Probing the Molecular and Macroscopic Structure of Solid Solutions by Dynamic Nuclear Polarization (DNP) Enhanced 13C and 15N Solid-State NMR Spectroscopy.

Crystallization is a widely used purification technique in the manufacture of active pharmaceutical ingredients (APIs) and precursor molecules. However, when impurities and desired compounds have similar molecular structures, separation by crystallization may become challenging. In such cases, some impurities may form crystalline solid solutions with the desired product during recrystallization. Understanding the molecular structure of these recrystallized solid solutions is crucial to devise methods for effective purification. Unfortunately, there are limited analytical techniques that provide insights into the molecular structure or spatial distribution of impurities that are incorporated within recrystallized products. In this study, we investigated model solid solutions formed by recrystallizing salicylic acid (SA) in the presence of anthranilic acid (AA). These two molecules are known to form crystalline solid solutions due to their similar molecular structures. To overcome challenges associated with the long 1H longitudinal relaxation times (T1(1H)) of SA and AA, we employed dynamic nuclear polarization (DNP) and 15N isotope enrichment to enable solid-state NMR experiments. Results of solid-state NMR experiments and DFT calculations revealed that SA and AA are homogeneously alloyed as a solid solution. Heteronuclear correlation (HETCOR) experiments and plane-wave DFT structural models provide further evidence of the molecular-level interactions between SA and AA. This research provides valuable insights into the molecular structure of recrystallized solid solutions, contributing to the development of effective purification strategies and an understanding of the physicochemical properties of solid solutions.

Design, synthesis, biological evaluation and molecular docking studies of quinoline-anthranilic acid hybrids as potent anti-inflammatory drugs.

Despite the high global prevalence, rheumatoid arthritis lacks a satisfactory treatment. Hence, the present study is undertaken to design and synthesize novel anti-inflammatory compounds. For this, quinoline and anthranilic acid, two medicinally-privileged moieties, were linked by pharmacophore hybridization, and following their computational assessments, three hybrids 5a-c were synthesized in good over all yields. The in vitro and in vivo anti-inflammatory potential of these hybrids was determined by anti-denaturation and anti-proteinase, and carrageenan-induced paw edema models. The computational studies of these hybrids revealed their drug-likeness, optimum pharmacokinetics, and less toxicity. Moreover, they demonstrated high binding affinity (-9.4 to -10.6 kcal mol-1) and suitable binding interactions for TNF-α, FLAP, and COX-II. A three-step synthetic route resulted in the hybrids 5a-c with 83-86% yield of final step. At 50 µg mL-1, the antiprotease and anti-denaturation activity of compound 5b was significantly higher than 5a and 5c. Furthermore, 5b significantly reduced the edema in the right paw of the rats that received carrageenan. The results of this study indicate the medicinal worth of the novel hybrids in treating inflammatory disorders such as rheumatoid arthritis.

Anti-inflammatory effect of covalent PPARγ ligands that have a hybrid structure of GW9662 and a food-derived cinnamic acid derivative.

Peroxisome proliferator-activated receptor γ (PPARγ) belongs to the nuclear receptor superfamily and is involved in the inflammatory process. Previously, we synthesized the ligands of PPARγ that possess the hybrid structure of a food-derived cinnamic acid derivative (CA) and GW9662, an irreversible PPARγ antagonist. These ligands activate the transcription of PPARγ through the covalent bond formation with the Cys285 residue of PPARγ, whereas their anti-inflammatory effect has not been examined yet. Here, we show the anti-inflammatory effect of the covalent PPARγ ligands in RAW264 cells, murine macrophage-like cells. GW9662 suppressed the production of nitric oxide (NO) stimulated by lipopolysaccharide and exerted a synergistic effect in combination with CA. The compounds bearing their hybrid structure dramatically inhibited NO production and transcription of proinflammatory cytokines. A comparison study suggested that the 2-chloro-5-nitrobenzoyl group of the ligands is important for anti-inflammation. Furthermore, we synthesized an alkyne-tagged analogue that becomes an activity-based probe for future mechanistic study.

