Anticancer and antimicrobial effects of novel ciprofloxacin fatty acids conjugates

Journal Pre-proof Anticancer and antimicrobial effects of novel ciprofloxacin fatty acids conjugates Alicja Chrzanowska, Piotr Roszkowski, Anna Bielenica, Wioletta Olejarz, Karolina Stępień, Marta Struga PII:

S0223-5234(19)30962-6

DOI:

https://doi.org/10.1016/j.ejmech.2019.111810

Reference:

EJMECH 111810

To appear in:

European Journal of Medicinal Chemistry

Received Date: 26 July 2019 Revised Date:

18 October 2019

Accepted Date: 21 October 2019

Please cite this article as: A. Chrzanowska, P. Roszkowski, A. Bielenica, W. Olejarz, K. Stępień, M. Struga, Anticancer and antimicrobial effects of novel ciprofloxacin fatty acids conjugates, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111810. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

Graphical abstract

Cytotoxicity against PC3 cells: 7.7 ‒ 11.7 µM LDH release from normal HaCaT cells: 4.1 ‒ 7.5 % LDH release from PC3 cells at 10 µM: 47.2 ‒ 67.2 % Apoptosis-inducing effect in PC3 cells: 63.5 ‒ 81.5% Inhibition of IL-6 release in PC3 cells: 2.5-4 times

Submitted to: European Journal of Medicinal Chemistry

Anticancer and antimicrobial effects of novel ciprofloxacin fatty acids conjugates Alicja Chrzanowskaa, Piotr Roszkowskib, Anna Bielenicaa,*, Wioletta Olejarzc, Karolina Stępieńd, Marta Strugaa

a

Medical University of Warsaw, Chair and Department of Biochemistry, Banacha 1, 02-097 Warsaw, Poland

b

Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland

c

Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw, 02-097 Warsaw, Poland

d

Department of Pharmaceutical Microbiology, Medical University, 02-007 Warsaw, Poland

*

Corresponding author: Anna Bielenica, Chair and Department of Biochemistry, Medical

University of Warsaw, 02-097 Warsaw,

Poland, Phone: +48225720693, e-mail:

[email protected]

Keywords: ciprofloxacin; fatty acids conjugates; cytotoxicity; apoptosis; interleukin-6; antimicrobial activity

1

Abstract: Ciprofloxacin (CP) has a confirmed cytotoxic action on some cancerous cells, but in high, non-pharmacological concentrations. Considering features of natural fatty acids, such as biocompatibility, biodegradability and their increased cellular uptake by cancer cells, it seems that combining them with a drug could improve its bioavailability, and thus cytotoxicity. Therefore, the aim of this study was coupling of CP with saturated and unsaturated fatty acids, and evaluation of their cytotoxicity, apoptosis-inducing effects and inhibition of IL-6 release in human primary (SW480) and metastatic (SW620) colon cancer, metastatic prostate cancer (PC3) and normal (HaCaT) cell lines. The PC3 cell line was the most sensitive to the presence of the obtained conjugates. The value of IC50 for oleic acid conjugate (4) was 7.7 µM, and it was 12 times lower than for CP alone (101.4 µM). The studied derivatives induced late apoptosis in all cancer cell lines, but not in normal cells. The most potent apoptosis inducer was conjugate 4, that resulted in the highest percentage of PC3 cells in late apoptosis (81.5%±3.9), followed by elaidic acid amide 5 (75%±4.8). The strongest pro-apoptic effects on SW480 cells were demonstrated by conjugates of DHA (8) and sorbic (2) acids, whereas in SW620 cell lines, compounds 2 and 5 appeared to be the most effective. To establish the mechanism of cytotoxic action of derivatives 2, 4, 5, the level of interleukin-6 (IL-6) was measured. The compounds with the highest cytotoxic potential significantly decreased the release of IL-6 by cancer cells. Additionally, all conjugates were evaluated for their in vitro antimicrobial activity. Short chain amides - crotonic (1) and sorbic (2) - were the most active against

Staphyloccoci.

The

second-mentioned

amide

has

shown

both

strong

antistaphylococcal and antitumor properties.

2

1. Introduction Prostate cancer (PC) is one of the most common cancers in men [1]. Usually, it depends on androgens in the first stage of development, but finally progresses to an androgenindependent phenotype. Unfortunately, this phenotype becomes resistant to secondary endocrine therapy and chemotherapy [2,3]. As an androgen-independent tumor, it is affected by several molecular changes, especially a reduced rate of apoptosis or/and a serum interleukin-6 (IL-6) rise [4,5]. Various studies have reported that IL-6 is involved in the transition of PC from an androgen-dependent to an androgen-independent state. It has been proved that human tumors became more invasive and metastatic through the activation of both autocrine and paracrine IL-6 signaling pathways [6-8]. Interestingly, development of many solid tumors is based on inflammatory states [9]. If there is a link relating prostatitis and prostate cancer, it is possible to prevent, or even treat prostate cancer by restraining an inflammation of the prostate gland. In most cases, prostatitis is observed in coincidence with a bacterial infection [10,11]. The most commonly used treatment for this inflammatory condition are antibiotics, mainly of the fluoroquinolone group, e.g. ciprofloxacin (CP) [12]. It has the ability to inhibit eukaryotic cell proliferation via mitochondrial DNA damage and reacts with the mitochondrial topoisomerase II isoform [13,14]. CP-induced cytotoxicity in cells may be also a result of free radicals generation [15]. These properties result in its pro-apoptotic effects on mammalian tumor cell lines, including human cells, and introduce the possibility of using CP as a potential anticancer drug. However, in vitro antiproliferative and apoptosis-inducing activities of CP on human prostate cancer cell lines were observed at non-clinically achievable concentrations of 50-400 µg/ml [16,17]. Colorectal carcinoma cells were also sensitive to cytotoxic effects of CP, when it was administrated at even higher doses (200-500 µg/ml) [18]. Many efforts have been employed to overcome the limitation of rapid drug metabolism in vivo, which results in a short circulation time and a poor efficacy of known anticancer agents. One of them, gemcitabine, has been covalently conjugated at the N4amino group with linoleic acid (CLA). The resulting amide exhibited a longer plasma half-life and a higher bioavailability [19]. Moreover, the synthesis of a gemcitabine derivative with a cardiolipin moiety allowed to obtain a less toxic compound that binds to biological membranes and overcomes resistance of cancer cells to the parent drug [20]. To increase the therapeutic index of another known chemotherapeutic, doxorubicin (DOX), and to attenuate 3

its toxicity towards normal tissues, conjugates with α-linolenic (LNA) and palmitic acids (PA) were produced. Among them, DOX-LNA hydrazone improved the survival time, and decreased cancer growth in tumor-bearing nude mice [21]. On the other hand, the presence of a ω-3 polyunsaturated fatty acids (PUFA) branch in conjugates of flavonoids, improved the lipophilic properties of the whole molecule, hence increased its penetration through the cell membrane [22]. Some attempts at modification of the CP structure have also been made. Within 7-(4-substitued)piperazin-1-yl) derivatives of CP, those bearing decanoyl and chloroacetyl moieties displayed IC50 values 13-93-fold lower than the parent drug, when tested on a panel of human cancer cell lines [23]. A series of other CP analogs, obtained by a condensation with N-substituted pipemidic acid, revealed both antiproliferative and antibacterial properties [24]. Other authors have attempted to encapsulate CP in chitosan-fatty acid nanomicelles that improved considerably an antimicrobial effect of that antibiotic. In consequence, the MIC values towards Gram-negative pathogens, applied in the presence of a long-chain carboxylic acid-grafted chitosan conjugate, were 2-4 times lower in comparison with the free drug [25]. Based on these reports, it seems that modifications of the CP molecule by conjugation with fatty acids of different length and saturation, could have a higher therapeutic value for treating cancers than CP itself. Additionally, these compounds may exhibit protective effects for normal cells [21]. It was proved that the composition of membrane lipids in cancer cells is altered [26,27]. Therefore, it is obvious that the bioactivity of various anticancer drugs is associated with their ability to penetrate the membrane lipid structure. Thus, their conjugation with fatty acids shows multifaceted effects [26-28]. In the up-to-date literature there are no evidences concerning CP and unsaturated fatty acid connections, which combine cytostatic and antibacterial activity. In the present study, we have synthetized novel CP-amides with representative fatty acids. The new derivatives were evaluated for their cytotoxic activity, apoptosis induction and interleukin-6 release on primary and metastatic cancer cell lines, as well as on normal cells. Additionally, all studied compounds were tested for their preliminary antimicrobial potency, in order to compare their properties with the reference antibiotic.

