Name: 1334177-86-4|1-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oic acid|95%
Brand: Ambeed
SKU: A680616
Rating: 96 (22 reviews)

1
[ 1599447-65-0 ]
[ 1334177-86-4 ]
tert-butyl (11S)-8-(3-(((11S)-10-(tert-butoxycarbonyl)-11-((tert-butyldimethylsilyl)oxy)-2-(4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)phenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-((tert-butyldimethylsilyl)oxy)-2-cyclopropyl-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
82%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 1h;Inert atmosphere;	(ii) tert-butyl (11S)-8-(3-(((11S)-10-(tert-butoxycarbonyl)-11-((tert-butyldimethylsilyl)oxy)-2- (4-((2S, 5S)-37-(2, 5-dioxo-2, 5-dihydro- 1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4, 7, 35-trioxo- 10, 13, 16, 19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)phenyl)-7-methoxy-5- oxo-5, 10, 11, 11 a-tetrahydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8-yl)oxy)propoxy)-11- ((tert-butyldimethylsilyl)oxy)-2-cyclopropyl-7-methoxy-5-oxo-11, 11a-dihydro-1H- benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepine-10(5H)-carboxylate (55) In a dry round bottom flask previously flushed three times with argon, dimer 54 (270 mg, 0.021 mmol) was solubilised in dry CH2CI2 (6 mL). EDCI hydrochloride (40 mg, 0.021 mmol) and maleimide-PEG8-OH (123 mg, 0.021 mmol) were subsequently added to the solution which was left to stir at room temperature until complete (=1 hour, followed by LCMS). Upon completion, the reaction was diluted with CH2CI2 (50 mL) and the organic phase was washed with H20 (50 mL) and brine (50 mL) before being dried over MgS04, filtered and the volatiles removed by rotary evaporation under reduced pressure. The crude material was purified by silica gel chromatography (CHCI3/MeOH 100% to 97:3) and pure product 55 was isolated as a light yellow foam (318.8 mg, 82% yield). 1H-NMR (400 MHz, CDCI3) 8.64 (s, 1 H), 7.69 (d, J = 15.0 Hz, 2H), 7.39 (s, 1 H), 7.28 (d, J = 15.0 Hz, 2H), 7.26, (s, 1 H), 7.22 (s, 1 H), 7.20 (s, 1 H),7.03 (d, J = 4.5 Hz, 1 H), 6.92 (d, J = 7.5 Hz, 1 H), 6.69 (s, 2H), 6.65 (s, 1 H), 6.63 (s, 1 H), 6.37 (t, J = 4.7 Hz, 1 H), 5.89 (d, J = 6.8 Hz, 1 H), 5.81 (d, J = 8.4 Hz, 1 H), 4.67 (p, J = 7.2 Hz, 1 H), 4.27 - 4.12 (m, 5H), 3.88 (s, 3H), 3.87 (s, 3H), 3.84 (t, J = 5.6 Hz, 1 H), 3.73 - 3.56 (m, 46H), 3.53 (t, J = 5.0 Hz, 1 H), 3.41 (dd, J = 10.3, 5.2 Hz, 1 H), 3.35 - 3.22 (m, 1 H), 2.90 - 2.74 (m, 1 H), 2.67 (ddd, J = 13.6, 9.2, 4.1 Hz, 1 H), 2.52 (t, J = 7.2 Hz, 1 H), 2.48 - 2.45 (m, 1 H), 2.45 - 2.37 (m, 1 H), 2.37 - 2.22 (m, 1 H), 2.02 (t, J = 9.0 Hz, 1 H), 1.45 (d, J = 7.1 Hz, 3H), 1 .42 - 1.37 (m, 1 H), 1.30 (s, 9H), 0.99 (s, 6H), 0.89 (s, 9H), 0.86 (s, 9H), 0.78 - 0.67 (m, 2H), 0.58 - 0.49 (m, 1 H), 0.48 - 0.42 (m, 1 H), 0.28 - 0.20 (m, 12H) ; ES+ = 2.15 min, m/z 1891.60 [M+Na]+.

Reference： [1]Patent: WO2014/57073,2014,A1 .Location in patent: Page/Page column 149-151

2
[ 1595275-00-5 ]
[ 1334177-86-4 ]
[ 1428523-12-9 ]

Yield	Reaction Conditions	Operation in experiment
33%		[0477] Starting material 17 (154 mg, 0.185 mmol) and EDCI.HC1 (110 mg, 0.185 mmol) were solubilised in dry CH2CI2 (5 mL) in a round bottom flask purged and filled with argon. The mixture was left to stir at room temperature for 1 hour before PEGs-maleimide (35.6 mg, 0.185 mmol) was added and the reaction mixture stirred for a further 16 hours (or until the reaction is complete, monitered by LC/MS). The reaction solution was diluted with CH2CI2 (50 mL) and the organics were washed with 0 (50 mL) and brine (50 mL) before being dried with MgS04, filtered and the solvent removed by rotary evaporation under reduced pressure to afford the crude product. Purification on silica gel column chromatography (100% CHCI3 to 85/15 v/v CHCls/MeOH) gave the desired product (135mg), however remaining traces of unreacted PEGs- maleimide were observed (by LC/MS, 2.21 min, method 2). Automated reverse phase silica gel chromatography (H2O/CH3CN) (see general information for conditions) successfully removed the impurity affording pure final product (18, 37mg of pure product starting from l lOmg, 33%). Overall yield = 17%. Method 2 LC/MS (2.58 min (ES+) m/z (relative intensity) 1404.03 ([Mu+Eta]+·, 20), 702.63 (100)). XH NMR (400 MHz, CDC13) delta 7.91 (t, J = 3.5 Hz, 1H), 7.80 (d, J = 4.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.54 - 7.50 (m, 2H), 7.45 (s, 1H), 7.39 - 7.31 (m, 2H), 6.87 (d, J = 10.5 Hz, 2H), 6.76 (s, 1H), 6.72 - 6.68 (m, 2H), 4.74 - 4.62 (m, 1H), 4.45 - 4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67 - 3.58 (m, 34H), 3.54 (m, 2H), 178 3.42 (dd, / = 10.2, 5.2 Hz, 2H), 3.16 - 3.07 (m, IH), 2.92 (dd, J = 16.1, 4.1 Hz, IH), 2.62 - 2.49 (m, 4H), 2.48 - 2.39 (m, 2H), 2.37 - 2.25 (m, IH), 1.92 (s, IH), 1.52 - 1.44 (m, 3H), 1.10 - 0.93 (m, 6H), 0.79 (dd, / = 9.2, 5.3 Hz, 2H), 0.57 (dd, / = 9.2, 5.3 Hz, 2H), NH were not observed.
21.8 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 6h;Inert atmosphere;	(g) N-((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5, 11 a-dihydro-1 H- benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5, 11 a-dihydro-1 H- benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)ami oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)- 3, 6,9, 12, 15, 18,21,24-octaoxaheptacosan-27-amide (18) EDCI hydrochloride (14 mg, 0.0732 mmol) was added to a suspension of Maleimide-PEG8- acid (43.4 mg, 0.0732 mmol) in dry CH2CI2 (5 ml.) under argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 17 (60.7 mg, 0.0732 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9: 1 CHCI3/MeOH) followed by reverse phase chromatography to remove unreacted maleimide- PEGe-acid. The product 18 was isolated in 17.6% (21 .8 mg). LC/MS 2.57 min (ES+) m/z (relative intensity) 1405.30 ; 1 H NMR (400 MHz, CDCI3) delta 7.91 (t, J = 3.5 Hz, 1 H), 7.80 (d, J = 4.0 Hz, 1 H), 7.75 (d, J = 8.8 Hz, 1 H), 7.69 (d, J = 8.7 Hz, 1 H), 7.54 - 7.50 (m, 2H), 7.45 (s, 1 H), 7.39 - 7.31 (m, 2H), 6.87 (d, J = 10.5 Hz, 2H), 6.76 (s, 1 H), 6.72 - 6.68 (m, 2H), 4.74 - 4.62 (m, 1 H), 4.45 - 4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67 - 3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J = 10.2, 5.2 Hz, 2H), 3.16 - 3.07 (m, 1 H), 2.92 (dd, J = 16.1 , 4.1 Hz, 1 H), 2.62 - 2.49 (m, 4H), 2.48 - 2.39 (m, 2H), 2.37 - 2.25 (m, 1 H), 1 .92 (s, 1 H), 1 .52 - 1 .44 (m, 3H), 1 .10 - 0.93 (m, 6H), 0.79 (dd, J = 9.2, 5.3 Hz, 2H), 0.57 (dd, J = 9.2, 5.3 Hz, 2H), NH were not observed.
21.8 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 1h;Inert atmosphere;	EDCI hydrochloride (14 mg, 0.0732 mmol) was added to a suspension of Maleimide-PEG8- acid (43.4 mg, 0.0732 mmol) in dry CH2CI2 (5 mL) under argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 8 (60.7 mg, 0.0732 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9:1 CHCI3/MeOH) followed by reverse phase chromatography to remove unreacted maleimide- PEG8-acid. The product 9 was isolated in 17.6% (21 .8 mg). LC/MS 2.57 min (ES+) m/z (relative intensity) 1405.30 ; 1H NMR (400 MHz, CDCI3) delta 7.91 (t, J = 3.5 Hz, 1 H), 7.80 (d, J = 4.0 Hz, 1 H), 7.75 (d, J = 8.8 Hz, 1 H), 7.69 (d, J = 8.7 Hz, 1 H), 7.54 - 7.50 (m, 2H), 7.45 (s, 1 H), 7.39 - 7.31 (m, 2H), 6.87 (d, J = 10.5 Hz, 2H), 6.76 (s, 1 H), 6.72 - 6.68 (m, 2H), 4.74 - 4.62 (m, 1 H), 4.45 - 4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67 - 3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J = 10.2, 5.2 Hz, 2H), 3.16 - 3.07 (m, 1 H), 2.92 (dd, J = 16.1 , 4.1 Hz, 1 H), 2.62 - 2.49 (m, 4H), 2.48 - 2.39 (m, 2H), 2.37 - 2.25 (m, 1 H), 1.92 (s, 1 H), 1.52 - 1.44 (m, 3H), 1 .10 - 0.93 (m, 6H), 0.79 (dd, J = 9.2, 5.3 Hz, 2H), 0.57 (dd, J = 9.2, 5.3 Hz, 2H), NH were not observed.

