Development of β-amino alcohol derivatives that inhibit toll-like receptor 4 mediated inflammatory response as potential antiseptics

Article abstract of DOI:10.1021/jm2003365

Toll-like receptor 4 (TLR4) induced proinflammatory signaling has been directly implicated in severe sepsis and represents an attractive therapeutic target. Herein, we report our investigations into the structure-activity relationship and preliminary drug metabolism/pharmacokinetics study of β-amino alcohol derivatives that inhibit the TLR4 signaling pathway. Lead compounds were identified from in vitro cellular examination with micromolar potency for their inhibitory effects on TLR4 signaling and subsequently assessed for their ability to suppress the TLR4-induced inflammatory response in an ex vivo whole blood model. In addition, the toxicology, specificity, solubility, brain-blood barrier permeability, and drug metabolism of several compounds were evaluated. Although further optimizations are needed, our findings lay the groundwork for the future drug development of this class of small molecule agents for the treatment of severe sepsis.

Full text of DOI:10.1021/jm2003365

ARTICLE
pubs.acs.org/jmc
Development of β-Amino Alcohol Derivatives That Inhibit Toll-like
Receptor 4 Mediated Inflammatory Response as Potential Antiseptics
||
||
Sherry A. Chavez,†, Alexander J. Martinko,†, Corinna Lau,‡ Michael N. Pham,† Kui Cheng,†
Douglas E. Bevan,† Tom E. Mollnes,‡,§ and Hang Yin*,†
†Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309, United States
‡Research Laboratory, Nordland Hospital, Bodø NO-8092, Norway
§University of Tromsø, Tromsø, NO-9037, Norway
S Supporting Information
b
ABSTRACT: Toll-like receptor 4 (TLR4) induced proinflammatory signal-
ing has been directly implicated in severe sepsis and represents an attractive
therapeutic target. Herein, we report our investigations into the structureꢀ
activity relationship and preliminary drug metabolism/pharmacokinetics
study of β-amino alcohol derivatives that inhibit the TLR4 signaling path-
way. Lead compounds were identified from in vitro cellular examination with
micromolar potency for their inhibitory effects on TLR4 signaling and
subsequently assessed for their ability to suppress the TLR4-induced
inflammatory response in an ex vivo whole blood model. In addition, the
toxicology, specificity, solubility, brainꢀblood barrier permeability, and drug
metabolism of several compounds were evaluated. Although further opti-
mizations are needed, our findings lay the groundwork for the future drug development of this class of small molecule agents for the
treatment of severe sepsis.
’ INTRODUCTION
has been directly implicated in a myriad of diseases including
sepsis and neuropathic pain.6ꢀ9 Severe sepsis poses a particular
threat as a serious medical condition characterized by a systemic
inflammation state that results in over 200 000 attributed deaths
annually in the United States.10 Several groups have attempted to
design potent antisepsis agents by employing the strategy of
developing LPS-mimicking antagonists of TLR4.11,12 One ad-
vantage of deriving inhibitors by this method is that antagonists
can be developed by rationally modifying the active glycolipid
component of LPS to optimize drug efficacy. The disadvantage
posed is that these LPS mimetics are typically too large to render
the desired pharmacological properties.13 A recent example of
such a design is eritoran, a lipid-A mimetic with high affinity for
TLR4. Although it reached phase III clinical trials as an antisepsis
agent, eritoran failed to demonstrate sufficient efficacy in late
stage human trials. An alternative strategy, which has the poten-
tial for high specificity and bioavailability, is to design low-molecular-
weight inhibitors.14,15 By use of this strategy, several small mole-
cules have been developed to target and antagonize TLR4. To
the best of our knowledge, none have successfully advanced
through late stage clinical trials.16ꢀ18 TAK-242 (resatorvid), the
most successful TLR4-binding small molecule antagonist, reached
phase III clinical trials as an antisepsis agent, but studies were
recently discontinued because of a failure to suppress cytokine
The protective functions of innate immunity rely principally
on the ability of an organism to discriminate between endogen-
ous signals and exogenous danger signals of microbial origin.1,2
Toll-like receptors (TLRs) are a conserved family of integral
membrane proteins, which maintain this essential role of innate
immunity. At least 10 TLRs have been identified in human and
13 in mice, each of which recognizes its own group of conserved
pathogen associated molecular patterns (PAMPs).
Toll-like receptor 4 (TLR4), the first of the TLR family to be
identified, along with its accessory protein myeloid differentia-
tion factor 2 (MD2), forms a heterodimeric complex. This
complex specifically recognizes lipopolysaccharide (LPS), a
structurally variant component of Gram-negative bacterial cell
walls.3,4 Once LPS is recognized, a homoheterodimer consisting
of two TLR4ꢀMD2ꢀLPS complexes is formed. This homo-
heterodimer confers an intracellular signaling cascade that results
in downstream activation and nuclear translocation of the
transcription factor, nuclear factor kB (NF-kB).5 Nuclear trans-
location of NF-kB results in subsequent transcriptional up-
regulation of proinflammatory cytokines such as interleukin 1 (IL-1),
interleukin 6 (IL-6), and tumor necrosis factor-R (TNF-R).2,5
Given the key role TLR4 plays in regulating cytokines, inhibition of
TLR4-mediated signaling is an appealing target for therapeutic
development.
Although TLR4-induced cytokine signaling is generally ad-
vantageous in fighting infection, TLR4 signaling dysregulation
Received: March 23, 2011
Published: May 17, 2011
r
2011 American Chemical Society
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levels in patients despite showing promising preclinical efficacy in
animal models.19ꢀ21 These results might suggest that the current
approaches to targeting the TLR4 receptor are flawed. There is
an urgent need to develop and validate novel strategies to target
the TLR4 pathway for antisepsis therapeutic development.
Using an in silico screen, we have identified β-amino alcohol
derivatives (1) as generic inhibitors that disrupt the TLR4/MD2
complex formation.22,23 These inhibitors were shown to be
effective at suppressing NF-kB activation in TLR4-overexpres-
sing human embryonic kidney (HEK) 293.22 Herein, we further
these studies on the preparation, optimization, and biological
evaluation of β-amino alcohol derivatives that inhibit LPS-
induced inflammation in whole blood. Our findings may lay
the groundwork for a new drug development strategy for the
treatment of severe sepsis by targeting the TLR4/MD2 interface
and suggest a class of prototype small molecule agents.
fragment of type 3, a Mannich-type reaction was utilized to
further functionalize a 3,5-dimethyl-1H-pyrazole intermediate
where paraformaldehyde provided the methylene source and
methylamine hydrochloride.22
The desired arylalkoxyoxiranes (2) were synthesized via the
reaction of the sodium salt of substituted phenols (4) with
epichlorohydrin (Scheme 1).24 Selective incorporation of an
allyl ether group was accomplished starting from hydroquinone
(R = OH, K2CO3, allyl bromide, acetone) to yield the epoxide 2c
after addition to epichlorohydrin.25
The functionalized heterocycles of type (3) were synthesized
using previously established methodology.21Preparation of ana-
logues containing a styrene functionality was achieved using
(2-bromobenzyl)pyrazole (6f). A palladium-catalyzed Stille cou-
pling, using Pd(PPh3)4 and tributylvinyltin in toluene, generated
the styrenepyrazole intermediate (6g).26 A Mannich coupling
with methylamine and paraformaldehyde formed the amine
derivative 3f.
’ INHIBITOR PREPARATION
Preparation of the β-aminohydroxyl compounds was carried
out by condensing amino substituted pyrazoles with the arylox-
yoxiranes. We found that by subjecting 1 equiv of an arylalk-
oxyoxirane (2) and a slight excess (1.1 equiv) of the
functionalized secondary amine (3) to different reaction condi-
tions, we could improve the isolated yields of the β-aminohy-
droxyl compounds. Because of the limited solubility of many of
the starting materials, ethanol was commonly used as the solvent.
Unfortunately, we frequently observed the addition of ethanol to
the oxirane under refluxing conditions. We successfully sup-
pressed this competing reaction in several reactions with the use
of either acetonitrile27 or toluene.28
Synthesis of β-Amino Alcohol Derivatives. A β-amino
alcohol of type 1 can be derived from a substituted arylalkoxy
epoxide of type 2 and a functionalized pyrazole compound such
as 3 (Figure 1). The epoxide fragments 2 were derived from
epichlorohydrin and a phenol derivative, 4. To generate an amine
Flitsch and co-workers reported good yields of amino alcohols
via the ring-opening of epoxides with a variety of amines under
microwave irradiation conditions.29 Conducting the coupling
reaction under similar conditions (140 °C, 200 W) using a variety
of solvent systems resulted in a significant decrease in reaction
times (24ꢀ48 h to less than 1 h) and generated excellent yields.
