The discovery and optimization of a series of 2-aminobenzoxazole derivatives as ChemR23 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115091

A structural class of 2-aminobenzoxazole derivatives possessing biphenyltetrazole was discovered to be potent human ChemR23 inhibitors. We initially tried to improve the potency of compound 1, which was found through in-house screening using the human pla

Full text of DOI:10.1016/j.bmc.2019.115091

Bioorganic&MedicinalChemistry27(2019)115091
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The discovery and optimization of a series of 2-aminobenzoxazole
derivatives as ChemR23 inhibitors
⁎
Takamichi Imaizumia, , Atsuko Kobayashia, Shigeki Otsubob, Masato Komaib,
Megumiko Magarac, Nobumasa Otsuboa
a Small Molecule Drug Research Laboratories, R&D Division, Kyowa Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan
b Immunology & Allergy Research Laboratories, R&D Division, Kyowa Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan
c Research Core Function Laboratories, R&D Division, Kyowa Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
SLE
A structural class of 2-aminobenzoxazole derivatives possessing biphenyltetrazole was discovered to be potent
human ChemR23 inhibitors. We initially tried to improve the potency of compound 1, which was found through
in-house screening using the human plasmacytoid dendritic cell (pDC)-like cell line CAL-1. The introduction of a
chiral methyl moiety at a benzylic position in a center of compound 1 showed a large impact on the inhibitory
activity against calcium signaling of ChemR23 induced by the natural ligand chemerin. As a result of further
investigations at the benzylic position, (R)-isomer 6b was found to show a 30-fold increased potency over
desmethyl compound 1. In addition, an extensive structure-activity relationship study on the benzoxazole
moiety successfully led to a further increase in the potency. The antagonistic effect of the compounds was based
on the induction of ChemR23 internalization. In addition, we observed that compound 31, which contained an
amide moiety on benzoxazole, inhibited chemotaxis of CAL-1 cells induced by chemerin in vitro. These results
suggest that our ChemR23 inhibitors are attractive compounds for the treatment of pDC-related autoimmune
diseases, such as systemic lupus erythematosus and psoriasis.
pDC
Interferon
ChemR23
Internalization
1. Introduction
discussed.15
ChemR23, also known as chemokine-like receptor 1 (CMKLR1), is a
Systemic lupus erythematosus (SLE) is an autoimmune disease in
which the immune system mistakenly attacks healthy tissues.1 The
choice of therapy for this disease is highly individualized based on
symptom manifestations, severity and organ condition.2 Accumulating
evidence has suggested a relationship between SLE and type I interferon
(IFN), and the regulation of IFN has received attention as a potential
therapeutic approach for this disease.3–5 In fact, the newly developed
biologics Sifalimumab and Anifrolumab, which are an anti-IFN-α
monoclonal antibody and monoclonal antibody against IFN receptor,
respectively, both met the primary endpoint in phase II studies.6,7 These
positive results also support research programs for developing new
agents capable of controlling the IFN level.8
G–protein coupled receptor (GPCR) expressed on macrophages, natural
killer cells and pDCs.16,17 Chemerin was reported as a natural ligand for
ChemR23 in 2003.18 Subsequently, chemerin short peptide and resolvin
E1 were also identified as ligands for ChemR23.19,20 Chemerin performs
a pro-inflammatory role,21,22 whereas chemerin short peptide and re-
solvin E1 show anti-inflammatory properties.23,24 Fibroblasts cultured
from human psoriatic skin lesions express high levels of chemerin, and
a previous report found that the migration of pDCs induced by fibro-
blast-derived chemerin was blocked by anti-ChemR23 antibody in
vitro.25 In addition, this inhibition was not observed in an experiment
using anti-CXCR3 and anti-CXCR4 antibodies.25 Furthermore, pDCs
infiltrating skin lesions are known to be associated with the patho-
genesis of psoriasis through IFN-α production.26
Plasmacytoid dendritic cells (pDCs) are a main type I interferon-
producing cell,9–13 and a number of new biologics focusing on depleting
or inhibiting pDCs are now in clinical trials.8,14 Given the above-men-
tioned previous findings, the inactivation of pDCs has been considered
an attractive approach for the treatment of SLE, and the molecular
mechanism underlying the development and function of pDCs has been
Based on these reports, we assumed that small molecules capable of
suppressing the migration of pDCs by disturbing ChemR23 signaling
might be promising agents for treating SLE or psoriasis. With this hy-
pothesis, we created a discovery program searching for ChemR23 in-
hibitors. We herein report the initial structure–activity relationship
⁎ Corresponding author.
E-mail address: takamichi.imaizumi.6p@kyowakirin.com (T. Imaizumi).
https://doi.org/10.1016/j.bmc.2019.115091
Received 12 July 2019; Received in revised form 5 September 2019; Accepted 5 September 2019
Availableonline06September2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
primary amine 11 was selected as the nucleophile for the SNAr reac-
tion. The next alkylation reaction was attempted using alkyl halide
under basic conditions and provided the desired bromide 13 along with
a byproduct. The structures of these compounds were confirmed by
measuring both Heteronuclear Multiple Bond Coherence (HMBC) and
Nuclear Overhauser Effect (NOE) after the separation using silica gel
column chromatography. Because the substrate with nitrile moiety
obtained from bromide 13 was decomposed during the treatment with
trimethylsilyl azide and dibutyltin oxide, a commercially available 2-
(tetrazol-5-yl)phenylboronic acid was used for the coupling reaction
with bromide 13. The cyclopropyl-containing amine 16 was prepared
from nitrile 15 using Dejaegher’s method.27 The desired tetrazole 18
was synthesized by a similar route as compound 14.
Figure 1. Structure of screening hit compound 1.
The synthesis of benzoxazole analogues substituted with an amide
or urea group at the 5-position is shown in Scheme 2. Modifications of
this position were conducted from acetic acid units, methoxy carbonyl
units and nitro units. A series of 2-mercaptobenzoxazoles 20a–c was
prepared from aminophenols 19a–c with potassium ethylxanthate.
After the conversion of these thiols to chlorides, coupling reaction with
the chiral amine 3b afforded bromides 21a–c. The amide 23 was syn-
thesized from carboxylic acid 22, which was obtained by hydrolysis of
ester 21a. For the formation of the methyl urea moiety, Curtius re-
arrangement with diphenylphosphoryl azide followed by methylamine
treatment was performed. The targeted tetrazoles 25 and 28 were ob-
tained in a two-step manner from bromides 23 and 26, respectively.
The same strategy was applied to the methoxy carbonyl compound 21b
to obtain amide 31 and ureas 33a–c. Compound 21c possessing a nitro
group was first converted to aromatic amine 34, which was treated with
acetyl chloride to give the N-acetylated compound 35. Mono-methy-
lation was conducted using iodomethane and potassium carbonate. For
the formation of the urea moiety from amine 37, the reaction was
executed with trichloroacetyl isocyanate and successive basic treatment
to obtain the urea 38. Finally, bromides 35 and 38 were converted to
the desired compounds 36 and 39 by coupling reaction in the presence
of palladium catalyst.
(SAR) of novel ChemR23 inhibitors derived from screening hit com-
pound 1 (Figure 1). In addition, the mode of action for the inhibitory
activity against ChemR23 is discussed.
2. Chemistry
Synthetic routes employed to prepare alkyl-substituted analogues at
the benzylic position are described in Scheme 1. The commercially
available racemic or chiral primary amines 2a–c were used to synthe-
size target compounds possessing a methyl substituent. Reductive
amination with butylaldehyde produced the secondary amines 3a–c.
Nucleophilic substitution reaction with 2-chlorobenzoxazole was exe-
cuted in refluxing 1,4-dioxane, affording compounds 4a–c. Suzuki
coupling reaction was conducted with pinacol boronic ester to provide
nitriles 5a–c in good yields. The desired tetrazoles 6a–c were synthe-
sized with nitriles 5a–c and trimethylsilyl azide in the presence of di-
butyltin oxide. The ethyl-branched compound was prepared from the
ketone 7. Acetic acid was a good additive to obtain the secondary amine
8 in the reductive amination step. The desired tetrazole 10 was ob-
tained by the same method as above, although the introduction of
benzoxazole resulted in a low yield. Some optimization was needed for
the synthesis of geminally dimethylated compound 14 because of the
bulkiness of this moiety. With the goal of reducing steric hindrance,
Scheme 1. aReagents and conditions: (a) 1-butanal, NaBH(OAc)3, MeOH, rt; (b) 2-chlorobenzoxazole, DIPEA, 1,4-dioxane, 120 °C; (c) 2-cyanophenylboronic acid
pinacol ester, Pd(PPh3)4, Cs2CO3, 1,4-dioxane, 100 °C; (d) TMSN3, dibutyltin oxide, toluene, 120 °C; (e) butylamine, NaCNBH3, AcOH, toluene, 120 °C; (f) iodobutane,
NaH, DMF, rt; (g) 2-(tetrazol-5-yl)phenylboronic acid, Pd(PPh3)4, Cs2CO3, 1,4-dioxane (for 14) or DMF (for 18), H2O, 100 °C; (h) c-PrMgBr, THF, then NaBH4, MeOH,
rt.
2

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
Scheme 2. aReagents and conditions: (a) potassium ethylxanthate, MeOH, reflux; (b) SOCl2, DMF, 70 °C; (c) 3b, DIPEA, 1,4-dioxane, 120 °C; (d) 3 M NaOH, EtOH,
50 °C; (e) MeNH2 in 2.0 M THF, EDCI·HCl, HOBt·H2O, DMF, rt; (f) 2-cyanophenylboronic acid pinacol ester, Pd(PPh3)4, Cs2CO3, 1,4-dioxane, 100 °C; (g) TMSN3,
dibutyltin oxide, toluene, 120 °C; (h) DPPA, Et3N, toluene, 110 °C then amines, rt; (i) 2-(tetrazol-5-yl)phenylboronic acid, Pd(PPh3)4, Cs2CO3, DMF, H2O, 100 °C; (j)
Fe, AcOH, 40 °C; (k) AcCl, pyridine, 0 °C; (l) MeI, K2CO3, MeCN, rt; (m) trichloroacetyl isocyanate, CH2Cl2, 0 °C; (n) K2CO3, MeOH, 50 °C.
