Benzylmorpholine analogs as selective inhibitors of lung cytochrome P450 2A13 for the chemoprevention of lung cancer in tobacco users

Article abstract of DOI:10.1007/s11095-013-1054-z

Purpose: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. Methods: Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is a major challenge in compound design. Results: A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. Conclusions: Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure.

Full text of DOI:10.1007/s11095-013-1054-z

Pharm Res
DOI 10.1007/s11095-013-1054-z
RESEARCH PAPER
Benzylmorpholine Analogs as Selective Inhibitors of Lung
Cytochrome P450 2A13 for the Chemoprevention of Lung
Cancer in Tobacco Users
Linda C. Blake & Anuradha Roy & David Neul & Frank J. Schoenen & Jeffrey Aubé & Emily E. Scott
Received: 14 January 2013 /Accepted: 2 April 2013
#
Springer Science+Business Media New York 2013
. .
KEY WORDS chemopreventative cytochromeP4502A13
cytochrome P450 2A6 lung cancer tobacco
ABSTRACT
.
.
Purpose 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK), one of the most prevalent and procarcinogenic
compounds in tobacco, is bioactivated by respiratory cytochrome
P450 (CYP) 2A13, forming DNA adducts and initiating lung
cancer. CYP2A13 inhibition offers a novel strategy for chemopre-
vention of tobacco-associated lung cancer.
Methods Twenty-four analogs of a 4-benzylmorpholine scaffold
identified by high throughput screening were evaluated for binding
and inhibition of both functional human CYP2A enzymes,
CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibi-
tion is undesirable. Thus, selectivity is a major challenge in com-
pound design.
ABBREVIATIONS
CYP
Cytochrome P450
DMSO Dimethyl sulfoxide
hERG
Kd
Ki
Human ether-a-go-go-related gene
Equilibrium dissociation constant
Inhibition constant
NNK
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone
PEITC Phenethyl isothiocyanate
POR
SAR
NADPH-cytochrome P450 oxidoreductase
Structure activity relationship
Results A key feature resulting in CYP2A13-selective binding
and inhibition was substitution at the benzyl ortho position, with
three analogs being >25-fold selective for CYP2A13 over
CYP2A6.
INTRODUCTION
Conclusions Two such analogs were negative for genetic and
hERG toxicities and metabolically stable in human lung micro-
somes, but displayed rapid metabolism in human liver and in
mouse and rat lung and liver microsomes, likely due to CYP2B-
mediated degradation. A specialized knockout mouse mimicking
the human lung demonstrates compound persistence in lung and
provides an appropriate test model. Compound delivered by
inhalation may be effective in the lung but rapidly cleared otherwise,
limiting systemic exposure.
The cytochrome P450 (CYP) superfamily of heme-
containing monooxygenase enzymes metabolizes a variety
of exogenous and endogenous substrates which can be
manipulated for therapeutic benefit (1). For example, the
selective inhibition of androgen and estrogen biosynthetic
CYP enzymes has afforded significant progress in the treat-
ment of prostate and breast cancer (2,3). In contrast, inhibition
of xenobiotic-metabolizing CYP enzymes has often been
viewed as an unproductive approach to drug discovery due
:
:
L. C. Blake J. Aubé E. E. Scott (*)
Department of Medicinal Chemistry, University of Kansas
1251 Wescoe Hall Dr.
D. Neul
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc. La Jolla
California 92121, USA
Lawrence Kansas 66045, USA
e-mail: eescott@ku.edu
:
F. J. Schoenen J. Aubé
University of Kansas Specialized Chemistry Center
University of Kansas, 2034 Becker Drive
Lawrence Kansas 66047, USA
A. Roy
High Throughput Screening Laboratory, University of Kansas
2034 Becker Drive
Lawrence Kansas 66047, USA

Blake et al.
to selectivity issues expected for such agents. Despite this, a
selective CYP3A4 inhibitor has now been deliberately devel-
oped from retonavir for explicit use as pharmacokinetic booster
(4–6). Similarly, inhibition of CYP enzymes involved in the
activation of procarcinogens can offer chemopreventative op-
portunities; such is the case in tobacco-associated lung cancer.
Lung cancer is the leading cause of all cancer-related deaths (7),
and >80% of all lung cancer incidence results from tobacco use
(8). According to the CDC, 69% of tobacco users desire to quit
(9), but due to the addictive properties of nicotine and ineffec-
tiveness of current tobacco cessation techniques, only 5% of
users successfully cease tobacco use (10). A complementary
strategy to reduce lung cancer prevalence is to decrease the in
vivo formation of tobacco carcinogens. 4-(Methylnitrosamino)-
1-(3-pyridyl)-1-butanone (NNK) is one of the most prevalent
and, when activated, one of the most carcinogenic compounds
in tobacco. NNK is selectively biotransformed by cytochrome
P450 2A13 (CYP2A13) in the respiratory tract to form one of
two diazonium ions that generate DNA adducts in genes like
p53 and ras and can initiate lung cancer (11,12).
A CYP2A13 inhibitor, therefore, offers a novel therapeu-
tic strategy to reducing the risk of lung cancer in tobacco
users. This target is validated by CYP2A13 polymorphisms
and animal studies. Tobacco users with natural-occurring
polymorphisms resulting in reduced CYP2A13 activity
exhibit normal physiological characteristics but have a
decreased risk for lung adenocarcinoma (13,14). Mouse
studies have substantiated the role of the respiratory
tract (15) and the CYP2A13 ortholog, CYP2A5 (16),
in NNK bioactivation. NNK-induced tumors were also
reduced from 70% to 5% in rats with the known pan-
CYP2A inhibitor, phenethyl isothiocyanate (PEITC)(17).
Finally, CYP2A13 inhibition has been correlated to a
decrease in NNK bioactivation and an increase in NNK
clearance via nontoxic glucuronidation and excretion
(18). Thus, once CYP2A13 is inhibited, an increased
amount of NNK is cleared via nontoxic, non-
carcinogenic metabolites.
drug metabolism are also much less likely to have adverse
effects, so long as the inhibitors are selective and do not
inhibit other CYP enzymes.
