Neutral sphingomyelinase 2 inhibitors based on the 4-(1H-imidazol-2-yl)-2,6-dialkoxyphenol scaffold

Article abstract of DOI:10.1016/j.ejmech.2019.03.015

Neutral sphingomyelinase 2 (nSMase2), a key enzyme in ceramide biosynthesis, is a new therapeutic target for the treatment of neurological disorders and cancer. Using 2,6-dimethoxy-4-[4-phenyl-5-(2-thienyl)-1H-imidazol-2-yl]phenol (DPTIP), our initial hit compound (IC₅₀ = 30 nM) from nSMase2 screening efforts, as a molecular template, a series of 4-(1H-imidazol-2-yl)-2,6-dialkoxyphenol derivatives were designed, synthesized, and evaluated. Systematic examination of various regions of DPTIP identified the key pharmacophore required for potent nSMase2 inhibition as well as a number of compounds with the 4-(1H-imidazol-2-yl)-2,6-dialkoxyphenol scaffold with similar or higher inhibitory potency against nSMase2 as compared to DPTIP. Among them, 4-(4,5-diisopropyl-1H-imidazol-2-yl)-2,6-dimethoxyphenol (25b) was found to be metabolically stable against P450 metabolism in liver microsomes and displayed higher plasma exposure following oral administration as compared to DPTIP. Analysis of plasma samples identified an O-glucuronide as the major metabolite. Blockade of the phase II metabolism should further facilitate our efforts to identify potent nSMase2 inhibitors with desirable ADME properties.

Full text of DOI:10.1016/j.ejmech.2019.03.015

European Journal of Medicinal Chemistry 170 (2019) 276e289
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Neutral sphingomyelinase 2 inhibitors based on the 4-(1H-imidazol-2-
yl)-2,6-dialkoxyphenol scaffold
,
Ondrej Stepanek a, Niyada Hin a, Ajit G. Thomas a, Ranjeet P. Dash a b, Jesse Alt a,
,
,
,
, b, *
Rana Rais a b, Camilo Rojas a c, Barbara S. Slusher a b, Takashi Tsukamoto a
a Johns Hopkins Drug Discovery, Johns Hopkins University, Baltimore, MD, 21205, USA
b Department of Neurology, Johns Hopkins University, Baltimore, MD, 21205, USA
c Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD, 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Neutral sphingomyelinase 2 (nSMase2), a key enzyme in ceramide biosynthesis, is a new therapeutic
target for the treatment of neurological disorders and cancer. Using 2,6-dimethoxy-4-[4-phenyl-5-(2-
thienyl)-1H-imidazol-2-yl]phenol (DPTIP), our initial hit compound (IC50 ¼ 30 nM) from nSMase2
screening efforts, as a molecular template, a series of 4-(1H-imidazol-2-yl)-2,6-dialkoxyphenol de-
rivatives were designed, synthesized, and evaluated. Systematic examination of various regions of DPTIP
identified the key pharmacophore required for potent nSMase2 inhibition as well as a number of
compounds with the 4-(1H-imidazol-2-yl)-2,6-dialkoxyphenol scaffold with similar or higher inhibitory
potency against nSMase2 as compared to DPTIP. Among them, 4-(4,5-diisopropyl-1H-imidazol-2-yl)-2,6-
dimethoxyphenol (25b) was found to be metabolically stable against P450 metabolism in liver micro-
somes and displayed higher plasma exposure following oral administration as compared to DPTIP.
Analysis of plasma samples identified an O-glucuronide as the major metabolite. Blockade of the phase II
metabolism should further facilitate our efforts to identify potent nSMase2 inhibitors with desirable
ADME properties.
Received 4 February 2019
Received in revised form
5 March 2019
Accepted 6 March 2019
Available online 9 March 2019
Keywords:
Phosphodiesterase
Neutral sphingomyelinase 2
Sphingomyelin
Ceramide
Glucuronidation
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
encapsulation and transfer of diverse types of substances (proteins,
lipids, and RNAs) [6,7]. For instance, exosomes have been shown to
Neutral sphingomyelinase 2 (nSMase2) is a membrane associ-
ated enzyme that catalyzes the hydrolysis of sphingomyelin (SM)
into phosphorylcholine and ceramide (Fig. 1) in response to
extracellular stimuli [1,2]. Formation of ceramide enriched areas
due to the action of nSMase2 is associated with changes in me-
chanical properties of the membrane [3] as well as organization
and function of various membrane bound receptors [4,5]. More-
over, ceramide is involved in formation of exosomes, a specific type
of extracellular vesicle, which participate in intercellular commu-
nication in both physiological and pathological processes through
carry pathogenic molecules such as amyloid-b [8] and tau protein
[9] and upregulated ceramide production has been implicated in
various neurological disorders including Alzheimer's disease, HIV-
associated neurocognitive disorders, Parkinson's disease, and ALS
[1,10e12]. Furthermore, nSMase2-regulated release of miRNAs was
reported to play an important role in cancer cell metastasis [13,14]
while production of extracellular vesicles containing hepatitis B
virus (HBV) DNA was implicated as a separate pathway for trans-
mission of HBV [15]. These findings collectively suggest that path-
ogenic extracellular vesicles generated through the action of
nSMase2 are involved in a broad range of diseases. Thus, inhibition
of nSMase2 may offer unique therapeutic opportunity in these
diseases by reducing ceramide levels and subsequent exosome
formation.
Abbreviations: ADME, absorption, distribution, metabolism, and excretion; ALS,
amyotrophic lateral sclerosis; AP, alkaline phosphatase; nSMase2, neutral sphin-
gomyelinase 2; DPTIP, 2,6-dimethoxy-4-[4-phenyl-5-(2-thienyl)-1H-imidazol-2-yl]
phenol; SAR, structure-activity relationship; UDPGA, uridine diphosphate glucur-
onic acid.
To date, a number of nSMase2 inhibitors have been reported.
Unfortunately, these compounds inhibit nSMase2 only at micro-
molar concentrations and display unfavorable physicochemical and
molecular properties, limiting their ability to serve as molecular
* Corresponding author. Johns Hopkins Drug Discovery, Johns Hopkins Univer-
sity, Baltimore, MD 21205, USA.
E-mail address: ttsukamoto@jhmi.edu (T. Tsukamoto).
https://doi.org/10.1016/j.ejmech.2019.03.015
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
277
Fig. 1. Reaction catalyzed by nSMase2.
Fig. 2. Examples of nSMase2 inhibitors.
templates for lead optimization efforts [16e18]. For instance, the
most widely used tool inhibitor GW4869 (Fig. 2) [19], exhibits low
nSMase2 inhibition. Herein, we report the design, synthesis, SAR,
and ADME profile of DPTIP derivatives as potent nSMase2
inhibitors.
nSMase2 inhibitory activity (IC50 ¼ 1
mM) in biochemical assays. In
addition, it has negligible aqueous solubility and very poor solu-
bility in organic solvents (e.g. 0.2 mg/ml in DMSO) which has
limited its potential as a lead nSMase2 inhibitor. Cambinol, an in-
hibitor identified by our group from a pilot screening of commer-
cially available small chemical libraries (Fig. 2) [18], showed better
solubility compared to GW4869, but it was metabolically unstable
2. Results and discussion
2.1. Chemistry
Compounds 3a-n containing various substituents at the 4 and 5-
positions of the imidazole ring were prepared by condensation of
and exhibited low potency (IC50 ¼ 5
mM).
Our large-scale screening efforts have recently identified a
highly potent nSMase2 inhibitor (IC50 ¼ 30 nM), 2,6-dimethoxy-4-
[4-phenyl-5-(2-thienyl)-1H-imidazol-2-yl]phenol (DPTIP, Fig. 2),
which is structurally distinct from the existing nSMase2 inhibitors
[20]. Although the phenol moiety in DPTIP raises concerns about
the promiscuous activity towards multiple targets, DPTIP showed
syringaldehyde (1) and
a-dicarbonyl compounds 2a-n in the
presence of ammonium acetate (Scheme 1) as previously reported
[21] with some minor modifications.
DPTIP derivatives 6a-e containing an altered phenolic moiety
were prepared from aldehydes 5a-e and 1-phenyl-2-(thiophen-2-
yl)ethane-1,2-dione 4 (Scheme 2) using condensation reactions as
described in Scheme 1. Additional DPTIP derivatives 9a-d retaining
weak activity (2e50 mM) in only 19 (2.5%) of 759 different bioassays
conducted at NCATS [20]. In addition, DPTIP was found to dose-
dependently inhibit extracellular vesicle release in primary astro-
cyte cultures, an orthogonal assay of high physiological relevance
the
para-phenol
group
were
prepared
from
3,4,5-
trihydroxybenzaldehyde 5d in three steps. After conversion of 5d
to its 4-(p-methoxybenzyl) (PMB) derivative 7 using modified
literature procedure [22], Mitsunobu reaction was performed to
afford 3,5-dialkoxy derivatives 8a-d. The advantage of using PMB
protecting group was its spontaneous cleavage during the subse-
quent condensation with 4, providing the desired final products 9a-
d (Scheme 3).
Di-2,2,2-trifluoroethoxy derivative 12 could not be obtained by
the Mitsunobu method described above. Instead, we converted
dibromide 10 into 4-hydroxy-3,5-bis(2,2,2-trifluoroethoxy)benz-
aldehyde 11 using freshly prepared sodium 2,2,2-trifluoroethoxide
in the presence of CuCl2 (Scheme 4) [23]. Compound 11 was sub-
sequently condensed with 4 in the presence of ammonium acetate
to give compound 12.
[20]. Furthermore, DPTIP was reported to attenuate IL-1b-induced
astrocyte-derived extracellular vesicle release in an animal model
of brain injury conducted in GFAP-GFP mice [20]. In order to further
establish structure-activity relationship (SAR) of this new prom-
ising series of nSMase2 inhibitors, we conducted a systematic ex-
amination of various regions of DPTIP with the primary focus on
determining the key pharmacophore required for potent nSMase2
inhibition. Since DPTIP is already highly potent in inhibiting
nSMase2, the objective of the lead optimization efforts was to
improve ADME properties rather than to further improve inhibitory
potency. We divided the molecule into three distinct components,
namely, the para-substituted phenol, the central imidazole ring,
and two substituents at the 4- and 5-positions of the imidazole
ring. Performing chemical modifications in each of the three areas
independently allowed us to evaluate its relative importance for
Di-tert-butoxy derivative 16 was synthesized using 3,5-
dihydroxy-4-methoxybenzaldehyde 13 [24] as a starting material
(Scheme 5). Reaction of 13 with N,N-dimethylformamide di-tert-
Scheme 1. Synthesis of compounds 3a-3n. Reagents and conditions: (a) NH4OAc, AcOH, reflux, 3e11%; (b) NH4OAc, EtOH, 60e85 ꢀC, 10e72%; (c) NH4OAc, EtOH, rt, 11e26%.

