View
5
Download
0
Category
Preview:
Citation preview
(I~~ @Nyyj~y~gqg~&~yg N@qg yt"1tit+@tit~@Nt ~~IIII II II I I III I II IIII I II II I II III II I II
(10) NO~i.'4'42014 43 8 6 8 (06.03.2014) ~(po WO 2014/032257 A1
(5» Hl@+@J&4%:C07K 1/04 (2006.01) C07K 7/08 (2006.01)
(2» HR@%8: PCT/CN2012/080792
(22) gg'@gg: 2012 Q 8 Q 30 H (30.08.2012)
(25) @%%n: 03C
(26) /h~+mn: 03C
(7» @%A (~et~%~~&j.e%@18e~~):8@II4k+%++@'g@/~%+~ (HYBIO PHARMACEUTICAL COLTD) IcN/cN]; +~~)' 4gi@&JII4i4iiJJ x Akron+j-+K + x N WC f/3 &F7 K 4' & g48I )z, Guang-
dong 518057 (CN).
(72) %%A.; 2k(75) RINA/@@A. (%30% ~~): 455 (MI,
Pengcheng) IcN/cN]; + +) r+igj@JJII4NIJJ x%I
/t' j:Jk~~+ x NACA &Fi~~h &%@I/IIF~,Guangdong 518057 (CN) . 3g@@ (TAN, Liang-zheng) IcN/cN]; 0 + )44K&JII4NIJJ x %rÃfR/t' j:Jk ~~ 0 x N 9C 8 & Fi ~~ 4 &%@I/II F~,Guangdong 518057 (CN) . 9X+ (MA, Yaping)IcN/cN]; + + )' 45XJJII4@IJJ x A@R4j Jk~~ + x N 9C8 & &7 ~~ 4 &AC I/II Ez, Guangdong
518057 (CN). ggg (YUAN, Jiancheng) ICN/CN];
44K&JII4NIJJ x NRW j-4.~~ + x VN
9%8 X F7 ~~ 4' &g4 I/II)~, Guangdong 518057(CN) .
(74) RNA. 8NII4%%@-Wh@RN4%% (%%a 9'. ) (SHENPAT INTELLECTUAL PROPERTYACENCY); + ~~)' 45K&JII4~~%%% 15 CiJIjP&1521 %, Guangdong 518014 (CN) .
(s» SRN (IIJ~A NHJJ, %% —8 Un(@@%~~%%fP ): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG,BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR,CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB,GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP,KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT,LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY,MZ, NA, NG, NI, NO, NZ, OM, PE, PG, PH, PL, PT, QA,RO, RS, RU, RW, SC, SD, SE, SG, SK, SL, SM, ST, SV,SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC,VN, ZA, ZM, ZW.
(84) @gw (IIJ-n@Pia HJJ, %Q —$f ~7gl++ftIt x%fP): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA,RW SD SL SZ TZ UG ZM ZW) gA+) (AM AZ
BY, KG, KZ, RU, TJ, TM), 5 /)'I'I (AL, AT, BE, BG, CH,CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE,IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO,RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM,GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
48@/i~4':818+ PY~CN'h@1%HJJW)itiJk&(5lr~iJ 5.2( )).
(54) Title: METHOD FOR PREPARING BIVALIRUDIN
W (54) RN48': —8 ESPR%5iJk&/A(57) Abstract: Provided is a method for preparing bivalirudin. The method comprises preparing polypeptide fragments, and coup-ling the polypeptide fragments, amino acid and solid state carrier into bivalirudin peptide resin, and then cracking and purifying it toobtain bivalirudin. The method utilizes the characteristic that there are hydrophobic and charged amino acid residues at both sides ofGly-Gly-Gly-Gly in the bivalirudin to prepare the polypeptide fragments as the raw material, and effectively separating impurities bythe differences of polarity and charge to avoid the occurrence of impurity in the bivalirudin in which I or 2 Gly are lost or added.
~~ (57)%%:%&A X—8'&I'-6/ RYJ5iJkx/A. AW/A8, $85iJkkh P~ P4~kh P~ke, %% U~KN@t'rIj@@iJ&gPI:gPggyJg, , PJ,;ggtgy~gyg&ggiJPI:gPR. 4%%/AWIJR kgPR0 Gly Gly Gly GlyW/rltfk~iii7k'l0~ WIj-
i've.
e=+ee+e6,+W+eira&image ee, 'm'~ere. WIj e, ~ eX2 AI Gly 5 kN/ R.i+i)&A O' r
WO 2014/032257 PC T/CN2012/080792
—Rk&4, 484]kmk
gk+e4+&~5 i+f8 $ N. Fq 444~j4-w &@@„Pt.~ i+f8 —Q re@,P ~h3 4~j4-~
10
15
20
25
~v. $~4
PKA, P & ( Bivalirudin, Angiomax) a) ~~~4,N»'NF)4k, w—~S8 0~I-r~h0.~~5M«W-. j, ~~4~~~+~08&~;jeke«rh~~~"~aa~I.h3 IA 4+I ) k5-' ~ %444$I'4j'N&i@hhhPR. ~I. )+~' ~ w: 44&44 'N.
FC4k4H t4, tlat, r" ~h3 VCR&'FR $~FC ~AN ~+44k R45P~&&, 444~/T+4k~j' h0)&R O'I fbi~/i.
khan, r" Ww~WH 4 &iT&444444 ( CN ) h3 5 N. , 7i& 5 N. ~e 4-20+
4P~ 548k~, 5+4~J+0~j 4+SEQ ID NO: 6J'Jtrw.
k4t4A, r" wh34~j4-wk, g+ @P)E4&~N~W:
f 9++4'j200910028793.7Nikf f 5+A&4H&4~~4, LKA, )"w. 4a 4,:-+g 5'j ~ 6, 6, 8 ] 4,P~ Ak.+$w4j g g. , '„'Pc+ik]HQ]g =
] ggir" %Xi. f 9++4'j200980152943.3 fNik 7 4 Xm~+4'jgH/&J h3+T&4H
~4,w& ~3-rh N.~ 4 Y5+ +5k% Y re@,r" wh0i
f g++ 4'j 200910051311~~~47 —9F4' r" wh')Fmoc I4H)Ir~+~~4 &
f5 Wangk~j 884K&46$~$88, Air" 44P.%.4)'4,P~ Ak. , j'44~$ N.k~j 88f8:-4,L 6klkiVr-R-J] r~A, r" ~4~, &44~j4-~~fxR)44. -@~ICE'J
WO91/02750%it 7 —~f 5 Boc&4 4~j 4-F4', )" ~ hl W 4,Merrifiledk~j 884 g4H@,g aW&T4-4„, ir" dkP. $~4)'4,$.5k. , g.k&f g 4,g, 5k. (HF) 5k.@.
40~~A )"&~+4'J 0~~4—+]Gly-Gly-Gly-Glyd @., 4is~ %gi-Glym4$X. Q, 5.NGly fJ Pf4 5., $4444,kP+A, ~~ 4t 1A,2 ] Glyh348g. ,
[-Gly]-Bivalirudin[-2Gly]-Bivalirudin[+Gly]-Bivalirudin[+2Gly]-Bivalirudin.
WO 2014/032257 PC T/CN2012/080792
10
—2—
%~~ ~~Bi. 4Bivalirudin fJ P h3$k 54HmR, +4k'+ ~~ 4 &4.54h3mtgX f 84$~$
*I'0„+8,-".-4~8.p: &4.i t'+„, 4- —;," AH448, .—~, ~.'4.R H%t'~.
4@iPK+1 Im~ +4'jWO20117725f 5 Fmoc-Gly-Gly-OH444$m~
&T'-Gly-Gly-Gly-Gly ~ N. h l ~~4„, 7& K5X5X@iP [-Gly]-Bivaliruding&
[+Gly]-Bivalirudinh3~~8r. hl 1 Im Pt Ai@iP44)SHJ'fr I' %hi
[-2Gly]-Bivaliruding& [+2Gly]-Bivalirudin h3 ~~Bi. .f EJ+4'j 201110144317 f~~] f 5 7 Fmoc-Gly-Gly-Gly-Gly-OH 3(48
J~@Hf@iPZm~ Gly w~Sr. hl llew. 7~m fi i~~mf 8 Gly 4,+ Gly-Gly
Gly-Gly-Gly-Gly-w~&T~~4 4~~44 Hh'l 4~j 4 m~5 f 5 I P-2~~~&8-, r).++@44-h3~t:A,P&fZ~~~&8-h)4-&~.')..
4-.": 4~I-, 4X~PJ ~gt--k@-.P.h08.f-H~~44$44, —8~t=A, P ~hh4-, m@P~44 H4~j 4-m~$X. f 5 I P.[-Gly]-Bivalirudin, [-2Gly]-Bivalirudin,
15 [+Gly]-Bivalirudink, [+2Gly]-Bivalirudin~~8i. .+X~PJkkk. hilt AJ X hl4~j4-&k, ~e 44&J. F+~4g:
+r4w 1: 4ij4-$ N. g g.'
+r4P- 2: gxPfrm4$ N. g 4~44%5k. , ggH4@%4i4~jl~rt A)" mN$~f
20
-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Gl
u(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang 5'+ 4~J h l 5 N.$~f
88.
+~47. 3: W~Pfr~4&t:A, P ~N.5~$884'.@-5~4.&K~I &zest:A, P ~;j'gm~P N. g g.h34,%5k.+0~j+ SEQ ID NO: 1 4, SEQ ID NO: 2 4,
SEQ ID NO: 3 4, SEQ ID NO: 4 4, SEQ ID NO: 5 Pgw.