2. Result and discussion 2.1 Chemistry 4

The aim of this study was to obtain a series of amide derivatives of the chemotherapeutic CP, by its conjugation with exemplary fatty acids (Scheme 1). The synthesis was carried out under mild conditions at room temperature with satisfactory yields. In order to ensure structural variability, acids of different chain length, degree of unsaturation and geometrical isomerism were chosen (Table 1). The introduced acyl moieties were either unsaturated (1-8) or saturated (9), as well as belonged to the group of short- (1, 2), middle- (3) or long-chain (4-9) components. Some of them represented geometrical E-isomers (1-3, 5), whereas the other formed naturally occurring Z-isomers (4, 6-8). In consequence, the tested group presented a variable, but comparable collection. [Scheme 1] [Table 1]

2.2 Biological studies

2.2.1

Anticancer activity

2.2.1.1 MTT assay

To establish the cytotoxic effect of the compounds, they were screened for their in vitro antiproliferative activity against a panel of different human cancer cell lines, namely prostate (PC3) and colon (SW480, SW620), in contrast to normal (HaCaT) cell lines, by using the MTT method (Table 2). The anticancer profile of all tested derivatives, expressed as IC50 value, was incomparably stronger than the reference CP. The drug itself remained inactive towards all tested cancer cell lines, with IC50 factors exceeding 100 µM. The highest growth-inhibitory potency on PC3 cells was denoted for monounsaturated oleic 4 (IC50=7.7±2.1µM) and diunsaturated sorbic 2 (IC50 =11.7±1.8µM) acids conjugates, as well as for elaidic acid amide (IC50=15.3±5.3 µM). The replacement of the double bond orientation from Z- (oleic, 4) to E(elaidic acid derivative, 5) resulted in 2-fold reduction of the activity. As compared to CP alone, the potency of its conjugates was found to be 7-13 times stronger. Z-polyunsaturated long-chain fatty acids amides, such as DHA (8) and linolenic (6), behave as moderate inhibitors of the growth of PC3 cancer cells. On the other hand, lower cytotoxicity was found 5

for the saturated palmitic acid derivative (9) and other tested connections bearing an unsaturated hydrocarbon linker (3, 7). Furthermore, the highest selectivity index (SI) was achieved also by the most potent conjugates: oleic (17.8), sorbic (9.3) and elaidic (9.2). Similar SI factors were observed for compounds of moderate cytotoxic activity – DHA (8) and linolenic (6) acids derivatives (SI = 7.8-8.5). In addition, a 72h exposure of normal human keratinocytes (HaCaT) on newly synthesized CP-amides have not caused any cytotoxic effect. The lengthening of an alkyl chain of the polyunsaturated amide derivative 2 resulted in loss of the growth – inhibitory potency of its analogue (3) in both SW480 and SW620 cells, and an over 6-fold reduction of cytotoxic activity in PC3 cell lines. However, the influence of the degree of unsaturation of long-chain fatty acid conjugates on their bioactivity seems to be ambiguous. Comparing 18-carbon derivatives 4 and 6, an increase in the double bonds amount caused a 4,5-fold reduction in anticancer activity of the compound. Contrarily, the higher number of double bonds in 22-carbon amides (7, 8) caused a 3- or 4.5-fold enhancement of the growth-inhibitory effect in PC3 and SW480 cells, respectively. The most promising inhibitory effect on primary colon cancer cell lines (SW480) was exerted by derivatives conjugated with polyunsaturated fatty acids of short (sorbic 2), middle (geranic 3) and long (DHA 8, elaidic 5) hydrocarbon chains. Their IC50 values varied from 20.1 µM to 35.7 µM, and were higher than those denoted for PC3 cells. Nearly the same group of CP- compounds suppressed the growth of metastatic SW620 colon cancer cell lines. The types of acid residues can be arranged in order of their decreasing impact as follows: sorbic (IC50 = 21.5 µM), elaidic, oleic, geranic, DHA (40 µM). The lowest cytotoxic potency was observed for the saturated acid derivative (9) and for unsaturated crotonic (1) and erucic (7) acid amides. The most cytotoxic sorbic acid compound (2) acted against both colon cancer cells with 5-fold higher selectivity, compared to HaCaT cultures. Similarly, the selectivity of the DHA derivative (8) was 8.8-fold higher for the SW480 cell line, and 5.8-times higher vs the SW620 culture. Obtained results indicated that the tested conjugates are selective and, in the contrast to the reference drug – doxorubicin, damaged cancer cells without causing toxic effects on normal cells. Moreover, a link between anticancer potency and calculated logP values of newly synthesized compounds was also noticed. Considerably cytotoxic monounsaturated CPderivatives 4 and 5 are characterized by high octanol-water partition coefficients (7.76). 6

However, highly lipophilic conjugates 7 and 9 expressed weak antiproliferative potency. The sorbic acid amide (2), with logP descriptor equalled 1.15, is an exception – it exerted both anticancer and antimicrobial activities. Compounds 1 and 3, found as the least lipophilic, were moderately cytotoxic towards tested cancer cell lines. [Table 2] Interestingly, Zhao et al. proved that the loading of the doxorubicin - oleic acid conjugate into the nanostructured lipid carriers increased its uptake by colon carcinoma cells [29]. What is more, it has been shown that some fatty acids regulated the lipid structure of membranes, facilitating the binding and activation of important membrane signaling proteins [30]. The in vitro study of human cancer cell lines provided by Breistøl et al. revealed a diverse cytostatic activity of various cytarabine and fatty acids hybrids. Among long-chain acyl moieties with a different degree of saturation, the most promising was a drug connection with elaidic acid [31]. Our results report similar findings, since coupling with oleic or elaidic acids significantly increased the anticancer activity of CP. In contrast, free sorbic acid has no cytotoxic properties [32], but may exert a negative impact on the lipid oxidative stability, leading to formation of hydroperoxides and other oxidation products [33]. In respect of known prooxidative activities of CP [15], it implies a synergistic action during treatment with sorbic acid – CP conjugation. 2.2.1.2 Lactate dehydrogenase assay LDH assay as a marker of a cell death was performed for all synthesized compounds (Table 3). Tested conjugates and CP alone were examined at four concentrations of 10, 20, 40 and 60µM. Oleic acid amide (4) was studied at 5, 10, 20 and 40µM, because of its high cytotoxicity. As expected, the LDH release from HaCaT cells was low, even if high concentrations of conjugates were used (60µM), and ranged from 3.2 to 7.5%. Similarly, when a dose of 10µM was applied, it varied from 3.2% to 4.1%. All studied derivatives expressed higher cytotoxic effect against tumor cell lines than against normal keratinocytes (HaCaT). However, the effect was stronger in the prostate cancer cell line (PC3), compared to colon carcinoma cell lines (SW480, SW620). The cytotoxic actions of the most promising conjugates 2, 4 and 5 are shown in Fig.1. The derivative 4, used at 10µM, showed the highest response against the PC3 cell line (LDH secretion was 71%), comparing to conjugates 2 and 5, where it accounted 47.2% and 41%, respectively. However, the compound 4 was less cytotoxic for SW480 and SW620 cells, achieving 22.3% and 32% LDH release at 10µM, and 7

