Bis-Cinnamamide Derivatives as APE/Ref-1 Inhibitors for the Treatment of Human Melanoma

Human malignant melanoma exhibits imbalances in redox status, leading to activation of many redox-sensitive signaling pathways. APE/Ref-1 is a multifunctional protein that serves as a redox chaperone that regulates many nuclear transcription factors and is an important mechanism in cancer cell survival of oxidative stress. Previous studies showed that APE/Ref-1 is a potential druggable target for melanoma therapy. In this study, we synthesized a novel APE/Ref-1 inhibitor, bis-cinnamoyl-1,12-dodecamethylenediamine (2). In a xenograft mouse model, compound 2 treatment (5 mg/kg) significantly inhibited tumor growth compared to the control group, with no significant systemic toxicity observed. We further synthesized compound 2 analogs to determine the structure-activity relationship based on their anti-melanoma activities. Among those, 4-hydroxyphenyl derivative (11) exhibited potent anti-melanoma activities and improved water solubility compared to its parental compound 2. The IC50 of compound 11 was found to be less than 0.1 μM. Compared to other known APE/Ref-1 inhibitors, compound 11 exhibited increased potency in inhibiting melanoma proliferation. As determined by luciferase reporter analyses, compound 2 was shown to effectively inhibit H2O2-activated AP-1 transcription activities. Targeting APE/Ref-1-mediated signaling using pharmaceutical inhibitors is a novel and effective strategy for melanoma treatment with potentially high impact.


Introduction
Melanoma arising from melanocytes makes up only 2% of all skin cancers, while it causes over 80% of all skin cancer deaths [1]. In recent years, melanoma incidence rates have increased more rapidly than other cancer types in the United States, and it is expected that 7650 patients will die due to melanoma in 2022 alone [2]. Ultraviolet radiation (UVR,) which can generate reactive oxygen species (ROS) and nitric oxide (NO) in the skin [3][4][5], has been implicated as a significant environmental contributor to the development of melanoma [6,7]. The contributions of UVR to melanomagenesis and malignant development may, at least partially, be due to ROS-or NO-induced DNA damage, which has been studied in our laboratory and by other groups [8][9][10][11].

