New γ-Halo-δ-lactones and δ-Hydroxy-γ-lactones with Strong Cytotoxic Activity

This paper presents the synthesis of γ-halo-δ-lactones, δ-iodo-γ-lactones and δ-hydroxy-γ-lactones from readily available organic substrates such as trans-crotonaldehyde and aryl bromides. Crystal structure analysis was carried out for lactones that were obtained in crystalline form. All halo-δ-lactones and δ-hydroxy-γ-lactones were highly cytotoxic against gastric cancer AGS cells with IC50 values in the range of 0.0006–0.0044 mM. Some lactones showed high bactericidal activity against E. coli ATCC 8739 and S. aureus ATCC 65389, which reduced the number of CFU/mL by 70–83% and 87% respectively.


Introduction
Lactones are compounds with unusual biological activities. The natural source of lactones are plants [1,2] but they are also found in microorganisms [3] and animals [4]. They exhibit, among others, anti-cancer activity [5,6], cytotoxic [7][8][9], bacterio- [10][11][12] and fungistatic [13,14], antiviral [15,16] and deterrent properties [17,18]. This biological potential is widely used in medicine and agriculture. Lactones also have sensory properties [19] used in the cosmetics and food industries but they can also be toxic, such as mycotoxins produced by mold fungi [20]. The properties and application possibilities of lactones are they reason they are still intensively studied by many groups. New natural derivatives are still isolated, their properties are examined and new synthetic analogs with desirable properties are sought. This interesting group of compounds also attracted our attention. Thus far, we have developed a synthesis of β-ayl-δ-iodo-γ-lactones [21], aryl-δ-hydroxy-γ-lactones [22], and trans-γ-halo-δ-lactones [23], and tested their cytotoxic and bactericidal activities. Here, we present stereoselective substrate-controlled synthesis of new γ-halo-δ-lactones. Some of the obtained iodo-δ-lactones can easily undergo translactonization reactions leading to δ-hydroxy-γ-lactones with high yields. Their biological activities were also evaluated: antimicrobial activity against Escherichia coli strain ATCC 8739 and Staphylococcus aureus strain ATCC 65389, and cytotoxicity against L929 cell lines (mouse fibroblasts) and human gastric cancer cell line AGS.

Synthesis
Halolactones were obtained as a result of the four-step reaction [23] shown in Figure 1. The first step of the synthesis was a Grignard reaction between selected aryl bromides 1a-c and trans-crotonaldehyde, which gave us unsaturated allyl alcohols 2a-c with yields 48-62% [24]. The Johnson-Claisen rearrangement [25] of these alcohols with triethyl orthoacetate and catalytic amount of propionic acid obtained γ,δ-unsaturated ethyl esters 3a-c with yields 49-63%. In the third step, the esters were subjected to alkaline hydrolysis in ethanol to obtain γ,δ-unsaturated carboxylic acids 4a-c. Thus obtained substrates, with suitably substituted double bonds, enforced selectivity of halolactonization in the last step of the synthesis. The yields and products distribution are shown in Table 1. Only two of these compounds have been described thus far in the literature [26].  According to the literature [27,28], halolactonization of γ,δ-unsaturated carboxylic acids, carried out with N-chlorosuccinimide (NCS) or N-bromosuccinimide (NBS), leads to a mixture of γ-and δ-lactones, with an excess of 6-endo cyclization products. The regioselectivity of the reaction is influenced by steric hindrance at carbon atoms adjacent to the double bond. In the case of acids with substituents at the C-3, nucleophilic attack at the γ-position is hindered and the amount of products formed by 5-exo cyclization decreases. This is also confirmed by our results. The chlorolactonization of unsaturated carboxylic acids 4a-c with N-chlorosuccinimide (NCS) in THF, only proceeded through a 6-endo cyclization mechanism and gave new γ-halo-δ-lactones 5a-c and 6a-c. The analysis of post-reaction mixtures showed that in each case only isomers trans,trans and cis,trans were formed ( Figure 2). The thermodynamically more stable isomers trans,trans were always present in large excess, up to 80% (Tables 1 and 2). An analogous synthesis using γ,δ-unsaturated carboxylic acids with a methyl group in the β-position led to a mixture of products. Lactonization of unsaturated carboxylic acids with two methyl groups in the β-position have only one product, trans-γ-chloro-δ-lactone [23].  Table 2. The yields and products distribution from iodolactonization of unsaturated carboxylic acids 4a-c (according to GC).

