US 20190127401A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2019 /0127401 A1 ZHAO et al. (43 ) Pub . Date : May 2 , 2019 ( 54 ) CHARGED LINKERS AND THEIR USES FOR Publication Classification CONJUGATION (51 ) Int. Ci. CO7F 9 / 30 (2006 . 01 ) (71 ) Applicant : HANGZHOU DAC BIOTECH CO . , CO7F 9 /572 ( 2006 . 01 ) LTD ., Hangzhou ( CN ) C07F 9 /6558 ( 2006 .01 ) A61K 47/ 68 ( 2006 . 01 ) (72 ) Inventors : Robert Yongxin ZHAO , Hangzhou C07F 9 /6561 ( 2006 . 01 ) ( CN ) ; Xing LI, Hangzhou (CN ) ; CO7F 9 / 32 ( 2006 .01 ) Yuangyuang HUANG , Hangzhou A61K 38 /07 ( 2006 . 01 ) (CN ) ; Qingliang YANG , Hangzhou CO7F 9 /58 ( 2006 . 01) ( CN ) .) U . S . CI. CPC . C07F 9 / 301 ( 2013 .01 ) ; CO7F 9 / 58 ( 2013 .01 ) ; CO7F 9 /65583 ( 2013 .01 ) ; A61K (73 ) Assignee : HANGZHOU DAC BIOTECH CO ., 47/ 6817 ( 2017 .08 ) ; A61K 47 /6851 ( 2017 .08 ) ; LTD . , Hangzhou (CN ) A61K 47 /6811 ( 2017 .08 ) ; A61K 47 /6809 ( 2017 .08 ) ; A61K 47/ 6807 ( 2017 .08 ) ; A61K (21 ) Appl . No. : 16 / 228 , 437 47 /6803 (2017 .08 ) ; A61K 47 /6889 ( 2017 . 08 ) ; CO7F 9 /65616 (2013 .01 ) ; CO7F 9 /3211 ( 2013 . 01 ) ; CO7F 9 / 3217 ( 2013 .01 ) ; A61K ( 22 ) Filed : Dec. 20 , 2018 38 /07 ( 2013 . 01 ) ; C07F 9 / 302 ( 2013 . 01) ; C07F 9 / 6561 ( 2013 .01 ) ; C07F 9 /572 ( 2013 .01 ) Related U . S . Application Data (57 ) ABSTRACT (62 ) Division of application No . 15 / 118 , 726 , filed on Aug . Cell binding agent -drug conjugates comprising phosphinate 12 , 2016 , filed as application No . PCT/ CN2014 / based charged linkers and methods of using such linkers and 072769 on Feb . 28 , 2014 . conjugates are provided . Patent Application Publication May 2 , 2019 Sheet 1 of 22 US 2019 /0127401 A1 H - P - H HMDS C OSiMe3 2 COOEt MezSi . , BrBr ? NH * 120 °C, 2h | OSiMe, J 50 °C, 2h . 120 °C , 5h |Mezsio 42 % from 1 Brave CH (OEt ) ; BrenP 0 OH 140 °C OEt 5 HSAC Acs 1 ). IN NaOH DIPEA VET 2 ) , pH 7 Na OH OEt PySSPy , 85 % OH NHS/ EDC Drug - SH Drug HCI/ DMA N pH 4 ~ 7 Antibodies Drug or Proteins NA MAL j n = 1 - 20 Antibodies or Proteins I ' N SL hmAb OH e Qeven in g ehele11a omas 11 Fig . 1 . Patent Application Publication May 2 , 2019 Sheet 2 of 22 US 2019 / 0127401 A1 HSAC 1 ) . NaOH ACS . Et 2 ), pH 7 OH 4 DIPEA PySSPy , 85 % NHS /EDC Drug -SH 82 % pH 4 ~ 7 Drug Antibodies Drug - S. or Proteins fu mAh mesinin bir pa r ently Jnunder = 1 ~ 20 Fig. 2 . Et f 120 °C NaH / DMF then 5 , 6 OEt DMA 18 Drug - SH Drug Antibodies or Proteins S pH 4 ~ 7. 5 OH 19 pH 5 .5 ~ 8 .5 Drug mAb N4 OHHIJn = 1 - 20 20 Fig . 