Full text of DOI:10.1039/d0md00165a

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RESEARCH ARTICLE
Hit-to-lead optimization of a benzene
sulfonamide series for potential antileishmanial
Cite this: DOI: 10.1039/d0md00165a
agents†
Paul J. Koovits,a Marco A. Dessoy,a An Matheeussen,b Louis Maes,b Guy Caljon,b
c
Leonardo L. G. Ferreira,
Rafael C. Chelucci,c Simone Michelan-Duarte,c
Adriano D. Andricopulo, c Simon Campbell,d Jadel M. Kratz,
d
Charles E. Mowbray d and Luiz C. Dias
*
a
A series of benzene sulphonamides with good potency and selectivity against Leishmania spp. intracellular
amastigotes was identified by high-throughput screening. Approximately 200 compounds were
synthesized as part of a hit-to-lead optimization program. The potency of the series appears to be strongly
dependent on lipophilicity, making the identification of suitable orally available candidates challenging due
to poor pharmacokinetics. Despite not identifying a clinical candidate, a likely solvent exposed area was
found, best exemplified in compound 29. Ongoing detailed mode-of-action studies may provide an
opportunity to use target-based medicinal chemistry to overcome the issues with the current series.
Received 20th May 2020,
Accepted 7th August 2020
DOI: 10.1039/d0md00165a
rsc.li/medchem
Leishmaniasis is a neglected parasitic protozoal disease that
causes an estimated 20 000–30 000 deaths annually and
infects up to 12 million people, leaving up to a billion at risk
across Latin America, Africa, Asia and the Middle East.1,2 The
disease is caused by the kinetoplastid Leishmania,3 of which
there are over 20 species, and is transmitted by the female
phlebotomine sand fly. There are several clinical forms, the
most common being cutaneous leishmaniasis (CL) and
visceral leishmaniasis (VL). Annually, there are an estimated
700 000–1 200 000 new cases of CL,4 which presents itself as
skin lesions on exposed body parts such as the arms, legs,
and face, leaving permanent scars and severe disabilities. VL
or kala-azar is the more serious form and is fatal without
treatment. It is characterized by anaemia, enlarged organs
(spleen and liver), fever and weight loss. VL is specifically
caused by L. infantum (L. inf.) or L. donovani (L. don.). L. inf. is
zoonotic in Latin America, Europe, and Central Asia, where
canines act as the major reservoir. In South Asia and East
Africa, L. don. is considered anthroponotic and is responsible
for 95% of VL cases worldwide.5 Whilst the numbers of new
infections has been decreasing annually and dropped below
20 000 in 2018,6 it is estimated that only 20–45% of cases are
reported to the WHO.7,8 Despite these large numbers,
infections by L. inf. generally remain sub-clinical and VL only
develops in cases where the patient is immunosuppressed,
such as patients with HIV or severe malnourishment.9 The
latter is one of the reasons why leishmaniasis particularly
affects poor populations in rural areas.
Today, there are insufficient treatment options (Fig. 1),5,10
all of which are associated with severe side-effects and/or
other limitations, often requiring intravenous or painful
intramuscular injections as well as monitoring in hospital.
Miltefosine (MIL) is presently the only approved oral drug for
VL and CL. Although MIL has good efficacy, it has a long
treatment regimen (28 days); has dose-limiting side-effects
(due to vomiting and diarrhoea); is teratogenic which limits
use in pregnancy;11 and there are reports of resistance and
variable efficacy in different geographical areas.12–14 Whilst
the current pipeline looks more promising than in previous
years, with three candidates in phase I and several more in
pre-clinical phases,15–17 due to the pressing need for new
medicines it is imperative that we continue to develop novel,
affordable, effective and safe drugs for leishmaniasis.
a Institute of Chemistry, University of Campinas (UNICAMP), Rua Josué de Castro,
S/N, Cidade Universitária, Campinas, SP, 13083-861, Brazil.
