Cytochrome P450 2C9 type II binding studies on quinoline-4-carboxamide analogues

Article abstract of DOI:10.1021/jm8011257

CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic compounds in drug design, a rapid and efficient predrug screening of these potential type II binding compounds is essential to avoid adverse drug

Full text of DOI:10.1021/jm8011257

8000
J. Med. Chem. 2008, 51, 8000–8011
Cytochrome P450 2C9 Type II Binding Studies on Quinoline-4-Carboxamide Analogues
Chi-Chi Peng,† Jonathan L. Cape, Tom Rushmore,‡ Gregory J. Crouch,† and Jeffrey P. Jones*,†
Department of Chemistry, Washington State UniVersity, P.O. Box 644630, Pullman, Washington 99164-4630, and Department of Drug
Metabolism, Merck Research Laboratories, West Point, PennsylVania 19486
ReceiVed September 9, 2008
CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic
compounds in drug design, a rapid and efficient predrug screening of these potential type II binding compounds
is essential to avoid adverse drug reactions. To understand binding modes, we use quinoline-4-carboxamide
analogues to study the factors that determine the structure-activity relationships. The results of this study
suggest that the more accessible pyridine with the nitrogen para to the linkage can coordinate directly with
the ferric heme iron, but this is not seen for the meta or ortho isomers. The π-cation interaction of the
naphthalene moiety and Arg 108 residue may also assist in stabilizing substrate binding within the active-
site cavity. The type II substrate binding affinity is determined by the combination of steric, electrostatic,
and hydrophobicity factors; meanwhile, it is enhanced by the strength of lone pair electrons coordination
with the heme iron.
Introduction
in the Soret band of the absolute UV spectrum. Type I binding
makes the heme more easily reduced by P450 reductase, starting
the catalytic cycle of oxidative metabolism. A type II binding
mode describes a situation where a substrate binds as a sixth
ligand to the heme iron. In addition to the displacement of the
coordinated water, the compound itself serves as a ligand. The
heme iron remains in the low-spin state, which makes reduction
by P450 reductase more difficult. Type II substrates typically
contain an sp2 hybridized nitrogen atom as part of a heteroaro-
matic system where the nitrogen lone pair coordinates with the
heme iron. Type II substrates show an increase in binding
affinity and results in an overall rate decrease in the catalytic
cycle of oxidative metabolism. Even though binding modes are
divided into two categories, the existence of mixed or multiple
binding modes is inevitable. There could be an equilibrium
between type I and type II binding.10
Cytochrome P450 enzymes (P450sa) constitute a superfamily
of heme-containing monooxygenases that are responsible for a
large range of metabolic reactions on endogenous and exogenous
substrates. Three of the major P450 isoforms involved in drug
metabolism are CYP2C9, CYP2D6, and CYP3A4, which
typically metabolize anions, cations, and large lipophilic
molecules, respectively. Given the structural diversity of drugs,
broad substrate selectivity is required for these isoforms to
mediate metabolism. This makes the metabolic P450 enzymes
different than other enzymes, including P450 enzymes that
catalyze endogenous reactions, in that multiple metabolites are
often formed from very different binding orientations.1,2 Even
very tight binding inhibitors have been shown to bind with
multiple orientations in the catalytic sites of metabolic P450
enzymes.3 Given this broad substrate selectivity, it is not
surprising that potentially severe drug-drug interactions can
occur due to P450s binding multiple compounds with high
affinity.4-6 Additionally, given that the physiological concentra-
tions of administered drugs are normally near 1 µM, it is
generally accepted that compounds that bind with a Ki less than
10 µM may cause drug-drug interactions.
To minimize these interactions, it is important to understand
the mechanisms involved in substrate binding to P450s, which
are described by two binding modes called type I and type II.7
Type I and type II substrate binding modes have been
extensively studied using UV spectroscopy,8-12 which charac-
terizes spectral changes in the heme-Soret absorption band. In
type I binding, the substrate binds in a hydrophobic pocket
located near the catalytic site of P450. This binding displaces a
water molecule that was coordinated to the catalytic face of the
heme iron. The resulting five-coordinated heme iron shifts from
a low-spin to high-spin state with an accompanying blue shift
In addition to UV spectroscopy, electron paramagnetic
resonance (EPR) spectroscopy has been used to differentiate
between type I and type II binding modes by directly probing
spin state changes in the heme iron for both bacterial and
mammalian P450 enzymes.8,12-14 This technique provides
information regarding the spin state of ferric iron, which has as
many as five unpaired electrons. With dependence on substrate
binding, different spin state heme signals will appear on the
spectra as a result of ligand binding. A high-spin state ferric
iron (S ) 5/2) gives EPR signals as g ) 7.95, 3.78, and 1.74; a
1
low-spin state ferric iron (S ) /2) gives signals as g ) 2.42,
2.25, and 1.92.
In order to improve metabolic stability and inhibitory potency,
current practices in drug design incorporate nitrogen hetero-
cycles (pyridines, imidazoles, and triazoles) moieties into drug
molecules.11,15 Nitrogen incorporation has the potential of
creating a type II binder that, in addition to affecting metabolic
rate, could potentially inhibit coadministered drugs and lead to
toxicity. Therefore, to facilitate rational drug design, it is
necessary to understand and be able to predict the binding mode
of a given compound as well as to balance the inhibitory potency
with the metabolic stability.
* To whom correspondence should be addressed. Jeffrey P. Jones, Fulmer
455, Department of Chemistry, Washington State University, Pullman, WA
99164-4630. Phone: (509) 335-5983. Fax: (509) 335-8867. E-mail:
jpj@wsu.edu.
†
Washington State University.
Merck Research Laboratories.
‡
Finally, some P450 enzymes are therapeutic targets. Examples
include aromatase for breast cancer,16 CYP2A6 for smoking
cessation,17,18 CYP51 for high cholesterol,19,20 yeast infections,
a Abbreviations: P450s, cytochrome P450 enzymes; EPR, electron
paramagnetic resonance; 2C9, CYP2C9; 4-PI, 4-phenylimidazole; FLU,
fluconazole, PDB, Protein Data Bank.
10.1021/jm8011257 CCC: $40.75
2008 American Chemical Society
Published on Web 11/19/2008

