Jordan Journal of Chemistry Vol. 9 No.3, 2014, pp. 170-186 JJC

Green Synthesis, Crystal Structure and Bioactivity of C-(p- substituted phenyl)calix[4]resorcinarenes-DMSO Inclusion Complexes

Solhe F. Alshahateeta, Salah A. Al-Trawneha, Wael A. Al-Zereinib, Saad S. Al-Sarhana

aDepartment of Chemistry, Mutah University, P.O. BOX 7, Mutah 61710, Alkarak, Jordan bDepartment of Biological Sciences, Mutah University, P.O. BOX 7, Mutah 61710, Alkarak, Jordan

Received on April 24, 2014 Accepted on Jun. 26, 2014

Abstract Green synthesis of calix[4]resorcinarene hosts with p-substituted phenyl group at their methine carbons was achieved. Fluorine, chlorine and bromine atoms, as well as methoxy group, were selected as substituents at the para position of the benzaldehyde which was condensed with resorcinol in presence of solid p-toluenesulfonic acid. The solid state structures of the newly prepared C-(p-fluorophenyl)calix[4]resorcinarene-DMSO and C-(p-chlorophenyl)- calix[4]resorcinarene-DMSO inclusion complexes were determined by single-crystal X-ray diffraction and found to form different structural conformations; chair (C2h) and boat (C2v), respectively. In addition, both crystal structures adopted several intermolecular noncovalent supramolecular interactions which were carefully investigated and presented in terms of crystal engineering and supramolecular chemistry. The synthesized C-(p-substitutedphenyl)calix[4]- resorcinarene hosts inhibited the growth of Gram-positive bacteria with the C-(p-bromophenyl)- calix[4]resorcinarene derivative being the most potent agent (MIC=15.6-125 μg/ml).

Keywords: C-(p-substituted phenyl)calix[4]resorcinarenes-DMSO inclusion complex; Boat conformation; Chair conformation; Crystal structure; Bioactivity; Non-covalent interactions.

Introduction Non-covalent intermolecular interactions of multicomponent crystalline compounds are of great interest to chemists due to their wide-range uses and application areas such as energy storage, drug delivery and separation technology.[1] Calix[4]resorcinarenes represent a class of macrocyclic molecules used as potential lattice hosts for different guests.[2] In crystal engineering and supramolecular chemistry, understanding the intermolecular interactions between molecules is very important and crucial to understanding many biological processes.[3-11] Through this understanding, chemists hope to predict both the industrial applications and the properties of any given crystalline solid material. Research work on different calix[4]resorcinarenes and proposing new efficient and green methods for their

 Corresponding author: e-mail: [email protected] 170 preparation is worldwide conducted.[12-21] Design and preparation of different types of lattice host molecules and investigating their ability to form different forms of crystalline materials with different potential applications are, therefore, of our main interest.[22-38] Compounds 6-9 were reported in literature to be prepared in alcoholic acid catalyzed solutions as a mixture of two conformers; chair and boat were formed.[13,15-20] In this work, the selective solvent-free formation of one isomeric product and the bioactivity of C-(p-substituted phenyl)calix[4]resorcinarenes hosts 6-9 (Scheme 1) is reported. In addition, the new 6·(DMSO)8 and 8·(DMSO)8 lattice inclusion complexes were prepared and their crystal structures were determined using X-ray crystallography. The intermolecular interactions were carefully discussed and presented in terms of supramolecular chemistry. R R c HO d OH O H b a OH HO OH

+

OH R HO OH

HO OH

1 R = Cl R R 2 R = Br 3 R = F 5 6-9 4 R = OCH3 Scheme 1: Synthetic route for C-(p-substitutedphenyl)calix[4]resorcinarene 6-9.[36]

Experimental

Materials and physical measurements All solvents were purchased as analytical reagent grade. Melting points were measured on a Stuart scientific melting point apparatus in open capillary tubes. The infrared spectra were recorded over the range of 4000-500 cm-1 on a Maltson 5000 FTIR spectrometer. 1H-NMR and 13C-NMR measurements were conducted on a Bruker 500 MHz. Chemical shifts were referenced to TMS as the internal standard and deuterated dimethylsulfoxide (DMSO-d6) as the solvent. In all of the compounds reported, the internal aromatic proton in the cavity could not be observed by NMR in the indicated solvent; this might be attributed to ring current effects. Similar findings were observed in our previous related work.[38] X-ray single crystal structure determination was accomplished using a Bruker SMART APEX-1000 diffractometer.

