Bioorthogonal release of anticancer drugs via gold-triggered 2-alkynylbenzamide cyclization

Metal-based uncaging of biomolecules has become an emerging approach for in vivo applications, which is largely due to the advantageous bioorthogonality of abiotic transition metals. Adding to the library of metal-cleavable protecting groups, this work introduces the 2-alkynylbenzamide (Ayba) moiety for the gold-triggered release of secondary amines under mild and physiological conditions. Studies were further performed to highlight some intrinsic benefits of the Ayba protecting group, which are (1) its amenable nature to derivatization for manipulating prodrug properties, and (2) its orthogonality with other commonly used transition metals like palladium and ruthenium. With a focus on highlighting its application for anticancer drug therapies, this study successfully shows that gold-triggered conversion of Ayba-protected prodrugs into bioactive anticancer drugs (i.e. doxorubicin, endoxifen) can proceed effectively in cell-based assays.


HPLC Methods
Reverse-phase HPLC was used to analyze and purify compounds as indicated. The Shimadzu system (Kyoto, Japan) employed two LC-20AP pumps outfitted with a SPD-20AV photodiode array detector, a RF-10AXL fluorescence detector, and a SIL-20A autosampler. The columns used were a semipreparative 10 × 250 mm Cosmosil 5C18-Ar-300 and an analytical 4.6 × 250 mm Cosmosil 5C18-Ar-300 from Nacalai Tesque (Kyoto, Japan). Samples were eluted using a combination of mobile phases A (100% H2O), B (100% acetonitrile), C (H2O with 0.1% TFA), and D (acetonitrile with 0.1% TFA). The detector was set to 214 and 254 nm. The different elution methods that were employed in this study are shown in Table S1.

HPLC Analysis (Product Standard Curve and Calibration)
To determine yields from HPLC analysis, calibration curves were constructed using product standards of known amounts (shown in Figures S1-S10).
Product Structure HPLC Method (see Table S1) Calibration Curve  Product Structure HPLC Method (see Table S1) Calibration Curve  Product Structure HPLC Method (see Table S1) Calibration Curve  Product Structure HPLC Method (see Table S1) Calibration Curve

Protocols and Yield Determination
In general, product identification was determined by MS analysis and comparison with retention times of known product standards. Yields were obtained via HPLC analysis (unless otherwise noted). This was carried out via peak integration, followed by calculations based on product calibration curves shown in Supplementary Section 1.3.
For the model reactivity and derivatization studies, compounds 1a-t (0.01 mmol) and Au-1 (concentration adjusted as indicated) were dissolved in solvent (1 ml). The mixtures (in glass test tubes) were then placed on a block temperature-controlled hotplate stirrer (Asynt, UK). Reactions were stirred at the indicated times and temperature. To workup, mixtures were diluted with acetonitrile containing 0.1% TFA (1 ml), filtered, and then injected (50 µl) onto a HPLC with an autosampler. Depending on the desired product, HPLC methods 1-4 were used. For the individual reactivity studies, prodrugs 3a, 3m, 6a, 6m, or 7 (30 nmol) and metal catalyst (30 nmol) were dissolved in 50% THF in aqueous solvent (50 µl).
In general, the mixtures (in eppendorf tubes) were placed in a MD-MINI water bath incubator (Major Science, USA). Without stirring, reactions were left to incubate for 12 hr at 37°C. To workup, mixtures were diluted with 1 mM dodecanethiol in THF (100 µl) to quench the metal catalysts. Since doxorubicin is insoluble unless its primary amine is protonated, 1M HCl (50 µl) was added to ensure the released compound would match the commercial product standards (i.e. doxorubicin

B)
A) hydrochloride). The solutions were then filtered, and injected (50 µl) onto a HPLC with an autosampler using HPLC method 5.

