Design, Synthesis and In Vitro Analysis of New Vemurafenib Analogs

1. Introduction

Metastatic melanoma patients have a poor prognosis with poor responsiveness to chemotherapy. Metastasis is the major cause of death for patients with cancer and this complex multistep process involves cell adhesion, motility, proteolysis degradation of ECM, angiogenesis, and invasion [1]. About 50% of melanomas harbor activating BRAF mutations (over 90 % V600E). The BRAF V600 mutated gene product is translated to a constitutively activated BRAF protein that dysregulates the downstream mitogen-activated protein kinase (MAPK) signaling transduction [2]. This pathway activation is required in melanoma proliferation, apoptosis inhibition, and progression.

Vemurafenib is an oral agent licensed for patients with BRAF V600E mutation-positive inoperable and metastatic melanoma. Exhibits dose-dependent anti-proliferative and apoptotic effects in melanoma cells via downstream inactivation of the MAPK signaling cascade [3]. Vemurafenib provides substantial benefits for melanoma patients, but a major challenge in melanoma treatment with mitogen-activated protein kinase (MAPK)-targeted therapy is an almost universal emergence of resistance that leads to patient relapse and the serious side effects like fatigue, arthralgia, headache, cutaneous toxicities and growth of secondary skin neoplasms [4]. So, finding new molecules, inhibitors of BRAF V600E, as a starting point the compound vemurafenib, that can present good activity and less side effects, may represent a big step for the treatment of metastatic melanoma.

Our group synthesized 5 new vemurafenib analogs, 2a (RF-86A), 2d (RF-87A), 2c (RF-94A), 2b (RF-94B) and 2e (RF-96B), with structural improvements, to increase the anti-proliferative and anti-metastatic effect on human melanoma. In this work, we describe the structural design, synthesis, and in vitro pharmacological profile of an unheard class of Vemurafenib analogs, against human melanoma cells, presenting evidence for cytotoxicity, anti-invasive and anti-metastatic effects.

2. Results

2.1. Analogs Synthesis

The synthetic methodology for obtaining the molecules in this work is a convergent synthesis of 5 steps and is presented in Scheme 1 and Scheme 2. Initially, the carboxylic acids (3a-e) were converted to their respective methyl esters (4a-e) through the Fischer esterification reaction in methanol, toluene in acid catalysis promoted by sulfuric acid and refluxing at 100°C with varying yields [5]. Then, the methyl esters (4a-e) were added to 1M ethanolic solution of hydrazine (NH2NH2) at reflux for 48 hours to obtain the corresponding hydrazides (5a-e) in yields of 50 to 63% (Scheme 1) [6,7].

In parallel, the commercial reagent 5-bromo-7-azaindole (6) was regioselectively formylated at position 3 through the Duff reaction by adding hexamethylenetetramine (HMTA) in acetic acid (HOAc) and water at reflux for 16 hours to obtain 5-bromo-3-carboxyaldehyde-7-azaindole (7) in 66% yield [8]. Finally, the synthesized aldehyde (7) was added, separately, to the different hydrazides (5a-e) obtained in Scheme 1 in the presence of ethanol and hydrochloric acid catalysis at room temperature for 6 hours to form the respective N-acylhydrazones (2a-e) in good yields [7].

For the final products, it was possible to state that N-acylhydrazone (NAH) was formed through the presence of two characteristic signals in the 1H-NMR spectrum: amidic hydrogen (RCONH-CH=) as a broad signal with integral 1 by around 12 ppm, and the imine hydrogen (RCONH-CH=) as a simplet with integral 1 at approximately 8.5 ppm. In the 13C-NMR spectra, the two carbons of the NAH group were visualized at approximately 160 ppm (RCONH-CH=) and around 144 ppm (RCONH-CH=), confirming the obtaining of the molecules. In this scenario, it is worth highlighting that despite NAH being able to present two different diastereoisomers: the E-diastereoisomer and the Z-diastereoisomer [9], only E-diastereoisomer was formed in four molecules (2a-d) in this series. We can strongly suggest the stereoselective formation of the reaction due to the absence of duplication of signals in all 1H-NMR spectra of the molecules in this series. This fact is already well described in the literature by our research group and other researchers [10,11,12].