General procedure for the synthesis of compounds 6-9

171 A solvent-free procedure was applied to synthesize compounds 6-9 in very good yields.[36] Equimolar amounts of resorcinol (1 mol) and p-substituted benzaldehyde (1 mol) 1-4 were ground in the presence of solid p-toluenesulfonic acid (0.05 mol) as a catalyst using a mortar and pestle. The reactants melted on grinding but thereafter the contents solidified. The reaction mixture was ground again, washed with water, filtered and dried to give compounds 6-9 in excellent yields (90-95 %). The products obtained were identical with those obtained through reactions in acidic solutions.[13,15-20]

Preparation of C-(4-chlorophenyl)calix[4]resorcinarene 6 4-Chlorobenzaldehyde and resorcinol (1:1) were mixed together in presence of a catalytic amount of p-toluenesulfonic acid (0.05 mol) and processed as described above. Spectral data were: IR (KBr), cm-1: 3435, 2912, 1620, 1510, 1489, 1429, 1209, 1 3 1078, 1014, 552; H-NMR (500 MHz, DMSO-d6), (ppm) 8.69 (br, 8H, OH), 7.05(d, J 3 = 8.5 Hz, 8H, Ha), 6.63 (d, J = 8.5 Hz, 8H, Hb), 6.19 (br, 4H, Hd), 5.62 (s, 4H, H- 13 benzylic).; C-NMR (125 MHz, DMSO-d6), δ (ppm): 41.4, 102.5, 120.3, 127.5, 129.9,

130.4, 145.3, 155.3. Elem. Anal. Calcd for C52H36Cl4O8: C, 67.11; H, 3.90. Found: C, 67.34; H, 3.92. Direct crystallization of compound 6 from fresh DMSO yielded crystals of the lattice inclusion compound that were suitable for single-crystal X-ray study.

Preparation of C-(4-bromophenyl)calix[4]resorcinarene 7 4-Bromobenzaldehyde and resorcinol (1:1) were mixed together in presence of a catalytic amount of p-toluenesulfonic acid (0.05 mol) and processed as described above. Spectral data were: IR (KBr), cm-1: 3441, 2901, 1618, 1508, 1485, 1429, 1402, 1 1240, 1209, 1078, 1011, 550m; H-NMR (500 MHz, DMSO-d6), (ppm) 8.70 (s, 8H, 3 3 OH), 7.19 (d, J = 8.5 Hz, 8H, Ha), 6.58 (d, J = 8.5 Hz, 8H, Hb), 6.18 (br, 4H, Hd), 5.61 13 (s, 4H, H-benzylic); C-NMR (125 MHz, DMSO-d6), δ (ppm): 41.4, 102.6, 118.3, 120.2,

128.6, 130.5, 130.9, 145.8, 153.7. Elem. Anal. Calcd for C52H36Br4O8: C, 56.34; H, 3.27. Found: C, 56.17; H, 3.28.

Preparation of C-(4-fluorophenyl)calix[4]resorcinarene 8 4-Fluorobenzaldehyde and resorcinol (1:1) were mixed together in presence of a catalytic amount of p-toluenesulfonic acid (0.05 mol) and processed as described above. Spectral data were: IR (KBr), cm-1: 3412, 2930, 1605, 1508, 1431, 1213, 1159, 1 3 1076, 553; H-NMR (500 MHz, DMSO-d6), (ppm) 8.63 (s, 8H, OH), 6.80 (t, JH-H = 8.4 3 3 Hz, 8H, Ha), 6.65 (dd, JH-F = 8.5 Hz, JH-H = 8.4 Hz, 8H, Hb), 6.17 (br, 4H, Hd), 5.63 (s, 13 2 4H, H-benzylic).; C-NMR (125 MHz, DMSO-d6), δ (ppm): 41.3, 102.6, 114.1 (d, JC-F 3 1 = 21 Hz), 120.8, 130.3 (d, JC-F = 8 Hz), 142.3, 153.2, 160.5 (d, JC-F = 239 Hz). Elem.

Anal. Calcd for C52H36F4O8: C, 72.22; H, 4.20. Found: C, 72.50; H, 4.23. Direct crystallization of compound 8 from fresh DMSO yielded crystals of the lattice inclusion compound that were suitable for single-crystal X-ray study.

Preparation of C-(4-methoxyphenyl)calix[4]resorcinarene 9

172 4-Methoxybenzaldehyde and resorcinol (1:1) were mixed together in presence of a catalytic amount of p-toluenesulfonic acid (0.05 mol) and processed as described above. Spectral data were: IR (KBr), cm-1: 3408, 2891, 1609, 1510, 1429, 1246, 1208, 1 1180,1147, 1076, 1032; H-NMR (500 MHz, DMSO-d6), (ppm) 8.42 (s, 8H, OH), 7.19 3 3 (d, J = 8.4 Hz, 8H, Ha), 6.58 (d, J = 8.4 Hz, 8H, Hb), 6.24 (br, 4H, Hd), 5.61 (s, 4H, H- 13 benzylic); 3.72 (s, 3H, -OCH3); C-NMR (125 MHz, DMSO-d6), δ (ppm): 41.7, 55.1,

102.5, 113.0, 122.0, 129.9, 139.1, 153.8, 157.9. Elem. Anal. Calcd for C56H48O12: C, 73.67; H, 5.30. Found: C, 74.00; H, 5.32.

