b College of Chemistry and Life Sciences, China Three Gorges University, Yichang 443002, China;
c Chongqing Center for Biomedicines and Medical Equipment, Chongqing Academy of Science and Technology, Chongqing 401123, China
HIV-1 integrase (IN),belongs to the superfamily of polynucleotidyl transferases,which insert a double-stranded DNA copy of the viral RNA genome into the chromosomes of an infected cell through a multistep process that involves 30-processing (30-P) and strand transfer (ST) or integration . Considering that HIV-IN plays a key role in stable infection and that there is no known human counterpart of HIV-IN,it is understandable why this enzyme is an attractive therapeutic target . Numerous smallmolecule HIV-1 integrase inhibitors have been described,with the most predominant class of inhibitors being the diketo acids (DKA) . The structures of the DKA moiety can be replaced by various bioisosteres [3, 4, 5]. The structure of the HIV-1 integrase core domain complexed with the inhibitor 5-CITEP (Fig. 1) has been described as a platform for the structure-based design of novel HIV-1 integrase inhibitors . It is believed that their IN-binding mechanism is connected to the presence of the DKA pharmacophoric motif,which could be involved in functional sequestration of one or both divalent metal ions in the enzyme catalytic site to form a ligand-M2+-IN complex. This would subsequently block the transition state of the IN-DNA complex by competing with the target DNA substrate,acting as an "interfacial inhibitor" . The global research efforts to identify drugs that inhibit HIV integrase recently led to new strand-transfer inhibitors MK-0518 and GS9137 (Fig. 1),which have been approved by the FDA.
Calixarenes,which constitute a major class of supramolecular organic host compounds,have been paid special attention as new molecular platforms for the design and development of new drugs [7, 8, 9]. During the last 10 years,calixarene derivatives have been the subject of growing interest in the biology. Many pharmacological properties are described for calixarenes (antiviral,antibacterial, antifungal,and anticancer activities) [10, 11, 12]. In particular, calixarenes have been increasingly studied for their anti-HIV activity [13, 14]. The 1,2,3-triazole ring is not only a hydrogen-bond donor but also as a linking unit in the structure scaffold. Its planar structure may facilitate the p stacking interaction with target enzymes similar to phenyl rings. 1,2,3-Triazole moiety can be synthesized by application of a synthetic strategy using organoazide and acetyl acetone . Further,many known 1,2,3-triazoles have anti-HIV properties [16, 17].
In recent years,we also engaged in the development of new anti-IN inhibitors [18, 19, 20]. To synthesize novel molecules with potential biological activities,we used the calixarene skeleton as a platform to design a new class of integrase inhibitors in the present study. Therefore,we detail the synthesis of a series of new calixarene derivatives 4a-h (Scheme 1) incorporating two triazolyl 1,3-diketo subunits in alternate positions at the lower rim, and evaluation of their anti-IN activities against HIV-1 with baicalein as a reference compound.
|Scheme 1.Synthetic route for calixarene derivatives 4a-h. Reagents and conditions: (i) BrCH2COOEt,K2CO3,DMF,70-90 ℃,12-24 h (86% for 2a and 78% for 2b); (ii) NaH, THF,reflux,1.5-2 h (33%-52%).|
Unless otherwise noted,all materials were obtained from commercial suppliers and dried and purified by standard procedures. The melting point was measured on an SGW X-4 monocular microscope melting point apparatus with an unadjusted thermometer. 1H NMR and 13C NMR spectra were acquired on a Bruker Avance-400 MHz spectrometer with CDCl3 as the solvent and tetramethylsilane (TMS) as the internal standard. The chemical shifts were reported in d (ppm). Mass spectra (MS) data were obtained using an Esquire 6000 Mass Spectrometer. Column chromatography was performed with silica gel (200-300 mesh, Qingdao Haiyang Chemical Co.,Ltd.,China).
