Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell lines

Accepted Manuscript Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell lines George Mihai Nitulescu, Constantin Draghici, Octavian Tudorel Olaru, Lilia Matei, Aldea Ioana, Laura Denisa Dragu, Coralia Bleotu PII: DOI: Reference:

S0968-0896(15)00577-5 http://dx.doi.org/10.1016/j.bmc.2015.07.010 BMC 12439

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

21 April 2015 3 July 2015 4 July 2015

Please cite this article as: Nitulescu, G.M., Draghici, C., Olaru, O.T., Matei, L., Ioana, A., Dragu, L.D., Bleotu, C., Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell lines, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.010

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Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell lines George Mihai Nitulescua*, Constantin Draghicib, Octavian Tudorel Olarua, Lilia Mateic, Aldea Ioana c, Laura Denisa Draguc, Coralia Bleotuc a

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, Traian Vuia 6, Bucharest 020956, Romania b C.D. Nenitzescu Institute of Organic Chemistry, 202B Spl. Independentei, Bucharest 060023, Romania c Stefan S Nicolau Institute of Virology, 285 Mihai Bravu Avenue, Bucharest, 030304, Romania E-mail: [email protected] Tel: +40 213180739 Abstract We designed and synthesized new pyrazole thiourea chimeric derivatives and confirmed their structures by NMR and IR spectra. Apoptotic effects were studied in human cancer cells. The N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N’-(3-bromophenyl)-thiourea compound (4b) exhibited the highest apoptosis-inducing effect. Compound 4b and the thiazole derivatives, 5b and 6b, increased the expression of tumor necrosis factor receptors TRAIL-R2 and TRAILR1, accompanied by down-modulation of pro-caspase 3 levels, and the augmentation of cleaved caspase 3. They also reduced the levels of apoptosis inhibitory proteins and the expression of the heat-shock proteins Hsp27 and Hsp70. All the tested pyrazole derivatives induced a concentration-dependent increase of cells in G2/M phases. The analysis of the experimental data indicates the reduction of Akt phosphorylation as the most probable cellular mechanism of action for the tested compounds. The in vitro study indicated that compound 4b could be a promising anti-cancer drug, to be further developed in animal models of cancer. Keywords: pyrazole; apoptosis; TRAIL-R2; IAP; Akt; cell cycle Abbreviations: AMPK, AMP-activated protein kinase; DISC, death-inducing signaling complex; DMSO, dimethyl sulfoxide; mp, melting point; MTS, 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); NMR, nuclear magnetic resonance; ppm, parts per million; PBS, phosphate buffered saline; PI, propidium iodide; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

1. Introduction Apoptosis is a highly regulated cellular process, in which the cell actively pursues a course towards death upon receiving either extrinsic or intrinsic signals [1]. Cancer research proved that apoptosis and its regulatory mechanisms have a major effect on the malignant phenotype, and that oncogenic mutations that disrupt apoptosis can lead to tumor initiation, progression or metastasis [2]. Many cancer cells develop mechanisms to evade this tightly regulated process by manipulating the levels of anti-apoptotic molecules or inactivating proapoptotic cell death components [3]. Drugs that can restore the normal apoptotic pathways in these types of cancer cells have the potential to become efficient cancer therapies [4]. Targeting the apoptotic defects in cancer emerged as a promising treatment option, and series of targets are being exploited in the drug development process. The most important molecular

targets are the pro-apoptotic factors such as p53 and caspases and anti-apototic factors like Bcl-2, XIAP and Survivin [5]. The pyrazole scaffold is a highly versatile drug-like template that is being used extensively in the design of cancer therapies [6,7], especially in the structure of various protein kinase inhibitors [8,9]. Some pyrazole derivatives progressed to clinical development, such as ruxolitinib, a selective JAK1 and JAK2 inhibitor approved for the treatment of myelofibrosis [10], crizotinib a c-Met and ALK inhibitor used in the treatment of non-small cell lung carcinoma [11], AT7519, an inhibitor of cyclin-dependent kinases used in refractory solid tumors [12], and tozasertib, a pan-Aurora kinase inhibitor [13]. The pyrazole template proved to be favorable also in the design of drugs targeting cellular apoptosis. Shaw et al. (2010) synthesized a series of 3,5-diaryl-1H-pyrazole derivatives with apoptosis-inducing effect on OVCA cells through caspase-dependent pathways and inactivation of protein kinase B/Akt activity [14]. Balbi et al. (2011) developed several pyrazole derivatives with anti-proliferative and pro-apoptotic activity through disassembly of microtubules [15]. A large series of 3-aryl pyrazole-5-carbohydrazide hydrazone derivatives demonstrated potent pro-apoptotic effects in A549 lung cancer cells [16-20]. Based on these compounds, a series of 1H-pyrazole-5-carboxamide [21], some oxime containing pyrazole [22] derivatives and several pyrazolyl hydroxamic acid derivatives [23] were designed and demonstrated apoptosis- and autophagy-inducing effects in A549 lung carcinoma cells [2123]. The literature provides compelling information on the usefulness of pyrazole-5carboxamide [24] and pyrazole-5-carboxylate scaffolds in the development of pro-apoptotic agents [25]. Cankara Pirol and co-workers (2014) used the pyrazole-carboxamide template to obtain a series of 1-(quinolin-2-yl)-5-(4-methylphenyl)pyrazole-3-carboxamides derivatives with potent apoptotic effect on Mahlavu and Huh7 cells [26]. Lv et al. (2012) transformed the pyrazole-5-carboxamide moiety in a pyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione scaffold, and obtained potent apoptosis inducers in H322 lung cancer cells [27]. Tenovin-1, and its more soluble analog tenovin-6, are thiourea derivatives that induce p53-mediated apoptosis [28]. The effectiveness of the thiourea fragment in pro-apoptotic agents can be also seen in cambinol, a molecule that induces apoptosis by increasing the level of p53 [29]. Ryu (2012) used the thiourea moiety, along with the pyrazole template, and obtained AW00178, a compound that induces sensitization to TRAIL-mediated apoptosis [30]. In a previous study [31] we designed and synthesized some chimeric thiourea-pyrazole derivatives and evaluated the in vitro anti-proliferative effects on the NCI-60 panel of human cancer cells. The compound N-benzoyl-N’-(3-(4-bromophenyl)-1H-pyrazol-5-yl)-thiourea exerted the highest anti-proliferative effect registering logGI50 values under -4 in 43 of the 60 investigated cancer lines, values ranging from -4.12 to -5.75. The related N-(1-methyl-1Hpyrazole-4-carbonyl)-N’-(aryl)-thiourea derivatives demonstrated low anti-proliferative effects. The close structural similarity of these pyrazole derivatives with AW00178 and tenovins (Fig. 1), focused our research to optimize this chemical series and to explore the compounds’ pro-apoptotic effects.

