Synthesis and structural assignment of two major metabolites of the LTA4H inhibitor DG-051

Article abstract of DOI:10.1016/j.bmcl.2009.09.097

The same two major CYP mediated metabolites of DG-051 were produced in the presence of rat, dog, monkey and human liver microsomes. Their respective structures were hypothesized based on mass spectrometry data correlated with the parent structure and conf

Full text of DOI:10.1016/j.bmcl.2009.09.097

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6275–6279
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structural assignment of two major metabolites
of the LTA4H inhibitor DG-051
*
Livia A. Enache , Jun Zhang, David W. Sullins, Isaac Kennedy, Emmanuel Onua, David E. Zembower,
Frank W. Muellner, Jasbir Singh, Alex S. Kiselyov
DeCODE Chemistry, Inc., 2501 Davey Road, Woodridge, IL 60517, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The same two major CYP mediated metabolites of DG-051 were produced in the presence of rat, dog,
monkey and human liver microsomes. Their respective structures were hypothesized based on mass
spectrometry data correlated with the parent structure and confirmed by comparison with authentic syn-
thetic samples. The number of regioisomers synthesized as candidates for metabolite M1 was narrowed
down using a metabolic study of a selectively deuterated DG-051 analogue.
Received 28 August 2009
Revised 24 September 2009
Accepted 25 September 2009
Available online 29 September 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Metabolite
DG-051
LTA4H inhibitor
DG-051 (Fig. 1) is a first-in-class small molecule inhibitor of leu-
kotriene A4 hydrolase (LTA4H), currently in Phase II clinical devel-
opment for the prevention of heart attack. LTA4H is a zinc
metalloprotease encoded by a gene that deCODE studies have
linked to increased risk of heart attack.1 It is directly involved in
the synthesis of the pro-inflammatory molecule leukotriene B4
(LTB4).
liver microsome (RLM) gave m/z 406, 16 amu higher than that of
the parent DG-051 (m/z 390). A prominent fragment ion observed
from either M1 or M2, m/z 320, was absent from the DG-051 prod-
uct ion spectrum [m/z 390?170 (major)?84 or 390?304 (min-
or)?84] and 16 amu higher than the m/z 304 DG-051 fragment.
This disparity, consistent with addition of an oxygen atom to the
m/z 304 ion in Figure 2, was attributed to either hydroxylation of
an aromatic ring or N-oxidation of the pyrrolidine moiety.
Understanding the metabolite(s) formed from a new chemical
entity (NCE), especially from a development candidate, is an
important requirement of the drug development process. In this
Letter we describe the identification and synthesis of two major
metabolites of DG-051 produced by CYP mediated pathway. In
vitro evaluation of metabolism of DG-051 was conducted using
rat, dog, monkey, and human liver microsomes, while in vivo
metabolism studies were conducted in rat and dog. In all tested
species, incubation of DG-051 for 60 min in the presence of an
NADPH-generating system2 yielded the same two major metabo-
lites, designated as M1 and M2. These were analyzed using a high
performance liquid chromatograph interfaced with a Q-trap mass
spectrometer.3 To help elucidate the biotransformation site, molec-
ular ions of both DG-051 (m/z 390) and metabolites (m/z 406 for
either M1 or M2) were further analyzed via multiple reaction mon-
itoring (MRM).4 The product ion spectrum of DG-051 served as a
template and was compared with that of the metabolites. The frag-
ment ions observed for DG-051 are shown in Figure 2. The product
ion scan for both M1 and M2 formed following incubation with rat
Interestingly, the fragmentation pathway of M1 showed
406?320?84, whereas the M2 pathway also showed 320?302.
CO2H
c
b
O
d
a
.HCl
or
.TsOH
N
Cl
O
Figure 1. DG-051 (HCl salt) or DG-051B (TsOH salt).
170
304
219
CO2H
O
86
N
Cl
O
* Corresponding author. Tel.: +1 630 783 4697; fax: +1 630 783 4689.
E-mail address: lenache@decode.com (L.A. Enache).