We found that either acetonitrile or EtOH worked equally well
under the microwave irradiation conditions with only minor
traces of the EtOH addition products.
In order to explore the role the alcohol group of 1 plays in
binding to TLR4, compound 1j was methylated utilizing the
standard conditions (MeI, NaH) to generate the methyl ether 7.
Figure 1. Retrosynthesis of pyrazole containing β-amino alcohols.
Scheme 1. Synthesis of β-Amino Alcohol Derivativesa
a Method A = EtOH, reflux. Method B = MeCN, K2CO3, reflux. Method C = toluene, reflux. Method D = EtOH, microwave 200 W, 140 °C. Method
E = MeCN, K2CO3, microwave 200 W, 140 °C. See Experimental Section for specific methods and yields.
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Synthesis of Chiral Derivatives. In order to determine if the
stereochemistry of the hydroxyl group was essential to the
ligand/receptor interaction, the enantiomers of 1s (R = p-Cl,
R1 = o-Cl) were synthesized (Scheme 2). Previously synthesized
enantiomers of 1j (R = p-OEt, R1 = o-Cl) were also prepared for
direct comparison in the current biological assays. Published
reports utilized Jacobsen’s hydrokinetic resolution methodology
to generate enantioenriched epoxides that could then be opened
with the secondary amine nucleophile.30 The active (R,R)-
(salen)Co(III)OAc catalyst was generated in situ by the aerobic
oxidation of [(R,R)-N,N0-bis(3,5-di-tert-butylsalicylidene)-1,2-
cyclohexanediamino)]cobalt(II) in the presence of acetic acid.
Treatment of the desired racemic epoxides (2b and 2d) with
(R,R)(salen)Co(III)OAc catalyst and water yielded the (S)-
enantioenriched epoxides (2h, R = p-OEt and 2j, R = p-Cl)
and the ring opened (R)-diol products. The corresponding
(S,S)(salen)Co(III)OAc catalyst was utilized to generate the
desired (R)-epoxides (2i, R = p-OEt and 2k, R = p-Cl).31,32
We later found that quenching the reaction with pyridinium
p-toluenesulfonate (PPTS) (4 equiv of PPTS/mmol of cat-
alyst) in a 1:1 mixture of MeCN and CH2Cl2 removed the
catalyst prior to purification and increased the chiral epoxide
yields.33,34
Generation of the chiral β-amino alcohols (1l, 1k, 1u, and
1t) was accomplished by using similar reaction conditions to
their racemic counterparts. We found that both toluene and
acetonitrile were effective solvents for these coupling. The
addition of a heterogeneous base K2CO3 increased the overall
yields, and the use of microwave conditions did not affect the
enantioselectivity or the yields (Scheme 3). The enantiomeric
excess was determined to be greater than 96% by chiral HPLC
analysis for each of the derivatives regardless of the reaction
conditions.
one of our more active linear compounds, 1q, and is properly
substituted to undergo ring closing metathesis through a con-
venient annulation method (Scheme 4). The desired macrocyclic
compound 8 was synthesized by cyclizing the linear precursor 1q
using Grubbs II catalyst with a 52% yield and greater than 1:10 E/
Z selectivity after purification.36
’ BIOLOGICAL EVALUATION
Inhibition of the LPS-Induced TLR4 Activation in Macro-
phage Cells. The potency of the β-amino alcohol derivatives as
inhibitors of TLR4 signaling was determined by monitoring the
reduction of LPS-induced production of nitric oxide (NO) in
RAW 264.7 macrophage cells. The IC50 values and the margin of
error were determined by a modified Kou-type method from
experiments conducted in quadruplicate.37
The compound that lacked substitution on either of the aryl
rings (e.g., 1a, R = R1 = H) was ineffective at suppressing NO
signaling (Table 1). For this reason, compound 1a was utilized as
a negative control for this assay. Mild suppression of NO
production was observed when substitutions were made to either
of the benzyl rings (1e IC50 = 51.8 ( 5.8 μM and 1b IC50
=
64.3 ( 0.1 μM), suggesting that substitution of both aryl rings is
needed for activity.
The small structural variations between compounds 1b, 1c, 1e,
and 1j and the corresponding biological activity gave preliminary
indications that a combination of electronic properties and
lipophilicity may play a role in improving the biological activity.
Several additional compounds were synthesized to further ex-
plore the effect of substitution located on the aromatic ring
derived from the epoxide fragment.
By use of 1j as the prototype compound (R = p-OEt, R1 = o-Cl:
IC50 = 27.8 ( 0.3 μM), several analogues were prepared in which
the R group was modified to explore the influence of electronics
and lipophilicity. Synthesis of derivative 1v, which incorporated
an additional chloride substitution (R = 3,4-dichloro-, R1 = o-Cl),
resulted in a slight decrease of activity from compound 1s (R =
p-Cl, R1 = o-Cl: IC50 = 16.1 ( 1.1 μM). Increasing the
lipophilicity (R = p-CF3, R1 = o-Cl, 1y) generated a slight loss
of NO suppression compared with that of 1s. Interestingly,
significant loss of activity was observed with compound 1z where
R = p-CN (IC50 = 52.7 ( 2.6 μM).
Synthesis of Cyclic Derivatives. A commonly used strategy
to improve the potency of flexible small molecule inhibitors is to
prepare the cyclic derivatives with increased rigidity, which
reduces the entropic penalty upon binding to their protein
receptor.35 The macrocycle scaffold (8) was designed based on
Scheme 2. Synthesis of Chiral Epoxides
On the basis of the current SAR set, it would appear that an
electron withdrawing group verses an electron donating group
on the phenoxy ring at the para position favors activity (e.g., 1s
[R = p-Cl] > 1j [R = p-OEt] > 1a [R = H]). Although there
seems to be a slight dependence on the electronic nature of
the aromatic ring, activity appears to be influenced more by
increased lipophilicity.
Scheme 3. Synthesis of Enantioenriched β-Amino Alcohol
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Scheme 4. Synthesis of a Cyclic Derivative
Table 1. Suppression of the LPS-Induced NO Production in
RAW 264 Macrophage Cells
R1 = H). Addition of an o-OMe group (1g) gave a compound
with no detectable NO suppression. Although relocating the
methoxy group to the para position (1h) regenerated some
activity (IC50 = 67.6 ( 0.3 μM) the increase in potency was not
significant enough to justify further exploration of electron
donating groups on the amine fragment.
Modification of o-R1 to a fluorine substituent (1i) also did not
improve activity. However, incorporation of a more lipophilic,
electron withdrawing group such as chlorine (1j) reduced the
IC50 to nearly half of that seen with fluorine (47.7 ( 2.7 μM
compared to 27.8 ( 0.3 μM). Relocating the chlorine substituent
to the meta position (1m) further improved the IC50 to 16.5 (
0.8 μM, which is comparable to the IC50 of compound 1s.
Introducing disubstitution, e.g., R1 = 3,4-dichloro (1v), however,
did not significantly affect the NO suppression compared to the
monochloro derivative.
As seen with the modifications made to phenol group, the
addition of an o-CF3 group resulted in a modest decrease in
activity (1p, IC50 = 31.1 ( 2.2 μM) compared to that of 1j.
Interestingly, when a vinyl group (1o) was incorporated into the
structure, the inhibitory potency was reduced to an IC50 of
24.7 ( 9.0 μM. These results imply that the ortho position may
be more sensitive to sterics than to electronics.
The effect of the hydroxyl group’s stereochemistry on NO
production was also investigated. Surprisingly, no significant
difference in activity between the racemic derivatives and their
stereoisomers (1j, 1k, and 1l) and (1s, 1t, and 1u) was observed.
The results suggest that the role of the alcohol in the small
moleculeꢀprotein interaction remains unclear and may not be
critical for potency. This hypothesis was supported by the methyl
ether analogue 7. The ether derivative demonstrated reasonable
activity with an IC50 of 19.8 ( 0.1 μM, implying that an alcohol
substitution may not be essential but the CꢀOꢀ functionality
may be important.