The largest challenge in developing CYP2A13 inhibition
as a chemopreventative approach is likely to be nonselective
inhibition of the only other functional human CYP2A en-
zyme, the 94% identical hepatic CYP2A6. CYP2A6 shares
a number of substrates and inhibitors with CYP2A13, but is
primarily involved in vivo in the metabolism of drugs in
systemic circulation, including nicotine, and does not effec-
tively bioactivate NNK or contribute to DNA adduct for-
mation in the respiratory tract (20,21). The emphasis of this
work was the identification and evaluation of small molecule
inhibitors of CYP2A13 that are selective vs. CYP2A6. High
throughput screening was initially used to identify a 4-
benzylmorpholine scaffold that might have the potential to
effectively and selectively inhibit CYP2A13. A library of
twenty-four 4-benzylmorpholine analogs was then generated.
Analysis of differential binding to and inhibition of recombi-
nant, purified CYP2A13 and CYP2A6 enzymes in vitro led to
the determination of structure-activity relationships for
the 4-benzylmorpholine scaffold with the human
CYP2A enzymes that may form the basis for future
studies toward lung cancer chemoprevention.
MATERIALS AND METHODS
Protein Expression and Purification
Human CYP2A enzymes were truncated, His-tagged,
expressed, and purified as previously described (22,23).
Rat cytochrome P450 oxidoreductase (POR) was purified
using published procedures (24).
High Throughput Screening
The most significant difficulty in developing effective
P450 inhibitors arises in designing selectivity for an individ-
ual enzyme in this superfamily. In contrast to biosynthetic
CYP enzymes like aromatase that are fairly specific for their
substrates, most xenobiotic-metabolizing CYP enzymes
have evolved to recognize and act upon a wide range of
chemically diverse small molecules. This is often accom-
plished via large and/or flexible active sites that frustrate
structure-based predictions of ligand binding. For example,
the large active site of the hepatic CYP3A4 enzyme is
responsible for the metabolism of 34% (19) of clinically used
drugs, so that even a selective CYP3A4 inhibitor such as the
pharmacokinetic booster cobicistat can frequently have sub-
stantial adverse drug-drug interactions. Extrahepatic CYP
enzymes such as CYP2A13 that are much less involved in
The spectral shift assay (vide infra) reflecting interactions of
compounds with CYP2A13 was adapted to HTS format for
screening ChemBridge library of 35,200 compounds. Each
384-well screening plate contained 320 library compounds
(20 μM in 0.71% DMSO) and a set of standards com-
prising 32 wells: 16-wells each of 0.71% DMSO (negative
control), and 16-wells of positive control (30 μM phenethyl
isothiocyanate or PEITC). Purified CYP2A13 (3 μM) was
added to all the wells of the assay plates and the
protein/compound mixtures were allowed to equilibrate at
room temperature for 5 min. The plates were read on
SpectraMax (Molecular Devices, CA) at 385 nm and
420 nm. Primary screen actives were identified as compounds
that resulted in difference of 0.1 (plate median + 5 standard
deviation) in raw absorbance values at 385 nm and 420 nm.

Benzylmorpholine Inhibitors of Cytochrome P450 2A13
Spectral Ligand Binding Assay
Inhibitors
Ligand binding was monitored by following spectral
changes upon titration with increasing concentrations
of ligand and the data analyzed as previously described
(25), except that all compounds were dissolved in
DMSO. However, the imidazole-containing compounds
bound tightly enough to require a reduction in protein
concentration to 0.1 μM, use of a 5 cm path length
cuvette, and analysis with the tight binging equation as
described previously (26).
The benzylmorpholine analogs 4-(2-fluorobenzyl)
morpholine (2), 4-(2-chlorobenzyl)morpholine (3),
4-(2-bromobenzyl)morpholine (4), 4-(2-methylbenzyl)
morpholine (5), 4-(2-chloro-6-fluorobenzyl)
morpholine (7), 4-(2,6-dichlorobenzyl)morpholine
(8), 4-(2,3-dichlorobenzyl)morpholine (12), 4- (2,4-
dichlorobenzyl)morpholine (13), 4-(2,3-
dichlorobenzyl)-2,6-dimethylmorpholine (14),
4-(naphthalen-1-ylmethyl)morpholine (15), and 4-(2-
chlorobenzyl)thiomorpholine (16) were purchased from
ChemBridge. The stereochemistry of the methyl substitu-
ents on 14 is unknown but the supplier indicates it is be-
lieved to be racemic.
Coumarin Metabolism Assay (27,28)
The 50 pmol of purified CYP2A enzyme was reconstituted
with 200 pmol of POR by coincubation on ice for 50 min
before addition to the reaction samples containing buffer
(50 mM Tris, HCl, pH 7.4, 5 mM MgCl), 0–90 μM cou-
marin substrate, and varying inhibitor concentrations.
Samples were warmed for 3 min at 37°C before addition
of 1 mM NADPH to initiate the 250 μl reaction. Catalysis
proceeded for 10 min before termination with 75 μl of 20%
trichloroacetic acid (TCA) to precipitate protein. TCA was
added to all standard samples prior to NADPH addition.
Additional buffer (200 μl) and 100 μl of 1.6 M glycine were
added to each tube before centrifugation at 4,400 ×g for
6 min. CYP2A6 samples required an additional 5-fold dilu-
tion due to detector limitations. Formation of the 7-
hydroxycoumarin metabolite was monitored via fluores-
cence detection with an excitation wavelength of 355 nm
and an emission wavelength of 460 nm following chromato-
graphic separation with a reverse phase C18 column on an
HPLC. A mobile phase of 40% methanol and 0.12% acetic
acid was used with a flow rate of 0.8 mL min−1. A small
amount of a second product, likely the 3,4-epoxide metab-
olite (29), was observed at the lowest substrate concentration
(5 μM) in the absence of inhibitor and automated outlier
exclusion analysis with the data analysis software Prism 5
was used to handle this data point. Prism 5 was also used for
data analysis of the six substrate and four inhibitor concen-
trations resulting in the kcat and Km values that were used to
determine the mode of inhibition and ultimately Ki value for
each compound with the CYP2A enzymes with the appro-
Benzylmorpholine (1),(30) 4-(2-ethylbenzyl)
morpholine (6), 4-(2-chloro-6-methylbenzyl)
morpholine (9), 4-(3-chlorobenzyl)morpholine (10),
4-(4-chlorobenzyl)morpholine (11),(31) 1-(2-
chlorobenzyl)-4-methylpiperazine (17), 1-(2,6-
dichlorobenzyl)-4-methylpiperazine (18), 4-
(thiophen-2-ylmethyl)morpholine (19), 4-((3-
chlorothiophen-2-yl)methyl)morpholine (20), 1-(2-
chlorobenzyl)-4-methylpiperazine (21), and 1-(2,6-
dichlorobenzyl)-4-methylpiperazine (22) were syn-
thesized in a MiniBlock® apparatus using published pro-
tocols (32). Synthesis of 4-(1-(2-chlorophenyl)
ethyl)morpholine (23) followed the same procedure(32)
in a reaction flask, which resulted in a racemic mixture.
