Design and synthesis of selective, high-affinity inhibitors of human cytochrome P450 2J2

Article abstract of DOI:10.1016/j.bmcl.2006.02.004

The active site topology, substrate specificity, and biological roles of the human cytochrome P450 CYP2J2, which is mainly expressed in the cardiovascular system, are poorly known even though recent data suggest that it could be a novel biomarker and potential target for therapy of human cancer. This paper reports a first series of high-affinity, selective CYP2J2 inhibitors that are related to terfenadine, with K_i values as low as 160 nM, that should be useful tools to determine the biological roles of CYP2J2.

Full text of DOI:10.1016/j.bmcl.2006.02.004

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2777–2780
Design and synthesis of selective, high-affinity inhibitors
of human cytochrome P450 2J2
Pierre Lafite,a Sylvie Dijols,a Didier Buisson,a Anne-Christine Macherey,a
Darryl C. Zeldin,b Patrick M. Dansettea and Daniel Mansuya,*
aLaboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite Paris 5 Rene´ Descartes,
`
45 Rue des Saints-Peres, 75270 Paris Cedex 06, France
bNIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA
Received 11 January 2006; revised 1 February 2006; accepted 1 February 2006
Available online 21 February 2006
Abstract—The active site topology, substrate specificity, and biological roles of the human cytochrome P450 CYP2J2, which is
mainly expressed in the cardiovascular system, are poorly known even though recent data suggest that it could be a novel biomarker
and potential target for therapy of human cancer. This paper reports a first series of high-affinity, selective CYP2J2 inhibitors that
are related to terfenadine, with Ki values as low as 160 nM, that should be useful tools to determine the biological roles of CYP2J2.
Ó 2006 Elsevier Ltd. All rights reserved.
In the human genome, 57 genes have been found to code
for cytochromes P450 (CYPs) that are involved in the
oxidative metabolism of endogenous compounds and
xenobiotics.1 The main CYPs implicated in drug metab-
olism, such as CYP3A4, CYP2C9 or CYP2D6, and
those responsible for the biosynthesis of steroid
hormones have been extensively studied,1 and several
X-ray structures of human CYPs have been recently
published.2 Much less is known about recently discov-
ered human CYPs such as CYP2J2.1,3 This cytochrome
seems to be primarily expressed in heart3; it has also
been found in kidney, liver, lung,4 and the gastrointesti-
nal tract.5 Its biological role is presently unclear, even
though it has been found to catalyze the oxidation of
a few drugs such as ebastine in the intestine.6,7 More-
over, recombinant CYP2J2 catalyzes the epoxidation
of arachidonic acid to four cis-epoxyeicosatrienoic acids
(EETs), with regio- and stereo-selectivities that match
those of the EETs isolated from heart tissue.3 Some
EET-derived metabolites play important roles in regula-
tion of vascular tone8 and in a host of processes related
to cancer cell behavior, angiogenesis, and tumor patho-
genesis.9 Very recent data suggest that CYP2J2 pro-
motes the neoplastic phenotype of carcinoma cells and
may represent a novel biomarker and potential target
for therapy of human cancers.10 However, little data
are presently available on the active site topology and
substrate specificity of CYP2J2.1 This communication
reports the design and synthesis of a first series of
high-affinity inhibitors of human CYP2J2. These inhib-
itors should be useful tools to determine the biological
roles of this cytochrome.
Hydroxylation of the drug ebastine by recombinant
CYP2J27 expressed in baculovirus-infected Sf9 insect
cells3 was used as an assay to find CYP2J2 inhibitors.