/
R
R1
R2
IC50 (μM)
1a
1b
1c
1d
1e
1f
(
H
H
H
H
>100
(
o-Cl
o-Cl
o-Cl
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
64.3 ( 0.1
57.3 ( 2.8
92.5 ( 0.8
51.8 ( 5.8
47.7 ( 3.7
>100
(
p-CH3OCH2CH2-
o-CH3O-
(
(
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH3CH2O-
p-CH2dCHCH2O-
p-CH2dCHCH2O-
p-Cl-
(
o-CH3
o-OCH3
p-OCH3
o-F
1g
1h
1i
(
(
67.6 ( 0.3
47.7 ( 2.7
27.8 ( 0.3
24.8 ( 3.0
24.3 ( 3.7
19.8 ( 0.1
16.5 ( 0.8
21.8 ( 3.1
24.7 ( 9.0
32.1 ( 2.2
23.1 ( 1.1
44.6 ( 7.0
16.1 ( 1.1
15.5 ( 0.9
15.6 ( 0.6
20.1 ( 0.1
23.2 ( 0.7
27.3 ( 0.0
23.7 ( 2.6
52.7 ( 2.6
(
1j
(
o-Cl
1k
1l
(R)
(S)
(
o-Cl
o-Cl
7
o-Cl
1m
1n
1o
1p
1q
1r
1s
1t
(
m-Cl
(
3,4-dichloro
o-CHdCH2
o-CF3
o-CHdCH2
o-Cl
(
(
(
(
(
o-Cl
(R)
(S)
(
p-Cl-
o-Cl
1u
1v
1w
1x
1y
1z
p-Cl-
o-Cl
3,4-dichloro
p-Cl-
o-Cl
An interesting result was obtained with the macrocyclic
(
o-CHdCH2
o-CF3
o-Cl
compound 8. Compared to the acyclic system (1q, IC50
23.1 ( 1.1 μM), the macrocycle retained its potency (IC50
=
=
(
p-CF3-
(
p-CF3-
16.0 ( 2.0 μM), suggesting the possibility of an alternative scaffold.
Inhibition of Inflammatory Response in Human Whole
Blood. The biological properties of compounds 1j and 1a were
further investigated in an effort to determine if this class of
compounds would be suitable for further studies as antisepsis
agents.38 In human whole blood, 1j and the control compound
1a showed differential efficiency in inhibiting the LPS-induced
cytokine release. Compound 1j was more effective at reducing
IL-8 secretion than 1a, and this difference was significant at
(
p-CN-
o-Cl
The effect of substitution on the aromatic ring attached to
the pyrazole ring was also studied. Starting again from 1j as a
baseline, the R group was maintained as the p-OEt functionality
and the R1 substituent was varied. The use of a methyl group at
the ortho position (1f) resulted in a compound with activity
similar to that of the monosubstituted compound 1e (R = p-OEt,
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Figure 2. (A) Dose-dependent inhibitory effects of 1a and 1j on LPS-mediated cytokine release in human whole blood. (B) Inhibitory effects of
compounds 1a and 1j at a fixed concentration of 250 μM on LPS-induced cytokine release in human whole blood. Significant difference between the two
compounds was observed for inhibiting IL-8 and TNFR but not for IL-6. (** p < 0.01, * p < 0.05, t test).
Figure 3. Kinase selectivity screen for 1j (28 μM) and 1s (16 μM). Both compounds were profiled against a panel of 12 kinases using the KinaseSeeker
assay. (A) Treatment with 1j resulted in retention of >95% activity for a majority of the kinases, and the most inhibited kinase, PKC-γ, was inhibited by
22.4%. (B) Treatment with 1s also resulted in the retention of 95% activity for a majority of the kinases, and the most inhibited kinase, MARK1, was
inhibited by 26.7%.
concentrations of 50 and 250 μM (p < 0.05, t test) (Figure 2A and
Figure 2B). Suppression of TNFR release was also observed for 1j at
a dose of 250 μM. However, at 500 μM, both compounds reduced
the levels of all of the studied cytokines equally well, suggesting that
nonspecific inhibition becomes prevalent at this concentration.
These inhibitory affects by 1j have also been shown to be
cytokine-specific. For example, 1j and 1a had no observable
differences in their inhibitory effects on IL-6 cytokine release.
Compound 1j reduced the plasma levels of IL-8 to 35% and
those of TNFR and IL-6 to 57% and 50%, respectively, at
250 μM. These data are consistent with the estimated IC50
values for compound 1j of 110.5 μM for IL-8, 315.6 μM for
TNF-R, and 318.4 μM for IL-6 using the human whole blood
model and the secretion of the respective cytokines. The elevated
IC50 values seen in the whole blood assay compared to the results
derived from the cell-culture based assays are perhaps due to the
complexity of the ex vivo whole blood system. Consistent with
the in vitro assay results, the control compound 1a showed a
significantly lower potency to inhibit the inflammatory response
in the whole blood model. Importantly, no agonistic effects with
respect to cytokine release or hemolysis in the presence of up to
1 mM of either compound were observed. Taken together, our
observations imply that 1j is effective to suppress LPS-induced
inflammatory response in human whole blood.
Drug Metabolism/Pharmacokinetics (DMPK). To prelimi-
narily evaluate the drug potential of 1j, the metabolic stability,
bloodꢀbrain barrier (BBB) permeability, and lipophilicity were
investigated. To determine the lipophilicity of 1j, the octanol/
water distribution coefficient was measured at the physiologically
relevant pH of blood serum (pH 7.4). The log D of a drug at
pH 7.4 is known as a predictor of ADME/TOX properties. The
log D of 1j was determined to be 3.36, a value marginally above
the optimum for drug development but still within a functional
range, showing potential for a balance of permeability and
solubility.39,40
The BBB permeability of 1j was evaluated using the MDR1-
MDCK permeability assay, a cell culture model system shown to
be an accurate predictor of BBB penetration potential. To
determine penetration efficiency, the ability of 1j to pass through
an MDR1-MDCK monolayer in both the apical to basolateral
and basolateral to apical directions was measured. At 5 μM, 1j
was determined to have a brainꢀblood barrier permeability
classification of “high”.
The metabolic stability of 1j was also evaluated. The intrinsic
clearance (CLint) values of 1j (1 μM) were determined in the
presence of human liver microsomes and human S9 fractions as
well as rat S9 fractions. The observed values of CLint in each
system were determined to be >0.70, 0.3, and >0.35 (mL/min)/mg
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protein, respectively. These data suggest that 1j is likely to be
metabolized in the liver at a relatively rapid rate. Generally, rapid
metabolism for a drug candidate is undesirable, and this issue
should be addressed as SAR studies continue.
Further, a preliminary cytochrome P450 (CYP) screening was
also conducted. Compound 1j was tested for its ability to inhibit
four key cytochrome enzymes involved in drug metabolism. The
IC50 values for CYP1A2, CYP2C9, CYP2C19, and CYP2D6
were found to be greater than 100, 43, 20, and 32 μM,
respectively. These data, in conjunction with the metabolism
results, indicate that further optimization of the specificity of 1j is
needed before advancement to more clinically relevant in vivo
models can be considered.
RAW264.7 cells were then planted in 96 -well plates at 100 000 cells per
well and grown for 24 h in the medium described previously.43,44 After
24 h, the medium was removed and replaced with unsupplemented
RPMI (Invitrogen, Carlsbad, CA). Lanes were doped with the TLR
agonists and simultaneously doped with small molecule drugs as specified
for each experiment. Plates were then incubated at 37 °C for 18ꢀ24 h.
Following 18ꢀ24 h of incubation, 100 μL of medium was removed and
added to flat black 96-well Microfluor plates (ThermoScientific, Hudson,
NH). An amount of 10 μL of 2,3-diaminonaphthalene (0.05 mg/mL in
0.62M HCl) wasadded to each well and incubatedfor 15 min. Then 5 μL
of 3 M NaOH was added to stop the reaction and the plate was read on a
Beckman Coulter DTX880 reader with excitation set at 365 nm and
emission analyzed at 450 nm. Nitrite concentration was determined from
nitrite standard curve.
Specificity and Toxicity Tests. The β-amino alcohol deriva-
tives were further examined for their specificity to TLR4 and
screened against 12 representative kinases (AKT1, CAMK1,
DDR2, GSK-3R, MAPK1, MET, PAK1, PDGFRB, PIM1, PKC-γ,
PLK4, and SRC). The two active lead compounds, 1j and 1s,
were screened (Figure 3) using a KinaseSeeker assay (a luciferase
fragment complementation assay that provides a direct measure-
ment of their interactions with each respective kinase). Neither
1j nor 1s inhibited a kinase by more than 27% at their respective
IC50 concentrations determined in the RAW264.7 macrophage
cells, while the majority of the kinases examined retained >95% of
their activity. The results suggest that the active dose of 1j or 1s
required for TLR4 inhibition does not substantially perturb the
activity of these biologically essential enzymes.
