Synthesis, Crystal Structure and Anti-integrase Activity of 25,27-Bis[(Z)-4-(p-methoxyphenyl)-4-hydroxybut-3-en-2-one-1-methyl]-26,28-dihydroxycalix[4]arene①

LUO Zai-Gang ZHAO Yu MA Chao CAO Lu AI Shao-Hua HU Jin-Song XU Xue-Mei

(College of Chemical Engineering, Anhui University of Science & Technology, Huainan, Anhui 232001, China)

1 INTRODUCTION

Calixarenes, synthetically available macrocyclic compounds obtained by precise cyclo-condensation of p-substituted phenols and formaldehyde, possess intramolecular lipophilic cavities formed by aromatic rings of the macrocyclic skeleton[1]. In the past three decades, calixarenes have been the targets of basic and applied sciences[2]. Numerous efforts have been directed toward such molecules as new platforms for new drug design in recent years[3-5].Many pharmacological properties are described for calixarenes (antiviral, antibacterial, anticancer and so on)[6-8]. Especially, using calixarene as new chemical entities of distinct anti-HIV activities has been an increasingly active and newly rising highlight field[9,10].

Over the last two decades, numerous small-molecule HIV-1 integrase (IN) inhibitors have been described. The most predominant class of inhibitors bear the diketo acid moiety[11,12]which was believed to be the most crucial pharmacophore for the inhibition of IN inhibitors[13]. In the present study, we have used calix[4]arene skeleton as a platform to design a new class of integrase inhibitors in order to gain new molecular entity with potential biological activities. Herein, we report the synthesis, crystal structure and anti-integrase activity of a novel calix[4]arene derivative with 1,3-diketo subunit disposed in alternate position at the lower rim (Scheme 1).

Scheme 1. Synthetic procedure of the title compound 3

2 EXPERIMENTAL

2. 1 Instruments and reagents

The melting point was measured on a SGW X-4 monocular microscope melting point apparatus with thermometer unadjusted.1H NMR and13C NMR spectra were acquired on a Bruker Avance III 400 MHz spectrometer with CDCl3as solvent. Mass spectrum was obtained on an Esquire 6000 mass spectrometer. X-ray diffraction was performed using a Bruker Smart Apex CCD diffractometer.

Unless otherwise noted, all materials were obtained from commercial suppliers and purified by standard procedures. Column chromatography was performed with silica gel (200～300 mesh, Qingdao Haiyang Chemical Co., Ltd, China).

2. 2 Synthesis of the title compound 3

Compound 1 was synthesized according to the corresponding literature[14]. The title compound was synthesized according to our previous work[15], as shown in Scheme 1. To a suspension of 0.09 g of NaH (60% dispersion in oil) (2.1 mmol) in 5 mL of dried THF was slowly added 0.30 g of 4-methoxyacetophenone 2 (2 mmol) in 5 mL of dried THF at 0 ℃, and the mixture was stirred for 10 min.After that, 0.60 g of calix[4]arene 1 (1 mmol) in dried THF (5 mL) was added to the above solution at 0 ℃ and then the reaction mixture was slowly heated to reflux for about 50 min with stirring till TLC confirmed the reaction had finished. The cooled mixture was poured into a mixture of ice-water (20 mL) and concentrated HCl (5 mL),and extracted with EtOAc. The organic layer was dried over MgSO4and evaporated to give the crude product, and product 3 was obtained by column chromatography on silica gel eluting with petroleum ether/ethyl acetate (10:1), obtaining 0.34 g of fine product as white powder. Yield: 42%. m.p.: 193～196 ℃.1H NMR (CDCl3, 400 MHz, δ ppm) 3.52 (d,4H, J = 13.2 Hz, Ar CH2Ar), 3.89 (s, 6H, ArO CH3),4.36 (d, 4H, J = 13.2 Hz, Ar CH2Ar), 4.56 (s, 4H,ArO CH2), 6.75～6.81 (m, 4H, Ar H), 6.87 (d, 4H, J= 8.4 Hz, Ar H), 6.94 (d, 4H, J = 7.6 Hz, Ar H), 7.15(d, 4H, J = 7.6 Hz, Ar H), 7.49 (s, 2H, CO CH), 7.75(s, 2H, Ar OH), 7.88 (d, 4H, J = 8.4 Hz, Ar H), 15.45(s, 2H, OH).13C NMR (CDCl3, 100 MHz, δ ppm)189.4, 184.3, 163.5, 153.0, 151.3, 132.7, 129.8,129.5, 128.9, 127.9, 126.7, 126.2, 119.8, 113.6, 93.3,55.6, 31.4; ESI-MS (m/z): 803.3 [M–H]–. The solid of compound 3 was re-crystallized from acetonitrile to give colorless single crystals of 3 suitable for single-crystal X-ray diffraction. The crystal of the title compound is colorless and stable in air at room temperature.

