Synthesis, characterization, and in vitro antimicrobial activities of 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles

Article abstract of DOI:10.1016/j.bmcl.2010.01.126

The long-chain alkenoic acid hydrazides (1a-d) on reaction with phenylisocyanate and phenylthiocyanate gave their corresponding semicarbazides (2a-d) and thiosemicarbazides (4a-d), which on further refluxing with POCl₃ and Ac₂O yield

Full text of DOI:10.1016/j.bmcl.2010.01.126

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1933–1938
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization, and in vitro antimicrobial activities of
5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles
Nida N. Farshori a, Mudasir R. Banday a, Anis Ahmad b, Asad U. Khan b, Abdul Rauf a,
*
a Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India
b Interdisciplinary Biotechnology Unit, Aligarh Muslim University Aligarh 202 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The long-chain alkenoic acid hydrazides (1a–d) on reaction with phenylisocyanate and phenylthiocya-
nate gave their corresponding semicarbazides (2a–d) and thiosemicarbazides (4a–d), which on further
refluxing with POCl3 and Ac2O yielded corresponding 1,3,4-oxadiazoles (3a–d) and thiadiazoles (5a–d),
respectively.
The structure elucidation of synthesized compounds is based on the elemental analysis and spectral data
(IR, 1H NMR, 13C NMR and MS). The synthesized oxadiazoles and thiadiazoles have been screened for anti-
bacterial and antifungal activities. The investigation of antimicrobial screening revealed that compounds
3c, 3d, 5c, 5d and compounds 3b, 5b, showed good antibacterial and antifungal activities, respectively.
Ó 2010 Published by Elsevier Ltd.
Received 29 October 2009
Revised 9 January 2010
Accepted 27 January 2010
Available online 1 February 2010
Keywords:
1,3,4-Oxadiazoles and thiadiazoles
Synthesis
Elemental analysis
Spectral data
Antifungal and antibacterial activities
A wide variety of heterocyclic systems have been explored for
developing pharmaceutically important molecules. Among them
the derivatives of oxadiazoles and thiadiazoles have played an
important role in the medicinal chemistry. These heterocycles have
been found to possess broad-spectrum antimicrobial activity and
many other uses.1,2 The therapeutic effects of compounds contain-
ing 1,3,4-thiadiazole rings have been studied for a number of
pathological conditions including inflammation3,4, pain,5–7 and
hypertension.8 Furthermore, the synthesis of thiadiazoles and
oxadiazoles has attracted wide attention due to the diversity of
their applications as antibacterial9, antimycobacterial10,11, anti-
fungal,12–14 and antidepressant agents.15
chain substituent at C-5. To the best of our knowledge the synthe-
sis and antimicrobial activity of these newly synthesized com-
pounds has not been reported so far.
The long-chain alkenoic acid hydrazides (1a–d), used as the
starting material were prepared from corresponding long-chain
alkenoic acids by esterification and further treatment with hydra-
zine hydrate.21 Reacting 1a–d with phenyl isocyanate in dry ben-
zene under reflux and removing excess of solvent under reduced
pressure gave semicarbazides (2a–d). The treatment of 2a–d with
POCl3 yielded 2,5-disubstituted- 1,3,4-oxadiazoles (3a–d) in good
yields. The reaction of 1a–d with phenyl isothiocyanate gave corre-
sponding thiosemicarbazides (4a–d) which on dehydrative cycliza-
tion by Ac2O produced 2,5-disubstituted- 1,3,4-thiadiazoles (5a–d)
in excellent yields. The reaction sequences are outlined in Schemes
1 and 2.
Various biological applications such as antimicrobial16,17, anti-
fungal18, pesticidal,19 and anticancer activities20 have also been
reported for seed oils, long-chain alkenoic acids and their
derivatives.
The semicarbazide 2a showed IR bands at 3236 cmꢀ1 (NH, NH–
NH) and 1667 cmꢀ1 (C@O). 1H NMR was more informative, charac-
teristic peaks were observed at d 13.04 (1H, s, CO–NH-Ar), 9.16 (2H,
br s, CO–NHNH–CO), 8.17 (2H, d, J = 7.2 Hz, Ar-H-200/600), 7.60 (1H, t,
J = 7.4 Hz, Ar-H-400) and 7.51 (2H, t, J = 7.4 Hz, Ar-H-300/500). In 13C
NMR peaks at d 168.2 and 165.4 were observed.
The build up of 3a–d is evident from their spectral data.
Compound 3a, 5-(Dec-90-enyl)-2-phenylamine-1,3,4-oxadiazole,
showed IR absorption bands at 3228 cmꢀ1, 1504 cmꢀ1, 1258 cmꢀ1
due to stretching vibrations of NH, C@N and C–O–C functions. The
1H NMR was more informative in assigning the structure. Diagonas-
tic peaks at d 9.07 (1H, s, NH), 7.27 (2H, d, J = 7.6 Hz, Ar-H-200/600), 7.18
(1H, t, J = 7.5 Hz, Ar-H-400) and 6.98 (2H, t, J = 7.3 Hz, Ar-H-300/500)
The wide range of therapeutic values of alkenoic acids and oxa-
diazoles/thiadiazole ring systems prompted us to synthesize the
title compounds and screen them for various antimicrobial activi-
ties. The basic idea was to append the long-chain alkenyl/hydrox-
yalkenyl moiety of the long-chain alkenoic acid to the oxadiazole/
thiadiazole nucleus so as to combine the beneficial effects in a
single structure with expected biological activities.
In this communication we are reporting the synthesis of 1,3,4-
oxadiazoles/thiadiazoles bearing a long alkenyl/hydroxyalkenyl
* Corresponding author. Tel.: +91 09412545345.
E-mail address: abduloafchem@gmail.com (A. Rauf).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.01.126

1934
N. N. Farshori et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1933–1938
were observed. A triplet at d 2.24 was observed for CH2 protons
a to
O
S
O
oxadiazole ring. In 13C NMR peaks at d 172.9 and 155.6 were ob-
served for ring carbon atoms. The mass spectra showed characteris-
tic molecular ion peak which were in accordance with the molecular
formula.
