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Hematopoietic prostaglandin D synthase (HPGDS) is primarly expressed in mast cells, antigen-presenting cells, and Th-2 cells. HPGDS converts PGH2 into PGD2, a mediator thought to play a pivotal role in airway allergy and inflammatory processes. In this letter, we report the discovery of an orally potent and selective inhibitor of HPGDS that reduces the antigen-induced response in allergic sheep.

Herein we describe the design and synthesis of a novel series of potent thienopyrimidine P2Y12 inhibitors and the negative impact protein binding has on the inhibition of platelet aggregation.

[reaction: see text] A general demonstration of orthogonal selectivity of the Liebeskind-Srogl cross-coupling protocol compared to the Suzuki-Miyaura and Stille variants is reported.

Imidazole glycerol phosphate dehydratase (IGPD) has become an attractive target for herbicide discovery since it is present in plants and not in mammals. Currently no knowledge is available on the 3-D structure of the IGPD active site. Therefore, we used a pharmacophore model based on known inhibitors and 3-D database searches to identify new active compounds. In vitro testing of compounds from the database searches led to the identification of a class of pyrrole aldehydes as novel inhibitors of IGPD.

We describe the synthesis and enzymatic activity of a library of beta-carboxamido phosphonates as inhibitors of imidazole glycerol phosphate dehydratase (IGPD). Biological results suggest the presence of an enzymatic interaction site not previously observed for other inhibitors of IGPD.

Herein we report the identification of two new fatty acid amide hydrolase (FAAH) inhibitor lead series with FAAH k(inact)/K(i) potency values greater than 1500M(-1)s(-1). The two novel spirocyclic cores, 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane, clearly distinguished themselves from the other spirocyclic cores on the basis of their superior potency for FAAH. Lead compounds from these two series have suitable FAAH potency and selectivity for additional medicinal chemistry optimization.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.

Reduction of 5-iminodaunomycin with dithionite in anaerobic methanol followed by lowering the pH to 3 and saturating with air led to deamination without glycosidic cleavage to yield 89% 5-deoxydaunomycin. An intermediate observed during the reaction is proposed to be the hydroquinone tautomer, 8-acetyl-12-amino-10-[(3-amino-2,3,6- trideoxy-α-lyxo-hexopyranosyl)oxy]-1-methoxy-7,9, 10, 12-tetrahydro-6,8,11-trihydroxy-5(8H)-naphthacenone hydrochloride (1), which loses ammonia with a half-life of 49 min. Anaerobic reduction of 5-deoxydaunomycin with bi(3,5,5-trimethyl-2-oxomorpholin-3-yl) (TM-3 dimer) in methanol buffered to an apparent pH of 8 yielded 26% recovered 5-deoxydaunomycin, 56% 5,7-dideoxydaunomycinone, and 18%2-acetyl-11-hydroxy-7-methoxy-5,12-naphthacenedione (5) after 42 h and subsequent exposure to molecular oxygen

Saponification of the heptaacetate of daunomycin hydroquinone (6) with 7 equiv of lithium hydroxide to N-acetyldaunomycin hydroquinone (7) in argon-degassed tetrahydrofuran (THF)/water medium was reported earlier not to give rise to glycosidic cleavage to yield 7-deoxydaunomycinone (5) (J. Am. Chem. Soc. 1991, 113, 1373). The only product characterized after exposure to air was N-acetyldaunomycin (8). The result was interpreted as possible evidence against glycosidic cleavage of daunomycin at the hydroquinone redox state. We now report that performing the saponification in freeze-thaw-degassed THF/water in vacuo yielded 10% (5), 17% 8, 28% N-acetyl-5-deoxydaunomycin (12), 7% 7-epidaunomycinone (13), 2% daunomycinone (14), 9% 2-acetyl-11-hydroxy-7-methoxy-5,12-naphthacenedione (15), and 14% distributed between three unidentified products

