Discovery of a novel binding pocket for CYP 2C9 inhibitors: Crystallography, pharmacophore modelling and inhibitor SAR

Article abstract of DOI:10.1039/c6md00011h

Herein, we describe the discovery of a novel binding pocket for CYP 2C9 inhibitors. Trifluoromethanesulfonamide compounds 1 and 2, identified within a Pfizer progesterone receptor antagonist program, were found to strongly inhibit the drug metabolizing cytochrome P450 enzyme CYP 2C9. Homology modelling and subsequent X-ray co-crystal data have elucidated the binding orientation of compounds 1 and 2 in CYP 2C9. Compound 2 adopts a previously unreported binding mode. Less acidic sulfonamide analogues within these series have reduced CYP 2C9 activity, and we postulate this is due to a reduced hydrogen bonding potential with key interacting residues within CYP 2C9. This work shows that CYP 2C9 has a more flexible active site than previously reported; therefore multiple binding modes and alternative pharmacophore models must be considered when predicting CYP 2C9 affinity.

Full text of DOI:10.1039/c6md00011h

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Discovery of a novel binding pocket for CYP 2C9 inhibitors:
†
Crystallography, pharmacophore modelling and inhibitor SAR
Cite this: DOI: 10.1039/x0xx00000x
a*
b
c
Sarah. E. Skerratt, Marcel J. de Groot and Chris Phillips
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
†The authors declare no competing
interests.
Herein, we describe the discovery of a novel binding pocket industry. In order to identify chemical matter for the Pfizer
for CYP 2C9 inhibitors.
Trifluoromethanesulfonamide progesterone receptor antagonist (PRA) program, a highꢀthroughput
compounds 1 and 2, identified within a Pfizer progesterone screening campaign was performed. [1] From this work, a number
receptor antagonist program, were found to strongly inhibit of chemotypes were selected for lead optimisation, including a
the drug metabolizing cytochrome P450 enzyme CYP 2C9. cyclohexyl
ether
and
biaryl
ether
series.
Homology modelling and subsequent X-ray co-crystal data Trifluoromethanesulfonamide analogues made within these series
have elucidated the binding orientation of compounds 1 and 2 were found to be potent antagonists of the progesterone receptor,
in CYP 2C9. Compound 2 adopts a previously unreported however they were also found to inhibit the drug metabolizing
binding mode. Less acidic sulfonamide analogues within cytochrome P450 enzyme, CYP 2C9. [2] Examples are shown in
these series have reduced CYP 2C9 activity, and we postulate Figure 1. Compound 1, from the cyclohexyl ether series, is a potent
this is due to a reduced hydrogen bonding potential with key PR antagonist (PR IC50 43 nM), but is also a strong inhibitor of CYP
interacting residues within CYP 2C9. This work shows that 2C9 (77% I in a CYP 2C9 biochemical assay run at a concentration
CYP 2C9 has a more flexible active site than previously of 3 µM). Compound 2 from the biaryl ether series is also active at
reported; therefore multiple binding modes and alternative the progesterone receptor (PR IC50 of 175 nM) and strongly inhibits
pharmacophore models must be considered when predicting CYP 2C9 (95% I in a CYP 2C9 biochemical assay run at a
CYP 2C9 affinity.
concentration of 3 µM).
CN
Cl
CN
Introduction
Cl
Compound 1
Compound 2
OMe
The progesterone receptor (PR) is a member of the family of ligandꢀ
activated transcription factors that includes the estrogen (ER),
androgen (AR), glucocorticoid (GR) and mineralocorticoid (MR)
receptors. The use of progesterone antagonists for the treatment of a
O
O
PR IC50 43 nM
CYP 2C9 77% I
PR IC50 175 nM
CYP 2C9 95% I
NH
MeO
NH
O
S
O
O
S
O
CF3
CF3
variety of progesteroneꢀrelated diseases and disorders, such as Figure 1: Structure and properties of compounds 1 and 2. The CYP 2C9 %I
endometriosis, is of considerable interest to the pharmaceutical data were generated at a 3 µM concentration.
