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Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enable the design of efficient allosteric drugs targeting the polymerase of hepatitis C virus (NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on its interaction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activity of VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5B inhibitors.

A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H(c))], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(H(c)) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(H(c)) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel™), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5-100mL columns and the process was performed at the pilot scale using 0.6-1.6L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H(c))/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(H(c))/kg WCW at the pilot scale. The purified rBoNTC(H(c)) was stable for at least 3 months at 5 and -80°C as determined by reverse phase-HPLC and SDS-PAGE and was stable for 24 months at -80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H(c)) was intact.

The one-pot, microwave-assisted synthesis of 5-thia-4b,10-diaza-indeno[2,1-a]indene-5,5-dioxide (6) and 10H-11-thia-5,10a-diaza-benzo[b]fluorene-11,11-dioxide (7) from 1,2-phenylenediamine and 2-aminobenzylamine, respectively, is described. Generation of a sulfonamide through reaction of the appropriate diamine with 2-cyanobenzenesulfonyl chloride is followed by a multistep intramolecular transformation and subsequent elimination of ammonia to provide the corresponding tetracycle in good yield.

The adsorption of recombinant botulinum neurotoxin (BoNT) protein-derived vaccine antigens to aluminum salt adjuvants has been previously studied for the development of a trivalent vaccine against the neurotoxins (Vessely et al., in press, J Pharm Sci). The current paper describes an investigation of the stability of recombinant BoNT antigens adsorbed to aluminum salt adjuvants during storage in aqueous solution. Both chemical and physical changes occurred during storage. Phosphate groups in the buffer exchanged with hydroxyl groups on the adjuvant surface. The resulting changes in solution pH and adjuvant surface chemistry promoted more favorable electrostatic interaction between the BoNT proteins and the surface, possibly increasing the affinity of the proteins for the surface during storage. Fluorescence and UV spectroscopy suggested changes to protein structure during storage, whereas differential scanning calorimetry showed changes to thermal processes related to protein conformation and/or surface adsorption. The consequence of the chemical and physical changes to the proteins was a decrease in the ability to desorb protein from the adjuvant surface during storage. Overall, the results of this study emphasize the utility of a thorough characterization of the interactions between protein antigens and aluminum salt adjuvants.

We identified a series of structurally novel SCD (Delta9 desaturase) inhibitors via high-throughput screening and follow-up SAR studies. Modification of the central bicyclic scaffold has proven key to our potency optimization effort. The most potent analog (8g) had IC(50) value of 50 pM in a HEPG2 SCD assay and has been shown to be metabolically stable and selective against Delta5 and Delta6 desaturases.

A general solid-phase synthesis of 1,2,5-trisubstituted imidazolidin-4-ones is described. The key synthetic transformation incorporates a microwave-assisted condensation of an α-amino amide on solid support with an aldehyde in solution to give the corresponding resin-bound imidazolidin-4-one in a simple one-pot procedure.

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.

The identification and evaluation of aryl-[1,4]diazepane ureas as functional antagonists of the chemokine receptor CXCR3 are described. Specific examples exhibit IC(50) values of approximately 60 nM in a calcium mobilization functional assay, and dose-dependently inhibit CXCR3 functional response to CXCL11 (interferon-inducible T-cell alpha chemoattractant/I-TAC) as measured by T-cell chemotaxis, with a potency of approximately 100 nM.

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.

A chemoenzymatic approach for the synthesis of homoazasugars is described, utilizing an aldolase to catalyze the key asymmetric aldol addition reaction. This approach is illustrated by the synthesis of β-D-homomannonojirimycin (11) using D-fructose diphosphate (FDP) aldolase.

Two methods are presented for kinetic resolutions of compounds containing ester and sulfoxide functionalities (sulfinylalkanoates). In the first a crude lipase preparation from Pseudomonas sp. (K10) mediates enantioselective hydrolysis of these esters in an aqueous environment. The second method uses the same lipase preparation to promote enantioselective transestesterifications with alcohols in hexane

The one-pot, microwave-assisted synthesis of 5-thia-4b,10-diaza-indeno[2,1-a]indene-5,5-dioxide (6) and 10H-11-thia-5,10a-diaza-benzo[b]fluorene-11,11-dioxide (7) from 1,2-phenylenediamine and 2-aminobenzylamine, respectively, is described. Generation of a sulfonamide through reaction of the appropriate diamine with 2-cyanobenzenesulfonyl chloride is followed by a multistep intramolecular transformation and subsequent elimination of ammonia to provide the corresponding tetracycle in good yield.

