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Design and Evaluation of Novel Antimicrobial and Anticancer Agents Among Tetrazolo[1,5-c]quinazoline-5-thione S-Derivatives

The novel heterocyclization of 5-(2-aminophenyl)-1H-tetrazole with potassium ethylxanthogenate or carbon disulfide was proposed. The potassium salt of the tetrazolo[1,5-c]quinazoline-5-thione was subsequently modified by alkylation with proper halogen derivatives to (tetrazolo[1,5-c]quinazolin-5-ylthio)alkyls, N,N-dialkylethylamines, 1-aryl-2-ethanones, 1-(alkyl)aryl-2-ethanols, carboxylic acids, and esters. The structures of all newly synthesized compounds were confirmed by FT-IR, UV-vis, LC-MS, 1H, 13C NMR, and elemental analysis data. The substances were screened for antibacterial and antifungal activities (100 μg) against Escherichia coli, Staphylococcus aureus, Enterobacter aerogenes, Entrococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Preliminary bioluminescence inhibition tests against Photobacterium leiognathi Sh1 showed that substances 5.2–5.4, 6.1, 7.1 with ethanone or carboxylic acid substituents showed toxicity against bacteria cells. The substances chosen by the US National Cancer Institute (NCI) were screened for their ability to inhibit 60 different human tumor cell lines, where 2-(tetrazolo[1,5-c]quinazolin-5-ylthio)-1-(4-tolyl)ethanone (5.2), 3-(tetrazolo[1,5-c]quinazolin-5-ylthio)propanoic and related 3-metyl-butanoic acids (6.2, 6.3), and ethyl tetrazolo[1,5-c]quinazolin-5-ylthio)acetate (7.2) showed lethal antitumor activity (1.0 μM) against the acute lymphoblastic leukemia cell line (CCRF-CEM), and substances 5.2 and 6.3 exhibited moderate anticancer properties inhibiting growth of the leukemia MOLT-4 and HL06-(TB) cell lines. The moderate antitumor activity was demonstrated in 1-(2,5-dimethoxyphenyl)-2-(tetrazolo[1,5-c]quinazolin-5-ylthio)ethanone (5.4) against the CNS cancer cell line SNB-75. Comparing the docking mode of the Gefitinib and synthesised substances on the ATP binding site of EGFR, it could be assumed that these compounds might act in the same way. The results of the investigation could be considered as a useful base for future development of potent antimicrobials and antitumor agents among tetrazolo[1,5-c]quinazoline-5-thione S-derivatives.

It is known that tetrazoloquinazolines are capable of tetrazole-azide-azomethine tautomeric transformations due to the electrono-donative influence of the condensed quinazoline ring to the tetrazole one [15]. FT-IR spectral analysis of the synthesized substances (3.1-3.5) in solid form and in chloroform solution showed the absence of the azide group vibration at 2100 сm −1 , which indicates the prevalence of the tetrazole form of the molecule. The low intensity vibrations of the azide tautomer at 2145 сm −1 and its overtone at 2198 сm −1 were detected when trifluoroacetic acid was added to the substances (3.1-3.5) in chloroform solution.
In the 1 Н NMR-spectra, the tetrazolo [1,5-c]quinazoline system of all synthesized substances was characterized according to the proposed structure and available literature data: the one-proton doublet of Н-10 at 8.34-8.55 ppm, Н-7 and Н-9 as the two-proton multiplet at 8.06-7.74, and H-8 as the one-proton triplet at 7.60-7.97 ppm were observed [8]. The signal of SCH 3 was seen in a strong field of spectra at 2.86 ppm as the threeproton singlet for substance 2.1. The proton of the OH group was shown as a wide oneproton singlet at 14.39 ppm for alcohol 4.1 and at 13.54-12.35 ppm for acids 6.1-6.2, and shifted to a stronger field to 11.68 ppm for 4.2. The signal of the SCH 2 -group appeared in a strong field of the spectra as the two-proton triplet at 3.72 ppm for 2.2, at 3.62 ppm for 2.3, and shifted to a low field as the two-proton singlet at 5.22-5.06 ppm for alcohols 4.1, 4.2, аnd to 4.72-3.40 ppm for ethanones 5.1-5.4, and was also observed at 4.85-4.26 ppm for acid 6.1 and acid esters 7.1, 7.3, 7.4, 7.8. The SCH-group was observed as the one-proton triplet at 5.17-5.03 ppm for 7.5-7.7 and as the one-proton singlet at 4.80 ppm for 7.2. The signals of aliphatic substituents of the synthesized substances were registered in a strong field (2.89-0.86 ppm) and aromatic substituent signals were observed in a low field (7.98-7.22 ppm).
In 13 C NMR spectra of substances 7.2 and 7.6, the characteristic signal of the carboxylic group was registered at 170.55 ppm and 170.18 ppm respectively, and the carbonyl group of compound 5.4 at 191.50 ppm. The quinazoline С-5 was detected at 149.32-149.07 ppm, and the peak of the SСН 2 -group was registered at 38.80 ppm for 5.3, and at 49.87-47.84 ppm for 7.2 and 7.6, which definitely confirmed the tetrazolo [1,5-c]quinazoline-5thione formation and S-regioselectivity of alkylation.

Values of BL in acute action test (%)
It is worth mentioning that substances 2.1, 3.2, 3.3, 3.5, 5.4, and 7.3 showed the effect of the hormesis phenomenon (stimulation of activity with a lower concentration) for 0.025 and 0.1 mg/mL doses. Compounds 2.2, 3.1, 7.4, 7.5 possessed moderate inhibition. The highest toxicity (inhibition Bl and growth to 0%) was shown by 4-nitro-2-((tetrazolo [1,5c] Thus, the SAR study revealed that the introduction of the aliphatic acid ester group resulted in the initiation of luminescent bacteria growth in the chronic test. But replacement of the alkyl group with carboxylic acid or the aromatic acid ester, 2-methyl-4-nitrophenol and ethanone substituents in the 5 th position of the 5-thio-tetrazolo [1,5-c]quinazoline led to increased cytotoxic activity against Photobacterium leiognathi Sh1. The high levels of growth inhibition served as a cytotoxicity marker of potential possession of the antitumor activity. 0 DMSO (control) 100.0 74.5 127.7 127.7 a substances 4.1, 6.2, 6.3, 7.2 were not tested.

Antimicrobial and antifungal activities
All of the newly synthesized compounds were evaluated for their in vitro antibacterial activity against Gram positive bacteria (Staphylococcus aureus, Enterococcus faecalis), Gram negative bacteria (Enterobacter aerogenes, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae), and antifungal properties against Candida albicans. The agar-diffusion method was used for the determination of preliminary activity compared to well-known reference antimicrobials. All of the compounds were dissolved in DMSO in a concentration of 100 µg/disk, using inhibition zone diameter (IZD, mm) as a measure for the antimicrobial activity ( Table 3).
The SAR determined that the introduction of the chloropropyl substituent in substance 2.3 was essential for its antifungal activity against Candida albicans like Nystatin action in the same concentration, but wasn't enough to possess antibacterial properties. Replacing the alkyl substituent by ethylaminodialkyl for substances 3.1-3.3 broadened the range of their antimicrobial activity. Also, shortening of the dialkylamino fragment of substances 3.1-3.3 led to a moderate decrease in antimicrobial activity against Enterococcus faecalis, and otherwise led to an increase in antibacterial properties against Staphylococcus aureus and Escherichia coli, and antifungal properties against Candida albicans. Derivatives with ethylaminohetaryl radicals (3.4, 3.5) showed no antimicrobial properties. 2-(Tetrazolo [1,5-c]quinazolin-5-ylthio)ethanol (4.1) had some antimicrobial activity, but the presence of the 4-nitrophenol fragment of substance 4.2 led to the demonstration of light inhibition of Staphylococcus aureus growth. Introduction of the ethanone moiety into the structure of the synthesized compounds (5.1, 5.2, 5.4) had no influence on the mentioned activity. An exception was substance 5.3 with a 4-methoxyphenyl radical at the 5 position of tetrazolo [1,5-c]quinazoline-5-thione, which demonstrated the best results for the antibacterial and antifungal properties among all the synthesized compounds against Staphylococcus aureus, Escherichia coli, and Candida albicans, and was the only one that inhibited growth of Pseudomonas aeruginosa and Klebsiella pneumoniae. The impact of the halogenocarboxylic acid and ester fragments on substances 6.1-6.3 and 7.1-7.8 led to the disappearance of all their antimicrobial activities. Enterococcus faecalis was nonsensitive to all of the synthesized compounds.

Anticancer assay for preliminary in vitro testing
Considering non-small-cell lung, colon cancer, melanoma, and ovarian cancer, they were practically insensitive to the synthesized substances except for the demonstration of the light antitumor activity by 1-(4-methoxyphenyl)-2-(tetrazolo [1,5-c] So, according to the SAR, introduction of the (4-tolyl)ethanone (5.2), acid (6.2, 6.3), and ester (7.1) fragments into the molecule of synthesized compounds imparted them the lethal activity against acute lymphoblastic leukemia CCRF-CEM cell line, among which ethyl 2-(tetrazolo [1,5-c]quinazolin-5-ylthio)acetate (7.1) was the most active. The substitution of the 4-methyl group of substance 5.2 with the 2,5-dimethoxy group for 5.4 changedthe range of its antitumor activity greatly to the widest moderate one among all investigated compounds with the best results inhibiting the CNS cancer SNB-75 cell line. But, unfortunately all of these compounds haven't satisfied the predetermined threshold inhibition criteria to progress to the 5-dose screen, which was designed to efficiently capture compounds with antiproliferate activity.

