Synthesis, characterization, antimicrobial activity, and QSAR studies of some new 6-substituted phenyl 3-(4-chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino [5,6-d]isoxazoles

Article abstract of DOI:10.1002/hc.21363

3-(4-Chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino[5,6-d] isoxazoles were synthesized from the reaction of (3-(4-chlorophenyl)-4,5-dihydroisoxazole-4,5-diyl)dimethanol with substituted phenylboronic acids. Crystal structure of 1-(4-(3-(4-chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazol-6-yl)phenyl) ethanone was studied and the values of selected bond distances (?), bond angles (°), and dihedral angles (°) were found in agreement with the calculated (DFT, B3LYP/6-311++G(d,p)) values. Antimicrobial activity of these new compounds was also studied against a panel of microorganisms including Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, and Candida albicans. Most of the dioxaborepines exhibited fair activities against these microorganisms. The pMIC values of the compounds were first correlated with Hammett polar substituent constant (σ) together with lipophilic constant (π) and statistically significant 2D correlations were obtained. In addition, the pMIC values of the compounds were correlated with some theoretical descriptors and fair 2D-QSAR models with clogP, SAA, and μ as independent variables were obtained.

Full text of DOI:10.1002/hc.21363

Received: 5 May 2016
DOI: 10.1002/hc.21363
Revised: 26 October 2016
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R E S E A R C H A R T I C L E
Synthesis, characterization, antimicrobial activity, and QSAR
studies of some new 6-substituted phenyl 3-(4-chlorophenyl)-
3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino [5,6-d]isoxazoles
Meryem Pir1
Hikmet Agirbas1
Fatma Budak2
Onur Sahin3
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1Department of Chemistry, Kocaeli
University, Izmit, Turkey
2Department of Microbiology, Kocaeli
University, Izmit, Turkey
3Department of Scientific and Technological
Research Application and Research
Center, Sinop University, Sinop, Turkey
Abstract
3-(4-Chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino[5,6-d] isoxazoles were
synthesized from the reaction of (3-(4-chlorophenyl)-4,5-dihydroisoxazole-4,5-diyl)
dimethanol with substituted phenylboronic acids. Crystal structure of 1-(4-(3-(4-chlo
rophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazol-6-yl)phenyl)
ethanone was studied and the values of selected bond distances (Å), bond angles (°),
and dihedral angles (°) were found in agreement with the calculated (DFT, B3LYP/6-
311++G(d,p)) values. Antimicrobial activity of these new compounds was also stud-
ied against a panel of microorganisms including Staphylococcus aureus, Enterococcus
faecalis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, and
Candida albicans. Most of the dioxaborepines exhibited fair activities against these
microorganisms. The pMIC values of the compounds were first correlated with
Hammett polar substituent constant (σ) together with lipophilic constant (π) and sta-
tistically significant 2D correlations were obtained. In addition, the pMIC values of
the compounds were correlated with some theoretical descriptors and fair 2D-QSAR
models with clogP, SAA, and μ as independent variables were obtained.
Correspondence
Hikmet Agirbas, Department of Chemistry,
Kocaeli University, Izmit, Turkey.
Email: agirbash@yahoo.com
1
INTRODUCTION
agent),[9] oxazaborolidines (antibacterial agent),[10] difluoro-
boron complexes (potential fungicides).[11]
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Microbial infections are among the oldest health problems in
the human history. In the past few decades, scientific research
has led to the discovery of antimicrobial agents. However,
treatments of the resulting diseases have been threatened by
the spreading and resurgence of drug-resistant microbes.[1]
Boron compounds, which have limited studies in the field of
medicinal chemistry, have high potential in drug discovery.
Some biological activities of boron-containing compounds
are reported in the literature.[2] Among these are heterocyclic
aminoboron compounds (antituberculosis agent),[3] boron-
containing GSK2251052 (antimicrobial agent),[4] oxaborole
compounds (antibacterial prototypes),[5] α-amino cyclic bo-
ronates (inhibitors of HCV NS3 protease),[6] benzoxaborole
compounds (anti-inflammatory agent),[7] boronic acid pico-
linate esters (antibacterial agent with anti-inflammatory ac-
tivity),[8] boron-containing thiosemicarbazones (antifungal
The presence of B-O bond in the heterocyclic systems
also shows biological activities.[2,6] Dioxaborepines possess
two B-O bonds, therefore they should be interesting can-
didates for biological activity. Some dioxaborepines were
synthesized from naphthol derivatives and borane com-
pounds.[12,13] However, to our knowledge, no synthetic pro-
cedure of 6-substituted phenyl 3-(4-chlorophenyl)-3a,4,8,8a-
tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazole derivatives
(4) was reported. Considering the structural characteristics
of the [1,3,2]dioxaborepines, the existence of oxygen- and
boron-containing seven-membered non-aromatic heterocy-
cles system, these compounds should possibly show phys-
iological activities. Therefore, we synthesized some new
dioxaborepine derivatives (4) (Scheme 1) and determined
their antimicrobial activities against several bacteria and
fungi.
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Heteroatom Chemistry 2017; 00: e21363;
DOI: 10.1002/hc.21363
wileyonlinelibrary.com/journal/hc
© 2017 Wiley Periodicals, Inc.
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O
NOH
Na2CO3
H
H
+
NH2OH.HCl
r.t.
Cl
Cl
(2)
(1)
Cl2
NOH
C
N
O
Cl
Et3N
r.t.
Cl
Cl
-Et3N.HCl
HO
OH
Cl
R
a:p-OCH3
b:p-CH3
c:m-CH3
d:-H
e:m-OCH3
f:p-Br
g:p-Cl
h:m-Cl
i:m-Br
Cl
N
OH
O
B
N
OH
R
O
Toluene, reflux
O
O
B
j:p-COCH3
k:m-CN
l:p-CN
OH
HO
m:m-NO2
(3)
R
SCHEME 1 Synthesis of
compounds (4a-m)
(4)
Quantitative structure-activity relationship (QSAR) is a
significant tool to correlate biological activity with the struc-
tural properties of the compounds that helps in the process
of development of new molecules and rational drug design.
