By utilization of three-dimensional structure information of rifamycins bound to RNA polymerase (RNAP) and the human pregnane X receptor (hPXR), representative examples (2b-d) of a novel subclass of benzoxazinorifamycins have been synthesized. Relative to rifalazil (2a), these analogues generally display superior affinity toward wild-type and Rif-resistant mutants of the Mycobacterium tuberculosis RNAP but lowered antitubercular activity in cell culture under both aerobic and anaerobic conditions. Lowered affinity toward hPXR for some of the analogues is also observed, suggesting a potential for reduced Cyp450 induction activity. Mouse and human microsomal studies of analogue 2b show it to have excellent metabolic stability. Mouse pharmacokinetics in plasma and lung show accumulation of 2b but with a half-life suggesting nonoptimal pharmacokinetics. These studies demonstrate proof of principle for this subclass of rifamycins and support further expansion of structure-activity relationships (SARs) toward uncovering analogues with development potential.

Background Renin-angiotensin system blockade reduces inflammation in several organ systems. Having found a fourfold increase in angiotensin II type Ia receptor expression in a dextran sodium sulfate colitis model, we targeted blockade with angiotensin II type Ia receptor antagonists to prevent colitis development. Because hypotension is a major complication of angiotensin II type Ia receptor antagonists use, we hypothesized that use of angiotensin II type Ia receptor antagonists compounds which lack cell membrane permeability, and thus enteric absorption, would allow for direct enteral delivery at far higher concentrations than would be tolerated systemically, yet retain efficacy. Methods Based on the structure of the angiotensin II type Ia receptor antagonist losartan, deschloro-losartan was synthesized, which has extremely poor cell membrane permeability. Angiotensin II type Ia receptor antagonist efficacy was evaluated by determining the ability to block NF-κB activation in vitro. Dextran sodium sulfate colitis was induced in mice and angiotensin II type Ia receptor antagonist efficacy delivered transanally was assessed. Results In vitro, deschloro-losartan demonstrated near equal angiotensin II type Ia receptor blockade compared to losartan as well as another angiotensin II type Ia receptor antagonist, candesartan. In the dextran sodium sulfate model, each compound significantly improved clinical and histologic scores and epithelial cell apoptosis. Abundance of TNF-α, IL-1β, and IL6 mRNA were significantly decreased with each compound. In vitro and in vivo intestinal drug absorption, as well as measures of blood pressure and mucosal and colonic blood flow, showed significantly lower uptake of deschloro-losartan compared to losartan and candesartan. Conclusions This study demonstrated efficacy of high-dose angiotensin II type Ia receptor antagonists in this colitis model. We postulate that a specially designed angiotensin II type Ia receptor antagonist with poor oral absorption may have great potential as a new therapeutic agent for inflammatory bowel disease in the future.

Regulators of G Protein Signaling (RGS) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation and structure-activity relationships (SAR) of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-ValLys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH 2 , S-S) (Roof, et al. Chem Biol Drug Des 2006; 67:266-274). Using a focused one-bead, one-compound (OBOC) peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2, (Gly-Thrc[Cys-Phe-Gly-Thr-Cys]-Trp-NH 2 , S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated Gα o GTPase activity at 25-50 μM. They selectively inhibit RGS4 but not RGS7, RGS16 and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.