PhNCS
R
NH NH
NH
R
NH
NH2
4a-d
1a-d
5-(Dec-90-enyl)-2-phenylamine-1,3,4-thiadiazole 5a gave sig-
nificant IR bands at 3221 cmꢀ1 (NH), 1488 cmꢀ1 (C@N), and
707 cmꢀ1 (C–S–C). 1H NMR peak at d 12.20 (1H, s, NH), 8.16 (2H,
d, J = 8.5 Hz, Ar-H-200/600), 7.55 (1H, t, J = 7.3 Hz, Ar-H-400), and 7.44
(2H, t, J = 7.8 Hz, Ar-H-300/500) were observed in addition to peaks
of normal fatty acid chain. In 13C NMR peaks at d 165.2 and
153.0 were observed for ring carbon atoms. The mass spectra
showed characteristic molecular ion peak which were in accor-
dance with the molecular formula. The newly synthesized com-
Ac2O
N
N
R
NH
S
5a-d
Scheme 2. Synthesis of 2,5-disubstituted-1,3,4-thiadiazoles 5a–d.
pounds have been confirmed by their spectral data28
.
The characterization data of semicarbazides (2a–d), oxadiazoles
(3a–d), and thiadiazoles (5a–d) is given in Table 1.
medium. Solvent and growth controls were kept. Chloramphenicol
(30 g) was used as positive control and the disk poured in DMSO
Undec-10-enoic (purity, 98%) and (Z)-octadec-9-enoic (97%)
acids were purchased from Fluka chemicals (Buck: Switzerland).
(9Z,12R)-12-hydroxyoctadec-9-enoic acid (ricinoleic acid, 98%)
and (9R,12Z)-9-hydroxyoctadec-12-enoic acid (isoricinoleic acid,
98%) were isolated from Ricinus communis and Wrightia tinctoria
seed oils, respectively, following Gunstone’s partition procedure.26
Phenyl thiocyanate, phenyl isocyanate, phosphorous trichloride,
and acetic anhydride were purchased from Merck, Mumbai, India.
Thin layer chromatography (TLC) was done on glass plates
(20 ꢁ 5 cm) with a layer of silica gel G (Merck, Mumbai, India
0.5 mm thickness). Mixtures of petroleum ether/diethyl ether/ace-
tic acid (70:30:1; v/v) were used as developing solvent. The col-
umn chromatography was carried out with silica gel (Merck,
Mumbai, India, 60–120 mesh).IR spectra were recorded on Shima-
dzu 8201 PC spectrophotometer. 1H NMR spectra were recorded
with a Bruker, DRX 400 spectrometer (400 MHz) in CDCl3, using
TMS as internal standard. Chemical shifts (d) are quoted in ppm
and coupling constants (J) are given in Hz. 13C NMR spectra were
recorded at Bruker DRX 400 spectrometer in CDCl3 with CDCl3
(d = 77.00).
Antibacterial studies. The newly prepared compounds were
screened for their antibacterial activity against Escherichia coli
(ATCC-25922), Staphylococcus aureus (ATCC-25923), Pseudomonas
aeruginosa (ATCC-27853), Streptococcus pyogenes, and Klebsiella
pneumoniae (Clinical isolate) bacterial strains by disc diffusion
method.22,23 A standard inoculums (1–2 ꢁ 107 c.f.u./ml 0.5 McFar-
land standards) were introduced on to the surface of sterile agar
plates, and a sterile glass spreader was used for even distribution
of the inoculums. The disks measuring 6 mm in diameter were pre-
pared from Whatman no. 1 filter paper and sterilized by dry heat at
140 °C for 1 h. The sterile disks previously soaked in a known con-
centration of the test compounds were placed in nutrient agar
l
was used as negative control. The plates were inverted and incu-
bated for 24 h at 37 °C. The susceptibility was assessed on the basis
of diameter of zone of inhibition against Gram-positive and Gram-
negative strains of bacteria. Inhibition zones were measured and
compared with the controls. The bacterial zones of inhibition val-
ues are given in Table 2.
Minimum inhibitory concentrations (MICs) were determined by
broth dilution technique. The nutrient broth, which contained log-
arithmic serially two fold diluted amount of test compound and
controls were inoculated with approximately 5 ꢁ 105 c.f.u./ml of
actively dividing bacterial cells. The cultures were incubated for
24 h at 37 °C and the growth was monitored visually and spectro-
photometrically. The lowest concentration (highest dilution)
required to arrest the growth of bacteria was regarded as MIC.
To obtain the minimum bacterial concentration (MBC), 0.1 ml
volume was taken from each tube and spread on agar plates. The
number of c.f.u. was counted after 18–24 h of incubation at
35 °C. MBC was defined as the lowest drug concentration at which
99.9% of the inoculums were killed. The minimum inhibitory con-
centration and minimum bactericidal concentration are given in
Table 3. The PBE (percentual bacteriostatic efficiency, %) was ob-
tained as,
PBE = 100/MIC
The results have been reported in Table 6.
The investigation of antibacterial screening data revealed that
all the tested compounds showed moderate to good bacterial inhi-
bition. Compounds 3a, 3b, 3c, 3d, 5c, 5d showed good inhibition
against all Gram-positive and Gram-negative bacterial strains at
6.25 lg/ml concentrations. The compounds 3c, 3d, 5c and 5d were
found to be almost equally potent as the reference drug, chloram-
phenicol, in case of K. pneumoniae. The MBC of few compounds was
found to be the same as MIC but in most of the compounds it was
two or three folds higher than the corresponding MIC results.
Antifungal studies. Antifungal activity was also done by disk dif-
fusion method. For assaying antifungal activity Candida albicans,
Aspergillus fumigatus, Penicillium marneffei, and Trichophyton ment-
agrophytes (recultured) in DMSO by agar diffusion method.24,25
Sabourauds agar media was prepared by dissolving peptone (1 g),
O
O
O
PhNCO
R
NH NH
NH
R
NH
NH2
2a-d
1a-d
D
-glucose (4 g), and agar (2 g) in distilled water (100 ml) and
POCl3
adjusting pH to 5.7. Normal saline was used to make a suspension
of spore of fungal strain for lawning. A loopful of particular fungal
strain was transferred to 3 ml saline to get a suspension of corre-
sponding species. Twenty millilitres of agar media was poured into
each petri dish. Excess of suspension was decanted and the plates
were dried by placing in an incubator at 37 °C for 1 h using an agar
punch, wells were made and each well was labeled. A control was
also prepared in triplicate and maintained at 37 °C for 3–4 days.