5P~,AH„P4m~%4&-JL 444j'4m~ R14H4, 4Z%ir" %4i Leu, Tyr, Glu,
Glu, Pro, Ile, Glu, Glu, Phe, Asp, I'=L4 SEQIDNO: 2 j'4wk'+P~Jhl$
25 N. g g. , -I'=L SEQ ID NO: 1 j'4wk+P~J h3 5 N. g g., Arg, Pro, Phe, 4~j
4~-.I'-L4 D-Phe-Pro
WO 2014/032257 PC T/CN2012/080792
—3—
5P~,AH„P4m~%4&-JL 444j'4m~ R14H4, 4Z%ir" %4i Leu, Tyr, Glu,
Glu, Pro, Ile, Glu, Glu, Phe, Asp, Gly, Asn, I'=L4 SEQ ID NO: 3
j'g w k'4' 4~J h3 $ N. g g., Arg, Pro, Phe, 4~j d~-I'=L4
D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly
5 -Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu
(OtBu)-Tyr(tBu)-Leu-Wang 5'+4~J h l 5 N.k~j 88.
5P~,AH„P4m~%4&-JL 444j'4m~ R14H4, 4Z%ir" %4i Leu, Tyr, Glu,
Glu, Pro, Ile, Glu, Glu, Phe, Asp, I'=L4 SEQIDNO: 5 j'4wk'+P~Jhl$
N. g g. , -I'=L SEQ ID NO: 4 j'4wk+P~J h3 5 N. g g., Arg, Pro, Phe, 4~j
10 4~-I'=L D-Phe-Pro
-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Gl
u(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang 5'+ 4~J h l 5 N.k~j
88.
tBu
OtBu
Pbf 2,2,4,6,7-K 7k.W+~A 4-5-~Pi%%
Tyr
Asp, Glu
Arg
Asn
11 ) 4~j4- Fmoc-Gly-CTC $~$88A, Fmoc-Gly-Gly-CTC $~j 88;
12 ) %.t'~t, j'4m~ Fmoc-Gly-CTC $~j 884, Fmoc-Gly-Gly-CTC $~j 88Th )
20 Fmoc 3+0)'A, s&4~%.l'~t Fmoc %4) P~ h l CTC $~j 88;
13 ) 44j'4m~%.*Fmoc %4)'Ah) CTC $~j 88, Air" %4&44 Fomc %.
4j'P~ h34,P~ Ak. , ~ 5'j4g~. .+ SEQ ID NO: 1-5 Pgw4, P~ Ak.+Njh34, P~
Ak.f~j gal ',
14 ) $4j'$~4-JL+ SEQ ID NO: 1-5 j'gw4%5k+0tj h34%5kk~j88%.
25 @, 4~jg~-I'=L+ SEQ ID NO: 1-5 Pgw4P~ Ak+0~j hap N. g g..
WO 2014/032257 PC T/CN2012/080792
j'4m~ Fmoc-Gly-CTC $~$88A, Fmoc-Gly-Gly-CTC 5~$88 h 14+8,
0.8mmoVg-1. 2mmol/g, P 5P~A h l 4 0.9 mmoVg -1.1mmol/g, 45P~A h3
1.0 mmoVg.
+r4P- 13 ) QPfr~~h344 Fomc@4)'Ah34%5kg
5 Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-Gly-OH, Fmoc-Gly-OH fh3 —~A, P9~.
tb 4+~~4, h35 N. g g.f (t P. 7 -Pro g~-Asn KP9~kt A, P mN. kk. f~~~hq4P R rZWP&K&e~+g. + V r, kjR@ 554 44, 5hQ+~d-rkP-
4A, ~", 4~ 1 4, 2 + Gly h3 tt A, r" w4 8i & A*t'k.+~4m 2 QPfrm4h3 Rgb@,g 4 Wang 5~$88.
5P~AH, +r4P- 3 f j'4m~%@fRik, i~i~j h')AL44%4$~ i'=i ~& kt ~44654. 90%-95%, :-~AP~ +k.N, 2%-4%, +~ 4
JJ, ml/g ifj'gm4ik, jI~j4Pgm4Lt A, r MN$~$88h38i ag $. Lt 4
10
10:1.15 5P AH„+&4& 3 flfr~~hh l4.&Ef-5 ~-'kA~4r K-i'.:4 .
4srL4$4. f-4S ~t:, 4&%$k4A 7 —H~t.-A, P ~h344-wk, -i~wk4~ 4,h'l-Gly-Gly-Gly-Gly4HW N.f (I P- 7 kt A, )"&5 krak. f14' ~h'l4&&4,AR r~@w~r. fjg N~5]kf 44, ]kh3+ ~gfkXN. + h344~7+Yh3~
A*t'~w, m@P& Y&I&A,&Hat 1 4, 2+ Gly hatt A, r" ww~@r. hei' &. 8 Iltk20 X~I]kkkkhhwC 4,+M,+, ~8~~1 i44. j™".-Ck.~PJ: 34,~%+uk W4-h3
Lt A, P wkX &4, 4 4 77.1% — 87.1%, &4, &E4 Lt A, P w
[-2Gly]-Bivalirudin 4& [+2Gly]-Bivalirudin ~~ 8i. , [-Gly]-Bivalirudin A,
[+Gly]-Bivalirudin P—~~Bi.~~ J'4 0.05.
25 8- I%4.%
5 1 4 j™".-&&~J 25 WR-h3 ~t.-A, P ~4& N. K~.:5.
A4gAmA,
WO 2014/032257 PC T/CN2012/080792
~@..-, ~~% ~X~~~K444%~r~. 44xj ~-kP. k h3~, Pgt-44&v, h3-4&kkf
p4~3A4+4Q, $k 4 W7q +i%,mX rR 4 A~ h'l, i fl'] WHklf~~k ~e 444+Xi-tlat .44% h3 wk8. )~R E ~~~4m445k+ 4,&~~J~~&z 7 54~4, 4H kf j~ 5X~RA
XR @4&i-tP] @~,gafg~~t. l% @st+uf'fr~4h3w kg~)2 5 ~~a(k~kk, m~
8--P48t4-, 4%~r&$ r~RWX~PJ8. 4-.+&i'tPJkk4, h3 rt-A, r" wh34~j4-uk, 8.44~'r. T+r 4k:
+r4w 1: 4ij4-$ N. g g.'
+r4P- 2: gxPfrm4$ N. g 4~44%5k. , ggH4@%4i4~jl~rt A)" mNk~j
10
15
20
25
+~47. 3: W~Pfr~4&t:A, P ~N.5~$884'.@-5~4.&K~I &zest:A, P ~;~f$ N. g g.h34,P~ Ak.+Nij+ SEQ ID NO: 1 4, SEQ ID NO: 2 4,
SEQ ID NO: 3 4, SEQ ID NO: 4 4, SEQ ID NO: 5 Pgw.
4+XitP]hl —4~+4,&P~Jf, 8f5
hler%.
gg 4-I'=L4 SEQ ID NO: 1
j'/we'+4~J h3 $ N. g g.$&-I'=L SEQ ID NO: 2 j'gw k'+4~J h3 $ N. g g.84, +I4I='t 4, 4m~&T'%4i4P~ i'=i%. h3)Ir~+ 4: Leu, Tyr, Glu, Glu, Pro, Ile, Glu,
Glu, Phe, Asp, I'=L SEQIDNO: 2 j'4wk+P~Jh35N. gg. , -I'=L SEQID
NO: 1 j'/we'4'4~Jh3$ N. g g. , Arg, Pro, Phe, 4~jd~-I'=L4
D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly
-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(Ot
Bu) -Glu(OtBu)-Tyr(tBu)-Leu-Wang 5'+4~J h l 5 N.k~j 88.44Xi'tP]h'l5 —4~+4,5%jf, 8f Rh'&AN. gg 4-I'=L4 SEQIDNO:
3 Pgt--rk. khh3$ N. W SRIlt, 4IW4&4, 4~~~&%8~44)4'/IF+4: I.eu,
Tyr, Glu, Glu, Pro, Ile, Glu, Glu, Phe, Asp, Gly, Asn, I'=L4 SEQID
NO: 3 j'4wk+4~J h3 5 N. g g. , Arg, Pro, Phe, 4~jd~-I'=L
D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly
-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(O
tBu) -Glu(OtBu)- Tyr(tBu)-Leu- Wang 5'+4~J h l 5 N.k~j 88.
A, 4+XitP]h'l5 —4+4/PJf, 8fRh3$N. gg 4-I'=L SEQID
NO: 4 Pfrwk'+P~J h l 5 N. g N.g&-Ij-4 SEQ ID NO: 5 Pfrwk'+P~J h l 5 N. g N.
WO 2014/032257 PC T/CN2012/080792
4 I~1 4I='1 4, 4m~&T'%4&45~ i'=i%. h3)Ir~+ 4: Leu, Tyr, Glu, Glu, Pro,
Ile, Glu, Glu, Phe, Asp, I'=L SEQ ID NO: 5 j'4wk+P~Jh3$ N. g g.,
-I'=L SEQ ID NO: 4 j'4wk+4~J h3 5 N. g g., Arg, Pro, Phe, 4~jd~-I'=L
D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly
5 -Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)
-Glu (OtBu)- Tyr(tBu)-Leu- Wang 5'+4~J h l 5 N$~f88.
