Jean-Pierre Wolf 

Stephan Burkhardt 

CE707 

R-1059.5.13/31 Basel, 26.6.2003 

Investigation of hydrolytic stability of CGI 827 (CG39-0827) at pH 

4.00 (phthalate buffer) 

1. Experimental procedure 

Preparation of test solution: 

1.00 g of CGI 827 (Batch Nr. RH 2017-13) was dissolved in 100 ml acetonitrile 

(UV grade). 

Test solution: 50 ml of above clear solution was added to 50 ml of a phthalate 

buffer, pH 4.00 (Fluka, Art Nr. 72655). 

Storage of test solution: 

100 ml of above test solution containing 500 mg of CGI 827 dissolved in 100 ml 

acetonitrile/phthalate buffer 50:50 was stored in a closed flask at 50°C in the dark 

for 4 days (approximately 90 hours). 

UV measurements: 

Test solution, diluted with acetonitrile to a concentration of 10 mg/liter before storage 

Test solution, diluted with acetonitrile to a concentration of 10 mg/liter after storage at 50°C for 4 

days 

1.0-

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200 300 < 400 500 % ; 

Wavelength (nm) 

• Graph4-90ha)C f^ '''11^ ' ^} ^-!? ^ ^- °?303552 

blue line : solution before stability testing 

red line : solution after 90 h at 50°C 

black line : after extraction with ether (see Section 2. Conclusions) 
Extraction experiments: 

20 ml of the solution stored at 50°C for 4 days (corresponding to 100 mg CGI 827 

in 100 ml acetonitrile/phthalate buffer 50:50) was evaporated almost to dryness. 

The residue was extracted between diethyl ether and water. The water phase 

was separated and extracted with diethyl ether. The combined ether phases 

were dried over MgS04 and evaporated to dryness. 

The residue, a light oil, crystallized upon standing at room temperature to give 

102 mg of a white solid. 

The ^H-NMR of this white solid was identical with a fresh sample of CGI 827. 

The UV spectrum of above solid residue (10 mg/l acetonitrile) was identical with 

a fresh sample of CGI 827. 

2. Conclusions 

The disappearance ofthe UV signal of CGI 827 (lambda max at 260 nm) upon 

storage at 50°C in acetonitrile/phthalate buffer 50:50 must be attributed to an 

equilibrium reaction. 

Isolation ofthe sample stored at 50°C by extraction quantitatively renders CGI 

827 (as unambiguously identified by ^H-NMR and UV spectra). 

If hydrolytic decomposition or fragmentation of CGI 827 had occurred, a 

quantitative back extraction of CGI 827 would have been impossible. 

^H-NMR of back isolated CGI 827 (sample contains low amounts of diethyl ether) 

^H-NMR of fresh CGI 827 

UV spectra of back isolated CGI 827 (at a concentration of 10 mg/l acetonitrile) 

3. Possible equilibrium reaction 

Formation of a half acetale 

in acetonitrile/phthalate 

buffer pH 4.00 

at 50°C 

^ HO 

o o 

in organic solvent 

no water 
In the acetonitrile/phthalate buffer media the equilibrium is strongly shifted to the 

half-acetale side. In absence of phthalate buffer (back extraction with ether), the 

equilibrium is clearly on the CGI 827 side. 

Similar equilibrium reactions may occur with a phosphate buffer at pH 7.00 and a 

borate buffer at pH 9.00. 

4. Solubility and stability of CGI 827 in pure water 

10 mg of CGI 827 (Batch Nr. RH 2017-13) were suspended in 1 liter of deionized water and stirred for 6 hours at 50°C. After filtration of a small nondissolved portion of CGI 827, the clear filtrate was cooled to room temperature. 

UV comparison of above solution of approximately 10 mg CGI 827 in water with 

a reference solution of 10 mg of CGI 827 (Batch Nr. RH 2017-13) in 1 liter 

acetonitrile clearly demonstrates that CGI 827 is stable under these conditions in 

water. The result also suggests that solubility of CGI 827 in water is close to 10 

mg/l. 

UV measurements: 

10 mg of CGI 827 in 1 liter acetonitrile 

Ca. lOmgo f CGI 827 in 1 liter de-ionized water (SO'C for 6 hours) 

200 300 400 

Vtfavelength (nm) 

Cell2-827lnWisser^ 

blue line : 10 mg CGI 827 dissolved in 1000 ml acetonitril 

red line : approx. 10 mg CGI 827 dissolved in 1000 ml water (after filtration) 

500 

X: 445.4023, Y: 0.7698125 
NMR spectral data 

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