3. Results and discussion
chemerin/ChemR23 signaling was not found to be dramatically im-
proved, although some carbonyl-contained compounds on benzoxazole
ring maintained their selectivity against SDF-1a/CXCR4 (Tables S1 and
S2). Only the compound with N-methylacetamide in the 5-position of
the benzoxazole ring provided slightly improved potency and se-
lectivity at the initial attempts. We therefore focused on the benzylic
position in the center of the molecule.
Compound 1 was identified as an antagonist of ChemR23 via
screening based on the inhibitory activity against calcium signaling
induced by chemerin. This screening was conducted using natively
ChemR23-expressing CAL-1 cells, a pDC-like cell line, established from
a patient with blastic natural killer cell lymphoma.28 CXCR4, which was
also expressed on CAL-1 cells, was used for the counter screening.
Compound 1 showed ChemR23 inhibitory activity with a single digit
micromolar IC50 value, and the selectivity against CXCR4 inhibitory
activity was admitted.
The results of alkyl modification at the benzylic position are shown
in Table 1. Compared with the potency of unsubstituted
1
(IC50 = 3.3 μM), that of the methylated 6a was increased by > 10-fold
(IC50 = 0.25 μM). Both the ethylated 10 (IC50 = 0.94 μM) and the cy-
clopropylmethylated 18 (IC50 > 3 μM) showed decreased activity due
to their bulkiness. Encouraged by the findings for methylated 6a, a
further examination was performed for each enantiomer. As a result,
(R)-isomer 6b exhibited 2-fold increased inhibitory potency
To confirm the potency of the compounds structurally similar to hit
compound 1, we first examined the SAR of both the n-butyl moiety
(Table S1 and Scheme S1) and benzoxazole moiety (Table S2 and
Scheme S2) as
a hit-follow-up study. The inhibitory potency of
3

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
Table 1
The structure–activity relationship at the benzylic position.
(IC50 = 0.11 μM), while (S)-isomer 6c showed an impaired potency
(IC50 = 7.6 μM). Given the low bioactivity of geminally dimethylated
14 (IC50 = 8.4 μM), the orientation of methyl substituent appears to
play a significant role in the activity against ChemR23. Furthermore,
this methyl substituent is also expected to affect the conformation of the
two substituents on adjacent nitrogen atoms. That is, it is presumed that
the significant improvement of the in vitro inhibitory activity is also due
to the stable conformation induced by the methyl moiety.
With the initial lead compound 6b in hand, a mechanistic study was
conducted regarding the antagonistic effect against ChemR23.
Compound 6b showed calcium mobilization in CAL-1 cells
(EC50 = 1.2 μM), and the natural ligand chemerin for ChemR23 also
induced this phenomenon. In addition, given the reports that receptor
internalization was related to intracellular signaling,29–31 we assumed
that the inhibitory effect of these synthetic compounds was based on
ChemR23 internalization. We therefore measured the surface expres-
sion of ChemR23 by flow cytometry after treatment with our ChemR23
inhibitors. The results are shown in Figure 2. CAL-1 cells were labeled
with anti-ChemR23 antibody after incubation with chemerin (50 ng/
mL), compound 1 (10 μM), 6b (3.0 μM) or 6c (3.0 μM) at 37 °C or 4 °C
for 30 min. As expected, both chemerin and our compounds led to the
internalization of ChemR23 at 37 °C, while these phenomena were not
induced at 4 °C.
Figure 2. Effects of chemerin and compounds on the surface expression of
ChemR23 in CAL-1 cells. The cells were incubated with chemerin (50 ng/mL),
compound 1 (10 μM), compound 6b (3.0 μM) or compound 6c (3.0 μM) at 37 °C
or 4 °C for 30 min. The surface expression of ChemR23 was measured by flow
cytometry (N = 1).
pro-inflammatory ligand, and pDC migration to inflammatory lesions is
promoted by this ligand. Therefore, the inhibition of pDC migration
might be observed in vivo if ChemR23 internalization could be induced
by these ligands in plasma.
Furthermore, the concentration range indicating receptor inter-
nalization showed a good correlation with the inhibition curve for
calcium signaling. (Figure 3). The obtained compounds were therefore
characterized as receptor-internalization inducers of ChemR23, leading
to Chemerin-ChemR23 signal inhibition. Chemerin is recognized as a
To this end, we conducted an additional SAR study for the 5-posi-
tion on the benzoxazole ring to improve the potency before performing
the chemotaxis assay. At this stage, we also decided to evaluate the
inhibitory activity against mouse ChemR23 to confirm the above hy-
pothesis in vivo. However, no information was available on the mouse
4

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
the 5-position on benzoxazole slightly improved its potency (Table S2).
Based on our preliminary study, we first synthesized and evaluated the
corresponding N-methylacetoamide 25 possessing a chiral methyl
moiety at the benzylic position. However, compound 25 did not exhibit
increased potency compared to human ChemR23 (hIC50) from un-
substituted 6b (25: hIC50 = 0.12 μM, 6b: hIC50 = 0.11 μM). We next
explored other functional groups at this position. The inhibitory activity
of urea analogues (28: hIC50 = 0.048 μM, 33a: hIC50 = 0.034 μM, 33b:
hIC50 = 0.032 μM, 33c: hIC50 = 0.037 μM) to human receptor was
higher than that of unsubstituted 6b, while the bioactivity against
mouse ChemR23 (mIC50) was decreased (28: mIC50 = 13 μM, 33a:
mIC50 = 0.94 μM, 33b: mIC50 = 0.51 μM, 33c: mIC50 = 0.43 μM). Both
N-methylated urea 39 and N-acylamide 36 showed decreased activity
against human receptor (39: hIC50 > 30 μM, 36: hIC50 = 0.44 μM).
The attachment of N-methylamide improved the activity against human
ChemR23 by 3-fold (31: hIC50 = 0.038 μM); however, a similar im-
provement was not observed for mouse ChemR23 (mIC50 = 0.15 μM).
At this stage, our compounds encountered species difference be-
tween human and mouse receptor in the inhibitory assay, and this
appeared to be derived from a relatively low homology of ChemR23
between humans and mice (80% identity).32 Therefore, we evaluated
our compound against chemerin-induced chemotaxis using human
ChemR23-expressing CAL-1 cells. As a result, compound 31 blocked the
cell migration induced by chemerin (chemotaxis IC50 = 0.38 μM). The
slight decrease in the potency in chemotaxis assay might be due to the
influence of bovine serum albumin, which was included in the assay
Figure 3. The correlation between calcium signaling inhibition and ChemR23
internalization induced by compound 6b; the activity of this compound was
measured by a calcium signaling assay (mean of four measurements) and in-
ternalization assay (N = 1), respectively.
pDC line. Therefore, we used the Ba/F3 cell line, in which mouse
ChemR23 was stably expressed. The results are shown in Table 2. In our
initial SAR study from hit compound 1, the N-methylacetoamide unit at
Table 2
The structure–activity relationship at the 5-position of the benzoxazole ring.
5

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
buffer. These results obtained with the CAL-1 cell suggest that a series
of compounds may block the infiltration of human pDCs into in-
flammatory lesions and may inhibit the production of cytokines, espe-
cially IFN-α, in the inflamed tissue. The results of our ChemR23 in-
hibitors may be useful for further examinations to identify new
therapeutics for SLE or psoriasis patients.
5.1.2. (R)-N-[1-(4-bromophenyl)ethyl]-N-butylamine (3b)
(R)-1-(4-bromophenyl)ethanamine (2b) (2.00 g, 10.0 mmol) was
dissolved in MeOH (50 mL). To this solution was added 1-butanal
(1.78 mL, 20.0 mmol) and NaBH(OAc)3 (4.24 g, 20.0 mmol). After
stirring for 18 h at room temperature, 5% NaOH was added. The mix-
ture was extracted with CHCl3, dried over MgSO4, filtered, and con-
centrated under reduced pressure. The crude material was purified by
silica gel column chromatography eluted with 5–10% MeOH in CHCl3
to afford the titled compound (2.17 g, 85%) as a colorless oil. 1H NMR
(400 MHz, CDCl3) δ: 7.44 (d, J = 8.8 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H),
3.72 (q, J = 6.8 Hz, 1H), 2.50–2.44 (m, 1H), 2.41–2.34 (m, 1H),
1.47–1.38 (m, 2H), 1.35–1.24 (m, 5H), 0.87 (t, J = 7.3 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ: 143.5, 131.6(2C), 128.5(2C), 120.8, 57.9,
4. Conclusion
We discovered a novel series of potent ChemR23 inhibitors capable
of suppressing the ChemR23 function through receptor internalization.
A methyl substituent at the benzylic position greatly improved the in-
hibitory potency compared to screening hit compound 1. A further
improvement in the potency against human ChemR23 was achieved by
the modification of the 5-position on the benzoxazole ring. Since a
species difference was observed between human and mouse receptor in
the calcium inhibitory assay, we examined the chemotaxis inhibition
assay using CAL-1 cells, which natively express human ChemR23, and
observed that chemerin-induced migration was blocked by re-
presentative compound 31. Since CAL-1 cells are a line of pDCs, which
can infiltrate skin lesions and induce IFN-α production, our results
suggest that a series of ChemR23 inhibitors may be viable therapeutic
agents for SLE or psoriasis. However, our compounds were not active
against mouse-derived ChemR23. The further study of our ChemR23
inhibitors with a focus on non-rodent animal species is currently being
performed.
47.2, 31.7, 23.7, 20.3, 13.9. LC-MS (ESI) m/z = 256 [M + H]+
.