Toxicity assessments for the β-amino alcohol derivatives were
carried out using a variety of methods. We have observed that the
cell toxicity was variable and cell line dependent (Supporting
Information Figures 1 and 2). Most importantly, negligible
hemolysis was observed at concentrations as high as 1 mM in
human whole blood.
Trypan Blue Toxicity Assay. Human embryonic kidney 293
(HEK293) cells were stably transfected with TLR4 and necessary
assembly and signaling proteins (MD2, CD-14, LPSBP, etc.). Cells
were cultured in DMEM supplemented with 10% FBS, penicillin (100
U/mL), streptomycin (100 mg/mL), L-glutamine (2 mM), 1 mg/mL
normocin (InvivoGen, San Diego, CA), and 2 units of HEK-Blue
Selection solution (InvivoGen, San Diego, CA). Cells were implanted
in 6 cm plates and grown to 65ꢀ85% confluency by incubating at 37 °C
prior to drug treatment. On the day of treatment, the medium was
removed from the 6 cm plate and replaced with CSF buffer supple-
mented with drug treatment. After 24 h of incubation at 37 °C, CSF was
removed and cells were agitated with 1ꢁ trypsin and resuspended in
fresh DMEM supplemented medium. After resuspension, a 100 μL
sample was taken from each 6 cm plate, mixed gently with 100 μL of
Trypan blue, and allowed to sit for 5 min. The ratio of blue stained cells
to total cells was then quantified using a Bright Line 0.1 mm depth
hemocytometer under a Nikon TMS light microscope.
Whole Blood Cytokine Release Assay. The following studies
were approved by the regional ethics committee in Norway. Human
whole blood was drawn from healthy individuals after giving their
written consent. The whole blood model of sepsis has been described
in detail previously.38 Briefly, blood was drawn from healthy human
donors into 4.5 mL cryotube vials (Nunc, Roskilde, Denmark) contain-
ing lepirudin (Refludan, Pharmion, Copenhagen, Denmark) in a final
concentration of 50 μg/mL. Lepuridin is a highly specific thrombin
inhibitor preventing coagulation only, not interfering with other biolo-
gical activities. Small molecule drugs or PBS with Ca2þ and Mg2þ
(Sigma, D8662) were administered to 1.8 mL cryotube vials prior
to blood sampling. Immediately after the sample was obtained,
blood was added to each of the respective tubes and preincubated
with inhibitors at 37 °C for 5ꢀ10 min. Afterward they were supplied
with 100 ng/mL phenol-extracted ultrapure LPS (upLPS) from E. coli
0111:B4 (InvivoGen, San Diego, CA, U.S.) or endotoxin-free PBS with
Ca2þ and Mg2. Samples were incubated, rotating at 37 °C for 2 h. Then
ethylenediaminetetraacetic acid (EDTA) (Sigma) was added to a final
concentration of 10 mM, and the samples were centrifuged 15 min at
3220g, 4 °C. Plasma was obtained and stored at ꢀ80 °C until further
analysis.
’ CONCLUSIONS
We have reported initial results regarding the development of
a series of low molecular weight β-amino alcohol derivatives that
appear to suppress TLR4-mediated inflammation response.
Preliminary structureꢀactivity relationship studies revealed that
variation of functionality on both aryl rings could increase the
potency. As part of our evaluation process, we have also demon-
strated that these compounds are capable of suppressing LPS-
induced inflammatory response in both macrophage cells and a
human whole blood ex vivo model.
Although the potency values of the compounds described
herein are modest, it should be noted that they presumably
function by disrupting the association of TLR4 and MD2.23
-
Energetically, it is a challenging goal to compete with two protein
binding partners and block their association with small molecule
agents.15,41,42 The micromolar inhibitory activities observed with
the β-amino alcohol derivatives are promising starting point for
further drug development optimization.
It is anticipated that as improvements in potency, metabolic
stability, and safety occur, these β-amino alcohol derivatives will
serve as useful probes in studying TLR4-mediated inflammation
and could pave the way for future antisepsis therapeutics.
Plasma levels of TNF-R, IL-8, and IL-6 were determined using
singleplex Bio-plex cytokine kits from BioRad (Hercules, CA, U.S.).
The analyses were performed according to the manufacturer’s recom-
mendations. The plasma cytokine concentration measured upon LPS
stimulation in the absence of inhibitor was set to 100%. For statistical
analyses, based on the data set of n = 3 (Figure 2A) or n = 9 (Figure 2B),
a two-sided, paired Student’s t test was performed based on single data
points of % plasma cytokine levels.
Metabolic Stability. Metabolic Stability in Human Liver Micro-
somes. Compound 1j (1 μM) with <0.25% DMSO was incubated at
37 °C in buffer with 0.5 mg/mL microsomal protein. The reaction was
initiated by the addition of cofactors at five different time points (0, 10,
’ EXPERIMENTAL SECTION
Secreted Nitric Oxide Assay. RAW264.7 cells were grown in RPMI
supplemented with 10% FBS, penicillanꢀstreptomycin, and L-glutamine.
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20, 30, and 60 min). A positive control (5 μM testosterone) was run in
parallel with sampling at 1, 10, and 30 min. After the final time point,
fluorimetry was used to confirm the addition of NADPH to the reaction
mixture. LCꢀMS/MS was used to determine the peak area response
ratio (peak area corresponding to 1j divided by that of an internal
standard) without running a standard curve. The in vitro intrinsic
chromatography (TLC) and nuclear magnetic resonance (NMR). TLC
was performed on Merck Kieselgel 60 F254 plates, eluting with the
solvent indicated, visualized by a 254 nm UV lamp, and stained with an
ethanolic solution of phosphomolybdic acid hydrate. Glassware for
reactions was oven-dried at 125 °C prior to use. Column flash
chromatography was performed using silica gel (SiO2) Premium Rf,
60 Å, 200 ꢁ 400 mesh from Sorbent Technologies. Nuclear magnetic
resonance spectra were acquired on a Bruker spectrometer (300 MHz
for 1H and 75 MHz for 13C) or a Varian Inova-400 (400 MHz for 1H and
100 MHz for 13C). Chemical shifts for 1H NMR spectra are reported in
parts per million (ppm) and referenced to the signal of residual CDCl3 at
7.26 ppm. Chemical shifts for 13C NMR and DEPT spectra are reported
in parts per million (ppm) and referenced to the center line of the
residual CDCl3 triplet at 77.23 ppm. Chemical shifts of the unproto-
nated carbons (“C”) for DEPT spectra were obtained by comparison
with the 13C NMR spectrum. Enantiomeric excess was determined by
chiral HPLC analysis using a Daicel Chiralcel AD-H silica column
(length = 25 cm), eluting with a mobile phase of an indicated percentage
of i-PrOH/hexanes with 0.1% Et2NH and at a flow rate of 0.5 mL/min.
Retention times for the major and minor enantiomers were detected
with Shimdzu SPD-6A UV spectrometric detector at 254 nm. Optical
rotations were obtained on a Jasco P1038 polarimeter (Na D line) using
a microcell with a 1 dm path length. Specific rotations ([R]2D0, unit,
deg cm2/g) are based on the equation R = (100R)/(lc) and are reported
as unitless numbers where the concentration c is in g/100 mL and the
path length l is in decimeters. High resolution mass spectrometry data
were obtained at the mass spectrometer facility of the University of
Colorado Boulder, Department of Chemistry and Biochemistry, on an
ESI-qTOF-MS (electrospray triple quadrupole time-of-flight mass spec-
trometry) spectrometer from Applied Biosystems, PE SCIEX/ABI API
QSTAR Pulsar i Hybrid LC/MS/MS. All compounds tested have a
purity of g95% as determined by TLC, NMR, HRMS, and chiral HPLC
for chiral compounds. Compounds were named using ChemBioDraw
Ultra 11.0.
General Synthetic Procedures for Racemic Epoxides of
Type 2. 2-((4-Chlorophenoxy)methyl)oxirane (2d). Into a
50 mL round-bottom flask, (4-chloro)phenol (1.00 g, 7.78 mmol, 1
equiv) was taken up into acetone (20 mL, 0.4 M), and K2CO3 (3.22 g,
23.3 mmol, 3 equiv) and epichlorohydrin (2.34 mL, 31.1 mmol, 4 equiv)
were added consecutively. The reaction mixture was set to stir at reflux
for 24 h. At this time an additional 4 equiv of epichlorohydrin was added
and the solution was allowed to stir at reflux for an additional 24 h.