Cytochrome P450 2C9 Type II Binding Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8001
rates of 4-hydroxydiclofenac. Four different final concentrations
of diclofenac (5, 10, 20, and 30 µM) in 100 mM potassium
phosphate buffer at pH 7.4 were used. Three different inhibitor
concentrations were added to each different concentration of
diclofenac. 2C9 baculosomes (1 pmol) were added to the incubation
mixtures containing the substrate and selected inhibitor and followed
by preincubation for 5 min at 37 °C. NADPH was added to a final
concentration of 1 mM to initiate the reaction. The final volume of
all incubations was 500 µL. Reactions were quenched after 5 min
by adding 200 µL of 6% acetic acid in acetonitrile containing an
internal standard (0.05 mM phenacetin). The enzyme was removed
by centrifugation, and product formation was analyzed using HPLC
separation with UV detection. The HPLC system used reversed-
phase chromatography beginning with 30% of mobile phase A
(MeOH) and 70% of mobile phase B (30% acetonitrile/70% water
containing 1 mM perchloric acid) with a linear gradient to 95% of
MeOH over 6 min.26 Each compound’s Ki was determined from
eq 1 using GraphPad Prism4 (San Diego, CA) graphing software.
The low enzyme concentration of 2 nM means that substrate
depletion does not need to be corrected for even for the tightest
binding inhibitors.
Figure 1. Quinoline-4-carboxamide analogues.
and fungal infections (tinea pedis, tinea cruris, and candida in
HIV-AIDS).21 CYP51 is also a target for parasitic diseases
including American trypanosomiasis (Chagas disease) and
African trypanosomiasis (sleeping sickness). Many of the
currently available therapeutic agents used to treat these diseases
are high affinity type II binders.21 While type II binding
enhances affinities for P450 enzymes relative to other non-P450
targets, the nonspecific inhibition of P450 enzymes using these
therapeutics leads to toxicity. For example, azole antifungal
agents that target CYP51 can cause birth defects similar to those
observed with a phenotypic variation in the P450 reductase
function that is related to a decrease in steroid metabolism.22
In order to design a viable therapeutic, the compound must
specifically target P450 disease related isoforms while not
inhibiting drug metabolizing P450 isoforms. Since type II
binding potentially enhances binding to all P450 isoforms, it is
particularly important to understand and be able to attenuate
this binding interaction.
CYP2C9 (2C9) is one of the most significant drug metaboliz-
ing P450 isoforms, metabolizing many nonsteroidal anti-
inflammatory drugs including ibuprofen, flurbiprofen, diclofenac,
theantidiabeticsulfonylureas,andtheanticoagulant(S)-warfarin.4,23,24
During routine screening of diverse inhibitors of 2C9 we found
a quinoline-4-carboxamide compound that bound with a low
Ki value. In an effort to elucidate the role that nitrogen
coordination to the heme plays in type II substrate binding
interactions with 2C9, we have synthesized a series of quinoline-
4-carboxamide analogues (Figure 1 and Table 1). In addition
to other structural changes, we have intentionally varied the
position of the sp2 nitrogen atom in the ring system to study
heme coordination in type II binding interactions. Binding
affinity, spectroscopy, computational models, and docking
experiments to the known crystal structure of 2C9 are used to
determine the strength and nature of these interactions. Our
analogues were designed to probe binding interactions as a result
of both apoprotein interactions and type II coordination while
maintaining the core binding motif in order to decrease
differences in binding not related to coordination to the heme
iron.
[ ]
S
Vm
V )
(1)
[I]
[ ]
1 + Km + S
Ki
Ki Determination from IC50 Measurements. Measurements
were performed using diclofenac at Km concentrations. Inhibitors
at three different concentrations were added across to the reaction
mixtures containing the same substrate concentration and P450
enzyme (1 pmol). The same incubation procedure and HPLC
analysis (detailed above) were used to measure product formation
rates. IC50 and Ki values were calculated by GraphPad Prism4
graphing software using eq 2:
IC50
Ki )
(2)
[S]
1 +
Km
Reconstituted 2C9 System. The 2C9 protein premix was
prepared as described previously27 using purified 2C9 protein.28
The incubation conditions were the same as the baculosomes
incubation assay except 20 pmol of purified 2C9 protein was used
in each sample with an incubation time of 10 min.
Synthesis of Proposed Type II Binders Based on
Quinoline-4-carboxamide. All compounds were synthesized as
outlined in Scheme 1 with slight modifications for each analogue.
The general procedure for quinoline-4-carboxamide analogues
is given with compound 7: To a 100 mL round-bottom flask
equipped with a water condenser and stir bar were combined
potassium hydroxide (1.71 g, 0.030 mol) and ethanol (8 mL). The
reaction mixture was stirred at 80 °C until the potassium hydroxide
was completely dissolved. At this time, isatin (1.61 g, 0.011 mol)
was added followed by a dropwise addition of 4-acetylpyridine (1.57
g, 0.013 mol). The mixture was refluxed with stirring at 80 °C for
3 days. At the end of this time, the reaction solvent was evaporated
using a rotary evaporator, and the residue was dissolved in 50 mL
of water and neutralized with 1 N HCl to pH ∼6. The resulting
solid was collected by vacuum filtration and washed with ethanol
(25 mL × 2), acetone (25 mL × 2), and pentane (25 mL × 2) to
give crude 2-(pyridin-4-yl)quinoline-4-carboxylic acid, which was
used directly to generate the acyl chloride in the next step. The
acyl chlorides were formed by combining the crude 2-(pyridin-4-
yl)quinoline-4-carboxylic acid (formed above, 500 mg, 2.00 mmol)
with neat thionyl chloride (10 mL) in a 100 mL round-bottom flask
fitted with an air condenser under reflux for 2 h. At the end of this
time, the condenser was removed and the excess thionyl chloride
was evaporated using a stream of argon to give 2-(pyridin-4-
yl)quinoline-4-carbonyl chloride. The crude 2-(pyridin-4-yl)quino-
line-4-carbonyl chloride (200 mg, 0.744 mmol) was dissolved in a
1:1:1 mixture of acetone/dichloromethane/triethylamine (15 mL)
Experimental Methods
Enzymes, Chemicals, and Instruments. 2C9 baculosomes were
prepared as described previously.25 Diclofenac and ꢀ-NADPH were
purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade
solvents were purchased from EMD Chemicals Inc. (Gibbstown,
NJ) and used without purification. All other chemicals were
purchased from commercial sources and used without purification.
The1H and 13C NMR spectra were gathered on a Varian Mercury
Vx 300 MHz spectrometer using TMS as an internal standard except
the 13C NMR spectra of compounds 19, 20, 21, and 22, which
were collected by a Varian Inova 500 MHz. A Hewlett-Packard
8453 single-beam spectrophotometer with an HP 845x UV-visible
system (Hewlett-Packard, Palo Alto, CA) was used to determine
substrate concentrations. The HPLC system was from Agilent 1100
series with an Alltima C18 5 µm, 3.2 mm × 150 mm column from
Alltech. Values of pKa were estimated using MarvinSketch (Che-
mAxon Kft. Budapest, Hungary).
Baculosomes Incubation Conditions. Diclofenac was used as
a 2C9 substrate, and Ki values were determined from the formation

8002 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Table 1. Ki Values of Type II Binding Analogues
Peng et al.
a Compounds not available.

Cytochrome P450 2C9 Type II Binding Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8003
Scheme 1
in a 100 mL round-bottom flask. To this flask, 2-naphthylamine
(117.2 mg, 0.818 mmol) was added and the reaction mixture
refluxed for 2 h. Following solvent evaporation, the crude compound
was purified by chromatography (30 g, silica gel 60, 0.063-0.200
mm) using ethyl acetate. Crystallization from diisopropyl ether gave
pure N-(naphthalen-2-yl)-2-(pyridin-4-yl)quinoline-4-carboxamide
in 14.67% yield overall.
130.96, 130.65, 128.80, 124.83, 123.97, 121.70, 115.37, 61.84,
51.75, 45.93, 40.46, 31.79, 30.52, 23.33; ESI-MS m/z 387.5 (M +
1).
(2-(Pyridin-3-yl)quinolin-4-yl)(4-(pyrrolidin-1-yl)piperidin-
1-yl)methanone (5). The reaction conditions were the same as
above except 4-acetylpyridine was substituted with 3-acetylpyridine
and 4-(1-pyrrolidinyl)piperidine was used instead of 2-naphthy-
lamine to form the amide. The crude compound was purified by
chromatography using 10% MeOH in CH2Cl2. The overall yield
Piperidin-1-yl(2-(pyridin-4-yl)quinolin-4-yl)methanone (1).
The reaction conditions were the same as above except piperidine
was used instead of 2-naphthylamine to form the amide to give an
overall yield of 69.5%; mp 136-137 °C; 1H NMR (CDCl3) δ 8.79
(dd, J ) 1.5, 4.8 Hz, 2H), 8.23 (d, J ) 8.4 Hz, 1H), 8.06 (dd, J )
1.5, 4.5 Hz, 2H), 7.87-7.78 (m, 3H), 7.63 (m, 1H), 3.90 (m, 2H),
3.17 (t, J ) 5.7 Hz, 2H), 1.83-1.68 (m, 4H), 1.49-1.43 (m, 2H);
13C NMR (CDCl3) δ 166.78, 154.51, 150.84, 148.64, 146.27,
144.61, 130.95, 130.77, 128.37, 124.86, 124.12, 121.75, 115.38,
48.54, 43.00, 26.91, 25.97, 24.61; ESI-MS m/z 318.5 (M + 1).
1
was 50.2%; mp 164-165 °C; H NMR (CDCl3) δ 9.37 (dd, J )
1.8, 18.6 Hz, 1H), 8.73-8.71 (m, 1H), 8.53-8.47 (m, 1H),
8.23-8.19 (m, 1H), 7.92-7.77 (m, 3H), 7.67-7.56 (m, 1H),
7.49-7.45 (m, 1H), 4.90 (m, 1H), 3.46 (d, J ) 14.4 Hz, 1H),
3.14-2.98 (m, 2H), 2.75 (m, 4H), 2.17 (m, 1H), 2.03-1.49 (m,
7H). 13C NMR (CDCl3) δ 166.72, 154.59, 150.73, 148.99, 148.90,
144.17, 135.04, 134.99, 130.89, 130.48, 128.09, 124.78, 123.91,
123.57, 115.37, 61.78, 51.84, 46.06, 40.54, 32.56, 31.63, 23.37.
ESI-MS m/z 387.4 (M + 1).
Piperidin-1-yl(2-(pyridin-3-yl)quinolin-4-yl)methanone (2).
The reaction conditions were the same as above except 4-acetylpy-
ridine was substituted with 3-acetylpyridine and piperidine was used
instead of 2-naphthylamine to form the amide. The overall yield
(2-(Pyridin-2-yl)quinolin-4-yl)(4-(pyrrolidin-1-yl)piperidin-
1-yl)methanone (6). The reaction conditions were the same as
above except 4-acetylpyridine was substituted with 2-acetylpyridine
and 4-(1-pyrrolidinyl)piperidine was used instead of 2-naphthy-
lamine to form the amide. The crude compound was purified by
chromatography using 10% MeOH in CH2Cl2. The overall yield
was 60.3%; mp 81-82 °C; 1H NMR (CDCl3) δ 8.73 (m, 1H), 8.65
(m, 1H), 8.51 (dd, J ) 18.9 Hz, 1H), 8.21 (dd, J ) 8.4 Hz, 1H),
7.92-7.75 (m, 3H), 7.65-7.58 (m, 1H), 7.40-7.35 (m, 1H), 4.92
(m, 1H), 3.49 (d, J ) 12.9 Hz, 1H), 3.07-2.93 (m, 2H), 2.79 (m,
4H), 2.62-2.53 (m, 1H), 2.20-2.13 (m, 1H), 1.88-1.77 (m, 6H),
1.68-1.55 (m, 1H); 13C NMR (CDCl3) δ 167.22, 156.22, 155.84,
149.51, 148.26, 143.59, 137.24, 130.49, 130.32, 128.07, 127.85,
124.96, 124.56, 121.97, 115.96, 61.98, 51.86, 46.18, 40.53, 32.67,
31.67, 23.44; ESI-MS m/z 387.4 (M + 1).
1
was 71.4%; mp 146-147 °C; H NMR (CDCl3) δ 9.36 (dd, J )
0.9, 2.1 Hz, 1H), 8.72 (dd, J ) 1.8, 4.8 Hz, 1H), 8.50 (m, 1H),
8.21 (d, J ) 8.1 Hz, 1H), 7.86-7.76 (m, 3H), 7.60 (m, 1H), 7.47
(m, 1H), 3.90 (m, 2H), 3.18 (t, J ) 5.4 Hz, 2H), 1.82-1.68 (m,
4H), 1.49-1.44 (m, 2H); 13C NMR (CDCl3) δ 166.86, 154.67,
150.79, 149.03, 148.73, 144.48, 135.13, 134.81, 130.84, 130.54,
127.93, 124.88, 123.96, 123.67, 115.37, 48.54, 42.99, 26.91, 25.98,
24.63; ESI-MS m/z 318.5 (M + 1).
Piperidin-1-yl(2-(pyridin-2-yl)quinolin-4-yl)methanone (3).
The reaction conditions were the same as above except 4-acetylpy-
ridine was substituted with 2-acetylpyridine and piperidine was used
instead of 2-naphthylamine to form the amide. The overall yield
N-(Naphthalen-2-yl)-2-(pyridin-4-yl)quinoline-4-carboxa-
mide (7). The reaction conditions were described as above. mp
1
was 33.9%, mp 157-158 °C; H NMR (CDCl3) δ 8.73 (m, 1H),
1
8.66 (m, 1H), 8.51 (s, 1H), 8.21 (d, J ) 8.7 Hz, 1H), 7.91-7.85
(m, 2H), 7.76 (m, 1H), 7.58 (m, 1H), 7.37 (m, 1H), 3.89 (m, 2H),
3.21 (t, J ) 5.7 Hz, 2H), 1.82-1.66 (m, 4H), 1.56-1.39 (m, 2H);
13C NMR (CDCl3) δ 167.20, 156.10, 155.93, 149.44, 148.32,
143.88, 137.22, 130.54, 130.33, 127.84, 125.02, 124.62, 124.54,
121.99, 115.91, 48.56, 42.90, 26.90, 26.00, 24.70; ESI-MS m/z
318.3 (M + 1), 233.1 (M - 85), 207.2 (M - 111).
251-252 °C; H NMR (DMSO) δ 11.07 (s, 1H), 8.79 (dd, J )
1.8, 4.8 Hz, 2H), 8.56-8.54 (m, 2H), 8.33 (dd, J ) 1.5, 4.5 Hz,
2H), 8.24 (d, J ) 8.4 Hz, 2H), 7.96-7.88 (m, 4H), 7.78-7.71 (m,
2H), 7.54-7.43 (m, 2H); 13C NMR (DMSO) δ 165.98, 154.22,
151.25, 148.58, 145.67, 144.12, 137.04, 134.01, 131.45, 130.91,
130.61, 129.22, 129.08, 128.24, 127.32, 125.89, 125.78, 124.60,
122.06, 121.08, 117.66, 117.09; ESI-MS m/z 376.3 (M + 1), 348.4
(M - 28), 297.4 (M - 79), 233.1 (M - 143), 205.3 (M - 171).
(2-(Pyridin-4-yl)quinolin-4-yl)(4-(pyrrolidin-1-yl)piperidin-
1-yl)methanone (4). The reaction conditions were the same as
above except 4-(1-pyrrolidinyl)piperidine was used instead of
2-naphthylamine to form the amide. The crude compound was
purified with chromatography using 10% MeOH in CH2Cl2. The
N-(Naphthalen-2-yl)-2-(pyridin-3-yl)quinoline-4-carboxa-
mide (8). The reaction conditions were the same as above except
4-acetylpyridine was substituted with 3-acetylpyridine. The overall
yield was 19.8%; mp 215-216 °C; 1H NMR (CDCl3) δ 9.19 (d, J
) 1.8 Hz, 1H), 9.06 (s, 1H), 8.52-8.50 (m, 2H), 8.37 (m, 1H),
8.28 (dd, J ) 0.6, 8.1 Hz, 1H), 8.16 (d, J ) 8.4, 1H), 7.97 (s, 1H),
7.91-7.76 (m, 4H), 7.72 (dd, J ) 2.1, 8.7 Hz, 1H), 7.62 (m, 1H),
7.50 (m, 2H), 7.33-7.29 (m, 1H); 13C NMR (CDCl3) δ 165.81,
154.05, 150.37, 148.98, 148.55, 143.53, 135.46, 135.04, 134.41,
134.05, 131.27, 130.94, 130.40, 129.37, 128.34, 128.11, 127.92,
1
overall yield was 39.4%; mp 164-165 °C; H NMR (CDCl3) δ
8.79 (s, 2H), 8.23 (d, J ) 8.7 Hz, 1H), 8.06 (m, 2H), 7.91-7.78
(m, 3H), 7.67-7.59 (m, 1H), 4.85 (m, 1H), 3.42 (d, J ) 13.8 Hz,
1H), 3.16-2.99 (m, 2H), 2.69 (m, 4H), 2.45-2.38 (m, 1H),
2.19-2.13 (m, 1H), 1.86-1.76 (m, 6H), 1.60-1.49 (m, 1H); 13C
NMR (CDCl3) δ 166.72, 154.41, 150.80, 148.50, 146.09, 144.11,