5.1.3. (R)-N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]oxazole
(4b)
Compound 3b (850 mg, 3.32 mmol) was dissolved in 1,4-dioxane
(10 mL). To this solution was added DIPEA (3.48 mL, 19.9 mmol) and 2-
chlorobenzoxazole (1.16 mL, 9.95 mmol). After stirring for 2 days at
120 °C, saturated NaHCO3 was added. The mixture was extracted with
AcOEt, dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude material was purified by silica gel column chro-
matography eluted with 0–5% AcOEt in heptane to afford the titled
compound (815 mg, 66%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ:
7.46 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.8 Hz, 1H), 7.28–7.24 (m, 3H),
7.17 (dd, J = 7.8, 7.8 Hz, 1H), 7.01 (dd, J = 7.8, 7.8 Hz, 1H), 5.67 (q,
J = 6.9 Hz, 1H), 3.33–3.25 (m, 1H), 3.17–3.09 (m, 1H), 1.67 (d,
J = 6.9 Hz, 3H), 1.59–1.45 (m, 2H), 1.30–1.20 (m, 2H), 0.86 (t,
J = 7.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ: 162.8, 148.8, 143.3,
139.6, 131.6(2C), 129.0(2C), 123.9, 121.6, 120.2, 116.0, 108.7, 55.3,
5. Experimental section
5.1. General statement
44.5, 31.6, 20.1, 17.1, 13.8. LC-MS (ESI) m/z = 373 [M + H]+
.
Unless otherwise indicated, all reagents and solvents were pur-
chased from commercial sources and used without further purification.
Moisture- or oxygen-sensitive reactions were conducted under an at-
mosphere of argon or nitrogen gas. Unless otherwise stated, 1H NMR
and 13C NMR spectra were recorded at 300, 400 or 500 MHz using
JNM-AL300, JNM-AL400, Bruker Avance500 or Bruker DRX500 or in-
struments operating at the indicated frequencies. Chemical shifts are
expressed in ppm (δ) relative to an internal standard: tetramethylsilane.
The following abbreviations are used: br = broad signal, s = singlet,
d = doublet, dd = double doublet, ddd = double double doublet,
t = triplet, q = quartet, m = multiplet. Purification by silica gel
column chromatography was carried out using Moritex systems
(MORITEX Corporation) with prepacked cartridges. Melting points
(m.p.) were measured by MPA100 [Stanford Research Systems]. Liquid
chromatography with tandem mass spectrometry (LC-MS) analyses
were generally recorded using Waters Micromass ZQ (ESI) [Nihon
Waters K.K.]. High-resolution (HR)-LC-MS was conducted using a
SYNAPT G2 (ESI) [Nihon Waters K.K.]. Chemical purities were assessed
by HPLC at UV 254 nm. Chemical yields were not optimized. (R)-1-(4-
bromophenyl)ethanamine (99%, 98% ee) and (S)-1-(4-bromophenyl)
ethanamine (99%, 98% ee) were purchased from Alfa aesar.
5.1.4. (R)-N-butyl-N-[1-(2′-cyano-[1,1′-biphenyl]-4-yl)]ethyl-2-
aminobenzo[d]oxazole (5b)
Compound 4b (400 mg, 1.07 mmol) was dissolved in 1,4-dioxane
(5.0 mL). To this solution was added 2-cyanophenylboronic acid pi-
nacol ester (437 mg, 2.14 mmol), Cs2CO3 (1.05 g, 3.21 mmol) and Pd
(PPh3)4 (124 mg, 0.107 mmol). The flask was evacuated and backfilled
with argon. The mixture was stirred at 100 °C for 6 h. After cooling to
room temperature, saturated NaHCO3 was added. The mixture was
extracted with AcOEt, dried over MgSO4, filtered through celite bed,
and concentrated under reduced pressure. The crude material was
purified by silica gel column chromatography eluted with 10–15%
AcOEt in heptane to afford the titled compound (420 mg, 99%) as a
yellow oil. 1H NMR (400 MHz, CDCl3) δ: 7.76 (d, J = 7.8 Hz, 1H), 7.63
(dd, J = 7.8, 7.8 Hz, 1H), 7.57–7.48 (m, 5H), 7.43 (dd, J = 7.8, 7.8 Hz,
1H), 7.40 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.17 (dd,
J = 7.8, 7.8 Hz, 1H), 7.01 (dd, J = 7.8, 7.8 Hz, 1H), 5.79 (q, J = 6.8 Hz,
1H), 3.42–3.33 (m, 1H), 3.26–3.18 (m, 1H), 1.74 (d, J = 6.8 Hz, 3H),
1.65–1.49 (m, 2H), 1.32–1.22 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). 13C
NMR (100 MHz, CDCl3) δ: 162.8, 148.7, 144.8, 143.4, 141.1, 137.4,
133.7, 132.8(2C), 129.9, 128.8, 127.6(2C), 127.6, 123.8, 120.1, 118.6,
115.9, 111.1, 108.6, 55.6, 44.6, 31.6, 20.1, 17.2, 13.7.LC-MS (ESI) m/
5.1.1. 5.1.1.
N-butyl-N-{[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
z = 396 [M + H]+
.
methyl}-2-aminobenzo[d]oxazole (1)
The synthesis of screening hit compound (white solid) was de-
scribed in supporting information (Scheme S1). 1H NMR (300 MHz,
DMSO‑d6) δ: 7.63–7.58 (m, 2H), 7.53–7.47 (m, 2H), 7.41–7.26 (m, 4H),
7.23–6,99. (m, 4H), 4.74 (s, 2H), 3.48 (t, J = 7.2 Hz, 2H), 1.63–1.58
(m, 2H), 1.33–1.26 (m, 2H), 0.88 (t, J = 7.5 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ: 162.6, 156.1, 148.5, 142.5, 140.8, 139.0, 136.3,
130.8(2C), 130.7, 129.4(2C), 128.0, 127.3, 125.1, 124.1, 123.8, 120.7,
115.7, 108.8, 51.4, 48.4, 29.8, 19.9, 13.8. m.p.:80–82 °C. LC-MS (ESI)
m/z = 425 [M + H]+ . Purity: 99.9%. HR-LC-MS (ESI) m/z [M + H] +
calcd, 425.2090; found, 425.2099.
5.1.5. (R)-N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
ethyl}-2-aminobenzo[d]oxazole (6b)
Compound 5b (316 mg, 0.799 mmol) was dissolved in toluene
(10 mL). To this solution was added TMSN3 (0.636 mL, 4.79 mmol),
dibutyltin oxide (199 mg, 0.799 mmol) and the mixture was stirred at
120 °C for 24 h. After cooling to room temperature, MeOH was added
and concentrated under reduced pressure. The crude material was
purified by silica gel column chromatography eluted with 5–10%
MeOH in CHCl3 to afford the titled compound (306 mg, 87%) as a white
solid. 1H NMR (300 MHz, DMSO‑d6) δ: 7.71–7.63 (m, 2H), 7.60–7.51
6

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
(m, 2H), 7.41–7.27 (m, 4H), 7.17–7.08 (m, 3H), 6.99 (dd, J = 7.3,
7.3 Hz, 1H), 5.54 (q, J = 7.0 Hz, 1H), 3.39–3.18 (m, 2H), 1.65 (d,
J = 7.0 Hz, 3H), 1.52–1.35 (m, 2H), 1.28–1.17 (m, 2H), 0.88–0.80 (m,
3H). 13C NMR (100 MHz, CDCl3) δ: 162.5, 155.6, 148.3, 142.0, 141.0,
139.7, 138.8, 131.1, 130.8, 130.8, 129.2(2C), 128.0, 127.4(2C), 124.0,
123.2, 120.7, 115.3, 108.8, 55.9, 44.8, 31.4, 20.0, 17.0, 13.7.
5.1.10. N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]propyl}-
2-aminobenzo[d]oxazole (10)
The titled compound was synthesized from compound 9 (18 mg,
0.044 mmol) by a process analogous to the preparation of 6b. The crude
material was purified by silica gel column chromatography eluted with
2–5% MeOH in CHCl3 to afford the titled compound (4.2 mg, 21%) as a
white solid. 1H NMR (400 MHz, CDCl3) δ: 8.07 (d, J = 7.8 Hz, 1H),
7.62–7.51 (m, 2H), 7.41 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 2H),
7.30–7.26 (m, 1H), 7.22–7.11 (m, 4H), 7.02 (dd, J = 7.8, 3.9 Hz, 1H),
5.33–5.29 (m, 1H), 3.27–3.21 (m, 2H), 2.16–2.08 (m, 2H), 1.61–1.39
(m, 2H), 1.29–1.20 (m, 2H), 1.02 (t, J = 7.3 Hz, 3H), 0.86 (t,
J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 453 [M + H]+. Purity: 90.8%. HR-
m.p.:82–84 °C. LC-MS (ESI) m/z = 439 [M + H]+ . Purity: 99.9%.
+
[α]24 = +181.2 (c 0.33, CHCl3), HR-LC-MS (ESI) m/z [M + H]
D
calcd, 439.2246; found, 439.2233.
5.1.6. N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]ethyl}-2-
aminobenzo[d]oxazole (6a)
+
The titled compound (92.2 mg, white solid) was synthesized from
commercially available 1-(4-bromophenyl)ethanamine (2a) by a 4-step
process (60%, 59%, quantitative and 46% yield) that was analogous to
the preparation of 6b. 1H NMR (400 MHz, DMSO‑d6) δ: 7.71–7.63 (m,
2H), 7.60–7.51 (m, 2H), 7.41–7.27 (m, 4H), 7.17–7.08 (m, 3H), 6.99
(dd, J = 7.3, 7.3 Hz, 1H), 5.54 (q, J = 7.0 Hz, 1H), 3.37–3.19 (m, 2H),
1.65 (d, J = 7.0 Hz, 3H), 1.52–1.35 (m, 2H), 1.28–1.17 (m, 2H),
0.88–0.80 (m, 3H). LC-MS (ESI) m/z = 439 [M + H]+. Purity: 99.7%.
LC-MS (ESI) m/z [M + H] calcd, 453.2403; found, 453.2402.
5.1.11. N-[2-(4-bromophenyl)propan-2-yl]-2-aminobenzo[d]oxazole (12)
The titled compound was synthesized from 2-(4-bromophenyl)
propan-2-amine (11) (118 mg, 0.551 mmol) by a process analogous to
the preparation of 4b. The crude material was purified by silica gel
column chromatography eluted with 20–33% AcOEt in heptane to af-
ford the titled compound (80.0 mg, 44%) as a white solid. 1H NMR
(400 MHz, CDCl3) δ: 7.43 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H),
7.26 (d, J = 7.8 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.11 (dd, J = 7.8,
7.8 Hz, 1H), 6.97 (dd, J = 7.8, 7.8 Hz, 1H), 1.82 (s, 6H). LC-MS (ESI)
+
HR-LC-MS (ESI) m/z [M + H] calcd, 439.2246; found, 439.2246.
