Preclinical Characterization of 3Î²-(N-Acetyl l-cysteine methyl ester

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Preclinical characterization of 3#-(N-acetyl L-cysteine methyl ester)-2a#,3-dihydrogaliellalactone (GPA512), a prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer Zilma Escobar, Anders Bjartell, Giacomo Canesin, Susan Evans-Axelsson Evans-Axelsson, Olov Sterner, Rebecka Hellsten, and Martin Hans Johansson J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01814 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 27, 2016

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Journal of Medicinal Chemistry

TITLE Preclinical characterization of 3β-(N-acetyl L-cysteine methyl ester)-2aβ,3dihydrogaliellalactone (GPA512), a prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer

AUTHOR NAMES Zilma Escobar1, Anders Bjartell2, Giacomo Canesin2, Susan Evans-Axelsson2, Olov Sterner1, Rebecka Hellsten2#, Martin H Johansson3*#

AUTHOR AFFILIATIONS 1

Center for Analysis and Synthesis, Lund University, Lund, Sweden; 2Division of Urological

Cancers, Department of Translational Medicine, Lund University, Malmo, Sweden; 3Glactone Pharma Development AB, Helsingborg, Sweden. *corresponding author #

co-senior authors

1 Environment ACS Paragon Plus


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ABSTRACT The transcription factor STAT3 is a potential target for the treatment of castration-resistant prostate cancer. Galiellalactone (1), a direct inhibitor of STAT3, prevents the transcription of STAT3 regulated genes. In this study we characterized 6 (GPA512, Johansson, M.; Sterner, O. Patent WO 2015/132396 A1, 2015), a prodrug of 1. In vitro studies showed that 6 is rapidly converted to 1 in plasma and is stable in a buffer solution. The pharmacokinetics of 6 following a single oral dose indicated that the prodrug was rapidly absorbed and converted to 1 with a tmax of 15 min. Oral administration of 6 in mice increased the plasma exposure of the active parent compound 20-fold compared to when 1 was dosed orally. 6 treated mice bearing DU145 xenograft tumors had significantly reduced tumor growth compared to untreated mice. The favorable drug-like properties and safety profile of the 6, warrant further studies of 6 for the treatment of castration-resistant prostate cancer.

INTRODUCTION The transcription factor signal transducer and activator of transcription 3 (STAT3) is classified as an oncogene and is implicated in drug resistance, progression of androgen independent growth, metastatic spread, tumor growth and immune-avoidance.1-3 STAT3 is thus a promising target for the treatment of castration-resistant prostate cancer as well as other cancers expressing active STAT3.

Eventually most prostate cancers advance to become metastatic and resistant to anti-androgen therapies making it important to develop novel treatment strategies for this patient group. Targeted therapy towards STAT3 may reverse or overcome this resistance and STAT3 inhibitors may be used alone or in combination with other prostate cancer treatments such as


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chemotherapy and anti-androgens. Furthermore, STAT3 is involved in the tumor microenvironment suggesting a role for STAT3 inhibitors in immunotherapy.

STAT3 may be activated by numerous extracellular factors, with one main factor being interleukin-6 (IL-6). Constitutive activation of STAT3 may be caused by aberrant cytokine signaling, increased tyrosine kinase activation and loss of suppressor proteins.4 In liver cancer, mutations in upstream activators of STAT3 as well as somatic STAT3 mutations have been found, all promoting constitutive activation of STAT3.5 Furthermore, activating mutations have also been found in large granular lymphocyte leukemia and auto-immune disorders.6-7 Due to the numerous activating pathways converging on STAT38 a direct inhibitor of STAT3 would likely be effective against STAT3 related pathologies regardless of activating process.

Direct STAT3 inhibitors have been difficult to find due to the lack of enzymatic activity of STAT3.9-10 The most investigated strategy for inhibiting STAT3 signaling has been targeting the SH2 domain, thereby blocking the STAT3 dimerization process.11 Galiellalactone (1) is a fungal metabolite that was discovered to inhibit the IL-6/STAT3 pathway12 and later shown to directly bind to STAT3 thereby preventing the binding to DNA.13 Like many naturally occurring transcription factor inhibitors, 1 is a cysteine reactive Michael acceptor and 1 has been shown to react with cysteine-468 in the DNA binding domain of STAT3.13 Binding to this residue has previously been shown to block STAT3 signaling.14 Due to the promise of STAT3 inhibition as a new modality of targeting cancer growth and resistance new chemical starting points are highly desired.15 Over the last 10 years there has been a renewed interest in covalent inhibitors. Several covalent inhibitors have been approved and covalent inhibitors



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might prove to be highly suitable for targeting difficult targets, including blocking proteinprotein and protein-DNA interactions and achieving selectivity.16-19

When studying the ability of 1 to act as a Michael acceptor and its reactivity towards nucleophiles we observed that cysteine adds rapidly to 1 but that the addition of cysteine is reversible (Scheme 1). We have previously discovered that secondary amines add to 1 and that this addition is also reversible which led us to investigate if these amine adducts could be used as prodrugs of 1 (unpublished results). This prodrug strategy, utilizing the reversibility of the Michael addition reaction, has previously been used for preparing orally bioavailable prodrugs of cysteine reactive sesquiterpene lactones.20-23 As the chemical stability of the amine adducts of 1 was less than optimal under physiological conditions and oral administration of an amine adduct of 1 did not increase the exposure of the active compound, we investigated the possibility to instead use thiol adducts of 1 as prodrugs.

REULTS AND DISCUSSION In vitro and in vivo ADME characterization of galiellalactone (1) Initial preclinical ADME studies revealed that 1 had a solubility of 143 µM in phosphate buffered saline (PBS; pH 7.4). Furthermore, 1 showed good chemical stability in 0.1 M PBS, with a half-life of more than 9.8 h but only moderate stability when incubated at 37 ºC in mouse plasma (a half-life value of 74 min).

The cellular permeability of 1 was evaluated using Caco-2 cell monolayers. The absorptive (apical to basal) and secretory (basal to apical) permeability were studied in the absence of chemical inhibitors and cellular uptake followed by efflux to buffers was measured in the presence and absence of 50 µM MK-571, an inhibitor of multidrug resistance-associated


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protein efflux transporters. The apparent absorptive permeability of 1 over Caco-2 cell monolayers was high (mean A to B Papp 65.9 cm/s*10-6) and approximately 2 fold higher in the absorptive direction than to secretory direction suggesting a potential involvement of apical uptake or basolateral efflux of this compound in Caco-2 cells. A glutathione adduct of 1 was observed in both apical and basolateral compartments in all incubations. Rapid glutathione conjugation of 1 was also observed in intestinal S9 fractions. The in vitro metabolic stability of 1 was studied using mouse hepatocytes. 1 disappeared to about 45% in 2 h incubation with mouse and no disappearance was observed without cells.

As a next step evaluating the drug-like properties of 1, the plasma exposure in mice following single oral, intravenous and intraperitoneal administrations at 10, 1.25 and 5 mg/kg, respectively was determined (Table 1). Following oral administration, circulating concentrations of 1 were low, with an average Cmax of 52 ng/mL. The drug was measurable in plasma out to 4 h post-dose from all animals. However, absorption appeared to be quite rapid, Tmax being at 30 min after dosing and concentrations declining with a modest apparent elimination half-life of around 90 min. Following intravenous administration, quantifiable concentrations of 1 were detected up to the last time-point taken at 8 h post-dose (63 ng/mL). A moderate half-life was obtained (2.2 h) due to a low clearance (7.3 mL/min/kg) and a moderate volume of distribution (1.4 L/kg). Following intraperitoneal dosing, 1 could also be detected at 8 h post-dose (98 ng/mL). A similar moderate half-life was obtained (2.4 h) following intraperitoneal dosing.