19% and 23% at 5µM. The primary colon cancer cells (SW480), treated with amides 2 and 8 at the dose of 10µM, expressed the highest LDH release of 37.2% and 31%, respectively. This secretion was much lower in metastatic colon carcinoma cells (SW620), incubated with conjugates 2 and 8 at the same concentrations, than in PC3 and SW480 cells (28% and 7.3%, respectively). The similar level of LDH release (23% and 21.6%) was observed after treatment of SW620 cells with compounds 4 and 5. The results obtained by the LDH cytotoxic assay are in accordance with the data achieved for all mentioned CP-derivatives by the MTT method.

[Table 3] [Fig.1.]

2.2.1.3

Apoptotic activity To assess in vitro the anticancer mechanism of action, the effect of CP-conjugates on

early and late apoptosis was provided by flow cytometry analysis (Fig.2.). The incubation of SW480, SW620 and PC3 cells with compounds 2, 4, 5 and 8 showed significantly higher percentage of cells in late apoptosis, as compared to the controls. The conjugates of longchain monounsaturated geometrical isomers: oleic (4) and elaidic (5) acids had the strongest late apoptosis-inducing effect in PC3 cells. The most potent activator was the amide 4 that showed visibly higher percentage of PC3 in late apoptosis (81.5%±3.9), in comparison to the product 5 (75%±4.8). Other CP-derivatives, such as polyunsaturated sorbic (2) or DHA (8) acids compounds, also influenced considerably prostate cancer cells apoptosis, and induced the late apoptosis in 63.5%±3.8 and 61.2%±6.7 of cells, respectively. On the other hand, the strongest pro-apoptotic inducing properties in SW480 cell lines were exerted by conjugates of DHA (8), sorbic (2) and elaidic (5) fatty acids. Amides 2 and 8 significantly affected the level of late apoptosis in primary colon carcinoma cells, and gave a similar result (52.6%±2.7 and 48.6%±3.3, respectively). Late apoptosis in metastatic SW620 cells was not so spectacular, however, conjugates 2, 4, 5 and 8 indicated noticeable pro-apoptic inducing properties. The apoptotic activity of those compounds varied from 31.4%±2.8 to 39.2%±2.8. Beside it, a treatment of SW480 cells with the conjugate 4, and also an incubation of SW620 cells with amides 2 or 8, increased both, early and late apoptosis. In contrary, the test performed in HaCaT cells, cultured with studied compounds, revealed the late apoptosis at similar low

8

levels as in untreated cancerous controls, where it counted from 6.2% to 12%. The obtained results comply with IC50 values assigned to mentioned cancer cell lines (Fig. 3). [Fig.2.] [Fig.3.] As described previously [15-17], the antitumor activity of CP was determined to act through its pro-apoptotic effect. However, this ability was observed at a 600–1500 µM concentration range, while in vitro growth inhibition activity often occurs at lower doses [16,18,23,34]. It was suggested that CP expresses its antitumor activity through a apoptoticindependent

mechanisms,

such

as

pro-autophagic

and/or

lysosomal

membrane

permeabilization-related cell death [23]. Moreover, in vitro and in vivo studies on tongue squamous cell carcinomas revealed that oleic acid has valuable anticancer effects manifested by a cell cycle G0/G1 arrest and by induction of cell death via autophagy [35]. Some investigations have shown that an influence of unbound oleic acid on cancer cells included effects on the cell membrane, apoptosis, autophagy, mitochondria, proteasome inhibition, cell adhesion and glycolysis [36,37,38]. Indeed, in our study we have indicated that the conjugate of CP with oleic acid (4) exhibited highest pro-apoptotic activity. 2.2.1.4 IL-6 assay Among the few proposed mechanisms of action of CP-derivatives are the decreased production of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), lymphotoxin and granulocyte-macrophage colony stimulating factor (GM-CSF) by human lymphocytes. It is also known that CP can reduce IL-6 and IL-8 levels and influence host defense mechanisms [39,40]. All studied human cell lines were treated with IC50 doses of the most promising CP amides: sorbic (2) oleic (4) and elaidic (5), CP alone (control) and the reference drug – doxorubicin (Fig.4.). A significant inhibition of IL-6 was observed in all studied cancerous cells lines. Generally, the PC3 cell line seems to be the most sensitive for the inhibition of IL6 release, evoked by the presence of fatty acids conjugates and the standard CP. The strongest effect in PC3 cells was observed for oleic acid amide 4, which inhibited interleukin release fourfold. This response was greater than the effect of the reference compound – doxorubicin, which decreased IL-6 secretion only twice. However, the treatment with elaidic acid amide (5) was more effective for both SW480 and SW620 cell lines. The mentioned compounds 9

inhibited IL-6 secretion 3 and 4 times, respectively. The values of inhibition of IL-6 release in PC3 line were very similar for the sorbic acid conjugate 2 and the reference CP. Both substances decreased IL-6 concentration 2.5 times, however the compound 2 was more effective for colon carcinoma cells. It is commonly known that IL-6 is a strong pro-inflammatory molecule [5,6]. As inflammation may precede and is associated with both prostate and colon cancers, the IL-6 based mechanism may be involved in tumor development. More importantly, it was found that IL-6 is able to convert differentiated prostate and colon cancer cells to cancer stem cells resistant to conventional chemo- and radiotherapy [41,42]. Thus, IL-6 inhibition by CPconjugates seems to give the triple benefit, suppressing precancerous inflammatory state, tumor development and progression. [Fig. 4.]

2.2.2 Antibacterial activity New CP-conjugates were tested in vitro against a series of standard Gram-positive cocci and Gram-negative-rods. Representative bacterial strains used in this study have common applications in antimicrobial assays of antibiotics, antiseptics and in search for new disinfectants. The preliminary investigations based on the determination of the minimal inhibitory concentration (MIC) by the twofold serial microdilution method. The observed data are presented in Table 4. [Table 4] The best antibacterial activity was shown by E-isomers conjugated with short- and medium-chain fatty acids derivatives. Among them, the highest potency against a series of Staphylococcal isolates exerted short-chain compounds, represented by crotonic (1) and sorbic (2) acid amides (MIC 0.5-1 µg/ml). With the same strength they both inhibited the growth of Gram-negative Escherichia coli strains. Their activity against Staphylococci and E. coli isolates was comparable to the potential of the reference CP (MIC ≤ 0.5 µg/ml). The standard antibiotic was more effective only against Enterococcus hirae and Pseudomonas aeruginosa rods. Medium-chain geranic acid derivative (3) also exhibited high antimicrobial properties towards standard strains of Staphylococcus aureus and Staphylococcus epidermidis (MIC ≤ 1 µg/ml), and moderate towards E. coli isolates (MIC = 8 µg/ml).

