Synthesis, Structure Revision, and Cytotoxicity of Nocarbenzoxazole G

Article abstract of DOI:10.1021/acs.jnatprod.9b00072

The total synthesis of nocarbenzoxazoles F (1) and G (2), originally obtained from the marine-derived halophilic bacterial strain Nocardiopsis lucentensis DSM 44048, was achieved via a simple and versatile route involving microwave-assisted construction of a benzoxazole skeleton, followed by carbon-carbon bond formation with the corresponding aryl bromides. Unfortunately, the ¹H and ¹³C NMR spectra of natural nocarbenzoxazole G did not agree with those of the synthesized compound. In particular, the spectra of the isolated and synthesized compounds showed considerable differences in the signals from the protons and carbons in the aryl group. The revised structure was validated by the total synthesis of the actual nocarbenzoxazole G (8c) molecule, which is a regioisomer of the compound that was reported earlier as nocarbenzoxazole G. The synthesized derivatives showed specific cytotoxicity to the human cervical carcinoma cell line, HeLa, but did not have any remarkable effect on the other cell lines.

Full text of DOI:10.1021/acs.jnatprod.9b00072

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Synthesis, Structure Revision, and Cytotoxicity of Nocarbenzoxazole
G
Taejung Kim,† Sin-Ae Lee,† Taesub Noh,† Pilju Choi,† Seon-Jun Choi,† Bong Geun Song,†
Youngseok Kim,† Young-Tae Park,† Gyuwon Huh,†,‡ Young-Joo Kim,† and Jungyeob Ham*,†,‡
†Natural Products Research Institute, Korea Institute of Science and Technology (KIST), 679 Saimdang-ro, Gangneung 25451,
Republic of Korea
‡Division of Bio-Medical Science & Technology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu,
Daejeon 34113, Republic of Korea
S
* Supporting Information
ABSTRACT: The total synthesis of nocarbenzoxazoles F (1)
and G (2), originally obtained from the marine-derived
halophilic bacterial strain Nocardiopsis lucentensis DSM
44048, was achieved via a simple and versatile route involving
microwave-assisted construction of a benzoxazole skeleton,
followed by carbon−carbon bond formation with the
1
corresponding aryl bromides. Unfortunately, the H and 13C
NMR spectra of natural nocarbenzoxazole G did not agree
with those of the synthesized compound. In particular, the
spectra of the isolated and synthesized compounds showed
considerable differences in the signals from the protons and
carbons in the aryl group. The revised structure was validated
by the total synthesis of the actual nocarbenzoxazole G (8c)
molecule, which is a regioisomer of the compound that was reported earlier as nocarbenzoxazole G. The synthesized derivatives
showed specific cytotoxicity to the human cervical carcinoma cell line, HeLa, but did not have any remarkable effect on the
other cell lines.
2-Arylbenzoxazole-based compounds are one of the most
important classes of heterocyclic scaffolds that exhibit
remarkable biological properties such as anticonvulsant,1
anti-inflammatory,2 and anticancer activities.3 These com-
pounds show excellent activities against Gram-positive
bacteria, Gram-negative bacteria, and the yeast Candida
albicans.4 Interestingly, a slight change in the substitution
pattern of the aryl group at the C-2 position of 2-
arylbenzoxazoles results in distinguishable differences in
biological activities. Accordingly, various functionalized 2-
arylbenzoxazoles have been studied in the field of pharma-
ceuticals.5
Figure 1. (Top) Originally proposed structures of nocarbenzoxazole
F (1) and nocarbenzoxazole G (2). (Bottom) Retrosynthesis of 2-
arylbenzoxazole.
In 2015, Lu and co-workers isolated a new series of
compounds from the halophilic bacterial strain Nocardiopsis
lucentensis DSM 44048 and tested their cytotoxicity against a
panel of human tumor cell lines.6 These compounds were
called nocarbenzoxazoles, and their structures were based on
that of 2-arylbenzoxazole. Among the compounds isolated,
nocarbenzoxazole F (1) and nocarbenzoxazole G (2) showed
selective activities against HepG2 and HeLa cell lines. The
compounds (Figure 1) were characterized using NMR
spectroscopy and HRESIMS.
