Calix[4]arene based 1,3,4-oxadiazole and thiadiazole derivatives: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1039/c2ob06730g

In the present investigation, we describe some novel calixarene based heterocyclic compounds (5a-5i) in which 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives have been coupled with 5,11,17,23-tetra-tert-butyl-25,27- bis(chlorocarbonyl-methoxy)-26,28-dihydroxy calix[4]arene. All the newly synthesized calixarene based heterocyclic compounds have been characterized by elemental analysis and various spectroscopic methods like FTIR, ¹H NMR, ¹³C NMR, and FAB-MS. All the final scaffolds have been subjected to antioxidant activity, in vitro antimicrobial screening against two gram (+ve) bacteria (S. aureus, S. pyogenes), two gram (-ve) bacteria (E. coli, P. aeruginosa) and two fungal strains (C. albicans, A. clavatus) and also have been screened for their antitubercular activity against Mycobacterium tuberculosis H₃₇Rv.

Full text of DOI:10.1039/c2ob06730g

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PAPER
Calix[4]arene based 1,3,4-oxadiazole and thiadiazole derivatives: Design,
synthesis, and biological evaluation
Manishkumar B. Patel,a Nishith R. Modi,a Jignesh P. Ravalb and Shobhana K. Menon*a
Received 13th October 2011, Accepted 5th December 2011
DOI: 10.1039/c2ob06730g
In the present investigation, we describe some novel calixarene based heterocyclic compounds (5a–5i) in
which 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives have been coupled with 5,11,17,23-tetra-tert-
butyl-25,27-bis(chlorocarbonyl-methoxy)-26,28-dihydroxy calix[4]arene. All the newly synthesized
calixarene based heterocyclic compounds have been characterized by elemental analysis and various
spectroscopic methods like FTIR, 1H NMR, 13C NMR, and FAB-MS. All the final scaffolds have been
subjected to antioxidant activity, in vitro antimicrobial screening against two gram (+ve) bacteria
(S. aureus, S. pyogenes), two gram (−ve) bacteria (E. coli, P. aeruginosa) and two fungal strains
(C. albicans, A. clavatus) and also have been screened for their antitubercular activity against
Mycobacterium tuberculosis H37Rv.
focusing on the development of calixarene platforms designed
Introduction
molecular drug dispensers offering penicillin and quinolone moi-
eties at the lower rim.22–25 Ionic calixarene derivatives exhibit
The development of biologically active molecules based on mol-
intrinsic antimicrobial activity,26–29 while p-guanidino ethyl
ecular recognition is an attractive and challenging research topic
in medicinal and supramolecular chemistry.1 The calixarenes
calixarene and parent phenol derivatives exhibited antibacterial
activities.30 Hydroxycinnamic acid based derivatives also had
have often been employed in recent years as carriers and spatial
organizers of various kinds of active substituent, displaying
properties dealing with recognition of organic substrates or met-
allic cations. Efforts have also been made to use their specific
conformations to prepare, notably in the case of the tensed calix
[4]arene, highly ordered and organized molecular devices.2–5 As
assessed by recent reviews,6–10 the calixarenes have been investi-
gated in the medicinal field, some patents, have been devoted to
their use in this domain, for example, hydrophilic derivatives
been reported as radical scavenging agents having antioxidant
activity.31
Heterocyclic compounds containing five-membered rings
gained importance because of their versatile biological proper-
ties. In particular, compounds bearing 1,3,4-oxadiazole nucleus
has been found to exhibit diverse biological activities such as
37
antimicrobial,32–34 anti-HIV,32 antitubercular,35,36 antioxidant
and antimalarial.38
have shown interesting levels of activity against bacteria,11 fungi,
The emergence of resistance in pathogenic microorganisms to
commercial antibiotic calls for the development of specific fields
of research dedicated to the discovery of new drugs.39 Tubercu-
losis (TB) is one of the most common infectious diseases known
to mankind. Around 32% of the world’s population is infected
by Mycobacterium tuberculosis, the main causal agent of TB.
Problems in the treatment of tuberculosis arise when bacteria
develop resistance to the first-line drugs like isoniazid, rifampicin,
ethambutol, streptomycin, and pyrazinamide.40
Isoniazid is a prodrug and must be activated by bacterial cata-
lase. It is activated by catalase–peroxidase hemoproteins, KatG,
which couples the isonicotinic acyl with nicotinamide adenine
dinucleotide (NADH) to form the isonicotinic acyl-NADH
complex. This complex indirectly inhibits the synthesis of
mycolic acid required for the mycobacterial cell wall.41 It is bac-
tericidal to rapidly-dividing mycobacteria but is bacteriostatic if
the Mycobacterium is slow-growing.42 Isoniazid is metabolized
by the liver, mainly by acetylation and dehydrogenation. The
13
cancerous cell, enveloped virus,12 thrombosis
diseases.14
and fibrosis
15
In the mid-1950s, the calixarene derivative ‘Macrocyclon’
16,17
and more recently some parent structures
have been studied
in the treatment of tuberculosis and other mycobacterioses.
The building of designed calixarenic mimics of vancomycin
has also been studied, with the aim of focusing on an antimicro-
bial activity,18 and some biological studies related to plasmid
DNA binding, and cell transfection have notably been reported
by Ungaro and co-workers.19–21 Regnouf and coworkers,
aDepartment of Chemistry, School of Sciences, Gujarat University,
Navrangpura, Ahmedabad, 380009 Gujarat, India. E-mail: shobhana-
menon07@gmail.com; Fax: +91 79 26308545; Tel: +91 79 26302286
bDepartment of Pharmaceutical Chemistry, Ashok & Rita Patel Institute
of Integrated Study and Research in Biotechnology and Allied Sciences,
New Vallabh Vidyanagar, 388121 Gujarat, India
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N-acetyl hydrazine metabolite is believed to be responsible for
the hepatotoxicity effects seen in patients treated with isoniazid.
It is established that CYP2E1 (cytochrome P450, family 2, sub-
family E polypeptide 1) is reportedly involved in INH-induced
hepatotoxicity.43
For this reason, mentioned above in our study, we tried to
eliminate in vivo acetylation by arylamine N-acetyltransferase
(NAT) to form an inactive acetylated drug, by replacing the
hydrazide moiety of INH (isonicotinic acid hydrazide) with
1,3,4-thiadiazole and 1,3,4-oxadiazole heterocycles.
We report here the synthesis and characterization of novel
calix[4]arene derivatives incorporating two oxadiazole and thia-
diazole subunits at the lower rim. The pharmacophores oxa-
diazole and thiadiazole subunits were incorporated into
functionalized calix[4]arene anticipating an enhancement of lipo-
philicity and a cooperative effect of the subunits by bringing
them together at the lower rim, resulting from the organizational
role of the calixarene core. We also have evaluated their antio-
xidant activity, antibacterial activity against gram +ve and gram
−ve strains, antifungal activity against fungal strains and antitu-
bercular activity against H37RV bacteria.
