2D NMR Studies Research Articles

The oligonucleotides containing N7-regioisomer of guanosine. Influence on thermodynamic properties and structure of RNA duplexes.

During chemical synthesis of the purine riboside, the N7-regioisomer is kinetically formed whereas the N9-regioisomer is a thermodynamically formed product. We have studied the effect of substituting the N9-regioisomer of guanosine with its N7-regioisomer (N7-guanosine, 7G) at a central position of several RNA duplexes. We found that this single substitution by 7G severely diminished their thermodynamic stabilities when 7G paired with C and U, but remarkably, led to a significant amount of stabilization in most of the duplexes when forming mismatches with G and A. The extent of stabilization was observed to be dependent on the sequence and orientation of neighboring base pairs of N7-guanosine. 1D and 2D NMR studies on the duplexes along with extensive molecular dynamics simulations revealed the conformational differences occurring due to substitution of G by 7G and it was observed that the thermodynamic results were largely explainable by considering the formation of stable non-canonical hydrogen bonding interactions, although other interactions such as stacking and electrostatic interactions could also play a role. These observations can have important applications in the design of RNA-based disease diagnostics and therapeutics.

Modulating Helix-Preference of an Axially-Twisted Molecular Scaffold Through Diastereomeric Salt Formation with Tartaric Acid Stereoisomers.

Herein, we designed a chiral, axially-twisted molecular scaffold (ATMS) using pyridine-2,6-dicarboxamide (PDC) unit as pivot, chiral trans-cyclohexanediamine (CHDA) residues as linkers, and pyrene residues as fluorescent reporters. R,R-ATMS exclusively adopted M-helicity and produced differential response in UV-vis, fluorescence, and NMR upon addition of tartaric acid (TA) stereoisomers allowing naked-eye detection and enantiomeric content determination. Circular dichroism (CD) profile of R,R-ATMS underwent unique changes during titration with TA stereoisomers - while loss of CD signal at 345 nm was observed with equimolar D-TA and meso-TA, inversion was seen with equimolar L-TA. Temperature increase weakened these interactions to partially recover the original CD signature of R,R-ATMS. 2D NMR studies also indicated the significant structural changes in R,R-ATMS in the solution state upon addition of L-TA. Single crystal X-ray diffraction (SCXRD) studies on the crystals of the R,R-ATMS⊃D-TA salt revealed the interacting partners stacked in arrays and ATMS molecules stabilized by π-π stacking between its PDC and pyrene residues. Contrastingly, tightly-packed supramolecular cages comprised of four molecules each of R,R-ATMS and L-TA were seen in R,R-ATMS⊃L-TA salt, and the ATMS molecules contorted to achieve CH-π interactions between its pyrene residues. These results may have implications in modulating the helicity of topologically-similar larger biomolecules.

Synthesis and Studies of Meso‐Triaryl Trithiapentaphyrin(2.2.1.1.1)s

AbstractThree examples of meso‐triaryl trithiapentaphyrin(2.2.1.1.1)s 1–3 (Figure 1) that contains three thiophenes and two pyrrole rings connected via seven meso‐carbons were synthesized by condensing newly synthesized benzo‐fused trithiatripyrrane diol and appropriate meso‐aryl dipyrromethane under mild acid catalysed conditions in 5–8 % yields. In these macrocycles, two of the meso‐positions were fused with the benzene rings and the DFT optimized structural analysis revealed that the macrocycles adopt saddled conformation with the two thiophene rings oriented towards outside the macrocyclic core. HR‐MS and detailed 1D & 2D NMR studies were used in deducing the molecular structures of the macrocycles and NMR studies support their nonaromatic nature. The macrocycles exhibit one strong sharp absorption band at ~430 nm and a broad band in the region of 550–850 nm which were further red‐shifted to NIR region in their protonated derivatives. The macrocycles exhibit two reversible oxidations and one reduction and the redox data revealed that the macrocycles were easier to oxidize and were electron‐rich.