RESULTS AND DISCUSSION
Our synthesis strategy was based on the practical and efficient
construction of the 2-arylbenzoxazole framework. The
■
Received: January 25, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00072
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Article
Scheme 1. Syntheses of Nocarbenzoxazoles F and G and Their Derivatives
retrosynthetic analysis of structure A predicts the formation of
a benzoxazole core B and the corresponding 4-bromophenols
D. Further retrosynthesis of B suggests a 2-aminophenol
precursor C. In this study, the C−C bond formation between
the benzoxazole core B and the aryl moiety D to produce the
2-arylbenzoxazole was achieved using a microwave (MW)-
assisted Pd-catalyzed reaction. Owing to the efficient synthesis
of the natural 2-arylbenzoxazole, this compound can contribute
to the library of compounds for a structure−activity relation-
ship (SAR) study for the investigation of the antitumor
activities of these types of compounds.
The syntheses of 1, 2, and their derivatives (8c−8h) are
described in Scheme 1. First, we focused on the synthesis of 1.
Methyl benzoate 5 was prepared from the commercially
available 4-hydroxy-3-nitrobenzoic acid (3) using the protocol
available in the literature.7 Then, 5 was dissolved in trimethyl
orthoformate and heated under microwave irradiation at 160
°C for 30 min without any additional oxidant, reductant, or
catalyst. This gave 2-H benzoxazole 6 in 95% yield (Scheme
1). Next, 2-H benzoxazole 6 was treated with the
corresponding bromophenol in the presence of Pd(OAc)2,
Cu(OAc)2·H2O, triphenylphosphine (PPh3), and K2CO3 in
toluene under microwave irradiation at 160 °C for 30 min.
This produced a trace amount of 7a along with an unexpected
adduct, S1, as the major product.8 When the phosphine ligand
was changed to tricyclohexylphosphine (PCy3), the desired
product 7a was obtained in 67% yield. Finally, the reduction of
the methyl ester in 7a with lithium aluminum hydride afforded
nocarbenzoxazole F (1) in 59% yield over five steps. The
reported nocarbenzoxazole G (2) and the other derivatives
(8c−8h) were synthesized similarly. Their syntheses required
only two additional steps after the formation of the common
intermediate 6: (i) Pd-catalyzed C−H activation/aryl−aryl
coupling with various aryl bromides (b−h) for the formation
of the C−C bond between 2-H benzoxazole and the aryl
moiety and (ii) reduction of the methyl ester to primary
alcohol. The overall yields ranged from 54% to 90%.
The structures of 1, 2, and 8c−8h synthesized here were
determined by NMR, IR, and mass spectrometric analysis.
Additionally, the data of 1 and 2 were compared with the
reported data of the natural nocarbenzoxazoles F and G. The
data for synthetic 1 matched the data for natural 1. However,
the spectroscopic properties of 2 synthesized in this study were
different from those of natural nocarbenzoxazole G. In
particular, the chemical shifts and coupling constants of the
protons of the aryl group around C-2 of 2 (Figure 2, A) were
clearly different in the natural compound and synthesized
compound (Figure 2, B): natural δH 6.97 (d, 8.2), 7.74 (dd, 1.9
and 8.3), and 7.77 (d, 1.9); synthetic δH 6.56 (dd, 2.4 and 8.4),
6.61 (d, 2.4), and 7.94 (d, 8.4). Moreover, upon reexamining
the previously reported NMR data of 2,6 we found that the
natural product contained two signals at δH 7.74 (dd, 1.9 and
8.3) and 7.77 (d, 1.9) that corresponded to the C-2 carbon
peak at δC 165.7 in the HMBC spectrum. This led us to
conclude that the actual structure of the aryl group in the
natural nocarbenzoxazole G should be a regioisomer with
respect to the 2-methoxy group, thereby suggesting a 4-
hydroxy-3-methoxyphenyl at the C-2 position. Consequently,
the coupling of 6 with 4-bromo-2-methoxyphenol (c) followed
by the reduction afforded the derivative 8c, whose 1H and 13
C
NMR spectra were identical with those of natural nocarben-
zoxazole G (Figure 2, C). Therefore, we achieved the total
synthesis of nocarbenzoxazole G (8c). The revised structure of
natural nocarbenzoxazole G (8c) is a regioisomer (with respect
B
DOI: 10.1021/acs.jnatprod.9b00072
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derivatives (1, 2, and 8c−8h). The reexamination of the
NMR data and the study of the synthesis have provided a
reliable and practical route to obtain the revised structure of
nocarbenzoxazole G (8c). Furthermore, the toxicity studies
show that 8h, containing the 2-hydroxynaphth-6-yl group, has
the maximum inhibitory effect on the growth of HeLa cells. As
a result, these compounds can also be investigated for their
antitumor activities or other biological activities.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
obtained on a OptiMelt MPA100 and are uncorrected. FTIR spectra
were recorded on an ATR plate. 1H NMR spectra were recorded on a
Bruker AVANCE 400 spectrometer, and the chemical shifts are
reported relative to the internal standard tetramethylsilane (0 ppm)
for CDCl3 and internal solvent MeOH (3.31 ppm) for CD3OD. 13C
NMR spectra were obtained on the same spectrometer and referenced
to the internal solvent signals (central peak is 77.16 or 49.00 ppm in
CDCl3 or CD3OD, respectively). Mass spectra were recorded on a
LCMS-2020 (Shimadzu Corporation) for ESIMS and an Accu TOF
JMS-T100LCS (JEOL) for HRESIMS. Flash column chromatography
was performed over silica gel 200−300. All reagents were weighed and
handled in air at room temperature, and all reactions were performed
under an air atmosphere. Unless otherwise noted, all reagents were
purchased from commercial suppliers without further purification.
