Next generation maleimides enable the controlled assembly of antibody–drug conjugates via native disulfide bond bridging

Highly homogeneous ADCs are generated by the efficient bridging of interchain disulfide bonds in trastuzumab, using next generation maleimides.


Synthesis General Remarks
All reactions were carried out at atmospheric pressure with stirring at 20 °C unless otherwise stated. Reagents and solvents were purchased from Sigma Aldrich and Alfa Aesar and used as supplied. Reactions were monitored by TLC analysis carried out on silica gel SIL G/UV254 coated onto aluminium plates purchased from VWR. Visualization was carried out under a UV lamp operating at 254 nm wavelength and by staining with a solution of potassium permanganate (3 g) and potassium carbonate (20 g) in 5% aqueous sodium hydroxide (5 mL) and water (200 mL), followed by heating. Flash column chromatography was carried out with silica gel 60 (0.04-0.063 mm, 230-400 mesh) purchased from Merck, using solvents dichloromethane (DCM), methanol (MeOH), ethyl acetate (EtOAc) and petroleum ether 40 °C -60 °C boiling range, purchased from Fisher Scientific. Nuclear magnetic resonance spectra were recorded in either CDCl 3 , MeOD-d 4 or DMSO-d 6 (unless another solvent is stated) on Bruker NMR spectrometers operating at ambient 20 °C probe. 1 H spectra were recorded at 400, 500 or 600 MHz and 13 C spectra were recorded at 125 or 150 MHz, using residual solvents as internal reference. Where necessary, DEPT135, COSY, HMQC, HMBC and NOESY spectra have been used to ascertain structure. Data is presented as follows for 1 H: chemical shift in ppm (multiplicity, J coupling constant in Hz, nº of H, assignment on structure); and on 13 C: chemical shift in ppm (assignment on structure). Multiplicity is reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), quint. (quintet), sext. (sextet), oct. (octet), m (multiplet), br (broad), dd (doublet of doublet), dt (doublet of triplets), ABq (AB quartet). Infrared spectra were recorded on a Perkin Elmer Spectrum 100 FTIR spectrometer operating in ATR mode. Melting points were measured on a Gallenkamp apparatus and are uncorrected. Experimental procedures for all isolated compounds are presented. All yields quoted are isolated yields, unless otherwise stated, and when multiple products are obtained, data are presented in terms of order isolated. Dibromomaleimide 1 was purchased from Sigma Aldrich and dithiophenolmaleimide 2 was prepared as reported by Schumacher et al. 1

Synthesis of compounds 3,4-Dibromo-maleimide-N-hexanoic acid 9
In a 10 mL round-bottom flask, 3,4-dibromomaleic anhydride (256 mg, 1.0 mmol), prepared as reported, 2 and 6-aminocaproic acid (131 mg, 1.0 mmol, 1 eq.) were added. Next, AcOH (2 mL) was added and the mixture was heated at 120 °C with stirring for 3 h. Then, the mixture was allowed to cool to 20 °C. AcOH was removed by concentrating under vacuum at 80 °C and traces of AcOH were removed by adding toluene (10 mL) and concentrating once more to yield a yellow white solid which was purified by flash chromatography on silica with petroleum ether:EtOAc (1:1 v/v) to afford 9 as a white solid (311 mg, 0.84 mmol, 84%). Data for 9:

N-(p-Benzoic acid)-3,4-dibromo-maleimide 10
In a 25 mL round-bottom flask, 3,4-dibromomaleic anhydride (1.02 g, 4.0 mmol), prepared as reported, 2 and p-amino benzoic acid (0.549 g, 4 mmol, 1 eq.) were added. Next, AcOH (12 mL) was added and the mixture was heated at 120 °C with stirring for 40 minutes. The product crashes out from solution in the meantime. Then, the mixture was allowed to cool to 20 °C and filtered. The filter cake was washed with cold MeOH (2 mL) and DCM and dried under vacuum to afford 10 as an off-yellow solid (

Maleimide-N-hexanoic acid 11 3, 4
In a 10 mL round-bottom flask, maleic anhydride (196 mg, 2.0 mmol) and 6-aminocaproic acid (262 mg, 2 mmol, 1 eq.) were suspended in AcOH (4 mL). Next, the mixture was heated to 120 °C, dissolving all reagents. The mixture was stirred at 120 °C for 3 h. Afterwards, cooled down to 20 °C and concentrated under vacuum, redissolved in toluene and concentrated once more to dryness, affording the crude as a yellow oil. The crude oil was purified by flash chromatography on silica with DCM:MeOH
Then, silica was added and the resulting mixture was stirred overnight. Next, the mixture was filtered and concentrated under vacuum to yield a yellow oil that was purified by column chromatography on silica gel 60 in petroleum ether:EtOAc

