Fabrication of architectonic nanosponges for intraocular delivery of Brinzolamide: An insight into QbD driven optimization, in vitro characterization, and pharmacodynamics.

The intricate structure of the eye poses difficulties in drug targeting, which can be surmounted with the help of nanoformulation strategies. With this view, brinzolamide nanosponges (BNS) were prepared using the emulsion solvent evaporation technique and optimized via Box-Behnken statistical design. The optimized BNS were further incorporated into a poloxamer 407 in situ gel (BNS-ISG) and evaluated. The optimized BNS showed spherical morphology, entrapment efficiency of 83.12 ± 1.2 % with particle size of 114 ± 2.32 nm and PDI of 0.11 ± 0.01. The optimized BNS-ISG exhibited a pseudoplastic behavior and depicted a gelling temperature and gelation time of 35 ± 0.5 °C and 10 ± 2 s respectively. In-vitro release and ex- vivo permeation studies of BNS-ISG demonstrated a sustained release pattern as compared to Brinzox®. Additionally, the HET-CAM and in vitro cytotoxicity studies (using SIRC cell line) ensured that the formulation was non-irritant and nontoxic for ophthalmic delivery. The in vivo pharmacodynamic study using rabbit model depicted that BNS-ISG treatment significantly lowers the intra ocular pressure for prolonged period of time when compared with Brinzox®. In conclusion, the BNS-ISG is an efficient and scalable drug delivery system with significant potential as the targeted therapy of posterior segment eye diseases.

Synthesis, structures, reactivity and medicinal chemistry of antitubercular benzothiazinones.

Tuberculosis is the leading bacterial killer worldwide. 8-Nitro-4H-benzo[e][1,3]thiazin-4-ones are a potent class of antitubercular agents with a new mechanism of action. BTZ043 and PBTZ169 (macozinone) have progressed to clinical studies. Herein, we give a comprehensive account of this important class of potential new drugs to treat tuberculosis. We present an overview of recent developments in the field of antitubercular benzothiazinones (BTZs) and summarize our own contributions. The review covers synthesis, structures and reactivity, mechanism of action, in vitro activity and structure activity relationships (SARs), physicochemical and pharmacokinetic properties as well as a brief summary of in vivo models and clinical studies. We address bioavailability issues and the challenge of the potentially toxic nitroaromatic moiety, including reactivity towards nucleophiles in vivo and highlight possible directions of further research into BTZs through chemical modification.

Chitosan Encapsulated Meloxicam Nanoparticles for Sustained Drug Delivery Applications: Preparation, Characterization, and Pharmacokinetics in Wistar Rats.

Meloxicam (MLX) is currently used in the therapeutic management of both acute and chronic inflammatory disorders such as pain, injuries, osteoarthritis, and rheumatoid arthritis in both humans and animals. Gastrointestinal toxicity and occasional renal toxicity were observed in patients taking it for a long-term period. Meloxicam's late attainment of peak plasma concentration results in a slow onset of action. The goal of the current study was to prepare and characterize chitosan encapsulated meloxicam nanoparticles (CEMNPs) with high bioavailability and less gastro intestinal toxicity in order to prevent such issues. The size of the prepared CEMNPs was approximately 110-220 nm with a zetapotential of +39.9 mV and polydispersity index of 0.268, suggesting that they were uniformly dispersed nanoparticles. The FTIR and UV-Vis spectroscopy have confirmed the presence of MLX in the prepared CEMNPs. The pharmacokinetics have been studied with three groups of male Wistar rats receiving either of the treatments, viz., 4 mg·kg-1 of MLX and 1 or 4 mg·kg-1 of CEMNPs. Plasma samples were collected until 48 h post administration, and concentrations of MLX were quantified by using reverse (C18) phase HPLC. Non-compartmental analysis was applied to determine pharmacokinetic variables. Upon oral administration, the maximum concentration (Cmax) was reached in 4 h for CEMNPs and 6 h for MLX. The mean area under the plasma MLX concentration-time curve from 'zero' to infinity (AUC0-∞), half-life (t1/2ß), and mean resident time (MRT) of 1 mg·kg-1 of CEMNPs was 1.4-, 2-, and 1.8-fold greater than 4 mg·kg-1 of MLX. The prepared CEMNPs demonstrated quicker absorption and prolonged release along with a significant improvement in the bioavailability of MLX, paving a prospective path for the development of drugs with enhanced bioavailability with less side effects.

