Intrinsic electrophilicity of the 4-methylsulfonyl-2-pyridone scaffold in glucokinase activators: Role of glutathione-S-transferases and in vivo quantitation of a glutathione conjugate in rats

Article abstract of DOI:10.1016/j.bmcl.2010.08.095

Previous studies on the in vitro metabolism of 4-alkylsulfonyl-2-pyridone- based glucokinase activators revealed a facile, non-enzymatic displacement of the 4-alkylsulfonyl group by glutathione. In the present studies, a role for glutathione-S-transferases (GST) as catalysts in the desulfonylation reaction was demonstrated using a combination of human liver microsomes, human liver cytosol and human GSTs. The identification of a glutathione conjugate in circulation following intravenous administration of a candidate 4-methylsulfonyl-2-pyridone to rats confirmed the relevance of the in vitro findings.

Full text of DOI:10.1016/j.bmcl.2010.08.095

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6262–6267
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Intrinsic electrophilicity of the 4-methylsulfonyl-2-pyridone scaffold
in glucokinase activators: Role of glutathione-S-transferases and in vivo
quantitation of a glutathione conjugate in rats
John Litchfield, Raman Sharma, Karen Atkinson, Kevin J. Filipski, Stephen W. Wright, Jeffrey A. Pfefferkorn,
⇑
Beijing Tan, Rachel E. Kosa, Benjamin Stevens, Meihua Tu, Amit S. Kalgutkar
Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous studies on the in vitro metabolism of 4-alkylsulfonyl-2-pyridone-based glucokinase activators
revealed a facile, non-enzymatic displacement of the 4-alkylsulfonyl group by glutathione. In the present
studies, a role for glutathione-S-transferases (GST) as catalysts in the desulfonylation reaction was dem-
onstrated using a combination of human liver microsomes, human liver cytosol and human GSTs. The
identification of a glutathione conjugate in circulation following intravenous administration of a candi-
date 4-methylsulfonyl-2-pyridone to rats confirmed the relevance of the in vitro findings.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 20 July 2010
Revised 16 August 2010
Accepted 18 August 2010
Available online 21 August 2010
Keywords:
Reactive metabolite
Electrophile
Glutathione
Microsomes
Cytosol
Glutathione transferase
Pharmacokinetics
Innately electrophilic compounds represent a significant liabil-
ity in drug discovery and development because they possess chem-
ical reactivity similar to affinity labeling agents (e.g., N-substituted
maleimides and/or alkyl halides); such compounds can alkylate
proteins, DNA and/or the endogenous antioxidant glutathione
(GSH), leading to toxicological outcomes.1–3 Consequently, electro-
philic functional groups (e.g., alkyl halides, Michael acceptors, etc.)
are generally avoided in drug design.4 Despite the necessary dili-
gence, there have been reports in the medicinal chemistry litera-
ture on seemingly ‘chemically inert’ compounds, which are prone
to nucleophilic displacement by GSH under non-enzymatic condi-
tions (pH 7.4, phosphate buffer, 37 °C) and/or enzyme-catalyzed
conditions.5–8 In the case of enzyme-assisted reactions, GSH conju-
gation to electrophilic centers is mediated by microsomal, cytosolic
and/or mitochondrial glutathione-S-transferase (GST).9–11
GSH at the electrophilic center on the pyridone ring to yield the
negatively charged Meisenheimer complex followed by elimina-
tion of the methylsulfonyl group (Fig. 1). Analogs 3–7 (Fig. 1) with
increasing steric bulk either directly on the sulfone or in the adja-
cent position on the ring also reacted readily with GSH suggesting
that chemical reactivity with GSH was not sensitive to steric
factors.
While the results from our previous work established the elec-
trophilic nature of 1, the role of GST isozymes in catalyzing the
nucleophilic displacement reaction and the relevance of this over-
all liability in vivo remained unclear. In the present work, we
undertook a side-by-side characterization of the in vitro reactivity
of 1 and related 2-pyridone analogs 24–27 (Scheme 2) with GSH
under non-enzymatic (phosphate buffer, pH 7.4, 37 °C) and enzy-
matic (human liver microsomes, human liver cytosol and human
GST enzymes) conditions. In vivo studies on 1 were also conducted
in the rat to quantitatively assess the presence of 2 in circulation
(plasma and bile). The collective findings are summarized, herein.