Although 1 displays good solubility and permeability and moderate plasma and hepatocyte stability, oral administration in mice provided very low plasma exposure levels which severely limits its usefulness as an oral STAT3 inhibitor. The plasma levels following



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intraperitoneal administration supports the positive results obtained in the previously described DU145 xenograf studies. 24-25.The lack of adequate exposure following oral administration illustrates the need for a prodrug approach to develop 1 into an oral STAT3 inhibitor.

Chemical synthesis of thiol adducts 1was reacted with simple thiols and cysteine derivatives in MeOH containing catalytic amounts of triethyl amine (Scheme 2). For most thiols the reaction proceeded smoothly and the adducts were obtained in good to high yields following flash chromatography. One notable exception was the addition of cystamine (2-amino-ethane thiol), which gave a complex mixture of products. The chemical purity of the novel adducts produced was determined to be at least 98 %, by 1H NMR and integration of eventual peaks of impurities. The addition of thiols was completely stereoselective with thiols adding exclusively from the least hindered face to give just one stereoisomeric product (Figure 1).

In vitro efficacy of 1 and thiol adducts The inhibitory effect of 1 on prostate cancer cell lines with varying levels of STAT3 expression and activity was studied. As expected, 1 inhibited the proliferation and viability of the prostate cancer cell lines DU145 and LNCaP-IL6+, which express constitutively active STAT3 (pSTAT3), but had no effect on PC3 cells which lack STAT3 or LNCaP cells which do not express constitutively active STAT3 (Figure 2A-B).

The synthesized thiol adducts were tested for their ability to inhibit the proliferation of pSTAT3 expressing DU145 prostate cancer cells. We have previously shown that STAT3 inhibition by 1 inhibits proliferation and induces apoptosis in DU145 cells by downregulating


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STAT3 target genes and proteins .25 Figure 2C and Table 2 show that all of the thiol adducts tested could reduce the proliferation of STAT3 dependent DU145 prostate cancer cells. Since all these thiol adduct compounds lack a conjugated double bond that can act as Michael acceptor, which is an absolute requirement for 1 and galiellalactone analogues to inhibit STAT3 26, they must be undergoing a retro-Michael addition under the assay conditions. Furthermore, the ability to block DU145 cell proliferation follows the stability of the thiol adducts at pH 7.4 where more stable compounds (e.g. 5) have reduced activity compared to the compounds displaying a lower stability at pH 7.4. Notable is that 17, the glutathione adduct displays activity. This indicates that even in the presence of glutathione, the active compound 1 will not be completely inactivated due to it being in equilibrium with added glutathione and free 1.

Chemical stability Thiol adducts 2-6, 8-10, 14, 15 and 17 were assayed for their stability in PBS buffer, and the half-lives of the thiol adducts were monitored by LC/MS and determined by the formation of 1 (Table 3). Notably, thiol adducts containing free –NH2 or -COOH groups (2-4 with the exception of 10 and 14) showed poor stability at pH 7.4. However, these compounds showed increased stability in acidic buffer at pH 1.2 (Table 3). This is consistent with previous reports on the stability of Michael addition products where a pH dependency has been observed.20, 2728

Compounds 2-4 that contain ionizable functional groups (free amine (4), carboxylic acid (3)

or both free amine and carboxylic acid (2)) showed decreased stability at pH 7.4 and are more rapidly converted to 1. It is possible that these ionizable groups, -NH2 or –COOH, could assist or catalyze a retro-Michael reaction thus liberating the parent drug. The remaining compounds showed chemical stabilities at pH 7.4 that made them amendable to in vivo pharmacokinetic (PK) assessment (vide infra).



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Based on the PK assessment, thiol adduct 6 was chosen for a study of its stability in plasma, and was found to be highly unstable in plasma of all tested species (see Table 4). This instability in plasma is in contrast to the chemical stability in PBS buffer indicating that the prodrug 6 is metabolized to an instable intermediate, similar to compounds 2-4, that is converted to the active parent (metabolic activation). In the plasma stability studies, 1 was present in all samples in all species, including early time-points, indicating a rapid conversion of prodrug 6 to the parent compound (data not shown). In mouse plasma 1 displays moderate stability with a half-life of 74 min.

Pharmacokinetic study in mouse To investigate if oral administration of thiol adducts could increase the plasma exposure of 1 compared to oral administration of 1 itself and thereby function as prodrugs of 1, mice were given an oral gavage of 10 mg/kg of 10 thiol adducts (Table 5). The plasma levels of 1 were analyzed for up to 24 h post dosing (Table 5 and Figure 3). All compounds tested had rapid Tmax values (15-30 min) and all thiol adducts in the PK study, except 8 and 16, gave an increased total exposure of 1 as measured by AUC, and greatly enhanced Cmax. The PK results indicate that thiol adducts of 1 have increased uptake and absorption following oral dosing compared to 1 itself and that they are converted to 1 in vivo, thereby acting as prodrugs. Assuming that Cmax is more important than AUC for covalently acting drugs for ensuring target inhibition, 6 was selected for a proof of concept efficacy study using a prostate cancer cell xenograft mouse model. Oral dosing of 6 significantly increased the plasma exposure of 1; Cmax and AUC of 1 was increased 36-fold and 20-fold respectively, compared to when 1 was dosed orally. Dose escalation PK studies using 40 and 100 mg/kg 6 showed that the exposure of 1 was fairly linear across the dose levels tested, in terms of both area


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under the curve (AUC) and maximal concentrations (Cmax) (Table 6). The PK results show that an oral dose of 40 mg/kg 6 would give comparable plasma exposure levels of 1 as the previously used intraperitoneal dose of 3 mg/kg.24

In vitro ADME characterization of thiol adduct 6 To study potential metabolites, 6 was incubated with cryopreserved mouse hepatocytes for 2 h with an initial concentration of 10 µM. Samples were analysed using UPLC/Q-TOF-MS. 6 was completely metabolized in 2 h with mouse hepatocytes. The major metabolite was 3, the result of ester hydrolysis of 6. 1 was detected as the second most abundant metabolite. This suggests that the initial step in the conversion of the prodrug 6 to the active parent 1 is metabolic ester hydrolysis to the carboxylic acid 3, an enzymatic step, followed by spontaneous conversion of 3 to 1 as the 3 was determined (vide supra) the chemically instable. The conversion of 3 to 1 most likely involves a retro-Michael addition. In the Caco-2 assay 6 showed intermediate apparent permeability to both directions without difference between absorptive and secretory directions.

6 inhibits prostate cancer xenograft growth in vivo The in vivo effect of 6, was investigated in mice bearing DU145 prostate cancer cell xenograft tumors expressing pSTAT3 (Figure 4A). The mice were orally treated with 6 (40 mg/kg) or vehicle daily five times a week for four weeks. The tumor growth rate was significantly reduced in 6 treated mice after 26 and 29 days compared to vehicle treated mice (Figure 4A). 6 was well tolerated and no weight loss (Figure 4B), apparent behavioral alterations or other side effects were observed. All mice survived the treatment.



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Molecular analysis of DU145 xenograft tumors showed decreased proliferation and increased apoptosis in 6 treated tumors compared to vehicle treated, as measured by decreased Ki67 ratio and increased amount of cleaved caspase-3 positive cells (Figure 5 A-B). The mRNA expression of STAT3 regulated anti-apoptotic genes MCL1 and BCL2 were reduced by 6 treatment indicating an effect via STAT3 (Figure 5C). This is in line with previous findings showing that 1 induces apoptosis via caspase-3 cleavage, decreased MCL1 gene expression and BCL2 protein down regulation.24-25 The pSTAT3 expression was not altered by 6 (Figure 5D) which is in accordance with the fact that 1 is a direct inhibitor that does not effect the phosphorylation of STAT3 but prevents DNA binding and transcription of STAT3 regulated genes.13, 25 Taken together, these results show that oral administration of the prodrug 6 is well tolerated and shows similar anti-tumor effects via STAT3 inhibition as 1 given intraperitonealy,25 thus confirming that 6 functions as a prodrug.