The synthesis of our target calixarene derivatives is outlined in Scheme 1. Compounds 1 ,2 ,and 3  were prepared according to the corresponding literature. Compound 2 underwent Claisen condensation with variable 4-acetyl-5-methyl-1,2,3-triazoles 3 to afford the novel calixarene derivatives 4a-h incorporating two triazolyl 1,3-diketo subunits in alternate positions at the lower rim in the alkaline medium (NaH) with a moderate yield (33%-52%).
General procedure for the synthesis of calixarene derivatives 4a-h: To a suspension of sodium hydride (60% dispersion in oil), 30 mmol in dry THF (10 mL) was slowly added to variable 1,2,3- triazoles 3 (22 mmol) in dry THF (10 mL) at 0 8C,and the mixture was stirred for 10 min. After that,calixarenes 2 (10 mmol) in dry THF (15 mL) was added to the above solution at 0 8C,and then the reaction mixture was slowly heated to reflux for about 90 min with stirring until TLC confirmed that the reaction had finished. The cooled mixture was poured into a mixture of ice-water (20 mL) and concentrated HCl (5 mL),extracted with EtOAc,and purified by flash chromatography on silica gel eluted with petroleum ether/ ethyl acetate (15:1-8:1). 3. Results and discussion
All of the final products were new compounds and their structures were fully confirmed by MP,1H NMR,13C NMR,and mass spectra (ESI-MS). The data of the NMR spectra of the title compounds revealed that the calixarene moiety maintains the cone conformation. The bridging methylene groups of the compounds 4a-h exhibit two sets of doublets (absorptions of the doublets near δ 3.4 and 4.5 ppm,J = 13.2 Hz for ArCH2Ar protons) in their 1H NMR spectra and absorptions near 31 ppm in their 13C NMR spectra,respectively. The chemical shift values are close to those reported for other calixarene molecules in the same conformations [24, 25]. Moreover,the 1,3-diketo moieties of the title compounds possess enol-keto tautomerism in the solvent [23, 26],and the conformation of the 1,3-diketo moieties of the title compounds exist almost completely with an enol configuration in CDCl3 ,which was readily proven by their 1H NMR absorptions near 7 or 8 ppm for the methylene proton of the 1,3-diketo moiety and 13C NMR absorptions for the methylene carbon of the 1,3- diketo moiety near 97 ppm,respectively. In the negative ion MS (ESI) spectrum,the loss of hydrogen ion substances [M-H]- were usually observed as the base peak ion for all target compounds. Selected characterization data of the targeted compounds are listed below.
4a: White solid,yield: 41%; mp: 155-158 8C; 1H NMR (400 MHz,CDCl3): δ 0.96 (s,18H,C(CH3)3),1.33 (s,18H, C(CH3)3),2.44 (s,6H,CH3),3.42 (d,4H,J = 13.2 Hz,ArCH2Ar), 4.45 (d,4H,J = 13.2 Hz,ArCH2Ar),4.75 (s,4H,ArOCH2),5.56 (s,4H, ArCH2),6.85 (s,2H,COCH),6.90 (s,4H,ArH),7.12 (s,4H,ArH),7.18- 7.21 (m,4H,ArH),7.30-7.36 (m,6H,ArH),7.78 (s,2H,ArOH),15.31 (s,2H,OH); 13C NMR (100 MHz,CDCl3): d 187.7,181.5,150.8, 149.8,147.3,141.3,141.1,136.4,134.3,132.5,129.0,128.3,127.4, 127.1,125.7,125.0,96.7,75.7,51.6,33.9,33.8,31.8,31.6,30.9, 9.36; MS(ESI): m/z 1157.8 [M-H]-.
4b: White solid,yield: 52%; mp: 182-185 8C; 1H NMR (400 MHz,CDCl3): d 0.93 (s,18H,C(CH3)3),1.31 (s,18H, C(CH3)3),2.45 (s,6H,CH3),3.49 (d,4H,J = 13.2 Hz,ArCH2Ar), 4.50 (d,4H,J = 13.2 Hz,ArCH2Ar),4.75 (s,4H,ArOCH2),5.52 (s,4H, ArCH2),6.68-6.73 (m,4H,ArH),6.83 (s,2H,COCH),7.01 (d,4H, J = 7.6 Hz,ArH),7.12-7.17 (m,8H,ArH),7.36 (d,4H,J = 8.4 Hz,ArH), 7.80 (s,2H,ArOH),15.30 (s,2H,OH); 13C NMR (100 MHz,CDCl3): d 187.6,181.6,150.7,151.3,151.0,147.1,136.4,134.5,133.2,132.7, 129.3,128.6,127.6,125.8,119.0,118.7,97.0,75.7,51.0,34.0,33.8, 31.8,31.5,30.9,9.30; MS (ESI): m/z 1225.6 [M-H]-.