Docking, scoring, and visual inspection of synthesized substances into the ATP binding site of EGFR
To help us understand the anticancer activity of the observed compounds and guide further SAR studies, molecular docking of compounds 2.1, 2.2, 5.1-5.4, 6.1-6.3, and 7.1 into the ATP binding site of EGFR (2ITY.pdb) was performed [31]. The crystal structure of the enzyme with Gefitinib was obtained from the protein data bank [32]. The selected molecular targets cover some of the basic molecular mechanisms of antitumor effects. Also, the choice of the biological target was due to the literature data on the mechanism of action of 4-R-anilinoquinazolines -known anticancer products [17].
According to Consensus score, compound 6.2 showed the highest affinity for EGFR, but was still lower compared to other known drugs (Gefitinib, Lapatinib). The calculated values are presented in Table 5. Thus, after comparing the docking mode, it could be postulated that the designed compounds might act on the same enzyme target where Gefitinib acted.
Although the results of the antimicrobial activity are not very potent, the anticancer results are quite promising. Compared to the docking mode, it could be assumed that the synthesized substances might act on the ATP binding site of EGFR like Gefitinib does. And they could be used for advanced QSAR analysis based on in vitro biological activity investigations for the further purposeful optimization of the leading compounds in the more effective antimicrobials because of the ever-mounting problem of microorganisms' resistance as well as novel anticancer agents.

Experimental
Melting points were determined in open capillary tubes in a Thiele's apparatus and were uncorrected. The elemental analyses (C, H, N, S) were performed using the ELEMENTAR vario EL cube analyzer. UV-vis spectra (190-400 nm) were recorded on the Analytic Jena UV-vis spectrophotometer Specord 200 in methanol. The IR spectra (4000-600 cm −1 ) were recorded on a Bruker ALPHA FT-IR spectrometer using a module ATR eco ZnSe. 1 H NMR spectra (400 MHz and 500 MHz) were recorded on the Varian-Mercury 400 and Bruker Avance DRX-500 spectrometers with SiMe 4 as the internal standard in DMSO-d 6 solution. The LC-MS was recorded using a chromatography/mass spectrometric system which consisted of the high-performance liquid chromatograph "Agilent 1100 Series" equipped with the diode-matrix and mass-selective detector "Agilent LC/MSD SL" (atmospheric pressure chemical ionization -APCI). The purity of all obtained compounds was checked by 1 H NMR and LC-MS.
Other starting materials and solvents were obtained from commercially available sources and used without additional purification.

Bioluminescence inhibition test
The marine luminescent bacteria Photobacterium leiognathi strain Sh1, isolated from the Azov Sea Shrimp, were used for the bioluminescence analysis. Bacteria were cultivated on a nutrient environment containing (g/L): pepton -5, yeast extract -1.5, meat extract -1.5, sodium chloride -30, pH 7.4. In the acute action test (inhibiting luminescence of bacteria), bacteria were diluted with the 3% sodium chloride solution up to the concentration 10 5 cell/mL. The 5 -50 µg/mL of the studied substances suspended in DMSO were mixed with 1 mL of the diluted bacterial suspension. Vials were incubating for 10 minutes at 25 °C, then the intensity of bioluminescence was measured in % relatively to the control tests which were prepared without the studied compounds. In the chronic action test (inhibiting growth and luminescence of bacteria), growth environment was added to the eventual breeding 1:50 and was incubated for 16-18 hours at 30 °С, whereupon the intensity of bioluminescence was measured as well as in the previous method. Tetracycline was used as a reference. The bacterial luminescence was measured with the Bioluminometer BLM-8801 («Science», Krasnoyarsk, Russia).

Antimicrobial and antifungal test
All the newly synthesized compounds were evaluated for their in vitro antibacterial activity in the Zaporozhye Regional Hospital Bacterial Laboratory against Gram positive bacteria (Staphylococcus aureus ATCC 25923, Enterobacter aerogenes, E. faecalis ATCC 29212), Gram negative bacteria (Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922, Klebsiella pneumoniae 68). They were also evaluated for their in vitro antifungal potential against Candida albicans ATCC 885653. The amount of microbial cells was 1.5 * 10 8 c.f.u./mL. The incubation period of bacteria was 24 h at 35 °C, yeast -48-72 h at 28-30°С. The agar-diffusion method was used for the determination of the preliminary antibacterial and antifungal activity. Standard sterilized filter paper discs (6 mm diameter) impregnated with a solution of the test compound in DMSO (100 µg/disk) were placed on an agar (Müller-Hinton Broth (Oxoid)) plate seeded with the appropriate test organism in triplicates. DMSO alone was used as the control at the same above-mentioned concentration. Ampicillin, Ceftazide, Amikacin, Gentamycin, Ceftriaxone, and Nystatin were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones diameters (IZD) of bacterial or fungal growth around the discs in mm.

Antitumor activity
The primary anticancer assay was performed against the human tumor cell lines panel derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [28][29][30]. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated in 96 well microtiter plates in 100 mL assay volume, at plating densities ranging from 5000 to 40000 cell/well. After cell inoculation, the microtiter plates were incubated at 37°C under an atmosphere of 5:95 CO 2 :air (v/v) at 100% relative humidity, for 24 h prior to the addition of drugs under assessment. Following drug addition (1 µM), the plates were incubated for an additional 48 h, under the same conditions. Sulforhodamine B (SRB) solution (100 µL, 0-4% w/v in 1% aq. acetic acid) was added to each well and plates were incubated for 10 min at room temperature. The percentage growth was evaluated spectrophotometrically versus the controls not treated with test agents.

Docking, scoring, and visual inspection of synthesized substances into ATP binding site of EGFR
Research was conducted by flexible molecular docking, as an approach for finding molecules with affinity for a specific biological target using the software package OpenEye, including related utilities: Fred Receptor2.2.5, Vida4. 1.1,Flipper,Babel3,Omega2.4.3,and Fred2.2.5 [33]. The crystal structure of the enzyme EGFR (2ITY.pdb) with Gefitinib was obtained from the protein data bank [32].
The methodology of research consisted of the following steps: o generation of R-, S-and cis-, trans-isomers of ligands (the studied compounds and relevant drugs, program Flipper), which allowed the production isomer's range of studied compounds; o generation of 3D-structure of the obtained isomeric forms -molecular modelling (Hyper Chem 7.5) using the method of molecular mechanics (MM +) and semiempirical quantum mechanical method with Polak-Ribiere algorithm (PM3); o generation of conformations of ligands (Omega2.4.3). The number of conformations obtained wasn't significant due to the further selection by program Fred2.2.5 most optimal conformer; o carrying out molecular docking (Fred2.2.5).
A number of scoring functions (Shapegauss, PLP, Chemgauss2, Chemgauss3, Chemscore, OEChemscore, Screenscore, CGO, CGT, Zapbind, Consensus Score) was obtained as a result of the studies, values of which assess specific characteristics of the ligand-protein complex, indicating the possibility of their matching.


extfieldsofstudy: Chemistry Medicine

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Chemistry of Fullerene Epoxides: Synthesis, Structure, and Nucleophilic Substitution-Addition Reactivity

Fullerene epoxides, C60On, having epoxide groups directly attached to the fullerene cage, constitute an interesting class of fullerene derivatives. In particular, the chemical transformations of fullerene epoxides are expected to play an important role in the development of functionalized fullerenes. This is because such transformations can readily afford a variety of mono- or polyfunctionalized fullerene derivatives while conserving the epoxy ring arrangement on the fullerene surface, as seen in representative regioisomeric fullerene polyepoxides. The first part of this review addresses the synthesis and structural characterization of fullerene epoxides. The formation of fullerene epoxides through different oxidation reactions is then explored. Adequate characterization of the isolated fullerene epoxides was achieved by concerted use of NMR and LC-MS techniques. The second part of this review addresses the substitution of fullerene epoxides in the presence of a Lewis acid catalyst. Most major substitution products have been isolated as pure compounds and their structures established through spectroscopic methods. The correlation between the structure of the substitution product and the oxygenation pattern of the starting materials allows elucidation of the mechanistic features of this transformation. This approach promises to lead to rigorous regioselective production of various fullerene derivatives for a wide range of applications.