Hyaluronic acid conjugates of glycine peptides and L-tryptophan

This work reports about the conjugation of glycine C-terminal ethyl and methyl ester peptides and L-tryptophan methyl ester with sodium hyaluronate in aqueous solutions using the peptide coupling agent DMTMM (or short DMT, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride). Detailed infrared (IR) absorbance and 1H and 13C (2D) NMR studies (heteronuclear multi-bond correlation spectroscopy, HMBC) confirmed covalent and regioselective amide bonds with the D-glucuronate, but also proves the presence of DMT traces in all conjugates. The ethyl ester`s methyl protons on the peptides` C-terminal could be used to quantify the degree of substitution of the peptide on the hyaluronate scaffold by NMR. The ester group also proved stable during conjugation and work-up, and could in some cases be selectively cleaved in water whilst leaving the amide bond intact as shown by potentiometric charge titration, NMR and IR. The conjugates did not influence the capability of human umbilical vein endothelial cells (HUVECs) to reduce MTS (5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) to a formazan dye, which points towards a low cytotoxicity for the obtained products. The conjugation method and products could be tested for tissue engineering gels or drug delivery purposes with alternative, biologically active peptides.

Phenothiazine Embedded Dithiasmaragdyrins.

Two different types of novel phenothiazine-embedded dithiasmaragdyrins containing one phenothiazine ring, two thiophene rings and two pyrrole rings connected via three meso carbons and two direct bonds in the macrocyclic framework were synthesized over the sequence of synthetic steps starting with phenothiazine. Three examples of phenothiazine-embedded dithiasmaragdyrins were synthesized by condensing appropriate phenothiazine-based pentapyrrane with pentafluorobenzaldehyde and two examples of phenothiazine sulfone embedded dithiasmaragdyrins were synthesized by condensing phenothiazine-based diol with appropriate meso-aryl dipyrromethane under mild acid-catalysed conditions. 1D&2D NMR studies revealed that the thiophene rings adopted inverted orientation in phenothiazine sulfone embedded dithiasmaragdyrins whereas in phenothiazine-embedded dithiasmaragdyrins, the thiophene rings were in normal orientation. Both types of macrocycles exhibit nonaromatic absorption features and showed panchochromic absorption features in its neutral and protonated forms. The electrochemical studies indicated that the phenothiazine-embedded dithiasmaragdyrins were more electron-rich compared to phenothiazine sulfone embedded dithiasmaragdyrins. DFT studies revealed that both types of dithiasmaragdyrins exhibit significantly distorted structures and TD-DFT studies support the experimental observations.

In Search of Better Peptide-(Derived from PD-L2)-Based Immune Checkpoint Inhibitors.

Current anti-cancer immune checkpoint therapy relies on antibodies that primarily target the PD-1/PD-L1(-L2) negative regulatory pathway. Although very successful in some cases for certain cancers, these antibodies do not help most patients who, presumably, should benefit from this type of therapy. Therefore, an unmet clinical need for novel, more effective drugs targeting immune checkpoints remains. We have developed a series of high-potency peptide inhibitors interfering with PD-1/PD-L1(-L2) protein-protein interaction. Our best peptide inhibitors are 12 and 14 amino acids long and show sub-micromolar IC50 inhibitory activity in the in vitro assay. The positioning of the peptides within the PD-1 binding site is explored by extensive modeling. It is further supported by 2D NMR studies of PD-1/peptide complexes. These results reflect substantial progress in the development of immune checkpoint inhibitors using peptidomimetics.

Crystal structure of the photosensory module from a PAS-less cyanobacterial phytochrome as Pr shows a mix of dark-adapted and photoactivated features

Phytochromes (Phys) are a diverse collection of photoreceptors that regulate numerous physiological and developmental processes in microorganisms and plants through photointerconversion between red-light absorbing Pr and far-red light-absorbing Pfr states. Light is detected by an N-terminal photosensing module (PSM) sequentially comprised of Period/ARNT/Sim (PAS), cGMP-phosphodiesterase/adenylyl cyclase/FhlA (GAF), and Phy-specific (PHY) domains, with the bilin chromophore covalently-bound within the GAF domain. Phys sense light via the Pr/Pfr ratio measured by light-induced rotation of the bilin D-pyrrole ring that triggers conformational changes within the PSM, which for microbial Phys reaches into an output region. A key step is a β-stranded to α-helical reconfiguration of a hairpin loop extending from the PHY domain to contact the GAF domain. Besides canonical Phys, cyanobacteria express several variants, including a PAS-less subfamily that harbors just the GAF and PHY domains for light detection. Prior 2D-NMR studies of a model PAS-less Phy from Synechococcus_sp._JA-2-3B'a(2-13) (SyB-Cph1) proposed a unique photoconversion mechanism involving an A-pyrrole ring rotation, while magic-angle-spinning NMR probing the chromophore proposed the prototypic D-ring flip. To help solve this conundrum, we determined the crystallographic structure of the GAF-PHY region from SyB-Cph1 as Pr. Surprisingly, this structure differs from canonical Phys by having a Pr ZZZsyn,syn,anti bilin configuration but shifted to the activated position in the binding pocket with consequent folding of the hairpin loop to α-helical, an architecture common for Pfr. Collectively, the PSM of SyB-Cph1 as Pr displayed a mix of dark-adapted and photoactivated features whose co-planar A-C pyrrole rings supports a D-ring flip mechanism.