To enable analytical quantitation of GSH conjugate 2 in biolog-
ical matrices, an authentic standard was synthesized via the base
mediated reaction of GSH with 1 in 63% yield (Scheme 1).13
Substitutedpyridones 24–27 were synthesized via a pyridone
N-alkylation strategy depicted in Scheme 2. The preparation of
requisite 2-pyridones (compounds 9, 12 and 16) that were utilized
in the N-alkylation reaction is also shown in Scheme 2.
In the course of efforts to optimize 4-sulfonyl-2-pyridone-based
glucokinase activators as anti-diabetic agents, a unique metabolic
liability of the 4-sulfonyl-2-pyridone scaffold in the lead com-
pound 1 was identified wherein the heterocycle readily adducted
to GSH in phosphate buffer to form conjugate 2 (Fig. 1).12 The
mechanism involves nucleophilic attack by the thiolate anion of
⇑
Corresponding author.
E-mail address: amit.kalgutkar@pfizer.com (A.S. Kalgutkar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.095

J. Litchfield et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6262–6267
6263
3: R1 = H, R2 = Et
4: R1 = H, R2 = iPr
5: R1 = H, R2 = cPr
6: R1 = H, R2 = cBu
7: R1 = Me, R2 = Me
H
N
H
N
R1
S
N
N
N
N
N
N
R2
O
O
Me
Me
O
S
O
Me
O O
O O
1
GSH
37 ºC, Buffer (pH 7.4)
NH2
HOOC
H
N
O
S
GS
O
N
N
O
N
HN
O
H3C
O
S
O
N
Me
O
NH
COOH
O
H3C
S
2
OH
Figure 1. Chemical reactivity of 4-sulfonyl-2-pyridone-based glucokinase activator 1 with glutathione.
N
b
NH
a
O
OH
NH
Br
OMe
Br
O
H
N
N
N
8
N
9
H
N
N
N
O
a
N
O
S
O
O
MeO2S
MeO2S
MeO2S
c
O
HN
N
N
NH
S
O
O
2
O
Br
OMe
O
1
10
12
11
O
H2N
OH
N
NH
N
d
e
f
N
Scheme 1. Reagents and conditions: (a)
MeOH, 55 °C, 72 h, 63%.
L-glutathione (1.7 equiv), Et3N (3.0 equiv),
F
O
F
F
SO2Me
SO2Me
SMe
13
14
15
16
2-methoxyl-5-methylsulfonylpyridine (11) was prepared from
2-bromo-5-methylsulfonylpyridine (10)14 via a Pd-catalyzed cou-
pling reaction.15 O-Demethylation of 8 or 11 with iodotrimethyl
silane (generated in situ from chlorotrimethylsilane and sodium
iodide) afforded 4-bromo-2-pyridone (9) and 5-methylsulfonyl-2-
pyridone (12), respectively. Thioanisole 14 was synthesized from
13 through a base mediated reaction with methylthiocyanate.
S-Oxidation of 14 in the presence of mCPBA generated sulfone
15. Nucleophilic displacement of the 2-fluoro group in 15 by water
afforded 3-methylsulfonyl-2-pyridone (16). Hydroxymethyl ester
18 was prepared via nucleophilic opening of epoxide 17.16 Reaction
of 18 with trifluoromethanesulfonic anhydride in the presence of
2,6-lutidine generated triflate 19. Alkylation of pyridone and its
derivatives (compounds 9, 12, 16) was accomplished via deproto-
nation with lithium bis(trimethylsilyl)amide followed by reaction
with 19 to afford the N-alkylated derivatives (20–23). Trimethyl-
aluminum-mediated transamidation of ester derivatives 20–23
with 2-amino-5-picoline provided the title compounds 24–27,
respectively.