Introduction
Since the first detection of fullerene epoxides via mass spectrometry in a fullerene mixture generated by the arc discharge of graphite in 1991, many studies on fullerene oxides have been performed for the purpose of developing new materials. The epoxidation of fullerene can proceed readily in the presence of oxidants such as ozone [1], organic peroxide [2], dimethyldioxirane [3], methyltrioxorhenium-hydrogen peroxide [4], and cytochrome P450 [5]. Under several circumstances, in different oxidations fullerene C 60 has been shown to give higher oxides (C 60 O n , n ≥ 2) that possess only a few regioisomers. For instance, although there are indeed eight possible regioisomers of C 60 O 2 and 43 isomers of C 60 O 3 given the multiple reaction sites available on the C 60 cage [6], the actual products of most oxidations are only two isomers of C 60 O 2 and three isomers of C 60 O 3 . However, few C 60 O n isomers have been isolated and identified thus far, and experimental data on the regioselective epoxidation of C 60 are also scarce.
Meanwhile, the structures of C 60 O n isomers were first studied theoretically on the basis of the thermodynamic stability of ground state molecules and on the dynamic behavior of the molecules via the transition states. Those results regarding the structures of C 60 O and the predominant isomers of C 60 O 2 can explain these experimental observations. Manoharan showed computationally that multiple epoxidations of C 60 preferentially proceeds at the adjacent rather than distant double bonds of the existing epoxide group, and predicted that multiple epoxidations should occur on one benzenoid ring of C 60 to form the C 60 O 3 isomer with C 3v symmetry. Feng et al. studied three isomers of C 60 O 3 by using the semiempirical quantum mechanical INDO method, and predicted that the three types of C 60 O 3 isomers with C 3v , C s and C 2 symmetries, respectively, should be very stable, and reported their calculated electronic spectra [7]. Curry et al. predicted that the three lowest-energy isomers (C 3v , C s and C 2 ) of C 60 O 3 should exist in equilibrium at room temperature by using a modified and extended Hűckel method [8].
Previously, we found three types of C 60 O 3 isomers in a reaction solution of C 60 with m-CPBA by means of a chromatographic technique involving the use of two different columns [9]. Electronic spectroscopy and mass-spectroscopy examinations of these isomers were mostly consistent with the calculated results by Feng et al. However, the precise structures of these isomers could not be confirmed by 13 C-NMR and X-ray studies, which are unavailable due to the low solubility and poor crystallinity. We carried out measurements of FT-IR and 13 C-NMR spectra precisely after the isolation and further purification of two types of C 60 O 2 isomers and three types of C 60 O 3 isomers. Simultaneously, we demonstrated experimentally the regioselectivity of the epoxidation of C 60 by means of the identification of products from each oxidation of the isolated isomers.
Fullerene epoxides exhibit interesting properties applicable to new materials development. However, the chemical transformations of fullerene epoxides have been studied sparingly, despite the general recognition that they could serve as convenient starting materials for the synthesis of functionalized fullerene derivatives [10]. We first succeeded in converting fullerene epoxide stoichiometrically into a 1,3-dioxolane derivative [11]. Reaction of C 60 O with benzaldehyde in the presence of a Lewis acid led to the formation of a 1,3-dioxolane derivative of C 60 in high yield. This implies the possibility of other nucleophilic substitutions of the epoxy rings on a fullerene cage. The chemical transformation of fullerene epoxide is expected to play an important role in the development of functionalized fullerenes, because such transformations can readily afford a variety of mono-or polyfunctionalized fullerene derivatives, such as regioisomeric fullerene polyepoxides, while conserving the epoxy ring arrangement on the fullerene surface. The recent development of large-scale production techniques for fullerene epoxide [12] thus prompted us to develop a new methodology to synthesize polyfunctionalized fullerene derivatives by means of efficient chemical transformation of regioisomerically pure fullerene polyepoxides. Then, we also achieved the efficient formation of fullerene derivatives from C 60 O with aromatic nucleophilic compounds by Lewis acid-assisted nucleophilic substitution of the epoxy ring. The direct substitution of epoxide oxygen atoms on the fullerene epoxide-a versatile and advantageous synthetic methodology we report here-provides highly regioselective access to a variety of fullerene adducts.

Epoxidation of Fullerene C 60 and Their Regioisomeric Structure
The epoxidation of C 60 generally affords the higher fullerene epoxides (C 60 O n , n ≥ 2), which have only a few types of regioisomers. For instance, the epoxidation of C 60 with m-chloroperoxybenzoic acid (m-CPBA) forms a few C 60 O 2 regioisomers (see Figure 1 for positional notation of C 60 O) that can be separated into two fractions by high-performance liquid chromatography (HPLC) (peaks A or B in Figure 2), although from the standpoint of the multiple reacting sites available on a C 60 cage there are eight possible regioisomers of C 60 O 2 [6]. A major C 60 O 2 fraction is known to be composed of only one isomer with both oxygen atoms positioned over 6:6 ring junctions on a common six-membered ring of the carbon cage, namely the cis-1 adduct [5]. Meanwhile, a minor fraction of C 60 O 2 has been considered to contain more than one isomer [13]. These isomers could not be separated by any HPLC column and their precise structures have hardly been ascertained by 13 C-NMR or X-ray studies because of the low yield and the non-crystalline nature of the products.  The splitting pattern of each fraction in HPLC varies with both the position of the epoxy group on the C 60 cage and its structural symmetry. A detailed comparison of these patterns is informative in relation to the structure of existing C 60 O 2 isomers. In order to explain the isomeric structure of the preferentially formed C 60 O 2 , we also performed calculation of the static reaction index of oxygen addition sites on the C 60 cage, and compared the experimental observations with the calculated results [14].
In the past, the structure of C 60 O n isomers was studied on the basis of the thermodynamic stability of ground state molecules and the dynamic behavior of the molecules via the transition states. The results of these studies can explain the experimental observations regarding the structures of C 60 O and the predominant isomer of C 60 O 2 . Manoharan demonstrated computationally that the multiple epoxidations of C 60 preferentially proceeds at the adjacent rather than distant double bonds of the existing epoxide group [15]. By using a modified extended Hűckel method, Curry et al. also correctly predicted for C 60 O 2 that the cis-1 adduct has exceptionally low energy compared to the next stable isomers [8]. Feng et al. studied the possible structure of C 60 O 2 isomers by using the semiempirical quantum mechanical INDO method, and showed that three types of C 60 O 2 with the regioisomeric structures of cis-1, cis-2, and trans-4 adduct, respectively, should be very stable [7]. A more detailed theoretical elucidation of the regioselective epoxidation on fullerene, however, is required to provide information concerning the possible structures of other experimentally obtained C 60 O 2 isomers.
The reactivity for regioselective epoxidation on fullerene is assumed to be predicted by calculating the static reaction index for addition sites. On the basis of theoretical studies on the epoxidation of alkenes with peroxide [16,17], we hypothesized that the reaction index should fall between fullerene and m-CPBA. The electrophilicity of peroxide was attributed to its relatively weak O-O bond, which can provide an empty * orbital that can mix with the nucleophilic -bond of fullerene. The electrophilic oxygen tends to attack the bonds with a high electron density. In the same manner, the epoxidation of fullerene by the electrophilic oxygen of m-CPBA is likely to occur on electron rich double bonds. Thus the electron density of a bond is expected to give a good index of a probable reaction site. A similar calculation has been reported for the first oxygen addition sites on fullerenes [18].
Since the epoxidation of fullerene is a step-wise reaction, the yield for C 60 O n (n ≥ 2) can be calculated by considering the yield of the parent isomer C 60 O n−1 . The electron density of a bond has been determined by semiempirical molecular orbital calculations (PM3). The yield of the j-th isomer of C 60 O n , Y (n,j) , can then be determined by the summation of the product of the yield of i-C 60 O n−1 , Y (n−1,i) , and the branching probability from i-C 60 O n-1 to j-C 60 O n , p (i,j) as follows: where m denotes the number of the parent isomer. If the branching probability increases linearly with the difference in electron density, p (i,j) is defined by utilizing the difference between the electron density of the bond of the addition site and the minimum electron density of the bond that undergoes epoxidation as follows: where  j and N j denotes the electron density of the bond of the addition site j, and the number of the symmetrically equivalent bond of j, respectively. The constant c is the minimum electron density that undergoes epoxidation. The variables l denotes the number of equivalent bonds of the possible addition sites. Table 1 shows the calculated yield for the C 60 O 2 isomers, and the candidates for the dominant isomers based on the calculated yield. The cis-1 adduct overwhelmingly dominated the other isomers. The next dominant isomer of C 60 O 2 was predicted to be the equatorial adduct. The structure of the predominant isomer, the cis-1 adduct (1b in Figure 3), agreed with the structure actually confirmed by X-ray analysis [2], whereas the next dominant isomer is differed from a previous suggestion [1] that the C 60 O 2 isomers in fraction B are probably the cis-2 or trans-4 adducts. From both our experiment and calculations, at a minimum, we expect that the main product in fraction B is not the trans-1 but rather the equatorial adduct of C 60 O 2 (1c in Figure 3).

Structure
Symmetry

Lewis Acid-Assisted Reaction of Fullerene Epoxides with Nucleophiles
Epoxides are well-known to undergo heterolytic C-O bond cleavage in the presence of an acid catalyst to generate active carbocationic species for further reactions with nucleophiles. These active species react with various types of nucleophile such as carbonyl compounds, alcohols, and amines to afford 1,3-dioxolanes, 1,2-hydroxyethers, and 1,2-aminoalcohols, respectively. We have studied the reaction of fullerene epoxide 1a with various nucleophiles in the presence of a Lewis acid. Reaction with a carbonyl compound led to the formation of a 1,3-dioxolane derivative in high yield; interestingly, reaction with a phenol or aniline derivative afforded an O-or N-heterocycle-fused fullerene derivative. These reactions are considered to proceed via a nucleophilic reaction on the carbocationic active species generated on the carbon atom of fullerene epoxide 1a (Scheme 1). The formation of the heterocycle-fused fullerene derivative can be accounted for by the ring closure reaction induced by intramolecular dehydration of the initially formed 1,2-substituted product. In contrast, a Lewis acid-assisted nucleophilic reaction of C 60 O with an aromatic nucleophile such as toluene gave a 1,4-bisadduct. Application of these reactions to a regioisomeric fullerene polyepoxide would provide a convenient synthetic route to a variety of polyfunctionalized fullerene derivatives with a regioisomerically pure structure. These chemical transformations of fullerene epoxides are expected to play an important role in the development of functionalized fullerene cages. In this section, we review our recent progress in the work on the acid-assisted reaction of fullerene epoxides with various types of nucleophile.

