Synthesis, characterization, X-ray structure and biological activities of c-5-bromo-2-hydroxyphenylcalix[4]-2-methyl resorcinarene

Article abstract of DOI:10.3390/molecules181113369

Abstract: C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I) was synthesized by cyclocondensation of 5-bromo-2-hydroxybenzaldehyde and 2-methylresorcinol in the presence of concentrated HCl. Compound I was characterized by infrared and nuclear magnetic resonance spectroscopic data. X-ray analysis showed that this compound crystallized in a triclinic system with space group of Pi, a = 15.9592(16)A, b = 16.9417(17)A, c = 17.0974(17)A, a = 68.656(3)°, β = 85.689(3)°, ? = 81.631(3)°, Z = 2 and V = 4258.6(7)A3. The molecule adopts a chair (C2h) conformation. The thermal properties and antioxidant activity were also investigated. It was strongly antiviral against HSV-1 and weakly antibacterial against Gram-positive bacteria. Cytotoxicity testing on Vero cells showed that it is non-toxic, with a CC50 of more than 0.4 mg/mL.

Full text of DOI:10.3390/molecules181113369

Molecules 2013, 18, 13369-13384; doi:10.3390/molecules181113369
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Characterization, X-ray Structure and Biological
Activities of C-5-Bromo-2-hydroxyphenylcalix[4]-2-methyl
resorcinarene
Hamza M. Abosadiya 1, Siti Aishah Hasbullah 1, Mukram Mohamed Mackeen 1,2, Seow Chew Low 3,
Nazlina Ibrahim 3, Mamoru Koketsu 4 and Bohari M. Yamin 1,*
1
School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia,
Bangi 43600, Selangor, Malaysia; E-Mails: hamza_inorg@yahoo.com; (H.M.A.);
aishah80@ukm.my (S.A.H.); mukram@ukm.my (M.M.M.)
2
Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, Bangi 43600, Selangor,
Malaysia
3
School of Biosciences and Biotechnology, Universiti Kebangsaan Malaysia, Bangi 43600,
Selangor, Malaysia; E-Mails: alicia.w05@gmail.com (S.C.L.); nazlina@ukm.my (N.I.)
4
Department of Chemistry and Biomolecular Sciences, Gifu University, 1-1 Yanagido,
Gifu 501-1193, Japan; E-Mail: koketsu@gifu-u.ac.jp
* Author to whom correspondence should be addressed; E-Mail: bohari@ukm.my;
Tel.: +60-389-215-424; Fax: +60-389-215-410.
Received: 23 September 2013; in revised form: 14 October 2013 / Accepted: 14 October 2013 /
Published: 29 October 2013
Abstract: C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I) was synthesized
by cyclocondensation of 5-bromo-2-hydroxybenzaldehyde and 2-methylresorcinol in the
presence of concentrated HCl. Compound I was characterized by infrared and nuclear
magnetic resonance spectroscopic data. X-ray analysis showed that this compound
crystallized in a triclinic system with space group of Pī, a = 15.9592(16)Å, b =
16.9417(17)Å, c = 17.0974(17)Å, α = 68.656(3)°, β = 85.689(3)°, γ = 81.631(3)°, Z = 2
and V = 4258.6(7)Å3. The molecule adopts a chair (C2h) conformation. The thermal
properties and antioxidant activity were also investigated. It was strongly antiviral against
HSV-1 and weakly antibacterial against Gram-positive bacteria. Cytotoxicity testing on
Vero cells showed that it is non-toxic, with a CC50 of more than 0.4 mg/mL.
Keywords: C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene; biological activities;
X-ray structural study