3 . Patent Application Publication May 2 , 2019 Sheet 3 of 22 US 2019 / 0127401 A1 HMDS I OSIMez 1 Br H - PH HPOSiMe3 1 a coo?t Mez Sio Br ONH , + 120 °C 1 Mez / 50 °C , 26 MezSio " 120 °C , 5h 1 2 43 % in 3 steps CH (OEt ) ; OEt minh OH cong140 °C ngÓEt mga HSAC Br 1 ) . NaOH DIPEA 2 ) , pH 7 OH 72 % from 4 ÓFt PySSPy, 85 % NHS/ EDC Drug - SH Drug 82 % ??? ( - ? pH? 4 -? 7 ?OH ? Antibodies or Proteins 2 .win in Inthe = more1 20 Fig . 4 . HMDS OSiMe3 H - P - H HP a cooEt | MezSio Br Br O?NH4+ 120 °C OSiMe3 50 °C , 2h TOEt 120 °C , 5h Mezsio 43 % in 3 steps when one or more thanOEt HSAC P 1 ). NaOH DIPEA ÁCS OEt 2 ), pH 7 T OH homewhere to PySSPy , 85 % ripulinkyOH 33 NHS/ EDC Drug -SH Drug S 82 % pH 4 ~ 7 OH 35 1 2 Fig 5 . Patent Application Publication May 2 , 2019 Sheet 4 of 22 US 2019 /0127401 A1 HMDS I ?? - ? HP OSiMe3 2 COOEE (MezSio Br Br ?NH + 120 °C | OSiMe31750°C , 2h Mezsio 120 °C , 5h 43 % in 3 steps CH (OEt ) ; Br OEt 140 °C Br OEt at the37 end 38 of the HSAC 1 ) . NaOH DIPEA OEt 2 ) , pH 7 S - OH PySSPy NHS /EDC Drug -SH Drug 83 % pH 4 7 So Antibodies Drug S . or Proteins ???? mAh * OHfententiae Jnr 1 - 20 Fig. 66 . X CH (OET ) ; oEt 140 °C VOET OH 37 OEt 38 OEt C2H30CSSK 1 ) . NaOH OEt SO C2H5OH 2 ), pH 7 N S- S OH bet 45 PySSPy 411 NHS/ EDC Drug - SH Drug ons83 % herseling og pH 4 - 7kontinuing Antibodies rug nu mAb or Proteins 44 In = 1 ~ 20 Fig . 7 . Patent Application Publication May 2 , 2019 Sheet 5 of 22 US 2019 / 0127401 A1 ÇO2Et H - P - H AIBN /MeOH ÇO Et ÓH CO2Et 80°C , 5h HP CO , Et H1 CCO Et 46 47 OH 48 HMDS Br - Bi CH (OET ) 120 °C , 9h 140 °C 49 OEt 50 HSAC ACS . 1 ) . NaOH DIPEA 2 ), pH 7 .5 Et 51 PySSPy OH 52 0 O NHS /EDC 0 Drug- SH Drug sr 82 % ? pH 4 - 7 's -Sriwi o Nog inn i en OH 54 Antibodies mAb or Proteins Nam Jn = 1 ~ 20 Fig . 8 . Patent Application Publication May 2 , 2019 Sheet 6 of 22 US 2019 /0127401 A1 CO2Et AIBN /MeOH ÇO2Et H - P - H HP HB OH CO2Et - 80 °C , 5h | CO, Et CO Et 1 46 OH 48 HMDS Br - brBr CH (OET ) 3 Br OEt 120 °C , 9h 140°C OEt 56 EtOCSSK 1 ) . NaOH OEt 2 ) , pH 7 . 5 OH InTfz / EtOH OEt 58 PySSPy OH 59 NHS / EDC Drug- SH Drugs 82 % pH 4 - 7 OH 61 Antibodies Dru or Proteins Normab OH n = 1 - 20 Fig 9 . EtOCSSK 1 ) . NaOH EtOH 2 ), pH 7 .5 mylimahora en verlinktenPySSPy OH 59 Drug -SH Drug Antibodies pH 4 ~ 7 OM or Proteins 1 61 Drug VAL [user findestensn = 1 - 20 Fig 10 . Patent Application Publication May 2 , 2019 Sheet 7 of 22 US 2019 /0127401 A1 Bn OH /DCC /DMAP H - P - H HMDS ( ., OSiMe3 | Br?cook int O -NH ,+ 120°C , 2h ! SiMe3 / 50 °C , 2h Ôu THF , RT , 16h 2 Hp BrBr Brno 120 °C , 5h got OBA 65 OBn 66 HSAC Acs 1 ). 5N HCI, 100°C 2 ) , pH 7 OH DIPEA OBn PySSPy , 85 % NHS/ EDC Drug -SH 82 % pH 4 - 7 Antibodies Drug S or Proteins ÓH NAn = 1 ~ 20 or : oynamaktan olimline 69 2 11 Fig . 