Exploring of novel 4-hydroxy-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide derivative as a dual inhibitor of α-glucosidase and α-amylase: Molecular docking, biochemical, enzyme kinetic and in-vivo mouse model study.

Diabetes mellitus (DM) is a metabolic disorder that leads to hyperglycemia due to improper insulin secretion. The study aims to investigate the anti-diabetic potential of benzothiazine derivatives. Molecular docking and Molecular Dynamics simulation study revealed that Compound S6 (4-hydroxy-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide) and S7 (4-Hydroxy-2-methyl-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide) had less conformational changes during MD simulation analysis at 100 ns. Compound S6 and S7 showed potent activity with IC50 values of 5.93 µM, 6.91 µM and 75.17, 29.10 µM for α-glucosidase and α-amylase respectively and competitive type of inhibition was observed during enzyme kinetic study with a low value of Ki and Ki' for α-glucosidase and α-amylase, respectively. S6 has the lowest Ki (0.0736) and Ki' (-0.0982) for α-glucosidase. Furthermore, in vivo studies were carried out to distinguish the effects of the drug on the body. Histology analysis on mice model showed that compound S6 has a low necrosis rate in the liver, kidney, and pancreas compared to S7. Biochemical results of S6 revealed lower sugar level (112 mg/dL), increase insulin secretion (23, 25 µM/L), and low level of cholesterol (80, 85 mg/dL) and creatinine (1.6, 1.4 mg/dL). The results conclude that compound S6 is a new anti-diabetic agent that minimizes hyperglycemia complications.

New Insight into Dearomatization and Decarbonylation of Antitubercular 4H-Benzo[e][1,3]thiazinones: Stable 5H- and 7H-Benzo[e][1,3]thiazines.

8-Nitro-4H-benzo[e][1,3]thiazinones (BTZs) are potent in vitro antimycobacterial agents. New chemical transformations, viz. dearomatization and decarbonylation, of two BTZs and their influence on the compounds' antimycobacterial properties are described. Reactions of 8-nitro-2-(piperidin-1-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one and the clinical drug candidate BTZ043 with the Grignard reagent CH3 MgBr afford the corresponding dearomatized stable 4,5-dimethyl-5H- and 4,7-dimethyl-7H-benzo[e][1,3]thiazines. These methine compounds are structurally characterized by X-ray crystallography for the first time. Reduction of the BTZ carbonyl group, leading to the corresponding markedly non-planar 4H-benzo[e][1,3]thiazine systems, is achieved using the reducing agent (CH3 )2 S ⋅ BH3 . Double methylation with dearomatization and decarbonylation renders the two BTZs studied inactive against Mycobacterium tuberculosis and Mycobacterium smegmatis, as proven by in vitro growth inhibition assays.

Sultam based Carbonic Anhydrase VII inhibitors for the management of neuropathic pain.

We report a series of compounds 1-17 derived from the antiepileptic drug Sulthiame (SLT) from which both the benzenesulfonamide and the sultam moiety were retained. All compounds were tested in vitro for their inhibition activity against the human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) I, II, VII, IX and XII isoforms. Among the series, derivatives 1 and 11 showed great enhancement of both inhibition potency and selectivity towards the hCA VII isoform, when compared to the reference SLT drug. The binding mode of 11 within the hCA VII active site was deciphered by means of X-ray crystallography and revealed the sultam moiety being exposed to the rim of the active site. In vivo experiments on a model of neuropathic pain induced by oxaliplatin clearly showed 11 being an effective pain relieving agent and therefore worth of further exploitation towards the validation of the hCA VII as new target for the management of neuropathies.