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DOI: 10.1039/C6MD0001
Journal Na1Hme
2
C5 (rabbit)
3.00
1dt6 [10]
1n6B [16]
1nr6 [17]
None
DMZ
2
2
.30
.10
CYP 2C9 is a member of the P450 enzyme superfamily of
membraneꢀbound hemeꢀproteins that catalyse the oxidative
metabolism of structurally diverse molecules.[2] Of the fifty seven
known human P450 isoforms, CYP 2C9 is one of seven isoforms
responsible for more than 90% of the metabolism of compounds in
current clinical use.[3] However, CYP 2C9 has known human
polymorphisms that can lead to rapid or very poor metabolism in
specific patient populations.[4ꢀ6] In addition, the inhibitory binding
of molecules to P450 enzymes such as CYP 2C9 can lead to drugꢀ
drug interactions, and may cause severe side effects resulting in the
early termination of candidates in development, refusal of approval,
prescription limitations or withdrawal of drugs from the market.[7]
Diclofenac
2C8 (human)
2.70
2.60
1pq2 [18]
2nnh
None
9ꢀCisꢀretinoic acid
Montelukast
Felodipine
2
2
2
.80
.28
.70
2nni
2nnj
2vn0
Troglitazone
2C9 (human)
2.60
1og2 [19]
1og5 [19]
1r9O [20]
4nz2
None
2
2
2
2
.55
.00
.45
.20
SꢀWarfarin
Flurbiprofen
Inhibitor 2QJ
NAMPT Inhibitors
Inhibitor OVX
4jnm
2
C19 (human)
2.87
4gqs
Theoretical models that can predict for the possible interaction of
drugs or drug candidates with P450 enzymes can be valuable tools in
Table 1: Available crystal structures of the human P450 2C subfamily
the drug discovery process.[8,9] In order to determine the nature of With the known risks associated with CYP 2C9 inhibition, [21] the
the protein binding site that interacts with a substrate, a three project team sought to design PRA analogues of compounds 1 and 2
dimensional representation of the entire active site is required. For with reduced CYP 2C9 liability. To achieve this we desired an
example, rabbit CYP2C5/3, the first mammalian P450 structure to be understanding of the binding site interactions of compounds 1 and 2
solved,[10] became the basis of several CYP 2C9 homology models. in the CYP 2C9 ligand binding site. In the absence of CYP 2C9 coꢀ
The publication of crystal structures of the human P450 2C crystal structure information, we elected to utilise a CYP 2C9
subfamily (Table 1) over recent years has enabled the scientific homology model to guide analogue design.
community to compare previously reported homology models and
establish model validity. For example, CYP 2C8 and CYP 2C9 Results and Discussion
models have been shown to exhibit αꢀcarbon RMS distances of 1.2Å
and 1.5Å respectively, demonstrating the utility of the CYP 2C5
crystal structure as a template for generating threeꢀdimensional
models for other CYP 2C subfamily enzymes.[11ꢀ13] Modelling the
CYP 2C9 Homology Modelling
De Groot et al. previously described a CYP 2C9 homology model
based on the rabbit CYP2C5/3 crystal structure.[11] This model,
and the pharmacophore embedded within, was initially used to
predict the potential binding modes of compounds 1 and 2. In order
to dock compounds 1 and 2 into the CYP 2C9 model, the assumption
was made that they bind at the same location, and with the same
orientation, as substrates in the existing model, such as Flurbiprofen.
This orientation consists of a polar/acidic region of the substrate
3
D structure of P450s to allow docking of potential substrates can be
a useful method for predicting and rationalising substrate selectivity.
It can however be complicated by the fact that multiple ligand
binding modes may be available. Several years ago, evidence began
to emerge that CYP 3A4 could bind multiple substrates
simultaneously.[14]
This observation was later extended to
1
08
additional P450 enzymes.[15]
interacting with a positively charged region of the protein (Arg ), a
hydrophobic region of the substrate interacting with several
476
362
Available Crystal Structures of the CYP 2C subfamily
Resolution
hydrophobic protein residues (Phe and Leu ), and a site of ligand
oxidation in close proximity to the heme.