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O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
Scheme 2. Synthesis of compounds 6a-e. Reagents and conditions: (a) NH4OAc, AcOH, 120 ꢀC, 19e35%.
Scheme 3. Synthesis of compounds 9a-c. Reagents and conditions: (a) NaH, PMB-Cl, DMF, 0 ꢀC to rt, 44%; (b) DIAD, PPh3, ROH, THF, 0 ꢀC to rt, 20e90%; (c) 4, NH4OAc, AcOH, 120 ꢀC,
13e44%.
were not separated. They were characterized and tested as mixture.
Thiazole analog 19 lacking substituents at the 4- and 5-positions
was synthesized by demethylation of the central methoxy group of
compound 18 [28] using boron trichloride (Scheme 7) [29].
Imidazoline analog 22 lacking 4- and 5-substituents was syn-
thesized from aldehyde 1. Aldehyde 1 was first converted into the
corresponding methanesulfonate 20, which was subsequently
condensed with ethylenediamine under oxidative conditions [30]
to give compound 21. Hydrolysis of the methanesulfonate ester 21
under basic conditions [31] gave compound 22 (Scheme 8).
Scheme 4. Synthesis of compound 12. Reagents and conditions: (a) CF3CH2OH, Na,
CuCl2, DMF, 115 ꢀC, 53%; (b) 4, NH4OAc, AcOH, 120 ꢀC, 36%.
Compounds 25a and 25b were prepared from
a-hydroxy ke-
tones 24a and 24b, respectively (Scheme 9) [32]. The oxidative
formation of the imidazole ring appears to be mediated by dis-
solved oxygen in the solvent as the products did not form in the
absence of oxygen.
Synthesis of compound 29 is illustrated in Scheme 10. First,
aldehyde 20 was converted to nitrile 26 with sulfamic acid [33].
Treatment of nitrile 26 with dry HCl followed by ammonolysis [34]
afforded amidine 27, which was subsequently reacted with 2-
bromo-1-(thiophen-2-yl)propan-1-one [35] to form 28. The
methanesulfonyl protective group of 28 was removed by LDA [36]
butyl
acetal
[25]
yielded
3,5-di-tert-butoxy-4-
methoxybenzaldehyde 14, which was subsequently condensed
with 4 in the presence of ammonium acetate to form compound 15.
Demethylation of 15 was achieved by heating in a mixture of water
and piperidine [26], giving the desired product 16.
N-Methyl analogues of DPTIP (a mixture of two regioisomers
17a and 17b) were prepared by condensation of aldehyde 1 and
diketone 4 in the presence of ammonium acetate and methylamine
as previously described (Scheme 6) [27]. Compounds 17a and 17b
Scheme 5. Synthesis of compound 16. Reagents and conditions: (a) N,N-Dimethylformamide di-t-butyl acetal, PhMe, 85 ꢀC, 30%; (b) 4, NH4OAc, AcOH, 120 ꢀC, 19%; (c) piperidine-
H2O (v/v 50%), sealed tube, 150 ꢀC, 67%.
Scheme 6. Synthesis of compounds 17a and 17b. Reagents and conditions: (a) NH4OAc, NH2Me, NaH2PO4, THF, sealed tube, 150 ꢀC, 17%.

O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
279
glucuronide formation was provided by the chemical shift of C-10
anomeric carbon (101.4 ppm) of compound 30, which is in good
agreement with those of other O-glucuronides (~100 ppm) derived
from phenols [37] and a downfield shift of approximately 15 ppm
with respect to the C-1’ (~85 ppm) of N-glucuronides derived im-
idazoles [38].
2.2. Evaluation of nSMase2 inhibitory potency
The inhibitory potencies of the synthesized compounds were
determined using a fluorescence-based coupled assay involving
human nSMase2, alkaline phosphatase (AP), choline oxidase, and
horseradish peroxidase as previously reported [18]. In order to
assure that potent inhibitors identified in this coupled assay
directly inhibit nSMase2, we performed a counter screen assay in
which test compounds were incubated with AP, choline oxidase,
and horseradish peroxidase in the presence of phosphorylcholine.
Some caution needs to be taken in interpreting the inhibitory po-
tency data when IC50 values from the primary assay are comparable
to those from the counter screening as that may indicate interfer-
ence of AP, choline oxidase, and/or horseradish peroxidase in
addition to (or instead of) nSMase2 inhibition. We considered that
compounds with greater than 5-fold separation in IC50 values in
favor of nSMase2 assay over the counter assay predominantly
Scheme 7. Synthesis of compound 19. Reagents and conditions: (a) BCl3, DCM, rt, 15%.
to yield compound 29.
Glucuronide 30 was prepared from compound 25b by following
a previously reported procedure (Scheme 11) [37]. Reaction affor-
ded the b-anomer of O-glucuronide as the sole product. The low
overall yield (two steps) was presumably caused by the poor
reactivity of the sterically hindered phenolic OH of compound 25b
in the first step. The formation of b
-anomer was confirmed by 1H
NMR coupling constant of anomeric hydrogen (J ¼ 7.5 Hz). Equiva-
lence of C4 and C5 carbons of imidazole ring (one signal in 13C
NMR) suggests the symmetricity of the imidazole ring and rules out
the formation of N-glucuronide. Another evidence supporting O-
Scheme 8. Synthesis of compound 22. Reagents and conditions: (a) MsCl, Et3N, DCM, 0 ꢀC to rt, 71%; (b) ethane-1,2-diamine, NBS, DCM, 0 ꢀC to rt, 89%; (c) KOH, MeOH, rt, 15%.
Scheme 9. Synthesis of compounds 25a and 25b. Reagents and conditions: (a) (diacetoxyiodo)benzene, KOH, MeOH, rt; (b) 1, NH4OAc, O2, EtOH, 65 ꢀC, 16% for 25a, 43% for 25b.
Scheme 10. Synthesis of compound 29. Reagents and conditions: (a) H2NSO3H, AcOH, H2O, 100 ꢀC, 98%; (b) (i) HCl/dioxane, MeOH, rt, (ii) NH3/MeOH, rt, 20%; (c) 2-bromo-1-
(thiophen-2-yl)propan-1-one, K2CO3, EtOH, 78 ꢀC, 48%; (d) (i) LDA, THF, ꢁ78 ꢀC to 0 ꢀC, (ii) HCl, H2O, 0 ꢀC, 14%.
Scheme 11. Synthesis of compound 30. Reagents and conditions: (a) (i) BF3∙OEt2, DCM, ꢁ10 ꢀC; (ii) Na2CO3, MeOH, H2O, rt, 9%.

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O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
Table 2
inhibit nSMase2. The nSMase2 IC50 values for those with less than
5-fold separation from the counter assay are indicated with a
footnote, suggesting that those compounds may have a higher
intrinsic IC50 value against nSMase2 than the listed value. The assay
results are summarized in Tables 1e3.
Inhibition of nSMase2 by compounds 3a, 17a and 17b, 19, 22.
Table 1 shows biological data obtained from DPTIP analogs in
which its 4-hydroxy-3,5-dimethoxyphenyl group is modified.
Compounds 6f and 6g bearing plain benzene ring and 3,4,5-
trimethoxybenzene respectively, were shown to be completely
inactive in the nSMase2 assay. Previously, we reported that 3,5-
dimethoxyphenyl derivative 6h was also inactive as a nSMase2
inhibitor [20], clearly indicating the essential role played by the
phenolic hydrogen of DPTIP in binding to nSMase2. The removal of
one methoxy group from DPTIP resulted in 10-fold loss of inhibitory
potency as seen in compound 6a. When both methoxy groups were
removed (compound 6b), not only more profound loss of potency
(100-fold) was observed but also IC50 values in nSMase2 and
counter screen assay were no longer distinguishable. Moving the
hydroxyl group of 6b to the position 2 or 3 (compounds 6i and 6j)
Cmpd
Y
IC50
(
m
M)a
Cmpd
Y
IC50 (m
M)a
DPTIP
0.03 0.01 3a
0.42 0.11
17a-b
40 20b
19
22
20 4b
>100
a
Values are mean SD of at least three experiments.
Separation of IC50 values in nSMase2 assay and counter assay is ꢂ 5-fold.
Table 1
b
Inhibition of nSMase2 by compounds 6a-6j, 9a-9c, 12 and 16.
led to complete loss of potency, underscoring the importance of
para-hydroxyl relative to the imidazole ring. Replacing one or both
methoxy substituents of DPTIP by hydroxyl group(s) (compounds
6c and 6d) led to moderate loss of potency compared to DPTIP.
These compounds also displayed potent inhibition in the counter
assay. Replacement of the two methoxy groups of DPTIP by chlorine
atoms (compound 6e) caused complete loss of activity. In contrast,
some of the compounds with methoxy moieties replaced by
different alkoxy groups exhibited very potent nSMase2 inhibitory
activity, including diethoxy derivative 9a with an IC50 value of
10 nM. Small alkyl chains were well tolerated though decrease in
potency was seen with more sterically demanding branched alkyl
groups such as cyclopentyl (compound 9c) and t-butyl (compound
16).
Cmpd
X
IC50
(
m
M)a
Cmpd
X
IC50 (m
M)a
DPTIP
0.03 0.01 6c
0.56 0.10b
6f
>100
>100
6d
6e
0.84 0.16b
6g
>100
Table 2 summarizes inhibitory data of DPTIP analogs where its
imidazole core is altered. Significant loss of potency was observed
upon methylation of the imidazole NH (compounds 17a and 17b,
tested as a mixture). On the other hand, the removal of the two
aryl substituents from the imidazole core of DPTIP resulted in
only 10-fold decrease in potency as seen in compound 3a.
Replacement of the imidazole ring of 3a with a thiazole ring
(compound 19) led to substantial decrease in potency presumably
due to the loss of the hydrogen bond donor. Interestingly, con-
version of the imidazole ring of 3a into an imidazoline 22 led to a
complete loss of potency despite the presence of the critical
hydrogen donor. These SAR findings indicate that 3a represents
the key pharmacophore required for potent nSMase2 inhibition.
Therefore, subsequent efforts were focused on analogs of DPTIP,
where its two aryl substituents are altered while retaining the 3a
pharmacophore.
6h
6a
6b
>100
9a
0.01 0.00
0.07 0.03
0.04 0.01
0.32 0.06 12
2.1 0.6b
9b
9c
6i
>100
0.12 0.02
0.52 0.19
Table 3 shows inhibitory data of DPTIP analogs in which its
phenyl and thienyl substituents at positions 4 and 5 of imid-
azole ring are altered. All of the compounds within this struc-
tural series potently inhibited nSMase2 with IC50 values ranging
6j
>100
16
from 0.02 to 0.5 mM although compounds 3b-3d displayed
comparable inhibitory potency in the counter assay. In partic-
ular, di-substituted derivatives, including hybrid of alkyl and aryl
groups, are consistently potent and it appears that a variety of
substituents are tolerated at the 4- and 5-positions of the
imidazole ring.
a
Values are mean SD of at least three experiments.
Separation of IC50 values in nSMase2 and counter assays is ꢂ 5-fold.
b

