CC([C@@H]1[C@@]2(C)CC[C@@H]3[C@:21]4([CH3:22])[C:15](=[CH:16][C:17]([CH2:19][CH2:20]4)=[O:18])CC[C@H]3[C@@H]2CC1)=O.CN1C[CH2:29][O:28]CC1.C(O[C:36](Cl)=[O:37])C(C)C.C1C2C(=[NH+:44]C3C(C=2)=CC=CC=3)C=CC=1>CN(C=O)C.C(=O)=O.C(Cl)(Cl)(Cl)Cl.CS(C)=O>[CH3:36][O:37][C:16]1[CH:15]=[C:21]([CH2:22][NH2:44])[CH:20]=[C:19]([O:28][CH3:29])[C:17]=1[OH:18]
Progesterone hemisuccinate (90 mg, 0.209 mmole) and N-methylmorpholine (22 μl, 209 mmole) were dissolved in anhydrous DMF (2 ml). The solution was chilled in dry ice/CCl4 bath and isobutylchloroformate (30 μl, 0.229 mmole) was added. After 2 minutes a solution of the acridinium (42) (101 mg, 0.143 mmole) in dimethylsulfoxide (2 ml) containing N-methylmorpholine (3.14 μl, 0.28 mmole) was added. Stirring was continued at -20° C. for 10 minutes and the cooling bath was removed. After stirring at room temperature for 7 hours, 3 drops of water were added. The solvents were removed in vacuo and ethyl acetate was added to the residue. The oily precipitate was washed repeatedly with ethyl acetate. Upon trituration with acetonitrile (2 ml) the oil separated as solids. The product was purified on HPLC using C18Dynamax semi-prep column (10 mm×250 mm) (commercially available from Rainin Instrument Co., Inc., Woburn, Mass.) using CH3CN/H2O (0.1% trifluoroacetic acid), 55/45 as mobile phase at a flow rate of 2.75 ml/min. The peak appearing at retention time of 6.00 minutes was collected. Evaporation of solvents gave the conjugate (43) (yield=30%). MS: FAB, thioglycerol matrix, 895 (M+, without any counterions).
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