CYP
PDB
Substrate/Inhibitor
(Å)
After superposition, the compounds were relaxed using
a
Macromodel[22] optimization within the confines of the homology
model binding pocket. The main binding interactions between
2
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6MD00011H
compounds 1 and 2 and CYP 2C9 were predicted to be an interaction Figure 3: Predicted hydrogen bonding interactions between sulfonamide
108
204
oxygens of compound 1 and Arg and Asn in the in CYP 2C9 homology
between the acidic trifluoromethanesulfonamide unit of either
1
08
204
model
compound with CYP 2C9 Arg and Asn , as well as hydrophobic
4
76
interactions between the chlorocyanoꢀphenyl moiety and Phe and
3
62
Compound analogues were designed to perturb the strength of the
hydrogen bond interaction network with the aim of reducing CYP
Leu
.
The binding orientation also directed the chlorocyanoꢀ
phenyl groups towards the CYP 2C9 heme moiety (Figure 2).
2
C9
inhibition.
Specifically,
unit
replacement
with the less
of
the
trifluoromethanesulfonamide
acidic
methanesulfonamide group was explored (Figure 4). Compound 3,
the methanesulfonamide analogue of compound 1, gratifyingly
retained activity at (PR IC50 107 nM) but, unlike compound 1, was
devoid of activity at CYP 2C9 (0% I at 3µM 2C9). Similarly,
compound 4, the methanesulfonamide analogue of compound 2
maintained activity at the PR receptor (PR IC50 26 nM) and was
inactive at CYP 2C9 (9% I at 3µM 2C9).
CN
CN
Cl
Cl
Compound 3
Compound 4
PR IC50 26 nM
CYP 2C9 9% I
OMe
O
O
PR IC50 107 nM
CYP 2C9 0% I
NH
MeO
NH
O
S
O
O
S
O
Figure 2: Superposition of compound 1 (green), compound 2 (yellow) and
CH3
CH3
Flurbiprofen (cyan) in the CYP 2C9 homology model binding pocket.
Figure 4: Structure and properties of compounds 3 and 4. The CYP 2C9 %I
476
Ligand interactions are predicted to be with hydrophobic amino acids Phe
data were generated at a 3 µM concentration.
3
62
108
204
and Leu , polar residues Arg and Asn , and heme group
The difference in CYP 2C9 activity between the
CYP 2C9 structure-activity-relationships
trifluoromethanesulfonamide
compounds
and
their
It is known that CYP 2C9 preferentially binds to hydrophobic
molecules that contain an acidic group.[23] A key binding
interaction predicted in the homology modelling of compounds 1
and 2 in CYP 2C9 is a hydrogen bond network between the oxygen
methanesulfonamide counterparts is intriguing. Each motif is
capable of making hydrogen bonding interactions between the
108
204
sulfonamide oxygen atoms and CYP 2C9 Arg
and Asn
residues, but we postulate that the difference in affinity may be
driven by their disparate pKa values. For example, the measured
pKa of the trifluoromethanesulfonamide unit in compound 1 is 7.0,
whereas the pKa of the methanesulfonamide group in compound 3 is
1
08
atoms of the acidic sulfonamide group and polar residues Arg and
204
Asn (Figure 3, compound 1 highlighted only for clarity).
1
0.5. As shown in the electrostatic potential plot (Figure 5) there is
a significant increase in negative electrostatic potential on the
sulfonamide oxygen atoms when compound 1 exists in the
deprotonated (ionized) form relative to the neutral form, the same is
true for compound 3 (electrostatic plot not shown). Hence, the
increased propensity of compound 1 to exist in the ionic form at
neutral (physiologically relevant) pH relative to compound 3, and
1
08
drive stronger (charge reinforced) hydrogen bonding with Arg and
204
Asn , could explain the enhanced CYP 2C9 activity (the CYP 2C9
assay is run at pH 7.4). The pKa of compounds 2 and 4 are 4.5 and
8
.5 respectively.
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Jou al Na1Hme
rn
Comparison of Crystal Structures with the Homology Model
Cl
The homology model [12] utilised in this work was, in general, in
agreement with the CYP 2C9 coꢀcrystal structures generated for
compound 2. One difference noted was the location of the BꢀC loop
H
F3C
N
CN
.
