Discovery of Benzamidine- And 1-Aminoisoquinoline-Based Human MAS-Related G-Protein-Coupled Receptor X1 (MRGPRX1) Agonists

Article abstract of DOI:10.1021/acs.jmedchem.9b01003

Mas-related G-protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and has been actively investigated as a therapeutic target for the treatment of pain. By use of two HTS screening hit compounds, 4-(4-(benzyloxy)-3-methoxybenzylamino)benzimidamide (5a) and 4-(2-(butylsulfonamido)-4-methylphenoxy)benzimidamide (11a), as molecular templates, a series of human MRGPRX1 agonists were synthesized and evaluated for their agonist activity using HEK293 cells stably transfected with human MrgprX1. Conversion of the benzamidine moiety into a 1-aminoisoquinoline moiety carried out in the later stage of structural optimization led to the discovery of a highly potent MRGPRX1 agonist, N-(2-(1-aminoisoquinolin-6-yloxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (16), not only devoid of positively charged amidinium group but also with superior selectivity over opioid receptors. In mice, compound 16 displayed favorable distribution to the spinal cord, the presumed site of action for the MRGPRX1-mediated analgesic effects.

Full text of DOI:10.1021/acs.jmedchem.9b01003

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Discovery of Benzamidine- and 1‑Aminoisoquinoline-Based Human
MAS-Related G‑Protein-Coupled Receptor X1 (MRGPRX1) Agonists
†,‡
Eva Prchalova, Niyada Hin,† Ajit G. Thomas,† Vijayabhaskar Veeravalli,†,‡ Justin Ng,† Jesse Alt,†
́
Rana Rais,†,‡ Camilo Rojas,†,§ Zhe Li,∥ Hiroe Hihara,⊥ Mika Aoki,⊥ Kyoko Yoshizawa,⊥
Tomoki Nishioka,⊥ Shuichi Suzuki,⊥ Theresa Kopajtic,# Sheena Chatrath,∇ Qin Liu,∇ Xinzhong Dong,∥
Barbara S. Slusher,†,‡,∥ and Takashi Tsukamoto*,†,‡
‡
§
†Johns Hopkins Drug Discovery, Department of Neurology, Department of Molecular and Comparative Pathobiology, and
∥Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland 21205, United States
⊥Tsukuba Research Laboratories, Eisai Co., Ltd., Tsukuba, Ibaraki 300-2635, Japan
#Biobehavioral Imaging and Molecular Neuropsychopharmacology Unit, National Institute on Drug Abuse-Intramural Research
Program, National Institutes of Health, Baltimore, Maryland 21224, United States
∇Department of Anesthesiology and Center for the Study of Itch, Washington University School of Medicine, St. Louis, Missouri
63110, United States
S
* Supporting Information
ABSTRACT: Mas-related G-protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and has
been actively investigated as a therapeutic target for the treatment of pain. By use of two HTS screening hit compounds, 4-(4-
(benzyloxy)-3-methoxybenzylamino)benzimidamide (5a) and 4-(2-(butylsulfonamido)-4-methylphenoxy)benzimidamide
(11a), as molecular templates, a series of human MRGPRX1 agonists were synthesized and evaluated for their agonist
activity using HEK293 cells stably transfected with human MrgprX1. Conversion of the benzamidine moiety into a 1-
aminoisoquinoline moiety carried out in the later stage of structural optimization led to the discovery of a highly potent
MRGPRX1 agonist, N-(2-(1-aminoisoquinolin-6-yloxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (16), not only devoid
of positively charged amidinium group but also with superior selectivity over opioid receptors. In mice, compound 16 displayed
favorable distribution to the spinal cord, the presumed site of action for the MRGPRX1-mediated analgesic effects.
clinical translation. Human MRGPRX1 agonists 1 and 2
(Figure 1) reported by GSK7 and ACADIA,8 respectively, may
have a potential to be explored as new leads though their high
molecular weight (>500) likely contributes to unfavorable
CNS drug-like molecular properties and solubility. Indeed,
compound 1 could not be tested in vitro at concentrations
above 2 μM due to its limited solubility.6
INTRODUCTION
■
A subset of Mas-related G-protein-coupled receptors (Mrgprs)
is expressed specifically in the small diameter DRG sensory
neurons and is implicated in the modulation of nociception.1
Among these, human homologue MRGPRX1 has gained
increased interest as a promising therapeutic target partly
owing to the analgesic effects of BAM8-22 (Figure 1),2
a
This prompted us to conduct high throughput screening of
Eisai’s compound library in HEK293 cells stably transfected
with human MRGPRX1 by using FLIPR calcium 4 assay kit
(Molecular Devices) to measure intracellular calcium re-
sponses.9 We identified two submicromolar hits 5a and 11a
(Figure 2) as full agonists with EC50 values of 0.51 μM and
0.92 μM, respectively. The common structural feature of the
two lead compounds is obviously the presence of benzamidine
moiety, which is known as a bioisostere of a guanidyl group of
proteolytic product derived from proenkephalin A with full
agonist activity at human MRGPRX1 and its closest rodent
orthologues, mouse MRGPRC11 and rat MRGPRC.3 For
example, intrathecal (spinal cord) injection of BAM8-22 was
found to attenuate both mechanical and thermal hyperalgesia
in mice4 and rats.5 We have also found that JHU-58 (Figure
1), an Arg-Phe-NH2 peptidomimetic with full agonist activity
at mouse MRGPRC11 and rat MRGPRC,6 exhibits analgesic
effects in rodent models of neuropathic pain.5 JHU-58,
however, displayed negligible agonist activity at human
MRGPRX1, hindering its ability to serve as a molecular
template for further structural optimization efforts aimed at
Received: June 24, 2019
© XXXX American Chemical Society
A
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Figure 1. Representative Mrgpr agonists.
a
Scheme 1. Synthesis of Compounds 5a−h
Figure 2. New lead human MRGPRX1 agonists 5a and 11a identified
by HTS.
arginine. Thus, it is conceivable that the benzamidine portion
of these lead compounds shares a binding site with the Arg
residue of BAM8-22. While compound 1 is more potent than
5a and 11a, what differentiates these HTS hits from the
existing human MRGPRX1 agonists 1 and 2 is their higher
ligand efficiency10 (LE = 0.32−0.33) values owing to their
lower molecular weight (MW = 361) as compared to those of
1 (LE = 0.27) and 2 (LE = 0.19).
Here, we describe the design, synthesis, and structure−
activity relationships (SARs) of a new series of human
MRGPRX1 agonists using the new leads 5a and 11a as
molecular templates. Our optimization efforts aimed at not
only improving agonist potency but also replacing the
benzamidine group with other functional groups with reduced
basicity in an attempt to improve CNS permeability. These
efforts led to the discovery of a highly potent and CNS-
penetrant agonist potentially valuable in exploring the
therapeutic utility of human MRGPRX1 activation in neuro-
pathic pain.
a
Reagents and conditions: (a) NaCNBH3, DIEA, AcOH, EtOH, rt.
1,6-diamine 12 mediated by NaCNBH3. Sulfonamide-based
agonists 16 and 19 were obtained from 6b, which was first
coupled with 14 or 17 in the presence of potassium carbonate
to give 15 or 18, respectively. Catalytic hydrogenation of the
nitro group of 15 and 18 followed by reaction with 2-
methoxybenzene-1-sulfonyl chloride in pyridine provided the
desired compounds 16 and 19.
RESULTS AND DISCUSSION
■
In vitro experiments measuring agonist activity at MRGPRX1
were performed in a FLIPR assay using HEK293 cells stably
transfected with human MrgprX1. The results are summarized
in Table 1. All of the compounds acted as full MRGPRX1
agonists (Emax > 95% of BAM8-22) except for compounds 5c
and 19, which showed negligible agonist activity at
concentrations up to 100 μM. It appears that the position of
the amidine group has significant impact on the agonist
activity. Meta- (compound 5b) and ortho-substitution
(compound 5c) resulted in 6-fold and >100-fold decreases in
potency, respectively, as compared to 5a. This suggests the
critical role played by the para-amidine group of compound 5a
in strong interaction with MRGPRX1. Replacement of the
terminal benzyl group with a 3-pyridinylmethyl group led to
10-fold loss of potency (compound 5d). In contrast,
substitution with 3-thiophenylmethyl group (compound 5e)
and [4-(1H-pyrrol-1-yl)phenyl]methyl (compound 5f) re-
sulted in similar potency to 5a. Meanwhile, both compound
5g containing an aliphatic group instead of the benzyl group
and compound 5h devoid of the methoxy group from the
linker region exhibited decreased agonist potency.