F

F

F

N N

H3C

H3 C H3 C

O

H3C

N H

H N N H

H N

H N

S

O

C H3

Cl

S

AW00178

tenovin-1 O

N N

H N

N H

H3C

R S

4a-e

Fig. 1. The chemical structure of tenovin-1 and AW00178 used as pattern for the design of the new acylthiourea derivatives as apoptosis-inducing agents. 2. Results and Discussion 2.1. Chemistry The new 1H-pyrazol-4-ylcarbonyl-thiourea derivatives were synthesized by the general method presented in Scheme 1 starting from the 1-methyl-1H-pyrazole-4-carboxylic acid (1) which was converted into the corresponding acid chloride (2) using thionyl chloride as chlorination reagent and treated afterwards with ammonium isothiocyanate to afford 1methyl-1H-pyrazole-4-carbonyl isothiocyanate (3). PEG-400 was used as solid-liquid phase transfer catalyst. The reaction of 3 with suitable substituted primary aromatic amines afforded the desired N-(1-methyl-1H-pyrazole-4-carbonyl)-N’-(R-phenyl)-thiourea (4a-e). S O

O

C OH

O

C Cl

a N

O

C N C

b N

N

c S

N

N

N

N

N

C H3

C H3

C H3

C H3

3

4a-e

1

2 4a. R= 2-Br

4b. R= 3-Br 4d. R= 2-I

C NH

C N H R

4c. R= 4-Br 4e. R= 4-I

Scheme 1. Reagents: (a) SOCl2, C2H4Cl2, reflux; (b) NH4SCN, PEG-400, (CH3)2CO, reflux; (c) R-C6H4-NH2, (CH3)2CO, reflux. The synthesis of the 1,3-thiazolidine derivatives was carried out by pyridine catalyzed cyclization of N-(1-methyl-1H-pyrazole-4-carbonyl)-N’-(R-phenyl)-thiourea with αbromoacetone. The corresponding thiazole derivatives were obtained by hydrobromic acid dehydration. The synthesis procedures are presented in Scheme 2.

