All-in-one disulfide bridging enables the generation of antibody conjugates with modular cargo loading

Antibody–drug conjugates (ADCs) are valuable therapeutic entities which leverage the specificity of antibodies to selectively deliver cytotoxins to antigen-expressing targets such as cancer cells. However, current methods for their construction still suffer from a number of shortcomings. For instance, using a single modification technology to modulate the drug-to-antibody ratio (DAR) in integer increments while maintaining homogeneity and stability remains exceptionally challenging. Herein, we report a novel method for the generation of antibody conjugates with modular cargo loading from native antibodies. Our approach relies on a new class of disulfide rebridging linkers, which can react with eight cysteine residues, thereby effecting all-in-one bridging of all four interchain disulfides in an IgG1 antibody with a single linker molecule. Modification of the antibody with the linker in a 1 : 1 ratio enabled the modulation of cargo loading in a quick and selective manner through derivatization of the linker with varying numbers of payload attachment handles to allow for attachment of either 1, 2, 3 or 4 payloads (fluorescent dyes or cytotoxins). Assessment of the biological activity of these conjugates demonstrated their exceptional stability in human plasma and utility for cell-selective cytotoxin delivery or imaging/diagnostic applications.


General Experimental Details
All solvents and reagents were used as received unless otherwise stated. Ethyl acetate, methanol, dichloromethane, acetonitrile and toluene were distilled from calcium hydride. Diethyl ether was distilled from a mixture of lithium aluminium hydride and calcium hydride. Petroleum ether (PE) refers to the fraction between 40 -60 °C upon distillation. Tetrahydrofuran was dried using Na wire and distilled from a mixture of lithium aluminium hydride and calcium hydride with triphenylmethane as indicator.
Non-aqueous reactions were conducted under a stream of dry nitrogen using oven-dried glassware. Temperatures of 0 °C were maintained using an ice-water bath. Room temperature (rt) refers to ambient temperature.
High resolution mass spectrometry (HRMS) measurements were recorded with a Micromass Q-TOF mass spectrometer or a Waters LCT Premier Time of Flight mass spectrometer. Mass values are reported within the error limits of ±5 ppm mass units. ESI refers to the electrospray ionisation technique.
Protein LC-MS was performed on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1 × 50 mm). H 2 O with 0.1% formic acid (solvent A) and 95% MeCN and 5% H 2 O with 0.1% formic acid (solvent B) were used as the mobile phase at a flow rate of 0.2 mL/min. The gradient was programmed as follows: 95% A for 0.93 min, then a gradient to 100% B over 4.28 min, then 100% B for 1.04 minutes, then a gradient to 95% A over 1.04 min. The electrospray source was operated with a capillary voltage of 2.0 kV and a cone voltage of 190 V. Nitrogen was used as the desolvation gas at a total flow of 850 L/h. Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (v4.2 from Waters) according to the manufacturer's instructions. Trastuzumab samples were deglycosylated with PNGase F (New England Biolabs) prior to LC-MS analysis.

Chemical Synthesis
Scheme S1. Synthesis of DVP 1.  These data are consistent with those previously reported. 1

O Br N H
A solution of propargylamine (1.16 mL, 18.2 mmol) in CH 2 Cl 2 (33 mL) and sat. NaHCO 3 (33 mL) at maximum stirring was cooled to -10 C and 2-bromoacetyl bromide (2.42 mL, 27.2 mmol) was added dropwise over 15 min. The reaction mixture was allowed to slowly reach rt, and upon completion the reaction mixture was concentrated. Following addition of water (30 mL), the aqueous solution was extracted with EtOAc (2x80 mL) and the combined organic phases were washed with sat. NaHCO 3 (30 mL), 5% HCl (30 mL) and brine (30 mL). Combined organic phases were dried over Na 2 SO 4 and concentrated in vacuo to give the title compound as a light yellow solid (2.97 g, 16.9 mmol, 93%).   The amine hydrochloride salt was re-dissolved in CH 2 Cl 2 (1 mL) and cooled to 0 °C. To this solution was added a solution of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid 1 (72. 8   In a pre-dried microwave vial, a suspension of 31 (184 mg, 0.21 mmol) and K 2 CO 3 (174 mg, 1.26 mmol) in anhydrous MeCN (0.26 mL) was cooled to 0 C by means of an ice-bath. A solution of 26 (anh. MeCN, 0.5 M, 1.58 mL) was slowly added to the stirring suspension, after which the reaction was brought to rt and stirred overnight under nitrogen. The reaction mixture was concentrated, re-dissolved in CH 2 Cl 2 (40 mL), the organic phase washed with water (2x10 mL), brine (20 mL), dried over Na 2 SO 4 , and purified by flash chromatography (5-10% MeOH/CH 2 Cl 2 ) to give the title compound as a white foam (119 mg, 0.102 mmol, 49%).