10

Modest antibacterial potency against Staphylococci was observed for polyunsaturated long-chain conjugates, namely amides of linolenic (6) (MIC = 4 µg/ml) and DHA (8) (MIC = 8-16 µg/ml) acids. In addition, the derivative 8 was effective also against E. coli NCTC 8196 isolate (MIC = 2 µg/ml). Other Gram-negative strains were only weakly susceptible for the presence of these fatty acids compounds. No evident activity was observed for saturated palmitic (9) and long-chain monounsaturated acids amides 4, 5, 7. An elongation of a hydrocarbon linker of the diunsaturated derivative (2 to 3) has not influenced its activity against tested Gram-positive rods, however it diminished 16-fold the potency of the compound towards Gram-negative E. coli strains. Similarly, the change of geometrical isomerism between amides 4 and 5 led to the complete loss of the antibacterial activity. On the contrary, in the group of the long-chain CP-amides (4, 6-8), as the degree of unsaturation rised, a noticeable increase in their antimicrobial potency was observed. Triunsaturated (6) and hexaunsaturated (8) fatty acid conjugates showed antistaphylococcal activity up to 128- and 64-fold higher than that observed for their monounsaturated analogues 4 and 7, respectively. They exerted also 4-256-fold higher inhibitory activity against E. coli rods as compared to their less unsaturated counterparts. What is more, the antibacterial potency of uncoupled fatty acids towards Gram-positive cocci was slight in comparison with synthesized CP-conjugates. Their MIC values increased from 64 to 512 µg/ml. In addition, they were inactive against investigated Gram-negative isolates (Table 1S, Supplementary material). It is worth to note that generally the CP-derived amides with the lowest lipophilicity (Table 1) exhibited the highest antibacterial activity. The logP values of the most potent antistaphylococcal agents, short-chain derivatives 1-3, ranged from 0.63 to 3.46. On the other hand, the long-chain fatty acids derivatives 4, 5, 7, 9, that are the most lipophilic, were devoid of antimicrobial potency. Antibacterial properties of sorbic acid are widely described in literature [43,44]. It was established that an inhibition of bacterial growth by sorbate may result from alteration of cell membranes, or restraining of the transport systems and key enzymes in bacteria’s metabolism. Usually, the concentration of this acid used as antimicrobial agent was 0.5-1%. Similarly, its antibacterial effects against S. aureus and E. coli was shown with MIC ≥ 1250 µg/ml [45], and it was much higher as compared to the dose established for the CP-conjugates in our study (0.5-1 µg/ml). 11

3. Conclusion

To sum up, we have presented the cytotoxic and antimicrobial properties of diversified series of CP-conjugates, derived from fatty acids of various length, degree of unsaturation and double bonds spatial orientation. Studied conjugates did not express cytotoxic effects in normal cells (HaCaT) at clinically achievable concentrations. The most active (poly)unsaturated compounds 2, 4, 5 showed higher anticancer potential against PC3, comparing to SW480 and SW620 cell lines. Additionally, these conjugates reduced secretion of IL-6 by cancer cells. It may result in a decrease of known IL-6 tumor- promoting effects. Thus, obtained data indicate possible suppressive effect of CP-amides on cancer cells. Moreover, short-chain geometric E-isomers of crotonic (1) and sorbic (2) acid amides were the most active against a series of Staphylococci and Escherichia coli strains (MIC 0.5-1 µg/ml), being equally potent as the reference antibiotic. The structural modification of CP by conjugation resulted in a higher therapeutic value for treating cancers than CP itself. Due to the fact that sorbic, oleic and elaidic acids themselves can exhibit antitumor or antibacterial properties, they can act synergistically by enhancing cytotoxic or growth-inhibitory effects of a parent drug on bacteria. Since it is known that prostatitis precedes prostate cancer, dual anticancer and antibacterial activities of the sorbic acid-CP conjugate (2) can represent a novel combined preventive and therapeutic approach.

4. Experimental

4.1. Chemistry

4.1.1 General procedure

Dichloromethane and methanol were supplied from Sigma Aldrich. The trans-crotonic (98%), sorbic (99%), geranic (90+%) and erucic acids, tech. (90%) were purchased from Alfa Aesar company. DHA (98%) and CP (98%) were supplied from Acros Organics. Linolenic (≥99%), elaidic (≥99%) and palmitic acids (≥99%) were purchased from Sigma Aldrich. All other chemicals were of analytical grade and were used without any further purification. The NMR spectra were recorded on a Bruker AVANCE spectrometer operating at 300 MHz for 1

H NMR and at 75 MHz for 13C NMR. The spectra were measured in CDCl3 and are given as 12

δ values (in ppm) relative to TMS. Mass spectral ESI measurements were carried out on LCT

Micromass TOF HiRes apparatus. TLC analyses were performed on silica gel plates (Merck Kiesegel GF254) and visualized using UV light or iodine vapour. Column chromatography was carried out at atmospheric pressure using Silica Gel 60 (230-400 mesh, Merck) and using dichloromethane/methanol (0-2%) mixture as eluent.

4.1.1.1 General method for the synthesis of derivatives 1-9

To a suspension of CP (0.20 g; 0.60 mmol) and an appropriate fatty acid (0.60 mmol) in dried CH2Cl2 (24 mL), the BOP reagent (benzotriazol-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate) (0.27 g, 0.60 mmol) and triethylamine (0.13 mL; 0.91 mmol) were added. The resulting solution was stirred for 2 h at 22-23 oC. To the obtained reaction mixture, 0.67% HClaq solution (15 mL) was added. Next, it was extracted with CH2Cl2 (2x25 mL). The combined organic layers were washed with 1% HClaq solution (4x25 mL) and distilled water (2x25 mL), and then dried over anhydrous MgSO4. After the solvent evaporation, the product was isolated using column chromatography on silica gel (with CH2Cl2:MeOH mixture as an eluent). The final product was washed with diethyl ether (2x2 mL). The structures of all compounds were confirmed by 1H NMR, 13C NMR and MS spectra (see Supplementary material).

7-[4-(2E)-but-2-enoyl-piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-quinoline3-carboxylic acid (1) Pale yellow solid, 135 mg (56%). Mp. 325°C. Insoluble in solvent used to NMR analysis. HRMS (ESI) m/z 422.1508 (calcd for C21H21FN3O4Na [M+Na]+, 422.1492).

7-[4-(2E,4E)-hexa-2,4-dienoyl-piperazin-1-yl])-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2) Pale yellow solid, 150 mg (58%). Mp. 240°C. 1H NMR (CDCl3\CD3OD, 9:1 mixture, 300 MHz) δ (ppm): 1.16-1.22 (m, 2H), 1.36-1.43 (m, 2H), 1.62 (s, 3H), 1.84 (d, J = 5.7 Hz, 3H), 3.32-3.36 (m, 4H), 3.52-3.59 (m, 1H), 3.86 (d, J = 19.5 Hz, 4H), 6.07-6.26 (m, 3H), 7.23-7.32 (m, 1H), 7.36 (d, J = 6.9 Hz, 1H), 7.96 (d, J = 12.9 Hz, 1H), 8.72 (s, 1H).

13

C NMR

(CDCl3\CD3OD, 9:1, 75 MHz) δ (ppm): 8.2 (2xC), 18.6, 35.4, 41.6, 45.5, 49.3, 50.1, 105.2 (d, 3

JC-F = 3.0 Hz), 107.8, 112.4 (d, 2JC-F = 23.3 Hz), 116.8, 120.1 (d, 3JC-F = 8.3 Hz), 129.9,

13

138.9, 139.0, 144,3, 145.4 (d, 2JC-F = 9.8 Hz), 147.7, 153.6 (d, 1JC-F = 249.8 Hz), 166.3, 167.2, 177.0 (d, 4JC-F = 2.3 Hz). HRMS (ESI) m/z 448.1662 (calcd for C23H24FN3O4Na [M+Na]+, 448.1649).

