Identification of diphenylalkylisoxazol-5-amine scaffold as novel activator of cardiac myosin

Article abstract of DOI:10.1016/j.bmc.2020.115742

To identify novel potent cardiac myosin activator, a series of diphenylalkylisoxazol-5-amine compounds 4–7 have been synthesized and evaluated for cardiac myosin ATPase activation. Among the 37 compounds, 4a (CMA at 10 μM = 81.6%), 4w (CMA at 10 μM = 71.2%) and 6b (CMA at 10 μM = 67.4%) showed potent cardiac myosin activation at a single concentration of 10 μM. These results suggested that the introduction of the amino-isoxazole ring as a bioisostere for urea group is acceptable for the cardiac myosin activation. Additional structure–activity relationship (SAR) studies were conducted. Para substitution (-Cl, –OCH₃, -SO₂N(CH₃)₂) to the phenyl rings or replacement of a phenyl ring with a heterocycle (pyridine, piperidine and tetrahydropyran) appeared to attenuate cardiac myosin activation at 10 μM. Additional hydrogen bonding acceptor next to the amino group of the isoxazoles did not enhance the activity. The potent isoxazole compounds showed selectivity for cardiac myosin activation over skeletal and smooth muscle myosin, and therefore these potent and selective isoxazole compounds could be considered as a new series of cardiac myosin ATPase activators for the treatment of systolic heart failure.

Full text of DOI:10.1016/j.bmc.2020.115742

Bioorganic&MedicinalChemistry28(2020)115742
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of diphenylalkylisoxazol-5-amine scaffold as novel activator
of cardiac myosin
Pulla Reddy Boggua,b, Eeda Venkateswararaoa, Manoj Manickama, Niti Sharmaa,
⁎
⁎
Jong Seong Kanga, , Sang-Hun Junga,
a College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 34134, Republic of Korea
b School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 16419, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
To identify novel potent cardiac myosin activator, a series of diphenylalkylisoxazol-5-amine compounds 4–7
have been synthesized and evaluated for cardiac myosin ATPase activation. Among the 37 compounds, 4a (CMA
at 10 µM = 81.6%), 4w (CMA at 10 µM = 71.2%) and 6b (CMA at 10 µM = 67.4%) showed potent cardiac
myosin activation at a single concentration of 10 µM. These results suggested that the introduction of the amino-
isoxazole ring as a bioisostere for urea group is acceptable for the cardiac myosin activation. Additional
structure–activity relationship (SAR) studies were conducted. Para substitution (-Cl, –OCH3, -SO2N(CH3)2) to the
phenyl rings or replacement of a phenyl ring with a heterocycle (pyridine, piperidine and tetrahydropyran)
appeared to attenuate cardiac myosin activation at 10 µM. Additional hydrogen bonding acceptor next to the
amino group of the isoxazoles did not enhance the activity. The potent isoxazole compounds showed selectivity
for cardiac myosin activation over skeletal and smooth muscle myosin, and therefore these potent and selective
isoxazole compounds could be considered as a new series of cardiac myosin ATPase activators for the treatment
of systolic heart failure.
Diphenylalkylisoxazol-5-amine
Cardiac myosin ATPase activator
SAR
Systolic heart failure
1. Introduction
Omecamtiv mecarbil (OM, Fig. 1), a cardiac-selective myosin acti-
vator, is a first in class and effective drug for systolic HF treatment and
Heart failure (HF) is a global widespread in health care and a
leading cause of mortality and morbidity worldwide1,2. The health
expenditures for regulating HF are significant and will increase dra-
matically with an ageing population1,3. The weakening of cardiac
contractions is expected due to HF. Of the millions of patients with HF
worldwide, approximately 50% have HF with reduced ejection fraction
(HFrEF). Inotropes have shown advantage in maintaining hemody-
namic stability and relieving the patient’s pain in systolic HF pa-
the achievement of this drug has been tested in small and large animal
models, as well as in clinical studies4,16. In contrast to calcitropes that
increased intracellular concentrations of calcium and cAMP and de-
crease ejection time, OM is a myotrope6 that improves the systolic
function by increasing myocardial contraction and stroke volume
without consuming ATP energy, oxygen, or changing intracellular cal-
cium levels4,13,15. Clinical investigations of OM on humans have shown
there is a linear association between dose and systolic ejection
time17–20. Even though the clinical examinations results presented that
OM is highly effective in relieving symptoms and improve the quality of
life of systolic HF patients, additional drugs to improve cardiac function
could be beneficial to patients with HFrEF.
tients4,5
.
Currently accessible calcitropes6 such as phosphodiesterase in-
hibitors and adrenergic receptor agonists are used in the treatment of
systolic HF5. Inappropriately, these drugs have adverse effects, for in-
stance, increased intracellular concentrations of calcium and cAMP
contributing to increased heart rate, hypotension, and mortality7–10. To
overcome these limitations cardiac myosin activators are being devel-
oped, which directly work at the level of the cardiac sarcomere acti-
vating the actin-myosin cross-bridges, the smallest force-producing unit
Our group identified flexible dialkyl substituted urea scaffold as a
novel cardiac myosin activator with potent cardiac myosin ATPase
activation (CMA)21. As shown in Figure 1, the urea compound 1, (CMA
at 10 µM = 53.3%) exhibited robust activity at a single concentration
of 10 µM21. In another study, the lead compound 1 was optimized to
enhance the activity and found dimethylsulfonamidediphenylalkylurea
involved in the contraction mechanism4,11–15
.
⁎ Corresponding authors.
E-mail addresses: kangjss@cnu.ac.kr (J.S. Kang), jungshh@cnu.ac.kr (S.-H. Jung).
https://doi.org/10.1016/j.bmc.2020.115742
Received 30 June 2020; Received in revised form 26 August 2020; Accepted 29 August 2020
Availableonline05September2020
0968-0896/©2020PublishedbyElsevierLtd.

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
Figure 1. Reported urea analogs and oxadiazole isostere as cardiac myosin
activators.
Scheme 1. Synthesis of isoxazol-5-amine compounds (substituents are listed in
Table 1). Reagents and Conditions: (a) 60% NaH, CH3CN, THF, reflux, 3 h; (b)
NH2OH.HCl, NaOH, H2O, reflux, 2 h, 40–82%; (c) Alkyl halide 11a-f, Cs2CO3 or
60% NaH, DMF, 60 °C, 2 h, 30–72%; (d) 60% NaH, MeI, THF, rt, 8 h, 58%.
2 (CMA at 10 µM = 91.6%) as a novel cardiac myosin activator (Figure
1)22. Further to enhance the scope of the cardiac myosin activators with
new heterocyclic analogs, the urea scaffold was switched over to ox-
adiazole isostere. However, oxadiazole 3 (CMA at 10 μM = 62.9%) had
reduced activity at 10 µM when compared to dimethylsulfonamidedi-
phenylalkylurea 2.23 Continuous exploration of new isosteres of urea,
parallelly without diminishing the activity of the dimethylsulfonami-
dediphenylalkylurea 2 is the main objective of this study.
alkyl halide in presence of Cs2CO3 or 60% NaH in N,N-di-
methylformamide at 60 °C to give desired final amino-isoxazole com-
pounds 4a-z and 4ab. The N-methylated compound 5 was synthesized
from 4a by alkylation with methyl iodide in the presence of 60% NaH in
58% yield (Scheme 1).
In this respect, the current work aim to investigate possible bioi-
sosteric replacement of the urea scaffold with isoxazole ring. Thus, rigid
amino-isoxazoles (4 and 5) have been designed and synthesized as se-
lective cardiac myosin activators for the treatment of systolic heart
failure. In addition, we also synthesized amide (6) and urea (7) iso-
xazole compounds by introduction of additional hydrogen bonding and
evaluated cardiac myosin activation (Figure 2).
As shown in Scheme 2, the amide compounds 6a, 6b and 6e were
synthesized from the amine intermediate 10a and 10 l by coupling with
appropriate carboxylic acid 12 in the presence of EDC.HCl and DMAP in
DMF at 50 °C for 16 h. The amide compounds 6c, 6d and 6f were
synthesized from intermediate 10a and 10d by reaction with the ap-
propriate acid chloride 12 in pyridine at 80 °C for 16 h. Finally, the urea
Figure 2. Introduction of isoxazol-5-amine ring as a bioisostere for urea group as cardiac myosin activator.
2. Chemistry
The synthetic routes to acquire the target amino-isoxazole com-
compounds 7a-c was synthesized from 10a in the presence of tri-
phosgene and DIPEA in THF at 0 °C to ambient temperature for 16 h.
Scheme 3 represents the synthesis of intermediate compounds 10 m
and 11f. The intermediate 10 m was synthesized similar to that of the
preparation of 10a. The addition of 9 m to hydroxylamine hydro-
chloride in the presence of sodium hydroxide in water at reflux tem-
perature yielded the de-protected product 10 m-In in 62% yield. Later,
a conversion of 10 m-In to 10 m in 66% yield was smoothly conducted
by the reaction with the methyl chloroformate in the presence of
DIPEA. The intermediate 11f was synthesized by mixing starting ma-
terial 14 with chlorosulfonic acid to give 15; to 15 was added di-
methylamine hydrochloride and TEA in DCM resulting in 11f.
pounds 4–7 were outlined in Scheme 1 and Scheme 2. Commercially
available various carboxylic esters 8a-m were mixed with acetonitrile
in presence of 60% sodium hydride in tetrahydrofuran under reflux
temperature to give nitrile compounds 9a-m in quantitative yields by
using the reported synthetic procedure 24,25. The reaction of appro-
priate nitrile compound 9 with hydroxylamine hydrochloride in the
presence of sodium hydroxide in water at reflux temperature afforded
the key isoxazol-5-amine intermediates 10a-m25–31 in excellent yields.
The resulted isoxazol-5-amines 10a-m were stirred with appropriate
2

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
Scheme 2. Synthesis of amide and urea
isoxazole compounds (substituents are listed
in Table 1). Reagents and Conditions: (a)
Carboxylic acid, EDC.HCl, DMAP, DMF,
50 °C, 16 h, 41–52%; (b) Acid chloride,
pyridine, 80 °C, 16 h, 47–67%; (c) Tri-
phosgene, DIPEA, THF, 0 °C to rt, 16 h,
45–58%.
