Fluorescence Sensing of Caffeine in Tea Beverages with 3,5-diaminobenzoic Acid.

A rapid, selective and sensitive method for the detection of caffeine in tea infusion and tea beverages are proposed by using 3,5-diaminobenzoic acid as a fluorescent probe. The 3,5-diaminobenzoic acid emits strong fluorescence around 410 nm under the excitation of light at 280 nm. Both the molecular electrostatic potential analysis and fluorescent lifetime measurement proved that the existence of caffeine can quench the fluorescence of 3,5-diaminobenzoic acid. Under the optimal experimental parameters, the 3,5-diaminobenzoic acid was used as a fluorescent probe to detect the caffeine aqueous solution. There exists a good linear relationship between the fluorescence quenching of the fluorescent probe and the concentration of caffeine in the range of 0.1-100 µM, with recovery within 96.0 to 106.2%, while the limit of detection of caffeine is 0.03 µM. This method shows a high selectivity for caffeine. The caffeine content in different tea infusions and tea beverages has been determined and compared with the results from HPLC measurement.

Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.

Orthogonal Cysteine Protection Enables Homogeneous Multi-Drug Antibody-Drug Conjugates.

A strategy for the preparation of homogeneous antibody-drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site-specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug-linkers that have differing physiochemical properties and exert complementary anti-cancer activities. Dual-auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual-drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site-specific protein modification.

Crystal structures of the pyrazinamide-p-aminobenzoic acid (1/1) cocrystal and the transamidation reaction product 4-(pyrazine-2-carboxamido)benzoic acid in the molten state.

The synthesis of pharmaceutical cocrystals is a strategy to enhance the performance of active pharmaceutical ingredients (APIs) without affecting their therapeutic efficiency. The 1:1 pharmaceutical cocrystal of the antituberculosis drug pyrazinamide (PZA) and the cocrystal former p-aminobenzoic acid (p-ABA), C7H7NO2·C5H5N3O, (1), was synthesized successfully and characterized by relevant solid-state characterization methods. The cocrystal crystallizes in the monoclinic space group P21/n containing one molecule of each component. Both molecules associate via intermolecular O-H···O and N-H···O hydrogen bonds [O···O = 2.6102 (15) Šand O-H···O = 168.3 (19)°; N···O = 2.9259 (18) Šand N-H···O = 167.7 (16)°] to generate a dimeric acid-amide synthon. Neighbouring dimers are linked centrosymmetrically through N-H···O interactions [N···O = 3.1201 (18) Šand N-H···O = 136.9 (14)°] to form a tetrameric assembly supplemented by C-H···N interactions [C···N = 3.5277 (19) Šand C-H···N = 147°]. Linking of these tetrameric assemblies through N-H···O [N···O = 3.3026 (19) Šand N-H···O = 143.1 (17)°], N-H···N [N···N = 3.221 (2) Šand N-H···N = 177.9 (17)°] and C-H···O [C···O = 3.5354 (18) Šand C-H···O = 152°] interactions creates the two-dimensional packing. Recrystallization of the cocrystals from the molten state revealed the formation of 4-(pyrazine-2-carboxamido)benzoic acid, C12H9N3O3, (2), through a transamidation reaction between PZA and p-ABA. Carboxamide (2) crystallizes in the triclinic space group P1̅ with one molecule in the asymmetric unit. Molecules of (2) form a centrosymmetric dimeric homosynthon through an acid-acid O-H···O hydrogen bond [O···O = 2.666 (3) Šand O-H···O = 178 (4)°]. Neighbouring assemblies are connected centrosymmetrically via a C-H···N interaction [C···N = 3.365 (3) Šand C-H···N = 142°] engaging the pyrazine groups to generate a linear chain. Adjacent chains are connected loosely via C-H···O interactions [C···O = 3.212 (3) Šand C-H···O = 149°] to generate a two-dimensional sheet structure. Closely associated two-dimensional sheets in both compounds are stacked via aromatic π-stacking interactions engaging the pyrazine and benzene rings to create a three-dimensional multi-stack structure.

