Diindolylmethane Derivatives: New Selective Blockers for T-Type Calcium Channels

The natural product indole-3-carbinol (I3C) and its major digestive product 3,3′-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3′-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.

T-type calcium channels (Ca V 3.x) are recognized as a potential target for novel cancer therapies as they are aberrantly expressed in various cancer cells or tumors [17,18] and are implicated in cancer cell proliferation [19][20][21]. Our previous study identified two bisindole alkaloid analogs of marine fungal product pseudellone C as novel and selective T-type blockers [22], suggesting the indole moiety may contribute to Ca V 3.x inhibition. In this

T-Type Calcium Channel Window Current FLIPR Assays
HEK 293 cells stably expressing Ca V 3.2 or Ca V 3.3 were seeded into 384-well black wall clear bottom plates (Corning) at a density of 30,000 cells per well. Transiently transfected Ca V 3.1HEK293 T cells were seeded into 384-well black wall clear bottom plates at a density of 60,000 cells per well. Once the cells reached 90-95% confluence after 24 h, the media were removed from the wells and replaced with 20 µL of 10% calcium 4 dye (Molecular Devices, Sunnyvale, CA, USA) in HBSS-HEPES (containing 5 mM KCl, 10 mM HEPES, 140 mM NaCl, 10 mM glucose, and 0.5 mM CaCl 2 , pH 7.4) with 0.1% bovine serum albumin (BSA). The cells were incubated for 30 min at 37 • C in the presence of 5% carbon dioxide. Each well on the reagent plates for the first addition contained 15 µL different concentrations of compounds dissolved in HBSS-HEPES containing 0.1% BSA and <0.5% DMSO and was incubated for 20 min after loaded. Positive and negative controls contained 15 µL of HBSS-HEPES (0.1% BSA) alone. The plates were placed in the FLIPR TETRA (Molecular Devices, Sunnyvale, CA, USA) programmed to measure maximum fluorescence intensity following a second addition of the agonist 5 mM CaCl 2 . The fluorescence readings were recorded and converted as described previously [22], and HBSS-HEPES (0.1% BSA) was used in the second addition as a negative control.

HVA Calcium Channel FLIPR Assays
SH-SY5Y cells were seeded into 384-well black wall clear bottom plates at a density of 15,000 cells per well, resulting in 90-95% confluence after 24 h. The media were then removed from the wells and replaced with 20 µL of 10% calcium 4 dye (Molecular Devices) in physiological salt solution (PSS) (containing 5.9 mM KCl, 1.4 mM MgCl 2 , 10 mM HEPES, 1.2 mM NaH 2 PO 4 , 5 mM NaHCO 3 , 140 mM NaCl, 11.5 mM glucose, and 1.8 mM CaCl 2 , pH 7.4) with 0.1% BSA. As reported [25], for N-type calcium channel FLIPR assays the cells were pre-incubated with 10 µM nifedipine added in the dye to ensure full inhibition of L-type calcium responses. For L-type calcium channel FLIPR assays, the cells were pre-incubated with 1 µM CVID added in the dye to ensure full inhibition of N-type calcium responses. Positive control on the first reagent plate contained 15 µL of PSS (0.1% BSA), whereas PSS (0.1% BSA) containing 1 µM CVID and 10 µM nifedipine (final concentration) was used as a negative control. The fluorescence readings were recorded and converted as described previously [25], and agonist containing 90 mM KCl + 5 mM CaCl 2 was used in the second addition.