Scheme 3. Synthesis of intermediate 10 m and 11f. Reagents and conditions: (a) 60% NaH, CH3CN, THF, reflux 3 h; (b) NH2OH.HCl, NaOH, H2O, reflux, 2 h, 62%;
(c) ClCOOCH3, DIPEA, DCM, 0 °C to rt, 1 h, 66%; (d) ClSO3H, DCM, rt, 18 h; (e) (CH3)2N.HCl, TEA, DCM, rt, 2 h, 75%.
3. Pharmacology
muscle type was estimated by comparing the effect of the compound on
actin stimulated ATPase activity of a panel of myosin isoforms in-
cluding cardiac (bovine (10 µM))32, smooth muscle (chicken gizzard
(100 µM))33,34 and skeletal (rabbit (100 µM))35,36 at a single dose of the
compound. Omecamtiv mecarbil (1) was taken as a positive control for
cardiac myosin ATPase activity and (-)-blebbistatin was taken as a ne-
gative control for measurement of smooth or skeletal muscle myosin
ATPase activity37. The selectivity data is shown in Table 2.
In the sarcomere, force generation is directly coupled to ATP hy-
drolysis by myosin. The Actin stimulated ATPase activity of myosin was
evaluated spectrophotometrically using a modified version of a pre-
viously reported assay.32 Compounds 4–7 were tested at a single con-
centration of 10 µM and were reported for the % increase from baseline
of the myosin ATPase at pCa concentration of 6.5. The results from this
assay are shown in Table 1. Compound specificity with respect to
Table 1
List of the synthesized diphenylalkylisoxazolamine compounds (4a-z, 4ab, 5, 6a-f and 7a-c) as cardiac myosin activator.
Comp. No.
Substituents
R1
CLogP
Myosin ATPase activity(% at 10 µM)
Rnn
Rn
m
n
4a
4b
4c
4d
4e
4f
H
H
0
2
5.139
5.668
4.760
4.111
4.878
4.229
81.6
44.2
48.5
21.8
46.5
15.0
H
H
H
H
H
H
H
H
H
H
0
0
0
1
2
3
1
0
2
0
4 g
4 h
H
H
H
H
3
0
0
2
4.758
3.839
53.9
25.2
(continued on next page)
3

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
Table 1 (continued)
Comp. No.
Substituents
R1
CLogP
5.869
5.490
5.155
4.776
5.685
5.431
Myosin ATPase activity(% at 10 µM)
Rnn
Rn
m
n
4i
H
H
0
2
27.2
31.4
35.2
11.5
22.4
10.3
4j
H
H
H
H
H
H
H
H
H
H
0
0
0
0
0
1
2
1
3
2
4 k
4 l
4 m
4n
4o
4p
4q
4r
H
H
H
H
H
H
H
H
2
1
0
0
2
2
2
1
4.368
3.839
3.310
2.653
15.0
10.2
5.1
45.6
4 s
4 t
4u
H
H
H
H
H
H
0
0
0
2
3
2
3.032
3.561
3.708
41.2
41.8
35.2
4v
4w
4x
4y
4z
–OCH3
H
H
H
H
H
0
0
0
0
0
2
2
2
2
2
5.058
4.334
5.058
3.035
3.627
26.2
71.2
20.3
43.2
12.0
-SO2N(CH3)2
–OCH3
-SO2N(CH3)2
–OCH3
4ab
-SO2N(CH3)2
H
0
2
2.903
33.1
5
H
H
H
H
H
–CH3
H
0
0
0
0
0
2
1
2
3
4
5.394
3.581
4.140
4.519
5.048
18.0
56.6
67.4
43.2
40.8
6a
6b
6c
6d
H
H
H
(continued on next page)
4

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
Table 1 (continued)
Comp. No.
Substituents
R1
CLogP
Myosin ATPase activity(% at 10 µM)
Rnn
Rn
m
n
6e
6f
H
H
0
2
2.033
4.179
3.891
4.220
4.599
3.266
42.2
24.6
15.0
30.0
39.4
55.3
H
H
H
H
H
H
H
H
3
0
0
0
0
1
2
3
7a
7b
7c
STD
Omecamtiv mecarbil (OM)
Table 2
group R1 to the isoxazol-5-amine ring were sequentially varied as
shown in 4e (m = 1, n = 2, CMA at 10 µM = 46.5%; cLogP = 4.878),
4f (m = 2, n = 0, CMA at 10 µM = 15.0%; cLogP = 4.229) and 4 g
(m = 3, n = 0, CMA at 10 µM = 53.3%; cLogP = 4.758), which did
not improve the ATPase activity. These results show that no spacer
between the R1 (phenyl) and isoxazol-5-amine ring “m = 0” is optimum
for the activity at 10 µM.
Selectivity study of compounds 4a, 4e, 4w and 6b in myosins.
Compound No.
ATPase % activity
Cardiac myosin(at
10 µM)
Skeletal myosin(at
100 µM)
Smooth myosin(at
100 µM)
4a
81.6
46.5
71.2
67.4
55.3
N.D.
−2.5
3.2
1.1
4e
2.5
The change of phenyl ring (R1) of 4a with pyridine which could
improve the water solubility as represented in analog 4 h (m = 0,
n = 2, CMA at 10 µM = 25.2%; cLogP = 3.839) showed a decrement
in the activity at 10 µM. Further, exploration of the effect of sub-
stituents on the 3-phenyl ring (left side) was attempted. Accordingly,
we prepared 4-chloro analogs such as 4i (m = 0, n = 2, CMA at
10 µM = 27.2%; cLogP = 5.869) and 4j (m = 0, n = 1, CMA at
10 µM = 31.4%; cLogP = 5.490), and 4-methoxy analogs such as 4 k
(m = 0, n = 2, CMA at 10 µM = 35.2%; cLogP = 5.155), 4 l (m = 0,
n = 1, CMA at 10 µM = 11.5%; cLogP = 4.776) and 4 m (m = 0,
n = 3, CMA at 10 µM = 22.4%; cLogP = 5.685) which showed
moderate to minimal ATPase activation. These results suggested that
this substitution on the phenyl ring is not tolerated for robust activation
at 10 µM.
4w
−1.3
1.8
2.1
6b
3.2
OM
3.5
−2.6
−36.4
(-)Blebbistatin
−52.3
N.D.: Not determined.
4. Results and discussion
In the first set of experiments, diphenylalkylisoxazol-5-amines 4a-z
and 4ab were synthesized and determined for their cardiac myosin
ATPase activation at a single compound concentration of 10 µM
(Table 1). Initially, we synthesized analog 4a with three carbon spacer
between the amine functional group of the isoxazole to the phenyl ring
and evaluated for cardiac myosin activation. Surprisingly, analog 4a
(m = 0, n = 2, CMA at 10 µM = 81.6%; cLogP = 5.139) showed
greater activity at 10 µM than the previously reported analog 2 (CMA at
10 µM = 53.3%) and OM (CMA at 10 µM = 55.3%). This outcome
suggested that the bioisosteric replacement of the urea scaffold could be
useful for discovery of novel rigid urea molecules such as isoxazol-5-
amines as cardiac myosin ATPase activator.
Replacement of the phenyl ring (R1) in 4a with hydrophobic cy-
clohexyl ring as denoted in 4n (m = 0, n = 2, CMA at 10 µM = 15.0%;
cLogP = 4.368) revealed reduced activity at 10 µM. Further we pre-
pared 4o (m = 2, n = 2, CMA at 10 µM = 10.3%; cLogP = 5.431), 4p
(m = 1, n = 2, CMA at 10 µM = 10.2%; cLogP = 3.839) and 4q
(m = 0, n = 2, CMA at 10 µM = 5.1%; cLogP = 3.310) with the R1
phenyl was replaced with a methyl group and tested for the CMA. The
activity of these analogs at 10 µM is dramatically reduced. In addition,
replacement of the cyclohexyl ring in 4n with hydrogen bonding tet-
rahydropyrane ring as denoted in 4 s (m = 0, n = 2, CMA at
10 µM = 41.2%; cLogP = 3.032) showed 4-fold more activity at 10 µM
than 4n. However, variation of the chain length as in 4r (m = 0, n = 1,
CMA at 10 µM = 45.6%; cLogP = 2.653) and 4 t (m = 0, n = 3, CMA
at 10 µM = 41.8%; cLogP = 3.561) did not enhance the activity.
Moreover, we also synthesized methyl carbamate analog 4u (m = 0,
n = 2, CMA at 10 µM = 35.2%; cLogP = 3.708). Hence, all of these
compounds did not show the better activity than the phenyl analog 4a.
Next, introduction of the methoxy and N,N-dimethylsulfonamide
The above result encouraged us to study SAR of the isoxazol-5-
amine scaffold. Next, extension and contraction tactics were used to
confirm the length of the methylene chain between isoxazol-5-amine
and the phenyl ring. Accordingly, analogs 4b, 4c and 4d were prepared.
Among them, 4b (m = 0, n = 3, CMA at 10 µM = 44.2%;
cLogP = 5.668), 4c (m = 0, n = 1, CMA at 10 µM = 48.5%;
cLogP = 4.760) showed similar activation at 10 µM when compared to
OM (CMA = 55.3%) whereas analog 4d (m = 0, n = 0, CMA at
10 µM = 21.8%; cLogP = 4.111) decreased the activity at 10 µM.
These results confirmed that the three carbon spacer “n = 2” is ideal for
the activation at 10 µM. On the other side, the methylene spacer to aryl
5

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
substituents on the para position (R2) of the phenyl ring as in 4v
(R2 = –OCH3, CMA = 26.2%at 10 µM; cLogP = 5.058) showed de-
creased CMA activation at 10 µM and 4w (R2 = -SO2N(CH3),
CMA = 71.2%at 10 µM; cLogP = 4.334) showed good CMA activity.
Variation of R1 groups with methoxy and N,N-dimethylsulfonamide
substituents as shown in 4x (CMA = 20.3% at 10 µM; cLogP = 5.058),
4y (CMA = 43.2% at 10 µM; cLogP = 3.035), 4z (CMA = 12.0% at
10 µM; cLogP = 3.627) and 4ab (CMA = 33.1% at 10 µM;
cLogP = 2.903) did not enhance the activity. These results suggested
that the N,N-dimethylsulfonamide group on phenyl ring play an im-
portant role in the CMA activation for this set of analogs.
