A structurally novel series of adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channel openers is described. As part of our efforts directed toward identifying novel, bladder-selective potassium channel openers (KCOs) targeted for urge urinary incontinence (UUI), we found that bioisosteric replacement of the N-cyanoguanidine moiety of pinacidil (1, Figure 1) with a diaminocyclobutenedione template afforded squaric acid analogue 2, the prototype of a novel series of K(ATP) channel openers with unique selectivity for bladder smooth muscle in vivo. Further modification of the heterocyclic ring to give substituted aryl derivatives (3) afforded potent KCOs that possessed the desired detrusor selectivity when administered orally. The effects of these potassium channel agonists on bladder contractile function was studied in vitro using isolated rat detrusor strips. Potent relaxants were evaluated in vivo in a rat model of bladder instability. Lead compounds were evaluated concomitantly in normotensive rats for their effects on mean arterial blood pressure (MAP) and heart rate as a measure of in vivo bladder selectivity. (R)-4-[3,4-Dioxo-2-(1,2, 2-trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzo nitrile (79) met our potency and selectivity criteria and represents an attractive development candidate for the treatment of UUI. Electrophysiological studies using isolated rat bladder detrusor myocytes have demonstrated that compound 79 produces significant hyperpolarization which is glyburide-reversed, thus consistent with the activation of K(ATP). The design, synthesis, structure-activity relationships (SAR), and pharmacological activity associated with this series of novel KCOs will be discussed.

No abstract

Trypanosomiasis remains a significant disease across the sub-Saharan African continent, with 50,000 to 70,000 individuals infected. The utility of current therapies is limited by issues of toxicity and the need to administer compounds intravenously. We have begun a program to pursue lead optimization around MDL 73811, an irreversible inhibitor of S-adenosylmethionine decarboxylase (AdoMetDC). This compound is potent but in previous studies cleared rapidly from the blood of rats (T. L. Byers, T. L. Bush, P. P. McCann, and A. J. Bitonti, Biochem. J. 274:527-533). One of the analogs synthesized (Genz-644131) was shown to be highly active against Trypanosoma brucei rhodesiense in vitro (50% inhibitory concentration, 400 pg/ml). Enzyme kinetic studies showed Genz-644131 to be approximately fivefold more potent than MDL 73811 against the T. brucei brucei AdoMetDC-prozyme complex. This compound was stable in vitro in rat and human liver microsomal and hepatocyte assays, was stable in rat whole-blood assays, did not significantly inhibit human cytochrome P450 enzymes, had no measurable efflux in CaCo-2 cells, and was only 41% bound by serum proteins. Pharmacokinetic studies of mice following intraperitoneal dosing showed that the half-life of Genz-644131 was threefold greater than that of MDL 73811 (7.4 h versus 2.5 h). Furthermore, brain penetration of Genz-644131 was 4.3-fold higher than that of MDL 73811. Finally, in vivo efficacy studies of T. b. brucei strain STIB 795-infected mice showed that Genz-644131 significantly extended survival (from 6.75 days for controls to >30 days for treated animals) and cured animals infected with T. b. brucei strain LAB 110 EATRO. Taken together, the data strengthen validation of AdoMetDC as an important parasite target, and these studies have shown that analogs of MDL 73811 can be synthesized with improved potency and brain penetration.