cis-δ-Iodo-δ-lactones γ-Iodo-δ-lactones δ-Hydroxy-γ-lactones yield [%] trans cis trans,trans cis,trans trans cis
Bromolactones 7a-c and 8a-c were obtained by reacting 4a-c with N-bromosuccinimide (NBS) in THF with a yield of 72-78%. Mechanisms for lactonization of γ,δ-unsaturated carboxylic acids with NCS and NBS are similar [27] (Figure 2) and lead to the same products of 6-endo cyclization. The analysis of post-reaction mixtures showed that in bromolactonization of unsaturated carboxylic acids 4a-c in each case only isomers trans,trans and cis,trans were formed. The yield of trans,trans-γ-bromo-δ-lactones was 76-82% and yield of cis,trans-γ-bromo-δ-lactones was only 18-24%. The previously described reactions of γ,δ-unsaturated carboxylic acids with I 2 /KI in saturated NaHCO 3 solution, carried out under kinetic control, resulted in mixtures of γ-and δ-iodolactones, in which the products formed by 5-exo cyclization predominated. The regioselectivity-determining step for the reaction is the attack of the negatively charged oxygen atom of the carboxyl group on the iodine-double bond complex. The presence of substituents in the β-position of unsaturated carboxylic acids hindered nucleophilic attack at the C-4 position. This limited the formation of five-membered lactones. The effect of steric hindrance at the C-6 negatively influenced the formation of δ lactones and favored γ-lactones [27].
Iodolactonization of 4a-c acids was carried out with I 2 /KI and NaHCO 3 in a water/diethyl ether mixture [29]. Analysis of the reaction mixtures unexpectedly showed the formation of two new isomers of δ-hydroxy-γ-lactone 12a and 13a. The yield of reactions was in the range of 69-85%. The reaction conditions prevented the direct synthesis of these compounds because the reaction was carried out in an alkaline solution, whereas hydroxylactonization requires an acidic environment [22]. Cis-isomers are more thermodynamically stable than trans-isomers and predominantly formed in this reaction. The excess of trans-isomer is usually observed in the synthesis of γ-lactones. However, the reaction initially gave δ-iodo-γ-lactones, which, through a translactonization reaction, were transformed into the final products-hydroxylactones 12a and 13a. This was confirmed by observations of purified isomers trans,trans and cis,trans γ-iodo-δ-lactones 9b and 10b, and 9c and 10c. These isomers underwent rapid decomposition with release of iodine. The analysis of residues showed that pure isomers trans,trans or cis,trans transformed into an equimolar mixture of isomers trans and cis hydroxylactones ( Figure 3). These results indicate that the translactonization of γ-iodo-δ-lactones involved elimination of the iodine, which was a good leaving group and rearrangement of the six-membered lactone ring into the five-membered one, according to the SN1 mechanism. The carbocation was stabilized by the electron lone pairs of the oxygen atom present in the water molecule. The preparation of hydroxylactones from iodolactones has been described thus far only for biotransformation experiments. The synthesis described here allows obtaining these compounds without microorganisms. Iodolactonization of γ,δ-unsaturated carboxylic acids 4b and 4c also led to results different from previous studies (Table 1). In both reactions, five products with the lactone ring were identified. The main products were isomers trans,trans-γ-iodo-δ-lactones 9b (yield 42%) and 9c (yield 37%). Isomers cis,trans-γ-iodo-δ-lactones 10b,c were formed with yields of 30% and 33% and isomers cis-δ-iodo-γ-lactones 11b,c only with yields of 10% and 8%. This result was new and unexpected, because iodolactonization of γ,δ-unsaturated carboxylic acid usually gives a mixture of γ-lactones as the main product and only a trace of δ-lactones [27]. This reaction also produced δ-hydroxy-γ-lactones. Isomers trans 12b,c were formed with yields of 5% and 7% and isomers cis 13b,c with yields of 14% and 18%.