Figure 2. Fragment ions derived from DG-051.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.097

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L. A. Enache et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6275–6279
ated using the trimethylsilyl chloride/sodium iodide protocol.8
CO2H
Reaction of the phenoxide derived from phenol 4-d4-a with tosyl-
ate 5 (easily accessible from commercially available tert-butyl (2S)-
2-(hydroxymethyl)pyrrolidine-1-carboxylate 6) produced key
intermediate 7-d4-a. De-protection of the pyrrolidine nitrogen,
N-alkylation with ethyl 4-bromobutyrate, saponification, and acid-
ification resulted in the desired tetradeuterated analogue DG-051-
d4-a (Scheme 1).
O
O
N+
Cl
O
Figure 3. Chemical structure of DG-051 N-oxide (metabolite M2).
Both DG-051 and DG-051-d4-a were subjected to RLM incuba-
tion, with and without the addition of NADPH-generating system.
The analysis of the reaction product by LC/MS/MS indicated that
the M1 metabolite, while absent in the mixtures lacking NADPH
or in the mixtures quenched at 0 min incubation, was present in
the RLM mixtures after 60 min incubation, confirming its forma-
tion from the CYP mediated metabolic process. The analysis of
RLM incubated DG-051 showed a molecular ion of M1 at m/z
406. The analysis of RLM incubated DG-051-d4-a showed a molec-
ular ion of M1 at m/z 409, indicating that M1 was a derivative
resulting from hydroxylation at the deuterated/halogenated ring
(a molecular ion for M1 of m/z 410 was expected from the same
deuterated substrate if the attack had occurred in the non-deuter-
ated ring). This prompted us to conclude that positions ‘c’ and ‘d’
(Fig. 1) are likely metabolic sites for DG-051 to form M1.
The corresponding possible metabolite structures were desig-
nated M1-A and M1-B (Fig. 4). Both M1-A and M1-B were synthe-
sized as p-toluenesulfonate (tosylate) salts, generally easier to
isolate than their hydrochloride counterparts or the respective zwit-
terion forms. The preparation of M1-A is illustrated in Scheme 2.
We had previously prepared intermediate 12 via the reaction of
tosylate 5 derived from N-Boc-(S)-prolinol 6 (Scheme 1) with the
phenolate of 4-iodophenol 11. An Ullmann reaction between the
iodobenzene derivative 12 and 4-chloro-2-methoxyphenol 13
produced the key intermediate 14 which was de-protected and
N-alkylated with ethyl 4-bromobutyrate. The selective de-methyl-
The latter pathway suggested loss of a water molecule and sup-
ported the idea of the N-oxide as the M2 metabolite. Gratifyingly,
comparison of the LCMS data for M2 with that of an available sam-
ple of the respective synthetic N-oxide derivative of DG-051 (Fig. 3)
provided a positive match. Notably, under liquid chromatography,
the synthetic sample of DG-051 N-oxide gave two peaks in 9:1
ratio, corresponding to a mixture of two diastereomers. The major
peak (diastereomer) co-eluted with the M2 peak of the microsomal
incubation sample. The absolute configuration of M2 at the
N-oxide center has not yet been established.
The identification of M1, a phenolic metabolite, was less
straightforward. This was due to the relatively close estimated
reactivity of the four potential metabolic sites in the diaryl ether
pharmacophore (a–d, Fig. 1). To unambiguously determine the
M1 structure, one possible strategy was to synthesize all four pos-
sible phenolic derivatives of DG-051 and compare these synthetic
molecules with a metabolized specimen. However, both synthetic
involvement and availability of starting materials for this endeavor
prompted us to design an alternative approach. In order to narrow
down potential metabolic site(s), we prepared a D4 DG-051 ana-
logue featuring exhaustive replacement of hydrogens with deute-
rium atoms within a single aromatic ring (Scheme 1).5,6
The Ullmann type condensation7 of commercially available
1-bromo-4-chlorobenzene-d4 (1-d4) with 4-methoxyphenol 2 pro-
vided the diphenyl ether derivative 3-d4-a which was de-methyl-
COOtBu
N
HO
Ts = H3C
SO2
6
c
HO
COOtBu
N
TsO
O
D
D
D
D
D
D
2
O
O
5
D
O
D
D
O
D
D
Br
D
D
O
D
N
Cl
OH
Cl
O
Cl
Cl
O
b
d
a
D
D
7-d4-a
4-d4-a
3-d4-a
1-d4
CO2Et
CO2H
Br
COOEt
D
D
D
D
9
D
D
O
D
D
O
D
.TsOH
or
.HCl
D
O
D
N
H
N
Cl
O
N
Cl
O
e
Cl
O
g
f
D
DG-051-d4-a (HCl salt)
DG-051B-d4-a (TsOH salt)
10-d4-a
8-d4-a
Scheme 1. Reagents and conditions: (a) CuI, N,N-dimethylglycineÁHCl, Cs2CO3, 1,4-dioxane, 110 °C, 1.5 h; (b) TMSCl, NaI, acetonitrile, reflux 20 h; (c) TsCl, TEA, DMAP, DCM,
rt, 16 h, 97%; (d) KOtBu, DMF, 55 °C, 16 h, 61% (three steps); (e) 4 M HCl in 1,4-dioxane, rt, 16 h, 79%; (f) K2CO3, acetonitrile, 50 °C, 16 h; (g) NaOH, EtOH, H2O, rt, 16 h, then HCl
or TsOHÁH2O, 80–90% (two steps).