Reactivity Data
In Table S2, the model reaction from 1a to 2a was shown to be time-dependent (entries 1-3) and temperature-dependent (entries 4-5).  1  1  37  19  2  3  37  44  3  6  37  54  4  16  rt  46  5 16 70 69 a Yields determined by HPLC (peak retention times compared to product standards, followed by MS analysis for confirmation, and then calculation of yields based on product standard curves). b Isolated yields obtained by column chromatography purification. All reactions were standardized to 10 µmol of 1a in 1 ml of solvent (10 mM).
In Table S3, derivatives 1b-t were prepared and tested to investigate the factors that could possibly influence amine release.
Alkyne substitution (R1 position): Pentyl, phenyl, and anisole derivatives 1q-s were tested, which all largely showed an increase in reactivity. The only example that falls from this pattern is 1t (entry 20), which may possibly be due to the amine substituent perturbing reactivity by interacting with the Aucatalyst.
Benzyl substitution (R2, R3, and R4 positions): Electron-withdrawing group were shown to increase yields of amine release. From comparing simple fluoro substitutions either at the R2, R3, and R4 positions (entries 9-11), it can be observed that the R3 position (para to the alkyne moiety) gave slightly better yields. Subsequently stronger electron-withdrawing groups at the R3 position, such as nitro 1l (56%, entry 12) and trifluoromethyl 1m (74%, entry 13), gave higher yields of amine release. No significant changes in reactivity were seen with substitutions that were weakly electron-donating (methyl, entry 14) and moderately electron-donating (methoxy, entries [15][16]. A possible explanation for these observations is that para-positioned electron withdrawing groups likely has a stronger effect in pulling electron density away from the alkyne, thereby better activating it for Au-catalyzed cyclization. N-substitution (R5 position): Unsubstituted amide 1b showed no detectable levels of Au-triggered amine release (entry 2). Longer and bulkier alkyl groups generally led to lower yields of amine release (c-hexyl, hexyl, isopropyl, and t-butyl, entries 4-7). The same observation was also seen by replacement with an aromatic group (phenyl, entry 3) and an intramolecularly-linked alkane (prolinebased amine, entry 8). Likely, the observed reductions in amine release are the outcome of steric bulk outweighing the alpha effects of N-substitution.  Table S4, the reactivity of the tested metal catalyst complexes (Au-2, Au-3, Au-4) were compared under aqueous conditions containing either pure water or PBS buffer pH 7.4. Due to the instability of Au-2 and Au-3 complexes, both 3a and 3m experienced a noticeable drop in reactivity when switching to buffered conditions.  -<1  <1  6  3m  Au-2  60  3  7  3m  Au-3  62  22  8 3m Au-4 60 60 a Yields determined by HPLC (peak retention times compared to product standards, followed by MS analysis for confirmation, and then calculation of yields based on product standard curves). All reactions were standardized to 30 nmol of 3a,m and 30 nmol of catalyst in 50 µl of solvent (600 µM).

By-product Identification
For Ayba-decaging to occur, the initial transformation involves a nucleophilic cyclization process where a bond-forming interaction occurs between the lone-pair orbital of the carbonyl-O and the inplane alkyne π* orbital. Depending on the type of catalyst (and its ligand), either a 5-exo-dig or 6-endodig cyclization will proceed. As highlighted in Scheme S1, the resultant by-product 46 was purified from a model substrate 1q. Differentiation between the exo and endo forms can be made by NMR analysis, where protons at the C-1 position of 46 are expected to be down-shifted compared to the substrate.
Scheme S1. Identification of the by-product 46

Reactivity Study Substrates
Scheme S2. Synthesis of 2a-c Preparation of 9a : N-methylglycine 8a (593 mg, 6.66 mmol) was dissolved in dioxane (10 ml) and H2O (6 ml). NaOH (1.2 g, 30 mmol) dissolved in H2O (2 ml) was added and the mixture was cooled to 0°C. Boc anhydride (1.73 g, 7.92 mmol) dissolved in dioxane (2 ml) was then added dropwise and the mixture was stirred for 16 hr at room temperature. To workup, dioxane in the mixture was evaporated under vacuum and the residual solution was washed with EtOAc. The aqueous layer was then acidified to pH~3 with 1N HCl, which allowed the product to be extracted with CHCl3.The combined organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum to give the desired pure compound. Yield: 1250 mg, quan. Characterization matched a previous report of this known compound. N-Phenylglycine 8c (500 mg, 3.31 mmol) was dissolved in 1:1 acetone/H2O (6ml). Boc anhydride (2.16 g, 9.89 mmol) dissolved in acetone (1 ml) was then added dropwise and the mixture was stirred for 16 hr at room temperature. To workup, acetone in the mixture was evaporated under vacuum and the residual solution was washed with ether. The aqueous layer was then acidified to pH~3 with 1N HCl, which allowed the product to be extracted with CHCl3.The combined organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 0-10% MeOH in CHCl3 was used to purify the desired compound. Yield: 804 mg, 97%. Rf =0.29 (MeOH/CHCl3, 1:10). Characterization matched a previous report of this known compound. 2  N-Boc-glycine 9b (100 mg, 0.571 mmol), benzylamine (63 μl, 0.571 mmol), EDC (177 mg, 1.14 mmol), HOBt (154 mg, 1.14 mmol) were dissolved in CHCl3 (10 ml). NEt3 (160 μl, 1.14 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the solvent was evaporated under vacuum and the mixture redissolved in EtOAc. The organic layer was then washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 10-50% EtOAc in Hex was used to purify the desired compound. Yield: 538 mg, 89%. Rf =0. 21  Compound 10a (1293 mg, 4.65 mmol) was dissolved in a mixture of TFA:DCM (1 ml:1 ml) and stirred for 60 min at room temperature. Completion of the reaction was determined by disappearance of starting material spot on TLC (100% EtOAc). To workup, the reaction was evaporated under vacuum to an oil. Flash column chromatography using a gradient of 0-10% MeOH in CHCl3 was used to purify the desired compound. Yield: 828 mg, quan. Rf =0. 12  Compound 10c (225 mg, 0.661 mmol) was dissolved in a mixture of TFA:DCM (1 ml:1 ml) and stirred for 60 min at room temperature. Completion of the reaction was determined by disappearance of starting material spot on TLC (MeOH/CHCl3, 1:10). To workup, the reaction was evaporated under vacuum to an oil. Flash column chromatography using a gradient of 0-5% MeOH in CHCl3 was used to purify the desired compound. Yield: 158 mg, quan. Compound 12 (209 mg, 0.920 mmol) was dissolved in DCM (2 ml). n-Hexylamine (1.2 ml, 9.920 mmol) was then added and the mixture was stirred for 16 hr and at room temperature under N2. To workup, the reaction was evaporated under vacuum to an oil. Flash column chromatography using a gradient of 0-5% MeOH in CHCl3 was used to purify the desired compound. Yield: 194 mg, 85%.

