Synthesis , Crystal Structure and Biological Activity of Novel Diester Cyclophanes

Epoxyethane (5 mL) was added dropwise to a stirred mixture of substituted aniline 2 (0.02 mol), H2O (10 mL) and propanoic acid (1 mL) at 0 °C over a period 30 min. The reaction mixture was stirred for 24 h at room temperature. After, 50 mL of dichloromethane was added to the mixture and the organic phase was separated. Then the organic layer was washed with 20 mL of saturated sodium carbonate and 5mL water prior to anhydrous MgSO4. The solvent was removed to leave the crude product, which was purified by chromatography on silica gel (petroleum ether/ethyl acetate = 1:1→1:3).


Introduction
Cram and Steinberg 1 opened the field of cyclophane chemistry by their work in 1951.8][9] In some reaction systems, the host-guest complex can simulate the high efficiency of enzyme catalysis, which means the reaction yield can be greatly improved. 10,11Besides, some ester cyclophanes were also used in the perfume industry because of their aromatic properties.Cyclophanes have so many important applications that it can be predicted a promising future in this field.However, the biological activities of cyclophanes were scarcely reported. 12,13herefore, there is a continuous need to synthetize novel cyclophanes through efficient and simple routes and to study their biological activities.
So far, scientists have synthesized a wide variety of cyclophanes with numerous modes of ring attachments.Medium-sized ring cyclophanes generally have a ring size of 8 to 11 atoms. 14Herein, it is reported the synthesis and biological activity of several diester cyclophanes.In continuation with our interest in cyclophane synthesis, 15 an effective method for the synthesis of cyclophane compounds was designed, and a series of medium-sized cyclophanes 4a-4h was obtained by esterification of 1,2-benzenedicarbonyl chloride with some diols under high dilution conditions.Their antibacterial and antifungal activities are also described.

Results and Discussion
O-Phthalic acid undergoes a reaction with SOCl 2 and catalytic DMF (dimethylformamide) to form the diacid chlorides 1 (Scheme 1). 16Diols 3a-3h were also prepared and used for the synthesis of cyclophanes.4-Methylaniline was treated with oxirane and catalytic propanoic acid by stirring at room temperature for 24 h.Diol 3a was obtained as white solid. 17Diols 3b, 3c, 3d, 3e and 3f were also prepared by similar procedures.Compounds 3g and 3h were obtained using CaCO 3 , KI and 2-chloroethanol according to Lin et al. 18 Different catalysts showed different catalytic effect in the cyclization procedure.The inorganic base K 2 CO 3 showed no catalytic activity.The reaction could be improved by adding pyridine, but the product yield was still low.It was found that a high cyclization yield was achieved in the presence of pyridine and 4-dimethylaminopyridine (DMAP).0][21][22][23][24] Using the new dual catalyst system not only increased the yield of the product, but also improved the reaction rate.The 1 H NMR spectrum of cyclophane 4a displayed the aromatic methyl protons as a singlet at d 2.27, OCH 2 CH 2 protons as two triplet at d 3.80 and 4.55 with J 4.0 Hz, in addition to aromatic protons.Cyclophanes 4b-4h were also accessed by a familiar method from the reaction of diols 3b-3h with diacid chloride 1.In order to avoid polymerization, the cyclization procedure was run under high dilution conditions and the reaction solution had to be vigorously stirred.
The carbonyl absorption of cyclophanes in the IR was in 1700-1720 cm -1 region, at lower frequency than that of normal esters due to the formation of a large p bonding.This peak was the strongest peak compared with all other peaks in the IR spectrum.

Crystal and molecular structures
Crystals of compounds 4g and 4h were obtained from a mixture of petroleum ether and ethyl acetate (5:1).Crystal data collection and refinement parameters for the two compounds are shown in Table 1.The structures of compounds are presented in Figures 1a and 2a.The crystal structure X-ray analysis of 4g showed that there are six intermolecular CH … OH-bonding interactions (Figure 1c): The crystal structure X-ray analysis of 4h revealed cyclophanes connected by four intermolecular CH … O hydrogen bonding (Figure 2c):  1b and 2b).