The in vivo formation of 2 was examined in rats after adminis-
tration of a single intravenous bolus dose (5 mg/kg) of 1. A repre-
sentative extracted ion chromatogram of plasma from rats
administered with 1 is shown in Figure 2. The retention time (Rt)
and mass spectral characteristics (MH+ = 632, m/z + H+ = 523,
394) of the peak that eluted at 1.8 min were identical to those ob-
served for the synthetic standard of 2. Figure 3 depicts the time
course profile of mean rat plasma and biliary concentrations of 1
and GSH conjugate 2.17 The rapid appearance of 2 in rat plasma
(as early as 5 min with peak total plasma concentrations of
g
h
O
CO2Me
17
HO
CO2Me
18
TfO
CO2Me
19
i
H
N
j
N
N
N
N
CO2Me
R
R
O
O
Me
O
20-23
24: R = H; 25: R = 5-SO2Me
26: R = 3-SO2Me; 27: R = 4-Br
Scheme 2. Reagents and conditions: (a) TMSCl (1.5 equiv), NaI (1.5 equiv), MeCN,
23 °C, 5 h, 29%; (b) Pd2(dba)3 (0.005 equiv), bippyphos (0.02 equiv), KOH
(1.5 equiv), MeOH, reflux, 16 h, 91%; (c) TMSCl (1.8 equiv), NaI (1.8 equiv), MeCN,
23 °C, 4 h, 100%; (d) LDA (1.0 equiv), MeSCN (1.0 equiv), THF, ꢁ78 to 0 °C, 2.5 h,
92%; (e) mCPBA (2.5 equiv), DCM, 0 °C, 2 h, 94%; (f) H2O, cat. acid; (g) Li2CuCl4
(0.1 equiv prepared from CuCl2 [0.1 equiv], LiCl [0.2 equiv], THF), cyclopentylmag-
nesium bromide (2.0 M in Et2O, 1.1 equiv), THF, ꢁ78 to ꢁ50 °C, 30 min, 70%; (h)
Tf2O (1.8 equiv), 2,6-lutidine (2.1 equiv), DCM, 0 °C, 30 min; (i) (1) pyridone
(1.0 equiv), LiHMDS (1.0 M in THF, 0.9 equiv), THF, 23 °C, 45 min, (2) 19 (1 equiv),
THF, 23 °C, 2 h, 44–85%; (j) 5-methylpyrazin-2-amine (2–4 equiv), Me3Al (2.0 M in
toluene, 2.2–4.4 equiv), 1,2-DCE, 80 °C, 1–2 d, 53–71%.
in rats. GSH conjugate 2 appeared to be the predominant metabo-
lite of 1 in circulation; oxidative metabolites (mono-hydroxylation
on the cyclopentyl and methylpyrazine ring systems) represented
a very minor component of drug-related material in circulation.
The in vivo observation is consistent with the low oxidative
ꢀ8.0
lM) indicates the efficiency of reaction between 1 and GSH

6264
J. Litchfield et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6262–6267
1.8e5
1.7e5
1.6e5
1.5e5
1.4e5
1.3e5
1.2e5
1.1e5
1.0e5
9.0e4
8.0e4
7.0e4
6.0e4
5.0e4
4.0e4
3.0e4
2.0e4
1.0e4
0.0
1
2
1.80
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
Time (min)
Figure 2. Extracted ion chromatogram of pooled rat plasma following a single intravenous bolus dose of 1 at 5 mg/kg.
Under the present experimental conditions, the possibility of di-
rect Michael addition of GSH on the 2-pyridone moiety was exam-
ined with 24 (R = H) and the methylsulfone regioisomers of 1, that
is, compounds 25 (R = 5-SO2Me) and 26 (R = 3-SO2Me). Likewise,
the 4-bromo-2-pyridone derivative 27, which is a harder, less elec-
trophilic substrate (relative to 1) was also included in this analysis.
Following incubations of compounds 24–26 and GSH in buffer,
microsomes, cytosol and/or GSTs, no GSH conjugates derived from
Michael addition were observed. Likewise, no nucleophilic dis-
placement of the 4-bromo substituent by GSH was observed in
incubations of 27 and GSH in phosphate buffer or human liver
microsomes (in the absence and/or presence of NADPH co-factor)
(Fig. 5, panels A–C). In human liver microsomal incubations forti-
fied with NADPH, only metabolites derived from P450-catalyzed
oxidation on the cyclopentylmethylene and pyrazinylmethyl re-
gions in 27 were discerned (Fig. 5, panel B). In contrast with the
findings in liver microsomes, incubations of 27 and GSH in human
liver cytosol and human GSTs led to the formation of a single
metabolite (Fig. 5, panels D and E), which was unambiguously pro-
ven to be GSH conjugate 2 by virtue of identical LC Rt and mass
spectral characteristics with those of the authentic standard. Thus,
while 27 was stable towards reaction with GSH under non-enzy-
matic conditions, a facile displacement reaction occurred upon
inclusion of cytosolic fraction and/or GST enzymes. Lack of analo-
gous reactivity in liver microsomes suggests that GSH displace-
ment of the 4-bromo substituent in 27 (see Fig. 5) is exquisitely
mediated by cytosolic GST enzymes.