However, molecule 2e (RF-96B) was formed as a mixture of E/Z diastereoisomers since some specific signals close to the double bond were seen duplicated in NMR-1H. These signals were the imine hydrogen (8.27 and 8.11 ppm, integral = 1) and amidic hydrogen (11.17 and 11.05 ppm, integral = 1) and the methylene group (2.83 and 2.26 ppm, integral = 2) neighboring the carbonyl of the NAH fragment. In NMR-13C all signals referring to methylene carbons are duplicated. The formation of this mixture of diastereoisomers in NAH is due to the large conformational degree of freedom in hydrazide (5e). It has already been reported in the literature that hydrazides containing hybridized carbons such as sp3 near the carbonyl will form diastereoisomeric mixtures in NAH [11]. However, it should be noted that the 1H-NMR and 13C spectra of the methyl ester (4e) and hydrazide (5e) (supplementary material) do not present duplicate signals, since there is no possibility of forming diastereoisomers.

Reagents and solvents were purchased from suppliers and were used without prior treatment. NMR spectra were obtained on Bruker AVIII500 (11.7 T) operating at 500 MHz and on Bruker AVHD400 (9,4 T) operating at 400 MHz. Chemical shifts (δ) were given in parts per million (ppm) from tetramethylsilane (TMS) as internal standard, and values of coupling constant (J) were given in Hertz (Hz). The deuterated solvent used to obtain the spectra was dimethyl sulfoxide-d6 (DMSO-d6) using specific NMR glass tubes. The peak areas of the NMR spectra were obtained by electronic integration and the multiplicities were described as follows: s (simple); d (doublet); t (triplet), m (multiplet) and dd (double doublet). The spectra of the Infrared region were generated on the Varian FT-IR 660 Spectrometer.

Purity of the final products was quantified through the analysis of chromatograms obtained by High Performance Liquid Chromatography (HPLC) on the Shimadzu LC-20AD device, with a Kromasil 100-5 C18 column (4.6 mm x 250 mm) and SPD-M20A detector (Diode Array). Quantification of purity was performed at a wavelength of 300 nm, and the mobile phase used was acetonitrile and water (80%) with running time of 20 minutes.

Synthetic methodologies used to prepare methyl esters (4a-e) have been carefully described in a published paper [12,13,14,15,16]. In a similar way, hydrazides (5a-e) were synthesized using methods described in the literature [12,13,15,16,17]. And 5-bromo-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (7) was synthesized as described by [8].

2.2. General mechanism for obtaining N-acylhydrazones (2a-e)

To the 100 mL round bottom flask was added picolinohydrazide (5d, 0.18g, 1.3 mmol, 1 equivalent) and 5-bromo-1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde (7, 0,35 g, 1.6 mmol, 1.2 equivalents) and 30 mL of ethanol. After turning on the magnetic stirring, 5 drops of hydrochloric acid were added to the reaction mixture. The reaction was then stirred for 6 hours at room temperature, with precipitates forming during the first minutes of the reaction. After this time, the volume of ethanol was reduced under reduced pressure, and the solid was isolated through filtration using a porcelain funnel to obtain the desired final product (2d).

• (E)-N-((5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-5,6,7,8-tetrahydronaphthalene-2-carbohydrazide (2a or RF-86B)

Pinkish amorphous solid; 70%; mp 260-261 °C. Purity (HPLC): 97.1%

IR (cm-1): 626 (ʋ C-Br), 1613 (ʋ C=C), 1651 (ʋ C=O), 3102 (ʋ N-H). 1H-NMR (500 MHz, DMSO) δ 12.34 (s, 1H, H7), 11.58 (s, 1H, H13), 8.74 (d, J = 2.2 Hz, 1H, H2), 8.56 (s, 1H, H11), 8.39 (d, J = 2.3 Hz, 1H, H6), 8.04 (d, J = 2.7 Hz, 1H, H17), 7.64 (d, J = 4.2 Hz, 2H, H8, H21), 7.21-7.17 (m, 1H, H18), 2.81-2.76 (m, 4H, H22, H25), 1.79-1.74 (m, 4H, H23, H24). 13C-NMR (126 MHz, DMSO) δ 162.6 (C14), 147.7 (C4), 144.0 (C11), 143.3 (C2), 140.6 (C20), 136.7 (C19), 131.9 (C8), 131.8 (C6), 130.8 (C16), 128.9 (C21), 128.0 (C18), 124.6 (C17), 118.3 (C5), 112.0 (C9), 110.4 (C1), 28.83 (C25) 28.81 (C22) 22.4 (C23), 22.5 (C24).