Acetalization of Fullerene Epoxides
Several methods have been reported for the preparation of fullerene-fused 1,3-dioxolane derivatives, such as the reactions of C 60 fullerene with dioxiranes [3], acyl hypohalogenites [19], and 4a (0. 29) 4b (0. 28) diacyl peroxides [20], or the reaction of C 60 fullerene with a sodium alkoxide in the presence of air [21]. However, all of these reactions afforded fullerene-fused 1,3-dioxolane derivatives in only low to moderate yields, (i.e., <50%). As described in the previous section, we recently developed a preparative HPLC isolation method for some regioisomeric fullerene epoxides (Figure 3) [7]. Isolation of these epoxides prompted us to employ them as a starting material for efficient synthesis of fullerene-fused 1,3-dioxolane derivatives. We have found that the acetalization reactions of fullerene epoxide 1a with various carbonyl compounds occur in the presence of a Lewis acid catalyst, an ionexchange resin such as Amberlite, and a clay mineral such as montmorillonite to afford the corresponding 1,3-dioxolane derivatives in very high yields (  The reaction of a toluene solution of fullerene epoxide 1a with excess amounts (200 equiv.) of a benzaldehyde derivative at 75 °C in the presence of a Lewis acid catalyst led to the formation of 1,3-dioxolane 3 in very high yield. Table 2 also shows that a similar acetalization of 1a with other ketone compounds such as acetophenone, methyl ethyl ketone or cyclohexanone took place to give the corresponding 1,3-dioxolanes in moderate yields. Furthermore, a similar reaction of 1a with γ-butyrolactone in benzene afforded an ortho-ester type 1,3-dioxolane derivative in high yield, although in toluene, it resulted in the formation of a 1,4-addition product of toluene to 1a. This fact suggests that the nucleophilicity of a carbonyl group of an ester compound is not as strong as that of an aldehyde or a ketone, such that toluene instead acts preferentially as a nucleophile to the carbocationic species to yield a 1,4-addition product (1,4-bis(p-tolyl)-1,4-dihydro[60]fullerene). The Lewis acid catalyzed 1,4-addition reaction of aromatic nucleophiles to 1a will be discussed in Section 3. 2. Recently Gan and Zhang et al. reported that a Lewis acid such as BF 3 ·Et 2 O catalyzes the acetalization reaction of the fullerene epoxide bearing t-butylperoxo groups to yield the corresponding 1,3-dioxolane derivatives in moderate yields [23].
An epoxy ring is known to undergo a Lewis acid catalyzed acetalization to afford 1,3-dioxolane [24][25][26]. Generally the acetalization is assumed to follow a concerted S N 2-like mechanism involving backside attack of a carbonyl oxygen on an epoxide carbon followed by rotation of the C-C bond of the epoxide and then the second C-O bond formation to produce a 1,3-dioxolane [26]. However, such a backside attack on a fullerene epoxide is not expected to occur owing to occupation of the entire side opposite the epoxy ring by the fullerene cage. According to this view, an acetalization of a fullerene epoxide is not expected to occur unless an alternative mechanism is involved in a front side attack of a carbonyl compound on the epoxy ring of the fullerene epoxide. The acetalization reaction of fullerene epoxide 1a with benzaldehyde at 75 °C for 1 d did not proceed at all in the absence of a Lewis acid and resulted in the complete recovery of 1a. This result suggests that the Lewis acid catalyzes the acetalization reaction to proceed by a stepwise S N 1-like mechanism (Scheme 2). A Lewis acid induces a catalytic C-O bond cleavage of the epoxy ring to generate a carbocation on the carbon atom of the fullerene moiety. This is followed by nucleophilic attack of a carbonyl compound on the carbocation. An energetically unfavorable S N 1-like attack of a carbonyl compound on the carbocation from the fullerene surface would be forced to occur with the requirement of relatively high activation energy for the C-O bond cleavage of the epoxy ring.
The activation energy for the reaction of 1a with benzaldehyde was determined to be 112.7 kJ·mol −1 from the Arrhenius plots of ln k vs. 1/T, where k is the pseudo-first order rate constant and T is the reaction temperature. Based on the activation energy determined, the rate constant at room temperature (293 K) can be calculated to be 9.5 × 10 −7 s −1 , which is more than 10 3 times smaller than that at 75 °C (k = 1.43  10 −3 s −1 ). Therefore, that the Lewis acid catalyzed acetalization of 1a with benzaldehyde did not occur at room temperature is unsurprising, although a similar acetalization of oxiranes such as but-2-ene epoxide or styrene oxide readily proceeds at room temperature and even at 0 °C to yield 1,3-dioxolanes according to an energetically favorable S N 2-like mechanism [24][25][26].
The application of the above acetalization reaction to the regioisomerically pure fullerene di-epoxide 1b (cis-isomer) or 1c (equatorial isomer) would lead to chemical transformation of the two epoxy rings while conserving their arrangement on the fullerene cage. Under reaction conditions similar to those for mono-epoxide 1a, the fullerene di-epoxides 1b and 1c were also subjected to acetalization with benzaldehyde derivatives to give bis-1,3-dioxolanes 5a and 5b, respectively in high yields (Table 3) [9,21]. The visible absorption spectra of 5a and 5b show the bands characteristic to those of the di-epoxides 1b and 1c, respectively, that is, 5a showed a sharp band at 423 nm, which is the characteristic band for the cis-isomer 1b [11,15]. This fact suggests that during the acetalization reaction, no migration of the carbocation on the fullerene moiety occurs and that the acetalization reaction of the fullerene di-epoxide proceeds while the rearrangement of the two epoxy rings on the fullerene cage is conserved. The 1 H-NMR spectrum of 5a (R 3 = n-BuO) showed the two unequivalent acetal methine protons of equal intensity. In addition, it showed two sets of four phenyl protons and nine n-butoxy protons, also of equal intensity. Thus, the 1 H-NMR spectrum of 5a clearly demonstrates that the two phenyl groups are unsymmetrically disposed with respect to the fullerene moiety. The 13 C-NMR spectrum of 5a showed 51 signals for the fullerene sp 2 carbons, indicating a lack of symmetry of the fullerene cage. Based on the above NMR analysis, bis-1,3-dioxolane 5a is confirmed to have the structure of the stereoisomer depicted in Table 3. However, the configuration of the two phenyl groups in 5b remains undetermined because no definitive information suggesting their configuration could be obtained from the 1 H-NMR spectra of 5b. We assume that 5b is likely to be a mixture of two stereoisomers.
The fullerene di-epoxide 1b (cis-isomer) or 1c (equatorial isomer) also reacts with a ketone compound such as cyclohexanone in the presence of a Lewis acid catalyst or Amberlyst 15 ® to yield the corresponding bis-1,3-dioxolane derivative in moderate yields (50-70%) [27]. These acetalization reactions are presumed to proceed with conservation of their arrangement on the fullerene cage.
The present work has clearly shown that the epoxy moiety of fullerene epoxides can be readily converted to 1,3-dioxolane derivatives in high yield by treatment with a carbonyl compound in the presence of a Lewis acid, ion-exchange resin or clay mineral. The acetalization reaction proceeds by a stepwise S N 1-like mechanism which involves the nucleophilic attack of a carbonyl moiety at the carbocationic active species generated on the carbon atom of fullerene epoxides. Furthermore, the efficient bis-acetalization of a regioisomerically pure fullerene di-epoxide occurs under conservation of the arrangement of the two epoxy moieties to yield bis-1,3-dioxolane derivatives in high yield. These results could lead to development of a new methodology for facile synthesis of a variety of regioisomeric polyfunctionalized fullerene derivatives by means of efficient chemical transformation of a specific regioisomeric fullerene polyepoxide. In order to elucidate the reactivity of the carbocationic active species generated from fullerene epoxides, the next part of this section is oriented toward studies on the reaction of fullerene epoxides with nucleophiles other than carbonyl compounds.