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1. Introduction
Calixarenes are now important technology materials. Their unique size and cup structure cavities
can accommodate some ions and neutral molecules via host-guest interaction and have driven
supramolecular chemistry applications in many fields [1–5]. Calixarenes have been used as additives
in capillary electrophoresis, liquid membranes, extraction process, chemical sensing and HPLC
stationary phases. A number of calix[4]resorcinarene compounds have shown a potential as adsorbents
for heavy metal separation [6,7]. Among all known resorcinarenes, calix[4]resorcinarenes are the most
widely investigated group of compounds. Basically, they can be synthesized via the acid-catalyzed
condensation reaction of resorcinol with aldehydes [8]. One of the important properties of
resorcinarenes is their ability to bind strongly with solvent molecules that has great influence on their
stability. TGA studies have shown that the solvate molecules such as alcohol, pyridine and DMF were
removed at about 300 °C [9]. Therefore, most of the crystal structures of calix[4]resorcinarenes
reported so far have 1 to 3 or more solvated molecules. Many of the calix[4]resorcinarenes that we
have attempted to crystallize changed their crystalline forms into whitish solids or powders after
standing for few hours or even minutes at room temperature. In fact for some compounds, instability
was observed while in solution. There is interest in investigating in more detail the flexibility among
the possible conformers (crown, boat, chair, diamond and saddle) in the crystalline state and the role of
solvents. In the present study, C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I) was
synthesized and recrystallized from DMF. The crystal was quickly coated with the Princeton-H oil and
mounted on a glass fiber and the X-ray experiment was conducted at low temperature. After a number
of experiments, a reasonably good data set was obtained. This article describes the synthesis,
characterization, crystal structure, thermal and biological properties of C-5-bromo-2-hydroxyphenyl-
calix[4]-2-methylresocinarene (I).
2. Results and Discussion
2.1. Synthesis and Characterization
The synthesis of C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I) was accomplished
by refluxing mixture of equal molar amounts of 2-methylresorcinol with 5-bromo-2-
hydroxybenzaldehyde in the presence of concentrated HCl in EtOH at 80 °C (Scheme 1).
1
The H-NMR spectrum of compound I (Figure 1) showed a clean two sets of phenolic protons at
7.48 and 8.94 ppm, respectively, belonging to the resorcinarene rings. The phenolic proton of the
p-bromophenyl ring appeared upfield at 6.38 ppm. The four methyl groups of the resocinarene also
appeared in two sets of signals at 1.94 and 2.11 ppm, respectively. It is known that the phenolic
protons of the resorcinarene and methine protons are sensitive to the chemical environment of the
different conformations of the resorcinarene [10–12]. Although mixed conformations are quite
common in solution, for the present compound, the two sets of the hydroxyl protons of resorcinol
(7.48 and 8.94 ppm) and the methine protons (5.37 and 6.10 ppm) indicate a high degree of co-planarity
between the resorcinol rings and lead to the adoption of a chair conformation. The 13C-NMR spectrum
showed the phenolic carbon of the bromohydroxybenzene fragment at 153.8 ppm whereas the two sets
of resorcinol carbons appeared at 150.9 and 151.2 ppm. The corresponding two sets of methyl carbons

MMolecules 2013, 18
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a
r
r
e at 10.4 and 10.6 ppm, respectively. The four tertiary methine carbons appeared at 36.8 ppm. The
1H-NMR spectrum of the crystallized calix is very similar to that of the solid precipitate product,
cept for the presence of extra signals at 2.73, 2.89 and 7.95 ppm, respectively, due to the two
methyls and the aldehyde proton of the DMF solvent.
H
exx
m
Schemme 1. Synthesis of C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I).
Fii NNMR spectrum of C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene in DMSO-d6.
gure 1. 1H-
2
.
.
2. X-Ray Structure
The X-ray investigation showed that compound (I), crystallized in DMF, possesses a triclinic
stem with the space group Pī, a = 15.9592(16) Å, b = 16.9417(17) Å, c = 17.0974(17) Å,
= 68.656(3)°, β = 85.689(3)°, γ = 81.631(3)°, Z = 2 and V = 4258.6(7) Å3. The asymmetric unit
syy
α
coonsists of one C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene at special position, eight

Molecules 2013, 18
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DMF and four water molecules of crystallization. The calix molecule adopts a chair conformation
(C2h) with two opposite resorcinol groups almost coplanar to each other and a pair of them that are
anti-parallel (Figure 2).
Figure 2. The molecular structure of C-5-bromo-2-hydroxyphenylcalix[4]-2-methyl-
resorcinarene drawn at 50% probability ellipsoid. The hydrogen atoms and solvent
molecules are not shown for clarity.
The dihedral angle between the opposite resorcinol rings (C15–C20) and (C43–C48) is 5.8(2)°
whereas the anti-parallel rings (C1–C6) and (C29–C34) have a dihedral angle of 1.4(2)°. The alternate
resorcinol rings (C1–C6) and (C15–C20) are perpendicular with a dihedral angle of 77.2(2)°. The two
bromo-hydroxyphenyl linkage groups that are attached to both sides of the co-planar resorcinol rings
are in opposite directions, whereas two rings face upwards and the other two rings directly downwards.
A similar conformation with approximately C2h symmetry due to the presence of a crystallographic
inversion center has also been observed for tetraarylboronic acid resorcinarene [13]. The bond lengths
and angles (Table 1) are in normal ranges and comparable to those of other resorcinarenes [14]. The
co-crystallized solvent molecules are located outside the calix molecule and therefore the cavity
remains empty, which enables it to participate in the host-guest activity.

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Table 1. Selected bond lengths and angles of C-5-bromo-2-hydroxyphenylcalix[4]-2-
methylresorcinarene (I).
Bond
Length Å
1.905(6)
1.902(5)
1.904(6)
1.905(5)
1.381(6)
1.390(6)
1.359(7)
1.366(6)
1.319(8)
1.314(9)
1.325(8)
1.327(8)
Bond
Angles °
120.0(4)
120.6(4)
118.8(4)
119.8(4)
116.9(5)
117.2(5)
122.6(5)
116.0(5)
120.6(6)
120.6(6)
121.5(5)
120.8(6)
Br1-C13
Br2-C27
Br3-C41
Br4-C55
O1-C3
C12-C13-Br1
C26-C27-Br2
C40-C41-Br3
C54-C55-Br4
O1-C3-C2
O2-C5
O2-C5-C4
O3-C10
O4-C16
N1-C59
N2-C62
N3-C65
N4-C68
O3-C10-C11
O4-C16-C15
C59-N1-C58
C62-N2-C60
C65-N3-C64
C68-N4-C67
There are no significant intramolecular hydrogen bonds in the calix moiety. In the crystal structure,
two calix molecules are connected by C—H…O hydrogen bonds between the methyl hydrogen atoms
and the resorcinol oxygen atoms to form dimers (Figure 3) which then connected to the water and
DMF solvent molecules by O—H…O and O...H—C hydrogen bonds, respectively (symmetry codes as
shown in Table 2).
Figure 3. Molecular packing of C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene,
viewed down the b axis. Dashed lines denote O—H….O and C—H….O hydrogen bonds and the
non-hydrogen bonded atoms are omitted for clarity.