		(f) N-((R)- 1-(((S)- 1-((4-((S) -8-(3-(((S)-2-cyclopropyl- 7-met hoxy-5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-8-yI) oxy) pro poxy) - 7-met hoxy-5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-2-yI)phenyl)amino) - 1-oxopropan-2-yI)amino)-3-methyl- 1- oxobutan-2-yI) - 1-(3-(2, 5-dioxo-2, 5-dihydro- 1H-pyrrol- 1-yI)propanamido)-3,6,9, 12,15, 18,21, 24-octaoxaheptacosan-27-amide (18)Starting material 17(154mg, 0.185 mmol) and EDCI.HCI (110 mg, 0.185 mmol) were solubilised in dry CH2CI2 (5 mL) in a round bottom flask purged and filled with argon. The mixture was left to stir at room temperature for 1 hour before PEG8-maleimide (35.6 mg, 0.185 mmol) was added and the reaction mixture stirred for a further 16 hours (or until the reaction is complete, monitered by LC/MS). The reaction solution was diluted with CH2CI2 (50 mL) and the organics were washed with H20 (50 mL) and brine (50 mL) before being dried with Mg504, filtered and the solvent removed by rotary evaporation under reduced pressure to afford the crude product. Purification on silica gel column chromatography (100% CHCI3 to 85/15 v/v CHCI3/MeOH) gave the desired product (1 35mg), however remaining traces of unreacted PEG8-maleimide were observed (by LC/MS, 2.21 mm, method 2). Automated reverse phase silica gel chromatography (H20/CH3CN) (see general information for conditions) successfully removed the impurity affording pure final product (18, 37mg of pure product starting from 110mg, 33%). Overall yield = 17%. Method 2 LC/MS (2.58 mm (ES+) m/z (relative intensity) 1404.03 ([M+H], 20), 702.63 (100)). 1H NMR (400 MHz, ODd3) O 7.91 (t, J= 3.5 Hz, 1H), 7.80 (d, J= 4.0 Hz, 1H), 7.75 (d, J= 8.8 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1 H), 7.54 - 7.50 (m, 2H), 7.45 (s, 1 H), 7.39 - 7.31 (m, 2H), 6.87 (d, J =10.5 Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H),3.95 (s, 3H), 3.94 (s, 3H), 3.67 - 3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J = 10.2, 5.2 Hz,2H), 3.16-3.07(m, 1H), 2.92 (dd, J= 16.1, 4.1 Hz, 1H), 2.62-2.49(m, 4H), 2.48-2.39(m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79(dd, J = 9.2, 5.3 Hz, 2H), 0.57 (dd, J = 9.2, 5.3 Hz, 2H), NH were not observed.
	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 1h;Inert atmosphere;	(g) N-((2S)- 1-(((2S)- 1-((4-(8-(3-((2-cyclopropyl- 7-methoxy-5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-8-yI) oxy)propoxy) - 7-met hoxy-5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-2-yI)phenyl)amino) - 1-oxopropan-2-yI)amino)-3-methyl- 1- oxobutan-2-yI) - 1-(3-(2, 5-dioxo-2, 5-dihydro- 1H-pyrrol- 1-yI)propanamido)-3,6,9, 12,15, 18,21, 24-octaoxaheptacosan-27-amide (18)EDCI hydrochloride (14 mg, 0.0732 mmol) was added to a suspension of Maleimide-PEG8- acid (43.4 mg, 0.0732 mmol) in dry CH2CI2 (5 mL) under argon atmosphere. The mixture was stirred for 1 hour at room temperature before PBD 17 (60.7 mg, 0.0732 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over Mg504, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9:1 CHCI3/MeOH) followed by reverse phase chromatography to remove unreacted maleimidePEG 8-acid. The product 18 was isolated in 17.6% (21.8 mg). LC/MS 2.57 mm (ES+) m/z (relative intensity) 1405.30; 1H NMR (400 MHz, ODd3) O 7.91 (t, J= 3.5 Hz, 1H), 7.80 (d, J = 4.0 Hz, 1H), 7.75 (d, J= 8.8 Hz, 1H), 7.69 (d, J= 8.7 Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (5, 1 H), 7.39- 7.31 (m, 2H), 6.87 (d, J = 10.5 Hz, 2H), 6.76 (5, 1 H), 6.72 -6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (5, 3H), 3.94 (5, 3H), 3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J= 10.2, 5.2 Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J= 16.1, 4.1 Hz, 1H),2.62 -2.49 (m, 4H), 2.48 -2.39 (m, 2H), 2.37 - 2.25 (m, 1 H), 1.92 (5, 1 H), 1.52 - 1.44 (m, 3H), 1.10-0.93(m, 6H), 0.79 (dd, J= 9.2, 5.3 Hz, 2H), 0.57(dd, J= 9.2, 5.3 Hz, 2H), NH were not observed.

Reference： [1]Patent: WO2015/155345,2015,A1 .Location in patent: Paragraph 0477
[2]Patent: WO2014/57073,2014,A1 .Location in patent: Page/Page column 123; 127
[3]Patent: WO2014/57072,2014,A1 .Location in patent: Page/Page column 46; 47
[4]Patent: WO2015/159076,2015,A1 .Location in patent: Page/Page column 157; 160
[5]Patent: WO2015/159076,2015,A1 .Location in patent: Page/Page column 162; 166

3
[ 1334177-86-4 ]
[ 1595275-37-8 ]
[ 1428523-14-1 ]

Yield	Reaction Conditions	Operation in experiment
18%	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; chloroform; at 20℃; for 96h;Inert atmosphere;	The imine 32 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHC13 (6 mL) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG8-acid (53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14 mmol, 1.5 equiv.). This wasleft to stir vigorously at room temperature under Ar for 4 days until LC/MS analysis showed majority product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHC13 with 1% to 10% MeOH gradient) yielding 33 (8 1mg). The material was purified further by preparative HPLC to give 33 as a yellow solid (26.3 mg, 18%). Fast Formic run: LC/MS (1.39 mm(ES+) mlz (relative intensity) 1485.00 ([M + H]+., 64).
26.3 mg	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; chloroform; at 20℃; for 96h;Inert atmosphere;	(e) N-((S)- 1-(((S)- 1-((4-((S)-8-(3-(((S)-2-(Benzo[d][1, 3]dioxol-5-yl)-7-methoxy-5-oxo-5, 11a- dihydro-1H-pyrrolo[2, 1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5, 11a- dihydro-1H-pyrrolo[2, 1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3- methyl- 1 -oxobutan-2-yl)- 1-(3-(2, 5-dioxo-2, 5-di hydro- 1H-pyrrol- 1 -yl)propanamido)- 3,6,9, 12, 15, 18,21 ,24-octaoxaheptacosan-27-amide (33) The imine 32 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHCI3 (6 mL) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG8-acid (53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14 mmol, 1 .5 equiv.). This was left to stir vigorously at room temperature under Ar for 4 days until LC/MS analysis showed majority product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHCI3 with 1 % to 10% MeOH gradient) yielding 33 (81 mg). The material was purified further by preparative HPLC to give 33 as a yellow solid (26.3 mg, 18%). Fast Formic run: LC/MS (1.39 min (ES+) m/z (relative intensity) 1485.00 ([M + H]+., 64).
26.3 mg	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; chloroform; at 20℃; for 96h;Inert atmosphere;	The imine 17 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHCI3 (6 ml.) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG8-acid (53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14 mmol, 1 .5 equiv.). This was left to stir vigorously at room temperature under Ar for 4 days until LC/MS analysis showed majority product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHCI3 with 1 % to 10% MeOH gradient) yielding 18 (81 mg). The material was purified further by preparative HPLC (method 2) to give 18 as a yellow solid (26.3 mg, 18%). Fast Formic run: LC/MS (method 3)(1 .39 min (ES+) m/z (relative intensity) 1485.00 ([M + H]+., 64).

26.3 mg	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; chloroform; at 20℃; for 96h;Inert atmosphere;	[0496] The imine 32 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHCI3 (6 mL) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEGs-acid (53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14 mmol, 1.5 equiv.). This was left to stir vigorously at room temperature under Ar for 4 days until LC/MS analysis showed majority product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHC13 with 1% to 10% MeOH gradient) yielding 33 (81mg). The material was purified further by preparative HPLC to give 33 as a yellow solid (26.3 mg, 18%). Fast Formic run: LC/MS (1.39 min (ES+) m/z (relative intensity) 1485.00 ([M + H]+., 64).
81 mg	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In methanol; chloroform; at 20℃; for 96h;Inert atmosphere;	(e) N-((S)- 1-(((S)- 1-((4-((S)-8-(3-(((S) -2-(Benzo[d][1, 3]dioxol-5-yI) - 7-methoxy-5-oxo-5, 1 ladihydro- 1H-pyrrolo[2, 1-c][1, 4]benzodiazepin-8-yI) oxy) propoxy)- 7-methoxy-5-oxo-5, 1 ladihydro- 1H-pyrrolo[2, 1-c][1, 4]benzodiazepin-2-yI)phenyl)amino)- 1-oxopropan-2-yI)amino)-3- methyl- 1-oxobutan-2-yl)- 1-(3-(2, 5-dioxo-2, 5-dihydro- 1H-pyrrol- 1-yl)propanamido)- 3,6,9, 12,15, 18,21, 24-octaoxaheptacosan-2 7-amide (33)The imine 32 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHCI3 (6 mL) with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG8-acid (53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14 mmol, 1.5 equiv.). This was left to stir vigorously at room temperature under Ar for 4 days until LC/MS analysis showed majority product formation. The solvent was removed in vacuo and the crude product was partially purified by silica gel column chromatography (CHCI3 with 1% to 10% MeOH gradient) yielding 33 (81mg). The material was purified further by preparative HPLC to give 33 as a yellow solid (26.3 mg, 18%). Fast Formic run: LC/MS (1.39 mm (ES+) mlz (relative intensity) 1485.00 ([M + H]+., 64).

Reference： [1]Patent: WO2014/130879,2014,A2 .Location in patent: Page/Page column 133; 136
[2]Patent: WO2014/57073,2014,A1 .Location in patent: Page/Page column 132; 134
[3]Patent: WO2014/57072,2014,A1 .Location in patent: Page/Page column 53
[4]Patent: WO2015/155345,2015,A1 .Location in patent: Paragraph 0496
[5]Patent: WO2015/159076,2015,A1 .Location in patent: Page/Page column 171; 173

4
[ 1595275-15-2 ]
[ 1334177-86-4 ]
[ 1428523-12-9 ]
[ 1616467-39-0 ]
[ 1616467-40-3 ]
[ 1616467-41-4 ]