Chemistry
In this study, a series of symmetrical and unsymmetrical dicinnamoyl derivatives conjugated through hydrophobic central units of 1,12-diaminododecane and 1,6-diaminohexane were synthesized as depicted in Schemes 1 and 2. Different bolaamphiphiles were prepared based on increasing the hydrophilicity of these compounds by introducing hydrophilic hydroxyl groups to the aromatic rings and changing the hydrophobic chain length. First, bis-cinnamoyl-1,12-dodecamethylenediamine (2) was synthesized by the reacting cinnamoyl chloride (1) with 1,12-dodecamethylenediamine in dry THF in the presence of potassium carbonate as a base (Scheme 1). The structure of the product was confirmed by NMR and mass spectrometry. The purity was confirmed by analytical HPLC, showing a retention time (R t ) of 26 min.
To improve water solubility, diamide bolaamphiphiles containing two hydroxyl groups were prepared using N-methylmorpholine (NMM) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b]pyridinium 3-oxide hexafluorophosphate (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium, HATU) as a coupling reagent. Symmetrical bolaamphiphile derivatives (4, 5, and 7) were prepared through a coupling reaction of one equivalent of 1,12-dodecamethylenediamine with two equivalents of the cinnamic acid derivative under basic conditions; 4-Hydroxycinnamic acid (3) and 3-hydroxycinnamic acid (6) were activated by HATU, followed by a coupling reaction with 1,12-dodecamethylenediamine in the presence of NMM as a basic catalyst to generate the corresponding diamide, bis-(4hydroxy-cinnamoyl)-1,12-dodecamethylenediamine (4) and bis-(3-hydroxy-cinnamoyl)-1,12-dodecamethylenediamine (7), respectively (Scheme 2a,b). Analytical HPLC confirmed the purity of compounds 4 and 7, showing (R t ) = 21.89 and 22.19 min, respectively; moreover, to prepare a more hydrophilic bolaamphiphile derivative, a shorter aliphatic 1,6-dihexylamine was used for the reaction with compound 3 to prepare bis-(4-hydroxycinnamoyl)-1,6-hexanemethylenediamine (5) under the same conditions, as mentioned above (Scheme 2a). The crude products were purified by silica gel column chromatography and the purity was confirmed using analytical HPLC (Supplementary Materials). Unsymmetrical bis-cinnamamide derivatives 9 and 11 were synthesized in two steps. First, the reaction of equimolar amounts of the appropriate acid derivative and the diamine was used to afford the corresponding monocinnamamide. It should be noted that optimizing the reaction conditions, such as using a mild activating reagent and reaction time control, was required to drive the reaction towards a single coupling and create the desired compound containing a free amine group for the subsequent coupling. As depicted in Scheme 2b,c, N-(12-aminododecyl)-3-(3-hydroxyphenyl) acrylamide (8) and N-(12aminododecyl)-cinnamamide (10) were constructed from the reaction of 3-hydroxycinnamic acid (6) and cinnamic acid (1), respectively, with diamine in the presence of HBTU and DIPEA over 2 h. As a representative example, the novel monoamide (8) was identified via mass spectrometry, exhibiting a mass of 347.1 [M + H] + ; moreover, the 1 H NMR spectra showed broad singlet and triplet peaks at 7.69 and 8.08 ppm, which are characteristics of NH 2 and NH, respectively. The singlet peak at 9.57 ppm represents the phenolic hydroxyl group ( Figure S13, Supplementary Materials).
Second, using similar conditions as used in the synthesis of compound 4, monocinnamoylfatty acyl amide conjugates (8 and 10) containing free amino groups were reacted with the additional cinnamic acid derivatives (1 and 3), respectively. As shown in Scheme 2b,c, these reactions afforded the corresponding unsymmetrical bis-cinnamoyl derivatives, N-(12cinnamamidododecyl)-3-(3-hydroxyphenyl) acrylamide (9) and N-(12-cinnamamidododecyl)-3-(4-hydroxyphenyl) acrylamide (11), with each possessing only one hydroxyl group. As a representative example, the 1 H NMR spectrum fits well with structure 9 and indicated that the molecule has unsymmetrical imide protons, showing two triplet signals at 7.92 and 8.08 ppm.

Molecular Docking Study of the Synthesized Compound Candidates
Using molecular modeling, we further evaluated the new compounds by docking the compounds to the APE/Ref-1 crystal structure, downloaded from PDB (1BIX) ( Table 1) [15]. Figure 2 shows the binding of compound 2 into its binding site of APE/Ref-1, resulting in an ICM docking score of −9.23. Compound 11 docked into the same binding cavity, with an ICM docking score of −16.31. The best docking score among all the synthesized candidate compounds was observed with Compound 4 (−20.14), as shown in Table 1.

