Simple and convenient preparation of some bicyclic alcohols and epoxide derivatives: Promising antibiotic activities of (rac)-dimethyl 3,8-dioxatricyclo[3.2.1.02,4]oct-6-ene-6,7-dicarboxylate

Article abstract of DOI:10.1007/s00044-015-1324-x

The biological activity of alcohol and epoxide groups containing bicyclic skeletal structures was investigated in terms of antibiotic and toxic effects at cellular level. The present protocol, which utilizes readily available starting materials and mild reaction conditions, provides an attractive approach to a series of functionalized bicyclic alcohol and epoxide derivatives with promising biological applications. In this protocol, while some of the bicyclic compounds were synthesized as in the literature, the other bicyclic compounds were synthesized by new synthetic methods. The antibiotic and toxic properties of the synthesized bicyclic compounds were investigated, and some of their biological activities were tested in prokaryotic and eukaryotic systems. (rac)-Dimethyl 3,8-dioxatricyclo[3.2.1.0^2,4]oct-6-ene-6,7-dicarboxylate, one of the synthesized molecules that showed antibiotic activity, was tested in two different living systems (prokaryotic and eukaryotic) in terms of biological and toxic effects at cellular level using a peripheral lymphocyte culture assay. Mitotic index for this compound was calculated in lymphocyte cultures.

Full text of DOI:10.1007/s00044-015-1324-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1324-x
ORIGINAL RESEARCH
Simple and convenient preparation of some bicyclic alcohols
and epoxide derivatives: promising antibiotic activities of (rac)-
dimethyl 3,8-dioxatricyclo[3.2.1.02,4]oct-6-ene-6,7-dicarboxylate
¨
•
•
•
Ozgu¨r Yilmaz Nermin S¸ims¸ek Kus¸ Pinar Ku¨ce
•
Gokhan Coral Ayla C¸ elik Mehmet Serdar Gultekin
•
¨
¨
Received: 21 October 2014 / Accepted: 5 January 2015
Ó Springer Science+Business Media New York 2015
Abstract The biological activity of alcohol and epoxide
groups containing bicyclic skeletal structures was investi-
gated in terms of antibiotic and toxic effects at cellular
level. The present protocol, which utilizes readily available
starting materials and mild reaction conditions, provides an
attractive approach to a series of functionalized bicyclic
alcohol and epoxide derivatives with promising biological
applications. In this protocol, while some of the bicyclic
compounds were synthesized as in the literature, the other
bicyclic compounds were synthesized by new synthetic
methods. The antibiotic and toxic properties of the syn-
thesized bicyclic compounds were investigated, and some
of their biological activities were tested in prokaryotic and
eukaryotic systems. (rac)-Dimethyl 3,8-dioxatricy-
clo[3.2.1.02,4]oct-6-ene-6,7-dicarboxylate, one of the syn-
thesized molecules that showed antibiotic activity, was
tested in two different living systems (prokaryotic and
eukaryotic) in terms of biological and toxic effects at cel-
lular level using a peripheral lymphocyte culture assay.
Mitotic index for this compound was calculated in lym-
phocyte cultures.
Keywords Cyclitol Á Bicyclic cyclitol Á
Bicyclic epoxide Á Antibiotic effect Á Toxic effect
Introduction
Cyclitols and bicyclic cyclitols, which are structures con-
taining one hydroxyl group on each carbon atom of three or
more ring atoms (Scheme 1), are useful molecules with
anticancer, antibiotic, antifeedant, and antileukemic activ-
ity and versatility as synthetic material and intermediates
(Kilbas and Balci, 2011; Mehta and Reddy, 1999). For this
reason, they have been intensively investigated for bioac-
tive properties as pharmaceutical materials (Ferguson and
Williams, 1988; Varki, 1993; Schultz et al., 1999;
Heightman and Vasella, 1999; Asano, 2003; Irvine, 2003).