Solution and refinement of the crystal structures Reflection data were measured at 223(2) K on a Bruker SMART APEX-1000 diffractometer equipped with a CCD detector and Mo-Kα sealed tube. SMART was used for collecting frame data, indexing reflection, determination of lattice parameters, integration of intensity of reflections and scaling. SADABS was used for absorption correction and SHELXTL for space group, structure determination, and least-square refinements on F2.[39-41] All non-hydrogen atoms were assigned anisotropic displacement parameters in the refinement. All hydrogen atoms were added at calculated positions and refined using a riding model.

Supplementary data Crystallographic data for the structural analysis reported in this paper have been deposited in the Cambridge Structural Data Centre, CCDC, Numbers (915290- 915291). Copies of the information may be obtained free of charge from Director, CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033; email: [email protected].

In vitro antibacterial activity Inhibition zones caused by four different concentrations (0.1, 0.25, 0.5, 1 mg/disk) of compounds 6-9 were measured in agar diffusion test. 1 mg/ml of test compounds and 100 μg/ml of positive control (chloramphenicol) were used in serial dilution assay as starting concentration to deduce the minimum inhibitory concentration (MIC) values.[42] The test microorganisms used in this study were Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 43300), Escherichia coli (ATCC 25922), Enterobacter aerogenes (ATCC 13048), and Micrococcus luteus (ATCC 10240).

Results and Discussion

Infrared measurements and NMR spectra The important bands of the prepared calix[4]resorcinarenes were assigned by comparing them with those reported previously.[43-50] The IR spectra of compounds 6-9 show a stretching aliphatic C-H absorption band in the range of 2891-2930 cm-1 and display a broad band in the range of 3401-3447 cm-1 corresponding to phenolic O-H bond. The absorption band corresponding to the stretching of aromatic C=C was 173 observed in the range of 1605-1620 cm-1 in addition to the absorption band in the range of 546-849 cm-1 due to carbon- halogen (Cl, Br, F) bond and at 1250 cm-1 due to O-C bond of methoxy group in compound 9. The absence of a υ(C=O) band in the range of 1740-1720 cm-1 confirms that the p-substituted benzaldehyde has completely reacted. The 1H-NMR spectra for the prepared calix[4]resorcinarenes give signals corresponding to the aromatic protons (belonging to the resorcinol ring) around 6.20 ppm. The methine protons resonate as a singlet in the range of 5.60-5.63 ppm, while the phenolic protons resonate in the range of 8.41-8.70 ppm. The H-a and H-b protons (scheme 1) resonate as doublets in the range of 6.58- 7.19 ppm (3J = 8.5Hz) due to spin-spin coupling between each other. In all of the compounds reported, the internal aromatic proton in the cavity could not be observed by NMR in the indicated solvent; this might be due to ring current effects. Similar findings were observed in our previous related work.[38] The 13C-NMR spectral data support the formation of calix[4]resorcinarene through the absence of carbonyl signals of the functionality at 191 ppm and the formation of methylene bridge (CH-aliphatic) signals in the range of 41.2-41.4 ppm. The aromatic carbons are resonating in the range of 102.5-161.4 ppm. The 13C- NMR spectra for compound 8, in contrast to the other compounds, showed that the C–a, C-b and C–c carbons are distinguishable due to spin–spin coupling with the fluorine atom at C–a position.

Crystal structure of 6·(DMSO)8 lattice inclusion complex It was reported that calix[4]resorcinarene forms two different structural 1 conformations, chair (C2h) and boat (C2v), which are distinguishable by H-NMR spectra.[12-14,16] Host 6 crystallized from fresh DMSO formed a new lattice inclusion complex 6·(DMSO)8 in a triclinic system with P-1 space group. Table 1 illustrates the crystal data and structure refinements of 6·(DMSO)8 lattice inclusion complex.