O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
281
Table 3
Inhibition of nSMase2 by compounds 3b-n, 25a-b, and 29.
Cmpd
Z1
Z2
IC50
(
m
M)a
Cmpd
Z1
Z2
IC50 (m
M)a
DPTIP
3b
3c
3d
3e
3f
25b
3g
Ph
H
CH3
2-Thienyl
CH3
CH3
0.03 0.01
0.22 0.06b
0.52 0.14b
0.42 0.08b
0.12 0.03
0.06 0.01
0.09 0.03
0.07 0.02
0.13 0.03
3i
3j
Ph
Ph
0.02 0.00
0.02 0.00
0.04 0.02
0.07 0.02
0.02 0.00
0.02 0.01
0.07 0.01
0.02 0.01
2-Thienyl
2-Furyl
p-Tolyl
p-CH3OPh
CH3
2-Thienyl
2-Furyl
p-Tolyl
p-CH3OPh
Ph
3k
3l
3m
3n
25a
29
-(CH2)4-
CH3CH2
CH3
(CH3)2CH
H
H
CH3CH2
n-Butyl
(CH3)2CH
Ph
(CH3)2CH
CH3
Ph
2-Thienyl
3h
p-Tolyl
a
Values are mean SD of at least three experiments.
Separation of IC50 values in nSMase2 and counter assays is ꢂ 5-fold.
b
2.3. Evaluation of metabolic stability in liver microsomes
(Fig. 3). The brain/plasma ratio of 25b at 2 h, however, was only 9%
(17% for oral DPTIP).
Selected compounds were tested in mouse and human liver
microsomes for phase I metabolic stability (Table 4). As previously
reported DPTIP was found to be stable against phase I metabolism
in both human and mouse microsomes. The majority of DPTIP de-
rivatives showed good metabolic stability in both mouse and hu-
man microsomes (>70% after 1 h incubation). The two exceptions
are compounds 9a and 9b containing two ethoxy or isopropoxy
groups instead of methoxy groups of DPTIP. Therefore, we suspect
that the oxidation took place at the modified alkoxy groups of these
compounds. Indeed, when trifluoroethoxy moieties were intro-
duced in an attempt to enhance resistance to oxidation, metabolic
stability was completely regained as seen in compound 12.
2.5. Identification of O-glucuronide metabolite from plasma
samples
Subsequent metabolite identification analysis of the plasma
samples identified a glucuronide as the major metabolite. Glucur-
onidation of 25b can possibly take place at either its phenolic OH or
imidazole NH group [39]. We found that the retention time and
mass spectrum of the glucuronidated metabolite of 25b from
mouse plasma samples (2 h after oral administration of 25b)
matched with those of O-glucuronide 30 chemically synthesized
from 25b (Figure S1 in Supporting Information). Subsequently, we
assessed the metabolic stability of 25b in mouse and human liver
microsomes in the presence of UDPGA. Consistent with the mouse
plasma pharmacokinetic data, compound 25b was found to be
substantially glucuronidated with less than 20% of the parent
compound remaining intact after 1 h incubation in mouse liver
microsomes. Compound 25b also underwent substantial glucur-
onidation in human microsomes (32% of the parent compound
remaining intact after 1 h incubation). In order to investigate
whether the O-glucuronidation can be minimized with bulky
alkoxyl groups introduced in the proximity of the phenol group, we
assessed the stability of DPTIP and its analog 9b containing two
isopropoxy groups in liver microsomes in the presence of UDPGA.
As shown in Table 5, DPTIP was found to undergo a moderate de-
gree of glucuronidation, resulting in the loss of the parent com-
pound by 33% (mouse liver microsomes) and 50% (human liver
microsomes), respectively. In contrast, compound 9b was
completely resistant to glucuronidation in mouse liver microsomes,
demonstrating that two sterically hindered alkoxy groups adjacent
to the phenolic OH group can completely block the
glucuronidation.
2.4. Evaluation of oral pharmacokinetics in mice
Subsequent to the in vitro metabolic stability studies, we
selected compound 25b for preliminary in vivo pharmacokinetics
studies in mice. Although compound 25b is not the most potent
nSMase2 inhibitor, it exhibits a substantial structural difference
from DPTIP with the two aliphatic substituents on the imidazole
ring (as opposed to two aryl groups in DPTIP). Comparison of these
two compounds allowed us to assess the impact of the two sub-
stituents on the pharmacokinetic properties. Fig. 3 shows plasma
levels of DPTIP and 25b at 0.5 and 2 h following oral administration
(10 mg/kg). Interestingly, plasma levels of compound 25b were
higher than those of DPTIP at both of the time points (3-fold at 0.5 h
and 6-fold at 2 h). Compound 25b also exhibited 3-fold higher brain
levels at 2 h following oral administration compared to DPTIP
Table 4
Metabolic stability of selected nSMase2 inhibitors in mouse and human liver
microsomes.
Compd
% Remaining at 1 h
mouse human
Compd
% Remaining at 1 h
mouse human
3. Conclusion
DPTIP
3e
3i
3l
3m
3n
95
75
>95
>95
73
4
0
95
7
9a
9b
12
25a
25b
29
35
4
>95
94
82
95
0
NDa
NDa
94
>95
>95
73
Cumulative evidence indicates that ceramide plays a key role in
the pathophysiology of various neurological disorders and cancer.
Development of small molecule inhibitors of nSMase2 will enable
us to further investigate the pathogenic role played by ceramide as
well as the therapeutic utility of blocking nSMase2-mediated pro-
duction of ceramide pharmacologically. DPTIP represents one of the
>95
>95
>95
75
0
3
1
1
2
1
1
1
1
1
82
95
a
Not Determined.

282
O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
Fig. 3. Plasma and brain levels of DPTIP and 25b in mice following oral administration of DPTIP (10 mg/kg) and 25b (10 mg/kg), respectively.
Table 5
from InterBioScreen Ltd. Compound 6g was obtained from Chem-
Metabolic stability of DPTIP, 9b and 25b in mouse and human liver microsomes in
Bridge Corp. Melting points were obtained on a Mel-Temp appa-
ratus and are uncorrected. 1H NMR spectra were recorded at 400 or
500 MHz. 13C NMR spectra were recorded at 100 or 125 MHz. In
many cases signals of quaternary carbons in 13C NMR were not
observable. With unsymmetrically substituted imidazoles, tauto-
mers could be observed in NMR leading to difficulties during
spectra interpretation. This could be overcome by converting given
sample to protonated form (HCl or TFA-d salt) where only one
protonated species exists. In such case “NMR measured as salt” is
noted prior experimental data. The HPLC solvent system consisted
of deionized water and acetonitrile, both containing 0.1% formic
acid. Preparative HPLC purification was performed on an Agilent
1200 series HPLC system equipped with an Agilent G1315D DAD
the presence of UDPGA.
Compd
% Remaining at 1 h
mouse
human
25b
DPTIP
9b
14
67
0
0
32
0
7
50
>95
NDa
a
Not Determined.
most potent nSMase2 inhibitors and our systematic SAR studies on
this scaffold presented herein revealed the key pharmacophore
essential for the potent nSMase2 inhibition as well as the structural
modifications that can be tolerated by the enzyme. In particular, the
4- and 5-positions of the imidazole ring and the two alkoxy groups
offer ample opportunities to introduce structural diversity in an
effort to improve ADME properties while retaining potent nSMase2
inhibitory activity. The binding mode of DPTIP and its analogs
presented herein has not yet been determined. While the crystal
structure of the human nSMase2 catalytic domain recently deter-
mined at 1.85 Å resolution is highly informative [2], further struc-
tural investigation is required to identify the binding site given the
noncompetitive mechanism of inhibition shown by DPTIP with
respect to sphingomyelin [20]. In this regard, the allosterically
gated “DK switch” of nSMase2 is of particular interest as a potential
binding site and has been recently implicated as the target of
cambinol by docking studies [40]. Despite the substantial degree of
glucuronidation, compound 25b displayed superior plasma phar-
macokinetics as compared to DPTIP following oral administration.
Given that compound 9b showed complete resistance to glucur-
onidation, this metabolic liability can be circumvented while
retaining the key pharmacophore. Future efforts will be centered
on identifying alkoxy groups that would be metabolically stable
against oxidation yet prevent glucuronidation of the phenolic OH
group. Although compound 25b exhibited negligible CNS pene-
tration, its therapeutic potential can be realized in non-CNS disease
in which a minimum brain exposure is preferred.
detector using
a
Phenomenex Luna
5
mm
C18 column
(21.2 mm ꢃ 250 mm, 5
m
m). Analytical HPLC was performed on an
Agilent 1200 series HPLC system equipped with an Agilent G1315D
DAD detector (detection at 220 nm) and an Agilent 6120 quadru-
pole MS detector. Unless otherwise specified, the analytical HPLC
conditions involve: (A) for nonpolar compounds 20% acetonitrile/
80% water for 0.25 min followed by gradient to 85% acetonitrile/15%
water over 1.5 min and continuation of 85% acetonitrile/15% water
for 2.25 min with a Luna C18 column (2.1 mm ꢃ 50 mm, 3.5
mm) at a
flow rate of 1.25 mL/min; (B) for polar compounds 5% acetonitrile/
95% water for 0.25 min followed by gradient to 40% acetonitrile/60%
water over 1.5 min and continuation of 85% acetonitrile/15% water
for 2.25 min with a Luna C18 column (2.1 mm ꢃ 50 mm, 3.5
mm) at a
flow rate of 1.25 mL/min. Unless otherwise noted, all final com-
pounds biologically tested were confirmed to be of ꢄ95% purity by
the HPLC methods described above.
4.1.2. 4-(1H-Imidazol-2-yl)-2,6-dimethoxyphenol (3a)
A mixture of 4-hydroxy-3,5-dimethoxybenzaldehyde (0.50 g,
2.74 mmol), ammonium acetate (2.12 g, 27.45 mmol) and glyoxal
(40% solution in water, 5.49 mmol, 0.63 mL) in glacial acetic acid
(15 mL) was stirred at 120 ꢀC for 16 h. The solvent was removed in
vacuo and the residue was partitioned between 10% aq. NaHCO3
(15 mL) and EtOAc (15 mL). The water layer was washed with EtOAc
(2 ꢃ 10 mL) and combined organic layers were washed with brine
(30 mL), dried over MgSO4, filtered and solvents evaporated. The
residue was purified using a Biotage Isolera One flash purification
system with a silica gel cartridge (5e10% MeOH/DCM). Dark red
product was further purified using preparative HPLC (0% MeCN/
100% water for 5 min followed by an increase to 20% MeCN over
35 min and an increase to 40% MeCN over 10 min; flow rate 10 mL/
min) to give 18 mg (3%) of title compound as brown solid; mp 99 ꢀC.
4. Experimental
4.1. Chemical synthesis
4.1.1. General
All solvents were reagent grade or HPLC grade. Unless otherwise
noted, all materials were obtained from commercial suppliers and
used without further purification. Compounds 6f and 6h were
obtained from ChemDiv Inc. Compounds 6i and 6j were obtained
1H NMR (400 MHz, DMSO‑d6)
8.15 (s, 1H); 13C NMR (101 MHz, DMSO‑d6)
d
3.81 (s, 6H), 7.07 (s, 2H), 7.23 (s, 2H),
56.0, 102.5, 121.2, 135.7,
d