Nucleophilic Substitution of Fullerene Epoxide with Aromatic Compounds
The reaction of a toluene solution of fullerene epoxide 1a in the presence of 5 equiv. of boron trifluoride etherate (BF 3 OEt 2 ) at r.t. for 60 min led to the formation of 1,4-bis(p-tolyl)-1,4-dihydro [60]fullerene (7a) in 80.8% yield together with a very small amount of pristine fullerene C 60 based on HPLC analysis [28]. Compound 7a was purified by flash column chromatography on a silica gel with n-hexane as an eluent. A detailed spectral characterization of 7a revealed that two p-tolyl moieties were introduced to the C 60 core in a 1,4-addition pattern. The FT-IR spectrum was in good agreement with that in an earlier report [29,30]. The characteristic vibrations for the 1,4-adduct were found at 1,430, 1,187, 573, and 527 cm −1 , and the C-H vibrations for toluene showed strong vibrations at 2,963 and 810 cm −1 . The atmospheric pressure photochemical ionization (APPI) mass spectrum showed a molecular ion peak at m/z 902 (i.e., 166 units more than 1a), corresponding to a 1:2 adduct of C 60 and toluene-H. In the 1 H-NMR spectrum, in addition to two doublets at 7.94 (2H), 7.93 (2H), 7.28 (2H), and 7.27 (2H) ppm for eight phenyl protons, one singlet methyl proton was observed at 2.46 (6H) ppm. The 13 C-NMR showed the characteristic absorption of aromatic carbons in the p-tolyl group at 138.25 (2C), 136.91 (2C), 129.52 (4C), and 126.94 (4C) ppm, and two quaternary carbons of the C 60 moiety were observed at 60.86(2C) ppm. The UV-VIS spectrum showed a characteristic absorption band around 450 nm. The 1,4-bisadduct structure for 7a was evidently confirmed by comparing these spectra with FT-IR, 1 H-NMR and 13 C-NMR spectra for similar compounds [29,30].
Reactions of 1 with other aromatic compounds were carried out under the same conditions as described above, the results of which are listed in Table 4. In anisole, o-xylene and m-xylene the reaction produced the corresponding 1,4-bisadducts (7b, 7c and 7d) in 45-95% yields, whereas neither the 1,2-(6e) nor 1,4-bisadduct (7e) was produced in p-xylene.  35.3 Trace g chlorobenzene 0 0 h benzene 0 0 a All reactions were performed according to the procedure described in the text; b,c All reactions were continued until complete disappearance of the starting material 1; d Overall yield of the structural isomeric mixture of 1,4-adducts.
Although the purification of 1,4-bisadducts 7a, 7b, and 7c was easily carried out with the HPLC apparatus, 3d was not well separated owing to the formation of structural isomers. In the case of 1,3,5-trimethylbenzene, the corresponding 1-(substituted phenyl)-2-hydroxyl-1,2-dihydro[60]fullerene (6f) was dominantly formed instead of the 1,4-bisadduct, as identified by UV-VIS, APPI mass, 1 H-and 13 C-NMR spectra. Usually the sp 3 carbons of the C 60 cage were observed to shift downfield ca. 10 ppm for 1,2-adducts relative to the 1,4-adducts. The two peaks at 86.9 and 72.0 ppm for the two sp 3 carbons of the fullerene core in 6f indicate a typical structure of 1,2-adduct. This suggests that the formation of 1,4-bisadduct from 1a proceeds stepwise via 1,2-adduct 6 as an intermediate. In fact, the formation of 6d can be observed as a transient intermediate in the course of the reaction in m-xylene. With respect to the UV-VIS spectrum beyond wavelengths of 300 nm, 6d is identical to 6f. The substitution reaction scarcely proceeded in benzene and chlorobenzene, even above 75 C, and pristine C 60 was observed instead of 1,4-adducts. The formation of pristine C 60 without epoxy group was observed. Thus, it appears that the progress of the reaction depends considerably on the nucleophilicity of the aromatic compound. In the absence of BF 3 OEt 2 , no reaction was observed in any solvent. From this general behavior, we may conclude that the first substitution of 1a to obtain 6 occurs via a carbocationic intermediate generated with the assistance of the Lewis acid, followed by a nucleophilic attack of an aromatic compound on the cation. In the transformation of 6 into 7, a reasonable assumption is that the substitution of the hydroxyl group proceeded by an S N 1-or S N 2-type mechanism with allylic rearrangement (Scheme 3). Scheme 3. Proposed mechanism for the substitution of C 60 O with aromatic compounds. Part of the fullerene surface is shown.
Generally, the Lewis acid-assisted substitution of the epoxy group is assumed to follow a concerted S N 2-like mechanism involving a backside attack of a nucleophilic reagent on an epoxide carbon atom [26]. However, such an attack is rather unrealistic, because the fullerene cage occupies the entire side opposite the epoxy ring. The first substitution of the epoxy ring on a fullerene, therefore, would occur with an energetically unfavorable S N 1-like attack of a nucleophilic reagent on the carbocation generated by the CO bond cleavage of the epoxy ring. On the other hand, the second substitution for 6 might occur by an S N 2'-type mechanism, in which the nucleophilic attack on the residual hydroxyl group occurs first, followed by the elimination of -OH on the side with an allylic rearrangement [31]. The fact that no reaction proceeded in the absence of the Lewis acid catalyst suggests that the second substitution occurs by an S N 1-or S N 2-type mechanism with allylic rearrangement, where 7 is formed via a carbocationic intermediate with an allylic resonance [32][33][34][35][36], generated with the assistance of BF 3 OEt 2 . In no case was the formation of 1,2-bis(substituted phenyl)adduct observed, presumably because of the steric hindrance between the two substituents.

Formation of Indolino[2',3':1,2][60]fullerene Derivatives from C 60 O and Aniline Derivatives
As shown in section 3.1, the reactions of C 60 O x with nucleophiles in the presence of acidic compounds have uncovered a means to achieve the regioselective functionalization of various fullerene derivatives. In extending this procedure, we found that a reaction of C 60 O with aniline derivatives in the presence of acidic compounds afforded the indoline-fused fullerene derivatives: indolino[2',3':1,2]-1,2-dihydro[60]fullerene (Figure 4), in moderate to excellent isolated yields (ca. 60-80%) [37]. To the best of our knowledge, only a few cases have thus far been reported on the preparation of indolino[60]fullerene analogues [38][39][40][41][42]. This method enabled access to a wide variety of indolino[60]fullerene derivatives because numerous anilines are commercially available. First, we examined the catalytic activities of different types of acidic compound for this reaction by using 4-dodecylaniline as a model compound. The examined acidic compounds were BiCl 3 , BF 3 ·OEt 2 , Montmorillonite K10, Sepiolite, TsOH, and Amberlyst 15 ® . The Brønsted acids TsOH and Amberlyst 15 ® did not exhibit catalytic activities for the reactions, and all of the C 60 O was converted to pristine C 60 . Although the Lewis acid compound; BiCl 3 gave products with moderate production yields (−50%), BF 3 OEt 2 did not give products and only the pristine C 60 was obtained. The clay compounds, Montmorillonite K10 and Sepiolite exhibited excellent catalytic activities toward the cyclization reactions (Table 5). The lack of catalytic activity of the Brønsted acid may be attributed to a neutralization process between the Brønsted acid and anilines. For BiCl 3 , the Lewis acidity may be slightly strong; thus, the epoxy oxygen may have been removed from the C 60 core more quickly than with the nucleophilic addition of anilines. In contrast, BF 3 OEt 2 forms a Lewis acid-base complex with aniline. The capped aniline may have lost its nucleophilicity, and thus completely prevented the reaction from proceeding. Cray minerals presumably have moderate Lewis acidity relative to BF 3 , and may thus be appropriate for the reaction.
Subsequently, in order to examine the versatility of this reaction, we carried out the reactions with a wide variety of anilines. The examined anilines and results are summarized in Table 6. Each aniline examined gave the corresponding indolino[60]fullerene derivative in good yield except for the reaction with N-Me-p-toluidine (Entry 8f). For 8f, the steric hindrance of the methyl group on the nitrogen atom might inhibit nucleophilic addition to the carbon atom of the fullerene core. Next, we attempted to reveal the reaction mechanism of the formation of indolino[60]fullerene from C 60 O and anilines. By tracing the reaction with an HPLC-MS system, we revealed that the cyclization reaction proceeded via 1,2-anilinoalcohol fullerene derivative. The time course of the HPLC chart and the change in the relative peak area for each compound are shown in Figure 5. At the start time, peaks corresponding to C 60 O at 9.3 min and slightly impure C 60 at 11.8 min appeared. At first, a new peak (9a) appeared at 5.4 min in the LC chart, and then, a peak corresponding to 8a at 6.8 min increased as the peak area of 9a decreased. Finally, the peaks of the starting C 60 O and 9a were completely consumed and the peak 8a and a small peak corresponding to pristine C 60 as a decomposed compound at 11.8 min were observed. Therefore, we surmised that 9a was an intermediate of the indolino[60]fullerene derivative. To confirm our assumption, isolated 9a with the catalyst under similar reaction conditions, whereupon the reaction gave 8a.
In order to assign each peak, we isolated and characterized compound 9a by means of IR, APPI-MS, and NMR spectra. In the IR spectrum, an absorption corresponding to O-H stretching was observed at 3,793 cm -1 . The MS spectrum of 9a showed a peak at m/z = 998. The MS of the peak corresponds to the summation of C 60 O (mw = 736) and dodecylaniline (mw = 262). As a result of 1 H-NMR, we observed in the range of 0.87-2.60 ppm, two doublet peaks of aryl protons corresponding to four aniline protons at 7.19 and 7.61 ppm, and one singlet peak of NH at 5.93 ppm. Taking these results into consideration, we assigned 9a to a 1-anilino-2-hydroxy[60]fullerene derivative (Scheme 4). Based on the experimental results, we proposed a plausible mechanism for the formation of indolino[60]fullerene (8) via 1,2-anilinohydroxy fullerene (9) (Scheme 4). At first, the epoxy ring is opened by adding a Lewis acid to the oxygen of the epoxide. Then, nucleophilic addition of anilines to the carbon atom underlying the epoxy oxygen forms a 1,2-anilinohydroxy fullerene derivatives. Consequently, the Lewis acid again adds to the oxygen atom of the hydroxyl group, and a C-C bond forms between the carbon of C 60 and the ortho-position of the anilino group to give the indoline-fused fullerene derivative.