2. 3 Structure determination

A colorless block crystal 3 with dimensions of 0.25mm × 0.22mm × 0.21mm was selected and mounted on a glass fiber for measurement. X-ray crystallographic data were collected at 296 K. All measurements were made on a Bruker Smart Apex CCD diffractometer equipped with a graphitemonochromated Mo Ka radiation (λ = 0.71073 ?).The structure was solved by direct methods, and the non-hydrogen atoms were located from the trial structure and then refined anisotropically with SHELXTL using a full-matrix least-squares procedure based on F2values[16]. The hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on the parent atoms. A total of 7279 reflections with 3172 unique ones (Rint=0.0674) were collected in the range of 1.89≤θ≤25.00o (h: -19～19, k: -15～15, l: -23～22) by using a ψ-ω scan mode. The final R = 0.0520, wR =0.1203 (w = 1/[σ2(Fo2) + (0.0784P)2+ 0.0000P],where P = (Fo2+ 2Fc2)/3), GOF = 0.928, (Δρ)max=0.186, (Δρ)min= -0.182 e/?3and (?/σ)max= 0.000.Data collection was controlled by Rapid Auto program. The hydrogen atoms bound to carbon were calculated theoretically. And the non-hydrogen atoms were located from the trial structure and then refined anisotropically with SHELXL-97 using a full-matrix least-squares procedure on F2[17-19]. The selected bond lengths and bond angles are listed in Table 1.

Table 1. Selected Bond Lengths (?) and Bond Angles (°)

2. 4 HIV-1 integrase inhibitory assay

The inhibitory activity of the calix[4]arene derivative 3 was measured by HIV-1 integrase strand transfer activity assay, which was carried out as described previously with some minor modifications[20]. Compounds diluted in DMSO were preincubated with 800 ng of integrase at 37.8 ℃ in the reaction buffer in the absence of Mn2+for 10 min.Subsequently, 1.5 pmol of donor DNA and 9 pmol of target DNA were added and the reaction was initiated by the addition of 10 mmol/L Mn2+into the final reaction volume. The reactions were carried out at 37.8 ℃ for 1 h and subsequent detection procedure was applied to detect the assay signals.Baicalein was used as the control compound(positive control), whereas no compound but only DMSO in the reaction mixture was set as the drug-free control (negative control). The inhibitory activity of compounds 3 was calculated based on the positive and negative controls.

3 RESULTS AND DISCUSSION

3. 1 X-ray crystallographic analysis

We report herein mainly the X-ray diffraction study of compound 3 and demonstrated the molecular structure of the macrocycle (Fig. 1).

In general, the calixarene moiety maintains the symmetric cone conformation through intramolecular hydrogen bonds (Table 2)[21], which exist between the unsubstituted phenolic hydroxyl groups and the oxygen atoms coupled to two 1,3-diketo arms. The intramolecular hydrogen bonds of O(1)–H(1)··O(3) and O(2)–H(2)··O(4) are within the expected range for such interactions, 1.8～2.6 ?[21],and the bond angles of them (167° and 163°,respectively) are close to 180°. Such strong interactions result in that the dihedral angle of unsubstituted benzene rings of the calixarene moiety is much wider than that of benzene rings with two 1,3-diketo arms (76.5° and 27.1°, respectively). Obviously,another two intramolecular hydrogen bonds of the two 1,3-diketo moieties, which possess enol-keto tautomerism in the solvent[22], were also presented(Table 2). And the two 1,3-diketo arms are almost anti-parallel and form a large cavity at the lower rim of the macrocyclic skeleton (Fig. 1). However, π··π interactions between the two benzene rings of 1,3-diketo subunits disposed in alternate positions at the lower rim were not observed.

Table 2. Hydrogen Bonds for the Title Compound (?, °)

Fig. 1. Coordination environment of compound 3.The selected atoms are omitted for clarity.

Moreover, the crystal packing of 3 illustrated in Fig. 2 reveals that the overall packing is staggered parallelly and the cavities formed by the antiparallel 1,3-diketo arms at the lower rim of the carlixarenes were clearly seen along the c axis.When viewed the packing diagram of the title compound from the parallel side of the c direction(Fig. 3), the two adjacent molecules are essentially vertical to each other, where one is along the b axis alignment while the other is nearly along the a axis alignment, but there are no clear interactions between them.

3. 2 Inhibition of HIV integrase activity

Compound 3 and positive control baicalein were tested against purified integrase and the data are summarized in Table 3.

Table 3. Inhibition of HIV-1 Integrase Strand Transfer Catalytic Activitiesa

Fig. 2. View of the packing diagram of the title complex along the c axis

Fig. 3. View of the packing diagram of the title complex from the parallel side of the c axis

As shown in Table 3, compound 3 is proved to be active in the strand transfer assay at the concentration of 50 μM (inhibitory ratio 79.61%), while it presented anti-integrase activity with a low inhibitory activity at the concentration of 25 μM (inhibitory ratio 29.12%). This experimental observation seems to indicate that the inhibitory activity of 3 might be attributed to the dissolubility, which means an appropriate water-soluble anionic group incorporated at the upper rim, including carboxylates, sulfonates, or phosphonates, seems essential to enhance the potential anti-integrase activity[9].

4 CONCLUSION

We have synthesized a novel calix[4]arene derivative with 1,3-diketo subunits disposed in the alternate position at the lower rim and structurally characterized by single-crystal X-ray diffraction method. The calixarene moiety maintains the symmetric cone conformation through intramolecular hydrogen bonds. The two 1,3-diketo arms are almost anti-parallel and form a large cavity at the lower rim of calixarene. Also, the overall crystal packing of 3 is staggered parallelly. The HIV-1 integrase strand transfer activity assay results showed potent inhibitory activities for compound 3. And further work based on this structure is in progress.

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