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

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

LUO Zai-Gang②ZHAO Yu MA Chao CAO Lu AI Shao-Hua HU Jin-Song XU Xue-Mei②

(232001)

The title compound (C50H44O10) was synthesized and structurally determined by single-crystal X-ray diffraction method. It crystallizes in monoclinic, space group21/with= 16.713(4),= 13.189(3),= 19.434(5) ?,= 104.411(4)°,M= 804.85,D= 1.288 g/cm3,= 4149.2(17) ?3,= 4,(000) = 1696,(Mo) = 0.089 mm-1,= 296(2) K, 7279 independent reflections with 3172 observed ones (> 2()),= 0.0520 and= 0.1203 with= 0.928 (= 0.1464 and= 0.1657 for all data).The calixarene moiety maintains the symmetric cone conformation through intramolecular O–H···O hydrogen bonds.Preliminary bioassays indicated that thetitle compound has a potent inhibitory activityagainst the strand transfer process of HIV-1 integrase.

calix[4]arene derivative, 1,3-diketo, HIV-1 integrase, crystal structure

1 INTRODUCTION

Calixarenes, synthetically available macrocyclic compounds obtained by precise cyclo-condensation of-substituted phenols and formaldehyde, possess intramolecular lipophilic cavities formed by aroma- tic 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 che- mical 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-mo- lecule 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 inhi- bitors 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-diketosubunitdisposed 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 0.09g of NaH (60% dispersion in oil) (2.1 mmol) in 5 mL of dried THF was slowly added 0.30 gof 4-me- thoxyacetophenone 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]arene1 (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,ppm) 3.52 (d, 4H,= 13.2 Hz, ArAr), 3.89 (s, 6H, ArOCH), 4.36 (d, 4H,= 13.2 Hz, ArAr), 4.56 (s, 4H, ArOCH), 6.75～6.81 (m, 4H, Ar), 6.87 (d, 4H,= 8.4 Hz, Ar), 6.94 (d, 4H,= 7.6 Hz, Ar), 7.15 (d, 4H,= 7.6 Hz, Ar), 7.49 (s, 2H, CO), 7.75 (s, 2H, Ar), 7.88 (d, 4H,= 8.4 Hz, Ar), 15.45 (s, 2H,).13C NMR (CDCl3, 100 MHz,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 fromacetonitrile to give colorless single crystals of3suitable 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 graphite- monochromated Moradiation (= 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 proce- dure based on2values[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 (int= 0.0674) were collected in the range of 1.89≤≤25.00o (: ?19～19,: ?15～15,: ?23～22) by using a-scan mode. The final= 0.0520,= 0.1203 (= 1/[2(F2) + (0.0784)2+ 0.0000], where= (F2+ 2F2)/3),= 0.928, (Δ)max= 0.186, (Δ)min= ?0.182 e/?3and ()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 on2[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 deri- vative 3was measured by HIV-1 integrase strand transfer activity assay, which was carried out as described previously with some minor modifica- tions[20].Compounds diluted in DMSO were pre- incubated 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 3was 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 mole- cular structure of the macrocycle (Fig. 1).

In general, the calixarene moiety maintains the symmetric cone conformation through intramole- cular 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 interac- tions result in that the dihedral angle of unsubstitu- ted 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,×××interactions between the two benzene rings of 1,3-diketosubunits 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 stagge- red parallelly and the cavities formed by the anti-parallel 1,3-diketo armsat the lower rim of the carlixarenes were clearly seen along theaxis. When viewed the packing diagram of the title compound from the parallel side of thedirection (Fig. 3), the two adjacentmolecules are essentially vertical to each other, where one is along theaxis alignment while the other is nearly along theaxis alignment, but there are no clear interactions between them.

3.2 Inhibition of HIV integraseactivity

Compound 3and 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

aHIV-1 IN inhibitory activities were measured according to the procedure described in Ref. 20

Fig. 2. View of the packing diagram of the title complex along theaxis

Fig. 3. View of the packing diagram of the title complexfrom the parallel side of theaxis

As shown in Table 3, compound 3 is proved to be active in the strand transfer assay at the concentra- tion of 50 μM (inhibitory ratio 79.61%), while it presented anti-integrase activity with a low inhibi- tory activity at the concentration of 25 μM (inhibi- tory 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 carboxyla- tes, sulfonates, or phosphonates, seems essential to enhance the potential anti-integrase activity[9].

4 CONCLUSION

We have synthesized a novel calix[4]arene deriva- tive with 1,3-diketosubunitsdisposed in the alter- nate position at the lower rim and structurally characterized by single-crystal X-ray diffraction method. The calixarene moiety maintains the sym- metric 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 inhibi- tory activities for compound 3. And further work based on this structure is in progress.

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8 January 2014;

9 April 2014 (CCDC976689)

① This work was supported by the National Natural Science Foundation of China (No. 21102003),National Students' Innovation and Entrepreneurship Training Program (No. 201210361094), Scientific Research Foundation for the Introduction of Talent and Young Teachers Scientific Research Foundation of Anhui University of Science & Technology (11214, 2012QNY27)

. Luo Zai-Gang, majoring in drug design and synthesis. E-mail: luozi139@163.com Xu Xue-Mei, majoring in drug design and synthesis. E-mail: littlekitty@126.com