Preparation of Methyl 1,3-Benzoxazole-5-carboxylate (6).
Methyl 1,3-Benzoxazole-5-carboxylate (6). The methyl 3-amino-4-
hydroxybenzoate 38 (5.50 g, 32.7 mmol) was dissolved in trimethyl
orthoformate (32.7 mL) in a microwave tube. The mixture was heated
under microwave irradiation at 160 °C for 30 min (200 w). After
completion of the reaction, the mixture was cooled to room
temperature, and the solvent was evaporated under reduced pressure.
The crude residue was purified by column chromatography on silica
gel to afford 6 (5.50 g, 95%) as a white solid; mp 108−109 °C; FTIR
(ATR) 3102, 2999, 2951, 1701, 1604, 1434, 1288, 1116, 1049, 751,
Figure 2. 1H NMR spectra (δH 6.4−8.0) of natural nocarbenzoxazole
G isolated from Nocardiopsis lucentensis DSM 44048 (A). Previously
reported structure 2 (B) and revised structure of nocarbenzoxazole G
8c (C).
to the methoxy group) of the originally proposed structure (2)
of this compound.
Next, the synthesized compounds (1, 2, and 8c−8h) were
evaluated for their cytotoxicities against a panel of human
cancer cell lines, namely, HepG2 and HepB3 (liver), HeLa
(cervical), MDA-MB-231 and MCF 7 (breast), and T98G
(glioblastoma). Interestingly, all of the synthesized compounds
specifically inhibited the growth of HeLa cells. Table 1 shows
that the IC50 values in the CCK-8 assay with synthetic
nocarbenzoxazoles 1 and 8c were 4.7 and 5.7 μM, respectively,
compared with the IC50 values of 14 and 1 μM in the MTT
assay with the corresponding natural compounds.9 In
particular, 8h, containing the 2-hydroxynaphth-6-yl group,
showed a high cytotoxicity against the HeLa cells, with an IC50
value of 0.56 μM. The cytotoxic effects on the other cell lines
were not remarkable because the IC50 values were higher than
20 μM. Further studies on the structure optimization and
mechanism of their biological activities are currently in
progress in our laboratory.
1
769 cm−1; H NMR (400 MHz, CDCl3) δ 8.46 (d, 1H, J = 1.2 Hz),
8.15 (s, 1H), 8.12 (dd, 1H, J = 1.6 and 8.4 Hz), 7.60 (d, 1H, J = 8.8
Hz), 3.93 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.6, 153.7,
152.9, 140.2, 127.5, 127.2, 122.8, 110.9, 52.4; ESIMS m/z 178.10 [M
+ H]+ (calcd for C9H8NO3,178.05).
Syntheses 7a−7h from 2-H Benzoxazole 6. Methyl 2-(4-
Hydroxyphenyl)benzo[d]oxazole-5-carboxylate (7a). To a solution
of 6 (354 mg, 2.00 mmol) in toluene (20.0 mL) were added 4-
bromopheneol (381 mg, 2.20 mmol), Pd(OAc)2(4.50 mg, 1 mol %),
Cu(OAc)2·H2O (72.5 mg, 20 mol %), PCy3 (280 mg, 50 mol %), and
K2CO3(553 mg, 4.00 mmol) in a microwave tube. The mixture was
heated under microwave irradiation at 160 °C for 30 min (200 W).