N O O Boc
A solution of diethanolamine (2.44 g, 23.0 mmol) in CH 2 Cl 2 (25 mL) was cooled to 0 C by means of an ice-bath and a solution of di-tert-butyl dicarbonate (6.33 g, 29.0 mmol) in anh. CH 2 Cl 2 (5 mL) as slowly added. The reaction was stirred for 12 h at rt, diluted in CH 2 Cl 2 (40 mL), added water (40 mL), and the aqueous phase was extracted twice with CH 2 Cl 2 (2x20 mL). The combined organic phases were washed with brine, dried over Na 2 SO 4 , and concentrated to give tert-butyl bis(2-hydroxyethyl)carbamate as a clear oil, which was used directly in the next step without further purification.
To a 50% NaOH (aq) solution (11 mL) was sequentially added tert-butyl bis(2hydroxyethyl)carbamate in toluene (11 mL), tetrabutylammonium bisulfate (16.0 mg, 47.0 mol), and propargyl bromide (80 w% in toluene, 8.70 mL, 78.0 mmol), and the reaction was stirred at rt for 48 h under nitrogen atmosphere. The organic layer was isolated, concentrated and the desired compound was purified by flash chromatography (50% EtOAc/hexane) to give the title compound as a yellow viscous oil (2.30 g, 7.57 mmol, 35% over 2 steps). R f 0.60 (SiO 2 , 50% EtOAc/hexane); ν max (neat/cm A solution of 33 (281 mg, 1.00 mmol) in CH 2 Cl 2 (1.5 mL) was cooled to 0 C, followed by addition of HCl (4 M in dioxane, 3 mL). The reaction was brought to rt and stirred for 2 h. The reaction mixture was concentrated to give the desired amine hydrochloride salt as a white solid.
The solid was re-suspended in MeCN (10 mL). Sodium carbonate (1.06 g, 10.0 mmol) was added. After stirring for 5 min, a solution of tert-Butyl (5-bromopentyl)carbamate (399 mg, 1.50 mmol) in MeCN (2 mL) was added. The reaction mixture was then refluxed at 70 °C for 48 h. Subsequently, the reaction mixture was diluted with brine and extracted with EtOAc (3x). The combined organic layers were dried over Na 2 SO 4 , concentrated, and purified by flash chromatography (30-50% EtOAc/hexane) to give the title compound as a pale yellow oil (242 mg, 66% over 2 steps).
The hydrochloride salt was re-dissolved in a mixture of CH 2 Cl 2 (1 mL) and sat. NaHCO 3 (1 mL) and cooled to -10 C, followed by dropwise addition of 2-bromoacetyl bromide (35.3 µL, 0.405 mmol). The reaction mixture was stirred for 1 h, and then diluted with brine and extracted with CH 2 Cl 2 (x3). Combined organic phases were dried over Na 2 SO 4 and concentrated in vacuo. Purification by flash chromatography (0-5% MeOH/CH 2 Cl 2 ) yielded the title compound as a pale yellow oil (57.3 mg, 0.148 mmol, 55% over 2 steps).

Reaction of trastuzumab with DVP 1
To a solution of trastuzumab (11.9 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of 1 (10 mM in DMSO) was added (final concentration of 340 μM, 20 equiv.) and the reaction mixture incubated at 37 °C for 4 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (7,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the bridged conjugate.

Reaction of trastuzumab with BisDVP 2
To a solution of trastuzumab (11.9 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of 2 (10 mM in DMSO) was added (final concentration of 170 μM, 10 equiv.) and the reaction mixture incubated at 37 °C for 4 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (7,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the bridged conjugate.

Reaction of trastuzumab with TetraDVP 3
To a solution of trastuzumab (198 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of 3 (10 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37 °C for 4 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the bridged conjugate.