Hence, we also studied the substituent effects on the anti-
microbial activity of the dioxaborepines to obtain a better
understanding of their structure-antimicrobial activity rela-
tionships. In addition, we applied 2D-QSAR analysis to see
the relations of the molecular descriptors with the activities.
form 4-chloro-N-hydroxybenzimidoyl chloride. The solu-
tion was kept in the fridge overnight, upon which the sol-
vent was removed under reduced pressure. The residue
4-chloro-N-hydroxybenzimidoyl chloride was reacted with
triethylamine and cis-2-butene-1,4-diol in dichloromethane
at room temperature to give (3-(4-chlorophenyl)-4,5-dihyd
roisoxazole-4,5-diyl)dimethanol (3). The residual solid (3)
was subjected to flash column chromatography and separated
from dimerization products (yield: 38%). Then, compound
(3) was reacted with substituted phenylboronic acid in tolu-
ene to yield corresponding 6-substituted phenyl 3-(4-chlor
ophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino[5,6-d]
isoxazoles (4a-m). The yields were between 34% and 94%.
IR stretching frequencies of new compounds (4a-m) were
found at about 1590 cm−1 for C=N bonds and about 1300 cm−1
for B–O bonds. In the 1H NMR spectra of the dioxaborepine
rings, both OCH2 protons gave multiplets between 4.60 and
4.30 ppm and also both CH protons gave multiplets between
5.10 and 4.10 ppm. Only one group of OCH2 protons of com-
pounds (4c) and (4j) gave doublets at 4.60 ppm (J=4.44 Hz)
2
RESULTS AND DISCUSSION
Chemistry
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2.1
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For the synthesis of compounds (2-4) (Scheme 1), p-
chlorobenzaldehyde (1) was reacted with hydroxylamine hy-
drochloride to give p-chlorobenzaldehyde oxime (2) in 56%
yield.Then,chlorinationwasfollowed:p-chlorobenzaldehyde
oxime was reacted with chlorine gas in anhydrous chloro-
form at 0°C, until a certain weight increase was obtained to

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and at 4.63 ppm (J=3.50 Hz), respectively. In the 13C NMR
spectra of the dioxaborepine rings, the peaks of C=N, OCH,
OCH2, and CH carbons were observed at about 156, 82, 63-
61, and 52 ppm, respectively. As an example, 11B NMR spec-
trum of compound (4h) was taken and a singlet at 26.78 ppm
was observed. The purity of all the new compounds (4a-m)
was checked by elemental analysis. The structure of com-
pound (4j) was confirmed by single crystal X-ray diffraction.
Bands of NOH and C=N groups of oxime (2) and C=N
and OH groups of compound (3) were followed by IR spectra.
bond angles (°) and dihedral angles (°) were found in agree-
ment with the calculated (DFT, B3LYP/6-311++G(d,p))
values.
The molecular structure of 4j with the atom labeling is
shown in Figure 1. The isoxazole ring make dihedral angles
of 31.97(13)° and 55.39(14)° with the two phenyl rings. The
dihedral angle of the phenyl rings is 31.12(20)°. Molecules
of 4j are linked into sheets by the combination of C–H···O
hydrogen bonds and C–H···π interactions. Atom C9 atom acts
as hydrogen-bond donor, via atom H9B, to atom O4 in the
molecule at (−x, −y+2, −z+1), forming a centrosymmet-
2
ricR2 (20) ring centered at (0, 1, 1/2). Compound (4j) also
2.2
Computational details
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contains two C–H···π interactions. The C19-H19B···Cg(2)ii
2
For the geometrical optimization of compound (4j), the
calculations have been carried out by DFT method on the
basis of B3LYP exchange-correlation functional with
6-311++G(d,p) basis set. All the calculations were carried
out with the restricted closed-shell formalism. Gaussian 03W
program[14] was used for the calculations.
interaction produce centrosymmetricR2 (28) ring, while
the C19-H19C···Cg(3)iii interaction produce centrosymmet-
2
ricR2 (10) ring ((ii) 1-x, 1-y, 1-z; (iii) –x, 1-y, 1-z). The com-
bination of these interactions produce 2D supramolecular
network.
2.4
Biological activity
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2.3
X-ray diffraction characterization
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The antimicrobial activity of compounds (4a-m) have been
Compound (4j) was crystallized from dichloromethane,
yielding single crystals suitable for X-ray diffraction analy-
sis. Details of data collection and crystal structure deter-
minations and selected atomic parameters are given in
Tables 1 and 2. The values of selected bond distances (Å),
evaluated against a panel of microorganisms including three
TABLE 2 Selected bond distances (Å), bond angles (°), and
dihedral angles (°) for 4j
Parameters
X-ray
DFT
TABLE 1 Crystal data and structure refinement parameters for 4j
Bond distances (Å)
B1–O2
Empirical formula
C19H17BClNO4
1.351(5)
1.371
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
369.60
Triclinic
P-1
C18–O4
C8–O1
B1–O3
C12–B1
C1–Cl1
N1–O1
C9–O2
Bond angles (°)
O2–B1–O3
C7–N1–O1
B1–O3–C10
O2–B1–C12
N1–O1–C8
O3–B1–C12
B1–O2–C9
Dihedral angles (°)
C13–C12–B1–O3
C17–C12–B1–O2
C5–C4–C7–N1
C3–C4–C7–C11
1.214(5)
1.453(5)
1.362(5)
1.565(6)
1.743(4)
1.409(4)
1.422(5)
1.217
1.443
1.370
1.573
1.754
1.399
1.423
6.1173 (6)
10.0797 (11)
15.1193 (17)
108.899 (5)
97.061 (4)
94.260 (5)
868.83 (16)
2
1.413
0.25
3.4-24.3
21433
3641
α (°)
β (°)
γ (°)
126.7(4)
109.5(3)
124.9(3)
116.4(3)
110.1(3)
116.8(4)
127.3(3)
128.7
V (Å3)
110.1
132.7
115.4
108.0
115.7
133.4
Z
Dc (g cm−3
)
μ (mm−1
θ range (°)
)
Measured refls.
