Imidazopyridines as selective CYP3A4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.12.125

Cytochrome P450s are the major family of enzymes responsible for the oxidative metabolism of pharmaceuticals and xenobiotics. CYP3A4 and CYP3A5 have been shown to have overlapping substrate and inhibitor profiles and their inhibition has been demonstrated to be involved in numerous pharmacokinetic drug-drug interactions. Here we report the first highly selective CYP3A4 inhibitor optimized from an initial lead with ≈30-fold selectivity over CYP3A5 to yield a series of compounds with greater than 1000-fold selectivity.

Full text of DOI:10.1016/j.bmcl.2011.12.125

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1611–1614
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Imidazopyridines as selective CYP3A4 inhibitors
⇑
Xinyi Song, Xiaohai Li, Claudia H. Ruiz, Yan Yin, Yangbo Feng, Theodore M. Kamenecka ,
Michael D. Cameron
⇑
Department of Molecular Therapeutics, and Translational Research Institute, The Scripps Research Institute, Florida, 5353 Parkside Drive, RF-2, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytochrome P450s are the major family of enzymes responsible for the oxidative metabolism of pharma-
ceuticals and xenobiotics. CYP3A4 and CYP3A5 have been shown to have overlapping substrate and
inhibitor profiles and their inhibition has been demonstrated to be involved in numerous pharmacoki-
netic drug–drug interactions. Here we report the first highly selective CYP3A4 inhibitor optimized from
an initial lead with ꢀ30-fold selectivity over CYP3A5 to yield a series of compounds with greater than
1000-fold selectivity.
Received 30 November 2011
Revised 22 December 2011
Accepted 27 December 2011
Available online 4 January 2012
Keywords:
Cytochrome P450
CYP3A4
Ó 2012 Elsevier Ltd. All rights reserved.
CYP3A5
Imidazopyridines
Prescription pharmaceuticals are the most common treatment
option for physicians. A recent study found 10% of individuals
age 75–85 were taking drug combinations that were likely to have
drug–drug interactions and that 37% of these patients were con-
currently taking 5 or more prescription medications.1 Because of
their central role in drug metabolism, inhibition of cytochrome
P450s (CYPs) can significantly alter drug levels in patients taking
medications that are metabolized by the inhibited enzyme, occa-
sionally inducing toxicities similar to drug-overdose. During the
drug-discovery process, in vitro assays are utilized to determine
the risk of a compound to alter the concentrations of concomi-
tantly administered drugs or having its own concentration altered.
While great progress has been made in this area, there is a major
deficiency with respect to CYP3A4 and CYP3A5. These two en-
zymes have overlapping substrate and inhibitor profiles and are
arguably the most important cytochrome P450 involved in the
metabolism of pharmaceuticals based on their overall abundance
and the percentage of drugs they metabolize. Suitable chemical
tools are not available to differentiate these two enzymes (only
pan-substrates and pan-inhibitors are available) and activity is
typically expressed as a summation of the activities of the two
enzymes.
numerous reports of serious drug–drug interactions. Terfenadine
(SeldaneÒ) and Cisapride (PropulsidÒ) were withdrawn after pa-
tients taking the recommended dose along with CYP3A4 inhibitors
such as ketoconazole developed heart arrhythmia leading to heart
attack or death.2
To date, no substrates have been identified that are metabolized
exclusively by only one of the enzymes. However, using recombi-
nantly expressed enzymes, several substrates have been shown
to have higher catalytic efficiency for either CYP3A4 or CYP3A5.3
Similarly, no highly selective inhibitors have been identified, but
many do show two to fivefold preference for one of the two en-
zymes.4 Many of these have been associated with time-dependent
inactivation where CYP3A4 appears to be more susceptible to
time-dependent inactivation than does CYP3A5.5
This has been shown to have clinical importance because of the
polymorphic expression of CYP3A5. CYP3A5ꢁ1 leads to the expres-
sion of active, full length CYP3A5, but the CYP3A5ꢁ3 (22893A?G)
allele in intron 3 leads to a frame shift resulting in the majority of
the CYP3A5 mRNA coding for inactive protein and loss of CYP3A5
expression.6 The frequency of having at least one functional
CYP3A5ꢁ1 allele has varied with different reports but averages
approximately 10–30% in the Caucasian population and raises to
50–70% in African Americans.6 This has been shown to have clini-
cal implications with the immunosuppressant tacrolimus. Plasma
concentrations of tacrolimus are vitally important and are regu-
larly monitored with dose adjustment to achieve the desired drug
level. Patients that expressed active CYP3A5 (ꢁ1/ꢁ1 or ꢁ1/ꢁ3)
required approximately twice the dose as patients that did not ex-
press CYP3A5 to maintain appropriate tacrolimus concentration.7
Additionally, clinical drug interactions between the pan-CYP3A4/
Associated toxicities due to CYP3A4 related drug–drug interac-
tions have led to clinical failure and withdrawal from market of
previously approved pharmaceuticals. Mibefradil (PosicorÒ), a
potent inhibitor of CYP3A4, was withdrawn from the market after
⇑
Corresponding authors.
E-mail addresses: kameneck@scripps.edu (T.M. Kamenecka), cameron@scrip
ps.edu (M.D. Cameron).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.125

