Structural analysis and antitumor activity of androstane D-seco-mesyloxy derivatives

ARTICLE

Structural analysis and antitumor activity of androstane D-seco-mesyloxy derivatives

Aleksandar M. Oklješa^I; Suzana S. Jovanović-Šanta^I,*; Olivera R. Klisurić^II; Marija N. Sakač^I; Evgenija A. Djurendić^I; Dimitar S. Jakimov^III; Lidija D. Aleksić^III; Katarina M. Penov Gaši^I

^IDepartment of Chemistry, Biochemistry and Environmental Protection and Department of Physics, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia

^IIDepartment of Physics, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia

^IIIOncology Institute of Vojvodina, Put Dr Goldmana 4, 21204 Sremska Kamenica, Serbia

ABSTRACT

The study of the influence of steroidal compounds on tumor cell cultures, cell growth, induction of apoptosis and/or cell cycle changes, is a common way of discovering potential therapeutics for treating people suffering from hormone-dependent problems and diseases. Because of the very high mortality rate associated with this class of disease, therapeutics for treating different types of cancers are among the most important. This work presents the synthesis of two stereoisomeric 16,17-secoandrostane mesyloxy derivatives and their 17-hydroxy precursors. Compounds were structurally analyzed by X-ray crystallography, and their antiproliferative activity, influence on the cell cycle and potential to induce apoptosis were investigated.

Keywords: 16,17-seco mesyloxy androstane derivatives, X-ray crystallography, tumor cell cytotoxicity, apoptosis, cell cycle

RESUMO

Neste artigo, a influência dos compostos esteróides nas culturas de células tumorais foi estudada: o crescimento de células, a indução de apoptose e/ou alterações do ciclo celular. Esta é uma forma comum na descoberta de compostos terapêuticos potenciais para o tratamento de pessoas que sofrem de doenças dependentes de hormônios. Os tipos de terapêutica para o tratamento de diferentes tipos de câncer são os mais importantes devido à elevada taxa de mortalidade relacionada com esta classe de doenças. O trabalho apresenta a síntese de dois estereoisómeros 16,17-secoandrostane mesilados e seus precursores 17-hidroxi, e o estudo da sua atividade antiproliferativa, influência sobre o ciclo celular e indução de apoptose, assim como a análise cristalográfica destes compostos.

Introduction

Androgens play an important role in the development of diseases such as benign prostate hyperplasia and prostate cancer.^1,2 On the other hand, estrogen-dependent breast cancer is the most common cancer among women and continues to be a major cause of cancer-related deaths.³ The efficacy of adjuvant endocrine therapy in hormone receptor-positive early breast cancer is constantly examined.⁴ Since many estrogen and androgen derivatives exhibit anti-hormone and/or cytotoxic activity, a large number of steroidal derivatives have been synthesized from appropriate estrogen or androgen precursors in order to obtain potential therapeutics for the treatment of steroiddependent cancers.^5-15

It is known that many sulfur-containing compounds display biological potency.^16-19 In addition, some steroidal compounds induce apoptosis or cause changes in the cell cycle of tumor cells.^20-22 In light of the above, as a continuation of our ongoing efforts on D-seco steroid synthesis as potential anticancer agents, in the present study, we report the structural analysis of recently (5)²³ and newly (6) synthesized stereoisomeric D-seco-mesyloxy derivatives, the study of their antiproliferative activity against tumor cell lines and their impact on the cell cycle and apoptosis of tumor cells.

Experimental

Chemical synthesis

General

Melting points were measured on a Nagema Rapido melting point microscope. Infrared (IR) spectra were recorded on a Nexus 670 FTIR spectrometer. ¹H (250 MHz) and ¹³C (62.5 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AC 250 apparatus using tetramethylsilane as an internal standard. High-resolution mass spectroscopy (HRMS) data (TOF) were recorded on a 6210 time-of-flight LC/MS Agilent Technologies (ESI⁺) instrument. Organic solutions were dried with Na₂SO₄. Column chromatography (CC) was performed on silica gel 60 (0.04-0.063 µm, Merck).