Results and discussion
Chemistry
As part of our research program on developing new antimyco-
bacterial and antimicrobial agents, we designed several functio-
nalized fullerenes,44,45 and now the work is extended to
functionalized calixarenes. For the synthesis of functionalized
calixarenes the hydrazides of nicotinic acid, benzoic acid and
cis-cinnamic acid were prepared by refluxing the above acids
with sulphuric acid and methanol for 10 h to form the corre-
sponding acid ester and this ester was condensed with hydrazine
hydrate by maintaining the reaction temperature at 0 °C to form
nicotinic acid, benzoic acid and cis-cinnamic acid hydrazide.
Intramolecular cyclization of nicotinic acid, benzoic acid and
cis-cinnamic acid hydrazide with carbon disulfide and potassium
hydroxide in the presence of ethanol resulted in 5-(substituted
pyridyl)-1,3,4-oxadiazole-2-thiols (A, D, F & H). The corre-
sponding acid hydrazide was refluxed with ammonium thiocya-
nate in an acidic medium to obtain acid thiosemicarbazides,
which gave 2-amino-5-(substituted pyridyl)-1,3,4-thiadiazoles
(B, E, G & I) on cyclization in the presence of conc. sulfuric
acid. Isoniazid, on stirring and heating with cyanogen bromide,
gave 2-amino-5-(pyridin 4-yl)-1,3,4-oxadiazole (C). (Scheme 1)
The tetra-tert-butylcalix[4]arene 1 was prepared according
to Gutche’s procedure.46 The synthesis of 5,11,17,23-tetra-tert-
butyl-25,27-diethoxycarbonylmethoxy-26,28-dihydroxy calix-[4]
arene 2 was carried out by the reaction of tetra-p-tert-butylca-
lix[4]arene 1 and bromo ester in refluxing acetone in the pres-
ence of K2CO3 as a base, and gave 78% yield. Ester 2 was
hydrolyzed in a hydroalcoholic medium to give the correspond-
ing diacid 3, with 97% yield. The diacid 3, when refluxed in the
presence of thionyl chloride, afforded the corresponding diacid
chloride compound 4 with a quantitative yield. Compound 4 was
then refluxed in THF with compound (A, D, F & H) in the pres-
ence of pyridine to give the corresponding 1,3,4-oxadiazole
coupled calix[4]arenes (5a, 5d, 5f & 5h). Compound 4 when
Scheme 1 Synthesis of oxadiazole & thiadiazole derivatives of isonia-
zid, nicotinic, benzoic and cis-cinnamic acid hydrazide.
refluxed in THF with the 2-amino-5-(substituted pyridyl)-1,3,4-
thiadiazoles compounds (B, E, G & I) in a presence of pyridine
gave the corresponding 1,3,4-thiadizole coupled calix[4]arenes
(5b, 5e, 5g & 5i), Compound 4 when refluxed in THF with C
in the presence of pyridine gave corresponding 5,11,17,23-tetra-
tert-butyl-25,27-bis(5-(pyridin-4-yl)-1,3,4-oxadiazole-2-amide-
methyleneoxy) 26,28-dihydroxy calix[4]arene 5c. (Scheme 2)
Biological evaluation
In vitro evaluation of antimicrobial activity
The Minimum Inhibitory Concentration (MIC) of synthesized
compounds 5a–5i was carried out by broth micro dilution
method as described by Rattan.47 Antibacterial activity was
screened against two +ve (gram positive) bacteria (S. aureus
MTCC 96, S. pyogenes MTCC 442) and two −ve (gram nega-
tive) bacteria (E. coli MTCC 443, P. aeruginosa MTCC 1688).
Ampicillin was used as a standard antibacterial agent. The anti-
fungal activity of the synthesized compounds 5a–5i was
screened against two fungal species (C. albicans MTCC 227,
A. clavatus MTCC 1323). Ampicillin and Griseofulvin were
used as standard drugs.
All MTCC (Microbial Type Culture Collection) cultures were
provided by the Institute of Microbial Technology, Chandigarh
and tested against known drugs ampicillin and griseofulvin.
Mueller–Hinton broth was used as the nutrient medium to grow
and to dilute the drug suspension for the test. Inoculum size for
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Scheme 2 Synthesis of calix[4]arene-based-1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives of isoniazid, nicotinic acid, benzoic acid, and cis-cin-
namic acid.
test strain was adjusted to 108 CFU (Colony Forming Unit) per
millilitre by comparing the turbidity. DMSO was used as dilu-
ents to get desired concentration of drugs to test upon standard
bacterial strains. Serial dilutions were prepared for primary and
secondary screening. The control tube, containing no antibiotic,
was immediately subcultured (before inoculation) by spreading a
loopful evenly over a quarter of plate of a medium suitable for
the growth of the test organism and put for incubation overnight
at 37 °C. The MIC of the control organism was read to check the
accuracy of the drug concentrations. The lowest concentration
inhibiting growth of the organism was recorded as the MIC. All
the tubes not showing visible growth (in the same manner as a
control tube described above) was subcultured and incubated
overnight at 37 °C. The amount of growth from the control tube
before incubation (which represents the original inoculum) was
compared. Subcultures might show similar number of colonies
indicating bacteriostatic, a reduced number of colonies indicating
a partial or slow bactericidal activity, or no growth if the whole
inoculum has been killed. The test included a second set of the
same dilutions inoculated with an organism of known sensitivity.
A stock solution of 2000 μg mL−1 of each of the synthesized
compounds was prepared and diluted as and when required. In
the primary screening 500, 250, 200 and 125 μg mL−1 concen-
trations of the synthesized drugs were taken. The active syn-
thesized drugs found in this primary screening were further
tested in a second set of dilution against all microorganisms. The
drugs found active in primary screening were similarly diluted to
obtain 100, 50, 25, 12.5, 6.250, 3.125 and 1.5625 μg mL−1 con-
centrations. The highest dilution showing at least 99% inhibition
is taken as MIC.