Yield	Reaction Conditions	Operation in experiment
2 mg; 3.8 mg; 2.7 mg; 2.2 mg		A solution of Super-Hydride (95 mu, 1 eq, 1 M in THF) was added dropwise to a solution of bis imine 7 (100 mg, 0.095 mmol) in THF (10 mL) at -78C under an argon atmosphere. After 20 minutes, an aliquot was quenched with water for LC/MS analysis, which revealed that the reaction was complete, with some over-reduction towards the bis amine 10.(Observed LC: Bis-imine 7 19%, Imine-Amine 8 + 9 36%, bis-amine 10 45%; theoretical target for 1 eq of reducing agent is 7 25%, 8+9 50%, 10 25%). Water (20 mL) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with chloroform (40 mL) and the combined organics were washed with water (1 x 40 mL), brine (50 mL), dried with Na2S04, filtered and the solvent removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (100% CHCI3 to 96% CHCI3/ 4% MeOH) improved the ratio of 8+9 to: 7 25%, 8+9 50%, 10 25% (20 mg, 25%, as a mixture). LC/MS (Fast Formic, 2.5 min system) Bis-lmine 7 1 .66 min (ES+) m/z (relative intensity) 1052.15 ; Hybrids Amine-lmine 8+9 (no separation on the 2.5 min system) 1.71 min (ES+) m/z (relative intensity) 1054.45; Bis-Amine 10 1.66 min (ES+) m/z (relative intensity) 1056.95, in a 1/2/1 ratio. (g) Fmoc deprotection Excess piperidine was added (0.1 mL, 1 mmol) to a 1/2/1 mixture of Fmoc protected 7, 8+9 and 10 (20 mg, 0.019 mmol) in DMF (1 mL). The mixture was allowed to stir at room temperature for 20 minutes, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH2CI2 (30 ml.) and the organic phase was washed with H20 (2x30 ml.) until complete piperidine removal. The organic phase was dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude products 11 , 12 + 13 and 14 (1/2/1 ratio), which were used as such in the next step. LC/MS (Fast Formic, 2.5 min system) Bis-lmine 11 1.12 min (ES+) m/z (relative intensity) 830.45 ; Hybrids Amine-lmine 12+13 (no separation) 1 .15 min (ES+) m/z (relative intensity) 832.35; Bis-Amine 14 1.19 min (ES+) m/z (relative intensity) 834.35, in a 1/2/1 ratio. Example 2 PEG maleimide coupling EDCI hydrochloride (5.46 mg, 0.028 mmol, 1 .5 eq) was added to a suspension ofMaleimide-PEGs-acid (1 1 .3 mg, 0.019 mmol, 1 eq) in chloroform (5 mL) under argon atmosphere. The mixture was stirred for 1 h at room temperature before crude PBD mixture 11 +12+13+14 (20 mg, 0.019 mmol, 1 eq) was added. Stirring was maintained until the reaction was complete (usually 2 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure. The four products were purified and separated individually by reverse phase chromatography (see method below). The bis-imine 15 was isolated (2.0 mg, 7.5%), followed by the two separable amine-imine hybrids 16 (3.8 mg, 14.2%) and 17 (2.7 mg, 10.1%), and the bis-amine 18 (2.2 mg, 8.2%).1H NMR analysis unambiguously identified 16 from 17. One key feature is the imine proton (d, J = 4.0 Hz, 1 H), which is at 7.78 ppm on the cyclopropyl side, and 7.88 ppm on the aromatic side of the molecule.LC/MS C1815 min formic:Bis-lmine 155.23 min (ES+) m/z (relative intensity) 703.20 (100, (M+2H)/2), 1404.55 (10, M+H); 1H NMR (400 MHz, CDCI3) delta 8.96 (s, 1 H), 7.89 (d, J = 4.0 Hz, 1 H), 7.78 (d, J = 4.0 Hz, 1H), 7.77 - 7.64 (m, 2H), 7.53 - 7.47 (m, 2H), 7.44 (s, 1H), 7.33 (d, J = 8.7 Hz, 2H), 7.12 (s, 1 H), 6.89 - 6.81 (m, 2H), 6.74 (s, 1 H), 6.69 (d, J = 1.6 Hz, 2H), 6.50 (s, 1 H), 4.72 - 4.57 (m, 1 H), 4.43 - 4.14 (m, 7H), 4.11 - 4.03 (m, 1 H), 3.93 (d, J = 4.6 Hz, 6H), 3.83 (t, J = 7.2 Hz, 2H), 3.80 - 3.71 (m, 2H), 3.68 - 3.56 (m, 28H), 3.55 - 3.48 (m, 3H), 3.44 - 3.34(m, 3H), 3.16-3.04 (m, 1H), 2.95-2.84 (m, 1H), 2.58-2.47 (m, 4H), 2.47-2.37 (m, 2H), 2.33-2.16 (m, 1H), 1.51 - 1.41 (m, 4H), 1.05-0.94 (m, 6H), 0.77 (dt, J = 5.5, 4.8 Hz, 2H), 0.55 (dd, J = 9.3, 4.9 Hz, 2H). Hybrid Amine-imine 165.48 min (ES+) m/z (relative intensity) 704.20 (100, (M+2H)/2), 1406.70 (5, M+H); 1H NMR (400 MHz, CDCI3) delta 8.97 (s, 1H), 7.88 (d, J = 3.9 Hz, 1H), 7.78 - 7.64 (m, 2H), 7.55 - 7.48 (m, 2H), 7.43 (s, 1 H), 7.36 - 7.29 (m, 2H), 7.12 (s, 1 H), 6.93 - 6.80 (m, 3H), 6.69 (d, J= 1.7 Hz, 2H), 6.49 (s, 1H), 6.10 (s, 1H), 4.72-4.58 (m, 1H), 4.41 -4.32 (m, 1H), 4.32-4.24 (m, 2H), 4.24-4.16 (m, 2H), 4.15-4.02 (m, 2H), 3.94 (s, 3H), 3.87 - 3.80 (m, 5H), 3.79 - 3.71 (m, 2H), 3.69 - 3.55 (m, 28H), 3.55 - 3.49 (m, 2H), 3.46 - 3.34 (m, 4H), 2.93 - 2.81 (m, 2H), 2.60 - 2.46 (m, 4H), 2.42 - 2.34 (m, 2H), 2.24 (dd, J = 14.0, 6.5 Hz, 2H), 1.52 - 1.38 (m, 4H), 1.07 - 0.92 (m, 6H), 0.75 - 0.66 (m, 2H), 0.53 - 0.44 (m, 2H). Hybrid Imine-Amine 175.41 min (ES+) m/z (relative intensity) 704.25 (100, (M+2H)/2), 1406.45 (3, M+H); 1H NMR (400 MHz, CDCI3) delta 8.90 (s, 1 H), 7.78 (d, J = 4.0 Hz, 1 H), 7.74 - 7.58 ...

Reference： [1]Patent: WO2014/96365,2014,A1 .Location in patent: Page/Page column 204-207

5
[ 1334177-86-4 ]
C₇₁H₈₇N₇O₁₄Si [ No CAS ]
[ 1616467-44-7 ]

Yield	Reaction Conditions	Operation in experiment
58%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃;Inert atmosphere;	To a solution of 34 (337mg, 0.31 mmol) in dry CH2CI2 (5 mL) was added the PEG moiety (186 mg, 0.31 mmol) and EDCI.HCI (60 mg, 0.31 mmol). The mixture was stirred at room temperature under an atmosphere of argon until completion. The mixture wassubsequently diluted with CH2CI2 (50 mL) and washed with H20 (50 mL) and brine (50 mL) before removing the volatiles in vacuo. . The crude material was purified by silica gel column chromatography (CHCI3/MeOH ; 100% to 95:5) to afford pure product 35 as a light yellow foam (408.8 mg, 58% yield). Analytical Data: RT 1 .75 min; MS (ES+) m/z (relative intensity) 1643.15 [M + H]+ , 10) 822.25 ([M + 2H]2+, 100).

Reference： [1]Patent: WO2014/96365,2014,A1 .Location in patent: Page/Page column 216

6
[ 1334177-86-4 ]
(11S)-4-(2-(1-((1-amino-3-methyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl 11-hydroxy-7-methoxy-8-((5-((7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
(11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1h-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-7-methoxy-8-((5-(((s)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)-oxy)-2-methyl-5-oxo-11,11a-dihydro-1h-benzo[e]pyrrolo-[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
25%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 2h;	1-ethyl-3-(3?-dimethylaminopropyl)carbodiimide (EDCI, 33 mg, 0.172 mmol) was added to a solution of crude 63 (0.172 mmol) and Mal-(PEG)8-acid (100 mg, 0.172 mmol) in dry dichloromethane (10 mL). The reaction was stirred for 2 hours and the presence ofstarting material was no longer observed by LC/MS. The reaction was diluted withdichloromethane and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 100% chloroform to 10% methanol in chloroform). Pure fractions were collected and combined and excess eluent was removed by rotaryevaporation under reduced pressure to give 64 or DL5 (60 mg, 25% over 3 steps).
60 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 2h;	l-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDCI, 33 mg, 0.172 mmol) was added to a solution of crude 63 (0.172 mmol) and Mal-(PEG)g-acid (100 mg, 0.172 mmol) in dry dichloromethane (10 mL). The reaction was stirred for 2 hours and the presence ofstarting material was no longer observed by LC/MS. The reaction was diluted with dichloromethane and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 100% chloroform to 10% methanol in chloroform). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give 64 (E) (60 mg, 25% over 3 steps).

Reference： [1]Patent: WO2014/130879,2014,A2 .Location in patent: Page/Page column 152; 154
[2]Patent: WO2015/155345,2015,A1 .Location in patent: Paragraph 0522
[3]Organic Process Research and Development,2018,vol. 22,p. 1241 - 1256

7
[ 1595275-00-5 ]
[ 1334177-86-4 ]
N-((R)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
17.6%		EDCI hydrochloride (14 mg, 0.0732 mmol) was added to a suspension ofMaleimide-PEG8-acid (43.4 mg, 0.0732 mmol) in dry CH2C12 (5 mL) under argonatmosphere. The mixture was stirred for 1 hour at room temperature before PBD 17 (60.7 mg, 0.0732 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH2C12 and the organic phase was washed with H20 and brine before being dried over MgSO4, filtered and excess solvent removedby rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHC13 up to 9:1 CHC13/MeOH) followed by reverse phase chromatography to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 18 (DL1) was isolated in 17.6% (21.8 mg). LC/MS 2.57 mm (ES+) m/z (relative intensity) 1405.30 ; ?HNMR (400 MHz, CDC13) oe 7.91 (t, J 3.5 Hz, 1H), 7.80 (d, J 4.0 Hz, 1H), 7.75 (d, J8.8 Hz, 1H), 7.69 (d, J 8.7 Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H), 7.39-7.31 (m,2H), 6.87 (d, J = 10.5 Hz, 2H), 6.76 (s, 1H), 6.72 - 6.68 (m, 2H), 4.74 - 4.62 (m, 1H),4.45 - 4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67 - 3.58 (m, 34H), 3.54 (m, 2H), 3.42(dd, J 10.2, 5.2 Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J= 16.1, 4.1 Hz, 1H), 2.62- 2.49 (m, 4H), 2.48 - 2.39 (m, 2H), 2.37 - 2.25 (m, 1H), 1.92 (s, 1H), 1.52 - 1.44 (m, 3H),1.10-0.93 (m, 6H), 0.79 (dd, J= 9.2, 5.3 Hz, 2H), 0.57 (dd, J= 9.2, 5.3 Hz, 2H), NH were not observed.

Reference： [1]Patent: WO2014/130879,2014,A2 .Location in patent: Page/Page column 119; 128; 129

8
[ 1334177-86-4 ]
(S)-2-amino-N-((S)-1-((4-((R)-7-methoxy-8-(3-(((R)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4] benzodiazepin8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide [ No CAS ]
[ 1428523-15-2 ]

Yield	Reaction Conditions	Operation in experiment
	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid(25 mg, 0.042 mmol) in dry CH2CI2 (4 mL) under argon atmosphere. PBD 19 (42 mg, crude)was added straight away and stirring was maintained until the reaction was complete (3hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100%CHCI3 up to 9:1 CHCI3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 20 was isolated in 10% over two steps (6.6 mg). LC/MS 1.16 mm (ES+) m/z (relative intensity) 770.20 ([M + 2H], 40%).

Reference： [1]Patent: WO2015/52533,2015,A1 .Location in patent: Page/Page column 51; 52

9
[ 1334177-86-4 ]
C₅₆H₆₅N₉O₈ [ No CAS ]
1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((S)-1-(((S)-1-((4-((S)-7-methoxy-8-((5-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yI)-3, 6,9,12,15,18,21,24-octaoxaheptacosan-27-amide [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
43%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; at 20℃; for 3h;	Piperidine (0.2 mL) was added to a solution of 25 (77 mg, 63.4 pmol) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefullydiluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over Mg504, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 pmol) and EDCI (18 mg, 95 pmol) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS(1.19 mm (ES+) m/z (relative intensity) 784.25 (([M + 2H]2)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgSO4), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCI3/MeOH to 90:10 v/v CHCI3/MeOH) gave 26 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3)58.73 (s, IH), 7.88 (dd, J= 7.6, 3.9 Hz, 2H), 7.75 (d, J= 8.6 Hz, 2H), 7.52 (d, J= 2.0 Hz,2H), 7.44 (s, I H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H),6.48 (s, I H), 4.72 - 4.63 (m, I H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84(dd, J= 17.2, 10.1 Hz, 4H), 3.72-3.46 (m, 30H), 3.44-3.32 (m, 4H), 3.30-3.20 (m, 4H),2.75 - 2.63 (m, I H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7Hz, IH), 2.03-1.89 (m, 4H), 1.72 (d, J= 22.7 Hz, 8H), 1.46 (d, J= 7.2 Hz, 3H), 1.01 (dd, J = 11 .5, 6.9 Hz, 6H). MS (ES) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2 100).
43%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; at 20℃; for 3h;	Piperidine (0.2 mL) was added to a solution of 25 (77 mg, 63.4 pmol) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefullydiluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over Mg504, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 pmol) and EDCI (18 mg, 95 pmol) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS(1.19 mm (ES+) m/z (relative intensity) 784.25 (([M + 2H]2)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgSO4), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCI3/MeOH to 90:10 v/v CHCI3/MeOH) gave 26 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3)58.73 (s, IH), 7.88 (dd, J= 7.6, 3.9 Hz, 2H), 7.75 (d, J= 8.6 Hz, 2H), 7.52 (d, J= 2.0 Hz,2H), 7.44 (s, I H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H),6.48 (s, I H), 4.72 - 4.63 (m, I H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84(dd, J= 17.2, 10.1 Hz, 4H), 3.72-3.46 (m, 30H), 3.44-3.32 (m, 4H), 3.30-3.20 (m, 4H),2.75 - 2.63 (m, I H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7Hz, IH), 2.03-1.89 (m, 4H), 1.72 (d, J= 22.7 Hz, 8H), 1.46 (d, J= 7.2 Hz, 3H), 1.01 (dd, J = 11 .5, 6.9 Hz, 6H). MS (ES) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2 100).
43%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; chloroform; at 20℃; for 3h;	(d) 1-(3-(2, 5-dioxo-2 , 5-dihydro- 1H-pyrrol- 1-yl)propanamido)-N-((S)- 1-(((S)- 1-((4-((S)-7- methoxy-8-((5-(((S)- 7-methoxy-2-(4-(4-methylpiperazin- 1-yl)phenyl)-5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-8-yl) oxy)pentyl) oxy)-5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-2-yl)phenyl)amino) - 1-oxopropan-2-yl)amino)-3-methyl- 1- oxobutan-2-yl)-3, 6,9, 12,15, 18,21, 24-octaoxaheptacosan-2 7-amide (91)Piperidine (0.2 mL) was added to a solution of 90 (77 mg, 63.4 pmol) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over Mg504, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 pmol) and EDCI (18 mg, 95 pmol) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1.19 mm (ES+) m/z(relative intensity) 784.25 (([M+ 2H]2j/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgSO4), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCI3/MeOH to 90:10 v/v CHCI3/MeOH) gave 91 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, ODd3) O 8.73 (5, 1H), 7.88 (dd, J= 7.6, 3.9 Hz, 2H), 7.75 (d, J= 8.6 Hz, 2H), 7.52 (d, J= 2.0 Hz, 2H), 7.44 (5, 1H), 7.40-7.28 (m, 4H), 6.91 (d, J= 8.8 Hz, 2H), 6.81 (5, 2H), 6.69 (5, 2H),6.48 (5, 1 H), 4.72 -4.63 (m, 1 H), 4.46 -4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (5, 4H), 3.84 (dd, J = 17.2, 10.1 Hz, 4H), 3.72 -3.46 (m, 30H), 3.44 -3.32 (m, 4H), 3.30-3.20 (m, 4H), 2.75-2.63 (m, 1H), 2.59 (5, 4H), 2.55-2.43 (m, 3H), 2.37 (5, 3H), 2.29 (dd, J= 12.7, 6.7 Hz, 1H), 2.03-1.89 (m, 4H), 1.72 (d, J= 22.7 Hz, 8H), 1.46 (d, J= 7.2 Hz, 3H), 1.01 (dd, J = 11.5, 6.9 Hz, 6H). MS (ES) m/z (relative intensity) 784.25 (([M+ 2H]2)/2, 100).