8004 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Peng et al.
127.04, 125.75, 125.26, 123.95, 123.71, 120.00, 117.52, 116.29;
ESI-MS m/z 376.5 (M + 1).
1H), 7.24 (t, J ) 7.8 Hz, 1H); 13C NMR (CDCl3) δ 165.64, 154.04,
150.37, 148.95, 148.53, 143.56, 138.01, 135.02, 134.39, 130.91,
130.37, 129.55, 128.29, 125.45, 125.23, 123.94, 123.67, 120.40,
116.25; ESI-MS m/z 326.5 (M + 1).
N-(Naphthalen-2-yl)-2-(pyridin-2-yl)quinoline-4-carboxa-
mide (9). The reaction conditions were the same as above except
4-acetylpyridine was substituted with 2-acetylpyridine. The crude
compound was purified by chromatography using a mixture of
hexanes and ethyl acetate (2:1). The overall yield was 23.9%; mp
261-262 °C; 1H NMR (CDCl3) δ 8.80 (s, 1H), 8.74-8.68 (m,
2H), 8.48-8.42 (m, 2H), 8.26-8.20 (m, 2H), 7.95-7.78 (m, 5H),
7.66-7.61 (m, 2H), 7.55-7.39 (m, 3H); 13C NMR (CDCl3) δ
165.65, 155.64, 155.47, 149.16, 148.62, 142.34, 137.25, 135.03,
133.81, 131.03, 130.36, 130.25, 129.06, 128.13, 127.88, 127.66,
126.73, 125.44, 124.57, 124.46, 121.87, 119.89, 117.31, 116.18;
ESI-MS m/z 376.5 (M + 1).
N-(Naphthalen-1-ylmethyl)-2-(pyridin-4-yl)quinoline-4-carboxa-
mide (10). The reaction conditions were the same as above except
1-naphthylmethylamine was used instead of 2-naphthylamine to form
the amide. The overall yield was 72.3%; mp 188-189 °C; 1H NMR
(CDCl3) δ 8.71 (d, J ) 6 Hz, 2H), 8.25-8.15 (m, 3H), 7.96-7.86
(m, 5H), 7.77 (m, 1H), 7.67-7.54 (m, 4H), 7.50-7.45 (m, 1H), 6.42
(m, 1H), 5.24 (d, J ) 5.4 Hz, 2H); 13C NMR (CDCl3) δ 167.06,
154.15, 150.76, 148.93, 145.93, 143.35, 134.26, 132.88, 131.60,
130.89, 130.64, 129.45, 129.29, 128.57, 127.61, 127.18, 126.53,
125.71, 125.24, 124.23, 123.58, 121.62, 116.23, 42.81. ESI-MS m/z
390.5 (M + 1).
N-Phenyl-2-(pyridin-2-yl)quinoline-4-carboxamide (15). The
reaction conditions were the same as above except 4-acetylpyridine
was substituted with 2-acetylpyridine and aniline was used instead of
2-naphthylamine to form the amide. The crude compound was purified
by chromatography using a mixture of hexanes and ethyl acetate (2:
1). The overall yield was 32.4%; mp 207-208 °C; 1H NMR (CDCl3)
δ 8.75-8.66 (m, 3H), 8.38 (d, J ) 7.8 Hz, 1H), 8.23 (d, J ) 8.4 Hz,
1H), 8.02 (s, 1H), 7.91 (m, 1H), 7.82-7.73 (m, 3H), 7.62 (m, 1H),
7.46-7.38 (m, 3H), 7.22 (t, J ) 7.5 Hz, 1H); 13C NMR (CDCl3) δ
165.74, 155.83, 155.67, 149.37, 148.79, 142.62, 137.81, 137.46,
130.56, 130.44, 129.48, 128.31, 125.62, 125.37, 124.78, 124.65,
122.08, 120.49, 116.37; ESI-MS m/z 326.3 (M + 1).
6,8-Dimethyl-N-(naphthalen-2-yl)-2-(pyridin-4-yl)quinoline-
4-carboxamide (16). The reaction conditions were the same as
above except isatin was substituted with 5,7-dimethylisatin. The
crude compound was purified by chromatography using a mixture
of hexanes and ethyl acetate (1:2). The overall yield was 35.6%;
1
mp 233-234 °C; H NMR (CDCl3) δ 8.74 (s, 1H), 8.61 (dd, J )
1.5, 4.8 Hz, 2H), 8.51 (d, J ) 2.1 Hz, 1H), 7.98 (dd, J ) 1.8, 4.8
Hz, 2H), 7.90-7.84 (m, 5H), 7.70 (dd, J ) 2.1, 9 Hz, 1H),
7.55-7.45 (m, 3H), 2.84 (s, 3H), 2.48 (s, 3H); 13C NMR (CDCl3)
δ 166.35, 150.96, 150.46, 146.60, 146.27, 142.61, 138.88, 138.29,
135.46, 134.05, 133.42, 131.25, 129.35, 128.09, 127.91, 127.04,
125.74, 124.20, 121.72, 121.42, 119.99, 117.46, 115.59, 22.24,
18.31; ESI-MS m/z 404.5 (M + 1).
N-(Naphthalen-1-ylmethyl)-2-(pyridin-3-yl)quinoline-4-carboxa-
mide (11). The reaction conditions were the same as above except
4-acetylpyridine was substituted with 3-acetylpyridine and 1-naph-
thylmethylamine was used instead of 2-naphthylamine to form the
1
6,8-Dimethyl-N-(naphthalen-2-yl)-2-(pyridin-3-yl)quinoline--
4-carboxamide (17). The reaction conditions were the same as
above except 4-acetylpyridine was substituted with 3-acetylpyridine
and 5,7-dimethylisatin was used instead of isatin. The crude
compound was purified by chromatography using a mixture of
hexanes and ethyl acetate (1:2). The overall yield was 13.9%; mp
amide. The overall yield was 10.3%; mp 175-176 °C; H NMR
(CDCl3) δ 9.15 (s, 1H), 8.59 (d, J ) 3.3 Hz, 1H), 8.38 (m, 1H),
8.21 (d, J ) 8.1 Hz, 2H), 8.11 (d, J ) 8.7 Hz, 1H), 7.93-7.82 (m,
3H), 7.74 (m, 1H), 7.66-7.53 (m, 4H), 7.49-7.44 (m, 1H),
7.39-7.35 (m, 1H), 6.61 (m, 1H), 5.23 (d, J ) 5.1 Hz, 2H); 13C
NMR (CDCl3) δ 167.17, 154.04, 150.53, 148.86, 148.53, 143.27,
134.96, 134.34, 134.23, 133.04, 131.63, 130.72, 130.24, 129.35,
129.25, 128.06, 127.56, 127.11, 126.50, 125.70, 125.29, 123.86,
123.76, 123.66, 116.16, 42.76; ESI-MS m/z 390.3 (M + 1), 262.1
(M - 128), 233.0 (M - 157), 207.2 (M - 183), 141.1 (M - 249).
N-(Naphthalen-1-ylmethyl)-2-(pyridin-2-yl)quinoline-4-carbox-
amide (12). The reaction conditions were the same as above except
4-acetylpyridine was substituted with 2-acetylpyridine and 1-naphth-
ylmethylamine was used instead of 2-naphthylamine to form the amide.
The crude compound was purified by chromatography using a mixture
of hexanes and ethyl acetate (2:1). The overall yield was 23%; mp
198-199 °C; 1H NMR (CDCl3) δ 8.66-8.57 (m, 3H), 8.37 (d, J )
7.8 Hz, 1H), 8.24-8.14 (m, 2H), 7.91-7.82 (m, 3H), 7.75 (m, 1H),
7.64-7.44 (m, 5H), 7.37-7.32 (m, 1H), 6.54 (m, 1H), 5.22 (d, J )
5.4 Hz, 2H); 13C NMR (CDCl3) δ 167.40, 155.76, 155.69, 149.29
148.65, 142.55, 137.33, 134.18, 133.07, 131.65, 130.36, 129.24,
129.13, 128.12, 127.41, 127.09, 126.40, 125.67, 124.79, 124.63,
123.73, 122.01, 116.35, 42.60; ESI-MS m/z 390.5 (M + 1).
1
224-225 °C; H NMR (CDCl3) δ 9.23 (d, J ) 1.8 Hz, 1H), 9.11
(s, 1H), 8.52-8.46 (m, 2H), 8.35 (m, 1H), 7.90-7.83 (m, 5H),
7.72 (dd, J ) 2.1, 8.7 Hz, 1H), 7.54-7.43 (m, 3H), 7.29-7.25 (m,
1H), 2.75 (s, 3H), 2.46 (s, 3H); 13C NMR (CDCl3) δ 166.53, 151.11,
149.90, 148.45, 146.54, 142.74, 138.20, 137.99, 135.63, 134.63,
134.06, 133.23, 131.20, 129.29, 128.10, 127.88, 126.97, 125.65,
123.80, 123.67, 121.70, 120.08, 117.46, 115.50, 22.17, 18.29; ESI-
MS m/z 404.4 (M + 1).
6,8-Dimethyl-N-(naphthalen-2-yl)-2-(pyridin-2-yl)quinoline--
4-carboxamide (18). The reaction conditions were the same as
above except 4-acetylpyridine was substituted with 2-acetylpyridine
and 5,7-dimethylisatin was used instead of isatin. The crude
compound was purified by chromatography using a mixture of
hexanes and ethyl acetate (1:2). The overall yield was 25.1%; mp
1
226-227 °C; H NMR (CDCl3) δ 8.74-8.70 (m, 3H), 8.46 (d, J
) 2.1 Hz, 1H), 8.16 (s, 1H), 8.00 (s, 1H), 7.92-7.82 (m, 4H),
7.63 (dd, J ) 2.4, 8.7 Hz, 1H), 7.48 (m, 3H), 7.39-7.35 (m, 1H),
2.89 (s, 3H), 2.50 (s, 3H); 13C NMR (CDCl3) δ 166.28, 155.94,
152.95, 148.91, 146.08, 141.72, 137.97, 137.58, 137.02, 135.18,
133.81, 132.69, 130.95, 128.97, 127.86, 127.62, 126.63, 125.32,
124.37, 124.14, 121.91, 121.65, 119.96, 117.25, 115.57, 21.95,
18.13; ESI-MS m/z 404.3 (M + 1).
N-Phenyl-2-(pyridin-4-yl)quinoline-4-carboxamide (13). The
reaction conditions were the same as above except aniline was used
instead of 2-naphthylamine to form the amide. The crude compound
was purified by chromatography using a mixture of hexanes and
ethyl acetate (2:1). The overall yield was 67.8%; mp 202-203 °C;
1H NMR (CDCl3) δ 8.86 (s, 1H), 8.57 (d, J ) 4.2 Hz, 2H),
8.24-8.16 (m, 2H), 7.88 (d, J ) 6 Hz, 2H), 7.81-7.76 (m, 4H),
7.60 (m, 1H), 7.44 (t, J ) 8.1 Hz, 2H), 7.24 (t, J ) 7.8 Hz, 1H);
13C NMR (CDCl3) δ 165.55, 153.70, 150.48, 148.89, 145.93,
143.33, 138.01, 131.02, 130.61, 129.56, 128.75, 125.50, 125.25,
124.12, 121.64, 120.39, 116.25; ESI-MS m/z 326.5 (M + 1).
N-Phenyl-2-(pyridin-3-yl)quinoline-4-carboxamide (14). The
reaction conditions were the same as above except 4-acetylpyridine
was substituted with 3-acetylpyridine and aniline was used instead
of 2-naphthylamine to form the amide. The overall yield was 41.3%;
6-Chloro-N-(naphthalen-2-yl)-2-(pyridin-4-yl)quinoline-4-
carboxamide (19). The reaction conditions were the same as above
except isatin was substituted with 5-chloroisatin. The overall yield
1
was 8.3%; mp 225-226 °C; H NMR (DMSO) δ 11.10 (s, 1H),
8.81 (d, J ) 5.1 Hz, 2H), 8.64 (s, 1H), 8.56 (s, 1H), 8.34-8.31
(m, 3H), 8.26 (d, J ) 9 Hz, 1H), 7.97-7.88 (m, 4H), 7.77 (d, J )
8.7 Hz, 1H), 7.54-7.45 (m, 2H); 13C NMR (DMSO) δ 164.6,
154.0, 150.5, 146.4, 144.6, 142.0, 136.1, 133.2, 132.8, 132.0, 131.2,
130.2, 128.4, 127.5, 126.5, 125.1, 124.6, 123.9, 121.3, 120.4, 118.2,
116.6; ESI-MS m/z 410.3 (M + 1), 382.4 (M - 28), 267.0 (M -
143), 239.2 (M - 171).
1
mp 215-216 °C; H NMR (CDCl3) δ 9.16 (d, J ) 1.8 Hz, 1H),
8.82 (s, 1H), 8.52 (dd, J ) 1.5, 4.5 Hz, 1H), 8.36 (m, 1H), 8.23 (d,
J ) 7.5 Hz, 1H), 8.15 (d, J ) 8.4 Hz, 1H), 7.92 (s, 1H), 7.81-7.75
(m, 3H), 7.60 (m, 1H), 7.44 (t, J ) 8.1 Hz, 2H), 7.34-7.30 (m,
6-Chloro-N-(naphthalen-2-yl)-2-(pyridin-3-yl)quinoline-4-
carboxamide (20). The reaction conditions were the same as above
except 4-acetylpyridine was substituted with 3-acetylpyridine and