Independent refls.
Rint
0.099
1.07
0.071/0.226
0.36/−0.31
3.6(6)
3.4(6)
−31.0(5)
−33.0(5)
2.4
2.5
26.1
22.6
S
R1/wR2
Δρmax/Δρmin (eÅ−3
)

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optimization of all the compounds (4a-m) has been car-
ried out by ab initio (RHF/3-21G) method incorporated in
the Hyperchem package.a Theoretical descriptors: surface
area approx (SAA), molecular volume (MV), molar refrac-
tivity (MR), polarizability (polar), magnitude of dipolar
moment (μ), and the calculated log of octanol-water parti-
tion coefficient (clogP) of the compounds were also com-
puted by Hyperchem software (RHF/3-21G) method
(Table 5).
When SAA, clogP and μ were used as independent
variables, three significant correlations with pMIC val-
ues were obtained (Table 4; eqs. 3, 4, and 5). The gen-
eral quality of 2D-QSAR models was given by r and r2
(correlation coefficients), s (standard deviations) of the
regression equations, F value (F-statistical analysis;
Fischer test), P (probability value) and n (number of data
points). The predictability of each model was assessed by
the cross-validated correlation coefficient (r2 adj). For the
structure-reactivity models, a value of r2 adj above .38
was considered.
To evaluate the predictive power of the models, the MIC
values have been separated into the training and test sets. The
regression equations of the training sets gave fair internal
cross-validation (r2 adj) values and good coefficient of deter-
mination (r2) values (Table 6). A good predicted values of the
test sets have also been obtained.
FIGURE 1 The molecular structure of (4j) showing the atom
numbering scheme
Grampositivebacteria(Staphylococcusaureus, Enterococcus
faecalis, and Streptococcus mutans), two Gram negative bac-
teria (Pseudomonas aeruginosa and Escherichia coli) and
one fungi (Candida albicans) by their minimal inhibitory
concentrations (MIC) via broth microdilution susceptibil-
ity tests.[15–17] The results are given in Table 3. Among the
tested compounds, (4h), (4i), (4k) and (4m) have been found
to be the most active derivatives against fungi (C. albicans)
with the MIC value of 50 μg/mL. All the compounds ex-
hibited antimicrobial activity with MIC values between 50
and 100 μg/mL against S. aureus, E. coli, E. faecalis, S. mu-
tans and C. albicans. (4h) and (4i) are the most active de-
rivatives against all microorganisms. The results showed that
the compounds (4) have the most effective activity against
Pseudomonas aeruginosa among the microorganisms (MIC:
25-50 μg/mL).
The correlation matrix for the descriptors has been ob-
tained and cross-relations between the descriptors have not
been observed. Hence, the values of the descriptors are safe
to be used in the multilinear regression relationship.[21,22]
The statistical calculations were performed by SigmaPlot
program package.
3
CONCLUSIONS
|
In this work, thirteen 6-substituted phenyl 3-(4-chlorop
henyl)-3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino[5,6-d]
isoxazoles (4) were synthesized successfully from the reac-
tions of (3-(4-chlorophenyl)-4,5-dihydroisoxazole-4,5-diyl)
dimethanol (3) with phenylboronic acid derivatives and their
antimicrobial activities were evaluated. Antibacterial and an-
tifungal activities of the dioxaborepine derivatives have been
screened against three Gram-positive bacteria, two Gram-
negative bacteria, and one fungi. Interestingly, all the com-
pounds exhibited better results against P. aeruginosa (MIC
of 25-50 μg/mL) than S. aureus, E. faecalis, E. coli, S. mu-
tans, and C. albicans. P. aeruginosa bacteria shows the most
dramatic resistance problems related to nosocomial infec-
tions and multiresistant strains.[23] In addition, quantitative
2.5
QSAR analysis
|
We have studied linear regression of molecular descrip-
tors against antimicrobial activities of compounds (4a-m).
The biological activities, reported as MIC values (Table 3),
were converted to pMIC (−logMIC) on a molar basis and
used as dependent variables to obtain the linear relation-
ship. The pMIC values were first correlated with Hammett
polar substituent constant (σ)[18] together with lipophilic
constant (π)[19] as independent variables and gave four sta-
tistical significant 2D-QSAR models (Table 4; eqs. 1, 2, 6,
and 7).
Computational theoretical studies range from molecu-
lar design to architectural design[20] and this type of stud-
ies assist to control the experimental data. Hence, we
decided to add theoretical descriptors to the structure-
antimicrobial activity relationship study. The geometrical
aHyperChem 7.2 for Windows, Hypercube Inc., USA,
2002.

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TABLE 4 Significant 2D-QSAR models obtained for antimicrobial activity of 6-substituted phenyl 3-(4-chlorophenyl)-3a,4,8,8a-
tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazoles (4a-m)
Statistic parameter
Microorganisms
Equations
Regression equation
n
r
r2
r2 adj.