After completion of the reaction, the mixture was cooled to room
temperature (rt), and the mixture was quenched with saturated
NH4Cl solution. The resulting mixture was extracted with EtOAc.
The organic layer was washed with a saturated aqueous solution of
NaCl and dried with Na2SO4. After filtration, the mixture was
In summary, we have developed a facile method for the
synthesis of 2-arylbenzoxazoles via a Pd-catalyzed direct
arylation of 2-H benzoxazole 6 with the corresponding aryl
bromides using microwave irradiation. The reduction of the
methyl ester afforded the nocarbenzoxazoles and their
Table 1. IC50 Values (μM) of Nocarbenzoxazoles and Their Derivatives against a Panel of Human Tumor Cell Lines
a
IC50 (μM)
compound
HepG2
HepB3
HeLa
MDA-MB-231
MCF7
T98G
b
b
1
2
>20 (16)
>20
>20 (3)
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
4.7 0.1 (14)
2.9 0.2
5.7 0.1 (1)
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
b
a
8c
8d
8e
8f
8g
8h
11
16
19
1
1
1
8.9 0.3
0.56 0.01
a
b
Data presented as a mean SD of three replicate measurements of the same sample. Values in parentheses are from ref 6.
C
DOI: 10.1021/acs.jnatprod.9b00072
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1
concentrated under reduced pressure, and the crude residue was
purified by column chromatography on silica gel to afford 7a (361 mg,
67%) as a white solid: mp 270−274 °C; FTIR (ATR) 3059, 2921,
2850, 1722, 1606, 1433, 1290, 1120, 1170, 1085, 1064, 749 cm−1; 1H
NMR (400 MHz, CD3OD) δ 8.30 (d, 1H, J = 1.6 Hz), 8.11 (d, 1H, J
= 8.8 Hz), 8.06 (d, 2H, J = 1.6 Hz), 7.71 (d, 1H, J = 8.8 Hz), 6.98 (d,
2H, J = 8.8 Hz), 3.95 (s, 3H); 13C NMR (100 MHz, CD3OD
+CDCl3) δ 168.0, 166.6, 163.0, 154.8, 143.2, 130.8 (2C), 128.2,
127.7, 121.6, 118.3, 117.0 (2C), 111.4, 52.8; ESIMS m/z 270.10 [M +
H]+ (calcd for C15H12NO4, 270.08).
2851, 1721,1617, 1483, 1286, 1227, 1172, 1093, 980, 746 cm−1; H
NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 8.04 (dd, 1H, J = 1.6 and
8.4 Hz), 7.87 (s, 2H), 7.55 (d, 1H, J = 8.4 Hz), 3.95 (s, 3H), 2.26 (s,
6H); 13C NMR (100 MHz, CDCl3) δ 167.1, 165.6, 153.7, 146.9,
142.7, 128.4 (2C), 126.8, 126.3, 121.6, 121.1, 115.4, 110.0 (2C), 52.3,
17.6 (2C); ESIMS m/z 297.20 [M + H]+ (calcd for C17H17N2O3,
297.13).
Methyl 2-(4-hydroxy-2-methoxyphenyl)benzo[d]oxazole-5-car-
boxylate (7g). By following the procedure described above for the
preparation 7a, the reaction with 5-bromoindole (431 mg, 2.20
mmol) instead of 4-bromophenol was performed. Purification by
column chromatography afforded 7g (543 mg, 93%) as a light yellow
solid: mp 180−183 °C; FTIR (ATR) 3239, 2922, 2852, 1718, 1623,
Methyl 2-(4-Hydroxy-2-methoxyphenyl)benzo[d]oxazole-5-car-
boxylate (7b). By following the procedure described above for the
preparation of 7a, the reaction with 4-bromo-2-methoxyphenol (447
mg, 2.20 mmol) instead of 4-bromophenol was performed.
Purification by column chromatography afforded 7b (389 mg, 65%)
as a white solid: mp 176−179 °C; FTIR (ATR) 3222, 2921, 2845,
1434, 1334, 1286, 1206, 1084, 762, 730 cm−1; H NMR (400 MHz,
1
CDCl3) δ 8.51 (s, 1H), 8.43 (d, 1H, J = 1.2 Hz), 8.14 (dd, 1H, J = 1.2
and 8.4 Hz), 8.09 (dd, 1H, J = 1.6 and 8.8 Hz), 7.61 (d, 1H, J = 8.4
Hz), 7.53 (d, 1H, J = 8.4 Hz), 7.32 (t, 1H, J = 1.6 Hz), 6.70 (s, 1H),
3.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 167.1, 166.1, 153.9,
142.7, 138.0, 128.1, 126.9, 126.5, 125.8, 122.0, 121.7, 121.5, 118.5,
111.8, 110.2, 104.1, 52.4; ESIMS m/z 293.20 [M + H]+ (calcd for
C17H13N2O3, 293.09).
