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. Author manuscript; available in PMC: 2007 Jun 26.
Published in final edited form as: Bioorg Med Chem Lett. 2006 Dec 3;17(5):1362–1368. doi: 10.1016/j.bmcl.2006.11.094

Synthesis and HIV-1 Integrase Inhibitory Activity of Spiroundecane(ene) Derivatives

Elvira E Shults a,#,*, Elena A Semenova b,#, Allison A Johnson b, Svetlana P Bondarenko a, Irina Y Bagryanskaya a, Yuri V Gatilov a, Genrikh A Tolstikov a, Yves Pommier b,*
PMCID: PMC1899478  NIHMSID: NIHMS19138  PMID: 17189685

Abstract

Fifteen 2,4-dioxaspiro[5.5]undecane ketone and 2,4-dioxa-spiro[5.5]undec-8-ene (spiroundecane(ene)) derivatives were synthesized using the Diels-Alder reaction. Inhibition of human immunodeficiency virus integrase (IN) was examined. Eight spiroundecane(ene) derivatives inhibited both 3’-processing and strand transfer reactions catalyzed by IN. SAR studies showed that the undecane core with at least one furan moiety is preferred for IN inhibition. Moreover, crosslinking experiments showed that spiroundecane derivatives did not affect IN-DNA binding at concentrations that block IN catalytic activity, indicating spiroundecane derivatives inhibit preformed IN-DNA complex. The moderate toxicity of spiroundecane(ene) derivatives encourages the further design of therapeutically relevant analogues based on this novel chemotype of IN inhibitors.

Human immunodeficiency virus (HIV) enzymes are targets for antiretroviral therapy due to their requirement for the HIV life cycle. At the present time, only inhibitors of HIV reverse transcriptase and HIV protease are approved for AIDS therapy.1 However, the third viral enzyme – integrase (IN), is also a promising target because of its non-homology to mammalian enzymes.2 In contrast, toxicity of compounds that inhibit HIV reverse transcriptase and protease is believed to be due to their homology to the host cell’s enzymes. Promising results of clinical trials for two new IN inhibitors – a derivative from quinolone antibiotics (JTK-303/GS-9137, Gilead Sciences, Inc) and the compound MK-0518 from “Merck & Co”, were announced recently, providing the proof of concept for IN inhibitors as antiretroviral therapy.3

The joining (integration) of the viral cDNA to host cellular DNAs is performed by IN whose catalytic site is characterized by the D,D-35-E motif.4 The first IN-catalyzed reaction, 3’-processing (3’-P), consists of the cleavage of the viral cDNA immediately 3’ from the conserved CA-sequences at the 3’-ends of the HIV long terminal repeats (LTRs). 3’-P occurs in the cytoplasm after viral reverse transcription. It is still unclear whether 3’-P takes place before or after preintegration complex (PIC) formation and whether PIC formation requires the catalytic activity of IN. As almost all viral cDNA within the PICs consists of 3’-processed ends and viral DNA is not protected from nucleases after isolation of PICs with mutant IN5, 3’-P probably precedes and may be required for the formation of PICs. The second IN-catalyzed reaction, strand transfer (ST), consists of joining of viral cDNA to cellular DNA. ST is therefore contingent of the 3’P and migration of the PIC into the nucleus.

Recently the development of HIV integration inhibitors has focused on inhibitors of the ST reaction.3 However, equal importance of 3’-P for HIV integration as well as its possible involvement in PIC formation make 3’-P a rational approach to inhibit HIV integration. It might also be logical to combine 3’-P inhibitors with the currently developed ST inhibitors.

In our systematic search for novel IN inhibitors, we have identified spirocyclic ketone derivatives (Scheme 1) as compounds that effectively block recombinant HIV IN. Spirocyclic ketones are employed as intermediates in the total stereoselective synthesis of natural products such as gymnodimine (marine toxin from oysters) and laxaphycins A (cytotoxic compounds from marine cyanobacterium).6

Scheme 1.

Scheme 1

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Reagents and conditions: (a) L-proline (0.2 mmol), MeCN, r.t., 13–35 h; (b) L-proline (0.2 mmol), MeCN, r.t., 1.5 h.

The synthesis of the above compounds 1-11 is outlined in Scheme 1. Diels-Alder reaction of 1-(2-furyl)-3-trimethylsiloxy-butadiene 127 and 5-aryl(hetaryl)methylene-2,2-dimethyl-1,3-dioxane-4,6-diones (13a-k) with a catalytic amount of L-proline in acetonitrile at ambient temperature proceeds in the regioselective fashion and furnished the corresponding spirodioxane triones (1-11) (yield 63–92%).8 Compound 4 was also obtained by the three-component reaction of diene 12, 4-methoxy-benzaldehyde, and Meldrum's acid 14 in the presence of L-proline in methanol solution (yield 56%). The reaction of 1-(2-furyl)-2-ethoxycarbonyl-3-trimethylsiloxy-butadiene 159 with methylene Meldrum's acid (13l R1=H) (obtained in situ from Meldrum's acid 14 and formaldehyde with L-proline in eq. acetonitrile) gave 7-(furan-2-yl)-9-hydroxy-3,3-dimethyl-1,5-dioxa-spiro[5,5]undec-8-ene-8-carboxylic acid ethyl esters 16.10 Compounds 17-19 10, containing aryl substituents in the C-7 position, were obtained as follows. Cycloaddition reaction of 1-(2-methoxyphenyl)-3-trimethylsiloxy-butadiene 2011 with 5-[1-(3-hydroxy-4-methoxyphenyl)-ethylidene]-2,2-dimethyl-[1,3]dioxane-4,6-dione 13h leads to compound 17. By three-component reaction of 1-(4-methoxyphenyl)-2-ethoxycarbonyl-3-trimethylsiloxy-butadiene 219 with Meldrum's acid and formaldehyde compound 18 was obtained. The reaction of 1-(2-methoxyphenyl)-2-ethoxycarbonyl-3-trimethylsiloxy-butadiene 2211 with Meldrum's acid and formaldehyde yilded the dioxaspiro-undec-8-ene derivative 19.