Whole-Cell Patch-Clamp Electrophysiology
Whole-cell patch-clamp experiments were performed on an automated electrophysiology platform QPatch 16 X (Sophion Bioscience A/S, Ballerup, Denmark) in single-hole configuration using 16-channel planar patch chip QPlates (Sophion Bioscience A/S). The extracellular recording solution contained, in mM: TEACl 157, MgCl 2 0.5, CaCl 2 5, and HEPES 10; pH 7.4 adjusted with TEAOH; and osmolarity 320 mOsm. The intracellular pipette solution contained, in mM: CsF 140, EGTA 1, HEPES 10, and NaCl 10; pH 7.2 adjusted with CsOH; and osmolarity 325 mOsm. Compounds were diluted in extracellular recording solution with 0.1% BSA at the concentrations stated (DMSO ≤ 0.1%), and the effects of compounds were compared to the control (extracellular solution with 0.1% BSA) parameters within the same cell. Compounds' incubation time varied from two (for the highest concentration) to five (for the lowest concentration) minutes by applying the voltage protocol 10-30 times at 10 s intervals to ensure steady-state inhibition was achieved. The effects of compounds were obtained using 200 ms voltage steps to peak potential from a holding potential of −90 mV. Current-voltage (I-V) relationships were obtained by holding the cells at a potential of −100 mV before applying 50 ms pulses to potentials from −75 to +50 mV every 5 s in 5 mV increments. Data were fitted with a single Boltzmann distribution: where V 50 is the half-availability voltage and k is the slope factor. Off-line data analysis was performed using QPatch Assay Software v5.6 (Sophion Bioscience A/S) and Excel 2013 (Microsoft Corporation, Redmond, WA, USA).

Cell Viability MTT Assay
Seeded cells were treated with various concentrations of compounds for desired time period (24,48 and 72 h). After the treatments, the media were removed from each well, and replaced with 25 µL of serum-free media and 25 µL of MTT Reagent (cat. no., ab211091; Abcam, Cambridge, MA, USA). The plate was then incubated at 37 • C for 3 h, and 75 µL of MTT Solvent (cat. no., ab211091; Abcam) was added into each well after incubation. The absorbance was evaluated at 590 nm. Cell-wells treated with 0.1% DMSO were used as positive control and no cell-wells were used as background control.

Data Analysis
Data were plotted and analyzed using GraphPad Prism v7.0 (GraphPad Software Inc., San Diego, CA, USA). A four-parameter logistic Hill equation with variable Hill coefficients was fitted to the data for concentration-response curves. Data are means ± SEM of n independent experiments. Statistical analysis was performed with Two-way analysis of variance (ANOVA) with statistical significance at p < 0.05.

Synthesis of 3,3 -Diindolylmethanone and Related Analogs
The synthesis of DIM-one (II) (60% yield) was achieved by Friedel-Crafts acylation of indole, using 1H-indole-3-carboxylic acid in dry dichloroethane (DCE) in the presence of ZrCl 4 , a method first reported by Guchhait et al. [23]. To better explore the structureactivity relationship (SAR) of the synthetic DIM analogs on VGCC, sulfonation [24,26] has been applied to achieve analogs III and V (revised structure of the rare sponge metabolite echinosulfone A [26]) (Scheme 1), and tosyl and benzyl protection [26] have been applied to achieve analogs IX-XI (Figure 1), resulted in symmetric and asymmetric substitutions on the indole rings. Three I3C analogs VI-VIII were also made, and VIII, previously reported with weak pyruvate kinase inhibitory activities [27], was achieved by Friedel-Crafts acylation of indole and oxalyl chloride. Seeded cells were treated with various concentrations of compounds for desired time period (24, 48 and 72h). After the treatments, the media were removed from each well, and replaced with 25 μL of serum-free media and 25 μL of MTT Reagent (cat. no., ab211091; Abcam, Cambridge, MA, USA). The plate was then incubated at 37°C for 3 h, and 75 μL of MTT Solvent (cat. no., ab211091; Abcam) was added into each well after incubation. The absorbance was evaluated at 590 nm. Cell-wells treated with 0.1% DMSO were used as positive control and no cell-wells were used as background control.

Data Analysis
Data were plotted and analyzed using GraphPad Prism v7.0 (GraphPad Software Inc., San Diego, CA, USA). A four-parameter logistic Hill equation with variable Hill coefficients was fitted to the data for concentration-response curves. Data are means ± SEM of n independent experiments. Statistical analysis was performed with Two-way analysis of variance (ANOVA) with statistical significance at p < 0.05.

