Structure-activity relationship studies and in vivo activity of guanidine-based sphingosine kinase inhibitors: Discovery of SphK1- and SphK2-selective inhibitors

Article abstract of DOI:10.1021/jm501760d

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that acts as a ligand for five G-protein coupled receptors (S1P_1-5) whose downstream effects are implicated in a variety of important pathologies including sickle cell disease, cancer, inflammation, and fibrosis. The synthesis of S1P is catalyzed by sphingosine kinase (SphK) isoforms 1 and 2, and hence, inhibitors of this phosphorylation step are pivotal in understanding the physiological functions of SphKs. To date, SphK1 and 2 inhibitors with the potency, selectivity, and in vivo stability necessary to determine the potential of these kinases as therapeutic targets are lacking. Herein, we report the design, synthesis, and structure-activity relationship studies of guanidine-based SphK inhibitors bearing an oxadiazole ring in the scaffold. Our studies demonstrate that SLP120701, a SphK2-selective inhibitor (K_i = 1 μM), decreases S1P levels in histiocytic lymphoma (U937) cells. Surprisingly, homologation with a single methylene unit between the oxadiazole and heterocyclic ring afforded a SphK1-selective inhibitor in SLP7111228 (K_i = 48 nM), which also decreased S1P levels in cultured U937 cells. In vivo application of both compounds, however, resulted in contrasting effect in circulating levels of S1P. Administration of SLP7111228 depressed blood S1P levels while SLP120701 increased levels of S1P. Taken together, these compounds provide an in vivo chemical toolkit to interrogate the effect of increasing or decreasing S1P levels and whether such a maneuver can have implications in disease states.

Full text of DOI:10.1021/jm501760d

Article
pubs.acs.org/jmc
Structure−Activity Relationship Studies and in Vivo Activity of
Guanidine-Based Sphingosine Kinase Inhibitors: Discovery of SphK1-
and SphK2-Selective Inhibitors
Neeraj N. Patwardhan,†,# Emily A. Morris,†,# Yugesh Kharel,§ Mithun R. Raje,† Ming Gao,†
Jose L. Tomsig,§ Kevin R. Lynch,§ and Webster L. Santos*,†
†Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24061, United States
§Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, United States
S
* Supporting Information
ABSTRACT: Sphingosine 1-phosphate (S1P) is a pleiotropic
signaling molecule that acts as a ligand for five G-protein
coupled receptors (S1P1−5) whose downstream effects are
implicated in a variety of important pathologies including
sickle cell disease, cancer, inflammation, and fibrosis. The
synthesis of S1P is catalyzed by sphingosine kinase (SphK)
isoforms 1 and 2, and hence, inhibitors of this phosphorylation
step are pivotal in understanding the physiological functions of
SphKs. To date, SphK1 and 2 inhibitors with the potency,
selectivity, and in vivo stability necessary to determine the
potential of these kinases as therapeutic targets are lacking. Herein, we report the design, synthesis, and structure−activity
relationship studies of guanidine-based SphK inhibitors bearing an oxadiazole ring in the scaffold. Our studies demonstrate that
SLP120701, a SphK2-selective inhibitor (Ki = 1 μM), decreases S1P levels in histiocytic lymphoma (U937) cells. Surprisingly,
homologation with a single methylene unit between the oxadiazole and heterocyclic ring afforded a SphK1-selective inhibitor in
SLP7111228 (Ki = 48 nM), which also decreased S1P levels in cultured U937 cells. In vivo application of both compounds,
however, resulted in contrasting effect in circulating levels of S1P. Administration of SLP7111228 depressed blood S1P levels
while SLP120701 increased levels of S1P. Taken together, these compounds provide an in vivo chemical toolkit to interrogate
the effect of increasing or decreasing S1P levels and whether such a maneuver can have implications in disease states.
isoenzyme has been reported to result in opposing biological
effects, implying conflicting roles by these enzymes. For example,
SphK1 overexpression increases cell survival and proliferation8
whereas SphK2 overexpression induces cell cycle arrest and
apoptosis.9,10 The latter effect can be explained through the
interaction of Bcl-XL with the BH3 domain within SphK2.11 In
addition, there is also a difference in their subcellular localization:
SphK1 is mostly in the cytoplasm and migrates to the plasma
membrane upon phosphorylation12,13 while SphK2 can localize
in the nucleus to inhibit DNA synthesis and regulate HDAC1/2
activity.14,15 Gene deletion studies in mice indicate that there is
some functional redundancy between the two enzymes, as
Sphk1−/− and Sphk2−/− mice are viable, fertile, and phenotypi-
cally unremarkable.16,17 However, germ line inactivation of all
four SphK alleles is embryonically lethal (day E12.5−13.5) as a
result of impaired neurological and vascular development.18 The
circulating level of S1P in SphK1-null mice is reduced by about 2-
fold while, curiously, SphK2-null mice have more than double
wild type S1P levels in blood and plasma.18−21
INTRODUCTION
■
The lysophospholipid sphingosine 1-phosphate (S1P) is a
pleiotropic signaling molecule that regulates growth, survival,
and migration of many cell types. S1P acts as an extracellular
mediator by binding to five G-protein coupled receptors
(S1P1−5), leading to diverse physiological and pathophysiological
processes.1 Biosynthesis of S1P is realized only by phosphor-
ylation of sphingosine, which is generated by the catabolism of
more complex sphingolipids such as sphingomyelin and
ceramide or taken up by cells from their environment. The
enzyme responsible for this phosphoryl transfer exists as two
isoforms encoded by unlinked genes: sphingosine kinase 1
(SphK1) and 2 (SphK2). SphKs have been implicated in a variety
of disease states including sickle cell disease,2 cancer,1,3
atherosclerosis,4 and asthma,5 among others. SphK1’s role in
cancer is widely studied, where correlations between expression
and severity of disease, drug resistance, and/or reduced patient
survival have been reported.3,6 However, pharmacological
intervention to lessen SphK1 activity and control the “SphK
rheostat”, i.e., changing the equilibrium of S1P/Sph ratio, failed
to demonstrate statistically significant effects on cell viability,
suggesting that S1P’s role in oncology or other diseases is
complex.7 Interestingly, the S1P generated by each SphK
Received: November 13, 2014
© XXXX American Chemical Society
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Figure 1. Structures, inhibitory activity, and half-lives of sphingosine kinase inhibitors.
Because of the potential role of S1P in a variety of diseases,
pharmacological inhibition of SphKs with small molecules has
been a subject of interest both in academia and pharmaceutical
industry. Figure 1 illustrates the structures of reported SphK
inhibitors.22 SphK1, for which selective inhibitors have been
developed, has been the focus of most studies. Because early
inhibitors used to interrogate the function of SphK1 had Ki’s in
the mid micromolar range (e.g., SK1-I, SKI-II, dimethyl-
sphingosine), some of the biological effects attributed to
SphK1 activity may be due to off-target interactions. Indeed,
these compounds are best described as molecules that include in
their properties “inhibition of SphK1” rather than the commonly
used “SphK1 inhibitors”. The discovery of selective, nanomolar
inhibitors such as PF-543, VPC96091, and compound 51 will be
useful chemical tools to better understand SphK1 function,
although some of these have limited in vivo stability23,24 or their
effect on S1P levels in vivo have not been documented.25 The
potent, dual SphK1/2 inhibitor, 82, was recently described to
decrease S1P concentration when administered in mice.7,26 In
contrast to SphK1, there is a paucity of SphK2-selective
inhibitors;27 indeed with SphK2, we have found it challenging
to duplicate the selectivity and potency readily achieved with
SphK1 inhibitors. ABC29464028 (Ki 10 μM) was the first
compound with SphK2 inhibitor properties reported, and it has
been deployed in a variety of disease models where it has been
reported to have remarkable efficacy. These models include
ulcerative colitis,29 Crohns disease,30 ischemia/reperfusion
injury,31 osteoarthritis,32 and colon cancer.33 However,
ABC294640 has at least one reported additional mode of action,
that is binding to the estrogen receptor and acting as a partial
agonist similar to tamoxifen.34 Hence, attributing ABC294640
effects to SphK2 is difficult. Other inhibitors such as SG-12,35
(R)-FTY720-OMe,36 K145,37 and trans-12b38 are reported as
SphK2 inhibitors, albeit with moderate potency and selectivity.
Hence, new scaffolds with the potency and selectivity are needed
to investigate SphK1 and 2 functions in vivo.39
Recently, we reported a novel guanidine-based compound,
SLR080811, as a selective SphK2 inhibitor.40 It was the most
potent and selective inhibitor of SphK2 at that time with in vitro
activity on U937 cells and in vivo activity in mice. In continuation
of our investigations in understanding SphKs, we herein describe
the design, synthesis, and structure−activity relationship studies
of guanidine-based SphK inhibitors. Our studies reveal SphK1
and SphK2 inhibitors based on the same scaffold but with the
distinction of a key methylene unit that induces a switch in
isoform selectivity. These inhibitors are the most potent and
selective inhibitors reported with in vitro and in vivo effects of
altering cellular and blood S1P levels.
B
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and Hunig’s base over a period of 3 days. Standard deprotection
conditions afforded compounds 7a−k.
RESULTS AND DISCUSSION
■
Design of Inhibitors. Previous reports detailed our efforts to
develop SphK2-selective inhibitors that led to the identification
of quaternary ammonium salts that were fairly potent and
moderately selective.38,41 Compound trans-12b was shown to be
∼8-fold selective toward SphK2 with a Ki of 8 μM (Figure 1).
The inhibition data validated our hypothesis that a positive
charge is essential for electrostatic interaction, perhaps with the
catalytic Asp residues on SphKs, which has been shown to be
important for recognition of Sph by the enzyme.42 Furthermore,
recent high resolution structure studies on SphK1 indicated
involvement of the catalytic Asp81 residue on the enzyme in the
activation of the primary hydroxyl of sphingosine.43 We thus
concluded that a polar, and preferably charged, species was most
likely to exhibit the desired inhibition activity toward these
enzymes.
To mimic the hydroxyl moieties in sphingosine, hydroxyl
groups were installed on the pyrrolidine ring as presented in
Scheme 2. Following the standard synthetic sequence of
coupling−guanidylation−deprotection, (S)-3-hydroxy- and
(R)-4-hydroxy derivatives 11 and 15, respectively, were
synthesized. Further, we reversed the stereochemistry of the
hydroxyl group on the 4-position of the pyrrolidine ring (Scheme
3). Inversion was accomplished under Mitsunobu conditions
using benzoic acid as the nucleophile to provide compound 16.
Subsequent saponification afforded the corresponding alcohol,
which was converted to product 17. Derivatives possessing
nitrogen in different positions of four (20a) or five (20b)
membered ring heterocycles were synthesized as shown in
Scheme 4. Finally, constitutional isomers of the oxadiazole
moiety were synthesized. As illustrated in Scheme 5, 1,2,4-
oxadiazole 23, wherein the position of the 4-octylphenyl and
pyrrolidine rings are reversed, was generated by coupling of 4-
octylbenzoic acid with the amidoxime of Boc-proline to afford
compound 22, which was an intermediate that led to product 23.
The alternative structure, 1,3,4-oxadiazole 26, was synthesized as
depicted in Scheme 6. Key intermediate tetrazole 24 was
produced by reacting benzonitrile 3 with NaN3 at reflux
temperature.45 Under DCC coupling conditions at elevated
temperature, compound 25 was isolated and converted to
guanidine 26 using standard procedures.
To define the structure−activity relationships of SphK
inhibitors, we divided the structure of trans-12b into three
regions: the quaternary ammonium group as the head, the
cyclohexyl ring as the linker, and the 4-octylphenyl group as the
tail (Figure 2). In this paper, we highlight our studies toward the
To determine whether amides are a sufficient surrogate for the
oxadiazole linker, we synthesized compounds 29−34. Activation
of Boc-proline with ethyl chloroformate followed by addition of
4-decylaniline afforded 27a,b as single enantiomers (Scheme 7).
Deprotection, guanidylation, and deprotection yielded guani-
dines 29a,b. A homologated derivative was achieved by the
reduction of benzonitrile 2, which after subsequent reactions
afforded compound 31 (Scheme 8). Further, a reversed amide
analogue was synthesized using amide coupling chemistry with 4-
ocylbenzoic acid 32, and following standard protocols yielded
guanidine 34 (Scheme 9).
Figure 2. Scaffold modifications toward second generation SphK2
inhibitors.
second generation of SphK inhibitors with a focus on the linker
and headgroup region, which features a guanidine functional
group. The guanidine group as the warhead is attractive because
it is charged under physiological conditions and possesses the
ability for electrostatic interaction with the active site Asp
residue, an interaction akin to that of quaternary amine group in
trans-12b. We were further encouraged with this strategy because
compounds with carboximidamide functionalities (amidines) are
present in SphK1 inhibitors.24,44 As further improvement, we
replaced the flexible cyclohexyl ring with a rigid heteroaromatic
1,2,4-oxadiazole linker to minimize the entropic cost of binding
and introduce potential hydrogen bond acceptor moieties. The
4-octylphenyl tail was kept constant as previously defined by a
prior chain length study.41
Chemical Synthesis. The synthesis of guanidine derivatives
is shown in Scheme 1. 4-Octyl benzonitrile 2 was synthesized via
hydroboration of 1-octene (1) with 9-borabicyclo[3.3.1]nonane
(9-BBN) followed by a Suzuki−Miyaura cross-coupling reaction
of 4-iodobenzonitrile. Treatment of benzonitrile 2 with
hydroxylamine hydrochloride and triethylamine in refluxing
ethanol affords the common intermediate amidoxime 3 in
excellent yield. Subsequent coupling with the desired amino acid
using HCTU followed by dehydration at 80 °C generates 1,2,4-
oxadiazole 4. Removal of the Boc group was accomplished with
trifluoroacetic acid or by bubbling HCl gas, which produced the
desired amine 5 that is converted to bis-Boc protected guanidine
6 by reacting with N,N′-di-Boc-1H-pyrazole-1-carboxamidine
Finally, we inserted carbon spacers between the oxadiazole and
pyrrolidine rings. Employing Boc-homoproline and a further
homologated derivative afforded compounds 36a,b as shown in
Scheme 10.
Structure−Activity Relationships of Analogues and in
Vivo Activity. Analysis of reported SphK inhibitors suggests an
optimal length, which is a positive charge 18−21 atoms from the
omega carbon of the lipid tail. Hence, compounds bearing the
guanidine headgroup with a 1,2,4-oxadiazole linked to a 4-
octylphenyl chain were synthesized and screened for their
inhibitory activity against recombinant Sphk1 and SphK2 at a
concentration of 1 μM. Most of these compounds contained a
diversifying moiety on the α carbon or on the internal nitrogen
because the coupling partners were derived from amino acids.
As shown in Table 1, glycine-derived guanidine 7a and others
bearing methyl (7c), isopropyl (7d), and hydroxymethyl (7e) on
the α carbon demonstrated modest inhibition (less than 20%
when present at 1 μM). The analogue methylated on the internal
nitrogen (7b, derived from sarcosine) was inactive. Surprisingly,
compound 7f, a cyclopropyl-containing derivative, showed little
activity although cyclopropylamidines were previously found to
be nanomolar inhibitors of SphK1.24,44 Compounds 7a−f appear
to be slightly SphK1 selective. However, when a pyrrolidine ring
was introduced (7g, SLR080811), a sharp increase in inhibitory
activity was observed along with a reversal in selectivity toward
C
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a
Scheme 1. Synthesis of Analogues 7a−k
a
Reagents and conditions; (a) 9-BBN, THF, rt, 18 h; (b) 4-iodobenzonitrile, Cs2CO3, Pd(dppf)Cl2, DMF, 80 °C, 18 h, 62%; (c) NH2OH·HCl,
Et3N, EtOH, reflux, 2 h, 92%; (d) Boc-protected amino acid, DIEA, HCTU·PF6, DMF, 80 °C, 18 h, 20−82%; (e) HCl(g), MeOH, 5 min, 59−100%;
(f) CF3COOH, CH2Cl2, 2−12 h, 59−100%; (g) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, rt, 3−6 days, 18−81%.
a
Scheme 2. Synthesis of Derivatives 11 and 15
a
Reagents and conditions: (a) Boc-amino acid, DIEA, HCTU·PF6, DMF, 80 °C, 18 h; (b) TFA, CH2Cl2, 3−12 h; (c) N,N′-di-Boc-1H-pyrazole-1-
carboxamidine, DIEA, CH3CN, rt, 3 days; (d) HCl(g), MeOH.
a
Scheme 3. Synthesis of 17
a
Reagents and conditions: (a) PPh3, DIAD, PhCOOH, THF, 0 °C, rt, 18 h, 75%; (b) 2 N NaOH, H2O: THF (1:1), rt, 18 h, 95%; (c) TFA, CH2Cl2,
45%; (d) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, rt, 3 days, 79%; (e) HCl(g), MeOH, 22%.
D
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a
Scheme 4. Synthesis of 18a,b
a
Reagents and conditions: (a) Amino acid, DIEA, HCTU·PF6, DMF, 80 °C, 18 h; (b) TFA, CH2Cl2 3−12 h; (c) HCl(g), MeOH; (d) N,N′-di-Boc-
1H-pyrazole-1-carboxamidine, DIEA, CH3CN, rt, 3 days.
a
Scheme 5. Synthesis of Reverse 1,2,4-Oxadiazole 23
a
Reagents and conditions: (a) DIEA, HCTU·PF6, DMF, 80 °C, 18 h, 19%; (b) TFA, CH2Cl2, 89%; (c) N,N′-di-Boc-1H-pyrazole-1-carboxamidine,
DIEA, CH3CN, rt, 3 days, 19%; (d) HCl(g), MeOH, 28%.
a
Scheme 6. Synthesis of 1,3,4-oxadiazole 26
a
Reagents and conditions: (a) NaN3, NH4Cl, DMF, reflux, 4 h, quant; (b) Boc-proline, DCC, toluene, 110 °C, 4 h, 67%; (c) TFA, CH2Cl2, quant;
(d) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, rt, 3 days.
a
Scheme 7. Synthesis of Amide Analogues 29a,b
a
Reagents and conditions: (a) Ethyl chloroformate, TEA, 0 °C, 30 min, then 4-decylaniline, 0 °C; (b) TFA, CH2Cl2; (c) N,N′-di-Boc-1H-pyrazole-
1-carboxamidine, DIEA, CH3CN, rt.
a
Scheme 8. Synthesis of Amide Analogue 31
a
Reagents and conditions: (a) Lithium aluminum hydride, 0 °C to rt, 1 h; (b) ethyl chloroformate, TEA, Boc-proline, 0 °C to reflux; (c) TFA,
CH2Cl2; (d) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, rt.
a
Scheme 9. Synthesis of Amide Analogue 34
a
Reagents and conditions: (a) EDC, TEA, NHS, CH2Cl2, rt then tert-butyl (S)-2-(aminomethyl)pyrrolidine-1-carboxylate, 85%; (b) TFA, CH2Cl2,
quant; (c) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA, CH3CN, rt, 47%.