Investigation of the solid state structure of 6·(DMSO)8 inclusion complex revealed that host 6 adopted the boat conformation (C2v). The asymmetric unit contains one titled host molecule 6 and eight guest molecules (DMSO); two of the eight DMSO guest molecules have some degree of disorder (Figure 1). In addition, there are a number of supramolecular noncovalent interactions such as H-bonding between the OH of the host molecule and the oxygen and sulfur atoms of the guest molecule. Host-guest interactions are clearly observed in the crystal structure of 6·(DMSO)8 inclusion complex (i.e. chlorine atom (Cl8) of one host molecule is interacting with an oxygen atom (O3S) of a guest molecule with a contact distance of 3.23 Å). In addition, hydrogen atom (H8) of a hydroxy group of host molecule is interacting with an oxygen atom (O2S) of another guest molecule with a contact distance of 1.84 Å (Figure 2). Different types of angles and lengths that existed in the crystal structure of 6·(DMSO)8 lattice inclusion complex are presented in table 2. 174 Centrosymmetric dimer was adopted by the crystal structure of 6·(DMSO)8 in which chlorine atom of one host is hydrogen bonded with hydrogen atom of another host molecule with bond distances of 2.67 Å and 2.78 Å as illustrated in figure 3. The presence of three hetero atoms in the crystal structure of 6·(DMSO)8 led to very interesting intermolecular noncovalent interactions such as; S…S (3.54 Å), S…O (3.28 Å), S…Cl (3.90 Å), O…Cl (3.23 and 3.93 Å), Cl…Cl (3.95 Å) and O…O (3.85 and 3.75

Å). In addition, careful analyses for the crystal packing of 6·(DMSO)8 revealed that sulfur and oxygen atoms of both host and guest are involved in the network of intermolecular hydrogen bondings in terms of host-guest (S…H-O-Ar, 2.87 Å), guest- guest ((CH3)2OS…H-CH2-SO-CH3, 2.96 Å), guest-guest ((CH3)2SO…H-CH2-SO-CH3,

2.57 Å), and host-guest ((CH3)2SO…H-O-Ar, 1.81 and 1.85 Å). These interactions were in good agreement with what we have observed previously in similar [37-38, 51] compounds. Crystal packing of 6·(DMSO)8 lattice inclusion complex is illustrated in figure 4.

Table 1: Numerical Details of the Solution and Refinement of the Crystal Structures of 6·(DMSO)8 and 8·(DMSO)8.

Compound 6·(DMSO)8 8·(DMSO)8

Formula C68H84Cl4O16S8 C68H84F4O16S8 Formula mass 1555.63 1489.83 Crystal system Triclinic Triclinic Space group P-1 P-1 a / Å 13.0931(6) 10.2575(5) b / Å 15.0868(7) 13.9642(7) c / Å 20.9627(9) 15.3376(8)  / o 82.7770(10) 105.1780(10)  / o 79.5100(10) 107.1530(10)  / o 70.7850(10) 107.2570(10) V / Å3 3835.1(3) 1852.01(16) T / oK 223(2) 223(2) Z 2 1 Dcalc. / g cm-3 1.347 1.336 Radiation, / Å MoK, 0.71073 MoK, 0.71073 / mm-1 0.434 0.314 Scan mode /2 /2 F(000) 1632 784 Goodness-of-fit on F2 1.047 1.147 Theta range for data 1.68 to 27.50o 1.50 to 25.00o collection Completeness to theta 99.4% 100% max. Final R indices R1 = 0.0682, R1 = 0.0799, [I>2sigma(1)] wR2 = 0.1847 wR2 = 0.1693 R indices (all data) R1 = 0.0872, R1 = 0.1034, wR2 = 0.1990 wR2 = 0.1802 Data / restrains / 17523 / 16 / 900 6540 / 0 / 449 parameters CCDC number 915290 915291

175

Figure 1: ORTEB diagram of 6·(DMSO)8 lattice inclusion complex with the ellipsoids drawn at the 50% probability level.

Figure 2: Host-guest interactions between one molecule of host 6 and two guest molecules involving Cl…O and H…O supramolecular interactions.

176

Figure 3: Centrosymmetric dimer resulting from double Ar-Cl…H-Ar interactions between two molecules of host 6.

Table 2: Hydrogen bond lengths and angles for 6·(DMSO)8 lattice inclusion complex (Å and °). ______D-H...A d(D-H) d(H...A) d(D...A) <(DHA) ______O(1)-H(1)...O(7S)#1 0.83 1.81 2.636(3) 173.4 O(2)-H(2)...O(8S)#1 0.83 1.82 2.622(9) 161.3 O(2)-H(2)...O(8SA)#1 0.83 1.88 2.663(6) 156.6 O(3)-H(3)...O(4S)#2 0.83 1.85 2.673(3) 169.3 O(4)-H(4)...O(6S)#3 0.83 1.87 2.695(3) 176.4 O(5)-H(5)...O(1S) 0.83 1.85 2.679(3) 174.4 O(6)-H(6)...O(5S) 0.83 1.81 2.611(3) 161.0 O(7)-H(7)...O(3S)#4 0.83 1.84 2.659(3) 167.0 O(8)-H(8)...O(2S)#5 0.83 1.84 2.666(3) 171.8 ______Symmetry transformations used to generate equivalent atoms: #1 -x+1,-y+1,-z+1 #2 x+1, y, z #3 -x+1,-y+1,-z #4 x-1, y+1, z #5 -x+1,-y+2,-z+1

177

Figure 4: Crystal packing of 6·(DMSO)8 lattice inclusion complex. Hydrogen atoms of host 6 and DMSO were omitted for clarity, non-hydrogen atoms were labeled.