The mixture was cooled to room temperature, and the solids were
filtered off. The solvent was removed under reduced pressure and the
resulting oil was taken up in toluene (20 mL). The organic layer was
washed with H2O (1 ꢁ 20 mL), 1 M aqueous NaOH solution (1 ꢁ
20 mL), and H2O (1 ꢁ 30 mL). The organic layer was dried over
Na2SO4, filtered, and the solvent was removed under reduced pressure.
The resulting yellow oil was purified via flash SiO2 chromatography
(4.5 cm ꢁ 5 cm, 10% EtOAc/hexanes) to yield the desired epoxide 2d as
a yellow oil (1.19 g, 84%); Rf = 0.22 (10% EtOAc/hexanes); 1H NMR
(400 MHz, CDCl3) δ 7.25ꢀ7.19 (m, 2H), 7.25ꢀ7.19 (m, 2H),
6.87ꢀ6.81 (m, 2H), 6.86ꢀ6.81 (m, 2H), 4.20 (dd, J = 2.95, 11.03 Hz,
1H), 3.88 (dd, J = 5.82, 11.03 Hz, 1H), 3.33 (ddt, J = 2.81, 2.81, 4.13,
5.72 Hz, 1H), 2.89 (dd, J = 4.18, 4.86 Hz, 1H), 2.73 (dd, J = 2.66, 4.90
Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 157.20 (C), 129.49 (CH),
126.19 (C), 116.04 (CH), 69.17 (CH2), 50.17 (CH), 44.70 (CH2);
HRMS (ESIþ), calcd C9H9ClO2Li (M þ Liþ) = 191.0445, found =
191.0440.
clearance CLint in vitro = k/P (mL/min mg protein) was determined,
3
where k is the elimination rate constant (minꢀ1) and P is the protein
concentration (mg/mL).
Metabolic Stability in Liver S9 Fractions. Compound 1j (1 μM)
in <0.25% DMSO was incubated at 37 °C in buffer with 1 mg/mL of
pooled S9 protein from both rat and human and 1 mM NADPH,
UDPGA, PAPS, and GSH. The reaction was initiated by the addition of
cofactors at five different time points (0, 10, 20, 30, and 60 min). A
positive control (20 μM testosterone and 20 μM 7-hydroxycoumarin)
was run in parallel with sampling at 1, 10, 30, and 60 min. After the final
time point, fluorimetry was used to confirm the addition of NADPH to
the reaction mixture. LCꢀMS/MS was used to determine the peak area
response ratio (peak area corresponding to 1j divided by that of an
internal standard) without running a standard curve. The in vitro
intrinsic clearance CLint
= k/P (mL/min mg protein) was
in vitro
determined, where k is the elimination rate constant (minꢀ1) and P is
the protein concentration (mg/mL).
3
CYP IC50 in Human Liver Microsomes. The IC50 values of 1j for
five CYP enzymes (1A2, 2C9, 2C19, 2D6, and 3A4) were determined
using a pool of mixed-gender human liver microsomes (minimum of 10
donors). Compound 1j was incubated with pooled human liver micro-
somes (0.25 mg/mL) at 37 °C in the presence of phosphate buffer
(100 mM, pH 7.4), MgCl2 (5 mM), CYP-specific probe (at approxi-
mately Km), and NADPH (1 mM) at eight concentrations (0ꢀ100 μM).
After a period of incubation, the samples were treated by the addition of
protein precipitation solvent and centrifuged. The CYP enzyme activ-
ities were measured by determining the formation of the CYP probe
metabolites by LCꢀMS/MS. The individual CYP isoforms (specific
probe/metabolite formed/known inhibitor used as the positive control)
tested were the following: CYP 1A2 (phenacetin (150 μM)/acetami-
nophen/R-naphthoflavone); CYP 2C9 (diclofenac (6 μM)/40-OH
diclofenac/sulfaphenazole); CYP 2C19 (S-mephenytoin (40 μM)/40-
OH mephenytoin/(þ)-N-3-benzylnirvanol); CYP 2D6 (bufuralol
(7 μM)/10-OH bufuralol/quinidine); CYP 3A4 (testosterone (75 μM)/
6β-OH testosterone/Kketoconazole); CYP 3A4 (midazolam (1.4 μM)/
10-OH midazolam/ketoconazole). The IC50 was estimated by fitting the
experimental data to a sigmoidal model and nonlinear regression analysis
using GraphPad Prism software.
Kinase Profiling. The kinase profiling was performed by Luceome
Biotechnologies, Tucson, AZ. Compounds 1j and 1s were dissolved in
DMSO and tested at concentrations of 28 and 16 μM, respectively. Each
compound was first evaluated for false positive against split luciferase. If
they did not inhibit luciferase control, then they were profiled in
duplicate against the following kinases: AKT1, CAMK1, DDR2,
GSK3R, MARK1, MET, PAK1, PDGFRB, PIM1, PKC-γ, PKL4, and
SRC using the protocol described by Ghosh and co-workers.45 The
percent inhibition and percent activity remaining was calculated using
the following equations:
% inhibition ¼ ½ðALUcontrol ꢀ ALUsampleÞ=ALUcontrolꢂ ꢁ 100
% activity remaining ¼ 100 ꢀ % inhibtion
General Chemistry Methods. All reactions were run under an
inert atmosphere of either N2 or Ar gas. Reaction solvents were
purchased anhydrous and of HPLC quality. All other reagents were
purchased from Sigma Aldrich and used without further purification.
Yields were calculated for material judged homogeneous by thin layer
General Synthetic Procedure for the Pyrazole Fragments
of Type 6. 1-(3-Chlorobenzyl)-3,5-dimethyl-1H-pyrazole (6c).
In a 25 mL round-bottom flask with DMSO (14.5 mL), KOH (0.88 g,
15.6 mmol, 1.5 equiv) and 3,5-dimethylpyrazole (1.00 g, 10.4 mmol, 1
equiv) were combined and set to stir at room temperature. This mixture
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was then heated at 80 °C for 1 h. The mixture was then cooled to
room temperature, and 3-chlorobenzyl chloride (1.32 mL, 10.4 mmol,
1 equiv) was added. The reaction mixture was allowed to stir for an
additional 2 h.
1H), 2.48 (dd, J = 4.77, 11.80 Hz, 1H), 2.25 (s, 1H), 2.23 (s, 1H), 2.11 (s,
1H); 13C NMR (75 MHz, CDCl3) δ 158.96 (C), 147.50 (C), 138.18
(C), 137.54 (C), 129.65 (CH), 128.93 (CH), 127.67 (CH), 126.69
(CH), 121.14 (CH), 114.73 (CH), 113.62 (C), 70.53 (CH2), 66.29
(CH), 59.58 (CH2), 53.02 (CH2), 51.73 (CH2), 42.05 (CH3),
12.35 (CH3), 10.05 (CH3); HRMS (ESIþ), calcd C23H29N3O2Na
(M þ Naþ) = 402.2152, found = 402.2168.
Upon completion, the mixture was poured into H2O (50 mL) and the
solution was extracted with CHCl3 (4 ꢁ 50 mL). The resulting organic
layers were washed with H2O (4 ꢁ 100 mL) to remove DMSO, dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
resulting material was purified via flash SiO2 column chromatography
(5.5 cm ꢁ 5 cm, 5% EtOAc/hexanes) to yield the desired pyrazole 6c as
an yellow oil (2.11 g, 92%); Rf = 0.139 (10% EtOAc/hexanes); 1H NMR
(300 MHz, CDCl3) δ 7.23ꢀ7.18 (m, 2H), 7.05ꢀ7.01 (m, 1H),
6.96ꢀ6.89 (m, 1H), 5.85 (s, 1H), 5.16 (s, 2H), 2.24 (s, 3H), 2.14 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 148.06 (C), 139.62 (C), 139.34
(C), 134.78 (C), 130.13 (CH), 127.83 (CH), 126.81 (CH), 124.85
(CH), 105.93 (CH), 52.06 (CH2), 13.69 (CH3), 11.24 (CH3); HRMS
(ESIþ), calcd C12H13ClN2Na (M þ Naþ) = 243.0659, found =
243.0652.