Synthesis of 3,3'-diindolylmethanone and related analogs
The synthesis of DIM-one (II) (60% yield) was achieved by Friedel-Crafts acylation of indole, using 1H-indole-3-carboxylic acid in dry dichloroethane (DCE) in the presence of ZrCl4, a method first reported by Guchhait et al. [23]. To better explore the structureactivity relationship (SAR) of the synthetic DIM analogs on VGCC, sulfonation [24,26] has been applied to achieve analogs III and V (revised structure of the rare sponge metabolite echinosulfone A [26]) (Scheme 1), and tosyl and benzyl protection [26] have been applied to achieve analogs IX-XI (Figure 1), resulted in symmetric and asymmetric substitutions on the indole rings. Three I3C analogs VI-VIII were also made, and VIII, previously reported with weak pyruvate kinase inhibitory activities [27], was achieved by Friedel-Crafts acylation of indole and oxalyl chloride.  Seeded cells were treated with various concentrations of compounds for desired time period (24, 48 and 72h). After the treatments, the media were removed from each well, and replaced with 25 μL of serum-free media and 25 μL of MTT Reagent (cat. no., ab211091; Abcam, Cambridge, MA, USA). The plate was then incubated at 37°C for 3 h, and 75 μL of MTT Solvent (cat. no., ab211091; Abcam) was added into each well after incubation. The absorbance was evaluated at 590 nm. Cell-wells treated with 0.1% DMSO were used as positive control and no cell-wells were used as background control.

Data Analysis
Data were plotted and analyzed using GraphPad Prism v7.0 (GraphPad Software Inc., San Diego, CA, USA). A four-parameter logistic Hill equation with variable Hill coefficients was fitted to the data for concentration-response curves. Data are means ± SEM of n independent experiments. Statistical analysis was performed with Two-way analysis of variance (ANOVA) with statistical significance at p < 0.05.

Synthesis of 3,3'-diindolylmethanone and related analogs
The synthesis of DIM-one (II) (60% yield) was achieved by Friedel-Crafts acylation of indole, using 1H-indole-3-carboxylic acid in dry dichloroethane (DCE) in the presence of ZrCl4, a method first reported by Guchhait et al. [23]. To better explore the structureactivity relationship (SAR) of the synthetic DIM analogs on VGCC, sulfonation [24,26] has been applied to achieve analogs III and V (revised structure of the rare sponge metabolite echinosulfone A [26]) (Scheme 1), and tosyl and benzyl protection [26] have been applied to achieve analogs IX-XI (Figure 1), resulted in symmetric and asymmetric substitutions on the indole rings. Three I3C analogs VI-VIII were also made, and VIII, previously reported with weak pyruvate kinase inhibitory activities [27], was achieved by Friedel-Crafts acylation of indole and oxalyl chloride.

Evaluation of VGCC Activities of the Synthetic Compounds Using FLIPR Cell-Based Assays
DIM (I), DIM-one (II), and its analogs III-XI were evaluated for activity on VGCC using FLIPR cell-based assays. Their IC 50 values as well as structures were summarized in Table 1. The mono-indole VII showed moderate inhibition against Ca V 3.2 and Ca V 3.3 current measured in T-type window current assays with similar IC 50 s of 17.07 ± 1.87 µM (n = 3) and 13.84 ± 2.02 µM (n = 3), respectively. Among the tested bis-indole compounds, VIII is highly cytotoxic, which caused abnormal current in cells with aberrant partterns, while XI is highly insoluble in buffer with 0.1% BSA. III showed moderate inhibition against Ca V 3.3, while compounds IV, V, VI, and X showed poor inhibition of all the Ca 2+ responses. In contrast, DIM, DIM-one and IX were identified to be selective Ca V 3.x blockers with good potency. DIM had the best potency and selectivity against Ca V 3.3 window current, with an IC 50 value of 2.09 ± 0.43 µM (n = 3), which was >27-fold better than its potency at high voltageactivated (HVA) Ca V s, and~25-fold better potency at Ca V 3.1 and Ca V 3.2. Comparatively, DIM-one, which has an oxidized methylene group, showed a 4.3-fold reduced potency for Ca V 3.3 window current with an IC 50 value of 9.07 ± 0.69 µM (n = 3) and a~1.4-fold reduced potency for Ca V 3.2 window current compared to DIM, with an IC 50 value of 73.85 ± 2.48 µM (n = 3).
DIM-one analog, IX potently blocked both Ca V 3.2 and Ca V 3.3 responses with IC 50 values of 3.05 ± 0.51 µM (n = 3) and 6.68 ± 0.79 µM (n = 3), respectively, while it was >7-fold less active at high voltage-activated (HVA) Ca V s. In general, compounds with electrondonating substituents had better Ca V 3.x activity, while electron-withdrawing substituents compromised activity. The fluorescent Ca V 3.2 and Ca V 3.3 Ca 2+ responses before and after addition of DIM, DIM-one and IX, and their representative concentration-response curves, are presented in Figure 2