Effects of Novel APE/Ref-1 Inhibitors on Melanoma Proliferation Using MTT Colorimetric Assay
APE/Ref-1 inhibitors have exhibited anti-proliferation and anti-angiogenesis activity against several human malignancies; therefore, we have chosen anti-tumor efficacy as an endpoint to rank the candidate compounds for bio-evaluation. MTT assays were conducted to evaluate the in vitro anti-melanoma activities of the synthesized compounds in human melanoma cell lines and immortalized melanocytes. Cells were incubated with compounds at various concentrations for 72 h. Cell viabilities after treatments were then analyzed in comparison to that of control.
As shown in Figure 3a, lead compound 2 exhibited potent cytotoxicity in human melanoma A375 cells at 0.1 µM as the surviving cells were reduced to less than 50% of control after 72 h treatment; however, with increasing doses, the cellular toxicity was not increased accordingly in both cell lines, which may be explained by the limited water solubility and stability of compound 2 ( Figure 3a). Apparent drug precipitation was evident in the media at higher concentrations (≥2.5 µM). Of note, no significant cytotoxicity of compound 2 was observed among two melanoblast cell lines after 72 h treatment (Hermes 3A and Hermes 4A, Figure 3b). Hermes 1 cells were more sensitive to compound 2, but the cytotoxicity was not elevated with increasing concentrations.
Compounds 4 and 7 are analogs of lead compound 2 in which two hydrophilic hydroxyl groups (−OH) were added to the aromatic rings. The virtual docking scores of compounds 4 and 7 analyzed by the ICM-Pro program are −20.14 and −11.74, respectively. Compared to compound 2 (−9.23), compound 4 exhibited improved docking to the druggable pocket localized in the redox domain of APE/Ref-1; however, the MTT results showed these modifications led to a significant loss of anti-tumor activity compared to compound 2.
Similar to compound 4, compound 5 has two hydrophilic groups (−OH) but includes a modified chain length to increase the water solubility and was found to have a virtual docking score of −12.91. MTT analysis demonstrated that such modification also resulted in the loss of anti-melanoma activity compared to compound 2, and 50% cell death was not observed up to 50 µM (Table 1).
Compounds 9 and 11 are analogs of lead compound 2, in which one hydrophilic hydroxyl group (−OH) was added to the metaor para-position, respectively. The corresponding virtual docking scores of compounds 9 and 11 are −5.97 and −16.31 (Table 1). Compound 9 showed poor anti-melanoma activity, while compound 11 exhibited a potent cytotoxic effect in melanoma cells ( Figure 3). The virtual docking profiles align well with their anti-melanoma activities, suggesting the position of the −OH group plays a vital role in their interactions with the APE/Ref−1 protein. The binding profile model may guide future modifications of the aromatic ring and predict the compounds' anti-melanoma activities; moreover, compound 11 exhibits improved water solubility compared to compound 2 due to the hydrophilic hydroxyl group. Significant precipitation was evident at~20 µM, approximately 8-folds higher than compound 2 (2.5 µM, data not shown).  Table 1), which are more potent than commercially available APE/Ref-1 redox inhibitors. At 1 µM, cell viability reduced to 19% of control (vehicle only, 0.1% DMSO) after treatment with compound 11, while no significant cytotoxicity was observed with E2009 and E3330 treatment at the same concentration (96.7% and 102.6% of control, respectively) ( Figure 3c). The IC 50s of E3330 and E2009 in A375 melanoma cells were 6.6 µM and 5.3 µM, respectively, which were 75-fold and 60-fold higher compared to that of compound 11.
The selectivity of compound 11 was also determined by comparing its cytotoxicity in melanoma cells to that in normal melanoblast Hermes 1 cells. As shown in Figure 3d, the IC 50 of compound 11 in Hermes 1 is 0.274 µM, which is much higher than the IC 50s observed in A375 and SK-Mel-28 melanoma cells.

Compound 2 Suppressed H 2 O 2 -Induced AP-1 Transactivation in Human Melanoma Cells
An AP-1 transactivation assay was conducted in melanoma cells transfected with a luciferase reporter construct as described previously [39]. Our study showed that compound 2 at 0.5 µM effectively inhibited AP-1 transactivation induced by H 2 O 2 in A375 melanoma cells compared with H 2 O 2 alone (p < 0.05) ( Figure 4). The H 2 O 2 -activated AP-1 activity was reduced from 2.3-fold of control to approximately basal levels (95.7% of control); however, compound 2 did not display any inhibition of the basal AP-1-dependent transcription. Co-treatment with E3330, an APE/Ref-1 redox inhibitor, at 10 µM concentration, reduced AP-1 activity marginally. Compound 11 at 10 µM also failed to inhibit H 2 O 2 -activated AP-1 transactivation (p > 0.05).

