Fluoroaryl analogs of sulforaphane – A group of compounds of anticancer and antimicrobial activity

Journal Pre-proofs Fluoroaryl analogs of sulforaphane – a group of compounds of anticancer and antimicrobial activity Tomasz Cierpiał, Piotr Kiełbasiński, Małgorzata Kwiatkowska, Piotr Łyżwa, Katarzyna Lubelska, Dominika Kuran, Aleksandra Dąbrowska, Hanna Kruszewska, Lidia Mielczarek, Zdzisław Chilmonczyk, Katarzyna Wiktorska PII: DOI: Reference:

S0045-2068(19)31183-6 https://doi.org/10.1016/j.bioorg.2019.103454 YBIOO 103454

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

24 July 2019 24 September 2019 18 November 2019

Please cite this article as: T. Cierpiał, P. Kiełbasiński, M. Kwiatkowska, P. Łyżwa, K. Lubelska, D. Kuran, A. Dąbrowska, H. Kruszewska, L. Mielczarek, Z. Chilmonczyk, K. Wiktorska, Fluoroaryl analogs of sulforaphane – a group of compounds of anticancer and antimicrobial activity, Bioorganic Chemistry (2019), doi: https://doi.org/ 10.1016/j.bioorg.2019.103454

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Fluoroaryl analogs of sulforaphane – a group of compounds of anticancer and antimicrobial activity Tomasz Cierpiał1*, Piotr Kiełbasiński1, Małgorzata Kwiatkowska1, Piotr Łyżwa1, Katarzyna Lubelska2, Dominika Kuran2, Aleksandra Dąbrowska2, Hanna Kruszewska3, Lidia Mielczarek2, Zdzisław Chilmonczyk2, Katarzyna Wiktorska2* AUTHOR ADDRESSES 1 Division

of Organic Chemistry, Centre of Molecular and Macromolecular Studies, Polish

Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland. 2Department

of Drug Biotechnology and Bioinformatics. National Medicines Institute,

Chełmska 30/34, 00-725 Warszawa, Poland. 3Department

of Antibiotics and Microbiology. National Medicines Institute, Chełmska 30/34,

00-725 Warszawa, Poland.

KEYWORDS Sulforaphane, isothiocyanates, polyfluoroaryl derivatives, anticancer, antibacterial, antifungal and antiviral activity

1

ABSTRACT

A series of new sulforaphane analogs bearing various (poly)fluoroaryl substituents bonded to the sulfinyl sulfur atom in place of the original methyl group and having different number of methylene groups in the central alkyl chain were synthesized and fully characterized. The new compounds were tested in vitro for their anticancer, antibacterial, antifungal and antiviral properties. Some of them demonstrated a much higher anticancer activity against selected lines of cancer: skin (MALME-3M), colon (HT-29) and breast (MCF7 and MDA-MB-231) cells than that exhibited by native sulforaphane (SFN). Related lines of untransformed (normal) cells, taken from the same organs as the cancer ones, i.e. MALME3, CRL-1790 and MCF10, respectively, were checked, which allowed for the determination of the selectivity indexes (SI). In certain cases, the latter exceeded 3.2. Concerning the antibacterial activity, gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) were susceptible to some newly synthesized SFN analogs, while the selected probiotic strains were from 10 to 100 fold more resistant to them, which gives a possibility of protection of symbiont strains during a potential therapy with such compounds. The antifungal activity of the new compounds possessing the fluorophenyl substituent was found to be higher than the activity of the parent SFN. In turn, most of the new compounds showed generally no anti-HIV activity. The influence of the particular structural differences in the new molecules, analogs of SFN, on their biological activity is discussed. INTRODUCTION Sulforaphane (SFN) 1 (Figure 1) is a well-examined isothiocyanate (ITC) present in the cruciferous plant family with the highest concentration in broccoli.1 Recent epidemiological, laboratory and animal studies suggest that sulforaphane exhibits chemopreventive and therapeutic

2

activities in cancer. It induces apoptosis, cell cycle arrest, and it has an anti-inflammatory activity. SFN exhibits a pleiotropic mechanism of action involving e.g. multiple oncogenic signaling pathways related to STAT, NfkB, Wnt/-catenin transcriptional factors. SFN was also shown to inhibit histone deacetylases (HDACs) activity and regulate reactive oxygen species (ROS) level via the Nrf2-dependent manner. On the other hand, SFN exhibits low toxicity and good distribution and bioavailability. This feature makes sulforaphane activity different from conventional chemotherapy.2 While the perspectives for SFN and its analogs to be u sed in prevention or treatment of cancer have been discussed for over 20 years, only recently the action of these compounds to counteract human bacteria-related infections has been assessed.3-5 The recent comprehensive review of antibacterial properties of diverse isothiocyanates (ITCs) clearly shows that SFN belongs to the most effective ITCs against the human pathogens, in particular against H. pylori. The proposed antimicrobial mechanism of the SFN action includes the affecting of membrane integrity and enzymes involved in the redox balance and metabolism which leads to the bacteria death.5 The published data revealed the structural changes which yielded the molecule of significantly better anticancer and antibacterial activity.5,6-10 Nowadays, due to the still insufficient availability of effective and safe anticancer therapy and a lack of an effective treatment of infections caused by an increasing number of antibiotic-resistant bacteria phenotypes, the search for the appropriate medicines becomes a burning issue. Therefore, investigations of the synthesis and evaluation of various biological properties of SFN itself and various types of its new analogs and derivatives still continue unabated.6-10

3

H3C

O S

(CH2)4 1

N CS

Figure 1. Sulforaphane (SFN) In search of new sulforaphane analogs, we synthesized a series of enantiomeric fluoroalkyl analogs of sulforaphane 2, 3 and 4 (Figure 2). All the new compounds were tested in vitro for their cytotoxicity against melanoma cells to show increased activity in comparison with the natural sulforaphane. Interestingly, in no case a notable difference in activity was observed between enantiomers.11 This is in contrast to the natural sulforaphane whose (R) enantiomer exhibits significantly higher anticancer activity than the (S) enantiomer.

F3C

O S 2

NCS

(CH2)4

F3CCH2

O S

N C S

(CH2)4

3

F3CCH2

O S

N C S (CH2)5

4

Figure 2. Fluoroalkyl analogs of sulforaphane Continuing our research, we came across the publication which showed that the analog of sulforaphane, in which the isothiocyanate group was replaced by the isoselenocyanate moiety was more effective in killing certain cancer cells, yet was less toxic to non-cancer cells, than original sulforaphane.12 This prompted us to synthesize a new type of sulforaphane derivatives, in which the methyl group was replaced by various organofluorine substituents and the isothiocyanate group – with the isoselenocyanate group 5 – 9 (Figure 3).13