The following three stereoisomers of castanospermine have been prepared: (1R,6R,7R,8S,8aR)-1,6,7,8-tetrahydroxyindolizidine (1), (1R,6S,7S,8S,8aR)-1,6,7,8-tetrahydroxyindolizidine (2), and (1R,6R,7S,8S,8aR)-1,6,7,8-tetrahydroxyindolizidine (3). Each of these compound was synthesized via asymmetric allylation of pentose derivatives with [(Z)-γ-(methoxyinethoxy)allyl]diisopinocampheylborane, an approach which is, in principle, suitable for preparations of a total of eight stereoisomers of castanospermine

Two methods are presented for kinetic resolutions of compounds containing ester and sulfoxide functionalities (sulfinylalkanoates). In the first a crude lipase preparation from Pseudomonas sp. (K10) mediates enantioselective hydrolysis of these esters in an aqueous environment. The second method uses the same lipase preparation to promote enantioselective transestesterifications with alcohols in hexane

We discovered a structurally novel SCD (Delta9 desaturase) inhibitor 4a (CVT-11,563) that has 119 nM potency in a human cell-based (HEPG2) SCD assay and selectivity against Delta5 and Delta6 desaturases. This compound has 90% oral bioavailability (rat) and excellent plasma exposure (dAUC 935 ng h/mL). Additionally, 4a shows moderately selective liver distribution (three times vs plasma and adipose tissue) and relatively low brain penetration. In a five-day study (high sucrose diet, rat) compound 4a significantly reduced SCD activity as determined by GC analysis of fatty acid composition in plasma and liver. We describe the discovery of 4a from HTS hit 1 followed by scaffold replacement and SAR studies focused on DMPK properties.

4-Chlorophenylsulfinylacetates of defined stereochemistry at sulfur were prepared from monochiral alcohols: compound 2 from (+)-menthol; 3 and 4 from (−)-8-phenylmenthol; 5 and 6 from (+)-trans-2-phenylcyclohexanol; and, 7 and 9 from (-)-10-dicyclohexylsulfamoyl-D-isoborneol. Reagents for which the sulfoxide asymmetry is matched with the inducing effect of the auxiliary give good diastereoselection in SPAC reactions affording γ-hydroxy-α,β-unsaturatod alcohols of high optical purity. Asymmetry at the sulfoxide has a greater effect than the auxiliaries on the stereochemical outcome of these reactions.

Factors that govern chemical and optical yields of methyl γ-hydroxy-α,β-unsaturated esters 1 formed in reactions of optically active sulfinylacetates 2 with aldehydes (the «SPAC» reaction) are defined. Racemic samples of these chirons (1) can be resolved via acylations mediated by crude preparations of the lipase Pseudomonas K-10 in organic solvents. Combinations of asymmetric SPAC reactions with these biocatalytic resolutions provide routes to highly optically active esters 1 in good yields. This methodology is applied in a completely asymmetric synthesis of (4S-cis)-5-(cyclohexylmethyl)-4-hydroxy-2-pyrrolidinone (15), a cyclic derivative of (3S,4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA)

A series of trisubstituted purinones was synthesized and evaluated as A(2A) receptor antagonists. The A(2A) structure-activity relationships at the three substituted positions were studied and selectivity against the A(1) receptor was investigated. One antagonist 12o exhibits a K(i) of 9nM in an A(2A) binding assay, a K(b) of 18nM in an A(2A) cAMP functional assay, and is 220-fold selective over the A(1) receptor.

The discovery and synthesis of a series of 2-amino-5-benzoyl-4-(2-furyl)thiazoles as adenosine A2A receptor antagonists from a small-molecule combinatorial library using a high-throughput radioligand-binding assay is described. Antagonists were further characterized in the A2A binding assay and an A1 selectivity assay. Selected examples exhibited excellent affinity for A2A and good selectivity versus the A1 receptor.