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Table 1 In vitro antibacterial activity of compounds (A–I) and newly synthesized calix[4]arene based 1,3,4-oxadiazoles and 1,3,4-thiadiazole
derivatives of isoniazid, nicotinic acid hydrazide, benzoic acid hydrazide and cis-cinnamic acid hydrazide (5a–5i)
In vitro activity-zone of inhibition in mm (MIC in μg mL−1 a
)
Gram+ve
Gram−ve
E.coli
MTCC 443
P. aeruginosa
MTCC 1688
S. aureus
MTCC 96
S. pyogenes
MTCC 442
Compound no
Concentration μg/disc
A
B
C
D
E
F
G
H
I
5a
5b
5c
5d
5e
5f
5g
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
14.00 0.04 (200)
12.00 0.01 (250)
11.00 0.13 (250)
10.35 0.18 (250)
10.50 0.34 (250)
11.40 0.29 (250)
8.00 0.74 (500)
11.34 0.59 (250)
12.10 0.02 (200)
19.00 0.01 (200)
17.00 0.01 (100)
18.00 0.02 (100)
17.35 0.18 (200)
12.20 0.34 (250)
16.40 0.29 (200)
12.00 0.74 (250)
17.32 0.29 (100)
15.10 0.02 (200)
19.00 0.00 (100)
—
13.00 0.02 (200)
12.40 0.04 (200)
12.67 0.01 (250)
13.00 0.43 (200)
11.45 0.94 (200)
12.15 0.95 (250)
8.40 0.03 (200)
12.00 0.45 (250)
11.50 0.35 (250)
19.00 0.02 (200)
18.00 0.04 (200)
18.00 0.02 (100)
19.00 0.43 (200)
17.46 0.94 (250)
17.15 0.95 (200)
12.40 0.03 (250)
18.00 0.25 (100)
16.50 0.15 (100)
20.00 0.00 (100)
—
13.00 0.02 (100)
14.50 0.05 (200)
14.00 0.02 (250)
11.00 0.03 (250)
12.00 0.25 (250)
11.25 0.40 (200)
8.30 0.15 (250)
14.30 0.78 (250)
12.40 0.15 (250)
17.00 0.02 (100)
16.00 0.05 (200)
17.00 0.04 (250)
16.00 0.33 (100)
15.00 0.25 (250)
14.20 0.40 (100)
11.00 0.15 (250)
17.30 0.78 (100)
13.00 0.15 (200)
18.00 0.00 (250)
—
14.50 0.03 (200)
13.00 0.03 (200)
12.00 0.44 (250)
10.40 0.20 (250)
10.25 0.50 (250)
11.12 0.40 (250)
6.55 0.90 (250)
13.00 0.57 (250)
13.20 0.60 (250)
19.00 0.03 (200)
16.00 0.03 (200)
18.00 0.04 (250)
17.40 0.02 (250)
15.00 0.05(200)
15.15 0.68 (200)
14.55 0.90 (250)
19.00 0.47 (200)
16.30 0.60 (200)
19 0.00 (100)
5h
5i
Ampicillinb
DMSO
—
a MIC values are given in brackets. MIC (μg mL−1) = Minimum inhibitory concentration, mean of five replicates standard deviation. b Standard
Table 2 In vitro antifungal activity of compounds (A–I) and the newly
synthesized calix[4]arene based 1,3,4-oxadiazoles and 1,3,4-thiadiazole
derivatives of isoniazid, nicotinic acid hydrazide, benzoic acid
hydrazide, cis-cinnamic acid hydrazide (5a–5i)
In vitro activity-zone of inhibition in mm (MIC in μg mL−1 a
)
C.albicans
A.clavatus
Compound
no
Concentration
μg/disc
MTCC 227
MTCC 1323
A
B
C
D
E
F
G
H
I
5a
5b
5c
5d
5e
5f
5g
5h
5i
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
16.24 0.32 (250)
12.00 0.01 (500)
14.25 0.04 (500)
14.36 0.65 (250)
13.07 0.12 (500)
10.10 0.15 (500)
6.50 0.70 (500)
14.30 0.56 (250)
13.20 0.58 (500)
23.30 0.02 (250)
21.50 0.01 (500)
22.00 0.04 (500)
22.36 0.79 (250)
12.07 0.22 (500)
18.00 0.15 (500)
9.00 0.07 (500)
21.36 0.79 (250)
17.00 0.48 (500)
24.00 0.00 (500)
—
14.00 0.35 (500)
12.33 0.05 (500)
11.45 0.15 (500)
13.00 0.43 (500)
14.35 0.42 (500)
14.00 0.05 (500)
10.30 0.12 (500)
16.37 0.31 (500)
14.22 0.25 (500)
24.25 0.03 (500)
20.45 0.05 (500)
21.50 0.01 (500)
23.00 0.23 (500)
18.45 0.22 (500)
19.00 0.35 (500)
11.30 0.12 (500)
22.00 0.01 (250)
18.32 0.30 (500)
24.00 0.00 (100)
—
Fig. 1 Antibacterial activity of compounds (A–I) and (5a–5i).
Griseofulvinb 250
DMSO
250
a MIC values are given in brackets. MIC (μg mL−1) = Minimum
inhibitory concentration. Mean of five replicates: Standard deviation.
b Standard
Fig. 2 Antifungal activity of compounds (A–I) and (5a–5i).
Reviewing of the antibacterial activities (Fig. 1), and antifun-
gal activity (Fig. 2), of compounds A–I, and 5a–5i are presented
in Table 1 & Table 2. The results revealed that compounds A
and I displayed good activity compare to others against gram
−ve bacteria E.coli (growth inhibition zones 14
0.04 and
12.10 0.02 mm) at MIC 200 μg mL−1 and compound A and D
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showed good inhibition compared to others against gram −ve
bacteria P.aeruginosa (growth inhibition zones 13 0.02 and
13.00
concentration of each test compound was prepared in dimethyl
sulfoxide (DMSO). These tubes were then incubated at 37 °C
for 24 h followed by streaking of M.tuberculosis H37Rv (5 × 104
bacilli per tube). These tubes were then incubated at 37 °C.
Growth of bacilli was seen after 12, 22, and finally 28 days
of incubation. Tubes having the compounds were compared
with control tubes where the medium alone was incubated with
M.tuberculosis H37Rv. The concentration at which no develop-
ment of colonies occurred or <20 colonies was taken as
the MIC concentration of the test compound. The standard strain
M. tuberculosis H37Rv was tested with known drug isoniazid.
In vitro antimycobacterial activity (Fig. 3) showed that all the
compounds are bioactive against M. tuberculosis H37Rv compar-
able to the standard. Compounds A, C and H showed good anti-
mycobacterial activity (% inhibition 82.27 0.04, 71.50 0.03
and 63.23 0.90) at MIC 50 μg mL−1 and 100 μg mL−1. Also
compounds 5a and 5c exhibited excellent antimycobacterial
activity (% inhibition 94 0.01, 96.54 0.90) at MIC 50 μg
mL−1 and 62.5 μg mL−1 against M.tuberculosis H37RV. The
compounds 5b, 5d and 5h showed moderate to good antimyco-
bacterial activity (% inhibition 92.80 0.07, 89.42 0.8 and
91.23 0.92) at MIC 150, 100 & 62.5 μg mL−1 against M.tuber-
culosis H37RV. Compounds 5f, 5i and 5e displayed less antimy-
cobacterial activity (% inhibition 45 0.06, 57.10 0.41 and
0.43 mm) at MIC 200 μg mL−1, also compound A
showed good inhibition against gram +ve S.aureus (% inhibition
13 0.02 mm) at MIC 100 μg mL−1 and compound A showed
good inhibition against gram +ve S.pyogenes (growth inhibition
zones 14.50 0.03 mm) at MIC 200 μg mL−1. Compounds 5c,
5h and 5b displayed excellent activity against both gram −ve
bacteria E.coli (growth inhibition zones 18 0.02, 17.32 0.29
and 17
0.01 mm) at MIC 100 μg mL−1 and P.aeruginosa
(growth inhibition zones 18 0.02, 18.00 0.25 and 16.50
0.15 mm) at MIC 100 μg mL−1. Compounds 5a & 5h exhibited
excellent activity against S.aureus gram +ve bacteria (growth
inhibition zones 17 0.02 and 17.30 0.78 mm) at MIC 100 μg
mL−1 and S.pyogenes (gram +ve bacteria) (growth inhibition
zones 19 0.03 and 19.00 0.47) at MIC 200 μg mL−1. Com-
pounds 5d, 5f and 5i displayed a moderate to good inhibition
zone against both gram −ve bacteria (growth inhibition zones
17.35
0.18 and 16.40
0.29, 15.10
0.02 mm) at MIC
200 μg mL−1 and compounds 5b and 5c also showed good
activity against both gram +ve bacteria (growth inhibition zones
16 0.05, and 16 0.03 mm) at MIC 200 μg mL−1 and (growth
inhibition zones 17 0.04 and 18 0.04 mm) at MIC 250 μg
mL−1. Compounds 5e and 5g, showed weak activity against
E.coli (gram −ve) bacteria (growth inhibition zones <12.00
0.74, 12.40 0.03 mm) at MIC 250 μg mL−1 while compound
5g showed weak activity against both gram +ve bacteria (growth
inhibition zones 11.00 0.15 and 14.55 0.99 mm) at MIC
250 μg mL−1 respectively. Compounds A and D displayed a
moderate to good inhibition zone against C.albicans (growth
inhibition zones 16.24 0.32 and 14.36 0.65 mm) at MIC
250 μg mL−1, and also compound H showed good inhibition
against A. clavatus (growth inhibition zones 16.37 0.31 mm)
at MIC 500 μg mL−1. All the tested compounds 5a, 5d, and 5h
displayed excellent activity against fungi C.albicans (growth
38.24
0.57) at MIC 200, μg mL−1 towards M.tuberculosis
H37RV. Compound 5g showed no inhibition, as displayed in
Table 3. The presence of the 1,3,4-oxadiazole and 1,3,4-thiadia-
zole ring as pharmacophores and the increase in the lipophilic
character of the molecule due to the presence of substituted calix
[4]arene, which facilitates the crossing through the biological
membrane of the micro-organism thereby inhibiting their
growth.