During a first screening for such inhibitors, compound
1, derived from the drug terfenadine by oxidation of
its benzylic alcohol function, was found to inhibit
CYP2J211 with an IC50 value of 0.7 0.1 lM. This val-
ue was much lower than the IC50 found for terfenadine
itself (8.1 0.4 lM) (Table 1), that was previously de-
scribed as an inhibitor of CYP2J2.12 Then, compound
1, called terfenadone in the following, was used as a
starting point for the design of high-affinity inhibitors
of CYP2J2. Ebastine,7 terfenadine,13 and compound 1
are all hydroxylated by CYP2J2 at a site that is weakly
reactive from a chemical standpoint, a CH3 of the t-bu-
tyl group (Scheme 1). This regioselectivity in favor of the
least reactive part of these substrates implies their strict
positioning in the CYP2J2 active site in order to main-
tain the t-butyl group in close proximity of the heme
iron for transfer of an oxygen atom from O2. Therefore,
a series of compounds derived from 1 by replacement of
Keywords: Cardiovascular system; Terfenadine; Ebastine; Hydroxyl-
ation; Suicide substrates; Monooxygenases.
*
Corresponding author. Tel.: +33 (0)1 42 86 21 69; fax: +33 (0)1 42 86
83 87; e-mail: daniel.mansuy@univ-paris5.fr
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.004

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P. Lafite et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2777–2780
Table 1. Inhibitory effects of terfenadone derivatives toward recombi-
nant CYP2J2
from P450-catalyzed oxidation of the substrate double
bond.14 The choice of the CHF2 and benzodioxole func-
tions of compounds 12 and 13 was also made on the ba-
sis of literature data on suicide substrates of cytochrome
P450.14 In situ hydroxylation of the C–H bond of CHF2
groups leads to an electrophilic intermediate able to
acylate the P450 protein, whereas inactivation of cyto-
chrome P450 by benzodioxole derivatives is due to the
formation of an iron–carbene bond after oxidation of
the benzodioxole CH2 group.14 The structure of com-
pounds 5, 12, and 13 has been chosen in order that
CYP2J2-catalyzed oxidations occur at the site leading
to inactivating metabolites, assuming that hydroxylation
of 5, 12, and 13 should occur on the homobenzylic
position as the hydroxylation of compound 1 and
terfenadine.
R
HO
N
O
Inhibitor
–R
IC50a(lM)
Terfenadine
Terfenadone, 1
8.1 0.4
0.7 0.1
0.7 0.1
0.6 0.1
0.4 0.1
0.4 0.2
0.7 0.2
1.3 0.8
1.9 0.3
2.5 0.5
7.6 0.6
4.2 0.6
2.2 0.9
–C(CH3)3
–CH3
–CH2–CH3
2
3
4
–CH2–CH2–CH3
–CH2–CH@CH2
–(CH2)3CH3
–CH2–CH2–OH
–CH2–CH2–CH2–OH
–CH2–CH2–CH2–OAc
–O–CH3
5
6
7
8
The general synthetic route used for the preparation
of terfenadone derivatives (Scheme 2) with
R = (CH2)nCH3 (with n = 0–3), (CH2)nOH and
(CH2)nOCOCH3 (with n = 2 or 3), Br, CH2CHF2,
OCH2O–, and OCH3 involved an acylation of the ben-
zenic starting compound with 4-chlorobutanoic acid
chloride in the presence of FeCl3, AlCl3 or SnCl4 and
reaction of the corresponding product with a,a-diphen-
yl-4-piperidinomethanol. In the case of 7 and 8, the
starting compounds were the acetates of 2-phenyletha-
nol and 3-phenylpropanol, respectively. Deprotection
of the alcohol function was done as the last step of
the synthesis. Compound 5 was obtained from reaction
of 11 with allyltributyltin, in the presence of
tetrakis(triphenylphosphine)palladium(0).
9
10
11
12
–Br
–CH2–CHF2
O
O
13
Arb =
6.7 2.3
a Compound concentration leading to 50% inhibition of the hydrox-
ylation of ebastine by CYP2J2 expressed in baculovirus-infected Sf9
insect cells.3 Conditions as described in Ref. 15. Under these condi-
tions, the Km of ebastine hydroxylation was 0.5 lM. Values are
means SD from three to four experiments.
b Ar = –C6H5–R.
H3C
CH3
CH3
Ebastine
The structures of all the terfenadone derivatives listed in
Table 1 were completely established from their 1H NMR
and mass spectra; 1H NMR spectroscopy analysis in the
presence of an internal standard showed that all these
compounds were more than 95% pure.
