US 20150322155A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0322155A1 Zhao (43) Pub. Date: Nov. 12, 2015 (54) ACETYLENEDICARBOXYL LINKERS AND A647/48 (2006.01) THEIR USES IN SPECIFIC CONUGATION C07C 237/52 (2006.01) OFA CELL-BINDING MOLECULE A6II 45/06 (2006.01) A 6LX39/395 (2006.01) (71) Applicants: Dr. Robert Yongxin Zhao, Lexington, C07D 207/216 (2006.01) MA (US); Suzhou M-conj Biotech Co., C07D 417/4 (2006.01) Ltd, Suzhou (CN) (52) U.S. Cl. CPC .......... C07K 16/2863 (2013.01); C07D 207/46 (72) Inventor: Robert Yongxin Zhao, Lexington, MA (2013.01); C07C 59/76 (2013.01); C07D (US) 417/14 (2013.01); C07C 237/52 (2013.01); A 6LX 45/06 (2013.O1): 46K.39/3955 (73) Assignees: Sushou M-conj Biotech Co., Ltd, (2013.01); A61 K %2, psets, A61 K Suzhou City (CN); Robert YongXin 2039/505 (2013.01) Zhao, Lexington, MA (US) (57) ABSTRACT (21) Appl. No.: 14/799,666 The present invention relates to novel acetylenedicarboxyl (22) Filed: Jul. 15, 2015 linkers used for the specific conjugation of compounds/cyto toxic agents to a cell-binding molecule, through bridge link Publication Classification ing pairs of thiols on the cell-binding molecule. The invention also relates to methods of making such linkers, and of using (51) Int. Cl. Such linkers in making homogeneous conjugates, as well as of C07K 6/28 (2006.01) application of the conjugates in treatment of cancers, infec C07C 59/76 (2006.01) tions and autoimmune disorders. Patent Application Publication Nov. 12, 2015 Sheet 1 of 11 US 2015/0322155A1 O =N-k O MISC ch"). Na/THFOS2 nohn')^ 'e, -N- soh-)^'eO NN - Nh-)^^eO, triH/Pd/C 4 5 H.Nhn- ^^e 20%HCIDioxane H.Nhn- ^"O 6 O O NHS/EDC O O. O. HO - E-I-OHOH by is QNNO i = it O-N 8 O 9 O 7 O O OH - pH 6-8.5> to YS/O \) N - E-l kn-O ^? O H 10 H O Drug-NHrug-IN 12 NH O knO w -o-EcoMA' Drug 1 ?t- m 11 ^ Drug H O O -Antibody be Drug e N YS/O M. 1, \l h O ^ N D rug O - S.a 2S N M mAb 12 n = 1-20, m = 0-50 Figure 1. Patent Application Publication Nov. 12, 2015 Sheet 2 of 11 US 2015/0322155A1 O C N y 1, (5 mol%) '. E-é' c'Yn + -Si-E-Si- a O / V 0 °C-RT, DCM 15 13 14 90% 07 OH HO1 NO O O O c'Yn CI BrMg-E-MgBr THFHe 13 O 16 OY OH HO1SO 15 H R 8 H Drug-NH N M S-E-4 M N antibody EDC/DMA Drug? O O YDrug 17 TCEP H R 9 H Drug N N Drug O s Z O n 18 mAb O H R 9 H -H.N-S)-R R-e 19 O Ray = Q C EDC/DMA O O 2O H Q 9 H - antibodyPle O N N O Drug-O-NHAESA TCEP O S S O buffer AZ 21 mAb H O O H N N N Dru O S S O n = 1-20 mAb 22 Figure 2. Patent Application Publication Nov. 12, 2015 Sheet 3 of 11 US 2015/0322155A1 O O NHS/EDC O O O HO-Is I-OH - = - 8 O H O HN Noh O H O O HO 23 - El-N N-oh --e O H H 6 NG) 24 O E O EDC NHS/EDCe N-O DMA H O E. Q-) 25 H 9 H Drug-NH2 Drug-N He EDC/DMA O Q- HBOc 20%HC O H -e Drug EtOAc O H O N Nsu- Drug O H ) \ H O NH S S (-a NH mAb Figure 3. Patent Application Publication Nov. 12, 2015 Sheet 5 of 11 US 2015/0322155A1 mAb 49 n=1-20 Figure 5. Patent Application Publication Nov. 12, 2015 Sheet 6 of 11 US 2015/0322155A1 HO O N.H R. Q O H O O OH NHS Y\-OVNO 3 H 5 HN-life if-N-N-NY'NY'y?H H O EDC/DMANS NHBOc (^NiBoe O OH OAc O % 2N NY UN / N OH NH W S H O O O O Rui alo VN3 H YN-I(5 H = INH 5 NH 3 O O2V HO NHBO S^NHBoe 52 HN s N Ac92 Y. Q 'O to-vy.'O H S O N 20%TFA CH.Cl HN Cls N, Acga Y YO O H S OH H. O. OAc O O y 2% N Ny OH NH OS S H O O H. E. O N O O H O ov'Y^i3 H 5 H = f^N'Y',H H NH S^N, 53 Figure 6. Patent Application Publication Nov. 12, 2015 Sheet 7 of 11 