Scheme S4. Synthesis of 2h
Preparation of 9h : L-Proline 8h (766 mg, 6.66 mmol) was dissolved in dioxane (6 ml) and H2O (10 ml). NaOH (1.2 g, 30 mmol) dissolved in H2O (2 ml) was added and the mixture was cooled to 0°C. Boc anhydride (1.73 g, 7.92 mmol) dissolved in dioxane (2 ml) was then added dropwise and the mixture was stirred for 16 hr at room temperature. To workup, dioxane in the mixture was evaporated under vacuum and the residual solution was washed with EtOAc. The aqueous layer was then acidified to pH~3 with 1N HCl, which allowed the product to be extracted with CHCl3.The combined organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum to give the desired compound. Yield: 1220 mg, 85%. Characterization matched a previous report of this known compound. Compound 9h (411 mg, 1.91 mmol), benzylamine (210 μl, 1.91 mmol), EDC (592 mg, 3.82 mmol), HOBt (516 mg, 3.82 mmol) were dissolved in CHCl3 (10 ml). NEt3 (534 μl, 3.82 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the solvent was evaporated under vacuum and the mixture redissolved in EtOAc. The organic layer was then washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 25-50% EtOAc in Hex was used to purify the desired compound. Yield: 543 mg, 93%. Rf =0. 16  Compound 10h (875 mg, 2.88 mmol) was dissolved in a mixture of TFA:DCM (1 ml:1 ml) and stirred for 60 min at room temperature. Completion of the reaction was determined by disappearance of starting material spot on TLC (EtOAc/Hex, 1:1). To workup, the reaction was evaporated under vacuum to an oil. Flash column chromatography using a gradient of 0-10% MeOH in CHCl3 was used to purify the desired compound. Yield: 588 mg, quan. Rf =0. 18  2-Iodobenzoic acid 13a (1 g, 4.03 mmol) was dissolved in MeOH (20 ml). Sulfuric acid (2.7 ml) was then slowly added and the mixture was stirred for 16 hr and heated at reflux under N2. To workup, the reaction was cooled to room temperature, before diluting with ether. The organic layer was then washed with H2O, brine, dried over magnesium sulfate, and evaporated under vacuum to give the desired pure compound. Yield: 847 mg, 80%. Characterization matched a previous report of this known compound. 5  Preparation of 14j : 5-Fluoro-2-iodobenzoic acid 13j (300 mg, 1.13 mmol) and K2CO3 (234 mg, 1.69 mmol) were dissolved in DMF (10 ml) and stirred for 5 min. CH3I (105 μl, 1.69 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum to give the desired pure compound. Yield: 300 mg, quan. Rf =0.60 (EtOAc/Hex, 1:4). Characterization matched a previous report of this known compound. Preparation of 14k : 6-Fluoro-2-iodobenzoic acid 13k (300 mg, 1.13 mmol) and K2CO3 (234 mg, 1.69 mmol) were dissolved in DMF (10 ml) and stirred for 5 min. CH3I (105 μl, 1.69 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum to give the desired pure compound. Yield: 299 mg, quan. Rf =0.59 (EtOAc/Hex, 1:4). Characterization matched a previous report of this known compound. 8  Preparation of 14n : 4,5-Dimethyl-2-iodobenzoic acid 13n (300 mg, 1.09 mmol) and K2CO3 (225 mg, 1.63 mmol) were dissolved in DMF (10 ml) and stirred for 5 min. CH3I (101 μl, 1.63 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum to give the desired pure compound. Yield: 299 mg, quan. Rf =0.60 (EtOAc/Hex, 1:4). Characterization matched a previous report of this known compound. 11  Compound 15i (361 mg, 1.08 mmol) was dissolved in MeOH (6 ml) and THF (6 ml) at 0°C. KOH (607 mg, 10.83 mmol) dissolved in H2O (6 ml) was then added and the mixture was stirred for 3 hr at rt under N2. To workup, the mixture was acidified to pH~3 with 1M HCl solution, which allowed the product to be extracted with EtOAc. The organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 0-20% EtOAc in Hex was used to purify the desired compound. Yield: 292 mg, 84%. Rf =0. 