4X&PJ f-8 F4,4%4i k8.Pr%4/-'F4, 4+4. 1 fft -r. :
4 1 4X&PJ f-8 F4,4%4/'k8. ~gt-%4/'F4, ktBu
OtBu
Pbf
Tyr
Asp, Glu
2,2,4,6,7-K 'P %~+&4'4-5-&4i%% Arg
Asn
Y 4~j4-5 N. W N. , 44~j4- Fmoc-Gly-CTC $~f88A,
10 Fmoc-Gly-Gly-CTC 5~$88; &+4, 4'44 Fmoc-Gly-CTC 5~$88 A,
Fmoc-Gly-Gly-CTC$~$88, 4t P.R14H8 J& 8 f,5 N,N:7A7 iR%( DMF )
ikPg;, 5 DMF 'i~jjgf~fgg, &+Cg 20%&i 4,&LI"m/DMF i'~i@ ( DBLK) %.
I'~t Fmoc 3+4)'P~, WC45 DMF A, Pt;, WR:-4~'PN, (DCM) A, Pt,
4, 4~44 Fmoc Ski'Ah)4Ãlkg~gHPi h)5)&I$4%4)'kimgg g„rt-4 1:1
15 hg DCM g~ DMF i~a,~~i~if&f, i&golgi~~ P4~P-:~45~4.:3P%- ( DIC )~
i4 &EC)~P.@488)24+ f, X~X8 )2 2 h 4Q+Sk%4a F—-KP-Ax.
~ 5'j4~jd~-I'=L+ SEQ ID NO: 1-5 Pfrw4P~ Ak+0ij hl4P~ 5%5~$88; 44,44-I'=L+ SEQ ID NO: 1-5 Pfrw44. 5kFj 0ij h344. 54$~fggkt P.+~@8)i g
4&P.~:—g, 6 illa'- ( TFE ) $&—-4~ 'P 4 ( DCM ) h3 +~@-ik ( 4 $. Lt,
20 TFE: DCM=1:4), X.' 8 8 2h. 8 Sk'+4. a~'j -k~f88 @.4'j -At. 3s'j-
K&g 8~4~7~~~( 25vo1%4, /iij4t ~~in X.~E, ~ii j f ( 4 $~Lt, iE- 2 N, : 65&
=1:4), 4 ~45/t, ~565kikPt;, jjKg~+)~, g~Pj-I'=L4+ SEQIDNO:
1-5 j'gw4%5k. +0ij h3 5 N. 8 N. .4-0, 4%%44~j4-44+ SEQ ID NO: 1-5 Pfr-r4, 46kFj 0ij Ittf5
25 h l Fmoc -Gly-CTC $~$88A, Fmoc-Gly-Gly-CTC 5~$88 h l ~&+A,44 0.8mmol/g
WO 2014/032257 PC T/CN2012/080792
—1.2mmol/g, ~44'.gk h3 4 1.0mmol/g.
4 Y %RE~& A*1'k&P4A, && 4t 1 4, 2 + Gly h0 tt A, )"wf hhk@i. ,
+XitPJ f5 h l 44 Fomc %4)'Ah34+i'=i%4 Fmoc-Pro-OH,
Fmoc-Asn(Trt)-OH, Fmoc-Gly-Gly-OH, Fmoc-Gly-OH f h3 —~A, P9~,~~4,-I'=L+ SEQ ID NO: 1-5 Pfrv4P~ Ak+ Nij h l 4P~ I'=i%. W N. . 04@~+4,h3 5 Ng gf gl P. 7 Pro $~ Asn, )AA+&4&4~4 jX f .I&J. 4'j 5 Si.75 5$~
+0, 5.h3+ )-t-]-rkPAA, ~gkt 1 4, 2 ~] Gly hl kt A, )"w~~))) ~& A*l'~w
eI'Pl, 4%%4-4, ~t A, )"~$ N$~$88I14 f-5 h3 IWSI='14, 44 Wang f~$
10 ~~W~T~t A, )"~P~JS:&"@-Itt, ~r, mVg 't&" @-r~6 ~~A, )"~Xk~tS:h0
10:1. 4 f4'.@i~h)At.4,@@8;,W~~ st ~44::-4,56k90%-95%, :- )-t- AP~ jk.N, 2%-4%, +~ 4
4eI'%~AX, 4&itP]~t~t A, )"~IX. hall. &Ef-5 ~-'kA~4r K-i'.:4, l4.
&eh~.:8~f-R8 4I='1/N ~H 4+~$4'"~@A.@~4~&4H, P4-» K~.:8~f-R
20
15 8 4I='1 C18 Ki~g34H.
+&its]kkkkhh ~t A)" ~h344-wC, 44-4, hh Giy-Giy-Giy-Giy 4Hh 4~0 Pl ~7 ~~A, )" ~$ N.kkf J'Jtr~a. ~hh~&44Ãrk ~eP 7kP.AA, ~ 0
1 4, 2 + Gly h3 Pt A, )"m~~))). ~~))). h3 j' 4.K4X i'13 $44, h3 %44,4/8, +,
4X%ff5 hl Wang $~f88g& 2-CTC 5~$88)4* fJ Aigr--'444~4, 4I'k~~~
J, 8 SI'1/Nl'8 0+~$4'"4-NA. W~4&)W* 0 0 K18f~+t'='A&44kPr, 0-
H&+4) mP~-i'=i&)4* fJ ~ ~ &&4@I'P&~ ~J, 4 i- imii~jg~~A, ii~jg '~ mme~~
V 45+4-4 4i4, &~J, ~~—+r tW ~~+&i-tP].
25 +A@~J 1: +4M4 0.8mmoVg W Fmoc-Gly-Gly-CTC Resin W4]4.4'44~~+A, V~4 0.85mmol/g h3 2-CTC$~f 88300g ( 255mmol ), A~P.Pj I$H
8 )28+ 0 ~ R DMFA Pt. 2'", $ DMFg-5(s) )530~g If', (%180.7g
Fmoc-Gly-Gly-OH (510mmol) 5DMF'i~@, i~k75P~ FA~P. 177.3mL N, N
:)-t-AAZ%- (DIPEA) (1020mmol) 4&4)=, 4~P-%~444$~$88h38)~
WO 2014/032257 PC T/CN2012/080792
5f, 8 g2 J IlfP )~&243mLf~~57Pi+kgA]30m1n. RDCMA, 3.~, 7t'tj-@.~%4@+, S~SI]Fmoc-Gly-Gly-CTC$~$88, $~ i)~'J~&+A,44 0.812mmol/g.
10
+A4~J 2: +4M4 1.0mmoVg i' Fmoc-Gly-Gly-CTC Resin iYJ@J4-
4'44~+5,P 41.0 mmol/gh32-CTC$~$88300g ( 300mmol ), At P.Pj I$H
8 f28+ 0, R DMFA, Pt;2'~, $ DMF'iwgfsf gg 30~g 4/4, 4%212.6g
Fmoc-Gly-Gly-OH(600mmol) 5 DMF 'im @, i~W 75 P~ F 4t P. 208.6mL
DIPEA(1200mmol)'i4&EC, 4t P.Za~44$~$885'll Ji5 f, 8 Ji2 J'Ills,AvP. 243mL/t, rg $ t'tj4$ A3 30min. 5 DMFikPt;3'~, DCMi43ir~,
444, 4~SI]Fmoc-Gly-Gly-CTC$~$88, $~ ')~'J ~&+A,44 0.976mmol/g.
15
+A4~J 3: +4M4 1.2mmoVg W Fmoc-Gly-Gly-CTC Resin W@J4.
4'44~~+A, 44 1.25 mmol/gh32-CTC$~$88300g(375mmol), At P.Pj I$H
8 f28+ 0 ~ R DMFA Pt. 2'~, $ DMFg-5(s))530~g If', (%265.8 g
Fmoc-Gly-Gly-OH( 750mmol )5DMF'im@, i~k75P~ Fkt P.260.8 mL DIPEA
(1500mmol) '4544, )t P.Za4 f $Af8gh38 Ji5), 8 Ji2 J 844, )t
P.243mL/t, 75$ t'tj-4$ A3 30min. 5 DMFikPt;3'~, DCMi43ir~, 'P i"-ij-@.&%44
d~P] Fmoc-Gly-Gly-CTC$~$88, $~~i)~'J ~&+A,44 1.113mmol/g.
20
25
+A4~J 4: +4M4 0.8mmoVg i' Fmoc-Gly-CTC Resin g@J4-4'44~~+A, V~40.85mmol/gh32-CTC$~$88300g ( 255mmol ), At P.Pj g$H
8 f25 f, g DMFA, Pt;2'~, g DMFg 55~$8 30g~4$4, 4~151.6g
Fmoc-Gly-OH ( 510mmol ) 5 DMF'im@, i~k75P~ F At P.177.3mL DIPEA
(1020mmol) '4544, ) P.Za4 f $Af8 h38 Ji5), 8 Ji2 J 844, )P.243mL/t, 75$ t'tj-4$ A3 30min. 5 DMFikPt;3'~, DCMi43ir~, 'P i"-ij-@.&%44
4~SI]Fmoc-Gly-CTC$~$88, $~ i)~'J ~&+A,44 0.798mmol/g.