SphK2. The calculated Ki for SLR080811 was 13 μM and 1.3 μM
for SphK1 and 2, respectively, and it had a 10-fold selectivity
toward SphK2 (Table 2). The inhibitory effect of SLR080811
was highly dependent on the (S)-stereochemistry at α carbon
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a
Scheme 10. Synthesis of Homologated Compounds 36a,b
a
Reagents and conditions: (a) DIEA, HCTU·PF6, DMF, 80 °C, 18 h; (b) HCl(g), MeOH; (c) N,N′-di-Boc-1H-pyrazole-1-carboxamidine, DIEA,
CH3CN, rt, 3 days.
center of the pyrrolidine ring as the (R)-enantiomer 7h was
markedly less potent as a SphK inhibitor.
inhibition at SphK2. The calculated Ki of SLP7111228 for SphK1
is 48 nM and >10 μM for SphK2 (Table 2), affording >200-fold
selectivity. This compound is the most potent SphK1-selective
compound reported to date. Further extension of the linker in
36b resulted in substantial decrease in both SphK1 and SphK2
inhibitory activity.
As azetidine derivative SLP120701 had a similar binding
constant (Ki = 1.2 μM) but was more selective toward SphK2
than the pyrrolidine analogue SLR080811, we performed
biochemical characterization to determine its in vitro and in
vivo properties (Table 2 and Figure 3). First, U937 cells (a
myeloid cell line isolated from a patient with histiocytic
lymphoma), which express both SphK1 and SphK2, were
incubated with SLP120701. After cell lysis and sample
preparation, the inhibitor, sphingosine, and S1P were quantified
using LC-MS-MS. As shown in Figure 3, a dose-dependent
accumulation of SLP120701 was observed with a concomitant
decrease of S1P and increase in Sph levels. These results suggest
that SphKs were inhibited in whole cells and that SLP120701
penetrates these cells effectively.
To determine whether SLP120701 was inhibiting SphK2 in
these cells, the cultures were treated with the SphK2-selective
substrate, FTY720, and the resulting phosphorylated FTY720
(FTY720-P) was monitored (Figure 4).47,48 Addition of FTY720
to U937 cells resulted in accumulation of FTY720-P, which
should be dampened in the presence of an inhibitor. As expected,
increasing SLP120701 from 0.1 to 3 μM was accompanied by
decreased amounts of FTY720-P, a trend that reached a
minimum at 3 μM. These in vitro studies indicate that the
decrease in S1P and FTY720-P as a function of SLP120701
concentration may be attributed to inhibition of SphK2. Note
that the IC50 of SLP120701 estimated from this data appears to
be significantly less than the Ki value, which in our experience
results from the avid accumulation of compound in cells and
consistent with other studies.7,40
In an effort to improve the activity of SLR080811 further, a
more conformationally restricted analogue, 7i, wherein a double
bond was introduced on the aliphatic ring, was synthesized.
However, this compound was significantly less potent (Table 1).
Contraction of the pyrrolidine to a four-membered azetidine
ring, 7j (SLP120701), afforded a compound that was equipotent
with SLR080811. In contrast, ring expansion of the pyrrolidine to
generate the piperidine analogue, 7k, resulted in a compound
with severely diminished activity, presumably as a result of the
change in dihedral angle that orients the guanidine group in a
suboptimal position. We next explored the effect of hydroxyl
groups on the pyrrolidine ring in an attempt to mimic the
hydroxyl groups on Sph. Such a strategy has recently been used
with SphK1 inhibitors.23,26 Compound 11, which has a (3S)-
hydroxyl group, was equipotent at both the SphK1 and Sphk2
enzymes, albeit with less potency than the parent compound
(Table 1). (4R)-15 retained SphK2 selectivity but was less potent
than SLP120701 while its stereoisomer (4S)-17 was significantly
less active (Tables 1 and 2). A compound with (3R)-hydroxyl
group was not tested, as our attempts to synthesize it failed.
To determine the optimal position of the guanidine group
around the heterocyclic ring, we installed the nitrogen atom away
from the α carbon to generate azetidine (20a) and pyrrolidine
(20b). Both of these compounds were essentially inactive in our
assay. We also investigated effect of heteroatoms of the
oxadiazole ring, as positional isomers of oxadiazoles are known
to possess varying pharmacokinetic properties.46 Hence,
reversed 1,2,4-oxadiazole 23 and 1,3,4-oxadiazole 26 were
synthesized. While (5-phenyl)-1,2,4-oxadiazole 23 had inhib-
itory activity indistinguishable from SLP120701, 26 did not
inhibit at 1 μM (Tables 1 and 2). These studies suggest that the
isolated nitrogen atom interacts with the target protein, possibly
via hydrogen bonding.
In an effort to discover a surrogate for the oxadiazole ring, we
installed an amide bond between the phenyl and pyrrolidine
rings. Anilide 29a and 29b possessing opposite stereochemistry
at the α carbon were both inactive at SphK1 and -2. As the amide
bonds in these compounds are approximately one bond shorter
than the corresponding oxadiazole and can be “off register” in the
enzyme binding pocket, we synthesized the homologated
benzylamide 31 and found that it possessed no significant
inhibitory effect. To perform a thorough analysis, we also
synthesized the reversed homologated analogue 34 and found a
similar activity. Hence, we conclude that an amide bond is not a
sufficient replacement for the oxadiazole ring.
We next focused our attention on the spacing between the
oxadiazole and pyrrolidine rings; compound 36a (SLP7111228)
and 36b contain one or two methylene units, respectively. To our
surprise, we observed a complete switch in isoform selectivity as
36a showed 88% inhibition at SphK1 while exhibiting 20%
To ascertain whether inhibition of SphK2, via enzymatic
blockade using SLP120701, has an effect on the levels of
sphingoid bases in vivo, we treated C57BL/6 mice with a single
intraperitoneal dose of the compound (10 mg/kg) and
monitored S1P, Sph, and inhibitor concentration in blood
using LC-MS-MS. In contrast to our in vitro studies, S1P levels
increased to approximately 2-fold following injection of the
SphK2-selective inhibitor (Figure 5). This phenomenon is
consistent with S1P levels observed in Sphk2−/− mice where the
blood S1P level is 3−4 times higher than wild type control
mice.18−21 We also observed a modest increase in Sph levels
(Figure 5B). Further monitoring of compound disappearance
from blood suggests a half-life of approximately 8 h, which is
significantly longer than that of SLR080811.40
To compare the effects of SphK1 inhibition, we similarly
subjected U937 cells with SphK1-selective inhibitor
SLP7111228. As expected, a dose-dependent accumulation of
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a
Table 1. SphK1 and SphK2 Inhibitory Activity
a
SphK activity is represented as % of control (without inhibitor). Human SphK expression was forced in insect cells infected with recombinant
baculovirus, and activity in a cleared lysate was measured using 5 μM (for SphK1) or 10 μM (for SphK2) sphingosine and 250 μM [32P]-ATP. Each
compound was assayed at a concentration of 1 μM in triplicate, except for 36a (assayed at 3 μM with SphK2). Control = activity without inhibitor.
SLP7111228 in these cells is observed (Figure 6A). This
property is accompanied by a marked decrease in S1P level in a
dose-dependent fashion (Figure 6B). Interestingly, addition of
100 nM of this compound resulted in approximately 90%
depression of cellular S1P, which is significantly more compared
to SphK2-selective inhibitor SLP120701. When Sph levels were
measured as a function of inhibitor concentration, no statistical
change was observed (Figure 6C), which is again in contrast with
Sphk2 inhibitor. We further determined the effect of SLP120701
on the viability of U937 cells, as recent reports suggested that
SphK1-selective inhibitors such as PF-54323 and compound A/
B7 had no effect on the viability of cancer cells (Figure 6D). Our
studies similarly demonstrate that inhibition of SphK1 via
SLP120701 up to 3 μM over 24 h has no cytotoxicity effects on
cancer cell growth.
G
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Table 2. Ki of Selected Inhibitors at SphK1 and SphK2
To determine the effect of SLP7111228 in vivo, we injected
increasing concentrations of this compound in rats intra-
peritoneally. As shown in Figure 7A, plasma S1P levels decreased
slightly (up to 25% of control) as the inhibitor concentration
increases. S1P levels in plasma are known to be significantly
lower than in blood. In contrast, blood S1P levels shows a marked
dose-dependent depression, which reaches a minimum at about
80% when compared to vehicle (Figure 7B). Finally, our studies
suggest that SLP7111228 has an in vivo half-life of over 4 h and
that its effect of lowering S1P level is sustained over a period of at
least 6 h (Figure 7C,D).
cells. Curiously, SphK1 inhibitors decrease S1P levels further
compared to SphK2 inhibitors in vitro. Whether this an activity
associated with the potency of these compounds is a possibility,
but we note that these compounds are avidly taken up by these
cells. Second, in vivo administration of these inhibitors in mice
and rats resulted in opposite effects on blood S1P levels. That is, a
decrease in blood S1P level is observed with SphK1 inhibitor
while there is an increase with SphK2 inhibitors. While these
observations are consistent with mouse gene “knock out” studies,
our results are notable in that (1) SLP120701 is the second
SphK2 inhibitor profiled to result in increased circulating levels
of S1P;40 ABC294640 (a low potency compound that has SphK2
inhibitor properties) had the opposite effect of decreased S1P
levels.49 Our result suggest that increased blood S1P levels are a
bona fide property of SphK2-selective inhibitors. (2) SLP120701
has an improved in vivo half-life as compared to the pyrrolidine
analogue, SLR080811. (3) SLP7111228 is the most potent and
selective (>200 fold) SphK1 inhibitor reported to date with
favorable in vivo stability. Interestingly, a single dose of 10 mg/kg
in rats decreases blood S1P levels by about 80%. With these two
chemical tools in hand, we have the capacity to increase or
decrease blood S1P levels, and such studies will aid in
determining the pathophysiological effects of such maneuvers
in live animals.
CONCLUSIONS
■
The second generation inhibitors reported herein feature a
guanidine moiety as a warhead and an oxadiazole heterocycle as
the linker. Structure−activity studies with this scaffold suggest
that conformational restriction of the guanidine group is essential
for potent kinase inhibitory activity. Indeed, acyclic analogues
along with six membered piperidine or a dehydropyrrolidine
derivatives are weak inhibitors of SphK2. Further, decoration of
the pyrrolidine ring with a hydroxyl group to mimic the hydroxyl
moiety in sphingosine does not increase potency of the
compounds, although the compound bearing a (3R)-hydroxyl
group may have beneficial effect. Our studies also demonstrate
that amides are not a good replacement for oxadiazole rings in
our scaffold and that the position of the isolated nitrogen of the
oxadiazole ring is important; we suspect that this nitrogen atom
forms a hydrogen bond with a key amino acid in SphK. A
surprising finding in these investigations is that the insertion of a
single methylene unit as a spacer between the oxadiazole and
pyrrolidine rings resulted in a SphK inhibitor with reversed
selectivity, favoring SphK1.
EXPERIMENTAL SECTION
■
Sphingosine Kinase Assays. Recombinant baculovirus encoding
either SphK1 or SphK2 was expressed in Sf9 insect cells, cleared lysates
were prepared after 48 h, and 1−2 μL (0.02−0.03 mg protein) was used
in each assay. Alternately, plasmids encoding SphK1 or SphK2 were
used to transfect HEK293T cells, and cleared lysates were prepared after
48 h. SphK activity was measured in kinase assay buffer that consisted of
20 mM Tris-Cl (pH 7.4), 1 mM 2-mercaptoethanol, 1 mM EDTA, 5
mM sodium orthovanadate, 40 mM β-glycerophosphate, 15 mM NaF, 1
mM phenylmethylsulfonyl fluoride, 10 mM MgCl2, 0.5 mM 4-
deoxypyridoxine, 10% glycerol, and 0.01 mg/mL each of leupeptin,
Profiling of SphK1 (SLP7111228) and SphK2 (SLP120701)
both in vitro and in vivo reveal interesting observations. First,
both inhibitors have an effect of depressing S1P levels in U937
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Figure 5. S1P and SLP120701 levels in the blood of mice injected with
SLP120701. Wild-type mice were injected (ip) with a single dose (10
mg/kg) and blood was drawn at indicated time points. Levels of S1P (a),
sphingosine (b), and SLP120701 (c) from blood samples of WT mice
were measured by LC-MS-MS. The standard deviations are values from
a group of three to five mice.
compounds, the assay was supplemented with substrate (D-erythro-
sphingosine, (10 μM for SphK1 and 5 μM for SphK2), an appropriate
amount of compounds (to achieve 10−10 000 nM); γ-[32P]ATP (10
μM, specific activity = 8.3 Ci/mmol), and recombinant enzyme (0.02−
0.03 mg of total protein). After 20 min at 37 °C, the reaction mixture was
extracted with 2 volumes of chloroform/methanol/1 N HCl
(100:200:1), and the components in the organic phase were separated
by thin layer chromatography using a 1-butanol/glacial acetic acid/water
(3:1:1) solvent system. Radiolabeled enzyme products were detected by
autoradiography and identified by migration relative to authentic
standards. For quantification, the silica gel containing radiolabeled lipid
was scraped into a scintillation vial and measured by liquid scintillation
counting.
Figure 3. Effect of SLP120701 on sphingolipids in U937 cells. After 2 h
of incubation, cells were harvested by centrifugation, lysed, and levels of
(A) SLP120701, (B) S1P, and (C) sphingosine were measured using
LC-MS-MS. Amounts associated with cells are expressed as the number
of pmoles per 106 cells. Each value was determined in triplicate.
Sample Preparation and LC-MS-MS Analysis. To analyze the
lipids and compounds by LC/MS, sample preparation protocols were
adapted from a literature procedure,50 with minor modifications. Cell
pellets ((2−4) × 106 cells), whole blood (20 μL), or plasma (50 μL) was
mixed with 2 mL of a methanol:chloroform solution (3:1) and
transferred to a capped glass vial. Suspensions were supplemented with
10 μL of internal standard solution containing 10 pmol each of
deuterated (D7) S1P and deuterated (D7) sphingosine. The mixture
was placed in a bath sonicator for 10 min and incubated at 48 °C for 16 h.
The mixture was then cooled to ambient temperature and mixed with
200 μL of 1 M KOH in methanol. The samples were again sonicated and
incubated a further 2 h at 37 °C. Samples were then neutralized by the
addition of 20 μL of glacial acetic acid and transferred to 2 mL
microcentrifuge tubes. Samples were then centrifuged at 12 000g for 12
min at 4 °C. The supernatant fluid was collected in a separate glass vial
and evaporated under a stream of nitrogen gas. Immediately prior to LC-
MS analysis, the dried material was dissolved in 0.3 mL of methanol and
centrifuged at 12 000g for 12 min at 4 °C. Fifty microliters of the
resulting supernatant fluid was analyzed.
Figure 4. Sphingosine kinase 2 is inhibited by SLP120701. Cultured
U937 cells were incubated with 1 μM of FTY720 and increasing
concentrations of SLP120701. After 2 h of exposure, cells were
harvested by centrifugation, lysed, and levels of FTY720 and FTY720-
phosphate (FTY720-P) were quantified using LC-MS-MS. Amounts
associated with cells are expressed as the number of pmoles per 106 cells.
Each value was measured in triplicate.
aprotinin, and soybean trypsin inhibitor. To achieve optimal activity of
SphK1 or SphK2, the buffer was supplemented with either 0.5% Triton
X-100 or 1 M KCl, respectively. To ascertain any inhibitory effect of
Analyses were performed using liquid chromatography−ESI mass
spectrometry (LC-MS) using a triple quadrupole mass spectrometer
(AB-Sciex 4000 Q-Trap) coupled to a Shimadzu LC-20AD LC system.
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Figure 6. Effect of SLP7111228 on sphingolipids and viability of U937 cells. After 2 h of incubation, cells were harvested by centrifugation and lysed, and
levels of (A) SLP7111228, (B) S1P, and (C) sphingosine were measured using LC-MS-MS. Amounts associated with cells are expressed as the number
of pmoles per 106 cells. Each value was determined in triplicate. (D) Cell viability. Increasing concentration of inhibitor was added to U937 cells and cell
growth was measured after 24 h. Each value was determined in duplicate.
Figure 7. Effect of SLP7111228 upon IP administration in rats. Rats were injected with indicated dose and blood was drawn 2 h postinjection. Levels of
S1P in (a) plasma, (b) blood, and (c) SLP7111228 are shown at indicated inhibitor concentration. (d) Time-course experiment with 10 mg/kg
SLP7111228 showing blood S1P levels. Samples were analyzed by LC-MS-MS. The standard deviations are values from a group of three to five rats.
A binary solvent gradient with a flow rate of 1 mL/min was used to
separate sphingolipids and drugs by reverse phase chromatography
using a Supelco Discovery C18 column (50 mm × 2.1 mm, 5 μm bead
size). Mobile phase A consisted of water:methanol:formic acid
(79:20:1) while mobile phase B was methanol:formic acid (99:1).
The run started with 100% A for 0.5 min. Solvent B was then increased
linearly to 100% B in 5.1 min and held at 100% for 4.3 min. The column
was finally re-equilibrated to 100% A for 1 min. Natural sphingolipids
were detected using multiple reaction monitoring (MRM) protocols
previously described as follows: S1P (380.4 → 264.4); deuterated
(D7)C18S1P (387.4 → 271.3); sphingosine (300.5 → 264.4);
deuterated (D7) sphingosine (307.5 → 271.3).50 Fragmentation of
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compound SLP120701 was analyzed by direct infusion of a 1 μM
solution in methanol:formic acid (99:1), and it was found that the
transition (356.3→ 126.1) in positive mode provided the most intense
signal at the following voltages, DP: 106; EP: 10; CE: 29; CXP: 8. All
analytes were analyzed simultaneously using the aforementioned
MRMs. Retention times for all analytes under our experimental
conditions were between 5.1 and 5.6 min. Quantification was carried out
by measuring peak areas using commercially available software (Analyst
1.5.1).
U937 Cell Culture/Viability Assay. U937 cells were grown in
RPMI 1640 media supplemented with L-glutamate, 10% fetal bovine
serum (FBS), and 1% penicillin/streptomycin at 37 °C in an atmosphere
containing 5% CO2.51 Twenty-four hours before adding inhibitors, the
growth media was replaced with media containing 0.5% FBS. For
viability studies, inhibitors were added at the indicated concentrations
(0.1, 0.3, 1.0, and 3.0 μM) and after 24 h, trypan blue dye was added to
count cells according to manufacturer instructions (Bio-RAD TC10
Automated Cell Counter).
Pharmacokinetic Analysis. Groups of 8−20 week old mice (strain:
C57BL6/j) were injected (intraperitoneal route) with either
SLP120701 (dose: 10 mg/kg) or an equal volume of vehicle (2%
solution of hydroxypropyl-β-cyclodextrin (Cargill Cavitron 82004)).