The synthesis and identification of sulfonamido-aryl ethers as potent bradykinin B1 receptor antagonists from a approximately 60,000 member encoded combinatorial library are reported. Two distinct series of compounds exhibiting different structure-activity relationships were identified in a bradykinin B1 whole-cell receptor-binding assay. Specific examples exhibit K(i) values of approximately 10nM.

(1S, 2R, 7R, 7aR)- 1,2,7-Trihydroxypyrrolizidine ( 1 ) is prepared by elaboration of an erythrose derivative ( 4 ) via an asymmetric allylation reaction; this approach promises to be widely applicable to syntheses of polyhydroxylated indolizidine and pyrrolizidine alkaloids.

Botulinum neurotoxin (BoNT) is a biological warfare threat. Protein antigens have been developed against the seven major BoNT serotypes for the development of a recombinant protein vaccine. This study is an evaluation of adsorption profiles for three of the recombinant protein antigens to aluminum salt adjuvants in the development of a trivalent vaccine against BoNT. Adsorption profiles were obtained over a range of protein concentrations. The results document that charge-charge interactions dominate the adsorption of antigen to adjuvant. Optimal conditions for adsorption were determined. However, potency studies and solution stability studies indicated the necessity of using aluminum hydroxide adjuvant at low pH. To improve the adsorption profiles to AlOH adjuvant, phosphate ions were introduced into the adsorption buffers. The resulting change in the adjuvant chemistry led to an improvement of adsorption of the BoNT antigens to aluminum hydroxide adjuvant while maintaining potency. Competitive adsorption profiles were also determined, and showed changes in maximum adsorption from mixed solutions compared to adsorption from individual protein solutions. The adsorption profiles for each protein varied due to differences in adsorption mechanism and affinity for the adjuvant surface. These results emphasize the importance of evaluating competitive adsorption in the development of multivalent vaccine products.

The discovery and evaluation of 5-(4-phenylbenzyl)oxazole-4-carboxamides as prostacyclin (IP) receptor antagonists is described. Analogs disclosed showed high affinity for the IP receptor in human platelet membranes with IC50 values of 0.05–0.50 μM, demonstrated functional antagonism by inhibiting cAMP production in HEL cells with IC50 values of 0.016–0.070 μM, and exhibited significant selectivity versus other prostanoid receptors.

The p38 mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play important roles in cellular responses to inflammation and external stress. Inhibitors of the p38 MAP kinase have shown promise for potential treatment of inflammatory disorders such as rheumatoid arthritis, acute coronary syndrome, psoriasis, and Crohn's disease. We identified a novel class of p38 inhibitors via high-throughput screening. PS200981 [3-(4-(1,4-diazepan-1-yl)-6-(((1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)methylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide], a representative compound identified from screening a collection of combinatorial libraries, amounting to 2.1 million compounds, inhibits p38alpha kinase and the lipopolysaccharide (LPS)-induced increase in tumor necrosis factor (TNF) alpha levels in cell media of human monocytes with IC50 values of 1 microM. The screening data revealed a preferred synthon, 3-amino-4-methyl benzamide, which is critical for the activity against p38. This synthon appeared almost exclusively in screening hits including PS200981, and slight variations of this synthon including 3-amino benzamide and 2-amino-4-methyl benzamide also contained in the library were inactive. PS200981 is equally potent against the alpha and beta forms of p38 but did not inhibit p38 gamma and is25-fold selective versus a panel of other kinases. PS200981 inhibited the LPS-induced increase in TNFalpha levels when administered at 30 mg/kg to mice. Selectivity and in vivo activity of this class of p38 inhibitors was further demonstrated by PS166276 [(R)-3-(4-(isobutyl(methyl)-amino)-6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide], a highly structurally related but more potent and less cytotoxic inhibitor, in several intracellular signaling assays, and in LPS-challenged mice. Overall, this novel class of p38 inhibitors is potent, active in vitro and in vivo, and is highly selective.