Cytochrome P450 2C9 Type II Binding Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8005
5-chloroisatin was used instead of isatin. The overall yield was
3.2%; mp 228-229 °C; H NMR (DMSO) δ 11.07 (s, 1H), 9.54
with GraphPad Prism4 using equation ∆A ) (Bmax[S])/(Ks + [S]),
where A is the absorbance difference, Bmax is the maximum
absorbance difference, [S] is the substrate concentration. The types
of substrate-induced binding spectra with P450 heme were deter-
mined by the positions of wavelengths for peaks (λmax) and minima
(λmin) on the spectra.29
EPR Studies on Binding Modes. EPR spectra were recorded
on a Bruker ESP-300e X-band spectrometer fitted with a liquid
helium flow cryostat (ESR-9, Oxford Instruments). The ferric 2C9
samples were prepared in KPi buffer (100 mM, pH 7.4, 20%
glycerol) and frozen in liquid nitrogen prior to spectral acquisition.
The protein concentrations of samples were 18.6 µM (substrate-
free form) and 18.5 µM (substrate-bound form), respectively, with
a substrate concentration of 1 mM. Spectra were recorded at 20 K
with microwave frequency of 9.614 GHz, microwave power of 10
mW, modulation amplitude of 16 G, modulation frequency of 100
kHz, receiver gain of 1 × 104, conversion time of 168 ms, and
time constant of 20 ms.
pKa Measurement. Data were collected on a diode array HP
845 UV-vis spectrophotometer. Samples were prepared by adding
500 µL of 0.1 mM substrate stock solution in DMSO to 500 µL of
0.1 M KPi buffer prepared at pH values between 1.04 and 10.03.
As the pyridine nitrogen is protonated at lower pH, peaks emerged
with λmax at 287 nm. Absorbance was plotted versus pH and fit to
a sigmoidal function using GraphPad Prism4. Results agreed with
MarvinSketch calculated values.
Automated Docking. Substrates were docked into the crystal
structure of 2C9 1r9o30 using MOE 2006.08 (Chemical Computing
Group Inc. Montreal, Quebec, Canada). Compounds were sketched
and underwent energy minimization as entries. Selected substrates
were then fit into the active site cavity using the automatic docking
program, exploring the conformational space associated with each
rotatable bond. The parameters were as follows: receptor, receptor
atoms; site, selected atoms (glycerol + flurbiprofen); ligand,
sketched substrates; placement, alpha triangle; scoring, London dG;
retain, 100. The parameters for inclusion of the residues by ligand
interactions calculations in MOE were fields of H-bond, ionic, and
solvent with a minimum score 10% prospectively. Only the substrate
and amino acid residues within 4.5 Å were allowed to move during
the minimization. The protein was frozen when the autodocking
was performed. The water molecules were modeled within the
crystal structure before the docking process was initiated. The force
field for both docking and energy minimization were Hamiltonian,
MMFF94x in MOE.
1
(s, 1H), 8.73 (d, J ) 7.5 Hz, 2H), 8.61 (s, 1H), 8.56 (s, 1H), 8.30
(d, J ) 2.7 Hz, 1H), 8.23 (d, J ) 9 Hz, 1H), 7.97-7.88 (m, 4H),
7.78 (d, J ) 8.4 Hz, 1H), 7.64-7.60 (m, 1H), 7.54-7.43 (m, 2H);
13C NMR (DMSO) δ 164.8, 154.5, 150.9, 148.6, 146.6, 141.9,
136.2, 134.9, 133.3, 132.4, 131.9, 131.2, 130.3, 128.6, 127.6, 126.7,
125.2, 124.2, 124.0, 120.6, 118.4, 116.7; ESI-MS m/z 410.2 (M +
1), 382.4 (M - 28), 267.0 (M - 143), 239.2 (M - 171).
6-Bromo-N-(naphthalen-2-yl)-2-(pyridin-4-yl)quinoline-4-
carboxamide (21). The reaction conditions were the same as above
except isatin was substituted with 5-bromoisatin. The overall yield
1
was 5.6%; mp 208-209 °C; H NMR (DMSO) δ 11.10 (s, 1H),
8.81 (d, J ) 3.9 Hz, 2H), 8.63 (s, 1H), 8.56 (s, 1H), 8.47 (d, J )
2.1 Hz, 1H), 8.33 (d, J ) 5.7 Hz, 2H), 8.18 (d, J ) 8.7 Hz, 1H),
8.04 (dd, J ) 2.1, 9.3 Hz, 1H), 7.92 (m, 3H), 7.77(dd, J ) 2.1, 8.7
Hz, 1H), 7.54-7.43 (m, 2H); 13C NMR (DMSO) δ 164.7, 154.1,
150.3, 146.6, 144.9, 141.9, 136.2, 133.9, 133.3, 132.1, 130.3, 128.5,
127.6, 127.5, 127.3, 126.6, 125.2, 125.1, 121.7, 121.5, 120.5, 118.4,
116.6; ESI-MS m/z 454.2 (M + 1), 426.3 (M - 28), 311.0 (M -
143), 283.2 (M - 171).
6-Bromo-N-(naphthalen-2-yl)-2-(pyridin-3-yl)quinoline-4-
carboxamide (22). The reaction conditions were the same as above
except 4-acetylpyridine was substituted with 3-acetylpyridine and
5-bromoisatin was used instead of isatin. The overall yield was
1
6.1%; mp 240-241 °C; H NMR (DMSO) δ 11.07 (s, 1H), 9.54
(s, 1H), 8.74-8.71 (m, 2H), 8.61 (s, 1H), 8.56 (s, 1H), 8.45 (d, J
) 2.1 Hz, 1H), 8.15 (d, J ) 9 Hz, 1H), 8.03-7.88 (m, 4H), 7.77(dd,
J ) 1.5, 8.7 Hz, 1H), 7.64-7.60 (m, 1H), 7.54-7.43 (m, 2H); 13
C
NMR (DMSO) δ 164.7, 154.5, 150.9, 148.6, 146.7, 141.7, 136.2,
134.8, 133.7, 133.3, 131.9, 130.3, 128.5, 127.6, 127.2, 126.6, 125.2,
124.6, 124.0, 121.1, 120.5, 118.3, 116.7; ESI-MS m/z 454.2 (M +
1), 426.3 (M - 28), 311.0 (M - 143), 283.1 (M - 171).
N-(Naphthalen-1-yl)-2-(pyridin-3-yl)quinoline-4-carboxamide
(23). The reaction conditions were the same as above except
4-acetylpyridine was substituted with 3-acetylpyridine and 1-naph-
thylamine was used instead of 2-naphthylamine to form the amide.
The overall yield was 35.8%; mp 201-202 °C; 1H NMR (CDCl3)
δ 9.27 (s, 1H), 8.67-8.61 (m, 2H), 8.47 (d, J ) 7.8 Hz, 1H), 8.36
(d, J ) 8.1 Hz, 1H), 8.21 (t, J ) 7.8 Hz, 2H), 8.11 (s, 1H),
7.95-7.91 (m, 2H), 7.85-7.80 (m, 2H), 7.67-7.52 (m, 4H),
7.42-7.38 (m, 1H); 13C NMR (CDCl3) δ 166.05, 154.16, 150.44,
148.93, 148.57, 143.31, 134.86, 134.27, 134.21, 131.71, 130.76,
130.35, 128.98, 128.19, 127.11, 126.75, 126.30, 125.77, 124.98,
123.74, 123.59, 121.33, 120.53, 116.19; ESI-MS m/z 376.5 (M +
1).
DFT Calculations. Density functional calculations were per-
formed using Gaussian 03.31 The TPSS functional32 was used with
the cc-pVDZ basis set with the effective core potential on iron,33
the aug-cc-pVDZ on sulfur34 and nitrogen35 with diffuse d functions
removed, and the cc-VDZ basis set on carbon and hydrogen.36 The
basis set and optimized geometries are available as Supporting
Information. Solvation energies were calculated for the gas phase
optimized geometries using the polarized continuum model self-
consistent reaction field with the solvent set to water’s dielectric
constant. The heme model was the abbreviated heme with an S-H
N-(Naphthalen-1-yl)-2-(pyridin-2-yl)quinoline-4-carboxamide
(24). The reaction conditions were the same as above except
4-acetylpyridine was substituted with 2-acetylpyridine and 1-naph-
thylamine was used instead of 2-naphthylamine to form the amide.
1
The overall yield was 3.8%; mp 207-208 °C; H NMR (DMSO)
δ 10.99 (s, 1H), 8.87-8.81 (m, 2H), 8.68 (d, J ) 7.8 Hz, 1H),
8.31-8.23 (m, 2H), 8.13-7.99 (m, 3H), 7.92-7.86 (m, 3H), 7.75
(t, J ) 7.2 Hz, 1H), 7.64-7.57 (m, 4H); 13C NMR (DMSO) δ
167.15, 155.84, 155.32, 150.18, 148.39, 143.90, 138.35, 134.51,
133.63, 131.20, 130.51, 129.20, 128.93, 128.80, 127.21, 126.97,
126.92, 126.35, 126.02, 125.79, 125.04, 124.11, 123.53, 121.92,
117.14; ESI-MS m/z 376.5 (M + 1).
37
fifth ligand used by Shaik and co-workers.
Results
All quinoline-4-carboxamide analogues were synthesized by
the route shown in Scheme 1. Results of the inhibition assays
are shown in Table 1. From the experimental results, we saw a
trend in binding affinity related to the positions of the type II
accessible nitrogen in the pyridine moiety (Figure 1). Com-
pounds with a pyridine nitrogen in the para position (relative
to the linkage) generally exhibit higher binding affinity than
those in the meta and ortho positions. In addition, compounds
with an aromatic ring system at the carboxyamide moiety bind
more tightly than those that lack a ring system (Figure 1, R )
aromatic).
Difference Spectroscopy of Binding and Substrate
Titrations. Difference spectra were collected using a double-beam
Olis upgraded Aminco DW-2000 spectrophotometer (Olis, Inc.,
Bogart, GA). The wavelength range encompassed 350-500 nm.
Both sample and reference cuvettes contained purified 2C9 in 100
mM KPi buffer with 20% glycerol, pH 7.4.10 To obtain difference
spectra, a baseline was recorded with 2C9 enzyme and buffer in
both the sample and reference cuvettes followed by substrate
titratation into the sample cuvette, and the same volume of solvent
was added to the reference cuvette. The difference spectra of
samples containing purified 2C9 0.49 µM and substrates were
scanned at 30 °C. Substrate concentrations were between 0.107 and
109.9 µM. Substrate binding curves were fit by nonlinear regression
UV difference spectra were used to determine substrate
binding modes and spectral dissociation constants Ks for the