• (E)-N-((5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)picolinohydrazide (2b or RF-87A)

Light yellow amorphous solid; 91%; mp 245-246 °C. Purity (HPLC): 98.6%

IR (cm-1): 619 (ʋ C-Br), 1615 (ʋ C=C), 1671 (ʋ C=O), 3099 (ʋ N-H). 1H-NMR (500 MHz, DMSO) δ 12.36 (s, 1H, H7), 12.04 (s, 1H, H13), 8.76 (d, J = 2.2 Hz, 1H, H20), 8.74 (s, 1H, H11), 8.71 (d, J = 4.6 Hz, 1H, H2), 8.40 (d, J = 2.3 Hz, 1H, H17), 8.13 (d, J = 7.8 Hz, 1H, 6H), 8.06 (t, J = 7.7 Hz, 1H, H18), 8.02 (d, J = 2.7 Hz, 1H, H8), 7.68-7.64 (m, 1H, H19). 13C-NMR (126 MHz, DMSO) δ 160.0 (C14), 149.9 (C16), 148.4 (C20), 147.7 (C4), 144.9 (C2), 144.1 (C11), 138.01 (C18), 132.0 (C8), 131.9 (C6) 126.8 (C19), 122.5 (C17), 118.4 (C5), 112.1 (C9), 110.3 (C1).

• (E)-N-((5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)pyrazine-2-carbohydrazide (2c or RF-94A)

Pinkish amorphous solid; 88%; mp 304 - 306 °C. Purity (HPLC): 98.2%

IR (cm-1): 673 (ʋ C-Br), 1611 (ʋ C=C), 1643 (ʋ C=O), 3085 (ʋ N-H). 1H-NMR (500 MHz, DMSO) δ 12.39 (s, 1H), 12.20 (s, 1H), 9.27 (d, J = 1.3 Hz, 1H), 8.92 (d, J = 2.4 Hz, 1H), 8.79 (dd, J = 2.3, 1.5 Hz, 1H), 8.76 (d, J = 2.2 Hz, 1H), 8.74 (s, 1H), 8.40 (d, J = 2.3 Hz, 1H), 8.06 (d, J = 2.8 Hz, 1H). 13C-NMR (126 MHz, DMSO) δ 159.1 (C14), 147.7 (C4), 147.6 (C20), 145.6 (C19), 144.9 (C16), 144.1 (C11), 144.0 (C17), 143.3 (C2), 132.4 (C8), 131.9 (C6), 118.4 (C5), 112.2 (C9), 110.2 (C1).

• (E)-N-((5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)quinoxaline-2-carbohydrazide (2d or RF-94B)

Yellow amorphous solid; 84%; mp 286 - 288°C. Purity (HPLC): 98.5%

IR (cm-1): 628 (ʋ C-Br), 1610 (ʋ C=O), 1668 (ʋ C=C), 3099 (ʋ N-H). 1H-NMR (500 MHz, DMSO) δ 12.41 (s, 1H, H7), 12.27 (s, 1H, H13), 9.53 (s, 1H, H17), 8.80 (s, 1H, H11), 8.78 (d, J = 2.3 Hz, 1H, H2), 8.41 (d, J = 2.3 Hz, 1H, H6), 8.27 (dd, J = 6.1, 3.7 Hz, 1H, H22), 8.21 (dd, J = 6.7, 3.2 Hz, 1H, H25), 8.08 (d, J = 2.4 Hz, 1H, H8), 8.03-7.99 (m, 2H, H23, H24). 13C-NMR (126 MHz, DMSO) δ 159.5 (C14), 147.8 (C4), 145.9 (C11), 144.6 (C19), 144.2 (C2), 144.1 (C17), 143.0 (C16), 139.8 (C20), 132.6 (C8), 132.1 (C6), 132.0 (C23), 131.4 (C25), 129.5 (C24), 129.2 (C22), 118.4 (C5), 112.3 (C9), 110.2 (C1).