6. Materials and methods
6.1. Chemistry
All commercial chemicals were used as obtained and all solvents
were purified by distillation prior to use applying the standard proce-
dures38. Thin layer chromatography (TLC) was performed on E Merck
silica gel GF-254 pre-coated plates, identification was performed under
UV illumination (λ = 254 nm), and colorization with Iodine and
KMnO4. All compounds were purified by flash column chromatography
which was performed on E Merck silica gel (230–400 mesh). 1H NMR
and 13C NMR spectra were measured against the peak of tetra-
methylsilane using a Bruker Fourier 300 NMR (300 MHz) and JEOL,
JNM-AL400 NMR (400 MHz) spectrometer. Infrared (IR) spectra were
recorded on a Nicolet 380 model FTIR. Melting points (mp) were de-
termined on an Electro thermal 1A 9100 MK2 apparatus and are un-
corrected. High resolution mass spectrum (HRMS) were measured in
ESI ionization using AB Sciex Triple TOF 5600 LCMS instrument.
Introduction of methyl group on nitrogen of 4a as represented in
compound 5 (CMA = 18.1% at 10 µM; cLogP = 5.394) showed dra-
matic decreased in the in vitro activity at 10 µM. This outcome sug-
gested us substitution on nitrogen is not necessary.
Next objective of our work was to introduce additional hydrogen
bonding property next to the amine group. Therefore, we synthesized
amide analogs 6a-f and urea analogs 7a-c. Among them 6a, 6b and 6c
showed greater activation at 10 µM than the OM (Table 1). However,
these compounds did not improve the CMA activity when compared
with amine compound 4a. These results suggested that the amine group
is important for the CMA activity.
6.1.1. General procedure for the preparation of compounds 4a-c, 4e-z and
4ab
To
a
stirred solution of 3-substituted-isoxazol-5-amine 10
Additional experiments were carried out to determine the cardiac
myosin selectivity, the potent isoxazole compounds 4a, 4e, 4w and 6b
were tested in skeletal and smooth myosins. As shown in Table 2, all of
them did not activate myosin ATPase of skeletal and smooth myosin S1
which clearly indicate that these isoxazole compounds are selective
cardiac myosin activators from these in vitro systems.
(1.87 mmol) in DMF (5 mL), Cs2CO3 (1.87 mmol) and appropriate
bromo compound 11 (1.87 mmol) were added at room temperature.
The resulting solution was stirred at 60 °C for 16 h. The mixture was
cooled, quenched with ice cold water (30 mL), and then extracted with
EtOAc (2x times). The combined organic extracts were dried over an-
hydrous Na2SO4 and then concentrated under reduced pressure. The
crude mixture was subjected to flash silica gel (230–400 mesh) column
chromatography (eluting with 5–10% Ethyl acetate in hexanes) to af-
ford the title compounds 4.
5. Conclusion
In summary, novel diphenylalkylisoxazol-5-amines 4–7 have been
synthesized and evaluated for cardiac myosin ATPase activation at
10 µM. Among them, compounds 4a, 4w and 6b showed high cardiac
myosin activation at 10 µM. These results suggested that the in-
troduction of the amino-isoxazole ring as a bioisostere for urea group is
acceptable for the cardiac myosin activation. In addition, SAR (Fig. 3)
studies were conducted. Addition of simple functional groups like
chlorine, methoxyl and N,N-dimethyl sulfonamide on para position of
either phenyl rings or change of one phenyl ring to other heterocycles
like pyridine, piperidine and tetrahydropyran are not tolerated for the
cardiac myosin activation at 10 µM. Additional HBA next to the amino
group of the isoxazoles did not enhance the activity. The highly active
isoxazole compounds showed selectivity for cardiac myosin activation
over skeletal and smooth muscle myosin, and therefore these isoxazole
compounds could be considered a new series of cardiac myosin ATPase
activators for the treatment of systolic HF.
6.1.1.1. 3-Phenyl-N-(3-phenylpropyl)isoxazol-5-amine (4a).. Yield 59%;
off white solid; mp 54–57 °C; IR (neat) 3265, 2935, 2849, 1611, 1403,
1258, 1176, 1081, 763, 698 cm−1
;
1H NMR (400 MHz, CDCl3) δ
7.71–7.77 (m, 2H), 7.40–7.46 (m, 3H), 7.29–7.36 (m, 2H), 7.18–7.26
(m, 3H), 5.23 (s, 1H; isoxazole ring proton), 4.55 (t, J = 6.22 Hz, 1H),
3.26 (q, J = 6.99 Hz, 2H), 2.75 (t, J = 7.56 Hz, 2H), 1.94–2.05 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 170.4, 163.7, 141.0, 130.0, 129.7,
128.7, 128.6, 128.5, 126.6, 126.2, 75.1, 44.0, 32.9, 30.9; HRMS (ESI)
calculated for C18H18N2O [M + H]+ 279.1497, found 279.1521.
6.1.1.2. 3-Phenyl-N-(4-phenylbutyl)isoxazol-5-amine (4b).. Yield 61%;
white solid; mp 75–78 °C; IR (neat) 3267, 2935, 2849, 1611, 1403,
1258, 1176, 1082, 763, 697 cm−1
;
1H NMR (400 MHz, CDCl3) δ
7.72–7.78 (m, 2H), 7.40–7.47 (m, 3H), 7.27–7.33 (m, 2H), 7.17–7.24
(m, 3H), 5.26 (s, 1H; isoxazole ring proton), 4.52 (t, J = 5.98 Hz, 1H),
3.21–3.30 (m, 2H), 2.68 (t, J = 7.20 Hz, 2H), 1.64–1.80 (m, 4H); 13
C
NMR (100 MHz, CDCl3) δ 170.5, 163.7, 141.9, 130.0, 129.7, 128.7,
128.5, 128.4, 126.6, 126.0, 75.1, 44.5, 35.4, 29.0, 28.4; HRMS (ESI)
calculated for C19H20N2O [M + H]+ 293.1654, found 293.1675.
6.1.1.3. N-Phenethyl-3-phenylisoxazol-5-amine
(4c)39.. Yield
30%;
colorless oil; IR (neat) 3266, 2935, 2849, 1611, 1403, 1258, 1176,
1081, 763, 698 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.71–7.82 (m, 2H),
7.41–7.47 (m, 3H), 7.32–7.41 (m, 2H), 7.27–7.31 (m, 1H), 7.21–7.26
(m, 2H), 5.31 (s, 1H), 4.57 (br. s., 1H), 3.52 (q, J = 6.67 Hz, 2H), 2.96
(t, J = 6.83 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 170.2, 163.7, 138.2,
129.9, 129.7, 128.9, 128.8, 128.7, 126.9, 126.6, 75.5, 45.7, 35.5;
HRMS (ESI) calculated for C17H16N2O [M + H]+ 265.1341, found
265.1361.
6.1.1.4. 3-Benzyl-N-(3-phenylpropyl)isoxazol-5-amine (4e).. Yield 51%;
Pale yellow solid; mp 63–65 °C; IR (neat) 3253, 3077, 2937, 2918,
1608, 1548, 1494, 1452, 1094, 992, 695 cm−1
;
1H NMR (400 MHz,
Figure 3. SAR of isoxazoles as a selective cardiac myosin activator.
CDCl3) δ 7.27–7.35 (m, 5H), 7.13–7.27 (m, 5H), 4.69 (s, 1H), 4.41 (t,
6

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
J = 5.12 Hz, 1H), 3.85 (s, 2H), 3.12 (q, J = 6.83 Hz, 2H), 2.68 (t,
J = 7.56 Hz, 2H), 1.85–1.97 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
170.1, 164.6, 141.0, 137.8, 128.9, 128.6, 128.6, 128.4, 126.7, 126.2,
77.1, 43.9, 32.8, 30.9; HRMS (ESI) calculated for C19H20N2O [M + H]+
293.1654, found 293.1674.
6.1.1.11. 3-(4-Methoxyphenyl)-N-phenethylisoxazol-5-amine (4 l).. Yield
58%; off white solid; mp 95–98 °C; IR (neat): 3281, 3234, 2938, 1633,
1610, 1477, 1252, 1032, 953, 838, 743, 696 cm−1; 1H NMR (400 MHz,
CDCl3) δ 7.66–7.74 (m, 2H), 7.31–7.39 (m, 2H), 7.27–7.30 (m, 1H),
7.21–7.27 (m, 2H), 6.91–7.00 (m, 2H), 5.24–5.28 (m, 1H), 4.49–4.57
(m, 1H), 3.84–3.87 (m, 3H), 3.47–3.54 (m, 2H), 2.96 (t, J = 6.95 Hz,
2H); 13C NMR (100 MHz, CDCl3) δ 170.1, 163.3, 160.9, 138.3, 128.9,
128.8, 128.0, 126.9, 122.5, 114.1, 75.3, 55.3, 45.7, 35.6; HRMS (ESI)
calculated for C18H18N2O2 [M + H]+ 295.1446, found 295.1467.
6.1.1.5. N-Benzyl-3-phenethylisoxazol-5-amine (4f).. Yield 48%; off
white solid; mp 58–61 °C; IR (neat) 3253, 3077, 2937, 2918, 1608,
1548, 1494, 1452, 1094, 823, 693 cm−1; 1H NMR (400 MHz, CDCl3) δ
7.27–7.40 (m, 7H), 7.19–7.24 (m, 3H), 4.81 (s, 2H), 4.34 (d,
J = 5.85 Hz, 2H), 2.90–3.00 (m, 2H), 2.80–2.87 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 169.7, 165.0, 141.1, 137.6, 128.8, 128.5, 128.4,
127.9, 127.4, 126.2, 77.5, 48.5, 34.3, 28.2; HRMS (ESI) calculated for
6.1.1.12. 3-(4-Methoxyphenyl)-N-(4-phenylbutyl)isoxazol-5-amine
(4 m).. Yield 68%; off white solid; mp 102–104 °C; IR (neat): 3281,
3234, 2938, 1633, 1610, 1477, 1252, 1032, 953, 838, 743, 696 cm−1
;
C
18H18N2O [M + H]+ 279.1497, found 279.1521.
