Efficient Screening of Pharmaceutical Cocrystals by Microspacing In-Air Sublimation.

Cocrystal screening and single-crystal growth remain the primary obstacles in the development of pharmaceutical cocrystals. Here, we present a new approach for cocrystal screening, microspacing in-air sublimation (MAS), to obtain new cocrystals and grow high-quality single crystals of cocrystals within tens of minutes. The method possesses the advantages of strong designable ability of devices, user-friendly control, and compatibility with materials, especially for the thermolabile molecules. A novel drug-drug cocrystal of favipiravir (FPV) with salicylamide (SAA) was first discovered by this method, which shows improved physiochemical properties. Furthermore, this method proved effective in cultivating single crystals of FPV-isonicotinamide (FPV-INIA), FPV-urea, FPV-nicotinamide (FPV-NIA), and FPV-tromethamine (FPV-Tro) cocrystals, and the structures of these cocrystals were determined for the first time. By adjusting the growth temperature and growth distance precisely, we also achieved single crystals of 10 different paracetamol (PCA) cocrystals and piracetam (PIR) cocrystals, which underscores the versatility and efficiency of this method in pharmaceutical cocrystal screening.

Structure Activity Relationships for a Series of Eticlopride-Based Dopamine D2/D3 Receptor Bitopic Ligands.

The crystal structure of the dopamine D3 receptor (D3R) in complex with eticlopride inspired the design of bitopic ligands that explored (1) N-alkylation of the eticlopride's pyrrolidine ring, (2) shifting of the position of the pyrrolidine nitrogen, (3) expansion of the pyrrolidine ring system, and (4) incorporation of O-alkylations at the 4-position. Structure activity relationships (SAR) revealed that moving the N- or expanding the pyrrolidine ring was detrimental to D2R/D3R binding affinities. Small pyrrolidine N-alkyl groups were poorly tolerated, but the addition of a linker and secondary pharmacophore (SP) improved affinities. Moreover, O-alkylated analogues showed higher binding affinities compared to analogously N-alkylated compounds, e.g., O-alkylated 33 (D3R, 0.436 nM and D2R, 1.77 nM) vs the N-alkylated 11 (D3R, 6.97 nM and D2R, 25.3 nM). All lead molecules were functional D2R/D3R antagonists. Molecular models confirmed that 4-position modifications would be well-tolerated for future D2R/D3R bioconjugate tools that require long linkers and or sterically bulky groups.

Synthesis and activities of acetylcholinesterase inhibitors.

Cholinesterase (ChE) inhibitors can be divided into two categories: acetylcholinesterase (AChE) inhibitors and butylcholinesterase (BuChE) inhibitors. Therefore, the development of selective inhibition of AChE and BuChE activities is the central content of ChE pharmacochemistry research. In order to clarify the progress of AChE inhibitor-based design, synthesis, and activity studies, we reviewed the pharmacochemical and pharmacological properties of selective AChE inhibitors over the past decade. We hope that this review will make it easier for readers to understand the development of new drug chemistry methods for AChE inhibitors in order to develop more effective and selective AChE inhibitors.

Solvates of New Arylpiperazine Salicylamide Derivative-a Multi-Technique Approach to the Description of 5 HTR Ligand Structure and Interactions.

A new ligand for 5-HT1A and 5-HT7 receptors, an arylpiperazine salicylamide derivative with an inflexible spacer, is investigated to identify preferred fragments capable of creating essential intermolecular interactions in different solvates. To fully identify and characterize the obtained crystalline materials, various methods including powder and single-crystal X-ray diffraction, solid-state NMR, and thermal analysis were employed, supplemented by periodic ab initio calculations. The molecular conformation in different solvates, types, and hierarchy of intermolecular interactions as well as the crystal packing were investigated to provide data for future research focused on studying protein-ligand interactions. Based on various methods of crystal structure analysis, including the interaction energy calculation and programs using an artificial neural network, a salicylamide fragment was found to be crucial for intermolecular contacts, mostly of dispersion and electrostatic character. A supramolecular 2D kite-type layer of {4,4} topology was found to form in crystals. The closed voids between layers contain disordered solvents, very weakly interacting with the molecule and the layer. It has been postulated that the separation of the layers might be influenced by an increase in temperature or the size of the solvent; hence, only methanol and ethanol hemi-solvates could be obtained from a series of various alcohols.

Discovery of a Small Molecule Inhibitor of Human Adenovirus Capable of Preventing Escape from the Endosome.

