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HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, 1), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug-drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of 1 but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of 1 when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with 1, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs.

Analysis and purification of boronic acid pinacol esters by RPLC is very challenging due to their degradation in aqueous and alcoholic solvents. These compounds are difficult to purify by SFC too as they are equally sensitive to traditional co-solvents like methanol, ethanol, and 2-propanol. 2,2,2 trifluoroethanol (TFE), which has been reported for the purification of a few alcohol sensitive compounds, was evaluated as a co-solvent in this study for the purification of chiral and achiral boronate esters by SFC. Examples of twelve compounds were presented in this paper where degradation of boronic acid pinacol esters was successfully controlled by replacing methanol with TFE as the co-solvent in SFC. A separate study showed that TFE can also control the epimerization of the enantiomers of 3 substituted 1,4 benzodiazepine analogues during the purification process. In addition to above benefits, 2,2,2trifloroethanol showed improved selectivity and resolution for most of the compounds. With its stronger solvent strength compared to other alcohols, TFE could also be used to reduce the co-solvent percentage needed for elution and to shorten retention time of highly polar samples which did not elute even in 50% of other co-solvents in SFC. A case study of compound B demonstrated that TFE provided a reduced co-solvent percentage and a shorter cycle time with much improved resolution as compared to methanol, thus resulting in higher loading and throughput with reduction of total solvent consumption., (Copyright © 2017 Elsevier B.V. All rights reserved.)

During a preparative separation of the cis enantiomeric pair of benzyl-2,3-dihydroxypiperidine-1-carboxylate using supercritical-fluid chromatography (SFC) with methanol modifier, significant degradation of the products in the collected fractions was observed when a Waters SFC-350 ® (Milford, MA, USA) was used, but same was not observed when a Waters SFC-80q ® (Milford, MA, USA) was used. Through a systematic investigation, we discovered that the compound degraded over time under an acidic condition created by the formation of methyl carbonic acid from methanol and CO 2. The extent of the product degradation was dependent on the time and the concentration of CO 2 remained in the product fraction, which was governed by the efficiency of CO 2 -methanol separation during the fraction collection. Hence, we demonstrated that the different designs of CO 2 -solvent separator (high pressurized cyclone in Waters SFC-350 ® and low-pressurized vortexing separator in Waters SFC-80q ®® ) had a significant impact on the degradation of an acid-sensitive compound. The acidity caused by CO 2 in methanol was supported by diminished degradation after a nitrogen purging or after neutralizing the collected fractions with a base. Three different solutions to overcome the degradation problem of the acid sensitive compounds using SFC-350 ® with the high pressurized separator were investigated and demonstrated. The degraded products were isolated as four enantiomers and their relative stereochemistry were established based on 2D NMR data along with the plausible mechanism of degradation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Metabolites of new chemical entities can influence safety and efficacy of a molecule and often times need to be quantified in preclinical studies. However, synthetic standards of metabolites are very rarely available in early discovery. Alternate approaches such as biosynthesis need to be explored to generate these metabolites. Assessing the quantity and purity of these small amounts of metabolites with a nondestructive analytical procedure becomes crucial. Quantitative NMR becomes the method of choice for these samples. Recent advances in high-field NMR (>500 MHz) with the use of cryoprobe technology have helped to improve sensitivity for analysis of small microgram quantity of such samples. However, this type of NMR instrumentation is not routinely available in all laboratories. To analyze microgram quantities of metabolites on a routine basis with lower-resolution 400 MHz NMR instrument fitted with a broad band fluorine observe room temperature probe, a novel hybrid capillary tube setup was developed. To quantitate the metabolite in the sample, an artificial signal insertion for calculation of concentration observed (aSICCO) method that introduces an internally calibrated mathematical signal was used after acquiring the NMR spectrum. The linearity of aSICCO signal was established using ibuprofen as a model analyte. The limit of quantification of this procedure was 0.8 mM with 10 K scans that could be improved further with the increase in the number of scans. This procedure was used to quantify three metabolites-phenytoin from fosphenytoin, dextrophan from dextromethorphan, and 4-OH-diclofenac from diclofenac-and is suitable for minibiosynthesis of metabolites from in vitro systems., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Purification of many pharmaceutical compounds by supercritical fluid chromatography (SFC) has always been challenging because of degradation of compound during the isolation step in the presence of acidic or basic modifiers in the mobile phase. Stability of such acid or base-sensitive compounds could be improved by post-column addition of a solvent containing base or acid modifier as counter ion through a make-up pump respectively to neutralize the compound fraction without affecting the resolution. One such case study has been presented in this work where the stability of a base-sensitive compound was addressed by the addition of acidic co-solvent through the make-up pump. Details of this setup and the investigation of degradation of the in-house base-sensitive compound are discussed in this paper. In addition, poor retentivity and low recovery of many non-polar compounds in SFC eluting under low co-solvent percentage is another major concern. Even though the desired separation could be achieved with low percentage of co-solvent, it's difficult to get the proper recovery after purification due to precipitation of the sample and significant aerosol formation inside the cyclone. We have demonstrated the first-time use of a post-column make-up pump on SFC 350 system to introduce additional solvent prior to cyclone to avoid the precipitation, reduce the aerosol formation and thus improve the recovery of non-polar compounds eluting under less than 10% of co-solvent., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To resolve the metabolite redox cycling associated with our earlier clinical compound 2, we carried out lead optimization of lead molecule 1. Compound 4 showed improved lipophilic ligand efficiency and demonstrated robust glucose lowering in diet-induced obese mice without a liability in predictive preclinical drug safety studies. Thus, it was selected as a clinical candidate and further studied in type 2 diabetic patients. Clinical data suggests no evidence of metabolite cycling, which is consistent with the preclinical profiling of metabolism.