General Synthetic Procedure for Methyl Amine Fragment
of Type 3. 1-(1-(3-Chlorobenzyl)-3,5-dimethyl-1H-pyrazol-
4-yl)-N-methylmethanamine (3c). In a 25 mL round-bottom flask,
methylamineꢀHCl (0.92 g, 13.6 mmol, 3 equiv) and paraformaldehyde
(0.82 g, 27.2 mmol, 6 equiv) were combined in EtOH (9 mL). This
solution was stirred for 2 h at 60 °C. At this point, the pyrazole derivative
1-(3-chlorobenzyl)-3,5-dimethyl-1H-pyrazole (6c) (1.00 g, 4.5 mmol,
1 equiv) was added, and the mixture was heated to 75 °C and stirred
for 21 h.
Method B: 1-(((1-(2-Chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)-
methyl)(methyl)amino)-3-(4-ethoxyphenoxy)propan-2-ol (1j). Into a
25 mL round-bottom flask, the epoxide 2-((4-ethyoxyphenoxy)methyl)-
oxirane) (2b) (0.300 g, 1.54 mmol, 1 equiv), amine 1-(1-(2-chloro-
benzyl)-3,5-dimethyl-1H-pyrazol-4-yl)-N-methylmethanamine (3b)
(0.45 g, 1.70 mmol, 1.1 equiv), and K2CO3 (0.23 g, 1.62 mmol, 1
equiv) were combined in acetonitrile (5.7 mL, 0.27 M). This solution
was heated to reflux for 24 h, at which time the reaction was complete.
The solvent was removed under reduced pressure, and the resulting
yellow oil was taken up into 10 mL of CHCl3 and partitioned into a
saturated aqueous solution of NaHCO3 (10 mL) and CHCl3 (10 mL).
The layers were separated, and the aqueous layer was back-extracted
with CHCl3 (4 ꢁ 30 mL). The organic layers were combined and dried
over Na2SO4, filtered, and the solvent was removed under reduced
pressure.
The resulting yellow oil was purified via flash SiO2 column chroma-
tography (2.5 cm ꢁ 4 cm, 40% EtOAc/hexanes with 2% Et3N) to yield
the desired alcohol 1j as a white solid (0.612 g, 87%); Rf = 0.300 (60%
EtOAc/hexanes with 2% Et3N); 1H NMR (300 MHz, CDCl3) δ
7.38ꢀ7.33 (m, 1H), 7.22ꢀ7.09 (m, 2H), 6.87ꢀ6.78 (m, 4H),
6.51ꢀ6.47 (m, 1H), 5.30 (s, 2H), 4.12ꢀ4.03 (m, 1H), 3.98 (q, J =
6.96, 6.98, 6.98 Hz, 2H), 3.90 (d, J = 4.98 Hz, 2H), 3.40 (ABq, J = 13.19,
Δv = 47.60 Hz, 2H), 2.61 (dd, J = 9.58, 12.25 Hz, 1H), 2.47 (dd, J = 4.18,
12.24 Hz, 1H), 2.26 (s, 3H), 2.25 (s, 2H), 2.12 (s, 2H), 1.39 (t, J = 6.99
Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 153.52 (C), 153.09 (C), 148.08
(C), 138.70 (C), 135.36 (C), 131.97 (C), 129.47 (CH), 128.82 (CH),
127.63 (CH), 127.53 (CH), 115.68 (CH), 115.58 (CH), 113.66 (C),
71.30 (CH2), 66.40 (CH), 64.20 (CH2), 59.57 (CH2), 51.74 (CH2),
50.33 (CH2), 42.12 (CH3), 15.16 (CH3), 12.40 (CH3), 9.86 (CH3);
HRMS (ESIþ), calcd C25H33ClN3O3 (M þ Hþ) = 458.2205, found =
458.2199.
Method C: (S)-1-(((1-(2-Chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-
yl)methyl)(methyl)amino)-3-(4-ethoxyphenoxy)propan-2-ol (1l). Into
a 5 mL reaction vial, the epoxide (S)-2-((4-ethoxyphenoxy)methyl)-
oxirane (2h) (0.017 g, 0.087 mmol, 1 equiv) and the amine, 1-(1-(2-
chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)-N-methylmethanamine (3b)
(0.023 g, 0.087 mmol, 1.1 equiv) were taken up into toluene (0.3 mL,
0.3 M) and set to reflux for 48 h.
Upon completion, the mixture was cooled to room temperature and
the solvent was removed under reduced pressure. The resulting oil was
taken up into 50 mL of CHCl3 and washed with a saturated aqueous
solution of NaHCO3 (1 ꢁ 20 mL). The resulting aqueous layer was then
extracted with CHCl3 (3 ꢁ 30 mL) and then dried over Na2SO4, filtered,
and concentrated under reduced pressure. The resulting yellow oil was
purified via flash SiO2 chromatography (5 cm ꢁ 4 cm, 40% EtOAc/
hexanes with 2% Et3N) to yield the desired methylamine 3c as a yellow
1
3
oil (0.467 g, 40%); Rf = 0.09 (60% EtOAc/hexanes with 2% Et N); H
NMR (300 MHz, CDCl3) δ 7.23ꢀ7.18 (m, 2H), 7.01ꢀ6.96 (m, 1H),
6.95ꢀ6.88 (m, 1H), 5.18 (s, 2H), 3.27 (s, 2H), 2.87 (s, 1H), 2.23 (s,
3H), 2.12 (s, 3H), 2.11 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 147.92
(C), 139.82 (C), 137.98 (C), 134.82 (C), 130.13 (CH), 127.81 (CH),
126.75 (CH), 124.79 (CH), 114.56 (C), 52.21 (CH2), 49.02 (CH2),
40.64 (CH3), 12.28 (CH3), 9.89 (CH3); HRMS (ESIþ), calcd
C14H19ClN3 (M þ Hþ) = 264.1262, found =264.1263.
Methods for the Synthesis of β-Hydroxyamide Com-
pounds of Type 1. Method A: 1-(((1-Benzyl-3,5-dimethyl-1H-pyr-
azol-4-yl)methyl)(methyl)amino)-3-phenoxypropan-2-ol (1a). To a
10 mL round-bottom flask, 3.33 mL of absolute EtOH was added. To
this the amine, 1-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)-N-methyl-
methanamine (3a) (0.17 g, 0.73 mmol, 1.1 equiv) and the commercially
available epoxide 2a (0.10 g, 0.66 mmol, 1.0 equiv) were combined. The
reaction mixture was set to stir for 22 h at reflux.
The reaction mixture was cooled to room temperature, transferred to
a 25 mL round-bottom flask, and the solvent was removed under
reduced pressure. The resulting orange oil was purified via flash SiO2
column chromatography (1.5 cm ꢁ 4 cm, 60% EtOAc/hexanes with 2%
Et3N) to yield the desired chiral alcohol (1l) as a clear oil (0.025 g, 63%);
3
Rf = 0.210 (50% EtOAc/hexanes with 2% Et N). Assay of enantiomeric
excess: HPLC (Chiralcel AD 25 cm column, 20% i-PrOH/hexanes with
0.1% Et2NH; 1.0 mL/min) tR (major) = 4.56 min, tR (minor) = 5.7 min;
>96% ee; [R]2D5 ꢀ15.06 (c 0.649, CHCl3); 1H NMR (400 MHz, CDCl3)
δ 7.35 (dd, J = 1.42, 7.79 Hz, 1H), 7.15 (dtd, J = 1.55, 7.43, 7.45, 20.61
Hz, 1H), 6.85ꢀ6.79 (m, 2H), 6.51ꢀ6.43 (m, 1H), 5.30 (s, 1H), 4.08
(dddd, J = 5.33, 5.33, 5.33, 9.28 Hz, 1H), 3.97 (q, J = 6.97, 6.98, 6.98 Hz,
1H), 3.90 (d, J = 4.96 Hz, 1H), 3.42 (s, 1H), 3.40 (ABq, J = 13.19 Hz,
Δv = 63.80 Hz, 1H), 2.61 (dd, J = 9.70, 12.18 Hz, 1H), 2.47 (dd, J = 4.13,
12.24 Hz, 1H), 2.26 (s, 1H), 2.25 (s, 1H), 2.12 (s, 1H); 13C NMR (101
MHz, CDCl3) δ 153.45 (C), 153.03 (C), 148.05 (C), 138.68 (C),
135.32 (C), 131.91 (C), 129.43 (CH), 128.79 (CH), 127.55 (CH),
127.51 (CH), 115.61 (CH), 115.50 (CH), 113.70 (C), 71.22 (CH2),
66.33 (CH), 64.13(CH2), 59.50 (CH2), 51.69 (CH2), 50.30 (CH2),
When the reaction was complete, the mixture was cooled to room
temperature and the solvent was removed under reduced pressure. The
resulting oil was taken up in CHCl3 (30 mL) and washed with a
saturated aqueous solution of NaHCO3 (1 ꢁ 20 mL). The resulting
aqueous layer was extracted with CHCl3 (3 ꢁ 30 mL). The combined
organic layers were then dried over Na2SO4, filtered, and the solvent was
removed under reduced pressure. The resulting oil was purified via flash
SiO2 column chromatography (3.0 cm ꢁ 4.0 cm, 50% EtOAc/hexanes
with 2% Et3N) to yield the desired alcohol 1a as a clear oil (0.13 g, 50%);
Rf = 0.46 (50% EtOAc/hexanes with 2% Et N); H NMR (300 MHz,
CDCl3) δ 7.33ꢀ7.19 (m, 4H), 7.08ꢀ7.00 (m, 2H), 6.98ꢀ6.86 (m, 4H),
5.21 (s, 2H), 4.16ꢀ4.01 (m, 2H), 3.95 (s, 2H), 3.53 (br s, 1H), 3.37
(ABq, J = 12.90 Hz, Δv = 45.41 Hz, 2H), 2.60 (dd, J = 9.41, 12.24 Hz,
1
3
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42.07 (CH3), 15.15 (CH3), 12.39 (CH3), 9.84 (CH3); HRMS (ESIþ),
calcd C25H33ClN2O3 (M þ Hþ) = 458.2205, found = 458.2191.