8006 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Peng et al.
Figure 2. Binding spectrum and titration curves of compound 13, Ks ) 0.325 µM.
Figure 3. EPR spectra of 2C9 in the presence of substrates 7 (para), 8 (meta), 9 (ortho), 16 (para), 17 (meta), and 18 (ortho).
quinoline-4-carboxamide analogues shown in Table 1 whose
absorbance did not interfere with the UV absorption of the
titration. Of those analogues, 10 and 13 gave clear type II
binding spectra (representative titrations results are shown in
Figure 2). Since the Ki values reported in Table 1 were obtained
using baculosomes, we used the reconstituted 2C9 system to
determine if purified enzyme gave different Ki values relative
to the baculosomes preparations. The results gave Ki values for
compound 10 of 113 ( 6.85 nM in the purified system and
125 nM in the baculosomes. For compound 13, the Ki was 79.8
( 5.91 nM in the purified system and 100 nM in the baculosome
system. The results indicate that for these substrates the purified
reconstituted system and the baculosomes system gave similar
results. Compound 14 showed a peak at ∼415 nm but no
obvious trough in the difference spectra, which implies that 14
exhibits mixed type II and type I binding modes.
Since EPR measurements are not hindered by the substrate
absorbing in the Soret band range, additional compounds could
be examined for the binding mode. Compounds 7, 8, 9, 16, 17,
18, 19, and 21 were studied by EPR to probe binding modes
(Figure 3 and 4). From these results, analogues with the pyridine
nitrogen in the para position to the linkage (7, 16, 19, and 21),
EPR spectra showed a low-spin shift which was absent with
compounds having nitrogen in the meta or ortho positions. The
EPR spectrum of substrate-free 2C9 had g-values at 2.42/2.38,
2.24, and 1.92 (data not shown), and the type II binding substrate
bound 2C9 spectrum had g-values at 2.51, 2.26, and 1.88. This
result suggests that the para nitrogen is coordinated to the heme
iron, while the meta and ortho compounds are not.
To determine which portion of the total binding energy
depends on pure type II interaction (as opposed to apoprotein
interactions), the density functional method TPSS was used for
the core structure of the three pyridinylquinolines (ortho, meta,
and para pyridines, Figure 5), and the differences in energies
for nitrogen substitution ortho, meta, and para from the linkage
to quinoline were determined. The results indicate that the ortho
nitrogen linkage has a much longer N-Fe bond and significantly
distorts the heme from planarity. In fact, this structure was so
distorted that the geometry could not be minimized. The meta
and para linkage both have 2.1 Å N-Fe bond lengths with only
a slight distortion in the planarity of the heme. Electronically,
the para linkage has the strongest interaction energy by 0.6 kcal/
mol; however, this is not a significant difference given the level
of computation.
To determine if differences in solvation energy might
influence the overall binding energy, a series of ortho, meta,
and para linkages were used to calculate the solvation energies
for each core structure using a polarized continuum self-