s
P
F
S. aureus
1
pMICS.a.=0.38( 0.10)σ+0.16( 0.05)
13
.810
.656
.587
0.112
.0048
9.561
16.977
17.294
7.807
6.401
4.669
6.057
π+0.57( 0.04)
P. aeruginosa
2
3
4
5
6
7
pMICP.a.=−0.11( 0.08)σ+0.24( 0.04)
π+0.97( 0.03)
pMICP.a.=−0.08( 0.02)μ−0.006( 0.002)
SAA+3.22( 0.89)
pMICP.a.=−0.009( 0.003)
SAA+0.03( 0.02) clogP+4.35( 1.04)
pMICE.c.=−0.06( 0.03)μ−0.006( 0.003)
SAA+2.95( 1.25)
pMICS.m.=0.21( 0.08)σ+0.10( 0.05)
π+0.73( 0.04)
pMICC.a.=0.32( 0.10)σ+0.12( 0.06)
π+0.53( 0.04)
13
13
13
13
13
13
.878
.880
.780
.749
.694
.740
.772
.775
.609
.561
.483
.547
.727
.730
.531
.473
.380
.457
0.086
0.086
0.113
0.119
0.095
0.116
.0006
.0006
.0091
.0162
.0370
.0189
E. coli
S. mutans
C. albicans
TABLE 5 Theoretical descriptors of
6-substituted phenyl 3-(4-chlorophenyl)-
3a,4,8,8a-tetrahydro-[1,3,2]
Compound
clogP
SAA (Ǻ2)
MV (Ǻ3)
MR (Ǻ3)
Polar (Ǻ3)
μ (D)
4a
4b
4c
4d
4e
4f
4g
4h
4i
1.21
2.36
2.36
2.20
1.21
2.25
1.98
1.98
2.25
2.13
1.92
1.92
−2.48
351.86
339.36
339.37
315.70
352.08
337.42
333.06
333.00
337.36
360.84
344.35
344.38
345.57
305.85
298.20
298.24
281.55
306.22
303.59
296.69
296.87
303.73
315.88
299.54
299.47
300.60
98.75
96.65
96.65
92.37
98.75
99.90
97.09
97.09
99.90
101.92
97.35
97.35
97.59
36.63
36.00
36.00
34.16
36.63
36.79
36.09
36.09
36.79
37.92
36.01
36.01
36.00
2.75
1.51
1.42
1.15
2.12
1.62
2.47
1.87
1.04
3.65
3.69
4.41
4.41
dioxaborepino[5,6-d]isoxazoles (4a-m) used
for the regression analyses
4j
4k
4l
4m
structure-activity relationship studies were carried out and
this allowed us to draw the following conclusions about the
antimicrobial activities of the dioxaborepine derivatives:
(i) 6-substituted phenyl 3-(4-chlorophenyl)-3a,4,8,8a-tetra
hydro-[1,3,2]dioxaborepino[5,6-d]isoxazoles (4a-m) gave
a fair 2D correlations with the electronic (σ) and lipophilic
(π) descriptors against S. aureus, P. aeruginosa, S. mutans,
and C. albicans; (ii) all the dioxaborepine derivatives gave a
fair 2D correlations with some theoretical descriptors (clogP,
SAA, and μ) against P. aeruginosa and E. coli. The QSAR
study has given us critical information regarding the struc-
tural properties of 6-substituted phenyl 3-(4-chlorophenyl)-
3a,4,8,8a-tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazoles.
Therefore, such compounds can be improved and used for
the design of more potent and a new class of antibacterial and
antifungal agents in the future.
4
4.1
EXPERIMENTAL
General
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|
Melting points were determined on Electrothermal 9200 ap-
paratus and are uncorrected. The FT IR spectra (ν, cm−1)
were recorded on Bruker Alpha-P spectrometer in the re-
gion of 4000-400 cm−1. H and 13C NMR spectra (δ, ppm)
1
were recorded on Varian Mercury Plus (300 MHz) High
Performance Digital FT-NMR Spectrometer using CDCl3
with TMS as an internal standard. Chemical shifts (δ) are

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TABLE 6 The results of the application of training and test sets to 2D−QSAR models (Eqs. 1-7 in Table 4)
Statistic parameter
Regression equation of
training set
Predicted value
(MIC)
Eqs.
Training set
Test set (MIC)
r2
r2 adj.
s
1
4a, 4c, 4e, 4g, 4i, 4k, 4m
pMICS.a.=0.42σ+0.11π+0.60
4b(100)
4d(100)
4f(50)
4h(50)
4j(50)
4l(100)
4b(25)
4d(25)
4f(25)
4h(25)
4j(50)
4l(50)
4b(25)
4d(25)
4f(25)
4h(25)
4j(50)
4l(50)
4b(25)
4d(25)
4f(25)
4h(25)
4j(50)
4l(50)
4b(100)
4d(50)
4f(50)
4h(50)
4j(100)
4l(100)
4b(50)
4d(50)
4f(50)
4h(50)
4j(50)
4l(50)
89
82
63
49
63
51
29
40
26
24
62
59
280
85
310
234
1149
580
61
20
66
49
179
81
558
170
619
468
2294
1157
70
72
54
38
66
0.827
0.741
0.091
0.078
0.042
0.093
0.042
0.107
0.118
2
3
4
5
6
7
4a, 4c, 4e, 4g, 4i, 4k, 4m
4a, 4c, 4e, 4g, 4i, 4k, 4m
4a, 4c, 4e, 4g, 4i, 4k, 4m
4a, 4c, 4e, 4g, 4i, 4k, 4m
4a, 4c, 4e, 4g, 4i, 4k, 4m
4a, 4c, 4e, 4g, 4i, 4k, 4m
pMICP.a.=−0.06σ+0.
29π+0.91
0.859
0.959
0.802
0.959
0.693
0.717
0.788
0.939
0.703
0.939
0.540
0.576
pMICP.a.=−0.07μ
− 0.02SAA+6.98
pMICP.a.=−0.02SAA+0.02
clogP+7.49
pMICE.c.=−0.07μ
− 0.02SAA+6.68
pMICS.m.=0.29σ+0.15π+0.66
pMICC.a.=0.43σ+0.05π+0.57
55
102
88
77
60
4b(100)
4d(100)
4f(100)
4h(50)
4j(100)
4l(100)
63
51
r2 coefficient of determination, r2 adj internal cross-validation, s the standard deviation.

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reported in ppm. Silica Gel (Fluka or Merck) was used for
column chromatography.