CHEMISTRY
■
Compounds 5a−h were synthesized by reductive amination of
substituted benzaldehydes 3a−f with aminobenzamidines 4a−
c mediated by NaCNBH3 (Scheme 1).
Compounds 11a−i were synthesized in four steps starting
with nucleophilic aromatic substitution of 1-fluoro-2-nitro-
benzene derivatives 6a−c with hydroxybenzonitriles 7a−c in
the presence of potassium carbonate as a base (Scheme 2).11
The nitro group of resulting 2-nitrodiphenyl ethers 8a−e
underwent catalytic hydrogenation, yielding the corresponding
amines 9a−e. Reaction of 9a−e with sulfonyl chlorides
afforded sulfonamides 10a−i. Conversion of the nitrile group
of 10a−i into amidine group via Pinner reaction followed by
treatment with ammonia12 gave the desired compounds 11a−i.
As shown in Scheme 3, we also synthesized analogs of
compounds 5a and 11a, in which the highly basic amidine
moiety is replaced by less basic bioisosteres. Compound 13
containing an 1-aminoisoquinoline was synthesized by
reductive amination of benzaldehyde 3a with isoquinoline-
Given the preference for para-amidine substitution observed
in analogs of 5a, SAR studies on lead compound 11a focused
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a
Scheme 2. Synthesis of Compounds 11a−i
a
Reagents and conditions: (a) K2CO3, DMF, 50 °C; (b) H2, 10% Pd/C, MeOH, 20−30 psi, 30 min; (c) R2-SO2Cl, pyridine, 50 °C, 1.5 h; (d) (i)
HCl (gas), MeOH, 0 °C to rt, (ii) 7 N NH3 in MeOH, 65 °C, 1 h.
a
Scheme 3. Synthesis of Compounds 13, 16, and 19
a
Reagents and conditions: (a) NaCNBH3, DIEA, AcOH, EtOH, rt; (b) ArOH, K2CO3, DMF, 50 °C; (c) (i) H2, Pd/C, MeOH, 20−30 psi, 30 min;
(ii) 2-methoxybenzene-1-sulfonyl chloride, pyridine, 50 °C, 1.5 h.
on its non-benzamidine portion. Structural changes in the
internal linker and/or the terminal alkylsulfonamide group had
various impact in MRGPRX1 agonist potency as seen in
compounds 11a−g. The most potent agonists within this
series, compounds 11e and 11g, exhibited EC50 values of 150
and 100 nM, respectively. Using compound 11g as a template,
we moved the amidine group to the meta (compound 11h)
and the ortho (compound 11i) positions. Both compounds
exhibited decreased agonist potency compared to 11g,
confirming the critical role played by the para-amidine group
in MRGPRX1 agonist potency.
Compounds 13, 16, and 19 possess a potential bioisostere of
the benzamidine group, an effective strategy often used in
inhibitors of serine proteases involved in the coagulation
cascade.13−16 Compound 13 contains a 1-aminoisoquinoline
instead of the benzamidine group of compound 5a. This
modification resulted in decline of agonist potency from 0.51
μM (compound 3a) to 3.7 μM (compound 13). In contrast,
similar modification to compound 11g resulted in the most
potent agonist 16 with an EC50 value of 0.05 μM. Compound
19 containing a 2-aminobenzothiazole, however, showed no
agonist activity at concentrations up to 100 μM. The discovery
of agonist 16 is significant not only because of its high agonist
potency but also because of its substantially lower pKa value for
the 1-amino group of the aminoisoquinoline (pKa ∼ 7.5) as
compared to that of the benzamidine moiety (pKa ∼ 12.5) of
compound 11g.13−16
Given the complete loss of agonist activity for ortho-amidine
analog 5c, structurally close compounds 5a and 5c served as
ideal positive and negative controls in target engagement
studies. Activation of MRGPRX1 is known to inhibit high-
voltage-activated (HVA) Ca2+ currents, which reduces neuro-
transmitter release and attenuates spinal nociceptive trans-
mission.17 To this end, we assessed BAM8-22, potent agonist
5a, and compound 5c devoid of MRGPRX1 agonist activity for
their inhibitory effect on HVA Ca2+ currents in cultured
human MRGPRX1-expressing DRG neurons obtained from
humanized MrgprX1 mice.17 As shown in Figure 3, both
BAM8-22 and compound 5a inhibited HVA Ca2+ currents
while compound 5c showed no inhibitory effects, indicating
the MRGPRX1-dependent mechanism involved in inhibition
of HVA Ca2+ currents.
Given the close relation between opioid and MrgC receptors
in regard to ligand pharmacology2,3 and function,18,19 we
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Table 1. In Vitro MRGPRX1 Agonist Potency of Compounds 5a−h, 11a−i, 13, 16, and 19
a
Values are the mean SD of at least four experiments.
assessed the selectivity of the two most potent MRGPRX1
agonists 11g and 16 over δ-, μ-, and κ-opioid receptors by
radioligand binding assays. As shown in Table 2, benzamidine-
based agonist 11g exhibited substantial affinity to δ- and μ-
opioid receptors. Compound 16, however, exhibited 50-fold or
greater selectivity over all three receptors, unmasking addi-
tional advantage brought by the 1-aminoisoquinoline moiety.
Compound 16 was also found to be inactive against mouse
MRGPRC11 even at concentration of 40 μM (see the
Supporting Information).
Given its reduced basicity, potent MRGPRX1 agonist
activity, and good selectivity over opioid receptors, we selected
compound 16 for in vivo pharmacokinetics studies in mice. In
addition to plasma levels, we measured drug levels in spinal
cord, which is the site of action for MRGPRX1 agonists to
attenuate spinal nociceptive transmission. As shown in Figure
4, substantial levels of 16 were detected in plasma and spinal
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Figure 3. Traces of HVA Ca2+ currents evoked by depolarization before (black) and after (red) bath application of 5 μM BAM8-22 (A), 10 μM 5a
(B), and 10 μM 5c (C). Currents were recorded by whole-cell patch clamp recording of DRG neurons from MrgprX1 mice. HVA Ca2+ currents are
inward currents (negative nA) evoked by −10 mV depolarization.
MRGPRX1 agonist 16 not only devoid of positively charged
amidinium group but also with superior selectivity over opioid
receptors. Compound 16 displayed favorable distribution to
the spinal cord, presumably due to the reduced pKa value for
the 1-amino group of the aminoisoquinoline as compared to
that of the benzamidine moiety. Although compound 16
showed a high degree of clearance, the preferential distribution
to the spinal cord over the circulatory system is particularly
appealing since it will minimize the itch side effect caused by
peripheral MRGPRX1 activation. Given its high selectivity
over opioid receptors, compound 16 appears to be a promising
agent that merits further investigation. Compound 16,
however, failed to act as an agonist at mouse MrgprC11,
limiting its utility in conventional mouse models of pain. It
should be noted, however, that humanized MrgprX1 mice have
been developed to overcome the limited cross-species
reactivity of human MRGPRX1 agonists on the murine
receptors.17 These mice lack a cluster containing most MrgprA
and MrgprC genes including MrgprC11 and are well suited for
studying the pharmacology of human MRGPRX1 modulation.
Indeed, intrathecal injection of BAM8-22 was found to inhibit
heat hypersensitivity and spontaneous pain in MrgprX1 mice
after peripheral nerve injury,17 demonstrating the utility of this
strain in evaluating human MRGPRX1 agonists. Studies are
underway to evaluate our human MRGPRX1 agonists in the
humanized mice and improve the metabolic stability in liver
microsomes to achieve sustained exposure. Collectively, these
efforts may lead to the development of viable treatment
options for chronic pain.
Table 2. Binding Affinities (Ki) of 11g and 16 to Opioid
Receptors
Ki (nM)
compd
δ ([3H]DADLE)
μ ([3H]DAMGO)
κ ([3H]U-69,593)
11g
16
220
2470
72.7
3730
1430
12100
Figure 4. Pharmacokinetics of 16 in mice (10 mg/kg, iv).
cord (AUC: 0.90 0.09 nmol·h/mL plasma vs 5.66 0.33
nmol·h/g spinal cord) following intravenous administration of
16 at 10 mg/kg dose in male CD1 mice (n = 3 for each time
point), resulting in a spinal cord to plasma ratio of 6.3. While
this is encouraging, 16 had a half-life of approximately 0.33 h.
Subsequent studies revealed that 16 is extensively metabolized
(t1/2 < 15 min) in mouse liver microsomes (results not
shown), which likely contributed to the short half-life observed
in mice after intravenous administration.
