Synthesis and antimicrobial evaluation of new 2-substituted 5,7-di-tert-butylbenzoxazoles

Article abstract of DOI:10.1016/j.bmc.2006.05.030

Various synthetic pathways of the 30 novel 2-substituted 5,7-di-tert-butylbenzoxazoles as new potential antimicrobial drugs are discussed. The 28 intermediates are described as well. The compounds were characterized by ¹H and ¹³C NMR spectra, MS spectra, IR/UV spectra and by means of CHN analysis. The purity of the final compounds was checked by HPLC and their lipophilicity (log K) was also determined by means of RP-HPLC. In the present study, the correlation between RP-HPLC retention parameter log K (the logarithm of capacity factor K) and various calculated log P data is shown. The target compounds were tested for their in vitro antimycobacterial activity. Several compounds showed antituberculotic activity comparable with or higher than the standard isoniazide. In vitro cytotoxicity testing of the most active benzoxazoles and isoniazide as a reference drug was performed using MTT assay and compared with isoniazide as a reference drug. Structure-activity relationships among the chemical structures, the physical properties and the biological activities of the evaluated compounds are discussed in the article.

Full text of DOI:10.1016/j.bmc.2006.05.030

Bioorganic & Medicinal Chemistry 14 (2006) 5850–5865
Synthesis and antimicrobial evaluation of new 2-substituted
5,7-di-tert-butylbenzoxazoles
Jarmila Vinsova,a,* Katerina Cermakova,a Alexandra Tomeckova,a Martina Ceckova,b
Josef Jampilek,c Pavel Cermak,d Jiri Kunes,a Martin Dolezale and Frantisek Staudb
aDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, 500 05 Hradec Kralove, Czech Republic
bDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, 500 05 Hradec Kralove, Czech Republic
cZentiva a.s., U kabelovny 130, 102 37 Prague, Czech Republic
dDepartment of Clinical Microbiology, University Hospital and Faculty of Medicine, 500 05 Hradec Kralove, Czech Republic
eDepartment of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University,
500 05 Hradec Kralove, Czech Republic
Received 3 April 2006; revised 9 May 2006; accepted 15 May 2006
Available online 19 June 2006
Abstract—Various synthetic pathways of the 30 novel 2-substituted 5,7-di-tert-butylbenzoxazoles as new potential antimicrobial
1
drugs are discussed. The 28 intermediates are described as well. The compounds were characterized by H and 13C NMR spectra,
MS spectra, IR/UV spectra and by means of CHN analysis. The purity of the final compounds was checked by HPLC and their
lipophilicity (logK) was also determined by means of RP-HPLC. In the present study, the correlation between RP-HPLC retention
parameter logK (the logarithm of capacity factor K) and various calculated logP data is shown. The target compounds were tested
for their in vitro antimycobacterial activity. Several compounds showed antituberculotic activity comparable with or higher than the
standard isoniazide. In vitro cytotoxicity testing of the most active benzoxazoles and isoniazide as a reference drug was performed
using MTT assay and compared with isoniazide as a reference drug. Structure–activity relationships among the chemical structures,
the physical properties and the biological activities of the evaluated compounds are discussed in the article.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Benzoxazoles belong to biologically very active skele-
tons.1 Various benzoxazole derivatives were extensively
studied for their antibacterial and antifungal activity2–5
anticancer activity6,7 also as new non-nucleoside topoi-
somerase I poisons8 and HIV-1 reverse transcriptase
inhibitors.9,10
Tuberculosis (TB), the world’s leading infectious dis-
ease, caused by Mycobacterium tuberculosis represents
a major global health problem. One-third of the world’s
population is currently infected; more than 5000 people
die of TB every day. A great number of people are car-
riers of the latent form that creates dangerous source of
illness for the future. Therefore, there is an urgent need
to develop new structural classes of anti-tuberculosis
drugs. The development stimulates also a problem of
multi-drug resistant strains (MDR-TB) that challenge
preparation of new type of compounds with unique
mechanism of action, different from antitubercular
drugs currently used.
Benzoxazoles are also interesting fluorescent probes
which show high Stokes shift and present thermal and
photophysical stability due to an excited state intramo-
lecular proton transfer mechanism.11,12 Since they inter-
fere with biosynthesis of coloured carotenoids by
inhibiting the enzyme phytoene desaturase, they are
studied as potential bleaching herbicides.13
Benzoxazoles can be considered as structural bioiso-
sters of naturally occurring nucleotides such as adenine
and guanine, which allow them to interact easily with
the biopolymers of a living system. They have shown
low toxicity in warm-blooded animals.14 In a previous
paper we reported the synthesis of a series of lipophilic
2-substituted 5,7-di-tert-butylbenzoxazoles, which were
Keywords: 5,7-Di-tert-butylbenzoxazole; In vitro antimycobacterial
activity; Lipophilicity determination; Cytotoxicity; Structure–activity
relationships.
*
Corresponding author. Tel.: +420 49 5067343; fax: +420 49
5514330; e-mail: vinsova@faf.cuni.cz
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.030

J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5851
This presented study is a follow-up paper to the previous
articles dealing with the N-heterocyclic derivatives as
potential drugs.18–20
N
O
N
O
R
R
R = CH2OH, styryl, 5-, 6-membered heterocycle,
substituted heterocycle
R = H, CH3, Cl, Br, CF3, OH, NO2,
N(CH3)2, SCH3, 4-F-3-OC6H5
2. Results and discussion
2.1. Chemistry
Figure 1. General formula of the target 2-substituted 5,7-di-tert-
butylbenzoxazoles.
There are three general methods for synthesis of target
2-substituted benzoxazoles: (i) consists of coupling
2-aminophenols with carboxylic acid derivatives,
which is either catalyzed by strong acids21 or requires
microwave conditions;22 (ii) is oxidative cyclization of
phenolic Schiff bases obtained from 2-amino-4,6-di-
tert-butylphenol (3) condensation with the appropriate
aromatic aldehydes; (iii) is the reaction of 3,5-di-tert-
butyl-1,2-benzoquinone (DTBBQ) with amino acids.15
prepared by the reaction of 3,5-di-tert-butyl-1,2-benzo-
quinone with amino acids and dipeptides carrying N-
terminal glycine.15 They have shown promising activity
against M. tuberculosis and some non-tuberculous
strains where isoniazide, the first line antituberculous
drug, has been inactive. This fact has prompted us to
turn our attention to the preparation of a new lipophil-
ic series of 5,7-di-tert-butylbenzoxazoles substituted in
the C(2) position with aromatic substituent. The first
series of benzoxazoles possess in the C(2) position ben-
zene ring substituted by various electron-accepting or
donor properties and lipophilic–hydrophilic balance,
see Figure 1. In particular the electronegative substitu-
ents OH, Cl, Br and the most electron-withdrawing
groups NO2, CF3 were compared with unsubstituted
terms or with those bearing electron-releasing groups
such as CH3. The substitution on the benzene ring
was chosen in accordance with Topliss.16 The other
series of prepared compounds possess in the C(2) posi-
tion of benzoxazole the heterocyclic moiety, see Figure
1. Tertiary butyl groups increase lipophilicity of the
molecule which is very important for passing through
the extraordinary thick and tight mycobacterial cell
wall. Unusually low cell membrane permeability also
contributes to the resistance to therapeutic agents.
2-Amino-4,6-di-tert-butylphenol (3) is not commer-
cially available and was therefore synthesized via
several possible procedures. Nitration of 2,4-di-tert-
butylphenol gave according to TLC a mixture of
two yellow components. The large excess of 2,4-di-
tert-butyl-6-nitrophenol (1), the less polar compound,
was isolated by continuous column chromatography
on silica gel with hexane as eluent. The second more
polar compound was identified as 2-tert-butyl-4,6-di-
nitrophenol (2). Hydrogenation of nitrophenol 1 on
5% Pd/C gave starting 2-amino-4,6-di-tert-butylphenol
(3). Further synthetic pathways of aminophenol 3
generation, deamination of ethylene-1,2-diamine with
3,5-di-tert-butyl-1,2-benzoquinone in boiling propan-
2-ol23, gave on the contrary to the good yields pre-
sented in the literature23 a mixture of required ami-
nophenol 3 with fluorescent 5,7,50,70-tetra-terc-butyl-
2,20-bi-benzoxazole (4). More convenient one-pot
preparation was reduction of 3,5-tert-butyl-1,2-benzo-
quinone monoimine by means of complex hydride
NaBH4.24
One of the major prerequisites for pharmacological
screening and drug development is the prediction of
permeability, that is the transport of a molecule
through cellular membranes. Drugs cross biological
barriers most frequently through passive transport,
which strongly depends on their lipophilicity. There-
fore, hydrophobicity is one of the most important
physical properties of biologically active compounds.
This thermodynamic parameter describes the partition-
ing of a compound between an aqueous and an
organic phase and is characterized by the partition
coefficient (logP). Reversed phase high performance li-
quid chromatography (RP-HPLC) methods have be-
come popular and are widely used for lipophilicity
measurement. The general procedure consists of the
measurement of the directly accessible retention time
under isocratic conditions with varying amounts of
an organic modifier in the mobile phase using end-
capped non-polar C18 stationary RP columns and cal-
culating the capacity factor K. LogK, calculated from
the capacity factor K, is used as the lipophilicity index
converted to logP scale.17 The hydrophobicity evalua-
tion of the target compounds by means of RP-HPLC
and computational techniques is reported in this
communication.
Synthesis of 2-substituted 5,7-di-tert-butylbenzoxazoles
7a–7q and 8a–8m is shown in Scheme 1. The majority
of the target compounds were prepared via cyclization
of the Schiff bases (method A), which were generated
by the reaction of aminophenol 3 with the appropriate
aromatic aldehydes. Schiff bases 5a–5q and 6a–6l were
isolated and characterized. The target phenyl substi-
tuted derivatives 7a–7q were obtained by means of
5a–5q catalytic cyclization using PbO2 in glacial
AcOH, whereas the final heterocyclic substituted
derivatives 8a–8l were prepared through cyclization
of 6a–6l under (AcO)4Pb catalysis in glacial AcOH.
(5,7-Di-tert-butyl-benzoxazol-2-yl)-methanol (8m) was
obtained by the reaction of serine with DTBBQ,21
method C. Direct condensation by PPA (method B)
was tried out only for the preparation of 2-(5,7-di-
tert-butylbenzoxazol-2-yl)-phenol (7l) with a much
lower yield (15%) than by classical Shiff base oxidative
cyclization (75%), method A. Straightforward method
taking aminophenol 3 and carboxylic acid in the pres-
ence of polyphosphoric acid (PPA)21 has not been
found optimal.

5852
J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
H
R
NH
N
2
N
O
O
(AcO) Pb
4
R
R
+
or PbO
2
H
OH
OH
3
5a-5q
6a-6l
7a-7q
8a-8m
PPA
NH
COOH
OH
2
serine
+
OH
O
O
3
5a-5q, 7a-7q R = phenyl, substituted phenyl
6a-6l, 8a-8m R = styryl, 5-, 6-heterocycles, substituted heterocycles, CH OH
2
Scheme 1. Synthesis of the target 2-substituted 5,7-di-tert-butylbenzoxazoles 7a–7q and 8a–8m.
2.2. Lipophilicity
program. Nevertheless, logP values calculated by the
ACD/Log P program agree better with target benzoxaz-
oles 8a–8m. All the showed differences between experi-
mental and calculated lipophilicity values are probably
caused by interactions of the substituents with heteroa-
toms of benzoxazole nucleus in individual compounds.
Hydrophobicities (logP/ClogP values) of the studied
compounds 7a–7q and 8a–8m were calculated using
two commercially available programs and measured by
means of RP-HPLC determination of capacity factors
K with a subsequent calculation of log K. The results
are shown in Tables 1, 2 and illustrated in Figures 2
and 3.
5,7-Di-tert-butyl-2-(2-nitrophenyl)-benzoxazole
(7m)
possesses the lowest lipophilicity, as expected. The phe-
nolic derivative 7l shows the highest lipophilicity in con-
trast with our expectations, see Figure 2. Great
differences between experimental logK value and all
computed data log P/ClogP were also observed for
The results show that the experimentally determined logK
values correlate relatively poorly with all computed lipo-
philicity data using ChemOffice software or ACD/Log P
Table 1. Comparison of the determined logK values with the calculated lipophilicities (logP/ClogP) of the synthesized ring substituted 2-phenyl-5,7-
di-tert-butylbenzoxazoles 7a–7q
N
O
R
Compound
R
logK
logP/ClogPChemOffice
logPACD/Log P
7a
7b
7c
7d
7e
7f
H
3-Me
0.7515
0.8370
0.8350
0.9254
0.9118
1.0021
1.0005
0.9981
0.8507
0.8487
0.8696
1.0221
0.5331
0.7543
0.7555
0.8445
0.9245
6.35/7.165
6.83/7.664
6.83/7.664
6.90/7.8948
6.90/7.8948
7.17/7.7448
7.17/8.0448
7.17/8.0448
7.27/8.0774
7.27/8.0774
7.27/8.0774
5.96/6.1888
5.00/3.873
5.04/3.873
6.63/7.4398
6.79/7.76
7.39 0.59
7.85 0.59
7.85 0.59
8.18 0.60
8.15 0.60
7.74 0.63
8.36 0.63
8.33 0.63
8.36 0.62
8.44 0.62
8.36 0.62
7.66 0.61
6.89 0.60
7.35 0.60
7.81 0.60
8.23 0.61
9.81 0.66
4-Me
3-Cl
4-Cl
2-Br
7g
7h
7i
3-Br
4-Br
2-CF3
3-CF3
4-CF3
2-OH
2-NO2
4-NO2
4-N(CH3)2
4-SCH3
4-F-3-OC6H5
7j
7k
7l
7m
7n
7o
7p
7q
8.04/9.2228