4.4
Synthesis of 3-(4-chlorophenyl)-
|
6-phenyl-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4d) (general
procedure for 4a-m)
4.2
Synthesis of p-chlorobenzaldehyde
|
oxime (2)
(3-(4-Chlorophenyl)-4,5-dihydroisoxazole-4,5-diyl)dimetha-
nol 3 (0.50 g, 2.07 mmol) and phenylboronic acid (0.25 g,
2.07 mmol) were dissolved in toluene (50 mL) and the
solution was refluxed for 16 hour. After the solvent was
evaporated under vacuum. The residual was crystallized
from dichloromethane-petroleum ether mixture to give
0.49 g of 3-(4-chlorophenyl)-6-phenyl-3a,4,8,8a-tetrahyd
ro-[1,3,2]dioxaborepino[5,6-d]isoxazole (4d), yield 73%,
mp 171-173.5°C. IR (ATR), υ (cm−1): 1597 (C=N), 1303
A solution of hydroxylamine hydrochloride (3.93 g,
56 mmol) in water (11 mL) and a solution of anhydrous
sodium carbonate (3.05 g, 28 mmol) in water (17 mL)
were mixed and stirred. p-Chlorobenzaldehyde 1 (8.00 g,
57 mmol) in chloroform (22 mL) was added to this mixture.
The reaction was stirred at room temperature for 24 hour.
After the reaction was complete, chloroform phase was sep-
arated and the water phase was extracted with chloroform
(15 mL) three times, then combined with the chloroform
phase. The collected chloroform solution was dried with
anhydrous CaCl2 overnight. The solvent was evaporated
under vacuum. The precipitate was crystallized from ethyl
acetate-petroleum ether (1:4) mixture to give 4.96 g of p-
chlorobenzaldehyde oxime 2, yield 56%, mp 107-110.5°C
(Lit.[24]: 105.5-108°C); IR (ATR), υ (cm−1): 3302 (NOH),
1595 (C=N).
1
(B-O). H NMR (CDCl3), δ (ppm): 7.73-7.56 (m, aromatic,
4H), 7.43-7.38 (m, aromatic, 3H), 7.32-7.26 (m, aromatic,
2H), 5.16-5.09 (m, 1H, OCH), 4.66-4.54 (m, 2H, OCH2),
4.51-4.37 (m, 2H, OCH2), 4.23-4.16 (m, 1H, CH). 13C NMR
(CDCl3), δ (ppm): 156.0 (C=N), 136.6, 134.5, 131.1, 129.5,
128.4, 127.7, 127.0, 82.4 (OCH), 62.7, 61.1 (OCH2), 52.1
(CH). Anal. calcd for C17H15BClNO3: C, 62.33; H, 4.62; N,
4.28; found: C, 62.71; H, 4.94; N, 4.25.
4.5
Spectroscopic and analytical data of
4.3
Synthesis of (3-(4-chlorophenyl)-4,5-
|
|
compounds (4)
dihydroisoxazole-4,5-diyl)dimethanol (3)
p-Chlorobenzaldehyde oxime (2) (2.01 g, 13 mmol) was
dissolved in anhydrous chloroform (50 mL). Cooling the
solution in an ice bath, chlorine gas was passed through
the solution until the determination of the 1.00 g weight
increase. The solution was kept in the fridge over night.
Then, the solution was evaporated under reduced pressure at
40°C. The residue of 4-chloro-N-hydroxybenzimidoyl chlo-
ride (2.32 g, 12.2 mmol) was dissolved in dichloromethane
(50 mL). The solution of 4-chloro-N-hydroxybenzimidoyl
chloride was added dropwise to the solution of triethyl-
amine (1.70 g, 17 mmol) and cis-2-butene-1,4-diol (1.51 g,
17 mmol) in dichloromethane (50 mL). The mixture was
stirred in an ice bath for 3 days and the product formation
was followed by TLC. The precipitated salt was removed by
filtration. Dichloromethane was evaporated under reduced
pressure at 40°C. The residual solid was subjected to flash
column chromatography (eluant: ethyl acetate:petroleum
ether (1:5)). The crude product was crystallized from ethyl
acetate:petroleum ether (1:3.5) mixture to give 1.13 g of (3-
(4-chlorophenyl)-4,5-dihydroisoxazole-4,5-diyl)dimethanol
(3), yield 38%, mp 116-119°C. IR (ATR), υ (cm−1): 3292
(O–H), 1575 (C=N). 1H NMR (CDCl3), δ (ppm): 7.66-7.26
(m, aromatic, 4H), 4.81-4.76 (m, 1H, OCH), 4.26-4.08 (m,
2H, OCH2), 3.90-3.83 (m, 3H, CH and OCH2), 3.17 (s, 2H,
OH).
4.5.1
3-(4-Chlorophenyl)-6-(4-
|
methoxyphenyl)-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4a)
Yield 34%, mp 140-145°C. IR (ATR), υ (cm−1): 1599 (C=N),
1294 (B-O). H NMR (CDCl3), δ (ppm): 7.67-7.58 (m, aro-
1
matic, 4H), 7.43-7.39 (m, aromatic, 2H), 6.83 (d, J=8.17 Hz,
aromatic, 2H), 5.15-5.09 (m, 1H, OCH), 4.60-4.56 (m, 2H,
OCH2), 4.44-4.40 (m, 2H, OCH2), 4.22-4.17 (m, 1H, CH),
3.81 (s, 3H, OCH3). 13C NMR (CDCl3), δ (ppm): 162.1 (Ar-
O), 156.0 (C=N), 136.6, 136.3, 129.5, 128.4, 127.1, 113.2,
82.4 (OCH), 62.6, 61.0 (OCH2), 55.3 (OCH3), 52.2 (CH).
Anal. calcd for C18H17BClNO4: C, 60.46; H, 4.79; N, 3.92;
found: C, 60.84; H, 4.52; N, 4.38.
4.5.2
3-(4-Chlorophenyl)-6-p-
|
tolyl-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4b)
Yield 61%, mp 155-157°C. IR (ATR), υ (cm−1): 1595 (C=N),
1
1307 (B-O). H NMR (CDCl3), δ (ppm): 7.62-7.58 (m, aro-
matic, 4H), 7.42-7.40 (m, aromatic, 2H), 7.13 (d, J=7.54 Hz,
aromatic, 2H), 5.16-5.09 (m, 1H, OCH), 4.61-4.57 (m, 2H,
OCH2), 4.45-4.42 (m, 2H, OCH2), 4.24-4.17 (m, 1H, CH),
2.35 (s, 3H, CH3). 13C NMR (CDCl3), δ (ppm): 155.9 (C=N),
141.2, 136.6, 134.6, 129.5, 128.6, 128.4, 127.1, 82.4 (OCH),
62.6, 61.0 (OCH2), 52.2 (CH), 21.9 (CH3). Anal. calcd for

PIR et al.