Human adenoviruses (HAdVs) display a wide range of tissue tropism and can cause an array of symptoms from mild respiratory illnesses to disseminated and life-threatening infections in immunocompromised individuals. However, no antiviral drug has been approved specifically for the treatment of HAdV infections. Herein, we report our continued efforts to optimize salicylamide derivatives and discover compound 16 (JMX0493) as a potent inhibitor of HAdV infection. Compound 16 displays submicromolar IC50 values, a higher selectivity index (SI > 100) and 2.5-fold virus yield reduction compared to our hit compound niclosamide. Moreover, unlike niclosamide, our mechanistic studies suggest that the antiviral activity of compound 16 against HAdV is achieved through the inhibition of viral particle escape from the endosome, which bars subsequent uncoating and the presentation of lytic protein VI.

Investigation of Preprocessing and Validation Methodologies for PAT: Case Study of the Granulation and Coating Steps for the Manufacturing of Ethenzamide Tablets.

After the Food and Drug Association in the USA published guidelines on the enhanced use of process analytical technology (PAT) and continuous manufacturing, many studies regarding PAT and continuous manufacturing have been published. This paper describes a case study involving granulation and coating steps with ethenzamide to investigate interference for PAT model construction and model management. We investigated what factors should be considered and addressed when PAT is implemented for continuous manufacturing and how predictive models should be constructed. The product qualities that were monitored were moisture content and particle size in the granulation step and tablet weight and moisture content in the coating step. We have constructed models for the granulation step and validated the predictive capability of the models against an external dataset. A partial least squares (PLS) model with manual wavelength selection had the best predictive accuracy for loss on drying against the external validation set. We found that the prediction of loss on drying was accurate, but the prediction of particle size was not sufficiently accurate. In the coating step, because of the small amount of data, we performed three-fold cross-validation and y-scrambling 10 times, to select the optimal hyper-parameters and to check if the models were fitted to chance correlations. We confirmed that the coating agent weights, tablet weights, and water content could be accurately predicted based on the mean of the R2 score for cross-validation. Addition of other variables, as well as the absorbance, slightly improved the predictive accuracy.

Determining key parameters of continuous wet granulation for tablet quality and productivity: A case in ethenzamide.

This paper aims to determine key parameters that affect tablet quality and productivity in continuous tablet manufacturing. Experiments were performed based on design of experiments using a continuous high-shear granulator and ethenzamide as the active pharmaceutical ingredient. To guide a systematic and comprehensive parameter analysis, a parameter framework was defined that comprised five input parameters on raw material properties and process parameters, 11 intermediate parameters on granule properties, and 11 output parameters on tablet quality and productivity. The interrelationships were analyzed statistically and were described as matrix functions. The liquid/solid ratio was the key parameter that affected circularity, density, and flowability as the granule properties, and disintegration and dissolution as the tablet quality. The maximum acceptable manufacturing rate that governs productivity was also affected by the liquid/solid ratio. Circularity was found to affect disintegration and dissolution. This result was specific to the setup of the study, but suggested development opportunities for a new process analytical technology system/quality-by-design application based on circularity. In addition, practical findings were obtained as follows: (1) high-speed manufacturing favored a lower liquid/solid ratio, and (2) high circularity slowed down disintegration/dissolution. This obtained knowledge will enhance the applicability of continuous technology in an actual manufacturing environment.

Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism.

By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands.

Almost a third of prescribed drugs work by acting on a group of proteins known as GPCRs (short for G-protein coupled receptors), which help to transmit messages across the cell's outer barrier. The neurotransmitter dopamine, for instance, can act in the brain and body by attaching to dopamine receptors, a sub-family of GPCRs. The binding process changes the three-dimensional structure (or conformation) of the receptor from an inactive to active state, triggering a series of molecular events in the cell. However, GPCRs do not have a single 'on' or 'off' state; they can adopt different active shapes depending on the activating molecule they bind to, and this influences the type of molecular cascade that will take place in the cell. Some evidence also shows that classes of GPCRs can have different inactive structures; whether this is also the case for the dopamine D₂ and D₃ receptors remained unclear. Mapping out inactive conformations of receptors is important for drug discovery, as compounds called antagonists can bind to inactive receptors and interfere with their activation. Lane et al. proposed that different types of antagonists could prefer specific types of inactive conformations of the dopamine D₂ and D₃ receptors. Based on the structures of these two receptors, the conformations of D₂ bound with the drugs risperidone and eticlopride (two dopamine antagonists) were simulated and compared. The results show that the inactive conformations of D₂ were very different when it was bound to eticlopride as opposed to risperidone. In addition D₂ and D₃ showed a very similar conformation when attached to eticlopride. The two drugs also bound to the inactive receptors in overlapping but different locations. These computational findings, together with experimental validations, suggest that D₂ and D₃ exist in several inactive states that only allow the binding of specific drugs; these states could also reflect different degrees of inactivation. Overall, the work by Lane et al. contributes to a more refined understanding of the complex conformations of GPCRs, which could be helpful to screen and develop better drugs.