+A4~J 5: +4M4 1.0mmoVg i' Fmoc-Gly-CTC Resin iYJ@J4-
4'44~+5,P 4 1.0mmol/gh32-CTC$~$88300g ( 300mmol ), At P.Pj g gH
WO 2014/032257 PC T/CN2012/080792
8 f28+ 0 ~ R DMFA Pt. 2'~, $ DMFg-5(s))530~g If', (%178.4g
Fmoc-Gly-OH ( 600mmol ) 5 DMF'im@, i~k75P~ F At P.208.6mL DIPEA
(1200mmol) '4544, ) P.Za4 f $Af8 h38 Ji5), 8 Ji2 J 844, )P.243mL/t, 75$ t'ij-4$9330min. 5DMFikPt;3'~, DCMi43ir~, 'P t'ij-@.&%44
4~Pl] Fmoc-Gly-CTC$~$88, $~ i)~'J ~&+A,44 0.956mmol/g.
10
+4,4~J 6: +4M4 1.2mmoVg W Fmoc-Gly-CTC Resin W@J4-
4'44~+AP 41.25mmol/gh32-CTC$~$88300g ( 375mmol ), At P.Pj g$H
8 /i 5 f, g DMFikPt;2'~, g DMF'iwgf~f 8g 30~g 4/4, 4~223.0g
Fmoc-Gly-OH ( 750mmol ) 5 DMF'im@, i~k75P~ F At P.260.8mL DIPEA
(1500mmol) '4544, )t P.Za4 f $Af8gh38 Ji5), 8 Ji2 J 844, )t
P.243mL/t, 75$ t'ij-4$ A3 30min. 5 DMFikPt;3'~, DCMi43ir~, 'P t'ij-@.&%44
4~Pl] Fmoc-Gly-CTC$~$88, $~ ')~'J ~&+A,44 1.082mmol/g.
15
20
25
NASL 7: + SEQ ID NO 1 P)T4 i' 5 )kW R Fmoc-Pro-Gly-Gly-OH i' @J
4-4'44+ 4 5P~J 2 f 4~J 4-h3 ~@+A,~m 4 0.976mmol/g h3 Fmoc-Gly-Gly-CTC$~f
8892.2 g ( 90mmol ), 4t P.@488)i5 f, R DMFA, Pt-2'", R DMF'iaaf
Fmoc-Gly-Gly-CTC$~$8830~~~444, 5DBLK%.l'~tFmocf+0)', &+45 DMF
A, Pt;4'", DCMA, 2'". gr91.1g Fmoc-Pro-OH (270mmol), 43.8 g HOBt
( 324 mmol ) 'i~f@g~ Lt $1:1h)DCMJ~DMFZ~~'i~i@, ng75X- F)&P.
40.8g DIC (324mmol), '4545min4)t P.RJ]H8)25), Xi 8)22h (8re@») 8p = Kk$~'r~J44, +Q~$88f~K4~P], )~]8 y~K~~, k~f88~K,
4.v8 Jita+, g-P}%448 )21h ). Sgtj120.7g +SEQ ID NO 1Pfrvh)
5 N. g g.h3CTC$~$88.
3~120.7g+SEQ ID NO 1Pgw h'3 5 N. g N.h'3CTC$~$884t'P-%@8 Ji
f, ).~1207ml~@-r~ (g$;,k, TFE: DCM=1:4), i-'88.2h. 88.~~iJ, -k~f88, @.@iJ, -8&. 3&iJ, -8& 4$„4.~Y~~ & 25vo1%4, ii(kt ~5L
iKX.~E,iij f (g$„rt, KC N, : 651=1:4), 4 ~, Pt, ~565kikP+;, +K
WO 2014/032257 PC T/CN2012/080792
—10—
4-~4)4, d~Pj+SEQ ID NO 1Pfrwh3$ N. g g.39.8 g. @.+498.1%, &4,
C.4 96.9%.
10
15
20
g+giJ 8: + SEQ ID NO 2 Hj+ +Al +g +Fmoc-Gly-Gly-Asn(Trt)-Gly-OH g )iJ+
@~ +'+&&'J5044 h'1 z AC40. 956mmol/ghgFmoc Gly CTC$pf$@
~ P- t-1 SI-.14880, R DMFA, P~-2l~, g DMFg PFmoc-Gly-CTC$~f8g 30iw 444, 5DBLKg, f~tpmocf+gj', p4@g DMF-'+pg;
4'", DCMA, 2'" . gr179.0g Fmoc-Asn(Trt)-OH ( 300mmol ), 48.6 g HOBt
( 360 mmol ) 'imper $„Lt P 1:1h)DCMJi DMFiÃ~'i~ij&, i~/)J&-5- P)vP.
45.4g DIC (360 mmol), 'i4545minC)i P.@488)i5),i iX8 )i 2 h (8re@») 8p = K k)~'r~] 4 4, +Q~$88f~ K4~P], )~]8 y~ K~~, k~f88~ K,4.v8 )i7a+, ~g-P}%4i8 )i1h ). 5(r".8kj-i k%4i7 —+4,P~-6k.
Fmoc-Gly-Gly-OH. S~P]163.3g+SEQ ID NO 2j'4mh35 N. ~@h3CTC$~$
88.
3~163.3g+SEQ ID NO 2P4w h'3 5 N. g N. h'3 CTC$~$884i'P-%@8 Ji
f, ).P.1663ml ~@-r~ (g$;,k, TFE: DCM=1:4), i-'88.2h. 88.~~iJ, -k~f88, @.@iJ, -8&. 3&iJ, -8& 4$„4.~Y~~ & 25vo1%4, ii(ki ~5L
iKX.~E,iiij f (g$„rt, KC N, : 651=1:4), 4 ~, Pt, ~J&-65kikP+;, +K4~Sj+SEQ ID NO 2Pfrwh3$ N. g g.74.32 g. @.+496.7%,
&4,V~4 95.8%.
25
@iJ 9: + SEQ ID NO 3 PET 4 8 A Jk h RFmoc-Pro-Gly-Gly-Gly-Gly-OH K0J4.
@$g++gpiJ 2 f piJ 4 hg ~~&ggP~P 0.976mmol/g hg Fmoc-Gly-Gly-CTC$~f
$g92 2 g ( 90mmpl ), )~P.@ggg)ig. f, g DMFikPg;2', g DMF@$Fmoc-Gly-Gly-CTC$1$%t 30~ 4fPi ~ 8DBLK%,l+Fmocf+4) ~ )+Pi 8 DMF
4, 'rt. 4'", DCMik2ir" . 3-r95.7g Fmoc-Gly
-Gly-OH ( 270mmol ), 43.8 g HOBt ( 324 mmol ) 'im4 4$„kt 4 1:1h3DCM
WO 2014/032257 PC T/CN2012/080792
10
—11—
g~DMFZ~~~'i'd&&, i~j&-7j&-P~ Fkt P.40.8g DIC ( 324 mmol ), 'i4 545minCkt
P.RJ]H8)i5), i.' 8)i2 h (8 Ji ~~4 ') 8p = 6ttk$~'r, j4'g,% fg A~i'ted], )~F8 8~+ if'~(% fr g, f p8 )gi' 7x~+, y 0%4&8 )gi' 1h ),
Air" g 4+%4%4& F—+4,P~ Ak.Fmoc-Pro-OH. S~Pj130.9g+SEQ ID NO
3/fr-r h3 5 N. g gkhh CTC$~$88.
3~130.9g+SEQ ID NO 3Pgwh'l5 N. g Nh'JCTC$~$884t'P-%@8 Ji
f, ).~1309ml ~@-r~ (g$;,k, TFE: DCM=1:4), i-'88.2h. 88.~~iJ, -k~f88, @.@iJ, -8&. 3&iJ, -8& 4$„4.~Y~~ & 25vo1%4, ii(kt ~5L
iKX.~E,iiij f (g$„rt, KC N, : 651=1:4), 4 ~, Pt, ~j&-65kikP+;, +K4~Sj+SEQ ID NO 1Pfrwh35 N. g @46.3 g. @.+491.0%%,
&4,44 97.1%.
15
20
25
+4,@iJ 10: + SEQ ID NO 4 Pf4 $ lk~ 4.Fmoc-Pro-Gly-Gly-Gly-OH
84i]k4'44+ 4 5%ij 2 f 4' 4-h3 ~@+A,~m 4 0.976mmol/g h3 Fmoc-Gly-Gly-CTC$~f
8892.2 g ( 90mmol ), 4t P.@488)i5 f, R DMFA, Pt;2'", R DMF'iaaf
Fmoc-Gly-Gly-CTC$~$8830i~~444, 5DBLK%.l'~tFmocf+0)', &+45 DMF
A, Pt;4'", DCMA, 2'" . 3-r80.3g Fmoc-Gly-OH ( 270mmol ), 43.8 g HOBt
( 324 mmol ) 'i~f@g~ Lt P 1:1h)DCMJ~DMFZ~~'i~i@, nj&-7j&-X- F)&P.
40.8g DIC (324mmol), '4545min4)t P.RJ]H8)25), Xi 8)22h (8re@») 8p = Kk$~'r~j44, +Q~$88f~K4~P], )~j8 y~K~~, k~f88~K,
4.v8 )i7a+, ~g-P}%4i8 )i1h ). 5(r".8 kj-i k%4i7 —+4,P~-5k.
Fmoc-Pro-OH. 4~Pj 125.9g+SEQ ID NO 4j'gw h3 5 N. g g.h3CTC$~$88.