9 of 12
|
C18H17BClNO3: C, 63.29; H, 5.02; N, 4.10; found: C, 63.41;
H, 4.95; N, 4.09.
(d, J=7.86 Hz, aromatic, 2H), 5.14-5.08 (m, 1H, OCH),
4.60-4.48 (m, 2H, OCH2), 4.41-4.35 (m, 2H, OCH2), 4.21-
4.16 (m, 1H, CH). 13C NMR (CDCl3), δ (ppm): 156.0
(C=N), 137.3, 136.7, 136.0, 129.5, 128.3, 128.0, 127.0,
82.4 (OCH), 62.8, 61.2 (OCH2), 52.0 (CH). Anal. calcd
for C17H14BCl2NO3: C, 56.40; H, 3.90; N, 3.87; found: C,
56.68; H, 4.20; N, 3.86.
4.5.3
3-(4-Chlorophenyl)-6-m-
|
tolyl-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4c)
Yield 83%, mp 120-123°C. IR (ATR), υ (cm−1): 1592
1
(C=N), 1307 (B-O). H NMR (CDCl3), δ (ppm): 7.61-7.51
4.5.7
6-(3-Chlorophenyl)-3-(4-
|
(m, aromatic, 4H), 7.41 (d, J=8.34 Hz, aromatic, 2H), 7.26-
7.22 (m, aromatic, 2H), 5.14-5.07 (m, 1H, OCH), 4.60 (d,
J=4.44 Hz, 2H, OCH2), 4.50-4.40 (m, 2H, OCH2), 4.20-4.13
(m, 1H, CH), 2.33 (s, 3H, CH3). 13C NMR (CDCl3), δ (ppm):
156.0 (C=N), 137.1, 136.6, 135.2, 131.9, 131.6, 129.5,
128.4, 127.7, 127.0, 82.5 (OCH), 62.7, 61.1 (OCH2), 52.1
(CH), 21.6 (CH3). Anal. calcd for C18H17BClNO3: C, 63.29;
H, 5.02; N, 4.10; found: C, 63.00; H, 5.24; N, 4.15.
chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4h)
Yield 85%, mp 156.5-158°C. IR (ATR), υ (cm−1): 1595
(C=N), 1303 (B-O). 1H NMR (CDCl3), δ (ppm): 7.66-7.55
(m, aromatic, 4H), 7.43-7.35 (m, aromatic, 3H), 7.27-7.21
(m, aromatic, 1H), 5.13-5.07 (m, 1H, OCH), 4.61-4.52 (m,
2H, OCH2), 4.49-4.34 (m, 2H, OCH2), 4.19-4.13 (m, 1H,
CH). 13C NMR (CDCl3), δ (ppm): 156.0 (C=N), 136.7,
134.4, 134.0, 132.5, 131.1, 129.6, 129.2, 128.3, 126.9,
82.4 (OCH), 62.8, 61.2 (OCH2), 52.0 (CH). 11B NMR
(CDCl3), δ (ppm): 26.78. Anal. calcd for C17H14BCl2NO3:
C, 56.40; H, 3.90; N, 3.87; found: C, 56.56; H, 4.10; N,
3.85.
4.5.4
3-(4-Chlorophenyl)-6-(3-
|
methoxyphenyl)-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4e)
Yield 92%, mp 144-148°C. IR (ATR), υ (cm−1): 1597 (C=N),
1
1301 (B-O). H NMR (CDCl3), δ (ppm): 7.61-7.40 (m, aro-
matic, 4H), 7.31-7.22 (m, aromatic, 3H), 6.98-6.95 (m,
aromatic, 1H), 5.15-5.10 (m, 1H, OCH), 4.61-4.58 (m, 2H,
OCH2), 4.46-4.41 (m, 2H, OCH2), 4.23-4.19 (m, 1H, CH),
3.79 (s, 3H, OCH3). 13C NMR (CDCl3), δ (ppm): 159.1 (Ar-
O), 155.9 (C=N), 136.6, 129.5, 128.9, 128.4, 127.0, 126.9,
118.7, 117.5, 82.4 (OCH), 62.7, 61.1 (OCH2), 55.4 (OCH3),
52.1 (CH). Anal. calcd for C18H17BClNO4: C, 60.46; H, 4.79;
N, 3.92; found: C, 60.80; H, 4.49; N, 4.43.
4.5.8
6-(3-Bromophenyl)-3-(4-
|
chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4i)
Yield 62%, mp 161-165°C. IR (ATR), υ (cm−1): 1594 (C=N),
1303 (B-O). 1H NMR (CDCl3), δ (ppm): 7.82-7.50 (m, aro-
matic, 5H), 7.42-7.39 (m, aromatic, 2H), 7.26-7.15 (m, ar-
omatic, 1H), 5.12-5.05 (m, 1H, OCH), 4.60-4.52 (m, 2H,
OCH2), 4.49-4.33 (m, 2H, OCH2), 4.18-4.12 (m, 1H, CH).
13C NMR (CDCl3), δ (ppm): 156.0 (C=N), 137.4, 136.7,
134.0, 133.0, 129.6, 128.3, 126.9, 122.5, 82.4 (OCH), 62.8,
61.3 (OCH2), 52.0 (CH). Anal. calcd for C17H14BBrClNO3:
C, 50.23; H, 3.47; N, 3.45; found: C, 50.24; H, 3.80; N,
3.48.