J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5853
Table 2. Comparison of the determined logK values with the calculated lipophilicities (logP/ClogP) of the prepared compounds 8a–8m
N
R
O
Compound
R
logK
logP/ClogPChemOffice
logPACD/Log P
8a
8b
8c
8d
8e
8f
Styryl
0.8325
0.4065
0.5044
0.5726
0.6473
0.5726
0.6064
0.6234
0.6783
0.6972
0.6723
0.6870
0.3281
7.13/7.759
4.87/5.669
5.43/5.986
6.14/6.485
5.01/5.776
6.14/6.485
5.89/7.299
4.84/6.551
5.30/7.050
5.78/7.579
6.33/7.060
6.27/6.8500
4.37/3.824
7.93 0.59
5.29 0.61
6.31 0.60
6.77 0.60
6.13 0.60
6.77 0.60
7.31 0.84
6.18 0.61
6.64 0.61
7.17 0.61
6.99 0.61
7.07 0.84
4.40 0.60
1H-Pyrazol-3-yl
Pyridin-2-yl
Pyridin-3-yl
pyridin-4-yl
6-CH3-pyridin-2-yl
1H-Indol-3-yl
Furan-2-yl
8g
8h
8i
5-CH3-furan-2-yl
5-C2H5-furan-2-yl
Thiophen-2-yl
Thiophen-3-yl
ACH2OH
8j
8k
8l
8m
12.0
10.0
8.0
6.0
4.0
2.0
0.0
7m 7a 7n 7o 7c 7b 7p 7j
7i 7k 7e 7q 7d 7h 7g 7f
7l
Compounds
Log K Log P (ChemOffice) Clog P (ChemOffice) Log P (ACD/Log P)
Figure 2. Comparison of the calculated log P/ClogP data using the two programs with the experimentally found logK values of compounds 7a–7q.
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
8m
8b
8c
8f
8d
8g
8h
8e
8k
8i
8l
8j
7a
8a
Compounds
Log K Log P (ChemOffice) Clog P (ChemOffice) Log P (ACD/Log P)
Figure 3. Comparison of the calculated log P/ClogP data using the two programs with the experimentally found logK values of compounds 8a–8l
and 7a.

5854
J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5,7-di-tert-butyl-2-(4-fluoro-3-phenoxyphenyl)-benzoxaz-
ole (7q). According to the calculations compound 7q
shows the highest lipophilicity of all compounds in Fig-
ure 2, but according to the experiment this disubstituted
derivative shows lipophilicity comparable with chloro
derivatives 7e and 7d, it seems to be much more
hydrophilic.
pyridine series, but in accordance with logK is less
lipophilic than 5,7-di-tert-butyl-2-(pyridin-3-yl)-benz-
oxazole (8d), see Figure 3. Contrary to our expecta-
tions, all computed data for 5,7-di-tert-butyl-2-(1H-
indole-3-yl)-benzoxazole (8g) show great differences
from experimental logK value. According to the calcu-
lations, the compound 8g shows the highest lipophilic-
ity of all heterocyclic substituents, but in accordance
with the experiment the compound 8g shows medium
lipophilicity in the series of benzoxazoles 8a–8m.
The program ChemOffice does not resolve C(2)/C(3)/C(4)
substitutions in phenyl ring. According to the program
ACD/Log P lipophilicity of the compounds substituted
by CF3, Cl, Br groups in phenyl ring increases
C(2) < C(4) < C(3), but experimental logK for these benz-
oxazole derivatives increases C(4) < C(3) < C(2). This fact
is not in good agreement with calculated data accord-
ing to the program ACD/Log P. On the other hand,
position derivatives of CH3 or NO2 moieties possess
similar log P (ACD/LogP) to log K. As mentioned
above, great differences were found for 2-OH moiety
probably because of intramolecular interactions of
C(2) phenolic moiety with heteroatoms of benzoxazole
nucleus.
2.3. Biological activity
All the prepared benzoxazole derivatives were tested
for their antituberculotic activity at the GWL Han-
sen’s Disease Center (Colorado State University) with-
in the Tuberculosis Antimicrobial Acquisition and
Coordinating Facility (TAACF) screening program
for the discovery of novel drugs for treatment of
TB. Primary screening was conducted at a single con-
centration, 6.25 lg/mL against M. tuberculosis H37Rv
(ATCC2729) in BACTEC 12B medium using a broth
microdilution assay, the Microplate Almar Blue Assay
(MABA). Compounds affecting >90% inhibition in the
primary screening (MIC < 6.25 lg/mL) were evaluated
further. The active compounds were re-tested by serial
dilution beginning at the concentration of 6.25 lg/mL
against M. tuberculosis H37Rv to determine the actual
minimum concentration (MIC) in the BACTEC 460
radiometric system and BACTEC 12B medium. The
MIC is defined as the lowest concentration affecting
a reduction in fluorescence of 90% relative to controls.
Figure 3 shows that (5,7-di-tert-butyl-benzoxazol-2-yl)-
methanol (8m) possesses the lowest logK, whereas styryl
derivative 8a possesses the highest lipophilicity, as
expected. The pyrazole derivative 8b shows the lowest
lipophilicity parameter logK of all heteroaromatic sub-
stituents, whereas 5,7-di-tert-butyl-2-(5-ethylfuran-2-yl)-
benzoxazole (8j) possesses the highest lipophilicity
among heterocycle substituted benzoxazoles 8b–8l dis-
cussed in Figure 3. Great differences between logK
and calculated logP/ClogP were also observed for 8d,
8e and 8g.
The results are reported in Table 3 and show an interest-
ing activity for several compounds. Compounds 7f, 7h,
7l, 7o, 8a–8c, 8e exhibited an inhibitory effect against
M. tuberculosis at a concentration of 6.25 lg/mL or low-
er in the range 50–100%. Five most active compounds
7f, 7h, 8a, 8c, 8e were evaluated to determine the MIC
and their activity against opportunistic pathogen Myco-
bacterium kansasii and two strains of Mycobacterium
avium in comparison with isoniazide (INH).15 Results
of the latter tests are also reported in Table 3. Schiff
bases 5f, 5h, 6a, 6c, 6e of the most active benzoxazoles
showed much lower activity against M. tuberculosis,
the range 14–21% indicates benzoxazole structure’s
necessity.
As expected, the dependence between logK and the
length of the alkyl substituents in compounds 8h–8j
and 8c, 8f (H, CH3, C2H5) is approximately linear.
LogK values of 2-pyridinyl 8c, 3-pyridinyl 8d and 4-
pyridinyl 8e derivatives show also approximate linear-
ity as well as 2-thiophenyl 8k and 3-thiophenyl 8l
derivatives. The 4-pyridinyl derivative 8e is the most
lipophilic compound of the pyridinyl benzoxazoles in
contrast to computed data. Great differences were
observed for lipophilicity of 6-methylpyridin-2-yl
derivative 8f. According to the computed data
this compound shows the highest lipophilicity of
Table 3. In vitro biological properties of the selected 2-substituted 5,7-di-tert-butylbenzoxazoles: antimycobacterial activities against Mycobacterium
tuberculosis and atypical strains Mycobacterium kansasii and Mycobacterium avium in comparison with standard isoniazide (INH) as well as
cytotoxicity of the compounds on HCT-8 cells
Compound
M. tuberculosis H37Rv
M. kansasii 235/80
M. avium 80/72
M. avium
152/74
Cytotoxicity
IC50 (lg/mL)
MIC (lg/mL)
Inhibition (%)
7d
14d
21d
21d
28d
21d
28d
7f
6.25
6.25
99
94
>128
>128
—
>128
>128
—
>128
>128
—
>128
>128
—
>128
>128
—
>128
>128
—
>128
>128
—
53.6
80.4
—
7h
7l
>6.25
>6.25
3.13
70
54
7o
8a
8b
8c
—
—
—
—
—
—
—
—
902.2
—
100
72
>128
—
32
>128
—
64
>128
—
64
>128
—
64
>128
—
64
>128
—
64
>128
—
64
>6.25
>6.25
6.25
79
32.9
39.3
>100
8e
INH
100
100
32
>128
32
>128
32
>128
32
>128
32
>128
32
>128
32
>128
0.02

J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5855
120
100
80
60
40
20
0
7f
8e
8a
7h
8c
8b
7l
7o
0
0.2
0.4
0.6
Log K value
0.8
1
1.2
Figure 4. Dependence between growth inhibition (%) of M. tuberculosis H37Rv and the logarithm of the retention factor (logK) of the studied most
active compounds.
The cytotoxicity to human intestinal cells HCT-8 of the
most active benzoxazoles 7f, 7h, 8a, 8c, 8e and isoniazide
as a reference drug was determined using MTT assay.
The IC50 values of all the evaluated compounds are
shown in Table 3. Among the tested compounds, 8e
and 8c were the most toxic to the HCT-8 cells with
low IC50 values. In contrast, the compound 8a was the
least toxic, revealing considerably higher IC50 value than
the others. Toxicity of isoniazide to HCT-8 cells was not
observed up to a concentration of 100 lg/mL. These
data show for the most active styryl derivative 8a MIC
3.13 lg/mL and the lowest toxicity to human intestinal
cells HCT-8 (IC50 = 902.2 lg/mL).
3. Conclusion
The 30 2-substituted 5,7-di-tert-butylbenzoxazoles were
prepared by means of various synthetic pathways. In
addition, 28 intermediates were described. All final
compounds and some representative Schiff bases were
characterized by analytical and spectroscopic data.
Lipophilicity of the final compounds was determined
by means of RP-HPLC and the correlation between
RP-HPLC retention parameter logK and logP data
calculated in various ways was discussed. Several com-
pounds showed high antituberculotic activity. 5,7-Di-
tert-butyl-2-(pyridin-4-yl)-benzoxazole (8e) and espe-
cially 5,7-di-tert-butyl-2-styrylbenzoxazole (8a) showed
the highest in vitro antituberculotic activities. The anti-
mycobacterial activity seems to be independent of lipo-
philicity and dependent on chemical structure.
From analysis of the data reported in Table 3 it can be
generally deduced that bromine substituent in positions
C(2) and C(4) has been found as the most active phenyl
derivative. Hydrophilic electron-withdrawing groups
CF3, NO2, OH, SCH3 or electron-releasing group CH3,
N(CH3)2 with both lipophilic and hydrophilic properties
are ineffective. 5,7-Di-tert-butyl-2-(pyridin-4-yl)-benzox-
azole (8e) has been found as the most efficient heterocyclic
derivative with 100% of inhibition of M. tuberculosis
growth at 6.25 lg/mL as well as with the best activity also
against M. kansasii and both strains of M. avium. In the
light of the above results we can conclude that the 5,7-
di-tert-butyl-benzoxazoles appear to be useful for antitu-
bercular agents, the most active 8a has even the lowest
cytotoxicity. The highest cytotoxicity was found to corre-
late with the activity against nontuberculotic mycobacte-
rial strains.
4. Experimental
4.1. Chemistry
4.1.1. Instrumentation and chemicals. The chemicals
were purchased from Aldrich. Melting points (uncor-
rected) were determined on a Kofler block. Elemental
analyses were performed on an automatic microanaly-
ser CHNS-O CE instrument (FISONS EA 1110,
Milano, Italy). UV spectra (k, nm) of intermediates
were measured on a Polarimeter ADP 220 (BS Belling-
ham Stanley Ltd) in ethanol. UV spectra (k, nm) of the
final compounds were determined on a Waters Photo-
diode Array Detector 2996 (Waters Corp., Milford,
MA, USA) in ca 8 · 10ꢀ4 mol methanolic solution
and loge (the logarithm of molar absorption coefficient
e) was calculated for the absolute maximum kmax of
individual target compounds. Infrared spectra were
recorded in Nicolet Impact 400 spectrometer in KBr
pellets, Nujol mulls or CHCl3 solutions. NMR spectra
were measured in CDCl3 or DMSO-d6 solutions at
ambient temperature on a Varian Mercury-VxBB 300
Figure 4 describes the dependence between in vitro
growth inhibition (%) of M. tuberculosis and the loga-
rithm of the retention factor (logK) of the most active
compounds 7f, 7h, 7l, 7o and 8a–8c, 8e. Generally, it
could be assumed that the higher lipophilicity of the
compounds results in positive effect for antituberculotic
activity, but a straight correlation between the activity
and lipophilicity of 2-substituted 5,7-di-tert-butylbenz-
oxazoles was not found.