A Novel Binary Supercooled Liquid Formulation for Transdermal Drug Delivery.

The aim of this study was to prepare binary supercooled liquid (SCL) by intermolecular interaction and apply this formulation to transdermal drug delivery. Ketoprofen (KET) and ethenzamide (ETH) were selected as binary SCL component. Thermal analysis of physical mixtures of KET and ETH showed decreases in melting points and glass transition below room temperature, thereby indicating formation of KET-ETH SCL. Intermolecular interactions between KET and ETH in the SCL were evaluated from Fourier transform (FT)-IR spectra. KET-ETH SCL maintained SCL state at 25°C with silica gel over 31 d and at 40°C/89% relative humidity (RH) over 7 d. KET SCL and KET-ETH SCL showed similar permeability of KET for hairless mice skin, which was two-fold higher than that of KET aqueous suspension. Our findings suggest that the SCL state could enhance the skin permeation of drugs and the binary SCL formed by intermolecular interaction could also improve the stability of the SCL. The binary SCL system could become a new drug form for transdermal drug delivery.

Organocatalytic Asymmetric Conjugate Addition of Aldehydes to Maleimides and Nitroalkenes in Deep Eutectic Solvents.

A chiral primary amine-salicylamide is used as an organocatalyst for the enantioselective conjugate addition of α,α-disubstituted aldehydes to maleimides and nitroalkenes. The reactions are performed in deep eutectic solvents as reaction media at room temperature, leading to the corresponding adducts with enantioselectivities up to 88% (for maleimides) and 80% (for nitroalkenes). Catalyst and solvent can be recovered and reused.

SAR optimization studies on modified salicylamides as a potential treatment for acute myeloid leukemia through inhibition of the CREB pathway.

Disruption of cyclic adenosine monophosphate response element binding protein (CREB) provides a potential new strategy to address acute leukemia, a disease associated with poor prognosis, and for which conventional treatment options often carry a significant risk of morbidity and mortality. We describe the structure-activity relationships (SAR) for a series of XX-650-23 derived from naphthol AS-E phosphate that disrupts binding and activation of CREB by the CREB-binding protein (CBP). Through the development of this series, we identified several salicylamides that are potent inhibitors of acute leukemia cell viability through inhibition of CREB-CBP interaction. Among them, a biphenyl salicylamide, compound 71, was identified as a potent inhibitor of CREB-CBP interaction with improved physicochemical properties relative to previously described derivatives of naphthol AS-E phosphate.

Anti-trypanosomal activity of doubly modified salinomycin derivatives.

As a group of biologically active compounds, polyether antibiotics (ionophores) show a broad spectrum of interesting pharmacological properties, ranging from anti-bacterial to anti-cancer activities. There is increasing evidence that ionophores, including salinomycin (SAL), and their semi-synthetic analogues are promising candidates for the development of drugs against parasitic diseases. Our previous studies have shown that esterification and amidation of the C1 carboxylate moiety of SAL provides compounds with potent activity against Trypanosoma brucei, protozoan parasites responsible for African trypanosomiasis. In this paper, we present the synthetic pathways, crystal structures and anti-trypanosomal activity of C1 esters, amides and hydroxamic acid conjugates of SAL, its C20-oxo and propargylamine analogues as well novel C1/C20 doubly modified derivatives. Evaluation of the trypanocidal and cytotoxic activity using bloodstream forms of T. brucei and human myeloid HL-60 cells revealed that the single-modified C20-oxo and propargylamine precursor molecules 10 and 16 were the most anti-trypanosomal and selective compounds with 50% growth inhibition (GI50) values of 0.037 and 0.035 µM, and selectivity indices of 252 and 300, respectively. Also the salicylhydroxamic acid conjugate of SAL (compound 9) as well as benzhydroxamic acid and salicylhydroxamic acid conjugates of 10 (compounds 11 and 12) showed promising trypanocidal activities with GI50 values between 0.032 and 0.035 µM but less favorable selectivities. The findings confirm that modification of SAL can result in derivatives with improved trypanocidal activity that might be interesting lead compounds for further anti-trypanosomal drug development.