43%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; chloroform; at 20℃; for 3h;	Piperidine (0.2 mL) was added to a solution of 90 (77 mg, 63.4 pmol) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over Mg504, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 pmol) and EDCI (18 mg, 95 pmol) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1.19 mm (ES+) m/z(relative intensity) 784.25 (([M+ 2H]2)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgSO4), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCI3/MeOH to 90:10 v/v CHCI3/MeOH) gave 91 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, ODd3) 6 8.73 (s, 1 H), 7.88 (dd, J = 7.6, 3.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 2.0 Hz, 2H), 7.44 (s, 1 H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H),6.48 (s, 1 H), 4.72 - 4.63 (m, 1 H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84 (dd, J 17.2, 10.1 Hz, 4H), 3.72-3.46 (m, 30H), 3.44-3.32 (m, 4H), 3.30-3.20 (m, 4H), 2.75 - 2.63 (m, 1 H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7 Hz, 1 H), 2.03 - 1.89 (m, 4H), 1.72 (d, J = 22.7 Hz, 8H), 1.46 (d, J = 7.2 Hz, 3H), 1.01 (dd, J = 11 .5, 6.9 Hz, 6H). MS (ES) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100).
43 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; at 20℃; for 3h;	Piperidine (0.2 mL) was added to a solution of 25 (77 mg, 63.4 pmol) in DM F (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over MgS04, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 ml_). Mal(Peg)8-acid (56 mg, 95 pmol) and EDCI (18 mg, 95 pmol) were added, followed by methanol (0.1 ml_). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1.19 min (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100)). The reaction mixture was diluted with chloroform (50 ml_), washed with water (100 ml_), dried (MgS04), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCI3/MeOH to 90:10 v/v CHCIs/MeOH) gave 26 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3) delta 8.73 (s, 1 H), 7.88 (dd, J = 7.6, 3.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 2.0 Hz, 2H), 7.44 (s, 1 H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H), 6.48 (s, 1 H), 4.72 - 4.63 (m, 1 H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84 (dd, J = 17.2, 10.1 Hz, 4H), 3.72 - 3.46 (m, 30H), 3.44 - 3.32 (m, 4H), 3.30 - 3.20 (m, 4H), 2.75 - 2.63 (m, 1 H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7 Hz, 1 H), 2.03 - 1 .89 (m, 4H), 1 .72 (d, J = 22.7 Hz, 8H), 1.46 (d, J = 7.2 Hz, 3H), 1 .01 (dd, J = 1 1 .5, 6.9 Hz, 6H). MS (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100).
	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; chloroform; at 20℃; for 3h;	Piperidine (0.2 mL) was added to a solution of 25 (77 mg, 63.4 pmol) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefullydiluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over Mg504, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 pmol) and EDCI (18 mg, 95 pmol) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS(1.19 mm (ES+) m/z (relative intensity) 784.25 (([M + 2H]2)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgSO4), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCI3/MeOH to 90:10 v/v CHCI3/MeOH) gave 26 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3)58.73 (s, IH), 7.88 (dd, J= 7.6, 3.9 Hz, 2H), 7.75 (d, J= 8.6 Hz, 2H), 7.52 (d, J= 2.0 Hz,2H), 7.44 (s, I H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H),6.48 (s, I H), 4.72 - 4.63 (m, I H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84(dd, J= 17.2, 10.1 Hz, 4H), 3.72-3.46 (m, 30H), 3.44-3.32 (m, 4H), 3.30-3.20 (m, 4H),2.75 - 2.63 (m, I H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7Hz, IH), 2.03-1.89 (m, 4H), 1.72 (d, J= 22.7 Hz, 8H), 1.46 (d, J= 7.2 Hz, 3H), 1.01 (dd, J = 11 .5, 6.9 Hz, 6H). MS (ES) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2 100).
43 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	Piperidine (0.2 mL) was added to a solution of 90 (77 mg, 63.4 mumomicronIota) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over MgSO4, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 mumomicronIota) and EDCI (18 mg, 95 mumomicronIota) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1 .19 min (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgS04), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCl3/MeOH to 90:10 v/v CHCl3/MeOH) gave 91 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3) delta 8.73 (s, 1 H), 7.88 (dd, J = 7.6, 3.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 2.0 Hz, 2H), 7.44 (s, 1 H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H), 6.48 (s, 1 H), 4.72 - 4.63 (m, 1 H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84 (dd, J = 17.2, 10.1 Hz, 4H), 3.72 - 3.46 (m, 30H), 3.44 - 3.32 (m, 4H), 3.30 - 3.20 (m, 4H), 2.75 - 2.63 (m, 1 H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7 Hz, 1 H), 2.03 - 1 .89 (m, 4H), 1 .72 (d, J = 22.7 Hz, 8H), 1 .46 (d, J = 7.2 Hz, 3H), 1 .01 (dd, J = 1 1 .5, 6.9 Hz, 6H). MS (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100).
43 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	Piperidine (0.2 mL) was added to a solution of 90 (77 mg, 63.4 mumomicronIota) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over MgSO4, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 mumomicronIota) and EDCI (18 mg, 95 mumomicronIota) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1 .19 min (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgS04), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCl3/MeOH to 90:10 v/v CHCl3/MeOH) gave 91 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3) delta 8.73 (s, 1 H), 7.88 (dd, J = 7.6, 3.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 2.0 Hz, 2H), 7.44 (s, 1 H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H), 6.48 (s, 1 H), 4.72 - 4.63 (m, 1 H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84 (dd, J = 17.2, 10.1 Hz, 4H), 3.72 - 3.46 (m, 30H), 3.44 - 3.32 (m, 4H), 3.30 - 3.20 (m, 4H), 2.75 - 2.63 (m, 1 H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7 Hz, 1 H), 2.03 - 1 .89 (m, 4H), 1 .72 (d, J = 22.7 Hz, 8H), 1 .46 (d, J = 7.2 Hz, 3H), 1 .01 (dd, J = 1 1 .5, 6.9 Hz, 6H). MS (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100).
43 mg	With 1,1'-[[(pentane-1,5-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione]; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	Piperidine (0.2 mL) was added to a solution of 90 (77 mg, 63.4 mumomicronIota) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over MgSO4, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 mumomicronIota) and EDCI (18 mg, 95 mumomicronIota) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1 .19 min (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgS04), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCl3/MeOH to 90:10 v/v CHCl3/MeOH) gave 91 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3) delta 8.73 (s, 1 H), 7.88 (dd, J = 7.6, 3.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 2.0 Hz, 2H), 7.44 (s, 1 H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H), 6.48 (s, 1 H), 4.72 - 4.63 (m, 1 H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84 (dd, J = 17.2, 10.1 Hz, 4H), 3.72 - 3.46 (m, 30H), 3.44 - 3.32 (m, 4H), 3.30 - 3.20 (m, 4H), 2.75 - 2.63 (m, 1 H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7 Hz, 1 H), 2.03 - 1 .89 (m, 4H), 1 .72 (d, J = 22.7 Hz, 8H), 1 .46 (d, J = 7.2 Hz, 3H), 1 .01 (dd, J = 1 1 .5, 6.9 Hz, 6H). MS (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100).
43 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;Inert atmosphere;	Piperidine (0.2 mL) was added to a solution of 90 (77 mg, 63.4 mumomicronIota) in DMF (1 mL). The reaction mixture was allowed to stir for 20 minutes. The reaction mixture was carefully diluted with DCM (50 mL) and washed with water (50 mL). The organic layers was washed with brine (100 mL), dried over MgSO4, filtered and evaporated under reduced pressure to provide the unprotected valine intermediate. The crude residue was immediately redissolved in chloroform (5 mL). Mal(Peg)8-acid (56 mg, 95 mumomicronIota) and EDCI (18 mg, 95 mumomicronIota) were added, followed by methanol (0.1 mL). The reaction was allowed to stir for 3 hours at room temperature at which point completion was observed by TLC and LC/MS (1 .19 min (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100)). The reaction mixture was diluted with chloroform (50 mL), washed with water (100 mL), dried (MgS04), filtered and evaporated in vacuo, followed by high vacuum drying, to provide the crude product. Purification by flash chromatography (gradient elution: HPLC grade 96:4 v/v CHCl3/MeOH to 90:10 v/v CHCl3/MeOH) gave 91 as a yellow solid (43 mg, 43%). 1H NMR (400 MHz, CDCI3) delta 8.73 (s, 1 H), 7.88 (dd, J = 7.6, 3.9 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 2.0 Hz, 2H), 7.44 (s, 1 H), 7.40 - 7.28 (m, 4H), 6.91 (d, J = 8.8 Hz, 2H), 6.81 (s, 2H), 6.69 (s, 2H), 6.48 (s, 1 H), 4.72 - 4.63 (m, 1 H), 4.46 - 4.34 (m, 2H), 4.25 - 4.03 (m, 6H), 3.95 (s, 4H), 3.84 (dd, J = 17.2, 10.1 Hz, 4H), 3.72 - 3.46 (m, 30H), 3.44 - 3.32 (m, 4H), 3.30 - 3.20 (m, 4H), 2.75 - 2.63 (m, 1 H), 2.59 (s, 4H), 2.55 - 2.43 (m, 3H), 2.37 (s, 3H), 2.29 (dd, J = 12.7, 6.7 Hz, 1 H), 2.03 - 1 .89 (m, 4H), 1 .72 (d, J = 22.7 Hz, 8H), 1 .46 (d, J = 7.2 Hz, 3H), 1 .01 (dd, J = 1 1 .5, 6.9 Hz, 6H). MS (ES+) m/z (relative intensity) 784.25 (([M + 2H] 2+)/2, 100).