5856
J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
1
spectrometer (299.95 MHz for H and 75.43 MHz for
13C), Varian Comp. (Palo Alto, CA, USA). The chem-
ical shifts d are given in ppm related to tetramethylsi-
lane (TMS) as internal standard. The coupling
constants (J) are reported in Hz. Mass spectra were
measured on ABI/MSD SCIEX API 3000TM LC/MS/
MS System (MSD SCIEX, Concord, Ont., Canada).
The reactions were monitored and the purity of the
products was checked by TLC (Silufol UV 254, Kava-
lier Votice, Czech Republic and Merck TLC plates Sil-
ica gel 60 F254, aluminium back) using two types of
developing solvents S1 (petroleum ether/EtOAc 9:1)
and S2 (toluene/EtOAc 4:1). The plates were visualized
using UV light. Preparative thin-layer chromatography
was carried out on 20 · 20 cm plates coated by silica
gel. Silica gel 60 (0.015–0.040 mm, Merck, Darmstadt,
Germany) was used for column chromatography.
4.1.4. Data of prepared compounds
4.1.4.1. 2,4-Di-tert-butyl-6-nitrophenol (1). 2,4-Di-tert-
butylphenol 19.2 g (0.093 mol) was dissolved in glacial
AcOH (240 mL) and concd nitric acid (6.6 mL,
0.183 mol) was added with stirring at 25 ꢁC. A rapid
change from yellow to deep red was observed. After
50 min of stirring, water (150 mL) was added. NaHCO3
was added to the orange solution until pH was raised to
5; a brown-red oily precipitate was extracted into CHCl3,
dried over anhydrous Na2SO4 and concentrated to the
red oil. Continuous column chromatography (silica gel,
hexane) separated the product mixture into two main
components. Yield 90%. Anal. Calcd for C14H21NO3
(251.32): 66.91% C, 8.42% H, 5.57% N. Found: 66.79%
C, 8.18% H, 5.42% N. Mp 59–60 ꢁC, mp 59–60 ꢁC.25
UV (nm), kmax: 220, 289, 372. IR (KBr) cmꢀ1: 2955,
2911, 2871 (C–H), 1541 (N–O), 1460 (C@C), 1366
(CH3), 1317, 1272, 1236, 1202, 1179, 1139, 924, 886,
773, 709, 641. MS: the peak 251.5 was found.
The purity of the final compounds was checked by
HPLC, see Section 2.2. The detection wavelength
210 nm was chosen. Peaks in the chromatogram of
the solvent (blank) were deducted from peaks in the
chromatogram of the sample solution. A purity of
the individual compounds was determined from area
peaks in the chromatogram of the sample solution.
4.1.4.2. 2-tert-Butyl-4,6-dinitrophenol (2). Yield 14%.
Anal. Calcd for C10H12N2O5 (240.22): 50.00% C,
5.04% H, 11.66% N. Found: 49.78% C, 4.98% H,
11.50% N. Mp 124–126 ꢁC. Mp 124–125 ꢁC.26 TLC: Rf
(S1) 0.07. IR (KBr) cmꢀ1: 2975, 2907, 2880 (C–H),
1600, 1550, 1460, 1440 (C@C), 1395, 1365 (CH3),
1335, 1270 (NO2), 1150 (C–O), 935, 920, 735, 710 (Ar–
4.1.2. General procedure for Schiff base synthesis (com-
pounds 5a–5q and 6b–6l). The appropriately substituted
aldehyde (0.01 mol) was added to the mixture of amino-
phenol 3 (0.01 mol) and boiled for 2 h. After evapora-
tion, residue was crystallized from the mixture EtOH/
water.
1
H). MS: the peak 240.3 was found. H NMR (CDCl3),
d: 12.01 (s, 1H, OH), 8.96 (d, 1H, J = 2.80 Hz, Ar),
8.46 (d, 1H, J = 2.80 Hz, Ar), 1.50 (s, 9H, CH3). 13C
NMR (CDCl3), d: 158.8, 142.8, 139.1, 133.0, 128.5,
119.5, 36.1, 29.0.
4.1.3. General procedure for benzoxazole synthesis (com-
pounds 7a–7q and 8a–8m). Method A—Schiff base cycliza-
tion. (AcO)4Pb or PbO2 (0.003 mol) was added to the
mixture of the corresponding Schiff base (0.003 mol) in
glacial AcOH (12 mL) and after 1 h stirring at the labora-
tory temperature the reaction mixture was evaporated,
dissolved in EtOAc (25 mL) and extracted with water
(25 mL), 5% NaHCO3 (15 mL) and water (25 mL).
EtOAc extract was dried over anhydrous Na2SO4 and
evaporated to dryness. Purification was done by column
chromatography on Silica gel using EtOAc/petroleum
ether 1:9 as an eluent.
4.1.4.3. 2-Amino-4,6-di-tert-butylphenol (3). Prepared
from 3,5-di-tert-butyl-1,2-benzoquinone (0.01 mol) by
reaction with ammonia (20 mL) and subsequent addi-
tion of powdered NaBH4 in MeOH (50 mL) according
to the Ref. 27 Yield 80%. Anal. Calcd for C14H23NO
(221.35): 75.97% C, 10.47% H, 6.33% N. Found:
75.63% C, 10.15% H, 6.34% N. Mp 171–172 ꢁC. Mp
170 ꢁC.28 TLC: Rf (S1) 0.26. UV (nm), kmax: 210,
287, 342. (KBr) cmꢀ1: 3395, 3383 (N–H), 2950,
2900, 2860 (C–H), 1590, 1482 (C@C), 1392, 1364
(CH3), 909, 869, 833 (Ar–H). MS: the peak 221.5
was found. 1H NMR (CDCl3), d: 6.90 (d, 1H,
J = 2.30 Hz, Ar), 6.79 (d, 1H, J = 2.30 Hz, Ar), 1.40
(s, 9H, CH3), 1.27 (s, 9H, CH3). 13C NMR (CDCl3),
d: 144.2, 142.4, 135.5, 132.2, 116.0, 117.1, 34.7, 34.3,
31.7, 29.9.
Method B—used for synthesis of 7l. The mixture of ami-
nophenol 3 (0.01 mol) and salicylic acid (0.02 mol) was
heated in PPA (12 g) while stirring for 2.5 h. At the
end of the reaction period, the residue was poured into
ice-water and neutralized with excess of 10% NaOH
solution. After extraction with EtOAc, the EtOAc solu-
tion was dried over anhydrous Na2SO4 and evaporated
under reduced pressure. The residue was recrystallized
from EtOH/water.
4.1.4.4. 5,7,50,70-Tetra-tert-butyl-2,20-bi-benzoxazole
(4). Yield 13%. Anal. Calcd for C30H40N2O2 (460.66):
78.22% C, 8.75% H, 6.08% N. Found: 78.00% C,
8.74% H, 5.89% N. Mp 210–212 ꢁC. TLC: Rf (S2)
0.13. UV (nm), kmax: 202, 241, 333. IR (KBr) cmꢀ1
:
2961, 2908, 2871 (C–H), 1617 (C@N), 1482 (C@C),
1392, 1364 (CH3), 909, 869, 833 (Ar–H). MS: the peak
460.8 was found. H NMR (CDCl3), d: 7.80 (d, 2H,
J = 1.92 Hz, Ar), 7.44 (d, 2H, J = 1.92 Hz, Ar), 1.58 (s,
18H, CH3), 1.41 (s, 18H, CH3). 13C NMR (CDCl3), d:
151.9, 148.8, 147.4, 141.7, 134.6, 121.7, 115.3, 35.2,
34.6, 31.7, 30.0.
Method C—used for synthesis of 8m. Serine (1 mmol)
and DTBBQ (1 mmol) were dissolved in EtOH
(50 mL, 60%) and heated for 5 h at 50 ꢁC. The solvent
was removed and the residue separated by column chro-
matography with mixture of EtOAc/petroleum ether in
the appropriate ratio.
1

J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5857
4.1.4.5. 2,4-Di-tert-butyl-6-[(phenylmethylene)amino]-
phenol (5a). Yield 86%. Anal. Calcd for C21H27NO
(309.45): 81.51% C, 8.79% H, 4.53% N. Found:
81.43% C, 8.43% H, 8.67% N. Mp 85 ꢁC. Mp 85 ꢁC.28
TLC: Rf (S1) 0.46, RF (S2) 0.75. UV (nm), kmax: 209,
271, 364. IR (KBr) cmꢀ1: 3351 (O–H), 3066, 3034
(@C–H), 2961, 2904, 2869 (C–H), 1624, 1577 (C@N),
1475 (C@C), 1391, 1363 (CH3), 1075 (O–H), 874, 857,
751 (Ar–H). MS: the peak 309.6 was found.
Found: 65.18% C, 6.76% H, 3.56% N. Mp 124–126 ꢁC.
TLC: Rf (S1) 0.71, RF (S2) 0.93. UV (nm), kmax: 210,
270, 369. IR (KBr) cmꢀ1: 3393 (O–H), 2955, 2904,
2866 (C–H), 1619, 1562 (C@N), 1480 (C@C), 1390,
1362 (CH3), 1068 (O–H), 786 (Ar–H). MS: the peak
388.4 was found.
4.1.4.12. 2,4-Di-tert-butyl-6-{[(4-bromophenyl)methyl-
ene]amino}-phenol (5h). Yield 90%. Anal. Calcd for
C21H26BrNO (388.34): 64.95% C, 6.75% H, 3.61% N.
Found: 64.83% C, 6.38% H, 3.54% N. Mp 144–146 ꢁC.
TLC: Rf (S1) 0.64, RF (S2) 0.87. UV (nm), kmax: 208,
279, 369. IR (KBr) cmꢀ1: 3416 (O–H), 2957, 2904,
2867 (C–H), 1623, 1566 (C@N), 1478 (C@C), 1390,
1361(CH3), 1068 (O–H), 820 (Ar–H). MS: the peak
388.5 was found.
4.1.4.6. 2,4-Di-tert-butyl-6-{[(3-methylphenyl)methyl-
ene]amino}-phenol (5b). Yield 65%. Anal. Calcd for
C22H29NO (323.48): 81.69% C, 9.04% H, 4.33% N.
Found: 81.45% C, 8.98% H, 3.95% N. Mp 98–99 ꢁC.
TLC: Rf (S1) 0.77, RF (S2) 0.82. UV (nm), kmax: 206,
274, 361. IR (KBr) cmꢀ1: 3424 (O–H), 2955, 2904,
2866 (C–H), 1619, 1583 (C@N), 1478 (C@C), 1390,
1361 (CH3), 1120 (O–H), 865, 788, (Ar–H). MS: the
peak 323.6 was found.
4.1.4.13. 2,4-Di-tert-butyl-6-{[(2-trifluoromethylphe-
nyl)methylene]amino}-phenol (5i). Yield 73%. Anal.
Calcd for C22H26F3NO (377.20): 70.01% C, 6.94% H,
3.71% N. Found: 69.78% C, 6.80% H, 3.80% N. Mp
83–85 ꢁC. TLC: Rf (S1) 0.51, Rf (S2) 0.95. UV (nm),
4.1.4.7. 2,4-Di-tert-butyl-6-{[(4-methylphenyl)methyl-
ene]amino}-phenol (5c). Yield 79%. Anal. Calcd for
C22H29NO (323.48): 81.69% C, 9.04% H, 4.33% N.
Found: 81.5% C, 8.88% H, 4.05% N. Mp 137–139 ꢁC.
TLC: Rf (S1) 0.77, RF (S2) 0.82. UV (nm), kmax: 206,
279, 357. IR (KBr) cmꢀ1: 3408 (O–H), 2954, 2903,
2866 (C–H), 1620, 1570 (C@N), 1480 (C@C), 1390,
1362 (CH3), 1121 (O–H), 811, (Ar–H). MS: the peak
323.5 was found.
k
max: 230, 268, 364. IR (KBr) cmꢀ1: 3355 (O–H), 2965,
2906, 2870 (C–H), 1625 (C@N), 1482 (C@C), 1393,
1364 (CH3), 1332 (CF3), 1067 (O–H), 866, 799 (Ar–H).
MS: the peak 377.3 was found.
4.1.4.14. 2,4-Di-tert-butyl-6-{[(3-trifluoromethylphe-
nyl)methylene]amino}-phenol (5j). Yield 38%. Anal.
Calcd for C22H26F3NO (377.20): 70.01% C, 6.94% H,
3.71% N. Found: 69.81% C, 6.56% H, 3.53% N. Mp
89–94 ꢁC. TLC: Rf (S1) 0.78, Rf (S2) 0.85. UV (nm),
4.1.4.8. 2,4-Di-tert-butyl-6-{[(3-chlorophenyl)methyl-
ene]amino}-phenol (5d). Yield 75%. Anal. Calcd for
C21H24ClNO (341.87): 73.78% C, 7.08% H, 4.10% N.
Found: 73.67% C, 6.87% H, 4.02% N. Mp 106–108 ꢁC.
TLC: Rf (S1) 0.71, RF (S2) 0.93. UV (nm), kmax: 208,
270, 371. IR (KBr) cmꢀ1: 3386 (O–H), 2955, 2904,
2866 (C–H), 1618, 1569 (C@N), 1478 (C@C), 1390,
1361 (CH3), 1074 (O–H), 788, 683 (Ar–H). MS: the peak
341.9 was found.
k
max: 219, 268, 364. IR (KBr) cmꢀ1: 3405 (O–H), 2964,
2907, 2870 (C–H), 1625 (C@N), 1481 (C@C), 1393,
1363 (CH3), 1332 (CF3), 1068 (O–H), 866, 800 (Ar–H).
MS: the peak 377.3 was found.
4.1.4.15. 2,4-Di-tert-butyl-6-{[(4-trifluoromethylphe-
nyl)methylene]amino}-phenol (5k). Yield 64%. Anal.
Calcd for C22H26F3NO (377.20): 70.01% C, 6.94% H,
3.71% N. Found: 70.06% C, 6.67% H, 3.46% N. Mp
124–126 ꢁC. TLC: Rf (S1) 0.80, Rf (S2) 0.88. UV (nm),
4.1.4.9. 2,4-Di-tert-butyl-6-{[(4-chlorophenyl)methyl-
ene]amino}-phenol (5e). Yield 89%. C21H24ClNO
(341.87): 73.78% C, 7.08% H, 4.10% N. Found:
73.54% C, 7.17% H, 3.79% N. Mp 138–140 ꢁC. TLC:
Rf (S1) 0.71, Rf (S2) 0.92. UV (nm), kmax: 210, 277,
364. IR (KBr) cmꢀ1: 3424 (O–H), 2958, 2905, 2868
(C–H), 1624, 1591 (C@N), 1478 (C@C), 1362 (CH3),
1087 (O@H), 824, (Ar–H). MS: the peak 341.9 was
found.
k
max: 209, 230, 287, 378. IR (KBr) cmꢀ1: 3397 (O–H),
2960, 2906, 2869 (C–H), 1624 (C@N), 1479 (C@C),
1391, 1362 (CH3), 1065 (O–H), 836, (Ar–H). MS: the
peak 377.4 was found.
4.1.4.16. 2,4-Di-tert-butyl-6-{[(2-hydroxyphenyl)meth-
ylene]amino}-phenol (5l). Yield 78%. Anal. Calcd for
C21H27NO2 (325.45): 77.50% C, 8.36% H, 4.30% N.
Found: 77.15% C, 8.06% H, 4.15% N. Mp 124–125 ꢁC.
Mp 114–115 ꢁC.29 TLC: Rf (S1) 0.52, RF (S2) 0.82. UV
(nm), kmax: 207, 269, 358. IR (KBr) cmꢀ1: 3503 (O–
H), 2957, 2906, 2869 (C–H), 1608, 1571 (C@N), 1479
(C@C), 1391, 1362 (CH3), 1151 (O–H), 866, 756
(Ar–H). MS: the peak 325.5 was found.
4.1.4.10. 2,4-Di-tert-butyl-6-{[(2-bromophenyl)methyl-
ene]amino}-phenol (5f). Yield 58%. Anal. Calcd for
C21H26BrNO (388.34): 64.95% C, 6.75% H, 3.61% N.
Found: 65.68% C, 6.46% H, 3.33% N. Mp 122–124 ꢁC.
TLC: Rf (S1) 0.75, RF (S2) 0.90. UV (nm), kmax: 207,
275, 370. IR (KBr) cmꢀ1: 3374 (O–H), 2953, 2906,
2865 (C–H), 1614, 1560 (C@N), 1480 (C@C), 1387,
1361 (CH3), 1027 (O–H), 867 (Ar–H). MS: the peak
388.5 was found.
4.1.4.17. 2,4-Di-tert-butyl-6-{[(2-nitrophenyl)methy-
lene]amino}-phenol (5m). Yield 87%. Anal. Calcd for
C21H26N2O3 (354.44): 71.16% C, 7.39% H, 7.90% N.
Found: 71.06% C, 7.15% H, 7.96% N. Mp 127–128 ꢁC.
TLC: Rf (S1) 0.32, Rf (S2) 0.88. UV (nm), kmax: 204, 275,
379. IR (KBr) cmꢀ1: 3425 (O–H), 2957, 2907, 2868
4.1.4.11. 2,4-Di-tert-butyl-6-{[(3-bromophenyl)methyl-
ene]amino}-phenol (5g). Yield 56%. Anal. Calcd for
C21H26BrNO (388.34): 64.95% C, 6.75% H, 3.61% N.