5.1.7. (S)-N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
ethyl}-2-aminobenzo[d]oxazole (6c)
m/z = 331 [M + H]+
.
The titled compound (470 mg, white solid) was synthesized from
commercially available (S)-1-(4-bromophenyl)ethanamine (2c) by a 4-
step process (86%, 90%, 94% and 78% yield) that was analogous to the
preparation of 6b. 1H NMR (300 MHz, DMSO‑d6) δ: 7.71–7.63 (m, 2H),
7.60–7.51 (m, 2H), 7.41–7.27 (m, 4H), 7.17–7.08 (m, 3H), 6.99 (dd,
J = 7.3, 7.3 Hz, 1H), 5.54 (q, J = 7.0 Hz, 1H), 3.39–3.18 (m, 2H), 1.65
(d, J = 7.0 Hz, 3H), 1.52–1.35 (m, 2H), 1.28–1.17 (m, 2H), 0.88–0.80
(m, 3H). 13C NMR (100 MHz, CDCl3) δ: 161.4, 155.5, 147.6, 140.9,
139.7, 139.0, 139.0, 131.1, 130.8, 130.7, 129.3(2C), 128.0, 127.2(2C),
124.6, 123.2, 121.4, 115.1, 109.1, 56.5, 45.3, 31.2, 19.9, 17.1, 13.6.
5.1.12. N-[2-(4-bromophenyl)propan-2-yl]-N-butyl-2-aminobenzo[d]
oxazole (13)
Compound 12 (83.0 mg, 0.251 mmol) was dissolved in DMF
(2.5 mL). To this solution was added NaH (60% in mineral oil, 30.0 mg,
0.752 mmol). After stirring for 1 h at 0 °C, 1-iodobutane (0.086 mL,
0.752 mmol) was added. After stirring for 2 h at room temperature,
10% NaOH was added. The mixture was extracted with CHCl3, dried
over MgSO4, filtered, and concentrated under reduced pressure. The
crude material was purified by preparative thin-layer chromatography
(TLC) with 10% AcOEt in heptane to afford the titled compound
(48.1 mg, 50%) as a white solid. 1H NMR (500 MHz, CDCl3) δ:
7.40–7.38 (m, 2H), 7.31 (d, J = 7.8 Hz, 1H), 7.26–7.23 (m, 2H), 7.08
(ddd, J = 7.8, 7.8, 1.0 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.91 (ddd,
J = 7.8, 7.8, 1.0 Hz, 1H), 3.72–3.69 (2H m), 1.81–1.75 (m, 2H), 1.80 (s,
6H), 1.43–1.36 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ: 162.4, 148.5, 147.8, 142.8, 131.4 (2C) 126.7 (2C), 123.6,
120.2, 120.2, 116.3, 108.5, 61.9, 47.3, 32.4, 29.3 (2C), 20.2, 13.9. LC-
MS (ESI) m/z = 387 [M + H]+. 13-byproduct (28.1 mg, 29%, white
solid). 1H NMR (500 MHz, CDCl3) δ: 7.47–7.44 (m, 2H), 7.41–7.38 (m,
2H) 7.01 (ddd, J = 7.7 Hz, 7.7 Hz, 1.0 Hz, 1H), 6.92 (ddd, J = 7.7, 7.7,
0.9 Hz, 1H), 6.84 (ddd, J = 7.7, 7.7, 0.9 Hz, 1H), 6.75 (ddd, J = 7.7,
7.7, 1.0 Hz, 1H), 3.78 (t, J = 7.1 Hz, 2H), 1.78–1.71 (m, 2H), 1.65 (s,
6H), 1.46–1.38 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ: 150.7, 144.9, 147.2, 133.1, 130.8 (2C), 127.3 (2C), 123.0,
119.5, 119.3, 108.5, 106.3, 57.1, 42.0, 30.7 (2C), 29.3, 20.0, 13.8. LC-
m.p.:82–84 °C. LC-MS (ESI) m/z = 439 [M + H]+
. Purity: 99.9%.
[α]24D = -174.1 (c 0.32, CHCl3). HR-LC-MS (ESI) m/z [M + H] + calcd,
439.2246; found, 439.2234.
5.1.8. N-[1-(4-bromophenyl)propyl]-N-butylamine (8)
1-(4-bromophenyl)propan-1-one (7) (2.05 g, 9.62 mmol) was dis-
solved in toluene (48 mL). To this solution was added 1-butylamine
(1.90 mL, 19.2 mmol), AcOH (0.551 mL, 9.62 mmol) and NaCNBH3
(1.21 g, 19.2 mmol). The mixture was stirred at 120 °C for 3 h. After
cooling to room temperature, 10% NaOH was added. The mixture was
extracted with CHCl3, dried over MgSO4, filtered, and concentrated
under reduced pressure. The crude material was purified by silica gel
column chromatography eluted with 10–20% MeOH in CHCl3 to afford
the titled compound (1.36 g, 52%) as a colorless oil. 1H NMR (400 MHz,
CDCl3) δ: 7.43 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 3.46–3.42
(m, 1H), 2.46–2.32 (m, 2H), 1.75–1.66 (m, 1H), 1.62–1.53 (m, 1H),
1.48–1.36 (m, 2H), 1.34–1.24 (m, 2H), 0.86 (t, J = 6.8 Hz, 3H), 0.78 (t,
MS (ESI) m/z = 387 [M + H]+
.
J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 270 [M + H]+
.
5.1.13. N-butyl-N-{2-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]propan-
2-yl}-2-aminobenzo[d]oxazole (14)
5.1.9. N-butyl-N-[1-(2′-cyano-[1,1′-biphenyl]-4-yl)]propyl-2-aminobenzo
[d]oxazole (9)
Compound 13 (68.0 mg, 0.176 mmol) was dissolved in 1,4-dioxane
(5 mL). To this solution was added 2-(tetrazol-5-yl)phenylboronic acid
(67.0 mg, 0.351 mmol), Cs2CO3 (172 mg, 0.527 mmol), Pd(PPh3)4
(20.0 mg, 0.018 mmol) and H2O (2.5 mL). The flask was evacuated and
backfilled with argon. The mixture was stirred at 100 °C for 20 h. After
cooling to room temperature, saturated NaHCO3 was added. The mix-
ture was extracted with CHCl3, dried over MgSO4, filtered, and con-
centrated under reduced pressure. The crude material was purified by
silica gel column chromatography eluted with 5–10% MeOH in CHCl3
and preparative TLC with 10% MeOH in CHCl3 to afford the titled
compound (7.9 mg, 10%) as a white solid. 1H NMR (300 MHz, CDCl3) δ:
8.13 (d, J = 7.0 Hz, 1H), 7.58–7.46 (m, 2H), 7.43 (dd, J = 7.7, 1.3 Hz,
The titled compound was synthesized from compound 8 (300 mg,
1.11 mmol) by a process analogous to the preparations of 4b and 5b.
The crude material was purified by silica gel column chromatography
eluted with 5–15% AcOEt in heptane to afford the titled compound
(18 mg, 4%). 1H NMR (400 MHz, CDCl3) δ: 7.76 (d, J = 7.8 Hz, 1H),
7.64 (dd, J = 7.8, 7.8 Hz, 1H), 7.56–7.37 (m, 7H), 7.29 (d, J = 7.8 Hz,
1H), 7.18 (dd, J = 7.8, 7.8 Hz, 1H), 7.01 (dd, J = 7.8, 7.8 Hz, 1H),
5.54–5.50 (m, 1H), 3.27 (t, J = 7.8 Hz, 2H), 2.25–2.12 (m, 2H),
1.62–1.52 (m, 1H), 1.44–1.35 (m, 1H), 1.29–1.21 (m, 2H), 1.08 (t,
J = 7.3 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 410
+
[M + H]
.
7

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
+
1H), 7.37 (d, J = 8.4 Hz, 2H), 7.31–7.22 (m, 2H), 7.16–7.09 (m, 3H),
6.99 (dd, J = 7.7, 7.7 Hz, 1H), 3.83–3.77 (m, 2H), 1.84 (s, 6H),
1.53–1.40 (m, 2H), 1.33–1.21 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H). LC-MS
m/z = 465 [M + H]+. Purity: 96.8%. HR-LC-MS (ESI) m/z [M + H]
calcd, 465.2403; found, 465.2413.
(ESI) m/z = 453 [M + H]+
.
Purity: 99.9%. HR-LC-MS (ESI) m/z
5.1.17. Methyl 2-(2-mercaptobenzo[d]oxazol-5-yl)acetate (20a)
+
[M + H] calcd, 453.2403; found, 453.2399.
Methyl
2-(3-amino-4-hydoxyphenyl)acetate
(19a)
(5.09 g,
28.1 mmol) was dissolved in MeOH (140 mL). To this solution was
added potassium O-ethyl carbonodithioate (7.66 g, 47.8 mmol). The
mixture was stirred under reflux conditions for 6 h. After cooling to
0 °C, AcOH (25 mL) and H2O (500 mL) was added. The reaction mixture
was stirred at that temperature for 1 h. The resulting precipitate was
collected by filtration and dried to afford the titled compound (5.05 g,
81%) as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ: 13.87 (br s, 1H),
7.45 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 3.78 (s,
2H), 3.62 (s, 3H).