inhibition zones 23.30
0.02, 22.36
0.79 and 21.36
0.79 mm) at MIC 250 μg mL−1 while compound 5h, exhibited
excellent activity against fungi A.clavatus (growth inhibition
zones 22 0,01) at MIC 250 μg ml−1. Compounds 5c, 5b, 5f
and 5i showed moderate to good inhibitory effects towards tested
fungi (growth inhibition zones >17 mm) at MIC 500 μg ml−1
.
Compounds 5e and 5g showed weak inhibitory effect towards C.
albicans (growth inhibition zones 12.07
0.22, and 9
0.07 mm) at MIC 500 μg mL−1 and 5g exhibited weak activity
against A.clavatus (growth inhibition zones 11.30 0.12 mm) at
MIC 500 μg mL−1
.
Fig. 3 Antimycobacterial activity (% inhibition) of compounds (A–I)
and (5a–5i).
In vitro evaluation of antimycobacterial activity
Antioxidant activity
Drug susceptibility and determination of MIC of the test com-
Free radical scavenging activity of the tested compounds 5a–5i
was studied by the DPPH (diphenyl picryl hydrazyl) assay
method.51 The drug stock solution (1 mg mL−1) was diluted to
final concentrations of 2, 4, 6, 8 and 10 μg mL−1 in methanol.
DPPH in methanol solution (1 mL, 0.3 mmol) was added to
2.5 mL of drug solutions of different concentrations and were
then allowed to react at room temperature. After 30 min, the
absorbance values were measured at 518 nm and were converted
pounds against Mycobacterium tuberculosis H37Rv were per-
48–51
formed by Lowenstein–Jensen (LJ) MIC method
where
primary 1000, 500, 250, and secondary 200, 100, 62.5, 50, 25,
12.5, 6.25, 3.25 μg mL−1 dilutions of each test compound was
added to liquid Lowenstein–Jensen medium and then media
were sterilized by inspissation. A culture of M. tuberculosis
H37Rv growing on Lowenstein–Jensen medium was harvested in
0.85% saline in bijou bottles. A solution of 2000 mg L−1
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Table 3 In vitro antituberculosis activity
% inhibition against
M.tuberculosis H37Rv at concentration 250 μg mL−1 at (MIC μg mL−1
)
of compounds (A–I) and newly synthesized calix[4]arene based 1,3,4-
oxadiazoles and 1,3,4-thiadiazole derivatives of isoniazid, nicotinic acid
hydrazide, benzoic acid hydrazide, cis-cinnamic acid hydrazide (5a–5i)
In vitro antimycobacterial activity
Compound no
MIC μg mL−1a
% Inhibition
A
B
C
D
E
F
G
H
I
5a
5b
5c
5d
5e
5f
5g
50
100
100
100
250
100
200
100
125
50
150
62.5
100
200
200
—
82.27 0.04
62.00 0.02
71.50 0.03
62.21 0.80
38.24 0.57
45.00 0.60
35.00 0.64
63.23 0.90
34.10 0.51
94.00 0.01
92.80 0.07
96.54 0.90
89.42 0.8
38.24 1.57
45.00 0.06
—
Fig. 4 Antioxidant activity of compounds 5a–d.
5h
62.5
200
0.2
91.23 0.92
57.10 0.41
99.00
5i
Isoniazidb
a MIC (μg mL−1) = Minimum inhibitory concentration. Mean of five
replicates: Standard deviation (−) No activity. b Standard.
Fig. 5 Antioxidant activity of compounds 5e–i.
(IC50 >10 μg mL−1). The results revealed that the compounds
with an oxadiazole moiety exhibited good antioxidant properties,
compared to those having a thiadiazole moiety. The results are
presented in Fig. 4 and Fig. 5.
Table 4 Antioxidant data for the newly synthesized calix[4]arene
based 1,3,4-oxadiazoles and 1,3,4-thiadiazole derivatives of isoniazid,
nicotinic acid hydrazide, benzoic acid hydrazide and cis-cinnamic acid
hydrazide
Antioxidant activity
Conclusion
Compound no
DPPH scavenging (%)a
IC50 μg mL−1
We have synthesized a novel calix[4]arene assembly incorporat-
ing isoniazid derivatives with 1,3,4-thiadiazole and 1,3,4-oxadia-
zole heterocycles. In vitro antibacterial, antifungal and
antitubercular activities showed that all compounds were
efficient on two gram negative (E. coli, P.aeruginosa), two gram
positive (S. aureus, S. pyogenes) reference strains, two fungi
species (C. albicans, A. clavatus) and H37Rv bacteria compared
to standard drugs. Compounds 5a, 5,11,17,23-tetra-tert-butyl-
25,27-bis(5-(pyridin-4-yl)-1,3,4-oxadiazole-2-thiacarbonyl-
methoxy)-26,28-dihydroxycalix[4]areneand, and 5b, 5,11,17,
23-tetra-tert-butyl-25,27-bis(5-(pyridin-4-yl)-1,3,4-thiadiazole-
2-amidemethyleneoxy)-26,28-dihydroxycalix[4]arene, exhibited
even higher bioactivity, especially towards M. tuberculosis, than
the standard drug (isonazid). The presence of the 1,3,4-oxadia-
zole and thiadiazole ring as pharmacophores and the increase in
the lipophilic character of the molecule due to the presence of
substituted calix[4]arene in the molecule facilitated the crossing
through the biological membrane of the micro-organism thereby
inhibiting their growth. Moreover, the results also confirm the
organizational role of calixarene in bringing together the oxadia-
zole groups for the genesis of antimycobacterial activity. The
enhancement of activity of oxadiazole and thiadiazole functiona-
lized calix[4]arenes compared to the unsubstituted oxadiazole
and thiadiazole (A–I) is attributed to the cooperative effect
of the pharmacophores. Furthermore, compounds having an
5a
5b
5c
5d
5e
5f
5g
5h
62.0
50.0
75.0
72.3
54.6
68.4
48.6
82.6
64.4
92.0
8.0 0.05
c
6.45 0.11
7.23 0.1
9.34 0.17
7.42 0.04
c
6.1 0.04
8.16 0.10
5.70 0.00
5i
Ascorbic acidb
a Results are mean of three different experiments. b Standard mean of
three replicates: Standard deviation. c Low antioxidant activity (IC50
10 μg mL−1
>
)
into the percentage antioxidant activity. Methanol was used as
the solvent and ascorbic acid as the standard. The trials were
done in triplicate. The inhibitory concentration (IC50) value, rep-
resents the concentration required to exhibit 50% antioxidant
activity (Table 4). In the synthesized compounds, compounds 5h
and 5c were found to possess maximum antioxidant activity
82.6%, 75.0% and their inhibitory concentration (IC50-6.1 μg
mL−1, IC50-6.45 μg mL−1) against the standard drug ascorbic
acid 92% (IC50-5.7 μg mL−1). However compound 5f
showed moderate antioxidant activity 68.4% (IC50-7.42 μg
mL−1), and 5b and 5g showed minimum antioxidant activity
1790 | Org. Biomol. Chem., 2012, 10, 1785–1794
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5-(pyridin-3-yl)-1,3,4-oxadiazole-2-thiol (D)
oxadiazole moiety showed good antioxidant activity. Further,
optimization and pharmacokinetic characterization of this series
are in progress in our laboratory.