5858
J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
(C–H), 1610, 1569 (C@N), 1527 (NO2), 1481 (C@C),
1390, 1362 (CH3), 1345 (NO2), 1065 (O–H), 864, 785,
4.1.4.23. 2,4-Di-tert-butyl-6-[(1H-pyrazol-3-ylmethyl-
ene)amino]-phenol (6b). Yield 80%. Anal. Calcd for
C18H25N3O (299.41): 72.21% C, 8.42% H, 14.03% N.
Found: 72.08% C, 8.06% H, 13.88% N. Mp 174–
1
744 (Ar–H). MS: the peak 354.6 was found. H NMR
(300 MHz, CDCl3), d: 9.16 (s, 1H, OH), 8.28–8.24 (m,
1H, H30), 8.09–8.04 (m, 1H, H60), 7.78–7.71 (m, 1H,
H50), 7.66–7.51 (m, 2H, 4H0, NCH), 7.33 (d, 1H,
J = 2.06 Hz, H3), 7.23 (d, 1H, J = 2.06 Hz, H5), 1.46 (s,
9H, CH3), 1.34 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 150.7, 149.4, 141.8, 135.7, 133.9, 133.4,
131.1, 130.8, 129.5, 124.8, 124.7, 110.3, 35.0, 34.6, 31.5,
29.4.
176 ꢁC. TLC: Rf (S1) 0.04, Rf (S2) 0.32. UV (nm), kmax
:
201, 227, 273, 356. IR (KBr) cmꢀ1: 3428 (O–H), 2958,
2907, 2868 (C–H), 1629 (C@N), 1481 (C@C), 1391,
1362 (CH3), 1052 (O–H), 868, 821, 794, 764 (Ar–H).
MS: the peak 299.6 was found.
4.1.4.24. 2,4-Di-tert-butyl-6-[(pyridin-2-ylmethylene)-
amino]-phenol (6c). Yield 56%. Anal. Calcd for
C20H26N2O (310.44): 77.38% C, 8.44% H, 9.02% N.
Found: 77.22% C, 8.04% H, 8.86% N. Mp 159–161 ꢁC.
TLC: Rf (S1) 0.15, Rf (S2) 0.35. UV (nm), kmax: 209,
226, 295, 381. IR (KBr) cmꢀ1: 3424 (O–H), 2955,
2904, 2866 (C–H), 1623, 1590 (C@N), 1481 (C@C),
1389, 1363 (CH3), 1151 (O–H), 881, 864 (Ar–H). MS:
the peak 310.6 was found. 1H NMR (300 MHz, CDCl3),
d: 8.87 (s, 1H, NCH), 8.72 (ddd, 1H, J = 4.94 Hz,
J = 1.65 Hz, J = 1.10 Hz, Ar), 8.22 (dt, 1H,
J = 7.69 Hz, J = 1.10 Hz, Ar), 7.90-7.81 (m, 2H, Ar,
OH), 7.39 (ddd, 1H, J = 7.69 Hz, J = 4.94 Hz,
J = 1.10 Hz, Ar), 7.34–7.30 (m, 2H, Ar), 1.46 (s, 9H,
CH3), 1.33 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3),
d: 155.1, 154.1, 149.4, 149.3, 141.6, 137.2, 135.7 133.5,
125.1, 124.7, 121.4, 110.3, 35.0, 34.6, 31.5, 29.4.
4.1.4.18. 2,4-Di-tert-butyl-6-{[(4-nitrophenyl)methyl-
ene]amino}-phenol (5n). Yield 77%. Anal. Calcd for
C21H26N2O3 (354.44): 71.16% C, 7.39% H, 7.90% N.
Found: 70.96% C, 7.08% H, 7.87% N. Mp 185–187 ꢁC.
Mp 169–170 ꢁC.29 TLC: Rf (S1) 0.39, Rf (S2) 0.87. UV
(nm), kmax: 202, 285, 346, 400. IR (KBr) cmꢀ1: 3424
(O–H), 2957, 2906, 2869 (C–H), 1623, 1599 (C@N),
1522 (NO2), 1479 (C@C), 1390, 1362 (CH3), 1343
(NO2), 1107 (O–H), 842, 747 (Ar–H). MS: the peak
354.6 was found.
4.1.4.19. 2,4-Di-tert-butyl-6-{[(4-dimethylaminophe-
nyl)methylene]amino}-phenol (5o). Yield 85%. Anal.
Calcd for C23H32N2O (352.51): 78.36% C, 9.15% H,
7.95% N. Found: 78.34% C, 9.04% H, 7.75% N. Mp
156–158 ꢁC. Mp 158–159 ꢁC.29 TLC: Rf (S1) 0.32, RF
(S2) 0.75. UV (nm), kmax: 204, 242, 379. IR (KBr)
cmꢀ1: 3425 (O–H), 2957, 2904, 2867 (C–H), 1598,
1552 (C@N), 1478 (C@C), 1363 (CH3), 1166 (O–H),
816, 764 (Ar–H). MS: the peak 352.6 was found.
4.1.4.25. 2,4-Di-tert-butyl-6-[(pyridin-3-ylmethylene)-
amino]-phenol (6d). Yield 80%. Anal. Calcd for
C20H26N2O (310.44): 77.38% C, 8.44% H, 9.02% N.
Found: 76.98% C, 8.32% H, 8.79% N. Mp 180–182 ꢁC.
TLC: Rf (S1) 0.20, Rf (S2) 0.65. UV (nm), kmax: 228,
269, 375. IR (KBr) cmꢀ1: 3424 (O–H), 2957, 2906,
2868 (C–H), 1623 (C@N), 1481 (C@C), 1391, 1362
(CH3), 1026 (O–H), 806 (Ar–H). MS: the peak 310.6
was found. 1H NMR (300 MHz, CDCl3), d: 9.07 (d,
1H, J = 1.64 Hz, Ar), 8.75 (s, 1H, NCH), 8.71 (dd,
1H, J = 4.67 Hz, J = 1.64 Hz, Ar), 8.32 (dt, 1H,
J = 7.97 Hz, J = 1.65 Hz, Ar), 7.66 (br s, 1H, OH),
7.46 (dd, 1H, J = 7.96 Hz, J = 4.67 Hz, Ar), 7.31 (d,
1H, J = 2.19 Hz, Ar), 7.22 (d, 1H, J = 2.20 Hz, Ar),
1.46 (s, 9H, CH3), 1.35 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d: 152.1, 151.3 150.2, 149.0, 141.6,
135.6, 135.0 133.9, 131.9, 124.4, 124.0, 109.8, 35.0,
34.6, 31.6, 29.4.
4.1.4.20. 2,4-Di-tert-butyl-6-{[(4-methylsulfanylphe-
nyl)methylene]amino}-phenol (5p). Yield 68%. Anal.
Calcd for C22H28NOS (354.53): 74.53% C, 7.96% H,
3.95% N. Found: 74.33% C, 7.80% H, 3.84% N. Mp
139–140 ꢁC. TLC: Rf (S1) 0.32, RF (S2) 0.75. UV (nm),
k
max: 207, 324, 364. IR (KBr) cmꢀ1: 3443 (O–H), 2957,
2918, 2867 (C–H), 1618, 1592 (C@N), 1478 (C@C),
1390, 1361 (CH3), 1062 (O–H), 877, 815 (Ar–H). MS:
the peak 354.6 was found.
4.1.4.21. 2,4-Di-tert-butyl-6-{[(3-fluoro-4-phenoxyphe-
nyl)methylene]amino}-phenol (5q). Yield 62%. Anal.
Calcd for C27H30FNO2 (419.54): 77.30% C, 7.21%
H, 3.34% N. Found: 77.06% C, 6.89% H, 3.01% N.
Mp 114 ꢁC. TLC: Rf (S1) 0.18, Rf (S2) 0.94. UV
(nm), kmax: 204, 269, 361. IR (KBr) cmꢀ1: 3397 (O–
H), 2957, 2905, 2867 (C–H), 1628, 1586 (C@N),
1509, 1490, 1425 (C@C), 1391, 1362 (CH3), 1105
(O–H), 870, 818, 750 (Ar–H). MS: the peak 419.6
was found.
4.1.4.26. 2,4-Di-tert-butyl-6-[(pyridin-4-ylmethylene)-
amino]-phenol (6e). Yield 79%. Anal. Calcd for
C20H26N2O (310.44): 77.38% C, 8.44% H, 9.02% N.
Found: 77.40% C, 8.26% H, 8.76% N. Mp 138–140 ꢁC.
TLC: Rf (S1) 0.08, Rf (S2) 0.30. UV (nm), kmax: 228,
290, 384. IR (KBr) cmꢀ1: 3424 (O–H), 2958, 2906,
2868 (C–H), 1636, 1601 (C@N), 1480 (C@C), 1391,
1362 (CH3), 1052 (O–H), 814 (Ar–H). MS: the peak
310.6 was found. 1H NMR (300 MHz, CDCl3), d:
8.79–8.74 (m, 2H, Ar), 8.69 (s, 1H, NCH), 7.82–7.77
(m, 2H, Ar), 7.70 (br s, 1H, OH), 7.34 (d, 1H,
J = 2.00 Hz, Ar), 7.23 (d, 1H, J = 2.00 Hz, Ar), 1.45 (s,
9H, CH3), 1.35 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 152.5, 150.1, 149.5, 143.1, 141.7, 135.9,
133.4, 125.1, 122.1, 109.9, 35.0, 34.6, 31.5, 29.3.
4.1.4.22. 2,4-Di-tert-butyl-6-[(3-phenylallylidene)ami-
no]-phenol (6a). Yield 80%. Anal. Calcd for C23H29NO
(335.49): 82.34% C, 8.71% H, 4.18% N. Found:
82.30% C, 8.54% H, 3.97% N. Mp 143 ꢁC. TLC: Rf
(S1) 0.56, Rf (S2) 0.75. UV (nm), kmax: 207, 306, 375.
IR (KBr) cmꢀ1: 3259 (O–H), 2953, 2906, 2866 (C–H),
1625, 1586 (C@N), 1478 (C@C), 1395, 1360 (CH3),
1162 (O–H), 865 (Ar–H). MS: the peak 335.6 was found.