Cyclitols, which have cyclic and bicyclic skeleton struc-
tures, are widespread in nature (Posternak, 1965; Pigman
and Horton, 1972; Balci, 1997; Gu¨ltekin et al., 2003, 2004;
Atsumi et al., 1990). Synthetic and natural cyclitol struc-
tures are well known for their wide application fields. In
addition, bicyclic cyclitols were very useful and convenient
molecules for the synthesis of large functionalized mac-
rocycles (Baran et al., 2012; Baran and Balci, 2009;
Hudlicky and Cebulak, 1993; Hudlicky et al., 1996)
Cytotoxic activity studies of epoxide derivatives of 7-oxa-
bicyclo[2.2.1]heptane were performed in the 9 KB tissue
culture assay in the literature (Anderson and Dewey, 1977).
Several bicyclic and tricyclic conduritol derivatives have
also been synthesized and evaluated for their efficacy
against various glycosidases (Mehta and Ramesh, 2005).
Although several methods for the synthesis of cyclitol
derivatives are known, new methods are still being devel-
oped. In our initial studies, we intended to synthesize
bicyclic cyclitols in easy and practical steps with the
¨
O. Yilmaz Á N. S¸ims¸ek Kus¸ (&)
Department of Chemistry, Mersin University, 33343 Mersin,
Turkey
e-mail: simner@gmail.com
P. Ku¨ce Á G. Coral Á A. C¸ elik
Department of Biology, Mersin University, 33343 Mersin,
Turkey
M. S. Gu¨ltekin
Department of Chemistry, Ataturk University, 25240 Erzurum,
Turkey
123

Med Chem Res
Scheme 1 Some cyclitol
derivatives
OH
OH
OH
OH
( )n
OH
OH
OH
OH
OH
HO
HO
OH
OH
HO
HO
HO
HO
O
OH
OH
OH
OH
4
1
2
3
n=1,Hydrindanes
n=2,Dekalins
Tetracyclo dodekantetrol
Cyclophellitol
Inositol
O
known oxidation methods, epoxidation and cis-dihydr-
oxylation as in the literature (Kilbas and Balci, 2011). We
performed a method for the synthesis of some bicyclic
cyclitols, which can serve as the key components for the
preparation of other important compounds such as bishomo
inositols and macrocyclic molecules. For this synthesis,
dimethyl bicyclo[2.2.1] hepta-2,5-diene-2,3-dicarboxylate
(6) (Danishefsky et al., 1979; Renold and Tamm, 1995)
and dimethyl 7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2,3-
dicarboxylate (Simsek et al., 2001) (7) were used as
starting materials, which are inexpensive and commercially
available. In this study, our aim was to synthesize bicyclic
alcohols and epoxides, which are known and unknown
bicyclic derivatives in the literature, and the conduct bio-
logical experiments on the antibiotic and toxic properties of
these synthesized compounds. Despite the literature on the
metabolic fate and biological activity of polycyclic
hydrocarbons and their metabolities, few studies have been
reported for synthetically derived epoxides (Anderson and
Dewey, 1977; Wood et al., 1983).
R
R
R
O
, heat
, heat
90%
85%
R
R
5
R
6
7
R=-CO2CH3
Scheme 2 The Diels–Alder addition reaction of the dienophiles (6,
7) with dimethyl acetylene dicarboxylate 5
temperature. cis-Diol 8 was obtained from bicyclic diene 6
in 91 % yield (Anderson and Dewey, 1977; Danishefsky
et al., 1979). Immediately after, the same reaction condi-
tions were applied to bicyclic diene 7 with a catalytic
amount of OsO4 and 1 eq. NMO, the substituted double
bond was preferentially oxidized, and this reaction gave
cis-diol 10 in 95 % and cis-diol 11 in 5 % yields. The
bicyclic diene 6 was treated with a catalytic amount of
OsO4 and 1 eq. NMO, and as a result of the reaction, cis-
diol 8 was obtained as the sole product; molecule 15 was
not obtained (Table 1). The structures of cis-diols 8, 10,
and 11 were determined by 1H-NMR and 13C-NMR
spectroscopy.
After the successful separation and characterization of
the bicyclic cis-diols 8, 10, and 11 derived from cis-
hydroxylation of only one of the double bonds in 6 and 7,
we synthesized the bicyclic tetrols 9 and 12 with a catalytic
amount of OsO4 in the presence of 2 eq. NMO as co-
oxidant. When we used 2 eq. NMO in the same reaction
conditions, molecules 9 and 12 were synthesized as the sole
products; molecule 15 was not obtained. This result was
very surprising in these reaction conditions; therefore, we
think that synthesis of molecule 9 was obtained over syn-
thesis of molecule 8.