EXPERIMENTAL SECTION
■
General. All solvents were reagent grade or HPLC grade. Unless
otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Syntheses of
compounds 3,20 4c,21 and 1422 were previously reported. Compounds
3c, 3d, and 3e were obtained by Mitsunobu reactions of 4-hydroxy-3-
methoxybenzaldehyde with the corresponding alcohols (ROH) by
following the method previously reported for similar reactions.23
Melting points were obtained on a Mel-Temp apparatus and are
CONCLUSIONS
■
Chronic pain remains one of the most prevalent yet
undertreated health problems. Furthermore, given the ongoing
opioid crisis, there is a critical unmet medical need for new
chronic pain therapies devoid of abuse potential and adverse
effects. Restricted expression of human MRGPRX1 in primary
nociceptive neurons could provide a substantial advantage to
its agonists over opioid-based analgesics in treating pain. By
use of two amidine-based hit compounds 5a and 11a as
molecular templates, systematic lead optimization was carried
out in an attempt to improve potency and drug-like molecular
properties. In particular, successful conversion of the
benzamidine moiety of 11g into a 1-aminoisoquinoline moiety
without loss of agonist potency led to a highly potent
uncorrected. 1H NMR spectra were recorded at 400 or 500 MHz. 13
C
NMR spectra were recorded at 101 or 125 MHz. 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 detector using a Phenomenex Luna 5 μm C18 column (21.2
mm × 250 mm, 5 μm). 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 quadrupole
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MS detector. Unless otherwise specified, the analytical HPLC
conditions involve the following: (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 × 50 mm, 3.5 μm) 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 × 50 mm, 3.5 μm) at a flow rate of 1.25 mL/min. Unless
otherwise noted, all final compounds biologically tested were
confirmed to be of ≥95% purity by the HPLC methods described
above.
benzaldehyde 3b was used in place of 4-(benzyloxy)-3-methox-
ybenzaldehyde 3a and the compound was purified using preparative
HPLC (method 20−70%: flow rate of 25 mL/min; gradient of 20%
acetonitrile/80% H2O for 5.0 min followed by an increase to 70%
acetonitrile/30% H2O over 40 min and continuation of 70%
acetonitrile/30% H2O until 50 min). White fluffy solid (35% yield);
mp 87 °C. 1H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 4.29 (d, J
= 6.1 Hz, 2H), 5.09 (s, 2H), 6.69 (d, J = 9.1 Hz, 2H), 6.83 (dd, J =
2.0, 7.8 Hz, 1H), 7.00 (dd, J = 2.5, 4.3 Hz, 2H), 7.32 (d, J = 6.3 Hz,
1H), 7.41 (dd, J = 4.8, 7.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.83 (d,
J = 8.1 Hz, 1H), 8.32 (bs, 2H), 8.53 (dd, J = 1.5, 4.8 Hz, 1H), 8.63 (d,
J = 2.0 Hz, 1H), 8.74 (bs, 1H). 13C NMR (125 MHz, DMSO-d6) δ
45.5, 55.6, 67.8, 111.6, 113.0, 113.9, 119.2, 123.6, 129.6, 132.2, 132.8,
135.7, 146.5, 149.1, 149.2, 153.3, 164.3. LCMS: (C) retention time
1.63 min, m/z = 363 [M + H]+.
4-(3-Methoxy-4-(thiophen-3-ylmethoxy)benzylamino)-
benzimidamide (5e). Compound 5e was prepared as described for
the preparation of 5a except 3-methoxy-4-(thiophen-3-ylmethoxy)-
benzaldehyde 3c was used in place of 4-(benzyloxy)-3-methoxyben-
zaldehyde 3a and the compound was purified using preparative HPLC
(method 20−70%: flow rate of 25 mL/min; gradient of 20%
acetonitrile/80% H2O for 5.0 min followed by an increase to 70%
acetonitrile/30% H2O over 40 min and continuation of 70%
acetonitrile/30% H2O until 50 min). White fluffy solid (28% yield);
mp 112 °C. 1H NMR (400 MHz, DMSO-d6) δ 3.74 (s, 3H), 4.29 (d,
J = 5.6 Hz, 2H), 5.02 (s, 2H), 6.69 (d, J = 8.8 Hz, 2H), 6.82 (dd, J =
2.3, 5.6 Hz, 1H), 6.98 (m, 2H), 7.14 (dd, J = 2.0, 3.8 Hz, 1H), 7.31 (t,
J = 5.8 Hz, 1H), 7.53 (m, 2H), 7.58 (d, J = 8.8 Hz, 2H), 8.41 (bs,
2H), 8.72 (bs, 2H). 13C NMR (125 MHz, DMSO-d6) δ 45.8, 55.7,
65.7, 111.8, 113.0, 113.8, 119.4, 124.3, 126.8, 128.0, 129.9, 132.0,
138.3, 146.9, 149.4, 153.6, 164.5. LCMS: (A) retention time 1.40 min,
m/z = 368 [M + H]+.
4-(4-(4-(1H-Pyrrol-1-yl)benzyloxy)-3-methoxy-
benzylamino)benzimidamide (5f). Compound 5f was prepared as
described for the preparation of 5a except 4-(4-(1H-pyrrol-1-
yl)benzyloxy)-3-methoxybenzaldehyde 3d was used in place of 4-
(benzyloxy)-3-methoxybenzaldehyde 3a and the compound was
purified using preparative HPLC (method 20−70%: flow rate of 25
mL/min; gradient of 20% acetonitrile/80% H2O for 5.0 min followed
by an increase to 70% acetonitrile/30% H2O over 40 min and
continuation of 70% acetonitrile/30% H2O until 50 min). White solid
(45%); mp 225 °C (dec). 1H NMR (400 MHz, DMSO-d6) δ 3.76 (s,
3H), 4.28 (d, J = 5.8 Hz, 2H), 5.06 (s, 2H), 6.26 (t, J = 2.3 Hz, 2H),
6.67 (d, J = 8.8 Hz, 2H), 6.83 (dd, J = 1.5, 7.8 Hz, 1H), 6.98 (m, 2H),
7.25 (t, J = 6.1 Hz, 1H), 7.37 (t, J = 2.3 Hz, 2H), 7.48 (m, 2H), 7.57−
7.60 (m, 4H), 8.43 (s, 1H), 8.52 (bs, 2H), 10.22 (bs, 2H). 13C NMR
(125 MHz, DMSO-d6) δ 45.6, 55.6, 69.4, 110.5, 111.5, 113.6, 119.0,
119.2, 129.2, 131.9, 134.1, 139.5, 146.7, 149.2, 153.1, 164.8, 167.6.
LCMS: (A) retention time 1.73 min, m/z = 427 [M + H]+.
4-(4-(Benzyloxy)-3-methoxybenzylamino)benzimidamide
(5a). 4-(Benzyloxy)-3-methoxybenzaldehyde 3a (300 mg, 1.24
mmol), 4-aminobenzimidamide dihydrochloride 4a (184 mg, 1.36
mmol), DIEA (2.48 mmol, 0.43 mL), and a trace of bromocresol
green were dissolved in ethanol (5 mL) at rt, and the mixture was
stirred for 15 min. Acetic acid was added dropwise until the blue
solution turned slightly green and the resulting mixture was stirred at
rt for 15 min. Sodium cyanoborohydride (311 mg, 4.95 mmol) was
added portionwise at rt, and the reaction was stirred overnight.
Volatiles were removed under reduced pressure, and the residue was
dissolved in EtOAc, washed with 2 N NaOH, water and brine. The
organic layer was dried over magnesium sulfate, filtered and solvents
were evaporated to give a white solid which was purified using
preparative HPLC (method 20−70%: flow rate of 25 mL/min;
gradient of 20% acetonitrile/80% H2O for 5.0 min followed by an
increase to 70% acetonitrile/30% H2O over 40 min and continuation
of 70% acetonitrile/30% H2O until 50 min) to give 94 mg (21%
1
yield) of the title compound as white solid; mp 181 °C. H NMR
(400 MHz, DMSO-d6) δ 3.75 (s, 3H), 4.28 (d, J = 6.1 Hz, 2H), 5.04
(s, 2H), 6.69 (d, J = 9.1 Hz, 2H), 6.82 (dd, J = 1.8, 8.2 Hz, 2H), 6.96
(d, J = 8.1 Hz, 1H), 7.00 (d, J = 1.8, 1H), 7.32 (m, 1H), 7.36−7.43
(m, 5H), 7.61 (d, J = 9.1 Hz, 2H), 8.74 (bs, 3H). 13C NMR (125
MHz, DMSO-d6) δ 45.6, 55.5, 69.9, 111.5, 113.5, 113.7, 119.2, 127.7,
128.4, 129.2, 131.9, 137.2, 146.7, 149.1, 153.0, 164.9. LCMS: (A)
retention time 1.53 min, m/z = 362 [M + H]+.