Production of 1-(2-chlorobenzyl)-1H-imidazole (17)
and 1-(2,6-dichlorobenzyl)-1H-imidazole (18) also
used previously established methods (33). 4-(2-
Chlorophenethyl)morpholine (24) was synthesized
by combining 2-chlorophenethyl bromide (1.3 mmol),
morpholine (1.0 mmol), potassium carbonate (2.0 mmol)
in 4 mL of dry acetonitrile and stirred at 80°C over-
night. Product was extracted with ethyl acetate, washed
with sodium bicarbonate, dried with magnesium sulfate,
and solvent was removed under vacuum.
Compound analysis by 1H and 13C NMR was performed
on a Bruker 400 MHz instrument. TopSpin and MestReNova
were used to analyze the NMR data, which is given in ppm.
LCMS was completed with a Waters 2795 HPLC and Waters
LCT Premier TOF mass spectrometer. Compound purity
was determined to be >95% by HPLC reverse phase chro-
matography. The HPLC mobile phase consisted of a 5–95%
organic gradient with solvents A (99% water (pH 9.8 ammo-
nium hydroxide), 1% acetonitrile) and B (99% acetonitrile,
1% water) and a flow rate of 0.6 mL/min.
priate formula shown below.
Vmaxꢀ½Sꢁ
Competitive inhibition: Y ¼
and Km Obs ¼ Km
Km Obsþ½Sꢁ
ꢀ
ꢁ
½Iꢁ
1 þ K where Km Obs is the observed Km, [I] is the
i
inhibitor concentration, Ki is the inhibition constant, and
[S] is the substrate concentration. Mixed model inhibition:
ꢀ
ꢀ
ꢁ
ꢁ
½Iꢁ
1þKi
4-(2-Ethylbenzyl)morpholine (6). The synthesis afforded a
Vmax app
Km appþ½Sꢁ
Vmax
ꢀ
ꢁ
Y ¼
ꢀ½Sꢁ , Km app ¼ Km ꢀ
, and Vmax app
¼
clear oil with a yield of 77.9 mg (35%). HRMS (m/z):
½Iꢁ
½Iꢁ
1þaK
1þaK
1
i
i
[M+H] 206.15; calculated 206.15. H NMR (400 MHz,

Blake et al.
chloroform-d) δ 7.35−7.12 (m, 4H), 3.79−3.67 (m, 4H),
3.52 (s, 2H), 2.78 (q, J=7.5 Hz, 2H), 2.48 (dd, J=5.8,
3.7 Hz, 4H), 1.27 (t, J=7.5 Hz, 3H).
4-(1-(2-Chlorophenyl)ethyl)morpholine (23). The synthesis
afforded a yield of 48.1 mg (21%) of an oil. HRMS (m/z):
1
[M+H] 226.10; calculated 226.09. H NMR (400 MHz,
chloroform-d) δ 7.60 (dd, J=7.8, 1.8 Hz, 1H), 7.35 (dd, J=
7.9, 1.4 Hz, 1H), 7.31−7.23 (m, 1H), 7.18 (dd, J=7.7,
1.8 Hz, 1H), 3.89 (q, J=6.6 Hz, 1H), 3.72 (dt, J=5.8,
3.6 Hz, 4H), 2.57 (dt, J=9.8, 4.6 Hz, 2H), 2.41 (ddd, J=
11.3, 5.8, 3.3 Hz, 2H), 1.31 (d, J=6.6 Hz, 3H).
4-(2-Chloro-6-methylbenzyl)morpholine (9). The synthesis
yielded 55.6 mg (27%) of an oily product. HRMS (m/z):
1
[M+H] 226.10; calculated 226.09. H NMR (400 MHz,
chloroform-d) δ 7.31−7.19 (m, 1H), 7.14−7.07 (m, 2H),
3.73−3.60 (m, 6H), 2.57−2.49 (m, 4H), 2.47 (s, 3H).
4-(2-Chlorophenethyl)morpholine (24). The synthesis
4-(3-Chlorobenzyl)morpholine (10). The synthesis resulted in
25 mg (44.7%) of a yellow oil. HRMS (m/z): [M+H] 212.09;
calculated 212.08. 1H NMR (400 MHz, chloroform-d) δ 7.37
(d, J=2.0 Hz, 1H), 7.33−7.05 (m, 3H), 3.81−3.62 (m, 4H),
3.49 (s, 2H), 2.46 (dd, J=5.7, 3.7 Hz, 4H).
resulted in 129 mg (58%) of a yellow oil. HRMS (m/z):
[M+H] 226.10; calculated 226.09. H NMR (400 MHz,
Chloroform-d) δ 7.39−7.03 (m, 4H), 3.88−3.62 (m, 4H),
3.00−2.83 (m, 2H), 2.65−2.40 (m, 6H).
1
Compound Solubility
1-(2-Chlorobenzyl)-1H-imidazole (17). The synthesis pro-
duced 30.7 mg (17%) of a yellow oil. HRMS (m/z): [M+H]
193.05; calculated 193.05. 1H NMR (400 MHz, chloroform-
d) δ 7.57 (s, 1H), 7.41 (dd, J=7.7, 1.6 Hz, 1H), 7.33−7.18 (m,
2H), 7.10 (s, 1H), 6.99−6.88 (m, 2H), 5.23 (s, 2H).