1H- and 13C-NMR spectra are in good agreement with
the structures of 9 and 12, especially the measured cou-
pling constants between the bridgehead protons and the
alkoxy protons, J1,2en = J5,4en = 0 Hz, clearly showing the
exo-configuration of the hydroxyl groups.
Results and discussion
Synthesis of bicyclic cyclitols and bicyclic epoxides
The starting materials dimethyl bicyclo[2.2.1]hepta-2,5-
diene-2,3-dicarboxylate (6) (Danishefsky et al., 1979; Re-
nold and Tamm, 1995) and dimethyl 7-oxa-bicy-
clo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate (7) (Simsek
et al., 2001) were prepared through the Diels–Alder reac-
tion between dimethyl acetylene dicarboxylate (5) and
furan to yield 7, and dimethyl acetylene dicarboxylate (5)
and cyclopentadiene to yield 6 in high yields as in the
literature, followed by both cis-dihydroxylation reaction
and epoxidation reaction (Scheme 2).
After the synthesis of the starting materials, the next step
was the functionalization with hydroxyl groups of the
double bonds in 6 and 7. For this reason, firstly, each of the
starting molecules (6 and 7) was treated separately with a
catalytic amount of OsO4 in the presence of 1 eq. NMO as
co-oxidant in a mixture of acetone/H2O (1:1) at room
Furthermore, bicyclic dienes 6 and 7 were reacted with
MCPBA to give epoxides 13 (Niwayama, 2000a, b; Ni-
wayama et al., 1988, 1994) and 14 (Ito et al., 1981) in high
yields (82 and 85 %, respectively). The configurations of
123

Med Chem Res
Table 1 The synthesis of bicyclic alcohols (8–12) and bicyclic epoxides (13, 14)
X
X
X
X
(OH)2
H
O
H
O
R
R
R
O
M PBA
C
s
H
O
NM
O O4 /
O
H
O
or
R
R
R
R
R
=
C
X
R
H
, O
2
=-
H
CO2C
3
Bicyclic
Bicyclic
dien
OsO4/
NMOa
Bicyclic
Alcohols (%)
Bicyclic
dien
MCPBA
Entry
1
Entry
5
epoxides
(%)
b
O
R
R
H
O
O
R
0.02
mmol
/1eq
H
R
13
R
8
R
6
R
R
82
%
91
%
R
2 eq.
O
H
H
O
H
O
6
O
R
O
H
O
O
0.02
mmol
/2eq
R
R
9
14
2
3
4
6
R
R
7
81
%
85
%
O
H
O
R
H
O
10
R
O
0.02
mmol
/1eq
95
%
O
H
H
O
H
O
R
O
H
O
R
R
11
R
15
R
7
5
%
R
Not obtained
O
H
O
H
O
0.02
mmol
/2eq
H
O
H
O
R
12
R
83
%
a
Conditions: 4,8 mmol diene (6 or 7), OsO4/NMO, acetone:water (1:1), rt, 24 h
b
MCPBA, CHCl3, 72 h
the formed epoxides 13 and 14 were also determined as
exo-isomer on the basis of the absence of coupling between
the bridgehead and epoxide protons.
against Salmonella typhimurium ATCC 14028, Pseudo-
monas aeruginosa ATCC 9027, Bacillus Spizizenii ATCC
6633, Escherichia coli ATCC 25922, and Staphylococcus
Aureus ATCC 25923. The resulting inhibition zones on the
plates were measured in mm by using ampicillin (10 lg)
(Bioanalyse) and tobramycin (10 lg) (Bioanalyse) as
standard drugs for the antibacterial activity (Table 2).
