), 1.01 (d, J = 6.6 Hz, 6H),
0.89 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 140.9, 139.0,
129.0, 127.2, 71.7, 63.9, 44.7, 35.6, 34.2, 32.1, 31.7, 29.7, 29.6,
4.1.8. General procedure for the synthesis of quaternary
ammonium salts from secondary amines
29.5, 29.1, 22.9, 22.4, 17.8, 14.3; ESI-MS m/z calcd for C24H41
N
[M+H]+ 344.32, found 344.32.
Methyliodide(10 equiv)was addedto a solutionof thesecondary
amine (1 equiv) and K2CO3 (3 equiv) in acetonitrile. The reaction
mixture was refluxed for 2 h. The organic solvent was evaporated
under reduced pressure and the residue dissolved in diethyl ether.
The precipitate was collected by filtration and washed three times
with diethyl ether. The precipitate was dissolved in CHCl3, the inor-
ganic precipitates filtered and the filtrate concentrated under re-
duced pressure to give the pure quaternary ammonium salt.
4.1.7. General procedure for the synthesis of quaternary
ammonium salts from tertiary amines
Methyl iodide (10 equiv) was added to a solution of the tertiary
amine (1 equiv) in acetonitrile. The reaction mixture was refluxed
for 2 h. The organic solvent was evaporated under reduced pres-
sure and the residue dissolved in diethyl ether. The precipitate
was collected by filtration and washed 3 times with diethyl ether
to give the pure quaternary ammonium salt.
4.1.8.1.
(1r,4r)-N-Isopropyl-N,N-dimethyl-4-(4-octylphe-
68% Yield, white
nyl)cyclohexanaminium iodide (trans-12c).
4.1.7.1. (1s,4s)-N,N,N-Trimethyl-4-(4-octylphenyl)cyclohexana-
solid; 1H NMR (400 MHz, CDCl3) d 7.10 (s, 4H), 4.20–4.08 (m,
1H), 3.72 (tt, J = 2.8 Hz, 11.7 Hz, 1H), 3.13 (s, 6H), 2.64–2.52 (m,
3H), 2.45–2.35 (m, 2H), 2.20–2.11 (m, 2H), 1.91–1.66 (m, 4H),
1.65–1.49 (m, 8H), 1.39–1.19 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H);
13C NMR (101 MHz, CDCl3) d 141.6, 141.2, 128.5, 126.5, 71.2,
63.7, 44.7, 42.4, 35.5, 32.7, 31.9, 31.5, 29.5, 29.4, 29.2, 26.8, 22.7,
17.4, 14.1; ESI-MS m/z calcd for C25H44N+ 358.35, found 358.32.
minium iodide (cis-12a).
69% Yield, pink solid, mp 238.4–
239.8 °C; 1H NMR (500 MHz, CDCl3) d 7.22–7.17 (m, 2H), 7.17–
7.12 (m, 2H), 4.04 (tt, J = 3.3 Hz, 12.1 Hz, 1H), 3.31 (s, 9H), 3.10
(s, 1H), 2.62–2.52 (m, 2H), 2.51–2.43 (m, 2H), 2.16–2.00 (m, 4H),
1.69–1.50 (m, 4H), 1.37–1.18 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H);
13C NMR (126 Hz, CDCl3) d 140.9, 138.7, 128.9, 127.0, 74.6, 51.6,
35.5, 33.8, 32.0, 31.5, 29.6, 29.5, 29.3, 28.5, 22.7, 22.1, 14.2; ESI-
MS m/z calcd for C25H44N+ 330.32, found 330.31.
4.1.8.2. (1r,4r)-N-Benzyl-N,N-dimethyl-4-(4-octylphenyl)cyclo-
hexanaminium iodide (trans-12e).