a

b

5a. R= 4-Br

6a. R= 4-Br

5b. R= 4-I

6b. R= 4-I

Scheme 2. Reagents: (a) C5H5N, BrCH2COCH3; (b) HBr. The structures of the synthesized compounds were confirmed by NMR and IR spectral data. For the compounds 4a-e the 1H NMR spectra presents two broad singlets for the thiourea moiety, 12.73-12.37 ppm (NH-C=S) and 11.75-11.37 ppm (NH-C=O). The hydrogens of the pyrazole ring present two singlets, around 8.60 ppm and 8.21 ppm, values indifferent to the aryl substitution. In the 13C NMR spectra are characteristic the thiourea carbon (C=S), 181.24-179.37 ppm, and the carbonyl (C=O) group, 163.80-163.02 ppm. The pyrazole ring signals are 141.09-140.40 ppm (C-5), 135.35-134.65 ppm (C-3) and 116.07115.40 ppm (C-4). The methyl group presented a signal in the range of 39.70-39.09 ppm. In comparison with the spectra of the thiourea precursors of the 4a-e series, the 5a-b derivatives do not present the two characteristic signals of the -NH- groups. The hydrogen atoms of the 1,3-thiazolidine ring present two doublet signals because of the germinal methyl and hydroxyl groups. In the 13C NMR spectra the thiazolidine carbons present signal at 170.21-169.16, 90.61-90.09 and 41.95-41.04. The transformation of the compounds 5a-b into the corresponding 6a-b derivatives is demonstrated by the significant augmentation of the methyl 1 H NMR shift from 1.36-1.39 to 1.98-2.00. The new acylthiourea are characterized by IR absorptions in the ranges of 33503300, 3250-3200 cm-1 for the free and associated NH. These bands disappear in the 5a-b and 6a-b compounds. For the 5a-b compounds the most characteristic band is that produced by the C-O bond at approximately 1160 cm-1. The compound's purity was certified by elemental analyses, the results being within ±0.4 of the theoretical values. 2.2. Evaluation of biological activity 2.2.1. Cell viability assay The evaluation of growth-inhibitory activity of the synthesized compounds was performed on a panel of human carcinoma cell lines represented by THP-1 (monocytic leukemia), HCT-8 (colorectal adenocarcinoma) and HEp-2 (cervix carcinoma) cells, we performed a MTS assay. The data obtained showed inhibitory effects on the growth of all cells in dose-dependent manner (Table 1). Exposure of THP-1 cells to the new compounds at 50 µg/mL for 24 h resulted in cell viability decrease from 100% to 41.6-31.3%. In the case of the HEp-2 cells, the viability reduced at 93.0-70.0%. The compounds produced a small effect on the HCT-8 cells, diminishing their viability at 91.8-78.2%. The compound 4b was the most effective compound in suppressing cell growth. Table 1 Results of cell viability after 24 h exposure to 12.5 µg/mL, 25 µg/mL, and 50 µg/mL of the new compounds using the MTS assay.

THP-1 sample

12.5

4a

98.0±2.0

4b

77.0±5.2

4c

100±1.0

4d

100±1.0

4e

100±1.0

5a

25 µg/mL 59.4±6.3

HCT-8 50

12.5

31.9±5.2

55.8±7.0 72.9±3.0 84.0±1.9 79.3±4.0

95.0±3.2

5b 6a 6b

HEp-2 50

12.5

100±1.1

25 µg/mL 100±1.0

91.5±2.4

100±1.4

25 µg/mL 100±1.0

50

32.0±8.2

94.0±2.0

89.8±2.1

78.2±3.3

100±1.3

82.0±1.7

70.0±3.2

37.0±2.5

100±1.0

100±1.5

91.7±1.0

100±3.0

100±3.0

90.0±1.2

31.3±4.3

100±1.0

100±3.2

91.8±1.6

100±1.3

100±1.0

90.0±1.0

35.2±3.3

100±4.3

100±1.0

93.3±5.7

100±2.1

100±2.1

93.0±2.5

67.4±7.3

33.9±6.2

98.0±3.3

95.0±4.5

85.7±7.3

100±2.3

92.0±2.4

91.0±6.5

97.0±4.0

84.1±3.0

38.4±5.2

100±8.2

100±1.0

90.2±1.0

100±2.6

100±4.6

89.0±3.6

97.0±2.0

87.6±3.4

41.6±9.3

100±1.0

97.0±5.0

89.4±4.8

100±5.0

95.0±2.0

85.0±3.6

99±1.4

86.9±1.0

37.2±6.2

100±3.0

100±1.3

91.8±2.4

100±2.7

100±7.2

90.0±4.0

80.0±2.0

2.2.2. Flow cytometry analysis of cellular apoptosis We performed a cytofluorimetry analysis, using propidium iodide (PI) in conjunction with Annexin-V-FITC, to measure the apoptosis-inducing activity of the new compounds in HCT-8 cells. Both late and early apoptosis were evaluated. The compound 4b has the highest apoptosis- and necrosis- inducing effect, whilst compound 5a elicited apoptosis, but not significant necrosis of HCT-8 cells. The rest of the tested compounds produced only modest effects on apoptosis/necrosis (Table 2). Table 2 Apoptosis/necrosis evaluation in HCT-8 cells treated for 24 h with 50 µg/mL pyrazole derivatives. Apoptosis/necrosis was measured by flow cytometry using the Annexin V-PI test. Sample

Necrosis

Early apoptosis

Late apoptosis

Viable cells

control 4a 4b 4c 4d 4e 5a 5b 6a 6b

1.12 1.87 7.58 1.73 2.49 1.25 1.34 1.44 2.66 1.41

0.75 1.23 7.07 2.78 2.01 2.31 6.22 4.96 4.47 2.91

4.80 5.41 7.11 3.83 3.75 3.10 6.76 3.37 3.44 3.89

93.30 91.50 78.20 91.70 91.80 93.30 85.70 90.20 89.40 91.80

Flow cytometry diagrams of apoptosis/necrosis in HCT-8 cells treated for 24 h with 4a-e, 5a-b and 6a-b compounds using Annexin V-FITC and PI double-staining are presented. Dot plots are representation of logarithmic Annexin V fluorescence versus PI fluorescence. The cells in region Q4 represent living cells, Q3 early apoptotic cells, Q2 late apoptotic cells and in Q1 those in necrosis (Fig. 2).