Toxicity To assess the tolerability and eventual toxicity of 6, the maximum tolerated dose (MTD) of 6 was determined in male CD1 mice following a single oral dose. 6 was administered as single dose orally (by gavage) at 40 mg/kg, 200 mg/kg and 400 mg/kg with 5 male mice per group. One group received the vehicle alone (50 mM citrate buffer, pH 4.0, 5% DMSO) as a control. Under the experimental conditions of the study, when administered as a single dose orally to male CD1 mice, 6 showed no toxicologically relevant clinical signs, no changes in body weight (Table 7) and no adverse events in clinical pathology and histopathology. Thus, the MTD was considered to be 400 mg/kg.

CONCLUSION By exploiting the reversible nature of the Michael addition of thiols to a reactive electrophilic


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double bond, an orally bioavailable prodrug of the STAT3 inhibitor 1 was discovered. 1 itself is not suitable for oral administration, having very low plasma exposure following oral administration, but by conjugating 1 with a thiol, a stable adduct was obtained that functioned as an oral prodrug. Therefore, the prodrug 6 is an orally active STAT3 inhibitor whereas the parent compound only displays in vivo activity following intraperitoneal administration.

The reason for the poor oral bioavailability of 1 could not be determined from these studies. The permeability per se does not seem to be limiting as observed from the quick Tmax. However, the instability towards intestinal S9 fractions suggests extensive intestinal metabolism and it cannot be excluded that 1 is conjugated with glutathione in the intestinal membrane and then effluxed. Preconjugation of 1 with N-acetyl cysteine methyl-ester, as in prodrug 6, would protect the compound from this initial conjugation and allow it to pass over the intestinal membrane.

The proposed two-step conversion of the prodrug 6 to the active parent is shown in Figure 6. The data suggest that the chemically stable N-acetyl cysteine methyl ester prodrug is metabolically hydrolyzed to the corresponding free carboxylic acid which is less stable under physiological conditions and undergoes a spontaneous reversible Michael addition reaction to liberate the active parent drug 1.

STAT3 as a drug target in cancer is supported by a vast number of in vitro and in vivo studies. However, the discovery and development of selective and orally bioavailable STAT3 inhibitors has been slow and only few small molecules have made it into the clinic.29-30 1 is a promising lead for generating an efficacious STAT3 inhibitor. The direct inhibition of STAT3 through the alkylation of cysteine-468 in the DNA binding domain makes it possible for a



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small molecule to block the binding interactions between the protein and DNA. Furthermore, 1 targets a cysteine residue unique to the STAT3 sequence, and covalent inhibition offers the possibility of decoupling pharmacokinetics from pharmacodynamics thus giving sustained inhibition even when systemic exposure has decreased.

The favorable drug-like properties and safety profile of the prodrug 6 and the direct inhibition of STAT3 by 1, warrant further studies of 6 (GPA512) as a drug candidate for treatment of patients with castration-resistant prostate cancer. Finally, it is clearly demonstrated that 1 reacts reversibly with thiols thus making it a reversible covalent inhibitor and therefore has less potential for toxic effects caused by irreversible binding to cysteine residues and thiols.

EXPERIMENTAL SECTION General information Reagents was purchased from commercial suppliers and used without further purification unless otherwise noted. All reactions were carried out in standard dry glassware and atmospheric surroundings, unless otherwise stated.

1

H NMR (500 MHz) and 13C NMR (125 MHz) were recorded at room temperature with a

Bruker DRX500 spectrometer with an inverse multinuclear 5 mm probehead equipped with a shielded gradient coil. The spectra were recorded in CD3OD, CDCl3 and CD2Cl2, and the solvent signals (3.31/49.15 ppm, 7.27/77.00 ppm, 5.34/54.00 ppm, respectively) were used as reference. The chemical shifts (δ) are given in ppm, and the coupling constants (J) in Hz. COSY, HMQC and HMBC experiments were recorded with gradient enhancements using sine shaped gradient pulses. For the 2D heteronuclear correlation spectroscopy the refocusing delays were optimized for 1JCH=145 Hz and nJCH=10 Hz. The raw data were transformed and


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the spectra were evaluated with the standard Bruker XWIN-NMR software (rev. 010101). Mass spectra (HREI, HRFAB) were recorded with a Jeol SX102 spectrometer while HRESI spectra were recorded with a Micromass Q-TOF Micro. The chemical purity of the novel adducts was determined to be at least 95 %, by analytical HPLC. HPLC analyses were performed using an Agilent 1260 Infinity system with an Agilent C18 (3.5 µm, 4.6 × 100 mm column. A linear gradient was applied from 0-20 min using 0.1% of TFA in a H2O/MeCN gradient 5-95 % and a flow rate of 1.6 mL/min. UV detection was performed at 220 and 254 nm.

Chemistry 3β-(L-cysteine)-2aβ,3-dihydrogaliellalactone (2) To a solution of galiellalactone (1) (10 mg, 0.053 mmol) in methanol-d4 (0.5 mL) was added L-cysteine (7 mg, 0.058 mmol) and triethylamine (1 µL, 0.007 mmol). The reaction was followed by NMR and was complete after 24 h, the excess of solvent and the triethylamine was removed in vacuum and 2 was obtained in quantitative yield. δ 1H (400 MHz, CD3OD) 4.58 (1 H, brs), 3.82 (1 H, dd, J 8.8, 3.8), 3.39 (2 H, s), 3.11 (2 H, m), 2.88 (1 H, d, J 10.4), 2.19 (2 H, m), 2.01 (3 H, m), 1.79 (1 H, m), 1.36 (1 H, m), 1.21 (3 H, d, J 6.6), 0.74 (1 H, m). δ 13C (101 MHz, CD3OD) 178.3, 173.5, 91.2, 85.3, 55.9, 49.0, 48.5, 47.1, 38.2, 35.2, 34.6, 33.0, 30.6, 21.2. HRMS calcd for C14H22NO5S [M+H]: 316.1219, found: 316.1225.

3β-(N-acetyl L-cysteine)-2aβ,3-dihydrogaliellalactone (3) To a solution of galiellalactone (1) (10 mg, 0.053 mmol) in methanol-d4 (0.5 mL) was added N-acetyl L-cysteine (10 mg, 0.058 mmol) and triethylamine (1 µL, 0.006 mmol). The reaction was followed by NMR and was complete after 24 h, the excess of solvent and the triethylamine was removed in vacuum and 3 was obtained in quantitative yield. δ 1H (400



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MHz, CD3OD) 4.64 (1 H, dd, J 7.8, 4.6), 4.51 (1 H, brs), 3.21 (2 H, q, J 7.9), 3.03 (1 H, dd, J 13.6, 7.9), 2.88 (2 H, d, J 10.1), 2.18 (1 H, m), 2.10 (1 H, m), 1.96 (3 H, s), 1.94 (2 H, m), 1.90 (1 H, m), 1.65 (1 H, m), 1.14 (3 H, d, J 6.6), 0.63 (1 H, dt, J 13.7, 12.2). δ 13C (101 MHz, CD3OD) 178.1, 174.3, 173.4, 91.0, 85.4, 54.7, 49.0, 48.5, 48.0, 38.6, 35.3, 34.6, 32.9, 30.2, 22.6, 21.6. HRMS calcd for C16H23DNO6S [M+H]: 359.1387, found: 359.1388.