7-[4-(2E)-3,7-dimethylocta-2,6-dienoyl-piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydro-quinoline-3-carboxylic acid (contaminated with (Z)-isomer) (3) White solid, 140 mg (48%). Mp. 150°C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 1.19-1.24 (m, 2H), 1.38-1.44 (m, 2H), 1.62 (s, 3H), 1.68-1.71 (m, 3H), 1.87-1.93 (m, 3H), 2.13-2.39 (m, 4H), 3.34 (t, J = 5.1 Hz, 4H), 3.54-3.61 (m, 1H), 3.76 (bs, 2H), 3.90 (bs, 2H), 5.08-5.15 (m, 1H), 5.81-5.83 (m, 1H), 7.36 (d, J = 6.9 Hz, 1H), 7.92 (d, J = 12.9 Hz, 1H), 8.68 (s, 1H), 14.87-14.89 (m, 1H).

13

C NMR (CDCl3, 75 MHz) δ (ppm): 8.2 (2xC), 17.7, 18.7, 23.7 (minor

isomer), 25.7, 25.9, 26.5 (minor isomer), 33.8 (minor isomer), 35.3, 39.6, 40.8, 45.9, 49.4, 50.3 (d, 4JC-F = 4.5 Hz), 105.1 (d, 3JC-F = 3.7 Hz), 107.9, 112.3 (d, 2JC-F = 22.5 Hz), 117.0, 117.8 (minor isomer), 119.9 (d, 3JC-F = 8.3 Hz), 123.4, 123.6 (minor isomer), 132.1 (minor isomer), 132.3, 138.9, 145.4 (d, 2JC-F = 10.5 Hz), 147.4, 150.0, 151.9 (minor isomer), 153.5 (d, 1JC-F = 249.8 Hz), 166.7, 167.0 (minor isomer), 167.4, 176.8 (d, 4JC-F = 2.3 Hz). HRMS (ESI) m/z 504.2291 (calcd for C27H32FN3O4Na [M+Na]+, 504.2275). 7-[4-(9Z)-octadec-9-enoyl-piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (4) White solid, 170 mg (47%). Mp. 108°C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, J = 6.9 Hz, 3H), 1.20-1.42 (m, 24H), 1.65 (quint, J = 7.5 Hz, 2H), 1.93-1.99 (m, 4H), 2.37 (t, J = 7.8 Hz, 2H), 3.29 (t, J = 4.5 Hz, 2H), 3.36 (t, J = 4.5 Hz, 2H), 3.51-3.59 (m, 1H), 3.71 (t, J = 4.5 Hz, 2H), 3.86 (t, J = 4.8 Hz, 2H), 5.27-5.38 (m, 2H), 7.33 (d, J = 7.2 Hz, 1H), 7.90 (d, J = 12.9 Hz, 1H), 8.65 (s, 1H), 14.85 (s, 1H). 13C NMR (CDCl3, 75 MHz) δ (ppm): 8.2 (2xC), 14.1, 22.6, 25.2, 27.1, 27.2, 29.1, 29.3 (2xC), 29.3, 29.4, 29.5, 29.7, 29.7, 31.9, 33.2, 35.3, 41.0, 45.3, 49.3 (d, 4JC-F = 3.0 Hz), 50.2 (d, 4JC-F = 6.0 Hz), 105.0 (d, 3JC-F = 3.0 Hz), 108.0, 112.3 (d, 2JC-F = 24.7 Hz), 119.9 (d, 3JC-F = 7.5 Hz), 129.7, 130.0, 138.9, 145.4 (d, 2JC-F = 10.5 Hz), 147.4, 153.5 (d, 1JC-F = 249.7 Hz), 166.7, 171.8, 176.8 (d, 4JC-F = 3.0 Hz). HRMS (ESI) m/z 618.3696 (calcd for C35H50FN3O4Na [M+Na]+, 618.3683).

7-[4-(9E)-octadec-9-enoyl-piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (5) White solid, 200 mg (56%). Mp. 125°C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.86 (t, J = 6.9 Hz, 3H), 1.18-1.40 (m, 24H), 1.65 (quint, J = 7.2 Hz, 2H), 1.96-2.02 (m, 4H), 2.39 (t, J = 14

7.8 Hz, 2H), 3.31 (t, J = 5.1 Hz, 2H), 3.38 (t, J = 5.1 Hz, 2H), 3.53-3.61 (m, 1H), 3.73 (t, J = 5.1 Hz, 2H), 3.88 (t, J = 4.8 Hz, 2H), 5.32-5.45 (m, 2H), 7.35 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 12.9 Hz, 1H), 8.68 (s, 1H), 14.88 (s, 1H). 13C NMR (CDCl3, 75 MHz) δ (ppm): 8.2 (2xC), 14.1, 22.6, 25.2, 29.0, 29.1, 29.3 (2xC), 29.4, 29.4, 29.6, 29.6, 31.9, 32.5, 32.6, 33.2, 35.3, 41.0, 45.3, 49.4 (d, 4JC-F = 2.2 Hz), 50.2 (d, 4JC-F = 6.0 Hz), 105.0 (d, 3JC-F = 3.0 Hz), 108.0, 112.3 (d, 2JC-F = 23.2 Hz), 120.0 (d, 3JC-F = 8.2 Hz), 130.1, 130.4, 138.9 (d, 4JC-F = 0.7 Hz), 145.4 (d, 2JC-F = 10.5 Hz), 147.4, 153.5 (d, 1JC-F = 249.7 Hz), 166.7, 171.8, 176.9 (d, 4JC-F = 2.3 Hz). HRMS (ESI) m/z 618.3698 (calcd for C35H50FN3O4Na [M+Na]+, 618.3683).

7-[4-(9Z,12Z,15Z)-octadec-9,12,15-trienoyl-piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo1,4-dihydro-quinoline-3-carboxylic acid (6) White solid, 200 mg (56%). Mp. 104°C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.92-0.98 (m, 3H), 1.18-1.22 (m, 2H), 1.32-1.40 (m, 10H), 1.65 (quint, J = 7.2 Hz, 2H), 2.00-2.08 (m, 4H), 2.37 (t, J = 7.5 Hz, 2H), 2.76-2.80 (m, 4H), 3.29 (t, J = 5.1 Hz, 2H), 3.60 (t, J = 4.8 Hz, 2H), 3.51-3.59 (m, 1H), 3.71 (t, J = 4.8 Hz, 2H), 3.86 (t, J = 5.1 Hz, 2H), 5.27-5.42 (m, 6H), 7.33 (d, J = 7.2 Hz, 1H), 7.90 (d, J = 12.9 Hz, 1H), 8.65 (s, 1H), 14.85 (s, 1H). 13C NMR (CDCl3, 75 MHz) δ (ppm): 8.2 (2xC), 14.2, 20.5, 25.2, 25.5, 25.6, 27.2, 29.1, 29.3, 29.4, 29.5, 33.2, 35.3, 41.0, 45.3, 49.4 (d, 4JC-F = 3.7 Hz), 50.2 (d, 4JC-F = 5.2 Hz), 105.0 (d, 3JC-F = 3.0 Hz), 107.9, 112.3 (d, 2JC-F = 23.2 Hz), 119.9 (d, 3JC-F = 8.2 Hz), 127.0, 127.7, 128.2, 128.2, 130.2, 131.9, 138.9, 145.4 (d, 2JC-F = 10.5 Hz), 147.4, 153.5 (d, 1JC-F = 249.7 Hz), 166.7, 171.8, 176.8 (d, 4J = 2.3 Hz). HRMS (ESI) m/z 614.3387 (calcd for C35H46FN3O4Na [M+Na]+, 614.3370).