Broth macrodilution was performed for detecting mini-
mal inhibitory concentration (MIC) of the test solution
bicyclic epoxide 14. The tubes contained dilutions, ranging
from 5 to 75 lg/mL, in LB medium. The macro broth
dilutions were incubated at 37 °C and measured spectro-
photometrically (OD600 nm) after 24 h to verify the pre-
sence or absence of growth (Fig. 1). MIC values of 75 lg/
All of the synthesized molecules were subjected to
bioactivity examinations in prokaryotic and eukaryotic
systems. The dioxa-tricyclo compound 14, one of the
synthesized molecules, showed antibiotic activity.
The antimicrobial activity of (rac)-dimethyl 3,8-
dioxatricyclo[3.2.1.02,4]oct-6-ene-6,7-dicarboxylate
(14)
The antimicrobial activity of (rac)-dimethyl 3,8-dioxatri-
cyclo[3.2.1.02,4]oct-6-ene-6,7-dicarboxylate (14) was tested
123

Med Chem Res
Table 2 Inhibitory effects of bicyclic epoxide 14 on the tested bacterial strains
Bacteria
Test (mm) (epoxide 14)
Controls (mm)
15 lg
30 lg
45 lg
50 lg
100 lg
Tobramycin (10 lg)
Ampicillin (10 lg)
Ethanol (96 %)
–
S. aureus
9.66
12
16.33
16
17.5
21
37
S. typhimurium
B. spizizenii
E. coli
8.66
11
10.4
12.25
19
12.3
15.8
12.8
13
17
–
–
–
25
13.83
11.66
17.5
13
19.4
20.1
19.8
20
8.16
12.25
Radii of the inhibition zones on the plates were measured in mm. Ampicillin and tobramycin were used as standard drugs for the antibacterial
activity
mL for E. coli, 45 lg/mL for S. aureus, 15 lg/mL for B.
spizizenii, and 12 lg/mL for S. typhimurium were obtained.
mL penicillin and 10,000 IU/mL streptomycin). Lympho-
cytes were cultured for 72 h, and metaphases were blocked
during the last 1.5 h with colcemid at a final concentration
of 0.2 lg/mL. Mitomycin C (2 lg/mL) was used as posi-
tive control. The cells were harvested by replacing the
culture medium with KCl (0.075 M) in which cells were
incubated for 20 min at 37 °C. The cells were fixed in
Carnoy’s fixative (methanol: acetic acid, 3:1 v:v) five
times, and the slides were kept at room temperature over-
night. The air-dried slides were stained with 5 % Giemsa
stain.
Eukaryotic system assay
The cytotoxicity of bicyclic epoxide derivative 14 was
tested by peripheral blood lymphocytes culture assay.
In vitro analysis was performed in human blood lympho-
cytes from two people at six different concentrations of 14
(5, 7.5, 10, 12.5, 15, and 30 lg/mL). Cytotoxic potentials
of the bicyclic cyclitol derivative were evaluated by using
mitotic index (MI), the parameter of cytotoxicity in human
peripheral blood lymphocyte cultures. MI is commonly
used as the cell kinetic parameter in many studies
(Anderson et al., 1988; Scott et al., 1991; C¸ elik and Akbas¸,
2005; C¸ elik and Aras, 2006; Moorhead et al., 1960; Butler
and Munshaw, 1981; Ozturk et al., 2012; Turker, 2013).
Microscopic evaluation
The MI was calculated as the proportion of metaphases
among the total cell population by counting a total of 1,000
cells for each dose treatment.
Lymphocyte cultures in peripheral blood lymphocytes
and mitotic index
Statistical evaluation
The study was carried out using blood samples from two
healthy non-smoking male donors, aged 26 and 24 years.
In both donors, the results of clinical routine laboratory
analyses were within the normal ranges, and the absence of
exposure to known genotoxicants was assumed. In order to
measure the MI in lymphocytes cultures, the biological
activity of the dose range was selected according to inhi-
bition zones in bacteria of bicyclic epoxide derivative 14.
Six different concentrations of bicyclic epoxide 14 (5, 7.5,
10, 12.5, 15 and 30 lg/mL) were prepared in ethanol.
Lymphocyte cultures were prepared according to the
technique of Moorhead et al. (1960) with slight modifica-
tions. Heparinized whole blood (0.8 mL) was added to
6 mL of culture medium F 10 (Gibco, USA), supplemented
with 20 % et al calf serum (Gibco, USA), 0.1 % mL
phytohemagglutinin (Gibco), and antibiotics (10,000 IU/
The data were compared by one-way variance analysis.