All compounds were formed as single diastereomers. The stereochemistry of products was established by NMR analysis. Relative stereochemistry of cyclohexanone derivatives 1-11 and 17 was determined by analysis of the vicinal coupling constants for protons at C-7 and C-11. The syn-arrangement of aryl(hetaryl) and furyl substituents follows from the axial-axial coupling constants between 7-H and 8-H (J = 13.4 – 14.8 Hz) and 10-H and 11H (J = 13.3 – 15.0 Hz). The axial-axial coupling constants between 7-H and 8-H were also observed in the case of compound 18.

The structure of compound 1 was determined by single crystal X-ray diffraction analysis (Fig. 1,12). The bond lengths in the molecule are close to the statistical mean values. In the Cambridge structural database (University of Cambridge, UK. Version 5.26) we found only two compounds6a,13 in those the cyclohexane ring was spiro fused to the 1,3-dioxane ring. The most closest structural analog was 3,3-dimethyl-7-(4-nitrophenyl)-11-phenyl-2,4-dioxaspiro[5,5]undecane-1,5,9-trione6a.

Figure 1.

Figure 1

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Single-crystal X-ray structure of 3,3-dimethyl-7,11-bis(furan-2-yl)-2,4-dioxa-spiro[5,5]undecane-1,5,9-trione (compound 1).

All compounds were tested against recombinant IN using a 21 bp substrate that allows determination of 3’-P, as the release of the terminal dinucleotide, and ST, as the generation of DNA molecules larger than the starting substrate (Fig. 2 and Table 1)14. Substitutions on the R1 position of the spiroundecane core (Scheme 1, Table 1) highlight the importance of this position for IN inhibition. The most active spiroundecane ketone derivative (2) contains 3-indolyl moiety at R1, and is approximately twice more active than the symmetric 7,11-bis-furan-2yl substituted compound (1). Substitution to the dimethoxyphenyl moiety (11) at R1 failed to increase potency compared to the symmetrical compound (1). Comparison of four compounds with different substitutions on the phenyl ring (11, 9, 4, 3) demonstrates the importance of the ortho-methoxy substitution in compound 11 for 3’-P inhibition. Additionally, halogen (5, 6, 7) or para-hydroxy (17, 8) substitutions to R1 tend to decrease inhibitory potency with exception for compound 4.

Figure 2.

Figure 2

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Inhibition of HIV-1 IN 3’-P, ST and disintegration activities by spiroundecane(ene) derivatives (A) Left: Schematic representation of the integrase reactions using the 21 bp oligonucleotide duplex that corresponds to the terminal U5 sequence of the HIV-1 LTR. Arrowhead represents the 3’-P site. The initial step involves cleavage of two bases from the 3’-end resulting in a 19 bp product. ST products (STP) result from the covalent joining of the 3’-processed duplex into another identical duplex that serves as the target DNA. Right: PAGE analysis of IN inhibition by investigated derivatives. (B) Left: Schematic representation of the ST assay using the preprocessed (19/21) oligonucleotide duplex. Right: PAGE analysis of IN inhibition by the indicated compounds. (C) Left: Schematic representation of the disintegration reaction with a Y-oligomer substrate. The disintegration product results from cleavage of the 34-mer oligonucleotide, allowing accumulation of radiolabeled 19-bp oligonucleotide. Right: PAGE analysis of the IN-mediated disintegration reactions in the presence of the indicated compounds. Drug concentrations are shown above each lane. Asterisks represent the 5’-[32P]-label.

Table 1.

Inhibition of HIV-1 IN by spiroundecane(ene) derivatives using 21 bp duplex as a substrate.

Compounds 3-P IC50, μMa ST IC50, μMa
2 11.1 ± 6.0 9.6 ± 1.6
1 32.9 ± 8.1 17.6 ± 5.9
11 44.8 ± 18.3 44.2 ± 16.8
16 67.8 ± 9.5 43.8 ± 2.8
9 111.3 ± 22.4 91.5 ± 15.7
4 130.5 ± 50.5 36.3 ± 8.5
3 141.1 ± 17.4 131.7 ± 10.9
18 142.3 ± 37.6 97.5 ± 25.4
19 na 281.0 ± 41.3
6 na na
7 na na
5 na na
10 na na
17 na na
8 na na
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a

All data represent mean values and standard deviations for at least three independent experiments (na = not active).

We tested two spiroundecene derivatives (16, 19) and found that the furan moiety is preferred for IN inhibition compared with methoxyphenyl for the spiroundecene core (compare 16 and 19). We also found that the spiroundecane core is more potent as a scaffold for IN inhibitors than the spiroundecene core (compare 18 and 19) (Table 1).

To characterize IN inhibition by spiroundecane(ene) derivatives, we compared the effect of the two most inhibitory spiroundecane (2, 1) and of one spiroundecene (16) derivatives on the three reactions catalyzed by IN (3’-P, ST and disintegration). As shown in Figure 2 these compounds show similar inhibition for 3’-P (21 bp duplex as a substrate, Fig. 2A, Table 1) and ST (precleaved substrate, Fig. 2B, IC50 are 10.3 ± 5.0 μM (compound 2), 13.7 ± 4.4 μM (compound 1) and 35.7 ± 16.3 μM (compound 16). Therefore spiroundecane(ene) derivatives are dual inhibitors of IN-mediated 3’P and ST. Spiroundecane(ene) derivatives also inhibit disintegration, which corresponds to the reverse reaction of strand transfer3, with comparable potency as for 3’-P or ST for undecane derivatives (Fig. 2C, IC50 are 19.2 ± 1.2 μM (compound 2), 81.4 ± 10.8 μM (compound 1) and 213.0 ± 11.2 μM (compound 16).