We report the use of thermodynamic measurements in a self-complementary DNA duplex (5'-dXCGCGCG)(2), where X is an unpaired natural or nonnatural deoxynucleoside, to study the forces that stabilize aqueous aromatic stacking in the context of DNA. Thermal denaturation experiments show that the core duplex (lacking X) is formed with a free energy (37 °C) of -8.1 kcal·mol(-1) in a pH 7.0 buffer containing 1 M Na(+). We studied the effects of adding single dangling nucleosides (X) where the aromatic "base" is adenine, guanine, thymine, cytosine, pyrrole, benzene, 4-methylindole, 5-nitroindole, trimethylbenzene, difluorotoluene, naphthalene, phenanthrene, and pyrene. Adding these dangling residues is found to stabilize the duplex by an additional -0.8 to -3.4 kcal·mol(-1). At 5 μM DNA concentration, T(m) values range from 41.7 °C (core sequence) to 64.1 °C (with dangling pyrene residues). For the four natural bases, the order of stacking ability is AG ≥ T = C. The nonpolar analogues stack more strongly in general than the more polar natural bases. The stacking geometry was confirmed in two cases (X = adenine and pyrene) by 2-D NOESY experiments. Also studied is the effect of ethanol cosolvent on the stacking of natural bases and pyrene. Stacking abilities were compared to calculated values for hydrophobicity, dipole moment, polarizability, and surface area. In general, hydrophobic effects are found to be larger than other effects stabilizing stacking (electrostatic effects, dispersion forces); however, the natural DNA bases are found to be less dependent on hydrophobic effects than are the more nonpolar compounds. The results also point out strategies for the design nucleoside analogues that stack considerably more strongly than the natural bases; such compounds may be useful in stabilizing designed DNA structures and complexes.

Fatty acid amide hydrolase (FAAH) has attracted significant attention due to its promise as an analgesic target. This has resulted in the discovery of numerous chemical classes as inhibitors of this potential therapeutic target. In this paper we disclose a new series of novel FAAH irreversible azetidine urea inhibitors. In general these compounds illustrate potent activity against the rat FAAH enzyme. Our SAR studies allowed us to optimize this series resulting in the identification of compounds 13 which were potent inhibitors of both human and rat enzyme. This series of compounds illustrated good hydrolase selectivity along with good PK properties.

We describe the structure-based design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors contain substituents meta to the P1 amidine designed to explore additional interactions with the VIIa residues in the so-called ‘S1 side pocket’. A crystal structure of the designed inhibitors demonstrates the ability of the P1 side pocket moiety to engage Lys192 and main chain of Gly216 via hydrogen bond interactions, thus, providing additional possibility for chemical modification to improve selectivity and/or physical properties of inhibitors.

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.

We report the properties of hydrophobic isosteres of pyrimidines and purines in synthetic DNA duplexes. Phenyl nucleosides 1 and 2 are nonpolar isosteres of the natural thymidine nucleoside, and indole nucleoside 3 is an analog of the complementary purine 2-aminodeoxyadenosine. The nucleosides were incorporated into synthetic oligodeoxynucleotides and were paired against each other and against the natural bases. Thermal denaturation experiments were used to measure the stabilities of the duplexes at neutral pH. It is found that the hydrophobic base analogs are nonselective in pairing with the four natural bases but selective for pairing with each other rather than with the natural bases. For example, compound 2 selectively pairs with itself rather than with A, T, G, or C; the magnitude of this selectivity is found to be 6.5-9.3 °C in Tm or 1.5-1.8 kcal/mol in free energy (25 °C). All possible hydrophobic pairing combinations of 1, 2, and 3 were examined. Results show that the pairing affinity depends on the nature of the pairs and on position in the duplex. The highest affinity pairs are found to be the 1-1 and 2-2 self-pairs and the 1-2 heteropair. The best stabilization occurs when the pairs are placed at the ends of duplexes rather than internally; the internal pairs may be destabilized by imperfect steric mimicry which leads to non-ideal duplex structure. In some cases the hydrophobic pairs are significantly stabilizing to the DNA duplex; for example, when situated at the end of a duplex, the 1-1 pair is more stabilizing than a T-A pair. When situated internally, the affinity of the 1-1 pair is the same as, or slightly better than, the analogous T-T mismatch pair, which is known to have two hydrogen bonds. The studies raise the possibility that hydrogen bonds may not always be required for the formation of stable duplex DNA-like structure. In addition, the results point out the importance of solvation and desolvation in natural base pairing, and lend new support to the idea that hydrogen bonds in DNA may be more important for specificity of pairing than for affinity. Finally, the study raises the possibility of using these or related base pairs to expand the genetic code beyond the natural A-T and G-C pairs.