178 Crystal structure of 8.(DMSO)8 lattice inclusion complex

The X-ray crystal structure of compound 8 was obtained as 8·(DMSO)8 lattice inclusion complex through the crystallization of host 8 from fresh DMSO. The structure revealed that host 8 adopted the chair conformation (C2h). The newly prepared

8·(DMSO)8 lattice inclusion complex crystallizes in triclinic system with P-1 space group. Similar conformation was recently observed in our group when the formation, supramolecularity and the bioactivity of the ternary lattice inclusion compound (C- phenylcalix[4]resorcinarene)·(DMSO)8·(H2O)3 were examined and investigated; the [51] host molecules adopted the chair conformation with C2h symmetry. Table 1 illustrates the crystal data and structure refinements of compound (8).(DMSO)8 lattice inclusion complex. Carful analyses of the solid state structure of the above inclusion complex revealed that the asymmetric unit contains one half of the titled molecule and four DMSO molecules; two of the DMSO show small degree of disorder (flipping over of the S atom) (Figure 5). There are a number of supramolecular noncovalent interactions such as H-bonding between the OH of the host molecule and the O and S atoms of the guest molecule. Host-guest interactions are clearly existing in the crystal structure of (8).(DMSO)8 inclusion complex (i.e. hydrogen atom (H2) of hydroxyl group of one host molecule is interacting with an oxygen atom (O3S) of a guest molecule with a contact distance of 1.88 Å). In addition, hydrogen atom (H1) of a hydroxyl group of the same host molecule is interacting with an oxygen atom (O2S) of another guest molecule with a contact distance of 1.86 Å (Figure 6), different types of hydrogen bond angles and lengths that existed in the crystal structure of (8).(DMSO)8 lattice inclusion complex are presented in table 3. Sulfur atom of the guest molecule is interacting with hydrogen atom of host molecule with bond distances of 2.71 and 2.82 Å; no sulfur- sulfur interaction is detected in the crystal packing of (8).(DMSO)8. Bifurcated host- guest interactions in form of O1S…H11 and O1S…H3 are observed with bond distances of 2.60 and 1.85 Å, respectively (Figure 7). Furthermore, crystal packing of

(8).(DMSO)8 lattice inclusion complex showed that two DMSO guest molecules are intermolecularly hydrogen-bonded with host molecule with bond distances of 1.85, 1.88, and 2.61 Å as illustrated in figure 8. Fluorine atom of the host molecule is interacting with hydrogen atom of the guest molecule ((CH3SOCH2-H…F-Ar, 2.47 Å) as shown in figure 9. Other hetero atoms existing in the molecular structure of both host and guest molecules are interacting via different motives such as; Ar-F…F-Ar

(3.48 Å), Ar-F…O=S(CH3)2 (4.15, 4.42, 4.79, 4.76 Å), Ar-F…SO(CH3)2 (3.85 and 3.58 Å), S…S (3.6 Å). These interactions were in good agreement with what we have [37-38,51] observed previously in similar compounds. Crystal packing of 8·(DMSO)8 lattice inclusion complex is illustrated in figure 10.

179

Figure 5: ORTEB diagram of 8·(DMSO)8 lattice inclusion complex with the ellipsoids drawn at the 50% probability level.

Figure 6: Host-guest interactions between one host molecule 8 and four guest molecules including H…O supramolecular interactions.

180

Figure 7: Bifurcated host-guest interactions with O1S…H11 and O1S…H3 bond distances of 2.60 and 1.85, respectively, existing in the crystal packing of (8).(DMSO)8 lattice inclusion complex.

Figure 8: Different guest-guest-host intermolecular motives existing in the crystal structure of (8).(DMSO)8.

181

Figure 9: Host-guest interaction in which fluorine atom of one host molecule 8 is interacting with a hydrogen atom of a guest molecule.

Figure 10: Crystal packing of 8·(DMSO)8 lattice inclusion complex. Hydrogen atoms of host 8 and DMSO were omitted for clarity, non hydrogen atoms were labeled.