1H NMR (400 MHz, CDCl3) δ 7.65–7.72 (m, 2H), 7.27–7.34 (m, 2H),
7.16–7.23 (m, 3H), 6.92–6.99 (m, 2H), 5.16–5.24 (m, 1H), 4.42–4.51
(m, 1H), 3.82–3.89 (m, 3H), 3.18–3.30 (m, 2H), 2.68 (t, J = 7.20 Hz,
2H), 1.61–1.81 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 170.4, 163.3,
160.9, 141.9, 128.5, 128.4, 128.0, 126.0, 122.6, 114.1, 74.8, 55.3,
6.1.1.6. N-Benzyl-3-(3-phenylpropyl)isoxazol-5-amine (4 g).. Yield 50%;
pale yellow oil; IR (neat) 3275, 3025, 2950, 1610, 1420, 1235, 1156,
1089, 885, 695 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.26–7.40 (m, 7H),
7.15–7.21 (m, 3H), 4.84 (s, 1H), 4.79 (t, J = 6.22 Hz, 1H), 4.34 (d,
J = 5.85 Hz, 2H), 2.63–2.70 (t, J = 7.6 Hz, 2H), 2.51–2.57 (t,
J = 7.6 Hz, 2H), 1.89–2.00 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
169.7, 165.4, 141.8, 137.6, 128.8, 128.5, 128.4, 127.8, 127.4, 125.9,
77.3, 48.5, 35.2, 29.7, 25.93; HRMS (ESI) calculated for C19H20N2O
[M + H]+ 293.1654, found 293.1675.
44.6, 35.4, 29.0, 28.4; HRMS (ESI) calculated for
[M + H]+ 323.1696, found 323.1721.
C20H22N2O2
6.1.1.13. 3-Cyclohexyl-N-(3-phenylpropyl)isoxazol-5-amine (4n).. Yield
71%; pale yellow oil; IR (neat) 3267, 3026, 2926, 2852, 1613, 1471,
1451, 1162, 1090, 740, 693 cm−1
;
1H NMR (400 MHz, CDCl3) δ
7.28–7.34 (m, 2H), 7.16–7.25 (m, 3H), 4.77 (s, 1H), 4.40 (t,
J = 6.46 Hz, 1H), 3.17 (q, J = 6.75 Hz, 2H), 2.71 (t, J = 7.56 Hz,
2H), 2.52–2.63 (m, 1H), 1.94 (quin, J = 7.32 Hz, 4H), 1.68–1.84 (m,
3H), 1.20–1.47 (m, 5H); 13C NMR (100 MHz, CDCl3) δ 170.2, 169.7,
141.1, 128.6, 128.4, 126.2, 75.2, 43.9, 36.2, 32.8, 32.0, 30.9, 26.0,
25.9; HRMS (ESI) calculated for C18H24N2O [M + H]+ 285.1967,
found 285.1988.
6.1.1.7. N-(3-Phenylpropyl)-3-(pyridin-4-yl)isoxazol-5-amine
(4 h).. Yield 57%; Pale yellow oil; IR (neat) 3265, 2935, 2849, 1611,
1560, 1508, 1403, 1258, 1324, 1176, 763, 698 cm−1
;
1H NMR
(400 MHz, CDCl3)
δ 8.67–8.72 (m, 2H), 7.59–7.64 (m, 2H),
7.29–7.37 (m, 2H), 7.16–7.26 (m, 3H), 5.26 (s, 1H), 4.71 (t,
J = 5.24 Hz, 1H), 3.27 (q, J = 6.67 Hz, 2H), 2.75 (t, J = 7.44 Hz,
2H), 2.01 (quin, J = 7.26 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 171.0,
161.6, 150.4, 140.9, 137.5, 128.6, 128.4, 126.3, 120.9, 74.9, 43.9,
32.8, 30.8; HRMS (ESI) calculated for C17H17N3O [M + H]+ 280.1450,
found 280.1473.
6.1.1.14. N-(3-Phenylpropyl)-3-propylisoxazol-5-amine
(4o).. Yield
42%; colorless oil; IR (neat) 3272, 3028, 2927, 2857, 1613, 1537,
1453, 1369, 1029, 697 cm−1
;
1H NMR (400 MHz, CDCl3) δ 7.27–7.35
(m, 2H), 7.15–7.25 (m, 3H), 4.77 (s, 1H), 4.33–4.48 (m, 1H), 3.16 (q,
J = 6.67 Hz, 2H), 2.70 (t, J = 7.56 Hz, 2H), 2.48 (t, J = 7.56 Hz, 2H),
1.88–2.00 (m, 2H), 1.65–1.70 (m, 1H), 1.59–1.64 (m, 1H), 0.92–1.04
(m, 3H); 13C NMR (100 MHz, CDCl3) δ 169.9, 165.7, 141.1, 128.6,
128.4, 126.2, 76.6, 43.9, 32.8, 30.9, 28.3, 21.5, 13.7; HRMS (ESI)
calculated for C15H20N2O [M + H]+ 245.1654, found 245.1674.
6.1.1.8. 3-(4-Chlorophenyl)-N-(3-phenylpropyl)isoxazol-5-amine
(4i).. Yield 61%; off white solid; mp 87–88 °C; IR (neat) 3276, 3029,
2936, 1613, 1538, 1407, 1255, 1201, 1172, 1081, 821, 760, 690 cm−1
;
1H NMR (400 MHz, CDCl3) δ 7.63–7.71 (m, 2H), 7.36–7.43 (m, 2H),
7.28–7.35 (m, 2H), 7.15–7.25 (m, 3H), 5.18 (s, 1H), 4.56 (t,
J = 6.34 Hz, 1H), 3.25 (q, J = 6.83 Hz, 2H), 2.74 (t, J = 7.44 Hz,
2H), 1.99 (quin, J = 7.26 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 170.6,
162.7, 141.0, 135.6, 129.0, 128.6, 128.5, 128.5, 127.9, 126.3, 75.0,
44.0, 32.8, 30.9; HRMS (ESI) calculated for C18H17ClN2O [M + H]+
313.1107, found 313.1128.
6.1.1.15. 3-Ethyl-N-(3-phenylpropyl)isoxazol-5-amine (4p).. Yield 61%;
colorless oil; IR (neat) 3275, 3026, 2923, 2856, 1613, 1538, 1453,
1369, 1029, 697 cm−1
;
1H NMR (400 MHz, CDCl3) δ 7.27–7.34 (m,
2H), 7.15–7.24 (m, 3H), 4.79 (s, 1H), 4.42 (br. s., 1H), 3.17 (q,
J = 6.75 Hz, 2H), 2.70 (t, J = 7.56 Hz, 2H), 2.54 (q, J = 7.72 Hz, 2H),
1.94 (td, J = 7.20, 14.88 Hz, 2H), 1.22 (t, J = 7.68 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 169.9, 167.0, 141.1, 128.6, 128.4, 126.2, 76.3,
43.9, 32.8, 30.9, 19.8, 12.5; HRMS (ESI) calculated for C14H18N2O
[M + H]+ 231.1497, found 231.1519.
6.1.1.9. 3-(4-Chlorophenyl)-N-phenethylisoxazol-5-amine
(4j).. Yield
35%; off white solid; mp 88–90 °C; IR (neat)3276, 3029, 2936, 1613,
1538, 1407, 1255, 1201, 1172, 1081, 821, 760, 690 cm−1
;
1H NMR
(400 MHz, CDCl3) δ 7.65–7.72 (m, 2H), 7.38–7.44 (m, 2H), 7.32–7.38
(m, 2H), 7.27–7.31 (m, 1H), 7.21–7.26 (m, 2H), 5.27 (s, 1H), 4.52–4.62
(m, 1H), 3.52 (q, J = 6.67 Hz, 2H), 2.96 (t, J = 6.95 Hz, 2H); 13C NMR
(100 MHz, CDCl3) δ 170.4, 162.7, 138.2, 135.7, 129.0, 128.9, 128.8,
128.5, 127.9, 126.9, 75.4, 45.7, 35.6; HRMS (ESI) calculated for
6.1.1.16. 3-Methyl-N-(3-phenylpropyl)isoxazol-5-amine
(4q).. Yield
72%; pale yellow oil; IR (neat): 3272, 3025, 2927, 2857, 1613, 1538,
1453, 1369, 1029, 697 cm−1
;
1H NMR (400 MHz, CDCl3) δ 7.27–7.34
C
17H15ClN2O [M + H]+ 299.0951, found 299.0973.
(m, 2H), 7.16–7.25 (m, 3H), 4.77 (s, 1H), 4.38–4.48 (m, 1H), 3.16 (q,
J = 6.75 Hz, 2H), 2.70 (t, J = 7.56 Hz, 2H), 2.15 (s, 3H), 1.88–2.00 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 170.0, 161.5, 141.1, 128.6, 128.4,
126.2, 77.7, 43.9, 32.8, 30.9, 11.7; HRMS (ESI) calculated for
6.1.1.10. 3-(4-Methoxyphenyl)-N-(3-phenylpropyl)isoxazol-5-amine
(4 k).. Yield 64%; off white solid; mp 110–112 °C; IR (neat): 3281,
3234, 2938, 1633, 1610, 1477, 1252, 1032, 953, 838, 743, 696 cm−1
;
C
13H16N2O [M + H]+ 217.1341, found 217.1363.
1H NMR (400 MHz, CDCl3) δ 7.64–7.71 (m, 2H), 7.28–7.35 (m, 2H),
7.17–7.26 (m, 3H), 6.92–6.98 (m, 2H), 5.18 (s, 1H), 4.52 (s, 1H), 3.85
(s, 3H), 3.21–3.29 (m, 2H), 2.74 (t, J = 7.56 Hz, 2H), 1.94–2.04 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 170.3, 163.3, 160.8, 141.1, 128.6,
128.5, 128.0, 126.2, 122.6, 114.1, 75.0, 55.3, 44.0, 32.9, 30.9; HRMS
(ESI) calculated for C19H20N2O2 [M + H]+ 309.1603, found 309.1625.
6.1.1.17. N-Phenethyl-3-(tetrahydro-2H-pyran-4-yl)isoxazol-5-amine
(4r).. Yield 30%; colorless oil; IR (neat) 3276, 2931, 2849, 1611, 1444,
1238, 1126, 1086, 981, 890,695 cm−1
;
1H NMR (400 MHz, CDCl3) δ
7.31–7.36 (m, 2H), 7.19–7.29 (m, 3H), 4.85 (s, 1H), 4.48 (br. s., 1H),
3.98–4.09 (m, 2H), 3.39–3.56 (m, 4H), 2.79–3.04 (m, 3H), 1.75–1.89
7

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(m, 4H); 13C NMR (100 MHz, CDCl3) δ 169.8, 168.6, 138.3, 128.8,
[M + H]+ 310.1555, found 310.1576.