S
O
O
O
88
115
region, i.e. residues Ile ꢀSer ,which resided in a more “inward”
orientation than in the homology model, and caused by the different
conformation of this region in the template used (Figure 6 shows the
neutral form
Cl
F3C
N
CN
location of the BCꢀloop from crystallographic data).
consequence, the embedded pharmacophore needed
As a
slight
S
O
O
a
O
reorientation. This was achieved by a pivot around the site of
oxidation along the BꢀC loop movement vector. Interestingly,
despite the movement of the BꢀC loop, all predicted interactions
ionized form
Figure 5: Electrostatic potential plot of compound 1 in the neutral and between the protein and compound 1 were maintained and the results
ionised state†
consistent with binding mode 1. As a consequence, the existing
homology model could be effectively utilised to predict binding
CYP 2C9 Crystal Structures
modes of analogues of compound 1, and guide medicinal chemistry
Coꢀcrystal structures of compounds 1 and 2 with CYP 2C9 were
efforts within this series. The binding mode of compound 2 was not
subsequently generated. Compound 1 was shown to bind in a
correctly predicted by the existing homology model, and
conformation similar to that predicted by the homology model,
demonstrates CYP 2C9 has a more flexible active site than
whereas compound 2 adopted a novel binding conformation. In each
previously assumed. However, the availability of crystallographic
108
case, Arg interacts with the sulfonamide group, but the orientation
of the cyanophenoxy moieties differ. The cyanophenoxy motif of
compound 1 faces the heme oxyꢀiron (binding mode 1), a binding
mode consistent with homology modelling data. However, in the
coꢀcrystal structure of CYP 2C9 and compound 2, the cyanophenoxy
motif of compound 2 resides in a newly opened hydrophobic pocket,
data for this newly observed binding site should enable orthogonal
homology models to generated, and enable additional binding modes
to be taken into account when predicting potential CYP 2C9 affinity.
Comparison with Available Site Directed Mutagenesis Data
The influence of various amino acids on the metabolism of CYP 2C9
substrates has previously been examined using site directed
4
76
forming a piꢀstacking interaction with Phe (Figure 6).
1
05
mutagenesis (SDM). Mutagenesis in the region of Arg indicates
that this residue is not an important anionic binding site.[24]
97
Mutation of Arg has an influence on diclofenac metabolism, but
97
Arg is not predicted to reside in the active site. In our crystal
97
structures, Arg (Figure 7) is involved in stabilising the heme
108
propionate moieties.[10, 24] Mutation of Arg to alanine reduces
the formation of 4’ꢀhydroxydiclofenac by 100ꢀfold compared with
CYP 2C9 wild type. A recent homology model based on the crystal
108
structure of bacterial CYP 102 suggested Arg is not in the active
site,[24] however, in the model for CYP 2C9 used in this work,
108
Arg is an essential part of the CYP 2C9 active site.[12] Our
108
current crystal structures confirm that Arg plays a key role in the
interaction with the acidic part of the inhibitors used in this study.
Figure 6: CYP 2C9 coꢀcrystal structures of compound 1 (green) in binding
mode 1 (CYP 2C9 in green) and compound 1 (yellow) in binding mode 2
Recent SDM experiments designed to confer (S)ꢀmephenytoin
(CYP 2C9 in yellow)
activity at CYP 2C9 suggested activity is dictated by amino acids
4
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CN
F
OH
CN
that influence the packing of structural elements or influence
substrate access to the channel, rather than altering the active site
Cl
Cl
iv)
+
99
220
221
directly (Ile in the B’ꢀregion, Ser and Pro in the FꢀGꢀloop and
NH .HCl
O
2
286
292
295
Ser , Val and Phe in the Iꢀhelix, none of which are in direct
contact with (S)ꢀmephenytoin).[25] Our crystal structures agree that
none of these residues seem to be directly interacting with our coꢀ
crystallised inhibitors, or the modelled locations of the substrates.
5
6
7
NH2
v)
vi)
CN
CN
O
Cl
Cl
Mutagenesis experiments guided by homology modelling and
O
1
14
113
CoMFA analysis suggest B'ꢀC helix loop residues Phe and Val
NH
NH
can make hydrophobic interactions with substrates.[26] Our crystal
structures support these data.
O
S
O
O S O
1
3
CF3
Scheme 1: Synthesis of ethers 1 and 3. Reagents and conditions: iv) NaH,
2 3 3
THF, 25 ºC, 79%. v) Tf O, Et N, DCM, -60 ºC, 5 min, 92%; v) MsCl, Et N,
DCM, 25 ºC, 1h, 95%.