3~125.9g+SEQ ID NO 4Pgwh'l5 N. g Nh'JCTC$~$884t'P-%@8 Ji)t P.1259ml 4.@k (g$., kt, TFE: DCM=1:4), i iX8)i 2h. 8)i
~~iJ, -k~f88, @.@iJ, -8&. 3&iJ, -8& 4$„4.~Y~~ & 25vo1%4, ii(kt ~5LiKX.~E,iiij f (g$„rt, KC N, : 651=1:4), 4 ~, Pt, ~j&-65kikP+;, +K4-~4 jw, 4~$ij+SEQ ID NO 1Pfrwh3$ N. g g.42.9 g. @.+493.8%, k4,
C4 96.8%.
WO 2014/032257 PC T/CN2012/080792
—12—
10
15
+A4~J 11:+SEQ ID NO 5 Pf4 $ )Rgb. Fmoc-Gly-Asn(Trt)-Gly-OH
KA~J4-
O'44+ 4 &P~J 5 f 4~] 4- h l ~&+A, V~ 4 0.956mmol/g h l Fmoc-Gly-CTC$~f 88
103.6 g ( 100mmol ), )t P.@488)25 f, R DMFA, Pt;2'r", R DMFXP-
Fmoc-Gly-CTC$~$88 30~a 444, 5DBLK%.l'~tFmocf+0)', &+45DMFA, Pt;
4'", DCMA, 2'" . gr179.0g Fmoc-Asn(Trt)-OH ( 300mmol ), 48.6 g HOBt
( 360 mmol ) 'imper $„L4$1:1h)DCMJ~DMFiÃ~~'i~ijt, i)g)J&-P~ P)vP.45.4g DIC (360 mmol), 'i4545minC)t P.@488)i5),i iX8 )i 2 h (8re@») 8p = K k)~'r~] 4 4, +Q~$88f~ K4~P], )~]8 F~ K~~, k~f88~ K,4.v8 )i7a+, ~g-P}%4i8 )i1h ). 5(r".8 kj-i k%4i7 —+4,P~-5k.
Fmoc-Gly-OH. d~P] 152.8g+SEQ ID NO 5j'4w h3 5 N. ~ @.h3CTC$~$88.
3~152.8g+SEQ ID NO 5j'4w h 3 5 N. g N. h 3 CTC$~$884t P-%@8Ji)t P.1528ml 4.@k (g$., k4, TFE: DCM=1:4), i iX8)i 2h. 8)i
~~iJ, -k~f88, @.@iJ, -8&. 3&iJ, -8& 4$„4.~Y~~ & 25vo1%4, ii(kt ~5LiKX.~E,iiij f (g$„r4, KC N, : 651=1:4), 4 ~, Pt, ~J&-65kikP+;, +K4-~+PE, S~SIj+SEQ ID NO 1Pfrwh3$ N. g g.64.2 g. @.+490.3%, k4,
C.4 95.3%.
20
25
+A4~J 12: Fmoc-Leu-Wang Resin gj$i]+4:4&~~+A,8~4 1.0mmol/g hit Wang $s$gg 150g (150mmol) p, p ggg
&r ~ ~ ~ DMF-~@-2=, ~ DMF'''5)$3 30 %fan, g&1061g
Fmoc-Leu-OH (300mmol), 48.6g HOBt (360mmol), 3.7g 4-:74k~ &LI;
'&(DMAP) ( 30mmol ) 'iwfg $., F44 1:1 h3 DCM g~ DMF Z~~'iaaf, ng
rJ&-X- F4t P. 45.4g DIC ( 360 mmol ), '4 &4 5min 4 4t P- I~1 4I='l8 Ji 8f,pJVL4)2 2 h. 4)2 5+$4 W DMF '4Pg; 4 ir, DCM '4 2 ir . )+4) P. 237.3g
'k5'w$& 306.3g Bi=i%.@iR,~~A&kgA3$~$88 6h. 5 DMF A, Pt; 4 la~, DCM ikPt;
2 ir" g, $ 5j-@k(44+, qgPj Fmoc-Leu-Wang ]sf%~, @;gq~]W4q(P~P
0.582mmol/g.
WO 2014/032257 PC T/CN2012/080792
—13—
10
15
20
gA4J 13: ~ikJf. f]+ SEQ ID NO. 6 Pfr+ gD-Phe-Pro-Arg(Pbf)-Pro-Gly
-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pr
o-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang @Ii' O'J 4-
gr 34.4g +4,5%~J 12 f 4~j4-h3 Fmoc-Leu-Wang Resin( 20mmol )5 DMF
4'rt. 4 ir", DCM A, 2 ir", R DBLK%l'+t Fmoc $e4)', &+CR DMF 4'rt. 4
DCM A, 2 ir". 4'44 27.6g Fmoc-Tyr(tBu)-OH (60mmol), 9.7 g HOBt
(72 mmol), 136.1g DIC(1080 mmol)'im4g $„kt 4 1:1 h3 DCM g~ DMF' 4-'+4, ) P.g]HA)i5), i.' 8)i 2h (8 Ji Ak ') 8p = 6ttk$~'r~j
, +Q&$88f~K@~PJ, )', ~]Are~~~, k~j88~6, 4.~8)it~+, g1h. 4 ~ka4% I'~t Fmoc $g4j'$~)t P.]H)i4P~ 5%%4ih)+r4P. ,
4%Pk Pt A, P mh3)Ir~+, Air" X,4, Fmoc-Glu(OtBu)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)
-OH, j™";4,5%~J 8 $4~j4-h3-I'=L SEQ ID NO 2 Pfrwk+4"Jh3$ N. g 4, j™";4,5%~J
7 $4~j4-h3-I'L SEQ ID NO 1 j'4wk+4~J h35 N. g g., Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-D-Phe-OH 8 /a 5'+4.CR 7 t-'-ij-@A~, k~j 884-~4Pw~~
4'44~$ij4%5k+N~j+ SEQ ID NO. 6 j'4wh3
D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly
-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(O
tBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j 88 76.7g.
25
gA+J 14: ~ikJf. f]+ SEQ ID NO. 6 Pfr4 i'D-Phe-Pro-Arg(Pbf)-Pro-Gly
-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pr
o-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang @Ii' O'J4-
gr 34.4g +4,5%~J 12 $4~j4-h3 Fmoc-Leu-Wang Resin( 20mmol )5 DMF
4'rt. 4 ir", DCM A, 2 ir", R DBLK%l'+t Fmoc $e4)', &+CR DMF 4'rt. 4
WO 2014/032257 PC T/CN2012/080792
10
15
—14—
DCM A, 2 ir". 4'4% 27.6g Fmoc-Tyr(tBu)-OH (60mmol), 9.7 g HOBt
(72 mmol), 136.1g DIC(1080 mmol)'im4g $„kt 4 1:1 h3 DCM g~ DMF' 4-'+4, ) P.g]HA)i5), i.' 8)i 2h (8 Ji Ak ') 8p = 6ttk$~'r~j
, +Q&$88f~K@~PJ, )ttij8ri~~~, k~j88~6, 4~8)it~+, g1h. 4 ~ka4% I'~t Fmoc $g4j'$~)i P.]H)i4P~ 5%%4ih)+r4P. ,
4%Pk Pt A, P mh3)~la~+, Air" X,4, Fmoc-Glu(OtBu)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)
-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, +4,5%ij 9 $4ij4-h3-I'=L4 SEQ ID
NO 3 j'4w k+4~J h3 5 N. g g., Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH,
Fmoc-D-Phe-OH 8 )& 5'+4@5 7 t'tj-@.~%, k~j 884- ' 4Pw~~4, 4'4 S~SIj
4P~ Ak+Nij+ SEQ ID NO. 6 j'4wh3
D-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)
-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wan
g k~j88 79.8g.
20
25
g+gi] 15: ~~~yij+r SEQ ID NO. 6 /f4 i'D-Phe-Pro-Arg(Pbf)-Pro-Gly
-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pr
o-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang WI i' 4']4.
gr 34.4g +4,5%ij 12 f 4ij4-h3 Fmoc-Leu-Wang Resin( 20mmol )5 DMF
4'rt. 4 ir", DCM A, 2 ir", R DBLK%l'+t Fmoc $e4)', &+CR DMF 4'rt. 4
DCM 4, 2 ir" . 4'4X 27.6g Fmoc-Tyr(tBu)-OH ( 60mmol ), 9.7 g HOBt
(72 mmol), 136.1g DIC(1080 mmol)'im4g $„kt 4 1:1 h3 DCM g~ DMF' 4-'+4, ) P.g]HA)i5), i.' 8)i 2h (8 Ji Ak ') 8p = 6ttk$~'r~j
, +Q&$88f~K@~PJ, )ttij8ri~~~, k~j88~6, 4.~8)it~+, g1h. 4 ~ka4% I'~t Fmoc $g4j'$~)i P.]H)i4P~ 5%%4ih)+r4P. ,
4%Pk Pt A, P mh3)~la~+, Air" X,4, Fmoc-Glu(OtBu)-OH,
Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH,
WO 2014/032257 PC T/CN2012/080792
—15—
Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)
-OH, +45%'J 11 $4~j4-h34 SEQ ID NO 5 Pfr~k+P~Jh3$ N. g 4, +45P'J 10 f 4~j4-h344 SEQ ID NO 4 j'4-rk'A'43~ h35 N. g 4~.,
Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-D-Phe-OH8 J& 5+4.45 'pillj-@.
4'%4&tjW4l=lk+N~j+ SEQ ID NO. 6 j'/t-~h jD-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly
-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(O
tBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j 88 73.9g.