Formation of Benzo[b]furano[2',3':1,2][60]fullerene Derivatives from C 60 O and Phenols
A similar nucleophilic addition to the fullerene core and the consequent cascade cyclization reaction with phenols via a 1-hydroxy-2-phenolic fullerene derivative as an intermediate was also observed  Unlike in the case of anilines, BF 3 ·OEt 2 exhibited superior catalytic activity for the reaction of C 60 O with phenols. The reaction of C 60 O with phenol derivatives in the presence of BF 3 ·OEt 2 was complete within 30 min for nearly all examined phenols. The different catalytic activities of the Lewis acids may be attributed to the Lewis acidity of a catalyst, the Lewis basicity of a nucleophile, or both. For the reaction with anilines, a complexation between BF 3 and the amine occurs preferentially over the addition of BF 3 to the epoxy oxygen. Therefore, either BF 3 or the aniline is completely consumed, and the reaction cannot proceed at all. On the other hand, for benzo[b]furano[60]fullerenes, the Lewis basicity of nearly all phenols is weaker than that of anilines. BF 3 thus effectively catalyzes the reaction of epoxy ring opening, nucleophilic addition, and cyclization.
The production yields of the benzo[b]furano[60]fullerene derivatives were subjected to the electrondonating and withdrawing properties of the substituent of the phenols. The isolated yields are summarized in Table 7. Unlike the case of anilines, the yields of benzo[b]furano[60]fullerene derivatives were strongly influenced by the type of substituent of the phenols. Figure 7 shows a plot of the isolated yields for benzo[b]furano[60]fullerene derivatives as a function of the Hammett constant for the 2-position of the precursor 4-substituted phenols [44]. Negative Hammett constants correspond to electron-withdrawing (EW) property, and positive ones correspond to the electron-donating (ED) property. The stronger ED property of the substituent increases the nucleophilicity of the oxygen atom of phenols and the carbon atoms of ortho-position of the phenols. In contrast, EW groups reduce the nucleophilicity of the phenolic oxygen and the carbon atoms of ortho-position. Therefore, ED functional groups improve the production yields of nucleophilic addition and cascade cyclization reactions. Conversely, 4-nitorophenol could not produce the benzo[b]furano[60]fullerene owing to the strong electronwithdrawing property of the nitro group. The low yield with pristine phenol (entry 10h) is presumably due to the competitive formation of the 1,4-bisadduct (Section 3.2), because the para-position of the phenol is not substituted.

Conclusions
In summary, this review presents an overview of our works on the nature and chemical transformation of fullerene epoxides. The structures of the formed fullerene epoxides, C 60 O n , were characterized on the basis of their LC-MS and 13 C-NMR spectral analyses. The number of carbon peaks and structural symmetry correlations enable the different oxygen addition sites on the fullerene framework to be distinguished. The obtained results show highly regioselective exohedral oxygenation of fullerene with oxidizing agents such as m-CPBA and ozone.
A variety of fullerene derivatives are easily prepared from fullerene epoxides, in which the epoxy groups on the fullerene framework are efficiently activated to undergo chemical transformations in the presence of a Lewis acid. We have found several Lewis acid-assisted reactions of fullerene epoxide with various types of nucleophiles. In all of the examined nucleophilic substitution reactions on fullerene epoxides, carbon-oxygen bond cleavage of the epoxy ring occurs with a front side attack of a nucleophile. Moreover, the substitution reaction of the fullerene di-epoxide proceeds while the rearrangement of the two epoxy rings on the fullerene cage is conserved. Fullerene epoxides are 0.39"materials in many industrial fields.

Acknowledgments
The author is deeply grateful to Kazuo Takeuchi


text:
Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel

Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-β-D-GlcpA and pentasaccharide β-D-Xylp-(1→3)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[β-D-Quip-(1→4)]-β-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

Introduction
Triterpenoid saponins [1] occur in many plant species and have a diverse range of properties [2]. Nowadays, a steadily growing number of publications [3][4][5][6][7][8][9][10] are aimed at research on saponins as potential adjuvants, with an urgent demand due to the fast development of immunotherapy methods. Among the most efficient saponin adjuvants are the components of a complex mixture of triterpenoids extracted from the bark of Quillaja saponaria Molina, which are used in veterinary vaccines [4]. One of the best known products of this origin is the less toxic and more stable fraction QS-21 [8], which is included now into a vast range of pilot vaccine compositions against viral infections [11][12][13] and cancer [14][15][16][17].
Meanwhile, the search for abundant, nontoxic, stable and individual saponin adjuvants is still urgent. This explains the enormous interest in investigations into saponins, particularly concerning the study of the relationships between their structure, adjuvant activity and toxicity [5,7]. In this paper we report the isolation and structural assessment of two saponins from the roots of A. gypsophiloides Rgl. (Turkestan soap root) with the further investigation of their toxicity, hemolytic activity, antiinflammatory, antifungal and adjuvant properties. The roots of A. gypsophiloides Rgl. are an easily available raw material, which was reported [18] to comprise a saponin with a structure that is close to that of the extremely efficient adjuvant QS-21.
A. gypsophiloides Rgl. is a member of the genus Caryophyllaceae (for other saponins see [19][20][21][22]), which is widely spread in mountain areas of central Asia. The crude saponin-containing fraction from the roots of A. gypsophiloides Rgl. has been known to be an excellent foaming agent for food and nutrition industry, and its composition has previously been under investigation [18].

Results and Discussion
The methanolic extract of the dried powdered roots of A. gypsophiloides was concentrated, and the crude mixture of saponins was precipitated from methanol by the addition of acetone and subjected to reversed-phase С18 HPLC. Compounds 1 and 2 (  The structures of both compounds 1 and 2 were confirmed on the basis of their 1 H NMR, 13 C NMR, APT, COSY, TOCSY, ROESY, HSQC, and HMBC spectra. In accordance with the earlier reports [18] on structures of saponins from A. gypsophiloides, the aglycons of compound 1 and 2 were supposed to comprise quillaic acid (16-α-hydroxygypsogenin) and gypsogenin, respectively. This assumption was in good agreement with the detection of characteristic signals for six methyl groups in the 1 H (Table 1) and 13 C NMR (Table 2) spectra of 1 and 2. Furthermore, the presence of these aglycons was unambiguously confirmed by the good agreement between 13 C NMR shifts of aglycon moieties of 1 and 2 and signals of aglycons for described bidesmosides comprising quillaic acid [21] and gypsogenin [21].