3-(4-(Benzyloxy)-3-methoxybenzylamino)benzimidamide
(5b). Compound 5b was prepared as described for the preparation of
5a except 3-aminobenzimidamide 4b was used in place of 4-
aminobenzimidamide 4a and the compound was obtained by
recrystallization in EtOAc/hexanes. Light blue solid (34% yield);
mp 153 °C. 1H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 4.22 (d,
J = 6.1 Hz, 2H), 5.04 (s, 2H), 6.49 (m, 1H), 6.78−6.88 (m, 3H),
6.94−7.00 (m, 3H), 7.17 (m, 1H), 7.32−7.41 (m, 4H), 8.24 (bs, 3H).
13C NMR (125 MHz, DMSO-d6) δ 46.1, 55.5, 69.9, 110.5, 111.6,
113.5, 114.5, 116.1, 119.3, 127.8, 128.4, 129.1, 132.5, 137.3, 146.7,
148.9, 149.1, 165.7. LCMS: (A) retention time 1.55 min, m/z = 362
[M + H]+.
4 - ( 4 - (I s o p e n t y l o x y ) - 3 - m e t h o x y b e n z y l a mi n o ) -
benzimidamide (5g). Compound 5g was prepared as described for
the preparation of 5a except 4-(isopentyloxy)-3-methoxy-
benzaldehyde 3e was used in place of 4-(benzyloxy)-3-methox-
ybenzaldehyde 3a and the compound was purified using preparative
HPLC (method 20−70%: flow rate of 25 mL/min; gradient of 20%
acetonitrile/80% H2O for 5.0 min followed by an increase to 70%
acetonitrile/30% H2O over 40 min and continuation of 70%
acetonitrile/30% H2O until 50 min). White hygroscopic solid (37%
2-(4-(Benzyloxy)-3-methoxybenzylamino)benzimidamide
(5c). Compound 5c was prepared as described for the preparation of
5a except 2-aminobenzimidamide dihydrochloride 4c was used in
place of 4-aminobenzimidamide dihydrochloride 4a and the residue
was purified using a Biotage Isolera One flash purification system with
a silica gel cartridge (EtOAc/hexanes, 2% Et3N), followed by a second
purification by preparative HPLC (method 20−70%: flow rate of 25
mL/min; gradient of 20% acetonitrile/80% H2O for 5.0 min followed
by an increase to 70% acetonitrile/30% H2O over 40 min and
continuation of 70% acetonitrile/30% H2O until 50 min). Off-white
solid foam (4% yield); mp 172 °C. 1H NMR (400 MHz, DMSO-d6) δ
3.76 (s, 3H), 4.28 (bs, 2H), 5.04 (s, 2H), 6.59 (dd, J = 8.8, 17.2 Hz,
2H), 6.89 (dd, J = 8.3, 19.7 Hz, 2H), 7.05 (d, J = 1.5, 1H), 7.23 (m,
2H), 7.31 (m, 1H), 7.36−7.43 (m, 4H), 8.44 (s, 1H). 13C NMR (125
MHz, DMSO-d6) δ 46.2, 55.5, 69.9, 111.4, 111.7, 113.4, 115.2, 115.5,
119.0, 127.8, 128.4, 129.0, 132.4, 137.3, 145.8, 146.6, 149.1, 166.4,
167.9. LCMS: (B) retention time 2.50 min, m/z = 362 [M + H]+.
4-(3-Methoxy-4-(pyridin-3-ylmethoxy)benzylamino)-
benzimidamide (5d). Compound 5d was prepared as described for
the preparation of 5a except 3-methoxy-4-(pyridin-3-ylmethoxy)-
1
yield). H NMR (400 MHz, DMSO-d6) δ 0.90 (d, J = 6.6 Hz, 6H),
1.55 (qt, J = 6.8, 13.4 Hz, 2H), 1.76 (m, 1H), 3.73 (s, 3H), 3.90 (t, J =
6.8 Hz, 2H), 4.27 (d, J = 5.8 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 6.82
(m, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 1.8 Hz, 1H), 7.28 (t, J
= 6.1 Hz, 1H), 7.57 (d, J = 9.1 Hz, 2H), 8.45 (s, 1H), 8.71 (bs, 1H),
9.33 (bs, 2H). 13C NMR (125 MHz, DMSO-d6) δ 22.5, 24.5, 37.6,
46.0, 55.5, 66.7, 111.5, 113.0, 113.5, 119.3, 129.3, 131.4, 147.2, 149.0,
153.1, 164.6, 168.0. LCMS: (A) retention time 1.57 min, m/z = 342
[M + H]+.
4-(4-(Benzyloxy)benzylamino)benzimidamide (5h). Com-
pound 5h was prepared as described for the preparation of 5a except
4-(benzyloxy)benzaldehyde 3f was used in place of 4-(benzyloxy)-3-
F
DOI: 10.1021/acs.jmedchem.9b01003
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
methoxybenzaldehyde 3a and the compound was purified using
preparative HPLC (method 20−70%: flow rate of 25 mL/min;
gradient of 20% acetonitrile/80% H2O for 5.0 min followed by an
increase to 70% acetonitrile/30% H2O over 40 min and continuation
of 70% acetonitrile/30% H2O until 50 min). White solid (57% yield),
3-(2-Amino-5-methylphenoxy)benzonitrile (9d). The title
compound was prepared as described for the preparation of 9a
except compound 8d was used in place of 8a. Beige solid (75%
yield);. H NMR (400 MHz, CDCl3) δ 2.25 (s, 3H), 3.62 (s, 2H),
6.72 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.87 (m, 1H), 7.18
1
1
mp 222 °C. H NMR (400 MHz, DMSO-d6) δ 4.28 (d, J = 5.8 Hz,
(m, 1H), 7.20 (m, 1H), 7.32 (m, 1H), 7.39 (m, 1H).
2H), 5.07 (s, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H),
7.25 (m, 3H), 7.32 (m, 1H), 7.36 (m, 2H), 7.42 (m, 2H), 7.56 (d, J =
8.8 Hz, 2H), 8.47 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 45.6,
69.6, 114.1, 115.2, 128.1, 128.3, 128.9, 128.9, 129.7, 131.6, 137.6,
153.4, 157.8, 165.3, 168.0. LCMS: (A) retention time 1.76 min, m/z
= 332 [M + H]+.
4-(4-Methyl-2-nitrophenoxy)benzonitrile (8a). A mixture of
1-fluoro-4-methyl-2-nitrobenzene 6a (7.5 g, 48.4 mmol), 4-hydrox-
ybenzonitrile 7a (5.76 g, 48.4 mmol), and potassium carbonate (20.0
g, 145.0 mmol) in DMF (100 mL) was heated at 50 °C overnight.
After cooling, solvent was removed under reduced pressure, 10%
KHSO4 solution in water and EtOAc were added, and the layers were
separated. The organic layer was washed with brine, dried over
magnesium sulfate, and concentrated. The residue was triturated in a
EtOAc−hexanes mixture to give 11 g of product as a yellow solid
(89% yield). 1H NMR (400 MHz, DMSO-d6) δ 2.43 (s, 3H), 7.12 (d,
J = 8.8 Hz, 2H), 7.33 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 0.8, 8.3 Hz,
1H), 7.85 (d, J = 9.1 Hz, 2H), 8.00 (d, J = 1.0 Hz, 1H).
2-(2-Amino-5-methylphenoxy)benzonitrile (9e). The title
compound was prepared as described for the preparation of 9a
except compound 8e was used in place of 8a. Light tan solid (87%
1
yield). H NMR (400 MHz DMSO-d6) δ 2.20 (s, 3H), 5.01 (s, 2H),
6.38 (m, 1H), 6.65 (m, 2H), 6.77 (d, J = 8.1 Hz, 1H), 7.13 (td, J =
1.0, 7.6 Hz, 1H), 7.56 (m, 1H), 7.80 (dd, J = 1.5, 7.3 Hz, 1H).