E-mail: ldias@iqm.unicamp.br; Tel: +55 19 3521 3097
b Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of
Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
c Laboratory of Medicinal and Computational Chemistry, Physics Institute of Sao
Carlos, University of Sao Paulo, Av. Joao Dagnone 1100, 13563-120, Sao Carlos-SP,
Brazil
As part of our collaboration with the Drugs for Neglected
Diseases initiative (DNDi), we have been examining new
chemical series with anti-trypanosomal activity as potential
leads for drug discovery programs.18 Sulphonamide hits 6–8
(Fig. 2)19 with moderate to good potency (pIC50 4.92–5.82)
and selectivity were identified in a cidal high-throughput
d Drugs for Neglected Diseases initiative (DNDi), 15 Chemin Louis-Dunant, 1202
Geneva, Switzerland
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00165a
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phenotypic screen of a commercial library against axenic L.
don. amastigotes (MHOM/SD/62/1S-CL2, LdBOB), as
previously described.20 Further profiling indicated that
intracellular amastigotes of L. inf. were slightly more sensitive
(pIC50 6.23–6.35). However, all these hits were highly
lipophilic (clog P ≥ 5) and were rapidly cleared in vitro in the
presence of both human and mouse microsomes (S9
fractions). To rapidly investigate their structure–activity
relationship (SAR), a library of approximately 100 commercial
analogues was purchased and tested in parallel against
intracellular amastigotes of both L. inf. and L. don. (see ESI†).
Active compounds could be clustered into two sub-sets, both
of which contained a 2-alkoxybenzene-sulphonamide moiety
(Fig. 2). The first sub-set consisted of a long alkyl chain (at
least
4 carbons) on the ortho-substituted oxygen and
possessed a secondary sulphonamide. The second sub-set
consisted of a simple methoxy group at the ortho position
and
a
tertiary sulphonamide. Of these sub-sets, no
compounds offered a significant advantage over the initial
hits in terms of potency or lower lipophilicity. The previous
heightened potency against L. inf. over L. don. (3 to 5-fold)
was consistently observed as well, although further
investigation of this discrepancy was not pursued in the
scope of this work. A hit-to-lead program was initiated
focusing primarily on the tertiary sulphonamide sub-set (a)
Fig. 1 Current available drugs for visceral leishmaniasis.
Fig. 2 Initial hits and preliminary structure activity relationship (SAR).
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as these compounds offered slightly higher potency and lower
lipophilicity, as well as ease of synthesis. It was also felt that
improved metabolic stability would be more readily achieved
without a large lipophilic alkyl chain in the molecule. The
initial hits were divided into two different subsets: tertiary
sulphonamides (hit 6) and secondary sulphonamides (hits 7
and 8). Attempts to reduce lipophilicity of the hit 6 by replacing
the tertiary sulphonamide moiety with a secondary one led to
complete loss of potency. The observed loss of potency most
likely is related to the drop in logD that such modification
implies. A mandatory requisite for the potency of secondary
sulphonamides (hits 7 and 8) is the presence of a fatty alkoxy
chain on the 2 position of the benzenesulfonyl core. Overall, this
requirement tends to increase the log D for this class of
analogues, leading to compounds with poor metabolic stability.
Initially, a variety of substituents in the parent aromatic
ring
was
explored
in
combination
with
the
dibenzylsulphonamide portion (Table 1). It was found that
the 4-methyl group from the original hit 6 was not necessary
for activity (compound 9), allowing
a
reduction in
Table 1 Variation of benzene sulphonamide substituents
c
Microsome Clint
(μL min−1 mg−1
)
L. inf.
pIC50
PMM
pIC50
elog
Compound
R1
R2
Human
Mouse
Dd
LLE
a
b
9
2-OMe
2-OMe
4-OMe
2-F
OMe
H
H
F
H
H
H
CN
CN
F
6.43 0.24
6.48 0.00
4.34 0.15
<4.19
<4.19
<4.19
<4.19
<4.19
<4.19
<4.19
<4.19
4.34 0.15
<4.19
<4.19
<4.19
<4.19
<4.19
242
n.a.
184
n.a.
699
417
92.9
27.4
304
955
407
<23
108
810
1300
n.a.
2412
n.a.
n.a.
2880
689
1100
n.a.
n.a.
1352
94.0
1670
2025
4.6
n.a
4.7
n.a.