Statistical analysis was performed using SPSS for Win-
dows 9.05. Post hoc analysis was performed by least sig-
nificant difference (LSD) test.
Table 3 shows the values of MI in lymphocytes cultures
treated with six different doses of bicyclic cyclitols sam-
ples. Values of MI as a parameter used to determine pos-
sible cytotoxic or mutagenic effects are presented in
Table 3. No significant decrease in the MI was detected for
treatment with bicyclic cyclitols under our experimental
conditions. There was no statistically significant difference
between the doses of bicyclic epoxide 14 (7.5, 10, 12.5,
15 lg/mL) and the negative control (p [ 0.05). A signifi-
cant difference (p \ 0.001) in the MI was observed
between the dose of 30 lg/mL of 14 and the negative
control.
123

Med Chem Res
Fig. 1 Effects of different
O
concentrations of (rac)-dimethyl
3,8-dioxatricyclo[3.2.1.02,4]oct-
6-ene-6,7-dicarboxylate (14) on
the bacterial strains tested
H
CO2C
3
O
H
CO2C
3
Experimental
The mixture was stirred at r.t. for 24 h. The reaction was
monitored by TLC. The solution was evaporated, and then the
crude residue was directly purified by column chromatogra-
phy on silica gel using EtOAc–n-hexane as eluent to give the
corresponding bicyclic alcohols (examples in Table 1).
Synthesis of dimethyl bicyclo[2.2.1]hepta-2,5-diene-
2,3-dicarboxylate (6) and 7-oxo-dimethyl
bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate (7)
Dimethyl bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate
(6) and dimethyl 7-oxo-bicyclo[2.2.1]hepta-2,5-diene-2,3-
dicarboxylate (7) were prepared as described in the related
literature (Danishefsky et al., 1979; Renold and Tamm,
1995; Simsek et al., 2001).
Typical procedure for the formation of bicyclic
epoxides with MCPBA (meta-Chloroperbenzoic acid)
Bicyclic diene (6 or 7) (4.8 mmol) was dissolved in
150 mL of chloroform, 1.66 g, 2 eq., MCPBA (9.62 mmol,
70 %) was added, and then the reaction was stirred at reflux
temperature for 3 days. The reaction mixture was added to
15 mL 50 % NaHSO3 solution, and mixture was stirred for
15 min. The organic layer was separated and then washed
with saturated aqueous NaHCO3 (100 mL), dried with
MgSO4?, and concentrated to give the corresponding
bicyclic epoxides (examples in Table 1).
Typical procedure for the formation of bicyclic alcohols
with OsO4/NMO oxidation
Bicyclic diene (6 or 7) (4.8 mmol) was dissolved in a mixture
of H2O–acetone (10 mL, 1:1) solution and NMO (4,8 or
9,6 mmol) and then, OsO4 (0.02 mmol, 5 mg) in acetone
(5 mL) was added to the stirring solution of bicyclic diene.
Table 3 The values of MI in lymphocytes cultures treated with five different doses of 14 numbered epoxide derivative in vitro
Groups
Donor 1
MI (%)
Donor 2
MI (%)
Mean SE
Negative control
6.60
6.50
6.60
6.60
6.60 0.00
6.55 0.50
Solvent control (ethanol)
Epoxide derivative 14 (lg/mL)
7.5
6.60
6.60
6.60
6.60
6.00
3.80
6.50
6.70
6.60
6.50
5.60
3.70
6.55 0.50
10
6.55 0.50
12.5
6.55 0.50
15
6.60 0.00
30
5.80 2.00***
3.75 0.50***
Positive control MMC (2 lg/mL)
MI mitotic index, MMC mitomycin C
*** p \ 0.001
123

Med Chem Res
(rac)-dimethyl 5,6-dihydroxy-bicyclo[2.2.1]hept-2-ene-2,3-
dicarboxylate (8): white solid IR (KBr, cm-1) 3,428,
s, H5 and H6), 3.71 (6H, s, –OCH3), 13C NMR (CDCl3,
100 MHz) d 173.3, 82.4, 69.8, 61.6, 55.9, MS m/z 201.9
(M?, –OH), 185, 184.0 (M?, –2 CH3), 155, 154, 153, 152
(M?, –C=O), 127, 126, 125, 124, 123 (M?, –C=O), 99, 98,
97, 96, 95), (M?, –OH), 84, 83.1, 82, 81, 79, (M?, OH), 70,
69.1, 68, 67, 66, 65 (M?, –OH), 57, 56, 55.1, 54, 53, 51
(M?, –OH), 43.1, 42, 41, 39, 38, 37. Anal. Calcd. for,
C10H14O9: C, 43.17; H, 5.07. Found: C, 44.27; H, 6.02.