4.5.5
6-(4-Bromophenyl)-3-(4-
|
chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4f)
Yield 94%, mp 183.5-184.5°C. IR (ATR), υ (cm−1): 1585
1
(C=N), 1303 (B-O). H NMR (CDCl3), δ (ppm): 7.66-7.39
(m, aromatic, 8H), 5.14-5.08 (m, 1H, OCH), 4.60-4.47 (m,
2H, OCH2), 4.41-4.35 (m, 2H, OCH2), 4.21-4.15 (m, 1H,
CH). 13C NMR (CDCl3), δ (ppm): 156.0 (C=N), 136.7,
136.2, 130.9, 129.6, 128.3, 126.9, 126.1, 82.4 (OCH), 62.8,
61.2 (OCH2), 52.0 (CH). Anal. calcd for C17H14BBrClNO3:
C, 50.23; H, 3.47; N, 3.45; found: C, 50.25; H, 3.76; N, 3.88.
4.5.9
1-(4-(3-(4-Chlorophenyl)-3a,4,8,8a-
|
tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazol-
6-yl)phenyl)ethanone (4j)
Yield 87%, mp 190-192.5°C. IR (ATR), υ (cm−1): 1595
(C=N), 1299 (B-O). H NMR (CDCl3), δ (ppm): 7.87-
1
7.76 (m, aromatic, 4H), 7.59-7.55 (m, aromatic, 2H), 7.42-
7.39 (m, aromatic, 2H), 5.15-5.10 (m, 1H, OCH), 4.63 (d,
J=3.50 Hz, 2H, OCH2), 4.57-4.37 (m, 2H, OCH2), 4.23-4.18
(m, 1H, CH), 2.59 (s, 3H, CH3). 13C NMR (CDCl3), δ (ppm):
199.0 (C=O), 156.0 (C=N), 138.8, 136.7, 134.7, 129.6,
128.3, 127.3, 126.9, 82.4 (OCH), 62.9, 61.3 (OCH2), 52.0
(CH), 27.1 (CH3). Anal. calcd for C19H17BClNO4: C, 61.74;
H, 4.64; N, 3.79; found: C, 62.03; H, 3.82; N, 3.93.
4.5.6
3,6-Bis(4-chlorophenyl)-3a,4,8,8a-
|
tetrahydro-[1,3,2]dioxaborepino[5,6-d]
isoxazole (4g)
Yield 65%, mp 162-163.5°C. IR (ATR), υ (cm−1): 1591
(C=N), 1302 (B-O). H NMR (CDCl3), δ (ppm): 7.63-7.55
1
(m, aromatic, 4H), 7.40 (d, J=8.39 Hz, aromatic, 2H), 7.27

10 of 12
PIR et al.
|
SHELXS-97[25] and refined by full-matrix least-squares
methods on F2 using SHELXL-97[25] from within the
WINGX[26] suite of software. Hydrogen atoms bonded to C
were refined using a riding model, with C–H=0.93-0.97 Å.
Molecular diagrams were created using MERCURY.b
Supramolecular analyses were made and the diagrams were
prepared with the aid of PLATON.[27]
Crystallographic data for the structural analysis has been
deposited with the Cambridge Crystallographic Data Centre,
CCDC No. 1456825. Copies of this information may be ob-
tained free of charge from the Director, CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (fax:+44-1223-336033;
e-mail:deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.
ac.uk).
4.5.10
3-(3-(4-Chlorophenyl)-3a,4,8,8a-
|
tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazol-
6-yl)benzonitrile (4k)
Yield 56%, mp 175-179°C. IR (ATR), υ (cm−1): 1596 (C=N),
1298 (B-O). 1H NMR (CDCl3), δ (ppm): 7.90 (d, J=8.79 Hz,
aromatic, 2H), 7.68-7.55 (m, aromatic, 3H), 7.44-7.37 (m,
aromatic, 3H), 5.12-5.09 (m, 1H, OCH), 4.65-4.55 (m, 2H,
OCH2), 4.42-4.36 (m, 2H, OCH2), 4.22-4.16 (m, 1H, CH).
13C NMR (CDCl3), δ (ppm): 155.9 (C=N), 138.7, 138.3,
136.8, 134.3, 129.6, 128.4, 128.3, 126.8, 119.4, 111.8 (CN),
82.4 (OCH), 62.9, 61.4 (OCH2), 51.9 (CH). Anal. calcd for
C18H14BClN2O3: C, 61.32; H, 4.00; N, 7.95; found: C, 61.06;
H, 3.03; N, 7.95.
4.5.11
4-(3-(4-Chlorophenyl)-3a,4,8,8a-
|
4.7
Biological assays
|
tetrahydro-[1,3,2]dioxaborepino[5,6-d]isoxazol-
6-yl)benzonitrile (4l)
The antibacterial activities of thirteen dioxaborepines (4a-m)
have been determined by broth microdilution susceptibil-
ity test, outlined by the Clinical and Laboratory Standards
Institute M7-A7.[17] For each compound, the minimal inhibi-
tory concentrations (MIC) were determined against S. aureus
(ATCC 25983), E. faecalis (ATCC 29212), P. aeruginosa
(ATCC 27853), and E. coli (ATCC 25922).
The MIC values for each compound were also determined
against S. mutans (ATCC 25175). For this, Mueller Hinton
broth was used with 2%-5% lyophilize horse blood for the
determination of microdilutions well containing the lowest
concentration that has completely inhibited visible bacterial
growth which is recommended standards of the Committee
Laboratory Standards Institute.[16]
The antifungal activities of the compounds against
Candida albicans (ATCC 90028) were also determined by
using broth microdilution susceptibility test outlined by
Clinical and Laboratory Standards Institute M27-A2.[15]
Multiwell microdilution plates (96 U-shaped wells) (ster-
ile and disposable) were used for broth microdilution pro-
cedures. The stock solutions were prepared in pure ethanol
(Sigma). Ethanol had no effect on the microorganisms in the
concentrations studied.