1
1730, 1618, 1501, 1429, 1292, 1192, 1083, 802, 746 cm−1; H NMR
(400 MHz, CD3OD) δ 8.42 (d, 1H, J = 1.6 Hz), 8.09 (dd, 1H, J = 1.6
and 8.4 Hz), 7.83 (dd, 1H, J = 1.6 and 8.4 Hz), 7.76 (d, 1H, J = 1.6
Hz), 7.59 (d, 1H, J = 8.4 Hz), 7.06 (d, 1H, J = 8.4 Hz), 4.03 (s, 3H),
3.96 (s, 3H); 13C NMR (100 MHz, MeOD-d3) δ 166.9, 164.6, 153.7,
149.5, 147.0, 142.4, 127.1, 126.8, 122.3, 121.6, 118.8, 115.0, 110.2,
110.0, 56.4, 52.4; ESIMS m/z 300.20 [M + H]+ (calcd for
C16H14NO5, 300.09).
Methyl 2-(4-Hydroxy-2-methoxyphenyl)benzo[d]oxazole-5-car-
boxylate (7c). By following the procedure described above for the
preparation 7a, the reaction with 4-bromo-3-methoxyphenol (447 mg,
2.20 mmol) instead of 4-bromophenol was performed. Purification by
column chromatography afforded 7c (532 mg, 89%) as a white solid:
mp 209−213 °C; FTIR (ATR) 3065, 2920, 2849, 1720, 1582, 1482,
1291, 1254, 1204, 1116, 1036, 749 cm−1; 1H NMR (400 MHz,
CD3OD) δ 8.31 (d, 1H, J = 1.6 Hz), 8.07 (dd, 1H, J = 1.6 and 8.4
Hz), 7.98 (d, 1H, J = 8.4 Hz), 7.68 (d, 1H, J = 8.4 Hz), 6.62 (d, 1H, J
= 2.0 Hz), 6.57 (dd, 1H, J = 2.0 and 8.4 Hz), 3.97 (s, 3H), 3.95 (s,
3H); 13C NMR (100 MHz, CD3OD) δ 168.1, 165.2, 164.5, 162.2,
154.1, 143.1, 133.5, 128.0, 127.6, 121.5, 111.2, 109.3, 107.1, 100.6,
56.1, 52.8; ESIMS m/z 300.00 [M + H]+ (calcd for C16H14NO5,
300.09).
Methyl 2-(4-Hydroxy-2-methoxyphenyl)benzo[d]oxazole-5-car-
boxylate (7h). By following the procedure described above for the
preparation 7a, the reaction with 6-bromo-2-naphthol (491 mg, 2.20
mmol) instead of 4-bromophenol was performed. Purification by
column chromatography afforded 7h (453 mg, 71%) as a light yellow
solid: mp 198−201 °C; FTIR (ATR) 3209, 2922, 2851, 1720, 1620,
1435, 1291, 1216, 1085, 1048, 763, 747 cm−1; H NMR (400 MHz,
1
CD3OD+CDCl3) δ 8.64 (s, 1H), 8.39 (d, 1H, J = 1.6 Hz), 8.16 (dd,
1H, J = 1.6 and 8.4 Hz), 8.10 (dd, 1H, J = 1.6 and 8.4 Hz), 7.87 (d,
1H, J = 8.4 Hz), 7.78 (d, 1H, J = 8.4 Hz), 7.66 (d, 1H, J = 8.8 Hz),
7.19−7.16 (m, 2H), 3.95 (s, 3H); 13C NMR (100 MHz, CD3OD
+CDCl3) δ 166.9, 156.9, 153.6, 142.1, 136.8, 130.8, 129.4, 128.6,
127.7, 127.1, 126.9, 126.8, 124.2, 121.5, 120.7, 119.5, 110.2, 109.3,
52.3; ESIMS m/z 320.20 [M + H]+ (calcd for C19H14NO4, 320.09).