73% Yield, white solid,
4.1.7.2. (1r,4r)-N,N,N-trimethyl-4-(4-octylphenyl)cyclohexana-
minium iodide (trans-12a).
mp 183.9–185.0 °C; 1H NMR (500 MHz, CDCl3) d 7.69–7.65 (m,
2H), 7.46–7.38 (m, 3H), 7.08–7.01 (m, 4H), 4.98 (s, 2H), 3.83 (tt,
J = 2.8 Hz, 12.0 Hz, 1H), 3.20 (s, 6H), 2.59–2.41 (m, 5H), 2.17–2.05
(m, 2H), 1.91–1.76 (m, 2H), 1.67–1.52 (m, 4H), 1.35–1.16 (m,
10H), 0.85 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) d 141.7,
141.3, 133.4, 130.9, 129.4, 128.6, 127.3, 126.6, 72.1, 65.1, 47.8,
42.2, 35.6, 32.5, 32.0, 31.6, 29.6, 29.5, 29.3, 27.1, 22.7, 14.2; ESI-
MS m/z calcd for C29H44N+ 406.35, found 406.31.
72% Yield, white solid; 1H NMR
(500 MHz, CD3OD) d 7.17–7.06 (m, 4H), 3.53 (m, 1H), 3.14 (s,
9H), 2.61–2.51 (m, 3H), 2.40–2.30 (m, 2H), 2.14–2.05 (m, 2H),
1.81–1.52 (m, 6H), 1.37–1.21 (m, 10H), 0.88 (t, J = 7.5 Hz, 3H);
13C NMR (126 MHz, CD3OD) d 144.5, 140.8, 128.2, 126.3, 74.1,
50.4, 42.2, 35.2, 32.5, 31.7, 31.4, 29.2, 29.1, 29.0, 26.1, 22.4, 13.1;
HRMS (FAB+) m/z calcd for C23H40N+ 330.3161, found 330.3168.
4.1.7.3. (1s,4s)-N,N-Dimethyl-4-(4-octylphenyl)-N-propylcyclo-
hexanaminium iodide (cis-12b).
NMR (400 MHz, CDCl3) d 7.23–7.18 (m, 2H), 7.17–7.13 (m, 2H),
3.98–3.84 (m, 1H), 3.48–3.39 (m, 2H), 3.21 (s, 6H), 3.12 (m, 1H),
2.61–2.54 (m, 2H), 2.54–2.45 (m, 2H), 2.12–2.00 (m, 4H), 1.90–
4.1.8.3.
(1r,4r)-N-Allyl-N,N-dimethyl-4-(4-octylphenyl)cyclo-
68% Yield, yellow solid; 1H
hexanaminium iodide (trans-12h).
72% Yield, white solid;
1H NMR (400 MHz, CDCl3) d 7.10–7.02 (m, 4H), 6.14–5.99 (m,
1H), 5.94–5.86 (m, 1H), 5.78–5.70 (m, 1H), 4.37 (d, J = 7.2 Hz,
2H), 3.65 (tt, J = 3.1 Hz, 12.0 Hz, 1H), 3.30 (s, 6H), 2.61–2.48 (m,

M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
193
3H), 2.43–2.32 (m, 2H), 2.18–2.08 (m, 2H), 1.87–1.50 (m, 6H),
1.36–1.16 (m, 10H), 0.85 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz,
CDCl3) d 141.6, 141.2, 130.0, 128.5, 126.5, 124.3, 71.8, 64.7, 48.6,
42.2, 35.5, 32.4, 31.9, 31.5, 29.5, 29.4, 29.2, 26.6, 22.7, 14.1; ESI-
MS m/z calcd for C25H42N+ 356.35, found 356.32.