1
2,956, 1,724, 1,438, 1,375, 1,250, 1,127, 1,070, 1,019. H
NMR (CDCl3, 400 MHz) d = 3.97 (1H, s, –OH), 3.86 (2H,
bs, H4 and H6), 3.71 (6H, s, –OCH3), 3.03 (2H, bs, H1 and
H4), 1.95–1.78 (AB system, dt, 1.95–1.78 ppm, J = 10.9,
J = 1.4 Hz,
H
7ab). 13C NMR (CDCl3, 100 MHz),
d = 164.7, 143.7, 68.3, 52.2, 51.5, 41.6. MS m/z 243 (M?,
–CH3), 227 (M?, –O), 211 (M?, –C=O), 183 (M?,
–OCH3), 152.9 (M?, –C=O), 126 (M?, –2 OH), 91 (M?,
–CH2) 81, 80, 79, 78, 77. Anal. Calcd. for C11H14O6:C,
54.54; H, 5.83. Found C, 55.45; H, 6.12 %)
(rac)-dimethyl 3-oxatricyclo[3.2.1.02,4]oct-6-ene-6,7-dicarbox-
ylate (13) m.p. = 182–183 °C IR (cm-1) 3,004, 2,955,
1,715, 1,614, 1,435, 1,370, 1,327, 1,256, 1,235, 1,193,
1,159, 1,104, 1,079. 1H NMR (CDCl3, 400 MHz) d = 3.72
(6H, s, –OCH3), 3.57 (2H, s, H2 and H3), 3.29 (2H, t, H1
and H4), 1.72–1.49 (2H, dt, –CH2, J = 9.1, J = 1.4 Hz),
13C NMR (CDCl3, 100 MHz) d = 163.6, 148.6, 57.1, 51.2,
45.9, 38.2. MS m/z 193.5, 192.4, 191.7 (M?, O, –CH2),
164.4, 163.3, 162.3, (M?, –2 CH3), 136.3, 133.3, 132.3
(M?, –C=O), 106.3, 105.3, 104.3 (M?, –C=O), 78.2, 77.2,
76.3, Anal. Calcd. for C11H12O5. C, 58.93; H, 5.39. Found:
C 60.12 H 6.92.