Noncovalent interactions between aromatic molecules are widely believed to be important contributing factors in the stabilization of organized structure in biological macromolecules.1,2 Among the most significant aromatic–aromatic interactions are those found in helical nucleic acid structures. Since the identity of the nearest neighbors to a given base pair is the best single predictor of thermodynamics in DNA duplexes,3 it is clear that aromatic π–π interactions are crucial to the stabilization of these structures.4 While there have been a considerable number of theoretical studies aimed at modeling the π–π interaction in DNA,5 there have been remarkably few experimental studies specifically addressing the thermodynamics of stacking (separate from base pairing) in DNA itself.6 For that reason we have undertaken a study of aromatic stacking in the context of duplex DNA, and we hope to begin to elucidate what are the important forces which stabilize this organized structure. We report here the first experimental comparison of the stacking abilities of natural DNA bases and of nonnatural aromatic analogs in double-stranded DNA. To separate stacking from pairing (hydrogen-bonding) interactions in duplex DNA we placed the natural or nonnatural nucleotide of interest in a “dangling” position (without a pairing partner) at the end of a base-paired duplex (Figure 1).7 The resulting stabilization of the duplex by the dangling base can be measured by thermal denaturation experiments, with comparison to the duplex lacking the added nucleotide. Figure 1 Structures and DNA sequences in this study. (A) Structures of natural (1, 3) and nonnatural (2, 4–8) nucleosides examined here. (B) Illustration of how placement of a single nucleotide residue at the 5′-end of a self-complementary strand ... Electrostatic effects resulting from such localized charge have been implicated both in the stabilization and in the geometry of aromatic stacking.5 To examine such effects we compared not only natural DNA bases but also nonpolar molecules with similar shape and surface area. Thus, we compared the DNA base thymine (1) and adenine (3) with their respective nonpolar isosteres difluorotoluene (2) and 4-methylindole (4).9 We also compared the stacking of the aromatic hydrocarbons benzene (5), naphthalene (6), phenanthrene (7), and pyrene (8). The synthesis of these nucleoside analogs has been reported.10–13 Results of the thermodynamic measurements made at pH 7.0 and 1 M NaCl are presented in Table 1. We measured melting transitions (Tm) as a function of concentration for the duplexes and calculated thermodynamic parameters by plotting 1/Tm vs ln([oligonucleotide]). The linear fits were quite good (r2 ≥ 0.97), with error in free energies of approximately ±2%. The unsubstituted core duplex under these conditions has a Tm (5 μM) of 41.0(±0.5) °C and a free energy (37 °C) of −8.05(±0.16) kcal/mol. Table 1 Comparison of Stacking Affinities of Natural DNA Bases and Simple Aromatic Analogs, As Measured by Dangling End Studies in a Self-Complementary DNA Duplex (Sequence d(XCGCGCG)2)a Measurement of the duplexes with dangling thymine and adenine residues shows, perhaps not surprisingly, that the purine stacks on the duplex more strongly than the smaller pyrimidine base. The two unpaired deoxyadenosines add 2.0 kcal of stabilizing interaction to the self-complementary sequence, and thymines add 1.1 kcal to the duplex stability. This relative stacking ability is as predicted from nearest-neighbor parameters3 and is consistent with dangling-end studies carried out in RNA.7 Interestingly, the data show that the nonpolar DNA base mimics stack considerably more strongly than their natural counterparts. Difluorotoluene raises the Tm of the duplex by 13.4 °C, about twice the effect of thymine, although the two have the same size, shape, and surface area.9 Similarly, the isosteric analog 4-methylindole increases the Tm by more than does the natural base adenine. The effects on free energies of the duplexes mirror these results (Table 1). These results show clearly that surface area alone is not a good predictor of stacking affinity. It seems likely that the increased hydrophobicity of these two nonpolar analogs contributes favorably to their enhanced stacking. An alternative explanation might be differences in polarizability, which would influence van der Waals forces. However, methylindole is calculated to be only slightly more polarizable than adenine, and difluorotoluene is equally as polarizable as thymine.14 Thus it appears that hydrophobicity differences exert the largest influence here. The superior stacking of the two purine-sized compounds relative to the pyrimidine-sized