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Table 2. Hydrogen geometric parameters (Å) of C-5-bromo-2-hydroxyphenylcalix[4]-2-
methylresocinarene.
D―H….A
D―H
0.82(6)
0.84
H….A
1.94(6)
1.86
H….A
D―H….A
O1W―H1WB.....O17
O3―H3….O20
2.756(8)
172(3)
2.690(6) 169
2.895(7) 173(6)
2.576(6) 146
2.696(7) 174
3.514(9) 155
3.400(9) 140
3.337(9) 172
3.440(8) 145
2.821(6) 148
2.815(7) 161(6)
2.718(7) 168(10)
2.668(6) 152
2.796(5) 145
2.756(7) 162(6)
2.718(7) 170
2.945(7) 174(9)
2.764(8) 174(7)
2.811(6) 159
2.778(10) 166(7)
2.706(7) 170
2.730(6) 165
2.786(5) 147
3.325(9) 165
3.305(7) 143
3.455(8) 158
3.255(7) 146
3.444(6) 146
3.441(8) 167
3.509(7) 160
3.453(8) 157
3.331(9) 133
3.487(8) 161
3.531(8) 171
O2W―H2WB…..O20
O7―H7….O15
0.82(4)
0.84
2.08(4)
1.83
O12―H12….O2W
C64―H64B….O1W
C66―H66B….O3W
C72―H72B….O4
0.84
1.86
0.98
2.60
0.98
2.59
0.98
2.36
C76―H76A….O18
O1―H1…..O19 ii
O1W―H1WA….O13 vii
O2W―H2WA….O16 ix
O4―H4….O3W iii
O5―H5….O7 iv
O3W―H3WA….O13 xi
O6―H6….O1W iv
O3W―H3WB....O14 x
O4W―H4WA….O17 viii
O8―H8….O18 v
O4W―H4WB....O14 iii
O9―H9….O4W vi
O10―H10….O18 v
O11―H11….O1 i
C21―H21A….O3W iii
C21―H21B….O7 iv
C21―H21C….O6 iv
C36―H36….O18 v
C49―H49B….O1 i
C59―H59….O4W vi
C61―H61B….O5 iv
C75―H75A….O15 iii
C78―H78A….O16 ix
C79―H79C….O3 ii
C80―H80….O16 iii
0.98
2.59
0.84
2.08
0.82(7)
0.82(3)
0.84
2.03(7)
1.91(4)
1.9
0.84
2.06
0.82(5)
0.84
1.96(6)
1.89
0.82(5)
0.82(6)
0.84
2.13(5)
1.94(6)
2.01
0.82(5)
0.84
1.98(5)
1.87
0.84
1.91
0.84
2.05
0.98
2.37
0.98
2.47
0.98
2.53
0.98
2.38
0.98
2.59
0.95
2.51
0.98
2.57
0.98
2.53
0.98
2.59
0.98
2.55
0.95
2.59
Symmetry codes: i = −x, 2 − y, 1 − z; ii = −x, 1 − y, 1 − z; iii = 1 − x, 1 − y, 1 − z; iv = 1 − x, 1 − y, 2 – z;
v = x, 1 + y, z; vi = x, 1 + y, 1 + z; vii = x, −1 + y, z; viii = 1 − x, −y, 1 – z; ix = −1 + x, y, z; x = 1 − x, 1 − y,
2 − z; xi = 1 − x, 2 − y, 1 − z.
2.3. Thermogravimetric Study
It has been reported based on TGA studies that the thermal decomposition of resorcinarene can be
very complicated due to the presence of large numbers of solvent molecules [15]. In many cases, the
solvent molecules were released at higher temperature than their boiling points, indicating strong