A novel class of 5-cyanopyrimidine-based inhibitors of p38alpha MAP kinase has been investigated. Analogues optimized through SAR iterations display low nanomolar enzymatic and cellular activity. The in vivo efficacy of this class of p38 inhibitors was demonstrated by 3a and 3b (>50% reduction in TNF levels when orally dosed at 5 mg/kg, 5 h prior to LPS administration in an acute murine model of inflammation). For 3a and 3b, the previously identified N-methoxybenzamide moiety (1) was replaced with N-(isoxazol-3-yl)benzamide, thereby providing increased metabolic stability. Cyanopyrimidine 3a demonstrated 100% oral bioavailability in mouse. High p38 kinase selectivity versus over 20 kinases was observed for analogue 3b. Direct hydrogen bonding of the cyano nitrogen of the 5-cyanopyrimidine core to the backbone NH of Met109 was confirmed by X-ray crystallographic analysis of 3a bound to p38alpha.

A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was puriWed from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7– 380 mL columns and the process was performed at the pilot scale using 0.5–3.1 L columns in preparation for technology transfer to cGMP manufacturing. The puriWcation process resulted in greater than 98% pure rBoNTE(Hc) based on HPLC and yielded up to 1.01 g of rBoNTE(Hc)/kg cells at the bench scale and 580 mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the puriWed rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50’s of BoNT/E.

A new structural class of triaminotriazine aniline amides possessing potent p38 enzyme activity has been discovered. The initial hit (compound 1a) was identified through screening the Pharmacopeia ECLiPS compound collection. SAR modification led to the identification of a short acting triaminotriazine aniline methoxyamide (compound 1m) possessing in vitro and in vivo oral activity in animal models of acute and chronic inflammatory disease. An X-ray crystal structure of compound 1m in this class, cocrystallized with unactivated p38 alpha protein, indicates that these compounds bind to the ATP binding pocket and possess key H-bonding interactions within a deeper cleft. Hydrogen bonding between one of the triazine nitrogens and the backbone NH of the Met109 residue occurs through a water molecule. The methoxyamide NH and carbonyl oxygen are within H-bonding distance of Glu71 and Asp168.

Laser milling of a variety of substrates is investigated with the intention of achieving high quality material removal to create three-dimensional shapes in the material. A high power Q-switched Diode Pumped Solid State Nd:YAG Laser at 1064nm is used in all cases. Materials investigated include Nickel Superalloys, Thermal Barrier Coatings, Steels, Tungsten Carbide and Polycrystalline Diamond. Multi-layer substrates are also considered. The effects of laser intensity, plasma formation, pulse duration, material properties, and resulting removal rate, recast and surface finish are explored for this process. This paper defines the findings of this study within the context of commercial imperatives.

Heavy chain fragments of botulinum neurotoxin serotypes A and B are being developed as a bivalent vaccine for botulism. To potentiate the immune response, an aluminum containing adjuvant will be formulated with the two antigens. The adsorption mechanisms of each antigen to aluminum phosphate and aluminum hydroxide adjuvants were studied. The adsorption of the serotype A antigen to each adjuvant, and the serotype B antigen to aluminum phosphate adjuvant, is dependent on electrostatic attractive forces. The serotype A antigen is basic, and pretreatment with phosphate anions is required for favorable adsorption conditions to aluminum hydroxide adjuvant. In contrast, the serotype B antigen displays a high affinity to aluminum hydroxide adjuvant even when the two species possess the same charge. It is proposed that the serotype B antigen is adsorbed to aluminum hydroxide adjuvant by a ligand exchange mechanism.

A general and efficient solid-phase synthesis of N-9-substituted 2,8-diaminopurines from 5-nitrouracil is described. The key synthetic transformation employs a carbodiimide-mediated cyclization of a thiourea. Thiourea formation on solid phase is performed using both thermal and microwave reaction conditions. Regiospecific solution-phase synthesis of key building blocks allows the incorporation of desired substituents at N-9 of the purine nucleus.

Double diastereoselection in the S ulfoxide P iperidine A nd C arbonyl ( SPAC ) reactions of sulfinyl acetate esters (2) with α-unsubstituted aldehydes (3) has been investigated to facilitate asymmetric syntheses of γ-hydroxy-α,β-unsaturated esters (1) .