To explore whether spiroundecane(ene) derivatives inhibitory properties were based on their ability prevent DNA binding to IN, we investigated the effects of compound 1 on IN-DNA binding using two crosslinking strategies. First, we evaluated the ability of compound 1 to inhibit a crosslinking reaction between the cytosine in the 5’-AC overhang of the viral DNA and glutamine 148 of IN (Fig. 3A). A Q148C mutant form of HIV-1 IN allows specific covalent interaction with a thiol-modified cytosine in the 5’-AC overhang.15 Figure 3 shows minimal interference with this specific IN-DNA contact. Marginal inhibition was only observed at the highest concentration (333 μM).

Figure 3.

Figure 3

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Compound 1 does not interfere with the formation of IN-DNA complexes at concentrations that inhibit IN catalytic activity. (A) IN-DNA disulfide crosslinking strategy. Left, schematic representation of the mutant IN used for crosslinking; Upper right, modified oligonucleotide used for crosslinking15 with a thioalkyl modification. The crosslinked complex forms between the cysteine residue 148 and the 5’-C from the DNA substrate (right). (B) SDS-PAGE analysis testing compound 1 in the disulfide crosslinking assay using the DNA substrate labeled with [32P] at the 5’-end of the top strand. 5-CITEP (5-chloroindolyltetrazolylpropenone) was used as a positive control for crosslinking inhibition15. (C) Principle of the Schiff base crosslinking assay. An abasic site is introduced by uracil DNA glycosylase in the DNA substrate containing uracil at the -1 (corresponding to the adenine in the conserved CA-dinucleotide), -12 or -6 positions. A nucleophile residue from IN (probably lysine) attacks the C1’-carbon of the abasic site16. Rearrangement of the initial enzyme-DNA complex leads to the formation of a Schiff base intermediate stabilized with NaBH4. (D) SDS-PAGE analysis showing the testing of compound 1 on the crosslinking reactions between IN and DNA. The asterisks indicate the 5’-[32P]-label.

To further determine whether compound 1 could interfere with the IN-DNA binding at other sites, we used the Schiff-base assay16. This assay measures crosslinking between IN and a DNA substrate mimicking the viral U5 LTR end containing a single abasic site. We examined the effects of compound 1 on IN crosslinking at three different positions in DNA substrates (Fig. 3C). Compound 1 only marginally blocked the Schiff base IN-DNA interactions (Fig. 3D), which is consistent with the results of the disulfide crosslinking assay. Together, the crosslinking results indicate that the spiroundecane derivative (compound 1) has no significant interference with IN-DNA binding at concentrations that block IN catalytic activity.

None of the compounds that inhibit HIV integrase displayed cytoprotective activity for MT-2 cells infected by HIVIIIB, but all of them had moderate toxicity in this type of cells (CC50>111 μM). Probably, low cell penetration ability leads to failure of antiviral activity. Therefore the design of analogues of this novel chemotype will be necessary for antiviral activity. Such work is currently in progress and will be reported elsewhere.

In conclusion, we have synthesized and evaluated a series of original spiroundecane(ene) derivatives as HIV-1 integrase inhibitors. Spiroundecane(ene) derivatives are dual inhibitors of both reactions (3’-processing and strand transfer) catalyzed by IN. An undecane core with at least one furan moiety is preferred for IN inhibition. Structure-activity comparison provides evidence that the presence of an oxygen-containing substitution in the benzene is important for inhibition of IN. Crosslinking data suggests that spiroundecane derivatives interfere with the IN catalytic activity without significant affecting IN-DNA binding. The moderate toxicity of spiroundecane(ene) derivatives encourages the further design of therapeutically relevant analogues based on of this novel chemotype of IN inhibitors.

Acknowledgments

We thank Dr. Robert Yarchoan and Dr. David A. Davis (HIV and AIDS Malignancy Branch, Center for Cancer Research, NCI, NIH) for advises with antiviral experiments. We thank Drs. Gregory Verdine and Webster Santos (Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA) for expertise with synthesis of the thio-modified DNA substrate. This research was supported by the Russian Foundation for Basic Research (grant N. 05-03-32365) and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Footnotes