The Structural Analysis of Compounds
Spectroscopic and spectrometric analyses were performed for all obtained compounds. The X-ray structural analysis was carried out for the solid products of lactonization. The 1 H NMR spectra of analogous isomers of γ-halo-δ-lactones are similar to each other in most respects. In cis,trans-γ-bromo-δ-lactone 8b, the H-6 proton was observed at 5.89 ppm as a doublet with coupling constants 2.5 Hz (Figure 4). The signal of proton H-5 appears at 4.46 ppm as a triplet due to coupling with protons H-4 and H-6 and the coupling constant 2.5 Hz. According to Karplus, the coupling constant of vicinal protons depends on the dihedral angle between them. The equatorial-equatorial and axial-equatorial coupling constants are in the range 1-5 Hz. The axial-axial coupling constant is normally large but these magnitudes are decreased (9-10 Hz) by the antiperiplanar arrangement of the hydrogens and oxygen atom of the carbonyl group [30,31]. This means that all the substituents are in the equatorial position, alternately above and below the lactone ring plane. The pyranose chair conformations with substituents in the equitorial position are more stable than in the axial position due to torsional strain and 1,3-diaxial interactions. This is reflected in the composition of the reaction mixture where trans isomers are dominant. The structure of the trans,trans-γ-halo-δ-lactones also have been confirmed by single crystal X-ray diffraction analysis. Figure 5a shows the crystalline structure of trans,trans-γ-chloro-δ-lactone 5a [32]. The dihedral angles C1 -C6-C5-Cl and C7-C4-C5-Cl being 67.01 • and 57.24 • respectively. Thus, the aryl substituent at C-6, chlorine atom at C-5 and methyl group at C4 are in the equatorial positions in gauche,gauche arrangement. Hydrogen atoms H-4, H-5 and H-6 occupy the axial position with the dihedral angles between H-4 and H-5 and between H-5 and H-6 equal to 175.72 • and 172.61 • , respectively. X-ray analysis of trans,trans-γ-chloroand trans,trans-γ-bromo-δ-lactones showed that the structure of the lactone rings of the same isomers is analogous. Figure 5b shows the crystalline structure of trans,trans-γ-bromo-δ-lactone 7a [33]. The crystalline structure of γ-hydroxylactone 13a [34], obtained as a result of iodolactonization of acid 4a in 90% yield, is shown in Figure 6a. The dihedral angles C7-C4-C5-C6 and H4-C4-C5-H5 are 33.36 • and 29.31 • , respectively. Thus, the substituents at C-4 and C-5 are in syn-periplanar orientation, on the same side of the lactone ring, forming the cis isomer.
The structure of γ-hydroxylactones was also confirmed by NMR spectroscopic analysis. In the 1 H NMR spectra of cis-γ-hydroxylactone 13a, the signal of proton H-5 appears at δ = 4.86 ppm as a double doublet, due to coupling with protons H-4 and H-6 (J H4/H5 = 7.2 Hz, J H5/H6 = 3.4 Hz). The analogous coupling constants for trans-γ-hydroxylactone 12a are smaller and equal to 5.5 Hz and 2.8 Hz, respectively ( Figure 7). This means that the substituents are in the equatorial position in trans-arrangement. The structure of the lactone rings was also confirmed by 13 C NMR and IR spectroscopy. The carbon signals at δ: 68.97 (13a) and 72.13 ppm (12a) are characteristic of carbon atoms bonded to the hydroxyl group. The typical chemical shift values for the ester carbon atoms in γ-lactones also have signals at 177.11 ppm (13a) and 175.98 ppm (12a). In the IR spectrum, the stretching bands of the C = O group appeared at 1747 cm −1 (13a) and 1774 cm −1 (12a). Spectroscopic analysis showed a similar structure of lactones 13b,c and 12b,c. The crystalline structures of γ-hydroxylactones 12b [35] and 12c [36] are shown in Figure 6b  The iodolactonization of γ,δ-unsaturated carboxylic acids 4b,c also gave δ-iodo-γ-lactones 11b and 11c in low yields (7% and 8%, respectively). Their structure was determined by spectroscopic methods. In the IR spectra, the absorption bands of the C = O group were observed at 1765 cm −1 (11b) and 1760 cm −1 (11c). This is the typical range for γ-lactones. The analysis of NMR spectra showed the signal of the carbonyl carbon at δ 176.00 ppm (11b) and 176.11 ppm (11c). The signals observed at δ: 28.38 ppm (11b) and 28.45 ppm (11c) were assigned to carbons connected to the iodine. The signal of proton H-5 appears as a double doublet, due to coupling with protons H-4 and H-6 (J H4/H5 = 11.2 Hz, J H5/H6 = 4.5 Hz 11b,c). This indicates the syn-periplanar orientation of the protons H-4 and H-5, and cis configuration of γ-lactones.