Table 3: Hydrogen bond lengths and angles for (8).(DMSO)8 lattice inclusion complex (Å and °). ______D-H...A d(D-H) d(H...A) d(D...A) <(DHA) ______O(1)-H(1)...O(2S)#2 0.83 1.85 2.669(4) 166.7 O(2)-H(2)...O(3S) 0.83 1.88 2.706(4) 172.8 O(3)-H(3)...O(1S)#3 0.83 1.85 2.677(4) 175.8 O(4)-H(4)...O(4S)#4 0.83 1.91 2.714(5) 162.8 ______Symmetry transformations used to generate equivalent atoms: #1 -x+1,-y+1,-z+2 #2 x-1, y, z #3 x, y+1, z+1 #4 x, y, z+1

182 In vitro antibacterial activity The bioactivity of compounds 6-9 are presented in tables 4 and 5. As shown in these tables, Gram negative bacteria were resistant to the applied compounds that showed only weak to moderate activity against the tested Gram positive ones. In the agar diffusion test, the microorganism were susceptible at concentrations starting from 0.5 mg/disc with the fluorinated derivative being more effective than the chloro- and bromo as well as the methoxy-derivatives (Table 4). Despite the concentrations required of the synthesized compounds in the agar diffusion test to affect the growth of tested bacteria, they were effective at MIC-values 2-10 fold lower than that in the agar diffusion assay (Table 5). Moreover, the brominated derivative was more potent than the other with MIC ranging between 15.6-125 μg/mL. The differences in activity in both antimicrobial assays may be attributed to their low solubility in water that renders their diffusion in agar plates. Such observation was noticed previously by Salem et al.[52] and agrees with what stated by Mokhtari and Pourabdollah.[53] They found that hydrophobic calixarenes lack solubility in biological media and thus they are unsuitable for biological evaluation as antimicrobial agents, while para-sulfonato-, phosphonato- or other hydrophilic analogues show bioactivity in different biological systems. Moreover, it was reported that peptide-calixarene exhibited moderate to good activity only against Gram positive bacteria with MIC (4-64 µg/ml),[54-55] while tetra-para-guanidinoethyl-calix[4]arene showed antibacterial activity with MIC (2-64 mg/ml).[56] Therefore, gaining hydro-solubility leads to access the biological activities and this bioactivity depends on the water-solubility and type of substitution group. In addition, the resistance of Gram negative bacteria to the tested compounds could be due to the low permeability of their outer membrane to the applied calixarene derivatives.

Table 4: Inhibition zone (mm) caused by synthesized compounds in agar diffusion test (conc. 0.5/1 mg/disc). Organisms Inhibition Zone (mm) 6 7 8 9 Bacillus subtilis 7/7 7/7 8/8 -/8 Staphylococcus aureus 7/7 7/8 10/11 -/8 Micrococcus luteus 8/8 9/10 9/10 9/10

Table 5: Minimal inhibitory concentration (MIC) of the synthesized compounds in the serial dilution assay.

Organisms MIC (μg/mL) 6 7 8 9 Chl. Bacillus subtilis 62.5 125 500 125 <0.8 Staphylococcus aureus 125 62.5 125 500 <0.8 Micrococcus luteus 31.2 15.6 62.5 62.5 <0.8 Chl.: Chloramphenicol

183 Conclusions A slight change in the molecular structure of any given crystalline compound will lead to a great change in the spectral data, bioactivity, supramolecularity and the conformation of the resulting compound. In the current study, compounds 6-9 were prepared as single isomeric product through an eco-friendly solvent-free approach. In addition, compounds 6 and 8 were predicted, and later confirmed, to form lattice inclusion complexes through direct crystallization from fresh DMSO.

The solid-state structure of the newly prepared 6·(DMSO)8 and 8·(DMSO)8 lattice inclusion complexes were determined by single crystal X-ray diffraction which revealed that their hosts adopted different structural conformation (boat and chair, respectively). The noncovalent supramolecular interactions involved in the crystal structures of these inclusion compounds have been carefully investigated and were presented in terms of crystal engineering and supramolecular chemistry. Compounds 6-9 were found to inhibit the growth of Gram positive bacteria with the C-(p-bromophenyl)calix[4]resorcinarene 7 derivative being the most potent agent (MIC) (15.6-125 μg/ml). The potency of calixarene derivatives as antimicrobial agents depends on their water-solubility and the type of substitution group.

Acknowledgements The authors would like to thank Mutah University, University of Jordan and the National University of Singapore for conducting the experiments needed to complete this work.