Cytochrome P450 2C9 Type II Binding Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8007
Figure 4. EPR spectra of 2C9 in the presence of substrates 7 (para), 16 (para), 19 (para), and 21 (para).
substrate metabolic stability as well as the potency of inhibition.
Therefore, elucidating how type II interactions can be used to
optimize drugs metabolic properties without increasing drug
toxicity is necessary to optimize drug design.
In this study we investigated the potential increase in binding
affinity that can be attributed to type II binding in the absence
of other binding features. To this end, we synthesized a series
of quinoline-4-carboxamide analogues that differ in the place-
ment of nitrogen capable of forming a type II complex with
the heme iron of 2C9 (Table 1). Each set of three compounds
differed only in the position of their linkage and are assumed
to have almost identical solvation energies allowing for the
determination of the nitrogen coordination binding. One caveat
is that solvation energy calculations and pKa measurements show
that interpretation of changes in binding affinity for the ortho
compounds may be complicated by differences in solvation
relative to the meta and para linkages.
Figure 5. Core structures used to determine nitrogen-iron coordination
energies and solvation energies.
consistent reaction field model as implemented in Gaussian 03.
Results indicate insignificant differences in solvation energy
between the meta and para isomer, while the ortho isomer was
around 2 kcal/mol less stabilized by solvent. This difference in
solvent interaction is also reflected in the measured differences
in pKa values for the ortho compound 18 of 3.05 versus nearly
identical values of 4.27 and 4.30 for para (16) and meta (17),
respectively. These results indicate that the difference in binding
affinity for the compounds with the ortho linkage may be
complicated by differences in solvation.
The effects of varying the position of the nitrogen atom in
the pyridine ring on the binding affinity of quinoline-4-
carboxamide analogues to 2C9 are shown in Table 1 and Figure
6. In general, compounds with a para nitrogen in the pyridine
ring bind more tightly than meta or ortho compounds; com-
pounds with meta nitrogens are better binders than ortho
compounds. Para compounds generally bind to 2C9 with Ki
between 9 and 125 nM except for compounds 1 and 4, which
have Ki values of 9.94 and 3.67 µM, respectively. The binding
affinity of para compounds are increased by a factor of up to
4200-fold compared with meta compounds (such as 21 and 22)
and a factor of up to 1900-fold compared with ortho compounds
(such as 16 and 18). Meta compounds overall have a larger
range of binding affinities to 2C9, having Ki values from 0.15
Discussion
Understanding the mechanism of type I and type II substrate
binding modes in P450 drug metabolizing isoforms is critical
to optimizing drug design and reducing drug toxicity. Metabolic
stability, enzyme inhibition, metabolite regioselectivity, and
formation of reactive metabolites are all functions of binding
modes. It has been shown that substrates which are capable of
type II binding are more potent inhibitors than those capable
of only type I binding.38 In addition, potent inhibition is possible
when coordination between the heteroatom (nitrogen atom) lone
pair electrons and the ferric heme iron is combined with
apoprotein interactions (lipophilic and electrostatic).11 Type II
binding slows the rate of P450-mediated oxidation, increasing

8008 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Peng et al.
EPR studies on para compounds 7, 16, 19, and 21 suggest
that the para nitrogen atom on the pyridine moiety is coordinated
to the P450 iron. The low-spin g-value shifts of 1.91 to 1.88
and 2.45 to 2.51 observed for these compounds are typical of
the low-spin shift commonly seen in type II binding substrates
(normal nitrogen donors) in other P450 enzymes.8,39 However,
compounds 16, 19, and 21 also show a low spin signal at 2.38
that is characteristic of a nonsubstrate bound EPR spectrum
(Figure 4). The coexistence of both the shifts and the unbound
signal at 2.38 for compounds 16, 19, and 21 suggests compound
7 is able to bind pure type II while the others bind in the mixed
mode. The difference in binding modes of these para compounds
may be attributed to the increase in steric bulk (and hydropho-
bicity) of compounds 16, 19, and 21 relative to compound 7.
While all are tight binders, increasing steric bulk may interfere
with formation of a nitrogen-ferric complex allowing for the
mixed binding mode seen in the EPR. Overall, UV and EPR
studies support the hypothesis that the pyridine rings with the
para-substituted nitrogen have strong type II binding interactions.
The meta compounds appear to be able to coordinate to the
heme to a lesser extent, while the ortho compounds do not
appear to coordinate to the heme.
Figure 6. Ki versus nitrogen atom in the para, meta, and ortho positions
toward the linkage of nine different groups of compounds indicating
that, in general, the order of tightness of binding is para > meta >
ortho (Table 1).
Table 2. B3LYP/cc-pVDZ Substrate Energies and Heme-Substrate
Complexes for Core Structures Shown in Figure 5
linkage
substrate energya
type II energyb
solvation energyc
para
meta
ortho
-649.207
-649.208
-649.344
-2135.127
-2135.126
not stable
-10.23
-9.87
-8.03
a Energies in Hartrees for the para, meta, and ortho linked core structures
in Figure 5. b Energies in Hartrees for each core structure coordinated to
the heme. c PCM solvation energies in kcal/mol for the different linkages
with the dielectric set to that of water.
Interestingly, the UV difference spectra appear to provide a
more sensitive test of substrate binding modes than EPR spectra.
UV difference spectrum gathered on meta compound 2 showed
what may be interpreted as mixed type I and type II binding
that was not apparent in the EPR spectrum. The disagreement
between the UV and EPR spectra of meta compounds could be
a sensitivity issue or other artifacts requiring additional
investigation.
Table 3. List of UV and EPR Spectral Results for Selected Compounds
binding type
compound
Ki (µM)
UV min (nm)/max (nm)
EPR
type II
type I
type II
type I
1
2
7
8
9
10
13
14
16
17
18
19
21
9.94
9.74
0.033
1.15
10.67
0.125
0.10
4.06
0.011
8.55
type II 410/429
mixed -/413
NDb
The spectral dissociation constants (Ks) for compounds 10
and 13 (1.28 and 0.325 µM) were higher than the inhibitor
dissociation constant, Ki, (0.125 and 0.1 µM) determined by
IC50 experiments. These discrepancies are not unusual40,41 and
may be attributed to the different methods used to measure each
value. Ks was measured as the type II binding substrate
dissociation constant when the heme iron was in the +3
oxidation state and the amount of spin state change between
high and low was dependent on the equilibrium of the nature
of the nonsubstrate bound protein. On the other hand, Ki was
determined as the overall inhibitor dissociation constant where
the catalytic cycle was completed and the iron is in equilibrium
between the +3 and +2 oxidation states, allowing the inhibitor
to bind in either type I or type II modes. Mixed substrate binding
and heme iron oxidation state equilibria further complicate the
elucidation of the substrate binding modes within the active site.
Another possible reason that the Ks was higher than Ki was that
the substrate heme coordination was incomplete. Incomplete
heme coordination has been documented by Locuson et al.
2007.10
NDa
NDa
type I
type II 413/428
type II 410/431
mixed -/417
NDc
NDa
NDa
NDa
type II/type I
type I
type I
type II/type I
type II/type I
NDc
>20.6
0.012
0.009
NDc
NDb
NDb
a Data not collected. b Substrate absorbance prevented characterization.
c Spectrum is not clear.
to 37.85 µM, and these compounds can be 300-fold tighter
binders than ortho compounds. In general, ortho compounds
bind less tightly than those of their para and meta counterparts.
Ki values of ortho compounds ranged from 76.1-1.32 µM. The
most likely explanation for these results is that the para
compounds are capable of stronger type II binding interaction
than the meta compounds, which are capable of stronger type
II interactions than the ortho compounds.
One potential complication of this interpretation would arise
if the para, meta, and ortho compounds had different pKa values.
To determine if the nitrogen atom in the pyridine ring was
differentially protonated for the ortho, meta and para substituted
pyridines in the physiological pH, the pKa values of all analogues
were predicted using MarvinSketch. To ensure that these
predictions were reliable, pKa values were experimentally
determined for compounds 16, 17, and 18. The results from
computational prediction/experimental determination were 4.15/
4.27, 3.44/4.30, and 3.36/3.05 for para, meta, and ortho,
respectively. All the compounds have a predicted pKa less than
4.3 for the pyridine nitrogen, which indicates that at physi-
ological pH, the analogues are free to coordinate with the heme
iron. In addition, a lower pKa corresponds to greater delocal-
To determine if type II binding is responsible for the
differences in binding affinity, we used both UV and EPR
spectroscopy (Table 3) to determine substrate binding modes
of several compounds from Table 1. Binding mode analysis
using UV spectroscopy was limited to only compounds that did
not absorb in the region of interest, 350-500 nm. UV difference
spectra results for para compounds (1, 10, and 13) show typical
type II binding spectra, with peaks at ∼430 nm and a trough at
∼410 nm. The meta compounds (2 and 14), however, show
what appears to be mixed type I and type II binding in the UV
difference spectra where peaks are at ∼418 nm but not an
obvious trough. The ortho compound (15) does not give either
a type I or type II binding spectra.