Yield: 74%, mp 231 °C; IR (KBr, υ cm−1): 1640 (CvN), 1510
(CvC aromatic), 1420 (C–O–C oxadiazole), 1167 (SH); 1H
NMR (400 MHz, DMSO-d6): δ 7.90, 8.72 (m, 4H, Py), 13.02
(s, 1H, SH); MS: m/z 179 (M+); Anal. Calcd for C7H5N3OS
(179.20): C, 46.92; H, 2.81; N, 23.45; O, 8.93; S, 17.89. Found:
C, 46.93; H, 2.83; N, 23.46; O, 8.91; S, 17.90%.
Materials and Method
All the chemicals and reagents were of analytical grade of BDH,
Aldrich and Merck unless and otherwise specified. The solvents
used for the analysis were purified by standard methods.52 The
melting points (°C, uncorrected) were taken using Veego Mel-
Temp apparatus. The FT-IR spectra were recorded on Bruker
Tensor 27 FT-IR spectrometer with KBr pellets. The FAB-MS
were recorded on a Jeol/SX/102/Da-600 mass spectrometer data
system using Argon/Xenon as the accelerating gas. m-Nitro
benzyl alcohol (NBA) was used as a matrix with the peak at m/z
136, 137, 154, 289 and 307. Elemental analyses system used
5-(phenyl)-1,3,4-oxadiazole-2-thiol (F)
Yield: 74%, mp 219 °C; IR (KBr, υ cm−1): 2940 (C–H), 1521
(CvC aromatic), 1425 (C–O–C oxadiazole), 1160 (SH); 1H
NMR (400 MHz, DMSO-d6): δ 7.94, 8.74 (m, 4H, Phenyl),
12.92 (s, 1H, SH); MS: m/z 178 (M+); Anal. Calcd for
C8H6N2OS (178.21): C, 53.92; H, 3.39; N, 15.72; O, 8.98; S,
17.99. Found: C, 53.94; H, 3.41; N, 15.75; O, 8.97; S, 17.89%.
1
was GmbH Vario Micro cube elementar analyzer. H NMR and
13C NMR spectra were recorded at 400 MHz & 500 MHz and
125 MHz respectively on a Bruker Avance II 400 spectropho-
tometer in DMSO-d6 with tetra methyl silane (TMS) as an
internal standard.
cis-5-(styryl)-1,3,4-oxadiazole-2-thiol (H)
Yield: 74%, mp 167 °C; IR (KBr, υ cm−1): 2980 (C–H), 1515
(CvC aromatic), 1430 (C–O–C oxadiazole), 1161 (SH); 1H
NMR (400 MHz, DMSO-d6): δ 7.94, 8.74 (m, 4H, Phenyl),
6.95–6.95 (dd, 2H, Styryl), 13.00 (s, 1H, SH); MS: m/z 204
(M+); Anal. Calcd for C10H8N2OS (204.04): C, 58.80; H, 3.95;
N, 13.72; O, 7.83; S, 15.70. Found: C, 58.81; H, 3.93; N, 13.70;
O, 7.81; S, 15.72%.
Synthesis of Nicotinic acid, benzoic acid and cis-cinnamic acid
hydrazide
Nicotinic acid, benzoic acid and cis-cinnamic acid hydrazide
were prepared as reported in the earlier paper.53,54
Synthesis of 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives of
isoniazid, nicotinic acid hydrazide, benzoic acid hydrazide and
cis-cinnamic acid hydrazide
General procedure for synthesis of B, E, G, and I
A mixture of various hydrazides (0.01 mol) was dissolved in a
minimum amount of 1 N HCl and ammonium thiocyanate
(0.02 mol) was added afterward. The reaction mixture was
heated under reflux for 8–10 h. After cooling, the product was
filtered, washed with water and recrystallized from absolute
alcohol to give the assorted thiosemicarbezides.
To a mixture of various thiosemicarbezides (0.01 mol) was
dissolved 4 mL of conc. sulphuric acid. Then, the solution was
kept at room temperature for 2 h, stirred occasionally and poured
over crushed ice. The resulting solid was kept in ammoniacal
water for 2 h. Then the solid product was filtered; washed with
water and dried. The crude was purified by crystallization from
absolute alcohol to afford the desired compounds (B, E, G,
and I).
These compounds A, B, D, E, F, G, H and I were prepared
according to a reported method.55–57
General procedure for the synthesis of compounds A, D, F
and H
Amixture of various acid hydrazides (0.005 mol), KOH
(0.005 mol) and carbon disulfide (5 mL) in ethanol (50 mL) was
refluxed on a steam bath for 12 h. The solution was then concen-
trated, cooled and acidified with dilute HCl. The solid mass that
separated out was filtered, washed with ethanol; dried solid was
purified by crystallization from absolute alcohol to afford the
desired compounds (A, D, F and H). The compounds (A, D, F
and H) were synthesized in an analogous manner and were
characterized as shown below.
The compounds (B, E, G, and I) were synthesized in an ana-
logous manner and were characterized as shown below.
5-(pyridin-4-yl)-1,3,4-oxadiazole-2-thiol (A)
2-Amino-5-(4′-pyridyl)-1,3,4-thiadiazole (B)
Yield: 74%, mp 166 °C; IR (KBr, υ cm−1): 1638 (CvN), 1521
(CvC aromatic), 1430 (C–O–C oxadiazole), 1164 (SH); 1H
NMR (400 MHz, DMSO-d6): δ 7.94, 8.74 (m, 4H, Py), 13.03
(s, 1H, SH); MS: m/z 179 (M+); Anal. Calcd for C7H5N3OS
(179.20): C, 46.92; H, 2.81; N, 23.45; O, 8.93; S, 17.89. Found:.