Electrophysiological characterization of the selective CaV3.x blockers in QPatch assays
We also examined the effects of DIM, DIM-one and IX on the CaV3.x by whole-cell patch-clamp using the automated electrophysiology platform QPatch 16 X (Figures 4-6), and their IC50s were summarized in Table 2

Electrophysiological characterization of the selective CaV3.x blockers in QPatch assays
We also examined the effects of DIM, DIM-one and IX on the CaV3.x by whole-cell patch-clamp using the automated electrophysiology platform QPatch 16 X (Figures 4-6), and their IC50s were summarized in Table 2

Electrophysiological Characterization of the Selective Ca V 3.x Blockers in QPatch Assays
We also examined the effects of DIM, DIM-one and IX on the Ca V 3.x by whole-cell patch-clamp using the automated electrophysiology platform QPatch 16 X (Figures 4-6), and their IC 50 s were summarized in Table 2. DIM modestly inhibitedCa V 3.2 and Ca V 3.3 whole-cell current, with IC 50 values of 21.09 ± 1.19 µM (n = 4) and 9.63 ± 0.97 µM (n = 5), respectively, and slightly better potency against Ca V 3.1 whole-cell current, with an IC 50 Membranes 2022, 12, 749 7 of 13 value of 8.32 ± 1.53 µM (n = 5). Compared to DIM, DIM-one had poor inhibition of Ca V 3.2 and Ca V 3.3 whole-cell current but potently inhibited Ca V 3.1 whole-cell current with an IC 50 value of 1.53 ± 1.06 µM (n = 3). Interestingly, low concentrations of DIM (49 nM-3.1 µM) weakly enhanced Ca V 3.1 channel current before inhibiting current at higher concentrations, without associated changes in current characteristics. Biphasic effects found in drugs, e.g., the chemotherapy medication doxorubicin [28] and the psychoactive cannabinoids THC [29] and CBD [30]  concentrations, without associated changes in current characteristics. Biphasic effects found in drugs, e.g., the chemotherapy medication doxorubicin [28] and the psychoactive cannabinoids THC [29] and CBD [30]     concentrations, without associated changes in current characteristics. Biphasic effects found in drugs, e.g., the chemotherapy medication doxorubicin [28] and the psychoactive cannabinoids THC [29] and CBD [30]

Effects of the synthetic compounds on cancer cell viability in MTT assay
DIM has been investigated extensively for anticancer activity in vitro and in vivo. However, the diindolemethanones, including DIM-one, have not been reported for anticancer activity previously. Based on our initial tests (data not shown), DIM and related analogs II-X were tested at 50 µ M on the human breast cancer cell lines MCF-7 (CaV3.

Effects of the synthetic compounds on cancer cell viability in MTT assay
DIM has been investigated extensively for anticancer activity in vitro and in vivo. However, the diindolemethanones, including DIM-one, have not been reported for anticancer activity previously. Based on our initial tests (data not shown), DIM and related analogs II-X were tested at 50 µ M on the human breast cancer cell lines MCF-7 (CaV3.