For SLP7111228, three to four Sprague−Dawley strain rats (200−300
g) were injected with 10 mg/kg drug or an equal volume of vehicle for
indicated time periods. After injection, animals were bled at the specified
time points (ASAP time points were 1−2 min after dosing). Whole
blood was processed immediately for LC-MS analysis as described
above. Animal protocols were approved prior to experimentation by the
University of Virginia’s School of Medicine Animal Care and Use
Committee.
organic extracts were washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The resulting brown residue was
purified by flash chromatography over silica gel (95/5 hexanes/EtOAc)
to give the title compound (2.3 g, 62%) as a colorless oil. Analytical data
matches with the literature.52
(Z)-N′-Hydroxy-4-octylbenzimidamide (3). Triethylamine (3.9 mL,
27.8 mmol) and hydroxylamine hydrochloride (1.7 g, 24.5 mmol) were
added to a solution of 2 (2.4 g, 11.1 mmol) in 95% ethanol (30 mL). The
colorless reaction mixture was then refluxed for 4 h. The organic solvent
was removed under reduced pressure, and the residue was purified by
flash chromatography on silica gel (65/35 hexanes/EtOAc) to give the
title compound (2.5 g, 92%) as a white solid. Analytical data matches
with the literature.53
General Procedure A: Coupling of Amidoxime 3 with Amino
Acids. DIEA (1.8 equiv) was added to a solution of 3 (1 equiv), and the
appropriate Boc-protected amino acid (1.2 equiv) in DMF (0.2 M
solution). HCTU (1.5 equiv) was then added to the resulting mixture at
rt and stirred at 80 °C for 18 h. At this time, TLC showed complete
conversion of starting material. The solution was partitioned between
ethyl acetate and water. The organic layer was collected and washed
twice with a satd LiBr. The aqueous solution was then back extracted
with ethyl acetate. The organic layers were then combined and washed
with satd NaHCO3 and brine, dried over Na2SO4, and concentrated
under reduced pressure. The resulting residue was purified by flash
chromatography on silica gel to yield the desired product.
General Procedure B: Deprotection of t-Boc Protecting
Groups Using HCl(g). Hydrochloric acid gas was bubbled through a
solution of the N-Boc protected compound in methanol for 2−5 min or
until complete consumption of starting material was observed by TLC.
The reaction mixture was concentrated under reduced pressure and
triturated with diethyl ether to yield the corresponding free amine
hydrochloride salt, which was further purified by trituration with diethyl
ether until satisfactory analytical data were obtained.
General Procedure C: Guanidylation of Amines. DIEA (3
equiv) was added to a solution of the corresponding amine hydrochloric
acid salt and the reagent (Z)-tert-butyl (((tert-butoxycarbonyl)imino)-
(1H-pyrazol-1-yl)methyl)carbamate (1.05 equiv) in acetonitrile (20%
vol/wt). The resulting reaction mixture was then stirred at RT until
acceptable conversion of the starting amine was observed using TLC.
The solvent was then removed under reduced pressure, and the resulting
colorless residue was purified by flash column chromatography over
silica gel to yield the pure product.
General Procedure D: Deprotection of t-Boc Protecting
Groups Using TFA. To a solution of Boc-protected intermediate in
CH2Cl2 was added a 1 N TFA solution in CH2Cl2. The resulting
solution was stirred at room temperature until complete consumption of
the starting material was observed using TLC. The reaction mixture was
concentrated under reduced pressure and triturated with diethyl ether to
yield the corresponding free amine TFA salt, which was purified by
trituration with diethyl ether/hexanes (1/1) until satisfactory analytical
data were obtained.
General Procedure E: Amide Coupling of Amino Acids and
Amines. The appropriate Boc-protected amino acid (1 equiv) and TEA
(1 equiv) were dissolved in tetrahydrofuran (0.2 M solution) and then
cooled to 0° C. Ethyl chloroformate (1 equiv) was added dropwise, and
the solution was stirred for 30 min at 0 °C. The appropriate amine (1
equiv) was added dropwise. The reaction was stirred another 1 h at 0 °C,
16 h at rt, and 3 h at reflux. After completion, the solution was
partitioned between ethyl acetate and water. The organic layer was
washed with brine, dried with Na2SO4, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
to yield the desired product.
tert-Butyl ((3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)methyl)-
carbamate (4a). Synthesized by general procedure A. 64% yield,
yellow oil; 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 8.1 Hz, 2H), 7.26
(d, J = 8.0 Hz, 2H), 5.38 (br s, 1H), 4.61 (d, J = 5.2 Hz, 2H), 2.64 (t, J =
8.4 Hz, 2H), 1.66−1.56 (m, 2H), 1.46 (s, 9H), 1.36−1.19 (m, 10H),
0.86 (t, J = 6.9 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 176.4, 168.5,
155.6, 146.8, 129.0, 127.5, 123.9, 80.7, 37.3, 36.0, 32.0, 31.3, 29.5, 29.4,
General Material and Synthetic Procedures. All reactions were
conducted in an oven-dried glassware under an inert atmosphere of
nitrogen or argon using magnetic stirring. All solvents were dried using
the PureSolv solvent purification system prior to use. All other chemical
reagents were purchased from commercial sources and were used
without further purification. Thin layer chromatography (TLC) was
performed either on aluminum-backed silica gel or aluminum oxide
(neutral) plates. Column chromatography was performed either on flash
grade silica gel (SiO2, 32−63 μm) or neutral, activated aluminum oxide
1
(Al2O3, ∼150 mesh, 58 Å) as solid phase. H NMR spectra were
recorded at 500 or 400 MHz; the corresponding 13C NMR resonant
frequencies were 126 and 101 MHz, respectively. Chemical shifts are
reported in ppm from tetramethylsilane (TMS) with the solvent
resonance as an internal standard (ex: CDCl3: 7.26 ppm). Data are
reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t
= triplet, q = quartet, br = broad, m = multiplet, quin = quintet), coupling
constants (Hz), and integration. In case of 13C NMR, 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
performed on a TSQ triple quadrupole mass spectrometer, equipped
with an ESI source, which was used in the positive ion mode. High-
resolution mass spectroscopy (HRMS) was performed on an LC/MS
time-of-flight mass spectrometer using either electrospray ionization
(ESI) or atmospheric pressure chemical ionization (APCI). All melting
points are reported without correction. HPLC analyses were performed
on an Agilent XDB-C8 reverse phase column using water (with 0.1% v/v
of TFA) and acetonitrile as eluents. Optical rotations of final
compounds are measured on a Jasco P-2000 polarimeter at room
temperature (25 °C). All compounds tested in biological assays are
>95% pure by 1H NMR and HPLC analyses unless noted otherwise.
4-Octylbenzonitrile (2). Oct-1-ene (3 mL, 19.2 mmol) was added to
a round-bottom flask containing THF (8 mL). 9-BBN (42 mL, 21.0
mmol) was added as a 0.5 M solution in THF, and the solution was
stirred overnight at rt. To the above borane solution was added a
solution of 4-iodobenzonitrile (4 g, 17.5 mmol) in DMF (50 mL). The
reaction mixture was degassed for 10 min by bubbling N2 through the
solution. Cs2CO3 (11.4 g, 34.9 mmol) and PdCl2(dppf) (383 mg, 0.52
mmol) were added together. The resulting reaction mixture was then
stirred at 80 °C for 18 h, after which it was poured into a saturated
solution of LiBr and extracted three times with EtOAc. The combined
K
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29.3, 28.4, 22.7, 14.2; HRMS (ESI+): Calcd for C22H33N3NaO3 [M +
Na]+: 410.2420, Found: 410.2451.
7.9 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 5.10−4.94 (m, 1H), 3.68 (m, 1H),
3.52 (m, 1H), 2.63 (t, J = 7.5 Hz, 2H), 2.36 (m, 1H), 2.12 (m, 2H),
2.02−1.87 (m, 1H), 1.61 (m, 2H), 1.44 (s, 3H), 1.34−1.15 (m, 16H),
0.85 (t, J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 180.4, 168.3,
153.5, 146.5, 128.9, 128.7, 127.4, 127.3, 124.0, 80.4, 60.3, 53.8, 46.3,
35.9, 32.4, 31.8, 31.4, 31.2, 29.4, 29.3, 29.2, 28.4, 28.1, 23.7, 22.6, 21.0,
14.1, 14.0; HRMS (ESI+): Calcd for C25H38N3O3 [M + H]+: 428.2913,
Found: 428.2908.
tert-Butyl Methyl((3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
methyl)carbamate (4b). Synthesized by general procedure A. 53%
yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.2 Hz,
2H), 7.26 (d, J = 7.9 Hz, 2H), 4.66 (d, J = 41.5 Hz, 2H), 3.03 (d, J = 13.2
Hz, 3H), 2.63 (t, J = 7.4 Hz, 2H), 1.62 (quin, J = 7.4 Hz, 2H), 1.46 (d, J =
29.3 Hz, 9H), 1.32−1.21 (m, 10H), 0.87 (t, J = 6.7 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 176.2, 168.6, 146.8, 129.0, 127.5, 124.0, 81.0, 45.5,
36.1, 35.3, 32.0, 31.3, 29.6, 29.4, 29.3, 28.4, 22.8, 14.2; HRMS (ESI+):
Calcd for C23H36N3O3 [M + H]+: 402.2756, Found: 402.2742.
(S)-tert-Butyl (1-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
ethyl)carbamate (4c). Synthesized by general procedure A. 82%
yield, yellow oil; 1H NMR (500 MHz, CDCl3) δ 7.96 (d, J = 8.0 Hz, 2H),
7.26 (d, J = 8.0 Hz, 2H), 5.29 (br s, 1H), 5.22−5.03 (m, 1H), 2.64 (t, J =
8.0 Hz, 2H), 1.68−1.53 (m, 5H), 1.45 (s, 9H), 1.36−1.17 (m, 10H),
0.86 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 180.0, 168.4,
154.9, 146.7, 129.0, 127.5, 124.0, 80.5, 44.3, 36.0, 31.9, 31.3, 29.5, 29.3,
29.3, 28.4, 22.7, 20.2, 14.2; HRMS: Calcd for C23H35N3NaO3 [M +
Na]+: 424.2576, Found: 424.2571.
(S)-tert-Butyl2- (3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-2,5-
dihydro-1H-pyrrole-1-carboxylate (4i). Synthesized by general
1
procedure A. 20% yield, yellow oil. H NMR (400 MHz, CDCl3) δ
7.97 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 6.09 (dd, J = 24.9, 4.3
Hz, 1H), 5.87, (d, J = 21.6 Hz, 1H), 5.87−5.77 (m, 1H), 4.49−4.30 (m,
2H), 2.65 (t, J = 8 Hz, 2H), 1.63 (quin, J = 7.6 Hz, 2H), 1.48 (s, 3H), 1.28
(d, J = 22.2 Hz, 16H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 178.28, 177.80, 168.69, 153.26, 146.79, 129.89, 129.08,
127.54, 124.99, 124.14, 81.00, 60.85, 53.46, 36.12, 32.01, 31.37, 29.85,
29.58, 29.38, 28.28, 22.81, 14.24; HRMS (ESI+): Calcd for
C25H35N3O3Na [M + Na]+: 448.2576, Found: 448.2595.
(S)-tert-Butyl,2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
azetidine-1-carboxylate (4j). Synthesized by general procedure A.
73% yield, yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.2 Hz,
2H), 7.38−7.15 (m, 2H), 4.60 (s, 1H), 4.19 (td, J = 9.4, 5.6 Hz, 1H), 2.8
(quin, J = 9.4 Hz, 2H), 2.72−2.40 (m, 2H), 1.83−1.61 (m, 3H), 1.42 (s,
9H), 1.33−1.19 (m, 10H), 1.18 (t, J = 6.9 Hz, 3H); 13C NMR (101
MHz, CDCl3) δ 179.0, 168.2, 155.9, 132.0, 127.3, 127.2, 125.2, 93.3,
80.2, 80.1, 31.3, 28.6, 28.5, 28.3, 25.7, 22.5, 19.5, 14.1; HRMS (ESI+):
Calcd for C24H35N3NaO3 [M + Na]+: 436.2576, Found: 436.2571.
(S)-tert-Butyl 2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
piperidine-1-carboxylate (4k). Synthesized by general procedure
A. 77% yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.1
Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 5.55 (s, 1H), 4.07 (s, 1H), 3.02 (s,
1H), 2.63 (t, J = 8.1, 6.7 Hz, 2H), 1.98−1.85 (m, 2H), 1.80−1.57 (m,
4H), 1.55−1.38 (m, 9H), 1.35−1.19 (m, 10H), 0.86 (t, J = 6.8 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 178.7, 168.3, 150.8, 146.4, 128.8, 127.4,
(S)-tert-Butyl (2-Methyl-1-(3-(4-octylphenyl)-1,2,4-oxadiazol-
5-yl)propyl)carbamate (4d). Synthesized by general procedure A.
77% yield, yellow oil; 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 8.1 Hz,
2H), 7.27 (d, J = 8.1 Hz, 2H), 5.28−5.17 (m, 1H), 5.01−4.92 (m, 1H),
2.68−2.60 (m, 2H), 2.33−2.17 (m, 1H), 1.67−1.53 (m, 2H), 1.45 (s,
9H), 1.34−1.16 (m, 10H), 0.98 (d, J = 6.8 Hz, 6H), 0.87 (t, J = 6.9 Hz,
3H); 13C NMR (126 MHz, CDCl3) δ 178.9, 168.3, 155.4, 146.7, 129.0,
127.5, 124.1, 80.4, 53.7, 36.0, 32.9, 31.9, 31.3, 29.5, 29.3, 28.4, 22.7, 18.7,
18.0, 14.2; HRMS: Calcd for C25H39N3NaO3 [M + Na]+: 452.2889,
Found: 452.2884.
(S)-tert-Butyl (2-Hydroxy-1-(3-(4-octylphenyl)-1,2,4-oxadia-
zol-5-yl)ethyl)carbamate (4e). Synthesized by general procedure
A. 28% yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.3
Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 5.56 (s, 1H), 5.18 (s, 1H), 4.17 (d, J =
8.9 Hz, 1H), 4.04 (d, J = 9.5 Hz, 1H), 2.65 (t, J = 7.6 Hz, 2H), 2.51 (s,
1H), 1.63 (quin, J = 7.6 Hz, 2H), 1.48 (s, 9H), 1.35−1.22 (m, 10H), 0.87
(t, J = 6.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 177.4, 168.4, 147.0,
129.1, 127.6, 123.8, 64.0, 50.2, 36.1, 32.0, 31.3, 29.6, 29.4, 29.3, 28.4,
22.8, 14.2; HRMS (Mixed ESI): Calcd for C23H36N3O4 [M + H]+:
418.5505, Found: 418.2692.
124.1, 80.5, 35.9, 31.8, 31.2, 29.4, 29.2, 29.2, 28.3, 27.9, 24.7, 22.6, 20.0,
14.0. HRMS (ESI+): Calcd for C26H39N3NaO3 [M + Na]+: 464.2889,
Found: 436.2874
(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)methanamine (5a).
1
Synthesized by general procedure D. 99% yield, off-white solid; H
tert-Butyl (1-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
cyclopropyl)carbamate (4f). Synthesized by general procedure A.
62% yield, yellow oil. 1H NMR (400 MHz, CDCl3) 1H NMR (400 MHz,
CDCl3) δ 7.91 (d, J = 7.5 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 5.53 (s, 1H),
2.70−2.57 (m, 2H), 1.77−1.73 (m, 2H), 1.67−1.56 (m, 2H), 1.49−1.45
(m, 11H), 1.35−1.18 (m, 10H), 0.92−0.82 (m, 3H); 13C NMR (101
MHz, CDCl3) δ 180.8, 168.5, 155.6, 146.3, 128.8, 127.3, 124.2, 80.5,
35.9, 31.8, 31.2, 30.9, 29.4, 29.3, 29.2, 28.2, 22.6, 19.6, 14.1; HRMS (ESI
+): Calcd for C24H35N3NaO3 [M + Na]+: 436.2576, Found: 436.2530.
(S)-tert-Butyl 2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
pyrrolidine-1-carboxylate (4g). Synthesized by general procedure
A. 48% yield, yellow oil; 1H NMR (500 MHz, CDCl3) δ 7.96 (d, J = 7.8
Hz, 2H), 7.33−7.18 (m, 2H), 5.21−5.14 (m, 1H, minor rotamer), 5.07−
5.01 (m, 1H, major rotamer), 3.75−3.67 (m, 1H, major rotamer), 3.67−
3.61 (m, 1H, minor rotamer), 3.59−3.50 (m, 1H, major rotamer), 3.50−
3.41 (m, 1H, minor rotamer), 2.66−2.58 (m, 2H), 2.44−2.25 (m, 1H),
2.20−2.05 (m, 2H), 2.05−1.89 (m, 1H), 1.68−1.54 (m, 2H), 1.44 (s,
3H), 1.37−1.16 (m, 16H), 0.85 (t, J = 6.8 Hz, 3H); 13C NMR (126
MHz, CDCl3, rotamers) δ 180.5 (major), 180.1 (minor), 168.5 (major),
154.3 (minor), 153.6 (major), 146.7 (major), 146.4 (minor), 129.0
(major), 128.9 (minor), 127.5 (minor), 127.5 (major), 124.4 (minor),
124.2 (major), 80.5 (major), 80.4 (minor), 53.9 (major), 46.7 (minor),
46.4 (major), 36.0 (major), 32.5 (major), 32.0 (major), 31.6 (minor),
31.3 (major), 29.5 (major), 29.3 (major), 29.2 (major), 28.5 (minor),
28.2 (major), 24.4 (minor), 23.8 (major), 22.7 (major), 14.2 (major).
HRMS (ESI+): Calcd for C25H37N3NaO3 [M + Na]+: 450.2733, Found:
450.2727.
NMR (500 MHz, CD3OD) δ 8.08−7.92 (m, 2H), 7.34 (d, J = 8.4 Hz,
2H), 4.86 (br s, 2H), 4.59 (s, 2H), 2.70−2.62 (m, 2H), 1.70−1.57 (m,
2H), 1.37−1.20 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR (126
MHz, CD3OD) δ 173.1, 168.5, 147.2, 128.9, 127.2, 123.4, 35.6, 34.9,
31.7, 31.1, 29.2, 29.1, 29.0, 22.4, 13.1; HRMS (ESI+): Calcd for
C17H26N3O+ [M+]: 288.2070, Found: 288.2067.
N-Methyl-1-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
methanamine (5b). Synthesized by general procedure B. 94% yield,
white solid. 1H NMR (400 MHz, CD3OD) δ 8.13 (d, J = 8.3 Hz, 2H),
7.48 (d, J = 8.3 Hz, 2H), 5.01 (s, 2H), 3.09 (s, 3H), 2.81 (t, J = 7.8, 2H),
1.78 (quin, J = 7.3 Hz, 2H), 1.51−1.38 (m, 10H), 1.01 (t, J = 6.7, 3H);
13C NMR (101 MHz, CD3OD) δ 173.2, 169.7, 148.5, 130.2, 128.5,
124.6, 44.4, 36.8, 34.2, 33.0, 32.4, 30.5, 30.3, 30.2, 23.7, 14.4; HRMS
(ESI+): Calcd for C18H28N3O [M + H]+: 302.2232, Found: 302.2224.
(S)-1-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)ethanamine
1
(5c). Synthesized by general procedure D. 89% yield, white solid; H
NMR (500 MHz, CDCl3) δ 8.06−7.89 (m, 2H), 7.35−7.19 (m, 2H),
4.33 (q, J = 6.9 Hz, 1H), 2.70−2.55 (m, 2H), 1.81 (br s, 2H), 1.66−1.52
(m, 5H), 1.36−1.17 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR (126
MHz, CDCl3) δ 183.0, 168.3, 146.6, 129.0, 127.5, 124.2, 45.0, 36.0, 32.0,
31.3, 29.5, 29.4, 29.3, 22.7, 21.8, 14.2. HRMS (ESI+): Calcd for
C18H28N3O+ [M+]: 302.2227, Found: 302.2219.