US 2015/0322155A1 53 TCEP mAb N HNHOV C % cy-g-NS N 8 ON OH O H O OA O o Mrs.5 wN S-Wy OH NH NH O s N 4.O N ... N alo 3 O H Whil NHO S S S^N, 54 mA OH O O H.Nf-')"54 O --pH 7.5 "(Y)O 2 H = H (-O-)-OH2 55 DCM/DMF(COC) 'r-\)O 2 = (-o-)c S6 S Q O OAc O NS C.N S ? OH D=1-20 mAb 59 Figure 7. Patent Application Publication Nov. 12, 2015 Sheet 8 of 11 US 2015/0322155A1 Y-Ml, CICO1. J. OCCl3 4.AsY-y Maytansinol visa 'Y'AA Orno O-SO ZnTr/DMF/EN 60 61 62 O O O "re- E ()." (COC)/DCM/DMF then 62/EtN Figure 8. Patent Application Publication Nov. 12, 2015 Sheet 9 of 11 US 2015/0322155A1 Hor-Yoh TsC/Pyr Tsor-shots NaN/DMF N-(--)N, Hyde 1 67 68 MeOH O 8 O - ? O MMAF ust \}, NH, EDC/DMA HN-- 9, the Sh, NH, EDC/DMA N s N NHBO V-n OCH 3 1). 20%HCI 2). TCEP 74 in Dioxane mAb mAh 75, n=1-20 Figure 9. Patent Application Publication Nov. 12, 2015 Sheet 10 of 11 US 2015/0322155A1 Oa-OneO O O LiB skif O O Li-S-Li - - - 76 77 HO 15 OH OH O Q H R Y W N t N O 51 > W N S W NH O | EDC/DMA OH H O OAce O N Qk N sy H O 79 N W N S / NH O OH OH Q O OAc O f ON Ny OH TCEP N Osn S H O - > | k OH Q 4.O OAe y O o,N S S H O 15 -NHS AD N-O O. O.I =l9 O y 28 EDC/DMA NO O pH 7.5 o O O 81 O H a O H. O. 51 HO N. ?ny VE &^YNO 'oh EDC/DMA NHBoc O O W^NHBoe 82 OH S) N O OAe O N.H is O Q.f N 2N // N OH O S1NO H S S H O NHBOc 1) TFA/DCM WN OH (S) HO Q O OAC O N-2' 2) TCEP/mAb 4.c S.yN OHGN-N-n Yo O W O NHBOc 83 OH S) N O NNN O f N OH O O H N O s ? N O S S H O NH mAb OH Q OAc O N- ? S 4. 2N H N-a O N O CN y // N OH O NYH r \S S H O NH 85 2 Figure 10. Patent Application Publication Nov. 12, 2015 Sheet 11 of 11 US 2015/0322155A1 Tumor volume of BALB/c Nude Mice Bearing NCI-N87 Xenograft Tumor 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 -O PBS 600 -- T-DM1(5 mg/kg) 500 -V Compound 91 (5 mg/kg) 400 -0 Compound 93 (5 mg/kg) 300 200 100 5-5- O 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 Days (after Initiation of Treatment) Figure 11 US 2015/03221 SS A1 Nov. 12, 2015 ACETYLENEDICARBOXYL LINKERS AND 0004. Therefore, biotechnology companies and academic THEIR USES IN SPECIFIC CONUGATION institutions are highly focusing on establishing novel reliable OFA CELL-BINDING MOLECULE methods for site-specific ADC conjugation. So far, there are several approaches developed in recent years for site selective FIELD OF THE INVENTION ADC preparation (Panowski, S, 2014, mabs 6, 34). They 0001. The present invention relates to the preparation of include incorporation of unpaired cysteines, e.g. engineered novel linkers used for the specific conjugation of compounds, reactive cysteine residues, called THIOMAB from Genen in particular, cytotoxic agents to a biological molecule. The tech (Junutula, J. R. et al 2010 Clin. Cancer Res. 16, 4769: present invention also relates to methods of making cell Junutula, J. R., et al 2008 Nat Biotechnol. 26,925-32; U.S. binding agent-drug (cytotoxic agent) conjugates in a specific Pat. Nos. 8.309,300; 7,855,275; 7,521,541; 7,723,485, manner comprising either modification of drugs with these WO2008/141044), genetically introduced glutamine tag with linkers first, followed by reaction with prepared cell-binding Streptoverticillium mobaraense transglutaminase (mTO) agents; or modification of cell-binding agents with these link (Strop, P., Bioconjugate Chem..., 2014, 25,855-862; Strop, P. ers first, followed by reaction with drugs. et al., 2013, Chem. Biol. 20, 161-167; U.S.