4.3
4.0
5.1
3.7
4.3
4.7
5.0
2.5
3.6
3.8
2.86
2.68
0.70
0.07
1.40
1.77
1.15
1.10
2.88
2.11
1.61
1.11
2.40
3.02
10
11
12e
13
14
15
16e
17
18
19
20
21
22
2-CN
4.95 0.00
4.49 0.06
5.96 0.21
4.51 0.05
6.36 0.04
5.86 0.04
5.86 0.04
4.24 0.05
5.13 0.04
6.34 0.04
2-SO2Me
2-OCF3
2-OH
2-OMe
2-OMe
2-OMe
2-OMe
2-OMe
2-OMe
Cl
CO2H
CONH2
4.34 0.15
23e
2-OMe
6.10 0.00
<4.19
178
395
4.0
3.11
24e
25e
2-OMe
2-OMe
6.28 0.02
6.49 0.00
<4.19
<4.19
49.4
421
3200
1400
4.3
4.2
2.84
2.98
26e
27
28
29
2-OMe
2-OMe
2-OMe
SO2Me
SO2NH2
SOMe
5.89 0.21
6.25 0.02
6.23 0.13
6.18 0.08
<4.19
<4.19
<4.19
<4.19
296
94.1
233
518
n.a.
884
929
1370
3.9
3.6
3.5
4.0
3.04
3.84
3.08
3.69
30
31
6.19 0.09
4.68 0.21
4.19
4.19
849
676
2567
2450
4.3
3.7
3.20
2.42
a
Geometric mean of a minimum of two experiments standard error. Assay was run according to experimental description (see ESI†) using
b
miltefosine as a positive control. Geometric mean of a minimum of two experiments
standard error. Assay was run according to
c
experimental description (see ESI†) using tamoxifen as a positive control. Intrinsic in vitro clearance calculated using liver microsomes.
d
HPLC measured log D. e Analogues with two p-fluorobenzyl groups on sulphonamide nitrogen instead of simple benzyl groups.
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lipophilicity as well as removing a possible metabolic soft
substituent was also not essential for potency (compound
spot. As observed in the initial hits 7 and 8, the 5-methoxy
10), allowing removal of the p-dimethoxyaryl moiety, which
Table 2 Variation of substituents on sulphonamide nitrogen
c
Microsome Clint,u
(μL min−1 mg−1
)
L. inf.
pIC50
PMM
pIC50
elog
Entry
R1
R2
R3
Human
n.a.
Mouse
n.a.
Dd
LLE
2.49
a
b
32
OMe
C6H4F
6.27 0.00
<4.19
4.7
33
34
OMe
OMe
C6H4F
C6H4F
4.94 0.25
4.27 0.08
<4.19
482
498
1560
3148
4.3
1.60
1.58
<4.19
3.8
35
36
OMe
OMe
C6H4F
C6H4F
5.28 0.26
5.11 0.05
4.34 0.15
651
350
5480
3054
3.5
3.5
3.49
3.03
<4.19
37
38
39
OMe
OMe
OMe
Me
Et
C6H4F
Ph
Ph
4.93 0.04
5.60 0.03
5.68 0.00
<4.19
<4.19
<4.19
414
534
253
n.a.
3497
n.a.
3.7
3.8
3.5
2.67
3.04
3.62
40
41
42
OCF3
CN
C6H4F
C6H5F
C6H5F
5.08 0.11
5.05 0.03
4.36 0.06
<4.19
<4.19
<4.19
194
<23
28
222
585
60
3.8
3.4
2.9
1.78
2.98
2.74
43
44
CN
C6H5F
C6H5F
<4.19
<4.19
<4.19
194
253
3.4
3.6
1.64
2.08
5.16 0.13
1050
1400
45
Ph
6.00 0.10
5.76 0.06
5.35 0.25
4.34 0.15
<4.19
<4.19
<4.19
<4.19
1600
265
73
2580
534
152
379
3.8
3.7
3.5
3.6
2.45
2.44
2.86
0.89
46e
47e
48
C6H5F
Ph
tBu
Ph
143
49
50
CN
CN
5.41 0.16
4.41 0.23
<4.19
<4.19
100
n.a.
95
3.8
3.8
2.76
1.13
<23
a
Geometric mean of a minimum of two experiments standard error. Assay was run according to experimental description (see ESI†) using
b
miltefosine as a positive control. Geometric mean of a minimum of two experiments
standard error. Assay was run according to
c
experimental description (see ESI†) using tamoxifen as a positive control. Intrinsic in vitro clearance calculated using liver microsomes.
d
HPLC measured log D. e Expected degradation product would be carboxylic acid 20 which was inactive.