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References and Notes

  • 1.(a) De Clercq E. J Med Chem. 2005;48:1297. doi: 10.1021/jm040158k. [DOI] [PubMed] [Google Scholar]; (b) Barbaro G, Scozzafava A, Mastrolorenzo A, Supuran CT. Curr Pharm Design. 2005;11:1805. doi: 10.2174/1381612053764869. [DOI] [PubMed] [Google Scholar]; (c) Dayam R, Deng J, Neamati N. Med Res Rev. 2006;26:271. doi: 10.1002/med.20054. [DOI] [PubMed] [Google Scholar]
  • 2.Pommier Y, Johnson AA, Marchand C. Nat Rev Drug Discov. 2005;4:236. doi: 10.1038/nrd1660. [DOI] [PubMed] [Google Scholar]
  • 3.(a) Sato M, Motomura T, Aramaki H, Matsuda T, Yamashita M, Ito Y, Kawakami H, Matsuzaki Y, Watanabe W, Yamataka K, Ikeda S, Kodama E, Matsuoka M, Shinkai H. J Med Chem. 2006;49:1506. doi: 10.1021/jm0600139. [DOI] [PubMed] [Google Scholar]; (b) Morales-Ramirez JO, Teppler H, Kovacs C, Steigbigel R, Cooper D, Liporace RL, Schwartz R, Wenning L, Zhao J, Gilde L, Isaacs RD, Nguyen B-N. Abstract of Papers, 10th European AIDS Conference; Dublin, Ireland. Paris, France: European AIDS Clinical Society; 2005. Abstract LBPS1/6. [Google Scholar]; (c) Semenova EA, Johnson AA, Marchand C, Pommier Y. Curr Opin HIV AIDS. 2006;1:380. doi: 10.1097/01.COH.0000239850.14991.f9. [DOI] [PubMed] [Google Scholar]; (d) Marchand C, Johnson AA, Semenova EA, Pommier Y. Drug Discovery Today: Disease Mechanism. 2006;3:253. doi: 10.1016/j.ddmec.2006.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]; (e) Hazuda DJ. Curr Opin HIV AIDS. 2006;1:212. doi: 10.1097/01.COH.0000221594.57035.55. [DOI] [PubMed] [Google Scholar]
  • 4.Bushman FD, Engelman A, Palmer I, Wingfield P, Craigie R. Proc Natl Acad Sci U S A. 1993;90:3428. doi: 10.1073/pnas.90.8.3428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.(a) Van Maele B, Debyser Z. AIDS Rev. 2005;7:26. [PubMed] [Google Scholar]; (b) Miller MD, Farnet CM, Bushman FD. J Virol. 1997;71:5382. doi: 10.1128/jvi.71.7.5382-5390.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]; (c) Chen H, Wei SQ, Engelman A. J Biol Chem. 1999;274:17358. doi: 10.1074/jbc.274.24.17358. [DOI] [PubMed] [Google Scholar]
  • 6.(a) Ramachary DB, Chowdari NS, Barbas CF. Angew Chem Int Ed Engl. 2003;42:4233. doi: 10.1002/anie.200351916. [DOI] [PubMed] [Google Scholar]; Kong K, Moussa Z, Romo D. Org Lett. 2005;7:5127. doi: 10.1021/ol051840r. [DOI] [PubMed] [Google Scholar]; Bonnard I, Rolland M, Francisco C, Banaigs B. Lett Pept Sci. 1997;4:289. [Google Scholar]
  • 7.Tolstikov GA, Shults EE, Safarova GM, Spirikhin LV, Panasenko AA. Russ J Org Chem. 1990;26:1283. [Google Scholar]
  • 8.General procedure for the synthesis of the 2,4-dioxa-spiro[5,5]undecane-1,5,9-triones 1-11. A solution of 3-trimethylsiloxy-1,3-butadiene 12 (1.14 g, 5.5 mmol) in 5 ml acetonitrile was added under stirring to a suspension of dienophile (5 mmol) and 0.03 g L-proline in 50 ml acetonitrile. The mixture was stirred at r.t. for 25–40 h. Upon evaporation of solvent, a residue was treated with 10 ml 3% NH4OH. The mixture was stirred for 10 min and then treated with 50 ml methylene chloride. The organic layer was separated, while the water one was extracted with methylene chloride. The collected extracts are washed (with water and brine) and dried (MgSO4). The solvent was evaporated and the residue was purified by column chromatograpies. The eluates, containing the products are evaporated, and residues are recrystallized from the corresponding solvent. 3,3-Dimethyl-7,11-di-(furan-2-yl)-2,4-dioxaspiro[5,5]undecane-1,5,9-trione 1. Yield 92%, m.p. 198–202 °C (from acetonitrile), 1H NMR (CDCl3, 400 MHz) δ: 1.02 (6H, s, 2xCH3-C3), 2.59 (2H, dd, J = 16.5, 4.5 Hz, H-8,10), 3.52 (2H, m, H1-8,10), 4.07 (2H, dd, J = 14.1, 4.7 Hz, H-7,11), 6.20 (1H, d, J = 3.5 Hz, H1-3′), 6.30 (1H, dd, J = 3.5, 2.1 Hz, H-4′), 7.33 (1H, dd, J = 2.1, 0.8 Hz, H1-5′). 