Yield 46%, mp 176-179°C. IR (ATR), υ (cm−1): 1596 (C=N),
1296 (B-O). 1H NMR (CDCl3), δ (ppm): 7.76 (d, J=8.34 Hz,
aromatic, 2H), 7.60-7.55 (m, aromatic, 4H), 7.44-7.36 (m,
aromatic, 2H), 5.15-5.09 (m, 1H, OCH), 4.69-4.55 (m, 2H,
OCH2), 4.42-4.36 (m, 2H, OCH2), 4.23-4.16 (m, 1H, CH). 13C
NMR (CDCl3), δ (ppm): 155.9 (C=N), 136.7, 134.9, 131.2,
129.6, 128.3, 126.9, 119.4, 114.2 (CN), 82.4 (OCH), 63.0,
61.4 (OCH2), 51.9 (CH). Anal. calcd for C18H14BClN2O3: C,
61.32; H, 4.00; N, 7.95; found: C, 61.07; H, 3.32; N, 8.00.
4.5.12
3-(4-Chlorophenyl)-6-(3-
|
nitrophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazole (4m)
Yield 71%, mp 196-200°C. IR (ATR), υ (cm−1): 1595
1
(C=N), 1294 (B-O). H NMR (CDCl3), δ (ppm): 8.24 (d,
J=8.19 Hz, aromatic, 1H), 8.00 (d, J=8.46 Hz, aromatic,
1H), 7.61-7.41 (m, aromatic, 6H), 5.17-5.10 (m, 1H, OCH),
4.67-4.55 (m, 2H, OCH2), 4.45-4.37 (m, 2H, OCH2), 4.24-
4.16 (m, 1H, CH). 13C NMR (CDCl3), δ (ppm): 155.9 (C=N),
147.9, 140.6, 136.8, 129.6, 129.3, 128.7, 128.3, 126.8, 122.5,
82.4 (OCH), 62.9, 61.4 (OCH2), 52.0 (CH). Anal. calcd for
C17H14BClN2O5: C, 54.80; H, 3.79; N, 7.52; found: C, 54.44;
H, 2.92; N, 7.62.
4.8
Dilutions of the compounds and
|
inoculum preparation
For the antibacterial activity studies, all the dilutions of di-
oxaborepine solutions were done in the wells of microdilu-
tion plates by Mueller-Hinton Broth (Oxoid). For S. mutans
antimicrobial activity tests, Mueller-Hinton Broth (Oxoid)
with lyophilized horse blood was used. The concentrations
were 1600, 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56,
4.6
X-ray diffraction analysis of 1-(4-(3-
|
(4-chlorophenyl)-3a,4,8,8a-tetrahydro-[1,3,2]
dioxaborepino[5,6-d]isoxazol-6-yl)phenyl)
ethanone (4j)
Suitable crystal of (4j) was selected for data collection
which was performed on a BrukerD8 QUEST diffractometer
equipped with a graphite-monochromatic Mo-Kα radiation at
296 K. The structure was solved by direct methods using
bMercury, Version 3.0; CCDC, available online via ccdc.
cam.ac.uk/products/mercury.

PIR et al.
11 of 12
|
0.78, 0.39, 0.19, 0.09, and 0.04 μg/mL. Ampicillin and cip-
rofloxacin were used as reference compounds, obtained from
the manufacturers.
For the antifungal activity studies, all the dilutions of the
compounds were done with RPMI medium with L-glutamine
buffered, pH 7, with MOPS (Sigma) in the wells of micro-
dilution plates. The concentrations were the same as above.
The fluconazole was used as a reference compound, obtained
from the manufacturer.
that each well contained 1×103 CFU/mL of inoculum. The
MIC plates were incubated at 37°C for 48 hour. The end point
was determined when the concentration produced optically
clear wells (MIC-0) compared with that of drug-free growth
control. To control the reliability of the results, fluconazole
was used as reference antifungal reagent. The parameter of
this reagent was compared with the data which was obtained
from the method applied in this study. Every experiment for
the antibacterial and antifungal assays was replicated twice.
MIC values for antimicrobial activities are given in Table 3,
respectively.
After the dilutions of dioxaborepine solutions, standard-
ized inoculum of each bacterium (S. aureus, E. faecalis,
P. aeruginosa, and E. coli) (0.5 Mc Farland standard unit,
1 × 108 CFU/mL; colony forming unit/mL) was prepared.
Then, the solutions were diluted once more (1/10), and final
concentrations became 1×107 CFU/mL. Five microliters from
each dilution was placed into each well containing 100 μL
of dilutions of the compounds, so that each well contained
5×105 CFU/mL of inoculum. All the inoculated plates were
incubated at 35°C for 16-20 hour. The lowest concentrations
of the compounds which prevents visible growth were con-
sidered to be the minimal inhibitory concentrations (MIC).
To control the reliability of the results, ampicillin and cipro-
floxacin were used as reference antimicrobial reagents. The
parameters of these reagents were compared with the data
which were obtained from the method applied in this study.
The bacteria (S. mutans) has been cultivated on sheep
agar plate for 36-48 hour at 37°C in 5%-10% CO2, so that
incubation in a candle extinction jar. After diluting the com-
pounds, standardized inoculum of each bacterium (0.5 Mc
Farland standard unit, 1×108 CFU/mL; colony forming unit/
mL) has been prepared in Brain Haert Infusion broth. Then,
the compounds have been diluted once more (1/10), and final
concentrations became 1×107 CFU/mL. Five microliters
from each dilution has been placed into each well contain-
ing 100 μL of dilutions of compounds, so that each well con-
tained 5×105 CFU/mL of inoculum. All the inoculated plates
have been incubated at 35°C for 36-48 hour with 5%-10%
CO2. The lowest concentration of compounds that prevents
visible growth has been considered to be the minimal inhibi-
tory concentration (MIC). Ampicillin has been used as refer-
ence antimicrobial reagent to compare its parameters with the
data which result from the method applied in this work and to
control the reliability of the latter.
ACKNOWLEDGMENTS
The authors thank Scientific and Technological Research
Application and Research Center, Sinop University, Turkey,
for the use of the Bruker D8 QUEST diffractometer.
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