Molecules 2013, 18
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binding among the molecules in the solid state. It is also possible that solvents such as DMF
decompose during the thermal process and the decomposition products react with each other. Such
complexity is also depicted by the thermogram of the present calix (Figure 4). At least five mass loss
steps occurred at about 80, 120, 165, 370 and 400 °C, respectively. The second derivative plot (DTA)
showed the possibility of more than two steps in the second mass loss step. The first mass loss is
normally due to the release of water molecules, but the calculated mole percentage of water (3.8%) is
very much lower than the experimental loss of 9.62%, indicating the release of a different product or
mixture of products. The final residue of decomposition is carbon. A more detailed study on the
thermal decomposition of the calix is necessary to further elucidate the multiple mass losses.
Figure 4. Thermogram of C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresocinarene (I).
2.4. Biological Studies
2.4.1. Antioxidant Properties
Antioxidant properties measured as radical scavenging activity are due to the transfer of electrons or
hydrogen atoms of the hydroxyl groups to an oxidizing agent. Compound I, being a polyphenolic
compound, could inhibit the oxidation of other molecules such as 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) by donating hydrogen atoms to form the stable non-radical form of DPPH as shown by the
formation of a pale yellow color. The antioxidant activity exhibited by compound I was 84.95%, which
is very close to that of the compound 5,11,17,23,-tetra-tert-butyl-25,27-bis(5-(hexanyl)-1,3,4-oxadiazole-
2-thiacarbonylmethoxy)-26,28-dihydroxycalix[4]arene [16] indicating high antioxidant capability.

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2.4.2. Antibacterial Activity
The antibacterial activity of C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I) on five
test bacteria (Gram-positive and -negative) is shown in Table 3. Results showed that compound I
only displayed antibacterial activity against Gram-positive bacteria, namely methicilin-resistant
Staphylococcus aureus (MRSA) (Figure 5) with inhibition zones between 10 and 15 mm. However, the
antibacterial activity was weaker than the antibiotic standards.
Figure 5. Inhibition zones of C-5-bromo-2-hydroxyphenylcalix[4]2-methylresorcinarene (I)
against MRSA using the disc diffusion assay tested at concentrations of two-fold dilution.
The highest concentration of tested would correspond to 250 μg.
Table 3. Diameter of inhibition zone for antibacterial screening of C-5-bromo-2-hydroxy
phenylcalix[4]-2-methylresorcinarene (I).
Diameter of inhibition zone (mm)
Dose (µg)
MRSA
13
Sa
13
12
11
11
10
22 b
6
Ef
15
13
11
11
10
23 b
6
Ea
6
Pa
6
250
125
12
6
6
62.5
31.25
12
6
6
11
6
6
15.63
10
15 a
6
26 b
6
16 b
Antibiotic control (30 µg)
DMSO (solvent control)
6
6
6
Notes: MRSA = methicillin-resistant Staphylococcus aureus; Sa = Staphylococcus aureus; Ef = Enterococcus
faecalis; Ea Enterobacter aerogenes; Pa Pseudomonas aeruginosa; vancomycin;
b = chloramphenicol (30 µg). SD inhibition zone = ± 1 mm (biological replicates, 3).
=
=
a
=
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of
compound I were also determined (Table 4). Consistent with the disc diffusion test results, compound I
only inhibited the Gram-positive bacteria. The MIC values revealed that the calix completely inhibited
the growth of the tested Gram-positives between 1.563 to 6.25 mg/mL which would be considered
weak. Therefore, it is interesting that the antibacterial activity against MRSA in the disc diffusion

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assay was comparable to the compared antibiotic standard, but was much weaker in the broth dilution
assay, thereby not showing low μg/mL MIC values. MBC/MIC value against MRSA is 16 which
indicate a bacteriostatic mode of action as opposed to the MBC/MIC values of 2 for both
Staphylococcus aureus and Enterococcus faecalis, which indicate bactericidal action. MBC to MIC
ratios of >4 have been defined as bacteriostatic [17].
Table 4. Minimum inhibition concentration (MIC) (mg/mL), minimum bactericidal
concentration (MBC) (mg/mL) and selectivity index (SI) of C-5-bromo-2-
hydroxophenylcalix[4]-2-methylresorcinarene (I).
Microorganism
MRSA (Gram-positive)
Sa (Gram-positive)
Ef (Gram-positive)
Ea (Gram-negative)
Pa (Gram-negative)
MIC mg/mL
1.563
6.25
MBC mg/mL
SI
0.256
0.064
0.064
-
25
12.5
12.5
-
6.25
>25
>25
-
-
Note: MRSA
=
methicillin-resistant Staphylococcus aureus); Sa
=
Staphylococcus aureus);
Ef = Enterococcus faecalis; Ea = Enterobacter aerogenes; Pa = Pseudomonas aeruginosa (-ve);
SI = selectivity index = CC50/MIC (refer to section 2.4.3).
2.4.3. Cytotoxicity Studies
The cytotoxicity test indicated that C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I)
is safe to be used as an antimicrobial therapeutic agent due to its non-toxicity towards Vero cells with a
CC50 value of 0.4 mg/mL. According to Zirihi et al. [18], a test compound is considered toxic if the
CC50 value is less than 0.02 mg/mL. CC50 value can be obtained directly from the graph of percentage
of cell survival viability versus compound concentration (Figure 6). However, when the CC50 value
was used to calculate the selectivity index (SI) of antibacterial activity using the equation,
SI = CC50/MIC, the SI values were lower than 1. Although the CC50 value is high indicating
non-cytotoxicity, the high MIC value shows weak antibacterial activity. This resulted in a low SI value
and suggests that compound I is unsuitable as a potential antibacterial agent [19].
2.4.4. Antiviral Activity towards HSV-1
Antiviral tests showed that the compound I was suitable as an antiviral agent because of its ability
to inhibit 100% plaque formation, even at the lowest concentration of 0.011 mg/mL (Figure 7). Thus,
the EC50, which is the concentration when the presence of test compound caused 50% reduction
of plaques or cytopathic effect, is much lower than the minimum inhibitory concentration of
0.011 mg/mL. The selectivity index (SI = CC50/EC50) of compound I is more than 36. This indicates
that compound I can be considered as a potentially safe antiviral agent with low cytotoxicity and high
potency. SI values greater than 10 indicate potential antiviral therapeutic safety and efficacy.