Design and discovery of C2-fluoroalkyl iminothiazine dioxides as BACE inhibitors.

This paper describes the structure-activity-relationships of novel fluoroalkyl substituents at the C2 position of iminothiazine dioxide beta secretase inhibitors. Key discoveries include reduced amidine basicity and its effect on Pgp, cell potency, and efficacy in various preclinical in vivo efficacy animal models. Findings from these structure-activity-relationships are discussed.

Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

Dual-high-frequency from single-piezoelectric crystal for ACE degradation by hybrid advanced oxidation UV-sonochemistry process.

This study investigates the combination of two waves emitted from a single-piezoelectric crystal by use of a dual-frequency generator in a sonochemical reactor. The dual-frequency configurations analyzed were the double-modulated fundamental frequency (376-376 kHz), resonant and second harmonic, termed 376D, 376R and 376H respectively. The effect of the phase shift (Φ) and the percentage of modulation between added waves were described by the total acoustic power distribution (Pt) measured inside the sonoreactor. Moreover, optimal angle alignment and modulation between dual-frequency waves for 376D, 376R and 376H cases were selected in order to evaluate the ultrasonic synergy by sonochemical reactivity in production of H2O2, in degradation rate of a model emerging pollutant ACE, and in the TOC and biodegradability evolution in the treated effluent. Phase shift and percentage of modulation had strong effect on the resulted waveform and on the sonochemical efficiency for all, harmonic and non-harmonic, dual-frequency combinations created. In the 376D case, the best reinforcement conditions are founded at 0° and 360°. In the 376H the maximum power distribution presents a 90° period. Shift phase does not determines any cyclic pattern in the total power distribution for the 376R case. The highest H2O2 production rate was observed for the 376H case followed for 376D and 376R configurations with 1.61, 1.12 and 0.58 µM/min by angle alignment in 105, 0 and 110° respectively. The highest initial degradation rate of ACE was observed for the 376D case followed for 376H and 376R with 0.56, 0.42 and 0.33 µM/min at 100% modulation. Reduced mineralization was observed in all dual-frequency configurations (8.54% for 376D and approximately 4.5% for 376R and 3756H modes). Contrasting results are observed regard to biodegradability ratio following the next sequence 376D < 376H≈376R with 0.9, 2.30 and 2.33 respectively. Relevant intensification in hydroxyl radicals production is observed by the UV-US system increasing up three folds the ACE removal and mineralization and two folds higher biodegradability of effluent in particular for 376R and 376H cases at optimal operation condition of dual-frequency signal.

Simultaneous CK2/TNIK/DYRK1 inhibition by 108600 suppresses triple negative breast cancer stem cells and chemotherapy-resistant disease.

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.

Synthesis of New Tricyclic 1,2-Thiazine Derivatives with Anti-Inflammatory Activity.

New, tricyclic compounds containing a sulfonyl moiety in their structure, as potential safer COX inhibitors, were designed and synthesized. New derivatives have three conjugated rings and a sulfonyl group. A third ring, i.e., an oxazine, oxazepine or oxazocin, has been added to the 1,2-benzothiazine skeleton. Their anti-COX-1/COX-2 and cytotoxic effects in vitro on NHDF cells, together with the ability to interact with model membranes and the influence on reactive oxygen species and nitric oxide, were studied. Additionally, a molecular docking study was performed to understand the binding interaction of the compounds with the active site of cyclooxygenases. For the abovementioned biological evaluation of new tricyclic 1,2-benzothiazine derivatives, the following techniques and procedures were employed: the differential scanning calorimetry, the COX colorimetric inhibitor screening assay, the MTT, DCF-DA and Griess assays. All of the compounds studied demonstrated preferential inhibition of COX-2 compared to COX-1. Moreover, all the examined tricyclic 1,2-thiazine derivatives interacted with the phospholipid model membranes. Finally, they neither have cytotoxic potency, nor demonstrate significant influence on the level of reactive oxygen species or nitric oxide. Overall, the tricyclic 1,2-thiazine derivatives are good starting points for future pharmacological tests as a group of new anti-inflammatory agents.