Fig. 2. Flow cytometry diagram of double-staining with Annexin V-FITC/PI after treatment with 50 µg/mL of the new compounds. 2.2.3. Quantitation of cellular proteins involved in apoptosis In order to identify the contribution of key apoptosis associated proteins, we performed a protein array analysis of major proteins involved in the extrinsic and intrinsic apoptotic pathways. For this study we chose the compounds 4b, 5b, and 6b.

Two major pathways of apoptosis have been described to activate aspartate-specific cysteine proteases representing major effectors of apoptosis: 1) the intrinsic pathway initiated by chemotherapeutic drugs, and intracellular signals such as DNA damage, mitochondria dysfunction and the tumor suppressor p53; 2) the extrinsic pathway initiated by death ligands binding to cell surface death receptors and subsequent formation of the death-inducing signaling complex (DISC) which triggers activation of the caspases cascade [32]. Treatment of HEp-2 cells with 50 µg/mL of 4b, 5b, 6b produced a series of modifications in the expression of apoptosis-related proteins. All 3 compounds increased the expression of TRAIL-R2, and to a lesser extent of TRAIL-R1, both tumor necrosis factor receptors. These receptors can be activated by tumor necrosis factor-related apoptosisinducing ligand (TRAIL), and deliver apoptosis signal. The levels of FADD and Fas/TNFRSF6 was also increased by the tested compounds. The initiation of DISC and subsequent activation of caspase-8 can lead to direct activation of pro-caspase 3 to initiate caspase 3 which leads to cell death. This mechanism can be observed the down-modulation pro-caspase 3 levels and the higher level of the cleaved caspase 3. The up-regulation of TRAIL pathway indicates future development for these new compounds in TRAIL synergic combinations as anticancer targeted therapies. Another mechanism by which the newly synthetized pyrazoles 4b, 5b and 6b induce apoptosis in HEp-2 cells is the reduction of the inhibitors of apoptosis proteins (IAP), like XIAP, c-IAP1, c-IAP2, Livin and Survivin. The tested compounds had the highest effect on XIAP, followed by Survivin and a lower effect on c-IAPs (Fig. 3). XIAP is considered the most potent mammalian IAP apoptotic regulator due to his ability to directly inhibit caspase activity [33]. The tested compounds reduced the expression of Hsp27 and Hsp70, two anti-apoptotic proteins with tumorigenic properties [34]. Phosphorylation of p53 at S15, S46, S392 was increased by 4b, 5b, and 6b compounds, and this might be associated with apoptosis activation via the p53 pathway [35] (Fig. 3).

Fig. 3. The effects exerted by 4b, 5b and 6b compounds (50 µg/mL) on the expression of proteins involved in apoptosis, assessed using the Human Apoptosis Array kit in HEp-2 cells. Results are expressed as log10-relative quantitation levels.

The effect of the new pyrazole compounds on major proteins of the apoptotic pathways is highly similar with that produced by AW00178. AW00178 sensitizes TRAILresistant human lung cancer H1299 cells to TRAIL-mediated apoptosis and reduces the expression of Survivin and XIAP, and up-regulates TRAIL-R2 expression by inhibiting Akt phosphorylation and c-Jun activation [30]. Wu et al. demonstrated that cholangiocarcinoma cells treated with celecoxib showed features of apoptosis through reduction of Akt phosphorylation [36]. Akt regulates cell survival and proliferation, cell migration, cancer progression and metastasis through phosphorylation of multiple downstream targets [37]. Both celecoxib and AW00178 reduces the Akt phosphorylation [38] and based on the high degree of structural similarity with our new pyrazole derivatives, they probably share the same apoptotic mechanism. 2.2.4. Flow cytometry analysis of cell cycle Analyses of the cell cycle distribution of HEp-2 cells after exposure for 24 h to 10 µg/mL and 50 µg/mL of each newly synthesized pyrazole derivatives showed a concentrationdependent increase of G2/M phase (Fig. 4, Table 3). The inhibition of Akt phosphorylation is correlated with induction of cell cycle arrest in the G/M phase, confirming the hypothesis of an Akt pathway inhibition [39]. The compound 4b had the highest anti-proliferative effect, correlated with a pro-apoptotic effect. The increase of G2/M was accompanied by a decrease in the number of cells in the G0/G1 indicating mitotic inhibition and anti-proliferative effects.

Fig. 4. HEp-2 cell cycle exposed for 24 h to 10 or 50 µg/mL of the new compounds. Evaluation by flow cytometry using the PI test and FlowJo 8.8.6 software. Table 3 Results of HEp-2 cell cycle quantification after 24 h exposure to 10 or 50 µg/mL of the new compounds. Evaluation by flow cytometry using the PI test. Sample control 4a 4b 4c 4d 4e 5a 5b 6a 6b