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has been associated with inherent toxicity risks. The 2-alkoxy
group, however, was confirmed to be crucial, since removal
resulted in a loss of potency (compounds 11–14). The
2-trifluoromethoxy analogue 15 retained some potency (pIC50
= 5.96), but with a large increase in lipophilicity (elog D = 5.1)
and therefore was not further pursued. Removal of the alkyl
ether group to give a phenol was investigated, and 5-cyano
analogue 16 was chosen because of ease of synthesis,21 but
resulted in loss of potency (pIC50 = 4.51). Re-installing a
potency (pIC50 = 5.68) as well as lower lipophilicity (elog D =
3.5) and slightly improved metabolic stability compared to
previous analogues. Replacing the 2,5-dimethoxybenzene
group of 38 with other substituents from Table 1, such as the
cyano and morpholine amide moieties, further improved the
in vitro clearance as exemplified by compounds 41 and 42,
but with a loss of potency (pIC50 = 5.05 and 4.36 respectively).
It is possible that the elog D of this series has a lower limit of
about 3.5, as no compounds have been observed with good
potency (pIC50 ≥ 6.0) below this value.
methoxy group (compound 17) restored potency (pIC50
=
6.36), confirming that the phenol group is not tolerated.
At this point, results from metabolite identification
(MetID) experiments (see ESI† Tables S2 and S3) suggested
that the benzyl CH2 was indeed the major metabolic soft spot
as previously postulated. Several analogues containing
sterically hindered aliphatic groups as lipophilic alternatives
to the benzyl group were synthesised to reduce clearance
whilst maintaining potency (Table 2, compounds 43–45). The
more polar 4-tetrahydropyranyl 43 was inactive and the
isobutyl analogue 44 only possessed moderate potency
against L. inf. (pIC50 = 5.16). The bulkier neopentyl analogue
45 showed good potency (pIC50 = 6.00) albeit without much
improvement in metabolic stability. The substituents which
led to the largest improvement in clearance without
completely compromising potency were trifluoro-pyridine
and trifluoro-pyrimidine analogues (Table 2, compounds
46–47). Replacement of a phenyl group with a trifluoro-
pyridine, as in 46, gave a 4-fold improvement in the in vitro
mouse clearance but suffered from a decrease in potency
(pIC50 = 5.76). The trifluoro-pyrimidine analogue 47 gave a
further 4-fold improvement in clearance but once more with
a drop in potency (pIC50 = 5.35). Attempts to switch the
morpholine amide for a nitrile group (compound 49) did not
lead to any improvement, and the replacement of the second
benzyl group with a neopentyl (compound 48) or another
trifluoropyridyl (compound 50) resulted in inactive
compounds, albeit with further improvements in metabolic
stability.
The substituent in the 5-position was further explored and
found to be tolerant towards almost all changes (see Table 1,
compounds 17–28), except for the carboxylic acid 20 (pIC50
=
4.24). Nitrile 17, amides 22 and 23, sulfone 26, sulphonamide
27, and sulfoxide 28 were the most promising analogues
offering the best ligand-lipophilicity efficiency (LLE) and
improved in vitro clearance (albeit still too high for in vivo
studies). Morpholine 25 was the most potent of these
analogues (pIC50 = 6.49) but was not further pursued due to
perceived toxicity risks associated with anilines, and the
likelihood of oxidation to
intermediate.
a reactive iminoquinone-like
It was hypothesized that the large degree of tolerability
around the 5-position was consistent with these substituents
pointing towards solvent. Compound 29 provides more
support for this idea, as the capped methoxyethoxymethyl
(MEM) derivative still possessed sub-micromolar potency,
raising the exciting prospect of using polyethyleneglycol
(PEG) based linkers as tool compounds to explore the
mechanism of action.
Finally, we investigated some heterocyclic aromatic
sulphonamides, such as compound 30, which retained
potency but led to poor metabolic stability. Pyridinone
analogues unfortunately showed a significant loss of potency
(see Table 1, compound 31 and ESI†).
The lack of improvement in in vitro clearance from
changes to the upper portion of the molecule suggested that
metabolic soft spots are likely to be present in the
substituents on the sulphonamide nitrogen. It was hoped
that changes here could result in improved metabolic
stability. The two suspected spots for metabolic oxidation
were the para-positions of the benzene ring and the benzyl
positions. Firstly, fluorination at the para-position was tested
and offered similar potency (pIC50 = 6.27 for 32 versus 6.43
for 9), although clearance data could not be obtained for this
analogue (failed in automatic detection high-throughput
assay). Replacement of the 4-fluorobenzyl group with a
benzene ring (or pyridine – see ESI†) reduced potency by
more than 1.5 log units (Table 2, compound 33, pIC50 = 4.94).