3β-(L-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (4) To a solution of galiellalactone (1) (10 mg, 0.051 mmol) in methanol-d4 (0.5 mL) was added L-cysteine methyl ester hydrochloride (10 mg, 0.057 mmol) and triethylamine (1 µL, 0.006 mmol). The reaction was followed by NMR and was complete after 24 h, the excess of solvent and the triethylamine was removed in vacuum and 4 was obtained in quantitative yield. δ 1H (400 MHz, CD3OD) 4.61 (1 H, s), 4.46 (1 H, t, J 5.8), 3.92 (3 H, s), 3.91 (1 H, s), 3.28 (2 H, dd, J 5.8, 2.3), 2.88 (1 H, d, J 10.3), 2.24 (1 H, ddd, J 9.2, 7.1, 3.0), 2.17 (2 H, m), 2.01 (3 H, m), 1.73 (1 H, m), 1.35 (1 H, m), 1.19 (3 H, d, J 6.6), 0.73 (1 H, dt, J 13.8, 12.1). δ 13

C (101 MHz, CD3OD) 178.1, 169.8, 91.3, 85.4, 54.1, 53.8, 51.8, 48.6, 47.1, 38.3, 35.1, 32.9,

32.7, 30.4, 21.4. HRMS calcd for C15H23DNO5S [M+H]: 331.1438, found: 331.1439.

3β-(N-acetyl L-cysteine amide)-2aβ,3-dihydrogaliellalactone (5) To a solution of galiellalactone (1) (13 mg, 0.064 mmol) in methanol (0.5 mL) was added Nacetyl L-cysteine amide (11 mg, 0.065 mmol) and triethylamine (1 µl, 0.006 mmol). The reaction was followed by NMR and was complete after 24 h, the excess of solvent and the triethylamine was removed in vacuum and 5 was obtained in quantitative yield. δ 1H (400 MHz, CDCl3) 7.21 (1 H, d, J 8.3), 4.82 (1 H, m), 4.61 (1 H, m), 3.43 (1 H, m), 3.18 (2 H, m), 2.19 (1 H, m), 2.09 (3 H, s), 2.05 (1 H, m), 2.01 (2 H, m), 1.88 (1 H, m), 1.64 (1 H, m), 1.42 (1 H, m), 1.38 (1 H, m), 1.11 (3 H, d, J 6.8), 0.70 (1 H, m). δ 13C (101 MHz, CDCl3) 176.6,


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173.7, 172.3, 89.8, 84.6, 53.1, 52.6, 47.4, 45.7, 36.7, 34.3, 33.5, 31.6, 29.3, 22.8, 21.1. HRMS calcd for C16H25N2O5S [M+H]: 357.0978, found: 357.0958.

3β-(N-acetyl L-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (6) Galiellalactone (1) (1.2 g, 6.18 mmol) of was dissolved in MeOH (30 mL), N-acetyl-Lcysteine methyl ester (1.2 g, 6.80 mmol) was added at room temperature, followed by triethylamine (86 µL, 0.62 mmol). The reaction mixture was stirred for 30 hours under N2, whereafter the solvent and the triethylamine was removed under reduced pressure. The resulting orange oil was passed through a Sephadex LH-20 column eluted with methanol. The crude product was passed through a very short SiO2 column, using EtOAc as eluent, affording 2.1 g (91 %) of 6 as a pale yellow oil that crystalized in CH2Cl2. δ 1H (400 MHz, CDCl3) 6.50 (1 H, d, J 7.2), 4.90 (1 H, dt, J 8.0, 5.8), 4.60 (1 H, t, J 2.8), 3.81 (3 H, s), 3.43 (1 H, dd, J 2.1, 1.3), 3.08 (2 H, dd, J 5.8, 0.6), 3.02 (1 H, dd, J 8.9, 2.1), 2.21 (1 H, m), 2.11 (1 H, m), 2.08 (3 H, s), 2.03 (2 H, m), 1.85 (1 H, ddd, J 14.3, 6.7, 3.2), 1.57 (1 H, ddd, J 8.7, 6.7, 3.3), 1.42 (1 H, ddd, J 17.5, 12.9, 8.0), 1.13 (3 H, d, J 6.7), 0.74 (1 H, dt, J 14.3, 12.3). δ 13C (101 MHz, CDCl3) 175.4, 171.2, 170.7, 89.4, 84.4, 52.9, 52.7, 52.6, 47.3, 46.1, 36.3, 34.4, 34.3, 31.5, 29.3, 23.2, 21.2. HRMS calcd for C17H26NO6S [M+H]: 372.1481, found: 372.1499.

3β-(N-acetyl L-cysteine ethyl ester)-2aβ,3-dihydrogaliellalactone (7) N-Acetyl L-cysteine (30 mg, 0.184 mmol) was dissolved in EtOH (2 mL) at room temperature and thionyl chloride (15 µL, 0.20 mmol) was added at 0 °C under N2, the reaction was stirred at room temperature and the residual solvent was removed under reduced pressure to yield Nacetyl L-cysteine ethyl ester that was used without further purification in the next step. To a solution of galiellalactone (25 mg, 0.129 mmol) in methanol (2 mL) was added the N-acetyl L-cysteine ethyl ester and triethylamine (2 µL, 0.013 mmol). The reaction was stirred at room temperature for 24 h, and the excess of solvent was removed under vacuum. The crude 15 Environment ACS Paragon Plus


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product was purified by SiO2 chromatography using EtOAc as eluent to yield compound 7 (22 mg, 44 %). δ 1H (500 MHz, CDCl3) 6.59 (1 H, dt, J 12.8, 8.0), 4.86 (1 H, dt, J 8.0, 5.6), 4.59 (1 H, t, J 2.9), 4.25 (2 H, qd, J 7.1, 1.7), 3.41 (1 H, dd, J 2.6, 1.8), 3.08 (2 H, dd, J 5.6, 1.7), 3.00 (1 H, dtd, J 7.7, 4.7, 2.6), 2.20 (1 H, dt, J 13.1, 5.2), 2.10 (1 H, dd, J 13.6, 5.2), 2.03 (2H, m), 2.02 (3 H, s), 1.85 (1 H, ddd, J 14.2, 6.8, 3.3), 1.64 – 1.54 (2 H, m), 1.32 (3 H, t, J 7.1), 1.12 (3 H, d, J 6.6), 0.73 (1 H, dt, J 14.2, 13.6). δ 13C (126 MHz, CDCl3) 175.4, 170.7, 170.7, 89.4, 84.5, 62.2, 52.8, 52.7, 47.4, 46.1, 36.4, 34.4, 34.3, 31.5, 29.3, 23.2, 21.1, 14.1. HRMS calcd for C18H28NO6S [M+H]: 386.1637, found: 386.1623.

3β-(N-(tert-butylcarbonate)-L-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (8) To a solution of galiellalactone (1) (22 mg, 0.113 mmol) in methanol (2 mL) was added N-( tert-butylcarbonate)-L-cysteine methyl ester (30 µL, 0.147 mmol) and triethylamine (2 µL, 0.011 mmol). The reaction was stirred at room temperature for 24 h, and the excess of solvent was removed under vacuum. The crude product was purified by SiO2 chromatography using heptane/EtOAc (3:7) as eluent to yield compound 8 (34 mg,70 %). δ 1H (500 MHz, CDCl3) 5.48 (1 H, d, J 7.3), 4.57 (1 H, dd, J 7.3, 2.5), 4.56 (1 H, d, J 1.7), 3.78 (3 H, s), 3.37 (1 H, m), 3.05 (2 H, d, J 5.8), 3.01 (1 H, dd, J 8.6, 1.9), 2.20 (1 H, m), 2.09 (1 H, m), 2.02 (2 H, m), 1.82 (1 H, ddd, J 14.2, 6.6, 3.4), 1.59 (1 H, m), 1.44 (9 H, s), 1.42 (1 H, m), 1.12 (3 H, d, J 6.7), 0.73 (1 H, dt, J 14.2, 12.3). δ 13C (126 MHz, CDCl3) 175.2, 171.3, 155.6, 89.3, 84.6, 80.6, 53.7, 52.7, 52.5, 47.0, 46.0, 36.2, 34.4, 34.2, 31.4, 29.3, 28.3, 21.2. HRMS calcd for C20H31NO7SNa [M+Na]: 452.1719, found: 452.1736.