126.8, 75.3, 67.6, 45.6, 35.6, 33.4, 31.5; HRMS (ESI) calculated for
C
16H20N2O2 [M + H]+ 273.1603, found 273.1626.
6.1.1.24. N,N-Dimethyl-4-(3-(3-(pyridin-4-yl)isoxazol-5-ylamino)propyl)
benzene sulfonamide (4y). . Yield 63%; Pale yellow solid; mp122-
125 °C; IR (neat): 3346, 2934, 1613, 1560, 1507, 1473, 1458, 1324,
6.1.1.18. N-(3-Phenylpropyl)-3-(tetrahydro-2H-pyran-4-yl)isoxazol-5-
1160, 953, 833, 742, 676 cm−1
;
1H NMR (400 MHz, CDCl3) δ
amine (4 s).. Yield 58%; off white solid; mp 80–84 °C; IR (neat) 3275,
2931, 2849, 1610, 1444, 1238, 1126, 1086, 981, 889,698 cm−1
;
1H
8.66–8.73 (m, 2H), 7.70–7.76 (m, J = 8.29 Hz, 2H), 7.59–7.63 (m,
2H), 7.34–7.40 (m, J = 8.29 Hz, 2H), 5.30 (s, 1H), 4.78 (t, J = 6.22 Hz,
1H), 3.31 (q, J = 6.83 Hz, 2H), 2.79–2.86 (m, 2H), 2.70–2.73 (m, 7H),
1.97–2.09 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 170.8, 161.7, 150.5,
146.3, 137.4, 133.6, 129.0, 128.2, 120.9, 75.1, 43.8, 37.9, 32.7, 30.6;
HRMS (ESI) calculated for C19H22N4O3S [M + H]+ 387.1491, found
387.1515.
NMR (400 MHz, CDCl3) δ 7.28–7.34 (m, 2H), 7.17–7.25 (m, 3H), 4.79
(s, 1H), 4.47 (t, J = 6.10 Hz, 1H), 3.98–4.07 (m, 2H), 3.51 (dt,
J = 3.17, 11.34 Hz, 2H), 3.13–3.22 (m, 2H), 2.79–2.91 (m, 1H), 2.71
(t, J = 7.56 Hz, 2H), 1.89–2.00 (m, 2H), 1.74–1.87 (m, 4H); 13C NMR
(100 MHz, CDCl3) δ 170.0, 168.6, 141.0, 128.6, 128.4, 126.2, 75.0,
74.9, 67.6, 43.9, 33.4, 33.4, 32.8, 31.5, 31.5, 30.9, 30.8; HRMS (ESI)
calculated for C17H22N2O2 [M + H]+ 287.1759, found 287.1779.
6.1.1.25. Methyl4-(5-(3-(4-methoxyphenyl) propylamino)isoxazol-3-yl)
piperidine-1-carboxylate (4z). . Yield 55%; colorless oil; IR (neat):
3269, 2938, 2847, 1725, 1611, 1560, 1508, 1403, 1278, 1324, 1176,
953, 833, 763, 676 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.05–7.14 (m,
2H), 6.81–6.87 (m, 2H), 4.75 (s, 1H), 4.46 (t, J = 6.22 Hz, 1H), 4.17
(br. s., 2H), 3.80 (s, 3H), 3.71 (s, 3H), 3.15 (q, J = 6.67 Hz, 2H),
2.83–2.97 (m, 2H), 2.76 (tt, J = 3.78, 11.59 Hz, 1H), 2.64 (t,
J = 7.56 Hz, 2H), 1.90 (quin, J = 7.26 Hz, 4H), 1.54–1.69 (m, 2H);
13C NMR (100 MHz, CDCl3) δ 170.0, 168.3, 158.1, 156.0, 133.0, 129.3,
114.0, 75.0, 55.2, 52.6, 43.8, 43.7, 34.3, 31.9, 31.1, 30.6; HRMS (ESI)
calculated for C20H27N3O4[M + H]+374.2080, found 374.2101.
6.1.1.19. N-(4-Phenylbutyl)-3-(tetrahydro-2H-pyran-4-yl)isoxazol-5-
amine (4 t).. Yield 61%; colorless oil; IR (neat) 3277, 2930, 2849, 1611,
1444, 1238, 1126, 1086, 981, 889, 697 cm−1
;
1H NMR (400 MHz,
CDCl3) δ 7.27–7.32 (m, 2H), 7.16–7.23 (m, 3H), 4.81 (s, 1H), 4.38–4.46
(m, 1H), 3.99–4.07 (m, 2H), 3.46–3.56 (m, 2H), 3.17 (q, J = 6.67 Hz,
2H), 2.79–2.90 (m, 1H), 2.66 (t, J = 7.32 Hz, 2H), 1.62–1.88 (m, 8H);
13C NMR (100 MHz, CDCl3) δ 170.0, 168.6, 141.9, 128.5, 128.4, 126.0,
74.9, 67.6, 44.4, 35.4, 33.4, 31.5, 31.4, 29.0, 28.4; HRMS (ESI)
calculated for C18H24N2O2 [M + H]+ 301.1916, found 301.1938.
6.1.1.20. Methyl 4-(5-(3-phenylpropylamino)isoxazol-3-yl)piperidine-1-
carboxylate (4u).. Yield 51%; colorless oil; IR (neat) 3265, 2935,
2849, 1698, 1611, 1560, 1508, 1403, 1278, 1324, 1176, 763,
6.1.1.26. Methyl 4-(5-(3-(4-(N,N-dimethylsulfamoyl)phenyl)propylamino)
isoxazol-3-yl)piperidine-1-carboxylate (4ab). . Yield 61%; colorless oil; IR
(neat): 3265, 2935, 2849, 1725, 1611, 1560, 1508, 1403, 1278, 1324,
1176, 953, 833, 763, 676 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.67–7.74
(m, 2H), 7.36 (d, J = 8.05 Hz, 2H), 4.78–4.82 (m, 1H), 4.53 (t,
J = 6.22 Hz, 1H), 3.69–3.72 (m, 3H), 3.21 (q, J = 6.67 Hz, 2H),
2.84–2.98 (m, 2H), 2.73–2.83 (m, 3H), 2.68–2.72 (m, 7H), 1.86–2.08 (m,
4H), 1.64–1.69 (m, 1H), 1.55–1.62 (m, 1H); 13C NMR (100 MHz, CDCl3) δ
169.8, 168.4, 156.0, 146.5, 133.5, 129.0, 128.1, 75.2, 52.6, 43.8, 43.7,
37.9, 34.3, 32.8, 30.6; HRMS (ESI) calculated for C21H30N4O5S [M + H]+
451.2015, found 451.2039.
698 cm−1
;
1H NMR (400 MHz, CDCl3) δ 7.28–7.34 (m, 2H),
7.16–7.25 (m, 3H), 4.75 (s, 1H), 4.48 (t, J = 6.10 Hz, 1H), 4.19 (br.
s., 2H), 3.71 (s, 3H), 3.17 (q, J = 6.75 Hz, 2H), 2.84–2.98 (m, 2H),
2.66–2.81 (m, 3H), 1.86–1.99 (m, 4H), 1.55–1.67 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 170.0, 168.3, 156.0, 141.0, 128.6, 128.4, 126.2,
75.0, 52.5, 43.9, 43.7, 34.3, 32.8, 30.9, 30.6; HRMS (ESI) calculated for
C
19H25N3O3 [M + H]+ 344.1974, found 344.1995.
6.1.1.21. N-(3-(4-Methoxyphenyl)propyl)-3-phenylisoxazol-5-amine
(4v).. Yield 48%; off white solid; mp 81–85 °C; IR (neat): 3285, 3234,
2938, 1610, 1477, 1262, 1032, 953, 838, 743, 696 cm−1
;
1H NMR
6.1.2. Preparation of 4d
(400 MHz, CDCl3) δ 7.70–7.78 (m, 2H), 7.38–7.46 (m, 3H), 7.08–7.15
(m, 2H), 6.82–6.90 (m, 2H), 5.22 (s, 1H), 4.55 (br. s., 1H), 3.81 (s, 3H),
3.24 (q, J = 6.67 Hz, 2H), 2.68 (t, J = 7.56 Hz, 2H), 1.90–2.00 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 170.4, 163.7, 158.1, 133.0, 130.0,
129.7, 129.4, 128.7, 126.6, 114.0, 75.1, 55.2, 43.9, 31.9, 31.1; HRMS
(ESI) calculated for C19H20N2O2 [M + H]+ 309.1603, found 309.1625.
To a stirred solution of 3-phenylisoxazol-5-amine (10a, 1.87 mmol)
in DMF (5 mL), 60% NaH (2.81 mmol) and benzyl bromide (1.87) were
added at 0 °C. The resulting solution was stirred at room temperature
for 16 h. The reaction mixture was quenched with ice cold water, and
extracted with EtOAc (2x times). The combined ethyl acetate layers
were dried over anhydrous Na2SO4 and then concentrated under re-
duced pressure. The crude mixture was subjected to flash silica gel
(230–400 mesh) column chromatography (eluting with 5–10% EtOAc
in hexanes) to afford the mono- and di-benzylated products.
6.1.1.22. N,N-Dimethyl-4-(3-(3-phenylisoxazol-5-ylamino)propyl)benzene
sulfonamide (4w).. Yield 59%; off white solid; mp 149–152 °C; IR
(neat): 3255, 3075, 2937, 2918, 1609, 1548, 1494, 1452, 1094, 953,
833, 742, 676 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.70–7.77 (m, 4H),
7.41–7.46 (m, 3H), 7.38 (d, J = 8.29 Hz, 2H), 5.26 (s, 1H), 4.60 (s, 1H),
3.30 (q, J = 6.59 Hz, 2H), 2.83 (t, J = 7.68 Hz, 2H), 2.71 (s, 6H),
1.96–2.09 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 170.2, 163.7, 146.5,
133.5, 129.8, 129.8, 129.0, 128.8, 128.1, 126.6, 75.3, 43.9, 37.9, 32.8,
30.6; HRMS (ESI) calculated for C20H23N3O3S [M + H]+ 386.1538,
found 386.1561.