• (EZ)-N-((1H-pyrrolo[2,3-b]pyridin-3-yl)methylene)-4-(1H-indol-3-yl)butanehydrazide (2e or RF-96B)

White amorphous solid; 75%; mp 218-220 °C. Purity (HPLC): 96.8 %

IR (cm-1): 689 (ʋ C-Br), 1619 (ʋ C=C), 1655 (ʋ C=O), 3292 (ʋ N-H). 1H-NMR (400 MHz, DMSO) δ 12.27 (s, 1H, NH indole), 11.17 (s) and 11.05 (s, 1H, H3), 10.7 (d, 1H, NH azaindole) 8.65 (d, J = 2.3 Hz) and 8.53 (d, J = 2.2 Hz, 1H, H10), 8.38 (dd, J = 5.9, 2.3 Hz, H20), 8.27 (s) and 8.11 (s, 1H, H5), 7.97 (d, J = 5.2 Hz, 1H, H13), 7.53 (d, J = 7.9 Hz, 1H, H11), 7.37- 7.29 (m, 1H, H24), 7.14 (d, J = 5.8 Hz, 1H, H27), 7.09-6.88 (m, 2H, H25 and H26), 2.83 (t, J = 7.4 Hz) and 2.26 (t, J = 7.4 Hz, 2H, H1) 2.73 (dd, J = 18.2, 7.8 Hz, 2H, H18), 2.00 (td, J = 14.7, 7.6 Hz, 2H, H17). 13C-NMR (100 MHz, DMSO) δ 173.76 (C12), 168.2 (C2), 147.6 (C8), 143.9 (C5), 141.8 (C13), 138.7 (C10), 136.3 (C22), 131.8 (C11), 131.4 (C20), 127.2 (C23), 122.3 (C25), 120.8 (C26), 118.3 (C27), 114.1 (C7), 112.0 (C6), 111.3 (C24), 110.3 (C19), 34.0 and 32.5 (C1), 25.9 and 25.4 (C18), 24.7 and 24.3 (C17).

2.3. Effects on Cell Viability

To study the role of new analogs of vemurafenib in human melanoma cells A375 we chose a range of concentrations since 0.01 to 10 μM. To analyze if these concentrations can reduce the viability of the cells we performed the MTS assay. The results showed that after 24h of treatment with all the analogs tested were able to reduce the viability of the A375 cells as well as the standard compound, vemurafenib (Figure 1). The mean IC50 values of vemurafenib and analogs are represented in Table 1. Noteworthy, the vemurafenib analog, RF-96B (2e), presented better cytotoxic activity between the other analogs (IC50 0.45±0.71 μM).

2.4. Vemurafenib and Analogs Caused DNA Fragmentation in A375 Cells

To understand the mechanisms involved in cell death, we carried out TUNEL assay–PI double staining. TUNEL assay is a well-known method for detecting such DNA fragments.

As shown in the Figure 2, normal cells had blue nucleus while treated cells with all compounds tested presented apoptotic cells revealed themselves as green-TUNEL-positive cells Indeed, all the compounds tested induced DNA fragmentation after 5 μM treatment during 24h, but the analogs RF-94A (2c) and RF-96B (2e) presented major number of apoptotic cells than the other analogs, as well as, the vemurafenib, at the same conditions.

2.5. Cell Apoptosis Detected by Cell Morphology Analysis

The apoptotic cells took on typical apoptotic morphology, apoptotic bodies namely, increasing intercellular distance, shrinking cell volume, chromatin condensation, rounding cell shape, nuclear pyknosis and plasma blebbing. By comparison to the control cells, treated cells with all compounds at 5 μM for 24h had a significant larger number of apoptotic cells, as shown in Figure 3. The red arrows indicate the apoptotic bodies.

2.6. Cell Migration Quantified by the Cell-Based Scratch Assay

To analyze whether vemurafenib and analogs could alter the migration of A375 cells, we performed a cell-based scratch assay followed by the quantification of the wounded area. Cells were cultured up to 90–100% confluence and then scratched wound lines were created with a micropipette tip. The cells were treated for 24h with 0.5 μM of vemurafenib and analogs. This concentration was chosen to have less interference in cell viability. Untreated cells were used as control. Interestingly, untreated cells nearly covered the scratched areas of the dish in 24 h, whereas in vemurafenib and all analogs treated cells relatively large empty areas were visible in the culture dishes (Figure 4A). Empty areas varied from 25% to 50%, as compared to control untreated cultures (Figure 4B).

2.7. Effects of Vemurafenib and Analogs on A375 Cell Secretion of MMP-2 and MMP-9

The potential effects of vemurafenib and analogs treatment on MMP-2 and MMP-9 activity by A375 cells were studied by gelatin zymography to detect the gelatinolytic activity in conditioned media. As shown in the Figure 5, conditioned media of A375 cells after 24h of the treatment with 1 and 5 μM vemurafenib and analogs, presented MMP-2 expression and activity reduced with 1 and 5 μM RF-86A (2a), RF-87A (2b) and RF-94A (2c)-treatment. The analog RF-87A reduced the MMP-2 activity only with the concentration of 1 μM. The analog RF-96B (2e) and the compound vemurafenib did not reduce the MMP-2 expression and activity. In the other side, the MMP-9 expression and activity were increased with the treatment with vemurafenib at 1 and 5 μM, which was not observed with any of the tested analogues.