Figure 3. Mean concentration versus time profile of 1 and its GSH conjugate 2 in
plasma and bile from rat administered an intravenous bolus dose of 1 (5 mgx/kg).
turnover of 1 in rat liver microsomes and rat hepatocytes.12 While
not directly assessed in this in vivo study, it is possible that re-
peated dosing of 1 could deplete the endogenous pool of GSH in
rats leading to toxicity in a similar fashion to that observed with
the anti-inflammatory agent acetaminophen.18,19
In order to examine whether GST enzymes catalyzed the nucle-
ophilic displacement reaction, the formation of 2 was monitored in
side-by-side in vitro incubations of 1 (10 lM) and GSH (5 mM) in
As metabolic activation is not a prerequisite to generation of a
transient reactive species for 4-substituted-2-pyridones, it would
be expected that the inherent electrophilicity and chemical soft-
ness parameters of these compounds would adequately predict
their relative reactivity with GSH. Indeed, 4-bromopyridone 27
was calculated to be a harder, less electrophilic substrate overall
when compared to the softer, more electrophilic 4-methylsulfonyl-
pyridone derivative 1 (Fig. 6);21,22 this corresponded well with the
observed reactivity of GSH with these compounds under non-enzy-
matic conditions. However, caution should be exercised when
applying the substrate electrophilicity number on an enzymatic
system such as GST, because of the synergetic interactions
between the enzyme and the substrate that stabilize the transition
state intermediate.
phosphate buffer (pH 7.4), human liver microsomes, human liver
cytosol and human placental GSTs.20 In each instance, the UV chro-
matogram (k = 300 nm) revealed the formation of a single metabo-
lite (Rt ꢀ16 min (Fig. 4). The Rt and mass spectral characteristics of
this metabolite were identical with those of the synthetic standard
of 2. The formation of 2 in microsomes that lacked NADPH (Fig. 4,
panel C) confirmed that prior metabolic activation to a reactive
species by cytochrome P450 isoforms was not required for the
nucleophilic displacement reaction. In incubations with human
placental GSTs, a quantitative conversion of 1 to 2 was observed
(Fig. 4, panel E). Furthermore, the detection of 2 in GSH-supple-
mented microsomes and cytosol (Fig. 4, panels B–D) suggested that
both microsomal and cytosolic GST enzymes were capable of con-
verting 1 to 2.

J. Litchfield et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6262–6267
6265
16.36
22.29
100
50
(A)
2
1
16.42
100
50
1
22.28
(B)
(C)
(D)
2
0
100
16.38
22.30
2
50
1
0
100
16.42
2
2
1
22.31
50
0
16.45
16
100
(E)
50
0
10
12
14
18
20
22
24
26
28
30
32
34
Time (min)
Figure 4. UV chromatograms of incubations of 1 (10 lM) in: phosphate buffer (pH 7.4) and GSH (5 mM) (panel A), NADPH-supplemented human liver microsomes and GSH
(5 mM) (panel B), human liver microsomes and GSH (panel C), human liver cytosol and GSH (5 mM), (panel D), and human placental GST and GSH (5 mM) (panel E). All
incubations were conducted at 37 °C for 60 min in a shaking water bath.
O
OH
NH
H
N
N
N
H
N
N
N
N
GST / GSH
Me
N
O
O
S
O
O
Me
O
HN
Br
O
2
27
O
H2N
OH
27.67
27.63
27.69
27.68
100
50
(A)
(B)
27
27
27
27
0
100
Oxidative metabolites
17.89
50
23.04
18.58
21.94
0
100
(C)
(D)
50
0
100
2
50
16.42
0
100
16.45
(E)
2
27
50
0
27.70
10
12
14
16
18
20
22
24
26
28
30
32
34
Time (min)
Figure 5. UV chromatograms of incubations of 27 (10 lM) in: phosphate buffer (pH 7.4) and GSH (5 mM) (panel A), NADPH-supplemented human liver microsomes and GSH
(5 mM) (panel B), human liver microsomes and GSH (panel C), human liver cytosol and GSH (5 mM) (panel D), and human placental GST and GSH (5 mM) (panel E). All
incubations were conducted at 37 °C for 60 min in a shaking water bath.
Overall, the results described herein provide a cautionary note
to drug discovery teams, which solely rely on reactive metabolite
trapping in liver microsomes as means of evaluating bioactivation
potential of new chemical entities. Based on the absence of a GSH
adduct(s) under non-enzymatic conditions and in human liver
microsomes ( NADPH), the 4-bromo-2-pyridone derivative 27

6266
J. Litchfield et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6262–6267
Figure 6. Electrophilicity prediction of 1 and 27.