N
N
R
NH
O
3a-d
Scheme 1. Synthesis of 2,5-disubstituted-1,3,4-oxadiazoles 3a–d.

N. N. Farshori et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1933–1938
1935
Table 1
Characterization data of synthesized compounds 2a–d, 3a–d, and 5a–d
Compounds
R
Mol. formula
Mp (°C)
Yield (%)
Analysis (%) found (calculated)
C
H
N
H
H
H
z
CH2
6
2a
2b
2c
C18H27O2N3
C25H41O2N3
C25H41O3N3
103–104
101–102
111–113
80
79
77
67.67(68.11)
8.44(8.56)
13.09(13.23)
10.02(10.11)
09.65(9.73)
H
6
E
CH2
71.95(72.25)
69.23(69.57)
9.86(9.93)
9.50(9.56)
5
CH
2
2
2
4
5
OH
OH
CH2
2d
C25H41O3N3
113–114
72
69.33(69.57)
9.20(9.56)
09.65(9.73)
3
6
z
CH2
6
3ª
C18H25ON3
C25H39ON3
C25H39O2N3
133–134
144–146
133–136
92
90
87
72.06(72.21)
75.23(75.52)
72.35(72.60)
8.11(8.40)
9.74(9.87)
9.41(9.49)
13.90(14.03)
10.44(10.56)
10.00(10.15)
H
E
CH2
3b
3c
6
4
5
CH
5
OH
OH
CH2
3d
C25H39O2N3
135–137
87
72.28(72.60)
9.36(9.49)
09.97(10.15)
3
6
z
CH2
6
5ª
C18H25SN3
C25H39SN3
C25H39OSN3
134–135
138–140
136–138
95
91
85
68.30(68.53)
72.23(72.59)
69.61(69.89)
7.78(7.98)
9.44(9.49)
9.04(9.14)
13.07(13.31)
10.10(10.15)
09.78(9.78)
H
E
CH2
5b
5c
6
4
5
CH
5
OH
OH
CH2
5d
C25H39OSN3
139–141
83
69.54(69.89)
9.02(9.14)
09.72(9.78)
3
6
Table 2
Antibacterial activity of 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles 3a–d and thiadiazoles 5a–d
Compounds
Diameter of zone of inhibition (mm)
Gram-positive bacteria
Gram-negative bacteria
K. pneumoniae
S. Pyogenes
S. aureus
P. aeruginosa
E. coli
3a
3b
3c
3d
5a
5b
5c
5d
18.0 0.5
15.6 0.2
21.2 0.3
20.1 0.2
13.5 0.3
13.1 0.7
22.9 0.8
21.1 0.2
23.0 0.2
—
17.7 0.6
15.3 0.2
20.2 0.4
21.1 0.3
13.1 0.5
12.9 0.3
21.6 0.3
20.8 0.4
22.0 0.2
—
17.3 0.8
19.3 0.5
25.6 0.2
19.4 0.2
17.2 0.4
16.3 0.6
29.2 0.2
25.9 0.6
32.0 0.3
—
16.8 0.8
14.9 0.3
19.7 0.2
20.2 0.1
12.8 0.2
12.1 0.5
22.7 0.6
19.9 0.9
19.0 0.2
—
19.8 0.6
17.5 0.3
22.8 0.3
21.6 0.2
15.2 0.4
14.8 0.4
25.5 0.5
22.9 0.2
27.0 0.2
—
Standard
DMSO
Positive control (standard); chloramphenicol and negative control (DMSO) measured by the Halo Zone Test (Unit, mm).
The fungal activity of each compound was compared with greseo-
fulvin as standard drug. Inhibition zones were measured and com-
pared with the controls. The fungal zones of inhibition values are
given in Table 4. The nutrient broth, which obtained logarithmic
serially two fold diluted amount of test compound and controls
was inoculated with approximately 1.6 ꢁ 104–6 ꢁ 104 c.f.u./ml.
The cultures were incubated for 48 h at 35 °C and the growth
was monitored. The lowest concentration (highest dilution) re-
quired to arrest the growth of fungus was regarded as minimum
inhibitory concentration (MIC).
To obtain the minimum fungicidal concentration (MFC), 0.1 ml
volume was taken from each tube and spread on agar plates. The
number of c.f.u. was counted after 48 h of incubation at 35 °C.
MFC was defined as the lowest drug concentration at which
99.9% of the inoculums were killed. The minimum inhibitory
concentration and minimum fungicidal concentration are given
in Table 5. The ratio MFC/MIC was calculated in order to determine
if the compound had a fungistatic (MFC/MIC P4) or fungicidal
(MFC/MIC 64) activity and the results have been summarized in
Table 6.

1936
N. N. Farshori et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1933–1938
Table 3
MIC and MBC results of 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles 3a–d and thiadiazoles 5a–d: positive control chloramphenicol
Compounds
Gram-positive bacteria
S. Pyogenes S. aureus
Gram-negative bacteria
P. aeruginosa
K. Pneumoniae
E. coli
MIC
MBC
MIC
6.25
12.5
6.25
12.5
12.5
12.5
MBC
MIC
MBC
MIC
MBC
MIC
MBC
3a
3b
3c
3d
5a
5b
5c
5d
12.5
25.0
50.0
25.0
12.5
25.0
50.0
100
12.5
25.0
12.5
25.0
50.0
12.5
12.5
25.0
25.0
12.5
12.5
12.5
12.5
12.5
50.0
50.0
50.0
50.0
25.0
25.0
12.5
25.0
12.5
12.5
12.5
25.0
50.0
6.25
25.0
50.0
50.0
6.25
6.25
12.5
12.5
12.5
50
50
25
25
100
50
12.5
12.5
12.5
6.25
6.25
25.0
12.5
6.25
6.25
6.25
12.5
12.5
6.25
6.25
6.25
25.0
25.0
6.25
6.25
6.25
25.0
25.0
6.50
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
Standard
MIC (
lg/ml) = minimum inhibitory concentration, that is, the lowest concentration of the compound to inhibit the growth of bacteria completely; MBC (lg/ml) = minimum
bacterial concentration, that is, the lowest concentration of the compound for killing the bacteria completely.