For compounds evaluated in binding and inhibition studies,
solubility was evaluated in buffer by monitoring turbidity in the
UV/Vis cuvettes. For test compounds advanced to toxicity and
metabolic stability studies, aqueous solubility was explicitly
determined in phosphate-buffered saline at pH 7.4 using the
shake-flask incubation method (24 h at room temperature).
The mean solubilities of 4-(2-chlorobenzyl)morpholine (3) and
4-(2-chloro-6-fluorobenzyl)morpholine (7) thus determined
were 180 and 184 μM, respectively. These values are near
the 200 μM maximum for the assay conditions and established
that solubility is generally high for these compounds.
1-(2,6-Dichlorobenzyl)-1H-imidazole (18). The resulting syn-
thetic product was a yellow oil with a yield of 171.3 mg
(75%). HRMS (m/z): [M+H] 227.01; calculated 227.01. 1H
NMR (400 MHz, chloroform-d) δ 7.61 (s, 1H), 7.39−7.24
(m, 2H), 7.17 (dd, J=8.8, 7.4 Hz, 1H), 6.98 (d, J=14.9 Hz,
2H), 5.33 (s, 2H).
4-(Thiophen-2-ylmethyl)morpholine (19). The synthesis
resulted in 130 mg (71%) of a yellow oil. HRMS (m/z):
[M+H] 184.08; calculated 184.07. H NMR (400 MHz,
Metabolic Stability Phenotyping with Recombinantly
Expressed CYPs
1
chloroform-d) δ 7.24 (dd, J=5.0, 1.3 Hz, 1H), 6.94 (ddd, J=
7.4, 4.4, 2.2 Hz, 2H), 3.75−3.68 (m, 6H), 2.53−2.45 (m, 4H).
The half-lives of select compounds were determined with a
panel of recombinantly expressed cytochrome P450 en-
zymes including 1A2, 2C9, 2D6, 3A4, and 2C19 (Pan
Vera Corp., Madison, WI) and 2C8 and 2B6 (BD Gentest,
San Jose, CA) in a plate assay format. Incubations consisted
of 100 pmol/ml of one of the above P450 enzymes and
1 μM of the inhibitor in 0.1 mM potassium phosphate,
pH 7.4, 3.33 mM MgCl2 in a total of 200 μl. These solu-
tions were prewarmed to 37°C for 10 min. prior to initiating
the reaction with the addition of 1 mM NADPH
regenerating system (NADP+, glucose-6-phosphate,
glucose-6-phosphate dehydrogenase in 0.1 mM postassium
phosphate). Aliquots of 25 μl were removed at various times
(0, 5, 10, 20, 30, 45 min) and immediately quenched by
addition to 75 μl of methanol containing buspirone as an
internal standard. Controls substituted potassium phosphate
buffer for the NADPH-generating system. Samples were
centrifuged at 3,000 rpm and 50 μl of the supernatant
diluted 1:1 with water. Mass spectrometry was used to
determine the peak areas for the inhibitors and internal
4-((3-Chlorothiophen-2-yl)methyl)morpholine (20). The syn-
thesis afforded a yellow oil with a yield of 150 mg (70%).
HRMS (m/z): [M+H] 218.04; calculated 218.03. 1H NMR
(400 MHz, Chloroform-d) δ 7.23 (d, J=5.3 Hz, 1H), 6.87
(d, J=5.3 Hz, 1H), 3.85−3.65 (m, 6H), 2.67−2.38 (m, 4H).
1-(2-Chlorobenzyl)-4-methylpiperazine (21). The synthesis of
21 yielded a yellow oil with a yield of 168.8 mg (75%).
HRMS (m/z): [M+H] 225.12; calculated 225.11. 1H
NMR (400 MHz, chloroform-d) δ 7.49 (dd, J=7.5,
1.9 Hz, 1H), 7.36 (dd, J=7.7, 1.5 Hz, 1H), 7.31−7.15 (m,
2H), 3.65 (s, 2H), 2.58 (s, 4H), 2.48 (s, 4H), 2.31 (s, 3H).
1-(2,6-Dichlorobenzyl)-4-methylpiperazine (22). The synthesis
resulted in 184.1 mg (71.0%) of an amorphous solid. HRMS
(m/z): [M+H] 259.08; calculated 259.07. 1H NMR (400 MHz,
chloroform-d) δ 7.26 (d, J=8.0 Hz, 2H), 7.09 (dd, J=8.5, 7.5 Hz,
1H), 3.73 (s, 2H), 2.60 (s, 5H), 2.43−2.34 (m, 3H), 2.24 (s, 3H).

Benzylmorpholine Inhibitors of Cytochrome P450 2A13
standard, from which the peak area ratios were calculated.
The ln of this ratio vs. time gave the slope, which was
converted to T1/2. All reactions were run in duplicate.
with both CYP 2A13 and the 94% identical CYP2A6, 93
compounds were found to bind to CYP2A13 specifically
(ΔAbs 385–419 nm >0.2) compared with CYP2A6 (ΔAbs
385–419 nm <0.03). Several CYP2A13-specific scaffolds were
tentatively identified. Analysis of few representative compounds
at varying concentrations resulted in identification of 4-(2-
chloro-6-fluorobenzyl) morpholine as a true selective ligand of
CYP2A13 vs. CYP2A6. As shown in the Fig. 1d, 4-(2-chloro-6-
fluorobenzyl) morpholine bound CYP2A13 with a Kd of ap-
proximately 2 μM, but caused no spectral shift with 2A6.
Thus, this study was designed to evaluate 4-benzylmorpholine
as a potential scaffold for CYP2A13-selective inhibitors. A pilot
library of twenty-four 4-benzomorpholine analogs was designed
to obtain structure–activity relationships in the parent scaffold
and constructed using standard chemistry. Both ligand binding
and enzyme inhibition were used to evaluate the interactions of
these analogs with purified, recombinant CYP2A enzymes.
Ligand binding is conveniently measured by observing, in differ-
ence mode, shifts in the electronic absorbance spectrum as
ligands enter the active site. Inhibition was measured as the
disruption of hydroxylation of the prototypical CYP2A substrate
coumarin to 7-hydroxycoumarin. The resulting Kd and Ki values
are shown in Table I, along with the selectivity, as calculated by
the ratio of the CYP2A6 value to the CYP2A13 value.
