US 20150250896A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0250896 A1 Zhao (43) Pub. Date: Sep. 10, 2015 (54) HYDROPHILIC LINKERS AND THEIR USES Publication Classification FOR CONUGATION OF DRUGS TO A CELL (51) Int. Cl BNDING MOLECULES A647/48 (2006.01) (71) Applicant: Yongxin R. ZHAO, Henan (CN) Ek E. 30.8 C07D 207/216 (2006.01) (72) Inventor: R. Yongxin Zhao, Lexington, MA (US) C07D 40/12 (2006.01) C07F 9/30 (2006.01) C07F 9/572 (2006.01) (73) Assignee: Hangzhou DAC Biotech Co., Ltd., (52) U.S. Cl. Hangzhou City, ZJ (CN) CPC ........... A61K47/48715 (2013.01); C07F 9/301 (2013.01); C07F 9/65583 (2013.01); C07F (21) Appl. No.: 14/432,073 9/5721 (2013.01); C07D 207/46 (2013.01); C07D 401/12 (2013.01); A61 K3I/454 (22) PCT Filed: Nov. 24, 2012 (2013.01) (86). PCT No.: PCT/B2O12/0567OO Cell(57) binding- agent-drugABSTRACT conjugates comprising hydrophilic- S371 (c)(1), linkers, and methods of using Such linkers and conjugates are (2) Date: Mar. 27, 2015 provided. Patent Application Publication Sep. 10, 2015 Sheet 1 of 23 US 2015/0250896 A1 O HMDS OSiMe 2n O Br H-B-H HPC 3 2 COOEt essiop-\5. E B to NH 120 °C, 2h OsiMe3 J 50 °C, 2h eSiO OEt 120 oC, sh 1 2 3. 42% from 1 Bra-11a1'oet - Brn 11-1 or a 1-1 or ÓH 140 °C ÓEt ÓEt 4 5 6 - --Messio. 8 B1a-Br aus 20 cc, hP-1}^-'ot Br1-Y. OEt 3. OEt 7 s a OH OEt 8 S O. O. O. 1). 1N NaOH EtOCSSK ka. He He Moss-a-NM-n1'oet 2), pH 7.5 EtOH OH OEt PySSPy 9 (lisN S1 n^^ --\ou9 -->NHS/EDC ÉllsN S1 Y}-\--\'o-'o' OH OH OH OH O 10 11 Drug-SH, pH 4-7, then O O O Antibodies,noodles, or Proteiroteins "S-S-1-N-N-NOH OH H n = 1 - 20 12 Fig. 1. The synthesis of a di-phosphinic acid linker. Br1Nile -/n aus,OEt -CH(OEt), > Br-al-M1'oetQ Q S-E-M1-\'oet OH OEt OE OEt is OEt OEt 14 2, 120 °C, 2h O O. O. O. EOCSSKw B1a-rB B^{-\-E-MB-n1'oetOH OEt OEt EtOH S 1). 1N NaOH O O. O. O. Fig. 2. The synthesis of a tri-phosphinic acid linker Patent Application Publication Sep. 10, 2015 Sheet 2 of 23 US 2015/0250896 A1 Q 9 O O CH(OEt) O O O O Br-a-ra--M. 1Soet --> B^-\l-MB-n-'oet OH OEt OEt is OEt OEt OEt 19 2, 120 °C, 2h O EtOCSSK Br1N1 - Bru-N-Pro-YrOH oEt OEt OEt OEt EtOH S 1). 1N NaOH 1oss?\-ra-rá-B-B-B-4-1. OEt Her2), pH 7.5 OH OEt OEt OEt PySSPy O O O I I O ÖH OH OH OH 23 O Fig. 3. The synthesis of a tetra-phosphinic acid linker Br1a--M.tallas OE n-i-M}^' of BizshO O Na2CO OEt OEt 13 OEt OEt - O --> DMF, 50 °C O O 8 NHC O O NHSFEDC pAZS-\-\-rottO OEt OEt O -reflux o- Nr-uo"ÖH oH HerDMA 24 25 O O O-N Drug-SH Drug O O e s Cr-y-O OH OH O pH 4 ~7.5 Cr-Y- 26 Antibodies or Proteins Drug O NH mAb pH 5.5-8.5 -N1\-1\--/n O OH OH O n = 1 - 20 Drug-SH 28 9 O O O O O O s Antibodies H O OH OH O pH 6.5-8.5 O OH OH O n = 1 - 20 Fig. 4. The synthesis of a di-phosphinic acid linker. Patent Application Publication Sep. 10, 2015 Sheet 4 of 23 US 2015/0250896 A1 Hs-a-o-O BY-1a-Br Bra-sa-to- 30% H.o2V2 Bra-9-n-to 51 DIPEA, 45°C 52 HOAc/KMnO(cat) 53 0.5 M NaOH { Drug-SH, then He -e as mAb or protein O Drug-YS 56 O O O O O Hs-n-to- Br1n 1B BMs-n-o 30%KMO HOV2 B-1S-1 51 DIPEA, 45°C 57 nu 58 HOA O O O O O HSAC M Y O.S M NaOH SS NHS/EDC DIPEA AcS S O PySSPy,w pH 7.2 NS-S2N, MNS OH HeDCM O 59 O 60 O Drug-SH then s^^ mAh or proteina Drug O O O Ms-\-y -eBr1N1Br, Br-1-s^^s^1/ 64 DIPEA, 45°C 65 30% HO R. HSAe it all it HOA - Br^1-a/n^1 HeDPEA Aesn1-a/n^1 / O O 66 O O 67 r O O O 0.5 MNaOH wa I NHS/EDC PySSPy,SMNne pH 7.2 (CaMaMa'ohNS-S NSEge -e then mAb I'll S N or protein Fig 6. The synthesis of mono-, and di-sulfonyl linkers Patent Application Publication Sep. 10, 2015 Sheet 5 of 23 US 2015/0250896 A1 O MSHw O Br1N1 Br O Br O1 Y - HS-sa-to- e Br1-sa/s-a-o- 51 DIPEA, 45°C 71 72 O O newN"- Brain/S-\'osiO 9 OS M NaOHNag s-Ma-onif Q NHS/EDC 18-crown-6 O 73 O 74 Drug-SH, then O O O -mAb or-o- protein Drug SMS-\-S^ O 0 n = 1 - 20 -e Br M S al O -30% H.O.> Br 1-allS O1 S1 DIPEA, 45°C 77 A. 78 HSA O O OS M NaOH C O O NHS/EDC -eDIPEA, 60°C As YS o? PySSPy, pH 7.2 NS-S a o- DCM D O O 80 Drug-SH,S thenT- Drug SS. Ms N mAb or protein O H 8a n = 1 - 20 B-1o-S-HS/N HS-s^1O o? BY-1N1 On AsaoO S1 DIPEA, 45°C 71 DIPEA, 45°C 82 O O O O 30% HO HSAc HOAc O 83 O O 0.5 M NaOH 1S O O O NHS/EDC PySSPy,-e pH 7.2 ClSNs-s NM-ul-\'oh - DM > O O 85 O O O Drug-SHES- fS Ys 19-v then mAh Drug N or proteino O 87O Hn Fig. 7. The synthesis of mono-, and di-sulfonyl linkers Patent Application Publication Sep. 10, 2015 Sheet 6 of 23 US 2015/0250896 A1 (ho^-oh - HO-o-o-e-C'sO Tso-hoa-o-O 88 m = m1-30 aro<O 89 90 O . no ->AcSH Acs-hoa-o-O in0,5 N NaOH Bry S \-hoo-k 30%HOA H2O, yr. 91 B1a-Br 92 y O O O O 1N1-S EtOCSSK'e MY s^-Sa-ho-ohs^-in-ho OH -o-SSNs Br i-hoon EtOH O O m PySSPy, pH 7.2 O O O O O -- (ys, Min-ho-oh SSPS- (ys, Min-ho-o- N s & 'm DMA S O O O O Br1-in-ho-oh FNN-a-ho-ohEtN y NSFPSNHS/EDC O "97 O m. DMA O O O azyan O Br^-sa-ho-o- YoDMF 7 Az O 100 O 101 30% HO O O 9 O O O. HOA, 60°Ca UN-NSy, r-i-hoon NHS/EDCDMA NYi-hoo O2 O 103 m = 1 - 30 O y HS101al O O1a-'-HO CB/PPh.4 3 Br. Br He Br aul Y ( O l, - DCM as OY pp. Ytors O 104 m = 1--30 105 106 30% HO, SRP R ww S R O HOA > Bra-to-vaul-EtOCSSk O to Mo'ss-to-vy-\'o m-1 107 m 108 '6) R NHS EDC pHpsp- 7.2 SNSSS Vtory-longm-1 DMA 110 m = 1 - 30 Fig. 8. The synthesis of mono-Sulfonyl linkers containing polyethylene glycols. Patent Application Publication Sep. 10, 2015 Sheet 7 of 23 US 2015/0250896 A1 O SH O Br B Tso-foot- HS-EtN Hsn-sa-ho-o- BryllO C-S-foot 90 m = 1 -30 11 EtN, 50°C 12 30% HO,SCS Br R O EtOCSSK J.S Oif 9 O 0.5 M NaOH HOA o'oh to Mo su/~\hoon PySSPy, 113 Ö O 114 pH 7.2 ( VS Qal O NHS/EDC FN 's-?s-in-hoon's 5 O 115 C. A.A ... is . O Sinho'ohO O ES-N-\-shoot NHSEDC 117 ", DMA O He NHS/EDC HOAc, 800C ONvvi{oons O DMA 121 O CBr/PPh B Br HSMSO M ( HO1n-NH DCM4f 13 OYi 138 p 104 m = 1--30 123 DIPEA 30% HO Q9 Q9 Q EtOCSSK S OW. V.O O -eHOAC Br-to SS-N-au-V// Y.EtOH S. M.O s/ton) SyllQ-M1 m-1 f m-1 125 126 0.5 M NaH2 O O. O. O '6)Pyssey, SN’s Svory-roll W pH 7.2 m-1 O 127 NHS/EDC/DMA Fig. 9. The Synthesis of di-sulfonyl linkers containing polyethylene glycols. Patent Application Publication Sep. 10, 2015 Sheet 8 of 23 US 2015/0250896 A1 O O O O 1nSS/NS.2 O O O O. O HaskOBn Br.120 °C, 5h Bill Bruin?O OBnn'o-e- N-S^^*o-8 ÖBn 129 13 131 S O O O EtOCSSK 20%oV CHSOHWDCM 13 SV3 O OBn PySSPy, pH 7.2 O allO O 20% CHSOH 9 O O O OBn 136 M 8 OH 137 DMA 139 m= 1 - 20 O O NaH/DMF RO Q Q Azisi,15 O thenen so,9 AlSz.--SabaoO O OBn 140 20then CHSoHDCy pH 7, 100'C O B-?tO NHS/EDC N1-\- OH - DMA e.