O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
283
146.1, 148.1. LCMS: (B) retention time 0.29 min, m/z ¼ 221 [M þ H]þ.
4.1.8. 2,6-Dimethoxy-4-(5-phenyl-1H-imidazol-2-yl)phenol
hydrochloride (3g)
Compound 3g was prepared as described for the preparation of
3b except phenylglyoxal monohydrate (0.167 g, 1.1 mmol) was used
in place of methylglyoxal. After workup the free base was converted
to HCl salt using excess of 4 N HCl in dioxane and purified using
preparative HPLC (10% MeCN/90% water for 5 min followed by an
increase to 50% MeCN over 35 min and an increase to 70% MeCN
over 10 min; flow rate 15 mL/min) to give 60 mg (18%) of title
compound as brown solid; mp > 110 ꢀC (dec). 1H NMR (500 MHz,
4.1.3. 2,6-Dimethoxy-4-(5-methyl-1H-imidazol-2-yl)phenol (3b)
A mixture of 4-hydroxy-3,5-dimethoxybenzaldehyde (0.182 g,
1.0 mmol), ammonium acetate (0.769 g, 10.0 mmol) and methyl-
glyoxal (35e45% solution in water, 0.226 g, ca. 1.1 mmol) in EtOH
(10 mL) was stirred at RT for 16 h. Solvents were evaporated and the
residue was partitioned between water (10 mL) and EtOAc (30 mL).
Organic phase was washed with saturated NaHCO3, brine, dried
over MgSO4, filtered and solvent was evaporated. Residue was
purified using Biotage Isolera One flash purification system with a
silica gel cartridge (5e10% MeOH/DCM) to give 25 mg (11%) of title
compound as brown solid; mp > 200 ꢀC (dec). NMR measured as
DMSO‑d6)
J ¼ 7.6 Hz, 2H), 7.65 (bs, 1H), 7.83 (d, J ¼ 7.6 Hz, 2H), 8.61 (bs, 1H),
12.46 (bs, 1H); 13C NMR (126 MHz, DMSO‑d6)
56.1, 102.7, 121.01,
d
3.85 (s, 6H), 7.20 (t, J ¼ 7.5 Hz, 1H), 7.30 (s, 2H), 7.37 (t,
d
HCl salt: 1H NMR (500 MHz, DMSO‑d6)
7.40 (s, 1H), 7.61 (s, 2H), 9.36 (bs, 1H), 14.59 (bs, 1H), 14.79 (bs, 1H);
13C NMR (126 MHz, DMSO‑d6)
9.7, 56.5, 104.4, 112.8, 115.7, 129.2,
d 2.34 (s, 3H), 3.86 (s, 6H),
128.5, 136.0, 148.1. LCMS: (B) retention time 1.89 min, m/z ¼ 297
[M þ H]þ.
d
4.1.9. 2,6-Dimethoxy-4-(4-p-tolyl-1H-imidazol-2-yl)phenol (3h)
Compound 3h was prepared as described for the preparation of
3b except 4-tolylglyoxal hydrate (95%, 0.183 g, 1.1 mmol) was used
in place of methylglyoxal. Obtained 35 mg (11%) of title compound
as yellow-green solid; mp > 130 ꢀC (dec). 1H NMR (500 MHz,
138.9, 143.4, 148.3. LCMS: (B) retention time 0.74 min, m/z ¼ 235
[M þ H]þ.
4.1.4. 4-(4,5-Dimethyl-1H-imidazol-2-yl)-2,6-dimethoxyphenol
hydrochloride (3c)
Methanol-d4)
d
2.35 (s, 3H), 3.94 (s, 6H), 7.21 (d, J ¼ 7.7 Hz, 2H), 7.29
A solution of 4-hydroxy-3,5-dimethoxybenzaldehyde (0.182 g,
1.0 mmol), ammonium acetate (0.769 g, 10.0 mmol) and dimethyl-
glyoxal (0.437 mL, 5.0 mmol) in glacial acetic acid (15 mL) was
stirred at 80 ꢀC. Workup as described for 3a. The product was
further purified by forced precipitation of HCl salt from MeOH so-
lution with Et2O to give 32 mg (11%) of title compound as beige
(s, 2H), 7.35 (s, 1H), 7.63 (d, J ¼ 7.7 Hz, 2H); 13C NMR (126 MHz,
Methanol-d4)
d 21.2, 56.8, 104.2, 122.3, 126.0, 130.3, 137.8, 149.6.
LCMS: (B) retention time 2.05 min, m/z ¼ 311 [M þ H]þ.
4.1.10. 4-(4,5-Diphenyl-1H-imidazol-2-yl)-2,6-dimethoxyphenol
(3i)
Compound 3i was prepared as described for the preparation of
3b except benzil (0.236 g, 1.10 mmol) was used in place of meth-
ylglyoxal and reaction mixture was stirred at 80 ꢀC for 6 h. Obtained
40 mg (15%) of title compound as pale brown solid; mp 293 ꢀC. 1H
powder; mp > 265 ꢀC (dec). 1H NMR (400 MHz, DMSO‑d6)
d 2.26 (s,
6H), 3.86 (s, 6H), 7.54 (s, 2H), 9.31 (s, 1H), 14.42 (s, 1H); 13C NMR
(101 MHz, DMSO‑d6)
d 8.6, 56.5, 104.0, 112.8, 124.5, 138.7, 141.7,
148.3. LCMS: (B) retention time 1.61 min, m/z ¼ 249 [M þ H]þ.
NMR (500 MHz, DMSO‑d6)
d
3.85 (s, 6H), 7.21 (t, J ¼ 7.3 Hz, 1H),
4.1.5. 2,6-Dimethoxy-4-(4,5,6,7-tetrahydro-1H-benzo[d]imidazol-
2-yl)phenol (3d)
7.28e7.31 (m, 2H), 7.37e7.39 (m, 3H), 7.43e7.50 (m, 4H), 7.54e7.55
(m, 2H), 8.62 (s, 1H), 12.44 (s, 1H); 13C NMR (126 MHz, DMSO‑d6)
The compound was prepared as described for 3b except cyclo-
hexane-1,2-dion (0.123 g, 1.10 mmol) was used instead of methyl-
glyoxal. Obtained 71 mg (26%) of title compound as yellow solid;
d
56.1, 102.8, 120.7, 126.4, 127.0, 127.5, 127.7, 128.1, 128.4, 128.7, 131.3,
135.3, 136.0, 136.6, 146.0, 148.1. LCMS: (B) retention time 2.38 min,
m/z ¼ 273 [M þ H]þ.
mp > 120 ꢀC (dec). 1H NMR (500 MHz, methanol-d4)
d
1.86 (bs, 4H),
2.61 (bs, 4H), 3.90 (s, 6H), 7.15 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
4.1.11. 4-(4,5-Di(thiophen-2-yl)-1H-imidazol-2-yl)-2,6-
dimethoxyphenol (3j)
d
23.2, 26.6, 101.9, 121.9, 135.4, 148.1. LCMS: (B) retention time
1.76 min, m/z ¼ 275 [M þ H]þ.
Compound 3j was prepared as described for the preparation of
3b except 2,20-thenil (0.250 g, 1.1 mmol) was used in place of
methylglyoxal and the reaction was stirred at 65 ꢀC. Product was
further purified by trituration (2 ꢃ 2 mL MeOH) to give 40 mg (10%)
of title compound as white solid; mp 218 ꢀC. 1H NMR (500 MHz,
4.1.6. 4-(4,5-Diethyl-1H-imidazol-2-yl)-2,6-dimethoxyphenol (3e)
Compound 3e was prepared as described for the preparation of
3b except hexane-3,4-dione (94%, 0.142 mL, 1.1 mmol) was used in
place of methylglyoxal and the reaction was stirred 16 h at 65 ꢀC.
Obtained 76 mg (28%) of title compound as brown solid. M.P. >
DMSO‑d6)
d
3.84 (s, 6H), 7.00 (t, J ¼ 4.1 Hz, 1H), 7.14 (d, J ¼ 3.5 Hz,
1H), 7.19e7.25 (m, 1H), 7.32 (s, 2H), 7.40 (dd, J ¼ 8.1, 4.3 Hz, 2H), 7.71
225 ꢀC (dec); 1H NMR (500 MHz, Acetone-d6)
d
1.18 (t, J ¼ 7.5 Hz,
(d, J ¼ 5.0 Hz, 1H), 8.69 (s, 1H), 12.63 (s, 1H); 13C NMR (101 MHz,
6H), 2.57 (q, J ¼ 7.5 Hz, 4H), 3.77 (s, 6H), 7.26 (s, 2H); 13C NMR
DMSO‑d6)
d 56.1, 102.9, 119.9, 120.1, 123.3, 124.6, 127.3, 127.5, 127.6,
(126 MHz, Acetone-d6)
d 15.5, 19.5, 56.5, 103.3, 123.2, 136.8, 144.9,
128.5, 131.0, 133.3, 136.4, 136.5, 146.3, 148.11. LCMS: (B) retention
148.9. LCMS: (B) retention time 1.84 min, m/z ¼ 277 [M þ H]þ.
time 2.47 min, m/z ¼ 385 [M þ H]þ.
4.1.7. 4-(5-Butyl-4-methyl-1H-imidazol-2-yl)-2,6-
4.1.12. 4-(4,5-Di(furan-2-yl)-1H-imidazol-2-yl)-2,6-
dimethoxyphenol (3f)
dimethoxyphenol hydrochloride (3k)
Compound 3f was prepared as described for the preparation of
3b except heptane-2,3-dione (97%, 0.158 mL, 1.1 mmol) was used in
place of methylglyoxal and the reaction was stirred 16 h at 65 ꢀC.
Obtained 52 mg (18%) of title compound as brown solid;
Compound 3k was prepared as described for the preparation of
3b except 2,20-furil (97%, 0.215 g, 1.1 mmol) was used in place of
methylglyoxal and the reaction was stirred 6 h at 85 ꢀC. Product was
further purified by forced precipitation with Et2O of HCl salt from
its MeOH solution. After filtration and drying 67 mg (17%) of title
compound was obtained as white solid; mp > 220 ꢀC (dec). 1H NMR
mp > 105 ꢀC (dec). 1H NMR (500 MHz, Acetone-d6)
d 0.89 (t,
J ¼ 7.4 Hz, 3H), 1.29e1.35 (m, 2H), 1.551e1.61 (m, 2H), 2.17 (s, 3H),
2.54 (t, J ¼ 7.6 Hz, 2H), 3.77 (s, 6H), 7.28 (s, 2H); 13C NMR (126 MHz,
(500 MHz, Methanol-d4)
d
3.97 (s, 6H), 6.69 (dd, J ¼ 3.6, 1.9 Hz, 2H),
Acetone-d6)
d
11.0, 14.2, 23.0, 25.8, 33.1, 56.5, 103.3, 122.7, 137.0,
7.15 (d, J ¼ 3.5 Hz, 2H), 7.44 (s, 2H), 7.80 (s, 2H); 13C NMR (126 MHz,
144.9, 148.9. LCMS: (B) retention time 2.02 min, m/z ¼ 291 [M þ
Methanol-d4) d 57.1, 106.4, 112.7, 113.3, 121.3, 142.5, 145.7, 147.4,
H]þ.
150.0. LCMS: (B) retention time 2.35 min, m/z ¼ 353 [M þ H]þ.