(br s, 1H) 5.72–5.85 (m, 1H) 6.39 (d, J = 7.41 Hz, 1H) 6.50 (br s, 1H) 7.68 (d,
J = 7.41 Hz, 1H) 8.27 (s, 1H) 9.14 (s, 1H). LC–MS: 632 (MH+).
14. Li, J.; Lynch, M. P.; DeMello, K. L.; Sakya, S. M.; Cheng, H.; Rafka, R. J.; Bronk, B.
S.; Jaynes, B. H.; Kilroy, C.; Mann, D. W.; Haven, M. L.; Kolosko, N. L.; Petras, C.;
Seibel, S. B.; Lund, L. A. Bioorg. Med. Chem. Lett. 2005, 13, 1805.
15. Withbroe, G. J.; Singer, R. A.; Sieser, J. E. Org. Process Res. Dev. 2009, 12, 480.
16. (a) Larcheveque, M.; Petit, Y. Tetrahedron Lett. 1987, 28, 1993; (b) Cryle, M. J.;
Matovic, N. J.; De Voss, J. J. Org. Lett. 2003, 5, 3341.
would have passed the reactive metabolite screen with flying col-
ors. Metabolism vectors other than liver microsomes need to be ta-
ken into consideration when evaluating bioactivation and/or
innate electrophilicity of lead chemical matter.
References and notes
17. All animal care and in vivo procedures conducted were in accordance with
guidelines of the Pfizer Animal Care and Use Committee. Compound 1 (5 mg/
kg) was administered intravenously via the jugular vein cannula of bile-duct
exteriorized male Wistar-Han rats (0.31–0.36 kg, n = 3). Compound 1 was
formulated in DMSO/propylene glycol/50 mM tris buffer (10:50:40, v/v/v).
After dosing, serial blood samples were collected at appropriate times, and
plasma was obtained via centrifugation and kept frozen at ꢁ20 °C until
analysis. Bile samples (0–7.0 and 7.0–24 h) were also collected after iv
administration to rats. Liquid-chromatography tandem mass spectrometry
(LC–MS/MS) was performed on a Sciex API4000 system equipped with a turbo-
ionspray source (Applied Biosystems, Foster City, CA, USA) operated in positive
ion mode. HPLC analysis was conducted on Shimadzu 10ADvp Binary HPLC
system (Shimadzu Scientific Instruments Columbia, MD, USA) with a CTC-PAL
(Thermo Scientific, Franklin, MA, USA) as the autosampler. Chromatographic
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S. P. Curr. Drug Metab. 2005, 6, 161.
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l
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Heinrikson, R. L.; Tomasselli, A. G. Drug Metab. Dispos. 1999, 27, 992.
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12. Pfefferkorn, J. A.; Lou, J.; Minich, M. L.; Filipski, K. J.; He, M.; Zhou, R.; Ahmed, S.;
Benbow, J.; Guzman-Perez, A.; Tu, M.; Litchfield, J.; Sharma, R.; Metzler, K.;
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HPLC column (Torrance, CA, USA) using mobile phase A (10 mM ammonium
acetate and 1% isopropyl alcohol in water) and mobile phase B (acetonitrile). A
linear gradient from 5% B to 95% B over 1.5 min with the flow rate of 0.3 ml/
min was performed to elute 1, 2 and the internal standard. Analyst (version
1.4.1, Applied Biosystems, Foster City, CA, USA) was employed to control the
instrument operation and acquire data in multiple reaction monitoring mode.
The ion transition for 1 and 2 were 405?296 and 632?523, respectively. The
dynamic range of the assay ranged from 5 ng/ml to 5000 ng/ml using linear
regression with a weighting of 1/x2.
13. Procedure for the synthesis of GSH conjugate 2. Sulfone 1 (80 mg, 0.2 mmol,
18. Mitchell, J. R.; Jollow, D. J.; Potter, W. Z.; Gillette, J. R.; Brodie, B. B. J. Pharmacol.
Exp. Ther. 1973, 187, 211.