(rac)-dimethyl 2,3,5,6-tetrahydroxybicyclo[2.2.1]heptane-
2,3-dicarboxylate (9) IR (KBr, cm-1) 3,388, 2,976,
1
1,726, 1,437, 1,254, 1,191, 1,165, 1,138, 1,096, 1,073. H
NMR (CDCl3, 400 MHz) d = 4.03 (2H, d, H5 and H6,
J = 1.7 Hz), 3.58 (6H, s, –OCH3), 2.11 (2H, t, J = 1.5 Hz,
H1 and H4), 1.94–1.87 (AB system, 2H, dt, J = 10.9 Hz,
7ab), 13C NMR (CDCl3, 100 MHz),173.3, 82.5, 69.8, 55.9,
52.5, 29.4.MS m/z 272.1 (M?, –OCH3), 240 (M?, –C=O,
214, 212 (M?, –CH3), 196, 195, 194, 193, 192 (M?,
–COO), 155, 153, 151, 154 (M?, –OH), 139, 138, 137, 136,
134, (M?, –2 OH), 107, 106, 105, 104, 103, (M?, –CH2),
93, 92, 91, 89, 87. Anal. Calcd. for C11H16O8: C, 47.83; H,
5.54. Found: C 49.3, H 6.12, %).
(rac)-dimethyl 3,8-dioxatricyclo[3.2.1.02,4]oct-6-ene-6,7-
dicarboxylate (14) mp. = 82–83 °C, IR (cm-1) 3,020,
2,957, 1,720, 1,628, 1,436, 1,367, 1,330, 1,228, 1,207,
1,117, 1,085 .1H NMR (CDCl3, 400 MHz) d = 5.05 (2H,
s, H1 and H4), 3.76 (6H, s, –OCH3), 3.70 (2H, s, H2 and
H3), 13C NMR (CDCl3, 100 MHz) d = 161.4,146.9, 78.3,
54.3, 51.6, MS m/z 226, (M?, –OCH3), 195, 194, (M?,
–OCH3), 167, 166, 165, 162.9, (M?, –C=O), 139, 137, 136,
135, 134 (M?, –C=O), 109, 108, 107, 106 (M?, –C–O), 81,
80, 79, 78, 77 (M?, –C–O), 55, 53, 52, 51, 50, Anal. Calcd.
for C10H10O6 C, 53.10; H, 4.46 (Found: C, 55.25; H, 5.34.
(rac)-dimethyl 5,6-dihydroxy-7-oxabicyclo[2.2.1]hept-2-
ene-2,3-dicarboxylate (10) IR (KBr, cm-1) 3,427, 2,956,
1
1,716, 1,643, 1,437, 1,268, 12,239, 1,129, 1,079. H NMR
(CDCl3, 400 MHz) d = 4.94 (2H, s, H1 and H4), 3.97 (2H,
s, H5 and H6), 3.76 (6H, s, –OCH3), 13C NMR (CDCl3,
100 MHz),162.4, 142.7, 86.2, 67.9, 52.6.MS m/z 245.1
(M?, –2 OH), 212, 211 (M?, –OCH3, –C=O), 153 (M?,
–OCH3), 125, 123, 121, 119 (M?, –C=O), 97, 96, 95, 93,
91 (M?, –O), 83, 82, 81, 79, 77. Anal. Calcd. for
C10H12O7: C, 49.18, H, 4.95. Found: C 52.52, H 5.92.
Preparation of microbial cultures for testing
antimicrobial activity of (rac)-dimethyl
3-oxatricyclo[3.2.1.02,4]oct-6-ene-6,7-dicarboxylate
(14)
(rac)-dimethyl 2,3-dihydroxy-7-oxabicyclo[2.2.1]hept-5-
ene-2,3-dicarboxylate (11) IR (KBr, cm-1) 3,412, 2,956,
1
1,732, 1,437, 1,263, 1,228, 1,077, 1,041, 1,028. H NMR
The antimicrobial activity of (rac)-dimethyl 3-oxatricy-
clo[3.2.1.02,4]oct-6-ene-6,7-dicarboxylate (14) was tested
against S. typhimurium ATCC 14028, P. aeruginosa ATCC
9027, B. spizizenii ATCC6633, E. coli ATCC 25922, and S.
aureus ATCC 25923. Bacteria strains were cultured in LBbroth
(Difco) and incubated for 24 h at 37 °C. According to the agar
disc diffusion method, bacteria were inoculated on Mueller–
Hinton Agar (Merck) (9). Samples were prepared by using
ethanol (96 %) as a solvent. Then, 10 lL of each test solution
was added to the 6-mm empty discs (Bioanalyse), and the discs
were then incubated at 37 °C for 24 h. Ethanol absorbed discs
were used only for control. The resulting inhibition zones on the
plates were measured in mm by using Ampicillin (10 lg)
(Bioanalyse) and Tobramycin (10 lg) (Bioanalyse) as standard
drugs for the antibacterial activity (Table 2).