284
O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
4.1.13. 4-(4,5-Di-p-tolyl-1H-imidazol-2-yl)-2,6-dimethoxyphenol
(3l)
129.08, 128.89, 128.37, 127.70, 127.10, 116.08. LCMS: (A) retention
time 1.35 min, m/z 319 [M þ H]þ.
Compound 3l was prepared as described for the preparation of
3b except 4,40-dimethylbenzil (95%, 0.276 g, 1.10 mmol) was used in
place of methylglyoxal and the reaction was stirred at 60 ꢀC. Crude
product was purified by trituration with Et2O/CHCl3 4/1 to give
0.287 g (72%) of title compound as pink solid; mp > 260 ꢀC (dec). 1H
4.1.18. 3-Methoxy-5-(4-phenyl-5-(thiophen-2-yl)-1H-imidazol-2-
yl)benzene-1,2-diol (6c)
Compound 6c was prepared as described for the preparation of
6a except benzaldehyde 5c was used in place of benzaldehyde 5a.
Dark brown solid (35% yield); mp 133 ꢀC. NMR measured as HCl
NMR (500 MHz, DMSO‑d6)
d 2.29 (s, 3H), 2.35 (s, 3H), 3.84 (s, 6H),
7.10 (d, J ¼ 7.7 Hz, 2H), 7.25 (d, J ¼ 7.8 Hz, 2H), 7.32e7.40 (m, 4H),
salt: 1H NMR (400 MHz, DMSO‑d6)
d
3.89 (s, 3H), 7.19 (dd, J ¼ 3.6,
7.43 (d, J ¼ 7.8 Hz, 2H), 8.59 (s, 1H), 12.33 (s, 1H); 13C NMR
5.1 Hz, 1H), 7.36 (d, 1H), 7.54 (dt, J ¼ 2.8, 4.2 Hz, 4H), 7.61e7.65 (m,
(126 MHz, DMSO‑d6)
d 20.8, 20.9, 56.1, 102.8, 120.9, 126.9, 127.1,
2H), 7.68 (dd, J ¼ 1.2, 5.2 Hz, 1H), 7.71 (d, J ¼ 3.6 Hz, 1H), 9.51 (bs,
128.2, 128.5, 128.7, 129.2, 132.5, 135.3, 136.0, 136.4, 136.8, 145.7,
2H), 14.73 (bs, 2H); 13C NMR (101 MHz, DMSO‑d6) d 56.4, 103.6,
148.1. LCMS: (B) retention time 2.70 min, m/z ¼ 401 [M þ H]þ.
108.8, 127.7, 128.9, 129.6, 129.9, 138.4, 144.6, 146.2, 148.6. LCMS: (A)
retention time 1.30 min, m/z 365 [M þ H]þ.
4.1.14. 4-(4,5-Bis(4-methoxyphenyl)-1H-imidazol-2-yl)-2,6-
dimethoxyphenol (3m)
4.1.19. 5-(4-Phenyl-5-(thiophen-2-yl)-1H-imidazol-2-yl)benzene-
1,2,3-triol (6d)
Compound 6d was prepared as described for the preparation of
6a except benzaldehyde 5d was used in place of benzaldehyde 5a.
Grey solid (20% yield); mp > 250 ꢀC. NMR measured as HCl salt: 1H
Compound 3m was prepared as described for the preparation of
2b except 4,40-dimethoxybenzil (0.303 g, 1.1 mmol) was used in
place of methylglyoxal and the reaction was stirred 6 h at 65 ꢀC.
Obtained 90 mg (21%) of title compound as pink solid; mp > 300 ꢀC.
1H NMR (500 MHz, DMSO‑d6)
d
3.74 (s, 3H), 3.80 (s, 3H), 3.84 (s,
NMR (400 MHz, DMSO‑d6)
3H), 7.59e7.62 (m, 3H), 7.68 (dd, J ¼ 1.2, 5.1 Hz, 1H), 9.54 (bs, 2H),
10.69 (bs, 1H), 14.61 (bs, 2H); 13C NMR (101 MHz, DMSO‑d6)
107.3,
d 7.14 (s, 2H), 7.19 (m, 1H), 7.50e7.54 (m,
6H), 6.87 (d, J ¼ 8.8 Hz, 1H), 7.01 (d, J ¼ 8.7 Hz, 2H), 7.34 (s, 2H), 7.39
(d, J ¼ 8.8 Hz, 2H), 7.45 (d, J ¼ 8.8 Hz, 2H), 8.58 (s, 1H), 12.27 (s, 1H);
d
13C NMR (126 MHz, DMSO‑d6)
d
55.0, 55.1, 56.1, 102.7, 113.6, 114.1,
126.8, 127.6, 127.7, 128.6, 128.8, 128.2, 129.9, 137.7, 144.8, 146.5.
121.0,123.8,126.4,128.0,128.1,129.7,135.8,135.9,145.3,148.1,157.8,
LCMS: (B) retention time 0.88 min, m/z 351 [M þ H]þ.
158.7. LCMS: (B) retention time 2.46 min, m/z ¼ 433 [M þ H]þ.
4.1.20. 2,6-Dichloro-4-(4-phenyl-5-(thiophen-2-yl)-1H-imidazol-
2-yl)phenol (6e)
4.1.15. 2,6-Dimethoxy-4-(4-methyl-5-phenyl-1H-imidazol-2-yl)
phenol (3n)
Compound 6e was prepared as described for 6a except benzal-
dehyde 5e was used in place of benzaldehyde 5a, and the com-
pound was purified by a reversed phase preparative HPLC (40%
MeCN/100% water followed by an increase to 100% MeCN over
60 min and maintaining 100% MeCN over 10 min; flow rate 15 mL/
min). Beige solid (31% yield); mp > 250 ꢀC. NMR measured as HCl
Compound 3n was prepared as described for the preparation of
3b except 1-phenylpropane-1,2-dione (98%, 0.166 g, 1.1 mmol) was
used in place of methylglyoxal and the reaction was stirred at 65 ꢀC.
Obtained 123 mg (35%) of title compound as brown solid;
mp > 135 ꢀC (dec). NMR measured as HCl salt: 1H NMR (400 MHz,
DMSO‑d6)
7.48e7.53 (m, 2H), 7.67e7.73 (m, 2H), 9.03 (s, 1H), 13.68 (bs, 2H);
13C NMR (101 MHz, DMSO‑d6)
11.0, 56.3, 102.7, 127.1, 127.4, 128.7,
d
2.46 (s, 3H), 3.87 (s, 6H), 7.32e7.41 (m, 1H), 7.47 (s, 2H),
salt: 1H NMR (400 MHz, DMSO‑d6)
7.49e7.59 (m, 4H), 7.61e7.68 (m, 3H), 8.34 (s, 2H), 11.16 (s, 1H); 13
NMR (101 MHz, DMSO‑d6) 122.8, 127.2, 127.7, 127.9, 128.9, 129.1,
d
7.16 (dd, J ¼ 3.6, 5.1 Hz, 1H),
C
d
d
137.6, 143.7, 148.2. LCMS: (B) retention time 2.02 min, m/z ¼ 353
129.5, 142.1, 151.4. LCMS: (A) retention time 1.99 min, m/z 388 [M þ
[M þ H]þ.
H]þ.
4.1.16. 2-Methoxy-4-(4-phenyl-5-(thiophen-2-yl)-1H-imidazol-2-
yl)phenol (6a)
4.1.21. 3,5-Dihydroxy-4-(4-methoxybenzyloxy)benzaldehyde (7)
To a cooled solution of 3,4,5-trihydroxybenzaldehyde (5d) (1.0 g,
6.49 mmol) in DMF (15 mL) was added sodium hydride (60% w/w,
0.26 g, 6.49 mmol). After 20 min stirring at 0 ꢀC, 4-methoxybenzyl
chloride (0.71 g, 4.54 mmol) was added. The mixture was stirred
at 0 ꢀC for an additional 20 min, then ice bath was removed and the
reaction was allowed to stir at rt over weekend. Water was added
followed by 3 mL of 10% aqueous KHSO4 solution. The product was
extracted with EtOAc (ꢃ2). The organic layer was washed with
brine, dried over sodium sulfate and concentrated. The resulting
residue was purified by Biotage Isolera One flash system with a
silica gel cartridge (30e50% EtOAc/hexanes with 2% AcOH) to give
0.78 g (44%) of title compound as a brown solid cake. 1H NMR
A mixture of aldehyde 5a (0.045 g, 0.30 mmol), ammonium ac-
etate (0.178 g, 2.31 mmol) and diketone 4 (0.050 g, 0.23 mmol) in
glacial acetic acid (5 mL) was stirred at 120 ꢀC for 16 h. The solvent
was removed in vacuo and the residue was purified using a Biotage
Isolera One flash purification system with a silica gel cartridge
(EtOAc/hexane) to give title compound 6a (0.015 g, 19%) as a beige
solid; mp 224 ꢀC. NMR measured as HCl salt: 1H NMR (400 MHz,
DMSO‑d6)
d
3.91 (s, 3H), 7.04 (d, J ¼ 8.4 Hz, 1H), 7.19 (dd, J ¼ 5.1,
3.6 Hz, 1H), 7.57e7.51 (m, 3H), 7.66 (ddd, J ¼ 7.8, 4.7, 1.9 Hz, 3H),
7.87e7.77 (m, 2H), 8.04 (d, J ¼ 2.2 Hz, 1H), 10.20 (s, 1H), 14.92 (s,
2H); 13C NMR (101 MHz, DMSO‑d6)
d 56.2, 111.5, 113.7, 115.9, 121.3,
123.2, 126.9, 127.7, 128.5, 128.8, 128.9, 129.56, 129.64, 129.9, 144.4,
(400 MHz, chloroform-d): d 3.83 (s, 3H), 5.10 (s, 2H), 6.91 (d,
J ¼ 8.8 Hz, 2H), 7.03 (s, 2H), 7.31 (d, J ¼ 8.6, 2H), 9.80 (s, 1H).
148.0, 150.5. LCMS: (A) retention time 1.38 min, m/z 349 [M þ H]þ.
4.1.17. 4-(4-Phenyl-5-(thiophen-2-yl)-1H-imidazol-2-yl)phenol
(6b)
4.1.22. 3,5-Diethoxy-4-(4-methoxybenzyloxy)benzaldehyde (8a)
To a solution of triphenylphosphine (0.96 g, 3.65 mmol) in THF
(10 mL) was slowly added DIAD (0.74 g, 3.65 mmol) via syringe at
0 ꢀC. White precipitate was formed. The mixture was allowed to stir
at 0 ꢀC for 1 h, upon which a solution of compound 7 (0.25 g,
0.91 mmol) and ethanol (0.16 mL, 2.73 mmol) in THF (5 mL) was
added via syringe. The reaction was stirred at 0 ꢀC, brought up to rt
and stirred overnight. After removal of solvent, the resulting res-
idue was purified by Biotage Isolera One (20% EtOAc/hexanes) to
Compound 6b was prepared as described for the preparation of
6a except benzaldehyde 5b was used in place of benzaldehyde 5a.
Yellow solid foam (23% yield); mp 122 ꢀC. NMR measured as HCl
salt: 1H NMR (400 MHz, DMSO‑d6)
d
7.02 (d, J ¼ 8.8 Hz, 2H), 7.19 (dd,
J ¼ 3.8, 5.1 Hz, 1H), 7.51e7.55 (m, 3H), 7.62e7.65 (m, 2H), 7.67 (d,
J ¼ 4.6 Hz, 2H), 8.09e8.13 (m, 2H), 10.58 (bs, 1H), 14.69 (bs, 1H); 13
C
NMR (101 MHz, DMSO‑d6)
d 161.05, 144.51, 129.84, 129.42, 129.11,