Titration of CYP2A13 and CYP2A6 with most of the 4-
benzylmorpholine analogs revealed binding primarily
resulted in type I spectral shifts in the UV/Vis difference
spectra, as indicated by (I) in the Kd column of Table I. This
transition from the Fe hexacoordinate low spin, water
bound state (λmin=~420 nm) to a pentacoordinate high spin
state (λmax=~385 nm), reflects ligand binding in the P450
active site so as to displace the water molecule normally coordi-
nated to the ferric heme iron in the resting state of the enzyme. In
contrast, binding of compounds 1, 10, and 18 to CYP2A6 and
binding of compounds 16 and 17 to both CYP2A13 and
CYP2A6 resulted in difference spectra with decreases in absor-
bance at 393–413 nm and increases in absorbance at ~433 nm
called type II spectra shifts. Such transitions upon ligand binding
indicate conversion from the hexacoordinate, low-spin, water-
bound iron to a hexacoordinate form in which the ligand typically
coordinates to the iron via its lone pair electrons. Thus, the spectra
of these compounds give an indication of the orientation of some
ligands with respect to the catalytic heme iron. Some compounds
yielded very limited or no spectral shifts with CYP2A6 which
indicates the compound is either not binding or not binding in the
active site in such a way that displaces water on the heme iron,
which are desirable features of a CYP2A13-selective inhibitor.
Inhibition studies indicated that all but seven 4-
benzylmorpholine analogs were purely competitive inhibi-
tors of both CYP2A13 and CYP2A6. However, mixed
inhibition resulting from inhibitor binding to both the free
and substrate-bound form of the enzyme was observed for
compounds 14, 16, and 17 with CYP2A13, and compounds
Metabolic Stability in Rat, Mouse, and Human Lung
and Liver Microsomes
The half-lives of select compounds were determined with
microsomes from pools of male B6C2F1 mouse liver, male
B6C2F1 mouse lung, male Sprague–Dawley rat liver, male
Sprague–Dawley rat lung, human liver and human lung
(Xenotech) and from transgenic B6 Cyp2a(4/5)bgs-Null mice
(35) as a gift from Dr. Xinxin Ding (Wadsworth Center). In
each case 1.0 or 0.1 mg protein/ml, respectively, was added to
50 mM potassium phosphate buffer, pH 7.4, with 3 mM
MgCl2, and 1 mM EDTA with and without the NADPH-
generating system and 1 μM test compound (in 0.1% DMSO)
to a total of 200 μl volume in a 96-well plate using a Tecan
Liquid Handling System at 37°C in triplicate. Reactions were
initiated by the addition of the NADPH-generating system
(1 mM NADP, pH 7.4, 5 mM glucose-6-phosphate, and 1
U/ml glucose-6-phosphate dehydrogenase) and were termi-
nated at 0, 5, 10, and 20 min by the addition of 200 μl
acetonitrile containing 0.1 μM antipyrene (v/v). Samples were
centrifuged at 920 x g for 10 min. at 10°C and the supernatant
analyzed by LC/MS/MS. The time course of disappearance
of the parent compound was evaluated as a single exponential
decay and used to determine the rate constant of elimination
values (kel, min−1) and converted to T1/2. The rate constant of
elimination was used to calculate the in vitro intrinsic clearance,
Clint microsomes : Clintꢂmicrosomesðμl= min =mg proteinÞ ¼
kel
ðminꢂ1Þꢃ incubation volumeðμlÞ=mg protein per incubation.
RESULTS AND DISCUSSION
To initiate a discovery effort to identify potential scaffolds for
selective inhibitors of human CYP2A13, a high throughput
screening campaign was conducted. A cuvette-based assay
based on UV/Vis absorbance changes upon ligand binding
(described in Materials and Methods) was miniaturized and
adapted for screening in 384-well format. Figure 1 summarizes
data from the high throughput screen. An average Z′ score of
0.89 0.08 was obtained from positive (CYP2A13+PEITC)
and negative control wells (CYP2A13+DMSO), which is an
acceptable dynamic range for a high throughput screening
assay. Primary screening of 35,200 Chembridge library com-
pounds resulted in a high hit rate of 1.4%. This relatively high
hit rate likely reflects the nature of the target. Xenobiotic-
metabolizing cytochrome P450 enzymes have evolved to be
promiscuous and would be expected to bind a higher number
of library compounds than most proteins. Of the 508 primary
screen actives that were repurchased and tested for binding

Blake et al.
Fig. 1 Identification of CYP2A13-specific ligands via high throughput screening. (a) Distribution of positive (PEITC) and negative (DMSO) controls. (b)
Scattergram of the UV/Vis spectral shifts observed for 35,200 compounds in the screen. (c) Schematic representation of compound workflow. (d)
Identification of 4-benzylmorpholine scaffold as a specific ligand for CYP2A13 vs. CYP2A6. While CYP2A13 (left) shows increasing absorbance changes in
response to ligand titration, no absorbance changes are observed upon titration with CYP2A6 (right).
1, 6, 15, and 18 with CYP2A6. In all cases the alpha value
was greater than 1, indicating a preference for inhibitor
binding to free enzyme over the enzyme/substrate complex.
Compounds 6 and 14 had the strongest preference for the
free enzyme with alpha values of 13.3 and 19.6, respectively,
while the remaining compounds demonstrating mixed inhi-
bition had alpha values between 2.0–3.9.
electronegativity, and overall inductive effects of the com-
pound. This modification did not have a significant impact on
the binding and inhibition of CYP2A13, but interactions with
CYP2A6 were greatly affected as no spectral shift, consistent
with no binding, and poor inhibition (Ki=≥1,000) was ob-
served with 2, illustrating an increase in selectivity for
CYP2A13. Exchanging the fluorine atom for a chlorine atom
(3) further enhanced the affinity and inhibition for CYP2A13
resulting in low micromolar Kd (5.9 3.4 μM) and Ki (39.7
4.3 μM) values, but without any apparent binding to CYP2A6.