One-Pot, Multi-Component Green Microwave-Assisted Synthesis of Bridgehead Bicyclo[4.4.0]boron Heterocycles and DNA Affinity Studies.

Anthranilic acids, salicylaldehydes and arylboronic acids reacted in EtOH/H2O (1/3) at 150 °C under microwave irradiation for 1 h to give, in excellent yields and purity, twenty-three bridgehead bicyclo[4.4.0]boron heterocycles via one-pot, three-component green synthesis. The scope and the limitations of the reactions are discussed in terms of the substitution of ten different anthranilic acids, three salicylaldehydes and three arylboronic acids. The replacement of salicylaldehyde with o-hydroxyacetophenone demanded a lipophilic solvent for the reaction to occur. Eight novel derivatives were isolated following crystallization in a toluene-containing mixture that included molecular sieves. The above one-pot, three-component reactions were completed under microwave irradiation at 180 °C within 1.5 h, thus avoiding the conventional prolonged heating reaction times and the use of a Dean-Stark apparatus. All derivatives were studied for their affinity to calf thymus DNA using proper techniques like viscosity and UV-vis spectroscopy, where DNA-binding constants were found in the range 2.83 × 104-8.41 × 106 M-1. Ethidium bromide replacement studies using fluorescence spectroscopy indicated Stern-Volmer constants between 1.49 × 104 and 5.36 × 104 M-1, whereas the corresponding quenching constants were calculated to be between 6.46 × 1011 and 2.33 × 1012 M-1 s-1. All the above initial experiments show that these compounds may have possible medical applications for DNA-related diseases.

In Vitro Selection of Foldamer-Like Macrocyclic Peptides Containing 2-Aminobenzoic Acid and 3-Aminothiophene-2-Carboxylic Acid.

Aromatic cyclic ß2,3-amino acids (cßAAs), such as 2-aminobenzoic acid and 3-aminothiophene-2-carboxylic acid, are building blocks that can induce unique folding propensities of peptides. Although their ribosomal elongation had been a formidable task due to the low nucleophilicity of their amino groups, we have recently overcome this issue by means of an engineered tRNAPro1E2 that enhances their incorporation efficiency into nascent peptide chains. Here we report ribosomal synthesis of a random macrocyclic peptide library containing aromatic and aliphatic cßAAs, and its application to de novo discovery of binders against human IFNGR1 and FXIIa as model targets. The potent binding peptides showed not only high inhibitory activity but also high protease resistance in human serum. Moreover, these cßAAs play a critical role in exhibiting their properties, establishing a discovery platform for de novo foldamer-like macrocycles containing such unique building blocks.

Structural basis for diamide modulation of ryanodine receptor.

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4-S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.

Structure Revision of Penipacids A-E Reveals a Putative New Cryptic Natural Product, N-aminoanthranilic Acid, with Potential as a Transcriptional Regulator of Silent Secondary Metabolism.

Reconsideration of the spectroscopic data for penipacids A-E, first reported in 2013 as the acyclic amidines 1-5 from the South China deep sea sediment-derived fungus Penicillium paneum SD-44, prompted a total synthesis structure revision as the hydrazones 6-10. This revision strongly supported the proposition that penipacids A-B (6-7) were artifact Schiff base adducts of the cryptic (undetected) natural product N-aminoanthranilic acid (11) with diacetone alcohol, induced by excessive exposure to acetone and methanol under acidic handling conditions. Likewise, the revised structures for penipacids C-D (8-9) and E (10) raise the possibility that they may also be artifact Schiff base adducts of 11 and the media constituents pyruvic acid and furfural, respectively. A review of the natural products literature revealed other Schiff base (hydrazone) natural products that might also be viewed as Schiff base adduct artifacts of 11. Having raised the prospect that 11 is an undetected and reactive cryptic natural product, we went on to establish that 11 is not cytotoxic to a range of bacterial, fungal or mammalian (human) cell types. Instead, when added as a supplement to microbial cultivations, 11 can act as a chemical cue/transcriptional regulator, activating and/or enhancing the yield of biosynthetic gene clusters encoding for other natural product chemical defenses. This study demonstrates the value of challenging the structure and artifact status of natural products, as a window into the hidden world of cryptic and highly reactive natural products.

Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Effect of Germination on the Avenanthramide Content of Oats and Their in Vitro Antisensitivity Activities.

In this study, a method, based on an ultraperformance liquid chromatography coupled with high-field quadrupole orbitrap high-resolution mass spectrometry (UHPLC-QE-HF-HRMS) platform, was established for the trace determination of three major avenanthramides (AVNs). The MS conditions for determining the AVNs were optimized, and the cracking methods of avenanthramides were analyzed. The linear range of the results and the correlation coefficient were 1−2000 µg/L and >0.996, respectively. Further, the established method was employed for the determination of the AVN contents of oats at different germination times, and the results indicated that the AVN contents of Zaohua and Bayou oats increased 19.26 and 6.09 times, respectively, after germination. The total AVN content of both oat varieties reached a maximum on the fifth day of germination (153.51 ± 4.08 and 126.30 ± 3.33 µg/g for the Zaohua and Bayou oats, respectively). Furthermore, this study investigated the antiallergic and antioxidant activities of the germinated oats via hyaluronidase inhibition and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging assays. The antiallergic and DPPH-scavenging abilities of the ungerminated forms of both oat varieties were weaker. However, on the fifth day of germination, the inhibition rate of anthranilamide hyaluronidase reached 72.7% and 67.3% for the Zaohua and Bayou oat varieties, respectively. The antiallergic abilities of the oats increased significantly on the fifth day of germination in terms of their antiallergic capacities and DPPH clearance (82.67% and 77.64% for the Zaohua and Bayou oats, respectively), and the two indicators exhibited similar trends. These findings demonstrated that AVNs exhibit good antisensitivity and antioxidation properties, and the antisensitivity effect correlated positively with the AVN content.

Profiling of glycosphingolipids with SCDase digestion and HPLC-FLD-MS.

Lipid components of cells and tissues feature a large diversity of structures that present a challenging problem for molecular analysis. Glycolipids from mammalian cells contain glycosphingolipids (GSLs) as their major glycolipid component, and these structures vary in the identity of the glycan headgroup as well as the structure of the fatty acid and sphingosine (Sph) tails. Analysis of intact GSLs is challenging due to the low abundance of these species. Here, we develop a new strategy for the analysis of lyso-GSL (l-GSL), GSL that retain linkage of the glycan headgroup with the Sph base. The analysis begins with digestion of a GSL sample with sphingolipid ceramide N-deacylase (SCDase), followed by labelling with an amine-reactive fluorophore. The sample was then analyzed by HPLC-FLD-MS and quantitated by addition of an external standard. This method was compared to analysis of GSL glycans after cleavage by an Endoglycoceramidase (EGCase) enzyme and labeling with a fluorophore (2-anthranilic acid, 2AA). The two methods are complementary, with EGCase providing improved signal (due to fewer species) and SCDase providing analysis of lyso-GSL. Importantly the SCDase method provides Sph composition of GSL species. We demonstrate the method on cultured human cells (Jurkat T cells) and tissue homogenate (porcine brain).

Formation and fragmentation of doubly and triply charged ions in the negative ion spectra of neutral N-glycans from viral and other glycoproteins.

Structural determination of N-glycans by mass spectrometry is ideally performed by negative ion collision-induced dissociation because the spectra are dominated by cross-ring fragments leading to ions that reveal structural details not available by many other methods. Most glycans form [M - H]- or [M + adduct]- ions but larger ones (above approx. m/z 2000) typically form doubly charged ions. Differences have been reported between the fragmentation of singly and doubly charged ions but a detailed comparison does not appear to have been reported. In addition to [M + adduct]- ions (this paper uses phosphate as the adduct) other doubly, triply, and quadruply charged ions of composition [Mn + (H2PO4)n]n- have been observed in mixtures of N-glycans released from viral and other glycoproteins. This paper explores the formation and fragmentation of these different types of multiply charged ions with particular reference to the presence of diagnostic fragments in the CID spectra and comments on how these ions can be used to characterize these glycans.