Reference： [1]Patent: WO2015/52533,2015,A1 .Location in patent: Page/Page column 56; 57
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10
[ 1334177-86-4 ]
(11S,11aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]
(11S,11aS)-4-((2R,5R)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;	N-(3-Dimethylaminopropyl)-N?-ethylcarbodiimide (28 mg, 0.146 mmol, I eq) was added to asolution of 42 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) inchloroform (5 mL). The reaction was stirred for 1.5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/I0% methanol] gave 43 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 mm; MS (ES+)m/z (relative intensity) 982.90 (100), 1963.70 (5).
72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;	N-(3-Dimethylaminopropyl)-N?-ethylcarbodiimide (28 mg, 0.146 mmol, I eq) was added to asolution of 42 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) inchloroform (5 mL). The reaction was stirred for 1.5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/I0% methanol] gave 43 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 mm; MS (ES+)m/z (relative intensity) 982.90 (100), 1963.70 (5).
72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;	N-(3-Dimethylaminopropyl)-N?-ethylcarbodiimide (28 mg, 0.146 mmol, I eq) was added to asolution of 42 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) inchloroform (5 mL). The reaction was stirred for 1.5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/I0% methanol] gave 43 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 mm; MS (ES+)m/z (relative intensity) 982.90 (100), 1963.70 (5).

72%

With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;

N-(3-Dimethylaminopropyl)-N?-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 105 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) in chloroform (5 mL). The reaction was stirred for 1 .5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/i0% methanol] gave 106 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 mm; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).

Reference： [1]Patent: WO2015/52534,2015,A1 .Location in patent: Page/Page column 64; 65
[2]Patent: WO2015/52533,2015,A1 .Location in patent: Page/Page column 65
[3]Patent: WO2015/52532,2015,A1 .Location in patent: Page/Page column 66
[4]Patent: WO2016/166299,2016,A1 .Location in patent: Page/Page column 212; 213

11
[ 1334177-86-4 ]
(11S,11 aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]
(11S,11aS)-4-((2R,5R)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;	N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 42 (203 mg, 0.146 mmol) and maleimide-PEGs acid (87 mg, 0.146 mmol) in chloroform (5 ml_). The reaction was stirred for 1 .5 h then diluted with chloroform (50 ml_), washed with water (50 ml_), brine (30 ml_), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/10% methanol] gave 43 as a pale yellow solid (205 mg, 72%). LC/MS, System 1 : RT 5.75 min; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).
72%	With N-(3-dimethylaminopropyl)-N-ethylcarbodiimide; In chloroform; for 1.5h;	[0573] N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 95 (203 mg, 0.146 mmol) and maleimide-PEGs acid (87 mg, 0.146 mmol) in chloroform (5 mL). The reaction was stirred for 1.5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/10% methanol] gave 96 as a pale yellow solid (205 mg, 72%). LC/MS, System 1: RT 5.75 min; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).
72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;Inert atmosphere;	N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 105 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) in chloroform (5 mL). The reaction was stirred for 1 .5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/10% methanol] gave 106 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 min; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).

72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;Inert atmosphere;	N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 105 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) in chloroform (5 mL). The reaction was stirred for 1 .5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/10% methanol] gave 106 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 min; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).
72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;Inert atmosphere;	N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 105 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) in chloroform (5 mL). The reaction was stirred for 1 .5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/10% methanol] gave 106 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 min; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).

Reference： [1]Patent: WO2015/52322,2015,A1 .Location in patent: Page/Page column 136; 137
[2]Patent: WO2015/155345,2015,A1 .Location in patent: Paragraph 0572; 0573
[3]Patent: WO2016/166298,2016,A1 .Location in patent: Page/Page column 209
[4]Patent: WO2016/166341,2016,A1 .Location in patent: Page/Page column 208
[5]Patent: WO2016/166304,2016,A1 .Location in patent: Page/Page column 223

12
[ 1334177-86-4 ]
[ 1595275-27-6 ]
[ 1428523-15-2 ]

Yield	Reaction Conditions	Operation in experiment
6.6 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid (25 mg, 0.042 mmol) in dry CH2CI2 (4 mL) under argon atmosphere. PBD 19 (42 mg, crude) was added straight away and stirring was maintained until the reaction was complete (3 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9: 1 CHCI3/MeOH) followed by reverse phase HPLC to remove unreacted maleimide-PEGs-acid. The product 20 was isolated in 10% over two steps (6.6 mg). LC/MS 1 .16 min (ES+) m/z (relative intensity) 770.20 ([M + 2H]+ , 40%).
	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid(25 mg, 0.042 mmol) in dry CH2CI2 (4 mL) under argon atmosphere. PBD 19 (42 mg, crude)was added straight away and stirring was maintained until the reaction was complete (3hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100%CHCI3 up to 9:1 CHCI3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 20 was isolated in 10% over two steps (6.6 mg). LC/MS 1.16 mm (ES+) m/z (relative intensity) 770.20 ([M + 2H], 40%).
6.6 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid(25 mg, 0.042 mmol) in dry CH2CI2 (4 mL) under argon atmosphere. PBD 19 (42 mg, crude)was added straight away and stirring was maintained until the reaction was complete (3hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100%CHCI3 up to 9:1 CHCI3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 20 was isolated in 10% over two steps (6.6 mg). LC/MS 1.16 mm (ES+) m/z (relative intensity) 770.20 ([M + 2H], 40%).

6.6 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid (25 mg, 0.042 mmol) in dry CH2CI2 (4 mL) under argon atmosphere. PBD 85 (42 mg, crude) was added straight away and stirring was maintained until the reaction was complete (3 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9:1 CHCI3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 86 was isolated in 10% over two steps (6.6 mg). LC/MS 1.16 mm (ES+) m/z(relative intensity) 770.20 ([M+ 2H], 40%).
6.6 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid (25 mg, 0.042 mmol) in dry CH2Cl2 (4 mL) under argon atmosphere. PBD 85 (42 mg, crude) was added straight away and stirring was maintained until the reaction was complete (3 hours). The reaction was diluted with CH2Cl2and the organic phase was washed with H2O and brine before being dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9:1 CHCl3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 86 was isolated in 10% over two steps (6.6 mg). LC/MS 1 .16 min (ES+) m/z (relative intensity) 770.20 ([M + 2H]+ , 40%).
6.6 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid (25 mg, 0.042 mmol) in dry CH2Cl2 (4 mL) under argon atmosphere. PBD 85 (42 mg, crude) was added straight away and stirring was maintained until the reaction was complete (3 hours). The reaction was diluted with CH2Cl2and the organic phase was washed with H2O and brine before being dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9:1 CHCl3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 86 was isolated in 10% over two steps (6.6 mg). LC/MS 1 .16 min (ES+) m/z (relative intensity) 770.20 ([M + 2H]+ , 40%).
6.6 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid (25 mg, 0.042 mmol) in dry CH2Cl2 (4 mL) under argon atmosphere. PBD 85 (42 mg, crude) was added straight away and stirring was maintained until the reaction was complete (3 hours). The reaction was diluted with CH2Cl2and the organic phase was washed with H2O and brine before being dried over MgS04, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9:1 CHCl3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 86 was isolated in 10% over two steps (6.6 mg). LC/MS 1 .16 min (ES+) m/z (relative intensity) 770.20 ([M + 2H]+ , 40%).

Reference： [1]Patent: WO2015/52321,2015,A1 .Location in patent: Page/Page column 119; 120
[2]Patent: WO2015/52532,2015,A1 .Location in patent: Page/Page column 52; 53
[3]Patent: WO2015/52535,2015,A1 .Location in patent: Page/Page column 51
[4]Patent: WO2016/166299,2016,A1 .Location in patent: Page/Page column 198; 200
[5]Patent: WO2016/166298,2016,A1 .Location in patent: Page/Page column 197
[6]Patent: WO2016/166341,2016,A1 .Location in patent: Page/Page column 196
[7]Patent: WO2016/166304,2016,A1 .Location in patent: Page/Page column 211

13
[ 1334177-86-4 ]
(11S,11aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl 11-hydroxy8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]
(11S,11aS)-4-((2R,5R)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-8-((5-(((11S,11aS)-11-hydroxy-10-(((4-((10R,13R)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
72%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;	N-(3-Dimethylaminopropyl)-N?-ethylcarbodiimide (28 mg, 0.146 mmol, I eq) was added to asolution of 42 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) inchloroform (5 mL). The reaction was stirred for 1.5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/I0% methanol] gave 43 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 mm; MS (ES+)m/z (relative intensity) 982.90 (100), 1963.70 (5).

Reference： [1]Patent: WO2015/52535,2015,A1 .Location in patent: Page/Page column 64

14
[ 1334177-86-4 ]
C₇₂H₉₄N₁₀O₁₈ [ No CAS ]
C₉₈H₁₃₆N₁₂O₃₀ [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 1.5h;	(C) (115,1 laS) -4-((2R, 5R) -3 7-(2, 5-dioxo-2 , 5-dihydro- 1H-pyrrol- 1-yI)-5-isopropyl-2-methyl- 4,7, 35-trioxo- 10, 13, 16, 19,22,25,28,3 1-octaoxa-3, 6, 34-triazaheptatriacontanamido)benzyl 11- hydroxy-8-((5-(((1 15, 1 laS)- 1 1-hydroxy- 1 0-(((4-((1OR, 13R)- lO-isopropyl- 13-met hyl-8, 11- dioxo-2, 5-dioxa-9, 12-diazatetradecanamido) benzyl) oxy)carbonyl)- 7-methoxy-2-methyl-5- oxo-5, 10, 11, 1 la-tetrahydro- 1H-pyrrolo[2, 1-c][1, 4]benzodiazepin-8-yI) oxy)pentyl) oxy) -7- methoxy-2-methyl-5-oxo- 11,1 la-dihydro- 1H-pyrrolo[2, 1-c][1, 4]benzodiaze pine- 10(5H)- carboxylate (106)N-(3-Dimethylaminopropyl)-N?-ethylcarbodiimide (28 mg, 0.146 mmol, 1 eq) was added to a solution of 105 (203 mg, 0.146 mmol) and <strong>[1334177-86-4]maleimide-PEG8 acid</strong> (87 mg, 0.146 mmol) in chloroform (5 mL). The reaction was stirred for 1.5 h then diluted with chloroform (50 mL), washed with water (50 mL), brine (30 mL), dried over magnesium sulphate, filtered and evaporated. Flash chromatography [gradient elution 100% DCM to 90% DCM/i0% methanol] gave 106 as a pale yellow solid (205 mg, 72%). LC/MS: RT 5.75 mm; MS (ES+) m/z (relative intensity) 982.90 (100), 1963.70 (5).

Reference： [1]Patent: WO2015/159076,2015,A1 .Location in patent: Page/Page column 219; 220

15
[ 1334177-86-4 ]
[ 1428523-15-2 ]

Yield	Reaction Conditions	Operation in experiment
6.6 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 3h;Inert atmosphere;	(e) 1-(3-(2, 5-dioxo-2 , 5-dihydro- 1H-pyrrol- 1-yI)propanamido)-N-((2S)- 1-(((2S) - 1-((4-(7- methoxy-8-(3-((7-methoxy-2-(4-(4-methylpiperazin- 1-yI)phenyl)-5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-8-yI) oxy)propoxy) -5-oxo-5, 1 la-dihydro- 1H- benzo[e]pyrrolo[1, 2-a][1, 4]diazepin-2-yI)phenyl)amino) - 1-oxopropan-2-yI)amino)-3-methyl- 1- oxobutan-2-yI)-3, 6,9, 12,15, 18,21, 24-octaoxaheptacosan-2 7-amide (86)EDCI hydrochloride (8 mg, 0.042 mmol) was added to a suspension of Maleimide-PEG8-acid (25 mg, 0.042 mmol) in dry CH2CI2 (4 mL) under argon atmosphere. PBD 85 (42 mg, crude) was added straight away and stirring was maintained until the reaction was complete (3 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (slow elution starting with 100% CHCI3 up to 9:1 CHCI3/MeOH) followed by reverse phase HPLC to remove unreacted <strong>[1334177-86-4]maleimide-PEG8-acid</strong>. The product 86 was isolated in 10% over two steps (6.6 mg). LC/MS 1.16 mm (ES+) m/z (relative intensity) 770.20 ([M+ 2H], 40%).