C, 46.95; H, 2.83; N, 23.48; O,8.90; S, 17.91%.
Yield 70%, m.p. 240 °C; IR (KBr, υ cm−1): 3340 (N–H str.),
1621–1433 (CvN), 660 (C–S–C); 1Η NMR (400 MHz, DMSO-
d6): δ 8.50 (d, 2H, Ar-H, pyridine), 6.51 (s, 2H, NH2); MS: m/z
178 (M+); Anal. Calcd. for C7H6N4S (178.21): C, 47.18; H,
3.39; N, 31.44; S,17.99. Found: C, 47.15; H, 3.40; N, 31.42; S,
17.97%.
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2-Amino-5-(3′-pyridyl)-1,3,4-thiadiazole (E)
was neutralized with 0.1 M HCl. The solid material was then
filtered and washed with 2 N HCl, NaHCO3 and distilled water
sequentially. The crude product was purified by crystallization
from ethanol–THF. The compounds (5a–5i) were synthesized in
an analogous manner and were characterized as shown below.
Yield 70%, m.p. 235 °C; IR (KBr, υ cm−1): 3344 (N–H str.),
1625–1430 (CvN), 664 (C–S–C); 1Η NMR (400 MHz, DMSO-
d6): δ 8.48 (d, 2H, Ar-H, pyridine), 6.45 (s, 2H, NH2); MS: m/z
178 (M+); Anal. Calcd. for C7H6N4S (178.21): C, 47.18; H,
3.39; N, 31.44; S,17.99. Found: C, 47.16; H, 3.41; N, 31.41; S,
17.98%.
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(pyridin-4-yl)-1,3,4-
oxadiazole-2-thiacarbonylmethoxy)-26,28-dihydroxycalix[4]
arene (5a)
2-Amino-5-(phenyl)-1,3,4-thiadiazole (G)
Yield 84%; mp > 250 °C; IR (KBr, υ cm−1), 3400 (-OH), 3080
(-Ar CH), 1710 (–CvO), 1731, 1025 (C–O–C linkage), 1598,
1434, 1366 (oxadiazole ring str);1H NMR (400 MHz, DMSO-
d6) δ 0.97 (s, 18H, tert-butyl), 1.20 (s, 18H, tert-butyl), 3.86 (d,
4H, J = 12.8 Hz, Ar-CH2-Ar), 4.25 (d, 4H, J = 12.8 Hz, Ar-
CH2-Ar), 4.17 (s, 4H, OCH2), 6.69–7.14 (m, 8H, ArH), 8.03
(s, 2H, OH), 7.25–8.50 (s, 8H, PyH); 13C NMR (125 MHz,
DMSO-d6) δ 31.2, 33.2 34.5, 32.2 (Me3C of tert-butyl), 32.2
(Ar-CH2-Ar), 122.4, 125, 126.4, 133.2, 141.7, 147.3, 150.2,
(-CH of Ar), 167.2 (SCO), 64.3 (OCH2), 120.3,137.4,150.2
(C1–C5 of pyridine ring), 164.5–167.5 (C6–C7 0f oxadiazole
ring); FAB-MS (m/z) 1088 (M+1); Anal. Calcd for C62H66N6
O8S2 (1087.35): C, 68.48; H, 6.12; N,7.73; O, 11.77; S, 5.90.
Found: C, 68.45; H, 6.14; N, 7.73; O, 11.76; S, 5.89%.
Yield 70%, m.p. 223 °C; IR (KBr, υ cm−1): 3342 (N–H str.),
1620–1438 (CvN), 656 (C–S–C); 1Η NMR (400 MHz, DMSO-
d6): δ 8.46 (d, 2H, Ar-H), 6.40 (s, 2H, NH2); MS: m/z 177 (M+);
Anal. Calcd. for C8H6N3S (177.04): C, 54.22; H, 3.98; N,
23.71; S,18.09. Found: C, 54.20; H, 3.97; N, 23.70; S, 18.11%.
cis-2-Amino-5-(styryl)-1,3,4-thiadiazole (I)
Yield 70%, m.p. 165 °C; IR (KBr, υ cm−1): 3342 (N–H str.),
2980 (C–H str.), 656 (C–S–C); 1Η NMR (400 MHz, DMSO-d6):
δ 8.46 (d, 2H, Ar-H), 6.40 (s, 2H, NH2); MS: m/z 203 (M+);
Anal. Calcd. for C10H9N3S (203.26): C, 59.09; H, 4.46; N,
20.67; S,15.78. Found: C, 59.11; H, 4.47; N, 20.70; S, 15.77%.
2-Amino-5-(pyridin-4-yl)-1,3,4-oxadiazole (C)
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(pyridin-4-yl)-1,3,4-
thiadiazole-2-amidemethyleneoxy)-26,28-dihydroxycalix[4]
arene (5b)
The mixture of isoniazid (1.37 g, 0.01 mol), the minimum
amount of methanol (20 mL) and cyanogen bromide (1.059 g,
0.01 mol) was stirred and refluxed at 55–56 °C for 2 h. The
resulting solution was cooled and neutralized with a sodium
bicarbonate solution. The solid thus precipitated was washed,
dried and recrystallized from ethanol.
Yield 82%: mp > 250 °C; IR (KBr, υ cm−1): 3412 (-OH), 2989
(-Ar-CH), 1670–1656 (NHCO), 696 (C–S–C linkage), 1240
(N–NvC thiadiazole ring str);1H NMR (400 MHz, DMSO-d6),
δ 0.94 (s, 18H, tert-butyl), 1.10 (s, 18H, tert-butyl), 3.80 (d, 4H,
J = 12.8 Hz, Ar-CH2-Ar), 4.10 (d, 4H, J = 12.8 Hz, Ar-CH2-
Ar), 4.39 (s, 4H, CH2O), 6.70–7.15 (m, 8H, ArH), 8.05 (s, 2H,
OH), 7.35 (s, 8H, pyH), 10.20 (d, 2H, NH), 13C NMR
(125 MHz, DMSO-d6) δ 31.4, 32.9, 34.2, 33.4 (Me3C), 32.2 (Ar
-CH2 -Ar), 124.4,125.9, 127.4, 133.2, 143.7, 147.3, 149.1, 151
(CH of Ar), 168.5 (NHCO of amide), 63.3 (OCH2),121.3, 137.2,
149.4 (C1–C5 of pyridine ring), 163.5–166.1 (C6–C7 0f thiadia-
zole ring); FAB-MS (m/z) 1086.(M+1); Anal. Calcd for
C62H68N8 O6S2 (1085.38): C, 68.61; H, 6.27; N,10.31; O, 8.82;
S, 5.91. Found: C, 68.59; H, 6.26; N, 10.28; O, 8.80; S, 5.89%.