N-(2-(4-Cyanophenoxy)-5-methylphenyl)butane-1-sulfona-
mide (10a). To a solution of compound 9a (298 mg, 1.33 mmol) in
pyridine (10 mL) was added butane-1-sulfonyl chloride (208 mg, 1.61
mmol) at rt. The reaction was stirred at rt for 10 min, then heated at
50 °C for 1.5 h. Pyridine was removed in vacuo, and the crude
material was partitioned between water and EtOAc. The organic layer
was washed with brine, dried over sodium sulfate, and concentrated in
vacuo. The residue was purified using a Biotage Isolera One flash
purification system with a silica gel cartridge (EtOAc/hexanes) to give
1
256 mg (56% yield) of title compound as a light brown solid. H
NMR (500 MHz, CDCl3) δ 0.87 (dd, J = 7.8, 6.9 Hz, 3H), 1.38 (q, J
= 7.4 Hz, 2H), 1.74 (td, J = 7.6, 7.0, 3.0 Hz, 2H), 2.38 (s, 3H), 3.16−
2.92 (m, 2H), 6.56 (s, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.95 (dd, J = 8.3,
2.0 Hz, 1H), 7.03 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 2.0 Hz, 1H), 7.64
(d, J = 8.7 Hz, 2H).
N-(2-(4-Cyanophenoxy)phenyl)butane-1-sulfonamide
(10b). The title compound was prepared as described for the
preparation of 10a except compound 9b was used in place of 9a and
the crude material was purified using DCM as mobile phase. Brown
oil (46% yield). 1H NMR (400 MHz, DMSO-d6) δ 0.76 (t, J = 7.3 Hz,
3H), 1.25 (m, 2H), 1.59 (m, 2H), 2.98 (t, J = 7.8 Hz, 2H), 7.03 (dd, J
= 1.5, 7.8 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.15 (m, 1H), 7.21 (m,
1H), 7.49 (dd, J = 1.8, 8.1 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 8.42 (s,
1H).
4-(2-Nitrophenoxy)benzonitrile (8b). Compound 8b was
prepared as described for the preparation of 8a except 1-fluoro-2-
nitrobenzene 6b was used in place of 1-fluoro-4-methyl-2-nitro-
1
benzene 6a. Yellow solid (85% yield). H NMR (400 MHz, DMSO-
d6) δ 7.16 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.3 Hz, 1H), 7.51 (m, 1H),
7.79 (m, 1H), 7.87 (d, J = 9.1 Hz, 2H), 8.15 (m, 1H).
4-(5-Methyl-2-nitrophenoxy)benzonitrile (8c). Compound 8c
was prepared as described for the preparation of 8a except 2-fluoro-4-
methyl-1-nitrobenzene 6c was used in place of 1-fluoro-4-methyl-2-
nitrobenzene 6a. Tan solid (76% yield). 1H NMR (400 MHz, CDCl3)
δ 2.45 (s, 3H), 6.99−7.04 (m, 3H), 7.18 (dd, J = 1.8, 8.3 Hz, 1H),
7.64 (m, 2H), 7.98 (d, J = 8.6 Hz, 1H).
3-(5-Methyl-2-nitrophenoxy)benzonitrile (8d). Compound
8d was prepared as described for the preparation of 8a except 2-
fluoro-4-methyl-1-nitrobenzene 6c was used in place of 1-fluoro-4-
methyl-2-nitrobenzene 6a and 3-hydroxybenzonitrile 7b in place of 4-
hydroxybenzonitrile 7a. Light yellow solid (80% yield). 1H NMR
(400 MHz, CDCl3) δ 2.44 (s, 3H), 6.93 (s, 1H), 7.15 (m, 1H), 7.20
(m, 1H), 7.24 (m, 1H), 7.43 (m, 1H), 7.48 (m, 1H), 7.97 (d, J = 8.6
Hz, 1H).
N-(2-(4-Cyanophenoxy)-4-methylphenyl)butane-1-sulfona-
mide (10c). The title compound was prepared as described for the
preparation of 10a except compound 9c was used in place of 9a. Dark
1
orange oil (57% yield). H NMR (500 MHz, CDCl3) δ 0.87 (td, J =
7.4, 1.1 Hz, 3H), 1.37 (q, J = 7.5 Hz, 2H), 1.79−1.69 (m, 2H), 2.30
(s, 3H), 3.08−3.02 (m, 2H), 6.50 (s, 1H), 6.78 (d, J = 1.8 Hz, 1H),
7.08−7.00 (m, 3H), 7.56 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.8 Hz,
1H).
2-(5-Methyl-2-nitrophenoxy)benzonitrile (8e). Compound 8e
was prepared as described for the preparation of 8a except 2-fluoro-4-
methyl-1-nitrobenzene 6c was used in place of 1-fluoro-4-methyl-2-
nitrobenzene 6a and 2-hydroxybenzonitrile 7c in place of 4-
N - ( 2 - ( 4 - C y a n o p h e n o x y ) - 4 - m e t h y l p h e n y l ) - 4 -
methylbenzenesulfonamide (10d). The title compound was
prepared as described for the preparation of 10a except compound
9c was used in place of 9a and 4-tolylsulfonyl chloride in place of
1
hydroxybenzonitrile 7a. Light tan solid (65% yield). H NMR (400
1
butane-1-sulfonyl chloride. Beige solid (98% yield). H NMR (400
MHz, CDCl3) δ 2.47 (s, 3H), 6.77 (d, J = 8.3 Hz, 1H), 7.09 (d, J =
8.3 Hz, 1H), 7.17 (td, J = 1.0, 7.8 Hz, 1H). 7.45 (m, 2H), 7.88 (dd, J
= 1.8, 7.8 Hz, 1H), 7.88 (d, J = 2.3 Hz, 1H).
MHz, CDCl3) δ 2.26 (s, 3H), 2.39 (s, 3H), 6.61 (m, 3H), 6.75 (s,
1H), 7.00 (dd, J = 1.5, 8.3 Hz, 1H), 7.10 (d, J = 7.8 Hz, 2H), 7.45 (m,
2H), 7.52 (m, 2H), 7.63 (d, J = 8.1 Hz, 1H).
4-(2-Amino-4-methylphenoxy)benzonitrile (9a). To a solu-
tion of 8a in methanol was added a spatula tip of 5% Pd/C, and the
mixture was hydrogenated for 40 min at 30 psi. The reaction was
filtered through Celite, and the filtrate was concentrated in vacuo.
Beige solid (90% yield). 1H NMR (400 MHz, CDCl3) δ 2.19 (s, 3H),
4.94 (s, 2H), 6.38 (d, J = 7.8 Hz, 1H), 6.64 (s, 1H), 6.76 (d, J = 7.8
Hz, 1H), 6.95 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 8.1 Hz, 2H).
4-(2-Aminophenoxy)benzonitrile (9b). The title compound
was prepared as described for the preparation of 9a except compound
N - ( 2 - ( 4 - C y a n o p h e n o x y ) - 4 - m e t h y l p h e n y l ) - 2 -
(trifluoromethoxy)benzenesulfonamide (10e). The title com-
pound was prepared as described for the preparation of 10a except
compound 9c was used in place of 9a and 2-(trifluoromethoxy)-
benzene-1-sulfonyl chloride in place of butane-1-sulfonyl chloride. Off
white solid (82% yield). 1H NMR (400 MHz, CDCl3) δ 2.23 (s, 3H),
6.65 (d, J = 1.8 Hz, 1H), 6.78 (m, 2H), 6.94 (m, 1H), 7.09 (s, 1H),
7.11 (m, 1H), 7.31 (m, 1H), 7.53 (m, 4H), 7.93 (dd, J = 1.8, 7.8 Hz,
1H).
1
8b was used in place of 8a. Light tan solid (quantitative yield). H
N-(2-(4-Cyanophenoxy)-4-methylphenyl)-4-fluorobenzene-
sulfonamide (10f). The title compound was prepared as described
for the preparation of 10a except compound 9c was used in place of
9a and 4-fluorobenzene-1-sulfonyl chloride in place of butane-1-
sulfonyl chloride. White solid (85% yield). 1H NMR (500 MHz,
CDCl3) δ 2.26 (s, 3H), 6.62 (s, 1H), 6.71−6.64 (m, 3H), 6.99−7.04
(m, 3H), 7.53 (dd, J = 7.8, 1.7 Hz, 2H), 7.60 (d, J = 8.4 Hz, 1H),
7.72−7.64 (m, 2H).
NMR (400 MHz, DMSO-d6) δ 5.04 (s, 2H), 6.58 (m, 1H), 6.84 (m,
2H), 6.98 (m, 3H), 7.79 (m, 2H).
4-(2-Amino-5-methylphenoxy)benzonitrile (9c). The title
compound was prepared as described for the preparation of 9a
except compound 8c was used in place of 8a. Yellow solid
(quantitative yield). 1H NMR (400 MHz, DMSO-d6) δ 2.15 (s,
3H), 4.80 (s, 2H), 6.72 (m, 2H), 6.81 (m, 1H), 6.96 (m, 2H), 7.77
(m, 2H).