10
15
+A+J i6 kAP CA KW4-)-,rj™~+fylj13 q~glj hg 25.5g ~+~5k+~~j+ SEQ ID NO. 6 j'4wh~
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j-88 X4%.f, &J. 10ml/g k~j 88 h3 t 4 &PJ 4t'P-%@.X,
'j[:—4, ~ i'=i%. ( TFA )::-~
AA+kN (TIS): 75=90: 2: 8 (V/V) ], ~i 4' 5$-2.5h. 8 Ji445+rl .-
20
4z4gg ~t'Pt AP md%- 13 86g. 4 ~4+4 95 4% HPLC &4P-4
80.6%. 4 / ~~Bi. [-Gly]-Bivalirudin 4 0.21%, [+Gly]-Bivalirudin 4 0.15%,
[-2Gly]-Bivalirudin 4 0.09%, [+2Gly]-Bivalirudin 4 0.12%.
25
4'] i7: ~~AJ CA~&KW43~r~4, &P'J 13 q~Pj h3 25.5g 4P~ l'=lk+~~j+ SEQ ID NO. 6 j'4~h3
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j-88 X4%.&J, 10ml/g k~j 88 h3 t 4&~J Wt'P-%@.X,
'j ( TFA: TIS: ~j =95: 4:
1 (V/V)), ai 4' 5$-2.5h. 8 Ji 4k5+i~=@+ ~:j.-, @~:j.-r~, &~&~:e
WO 2014/032257 PC T/CN2012/080792
—16—
~ TFA '4Pt; 3 i, 4-j-jp j&@i)4,fa kg . 0 P.ngi) hg gt 75 65~% '~,
R~t ~W+~~i4it. 3 ~ 4 '444C~IjA K484W 4 ~t'~t A, PX@~~12.93g. 4 ~@.+4 89.0%, HPLC &4,44 78.3%. 4 f48g.[-Gly]-Bivalirudin 4 0.32%, [+Gly]-Bivalirudin 4 0.13%,
[-2Gly]-Bivalirudin 4 0.10%, [+2Gly]-Bivalirudin 4 0.11%.
10
15
g@,@iJ 18 k&4,r" 44L~~W@]4.
3~r ~4,&tij 13 q~Pj h3 25.5 g 4P~ Ak+ Nij+ SEQ ID NO. 6 j'4w h3
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j 88 X4%.&J, 10ml/g k~j 88 h3 tt &~J Wi'P-%@~X,
'j ( TFA: TIS: uk=94: 3:
3 ( V/V ) ), w i 4' 5f- 2.5h. 8 Ji 4k% +i~-- @+~~'j -, @~ 'j -r~,
~ TFA '4Pt; 3 i, 4-j-jp j&@i)4,fa kg . 0 P.ngi) hg gt 75 65~% '~,
R~t ~W+~~i4it. 3 ~ 4 '444C~IjA K484W 4 ~t'~t A, PX@~~14.38g. 4 ~@.+4 99.0%, HPLC &4,44 86.7%. 4 f48g.[-Gly]-Bivalirudin 4 0.10%, [+Gly]-Bivalirudin 4 0.09%,
[-2Gly]-Bivalirudin 4 0.05%, [+2Gly]-Bivalirudin 4 0.03%.
20
25
QiJ 19' king +4K.~~IYJQ]4-
gr+4&lij 14 S~SIj h'l 26.6 g 4P~ Ak+Nij+ SEQ ID NO. 6 Pfrwh'l
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j 88 X4%.&J, 10ml/g k~j 88 h3 t t &~J Wi'P-%@~X,
'j ( TFA: TIS: uk=90: 2:
8 (V/V)), ai 4' 5$-2.5h. 8 Ji 445+r~---@+~~'j-, @~'j-r~,
~ TFA '4Pt; 3 i, 4-j-jp j&@i)4,fa kg . 0 P.ngi) hg gt 75 65~% '~,
R~t ~W+~~i4it. 3 ~ 4 '444C~IjA K484W 4 ~t'~t A, PX@~~13.98g. 4 ~@.+4 96.2%, HPLC &4,44 83.2%. 4 f48g.
WO 2014/032257 PC T/CN2012/080792
—17—
[-Gly]-Bivalirudin 4 0.10%, [+Gly]-Bivalirudin 4 0.05%,
[-2Gly]-Bivalirudin 4 0.02%, [+2Gly]-Bivalirudin 4 0.02%.
10
15
gA4'J 20: kAr" 4Ã~~g@J4-gr+4&lij 14 S~SIj h'l 26.6 g 4P~ Ak+Nij+ SEQ ID NO. 6 Pfrwh'l
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j 88 X4%.&i 10ml/g k~j 88 h3 t t &~J Wt'P-%@~X,
'j ( TFA: TIS: uk=95: 4:
1 ( V/V ) ), a i 4' 5f- 2.5h. 8 Ji 4k 5 +ii-- @+~~'j -, @~ 'j - r~,
~ TFA '4Pt; 3 i, 4-j-jp j&@i)4,fa kg . 0 P.ngi) hg gt 75 65~% '~,
R~t ~W+~~i4it. 3 ~ 4 '4444~ijA K484W 4 ~t'~t A, PX@~~13.65g. 4 ~@.+4 93.9%, HPLC &4,44 83.7%. 4 f48g.[-Gly]-Bivalirudin 4 0.11%, [+Gly]-Bivalirudin 4 0.07%,
[-2Gly]-Bivalirudin 4 0.02%, [+2Gly]-Bivalirudin 4 0.03%.
20
25
%%@iJ 21: kAP�CA~~KW-)4~�;4,5rij 14 q~fij hg 26.6 g 4+~54+ Nij+ SEQ ID NO. 6 Pfr w h'1
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j 88 X4%.&i 10ml/g k~j 88 h3 tt &~J Wt'P-%@~X,
'j ( TFA: TIS: uk=94: 3:
3 ( V/V ) ), w i 4' 5f- 2.5h. 8 Ji 4k 5 +ii-- @+~~'j -, @~ 'j - r~,
~ TFA '4Pt; 3 i, 4-j-jp j&@i)4,fa kg . 0 P.ngi) hg gt 75 65~% '~,
R~t ~W+~~i4it. 3 ~ 4 '4444~ijA K484W 4 ~t'~t A, PX@~~15.21g. 4 ~@.+4 104.7%, HPLC &4,44 87.1%. 4 f 48g.
[-Gly]-Bivalirudin 4 0.06%, [+Gly]-Bivalirudin 4 0.03%,
[-2Gly]-Bivalirudin 4 0.01%, [+2Gly]-Bivalirudin 4 0.02%.
WO 2014/032257 PC T/CN2012/080792
10
—18—
+ATE'J 22' R44p Q+zulYJQ]+
3~r~4, &P~J 15 q~Pjh3 24.6 g 4,P~ Ak.+N~j+ SEQ ID NO. 6 j'jt wh3
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang k~j 88 X4 %.
&J, 10ml/g k~j 88 h3 t t &~J Wt'P-%@~X,'
j ( TFA: TIS: uk=90: 2:
8 (V/V)), ai 4' 5f- 2.5h. 8 Ji 445+r~---@+~~'j-, @~'j-r~,
~ TFA '4Pt; 3 i, 4-j-jp j&@i)4,fa kg . 0 P.ngi) hg gt 75 65~% '~,4--e)~R-~I] h K4~ 4@@ &t ~t=A, P Xkx.
12.74g . 4 ~ @.+ 4 87.7%, HPLC &4, P~ 4 75.9% .[-Gly]-Bivalirudin 4 0.14%, [+Gly]-Bivalirudin 4 0.06%
[-2Gly]-Bivalirudin 4 0.04%, [+2Gly]-Bivalirudin 4 0.05%.
15
20
25
RAN'J 23' N4AJ CA&&KWk
3~r~4, &P~J 15 q~Pjh3 24.6 g 4,P~ Ak.+N~j+ SEQ ID NO. 6 j'jt wh3
Boc-D-Phe
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu-Wang k~j 88 X4%.&J, 10ml/g k~j 88 h3 t t &~J Wt'P-%@~X,
'j ( TFA: TIS: uk=95: 4:
1 (V/V)), ai 4' 5f- 2.5h. 8 Ji 445+i~--@+~~'j-, @~'j-r~,
~ TFA '4Pt; 3 i, 4-j-jP j&f= i)4,fakg. 0 P.ngi)hggt 7565~%'~,4--e)~R-~I] h K4~ 4@@ &t ~t=A, P Xkx.
13.15g . 4 ~ @.+ 4 90.5%, HPLC &4, 4 4 77.1% . 4 f 4 8g.
[-Gly]-Bivalirudin 4 0.13%, [+Gly]-Bivalirudin 4 0.08%
[-2Gly]-Bivalirudin 4 0.05%, [+2Gly]-Bivalirudin 4 0.05%.
geW 24: ~~AH &A--KWkj™~+$&ij 15 g~gij hg 24.6 g 4,+5k+ N~j+ SEQ ID NO. 6 j'4~ h'l
Boc-D-Phe
WO 2014/032257 PC T/CN2012/080792
—19—
-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu
)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)- Tyr(tBu)-Leu-Wang $2tf 88 X4 %.
&J, 10ml/g $2tf88 h3 t4&~J Wt'P-%@~X,'
j ( TFA: TIS: uk=94: 3:
3 (V/V)), Wi 4' 5f- 2.5h. 8 Ji 445+i~--@+2~'j-, @~'j-r~, &2t&~:e
10
15
13.94g . 4 ~ @.+ 4 95.9%, HPLC &4, 4 4 80.6% . 4 f 4 8g.