3β-Acetoxy-(17S)-mesyloxy-17-methyl-16,17-secoandrost5-ene-16-nitrile (5) and 3β-acetoxy-(17R)-mesyloxy17-methyl-16,17-secoandrost-5-ene-16-nitrile (6)

A mixture of compounds 3and4served as starting material for the synthesis of mesylates 5 and 6. Compounds 5 and 6 were separated by flash chromatography (petroleum ether-ethyl acetate 3:1; 2:1; 1.5:1) giving pure compound 5 (0.4938 g, 78%, white crystals, mp 158-160 ºC, after crystallization from ethyl acetate/hexane) and 6 (0.0579 g, 9%, white crystals, mp 160-161 ºC, after crystallization from ethyl acetate/hexane).

Compound 5: IR n/cm^-1 3019, 2943, 2243, 1728, 1247, 1173, 896, 755; ¹H NMR (250 MHz, CDCl₃) δ 1.041 (s, 3H, H19); 1.050 (s, 3H, H18); 1.412 (d, 3H, J 6.25 Hz, H20); 2.046 (s, 3H, Ac); 3.054 (s, 3H, Ms); 4.600 (m, 1H, H3); 4.782 (q, 1H, J 6.41 Hz, H17); 5.391 (d, 1H, J 4.54 Hz, H6); ¹³C NMR (62.5 MHz, CDCl₃) δ 15.07 (CH₂); 15.39 (C-19); 17.18 (C-18); 19.11 (C-20); 19.76 (CH₂); 21.39 (CH₃, Ac); 27.54 (CH₂); 31.00 (CH₂); 31.88 (CH₂); 31.91 (CH); 36.57 (CH₂); 36.76 (q); 37.65 (CH₂); 39.19 (CH₃, Ms); 40.53 (q); 42.09 (CH); 48.61 (CH); 73.56 (C-3); 82.83 (C-17); 119.04 (C-16); 121.30 (C-6); 139.26 (C-5); 170.53 (Ac); HRMS calculated (M + NH₄)⁺: 455.25742; measured: 455.25771.

Compound 6: IR, n/(cm^-1) 2941, 2243, 1727, 1241, 1172, 901, 800; ¹H NMR (250 MHz, CDCl₃) δ 1.045 (s, 3H, H19); 1.142 (s, 3H, H18); 1.410 (δ, 3H, J 6.50 Hz, H20); 2.047 (s, 3H, Ac); 3.051 (s, 3H, Ms); 4.600 (m, 1H, H3); 4.798 (q, 1H, J 6.50 Hz, H17); 5.380 (δ, 1H, J 4.54 Hz, H6); ¹³C NMR (62.5 MHz, CDCl₃), δ 15.38 (C-19); 15.52 (CH₂); 17.47 (C-18); 19.09 (C-20); 19.27 (CH₂); 21.36 (CH₃, Ac); 27.51 (CH₂); 30.39 (CH₂); 31.82 (CH₂); 32.93 (CH); 36.57 (CH₂); 36.84 (q); 37.65 (CH₂); 39.02 (CH₃, Ms); 40.52 (q); 44.23 (CH); 48.93 (CH); 73.50 (C-3); 85.43 (C-17); 118.68 (C-16); 120.99 (C-6); 139.55 (C-5); 170.50 (Ac); HRMS calculated (M + NH₄)⁺: 455.25742; measured: 455.25753.

Biological tests _ In vitro antitumor assay

Cell lines and cell culture

Six human tumor cell lines (ER+ breast adenocarcinoma, MCF-7; ER-breast adenocarcinoma, MDA-MB-231; prostate cancer, PC-3; cervical carcinoma, HeLa; myelogenous leukemia, K-562; colon adenocarcinoma, HT-29) and one human non-tumor cell line (normal fetal lung fibroblasts MRC-5) were used in this study. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5% of glucose (MCF-7; MDA-MB-231; PC-3; HeLa; HT-29; MRC-5) or RPMI 1640 medium (K562). Medium was supplemented with 10% of fetal calf serum (FCS, Sigma) and antibiotics: 105 IU mL^-1 of penicillin and 100 µg mL^-1 of streptomycin (ICN Galenika). All cell lines were cultured in flasks (Costar, 25 cm²) at 37 ºC in a 100% humidity atmosphere supplemented with 5% of CO₂. Only viable cells were used in the assays. Cell viability was determined by the trypan blue dye exclusion assay.