Yield 77%; mp 240 °C (dec); IR (KBr, υ cm−1): 3340 (str
1
NH), 1621–14330 (CvN), 1210 (C–O–C), H NMR (DMSO-
d6) δ 8.50 (m, 4H pyridine), 6.51 (s, 2H, NH2); 13C NMR
(DMSO-d6) δ 169.2–153 (C-2 and C-5 of oxadiazole): 149.8,
124.2, 147 (C-4 of pyridine); MS: m/z 178 (M+); Anal. Calcd
for C8H10N4 O4 (178.19): C, 53.92; H, 5.66; N, 31.44; O, 8.98.
Found: C, 53.90; H, 5.63; N, 31.44; O, 8.97%.
Synthesis of compounds 1, 2, 3 and 4
Compounds 1, 2, 3 and 4 were prepared as reported in the earlier
paper.58
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(pyridin-4-yl)-1,3,4-
oxadiazole-2-amidemethyleneoxy)-26,28-dihydroxycalix[4]
arene (5c)
General method for the synthesis of (5a–5i)
Compound 4, 5,11,17,23-tetra-tert-butyl-25,27-bis(chloro-
carbonyl-methoxy)-26,28-dihydroxycalix[4]arene, (2.07 mmol),
obtained in the previous step was dissolved in dry THF
(100 mL). The addition of pyridine (1 mL, 12.6 mmol) was
made dropwise and the solution of appropriate 1,3,4-oxadia-
zole,1,3,4- thiadiazole (A–I) (7.30 mmol) in THF (25 mL) was
added dropwise in about 1 h with continuous stirring room temp-
erature at 37 °C. The reaction mixture was then stirred and
refluxed for 5 h, after which most of the solvent was distilled off
under vacuum. The residue was diluted with 200 mL water and
Yield 85%; mp >250 °C(dec); IR (KBr, υ cm−1): 3410 (-OH),
2990 (-Ar-CH), 1670–1654 (NHCO), 1210, 1025 (C–O–C
linkage), 1598, 1434, 1366 (oxadiazole ring str);1H NMR
(400 MHz, DMSO-d6) δ 0.96 (s, 18H, tert-butyl)1.15 (s, 18H,
tert-butyl), 3.85 (d, 4H, J = 12.8 Hz, Ar-CH2-Ar), 4.15 (d, 4H, J
= 12.8 Hz, Ar-CH2-Ar), 5.18 (s, 4H, CH2O), 6.70–7.10 (m, 8H,
ArH), 8.20 (s, 2H, OH), 7.50 (s, 8H, pyH), 10.25 (d, 2H, NH),
13C NMR (125 MHz, DMSO-d6) δ 31.43, 32.2, 34.2, 34.30
(Me3C), 33.5 (Ar-CH2-Ar), 169.34 (NHCO of amide), 64.4
(OCH2), 125.4, 126.2, 127.9, 133.2, 141.7, 147.3, 148.4, 151.5
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(CH of Ar) 120, 137.2, 148.5 (C1–C5 of the pyridine ring),
153,169.4 (C6–C7 of an oxadiazole ring); FAB-MS (m/z) 1054
(M+1), Anal. Calcd for C62H68N8O8 (1053.26), C, 70.70; H,
6.51; N,10.64; O, 12.14; Found: C, 70.69; H, 6.48; N, 10.64; O,
12.15%.
(m/z) 1086 (M+1); Anal. Calcd for C62H66N6 O8S2 (1085.38):
C, 70.82; H, 6.31; N, 5.16; O, 11.79; S, 5.91. Found: C, 70.80;
H, 6.28; N, 5.14; O, 11.89; S, 5.92%.
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(phenyl)-1,3,4-
thiadiazole-2-amidemethyleneoxy)-26,28-dihydroxycalix[4]
arene (5g)
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(pyridin-3-yl)-1,3,4-
oxadiazole-2-thiacarbonylmethoxy)-26,28-dihydroxycalix[4]
arene (5d)
Yield 80%: mp > 250 °C; IR (KBr, υ cm−1): 3400 (-OH), 3025
(-Ar-CH), 1650–1666 (NHCO), 687 (C–S–C linkage), 1243
(N–NvC thiadiazole ring str);1H NMR (500 MHz, DMSO-d6),
δ 0.95 (s, 18H, tert-butyl), 1. (s, 18H, tert-butyl), 3.84 (d, 4H, J
= 12.8 Hz, Ar-CH2-Ar), 4.17 (d, 4H, J = 13.15 Hz, Ar-CH2-Ar),
4.12 (s, 4H, CH2O), 6.70–7.25 (m, 8H, ArH), 8.10 (s, 2H, OH),
7.35–9.24 (s, 8H, pyH), 10.10 (d, 2H, NH), 13C NMR
(125 MHz, DMSO-d6) δ 31.4, 32.9, 34.2, 33.4 (Me3C), 32.3
(Ar-CH2-Ar), 122.4,125.9, 127.4, 134.2, 143.7, 146.3, 149.1,
150 (CH of Ar), 167.4 (NHCO of amide), 66.40
(OCH2),122.3–168.4 (C1–C5 of pyridine the pyridine),
152.5–166.1 (C6–C7 0f thiadiazole ring); FAB-MS (m/z) 1084
(M+1); Anal. Calcd for C62H68N8 O6S2 (1083.41): C, 70.95; H,
6.51; N,7.76; O, 8.86; S, 5.92. Found: C, 70.93; H, 6.54; N,
7.74; O, 8.88; S, 5.94%.
Yield 81%: mp > 250 °C; IR (KBr, υ cm−1), 3410 (-OH), 3030
(-Ar-CH), 1710 (–CvO), 1731, 1015 (C–O–C linkage), 1600,
1424, 1356 (oxadiazole ring str),1H NMR (500 MHz, DMSO-
d6) δ 0.94 (s, 18H, tert-butyl), 1.10 (s, 18H, tert-butyl), 3.76
(d, 4H, J = 12.8 Hz, Ar-CH2-Ar), 4.35 (d, 4H, J = 12.8 Hz, Ar-
CH2-Ar), 4.14 (s, 4H, OCH2), 6.59–7.10 (m, 8H, ArH), 8.10
(s, 2H, OH), 7.20–9.10 (s, 8H, PyH), 13C NMR (125 MHz,
DMSO-d6) δ 31.2, 33.20, 34.5, 32.2 (Me3C of tert-butyl), 32.2
(Ar-CH2-Ar), 122.4, 125, 126.4 133.2 141.7 147.3. 150.2, (CH
of Ar), 167.5 (SCO), 64.3 (OCH2), 120.3–150.2 (C1–C5 of pyri-
dine ring), 165.5–167 (C6–C7 0f oxadiazole ring); FAB-MS
(m/z) 1088 (M+1); Anal. Calcd for C62H66N6 O8S2 (1087.35):
C, 68.48; H, 6.12; N, 7.73; O, 11.77; S, 5.90. Found: C, 68.47;
H, 6.14; N, 7.75; O, 11.76; S, 5.87%.