J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5859
4.1.4.27. 2,4-Di-tert-butyl-6-{[(6-methylpyridin-2-yl)-
methylene]amino}-phenol (6f). Yield 52%. Anal. Calcd
for C21H28N2O (324.46): 77.74% C, 8.70% H, 8.63%
N. Found: 77.56% C, 8.46% H, 8.35% N. Mp 176–
(CH3), 1018 (O–H), 801 (Ar–H). MS: the peak 325.6
was found.
4.1.4.32. 2,4-Di-tert-butyl-6-[(thiophen-2-ylmethylene)-
amino]-phenol (6k). Yield 62%. Anal. Calcd for
C19H25NOS (315.47): 72.34% C, 7.99% H, 4.44% N.
Found: 72.01% C, 7.50% H, 4.07% N. Mp 90–92 ꢁC.
TLC: Rf (S1) 0.68, RF (S2) 0.89. UV (nm), kmax: 211,
275, 307, 372. IR (KBr) cmꢀ1: 3406 (O–H), 2957,
2905, 2867 (C–H), 1612 (C@N), 1480 (C@C), 1391,
1362 (CH3), 1045 (O–H), 861, 813 (Ar–H). MS: the peak
178 ꢁC. TLC: Rf (S1) 0.11, Rf (S2) 0.39. UV (nm), kmax
:
208, 299, 379. IR (KBr) cmꢀ1: 3406 (O–H), 2952, 2903,
2867 (C–H), 1624, 1595 (C@N), 1462 (C@C), 1388, 1362
(CH3),1025 (O–H), 874 (Ar–H). MS: the peak 324.6 was
found. 1H NMR (300 MHz, CDCl3), d: 8.85 (s, 1H,
NCH), 8.05 (d, 1H, J = 7.69 Hz, Ar), 7.83 (s, 1H,
OH), 7.71 (t, 1H, J = 7.69 Hz, Ar), 7.34 (d, 1H,
J = 2.20 Hz, Ar), 7.30 (d, 1H, J = 2.20 Hz, Ar), 7.24
(d, 1H, J = 7.69 Hz, Ar), 2.64 (s, 3H, CH3), 1.46 (s,
9H, CH3), 1.32 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 158.4, 156.0, 154.0, 149.2, 141.6, 136.9,
135.4, 133.6, 124.8, 124.4, 118.2, 110.3, 34.9, 34.6,
31.6, 29.4, 24.3.
1
315.6 was found. H NMR (300 MHz, CDCl3), d: 8.80
(s, 1H, NCH), 7.67 (br s, 1H, OH), 7.53–7.49 (m, 2H,
Ar), 7.25 (d, 1H, J = 2.20 Hz, Ar), 7.18 (d, 1H,
J = 2.20 Hz, Ar), 7.14 (dd, 1H, J = 4.94 Hz,
J = 3.85 Hz, Ar), 1.46 (s, 9H, CH3), 1.35 (s, 9H, CH3).
13C NMR (75 MHz, CDCl3), d: 148.6, 148.5, 143.2,
141.3, 135.2, 134,1, 131.9, 130.3, 127.9, 123.3, 109.9,
34.9, 34.6, 31.6, 29.4.
4.1.4.28. 2,4-Di-tert-butyl-6-[(1H-indol-3-ylmethylene)-
amino]-phenol (6g). Yield 35%. Anal. Calcd for
C23H28N2O (348.49): 79.27% C, 8.10% H, 8.04% N.
Found: 79.06% C, 7.87% H, 7.76% N. Mp 187–189 ꢁC.
TLC: Rf (S1) 0.08, Rf (S2) 0.60. UV (nm), kmax: 207,
225, 271, 354. IR (KBr) cmꢀ1: 3435 (O–H), 2957,
2905, 2867 (C–H), 1618, 1577 (C@N), 1478, 1457
(C@C), 1362 (CH3), 1106 (O–H), 867, 766, 746 (Ar–
H). MS: the peak 348.6 was found. 1H NMR
(300 MHz, CDCl3), d: 8.86 (s, 1H, NCH), 8.55–8.43
(m, 2H, Ar), 7.68 (br s, 1H, OH), 7.46–7.39 (m, 1H,
Ar), 7.37–7.28 (m, 2H, Ar), 7.23 (d, 1H, J = 2.20 Hz,
Ar), 7.18 (d, 1H, J = 2.20 Hz, Ar), 1.49 (s, 9H, CH3),
1.37 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3), d:
151.8, 147.8, 141.3, 136.9, 136.6, 134.6, 131.0, 124.9,
123.9, 122.3, 122.1, 121.7, 116.8, 111.4, 109.9, 34.9,
34.6, 31.7, 29.5.
4.1.4.33. 2,4-Di-tert-butyl-6-[(thiophen-3-ylmethylene)-
amino]-phenol (6l). Yield 60%. Anal. Calcd for
C19H25NOS (315.47): 72.34% C, 7.99% H, 4.44% N.
Found: 72.40% C, 7.68% H, 4.12% N. Mp 68–71 ꢁC.
TLC: Rf (S1) 0.79, RF (S2) 0.90. UV (nm), kmax: 212,
249, 355. IR (KBr) cmꢀ1: 3424 (O–H), 2957, 2904,
2867 (C–H), 1618, (C@N), 1480 (C@C), 1391, 1362
(CH3), 1074 (O–H), 867, 831 (Ar–H). MS: the peak
315.6 was found.
4.1.4.34. 5,7-Di-tert-butyl-2-phenylbenzoxazole (7a).
Method A. Yield 57%. Anal. Calcd for C21H25NO
(307.43): 82.04% C, 8.20% H, 4.56% N. Found:
82.10% C, 8.26% H, 4.54% N. Mp 59–60 ꢁC. HPLC
purity 98.98%. TLC: Rf (S1) 0.52, Rf (S2) 0.80. UV
(nm), kmax/log e: 207/3.68, 239, 296. IR (KBr) cmꢀ1
:
4.1.4.29. 2,4-Di-tert-butyl-6-[(furan-2-ylmethylene)-
amino]-phenol (6h). Yield 61%. C19H25NO2 (299.41):
76.22% C, 8.42% H, 4.68% N. Found 75.97% C,
8.22% H, 4.44% N. Mp 88–90 ꢁC. TLC: Rf (S1) 0.47,
Rf (S2) 0.88. UV (nm), kmax: 208, 289, 364. IR (KBr)
cmꢀ1: 3424 (O–H), 2955, 2906, 2868 (C–H), 1629,
1574 (C@N), 1475 (C@C), 1391, 1361 (CH3), 1017 (O–
H), 884, 865, 820 (Ar–H). MS: the peak 299.5 was
found.
2957, 2907, 2868 (C–H), 1624, 1557 (C@N), 1482
(C@C), 1391, 1362 (CH3), 1060 (C–O–C), 863, 706
(Ar–H). MS: the peak 307.5 was found. 1H NMR
(300 MHz, CDCl3), d: 8.30–8.23 (m, 2H, Ar), 7.67 (d,
1H, J = 1.79 Hz, Ar), 7.57–7.50 (m, 3H, Ar), 7.32 (d,
1H, J = 1.79 Hz, Ar), 1.56 (s, 9H, CH3), 1.41 (s, 9H,
CH3). 13C NMR (75 MHz, CDCl3), d: 162.5, 147.7,
146.9, 142.3, 133.7, 131.2, 128.9, 127.5, 127.4, 119.5,
114.2, 35.1, 34.5, 31.8, 30.0.
4.1.4.30.
2,4-Di-tert-butyl-6-{[(5-methylfuran-2-yl)-
4.1.4.35. 5,7-Di-tert-butyl-2-(3-methylphenyl)-benzox-
azole (7b). Method A. Yield 56%. Anal. Calcd for
C22H27NO (321.46): 82.20% C, 8.47% H, 4.36% N.
Found: 82.14% C, 8.53% H, 4.29% N. Mp 69–72 ꢁC.
HPLC purity 99.92%. TLC: Rf (S1) 0.77, Rf (S2)
0.87. UV (nm), kmax/loge: 204/3.62, 243, 305. IR
(KBr) cmꢀ1: 2957, 2908, 2870 (C–H), 1608, 1592
(C@N), 1479 (C@C), 1401, 1363 (CH3), 1076 (C–O–
C), 868, 790, 723 (Ar–H). MS: the peak 321.6 was
found. 1H NMR (300 MHz, CDCl3), d: 8.11–8.02
(m, 2H, Ar), 7.66 (d, 1H, J = 1.92 Hz, Ar), 7.42 (t,
1H, J = 7.69 Hz, Ar), 7.37–7.32 (m, 1H, Ar), 7.31 (d,
1H, J = 1.92 Hz, Ar), 2.47 (s, 3H, CH3), 1.56 (s, 9H,
CH3), 1.40 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3),
d: 162.7, 147.7, 146.9, 142.2, 138.7, 133.7, 132.1, 128.8,
127.9, 127.3, 124.6, 119.5, 114.1, 35.1, 34.5, 31.8, 30.0,
21.4.
methylene]amino}-phenol (6i). Yield 98%. Anal. Calcd
for C20H25NO2 (311.42): 76.64% C, 8.68% H, 4.47%
N. Found: 76.25% C, 8.52% H, 4.14% N. Mp 98–
100 ꢁC. TLC: Rf (S1) 0.35, Rf (S2) 0.76. UV (nm), kmax
:
204, 249, 315. IR (KBr) cmꢀ1: 3405 (O–H), 2953, 2906,
2866 (C–H), 1623, 1577 (C@N), 1478 (C@C), 1389, 1362
(CH3), 1023 (O–H), 873, 821, 802 (Ar–H). MS: the peak
311.5 was found.
4.1.4.31. 2,4-Di-tert-butyl-6-{[(5-ethylfuran-2-yl)meth-
ylene]amino}-phenol
(6j).
Yield
40%.
C21H27NO2(325.44): 77.02% C, 8.93% H, 4.28% N.
Found: 76.84% C, 8.72% H, 3.96% N. Viscous oil.
TLC: Rf (S1) 0.51, Rf (S2) 0.75. UV (nm), kmax: 202,
316. IR (KBr) cmꢀ1: 3424 (O–H), 2965, 2906, 2869
(C–H), 1619, 1529 (C@N), 1479 (C@C), 1386, 1361