Table 4
Table 6
Antifungal activity of 5-Alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles
3a–d and thiadiazoles 5a–d: positive control (greseofulvin) and negative control
(DMSO) measured by the Halo Zone Test (Unit, mm)
PBE and fungicidal/fungistatic activity (MFC/MIC) of 5-alkenyl/hydroxyalkenyl-2-
phenylamine-1,3,4- oxadiazoles 3a–d and thiadiazoles 5a–d
Compounds
PBE = 100/MIC
Bacteria tested
MFC/MIC
Compounds
Diameter of zone of inhibition (mm)
Fungi tested
CA
AF
TM
PM
SP
SA
PA
KP
EC
CA
AF
TM
PM
3a
3b
3c
3d
5a
5b
5c
5d
24.2 0.4
26.1 0.3
23.3 0.9
23.2 0.2
19.5 0.5
27.2 0.2
25.1 0.7
24.9 1.4
30.0 0.2
—
20.8 0.3
23.1 0.2
20.7 0.4
21.6 0.4
16.5 0.4
23.8 1.2
22.4 0.3
21.8 0.2
27.0 0.2
—
16.9 0.9
20.2 1.2
17.5 0.2
18.7 0.2
14.1 0.3
20.3 0.7
18.9 0.5
19.0 0.2
24.0 0.3
—
15.2 1.2
15.9 0.3
13.9 0.3
16.3 0.2
10.2 0.5
16.1 0.2
15.1 0.9
15.2 1.2
20.0 0.5
—
3a
3b
3c
3d
5a
5b
5c
5d
8
4
16
16
4
16
8
16
8
8
8
16
16
16
—
8
8
16
16
8
8
8
16
16
4
8
8
16
16
4
2
4
4
2
2
2
2
2
—
2
4
2
4
2
4
4
4
2
—
2
4
2
4
2
2
2
4
4
—
2
2
4
8
4
4
2
2
4
—
2
8
8
4
4
16
16
16
—
16
16
16
—
16
16
16
—
15.4
16
16
—
Standard
DMSO
Chloramphenicol
Greseofulvin
CA = Candida albicans, AF = Aspergillus fumigatus, TM = Trichophyton mentagrophytes,
PM = Penicillium marneffei.
SP = S. pyogenes, SA = S. aureus, PA = P. aeruginosa, KP = K. Pneumonia, EC = E. Coli,
CA = C. albicans, AF = A. fumigatus, TM = T. mentagrophytes, and PM = P. Marneffei.
compounds showed good fungistatic activity against the fungal
strain C. albicans.
The antifungal screening data showed moderate to good activ-
ity. Among the screened compounds 3b and 5b were found to be
most active against all test fungal strains. The compound 5b
showed maximum activity against C. albicans, A. fumigatus, and T.
metagrophyte strains. The compound 3b was active against P. mar-
neffei. The MFC of most of the compounds was two or three folds
higher than the corresponding MIC results. Most of the synthesized
The present stratagem describes, the synthesis of new 4,5-
disubstituted-1,3,4-oxadiazoles and thiadiazoles using long-chain
alkenoic acids as the starting material. The described compounds
have been variously characterized and identified by IR, 1H NMR,
13C NMR, and mass spectral analysis. The potential antimicrobial
effects of the synthesized compounds were investigated against
S. pyogenes, S. aureus, P. aeruginosa, K. pneumoniae and E. coli bacte-
rial strains and C. albicans, A. fumigatus, T. mentagrophytes and P.
marneffei fungal strains. Among the synthesized oxadiazoles/thi-
adiazoles, the compounds with a hydroxyalkenyl chain substituent
at fifth position of oxa/thiadiazoles were found to increase the
antibacterial activity in compounds 3c, 3d, 5c and 5d. However
the position of the hydroxyl group had no significant effect on
the magnitude of the antibacterial activity. Further, the compounds
showed parallel activity against Gram-positive and Gram-negative
bacterial strains. The compounds with an internal double bond in
the long alkenyl substituent of synthesized oxa/thiadiazoles were
found to be potent antifungal agents. Contrary to the antibacterial
studies, the presence of the hydroxyl on the alkenyl side chain
turns out to be detrimental for the antifungal activity perhaps
due to pharmacokinetic reasons.
Table 5
MIC and MFC of 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles 3a–d
and thiadiazoles 5a–d, positive control greseofulvin
Compounds
CA
AF
TM
MFC
50.0 12.5
25.0
PM
MIC
25.0
MFC MIC
50.0 25.0
MFC MIC
100 12.5
MIC
MFC
25.0
3a
3b
3c
3d
5a
5b
5c
6.25 25.0
6.25 12.5 12.5
6.25 25.0
25.0
12.5
25.5
100
12.5
50.0 12.5
50.0 25.0
100
50.0 12.5
50.0 25.0
50.0 25.0
100
100
100
25.0 25.0
50.0 25.0
25.0
6.25
6.25 12.5
6.25 25.0
12.5
25.0 25.0
100 25.0
12.5
6.25 12.5
12.5
25.0
25.0 12.5
50.0 25.0
50.0 12.5
50.0 25.0
50.0
100
5d
Std
6.25 12.5
6.25 12.5
6.25
6.25 12.5
In conclusion the present study showed that the synthesized
compounds can be used as template for future development
through modification and derivatization to design more potent
and selective antimicrobial agents.
CA = Candida albicans, AF = Aspergillus fumigatus, TM = Trichophyton mentagrophytes,
PM = Penicillium marneffei; Std = standard. MIC g/ml) = minimum inhibitory
concentration, that is, the lowest concentration of the compound to inhibit the
growth of fungus completely; MFC ( g/ml) = minimum fungicidal.