13C NMR (CDCl3, 300 MHz) δC: 24.8 (2xCH3), 41.6 (C8,10), 43.8 (C7,11), 57.7 (C6), 106.3 (C3), 109.6 (C3′,3″), 111.7 (C4′,4″), 143.9 (C5′,5″), 152.4 (C2′,2″), 165.2 (C1), 168.5 (C5), 206.4 (C9). IR (cm−1): 898, 1500, 1589, 1636 (C=C),1285, 1760 (C=O). Calcd. for C19H18O7: C 63.69; H 5.03. Found: C 63.4; H 5.1. 11-(1H-Indol-3-yl)-3,3-dimethyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5] undecane-1,5,9-trione 2. Yield 65%, m.p. 167–169°C (from ethanol-ether). 1H NMR (CDCl3, 400 MHz) δ: 0.46 (3H, s, CH3-C3), 0.96 (3H, s, CH3-C3), 2.73 (2H, m, H-8,10), 3.71 (2H, m, H1-8,10), 4.19 (1H, dd, J = 14.0, 4.8 Hz, H-7), 4.32 (1H, dd, J = 14.2, 4.6 Hz, H-11), 6.20 (1H, d, J = 3.3 Hz, H1-3′), 6.30 (1H, dd, J = 3.3, 1.8 Hz, H-4′), ), 6.84 (1H, d, J = 1.5 Hz, H-2″), 7.01 (1H, dt, J = 7.5, 7.5, 1.1 Hz, H-6″), 7.15 (2H, m, H-7″,5″), 7.30 (1H, dd, J = 1.8, 0.8 Hz, H1-5′), 7.38 (1H, d, J= 7.8, H-4″), 8.9 (1H, s, NH). 13C NMR (CDCl3, 300 MHz) δC: 28.1 (CH3), 28.5 (CH3), 41.2 (C10), 41.9 C8), 43.6 (C7), 44.2 (C11), 60.5 (C6), 106.1 (C3), 108.5 (C3′), 110.7 (C4′), 111.0 (C7″), 113.6 (C3″), 119.6, 119.7 (C4″,5″), 122.7 (C2″), 123.3 (C6″), 125.4 (C3a″), 135.5 (C7a″), 142.5 (C5′), 151.1 (C2′), 167.0 (C1), 168.7 (C5), 206.8 (C9). Calcd. for C23H21NO6: C 67.81; H 5.16; N 3.44. Found: C 67.7; H 5.3; N 3.7. 3,3-Dimethyl-11-phenyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5]undecane-1,5,9-trione 3. Yield 81%, m.p. 186–187°C (from ethyl acetate). ). 1H NMR (CDCl3, 400 MHz) δ: 0.56 (3H, s, CH3-C3), 0.96 (3H, s, CH3-C3), 2.60 (2H, m, H-8,10), 3.57 (2H, m, H1-8,10), 3.85 (1H, dd, J = 13.6, 4.2 Hz, H-7), 4.09 (1H, dd, J = 13.5, 4.6 Hz, H-11), 6.17 (1H, d, J = 3.2 Hz, H1-3′), 6.27 (1H, dd, J = 3.2, 2.8 Hz, H-4′), 7.15 – 7.35 (6H, m, H5-Ph, H1-5′). IR (cm−1): 720, 900, 1497, 1589, 1610 (C=C), 1712, 1730, 1763 (C=O). Calcd for C21H20O6: 368.12598. Found: 368.12505. 3,3-Dimethyl-11-(p-methoxyphenyl)-7-(furan-2-yl)-2,4-dioxa-spiro[5.5] undecane-1,5,9-trione 4. Yield 71%, m.p. 165–167°C (ethyl acetate). 1H NMR (CDCl3, 400 MHz) δ: 0.67 (3H, s, CH3-C3), 0.98 (3H, s, CH3-C3), 2.59 (2H, m, H-8,10), 3.56 (2H, m, H1-8,10), 3.73 (3H, s, CH3O-C4″), 3.85 (1H, dd, J = 13.4, 4.5 Hz, H-7), 4.11 (1H, dd, J = 13.5, 4.6 Hz, H-11), 6.17 (1H, d, J = 3.0 Hz, H1-3′), 6.27 (1H, dd, J = 3.0, 2.6 Hz, H-4′), 6.82 (2H, d, J = 8.2 Hz, H-3″ and 5″), 7.11 (2H, d, J = 8.2 Hz, H-2″ and 6″), 7.31 (1H, d, J = 2.6 Hz, H-5′). 13C NMR (CDCl3, 300 MHz) δC: 28.4 (CH3), 28.2 (CH3), 41.1 (C10), 42.8 C8), 43.6 (C7), 48.6 (C11), 55.1 (OCH3), 60.8 (C6), 106.1 (C3), 108.5 (C3′), 110.6 (C4′), 114.3 (C3″,5″), 128.8 (C1″), 129.1 (C2″,6″), 142.4 (C5′), 150.9 (C2′), 159.5 (C4″), 164.7 (C1), 168.3 (C5), 206.4 (C9). IR (cm−1): 910, 1514, 1581, 1611 (C=C), 1724, 1759 (C=O). Calcd for C22H22O7: 398.13654. Found: 398.13605. 11-(p-Fluorophenyl)-3,3-dimethyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5]-undecane-1,5,9-trione 5. Yield 63%, m.p. 175–178°C (from ethyl acetate). 1H NMR (CDCl3, 400 MHz) δ: 0.70 (3H, s, CH3-C3), 0.97 (3H, s, CH3-C3), 2.61 (2H, m, H-8,10), 3.57 (2H, m, H1-8,10), 3.90 (1H, dd, J = 13.4, 4.5 Hz, H-7), 4.13 (1H, dd, J = 13.3, 4.7 Hz, H-11), 6.19 (1H, d, J = 3.0 Hz, H1-3′), 6.30 (1H, dd, J = 3.0, 2.8 Hz, H-4′), 7.01 (2H, d, J = 8.3 Hz, H-3″ and 5″), 7.19 (2H, d, J = 8.3 Hz, H-2″ and 6″), 7.33 (1H, d, J = 2.8 Hz, H-5′). 13C NMR (CDCl3, 300 MHz) δC: 28.3 (CH3), 28.5 (CH3), 41.3 (C10), 42.8 C8), 43.8 (C7), 48.8 (C11), 58.7 (C6), 106.3 (C3), 108.8 (C3′), 110.8 (C4′), 115.9 (C3″,5″), 130.1 (C1″), 130.3 (C2″,6″), 142.7 (C5′), 150.7 (C2′), 164.7 (C4″), 165.1 (C1), 168.0 (C5), 205.9 (C9). Calcd for C21H19O6F: 386.11655. Found: 386.11578. 11-(p-Bromophenyl)-3,3-dimethyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5]undecane-1,5,9-trione 6. Yield 81%, m.p. 145–147°C (from ethyl acetate). 1H NMR (CDCl3, 400 MHz) δ: 0.73 (3H, s, CH3-C3), 0.98 (3H, s, CH3-C3), 2.57 (1H, dd, J = 13.0, 4.4 Hz, H-10), 2.65 (1H, dd, J = 13.2, 4.0 Hz, H-8), 3.57 (2H, m, H1-8,10), 3.87 (1H, dd, J = 13.6, 4.0 Hz, H-7), 4.13 (1H, dd, J = 13.3, 4.4 Hz, H-11), 6.20 (1H, d, J = 3.3 Hz, H1-3′), 6.30 (1H, dd, J = 3.3, 2.8 Hz, H-4′), 7.09 (2H, d, J = 8.0 Hz, H-2″ and 6″), 7.46 (2H, d, J = 8.0 Hz, H-3″ and 5″), 7.33 (1H, d, J = 2.8 Hz, H-5′). 