Reference： [1]Patent: WO2015/159076,2015,A1 .Location in patent: Page/Page column 206; 208

16
[ 1334177-86-4 ]
(11S)-4-(2-(1-((1-amino-3-methyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl 11-hydroxy-7-methoxy-8-((5-((7-methoxy-2-methyl-5-oxo-5,11a-dihydro1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-diHydro₁H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
(11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1h-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-7-methoxy-8-((5-(((s)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)-oxy)-2-methyl-5-oxo-11,11a-dihydro-1h-benzo[e]pyrrolo-[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
60 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 2h;	(d) (ii S, i ia S)-4-((2S, 5S)-37-(2, 5-di oxo-2, 5-dihydro- iH-pyrrol- i-yl)-5-isopropyl-2-methyl- 4,7, 35-trioxo- 10, 13,16, 19,22,25,28,3 i-octaoxa-3, 6, 34-triazaheptatriacontanamido)benzyl iihydroxy- 7-methoxy-8-((5-(((S) - 7-methoxy-2-methyl-5-oxo-5, i ia-dihydro- iHbenzo[e]pyrrolo[i, 2-a][i, 4]diazepin-8-yl) oxy) pentyl) oxy) -2-met hyl-5-oxo- 11,1 ia-dihydro- iHbenzo[e]pyrrolo[i, 2-a][i, 4]diazepine- i 0(5H)-carboxylate (64) 1-ethyl-3-(3?-dimethylaminopropyl)carbodiimide (EDCI, 33 mg, 0.172 mmol) was added to a solution of crude 63 (0.172 mmol) and Mal-(PEG)8-acid (100 mg, 0.172 mmol) in dry dichloromethane (10 mL). The reaction was stirred for 2 hours and the presence ofstarting material was no longer observed by LC/MS. The reaction was diluted with dichloromethane and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 100% chloroform to 10% methanol in chloroform). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give 64 (E) (60 mg, 25% over 3 steps).

Reference： [1]Patent: WO2015/159076,2015,A1 .Location in patent: Page/Page column 188; 189; 190

17
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (3-(((S)-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)propyl)carbamate [ No CAS ]
4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (3-(((S)-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)propyl)carbamate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
3.7 g	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; for 2h;Inert atmosphere;	(d) 4-((2S, 5S) -37-(2, 5-dioxo-2, 5-dihydro- 1 H-pyrrol- 1 -yl) -5-isopropyl-2-methyl-4, 7, 35-trioxo- 10, 13, 16, 19, 22, 25, 28, 31 -octaoxa-3, 6, 34-triazaheptatriacontanamido)benzyl (3-(((S)-8-((5- (((S)- 7-methoxy-2-methyl-5-oxo-5, 11 a-dihydro- 1 H-benzo[e ]pyrrolo[1 , 2-a ][1 , 4 ]diazepin-8- yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5, 11 a-dihydro-1 H-benzo[e]pyrrolo[1 ,2-a][1 ,4]diazepin-7- yl)oxy)propyl)carbamate (4) 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDCI, 15 mg, 0.080 mmol, 1.1 eq) was added to a solution of crude 3 (0.077 mmol) and Mal-(PEG)8-acid (48 mg, 0.080 mmol, 1 .1 eq) in dry dichloromethane (2 ml_). The reaction was degassed three times with Argon and stirred for 2 hours and the presence of starting material was no longer observed by LC/MS. The reaction was diluted with dichloromethane and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 100% chloroform to 10% methanol in chloroform). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the desired product (40mg). This residue was then purified further by preparative HPLC (3.7 mg, 31 % over 2 steps). LC/MS 1.40 min, (ES+) m/z (relative intensity) 1521.95 [M + H]+. 1H NMR (400 MHz, CDCI3) delta 8.72 (s, 1 H), 7.83 (s, 1 H), 7.80 (d, J = 3.9 Hz, 1 H), 7.63 - 7.61 (m, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.26 (br, 2H), 6.77 - 6.75 (m, 4H), 6.69 (s, 2H), 5.02 (s, 2H), 4.64 - 4.62 (m, 2H), 4.24 - 4.21 (m, 4H), 4.19 - 3.95 (m, 4H), 3.89 (s, 2H), 3.86 - 3.75 (m, 4H), 3.63 (br, 32H), 3.53 - 3.33 (m, 8H), 3.18 - 3.14 (m, 2H), 2.98 - 2.94 (m, 2H), 2.53 - 5.49 (m, 3H), 2.03 (s, 2H), 1.83 (br, 10H), 1 .56 (br, 2H), 1.42 (s, 3H), 1.05 - 0.90 (m, 6H).

Reference： [1]Patent: WO2016/37644,2016,A1 .Location in patent: Page/Page column 164; 166

18
[ 1334177-86-4 ]
1-(3-bromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oic acid [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
		Procedure: Bromine (0.20 ml, 3.88 mmol) was added to a solution of 1 - (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4- azahentriacontan-31 -oic acid (1000 mg, 1 .69 mmol) in methylene chloride (17 ml). After stirring for 14 h, the solution was cooled to -10C in an ice/brine bath and diisopropylethylamine (1 .5 ml, 8.61 mmol) was slowly added dropwise. After stirring for an additional 24 h, during which time the solution warmed to ambient temperature, the solution was concentrated under reduced pressure to afford crude 1 -(3-bromo- 2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4- azahentriacontan-31 -oic acid. UPLC/MS 1 .18 min (5-95% acetonitrile/water + 0.1 % formic acid over 2 min, hold at 95% for 0.5 min, then 95-5% over 0.1 min, and hold at 5% for 0.4 min. Column used was Waters BEH C18 1 .7 muiotatauiota, 2.1 x 50 mm, flow rate was 0.8 mL/min.), m/z 671 .6 and 673.6 [M+H]+.

Reference： [1]Patent: WO2016/64749,2016,A2 .Location in patent: Paragraph 00362

19
[ 1334177-86-4 ]
(E)-34-(4-cyanophenoxy)-29, 33-dioxo-4,7,10,13,16,19,22,25-octaoxa-28,32-diazahexatriacont-34-enedioic acid [ No CAS ]

Reference： [1]Patent: WO2016/64749,2016,A2

20
[ 1334177-86-4 ]
1-(3-(4-cyanophenoxy)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oic acid [ No CAS ]

Reference： [1]Patent: WO2016/64749,2016,A2

21
[ 1334177-86-4 ]
(11S)-4-(2-(1-((1-amino-3-methyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl 11-hydroxy-7-methoxy-8-((5-((7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
(11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Reference： [1]ACS Medicinal Chemistry Letters,2016,vol. 7,p. 983 - 987

22
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (11S, 11aS)-8-(3-(((11s, 11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
tert-butyl (11S,11aS)-8-(3-(((115,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
82%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; at 20℃; for 1.5h;	EDCI .HCI (0.13 g, 0.66 mmol, 1 .1 eq.) was added to a cloudy solution of compound 21 (0.61 g, 0.6 mmol, 1 .0 eq.) and <strong>[1334177-86-4]Mal-dPEG8-OH</strong> (0.393 g, 0.66 mmol, 1 .1 eq.) in CHCI3 (25 mL). The clear solution was stirred at room temperature for 1 .5h., diluted with CHC (100 mL) washed with brine (2 x 100 mL), dried (MgS04) and evaporated under reduced pressure to give a yellow foam. Purification by flash column chromatography [CHCb/MeOH 0% to 6% in 1 % increments gave the product as a white foam (0.786 g, 82%). Analytical Data: RT 1 .44 min; MS (ES+) m/z (relative intensity) 1586 [M + H]+ ,40), 1609 [M + Na]+ , 100)
82%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; at 20℃; for 1.5h;	EDCI.HCI (0.13 g, 0.66 mmol, 1.1 eq.) was added to a cloudy solution of compound 21(0.61 g, 0.6 mmol, 1.0 eq.) and <strong>[1334177-86-4]Mal-dPEG8-OH</strong> (0.393 g, 0.66 mmol, 1.1 eq.) in CHCI3(25 mL). The clear solution was stirred at room temperature for 1 .5h., diluted with CHCI3(100 mL) washed with brine (2 x 100 mL), dried (Mg504) and evaporated under reducedpressure to give a yellow foam. Purification by flash column chromatography [CHCI3/MeOH0% to 6% in 1% increments gave the product as a white foam (0.786 g, 82%). AnalyticalData: RT 1.44 mm; MS (ES) m/z (relative intensity) 1586 ([M + H],40), 1609 ([M + Na],100)

Reference： [1]Patent: WO2017/137553,2017,A1 .Location in patent: Page/Page column 105; 107
[2]Patent: WO2017/137556,2017,A1 .Location in patent: Page/Page column 43; 45

23
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]
4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
40%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; chloroform; at 20℃; for 42h;	EDCI .HCI (0.203 g, 1 .06 mmol, 1 .1 eq.) was added to a solution of compound 32 (0.86 g, 0.96 mmol, 1 .0 eq.) and <strong>[1334177-86-4]Mal-dPEG8-OH</strong> (0.57 g, 0.96 mmol, 1 .1 eq.) in dry DCM (30 mL) and CHC (to give a clear solution). The clear solution was stirred at room temperature for 18h. then a further portion of EDCI.HCI (0.037 g, 0.19 mmol, 0.2 eq.) was added and reaction continued for a further 24h. The reaction mixture was diluted with DCM (70 mL) washed with water (100 mL), brine (100 mL), dried (MgS04) and evaporated under reduced pressure to give a yellow foam. Purification by flash column chromatography [CHC /MeOH 0% to 6% in 1 % increments] gave the product as an off white foam (0.56 g, 40%). Analytical Data: RT 6.13 min; MS (ES+) m/z (relative intensity) 1468 [M + H]+ , 20)
40%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; chloroform; at 20℃; for 42h;	EDCI.HCI (0.203 g, 1.06 mmol, 1.1 eq.) was added to a solution of compound 32 (0.86 g,0.96 mmol, 1.0 eq.) and <strong>[1334177-86-4]Mal-dPEG8-OH</strong> (0.57 g, 0.96 mmol, 1.1 eq.) in dry DCM (30 mL)and CHCI3 (to give a clear solution). The clear solution was stirred at room temperature for18h. then a further portion of EDCI.HCI (0.037 g, 0.19 mmol, 0.2 eq.) was added and reaction continued for a further 24h. The reaction mixture was diluted with DCM (70 mL) washed with water (100 mL), brine (100 mL), dried (MgSO4) and evaporated under reduced pressure to give a yellow foam. Purification by flash column chromatography[CHCI3/MeOH 0% to 6% in 1% increments] gave the product as an off white foam (0.56 g,40%). Analytical Data: RT 6.13 mm; MS (ES) m/z (relative intensity) 1468 ([M + H], 20)

Reference： [1]Patent: WO2017/137553,2017,A1 .Location in patent: Page/Page column 112; 115
[2]Patent: WO2017/137556,2017,A1 .Location in patent: Page/Page column 50; 52; 53

24
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-8-(3-(((11S,11aS)-11-hydroxy-10-(((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (11S,11aS)-11-hydroxy-8-(3-(((11S,11aS)-11-hydroxy-10-(((4-((10S,13S)-10-isopropyl-13-methyl-8,11-dioxo-2,5-dioxa-9,12-diazatetradecan-14-amido)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
63%	With methanol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 1.45h;	A solution of compound 39 (0.136 g, 0.1 mmol, 1 .0 eq.), <strong>[1334177-86-4]Mal-dPEG8-OH</strong> (0.066 g, 0.1 1 mmol, 1.1 eq.) and EDCI.HCI (0.022 g, 0.1 1 mmol, 1.1 eq.) in dry DCM (10 mL) and MeOH (1 drop) was stirred at room temperature for 1.45h. The reaction mixture was diluted with CHC and washed with water, brine, dried (MgS04) and evaporated under reduced pressure to give the crude product. Purification by flash column chromatography [CHC /MeOH 1 % to 9%] gave the product as a white solid (0.123 g, 63%). Analytical Data: [a]21D = +1 12.5 (c = 0.4, hplc CHCI3); RT 6.37 min; MS (ES+) m/z (relative intensity) 1936 [M + 1]+ , 35); 1958 [M + Na]+ , 15).