6.1.2.1. N-benzyl-3-phenylisoxazol-5-amine (4d)27. . Yield 35%; off
white solid; mp 91–93 °C; IR (neat) 3265, 2935, 2849, 1611, 1403,
1258, 1176, 1081, 763, 698 cm−1
;
1H NMR (400 MHz, CDCl3) δ
7.70–7.77 (m, 2H), 7.40–7.46 (m, 3H), 7.30–7.39 (m, 5H), 5.31 (s, 1H),
4.88–4.97 (m, 1H), 4.44 (d, J = 5.85 Hz, 2H); 13C NMR (100 MHz,
CDCl3) δ 170.3, 163.7, 137.5, 129.9, 129.7, 128.9, 128.7, 127.9, 127.4,
126.7, 76.0, 48.6; HRMS (ESI) calculated for C16H14N2O [M + H]+
251.1184, found 251.1205.
6.1.1.23. N-(3-(4-Methoxyphenyl)propyl)-3-(pyridin-4-yl)isoxazol-5-
amine (4x).. Yield 45%; pale yellow solid; mp 119–121 °C; IR (neat):
3285, 3234, 2938, 1610, 1560, 1508, 1477, 1262, 1032, 953, 838, 743,
696 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.66–8.72 (m, 2H), 7.58–7.64
(m, 2H), 7.08–7.15 (m, 2H), 6.82–6.90 (m, 2H), 5.24 (s, 1H), 4.72 (t,
J = 5.73 Hz, 1H), 3.80 (s, 3H), 3.26 (q, J = 6.99 Hz, 2H), 2.69 (t,
J = 7.44 Hz, 2H), 1.90–2.02 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
171.0, 161.6, 158.1, 150.4, 137.5, 132.9, 129.4, 120.9, 114.0, 74.9,
6.1.2.2. N,N-Dibenzyl-3-phenylisoxazol-5-amine (4da)40.. Yield 25%;
pale yellow solid; mp 77–79 °C; IR (neat): 3027, 2935, 2849, 1611,
1403, 1258, 1176, 1081, 763, 698 cm−1; 1H NMR (400 MHz, CDCl3) δ
7.72–7.78 (m, 2H), 7.39–7.45 (m, 3H), 7.25–7.39 (m, 10H), 5.30 (s,
1H), 4.53 (s, 4H); 13C NMR (100 MHz, CDCl3) δ 171.0, 164.0, 136.5,
130.1, 129.7, 128.8, 128.7, 127.8, 126.7, 126.6, 75.6, 52.3; HRMS (ESI)
calculated for C16H14N2O [M + H]+ 251.1184, found 251.1205.
55.2, 43.9, 31.9, 31.0; HRMS (ESI) calculated for
C18H19N3O2
8

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
6.1.3. Preparation of N-methyl-3-phenyl-N-(3-phenylpropyl)isoxazol-5-
amine (5).
the reaction was concentrated under reduced pressure to afford crude
compound. The crude compound was dissolved in pyridine (5 mL), and
added 3-phenylisoxazol-5-amine 10a (1.25 mmol). The resulting reac-
tion mixture was stirred at 80 °C for 16 h. After cooling to rt, the re-
action was concentrated under reduced pressure and then diluted with
EtOAc. The organic phase was washed with 1 N HCl solution (2x times),
dried over anhydrous Na2SO4 and then concentrated under reduced
pressure. The crude residue was subjected to flash silica gel (230–400
mesh) column chromatography (eluting with 10–15% EtOAc in hex-
anes) to afford the title compounds.
To a stirred solution of 3-phenyl-N-(3-phenylpropyl)isoxazol-5-
amine (4a) (0.36 mmol) in THF (5 mL), 60% NaH (0.716 mmol) and
MeI (0.716 mmol) were added at 0 °C. The resulting reaction mixture
was stirred at rt for 16 h. The reaction mixture was quenched with ice
cold water, and then extracted with EtOAc (2x times). The combined
organic layers were dried over anhydrous Na2SO4 and then con-
centrated under reduced pressure. The crude residue was subjected to
flash silica gel (230–400 mesh) column chromatography (eluting with
5–10% EtOAc in hexanes) to afford the title compound 5. Yield 58%;
colorless oil; IR (neat) 2938, 2848, 1611, 1403, 1258, 1176, 1081, 763,
698 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.67–7.81 (m, 2H), 7.37–7.49
(m, 3H), 7.28–7.35 (m, 2H), 7.16–7.26 (m, 3H), 5.11 (s, 1H), 3.29–3.45
(m, 2H), 2.97–3.05 (m, 3H), 2.69 (t, J = 7.68 Hz, 2H), 1.91–2.07 (m,
2H); 13C NMR (100 MHz, CDCl3) δ 171.0, 163.8, 141.3, 130.2, 129.6,
128.7, 128.5, 128.4, 126.6, 126.1, 74.6, 51.0, 36.4, 32.9, 28.7; HRMS
(ESI) calculated for C19H20N2O [M + H]+ 293.1654, found 293.1675.
6.1.5.1. 4-Phenyl-N-(3-phenylisoxazol-5-yl)butanamide
(6c).. This
compound was prepared by reaction of 10a with 4-phenylbutanoic
acid (12c). Yield 47%; off white solid; mp 89–92 °C; IR (neat) 3256,
3022, 2926, 1690, 1611, 1538, 1403, 1255, 1205, 1177, 1085, 763,
692 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.11 (br. s., 1H), 7.76–7.86 (m,
2H), 7.43–7.50 (m, 3H), 7.28–7.35 (m, 2H), 7.17–7.26 (m, 3H), 6.73 (s,
1H), 2.73 (t, J = 7.44 Hz, 2H), 2.44 (t, J = 7.44 Hz, 2H), 2.06–2.15 (m,
2H); 13C NMR (100 MHz, CDCl3) δ168.7, 163.9, 160.3, 140.9, 130.2,
129.0, 128.9, 128.6, 128.5, 126.9, 126.8, 126.8, 126.3, 87.0, 35.7,
6.1.4. General procedure for the preparation of 6a,6b, and 6e
To a stirred solution of the corresponding isoxazol-5-amine 10
(1.25 mmol) in DMF (10 mL), DMAP (1.5 mmol), appropriate acid 12
(1.25 mmol) and EDC.HCl (1.5 mmol) were added. The resulting so-
lution was stirred at 50 °C for 16 h. The reaction mixture was diluted
with EtOAc, washed with water (3x times), dried over anhydrous
Na2SO4 and then concentrated under reduced pressure. The crude re-
sidue was subjected to flash silica gel (230–400 mesh) column chro-
matography (eluting with 10–15% EtOAc in hexanes) to afford the title
compounds.
34.8, 26.2; HRMS (ESI) calculated for C19H18N2O2 [M
307.1446, found 307.1469.
+
H]+
6.1.5.2. 5-Phenyl-N-(3-phenylisoxazol-5-yl)pentanamide
(6d).. This
compound was prepared by reaction of 10a with 5-phenylpentanoic
acid (12d). Yield 56%; off white solid; mp 141–143 °C; IR (neat): 3255,
3030, 2937, 1693, 1613, 1538, 1403, 1255, 1201, 1176, 1082, 763,
690 cm−1;1H NMR (400 MHz, CDCl3) δ 8.21 (br. s., 1H), 7.78–7.85 (m,
2H), 7.43–7.50 (m, 3H), 7.27–7.32 (m, 2H), 7.16–7.23 (m, 3H), 6.73 (s,
1H), 2.67 (t, J = 7.32 Hz, 2H), 2.43–2.49 (t, J = 8 Hz, 2H), 1.67–1.84
(m, 4H); 13C NMR (100 MHz, CDCl3) δ 168.9, 163.9, 160.4, 141.8,
130.2, 129.1, 128.9, 128.5, 128.4, 126.9, 126.8, 126.8, 126.0, 87.0,
36.6, 35.5, 30.7, 24.6; HRMS (ESI) calculated for C20H20N2O2
[M + H]+ 321.1603, found 321.1624.
6.1.4.1. 2-Phenyl-N-(3-phenylisoxazol-5-yl)acetamide
(6a)41.. This
compound was prepared by reaction of 10a with 2-phenylacetic acid
(12a). Yield 41%; off white solid; mp 124–126 °C; IR (neat) 3257, 3028,
2937, 1695, 1613, 1538, 1403, 1255, 1222, 1202, 1176, 1082, 763,
690 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.07 (br. s., 1H), 7.75–7.83 (m,
2H), 7.36–7.49 (m, 6H), 7.34 (dd, J = 1.59, 7.93 Hz, 2H), 6.75 (s, 1H),
3.83 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 167.0, 163.9, 160.2, 132.9,
130.2, 129.6, 129.6, 129.0, 128.9, 128.3, 126.8, 87.1, 43.9; HRMS (ESI)
calculated for C17H14N2O2 [M + H]+ 279.1133, found 279.1156.
6.1.6. Preparation of N-(3-(3-phenylpropyl)isoxazol-5-yl)benzamide (6f)
To a stirred solution of 3-(3-phenylpropyl)isoxazol-5-amine 10d
(1.24 mmol) in pyridine (5 mL), benzoyl chloride 12e (1.48 mmol) was
added at 0 °C. The resulting reaction mixture was stirred at 80 °C for
16 h. After cooling to rt, the reaction was concentrated under reduced
pressure, and then diluted with EtOAc. The organic phase was washed
with 1 N HCl solution (2x times), dried over anhydrous Na2SO4 and
then concentrated under reduced pressure. The crude residue was
subjected to flash silica gel (230–400 mesh) column chromatography
(eluting with 10–15% EtOAc in hexanes) to afford the title compound.