Syntheses of 1, 2, and 8c−8h from 7a−7h. Nocarbenzox-
azole F (1). To a solution of 7a (161 mg, 0.60 mmol) in anhydrous
tetrahydrofuran (6.00 mL) was slowly added LiAlH4 (34.2 mg, 0.902
mmol) at 0 °C under nitrogen. The mixture was warmed to rt and
stirred for 2 h. After completion of the reaction, the mixture was
cooled to 0 °C and quenched with MeOH (20.0 mL). The mixture
was filtered through a Celite pad and washed with EtOAc. After
filtration, the mixture was concentrated under reduced pressure, and
the crude residue was purified by column chromatography on silica
gel to afford 1 (139 mg, 96%) as a white solid: mp 245−250 °C;
FTIR (ATR) 3094, 2922, 2851, 1614, 1438, 1368, 1234, 1174, 1011,
812, 742 cm−1; 1H NMR (400 MHz, CD3OD) δ 8.10 (tt, 2H, J = 4.8
and 9.6 Hz), 7.69 (d, 1H, J = 1.6 Hz), 7.61 (d, 1H, J = 8.4 Hz), 7.39
(dd, 1H, J = 1.6 and 8.4 Hz), 6.98 (tt, 2H, J = 4.8 and 9.6 Hz), 4.73
(s, 2H); 13C NMR (100 MHz, CD3OD) δ 165.6, 162.7, 151.2, 143.0,
140.0, 130.6 (2C), 125.2, 118.9, 118.3, 117.0 (2C), 111.2, 65.1;
HRESIMS m/z 242.0851 [M + H]+ (calcd for C14H12NO3,
242.0817).
Methyl 2-(4-Hydroxy-2-methoxyphenyl)benzo[d]oxazole-5-car-
boxylate (7d). By following the procedure described above for the
preparation of 7a, the reaction with 4-bromo-2-chlorophenol (456
mg, 2.20 mmol) instead of 4-bromophenol was performed.
Purification by column chromatography afforded 7d (491 mg, 81%)
as a white solid: mp 206−208 °C; FTIR (ATR) 3309, 2922, 2850,
1
1689, 1624, 1436, 1296, 1183, 1122, 1087, 1051, 765, 748 cm−1; H
NMR (400 MHz, CDCl3) δ 8.42 (t, 1H, J = 1.6 Hz), 8.27 (d, 1H, J =
2.0 Hz), 8.11 (d, 1H, J = 1.6 Hz), 8.09 (t, 1H, J = 2.0 Hz), 7.60 (d,
1H, J = 8.4 Hz), 7.18 (d, 1H, J = 8.4 Hz), 3.96 (s, 3H); 13C NMR
(100 MHz, CDCl3+CD3OD) δ 166.9, 163.4, 155.3, 153.7, 142.1,
129.3, 128.2, 127.2, 127.1, 121.7, 121.1, 119.8, 117.0, 110.4, 52.4;
ESIMS m/z 304.10 [M + H]+ (calcd for C15H11NO4, 304.04).
Methyl 2-(4-Hydroxy-2-methoxyphenyl)benzo[d]oxazole-5-car-
boxylate (7e). By following the procedure described above for the
preparation 7a, the reaction with 1-bromo-4-chlorobenzene (421 mg,
2.20 mmol) instead of 4-bromophenol was performed. Purification by
column chromatography afforded 7e (534 mg, 93%) as a white solid:
mp 164−166 °C; FTIR (ATR) 2956, 2850, 1730, 1622, 1434, 1295,
1216, 1086, 1047, 1011, 829, 748 cm−1; 1H NMR (400 MHz, CDCl3)
δ 8.45 (d, 1H, J = 1.6 Hz), 8.20 (dd, 2H, J = 1.6 and 6.4 Hz), 8.13
(dd, 1H, J = 1.6 and 8.8 Hz), 7.62 (d, 1H, J = 8.8 Hz), 7.53 (dd, 2H, J
= 1.6 and 6.4 Hz), 3.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
166.8, 163.5, 153.8, 142.2, 138.4, 129.5, 129.5, 129.2, 129.2, 127.4,
127.4, 125.2, 122.2, 110.5, 52.5; ESIMS m/z 288.10 [M + H]+ (calcd
for C15H11ClNO3, 288.04).
4-(5-(Hydroxymethyl)benzo[d]oxazol-2-yl)-3-methoxyphenol
(Reported Structure of Nocarbenzoxazole G, 2). By following the
procedure described above for the preparation of 1, the reaction with
7b (179 mg, 0.604 mmol) as the starting material was performed.