G0/G1

S

G2/M

10 µg/mL

50 µg/mL

10 µg/mL

50 µg/mL

10 µg/mL

50 µg/mL

74.51 74.41 66.12 75.28 76.40 71.62 62.35 74.73 74.24 66.94

74.51 70.17 15.40 67.63 67.60 74.24 37.33 43.62 63.34 50.70

21.06 19.09 20.85 16.97 21.88 17.26 19.20 17.85 21.45 23.56

21.06 21.87 34.70 22.60 18.14 24.79 28.71 27.01 24.20 27.45

7.82 10.28 18.43 9.91 6.86 11.98 19.88 14.92 7.76 9.07

7.82 15.93 52.33 18.75 15.92 15.88 39.46 33.40 19.45 25.92

2.2.5. Expression of genes involved in cell cycle We analysed the expression of genes involved in cell cycle of HEp-2 cells exposed for 24 h to the new pyrazole compounds. Results indicated different patterns, depending on compounds’ structure and concentration (Fig. 5). All tested compounds, with the exception of 4a, reduced the expression of PRKAA1 and PRKAA2 at 10 µg/mL and 50 µg/mL, genes which encode the catalytic subunits of the AMP-activated protein kinase (AMPK). The compound 4b decreases the expression of PRKAA1 and PRKAA2 at 10 µg/mL, but increases them at 50 µg/mL. The activation of AMPK can be triggered by metabolic stress, such as hypoxia and reactive oxygen species, indicating a possible implication of 4b in the oxidative stress at higher concentrations. Compounds 4a and 4b enhanced the expression of cyclin B (Fig. 5), correlated with the G2/M arrest [40].

A

B

Fig. 5. The influence of the tested compounds on the expression of genes involved in the cell cycle of HEp-2 cells (A- 10 µg/mL, B- 50 µg/mL) using the High-Capacity cDNA Reverse Transcription Kit. Results expressed as mean ± standard deviation for 3 independent experiments. 2.2.6. Structure-activity analysis The anti-proliferative, pro-apoptotic effects and the mitotic inhibition produced by the new compounds indicate as probable mechanism the inhibition of Akt phosphorylation. This hypothesis is based both on the biological data and on the high degree of structural similarity between celecoxib, AW00178 and the new pyrazole derivatives. Several X-ray and FT-IR studies [41-43] demonstrated that in acylthiourea derivatives a hydrogen bond occurs between the carbonyl and the thioamide group, resulting in the formation of a six-membered ring. In the case of the 4a-e compounds, the internal hydrogen bond forms a structural equivalent of the benzene ring found in AW00178 and celecoxib, the pyrazolylcarbonyl-thiourea scaffold functioning as a phenylpyrazole bioisoster (Fig. 6).

AW00178

celecoxib

4b

Fig. 6. The structure of celecoxib, AW00178 and 4b, highlighting the shared phenylpyrazole scaffold and its pyrazolylcarbonyl-thiourea bioisoster. In these series of compounds, only compound 4b induced significant apoptosis and G2/M arrest in cancer cell lines, indicating the importance of the substitution in meta. In previous research [31] we synthesized similar N-(1-methyl-1H-pyrazole-4-carbonyl)-N′(aryl)-thiourea derivatives that had growth inhibitory effects on a panel of cancer cells. The best anti-proliferative effects were recorded for the 4-chlorophenyl derivative and for the corresponding 2,4-dichlorophenyl compound, suggesting the usefulness of the substitution with halogens. The transformation of the thiourea group in a thiazolidin-2-ylidene or thiazol-2(3H)ylidene scaffold significantly improves the anti-proliferative and apoptosis-inducing effects. There was no significant difference between the cellular effects of the 5 and 6 series of compounds. 3. Conclusion We have described a simple approach to prepare N-(1-methyl-1H-pyrazol-4carbonyl)-N’-(R-phenyl)-thiourea as potential apoptosis triggering agents in cancer cells. Some compounds were used to obtain the corresponding 1,3-thiazolidine (5a-b) and thiazole derivatives (6a-b). Compounds 4b, 5b and 6b induced apoptosis of HEp-2 cells. The most effective apoptosis-inducer proved to be compound 4b. It induced TRAIL-mediated apoptosis signals and consequent activation of caspase 3. Concurrently, inhibitors of apoptosis proteins (mainly XIAP), along with Hsp27 and Hsp70 heat shock proteins, were down-regulated, thus sustaining the apoptotic action of compound 4b. Based on the high degree of structural similarity of our new pyrazole derivatives with celecoxib and AW00178, the pro-apoptotic effect possibly occurs through inhibition of Akt phosphorylation. Apoptosis of HEp-2 cancer cells induced by compound 4b was accompanied by a concentration-dependent arrest of cells in the G2/M phase of the cell cycle, which is also consistent with Akt pathway inhibition. Higher concentration of compound 4b induced in cancer cells activation of AMPK, possibly as cellular response to counteract metabolic stress. In preclinical in vitro study, compound 4b proved to be a promising anti-cancer agent to be further developed in animal model. 4. Experimental 4.1. Chemistry All starting materials and solvents were purchased from common commercial suppliers and used without purification, unless otherwise noted. The acetone was dried over 3Å molecular sieves (Sigma-Aldrich) and distillated. The melting points (mp) were measured in open capillary tubes on an Electrothermal 9100 apparatus and are uncorrected. The NMR spectra were recorded on a Varian Gemini 300BB instrument at room temperature, operating at 300 MHz for 1H and 75.075 MHz for 13C. The chemical shifts were recorded as δ values in ppm units downfield to tetrametylsilane, used as internal standard. The coupling constants values (J) are reported in hertz (Hz) and the splitting patterns are abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. The IR spectra were recorded on a JASCO FT/IR-4200 spectrometer with an ATR PRO450-S accessory. The elemental analyses were performed on a Perkin Elmer CHNS/O Analyser Series II 2400 apparatus. All reactions were followed by thin-layer chromatography analysis on