L. A. Enache et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6275–6279
6277
O
O
OH
OH
OH
HO
Cl
O
O
N
N
O
Cl
O
M1-B
M1-A
Figure 4. Proposed structures of the M1 metabolite of DG-051.
OMe
I
OMe
OMe
O
O
Cl
OH
O
O
O
O
I
O
O
11
H
13
OH
N
N
N
N
O
Cl
O
Cl
O
TsO
c
b
15
a
12
14
5
CO2Et
CO2Et
CO2H
OMe
OH
OH
O
O
O
N
N
N
e
f
d
. TsOH
Cl
O
Cl
O
Cl
O
17
16
M1-A
Scheme 2. Reagents and conditions: (a) KOtBu, DMF, 45–55 °C, 20 h, 92%; (b) CuI, N,N-dimethylglycineÁHCl, Cs2CO3, 1,4-dioxane, 95 °C, 22 h, 54%; (c) 4 M HCl in 1,4-dioxane,
rt, 2 h; (d) ethyl 4-bromobutyrate, K2CO3, acetonitrile, 60 °C, 16 h; (e) NaSEt, DMF, 95 °C, 1 h, 67% (three steps); (f) NaOH, EtOH, H2O, rt, 16 h, then TsOHÁH2O, 79%.
ation of intermediate 16 in the presence of the ester and the other
ether functionalities was not trivial. Boron tribromide in dichloro-
methane (DCM) afforded a complex product mixture. A complex
mixture was also obtained when using the TMSCl/NaI procedure
in similar fashion to its application per Scheme 1. Heating 16 with
a small excess of sodium ethanethiolate (1.6 equiv) in dimethyl-
formamide (DMF) at 95 °C for 1–3 h provided the desired interme-
diate 17 after concentration and aqueous work-up. An early pilot
showed very low recovery (29%). We attributed this to partial
hydrolysis of the ester functionality during a longer reaction time
(3 h) and subsequent loss of the free acid in the aqueous wash.
In a later installment, we both confirmed this hypothesis and
solved the low recovery issue by stopping the reaction after only
1 h (at which point TLC on silica gel with 1:9 (v/v) MeOH/DCM
showed a much less intense free acid spot at the baseline than in
the early experiment) and by refluxing the crude isolated product
in excess EtOH in the presence of catalytic amounts of hydrochloric
acid (to re-esterify the free acid traces). The de-methylated inter-
mediate 17 was thus isolated in 67% yield over three steps, after
chromatographic separation. Saponification and tosylate salt for-
mation completed the synthesis of M1-A.9
Preparation of M1-B (Scheme 3) started with the Ullmann reac-
tion between 5-bromo-2-chloroanisole 18 and hydroquinone 19. A
relatively large excess (6 equiv) of hydroquinone was required to
achieve a satisfactory conversion rate. The product (20) was con-
taminated with a significant amount of p-benzoquinone and was
used in the next step without further purification. The O-alkylation
of the phenolate derived from 20 with tosylate 5 (prepared as in
Scheme 1) produced the key intermediate 21 in a 14–21% yield
for the two step sequence including chromatography. Cleavage of
O
O
N
HO
OH
TsO
19
O
O
MeO
Cl
Br
MeO
Cl
O
MeO
O
MeO
Cl
O
5
H
N
N
a
b
c
O
Cl
O
OH
22
21
20
18
CO2H
CO2Et
CO2Et
HO
Cl
O
HO
Cl
O
MeO
Cl
O
. TsOH
N
N
N
d
f
e
O
O
O
M1-B
24
23
Scheme 3. Reagents and conditions: (a) CuI, N,N-dimethylglycineÁHCl, Cs2CO3, 1,4-dioxane, 95 °C, 70 h; (b) KOtBu, DMF, 55 °C, 48 h, 14–21% (two steps); (c) 4 M HCl in 1,4-
dioxane, rt, 16 h, 51%; (d) ethyl 4-bromobutyrate, K2CO3, acetonitrile, 55 °C, 16 h, 85%; (e) NaSEt, DMF, 95 °C, 1 h; (f) NaOH, EtOH, H2O, rt, 16 h, then TsOHÁH2O, 32% (two steps).