(CDCl3, 400 MHz) d = 6.42 (2H, bs, H5 and H6), 4.69
(2H, bs, H1 and H4), 4.54 (2H, s,–OH), 3.64 (6H, s,
–OCH3). 13C NMR (CDCl3, 100 MHz) d = 169.6, 133.6,
84.8, 81.9, 51.9, MS m/z 205 (M?, 2 –OH, –O), 154, 153,
152, 151, 150 (M?, –CH3), 139 (M?, –O) 125, 123, 122,
121 (M?, –C=O), 95, 93, 91 (M?, –CH3) 82, 81, 79, 77, 76,
(M?, –O) 59, 57, 56, 55, 54) (M?, –C=O) 32,31, Anal.
Calcd. for C10H12O7: C, 49.18; H 4.95. Found: C, 51.55; H,
5.62.
(rac)-dimethyl 2,3,5,6-tetrahydroxy-7-oxabicyclo[2.2.1]hep-
tane-2,3-dicarboxylate (12) IR (KBr, cm-1) 3,430, 2,957,
1
1,736, 1,438, 1,365, 1,266, 1,231, 1,132, 1,057. H NMR
(CDCl3, 400 MHz) d = 4.37 (2H, s, H1 and H4), 4.23 (2H,
123

Med Chem Res
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Conclusions
New bicyclic cyclitol and bicyclic epoxide derivatives
(8–14) were synthesized from bicyclo[2.2.1] heptadiene
derivatives (6 and 7) by easy methods in this study. In
addition, the antibiotic and toxic properties of the synthesized
molecules were examined. The in vitro antibacterial activity
of the synthesized compounds was determined against Gram-
positive and Gram-negative organism by broth macrodilution
method and compared with that of the antibiotics tobramycin
and ampicillin as controls. It was observed that dioxatricyclo
compound 14 showed antibiotic properties against S. ty-
phimurium ATCC 14028, P. aeruginosa ATCC 9027, B.
spizizenii ATCC 6633, E. coli ATCC 25922, and S. aureus
ATCC 25923. In today’s world, humans are exposed to a
variety of chemicals environmentally, occupationally, and
therapeutically or due to change in lifestyle. Studies carried
out on bacteria, mammalian systems, and cell cultures
showed that some chemicals have toxic effects on organisms
(Anderson et al., 1988; Scott et al., 1991; C¸ elik and Akbas¸,
2005; C¸ elik, 2006; Moorhead et al., 1960; Butler and Mun-
shaw, 1981; Ozturk et al., 2012; Turker, 2013). According to
the data presented in the present study, none of the different
doses of dioxatricyclo derivative 14, except for the dose of
30 lg/mL (p \ 0.001) affected the MI (which is considered
the cell kinetic parameter) or cytotoxic activity. These data
reveal that the dioxatricyclo compound 14 had no cytotoxic
effect at cellular level and did not negatively affect cell
growth in cultured lymphocytes.
Butler DN, Munshaw TJ (1981) The synthesis of 2,3,5,6-endo, endo,
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chromosomal aberration frequencies in peripheral blood lym-
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60:106–112
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treated with metronidazole in vitro. Drug Chem Toxicol 1:85–94
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Rev Biochem 57:285–320
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related compounds. Curr Org Chem 8:1159–1186
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of (1,4/2)-cyclohex-5-ene-triol and its conversion to ( )-proto-
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Hudlicky T, Cebulak M (1993) Cyclitols and derivatives. VCH, New
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83(6–7):581–594
This paper describes a useful method for the synthesis of
some bicyclic alcohol and epoxide derivatives useful
molecules with anticancer, antibiotic, antifeedant, and
antileukemic activity and versatility as synthetic interme-
diates (Posternak, 1965; Pigman and Horton, 1972; Balci,
1997; Gu¨ltekin et al., 2004; Atsumi et al., 1990). At the
same time, this paper provides information about antibiotic
and toxic effects of the synthesized bicyclic alcohol and
epoxide. New bicyclic cyclitols (9, 11, and 12) were syn-
thesized in this study. Dioxatricyclo compound 14 showed
antibiotic activity and was tested in two different living
systems in terms of biological and toxic effects at cellular
level using a peripheral lymphocyte culture assay.
Acknowledgments The authors are indebted to Prof. Dr Metin
Balci for his help during this work and Mersin University [BAP-FEF
¨
KB (NS¸K) 2010-3 A and BAP-FBE KA (OY) 2010-5 YL] for their
financial support to this work.
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