(S)-2-Methyl-1-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
propan-1-amine (5d). Synthesized by general procedure D. 59% yield,
white solid; 1H NMR (500 MHz, CDCl3) δ 8.01−7.92 (m, 2H), 7.30−
7.19 (m, 2H), 4.00 (d, J = 5.8 Hz, 1H), 2.61 (t, J = 8.0 Hz, 2H), 2.23−
2.11 (m, 1H), 1.73 (br s, 2H), 1.66−1.53 (m, 2H), 1.38−1.16 (m, 10H),
1.03−0.91 (m, 6H), 0.85 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz,
CDCl3) δ 182.2, 168.1, 146.6, 129.0, 127.5, 124.2, 55.1, 36.0, 33.6, 31.9,
(R)-tert-Butyl 2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
pyrrolidine-1-carboxylate ((R)-4h). Synthesized by general proce-
dure A. 73% yield, yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J =
L
DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
31.3, 29.5, 29.3, 22.7, 19.0, 17.9, 14.2. HRMS (ESI+): Calcd for
C20H32N3O+ [M+]: 330.2540, Found: 330.2532.
NMR (500 MHz, CDCl3) δ 11.47 (br s, 1H), 8.99 (t, J = 5.3 Hz, 1H),
7.99−7.95 (m, 2H), 7.28 (d, J = 8.0 Hz, 2H), 4.95 (d, J = 5.3 Hz, 2H),
2.64 (t, J = 7.7 Hz, 2H), 1.67−1.58 (m, 2H), 1.52 (s, 9H), 1.49 (s, 9H),
1.35−1.19 (m, 10H), 0.86 (t, J = 6.3 Hz, 3H); 13C NMR (126 MHz,
CDCl3) δ 175.4, 168.5, 163.2, 156.3, 153.1, 146.8, 129.0, 127.6, 123.8,
83.8, 79.9, 37.3, 36.1, 31.9, 31.3, 29.5, 29.3, 29.3, 28.3, 28.1, 22.7, 14.2.
HRMS (ESI+): Calcd for C28H44N5O5 [M + H]+: 530.3342, Found:
530.3302.
tert-Butyl (((tert-Butoxycarbonyl)amino)(methyl((3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)methyl)amino)carbamate (6b).
Synthesized by general procedure C. 34% yield, colorless oil. 1H
NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.7 Hz,
2H), 4.96 (s, 2H), 3.18 (s, 3H), 2.65 (t, J = 7.3 Hz, 2H), 1.67−1.59 (m,
2H), 1.49 (s, 18H), 1.36−1.21 (m, 10H), 0.87 (t, J = 7.3 Hz, 3H); 13C
NMR (101 MHz, CDCl3) δ 175.0, 168.6, 156.1, 146.8, 129.1, 127.6,
124.0, 77.4, 46.5, 38.0, 36.1, 32.0, 31.4, 29.9, 29.6, 29.4, 29.3, 28.2, 28.1,
22.8, 14.2; HRMS (ESI+): Calcd for C29H46N5O5 [M + H]+: 544.3499,
Found: 544.3453.
tert-Butyl N-[[(tert-Butoxy)carbonyl]amino([(1S)-1-[3-(4-oc-
tylphenyl)-1,2,4-oxadiazol-5-yl]ethyl]amino)methylidene]-
carbamate (6c). Synthesized by general procedure C. 81% yield,
yellow solid; 1H NMR (500 MHz, CDCl3) δ 11.50 (br s, 1H), 8.97 (d, J
= 8.1 Hz, 1H), 8.00−7.92 (m, 2H), 7.30−7.23 (m, 2H), 5.82−5.72 (m,
1H), 2.70−2.55 (m, 2H), 1.71−1.36 (m, 5H), 1.51 (s, 9H), 1.47 (s, 9H),
1.36−1.10 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz,
CDCl3) δ 179.2, 168.5, 163.4, 155.7, 153.1, 146.7, 129.0, 127.6, 124.0,
83.7, 79.7, 43.8, 36.0, 32.0, 31.3, 29.5, 29.3, 28.3, 28.2, 22.7, 20.1, 14.2.
HRMS (ESI+): Calcd for C29H46N5O5 [M + H]+: 544.3499, Found:
544.3477.
(S)-2-Hydroxy-1-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
ethanaminium chloride (5e). Synthesized by general procedure B.
74% yield, white solid. 1H NMR (400 MHz, CD3OD) δ 8.05 (d, J = 8.4
Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 4.99 (t, J = 4.5 Hz, 1H), 4.20 (d, J = 4.5
Hz, 2H), 2.73 (t, J = 6.5 Hz, 2H), 1.70 (quin, J = 7.3 Hz, 2H), 1.43−1.28
(m, 10H), 0.93 (t, J = 6.5 Hz, 3H); 13C NMR (101 MHz, CD3OD) δ
175.3, 169.8, 148.6, 130.2, 128.5, 124.7, 61.4, 51.4, 36.9, 33.0, 32.4, 30.5,
30.4, 30.3, 23.7, 14.4; HRMS (ESI+): Calcd for C18H28N3O2+ [M+]:
318.2181, Found: 318.2174.
1-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)cyclopropanamine
1
(5f). Synthesized by general procedure D. 100% yield, white solid. H
NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 8.0 Hz,
2H), 2.70−2.59 (m, 2H), 2.38 (s, 2H), 1.69−1.55 (m, 2H), 1.54−1.46
(m, 2H), 1.36−1.19 (m, 12H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (101
MHz, CDCl3) δ 183.8, 168.4, 146.4, 128.8, 127.3, 124.2, 35.9, 31.9, 31.8,
31.2, 29.4, 29.2, 29.2, 22.6, 19.8, 14.1; HRMS (ESI+): Calcd for
C19H28N3O [M + H]+: 314.2232, Found: 314.2208.
(S)-3-(4-Octylphenyl)-5-(pyrrolidin-2-yl)-1,2,4-oxadiazole
(5g). Synthesized by general procedure D. 82% yield, off white solid; 1H
NMR (500 MHz, CDCl3) δ 8.01−7.87 (m, 2H), 7.25−7.18 (m, 2H),
4.46 (dd, J = 8.3, 5.6 Hz, 1H), 3.20−3.09 (m, 1H), 3.09−2.94 (m, 1H),
2.60 (t, J = 8.0 Hz, 2H), 2.34 (br s, 1H), 2.29−2.17 (m, 1H), 2.14−2.00
(m, 1H), 1.96−1.76 (m, 2H), 1.65−1.51 (m, 2H), 1.34−1.13 (m, 10H),
0.83 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 181.9, 168.2,
146.5, 128.9, 127.5, 124.2, 54.4, 46.9, 36.0, 31.9, 31.3, 31.2, 29.5, 29.3,
29.3, 25.4, 22.7, 14.2; HRMS (ESI+) calcd for C20H30N3O [M+]:
328.2389, Found 328.2354.
(R)-3-(4-Octylphenyl)-5-(pyrrolidin-2-yl)-1,2,4-oxadiazole
(5h). Synthesized by general procedure D. 100% yield, off white solid.
1H NMR (500 MHz, CD3OD) δ 7.97 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.6
Hz, 2H), 4.65−4.44 (m, 1H), 3.34 (s, 1H), 3.12−3.18 (m, 2H), 2.67 (t, J
= 7.6 Hz, 2H), 2.35−2.31 (m, 1H), 2.18−2.14 (m, 1H), 2.05−1.91 (m,
2H), 1.68−1.62 (m, 2H), 1.38−1.25 (m, 10H), 0.90 (t, J = 6.8 Hz, 3H);
13C NMR (126 MHz, CD3OD) δ 182.8, 169.4, 148.0, 130.1, 128.4,
125.4, 55.3, 47.6, 36.9, 33.0, 32.4, 31.9, 30.6, 30.4, 30.3, 26.4, 23.7, 14.5;
HRMS (ESI+): Calcd for C20H30N3O [M + H]+: 328.2389, Found:
328.2383.
(S)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-2,5-dihydro-
1H-pyrrol-1-ium Chloride (5i). Synthesized by general procedure B.
72% yield, white solid. 1H NMR (400 MHz, CD3OD) δ 11.95 (d, J = 8.3
Hz, 2H), 11.31 (d, J = 8.3 Hz, 2H), 10.30 (d, J = 4.5 Hz, 1H), 10.20 (d, J
= 5.3 Hz, 1H), 9.97 (s, 1H), 8.37−8.20 (m, 2H), 6.64 (t, J = 7.0 Hz, 2H),
5.61 (quin, J = 7.4 Hz, 2H), 5.33−5.19 (m, 10H), 4.84 (t, J = 6.4 Hz,
3H); 13C NMR (101 MHz, CD3OD) δ 174.7, 170.1, 148.7, 130.3, 130.2,
128.5, 125.2, 124.5, 61.6, 54.0, 36.9, 33.0, 32.4, 30.5, 30.4, 30.3, 23.7,
14.4; HRMS (ESI+): Calcd for C20H28N3O+ [M+]: 326.2232, Found:
326.2240.
tert-Butyl N-[[(tert-Butoxy)carbonyl]amino({[(1S)-2-methyl-1-
[3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl]propyl]amino})-
methylidene]carbamate (6d). Synthesized by general procedure C.
43% yield, yellow oil; 1H NMR (500 MHz, CDCl3) δ 11.51 (br s, 1H),
9.05 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 7.3 Hz, 2H), 7.26 (d, J = 7.6 Hz,
2H), 5.59−5.55 (m, 1H), 2.64 (t, J = 7.6 Hz, 2H), 2.43−2.35 (m, 1H),
1.66−1.57 (m, 2H), 1.52 (s, 9H), 1.44 (s, 9H), 1.34−1.19 (m, 10H),
1.05−0.99 (m, 6H), 0.86 (t, J = 6.5 Hz, 3H); 13C NMR (126 MHz,
CDCl3) δ 178.2, 168.3, 163.4, 156.3, 153.2, 146.6, 129.0, 127.6, 124.2,
83.6, 79.6, 53.1, 36.0, 32.4, 31.9, 31.3, 29.5, 29.3, 29.3, 28.3, 28.2, 22.7,
18.6, 18.1, 14.2. HRMS (ESI+): Calcd for C31H50N5O5 [M + H]+:
572.3812, Found: 572.3802.
(S,E)-tert-Butyl (((tert-Butoxycarbonyl)amino)(2-hydroxy-1-
(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)ethyl)carbamate (6e).
Synthesized by general procedure C. 32% yield, colorless oil. 1H
NMR (400 MHz, CDCl3) δ 11.49 (s, 1H), 9.36 (d, J = 7.6 Hz, 1H), 7.98
(d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 5.77 (dt, J = 7.9, 4.1 Hz,
1H), 4.14 (qd, J=, 2H), 2.65 (t, J = 7.9 Hz, 2H), 1.68−1.59 (m, 2H), 1.51
(d, J = 14.0 Hz, 18H), 1.29 (d, J = 17.3 Hz, 10H), 0.88 (t, J = 6.9 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 176.5, 168.5, 163.0, 156.4, 153.0, 147.0,
129.1, 127.7, 123.7, 84.0, 80.0, 64.6, 50.6, 36.1, 32.0, 31.3, 29.6, 29.4,
29.4, 28.4, 28.2, 22.8, 14.2; HRMS (ESI+): Calcd for C29H46N5O6 [M +
H]+: 560.3448, Found: 560.3454.
(S)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)azetidin-1-ium
1
(5j). Synthesized by general procedure B. 94% yield, white solid. H
NMR (400 MHz, CD3OD) δ 8.00 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.5
Hz, 2H), 5.93 (t, J = 8.5 Hz, 1H), 4.39−4.26 (m, 1H), 4.17 (td, J = 10.0,
6.3 Hz, 1H), 3.21−2.96 (m, 3H), 2.67 (t, J = 7.9, 7.4 Hz, 2H), 1.64 (quin,
J = 7.3 Hz, 2H), 1.43−1.16 (m, 10H), 0.87 (t, J = 6.7 Hz, 3H). 13C NMR
(101 MHz, CD3OD) δ 173.8, 168.7, 147.2, 128.8, 127.1, 123.2, 52.8,
44.6, 35.4, 31.6, 31.0, 29.1, 28.9, 28.9, 23.9, 22.3, 13.0; HRMS (ESI+):
Calcd for C19H29ClN3O [M + H]+: 350.1999, Found: 350.2017.
(S)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-
ium Chloride (5k). Synthesized by general procedure B. 95% yield,
white solid. 1H NMR (500 MHz, CDCl3) δ 8.00 (d, J = 8.3 Hz, 2H), 7.27
(d, J = 8.8 Hz, 2H), 4.12 (dd, J = 9.8, 3.3 Hz, 1H), 3.20 (dt, J = 12.2, 3.6
Hz, 1H), 2.88−2.77 (m, 1H), 2.65 (t, J = 7.6 Hz, 2H), 2.29−2.10 (m,
2H), 1.94−1.76 (m, 2H), 1.72−1.54 (m, 5H), 1.30 (ddt, J = 24.7, 9.8, 3.9
Hz, 10H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ
180.3, 168.2, 146.5, 128.9, 127.4, 124.1, 53.5, 46.0, 36.0, 31.9, 31.6, 31.2,
30.3, 29.4, 29.3, 29.2, 25.7, 23.6, 22.7, 14.1; HRMS (ESI+): Calcd for
C21H32N3O+ [M+]: 342.2540, Found: 342.2554.
1,2-Di-Boc-3-(1-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
cyclopropyl)guanidine (6f). Synthesized by general procedure C.
35% yield, colorless oil. 1H NMR (500 MHz, CDCl3) δ 11.50 (s, 1H),
8.92 (s, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 2.57 (t, J =
7.5 Hz, 2H), 1.79 (dd, J = 5.5, 8.5 Hz, 2H), 1.60−1.53 (m, 2H), 1.51 (dd,
J = 5.5, 8.5 Hz, 2H), 1.45 (s, 9H), 1.32 (s, 9H), 1.26−1.12 (m, 10H),
0.80 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 180.0, 168.5,
163.3, 156.9, 153.2, 146.3, 128.8, 127.4, 124.3, 83.5, 79.6, 35.9, 31.9,
31.2, 30.7, 29.7, 29.4, 29.2, 28.2, 28.1, 22.7, 20.3, 14.1; HRMS (ESI+):
Calcd for C30H46N5O5 [M + H]+: 556.3499.
(S)-tert-Butyl (((tert-Butoxycarbonyl)imino)(2-(3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-1-yl)methyl)carbamate
1
(6g). Synthesized by general procedure C. 66% yield, yellow oil; H
NMR (500 MHz, CDCl3) δ 7.96 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 8.0 Hz,
2H), 5.62−5.54 (m, 1H), 3.93−3.83 (m, 1H), 3.83−3.71 (m, 1H), 2.64
(t, J = 7.9 Hz, 2H), 2.48−2.36 (m, 1H), 2.30−2.09 (m, 2H), 2.08−1.98
(m, 1H), 1.95−1.86 (m, 1H), 1.67−1.57 (m, 2H), 1.54−1.18 (m, 28H),
0.86 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 178.7, 168.3,
161.9, 153.5, 150.3, 146.5, 128.8, 127.4, 123.9, 82.2, 79.5, 55.3, 49.4,
tert-Butyl N-[[(tert-Butoxy)carbonyl]amino([3-(4-octylphen-
yl)-1,2,4-oxadiazol-5-yl]methyl amino)methylidene]carbamate
1
(6a). Synthesized by general procedure C. 78% yield, yellow oil; H
M
DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
35.9, 31.8, 31.2, 29.4, 29.2, 28.1, 23.9, 22.6, 14.1; HRMS (ESI+): Calcd
for C31H48N5O5 [M + H]+: 570.3655, found 570.3605.
23.7, 14.4; HRMS (ESI+): Calcd for C19H30N5O [M + H]+: 344.2450,
Found: 344.2416.
(R)-tert-Butyl (((tert-Butoxycarbonyl)amino)(2-(3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-1-yl)methylene)-
carbamate (6h). Synthesized by general procedure C. 55% yield,
colorless oil. 1H NMR (500 MHz, CDCl3) δ 10.10 (s, 1H), 7.99 (d, J =
8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 5.71−5.53 (m, 1H), 3.98−3.87 (m,
1H), 3.84−3.80 (m, 1H), 2.75−2.63 (m, 2H), 2.48−2.44 (m, 1H),
2.35−2.13 (m, 2H), 2.08−2.04 (m, 1H), 1.74−1.61 (m, 2H), 1.48 (s,
18H), 1.38−1.21 (m, 10H), 0.89 (t, J = 7.0 Hz, 3H); 13C NMR (126
MHz, CDCl3) δ 178.7, 168.4, 146.5, 129.6, 128.9, 127.5, 124.0, 82.2,
79.6, 55.3, 49.4, 35.9, 31.8, 31.4, 31.2, 29.7, 29.4, 29.2, 28.1, 23.9, 22.6,
14.1; HRMS (ESI+): Calcd for C31H48N5O5 [M + H]+: 570.3655,
Found: 570.3662.
(S,E)-tert-Butyl (((tert-Butoxycarbonyl)amino)(2-(3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)-2,5-dihydro-1H-pyrrol-1-yl)-
methylene)carbamate (6i). Synthesized by general procedure C. 22%
yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.2 Hz,
2H), 7.27 (d, J = 8.0 Hz, 2H), 6.42 (s, 1H), 6.13 (d, J = 4.6 Hz, 1H), 5.90
(dd, J = 6.4, 2.2 Hz, 1H), 2.65 (t, J = 7.5 Hz, 2H), 1.67−1.59 (m, 2H),
1.50 (d, J = 10.8 Hz, 18H), 1.35−1.22 (m, 10H), 0.88 (t, J = 6.8 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 176.7, 168.6, 129.0, 128.9, 127.6, 124.3,
(S)-1-(1-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)ethyl)-
guanidine Hydrochloride (7c). Synthesized by general procedure B.
82% yield, white solid; 1H NMR (500 MHz, CD3OD) δ 7.96 (d, J = 8.1
Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 5.28−5.14 (m, 1H), 2.75−2.56 (m,
2H), 1.75 (d, J = 6.9 Hz, 3H), 1.68−1.56 (m, 2H), 1.39−1.17 (m, 10H),
0.88 (t, J = 6.8 Hz, 3H); 13C NMR (126 MHz, CD3OD) δ 178.5, 168.4,
157.3, 147.0, 128.8, 127.1, 123.8, 45.2, 35.6, 31.7, 31.1, 29.2, 29.1, 29.0,
22.4, 18.0, 13.1; HRMS (ESI+) calcd for C19H30N5O [M + H]+:
344.2450, Found: 344.2407. [α]D = −148° (c = 0.0005, methanol).