Effects of the Synthetic Compounds on Cancer Cell Viability in MTT Assay
DIM has been investigated extensively for anticancer activity in vitro and in vivo. However, the diindolemethanones, including DIM-one, have not been reported for anticancer activity previously. Based on our initial tests (data not shown), DIM and related analogs II-X were tested at 50 µM on the human breast cancer cell lines MCF-7 (Ca V 3.2 enhanced) and T-47D (Ca V 3.2 and Ca V 3.3 enhanced), the lung cancer cell line A549 (Ca V 3.1 and Ca V 3.2 enhanced), and the colon cancer cell line HT-29 (No enhanced expression of Ca V 3.x) (Ca V 3.x status see the Human Protein Atlas: https://www.proteinatlas.org/, accessed on 16 July 2022). The anti-proliferative activities of the compounds are concluded in Figure 8. DIM had the strongest anti-proliferative activities on MCF-7 breast cancer cell line as previously found. In contrast, DIM-one (II), III, IV and V showed preferential anti-proliferative activity on the HT-29 colon cancer cell line, which does not have enhanced expression of Ca V 3.x. These data suggest the anti-proliferative activity of these compounds is unrelated to Ca V 3.x inhibition. Interestingly, compound IX had significant activity at Ca V 3.2 and Ca V 3.3 window currents, but did not affect cancer cell viability, whereas Compound III promoted A549 cell proliferation but reduced HT-29 cell viability. In future studies, the involvement of other cancer-related signaling pathways should be explored for these analogs.
Membranes 2020, 10, x FOR PEER REVIEW 9 of 13 anti-proliferative activities of the compounds are concluded in Figure 8. DIM had the strongest anti-proliferative activities on MCF-7 breast cancer cell line as previously found.
In contrast, DIM-one (II), III, IV and V showed preferential anti-proliferative activity on the HT-29 colon cancer cell line, which does not have enhanced expression of CaV3.x. These data suggest the anti-proliferative activity of these compounds is unrelated to CaV3.x inhibition. Interestingly, compound IX had significant activity at CaV3.2 and CaV3.3 window currents, but did not affect cancer cell viability, whereas Compound III promoted A549 cell proliferation but reduced HT-29 cell viability. In future studies, the involvement of other cancer-related signaling pathways should be explored for these analogs. Based on the promising data obtained from the one-concentration antiproliferative effects, we decided to examine the concentration response effects of DIM, DIM-one, IV and V. The concentration-dependent effects of the compounds on the four cancer cell lines after 72 h treatment are shown in Figure 9. Based on the promising data obtained from the one-concentration antiproliferative effects, we decided to examine the concentration response effects of DIM, DIM-one, IV and V. The concentration-dependent effects of the compounds on the four cancer cell lines after 72 h treatment are shown in Figure 9.
The MTT assay results showed that DIM had similar antiproliferative effects on breast cancer cell lines MCF-7 and T-27D and the colon cancer cell line HT-29, but was slightly less effective on the lung cancer cell line A549. Comparatively, all three methanone (DIM-one) compounds showed preferential antiproliferative effects on the non-Ca V 3.  The MTT assay results showed that DIM had similar antiproliferative effects on breast cancer cell lines MCF-7 and T-27D and the colon cancer cell line HT-29, but was slightly less effective on the lung cancer cell line A549. Comparatively, all three methanone (DIM-one) compounds showed preferential antiproliferative effects on the non-CaV3.x enhanced colon cancer cell line HT-29 compared with the other three CaV3.x enhanced cancer cell lines. DIM-one and V showed ≥ 2 fold better antiproliferative effect on HT-29 cell viability than their effects on the breast cancer cell lines and lung cancer cell line A549, whereas IV was ~ 3 fold more effective on HT-29 cell viability compared with its effects on the other three cancer cell lines. Compound IV also showed > 10 fold improved potency on HT-29 cell viability compared with the other three compounds. Further investigations revealed that instead of a continually increasing effectiveness with elongated treatment times, 48 h and 72 h treatment of IV showed similar antiproliferative effects on all four cell lines (see the Supplementary Materials).

Discussion
In this study, we demonstrated that the natural product DIM, along with its synthetic derivative DIM-one and IX, are potent and selective CaV3.x current blockers that showed preferential inhibition of CaV3.2 (IX) and/or CaV3.3 (DIM, DIM-one, and IX) window current measured in FLIPR assays, while DIM-one showed preferential inhibition of CaV3.1 whole-cell current QPatch assays. Unlike QPatch assays, where cells were held in hyperpolarized potentials, the FLIPR window current assay applied to CaV3.x was permissive of a window current resulting from incomplete inactivation [31], where most of the channels are in inactivated state. Inhibition of CaV3.x window current has been proposed