Thus, this compound is highly selective for CYP2A13. This
same trend with selective binding and inhibition was observed
for the remaining ortho substituted compounds (4–6), which
contained either electron withdrawing or electron donating
substituents. Compounds 4 and 6 were the most selective
inhibitors of CYP2A13 and were similar in both size and
lipophilicity but varied in electronic contributions from the
substituent. These results illustrated that size and lipophilicity
at the ortho position may be more important than electronics
in conferring selective binding and inhibition of CYP2A13.
Disubstitution at the ortho position resulted in compara-
ble Kd values or slight increases in affinity for CYP2A13 over
Ortho (C2) Substitution on the Benzene Ring
The parent, unsubstituted 4-benzylmorpholine compound
(1), demonstrated a type I spectral shift with CYP2A13 (Kd=
116 μM), but a definitive Kd value could not be determined for
CYP2A6. Binding of (1) to CYP2A6 resulted in spectral shifts
at concentrations that converted from type I to type II over the
course of the titration and were consistent with a Kd≥300 μM.
Not only did this parent compound bind poorly, it was also a
poor inhibitor of both CYP2A13 (Ki=477 μM) and CYP2A6
(Ki=675 μM) and was not selective for either enzyme.
Addition of a fluorine atom on the benzene ring (2)
would be expected to maintain the overall size of the analog
in comparison to 1, but enhance the lipophilicity,

Benzylmorpholine Inhibitors of Cytochrome P450 2A13
Table I Spectral Equilibrium Dissociation Constants (Kd) and Inhibition Constants (Ki) for the Benzylmorpholine Library with the Human CYP2A6 and
CYP2A13 Enzymes, Along with Selectivity Ratios for Eacha
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Blake et al.
Table I (continued).
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aStandard error is reported for data with ≥3 trials, but the majority of data is presented as the average of duplicate assays
bNA: Not applicable
cSpectral shift notes: (I), Type I spectral shift; (II), Type II spectral shift; (I→II), Change from type I to type II with a spectral shift at inhibitor concentrations ≥300 μM; ≥ Minimal Kd
value, except with 1 and 10 which had spectral shifts at ≥300 nm but no Kd value could be determined; + Kd value from average of duplicate samples with <4% variation, but an
additional assay reported a value of 121 μM; ** Evaluated concentrations greater than or equal to the maximum CYP2A13 concentration tested
dInhibition notes: (C), Competitive Inhibitor; (M), Mixed Inhibitor; *Outlier exclusion

Benzylmorpholine Inhibitors of Cytochrome P450 2A13
Steric bulk and/or addition of
a positive charge decreases
binding and inhbition of
CYP2A13
Disubstitution at the 2 and 6 positions enhances
affinity but also decreases selectivity for CYP2A13
3, with Kd values of 5.2 μM (7), 1.3 μM (8), and 1.1 μM (9),
while maintaining low micromolar inhibition with Ki values
between 18.1 μM and 38.1 μM with CYP2A13. While 7 did
not cause a spectral shift at all with CYP2A6, compounds
8 and 9 demonstrated some binding to CYP2A6 with Kd
values of ~500 μM but were still >400-fold selective for
binding to CYP2A13. Overall, the disubstituted compounds
were not as selective as the singly substituted compounds
with regard to enzyme inhibition. For CYP2A6, Ki values
ranged from 237 μM to 296 μM, thus reducing the selec-
tivity to 7.5- to 15-fold in comparison to ≥25-fold for (3).
These results were interesting as CYP2A6 has an overall
smaller active site volume than CYP2A13(22) and did not
demonstrate a spectral shift with 7 and weakly bound 8 and
9, yet competitive inhibition was observed with CYP2A6 and
the larger disubstituted analogs. Thus, while disubstitution
increased the overall lipophilicity of the compound and
maintained selective binding and inhibition of CYP2A13, it
enabled weak binding and inhibition of CYP2A6, perhaps via
a small conformational change in the CYP2A6 active site.
3 and/or 4 substitution
enhances affinity and
inhibition of CYP2A6
O
N
Substitution at the 2 position is
2-carbon linker decreases
inhibition of CYP2A13
required for selective binding to
CYP2A13
Br
Fig. 2 Structure-activity relationships for the 4-benzylmorpholine scaffold
with human CYP2A enzymes.
imidazole ring coordinated to the heme as demonstrated by
the type II spectral shift. Interestingly, both 16 and 17 were
mixed inhibitors with low α values with CYP2A13, but purely
competitive inhibitors with CYP2A6. While the type II bind-
ing and potent inhibition were anticipated for the imidazole
compounds (16 and 17) due to the accessibility of the nitrogen
for coordination to the heme iron, it was unexpected to
observe a type II spectral shift for 18 with CYP2A6 (Kd=
154 μM) versus a type I spectral shift with CYP2A13 (Kd=8.3
4.1 μM). It is at present unknown as to which active site
features resulted in the distinct binding modes. Additionally,
unlike the imidazole containing ligands, compound 18 was
still selective for CYP2A13 with both binding (18-fold) and
inhibition (8-fold).
Meta and Para (C3 and C4) Substitution on the Benzyl
Ring
Single substitutions at the meta (10) and para (11) positions
of the benzyl ring both decreased affinity (Kd=69.1
30.2 μM (10), 168 60.8 μM (11)) and inhibition (Ki=
260 μM (10), and 458 μM (11)) for CYP2A13 in compar-
ison to the ortho position (3). As with compound 1, defin-
itive equilibrium dissociation constants could not be
determined for 10 and 11 with CYP2A6, but weak spectral
shifts are consistent with Kd values of ≥400 μM (10) and
≥245 μM (11). Similarly both compounds weakly inhibited
CYP2A6 with Ki values of 352 μM (10) and 448 μM (11).
Evaluation of the binding and inhibition selectivity ratios indi-
cated that compounds 10 and 11 were not selective for either
CYP2A enzyme. However, disubstitution with a chlorine atom
at the ortho position (12, 13) rescued selectivity for CYP2A13
as illustrated by the inhibition results with an increase from ~1-
to ~9-fold selectivity for CYP2A13, further demonstrating the
importance of the ortho substitution. Additional di-methyl
substitution on the morpholine ring as shown in 14 had min-
imal effects on Kd values, but this change had a greater effect in
decreasing the inhibition of both CYP2A13 and CYP2A6.