Method D: 1-(4-(Allyloxy)phenoxy)-3-(((3,5-dimethyl-1-(2-vinyl-
benzyl)-1H-pyrazol-4-yl)methyl)(methyl)amino)propan-2-ol (1q). Into
a 5 mL Biotage microwave tube, the epoxide 2-((4-(allyloxy)phenoxy)-
methyl)oxirane (2c) (0.038 g, 0.182 mmol, 1 equiv) and the amine
1-(3,5-dimethyl-1-(2-vinylbenzyl)-1H-pyrazol-4-yl)-N-methylmethana-
mine (3f) (0.051 g, 0.20 mmol, 1.1 equiv) were combined in absolute
ethanol (0.455 mL). The tube was then sealed with the appropriate
crimp cap and the tube placed into the microwave. After a total of 15 min
(200 W, 140 °C, 4ꢀ5 bar) the reaction was compete.
(C), 148.02 (C), 138.66 (C), 135.29 (C), 132.07 (C), 129.67 (CH),
128.82 (CH), 127.56 (CH), 115.97 (C), 113.65 (C), 70.84 (CH2),
66.15 (CH), 59.26 (CH2), 51.70 (CH2), 50.59 (CH2), 42.07 (CH3),
12.39 (CH3), 9.74 (CH3); HRMS (ESIþ), calcd C23H28Cl2N3O2Na
(M þ Hþ) = 448.1538, found = 448.1553.
N-((1-(2-Chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)-
methyl)-3-(4-ethoxyphenoxy)-2-methoxy-N-methylpropan-
1-amine (7). Into a 10 mL round-bottom flask, the alcohol 1-(((1-(2-
chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)(methyl)amino)-
3-(4-ethoxyphenoxy)propan-2-ol (1j) (0.020 g, 0.044 mmol, 1 equiv)
was taken up in THF (2.2 mL). To this, NaH (60%, 0.023 g, 0.96 mmol,
22 equiv) was added, and the mixture was stirred for 10 min. MeI
(0.08 mL, 1.3 mmol, 30 equiv) was added dropwise. The solution was
allowed to stir at room temperature for 30 min, at which time, it
appeared complete by TLC. The reaction mixture was quenched by the
slow addition of 2 mL of a saturated aqueous solution of NH4Cl. Once
the effervescence ceased (about 1 h) the mixture was diluted with ether,
and the layers were separated. The aqueous layer was back-extracted
with ether (1 ꢁ 20 mL) and then CH2Cl2 2 ꢁ 20 mL). The combined
organic layers were dried over Na2SO4, filtered, and the solvent was
removed under reduced pressure. The resulting dark brown oil was
purified via flash SiO2 column chromatography (2 cm ꢁ 3 cm, dry
loaded on SiO2, 40% EtOAc/hexanes with 2% Et3N) to yield the desired
methy ether (7) as a clear oil (0.0078 g, 37%); Rf = 0.212 (60% EtOAc/
hexanes with 2% Et3N); 1H NMR (400 MHz, CDCl3) δ 7.34 (dd, J =
1.25, 7.87 Hz, 1H), 7.17 (td, J = 1.77, 7.66, 7.87 Hz, 1H), 7.10 (td, J =
1.38, 7.45, 7.53 Hz, 1H), 6.83ꢀ6.76 (m, 4H), 6.47 (d, J = 6.15 Hz, 1H),
5.25 (s, 2H), 4.03 (dd, J = 3.50, 9.98 Hz, 1H), 3.97 (q, J = 6.97, 6.98, 6.98
Hz, 2H), 3.87 (dd, J = 5.51, 10.00 Hz, 1H), 3.48 (s, 3H), 3.39ꢀ3.25 (m,
2H), 2.64ꢀ2.56 (m, 1H), 2.53ꢀ2.45 (m, 1H), 2.24 (s, 3H), 2.23 (s, 3H),
2.07 (s, 3H), 1.38 (t, J = 6.99 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ
153.34, 153.15, 148.16, 135.47, 131.91, 129.42, 128.75, 127.58, 127.47,
115.56, 115.49, 69.51, 64.15, 57.99, 57.40, 52.32, 50.21, 43.19, 29.92,
15.17, 12.33, 9.76; HRMS (ESIþ), calcd C26H35ClN3O3 (M þ Hþ) =
472.2382, found = 472.2361.
The reaction mixture was transferred to a round-bottom with ethyl
acetate, and the solvent was removed under reduced pressure. The
resulting oil was diluted with CHCl3 (20 mL) and washed with a
saturated aqueous solution of NaHCO3 (1 ꢁ 10 mL). The aqueous layer
was extracted with CHCl3 (2 ꢁ 20 mL), and the combined organic
layers were dried over Na2SO4, filtered, and the solvent was removed
under reduced pressure. The resulting material was purified via flash
SiO2 column chromatography (1.5 cm ꢁ 6.5 cm, 60% EtOAc/hexanes
with 2% Et3N) to yield the desired alcohol 1q (0.072 g, 86%); Rf = 0.25
(60% EtOAc/hexanes with 2% Et3N); 1H NMR (300 MHz, CDCl3) δ
7.46 (dd, J = 1.46, 7.61 Hz, 1H), 7.25ꢀ7.10 (m, 2H), 6.96 (dd, J = 10.95,
17.29 Hz, 1H), 6.83 (s, 4H), 6.47 (dd, J = 0.88, 7.59 Hz, 1H), 6.12ꢀ5.97
(m, 1H), 5.65 (dd, J = 1.37, 17.27 Hz, 1H), 5.39 (d, J = 1.33 Hz, 1H),
5.37ꢀ5.35 (m, 1H), 5.33 (dq, J = 1.42, 1.53, 1.53, 51.83 Hz, 1H),
5.30ꢀ5.27 (m, 2H), 4.48 (dt, J = 1.52, 1.52, 5.31 Hz, 2H), 4.14ꢀ4.02
(m, 1H), 3.90 (d, J = 5.03 Hz, 2H), 3.39 (ABq, J = 13.20 Hz, Δv = 47.40
Hz, 2H), 3.27 (br s, 1H), 2.60 (dd, J = 9.53, 12.19 Hz, 1H), 2.47 (dd, J =
4.21, 12.25 Hz, 1H), 2.25 (s, 2H), 2.24 (s, 2H), 2.07 (s, 2H); 13C NMR
(75 MHz, CDCl3) δ 153.29 (C), 153.14 (C), 147.68 (C), 138.56 (C),
135.92 (C), 134.59 (C), 133.76 (CH), 133.74 (CH), 128.44 (CH),
127.73 (CH), 126.33 (CH), 126.30 (CH), 117.71 (CH2), 117.38
(CH2), 115.86 (CH), 115.63 (CH), 113.60 (C), 71.28 (CH2), 69.67
(CH2), 66.36 (CH), 59.54 (CH2), 51.72 (CH2), 50.70 (CH2), 42.06
(CH3), 12.38 (CH3), 9.97 (CH3); HRMS (ESIþ), calcd C28H36N3O3
(M þ Hþ) = 462.2751 found = 462.2750.