4c: White solid,yield: 46%; mp: 189-192 8C; 1H NMR (400 MHz,CDCl3): d 0.95 (s,18H,C(CH3)3),1.32 (s,18H, C(CH3)3),2.44 (s,6H,CH3),3.43 (d,4H,J = 13.2 Hz,ArCH2Ar), 4.42 (d,4H,J = 13.2 Hz,ArCH2Ar),4.75 (s,4H,ArOCH2),5.52 (s,4H, ArCH2),6.85 (s,2H,COCH),6.90 (s,4H,ArH),7.10-7.16 (m,8H, ArH),7.30-7.37 (m,4H,ArH),7.85 (s,2H,ArOH),15.33 (s,2H,OH); 13C NMR (100 MHz,CDCl3): d 187.9,181.3,150.8,149.8,147.2, 141.4,141.0,136.3,134.4,132.7,132.4,129.2,128.5,127.3,125.7, 125.0,96.8,75.6,50.9,34.0,33.8,31.8,31.6,30.9,9.37; MS (ESI): m/z 1193.6 [M-H]-.
4d: White solid,yield: 43%; mp: 145-147 8C; 1H NMR (400 MHz,CDCl3): d 0.96 (s,18H,C(CH3)3),1.31 (s,18H, C(CH3)3),2.33 (s,6H,CH3),2.43 (s,6H,CH3),3.42 (d,4H, J = 13.2 Hz,ArCH2Ar),4.44 (d,4H,J = 13.2 Hz,ArCH2Ar),4.76 (s, 4H,ArOCH2),5.52 (s,4H,ArCH2),6.86 (s,2H,COCH),6.92 (s,4H, ArH),7.11-7.17 (m,7H,ArH),7.29-7.37 (m,4H,ArH),7.85 (s,2H, ArOH),15.33 (s,2H,OH); 13C NMR (100 MHz,CDCl3): d 187.6, 181.1,150.8,149.7,147.2,141.2,141.0,136.2,134.3,132.7,132.3, 129.1,128.2,127.3,125.5,125.0,97.0,75.2,50.9,34.1,33.7,31.5, 31.4,30.9,21.1,9.35; MS (ESI): m/z 1185.3 [M-H]-.
4e: White solid,yield: 38%; mp: 177-180 8C; 1H NMR (400 MHz,CDCl3): d 2.43 (s,6H,CH3),3.45 (d,4H,J = 13.2 Hz, ArCH2Ar),4.50 (d,4H,J = 13.2 Hz,ArCH2Ar),4.76 (s,4H,ArOCH2), 5.52 (s,4H,ArCH2),6.70 (t,4H,J = 7.2 Hz,ArH),7.00 (d,4H, J = 7.6 Hz,ArH),7.12 (d,4H,J = 7.6 Hz,ArH),7.19 (d,4H,J = 7.6 Hz, ArH),7.34-7.40 (m,6H,ArH),8.02 (s,2H,COCH),8.10 (s,2H,ArOH), 15.20 (s,2H,OH); 13C NMR (100 Hz,CDCl3): d 187.4,181.8,153.8, 152.1,141.5,136.6,134.7,133.6,129.6,129.0,128.5,128.3,127.7, 127.3,126.0,119.0,96.9,75.6,51.6,31.6,9.31; MS (ESI): m/z 933.4 [M-H]-.