Compounds 2 and 4 were prepared by a threeꢀstep sequence as
described in Scheme 2. Aniline 8 was derivatized under standard
conditions using trifluoromethanesulfonic anhydride to furnish
sulfonamide 9 in 76% yield. Regioselective demethylation of 9 with
23
trimethylsilyl iodide afforded phenol 11 in 88% yield. Subsequent
etherification of 11 via addition of aryl fluoride 5 under basic
conditions furnished compound 2 in an 85% yield. Compound 4
was prepared in a similar manner.
methanesulfonyl chloride in triethylamine and DCM afforded
sulfonamide 10 in 85% yield. Demethylation of 10 with
Mesylation of 8 with
Figure 7: Compound 1 (yellow) bound to CYP 2C9 (green). The CYP 2C9
residues for which siteꢀdirected mutagenesis data are available are
highlighted in purple.
trimethylsilyl iodide gave phenol 12 (89% yield) and subsequent
etherification via addition of aryl fluoride 5 furnished compound 4
(89% yield).
Compound Synthesis
OMe
OMe
OH
i)
ii)
MeO
OMe
MeO
OMe
MeO
O
OMe
The synthetic routes towards compounds 1 and 3 are described in
Scheme 1. Deprotonation of cyclohexanol 6 with NaH in THF
NH2
NH
O
NH
O
8
O
9, R = CF3
11, R = CF3
S
R
S
R
followed by addition of fluorobenzonitrile
cyclohexylamine 7 in good yield. Compound 1 was synthesised by
triflation of cyclohexylamine using triflic anhydride and
6
furnished
1
0, R = CH3
12, R = CH3
CN
Cl
Cl
CN
7
O
triethylamine in DCM at ꢀ60 °C (79% yield). Compound 3 was
made by addition of methanesulfonyl chloride and triethylamine to 7
in dichloromethane and pyridine (95% yield).
F
5
MeO
OMe
iii)
NH
O
O
2, R = CF3
S
R
4
, R = CH3
Scheme 2: Synthesis of aryl ethers 2 and 4. Reagents and conditions: Where
R = SO CF i) Tf O, Et N, DCM, -78 ºC, 3h, 76%. Where R = SO CH , i)
MsCl, Et N, DCM, 25 ºC, 2h, 85%; ii) TMSI, DCM, 25 ºC, 3h, 88-89%; iii)
Cs CO , NMP, 120 ºC, 10h, 85-89%.
2
3
2
3
2
3
3
2
3
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Journal Na1Hme
internal reference. Chemical shifts are expressed in parts per million (ppm),
multiplicity of the signals are indicated by lowerꢀcase letters (singlet s,
doublet d, triplet t, quadruplet q, multiple m, broad singlet br s), and
deuterated solvents are dimethylsulfoxide d6, methanol d4, and chloroform
d1. Mass spectral date were obtained using Waters ZQ ESCI or Applied
Biosystem's APIꢀ2000.
Conclusions
Trifluoromethanesulfonamide compounds 1 and 2, identified within
a progesterone receptor antagonist program, were found to strongly
inhibit the drug metabolizing cytochrome P450 enzyme CYP 2C9.
In order to design analogues with reduced CYP 2C9 activity, a CYP
2
C9 homology model previously described by De Groot et al. was 4-[(trans-4-aminocyclohexyl)oxy]-2-chlorobenzonitrile (7). To a stirred
suspension of 6 (486 mg, 3.22 mmol) in dry THF (200 mL) was added NaH
170 mg, 7.11 mmol) portionꢀwise. The resulting mixture was stirred at 25
used to predict the potential binding modes of compounds 1 and 2.
The modelling predicted a key binding determinant to be a hydrogen
bond network made between the two sulfonamide oxygen atoms of
(
ºC for 10 mins after which time 5 (500 mg, 3.22 mmol) was added. The
reaction mixture was heated to 70 ºC and stirred for 2 hrs. After cooling to
room temperature, the mixture was poured onto ice (100 g) and extracted
with EtOAc (3 x 50 mL). The combined organics were washed with 3N
HCl(aq.) (5 x 50 mL), the combined acidic extracts basified to pH 8 with 3N
1
08
204
the ligands and Arg and Asn of CYP 2C9.