Exploration on the drug solubility enhancement in aqueous medium with the help of endo-functionalized molecular tubes: a computational approach.

One major problem in the pharmaceutical industry is the aqueous solubility of newly developed orally administered drug candidates. More than 50% of newly developed drug molecules suffer from low aqueous solubility. The therapeutic effects of drug molecules are majorly dependent on the bioavailability and, in essence, on the solubility of the used drug molecules. Thus, enhancement of drug solubility of sparingly soluble drug molecules is a need of modern times. Considering the high importance of drug solubility, we have computationally shown the enhancement of drug solubility for seven class II (poorly water-soluble) drug molecules in a water medium. The uses of supramolecular macrocycles have immense importance in the same field. Thus, we have used two synthetic supramolecular receptors named host-1a and host-1b to enhance the water solubility of fluorouracil, albendazole, camptothecin, clopidogrel, indomethacin, melphalan, and tolfenamic acid drug molecules. Biomedical engagements of a supramolecular receptor commence with the formation of stable host-drug complexes. These complexations enhance the water solubility of drug molecules and sustain the release rate and bioavailability of drug molecules. Thus, in this work, we focus on the formation of stable host-drug complexes in water medium. Molecular dynamics simulation is applied to analyze the structural features and the energetics involved in the host-drug complexation process. The information obtained at the atomistic level helps us gain better insights into the key interactions that operate to produce such highly stable complexes. Thus, we can propose that these two supramolecular receptors may be used as drug solubilizing agents, and patients will benefit from this theragnostic application shortly.

Biotransformations of anthranilic acid and phthalimide to potent antihyperlipidemic alkaloids by the marine-derived fungus Scedosporium apiospermum F41-1.

A new diphenylamine derivative, scediphenylamine A (1), together with six phthalimide derivatives (2-7) and ten other known compounds (8-17) were obtained from the marine-derived fungus Scedosporium apiospermum F41-1 fed with synthetically prepared anthranilic acid and phthalimide. The structure and absolute configuration of the new compound were determined by HRMS, NMR, and X-ray crystallography. Evaluation of their lipid-lowering effect in 3T3-L1 adipocytes showed that scediphenylamine A (1), N-phthaloyl-tryptophan-methyl ester (4), 5-(1,3-dioxoisoindolin-2-yl) pentanamide (5), perlolyrine (10) and flazine (11) significantly reduced triglyceride level in 3T3-L1 cells by inhibiting adipogenic differentiation and synthesis with the EC50 values of 4.39, 2.79, 3.76, 0.09, and 4.52 µM, respectively. Among them, perlolyrine (10) showed the most potent activity, making it a candidate for further development as a potential agent to treat hyperlipidemia.

ß-Carboline tethered cinnamoyl 2-aminobenzamides as class I selective HDAC inhibitors: Design, synthesis, biological activities and modelling studies.

The effect of ß-carboline motif as cap for HDAC inhibitors containing cinnamic acid as linker and benzamides as zinc binding group was examined in this study. A series of ß-carboline-cinnamide conjugates have been synthesized and evaluated for their HDAC inhibitory activity and in vitro cytotoxicity against different human cancer cell lines. Almost all the compounds exhibited superior HDAC inhibitory activity than the standard drug Entinostat for in vitro enzymatic assay. Among the tested compounds, 7h displayed a noteworthy potency with an IC50 value of 0.70 ± 0.15 µM against HCT-15 cell line when compared to the standard drug Entinostat (IC50 of 3.87 ± 0.62 µM). The traditional apoptosis assays such as nuclear morphological alterations, AO/EB, DAPI, and Annexin-V/PI staining revealed the antiproliferative activity of 7h while depolarization of mitochondrial membrane potential by JC-1 was observed in dose-dependent manner. Cell cycle analysis also unveiled the typical accumulation of cells in G2M phase and sub-G1/S phase arrest. In addition, immunoblot analysis for compound 7h on HCT-15 indicated selective inhibition of the protein expression of class I HDAC 2 and 3 isoforms. Molecular docking analysis of compound 7h revealed that it can prominent binding with the active pocket of the HDAC 2. These finding suggest that the compound 7h can be a promising lead candidate for further investigation in the development of novel anti-cancer drug potentially inhibiting HDACs.