O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
285
give title compound in quantitative yield as a light yellow oil. 1H
NMR (400 MHz, chloroform-d):
4.11 (q, J ¼ 7.1, 13.9 Hz, 4H), 5.08 (s, 2H), 6.85 (d, J ¼ 8.6 Hz, 2H), 7.08
(s, 2H), 7.39 (d, J ¼ 8.6 Hz, 2H), 9.83 (s, 1H).
cut sodium (0.69 g, 30 mmol) was added to trifluoroethanol (10 mL)
at rt and the mixture was stirred until a complete dissolution of
sodium. The mixture was then heated at 80e90 ꢀC to distill off
portion of trifluoroethanol with the help of in house vacuum. Then,
d
1.47 (t, J ¼ 7.1 Hz, 6H), 3.81 (s, 3H),
a
solution of 3,5-dibromo-4-hydroxybenzaldehyde (10, 0.5 g,
4.1.23. 3,5-Diisopropoxy-4-(4-methoxybenzyloxy)benzaldehyde
(8b)
1.79 mmol) and copper (II) chloride (0.20 g, 1.49 mmol) in DMF
(4 mL) was added in one portion. The blue mixture was heated and
distilled at 110e115 ꢀC overnight. The reaction was cooled to RT,
then water was added and the undesired precipitate was filtered
off. The filtrate was partitioned between EtOAc and water. The
organic layer was washed with brine, dried over sodium sulfate and
concentrated in vacuo to give 0.30 g (53%) of title compound as a
brown solid, which was used without further purification. 1H NMR
Compound 8b was prepared as described for the preparation of
8a, with the exception that 2-propanol was used in place of ethanol.
Bright yellow oil (85%). 1H NMR (400 MHz, chloroform-d):
d 1.36 (d,
J ¼ 6.1 Hz, 12H), 3.81 (s, 3H), 4.62 (m, 2H), 5.04 (s, 2H), 6.86 (d,
J ¼ 8.6 Hz, 2H), 7.08 (s, 2H), 7.40 (d, J ¼ 8.6 Hz, 2H), 9.83 (s, 1H).
4.1.24. 3,5-Bis(cyclopentyloxy)-4-(4-methoxybenzyloxy)
benzaldehyde (8c)
(400 MHz, DMSO‑d6)
(s, 1H).
d
4.62 (q, J ¼ 9.6, 19.0 Hz, 4H), 6.96 (s, 2), 9.20
Compound 8c was prepared as described for the preparation of
8a, with the exception that cyclopentanol was used in place of
ethanol. Light yellow oil (90%). The compound was used as is
without further characterization.
4.1.29. 4-(4-Phenyl-5-(thiophen-2-yl)-1H-imidazol-2-yl)-2,6-
bis(2,2,2-trifluoroethoxy)phenol (12)
Diketone
4
(90 mg, 0.42 mmol), aldehyde 11 (155 mg,
0.49 mmol) and ammonium acetate (320 mg, 4.16 mmol) were
heated together in acetic acid (6 mL) at 120 ꢀC overnight. The re-
action was concentrated in vacuo. The resulting residue was parti-
tioned between EtOAc and water. The organic layer was washed
with brine, dried over sodium sulfate and concentrated to give a
brown oil. Purification by reverse phase preparative HPLC (40%
ACN/60% H2O for 5 min followed by an increase to 100% ACN/0%
H2O over 40 min and a continuation of 100% ACN/0% H2O until
50 min; flow rate 15 mL/min) afforded 75 mg (36%) of title com-
pound as a light purple solid; mp 205 ꢀC. NMR measured as HCl
4.1.25. 2,6-Diethoxy-4-(4-phenyl-5-(thiophen-2-yl)-1H-imidazol-
2-yl)phenol (9a)
Diketone 4 (0.2 g, 0.92 mmol), aldehyde 8a (0.31 g, 0.92 mmol)
and ammonium acetate (0.71 g, 9.25 mmol) were heated together
in acetic acid (10 mL) at 120 ꢀC overnight. The reaction was
concentrated in vacuo and the residue was partitioned between
EtOAc and water. The organic layer was washed with brine, dried
over sodium sulfate and concentrated. The crude material was
purified by Biotage Isolera One flash chromatography (EtOAc/hex-
ane) to give a solid, which was further triturated in 15% EtOAc/
hexanes to afford 50 mg (13%) of title compound as a purple solid;
mp 245 ꢀC. NMR measured as HCl salt: 1H NMR (400 MHz,
salt: 1H NMR (400 MHz, DMSO‑d6)
d
4.86 (q, J ¼ 8.9 Hz, 4H), 7.19 (dd,
J ¼ 3.6, 5.1 Hz, 1H), 7.53e7.56 (m, 3H), 7.64e7.68 (m, 3H), 7.79 (bs,
1H), 7.97 (s, 2H), 10.00 (s, 1H), 14.84 (s, 2H); 13C NMR (100 MHz,
DMSO‑d6)
d
1.38 (t, J ¼ 7.0 Hz, 6H), 4.16 (q, J ¼ 7.0 Hz, 4H), 7.19 (dd,
DMSO‑d6)
d
66.3 (q, J ¼ 34 Hz), 109.2, 123.9 (q, J ¼ 277 Hz), 127.7,
J ¼ 3.6, 5.1 Hz, 1H), 7.54 (dd, J ¼ 1.8, 5.0 Hz, 3H), 7.61e7.70 (m, 5H),
7.75 (d, J ¼ 3.6 Hz, 1H), 9.17 (bs, 1H), 17.75 (bs, 2H); 13C NMR
128.9, 129.5, 129.9, 140.3, 143.6, 146.7. LCMS: (A) retention time
2.06 min, m/z 515 [MþH]þ.
(101 MHz, DMSO‑d6)
d 14.8, 64.7, 106.1, 127.2, 127.7, 128.4, 128.9,
129.1, 129.3, 129.5, 129.9, 139.8, 144.5, 147.4. LCMS: (A) retention
time 1.69 min, m/z 407 [MþH]þ.
4.1.30. 3,5-Di-tert-butoxy-4-methoxybenzaldehyde (14)
Aldehyde 13 (0.2 g, 1.19 mmol) was heated at 85 ꢀC in toluene
(6 mL) for 30 min. N,N-Dimethylformamide di-tert-butyl acetal
(3 mL) was slowly added and heating continued at 85 ꢀC overnight.
The next day, additional 1.5 mL of N,N-Dimethylformamide di-tert-
butyl acetal was added and the reaction was completed after 3 h
stirring at 85 ꢀC. The reaction was concentrated in vacuo and the
residue was partitioned between EtOAc and water. The organic
layer was washed with brine, dried over sodium sulfate and
concentrated. The crude material was purified by Biotage Isolera
One flash chromatography (10% EtOAc/hexanes) to give 0.1 g (30%)
4.1.26. 2,6-Diisopropoxy-4-(4-phenyl-5-(thiophen-2-yl)-1H-
imidazol-2-yl)phenol (9b)
Compound 9b was prepared as described for the preparation of
9a, with the exception that compound 8b was used in place of 8a.
Purple solid (44%); mp 217 ꢀC. NMR measured as HCl salt: 1H NMR
(400 MHz, DMSO‑d6)
d
1.31 (d, J ¼ 6.8 Hz,12H), 4.72 (sept, J ¼ 6.0 Hz,
2H), 7.19 (t, J ¼ 4.3 Hz,1H), 7.51e7.58 (m, 3H), 7.61e762 (m, 5H), 7.77
(s, 1H), 8.94 (bs, 1H), 14.76 (bs, 2H); 13C NMR (126 MHz, DMSO‑d6)
d
22.0, 71.5, 108.4,127.1, 127.6, 128.4,128.9,129.6,129.9,144.4,146.4.
LCMS: (A) retention time 1.83 min, m/z 435 [MþH]þ.
of title compound as an oil. 1H NMR (400 MHz, chloroform-d)
d 1.39
(s, 18H), 3.95 (s, 3H), 7.31 (s, 2H), 9.83 (s, 1H).
4.1.27. 2,6-Bis(cyclopentyloxy)-4-(5-phenyl-4-(thiophen-2-yl)-1H-
imidazol-2-yl)phenol (9c)
Compound 9c was prepared as described for the preparation of
9a, with the exception that compound 8c was used in place of 8a.
Grey solid (32%); mp 230 ꢀC. NMR measured as HCl salt: 1H NMR
4.1.31. 2-(3,5-Di-t-butoxy-4-methoxyphenyl)-4-phenyl-5-
(thiophen-2-yl)-1H-imidazole (15)
Diketone
4
(80 mg, 0.37 mmol), aldehyde 14 (104 mg,
(400 MHz, DMSO‑d6)
d
1.52e1.65 (m; 4H), 1.78 (tdd, J ¼ 2.2, 6.3,
0.37 mmol) and ammonium acetate (285 mg, 3.70 mmol) were
heated together in acetic acid (6 mL) at 120 ꢀC for 2 h. The reaction
was concentrated in vacuo. The product was partitioned between
EtOAc and water. The organic layer was washed with brine, dried
over sodium sulfate and concentrated. The crude material was
purified by Biotage Isolera One flash chromatography (20e50%
EtOAc/hexanes with 2% AcOH) to give 35 mg (19%) of title com-
pound as a yellow solid cake. 1H NMR (400 MHz, chloroform-d)
9.4 Hz, 8H), 1.987e1.91 (m, 4H), 4.93e4.97 (m, 2H), 7.19 (dd, J ¼ 3.5,
5.1 Hz, 1H), 7.52e7.56 (m, 3H), 7.60 (s, 2H), 7.62e7.66 (m, 1H), 7.67
(dd, J ¼ 1.2, 5.1 Hz, 1H), 7.73 (d, J ¼ 3.6 Hz, 1H), 8.85 (bs, 1H), 14.71
(bs, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 23.6, 32.3, 80.4, 107.4,
127.7, 128.9, 129.6, 141.1, 144.4, 146.6. LCMS: (A) retention time
2.06 min, m/z 487 [MþH]þ.
4.1.28. 4-Hydroxy-3,5-bis(2,2,2-trifluoroethoxy)benzaldehyde (11)
In a flask equipped with a Claisen distillation apparatus, freshly
d
1.40 (s, 18H), 3.89 (s, 3H), 7.01 (m, 1H) 7.20 (m, 1H), 7.30 (s, 2H),
7.42 (m, 4H), 7.62 (m, 2H).