Electrostatic mapping showed a significant increase in negative
electrostatic potential on the sulfonamide oxygen atoms in the NaOH(aq.) and extracted with EtOAc (3 x 30 mL). The combined organics
ionized versus the neutral form, and we reasoned the interaction of were washed with water (30 mL), dried over MgSO and concentrated in
vacuo to afford the title compound as offꢀwhite solid (635 mg, 79% yield).
4
CYP 2C9 with the ionized form of the sulfonamide group would
lead to increased CYP 2CP binding through an enhanced hydrogen
35
+
37
+
m/z 251 [M+H, Cl] , 253 [M+H, Cl] .
bonding interaction.
We therefore synthesised less acidic
N-[trans-4-(3-chloro-4-cyanophenoxy)cyclohexyl]-1,1,1-
sulfonamide analogues such that, at physiological pH, the propensity
of the sulfonamide motif to exist in an ionized form would be
reduced. Methanesulfonamide compounds 3 and 4 were synthesised
trifluoromethanesulfonamide (1). To a stirred solution of 7 (250 mg, 1.00
mmol) and triethylamine (207 µL, 1.50 mmol) in DCM (25 mL) at ꢀ60 ºC
was added triflic anhydride (168 µL, 1.00 mmol). The reaction mixture was
and, gratifyingly, they retained activity at the progesterone receptor was stirred for 1 hr, then poured onto water (25 mL) and extracted with DCM
but lost their inhibitory effects at CYP 2C9.
(2 x 25 mL). The combined organics were washed with water (25 mL), dried
over MgSO and concentrated in vacuo. The residue was purified via silica
4
gel column chromatography (eluting with 25% EtOAc/75% hexane) to afford
Subsequent Xꢀray crystal structure work fully elucidated the binding
orientation of progesterone receptor antagonist compounds 1 and 2
in CYP 2C9. Significantly, the coꢀcrystal structure with compound
the title compound as a white crystalline solid (211m g, 79% yield). m/z 383
+
[M+H]
2
in CYP 2C9 identified a novel and unpredicted binding mode in
N-[trans-4-(3-chloro-4-cyanophenoxy)cyclohexyl]methanesulfonamide
3). To a stirred solution of 7 (130 mg, 0.52 mmol) in DCM (5 mL) and
476
which a conformational change is induced, reꢀordering Phe , and
(
opening up a hydrophobic pocket. This work shows that CYP 2C9 pyridine (1 mL) at 25 ºC, was added methaneulfonyl chloride (77 µL, 1
has a more flexible active site than previously assumed. The mmol). The resulting mixture was stirred at 25 ºC for 2 hrs, poured onto 2N
HCl(aq.) (10 mL) and extracted with DCM (2 x 10 mL). The combined
crystallographic data for the newly observed binding site should
organics were washed with water (10 mL), brine (10 mL), dried over MgSO
4
enable orthogonal homology models to be generated, and enable
additional binding modes to be taken into account when predicting
CYP 2C9 affinity.
and concentrated in vacuo. The residue was purified via silica gel column
chromatography (eluting with 45% EtOAc/55% hexane) to afford the title
+
4
compound as a white solid (162 mg, 95% yield). m/z 346 [M+NH ]
Experimental section
1,1,1-trifluoro-N-(3,4,5-trimethoxyphenyl)methanesulfonamide (9). To a
stirred solution of 8 (1.00 g, 5.46 mmol) and triethylamine (0.93 mL, 6.60
mmol) in DCM (20 mL) at ꢀ78 ºC was added triflic anhydride (0.11 mL, 6.60
mmol). The reaction mixture was stirred for 1 hr and then poured onto water
All commercially available chemicals and solvents were used without further
purification. All temperatures are in °C. Flash column chromatography was
carried out using Merck silica gel 60 (9385) or Redisep silica. NMR spectra
were obtained on a a Varian Mercury (400MHz) or a Bruker Avance
(
25 mL) and extracted with DCM (2 x 20 mL). The combined organics were
washed with brine (25 mL), dried over MgSO and concentrated in vacuo.