O
N
N
O
H3C
CH3
CH3
Terfenadine
HO
Table 1 compares the IC50 values measured for the
inhibition of ebastine hydroxylation catalyzed by
recombinant CYP2J2. It shows that most of the
synthesized terfenadone derivatives are good CYP2J2
inhibitors with IC50 values at the low lM range. Com-
pounds 4 and 5 had the highest affinity with an IC50
value of 0.4 lM. In fact, increasing the chain length
from R = methyl to R = propyl results in a gradual de-
crease of the IC50 value, whereas a further increase of
the chain length (R = butyl) leads to a loss of affinity.
Introduction of a polar function in the R substituent
generally leads to a decrease in the affinity of the
inhibitors. Compounds such as 10 and 13 in which
oxygen atoms have been introduced at benzylic posi-
tions exhibit IC50 values one order of magnitude
OH
Scheme 1. Formula and site of CYP2J2-dependent hydroxylation of
ebastine and terfenadine.
its t-butyl group with various R groups of different size
and polarity was synthesized (Scheme 2). This included
compounds bearing functions previously known to lead
to suicide inactivation of cytochromes P450 after in situ
oxidation.14 This is the case for the terminal double
bond of compound 5, since terminal alkenes act as
mechanism-based inhibitors of cytochromes P450 after
N-alkylation of the heme by an intermediate derived
R
R
NH
HOC(Ph)2
R
Cl(CH2)3COCl
HO
N
Cl
O
O
Scheme 2. General synthetic route used for the preparation of terfenadone derivatives (for the nature of R, see Table 1).

P. Lafite et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2777–2780
2779
Table 2. Comparison of the inhibitory effects of terfenadone deriva-
tives toward vascular cytochromes P450
References and notes
IC50 (lM)a
CYP2J2 CYP2B6 CYP2C8 CYP2C9 CYP3A4
1. Guengerich, F. P. In Cytochrome P450: Structure, Mech-
anism, and Biochemistry; Ortiz de Montellano, P. R., Ed.,
3rd ed.; Kluwer Academic/Plenum Publishers: New York,
2005; pp 377–530.
Compound
4
5
0.4 0.1 28
0.4 0.2 21
1
1
>100
>100
26
21
3
1
7.9 0.5
5.5 1.0
2. (a) Williams, P. A.; Cosme, J.; Ward, A.; Angove, H. C.;
Matak Vinkovic, D.; Jhoti, H. Nature 2003, 424, 464; (b)
Schoch, G. A.; Yano, J. K.; Wester, M. R.; Griffin, K. J.;
Stout, C. D.; Johnson, E. F. J. Biol. Chem. 2004, 279,
9497; (c) Wester, M. R.; Yano, J. K.; Schoch, G. A.; Yang,
C.; Griffin, K. J.; Stout, C. D.; Johnson, E. F. J. Biol.
Chem. 2004, 279, 35630; (d) Williams, P. A.; Cosme, J.;
Vinkovic, D. M.; Ward, A.; Angove, H. C.; Day, P. J.;
Vonrhein, C.; Tickle, I. J.; Jhoti, H. Science 2004, 305,
683; (e) Yano, J. K.; Wester, M. R.; Schoch, G. A.;
Griffin, K. J.; Stout, C. D.; Johnson, E. F. J. Biol. Chem.
2004, 279, 38091; (f) Yano, J. K.; Hsu, M. H.; Griffin, K.
J.; Stout, C. D.; Johnson, E. F. Nat. Struct. Mol. Biol.
2005, 12, 822(g) Rowland, P.; Blaney, F. E.; Smyth, M.
G.; Jones, J. J.; Leydon, V. R.; Oxbrow, A. K.; Lewis, C.
J.; Tennant, M. M.; Modi, S.; Eggleston, D. S.; Chenery,
R. J.; Bridges, A. M. J. Biol. Chem. 2005, in press.
3. Wu, S.; Moomaw, C. R.; Tomer, K. B.; Falck, J. R.;
Zeldin, D. C. J. Biol. Chem. 1996, 271, 3460.