Purification by column chromatography afforded 2 (135 mg, 83%) as
a white solid: mp 196−199 °C; FTIR (ATR) 3091, 2920, 2851, 1605,
1435, 1328, 1257, 1210, 1116, 1036, 838, 802 cm−1; H NMR (400
1
MHz, CD3OD) δ 7.94 (d, 1H, J = 8.4 Hz), 7.70 (s, 1H), 7.59 (d, 1H,
J = 8.4 Hz), 7.38 (d, 1H, J = 8.4 Hz), 6.61 (d, 1H, J = 2.4 Hz), 6.56
(dd, 1H, J = 2.4 and 8.4 Hz), 4.73 (s, 2H), 3.96 (s, 3H); 13C NMR
(100 MHz, CD3OD) δ 167.0, 164.0, 161.9, 143.0, 139.7, 133.2, 130.8,
125.1, 118.3, 110.9, 109.1, 107.8, 100.5, 65.1, 56.1; HRESIMS m/z
272.0921 [M + H]+ (calcd for C15H14NO4, 272.0923).
4-(5-(Hydroxymethyl)benzo[d]oxazol-2-yl)-3-methoxyphenol
(Revised Structure of Nocarbenzoxazole G, 8c). By following the
procedure described above for the preparation of 1, the reaction with
7c (179 mg, 0.602 mmol) as the starting material was performed.
Methyl 2-(4-Hydroxy-2-methoxyphenyl)benzo[d]oxazole-5-car-
boxylate (7f). By following the procedure described above for the
preparation 7a, the reaction with 4-bromo-2,6-dimethylaniline (440
mg, 2.20 mmol) instead of 4-bromophenol was performed.
Purification by column chromatography afforded 7f (503 mg, 85%)
as a light yellow solid: mp 232−233 °C; FTIR (ATR) 3364, 2922,
D
DOI: 10.1021/acs.jnatprod.9b00072
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Purification by column chromatography afforded 8c (153 mg, 94%) as
a white solid: mp 170−173 °C; FTIR (ATR) 3272, 2936, 2900, 1597,
139.8, 138.0, 131.6, 129.1, 128.9, 128.0, 125.4, 124.8, 121.9, 120.5,
118.4, 111.2, 110.0, 64.9; HRESIMS m/z 292.0973 [M + H]+ (calcd
for C18H14NO3, 292.0974).
1
1503, 1305, 1262, 1226, 1124, 1028, 811, 733 cm−1; H NMR (400
MHz, CD3OD) δ 7.75 (s, 1H), 7.73 (dd, 1H, J = 2.0 and 8.4 Hz),
7.68 (d, 1H, J = 1.2 Hz), 7.60 (d, 1H, J = 8.4 Hz), 7.38 (dd, 1H, J =
2.0 and 8.4 Hz), 6.97 (d, 1H, J = 8.4 Hz), 4.72 (s, 2H), 4.60 (brs,
1H), 3.98 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 165.5, 152.0,
151.1, 149.4, 142.9, 140.0, 125.2, 122.7, 119.1, 118.3, 116.7, 111.5,
111.2, 65.0, 56.5; HRESIMS m/z 272.0951 [M + H]+ (calcd for
C15H14NO4, 272.0923).
Cell Culture. The human hepatocellular carcinoma cell lines
HepG2 and Hep3B, the human cervical carcinoma cell line HeLa, the
human breast carcinoma cell lines MCF7 and MDA-MB-231, and the
human glioblastoma cell line T98G were purchased from the
American Type Culture Collection (ATCC). The cells were routinely
grown in DMEM (Gibco) and RPMI1640 (Gibco), respectively,
supplemented with 10% fetal bovine serum (Gibco), 100 U/mL
penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified
atmosphere with 5% CO2.
Cell Proliferation Assay. Cell Counting Kit-8 (Dojindo
Laboratories) was used to evaluate cell proliferation according to
the manufacturer’s recommendations. Briefly, exponentially growing
cells were seeded in a 96-well plate at a density of 1.0 × 104 cells/well
in triplicate. The next day, the cells were treated with compounds at
concentrations ranging from 1.25 to 20 μM. After incubation for 48 h,
10 μL of the kit reagent was added, and the cells were incubated for
an additional 1 h. Cell viability was measured by scanning with a
microplate reader at 450 nm. Control cells were exposed to culture
media containing 0.5% (v/v) DMSO.
2-Chloro-4-(5-(hydroxymethyl)benzo[d]oxazol-2-yl)phenol (8d).