Molecules 2013, 18
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Figure 6. Percentage of cell survival against concentration of compound C-5-bromo-2-
hydroxyphenylcalix[4]-2-methylresorcinarene (I).
Figure 7. Plaque formation to determine virus titer.
3. Experimental
3.1. Materials and Physical Measurements
All the compounds utilized in this work were commercially available high purity products purchased
from Acros Organics (Geel, Belgium) and Sigma-Aldrich (St Louis, MO, USA) and were used without
further purification. All solvents were distilled before use. The microelemental analysis for CHNS-O
was carried out using a Carlo Erba 1108 Elemental Analyzer (Milan, Italy). The infrared spectrum (IR)
of the product (KBr pellets) was recorded using a Perkin Elmer Spectrum GX spectrophotometer
(Perkin Elmer, Waltham, MA, USA) in the range of 400–4,000 cm−1. Nuclear Magnetic Resonance
13
(1H and C) experiments were performed on a Bruker 600 MHz instrument using DMSO-d6 as the
solvent. TGA was performed under flowing nitrogen at a heating rate of 10 °C min−1 using a Mettler
Thermogravimetric Analyzer (Mettler-Toledo, Poslfach, Switzerland).

Molecules 2013, 18
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3.2. Preparation of C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I)
Concentrated hydrochloric acid (7 mL) was added into a round-bottom flask containing a solution
of 5-bromo-2-hydroxybenzaldehyde (0.01 mol, 2.01 g) in absolute ethanol (60 mL). The mixture was
stirred for 30 minutes and a solution of 2-methylresorcinol (0.01 mol, 1.24 g) in absolute ethanol
(20 mL) was added. The mixture was refluxed for 24 hours at 80 °C. The yellow precipitate formed
was collected by filtration, washed with distilled water and acetone several times and dried under
vacuum. Yield (76%); FTIR (KBr, cm−1): 3340 (OH), 1433 (C=C), 1211 (C-O), 609 (C-Br);
1H-NMR (600 MHz; precipitate, DMSO-d6) δH: 1.94 (6H, s, Ar-CH3), 2.11 (6H, s, Ar-CH3), 5.37 (2H,
s, Ar-CH), 5.91 (4H, s, Ar-CH), 6.10 (2H, s, Ar-CH), 6.34 (4H, s, Ar-H), 6.38 (4H, d, J = 8.0,
OH-Ar-CH), 6.77 (4H, s, OH), 6.83 (4H, d, J = 8.0, Br-Ar-CH), 7.48 (4H, s, OH), 8.94 (4H, s, OH);
13C-NMR (150 MHz; DMSO-d6) δC: 10.4 (2 × CH3), 10.6 (2 × CH3), 36.8 (4 × CH), 110.5 (3 × Ar-
CH3), 110.8 (Ar-CH3), 112.1 (4 × Ar-H), 116.3 (4 × Ar-Br), 120.5 (4 × Ar-CH), 123.8 (4 × Ar-CH),
125.6 (2 × Ar-H), 127.5 (2 × Ar-H), 128.7 (4 × Ar-H), 132.9 (4 × Ar-CH), 133.2 (4 × Ar-H),
1
150.9 (4 × Ar--OH), 151.2 (4 × Ar-OH), 153.8 (4 × Ar-OH). H-NMR (600 MHz; crystallized,
DMSO-d6) δH: 1.95 (6H, s, Ar-CH3), 2.12 (6H, s, Ar-CH3), 2.73 (30H, s, 10 × CH3, DMF), 2.89 (30H,
s, 10 × CH3, DMF), 5.38 (2H, s, Ar-CH), 5.91 (4H, s, Ar-CH), 6.10 (2H, s, Ar-CH), 6.34 (4H, s, Ar-
H), 6.38 (4H, d, J = 8.0, OH-Ar-CH), 6.77 (4H, s, OH), 6.83 (4H, d, J = 8.0, Br-Ar-CH), 7.48 (4H, s,
OH), 7.95 (10H, s, 10 x COH, DMF), 8.95 (4H, s, OH). (Crystal). Anal. Calcd for (molecular
formula): C = 54.75 and H = 3.61 Found: C, 54.22 and H, 3.59. Suitable yellowish crystals for X-ray
investigation were obtained by recrystallization from DMF but changed to a powder after a few hours
exposure to air. Coating the fresh crystals with Princeton-H oil allowed the X-ray experiment to be
conducted for at least 10 hours.
3.3. X-ray Crystallography
Single-crystal X-ray experiment was performed on Bruker D-QUEST diffractometer (Bruker, AXS
Inc., Madison, WI, USA) using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). Intensity
data was measured at 100(2) K by the ω-scan. Accurate cell parameters and orientation matrix were
determined by the least-squares fit of 25 reflections. Intensity data was collected for Lorentz and
polarization effects. Empirical absorption correction was carried out using multi-scan. The structure
was solved by direct methods and least-squares refinement of the structure was performed by the
SHELXL-97 program [20]. All the non-hydrogen atoms were refined anisotropically. The
hydrogen atoms were placed in calculated positions, allowing them to ride on their parent C atom with
Uiso(H) = 1.2Ueq except the hydrogen atoms of the water solvates were located from Fourier maps and
refined isotropically. A summary of the data collections and details of the structure refinement is given
in Table 5. Crystallographic data for the structural determination has been deposited with the
Cambridge Crystallographic Data Center, CCDC No 959177. This information may be obtained free of
charge at http://www.ccdccam.ac.uk/const/retrieving.html or from the Cambridge Crystallographic Centre
(CCDC), 12 Union Road, Cambridge CB2, 1EZ, UK (Fax: +44(0)-1223-336033; E-Mail:
deposit@ccdc.cam.ac.uk.