An improved synthesis of 3-(substituted)pyrimido [4,5-c]pyridazine-5,7(1H,6H)-diones, a known subclass of 4-deazatoxoflavins, is reported. The approach involves treatment of 3-methyl-6-(1-methylhydrazinyl) uracil with representative phenyl and alkyl glyoxal monohydrates, which in turn are obtained by selenium dioxide oxidation of the corresponding phenyl and alkyl methyl ketones. The first entry into 4-monosubstituted isomers is also reported. KeywordsPyrimido [4,5-c]pyridazine-57(1H6H)-dione; 4-Deazatoxoflavin; Phenylglyoxal; Cyclizations; 6-(1-Methylhydrazinyl)uracil 1,6-Dimethylpyrimido[4,5-c]pyridazine-5,7(1H,6H)-dione, also known as 4-deazatoxoflavin, and its 3,4-disubstituted analogues have been shown to serve as biomimetics of flavin and 5-deazaflavin in their ability to oxidize amines to carbonyl compounds 1 and to abstract hydrogen equivalents from hydrogen donors under certain conditions (Fig. 1). 2 4-Deazatoxoflavin itself has also demonstrated both inhibitory activity against Pseudomonas and DNA-binding properties 3 while not displaying the generalized cytotoxicity that is characteristic of toxoflavin. 4 There are scattered reports of 3,4-disubstituted 3,5 and 3-mono-substituted 2,6 analogues of 4-deazatoxo flavin, but no reports of 4-monosubstituted analogues. The described syntheses of 3-monosubstitued analogues generally proceed in poor yields and via pathways that produce alternate products. Our goal was to develop an improved synthesis that would lead to clean products and possess generality for a wide range of analogues for biological testing.Our strategy is based on the known reaction of 6-(1-methylhydrazinyl) uracil with glyoxal and α-diketones to yield 3,4-unsubstituted-and 3,4-disubstituted-pyrimido[4,5-c]pyridazine-5,7 (1H,6H)-diones, respectively.3 Despite these prior examples, there are no reports of reaction between this or related 6-(hydrazinyl)uracils and unsymmetrical alkyl or aryl glyoxals. We imagined that reaction could occur by several manifolds. One could proceed via initial hydrazone formation onto either one or both of the aldehyde or ketone moieties, or as is often the case for 6-aminouracils, 7 by nucleophilic attack by the 5-position of the uracil onto either glyoxal carbonyl. Thus, we set out to evaluate a number of reaction conditions to determine if regiochemical preferences for the uracil and glyoxal reaction partners could be established. Initially, we treated 3-methyl-6-(1-methylhydrazinyl)uracil8 with phenylglyoxal monohydrate in refluxing ethanol or water and obtained the result shown in Scheme 1. Upon cooling a precipitate corresponding to ~85% of 3 was confirmed by 1 H NMR and mass spectrometry analysis, and a negative Tollens test for aldehyde. In addition to supporting spectral data, selective hydrazone formation onto the aldehyde moiety under these conditions is well precedented from prior studies on glyoxal hydrazones of methylhydrazine. 9,10 The ~15% cocrystallized impurity plus 5-10% additional material in the mother liquor corresponded to one of two...

A new synthesis of N 1 -(substituted)-pyrimido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-diones, which are analogues of the natural product toxoflavin, is reported. Condensation of preformed alkyl or aryl hydrazones with 6-chloro-3-methyl-5-nitrouracil efficiently provides pyrimidotriazinediones in a three-step process that broadens the scope of R 1 substituents. KeywordsPyrimido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-diones; Toxoflavin; Hydrazones; Cyclizations; Zinc Toxoflavin (1; Fig. 1), which was originally isolated from Pseudomonas cocovenenans, was first synthesized in 1961.1 Since then, a number of analogues have been prepared in order to explore the potent antibiotic and pharmacological properties of the pyrimido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-dione class represented by this natural product.2 Many of these analogues have the same methyl substituent found at the N 1 position of toxoflavin, partially due to the ease with which a methyl group can be incorporated via the use of methylhydrazine early in the synthesis.3 Other analogues have either a proton at N 1 or a variety of alkyl and benzyl groups. Conspicuously absent, however, are pyrimido [5,4-e]-1,2,4-triazine-5,7(1H,6H)-diones with an aryl substituent at the N 1 position. This can be attributed to the classical route that has been utilized, which requires that nucleophilic attack of 6-chloro-3-methyluracil proceed via the secondary nitrogen of the mono-substituted hydrazine. However, for mono-aryl hydrazines, such attack typically proceeds via the primary nitrogen. Herein, we report a novel and efficient route to pyrimido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-diones that allows for incorporation of aryl and alkyl substituents at N 1 of this ring system, thus broadening the scope of accessible analogues.Our strategy is shown in Scheme 1 and is exemplified with phenylhydrazine. First, phenylhydrazine (2) was treated with p-anisaldehyde (3a) under reflux conditions to form hydrazone 4a, which readily precipitated out of ethanol upon cooling. The resulting hydrazone (4a) was then treated with 6-chloro-3-methyl-5-nitrouracil (5), derived from nitration of commercially-available 6-chloro-3-methyluracil by the published method.4 The resultant 6-(2-(4-methoxybenzylidene)-1-phenyl)hydrazinyl-3-methyl-5-nitrouracil (6a) was cyclized to 3-(4-methoxyphenyl)-6-methyl-1-phenylpyrimido[5,4-e][1,2,4]triazine-5,7(1H, 6H)-dione (7a) upon treatment with zinc (4 equiv) and ammonium chloride (2 equiv) while exposed to air and with vigorous stirring in refluxing 50% aq ethanol. The N 1 -phenylpyrimidotriazinedione was isolated cleanly by treating the suspension with 1 N aq HCl to dissolve residual zinc and then washing the precipitate with 1 N aq HCl while filtering.5 The zinc-mediated cyclization was also tried in pure water or ethanol as the solvent, but the yields were considerably diminished. While hydrogenation over Pd/C has been shown to yield pyrimidotriazinediones previously from intermediates analogous to 6a, the reported yields are deplorable, usually only 2-5%.6 In star...