[-Gly]-Bivalirudin 4 0.11%, [+Gly]-Bivalirudin 4 0.05%
[-2Gly]-Bivalirudin 4 0.02%, [+2Gly]-Bivalirudin 4 0.02%.
gA+J z5: k,&XP&4aR.@&K
4&4&%+A&~~J 16 ~~ 24 6;.- —9h34~j4-WRA~JSzh34P~-6k+W~j+
SEQ ID NO. 6 Pfrw h3 Pt A, P ~4LN. 12.0g 5 &4,5475 600ml 'im@, mtiJ, -,
~@/j,.-i&4-5 .&4.&E Ki~+5$:K .:8: 50.250, @484H/Nl'HA+~PA:"~@A. R1~4H~444.
'ir i~it: 80ml/min.
t''r&J'kQ: 280nm.
'iri~~ggI='l A 4I='l: 4t 4 5% Z8q ( V/V ) h3 20mmol/L +~pl%:4,44~wif'rg A,20 5+45%.ig pH 4 2.5.
'iri~~kgH B 4H: 4t 50% Z8q ( V/V ) h3 20mmol/L +~j-lk.:4.44
+300mmol/L -4~5E4AA. if% 8&, 8 4.4,5448iJj pH 4 7.0.35min
80%A+20%B~ 50%A+50%B
25
&4+ x. .' 2.0g(100ml).
~454~~$X: 3-'r 6~~8+%- 5min 4%4'-, ~~&TgjP ~4,gQ, ~~+;y J+~~~W+
+» +4=4@"l=t h0+~N~ &+~~, &+4 ~N& l'k*4-~~& C@P&+I)~~i5~g~l'~w*k='3~g~ Z8q 4 ml g» .
~~~ r i rr+ ~kg+
$4-" K~~+&0:
WO 2014/032257 PC T/CN2012/080792
—20—
50 x 250mm, g4.8 $g C18 g j~g@
ir i~it: 80ml/min.
~~t+ ir~~J ikQ: 280nm.
'iri~~ggH A 4H: 0.05%TFA ( V/V ) 'iV-K&.
'ir~it. ~ggH B 4H: Ki~g~4 L8q .
95%A+5%B ' ":95%A+5%B '60%A+40%B:60%A+40%B
Xk+4$;, : 300ml.
l4.&4~4 fX: 3-.r K-i.':8+%- 5m1n 4&kj-, ~~vkyP~l4. &E, ~~- r~J+@410 Fl h3+~i4~~~ 3~~ Fl h3+~i4~~~')4, fake, ~%~~ 30ml 4 if'.
if g Pcr 4+ 8 K4s +34I 4r'I q N- 6.16g ~ ~g4~J 4+ hg L4A P X r'I q %~~4+~H g~~g~~'y~J g~~'y~J hq g~~g+g 1 Pg~ XP PgqgP gA+hq yA+Il
27.562min, 4 re@,P X g-."j~~ h3 k+84 1&1—f4, 'g Pf%.@.+i.~,'' ~ Pjt
f ale, ~ZA, P ~4~ZA, P ~g-.- h3%@+~~.' —f4. 4&1]~r 4«P]h3~&4
15 4-k Y~~A, P~ -' Pjt- ~)8—5' ~~~4 +XI]44-h)~~A, P~PE[-Gly]-Bivalirudin 8 [+Gly]-Bivalirudin ~+Bi. h3~ z 3:-'3 J' 4 0.05%,
[-2Gly]-Bivalirudin 8 [+2Gly]-Bivalirudin 3:-'3 +$~~ k, & i ~+Bi. h3~ z 3:-'J J
4 0.10%. 4@.++' 4 49.3%.
20
25
&~ »44X~PJ 8 Ikey. h0 —Ht4A, r" ~h04~J4-~4~&R- Y W'-m~rt ~8. 4~r- R 7 4'4+&~J~444% hh)8@&%4~A.~~A-Y W~~, ~r. S%4&~J h0
~X,&P]-.~R 4~~ sk JÃi@4«P] hhw&8. N~ ""=-~~.. ri 4 48 k, ~4448.f 4jhSgg f~jq +jjj, P+%,g ++OP]P Jghg jj $~ P, iT. +&f,~g++HP] ~k
&yg g p4~kg~&i~~~i$, it~~ (%~kg~&~~ ~i$4 g-p.+4~5 Ex''J+4-h3&g4j' 't'- l%
WO 2014/032257 PC T/CN2012/080792
—21—
1, —~rt A, P mh34~j4-wk, 8.44~1 T+r4P-:
+~'4~ 1: 4' 4-$ N. g g.;
+r4P- 2: gxPfrm4$ N. g 4~44%5k. , ggH@@%4i4~jl~rt A)" mN$~f
88;
+~47. 3: W~Pfr~4&t:A, P ~N.5~$884'.@-5~4.&K~I &zest:A, P ~;j'gm~P N. g g.h34%5k+0~j+ SEQ ID NO: 1 4, SEQ ID NO: 2 4,SEQ ID NO: 3 4, SEQ ID NO: 4 4, SEQ ID NO: 5 Pgw.
2. 5-Nf&f'j-k4- 1 Pfr~~h3WC
m~ Rl 4I='t 4, 4X%ir" %4& Leu, Tyr, Glu, Glu, Pro, Ile, Glu, Glu, Phe,
Asp, I'=L SEQ ID NO: 2 Pfrwk+P~Jh3$ N. g g., -I'=L SEQ ID NO: 1 j'g
~k'+P~Jhl 5 N. g g., Arg, Pro, Phe.
3. 5-8-k~fj-k4' 1 Pgt-~~h3~k, A-f4A44, Pgt-~~%4~-I=L'444Pgt-
15 m~ RJ 4H4, 4X%ir" %4& Leu, Tyr, Glu, Glu, Pro, Ile, Glu, Glu, Phe,
Asp, Gly, Asn, -I'=L SEQ ID NO: 3 j'4wk+4~J h3 5 N. ~@., Arg, Pro,
10
Phe.
4. 5-$54~4 j-k4' 1 Pfr~~h3~C, 4f4A4g, Pfr~~%4~-I-=L'44414
m~ Rl 4I='t 4, 4X%ir" %4& Leu, Tyr, Glu, Glu, Pro, Ile, Glu, Glu, Phe,
20 Asp, I'=L SEQ ID NO: 5 Pfrwk+P~Jh3$ N. g g., -I'=L SEQ ID NO: 4 j'g
~k'+P~Jhl 5 N. g g., Arg, Pro, Phe.
5, 5-$54%4'j-k4. 1 Pfrm4h3Wk, ~4gQPg, Pfrmk+~4g 1 I'=Lg P:11 ) 4~j4- Fmoc-Gly-CTC $~$88A, Fmoc-Gly-Gly-CTC 5~$88;
12 ) %.t'~t, j'4m~ Fmoc-Gly-CTC $~f88 4, Fmoc-Gly-Gly-CTC $~f88X h l
25 Fmoc 3+0)'A, s&4~%.l'~t Fmoc %4) P~ h l CTC 5~$88;
13 ) 44j'4m~%.*Fmoc %4)'Ah) CTC 5~$88, Air" %4&44 Fomc %.
4j'P~ h34,P~ Ak. , ~ 5'j4jg~. .+ SEQ ID NO: 1-5 Pgw4, P~ Ak.+Njh34, P~
Ak.f~fgal ',
14 ) $4j'$~4-JL+ SEQ ID NO: 1-5 j'gw4%5k+0tj h34%5kk~f88%.
WO 2014/032257 PC T/CN2012/080792
—22—
@, 4iI&z44+ SEQ ID NO: 1-5 Sf'-r446kFj 0i] h3$ N. g 4~.
6, 5-$%k4W'j-k4. 5 j'4m~h l W4, 4f4A44, j'4m~ Fmoc-Gly-CTC $~f
884, Fmoc-Gly-Gly-CTC 5~$88 h l ~&+3M~4 0.8mmoVg-1. 2mmol/g.
7, 5-$54~4)-k4 1@-~~Eh~&, A-AA4C, +re~ 2 0R-~~h0 W4~
Wang $~f88.
8, 5.$54~4]-k4 1 lfr~~hhwC, 4f4A4g, +~4& 3 fPfr~~~~@-f-8 iX~iiiI h'1~244%4$. i'=i w kt +44::—4, 65k. 90%-95%, :-~4+~kN,2%-4%, +~ 4
9, 5.$54%4'j-k4. 8 Pfrm~h')WAN, 4f4A44, &J. ml/g ifj'frm~ikiiii]4
Pfrm4kt A, P mN. $~$88h38g. ~g $., kt 4 10:1.
WO 2014/032257 PC T/CN2012/080792
~w CEII
' ~o p)N
0.00 5 QO 'i G.GG 15.00 2G.GG 25.00 30 OQ 35.00 40 QO 45.00K~livutes
INTERNATIONAL SEARCH REPORTInternational application No.
PCT/CN2012/080792
A. CLASSIFICATION OF SUBJECT MATTER
See the extra sheet
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
IPC: C07K
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
CNABS, CNKI and search terms: bivalirudin, synthesis
STN, DWPI, SIPOABS and search terms: bivalirudin, angiomax, synthesis
NCBI, National Bio-sequence Database of Chinese Patent and searched sequences: SEQ ID NOs: 1-5
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category" Citation of document, with indication, where appropriate, of the relevant passages
CN 102532274 A (CHENGDU SHENGNUO BIOPHARM CO, LTD.), 04 July 2012(04.07.2012), claim I, and description, paragraphs 12-43
CN 101475631A (CHINATECH PEPTIDE CO. , LTD.), 08 July 2009 (08.07.2009), claim I
Relevant to claim No.