for 5 min and then refluxed for 6 h. The reaction mixture was ex-
tracted with EtOAc, washed with brine, dried with MgSO4 and fil-
tered. After evaporation of the organic solvent under reduced
pressure, the resulting residue was purified by column chromatog-
raphy over silica gel (100% hexanes to 70/30 hexanes/EtOAc) to
provide a yellow oil (93% yield). 1H NMR (500 MHz, CDCl3) d 7.90
(d, J = 8.3 Hz, 2H), 7.58–7.50 (m, 1H), 7.23 (d, J = 7.6 Hz, 2H), 7.10
(d, J = 7.6 Hz, 1H), 6.57 (d, J = 8.5 Hz, 1H), 3.64–3.55 (m, 8H),
2.67–2.60 (m, 2H), 1.67–1.57 (m, 2H), 1.49 (s, 9H), 1.36–1.24 (m,
8H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR (126 MHz, CDCl3) d 158.9,
155.5, 155.0, 143.8, 138.3, 137.3, 128.7, 126.7, 109.9, 105.4, 80.0,
45.2, 35.8, 32.0, 31.5, 29.6, 29.4, 29.3, 28.5, 22.8, 14.2; HRMS
(ESI+) m/z calcd for C28H41N3O2 [M+H]+ 452.3199, found 452.3605.
The Boc-protected amine was dissolved in 10 mL methanol and
HCl (g) was bubbled through the solution for one minute. After
evaporation of the methanol, diethyl ether was added and the solid
filtered. It was washed with cold diethyl ether to yield the title
compound as orange oil (92% yield). 1H NMR (400 MHz, CD3OD)
d 8.09-8.03 (m, 1H), 7.80 (d, J = 7.3 Hz, 2H), 7.39 (d, J = 7.0 Hz,
2H), 7.33 (d, J = 7.0 Hz, 1H), 7.29–7.20 (m, 1H), 4.05–3.96 (m,
4H), 3.50–3.39 (m, 4H), 2.70 (t, J = 7.6 Hz, 2H), 1.71–1.59 (m, 2H),
1.40–1.20 (m, 8H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (126 MHz,
CD3OD) d 155.1, 151.8, 146.2, 143.6, 129.0, 127.7, 114.6, 113.0,
109.8, 44.0, 42.9, 35.4, 31.7, 31.2, 29.2, 29.1, 29.0, 22.4, 13.1; HRMS
(ESI+) m/z calcd for C23H34N3 [M+H]+ 352.2747, found 352.2742.
4.1.8.4. (1s,4s)-N-(2-Hydroxyethyl)-N,N-dimethyl-4-(4-octylphe-
nyl)cyclohexanaminium iodide (cis-12i).
52% Yield, white so-
lid; 1H NMR (400 MHz, CD3OD) d 7.38–7.24 (m, 2H), 7.22–7.07 (m,
2H), 4.09–3.91 (m, 2H), 3.91–3.68 (m, 1H), 3.56–3.43 (m, 2H),
3.15–2.95 (m, 7H), 2.56 (t, J = 7.7 Hz, 2H), 2.53–2.43 (m, 2H),
2.25–2.14 (m, 1H), 2.14–1.91 (m, 3H), 1.80–1.38 (m, 4H), 1.38–
1.13 (m, 11H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (126 MHz, CDCl3)
d 140.3, 138.7, 128.4, 126.7, 74.0, 63.4, 55.4, 35.0, 33.7, 31.5,
31.2, 29.1, 29.0, 28.9, 28.4, 22.3, 21.4, 13.4; HRMS (FAB+) m/z calcd
for C24H42NO+ 360.3266, found 360.3273.
4.1.8.5.