silica gel 60F254 aluminium sheets (Merck), mobile phase toluene: ethyl acetate: ethanol 3:1:1, and were visualized at 254 nm under UV lamp. 4.1.1. General synthesis procedure for compounds 4a-e A solution of 1-methyl-1H-pyrazole-4-carboxylic acid (0.1 mol) in anhydrous 1,2dichlorethane is refluxed for 3 h with thionyl chloride (14.5 mL, 0.2 mol). The solvent and the excess thionyl chloride were removed by reduced pressure distillation. The raw obtained 1methyl-1H-pyrazole-4-carbonyl chloride (10 mmol) is dissolved in acetone (30 mL) and added to a solution of ammonium thiocyanate (10 mmol) and PEG-400 (0.1 mL) in acetone and refluxed for 1 h. The ammonium chloride is removed by filtration and the suitable Rsubstituted aniline (10 mmol) is added while stirring. The mixture is heated under reflux for 1 h and then poured into ten times its volume of cold water when the N-(1-methyl-1H-pyrazol4-carbonyl)-N’-(R-phenyl)-thiourea (4a-e) precipitated. The compounds were recrystallized from isopropanol. 4.1.1.1. N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N’-(2-bromophenyl)-thiourea (4a). Yield 69%, mp 185-186 °C. IR (cm-1): 3154 (N-H), 3110 (N-H), 1657 (C=O). 1H NMR (DMSO-d6, ppm): 12.42 (s, NH), 11.75 (s, NH), 8.61 (s, 1H), 8.23 (s, 1H), 7.89 (dd, J = 7.8 Hz, J = 1.5 Hz, 1H), 7.72 (dd, J = 7.8 Hz, J = 1.5 Hz, 1H), 7.44 (td, J = 7.8 Hz, J = 1.5 Hz, 1H), 7.24 (td, J = 7.8 Hz, J = 1.5 Hz, 1H), 3.90 (s, 3H, CH3).13C NMR (DMSO-d6, ppm): 181.24 (C=S), 163.80 (C=O), 141.09, 137.44, 135.26, 129.45, 129.19, 128.42, 119.88, 115.97, 39.70 (CH3). Calcd. for C12H11BrN4OS: C, 42.49; H, 3.27; N, 16.52; S, 9.45. Found: C, 42.60; H, 3.19; N, 16.71; S, 9.57%. 4.1.1.2. N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N’-(3-bromophenyl)-thiourea (4b). Yield 71%, mp 174-175 °C. IR (cm-1): 3257 (N-H), 3103 (N-H), 1669 (C=O). 1H NMR (DMSO-d6, ppm): 12.73 (s, NH), 11.38 (s, NH), 8.59 (s, 1H), 8.20 (s, 1H), 8.05 (t, J = 1.9 Hz, 1H), 7.57 (dt, J = 7.9 Hz, J = 1.9 Hz, 1H), 7.48 (dt, J = 7.9 Hz, J = 1.9 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 3.89 (s, 3H, CH3). 13C NMR (DMSO-d 6, ppm): 180.12 (C=S), 163.54 (C=O), 141.03, 140,11, 135.27, 131.19, 129.58, 127,48, 124,16, 116.07, 39.69 (CH3). Calcd. for C12H11BrN4OS: C, 42.49; H, 3.27; N, 16.52; S, 9.45. Found: C, 42.59; H, 3.13; N, 16.68; S, 9.48%. 4.1.1.3. N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N’-(4-bromophenyl)-thiourea (4c). Yield 75%, mp 207-209 °C. IR (cm-1): 3263 (N-H), 3012 (N-H), 1660 (C=O). 1H NMR (DMSO-d6, ppm): 12.37 (s, NH), 11.39 (s, NH), 8.59 (s, 1H), 8.21 (s, 1H), 7.67-7.58 (m, 4H), 3.89 Hz (s, 3H, CH3). 13C NMR (DMSO-d 6, ppm): 179.53 (C=S), 163.02 (C=O), 140.42, 137.42, 134.66, 131.25, 126.55, 118.49, 115.45, 39.69 (CH3). Calcd. for C12H11BrN4OS: C, 42.49; H, 3.27; N, 16.52; S, 9.45. Found: C, 42.37; H, 3.29; N, 16.63; S, 9.41%. 4.1.1.4. N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N’-(2-iodophenyl)-thiourea (4d). Yield 67%, mp 167-168 °C. IR (cm-1): 3309 (N-H), 3132 (N-H), 1669 (C=O). 1H NMR (DMSO-d6, ppm): 12.53 (s, NH), 11.52 (s, NH), 8.62 (s, 1H), 8.23 (s, 1H), 7.93 (dd, J = 7.7 Hz, J = 1.4 Hz, 1H), 7.65 (dd, J = 7.7 Hz, J = 1.4 Hz, 1H), 7.44 (dd, J = 7.7 Hz, J = 1.4 Hz, 1H), 7.08 (dd, J = 7.7 Hz, J = 1.4 Hz, 1H), 3.90 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 180.75 (C=S), 163.09 (C=O), 140.40, 140.19, 138.88, 134.77, 128.95, 128.57, 115.40, 97.50, 39.70 (CH3). Calcd. for C12H11IN4OS: C, 37.32; H, 3.87; N, 14.51; S, 8.30; Found: C, 37.23; H, 2.90; N, 14.79; S, 8.24%. 4.1.1.5. N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N’-(4-iodophenyl)-thiourea (4e). Yield 68%, mp 201-202 °C. IR (cm-1): 3347 (N-H), 3128 (N-H), 1663 (C=O). 1H NMR (DMSO-d 6, ppm):