Glucokinase activators (GKAs) are being developed and clinically tested for potential antidiabetic therapy. The potential benefits and limitations of this approach continue to be intensively debated. To contribute to the understanding of experimental pharmacology and therapeutics of GKAs, we have tested the efficacy of one of these agents (Piragliatin) in isolated islets from humans with type 2 diabetes mellitus (T2DM), from mice with glucokinase (GK) mutations induced by ethyl-nitroso-urea (ENU) as models of Maturity Onset Diabetes of the Young linked to GK and Permanent Neonatal Diabetes Mellitus linked to GK (PNDM-GK) and finally of islets rendered glucose insensitive by treatment with the sulphonyl urea compound glyburide in organ culture. We found that the GKA repaired the defect in all three instances as manifest in increased glucose-induced insulin release and elevated intracellular calcium responses. The results show the remarkable fact that acute pharmacological activation of GK reverses secretion defects of β-cells caused by molecular mechanism that differ vastly in nature, including the little understood multifactorial lesion of β-cells in T2DM of man, the complex GK mutations in mice resembling GK disease and acute sulphonylurea failure of mouse β-cells in tissue culture. The implications of these results are to be discussed on the theoretical basis underpinning the strategy of developing these drugs and in light of recent results of clinical trials with GKAs that failed for little understood reasons., (© 2012 Blackwell Publishing Ltd.)

Glucokinase (GK) activation as a potential strategy to treat type 2 diabetes (T2D) is well recognized. Compound 1, a glucokinase activator (GKA) lead that we have previously disclosed, caused reversible hepatic lipidosis in repeat-dose toxicology studies. We hypothesized that the hepatic lipidosis was due to the structure-based toxicity and later established that it was due to the formation of a thiourea metabolite, 2. Subsequent SAR studies of 1 led to the identification of a pyrazine-based lead analogue 3, lacking the thiazole moiety. In vivo metabolite identification studies, followed by the independent synthesis and profiling of the cyclopentyl keto- and hydroxyl- metabolites of 3, led to the selection of piragliatin, 4, as the clinical lead. Piragliatin was found to lower pre- and postprandial glucose levels, improve the insulin secretory profile, increase β-cell sensitivity to glucose, and decrease hepatic glucose output in patients with T2D.

It was reported previously that isolated human islets from individuals with type 2 diabetes mellitus (T2DM) show reduced glucose-stimulated insulin release. To assess the possibility that impaired bioenergetics may contribute to this defect, glucose-stimulated respiration (Vo(2)), glucose usage and oxidation, intracellular Ca(2+), and insulin secretion (IS) were measured in pancreatic islets isolated from three healthy and three type 2 diabetic organ donors. Isolated mouse and rat islets were studied for comparison. Islets were exposed to a "staircase" glucose stimulus, whereas IR and Vo(2) were measured. Vo(2) of human islets from normals and diabetics increased sigmoidally from equal baselines of 0.25 nmol/100 islets/min as a function of glucose concentration. Maximal Vo(2) of normal islets at 24 mM glucose was 0.40 ± 0.02 nmol·min(-1)·100 islets(-1), and the glucose S(0.5) was 4.39 ± 0.10 mM. The glucose stimulation of respiration of islets from diabetics was lower, V(max) of 0.32 ± 0.01 nmol·min(-1)·100 islets(-1), and the S(0.5) shifted to 5.43 ± 0.13 mM. Glucose-stimulated IS and the rise of intracellular Ca(2+) were also reduced in diabetic islets. A clinically effective glucokinase activator normalized the defective Vo(2), IR, and free calcium responses during glucose stimulation in islets from type 2 diabetics. The body of data shows that there is a clear relationship between the pancreatic islet energy (ATP) production rate and IS. This relationship was similar for normal human, mouse, and rat islets and the data for all species fitted a single sigmoidal curve. The shared threshold rate for IS was ∼13 pmol·min(-1)·islet(-1). Exendin-4, a GLP-1 analog, shifted the ATP production-IS curve to the left and greatly potentiated IS with an ATP production rate threshold of ∼10 pmol·min(-1)·islet(-1). Our data suggest that impaired β-cell bioenergetics resulting in greatly reduced ATP production is critical in the molecular pathogenesis of type 2 diabetes mellitus.

GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A(2B) antagonists, due to its excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to impart superior potency) was prepared and evaluated for their selectivity against the A(2A) and A(1) receptors. This work resulted in the identification of compound 30, with excellent potency and high selectivity against both A(2A) and A(1) receptors., (Copyright © 2011. Published by Elsevier Ltd.)

Importance of the Field: Small molecule glucokinase activators (GKAs) continue to represent a potential strategy to treat type 2 diabetes (T2D). Glucokinase (GK) primarily exerts its effect through modulatory actions in pancreatic β-cells and hepatocytes. It couples insulin secretion in the pancreas with plasma glucose concentration and improves glucose utilization in the liver, thus, affecting two key aspects of glucose homeostasis. There has been an intense interest in GKAs within the pharmaceutical industry ever since the first report of a low molecular mass activator in 2003. The key drivers for this interest are the robust glucose lowering activity observed with GKAs in preclinical T2D animal models and early reports of efficacy in T2D patients., Areas Covered in This Review: The objective is to review GKA structures disclosed during the 2008 - 2010 period and classify them based on key structural features. For this purpose, only compound data from patent disclosures were used., What the Reader Will Gain: The reader would gain a detailed view of structural diversity of the GKA field disclosed during the review period., Take Home Message: There continues to be a high level of interest within the pharmaceutical industry in novel GKAs. Several new and highly potent structure types were reported for the first time in the past 3 years. Common features of all of them include a hydrogen bond donor-acceptor pair that makes contact with the backbone CO- and NH- bonds of Arg 63 residue on GK and two hydrophobic groups. During this review period, several GKAs progressed to Phase II clinical testing and the data on their safety and efficacy profiles are eagerly awaited.

Glucokinase Glucokinase (GK GK ; EC 2.7.1.1.) phosphorylates and regulates glucose metabolism in insulin-producing pancreatic beta-cells, hepatocytes, and certain cells of the endocrine and nervous systems allowing it to play a central role in glucose homeostasis glucose homeostasis . Most importantly, it serves as glucose sensor glucose sensor in pancreatic beta-cells mediating glucose-stimulated insulin biosynthesis and release and it governs the capacity of the liver to convert glucose to glycogen. Activating and inactivating mutations of the glucokinase gene cause autosomal dominant hyperinsulinemic hypoglycemia and hypoinsulinemic hyperglycemia in humans, respectively, illustrating the preeminent role of glucokinase in the regulation of blood glucose and also identifying the enzyme as a potential target for developing antidiabetic drugs antidiabetic drugs . Small molecules called glucokinase activators (GKAs) glucokinase activators (GKAs) which bind to an allosteric activator allosteric activator site of the enzyme have indeed been discovered and hold great promise as new antidiabetic agents. GKAs increase the enzyme's affinity for glucose and also its maximal catalytic rate. Consequently, they stimulate insulin biosynthesis and secretion, enhance hepatic glucose uptake, and augment glucose metabolism and related processes in other glucokinase-expressing cells. Manifestations of these effects, most prominently a lowering of blood glucose, are observed in normal laboratory animals and man but also in animal models of diabetes and patients with type 2 diabetes mellitus (T2DM T2DM ) type 2 diabetes mellitus (T2DM) . These compelling concepts and results sustain a strong R&D effort by many pharmaceutical companies to generate GKAs with characteristics allowing for a novel drug treatment of T2DM.

The phenylacetamide 1 represents the archtypical glucokinase activator (GKA) in which only the R-isomer is active. In order to probe whether the chiral center could be replaced, we prepared a series of olefins 2 and show in the present work that these compounds represent a new class of GKAs. Surprisingly, the SAR of the new series paralleled that of the saturated derivatives with the exception that there was greater tolerance for larger alkyl and cycloalkyl groups at R(2) region in comparison to the phenylacetamides. In normal Wistar rats, the 2,3-disubstituted acrylamide analog 10 was well absorbed and demonstrated robust glucose lowering effects., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A(2B) receptor antagonists because of its slight selectivity against A(1) and A(2A) receptors and modest A(2B) potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A(2B) potency, modest selectivity versus A(2A) and A(1) receptors, and good rodent PK properties., (2010 Elsevier Ltd. All rights reserved.)

Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

Glucokinase (GK) plays a key role in glucose homeostasis. Developments over the past decade, such as the determination of the function of GK regulatory protein, the discovery of GK mutations related to maturity onset of diabetes of the young, permanent neonatal diabetes mellitus and persistent hyperinsulinemia hypoglycemia of infancy, and the discovery of novel GK activators (GKAs) and their X-ray co-crystal structures with GK, have significantly enhanced our understanding of GK structure and function. This review discusses key publications on GKAs that report full characterization, key compound disclosures from patents, and a current hypothesis on the mechanism of GK activation based on the co-crystal structures of GK-GKA complexes.

Glucokinase (GK) plays a key role in whole-body glucose homeostasis by catalyzing the phosphorylation of glucose in cells that express this enzyme, such as pancreatic beta cells and hepatocytes. We describe a class of antidiabetic agents that act as nonessential, mixed-type GK activators (GKAs) that increase the glucose affinity and maximum velocity (Vmax) of GK. GKAs augment both hepatic glucose metabolism and glucose-induced insulin secretion from isolated rodent pancreatic islets, consistent with the expression and function of GK in both cell types. In several rodent models of type 2 diabetes mellitus, GKAs lowered blood glucose levels, improved the results of glucose tolerance tests, and increased hepatic glucose uptake. These findings may lead to the development of new drug therapies for diabetes.

Imidazole and oxazole derivatives 1 to 4 were designed and prepared as dipeptide mimetics to replace the Ser-Leu dipeptide sequence of Ro-25-9980 (Ac-(Cha)-RAMA-S-L-NH2), a peptidic inhibitor of antigen binding to major histocompatibility complex (MHC) class II DR molecules linked to rheumatoid arthritis (RA). The most potent analog in binding assays (IC50 = 30 nM in DRB1*0401 binding; 1.6 times as potent as Ro 25-9980) was 16, Ac-(Cha)RAMA-(S)S-psi(oxazole)-L-NH2. The SAR of peptide hybrids 10 to 24, prepared by incorporating the dipeptide mimetics 1 to 4 is discussed. Of these hybrids, 23 and 24, analogs that incorporated the imidazole and oxazole mimetics as well as optimized variants at positions 3 to 5, were found to have 70 to 80 nM binding affinity comparable to the parent peptide in DRB 1*0401 binding and were also active in DRB1*0101 binding, while being resistant to proteolysis by cathepsin B. Both of these compounds showed inhibitory activity in an antigen-stimulated T-cell proliferation assay, indicating their potential to suppress autoimmune responses and as leads for therapeutic agents to treat RA.

Molecular features of ligand binding to MHC class II HLA-DR molecules have been elucidated through a combination of peptide structure-activity studies and structure-based drug design, resulting in analogues with nanomolar affinity in binding assays. Stabilization of lead compounds against cathepsin B cleavage by N-methylation of noncritical backbone NH groups or by dipeptide mimetic substitutions has generated analogues that compete effectively against protein antigens in cellular assays, resulting in inhibition of T-cell proliferation. Crystal structures of four ternary complexes of different peptide mimetics with the rheumatoid arthritis-linked MHC DRB10401 and the bacterial superantigen SEB have been obtained. Peptide-sugar hybrids have also been identified using a structure-based design approach in which the sugar residue replaces a dipeptide. These studies illustrate the complementary roles played by phage display library methods, peptide analogue SAR, peptide mimetics substitutions, and structure-based drug design in the discovery of inhibitors of antigen presentation by MHC class II HLA-DR molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

The interleukin-1 proteins (IL-1 alpha and IL-1 beta) are key mediators of inflammatory and immunological responses, and several in vitro and in vivo studies with protein-based antagonists have demonstrated the potential usefulness of IL-1 receptor antagonists to treat various inflammation related diseases. Based on the X-ray crystal structures of IL-1 ligands and site-directed mutagenesis data, a noncontiguous binding epitope encompassing Arg4, Phe46, Ile56, Lys93, Lys103, and Glu105 for IL-1 beta was proposed. In this paper we describe the synthesis and binding assay results of small molecule IL-1 receptor antagonists designed on the basis of the three-dimensional structure of the binding epitope. Among these, the compound 45 was found to inhibit IL-alpha binding to the Type I receptor with an IC50 value of 3 microM. A hypothesis generated using BioCad CATALYST program is also presented to rationalize these observations.

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of cholecystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe33-NH2] (2) in which the phenyl ring of the C-terminal Phe-NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyl or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10-70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10(-11)-10(-14) M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.