5.1.14. N-[(4-bromophenyl)(cyclopropyl)methyl]-2-aminobenzo[d]
oxazole (16)
4-bromobenzonitrile (15) (210 mg, 1.15 mmol) was dissolved in
THF (3 mL). To this solution was added cyclopropylmagnesium bro-
mide (0.5 M in THF, 6.92 mL, 3.46 mmol) at 0 °C and stirred at the same
temperature for 5.5 h. To the resulting mixture was added MeOH (5 mL)
and NaBH4 (100 mg, 2.64 mmol) then stirred for 24 h at room tem-
perature. After diluting with 5% NaOH, the mixture was filtered
through celite bed and then extracted with CHCl3, dried over MgSO4,
filtered, and concentrated under reduced pressure. The crude material
was purified by silica gel column chromatography eluted with 5–10%
MeOH in CHCl3 to afford the titled compound (215 mg). An 1H NMR
analysis of this compound showed the presence of a cyclopropyl moiety
derived from the reagent, but the next step was conducted without
further purification. A solution of this compound, 2-chlorobenzoxazole
(0.086 mL, 0.737 mmol) and DIPEA (0.309 mL, 1.77 mmol) in 1,4-di-
oxane (3.7 mL) was stirred at 120 °C for 6 h. The mixture was diluted
with saturated NaHCO3, extracted with AcOEt, dried over MgSO4, fil-
tered, and concentrated under reduced pressure. The crude material
was purified by silica gel column chromatography eluted with 10–20%
AcOEt in heptane to afford the titled compound (210 mg, 53%) as a
white amorphous powder. 1H NMR (300 MHz, CDCl3) δ: 7.45 (d,
J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.26–7.20 (m, 2H), 7.15–7.09
(m, 1H), 7.04–6.97 (m, 1H), 6.38 (br s, 1H), 4.34 (d, J = 8.4 Hz, 1H),
1.31–1.19 (m, 1H), 0.68–0.62 (m, 2H), 0.58–0.41 (m, 2H). LC-MS (ESI)
5.1.18. Methyl (R)-2-N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo
[d]oxazol-5-yl-acetate (21a)
Compound 20a (1.53 g, 6.85 mmol) was dissolved in SOCl2
(5.00 mL, 68.5 mmol). To this solution was added DMF (0.027 mL,
0.343 mmol) slowly. The mixture was stirred at 70 °C for 4 h and then
concentrated. The crude compound was dissolved in 1,4-dioxane
(10 mL). To this solution was added DIPEA (2.39 mL, 13.7 mmol) and
compound 3b (1.17 g, 4.57 mmol) which was dissolved in 1,4-dioxane
(5 mL) in advance. After stirring for 90 h at 120 °C, saturated NaHCO3
was added at 0 °C. The mixture was extracted with AcOEt, dried over
MgSO4, filtered, and concentrated under reduced pressure. The crude
material was purified by silica gel column chromatography eluted with
15–20% AcOEt in heptane to afford the titled compound (1.32 g, 65%)
as a brown oil. 1H NMR (400 MHz, CDCl3) δ: 7.46 (d, J = 8.8 Hz, 2H),
7.30–7.24 (m, 3H), 7.20 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H),
5.65 (q, J = 6.8 Hz, 1H), 3.68 (s, 3H), 3.66 (s, 2H), 3.32–3.25 (m, 1H),
3.17–3.09 (m, 1H), 1.66 (d, J = 6.8 Hz, 3H), 1.56–1.45 (m, 2H),
1.29–1.22 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 445
m/z = 343 [M + H]+
.
5.1.15. N-[(4-bromophenyl)(cyclopropyl)methyl]-N-butyl-2-aminobenzo
[d]oxazole (17)
[M + H]+
.
The titled compound was synthesized from compound 16 (160 mg,
0.466 mmol) by a process analogous to the preparation of 13. The crude
material was purified by silica gel column chromatography eluted with
0–5% AcOEt in heptane to afford the titled compound (117 mg, 63%) as
a white amorphous powder. 1H NMR (400 MHz, CDCl3) δ: 7.45 (d,
J = 8.8 Hz, 2H), 7.40–7.34 (m, 3H), 7.26 (d, J = 7.8 Hz, 1H), 7.16 (dd,
J = 7.8, 7.8 Hz, 1H), 7.00 (dd, J = 7.8, 7.8 Hz, 1H), 4.67 (d, J = 9.8 Hz,
1H), 3.50–3.41 (m, 1H), 3.25–3.17 (m, 1H), 1.73–1.64 (m, 2H),
1.47–1.38 (m, 1H), 1.35–1.25 (m, 2H), 0.95–0.86 (m, 4H), 0.69–0.63
(m, 2H), 0.55–0.49 (m, 1H). LC-MS (ESI) m/z = 399 [M + H] + .
5.1.19. Methyl 2-mercaptobenzo[d]oxazole-5-carboxylate (20b)
The titled compound (3.52 g, 94%, white solid) was synthesized
from compound 19b (3.00 g, 18.0 mmol) by a process analogous to the
preparation of 20a. 1H NMR (400 MHz, DMSO‑d6) δ: 7.88 (d,
J = 8.8 Hz, 1H), 7.68 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 3.88 (s, 3H). LC-
MS (ESI) m/z = 210 [M + H]+
.
5.1.20. Methyl (R)-N-[1-(4-bromophenyl)ethyl] -N-butyl-2-aminobenzo
[d]oxazole-5-carboxylate (21b)
The titled compound was synthesized from compound 20b (2.00 g,
9.56 mmol) and compound 3b (1.63 g, 6.36 mmol) by a process ana-
logous to the preparation of 21a. The crude material was purified by
silica gel column chromatography eluted with 10–15% AcOEt in hep-
tane to afford the titled compound (1.95 g, 71%) as a brown oil.1H-
NMR (400 MHz, CDCl3) δ: 8.05 (s, 1H), 7.81–7.78 (m, 1H), 7.48 (d,
J = 8.8 Hz, 2H), 7.29–7.25 (m, 3H), 5.66 (q, J = 6.8 Hz, 1H), 3.92 (s,
3H), 3.35–3.11 (m, 2H), 1.68 (d, J = 6.8 Hz, 3H), 1.60–1.46 (m, 2H),
1.31–1.21 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 431
5.1.16. N-butyl-N-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
(cyclopropyl)methyl-2-aminobenzo[d]oxazole (18)
Compound 17 (115 mg, 0.288 mmol) was dissolved in DMF (4.5 mL)
and H2O (0.5 mL). To this solution was added Cs2CO3 (281 mg,
0.864 mmol),
2-(tetrazol-5-yl)phenylboronic
acid
(109 mg,
0.576 mmol) and Pd(PPh3)4 (33.0 mg, 0.029 mmol). The flask was
evacuated and backfilled with argon. The mixture was stirred at 100 °C
for 4 h. After cooling to room temperature, 1 M HCl was added. The
mixture was extracted with CHCl3, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude material was purified
by preparative TLC with 10% MeOH in CHCl3 to afford the titled
compound (127 mg, 95%) as a white solid. 1H NMR (400 MHz, CDCl3)
δ: 8.01 (d, J = 7.8 Hz, 1H), 7.58–7.47 (m, 2H), 7.42–7.38 (m, 3H),
7.28–7.24 (m, 1H), 7.18 (d, J = 6.8 Hz, 1H), 7.13–7.10 (m, 3H), 7.00
(dd, J = 8.3, 8.3 Hz, 1H), 4.56 (d, J = 9.8 Hz, 1H), 3.50–3.42 (m, 1H),
3.26–3.18 (m, 1H), 1.76–1.67 (m, 2H), 1.42–1.35 (m, 1H), 1.34–1.23
(m, 2H), 0.94–0.86 (m, 4H), 0.69–0.51 (m, 3H). 13C NMR (100 MHz,
CDCl3) δ: 162.8, 155.6, 148.3, 142.6, 140.8, 140.2, 138.7, 131.1, 130.8,
130.8, 129.1(2C), 128.1, 127.4(2C), 124.0, 123.0, 120.4, 115.6, 108.7,
66.2, 46.0, 31.9, 20.1, 13.8, 13.5, 4.2(2C). m.p.:96–98 °C.LC-MS (ESI)
[M + H]+
.
5.1.21. 2-mercapto-5-nitrobenzo[d]oxazole (20c)
The titled compound (3.52 g, 94%, yellow solid) was synthesized
from compound 19c (1.00 g, 6.49 mmol) and EtOH (32 mL) as a solvent
by a process analogous to the preparation of 20a.1H-NMR (400 MHz,
DMSO‑d6) δ: 8.19 (dd, J = 8.8, 2.0 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H),
7.74 (d, J = 8.8 Hz, 1H). LC-MS (ESI) m/z = 195 [M - H]-.
5.1.22. (R)-N-[1-(4-bromophenyl)ethyl]-N-butyl-2-amino-5-nitrobenzo[d]
oxazole (21c)
The titled compound was synthesized from compound 30c (1.15 g,
5.86 mmol) and compound 3b (1.59 g, 6.21 mmol) by
a process
8

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
analogous to the preparation of 21a. The crude material was purified by
silica gel column chromatography eluted with 10–20% AcOEt in hep-
tane to afford the titled compound (1.31 g, 54%) as a yellow oil. 1H
NMR (400 MHz, CDCl3) δ: 8.20 (d, J = 2.0 Hz, 1H), 8.00 (dd, J = 8.8,
2.0 Hz, 1H), 7.49 (d, J = 7.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 1H), 7.27 (d,
J = 7.8 Hz, 2H), 5.66 (q, J = 7.2 Hz, 1H), 3.37–3.29 (m, 1H), 3.24–3.15
(m, 1H), 1.70 (d, J = 6.8 Hz, 3H), 1.64–1.43 (m, 2H), 1.33–1.22 (m,
132.1, 131.0, 130.5, 130.5, 128.9(2C), 127.7, 126.9(2C), 123.5, 120.8,
116.1, 108.2, 55.4, 44.4, 42.4, 30.9, 25.6, 19.5, 17.1, 13.6.
m.p.:103–105 °C. LC-MS (ESI) m/z = 510 [M + H]+. Purity: 99.9%.
HR-LC-MS (ESI) m/z [M + H] + calcd, 510.2617; found, 510.2618.
5.1.27. (R)-1-({N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazol-5-yl}methyl)-3-methylurea (26)
2H), 0.88 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 418 [M + H]+
.
Compound 22 (300 mg, 0.696 mmol) was suspended in toluene
(5 mL). To this suspension was added Et3N (0.291 mL, 2.09 mmol) and
DPPA (0.449 mL, 2.09 mmol). After stirring for 5 h at 110 °C, methy-
lamine (2.0 M THF solution 1.74 mL, 3.48 mmol) was added at 0 °C.