(l
l

N. N. Farshori et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1933–1938
1937
3233 (NH, NH–NH) 1661 (C@O). 1H NMR (400 MHz, CDCl3, d, ppm): 13.23 (1H,
s, NH), 9.17 (2H, br s, NHNH), 8.12 (2H, d, J = 7.4 Hz, Ar-H-200/600), 7.65 (1H, t,
J = 7.4 Hz, Ar-H-400), 7.51 (2H, t, J = 7.2 Hz, Ar-H-300/500), 5.37 (2H, m, CH2–
CH@CH–CH2), 4.87 (1H, m, CH–OH), 2.55 (2H, t, J = 7.6 Hz, CH2–CO), 2.36 (4H,
m, CH2–CH2@CH–CH2), 1.83 (2H, m, CH2–CH2-CO), 1.76 (1H, m, CH–OH), 1.44–
1.28 (18H, br s, (CH2)9), 0.88 (3H, dist. t, terminus CH3). 13C NMR (100 MHz,
CDCl3, d, ppm): 166.2, 163.3, 138.7, 136.3, 132.2, 128.1, 128.0, 127.8, 70.7, 40.1,
39.9, 39.7, 39.4, 39.2, 31.3, 30.4, 29.4, 29.2, 29.0, 28.8, 26.1, 22.0, 14.1.
1-[(9R,12Z)-9-Hydroxy-octadec-12-enoyl]-5-phenylsemicarbazide (2d): off-white
Acknowledgments
The authors thank the Chairman, Department of Chemistry,
AMU, Aligarh, for providing necessary facilities and SAIF Punjab
University, Chandigarh for recording the spectra. One of the
authors (A.A.) is thankful to the University Grants commission
for financial support in part by a Grant from Major Research Project
[UGC-Scheme-F.No.33-263/2007 (SR)].
powder; yield 72%; mp 113–114 °C. IR (m
max, cmꢀ1, KBr): 3353 (OH), 3229 (NH,
NH–NH) 1668 (C@O). 1H NMR (400 MHz, CDCl3, d, ppm): 13.03 (1H, s, NH),
9.09 (2H, br s, NHNH), 8.16 (2H, d, J = 7.2 Hz, Ar-H-200/600), 7.61 (1H, t, J = 7.4 Hz,
Ar-H-400), 7.52 (2H, t, J = 7.2 Hz, Ar-H-300/500), 5.38 (2H, m, CH2–CH@CH–CH2),
4.89 (1H, m, CH–OH), 2.55 (2H, t, J = 7.6 Hz, CH2–CO), 2.26 (4H, m, CH2–
CH2@CH–CH2), 1.86 (2H, m, CH2–CH2–CO), 1.68 (1H, m, CH–OH), 1.40–1.25
(18H, br s, (CH2)9), 0.87 (3H, dist. t, terminus CH3). 13C NMR (100 MHz, CDCl3, d,
ppm): 167.6, 163.2, 138.4, 137.6, 131.2, 128.7, 128.3, 125.5, 70.56, 40.1, 39.9,
36.3, 24.8, 31.3, 29.1, 29.0, 28.9, 28.8, 28.5, 28.4, 25.1, 22.3, 14.01.
Synthesis of 5-(alkenyl/hydroxyalkenyl)-2-phenylamine 1,3,4-oxadiazoles (3a–d):
1-alkenoyl-5-phenyl semicarbazides (2a–d) (1.0 mmole) in POCl3 (6.0 ml)
were refluxed for 4 h. The resulting mixture was then poured into NaOH ice
water solution, resulting in deposition that was filtered, washed, dried and
recrystallized from aqueous ethanol and acetone (1:4 ml v/v) to give
compounds 3a–d. The characterization data of compounds 3a–d is given
below.
References and notes
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Sulfur Silicon Relat. Elem. 2001, 173, 223.
2. Wang, Z.; Shi, H.; Shi, H. J. Heterocycl. Chem. 2001, 38, 355.
3. Palaska, E.; Sahin, G.; Kelicen, P.; Durlu, N. T.; Altinok, G. Farmaco 2002, 57, 101.
4. Labanauskas, L.; Kalcas, V.; Udrenaite, E.; Gaidelis, P.; Brukstus, A.; Dauksas, V.
Pharmazie 2001, 56, 617.
5. Onkol, T.; Cakir, B.; Sahin, M. F. Turk. J. Chem. 2004, 28, 461.
6. Schenone, S.; Bruno, O.; Banise, A.; Bondavalli, F.; Filippeli, W.; Falcone, G.;
Giardano, L.; Vitelli, M. R. Bioorg. Med. Chem. 2001, 9, 2149.
7. Gokce, M.; Cakir, B.; Erol, K.; Sahin, M. F. Arch. Pharm. 2001, 334, 279.
8. Baldwin, J. J.; Engelhardt, E. L.; Hirschmann, R.; Ponticella, G. S.; Atkinson, J. G.;
Wasson, B. K.; Sweet, C. S.; Scriabini, A. J. Med. Chem. 1980, 23, 65.
9. Varvaresou, A.; Tsantili-Kakoulidou, A.; Siatra-Papastasikoudi, T.; Tiligada, E.
Arzneimittelforschung 2000, 50, 48.
10. Foroumadi, A.; Mirzaei, M.; Shafiee, A. Pharmazie 2001, 56, 610.
11. Mamola, M. G.; Falagiani, V.; Zanpieir, D.; Vio, L.; Banfi, F. Farmaco 2001, 56,
587.
12. Chen, H.; Li, Z.; Han, Y. J. Agric. Food. Chem. 2000, 48, 5312.
13. Zou, X. J.; Jin, G. Y.; Zang, Z. X. J. Agric. Food. Chem. 2002, 50, 1451.
14. Zou, X. J.; Lai, L. H.; Jin, G. Y.; Zhang, Z. K. J. Agric. Food. Chem. 2002, 50, 3757.
15. Clerici, F.; Pocav, D.; Guido, M.; Lochi, A.; Perline, V.; Brufani, M. J. Med. Chem.
2000, 44, 931.