Effects of APE/Ref-1 Inhibitor on Tumor Growth Using a Melanoma Xenograft Mouse Model
Given the limited water solubility, lead compound 2 (stocked in DMSO) was further diluted in normal saline. The drug suspension was sonicated at room temperature for 15 min before injection to nude mice. HPLC analysis shows no evidence of chemical structural change after compounding. The drug suspension was made fresh daily and injected into mice immediately after compounding. The control group was intraperitoneally injected (IP) with vehicle (0.1% DMSO in normal saline) daily based on the body weight (0.1 mL/10 g).
As shown in Figure 5, the study demonstrated that the APE/Ref-1 inhibitor, compound 2, effectively inhibited tumor growth in vivo. The effective dose of compound 2 was as low as 5 mg/kg IP daily without producing any apparent systemic toxicities ( Figure S27, Supplementary Materials). After 21-days of treatment, tumor growth was significantly inhibited, with the average tumor size reduced to 58.5% of control (p < 0.05) (Figure 5a). The average tumor weight in the treatment group was only 64.7% of that of the control group (p < 0.05) (Figure 5b). Future studies are warranted to determine the dose-response and pharmacokinetic profile. (a) Tumor volume was measured by digital calipers and determined using the formula (mm 3 ) = (L × W 2 )/2. Tumor growth was presented as a fold increase in size from day 3 (mean ± SD). (b) Comparison of measured tumor burden (via weight) after 21 days of therapy. The p values were determined using a two-tailed Student's t-test. * p < 0.05 compared to control group.