13C NMR (CDCl3, 300 MHz) δC: 28.3 (CH3), 28.5 (CH3), 41.2 (C10), 42.4 C8), 43.7 (C7), 48.9 (C11), 58.4 (C6), 106.4 (C3), 108.9 (C3′), 110.8 (C4′), 130.0 (C2″,6″), 122.8 (C1″), 132.3 (C3″,5″), 135.7 (C4″), 142.6 (C5′), 150.6 (C2′), 164.6 (C1), 167.9 (C5), 205.9 (C9). Calcd for C21H19O6Br: 446.03654. Found: 446.03711. 11-(o-Chlorophenyl)-3,3-dimethyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5]undecane-1,5,9-trione 7. Yield 71%, m.p. 174–177°C (from ethyl acetate). 1H NMR (CDCl3, 400 MHz) δ: 0.89 (3H, s, CH3-C3), 0.95 (3H, s, CH3-C3), 2.55 (1H, dd, J = 15.0, 4.4 Hz, H-10), 2.67 (1H, dd, J = 15.2, 4.6 Hz, H-8), 3.38 (1H, m, H1-8), 3.65 (1H, m, H1-10), 4.23 (1H, dd, J = 13.5, 4.6 Hz, H-7), 4.69 (1H, dd, J = 14.0, 4.4 Hz, H-11), 6.21 (1H, d, J = 3.1 Hz, H1-3′), 6.29 (1H, dd, J = 3.1, 2.0 Hz, H-4′), 7.20 – 7.41 (6H, m, H4-Ph, H1-5′). 13C NMR (CDCl3, 300 MHz) δC: 28.0 (CH3), 28.9 (CH3), 41.4 (C10), 43.3 (C8), 43.6 (C7), 44.6 (C11), 56.7 (C6), 106.3 (C3), 109.2 (C3′), 110.8 (C4′), 127.3 (C3″), 128.4 (C5″), 129.3 (C1″), 130.6 (C4″), 134.2 (6″), 134.7 (C2″), 142.5 (C5′), 150.8 (C2′), 165.2 (C1), 166.8 (C5), 205.8 (C9). ). Anal. Calcd. for C21H19ClO6. C 62.61; H 4.72; Cl 8.81. Found: C 62.8; H 4.9; Cl 9.1. 11-(3-Hydroxy-4-methoxyphenyl)-3,3-dimethyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5] undecane-1,5,9-trione 8. Yield 83%, m.p. 197–198°C (from acetonitrile). 1H NMR (CDCl3, 400 MHz) δ: 0.73 (3H, s, CH3-C3), 1.01 (3H, s, CH3-C3), 2.57 (1H, ddd, J = 15.5, 4.4, 1.0 Hz, H-10), 2.53 (1H, ddd, J = 15.2, 4.6, 1.2 Hz, H-8), 3.55 (2H, m, H1-8,10), 3.80 (1H, dd, J = 13.9, 4.4 Hz, H-7), 3.83 (3H, s, CH3O-C4″), 4.10 (1H, dd, J = 14.2, 4.7 Hz, H-11), 6.18 (1H, d, J = 3.3 Hz, H1-3′), 6.29 (1H, dd, J = 3.3, 2.0 Hz, H-4′), 6.68 (1H, dd, J = 8.4, 2.3 Hz, H-6″), 6.80 (1H, d, J = 2.3, H-2″), 6.98 (1H, d, J = 8.4, H-5″), 7.32 (1H, dd, J = 2.0, 0.9 Hz, H-5′), 10.8 (1H, s, OH). 13C NMR (CDCl3, 300 MHz) δC: 28.3 (CH3), 28.5 (CH3), 41.3 (C10), 43.0 (C8), 44.0 (C7), 48.8 (C11), 56.0 (OCH3), 58.6 (C6), 106.2 (C3), 108.6 (C3′), 110.8, 110.9 (C4′,6″), 114.5 (C5″), 120.2 (C2″), 130.1 (C1″), 142.4 (C2′), 146.1, 146.8 (C3″,4″), 151.0 (C2′), 165.8 (C1), 168.1 (C5), 206.5 (C9). Calcd for C22H22O8: 414.13145. Found: 414.13134. 11-(3,4-Dimethoxyphenyl)-3,3-dimethyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5] undecane-1,5,9-trione 9. Yield 90%, m.p. 145–146°C (from ether). 1H NMR (CDCl3, 400 MHz) δ: 0.70 (3H, s, CH3-C3), 0.99 (3H, s, CH3-C3), 2.61 (2H, m, H-8,10), 3.56 (2H, m, H1-8,10), 3.80 (1H, dd, J = 13.8, 4.5 Hz, H-7), 3.81 (6H, s, CH3O-C3″,4″), 4.11 (1H, dd, J = 15.0, 4.7 Hz, H-11), 6.18 (1H, d, J = 3.4 Hz, H1-3′), 6.29 (1H, dd, J = 3.4, 2.0 Hz, H-4′), 6.68 (1H, dd, J = 8.2, 2.0 Hz, H-6″), 6.88 (1H, d, J = 2.0, H-2″), 6.92 (1H, d, J = 8.2, H-5″), 7.32 (1H, dd, J = 2.0, 0.9 Hz, H-5′). 13C NMR (CDCl3, 300 MHz) δC: 28.4 (CH3), 28.5 (CH3), 41.2 (C10), 42.9 (C8), 43.9 (C7), 49.0 (C11), 55.8, 55.9 (OCH3), 58.7 (C6), 106.2 (C3), 108.6 (C3′), 110.7, 111.3, 111.5 (C4′,5″,6″), 120.2 (C2″), 129.2 (C1″), 142.5 (C2′), 149.1, 149.3 (C3″,4″), 150.8 (C2′), 165.5 (C1), 168.3 (C5), 206.4 (C9). Calcd for C23H24O8: 428.14710. Found: 428.14761. 3,3-Dimethyl-11-(2-methyl-4-methoxyphenyl)-7-(furan-2-yl)-2,4-dioxa-spiro[5.5] undecane-1,5,9-trione 10. Yield 78%, m.p. 172–175°C (from ether). 1H NMR (CDCl3, 400 MHz) δ: 0.86 (3H, s, CH3-C3), 1.00 (3H, s, CH3-C3), 2.27 (3H, s, CH3-C2″), 2.27 (1H, dd, J = 15.2, 4.4 Hz, H-10), 2.65 (1H, dd, J = 16.0, 4.0 Hz, H-8), 3.44 (1H, m, H-8), 3.63 (1H, m, H-10), 3.72 (3H, s, CH3O-C4″), 4.22 (1H, dd, J = 14.8, 4.0 Hz, H-7), 4.31 (1H, dd, J = 15.0, 4.4 Hz, H-11), 6.19 (1H, d, J = 3.2 Hz, H1-3′), 6.28 (1H, dd, J = 3.2, 2.0 Hz, H-4′), 6.67 (1H, d, J = 2.2 Hz, H-3″), 6.70 (1H, dd, J = 8.0, 2.2, H-5″), 7.18 (1H, d, J = 8.0, H-6″), 7.32 (1H, dd, J = 2.0, 0.8 Hz, H-5′). 