Comp.
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-   Analysis of COSY and TOCSY spectra of both 1 and 2 revealed the presence of the following residues: β-GlcpA (residue a), β-Quip (6-deoxy-β-Glcp, residue e), α-Rhap (residue f), β-Xylp (residues g and h). The HSQC spectrum confirmed the structures of the triterpene aglycon and showed the positions of the substitutions within the oligosaccharide fragments (Table 1 and Table 2). The ROESY spectra (identical for compounds 1 and 2) disclosed the sequence of the residues in two oligosaccharides and their location at the C-3 and C-28 of the aglycon. Thus, the location of GlcA (residue a) at the position 3 of the triterpene was established from the presence of a correlation peak 1a/3Agl ( Figure 2 and Figure 3). Correlation peaks 1b/2a and 1c/3a correspond to substitutions of the residue a by terminal b at the position 2 and by terminal c at the position 3. Esterification of the position 1 of Fuc (residue d) with the carboxy group of the triterpene was unambiguously shown by the high-field shift of C-1 (94.4 ppm), being indirectly confirmed with the long-range correlation peak in the ROESY spectra 16Agl/3d. The sequence of the other residues was disclosed from the presence of the correlation peaks 1e/4d, 1f/2d, 1g/4f and 1g/4h ( Figure 2). HMBC spectra finally confirmed the structure of the aglycons and the sequence of the residues. Thus, the correlation peak 1d/28Agl evidenced the location of Fuc (residue d) as the esterified substituent at C-28 of the triterpene ( Figure 4). The other inter-residue correlation peaks were in agreement with the structure of oligosaccharides established from analysis of the ROESY spectra.
Characteristic chemical shifts in the 13 C NMR spectrum of 2 (δ C 85.4 ppm for C-3 and δ C 176.4 ppm for C-28 of the aglycon) evidence the bidesmosidic nature of the genin, which is glycosydated at C-3 and esterified to an oligosaccharide. The structures of both the trisaccharide and pentasaccharide frag-     It should be noted that the elucidated structures of 1 and 2 are different from those reported earlier [18]. In the published structures β-D-Quip (residue e) is located at O-2 of the β-Fucp (residue d), and the trisaccharide moiety The acute-toxicity study of saponins 1 and 2 was carried out on albino mice. The median lethal dose (LD 50 ) was determined after a single dose administered through the oral or intraperitoneal route. The obtained data show that, in the case of oral administration of the studied compounds, LD 50 was in the range of 304 ± 55 mg/kg for compound 1 and 252 ± 57 mg/kg for compound 2 with p < 0.05 (t). In the case of intraperitoneal administration, the LD 50 was in the range of 15.1 ± 5.6 mg/kg for compound 1 and 5.4 ± 2.8 mg/kg for compound 2 with p < 0.05 (t). The immense difference between the values of LD 50 in oral and intraperitoneal tests evidences low or no absorption of saponins in the intestine. However, it remains to be studied whether traces or decomposition products of ingested saponins enter the blood stream through the permeable membranes of mucosal cells.
For compounds 1, 2 and saponin from Quillaja bark (Sigma) as a reference compound, the study on in vitro hemolysis was carried out. The obtained data confirmed high hemolytic activity of the Quillaja bark saponin, which caused 100% of hemolysis at a minimal hemolytic concentration of 5.5 μg/mL. Saponins 1 and 2 exhibited much lower hemolytic activity (Table 3): 50% hemolysis was observed at concentrations 11-18 μg/mL. Hemolysis of 85-95% for compounds 1 and 2 was observed at 62.5 μg/mL, whereas saponins QS-17, 18, and 21 from Quillaja bark were reported [23] to cause hemolysis at  concentrations of 7-25 μg/mL. These results are in good agreement with our expectations based on the factors that are known to accompany low hemolytic activity, i.e., the bidesmosidic nature of compounds 1 and 2, the presence of glucuronic acid at C-3 of the aglycone, and the absence of a lipid moiety.
Histamine-induced acute inflammation in the paws of the mice was used as a classical model of edema formation for the study of the anti-inflammatory activity of saponins. Two methods of saponin administration were used, namely oral (Table 4) and intraperitoneal (Table 5). In the first experiment, six groups of eight mice each were treated orally with compound 1 (20 mg/kg, 50 mg/kg), compound 2 (20 mg/kg, 50 mg/kg), indomethacin (20 mg/kg), and water (control). One hour after receiving the agents, each animal received a subcutaneous injection of 0.05 mL 0.1% histamine in the right, hind paw. The edema was measured 5 hours after the histamine injection. The anti-inflammatory effect was assessed by the decrease in the index of edema compared with the control group, which is defined as the percentage difference between the mass of the healthy and the inflamed paw, relative to the mass of the healthy paw.
In general, the anti-inflammatory effect of saponins 1 and 2 given intraperitoneally was dose-dependent, whereas that in the experiment with oral administration was not. Within the experiment based on oral administration, compound 1 did not show any reliable anti-inflammatory action. Data given in Table 5 evidences the more pronounced anti-inflammatory properties of compound 2 as compared to compound 1 in the experiment based on intraperitoneal administration.
The influence of saponins 1 and 2 on the vessel endothelium was assessed via determination of interleukin-6 (IL-6) production in primary human umbilical vein endothelial cells (HUVEC). These cells were found to express various patternrecognizing receptors (PRRs), including TLR4 [24], and can produce pro-inflammatory cytokines, including IL-6, IL-8 and IL-1β, upon stimulation with bacterial and viral components, such as lipopolysaccharides (LPS) and double-stranded RNA (dsRNA) [25].
HUVEC were cultivated in the presence of saponins 1 and 2 at nontoxic concentrations, i.e., 1 and 5 μg/mL, as determined by EZ4U test, and reference compounds LPS from E. coli and dsRNA analogue poly(I:C). Measurement of IL-6 production did not show any effect of saponins 1 and 2 on IL-6 secretion, either by testing intact endothelial cells or cells pre-stimulated with LPS or poly(I:C) ( Figure 5), indicating that at nontoxic concentrations saponins are not able to induce an innate immune response in endothelium. Thus we can conclude that compounds 1 and 2 are not prone to cause inflammation of the vessel endothelium. To estimate the adjuvant properties of compounds 1 and 2 a series of test immunizations was carried out by using the synthetic vaccine neoglycoconjugate α-NeuAc-(2→3)-β-Galp-(1→4)-β-Glcp-KLH (3'SL-KLH) on the basis of 3'-sialyllactoside (3'SL) ligands and keyhole limpet hemocyanin (KLH) protein carrier [26]. Four groups of mice were immunized with 40 µg of 3'SL-KLH together with 50 µg of compound 1 or 2, or saponin from Quillaja bark, or without an adjuvant, and the specific anti-3'SL IgM and IgG responses were evaluated. For saponins 1 and 2, a significant specific response was observed in comparison with the control vaccine formulation with antigen alone ( Table 6). High serum titers of IgM and IgG antibodies were registered in the vaccination with compound 1 as adjuvant, though the IgG level did not achieve the level measured in the experiment with saponin from Quillaja bark. Titers of those antibodies in the experiment with saponin 2 were rather low.
We can conclude, that in combination with 3'SL-KLH, antigen compound 1 showed significant adjuvant properties and, hence, can be considered as a prospective component of vaccine formulations.  Table 7 demonstrate that compounds 1 and 2 exhibit antifungal activity against both ascomycetous and basidiomycetous yeasts, including the medically important C. albicans and F. neoformans. Saponins are known to be more effective against basidiomycetous yeast and at acidic pH act similarly to natural detergents, such as cellobiose lipids [27]. Growth inhibition experiments showed ( Figure 6, Table 7), that the lower pH 4.0 favored antifungal activity of saponins 1 and 2. At pH 7.0 neither compound 1 nor 2 inhibited the growth of C. albicans and F. neoformans. However, at pH 4.0 both saponins exhibited suppressing properties against these two strains. Notably, compound 2 was totally inactive against S. cerevisiae at both pH values. The liquid medium test involving   F. neoformans showed only slight differences between saponins 1 and 2 (Table 8).

Conclusion
Thus, the structures of two easily available novel saponins 1 and 2 were elucidated, and they were shown to exhibit low hemolytic activity, low oral and intraperitoneal toxicity, and an inability to induce inflammation in the vessel endothelium. Meanwhile, compounds 1 and 2 exhibited prominent immunestimulating properties and can be considered as a prospective adjuvant in combination with KLH-based neoglycoconjugates. The antifungal activity of saponins 1 and 2 was also examined on four yeast species.

Experimental
General experimental procedures. Optical rotation values were measured on a JASCO DIP-360 polarimeter at 22 ± 2 °C. NMR spectra were recorded on Bruker DRX-500 and Bruker AM-300 instruments in D 2 O/pyridine-d 5 with TMS as internal reference. High-resolution mass spectra (HRMS) were measured on a Bruker micrоTOF II instrument by using electrospray ionization (ESI) [28]. The measurements were done in a positive-ion mode (interface capillary voltage: 4500 V) or in a negative-ion mode (3200 V); mass range from m/z 50-3000 Da; external or internal calibration was achieved with electrospray calibrant solution (Fluka). A syringe injection was used for solutions in acetonitrile, methanol, or water (flow rate 3 μL/min). Nitrogen was applied as a dry gas; the interface temperature was set to 180 °C. Monosaccharide analyses. Compounds 1 and 2 were hydrolyzed with 2 M CF 3 CO 2 H (120 °C, 2 h) and the absolute configurations of the monosaccharides were determined by GLC of acetylated (S)-(+)-2-octyl glycosides according to the published method [29]. GLC was performed using an Agilent 7820A chromatograph equipped with an HP-5 fused silica column (0.25 mm × 30 m) using a temperature program of 160 °C to 290 °C (7 °C min −1 ).
Oral route: The experiment involved 50 male mice. Animals were selected at random and divided into five groups, each consisting of 10 mice. The control group received only pelleted food and water. The other four groups received pelleted food and water along with varying doses of compounds 1 or 2, at either 50.0 mg/kg, 100.0 mg/kg, 250.0 mg/kg or 500.0 mg/kg. Saponins were given by single oral gavage in the prescribed doses by using a feeding cannula. The acute LD 50 toxicity of saponins 1 and 2 was calculated on the basis of the mortality data collected within seven days by using Probit Analysis 1.0 software with p < 0.05.
Intraperitoneal route. The experiment involved 50 male mice. Animals were selected at random and divided into five groups, each consisting of 10 mice. The control group received only pelleted food and water. The other four groups received pelleted food and water and were inoculated once intraperitoneally with solutions of varying doses of saponins 1 or 2, at either 0.50 mg/kg, 5.0 mg/kg, 10 mg/kg or 50.0 mg/kg in saline. The acute LD 50 toxicity of saponins 1 and 2 was calculated on the basis of the mortality data collected within seven days using Probit Analysis 1.0 software with p < 0.05.
Hemolysis assay. Red blood cells were obtained from Wistar sexually mature rats of both sexes from SPF-vivarium of SB RAS, weighing 200-250 g. Blood was collected from neck vessels in standard plastic tubes containing 3.8% solution of sodium citrate. Aliquots of 7 mL of citrated blood (volume ratio of blood to sodium citrate 9:1) were washed with sterile nonpyrogenic saline (0.89% sodium chloride). Washing was performed by adding an equal volume of saline solution to an aliquot of citrated blood and subsequent centrifugation at 180 g for 5 min, after which the supernatant was discarded, and the procedure repeated three times. Harvested erythrocytes were diluted with saline to obtain a suspension of 0.5% hematocrit.
Samples containing 0.5 mL of cell suspension were mixed with 0.5 mL of saline solutions (145 mM, isotonic conditions) containing the investigated saponins in concentrations of 5, 10, 15, 25, 50, 75, 100, 250, 500 μg/mL. Samples were stirred continuously for 30 min at 37 °C and then centrifuged at 70g for 10 min. The content of free hemoglobin in the supernatant was measured by spectrophotometric analysis at a wavelength of 412 nm (spectrophotometer Cary 50, Varian). Hemoglobin concentration in the supernatant was expressed as a percentage of hemoglobin concentration in the supernatant of cells, which were totally hemolysed by the addition of distilled water. The absorbance of samples with 0% hemolysis was registered for samples with saline and used as a blank measurement. The degree of hemolysis, depending on the concentration of saponin was calculated by using Probit Analysis 1.0 software.
Anti-inflammatory activity. Albino, nonbreeding, sexually mature male mice from SPF-vivarium of SB RAS weighing 20-25 g were used in the test. All research involving laboratory animals was carried out in accordance with The Guidelines for the Care and Use of Laboratory Animals. Mice were housed in appropriate caging facilities and allowed food and water ad libitum.
Oral route: Six groups of eight mice were treated orally with saponin 1 (20 mg/kg, 50 mg/kg), saponin 2 (20 mg/kg, 50 mg/kg), and indomethacin (20 mg/kg) in Twin-80 water solution (control). One hour after receiving the agents, each animal received a subcutaneous injection of 0.05 mL 0.1% histamine in the right hind paw. The edema was measured 5 h after the histamine injection as the difference in weight between the paw that was administered histamine, and a healthy paw. The anti-inflammatory effect was assessed by the decrease in index of edema compared with the control group. The index of edema is defined as the ratio of the difference between the masses of the inflamed and healthy paws to the mass of the healthy paw in percent: (Mi − Mh) / Mh × 100%; Mi: mass of inflamed paw, Mh: mass of healthy paw. A probability of p < 0.05 was considered significant.
Intraperitoneal route: Six groups of eight mice were treated intraperitoneally with saponin 1 (1.25 mg/kg, 2.5 mg/kg), saponin 2 (1.25 mg/kg, 2.5 mg/kg) and indomethacin orally (20 mg/kg) in Twin-80 water solution (control). One hour after receiving the agents, each animal received a subcutaneous injection of 0.05 mL 0.1% histamine in the right hind paw. The edema was measured 5 h after the histamine injection as the difference in weight between the paw that was administered histamine, and a healthy paw. The anti-inflammatory effect was assessed as described above. Glycoconjugate vaccine preparation: Conjugate (3'SL-KLH [28]) of α-NeuAc-(2→3)-β-Galp-(1→4)-β-Glcp ligands with keyhole limpet hemocyanin carrier (KLH), bearing 5% mass of indicated carbohydrate, was used as an immunogen, and saponins 1 and 2 and saponin from Quillaja bark were used as adjuvants. All samples were filtered through 0.22 μm Micro-pore® filters and kept at 4 °C prior to use.