21  Compound 15j (361 mg, 1.08 mmol) was dissolved in MeOH (6 ml) and THF (6 ml) at 0°C. KOH (607 mg, 10.83 mmol) dissolved in H2O (6 ml) was then added and the mixture was stirred for 2.5 hr at rt under N2. To workup, the mixture was acidified to pH~3 with 1M HCl solution, which allowed the product to be extracted with EtOAc. The organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 10-25% EtOAc in Hex was used to purify the desired compound. Compound 15k (361 mg, 1.08 mmol) was dissolved in MeOH (6 ml) and THF (6 ml) at 0°C. KOH (607 mg, 10.83 mmol) dissolved in H2O (6 ml) was then added and the mixture was stirred for 3 hr at 50°C under N2. To workup, the mixture was acidified to pH~3 with 1M HCl solution, which allowed the product to be extracted with EtOAc. The organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 10-25% EtOAc in Hex was used to purify the desired compound. Yield: 251 mg, 72%. Compound 15l (337 mg, 0.878 mmol) was dissolved in MeOH (6 ml) and THF (6 ml) at 0°C. KOH (492 mg, 8.78 mmol) dissolved in H2O (6 ml) was then added and the mixture was stirred for 1 hr at rt under N2. To workup, the mixture was acidified to pH~3 with 1M HCl solution, which allowed the product to be extracted with EtOAc. The organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 25-50% EtOAc in Hex was used to purify the desired compound. Yield: 296 mg, 97%. Rf =0.33 (EtOAc/Hex, 1:1 Compound 15p (379 mg, 1.10 mmol) was dissolved in MeOH (6 ml) and THF (6 ml) at 0°C. KOH (614 mg, 10.95 mmol) dissolved in H2O (6 ml) was then added and the mixture was stirred for 1 hr at rt under N2. To workup, the mixture was acidified to pH~3 with 1M HCl solution, which allowed the product to be extracted with EtOAc. The organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 0-50% EtOAc in Hex was used to purify the desired compound. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 10-25% EtOAc in Hex was used to purify the desired compound. Yield: 134  Compound 16k (143 mg, 0.447 mmol), 2a (95 mg, 0.536 mmol), HCTU (369 mg, 0.894 mmol), HOBt(6-Cl) (152 mg, 0.894 mmol) were dissolved in DMF (10 ml). DIPEA (303 μl, 1.79 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 15-30% EtOAc in Hex was used to purify the desired compound. Compound 16l (207 mg, 0.561 mmol), 2a (120 mg, 0.674 mmol), EDC (174 mg, 1.12 mmol), HOBt (152 mg, 1.12 mmol) were dissolved in DMF (5 ml). NEt3 (314 μl, 2.25 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 15-30% EtOAc in Hex was used to purify the desired compound. Yield: 207 mg, 73%.  139.1, 138.3, 133.6, 128.9, 127.8, 127.7, 126.1, 124.0, 121.7, 102.3 Compound 16n (168 mg, 0.509 mmol), 2a (109 mg, 0.610 mmol), EDC (158 mg, 1.02 mmol), HOBt (137 mg, 1.02 mmol) were dissolved in DMF (10 ml). NEt3 (345 μl, 2.03 mmol) was then added and the mixture was stirred for 16 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 25-50% EtOAc in Hex was used to purify the desired compound.  Compound 17a (236 mg, 0.510 mmol) was dissolved in DCM (2 ml). TBAF (764 μl of 1.0M solution, 0.764 mmol) was then added and the mixture was stirred for 1 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 50-100% EtOAc in Hex was used to purify the desired compound. Yield: 155 mg, quan. Compound 17b (134 mg, 0.299 mmol) was dissolved in DCM (2 ml). TBAF (450 μl of 1.0M solution, 0.448 mmol) was then added and the mixture was stirred for 1 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum to give a white precipitate, which was then washed with hexanes and filtered to give the desired compound. Yield: 42 mg, 48%. Rf =0.29 (EtOAc/Hex, 1:1 Compound 17e (213 mg, 0.400 mmol) was dissolved in DCM (2 ml). TBAF (655 μl of 1.0M solution, 0.655 mmol) was then added and the mixture was stirred for 1 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 25-50% EtOAc in Hex was used to purify the desired compound. Yield: 130 mg, 86%. Compound 17f (231 mg, 0.472 mmol) was dissolved in DCM (2 ml). TBAF (708 μl of 1.0M solution, 0.708 mmol) was then added and the mixture was stirred for 1 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 50-75% EtOAc in Hex was used to purify the desired compound. Yield: 156  Compound 17i (167 mg, 0.347 mmol) was dissolved in DCM (2 ml). TBAF (521 μl of 1.0M solution, 0.521 mmol) was then added and the mixture was stirred for 1 hr at room temperature under N2. To workup, the reaction was diluted with EtOAc and then the organic layer was washed with H2O, brine, dried over sodium sulfate, and evaporated under vacuum. Flash column chromatography using a gradient of 50-75% EtOAc in Hex was used to purify the desired compound. Yield: 87 mg, 77%. Rf =0.14 (EtOAc/Hex, 1:1 Compound 18 (81 mg, 0.199 mmol), CuI (8 mg, 0.040 mmol), and PdCl2(PPh3)2 (14 mg, 0.020 mmol) were dissolved in DMF (2 ml) and NEt3 (2 ml). The mixture was degassed with N2 for 5 min. 4-Ethynylaniline (47 mg, 0.397 mmol) dissolved in DMF (0.5 ml) was then added and the mixture was stirred for 16 hr at 30°C under N2.