After stirring for 18 h at room temperature, saturated NaHCO3 was
added. The mixture was extracted with CHCl3, dried over MgSO4, fil-
tered, and concentrated under reduced pressure. The crude material
was purified by silica gel column chromatography eluted with 5–10%
MeOH in CHCl3 to afford the titled compound (223 mg, 70%) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ: 7.45 (d, J = 8.8 Hz, 2H),
7.27 (s, 1H), 7.22 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 7.8 Hz, 1H), 6.90 (d,
J = 7.8 Hz, 1H), 5.64–5.56 (m, 1H), 5.44 (br s, 1H), 5.07 (br s, 1H),
4.33 (d, J = 3.9 Hz, 2H), 3.32–3.08 (m, 2H), 2.71 (d, J = 4.9 Hz, 3H),
1.65 (d, J = 6.8 Hz, 3H), 1.44–1.57 (m, 2H), 1.19–1.29 (m, 2H), 0.86 (t,
5.1.23. (R)-2-{N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazol-5-yl}-acetic acid (22)
Compound 21a (1.32 g, 2.96 mmol) was dissolved in EtOH (15 mL).
To this solution was added 3 M NaOH (5 mL). After stirring for 3 h at
50 °C, 3 M HCl was added at 0 °C. The mixture was extracted with
CHCl3, dried over MgSO4, filtered, and concentrated under reduced
pressure to afford the titled compound (1.16 g, 91%) as a brown solid.
1H NMR (400 MHz, DMSO‑d6) δ: 7.57 (d, J = 8.6 Hz, 2H), 7.41–7.36
(m, 3H), 7.23 (s, 1H), 6.95 (dd, J = 8.4, 1.6 Hz, 1H), 5.55–5.49 (m,
1H), 3.61 (s, 2H), 3.41–3.23 (m, 2H), 1.67 (d, J = 6.8 Hz, 3H),
1.59–1.15 (m, 4H), 0.82 (t, J = 7.2 Hz, 3H). LC-MS (ESI) m/z = 431
[M + H]+
.
J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 459 [M + H]+
.
5.1.24. (R)-2-{N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazol-5-yl}-N-methylacetamide (23)
Compound 22 (512 mg, 1.19 mmol) was dissolved in DMF (10 mL).
To this solution was added methylamine (2.0 M THF solution, 2.97 mL,
5.94 mmol), HOBt･H2O (545 mg, 3.56 mmol) and EDCI·HCl (683 mg,
3.56 mmol). After stirring for 24 h at room temperature, saturated
NH4Cl was added. The mixture was extracted with CHCl3, dried over
MgSO4, filtered, and concentrated under reduced pressure. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (491 mg, 93%) as
a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 7.47 (d, J = 8.8 Hz, 2H),
7.31–7.21 (m, 4H), 6.89 (d, J = 8.8 Hz, 1H), 5.67–5.61 (m, 2H), 3.62
(s, 2H), 3.36–3.11 (m, 2H), 2.74 (d, J = 4.9 Hz, 3H), 1.68 (d,
J = 6.8 Hz, 3H), 1.59–1.45 (m, 2H), 1.31–1.21 (m, 2H), 0.87 (t,
5.1.28. (R)-1-({N-butyl-N-[1-(2′-cyano-[1,1′-biphenyl]-4-yl)ethyl]-2-
aminobenzo[d]oxazol-5-yl}methyl)-3-methylurea (27)
The titled compound was synthesized from compound 26 (223 mg,
0.485 mmol) by a process analogous to the preparation of 5b. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (231 mg, 99%) as
a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 7.75 (d, J = 7.8 Hz, 1H),
7.66–7.62 (m, 1H), 7.55–7.41 (m, 6H), 7.31–7.26 (m, 1H), 7.17 (d,
J = 8.8 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 5.76–5.68 (m, 1H), 5.43 (br s,
1H), 5.06 (br s, 1H), 4.35 (d, J = 5.9 Hz, 2H), 3.40–3.32 (m, 1H),
3.25–3.17 (m, 1H), 2.71 (d, J = 4.9 Hz, 3H), 1.73 (d, J = 7.8 Hz, 3H),
1.60–1.48 (m, 2H), 1.31–1.21 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). LC-MS
J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 444 [M + H]+
.
(ESI) m/z = 482 [M + H]+
.
5.1.25. (R)-2-{N-butyl-N-[1-(2′-cyano-[1,1′-biphenyl]-4-yl)ethyl]-2-
aminobenzo[d]oxazol-5-yl}-N-methylacetamide (24)
5.1.29. (R)-1-[(N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
ethyl}-2-aminobenzo[d]oxazol-5-yl)methyl]-3-methylurea (28)
The titled compound was synthesized from compound 23 (490 mg,
1.10 mmol) by a process analogous to the preparation of 5b. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (511 mg, 99%) as
a yellow oil.1H-NMR (300 MHz, CDCl3) δ: 7.75 (dd, J = 7.7, 1.3 Hz,
1H), 7.63 (ddd, J = 7.7, 7.7, 1.3 Hz, 1H), 7.56–7.47 (m, 5H), 7.43 (ddd,
J = 7.7, 7.7, 1.3 Hz, 1H), 7.24–7.21 (m, 2H), 6.87 (dd, J = 8.2, 1.6 Hz,
1H), 5.75 (q, J = 7.1 Hz, 1H), 5.56 (br s, 1H), 3.61 (s, 2H), 3.43–3.16
(m, 2H), 2.72 (d, J = 5.1 Hz, 3H), 1.73 (d, J = 7.3 Hz, 3H), 1.62–1.47
(m, 2H), 1.32–1.20 (m, 2H), 0.86 (t, J = 7.1 Hz, 3H). LC-MS (ESI) m/
The titled compound was synthesized from compound 27 (220 mg,
0.457 mmol) by a process analogous to the preparation of 6b. The crude
material was purified by silica gel column chromatography eluted with
5–10% to afford the titled compound (123 mg, 51%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ: 7.82 (d, J = 6.8 Hz, 1H), 7.58–7.53 (m,
1H), 7.49–7.40 (m, 2H), 7.16–7.03 (m, 6H), 6.81 (d, J = 8.8 Hz, 1H),
5.57–5.51 (m, 1H), 5.38–5.31 (m, 2H), 4.24–4.09 (m, 2H), 3.43–3.33
(m, 1H), 3.23–3.14 (m, 1H), 2.56 (s, 3H), 1.63–1.52 (m, 5H), 1.32–1.24
(m, 2H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ:
162.5, 158.7, 155.1, 147.2, 143.3, 141.1, 140.0, 138.5, 137.0, 131.2,
130.7, 129.5, 129.0(2C), 127.9, 127.0(2C), 123.5, 119.1, 114.4, 108.3,
55.5, 44.4, 43.2, 31.0, 26.5, 19.6, 17.3, 13.7. m.p.:111–113 °C. LC-MS
z = 467 [M + H]+
.
5.1.26. (R)-2-(N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
ethyl}-2-aminobenzo[d]oxazol-5-yl)-N-methylacetamide (25)
(ESI) m/z = 525 [M + H]+
.
Purity: 96.9%. HR-LC-MS (ESI) m/z
+
[M + H] calcd, 525.2726; found, 525.2729.
The titled compound was synthesized from compound 24 (500 mg,
1.07 mmol) by a process analogous to the preparation of 6b. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (475 mg, 87%) as
a white solid. 1H NMR (400 MHz, CDCl3) δ: 7.93 (d, J = 7.8 Hz, 1H),
7.61 (dd, J = 7.8, 7.8 Hz, 1H), 7.53 (dd, J = 7.8, 7.8 Hz, 1H), 7.45 (d,
J = 7.8 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 7.8 Hz, 1H), 7.14
(d, J = 7.8 Hz, 2H), 6.94 (s, 1H), 6.86–6.83 (m, 1H), 5.60 (br s, 1H),
5.45 (q, J = 6.8 Hz, 1H), 3.48 (s, 2H), 3.39–3.31 (m, 1H), 3.20–3.13 (m,
1H), 2.67 (d, J = 4.9 Hz, 3H), 1.65 (d, J = 6.8 Hz, 3H), 1.60–1.48 (m,
2H), 1.32–1.22 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ: 170.8, 162.3, 157.1, 147.1, 143.2, 141.0, 139.9, 138.4,
5.1.30. (R)-N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazole-5-carboxylic acid (29)
The titled compound (1.54 g, quant., as a brown solid) was syn-
thesized from compound 21b (1.52 g, 3.52 mmol) by a process analo-
gous to the preparation of 22. 1H NMR (400 MHz, DMSO‑d6) δ: 7.80 (d,
J = 2.0 Hz, 1H), 7.68 (dd, J = 7.8, 2.0 Hz, 1H), 7.57 (d, J = 8.4 Hz,
2H), 7.50 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 5.56–5.49 (q,
J = 6.8 Hz, 1H), 3.41–3.23 (m, 2H), 1.67 (d, J = 6.8 Hz, 3H), 1.59–1.36
(m, 2H), 1.29–1.18 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/
z = 417 [M + H]+
.
9

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
5.1.31. (R)-N-methyl {N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo
[d]oxazole}-5-carboxamide (30)
1.20 mmol) by a process analogous to the preparation of 26. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (59.1 mg, 54%) as
a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 7.46 (d, J = 7.8 Hz, 2H),
7.27–7.24 (m, 3H), 7.16–7.09 (m, 2H), 6.31 (br s, 1H), 5.64 (q,
J = 6.8 Hz, 1H), 3.31–3.22 (m, 1H), 3.15–3.05 (m, 1H), 3.03 (s, 3H),
3.02 (s, 3H), 1.65 (d, J = 6.8 Hz, 3H), 1.56–1.44 (m, 2H), 1.29–1.18
The titled compound was synthesized from compound 29 (113 mg,
0.271 mmol) by a process analogous to the preparation of 23. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (95.6 mg, 82%) as
a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 7.71 (s, 1H), 7.53 (d,
J = 8.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 2H), 7.29–7.24 (m, 3H), 6.28 (br s,
1H), 5.65 (q, J = 6.8 Hz, 1H), 3.35–3.25 (m, 1H), 3.22–3.10 (m, 1H),
3.02 (d, J = 4.9 Hz, 3H), 1.68 (d, J = 6.8 Hz, 3H), 1.61–1.44 (m, 2H),
1.31–1.20 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 430
(m, 2H), 0.85 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 459 [M + H]+
.