Molecules 2013, 18
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Table 5. Crystallographic data and structural refinement of C-5-bromo-2-hydroxy
phenylcalix[4]-2-methylresorcinarene.
Crystal parameters
CCDC deposition number
Empirical formula
Moiety formula
Formula weight
Temperature
Data/values
959177
C80H108Br4N8O24
C56H44Br4O12, 8(C3H7NO), 4(H2O)
1885.38
100(2) K
0.71073 Å
Triclinic
Pī
Wavelength λ
Crystal system
Space group
a = 15.9592(16) Å
b = 16.9417(17) Å
c = 17.0974(17) Å
α = 68.656(3)°
β = 85.689(3)°
γ = 81.631(3)°
Unit cell dimensions
3
Volume
Z
Dcal (Mg/m3)
4258.6(7) Å
2
1.470
−1
Absorption coefficient
F(000)
1.969 mm
1952
Crystal dimension (mm)
Tmin/Tmax
0.42 × 0.37 × 0.24
0.4918, 0.6494
130536
Reflections measured
Ranges/indices (h,k,l)
θ limits (º)
−19, 19; −20, 20; −21, 21
2.8 to 26.0°
Unique reflections
Observed reflections (I>2σ(I))
16701
11455
Parameters
1101
Goodness of fit on F2
1.13
R1, wR2 (I≥2σ(I))
0.0661, 0.1652
0.1107, 0.1997
R1,wR2 indices (all data)
Largest diff. peak and hole
−3
2.669 and −0.977 e.Å
3.4. Antioxidant Test
A stock solution of DPPH was prepared by dissolving DPPH (0.4 g) in methanol (1 L) and the
solution was kept in the dark at 4 °C. A stock solution of the C-5-bromo-2-hydroxyphenylcalix[4]-2-
methylresorcinarene (I) was prepared at 10 mg/5 mL in DMSO. A volume of 100 µL from the stock
solution of the compound was added to 1 mL of DPPH. The mixture was shaken well and kept in the
dark at room temperature for 2 hours. The absorbance of the mixture was measured at 517 nm using a
spectrophotometer. The percentage of inhibition of radical scavenging ability was calculated as:
Inhibition % = [(ADPPH – ASample)/(ADPPH)] × 100 = [(1.012 − 0.1523)/1.012] × 100 = 84.95%.