(1r,4r)-N-(2-Hydroxyethyl)-N,N-dimethyl-4-(4-octyl-
39% Yield,
phenyl)cyclohexanaminium iodide (trans-12i).
white solid, mp 153.3–154.2 °C; 1H NMR (500 MHz, CDCl3) d
7.11–7.04 (m, 4H), 4.34–4.28 (m, 1H), 4.22–4.16 (m, 2H), 3.95–
3.87 (m, 1H), 3.81–3.76 (m, 2H), 3.30 (s, 6H), 2.52 (t, J = 7.5 Hz,
2H), 2.42–2.35 (m, 2H), 2.13–2.02 (m, 2H), 1.79–1.65 (m, 4H),
1.60–1.50 (m, 2H), 1.34–1.18 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H);
13C NMR (126 MHz, CDCl3) d 141.8, 141.3, 128.6, 126.7, 73.4,
64.1, 55.9, 50.1, 42.5, 35.6, 32.5, 32.0, 31.6, 29.6, 29.5, 29.4, 26.7,
22.8, 14.2; HRMS (FAB+) m/z calcd for C24H42NO+ 360.3266, found
360.3259.
4.1.8.8.
Pd(OAc)2 (0.232 mg, 1.032
1.032
50-(4-Octylphenyl)-2H-[1,20-bipyridin]-2-one
(15).
l
mol), and S-Phos (0.424 mg,
lmol) were added to a suspension of K2CO3 (35.7 mg,
4.1.8.6. 5-(4-Octylphenyl)pyrazin-2-aminium chloride (13).
Pd(OAc)2 (0.232 mg, 1.032 lmol), and S-Phos (2.89 mg, 7.03 lmol)
0.258 mmol), 1-bromo-4-octylbenzene (0.012 ml, 0.052 mmol),
50-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-[1,20-bipyri-
din]-2-one (20 mg, 0.067 mmol) in acetonitrile/water (1.5:1). The
suspension was degassed for 5 minutes and refluxed for 6 h. The
reaction mixture was extracted with EtOAc, washed with brine,
dried with MgSO4 and filtered. After evaporation of the organic sol-
vent under reduced pressure, the resulting residue was purified by
column chromatography over silica gel (100% hexanes to 40/60
hexanes/EtOAc) to provide the title compound as an off-white solid
(81% yield). 1H NMR (400 MHz, CDCl3) d 8.75 (t, J = 1.7 Hz, 1H), 8.00
(d, J = 1.6 Hz, 3H), 7.92 (dd, J = 2.1 Hz, 7.1 Hz, 1H), 7.52 (d,
J = 8.3 Hz, 2H), 7.41 (m, 1H), 7.31 (d, J = 8.3 Hz, 2H), 6.71–6.63
(m, 1H), 6.36–6.27 (m, 1H), 2.66 (t, J = 7.8 Hz, 2H), 1.71–1.59 (m,
2H), 1.37–1.22 (m, 8H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR
(101 MHz, CDCl3) d 162.3, 150.5, 147.0, 143.5, 140.2, 136.3,
136.0, 135.9, 134.1, 129.3, 127.0, 122.1, 121.1, 106.3, 35.7, 31.9,
31.5, 29.5, 29.4, 29.3, 29.2, 22.7, 14.1; HRMS (ESI+) m/z calcd for
were added to a suspension of K2CO3 (243 mg, 1.758 mmol), 5-
bromopyrazin-2-amine (61.2 mg, 0.352 mmol), and (4-octylphe-
nyl)boronic acid (107 mg, 0.457 mmol) (prepared according to
Ishi-I, T. et al.49) in acetonitrile/water (1.5:1). The suspension was
degassed for 5 min and then refluxed for 6 h. The reaction mixture
was extracted with EtOAc, washed with brine, dried with MgSO4
and filtered. After evaporation of the organic solvent under re-
duced pressure, the resulting residue was purified by column chro-
matography over silica gel (100% hexanes to 50/50 hexanes/EtOAc)
to provide an orange solid (95% yield). 1H NMR (400 MHz, CDCl3) d
8.45 (s, 1H), 8.04 (s, 1H), 7.78 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz,
2H), 4.82 (br s, 1H), 2.64 (t, J = 7.7 Hz, 2H), 1.63 (m, 2H), 1.38–1.20
(m, 8H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 152.8,
143.2, 143.0, 138.4, 134.3, 131.8, 128.9, 125.5, 35.7, 31.9, 31.4,
29.5, 29.3, 29.2, 22.7, 14.1; HRMS (ESI+) m/z calcd for C18H26N3
[M+H]+ 284.2121, found 284.2136.