Spectroscopic analysis of the influence of various external factors on ethenzamide-glutaric acid (1:1) cocrystal formation.

Cocrystal formation may affect manufacturability (flow, compaction and processability) as well as solubility/dissolution, hygroscopicity and stability properties of drugs. Therefore, cocrystallization could be used to improve the pharmaceutical properties of low-soluble drugs such as ethenzamide. In this project, solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopy studies were performed for ethenzamide-glutaric acid to obtain more information about the ethenzamide cocrystallization process. The impact of the grinding time of the physical mixture (ethenzamide-glutaric acid) on cocrystal formation and the further spontaneous cocrystallization was evaluated using spectroscopic methods and curve-fitting analysis of the spectra. The influence of pressure on the yield of cocrystal formation was also described. Additionally, studies on the effect of magic-angle spinning during solid-state nuclear magnetic resonance spectra collection on the initiation of cocrystal formation, have been performed. Based on this research, conclusions regarding the influence of the different external factors, such as pressure during the tableting process and grinding time, on the cocrystal formation have been drawn for ethenzamide cocrystals.

Configuration and molecular structure of 5-chloro-N-(4-sulfamoylbenzyl) salicylamide derivatives.

A systematic study on sulfonamide derivatives with salicylamide core is presented for possible use in pharmaceutical applications. The molecular structure of eight different compounds has been investigated by FTIR in the frequency range 4000-400 cm-1 to recognize the possible geometrical shape of the molecules needed to uniquely identify the activity of molecule in cancer cell. The electronic charge distribution of these different compounds is further illustrated by UV-Vis spectroscopy in the frequency range 190-1100 nm. The theoretical results obtained from molecular modeling calculations showed that the hydrogen bonds between the OH, CO, NH, and/or CH groups vary from one compound to the other regarding their number and bond length. This confirms the experimental FTIR results regarding the position and broadening of the OH and NH groups due to free rotation of the amide group because of changing the compounds structure by adding different groups to the last phenyl ring. The hydrogen bonds take different directions and values from one compound to the other, which seems to be the most important factor regarding the activity of these different compounds in cancer cell. Both theoretical molecular modeling calculations and FTIR experimental results have strongly evaluated the relation between the chemical structure of 5-chloro-N (4-sulfamoylbenzyl) salicylamide derivatives and their biological activities.

Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10-100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.

A large-scale experimental comparison of batch and continuous technologies in pharmaceutical tablet manufacturing using ethenzamide.

This paper compares batch and continuous technologies in terms of product quality and process performance in pharmaceutical tablet manufacturing using ethenzamide as the active pharmaceutical ingredient. Batch and continuous processes using wet granulation were investigated by performing experiments on the scale of 5 and up to 100 kg/lot, using the same raw materials. Three technologies were tested and compared: (i) batch technology using fluidized bed granulation, (ii) batch technology using high shear granulation, (iii) continuous technology using high shear granulation. In the full-scale experiment, in all three technologies including continuous technology, the quality of the tablets fulfilled the target values regarding hardness, active pharmaceutical ingredient content, and dissolution. The granules produced by different technologies, however, presented varying attributes regarding granule size distribution, loose bulk density, or scanning electron microscope images. The process performance, more specifically the yield, was slightly better for batch technologies than for the continuous technology, mainly due to losses in the start-up operation. Notably, this study has shown that continuous technology, which is generally believed to not entail scale-up procedures, could in fact, require parameter adjustment for prolonged operation. The results provided suggestions for improvements to implement large-scale continuous technologies in the pharmaceutical industry.

Novel salicylamide derivatives as potent multifunctional agents for the treatment of Alzheimer's disease: Design, synthesis and biological evaluation.

A series of salicylamide derivatives were designed, synthesized and evaluated as multifunctional agents for the treatment of Alzheimer's disease. In vitro assays demonstrated that most of the derivatives were selective AChE inhibitors. They showed good inhibitory activities of self- and Cu2+-induced Aß1-42 aggregation, and significant antioxidant activities. Among them, compound 15b exhibited good inhibitory activity toward RatAChE and EeAChE with IC50 value of 10.4 µM and 15.2 µM, respectively. Moreover, 15b displayed high antioxidant activity (2.46 Trolox equivalents), good self- and Cu2+-induced Aß1-42 aggregation inhibitory potency (42.5% and 31.4% at 25.0 µM, respectively) and moderate disaggregation ability to self- and Cu2+-induced Aß1-42 aggregation fibrils (23.4% and 27.0% at 25 µM, respectively). Furthermore, 15b also showed biometal chelating abilities, anti-neuroinflammatory ability and BBB permeability. These multifunctional properties indicated compound 15b was worthy of being chosen for further pharmacokinetics, toxicity and behavioral researches to test its potential for AD treatment.