Discussion
In this study, we demonstrated that the natural product DIM, along with its synthetic derivative DIM-one and IX, are potent and selective Ca V 3.x current blockers that showed preferential inhibition of Ca V 3.2 (IX) and/or Ca V 3.3 (DIM, DIM-one, and IX) window current measured in FLIPR assays, while DIM-one showed preferential inhibition of Ca V 3.1 whole-cell current QPatch assays. Unlike QPatch assays, where cells were held in hyperpolarized potentials, the FLIPR window current assay applied to Ca V 3.x was permissive of a window current resulting from incomplete inactivation [31], where most of the channels are in inactivated state. Inhibition of Ca V 3.x window current has been proposed to be privileged target for the development of new analgesic, antiepileptic, and anticancer drugs [32][33][34]. However, our data indicated that the anti-proliferative activities of the investigated compounds against cancer cells are unrelated to their Ca V 3.x inhibition.
As mentioned above, IX fully blocked Ca V 3.2 and Ca V 3.3 current with good potency in the FLIPR window current assay, but to our surprise, they showed only partial inhibition of both Ca V 3.2 and Ca V 3.3 whole-cell current in QPatch assays, where affinity is determined by interactions with the resting state of the channel. Both DIM and DIM-one showed good selectivity for Ca V 3.3 window current, while in QPatch assays, DIM showed good potency for both Ca V 3.1 and Ca V 3.3 activated current and DIM-one showed preferential inhibition of Ca V 3.1 activated current with an IC 50 value of 1.53 ± 1.06 µM (n = 3). Interestingly, low concentrations of DIM (49 nM-3.125 µM) have been observed to show a promoting effect of Ca V 3.1 channel opening, and started to show inhibitory effects with higher concentrations. However, neither low nor high concentrations of DIM showed voltage-dependent effect on I-V relationships of Ca V 3.1. Given overexpression and gene knockdown of Ca V 3.1 decrease MCF-7 cell proliferation [20,35], DIM may help clarify the role(s) of Ca V 3.1 in MCF-7 cell proliferation.
T-type Ca V 3.1 and Ca V 3.2 have been reported to play critical roles in neurological disorders and diseases like absence epilepsy [36][37][38][39], inflammatory pain [40], etc. As a rising anticancer agent under several clinical trials, DIM has been reported to show synergistic anti-colorectal cancer effect with capsaicin [8,9], which is an analgesic agent for peripheral nerve pain [41]. Combined with this work, the Ca V 3.x inhibitory activity of DIM may indicate its possible application in pain relief.
DIM has been explored for clinical application mainly in breast cancer therapy. In this work, cell viability MTT assay has also revealed that DIM has a preferable anti-proliferative activity on MCF-7 breast cancer cell line, while DIM-one along with its analogs III, IV, and V showed better anti-proliferative activity on HT-29 colon cancer cell line. Among them, IV and V also showed promising anti-proliferative activities on MCF-7, T-47D and A549 cell lines, which requires further studies to determine their targeting signaling pathways.
In summary, for the first time, the natural anticancer product DIM, along with its synthetic derivatives DIM-one and IX were characterized as promising and selective Ca V 3.x blockers, which could possibly guide a broader application of DIMs in clinical use. In general, the oxidation of DIM methylene group compromised its Ca V 3.x activities. However, these DIM derivatives, including DIM-one, III, IV and V, showed preferable antiproliferative activities on non-Ca V 3.x enhanced HT-29 cell line, highlighting their potential as early leads in the development of new colon cancer therapies.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/membranes12080749/s1, Synthetic procedures and NMR (DMSO-d 6 ) data for each compound; Figure S1: Cytotoxic effects of IV on the MCF-7, T-47D, A549 and HT-29 cell lines after 24 h, 48 h, and 72 h treatment measured in the MTT assay; Table S1: Effects of the synthetic compounds on cancer cell viability in MTT assay; Table S2: Concentration dependent effects of DIM, DIM-one, IV and V on cancer cell viability after 72 h treatment measured in the MTT assay; Table S3: Concentration dependent effects of IV on cancer cell viability after 24 h, 48 h, and 72 h treatment measured in MTT assay.