Spectroscopic Studies of Quinobenzothiazine Derivative in Terms of the In Vitro Interaction with Selected Human Plasma Proteins. Part 1.

Plasma proteins play a fundamental role in living organisms. They participate in the transport of endogenous and exogenous substances, especially drugs. 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium salts, have been synthesized as potential anticancer substances used for cancer treatment. Most anticancer substances generate a toxic effect on the human body. In order to check the toxicity and therapeutic dosage of these chemicals, the study of ligand binding to plasma proteins is very relevant. The present work presents the first comparative analysis of the binding of one of the 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium derivatives (Salt1) with human serum albumin (HSA), α-1-acid glycoprotein (AGP) and human gamma globulin (HGG), assessed using fluorescence, UV-Vis and CD spectroscopy. In order to mimic in vivo ligand-protein binding, control normal serum (CNS) was used. Based on the obtained data, the Salt1 binding sites in the tertiary structure of all plasma proteins and control normal serum were identified. Both the association constants (Ka) and the number of binding site classes (n) were calculated using the Klotz method. The strongest complex formed was Salt1-AGPcomplex (Ka = 7.35·104 and 7.86·104 mol·L-1 at excitation wavelengths λex of 275 and 295 nm, respectively). Lower values were obtained for Salt1-HSAcomplex (Ka = 2.45·104 and 2.71·104 mol·L-1) and Salt1-HGGcomplex (Ka = 1.41·104 and 1.33·104 mol·L-1) at excitation wavelengths λex of 275 and 295 nm, respectively, which is a positive phenomenon and contributes to the prolonged action of the drug. Salt1 probably binds to the HSA molecule in Sudlow sites I and II; for the remaining plasma proteins studied, only one binding site was observed. Moreover, using circular dichroism (CD), fluorescence and UV-Vis spectroscopy, no effect on the secondary and tertiary structures of proteins in the absence or presence of Salt1 has been demonstrated. Despite the fact that the conducted studies are basic, from the scientific point of view they are novel and encourage further in vitro and in vivo investigations. As a next part of the study (Part 2), the second new synthetized quinobenzothiazine derivative (Salt2) will be analyzed and published.

Design, Synthesis, and in†vitro Evaluation of Tubulin-Targeting Dibenzothiazines with Antiproliferative Activity as a Novel Heterocycle Building Block.

We prepared a series of free NH and N-substituted dibenzonthiazines with potential anti-tumor activity from N-aryl-benzenesulfonamides. A biological test of synthesized compounds (59 samples) was performed in vitro measuring their antiproliferative activity against a panel of six human solid tumor cell lines and its tubulin inhibitory activity. We identified 6-(phenylsulfonyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 6-tosyl-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide as the best compounds with promising values of activity (overall range of 2-5.4 µM). Herein, we report the dibenzothiazine core as a novel building block with antiproliferative activity, targeting tubulin dynamics.

Synthesis and α-Glucosidase Inhibition Activity of 2-[3-(Benzoyl/4-bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl]-N-arylacetamides: An In Silico and Biochemical Approach.

Diabetes mellitus (DM) is a chronic disorder and has affected a large number of people worldwide. Insufficient insulin production causes an increase in blood glucose level that results in DM. To lower the blood glucose level, various drugs are employed that block the activity of the α-glucosidase enzyme, which is considered responsible for the breakdown of polysaccharides into monosaccharides leading to an increase in the intestinal blood glucose level. We have synthesized novel 2-(3-(benzoyl/4-bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-arylacetamides and have screened them for their in silico and in vitro α-glucosidase inhibition activity. The derivatives 11c, 12a, 12d, 12e, and 12g emerged as potent inhibitors of the α-glucosidase enzyme. These compounds exhibited good docking scores and excellent binding interactions with the selected residues (Asp203, Asp542, Asp327, His600, Arg526) during in silico screening. Similarly, these compounds also showed good in vitro α-glucosidase inhibitions with IC50 values of 30.65, 18.25, 20.76, 35.14, and 24.24 µM, respectively, which were better than the standard drug, acarbose (IC50 = 58.8 µM). Furthermore, a good agreement was observed between in silico and in vitro modes of study.