Reference： [1]Patent: WO2017/137553,2017,A1 .Location in patent: Page/Page column 116-117; 120

25
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (3-(3,5-bis((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)carbamate [ No CAS ]
4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (3-(3,5-bis((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)carbamate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 2h;	(l) 4-a2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-Isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (3-(3,5-bis((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)carbamate 15 Mal-dPEG 8 acid (0.053 g, 0.09 mmol, 0.9 eq.) and EDCI (0.017 g, 0.09 mmol, 0.9 eq.) were added to a solution of crude amine 14 (0.094 g, 0.095 mmol, 1 eq.) in DCM. The solution was stirred at room temperature for 2 hours. The reaction mixture was washed with water, saturated brine, dried (MgSO4) and evaporated under reduced pressure. The residue was purified by column chromatography (Biotage Isolera) to give material which was further purified by prep HPLC (without the use of acid). This afforded two batches of the desired product 15 (5.47 mg, and 2.78 mg of purity 93% and 88% respectively).

Reference： [1]Patent: US2017/239365,2017,A1 .Location in patent: Paragraph 2112

26
[ 1334177-86-4 ]
tert-butyl (11S,11aS)-2-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)phenyl)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
tert-butyl (11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-(4-((2S,5S)-37-(2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16, 19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)phenyl)-7-methoxy-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10( 5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
75%		EDCI hydrochloride (39 mg, 0.197 mmol, leq.) was added to a suspension of <strong>[1334177-86-4]maleimide-PEG8-acid</strong> (117 mg, 0.197 mmol, leq.) in dry CH2CI2 (5 mL) under argon atmosphere. The mixture was stirredfor 30 mm at room temperature before PBD 13 (250 mg, 0.197 mmol) was added. Stirring was maintained until the reaction was complete (usually 5 hours). The reaction was diluted with CH2CI2 and the organic phase was washed with H20 and brine before being dried over Mg504, filtered and excess solvent removed by rotary evaporation under reduced pressure. The product was purified by careful silica gel chromatography (gradient elution: 100% CHCI3 to 9:1 v/v CHCI3/MeOH)to affordpure product 14(273.8mg, 75% yield). Analytical data: ES = 1.99 mi m/z 1863.95 [M+Na].

Reference： [1]Patent: WO2017/129652,2017,A1 .Location in patent: Page/Page column 139

27
[ 1334177-86-4 ]
C₁₆₄H₂₇₇N₃₅O₄₈ [ No CAS ]
C₁₄₀H₂₃₉N₃₇O₄₀ [ No CAS ]

Reference： [1]Patent: WO2017/218922,2017,A2 .Location in patent: Page/Page column 252; 253

28
[ 1334177-86-4 ]
C₄₉H₅₉N₇O₁₁ [ No CAS ]
(11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl 11-hydroxy-7-methoxy-8-(5-((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-8-yloxy)pentyloxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; In dichloromethane; N,N-dimethyl acetamide; at 20℃; for 18h;	EEDQ (12 mg, 4.8x105 mol, 1.1 eq.) was added to a suspension of amine dipeptide (12c) (40.3 mg 4.4x105 mol, 1.0 eq.) and <strong>[1334177-86-4]maleimide-8 Peg-acid</strong> (28 mg, 4.8x105 mol, 1.1 eq.) in dry DCM (S mE). Dry dimethylacetamide (0.OS mE) was added to give a pale yellow solution which was stirred at room temperature for 1 8h. The solvent was evaporated under reduced pressure and the residue was triturated with diethyl ethet The resultant solid product was purified by flash column chromatography. Analytical Data:RT 2.90 mm; MS (ES) mlz (relative intensity) 1496 ([M+ H], 40).

Reference： [1]Patent: US2018/134717,2018,A1 .Location in patent: Paragraph 1183

29
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-(3-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]
tert-butyl (11S,11aS)-8-(3-(((11S,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
277 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 2.5h;Inert atmosphere;	EDCI (56 mg, 0.29 mmol) was added to a stirred solution of MAL-dPEG8-acid (172 mg,0.29 mmol, Stratech Scientific Limited) and the amine 110 (261 mg, 0.26 mmol) in dry DCM(10 mL) at room temperature. The reaction mixture was stirred under an argon atmospherefor 2.5 hours at which point analysis by LC/MS showed complete conversion to desiredproduct at retention time 1 .38 minutes, ES+ mlz 1585 [M+ Na], 1563 [M+ H].Thereaction mixture was diluted with DCM (30 mL) and washed with H20 (20 mL), brine (2 x20 mL), dried (Mg504), filtered and evaporated in vacuo to provide the crude product. Purification by lsoleraTM (DCM/MeOH, SNAP Ultra 25 g, 75 mL per minute) gave the amideIll (eluting at 91% DCM/MeOH) as a white foam (277 mg, 67% yield).

Reference： [1]Patent: WO2018/192944,2018,A1 .Location in patent: Page/Page column 126; 127; 131; 132

30
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butoxycarbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
tert-butyl (11S,11aS)-8-((5-(((11S,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy) pentyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 24h;Inert atmosphere;	EDCI.HCI (117 mg, 0.29 mmol) was added to a stirred solution of MAL-dPEG8-acid (360mg, 0.6079 mmol, Stratech Scientific Limited) and amine 120 (608 mg, 0.5526 mmol) inCH2CI2 (15 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 24 hours, at which point analysis by LC/MS showed complete consumption of 120. The reaction mixture was diluted with CH2CI2 and washed successively with sat. NH4CI and sat. NaH 003, dried over Mg504, and concentrated in vacuo to provide thecrude product. Purification by lsoleraTM (4-16% MeOH in CH2CI2) gave amide 121 as a white solid (77 mg, 79% purity (UV integration 223 nm) 8.8% crude yield; 107 mg, 88% purity, 12% crude yield; 224 mg, 86% purity, 25% crude yield).

Reference： [1]Patent: WO2018/192944,2018,A1 .Location in patent: Page/Page column 133; 134; 137

31
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-8-((3-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8- yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
tert-butyl (11S,11aS)-8-((3-((((11S,11aS)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
485 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; for 2h;Inert atmosphere;	1-ethyl-3-(3?-dimethylaminopropyl)carbodiimide (94 mg, 0.79 mmol, 1 eq) was added to a solution of crude 137 (558 mg, 0.49 mmol, 1 eq) and Mal-(PEG)8-acid (292 mg, 0.49 mmol,1 eq) in chloroform (12 mL). The reaction was degassed three times with Argon and stirred for 2 hours and the presence of starting material was no longer observed by LC/MS. The reaction was diluted with dichloromethane and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resultingresidue was subjected to flash column chromatography (Biotage Isolera 50g Ultra; 98/2 to90/10 v/v DOM/methanol in 10 CV). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give 138 (485 mg, 58%). LC/MS, 3 mm method, 1.58 mm (ES+) rn/z (relative intensity) 1709.30 ([M+H], 100). 1H NMR (400 MHz, DMSO-d6) 69.88 (s, 1H), 8.13 (d, J 7.0 Hz, 1H), 8.06-7.92(m, 1 H), 7.85 (d, J = 8.6 Hz, 1 H), 7.68 - 7.04 (m, 9H), 6.99 (s, 2H), 6.89 (d, J = 15.0 Hz,2H), 6.52 (s, 1 H), 5.66 (d, J = 9.4 Hz, 1 H), 5.47 (d, J = 8.0 Hz, 1 H), 5.26 - 4.75 (m, 6H),4.49 - 4.31 (m, 1 H), 4.20 (t, J = 7.6 Hz, 1 H), 3.80 (d, J = 11.9 Hz, 6H), 3.59 (t, J = 7.2 Hz,4H), 3.55 - 3.41 (m, 32H), 3.41 - 3.30 (m, 11 H), 3.21 - 3.09 (m, 3H), 2.48 - 2.28 (m, 4H),2.18-1.08 (m, 24H), 0.84 (dd, J= 15.0, 6.7 Hz, 5H).

Reference： [1]Patent: WO2018/192944,2018,A1 .Location in patent: Page/Page column 139; 141; 147

32
[ 1334177-86-4 ]
4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate [ No CAS ]
4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-(3-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
34%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; at 20℃; for 2h;	EDCI.HCI (46 mg, 0.24 mmol) was added to a solution of 145 and Mal-PEG8-acid (130 mg,0.22 mmol) in CHCI3 (10 mL) and stirred at room temperature for 2 hr. LC/MS shows 78%starting material present. A further 2 eq EDCI.HCI was added in portions to push the reaction to completion. The reaction mixture was washed with water (10 mL), dried (Biotage PS) and evaporated to dryness, under reduced pressure, to leave a yellow solid which was purified by prep HPLC to leave the product I as an off-white solid (90 mg, 34%yield). LC/MS (method B): retention time 1.47 mins, (ES+) rnlz 1445.9 [M+ H].

Reference： [1]Patent: WO2018/192944,2018,A1 .Location in patent: Page/Page column 149; 150; 154

33
[ 1334177-86-4 ]
bis(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5-diyibis(oxy))(11S,11aS,11'S,11a'S)-bis(11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) [ No CAS ]
bis(4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl) 8,8'-(pentane-1,5-diylbis(oxy))(11S,11aS,11'S,11a'S)-bis(11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
20%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; at 20℃; for 0.5h;	EDCI.HCI (56 mg, 0.29 mmol, 3 eq.) was added to a stirred solution of bis-amine (9) (0.123 g, 98 IJmol, 1.0 eq.) and MaldPEGOH (0.128 g, 0.22 mmol, 2.2 eq.) in CHCb (15 ml). 15 The reaction mixture was stirred at room temperature for 30 min then diluted with CHCb (50 ml) washed with H20 (1 00 ml), saturated brine (1 00 ml), dried (MgS04) and evaporated under reduced pressure. Purification by preparative HPLC followed by lyophilisation gave the product as a white foam (0.047 g, 20%). Analytical Data: LC/MS, RT 6.61 min; MS (ES+) m/z (relative intensity) 1205 ([M + 2Ht·, 55).
20%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In chloroform; at 20℃; for 0.5h;	EDCI.HCI (56 mg, 0.29 mmol, 3 eq.) was added to a stirred solution of bis- amine (9) (0.123 g, 98 pmol, 1.0 eq.) and MaldPEGOH (0.128 g, 0.22 mmol, 2.2 eq.) in CHCI3 (15 mL). The reaction mixture was stirred at room temperature for 30 min then diluted with CHCI3 (50 mL) washed with H2O (100 mL), saturated brine (100 mL), dried (MgS04) and evaporated under reduced pressure. Purification by preparative HPLC followed by lyophilisation gave the product as a white foam (0.047 g, 20%). Analytical Data: LC/MS,RT 6.61 min; MS (ES+) m/z (relative intensity) 1205 ([ M + 2H]+, 55).