General Cell Culture Protocol
In this study, the following cell lines were obtained from the RIKEN Cell Bank. Cells were grown in 37 ºC incubators supplemented with 5% CO2 gas. Specific growth media used are indicated as follows:

MTS Assay Protocol
Cell viability was determined using an MTS assay, which is a colorimetric method to monitor the reduction of MTS tetrazolium salts to formazan via mitochondrial dehydrogenase of metabolically active cells. The commercial kit used in this study was the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Wisconsin, USA).
Based on cell titration experiments (data not shown), cells were plated and grown overnight on 96-well Falcon® microplates (1000 cells/well for HeLa; 1000 cells/well for PC3; 1000 cells/well for A549; 2000 cells/well for MCF7). The media was then removed, followed by the incubation of various concentrations of compounds used in this study. Generally, 10 μl of compounds were added to 90 μl of media. Following an incubation time of 4 days, cell viability was detected by first removing the media and replacing it with 20 µl MTS reagent and 80 µl media. Following incubation at 37 ºC for 2 hr, end-point absorbance was acquired at 490 nm, via a SpectraMax® iD3 Multi-Mode Microplate Reader (Molecular Devices, California, USA). The background control for this assay was the incubation of 20 µl MTS reagent and 80 µl media in the absence of cells. Obtained EC50 values were calculated via GraphPad Prism (version 7.0d) software using fitting based on the sigmoidal dose response equation.

Controls
Before cell toxicity studies, various controls experiments were performed. The first was to determine the toxicity of DMSO ( Figure S14), which was used to dissolve the compounds used in this study. This experiment led to the standardization of 1% DMSO. The next control was to determine the toxicity of Au-5 ( Figure S15). From the acquired growth curves, it was decided that the addition of Au-5 to a final concentration of 10 μM would be ideal for the tested cell lines (HeLa S3, PC3, A549, and MCF7).

Cell Toxicity Results
Cell growth curves to monitor toxicity of the tested compounds (3a, 3m, 6a, 6m, and 7) are shown in Figures S16-S19. A summary of the calculated EC50 values obtained in tests against MCF7 cells using prodrugs 3a,m are shown in Table S5.  Figure S16. Cell growth curves of MCF7 cancer cells aimed at exploring the effects of supplementation with A) prodrug 3a (orange) and mixture 3a/Au-5 (purple), as well as B) prodrug 3m (grey) and mixture 3m/Au-5 (turquoise). Endoxifen (brown) was used as a control.