Synthesis and cytotoxicity of some D-mannose click conjugates with aminobenzoic acid derivatives.

Two sets of new conjugates obtained from d-mannose derivatives and o-, m-, and p-substituted benzoic acid esters interconnected through a triazole ring were synthesized by Cu(I) catalyzed azide-alkyne cycloaddition. All synthesized compounds were tested for their in vitro cytotoxic activity against seven cancer cell lines with/without multidrug resistance phenotype as well as non-tumor MRC-5 and BJ fibroblasts. Butyl ester of 4-aminobenzoic acid 6c showed the highest activity among all tested compounds, however, it was active only against K562 myeloid leukemia cells. N-Glycosyltriazole conjugates, both acetylated and nonacetylated at mannose moiety, were almost completely inactive. In contrast, some of the acetylated O-glycosyl conjugates showed cytotoxic activity which was cell line dependent and strongly affected by position of benzoic acid substitution as well as a length of its ester alkyl chain; the most potent compound was acetylated mannoside conjugated with octyl ester of m-substituted benzoic acid. However, deacetylation resulting in hydrophilicity increase of the glycosides almost completely abolished their cytotoxic potency.

[Synthesis of 2-hydroxyl-5-butyramidobenzoic acid and its effect on acetic acid-induced colitis in rats].

OBJECTIVE: To study the method for synthesis of 2-hydroxyl-5- butyramidobenzoic acid and test its effect on acetic acid-induced colitis in rats. METHODS: 2-hydroxyl-5-butyramidobenzoic acid was synthesized from 5-aminosalicylic acid and butyric acid by amidation, esterification and hydrolization. The effect of 2-hydroxyl-5-butyramidobenzoic acid on acetic acid enema-induced colitis in rats was investigated. RESULTS: The structure of 2-hydroxyl-5-butyramidobenzoic acid was identified by IR and 1H-NMR. After treatment with acetic acid, the colon mucosal damage index (CMDI), fecal occult blood (OB) test, and activity of myelperoxidase (MPO) increased significantly in the rats as compared to the control levels. 2-hydroxyl-5- butyramidobenzoic acid obviously reduced the CMDI and OB, and reduced the level of MPO in the rats with colitis. CONCLUSION: The synthesis of 2-hydroxyl-5-butyramidobenzoic acid requires only mild conditions with simple procedures, and the synthesized 2-hydroxyl-5-butyramidobenzoic acid shows obvious therapeutic effects on mucosal damage of in rats with acetic acid-induced colitis.

Peroxidase-catalyzed oxidative coupling of paraphenylenediamine with 3-dimethylaminobenzoic acid: application in crude plant extracts.

This paper presents a novel spectrophotometric method to measure peroxidase activity using paraphenylenediamine dihydrochloride (PPDD) and 3-dimethylaminobenzoic acid (DMAB). The PPDD traps free radicals and becomes oxidized to electrophillic 1,4-diimine, which couples with DMAB to give an intense green-colored chromogenic species with maximum absorbance at 710 nm. This assay was adopted for the quantification of hydrogen peroxide between 5 and 45 microM. From the kinetic data, a two-substrate ping-pong mechanism of peroxidase was established. The catalytic efficiency and catalytic constant (k(cat)) of the proposed assay were 0.54 x 10(6) M(-1) min(-1) and 0.0436 x 10(3) min(-1), respectively. As a simple, rapid, precise, and sensitive technique, PPDD-DMAB stands as a potential replacement for the traditional guaiacol method. Application of this method in plant extracts opens its relevance in the field of biochemical analysis.

Synthesis and anti-platelet evaluation of 2-benzoylaminobenzoate analogs.