3β-(Benzylthio)-2aβ,3-dihydrogaliellalactone (9) To a solution of galiellalactone (1) (12 mg, 0.062 mmol) in methanol (0.5 mL) was added benzylthiol (8 µL, 0.068 mmol) and triethylamine (1 µL, 0.006 mmol). The reaction was


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stirred at room temperature for 24 h, and the excess of solvent and the triethylamine was removed in vacuum. The crude product was purified by SiO2 chromatography using heptane/EtOAc (15:85) as eluent to yield compound 9 (15 mg, 76 %). δ 1H (400 MHz, CD3OD) 7.42 (2 H, m), 7.33 (2 H, m), 7.25 (1 H, m), 4.54 (1 H, t, J 2.3), 3.86 (2 H, s), 3.45 (1 H, t, J 1.5), 2.84 (1 H, dd, J 10.1, 1.8), 2.22 (1 H, m), 2.12 (1 H, m), 2.00 (2 H, m), 1.89 (1 H, ddd, J 13.8, 7.0, 2.8), 1.64 (1 H, m), 1.33 (1 H, m), 1.00 (3 H, d, J 6.6), 0.61 (1 H, dt, J 13.8, 12.2). δ 13C (101 MHz, CD3OD) 178.0, 140.1, 130.4, 129.5, 128.0, 90.9, 85.5, 54.9, 47.4, 47.2, 38.6, 36.8, 34.8, 32.9, 30.2, 21.4. HRMS calcd for C18H23O3S [M+H]: 319.1368, found: 319.1365.

3β-(ethyl sulphonate)-2aβ,3-dihydrogaliellalactone (10) To a solution of galielallactone (1) (12 mg, 0.062 mmol) in methanol-d4 (0.5 mL) was added sodium 2-sulfanylethanesulfonate (Mesna) (11 mg, 0.068 mmol) and triethylamine (1 µL, 0.006 mmol). The reaction was followed by NMR at room temperature for 24 h, and after the reaction had finished the solvent and the triethylamine was removed in vacuum. 10 was obtained in quantitative yield. δ 1H (500 MHz, CDCl3) 4.52 (1 H, dd, J 5.3, 2.0), 3.40 (1 H, m), 3.15 (2 H, m), 3.03 (1 H, d, J 7.3), 3.01 (2 H, m), 2.20 (1 H, m), 2.11 (1 H, m), 1.98 (2 H, m), 1.92 (1 H, ddd, J 14.2, 6.2, 3.9), 1.63 (1 H, m), 1.32 (1 H, m), 1.14 (3 H, d, J 6.8), 0.64 (1 H, ddd, J 14, 12, 12) δ 13C (126 MHz, CDCl3) 178.0, 90.9, 85.4, 53.0, 54.2, 47.9, 47.1, 38.6, 35.0, 32.9, 30.2, 27.2, 21.6. HRMS calcd for C13H20O6NaS2 [M+H]: 359.0599, found: 359.0594.

3β-(Butyllthio)-2aβ,3-dihydrogaliellalactone (11) To a solution of galiellalactone (1) (33 mg, 0.170 mmol) in methanol (2 mL) was added 1butanethiol (20 µL, 0.187 mmol) and triethylamine (2 µL, 0.017 mmol). The reaction was



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stirred at room temperature for 24 h, and the excess of solvent was removed in vacuum. The crude product was purified by column chromatography using heptane/EtOAc (3:7) to yield compound 11 (35 mg, 72 %). δ 1H (500 MHz, CDCl3) 4.59 (1 H, dd, J 4.9, 2.1), 3.20 (1 H, m), 3.06 (1 H, dd, J 7.1, 1.9), 2.64 (1 H, tdd, J 12.6, 8.4, 7.3), 2.58 (1H, ddd, J 12.6, 8.4, 7.3), 2.23 (1 H, dtd, J 12.8, 8.2, 4.5), 2.12 (1 H, m), 2.08 (2 H, m), 2.00 (1 H, m), 1.78 (1 H, ddd, J 14.3, 6.1, 4.5), 1.64 (1H, m), 1.63 (2 H, m), 1.51 (1 H, m), 1.43 (1 H, dtd, J 14.3, 7.3, 1.3), 1.15 (3 H, d, J 6.8), 0.93 (3 H, t, J 7.3), 0.78 (1 H, dt, J 14.3, 12.4). δ 13C (126 MHz, CDCl3) 175.3, 88.9, 85.1, 51.7, 46.7, 44.8, 35.5, 34.8, 31.4, 31.4, 31.3, 29.5, 22.00, 21.7, 13.6. HRMS calcd for C15H25O3S [M+H]: 285.1524, found: 285.1527.

3β-(Phenylthio)-2aβ,3-dihydrogaliellalactone (12) To a solution of galiellalactone (1) (22 mg, 0.113 mmol) in methanol (2 mL) was added thiophenol (13 µL, 0.125 mmol) and triethylamine (2 µL, 0.011 mmol). The reaction was stirred at room temperature for 24 h, and the excess of solvent was removed under vacuum. The crude product was purified by SiO2 chromatography using heptane/EtOAc (3:7) as eluent to yield compound 12 (25 mg, 73 %). δ 1H (500 MHz, CDCl3) 7.45 (2 H, tt, J 8.5, 1.3), 7.34 (2 H, t, J 8.5), 7.25 (1 H, td, J 8.5, 1.3), 4.50 (1 H, dd, J 4.6, 1.9), 3.63 (1 H, dd, J 7.4, 1.6), 3.15 (1 H, m), 2.38 (1 H, m), 2.15 (1 H, m), 2.06 (1 H, dt, J 8.1, 4.6), 2.00 (1 H, m), 1.84 (1 H, ddd, J 14.3, 6.1, 3.8), 1.75 (1 H, m), 1.51 (1 H, m), 1.22 (3 H, d, J 6.7), 0.85 (1 H, dt, J 14.3, 12.4). δ 13C (126 MHz, CDCl3) 175.0, 134.4, 130.8, 129.4, 127.2, 88.9, 85.0, 50.6, 47.1, 46.8, 35.7, 34.1, 31.3, 29.3, 21.7. HRMS calcd for C17H21O3S [M+H]: 305.1211, found: 305.1219.