A novel series of benzylamine, potassium channel openers (KCOs) is presented as part of our program toward designing new, bladder-selective compounds for the treatment of urge urinary incontinence (UUI). We have found that the in vitro potency of (R)-4-[3,4-dioxo-2-(1,2, 2-trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzo nitrile 1 in the relaxation of precontracted rat detrusor strips can also be obtained with cyanobenzylamine derivative 4 (IC(50) = 0.29 microM) (Figure 3). Addition of a 2-Cl substituted benzylamine moiety and changing the alkylamino substituent of 4 to a t-Bu amine gives 31 (IC(50) = 0.14 microM)-a compound with similar in vitro potency as 4 as well as relaxant activity on bladder smooth muscle in vivo when administered orally (31, ED(50) = 3 mg/kg) in a rodent model of bladder instability. Further modifications, particularly the replacement of the t-Bu amino substituent with a tert-amylamine, gave a similarly active compound 60 (IC(50) = 0.10 microM) which shows excellent in vivo efficacy (ED(50) = 0.6 mg/kg). Moreover, 60, 3-(2,4-dichloro-6-methyl-benzylamino)-4-(1, 1-dimethyl-propylamino)-cyclobut-3-ene-1,2-dione (WAY-151616), shows excellent tissue selectivity for bladder K channels over arterial tissue (60, MAP ED(20) = 100 mg/kg; selectivity: MAP ED(20)/bladder ED(50) = 166). Other manipulations of the benzylamino cyclobutenediones, acylation of the benzylamine, conversion of the benzylamine substituent to a benzamide, homologation of the benzylamine to a phenethylamine, and incorporation of a methyl group at the benzyl carbon, all led to substantial loss of in vitro activity, although some in vivo activity was maintained in the acylated analogues. Compound 60 represents an attractive candidate for development in the treatment of UUI.

Sleeping sickness or human African trypanosomiasis infects between 50,000 and 150,000 people each year across subSaharan Africa and is fatal if left untreated. Yearly estimates of people at risk are 10 million on the African continent. Current drugs for late stage disease, such as melarsoprol, have significant toxicity and resistance to melarsoprol is increasing. Another drug, eflornithine, requires 2 weeks of intravenous infusion, which is highly impractical in rural Africa (10). A promising new combination regimen for latestage disease that appears to be effective uses eflornithine for 1 week plus oral nifurtimox for 10 days (15). This is a smallscale trial that needs to be reinforced with more data. Nevertheless, new therapies are urgently needed; because of the extreme poverty in countries with endemic disease, there has been little interest for many years within the pharmaceutical industry in discovering and developing new drugs to treat a disease that occurs primarily in developing countries (10).Polyamine metabolism of African trypanosomes has been shown to be a valid chemotherapeutic target for inhibitors aimed at critical points in the pathway such as ornithine decarboxylase (2), trypanothione synthase (11, 13), and S-adenosylmethionine decarboxylase (AdoMetDC) (6). AdoMetDC has been shown to be an essential enzyme in the trypanosome polyamine pathway, both by RNA interference knockdown (18,19) and through the use of inhibitors, such as MDL-73811 (8), which kill parasites both in vitro (1) and in vivo (4, 6). However, while potent, MDL-73811 lacks the pharmacokinetic and tissue (central nervous system [CNS]) distribution characteristics that are essential to meet the improved target product profile for a new late-stage antitrypanosomal drug (7). We therefore started a program to synthesize analogs with high potency and better pharmacodynamic properties. Genz-644131, the 8-methyl analog of MDL-73811, was significantly more potent in vivo against trypanosomes than other analogs made at that time (5). Some adenine C-8-substituted analogs were recently tested as inhibitors of human AdoMet decarboxylase but not against the trypanosome enzyme or versus trypanosomes in vitro. (14). MATERIALS AND METHODSChemical synthesis. MDL-73811 was synthesized as a benchmark using methods that have been previously described (15). The synthesis of Genz-644131 is described elsewhere in detail (see supplemental material for reference 17) and briefly outlined here. 8-Bromoadenosine was O protected with hexamethyldisilazane and subjected to palladium-catalyzed coupling with trimethylaluminum to install the 8-methyl group. The product was then deprotected and treated with thionyl chloride in pyridine to afford the 5Ј-chloro derivative. This intermediate was converted to the 5Ј-methylamino compound by treatment with excess methylamine and then alkylated with cis-1-(t-butoxycarbonylamino)-4-chloro-2-butene to furnish the protected product. The synthesis of Genz-644131 was completed by acid catalyzed cleavage of the terminal N-buto...