5-(Dec-90-enyl)-2-phenylamine-1, 3, 4-oxadiazole (3a): white powder; yield
92%; mp 133–134 °C. IR (m
max, cmꢀ1, KBr): 3228 (NH), 1504 (C@N), 1258(C–O–
C). 1H NMR (400 MHz, CDCl3, d, ppm): 9.07 (1H, s, NH), 7.27 (2H, d, J = 7.6 Hz,
Ar-H-200/600), 7.18 (1H, t, J = 7.5 Hz, Ar-H-400), 6.98 (2H, t, J = 7.3 Hz, Ar-H-300/500),
5.79 (1H, tdd, JHꢀ
= 6.6 Hz, JHꢀH = 10.1 Hz, JHꢀH = 16.9 Hz, CH2@CH–), 5.00
9 CH2
= 10.1 Hz, JH
Z
E
(1H, dd, JH
= 2.2 Hz, HZC@CH), 4.94 (1H, dd, J
= 16.9 Hz,
HE ꢀH
Z ꢀH
Z ꢀHE
JH
= 2.2 Hz, HEC@CH–), 2.24 (2H, t, J = 7.5 Hz, CH2 a to ring), 2.00 (2H, m,
E ꢀHZ
CH2@CH–CH2), 1.58 (2H, m, CH2 b to ring), 1.21 (10H, br s, (CH2)5). 13C NMR
(100 MHz, CDCl3, d, ppm): 172.9, 155.6, 139.4, 139.0, 128.7, 122.2, 118.5, 114.2,
40.6, 40.2, 39.8, 39.4, 33.9, 29.2, 28.9, 25.4. MS (ESI): m/z = 322.3 [M+Na]+,
calcd = 322.4.
5-[(80Z)-Heptatadecenyl]-2-phenylamine-1,3,4-oxadiazole (3b): white crystals;
yield 90%; mp 144–146 °C. IR (mmax, cmꢀ1, KBr): 3218 (NH), 1505 (C@N), 1242
(C–O–C). 1H NMR (400 MHz, CDCl3, d, ppm): 9.39 (1H, s, NH), 7.26 (2H, d,
J = 7.7 Hz, Ar-H-200/600), 7.17 (1H, t, J = 7.5 Hz, Ar-H-400), 6.98 (2H, t, J = 7.3 Hz,
16. Rauf, A.; Parveen, H. Indian J. Chem. 2005, 44B, 1273.
17. Rauf, A.; Banday, M. R.; Matto, R. M. Acta Chim. Slov. 2008, 55, 448.
18. Ahmed, S. M.; Ahmad, F.; Osman, S. M. J. Am. Oil Chem. Soc. 1985, 62, 1578.
19. Khan, M. W. Y.; Ahmad, F.; Ahmad, I.; Osman, S. M. J. Am. Oil Chem. Soc. 1983,
60, 949.
20. Mujeebur-Rahman, V. P.; Mukhtar, S.; Ansari, W. H.; Lemiere, G. Eur. J. Med.
Chem. 2005, 40, 173.
21. Rauf, A.; Sharma, S.; Gangal, S. ARKIVOC 2007, xvi, 137.
22. Cruickshank, R.; Duguid, J. P.; Marmion, B. P.; Swain, R. H. A., 12th ed.. In
Medicinal Microbiology; Churchill Livingstone: London, 1975; vol. 2.
23. Collins, A. H. Microbiological Methods, second ed.; Butterworth: London, 1976.
24. Z.K. Khan, In vitro and vivo screening techniques for bioactivity screening and
evaluation, Proc. Int. Workshop UNIDO-CDRI, 1997, 210.
Ar-H-300/500), 5.33 (2H, m, CH2–CH@CH–CH2), 2.23 (2H, t, J = 7.5 Hz, CH2
a to
ring), 2.01 (4H, m, CH2–CH@CH–CH2), 1.56 (2H, m, CH2 b to ring), 1.29 (20H, br
s, (CH2)10), 0.87 (3H, dist. t, terminus CH3). 13C NMR (100 MHz, CDCl3, d, ppm):
172.9, 152.2, 133.5, 130.4, 129.3, 125.6, 123.2, 114.2, 40.6, 40.2, 39.8, 39.4,
38.6, 38.4, 33.9, 31.8, 31.4, 29.2, 28.4, 28.2, 27.9, 22.6, 14.0. MS (ESI): m/
z = 420.3 [M+Na]+, calcd = 420.5.
(80Z,110R)-5-(110-Hydroxy-octadec-80-enyl)-2-phenylamine-1,3,4-oxadiazole (3c):
off-white powder; yield 87%; mp 133–136 °C. IR (mmax, cmꢀ1, KBr): 3358 (OH),
3224 (NH), 1518 (C@N), 1220 (C–O–C). 1H NMR (400 MHz, CDCl3, d, ppm): 9.31
(1H, s, NH), 7.62 (2H, d, J = 7.2 Hz, Ar-H-200/600), 7.53 (1H, t, J = 7.0 Hz, Ar-H-400),
7.41 (2H, t, J = 7.2 Hz, Ar-H-300/500), 5.37 (2H, m, CH2–CH@CH–CH2), 4.69 (1H, m,
25. Varma, R. S., Ed.Antifungal Agents: Past, Present and Future prospects; National
Academy of Chemistry & Biology: Lucknow, India, 1998.
26. Gunstone, F. D. J. Chem. Soc. 1954, 1611.
CH–OH), 2.38 (2H, t, J = 7.5 Hz, CH2
a to ring), 1.98 (4H, m, CH2–CH = CH–CH2),
1.78 (1H, m, CH–OH), 1.67 (2H, m, CH2 b to ring), 1.28 (18H, br s, (CH2)9), 0.88
(3H, dist. t, terminus CH3). 13C NMR (100 MHz, CDCl3, d, ppm): 172.1, 163.2,
138.7 ‘one signal hidden00, 132.2, 128.1, 128.0, 127.0, 70.7, 40.1, 39.9, 39.7, 39.5,
39.3, 31.3, 30.4, 29.1, 29.0, 28.8, 28.6, 25.1, 22.0, 13.6.167.2, 139.8, 139.0, 133.5,
130.0, 129.8, 127.9, 77.1, 38.6, 38.4, 33.5, 31.7, 30.2, 29.9, 29.6, 29.3, 28.7, 28.5,
28.4, 28.3, 22.4, 14.4. MS (ESI): m/z = 436.5 [M+Na]+, calcd = 436.59.