Discussion
In recent years, there have been dramatic developments in the treatment of advanced cutaneous melanoma (CM) using revolutionary immunotherapy, which is a powerful new approach yielding remarkable and durable responses in melanoma patients [40,41]; however, these checkpoint inhibitors are mainly indicated for patients with metastatic melanoma and disease progression following other treatments [42]. In addition, current and novel approaches have not slowed the worldwide melanoma epidemic, and the incidence of CM continues to rise in the United States [2]. As such, the development of novel therapeutic interventions to block melanomagenesis and disease progression to advanced stages will have both high impact and importance.
It has been well studied that APE/Ref-1 serves as an important mechanism facilitating cancer cell survival from oxidative stresses associated with radiation therapy and chemotherapy [15,20,43]. Novel APE/Ref-1 inhibitors have been shown to inhibit cancer cell metastasis and the development of drug resistance, suggesting that targeting APE/Ref-1 is a promising strategy to achieve a better clinical benefit for cancer patients [17,22,30,44].
In recent years, increasing efforts have been devoted to developing novel redox inhibitors of APE/Ref-1 for cancer treatment; however, to the best of our knowledge, only a limited number of compounds exhibit promising APE/Ref-1 inhibitory effects [24,30,43,45,46]. A recently developed small molecular inhibitor of apurinic/apyrimidinic (AP) endonuclease activity was shown to exacerbate the oxidative DNA damage following exposure to cisplatin at 10 µM [24,25]. To date, the most successful APE/Ref-1 redox inhibitors reported are E3330 (APX3330) and its analogs, which exhibited therapeutic effects on tumor angiogenesis and growth without interfering with its DNA repair activities [36,47]. E3330 was also shown to reduce the collective cell migration of human breast cancer cells and decrease chemo-invasion and colony formation when combined with docetaxel; however, such inhibitory activity was only evident at a higher concentration (50 µM) [30]. A recent Phase I trial of E3330 reported that six of nineteen cancer patients had disease stabilization for ≥4 cycles after treatment [23,28]. One melanoma patient presented with a stable disease with 245 days of treatment.
Beyond these studies, there has been little work on structure-based inhibitor design. As detailed in our previous studies [15,17], the redox function of APE/Ref-1 plays a vital role in melanoma progression, which is also consistent with other groups' observation of the anti-tumor activity of APE/Ref-1 redox inhibitors. We, therefore, selected the structure of the redox domain of APE/Ref-1 for screening and molecular modeling. In our study, we carried out virtual screenings using the ICM-Pro software package developed by MolSoft L.L.C [33,34]. The program uses an energetic scoring function to carry out virtual docking of molecules from a large database and ranks binding. Notably, the software incorporates ligand flexibility. We screened multiple drug libraries containing over 3-million compounds and identified the top-ranked candidate compounds. After chemical structure optimization and cell-based bioactivity screening, we have successfully developed our lead APE/Ref-1 inhibitor, compound 2, for further structure-activity studies.
More analogs of compound 2 were synthesized to develop the structure-activity relationship and improve the physicochemical properties. Our study demonstrated that incorporating one or two hydroxyl groups on the aromatic ring enhances the water solubility, but this comes at the expense of reducing anti-melanoma activity. Compound 11 was found to be more water-soluble than compound 2 and exhibited promising anti-melanoma activities compared to the reported APE-Ref-1 inhibitors, E3330 and E2009. Inhibition of cell viability was evident even at very low concentrations in vitro (<0.1 µM). Our in vivo mouse study showed that compound 2 treatment significantly inhibited tumor growth in a melanoma xenograft mouse model. However, given the hydrophobic nature of compound 2, its water solubility is limited, and increased precipitation was evident at concentrations above 2.5 µM. Shortening the chain from C12 to C6 markedly improved the compound's water solubility, but significantly reduced the anti-melanoma activity. One hypothesis is that the long-chain may contribute to binding by facilitating or stabilizing the interaction between the compound and the APE/Ref-1 protein. Shortening the chain may lead to an unstable binding profile and subsequent reduction of anti-tumor activity. This hypothesis may apply to compound 12 as well, which possesses a shortened chain and loss of anti-melanoma activity. To improve the bioavailability and drug delivery to melanoma cells, we will also consider using a cyclic peptide drug delivery system containing arginine and tryptophan residues developed by the Parang group [48]. These cell-penetrating peptides were shown to effectively enhance the uptake of anti-cancer drugs in tumor cells [49].
Structure-based virtual screening and docking have been widely used for drug discovery, such as in hit identification and lead optimization. As noted in Table 1, the virtual docking score of a candidate compound does not always align with its anti-melanoma activities. The poor water solubility of compound 2 significantly hindered the detection of direct interactions between compound 2 and the APE/Ref-1 protein using Surface Plasmon Resonance (SPR) analysis. Future crystallization studies of APE/Ref-1 protein-ligand complexes are warranted to optimize and refine the virtual modeling and ultimately improve structure-based drug design.

Materials
Trans-cinnamoyl chloride, trans-4-hydroxycinnamic acid, trans-cinnamic acid, 1,6hexamethylenediamine, 1,12-dodecamethylenediamine, all solvents, chemicals reagents, and HPLC-eluents were purchased from Sigma-Aldrich, St. Louis, MO, USA, and used as received without further purification. Analytical HPLC was used to confirm the purity of final products (≥95%). The analytical HPLC was conducted on the Shimadzu RP-HPLC system and C18 column (250 cm × 4.60 mm) over 80 min using water (0.1% TFA) as eluent A and acetonitrile (0.1% TFA) as eluent B. NMR spectra were recorded on a Bruker Avance III HDTM 400 NMR spectrometer using DMSO-d 6 or CD 3 OD as solvents and TMS as an internal reference. Mass spectra were obtained by a Bruker Impact 11, UHR-qTOF.

Three-Dimensional Virtual Docking Using Molsoft ICM-Pro System
The in silico molecular docking was performed using Molsoft ICM-Pro (x64) [33]. The druggable binding cavity located in the redox-regulatory domain of the APE/Ref-1 protein (1BIX, 10.2210/pdb1BIX/pdb) was identified and used for docking. The molecular docking conformation presenting the lowest binding energy was selected to visualize the possible compound-protein interaction. The virtual docking scores were collected and the ligands with lower observed ICM scores were chosen due to the higher chance of the ligand being a binder to the APE/Ref-1 cavity [50].