7-[4-(13Z)-docos-13-enoyl-piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7) White solid, 200 mg (54%). Mp. 110°C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, J = 6.9 Hz, 3H), 1.22-1.42 (m, 32H), 1.66 (quint, J = 7.5 Hz, 2H), 1.93-1.99 (q, J = 6.3 Hz, 4H), 2.39 (t, J = 7.5 Hz, 2H), 3.32 (t, J = 5.1 Hz, 2H), 3.39 (t, J = 5.1 Hz, 2H), 3.55-3.62 (m, 1H), 3.74 (t, J = 4.8 Hz, 2H), 3.88 (t, J = 4.8 Hz, 2H), 5.29-5.40 (m, 2H), 7.35 (d, J = 6.9 Hz, 1H), 7.86 (d, J = 12.9 Hz, 1H), 8.63 (s, 1H), 14.87 (s, 1H).

13

C NMR (CDCl3, 75 MHz) δ (ppm):

8.1 (2xC), 14.0, 22.6, 25.2, 27.1, 29.2, 29.4, 29.4, 29.5, 29.5, 29.6, 29.6, 29.7, 29.7, 31.8, 33.2, 35.3, 41.0, 45.3, 49.3, 50.1 (d, 4JC-F = 5.3 Hz), 105.0 (d, 3JC-F = 3.7 Hz), 107.8, 112.1 (d,

15

2

JC-F = 23.2 Hz), 119.7 (d, 3JC-F = 7.5 Hz), 129.7, 129.8, 138.9, 145.3 (d, 2JC-F = 10.5 Hz),

147.3, 153.4 (d, 1JC-F = 249.8 Hz), 166.6, 171.8, 176.7 (d, 4JC-F = 2.3 Hz). HRMS (ESI) m/z 674.4329 (calcd for C39H58FN3O4Na [M+Na]+, 674.4309).

7-[4-(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoyl-piperazin-1-yl]-1cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (8) White solid, 230 mg (59%). Mp. 105°C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.97 (t, J = 7.5 Hz, 3H), 1.18-1.24 (m, 2H), 1.37-1.44 (m, 2H), 2.02-2.12 (m, 2H), 2.46-2.47 (m, 4H), 2.79-2.89 (m, 10H), 3.31 (t, J = 5.1 Hz, 2H), 3.38 (t, J = 5.1 Hz, 2H), 3.53-3.61 (m, 1H), 3.73 (t, J = 5.1 Hz, 2H), 3.89 (t, J = 5.1 Hz, 2H), 5.26-5.45 (m, 12H), 7.50 (d, J = 7.2 Hz, 1H), 7.92 (d, J = 12.9 Hz, 1H), 8.67 (s, 1H), 14.87 (s, 1H). 13C NMR (CDCl3, 75 MHz) δ (ppm): 8.2 (2xC), 14.2, 20.5, 22.9, 25.5, 25.6, 25.6, 25.6 (2xC), 32.9, 35.3, 41.01, 45.2, 49.4 (d, 4JC-F = 3.0 Hz), 50.1 (d, 4JC-F = 6.0 Hz), 105.0 (d, 3JC-F = 3.0 Hz), 107.9, 112.3 (d, 2JC-F = 24.0 Hz), 119.9 (d, 3JC-F = 7.5 Hz), 126.9, 127.8, 128.0, 128.0, 128.0, 128.2, 128.2, 128.2, 128.2, 128.5, 129.1, 132.0, 138.9 (d, 4JC-F = 0.7 Hz), 145.4 (d, 2JC-F = 10.5 Hz), 147.4, 153.5 (d, 1JC-F = 249.7 Hz), 166.7, 170.9, 176.8 (d, 4JC-F = 3.0 Hz). HRMS (ESI) m/z 664.3545 (calcd for C39H48FN3O4Na [M+Na]+, 664.3527).

7-(4-hexadecanoyl-piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-quinoline-3carboxylic acid (8) Light beige solid, 152 mg (44%). Mp. 149°C. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, J = 6.9 Hz, 3H), 1.21-1.45 (m, 28H), 1.65 (quint, J = 7.5 Hz, 2H), 1.93-1.99 (m, 4H), 2.39 (t, J = 7.8 Hz, 2H), 3.31 (t, J = 5.1 Hz, 2H), 3.38 (t, J = 5.1 Hz, 2H), 3.54-3.61 (m, 1H), 3.74 (t, J = 4.8 Hz, 2H), 3.88 (t, J = 4.8 Hz, 2H), 7.36 (d, J = 6.9 Hz, 1H), 7.94 (d, J = 12.9 Hz, 1H), 8.69 (s, 1H), 14.88 (s, 1H). 13C NMR (CDCl3, 75 MHz) δ (ppm): 8.2 (2xC), 14.1, 22.7, 25.3, 29.4, 29.5, 29.5, 29.5, 29.6, 29.6, 29.7, 29.7 (3xC), 31.9, 33.3, 35.3, 41.1, 45.4, 49.4 (d, 4

JC-F = 3.0 Hz), 50.2 (d, 4JC-F = 6.0 Hz), 105.0 (d, 3JC-F = 5.5 Hz), 108.0, 112.3 (d, 2JC-F = 23.0

Hz), 112.0 (d, 3JC-F = 7.8 Hz), 139.0, 145.4 (d, 2JC-F = 10.4 Hz), 147.4, 153.5 (d, 1JC-F = 249.7 Hz), 166.7, 171.9, 176.8 (d,

4

JC-F = 2.7Hz).HRMS (ESI) m/z 592.3548 (calcd for

C33H48FN3O4Na [M+Na]+, 592.3527).

4.2. Biological studies 4.2.1. Cell culture 16

Primary and metastatic colon cancer (SW480, SW620), metastatic prostate cancer (PC3) and human immortal keratinocyte (HaCaT) cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, USA) and cultured in MEM (Minimal Essential Medium, Thermo Sci, USA), RPMI 1640 (Roswell Park Memorial Institute, Biowest SAS, France), and DMEM (Dulbecco’s Modified Eagle’s Medium, Biowest SAS, France), respectively. SW480 cell line was initiated by A. Leibovitz, et al. [46] and established from a human primary colon adenocarcinoma, whereas SW620 was derived from a lymph node metastasis, from the same patient as SW480. PC3 line (prostatic metastatic cells) was obtained from the bones. HaCaT cells were derived from an adult human skin. Cells were seeded in 6 ml medium in a tissue culture flask (50 ml) in a 37 °C/5% CO2 humidified incubator. Medium was supplemented with 10% heat-inactivated fetal bovine serum (Gibco Life Technologies,USA), penicillin (100 U/ml), streptomycin (100 µg/ml) and HEPES (20 mM). The cells were cultured until 80% confluency was reached, then harvested by treatment with 0.25% trypsin-0.02% EDTA (Gibco Life Technologies, USA), and used for experiments. 4.2.2. MTT assay The cell viability was assessed by using an enzymatic conversion of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) to insoluble formazan crystals by mitochondrial dehydrogenases, occurring in living cells. Cells were cultured in 96well plates at a density of lx104 cells per well, and cultured to adhere for 24 hours at 37°C in a CO2 incubator. After 24 hours of incubation, culture medium was replaced with a fresh medium, then cells were treated with various concentrations of the compounds (ranged from 5 to 140µM), and incubated for 72 hours at 37°C in a CO2 incubator. Untreated cells were used as the control. Subsequently, MTT solution (0,5 mg/mL in free-serum medium) was added, and samples were incubated for 4 hours at 37°C in a CO2 incubator. Then the medium was aspirated, and formed formazan crystals were solubilized by adding a isopropanol DMSO mixture (1:1 vol) The intensity of the dissolved crystals was measured using a UVM 340 reader (ASYS Hitech GmbH, Austria), at a wavelength of 570 nm. Cell viability was presented as a percent of MTT reduced in treated cells versus control cells (incubated without tested compounds). The relative MTT level (%) was calculated as [A]/[B] ×100, where [A] is the absorbance of the test sample, and [B] is the absorbance of the control sample containing the untreated cells. IC50 value was estimated using CompuSyn version 1.0.