27. Banday, M. R.; Rauf, A. Indian J. Chem. 2009, 48B, 97.
28. General procedure for the synthesis of long-chain alkenoic acid hydrazide (1a–d):
the hydrazides of long-chain alkenoic acids (1a–d) which are used as the
starting material were prepared by the previously reported methods.21
Synthesis of 1-(alkenoyl/hydroxyalkenoyl)-5-phenylsemicarbazide (2a–d):
hydrazide (1a–d) (1.0 mmole) was dissolved in abs. ethanol by heating to
make a clear solution. An equal molar amount of phenyl isocyanate was added
to it and the solution was refluxed for 5 h, cooled to get a precipitate. The
precipitate was filtered, washed with abs ethanol and dried to give analytically
pure compounds 2a–d. The characterization data of compounds 2a–d is given
below.
(80R,110Z)-5-(80-Hydroxy-octadec-110-enyl)-2-phenylamine-1,3,4-oxadiazole (3d):
off-white powder; yield 87%; mp 135–137 °C. IR (mmax, cmꢀ1, KBr): 3353
(OH), 3217 (NH), 1494 (C@N), 1225 (C–O–C). 1H NMR (400 MHz, CDCl3, d,
ppm): 9.88 (1H, s, NH), 8.20 (2H, d, J = 7.3 Hz, Ar-H-200/600), 7.63 (1H, t,
J = 7.4 Hz, Ar-H-400), 7.54 (2H, t, J = 7.9 Hz, Ar-H-300/500), 5.34 (2H, m, CH2–
CH@CH–CH2), 4.87 (1H, m, CH–OH), 2.50 (2H, t, J = 7.5 Hz, CH2
a to ring), 1.98
(4H, m, CH2–CH@CH–CH2), 1.77 (1H, m, CH–OH), 1.67 (2H, m, CH2 b to ring),
1.28 (18H, br s, (CH2)9), 0.89 (3H, dist. t, terminus CH3). 13C NMR (100 MHz,
CDCl3, d, ppm): d 172.1, 165.7, 139.1, 139.0, 133.0, 130.7, 129.4, 127.6, 77.3,
38.6, 36.4, 30.4, 31.8, 29.8, 29.6, 29.4, 29.2, 29.0, 28.9, 28.6, 25.9, 22.2, 14.3. MS
(ESI): m/z = 436.4 [M+Na]+, calcd = 436.59.
Synthesis of 1-(alkenoyl/hydroxyalkenoyl)-5-phenylthiosemicarbazide (4a–d): 1-
alkenoyl-5-phenylthiosemicarbazides (4a–d) were prepared by the reported
literature method.27
1-(Undec-10-enoyl)-5-phenylsemicarbazides (2a): white powder; yield 80%; mp
103–104 °C. IR (mmax, cmꢀ1, KBr): 3236 (NH, NH–NH), 1667 (C@O). 1H NMR
(400 MHz, CDCl3, d, ppm): 13.04 (1H, s, CO–NH-Ar), 9.16 (2H, br s, CO–NHNH–
CO), 8.17 (2H, d, J = 7.2 Hz, Ar-H-200/600), 7.60 (1H, t, J = 7.4 Hz, Ar-H-400), 7.51
(2H, t, J = 7.4 Hz, Ar-H-300/500), 5.82 (1H, tdd, JHꢀ
= 6.8 Hz, JHꢀH = 10.0 Hz,
9 CH2
Z
JHꢀH = 17.8 Hz, CH2=CH–), 5.01 (1H, dd, JH
= 10.0 Hz, JH
= 2.8 Hz,
Z ꢀHE
Z ꢀH
E
HZC@CH), 4.94 (1H, dd, J
= 17.8 Hz, JH
= 2.8 Hz, HEC@CH–), 2.45 (2H, t,
E ꢀHZ
HE ꢀH
J = 7.4 Hz, CH2–CO), 2.01 (2H, m, CH2@CH–CH2), 1.82 (2H, m, CH2CH2–CO), 1.45–
1.25 (10H, br s, (CH2)5). 13C NMR (100 MHz, CDCl3, d, ppm): 168.2, 165.4, 139.2,
133.2, 131.3, 128.9, 128.6, 114.2, 33.8, 29.9, 29.6, 29.3, 29.2, 29.1, 29.0, 28.9.
1-[(9Z)-Octadec-9-enoyl]-5-phenylsemicarbazides (2b): white powder; yield
79%; mp 101–102 °C. IR (mmax, cmꢀ1, KBr): 3222 (NH, NH–NH), 1665(C@O).
1H NMR (400 MHz, CDCl3, d, ppm): 13.18 (1H, s, NH), 9.18 (2H, br s, NHNH),
8.08 (2H, d, J = 7.2 Hz, Ar-H-200/600), 7.64 (1H, t, J = 7.4 Hz, Ar-H-400), 7.51 (2H, t,
J = 7.2 Hz, Ar-H-300/500), 5.31 (2H, m, CH2–CH@CH–CH2), 2.54 (2H, t, J = 7.8 Hz,
CH2–CO), 2.36 (4H, m, CH2–CH2@CH–CH2), 1.82 (2H, m, CH2–CH2–CO), 1.37–
1.25 (20H, br s, (CH2)10), 0.89 (3H, dist. t, terminus CH3). 13C NMR (100 MHz
CDCl3, d, ppm): 167.4, 164.7, 139.2, 137.3, 133.2, 131.5, 128.8, 129.0, 31.9, 30.7,
30.4, 30.1, 29.9, 29.7, 29.6, 29.4, 29.2 ‘two signals are hidden’, 29.1, 26.6, 22.7,
14.2.
Synthesis of 5-(alkenyl/hydroxyalkenyl)-2-phenylamine-1,3,4-thiadiazoles (5a–d):
1.0 mmole of compounds (4a–d) in acetic anhydride (Ac2O) (6.0 ml) was
refluxed for 5 hrs. The resulting mixture was poured into crushed ice (100gm)
with stirring. The product thus obtained was filtered, washed with cold water,
dried and recrystallized from aqueous ethanol and acetone (1:4 ml v/v) to give
analytically pure compounds 5a–d. The characterization data of compounds
5a–d prepared according to the above procedure is given below.