5860
J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
4.1.4.36. 5,7-Di-tert-butyl-2-(4-methylphenyl)-benzox-
1H, Ar), 7.36 (d overlapped, 1H, J = 1.92 Hz, Ar),
1.55 (s, 9H, CH3), 1.41 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d: 161.1, 147.8, 147.0, 141.8, 134.6,
134.1, 132.2, 131.8, 128.8, 127.5, 121.6, 119.9, 114.5,
35.1, 34.4, 31.8, 29.9.
azole (7c). Method A. Yield 42%. Anal. Calcd for
C22H27NO (321.46): 82.20% C, 8.47% H, 4.36% N.
Found: 82.28% C, 8.40% H, 4.23% N. Mp 92–93 ꢁC.
HPLC purity 99.85%. TLC: Rf (S1) 0.52, Rf (S2) 0.78.
UV (nm), kmax/loge: 215, 246, 293/3.63. IR (KBr)
cmꢀ1: 2954, 2907, 2869 (C–H), 1612, 1561 (C@N),
1482 (C@C), 1392, 1364 (CH3), 1068 (C–O–C), 867,
4.1.4.40. 5,7-Di-tert-butyl-2-(3-bromophenyl)-benzox-
azole (7g). Method A. Yield 33%. Anal. Calcd for
C21H24BrNO (386.33): 65.29% C, 6.26% H, 3.63% N.
Found: 65.20% C, 6.36% H, 3.52% N. Mp 100–101 ꢁC.
HPLC purity 99.47%. TLC: Rf (S1) 0.65, Rf (S2) 0.82.
1
825, 730 (Ar–H). MS: the peak 321.6 was found. H
NMR (300 MHz, CDCl3), d: 8.19–8.11 (m, AA0BB0,
2H, Ar), 7.65 (d, 1H, J = 1.92 Hz, Ar), 7.37–7.31 (m,
AA0BB0, 2H, Ar), 7.30 (d, 1H, J = 1.92 Hz, Ar), 2.44
(s, 3H, CH3), 1.55 (s, 9H, CH3), 1.40 (s, 9H, CH3).
13C NMR (75 MHz, CDCl3), d: 162.7, 147.7, 146.8,
142.2, 141.7, 133.6, 129.6, 127.4, 124.7, 119.3, 114.0,
35.1, 34.5, 31.8, 30.0, 21.6.
UV (nm), kmax/loge: 206/3.63, 290. IR (KBr) cmꢀ1
:
2961, 2907, 2868 (C–H), 1629, 1568 (C@N), 1481
(C@C), 1391, 1331 (CH3), 1082 (C@O@C), 868, 839,
768, 732 (Ar–H). MS: the peak 386.4 was found. H
1
NMR (300 MHz, CDCl3), d: 8.38 (t, 1H, J = 1.79 Hz,
Ar), 8.20–8.16 (m, 1H, Ar), 7.67–7.63 (m, 1H, Ar),
7.65 (d, overlapped, 1H, J = 1.92 Hz, Ar), 7.40 (t, 1H,
J = 7.69 Hz, Ar), 7.33 (d, 1H, J = 1.92 Hz, Ar), 1.55 (s,
9H, CH3), 1.40 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 160.9, 148.0, 147.0, 142.1, 134.1, 133.9,
130.4, 130.2, 129.4, 125.9, 122.9, 120.0, 114.3, 35.1,
34.5, 31.8, 30.0.
4.1.4.37. 5,7-Di-tert-butyl-2-(3-chlorophenyl)-benzox-
azole (7d). Method A. Yield 45%. Anal. Calcd for
C21H24ClNO (341.87): 73.78% C, 7.08% H, 4.10% N.
Found: 73.69% C, 7.15% H, 4.04% N. Mp 116–117 ꢁC.
HPLC purity 99.81%. TLC: Rf (S1) 0.79, Rf (S2) 0.82.
UV (nm), kmax/loge: 207/3.67, 308. IR (KBr) cmꢀ1
:
2954, 2906, 2869 (C–H), 1625, 1554 (C@N), 1475
(C@C), 1392, 1365 (CH3), 1059 (C–O–C), 864, 782,
719 (Ar–H). MS: the peak 342.0 was found. H NMR
4.1.4.41. 5,7-Di-tert-butyl-2-(4-bromophenyl)-benzox-
azole (7h). Method A. Yield 39%. Anal. Calcd for
C21H24BrNO (386.33): 65.29% C, 6.26% H, 3.63% N.
Found: 65.21% C, 6.19% H, 3.49% N. Mp 101–103 ꢁC.
HPLC purity 98.89%. TLC: Rf (S1) 0.82, Rf (S2) 0.85.
UV (nm), kmax/loge: 207/3.64, 249, 311. IR (KBr)
cmꢀ1: 2957, 2906, 2868 (C–H), 1620, 1596 (C@N),
1481 (C@C), 1397, 1363 (CH3), 1072 (C–O–C), 868,
1
(300 MHz, CDCl3), d: 8.24–8.21 (m, 1H, Ar), 8.14 (dt,
1H, J = 6.87 Hz, J = 1.65 Hz, Ar), 7.66 (d,
1H,J = 1.92 Hz, Ar), 7.50–7.43 (m, 2H, Ar), 7.33 (d,
1H, J = 1.92 Hz, Ar), 1.55 (s, 9H, CH3), 1.40 (s, 9H,
CH3). 13C NMR (75 MHz, CDCl3), d: 161.1, 148.0,
146.9, 142.1, 134.9, 133.9, 131.2, 130.2, 129.2, 127.3,
125.4, 120.0, 114.3, 35.1, 34.5, 31.8, 30.0.
1
730 (Ar–H). MS: the peak 386.5 was found. H NMR
(300 MHz, CDCl3), d: 8.14–8.08 (m, AA0BB0, 2H, Ar),
7.69–7.64 (m, AA0BB0, 2H, Ar), 7.65 (d, overlapped,
1H, J = 1.92 Hz, Ar), 7.32 (d, 1H, J = 1.92 Hz, Ar),
1.55 (s, 9H, CH3), 1.40 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d: 161.6, 147.9, 146.9, 142.2, 133.8,
132.2, 128.8, 126.5, 125.8, 119.9, 114.3, 35.1, 34.5,
31.8, 30.0.
4.1.4.38. 5,7-Di-tert-butyl-2-(4-chlorophenyl)-benzox-
azole (7e). Method A. Yield 39%. Anal. Calcd for
C21H24ClNO (341.87): 73.78% C, 7.08% H, 4.10% N.
Found: 73.87% C, 7.02% H, 4.03% N. Mp 109–111 ꢁC.
HPLC purity 99.91%. TLC: Rf (S1) 0.69, Rf (S2) 0.90.
UV (nm), kmax/loge: 204/3.64, 245, 309. IR (CHCl3)
cmꢀ1: 2957, 2908, 2869 (C–H), 1600, 1553 (C@N),
1483 (C@C), 1399, 1364 (CH3), 1064 (C–O–C), 868,
4.1.4.42. 5,7-Di-tert-butyl-2-(2-trifluoromethylphenyl)-
benzoxazole (7i). Method A. Yield 78%. Anal. Calcd for
C22H24F3NO (375.43): 70.38% C, 6.44% H, 3.73% N.
Found: 70.46% C, 6.58% H, 3.64% N. Mp 54-61 ꢁC.
HPLC purity 98.23%. TLC: Rf (S1) 0.52, Rf (S2) 0.78.
1
837, 733 (Ar–H). MS: the peak 341.9 was found. H
NMR (300 MHz, CDCl3), d: 8.21–8.15 (m, AA0BB0,
2H, Ar), 7.65 (d, 1H, J = 1.92 Hz, Ar), 7.53–7.47 (m,
AA0 BB0, 2H, Ar), 7.32 (d, 1H, J = 1.92 Hz, Ar), 1.55
(s, 9H, CH3), 1.40 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 161.5, 147.9, 146.9, 142.2, 137.4, 133.8,
129.2, 128.6, 126.0, 119.8, 114.3, 35.1, 34.5, 31.9, 30.0.
UV (nm), kmax/loge: 207/3.63, 306. IR (CHCl3) cmꢀ1
:
3023, 2967, 2908, 2871 (C–H), 1610, 1559(C@N), 1480,
1455 (C@C), 1393, 1364 (CH3). MS: the peak 375.6 was
1
found. H NMR (300 MHz, CDCl3), d: 8.53–8.49 (m,
4.1.4.39. 5,7-Di-tert-butyl-2-(2-bromophenyl)-benzox-
azole (7f). Method A. Yield 31%. Anal. Calcd for
C21H24BrNO (386.33): 65.29% C, 6.26% H, 3.63% N.
Found: 65.35% C, 6.32% H, 3.56% N. Mp 157–158 ꢁC.
HPLC purity 99.61%. TLC: Rf (S1) 0.73, Rf (S2) 0.82.
1 H, Ar), 8.45–8.40 (m, 1H, Ar), 7.81–7.75 (m, 1H,
Ar), 7.70–7.63 (m, 1H, Ar), 7.68 (d overlapped, 1H,
J = 1.93 Hz, Ar), 7.35 (d, 1H, J = 1.93 Hz, Ar), 1.56 (s,
9H, CH3), 1.40 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 161.0, 148.2, 147.0, 142.1, 133.0, 131.6 (q,
J = 32.9 Hz), 130.4 (q, J = 0.80 Hz), 129.5, 128.4, 127.6
(q, J = 3.50 Hz), 124.2 (q, J = 3.70 Hz), 123.7 (q,
J = 272.6 Hz), 120.2, 114.4, 35.1, 34.5, 31.8, 30.0.
UV (nm), kmax/loge: 210/3.63, 308. IR (KBr) cmꢀ1
:
2963, 2907, 2869 (C–H), 1624, 1551 (C@N), 1473
(C@C), 1392, 1364 (CH3), 1058 (C–O–C), 866, 841,
722 (Ar–H). MS: the peak 386.4 was found. H NMR
1
(300 MHz, CDCl3), d: 8.11 (dd, 1H, J = 7.69 Hz,
J = 1.37 Hz, Ar), 7.77 (dd, 1H, J = 7.69 Hz,
J = 1.37 Hz, Ar), 7.72 (d, 1H, J = 1.92 Hz, Ar), 7.47
(dt, 1H, J = 7.69 Hz, J = 1.37 Hz, Ar), 7.40-7.33 (m,
4.1.4.43. 5,7-Di-tert-butyl-2-(3-trifluoromethylphenyl)-
benzoxazole (7j). Method A. Yield 87%. Anal. Calcd for
C22H24F3NO (375.43): 70.38% C, 6.44% H, 3.73% N.
Found: 70.44% C, 6.56% H, 3.67% N. Mp 62 ꢁC. HPLC

J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5861
purity 99.86%. TLC: Rf (S1) 0.58, Rf (S2) 0.85. UV (nm),
max/loge: 211, 306/3.62. IR (CHCl3) cmꢀ1: 3022, 2967,
J = 1.92 Hz, Ar), 7.36 (d, 1H, J = 1.92 Hz, Ar), 1.47 (s,
9H, CH3), 1.40 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 158.2, 149.3, 148.2, 147.4, 141.7, 134.3,
132.1, 131.6, 131.2, 123.9, 121.4 120.5, 114.7, 35.1,
34.3, 31.8, 29.9.
k
2908, 2871 (C–H), 1610, 1559 (C@N), 1480, 1455
(C@C), 1393, 1364 (CH3). MS: the peak 375.5 was
1
found. H NMR (300 MHz, CDCl3), d: 8.53–8.49 (m,
1H, Ar), 8.45–8.40 (m, 1H, Ar), 7.81–7.75 (m, 1H,
Ar), 7.71–7.63 (m, 1H, Ar), 7.68 (d overlapped, 1H,
J = 1.92 Hz, Ar), 7.35 (d, 1H, J = 1.92 Hz, Ar), 1.56 (s,
9H, CH3), 1.40 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 161.0, 148.2, 147.0, 142.1, 134.0, 131.6 (q,
J = 32.9 Hz), 130.4, 130.3, 129.5, 128.4, 127.6 (q,
J = 3.50 Hz), 124.2 (q, J = 3.70 Hz), 123.7 (q,
J = 272.60 Hz), 120.2, 35.1, 34.5, 31.8, 30.0.
4.1.4.47. 5,7-Di-tert-butyl-2-(4-nitrophenyl)-benzoxaz-
ole (7n). Method A. Yield 78%. Anal. Calcd for
C21H24N2O3 (352.43): 71.57% C, 6.86% H, 7.95% N.
Found: 71.49% C, 6.95% H, 7.84% N. Mp 201–202 ꢁC,
Mp 199–200 ꢁC.29 HPLC purity 99.84%. TLC: RF (S1)
0.65, Rf (S2) 0.90. UV (nm), kmax/log e: 202, 238, 341/
3.67. IR (KBr) cmꢀ1: 2956, 2987, 2869 (C–H), 1622,
1606, 1558 (C@N), 1526 (NO2), 1483 (C@C), 1390,
1362 (CH3), 1345 (NO2), 1067 (C–O–C), 858, 707 (Ar–
H). MS: the peak 352.5 was found. 1H NMR
(300 MHz, CDCl3), d: 8.44–8.31 (m, 4H, Ar), 7.69 (d,
1H, J = 1.64 Hz, Ar), 7.38 (d, 1H, J = 1.64 Hz, Ar),
1.56 (s, 9H, CH3), 1.40 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d: 160.1, 149.1, 148.5, 147.3, 142.2,
134.1, 133.1, 128.1, 124.2, 120.9, 114.7, 35.2, 34.5,
31.7, 30.0.
4.1.4.44. 5,7-Di-tert-butyl-2-(4-trifluoromethylphenyl)-
benzoxazole (7k). Method A. Yield 37%. Anal. Calcd for
C22H24F3NO (375.43): 70.38% C, 6.44% H, 3.73% N.
Found: 70.31% C, 6.51% H, 3.62% N. Mp 79–81 ꢁC.
HPLC purity 99.68%. TLC: Rf (S1) 0.48, Rf (S2) 0.90.
UV (nm), kmax/loge: 202, 307/3.67. IR (KBr) cmꢀ1
:
2961, 2908, 2871 (C–H), 1627, 1562 (C@N), 1482
(C@C), 1393, 1365 (CH3), 1324, 1167, 1129 (CF3),
1073 (C–O–C), 851, 707 (Ar–H). MS: the peak 375.5
1
was found. H NMR (300 MHz, CDCl3), d: 8.40–8.52
4.1.4.48. 5,7-Di-tert-butyl-2-(4-dimethylaminophenyl)-
benzoxazole (7o). Method A. Yield 72%. Anal. Calcd for
C23H30N2O (350.50): 78.82% C, 8.63% H, 7.99% N.
Found: 78.90% C, 8.68% H, 7.91% N. Mp 72 ꢁC. Mp
149–150 ꢁC.29 HPLC purity 99.64%. TLC: Rf (S1) 0.17,
RF (S2) 0.72. UV (nm), kmax/loge: 206, 344/3.69. IR
(KBr) cmꢀ1: 2957, 2905, 2868 (C–H), 1611, 1512
(C@N), 1481 (C@C), 1391, 1366 (CH3), 1066 (C–O–
C), 823, 741 (Ar–H). MS: the peak 350.6 was found.
1H NMR (300 MHz, CDCl3), d: 8.17–8.05 (m AA0,
BB0, 2H, Ar), 7.60 (d, 1H, J = 1.65 Hz, Ar), 7.23 (d,
1H, J = 1.65 Hz, Ar), 6.83–6.74 (m AA0, BB0, 2H, Ar),
3.07 (s, 6H, NCH3), 1.54 (s, 9H, CH3), 1.39 (s, 9H,
CH3). 13C NMR (75 MHz, CDCl3), d: 163.6, 152.2,
147.2, 146.6, 142.7, 133.1, 128.8, 118.3, 114.7, 113.5,
111.6, 40.1, 35.0, 34.4, 31.8, 30.0.
(m, AA0BB0, 2H, Ar), 7.83–7.76 (m, AA0BB0, 2H, Ar),
7.68 (d, 1H, J = 1.92 Hz, Ar), 7.36 (d, 1H, J = 1.92 Hz,
Ar), 1.56 (s, 9H, CH3), 1.40 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d 160.9, 148.2, 147.1, 142.1, 134.0,
132.7 (q, J = 32.70 Hz), 130.8, 127.6, 125.9 (q,
J = 3.70 Hz), 120.3, 120.2 (q, J = 272.60 Hz), 114.5,
35.1, 34.5, 31.8, 30.0.
4.1.4.45. 2-(5,7-Di-tert-butylbenzoxazol-2-yl)-phenol
(7l). Method A. Yield 75%. Method B. Yield 15%. Anal.
Calcd for C21H25NO2 (323.43): 77.98% C, 7.79% H,
4.33% N. Found: 77.90% C, 7.88% H, 4.24% N. Mp
120–123 ꢁC, Mp 99–100 ꢁC.25 HPLC purity 99.97%.
TLC: Rf (S1) 0.65, Rf (S2) 0.90. UV (nm), kmax/loge:
208, 277, 290, 322/3.64. IR (CHCl3) cmꢀ1: 3432 (OH),
2964, 2906, 2870 (C–H), 1635, 1548 (C@N), 1488
(C@C), 1393, 1364 (CH3), 1063 (C–O–C), 867, 750
(Ar–H). MS: the peak 323.5 was found. 1H NMR
(300 MHz, CDCl3), d: 11.58 (br s, 1H, OH), 8.04 (dd,
1H, J = 7.28 Hz, J = 1.37 Hz, Ar), 7.61 (d, 1H,
J = 1.92 Hz, Ar), 7.47–7.40 (m, 1H, Ar), 7.34 (d, 1H,
J = 1.92 Hz, Ar), 7.13 (dd, 1H, J = 7.28 Hz,
J = 1.37 Hz, Ar), 7.06–6.99 (m, 1H, Ar), 1.56 (s, 9H,
CH3), 1.41 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3),
d: 162.3, 158.6, 148.3, 145.3, 140.2, 133.8, 133.2, 126.9,
119.9, 119.4, 117.3, 113.5, 110.9, 35.1, 34.5, 31.8, 30.0.
4.1.4.49. 5,7-Di-tert-butyl-2-(4-methylsulfanylphenyl)-
benzoxazole (7p). Method A. Yield 84%. Anal. Calcd for
C22H27NOS (353.52): 74.74% C, 7.70% H, 3.96% N.
Found: 74.83% C, 7.79% H, 3.82% N. Mp 111–112 ꢁC.
HPLC purity 99.51%. UV (nm), kmax/loge: 205, 231,
325/3.69. IR (KBr) cmꢀ1: 2963, 2905, 2869 (C–H),
1624, 1598 (C@N), 1483 (C@C), 1384, 1364 (CH3),
869, 828, 735 (Ar–H). MS: the peak 353.6 was found.
1H NMR (300 MHz, CDCl3), d: 8.23–8.11 (m, AA0BB0,
2H, Ar), 7.64 (d, 1H, J = 1.79 Hz, Ar), 7.40–7.33 (m,
AA0BB0, 2H, Ar), 7.30 (d, 1H, J = 1.79 Hz, Ar), 2.55
(s, 3H, SCH3), 1.55 (s, 9H, CH3), 1.39 (s, 9H, CH3).
13C NMR (75 MHz, CDCl3), d: 162.3, 147.7, 146.8,
143.2, 142.2, 133.6, 127.6, 125.8, 123.7, 119.4, 114.0,
35.1, 34.4, 31.8, 30.0, 15.0.
4.1.4.46. 5,7-Di-tert-butyl-2-(2-nitrophenyl)-benzoxaz-
ole (7m). Method A. Yield 32%. Anal. Calcd for
C21H24N2O3 (352.43): 71.57% C, 6.86% H, 7.95% N.
Found: 71.69% C, 6.98% H, 7.81% N. Mp 127–128 ꢁC.
HPLC purity 99.68%. TLC: RF (S1) 0.12, Rf (S2) 0.71.
UV (nm), kmax/loge: 211/3.65, 283, 311. IR (KBr)
cmꢀ1: 2961, 2908, 2870 (C–H), 1629 (C@N), 1536
(NO2), 1482, 1466 (C@C), 1392, 1365 (CH3), 1307
(NO2), 1061 (C–O–C), 869, 785, 756 (Ar–H). MS: the
4.1.4.50. 5,7-Di-tert-butyl-2-(4-fluoro-3-phenoxyphe-
nyl)-benzoxazole (7q). Method A. Yield 68%. Anal.
Calcd for C27H28FNO2 (417.52): 77.67% C, 6.76% H,
3.35% N. Found: 77.53% C, 6.84% H, 3.29% N. Mp
91–93 ꢁC. HPLC purity 99.33%. UV (nm), kmax/loge:
207/3.73, 308. IR (KBr) cmꢀ1: 2961, 2907, 2870 (C–
H), 1590 (C@N), 1491 (C@C), 1400, 1364 (CH3),
1
peak 352.5 was found. H NMR (300 MHz, CDCl3),
d: 8.23–8.19 (m, 1H, Ar), 7.86–7.82 (m, 1H, Ar), 7.77–
7.63 (m, 2H, Ar), 7.68 (d, overlapped, 1H,