Synthesis and characterization of a unique fused spiro-dihydropyran based chemodosimeter for selective visual and fluorescent detection of Cu2+ ion in aqueous medium by “turn-on” mode

A realistically newly designed Cu2+ ion specific fluorescent chemodosimeter has been synthesized from α,α'-(E,E)-bis(benzylidene)cyclohexanone having a fused spiro-dihydropyran moiety characterized by 2D NMR and X-Ray Crystallographic study. It also demonstrates an irreversible, instant direct visualisation by unaided eye chemosensing property in aqueous buffer (pH 7.4) with a colour change from yellow to orange upto 0.3 × 10−6 M.

N-9 methylated caffeine: An alternate potentially active pharmaceutical ingredient to caffeine and its complexation with β-CD

Caffeine is the most consumable bitter “energy-boosting” natural xanthine alkaloid that works on the central nervous system (CNS) and a good antagonist to adenosine receptors. Its partial aqueous solubility, however limits it to be used efficiently in novel drug formulations. To this endeavour, we have methylated the N-9 position of caffeine. The N-9 methylated caffeine thus formed is highly water soluble and showed attractive drug likeness and ADMET properties. It also docked at the identical orthosteric pocket of the A2A receptors showing H-bond interaction with ASN A:253 residues. It exhibited limited self-aggregation propensity in molecular dynamics simulation studies as against to natural caffeine. We propose this N-9 methylated caffeine as an alternate active pharmaceutical ingredient (API) of natural caffeine. Further, we have modified the texture of the caffeine API to powder form by complexing it with β-CD. A 1:1 inclusion complex (IC) was formed that was characterised by modern spectral techniques like, UV–vis, FT-IR, 1D and 2D NMR, WAXRD and SEM studies. The IC has enhanced thermal stability and is practically nontoxic as revealed in DNA interaction study. All the data indicated the applicability of β-CD as potential vehicle for caffeine-API which may open up new horizons to developing semi-synthetic alternative drug forms.

Alternative Approach to Access 5-Hydroxy-1H-pyrrol-2-(5H)-ones from Base-Induced Tandem Intramolecular Cyclization of Sulfur Ylide with Ketones and 1,3-Hydroxy Rearrangement.

An easily adaptable protocol for the preparation of 5-hydroxy-1H-pyrrol-2(5H)-ones from readily available starting materials has been reported. The reaction of sulfur ylides with carbonyl compounds is a common approach to synthesizing epoxides. Alternatively, we have developed a method with mild reaction conditions wherein sulfur ylide underwent an intramolecular cyclization with a ketonic carbonyl group in a highly efficient way and was followed by 1,3-hydroxy rearrangement to produce 5-hydroxy-1H-pyrrol-2(5H)-ones in excellent yields. The present method offers a straightforward approach to synthesize 5-hydroxy-1H-pyrrol-2(5H)-ones from sulfur ylides without the aid of transition metal in one-pot operation, which involves sequential cyclization and rearrangement reaction. The formation of 5-hydroxy-1H-pyrrol-2(5H)-ones is supported by different spectroscopic techniques, including X-ray crystallographic data and 2D NMR studies (COSY, HSQC, HMBC, and DEPT).

Synthesis, Spectral, Redox and Theoretical Studies of Stable Nonaromatic Dicarba Dithia Hexaphyrin(2.0.1.1.1.0)s with Two Inverted Thiophenes.