(S)-1-(2-Methyl-1-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
propyl)guanidine Hydrochloride (7d). Synthesized by general
procedure B. 80% yield, white solid; 1H NMR (500 MHz, CD3OD) δ
7.96 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 5.08−4.99 (m, 1H),
2.65 (t, J = 7.9 Hz, 2H), 2.51−2.40 (m, 1H), 1.68−1.56 (m, 2H), 1.38−
1.19 (m, 10H), 1.07 (dd, J = 15.1, 6.8 Hz, 6H), 0.87 (t, J = 6.8 Hz, 3H);
13C NMR (126 MHz, CD3OD) δ 177.4, 168.4, 157.9, 147.0, 128.9,
127.1, 123.7, 54.7, 35.6, 32.4, 31.7, 31.1, 29.2, 29.1, 29.0, 22.4, 17.6, 16.9,
13.1; HRMS (ESI+) calcd for C21H34N5O [M + H]+: 372.2763, Found:
372.2773. [α]D = −156° (c = 0.0005, methanol)
(S)-Amino((2-hydroxy-1-(3-(4-octylphenyl)-1,2,4-oxadiazol-
5-yl)ethyl)amino)methaniminium Chloride (7e). 12% yield, white
solid. 1H NMR (500 MHz, CD3OD) δ 8.00 (d, J = 8.4 Hz, 2H), 7.35 (d, J
= 8.4 Hz, 2H), 4.90 (d, J = 4.4 Hz, 1H), 4.15−4.10 (m, 2H), 2.68 (t, J =
7.6 Hz, 2H), 1.71−1.55 (m, 2H), 1.39−1.22 (m, 10H), 0.88 (t, J = 6.9
Hz, 3H); 13C NMR (126 MHz, CD3OD) δ 177.8, 169.8, 159.2, 148.3,
130.2, 128.4, 125.1, 63.4, 53.1, 36.9, 33.0, 32.4, 30.5, 30.4, 30.3, 23.7,
14.4; HRMS (ESI+): Calcd for C19H30N5O2+ [M+]: 360.2399, Found:
360.2386. [α]D = +118° (c = 0.00066, methanol).
115.6, 110.2, 53.6, 36.1, 32.0, 31.4, 29.9, 29.6, 29.4, 29.3, 28.3, 22.8, 14.2;
HRMS (ESI+): Calcd for C31H46N5O5 [M + H]+: 568.7286, Found:
568.3478.
(S)-tert-Butyl (((tert-Butoxycarbonyl)amino)(2-(3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)azetidin-1-yl)methylene)-
carbamate (6j). Synthesized by general procedure C. 58% yield,
colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 8.2 Hz, 2H),
7.29 (d, J = 8.1 Hz, 2H), 4.63 (s, 1H), 4.20 (td, J = 9.4, 5.6 Hz, 1H), 2.85
(quin, J = 9.4 Hz, 1H), 2.66 (t, J = 7.5 Hz, 2H), 2.56 (dq, J = 10.4, 5.0 Hz,
1H), 1.74 (s, 1H), 1.64 (quin, J = 7.4 Hz, 2H), 1.45 (s, 18H), 1.34−1.23
(m, 10H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ
177.1, 171.1, 168.5, 146.7, 128.9, 127.5, 123.8, 60.4, 36.0, 31.8, 31.2,
29.4, 29.2, 29.2, 28.0, 22.6, 22.4, 21.0, 14.2, 14.1; HRMS (ESI+): Calcd
for C30H46N5O5 [M + H]+: 556.3499, Found: 556.3488
(S)-tert-Butyl (((tert-Butoxycarbonyl)amino)(2-(3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methylene)-
carbamate (6k). To a microwave safe glass tube were added (S)-2-(3-
(4-octylphenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-ium 5k (0.04 g, 0.105
mmol), triethylamine (0.045 mL, 0.32 mmol), tert-butyl (((tert-
butoxycarbonyl)amino)(1H-pyrazol-1-yl)methylene)carbamate 9
(0.032 g, 0.105 mmol), and anhydrous acetonitrile (2 mL). The
resulting reaction mixture was sealed and heated in a microwave reactor
for 3 h at 60 W. After 3 h, the reaction flask was cooled, and the resulting
colorless solution was concentrated in vacuo. The resulting pale yellow
oil was then purified by flash chromatography to yield product 6k as
colorless oil in 18% yield. 1H NMR (500 MHz, CDCl3) δ 7.99 (d, J = 8.3
Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 5.98 (bs, 1H), 3.99 (bs, 1H), 3.67 (s,
1H), 3.35 (t, J = 12.0 Hz, 1H), 3.10 (s, 1H), 2.66 (t, J = 7.9, 7.5 Hz, 2H),
2.41 (d, J = 13.4 Hz, 1H), 2.11 (s, 1H), 1.77 (m, 2H), 1.72−1.60 (m,
4H), 1.56 (d, J = 11.9 Hz, 4H), 1.49 (s, 18H), 1.37−1.21 (m, 10H), 0.88
(t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 176.6, 167.3, 154.5,
145.5, 127.8, 126.5, 123.0, 52.8, 34.9, 30.8, 30.2, 28.4, 28.2, 27.1, 27.0,
23.7, 21.6, 19.1, 17.9, 16.6, 13.1; HRMS (ESI+): Calcd for C32H50N5O5
[M + H]+: 584.3734, Found: 584.3822
1-((3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)methyl)guanidine
Hydrochloride (7a). Synthesized by general procedure B. 84% yield,
white solid; 1H NMR (500 MHz, CD3OD) δ 7.96 (d, J = 8.2 Hz, 2H),
7.33 (d, J = 8.2 Hz, 2H), 4.84 (s, 2H), 2.67 (t, J = 7.6 Hz, 2H), 1.71−1.54
(m, 2H), 1.38−1.19 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR (126
MHz, CD3OD) δ 175.5, 168.4, 158.1, 147.0, 128.8, 127.1, 123.7, 37.4,
35.5, 31.7, 31.1, 29.2, 29.1, 29.0, 22.4, 13.1; HRMS (ESI+) m/z calcd for
C18H28N5O [M + H]+: 330.2294, Found: 330.2269.
1-Methyl-1-((3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)methyl)-
guanidine Hydrochloride (7b). Synthesized by general procedure B.
15% yield, white solid. 1H NMR (400 MHz, CD3OD) δ 8.00 (d, J = 8.4
Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 5.04 (s, 2H), 3.27 (s, 3H), 2.72 (t, J =
7.4 Hz, 2H), 1.69 (quin, J = 7.3 Hz, 2H), 1.43−1.26 (m, 10H), 0.93 (t, J
= 6.8 Hz, 3H); 13C NMR (101 MHz, CD3OD) δ 175.9, 169.8, 159.6,
148.4, 130.2, 128.4, 125.0, 47.4, 37.8, 36.8, 33.0, 32.4, 30.5, 30.4, 30.3,
1-(1-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)cyclopropyl)-
guanidine Hydrochloride (7f). Synthesized by general procedure B.
99% yield, white solid. 1H NMR (400 MHz, CD3OD) δ 8.63 (s, 1H),
7.92 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H),
1.92 (dd, J = 5.2, 8.4 Hz, 2H), 1.72−1.58 (m, 4H), 1.38−1.24 (m, 10H),
0.88 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CD3OD) δ 180.6, 169.9,
159.8, 148.2, 130.1, 128.3, 125.2, 36.8, 33.0, 32.4, 31.6, 30.5, 30.4, 30.3,
23.7, 21.1, 14.4; HRMS (ESI+): Calcd for C20H30N5O [M + H]+:
356.2445, Found 356.2439.
(S)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-
carboximidamide Hydrochloride (7g). Synthesized by general
procedure B. 80% yield, white solid; 1H NMR (500 MHz, CD3OD) δ
7.94 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 5.50−5.38 (m, 1H),
3.80−3.70 (m, 1H), 3.67−3.54 (m, 1H), 2.70−2.39 (m, 4H), 2.28−2.16
(m, 1H), 2.16−1.98 (m, 1H), 1.70−1.55 (m, 2H), 1.39−1.15 (m, 10H),
0.88 (t, J = 6.9 Hz, 3H); 13C NMR (126 MHz, CD3OD) δ 177.5, 168.4,
155.8, 147.0, 128.8, 127.1, 123.6, 55.1, 35.5, 31.7, 31.4, 31.1, 29.2, 29.0,
28.9, 23.0, 22.4, 13.1; HRMS (ESI+) Calcd for C21H32N5O [M + H]+:
370.2601, Found: 370.2607. [α]D = −74.6° (c = 0.005, methanol).
(R)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-
carboximidamide Hydrochloride (7h). Synthesized by general
procedure B. 99%, white solid. 1H NMR (400 MHz, CD3OD) δ 7.94 (d,
J = 7.8 Hz, 2H), 7.32 (d, J = 7.9 Hz, 2H), 5.46−5.42 (m, 1H), 3.79−3.75
(m, 1H), 3.65−3.61 (m, 1H), 2.66 (t, J = 7.5 Hz, 2H), 2.53−2.49 (m,
2H), 2.23−2.19 (m, 1H), 2.10−2.06 (m, 1H), 1.65−1.61 (m, 2H),
1.38−1.26 (m, 10H), 0.88 (t, J = 6.7 Hz, 3H); 13C NMR (126 MHz,
CD3OD) δ 178.9, 169.7, 157.1, 148.3, 130.2, 128.5, 125.0, 56.6, 36.9,
33.0, 32.8, 32.4, 30.6, 30.4, 30.3, 24.4, 23.7, 14.5; HRMS (ESI+): Calcd
for C21H32N5O [M+]: 370.2601, Found: 370.2596. [α]D = +78.8° (c =
0.00085, methanol).
(S)-Amino(2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-2,5-di-
hydro-1H-pyrrol-1-yl)methaniminium 2,2,2-Trifluoroacetate
(7i). Synthesized general procedure D. 98% yield, white solid. 1H
NMR (500 MHz, CD3OD) δ 7.95 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4
Hz, 2H), 6.31−6.28 (m, 1H), 6.19−6.13 (m, 2H), 4.57−4.43 (m, 2H),
2.68 (t, J = 5.9 Hz, 2H), 1.69−1.60 (m, 2H), 1.38−1.24 (m, 10H), 0.89
(t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CD3OD) δ 169.9, 157.0, 148.5,
130.2, 129.8, 129.7, 128.4, 125.6, 125.5, 124.8, 56.1, 36.9, 33.0, 32.4,
30.5, 30.4, 30.3, 23.7, 14.4; HRMS (ESI+): Calcd for C21H30N5O+
[M+]: 368.2450, Found: 368.2454. HPLC analysis shows that 7i is 70%
pure.
N
DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(S)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)azetidine-1-
carboximidamide Hydrochloride (7j). Synthesized by general
procedure B. 95% yield, white solid. 1H NMR (400 MHz, CD3OD) δ
7.98 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 5.83 (dd, J = 9.3, 5.2
Hz, 1H), 4.37 (q, J = 8.6 Hz, 1H), 4.27 (q, J = 8.7 Hz, 1H), 3.18−2.93
(m, 1H), 2.76−2.55 (m, 3H), 1.64 (q, J = 7.3 Hz, 2H), 1.40−1.19 (m,
12H), 0.88 (t, J = 7.2, 6.5 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ
178.2, 169.8, 158.6, 148.4, 130.2, 128.4, 124.9, 58.4, 50.9, 36.9, 33.0,
32.4, 30.4, 23.7, 23.0, 14.4; HRMS (ESI+): Calcd for C20H31N5O [M+]:
356.2450, Found: 356.2478. [α]D = −27.3° (c = 0.00055, methanol).
(S)-Amino(2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
piperidin-1-yl)methaniminium Chloride (7k). Synthesized bu
153.8 (major), 146.7 (major), 146.5 (minor), 129.0 (major), 128.8
(minor), 127.4 (major), 125.8 (minor), 123.9 (major), 81.0 (major),
80.8 (minor), 69.9 (minor), 69.3 (major), 54.8 (major), 52.6 (major),
40.9 (major), 40.2 (minor), 36.0 (major), 31.9 (major), 31.2 (major),
29.4 (major), 29.3 (major), 29.2 (major), 28.3 (minor), 28.1 (major),
22.7 (major), 14.1 (major). HRMS (ESI+) Calcd for C25H37N3NaO4
[M + H]+: 466.2682, Found: 466.2720.
(3R,5S)-5-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-
3-ol (13). Synthesized by general procedure D. 95% yield, yellow solid;
1H NMR (500 MHz, CDCl3) δ 7.89 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2
Hz, 2H), 4.79−4.63 (m, 1H), 4.57−4.43 (m, 1H), 3.22−3.10 (m, 1H),
3.08−2.95 (m, 1H), 2.77 (br s, 2H), 2.56 (t, J = 7.7 Hz, 2H), 2.35−2.18
(m, 2H), 1.63−1.48 (m, 2H), 1.33−1.10 (m, 10H), 0.80 (t, J = 6.9 Hz,
3H); 13C NMR (126 MHz, CDCl3) δ 181.3, 168.3, 146.6, 128.9, 127.4,
124.0, 72.2, 55.4, 53.0, 40.7, 36.0, 31.9, 31.2, 29.4, 29.3, 29.2, 22.7, 14.1.
1
general procedure B. 99% yield, white solid. H NMR (500 MHz,
MeOD) δ 7.87 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 5.44 (d, J =
4.0 Hz, 1H), 3.90−4.01 (m, 1H), 3.41−3.50 (m, 1H), 3.13 (d, J = 7.4 Hz,
1H), 2.59 (t, J = 7.6 Hz, 2H), 2.46 (d, J = 14.0 Hz, 1H), 2.10−1.98 (m,
1H), 1.75 (m, 2H), 1.63−1.51 (m, 4H), 1.37 (m, 1H), 1.23 (m, 11H),
0.79 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, MeOD) δ 176.5, 168.5,
158.5, 147.0, 128.8, 127.0, 127.0, 123.6, 54.5, 51.7, 43.4, 42.5, 37.3, 35.4,
31.6, 31.0, 29.1, 29.0, 28.9, 27.3, 23.9, 22.3, 19.1, 17.9, 17.4, 15.9, 13.0,
11.8. HRMS (ESI+): Calcd for C22H34N5O+ [M+]: 384.2758, Found:
384.2759. HPLC analysis indicates that compound 7k is 85% pure.
(2S,3S)-tert-Butyl 3-Hydroxy-2-(3-(4-octylphenyl)-1,2,4-oxa-
diazol-5-yl)pyrrolidine-1-carboxylate (8). Synthesized by general
procedure A. 46% yield, yellow oil. 1H NMR (500 MHz, CDCl3) δ 7.95
(d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 4.96 (s, 1H), 4.56 (s, 1H),
3.82−3.68 (m, 2H), 2.69−2.59 (m, 2H), 2.35−2.27 (m, 1H), 2.07−2.00
(m, 1H), 1.66−1.58 (m, 2H), 1.46 (s, 3H), 1.36−1.21 (m, 16H), 0.87 (t,
J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 178.3, 168.6, 154.0,
146.9, 129.1, 129.0, 127.5, 123.9, 81.0, 76.1, 62.4, 44.5, 36.1, 32.2, 32.0,
31.3, 29.5, 29.4, 29.3, 28.5, 28.3, 22.8, 14.2; HRMS (ESI+): Calcd for
C25H37N3O4 [M + H]+: 444.2862, Found: 444.2883.
+
HRMS (ESI+) m/z calcd for C20H30N3O2 [M+]: 344.2333, Found:
344.2336.
tert-Butyl (((tert-Butoxycarbonyl)imino)((2S,4R)-4-hydroxy-
2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-1-yl)-
methyl)carbamate (14). Synthesized by general procedure C. 53%
yield, yellow solid; 1H NMR (500 MHz, CDCl3) δ 7.99 (d, J = 8.3 Hz,
2H), 7.29 (d, J = 8.3 Hz, 2H), 5.83 (t, J = 8.2 Hz, 1H), 4.72−4.59 (m,
1H), 4.05 (dd, J = 12.5, 3.5 Hz, 1H), 3.85−3.69 (m, 1H), 2.67 (t, J = 7.6
Hz, 2H), 2.63−2.53 (m, 1H), 2.45−2.32 (m, 1H), 1.74−1.60 (m, 2H),
1.47 (s, 18H), 1.39−1.24 (m, 10H), 0.90 (t, J = 7.0 Hz, 3H); 13C NMR
(126 MHz,) δ 178.5, 168.5, 154.1, 146.6, 128.9, 127.5, 124.0, 69.3, 58.0,
53.7, 40.0, 36.0, 31.9, 31.2, 29.7, 29.4, 29.2, 28.1, 22.7, 14.1. HRMS (ESI
+) Calcd for C31H48N5O6 [M + H]+: 586.3605, Found: 586.3557.
Amino((2S,4R)-4-hydroxy-2-(3-(4-octylphenyl)-1,2,4-oxadia-
zol-5-yl)pyrrolidin-1-yl)methaniminium Chloride (15). Synthe-
1
sized by general procedure B. 79% yield, white solid; H NMR (500
MHz, CD3OD) δ 8.01−7.93 (m, 2H), 7.41−7.31 (m, 2H), 5.66−5.54
(m, 1H), 4.67−4.58 (m, 1H), 3.96−3.85 (m, 1H), 3.65−3.58 (m, 1H),
2.74−2.61 (m, 3H), 2.58−2.49 (m, 1H), 1.73−1.61 (m, 2H), 1.42−1.24
(m, 10H), 0.91 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CD3OD) δ
178.9, 169.8, 157.7, 148.4, 130.2, 128.4, 124.9, 69.8, 57.1, 54.9, 41.1,
36.9, 33.0, 32.4, 30.5, 30.4, 30.3, 23.7, 14.5; HRMS (ESI+) m/z calcd for
C21H32N5O2 [M + H]+: 386.2556, Found: 386.2596. [α]D = −45.5° (c =
0.00055, methanol).
(2S,3S)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-
1
3-ol (9). Synthesized by general procedure D. 59% yield, clear oil. H
NMR (500 MHz, CDCl3) δ 7.97 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 7.4 Hz,
2H), 4.70 (s, 1H), 3.35 (s, 1H), 2.74−2.52 (m, 2H), 2.43−2.17 (m, 3H),
1.97 (s, 1H), 1.72−1.49 (m, 2H), 1.43−1.13 (m, 10H), 0.87 (t, J = 6.9
Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 179.4, 168.4, 146.8, 129.0,
127.6, 124.0, 76.5, 63.3, 45.1, 36.1, 34.4, 32.0, 31.3, 29.6, 29.4, 29.3, 22.8,
14.2; HRMS (ESI+): Calcd for C20H30N3O2 [M + H]+: 344.2338,
Found: 344.2338.
(2S,4S)-tert-Butyl 4-(Benzoyloxy)-2-(3-(4-octylphenyl)-1,2,4-
oxadiazol-5-yl)pyrrolidine-1-carboxylate (16). PPh3 (112 mg,
0.43 mmol), benzoic acid (52 mg, 0.43 mmol), and (2S,4R)-tert-butyl
4-hydroxy-2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-car-
boxylate 15 were added to THF (1.0 mL) at room temperature. The
solution was cooled to 0 °C, and diisopropyl azodicarboxylate (0.08 mL,
0.43 mmol) was added. The solution was warmed to room temperature
and stirred overnight. At this time, TLC showed complete conversion of
the starting material. The organic solvent was removed under reduced
pressure, and the residue was purified by silica gel column
chromatography (15%, ethyl acetate/hexanes) to yield product 16 as
a colorless oil. 75% yield. 1H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 7.8
Hz, 2H), 7.77 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.36 (t, J = 7.9
Hz, 1H), 7.24 (t, J = 7.8 Hz, 2H), 7.13 (t, J = 7.0 Hz, 2H), 5.65 (s, 1H),
5.28 (d, J = 8.3 Hz, 1H), 4.00−3.80 (m, 2H), 2.80−2.63 (m, 4H), 1.67−
1.60 (m, 2H), 1.51 (s, 3H), 1.39 (s, 6H), 1.37−1.23 (m, 10H), 0.87 (t, J
= 6.9 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 179.7, 168.5, 165.8,
153.6, 146.6, 133.2, 129.7, 129.4, 129.0, 128.3, 127.5, 124.1, 81.1, 72.4,
53.5, 52.6, 38.0, 36.0, 32.0, 31.3, 29.5, 29.3, 28.5, 28.3, 22.7, 14.2; HRMS
(mixed+): Calcd for C32H41N3O5Na [M + Na]+: 570.2944, Found:
570.2903.