The residue was purified via silica gel column chromatography (eluting with
4
(400MHz) using the residual signal of the deuterated NMR solvent as the
5
0% EtOAc/50% hexane) to afford the title compound as a white crystalline
6
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1
solid (1.64 g, 79% yield). H NMR (400 MHz, CDCl
3 2 3
) δ: 3.85 (s, 9H), 6.50 mL) was added 5 (186 mg, 1.2 mmol) followed by Cs CO (715 mg, 2.2
(
s, 2H), 6.80 (br s, 1H).
mmol) and the resulting mixture heated to 120 ºC for 10 hrs. After this time
the mixture was portioned between water (10 mL) and EtOAc (10mL). The
To a stirred aqueous layer was further extracted with EtOAc (2 x 10 mL) and the
N-(3,4,5-trimethoxyphenyl)methanesulfonamide (10).
4
solution of 8 (5.00 g, 27.3 mmol) in DCM (10 mL) and and pyridine (2 mL) combined organics washed with brine (20 mL), dried over MgSO , and
at 25 ºC, was added methaneulfonyl chloride (2.1 mL, 27.3 mmol). The concentrated in vacuo. The residue was purified via silica gel column
resulting mixture was stirred at 25 ºC for 2 hrs, poured onto 2N HCl(aq.) (100 chromatography, eluting with 50% EtOAc/50% pentane) to afford the title
1
mL) and extracted with DCM (2 x 75 mL). The combined organics were compound as a white solid (343 mg, 89% yield). H NMR (400 MHz,
washed with water (100 mL), brine (100 mL), dried over MgSO
4
and CDCl
3
) δ: 3.10 (s, 3H), 3.80 (s, 6H), 6.40 (br s, 1H), 6.55 (s, 2H), 6.85 (dd,
ꢀ
concentrated in vacuo. The residue was purified via silica gel column 1H), 6.95 (d, 1H), 7.55 (d, 1H); m/z 381 [MꢀH]
chromatography (eluting with 30% EtOAc/70% hexane) to afford the title
1
compound as an offꢀwhite solid (6.06 g, 85% yield). H NMR (400 MHz,
Crystallography
3
CDCl ) δ: 3.00 (s, 3H), 3.90 (s, 9H), 6.40 (br s, 1H), 6.50 (s, 2H).
Following the methods of Wester et al (2004) an engineered construct
containing residues 23ꢀ489 of the catalytic domain of human 2C9 with the
first 22 residues replaced with the sequence MAKKT was overꢀexpressed in
E. coli and purified using the two column method previously described [17].
Crystals were grown using the hanging drop vapour diffusion method in
conditions essentially equivalent to those reported. The crystals are
isomorphous with pdb deposition 1R9O, belonging to space group R3 with a
unit cell of a=b=91.1Å, c=169.5Å and can be produced using a coꢀ
crystallization method, with ligands at a concentration of 5mM incubated
with the protein on ice for 30 minutes prior to crystallization setꢀup. Data
were collected at beamline ID23 of the ESRF to a resolution of 2Å for the
complex with compound 1 and on an ‘in house’ Rigaku FRD Xꢀray source
with a Saturn92 CCD camera for the complex with compound 2. Data were
1
,1,1-trifluoro-N-(4-hydroxy-3,5-dimethoxyphenyl)methanesulfonamide
11). To a stirred solution of 9 (1.30 g, 4.13 mmol) in DCM (10 mL) at 0 ºC
was added TMSI (1.76 mL, 12.4 mmol) and the resulting mixture warmed to
0 ºC and stirred for 3 hrs. 20 mL of a 1:1 water/acetone mix was then added
and the resulting mixture extracted with EtOAc (2 x 30 mL). The combined
organics were washed with brine (30 mL), dried over MgSO and
(
2
4
concentrated in vacuo to give the title compound as a white solid (3.28 g,
1
8
2
8%). H NMR (400 MHz, CDCl
3
) δ: 3.90 (s, 6H), 5.60 (br s, 1H), 6.55 (s,
H), 6.75 (br s, 1H).
N-(4-hydroxy-3,5-dimethoxyphenyl)methanesulfonamide (12).
