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Drug metabolism artifacts: formation of cytochrome P450 dependent amide metabonate from thioesters and
thiolactones
Patrick Dansette, Assia Hessani, Dan Levent, Daniel Mansuy
To cite this version:
Patrick Dansette, Assia Hessani, Dan Levent, Daniel Mansuy. Drug metabolism artifacts: formation of
cytochrome P450 dependent amide metabonate from thioesters and thiolactones. ”20th International
Conference on Cytochrome P450”., Aug 2017, Dusseldorf, Germany. �hal-02314472�
Drug metabolism artifacts: formation of cytochrome P450 dependent amide metabonate from thioesters and thiolactones.
Patrick M. Dansette a , Assia Hessani a , Dan Levent a and Daniel Mansuy a .
a
LCBPT, CNRS UMR 8601, Université Paris Descartes, Sorbonne Paris-Cité, 45 rue des Saints Pères, 75270 Paris Cedex 06, France. (patrick.dansette@parisdescartes.fr)
ABSTRACT:
Metabonate are false metabolites: after fortuitous introduction of small amount of ammonium (present in recent glucose-6-phosphate dehydrogenase commercial preparations) a series of new amide metabonates was found in the P450 catalyzed transformation of several thiolactones (thiolenones) and thioesters in place of the corresponding acids normal metabolites. Other amines like Tris buffer also lead to anormal Tris-subtituted amides.
INTRODUCTION
Thienopyridines anti-aggregants like ticlopidine, clopidogrel or prasugrel are metabolically transformed in a first step into 2-hydroxythiophene or their tautomeric thiolactone (thiolenone). In a second step, P450 enzymes catalyzes S- oxidation of the thiolactone to a reactive thiolactone S-oxide 2 and the opening of the ring to yield an acid and a sulfenic acid 3, which is subsequently reduced to the pharmacologically active thiol 4 (2,3) that can be stabilized by coupling with
8 10 12
Time (min) 0
100 0 100
RT: 11.00 AA: 781892 RT: 9.61 AA: 41488 RT: 8.81 AA: 61008
RT: 12.18 AA: 1115 RT: 9.23 AA: 807
345 350 355
m/z 0
2000 0
Relative Abundance 350.1218 C1 8H21O3N F S
349.1378 C18H22O2N2F S
354.2286 C22H30O2N2
505 510 515
m/z 0
0 100000
Relative Abundance 511.1367 C23H28O6N2F S2
510.15 29 C23H29O5N3F S2
8 10 12 14
Time ( min) 0
1 00 0 1 00
R T: 10.91 AA: 6145056
RT: 9.58 AA: 316694
RT: 10.03 AA : 1131 RT: 7.40
AA : 726
0 100
RT: 10.68 AA: 2657131 R T: 10.32 AA: 286541
RT: 9.94 0
50000
Relative Abundance 426.1200 C2 0H25O4N F S2 422.2898 C28H4 0N S
NAC 2mM, Thiols 4
NAC 2mM, Adducts 5
ME 2mM, Adducts 5
the pharmacologically active thiol 4 (2,3) that can be stabilized by coupling with N-ethylmaleimide (NEM) 6 or 3-methoxy-phenacyl bromide (MPB) 7. The sulfenic acid can also be trapped by a number of trapping agent like thiols 5, dimedone 8, indane-1,3-dione 9 (or BCN, KCN, NEM, not shown here).
High resolution mass spectrometry (HRMS) showed that these compounds were the amides instead of the acid. This was explained by ammonium ion present in the new G6PDH preparations. Although all thienopyridine studied gave this pattern we show here selected results using 2-oxo-prasugrel as an example.
Such false metabolite (also called metabonate) are also formed in P450 catalyzed thioester oxidations. We show here the case of dithiopyr.
METHOD: Incubation of 2-oxo-prasugrel (2OP) or dithiopyr (100µM) with human liver microsomes in the presence of NADPH (1mM) generating system and either ascorbate 2 mM or GSH 5mM as a reducing agent or of diverse
8 10 12 14
Time (min) 0
100
0 RT: 9.94
MA: 158359
RT: 13.19 AA: 879 10.35
420 425 430
m/z 0
5000 0
Relative Abundance
425.1360 C2 0H26O3N2F S2
Example 3 : In presence of 2mM ascorbate plus 2mM NEM the acid adduct (m/z = 475.1703) and amide adduct (m/z = 474.1865) were obtained.