5862
J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
1267, 1211 (–O–), 869, 803, 749, 731, 690 (Ar–H). MS:
the peak 417.6 was found. 1H NMR (300 MHz, CDCl3),
d: 8.06–7.93 (2H, m, Ar), 7.61 (1H, d, J = 1.70 Hz, Ar),
7.42–7.32 (3H, m, Ar), 7.31 (1H, d, J = 1.70 Hz, Ar),
7.19–7.11 (1H, m, Ar), 7.10-7.03 (2H, m, Ar), 1.52
(9H, s, CH3), 1.39 (9H, s, CH3). 13C NMR (75 MHz,
CDCl3), d: 159.4 (d, J = 237.30 Hz), 156.8, 154.5,
147.9, 147.0, 144.4, (d, J = 12.00 Hz), 142.1, 133.7,
129.9, 124.6 (d, J = 3.70 Hz), 123.9, 123.8 (d,
J = 22.60 Hz), 120.7 (d, J = 2.00 Hz), 119.8, 117.7 (d,
J = 22.60 Hz), 117.8, 114.2, 35.1, 34.4, 31.8, 30.0.
4.1.4.54. 5,7-Di-tert-butyl-2-(pyridin-3-yl)-benzoxaz-
ole (8d). Method A. Yield 77%. Anal. Calcd for
C20H24N2O (308.42): 77.89% C, 7.84% H, 9.08% N.
Found: 77.96% C, 7.78% H, 8.98% N. Mp 94–96 ꢁC.
HPLC purity 99.55%. TLC: Rf (S1) 0.20, Rf (S2) 0.38.
UV (nm), kmax/loge: 216, 306/3.68. IR (CHCl3) cmꢀ1
:
2965, 2906, 2869 (C–H), 1607, 1576, 1552 (C@N),
1480 (C@C), 1392, 1362 (CH3), 1076 (C–O–C), 865,
841 (Ar–H). MS: the peak 308.5 was found. H NMR
1
(300 MHz, CDCl3), d: 9.47 (d, 1H, J = 1.64 Hz, Ar),
8.75 (dd, 1H, J = 4.81 Hz, J = 1.64 Hz, Ar), 8.50 (dt,
1H, J = 7.97 Hz, J = 1.64 Hz, Ar), 7.67 (d, 1H,
J = 1.79 Hz, Ar), 7.47 (ddd, 1H, J = 7.97 Hz,
J = 4.81 Hz, J = 0.82 Hz, Ar), 7.34 (d, 1H, J = 1.79 Hz,
Ar), 1.55 (s, 9H, CH3), 1.40 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d: 160.1, 151.8, 148.6, 148.2, 147.0,
142.0, 134.4, 133.9, 123.8, 123.7, 120.2, 114.4, 35.1,
34.5, 31.8, 30.0.
4.1.4.51. 5,7-Di-tert-butyl-2-styrylbenzoxazole (8a).
Method A. Yield 67%. Anal. Calcd for C23H27NO
(333.47): 82.84% C, 8.16% H, 4.20% N. Found:
82.91% C, 8.08% H, 4.13% N. Mp 85–87 ꢁC. HPLC
purity 99.52%. TLC: Rf (S1) 0.43, Rf (S2) 0.88. UV
(nm), kmax/loge: 210, 327/3.73. IR (KBr) cmꢀ1: 2959,
2906, 2869 (C–H), 1639, 1578 (C@N), 1535, 1482
(C@C), 1392, 1364 (CH3), 1165 (C–O–C), 868, 842,
757 (Ar–H). MS: the peak 333.5 was found. 1H
NMR (300 MHz, CDCl3), d: 7.76 (d, 1H,
J = 16.35 Hz, CH), 7.66–7.60 (m, 2H, Ar), 7.60 (d,
1H, J = 1.92 Hz, Ar), 7.47–7.36 (m, 3H, Ar), 7.30 (d,
1H, J = 1.92 Hz, Ar), 7.11 (d, 1H, J = 16.35 Hz, CH),
1.54 (s, 9H, CH3), 1.39 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d: 162.3, 147.7, 146.6, 142.4, 138.4,
135.3, 133.5, 129.6, 128.9, 127.5, 119.7, 114.3, 114.0,
35.0, 34.4, 31.8, 29.9.
4.1.4.55. 5,7-Di-tert-butyl-2-(pyridin-4-yl)-benzoxaz-
ole (8e). Method A. Yield 78%. Anal. Calcd for
C20H24N2O (308.42): 77.89% C, 7.84% H, 9.08% N.
Found: 78.01% C, 7.98% H, 8.89% N. Mp 97–100 ꢁC.
HPLC purity 98.26%. TLC: Rf (S1) 0.06, Rf (S2) 0.41.
UV (nm), kmax/loge: 217, 309/3.65. IR (CHCl3) cmꢀ1
:
2958, 2907, 2870 (C–H), 1624, 1606, 1569 (C@N),
1482 (C@C), 1392, 1363 (CH3), 1063 (C–O–C), 868,
843 (Ar–H). MS: the peak 308.6 was found. H NMR
1
(300 MHz, CDCl3), d: 8.84–8.79 (m, 2H, Ar), 8.10–
8.06 (m, 2H, Ar), 7.68 (d, 1H, J = 1.79 Hz, Ar), 7.38
(d, 1H, J = 1.79 Hz, Ar), 1.55 (s, 9H, CH3), 1.40 (s,
9H, CH3). 13C NMR (75 MHz, CDCl3), d: 160.0,
150.7, 148.4, 147.1, 142.0, 134.6, 134.1, 120.8, 114.7,
35.1, 34.5, 31.7, 30.0.
4.1.4.52. 5,7-Di-tert-butyl-2-(1H-pyrazol-3-yl)-benz-
oxazole (8b). Method A. Yield 47%. Anal. Calcd for
C18H23N3O (297.39): 72.70% C, 7.80% H, 14.13% N.
Found: 72.62% C, 7.91% H, 14.04% N. Mp 177–
180 ꢁC. HPLC purity 99.55%. TLC: Rf (S1) 0.05, Rf
(S2) 0.65. UV (nm), kmax/loge: 203, 237, 293/3.65. IR
(KBr) cmꢀ1: 2961, 2908, 2870 (C–H), 1609 (C@N),
1482, 1464 (C@C), 1392, 1364 (CH3), 1050 (C–O–C),
867, 774, 740 (Ar–H). MS: the peak 297.5 was found.
1H NMR (300 MHz, CDCl3), d: 11.09 (br s, 1H, NH),
7.98 (d, 1H, J = 2.48 Hz, Ar), 7.71 (d, 1H,J = 1.92 Hz,
Ar), 7.35 (d, 1H, J = 1.92 Hz, Ar), 7.11 (d, 1H,
J = 2.48 Hz, Ar), 1.54 (s, 9H, CH3), 1.41 (s, 9H, CH3).
13C NMR (75 MHz, CDCl3), d: 157.0, 148.1, 146.6,
141.6, 138.4, 134.0, 133.0, 120.0, 114.2, 106.3, 35.1,
34.5, 31.8, 30.0.
4.1.4.56.
5,7-Di-tert-butyl-2-(6-methylpyridin-2-yl)-
benzoxazole (8f). Method A. Yield 37%. Anal. Calcd
for C21H26N2O (322.44): 78.22% C, 8.13% H, 8.69%
N. Found: 78.30% C, 8.19% H, 8.57% N. Mp 58–
60 ꢁC. HPLC purity 98.75%. TLC: Rf (S1) 0.13, Rf (S2)
0.48. UV (nm), kmax/loge: 208, 239, 309/3.73. IR
(CHCl3) cmꢀ1: 2961, 2908, 2870 (C–H), 1623, 1553
(C@N), 1457 (C@C), 1393, 1364 (CH3), 1082 (C–O–
C), 866, 804 (Ar–H). MS: the peak 322.6 was found.
1H NMR (300 MHz, CDCl3), d: 8.04 (d, 1H,
J = 7.69 Hz, Ar), 7.75 (t, 1H, J = 7.69 Hz, Ar), 7.71 (d,
1H, J = 1.92 Hz, Ar), 7.34 (d, 1H, J = 1.92 Hz, Ar),
7.28 (d, 1H, J = 7.69 Hz, Ar), 2.71 (s, 3H, CH3), 1.55
(s, 9H, CH3), 1.39 (s, 9H, CH3). 13C NMR (75 MHz,
CDCl3), d: 161.3, 159.7, 147.9, 147.1, 145.6, 142.2,
136.9, 133.9, 125.0, 120.2, 120.1, 115.0, 35.0, 34.5,
31.8, 30.0, 24.7.
4.1.4.53. 5,7-Di-tert-butyl-2-(pyridin-2-yl)-benzoxaz-
ole (8c). Method A. Yield 31%. Anal. Calcd for
C20H24N2O (308.42): 77.89% C, 7.84% H, 9.08% N.
Found: 77.99% C, 7.94% H, 8.91% N. Mp 75–77 ꢁC.
HPLC purity 98.44%. TLC: Rf (S1) 0.06, Rf (S2) 0.30.
UV (nm), kmax/loge: 215, 310/3.70. IR (CHCl3) cmꢀ1
:
2960, 2907, 2869 (C–H), 1623, 1556 (C@N), 1458
(C@C), 1392, 1364 (CH3), 1082 (C–O–C), 866, 839
(Ar–H). MS: the peak 308.6 was found. 1H NMR
(300 MHz, CDCl3), d: 8.85–8.81 (m, 1H, Ar), 8.30–
8.26 (m, 1H, Ar), 7.91–7.83 (m, 1H, Ar), 7.70–7.68 (m,
1H, Ar), 7.45–7.39 (m, 1H, Ar), 7.37–7.35 (m, 1H,
Ar), 1.56 (s, 9H, CH3), 1.40 (d, 9H, J = 0.55 Hz, CH3).
13C NMR (75 MHz, CDCl3), d: 161.1, 150.4, 148.0,
147.3, 146.5, 142.1, 136.8, 134.1, 125.1, 123.1, 120.4,
114.8, 35.1, 34.5, 31.8, 30.1.
4.1.4.57. 5,7-Di-tert-butyl-2-(1H-indol-3-yl)-benzoxaz-
ole (8g). Method A. Yield 51%. Anal. Calcd for
C23H26N2O (346.47): 79.73% C, 7.56% H, 8.09% N.
Found: 79.81% C, 7.63% H, 7.92% N. Mp 209–211 ꢁC.
HPLC purity 98.08%. TLC: Rf (S2) 0.20. UV (nm),
k
max/loge: 221/3.76, 267, 318. IR (KBr) cmꢀ1: 2961,
2907, 2869 (C–H), 1632 (C@N), 1482, 1458 (C@C),
1403, 1364 (CH3), 1041 (C–O–C), 865, 743 (Ar–H).
1
MS: the peak 346.6 was found. H NMR (300 MHz,
CDCl3), d: 9.46 (br s, 1H, NH), 8.45 (d, 1H,