Antiproliferative activity

Steroidal compounds 2 and 4-6 were evaluated for antiproliferative activity using the tetrazolium colorimetric MTT assay, after treating tumor cells with the study compounds for 48 h. The assay is based on conversion of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan by mitochondrial dehydrogenases in viable cells.²⁴ Doxorubicin (Dox) was used as a positive control. Exponentially growing cells were harvested, counted by the trypan blue exclusion test, and plated onto 96 well microplates (Costar) at an optimal seeding density of 10⁴ (K-562) or 5 × 10³ (other cell lines) cells per well, to assure a logarithmic growth rate throughout the assay period. Viable cells were plated in a volume of 90 µL per well, and pre-incubated in complete medium at 37 ºC for 24 h, to allow cell stabilization prior to the addition of test substances. Tested compounds were added in volume of 10 µL per well of appropriate concentration to all wells except controls in order to achieve the required final concentration in the growth medium (10^-8 to 10^-4 mol L^-1). Microplates were then incubated for 48 h. Wells containing cells without tested compounds were used as control.

After 48 h incubation, 10 µL of MTT solution were added to each well. MTT was dissolved in the medium at 5 mg mL^-1 and filtered sterilized to remove a small amount of insoluble residue present in some batches of MTT.Acidified 2-propanol (100 mL of 0.04 mol L^-1 HCl in 2-propanol) was added to each well and mixed thoroughly to dissolve the dark blue crystals. When all crystals were dissolved, plates were read on a spectrophotometer microplate reader (Multiscan MCC340, LabSystems) at 540/690 nm. Wells without cells containing complete medium and MTT only were used as a blank. Antiproliferative activity was expressed as the IC₅₀ (50% inhibitory concentration).

Data analysis

Two independent experiments were conducted in quadruplicate for each concentration of tested compound; mean values and standard deviations (SD) were calculated for each experiment. IC₅₀ defines the dose of compound that inhibits cell growth by 50%. The IC₅₀ value of each tested compound was determined by median effect analysis.²⁵

Morphological analysis of apoptosis and flow cytometric cell cycle analysis

The MDA-MB-231 human breast adenocarcinoma (ER-) cell line was used to examine induction of apoptosis by the steroidal test compounds. Apoptosis was monitored by observing morphological changes in cells using light microscopy. The same cell line was used for flow cytometry cell cycle analysis.

Cell culture and cell treatment

Cells were grown in the same conditions as for the MTT test. Each culture of both control and experimental groups was seeded in duplicate. Flasks with seeded cells were left at 37 ºC, in an atmosphere comprising 5% of CO₂, during the following 24 h to allow cells to adhere to the flask surface. After 24 h, test steroid compounds (4-6 and the reference formestane) were added to experimental cultures in 1 mL volume in order to achieve the appropriate final concentrations (equal to their IC₅₀ concentrations, which were estimated earlier by cytotoxicity assays; equitoxic doses). Cells were then again incubated in total darkness at 37 ºC in a 100% humidity atmosphere with 5% CO₂. Total incubation time with steroid compounds was 48 h, allowing cells to undergo two mitotic divisions, the same as in the control sample.

Cell harvesting

Cell harvesting and slide preparation were conducted under aseptic conditions, following a modified cytogenetic preparation procedure for micronuclei testing.²⁶ Each culture was harvested and processed separately. Cell suspension preparations involved hypotonic treatment in order to achieve adequate cell spreading and highquality cell preparation for scoring. Hypotonic treatment, fixation and centrifugation were modified to preserve the cell cytoplasm. The cell cytoplasm was retained to enable reliable detection and identification of all morphological features related to apoptosis.

After 48 h of treatment, cells were separated from culture flask walls by trypsinization (0.5% solution of trypsin). One part was used for slide preparation, and the other for cell cycle analysis.

Slide preparation for morphological analysis

Cells were gently centrifuged (1200 rpm) for 5 min and the supernatant culture medium was removed. Cells were then hypotonically treated with 7.0 mL of cold (4 ºC) 0.075 mol L^-1 KCl and centrifuged immediately at 1200 rpm for 8 min. The supernatant was removed and replaced with 5 mL of fixative, consisting of a mixture containing methanol and acetic acid (3:1) with 1% formaldehyde. The fixative was added while agitating the cells in order to prevent clump formation. The cells were then centrifuged again at 1100 rpm for 8 min, washed twice with fixative formaldehyde and gently resuspended. The final suspension was dropped onto clean glass slides and allowed to dry. Specimen staining (after 24 h) was performed using 2% Giemsa stain (Merck) in potassium phosphate buffer (pH 7.3).