460 480
m/z 0
100 0 100
Relative Abundance
475.1703 C24H28O5N2F S
474.1865 C24H29O4N3F S
8 9 10 11 12
Time (min) 0
100 0 100
Relative Abundance
RT: 9.45 AA: 3812180
RT: 8.64 AA: 369330
Trapping of sulfenic acid intermediate 3
Example 4 : In presence of dimedone 2mM the sulfenic acid gave the cis acid adducts (m/z = 488.1893) and cis amide adducts (m/z = 487.2055) Example 5 : In presence of 0.2 to 1 mM indandione the acid (m/z = 494.1426) and amide adduct (m/z = 493.1588) were found.
485 490
m/z 0
1000000 0 500000
Relative Abundance 488.1893 C26H31O5N F S
487.2055 C26H32O4N2F S
10 12 14 16
Time (min) 0
100 0 100
RT: 13.44 AA: 15578418
RT: 12.75 AA: 27100781
Relative Abundance
494.1426 100
RT: 13.07 AA: 6160024
2mM dimedone 8
0.5 mM indandione 9 and either ascorbate 2 mM or GSH 5mM as a reducing agent or of diverse
trapping agents (dimedone, indanedione, NEM) were stopped with AcCN/AcOH 9:1; after centrifugation (10 min, 13000g), HPLC-MS analysis of the supernatant using high resolution MS (Thermo Exactive) was performed (Shimadzu ODS 75 x 2.1 mm 2.6µ, gradient 0.1% formic acid/0.1% formic acid in AcCN) (4). In some case NADPH complemented with ammonium acetate or Tris buffer was used. The thiol metabolites were also derivatized using NEM or MPBr.
RESULTS: Incubation with liver microsomes in presence of pure NADPH and ascorbate leads to the formation of the two cis thiol diastereomers as major products and the endo isomer as a minor product (3,5,6). However in presence of NADPH generating system, a family of new peaks at one MS unit less was present. Accurate mass showed that one oxygen was replaced by NH, demonstrating the participation of ammoniac (ammonium ion) to the reaction.
Using derivatisation of the thiol by MPBr or NEM showed also a new family of amide derivatives. In presence of ascorbic acid 2mM, dithiothreitol 2mM or TCEP 1 mM only the cis-acid thiol was formed (m/z = 350.121) (not shown).
Effect of ammonium concentration: Incubation of 2OP in presence of NADPH and ammonium showed a correlation of the amide metabonate (7) with ammonium concentration (not shown) .
Example 1 : N-acetylcysteine (NAC) 2 mM :
In presence of 2 mM N-Acetylcysteine, 2-oxoP incubated with human liver microsomes gave a mixture of acid thiols (2 cis, 2 trans and one endo) (m/z = 350.1218), but only the cis amide thiol (m/z = 349. 1378).The NAC acid adduct (m/z = 511.1368) (7, 8) had also a corresponding amide (m/z = 510.1529). It represented about 5% of the acid form.
Example 2: Mercaptoethanol (ME) 2mM :
The acid thiols and amide thiols were present. The ME-acid adduct (7) (m/z
= 426.1200 had also a corresponding amide (m/z = 425.1360).
References: 1) Farid et al.
J Clin Pharmacol
2008, 50, 126-142; 2) Dansette et al.Chem Res Toxicol
2009, 22, 369-373; 3) Dansette et al.Chem Res Toxicol
2010, 23, 1268-1274; 4) Tuffal et al.Thromb Haemost.
2011, 105, 696-705; 5) Dansette et al.Chem Res
2012, 25, 348-56; 6) Dansette et al.Chem Res
2012, 25, 1058-65; 7) Dansette et al.Chem Res
2013, 26,794-802; 8) Dansetteet al. Chem Res
2015, 28, 1338-45; 9) Feng and Solsten.Xenobiotica
1991, 21, 1265-71. 10) de Montellano and KunzeArch Biochem Biophys.1981, 209, 710-2.
480 490 500
m/z 0
200000 0 200000
Relative Abundance
496.1468 478.1225492.1275
493.1588 495.1560 488.1439
10 12 14
Time (min) 0
100 0
100 AA: 6160024
RT: 12.56 AA: 5750900
Example 6 : Dithiopyr (a dithioester ) is metabolized by rat and human microsomes (9) into two mono acid isomers (ESI
-m/z = 370.054) as shown below. Again the amide were found at m/z = 369.070).
10 15
Time (min) 0
100 0 100
RT: 11.98 AA: 145534450
RT: 14.89 AA: 1214569
m/z370 0
100 0 100
Relative Abundance
370.0508 C14H13O3N F5S
369.0675 C14H14O2N2F5S