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L. A. Enache et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6275–6279
CO2Me
O
DG-051
N
b
a
Cl
O
25
CO2Me
CO2H
O
O
N+
O
N+
O
Cl
O
c
Cl
O
DG-051 N-oxide (M2)
26
Scheme 4. Reagents and conditions: (a) MeOH, 4 M HCl in 1,4-dioxane, reflux, 4 h, then Na2CO3, 100%; (b) MCPBA, DCM, À78 °C to rt, 16 h, 79%; (c) aq NaOH, rt, 16 h, then HCl
to pH 3.7–3.9, 99%.
the carbamate, alkylation, de-methylation, and saponification were
conducted similar to the protocol for the synthesis of M1-A to af-
ford M1-B.10
Finally, the N-oxide derivative of DG-051, previously identified
as the M2 metabolite, was re-synthesized via the route summa-
rized in Scheme 4.
Notably, DG-051 can be directly N-oxidized with 3-chloroper-
benzoic acid (MCPBA) or other oxidizing agents. However, the
resulting N-oxide was not stable on silica gel. It was not amenable
to re-crystallization due to both its glassy consistency and its low
melting point. In our hands, the more practical approach to high
purity N-oxide involved preparation of the DG-051 methyl ester
as a free base (25). Ester 25 was subsequently N-oxidized to gener-
ate intermediate 26 in reasonable yield (79%) after chromato-
graphic purification. Saponification of 26 followed by careful
acidification to mildly acidic pH and extraction work afforded
99.5% pure DG-051 N-oxide.11
DG-051 with dog liver microsomes (DLM) (Fig. 5). We re-con-
firmed the N-oxide to be the M2 metabolite and identified the
structure of M1 to be M1-A. Both M1 and M2 exhibited an increase
in abundance that was dependent on the incubation time.
Structure M1-A, hydroxylated at position ‘c’ (Fig. 1), is consistent
with the most favorable position for CYP mediated metabolic attack
resulting(albeitbased on onlyca. 1 kcal/molenergydifference) from
computational analysis that took into account (i) hydrogen abstrac-
tionenergy(lowestvaluedesired)and(ii)Connollysurfaceareaused
as surrogate for solvent accessible surface area (SASA) (highest value
desired).12a The respective calculated values (hydrogen abstraction
energy in kcal/mol/Connolly surface area in Å2) for positions a–d in
Figure 1 are (a) À56.59/7.65; (b) À60.00/7.25; (c) À61.16/7.61; and
(d) À58.17/6.89. All the absolute values of the calculated hydrogen
abstraction energies appear high relative to average calculated val-
ues available in literature,12c while the calculated Connolly surface
areas are all below 8 Å2. Due to these considerations and to the fact
that these types of calculations generally have a lower level of pre-
diction capability for hydroxylation at the sp2 carbon,12b,c we did
HPLC traces for M1-A, M1-B, and M2 were compared to that of
the authentic metabolite mixture formed during the incubation of
Figure 5. HPLC comparison of authentic standards with metabolites M1 and M2 from DLM incubated DG-051.

L. A. Enache et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6275–6279
6279
mixture contained 1.8 mg/mL microsomal protein and 25
reaction well.