A series of dicarba dithia hexaphyrin(2.0.1.1.1.0)s containing two p-phenylene rings, two thiophene rings, and two pyrrole rings connected via five meso carbons were synthesized by condensing the key precursor, hexapyrrane, which was prepared over a sequence of steps, with the appropriate aromatic aldehyde under acid catalytic conditions followed by alumina chromatographic purification. Detailed one-dimensional (1D) and two-dimensional (2D) NMR studies revealed that the two thiophene rings were inverted and facing outward from the macrocyclic core. Interestingly, one of the inverted thiophene rings adopts a normal orientation in the protonated derivatives of macrocycles generated by addition of trifluoroacetic acid to the appropriate macrocyclic solution. The spectroscopic studies support the non-aromatic nature of macrocycles, and the macrocycles exhibit a distinct sharp band at ∼425 nm along with a broad band in the range of 550-1000 nm, which experienced a red shift with a clear color change in the protonated derivatives. The redox studies showed lower oxidation potentials, indicating their electron-rich nature. The density functional theoretical (DFT) studies showed that the hexaphyrins adopt oval-shaped structures, and time-dependent-DFT (TD-DFT) studies parallelly matched the experimental observations of macrocycles.

Gwal Pahari Acid: Isolation, Characterization of a new Soil Based Plant Growth Promoting Humic Acid

There are many “Save the Soil” movements highlighting the degradation of the soil due to excessive use of fertilizers, ground water contamination and environmental pollution. An alternative strategy is the use of humic acids from Organic Matter (OM) in soils. Humic acids help grow better vegetables, quality fruits like (peas, potatoes, tomatoes, pomegranates, mangoes), cereals, and pulses. These create supramolecular self– assemblies capable of retaining water and not allowing loss of minerals and ions. Humic acids isolated from soil, lignite and city solid waste are emerging as attractive sources for developing value added products from them. There is much interest in exploiting the commercial aspects in the energy sector of the economy as these new technologies could help in cleaning the environment as well.
 In this paper, the isolation of a new humic acid from the soil of Gwal Pahari, Gurgaon, Haryana, India is described. This water soluble, Ninhydrin positive Gwal Pahari Acid (GPA) contains thirteen chiral centers and contains both partly rigid and dynamic systems capable of exhibiting pseudo rotation. The proposed structure of this new humic acid is based on spectroscopic studies (e.g. FT-IR, UV-visible spectroscopy), detailed mass spectrometry, and very challenging 1H- NMR and 2D-NMR studies. Ion leakage studies on Arabidopsis thaliana have shown that the new compound provides protection to the plant, and greenhouse studies demonstrate that Gwal Pahari Acid brings about substantial growth in the tomato plant.

Identification and enrichment of a UV-induced degradant of Anagrelide drug substance

Anagrelide (ANG) is a widely used drug for the treatment of essential thrombocytosis and myeloproliferative neoplasms. Recently, a new oxidative degradant was identified when the drug product capsule underwent stress testing. Full structural characterization of this previously unidentified degradant was conducted. Preliminary LC-MS analysis indicated the targeted degradant as a mono-oxygenated product of ANG. For the purpose of facile isolation and purification, various forced degradation conditions were screened to enrich the desired degradant, among which, pyridinium chlorochromate (PCC)-treatment effectively afforded a yield of 55 % unknown degradant. Following isolation by prep-HPLC, 1D and 2D NMR studies and HRMS characterization assigned the products as a pair of 5-hydroxy-Anagrelide (5-OH-ANG) enantiomers. A plausible mechanism of formation is proposed.

Insights into the Absorption of Hydrocarbon Gases in Phosphorus-Containing Ionic Liquids.