Oryza sativa class III peroxidase (OsPRX38) overexpression in Arabidopsis thaliana reduces arsenic accumulation due to apoplastic lignification.

ClassIII peroxidases are multigene family of plant-specific peroxidase enzyme. They are involved in various physiological and developmental processes like auxin catabolism, cell metabolism, various biotic, abiotic stresses and cell elongation. In the present study, we identified a class III peroxidase (OsPRX38) from rice which is upregulated several folds in both arsenate (AsV) and arsenite (AsIII) stresses. The overexpression of OsPRX38 in Arabidopsis thaliana significantly enhances Arsenic (As) tolerance by increasing SOD, PRX GST activity and exhibited low H2O2, electrolyte leakage and malondialdehyde content. OsPRX38 overexpression also affect the plant growth by increasing total biomass and seeds production in transgenics than WT under As stress condition. Confocal microscopy revealed that the OsPRX38-YFP fusion protein was localized to the apoplast of the onion epidermal cells. In addition, lignification was positively correlated with an increase in cell-wall-associated peroxidase activities in transgenic plants. This study indicates the role of OsPRX38 in lignin biosynthesis, where lignin act as an apoplastic barrier for As entry in root cells leading to reduction of As accumulation in transgenic. Overall the study suggests that overexpression of OsPRX38 in Arabidopsis thaliana activates the signaling network of different antioxidant systems under As stress condition, enhancing the plant tolerance by reducing As accumulation due to high lignification.

Investigation of activation mechanism and conformational stability of N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide and N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide in the: active site of p300 histone acetyl transferase enzyme by molecular dynamics and binding free energy studies.

The CBP (CREB-binding protein) and p300 are related to transcriptional coactivator family and are involved in several post-translational modifications, in which the acetylation is an important factor because it commences the transcription process. Experimental studies report that CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide) and CTB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide) are good activators of p300 HAT enzyme, but yet, the molecular mechanism of their activation is not explored. The present study pertains to determine the intermolecular interactions, stability and binding free energy of CTB and CTPB from the molecular docking, molecular dynamics (MD) simulation and binding free energy calculation. The docking studies of the molecules reveal that the docking score of CTPB (-15.64 kcal/mol) is higher than that of CTB (-12.30 kcal/mol); on the contrary, CTB forms a strong interaction with the key residues of catalytic site (Tyr1467 and Trp1436) compared with CTPB. The MD simulation shows the stability of both molecules in the active site of p300 and their interactions. Furthermore, both docking and MD simulation studies of CTB confirm that it forms expected key interactions and retain the interactions with the active site amino acid residues of p300 when compared with CTPB. For this reason, the CTB recruits more acetyl-CoA in the active site of p300 compared with CTPB; it leads to activate the acetylation process; hence, CTB may be a best activator than CTPB. The binding free energy value of CTPB (-24.79 ± 2.38 kcal/mol) is higher when compared with that of CTB (-12.14 ± 1.30 kcal/mol) molecule; perhaps, the interaction of pentadecyl chain of CTPB with p300, whereas in CTB, such a group is absent. Communicated by Ramaswamy H. Sarma.

Structural Characterization and Pharmaceutical Properties of Three Novel Cocrystals of Ethenzamide With Aliphatic Dicarboxylic Acids.

Ethenzamide (ET) was screened in cocrystallization experiments with pharmaceutically acceptable coformer molecules to discover materials of improved physicochemical properties, that is, higher solubility and better stability. Three novel cocrystals of ET with glutaric, malonic, and maleic acids were obtained by neat grinding and slow evaporation from solution. The purpose of the study was to notice the changes in the geometry and interactions of ET molecule in crystalline phase introduced by different acid and relate them to physicochemical properties of pure ET. Therefore, the crystal structure of the cocrystals was determined by single crystal X-ray diffraction analysis. The powder samples were characterized by differential scanning calorimetry, Fourier-transform infrared spectroscopy, and 13C and 15N solid-state nuclear magnetic resonance spectroscopy. Spectroscopic studies were supported by gauge including projector augmented wave calculations of chemical shielding constants. The high stability of cocrystals during direct compression was proved. The solubility in simulated gastric fluids for studied cocrystals appeared to be approximately 1.6 times-fold higher than ET. The dissolution rates of all ET cocrystals were not faster than the pure drug, but after 240 min, more drugs were released.