Spermine → Dexamethasone-Spermine

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The 21-hydroxy group of dexamethasone is not required for anti-inflammatory activity, and therefore is an ideal choice for conjugation to a polycation (FIG. 1A) (Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics, 1459-1485, eds. Hardman and Limbird, McGraw-Hill, New York). A one-pot reaction between spermine, 2-iminothiolane (Traut's reagent) and dexamethasone mesylate yielded the dexamethasone-spermine and disubstituted conjugates, (DS and D2S, respectively) (FIG. 1B). Traut's reagent is selectively ring-opened by the primary amines (Hermanson, 1996, Bioconjugate Techniques, Academic Press) on spermine, forming a hydrolytically sensitive amidimide bond (Hermanson, 1996, Bioconjugate Techniques, Academic Press) between spermine and iminothiolane and a reactive thiolate anion that reacts with the α-keto mesylate on the 21 position of dexamethasone mesylate (Simmons et al., 1980, J. Org. Chem. 45:084-3088), yielding an α-keto thioether linkage between the dexamethasone and iminothiolane. Dexamethasone mesylate (prepared or received from Steraloids, N.H.) or dexamethasone (Steraloids, N.H.), 2-iminothiolane (Traut's reagent) (Pierce), spermine (Sigma), and ethanol or DMSO (Aldrich) were used as received. A total of 105 mg (223 μmol) of dexamethasone-mesylate, and 28.4 mg (206 μmol) of Trout's reagent were dissolved together in either DMSO or ethanol prior to addition of 31.9 μl (145 μmol of spermine at room temperature. The conjugation reaction was complete in 45 minutes by TLC. HPLC purification (60/40 0.1% TFA/acetonitrile, Hamilton PRP-1 column), evaporative solvent removal, and freeze-drying yielded both DS and D2S as white powders, 1H NMR of DS confirmed the presence of the 3-bis-enone and 11- and 17-hydroxy groups required for glucocorticoid activity (FIG. 1A) as well as 1H signals from the conjugated spermine and the 21-α-keto thioether group. Hydrolysis of DS in 1M NaOH for 20 minutes resulted in the cleavage of the amidimide linkage between spermine and iminothiolane, forming a dexamethasone-amide (DA) (FIG. 1B), which has a 21-substituted butyl thioether amide side-chain on dexamethasone. For DA, FIG. 1B: Calc: C, 63.26; H, 7.35; N, 2.84. Found: C, 63.44; H, 7.27; N, 2.83. Water solubility: DS>100,000 mg/l; DA 60 mg/l; (dexamethasone 100 mg/l).

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