4f: White solid,yield: 36%; mp: 147-149 8C; 1HNMR (400 MHz, CDCl3): d 2.44 (s,6H,CH3),3.49 (d,4H,J = 13.2 Hz,ArCH2Ar),4.50 (d,4H,J = 13.2 Hz,ArCH2Ar),4.75 (s,4H,ArOCH2),5.52 (s,4H, ArCH2),6.68-6.73 (m,4H,ArH),7.01 (d,4H,J = 7.6 Hz,ArH),7.12- 7.17 (m,8H,ArH),7.36 (d,4H,J = 8.4 Hz,ArH),8.11 (s,2H,COCH), 8.15 (s,2H,ArOH),15.19 (s,2H,OH); 13C NMR (100 MHz,CDCl3): d 187.6,181.6,153.7,153.3,151.9,141.1,136.4,134.5,133.2,132.7, 129.3,128.6,127.6,125.8,119.0,118.7,97.0,75.7,51.0,31.5,9.30; MS (ESI): m/z 1001.3 [M-H]-.
4g:White solid,yield: 35%; mp: 170-172 8C; 1H NMR (400 MHz, CDCl3): d 2.43 (s,6H,CH3),3.45 (d,4H,J = 13.2 Hz,ArCH2Ar),4.54 (d, 4H,J = 13.2 Hz,ArCH2Ar),4.77 (s,4H,ArOCH2),5.50 (s,4H,ArCH2), 6.91 (s,4H,ArH),7.10-7.16 (m,8H,ArH),7.30-7.37 (m,4H,ArH), 8.09 (s,2H,COCH),8.13 (s,2H,ArOH),15.15 (s,2H,OH); 13C NMR (100 MHz,CDCl3): d 187.9,181.3,152.8,149.8,141.2,136.3,134.4, 132.5,132.4,129.2,128.5,127.3,125.7,125.0,119.3,119.0,96.8, 75.6,50.9,31.6,9.31; MS (ESI): m/z 970.1 [M-H]-.
4h: White solid,yield: 33%; mp: 149-151 8C; 1H NMR (400 MHz,CDCl3): d 2.32 (s,6H,CH3),2.39 (s,6H,CH3),3.46 (d, 4H,J = 13.2 Hz,ArCH2Ar),4.48 (d,4H,J = 13.2 Hz,ArCH2Ar),4.72 (s, 4H,ArOCH2),5.48 (s,4H,ArCH2),6.65 (t,4H,J = 7.6 Hz,ArH),6.95 (d,4H,J = 7.6 Hz,ArH),7.05-7.09 (m,8H,ArH),7.13 (d,4H, J = 7.6 Hz,ArH),7.97 (s,2H,COCH),8.06 (s,2H,ArOH),15.16 (s,2H, OH); 13C NMR (100 MHz,CDCl3): d 187.2,181.9,153.7,152.1, 141.1,138.2,136.4,133.2,131.3,129.7,129.3,128.5,127.7,127.2, 125.7,118.7,96.9,75.7,51.5,31.5,21.1,9.36; MS (ESI): m/z 961.4 [M-H]-.
The inhibition effects of the calixarene derivative 4a-h were measured by the HIV-1 integrase strand transfer activity assay, which was carried out as described previously  with some minor modifications. Compounds diluted in DMSO were preincubated with 800 ng integrase at 37.8 8C in the reaction buffer in the absence ofMn2+ for 10 min. Subsequently,1.5 pmol donor DNA and 9 pmol 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 8C for 1 h,and a subsequent detection procedure was applied to detect the assay signals. An integrase inhibitor,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 inhibition effects of compounds 4a-h were calculated based on the positive and negative controls,and the data are summarized in Table 1.