References [1] She, N-F.; Gao, M.; Meng, X-G.; Yang, G-F.; Elemans, J. A. A. W.; Wu, A-X.; Isaacs, L., J. Am. Chem. Soc., 2009, 131, 11695-11697. [2] Karami, B.; Khodabakhshi, S.; Saifikhani, N.; Arami, A., Bull. Korean Chem. Soc., 2012, 33, 123-127. [3] Hasenknopf, B.; Lehn, J-M.; Kneisel, B. O.; Baum, G.; Fenske, D., Angew. Chem., Int. Ed., 1996, 35, 1838-1840. [4] Day, A. I.; Blanch, R. J.; Arnold, A. P.; Lorenzo, S., Lewis; G. R., Dance, I., Angew. Chem., Int. Ed., 2002, 41, 275-277. [5] Bravo, J. A.; Raymo, F. M.; Stoddart, J. F.; White, A. J. P.; Williams, D. J., Eur. J. Org. Chem., 1998, 1998, 2565-2571. [6] Anderson, S.; Anderson, H. L.; Bashall, A.; McPartlin, M.; Sanders, J. K. M., Angew. Chem., Int. Ed., 1995, 34, 1096-1099. [7] Freeman, W. A., Acta Crystallogr., Sect. B: Struct. Sci., 1984, 40, 382-387. [8] Schmitt, J-L.; Stadler, A-M.; Kyritsakas, N.; Lehn, J-M., Helv. Chim. Acta, 2003, 86, 1598-1624. [9] Lehn, J.M., Science, 1993, 260, 1762-1763. [10] Lehn, J-M., “Supramolecular Chemistry: Concepts and Perspectives”, 1995. ISBN 978-3-527-29311-7. Wiley-VCH, Weinheim. [11] Oshovsky G. V.; Reinhoudt D. N.; Verboom W., Angew. Chem., Int. Ed., 2007, 46, 2366-2393. [12] Azov, V.A.; Beeby, A.; Cacciarini, M.; Cheetham, A.G.; Diederich, F.; Frei, M.; Gimzewski, J.K.; Gramlich, V.; Hecht, B.; Jaun, B.; Latychevskaia, T.; Lieb, A.; Lill, Y.; Marotti, F.; Schlegel, A.; Schlittler, R. R.; Skinner, P. J.; Seiler, P.; Yamakoshi, Y., Adv. Funct. Mater., 2006, 16, 147-156.

184 [13] Gerkensmeier, T.; Iwanek, W.; Agena, C.; Fröhlich, R.; Kotila, S.; Näther, C.; Mattay, J., Eur J. Org. Chem., 1999, 1999, 2257-2262. [14] Gomez-Benitez, V.; Toscano, R. A.; Morales-Morales, D., J. Inclusion Phenom. Macrocyclic Chem., 2004, 50, 199-202. [15] Tunstad, L. M.; Tucker, J. A.; Dalcanale, E.; Weiser, J.; Bryant, J. A.; Sherman, J. C.; Helgeson, R. C.; Knobler, C. B.; Cram, D. J., J. Org. Chem., 1989, 54, 1305-1312. [16] Roberts, B. A.; Cave, G. W. V.; Raston, C. L.; Scott, J. L., Green Chemistry, 2001, 3, 280-284. [17] Utomo, S. B.; Jumina, Siswanta; D.; Mustofa, Indo. J. Chem., 2012, 12, 49-56. [18] Karami, B.; Hoseini, S. J.; Nikoseresht, S.; Khodabakhashi, S., Chin. Chem. Lett., 2012, 23, 173-176. [19] Utomo, S. B.; Jumina, Siswanta, D.; Mustofa, Kumar, N., Indo. J. Chem., 2011, 11, 1-8. [20] Sardjono, R. E.; Kadarohman, A.; Mardhiyah, Procedia Chemistry, 2012, 4, 224- 231. [21] Funk, M.; Guest, D. P.; Cave, G. W. V., Tetrahedron Lett., 2010, 51, 6399-6402. [22] Alshahateet, S. F.; Bishop, R.; Craig, D. C.; Scudder, M. L., Cryst. Growth Des., 2011, 11, 4474-4483. [23] Alshahateet, S. F.; Rahman, A. N. M. M.; Bishop, R.; Craig, D. C.; Scudder, M. L., CrystEngComm, 2002, 4, 585-590. [24] MacNicol, D. D.; Downing, G. R. Symmetry in the Evolution of Host Design. In “Comprehensive Supramolecular Chemistry, Vol. 6 Solid-state Supramolecular Chemistry: Crystal Engineering”, MacNicol, D., Toda, F., Bishop, R., Eds.; Pergamon Press: Oxford, 1996, Ch. 14, pp. 421-464. [25] Alshahateet, S. F.; Bishop, R.; Craig, D. C.; Scudder, M. L., CrystEngComm, 2001, 3, 225-229. [26] Alshahateet, S. F.; Bishop, R.; Craig, D. C.; Scudder, M. L., Cryst. Growth Des., 2004, 4, 837-844. [27] Ghalib, R. M.; Hashim, R.; Alshahateet, S. F.; Mehdi, S. H.; Sulaiman, O.; Chan, K-L.; Murugaiyah, V.; Jawad, A., J. Chem. Crystallogr., 2012, 42, 783-789. [28] Ghalib, R. M.; Hashim, R.; Alshahateet, S. F.; Mehdi, S. H.; Sulaiman, O.; Murugaiyah, V.; Aruldass, C. A., J. Mol. Struct., 2011, 1005, 152-155. [29] Bishop, R.; Scudder, M. L.; Craig, D. C.; Rahman, A. N. M. M.; Alshahateet, S. F., Mol. Cryst. Liq. Cryst., 2005, 440, 173-186. [30] Alshahateet, S. F.; Bishop, R.; Craig, D. C.; Scudder, M. L., CrystEngComm, 2001, 3, 107-110. [31] Alshahateet, S. F.; Bishop, R.; Craig, D. C.; Scudder, M. L., Cryst. Growth Des., 2010, 10, 1842-1847. [32] Alshahateet, S. F.; Bishop, R.; Scudder, M. L.; Hu, C. Y.; Lau, E. H. E.; Kooli, F.; Judeh, Z. M. A.; Chow, P. S.; Tan, R. B. H., CrystEngComm, 2005, 7, 139-142. [33] Alshahateet, S. F.; Bishop, R.; Craig, D. C.; Scudder, M. L., CrystEngComm, 2001, 3, 265-269. [34] Alshahateet, S. F.; Bishop, R.; Craig, D. C.; Kooli, F.; Scudder, M. L., CrystEngComm, 2008, 10, 297-305. [35] Eloff, J. N., Planta Med., 1998, 64, 711-713. [36] Ahmed, T.; Gilani, A. H., Pharmacol., Biochem. Behav., 2009, 91, 554-559. [37] Alshahateet, S. F., J. Chem Crystallogr., 2010, 40, 191-194. [38] Alshahateet, S. F.; Kooli, F.; Messali, M.; Judeh, Z. M. A.; AlDouhaibi, A. S., Mol. Cryst. Liq. Cryst., 2007, 474, 89-110. [39] SMART and SAINT, Software Reference Manuals, Version 4.0, 1996, Siemens Energy and Automation, Inc., Analytical Instrumentation, Madison, WI, USA [40] Sheldrick, M. G., SADABS Software for empirical absorption correction, 1996, University of Goüttingen, Germany [41] SHELXTL, Reference Manuals Version 5.03, 1996, Siemens Energy and Automation Inc., Analytical Instrumentation, Madison, WI, USA. [42] Alshahateet, S. F.; Al-Zereini, W. A.; Alghezawi, N. M., J. Chem. Crystallogr., 2011, 41, 1807-1811. [43] Weinlet, F.; Schneider, H-J., J. Org. Chem., 1991, 56, 5527-5535. [44] Gutsche, C. D.; Iqbal, M., Org. Synth., 1990, 68, 234-237.