Analysis and scoring criteria

Prepared material was observed and analyzed by light microscopy (Olympus BX51). All specimens, including controls, were independently coded before microscopic analysis and analyzed with no prior information regarding the origin of the material. Cells were counted and at least 1000 cells were scored for each specimen. Scored features included normal cells and all forms of induced morphological changes that can be attributed to apoptosis.²⁷

Image capturing and data processing

Images were captured with a 12 megapixel digital camera (Canon 350D) attached to a computer. Data were processed with Microsoft Excel.

Flow cytometric cell cycle analysis

The cell cycle was investigated using a flow cytometer, by analyzing the DNA content of ethanol-fixed MDA-MB-231 cells stained with propidium iodide (PI). For this purpose, a Becton Dickinson (BD) Immunocytometry System²⁸ was used to analyze cell distributions.

After trypsinization, cell fixation (70% ethanol 30 min on ice) and centrifugation, a solution of ribonuclease A (RNase A, 100 units mL^-1) and propidium iodide (400 mg mL^-1) were added to the cell pellet. Cell suspensions were incubated in the dark at room temperature for 30 min. After incubation, and prior to analysis by flow cytometry, each FACSCalibur BD sample was filtered through a 35 µm grid. The cell excitation wavelength was 488-514 nm, and the fluorescence emission of PI was approximately 610 nm. The FL2 parameter of BD FACSCalibur was used for one parametric analysis.

Structure determination

Diffraction data for compounds 5 and 6 were collected using an Oxford Diffraction Gemini S four-circle goniometer equipped with a Sapphire CCD detector. The crystal to detector distance was 45.0 mm and graphite monochromated Mo K_α (λ = 0.71073 Å) radiation was used for the experiment. Data were reduced using the CrysAlis PRO program.²⁹ A semi-empirical absorptioncorrection, based upon the intensities of equivalent reflections, was applied, and the data were corrected for Lorentz, polarization and background effects. The structure was solved by direct methods using the Sir97 program,^30,31 and refined by full-matrix least-squares procedures on F² using SHELXL-97 programs,³² as implemented in the WinGX program suite.³³ Non-H atoms were refined anisotropically. Hydrogen atoms were treated by a mixture of independent and constrained refinement. The figures representing molecular structure were made using the ORTEP-3³⁴ and PLATON³⁵ programs.

Results and Discussion

Hydroxy oxime 1 by Beckmann fragmentation with acetic anhydride afforded D-seco derivative 2, which was reduced, giving a mixture of epimer alcohols 3 and 4. Mesylation of 3 and 4 resulted in D-seco mesyloxy derivatives 5 and 6 (Scheme 1).^36,23

The obtained mesyloxy derivatives 5 and 6 were successfully separated by column chromatography, and the crystallization afforded crystals suitable for X-ray structural analysis. X-ray structural analysis revealed that the major product, compound 5, has a (17S)-configuration, while the side-product 6 has a (17R)-configuration. Based on these results, it can indirectly be concluded that the reduction of D-seco ketone 2 with sodium boron hydride afforded a mixture of stereoisomeric alcohols 3 and 4, which could not be separated chromatographically, but from which the main product 4, with a (17S)-configuration, was isolated by crystallization (hexane/acetone, 3:1).³⁶

Biological tests

Antiproliferative activity

The primary tests for biological and anti-proliferative activities of compounds 2 and 4-6 were in vitro cytotoxicity assays against six human cancer cell lines: estrogen receptor negative (ER-) breast adenocarcinoma (MDA-MB-231); estrogen receptor positive (ER+) breast adenocarcinoma (MCF-7); prostate cancer (PC-3); human cervical carcinoma (HeLa); myelogenous leukemia (K-562); colon adenocarcinoma (HT-29); and normal fetal lung fibroblast (MRC-5) cells (Table 1 and Figure 1). Results were compared with the non-selective cytotoxic drug doxorubicin.