Ocular microemulsion of brinzolamide: Formulation, physicochemical characterization, and in vitro irritation studies based on EpiOcular™ eye irritation assay.

In recent years, the hydrophobic active substances have led researchers to develop new formulations to enhance bioavailability and dissolution rate; brinzolamide, a lipophilic drug belongs to carbonic anhydrase inhibitors, which cause reduction of intraocular pressure in patients suffering from glaucoma. Currently, the marketed product of brinzolamide is in the form of ocular drops; nonetheless, the conventional drops provide decreased therapeutic efficacy owing to their low bioavailability and pulsed drug release. Thus, the development of novel ocular formulations such as topical microemulsions is of high importance. In this work, the preparation of new microemulsions containing brinzolamide (0.2, 0.5 and 1% w/w) and comprised from isopropyl myristate, tween 80 and span 20 and Cremophor EL was performed. The obtained microemulsions were further characterized for their physicochemical properties. In addition, Fourier Transformed-Infrared spectroscopy was used touate the compatibility of active ingredients and components. In vitro release studies along with kinetic modeling were performed using the dialysis membrane method in simulated tear fluid. Bioadhesion studies were performed using Texture analysis. Finally, in vitro ocular irritation based on EpiOcular™ Eye Irritation Test and cytocompatibility studies was performed to examine any possible harm on ocular cells and predict in vivo safety profile.

Fused benzo[1,3]thiazine-1,2,3-triazole hybrids: Microwave-assisted one-pot synthesis, in vitro antibacterial, antibiofilm, and in silico ADME studies.

In this paper, we report an efficient one-pot three-component reaction sequences comprising Cu(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC) followed by Cu-catalyzed arylation of resulting 1,2,3-triazole in the presence of ionic liquid [Emim]BF4 under microwave conditions involving. The newly synthesized derivatives were screened for in vitro antibacterial inhibition potency against both gram +ve and gram -ve strains. Among the tested compounds, 4f exhibited significant inhibition activity with MIC value 3.12 µg/mL against B. subtilis and S. epidermidis which is two-fold higher than the standard ciprofloxacin (6.25 µg/mL) and also displayed equipotent activity to that of the positive control against S. aureus with MIC value 6.25 µg/mL. Conjugates of the series viz. 3f and 4b against S. aureus, and 4e against E. coli have also displayed promising results with MIC values 6.25 µg/mL which is comparable to the ciprofloxacin. Also we report the anti-biofilm profiles for the potent compounds and it was observed from the results that the active derivatives 4b and 4f were not only potent antibacterial agents but also efficient inhibitors of B. subtilis and S. aureus biofilm growth. Furthermore, in silico-ADME and pharmacokinetic profiles demonstrated the druggability of the hybrids.

Design, Synthesis and Anticancer Activity of New Polycyclic: Imidazole, Thiazine, Oxathiine, Pyrrolo-Quinoxaline and Thienotriazolopyrimidine Derivatives.