As shown in Table 1,p-tert-butylcalixarene derivatives 4a-d proved to be inactive in the ST assay at the concentration of 25 mmol/L. The calixarene derivatives 4e-h presented anti-IN activity in the low micromolar range (6.1-10.9 mmol/L). The most potent derivative was compound 4g,which had an IC50 value for strand transfer of 6.1 mmol/L. Moreover,the anti-IN activity of the compounds 4e-h with electron withdrawing groups or electron donating groups on the benzene rings of the 1,3-diketo subunits in alternate positions at the lower rim shows no clear difference. This experimental observation seems to indicate that the bulky tertbutyl groups reduced combination ability with integrase comparable to H atoms at the upper rim of calixarene. In addition,an appropriate water-soluble anionic group incorporated at the upper rim,including sulfonates,carboxylates,or phosphonates,seems essential to enhance potential anti-integrase activity . 4. Conclusion
In summary,a series of calixarene derivatives incorporating two 1,3-diketo subunits in alternate positions at the lower rim were synthesized and their preliminary bioassays were also evaluated. The biological results showed that the tert-butyl groups at the upper rim of calixarene have a negative effect on inhibiting HIV-1 integrase ST reaction. calixarene derivatives afforded more potent anti-IN activity in this series. Specifically, compound 4g was the most potent candidate among the tested compounds,which inhibited the ST step of IN at 6.1 mmol/L concentration. Further work based on these structures is in progress. Acknowledgment
The authors gratefully thank the National Natural Science Foundation of China (Nos.21102003,21102084,81202438), Scientific Research Foundation for the Introduction of Talent and Young Teachers Scientific Research Foundation of Anhui University of Science & Technology (Nos. 11214,2012QNY27) for the financial supports.
|||Y. Pommier, A.A. Johnson, C. Marchand, Integrase inhibitors to treat HIV/AIDS, Nat. Rev. Drug Discov. 4 (2005) 236-248.|
|||(a) R. Dayam, N. Neamati, Small-molecule HIV-1 integrase inhibitors: the 2001- 2002 update, Curr. Pharm. Des. 9 (2003) 1789-1802; (b) R. Dayam, J.X. Deng, N. Neamati, HIV-1 integrase inhibitors: 2003-2004 update, Med. Res. Rev. 26 (2006) 271-309; (c) R. Dayam, R. Gundla, L.Q. Al-Mawsawi, N. Neamti, HIV-1 integrase inhibitors: 2005-2006 update, Med. Res. Rev. 28 (2008) 118-154.|
|||Y. Goldgur, R. Craigie, G.H. Cohen, et al., Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: a platform for antiviral drug design, Proc. Natl. Acad. Sci. U.S.A. 96 (1996) 13040-13043.|
|||M.L. Barreca, S. Ferro, A. Rao, et al., Pharmacophore-based design of HIV-1 integrase strand-transfer inhibitors, J. Med. Chem. 48 (2005) 7084-7088.|
|||D.J. Hazuda, N.J. Anthony, R.P. Gomez, et al., A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 11233- 11238.|
|||A. Bacchi, M. Biemmi,M. Carcelli, et al., From ligand to complexes. Part 2. Remarks on human immunodeficiency virus type 1 integrase inhibition by β-diketo acid metal complexes, J. Med. Chem. 51 (2008) 7253-7264.|
|||Z.G. Luo, X.M. Xu, X.M. Zhang, L.M. Hu, Development of calixarenes, cyclodextrins and fullerenes as new platforms for anti-HIV drug design: an overview, Mini Rev. Med. Chem. 13 (2013) 1160-1165.|
|||Â. de Fátima, S.A. Fernandes, A.A. Sabino, Calixarenes as newplatforms for drug design, Curr. Drug Discov. Technol. 6 (2009) 151-170.|