5.1.36. (R)-3-{N-butyl-N-[1-(2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl)
ethyl]-2-aminobenzo[d]oxazol-5-yl}-1,1-dimethylurea (33b)
[M + H]+
.
The titled compound was synthesized from compound 32b
(51.0 mg, 0.111 mmol) by a process analogous to the preparation of 18.
The crude material was purified by preparative TLC with 10% MeOH in
CHCl3 to afford the titled compound (32.2 mg, 55%) as a yellow solid.
1H NMR (300 MHz, DMSO‑d6) δ: 8.17 (br s, 1H), 7.63–7.56 (m, 2H),
7.53–7.45 (m, 2H), 7.41 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H),
7.23 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 8.1 Hz, 2H), 7.02 (dd, J = 8.1,
2.0 Hz, 1H), 5.52 (q, J = 7.1 Hz, 1H), 3.43–3.11 (m, 2H), 2.92 (s, 6H),
1.64 (d, J = 7.0 Hz, 3H), 1.54–1.34 (m, 2H), 1.28–1.14 (m, 2H), 0.83 (t,
5.1.32. (R)-N-methyl (N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-
yl]ethyl}-2-aminobenzo[d]oxazole)-5-carboxamide (31)
The titled compound was synthesized from compound 30 (95.0 mg,
0.221 mmol) by a process analogous to the preparation of 18. The crude
material was purified by preparative HPLC to afford the titled com-
pound (80.6 mg, 74%) as a white solid. 1H NMR (300 MHz, CDCl3) δ:
7.92 (d, J = 6.6 Hz, 1H), 7.62–7.39 (m, 5H), 7.25–7.18 (m, 3H), 7.08
(d, J = 8.1 Hz, 2H), 6.68 (s, 1H), 5.44 (q, J = 7.0 Hz, 1H), 3.38–3.27
(m, 1H), 3.22–3.12 (m, 1H), 2.88 (d, J = 4.8 Hz, 3H), 1.63 (d,
J = 7.0 Hz, 3H), 1.59–1.48 (m, 2H), 1.32–1.20 (m, 2H), 0.87 (t,
J = 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ: 168.7, 163.2, 156.3,
150.4, 142.8, 140.9, 139.2, 139.0, 130.8, 130.6, 130.5, 130.5,
129.2(2C), 127.7, 126.8(2C), 124.1, 120.6, 113.9, 108.5, 56.0, 45.0,
31.6, 26.9, 20.0, 17.1, 13.7. m.p.:117–119 °C. LC-MS (ESI) m/z = 496
J = 7.3 Hz, 3H). m.p.:122–124 °C. LC-MS (ESI) m/z = 525 [M + H]+
.
Purity: 94.9%. HR-LC-MS (ESI) m/z [M + H] + calcd, 525.2726; found,
525.2730.
5.1.37. (R)-1-{N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazol-5-yl}-urea (32c)
The titled compound was synthesized from compound 29 (100 mg,
0.240 mmol) and ammonia (7 M in MeOH solution, 0.171 mL,
1.20 mmol) by a process analogous to the preparation of 28. The crude
material was purified by preparative TLC with 10% MeOH in CHCl3 to
afford the titled compound (52.9 mg, 51%) as a yellow oil. 1H NMR
(400 MHz, CDCl3) δ: 7.46 (d, J = 7.8 Hz, 2H), 7.27–7.22 (m, 3H),
7.20–7.14 (m, 2H), 6.99 (d, J = 7.8 Hz, 1H), 5.62 (q, J = 7.0 Hz, 1H),
4.95 (br s, 2H), 3.33–3.23 (m, 1H), 3.18–3.08 (m, 1H), 1.66 (d,
J = 7.0 Hz, 3H), 1.57–1.43 (m, 2H), 1.30–1.19 (m, 2H), 0.85 (t,
[M + H]+. Purity: 98.4%. [α]25 = +144.6 (c 0.52, CHCl3). HR-LC-
D
+
MS (ESI) m/z [M + H] calcd, 496.2461; found, 496.2458.
5.1.33. (R)-1-{N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazol-5-yl}-3-methylurea (32a)
The titled compound was synthesized from compound 29 (270 mg,
0.647 mmol) by a process analogous to the preparation of 26. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (117 mg, 41%) as
a colorless oil. 1H NMR (400 MHz, CDCl3) δ: 7.45 (d, J = 8.8 Hz, 2H),
7.26–7.20 (m, 3H), 7.19 (br s, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.01 (d,
J = 8.4 Hz, 1H), 5.65–5.58 (m, 1H), 5.24 (br s, 1H), 3.32–3.23 (m, 1H),
3.17–3.09 (m, 1H), 2.75 (d, J = 4.9 Hz, 3H), 1.65 (d, J = 7.8 Hz, 3H),
1.58–1.43 (m, 2H), 1.29–1.18 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H). LC-MS
J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 431 [M + H]+
.
5.1.38. (R)-1-(N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
ethyl}-2-aminobenzo[d]oxazol-5-yl)-urea (33c)
The titled compound was synthesized from compound 32c
(52.0 mg, 0.121 mmol) by a process analogous to the preparation of 18.
The crude material was purified by preparative TLC with 20% MeOH in
CHCl3 and preparative HPLC to afford the titled compound (20.7 mg,
37%) as a white solid. 1H NMR (400 MHz, DMSO‑d6) δ: 8.43 (br s, 1H),
7.71–7.64 (m, 2H), 7.60–7.53 (m, 2H), 7.40 (d, J = 2.0 Hz, 1H), 7.34
(d, J = 7.8 Hz, 2H), 7.22 (d, J = 8.8 Hz, 1H), 7.10 (d, J = 7.8 Hz, 2H),
6.94 (dd, J = 8.8, 2.0 Hz, 1H), 5.76 (br s, 2H), 5.52 (q, J = 6.8 Hz, 1H),
3.47–3.16 (m, 2H), 1.64 (d, J = 6.8 Hz, 3H), 1.55–1.33 (m, 2H),
(ESI) m/z = 445 [M + H]+
.
5.1.34. (R)-1-(N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
ethyl}-2-aminobenzo[d]oxazol-5-yl)-3-methylurea (33a)
The titled compound was synthesized from compound 32a (475 mg,
1.07 mmol) by a process analogous to the preparation of 18. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 and preparative HPLC to afford the titled
compound (389 mg, 71%) as a yellow solid. 1H NMR (300 MHz,
DMSO‑d6) δ: 8.39 (br s, 1H), 7.71–7.64 (m, 2H), 7.59–7.53 (m, 2H),
7.41 (d, J = 1.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.8 Hz,
1H), 7.10 (d, J = 8.4 Hz, 2H), 6.92 (dd, J = 8.8, 1.8 Hz, 1H), 5.93 (br s,
1H), 5.52 (q, J = 7.0 Hz, 1H), 3.39–3.14 (m, 2H), 2.63 (s, 3H), 1.64 (d,
J = 7.0 Hz, 3H), 1.55–1.31 (m, 2H), 1.29–1.13 (m, 2H), 0.83 (t,
J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 162.4, 157.1, 156.1,
143.2, 143.0, 141.1, 139.8, 138.4, 137.1, 131.1, 130.6, 130.6,
128.9(2C), 127.8, 127.0(2C), 123.4, 110.4, 108.2, 105.6, 55.4, 44.3,
1.26–1.16 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 497
+
[M + H]+. Purity: 99.5%. HR-LC-MS (ESI) m/z [M + H]
calcd,
497.2413; found, 497.2420.
5.1.39. (R)–N2-[1-(4-bromophenyl)ethyl]–N2-butyl-2,5-diaminobenzo[d]
oxazole (34)
Compound 21c (1.03 g, 2.46 mmol) was dissolved in AcOH (12 mL).
To this solution was added Fe (688 mg, 12.3 mmol). After stirring for
3 h at 40 °C, 3 M NaOH was added. The mixture was extracted with
CHCl3, dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude material was purified by silica gel column chro-
matography eluted with 20–50% AcOEt in heptane to afford the titled
compound (797 mg, 83%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ:
7.46 (d, J = 8.8 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.1 Hz,
1H), 6.73 (d, J = 2.2 Hz, 1H), 6.35 (dd, J = 8.1, 2.2 Hz, 1H), 5.64 (q,
J = 7.2 Hz, 1H), 3.56 (br s, 2H), 3.29–3.21 (m, 1H), 3.13–3.05 (m, 1H),
1.65 (d, J = 7.2 Hz, 3H), 1.56–1.44 (m, 2H), 1.28–1.18 (m, 2H), 0.85 (t,
30.9, 26.2, 19.5, 17.1, 13.6. m.p.:120–122 °C. LC-MS (ESI) m/z = 511
+
[M + H]+. Purity: 97.5%. HR-LC-MS (ESI) m/z [M + H]
calcd,
511.2570; found, 511.2568.
5.1.35. (R)-3-{N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazol-5-yl}-1,1-dimethylurea (32b)
The titled compound was synthesized from compound 29 (100 mg,
0.240 mmol) and dimethylamine (2.0 M THF solution, 0.600 mL,
J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 388 [M + H]+
.
10

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
5.1.40. (R)–N2-[1-(4-bromophenyl)ethyl]–N2-butyl-N5-acetyl-2,5-
diaminobenzo[d]oxazole (35)
2H), 3.27 (s, 3H), 1.69 (d, J = 6.8 Hz, 3H), 1.62–1.44 (m, 2H),
1.32–1.21 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 445
Compound 34 (0.123 g, 0.317 mmol) was dissolved in CH2Cl2
(5 mL). To this solution was added pyridine (0.077 mL, 0.950 mmol)
and AcCl (0.034 mL, 0.475 mmol) at 0 °C. After stirring for 1 h at the
same temperature, saturated NH4Cl was added. The mixture was ex-
tracted with CHCl3, dried over MgSO4, filtered, and concentrated under
reduced pressure. The crude material was purified by silica gel column
chromatography eluted with 50–75% AcOEt in heptane to afford the
titled compound (103 mg, 76%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) δ: 7.57 (br s, 1H), 7.49–7.43 (m, 3H), 7.27–7.21 (m, 3H), 7.17
(d, J = 8.4 Hz, 1H), 5.64 (q, J = 7.1 Hz, 1H), 3.34–3.07 (m, 2H), 2.17
(s, 3H), 1.66 (d, J = 7.1 Hz, 3H), 1.59–1.41 (m, 2H), 1.31–1.17 (m, 2H),
[M + H]+
.