11. Patent Application Publication May 2 , 2019 Sheet 8 of 22 US 2019 / 0127401 A1 I BnOH /DCC /DMAP PH HMDS HP OSiMe3 1 Brycod + 120°C , 2h O SIMez ) 50 °C , 2h THF, RT, 16h Br Br OBn 120 °C , 5h OBn a les66 est le 15 O 1 ) . 5N HCI, 100°C 2 ) , pH 7 NaH /DMF PySSPy , 85 % NHS / EDC Drug - SH Drug 82 % pH 4 - 7 eins or : Antibodies or Proteins INSHj n = 1 ~ 20 - Painting a 71 Drug -SH Drug pH 4 ~ 7 and| H the| n = 1 - 20 Fig . 12 . Patent Application Publication May 2 , 2019 Sheet 9 of 22 US 2019 / 0127401 A1 Quintocounter Jn = 1 - 20 Drug - - SSPy or DTT or TCEP Drug - SSPYNO2 pH 4 - 8 . 5 Jn = 1 - 20 YpH 5 - 8 .5 pH 5 - 8 . 5 / Drug Drug N 1 Jnr 1 - 20 11 presente ne DU 73 74 Fig . 13 . HMDS I I H - P - H cooEt MezSio . o Brio Br ONH + 120°C , 2h IHP .OSiMez :Mel ) 50 °C ,. 2h2h OEt Mezsio 3 120 °C , 5h m = 1 - 24 CH (OET ) OEt 3140 °C Et EtOCSSK Im OH 75 m OTt EtOH 1 ) . IN NaOH OEt 2) , pH 7 ' N OH OEt PySSPy , 85 % OH AtMatte 7 ?? NHS/ EDC N" S - S ' m = 1 ~ 24 82 % Im OH Fig . 14 . Patent Application Publication May 2 , 2019 Sheet 10 of 22 US 2019 / 0127401 A1 OH 4 OEt 5 87 Antibodies 3 or Proteins minimal things are comingOH Hj n = 1 ~ 20 pH 4 - 7 l Nan OHÀ Jn = 1 – 20 Fig . 15 . Patent Application Publication May 2 , 2019 Sheet 11 of 22 US 2019 / 0127401 A1 - H - - - - NaN3 IN NaOH Pd / C / H , DMF OEt OH CH3OH OEt 86 87 NHS /EDC HN OH OH und DMA geht DMAes Antibodies ? ) or Proteins= ( H )|. n = .1 - .20 DrugNHNH Drug pH 4 - 5 N NA se non presente trabalhando H Jn =come 1 ~ 20 EDC I UN DMA 0 0 Fig. 16 . Patent Application Publication May 2 , 2019 Sheet 12 of 22 US 2019 / 0127401 A1 0 H; ??? » NHNH 0 jH DMA NHS / EDC /DMA ??? ?? ? ?????????? ???? HCI/ Dioxane NHNH CNHNH, fyrir 100 ok noggly 6Hen ??? Drug Ri DrugN pH 4 5 OH R = H , Me, Et, orPh , .. »mAb + TCEP– ??????????? ?? ? : ?? ? mAb - - SH E yv42 0H n ? = 1? 20 ???????????? ??? ?????104 ?? ? - Fig . 17. Patent Application Publication May 2 , 2019 Sheet 13 of 22 US 2019 / 0127401 A1 an a word ener en en ?? ?? 1N NaOH O OEt - Na2CO3/ DMF OEt 105 106 OH NHS / EDC 0 . Antibodies or Proteins DMA OH 10107 ° DrugNHNH , H | n = 1 - 20 pH 4 - 5 Roman 108 to urdu N Hjn = 1 - 20 gourmalin hom op grond van het leven en hoe NHS /EDC DrugN DMA are not tomention Fig . 18 . Patent Application Publication May 2 , 2019 Sheet 14 of 22 US 2019 / 0127401 A1 HMDS OSIMez 1 Br ( 6 eq ) H - P - H Br Br CH (OEt ) ; O?NH + 120°C , 2h ! OSIM 120 °C , 5h ?? 140 °C 0 0 E1CNH( 0 . 8 eq ) O / NaCO3 + Eto OEt . N3 . in 14 DMF, then NaN3, miten113 ei38 % from112ole personenEt Ó ' 115 1 ) . PPh3/ DME /HCI ( 1M ) , 85°C H2N Drug - -COOH 2 ) . Tol.