ROS Inhibitory Activity and Cytotoxicity Evaluation of Benzoyl, Acetyl, Alkyl Ester, and Sulfonate Ester Substituted Coumarin Derivatives.

BACKGROUND: A number of non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, indomethacin, ibuprofen, flufenamic acid, and phenylbutazone are being clinically used to treat inflammatory disorders. These NSAIDs are associated with serious side effects such as gastric ulceration, nephrotoxicity, and bleeding. Therefore, the identification of potent and safe therapy for inflammatory disorders is still of great interest to the medicinal chemist. METHODS: A series of varyingly substituted benzoyl, acetyl, alkyl ester, and sulfonate ester substituted coumarins 1-64 were screened for the inhibition of ROS, generated from zymosan activated whole blood phagocytes, using luminol-enhanced chemiluminescence technique. RESULTS: Among all tested compounds, 8 (IC50 = 65.0 ± 3.1 µM), 24 (IC50 = 41.8 ± 1.5 µM), 26 (IC50 = 10.6 ± 2.8 µM), 28 (IC50 = 20.9 ± 1.5 µM), and 41 (IC50 = 4.6 ± 0.3 µM) showed good anti- inflammatory potential as compared to standard antiinflammatory drug ibuprofen (IC50 = 54.3 ± 1.9 µM). Specifically, compounds 24, 26, 28, and 41 showed superior activity than standard antiinflammatory drug. Furthermore, compounds 12 (IC50 = 219.0 ± 1.4 µM), 14 (IC50 = 216.5 ± 6.2 µM), 16 (IC50 = 187.4 ± 2.2 µM), and 20 (IC50 = 196.2 ± 2.0 µM) showed moderate ROS inhibitory activity. Limited SAR study revealed that the hydroxy-substituted compound showed better ROS inhibition potential in case of 3-benzoyl and 3-ethylester coumarin derivatives. Whereas, chloro substitution was found to be important in case of 3-acetyl coumarin derivatives. Similarly, in case of sulfonate ester, chloro, and nitro groups especially at positions -4 and -3 of ring "R" played vital role in ROS inhibition. Furthermore, cytotoxicity of all active compounds was also checked on NIH-3T3 cell line. Compounds 12, 14, and 20 were found to be non-cytotoxic. Whereas, 8, 16, 24, 26, 28, and 41 were found to be very weak cytotoxic as compared to standard cycloheximide (IC50 = 0.13 ± 0.02 µM). CONCLUSION: Identified ROS inhibitors offer the possibility of additional modifications that could give rise to lead structures for further research in order to obtain more potent, and safer antiinflammatory agent.

Benzophenone Esters and Sulfonates: Synthesis and their Potential as Antiinflammatory Agents.

BACKGROUND: Inflammation is a biological rejoinder of vascular tissues against destructive agents e.g. irritants, damaged cell or pathogens. During inflammation, respiratory burst occurs by activated phagocytes which help to destroy invading pathogens. Phagocytic cells such as neutrophils and macrophages are one of the major sources of reactive oxygen species (ROS) and nitric oxide (NO). Normally, the redox environment is maintained by various antioxidant defense systems, however, these reactive oxygen species may be destructive and can lead to various pathological conditions. METHODS: Benzophenone esters and sulfonates (1-18) were synthesized through one pot synthesis by reacting 4-hydroxy benzophenone either different benzoyl chloride or sulfonyl chloride. These synthetic compounds were evaluated for their in vitro immunosuppressive potential on two parameters of innate immune response including inhibition of intracellular reactive oxygen species (ROS) and nitric oxide (NO). ROS were induced in polymorphonuclear leukocytes (PMNs) isolated from human whole blood by serum opsonized zymosan stimulation, whereas NO were produced in J774.2 cells by lipopolysachharides (LPS) stimulation. Moreover, cytotoxicity of compounds was also determined using NIH-3T3 fibroblast cells (ATCC, Manassas, USA) was evaluated by using the standard MTT colorimetric assay. RESULTS: All compounds inhibited the production of ROS at various extent among which compounds 2, 5, 6, 8, 10, 13 and 16 were found to be the potent inhibitors of ROS with IC50 values ranging between (1.0 - 2.2 µg/mL) as compared to ibuprofen (IC50 = 2.5 ± 0.6 µg/mL) as the standard drug. Compounds 2, 7, 11, 13, 14 and 18 showed good inhibition of NO production with % inhibition values ranging between (63.6% - 76.7%) at concentration of 25 µg/mL as compared to NG-monomethyl-Larginine (L-NMMA 65.6 ± 1.1 µg/mL) as the standard. All other derivatives showed moderate to low level of inhibition on both tested parameters. Cytotoxicity activity also showed nontoxicity of synthetic compounds. Structures of all the synthetic compounds were confirmed through 1H-NMR, 13C-NMR, EI-MS and HREI-MS spectroscopic techniques. CONCLUSION: Compounds 2 and 13 were found to be good dual antiinflammatory (ROS and NO) agent. However, compounds 5, 6, 8, 10 and 16 were found to be selectively active for ROS inhibitory studies. Compounds 7, 11, 14 and 18 were discriminatory active at NO inhibition assay. These initial findings of antiinflammatory activity concluded that these compounds might have the potential to develop a novel non-steroidal antiinflammatory drugs (NSAIDs), non-acidic antiinflammatory agent. Most active compounds 2, 5-8, 10, 13, 14 and 16 showed nontoxicity of synthetic compounds.