3. LC/MS/MS was performed using a Finnigan LCQ spectrometer with ion trap
analyzer from Thermo Scientific.
l
M DG-051 per
not use the calculated results as a definitive tool at the structural
assignment stage. However, we find it useful to mention them for
comparison with the experimental findings. Furthermore, position
‘c’ as the preferred site of CYP mediated attack also matches our lat-
est computational results obtained with a mechanism based ap-
proach. The respective calculated values (activation energy/
reaction enthalpy, both in kcal/mol) for sites a–d in Figire 1 are (a)
À31.11/À39.95; (b) À33.95/À42.52; (c) À34.54/À50.53; and (d)
À32.46/À33.79.13a,b
4. (a) Kondrat, R. W.; McClusky, G. A.; Cooks, R. G. Anal. Chem. 1978, 50, 2017; (b)
Yu, X.; Cui, D.-H.; Davis, M. R. J. Am. Soc. Mass Spectrom. 1999, 10, 175; (c) Lim,
H. K.; Stellingweif, S.; Sisenwine, S.; Chan, K. W. J. Chromatogr. 1999, 831, 227;
(d) Anderson, L.; Hunter, C. L. Mol. Cell. Proteomics 2006, 5, 573.
5. When following the same route towards the preparation of the analogue DG-
051-d4-b (tetradeuterated at the two ‘a’ positions and the two ‘b’ positions from
Fig. 4) starting from 1-bromo-4-chlorobenzene (1) and 4-methoxyphenol-d4 (2-
d4), we noticed that the resulting product was contaminated with analogues
having a lower level of labeling (d3, d2). Most likely, deuterium to hydrogen
exchange had occurred under the conditions of the first step, via quino-phenolic
tautomerism of 2-d4.
6. There were no differences between the metabolite patterns of the
hydrochloride salt, DG-051-d4-a and the tosylate salt, DG-051B-d4-a.
7. Ma, D.; Cai, Q. Org. Lett. 2003, 5, 3799.
8. Olah, G. A.; Narang, S. C.; Gupta, B. G. B.; Malhotra, R. J. Org. Chem. 1979, 44,
1247.
In conclusion, we have successfully identified two key CYP med-
iated metabolites for the Phase II clinical candidate DG-051. The
evaluation sequence for M1 involved preparation of the tetradeu-
terated molecule DG-051-d4-a. Studies revealed two possible aro-
matic sites for metabolic hydroxylation. The specific reactive
center was further identified by comparison of the metabolized
mixture with authentic synthetic samples of the two possible can-
didate structures, each prepared via a six step synthetic sequence.
Positive identification of M2 was achieved by a tandem MSMS
analysis of the metabolite peak followed by chemical synthesis
and comparison with the metabolized sample.
9. Spectral data for M1-A (TsOH salt): 1H NMR (400 MHz, DMSO-d6) d 1.82 (m,
1H), 1.85–2.19 (m, 4H), 2.21 (m, 1H), 2.29 (s, 3H), 2.36 (t, J = 7.2 Hz, 2H), 3.14
(m, 2H), 3.42 (m, 1H), 3.57 (m, 1H), 3.88 (m, 1H), 4.10 (dd, J = 10.8, 8.0 Hz, 1H),
4.26 (dd, J = 10.8, 3.6 Hz, 1H), 6.83 (dd, J = 8.8, 2.0 Hz, 1H), 6.86 (d, J = 8.8 Hz,
1H), 6.88 (d, J = 9.2 Hz, 2H), 6.97 (d, J = 9.2 Hz, 2H), 6.98 (d, J = 2.0 Hz, 1H), 7.11
(d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 8.80 (br s, 1H), 10.02 (s, 1H), 12.00 (br
s, 1H) ppm; 13C NMR (500 MHz, DMSO-d6) d 20.73, 20.76, 22.40, 26.21, 30.47,
54.24 (2 signals), 65.90, 67.00, 115.63, 116.82, 118.07, 119.16, 121.72, 125.45,
127.85, 128.00, 137.52, 143.08, 145.76, 149.88, 151.52, 153.09, 173.58 ppm;
MS (APCI+) m/z 406 [MH+].
