Article Figures & Data
Figures
- Fig. 1.
Structures of eight mGluR5 PAMs.
- Fig. 2.
Positive-ion electrospray LC/MS extracted ion chromatogram (m/z 668) illustrating the elution of GSH conjugates (GSH1-1, GSH1-2, and GSH1-3) of compound 1 (A). HPLC-UV chromatograms of compound 1 and its GSH conjugates detected in GSH-fortified incubations without liver subcellular preparations (B), with rat liver microsomes (RLM) (C), human liver microsomes (HLM) (D), rat liver cytosol (RLC) (E), and human liver cytosol (HLC) (F).
- Fig. 3.
Positive-ion electrospray LC/MS extracted ion chromatogram (m/z 685) showing the elution of GSH conjugates (GSH2-1, GSH2-2, GSH2-3, and GSH2-4) of compound 2 (A). HPLC-UV chromatograms of compound 2 and its GSH conjugates detected in GSH-fortified incubations without liver subcellular preparations (B), with rat liver microsomes (RLM) (C), human liver microsomes (HLM) (D), rat liver cytosol (RLC) (E), and human liver cytosol (HLC) (F).
- Fig. 4.
Product-ion LC/MS/MS spectra of compound 1 (A) and GSH1-3 (B) annotated with proposed structures of fragment ions.
- Fig. 5.
Product ion liquid chromatography (LC)/MS/MS spectrum of GSH2-3 and proposed structures of fragment ions.
- Fig. 6.
Product ion LC/MS/MS spectrum of GSH3-1 and proposed structures of fragment ions.
- Fig. 7.
HMBC spectrum of GSH1-3, which was isolated from an incubation of compound 1 with GSH-fortified Aroclor-induced rat liver S9.
- Fig. 8.
ROESY spectrum of GSH1-3, which was isolated from an incubation of compound 1 with GSH-fortified Aroclor-induced rat liver S9.
- Fig. 9.
HMBC spectrum of GSH2-3, which was isolated from an incubation of 2 with GSH-fortified Aroclor-induced rat liver S9.
- Fig. 10.
ROESY spectrum of GSH2-3, which was isolated from an incubation of compound 2 with GSH-fortified Aroclor-induced rat liver S9.
- Fig. 11.
Proposed mechanism of GSH addition to the acetylene of compound 2. The thiolate anion (GS−), formed either spontaneously or as a result of catalysis by GST, initiates nucelophilic attack on either acetylenic carbon atom to produce four isomeric GSH conjugates. The negatively charged intermediate is stabilized by the nitrogen atoms of the heteroaryl rings adjacent to the triple bond. The structure and stereochemistry of GSH2-3 was confirmed by LC/MS and NMR results, and the conjugates GSH2-1, GSH2-2, and GSH2-4 were proposed as isomers.
Tables
- TABLE 1
Each numerical entry represents the relative abundance (percentage) of all GSH conjugates combined that were formed via conjugate addition during the reaction of the compound listed at left
The values were estimated based on the ratio of UV peak areas of the GSH conjugates detected following 30 minutes of incubation to that of the respective parent compound at 0 minute. The values represent the mean of the duplicate measurements for each of the incubations.
Liver Subcellular Fraction or Buffer Combined Amount of GSH Conjugates [Percentage of the Original Parent Drug Level (10 µM)] Potassium Phosphate Buffer Heat-Inactivated Liver S9 Liver Microsomes Liver Cytosol Species NA Human Rat Human Human Rat Rat Human Rat Cofactor(s) GSH GSH GSH GSH + NADPH GSH GSH + NADPH GSH GSH GSH Compound 1 2 3 3 4 4 6 10 5 48 Compound 2 1 1 1 3 4 8 9 16 56 Compound 3 MSa MS MS MS <1 <1 <1 <1 7 Compounds 4–8 ND ND ND ND ND ND ND ND ND NA, not applicable; ND, not detected.
↵a Detected only by MS with no UV response.
- TABLE 2
Proton (1H NMR) and carbon chemical shifts (13C NMR for parent and heteronuclear multiple quantum coherence for conjugates) of compound 1 and its two GSH conjugates (GSH1-2 and GSH1-3)
The proposed structure of GSH1-3 (Z) is shown below. The numbering system is for illustrative purposes only.
δH ppm δC ppm Position Compound 1 GSH1-2 GSH1-3 Compound 1 GSH1-2 GSH1-3 3 8.52 ND ND 4 5.00 4.95 5.00 60.9 61.8 61.7 5 5.54 5.45 5.50 82.8 83.2 83.1 7 7.24 7.26 7.23 122.6 123.4 122.9 8 7.44–7.58 7.55 7.45–7.55 132.0 131.8 131.6 9 7.26–7.37 7.26 7.25 116.9 116.6 116.4 11 7.26–7.37 7.30 7.35 113.6 113.8 113.6 
13 8.59 8.38 8.47 148.9 147.7 148.6 15 8.86 8.87 8.72 140.2 151.0 140.2 17 8.14 8.23 7.94 138.5 138.2 134.5 20 NA 7.60a NA 88.55 134.2a NA 21 NA 7.01 89.36 127.7 23 8.95 9.10 8.92 147.9 144.2 146.2 25 8.73 8.75 8.50 143.9 144.4 142.2 26 8.69 8.65 8.72 146.2 144.6 144.0 NA, not applicable; ND, not detected.
↵a The site could not be assigned because HMBC and ROESY spectra could not be acquired
- TABLE 3
Proton (1H NMR) and carbon chemical shifts (13C NMR for parent and heteronuclear multiple quantum coherence for conjugates) of compound 2 and its two GSH conjugates (GSH2-3 and GSH2-4)
The proposed structure of GSH2-3 (Z) is shown below. The numbering system is for illustrative purposes only and does not correspond to International Union of Pure and Applied Chemistry nomenclature.
δH ppm δC ppm Position Compound 2 GSH2-3 GSH2-4 Compound 2 GSH2-3 GSH2-4 3 8.59–8.47 8.54 8.53 NA NA NA 4 4.93 4.92 4.90 63.7 64.2 64.1 5 5.59 5.62 5.79 81.9 82.2 81.2 7 7.51 7.54 7.54 128.5 129.0 128.6 8 7.29 7.26 7.32 115.7 116.4 116.1 10 7.29 7.26 7.32 115.7 116.4 116.1 11 7.51 7.53 7.54 128.5 129.0 128.6 15 7.76 7.67 7.57 127.6 123.4 124.4 16 8.00 8.00 7.87 138.4 139.0 137.9 17 7.60–7.57 7.47 7.54 122.2 120.9 119.8 20 NA NA 81.9 NA 21 NA 7.15 6.84a 93.0 121.9 128.8a 24 8.59 – 8.47 8.55 8.53 146.7 145.1 145.8 25 7.64–7.61 7.40 7.54 126.4 124.1 125.6 26 7.76 7.72 7.88 124.1 123.4 125.2 NA, not applicable.
↵a The site could not be assigned because HMBC and ROESY spectra were unavailable.
In this issue
GSH Conjugation of Acetylene-Containing mGluR5 PAMs