Reference： [1]Patent: WO2019/34764,2019,A1 .Location in patent: Page/Page column 62; 63; 66
[2]Patent: WO2019/224340,2019,A1 .Location in patent: Page/Page column 64; 67

34
[ 1334177-86-4 ]
bis(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl) 8,8'-(propane-1,3-diylbis(oxy))(11S,11aS,11'S,11a'S)-bis(11-hydroxy-7-methoxy-5-oxo-2,3,11,11a--tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ No CAS ]
bis(4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl) 8,8'-(propane-1,3-diylbis(oxy))(11S,11aS,11'S,11a'S)-bis(11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
18.3%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; chloroform; at 20℃; for 0.75h;	Chloroform (1 0.00 ml) and methanol (0.4 ml) were added to crude 17 (307 mg, 0.254 30 mmol) followed by mal-amido-peg8-acid (339 mg, 0.561 mmol, 2.2 eq) and EDCI (1 07 mg, 0.558 mmol, 2.19 eq). The reaction was allowed to proceed at room temperature for 45 min when completion was observed by LCMS. Ammonium chloride in water (30 ml, 6 mass%) was added and the mixture was stirred vigorously. The mixture was decanted in a biotage phase separation cartridge. The DCM layer was evaporated to dryness under vacuum. 35 The volatiles were removed by rotoevaporation and the crude residue was purified by chromatography (50g Ultra, Biotage, gradient 30/70 to 100/0 of 16% MeOH in DCM I DCMin 1 OCV; Elution at more than 10% of MeOH). All fractions were analysed by TLC (1 0% MeOH in DCM). The pure fractions were pooled. The solvent was removed by evaporation to give 18 (200 mg). LCMS analysis showed traces of mal-pegS-acid, and the material was purified further by preparative HPLC, freeze-dried, aliquoted in dichloromethane, and dried 5 under high vacuum to give 18 as a white solid. The purity was 99.45%. (B, 110 mg, 0.0467 mmol, 18.3% Yield). Analytical Data: LC/MS, 15 min method, RT 5.90 min; MS (ES+) m/z (relative intensity) 1179.5 ([M + 2Hf+·, 100); 1H NMR (400 MHz, DMSO-d6) o 9.92 (s, 2H), 8.16 (d, J = 6.9 Hz, 2H), 7.99 (t, J = 5.7 Hz, 2H), 7.86 (d, J = 8.7 Hz, 2H), 7.55 (s, 4H), 7.18 (s, 4H), 7.07 (s, 2H), 7.00 (s, 4H), 6.79 (s, 2H), 6.50 (s, 2H), 5.48 (s, 2H), 5.23- 4.77 (m, 10 4H), 4.39 (t, J = 7.0 Hz, 2H), 4.22 (dd, J = 8.7, 6.6 Hz, 2H), 4.10 (s, 4H), 3.77 (s, 6H), 3.64 - 3.55 (m, 8H), 3.55- 3.42 (m, 56H), 3.37 (t, J = 5.9 Hz, 6H), 3.28 (t, J = 8.3 Hz, 2H), 3.15 (q, J = 5.8 Hz, 4H), 2.49-2.37 (m, 4H), 2.37-2.30 (m, 4H), 2.17 (s, 2H), 2.09- 1.73 (m, 1 OH), 1.30 (d, J = 7.0 Hz, 6H), 0.85 (dd, J = 15.3, 6.7 Hz, 12H). 15

Reference： [1]Patent: WO2019/34764,2019,A1 .Location in patent: Page/Page column 69; 71; 72

35
[ 1334177-86-4 ]
bis(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl) 8,8'-(propane-1,3-diylbis(oxy))(11S,11aS,11'S,11a'S)-bis(11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ No CAS ]
bis(4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl) 8,8'-(propane-1,3-diylbis(oxy))(11S,11aS,11'S,11a'S)-bis(11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
58.8%	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In methanol; chloroform; at 20℃; for 0.75h;	Chloroform (4.1 ml) and methanol (0.2 ml) were added to 23, followed by mal-amidopeg8-acid (238 mg, 0.394 mmol, 2.2 eq) and EDCI (85.0 mg, 0.443 mmol, 2.48 eq). The reaction was allowed to proceed at room temperature for 45 min when completion was observed by LCMS. The reaction mixture was concentrated (2 ml), loaded on a 3g biotage 20 silica samplet and dried under vacuum. The samplet was loaded on a 25g Ultra Biotage column, and eluted (gradient 10/90 to 58/42 of 20% MeOH in DCM I DCM in 12CV; Elution at around 55% of 20% MeOH). All fractions were analysed by TLC (10% MeOH in DCM). The pure fractions were pooled. The solvent was removed by evaporation to give 24 (250 mg, 0.105 mmol, 58.8% Yield). Analytical Data: LC/MS, 15 min method, RT 6.20 min; MS 25 (ES+) m/z (relative intensity) 1191.5 ([M + 2Hf+·, 1 00); 1H NMR (400 MHz, DMSO-d6) o 9.92 (s, 2H), 8.16 (d, J = 6.9 Hz, 2H), 7.99 (t, J = 5.5 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.68 - 7.42 (m, 4H), 7.39- 7.11 (m, 4H), 7.07 (s, 2H), 7.00 (s, 4H), 6.81 (s, 2H), 6.60 (s, 2H), 5.46- 5.30 (m, 2H), 5.21 -4.79 (m, 8H), 4.39 (t, J = 7.0 Hz, 2H), 4.22 (dd, J = 8.7, 6.7 Hz, 2H), 4.15- 3.88 (m, 8H), 3.77 (s, 6H), 3.65- 3.55 (m, 8H), 3.54- 3.40 (m, 58H), 3.37 (t, J 30 = 5.9 Hz, 4H), 3.15 (q, J = 5.8 Hz, 4H), 2.95-2.79 (m, 2H), 2.57-2.52 (m, 2H), 2.492.37 (m, 4H), 2.37-2.29 (m, 4H), 2.22-2.10 (m, 2H), 2.03- 1.88 (m, 2H), 1.30 (d, J = 7.0 Hz, 6H), 0.85 (dd, J = 15.3, 6.7 Hz, 12H).

Reference： [1]Patent: WO2019/34764,2019,A1 .Location in patent: Page/Page column 72; 73; 75

36
[ 1334177-86-4 ]
bis(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl) 8,8'-(pentane-1,5-diylbis(oxy))(2E,2'E,11S,11aS,11'S,11a'S)-bis(2-ethylidene-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ No CAS ]
bis(4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl) 8,8'-(pentane-1,5-diylbis(oxy))(2E,2'E,11S,11aS,11'S,11a'S)-bis(2-ethylidene-11-hydroxy-7-methoxy-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
516 mg	With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 4h;	DCM (10.00 ml) and methanol (0.4 ml) were added to 36 (393 mg, 0.305 mmol), followed by mal-amido-peg8-acid (380 mg, 0.628 mmol, 2.06 eq) and EDCI (128 mg, 0.668 mmol, 2.2 eq). The reaction was allowed to proceed at room temperature for 4h when completion 15 was observed by LCMS. Ammonium chloride in water (30 ml, 6 mass%) was added and the mixture was stirred vigorously. The mixture was decanted in a biotage phase separation cartridge. The DCM layer was evaporated to dryness under vacuum and the crude residue was purified by chromatography (25g Ultra gradient 15/85 to 100/0 of 20% MeOH in DCM I DCM in 12CV; hold at elution around 48%). The fractions were analysed 20 by TLC (1 0% MeOH in DCM). The pure fractions were pooled. The solvent was removed by evaporation. The residue was purified further by reverse phase preparative HPLC (gradient 15 to 75% water/acetonitrile + 0.01% formic acid) followed by freeze-drying and aliquoted from DCM to give 37 (516 mg, 0.212 mmol, 69.4% Yield) as a white foam. The purity was 97.65%. Analytical Data: LC/MS, 15 min method, RT 6.61 min; MS (ES+) m/z 25 (relative intensity) 1219.7 ([M + 2Hf+·, 100); 1H NMR (400 MHz, DMSO-d6) o 9.92 (s, 2H), 8.17 (d, J = 6.9 Hz, 2H), 8.01 (t, J = 5.6 Hz, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.72- 7.44 (m, 4H), 7.39- 7.10 (m, 4H), 7.05 (s, 2H), 7.00 (s, 4H), 6.76 (s, 2H), 6.66- 6.46 (m, 2H), 5.56 (d, J = 7.1 Hz, 2H), 5.34 (dd, J = 9.7, 5.9 Hz, 2H), 5.21 - 4.70 (m, 4H), 4.39 (t, J = 7.0 Hz, 2H), 4.22 (dd, J = 8.7, 6.7 Hz, 2H), 4.15-4.01 (m, 2H), 3.94 (d, J = 15.3 Hz, 4H), 3.8630 3.72 (m, 8H), 3.60 (t, J = 7.3 Hz, 8H), 3.55- 3.42 (m, 58H), 3.37 (t, J = 5.9 Hz, 4H), 3.15 (q, J = 5.8 Hz, 4H), 2.76-2.56 (m, 4H), 2.46 (t, J = 6.8 Hz, 2H), 2.40 (t, J = 6.5 Hz, 2H), 2.36- 2.29 (m, 4H), 1.96 (q, J = 6.7 Hz, 2H), 1.78 (s, 4H), 1.66 (d, J = 6.6 Hz, 6H), 1.57 (d, J = 8.6 Hz, 2H), 1.30 (d, J = 7.0 Hz, 6H), 0.85 (dd, J = 15.2, 6.7 Hz, 12H).

Reference： [1]Patent: WO2019/34764,2019,A1 .Location in patent: Page/Page column 76; 79; 83

37
[ 1334177-86-4 ]
(2S,2'S)-N,N'-((2S,2'S)-((((11aS,11a'S)-(propane-1.3-diylbis(oxy))bis(7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-8,2-diyl))bis(4,1-phenylene))bis(azanediyl))bis(1-oxopropane-1,2-diyl))bis(2-amino-3-methylbutanamide) [ No CAS ]
N,N-((2S,2'S)-(((2S,2'S)-((((11aS,11a'S)-(propane-1.3-diylbis(oxy))bis(7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-8,2-diyl))bis(4,1-phenylene))bis(azanediyl))bis(1-oxopropane-1,2-diyl))bis(azanediyl))bis(3-methyl-1-oxobutane-1.2-diyl))bis(1-(3-(2.5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18.21,24-octaoxaheptacosan-27-amide) [ No CAS ]

Yield	Reaction Conditions	Operation in experiment
		A solution of Super-Hydride (0.37 ml_, 1 M in THF) was added dropwise to a solution of SEM- dilactam 4 (200 mg, 0.15 mmol) in dry THF (5 ml_) at -78C under an argon atmosphere. The addition was completed over 5 minutes in order to maintain the internal temperature of the reaction mixture constant. After 40 minutes, an aliquot was quenched with water for LC/MS analysis, which revealed that the reaction was complete. Water (20 mL) was added to the reaction mixture and the cold bath was removed. The organic layer was extracted with CH2CI2 (3 x 50 mL) and the combined organics were washed with brine (100 mL), dried with MgS04, filtered and the solvent removed by rotary evaporation under reduced pressure. The crude product was dissolved in dry CH2CI2 in a round bottom flask purged with argon. Mal-dPEGs-OH acid (31.4 mg, 0.148 mmol) and EDCI.HCI (28.5 mg, 0.148 mmol) were added and the mixture was left to stir at room temperature until complete. The crude material was purified by reverse phase HPLC to afford product 6 with 82% purity (37 mg, 1 1 % yield). LC/MS 1.38 min (ES+) m/z = 1 101 .05 [Af + 2H]2+- ; LC/MSismin 5.84 min (ES+) m/z = 1 101.05 [Af + 2H]2+ .

Reference： [1]Patent: WO2019/96788,2019,A1 .Location in patent: Page/Page column 45; 47

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CAS No. :	1334177-86-4	MDL No. :	MFCD13184956
Formula :	C₂₆H₄₄N₂O₁₃	Boiling Point :	-
Linear Structure Formula :	-	InChI Key :	ARKUDJPBDMAVBL-UHFFFAOYSA-N
M.W :	592.63	Pubchem ID :	51340955
Synonyms :		Chemical Name :	1-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oic acid

Signal Word:	Warning	Class:	N/A
Precautionary Statements:	P261-P305+P351+P338	UN#:	N/A
Hazard Statements:	H302-H315-H319-H335	Packing Group:	N/A
GHS Pictogram:

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H412	Harmful to aquatic life with long-lasting effects
H413	May cause long-lasting harmful effects to aquatic life
H420	Harms public health and the environment by destroying ozone in the upper atmosphere

[ CAS No. 1334177-86-4 ] {[proInfo.proName]}

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[ 1334177-86-4 ] Synthesis Path-Downstream 1~37

Related Functional Groups of
[ 1334177-86-4 ]

Ethers

Amides

Carboxylic Acids

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[ 1334177-86-4 ]

Pyrrolines

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