13C NMR (CDCl3, 300 MHz) δC: 19.6 (CH3), 28.2 (CH3), 28.8 (CH3), 41.4 (C10), 43.9 (C8), 44.0 (C7), 44.5 (C11), 55.1 (OCH3), 57.3 (C6), 106.4 (C3), 108.8 (C3′), 110.7 (C4′), 112.2 (C5″), 116.3 (6″), 127.8 (C1″), 128.1 (C3″), 138.3 (C2″), 142.5 (C2′), 151.0 (C2′), 158.9 (C4″),165.4 (C1), 168.0 (C5), 206.7 (C9). Calcd for C23H24O7: 412.15219. Found: 412.15340. 11-(2,3-Dimethoxyphenyl)-3,3-dimethyl-7-(furan-2-yl)-2,4-dioxa-spiro[5.5] undecane-1,5,9-trione 11. Yield 74%, m.p. 127–128°C (from ethyl acetate). 1H NMR (CDCl3, 400 MHz) δ: 0.85 (3H, s, CH3-C3), 0.92 (3H, s, CH3-C3), 2.51 (2H, m, H-8,10), 3.57 (2H, m, H1-8,10), 3.76 (6H, s, CH3O-C2″,3″), 4.17 (1H, dd, J = 14.6, 4.2 Hz, H-7), 4.43 (1H, dd, J = 14.5, 4.5 Hz, H-11), 6.13 (1H, d, J = 3.0 Hz, H1-3′), 6.22 (1H, dd, J = 3.0, 2.0 Hz, H-4′), 6.76 (2H, d, J = 8.4 Hz, H-4″,6″), 6.92 (1H, m, H-5″), 7.27 (1H, dd, J = 2.0, 0.9 Hz, H-5′). 13C NMR (CDCl3, 300 MHz) δC: 27.9 (CH3), 29.0 (CH3), 41.1 (C10), 41.4 (C8), 43.2 (C7), 43.3 (C11), 55.6, 55.7 (OCH3), 57.1 (C6), 106.1 (C3), 108.8 (C3′), 108.9, 110.5, 110.7 (C4′,5″,6″), 112.3 (C4″), 130.3 (C1″), 142.5 (C2′), 151.2 (C2′), 152.7, 152.8 (C2″,3″), 165.5 (C1), 167.3 (C5), 206.7 (C9). Calcd for C23H24O8: 428.14770. Found: 428.14761. 11-(3-Hydroxy-4-methoxyphenyl)-3,3-dimethyl-7-(2-methoxyphenyl)-2,4-dioxa-spiro[5.5]undecane-1,5,9-trione 17 was obtained by the reaction of the diene 20 with 5-[1-(3-hydroxy-4-methoxyphenyl)-ethylidene]-2,2-dimethyl-[1,3]dioxane-4,6-dione 13h by the above method. Yield 73%, m.p. 192–194°C (from ethyl acetate). 1H NMR (CDCl3, 400 MHz) δ: 0.70 (3H, s, CH3-C3), 1.05 (3H, s, CH3-C3), 2.59 (2H, m, H-8,10), 3.59 (2H, m, H1-8,10), 3.77, 3.80 (6H, s, 2xCH3O-C2′,4″), 4.08 (2H, m, H-7,11), 6.70 (1H, dd, J = 8.4, 2.3 Hz, H-6″), 6.82 (2H, m, J = 2.3, H-3′,2″), 6.98 (2H, H-5″,6′), 7.23 (2H, m, H-4′,5′), 10.8 (1H, s, OH). Anal. Calcd. for C25H26O8. C 66.08; H 5.69. Found: C 66.5; H 5.9.
  • 9.Shults EE, Petrova TN, Rybalova TV, Gatilov YV, Tolstikov GA. Russ J Org Chem. 1998;34:895. [Google Scholar]
  • 10.General procedure for the synthesis of the 2,4-dioxa-spiro[5,5]undec-8-ene-1,5-diones 16, 19: To a solution of Meldrum's acid 14 (0.46 g, 3.2 mmol), formaldehyde (0.5 ml of 37%-eq. solution), and L-proline (0.03 g) in acetonitrile (10 ml) diene 15 (1.12 g, 4.0 mmol) (or 22) was added at ambient temperature in a period of 10 min. The reaction mixture was stirred 1.5 h, quenched with ice-water, the product extracted by methylene chloride, the combined organic layer was dried (MgSO4), evaporated in vacuum, and the residue was purified by chromatography on silica gel. The residues are recrystallized from the corresponding solvent. 9-Hydroxy-3,3-dimethyl-7-(furan-2-yl)-1,5-dioxo-2,4-dioxa-spiro[5,5]undec-8-ene-8-carboxylic acid ethyl ester 16. Yield 77%, m.p. 125–128°C (from ethyl acetate). IR (cm−1): 710, 750, 840, 1505, 1625 (C=C), 1720, 1760(C=O, 3090, 3420 (OH). 1H NMR (CDCl3, 400 MHz) δ: 0.98 (3H, t, J=7.0 Hz, CH3), 1.63 (3H, s, CH3-C3), 1.68 s (3H, s, CH3-C3), 2.20 (1H, m, H1-10), 2.41 (1H, m, H1-11), 2.78 (2H, m, H-10,11), 4.07, 4.11 (2H, dd, J=7.0 Hz, CH2), 4.68 (1H, s, H-7), 6.04 (1H, dd, J= 4.6, 1.6 Hz, H-3′) 6.25 (1H, dd, J= 4.6, 3.0, H-4′), 7.26 (1H, dd, J = 3.0, 1.0, H-5′), 12.4 s (1H, OH). Anal. Calcd. for C18H20O8: C 59.30; H5.49. Found: C 59.5; H 5.8. 9-Hydroxy-3,3-dimethyl-7-(2-methoxyphenyl)-1,5-dioxo-2,4-dioxa-spiro[5,5] undec-8-ene-8-carboxylic acid ethyl ester 19. Yield 86 %. M.p. 128–130°C (from ethyl acetate). IR (cm−1): 720, 750, 820, 880, 1040, 1080, 1120, 1260, 1360, 1500, 1560, 1580, 1620, 1710, 1720, 1740, 1780. 1H NMR (CDCl3, 400 MHz) δ: 0.84 (3H, t, J = 7.0 Hz, CH3), 1.64 (3H, s, CH3-C3), 1.67 (3H, s, CH3-C3), 2.14 (1H, m, H-5), 2.30 (1H, m, H-6), 2.72 (2H, m, H-5,6), 3.76 s (3H, OCH3), 3.91 (2H, q, J = 7.0 Hz, CH2), 4.99 (1H, s, H-7), 6.79 (1H, dd, J = 7.6, 1.0, H-3′), 6.85 (1H, dd, J = 7.2, 1.0, H-5′), 6.99 (1H, dd, J = 7.6, 1.8, H-6′), 7.18 (1H, dd, J = 7.2, 1.8, H-4′), 12.4 s (1H, OH). 