The sulfur atoms in the thiophene rings of compounds 19
and 20 differed from 18 in that only type I spectral shifts were
observed for these compounds. The thiophene ring mimicked a
benzene ring, and it was expected that 19 and 20 would
correlate with results from compounds 1 and 3, respectively.
Bioisosteric replacement of the benzene ring with a thiophene
ring, 19, increased the affinity and inhibition of CYP2A13 (Kd=
30.8 μM, Ki=131 μM) over the parent compound, 1, but had
similar results for CYP2A6 (Kd=≥464 μM, Ki=270 μM). The
ortho position of the sulfur may rationalize why 19 had a >3-
Table II Metabolic Stability of Compounds 3 and 7 with Human, Mouse,
and Rat Lung and Liver Microsomes. Data for (3) is in Normal Font, While
Data for (7) is in Bold
Species Test
system loss of
substrate (%)
Percent
Estimated Estimated in vitro
half life
(min)
intrinsic clearance
(μl/min/mg protein)
Human Liver
Lung
47.0/85.5
22.9/7.6 30.3/91.4
NA/NA
1.0a/0.8a 687/916
1.6a/1.3a 433/551
4.7a/4.0a 149/173
2.4a/1.5a 283/478
No Loss/No Loss NA/NA
Ring Substitutions
Mouse Liver
Lung
100/100
99.9/100
96.7/97.3
99.8/100
Multiple ring analogs demonstrated a wide range of binding
and inhibition constants. A naphthalene substitution for ben-
zene (15) weakly inhibited both CYP2A enzymes. Replacing
the morpholine ring with imidazole (16, 17) resulted in ex-
tremely potent but non-selective analogs with submicromolar
Kd and Ki values. Not surprisingly, the nitrogen of the
Rat
Liver
Lung
NA Not applicable due to the lack of substrate depletion
a Values were less than the shortest incubation time and are therefore
rough estimates

Blake et al.
Table III Results for the Average
Half-Life of Compounds 3 and 7
with a Panel of Human Recombi-
nant Hepatic Cytochromes P450
Compound
Average T1/2 (min)
CYP1A2
CYP2C8
CYP2C9
CYP2D6
CYP3A4
CYP2C19
CYP2B6
3
7
2317
1214
62
51
476
230
61
78
33
25
13
18
<5
<5
fold selectivity for CYP2A13 versus the parent compound (1)
even though 19 had the same spatial arrangement. As was
observed for the parent compound, addition of a chlorine
substituent at the ortho position on the thiophene analog, 20,
increased the selectivity for CYP2A13 in both binding (22.3-
fold) and inhibition (7.9-fold). This was achieved by decreasing
the equilibrium binding constant to 10.7 μM for CYP2A13 but
did not significantly impact the binding to CYP2A6 (Kd
=≥239 μM). Additionally, the chlorine atom increased selec-
tivity for CYP2A13 by reducing the Ki value to 54.7 μM (20)
while simultaneously decreasing inhibition of CYP2A6 (Ki=
433 μM).
but decreased selectivity since 23 also bound and inhibited
CYP2A6 (Kd=≥538 μM, Ki=98.5 μM). These trends were not
observed with 24 when the linker was extended to an ethyl
group between the morpholine and benzene ring. Instead, a
decrease in both affinity and inhibition with the CYP2A en-
zymes was observed. Decreased affinity for CYP2A13 could be
due to the poor fit of the longer molecule within the active site,
while the methyl group on the linker in 23 did not significantly
clash within the active site but rather may have had additional
van der Waals interactions with hydrophobic residues.
Summary
The remaining ring analogs had a methylpiperazine ring in
place of the morpholine ring (21, 22), which resulted in poor
affinity and inhibition of both CYP2A enzymes as the Kd
values were ≥300 μM for CYP2A13 with weak or no spectral
shifts with CYP2A6, and the Ki values were ≥1,000 μM. The
weak interactions between 21 and 22 with the CYP2A en-
zymes were due to either the disruption of optimal interactions
within the active site and/or charge addition. It was hypoth-
esized that the methyl group could disrupt a hydrogen bond
between the nitrogen atom and N297, one of two polar
residues within the active site. In addition, incorporation of
the methylpiperazine ring would have introduced a second
positive charge on the ligand at physiological pH. This charge
may not have been well tolerated, resulting in the poor bind-
ing and inhibition that was observed.
Compilation of the binding and inhibition studies has enabled
the determination of preliminary structure-activity relation-
ships (SAR) for 4-benzylmorpholine-based agents against
CYP2A13 (Fig. 2). We conclude that substitution at the ortho
position of the benzyl ring was the most vital feature of the
benzylmorpholine scaffold, promoting selective binding and
inhibition of CYP2A13. Disubstitution at both ortho positions
on the benzyl ring slightly enhanced affinity for both CYP2A
enzymes resulting in a reduction in the selectivity for
CYP2A13 versus the singly substituted analogs. Meta and para
substitutions on the benzyl ring, linkage extension between the
rings, and exchange of the morpholine ring for a
methylpiperazine ring proved to be detrimental alterations
in achieving selective binding and inhibition for CYP2A13.
Linker Substitutions
Toxicity and Metabolic Stability Evaluation
Attachment of a methyl group to the one carbon linker (23)
yielded a compound that maintained comparable affinity and
inhibition of CYP2A13 (Kd=7.6 2.8 μM, Ki=15.9 μM) as 3,
The in vitro results led to the conclusion that 4-benzylmorpholine
analogs could selectively inhibit CYP2A13. The next stage in the
development of these analogs as potential chemopreventative
Fig. 3 Metabolic stability of 7 in
(a) lung and (b) liver microsomes
generated from wild type and
transgenic CYP2A/CYP2B
knockout mice.

Benzylmorpholine Inhibitors of Cytochrome P450 2A13
agents was the determination of toxicity and metabolic stability.