12.52 (s, NH), 11.37 (s, NH), 8.59 (s, 1H), 8.20 (s, 1H), 7.75 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 3.90 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 179.37 (C=S), 163.02 (C=O), 140.42, 137.87, 137.38, 134.65, 126.55, 115.51, 91.07, 39.09 (CH3). Calcd. for C12H11IN4OS: C, 37.32; H, 3.87; N, 14.51; S, 8.30; Found: C, 37.30; H, 3.01; N, 14.71; S, 8.38%. 4.1.2. General synthesis procedure for compounds 5a-b To a solution of N-(1-methyl-1H-pyrazole-4-carbonyl)-N’-(R-phenyl)-thiourea (5 mmol) and pyridine (10 mmol) in acetone is added, while stirring, a solution of bromoacetone, prepared in situ, in the dropping funnel, from bromine (5 mmol) and acetone. The reaction mixture is stirred for 2 h, the solvent is removed by distillation and the solid is washed with water. The compounds were recrystallized from ethyl acetate. 4.1.2.1. N-(4-hydroxy-4-methyl-3-(4-bromophenyl)-1,3-thiazolidin-2-ylidene)-1-methyl-1Hpyrazole-4-carboxamide (5a). Yield 72%, mp 176-180 °C. IR (cm-1): 3203 (O-H), 1157 (CO), 1587 (C=N). 1H NMR (DMSO-d 6, ppm): 7.81 (s, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.50 (s, 1H, H-5), 7.32 (d, J = 8.5 Hz, 2H), 3.80 (s, 3H, CH3), 3.48 (d, J = 11.8 Hz, 1H), 3.28 (d, J = 11.8 Hz, 1H), 1.39 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 171.20 (C=O), 170.21 (C=N), 140.21, 137.46, 133.29, 131.55, 131.32, 121.74, 120.77, 90.61, 41.04, 39.35 (CH3), 25.98 (CH3). Calcd. for C15H15BrN4O2S: C, 45.58; H, 3.83; N, 14.17; S, 8.11; Found: C, 45.67; H, 3.80; N, 14.21; S, 8.04%. 4.1.2.2. N-(4-hydroxy-4-methyl-3-(4-iodophenyl)-1,3-thiazolidin-2-ylidene)-1-methyl-1Hpyrazole-4-carboxamide (5b). Yield 76%, mp 155-8 °C. IR (cm-1): 3211 (O-H), 1154 (C-O), 1585 (C=N). 1H NMR (DMSO-d 6, ppm): 7.78 (s, 1H), 7.93 (d, J = 7.7 Hz, 2H), 7.44 (s, 1H, H-5), 7.55 (d, J = 7.7 Hz, 2H), 3.78 (s, 3H, CH3), 3.45 (d, J = 11.6 Hz, 1H), 3.23 (d, J = 11.6 Hz, 1H), 1.36 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 171.24 (C=O), 169.16 (C=N), 140.40, 137.64, 133.29, 130.32, 128.32, 121.83, 101.87, 90.09, 41.95, 38.69 (CH3), 25.19 (CH3). Calcd. for C15H15IN4O2S: C, 40.74; H, 3.42; N, 12.67; S, 7.25; Found: C, 40.81; H, 3.49; N, 12.89; S, 7.23%. 4.1.3. General synthesis procedure for compounds 6a-b Compounds 5a-b (1 mmol) are dissolved in minimum amount of acetone and the mixture is stirred for 1 h, at room temperature, with hydrobromic acid (1 mmol). The solvent is removed under vacuum and the solid is washed with water to remove the hydrobromic acid. 4.1.3.1. N-(3-(4-bromophenyl)-4-methylthiazol-2(3H)-ylidene)-1-methyl-1H-pyrazole-4carboxamide (6a). Yield 90%, mp 216-218 °C. IR (cm-1): 1585 (C=N). 1H NMR (DMSO-d 6, ppm): 7.81 (s, 1H), 7.67-7.63 (m, 2H), 7.50 (s, 1H, H-5), 7.47-7.42 (m, 2H), 6.75 (q, J = 1.4 Hz, 1H), 3.79 (s, 3H, CH3), 2.00 (d, J = 1.4 Hz, CH3). 13C NMR (DMSO-d6, ppm): 169.30 (C=O), 168.53 (C=N), 139.88, 133.91, 133.29, 130.92, 130.57, 127.38, 122.24, 121.97, 104.24, 38.74 (CH3), 14.47 (CH3). Calcd. for C15H13BrN4OS: C, 47.76; H, 3.47; N, 14.85; S, 8.50; Found: C, 47.88; H, 3.41; N, 15.09; S, 8.39%. 4.1.3.2. N-(3-(4-iodophenyl)-4-methylthiazol-2(3H)-ylidene)-1-methyl-1H-pyrazole-4carboxamide (6b). Yield 85%, mp 201-204 °C. IR (cm-1): 1584 (C=N). 1H NMR (DMSO-d 6, ppm): 7.82 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.46 (s, 1H, H-5), 7.40 (d, J = 8.4 Hz, 2H), 6.75 (q, J = 1.2 Hz, 1H), 3.78 (s, 3H, CH3), 1.98 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 169.44 (C=O), 167.85 (C=N), 139.91, 133.09, 132.73, 132.30, 129.81, 129.72, 122.09, 104.34, 99.35, 38.75 (CH3), 14.31 (CH3). Calcd. for C15H13IN4OS: C, 42.47; H, 3.09; N, 13.21; S, 7.56; Found: C, 42.38; H, 2.98; N, 13.42; S, 7.63%.