G
DOI: 10.1021/acs.jmedchem.9b01003
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
N - ( 2 - ( 4 - C y a n o p h e n o x y ) - 4 - m e t h y l p h e n y l ) - 2 -
methoxybenzenesulfonamide (10g). The title compound was
prepared as described for the preparation of 10a except compound 9c
was used in place of 9a and 2-methoxybenzene-1-sulfonyl chloride in
place of butane-1-sulfonyl chloride. Light tan solid (quantitative
yield). 1H NMR (400 MHz, DMSO-d6) δ 2.18 (s, 3H), 3.65 (s, 3H),
6.73 (m, 3H), 6.96 (m, 3H), 7.25 (d, J = 8.1 Hz, 1H), 7.47 (m, 1H),
7.57 (m, 1H), 7.71 (m, 2H), 9.31 (s, 1H).
described for the preparation of 11a except compound 10d was
used in place of 10a. White powder (22% yield); mp 140 °C. H
1
NMR (400 MHz, DMSO-d6) δ 2.19 (s, 3H), 2.29 (s, 3H), 6.75 (m,
3H), 6.96 (dd, J = 2.0, 8.3 Hz, 1H), 7.20 (dd, J = 6.3, 8.6 Hz, 3H),
7.51 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 9.17 (bs, 2H), 9.71
(bs, 2H). 13C NMR (125 MHz, DMSO-d6) δ 20.3, 20.9, 116.9, 121.1,
121.9, 125.5, 126.0, 126.6, 129.3, 129.7, 138.3, 142.4, 147.2, 161.4,
165.0, 167.7. LCMS: (A) retention time 1.44 min, m/z 396 [M +
H]+.
N - ( 2 - ( 3 - C y a n o p h e n o x y ) - 4 - m e t h y l p h e n y l ) - 2 -
methoxybenzenesulfonamide (10h). The title compound was
prepared as described for the preparation of 10a except compound 9d
was used in place of 9a and 2-methoxybenzene-1-sulfonyl chloride in
4-(5-Methyl-2-(2-(trifluoromethoxy)phenylsulfonamido)-
phenoxy)benzimidamide (11e). The title compound was prepared
as described for the preparation of 11a except compound 10e was
1
1
place of butane-1-sulfonyl chloride. Tan solid (83% yield). H NMR
used in place of 10a. White fluffy solid (80% yield); mp 110 °C. H
(400 MHz, DMSO-d6) δ 2.17 (s, 3H), 2.28 (s, 3H), 3.61 (s, 3H),
6.68 (m, 2H), 6.75 (m, 2H), 6.94 (m, 1H), 6.98 (m, 1H), 7.27 (d, J =
8.1 Hz, 1H), 7.43 (dd, J = 17.8, 16.9 Hz, 2H), 7.51 (m, 1H), 9.15 (s,
1H).
N - ( 2 - ( 2 - C y a n o p h e n o x y ) - 4 - m e t h y l p h e n y l ) - 2 -
methoxybenzenesulfonamide (10i). The title compound was
prepared as described for the preparation of 10a except compound 9e
was used in place of 9a and 2-methoxybenzene-1-sulfonyl chloride in
place of butane-1-sulfonyl chloride. Tan solid (quantitative yield). 1H
NMR (400 MHz, DMSO-d6) δ 2.23 (s, 3H), 3.73 (s, 3H), 6.37 (d, J =
8.6 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.97 (m, 3H), 7.17 (td, J = 0.8,
7.6 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.48 (m, 2H), 7.66 (dd, J = 1.8,
7.8 Hz, 1H), 7.78 (dd, J = 1.8, 7.8 Hz, 1H), 9.55 (s, 1H).
NMR (400 MHz, DMSO-d6) δ 2.17 (s, 3H), 6.71 (s, 1H), 6.81 (dd, J
= 1.5, 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H),
7.33 (m, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.72 (m, 3H), 8.28 (s, 1H),
9.07 (bs, 2H), 9.61 (bs, 2H). 13C NMR (125 MHz, DMSO-d6) δ
20.3, 116.7, 118.6 120.7 121.1, 121.2, 121.4, 125.9, 126.8, 129.8,
130.1, 133.4, 145.2, 161.8, 164.9, 166.4. LCMS: (A) retention time
1.60 min, m/z 466 [M + H]+.
4-(2-(4-Fluorophenylsulfonamido)-5-methylphenoxy)-
benzimidamide (11f). The title compound was prepared as
described for the preparation of 11a except compound 10f was
1
used in place of 10a. White fluffy solid (43% yield); mp 185 °C. H
NMR (400 MHz, DMSO-d6) δ 2.10 (s, 3H), 6.61 (d, J = 2.3 Hz, 1H),
6.65 (m, 1H), 6.80 (m, 2H), 7.10 (m, 3H), 7.56 (dd, J = 5.7, 8.8 Hz,
2H), 7.71 (m, 2H), 8.91 (bs, 3H). 13C NMR (125 MHz, DMSO-d6)
20.4, 115.8, 116.1, 116.8, 121.3, 122.1, 125.9, 126.1, 128.3, 129.4,
129.5, 129.8, 135.4, 137.8, 137.8, 147.3, 161.4, 162.7, 165.2, 165.2.
LCMS: (A) retention time 1.35 min, m/z 400 [M + H]+.
4-(2-(Butylsulfonamido)-4-methylphenoxy)benzimidamide
(11a). A solution of 10a (230 mg, 0.668 mmol) in mixture of
anhydrous methanol (10 mL) and anhydrous dioxane (1 mL) was
subjected to a stream of bubbling HCl (gas) at 0 °C for 30 min. The
flask was capped with a septum and the mixture was stirred at rt
overnight. Methanol was removed in vacuo and the residue was dried
under high vacuum. The obtained material was redissolved in a 7 N
solution of ammonia in methanol (20 mL), and the mixture was
heated at 65 °C for 1 h before cooling to rt and stirring overnight. The
solvent was removed in vacuo. The resulting residue was purified
using reverse 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
4-(2-(2-Methoxyphenylsulfonamido)-5-methylphenoxy)-
benzimidamide (11g). The title compound was prepared as
described for the preparation of 11a except compound 10g was
1
used in place of 10a. White needles (58% yield); mp 149 °C. H
NMR (400 MHz, DMSO-d6) δ 2.17 (s, 3H), 3.67 (s, 3H), 6.72 (d, J =
1.3 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 6.94−7.01 (m, 3H), 7.23 (d, J
= 8.3 Hz, 1H), 7.47 (m, 1H), 7.58 (dd, J = 1.5, 7.8 Hz, 1H), 7.74 (d, J
= 8.8 Hz, 2H), 9.23 (bs, 2H), 9.78 (bs, 2H). 13C NMR (125 MHz,
DMSO-d6) δ 20.3, 55.8, 112.7, 117.2, 119.8, 120.8 122.9, 125.8,
129.4, 130.0, 134.7, 147.5, 156.3, 161.5, 164.8. LCMS: (A) retention
time 1.34 min, m/z 412 [M + H]+.
1
compound (15% yield) as a white solid; mp 82 °C. H NMR (400
MHz, DMSO-d6) δ 0.77 (t, J = 7.3 Hz, 3H), 1.24 (h, J = 7.4 Hz, 2H),
1.54−1.36 (m, 2H), 2.23 (s, 3H), 2.73−2.65 (m, 2H), 6.54 (dd, J =
8.2, 2.2 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.92−6.83 (m, 2H), 7.24
(d, J = 2.1 Hz, 1H), 7.77−7.68 (m, 2H), 9.18 (s, 4H). LCMS: (A)
retention time 1.40 min, m/z 362 [M + H]+. 13C NMR (101 MHz,
DMSO-d6) δ 14.0, 21.1, 21.3, 25.8, 52.2, 117.4, 121.3, 123.3, 126.9,
127.1, 128.5, 129.3, 130.4, 135.4, 162.3, 165.4. LCMS: (A) retention
time 1.40 min, m/z 362 [M + H]+.