To a
2
4
stirred solution of 10 (1.59 g, 6.1 mmol) in DCM (10 mL) at 0 ºC was added
TMSI (2.65 mL, 18.3 mmol) and the resulting mixture warmed to 20 ºC and
stirred for 3 hrs. 20 mL of a 1:1 water/acetone mix was then added and the
resulting mixture extracted with EtOAc (2 x 50 mL). The combined organics
reduced and scaled using the XDS and the CCP4 suite of programs ,
25
iterative rounds of model building preformed in COOT , and refined with
26
BUSTER . The coordinates and reflection data have been deposited with
the PDB with the accession codes: 5a5i and 5a5j respectively.
were washed with brine (50 mL), dried over MgSO
4
and concentrated in
1
vacuo to afford the title compound as a white solid (1.34 g, 89%). H NMR
Acknowledgements
3
(400 MHz, CDCl ) δ: 3.00 (s, 3H), 3.90 (s, 6H), 5.45 (br s, 1H), 6.30 (br s,
The authors would like to thank Mr. Toby Underwood and Dr. Paul
A Bradley for synthesis of the compounds described in this
publication, Dr. Kiyoyuki Omoto for generating the electrostatic
1
H), 6.55 (s, 2H)
N-[4-(3-chloro-4-cyanophenoxy)-3,5-dimethoxyphenyl]-1,1,1-
trifluoromethanesulfonamide (2). To a stirred solution of 11 (250 mg, 0.83 potential maps of compounds 2 and 3, Dr. Steve Irving for protein
mmol) in NMP (5 mL) was added 5 (142 mg, 0.91 mmol) followed by purification and Mr Alec Tucker for protein crystallization.
Cs
2 3
CO (809 mg, 2.49 mmol) and the resulting mixture heated to 120 ºC for
1
2 hrs. After this time the mixture was portioned between water (10 mL) and
Notes and references
EtOAc (10mL). The aqueous layer was further extracted with EtOAc (2 x 20
mL) and the combined organics washed with brine (10 mL), dried over
a
Pfizer Neusentis, The Portway Building, Granta Park, Cambridge, UK. Eꢀ
mail: sarah.skerratt@pfizer.com. Tel: +44 (0)1304644073
4
MgSO , and concentrated in vacuo. The residue was purified via silica gel
b
Current address: LEO Pharma A/S, 55 Industriparken, 2750 Ballerup,
column chromatography, eluting with 50% EtOAc/50% pentane) to afford
Denmark
c
1
the title compound as a white solid (307 mg, 85% yield). H NMR (400
Current address: Department of Discovery Sciences, AstraZeneca,
MHz, DMSOꢀd6) δ: 3.72 (s, 6H), 6.64 (s, 2H), 6.83ꢀ6.85 (m, 1H), 7.13ꢀ7.14
Darwin Building, Cambridge Science Park, Milton Road, Cambridge,
CB4 0WG, UK
+
ꢀ
(
m, 1H), 7.83ꢀ7.85 (m, 1H); m/z 239 [M+H] ; m/z 435 [MꢀH] .
N-[4-(3-chloro-4-cyanophenoxy)-3,5-dimethoxyphenyl]methane
sulfonamide (4). To a stirred solution of 12 (250 mg, 1.01 mmol) in NMP (3
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

MedChemComm
Page 8 of 9
View Article Online
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DOI: 10.1039/C6MD0001
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Atomic charges are estimated by a leastꢀsquare fitting of calculated
4
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| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 9 of 9
MedChemComm
View Article Online
DOI: 10.1039/C6MD00011H
Abstract:
Herein, we describe the discovery of a novel binding pocket for CYP 2C9 inhibitors. Trifluoromethanesulfonamide
compounds and , identified within a Pfizer progesterone receptor antagonist program, were found to strongly inhibit
the drug metabolizing cytochrome P450 enzyme CYP 2C9. Homology modelling and subsequent X-ray co-crystal
data have elucidated the binding orientation of compounds and in CYP 2C9. Compound adopts a previously
1
2
1
2
2
unreported binding mode. Less acidic sulfonamide analogues within these series have reduced CYP 2C9 activity, and
we postulate this is due to a reduced hydrogen bonding potential with key interacting residues within CYP 2C9. This
work shows that CYP 2C9 has a more flexible active site than previously reported; therefore multiple binding modes
and alternative pharmacophore models must be considered when predicting CYP 2C9 affinity.
CYP 2C9 co-crystal structures of compound 1 (green) in binding mode 1 (CYP 2C9 in green) and compound 2 (yellow) in binding mode 2
(
CYP 2C9 in yellow)