4.2. Evaluation of biological activity Human carcinoma HEp-2 cell line (ATCC CCL-23), human colorectal adenocarcionoma HCT-8 cell line (ATCC CCL-224) and THP-1 (ATCC TIB-202) were used. The adherent cell cultures were maintained in Dulbecco’s Modified Essential Medium (DMEM) (Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sigma, USA) at 37 ºC, 5% CO2, in a humid atmosphere. The monocytic cell culture, THP-1, was maintained in RPMI-1640 medium (Sigma, USA) and 10% fetal bovine serum. 4.2.1. Apoptosis Detection 4.2.2.1. Cell viability assay The adherent cells were seeded into 96-well plates at 5 x 103 cells/well (HEp-2 cells), and 7 x 103 cells/well (HCT-8 cells). After 24 h, binary dilutions of each compound were added and the cells were maintained for other 24 h at 37 ºC, 5% CO2, in a humid atmosphere. 5 x 10 3 THP-1 cells were added to binary dilutions of each compound and were maintained 24 h at 37 ºC, 5% CO2, in a humid atmosphere. The cell viability was evaluated using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega) measuring the absorbance at 490 nm in an ELISA reader. 4.2.2.2. Flow cytometry analysis of cellular apoptosis Apoptosis detection was made using Annexin V-FITC Apoptosis Detection Kit I (BD Bioscience Pharmingen, USA) according to manufacturer protocol. 3 x 105 HCT-8 cells were seeded in 3.5 cm diameter wells and treated with 50 µg/mL of the tested compounds for 24 h. Both adherent and detached cells were re-suspended in 100 µL of binding buffer and stained with 5 µL Annexin V-FITC and 5 µL propidium iodide for 10 min in dark. At least 10000 events from each sample were acquired using a Beckman Coulter EPICS XL flow cytometer (Fullerton, CA, USA). The percentage of treatment-affected cells was determined by subtracting the percentage of apoptotic/necrotic cells in the untreated population from percentage of apoptotic cells in the population. Early apoptosis was defined as Annexin V positive and PI negative, and late apoptosis, as Annexin V and PI positive. 4.2.2.3. Quantification of the proteins implicated in apoptosis The relative expression levels of 35 apoptosis-related proteins were evaluated using Human Apoptosis Array kit (R&D Systems, Abingdon, UK) in HEp-2 cells. Proteins were extracted according to the manufacturer’s protocol from cells treated for 24 h with the compounds 4b, 5b and 6b (50 µg/mL). 4.2.3. Cell Cycle Analysis 4.2.3.1. Flow cytometry analysis of cell cycle Cells were harvested after treatment with 50 µg/mL of tested compounds for 24 h, washed in cold solution of PBS (pH 7.5), then fixed in cold 70% ethanol and stored at -20 ºC overnight. Samples were then centrifuged, washed with PBS and then re-suspended in 100 µl PBS, treated with RNase A (1 mg/mL) and labelled with propidium iodide (100 µg/mL), incubated in the dark at room temperature for 30 min prior measurement. The DNA content of cells was quantified on a Beckman Coulter EPICS XL flow cytometer (Fullerton, CA, USA) and analysed using FlowJo 8.8.6 software (Ashland, Oregon, USA).

4.2.3.2. Quantification of the expression of genes involved in cell cycle Total RNA was extracted with Trizol Reagent (Invitrogen, USA) according to the manufacturer’s protocol from HEp-2 cells treated for 24 h with tested compounds (10 µg/mL, 50 µg/mL). For each sample, 2 µg of total RNA was used for reverse transcription with High Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Applied Biosystem), and 50 ng cDNA from each sample was used in real time PCR reaction. Real Time PCR was performed on an ABI 7300 Real Time PCR System using pre-validated Taqman Gene Expression Assays kits (Applied Biosystems). Human beta-actin was used as endogenous control. Each experiment was performed three times. Results were analysed with RQ study software (Applied Biosystems). The ∆∆CT method was used to compare the relative gene expression levels.

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