~~IIII [email protected]/pdf/wo/257/wo14032257.pdf · 2014-03-08 · 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,

(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)

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dong 518057 (CN).

(72) %%A.; 2k(75) RINA/@@A. (%30% ~~): 455 (MI,

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(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 [email protected]~~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—


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.


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

[email protected], ~~% ~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


-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


(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


(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



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


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


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


, +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,



-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


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


DCM 4, 2 ir" . 4'4X 27.6g Fmoc-Tyr(tBu)-OH ( 60mmol ), 9.7 g HOBt


, +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,


WO 2014/032257 PC T/CN2012/080792

—15—


-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


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


)-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%,


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


)-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~,


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%,


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


)-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~,


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%,


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


)-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~,


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%,


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


)-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~,


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%,


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


)-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%


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


)-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%


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—


)-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%


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

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1-9

1-9

1-9

1-9

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88 U%rL~R, %%, )%~&NNA, aiA'L', RIIAxBrL~4 H 4+ *I1rJ 4t~ HKJ&+%44IIMRK H IIAXB

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US 20110251372 AI

CN 102532274 A

CN 101475631 A

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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

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03.08.2011

24.08.2011

11.03.2010

11.03.2010

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11.03.2010

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16.04.2010

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11.03.2010

11.03.2010

22.06.2011

30.05.2011

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08.07.2010

08.07.2010

31.07.2011

12.10.2011

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30.11.2011

25.04.2012

21.06.2012

17.08.2011

PCT/ISA/210 4( )g+g~f Iffy) (2009 g 7 R)

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C07K 1/04 (2006.01) i

C07K 7/08 (2006.01) i

PCT/ISA/210 k(gf egg) (2009 g 7 R )