3β-(N-acetyl-L-homocysteine methyl ester)-2aβ,3-dihydrogaliellalactone (13) To a solution of galiellalactone (1) (30 mg, 0.154 mmol) in methanol (2 mL) was added homocysteine (23 mg, 0.170 mmol) and triethylamine (2 µL, 0.015 mmol). The reaction was


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stirred at room temperature overnight, and the excess of solvent was removed under vacuum. The crude was dissolved in dry THF (3 mL) and triethylamine (27 µL, 0.289 mmol) was added at 0°C followed by acetyl chloride (17 µL, 0.238 mmol), the reaction was stirred for 20 h at room temperature, the residual solvent was removed under vacuum. The intermediate was dissolved in MeOH (1.5 mL) and SOCl2 (28 µL, 0.385 mmol) was added at 0 °C, and stirred overnight. The crude product was purified by SiO2 chromatography using heptane/EtOAc (3:7) as eluent, to yield compound 13 (24 mg, 40 %) after three steps. δ 1H (500 MHz, CD2Cl2) 6.47 (1 H, d, J 7.9), 4.68 (1 H, td, J 7.9, 4.6), 4.55 (1 H, m), 3.73 (3 H, s), 3.42 (1 H, m), 2.20 (1 H, m), 2.17 (1 H, m), 2.10 (2 H, m), 2.01 (2 H, m), 2.00 (3 H, s), 1.98 (2 H, m), 1.87 (1 H, ddd, J 14.2, 7.0, 3.1), 1.67 (1 H, m), 1.36 (1 H, m), 1.12 (3 H, d, J 6.6), 0.69 (1 H, dt, J 14.2, 12.2). δ 13C (126 MHz, CDCl3) 176.2, 172.9, 171.1, 90.2, 85.1, 53.6, 53.1, 52.1, 47.5, 46.8, 37.4, 32.9, 32.2, 32.2, 29.9, 28.3, 23.6, 21.4. HRMS calcd for C18H27NO6SNa [M+Na]: 408.1457, found: 408.1454.

3β-(N-(L-valine)-L-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (14) To a solution of N-[(1,1-dimethylethoxy)carbonyl]-L-valine (0.50 g, 2.30 mmol) in dry THF (10 mL) was added N-methylmorpholine (0.30 g, 2.76 mmol) at room temperature, followed by isobutyl chloroformate (0.36 mL, 2.76 mmol) at -15 °C, after the reaction mixture had turned to a homogeneous solution it was stirred for an additional 30 min before addition of Lcysteine methyl ester (0.43 g, 2.53 mmol) in dry THF (2 mL) under N2. The reaction was quenched by addition of NaHCO3 (sat) and extracted with EtOAc (3 × 7 mL) and the organic phase concentrated under vacuum to yield N-[(1,1-dimethylethoxy)carbonyl]-L-valine-Lcysteine methyl ester that was used in the next step without further purification. To a solution of galiellalactone (40 mg, 0.201 mmol) in methanol (2 mL) was added the N-[(1,1dimethylethoxy)carbonyl]-L-valine-L-cysteine methyl ester, followed by triethylamine (3 µL,



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0.020 mmol). The reaction was stirred at room temperature overnight, and the excess of solvent was removed under vacuum. The reaction mixture was dissolved in Et2O (3 mL) and HCl in Et2O (1 N, 4 mL) was added at 0 °C, the reaction was stirred for 8 h. The crude product was purified by SiO2 chromatography using CH2Cl2/MeOH (1:9) as eluent, to yield compound 14 (30 mg, 34 %) after two steps. δ 1H (500 MHz, CDCl3) 8.00 (1 H, d, J 6.7), 4.89 (1 H, dd, J 12.4, 6.7), 4.73 (1 H, s), 4.14 (1 H, dd, J 7.5, 3.8), 3.79 (3 H, s), 3.72 (1 H, m), 3.18 (2 H, m), 2.93 (1 H, d, J 10.2), 2.33 (1 H, m), 2.17 (1 H, m), 2.10 (1 H, m), 1.99 (2 H, dd, J 7.3, 3.3), 1.85 (1 H, ddd, J 14.0, 6.6, 2.9), 1.63 (1 H, m), 1.36 (1 H, m), 1.14 (6 H, d, J 3.8), 1.13 (3 H, d, J 6.6), 0.69 (1 H, dd, J 14.2, 12.2). δ 13C (126 MHz, CDCl3) 176.4, 170.6, 89.5, 84.4, 58.8, 52.8, 52.7, 52.2, 47.1, 45.4, 36.8, 34.1, 33.4, 31.5, 30.4, 29.2, 21.1, 18.2, 18.1. HRMS calcd for C20H33N2O6S [M+H]: 429.2059, found: 429.2067.

3β-(N-isobutyryl L-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (15) To a solution of L-cysteine methyl ester hydrochloride (0.2 g, 1.17 mmol) in dry CH2Cl2 (5 mL) was added triethylamine (0.16 mL, 1.75 mmol) at room temperature. When the reaction mixture had turned into a homogeneous clear solution it was stirred for an additional 15 min before the addition of isobutyryl chloride (0.13 g, 1.28 mmol) at 0 °C under N2. The reaction was stirred overnight and quenched by NaHCO3 (sat), extracted with CH2Cl2 and the organic phase was concentrated under vacuum to yield N-isobutyryl-L-cysteine methyl ester that was used in the next step without further purification. To a solution of galiellalactone (1) (20 mg, 0.103 mmol) in methanol (2 mL) was added the N-isobutyryl-L-cysteine methyl ester (0.046 g, 0.224 mmol), followed by triethylamine (1 µL, 0.010 mmol). The reaction was stirred at room temperature overnight, and the excess of solvent was removed under vacuum. The crude product was purified by SiO2 chromatography using heptane/EtOAc (6:4) as eluent, to yield compound 15 (27 mg, 66 %) after two steps. δ 1H (500 MHz, CDCl3) 6.52 (1 H, d, J 8.1), 4.85


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(1 H, dd, J 8.1, 5.9), 4.59 (1 H, t, J 3.2), 3.79 (3 H, s), 3.47 (1 H, m), 3.09 (1 H, dd, J 14.3, 5.9), 3.03 (1 H, dd, J 14.3, 5.9), 2.99 (1 H, dd, J 9.2, 2.1), 2.47 (1 H, m), 2.19 (1 H, m), 2.09 (1 H, ddd, J 14.4, 12.3, 6.9), 2.01 (2 H, m), 1.84 (1 H, ddd, J 14.2, 6.9, 3.2), 1.57 (1 H, m), 1.39 (1 H, m), 1.18 (3 H, d, J 3.9), 1.16 (3 H, d, J 3.9), 1.11 (3 H, d, J 6.7), 0.71 (1 H, dt, J 14.2, 12.3). δ 13C (126 MHz, CDCl3) 177.7, 175.6, 171.3, 89.4, 84.5, 52.8, 52.6, 52.4, 47.5, 45.9, 36.6, 35.4, 34.2, 34.2, 31.5, 29.2, 21.1, 19.4, 19.3. HRMS calcd for C19H30NO6S [M+H]: 400.1794, found: 400.1780.

3β-(N-(2-Fluorobenzoyl)-L-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (16) To a solution of L-cysteine methyl ester hydrochloride (0.3 g, 1.75 mmol) in dry CH2Cl2 (5 mL) was added triethylamine (0.27 mL, 1.92 mmol) at room temperature. The reaction mixture was stirred for 15 min before the addition of 2-fluorobenzoyl chloride (0.23 g, 1.92 mmol) at 0 °C under N2. The reaction was stirred overnight and quenched by NaHCO3 (sat), extracted with EtOAc (3 × 10 mL) and the organic phase was concentrated under vacuum to yield N-(2-fluorobenzoyl)-L-cysteine methyl ester that was used in the next step without further purification. To a solution of galiellalactone (1) (20 mg, 0.103 mmol) in methanol (2 mL) was added the N-(2-fluorobenzoyl)-L-cysteine methyl ester (0.063 g, 0.257 mmol), followed by triethylamine (1 µL, 0.010 mmol). The reaction was stirred at room temperature overnight, and the excess of solvent was removed under vacuum. The crude product was purified by SiO2 chromatography using heptane/EtOAc (3:7) as eluent, to yield compound 16 (31 mg, 65 %). δ 1H (500 MHz, CDCl3) 8.04 (1 H, td, J 7.9, 1.5), 7.64 (1 H, dd, J 12.7, 7.5), 7.51 (1 H, m), 7.27 (1 H, ddd, J 8.6, 7.9, 1.5), 7.15 (1 H, ddd, J 12.7, 8.6, 1.5), 5.09 (1 H, dtd, J 7.5, 5.8, 2.1), 4.57 (1 H, dd, J 3.1, 2.5), 3.84 (3 H, s), 3.45 (1 H, m), 3.21 (2 H, qd, J 14.2, 5.8), 3.03 (1 H, dd, J 8.9, 1.9), 2.19 (1 H, ddd, J 12.4, 7.4, 4.4), 2.09 (1 H, m), 2.02 (2 H, ddd, J 8.1, 6.1, 3.2), 1.81 (1 H, ddd, J 14.2, 6.7, 3.3), 1.58 (1 H, m), 1.41 (1 H, m), 1.11 (3 H, d, J