Fifty-two 2-benzoylaminobenzoate analogs were synthesized and subjected to anti-platelet aggregation assay using arachidonic acid (AA), collagen (Col), thrombin (Thr), and U46619 as inducers. The results revealed that most of 2-benzoylaminobenzoic acid derivatives showed a selectively inhibitory effect on AA-induced platelet aggregation. As a result of the 2-benzoylaminobenzoic acid derivatives (18, 44, and 46), there were no inhibitory effects on platelet aggregation induced by U46619, but these elicited an inhibitory effect on thromboxane B(2) formation at 1.0microM. These 2-benzoylaminobenzoate analogs were therefore proposed as cyclooxygenase inhibitors.

Screening of telomerase inhibitors.

Shortening of telomeres prevents cells from uncontrolled proliferation. Progressive telomere shortening occurs at each cell division until a critical telomeric length is reached. Telomerase expression is switched off after embryonic differentiation in most normal cells, but it is expressed in a very high percentage of tumors of different origin. Thus, telomerase is regarded as the best tumor marker and a promising novel molecular target for cancer treatment. Therefore, different strategies to inhibit telomerase have been developed. However, systematic screening of telomerase inhibitors has not been performed to compare their therapeutic potential. We propose a suitable strategy for estimation of the therapeutic potential of telomerase inhibitors, which is based on a systematic screening of different inhibitors in the same cell system. From the long list of compounds discussed in the literature, we have selected four telomerase inhibitors of different structure and mode of action: BRACO19 (G-quadruplex-interactive compound), BIBR1532 (non-nucleosidic reverse transcriptase inhibitor), 2'-O-methyl RNA, and peptide nucleic acids (PNAs; hTR antisense oligonucleotides). To determine minimal effective concentrations for telomerase inhibition, telomerase activity was measured using the cell-free telomerase repeat amplification protocol (TRAP) assay. We also tested inhibitors in long-term cell-culture experiments by exposing A-549 cells to non-cytotoxic concentrations of inhibitors for a period of 99 days. Subsequently, telomerase activity of A-549 cells was investigated using the TRAP assay, and telomere length of samples was assessed by telomere restriction fragment (TRF) Southern blot analysis.

3,8-diazabicyclo--[3.2.1]-octane derivatives as analogues of ambasilide, a Class III antiarrhythmic agent.

Ambasilide, a representative of Class III antiarrhythmics, was reported to prolong the cardiac action potential duration in the dog, with little or no effect on Ca and Na currents. We synthesised a series of ambasilide analogues, having the 3,8-diazabicyclo-[3.2.1]-octane moiety instead of the 3,7-diazabicyclo-[3.3.1]-nonane present in ambasilide. The compounds were tested both in vitro extracellular electrophysiological assays and by the conventional microelectrode technique. Most of them lengthened the effective refractory period (ERP) with no change or slight increase on the impulse conduction time (ICT). Similarly some of the tested compounds lengthened the action potential duration (APD), a typical Class III feature, without exerting any significant effect on the maximal rate of depolarization, therefore apparently lacking Class I antiarrhythmic activity.

Colorimetric method for the determination of lipoxygenase activity.

A colorimetic assay for lipoxygenase activity has been developed. The assay is based on the detection of the lipoxygenase reaction product, linoleic acid hydroperoxide, by the oxidative coupling of 3-methyl-2-benzothiazolinone (MBTH) with 3-(dimethylamino)benzoic acid (DMAB) in a hemoglobin-catalyzed reaction. This test reaction is rapid and sensitive, and it offers advantages over other methods for detecting lipoxygenase activity. The assay is capable of detecting activity in a number of crude vegetable homogenates and should be particularly useful where a rapid visual determination of lipoxygenase activity is desired.

N-acetylation and N-formylation of m-aminobenzoic acid by cell suspension cultures of Solanum laciniatum.

Two new biotransformation products, N-acetyl-m-aminobenzoic acid and N-formyl-m-aminobenzoic acid were isolated from cell suspension cultures of Solanum laciniatum following administration of m-aminobenzoic acid, and their structures were elucidated using one- and two-dimensional 1H- and 13C-NMR data.