ACS Combinatorial Science
Research Article
treatment with a series of compounds involving scaffold VI,
such as 3e−5e, 6e−8e, and 13e−15e, compared to other
isoxazole derivatives. In the case of Griess assay in Raw264.7
murine macrophage cells, compounds of scaffold II, such as 4b,
6b, 10b, and 12b−14b, showed the significant reduction of
cellular nitric oxide (NO) release as a marker of anti-
inflammatory activity. Along with the difference in molecular
frameworks, we also observed the different patterns in
phenotypic changes according to the type of substituents.
Among the isoxazole derivatives containing quinoline sub-
structure, compounds of scaffold III (2c, 6c, and 15c) showed a
distinct display of polar surface area according to the type of
substituent in Figure 2 and inhibited the cellular NO release at
the different levels in the Griess assay (Figure 6b). In addition,
as shown in Figure 6c, isoxazoles containing cyanophenyl
moiety, such as 12a, 11b, 10e, 11e, and 11f, revealed the
cytotoxic effect against HeLa cells, while other isoxazoles
containing other moieties did not influence the cell viability.
under the reduced pressure, the residue was purified by silica-
gel flash column chromatography to obtain the desired
compounds. Characterization data for representative com-
pounds follow; for the full data set, see the Supporting
N,N-Dimethyl-6-(3-(3-(trifluoromethyl)phenyl)isoxazol-5-
1
yl)pyrimidin-4-amine 8d. Yield: 79%, white solid. H NMR
(400 MHz, CDCl3): δ 8.66 (s, 1H), 8.14 (s, 1H), 8.04 (d, J =
8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H),
7.33 (s, 1H), 7.05 (s, 1H), 3.22 (s, 6H). 13C NMR (100 MHz,
CDCl3): δ 169.6, 162.5, 162.2, 158.7, 150.8, 131.8, 131.5, 130.1,
129.76, 129.70, 127.0 (q, J = 3.8 Hz), 123.8 (q, J = 3.8 Hz),
101.7, 98.8, 37.5
2-(5-(6-(Dimethylamino)pyrimidin-4-yl)isoxazol-3-yl)-
benzonitrile 10d. Yield: 37%, white solid. 1H NMR (400 MHz,
CDCl3): δ 8.67 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.84 (d, J =
7.6 Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H),
7.50 (s, 1H), 7.06 (s, 1H), 3.22 (s, 6H). 13C NMR (100 MHz,
CDCl3): δ 162.4, 161.0, 158.7, 150.8, 134.49, 134.45, 133.3,
131.9, 130.3, 129.6, 117.9, 111.2, 103.2, 98.9, 37.5.
4-(5-(6-(Dimethylamino)pyrimidin-4-yl)isoxazol-3-yl)-
benzonitrile 12d. Yield: 74%, white solid. 1H NMR (400 MHz,
acetone-d6): δ 8.66 (s, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.79 (d, J
= 8.0 Hz, 2H), 7.86−7.75 (m, 3H), 7.32 (s, 1H), 7.05 (s, 1H),
3.22 (s, 6H). 13C NMR (100 MHz, DMSO-d6): δ 169.3, 161.9,
161.6, 158.4, 150.0, 132.2, 132.4, 127.6, 118.4, 112.9, 102.7,
99.0, 36.9.
CONCLUSION
■
In conclusion, we designed and synthesized a library of 3,5-
disubstituted isoxazoles via incorporating privileged substruc-
tures and various substituents, respectively, to make a diverse
arrangement of polar surface area within a similar 3-dimen-
sional molecular framework. We visualized the difference of
polar surface area in scaffolds I−VI using in silico analysis. To
access 3,5-disubstituted isoxazoles, we adopted 1,3-dipolar
cycloaddition between terminal acetylenes and nitrile oxides.