4

O F3C CH2-S-(CH2)4-N=C=Se

5 O CH2-S-(CH2)4-N=C=Se

F

O CH2-S-(CH2)5-N=C=Se

F

6 F3C

O S-(CH2)4-N=C=Se

F3C

7 F3C

O S-(CH2)5-N=C=Se

F3C 8

9

Figure 3. Organofluorine isoselenocyanate analogs of sulforaphane

The compounds were tested for their cytotoxicity against different subtypes of breast cancer, namely MCF7 and MDA-MB-231 cell lines and melanoma cells. Among a number of derivatives examined, compounds 6 - 9 shown in Figure 3 exhibited significantly higher cytotoxicity against the cancer cell lines than the original sulforaphane and the fluoroalkyl isothiocyanate derivatives (described previously11) and were, at the same time, less toxic for human nonmalignant CRL-1790 colon fibroblasts used as a model of a normal cell line. Finally, a recent overview should be mentioned which describes in detail syntheses and investigations of biological activities of isoselenocyanates and proves their very important properties.14 Since compounds 6 - 9 possessed the organofluorine substituents which belonged to the fluoroaryl or fluoroarylmethyl series and exhibited better anticancer properties than that bearing a fluoroalkyl group 5, we came to the conclusion that the fluoroaryl or fluoroarylmethyl substituents

5

might be to a higher extent responsible for the better biological activity of the compounds. For this reason, we considered it interesting to synthesize and examine a series of isothiocyanates bearing a broad variety of fluoroaryl and fluoroarylmethyl substituents. We have also broadened the assessment of their biological activity – not only of anticancer but also antifungal, antibacterial and antiviral properties. RESULTS AND DISCUSSION Chemistry To synthesize the required analogs of sulforaphane we decided to use the methodology described by us for the synthesis of the corresponding isoselenocyanates,13 which comprised the preparation of ω-aminoalk-1-yl fluoroaryl or fluoroarylmethyl sulfides 13 as crucial substrates. To obtain them, we performed the reaction between corresponding 1-bromo-ω-N-phthalimidoalkanes 10 and fluoroarene- or fluoroarylmethanethiols 11, followed by removal of the phthalimide moiety from ω-N-phthalimidoalk-1-yl fluoroaryl or fluoroarylmethyl sulfides 12 (Scheme 1). O Br (CH2)m

N

RFSH 11

O RF

S

(CH2)m

O 10

N O 12

H2N-NH2

RF

S

NH2 (CH2)m 13

m = 4, 5

Scheme 1. Synthesis of ω-aminoalk-1-yl fluoroaryl and fluoroarylmethyl sulfides 13 as crucial substrates.

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The sulfides 13 were then transformed in alternative ways – either they were treated with thiophosgene to give fluoroaryl or fluoroarylmethyl ω-isothiocyanatoalk-1-yl sulfides 14, followed by oxidation with m-CPBA, or first subjected to the oxidation with m-CPBA to ωaminoalk-1-yl fluoroaryl or fluoroarylmethyl sulfoxides 15, followed by the treatment with thiophosgene (Scheme 2). Using these procedures a series of the desired sulforaphane derivatives, fluoroaryl or fluoroarylmethyl ω-N-isothiocyanatoalk-1-yl sulfoxides 16(a-h) were obtained (Figure 4).

RF

S

(CH2)m

NH2

13 CSCl2 , 5 % NaOH, CH2Cl2

RF

S

(CH2)m 14

mCPBA, CH2Cl2

NC S

RF

mCPBA, CH2Cl2

RF

O S

O S

(CH2)m 15

NH2

CSCl2,, 5% NaOH, CH2Cl2 NC S (CH2)m 16 m = 4, 5

Scheme 2. Synthesis of fluoroaryl and fluoroarylmethyl analogs of sulforaphane

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F

O S

F

F F

(CH2)4 N=C=S

F

F

O S F

F3C

F

(CH2)5 N=C=S

F 16a O S

(CH2)4

16e

CH2

N=C=S

S

16b

O S

(CH2)5

N=C=S

O

F

F

(CH2)4

16f

N=C=S

O S

F3C

(CH2)4

N=C=S

F CF3

16c F F

O S

F3C

F

(CH2)4 N=C=S

F

O S

F3C

CF3 16d

16g

(CH2)5

N=C=S

16h

Figure 4. Structures of newly synthesized sulforaphane analogues 16(a-h) Additionally, to check whether the biological activity depends on the absolute configuration of particular enantiomers, we decided to resolve selected racemic products by preparative HPLC using chiral columns and the recycling HPLC instrument. Two compounds, namely 16d and 16e, were chosen and the data of the enantiomers are collected in Table 1.

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Table 1. Enantiomeric products. Enantiomers Comp.

Preparative-HPLC conditions

[α]D AcOEt

16d

Chirobiotic T2; hexane–(i-PrOH:MeOH 50%) 7%

+ 76.3 (c = 0.84)

99.9

- 76.1 (c = 1.52)

99.6

16e

Chirobiotic T2; hexane–(i-PrOH:MeOH 50%) 7%

+ 78.1 (c = 0.97)

99.8

- 65.3 (c = 0.55)

83.6

ee [%]

[α]D AcOEt

ee [%]

Biological testing The synthesized analogs were investigated for their anticancer, antibacterial, antiviral and antifungal activities. The study of the anticancer activity of the fluoroaryl and fluoroarylmethyl sulforaphane analogs is a continuation and also an extention of our previous research on the fluoroalkyl11 and organofluorine-isoselenocyanate13 analogs of SFN. The anticancer activity (cytotoxicity) was assessed as an ability of the compound to inhibit the growth of cancer cells which is expressed as the IC50 index (a concentration causing 50% inhibition of the untreated cells viability). Often the cells originating from different organs are used in the anticancer activity assessment to arrive at more general and complex conclusions.13,15 In this study, the set of the corresponding cancer and untransformed (normal) cells of three different organs: skin, colon and breast which allowed for evaluation of selectivity of action of the studied compounds. We calculated the selectivity index (SI) as a quotient: SI = IC50 of a compound in a normal cell line/IC50 of the same compound in a cancer cell line which indicated how many times cancer cells are more sensitive than normal cells to the compound investigated (Figure 7).16,17 Table 2 presents the calculated IC50 indexes in cancer and normal skin, colon and breast cells after 24, 48 and 72 h

9

incubation with SFN and the newly synthesized analogs. Table 3 contains the IC50 values in two cancer cell lines of the enantiomers of 16d and 16e. In turn, the corresponding SI indexes are presented in Figure 7.

Table 2. The IC50 [M] calculated for cancer and normal cells of skin, colon and breast incubated with SFN analogs (mean  SD, n=12). 16b