13C NMR (CDCl3, 300 MHz) δC: 13.9 (CH3), 25.8, 26.1 (C10,11), 27.7 (CH3), 29.8 (CH3), 51.4 (C7), 54.8 (C6), 55.4 (OCH2), 60.1 (CH2), 104.8 (C3), 109.9 (C3′), 120.6 (C6′), 127.4 (C1′), 128.5 (C4′), 129.2 (C5′), 130.9 (C8), 156.9 (C2′), 162.9 (C9), 166.0 (C1), 168.2 (C5), 172.7 (C=O). Anal. Calcd. for C21H24O8: C 62.38; H 6.0. Found: C 62.2; H 5.8. 3,3-Dimethyl-7-(4-methoxyphenyl)-2,4-dioxa-spiro[5,5]undecane-1,5,9-trione 18. To a solution of Meldrum's acid 14 (0.46 g, 3.2 mmol), formaldehyde (0.5 ml of 37%-eq. solution), and L-proline (0.03 g) in acetonitrile (10 ml) diene 21 (1.01 g, 4.0 mmol) was added at ambient temperature in a period of 10 min. The reaction mixture was stirred 1.5 h, quenched with ice-water, the product extracted by methylene chloride, the combined organic layer was dried (MgSO4) and evaporated in vacuum. Purification by silica gel chromatography eluting with 1% ethanole in chloroform yielded 0.82 g (62%) of the desired product. M.p. 112-114°C. 1H NMR (CDCl3, 400 MHz) δ: 1.58 (3H, s, CH3-C3), 1.72 (3H, s, CH3-C3), 2.38 (2H, m, H-10,11), 2.69 (1H, m, H-8), 3.30 (1H, dd, J 12.3, 5.0, H-7), 3.62 (2H, m, H-10,11), 3.78 (3H, s, OCH3-C4′), 4.26 (1H, dd, J 13.8, 5.0 H-8), 6.75 (1H, dd, J = 7.6, 1.0 Hz, H-3′), 6.88 (1H, dd, J 7.2, 1.0 Hz, H-5′), 7.08 (1H, dd, J = 7.2, 1.8 Hz, H-6′), 7.14 (1H, d, J = 7.6 Hz, H-2′) Anal. Calcd. for C18H20O6: C 65.06; H 6.02. Found: C 65.3; H 6.1.
  • 11.Nechepurenko IV, Shults EE, Tolstikov GA. Russ J Org Chem. 2001;37:1276. [Google Scholar]
  • 12.X-ray crystallographic analysis for compound 1: The data were collected on a Bruker P4 diffractometer with Mo Kα radiation. The cell parameters and the orientation matrix for data collection were obtained from least squares refinement in the range of 20–25° (2θ). For measurement a single crystal of 1 C19H18O7 [0.68 mm × 0.25 mm × 0.06 mm] was chosen. Monoclinic system with the following unit cell parameters: a = 9.362(2), b = 9.404(2), c = 19.889(3) Å, β = 92.48(1)°, V = 1749.4(6) Å3, space group P21/n, Z = 4, dcalc = 1.361 Γ/cM3, μ = 0.105 mm−1. A total of 3268 unique reflections were measured (among them 3066 independent R(int) = 0.0260). A correction for absorption was introduced empirically by psi-curves (transmission 0.84 – 0.93). The structure was solved by the direct method with the use of SHELXS-97 software. The refining of structural parameters was carried out by the least-squares procedure in the full-matrix anisotropic approximation applying the program SHELXL-97. Hydrogen atoms were included in the refinement restrained to ride on the atom to which they are bonded. The final refinement of the structure parameters was performed with respect to all F2 to wR2 = 0.1855, S = 1.010; 236 parameters (R = 0.0652 for 1535 F > 4σ). The crystal structure of compound 1 has been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 601465. Data Acquisition - the Cambridge crystallographic Data Centre deposit@ccdc.cam.ac.uk; http://www.ccdc.cam.ac.uk/deposit; Telephone: (44) 01223 762910; Facsimile: (44) 01223 336033; Address: CCDC, 12 Union Road, CAMBRIDGE CB2,1EZ, UK
  • 13.Jones PG, Kennard O. Cryst Struct Commun. 1977;6:97. [Google Scholar]
  • 14.The ratio 300 nM IN - 20 nM oligonucleotide substrate and the absence of β-mercaphtoethanol were used in all biological experiments. Experimental conditions for all biological procedures have been previously reported: Semenova EA, Johnson AA, Marchand C, Davis DA, Yarchoan R, Pommier Y. Mol Pharmacol. 2006;69:1454. doi: 10.1124/mol.105.020321.
  • 15.Johnson AA, Santos W, Pais GC, Marchand C, Amin R, Burke TR, Jr, Verdine G, Pommier Y. J Biol Chem. 2006;281:461. doi: 10.1074/jbc.M511348200. [DOI] [PubMed] [Google Scholar]
  • 16.Mazumder A, Pommier Y. Nucleic Acids Res. 1995;23:2865. doi: 10.1093/nar/23.15.2865. [DOI] [PMC free article] [PubMed] [Google Scholar]

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