19. Mitchell, J. R.; Jollow, D. J.; Potter, W. Z.; Davis, D. C.; Gillette, J. R.; Brodie, B. B. J.
Pharmacol. Exp. Ther. 1973, 187, 185.
prepared according to Ref. 12) and
in 1 mL of MeOH in a septum capped screw–cap vial. The mixture was placed
under argon and anhydrous Et3N (85 L, 0.6 mmol) was added via syringe. The
clear orange solution was heated at 55 °C for 3 days. The mixture was then
cooled, neutralized with 300 L of 1 M HCl, and chromatographed on a 12 g
L-GSH (102 mg, 0.3 mmol) were combined
l
20. Test compounds (10
the presence or absence of GSH (5 mM) for 60 min at 37 °C. Separately, test
compounds (10 M) were also incubated with human liver microsomes
lM) were incubated in phosphate buffer (0.1 M, pH 7.4) in
l
reverse-phase C18 column using a water–MeCN gradient. Fractions containing
the desired product were concentrated to a semisolid residue, which was
azeotropically dried with MeCN at reflux, then cooled and concentrated to
afford 2 as a white powder (79 mg, 63%). 1H NMR (400 MHz, CD3OD) d ppm
1.16 (dd, J = 11.80, 7.71 Hz, 2H) 1.23–1.33 (m, 2H) 1.41–1.57 (m, 2H) 1.57–1.70
(m, 3H) 1.70–1.84 (m, 2H) 2.05–2.24 (m, 3H) 2.49 (s, 3H) 2.52–2.59 (m, 1H)
3.24–3.29 (m, 2H) 3.49–3.59 (m, 1H) 3.62–3.77 (m, 1H) 3.84–3.95 (m, 1H) 4.73
l
(2.0 mg/ml) or human liver cytosol (2.0 mg/ml) or human placental GST
(2.6 U/ml) in 0.1 M potassium phosphate buffer (pH 7.4) containing MgCl2
(3.3 mM) and NADPH (1.3 mM) in the presence of GSH (5 mM). In the case of
microsomal and cytosol incubations, reactions were initiated with the addition
of the biological matrix (microsomes or cytosol). Control incubations were run
in parallel in the absence of NADPH and/or GSH. All incubations were

J. Litchfield et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6262–6267
6267
conducted in a shaking water bath maintained at 37 °C open to the air. After
21. The measure of a ligand’s electrophilicity is defined as Eq. 1. (Ref. 22):
60 min, the incubations were terminated by addition of ice-cold acetonitrile
containing 0.1% formic acid, mixed vigorously, and the precipitated materials
were removed by spinning in a centrifuge (3000g) for 5 min. Aliquots of the
supernatants were analyzed for metabolite formation by LC–MS/MS. The HPLC
system consisted of an Accela quaternary solvent delivery pump and
v2
x
¼
ð1Þ
2g
autoinjector,
Electron Corporation, Waltham, MA). Chromatography was performed on a
Phenomenex, Synergy RP column, 150 ꢂ 4.6 mm, (Phenomenex,
Torrance, CA). LC analysis was performed at a constant flow rate of 1000 l/
a Surveyor PDA Plus photodiode array detector (Thermo
where
v is electronegativity (Eq. 2), and g is hardness (Eq. 3)
5 lm
l
min using a binary solvent system: Solvent A, 5 mM ammonium formate buffer
(pH ꢀ3.0) with 0.1% formic acid and Solvent B, acetonitrile. The initial HPLC
gradient system was held at 5% B for 3 min and linearly increased to 80% B in
35 min, followed by a return to initial conditions for column re-equilibration.
Post-column flow passed through the PDA detector to provide UV (k = 200–
400 nm) detection prior to being split to the mass spectrometer such that
EHOME þ ELUMO
v
g
¼ ꢁ
ð2Þ
ð3Þ
2
¼ ELUMO ꢁ EHOMO
In our implementation, an input chemical structure was first neutralized and then
minimized with MacroModel module in Maestro software package (Maestro 9.0,
Schrodinger LLC, NYC, NY). After that EHOMO and ELUMO energies were calculated with
mobile phase was introduced into the electrospray source at a rate of 50 ll/
min. The LC system was interfaced to a Thermo Orbitrap mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany). Xcalibur software version 2.0
was used to control the HPLC/MS system. Mass spectroscopy analyses were
carried out in the positive ion mode using full-scan MS with a mass range of
100–1000 Da. Full scan data and data-dependent MS/MS acquisition on the
two most intense ions were collected at 15,000 resolution. All experimental
data were acquired using external calibration prior to data acquisition.
*
Jaguar module using HF/3-21G level of theory.
22. Parr, R. G.; Szentpaly, L. V.; Liu, S. J. Am. Chem. Soc. 1999, 121, 1922.