3. Discussion

Vemurafenib is a standard chemotherapy approved to treat metastatic malignant melanoma, which causes significant tumor regression in metastasized BRAFV600E mutated melanoma patients [18,19]. Since tumor regression is only transient in most cases, followed by acquired drug resistance and tumor progression, the search for alternative therapeutic strategies is warranted.

In this work, we synthesize, characterize e evaluate the activity of 5 new compounds analogs of vemurafenib, that were projected to further improve the results of that, leading to less metastasis formation, eradicating tumor cells as well as reducing side effects.

Our findings suggested that the toxicity in the A375 cells of the analogs was similar among then, under tested conditions, presenting morphological and molecular characteristics that cell death causes by apoptosis highlighting the compound RF-96B (2e) that presents an IC50 around 4X lower than the other analogues. Even though the tested analogues showed significant cytotoxicity against A375 human melanoma cells, the standard compound vemurafenib still presented an IC50 2X lower than the compound RF-96B (2e).

The great activity presented by the tested analogues was the inhibition of metalloproteinases such as MMP-2 and MMP-9. MMPs can degrade various protein components in the extracellular matrix (ECM). Meanwhile, they can also destroy the histological barrier of tumor cells and affect tumor migration, invasion, metastasis, and angiogenesis. These molecules are important in the growth and invasion of malignant tumors and their expression has an important impact on the prognosis of patients [20]. Interestingly, according to our results, vemurafenib presented great inhibition of the cell migration, as well as the analogs (2a-e) tested (Figure 5). On the other hand, our analogs (2a-e) were very effective in the MMP-2 and MMP-9 expression and activity, instead of vemurafenib which did not reduce the MMP-2 and MMP-9 activity (Figure 6). This result is very interesting because degradation and remodeling of the ECM and basement membranes by proteolytic enzymes are essential steps in the metastasis process.

In this context, it is worth highlighting MMP-9 is considered as an indicator of invasiveness in malignant melanoma [21]. MMP-2 can act as a pro-metastatic factor in melanoma [22]. During melanoma progression, in vivo, increased expression of MMPs was detected not only in tumor cells but also in stromal cells [23]. The treatment of melanoma cells A375 for 24h clearly suppressed MMP-2 and MMP-9, indicating that the action of the analogs (2a-e), comparing to the action of vemurafenib, may be longer lasting and with less metastatic potential than the standard compound vemurafenib.

4. Materials and Methods

4.1. Drug Design

Design concept of the molecules in this work is based on Molecular Simplification in the drug vemurafenib. The pyrrolopyridine or azaindole nucleus, considered pharmacophoric for the inhibitory action on kinases [24,25] was maintained in all 4 molecules. The amide function was replaced by the N-acylhydrazone subunit, deeply described in the literature as a privileged structure [26] generating the first published azaindole-N-acylhydrazone derivatives. And finally, the 2,6-difluorophenyl group was replaced by the tetrahydronaphthalene fragment, (2a) which maintains a similar lipophilic profile. In order to carry out new interactions through hydrogen bonding with the target enzyme, the 2,6-difluorophenyl group was also replaced by nitrogenous heterocyclics (2b-e, Figure 1).

4.2. Chemicals

Vemurafenibe was obtained from MedChemExpress (NJ, USA). Vemurafenib and vemurafenib analogs RF-86A (2a), RF-97A (2b), RF-94A (2c), RF-94B (2b) and RF-96B (2e) were dissolved in DMSO (dimethyl sulfoxide) and further diluted in sterile culture medium immediately before their use. DMSO did not exceed 0.3% v/v in the culture medium.

4.3. Cell culture

The human cutaneous melanoma cancer cell line A375 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), cultured in Dulbecco's Modified Eagle’s Medium (DMEM) supplemented with L-glutamine (2 mM), 10% fetal bovine serum (FBS), 50 IU/ml penicillin and 50 μg/ml streptomycin (Wisent, QC, Canada). Cells were maintained at 370 C in a humidified atmosphere containing 5% CO2. Low cell passages (between 5 and 20) were used in the present study.