Reaction of brentuximab with TetraDVP 3
To a solution of brentuximab (50 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of 3 (10 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37 °C for 2 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the bridged conjugate.   (Smaller proteins tend to ionise more strongly than big proteins under ESI ionisation conditions and thus appear as more abundant than they are.)

Reaction of trastuzumab with TetraDVP 7
To a solution of trastuzumab (198 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of 7 (10 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37 °C for 4 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the bridged conjugate. (Smaller proteins tend to ionise more strongly than big proteins under ESI ionisation conditions and thus appear as more abundant than they are.)

Reaction of trastuzumab with TetraDVP 8
To a solution of trastuzumab (198 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of 8 (10 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37 °C for 4 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the bridged conjugate. (Smaller proteins tend to ionise more strongly than big proteins under ESI ionisation conditions and thus appear as more abundant than they are.)

Reaction of trastuzumab with TetraDVP 9
To a solution of trastuzumab (198 μL, 17 μM, 2.5 mg/mL) in TBS (25 mM Tris HCl pH 8, 25 mM NaCl, 0.5 mM EDTA) was added TCEP (10 equiv.). The mixture was vortexed and incubated at 37 °C for 1 h. A solution of 9 (10 mM in DMSO) was added (final concentration of 34 μM, 2 equiv.) and the reaction mixture incubated at 37 °C for 4 h. The excess reagents were removed by use of a Zeba™ Spin Desalting Column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the bridged conjugate. (Smaller proteins tend to ionise more strongly than big proteins under ESI ionisation conditions and thus appear as more abundant than they are.)

Thermal stability
The thermal denaturation of proteins was monitored by differential scanning calorimetry (DSC), with a Malvern MicroCal VP-DSC instrument. 500 µL of protein at 0.5 mg mL -1 in 50 mM Tris pH 7.5 were used for each run. For each protein, the baseline was measured first, which consists of buffer in both cells (buffer versus buffer), followed by protein (buffer versus protein). Several clean-up cycles with water and suitability controls with lysozyme at 3 mg/mL in water were employed before, and after, the antibody experiment. Each protein sample was scanned twice to investigate the thermal reversibility. The temperature was ramped from 25 to 100 °C, increasing by 95 °C h -1 . The pre-scan thermostat was set to 2 min, no post-scan thermostat was employed. The data were processed with the Origin version 7.0 SR4 software. The baseline thermogram (buffer versus buffer) was subtracted from the thermogram of the protein (buffer versus protein). Two baselines, one at the beginning and one at the end of the thermogram, were placed to adjust the data, which was then normalized using the concentration of the protein. The unfolding peaks were selected and the thermogram was fitted to the "Non 2-state" model to obtain the melting temperatures (T m ) and the enthalpy of unfolding at the T m , . ∆

Thermodynamic stability
The thermodynamic stability of conjugation products was probed by chemical denaturation curves monitored by tryptophan fluorescence. The thermodynamic stability of each domain was determined for unmodified trastuzumab, TetraDVP conjugate 6, DVP conjugate 4, trastuzumab F(ab') 2 and TetraDVP-modified F(ab') 2 (produced as detailed above) by measuring the change in tryptophan fluorescence after incubation in various concentrations of guanidinium chloride (GdmCl). Each experiment was composed of forty-one points (120 μL total volume per point) of increasing concentrations of GdmCl from 0 to 3.7 M final. 110 μL of denaturant solution was mixed with 10 μL of protein to a final concentration of 1 μM. For the unfolding curves, the stock protein solution was made in 50 mM Tris pH 7.5. The solutions were dispensed with a liquid handling robot (Microlab®500 Series, ML541C, Hamilton Company). The denaturant solutions mixed with the protein were incubated at 25 °C at different time points until they reached equilibrium (7 days). Each of the forty-one denaturation points were measured in a 100 μL quartz cuvette (Hellma, Precision Cell in Quartz SUPRASIL®, Typ No: 105.250-QS, light path: 10x2 mm, centre: 20 mm). The fluorescence was recorded with a Cary 400 Eclipse Fluorescence Spectrophotometer (Agilent Technologies) thermostatted at 25 °C controlled by a heat block. The samples were excited at 280 nm, the emission was recorded from 300 to 400 nm, with a scan rate of 300 nm/min, excitation and emission band passes were set at 10 nm. The data were analyzed using an average emission wavelength (AEW), which is the arithmetic mean of the wavelengths weighted by the fluorescence intensity at each wavelength. It is calculated as shown in The following equation can be used normalize the native and denatured baselines: Eq. 1.4 The chemical denaturation curves of the full-length trastuzumab, TetraDVP conjugate 6 and DVP conjugate 4 clearly show that the additional multivalent linker has destabilised the protein, the modified variants unfolding at lower GdmCl concentrations than unmodified trastuzumab ( Figure S12A). This is reflected in, and can be attributed to, the destabilization of the native state of the Fab fragment and the C H 2 domain on conjugation ( Figure S12A-B). calculated for the single transition observed with the non-modified Fab ( Figure S12C). Given that the TetraDVP is covalently linked to the four canonical cysteines in the hinge and also to the two cysteines in each Fab arm originally forming the intrachain disulfide bridge between the heavy and light chains, it is unlikely that the linker has affected the stability of the variable domains which are distant from the conjugation sites. Thus, these data suggest that the TetraDVP linker has destabilised the constant domains within the Fab, i.e. the C L and C H 1 domains.