16c

16d

16e

16f

16h

SFN

MALME-3M

24h

malignant melanoma

11.9  3.5

10.1  3.1

3.7  0.9

4.7  0.4

15.6  4.1

7.9  1.7

40.6  3.0

48h

10.9  3.7

10.0  3.8

2.3  0.6

3.8  0.6

13.4  3.7

8.1  1.1

39.1  1.0

72h

10.5  1.9

9.3  1.7

2.7  0.7

4.3  0.7

12.9  1.8

11.0  1.3

33.7  0.6

72h

23.3  2.5

13.7  1.1

5.1  0.7

10.0  0.2

25.4  1.7

7.6  0.3

24.9  1.3

24h

6.8  2.6

6.1  0.8

2.0  0.4

2.5  0.8

6.1  1.0

4.5  0.3

30.5  8.1

4.5  0.2

4.1  0.4

1.2  0.1

1.7  0.2

5.1  1.0

3.7  0.6

16.3  3.9

72h

4.5  0.6

4.1  0.4

1.2  0.1

1.4  0.2

4.4  1.9

3.3  0.1

11.4  0.1

72h

12.9  2.8

11.7  0.4

4.0  0.7

4.0  0.7

12.9  2.5

7.4  0.8

17.1  2.3

24h

12.0  2.7

4.8  1.2

4.5  0.9

4.1  0.7

9.8  1.8

4.3  1.0

42.6  10.4

6.3  0.9

4.0  1.1

1.3  0.1

0.8  0.3

8.1  1.7

3.3  0.4

12.9  2.5

MALME3 skin fibroblasts HT-29

colorectal adenocarcinoma 48h cells

CRL-1790 non-tumorigenic, normal-like colon epithelial MCF7

breast adenocarcinoma 48h

10

MDA-MB-231

72h

4.9  1.3

3.4  1.0

0.9  0.1

0.7  0.1

5.8  1.5

2.4  0.3

11.9  2.0

24h

8.9  1.6

7.9  0.9

2.3  0.7

3.0  0.6

9.4  1.1

3.6  0.4

24.7  1.7

6.5  0.3

5.1  1.7

0.6  0.1

1.1  0.2

5.4  0.6

3.1  0.4

19.9  4.3

72h

4.0  0.9

3.1  0.8

0.5  0.1

1.2  0.1

4.3  0.4

2.6  0.3

11.3  0.7

72h

4.9  0.5

4.2  0.9

1.1  0.2

2.2  0.1

4.8  0.3

2.6  0.3

12.4  1.9

breast adenocarcinoma 48h

MCF10 non-tumorigenic, normal-like breast epithelial cells

Table 3. IC50 values [µM] of separated enantiomers, calculated for skin and breast cancer after 72 h incubation (mean  SD, n=12).

Comp. MALME-3M malignant melanoma MDA-MB-231 breast adenocarcinoma

16d

16e

(+)

(-)

racemate

(+)

(-)

racemate

2.2

1.4

2.7

4.8

2.3

4.3

 0.1

 0.1

 0.7

 0.1

 0.1

 0.7

0.5

0.5

0.5

1.4

0.7

1.2

 0.1

 0.1

 0.1

 0.1

 0.1

 0.2

Inspection of Table 2 clearly shows that the fluoroaryl SFN analogs exhibited the anticancer activity against MALME-3M melanoma cells, the most active being analogs 16d and 16e with the

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IC50 values after 72 h incubation equal to 2.7 and 4.3 M, respectively. A comparison of their IC50 in MALME-3M cells with those of the fluoroalkyl SFN analogs, previously studied by us11 (Figure 2) indicate that the new, fluoroaryl analogs are significantly more cytotoxic towards these cells. However, at the same time the fluoroaryl analogs exhibited significant cytotoxicity towards normal fibroblast MALME3. Hence, the calculated SI values were only moderate, being the highest (> 2) in case of compounds 16f, 16b and 16e, but yet almost 3 times higher than the value calculated for SFN (SI = 0.7) (Figure 7). The selectivity of the fluoroaryl SFN analogs was also better than the selectivity of the fluoroalkyl analogs of SFN described previously.11 These results indicate that the introduction of a benzene ring increases not only the anticancer activity but also the selectivity of the fluorine-containing SFN analogs. The cytotoxicity of our new SFN analogs in HT-29 colon cancer cell line was significantly higher than in melanoma cells. In case of the most active analogs 16d and 16e the calculated IC50 indexes reached after 72 h incubation the value of 1.2 and 1.4 M, respectively. Moreover, while the SI index calculated for SFN was 1.5, it was twice higher for analogs 16b-f and reached the value of 3.3. for analog 16d. The IC50 values of our new analogs 16 calculated in MCF7 and MDA-MB-231 breast cancer cell lines were similar to the value calculated in the HT-29 colon cancer cell line. Also in this case, they exhibited higher anticancer activity than original SFN. The IC50 values calculated for both breast cancer cell lines did not significantly differ for analogs 16b, 16c, 16h and 16f and were in the range of 2.4-5.8 M and 2.6-4.3 M in MCF7 and MDA-MB-231, respectively. The comparison of the calculated IC50 values of fluoroaryl analog 16h with structurally related organofluorine isoselenocyanate analog 9 (Figure 3)13 indicates that the analog 16h exhibits higher

12

cell growth inhibitory potency in breast cancer cell lines. Thus, replacement of the isoselenocyanate moiety in 9 by the isothiocyanate group as in 16h increased the anticancer activity of the molecule. Similarly to the MALME3M and HT-29, the highest potency of cell growth inhibitory was observed for compounds 16d and 16e in both breast cancer cell lines. Thus, the breast cancer cell lines exhibited the highest susceptibility to these analogs - the IC50 values reached a nanomolar range – 900 nM (16e) and 700 nM (16d) in MCF7 cells and 500 nM (16e) in MDA-MB-231 cells after 72 h of incubation. We conducted an introductory microscopic study of the mechanism of cell growth inhibition for these two analogs in MCF7 cells. Figure 5 shows that the number of MCF7 cells decreased drastically after incubation with 5 M of compounds 16d and 16e and the significant number of dead cells were visualized. In case of the same concentration of SFN only a moderate decrease in cell number and no dead cells were observed. Interestingly, recently SFN at 5 M was shown to reduce the breast cancer cell number by the induction of G2/M cell cycle arrest while the cytotoxic effect (cell death) was observed only after incubation with higher concentrations.18 The desired type of cell death is apoptosis called programmed cell death. As shown in Figure 6, in case of the most active analogs 16d and 16e the characteristic for early apoptosis AnnexinV staining with no PI fluorescence can be observed at concentration as low as 1 M. These results indicate that studied compounds have potency to induce apoptosis at low concentration, however to elucidate more precisely the ability of studied compounds to induce cell death further mechanistic study are required.

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Figure 5. The microscopic image of the cytotoxic activity of 5 M SFN analogs in the culture of MCF7 cells after 72 h of incubation. The image presents viable cells stained green with fluorescein – a product of enzymatic conversion of FDA occurring in living cells, dead cells stained red with propidium iodide (PI) – a DNA marker penetrating the dead cells with damaged cell membrane, and the transmitted light image of cell cultures. Magnification 10x, scale bar – 10 M.

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Figure 6. The microscopic image of the early apoptotic cells in Mcf7 cell culture after 72h of incubation with 1 M of 16d and 16e. The image presents cells stained green with FITC-AnnexinV – a probe for phosphatidylserine on the outer membrane of apoptotic cells, red with PI - a DNA marker penetrating the dead cells with damaged cell membrane, and the transmitted light image of cells. Magnification 60x, scale bar- 10M. The evaluation of the selectivity is one of the most important indicators to be considered during drug development (Lopez-Lazaro). Moreover in these cells analog 16e exhibited a desired selectivity of action towards cancer and normal cells of breast. The calculated SI value reached 3.1 and was the second record (after SI of analog 16d in colon cells) higher than 3 among all the SI’s obtained (Figure 7). One of the mechanisms of a selective cancer therapy is, as proposed by Sestili and Fimognari, the induction of oxidative stress19. It was proposed that the level of ROS is high in cancer cells and further elevation of ROS level, which is triggered by the exogenous inductor, leads to an induced ROS-dependent apoptosis. Normal cells are capable of a more effective elimination of ROS; additionally, the ROS basal level in normal cells is lower as compared to that in cancer cells. However, to fully elucidate the mechanism of action of our new SFN analogs, further study is needed.