We next examined smaller or more polar alternatives (see
Table 2, compounds 33–42 and ESI†). At best, these analogues
led to a 0.75log unit reduction in potency versus the parent
dibenzyl analogue 9, and in some cases even complete loss of
activity. The most promising analogue was the
methoxyethane derivative 38 which still possessed moderate
Despite not having identified a likely lead, the compounds
with the best compromise between potency and clearance,
trifluoro-pyridine 46 and trifluoro-pyrimidine 47, were
examined in more detail (Table 3). In addition to the poor
in vitro clearance it was found that these compounds had
extremely low aqueous solubility, which is likely to be due to
Table 3 Further DMPK assays of compounds 46 and 47
Compound
46
47
L. inf. pIC50
In vitro Clint (human)
5.76
5.35
265 μL min−1
mg−1
73 μL min−1 mg−1
In vitro Clint (mouse)
534 μL min−1
mg−1
152 μL min−1
mg−1
Plasma protein binding
(PPB)
95.9%
88.3%
Mouse plasma stability (6 h) 22% remaining
Kinetic solubility (pH 2.0)
Kinetic solubility (pH 7.4)
42% remaining
11.3 μg mL−1
6.2 μg mL−1
3.8 μg mL−1
3.2 μg mL−1
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their high lipophilicity. Furthermore, they possessed
surprisingly poor stability with only 22% and 42% (for 46
and 47, respectively) remaining after 6 h incubation in mouse
plasma. Amide hydrolysis may be responsible for poor
stability, although this has not been observed in microsomes,
but further analogues would need to be tested to confirm this
hypothesis.
Despite evaluating almost 200 compounds, no suitable
candidate with an acceptable balance of potency and
physicochemical properties could be identified, and it was
decided to halt further work on this series. In particular, the
potency of these sulphonamides appears to be dependent on
lipophilicity with almost all compounds with elog D < 3.5
losing activity. As can be seen in Fig. 3, it proved difficult to
synthesise compounds with LLE > 4. It should be noted that
attempts to return to the initially identified subset of
secondary sulphonamides (Fig. 2) and improve lipophilicity
also led to significant reduction in potency (see ESI†).
One of the reasons why drug discovery is notoriously
challenging in leishmaniasis is the difficulty of reaching the
parasites. The amastigotes reside in parasitophorous vacuoles
inside the hosts' macrophages,22,23 requiring molecules to
cross three cell membranes with differing pH gradients.10
There are several examples of target-based approaches for
leishmaniasis failing to translate into cell-based assays due
to this difficulty in permeating the parasite.24–26 This may be
Despite the difficulties in optimizing these compounds,
this series showed good potency and selectivity against
intracellular forms of Leishmania spp. covering several
species of CL and VL (see ESI†). The possibility of using these
tool compounds in detailed mode of action studies is
currently being explored. Such studies may provide an
opportunity to use target-based medicinal chemistry to
overcome the current issues with this series.
Material and methods
Parasite and cell cultures
Leishmania infantum MHOM/MA(BE)/67/ITMAP263 and L.
donovani MHOM/ET/67/L82 amastigotes were collected from
the spleen of an infected donor Golden hamster and used to
infect primary peritoneal mouse macrophages (PMM). PMM
were collected 2 days after peritoneal stimulation of Swiss
mice with a 2% potato starch suspension and maintained in
RPMI-1640 medium supplemented with 200 mM L-glutamine
and 5% FCS. Cultures and assays were maintained at 37 °C
under an atmosphere of 5% CO2.
Compound solutions/dilutions
Compound stock solutions were prepared in 100% DMSO at
20 mM. The compounds were serially pre-diluted (2-fold) in
100% DMSO to maintain maximal solubility, followed by a
one-step further dilution in demineralized water to achieve a
final in-test DMSO concentration of <1%. The compounds
were tested at 2-fold compound dilutions covering a range of
64 to 0.00024 μM.
responsible
for
the
loss
of
potency
in
the
benzenesulphonamide compounds with lower lipophilicities.
Additionally, given the species difference in drug sensitivity
observed during initial SAR exploration, we also examined
the activity of several analogues against L. inf. in a different,
immortalized, cell line (THP-1). We noticed significant
variability in potency (see ESI† Table S1), suggesting different
host cell characteristics and host–parasite interactions may
play an important role in the antileishmanial activity of this
series and ultimately impact hit discovery campaigns.27–29
This may be of relevance when considering host cell selection
for future screening efforts against leishmaniasis, particularly
given the ethical and logistical constraints of using primary
versus immortalized cells.