(2S,4S)-tert-Butyl 4-Hydroxy-2-(3-(4-octylphenyl)-1,2,4-oxa-
diazol-5-yl)pyrrolidine-1-carboxylate. Hydrolysis of 4-benzoyl
ester protecting group in 16. (2S,4S)-tert-Butyl 4-(benzoyloxy)-2-(3-
(4-octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate 16 (85
mg, 0.16 mmol) was dissolved in equal parts of methanol (0.38 mL) and
THF (0.38 mL) and 2 M NaOH (0.15 mL, 2 M) and stirred for 30 min
at room temperature under nitrogen. The solution was partitioned
between ethyl acetate and water. The aqueous solution was extracted
with ethyl acetate, and the combined organic layers were washed with
brine, dried over Na2SO4, and concentrated via vacuum to yield the
(E)-tert-Butyl (((tert-Butoxycarbonyl)amino)((2S,3S)-3-hy-
droxy-2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-1-
yl)methylene)carbamate (10). Synthesized by general procedure C.
89% yield, colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 8.2
Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H), 5.52 (s, 1H), 4.64 (s, 1H), 4.06−3.86
(m, 2H), 3.18 (bs, 1H), 2.63 (t, J = 7.1 Hz, 2H), 2.38−2.30 (m, 1H),
2.17−2.05 (m, 1H), 1.68−1.57 (m, 2H), 1.81 (bs, 1H), 1.49−1.40 (m,
18H), 1.33−1.23 (m, 10H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR (101
MHz, CDCl3) δ 177.1, 168.8, 147.2, 129.3, 127.9, 124.2, 75.1, 63.8, 47.4,
36.4, 32.3, 31.7, 30.2, 29.9, 29.7, 29.7, 28.5, 23.1, 14.6; HRMS (ESI+):
Calcd for C31H48N5O6 [M + H]+: 586.3604, Found: 586.3569.
Amino((1S,3S)-3-hydroxy-2-(3-(4-octylphenyl)-1,2,4-oxadia-
zol-5-yl)pyrrolidin-1-yl)methaniminium Chloride (11). Synthe-
1
sized by general procedure B. 52% yield, white solid. H NMR (500
MHz, CD3OD) δ 7.97 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 5.24
(s, 1H), 4.80 (d, J = 3.2 Hz, 1H), 3.85−3.82 (m, 2H), 2.70 (t, J = 7.2 Hz,
2H), 2.30−2.14 (m, 2H), 1.72−1.60 (m, 2H), 1.44−1.22 (m, 10H), 0.91
(t, J = 6.9 Hz, 3H); 13C NMR (126 MHz, CD3OD) δ 176.65, 169.7,
157.5, 148.4, 130.1, 128.4, 124.7, 76.0, 64.1, 47.3, 36.8, 33.0, 32.5, 32.4,
30.5, 30.4, 30.3, 23.7, 14.4; HRMS (ESI+): Calcd for C21H32N5O2 [M +
H]+: 386.2556, Found: 386.2576.
(2S,4R)-tert-Butyl 4-Hydroxy-2-(3-(4-octylphenyl)-1,2,4-oxa-
diazol-5-yl)pyrrolidine-1-carboxylate (12). Synthesized by general
procedure A. 63% yield, yellow oil; 1H NMR (500 MHz, CDCl3) δ 7.99
(d, J = 7.9 Hz, 2H), 7.31 (d, J = 7.9 Hz, 2H), 5.42−5.30 (m, 1H, minor
rotamer), 5.20 (m, 1H, major rotamer), 4.74−4.56 (m, 1H), 3.89−3.79
(m, 1H), 3.77−3.69 (m, 1H, major rotamer), 3.64−3.53 (m, 1H, minor
rotamer), 2.74−2.61 (m, 2H), 2.59−2.40 (m, 1H), 2.38−2.28 (m, 1H),
1.74−1.58 (m, 2H), 1.56−1.16 (m, 19H), 0.90 (t, J = 7.0 Hz, 3H); 13C
NMR (126 MHz, CDCl3, rotamers) δ 180.3 (major), 168.5 (major),
O
DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
product as colorless oil. 95% yield. 1H NMR (500 MHz, CDCl3) δ 7.92
(d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 5.11 (d, J = 9.1 Hz, 1H),
4.51 (s, 1H), 3.87−3.63 (m, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.59−2.50 (m,
1H), 2.35−2.25 (m, 1H), 1.66−1.57 (m, 2H), 1.43 (s, 3H), 1.37−1.19
(m, 16H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ
181.5, 168.1, 153.5, 147.1, 129.1, 129.0, 127.5, 123.4, 81.2, 70.6, 56.0,
52.4, 39.6, 36.1, 31.9, 31.3, 29.5, 29.3, 29.3, 28.2, 22.7, 14.2; HRMS (ESI
+): Calcd for C25H37N3O4Na [M + Na]+: 466.2681, Found: 466.2637.
(2S,4S)-2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-
4-ol. Deprotection of Boc group in (2S,4S)-tert-butyl 4-hydroxy-2-(3-
(4-octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate. Synthe-
33.0, 32.4, 30.5, 30.4, 30.3, 30.0, 23.7, 14.4. HRMS (ESI+): Calcd for
C19H28N3O+ [M+]: 314.2227, Found: 314.2221.
(R)-3-(4-Octylphenyl)-5-(pyrrolidin-3-yl)-1,2,4-oxadiazole
(19b). Synthesized by general procedure B. 79%, white solid. 1H NMR
(400 MHz, CDCl3) δ 7.95 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H),
3.57 (ddd, J = 12.7, 9.1, 6.3 Hz, 1H), 3.29 (d, J = 5.9 Hz, 2H), 3.25−3.16
(m, 1H), 3.06−2.96 (m, 1H), 2.67−2.57 (m, 2H), 2.34−2.08 (m, 3H),
1.68−1.55 (m, 2H), 1.27 (d, J = 19.6 Hz, 10H), 0.86 (t, J = 6.8 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 182.1, 168.4, 146.5, 129.0, 127.4, 124.3,
52.6, 47.5, 37.3, 36.0, 32.0, 32.0, 31.3, 29.8, 29.5, 29.3, 29.3, 22.7, 14.2;
HRMS (ESI+): Calcd for C23H34N2O2 [M + H]+: 328.2383, Found:
328.2386.
1
sized by general procedure D. 48% yield, colorless oil. H NMR (500
MHz, CDCl3) δ 7.92 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 7.6 Hz, 2H), 4.66
(d, J = 6.6 Hz, 1H), 4.53 (s, 1H), 3.91 (s, 2H), 3.27 (s, 2H), 2.67−2.60
(m, 2H), 2.56−2.50 (m, 1H), 2.29 (d, J = 13.8 Hz, 1H), 1.62 (quin, J =
7.5 Hz, 2H), 1.38−1.19 (m, 10H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR
(126 MHz, CDCl3) δ 181.1, 168.2, 147.0, 129.0, 127.6, 123.7, 72.0, 56.0,
53.1, 40.2, 36.1, 32.0, 31.3, 29.6, 29.4, 29.4, 22.8, 14.2; HRMS (mixed+):
Calcd for C20H30N3O2 [M + H]+: 344.2338, Found: 344.2315.
tert-Butyl (((tert-Butoxycarbonyl)amino)((2S,4S)-4-hydroxy-
2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-1-yl)-
methylene)carbamate. Guanylation of (2S,4S)-2-(3-(4-octylphen-
yl)-1,2,4-oxadiazol-5-yl)pyrrolidin-4-ol. Synthesized by general proce-
dure C. 79% yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J
= 8.2 Hz, 2H),7.26 (d, J = 8.2 Hz, 2H), 5.81 (t, J = 8.2 Hz, 1H), 4.63 (s,
1H), 4.02 (dd, J = 12.5 Hz, 1H), 3.79−3.70 (m, 1H), 2.65 (t, J = 7.3 Hz,
2H), 2.57 (dd, J = 13.3, 7.7 Hz, 1H), 2.41−2.29 (m, 1H), 1.62 (quin, J =
7.5 Hz, 2H), 1.45 (s, 18H), 1.35−1.20 (m, 10H), 0.87 (t, J = 6.9 Hz,
3H); 13C NMR (101 MHz, CDCl3) δ 178.6, 168.6, 154.2, 146.7, 129.0,
127.6, 124.1, 69.6, 58.1, 53.8, 40.2, 36.1, 32.0, 31.4, 29.6, 29.4, 28.2, 22.8,
14.2; HRMS (ESI+): Calcd for C31H48N5O6 [M + H]+: 586.3604,
Found: 586.3603.
tert-Butyl (((tert-Butoxycarbonyl)amino)(3-(3-(4-octylphen-
yl)-1,2,4-oxadiazol-5-yl)azetidin-1-yl)methylene)carbamate.
Guanylation of 3-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)azetidin-1-
ium chloride 19a. Synthesized by general procedure C. 57%, yellow
oil. 1H NMR (400 MHz, Chloroform-d) δ 11.01 (s, 1H), 7.95 (d, J = 8.2
Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 4.82−4.51 (m, 3H), 4.10 (ddd, J =
15.5, 8.9, 6.6 Hz, 1H), 2.64 (t, J = 8.0, 7.4 Hz, 2H), 1.61 (dt, J = 11.9, 7.1
Hz, 2H), 1.48 (s, 18H), 1.36−1.20 (m, 10H), 0.85 (t, J = 6.9 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 178.2, 168.6, 156.2, 146.7, 128.9, 127.4,
123.7, 35.9, 31.8, 31.1, 29.4, 29.2, 29.2, 28.1, 26.5, 22.6, 14.0; HRMS
(ESI+): Calcd for C30H46N5O5 [M + H]+: 556.3499, Found: 556.3455.
(R,Z)-tert-Butyl (((tert-Butoxycarbonyl)imino)(3-(3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)pyrrolidin-1-yl)methyl)-
carbamate. Guanylation of (R)-3-(4-octylphenyl)-5-(pyrrolidin-3-yl)-
1,2,4-oxadiazole 19b. Synthesized by general procedure C. 8% yield,
colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.3 Hz, 2H),
7.27 (d, J = 8.2 Hz, 2H), 4.05 (s, 2H), 3.87−3.68 (m, 3H), 2.69−2.62
(m, 2H), 2.45 (dt, J = 13.4, 6.5 Hz, 2H), 1.68−1.61 (m, 2H), 1.50 (s,
18H), 1.28 (d, J = 22.8 Hz, 10H), 0.92−0.83 (m, 3H); 13C NMR (101
MHz, CDCl3) δ 178.6, 171.3, 168.6, 154.4, 146.8, 129.1, 127.6, 124.0,
77.4, 66.0, 60.5, 36.1, 32.0, 31.7, 31.3, 29.8, 29.6, 29.4, 29.4, 28.3, 25.4,
22.8, 21.2, 15.4, 14.3, 14.3, 14.2, 11.6, 1.2; HRMS (ESI+): Calcd for
C31H48N5O5 [M + H]+: 570.3655, Found: 570.3690.
Amino((1S,4S)-4-hydroxy-2-(3-(4-octylphenyl)-1,2,4-oxadia-
zol-5-yl)pyrrolidin-1-yl)methaniminium Chloride (17). Synthe-
1
sized by general procedure B. 22% yield, white solid. H NMR (400
MHz, CD3OD) δ 7.97 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 5.59
(dd, J = 7.8, 6.7 Hz, 1H), 4.64 (quin, J = 4.4 Hz, 1H), 3.91 (dd, J = 10.7,
4.7 Hz, 1H), 3.62 (ddd, J = 10.7, 2.9, 1.3 Hz, 1H), 2.70 (t, J = 7.4 Hz,
2H), 2.65 (dtd, J = 8.0, 4.8, 4.1, 1.3 Hz, 1H), 2.54 (ddd, J = 13.3, 6.4, 4.9
Hz, 1H), 1.68 (quin, J = 7.3 Hz, 2H), 1.42−1.21 (m, 10H), 0.91 (t, J =
6.9 Hz, 3H); 13C NMR (101 MHz, CD3OD) δ 178.9, 169.8, 157.6,
148.4, 130.2, 128.4, 125.0, 69.7, 57.1, 54.9, 41.0, 36.8, 33.0, 32.4, 30.5,
30.4, 30.3, 23.7, 14.4; HRMS (ESI+): Calcd for C21H32N5O2 [M+]:
386.2556, Found: 386.2548. [α]D = −40.6° (c = 0.0015, methanol).
tert-Butyl 3-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
azetidine-1-carboxylate (18a). Synthesized by general procedure
A. 77% yield, yellow oil. 1H NMR (400 MHz, chloroform-d) δ 7.96 (d, J
= 8.4 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H), 4.48−4.21 (m, 4H), 4.15−3.92
(m, 1H), 2.64 (t, J = 7.5 Hz, 2H), 1.63 (td, J = 15.9, 15.1, 8.5 Hz, 2H),
1.45 (s, 9H), 1.36−1.19 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR
(101 MHz, Chloroform-d) δ 178.7, 168.6, 155.9, 146.7, 128.9, 127.4,
123.8, 80.1, 35.9, 31.8, 31.2, 29.4, 29.2, 29.2, 28.3, 25.7, 22.6, 14.0;
HRMS (ESI+): Calcd for C24H35N3NaO3 [M + Na]+: 436.2576, Found:
436.2558.
(R)-tert-Butyl 3-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
pyrrolidine-1-carboxylate (18b). Synthesized by general procedure
A. 77% yield, colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 8.2
Hz, 2H), 7.25 (d, J = 7.7 Hz, 2H), 3.93−3.37 (m, 5H), 2.68−2.57 (m,
2H), 2.36 (dq, J = 12.0. 6.9 Hz, 2H), 1.61 (quin, J = 7.4 Hz, 2H), 1.46 (s,
9H), 1.34−1.18 (m, 10H), 0.86 (t, J = 6.9 Hz, 3H); 13C NMR (126
MHz, CDCl3) δ179.3, 168.5, 154.3, 146.7, 129.0, 127.4, 124.0, 79.8,
49.4, 45.1, 36.7, 36.0, 31.9, 31.3, 29.7, 29.5, 29.3, 29.3, 28.6, 22.7, 14.2;
HRMS (ESI+): Calcd for C25H37N3O3Na [M + Na]+: 450.2732, Found:
450.2695.
Amino(3-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)azetidin-1-
yl)methaniminium Chloride (20a). Synthesized by general proce-
dure B. 82% yield, white solid. 1H NMR (400 MHz, CD3OD) δ 7.96 (d,
J = 8.3 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 4.63 (t, J = 8.9 Hz, 2H), 4.50−
4.33 (m, 3H), 2.66 (t, J = 7.5 Hz, 2H), 1.62 (quin, J = 7.3 Hz, 2H), 1.42−
1.18 (m, 10H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CD3OD)
δ 180.0, 169.8, 158.3, 148.2, 130.1, 128.4, 125.2, 55.8, 36.8, 33.0, 32.4,
30.5, 30.4, 30.3, 27.2, 23.7, 14.4; HRMS (ESI+): Calcd for C20H30N5O+
[M+]: 356.2445, Found: 356.2418.
(R)-Amino(3-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
pyrrolidin-1-yl)methaniminium Chloride (20b). Synthesized by
1
general procedure B. 72% yield, white solid. H NMR (400 MHz,
CD3OD) δ 7.98 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 4.13−3.87
(m, 3H), 3.74−3.65 (m, 2H), 2.75−2.62 (m, 3H), 2.53 (s, 1H), 1.72−
1.64 (m, 2H), 1.45−1.24 (m, 10H), 0.92 (t, J = 6.8 Hz, 3H); 13C NMR
(101 MHz, CD3OD) δ 180.3, 169.6, 156.4, 148.2, 130.1, 128.3, 125.2,
51.4, 47.7, 37.3, 36.8, 33.0, 32.4, 30.6, 30.5, 30.3, 30.3, 23.7, 14.4; MS:
Calcd for C21H32N5O [M+]: 370.2606, Found: 370.2586.
(S)-tert-Butyl 2-(5-(4-Octylphenyl)-1,2,4-oxadiazol-3-yl)-
pyrrolidine-1-carboxylate (22). Synthesized by general procedure
A. 19% yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 7.7
Hz, 2H), 7.32 (d, J = 7.8 Hz, 2H), 4.99 (d, J = 4.3 Hz, 1H), 3.73−3.52
(m, 2H), 2.68 (t, J = 7.6 Hz, 2H), 2.32 (s, 1H), 2.19−2.04 (m, 2H), 1.96
(d, J = 5.8 Hz, 1H), 1.69−1.59 (m, 2H), 1.46 (s, 3H), 1.37−1.22 (m,
16H), 0.91−0.85 (m, 3H); 13C NMR (101 MHz, CDCl3) δ172.9, 159.8,
156.0, 148.6, 129.3, 128.2, 107.7, 80.0, 77.4, 53.5, 46.6, 36.2, 32.0, 31.3,
29.6, 29.4, 29.4, 28.4, 23.5, 22.8, 14.2; HRMS (ESI+): Calcd for
C25H38N3O3 [M + H]+: 428.2913, Found: 428.2918.
(S)-2-(5-(4-Octylphenyl)-1,2,4-oxadiazol-3-yl)pyrrolidine-1-
carboximidamide Hydrochloride (23). Synthesized by general
3-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)azetidin-1-ium
Chloride (19a). Synthesized by general procedure B. 94% yield, white
solid. 1H NMR (400 MHz, CD3OD) δ 7.97 (d, J = 8.4 Hz, 2H), 7.33 (d, J
= 8.4 Hz, 2H), 4.64−4.31 (m, 5H), 2.67 (t, J = 7.5 Hz, 2H), 1.64 (quin, J
= 7.4 Hz, 2H), 1.42−1.17 (m, 10H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 178.9, 169.9, 148.3, 130.2, 128.4, 125.1, 51.0, 36.8,
1
procedure B. 89% yield, white solid. H NMR (400 MHz, CD3OD) δ
8.11 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 5.02 (t, J = 7.7 Hz, 1H),
3.63−3.46 (m, 2H), 2.78−2.72 (m, 2H), 2.62 (dtd, J = 12.8, 7.8, 5.2 Hz,
2H), 2.47−2.18 (m, 3H), 1.75−1.63 (m, 2H), 1.41−1.26 (m, 10H),
0.94−0.87 (m, 3H); 13C NMR (101 MHz, CD3OD) δ 178.7, 168.6,
P
DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
151.0, 130.6, 129.3, 122.1, 55.8, 47.2, 37.0, 33.0, 32.3, 30.5, 30.3, 30.3,
30.1, 24.5, 23.7, 14.4; HRMS (ESI+): Calcd for C20H30N3O [M + H]+:
328.2388, Found: 328.2386.