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6.7), 0.72 (1 H, dt, J 14.2, 12.4). δ 13C (126 MHz, CDCl3) 175.2, 170.9, 163.6, 160.8, 133.9, 131.8, 124.8, 120.2, 116.3, 89.3, 84.5, 53.1, 53.0, 52.6, 47.1, 46.1, 36.4, 34.2, 33.8, 31.5, 29.2, 21.1. HRMS calcd for C22H27NO6FS [M+H]: 452.1543, found: 452.1520.

3β-(glutathione)-2aβ,3-dihydrogaliellalactone (17) To a solution of galiellalactone (1) (11 mg, 0.054 mmol) in methanol-d4 and D2O (1:1, 0.5 mL) was added glutathione (17 mg, 0.059 mmol) and triethylamine (1 µL, 0.005 mmol). The reaction was followed by NMR and was complete after 24 h, and the excess of solvent was removed in vacuum. The crude product was purified on Sephadex LH-20 with MeOH:H2O 1:9 as eluent and then flash chromatography with CHCl3/MeOH (1:1) containing 3 % acetic acid to yield compound 17 (19 mg, 70 %). δ 1H (500 MHz, CD3OD/D2O 10%) 4.64 (1 H, dd, J 8.5, 5.6), 4.60 (1 H, m), 3.92 (2 H, s), 3.83 (1 H, t, J 7.6), 3.42 (1 H, m), 3.15/2.98 (2H, m), 2.87 (1 H, m), 2.55 (2 H, m), 2.39 (1 H, s), 2.19 (1 H, m), 2.15 (2 H, m), 2.11 (1 H, m), 1.99 (2 H, m), 1.92 (1 H, m), 1.65 (1 H, m), 1.27 (1 H, m), 1.22 (3 H, d, J 6.8), 0.62 (1 H, m) δ 13C (126 MHz, CD3OD/D2O 10 %) 175.2, 174.0, 173.2, 173.7, 173.2, 91.2, 85.5, 55.2, 55.2, 54.9, 48.6, 46.9, 42.5, 38.3, 35.2, 34.5, 32.8, 32.8, 30.2, 27.6, 21.6. HRMS calcd for C21H31DN3O9S [M+H]: 503.1922, found: 503.1926.

Cell culture The human prostate cancer cell lines DU145, PC3 and LNCaP from the American Type Culture Collection (ATCC) and long-term interleukin-6 (IL-6) stimulated LNCaP cells (LNCaP-IL6+ cells) 31 were used. DU145, LNCaP and LNCaP-IL6+ cells were cultured in RPMI-1640 medium and PC3 cells were cultured in in HAM’s F-12 medium, both supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. LNCaPIL6+ cells were maintained in the above medium supplemented with IL-6 (5 ng/mL; Sigma-


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Aldrich, St. Louis,MO). Cells were incubated at 37°C in a humidified atmosphere of 95% O2 and 5% CO2. Cells were used at low passages and not cultured for more than three months. The cells were routinely tested for and found free of mycoplasma. The molecular characterization of the cell line was performed by LGC Standards (Cologne, Germany) and the results were then evaluated by comparison with the ATCC database. Our batches of DU145, LNCaP and PC3 cells revealed a 100% match in comparison with ATCC standard for these cells.

WST-1 proliferation assay DU145 and LNCaP-IL6+ cells were seeded at a density of 2 000 cells/well and LNCaP and PC3 cells at a density of 5 000 cells/well in 96-well microplates and allowed to adhere for 24 h. The cells were incubated with the compounds for 72 h followed by cell proliferation measurement. At the end of the incubation time WST-1 reagent (Roche Applied Science) was added to each well and the absorbance was measured at a wavelength of 450 nm and 630 nm as a reference using ELISA reader. The result was calculated as percent of untreated cells after subtracting the background absorbance.

Western blot analysis For preparation of lysates, cells were washed and scraped in ice-cold PBS and centrifuged at 1400 rpm at 4 °C. The cells were lysed in mild cell lysis buffer containing 100 mM Tris HCl, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, Complete Mini EDTA-free protease inhibitor (Roche Applied Science), incubated for 30 min on ice, and centrifuged at 10 000 rpm for 30 min at 4 °C. The supernatant was collected, and protein content was determined using Bradford protein assay. Samples were separated on 7.5% precastgel (Mini-PROTEAN TGX; Bio-Rad). The gels were blotted onto PVDF membranes and blocked with 5% milk. Membranes were incubated with primary antibody diluted in 5% milk 1 h at room 23 Environment ACS Paragon Plus


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temperature antibodies raised against STAT3, and pSTAT3 Tyr-705 (Cell Signaling Technology). After incubation with secondary anti-rabbit antibody conjugated with horseradish peroxidase (GE Health-care), the membrane was treated with enhanced chemiluminescent reagent (Santa Cruz Biotechnology) followed by exposure to x-ray film or visualized using a ChemiDoc XRS system (Bio-Rad).

Chemical stability The chemical stability of the compounds was determined in 96-well plate format. Compounds were dissolved in DMSO, with the exception of 3 which proved insoluble in this solvent and was dissolved in MeOH instead. These stock solutions were diluted (n=2) in the required matrix (0.1 M PBS, pH 7.4) to a concentration of 10 µM (2% DMSO or methanol final) and mixed on an orbital shaker, with samples taken for analysis at a nominal 5, 15, 30 and 60 min. DMSO, containing an analytical internal standard, was added to the aliquots, vortex-mixed and analyzed immediately by LC-MS/MS. Equivalent T=0 samples were also included, with sample preparation staggered to allow sequential injections of timed aliquots and the T=0 samples. The amount of compound remaining (expressed as %) was determined from the MS response in each sample relative to that in the T=0 samples (normalized for internal standard). Samples were also monitored for the presence of parent compound, 1. The stability at pH 1.2, was performed as described above for stability assessment in PBS, but using a solution of 0.5 mM hydrochloric acid containing 2 mg/mL sodium chloride and adjusted to pH 1.2. As with testing in PBS, the amount of compound remaining (expressed as %) was determined from the MS response in each sample relative to that in the T=0 samples (normalized for internal standard). Samples were also monitored for the presence of parent compound, 1.


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For analysis of plasma stability, test and control compounds (simvastatin, eucatropine) were prepared in DMSO and incubated with the corresponding plasma at 37 ºC using an initial concentration of 5 µM in 1% DMSO (n=2). Aliquots were removed at 0, 5, 15, 30 and 60 min for termination of reactions and compound extraction with acetonitrile containing an analytical internal standard. Samples were centrifuged and the supernatant fractions analyzed for test compound and potential intermediates by mass spectrometry (LC-MS/MS). The amount of compound remaining (expressed as %) was determined from the MS response in each sample relative to that in the T=0 samples (normalized for internal standard). Ln plots of the % remaining were used to determine the half-life for compound disappearance using the relationship: t1/2 (min) = -0.693/λ where λ is the slope of the Ln % remaining vs time curve.