3-(2-(2-Methoxyphenylsulfonamido)-5-methylphenoxy)-
benzimidamide (11h). The title compound was prepared as
described for the preparation of 11a except compound 10h was
used in place of 10a. Brown solid (14% yield); mp 117 °C. 1H NMR
(400 MHz, DMSO-d6) δ 2.16 (s, 3H), 3.66 (s, 3H), 6.65 (d, J = 1.3
Hz, 1H), 6.88 (m, 2H), 6.97 (m, 2H), 7.15 (s, 1H), 7.18 (d, J = 8.3
Hz, 1H), 7.48 (m, 3H), 7.62 (m, 1H), 8.40 (bs, 1H). 13C NMR (101
MHz, DMSO-d6) δ 20.4, 55.8, 112.6, 117.1, 119.7, 120.2, 122.1,
125.3, 129.5, 130.4, 130.5, 134.5, 156.3, 157.0, 165.4. LCMS: (A)
retention time 1.62 min, m/z 412 [M + H]+.
4-(2-(Butylsulfonamido)phenoxy)benzimidamide (11b).
The title compound was prepared as described for the preparation
of 11a except compound 10b was used in place of 10a. White solid
(25% yield); mp 50 °C. 1H NMR (400 MHz, DMSO-d6) δ 0.75 (t, J
= 7.3 Hz, 3H), 1.26 (m, 2H), 1.58 (m, 2H), 2.97 (t, J = 7.8 Hz, 2H),
7.02 (dd, J = 1.5, 7.8 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 7.15 (m, 1H),
7.20 (m, 1H), 7.48 (dd, J = 1.8, 8.1 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H),
8.41 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.5, 20.9, 24.9,
117.1, 119.4, 122.9, 124.6, 126.0, 128.8, 130.4, 132.8, 146.3, 165.5.
LCMS: (A) retention time 1.45 min, m/z 348 [M + H]+.
2-(2-(2-Methoxyphenylsulfonamido)-5-methylphenoxy)-
benzimidamide (11i). The title compound was prepared as
described for the preparation of 11a except compound 10i was
1
used in place of 10a. Light tan solid (21% yield); mp 232 °C. H
NMR (400 MHz, DMSO-d6) δ 2.04 (s, 3H), 3.56 (s, 3H), 6.36 (d, J =
7.1 Hz, 1H), 6.81 (m, 1H), 6.92 (m, 4H), 7.16 (m, 1H), 7.29 (m,
1H), 7.47 (m, 1H), 7.54 (d, J = 7.1 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H),
8.49 (bs, 2H), 10.98 (bs, 1H). 13C NMR (125 MHz, DMSO-d6) δ
21.2, 55.6, 99.5, 112.3, 116.4, 116.6, 119.0, 120.0 122.0, 122.7, 129.7,
129.8, 131.5, 133.6, 134.1, 143.7, 156.6, 157.5, 163.7. LCMS: (A)
retention time 1.37 min, m/z 412 [M + H]+.
4-(2-(Butylsulfonamido)-5-methylphenoxy)benzimidamide
(11c). The title compound was prepared as described for the
preparation of 11a except compound 10c was used in place of 10a.
1
White solid (57% yield); mp 178 °C. H NMR (400 MHz, DMSO-
N6-(4-(Benzyloxy)-3-methoxybenzyl)isoquinoline-1,6-dia-
mine (13). The title compound was prepared as described for the
preparation of 5a except isoquinoline-1,6-diamine 12 was used in
place of 4-aminobenzimidamide 4a. Crude material was purified using
a Biotage Isolera One flash purification system with a silica gel
cartridge (CHCl3/MeOH, 0.5% Et3N). Tan powder (10% yield); mp
155 °C. 1H NMR (400 MHz, DMSO-d6) δ 3.76 (s, 3H), 4.26 (d, J =
5.6 Hz, 2H), 5.04 (s, 2H), 6.39 (s, 2H), 6.50 (d, J = 2.0 Hz, 1H), 6.55
d6) δ 0.77 (t, J = 7.6 Hz, 3H), 1.25 (m, 2H), 1.58 (m, 2H), 2.24 (s,
3H), 2.10 (m, 2H), 6.85 (d, J = 1.3 Hz, 1H), 7.01 (dd, J = 1.3, 8.5 Hz,
1H), 7.08 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.2 Hz, 1H), 8.44 (s, 1H).
13C NMR (125 MHz, DMSO-d6) δ 13.5, 20.4, 20.8, 25.3, 51.6, 117.4,
121.0, 122.7, 126.0, 128.7, 129.9, 165.1, 167.3. LCMS: (A) retention
time 1.60 min, m/z 362 [M + H]+.
4-(5-Methyl-2-(4-methylphenylsulfonamido)phenoxy)-
benzimidamide (11d). The title compound was prepared as
H
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Journal of Medicinal Chemistry
Article
(d, J = 5.8 Hz, 1H), 6.71 (t, J = 5.8 Hz, 1H), 6.87 (m, 2H), 6.96 (d, J
= 8.1 Hz, 1H), 7.04 (d, J = 1.5 Hz, 1H), 7.31 (m, 1H), 7.36−7.43 (m,
4H), 7.52 (d, J = 5.8 Hz, 1H), 7.84 (d, J = 9.1 Hz, 1H). 13C NMR
(125 MHz, DMSO-d6) δ 46.0, 55.5, 69.9, 102.0, 109.3, 109.4, 111.6,
113.5, 116.1, 119.3, 125.0, 127.8, 128.4, 132.3, 137.3, 139.1, 140.9,
146.7, 149.1, 149.7, 156.7. LCMS: (A) retention time 1.65 min, m/z
386 [M + H]+.
cells were incubated in 100 μL of HBSS with 2 μM Fluo 4AM and 1%
Trypan Red for 50 min at 37 °C. The cells were then equilibrated for
10 min at room temperature before imaging. Test compounds were
dissolved in HBSS and diluted in a serial dilution. Test compounds,
BAM 8-22 (positive control), or HBSS (negative control) were added
(50 μL into 100 μL), and cells were imaged on the FLIPR for 2 min.
Data were exported as maximum−minimum fluorescent signal.
DRG Sensory Neuronal Cultures. DRGs from 3- to 4-week-old
MrgprX1 mice were collected in cold DH10 medium (DMEM/F-12
with 10% fetal bovine serum and 1% penicillin/streptomycin, Gibco)
and treated with enzyme solution (5 mg/mL dispase and 1 mg/mL
collagenase type I in HBSS without Ca2+ and Mg2+, Gibco) at 37 °C.
After trituration and centrifugation, cells were resuspended in DH10
with nerve growth factor (50 ng/mL, Upstate Biotechnology, Lake
Placid, NY) and glial cell line-derived neurotrophic factor (25 ng/mL,
R&D Systems), plated on glass coverslips coated with poly-D-lysine
(100 μg/mL, Biomedical Technologies) and laminin (10 μg/mL,
Invitrogen), cultured at 37 °C, and used after 20−40 h.24
6-(5-Methyl-2-nitrophenoxy)isoquinolin-1-amine (15). The
title compound was prepared as described for the preparation of 8a
except 1-fluoro-2-nitrobenzene 6c was used in place of 1-fluoro-4-
methyl-2-nitrobenzene 6a and 1-aminoisoquinolin-6-ol 14 in place of
4-hydroxybenzonitrile 4a. Crude material was purified using a Biotage
Isolera One flash purification system with a silica gel cartridge
1
(DCM/EtOAc, 0.5% Et3N). Yellow powder (92% yield). H NMR
(400 MHz, DMSO-d6) δ 2.37 (s, 3H), 6.81 (m, 3H), 7.12 (d, J = 2.5
Hz, 1H), 7.15 (s, 1H), 7.21 (dd, J = 2.8, 9.1 Hz, 1H), 7.26 (m, 1H),
7.74 (d, J = 5.8 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 8.24 (d, J = 9.1 Hz,
1H).
Whole-Cell Recordings of Cultured DRG Neurons. Whole cell
currents of cultured DRG neurons from MrgprX1 mice with
MrgprA3-GFP marker were recorded with an Axon 700B amplifier
and pCLAMP 9.2 software (Molecular Devices, Sunnyvale, CA,
USA). Extracellular solution contained (in mM) 130 N-methyl-D-
glucamine chloride (NMDG-Cl), 5 BaCl2, 1 MgCl2, 10 HEPES, and
10 glucose, with pH of 7.4 adjusted with 1 M NMDG. Osmolality was
adjusted to 310 mOsm/kg with sucrose. Electrodes were pulled
(model pp-830; Narishige, East Meadow, NY, USA) from borosilicate
glass (World Precision Instruments, Sarasota, FL, USA) with
resistance of 2−4 MΩ. Pipette solution contained (in mM) 140
tetraethylammonium chloride, 10 EGTA, 1 MgCl2, 10 HEPES, 0.5
GTP, and 3 ATP, with pH of 7.4 and osmolality of approximately 300
mOsm/kg. The voltage protocol was modified from a previously
published method.3 Briefly, cells were held at −80 mV and evoked to
−40 mV for 20 ms (ms) to activate low-voltage Ca2+ channels and
then held to −60 mV for 20 ms and evoked to −10 mV for 40 ms to
activate HVA Ca2+ channels. Leak currents were subtracted with P/4
protocol. Liquid junction potentials and whole cell capacitances were
offset, and series resistances were compensated by 70%. All
experiments were performed at room temperature (21−23 °C).