Molecules 2013, 18
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3.5. Antibacterial Activity
Antibacterial activity was determined by the disc diffusion method [21] followed by minimum
inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests against two
Gramnegative and three Gram-positive bacteria. A stock solution at 25 mg/mL was prepared by
dissolving C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I, 25 mg) in dimethyl
sulfoxide (DMSO, 1 mL). Two fold-dilution of the stock solution was prepared to produce test
solutions at 12.5, 6.250, 3.125 and 1.563 mg/mL.
Test bacteria were grown in Mueller-Hinton broth (MHB) and bacterial suspensions standardized to
2 × 108 cells/mL were used to prepare bacterial lawn on Mueller-Hinton agar (MHA). After allowing
the disc to dry for 5 minutes, five sterile Whatman No: 1 filter paper discs (6 mm in diameter) were
placed on the bacterial lawn. Test solution (5 μL) at different concentrations was applied to the paper
discs using a micropipettor. Vancomycin or chloramphenicol was used as positive controls. DMSO
was used as a negative or solvent control. Plates were incubated at 37 °C for 24 hours of incubation
and the diameter of inhibition zone was measured (Figure 6).
The minimum inhibition concentration (MIC) determination was carried out on compound I using
the broth dilution method in 96 well microplate according to Andrews [22]. The minimum bactericidal
concentration (MBC) was determined by subculturing the broth in wells with test compound that
showed no growth on nutrient agar (NA). The concentration that showed no growth on the NA plates
was determined and the MBC.
Selectivity index is determined using the CC50 from the cytotoxicity evaluation and the MIC and
calculated as SI = CC50/MIC.
3.6. Cytotoxicity Evaluation
The cytotoxicity of the compound was first determined on uninfected Vero cells (African monkey
Cercophiteus aetiops kidney cells) with dilutions ranging from 5 mg/mL to 0.039 mg/mL in
Dulbecco’s Modified Eagle’s Medium (DMEM, Flowlab, North Ride, Australia). Cytotoxicity was
determined using the MTT assay [23]. The CC50 value that is the compound concentration that kills
50% of the cell population was determined by the optical density of solubilized formazan. The
percentage of growth inhibition was calculated using the following formula:
% Cell viability = 100 × (Abs/Ac)
where Abs = Absorbance value of test compound and Ac = Absorbance value of control (cells only).
3.7. Antiviral Activity
The plaque reduction assay was performed to study the presence of antiviral activity of compound I.
HSV-1 stock was prepared and the viral titre of the stock was determined as 5.7 × 108 pfu/mL. The
concentration of compound I used in this assay was set based on the concentration that allows growth
of 70% or more Vero cells. This is to ensure that cell death is not due to the toxicity of the test
compound which will affect the accuracy of the test results. In this study, the first test concentration of
C-5-bromo-2-hydroxophenylcalix[4]-2-methylresorcinarene (I) is 0.35 mg/mL, which is below the

Molecules 2013, 18
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CC50 value and continues with two fold serial dilutions until 0.011 mg/mL. Virus was infected to 80%
confluent cells and incubated for 48 hours for plaque formation. Plaques were stained with crystal
violet and the numbers of plaques were counted. The EC50 value was determined as the concentration
that inhibited plaque formation by 50% of the untreated cells [24,25].
4. Conclusions
C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I) was successfully synthesized by the
cyclocondensation of 5-bromo-2-hydroxybenzaldehyde and 2-methylresorcinol in the presence of
concentrated HCl. The X-ray structure was in good agreement with the NMR data and the calix
molecule adopted a chair C2h conformation. Compound I showed good anti-HSV-1 and antioxidant
activity at non-cytotoxic concentrations, but weak antibacterial activity.
Supplementary Material
Supplementary material can be accessed at: http://www.mdpi.com/1420-3049/18/11/13369/s1.
Acknowledgments
The authors thank the Ministry of Higher Education of Malaysia and Universiti Kebangsaan
Malaysia for the research grant DIP-2012-11. One of us HAMZA would like to thank the Ministry of
Higher Education of Libya for the scholarship.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Zhang, Z.; Luo, Y.; Chen, J.; Dong, S.; Yu, Y.; Ma, Z.; Huang, F. Formation of linear
supramolecular polymers that is driven by C–H⋅⋅⋅π interactions in solution and in the solid state.
Angew. Chem. Int. 2011, 50, 1397–1401.
2. Han, C.; Ma, F.; Zhang, Z.; Xia, B.; Huang, F. DIBPillar[n]arenes (n = 5, 6): Syntheses, X-ray
crystal structures, and complexation with n-Octyltriethyl ammonium hexafluorophosphate.
Org. Lett. 2010, 12, 4360–4363.
3. Zhang, M.; Xu, D.; Yan, X.; Chen, J.; Dong, S.; Zheng, B.; Huang, F. Self-healing
supramolecular gels formed by crown ether based host—Guest interactions. Angew. Chem. Int.
2012, 51, 7011–7015.
4. Zheng, B.; Wang, F.; Dong, S.; Huang, F. supramolecular polymers constructed by crown
ether-based molecular recognition. Chem. Soc. Rev. 2012, 41, 1621–1636.
5. Turshatov, A.A.; Melnikova, N.B.; Semchikov, Y.D.; Ryzhkina, I.S.; Pashirova, T.N.; Mobius, D.;
Zaitsev, S.Y. Interaction of monolayers of calix[4]resorcinarene derivatives with copper ions in
the aqueous subphase. Colloids Surf. A Physicochem. Eng. Aspects. 2004, 240, 101–106.