cases might be explained by increased surface area, which would allow for stronger interaction with the neighboring C–G base pair. To investigate further the effects of size and hydrophobicity on stacking interactions, we then examined the series of four nucleotides in which the “base” moiety is benzene, naphthalene, phenanthrene, and pyrene (Figure 1A). The results show (Table 1) that these compounds stack relatively strongly, and the interaction generally becomes more favorable with increasing size. The benzene nucleotide stacks with slightly greater affinity than thymine. The naphthalene compound shows a large Tm increase over the unsubstituted duplex of 15.2 °C and a total stacking energy of 2.9 kcal/mol, or twice that of benzene. Interestingly, the phenanthrene nucleoside, although larger, has a stacking propensity essentially the same as that of naphthalene (compare seventh and eighth entries in Table 1). Models indicate that the third ring in phenanthrene (which is absent in naphthalene) does not contact the adjacent bases and instead is likely to protrude toward the major groove of the DNA. We surmise that lack of added contact with the nearest neighbor precludes added favorable interactions. Since phenanthrene is significantly more polarizable than naphthalene14 but does not stack more strongly, the results are most easily explained by solvophobic effects rather than dipole-induced dipole effects. The amount of nonpolar surface area excluded from solvent by stacking is expected to be similar for naphthalene and phenanthrene in these structures. The pyrene compound interacts the most strongly of all the natural and nonnatural bases studied. The duplex substituted by this species has a melting transition 23.1 °C higher than the unsubstituted duplex and 12.5 °C higher than for the case with adenine. Addition of the two pyrene residues to the self-complementary duplex adds a quite large 3.4 kcal / mol of stabilization to the helix. Models indicate that a large fraction of one face of this molecule can stack with the neighboring base pair, and so the relatively high solvophobicity is apparently put to good effect. The overall order of stacking ability of these molecules is thus found to be thymine < benzene < adenine < difluorotoluene ≤ naphthalene ∼ phenanthrene ≤ methylindole < pyrene, at least in this sequence context. It seems significant that solvophobic effects appear to be the most important factor in determining this order. When molecules of the same size are compared, we find that the less polar structure stacks more strongly than one of higher polarity. When nonpolar molecules of different sizes are compared, we find that stacking propensity appears to correlate with the surface area excluded from solvent. A number of studies aimed at measuring aromatic–aromatic interactions in smaller model systems have been reported previously.15,16 A few of these have examined interactions of DNA bases;16 and mono- and dinucleotides have been studied as models for stacking interactions.17 The present results agree with the previously known superiority of purines over pyrimidines at stacking, which was also observed by Leonard et al. using nucleobases bridged by simple trimethylene linkers.16a In a comparison of adenines and naphthalenes linked by three-carbon bridges, Gellman found evidence that naphthalene–adenine interactions were qualitatively stronger than adenine–adenine interactions,16c a result in accord with our finding of superior stacking of naphthalene over adenine. In a simple receptor system, Rebek et al. found increasing interaction of adenine with aromatics of increasing size,16b also in agreement with the present results. Our results indicate that the natural DNA bases are not particularly effective at stacking, at least in a global comparison of aromatic structures. Evolution may not have favored bases which stack too strongly, since helix unwinding is a requirement for DNA replication. For designed nucleic acid structures, where thermodynamic stability is often desirable, our results indicate that one may gain stability simply by placing large nonpolar aromatics at the ends of helical structure. It will of course be useful to study this series of compounds in additional sequence contexts to explore the generality of the conclusions. It is worth noting that in a considerably different structure (a hairpin loop) we have noted similar stabilization by nonpolar DNA base analogs.11 Since thermodynamic rather than structural evidence of stacking is presented here, it will be of significant future interest to obtain structural information18 about dangling nucleotides to better evaluate the specific interactions involved.