Bactericidal Properties
The results of CFU counting method are shown in Table 3. The lactones 6c, 9c and 10c showed the highest bactericidal activity against E. coli ATCC 8739 (70-78% less CFU/mL). The other lactones exhibited moderate bactericidal properties. The highest bactericidal activity against S. aureus ATCC 65389 was found for lactone 5a which reduced the number of CFU/mL by 87%. The bactericidal properties against tested bacterial strain also showed lactones 6b, 8c and 7a (78-83% less CFU/mL). Lactones 9a and 12a did not show bactericidal activity against S. aureus ATCC 65389. The 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium Bromide ( Mtt) Reduction Effectiveness All lactones inhibited metabolic activity of the cell lines in the concentration range of 0.1-50 µg/mL. The ability of cells to reduce MTT to formazan decreased by 37-93% (Tables 4 and 5). The cytotoxicity of tested lactones against normal eukaryotic cells may exclude their use as antimicrobial agents in medicine but does not exclude their use in anti-cancer drugs. Our results showed that lactones 10b, 10c and 6b exhibit the lowest IC 50 values against L929 cells. All halo-δ-lactones and δ-hydroxy-γ-lactones were highly cytotoxic against gastric cancer AGS cells with IC 50 values in the range of 0.0006-0.0044 µM. These values are much lower than IC 50 = 0.025 µM of doxorubicin, the reference anticancer drug [37]. The differences between IC 50 values for AGS and L929 cell line were evaluated by non-parametric Mann-Whitney U test. Statistical significance (p ≤ 0.05) was observed for all tested lactones except 6b (p = 0.1).

Materials and Methods
All used chemicals were purchased from Sigma-Aldrich and Fluka and used without further purification. The NMR spectra were obtained in CDCl 3 on a Bruker Avance DRX 500 MHz. FTIR spectra were recorded using an attenuated total reflection technique on a Perkin Elmer Spectrum 400 spectrometer. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were acquired on a Bruker micrOTOF-Q II. Gas chromatography was performed using a Thermo Scientific-Trace 1310 chromatograph equipped with a TG-5HT column (30 m × 0.25 mm). Melting points were determined with a Boetius micro melting point apparatus and are uncorrected. The refractive index was determined with an Abbe refractometer (Atago RX-7000 CX). TLC analysis was performed on silica gel F254 plates (Merck). Column chromatography was performed on silica gel 60 (230-400 mesh, Merck) using mixtures of hexane, ethyl acetate and acetone as eluents. The crystal structure was determined by single-crystal X-ray diffraction (Xcalibur, Sapphire 2 CCD detector).

X-ray Study
The single crystal X-ray diffraction for lactones was carried out on a Rigaku Oxford Diffraction SuperNova diffractometer equipped with a micro focus Cu X-ray source. The crystals were maintained at 100 K by using an Oxford Cryosystems nitrogen gas-flow device. Data collection strategies were optimized using CrysAlisPro software package [41]. The crystal structures of lactones were solved using the charge-flipping method implemented in SUPERFLIP and refined with the JANA package [42]. The crystal data, data collection, and refinement parameters for lactones are given in Table 6. Crystallographic data, as CIF files, have been deposited with the Cambridge Crystallographic Data Centre. The solid-state structure of lactone was determined by single-crystal X-ray diffraction. The lactones 5a (Figure 8), 7a (Figure 9), and 13a ( Figure 10) crystallized in the centrosymmetric space group P2 1/c while lactones 12b ( Figure 11) and 12c ( Figure 12) in the orthorhombic space group Pbca. Only one molecule is present in the asymmetric unit. Experimental details of the crystallographic analysis are given in Table 5.

Claisen Rearrangement
The γ,δ-unsaturated ethyl esters 3a-c were obtained by Claisen rearrangement [25]. A mixture of triethyl orthoacetate (0.15 mol), ethanol (0.02 mol) and a catalytic amount (one drop) of propionic acid was heated at 138 • C for 5 h while distilling off alcohol. When the reaction was completed (TLC), the excess of triethyl orthoacetate was removed by evaporation. The crude product was purified by column chromatography on silica gel using a mixture of ethyl acetate and hexane (1:80). Spectral data are given below.

Bactericidal Activity
The bacterial cultures: Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 65389 were grown on Luria-Bertani Agar for 18 h at 37 • C. The strain suspension was prepared in 0.85% saline, with an optical density equivalent to a 0.5 McFarland standard. Next, the following mixtures were prepared: 180 µL of sterile enriched broth (BTL), 10 µL of the bacterial suspension and 10 µL of the tested compound (range 0.1-50 µg/mL) solubilized in DMSO. As a negative control, only DMSO was added. As a sterility control, 180 µL of BTL and 20 µL saline were used. The microtiter plate was incubated at 37 • C for 18 h. The colony forming unit (CFU) method was used to evaluate the microbial population. The experiment was repeated three times. Gentamicin was used as the reference antibiotic [23]