Caco-2 study The Caco-2 study was performed by AdmeScope (Finland). CacoReady-monolayers in 24transwell plates were used as provided by Readycell S.L. The apical side (donor) initial volume was 275 µl and the basolateral side (receiver) initial volume 775 µl. The buffer used was Hanks’ Balanced Salt Solution (BioWest L0612-500) buffered with 10 mM Hepes, pH 7.4. Briefly the studies were started by placing the transwell insert plate containing the apical solutions to a well plate containing the basolateral solutions. Test item concentrations used were 20 µM 1 and 5 µM 6. Both compartments were sampled at 0, 60 and 120 min. Samples were analyzed by UPLC-MS/MS (Waters Acquity UPLC + Waters XEVO- TQ-S triple quadrupole MS with a Waters Acquity HSST3 C18 (2.1 × 50 mm, 1.8 µm) column.

In vitro metabolism The in vitro metabolism study was performed by AdmeScope (Finland). Pooled cryopreserved mouse hepatocytes from Celsis IVT, were used. The incubation volume was



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300 µl in 48-well plate. Cell viability was determined using Trypan blue-method 1.0 million The initial test item concentration was 10 µM (stock solution in DMSO). The incubations were done at pH 7.4, in Celsis InVitro HI medium and at 37 °C. Sampling was done at 0 and 120 min. Termination of incubations was performed by adding an equal volume of acetonitrile. Samples were analyzed by UPLC/Q-TOF-MS (Waters Acquity UPLC + Waters Xevo G2 Q-TOF-MS with a Waters Acquity HSST3 C18 (2.1 × 50 mm, 1.8 µm) column).

Pharmacokinetic study The in-life pharmacokinetic study was performed by BioFocus DPI Limited (UK). Doses were prepared at a drug concentration of 0.5, 2 and 5 mg/mL in 5% DMSO in 50 mM citrate buffer (citric acid/sodium citrate), pH 4.0. Oral doses were administered in a volume of 20 mL/kg to a group of 24 mice and blood samples were taken under terminal barbiturate anesthesia, at eight time-points out to 8 h post-dose (n=3 mice per time-point). No adverse effects of dosing were observed. Blood samples were transferred to tubes containing EDTA as anticoagulant and, as soon as practicable after collection, samples were centrifuged to yield plasma which was immediately frozen awaiting analysis. All samples were frozen, prior to analysis using LC-MS/MS.

Plasma proteins were precipitated and compounds extracted by the addition of three volumes of acetonitrile containing analytical internal standard (reserpine). Samples were centrifuged for 30 min at 3452 g in a Sorvall bench centrifuge and the supernatant fractions removed for MS analysis. Quantification of 1 was by extrapolation from calibration lines prepared in control mouse plasma and analyzed concurrently with experimental samples and Quality Control (QC) samples prepared in control mouse plasma. Due to the instability of 1 in mouse


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plasma, calibration lines were constructed in water for qualitative MS comparison with any 1 signal seen in plasma samples.

Pharmacokinetic parameters of 1 were determined using the mean data from the n=3 mice at each time-point. Non-compartmental analysis was performed using the software package PK Solutions 2.0 from Summit Research Services. AUC values were calculated by the trapezoidal method.

In vivo efficacy of prodrug 6 using prostate cancer xenograft model Twenty NMRI-nude male mice were inoculated subcutaneously with 1.5 million DU145 cells in the flank. After four weeks the mice were divided in two groups with ten mice in each. The mice were orally dosed using a feeding needle with either 6 (40 mg/kg) or vehicle (50 mM citrate buffer pH 4.0 and 2% DMSO) daily five times per week for four weeks. The tumors were measured twice per week using a caliper. Tumor volume (mm3) = width (mm) x length x height x 0.52. After four weeks the mice were sacrificed by CO asphyxiation and the tumors were immediately dissected. Half of each tumor was snap frozen for mRNA extraction. The other half was fixed in formaldehyde for immunohistochemical analysis. The mice were kept on a 12 h light dark cycle with access to food and water ad libitum. Experimental procedures were carried out according to the guidelines set by the Malmo-Lund Ethical Committee for use and care of laboratory animals.

Toxicity study The study to assess maximum tolerated dose (MTD) was performed by Fidelta (Zagreb, Croatia) in an AAALAC I – approved Facility. The standard study plan relating to this study was reviewed by the Ethical Committee (CARE – Zagreb) as required by International



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Laws/Regulations and Croatian Low on Animal Welfare and Animal Welfare Officer. Test item 6 was administered to the animals as a single oral dose. 6 was given as a solution (50 mM citrate buffer, pH 4.0, 5% DMSO) by oral gavage, using a cannula of appropriate size (metal feeding tube). Male CD1 mice (Charles River) were used, aged 7 weeks at the time of administration with body weights: 27.0 – 38.5 g. Mice were housed under ambient conditions and 12 h light dark cycle with access to food and water ad libitum. Animals were weighed on day of randomization, on day 1, just before treatment, on day 4 and on day 8, at the end of study.

QPCR analysis The expression of the STAT3 related target genes MCL1 and BCL2 were investigated with quantitative real-time PCR (QPCR). Total RNA was isolated from DU145 xenograft tissue using RNeasy Plus Mini kit (Qiagen Sciences). Complementary DNA (cDNA) was synthesized using RevertAid Reverse Transcriptase (Thermo Fisher Scientific). Two micrograms of total RNA was obtained for the cDNA synthesis. QPCR was carried out using 6–20 ng cDNA, 250 nM forward and reverse primer in Maxima SYBR Green/ROX qPCR master mix (Thermo Fisher Scientific) in a 25 µl reaction. The cycling conditions were: 10 min at 95°C to activate the enzyme, then 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Relative expression levels were quantified by the comparative Ct method and normalized to the expression of two internal control genes (HMBS and SDHA). Primers sequences: HMBS 5´-GGCAATGCGGCTGCAA-3´ and 5´-GGGTACCCACGCGAATCAC-3´; SDHA 5´TGGGAACAAGAGGGCATCTG-3´ and 5´-CCACCACTGCATCAAATTCATG-3´; MCL1 5´-GGACATCAAAAACGAAGACG-3´and 5´-GCAGCTTTCTTGGTTTATGG-3´; BCL2 5´-GGGATGCCTTTGTGGAACTG-3´ and 5´-CAGCCAGGAGAAATCAAACAGA3´. All primers were synthesized by Invitrogen and checked for specificity before use.


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Immunohistochemical analysis of xenografts Tissue sections from the DU145 xenograft tumors were analyzed using immunohistochemistry (IHC) to evaluate the presence of actively proliferating cells with Ki67 staining and for apototic cells with cleaved caspase-3 staining. IHC staining was conducted using the EnVision Flex kit and Autostainer Plus (Dako, Glostrup, Denmark), according to the manufacturer’s instructions. The following antibodies were used: anti-Ki67 (RM-9106, clone SP6, Thermo Scientific, Sweden), anti-cleaved caspase-3 (#9661, Cell Signaling Technology) and anti-pSTAT3 (#9131, Cell Signaling Technology). For each stained slide, 10 x magnification pictures were taken using the Aperio ScanScope XT Slide Scanner (Aperio Technologies, Vista, CA, USA) system for bright field microscopy. The number of cleaved caspase-3 positive cells per section was calculated as mean cleaved caspase-3 positive cells per view from five views. The amount of Ki67 positive cells was calculated as the ratio between the number of Ki67-positive cells and the number of total cells.

Statistical Analyses Statistical analyses were performed using Graph Pad Prism software, and statistical significance was determined using ANOVA or Student's t-test. Statistical significance was considered when p =

Preclinical Characterization of 3Î²-(N-Acetyl l-cysteine methyl ester

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