Biological activity
The synthesized novel compounds 4a-4h were screened for their in vitro antibacterial activity against Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Salmonella typhi (ATCC 14028).The new compounds 4a-4h were also evaluated for their antifungal activity against Candida albicans (ATCC 10231).The results of preliminary antibacterial activity of the compounds are shown in Table 2.A control test was performed with sterile DMSO.This revealed no inhibiting activity.The compounds 4a, 4b, 4c and 4h were found to exhibit higher antifungal activity against C. albicans among the new compounds.
The minimum inhibitory concentration (MIC) for the active compounds is presented in Table 3. Chloramphenicol and fluconazole were chosen as standard drugs whose MIC values are also provided in Table 3.The antibacterial activity (MIC of 128 μg mL -1 ) of compound 4g against E. coli showed relatively better results than the other new compounds.This is Scheme 1. Synthesis of medium-sized cyclophanes 4a-4h.

Materials and equipment
All reagents and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd.The solvents were purified under conventional conditions or distilled immediately before use.The melting points were determined using an XT-4B micro-melting-point apparatus and are informed uncorrected.The elemental analyses for the compounds were carried out on a Vario EL III CHNOS instrument.The IR spectra (KBr) were recorded on an Equinox-55 spectrophotometer. 1 H NMR spectra were recorded on an INOVA-400 NMR spectrometer, using TMS as internal standard and CDCl 3 as the solvent.High resolution mass spectrometry (HRMS) were obtained with a MALDI-TOF mass spectrometer (Kratos Analytical Inc.).Column chromatography was performed on silica gel (200-300 mesh).

Antibacterial and antifungal assays
The bacteria and fungi were obtained from Shaanxi Province Institute of Microbiology (Xi'an, China) and maintained on Luria-Bertani (LB) medium consisting of the following: yeast extract 5.0 g, peptone 10.0 g, NaCl 10.0 g, distilled H 2 O 1 L, pH 7.5 in slants or Petri plates at room temperature.
Preliminary antimicrobial activities of compounds 4a-4h were tested as follows: the new compound was dissolved in DMSO at the concentration of 200 μg mL -1 and placed on the LB plates that had been previously inoculated separately with the microorganisms.The plates were incubated at 35 °C, and the growth-inhibition zones were measured after 12 h in the case of bacteria/fungi.
The MIC values of the compound assays were carried out using the broth microdilution method.The tested compounds were dissolved in DMSO to afford 128 μg mL -1 .Two fold    dilutions of the solution were prepared (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg mL -1 ).The bacterial cultures (ca. 10 8 CFU mL -1 ) in Müller-Hinton medium were added to the corresponding tubes.The plates were incubated at 35 °C for 12 h.Chloramphenicol and fluconazole were used as reference agents.Suitable solvent control (DMSO), positive growth control and standard drug control were also run simultaneously.The MIC values of the compounds were recorded as the lowest concentration of each chemical compound in the tubes with no turbidity of inoculated bacteria/fungus.
Crystal structure X-ray data of 4g and 4h All measurements of compounds 4g and 4h were made on a Bruker Smart APEX II CCD diffractometer with graphite monochromated Mo-K α radiation.The data was collected at a temperature of 296 K using the ω-2θ scan technique.Crystallographic data for the structure analysis of compounds 4g and 4h was deposited at the Cambridge Crystallographic Data Centre, CCDC No. 846714 and 846711, respectively. 25

Conclusions
Eight novel compounds (4a-4h) were synthesized.XRD studies were carried out for compounds 4g and 4h, which were further stabilized by H-bond stacking interaction.These new compounds 4a-4h represent a novel class of compounds that incorporate an 11-membered ring lactone type structure.Some of the synthesized cyclophanes have low antibacterial or antifungal activities, providing stimulus for further studies.

2 DataFigure 1 .
Figure 1.(a) Crystal structure of compound 4g, (b) packing diagrams of 4g and (c) the arrangement of 4g showing the hydrogen bonding interactions.

Figure 2 .
Figure 2. (a) Crystal structure X-ray analysis of 4h, (b) packing diagrams of 4h and (c) the arrangement of 4h showing the hydrogen bonding interactions.

Table 1
Crystallographic data and structure refinement parameters for the compounds 4g and 4h