The solubility of ethane, ethylene, propane, and propylene was measured in two phosphorus-containing ionic liquids, trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate, [P6,6,6,14][DiOP], and 1-butyl-3-methylimidazolium dimethylphosphate, [C4C1Im][DMP], using an isochoric saturation method. The ionic liquid [C4C1Im][DMP] absorbed between 1 and 20 molecules of gas per 1000 ion pairs, at 313 K and 0.1 MPa, while [P6,6,6,14][DiOP] absorbed up to 169 molecules of propane per 1000 ion pairs under the same conditions. [C4C1Im][DMP] had a higher capacity to absorb olefins than paraffins, while the opposite was true for [P6,6,6,14][DiOP], with the former being slightly more selective than the later. From the analysis of the thermodynamic properties of solvation, we concluded that in both ionic liquids and for all of the studied gases the solvation is ruled by the entropy, even if its contribution is unfavorable. These results, together with density measurements, 2D NMR studies, and self-diffusion coefficients suggest that the gases' solubility is ruled mostly by nonspecific interactions with the ionic liquids and that the looser ion packing in [P6,6,6,14][DiOP] makes it easier to accommodate the gases compared to [C4C1Im][DMP].

Synthesis of Acidiphilamide A–C: Secondary Metabolites from the Genus Streptacidiphilus

AbstractWe describe the efficient total syntheses of naturally occurring tripeptides acidiphilamides A–C and epi-acidiphilamides A–C, which were prepared from commercially available l-phenyl alanine using hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) as peptide coupling reagent. The structures of the natural acidiphilamides A, B and C were characterized by NMR, MS and SOR data, which match those of natural products, whereas the structures of epi-acidiphilamides A, B and C were confirmed by 2D NMR studies.

Design, synthesis, spectroscopic characterization, single crystal X-ray analysis, in vitro α-amylase inhibition assay, DPPH free radical evaluation and computational studies of naphtho[2,3-d]imidazole-4,9-dione appended 1,2,3-triazoles

In our quest to design and develop N/O-containing inhibitors of α-amylase, we have tried to synergize the inhibitory action of 1,4-naphthoquinone, imidazole and 1,2,3-triazole motifs by incorporating these structures into a single matrix. For this, a series of novel naphtho[2,3-d]imidazole-4,9-dione appended 1,2,3-triazoles is synthesized by a sequential approach involving [3 + 2] cycloaddition of 2-aryl-1-(prop-2-yn-1-yl)-1H-naphtho[2,3-d]imidazole-4,9-diones with substituted azides. The chemical structures of all the compounds are established with the help of 1D-NMR, 2D-NMR, IR, mass and X-ray studies. The developed molecular hybrids are screened for their inhibitory action on the α-amylase enzyme using the reference drug, acarbose. Different substituents present on the attached aryl part of the target compounds show amazing variations in inhibitory action against the α-amylase enzyme. Based on the type of substituents and their respective positions, it is observed that compounds containing –OCH3 and –NO2 groups show more inhibition potential than others. All the tested derivatives display α-amylase inhibitory activity with IC50 values in the range of 17.83 ± 0.14 to 26.00 ± 0.17 μg/mL. Compound 2-(2,3,4-trimethoxyphenyl)-1-{[1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl]methyl}-1H-naphtho[2,3-d]imidazole-4,9-dione (10y) show maximum inhibition of amylase activity with IC50 value 17.83 ± 0.14 μg/mL as compared to reference drug acarbose (18.81 ± 0.05 μg/mL). A molecular docking study of the most active derivative (10y) is performed with A. oryzae α-amylase (PDB ID: 7TAA) and it unveils favourable binding interactions within the active site of the receptor molecule. The dynamic studies reveal that the receptor-ligand complex is stable as the RMSD of less than 2 is observed in 100 ns molecular dynamic simulation. Also, the designed derivatives are assayed for their DPPH free radical scavenging ability and all of them exhibit comparable radical scavenging activity with the standard, BHT. Further, to assess their drug-likeness properties, ADME properties are also evaluated and all of them demonstrate worthy in silico ADME results.

Briavioid D, a rare 7β,8β-epoxybriarane from Briareum violaceum

A rare 7β,8β-epoxybriarane, briavioid D (1), has been isolated from a cultured octocoral Briareum violaceum. The structure of 1 was deduced by extensive 2D NMR studies. Single-crystal X-ray diffraction analysis was performed to identify the structure of 1 and this compound was found to reduce the release of iNOS to 85.77 %, at a concentration of 10 μM.