Kinetic stability
Unfolding kinetics provide information on the rate at which a native state of a domain unfolds. Native, folded protein was rapidly diluted into a range of chemical denaturant concentrations and the protein unfolding was monitored by the change in intrinsic fluorescence on a stopped-flow spectrophotometer. For these stopped-flow experiments, the native protein and the denaturant solutions were mixed in a 1:10 ratio respectively. Seven stock solutions of guanidinium chloride (GdmCl) were prepared in 50 mM Tris pH 7.5 so that the final concentrations range from 5.5 to 7.0 M GdmCl with an interval of 0.25 M. The stock solutions were 1.1-fold more concentrated than the final solutions, as solutions were diluted by a factor of 10/11 in the rapid mixing step, using 500 µL and 2.5 mL Hamilton syringes. Protein stock solutions of trastuzumab, DVP conjugate 4, TetraDVP conjugate 6, trastuzumab Fc, trastuzumab F(ab') 2 and TetraDVP-modified F(ab') 2 were prepared between 7 and 11 µM to achieve a final concentration between 0.5 to 1 µM after mixing with the denaturant solutions. The unfolding kinetics were monitored with a SX20 stopped-flow spectrometer from Applied Photophysics (software: SX Spectrometer Control Panel Application version 2.2.27). The temperature of the water bath was set to 25 °C, the excitation wavelength was set to 280 nm, both slit widths were 2 mm. A cut off filter of 320 nm was used. Three longer time traces (30 to 300s sec long depending on the unfolding rates) were acquired to measure the unfolding phases, both at each denaturant concentration.
The unfolding curves were fitted with the software Pro DataViewer version 4.2.27. Depending on the recorded traces and the number of domains unfolding, the fluorescence signal corresponding to the unfolding was fitted with a single (Equation 2.1), double-exponential (Equation 2.2) or triple-exponential function (Equation 2.3).

Eq. 2.3
where , and are the amplitudes, , and the respective unfolding rate  Figure S13 and Table S1). Overall, the unfolding kinetics data show that the modifications do not cause any significant change in kinetic stability. Error bars represent the standard deviation from triplicate measurements. In several instances, error bars are not visible because they are smaller than the size of the data points.