4.4. Cell Viability Assay

Cell viability was measured using an MTS method based on the conversion of the tetrazolium salt MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) to a purple formazan in the presence of phenazine methosulfate. The enzymes responsible are NADPH-dependent dehydrogenases, which are active in viable cells. Briefly, cells (5×104/well) were seeded in 96-well plates in medium with 10% FBS; after 24 h, the complete medium was replaced with fresh medium containing 1% FBS and incubated for 24h. Fresh stock solutions were prepared on the day of the experiment and the cells were treated with of different concentrations of vemurafenib and analogs (0.01, 0.1, 0.5, 1, 5 and 10 μM). Effects were evaluated after 24 h. Untreated cells were incubated as controls. Following drug exposure, MTS was added, and the absorbance was measured at 570 nm using a microplate reader (CLARIOStar Plus).

4.5. DNA Fragmentation Analyses

To detect DNA fragmentation in A375 cells, a modified apoptosis TUNEL method was used with the MEBSTAIN Apoptosis TUNEL Kit Direct (MBL, International Corporation, USA) according to the manufacturer’s protocol. Briefly, cells were grown in 8-well culture slides (Corning, USA) and incubated with the compounds (5 μM) for 24 h. Following treatments, the cells were washed with PBS and subsequently stained with TdT buffer, TdT solution and Propidium Iodide, according to the manufacturer’s instructions. Experiments were conducted by fluorescent microscopy. The images were collected by an EVOS M5000 microscope (Thermo Fisher Scientific).

4.6. Cell Morphology Analysis

In order to investigate cell apoptosis, morphological analysis was performed by contrast phase microscopy. Briefly, cells were grown in 8-well culture slides (Corning, USA) and incubated with the compounds (5 μM) for 24 h. Following treatments, the cells were washed with PBS and subsequently fixed with PFA (paraformaldehyde) 4% solution, followed by observation under a EVOS M5000 microscope (Thermo Fisher Scientific).

4.7. Cell Scratch Assay

Cellular migration was evaluated by scratch assay according to our previous studies [27,28]. Cells were cultured in 12-well culture plates for 24 h up to 90–100% confluence. The cells were incubated for 24 h with compounds-test at final concentrations of 1 μM. All cell-based scratch assays were performed in the presence of the anti-mitotic reagent cytosine arabinoside (AraC; Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 10–5 M (to inhibit cell proliferation). After treatment with the compounds, the wound areas were observed, and images were acquired with an Evos M5000 (Invitrogen, Thermo Fischer Scientific Inc., Waltham, MA, EUA). The filled area was quantified using the Fiji software (ImageJ, National Institutes of Health, Bethesda, MD, USA).

4.8. Gelatin Zymography

Gelatin zymography was carried out according to [29], using 7.5% SDS-polyacrylamide gels with 0.2% (w/v) gelatin. SDS-PAGE was run at 100 V at room temperature. Following electrophoresis, the gels were removed and placed in 2.5% (v/v) Triton X-100 to renature enzymes for 60 minutes. Then, gels were incubated in 50 mM Tris-HCl, 1 μM ZnCl2, 5 mM CaCl2, pH 7.5 overnight to allow sufficient time for genatinolytic activity to occur. Following overnight incubation at 37°C, gels were incubated in a staining solution (0.5% (v/v) Coomassie blue in a 40% methanol-10% acetic acid-water mixture) for 60 minutes. Following incubation the staining solution, the gels were washed with the destaining solution (without coomassie blue) until the time to start to see the bands. Then imaged with a ChemiDoc^TM MP (BIO-RAD, Germany) gel imaging system. Total protein and gelatinase activity were quantified using Image Lab™ Software (BIO-RAD, Germany).

4.9. Statistical Analysis

All the values of in vitro experiments are presented as the mean ± standard error of three independent experiments. Statistical analyses were performed via a one-way ANOVA with Dunnett´s post-hoc test, and statistical significance was defined as * p < 0.05. Statistical analyses were performed with GraphPad Prism 8.02 (GraphPad Software Inc., San Diego, CA USA).

5. Conclusions

Our results demonstrated for the first time that treatment with the new 5 vemurafenib analogs can significantly reduce the viability of the A375 melanoma cells and reduce the metastasis associated with MMP-2 and MMP-9 activity. New studies designed to interfere with specific MMP actions may be useful in the treatment of metastatic melanoma.