D-seco 17-oxo derivative 2 exhibited weak antiproliferative activity only against human cervical carcinoma cells (HeLa). However, its C-17 hydroxy derivative 4 showed strong antiproliferative activity against MDA-MB-231 breast cancer cells and moderate activity against HeLa cells, with no inhibition potency displayed against other cell lines. The introduction of a C-17 mesyloxy function significantly increased the antiproliferative activity of both epimers 5 and 6 against the majority of the studied tumor cells, compared to precursors 2 and 4. Namely, mesyloxy derivatives 5 and 6 displayed the strongest antiproliferative activity against MDA-MB-231 breast cancer cells. In addition, compounds 5 and 6 displayed respectively 16-and 8-fold greater antiproliferative activity against PC-3 cells than Dox, and also strongly inhibited HeLa and HT-29 tumor cell growth. (17R)-Stereoisomer 6 was a slightly less potent antiproliferative agent against the same cell cultures than its (17S)-stereoisomer 5.

Doxorubicin displayed significant cytotoxicity against nearly all cancer cell types, but was also highly toxic to healthy MRC-5 (control) cells, possibly explaining the severe side effects associated with Dox chemotherapy, including cardiotoxicity and nephrotoxicity.³⁷ In contrast, the new steroidal compounds investigated in the present study were selectively cytotoxic being non-toxic to healthy MRC-5 cells.

Effects of steroidal derivatives 4-6 and formestane on the cell cycle

Since compounds 4-6 expressed significant antiproliferative potential, especially against estrogen receptor negative MDA-MB-231 breast cancer cells, the following experiments in our research were aimed at identifying a preliminary mechanism for their action. Particular interest was focused on their effect on the cell cycle and their potential to induce apoptosis. We performed cell cycle analysis and studied the morphological changes in MDA-MB-231 cells treated with equitoxic doses (IC₅₀ concentrations) of the tested compounds over a 48 h time period.

Figure 2 shows the effect of the tested compounds on treated cells, while the percentages of cells in G1/M, S, G2/M and subG1 phases of the cell cycle were calculated and presented in Table 2. Formestane was used as a reference compound.

The treatment of MDA-MB-231 cells for 48 h with each of the newly synthesized compounds resulted in almost the same number of cells in G1/M1 phase, while the treatment with formestane resulted in a slight decrease, compared to control. The cell population in the synthetic (S) phase in cultures treated with compounds 4 and 5 was very similar to the control sample, and somewhat lower in the sample treated with compound 6. Formestane slightly increased the number of cells in S-phase. Concerning G2/M phase, compounds 4 and 5 exerted similar effects compared to the control sample, while compound 6 and formestane showed a modest increase. The number of hypodiploid (subG1) cells, which are generally regarded as an apoptotic population, was very low after 48 h incubation, and practically the same as the control sample (Table 3).

Induction of apoptotic cellular morphology by steroidal derivatives

Analysis of the morphology of MDA-MB-231 cells, which were treated for 48 h with equitoxic doses of compounds 4-6 and formestane (as a reference compound), was performed by visual observations using a light microscope. No significant difference was found concerning the quality of specimens between control and treated cells by light microscopy. The quality and clearness of the treated specimens were comparable to those of the control, regardless of the test compound.

It was found that the number of cells with apoptotic morphology increased after treatment with investigated steroidal derivatives. Compounds 4 and 5 slightly increased the number of cells featuring apoptotic cellular morphology (less than 4%, compared to control samples). Compound 6 modestly affected this ratio (9.5%), while formestane increased the number of cells with apoptotic morphology more than the other tested compounds (12.5%).

Apoptotic cells were identified by a series of typical morphological changes, and morphology still constitutes an important experimental proof of the underlying processes. To study the mechanism(s) behind the antiproliferative activity displayed by these novel seco-androstane derivatives, compounds 4-6 were selected and light microscopy was used to determine whether they induce apoptosis in MDA-MB-231 cells at the single cell level. As a reference molecule, the steroid formestane were used. Treated and untreated MDA-MB-231 cells were stained with Giemsa. Figure 3 shows the morphology of MDA-MB-231 cells following exposure to equitoxic doses of the tested compounds (4-6 and formestane) for 48 h, as well as untreated cells. The percentage of apoptotic cells, as estimated by visual observation of cell morphology, is given in Table 3. Numerous morphological changes indicative of apoptosis were detected: plasma membrane blebbing, cellular shrinkage, chromatin condensation and nuclear degradation (Figure 4). Most of the control cells appeared normal with round and homogeneous nuclei (Figure 3a).