(S)-tert-Butyl 2-((4-Decylphenyl)carbamoyl)pyrrolidine-1-
carboxylate (27a). Synthesized using general procedure E. 61%
yield, colorless oil; 1H NMR (500 MHz, CDCl3) δ 9.37 (s, 1H), 7.41 (d,
J = 8.4 Hz, 2H), 7.10 (s, 2H), 4.39 (d, J = 75.3 Hz, 1H), 3.68−3.21 (m,
2H), 2.54 (t, J = 7.5 Hz, 2H), 2.03−1.81 (m, 2H), 1.60−1.53 (m, 2H),
1.49 (s, 9H), 1.34−1.16 (m, 14H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 169.8, 156.5, 138.8, 136.1, 128.9, 119.8, 81.0, 60.6,
47.3, 35.5, 32.0, 31.7, 29.7, 29.6, 29.4, 29.4, 28.5, 27.2, 24.7, 22.8, 14.2.
HRMS (ESI+): Calcd for C26H43N2O3 [M + H]+: 431.3273, Found:
431.3240.
(R)-tert-Butyl 2-((4-Decylphenyl)carbamoyl)pyrrolidine-1-
carboxylate (27b). Synthesized using general procedure E. 95%
yield, colorless oil; 1H NMR (500 MHz, CDCl3) δ 9.39 (s, 1H), 7.40 (d,
J = 8.5 Hz, 2H), 7.07 (s, 2H), 4.38 (d, J = 85.0 Hz, 1H), 3.43 (t, J = 45.4
Hz, 2H), 2.59−2.48 (m, 2H), 2.21 (s, 1H), 2.05−1.84 (m, 3H), 1.52 (d,
J = 35.8 Hz, 11H), 1.27 (d, J = 19.0 Hz, 14H), 0.87 (t, J = 7.0 Hz, 3H);
13C NMR (126 MHz, CDCl3) δ 169.8, 156.5, 138.5, 136.1, 128.8, 119.7,
(S)-Amino(2-(5-(4-octylphenyl)-1,2,4-oxadiazol-3-yl)-
pyrrolidin-1-yl)methaniminium Chloride (23). Synthesized by
1
general procedure B. 28% yield, white solid. H NMR (400 MHz,
CD3OD) δ 8.05 (d, J = 6.9 Hz, 2H), 7.43 (d, J = 7.2 Hz, 2H), 5.29 (d, J =
7.3 Hz, 1H), 3.74−3.69 (m, 1H), 3.65−3.53 (m, 1H), 2.73 (t, J = 7.8 Hz,
2H), 2.52−2.39 (m, 2H), 2.19 (s, 2H), 1.70−1.63 (m, 2H), 1.32 (d, J =
20 Hz, 10H), 0.93−0.86 (m, 3H); 13C NMR (126 MHz, CD3OD) δ
178.2, 171.1, 156.9, 150.6, 130.5, 129.2, 122.4, 56.3, 37.0, 33.0, 32.4,
32.3, 30.5, 30.4, 30.3, 24.1, 23.7, 14.4; HRMS (ESI+): Calcd for
C21H32N5O [M + H]+: 370.2606, Found: 370.2617.
5-(4-Octylphenyl)-2H-tetrazole (24). To a solution of 4-
octylbenzonitrile 3 (120 mg, 0.557 mmol) in DMF (3 mL) were
added ammonium chloride (54 mg, 1.003 mmol) and sodium azide (65
mg, 1.003 mmol). The reaction was refluxed for ∼4 h. After the reaction
was completed, the solution was cooled to room temperature and
filtered, and the residue was washed with acetone. The solvent was then
evaporated in vacuo, and the residue was partitioned between equal
volumes of hexanes and water. The organic layer was then separated,
washed with satd LiBr and brine, dried over anhydrous sodium sulfate,
filtered, and concentrated in vaccuo to afford 34 in a quantitative yield as
colorless oil. The isolated product was sufficiently pure to use in the next
reaction. 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.3 Hz, 2H), 7.26
(d, J = 7.3 Hz, 2H), 2.65 (t, J = 8.1 Hz, 2H), 1.61 (quin, J = 7.4 Hz, 2H),
1.34−1.21 (m, 10H), 0.87 (t, J = 6.2 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 148.8, 132.2, 129.3, 119.3, 109.6, 36.3, 32.0, 31.1, 29.5, 29.3,
22.8, 14.2; HRMS (ESI+): Calcd for C15H23N4 [M + H]+: 259.1923,
Found: 259.1931.
(S)-tert-Butyl 2-(5-(4-Octylphenyl)-1,3,4-oxadiazol-2-yl)-
pyrrolidine-1-carboxylate (25). Synthesized by general procedure
A. 67% yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.3
Hz, 2H), 7.29 (d, J = 7.5 Hz, 2H), 5.21−5.05 (m, 1H), 3.71−3.34 (m,
2H), 2.65 (t, J = 7.7 Hz, 2H), 2.44−1.68 (m, 4H), 1.62 (q, J = 7.3 Hz,
2H), 1.44 (d, J = 8.6 Hz, 3H), 1.37−1.20 (m, 16H), 0.86 (t, J = 6.7 Hz,
3H); 13C NMR (101 MHz, CDCl3) δ 167.3, 164.8, 154.3, 147.4, 129.2,
126.9, 121.3, 80.3, 58.6, 53.0, 46.5, 36.1, 32.4, 32.0, 31.2, 29.5, 29.4, 29.3,
28.5, 28.4, 23.8, 22.8, 14.2; HRMS (ESI+): Calcd for C25H38N3O3 [M +
H]+: 428.2913, Found: 428.2930.
80.8, 60.5, 47.3, 35.4, 32.0, 31.6, 29.7, 29.7, 29.6, 29.4, 29.3, 27.4, 24.7,
28.5, 22.8, 14.2. HRMS (ESI+): Calcd for C26H42N2O3Na [M + Na]+:
453.3093, Found: 453.3126.
(S)-2-((4-Decylphenyl)carbamoyl)pyrrolidin-1-ium 2,2,2-Tri-
fluoroacetate (28a). Synthesized by general procedure D. 73%
yield, yellow oil; 1H NMR (400 MHz, CDCl3) δ 10.22 (s, 1H), 7.38 (s,
2H), 7.06 (d, J = 6.3 Hz, 2H), 4.6 (s, 1H), 3.39 (s, 2H), 2.53 (t, J = 8.0
Hz, 2H), 2.20−1.91 (m, 4H), 1.55 (s, 2H), 1.37−1.18 (m, 14H), 0.88 (t,
J = 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 166.6, 139.9, 135.0,
129.0, 120.3, 60.4, 46.7, 35.5, 32.1, 31.6, 30.3, 29.8, 29.8, 29.7, 29.5, 29.4,
24.9, 22.8, 14.3; HRMS (ESI+): Calcd for C21H35N2O+ [M + H]+:
331.2749, Found: 331.2716.
(R)-2-((4-Decylphenyl)carbamoyl)pyrrolidin-1-ium 2,2,2-Tri-
fluoroacetate 28b. Synthesized by general procedure D. Quantitative
yield, yellow oil; 1H NMR (400 MHz, CDCl3) δ 10.26 (s, 1H), 7.37 (d, J
= 7.9 Hz, 2H), 7.04 (d, J = 7.9 Hz, 2H), 4.80 (s, 1H), 3.34 (d, J = 26.2 Hz,
2H), 2.51 (t, J = 7.9 Hz, 2H), 2.44 (s, 1H), 2.08 (s, 1H), 1.95 (s, 2H),
1.54 (s, 2H), 1.32−1.22 (m, 14H), 0.87 (t, J = 6.6 Hz, 3H); 13C NMR
(126 MHz, CDCl3) δ 166.7, 140.0, 135.1, 128.9, 120.5, 60.5, 46.6, 35.6,
32.1, 31.6, 30.2, 29.8, 29.8, 29.7, 29.5, 24.6, 22.8, 14.2; HRMS (ESI+):
Calcd for C21H35N2O+ [M + H]+: 331.2749, Found: 331.2752.
(S,E)-tert-Butyl (((tert-Butoxycarbonyl)imino)(2-((4-
decylphenyl)carbamoyl)pyrrolidin-1-yl)methyl)carbamate.
Guanylation of (S)-2-((4-decylphenyl)carbamoyl)pyrrolidin-1-ium
2,2,2-trifluoroacetate. Synthesized by general procedure C. 55% yield,
colorless oil; 1H NMR (500 MHz, CDCl3) δ 9.75 (s, 1H), 7.57 (d, J = 8.5
Hz, 2H), 7.11 (d, J = 8.5 Hz, 2H), 5.10 (s, 1H), 3.71 (dt, J = 11.0, 7.3 Hz,
1H), 3.55−3.50 (m, 1H), 2.54 (t, J = 7.9 Hz, 2H), 2.41−2.34 (m, 1H),
2.22−2.15 (m, 1H), 2.06−1.99 (m, 1H), 1.91−1.83 (m, 1H), 1.65−1.53
(m, 2H), 1.51 (s, 18H), 1.31−1.21 (m, 14H), 0.87 (t, J = 7.0 Hz, 3H);
13C NMR (101 MHz, CDCl3) δ 171.0, 168.7, 161.9, 155.5, 138.7, 136.3,
(S)-2-(5-(4-Octylphenyl)-1,3,4-oxadiazol-2-yl)pyrrolidin-1-
ium 2,2,2-Trifluoroacetate (26). Deprotection of (S)-tert-butyl 2-(5-
(4-octylphenyl)-1,3,4-oxadiazol-2-yl)pyrrolidine-1-carboxylate 25. Syn-
thesized by general procedure D. 100% yield, white solid. 1H NMR (400
MHz, CD3OD) δ 8.00 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 5.17
(t, J = 7.7 Hz, 1H), 3.61−3.48 (m, 2H), 2.72 (t, J = 7.4 Hz, 2H), 2.68−
2.59 (m, 1H), 2.58−2.46 (m, 2H), 2.36−2.22 (m, 2H), 1.68 (quin, J =
7.3 Hz, 2H), 1.40−1.26 (m, 10H), 0.90 (t, J = 8.0 Hz, 3H); 13C NMR
(101 MHz, CD3OD) δ 167.8, 163.3, 149.7, 130.5, 128.2, 121.7, 54.7,
47.3, 36.9, 33.0, 32.3, 30.5, 30.4, 30.3, 29.8, 24.6, 23.7, 14.4; HRMS (ESI
+): Calcd for C20H30N3O+ [M + H]+: 328.2388, Found: 328.2389.
Guanylation of (S)-2-(5-(4-octylphenyl)-1,3,4-oxadiazol-2-yl)-
pyrrolidin-1-ium 2,2,2-trifluoroacetate. Synthesized by general proce-
dure C. 30% yield, colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.94 (d, J
= 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 5.64 (dd, J = 7.6, 5.3 Hz, 1H),
3.95−3.70 (m, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.48−2.39 (m, 1H), 2.34−
2.16 (m, 2H), 2.07−1.98 (m, 1H), 1.68−1.57 (m, 2H), 1.48 (d, J = 19.1
Hz, 18H), 1.31−1.24 (m, 10H), 0.87 (t, J = 7.5 Hz, 3H); 13C NMR (101
MHz, CDCl3) δ 171.7, 170.4, 165.9, 165.1, 147.3, 129.2, 127.1, 121.4,
54.3, 49.6, 36.1, 32.0, 31.3, 29.6, 29.4, 29.4, 28.3, 22.8, 14.2; HRMS (ESI
+): Calcd for C31H48N5O5 [M + H]+: 570.3655, Found: 570.3656.
128.8, 119.6, 61.8, 50.2, 50.2, 35.5, 32.0, 31.8, 29.8, 29.7, 29.7, 29.5, 29.4,
28.4, 28.3, 28.1, 24.7, 22.8; HRMS (ESI+): Calcd for C32H52N4O5 [M +
H]+: 573.4016, Found: 573.4011.
(R,E)-tert-Butyl (((tert-Butoxycarbonyl)imino)(2-((4-
decylphenyl)carbamoyl)pyrrolidin-1-yl)methyl)carbamate.
Guanylation of (R)-2-((4-decylphenyl)carbamoyl)pyrrolidin-1-ium
2,2,2-trifluoroacetate. Synthesized by general procedure C. 85% yield,
colorless oil; 1H NMR (500 MHz, CDCl3) δ 9.75 (s, 1H), 7.57 (d, J = 8.5
Hz, 2H), 7.11 (d, J = 8.5 Hz, 2H), 5.11 (s, 1H), 3.72 (dt, J = 10.9, 7.3 Hz,
1H), 3.60−3.49 (m, 1H), 2.54 (t, J = 7.1 Hz, 2H), 2.41−2.35 (m, 1H),
2.24−2.15 (m, 1H), 2.06−2.00 (m, 1H), 1.92−1.84 (m, 1H), 1.60−1.54
(m, 2H), 1.51 (s, 18H), 1.25 (s, 14H), 0.87 (t, J = 7.0 Hz, 3H); 13C NMR
(126 MHz, CDCl3) δ 168.8, 155.4, 138.7, 136.2, 128.8, 119.5, 61.8, 50.3,
35.5, 32.0, 31.7, 29.8, 29.7, 29.7, 29.5, 29.4, 28.3, 28.1, 24.7, 22.8, 14.3;
HRMS (ESI+): Calcd for C32H53N4O5 [M + H]+: 573.4016, Found:
573.4011.
1
Deprotection by general procedure D. 90% yield, white solid. H
NMR (400 MHz, CD3OD) δ 7.99 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4
Hz, 2H), 5.43 (dd, J = 7.3, 2.1 Hz, 1H), 3.80 (td, J = 9.2, 2.3 Hz, 1H),
3.63 (td, J = 9.8, 7.2 Hz, 1H), 2.75 (t, J = 7.2 Hz, 2H), 2.63−2.46 (m,
2H), 2.33−2.08 (m, 2H), 1.70 (quin, J = 7.3 Hz, 2H), 1.43−1.27 (m,
10H), 0.92 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz, CD3OD) δ 167.5,
165.8, 157.0, 149.4, 130.5, 128.0, 121.9, 55.7, 36.9, 33.0, 32.3, 32.1, 30.5,
30.4, 30.3, 24.1, 23.7, 14.4; HRMS (ESI+): Calcd for C21H32N5O+ [M +
H]+: 370.2606, Found: 370.2618.
(S)-Amino(2-((4-decylphenyl)carbamoyl)pyrrolidin-1-yl)-
methaniminium 2,2,2-Trifluoroacetate (29a). Synthesized by
1
general procedure D. 29% yield, white solid; H NMR (400 MHz,
CDCl3) δ 9.85 (s, 1H), 7.42 (d, J = 7.9 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H),
4.83 (s, 1H), 3.66 (s, 1H), 3.47 (s, 1H), 2.53 (t, J = 7.6 Hz, 2H), 2.31 (s,
2H), 2.19 (s, 1H), 2.00 (s, 1H), 1.55 (s, 2H), 1.25 (s, 14H), 0.88 (t, J =
Q
DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6.7 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 169.4, 155.4, 139.9, 135.2,
129.0, 120.3, 60.9, 48.4, 35.5, 32.0, 31.7, 31.4, 29.8, 29.8, 29.7, 29.5, 29.4,
24.1, 22.8, 14.3; HRMS (ESI+): Calcd for C22H37N4O+ [M + H]+:
373.2967, Found: 373.2963.
7.8 Hz, 2H), 1.33−1.20 (m, 10H), 0.87 (t, J = 6.8 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 171.2, 155.6, 142.3, 135.0, 128.8, 127.5, 60.5, 48.3,
43.5, 35.7, 32.0, 31.7, 31.3, 29.6, 29.5, 29.4, 24.1, 22.8, 14.2; HRMS (ESI
+): Calcd for C21H35N4O+ [M + H]+: 359.2810, Found: 359.2794.
(S)-tert-Butyl 2-((4-Octylbenzamido)methyl)pyrrolidine-1-
carboxylate (33). Dissolve 4-octylbenzoic acid 32 (50 mg, 0.21
mmol) in dichloromethane (1 mL). Add half the amount of TEA (0.04
mL, 0.32 mmol) and 1-hydroxypyrrolidine-2,5-dione (37 mg, 0.32
mmol) with stirring at room temperature. In a separate flask, dissolve
EDC (82 mg, 0.43 mmol) in dichloromethane (1 mL) with the other
half of TEA (0.04 mL, 0.32 mmol). After the EDC is dissolved, add that
into the reaction mixture dropwise over 15−20 min. Stir the reaction for
6 h at rt. After 6 h, add (S)-tert-butyl 2-(aminomethyl) pyrrolidine-1-
carboxylate (299 mg, 1.5 mmol) and stir 16−60 h. After completion, the
solution was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography to yield product 33 in an
85% yield as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H),
7.77 (d, J = 7.8 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 4.17 (s, 1H), 3.51 (d, J
= 13.3 Hz, 1H), 3.41−3.31 (m, 3H), 2.60 (t, J = 7.5 Hz, 2H), 2.08−1.79
(m, 3H), 1.72 (s, 1H), 1.59 (quin, J = 7.2 Hz, 2H), 1.45 (s, 9H), 1.32−
1.18 (m, 10H), 0.85 (t, J = 6.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
167.5, 157.1, 146.4, 131.7, 128.4, 127.2, 80.2, 56.2, 47.2, 47.1, 35.9, 31.9,
31.3, 29.6, 29.5, 29.3, 28.5, 24.0, 22.7, 14.2. HRMS (ESI+): Calcd for
C25H41N2O3 [M + H]+: 417.3117, Found: 417.3088.
(S)-Amino(2-((4-octylbenzamido)methyl)pyrrolidin-1-yl)-
methaniminium 2,2,2-Trifluoroacetate (34). (S)-2-((4-
Octylbenzamido)methyl)pyrrolidin-1-ium 2,2,2-Trifluoroace-
tate. Deprotection of Boc group in (S)-tert-butyl 2-((4-
octylbenzamido)methyl)pyrrolidine-1-carboxylate. Synthesized by gen-
eral procedure D. 63% yield, yellow oil; 1H NMR (400 MHz, CDCl3) δ
9.24 (s, 1H), 8.43 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.0 Hz,
2H), 3.80 (s, 1H), 3.72−3.62 (m, 1H), 3.56 (d, J = 14.0 Hz, 1H), 3.17 (s,
2H), 2.59 (t, J = 7.4 Hz, 2H), 2.05−1.93 (m, 2H), 1.92−1.82 (m, 1H),
1.73−1.64 (m, 1H), 1.57 (quin, J = 6.7 Hz, 2H), 1.33−1.20 (m, 10H),
0.87 (t, J = 6.7 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 169.8, 147.9,
130.4, 128.8, 127.5, 60.7, 45.2, 41.4, 36.0, 32.0, 31.3, 29.5, 29.4, 29.4,
27.8, 24.1, 22.8, 14.2; HRMS (ESI+): Calcd for C20H33N2O+ [M+]+:
317.2592, Found: 317.2564.