|||V.R. Roman, I.B. Vyacheslav, I.K. Vitaly, Calixarenes in bio-medical researches, Curr. Med. Chem. 16 (2009) 1630-1655.|
|||F. Perret, A.N. Lazar, A.W. Coleman, Biochemistry of the para-sulfonato-calix[- n]arenas, Chem. Commun. (2006) 2425-2438.|
|||E. Da Silva, A.N. Lazar, A.W. Coleman, Biopharmaceutical applications of calixarenes, J. Drug Deliv. Sci. Technol. 14 (2004) 3-20.|
|||A. Casnati, F. Sansone, R. Ungaro, Peptido- and glycocalixarenes: playing with hydrogen bonds around hydrophobic cavities, Acc. Chem. Res. 36 (2003) 246- 254.|
|||M. Mourer, N. Psychogios, G. Laumond, A.M. Aubertin, J.B. Regnouf-de-Vains, Synthesis and anti-HIV evaluation of water-soluble calixarene-based bithiazolyl podands, Bioorg. Med. Chem. 18 (2010) 36-45.|
|||L.K. Tsou, G.E. Dutschman, E.A. Gullen, et al., Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a tetrabutoxy-calixarene scaffold, Bioorg. Med. Chem. Lett. 20 (2010) 2137-2139.|
|||V.R. Kamalraj, S. Senthil, P. Kannan, One-pot synthesis and the fluorescent behavior of 4-acetyl-5-methyl-1,2,3-triazole regioisomers, J. Mol. Struct. 892 (2008) 210-215.|
|||M.J. Giffin, H. Heaslet, A. Brik, et al., A copper(I)-catalyzed 1,2,3-triazole azidealkyne click compound is a potent inhibitor of a multidrug-resistant HIV-1 protease variant, J. Med. Chem. 51 (2008) 6263-6270.|
|||R. Alvarez, S. Velazquez, A. San-Felix, et al., 1,2,3-Triazole-[2',5'-bis-O-(tertbutyldimethylsilyl)- β-D-ribofuranosyl]-3'-spiro-5"-(4"-amino-1",2"-oxathiole 2",2"-dioxide) (TSAO) analogs: synthesis and anti-HIV-1 activity, J. Med. Chem. 37 (1994) 4185-4194.|
|||Z.G. Luo, C.C. Zeng, L.F. Yang, et al., Synthesis of 6-sulfamoyl-4-oxoquinoline-3- carboxylic acid derivatives as integrase antagonists with anti-HIV activity, Chin. Chem. Lett. 20 (2009) 789-792.|
|||L.M. Hu, S.L. Zhang, X.Z. He, et al., Design and synthesis of novel β-diketo derivatives as HIV-1 integrase inhibitors, Bioorg. Med. Chem. 20 (2012) 177-182.|
|||L.M. Hu, S. Yang, Z.G. Luo, et al., Design, practical synthesis, and biological evaluation of novel 6-(pyrazolylmethyl)-4-quinoline-3-carboxylic acid derivatives as HIV-1 integrase inhibitors, Molecules 17 (2012) 10652-10666.|
|||M. Yukito, H. Osamu, N. Yasuyuki, Enantioselective discrimination by cage-type cyclophanes bearing chiral binding sites in aqueous media, J. Am. Chem. Soc. 116 (1994) 2611-2612.|
|||J. Guillon, J.M. Leger, P. Sonnet, C. Jarry, M. Robba, Synthesis of cone, partial-cone, and 1,3-alternate 25,27-bis[1-(2-ethyl)hexyl]- and 25,27-bis[1-(2-tert-butoxy) ethyl]calixarene-crown-6 conformers as potential selective cesium extractants, J. Org. Chem. 65 (2000) 8283-8289.|
|||Z.G. Luo, X.M. Xu, K. He, Synthesis of α,γ-diketo derivatives containing 1,2,3- triazole ring, Chem. Res. Appl. 25 (2013) 395-398.|
|||V.S. Talanov, R.A. Bartsch, Highly selective preparation of conformationally rigidstereoisomeric calixarenes with two carboxymethoxy groups, J. Chem. Soc., Perkin Trans. 1 (1999) 1957-1961.|
|||C. Jaime, J. de Mendoza, P. Prados, P. Nieto, C. Sanchez, Carbon-13 NMR chemical shifts. A single rule to determine the conformation of calixarenas, J. Org. Chem. 56 (1991) 3372-3376.|
|||Q.H. Chu, L.X. Gao, D.M. Wang, Y.H. Qi, M.X. Ding, Spectroscopy of several β-diketone compounds and their tautomers, Chem. J. Chin. Univ. 21 (2000) 439- 443.|
|||H.Q. He, X.H. Ma, B. Liu, W.Z. Chen, C.X. Wang, A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads, Acta Pharmacol. Sin. 29 (2008) 397-404.|