In Vitro Anti-Melanoma Activity Screening Using MTT Colorimetric Assay
Cell viability analysis was conducted based on the bio-reduction of a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTT) metabolized by live cells. Briefly, human melanoma cells were inoculated into 96 well microtiter plates in 100 µL at plating densities ranging from 5000 to 8000 cells/well depending on the doubling time of individual cell lines (A375: 5000 cells/well; Sk-Mel-28: 7500 cells/well; Hermes: 8000 cells/well). After cell inoculation, the microtiter plates are incubated for 24 h prior to the addition of experimental drugs. Various drugs dissolved in DMSO were added to a serum-free medium at different concentrations and incubated for 72 h, and negative controls were treated with the equivalent amounts of the DMSO solution. By the end of treatment, MTT solution was added to the wells to a final concentration of 0.5 mg/mL for an additional 2 h incubation. Solubilization solution (4 mM HCl, 0.1% NP-40 in isopropanol) was then added, and the absorbance was recorded at 595 nm on a plate reader (BioRad, Hercules, CA, USA). All readings were compared to the control, which represented 100% viability. Each experiment was performed in triplicate and independently repeated at least two times. 4.6. Luciferase Assay to Determine AP-1 Transactivation Activities 3 × AP1pGL3 (3 × AP-1 in pGL3-basic) was a gift from Alexander Dent (Addgene plasmid #40342), which contains three canonical AP-1 binding sites (TGACTCA) upstream of a minimal promoter fragment containing a TATA box in the luciferase reporter plasmid pGL3-basic [39]. 3 × AP1pGL3 was transfected to human melanoma A375 cells using Lipofectamine 2000 following the manufacturer's instructions (Invitrogen). 24 h after transfection, the cells were treated with H 2 O 2 (100 µM) for 48 h in the presence or absence of APE/Ref-1 inhibitors (10 µM).
After different treatments, the cells were rinsed twice with phosphate-buffered saline (PBS). Cells were then scraped from the plates in PBS and pelleted for 4 min at 4 • C in a microcentrifuge at 12,000 rpm. Cell pellets were resuspended in 100 µL luciferase lysis buffer as per manufacturer instructions (Promega, #E1500). Luciferase activity measured in relative light units reflects the AP-1 transcription rate and was detected by a SpectraMax M5 UV VIZ Plate Reader. A cell count was conducted for normalization. All samples were studied in duplicate, and readings were taken in triplicate. Each experiment was repeated at least two times.

In Vivo Xenograft Melanoma
The Institutional Animal Care and Use Committee approved all the animal procedures at Chapman University (IACUC #2020-1131). Male nude mice (Nu/Nu) were purchased from Charles River (Wilmington, MA, USA) and were housed and maintained in the Chapman University vivarium under pathogen-free conditions. Human metastatic melanoma A375 cells were injected subcutaneously into the flank (1 × 10 6 cells per mouse). Three days-post tumor cell injection, mice were randomized into different groups. The treatment group was injected with compound 2 (5 mg/kg/day, IP) for 21 days. The growth of the tumors was monitored three times a week and measured using digital Vernier calipers. The size of the tumors was calculated as tumor volume (mm 3 ) = (L × W 2 )/2. The mice were sacrificed at the end of the study via CO 2 euthanasia and cervical dislocation.

Conclusions
The novel APE/Ref-1 inhibitor developed in our study (compound 2) showed promising anti-melanoma activities in vitro and in vivo, suggesting that targeting APE/Ref-1mediated signaling might be a novel and effective strategy for melanoma therapy. More studies are warranted in the future to develop more bioavailable and potent APE/Ref-1 inhibitors with chemical modifications of the lead compound.