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1.
Bioorg Med Chem ; 20(6): 2152-7, 2012 Mar 15.
Article in English | MEDLINE | ID: mdl-22342267

ABSTRACT

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Influenza A virus/enzymology , Influenza in Birds/drug therapy , Neuraminidase/antagonists & inhibitors , Oseltamivir/analogs & derivatives , Oseltamivir/pharmacology , Animals , Antiviral Agents/chemical synthesis , Binding Sites , Birds/virology , Influenza A Virus, H7N1 Subtype/chemistry , Influenza A Virus, H7N1 Subtype/drug effects , Influenza A Virus, H7N1 Subtype/enzymology , Influenza A Virus, H7N3 Subtype/chemistry , Influenza A Virus, H7N3 Subtype/drug effects , Influenza A Virus, H7N3 Subtype/enzymology , Influenza A virus/chemistry , Influenza A virus/drug effects , Influenza in Birds/enzymology , Models, Molecular , Neuraminidase/chemistry , Neuraminidase/metabolism , Oseltamivir/chemical synthesis
2.
PLoS One ; 6(6): e20745, 2011.
Article in English | MEDLINE | ID: mdl-21673963

ABSTRACT

BACKGROUND: Mechlorethamine [ClCH(2)CH(2)N(CH(3))CH(2)CH(2)Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown. METHODOLOGY/PRINCIPAL FINDINGS: HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair) indicated formation of an interstrand crosslink at m/z 9222.088 [M-2H+Na](+). Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH(2)CH(2)N(CH(3))CH(2)CH(2)] at m/z 269.2 [M](2+) (expected m/z 269.6, exact mass 539.27) and its hydrolytic product dC-mech-OH at m/z 329.6 [M](+) (expected m/z 329.2). Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M](+), which are both due to loss of the 4-amino group of cytosine (as ammonia), in addition to dC and dC+HN(CH(3))CH = CH(2), respectively. The presence of m/z 269.2 [M](2+) and loss of ammonia exclude crosslink formation at cytosine N(4) or O(2) and indicate crosslinking through cytosine N(3) with formation of two quaternary ammonium ions. CONCLUSIONS: Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N(3) position of cytosine.


Subject(s)
Base Pair Mismatch , Chromatography, High Pressure Liquid/methods , Cytosine/chemistry , DNA/chemistry , Mass Spectrometry , Mechlorethamine/chemistry , Spectrophotometry, Ultraviolet/methods , Alkylating Agents/chemistry , Animals , Base Sequence , Cattle , DNA/genetics , DNA/metabolism , Phosphoric Diester Hydrolases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Snake Venoms/enzymology , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry
3.
Phytochem Anal ; 20(4): 314-9, 2009.
Article in English | MEDLINE | ID: mdl-19402187

ABSTRACT

INTRODUCTION: Turmeric (Curcuma longa) extracts contain three curcuminoids (curcumin, desmethoxycurcumin and bisdesmethoxycurcumin) as major bioactive substances. Previously reported HPLC-UV methods for the determination of curcuminoids have several disadvantages, including unsatisfactory separation times, poor resolution and/or complicated solvent mixtures with gradient elution. OBJECTIVE: To develop a simple isocratic HPLC-UV method for the simultaneous determination of individual curcuminoids for the quality control of turmeric extracts. METHODOLOGY: The sample was prepared by dissolving the extract in acetonitrile and subsequently diluting with 50% acetonitrile. This solution was analysed by reverse-phase chromatography on an Alltima C(18) column with isocratic elution of acetonitrile and 2% v/v acetic acid (40:60, v/v) at a flow rate of 2.0 mL/min, a column temperature of 33 degrees C, and UV detection at 425 nm. The method was validated and applied for quantification of individual curcuminoids in commercial turmeric extracts. RESULTS: The method allowed simultaneous determination of curcumin, desmethoxycurcumin and bisdesmethoxycurcumin in the concentration ranges of 10-60, 4-24 and 0.5-3.0 microg/mL, respectively. The limits of detection and quantification were, respectively, 0.90 and 2.73 microg/mL for curcumin, 0.84 and 2.53 microg/mL for desmethoxycurcumin and 0.08 and 0.23 microg/mL for bisdesmethoxycurcumin, and the percentage recoveries were, respectively, 99.16-101.75 (%RSD < or = 1.11%), 99.50-101.01 (%RSD < or = 1.74%) and 99.67-101.92 (RSD < or = 1.31%). CONCLUSION: The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of turmeric extracts containing the three curcuminoid compounds as the main principles in the herb.


Subject(s)
Chromatography, High Pressure Liquid/methods , Curcuma/chemistry , Curcumin/analogs & derivatives , Curcumin/chemistry , Plant Extracts/chemistry , Molecular Structure
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