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Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

(Aminoalkyl)carbamates of forskolin were synthesized at the 6- and 7-hydroxyl positions of forskolin with the length of the alkyl chain varying from ethyl to heptyl. Two of these derivatives, 7-[[(2-aminoethyl)amino]carbonyl]-7-desacetylforskolin (2) and 6-[[(2-aminoethyl)amino]carbonyl]forskolin (3), were used to synthesize iodinated derivatives of forskolin that bind with high affinity to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes, respectively. Hydroxyphenyl derivatives of forskolin were prepared from the (aminoalkyl)carbamates and tested for their ability to bind to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes. The 6-derivative (18) of forskolin had a Kd of 9 nM at adenylyl cyclase and was more potent than either the 7-derivatives or the 6-derivatives of 7-desacetylforskolin. The 7-derivatives were more potent at binding to the glucose transporter than forskolin. In contrast, the 6-derivatives had Kd's greater than 100 microM at the glucose transporter. Isothiocyanates and N-bromoacetyl derivatives were synthesized from 2 and 3 as potential alkylating agents for forskolin binding sites. The alkylating agents produced an irreversible loss of forskolin binding to adenylyl cyclase. In contrast, the alkylating agents bound reversibly to the glucose transporter.

Forskolin (7 beta-acetoxy-8,13-epoxy-1 alpha, 6 beta, 9 alpha-trihydroxylabd-14-en-11-one), a diterpene from the Indian plant Coleus forskohlii, activates cyclic AMP generating systems in a number of mammalian tissues in a rapid and reversible fashion. Derivatives of forskolin have been tested for their ability to stimulate membrane adenylate cyclase from rat brain and rabbit heart, as well as cyclic AMP generation in guinea pig brain vesicular preparations, a model system for intact cells. Derivatives at the 6 beta- and 7 beta-hydroxy functions retain activity, but none have greater activity than that of forskolin. Reduction of the 11-keto function affords an active 11 beta-hydroxy derivative. Reduction of the 14,15-vinyl (alpha) substituent reduces activity, while epoxidation abolishes activity. Derivatization or lack of the 1 alpha- and 9 alpha-hydroxy functions results in a marked reduction in activity, emphasizing the importance of the alpha aspect of the molecule. However, the 1 alpha, 6 beta-di-O-acetyl derivative does retain activity. None of the inactive derivatives, which include the 14,15-epoxy, the 1,9-dideoxy, and the 1,6-diketo derivatives, antagonize the stimulatory effects of forskolin.