Molecules 2013, 18
13383
6. Sarkar, A.; Krishnamurthy, S.S.; Nethaji, M. Calix[4]arene bisphosphite ligands bearing two
distal 2,2-biphenyldioxy conformational flexibility and allyl-palladium complexes. Tetrahedron.
2009, 65, 374–382.
7. Podyachev, S.N.; Burmakina, N.E.; Syakaev, V.V.; Sudakova, N.S.; Shagidullin, R.R.;
Konovalov, A.I. Synthesis IR and NMR characterization and ion extraction properties of
tetranonylcalix[4] resorcinol bearing acetylhydrazone groups. Tetrahedron. 2009, 65, 408–417.
8. Hedidi, M.; Hamdi, S.M.; Mazari, T.; Boutemeur, B.; Rabia, C.; Chemat, F.; Hamdi, M.
Microwave-assisted synthesis of calix[4]resorsinarene. Tetrahedron. 2006, 62, 5652–5655.
9. Ruderisch, A.; Pfeiffer, J.; Schurig, V. Synthesis of an enantiomerically pure resorcinarene with
pendant L-valine residues and its attachment to a polysiloxane(Chirasil-Calix). Tetrahedron
Asymmetry. 2001, 12, 2025–2030.
10. Makinen, M.; Jalkanen, J.P.; Vainiotalo, P. Conformational properties and intramolecular
hydrogen bonding of tetraethyl resorcinarene: An ab initio study. Tetrahedron. 2002, 58,
8591–8596.
11. Thondorf, I.; Brenn, J.; Bohme, V. Conformational properties of methylene-bridged resorcarenes.
Tetrahedron. 1998, 54, 12823-12828
12. Iwanek, W.; Wzorek, A. Introduction to the chirality of resorcinarenes. Mini Rev. Org. Chem.
2009, 6, 398–411.
13. Davis, C.J.; Lewis, P.T.; Billodeaux, D.R.; Fronczek, F.R.; Escobedo, J.O.; Strongin, R.M.
Solid-state supramolecular structures of resorcinol-arylboronic acid compounds. Org. Lett. 2001,
3, 2443–2445.
14. Knyanzeva, I.R.; Burilov, A.R.; Fazleeva, G.M.; Nuretdinov, I.A.; Gryanznova, T.V.;
Budnikova, Y.G.; Khrisanforova, V.V.; Gubaidullin, A.T.; Gabidullin, B.M.; Syakaey, V.V.; et al.
New Calix[4]resorcinols with Thiophosphoryl-containing fragments. Phosphorus Sulfur Silicon.
2011, 186, 1972–1980.
15. Aishahateet, S.F.; Kooli, F.; Messali, M.; Judeh, Z.M.A.; Eldouhaibi, A.S. Synthesis and
supramolecularity of C-phenylcalix[4]pyrogallolarenes: Temperature effect on the formation of
different isomers. Mol. Cryst. Liq. Cryst. 2007, 474, 89–110.
16. Patel, M.B.; Modi, N.R.; Raval, J.P.; Menon, S.K. Calix[4]arene based 1,3,4-oxadiazole and
thiadiazole derivatives: Design, synthesis and biological evaluation. Org. Biomol. Chem. 2012,
10, 1785–179.
17. Pankey, G.A.; Sabath, L.D. Clinical relevance of bacteriostatic versus bactericidal mechanisms of
action in the treatment of Gram-positive bacterial infections. Clin. Infect. Dis. 2004, 38, 864–870.
18. Al-Adiwish, W.M.; Tahir, M.I.M.; Siti-Noor-Adnalizawati, A.; Hashim, S.F.; Ibrahim, N.;
Yaacob, W.A. Synthesis, antibacterial activity and cytotoxicity of new fused pyrazolo [1,5-a]
pyrimidine and pyrazolo[5,1-c][1,2,4]triazine derivatives from new 5-aminopyrazoles. Eur. J.
Med. Chem. 2013, 64, 464–476.
19. Zirihi, G.N.; Mambu, L.; Guede-Guina, F.; Bodo, B.; Grellier, P. In vitro anti-plasmodial activity
and cytotoxicity of 33 West African plants used for treatment of malaria. J. Ethnopharmacol.
2005, 98, 281–285.

Molecules 2013, 18
13384
20. Sheldrick, G.M. SHELXTL. Version 6.14. Program for crystal structure determination University
of Gottingen: Gottingen, Germany, 1997.
21. Bauer, A.W.; Kirby, W.M.; Sherris, J.C. Antibiotic susceptibility testing by a standard single disk
method. Am. J. Clin. Pathol. 1966, 45, 493–496.
22. Andrews, J.M. Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother.
2001, 48, 5–16.
23. Andrighetti-Frohner, C.R; Antonio, R.; Creczynski-Pasa, T.; Barardi, C.; Simoes, C. Citotoxicity
and potential antiviral evaluation of violacein produced by Chromobacterium violaceum. Mem.
Inst. Oswaldo Cruz. 2003, 98, 843–848.
24. Jordao, A.K.; Ferreira, V.F.; Thiago, M.L.S.; Gabrielle, C.S.F.; Viviane, M.; Juliana, L.A.;
Maria, C.B.V.; Anna, C.C. Synthesis and anti-HSV-1 activity of new 1,2,3-triazole derivatives.
Biorg. Med. Chem. 2011, 19, 1860–865.
25. Macedo, N.R.P.V.; Ribeiro, M.S.; Villaca, R.C.; Ferreira, W.; Pinto, A.M.; Teixeira, V.L.;
Cirne-Santos, C.; Paixao, I.C.N.P.; Giongo, V. Caulerpin as a potential antiviral drug against
herpes simplex virus type 1. Braz. J. Pharmacogn. 2012, 22, 861–867.
Sample Availability: Samples of the compounds are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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