Biolayer interferometry (BLI)
Binding assays were performed on an OctetRED384 (ForteBio). All samples were prepared in DPBS (Sigma-Aldrich) with 0.1 % BSA (bovine serum albumin solution, 30% BSA in DPBS, sterile-filtered, BioXtra, Sigma-Aldrich) and 0.02% Tween 20 (TWEEN® 20 for molecular biology, Sigma-Aldrich). Serial dilutions were prepared for HER2: 0.9, 1.7, 3.4, 6.9 nM. Antihuman IgG Fc Capture (AHC) Biosensors (ForteBio) were used to immobilise trastuzumab conjugates at 32 nM. Before starting the BLI experiment, biosensors were incubated in DPBS with 0.1% BSA and 0.02% Tween 20 for 10 min. For the measurements, the biosensors were first incubated for one minute in DPBS with 0.1% BSA and 0.02% Tween 20 for the first baseline. Trastuzumab was then loaded onto the biosensors until reaching a displacement of 0.8-1 nm. The biosensors were then incubated in DPBS with 0.1% BSA and 0.02% Tween 20 for the second baseline for one minute, and subsequently moved to wells containing the serial dilutions of HER2 for the association step, and finally moved to DPBS with 0.1% BSA and 0.02% Tween 20 for the dissociation step. The association and dissociation results were fit to a 1:1 model using a global fitting.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS)
The HDX-MS experiments were conducted on full-length IgGs, unmodified, DVP and tetraDVP linked trastuzumab. For this experiment only, the unmodified trastuzumab was generated at AstraZeneca (expressed in CHO cells and purified on mAbSelect Sure columns, Cytiva -the validity of the use of this trastuzumab batch as the reference for the HDX-MS experiment was confirmed by the extremely high similarity of the deuterium exchange for peptides where no difference of exchange was observed between the unmodified, DVP and tetraDVP conjugated forms -see uptake plots). The peptide map was generated on trastuzumab in equilibration buffer 10 mM potassium phosphate pH 7. The peptide map was generated using BioPharmaFinder from equilibration data acquired with a MS 2 method. The recognition of the peptides was based in the fragmented b and y ions, to ensure confidence in the identification, and around the N-linked glycosylation in the C H 2 domain, the peptides including the asparagine at position 297 (in Eu numbering) were expected to carry the most abundant glycosylation expressed in CHO cells, i.e. G0F. The peptides obtained were filtered by confidence score higher than 80%. The exported csv file with the peptides as well as that same non-deuterated data were imported into HDExaminer, to operate a second filtration of the peptides: only the charge state with the highest intensity from the peptide map data was kept per peptide for comparative accuracy between the charge states, selected according to the highest intensity, the sharpest extracted ion chromatogram. After the peptide pool was curated, the labelled data were added, and the D incorporation per peptide data was then exported as a csv file. To identify which deuterium incorporations were significant and to observe the deuterium exchange for each time point separately and overall, the data processing method used was first described by Dobson

Size-Exclusion Chromatography (SEC) of TetraDVP conjugates
Size-exclusion chromatography (SEC) was carried out using a Superdex 200 10/300 GL column. Samples were injected at a concentration of 1 mg/mL and eluted with TBS buffer (25 mM Tris HCl pH 8, 200 mM NaCl, 0.5 mM EDTA) at a flow rate of 0.5 mL/min.
Fluorophore-to-antibody ratios (FAR) were determined by UV-vis spectroscopy. Sample buffer was used as blank for baseline correction with extinction coefficients ε 280 = 215,380 M -1 cm -1 for trastuzumab and ε 495 = 71,000 M -1 cm -1 for AlexaFluor 488™ (AF488). The correction factor for AF488 absorption at 280 nm is 0.11. FAR was calculated using the following formula:
Average DAR for each ADC was calculated as follows, where DAR n corresponds to the peak area at 280 nm for a given DAR species, with n representing the number of MMAE molecules per antibody for that DAR species.

Size-Exclusion Chromatography (SEC) of ADCs
Size-exclusion chromatography (SEC) was carried out on an Agilent 1200 Series system using a TSKgel G3000SWXL column (30 cm × 7.8 mm, 5 µm particle size) with a mobile phase of PBS (50 mM sodium phosphates, 100 mM NaCl, 0.02% sodium azide, pH 7.0) at a flow rate of 0.5 mL/min over 30 min. 10 µg of trastuzumab or ADC (1-2 mg/mL in PBS) was analysed per run. Samples were analysed via absorption at 280 nm.

Cell viability
Cells were seeded in 96-well plates for 24 h at 37 °C with 5% CO 2 . SKBR3 cells were seeded at 15,000 cells/well, BT474 cells were seeded at 20,000 cells/well, MCF7 cells were seeded at 7,500 cells/well and MDA-MB-468 cells were seeded at 10,000 cells/well. Serial dilutions of 17, 18, 19, 20 and trastuzumab were added to the cells in complete growth medium and incubated at 37 °C with 5% CO 2 for 96 h. Cell viability was measured using CellTiter-Glo viability assay (Promega) according to the manufacturer's instructions. Cell viability was plotted as a percentage of untreated cells. Each measurement was taken in triplicate and three independent repeats were performed.   (bis(2-(prop-2-yn-1-yloxy)