For the synthesis of nitrile oxides, we applied two
complementary routes and completed the library construction
in good to moderate yields. Through the biological evaluation
of our isoxazoles via three independent phenotypic assays, we
observed the different pattern in the biological activity that
exhibited the importance of molecular design via incorporating
privileged substructures and various substituents in a 3-
dimensionally similar conformer to maximize the diversity in
polar surface area.
N,N-Dimethyl-4-(6-(3-(3-(trifluoromethyl)phenyl)isoxazol-
1
5-yl)pyrimidin-4-yl)aniline 8f. Yield: 70%, yellow solid. H
NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 8.17−8.07 (m, 5H),
7.65 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.74 (s, 1H),
6.80 (d, J = 8.8 Hz, 2H), 3.09 (s, 6H). 13C NMR (100 MHz,
CDCl3): δ 169.2, 165.5, 162.3, 159.3, 152.9, 152.2, 130.2, 129.8,
129.5, 128.8, 127.8, 127.1, 127.0, 123.9 (q, J = 4.6 Hz), 122.9,
121.7, 111.9, 110.9, 102.7, 40.2.
2-(5-(6-(4-(Dimethylamino)phenyl)pyrimidin-4-yl)-
1
isoxazol-3-yl)benzonitrile 10f. Yield: 65%, yellow solid. H
NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.18 (s, 1H), 8.13
(d, J = 8.8 Hz, 2H), 8.01 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.6
Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.65−7.59 (m, 2H), 6.81 (d,
J = 8.8 Hz, 2H) 3.09 (s, 6H). 13C NMR (150 MHz, CDCl3): δ
168.6, 164.5, 129.0, 161.3, 159.7, 159.4, 125.3, 145.2, 134.5,
133.4, 130.6, 129.6, 1298.8, 121.8, 117.8, 112.4, 111.3, 103.8,
30.3.
EXPERIMENTAL PROCEDURES
■
General Procedure for the Synthesis of Representa-
tive Compound 8d, 10d, 12d, 8f, 10f, and 12f. Method A.
To a solution of oxime (1.0 equiv) in dimethylformamide
(DMF), N-chlorosuccinimide (1.1 equiv) was added. The
mixture was stirred at 60 °C until the starting materials were
fully consumed, which was monitored by thin layer
chromatography (TLC); then, TEA (1.2 equiv) and terminal
acetylene (1.2 equiv) were added to the reaction mixture. After
2 h (the reaction completion was monitored by TLC), the
reaction mixture was diluted with dichloromethane (DCM) and
washed with deionized water and brine. The combined organic
layer was dried with anhydrous Na2SO4(s). After the removal of
solvent under the reduced pressure, the residue was purified by
silica-gel flash column chromatography to obtain the desired
compounds.
Method B. To a solution of oxime (1.5 equiv) in
MeOH:H2O (5:1), PIFA (1.5 equiv), and terminal acetylene
(1.0 equiv) were added. The mixture was stirred at room
temperature until starting materials were consumed (the
reaction completion was monitored by TLC); then, the
reaction mixture was diluted with DCM and washed with
deionized water and brine. The combined organic layer was
dried with anhydrous Na2SO4(s). After the removal of solvent
4-(5-(6-(4-(Dimethylamino)phenyl)pyrimidin-4-yl)-
1
isoxazol-3-yl)benzonitrile 12f. Yield: 69%, yellow solid. H
NMR (400 MHz, CDCl3): δ 9.19 (s, 1H), 8.17−8.11 (m, 3H),
8.02 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.42 (s, 1H),
6.80 (d, J = 8.8 Hz, 1H), 3.90 (s, 6H). 13C NMR (100 MHz,
CDCl3): δ 169.5, 162.0, 159.3, 152.9, 152.1, 133.0, 128.8, 127.6,
122.8, 120.7, 118.4, 114.1, 111.9, 110.9, 102.2, 40.2.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
Detailed experimental procedures, full characterization
data, and the 1H and 13C NMR spectra for all synthesized
F
ACS Comb. Sci. XXXX, XXX, XXX−XXX