15

Interestingly, the literature data on the selectivity of standard cytostatics across the cancer cell lines report the SI values lower than 2.17,20 In this respect the newly synthesized SFN analogs 16d and 16e exhibit better properties than standard chemotherapeutics. However, the SI values may be not high enough to induce a desired effect in vivo. Hence, to consider these analogs as prospective candidates for cancer therapy, more studies are desirable.

Figure 7. The selectivity indexes (SI) calculated on the basis of IC50 indexes in skin, colon and breast cells after 72 h of incubation with SFN and its analogs. The results obtained assist in the discussion about the structure-activity relationship in the case of SFN analogs. Thus, compound 16f exhibited the lowest cytotoxicity in both cancer and normal

16

cells of skin, colon and breast. The highest activity was shown by compounds 16d and 16e which bear the highest number of fluorine atom. This can be due to a higher lipophilicity of these compounds21 since lipophilic compounds can more likely interact via non-specific binding with multiple target structures in the cell. Separation of the fluorophenyl ring from the sulfoxide moiety by a methylene group (as in 16f) decreased anticancer activity. An interesting relationship was observed concerning the length of the alkyl chain. Thus, for compounds bearing fluorine atoms that are attached directly to the phenyl ring, the potency to inhibit cell growth increased together with the increase of the length of the alkyl chain. On the contrary, substitution of the phenyl ring with the CF3 groups resulted in a higher cytotoxicity of the compounds with a shorter alkyl chain. To sum up, the effect of fluorine on the biological activity of organic compounds is rather difficult to predict22 and at this stage no ultimate conclusions can be drawn. Finally, the data collected in Table 3 clearly indicate that the absolute configuration of the enantiomers of 16d and 16e is not a key factor determining their cytotoxic activity. For this reason, we did not make any further efforts to separate enantiomers of the other analogs and to determine their absolute configurations. As mentioned earlier, this observation is in contrast to the known feature of original SFN and indicates the possible different mechanism of action of these SFN analogs, which was previously suggested by us.11 Apart from being cytotoxic to cancer cells, SFN exhibits also a chemopreventive activity by inducing Nrf-2 dependent detoxifying enzymes, including NQO1. The NQO1- mediated reduction of quinones is a part of chemoprotection/detoxification process. The toxic quinones reduced by NQO1 are conjugated and excreted from the cells. Therefore, the induction of the NQO1

17

expression by dietary compounds has emerged as a strategy for cancer prevention. SFN was shown previously to induce NQO1 in skin fibroblasts and thus protect these cells from ROS formation.23 The results obtained by us in normal skin fibroblast MALME3 show (Figure 8) that SFN and its analogs 16b, 16c and 16d induced moderately the NQO1 activity (up to 150% of the control cells activity). However, analog 16h (two CF3 groups) induced NQO1 activity more than two times in comparison to untreated cells, while compounds 16f and 16e induced NQO1 activity in MALME3 cells up to almost 400% of the activity of control cells which, at the same time, was more than twice the activity induced by SFN. Interestingly, these three analogs are structurally quite different. In this context, it would be reasonable to assume that the distance between the isothiocyanate group and the aromatic substituent, that is common for these compounds, plays here a crucial role. Such an influence of an additional methylene group on the isothiocyanate biological activity has already been described by us11,13 and for various isothiocyanate derivatives.24,25,26 It should be stressed that at the same time compound 16f exhibited the weakest cytotoxicity among all the compounds investigated towards skin fibroblast MALME3 cell line (Table 2). This makes the 16f an interesting structure for further study on its possible application in chemoprevention of skin diseases related to ROS action, including skin cancer.

18

Figure 8. The NQO1 activity in normal skin fibroblasts MALME3 line incubated with 0.5 M SFN and its analogs. The results are presented as a percentage of control (mean  SD, n=8). *significantly different from SFN, p<0.05.

The newly synthesized fluoroaryl SFN analogs were next expansively evaluated with respect to their antimicrobial, i.e. antibacterial, antifungal and antiviral properties (Table 4 and Table 5). Analog 16d (with beneficial anticancer properties, vide supra) and analog 16h did not exhibit any antiviral, antibacterial and antifungal activity. Concerning the anti-HIV activity, only compound 16e (exhibiting also beneficial anticancer properties) showed similar activity to SFN (0.5 M compound inhibited HIV replication in 9%), the other compounds proved inactive (Table 5). As shown in Table 4, SFN and its analogs were not active against gram-negative bacteria (E. coli, P. aeruginosa). On the contrary, gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) were susceptible to SFN and its analogues. Thus, the MIC values of analogs 16c and 16f were at average twice lower than those obtained for SFN. Compounds 16b

19

and 16e showed the highest activity against the tested gram-positive bacteria, except for B. subtilis and E. chirae strains. Only in case of E. coli SFN exhibited the highest activity which is in accordance with the previous reports.27 In fact, the antimicrobial activity of SFN was already shown in various pathogenic strains. 4,28 What is interesting for the therapy of antibiotic-resistant strains, compound 16b and in particular compound 16e exhibited a highly satisfactory activity against S. aureus strains, including MRSA. The MIC values were in the range of 0.031-0.065 while the susceptibility of Staphylococcus sp. to linezoid, an antibiotic used for the treatment of infections caused by gram-positive bacteria, was defined as <0.012-0.024 µmol/mL.29 There is also an increasing interest observed in recent years in antibacterial effect of SFN analogs and in their possible role in the design of novel antibiotics. To assess further perspectives for the proposed analogs we have additionally performed the study of the influence of SFN and analogs on the probiotic strains. The probiotic strains, which are our symbionts, induce beneficial effects for health of humans or animals.30 In the case of analogs 16f, 16b, 16c and 16e and SFN their activity against probiotic strains were from 10 to 100 fold lower than against the pathogenic strains, which gives a possibility of protection of symbiont strains during a potential therapy with such compounds. Such an action was previously shown for phenolic compounds31,32 or phytochemicals from berries which can selectively inhibit the growth of pathogenic bacteria by structural damage without affecting the viability of probiotics. What is

interesting, these four analogs exhibited at the same time the highest inhibitory activity against pathogenic strains. Among them, analog 16e showed no toxicity against probiotics. Taking into account its high activity against gram-positive bacteria, including antibiotic-resistant MRSA strains, it exhibits a selectivity of action and may be considered as a promising structure for further research towards potential medicines active against gram-positive bacteria, and in particular S.

20

aureus strains, including antibiotic-resistant MRSA. Surprisingly in case of antibacterial activity no reasonable structure- activity relationship could be noticed.

Table 4. The minimal inhibitory concentration (MIC) (mol/mL) of SFN and selected analogs against probiotic, gram-negative and gram-positive bacteria.