J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
5863
J = 7.69 Hz, Ar), 8.21–8.14 (m, 1H, Ar), 7.67-7.64 (m,
1H, Ar), 7.53–7.44 (m, 1H, Ar), 7.41–7.21 (m, 3H,
Ar), 1.60 (s, 9H, CH3), 1.41 (s, 9H, CH3). 13C NMR
(75 MHz, CDCl3), d: 160.9, 147.6, 146.0, 141.5, 136.4,
133.3, 127.9, 124.9, 123.4, 122.0, 121.2, 118.6, 113.1,
111.9, 105.1, 35.1, 34.4, 31.9, 30.1.
HPLC purity 99.08%. TLC: RF (S1) 0.43, Rf (S2) 0.86.
UV (nm), kmax/loge: 206, 251, 314/3.72. IR (CHCl3)
cmꢀ1: 2958, 2907, 2869 (C@H), 1624, 1581 (C@N),
1481 (C@C), 1392, 1363 (CH3), 1012 (C–O–C), 865,
1
858 (Ar–H). MS: the peak 313.6 was found. H NMR
(300 MHz, CDCl3), d: 7.90 (dd, 1H, J = 3.84 Hz,
J = 1.24 Hz, Ar), 7.62 (d, 1H, J = 1.79 Hz, Ar), 7.53
(dd, 1H, J = 4.95 Hz, J = 1.24 Hz, Ar), 7.29 (d, 1H,
J = 1.79 Hz, Ar), 7.18 (dd, 1H, J = 4.95 Hz,
J = 3.84 Hz, Ar), 1.53 (s, 9H, CH3), 1.39 (s, 9H, CH3).
13C NMR (75 MHz, CDCl3), d: 158.5, 147.9, 146.6,
142.1, 133.6, 130.1, 129.7, 129.3, 128.1, 119.5, 114.0,
35.1, 34.4, 31.8, 30.0.
4.1.4.58. 5,7-Di-tert-butyl-2-(furan-2-yl)-benzoxazole
(8h). Method A. Yield 95%. Anal. Calcd for
C19H23NO2 (297.39): 76.73% C, 7.80% H, 4.71% N.
Found: 76.84% C, 7.89% H, 4.63% N. Viscous oil.
HPLC purity 98.06%. TLC: Rf (S1) 0.51, Rf (S2) 0.77.
UV (nm), kmax/loge: 203, 248, 309/3.69. IR (KBr)
cmꢀ1: 2961, 2907, 2870 (C–H), 1637, 1600 (C@N),
1541, 1460 (C@C), 1393, 1364 (CH3), 1087 (C–O–C),
896, 867 (Ar–H). MS: the peak 297.5 was found.
1H NMR (300 MHz, CDCl3), d: 8.18 (dd, 1H,
J = 3.02 Hz, J = 1.10 Hz, Ar), 7.80 (dd, 1H,
J = 4.94 Hz, J = 1.10 Hz, Ar), 7.63 (d, 1H, J = 1.79 Hz,
Ar), 7.45 (dd, 1H, J = 4.94 Hz, J = 3.02 Hz, Ar), 7.30
(d, 1H, J = 1.79 Hz, Ar), 1.54 (s, 9H, CH3), 1.39 (s,
9H, CH3). 13C NMR (75 MHz, CDCl3), d: 159.2,
147.7, 146.5, 142.1, 133.6, 129.6, 127.4, 126.9, 126.6,
119.5, 114.1, 35.1, 34.5, 31.8, 30.0.
4.1.4.62. 5,7-Di-tert-butyl-2-(thiophen-3-yl)-benzoxaz-
ole (8l). Method A. Yield 42%. Anal. Calcd for
C19H23NOS (313.46): 72.80% C, 7.40% H, 4.47% N.
Found: 72.86% C, 7.51% H, 4.35% N. Viscous oil.
HPLC purity 98.95%. TLC: RF (S1) 0.52, Rf (S2) 0.78.
UV (nm), kmax/loge: 211/3.65, 301. IR (KBr) cmꢀ1
:
2961, 2907, 2869 (C–H), 1627, 1582 (C@N), 1481
(C@C), 1399, 1364 (CH3), 1064 (C–O–C), 865, 796
(Ar–H). MS: the peak 313.6 was found. 1H NMR
(300 MHz, CDCl3), d: 7.66 (dd, 1H, J = 1.92 Hz,
J = 0.82 Hz, Ar), 7.62 (d, 1H, J = 1.92 Hz, Ar), 7.30
(d, 1H, J = 1.92 Hz, Ar), 7.25 (dd, 1H, J = 3.57 Hz,
J = 0.82 Hz, Ar), 6.60 (dd, 1H, J = 3.57 Hz,
J = 1.64 Hz, Ar), 1.52 (s, 9H, CH3), 1.39 (s, 9H, CH3).
13C NMR (75 MHz, CDCl3), d: 154.9, 148.0, 146.3,
145.3, 143.0, 141.8, 133.7, 126.9, 119.7, 114.3, 113.5,
35.0, 34.4, 31.8, 30.0.
4.1.4.59.
5,7-Di-tert-butyl-2-(5-methylfuran-2-yl)-
benzoxazole (8i). Method A. Yield 98%. Anal. Calcd
for C20H25NO2 (311.42): 77.14% C, 8.09% H, 4.50%
N. Found: 77.20% C, 8.16% H, 4.41% N. Mp 98–
100 ꢁC. HPLC purity 98.83%. TLC: Rf (S1) 0.35, Rf
(S2) 0.76. UV (nm), kmax/loge: 204, 249, 315/3.64. IR
(KBr) cmꢀ1: 2967, 2917, 2868 (C–H), 1639, 1574
(C@N), 1482 (C@C), 1392, 1362 (CH3), 1021 (C–O–
C), 864, 837, 782 (Ar–H). MS: the peak 311.5 was
4.1.4.63. (5,7-Di-tert-butyl-benzoxazol-2-yl)-methanol
(8m). Method C. Yield 63%. Anal. Calcd for
C16H23NO2 (261.36): 73.53% C, 8.87% H, 5.36% N.
Found: 73.66% C, 8.98% H, 5.22% N. Mp 111–112 ꢁC.
HPLC purity 98.74%. UV (nm), kmax/loge: 210, 238,
276/3.61. IR (KBr) cmꢀ1: 2965, 2906, 2870 (C–H),
1610, 1578 (C@N), 1483 (C@C), 1392, 1363 (CH3),
1099 (C–O–C), 875, 839 (Ar–H). MS: the peak 261.5
1
found. H NMR (300 MHz, CDCl3), d: 7.60 (d, 1H,
J = 1.79 Hz, Ar), 7.27 (d, 1H, J = 1.79 Hz, Ar), 7.12
(d, 1H, J = 3.29 Hz, Ar), 6.21–6.18 (m, 1H, Ar), 2.45
(br s, 3H, CH3), 1.51 (s, 9H, CH3), 1.38 (s, 9H, CH3).
13C NMR (75 MHz, CDCl3), d: 156.2, 155.1, 147.8,
146.2, 142.0, 141.3, 133.5, 119.4, 114.8, 114.1, 108.4,
35.0, 34.4, 31.8, 30.0, 13.9.
1
was found. H NMR (300 MHz, CDCl3), d: 7.55 (d,
1H, J = 1.92 Hz, Ar), 7.28 (d, 1H, J = 1.92 Hz, Ar),
4.94 (s, 2H, CH2), 4.06 (br s, 1H, OH), 1.46 (s, 9H,
CH3), 1.37 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3),
d: 165.1, 147.8, 147.1, 140.6, 134.0, 119.6, 113.9, 58.0,
35.0, 34.4, 31.8, 29.9.
4.1.4.60. 5,7-Di-tert-butyl-2-(5-ethylfuran-2-yl)-benz-
oxazole (8j). Method A. Yield 40%. Anal. Calcd for
C21H27NO2 (325.44): 77.50% C, 8.36% H, 4.30% N.
Found: 77.61% C, 8.43% H, 4.22% N. Viscous oil.
HPLC purity 98.64%. TLC: Rf (S1) 0.51, Rf (S2) 0.75.
UV (nm), kmax/loge: 202, 316/3.64. IR (CHCl3) cmꢀ1
:
4.2. Lipophilicity HPLC determination (capacity factor
K/calculatedlogK)
2968, 2907, 2872 (C–H), 1648, 1567 (C@N), 1481
(C@C), 1392, 1365 (CH3). MS: the peak 325.6 was
1
found. H NMR (300 MHz, CDCl3), d: 7.60 (d, 1H,
The HPLC separation module Waters Alliance 2695 XE
and Waters Photodiode Array Detector 2996 (Waters
Corp., Milford, MA, USA) were used. The chromato-
graphic column Symmetryꢂ C18 5 lm, 4.6 · 250 mm,
Part No. WAT054275, (Waters Corp., Milford, MA,
USA) was used. The HPLC separation process was
monitored by Millennium32ꢂ Chromatography Manag-
er Software, Waters 2004 (Waters Corp., Milford, MA,
USA). The mixture of MeOH p.a. (90.0%) and H2O-
HPLC—Mili-Q Grade (10.0%) was used as a mobile
phase. The total flow of the column was 1.0 mL/min,
injection 30 lL, column temperature 45 ꢁC and sample
temperature 10 ꢁC. The detection wavelength 210 nm
J = 1.92 Hz, Ar), 7.27 (d, 1H, J = 1.92 Hz, Ar), 7.14
(d, 1H, J = 3.57 Hz, Ar), 6.22–6.19 (m, 1H, Ar), 2.80
(q, 2H, J = 7.42 Hz, CH2), 1.51 (s, 9H, CH3), 1.38 (s,
9H, CH3), 1.32 (t, 3H, J = 7.42 Hz, CH3). 13C NMR
(75 MHz, CDCl3), d: 161.8, 155.2, 147.8, 146.2, 142.0,
141.1, 133.5, 119.3, 114.7, 114.1, 106.8, 35.0, 34.4,
31.8, 29.9, 21.7, 11.9.
4.1.4.61. 5,7-Di-tert-butyl-2-(thiophen-2-yl)-benzoxaz-
ole (8k). Method A. Yield 54%. Anal. Calcd for
C19H23NOS (313.46): 72.80% C, 7.40% H, 4.47% N.
Found: 72.73% C, 7.48% H, 4.38% N. Mp 72–73 ꢁC.

5864
J. Vinsova et al. / Bioorg. Med. Chem. 14 (2006) 5850–5865
was chosen. The KI methanolic solution was used for
the dead time (TD) determination. Retention times
(TR) were measured in minutes.
without tested compound (positive control for cell viabil-
ity) and in the medium containing 5% DMSO (positive
control for cytotoxic effect). The ability of the com-
pounds to inhibit cellular growth was determined after
72 h by adding 10 lL MTT (5.5 mg/mL) solution (Sig-
ma-Aldrich, USA) to each well. After incubation for
4 h, the dark blue formazan crystal product was dissolved
in 100 lL lysis solution (10% SDS with 0.01 mol HCl).
The absorbance was read at 590 nm using multiplate
spectrofluorimeter GENios PlusTM (Tecan, Switzer-
land). Each concentration of the compounds was tested
in triplicate; the assays were repeated three times in sep-
arate experiments.
The capacity factors K were calculated using the Millen-
nium32ꢂ Chromatography Manager Software accord-
ing to the formula K = (TR ꢀ TD)/TD, where TR is the
retention time of the solute, whereas TD denotes the
dead time obtained via an unretained analyte. LogK,
calculated from the capacity factor K, is used as the lipo-
philicity index converted to logP scale. The logK values
of the individual compounds are shown in Tables 1
and 2.
4.3. Lipophilicity calculations
Acknowledgments
LogP, that is the logarithm of the partition coefficient
for n-octanol/water, was calculated using the programs
CS ChemOffice Ultra ver. 7.0 (CambridgeSoft, Cam-
bridge, MA, USA) and ACD/Log P ver. 1.0 (Advanced
Chemistry Development Inc., Toronto, Canada). ClogP
values (the logarithm of n-octanol/water partition coeffi-
cient based on established chemical interactions) were
generated by means of CS ChemOffice Ultra ver. 7.0
(CambridgeSoft, Cambridge, MA, U.S.A.) software.
The results are shown in Table 1 and 2.
This work was financially supported by the Ministry of
Health of the Czech Republic (No. 1A8238-3) and by
the Ministry of Education of the Czech Republic
(MSM 0021620822). Antimycobacterial data were pro-
vided by the Tuberculosis Antimicrobial Acquisition
and Coordinating Facility (TAACF) through a research
and development contract with the U.S. National
Institute of Allergy and Infectious Diseases.
4.4. Biological methods
References and notes
4.4.1. Antimycobacterial evaluation. Primary screening
was conducted at 6.25 lg/mL against M. tuberculosis
H37Rv (ATCC27294) in BACTEC 12B medium using
both microdilution assay and the Microplate Almar
Blue Assay (MABA).30 Compounds demonstrating at
least 90% inhibition in the primary screen were tested
at lower concentration against M. tuberculosis H37Rv
to determine the MIC testing by MABA. The MIC is de-
fined as the lowest concentration effecting a reduction in
fluorescence of 99% relative to controls.30 Antitubercu-
losis assays for atypical strains were evaluated against
M. kansasii CNCTC My 235/80, M. avium My 80/72
and M. avium My 152/74. The cultures were 10 days
old. The assay was carried out in the semisynthetic Sula
medium (Sevac, Prague, Czech Republic) at pH 6.0 and
37 ꢁC. Used method was the microdilution broth meth-
od. The ability of the compounds to inhibit mycobacte-
rial growth was determined after 7, 14 and 21 days.
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