Immunomodulatory activity and chemical characterization of fixed oils obtained from different parts of Oxytropis glabra DC.

Oxytropis glabra DC. is a plant with enormous therapeutic vitality. In the present study a comparison of lipophilic profiling of different parts of O. glabra has been carried out by using gas chromatography-mass spectrometry. A total of 32 compounds have been identified from this plant, amongst which 31 have been identified for the first time. These compounds have been further confirmed from their Van den Dool and Kratz (I) Indices. Out of these 32 compounds, 18 have been identified from flower (80.94%), 15 from fruit (85.36%), 11 from leaves (66.35%) and 11 from root (45.96%). The major class of metabolite identified from different parts is fatty acid. Hydrocarbons have also been detected in flower and fruit but not in root and leaves. The extracts were screened for their immunomodulatory activity on whole blood cells. The root oil was found to be moderately active (IC50 32.3 µg/mL). At present only limited data is available on the phytochemical composition of O. glabra.

Diclofenac 1,3,4-Oxadiazole Derivatives; Biology-Oriented Drug Synthesis (BIODS) in Search of Better Non-Steroidal, Non-Acid Antiinflammatory Agents.

BACKGROUND: Inflammation is defined as the response of immune system cells to damaged or injured tissues. The major symptoms of inflammation include increased blood flow, cellular influx, edema, elevated cellular metabolism, reactive oxygen species (ROS) nitric oxide (NO) and vasodilation. This normally protective mechanism against harmful agents when this normal mechanism becomes dysregulated that can cause serious illnesses including ulcerative colitis, Crohn's disease, rheumatoid arthritis, osteoarthritis, sepsis, and chronic pulmonary inflammation. METHOD: In this study synthetic transformations on diclofenac were carried out in search of better non-steroidal antiinflammatory drugs (NSAIDs), non-acidic, antiinflammatory agents. For this purpose diclofenac derivatives (2-20) were synthesized from diclofenac (1). All derivatives (2-20) and parent diclofenac (1) were evaluated for their antiinflammatory effect using different parameters including suppression of intracellular reactive oxygen species (ROS), produced by whole blood phagocytes, produced by neutrophils, and inhibition of nitric oxide (NO) production from J774.2 macrophages. The most active compound also evaluated for cytotoxicity activity. RESULTS: Diclofenac (1) inhibited the ROS with an IC50 of 3.9 ± 2.8, 1.2 ± 0.0 µg/mL respectively and inhibited NO with an IC50 of 30.01 ± 0.01 µg/mL. Among its derivatives 4, 5, 11, 16, and 20, showed better antiinflammatory potential. The compound 5 was found to be the most potent inhibitor of intracellular ROS as well as NO with IC50 values of 1.9 ± 0.9, 1.7 ± 0.4 µg/mL respectively and 7.13 ± 1.0 µg/mL, respectively, and showed good inhibitory activity than parent diclofenac. The most active compounds were tested for their toxic effect on NIH-3T3 cells where all compounds were found to be non-toxic compared to the standard cytotoxic drug cyclohexamide. CONCLUSION: Five derivatives were found to be active. Compound 5 was found to be the most potent inhibitor of ROS and NO compared to parent diclofenac 1 and standard drugs ibuprofen and L-NMMA, respectively. The most active compounds 1, 4, 5, 11 and 20 were found to be non-toxic on NIH-3T3 cells. Compound 4, 5, and 20 also showed good antiinflammatory potential, compound 11 and 16 showed moderate and low level of inhibition, respectively.

New Bis-Pyrazolones as Potential Leads for ROS Inhibition; Environment Friendly Green Synthesis, Structural Characterization, and In Vitro Studies.

BACKGROUND: Pyrazolones have identified as significant antioxidant agents and many marketed and clinically prescribed NSAIDs have pyrazolone ring as main scaffold. METHOD: Keeping in consideration the antioxidant potential of pyrazolone scaffold, new bispyrazolones 3-30 were synthesized by a green and enviroment friendly reaction route, in which two equivalents of 1-(4-chlorophenyl)-3-methyl-1H-pyrazol-5-ol were treated with one equivalent of benzaldehyde derivatives without any catalyst. All compounds were structurally characterzied by 1H-NMR and FAB analysis. 13C-NMR of selected compounds was also recorded. All compounds gave satisfactory elemental analyses and found in good agreement with calculated values. RESULTS: Synthetic bis-pyrazolones 3-30 were evaluated for their oxidative burst inhibitory effect of zymosan stimulated whole blood phagocytes by using luminol enhanced chemilluminescence technique. All molecules demonstrated the potent ROS inhibition activity in the range of IC50 = 1.2 ± 0.1-48.8 ± 3.9 µM as compared to the standard ibuprofen (IC50 = 54.2 ± 9.2 µM). The purity of active compounds was checked by HPLC. CONCLUSION: This study has identified a number of non-acidic lead molecules for future research on ROS inhibitors.