5.1.44. (R)-1-(N-butyl-N-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
ethyl}-2-aminobenzo[d]oxazol-5-yl)-1-methylurea (39)
The titled compound was synthesized from compound 38 (40.0 mg,
0.090 mmol) by a process analogous to the preparation of 18. The crude
material was purified by preparative TLC with 10% MeOH in CHCl3 to
afford the titled compound (35.5 mg, 77%) as a white solid. 1H NMR
(400 MHz, CDCl3) δ: 7.93 (d, J = 6.8 Hz, 1H), 7.57 (dd, J = 7.3, 7.3 Hz,
1H), 7.50 (dd, J = 7.3, 7.3 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.35–7.23
(m, 3H), 7.21–7.14 (m, 3H), 6.87 (d, J = 7.8 Hz, 1H), 5.59 (d,
J = 6.8 Hz, 1H), 4.54 (br s, 2H), 3.44–3.31 (m, 1H), 3.26–3.12 (m, 1H),
3.15 (s, 3H), 1.69 (d, J = 6.8 Hz, 3H), 1.63–1.49 (m, 2H), 1.36–1.21
0.85 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 430 [M + H]+
.
5.1.41. (R)–N2-butyl-N2-{1-[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]
(m, 2H), 0.89 (t, J = 7.3 Hz, 3H). LC-MS (ESI) m/z = 511 [M + H]+
.
ethyl}-N5-acetyl-2,5-diaminobenzo[d]oxazole (36)
Purity: 99.5%. HR-LC-MS (ESI) m/z [M + H] + calcd, 511.2570; found,
The titled compound was synthesized from compound 35 (102 mg,
0.237 mmol) by a process analogous to the preparation of 18. The crude
material was purified by silica gel column chromatography eluted with
10–15% MeOH in CHCl3 and preparative TLC with 20% MeOH in CHCl3
to afford the titled compound (77.6 mg, 66%) as a white solid. 1H NMR
(400 MHz, CDCl3) δ: 8.41 (br s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.55 (dd,
J = 7.3, 7.3 Hz, 1H), 7.46 (dd, J = 7.3, 7.3 Hz, 1H), 7.38 (d, J = 7.8 Hz,
1H), 7.22–7.09 (m, 4H), 7.05–6.99 (m, 2H), 5.45 (q, J = 6.8 Hz, 1H),
3.32–3.19 (m, 1H), 3.13–3.00 (m, 1H), 1.99 (s, 3H), 1.58–1.44 (m, 2H),
1.53 (d, J = 6.8 Hz, 3H), 1.32–1.16 (m, 2H), 0.83 (t, J = 7.3 Hz, 3H).
13C NMR (100 MHz, DMSO‑d6) δ: 168.0, 162.7, 155.4, 144.3, 143.3,
141.1, 139.8, 138.6, 135.9, 131.0, 130.7, 130.7, 129.0(2C), 127.8,
127.0(2C), 123.9, 111.4, 108.4, 106.9, 55.4, 44.4, 31.0, 24.0, 19.6,
511.2571.
5.2. Biological assay
5.2.1. Human ChemR23 assay
CAL-1 cells were suspended in RPMI 1640 containing 10 mM
HEPES, Calcium-3 (as described in the manufacturer's protocol) and
amaranth (final, 0.75 mg/mL). Cells were plated onto a 384-well plate
(1 × 104 cells/40 μL), and 5 μL of compounds dissolved in assay buffer
(final, 0–10 µM) was added. After incubation at 37 °C for 45 min, 5 μL of
human chemerin dissolved in assay buffer containing 1% (w/v) bovine
serum albumin (final, 50 ng/mL) was added, and the intracellular cal-
cium ion concentrations were measured using an FDSS6000
(Hamamatsu Photonics K.K.).
17.3, 13.7. m.p.:126–128 °C. LC-MS (ESI) m/z = 496 [M + H]+. Purity:
+
99.5%. HR-LC-MS (ESI) m/z [M + H]
calcd, 496.2461; found,
496.2464.
5.2.2. CXCR4 assay
CAL-1 cells were suspended in RPMI1640 containing 10 mM HEPES,
Calcium-3 (as described in the manufacturer's protocol) and amaranth
(final, 0.75 mg/mL). Cells were plated onto a 384-well plate (1 × 104
cells/40 μL), and 5 μL of compounds dissolved in assay buffer (final,
0–10 µM) was added. After incubation at 37 °C for 45 min, 5 μL of
human SDF-1α dissolved in assay buffer containing 1% (w/v) bovine
serum albumin (final, 5 × 10-3 µM) was added, and intracellular cal-
cium ion concentrations were measured using an FDSS6000
(Hamamatsu Photonics K.K.).
5.1.42. (R)–N2-[1-(4-bromophenyl)ethyl]–N2-butyl-N5-methyl-2,5-
diaminobenzo[d]oxazole (37)
Compound 34 (282 mg, 0.726 mmol) was dissolved in MeCN
(10 mL). To this solution was added K2CO3 (0.100 g, 0.726 mmol) and
MeI (0.050 mL, 0.799 mmol). After stirring for 24 h at room tempera-
ture, H2O was added. The mixture was extracted with CHCl3, dried over
MgSO4, filtered, and concentrated under reduced pressure. The crude
material was purified by silica gel column chromatography eluted with
5–10% MeOH in CHCl3 to afford the titled compound (68.7 mg, 24%) as
a colorless oil.1H-NMR (400 MHz, CDCl3) δ: 7.45 (d, J = 8.8 Hz, 2H),
7.25 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 7.8 Hz, 1H), 6.69 (d, J = 2.4 Hz,
1H), 6.29 (dd, J = 7.8, 2.4 Hz, 1H), 5.65 (q, J = 6.8 Hz, 1H), 3.30–3.22
(m, 1H), 3.13–3.06 (m, 1H), 2.84 (s, 3H), 1.65 (d, J = 6.8 Hz, 3H),
1.58–1.43 (m, 2H), 1.29–1.18 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H). LC-MS
5.2.3. Mouse ChemR23 assay
Ba/F3 cells stably expressing mouse ChemR23 were suspended in
RPMI1640 containing 10 mM HEPES, Calcium-3 (as described in the
manufacturer's protocol) and amaranth (final, 0.75 mg/mL). Cells were
plated onto a 384-well plate (1 × 104 cells/40 μL), and 5 μL of com-
pounds dissolved in assay buffer (final, 0–10 µM) was added. After in-
cubation at 37 °C for 45 min, 5 μL of mouse chemerin dissolved in assay
buffer containing 1% (w/v) bovine serum albumin (final, 2 ng/mL) was
added, and intracellular calcium ion concentrations were measured
using an FDSS6000 (Hamamatsu Photonics K.K.).
(ESI) m/z = 402 [M + H]+
.
5.1.43. (R)-1-{N-[1-(4-bromophenyl)ethyl]-N-butyl-2-aminobenzo[d]
oxazol-5-yl}-1-methylurea (38)
Compound 37 (68.0 mg, 0.169 mmol) was dissolved in CH2Cl2
(5 mL). To this solution was added trichloroacetyl isocyanate
(0.021 mL, 0.177 mmol) at 0 °C. After stirring for 1 h at the same tem-
perature, saturated NH4Cl was added. The mixture was extracted with
AcOEt, dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude material was dissolved in MeOH (5 mL). To this
solution was added K2CO3 (70.0 mg, 0.507 mmol). After stirring for 6 h
at 50 °C, saturated NH4Cl was added. The mixture was extracted with
CHCl3, dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude material was purified by silica gel column chro-
matography eluted with 5–10% MeOH in CHCl3 to afford the titled
compound (41.0 mg, 55%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
δ: 7.48 (d, J = 7.8 Hz, 2H), 7.29–7.24 (m, 4H), 6.93 (dd, J = 7.8,
2.0 Hz, 1H), 5.64 (q, J = 6.8 Hz, 1H), 4.50 (br s, 2H), 3.37–3.11 (m,
5.2.4. Agonist assay
CAL-1 cells were suspended in RPMI1640 containing 10 mM HEPES,
Calcium-3 (as described in the manufacturer's protocol) and amaranth
(final, 0.75 mg/mL). Cells were plated onto a 384-well plate (1 × 104
cells/45 μL) and incubated at 37 °C for 45 min. After incubation, 5 μL of
compounds dissolved in assay buffer (final, 0–10 µM) was added, and
intracellular calcium ion concentrations were measured using an
FDSS6000 (Hamamatsu Photonics K.K.).
5.2.5. Chemotaxis assay
CAL-1 cells were treated with various concentrations of compounds
dissolved in assay buffer (as above) containing 0.1% (w/v) bovine
11

T. Imaizumi, et al.
Bioorganic&MedicinalChemistry27(2019)115091
Appendix A. Supplementary data
serum albumin (final, 0–10 µM). Cells (2.0 × 105 cells, 200 µL) and
5 ng/mL of chemerin dissolved in assay buffer containing 0.1% (w/v)
bovine serum albumin (600 µL) were applied to the top and bottom
wells, respectively, of 8-μm-pore chambers. After incubation at 37 °C for
4 h, the numbers of cells in the bottom wells were counted using
CellTiter-Glo reagent.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.115091.
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
We are grateful to Hideyuki Onodera, Kazuo Kubo, Yutaka Kanda
and Katsuya Kobayashi for their helpful support and discussions. The
authors thank Kyoko Tsutsumi for performing the LC-MS analysis and
spectral measurements in this research. We sincerely thank Dr. T.
Maeda and Dr. S. Kamihira (Nagasaki University, Japan) for providing
the CAL-1 cells. This cell line invented at Nagasaki University was used
as a research material under license.
12