Cytochrome P450 2C9 Type II Binding Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8009
Figure 7. Proposed type II binding mode with compound 7 using the
flurbiprofen complex of 2C9dH (Protein Data Bank code 1r9o), on the
top. A 2D diagram of ligand-residue interactions is presented on the
bottom.
ization of the lone pair in the aromatic system. Delocalization
decreases the ability of an sp2 nitrogen to type II binding. The
piperidine nitrogen on compounds 4, 5, and 6, which are
predicted to have pKa values of ∼9.1, and since these values
are well above physiological pH, they would exist in their
protonated forms. In general, positively charged compounds do
not bind strongly to 2C9, which may explain the overall low
affinity of compounds 4, 5, and 6 for the enzyme.
To determine if differential interactions of the para, meta,
and ortho compounds result from different heme interactions,
quantum calculations of the binding energies of the different
compounds were determined (Table 2). The results indicate that
the ortho substituted pyridines do not strongly interact with the
heme. However, the para and meta compounds interact with
essentially the same energy. These results suggest the meta
compounds may have less favorable apoprotein interactions than
the para substituted compounds. Further, these computational
results suggest that active site residues play an important role
in determining the affinity for the meta and para compounds’
ability to form type II complexes. For meta and ortho com-
pounds, interactions with the apoprotein rather than just the
electrostatic interaction with the heme appear to have the most
significant influence on binding affinity versus para compounds.
Crystal structures that show type II binding interactions
include P450cam with nicotine, CYP51 with 4-phenylimidazole
Figure 8. Proposed implausible type II binding mode with compound
8 using the flurbiprofen complex of 2C9dH (Protein Data Bank code
1r9o).
(4-PI) and fluconazole (FLU), P450eryF with ketoconazole, and
CYP2B4 with bifonazole.42-45 Generally, these structures show
the heterocyclic substrates type II bound to the heme iron with
the remainder of the molecule bent away from the I-helix in
the active site pocket and the essential conformational changes
of apoprotein are necessary for substrates binding. With these
substrate-enzyme binding interactions in mind, we applied
computational modeling methods to further visualize the overall
substrate binding interactions.
In an attempt to ascertain specific substrate-protein interac-
tions, we used automated docking on two of our tight binders,
the quinoline-4-carboxamide analogues 7 and 8 (Figures 7 and
8). Both of these compounds contain naphthalene functionalized
amides and differ only in the position of the sp2 nitrogen in the
pyridine moiety. Indeed, all compounds from Table 1 that
contain aromatic amides (especially naphthalene) bind more
tightly than substrates with nonaromatic amides. This suggests
that in addition to type II coordination, the aromatic amide
systems may interact favorably with active site residues.
Docking of type II binder 7 into the crystal structure of 2C9 is
relatively straightforward since the nitrogen-heme coordination