Cytotoxicity, cell proliferation test and endotoxin test
Immunization: Four groups of seven mice each were immunized intramuscularly thrice, on days 0, 14, and 84 with a mixture of 40 μg of conjugate and 50 μg of different saponins (or without adjuvant for the control group) in PBS as a vehicle in a total vaccine volume of 200 μL.
ELISA: Sera from inoculated mice were collected on day 91 post-inoculation (p.v.) of the first dose of vaccine and pooled. The titers for IgG and IgM against α-NeuAc-(2→3)-β-Galp-(1→4)-β-Glcp were determined in an indirect ELISA as previously described [31]. ELISA plates (96-well, Nunc Maxisorp) were coated with a cover polyacrylamide antigen [28] with α-NeuAc-(2→3)-β-Galp-(1→4)-β-Glcp moieties. Coating was performed with a 10 μg/mL in 0.1 M bicarbonate buffer solution at 4 °C overnight. Wells were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and blocked with 1% solution of HSA in PBS-T. Diluted sera (100 µL) collected from the mice in PBS-T was added to wells and incubated overnight at 4 °C. The plates were washed three times with PBS-T, and goat anti-mouse IgG or IgM peroxidase conjugate (Jackson Immuno Research) in 1:1500 dilution (PBS-T) was added to the wells. Plates were then incubated for 1 h at 37 °C and washed, and substrate (o-phenylenediamine, 0.4 mg/mL in 0.1 M phosphate-citrate buffer with 0.0013% H 2 O 2 ) was added to each well. Plates were then incubated for 25 min at 37 °C, after which the reaction was terminated by adding 50 μL per well of 2 N H 2 SO 4 . The optical density (OD) was measured in an ELISA plate reader at 492 nm. Data were expressed as the mean OD value of the samples minus the mean OD value of the control wells. The value of the OD for the control group was less than 0.1 (dilution 1/100 and more). Antibody levels in the sera of all samples were higher than the control (p < 0.05).
Antifungal activity assay: Sterile 5 mm diameter glass microfiber filter discs GF/A (Whatman, UK) were placed onto the surface of a solid medium in Petri dishes inoculated with test cultures. Two media were used: YPD containing 0.5% glucose, 0.2% yeast extract, 0.25% peptone, 2% agar, and 0.04 M citrate-phosphate buffer (pH 4.0), and YPD with 2% agar (pH 7.0). Aliquots of saponin solutions in deionized water were pipetted onto discs. The plates were incubated at 24 °C for 2-3 days until growth of the lawn strain appeared, and the diameters of the growth inhibition zones were measured. For the assay of cell viability the suspension of the cells F. neoformans (3 × 10 6 cells mL −1 ) was treated with saponins in 0.01 M citrate buffer (pH 4.0) at room temperature for 1 h. Thereafter, the samples were diluted by the same buffer and inoculated on YPD agar. Three days later, the number of colonies was determined. The samples without saponins were used as a control. All experiments were repeated twice. Fresh solutions of saponins in deionized water were used.


text:
Bis{μ-2-[(dimethylamino)methyl]benzeneselenolato}bis[chloridopalladium(II)] dichloromethane hemisolvate

The asymmetric unit of the title compound, [Pd2(C9H12NSe)2Cl2]·0.5CH2Cl2, contains two half-molecules, each lying on a twofold axis; each molecule is chiral and of the same enantiomer. This is only possible as the molecule has a hinged cis arrangement about the Pd2+ coordination spheres. For this hinged dimeric structure, the angles between the two coordination planes in each molecule are 15.02 (5) and 14.91 (5)°. This hinged cis arragement also allows the two molecules to form pairs linked by secondary interactions between the Pd and Se atoms [3.4307 (9) and 3.4317 (9) Å] of adjoining molecules, leading to an overall tetrameric structure. During the refinement stages, it was noticed that there were dichloromethane solvent molecules present disordered about a twofold axis. After unsuccessful attempts were made to model this, they were removed using SQUEEZE.

The asymmetric unit of the title compound, [Pd 2 (C 9 H 12 NSe) 2 Cl 2 ]Á0.5CH 2 Cl 2 , contains two half-molecules, each lying on a twofold axis; each molecule is chiral and of the same enantiomer. This is only possible as the molecule has a hinged cis arrangement about the Pd 2+ coordination spheres. For this hinged dimeric structure, the angles between the two coordination planes in each molecule are 15.02 (5) and 14.91 (5) . This hinged cis arragement also allows the two molecules to form pairs linked by secondary interactions between the Pd and Se atoms [3.4307 (9) and 3.4317 (9) Å ] of adjoining molecules, leading to an overall tetrameric structure. During the refinement stages, it was noticed that there were dichloromethane solvent molecules present disordered about a twofold axis. After unsuccessful attempts were made to model this, they were removed using SQUEEZE.
HBS acknowledges the DST, New Delhi, for financial support. TC acknowledges the CSIR, New Delhi, for a fellowship. RJB acknowledges the NSF-MRI program (grant CHE-0619278) for funds to purchase the diffractometer.

Comment
The coordination chemistry of transition metal complexes with both organoselenide and organotelluride ligands is a rapidly growing area due to the ability of the resulting complexes to find applications in materials science (Morley et al., 2006;Ford et al., 2004), and investigations of oxidation additive to low valent transition metal centers. In addition to this, organotellurium compounds have been used in catalytic carbon-carbon formation. Bridged dimers of palladium mediated by Se (Nakata et al., 2009;Chakraborty et al., 2011;Oilunkaniemi et al., 1999;Oilunkaniemi et al., 2001;Brown & Corrigan, 2004;Dey et al., 2006) or Te (Oilunkaniemi et al., 2000;Kaur et al., 2009;Dey et al., 2006) have been previously reported. Such dimers involving two square planar coordination spheres can adopt either a coplanar or hinged arrangement. The arrangement of the donor ligands with respect to the bridging plane can be cis or trans. In the case of a hinged cis arrangemnt the possibility of chirality exisits. While the majority of previously determined Se/Te bridged Pd dimeric structures are both coplanar and trans, there have been a small number which exhibit either a hinged or cis arrangment of ligands about the bridging plane (Kaur et al., 2009;Oilunkaniemi et al., 2000). However, in no previous case has this resulted in a chiral structure.
C 18 H 24 Cl 2 N 2 Pd 2 Se 2 , crystallizes in the chiral orthorhombic space group, P2 1 2 1 2. The asymmetric unit contains 2 half molecules, each lying on a 2-fold axis and each molecule is chiral and of the same enantiomer. This is only possible as the molecule has a hinged cis arrangement about the Pd coordination spheres (Fig. 1). For this hinged dimeric structure the angles between the two coordination planes in each molecule are 15.02 (5) and 14.91 (5)° respectively. This hinged cis arragement also allows the two molecules to form pairs linked by secondary interactions between the Pd and Se of an adjoining molecule (Fig. 2) leading to a tetrameric overall structure. Apart from this the Pd-Se, Pd-Cl and Pd-N bond lengths are in the normal ranges.

Experimental
The ligand and complex were prepared using previously reported methods (Chakraborty et al., 2011). Crystallization of the selenolate was done at ambient temperature from dichloromethane/hexane (2:1).

Refinement
H atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with C-H distances of 0.95 -0.97 Å [U iso (H) = 1.2U eq (CH, CH 2 ) [U iso (H) = 1.5U eq (CH 3 )]. During the refinement stages it was noticed that there were disordered solvent molecules present. The solvent molecule is CH 2 Cl 2 and it is disordered about a 2-fold axis. After unsuccessful attempts were made to model this, it was removed using the SQUEEZE routine from PLATON (Spek, 2009 Fig. 1. The structure of one of the two molecules of the asymmetric unit showing the hinged cis arrangement of the two Pd coordination planes. The two halves of the molecule are related by 1 -x, 1 -y, z.

Special details
Experimental. The structure of the Te analog was also determined, but at low temperature. This compound is isostructural and isomorphous with the Se compound but in this case the solvent was ordered. An Acta E submission for this structure has been made and it is currently under review (jj2116). Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating Rfactors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.  (11) 117. Symmetry codes: (iv) x+1/2, −y+1/2, −z+2; (v) −x+1/2, y−1/2, −z+1.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 )
supplementary materials sup-9