4. Zeldin, D. C.; Foley, J.; Ma, J.; Boyle, J. E.; Pascual, J.
M.; Moomaw, C. R.; Tomer, K. B.; Steenbergen, C.; Wu,
S. Mol. Pharmacol. 1996, 50, 1111.
5. (a) Zeldin, D. C.; Foley, J.; Goldsworthy, S. M.; Cook, M.
E.; Boyle, J. E.; Ma, J.; Moomaw, C. R.; Tomer, K. B.;
Steenbergen, C.; Wu, S. Mol. Pharmacol. 1997, 51, 931; (b)
Scarborough, P. E.; Ma, J.; Qu, W.; Zeldin, D. C. Drug
Metab. Rev. 1999, 31, 205.
6. Matsumoto, S.; Hirama, T.; Matsubara, T.; Nagata, K.;
Yamazoe, Y. Drug Metab. Dispos. 2002, 30, 1240.
7. Hashizume, T.; Imaoka, S.; Mise, M.; Terauchi, Y.; Fujii,
T.; Miyazaki, H.; Kamataki, T.; Funae, Y. J. Pharmacol.
Exp. Ther. 2002, 300, 298.
8. Spector, A. A.; Fang, X.; Snyder, G. D.; Weintraub, N. L.
Prog. Lipid Res. 2004, 43, 55.
9. Wang, Y.; Wei, X.; Xiao, X.; Hui, R.; Card, J. W.; Carey,
M. A.; Wang, D. W.; Zeldin, D. C. J. Pharmacol. Exp.
Ther. 2005, 314, 522.
10. Jiang, J. G.; Chen, C. L.; Card, J. W.; Yang, S.; Chen, J.
X.; Fu, X. N.; Ning, Y. G.; Xiao, X.; Zeldin, D. C.; Wang,
D. W. Cancer Res. 2005, 65, 4707.
11. Pfister, S. L.; Spitzbarth, N.; Zeldin, D. C.; Lafite, P.;
Mansuy, D.; Campbell, W. B. Arch. Biochem. Biophys.
2003, 420, 142.
12. Matsumoto, S.; Hirama, T.; Kim, H. J.; Nagata, K.;
Yamazoe, Y. Xenobiotica 2003, 33, 615.
13. Patten, C.; Gagne, P.; Miller, V.; Crespi, C.; Thummel, K.
Drug Metab. Rev., 35, Suppl. 1, 8th European ISSX
Conference, Dijon, France 2003; International Society for
the Study of Xenobiotics, 2003.
a Compound concentration leading to 50% inhibition of CYP2B6-
catalyzed 7-benzyloxyresorufin O-deethylation,16 CYP2C8-catalyzed
6-a-hydroxylation of taxol,17 CYP2C9-dependent 40-hydroxylation
of diclofenac,18 and CYP3A4-catalyzed 6-b-hydroxylation of tes-
tosterone,19 respectively. Microsomes from W(R)fur yeast strain
expressing each of these cytochromes P45020 were incubated with the
corresponding substrate at a concentration equal to the Km value of
the studied reactions (0.5, 5, 10, and 20 lM, respectively) and a
NADPH-generating system. Incubations and analyses of the reaction
mixture were performed as described previously.16–19 Values are
means SD from three to four experiments.
greater than those observed for compounds bearing an
alkyl chain (R = Et or Pr, 3 or 4 for instance). Com-
pounds such as 7, 8, 9, and 12 in which an OH, OAc
or F substituent have been introduced in the R-chain
farther from the phenyl ring exhibit intermediate IC50
values, around 2 lM. Thus, the best inhibitors (in
terms of IC50 value) were compounds 4 and 5. Preli-
minary experiments showed that compound 4 is a
competitive inhibitor of CYP2J2-catalyzed hydroxyl-
ation of ebastine with a Ki of 160 30 nM and also
a competitive substrate of CYP2J2. Compound 5
seems to be a time-dependent inhibitor, as expected
for a compound bearing a terminal double bond.14
Interestingly, compounds 12 and 13 involving
a
CHF2 and benzodioxole function, respectively, also
led to time-dependent inhibitory effects that suggest
a mechanism-based type of inhibition.