Acknowledgment
10. Spectral data for M1-B (TsOH salt): 1H NMR (500 MHz, DMSO-d6) d 1.83 (m,
1H), 1.87–2.12 (m, 4H), 2.24 (m, 1H), 2.29 (s, 3H), 2.38 (t, J = 7.0 Hz, 2H), 3.18
(m, 2H), 3.48 (m, 1H), 3.63 (m, 1H), 3.93 (m, 1H), 4.14 (dd, J = 10.5, 8.5 Hz, 1H),
4.31 (dd, J = 10.5, 3.5 Hz, 1H), 6.35 (dd, J = 9.0, 3.0 Hz, 1H), 6.54 (d, J = 3.0 Hz,
1H), 7.05 (s, 4H), 7.11 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 1H), 7.47 (d,
J = 8.0 Hz, 2H), 9.44 (br s, 1H), 10.28 (s, 1H), 12.32 (br s, 1H) ppm; 13C NMR
(500 MHz, DMSO-d6) d 20.58, 20.72, 22.37, 26.15, 30.38, 54.36, 54.40, 66.22,
66.80, 105.59, 108.81, 113.48, 115.88, 120.92, 125.44, 127.96, 130.33, 137.46,
145.83, 149.71, 153.94, 154.07, 157.52, 173.52 ppm; MS (APCI-) m/z 404
[MÀ1].
The authors gratefully acknowledge computational chemistry
contributions from Dr. Rama K. Mishra.
References and notes
1. Sandanayaka, V.; Mamat, B.; Mishra, R. K.; Winger, J.; Krohn, M.; Zhao, L.-M.;
Keyvan, M.; Enache, L. A.; Sullins, D.; Onua, O.; Zhang, J.; Halldorsdottir, G.;
Sigthorsdottir, H.; Thorlaksdottir, A.; Sigthorsson, G.; Thorsteinnsdottir, M.;
Davies, D. R.; Stewart, L. J.; Zembower, D. E.; Andresson, T.; Kiselyov, A. S.;
Singh, J.; Gurney, M. E. J. Med. Chem., submitted for publication.
2. Liver microsomes (from Xenotech LLC, Lenexa, KS) were incubated with DG-
051 in a system consisting of microsomes and NADPH-generating system
(containing KH2PO4, K2HPO4, MgCl2, EDTA, and deionized water). For
11. Spectral data for DG-051 N-oxide: 1H NMR (400 MHz, CDCl3) d 2.00–2.18 (m,
3H), 2.18–2.45 (m, 4H), 2.52 (m, 1H), 3.68 (m, 2H), 4.00–4.14 (m, 3H), 4.19 (m,
1H), 4.70 (dd, J = 11.6, 8.0 Hz, 1H), 6.85 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 9.2 Hz,
2H), 6.94 (d, J = 9.2 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H) ppm; 13C NMR (500 MHz,
CDCl3) d 19.58, 20.28, 25.04, 32.54, 64.76, 65.13, 65.80, 75.60, 115.86, 118.91,
120.77, 127.55, 129.56, 150.64, 154.00, 156.77, 175.96 ppm; MS (APCI-) m/z
404 [MÀ1].
incubation, the diluted microsomes were mixed with DG-051 (1000
microsomes and 200 L of DG-051) in glass vials, and pre-incubated for 10 min
at 37 °C. The reaction was initiated by the addition of 1000 L of the NADPH-
lL of
l
12. (a) We used the Austin model-1 (AM1) Hamiltonian to calculate the hydrogen
abstraction energy. The Connolly surface area was calculated using the
MOLCAD module of SYBYL7.0.; (b) Singh, S. B.; Shen, L. Q.; Walker, M. J.;
Sheridan, R. P. J. Med. Chem. 2003, 46, 1330; (c) Sheridan, R. P.; Korzekwa, K. R.;
Torres, R. A.; Walker, M. J. J. Med. Chem. 2007, 50, 3173.
l
generating system, and the capped glass vials were set in a shaking incubator at
37 °C for up to 60 min. Samples were removed at 0 min (baseline) and at
appropriate intervals in order to track the formation of each metabolite and the
concomitant disappearance of the parent compound. At each sampling time,
13. (a) Jones, J. P.; Mysinger, M.; Korzekwa, K. R. Drug Metab. Dispos. 2002, 30, 7; (b)
Methoxy radical was added to the different positions of the aromatic rings. The
heat of reaction and the transition state were computed for each reaction using
the AM1 formalism.
200 lL of incubation mixture was removed and mixed with 200 lL ice-cold
acetonitrile. The samples were vortexed to mix for approximately 1 min, and
centrifuged at 3000g for 30 min. The supernatant was transferred into clean
plates, and analyzed using appropriate LC/MS/MS methods. Final incubation