Strain

Compound 16b

16c

16e

16f

SFN

L. rhamnosus ATCC 53103

>0.5

2

NA*

1

4

L. fermentum trivagia

>0.5

4

NA

4

NA

L. crispatus 8.1

0.5

2

NA

1

2

M. luteus ATCC 10240

0.0625

0.125

0.031

0.125

0.25

B. cereus ATCC 11778

0.0625

0.125

0.0625

0.125

0.5

B. subtilis ATCC 6633

0.125

0.125

0.125

0.125

0.25

S. epiderminis ATCC 12228

0.0625

0.0625

0.0625

0.125

0.25

S. aureus ATCC 6538

0.0625

0.125

0.0625

0.125

0.5

MRSA 876

0.0625

0.125

0.0625

0.125

0.5

MRSA ATCC 33392

0.0625

0.125

0.0625

0.0625

0.5

MRSA 854

0.125

0.0625

0.031

0.031

0.125

E. chirae ATCC 10541

0.125

0.25

0.25

0.125

1

E. coli ATCC 8739

NA

4

NA

2

0.5

P. aeruginosa ATCC 9027

NA

NA

NA

NA

NA

probiotic strains

bacterial strains

fungal strains

21

C. albicans ATCC 10231

0.25

0.5

NA

0.5

2

C. albicans ATCC 2091

0.25

0.5

NA

0.5

2

C. parapsilosis ATCC 22019

0.5

0.5

NA

1

2

C. tropicalis ATCC 750

0.25

0.5

1

0.5

2

A. brasiliensis ATCC 16404

0.125

1

0.5

0.5

2

*NA- not active in concentration over 4 µmol/mL, compounds 16d and 16h – not observed

The next group of microbes investigated were fungal strains which cause fungaemia: four candida strains - a major cause of morbidity and mortality in the healthcare setting and Aspergillus brasiliensis - responsible for pulmonary infections.33 As shown in Table 4, the antifungal activity of analogs 16b, 16c and 16f, possessing the same fluorophenyl substituent was higher than the activity of the parent SFN molecule. The derivative 16b with the lowest number of fluorine atoms exhibited similar activity against Candida sp. (MIC in range of 0.125-0.5) like fluconazole, an antifungal medication used for a number of fungal infections (MIC defined as <0.026-0.11 µmol/mL).34 Table 5. The inhibition of HIV replication (% of control)

Compound

Concentration [M] 0.1

0.2

0.5

1.2

2.5

5

SFN

NA*

NA

9.9 +/-0.2

16b, 16c, 16h, 16f

NA

NA

NA

-**

-**

-**

16d

NA

NA

5.2 +/-0.8

-**

-**

-**

16e

NA

3.0 +/-0.4

11.0 +/-0.1

-**

-**

-**

19.6 +/-0.5 20.5 +/-0.4 21.8 +/-2.4

*NA – not observed, ** – not studied due to cytotoxicity towards CEMT4 cells

22

CONCLUSION The new sulforaphane analogs, bearing fluoroaryl substituents bonded to the sulfinyl sulfur atom and having different number of methylene groups in the central carbon chain, which were synthesized and tested for their cytotoxicity against various cancer types, namely melanoma, colon and different subtypes of breast cancer, generally proved to exhibit significantly higher cytotoxicity against the cancer cell lines in comparison with the original sulforaphane, being at the same time less toxic for the corresponding normal cells. The selectivity indexes (SI) exceeded in certain cases the value of 3.2 (compounds 16d and 16e). Investigation of the activity of various compounds allowed to draw conclusions concerning dependence of their selectivity indexes on the compound structures. Selected new compounds exhibited antibacterial activity, particularly against gram-positive bacteria. Thus, the analog 16e was an effective agent against antibiotic resistant bacteria strains MRSA with MIC comparable to antibiotics used in clinic while being not toxic to probiotic beneficial strains. Finally, compound 16b which exhibited antifungal properties, may be considered as an effective agent against candidiasis. Such an activity is reported for the first time for the fluorine SFN analogs. As a result of the present study some new therapeutics area, apart from the anticancer one, were revealed which can be taken as a reason for the synthesis of new fluorine sulforaphane analogs as an addition to a broad variety of fluoroorganic pharmaceuticals.35,36 Hence, the studies will be continued.

23

EXPERIMENTAL SECTION Chemistry 1H, 13C,

and 19F NMR spectra were recorded on a Bruker instrument at 200, 504, and 188 MHz,

respectively, with CDCl3 as the solvent. Mass spectra, including HRMS, were measured on a Finnigan MAT instrument. Optical rotations were measured on a PerkineElmer 241MC polarimeter (c as indicated below). The enantiomeric excess (ee) values were determined by chiral HPLC (Varian Pro Star 210, Chirobiotic T) and the preparative enantiomer resolution by Chirobiotic T2 using Recycling Preparative HPLC instrument LC-9101, manufactured by Japan Analytical Industry Co., Ltd. Column chromatography was carried out with Merck 60 silica gel. TLC was performed on Merck 60 F254 silica gel plates. Preparation of ω-N-isothiocyanatoalk-1-yl fluoroaryl or ω-N-isothiocyanatoalk-1-yl fluoroarylalkyl sulfides 14. General procedure: To a stirred solution of appropriate ω-aminoalk1-yl fluoroaryl or fluoroarylalkyl sulfide 13 (1.85 mmol) in chloroform (10 mL), 2 mmol of thiophosgene was added dropwise, at 0 oC. After that, aqueous solution of NaOH (6 mmol, 2 mL H2O) was added. Solution was stirred for 1 h at 0 oC, and then at room temperature overnight. Layers were separated, aqueous layer was extracted with chloroform (2 x 10 mL). Combined organic layers were dried over anhydrous MgSO4, filtrated and volatile compounds were removed under reduced pressure. The crude product was purified by column chromatography (silica gel, dichloromethane/hexane in gradient) to give pure appropriate ω-N-isothiocyanatoalk-1-yl fluoroaryl or fluoroarylalkyl sulfide 14.

24

2’,3’,4’,5’,6’-pentafluorophenyl-4-isothiocyanato-1-butyl sulfide (14a): 41% yield. 1H NMR (200 MHz, CDCl3) δ 3.55 (t, J = 6.2 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H), 1.99 – 1.58 (m, 4H). 19F NMR (188 MHz, CDCl3) δ -131.38 – -132.37 (m), -151.23 – -151.76 (m), -159.65 – -160.19 (m). MS (CI/iso-butane): m/z = 314 [M+H]+. 4’-fluorophenyl-4-isothiocyanato-1-butyl sulfide (14b): 37% yield. 1H NMR (200 MHz, CDCl3) δ 7.43 – 7.29 (m, 2H), 7.06 – 6.92 (m, 2H), 3.53 (t, J = 6.2 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 1.93 – 1.61 (m, 4H). 1H{19F} NMR (200 MHz, CDCl3) δ 7.46 – 7.29 (m, 2H), 7.10 – 6.91 (m, 2H), 3.53 (t, J = 6.2 Hz, 2H), 2.89 (t, J = 6.7 Hz, 2H), 1.97 – 1.61 (m, 4H). 19F NMR (188 MHz, CDCl3) δ -114.40 – -114.61 (m). 19F{1H} NMR (188 MHz, CDCl3) δ -114.51 (s). 4’-fluorophenyl-5-isothiocyanato-1-pentyl sulfide (14c): 34% yield. 1H NMR (200 MHz, CDCl3) δ 7.40 – 7.28 (m, 2H), 7.06 – 6.93 (m, 2H), 3.50 (t, J = 6.3 Hz, 2H), 2.87 (t, J = 6.8 Hz, 1H), 1.80 – 1.42 (m, 6H). 1H{19F} NMR (200 MHz, CDCl3) δ 7.42 – 7.28 (m, 2H), 7.10 – 6.89 (m, 2H), 3.50 (t, J = 6.3 Hz, 2H), 2.87 (t, J = 6.8 Hz, 2H), 1.85 – 1.40 (m, 6H). 19F NMR (188 MHz, CDCl3) δ -114.80 – -115.07 (m). 19F{1H} NMR (188 MHz, CDCl3) δ -114.93 (s). 4’-(trifluorometyl)-2’,3’,5’,6’-pentafluorophenyl-4-isothiocyanato-1-butyl sulfide (14d): 25% yield. 1H NMR (200 MHz, CDCl3) δ 3.56 (t, J = 6.1 Hz, 2H), 3.08 (t, J = 6.7 Hz, 2H), 2.02 – 1.61 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 148.51 – 147.45 (m), 146.27 – 145.78 (m), 145.70 – 144.86 (m), 143.59 – 142.55 (m), 131.04 (s), 120.72 – 119.52 (m), 44.60 (s), 33.38 (s), 28.64 (s), 26.89 (s).