After treatment with test compounds, alterations in the size, shape and structure of treated cell nuclei were detected in slightly larger number. Chromatin condensation, cell shrinkage and nuclear fragmentation, as well as the formation of apoptotic bodies, were observed. In addition to these morphological changes, treated cells also showed signs of impairment of the plasma membrane and cell disintegration (Figure 3).

In order to study the origin of the differences between the biological activities of the two stereoisomeric mesyloxy derivatives 5 and 6, monocrystals of these two compounds were prepared, and their crystal structures by X-ray crystallography were analyzed. Comparing the structural features of compound 5 and 6, it is possible to notice that the bond lengths and bond angles, especially in the D-seco region, do not differ significantly (Table S11 in the Supplementary Information (SI) section). On the other hand, differences between conformations in the D-seco region were confirmed, as can be seen by inspection of selected torsion angles (Table S12 in the SI section).

A perspective view of the molecular structure of compounds 5 and 6 is shown in Figure 6. Selected bond lengths and bond angles are given in Table S11 (in the SI section). Torsion angles are given in Table S12 (in the SI section).

A structural alignment of the X-ray crystal structures of the two stereoisomeric mesyloxy derivatives 5 and 6 is shown in Figure 7. It can be seen that compound 5 has a (17S)-configuration, while 6 has a (17R)-configuration. Bond lengths and bond angles in the D-seco region do not differ significantly (Table S11), in agreement with the slight differences in their observed cytotoxic and pro-apoptotic potency. The differences between the conformations in the D-seco region are connected with the differences in the values of selected torsion angles (Table S12 in the SI section).

Conclusions

Starting from a 16,17-seco alcohol, two stereoisomeric mesyloxy derivatives were obtained (5 and 6). Both mesylates expressed strong antiproliferative activity against steroid receptor-independent tumor cell lines: breast adenocarcinoma (MDA-MB-231), prostate cancer (PC-3) and cervical carcinoma (HeLa), with no cytotoxicity against healthy cells (MRC-5). Their precursor, seco alcohol 4, strongly inhibited the growth of ER-breast adenocarcinoma cell line (MDA-MB-231). These three compounds did not influence the cell cycle of MDA-MB-231 cells, but slightly induced apoptosis in these cells. Slight differences in their cytotoxic and proapoptotic potency could be correlated with their molecular structures, i.e., with differences between their conformations in the D-seco region. Comparing cytotoxicity of D-seco ketone 2, (17S)-secocyanoalcohol 4 and both (17S) and (17R) D-seco mesyloxy derivatives 5 and 6, respectively, it may be concluded that compounds 4,5 and 6 were much more effective in reducing tumor cell culture growth. It can be presumed that this difference arises from the differences in distances of oxygen atom at C-17 from steroidal skeleton: in molecule 2, in which hybridization of C-17 is sp², it is less than in other compounds, in which hybridization of C-17 is sp³. On the other hand, antiproliferative effects of these three compounds, as well as their influences on the tumor cell cycle and inducing of apoptosis, are quite similar with each other, even though apoptosis induced by compound 6 is very slightly higher expressed.

Based on these results, it is propose that compounds 4,5 and 6 could be used to develop more potent inhibitors of breast and prostate cancer growth, with reduced cytotoxicity toward normal, non-cancerous cells. Synthesis of new steroidal compounds with mesyloxy function, but modified steroidal moiety, is being planned.

The time-and dose-dependencies of the processes by which the tested steroidal compounds influence cell cycle progression (if at all) remain to be clarified. Western-blot analysis would help determine the exact pathways of the compound mode of action. In order to estimate if these compounds induce apoptosis in treated cells, translocation of phosphatidylserine at the cell surface could be examined using an annexine binding assay.

Supplementary Information

Data concerning the IR, ¹H, ¹³C NMR, crystallographicand mass characterizations of the mesyloxy derivatives 5 and 6 are available free of charge at http://jbcs.sbq.org.br, or as a pdf file. CCDC 943744 and 943745 contains the supplementary crystallographic data for this work.

Acknowledgments

This project was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. 172021). The authors want to thank Dr Edward T. Petri for his kind revision of this manuscript, and for English language editing.

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Submitted: June 18, 2013

Published online: August 23, 2013

Supplementary Information

Publication Dates

• Publication in this collection
  02 Oct 2013
• Date of issue
  Oct 2013

History

• Received
  18 June 2013
• Accepted
  23 Aug 2013