1-9
1-9
1-9
US 20110251372Al (SOMMEN, G. et al), 13 October 2011 (13.10.2011), claim I
CN 102164609 A (SCINOPHARM TAIWAN. , LTD.), 24 August 2011 (24.08.2011), claim I
1-9
1-9
Further documents are listed in the continuation of Box C. g See patent family annex.
Special categories of cited documents:
"A" document defining the general state of the art which is not
considered to be of particular relevance
"E" earlier application or patent but published on or after the
international filing date
"L" document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
"0" document referring to an oral disclosure, use, exhibition or
other means
"P" document published prior to the international filing date
but later than the priority date claimed
Date of the actual completion of the international search
15 April 2013 (15.04.2013)
Name and mailing address of the ISA/CN:State Intellectual Property Office of the P. R. ChinaNo. 6, Xitucheng Road, JimenqiaoHaidian District, Beijing 100088, China
Facsimile No. : (86-10) 62019451
Form PCT/ISA/210 (second sheet) (July 2009)
"T" later document published after the international filing dateor priority date and not in conflict with the application butcited to understand the principle or theory underlying theinvention
"X" document of particular relevance; the claimed inventioncannot be considered novel or cannot be considered to involve
an inventive step when the document is taken alone"Y" document of particular relevance; the claimed invention
cannot be considered to involve an inventive step when thedocument is combined with one or more other suchdocuments, such combination being obvious to a personskilled in the art
"&" document member of the same patent family
Date of mailing of the international search report
23 May 2013 (23.05.2013)
Authorized officer
LIU, Rui
Telephone No. : (86-10) 62411107
INTERNATIONAL SEARCH REPORTInformation on patent family members
International application No.
PCT/CN2012/080792
Patent Documents referred
in the Report
CN 102164609 A
US 20110251372Al
CN 102532274 A
CN 101475631 A
Publication Date
24.08.2011
13.10.2011
04.07.2012
08.07.2009
Patent Family
EP 2349307 Al
CN 102164608 A
AU 2009288036 A2
AU 2009288027 A2
INCHENP 201101481E
JP 2012502044 A
JP 2012502045 A
INCHENP 201101490E
US 8252896 B2
US 2011288235 Al
US 2010081788 Al
US 2010056755 Al
WO 2010028122 Al
WO 2010028131 Al
AU 2009288027 Al
AU 2009288036 Al
TW 201014867 A
TW 201024316 A
CA 2736113Al
CA 2736126 Al
EP 2334314 Al
KR 20110056536A
KR 20110069048 A
WO 2010075983 Al
WO 2010075983 A8
TW 201024317 A
CA 2744627 Al
AU 2009335350 Al
MXPA 11006950A
KR 20110112385A
EP 2382232 Al
CN 102264757 A
ZA 201104195A
JP 2012514015 A
None
CN 101475631 B
Publication Date
03.08.2011
24.08.2011
11.03.2010
11.03.2010
02.12.2011
26.01.2012
26.01.2012
01.06.2012
28.08.2012
24.11.2011
01.04.2010
04.03.2010
11.03.2010
11.03.2010
11.03.2010
11.03.2010
16.04.2010
01.07.2010
11.03.2010
11.03.2010
22.06.2011
30.05.2011
22.06.2011
08.07.2010
26.08.2010
01.07.2010
08.07.2010
08.07.2010
31.07.2011
12.10.2011
02.11.2011
30.11.2011
25.04.2012
21.06.2012
17.08.2011
Form PCT/ISA/210 (patent family annex) (July 2009)
INTERNATIONAL SEARCH REPORT
CONTINUATION: A. CLASSIFICATION OF SUBJECT MATTER
C07K I/04 (2006.01) i
C07K 7/08 (2006.01) i
International application No.
PCT/CN2012/080792
Form PCT/ISA/210 (extra sheet) (July 2009)
W IIT, 4R8 ~+ IlrJ@N8
PCT/CN2012/OS0792
A. XIN&4r~il@lw R
fk,l:, + (T+W~& (IPC)N, e nii left, l:,+ %8~8 IPC A8'&
B. 4N~RIgi
Y- 4 h1R/R RCXi@(6%&%@Set~&AU)
IPC: C07K
8, -. 1~Y- 4'i~44+ h1@R/R RCXi@U Pl. h'I Y- %Xi@
1~ + IlrJcY~NK5kJh1475%$(MfhMA„@%%3'IY~NH (4I1%%) )
CNABS, CNKI $HYz4Hii: I.l:5Pg, I:I:5/ /7, ~~4; STN, DWPI, SIPOABS $H Yz4H~: bivalirudin, angiomax,
synthesis; NCBI, + + ~$~J%$PJI+Wl]Yi44~i9HYi4 3'JI+Wl]: SEQ ID NOs: 1-5
Cl@x/t, ~4K, NW@k~~i~
CN 102532274 A (44$~mgg@g~]gq, @@rziq~) 4 7 q 2012 (04 07 2012)
ggIJ+4 I, jgng+g 12-43 P~
cN 101475631 A (5)'I'I +HXHItz4+, Lc4@rz~q~)8 7 q 2009(08 07 2009
KNi44 I
US 20110251372 Al (Geoffroy Sommen et al) 13.10 H 2011 (13.10.2011)
KTIJ44 I
CN 102164609 A ( ~ ~~yP~Ir'gr @@rziqr) 24 8 q 2011 (24 08 2011)
KTIJ44 I
84h'I@TIJ44
1-9
1-9
1-9
1-9
1-9
ccAll
CCEll
ccE'll
CCOll
CCP'll
%%X/01+ C 4'- 31RR +%If5 .I%XBIIAW4~~
i) Ari 4~~5JTMIihk@7@A~W —hMkIIAXBrJC@4 HIIq~~i' ~rL@II~~g@4g~~]
U~aMNA844YJ4'8 NII03UV, NA4R% —4Vl@XBIIdrz', 4HRVI% II044. *NRN%1%8%9I%IIAX10 (/II1g'f@igWIIg)
88 U%rL~R, %%, )%~&NNA, aiA'L', RIIAxBrL~4 H 4+ *I1rJ 4t~ HKJ&+%44IIMRK H IIAXB
ccTll
CCX'll
CCY'll
CCg'll
r~ H%+TIJIN/t.
1+@1'H4@4$gHZj —L', 4 9@ipgggg JH4 f
1V@NWZ 1Vi.~%1A1W~F~ XB$~5JCWIIAXB, +Kkr.,4~+A, V Z44/Yk/'IIA
Q Eg +~@@yjhIig Q~g~g@Q~JQ
$~5J48%ÃJxjt 54~xjt 9%—444544~~~xjt4APIIRW'4A4+W, i~tl@WA&~ A~R@r~iIIV,
44%k/ ÃJN. %XA% Q~JK'I'%
-%+f~fIIQXB
+ IlrJc Y~NCIlrJc%4Ã1 H th)
15.4 8 2013 (15.04.2013)
ISA/CN II'J gj fr]i $Ij@g1$11I:
+%AX%%W WVR~~HPKP-0 + 3I: Y. tij 'A/rF ~ @~J I J68+@Itk 6 9 100088
3449: (86-10)62019451
PCT/ISA/210 k(g 2 g) (2009 Q 7 Q )
~~ IlrJc Y- 4N .Iiiil@ H NS
23.5 8 2013 (23.05.2013)
~K I- i~
(I]
4N849: (86-10) 62411107
W (54%85WT- —I+k~lN&=-. ,Q,
+ (rT@N8PC T/CN2012/OS0792
Y~NNh+CISÃJ
+k~lx/t
CN 102164609 A
US 20110251372 AI
CN 102532274 A
CN 101475631 A
'z'. 4 H NJ
24.08.2011
13.10.2011
04.07.2012
08.07.2009
H%+k~l
EP2349307A I
CN102164608A
AU2009288036A2
AU2009288027A2
INCHENP201101481E
JP2012502044A
JP2012502045A
INCHENP201 101490E
US 8252896B2
US2011288235AI
US2010081788AI
US2010056755AI
WO2010028122A I
WO201002813 IA I
AU2009288027A I
AU2009288036A I
TW201014867A
TW201024316A
CA2736113AI
CA2736126A I
EP2334314AI
KR20110056536A
KR20110069048A
WO2010075983AI
WO2010075983A8
TW201024317A
CA2744627A I
AU2009335350AI
MXPA11006950A
KR20110112385A
EP2382232AI
CN102264757A
ZA201104195A
JP2012514015A
CN101475631B
'z'. 4 H NJ
03.08.2011
24.08.2011
11.03.2010
11.03.2010
02.12.2011
26.01.2012
26.01.2012
01.06.2012
28.08.2012
24. 11.2011
01.04.2010
04.03.2010
11.03.2010
11.03.2010
11.03.2010
11.03.2010
16.04.2010
01.07.2010
11.03.2010
11.03.2010
22.06.2011
30.05.2011
22.06.2011
08.07.2010
26.08.2010
01.07.2010
08.07.2010
08.07.2010
31.07.2011
12.10.2011
02.11.2011
30.11.2011
25.04.2012
21.06.2012
17.08.2011
PCT/ISA/210 4( )g+g~f Iffy) (2009 g 7 R)
W (TUBS ~+ (rT@N8
PC T/CN2012/OS0792
C07K 1/04 (2006.01) i
C07K 7/08 (2006.01) i
PCT/ISA/210 k(gf egg) (2009 g 7 R )
Recommended