19F

NMR (188 MHz, CDCl3) δ -55.66 (t, J = 21.7 Hz), -131.58 – -132.06 (m), -

138.98 – -139.84 (m). MS (EI): m/z = 363 [M]+•. HRMS: m/z calcd for [M]+ C12H8F7NS2 362.99864, found 362.99773.

25

4’-(trifluorometyl)-2’,3’,5’,6’-pentafluorophenyl-5-isothiocyanato-1-pentyl sulfide (14e): 40% yield. 1H NMR (200 MHz, CDCl3) δ 3.53 (t, J = 6.2 Hz, 2H), 3.06 (t, J = 6.7 Hz, 2H), 1.86 – 1.44 (m, 6H). 13C NMR (126 MHz, CDCl3) δ 148.40 – 147.65 (m), 146.40 – 145.59 (m), 145.46 – 145.02 (m), 143.52 – 142.77 (m), 130.36 (s), 120.59 (s), 44.96 (s), 33.94 (s), 29.54 (s), 29.27 (s). 19F

NMR (188 MHz, CDCl3) δ -55.61 (t, J = 21.6 Hz), -131.23 – -132.89 (m), -138.50 – -140.81

(m). MS (EI): m/z = 377 [M]+•. HRMS: m/z calcd for [M]+ C13H10F7NS2 377.01429, found 377.01496. Preparation of ω-N-isothiocyanatoalk-1-yl fluoroaryl or ω-N-isothiocyanatoalk-1-yl fluoroarylalkyl sulfoxides 16. General procedure I: To a stirred solution of appropriate ωisothiocyanatoalk-1-yl fluoroaryl or fluoroarylalkyl sulfide 14 (0.8734 mmol) in dichloromethane (7 mL), solution of meta-chloroperbenzoic acid (m-CPBA) (0.8734 mmol) in dichloromethane (5 mL) was added dropwise, at -20 oC under argon. Solution was stirred for 0.5 h at -20 oC, and then additionally at room temperature for 1 h. The solution was washed with saturated aqueous NaHCO3 and the organic layer was separated and dried with anhydrous MgSO4. After filtration, the solvent was removed under vacuum. The crude product was purified by column chromatography (silica gel, dichloromethane/methanol in gradient) to give pure ω-Nisothiocyanatoalk-1-yl fluoroaryl or fluoroarylalkyl sulfoxides 16. 2’,3’,4’,5’,6’-pentafluorophenyl-4-isothiocyanato-1-butyl sulfoxide (16a): 75% yield. 1H NMR (500 MHz, CDCl3) δ 3.61 (t, J = 5.8 Hz, 2H), 3.56 – 3.15 (m, 2H), 2.00 – 1.86 (m, 4H). 19F NMR (188 MHz, CDCl3) δ -137.73 – -138.23 (m), -144.92 – -145.40 (m), -156.91 – -157.53 (m). MS (CI/iso-butane): m/z = 330 [M+H]+.

26

4’-fluorophenyl-4-isothiocyanato-1-butyl sulfoxide (16b): 86% yield. 1H NMR (200 MHz, CDCl3) δ 7.67 – 7.56 (m, 2H), 7.29 – 7.18 (m, 3H), 3.55 (t, J = 5.8 Hz, 2H), 2.81 (t, J = 6.5 Hz, 2H), 1.99 – 1.66 (m, 4H). 1H{19F} NMR (200 MHz, CDCl3) δ 7.69 – 7.53 (m, 2H), 7.32 – 7.17 (m, 3H), 3.55 (t, J = 5.8 Hz, 2H), 2.81 (t, J = 6.5 Hz, 2H), 2.01 – 1.67 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 164.45 (d, J = 251.9 Hz), 138.87 (s), 131.07 (s), 126.31 (d, J = 8.9 Hz), 116.81 (d, J = 22.6 Hz), 56.20 (s), 44.65 (s), 28.97 (s), 19.57 (s). 19F NMR (188 MHz, CDCl3) δ -107.42 – 107.74 (m). 19F{1H} NMR (188 MHz, CDCl3) δ -107.60 (s). MS (EI): m/z = 257 [M]+•. HRMS: m/z calcd for [M]+ C11H12F1NOS2 257.03444, found 257.03387. 4’-fluorophenyl-5-isothiocyanato-1-pentyl sulfoxide (16c): 84% yield. 1H NMR (200 MHz, CDCl3) δ 7.71 – 7.54 (m, 2H), 7.30 – 7.16 (m, 2H), 3.52 (t, J = 6.2 Hz, 2H), 2.78 (t, J = 7.3 Hz, 2H), 1.95 – 1.39 (m, 6H). 1H{19F} NMR (200 MHz, CDCl3) δ 7.70 – 7.51 (m, 2H), 7.26 – 7.18 (m, 2H), 3.52 (t, J = 6.2 Hz, 2H), 2.78 (t, J = 7.4 Hz, 2H), 1.99 – 1.39 (m, 6H). 13C NMR (126 MHz, CDCl3) δ 164.38 (d, J = 251.5 Hz), 139.12 (d, J = 2.9 Hz), 130.30 (s), 126.30 (d, J = 8.8 Hz), 116.71 (d, J = 22.5 Hz), 56.95 (s), 44.76 (s), 29.61 (s), 25.77 (s), 21.44 (s). 19F NMR (188 MHz, CDCl3) δ -107.70 – -108.05 (m). 19F{1H} NMR (188 MHz, CDCl3) δ -107.84 (s). MS (EI): m/z = 271 [M]+•. HRMS: m/z calcd for [M]+ C12H14F1NOS2 271.05009, found 271.04976. 4’-(trifluorometyl)-2’,3’,5’,6’-pentafluorophenyl-4-isothiocyanato-1-butyl sulfoxide (16d): 78% yield. 1H NMR (200 MHz, CDCl3) δ 3.62 (t, J = 5.8 Hz, 2H), 3.60 – 3.11 (m, 2H), 2.10 – 1.83 (m, 4H). 19F NMR (188 MHz, CDCl3) δ -55.99 (t, J = 21.9 Hz), -135.55 – -136.28 (m), 136.64 – -137.13 (m). MS (CI/izobutan): m/z = 381 [M+H]+. HRMS: m/z calcd for [M]+ C13H10F7NOS2 380.00138, found 380.00077. The racemic mixture of 16d was resolved into enantiomers using a recycling HPLC instrument equipped with chiral column Chirobiotic T2 with hexane:(i-PrOH:MeOH 50%) 7% as eluent.