By following the procedure described above for the preparation of 1,
the reaction with 7d (182 mg, 0.601 mmol) as the starting material
was performed. Purification by column chromatography afforded 8d
(155 mg, 94%) as a white solid: mp 223−226 °C; FTIR (ATR) 3049,
2921, 2852, 1604, 1505, 1410, 1261, 1227, 1071, 985, 813, 734 cm−1;
1H NMR (400 MHz, CD3OD) δ 8.17 (d, 1H, J = 2.0 Hz), 8.01 (dd,
1H, J = 2.0 and 8.8 Hz), 7.69 (s, 1H), 7.62 (d, 1H, J = 8.4 Hz), 7.40
(dd, 1H, J = 1.2 and 8.4 Hz), 7.09 (d, 1H, J = 8.8 Hz), 4.72 (s, 2H);
13C NMR (100 MHz, CD3OD) δ 164.2, 158.2, 151.2, 142.9, 140.2,
130.4, 128.6, 125.5, 122.6, 120.1, 118.5, 118.0, 111.3, 65.0; HRESIMS
m/z 276.0416 [M + H]+ (calcd for for C14H11ClNO3, 276.0427).
2-(4-Chlorophenyl)benzo[d]oxazol-5-yl)methanol (8e). By fol-
lowing the procedure described above for the preparation of 1, the
reaction with 7e (172 mg, 0.598 mmol) as the starting material was
performed. Purification by column chromatography afforded 8e (151
mg, 97%) as a white solid: mp 194−196 °C; FTIR (ATR) 3319,
2919, 2850, 1597, 1478, 1403, 1259, 1199, 1087, 1008, 810, 730
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00072.
1
cm−1; H NMR (400 MHz, CDCl3) δ 8.19 (dd, 2H, J = 2.0 and 7.2
Hz), 7.76 (s, 1H), 7.56 (d, 1H, J = 8.4 Hz), 7.51 (dd, 2H, J = 1.6 and
7.2 Hz), 7.40 (dd, 1H, J = 1.6 and 8.4 Hz), 4.82 (s, 2H); 13C NMR
(100 MHz, CDCl3) δ 142.4, 138.1, 138.0, 129.5 (2C), 129.1, 129.0
(2C), 127.8, 125.8, 124.8, 118.7, 110.7, 65.5; HRESIMS m/z
260.0498 [M + H]+ (calcd for C14H11ClNO2, 260.0478).
(2-(4-Amino-3,5-dimethylphenyl)benzo[d]oxazol-5-yl)methanol
(8f). By following the procedure described above for the preparation
of 1, the reaction with 7f (178 mg, 0.603 mmol) as the starting
material was performed. Purification by column chromatography
afforded 8f (140 mg, 87%) as a light yellow solid: mp 203−205 °C;
FTIR (ATR) 3335, 3225, 2920, 2851, 1727, 1642, 1455, 1377, 1338,
1264, 1024, 793 cm−1; 1H NMR (400 MHz, CD3OD) δ 7.75 (s, 2H),
7.62 (s, 1H), 7.54 (d, 1H, J = 8.4 Hz), 7.33 (dd, 1H, J = 1.2 and 8.4
Hz), 4.71 (s, 2H), 2.25 (s, 6H); 13C NMR (100 MHz, CD3OD
+CDCl3) δ 166.5, 150.8, 149.2, 143.0, 139.5, 128.7 (2C), 124.5, 122.5
(2C), 117.7, 115.2, 110.8, 65.1, 17.7 (2C); HRESIMS m/z 269.1275
[M + H]+ (calcd for C16H17N2O2, 269.1290).
Copies of NMR spectra of the compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: ham0606@kist.re.kr.
ORCID
■
Taejung Kim: 0000-0002-9449-4763
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Korea Institute of Science and
Technology (KIST) institutional program (Project No.
2Z05610) and by the Nano Convergence Industrial Strategic
Technology Development Program (No. 20000105) funded
by the Ministry of Trade, Industry & Energy (MOTIE, Korea).
(2-(1H-Indol-5-yl)benzo[d]oxazol-5-yl)methanol (8g). By follow-
ing the procedure described above for the preparation of 1, the
reaction with 7g (175 mg, 0.60 mmol) as the starting material was
performed. Purification by column chromatography afforded 8g (149
mg, 94%) as a light yellow solid: mp 233−236 °C; FTIR (ATR) 3246,
3000, 2950, 1611, 1460, 1556, 1375, 1335, 1259, 1027, 800, 735
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F
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