Yield 67%; White solid; mp: 122–125 °C; IR (neat): 3250, 3025, 2936,
6.1.4.2. 3-Phenyl-N-(3-phenylisoxazol-5-yl)propanamide
(6b).. This
compound was prepared by reaction of 10a with 3-phenylpropanoic
acid (12b). Yield 52%; off white solid; mp 136–138 °C; IR (neat): 3256,
3027, 2938, 1693, 1612, 1538, 1403, 1255, 1222, 1202, 1176, 1082,
763, 691 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.11–8.20 (m, 1H), 7.81
(m, 2H), 7.46 (m, 3H), 7.29–7.35 (m, 2H), 7.20–7.27 (m, 3H), 6.74 (s,
1H), 3.08 (t, J = 7.56 Hz, 2H), 2.77 (t, J = 7.68 Hz, 2H); 13C NMR
(100 MHz, CDCl3) δ 168.2, 163.9, 160.2, 139.8, 130.3, 129.0, 128.9,
128.8, 128.3, 126.8, 126.7, 87.1, 38.4, 30.9; HRMS (ESI) calculated for
1685, 1610, 1535, 1403, 1255, 1201, 1177, 1085, 763, 690 cm−1
;
1H
NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 7.87–7.93 (m, 2H), 7.60–7.66
(m, 1H), 7.50–7.57 (m, 2H), 7.28–7.33 (m, 2H), 7.21 (m, 3H), 6.43 (s,
1H), 2.71 (m, 4H), 1.99–2.10 (m, 2H); 13C NMR (100 MHz, CDCl3) δ
165.7, 163.2, 160.2, 141.5, 133.0, 132.4, 129.1, 128.5, 128.4, 127.4,
126.0, 88.8, 35.1, 29.5, 25.8; HRMS (ESI) calculated for C19H18N2O2
[M + H]+ 307.1446, found 307.1468.
C
18H16N2O2[M + H]+293.1290, found 293.1311.
6.1.4.3. 3-Phenyl-N-(3-(tetrahydro-2H-pyran-4-yl)isoxazol-5-yl)
propanamide (6e).. This compound was prepared by reaction of 10 l
with 3-phenylpropanoic acid (12b). Yield 51%; off white solid; mp159-
162 °C; IR (neat): 3256, 3029, 2929, 2849, 1613, 1403, 1237, 1201,
1175, 1081, 763, 690 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.26 (br. s.,
1H), 7.16–7.38 (m, 6H), 6.28 (s, 1H), 3.97–4.12 (m, 2H), 3.44–3.61 (m,
2H), 3.01–3.13 (m, 2H), 2.88–3.01 (m, 1H), 2.66–2.80 (m, 2H),
1.75–1.94 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 168.6, 159.9,
139.9, 128.8, 128.3, 126.7, 87.2, 67.5, 38.3, 33.5, 31.1, 30.9; HRMS
(ESI) calculated for C17H20N2O3 [M + H]+ 301.1552, found 301.1573.
6.1.7. General procedure for the preparation of 7
To a stirred solution of 3-phenylisoxazol-5-amine 10a (1.87 mmol)
in THF (10 mL), DIPEA (3.74 mmol) and triphosgene (0.75 mmol) were
added at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. The
appropriate amine 13 (1.87 mmol) was added and then the mixture was
allowed to warm to room temperature and stirred for 16 h. The reaction
mixture was quenched with 10% NaHCO3 solution and extracted with
EtOAc (3x times). The combined organic extracts were dried over an-
hydrous Na2SO4 and then concentrated under reduced pressure. The
crude residue was subjected to flash silica gel (230–400 mesh) column
chromatography (eluting with 2–5% MeOH in DCM) to afford the title
compounds.
6.1.5. General procedure for the preparation of 6c, and 6d
To a stirred solution of the corresponding acid 12 (1.25 mmol) in
DCM (5 mL), SOCl2 (3.75 mmol) was added at 0 °C. The resulting re-
action mixture was refluxed for 5 h. After cooling to room temperature,
9

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
6.1.7.1. 1-Benzyl-3-(3-phenylisoxazol-5-yl)urea (7a).. Yield 49%; off
6.1.8.3. 3-Phenethylisoxazol-5-amine (10c)27.. Yield 61%; Pale yellow
solid; mp 78 – 80 °C; 1H NMR (400 MHz, CDCl3) δ 7.28–7.35 (m, 2H),
7.19–7.26 (m, 3H), 4.94 (s, 1H), 4.38 (br. s., 2H), 2.90–3.01 (m, 2H),
2.79–2.89 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 168.4, 165.2, 141.0,
128.5, 128.4, 126.2, 79.9, 34.2, 28.1.
white solid; mp 148–150 °C; IR (neat) 3322, 3273, 3060, 2948, 1662,
1624, 1573, 1473, 1278, 1261, 752, 687 cm−1
;
1H NMR (400 MHz,
CDCl3) δ 8.65 (br. s., 1H), 7.66–7.73 (m, 2H), 7.34–7.46 (m, 3H),
7.27–7.30 (m, 1H), 7.18–7.27 (m, 4H), 6.41 (s, 1H), 6.08–6.17 (m, 1H),
4.38 (d, J = 5.85 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 164.1, 162.5,
152.6, 138.0, 130.3, 129.0, 128.8, 127.6, 127.4, 126.8, 84.7, 44.2;
HRMS (ESI) calculated for C17H15N3O2 [M + H]+ 294.1242, found
294.1266.
6.1.8.4. 3-(3-Phenylpropyl)isoxazol-5-amine (10d)28.. Yield 70%; Light
yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.28–7.32 (m, 2H),
7.16–7.23 (m, 3H), 4.99 (s, 1H), 4.37 (br. s., 2H), 2.67–2.70 (t,
J = 8 Hz, 2H), 2.54 – 2.58 (t, J = 7.56 Hz, 2H), 1.96 (quin,
J = 7.68 Hz, 2H).
6.1.7.2. 1-Phenethyl-3-(3-phenylisoxazol-5-yl)urea (7b).. Yield 58%; off
white solid; mp 110–114 °C; IR (neat): 3323, 3276, 3061, 2948, 1660,
6.1.8.5. 3-(Pyridin-4-yl)isoxazol-5-amine (10e)29.. Yield 61%; Pale
yellow solid; mp 186 – 188 °C; 1H NMR (400 MHz, DMSO‑d6) δ 8.66
(d, J = 5.37 Hz, 2H), 7.70 (d, J = 5.37 Hz, 2H), 6.93 (s, 2H), 5.52 (s,
1H); 13C NMR (100 MHz, DMSO‑d6) δ 171.7, 160.9, 150.4, 137.2,
120.6, 75.2.
1625, 1573, 1473, 1278, 1261, 752, 687 cm−1
;
1H NMR (400 MHz,
CDCl3) δ 8.39 (br. s., 1H), 7.70–7.78 (m, 2H), 7.40–7.51 (m, 3H),
7.27–7.30 (m, 1H), 7.25 (s, 1H), 7.13–7.23 (m, 3H), 6.38 (s, 1H), 5.64
(br. s., 1H), 3.45–3.56 (m, 2H), 2.80 (t, J = 6.95 Hz, 2H); 13C NMR
(100 MHz, CDCl3) δ 164.0, 162.4, 152.3, 138.5, 130.3, 129.1, 129.0,
128.8, 128.8, 126.8, 126.8, 84.7, 41.6, 35.8; HRMS (ESI) calculated for
6.1.8.6. 3-(4-Chlorophenyl)isoxazol-5-amine (10f)27.. Yield 75%; Pale
yellow solid; mp 168 – 170 °C; 1H NMR (400 MHz, CDCl3) δ 7.67 (d,
J = 8.54 Hz, 2H), 7.40 (d, J = 8.78 Hz, 2H), 5.42 (s, 1H), 4.55 (br. s.,
2H); 13C NMR (100 MHz, CDCl3) δ 169.0, 163.0, 135.8, 129.0, 128.3,
127.9, 78.1.
C
18H17N3O2 [M + H]+ 308.1399, found 308.1421.
6.1.7.3. 1-(3-Phenylisoxazol-5-yl)-3-(3-phenylpropyl)urea (7c).. Yield
45%; off white solid; mp 97–100 °C; IR (neat): 3323, 3276, 3061,
2948, 1660, 1625, 1573, 1473, 1278, 1261, 752, 687 cm−1
;
1H NMR
(400 MHz, CDCl3) δ 8.55 (br. s., 1H), 7.70–7.80 (m, 2H), 7.37–7.49 (m,
3H), 7.22–7.27 (m, 2H), 7.08–7.21 (m, 3H), 6.42 (s, 1H), 5.67–5.79 (m,
1H), 3.22–3.33 (m, 2H), 2.61 (t, J = 7.68 Hz, 2H), 1.79–1.86 (m, 2H);
13C NMR (100 MHz, CDCl3) δ 164.1, 162.6, 152.5, 141.2, 130.3, 129.0,
128.5, 128.4, 126.8, 126.8, 126.1, 84.6, 40.0, 33.0, 31.2; HRMS (ESI)
calculated for C19H19N3O2 [M + H]+ 322.1555, found 322.1578.
6.1.8.7. 3-(4-Methoxyphenyl)isoxazol-5-amine (10 g)27.. Yield 78%;
Pale yellow solid; mp 135 – 137 °C; 1H NMR (400 MHz, CDCl3) δ
7.63–7.73 (m, 2H), 6.90–6.99 (m, 2H), 5.40 (s, 1H), 4.50 (br. s., 2H),
3.85 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 168.8, 163.6, 160.9, 128.0,
122.3, 114.2, 78.1, 55.3.
6.1.8.8. 3-Cyclohexylisoxazol-5-amine (10 h)27.. Yield 81%; Off white
solid; mp 136 – 138 °C; 1H NMR (400 MHz, CDCl3) δ 4.99 (s, 1H), 4.37
(br. s., 2H), 2.50–2.65 (m, 1H), 1.86–1.99 (m, 2H), 1.67–1.85 (m, 3H),
1.19–1.45 (m, 5H); 13C NMR (100 MHz, CDCl3) δ 170.5, 168.1, 78.4,
36.2, 31.9, 25.9, 25.9.
6.1.8. General procedure for the preparation of 10
Step 1: A stirred suspension of NaH (15.6 mmol, 60% dispersion in
mineral oil) in THF was heated to 50 °C. To this was added a mixture of
ester 8 (10.40 mmol) and acetonitrile (15.60 mmol) dropwise over the
course of 15 min. The resulting suspension was heated at reflux tem-
perature for a further 16 h. After cooling to rt, the reaction mixture was
poured into H2O and then the resulting solution was washed with
diethyl ether (2x times) (ether extract discarded). The aqueous layer
was separated, acidified to pH ~ 2 with aqueous 2 M HCl, and extracted
with EtOAc (2x times). The combined EtOAc layers were dried over
anhydrous Na2SO4 and then concentrated under reduced pressure to
afford the title compounds 9, which is used for the next step without
further purification.