185 [45] Izatt, S. R.; Hawkins, R. T.; Christensen, J. J.; Izatt, R. M., J. Am. Chem. Soc., 1985, 107, 63-66. [46] Bocchi, V.; Foina, D.; Pochini, A.; Ungaro, R., Tetrahedron, 1982, 38, 373-378. [47] Böhmer, V.; Merkel, L.; Kunz, U., J. Chem.Soc., Chem. Commun., 1987, 896- 897. [48] Iwamoto, K.; Araki, K.; Shinkai, S., J. Org. Chem., 1991, 56, 4955-4962. [49] Guelzim, A.; Khrifi, S.; Baert, F.; Loeber, C.; Asfari, Z.; Matt, D.; Vicens, J., Acta Crystallogr., Sect. C: Cryst. Struct. Commun., 1993, 49, 72-75. [50] Hebbink, G. A.; Klink, S. I.; Ouda Alink, P. B.; Veggel, F. C., Inorg. Chim. Acta, 2001, 317, 114-120. [51] Alshahateet, S. F.; Al-Trawneh, S. A.; Al-Zereini, W. A.; ELDouhaibi, A. S., Mol. Cryst. Liq. Cryst., 2014, in press, DOI: 10.1080/15421406.2014.905011. [52] Salem, A.; Regnouf-de-Vains, J-B., Tetrahedron Lett., 2001, 42, 7033-7036. [53] Mokhtari, B.; Pourabdollah, K., J. Chil. Chem. Soc., 2012, 57, 1150-1154. [54] Casnati, A.C.; Fabbi, M.; Pelizzi, N.; Pochini, A.; Sansone, F.; Ungaro, R., Bioorg. Med. Chem. Lett., 1996, 6, 2699-2704. [55] de Fátima, Â.; Fernandes, S.; Sabino, A., Curr. Drug Discov. Technol., 2009, 6, 151-170. [56] Grare, M.; Mourer, M.; Fontanay, S.; Regnouf-de-Vains, J-B.; Finance, C.; Duval, R-E., J. Antimicrob. Chemother., 2007, 60, 575-580.

186