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(styryl)-1,3,4-oxadiazole-
2-thiacarbonylmethoxy)-26,28-dihydroxycalix[4]arene (5h)
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(pyridin-3-yl)-1,3,4-
thiadiazole-2-amidemethyleneoxy)-26,28-dihydroxycalix[4]
arene. (5e)
Yield 83%: mp > 250 °C; IR (KBr, υ cm−1), 3440 (-OH), 3010
(-Ar-CH), 1715 (–CvO), 1735, 1012 (C–O–C linkage), 1610,
1404, 1340 (oxadiazole ring str);1H NMR (500 MHz, DMSO-
d6) δ 0.96 (s, 18H, tert-butyl), 0.98 (s, 18H, tert-butyl), 3.90
(d, 4H, J = 12.7 Hz, Ar-CH2-Ar), 4.25 (d, 4H, J = 12.9 Hz, Ar-
CH2-Ar), 4.4 (s, 4H, OCH2), 6.59–7.24 (m, 8H, ArH), 8.10
(s, 2H, OH), 7.24–8.28 (s, 8H, PyH), 13C NMR (125 MHz,
DMSO-d6) δ 31.3, 33.2, 34.5, 32.3 (Me3C of tert-butyl), 33.8
(Ar-CH2-Ar), 121.2, 125, 126.4, 133.2, 141.7, 147.3, 150.2 (CH
of Ar), 161.5 (SCO), 65.4 (OCH2), 123.3–158.2 (C1–C5 of the
pyridine ring), 161.5–169 (C6–C7 0f oxadiazole ring); FAB-MS
(m/z) 1138 (M+1); Anal. Calcd for C62H66N6 O8S2 (1137.45):
C, 71.80; H, 6.38; N, 4.93; O, 11.25; S, 5.64. Found: C, 71.92;
H, 6.28; N, 4.82; O, 11.33; S, 5.60%.
Yield 80%: mp > 250 °C; IR (KBr, υ cm−1): 3420 (-OH), 3015
(-Ar-CH), 1670–1650 (NHCO), 685 (C–S–C linkage), 1230
(N–NvC thiadiazole ring str);1H NMR (500 MHz, DMSO-d6),
δ 0.92 (s, 18H, tert-butyl), 1.15 (s, 18H, tert-butyl), 3.82 (d, 4H,
J = 12.8 Hz, Ar-CH2-Ar), 4.10 (d, 4H, J = 13.2 Hz, Ar-CH2-
Ar), 4.39 (s, 4H, CH2O), 6.70–7.15 (m, 8H, ArH), 8.05 (s, 2H,
OH), 7.35–9.15 (s, 8H, pyH), 10.10 (d, 2H, NH), 13C NMR
(125 MHz, DMSO-d6) δ 31.4, 32.9, 34.2, 33.4 (Me3C), 32.2
(Ar-CH2-Ar), 124.4,125.9, 127.4, 133.2, 143.7, 147.3, 149.1,
151 (CH of Ar), 168.5 (NHCO of amide), 66.50 (OCH2),
121.3–167.4 (C1–C5 of pyridine ring), 153.5–164.1 (C6–C7 0f
thiadiazole ring); FAB-MS (m/z) 1086.(M+1); Anal. Calcd for
C62H68N8 O6S2 (1085.38): C, 68.61; H, 6.31; N,10.32; O, 8.84;
S, 5.91. Found: C, 68.58; H, 6.30; N, 10.32; O, 8.85; S, 5.88%.
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(styryl)-1,3,4-thiadiazole-
5,11,17,23-Tetra-tert-butyl-25,27-bis(5-(phenyl)-1,3,4-oxadiazole-
2-amidemethyleneoxy)-26,28-dihydroxycalix[4]arene (5i)
2-thiacarbonylmethoxy)-26,28-dihydroxycalix[4]arene (5f)
Yield 82%: mp > 250 °C; IR (KBr, υ cm−1): 3411 (-OH), 29285
(-Ar-CH), 1640–1661 (NHCO), 673 (C–S–C linkage), 1232
(N–NvC thiadiazole ring str);1H NMR (500 MHz, DMSO-d6),
δ 0.94 (s, 18H, tert-butyl), 1.00 (s, 18H, tert-butyl), 3.84 (d, 4H,
J = 12.8 Hz, Ar-CH2-Ar), 4.16 (d, 4H, J–13.15 Hz, Ar–CH2–
Ar), 4.41 (s, 4H, OCH2), 6.75–7.28 (m, 8H, ArH), 8.12 (s, 2H,
OH), 7.25–9.24 (s, 8H, pyH), 10.13 (d, 2H, NH); 13C NMR
(125 MHz, DMSO-d6) δ 31.2, 32.9, 34.2, 33.4 (Me3C), 32.3
(Ar-CH2-Ar), 121.4,125.9, 127.4, 134.2, 143.7, 146.3, 149.1,
153.(CH of Ar), 166.4 (NHCO of amide), 63.4 (OCH2),
125.3–168.4 (C1–C5 of pyridine ring), 151.5–168.1 (C6–C7 0f
thiadiazole ring); FAB-MS (m/z) 1136 (M+1); Anal. Calcd for
Yield 81%: mp > 250 °C; IR (KBr, υ cm−1), 3440 (-OH), 3010
(-Ar-CH), 1715 (–CvO), 1735, 1005 (C–O–C linkage), 1610,
1414, 1346 (oxadiazole ring str),1H NMR (500 MHz, DMSO-
d6) δ 0.95 (s, 18H, tert-butyl), 0.99 (s, 18H, tert-butyl), 3.96 (d,
4H, J = 12.7 Hz, Ar-CH2-Ar), 4.35 (d, 4H, J = 13.1 Hz, Ar-
CH2-Ar), 4.4 (s, 4H, OCH2), 6.59–7.24 (m, 8H, ArH), 8.10 (s,
2H, OH), 7.20–8.20 (s, 8H, PyH), 13C NMR (125 MHz,
DMSO-d6) δ 31.3, 33.2, 34.5, 32.3 (Me3C of tert-butyl), 32.4
(Ar-CH2-Ar), 122.4, 125, 126.4 133.2 141.7 147.3. 150.2, (CH
of Ar), 164.5 (SCO), 64.4 (OCH2), 121.3–153.2 (C1–C5 of the
pyridine ring), 162.5–168 (C6–C7 0f oxadiazole ring); FAB-MS
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 1785–1794 | 1793

View Article Online
27 M. Mourer, R. E. Duval, C. Finance and J. B. Regnouf-de-Vains, Bioorg.
Med. Chem. Lett., 2006, 16, 2960–2963.
C62H68N8 O6S2 (1135.48): C, 71.93; H, 6.57; N,7.40; O, 8.45;
S, 5.65. Found: C, 71.90; H, 6.54; N, 7.42; O, 8.48; S, 5.64%.
28 M. Grare, M. Mourer, S. Fontanay, J. B. Regnouf-de-Vains, C. Finance
and R. E. Duval, J. Antimicrob. Chemother., 2007, 60, 575–581.
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31 G. M. L. Consoli, E. Galante, C. Daquino, G. Granata, F. Cunsolo and
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33 S. L. Gaonkar, K. M. L. Rai and B. Prabhuswamy, Eur. J. Med. Chem.,
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Acknowledgements
We would like to thank SAIF, Chandigarh & CSMCRI, Bhavna-
gar for spectral data and Micro Care Laboratory Surat for bio-
logical studies. Manish Kumar would like to acknowledge UGC
New Delhi, for a Junior Research Fellowship.
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This journal is © The Royal Society of Chemistry 2012