Susceptibility assay
For the intracellular amastigote susceptibility assay, 3 × 104
PMM were seeded in a 96-well plate. After 24 h, 5 × 105
amastigotes per well were added and incubated for 2 h at 37
°C. The compound dilutions were added next and the plates
were further incubated for 5 days at 37 °C and 5% CO2. Total
parasite burdens were microscopically assessed after Giemsa
staining. In treated wells with high amastigote burdens, an
overall estimate of the total burden per well was made
without discrimination between the number of infected
macrophages and the number of amastigotes per infected
cell. In treated wells with low burdens, exact counting was
performed. The results were expressed as % reduction in
parasite burdens compared to control wells and an IC50 was
calculated. Miltefosine was included as the reference drug
(IC50 ∼5–10 μM).
Cytotoxicity assays
The evaluation of toxicity to PMM was part of the in vitro
susceptibility assay, determined by microscopic evaluation of
cell detachment, lysis, and granulation. Evaluation was done
by semi-quantitative scoring (no exact counting was
performed) of at least 500 cells distributed over adjacent
microscopic fields. The results were expressed as % reduction
Fig. 3 Plot of lipophilicity versus potency.
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in normal cells compared to untreated control wells and an Conflicts of interest
CC50 (50% cytotoxic concentration) was determined.
Tamoxifen was used as reference for cytotoxicity (CC50 ∼ 5–
10 μM).
The authors declare no competing interests.
Acknowledgements
We would like to thank FAPESP (Grants 2015/19495-5, 2015/
50655-9 and 2013/07600-3) as well as DNDi for funding;
UNICAMP NMR and technical staff for assistance; The
Thomson Mass Spectrometry Lab at UNICAMP for use of
HRMS equipment; the High-Throughput ADME team at
AbbVie for ADME testing and Dr. Dale Kempf (AbbVie) and
Dr. Michael Schrimpf (AbbVie) for helpful discussions; the
Translational Parasitology Group at the Drug Discovery Unit,
University of Dundee, for carrying out the primary screen and
initial analogue testing; DNDi is grateful to its donors, public
and private, who have provided funding for all DNDi activities
since its inception in 2003. A full list of DNDi's donors can be
found at http://www.dndi.org/donors/donors/.
ADME assays
For experimental determination of log D, test compounds
were prepared at a physiologically relevant pH of 7.4 at 200
nM and 2% DMSO in a 50/50 mix of mobile phase A (5%
methanol in 10 mM ammonium acetate and adjusted to pH
7.4) and B (100% methanol adjusted to pH 7.4) with an
appropriate internal standard at 4 nM, and injected onto an
Ascentis Express RP Amide column. Retention times were
compared to a standard curve of nine commercial drugs
covering a log D range of −1.86 to 6.1. The retention times of
each of the standards was plotted against the published log D
values. The resulting equation for this line (y = mx + b) was
used to calculate the log D values for the test compounds
where ‘x’ was the retention time in minutes of the test References
compound and the resultant “y” was the experimental log D
value.
For the experimental stability determination of test
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compounds in liver microsomes in the presence of NADPH,
a clearance rate was determined. Assay conditions were
0.25 mg mL−1 liver microsomal protein from the species of
interest (mouse and human), 0.5 μM test compound, pH
7.4 and 37 °C. The reagent was purchased commercially,
and the work did not involve the use of animals or
humans. Samples were taken at 0, 5, 10, 15, 20 and 30
min. The reaction was started after T0 was taken, with the
addition of NADPH at 0.5 μM. The reaction was stopped by
addition of 95% acetonitrile/5% methanol containing an
internal standard. Time point samples were combined in
compound groups of six that had been pre-sorted by mono
molecular weight and analyzed by LC/MS/MS. Peak area
ratios (analyte peak area/internal standard peak area) were
converted to % remaining using the area ratio at time 0 as
100%. Half-life (t1/2 = ln(2)/k) and intrinsic clearance (Clint
= k × 1000/(0.25)) in μL min−1 mg−1 were calculated from %
remaining versus incubation time. From this plot, the slope
(k) was determined. Data was qualified if t1/2 > 4X the last
time point.
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Author contribution
P. J. K. took the lead in writing the manuscript; P. J. K. and
M. A. D. carried out design and synthetic chemistry efforts; A.
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