17

4.2.3. LDH assay As a marker of cell death, the release of lactate dehydrogenase (LDH) from the cytosol to culture medium (cellular membrane integrity assessment) was used. The assay was performed after 72 h incubation of cells in 96-well plates with the investigated compounds, as described before [47]. The activity of lactate LDH released from the cytosol of damaged cells into the supernatant was measured according to the protocol of the LDH test, described by the manufacturer (Roche Diagnostics, Germany). Absorbance was measured at 490 nm using a microplate UVM 340 reader (ASYS Hitech GmbH, Austria). Compound-mediated cytotoxicity expressed as (%) of the LDH release was determined by the following equation: [(Atest sample−A low control)/(A high control−A low control)]×100% (A-absorbance); where “low control” were cells in DMEM, RPMI or MEM with 2% FBS, without tested compounds, and “high control” were cells incubated in DMEM, RPMI or MEM with 2% FBS and 1% Triton X-100 (100% LDH release). The detergent Triton X-100 is commonly used as the positive control in the LDH assay in determination of the maximum LDH release from the cells. 4.2.4. FITC Annexin V binding assay The SW480, SW620 and PC3 cells were cultured and harvested under the conditions mentioned in the Cell Culture section and seeded in 6-well plates (2 x 105 cells per well). After 24 h pre-incubation, cells were treated with IC50 concentrations of tested compounds and incubated for 72 hours. The apoptotic effect was determined by dual staining with Annexin V:FITC and propidium iodide (PI) using a detection kit (FITC:Annexin V Apoptosis Detection Kit I; BD Biosciences Pharmingen) according to manufacturer’s protocol, and analyzed by flow cytometry (Becton Dickinson). Cells which were Annexin V:FITC positive and PI negative were identified as early apoptosic, and Annexin V:FITC and PI positive as late apoptosic or necrotic. 4.2.5. IL-6 assay The level of IL-6 in primary and metastatic colon cancer cells, metastatic prostate cancer cells and immortal keratinocyte cell lines was measured by ELISA (Diaclon SAS Besancon Cedex, France). Cells were treated with IC50 concentrations of all studied conjugates for 72 h. The IL-6 level in a cell culture supernatant was measured using an enzyme-linked immunosorbent assay, in accordance with the manufacturer's protocol. 18

4.2.6. Statistical analysis The statistical analysis was performed using Statistica 13.0 (StatSoft, Inc, USA) program. Comparison between studied groups was performed by the paired Student’s t test. Results were expressed as means ± SD, and considered statistically significant at P < 0.05. All dates were calculated from 5 separate experiments. 4.2.7. Antimicrobial activity The antimicrobial assays were conducted using reference strains of bacteria derived from international microbe collections: American Type Culture Collection (ATCC) and National Collection of Type Culture (NCTC). Among the reference strains there were 5 Gram-positive isolates (Staphylococcus aureus: NCTC 4163, ATCC 29213 and ATCC 6538, S. epidermidis ATCC 12228, E. hirae ATCC 10541) and 4 Gram-negative rods (Pseudomonas aeruginosa ATCC 15442 and ATCC 27853, Escherichia coli NCTC 8196 and ATCC 25922). The antimicrobial activity of conjugates was expressed by minimum inhibitory concentration values (MICs), according to CLCI reference procedures with some modification. MIC was tested by the twofold serial microdilution method (in 96-well microtiter plates) on MH II liquid medium. The final inoculum of all studies bacteria was 106 CFU/ml (colony forming unit per ml). The stock solution of tested compounds was prepared in DMSO, and diluted in sterile medium (to maximum of 1 % of solvent content). The concentrations of compounds varied from 0.03125 to 512 µg/ml. The MIC value was the lowest concentration of the compound, at which bacterial growth was no longer observed after 18 h of incubation at 35 ◦C [48]. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the Medical University of Warsaw and carried out with the use of CePT infrastructure financed by the European Union - the European Regional Development Fund within the Operational Programme Innovative Economy for 2007-2013. Authors thank Agnieszka Filipek form the Department of Pharmaceutical Microbiology, Medical University of Warsaw, for flow cytometry analysis. 19

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24

Comp.

R

Name of fatty acid

Yields (%)

ClogPa

56

0.63

1

C3H5C(O)-

crotonic

Chain length: unsaturation 4:1 (E2)

2

C5H7C(O)-

sorbic

6:2 (E2, E4)

58

1.15

3

C9H15C(O)-

geranic

10:2 (E2, E6)

48

3.46

4

C17H33C(O)-

oleic

18:1 (Z9)

47

7.76

5

C17H33C(O)-

elaidic

18:1 (E9)

56

7.76

6

C17H29C(O)-

linolenic

18:3 (Z9, Z12, Z15)

56

6.02

7

C21H41C(O)-

erucic

22:1 (Z13)

54

9.02

8

C21H31C(O)-

DHA (Docosahexaenoic)

59

5.86

9

C15H31C(O)-

palmitic

22:6 (Z4, Z7, Z10, Z13, Z16, Z19) 16:0

44

7.24

Table 1. The structures, yields and ClogP values of CP-conjugates 1-9. aSoftware-predicted lipophilicity was estimated by means of the software ClogP (www.molinspiration.com).

Cancer cells c

Compound IC50a

SW480 SIb

Normal cells

d

e

SW620 IC50

PC3

SI

IC50

SI

HaCaTf IC50

1

51.9±4.7

1.25

54.5±5.2

1.19

93.9±3.4

0.69

64.9±2.9

2

20.1±2.1

5.43

21.5±3.1

5.07

11.7±1.8

9.32

109.1±2.8

3

32.8±2.4

3.06

38.2±3.1

2.63

73.4±2.7

1.36

100.5±2.6

4

79.5±5.4

1.66

37.9±4.7

3.49

7.7±2.1

17.20

132.5±5.3

5

35.7±2.6

3.95

33.9±1.8

4.16

15.3±5.3

9.22

141.2±4.4

6

64.6±3.1

4.13

45.8±5.6

5.83

34.4±2.4

7.75

266.9±4.7

7

118.6±5.6

1.31

65.8±4.3

2.36

76.4±3.3

2.03

155.7±4.3

8

26.2±4.3

8.83

40.1±3.7

5.82

27.8±1.9

8.32

231.4±3.4

9

82.4±6.3

1.53

93.3±6.9

1.35

51.1±4.5

2.48

126.7±2.9

160.4±6.7

1.38

200.4±4.9

1.11

101.4±3.6

2.19

222.1±5.2

0.75±0.1

0.38

0.26 ± 0.1

1.11

0.31±0.1

0.93

0.29 ± 0.1

CPg h

DOX

Table 2. Cytotoxic activity (IC50, µM) of studied compounds estimated by the MTT assay. Data are expressed as mean±SD, aIC50 (µM) - the concentration of the compound that corresponds to a 50% growth inhibition of a cell line as compared to the control, after 72 h-culture the cells with the individual compound, bThe SI (Selectivity Index) was calculated using formula: SI = IC50 for normal cell line/IC50 of a cancer cell line, c Human primary colon cancer (SW480), dHuman metastatic colon cancer (SW620), eHuman metastatic prostate cancer (PC3), fHuman immortal keratinocyte cell line from adult human skin (HaCaT), gCiprofloxacin, hDoxorubicin