Table 2 compares the inhibitory effects of the best inhib-
itors found for CYP2J2, compounds 4 and 5, toward the
other main human cytochromes P450 that are present
in the cardiovascular system, CYP2C8, CYP2C9,
CYP2B6, and CYP3A4.21 The data clearly show that
compounds 4 and 5 are selective inhibitors of CYP2J2,
as they are nearly inactive toward CYP2C8 and their
IC50 values for CYP2C9, CYP2B6, and CYP3A4 are
1–3 orders of magnitude higher than those observed
for CYP2J2.
In conclusion, the aforementioned results have led to
the first selective, high-affinity inhibitors of CYP2J2,
compounds 4 and 5, that exhibit IC50 values around
400 nM. Compound 4 is a competitive inhibitor char-
acterized by a Ki of 160 nM, a value that is remarkably
low for a human cytochrome P450 inhibitor.14 Addi-
tional studies are underway to determine the type of
inhibition exhibited by compounds 5, 12, and 13, and
to use these new inhibitors as tools to study the biolog-
ical roles of CYP2J2 in vitro and in vivo. In light of
the recent findings that CYP2J2 promotes the neoplas-
tic phenotype of carcinoma cells, these compounds are
also currently being investigated as potential anti-can-
cer therapeutics.
14. Correira, M. A.; Ortiz de Montellano, P. R. In Cyto-
chrome P450: Structure, Mechanism, and Biochemistry;
Ortiz de Montellano, P. R., Ed., 3rd ed.; Kluwer
Academic/Plenum Publishers: New York, 2005; pp 247–
322.
15. Ebastine (0.5 lM) and inhibitor were incubated at
37 °C, for either
2 or 4 min, in the presence of
microsomes from baculovirus-infected insect cells
(1 nM CYP2J2) in 0.1 M phosphate buffer, pH 7.4,
containing 0.1 mM EDTA and a NADPH-generating
system, for a total volume of 200 lL. The reaction was
stopped within a few seconds by treatment with 100 lL
of a cold CH3CN/CH3COOH (10:1) mixture and
vortexing of the incubate. Proteins were precipitated

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P. Lafite et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2777–2780
by centrifugation for 5 min at 10,000 rpm, and the
18. Mancy, A.; Antignac, M.; Minoletti, C.; Dijols, S.;
Mouries, V.; Duong, N. T.; Battioni, P.; Dansette, P.
M.; Mansuy, D. Biochemistry 1999, 38, 14264.
19. Brian, W. R.; Sari, M. A.; Iwasaki, M.; Shimada, T.;
Kaminsky, L. S.; Guengerich, F. P. Biochemistry 1990, 29,
11280.
supernatant aliquots were analyzed by HPLC, after
injection on a Hypersil C18 column (Thermo, Les Ulis,
France). The mobile phase was delivered at a rate of
1 mL/min with a gradient from A (0.1 M acetate, pH
4.6) to B (CH3CN/CH3OH/H2O, 7:2:1) (30% up to
100% B in 15 min). The column effluent was monitored
at 254 nm.
20. Truan, G.; Cullin, C.; Reisdorf, P.; Urban, P.; Pompon,
D. Gene 1993, 125, 49.
16. Ekins, S.; Vandenbranden, M.; Ring, B. J.; Gillespie, J. S.;
Yang, T. J.; Gelboin, H. V.; Wrighton, S. A. J. Pharmacol.
Exp. Ther. 1998, 286, 1253.
17. Rahman, A.; Korzekwa, K. R.; Grogan, J.; Gonzalez, F.
J.; Harris, J. W. Cancer Res. 1994, 54, 5543.
21. (a) Hoebel, B. G.; Steyrer, E.; Graier, W. F. Clin. Exp.
Pharmacol. Physiol. 1998, 25, 826; (b) Fisslthaler, B.; Popp,
R.; Kiss, L.; Potente, M.; Harder, D. R.; Fleming, I.; Busse,
R. Nature 1999, 401, 493; (c) Borlak, J.; Walles, M.; Levsen,
K.; Thum, T. Drug Metab. Dispos. 2003, 31, 888.