Analgesic, anti-inflammatory and anticancer activities of Combretin A and Combretin B isolated from Combretum fragrans F. HOFFM (Combretaceae) leaves.

Previous pharmacological and phytochemical studies showed that, Combretum fragrans F. HOFFM (Combretaceae) is a Cameroonian medicinal plant possessing numerous therapeutic virtues and rich in various active secondary metabolites. In this study, we investigate in vivo anti-nociceptive and anti-inflammatory activity and, in vitro anticancer, anti-TNFα, ROS and NO-inhibitory activities of Combretum A and Combretin B, two triterpenes cycloartane-type isolated from the leaves of Combretum fragrans. The effect on ROS, TNF-α and NO production, anticancer activity and cytotoxicity assay were done using chemiluminescence technique, ELISA kit, colorimetric method, MCF-7 cells and MTT assay, respectively. Antinociceptive and anti-inflammatory activities were estimated using a model of acetic acid, formalin and carrageenan. Combretin A and Combretin B significantly (p < 0.001) inhibited extracellular ROS production. These compounds also significantly (p < 0.001) reduced TNF-α and NO production. Moreover, these compounds decreased cell viability of MCF-7 cell lines. For acetic acid- or formalin-induced pain, as well as carrageenan-induced acute inflammation, Combretin A and Combretin B exhibited significant (p < 0.001) anti-nociceptive and anti-inflammatory activities. Anti-nociceptive, anti-inflammatory and anticancer potential associated with inhibitory effects on ROS, TNFα and NO production in this study show that, Combretin A and Combretin B could be considered as the promising chemotherapeutic agents in breast cancer treatment and inflammatory disease.

Immunomodulatory activities of isolated compounds from the root-bark of Cussonia arborea.

CONTEXT: Cussonia arborea Hochst. ex A. Rich (Araliaceae) is a folk medicine used to treat various diseases. However, there is no report of the root phytochemistry. OBJECTIVE: This study isolates and identifies the immunomodulatory compounds from root-bark of C. arborea. MATERIALS AND METHODS: The methanol extract (18 g) was subjected to repeated column chromatography resulting in isolation of five compounds (1-5). Structure determination was achieved by analysis of their 1 D and 2 D NMR, and mass spectroscopy. The compounds (100-1.0 µg/mL) were examined immunomodulatory for effect on production of reactive oxygen species (ROS) from whole blood phagocytes and on proliferation of T-cells. The compounds cytotoxicity (100-1.0 µg/mL) was evaluated on NIH-3T3 normal fibroblast cells. RESULTS: Three pentacyclic triterpenoids [3, 23-dihydroxy-12-oleanen-28-oic acid (1), 3ß-hydroxylolean-12-en-28-oic (2) and 23-hydoxy-oxo-urs-12-en-28-oic acid (5)], two phytosterols: [stigmasterol (3)] and [3-O-ß-d-glucopyranosyl stigmasterol (4)] were all isolated from the methanol soluble extract. All the tested compounds (1-4) were found to be nontoxic on NIH-3T3 cells. Compound 1 and 2 moderately inhibited the production of ROS (IC50 = 24.4 ± 4.3 and 37.5 ± 0.1 µg/mL, respectively) whereas compound 2 exhibited the highest inhibitory effect (IC50 = 12.6 ± 0.4 µg/mL) on proliferation of phytoheamagglutinin (PHA) activated T-cells. CONCLUSIONS: The isolated compounds (1-5) are reported for the first time from this species. In addition, compound 2 with suppressive potential on production of intracellular ROS and proliferation of T-cells could be of immense value in control of autoimmune diseases as well as in immune compromised patients.

Coumarin sulfonates: As potential leads for ROS inhibition.

Coumarin sulfonates 4-43 were synthesized by reacting 3-hydroxy coumarin 1, 4-hydroxy coumarin 2and6-hydroxy coumarin 3 with different substituted sulfonyl chlorides and subjected to evaluate for their in vitro immunomodulatory potential. The compounds were investigated for their effect on oxidative burst activity of zymosan stimulated whole blood phagocytes using a luminol enhanced chemiluminescence technique. Ibuprofen was used as standard drug (IC50=54.2±9.2µM). Eleven compounds 6 (IC50=46.60±14.6µM), 8 (IC50=11.50±6.5µM), 15 (IC50=21.40±12.2µM), 19 (IC50=5.75±0.86µM), 22 (IC50=10.27±1.06µM), 23 (IC50=33.09±5.61µM), 24 (IC50=4.93±0.58µM), 25 (IC50=21.96±14.74µM), 29 (IC50=12.47±9.2µM), 35 (IC50=20.20±13.4µM) and 37 (IC50=14.47±5.02µM) out of forty demonstrated their potential suppressive effect on production of reactive oxygen species (ROS) as compared to ibuprofen. All the synthetic derivatives 4-43 were characterized by different available spectroscopic techniques such as 1H NMR, 13C NMR, EIMS and HRMS. CHN analysis was also performed.