27

(+)-16d [α]D + 76.3 (c = 0.84, AcOEt), ee = 99.9% (–)-16d [α]D – 76.1 (c = 1.52, AcOEt), ee = 99.6% 4’-(trifluorometyl)-2’,3’,5’,6’-pentafluorophenyl-5-isothiocyanato-1-pentyl

sulfoxide

(16e): 89% yield. 1H NMR (200 MHz, CDCl3) δ 3.57 (t, J = 6.1 Hz, 2H), 3.59 – 3.07 (m, 2H), 2.00 – 1.62 (m, 6H). 19F NMR (188 MHz, CDCl3) δ -55.98 (t, J = 21.8 Hz), -135.78 – -136.56 (m), -136.76 – -137.11 (m). MS (EI): m/z = 393 [M]+•. HRMS: m/z calcd for [M]+ C13H10F7NOS2 393.00920, found 393.00864. The racemic mixture of 16e was resolved into enantiomers using a recycling HPLC instrument equipped with chiral column Chirobiotic T2 with hexane:(iPrOH:MeOH 50%) 7% as eluent. (+)-16e [α]D + 78.1 (c = 0.97, AcOEt), ee = 99.8% (–)-16e [α]D – 65.3 (c = 0.55, AcOEt), ee = 83.6% Preparation of ω-N-isothiocyanatoalk-1-yl fluoroaryl or ω-N-isothiocyanatoalk-1-yl fluoroarylalkyl sulfides 16. General procedure II: To a stirred solution of appropriate ωaminoalk-1-yl fluoroaryl or fluoroarylalkyl sulfoxide 15 (1.85 mmol) in chloroform (10 mL), 2 mmol of thiophosgene was added dropwise, at 0 oC. After that, aqueous solution of NaOH (6 mmol, 2 mL H2O) was added. Solution was stirred for 1 h at 0 oC, and then at room temperature overnight. Layers were separated, aqueous layer was extracted with chloroform (2 x 10 mL). Combined organic layers were dried over anhydrous MgSO4, filtrated and volatile compounds were removed under reduced pressure. The crude product was purified by column chromatography (silica gel, dichloromethane/methanol in gradient) to give chromatographically and

28

spectroscopically pure appropriate ω-N-isothiocyanatoalk-1-yl fluoroaryl or fluoroarylalkyl sulfoxide 16. 4’-fluorobenzyl-4-isothiocyanato-1-butyl sulfoxide (16f): 40% yield. 1H NMR (200 MHz, CDCl3) δ 7.35 – 7.19 (m, 2H), 7.17 – 7.01 (m, 2H), 3.96 (s, 2H), 3.55 (t, J = 6.1 Hz, 2H), 2.70 – 2.53 (m, 2H), 2.03 – 1.69 (m, 4H). 1H {19F} NMR (200 MHz, CDCl3) δ 7.35 – 7.21 (m, 2H), 7.14 – 6.99 (m, 2H), 3.96 (s, 2H), 3.55 (t, J = 6.1 Hz, 2H), 2.68 – 2.49 (m, 2H), 2.02 – 1.70 (m, 4H). 13C

NMR (126 MHz, CDCl3) δ 162.93 (d, J = 248.0 Hz), 131.83 (d, J = 8.3 Hz), 130.92 (s), 125.48

(d, J = 3.2 Hz), 116.18 (d, J = 21.7 Hz), 57.34 (s), 49.84 (s), 44.72 (s), 29.12 (s), 20.14 (s). 19F NMR (188 MHz, CDCl3) δ -112.13 – -112.48 (m). 19F {1H} NMR (188 MHz, CDCl3) δ -112.33 (s). MS (CI/iso-butane): m/z = 272 [M+H]+. 3’,5’-di-(trifluoromethyl)phenyl-4-isothiocyanato-1-butyl sulfoxide (16g): 42% yield. 1H NMR (200 MHz, CDCl3) δ 8.09 (s, 2H), 8.01 (s, 1H), 3.59 (t, J = 6.0 Hz, 2H), 3.08 – 2.71 (m, 2H), 2.19 – 1.75 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 147.43 (s), 133.09 (q, J = 34.3 Hz), 131.52 (s), 125.04 (dt, J = 6.9, 3.3 Hz), 124.40 (d, J = 2.6 Hz), 122.66 (q, J = 273.4 Hz), 56.28 (s), 44.60 (s), 28.86 (s), 19.75 (s). 19F NMR (188 MHz, CDCl3) δ -62.23 (s). MS (CI): m/z = 376 [M+H]+. HRMS: m/z calcd for [M]+ C13H11F6NOS2 375.01863, found 375.01812. 3’,5’-di-(trifluoromethyl)phenyl-5-isothiocyanato-1-pentyl sulfoxide (16h): 91% yield. 1H NMR (200 MHz, CDCl3) δ 8.08 (s, 2H), 8.01 (s, 1H), 3.55 (t, J = 6.1 Hz, 2H), 3.08 – 2.68 (m, 2H), 2.12 – 1.41 (m, 6H). 13C NMR (126 MHz, CDCl3) δ 147.79 (s), 133.14 (q, J = 34.2 Hz), 130.70 (s), 125.03 (s), 124.50 (d, J = 2.6 Hz), 122.79 (q, J = 273.5 Hz), 57.11 (s), 44.85 (s), 29.68 (s), 25.82 (s), 21.66 (s). 19F NMR (188 MHz, CDCl3) δ -62.23 (s). MS (EI): m/z = 389 [M]+•. HRMS: m/z calcd for [M]+ C14H13F6NOS2 389.03428, found 389.03395.

29

Biological testing Cell lines and microbial strains The human-origin cell lines used in the study MDA-MB-231 breast adenocarcinoma cells, MCF7 breast adenocarcinoma cells, HT-29 colorectal adenocarcinoma, MALME3M malignant melanoma and non-tumorigenic, normal-like MCF10 breast epithelial cells, CRL1790 colon epithelial cells and MALME3 skin fibroblasts were purchased from American Type Culture Collection (ATCC). Breast cancer cells and melanoma cells were grown in IMDM, both colon cells in MEM, media supplemented with fetal bovine serum, antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, 250 µg/mL amphotericin B) and non-essential amino acids. L-glutamine solution (2 mM) was added to MEM medium. MCF-10A cells were maintained in MEBM medium supplemented with 2 mL bovine pituitary extract, 0.5 mL human epidermal growth factor, 0.5 mL insulin, 0.5 mL hydrocortisone in ratio 1:1 with antibiotics mentioned above and non-essential aminoacids (0.1 mM). Malme-3 was cultured in the McCoy’s 5A Medium, supplemented with 15% heatinactivated fetal bovine serum. All cell lines were harvested at 37 oC in humidified incubator with 5% carbon dioxide atmosphere. The cells were routinely tested for the presence of mycoplasma using polymerase chain reaction (PCR) detection and microscopic examination.37 The following microbial strains were chosen from American Type Culture Collection (ATCC): Gram-positive bacteria strains: Micrococcus luteus 10240, Bacillus subtilis 6633, Staphylococcus