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O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
4.1.32. 2,6-Di-t-butoxy-4-(4-phenyl-5-(thiophen-2-yl)-1H-
imidazol-2-yl)phenol (16)
with saturated NaHCO3 solution and brine, dried over MgSO4,
filtered and solvent was evaporated. Crude product was crystalized
from MeOH/CHCl3 to give 10.10 g (71%) of title compound as beige
A solution of compound 15 (35 mg, 0.07 mmol) in a 1:1 mixture
of piperidine-water (10 mL) was heated at 150 ꢀC in a sealed tube
for 10 days. Upon completion, the reaction was concentrated in
vacuo. The product was partitioned between EtOAc and water. The
organic layer was washed with brine, dried over sodium sulfate and
concentrated. The crude material was purified by Biotage Isolera
One flash chromatography (10e30% EtOAc/hexanes) to give 20 mg
(67%) of title compound as a white solid; mp > 250 ꢀC. NMR
solid. 1H NMR (400 MHz, DMSO‑d6)
d 3.46 (s, 3H), 3.92 (s, 6H), 7.35
(s, 2H), 9.96 (s, 1H); m/z ¼ 261 [M þ H]þ.
4.1.36. 4-(4,5-Dihydro-1H-imidazol-2-yl)-2,6-dimethoxyphenyl
methanesulfonate (21)
To a solution of methansulfonate 20 (0.260 mg, 1.0 mmol) in
DCM (10 mL) cooled to 0 ꢀC was added 1,2-ethylenediamine (70
mL,
measured as TFA-d salt: 1H NMR (400 MHz, DMSO‑d6)
d 1.36 (s,
1.05 mmol) and reaction was stirred at 0 for 30 min, then N-bro-
mosuccinimide (0.187 g, 1.05 mmol) was added in one portion. The
reaction was allowed to reach RT and was stirred overnight. Reac-
tion was diluted with EtOAc, washed with sat. NaHCO3, dried over
MgSO4 and solvents were evaporated to give crude title compound
(0.267 g, 89%) which was used in next step without further purifi-
18H), 7.22 (dd, J ¼ 5.1, 3.6 Hz, 1H), 7.53e7.56 (m, 4H), 7.60 (m, 2H),
7.60e7.62 (m, 4H), 7.73 (dd, J ¼ 5.1, 1.2 Hz, 1H); 13C NMR (101 MHz,
DMSO‑d6)
d 28.7, 81.3, 111.9, 119.8, 123.2, 127.0, 127.4, 128.2, 129.4,
129.48, 129.54, 129.9, 130.2, 130.5, 144.0, 144.5, 150.8. LCMS: (A)
retention time 1.96 min, m/z 463 [MþH]þ.
cation. 1H NMR (400 MHz, chloroform-d)
d 3.30 (s, 3H), 3.79 (s, 4H),
3.90 (s, 6H), 7.05 (s, 2H).
4.1.33. 2,6-Dimethoxy-4-(1-methyl-4-phenyl-5-(thiophen-2-yl)-
1H-imidazol-2-yl)phenol and 2,6-dimethoxy-4-(1-methyl-5-
phenyl-4-(thiophen-2-yl)-1H-imidazol-2-yl)phenol (17a, 17b)
Diketone 4 (100 mg, 0.46 mmol), syringaldehyde (1, 84 mg,
0.46 mmol), 2 M methylamine solution in THF (0.23 mL,
0.46 mmol), ammonium acetate (36 mg, 0.46 mmol) and sodium
phosphate monobasic (17 mg, 0.14 mmol) were heated together in
a sealed tube at 150 ꢀC for 5 h. The reaction was cooled to rt and
stirred over weekend, then it was concentrated in vacuo. The res-
idue was purified by Biotage Isolera One flash chromatography
(40e50% EtOAc/hexanes) to afford 30 mg (17%) of mixture of title
compounds 17a and 17b as a light brown solid. Regioisomers 17a
and 17b were not separated and were characterized as mixture;
mp > 250 ꢀC. NMR of mixture of regioisomers in 1.5:1 ratio, *de-
4.1.37. 4-(4,5-Dihydro-1H-imidazol-2-yl)-2,6-dimethoxyphenol
hydrochloride (22)
Imidazoline 21 (0.150 g, 0.50 mmol) was dissolved in MeOH
(10 mL) and saturated solution of KOH (3.0 mL) was added in one
portion. After stirring at RT for 1 h the reaction was carefully
neutralized with 1.5 N HCl to pH ¼ 7e8 then it was diluted with
MeOH (250 mL) and formed precipitates were filtered off. Solvents
were evaporated and the residue was filtered through short column
of SiO2 with 30% MeOH in CHCl3. Solvents were evaporated and
crude product was purified using reversed phase preparative HPLC
(0% MeCN/100% water for 5 min followed by an increase to 20%
MeCN over 35 min and an increase to 40% MeCN over 10 min; flow
rate 10 mL/min). The product was further purified by forced pre-
cipitation with Et2O of its HCl salt from MeOH solution to give title
compound (20 mg, 15%) as white powder; mp > 250 ꢀC (dec). 1H
notes minor isomer: 1H NMR (400 MHz, DMSO‑d6)
d 3.44 (s, 1.8H),
3.51 (s, 1.2H)*, 3.84 (s, 6H), 6.77 (dd, J ¼ 3.6, 1.2 Hz, 0.6H), 6.88 (dd,
J ¼ 5.1, 3.6 Hz, 0.6H), 6.94 (s, 1.2H), 6.98 (s, 0.8H)*, 7.14e7.20 (m,
0.4H)*, 7.27 (dd, J ¼ 5.1,1.1 Hz, 0.6H), 7.23e7.31 (m,1.6H)*, 7.49e7.54
(m, 0.8H)*, 7.48e7.64 (m, 2H), 7.83 (dd, J ¼ 4.7, 1.7 Hz, 0.4H)*, 8.78
NMR (400 MHz, DMSO‑d6)
d
3.84 (s, 4H), 3.95 (s, 6H), 7.53 (s, 2H),
44.0,
9.78 (bs, 1H), 10.65 (bs, 2H); 13C NMR (101 MHz, DMSO‑d6)
d
(s, 1H); 13C NMR (100 MHz, DMSO‑d6)
d 32.7*, 33.0, 56.2, 106.6,
56.6, 106.9, 111.1, 141.5, 147.9, 164.5. LCMS: (B) retention time
120.3,120.5*,121.6,121.8*,123.6,126.3*,126.5*,127.2,128.1*,128.2*,
129.0, 129.1, 129.4*, 130.1, 130.6*, 130.8*, 130.9,131.7, 134.4*, 136.45*,
136.48, 138.4*, 138.5, 147.3, 147.8, 148.2*. LCMS: (A) retention time
1.64 min, m/z 393 [MþH]þ.
0.42 min, m/z ¼ 223 [M þ H]þ.
4.1.38. 2-Hydroxy-3-methyl-1-phenylbutan-1-one (24a)
To a solution KOH (0.308 g, 5.5 mmol) in MeOH (2 mL) was
added ketone 23 (0.171 g, 1.0 mmol) and the mixture was cooled to
0 ꢀC. Then (diacetoxyiodo)benzene (0.354 g, 1.1 mmol) was added
and reaction was stirred 48 h at RT. Reaction was diluted with water
(10 mL) and extracted with Et2O (3 ꢃ 5 mL). Combined organic
phases were sequentially washed with saturated NaHCO3 solution
and brine, dried over MgSO4 and solvent was evaporated to give
crude title compound (95 mg) which was used in next step without
further purification. m/z ¼ 179 [M þ H]þ, 161 [M ꢁ H2O þ H]þ.
4.1.34. 2,6-Dimethoxy-4-(thiazol-2-yl)phenol (19)
Compound 18 (0.032 g, 0.18 mmol) was dissolved in DCM
(1 mL), cooled to 0 ꢀC and BCl3/DCM solution (1 M, 0.42 mL,
0.42 mmol) was added dropwise. The reaction was allowed to
warm to rt and stirred at rt for 2 h. The reaction was quenched with
MeOH/water (1:1, 1 mL), then solvents were evaporated. The
resulting residue was purified using reversed phase preparative
HPLC (20% MeCN/80% water followed by an increase to 70% MeCN
over 40 min and an increase to 100% MeCN over 10 min; flow rate
15 mL/min) to give title compound (6 mg, 15%) as a brown solid; mp
4.1.39. 4-(5-Isopropyl-4-phenyl-1H-imidazol-2-yl)-2,6-
dimethoxyphenol (25a)
136 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
(d, J ¼ 3.2 Hz, 1H), 7.83 (d, J ¼ 3.3 Hz, 1H), 8.97 (s, 1H); 13C NMR
d
3.84 (s, 6H), 7.17 (s, 2H), 7.68
Crude compound 24a (0.095 g) was dissolved in EtOH (5 mL)
and aldehyde 1 (0.111 g, 0.6 mmol) and NH4OAc (0.457 g, 6.0 mmol)
were added. The reaction was stirred 16 h at 65 ꢀC. Workup as
described for 3b. Crude product was purified using column chro-
matography on silica (DCM þ 1e2% MeOH) to give 55 mg (16%) of
title compound as black solid. Purity (HPLC) ¼ 90%; mp 239 ꢀC.
(101 MHz, DMSO‑d6)
d 56.1, 103.7, 119.3, 123.7, 137.8, 143.3, 148.2,
167.6. LCMS: (A) retention time 0.95 min, m/z 238 [M þ H]þ.
4.1.35. 4-Formyl-2,6-dimethoxyphenyl methanesulfonate (20)
A solution of 4-hydroxy-3,5-dimethoxybenzaldehyde (10.0 g,
54.90 mmol) in DCM (200 mL) and Et3N (23.0 mL, 163.70 mmol)
was cooled to 0 ꢀC, then methanesulfonyl chloride (6.670 mL,
94.60 mmol) was added dropwise and reaction was stirred at RT
16 h. Reaction mixture was poured onto ice; after melting phases
were separated and water phase was extracted with DCM
(3 ꢃ 50 mL). Combined organic phases were sequentially washed
NMR measured as HCl salt: 1H NMR (400 MHz, DMSO‑d6)
d 1.44 (d,
J ¼ 7.2 Hz, 6H), 3.15e3.22 (m, 1H), 3.91 (s, 6H), 7.44e7.71 (m, 5H),
7.82 (s, 2H), 9.38 (bs, 1H), 14.44 (bs, 1H), 14.96 (bs, 1H); 13C NMR
(101 MHz, DMSO‑d6)
d 21.8, 24.4, 56.7, 105.4, 112.6, 126.9, 127.4,
129.0, 129.1, 129.2, 135.3, 139.1, 143.9, 148.2. LCMS: (B) retention
time 2.21 min, m/z ¼ 339 [M þ H]þ.