In this article, we showed the synthesis of new polycyclic aromatic compounds, such as thienotriazolopyrimidinones, N-(thienotriazolopyrimidine) acetamide, 2-mercapto-thienotriazolo-pyrimidinones, 2-(((thieno-triazolopyrimidine) methyl) thio) thieno-triazolopyrimidines, thieno-pyrimidotriazolo-thiazines, pyrrolo-triazolo-thienopyrimidines, thienopyrimido-triazolopyrrolo-quinoxalines, thienopyrimido-triazolo-pyrrolo-oxathiino-quinoxalinones, 1,4-oxathiino-pyrrolo- triazolothienopyrimidinones, imidazopyrrolotriazolothienopyrimidines and 1,2,4-triazoloimidazo- pyrrolotriazolothienopyrimidindiones, based on the starting material 2,3-diamino-6-benzoyl-5- methylthieno[2,3-d]pyrimidin-4(3H)-one (3). The chemical structures were confirmed using many spectroscopic ways (IR, 1H, 13C, -NMR and MS) and elemental analyses. A series of thiazine, imidazole, pyrrole, thienotriazolopyrimidine derivatives were synthesized and evaluated for their antiproliferative activity against four human cancer cell lines, i.e., CNE2 (nasopharyngeal), KB (oral), MCF-7 (breast) and MGC-803 (gastric) carcinoma cells. The compounds 20, 19, 17, 16 and 11 showed significant cytotoxicity against types of human cancer cell lines.

Design, synthesis and biological activity of N-(amino)piperazine-containing benzothiazinones against Mycobacterium tuberculosis.

A series of novel benzothiazinone derivatives containing a N-(amino)piperazine moiety, based on the structure of WAP-1902 discovered in our lab, were designed and synthesized as new anti-TB agents. Many of the compounds exhibited excellent in vitro activity against both drug-sensitive MTB strain H37Rv and multidrug-resistant clinical isolates (MIC: < 0.016 µg/mL), and good safety index (CC50: >64 µg/mL). Especially compound 1o displayed low hERG cardiac toxicity and acceptable oral pharmacokinetic profiles, indicating its promising potential to be a lead compound for future antitubercular drug discovery.

Sequence sensitivity and pH dependence of maleimide conjugated N-terminal cysteine peptides to thiazine rearrangement.

Thiazine formation during the conjugation of N-terminal cysteine peptides to maleimides is an underreported side reaction in the peptide literature. When the conjugation was performed at neutral and basic pH, we observed the thiazine isomer as a significant by-product. Nuclear magnetic resonance (NMR) spectroscopy confirmed the structure of the six-membered thiazine and ultra-high performance liquid chromatography (UHPLC) combined with tandem mass spectrometry (MS/MS) allowed for facile, unambiguous detection due to a unique thiazine mass fragment. Furthermore, substitution of various amino acids adjacent to the N-terminal cysteine in a tripeptide model system resulted in different rates of thiazine formation, albeit within the same order of magnitude. We also determined that varying the N-substitution of the maleimide affects the thiazine conversion rate. Altogether, our findings suggest that thiazine rearrangement for N-terminal cysteine-maleimide adducts is a general side reaction that is applicable to other peptide or protein systems. Performing the conjugation reaction under acidic conditions or avoiding the use of an N-terminal cysteine with a free amino group prevents the formation of the thiazine impurity.

Identification, separation by spiral high-speed counter-current chromatography, and quantification of 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one, an impurity in the thioindigoid color additive D&C Red No. 30 and its lakes.

An impurity in the color additives D&C Red No. 30 (R30) and D&C Red No. 30 lakes (R30L) was newly identified and characterized as 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one (BTZ), and its extent and level in certified batches of these color additives was determined. BTZ was extracted from the dye with ethanol, resulting in a crude extract enriched to a concentration of over 60%. BTZ was then separated from a portion of the enriched extract by high-speed counter-current chromatography using a spiral-tube assembly column with intermittently pressed tubing of 60 ml capacity. It was the first reported use of such a column to separate a small, moderately hydrophobic compound. The two-phase solvent system was also moderately hydrophobic, consisting of hexane-ethyl acetate-methanol-water (5:2:5:2), and the retention of the organic stationary phase measured after the separation was 83.3%. The separation yielded BTZ of two purity grades, the higher of which (~95.5%) was used as a standard to quantify the impurity in 37 batches of R30 and R30L using an HPLC method developed and validated for that purpose. Analyses revealed a wide range of BTZ levels across batches, <0.05 - 0.84%, and suggested that BTZ contamination could be reduced by appropriate adjustments in the manufacturing process. An explanation of the likely source of BTZ - as a side-reaction product in a particular step of the manufacturing process - was also presented.