6.1.8.9. 3-Propylisoxazol-5-amine (10i).. Yield 75%; Light yellow
crystals; mp 61 – 63 °C; 1H NMR (400 MHz, CDCl3) δ 4.99 (s, 1H),
4.38 (br. s., 2H), 2.49 (t, J = 7.56 Hz, 2H), 1.59–1.70 (m, 2H), 0.97 (t,
J = 7.44 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 168.3, 165.9, 79.8,
28.3, 21.4, 13.6.
6.1.8.10.3-Ethylisoxazol-5-amine (10j)30
.
Yield 68%; Light brown oil; 1H NMR (400 MHz, CDCl3) δ 5.00 (s,
1H), 4.39 (br. s., 2H), 2.55 (q, J = 7.64 Hz, 2H), 1.22 (t, J = 7.56 Hz,
4H); 13C NMR (100 MHz, CDCl3) δ 168.3, 167.2, 79.4, 29.6, 19.8.
Step 2: To a stirred solution of nitrile compound 9 (10.14 mmol) and
NaOH (20.80 mmol) in water (20 mL), hydroxylamine hydrochloride
(11.16 mmol) was added. The resulting mixture was heated at 100 °C
for 3 h. After cooling to rt, the precipitated compound was filtered and
washed with water and dried under vacuum to afford the title com-
pound 10. Those which did not obtain precipitation, the mixture was
diluted with EtOAc and the organic layer was separated. The aqueous
layer was further extracted with EtOAc (2x times), the combined or-
ganic layers were dried over anhydrous Na2SO4 and then concentrated
under reduced pressure to afford the title compound 10
6.1.8.10. 3-Methylisoxazol-5-amine (10 k)25.. Yield 71%; Off white
solid; mp 83 – 85 °C; 1H NMR (400 MHz, CDCl3) δ 4.98 (s, 1H), 4.39
(br. s., 2H), 2.16 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 168.5, 161.7,
80.7, 11.6.
6.1.8.11. 3-(Tetrahydro-2H-pyran-4-yl)isoxazol-5-amine (10 l)31.. Yield
70%; White solid; mp 156 – 158 °C; 1H NMR (400 MHz, DMSO‑d6) δ
6.52 (s, 2H), 4.85 (s, 1H), 3.82–3.91 (m, 2H), 3.35–3.43 (m, 2H), 2.71
(tt, J = 4.02, 11.46 Hz, 1H), 1.66–1.77 (m, 2H), 1.51–1.65 (m, 2H); 1H
NMR (400 MHz, CDCl3) δ 5.00 (s, 1H), 4.48 (br. s., 2H), 3.99–4.05 (m,
2H), 3.50 (dt, J = 2.93, 11.34 Hz, 2H), 2.81–2.89 (m, 1H), 1.75–1.86
(m, 4H); 13C NMR (100 MHz, CDCl3) δ 168.8, 168.4, 78.2, 67.6, 33.4,
31.4.
6.1.8.1. 3-Phenylisoxazol-5-amine (10a)25.. Yield 82%; Light brown
solid; mp 102 – 104 °C; 1H NMR (400 MHz, CDCl3) δ 7.70–7.79 (m,
2H), 7.36–7.50 (m, 3H), 5.46 (s, 1H), 4.54 (br. s., 2H); 13C NMR
(100 MHz, CDCl3) δ 168.9, 164.0, 129.8, 128.8, 126.7, 78.2 (one
quaternary peak could be merged with 128.8 peak).
6.1.8.12. Methyl 4-(5-aminoisoxazol-3-yl)piperidine-1-carboxylate (10 m)..
For the preparation of this compound we used dimethyl piperidine-1,4-
dicarboxylate(8 m) as a starting material. Surprisingly, we obtained
6.1.8.2. 3-Benzylisoxazol-5-amine (10b)26.. Yield 65%; Pale yellow
solid; mp 60 – 62 °C; 1H NMR (400 MHz, CDCl3) δ 7.29–7.34 (m,
2H), 7.21–7.28 (m, 3H), 4.89 (s, 1H), 4.30–4.42 (m, 2H), 3.86 (s, 2H);
13C NMR (100 MHz, CDCl3) δ 168.5, 164.9, 137.7, 128.8, 128.6, 126.7,
80.0, 32.7.
decarboxylated product 3-(piperidin-4-yl)isoxazol-5-amine (10 m-In). 1
H
NMR (400 MHz, CDCl3) δ 5.01 (s, 1H), 4.44 (br. s., 2H), 3.15 (td,
J = 3.38, 12.50 Hz, 2H), 2.66–2.78 (m, 3H), 0.78–0.91 (m, 4H).
10

P.R. Boggu, et al.
Bioorganic&MedicinalChemistry28(2020)115742
Furtherly, the compound 10 m-In was reacted with chloroformate at 0 °C
to afford the desired compound 10 m.
DMSO was used as a negative control.
Activity (%) = (Mean A
B) (NC
B)/(NC B) x100
6.1.8.12.1. Preparation of 10 m from 10 m to In.. To a solution of
10 m-In (2.99 mmol) in DCM (10 mL), DIPEA (4.49 mmol) and methyl
chloroformate (2.99 mmol) at 0 °C. The resulting solution was stirred at
room temperature for 3 h. The reaction solution was diluted with DCM,
washed with water, dried over anhydrous Na2SO4 and then
concentrated under reduced pressure to afford the title compound as
pale yellow solid. Yield 40%; Pale yellow solid; mp 96 – 98 °C; 1H NMR
(400 MHz, CDCl3) δ 4.97 (s, 1H), 4.48 (br. s., 2H), 4.16 (br. s., 2H), 3.70
(s, 3H), 2.90 (t, J = 12.20 Hz, 2H), 2.77 (tt, J = 3.72, 11.52 Hz, 1H),
1.90 (dd, J = 2.20, 13.17 Hz, 2H), 1.56–1.66 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 168.5, 168.5, 156.0, 78.1, 52.6, 43.7, 34.2, 30.6.
where, A = Absorbance; B = Blank; NC = Negative control
6.2.2. Selectivity studies
Compound specificity with respect to muscle type were assessed by
associating the effect of the compound on actin stimulated ATPase ac-
tivity of a panel of myosin isoforms including cardiac (bovine), smooth
muscle (chicken gizzard) and skeletal (rabbit) at a single higher dose
(100 µM) of the compound.
6.2.2.1. Smooth muscle. The standard reaction solution contained
20 mM Tris HCl (pH 7.5), 6 mM MgCl2, 15 mM KCl, 1 mM ATP, S1
myosin (CS-MYS05), actin (AD 99) and Tropomyosin (T2400) with
pCa = 6.5. The reaction was stopped by adding of Cytophos reagent
(Cytoskeleton BK054 kit) After 10 min incubation at rt, samples were
examined for inorganic phosphate liberated by taking absorbance at
650 nm on TECAN Infinite. Blebbistatin was used as a negative control
for selectivity assay.
6.1.9. Procedure for the preparation of 3-(4-methoxyphenyl)propyl
methanesulfonate 11e42
To a solution of 3-(4-methoxyphenyl)propan-1-ol (12.03 mmol) in
DCM (20 mL), TEA (24.06 mmol) and methanesulfonyl chloride
(14.43 mmol) were added at 0 °C. The solution was stirred for 3 h at rt,
diluted with DCM (50 mL), washed with water (2x50 mL), dried over
anhydrous Na2SO4 and then concentrated under reduced pressure to get
the title compound as brownish oil. The crude product was used for the
next step without further purification. Yield: 80%.
6.2.2.2. Skeletal muscle. The standard reaction solution contained
20 mM Tris HCl (pH 7.5), 6 mM MgCl2, 15 mM KCl, 1 mM ATP, S1
myosin (CS-MYS04) and actin thin filament complex (CS-TFC01) with
pCa = 6.5. The reaction was stopped by adding of Cytophos reagent
(Cytoskeleton BK054 kit) After 10 min incubation at rt, samples were
analyzed for inorganic phosphate liberated by taking absorbance at
650 nm on TECAN Infinite. Blebbistatin was used as a negative control
for selectivity assay.
6.1.10. Preparation of 4-(3-bromopropyl)-N,N-dimethylbenzenesulfonamide
(11f)
6.1.10.1. Preparation of 4-(3-bromopropyl)benzenesulfonyl chloride
(15).. Chlorosulfonic acid (6 mL) was taken into a 100 mL round
bottom flask, a solution of (3-bromopropyl)benzene14 (10.05 mmol) in
DCM (1 mL) was added at 0 °C. The resulting solution was stirred for
16 h at rt. The reaction solution was slowly poured into crushed ice, and
extracted with DCM (2x times). The combined organic layers were
dried over anhydrous Na2SO4 and then concentrated under reduced
pressure to afford the title crude compound 4-(3-bromopropyl)benzene-
1-sulfonyl chloride 15 as a brownish oil. The crude product was used
for the next step without further purification. Yield 70%; 1H NMR
(400 MHz, CDCl3) δ 7.96–8.01 (d, J = 8.54 Hz, 2H), 7.43–7.49 (d,
J = 8.54 Hz, 2H), 3.41 (t, J = 6.34 Hz, 2H), 2.94 (t, J = 7.56 Hz, 2H),
2.16–2.27 (m, 2H).
Declaration of Competing Interest
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.
Acknowledgement
This work was financially supported by the research fund of
Chungnam National University, South Korea.
6.1.10.2. Preparation
of
4-(3-bromopropyl)-N,N-dimethylbenzene-
sulfonamide (11f).. To a solution of 15 (6.73 mmol) in DCM (20 mL),
TEA (20.20 mmol) and dimethyl amine hydrochloride (10.10 mmol) were
added at 0 °C. The resulting solution was stirred for 3 h at rt. The reaction
solution was diluted with DCM, washed with water, dried over anhydrous
Na2SO4 and then concentrated under reduced pressure to afford the title
compound as light brown solid. The crude product was used for the next
step without further purification. Yield 90%; Light Brown solid; 1H NMR
(400 MHz, CDCl3) δ 7.69–7.74 (d, J = 8.29 Hz, 2H), 7.36–7.41 (d,
J = 8.29 Hz, 2H), 3.41 (t, J = 6.46 Hz, 2H), 2.88 (t, J = 7.44 Hz, 2H),
2.68–2.74 (s, 6H), 2.20 (m, 2H).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115742.
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