First, compounds 3 and 7, which represented both the singly
and disubstituted compounds, were evaluated for genetic and
cardiac toxicity (Cerep, Redmond, WA). The Ames test was
employed to determine genetic toxicity, and few or no revertants
were detected at compound concentrations ranging from 5–
100 μM. The potential of 3 and 7 to cause cardiac toxicity via
inhibition of the hERG channel was evaluated because this can
be a major reason for drug withdrawal or limitation. In a patch
clamp assay, compounds 3 and 7 inhibited the tail current by
13.9% and 24.8%, respectively, at the highest concentration
tested, 10 μM. These results suggest the hERG IC50 is
>10 μM, placing the potency in the low category and suggesting
that toxicity associated with long QT intervals is unlikely to be
arise from these compounds.
The metabolic stability of 3 and 7 were evaluated with
human, mouse, and rat lung and liver microsomes over a
20-minute incubation (Xenotech, Overland Park, KS).
Since the CYP2A13 enzyme targeted by these inhibitors
is located in the respiratory tract, it was reassuring to
observe that the benzylmorpholine analogs were very sta-
ble in human lung microsomes with minimal or no loss of
substrate observed (Table II). However, under the same
conditions in human liver microsomes, at the end of
20 min only 53% or 14% of the substrates (3 and 7,
respectively) remained and in vitro T1/2 values of 22.9 and
7.6 min were calculated. Corresponding in vitro intrinsic
clearance values were 30.3 and 91.4 μl/min/mg protein,
respectively. Thus the 4-benzylmorpholine compounds are
metabolized at substantially different rates between tissues.
Parallel evaluations from mouse and rat microsomes indi-
cated ≤3% compound remaining in either the lung or
liver microsomes after 20 min. Thus 4-benzylmorpholine
compounds are rapidly metabolized in mouse and rat
lung and liver and human liver microsomes, but persistent
in human lung, the target tissue. This data brought to
light substantial differences in metabolic stability of the
samples between species and tissues, suggesting that dosing
in human lung through inhalation would be optimal and that
systematic exposure might be low due to rapid hepatic clear-
ance. However, these results also brought to light metabolic
concerns over preclinical compound testing in rat or mice
model systems.
CYP1A2 (3). CYP2B6 most quickly reduced concentrations
of benzylmorpholine compounds with 0% of both com-
pounds remaining after only 5 min. Substantial metabolism
also occurred with CYP2C19 and to a lesser extent
CYP3A4 and CYP2C8.
The identification of CYP2B6 as a potentially significant
component in rapid 4-benzylmorpholine metabolism also
suggested a possible rationale for the differences between com-
pound metabolic stability in mouse lung and human lung.
While human lung tissues have lower expression levels of the
functional CYP2B enzyme, CYP2B6 (34), mice have five
CYP2B enzymes which might be active in the degradation of
4-benzylmorpholine compounds. We tested this hypothesis by
evaluating the metabolic stability of 7 in lung and liver micro-
somes from knockout mice vs. wild type mice (Fig. 3). These
knockout mice had a deletion of a DNA segment containing the
genes coding for the CYP2A4, CYP2A5, CYP2G1, CYP2S1,
and all five CYP2B enzymes (Cyp2a(4/5)bgs mice) (35). It should
be noted that these experiments were performed with 1/10 the
amount of protein (0.1 mg/ml) as the studies above due to
limited sample, so the clearance and t1/2 values can only be
compared with the side-by-side controls for wild type mice. In
these transgenic mouse lung microsomes metabolic stability of
7 was increased 5-fold compared to wild type lung micro-
somes (in vitro t1/2=669 min vs. 124 min for wild type mice; in
vitro intrinsic clearance 10.4 vs. 55.7 for wild type mice). In
liver microsomes from transgenic mice the metabolic stability
of 7 increased by 1.7-fold (in vitro t1/2=127 min vs. 73 min for
wild type mice; in vitro intrinsic clearance 54.8 vs. 95.3 for wild
type mice). The increased metabolic stability in the knockout
mouse lung microsomes was more similar to the metabolic
stability in the human lung, suggesting the knockout mouse as
an appropriate model for preclinical studies of inhibitor effects
on NNK bioactivation.
CONCLUSION
By altering various parts of the 4-benzylmorpholine small mol-
ecule scaffold, we were able to identify the key structural aspects
that contributed to the selective binding and inhibition between
the two closely related human CYP2A enzymes. This determi-
nation of structure-activity relationships demonstrated that
ortho-substituted 4-benzylmorpholine analogs have the most
promise as selective inhibitors of CYP2A13. Additionally test-
ing for genetic and cardiac toxicities revealed no obstacles to
further development and testing. Metabolic stability testing
demonstrated these compounds are stable in human lung mi-
crosomes but would be rapidly cleared upon circulation, which
would be expected to reduce systemic exposure and any side
effects. Administration through inhalation would reduce the
first pass effects while delivering the CYP2A13 inhibitors di-
rectly to the target tissue where they are designed to block NNK
In an effort to determine the enzyme(s) likely to be
involved in metabolism of these compounds, compounds 3
and 7 (1 μM) were screened against a panel of the major
human xenobiotic metabolizing CYP enzymes (CYP1A2,
CYP2C8, CYP2C9, CYP2D6, CYP3A4, CYP2C19, and
CYP2B6) and human liver microsomes (HLM) for in vitro
half-life determination (Table III). The percent compound
remaining was detected at 0, 5, 10, 20, 30, and 45 min. The
average in vitro half-life for the compounds ranged from
<5 min with CYP2B6 (3 and 7) to essentially no loss with

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ACKNOWLEDGMENTS AND DISCLOSURES
Thanks are due to Kelin Li, who provided synthetic training to
LB. We are grateful for purity analysis by Patrick Porubsky and
Ben Neuenswander at The University of Kansas Center for
Chemical Methodologies and Library Design. Toxicity studies
were undertaken at Cerep. Metabolic stability in microsomes
was undertaken at Xenotech, LLC. Mike Wester facilitated
metabolic screening with the major human xenobiotic metab-
olizing CYP enzymes. Transgenic microsomes were a gift from
Xinxin Ding at the Wadsworth Center, New York State De-
partment of Health and State University of New York at
Albany. This work was supported by the Kansas Masonic
Cancer Research Institute, the University of Kansas General
Research Fund, NIH grant NIGMS GM076343, and the
Institute for Advancing Medical Innovation.
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