5-(dec-90-enyl)-2-Phenylamine-1, 3, 4-thiadiazole (5a): white powder; Yield
95%; mp 134–135 °C. IR (mmax, cmꢀ1, KBr): 3221 (NH), 1488 (C@N), 707 (C–S–
C). 1H NMR (400 MHz, CDCl3, d, ppm): 12.20 (1H, s, NH), 8.16 (2H, d, J = 8.5 Hz,
Ar-H-200/600), 7.55 (1H, t, J = 7.3 Hz, Ar-H-400), 7.44 (2H, t, J = 7.8 Hz, Ar-H-300/500),
5.73 (1H, tdd, JHꢀ
= 6.7 Hz, JHꢀH = 10.1 Hz, JHꢀH = 16.9 Hz, CH2@CH–), 4.98
9 CH2
= 10.1 Hz, JH
Z
E
(1H, dd, JH
= 2.1 Hz, HZC@CH), 4.85 (1H, dd, J
= 16.9 Hz,
HE ꢀH
Z ꢀH
Z ꢀHE
1-[(9Z,12R)-12-Hydroxy-octadec-9-enoyl]-5-phenylsemicarbazides (2c): off-
JH
= 2.1 Hz, HEC@CH–), 2.98 (2H, t, J = 7.5 Hz, CH2 a to ring), 1.96 (2H, m,
white powder; yield 77%; mp 111–113 °C. IR (mmax, cmꢀ1, KBr): 3343 (OH),
E ꢀHZ

1938
N. N. Farshori et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1933–1938
CH2@CH–CH2), 1.76 (2H, m, CH2 b to ring), 1.25 (10H, br s, (CH2)5). 13C NMR
7.52 (2H, t, J = 7.8 Hz, Ar-H-300/500), 5.37 (2H, m, CH2–CH@CH–CH2), 3.59 (1H, s,
CH–OH), 3.00 (2H, t, J = 7.5 Hz, CH2 to ring), 2.38 (4H, m, CH2–CH2@CH–CH2),
(100 MHz, CDCl3, d, ppm): 165.2, 153.0, 139.1, 133.4, 131.3, 129.8, 127.9, 114.2,
33.7, 29.1, 28.9, 28.8, 28.7, 28.4, 26.0, 25.8. MS (ESI): m/z = 338.2 [M+Na]+,
calcd = 338.47.
a
1.83 (2H, m, CH2 b to ring), 1.71 (1H, m, CH–OH), 1.26 (18H, br s, (CH2)9), 0.87
(3H, dist. t, terminus CH3). 13C NMR (100 MHz, CDCl3, d, ppm): 170.9, 165.0,
133.3, 131.1, 130.7, 130.2, 128.8, 128.5, 73.9, 34.1, 31.5, 29.8, 29.7, 29.5, 29.3,
29.1, 28.9, 27.1, 25.2, 23.1, 22.5, 21.3, 14.6. MS (ESI): m/z = 452.4 [M+Na]+,
calcd = 452.6.
5-(80Z)(heptadec-80-enyl)-2-Phenylamine-1,3,4-thiadiazole (5b): white powder;
Yield 91%; mp 138–140 °C. IR (mmax, cmꢀ1, KBr): 3219 (NH), 1468 (C@N), 701
(C–S–C). 1H NMR (400 MHz, CDCl3, d, ppm): 12.10 (1H, s, NH), 8.14 (2H, d,
J = 8.5 Hz, Ar-H-200/600), 7.55 (1H, t, J = 7.4 Hz, Ar-H-400), 7.45 (2H, t, J = 7.8 Hz,
5-[(80R,110Z)-80-hydroxy-heptadec-110-enyl]-2-Phenylamine-1,3,4-thiadiazole (5d):
Ar-H-300/500), 5.30 (2H, m, CH2–CH@CH–CH2), 2.98 (2H, t, J = 7.7 Hz, CH2
a
to
off-white powder; Yield 83%; mp 139–141 °C. IR (m
max, cmꢀ1, KBr): 3322 (OH),
ring), 2.36 (4H, m, CH2–CH2@CH–CH2), 1.76 (2H, m, CH2 b to ring), 1.28 (20H, br
s, (CH2)10), 0.80 (3H, dist. t, terminus CH3). 13C NMR (100 MHz, CDCl3, d, ppm):
165.4, 158.0 ‘one signal hidden’, 139.1, 137.2, 133.2, 131.3, 128.8, 128.6, 31.9,
29.9, 29.7, 29.5 ‘two signals are hidden’, 29.4 ‘two signals are hidden’, 29.3,
29.1, 28.7, 26.4, 22.7, 14.2. MS (ESI): m/z = 436.3 [M+Na]+, calcd = 436.6.
5-[(80Z,110R)-110-hydroxy-heptadec-80-enyl]-2-Phenylamine-1,3,4-thiadiazole (5c):
white powder; Yield 85%; mp 136–138 °C. IR (mmax, cmꢀ1, KBr): 3310 (OH),
3219 (NH), 1467 (C@N), 695 (C–S–C). 1H NMR (400 MHz, CDCl3, d, ppm): 12.37
(1H, s, NH), 8.23 (2H, d, J = 8.5 Hz, Ar-H-200/600), 7.62 (1H, t, J = 7.4 Hz, Ar-H-400),
3236 (NH), 1461 (C@N), 699 (C–S–C). 1H NMR (400 MHz, CDCl3, d, ppm): 12.17
(1H, s, NH), 8.31 (2H, d, J = 8.4 Hz, Ar-H-200/600), 7.43 (1H, t, J = 7.4 Hz, Ar-H-400),
7.25 (2H, t, J = 7.4 Hz, Ar-H-300/500), 5.36 (2H, m, CH2–CH@CH–CH2), 3.57 (1H, s,
CH–OH), 2.81 (2H, t, J = 7.5 Hz, CH2
a to ring), 2.23 (4H, m, CH2–CH2@CH–CH2),
2.04 (2H, m, CH2 b to ring), 1.71 (1H, m, CH–OH), 1.33 (18H, br s, (CH2)9), 0.90
(3H, dist. t, terminus CH3). 13C NMR (100 MHz, CDCl3, d, ppm): 168.9, 163.1,
137.6, 133.2, 131.2, 128.7, 128.3, 125.5, 70.56, 40.1, 39.9, 36.3, 24.8, 31.3, 29.1,
29.0, 28.9, 28.8, 28.5, 28.4, 25.1, 22.3, 14.01. MS (ESI): m/z = 452.5 [M+Na]+,
calcd = 452.6.