Jean-Pierr e Wol f

Stepha n Burkhard t CE70 7 R-1059.5.13/3 1 Basel , 26.6.200 3 

### Investigatio n o fhydrolyti cstabilit yo fCG I 82 7 (CG39-0827 ) a t p H

4.0 0 (phthalat e buffer) 1 . Experimenta l procedur e Preparatio n o ftes t solution : 1.00 g o fCG I 82 7 (Batc h Nr . R H 2017-13 ) wa s dissolve d i n 10 0 m l acetonitril e (U V grade) . Tes t solution : 5 0 m l o fabov e clea rsolutio n wa s adde d t o 5 0 m l o fa phthalat e buffer , p H 4.0 0 (Fluka , Ar t Nr . 72655) . Storag e o ftes t solution : 10 0 m l o fabov e tes t solutio n containin g 50 0 m g o fCG I 82 7 dissolve d i n 10 0 m l acetonitrile/phthalat e buffe r50:5 0 wa s store d i n a close d flas k a t 50° C i n th e dar k fo r4 day s (approximatel y9 0 hours) . U V measurements : Tes t solution , dilute d wit h acetonitril e t o a concentratio n o f1 0 mg/lite rbefor e storag e Tes t solution , dilute d wit h acetonitril e t o a concentratio n o f1 0 mg/lite rafte rstorag e a t 50° C fo r4 day s 1.0 -

V) < 0. 5 0. 0 1 1 • •1 20 0 30 0 < 40 0 50 0 % ; Wavelengt h (nm ) •Graph4-90ha) C f

^ '''11^ ' ^ } ^-!? ^ ^ - °?30355 2 blu e lin e : solutio n befor e stabilit ytestin g re d lin e : solutio n afte r9 0 h at 50° C blac k lin e : afte rextractio n wit h ethe r(se e Sectio n 2 . Conclusions )Extractio n experiments :

2 0 m l o fth e solutio n store d at 50° C fo r4 day s (correspondin g t o 10 0 m g CG I 82 7 i n 100 m l acetonitrile/phthalat e buffe r50:50 ) wa s evaporate d almos t t o dryness . Th e residu e wa s extracte d betwee n diethy l ethe ran d water . Th e wate rphas e wa s separate d an d extracte d wit h diethy l ether . Th e combine d ethe rphase s wer e drie d ove rMgS0 4 an d evaporate d t o dryness . Th e residue , a ligh t oil , crystallize d upo n standin g at roo m temperatur e t o giv e 10 2 m g o fa whit e solid . Th e ^H-NM R o fthi s whit e soli d wa s identica l wit h a fres h sampl e o fCG I 827 . Th e U V spectru m o fabov e soli d residu e (1 0 mg/ l acetonitrile ) wa s identica l wit h a fres h sampl e o fCG I 827 . 2 . Conclusion s Th e disappearanc e ofth e U V signa l ofCG I 82 7 (lambd a maxat 26 0 nm ) upo n storag e at 50° C i n acetonitrile/phthalat e buffe r50:5 0 mus t b e attribute d t o a n equilibriu m reaction . Isolatio n ofth e sampl e store d at 50° C byextractio n quantitativel yrenders CG I 82 7 (a s unambiguousl yidentifie d by^H-NM R an d U V spectra) . I fhydrolyti cdecompositio n o rfragmentatio n o fCG I 82 7 ha d occurred , a quantitativ e bac k

extractio n ofCG I 82 7 woul d hav e bee n impossible . ^H-NM R o fbac k

isolate d CG I 82 7 (sampl e contain s lo wamount s o fdiethy l ether) ^H-NM R o ffres h CG I 82 7 U V spectr a o fbac k

isolate d CG I 82 7 (a t a concentratio n o f1 0 mg/ l acetonitrile ) 3 . Possibl e equilibriu m reactio n Formatio n o fa hal facetal e i n acetonitrile/phthalat e buffe rp H 4.0 0 a t 50° C ^ H O o o i n organi csolven t n o wate rI n th e acetonitrile/phthalat e buffe rmedi a th e equilibriu m i s strongl yshifte d t o th e

half-acetal e side . I n absenc e o fphthalat e buffe r(bac k extractio n wit h ether) , th e equilibriu m i s clearl yo n th e CG I 82 7 side . Simila requilibriu m reaction s mayoccu rwit h a phosphat e buffe rat p H 7.0 0 an d a borat e buffe rat p H 9.00 . 4 . Solubilit yan d stabilit yo fCG I 82 7 i n pur e wate r 1 0 m g o fCG I 82 7 (Batc h Nr . R H 2017-13 ) wer e suspende d i n 1 lite ro fde ionize d wate ran d stirre d fo r6 hours at 50°C . Afte rfiltratio n o fa smal l non dissolve d portio n o fCG I 827 , th e clea rfiltrat e wa s coole d t o roo m temperature . U V compariso n o fabov e solutio n o fapproximatel y1 0 m g CG I 82 7 i n wate rwit h a referenc e solutio n of1 0 m g o fCG I 82 7 (Batc h Nr . R H 2017-13 ) i n 1 lite r acetonitril e clearl ydemonstrate s tha t CG I 82 7 i s stabl e unde rthes e condition s i n water . Th e resul t als o suggest s tha t solubilit yo fCG I 82 7 i n wate ri s clos e t o 1 0 mg/l . U V measurements : 1 0 m g o fCG I 82 7 i n 1 lite racetonitril e Ca . lOmgo fCG I 82 7 i n 1 lite rde-ionize d wate r(SO' C fo r6 hours )

20 0 30 0 40 0 Vtfavelengt h (nm ) Cell2-827lnWisser ^ blu e lin e : 1 0 m g CG I 82 7 dissolve d i n 100 0 m l acetonitri l re d lin e : approx . 1 0 m g CG I 82 7 dissolve d i n 100 0 m l wate r(afte rfiltration ) 50 0 X : 445.4023 , Y : 0.769812 5NM R spectra l dat a

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