LDH realase (%)

Compounds Cell line

HaCaT

SW480

SW620

PC3

Control

CP

1

2

3

4*

5

6

7

8

9

3.1

4.6 4.2 3.9 3.2

3.7 3.5 3.3 3.2

7.3 6.2 4.9 4.1

4.4 3.4 3.2 3.0

7.5 6.2 4.9 4.1

6.2 4.8 4.1 3.3

4.7 4.5 3.9 3.3

4.3 3.5 3.1 2.9

5.3 4.1 3.6 3.3

6.3 5.3 4.1 3.4

15 14.5 14.2 12

29 22.4 19.5 13

68.3 56.4 42.3 37.2

29.2 21.6 17.4 14.4

36.8 28.4 22.3 19

63 52.3 33.3 29

39 32.4 29.5 27

29.3 23 14.7 11

59 41 47.7 31

39.3 33 24.6 15

9.3 9.1 8.9 8.1

20.3 14.3 12.6 11.7

44.4 37 31.7 28

30 28.4 25.3 19.7

42.4 34 32 23

37.7 34.5 29.4 21.6

30.2 27.5 24.9 18.7

13.8 11 8.4 7.3

32 25.3 24.4 22.6

13.5 11.8 6.5 7.3

18 17.5 16.5 14

29 22.3 19.4 17

73 66.4 51 47.2

32 25 14 12

83 76.4 71 67.2

71 67.8 44 41

49 33.2 29.5 17

25.3 15 11.3 5.8

62 45 39 32

25.3 21 17 15

4.9

4.2

5.3

Table 3. LDH release as a marker of a cell death in HaCaT, SW480, SW620 and PC3 cells, treated for 72 h with CP-conjugates at concentrations of 60, 40, 20 and 10 µM (for the compound 4* at 40, 20, 10 and 5 µM). The cytotoxicity was expressed as percentage of LDH release, as compared to the maximum release of LDH from Triton-X100-treated cells.

Compound 1

2

3

4

5

6

7

8

9

CP

512 512 512 512 512 512 512 512 512

4 4 4 4 64 32 32 512 256

256 256 256 512 256 512 512 512 512

8 8 16 8 64 32 2 16 32

512 512 512 512 512 512 512 512 512

0.25 0.5 0.5 0.25 8 0.031 0.031 0.25 0.5

Strain S. aureus NCTC 4163 S. aureus ATCC 29213 S. aureus ATCC 6538 S. epidermidis ATCC 12228 E. hirae ATCC 10541 E. coli ATCC 25922 E. coli NCTC 8196 P. aeruginosa ATCC 15442 P. aeruginosa ATCC 27853

0.5 0.5 1 256 0.5 0.5 1 512 0.5 0.5 0.5 128 0.5 1 1 512 128 256 128 128 1 0.5 8 128 1 0.5 8 128 512 128 128 512 512 64 256 512

Table 4. Activities of conjugates 1-9 against Gram-positive and Gram-negative bacteria – minimal inhibitory concentrations (MIC, µg/mL).

OH

O

OH F

O

BOP, Et3N, rt

+ R

N

O

O

N

OH NH

F O

CH2Cl2

N

N N

O R

Scheme 1. Synthesis of derivatives of CP and fatty acids (R = saturated or unsaturated carbon chain).

100

PC3 ***

90 ***

80

***

***

***

70

LDH release [%]

SW480 ***

60

***

SW620

***

***

HaCaT

***

50

*** ***

40 30 ***

***

20 10

**

***

**

*** ***

**

**

***

**

**

**

**

**

*

0 Control

60

40

20 CP

10

60

40

20 2

10

40

20

10 4

5

60

40

20 5

Fig. 1. LDH release as a marker of a cell death in the PC3, SW480, SW620 and HaCaT cells, treated for 72 h with various concentrations of tested compounds (60, 40, 20 and 10 µM for derivatives 2 and 5, and 40, 20, 10 and 5 µM for the compound 4. Data are expressed as the mean ± SD from three independent experiments, performed in triplicate. *** p≤0.001, ** p≤0.01, * p≤0.05 as compared to the control.

10

*** ***

***

% of apoptosis

***

***

*** ***

***

***

***

***

***

***

*** ******

*** *** ***

***

*** ***

**

***

*** **

***

*** ***

***

***

*** ***

***

***

Fig. 2. The effect of conjugates 2, 4, 5 and 8 on early and late apoptosis, or necrosis in PC3, SW480, SW620 and HaCaT cells. Cells were incubated for 72 h with compounds 2, 4, 5 and 8 used in their IC50 concentrations, then cells were harvested, stained with Annexin V-FITC and PI, and analyzed using flow cytometry. Data are expressed as % of cells at early stage of apoptosis, and as % of cells at late stage of apoptosis or necrosis. Data are expressed as means ± SD. *** p≤0.001, ** p≤0.01, p≤0.05, as compared to the control.

A.

B.

2.1%

1.7%

3.9%

31.1%

95.1%

1.1%

59.7%

5.3%

2.4%

85.1%

5.6%

6.9%

C.

D.

7.2%

61.2%

24.2%

7.4%

E.

F.

3.9%

77.6%

14.2%

4.3%

2.4%

63.8%

25.1%

8.7%

Fig. 3. The effect of conjugates 2, 4, 5 and 8 on early and late apoptosis or necrosis in PC3 cells detected with Annexin V-FITC/PI by flow cytometry. A.– control; B.- CP free; C.-compound 2; D.-compound 4; E.-compound 5; F.- compound 8. The lower right quadrant shows early apoptotic cells (Annexin V-FITC positive and PI negative staining). The upper right and upper left quadrants represent late stage of apoptotic or necrotic cells (Annexin V-FITC positive, PI positive, Annexin V-FITC negative and PI positive staining, respectively).

16 SW480 SW620

14

PC3

IL-6 concentration pg/ml

HaCaT

**

12

10 *** ***

***

8

** ***

6

**

*** ***

***

***

4

**

*

***

***

*** ***

***

***

*** 2

0

Control

2

4

5

Ciprofloxacin

Doxorubicin

Fig. 4. Effects of CP-conjugates (2, 4, 5), CP alone and doxorubicin on IL-6 levels. IL-6 levels in culture supernatant were measured by ELISA test. Data are expressed as the mean ± SD from three independent experiments performed in triplicate. *** p≤0.001, ** p≤0.01, * p≤0.05, as compared to the control.

Highlights: All conjugates exhibited higher anticancer effects than ciprofloxacin alone PC3 cell line was highly sensitive to substituted ciprofloxacin Compounds with sorbic, oleic and elaidic acids were the most effective Tested conjugates induced late apoptosis and inhibited IL-6 release in cancer lines Sorbic acid amide exhibited both high anticancer and antistaphylococcal activities