Evaluation of Radical Scavenging and Metal Chelating Potential of Cameroonian Propolis and Isolation of Some Chemical Constituents

Five extracts were prepared from different propolis samples and denoted PR1-PR5. They were subjected to two complementary antioxidant assays, DPPH radical scavenging and metal chelation. The propolis extracts PR4 and PR5 showed good radical scavenging power with IC50 20.45 ± 1.65 μg/mL and 17.23 ± 1.40 μg/mL respectively as well as appreciable chelating power of IC50 7.52± 0.61 µg/mL and 3.47± 0.61 µg/mL for PR4 and PR5 respectively. The PR1 extract showed a moderate radical scavenging activity with an IC50 = 230.08 ± 18.60 μg/mL and exhibited a high chelating power with an IC50 of 17.40 ± 1.41 μg /mL. This potent antioxidant effects of the propolis indicates its possible application in food science. Some compounds were isolated and characterized by 1D-, 2D-NMR studies as 7-O-ß–glucopyranosylapigenine, fridelin, lupeol, β-sitosterol, 3'-hydroxypentyltetratretracontanoate, 3'-hydroxybutyltetracontanoate, propylhexatetracontanoate, methyl-tetratretraconta-noate, 1′-O-eicosanylglycerol, and 2',3'-dihydroxypropyltetraeicosanoate which was further acetylated through hemi-synthesis to yield 2',3'-diacetylpropyltetraeicosanoate.

A Homogalacturonan from Peel of Winter Jujube (Zizyphus jujuba Mill. cv. Dongzao): Characterization and Protective Effects against CCl4-Induced Liver Injury.

A homogalacturonan pectin (HG, designated as WJP-F80) was extracted from the peel of winter jujube (Zizyphus jujuba Mill. Cv. Dongzao) and separated via ethanol-graded precipitation. The structural and conformational features were elucidated through HPAEC-PAD, GC-MS, 2D NMR, and HPSEC-MALLS studies. In vivo assessments were carried out to evaluate the hepatoprotective effects of WJP-F80 against CCl4-induced injury of mice. Results showed that WJP-F80 was a linear 1,4-α-galacturonan with partially methyl-esterified at O-6 of GalpA and occasionally acetylation. The Mw of WJP-F80 was determined as 45.3 kDa, the polydispersity was calculated as 1.56, and the Rg was measured as 22.7 nm in 0.1 M NaNO3. The conformational analysis revealed that WJP-F80 exhibited as rigid stiff chain in low Mw range, while aggregation by self-assembly of HG chains lead to high Mw and random coil conformation. In vivo studies indicated that WJP-F80 can protect the livers of mice from acute injury induced via CCl4 by decreasing the serum biochemical markers of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to normal levels. This work provides a theoretical basis for the value-added deep processing of winter jujube.

Characterization of potential degradation products of brexpiprazole by liquid chromatography/quadrupole-time-of-flight mass spectrometry and nuclear magnetic resonance, and prediction of their physicochemical properties by ADMET Predictor™.

Brexpiprazole (BRZ) was subjected to hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress degradation in solutions prepared in a mixture of acetonitrile-water (70:30 v/v). The oxidative study was additionally done in methanol-buffer mixture at pH3, 7 and 11. Also, compatibility of the drug with selectedexcipients was investigated in the solid state. Additionally, physicochemical and ADMET properties of BRZ and its degradation products (DPs) were predicted using ADMET Predictor™ software. It provides the conditions for quality control of BRZ and its derivatives during manufacturing, processing and storage conditions. The formed DPs were separated from the drug and among themselves on a C-18 column utilizing mobile phase composed of methanol and ammonium formate buffer (10mM, pH4.0), which was run in a gradient mode. Characterization of DPs was carried out by first establishing the mass fragmentation pathway of the drug based on its liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/Q-TOF-MS) data, followed by LC/Q-TOF-MS studies of DPs. Three DPs were isolated and, along with the drug, they were subjected to 1D (1 H, 13 C and DEPT-135) and 2D (COSY and HSQC) NMR studies for confirmation of their structures. BRZ was observed to be susceptible to hydrolytic (neutral, acid and alkali), photolytic and oxidative degradation conditions; it was stable on thermal exposure. A total of 12 DPs (BRZ-1 to BRZ-12) were formed in solution state. Mechanisms of BRZ degradation were postulated. The extent of degradation of BRZ in different stress conditions highlights that stability of BRZ in drug formulations can be improved (i) by using excipients that can impart a low-pH microenvironment, (ii) by addition of antioxidants and (iii) through protection from light.