8010 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Peng et al.
(7) Schenkman, J. B. Studies on the nature of the type I and type II spectral
changes in liver microsomes. Biochemistry 1970, 9, 2081–2091.
(8) Kumaki, K.; Sato, M.; Kon, H.; Nebert, D. W. Correlation of type I,
type II, and reverse type I difference spectra with absolute changes in
spin state of hepatic microsomal cytochrome P-450 iron from five
mammalian species. J. Biol. Chem. 1978, 253, 1048–1058.
(9) Schenkman, J. B.; Remmer, H.; Estabrook, R. W. Spectral studies of
drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol.
1967, 3, 113–123.
(10) Locuson, C. W.; Hutzler, J. M.; Tracy, T. S. Visible spectra of type
II cytochrome P450-drug complexes: evidence that ”incomplete” heme
coordination is common. Drug Metab. Dispos. 2007, 35, 614–622.
(11) Chiba, M.; Jin, L.; Neway, W.; Vacca, J. P.; Tata, J. R.; Chapman,
K.; Lin, J. H. P450 interaction with HIV protease inhibitors: relation-
ship between metabolic stability, inhibitory potency, and P450 binding
spectra. Drug Metab. Dispos. 2001, 29, 1–3.
(12) Roberts, A. G.; Campbell, A. P.; Atkins, W. M. The thermodynamic
landscape of testosterone binding to cytochrome P 450 3A4: Ligand
binding and spin state equilibria. Biochemistry 2005, 44, 1353–1366.
(13) LeLean, J. E.; Moon, N.; Dunham, W. R.; Coon, M. J. EPR
spectrometry of cytochrome P450 2B4: Effects of mutations and
substrate binding. Biochem. Biophys. Res. Commun. 2000, 276, 762–
766.
(14) Tsai, R.; Yu, C. A.; Gunsalus, I. C.; Peisach, J.; Blumberg, W.; Orme-
Johnson, W. H.; Beinert, H. Spin-state changes in cytochrome
P-450cam on binding of specific substrates. Proc. Natl. Acad. Sci.
U.S.A. 1970, 66, 1157–1163.
(15) Chiba, M.; Tang, C.; Neway, W. E.; Williams, T. M.; Desolms, S. J.;
Dinsmore, C. J.; Wai, J. S.; Lin, J. H. P450 interaction with farnesyl-
protein transferase inhibitors. Metabolic stability, inhibitory potency,
and P450 binding spectra in human liver microsomes. Biochem.
Pharmacol. 2001, 62, 773–776.
(16) Johnston, S. R.; Martin, L. A.; Head, J.; Smith, I.; Dowsett, M.
Aromatase inhibitors: combinations with fulvestrant or signal trans-
duction inhibitors as a strategy to overcome endocrine resistance. J.
Steroid Biochem. Mol. Biol. 2005, 95, 173–181.
(17) Denton, T. T.; Zhang, X.; Cashman, J. R. Nicotine-related alkaloids
and metabolites as inhibitors of human cytochrome P-450 2A6.
Biochem. Pharmacol. 2004, 67, 751–756.
(18) Sellers, E. M.; Kaplan, H. L.; Tyndale, R. F. Inhibition of cytochrome
P450 2A6 increases nicotine’s oral bioavailability and decreases
smoking. Clin. Pharmacol. Ther. 2000, 68, 35–43.
(19) Ekins, S.; Mankowski, D. C.; Hoover, D. J.; Lawton, M. P.; Treadway,
J. L.; Harwood, H. J., Jr. Three-dimensional quantitative structure-
activity relationship analysis of human CYP51 inhibitors. Drug Metab.
Dispos. 2007, 35, 493–500.
(20) Gibbons, G. F. The role of cytochrome P450 in the regulation of
cholesterol biosynthesis. Lipids 2002, 37, 1163–1170.
(21) Zhang, W.; Ramamoorthy, Y.; Kilicarslan, T.; Nolte, H.; Tyndale,
R. F.; Sellers, E. M. Inhibition of cytochromes P450 by antifungal
imidazole derivatives. Drug Metab. Dispos. 2002, 30, 314–318.
(22) Zarn, J. A.; Bruschweiler, B. J.; Schlatter, J. R. Azole fungicides affect
mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase
and aromatase. EnViron. Health Perspect. 2003, 111, 255–261.
(23) Evans, W. E.; Relling, M. V. Pharmacogenomics: translating functional
genomics into rational therapeutics. Science 1999, 286, 487–491.
(24) Rettie, A. E.; Jones, J. P. Clinical and toxicological relevance of
Cyp2c9: Drug-drug interactions and pharmacogenetics. Annu. ReV.
Pharmacol. Toxicol. 2005, 45, 477–494.
is known. This important feature of the nitrogen position on
pyridine is one of the major characteristics on substrate
structures that determine overall binding. For compound 7, a
direct coordination between the heme and the para pyridine
nitrogen is shown and this pyridine ring system is tilted away
from the I-helix. In this orientation, the naphthalene ring faces
Phe 114, allowing an edge-to-face π interaction. In addition,
the positively charged side chain of Arg 108 is oriented in such
a way to allow interaction with the naphthalene ring through
parallel stacking geometry. Presumably, a π-cation interaction
from the positively charged Arg 108 side chain with the aromatic
ring of amide allows for favorable enthalpic contributions of
van der Waals and electrostatic interactions. This type of
energetically favorable π-cation interaction has been described
in various biological systems and recognition processes.46,47 In
addition, the quinoline ring portion of 7 is located in a
hydrophobic pocket surrounded by Phe 476, Leu 208, Ile 205,
and Leu 362. This conformation allows for potential π-π
interactions between the quinoline ring and Phe 476.
The proposed binding orientation for compound 8 does not
have the nitrogen atom coordinated to the heme iron. This may
be a result of steric interference with the I-helix, even though
all the other ligand-residue interactions are similar to those
for compound 7. These docking results suggest the primary
difference in binding affinity between the meta and para
compounds is a result of steric interaction with the I-helix
resulting in diminished or nonexistent type II binding.
Conclusion
Type II interactions involving coordination of a pyridine
nitrogen to the heme results in an up to 4200-fold increase in
binding affinity. These significant increases in affinity can be
modulated by changing the position of the nitrogen relative to
the substituents and could be used to dial-in affinities for P450
enzymes to minimize drug-drug interactions, while maintaining
very similar reactivity and solubility in a potential drug.
Acknowledgment. This work was supported by National
Institutes of Health Grants GM032165 and GM084546. We
would like to thank Professor Kirk Peterson for providing the
basis set for iron.
Supporting Information Available: The cc-aug-pVDZ basis
set and effective core potential used for calculations and tables of
Cartesian coordinates for geometries used for Table 2 and the
chromatographic data for target compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(25) Rushmore, T. H.; Reider, P. J.; Slaughter, D.; Assang, C.; Shou, M.
Bioreactor systems in drug metabolism: synthesis of cytochrome P450-
generated metabolites. Metab. Eng. 2000, 2, 115–125.
References
(26) BD Biosciences. 4′-Hydroxydiclofenac standard. BD Biosciences: San
Jose, CA, 2005.
(27) Shaw, P. M.; Hosea, N. A.; Thompson, D. V.; Lenius, J. M.;
Guengerich, F. P. Reconstitution premixes for assays using purified
recombinant human cytochrome P450, NADPH-cytochrome P450
reductase, and cytochrome b5. Arch. Biochem. Biophys. 1997, 348,
107–115.
(28) Locuson, C. W.; Gannett, P. M.; Tracy, T. S. Heteroactivator effects
on the coupling and spin state equilibrium of CYP2C9. Arch. Biochem.
Biophys. 2006, 449, 115–129.
(1) Locuson, C. W.; Wahlstrom, J. L.; Rock, D. A.; Rock, D. A.; Jones,
J. P. A new class of CYP2C9 inhibitors: Probing 2C9 specificity with
high-affinity benzbromarone derivatives. Drug Metab. Dispos. 2003,
31, 967–971.
(2) Locuson, C. W.; Suzuki, H.; Rettie, A. E.; Jones, J. P. Charge and
substituent effects on affinity and metabolism of benzbromarone-based
CYP2C19 inhibitors. J. Med. Chem. 2004, 47, 6768–6776.
(3) Locuson, C. W.; Rock, D. A.; Jones, J. P. Quantitative binding models
for CYP2C9 based on benzbromarone analogues. Biochemistry 2004,
43, 6948–6958.
(29) Jefcoate, C. R. Measurement of substrate and inhibitor binding to
microsomal cytochrome P-450 by optical-difference spectroscopy.
Methods Enzymol. 1978, 52.
(30) Wester, M. R.; Yano, J. K.; Schoch, G. A.; Yang, C.; Griffin, K. J.;
Stout, C. D.; Johnson, E. F. The structure of human cytochrome
P4502C9 complexed with flurbiprofen at 2.0-angstrom resolution.
J. Biol. Chem. 2004, 279, 35630–35637.
(31) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin,
K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone,
V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
(4) Rettie, A. E.; Jones, J. P. Clinical and toxicological relevance of
CYP2C9: Drug-drug interactions and pharmacogenetics. Annu. ReV.
Pharmacol. Toxicol. 2005, 45, 477–494.
(5) Hudelson, M. G.; Jones, J. P. Line-walking method for predicting the
inhibition of P450 drug metabolism. J. Med. Chem. 2006, 49, 4367–
4373.
(6) Hudelson, M. G.; Ketkar, N. S.; Holder, L. B.; Carlson, T. J.; Peng,
C. C.; Waldher, B. J.; Jones, J. P. High confidence predictions of
drug-drug interactions: predicting affinities for cytochrome P450
2C9 with multiple computational methods. J. Med. Chem. 2008,
51, 648–654.

Cytochrome P450 2C9 Type II Binding Studies
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8011
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene,
M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev,
O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala,
P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;
Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas,
O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.;
Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen,
W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision
C.02; Gaussian, Inc.: Wallingford, CT, 2004.
(39) Dawson, J. H.; Andersson, L. A.; Sono, M. Spectroscopic investigations
of ferric cytochrome P-450-CAM ligand complexes. Identification of
the ligand trans to cysteinate in the native enzyme. J. Biol. Chem.
1982, 257, 3606–3617.
(40) Verras, A.; Kuntz, I. D.; Ortiz de Montellano, P. R. Computer-assisted
design of selective imidazole inhibitors for cytochrome p450 enzymes.
J. Med. Chem. 2004, 47, 3572–3579.
(41) Ahlstroem, M. M.; Zamora, I. Characterization of type II ligands in
CYP2C9 and CYP3A4. J. Med. Chem. 2008, 51, 1755–1763.
(42) Strickler, M.; Goldstein, B. M.; Maxfield, K.; Shireman, L.; Kim, G.;
Matteson, D. S.; Jones, J. P. Crystallographic studies on the complex
behavior of nicotine binding to P450cam (CYP101). Biochemistry
2003, 42, 11943–11950.
(43) Podust, L. M.; Poulos, T. L.; Waterman, M. R. Crystal structure of
cytochrome P450 14alpha-sterol demethylase (CYP51) from Myco-
bacterium tuberculosis in complex with azole inhibitors. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 3068–3073.
(44) Cupp-Vickery, J. R.; Garcia, C.; Hofacre, A.; McGee-Estrada, K.
Ketoconazole-induced conformational changes in the active site of
cytochrome P450eryF. J. Mol. Biol. 2001, 311, 101–110.
(45) Zhao, Y.; White, M. A.; Muralidhara, B. K.; Sun, L.; Halpert, J. R.;
Stout, C. D. Structure of microsomal cytochrome P450 2B4 complexed
with the antifungal drug bifonazole: iInsight into P450 conformational
plasticity and membrane interaction. J. Biol. Chem. 2006, 281, 5973–
5981.
(46) Pellequer, J.-L.; Zhao, B.; Kao, H.-I.; Bell, C. W.; Li, K.; Li, Q. X.;
Karu, A. E.; Roberts, V. A. Stabilization of bound polycyclic aromatic
hydrocarbons by a p-cation interaction. J. Mol. Biol. 2000, 302, 691–
699.
(32) Staroverov, V. N.; Scuseria, G. E.; Tao, J. M.; Perdew, J. P.
Comparative assessment of a new nonempirical density functional:
Molecules and hydrogen-bonded complexes. J. Chem. Phys. 2003, 119,
12129–12137.
(33) Peterson, K. A.; Dolg, M. Unpublished results.
(34) Woon, D. E.; Dunning, T. H. Gaussian-basis sets for use in correlated
molecular calculations 0.3. The atoms aluminum through argon.
J. Chem. Phys. 1993, 98, 1358–1371.
(35) Kendall, R. A.; Dunning, T. H.; Harrison, R. J. Electron-affinities of
the 1st-row atoms revisited-systematic basis-sets and wave-functions.
J. Chem. Phys. 1992, 96, 6796–6806.
(36) Dunning, T. H. Gaussian basis sets for use in correlated molecular
calculations. I. The atoms boron through neon and hydrogen. J. Chem.
Phys. 1989, 90, 1007–1023.
(37) Cho, K. B.; Moreau, Y.; Kumar, D.; Rock, D. A.; Jones, J. P.; Shaik,
S. Formation of the active species of cytochrome P450 by using
iodosylbenzene: A case for spin-selective reactivity. Chem.sEur. J.
2007, 13, 4103–4115.
(47) Dougherty, D. A.; Stauffer, D. A. Acetylcholine binding by a synthetic
receptor: implications for biological recognition. Science (Washington,
DC, U.S.) 1990, 250, 1558–1560.
(38) Schenkman, J. B.; Sligar, S. G.; Cinti, D. L. Substrate interaction with
cytochrome P-450. Pharmacol. Ther. 1981, 12, 43–71.
JM8011257