Conjugation of trastuzumab with NGM-DOX 3-5 by sequential protocol
To trastuzumab (22.9 μM, 300 μL, 0.0069 μmol) in borate buffer was added TCEP (22.9 mM, 1.8 μL, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. Next, NGM-DOX compound was prepared in dry DMF (9.16 mM) and added to the reduced trastuzumab (3.8 μL, 5 eq.). The concentration of DMF was corrected to 10% (v/v) and the reaction was incubated at 37 °C for 1 h. Afterwards, excess reagents were removed by ultrafiltration (10 kDa MWCO) with PBS to afford the modified trastuzumab ADC in PBS.

Conjugation of trastuzumab with M-DOX 6 by sequential protocol
To trastuzumab (22.9 μM, 300 μL, 0.0069 μmol) in borate buffer was added TCEP (22.9 mM, 0.6 μL, 2 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. Next, M-DOX 6 was prepared in dry DMF (9.16 mM) and added to the reduced trastuzumab (1.5 μL, 2 eq.). The concentration of DMF was corrected to 10% (v/v) and the reaction was incubated at 37 °C for 1 h. Afterwards, excess reagents were removed by ultrafiltration (10 kDa MWCO) with PBS to afford the modified trastuzumab ADC in PBS with yield 89%, DAR 3.7.

Conjugation of trastuzumab with NGM-DOX 3-5 by in situ protocol
To trastuzumab (22.9 μM, 300 μL, 0.0069 μmol) in borate buffer was added a 9.16 mM solution of NGM-DOX compound (3.8 μL, 5 eq.) in DMF and the amount of DMF was corrected to 10% (v/v). Next, added TCEP (22.9 mM, 1.8 μL, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. Afterwards, excess reagents were removed by ultrafiltration (10 kDa MWCO) with PBS to afford the modified trastuzumab ADC in PBS.

Conjugation of trastuzumab with compound 15 by in situ protocols, followed by coppercatalysed Huisgen 1,3-dipolar cycloaddition with compound 16
Trastuzumab (22.9 μM, 250 μL, 0.00573 μmol) in borate buffer was treated with one of the following protocols for targeting a specific DAR: DAR 1: Added DMF to a concentration of 12% (v/v) and the mixture was put on ice. Next, added benzeneselenol freshly prepared in dry DMF (22.9 mM, 2.5 μL, 10 eq.), followed by addition of NGM 15 in dry DMF (9.16 mM, 12.5 μL, 20 eq.) and corrected DMF to a concentration of 15% (v/v). The reaction was kept on ice for 1 h and purified by ultrafiltration (10 kDa MWCO). Yield 84%.

DAR 2:
Added DMF to a concentration of 12% (v/v) and the mixture was put on ice. Next, added benzeneselenol freshly prepared in dry DMF (22.9 mM, 2.5 μL, 10 eq.), followed by addition of NGM 15 in dry DMF (9.16 mM, 12.5 μL, 20 eq.) and corrected DMF to a concentration of 15% (v/v). The reaction was kept on ice for 30 minutes, after which added benzeneselenol freshly prepared in dry DMF (22.9 mM, 2.5 μL, 10 eq.). The reaction was kept on ice for another 30 minutes and purified by ultrafiltration (10 kDa MWCO). Yield 86%.

DAR 4:
Added NGM 15 in dry DMF (9.16 mM, 9.4 μL, 15 eq.) and corrected DMF to a concentration of 15% (v/v), followed by addition of TCEP in borate buffer (22.9 mM, 2.5 μL, 10 eq.). The reaction was incubated at 37 °C for 2 h under mild agitation after which the reaction was purified by ultrafiltration (10 kDa MWCO). Yield 86%.       Trastuzumab Fab was prepared through a reported protocol 10 and modified by an adaptation of the sequential and in situ protocols described above, using TCEP (3 eq.) and NGM-DOX 5 This instability of DOX has been previously reported. 10,11 Also, DAR values measured by UV-vis absorbance are in agreement with LCMS analysis, indicating that this is the likely cause for low DAR for conjugation with NGM-DOX 4 (Table S3)   Trastuzumab was modified using the pre-optimized conditions as described followed by copper-catalysed Huisgen 1,3-dipolar cycloaddition as described for various reaction times.