Opioid Receptor Binding Affinity. As previously reported in
detail,25 the binding affinities of compounds 11g and 16 to δ-, μ-, and
κ-opioid receptor sites were measured using [3H]DADLE, [3H]-
DAMGO, or [3H]U69,593 as ligands, respectively.
In Vivo Pharmacokinetic Studies in Mice. All pharmacokinetic
studies in mice were conducted according to protocols approved by
the Animal Care and Use Committee at Johns Hopkins University.
Male CD 1 mice between 25 and 30 g were obtained from Harlan,
Inc., and maintained on a 12 h light−dark cycle with ad libitum access
to food and water. Compound 16 was administered to mice (n = 3) as
a single intravenous dose of 10 mg/kg. The mice were sacrificed at
predetermined time points after drug administration. For collection of
plasma and spinal cord tissue, animals were euthanized with CO2, and
blood samples were collected in heparinized microtubes by cardiac
puncture. Spinal cords were dissected and immediately flash frozen
(−80 °C). Plasma was obtained by centrifugation of blood at 3000g
for 15 min and stored at −80 °C until LC/MS analysis.
N-(2-(1-Aminoisoquinolin-6-yloxy)-4-methylphenyl)-2-
methoxybenzenesulfonamide (16). To a solution of compound
15 (219 mg, 0.825 mmol) in pyridine (10 mL) was added 2-
methoxybenzenesulfonyl chloride (341 mg, 1.65 mmol) at rt. The
reaction was stirred at rt for 10 min, then heated at 65 °C overnight.
Pyridine was removed in vacuo, and the crude material was
partitioned between water and EtOAc. The organic layer was washed
with brine, dried over magnesium sulfate, and concentrated in vacuo.
The residue was purified using a Biotage Isolera One flash purification
system with a silica gel cartridge (CHCl3/MeOH) to give 16 as
1
yellow solid (98% yield); mp 238 °C (dec). H NMR (400 MHz,
DMSO-d6) δ 2.26 (s, 3H), 3.61 (s, 3H), 6.83 (d, J = 2.5 Hz, 1H), 6.85
(d, J = 8.6 Hz, 1H), 6.91 (m, 2H), 7.00 (m, 2H), 7.15 (dd, J = 2.5, 9.1
Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.36 (m, 1H), 7.58 (d, J = 7.1 Hz,
1H), 7.61 (dd, J = 1.8, 7.8 Hz, 1H), 8.43 (d, J = 9.3 Hz, 1H), 8.88 (bs,
2H), 9.42 (s, 1H), 12.93 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ
20.6, 55.8, 110.9, 111.3, 112.5, 119.5, 119.8, 121.1, 126.2, 127.2,
127.3, 127.8, 129.1, 129.3, 134.7, 135.1, 139.2, 144.5, 153.5, 156.2,
158.4, 162.2. LCMS: (A) retention time 1.65 min, m/z 436 [M +
H]+.
5-(5-Methyl-2-nitrophenoxy)benzo[d]thiazol-2-amine (18).
The title compound was prepared as described for the preparation
of 8a except 1-fluoro-2-nitrobenzene 6c was used in place of 1-fluoro-
4-methyl-2-nitrobenzene 6a and 2-aminobenzo[d]thiazol-5-ol 17 in
place of 4-hydroxybenzonitrile 4a. Crude material was purified using a
Biotage Isolera One flash purification system with a silica gel cartridge
1
(DCM/EtOAc, 0.5% Et3N). Yellow powder (84% yield). H NMR
(400 MHz, DMSO-d6) δ 2.30 (s, 3H), 6.85 (s, 1H), 6.95 (dd, J = 2.8,
8.8 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.49
(m, 3H), 7.94 (d, J = 8.3 Hz, 1H).
N-(2-(2-Aminobenzo[d]thiazol-5-yloxy)-4-methylphenyl)-2-
methoxybenzenesulfonamide (19). To a solution of compound
18 (228 mg, 0.840 mmol) in pyridine (10 mL) was added 2-
methoxybenzenesulfonyl chloride (347 mg, 1.68 mmol) at rt. The
reaction was stirred at rt for 10 min, then heated at 65 °C overnight.
Pyridine was removed in vacuo and the crude material was partitioned
between water and EtOAc. The organic layer was washed with brine,
dried over magnesium sulfate, and concentrated in vacuo. The residue
was purified using a Biotage Isolera One flash purification system with
a silica gel cartridge (CHCl3/MeOH) to give 19 as an orange solid
Prior to extraction, frozen samples were thawed on ice. The
calibration curves were developed using plasma and spinal cord from
naive animals as a matrix. Plasma samples (50 μL) were processed
using a protein precipitation method by addition of 300 μL of
acetonitrile as with internal standard (losartan [5 μM]), followed by
vortexing for 30 s and then centrifugation at 10 000 rcf for 10 min.
For spinal cord tissue, homogenized samples were vortexed and
centrifuged as above. A 10 μL aliquot of supernatant was diluted with
40 μL of water containing 0.5 μM losartan as internal standard.
Extracts were centrifuged at 10 000 rcf at 4 °C for 10 min.
Supernatants were transferred to 250 μL polypropylene autosampler
vials sealed with a Teflon cap. A volume of 3 μL was injected onto the
ultraperformance liquid chromatography (UPLC) instrument for
quantitative analysis by LC/MS/MS. Calibration curves over the
1
(97% yield), mp 198 °C. H NMR (400 MHz, DMSO-d6) δ 2.20 (s,
3H), 3.67 (s, 3H), 6.58 (d, J = 8.3 Hz, 1H), 6.65 (dd, J = 2.5, 8.6 Hz,
1H), 6.85 (dd, J = 2.0, 8.3 Hz, 1H), 6.96−7.01 (m, 3H), 7.20 (d, J =
2.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.47 (m, 1H), 7.64 (dd, J = 1.8,
7.8 Hz, 1H), 8.43 (bs, 2H), 9.07 (s, 1H). 13C NMR (125 MHz,
DMSO-d6) δ 20.4, 55.9, 111.6, 112.7, 116.8, 117.3, 118.1, 119.7,
125.7, 126.9, 127.2, 127.5, 129.4, 132.6, 134.7, 147.6, 150.8, 151.8,
156.5, 158.1, 167.2. LCMS: (A) retention time 1.78 min, m/z 442 [M
+ H]+.
In Vitro MrgV1 Receptor Assay. HEK293 cells stably trans-
fected with human MrgprX1 were plated in 96 well plates at 25 000
cell/well and incubated 2 days before imaging. On the day of imaging,
I
DOI: 10.1021/acs.jmedchem.9b01003
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range of 0.01−10 nmol/mL in plasma and spinal cord were
constructed from the peak area ratio of the analyte to the internal
standard using linear regression with a weighting factor of 1/(nominal
concentration). Correlation coefficient of greater than 0.99 was
obtained in all analytical runs for all analytes.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.9b01003.
1H and 13C spectra of the target compounds and agonist
activity of JHU-58 and compound 16 at MRGPRC11
(PDF)
Molecular formula strings and some data (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: (410) 614-0982. Fax: (410) 614-0659. E-mail:
ttsukamoto@jhmi.edu.
■
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Shimizu, K. D. Additivity of substituent effects in aromatic stacking
interactions. J. Am. Chem. Soc. 2014, 136, 14060−14067.
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S.; Cheng, Y.; Huang-Lionnet, J. H.; Geng, Y.; Xiao, B.; Peng, J.;
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ORCID
Rana Rais: 0000-0003-4059-2453
Barbara S. Slusher: 0000-0001-9814-4157
Takashi Tsukamoto: 0000-0002-0216-7520
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by NIH Grants R21NS101954
(T.T.), R01NS054791 (X.D.) and the Johns Hopkins Institute
for Clinical and Translational Research (ICTR), which is
funded in part by Grant UL1TR001079 from the National
Center for Advancing Translational Sciences (NCATS). X.D.
is an investigator of the Howard Hughes Medical Institute. E.P.
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.
ABBREVIATIONS USED
■
MRGPRX1, Mas-related G-protein-coupled receptor member
X1; DRG, dorsal root ganglion; HTS, high throughput
screening; HVA, high-voltage activated
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