O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
287
4.1.40. 4-(4,5-Diisopropyl-1H-imidazol-2-yl)-2,6-dimethoxyphenol
hydrochloride (25b)
increase to 70% MeCN over 10 min; flow rate 15 mL/min) and
further by forced precipitation with Et2O of HCl salt from its MeOH
solution. After filtration and drying 12 mg (14%) of title compound
as pale brown-gray solid was obtained. Purity (HPLC) ¼ 90%;
4-Hydroxy-2,5-dimethylhexan-3-one (0.152 g, 1.0 mmol) was
dissolved in EtOH (6 mL) and aldehyde 1 (0.185 g, 1.0 mmol) and
NH4OAc (0.770 g, 10.0 mmol) were added. The reaction was stirred
16 h at RT. Workup as described for 3b. Purification using Biotage
Isolera One flash chromatography (5e20% MeOH/DCM) followed by
forced precipitation with Et2O of HCl salt from MeOH solution
afforded title compound (0.147g, 43%) as deep purple solid;
mp > 125 ꢀC (dec). 1H NMR (500 MHz, Methanol-d4)
d 2.47 (s, 3H),
3.93 (s, 6H), 7.13e7.15 (m, 1H), 7.27 (s, 2H), 7.34 (d, J ¼ 3.6 Hz, 1H),
7.44 (d, J ¼ 5.0 Hz, 1H), 8.27 (s, 1H); 13C NMR (126 MHz, Methanol-
d4)
d 11.2, 56.9, 104.5, 119.4, 125.8, 125.9, 127.7, 128.5, 128.6, 134.7,
146.6,149.7. LCMS: (B) retention time 2.04 min, m/z ¼ 316 [M þ H]þ.
mp > 230 ꢀC (dec); NMR (400 MHz, DMSO‑d6)
12H), 3.19 (hept, J ¼ 6.9 Hz, 2H), 3.90 (s, 6H), 7.63 (s, 2H), 9.31 (s,
1H), 14.05 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
21.9, 24.0, 56.7,
d
1.34 (d, J ¼ 6.9 Hz,
4.1.45. (2S,3S,4S,5R,6S)-6-(4-(4,5-diisopropyl-1H-imidazol-2-yl)-
2,6-dimethoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-
carboxylic acid (30)
d
105.4, 112.8, 132.8, 139.0, 143.2, 148.2. LCMS: (B) retention time
2.43 min, m/z ¼ 305 [M þ H]þ.
To a solution of 25b (130 mg, 0.43 mmol) and 2,3,4-Tri-O-acetyl-
a
-D-glucuronide methyl ester trichloroacetimidate (362 mg,
4.1.41. 4-Cyano-2,6-dimethoxyphenyl methanesulfonate (26)
To a suspension of compound 20 (5.20 g, 20.0 mmol) in H2O
(100 mL) was added AcOH (1.21 mL, 21.10 mmol) and hydroxyl-
amine sulfonic acid (2.38 g, 21.1 mmol) and reaction was stirred
16 h at 100 ꢀC. The precipitate was filtered and thoroughly washed
with water to give 4.88 g (98%) of title compound as white solid. 1H
0.76 mmol) in dry DCM (6.0 mL) cooled to ꢁ10 ꢀC was under N2
atmosphere added BF3∙OEt2 complex (29 mL, 0.22 mmol) and the
reaction was stirred 4 h at ꢁ10 ꢀC until all starting material was
consumed as indicated by LCMS analysis. Then the reaction was
quenched by addition of Et3N (200 mL), solvents were evaporated
and the residue was purified using flash chromatography
(DCM þ MeOH, 0e15%). Obtained brow glassy solid (52 mg) was
dissolved in MeOH and solution of Na2CO3 (25 mg, 0.27 mmol) in
H2O (0.5 mL) was added. The reaction was stirred at RT 3 h until
complete consumption of intermediate was confirmed by LCMS
analysis. Solvents were evaporated and the residue was purified
using preparative HPLC (10% MeCN/90% water for 5 min followed
by an increase to 50% MeCN over 35 min and an increase to 70%
MeCN over 10 min; flow rate 15 mL/min) to give title compound
(18 mg, 9%) as white solid; mp > 180 ꢀC (dec). 1H NMR (500 MHz,
NMR (400 MHz, chloroform-d)
d 3.33 (s, 3H), 3.93 (s, 6H), 6.91 (s,
2H); m/z ¼ 258 [M þ H]þ.
4.1.42. 4-Carbamimidoyl-2,6-dimethoxyphenyl methanesulfonate
(27)
To a solution of compound 26 (4.88 g, 18.99 mmol) in dry MeOH
(30 mL) was added solution of HCl in dioxane (4 N, 60 mL) and
reaction was stirred at RT under N2 atmosphere 4 days. Solvents
were evaporated, residue was dried on high vacuum 4 h and then it
was dissolved in solution of NH3 in MeOH (7 N, 100 mL) and reac-
tion was stirred at RT under N2 atmosphere 7 days. LCMS showed
most of the product was converted to amide probably due to wet
solution of NH3/MeOH. Solvents were evaporated and residue was
chromatographed on silica (CHCl3/MeOH) to give 1.02 g (20%) of
Methanol-d4)
d
1.37 (dd, J ¼ 16.8, 7.0 Hz, 12H), 3.21 (hept, J ¼ 7.1 Hz,
2H), 3.42e3.58 (m, 3H), 3.53 (s, 3H), 3.64 (d, J ¼ 9.7 Hz, 1H), 5.76 (d,
J ¼ 7.4 Hz, 1H), 7.04 (s, 2H). 13C NMR (125 MHz, Methanol-d4)
d 22.5,
22.6, 26.1, 57.1, 73.4, 75.4, 75.7, 77.7, 101.4, 105.4, 118.7, 134.8, 136.7,
143.2, 153.2, 176.4. LCMS: (B) retention time 2.08 min, m/z ¼ 418
[M þ H]þ.
title compound as white foam. 1H NMR (500 MHz, DMSO‑d6)
d 3.46
(s, 3H), 3.93 (s, 6H), 7.30 (s, 2H), 9.40 (s, 3H); m/z ¼ 275 [M þ H]þ.
4.2. In vitro nSMase2 assay
4.1.43. 2,6-dimethoxy-4-(5-methyl-4-(thiophen-2-yl)-1H-
imidazol-2-yl)phenyl methanesulfonate (28)
The inhibitory potencies of the synthesized compounds were
determined using a fluorescence-based coupled assay involving
human recombinant nSMase2, alkaline phosphatase (AP), and
choline oxidase as previously reported [18]. Briefly, human
nSMase2 recombinant enzyme was used to catalyze the hydrolysis
of sphingomyelin into ceramide and phosphorylcholine. Phos-
phorylcholine undergoes dephosphorylation by AP to produce
choline which is subsequently oxidized by choline oxidase. H2O2
generated in this oxidative step is detected by horseradish
peroxidase-catalyzed conversion of Amplex Red® into resorufin
(ex/em 530/590 nm). In order to assure that potent inhibitors
identified in this coupled assay directly inhibit nSMase2, they were
subsequently assessed in a counter assay using AP, choline oxidase,
and horseradish peroxidase (but devoid of nSMase2 and sphingo-
myelin) in the presence of phosphorylcholine as previously
described [18].
To a solution of amidine 27 (0.274 g, 1.0 mmol) and NaHCO3
(0.252 g, 3.0 mmol) in EtOH (10 mL) was added 2-bromo-1-(thio-
phen-2-yl)propan-1-one (0.438 g, 2.0 mmol) and the reaction was
stirred 16 h at 60 ꢀC. Reaction was cooled to RT, solids were filtered
off and solvent was evaporated. The residue was purified using
Biotage Isolera One flash purification system with a silica gel car-
tridge (CHCl3) to give 0.190 g, (48%) of title compound as pale yel-
low solid. 1H NMR (400 MHz, DMSO‑d6)
d 2.47 (s, 3H), 3.41 (s, 3H),
3.91 (s, 6H), 7.09 (dd, J ¼ 5.1, 3.6 Hz, 1H), 7.19e7.22 (m, 1H), 7.31 (s,
2H), 7.38e7.41 (m, 1H), 12.57 (s, 1H); m/z ¼ 395 [M þ H]þ.
4.1.44. 2,6-Dimethoxy-4-(5-methyl-4-(thiophen-2-yl)-1H-
imidazol-2-yl)phenol hydrochloride (29)
To a solution of diisopropyl amine (0.092 mL, 0.64 mmol) in dry
THF (3 mL) cooled to ꢁ78 ꢀC was under N2 atmosphere added so-
lution of n-butyllithium in hexanes (2.5 M, 0.244 mL, 0.61 mmol)
and mixture was let to reach 0 ꢀC. Then solution of methansulfo-
nate 28 (0.096 g, 0.24 mmol) in dry THF (2 mL) was added in one
portion and reaction was stirred under N2 atmosphere 1 min at
0 ꢀC. Reaction was quenched by addition of HCl solution (5%,10 mL).
Reaction was diluted with water (20 mL) and extracted with EtOAc
(3 ꢃ 15 mL). Combined organic layers were washed with brine,
dried over MgSO4, filtered and solvents were evaporated. The res-
idue was purified using preparative HPLC (10% MeCN/90% water for
5 min followed by an increase to 50% MeCN over 35 min and an
4.3. In vitro metabolic stability studies
The metabolic stability was evaluated using mouse liver mi-
crosomes. For the cytochrome P450 (CYP)-mediated metabolism,
the reaction was carried out with 100 mM potassium phosphate
buffer, pH 7.4, in the presence of NADPH regenerating system
(1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl2, 0.4
U/mL glucose-6-phosphate dehydrogenase, 50
mM sodium citrate).
Reactions, in triplicate, were initiated by addition of the liver

288
O. Stepanek et al. / European Journal of Medicinal Chemistry 170 (2019) 276e289
microsomes to the incubation mixture (compound final concen-
tration was 5 M; 0.5 mg/mL microsomes). For the glucuronidation
R01AG059799 (CR), and P30MH075673 (BSS). OS is supported by a
joint postdoctoral fellowship from Johns Hopkins Drug Discovery
and the Institute of Organic Chemistry and Biochemistry (IOCB) of
the Czech Academy of Sciences.
m
reaction, a test compounds were added to TRIS-HCl buffer (50 mM,
pH 7.5) with microsomes (0.5 mg/mL), along with MgCl2 (8 mM)
and alamethicin (25 m
g/mL), and preincubated at 37 ꢀC. The reac-
tion was initiated (in triplicate) with UDPGA (2 mM; final concen-
tration). Controls in the absence of cofactors were carried out to
determine the specific cofactor-free degradation. After 60 min of
incubation, aliquots of the mixture were removed and the reaction
quenched by addition of three times the volume of ice-cold
acetonitrile spiked with the internal standard. Compound disap-
pearance was monitored over time using a liquid chromatography
and tandem mass spectrometry (LC/MS/MS) method.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.03.015.
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All procedures involving mice were approved by and conformed
to the guidelines of the Institutional Animal Care Committee of the
Johns Hopkins University. Male CD-1 mice between 25 and 30 g
were obtained from Harlan, and maintained on a 12-h light-dark
cycle with ad libitum access to food and water. Three animals
were used per time-point for each treatment group. DPTIP and 25b
were prepared immediately prior to dosing in 5% DMSO, 5% poly-
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oral dose by oral gavage of 10 mg/kg. The mice were sacrificed at
specified time points (0.5 and 2 h) post-drug administration. For
collection of plasma and brain tissue, animals were euthanized
with CO2, and blood samples were collected in heparinized
microtubes by cardiac puncture. Brains were dissected and imme-
diately flash frozen (ꢁ80 ꢀC). Blood samples were spun at 2000ꢃg
for 15 min, plasma was removed and stored at ꢁ80 ꢀC until LC-MS
analysis. Prior to extraction, frozen samples were thawed on ice.
The calibration curves were developed using plasma and brain from
naïve animals as a matrix. Plasma samples (50
using a single liquid extraction method by addition of 300
acetonitrile as with internal standard (losartan: 0.5 M), followed
mL), were processed
mL of
m
by vortex mixing for 30 s and then centrifugation at 10,000ꢃg for
10 min at 4 ꢀC. Fifty microliter of the supernatant is diluted with
50 mL of water and transferred to 250 mL polypropylene autosam-
pler vials sealed with a Teflon cap. For brain tissue analysis, brain
samples were weighed in the range of (80e100 mg) to which 2-vol
of acetonitrile as with internal standard (losartan: 0.5
added and homogenized for extraction of the analyte. Samples
were vortex mixed for 1 min and centrifuged as above. A 20
aliquot of supernatant was diluted with 20 L of water and trans-
ferred to 250 L polypropylene autosampler vials sealed with a
Teflon cap. A volume of was injected onto the ultra-
mM) was
mL
m
m
3 mL
performance liquid chromatography (UPLC) instrument for quan-
titative analysis by LC-MS/MS. Plasma and brain samples were
analyzed on a Thermo Scientific Accela UPLC system coupled to
Accela open autosampler at ambient temperature with an Agilent
Eclipse Plus column (100 ꢃ 2.1 mm i.d.) packed with a 1.8
mm C18
stationary phase. The autosampler was temperature controlled and
operated at 10 ꢀC. The mobile phase used for the chromatographic
separation was composed of acetonitrile/water containing 0.1%
formic acid and with a flow rate of 0.4 mL/min for 4.5 min using
gradient elution. The column effluent was monitored using TSQ
Vantage triple-quadrupole mass-spectrometric detector, equipped
with an electrospray probe set in the positive ionization mode.
Samples were introduced into the ionization source through a
heated nebulized probe (350 ꢀC).
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