(R)-Amino(2-((4-decylphenyl)carbamoyl)pyrrolidin-1-yl)-
methaniminium 2,2,2-Trifluoroacetate (29b). Synthesized by
1
general procedure D. 31% yield, white solid; H NMR (400 MHz,
CDCl3) δ 9.83 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H),
4.86 (s, 1H), 3.70−3.64 (m, 1H), 3.50−3.42 (m, 1H), 2.53 (t, J = 7.8 Hz,
2H), 2.40−2.16 (m, 3H), 2.06−1.98 (m, 1H), 1.61−1.50 (m, 2H),
1.32−1.19 (m, 14H), 0.87 (t, J = 6.2 Hz, 3H); 13C NMR (126 MHz,
CDCl3) δ 169.2, 155.3, 139.8, 135.3, 129.0, 120.3, 60.9, 48.3, 35.5, 32.1,
31.7, 31.4, 29.8, 29.8, 29.7, 29.5, 29.4, 22.8, 14.3; HRMS (ESI+): Calcd
for C22H37N4O+ [M + H]+: 373.2967, Found: 373.2949.
(S,Z)-tert-Butyl (((tert-Butoxycarbonyl)amino)(2-((4-
octylbenzyl)carbamoyl)pyrrolidin-1-yl)methylene)carbamate
(30). 4-Octylbenzonitrile 2 (200 mg, 0.93 mmol) was dissolved in
tetrahydrofuran and added dropwise to a solution of lithium aluminum
hydride (106 mg, 2.79 mmol) in tetrahydrofuran (9 mL) at 0 °C. After
the addition, the mixture was warmed to room temperature and stirred
for 0.5−1 h. After completion, the reaction was cooled to 0 °C and
diluted with diethyl ether. Next, 60 μL of water, 60 μL of 15% NaOH
solution, and 180 μL of water were added dropwise in that order. The
reaction was heated back up to room temperature and stirred for 0.5 h.
Next, the reaction was filtered through a Celite plug and the aqueous
layer was washed with diethyl ether 3× and separated. The organic layer
was then washed with brine, dried with sodium sulfate, and concentrated
in vacuo to give (4-octylphenyl)methanamine without further
purification. 1H NMR (400 MHz, CDCl3) δ 7.22 (d, J = 8.1 Hz, 2H),
7.15 (d, J = 8.1 Hz, 2H), 3.83 (s, 1H), 2.59 (t, J = 7.6 Hz, 2H), 1.66−1.54
(m, 2H), 1.37−1.24 (m, 10H), 0.89 (t, J = 6.8 Hz, 3H).
(S)-tert-Butyl 2-((4-Octylbenzyl)carbamoyl)pyrrolidine-1-car-
boxylate. Synthesized using general procedure E. Taken to the next
1
step without further purification, colorless oil; H NMR (500 MHz,
CDCl3) δ 7.16 (d, J = 7.9 Hz, 2H), 7.11 (d, J = 7.8 Hz, 2H), 4.62−16 (m,
3H), 3.50−3.26 (m, 2H), 2.55 (t, J = 7.4 Hz, 2H), 2.40−2.10 (m, 1H),
1.96−1.80 (m, 3H), 1.56 (quin, J = 7.4 Hz, 2H), 1.49−1.19 (m, 19H),
0.87 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 172.4, 155.8,
142.5, 135.5, 128.8, 128.0, 80.7, 61.4, 60.2, 47.2, 43.3, 35.7, 32.0, 31.6,
29.6, 29.4, 29.3, 28.4, 22.8, 14.2. HRMS (ESI+): Calcd for
C25H40N2O3Na [M + Na]+: 439.2936, Found: 439.2937.
(S,Z)-tert-Butyl (((tert-Butoxycarbonyl)amino)(2-((4-
octylbenzamido)methyl)pyrrolidin-1-yl)methylene)carbamate.
Guanylation of (S)-2-((4-octylbenzamido)methyl)pyrrolidin-1-ium
2,2,2-trifluoroacetate. Synthesized by general procedure C. 47% yield,
colorless oil; 1H NMR (400 MHz, CDCl3) δ 10.29 (s, 1H), 7.86 (d, J =
8.3 Hz, 2H), 7.71 (s, 1H), 7.20 (d, J = 8.2 Hz, 2H), 4.61−4.53 (m, 1H),
3.84 (dt, J = 14.0, 3.7 Hz, 1H), 3.76−3.68 (m, 1H), 3.62−3.41 (m, 2H),
2.62 (t, J = 7.6 Hz, 2H), 2.20−2.12 (m, 1H), 1.94−1.86 (m, 1H), 1.82−
1.67 (m, 3H), 1.59 (quin, J = 6.3 Hz, 2H), 1.50 (s, 18H), 1.36−1.18 (m,
10H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 167.6,
155.1, 146.6, 131.8, 128.4, 127.5, 58.6, 50.8, 42.6, 36.0, 32.0, 31.4, 29.6,
29.4, 29.4, 28.9, 28.3, 24.9, 22.8, 14.2; HRMS (ESI+): Calcd for
C31H51N4O5 [M + H] +: 559.3859, Found: 559.3813.
(S)-2-((4-Octylbenzyl)carbamoyl)pyrrolidin-1-ium 2,2,2-Tri-
fluoroacetate. Deprotection of the Boc group in (S)-tert-butyl 2-
((4-octylbenzyl)carbamoyl)pyrrolidine-1-carboxylate. Synthesized
using general procedure D. Taken to the next step without further
1
purification, yellow oil; H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H),
7.69 (s, 1H), 7.09 (dd, J = 8.5, 2.9 Hz, 4H), 4.58 (s, 1H), 4.33 (d, J = 5.2
Hz, 2H), 3.35−3.18 (m, 2H), 2.53 (t, J = 7.7 Hz, 2H), 2.37−2.25 (m,
1H), 2.00−1.84 (m, 3H), 1.55 (quin, J = 7.4 Hz, 2H), 1.33−1.20 (m,
10H), 0.86 (t, J = 6.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 168.62,
142.38, 134.87, 128.76, 127.52, 59.67, 46.33, 43.69, 35.73, 32.01, 31.68,
30.20, 29.60, 29.50, 29.40, 24.52, 22.80, 14.24; HRMS (ESI+): Calcd for
C20H33N2O+ [M + H]+: 317.2592, Found: 317.2591.
1
Deprotection by general procedure D. 47% yield, colorless oil; H
NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.21
(d, J = 8.1 Hz, 2H), 4.05 (s, 1H), 3.54 (s, 1H), 3.35−3.09 (m, 1H), 2.60
(t, J = 7.4 Hz, 2H), 2.13−1.92 (m, 3H), 1.58 (quin, J = 6.9 Hz, 2H),
1.50−1.36 (m, 1H), 1.32−1.22 (m, 10H), 0.87 (t, J = 6.9 Hz, 3H); 13C
NMR (101 MHz, CDCl3) δ 169.2, 155.5, 147.7, 130.5, 128.8, 127.3,
57.9, 47.0, 42.7, 36.0, 32.0, 31.3, 29.8, 29.6, 29.6, 29.4, 29.4, 27.8, 22.8,
22.7, 14.2; HRMS (ESI+): Calcd for C21H35N4O+ [M+]+: 359.2810,
Found: 359.2817.
Guanylation of (S)-2-((4-octylbenzyl)carbamoyl)pyrrolidin-1-ium
2,2,2-trifluoroacetate. Synthesized by general procedure C. 46% yield,
colorless oil; 1H NMR (400 MHz, CDCl3) δ 10.02 (s, 1H), 7.61 (s, 1H),
7.15 (d, J = 8.2 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 4.98 (t, J = 6.8 Hz, 1H),
4.45 (dd, J = 14.8, 6.0 Hz, 1H), 4.32 (dd, J = 14.8, 5.1 Hz, 1H), 3.63 (dt, J
= 11.1, 7.3 Hz, 1H), 3.51−3.44 (m, 1H), 3.54 (t, J = 7.4 Hz, 2H), 2.21 (q,
J = 7.0 Hz, 1H), 2.03−1.79 (m, 3H), 1.56 (quin, J = 6.9 Hz, 2H), 1.43 (s,
18H), 1.31−1.19 (m, 10H), 0.87 (t, J = 6.7 Hz, 3H); 13C NMR (101
MHz, CDCl3) δ 171.0, 155.1, 141.9, 135.4, 128.6, 127.7, 61.5, 49.8, 43.4,
35.7, 32.0, 31.6, 29.6, 29.5, 29.4, 29.0, 28.2, 24.6, 22.8, 14.2; HRMS (ESI
+): Calcd for C31H51N4O5 [M + H]+: 559.3859, Found: 559.3832.
(S)-Amino(2-((4-octylbenzyl)carbamoyl)pyrrolidin-1-yl)-
methaniminium 2,2,2-Trifluoroacetate (31). Synthesized by
general procedure D. 39% yield, yellow oil; 1H NMR (400 MHz,
CDCl3) δ 8.35 (s, 1H), 7.09 (s, 4H), 4.64 (s, 1H), 4.40 (dd, J = 14.5, 5.9
Hz, 1H), 4.20 (dd, J = 9.6 4.8 Hz, 1H), 3.64 (s, 1H), 3.44 (s, 1H), 2.54 (t,
J = 7.8 Hz, 2H), 2.32−2.05 (m, 3H), 2.01−1.93 (m, 1H), 1.56 (quin, J =
tert-Butyl (S)-2-((3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
methyl)pyrrolidine-1-carboxylate (35a). Synthesized by general
1
procedure A. 56% yield, colorless oil; H NMR (400 MHz, CDCl3) δ
7.95 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H), 4.39−4.16 (m, 1H),
3.48−3.22 (m, 3H), 3.15−2.94 (m, 1H), 2.63 (t, J = 7.9, 6.8 Hz, 2H),
2.14−1.95 (m, 1H), 1.85 (m, 3H), 1.60 (dq, J = 12.9, 6.4, 5.4 Hz, 2H),
1.45 (s, 9H), 1.36−1.17 (m, 18H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 177.0, 168.3, 154.1, 146.4, 128.8, 127.3, 124.1,
79.9, 55.1, 46.7, 46.3, 35.9, 31.8, 31.2, 29.4, 29.2, 29.2, 28.4, 22.6, 14.1.
R
DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
HRMS (ESI+): Calcd for C26H39N3NaO3 [M + Na]+: 464.2889, Found:
464.2905.
(S)-Amino(2-((3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
methyl)pyrrolidin-1-yl)methaniminium Chloride (36a). Synthe-
sized by general procedure B. 83% yield, white solid; H NMR (400
1
tert-Butyl (S)-2-(2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)-
MHz, CD3OD) δ 7.94 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 4.52
(m, 1H), 3.53 (m, 1H), 3.48−3.40 (m, 1H), 2.66 (t, J = 7.9, 7.4 Hz, 1H),
2.27 (m, 1H), 2.07 (m, 3H), 1.63 (m, 2H), 1.29 (m, 10H), 0.87 (t, J = 6.9
Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 176.5, 168.2, 155.0, 146.7,
128.7, 126.9, 123.8, 55.8, 35.4, 31.6, 31.0, 30.2, 29.1, 28.9, 28.9, 28.7,
22.3, 22.2, 13.0. HRMS (ESI+): Calcd for C22H34N5O [M + H]+:
384.2758, Found: 384.2743. [α]D = −89.6° (c = 0.005, methanol).
HPLC analysis indicates that compound 36a is 93% pure.
ethyl)pyrrolidine-1-carboxylate (35b). Synthesized by general
1
procedure A. 63% yield, colorless oil; H NMR (500 MHz, CDCl3) δ
7.97 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 3.94 (d, J = 51.5 Hz,
1H), 3.45 (s, 0.5 H), 3.34 (s, 1H), 3.12 (q, J = 6.3 Hz, 0.5 H), 2.95 (dq, J
= 19.8, 8.5, 7.6 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H), 2.37−2.15 (m, 1H),
2.09−1.79 (m, 4H), 1.64 (tq, J = 20.6, 13.1, 9.4 Hz, 4H), 1.45 (d, J = 6.8
Hz, 9H), 1.36−1.20 (m, 10H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (126
MHz, CDCl3) δ 179.6, 168.2, 154.6, 146.4, 128.9, 127.3, 124.3, 79.6,
56.4, 46.2, 40.4, 35.9, 31.8, 31.2, 29.9, 29.4, 29.2, 29.2, 28.7, 28.5, 28.4,
26.6, 26.5, 26.3, 22.6, 14.1. HRMS (ESI+): Calcd for C27H41N3NaO3 [M
+ Na]+: 478.3046, Found: 478.3042.
(S)-Amino(2-(2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)-
ethyl)pyrrolidin-1-yl)methaniminium Chloride (36b). Synthe-
1
sized by general procedure B. 95% yield, white solid; H NMR (500
MHz, CD3OD) δ 7.80 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 4.03
(m, 1H), 3.46 (m, 1H), 3.35−3.27 (m, 1H), 3.07−2.89 (m, 2H), 2.59 (t,
J = 7.5 Hz, 2H), 2.19 (m, 1H), 2.13−1.97 (m, 3H), 1.91 (m, 2H), 1.55
(m, 2H), 1.33−1.12 (m, 10H), 0.79 (t, J = 7.0 Hz, 3H). 13C NMR (126
MHz, CD3OD) δ 179.8, 167.8, 154.8, 146.7, 128.8, 126.8, 123.8, 57.6,
35.4, 31.6, 31.0, 29.3, 29.1, 29.0, 28.9, 28.1, 22.4, 22.3, 22.2, 13.0. HRMS
(ESI+): Calcd for C23H36N5O [M + H]+: 398.2914, Found: 398.2905.
HPLC analysis indicates that compound 36b is 89% pure.
(S)-2-((3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)methyl)-
pyrrolidin-1-ium Chloride. Deprotection of Boc group in tert-butyl
(S)-2-((3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidine-1-
carboxylate. Synthesized by general procedure B. 85% yield, white solid;
1H NMR (400 MHz, CD3OD) δ 8.00 (d, J = 7.9 Hz, 2H), 7.32 (d, J = 7.9
Hz, 2H), 4.18 (s, 1H), 3.61−3.47 (m, 2H), 3.42 (t, J = 6.7 Hz, 2H), 2.67
(t, J = 7.8 Hz, 2H), 2.47−2.36 (m, 1H), 2.12 (dd, J = 20.8, 13.5 Hz, 2H),
1.93 (d, J = 8.4 Hz, 1H), 1.71−1.57 (m, 2H), 1.39−1.22 (m, 10H), 0.88
(t, J = 7.2, 6.7 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 177.4, 169.5,
148.1, 130.1, 128.4, 125.1, 58.4, 47.1, 36.8, 33.0, 32.4, 31.5, 30.5, 30.3,
30.3, 29.8, 24.7, 23.7, 14.4. HRMS (ESI+): Calcd for C21H32N3O [M +
H]+: 342.2540, Found: 342.2537.
(S)-2-(2-(3-(4-Octylphenyl)-1,2,4-oxadiazol-5-yl)ethyl)-
pyrrolidin-1-ium Chloride. Deprotection of Boc group in tert-butyl
(S)-2-(2-(3-(4-octylphenyl)-1,2,4-oxadiazol-5-yl)ethyl)pyrrolidine-1-
carboxylate. Synthesized by general procedure B. 90% yield, white solid;
1H NMR (500 MHz, CD3OD) δ 7.96 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.1
Hz, 2H), 3.73 (q, J = 7.6, 6.9 Hz, 1H), 3.44−3.30 (m, 2H), 3.18 (t, J = 7.5
Hz, 2H), 2.98 (dt, J = 7.5 Hz, 1H), 2.68 (t, J = 7.8, 6.7 Hz, 2H), 2.36
(ddd, J = 14.1, 7.1 Hz, 2H), 2.10 (ddt, J = 37.7, 13.2, 8.2 Hz, 1.5H), 1.91
(q, J = 7.1 Hz, 0.5H), 1.84−1.57(m, 3.5H), 1.55−1.45 (m, 2H), 1.40−
1.23 (m, 10H), 0.90 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, CD3OD)
δ 180.2 (rotamer 1), 178.6 (rotamer 2), 168.1(rotamer 1), 168.0
(rotamer 2), 146.6 (rotamer 1), 146.5 (rotamer 2), 128.7 (rotamer 1),
128.7 (rotamer 2), 126.9 (rotamer 1), 126.9 (rotamer 2), 124.1
(rotamer 1), 124.0 (rotamer 2), 59.7, 45.0, 39.3, 35.5, 31.6, 31.1, 29.6,
29.1(rotamer 1), 29.0 (rotamer 2), 28.9, 28.2 (rotamer 1), 28.1 (rotamer
2), 26.9, 25.9, 25.7, 25.6, 23.2, 23.0, 22.3, 13.1. HRMS (ESI+): Calcd for
C22H34N3O [M + H]+: 356.2696, Found: 356.2703.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for compounds 2−36b. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 540-231-5742. E-mail: santosw@vt.edu.
Author Contributions
#These authors (N.N.P. and E.A.M.) contributed equally. The
manuscript was written through contributions of all authors. All
authors have given approval to the final version of the
manuscript.
Notes
The authors declare the following competing financial
interest(s): W.L.S. and K.R.L. are among the co-founders of
SphynKx Therapeutics LLC, which was created to commercialize
S1P-related discoveries, including SphK inhibitors, discovered
and characterized in their laboratories. The compounds
described in this manuscript are included in a patent application
licensed to SphynKx..
tert-Butyl (S,E)-(((tert-Butoxycarbonyl)amino)(2-((3-(4-octyl-
phenyl)-1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-yl)-
methylene)carbamate. Guanylation of (S)-2-((3-(4-octylphenyl)-
1,2,4-oxadiazol-5-yl)methyl)pyrrolidin-1-ium chloride. Synthesized by
1
general procedure C. 71% yield, colorless oil; H NMR (400 MHz,
CDCl3) δ 10.28 (s, 2H), 7.97 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 6.1 Hz,
2H), 4.76 (dt, J = 11.3, 5.7 Hz, 1H), 3.72−3.58 (m, 2H), 3.48 (s, 1H),
3.12 (dd, J = 15.1, 8.4 Hz, 1H), 2.63 (t, J = 8.2, 6.5 Hz, 2H), 2.29−2.17
(m, 1H), 1.89 (ddd, J = 10.1, 7.4, 4.6 Hz, 1H), 1.85−1.74 (m, 2H), 1.61
(quin, J = 7.3 Hz, 2H), 1.45 (s, 18H), 1.35−1.18 (m, 10H), 0.86 (t, J =
5.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 176.9, 168.2, 154.0, 146.3,
128.8, 127.4, 124.2, 56.4, 50.0, 35.9, 31.8, 31.6, 31.2, 30.5, 30.3, 29.4,
29.2, 28.1, 25.2, 22.6, 14.1, 14.1. HRMS (ESI+): Calcd for
C32H49N5NaO5 [M + Na]+: 606.3631, Found: 606.3700.
ACKNOWLEDGMENTS
■
The work was supported by NIH Grants R01 GM104366 and
R01 GM067958.
ABBREVIATIONS USED
■
Sph, sphingosine; S1P, sphingosine-1-phosphate; SphK, sphin-
gosine kinase
tert-Butyl (S,Z)-(((tert-Butoxycarbonyl)imino)(2-(2-(3-(4-oc-
tylphenyl)-1,2,4-oxadiazol-5-yl)ethyl)pyrrolidin-1-yl)methyl)-
carbamate. Guanylation of (S)-2-(2-(3-(4-octylphenyl)-1,2,4-oxadia-
zol-5-yl)ethyl)pyrrolidin-1-ium chloride. Synthesized by general
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DOI: 10.1021/jm501760d
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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