Synthesis of the quinazolinone derivatives using an acid-functionalized magnetic silica heterogeneous catalyst in terms of green chemistry.

In this research, the synthesis of the quinazolinone derivatives by the reaction of diaminoglyoxime with anthranilic acid or methyl 2-amino benzoate over an acetic acid-functionalized magnetic silica-based catalyst in water was described. The acetic acid-functionalized catalyst was prepared using a three-step procedure from magnetite NPs that initially coated with a layer of silica through the sol-gel process, modified with an aminosilane layer and functionalized with bromoacetic acid. The catalyst was characterized by means of spectroscopic and microscopic techniques, and its activity was investigated for the synthesis of the quinazolinones, bisquinazolinone and oxadiazole quinazolinones obtained from diaminoglyoxime in water at room temperature.

The ATPase mechanism of UvrA2 reveals the distinct roles of proximal and distal ATPase sites in nucleotide excision repair.

The UvrA2 dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA2 damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P2ATP-D2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA2 to transition through a different species, P2ADP-D2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P2ATP-Dempty and P2ATP-D2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA2 forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA2 utilizes ATP for lesion search, recognition and repair.

Medicinal chemistry of anthranilic acid derivatives: A mini review.

Anthranilic acid and its analogues present a privileged profile as pharmacophores for the rational development of pharmaceuticals deliberated for managing the pathophysiology and pathogenesis of various diseases. The substitution on anthranilic acid scaffold provides large compound libraries, which enable a comprehensive assessment of the structure activity relationship (SAR) analysis for the identification of hits and leads in a typical drug development paradigm. Besides, their widespread applications as anti-inflammatory fenamates, the amide and anilide derivatives of anthranilic acid analogues play a central role in the management of several metabolic disorders. In addition, these derivatives of anthranilic acid exhibit interesting antimicrobial, antiviral and insecticidal properties, whereas the derivatives based on anthranilic diamide scaffold present applications as P-glycoprotein inhibitors for managing the drug resistance in cancer cells. In addition, the anthranilic acid derivatives serve as the inducers of apoptosis, inhibitors of hedgehog signaling pathway, inhibitors of mitogen activated protein kinase pathway, and the inhibitors of aldo-keto reductase enzymes. The antiviral derivatives of anthranilic acid focus on the inhibition of hepatitis C virus NS5B polymerase to manifest considerable antiviral properties. The anthranilic acid derivatives reportedly present neuroprotective applications by downregulating the key pathways responsible for the manifestation of neuropathological features and neurodegeneration. Nevertheless, the transition metal complexes of anthranilic acid derivatives offer therapeutic applications in diabetes mellitus, and obesity by regulating the activity of α-glucosidase. The present review demonstrates a critical analysis of the therapeutic profile of the key derivatives of anthranilic acid and its analogues for the rational development of pharmaceuticals and therapeutic molecules.

Synthesis, Molecular Docking and Biological Characterization of Pyrazine Linked 2-Aminobenzamides as New Class I Selective Histone Deacetylase (HDAC) Inhibitors with Anti-Leukemic Activity.

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.

Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human-Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

The Crystal Structure and Intermolecular Interactions in Fenamic Acids-Acridine Complexes.

In order to improve pharmaceutical properties of drugs, complexes are synthesized as combinations with other chemical substances. The complexes of fenamic acid and its derivatives, such as mefenamic-, tolfenamic- and flufenamic acid, with acridine were obtained and the X-ray structures were discussed. Formation of the crystals is determined by the presence of the intermolecular O-H…N hydrogen bond that occur between fenamic acids and acridine. Intermolecular interactions stabilizing the crystals such as π…π stacking, C-H…X (X = O, Cl) intermolecular hydrogen bonds as well as C-H…π and other dispersive interactions were analyzed by theoretical methods: the quantum theory of atoms in molecules (QTAIM) and noncovalent interaction (NCI) approaches.