Macrocycle 8. To a 50 mL round-bottom flask, the β-amino alcohol
1q (0.05 g, 0.11 mmol, 1 equiv) was dissolved in toluene (21 mL). The
solution was degassed. Grubbs II catalyst (0.0009 g, 0.011 mmol, 0.1
equiv) was added, and reaction mixture was degassed again. This mixture
was stirred for 7 h at 110 °C. At this time, the mixture was cooled to
room temperature and the solvent was removed under reduced pressure.
The dark brown oil was purified via flash SiO2 column chromatography
(1.5 cm ꢁ 7.0 cm, 60% EtOAc/hexanes with 2% Et3N) to yield 8 as a
yellow oil (0.024 g, 52%); Rf = 0.38 (60% EtOAc/hexanes with 2%
Et3N); 1H NMR (300 MHz, CDCl3) δ 7.20ꢀ7.08 (m, 4H), 6.96ꢀ6.88
(m, 2H), 6.84 (d, J = 6.65 Hz, 1H), 6.74 (d, J = 5.27 Hz, 1H), 6.74ꢀ6.69
(m, 2H), 5.77 (dt, J = 5.83, 5.83, 16.01 Hz, 1H), 4.86 (d, J = 5.66 Hz,
2H), 4.62 (ABq, J = 16.50 Hz, Δv = 61.90 Hz, 2H), 3.98 (d, J = 7.24 Hz,
1H), 3.71ꢀ3.58 (m, 2H), 3.38 (d, J = 13.29 Hz, 1H), 3.34ꢀ3.19 (m,
2H), 2.56 (dd, J = 6.61, 12.46 Hz, 1H), 2.45 (s, 3H), 2.22 (s, 3H), 1.34 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 155.82, 153.37, 151.17, 147.10,
135.73, 134.78, 133.47, 130.00, 128.32, 128.23, 127.45, 127.08, 126.93,
119.18, 115.18, 69.31, 68.63, 66.64, 56.95, 52.03, 50.58, 12.13, 9.49;
HRMS (ESIþ) = calcd C26H32N3O3 (M þ Hþ) = 434.2438, found =
434.2423.
Method E: 1-(((1-(2-Chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)-
methyl)(methyl)amino-3-(4-chlorophenoxy)propan-2-ol (1s). Into a
5 mL Biotage microwave tube the epoxide 2-((4-chlorophenoxy)-
methyl)oxirane (2d, (0.05 g, 0.27 mmol, 1 equiv), the amine 1-(1-(2-
chlorobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl)-N-methylmethanamine (3b)
(0.078 g, 0.300 mmol, 1.1 equiv), and K2CO3 (0.036 g, 0.271 mmol, 1
equiv) were all taken up in acetonitrile (1 mL, 0.27 M). The tube was
then sealed with the appropriate crimp cap and the tube placed into the
microwave. After a total of 50 min (200 W, 180 °C, 9 bar) the reaction
was compete. The reaction tube was cooled to room temperature, the
cap was removed, and the reaction mixture was transferred to a 15 mL
round-bottom flask with EtOAc. The solvent was removed under
reduced pressure, and the resulting oil was diluted with CHCl3
(10 mL). This solution was partitioned into a separatory funnel
containing a 1:1 solution of CHCl3 and a saturated aqueous solution of
NaHCO3 (10 mL each). The layers were separated, and the organic
layer was extracted with CHCl3 (2 ꢁ 10 mL). The combined organic
layers were dried over Na2SO4, filtered, and the solvent was removed
under reduced pressure to yield an orange oil.
This oil was purified via flash SiO2 column chromatography (2.5 cm ꢁ
4 cm, 60% EtOAc/hexanes with 2% Et3N) to yield the desired alcohol
(1s) as a yellow oil (0.121 g, quant); Rf = 0.25 (60% EtOAc/hexanes
with 2% Et3N); 1H NMR (300 MHz, CDCl3) δ 7.33 (dd, J = 1.44, 7.71
Hz, 1H), 7.23ꢀ7.16 (m, 2H), 7.17ꢀ7.06 (m, 2H), 6.85ꢀ6.74 (m, 2H),
6.48 (dd, J = 1.60, 7.51 Hz, 1H), 5.28 (s, 2H), 4.18ꢀ3.96 (m, 1H),
3.97ꢀ3.79 (m, 2H), 3.38 (ABq, J = 13.18 Hz, Δv = 45.8 Hz, 3H), 2.58
(dd, J = 9.54, 12.17 Hz, 1H), 2.45 (dd, J = 4.29, 12.23 Hz, 1H), 2.24 (s,
3H), 2.24 (s, 3H), 2.10 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 157.55
Formation of β-Hydroxyamine HCl Salts. If required, the free
bases of the following compounds were made into the HCl salts via
the following procedure. The compound was dissolved in 0.5 M
Et2O. To this solution, an equal volume of 2 M HCl in Et2O was
added dropwise until the HCl salt precipitated out of solution. This
mixture was allowed to stir for an additional 10 min. Then the solvent
was evaporated off using a stream of N2 gas. The HCl salt was then
dried on high vacuum overnight and stored at room temperature over
Dryrite.
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Journal of Medicinal Chemistry
ARTICLE
’ ASSOCIATED CONTENT
(4) Matzinger, P. The danger model: a renewed sense of self. Science
2002, 296, 301–305.
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Supporting Information. Experimental data for all re-
(5) Lehnardt, S.; Massillon, L.; Follett, P.; Jensen, F. E.; Ratan, R.;
Rosenberg, P. A.; Volpe, J. J.; Vartanian, T. Activation of innate
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b
maining compounds and additional biological results. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Exploring the LPS/TLR4 signal pathway with small molecules. Biochem.
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receptor 4 modulation as a strategy to treat sepsis. Mediators Inflamma-
tion 2010, 2010, 568396.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ1-303-492-6786. Fax: þ1-303-492-5894. E-mail:
hang.yin@colorado.edu.
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of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5856–5861.
Author Contributions
These authors contributed equally to this work and should be
considered co-first-authors.
’ ACKNOWLEDGMENT
(10) Leon, C. G.; Tory, R.; Jia, J.; Sivak, O.; Wasan, K. M. Discovery
and development of Toll-like receptor 4 (TLR4) antagonists: a new
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We thank the National Institutes of Health (Grants DA026950,
DA025740, NS067425, RR025780, and DA029119) for financial
support of this work. K.C. thanks the China Scholarship Council
for a joint PhD scholarship (Grant No. 2009619072). Funding
for M.N.P. was supplied by CU Undergraduate Research Op-
portunities Program and HHMI Biosciences. We thank Dr. Jonel
Saludes, Dr. Peter Brown, and Dr. Greg Miknis for their sug-
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synthetic assistance.
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’ ABBREVIATIONS USED
AKT1, RAC-R serine/threonineꢀprotein kinase; BBB, bloodꢀ
brain barrier; CAMK1, calcium/calmodulin-dependent protein
kinase type 1; CSF, cerebrospinal fluid; CD14, cluster of
differentiation 14; CYP 450, cytochrome P450; DDR2, discoidin
domain-containing receptor 2; DMEM, Dulbecco’s modified
Eagle medium; DMSO, dimethylsulfoxide; FBS, fetal bovine
serum; GSK3R, glycogen synthase kinase-3R; HEK293, human
embryonic kidney 293 cells; IL-1, interleukin 1; IL-1β, interleu-
kin 1β; IL-6, interleukin 6; LPS, lipopolysaccharide; MARK1,
MAP/microtubule affinity-regulating kinase 1; MeCN, acetoni-
trile; MD2, myeloid differentiation factor 2; NADPH, nicotina-
mide adenine dinucleotide phosphate; NF-kB, nuclear factor
kB; NMR, nuclear magnetic resonance; NO, nitric oxide; PAPS,
30-phosphoadenosine 50-phosphosulfate; GSH, glutathione;
PAK1, P21/Cdc42/Rac1-activated kinase 1; PDGFβ, platelet-
derived growth factor-β; PBS, phosphate buffered saline; PIM1,
proto-oncogene serine/threonineꢀprotein kinase; PKC-γ, pro-
tein kinase γ; PLK4, polo-like kinase 4; PPTS, pyridinium
p-toluenesulfonate; RPMI, Roswell Park Memorial Institute;
SRC, sarcoma kinase; TLC, thin layer chromatography; TLR-4,
Toll-like receptor 4; TNF-R, tumor necrosis factor-R; UDPGA,
uridine 50-diphosphoglucuronosyltransferase
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