30

aureus (6538, 6538P), Enterococcus hirae 10541; Gram-negative bacteria strains: Escherichia coli 8739, Pseudomonas aeruginosa (9027, 15442); fungi (yeast strains): Candida albicans (10231, 2091), Candida parapsilosis 22019, Candida tropicalis 750; fungi (mold strain): Aspergillus brasiliensis 16404. The MRSA hospital strains (methicillin resistant S. aureus) from Clinical Hospital prof. Orlowski in Warsaw collection: A854 (isolated from throat mucosa, 30.03.2011) resistance: CIP, E, DA, AK and A876 (isolated from lower leg injury, 19.02.2012) resistance: E, DA, CIP, AK and MRSA ATCC strain 33392. The probiotic strains: Lactobacillus rhamnosus ATCC 53103 – the best characterized probiotic, Lactobacillus fermentum strain isolated from Trivagin capsules (dietary supplement), Lactobacillus crispatus strain isolated from cervical canal). Cell growth inhibition (MTT test) For cytotoxicity tests, after overnight incubation, cells were incubated with increasing concentrations of compounds. The sulforaphane and its analogs solutions were prepared in DMSO and were diluted at the ratio 1:1000 (v/v) in growth media. The MTT test used for the cytotoxicity assay is based on the ability of living cells dehydrogenases to convert water-soluble 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to non-soluble formazan. 50 µl of 0.25 mg/mL MTT-tetrazolium salts (Sigma-Aldrich Corp., St. Louis, MO, USA) in PBS was added to each well and after 3 hours of incubation, the formazan crystals were dissolved in 2-propanol. The absorbance was measured using a Power Wavex microplate spectrophotometer (Biotek Instruments) at 570 nm and 690 nm.38 All experiments were carried out in triplicate in at least two independent repetitions.

31

The IC50 values (the concentrations causing reduction of cell number to 50% of control) were determined with help of Prism 5 ver 5.03 (GraphPad Software, Inc.) according to the equation 1:

V

100 1  10(logIC50 c)h (1)

where: V - cell viability, c - concentration of the ITC, h - Hill coefficient. Selectivity index (SI) was determined as described previously as the ratio of IC50 of compound determined in a normal cell line and the IC50 of the same compound determined in cancer cell line.16,17 Cell cytotoxicity – cell viability and detection of apoptosis To determine the effect of the compounds on the viability of the cell culture the FDA/PI test was carried out. Fluorescein diacetate (FDA) is converted to fluorescent fluorescein by cellular esterases only in living cells, while propidium iodide (PI) stains dead cells with damaged membrane. The cells were stained with 0.125 µg/mL of FDA (fluorescein diacetate) and 0.5 µg/mL of PI (propidium iodide) for 15 min. The apoptosis was confirmed with help of Annexin V-FITC/PI test. The test employes two fluorescence probes AnnexinV-FITC which binds to phosphatidylserine on the outer membrane

32

of apoptotic cells and PI which stains dead cells. Cells were incubated for 15 minutes with propidium iodide (PI) (10ul / ml) and annexin (15 μl / ml). Then, the samples were analyzed using a confocal microscope with 10x UPlanApo or 60x PlanApoOil objectives. An Ar laser with a wavelength of 488 nm and a He-Ne laser with a wavelength of 543 nm were used as light sources. Fluorescence was recorded in the sequential mode with the use of 505-525 nm and 560-610 nm BP filters.39 The NQO1 activity The activity of NQO1 was determined by the Prochaska and Santamaria direct assay, measuring the NADPH-dependent menadiol-mediated reduction of MTT with modifications.40 Briefly, the cells were lysed and incubated for 10 minutes at 25 °C with NQO1 buffer containing 0.5M TRISCl at pH of 7.4, BSA, 1.5% Tween 20, 7.5 mM FAD, and 50 mM NADP (Sigma-Aldrich, USA). Immediately before the measurement, the buffer was charged with a 0.3 mg/mL of MTT, 50 mM acetonitrile solution of menadione, as well as glucose-6-phosphate dehydrogenase (200 U/mg of protein) (Sigma-Aldrich, USA). Formazan absorption was observed in a PowerWave X microplate spectrophotometer (Biotek Instruments, USA) at a wavelength of 610 nm. The activity of NQO1 was adjusted for the quantity of protein in a sample. Protein levels were quantified by the Bradford assay.41 Microbiological assays (MIC) The minimum inhibitory concentration (MIC) were assessed by the two fold broth microdilution method according to CLSI standard M07-A9.29 Sulforaphane derivatives were dispensed in 96well microplate. The compounds concentration range was from 0.004 to 4 µmol/mL. For probiotic

33

strains MRS broth (Merck) was used and for other strains Mueller Hinton II broth (bioMerieux) was used. Strain suspension at a density 0.5 McFarland units (Densimat, bioMerieux) were diluted 1:10 and 2 µL were added to each well. MIC was defined as the minimal concentration of the tested compound that completely inhibited visible strain growth. As a negative control the solution of the pure broth was used. All tests were performed in triplicate. Anti-HIV Activity CEM-T4 cells were preincubated (96 flat bottom wells culture plates) for 24 h in standard conditions (37 °C, 5% CO2) and in standard medium [RPMI, FCS 10%] enriched with tested compounds in concentrations: 0.12 M, 0.25 M, 0.5 M, 1.25 M, 2.5 M, 5 M. Compounds 16b, 16c, 16d, 16e, 16f, 16h, was tested only in concentration: 0.12 M, 0.25 M, 0.5 M because higher concentrations were toxic for CEMT4 cells. In each well, 20000 cells were suspended in the solution of the tested compound (200 L). For each concentration, cultures were run in triplicate. As a reference of maximal replication of HIV-1, culture in neat standard medium [RPMI/FCS 10%] was used. After 24 h of incubation in medium enriched with a tested compound, cells were inoculated with a known amount of HIV, and after 7 days, the amount of viral protein p24 was measured with the ELISA method. For each tested compound and for each concentration, the measurements of p24 were done in triplicate.

AUTHOR INFORMATION Corresponding Authors * T.C. e-mail: [email protected]; [email protected] * K.W. e-mail: [email protected]

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ORCID Tomasz Cierpiał: 0000-0003-2605-6945 Piotr Kiełbasiński: 0000-0002-0020-2492 Małgorzata Kwiatkowska: 0000-0001-6243-6561 Piotr Łyżwa: 0000-0002-6498-3750 Katarzyna Lubelska: 0000-0001-7546-8626 Dominika Kuran: 0000-0003-0502-0898 Aleksandra Dąbrowska: 0000-0002-0980-9418 Hanna Kruszewska: 0000-0002-0980-9418 Zdzisław Chilmonczyk: 0000-0002-5871-4458 Katarzyna Wiktorska: 0000-0002-2731-9535

Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

NOTES The authors declare no competing financial interest.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights    

A series of new sulforaphane analogs bearing various (poly)fluoroaryl substituents bonded to the sulfinyl sulfur atom were synthesized and fully characterized. In vitro tests revealed their enhanced activity against four cancer cell lines. In vitro investigations showed high activity against gram-positive bacteria and selected fungi. The antifungal activity was found to be higher than the activity of the parent SFN.

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