C
24H28N2O [M+Na]+ 383.2094, found 383.2113.
The above amine was dissolved in 10 mL methanol and HCl (g)
was bubbled through the solution for one minute. After evapora-
tion of the methanol, diethyl ether was added and the solid filtered.
It was washed with cold diethyl ether to yield the title compound
as a pale yellow solid (95% yield). 1H NMR (500 MHz, DMSO-d6) d
8.44(s, 1H), 8.29 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.26 (d,
J = 7.8 Hz, 2H), 2.59 (t, J = 7.3 Hz, 2H), 1.65–1.49 (m, 2H), 1.40–
1.10 (m, 8H), 0.84 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-
d6) d 151.6, 143.1, 139.5, 136.2, 133.8, 131.8, 129.3, 125.4, 35.4,
31.8, 31.4, 29.4, 29.3, 29.2, 22.6, 14.5; HRMS (ESI+) m/z calcd for
4.2. Biology
4.2.1. Sphingosine kinase assay
Human SphK1 and mouse SphK2 cDNAs were used to generate
recombinant baculoviruses that encoded the respective proteins.
Infection of Sf9 insect cells with the viruses for 72 h resulted in
>1000-fold increases in SphK activity in 10,000ꢂg supernatant
fluid from homogenized cell pellets. The enzyme assay conditions
were exactly as described,43 except infected Sf9 cell extract con-
C
18H26N3 [M+H]+ 284.2121, found 284.2119.
taining 2-3 lg protein was used as a source of enzyme.
4.1.8.7. 4-(6-(4-Octylphenyl)pyridin-2-yl)piperazin-1-ium chlo-
ride (14). Pd(OAc)2 (0.115 mg, 0.514 mol), and S-Phos
(0.211 mg, 0.514 mol) were added to a suspension of K2CO3
(17.75 mg, 0.128 mmol), 1-bromo-4-octylbenzene (6.12 l,
0.026 mmol), tert-butyl 4-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)pyridin-2-yl)piperazine-1-carboxylate (10 mg, 0.026
mmol), in acetonitrile/water (1.5:1). The suspension was degassed
4.2.2. LC/MS protocol
l
Analyses were performed by Liquid Chromatography/ESI Mass
Spectrometry (LC/MS) using a triple quadrupole mass spectrome-
ter (Sciex 4000 Q-Trap) coupled to a Shimadzu LC-20AD LC system.
A binary solvent gradient with a flow rate of 1 mL/min was used to
separate FTY720 and FTY720-P by reverse phase chromatography
l
l
using a Supelco Discovery C18 column (50 mm ꢂ 2.1 mm, 5
lm

194
M. R. Raje et al. / Bioorg. Med. Chem. 20 (2012) 183–194
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acid (79:20:1) while mobile phase B was methanol: formic acid
(99:1). The run started with 100% A for 0.5 minutes. Solvent B
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A for 1 min. Natural sphingolipids were detected using multiple
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lows: C17S1P (366.4?250.4); FTY720 (308.4?255.1); FTY720-P
(388.4?255.1); C17sphingosine (286.4?250.3); C17S1P and
C17sphingosine were used as the internal standards. All analytes
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were: 35, 10, 25, 6; and 156, 10, 25, 14 volts, respectively. Reten-
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Acknowledgments
We gratefully acknowledge support from Virginia Tech Depart-
ment of Chemistry. This work was supported in part by a grant
from the NIH (R01 GM067958) to K.R.L.
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Supplementary data (explanation of stereoselectivity, GC analy-
sis of reductive amination reactions, and 1H and 13C NMR spectral
data for all compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2011.11.011.
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