1612
X. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1611–1614
5 substrate midazolam and the mildly CYP3A4 selective (fivefold vs
3A5) inhibitor fluconazole were larger for individuals with the ꢁ3/
ꢁ3 CYP3A5 genotype.
An initial set of analogs meant to explore what portions of the
molecule were amenable to modification resulted in the discovery
that the corresponding 4-substituted phenyl analogs 3 and 4
showed similar IC50s for CYP3A4 inhibition but had reduced
Access to highly selective inhibitors of CYP3A4 will allow
researchers to isolate CYP3A5 activity in biologically relevant sam-
ples to better assess the potential for deleterious pharmacokinetic
drug–drug interactions and make higher quality predictions of
pharmacokinetic properties including the effect of CYP3A5ꢁ1, or
ꢁ3 genotype.
R
O
NH2
HN
N
N
H
We began a program to identify potent and selective CYP3A4
inhibitors mining data from 6000 in-house compounds that had
been previously tested in a single point CYP3A4/5 inhibition assay
using pooled (150-donor) human liver microsomes. The CYP3A5
content was assumed to be approximately 15% of the total
CYP3A4/5 so compounds that previously demonstrated between
Cl
N
N
N
N
N
a, b
c, d
N
H
N
H
N
80% and 90% inhibition at 10
tion using recombinant CYP3A4 and CYP3A5. After retesting at
lM were selected for further evalua-
5
6
7
MeO
10 lM, full IC50s were determined for compounds that appeared
to have a level of CYP3A4 versus CYP3A5 selectivity.8 This led us
to identify two imidazopyridines as promising leads (Fig. 1). These
compounds had modest selectivity with 1 having a CYP3A4
IC50 = 280 nM with 11-fold selectivity over CYP3A5 and 2 having
a CYP3A4 IC50 = 270 nM with 29-fold selectivity.
Scheme 1. (a) PMB-Cl, K2CO3, DMAc; (b) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)aniline, Pd(PPh3)4, K2CO3, THF, 80 °C; (c) TFA; (d) RCNO, toluene.
O
O
X
HN
N
N
H
CO2Me
HN
N
N
H
R
O
a,b
X
O
O
N
N
HN
N
N
H
HN
N
H
N
H
N
H
X
15
18-29
1
3
X=F
X=Cl
N
N
2 X=Br
4 X=Br
Scheme 2. (a) LiOH, methanol, THF, rt, 2 h, 90%; (b) (i) N(H)nR, HATU, N-
methylmorpholine, DMF, rt, 14 h, (n = 1–2).
N
H
N
H
N
Figure 1. Structure of imidazopyridines.
Table 2
Phenyl amide and ester derivatives
Table 1
P450 inhibition of substituted phenyl urea analogs
O
O
R
HN
N
H
R
HN
N
H
O
N
N
N
H
N
N
H
N
18-29
a,b
Compd
R
Enzyme IC50
(lM)
Fold-selective
a,b
Compd
R
Enzyme IC50
CYP3A4
(
l
M)
Fold-selective
CYP3A4
CYP3A5
CYP3A5
3
4
8
9
10
11
12
13
14
15
16
17
4-Cl
4-Br
2-Cl
3-F
3-Cl
3-Br
H
3-Me
3-NO2
3-CO2Me
4-OMe
4-Ph
0.31
0.21
0.60
0.71
0.48
0.092
1.2
34%
60
6.1
11.4
52
11.4
44
34%
31.5
8%
>190
290
10
15
18
19
20
21
22
23
24
25
26
27
28
29
OMe
OEt
OH
NHMe
NHEt
NHiPr
NMe2
NEt2
0.082
0.044
39%
0.098
0.055
0.090
0.11
0.047
0.060
0.053
0.070
0.079
0.29
8%
27%
33%
45%
20.3
41%
60
8.6
1.6
18%
6.9
5.5
>700
>1300
None
>600
368
>600
544
183
26
>1100
98
70
208
16
110
124
37
>40
518
>700
>300
>80c
1.5
0.061
0.082
0.20
0.026
NiPr2
39%
NHphenyl
NHcyclopentyl
Piperidine
Morpholine
27%c
a
Values are mean of two or more experiments using midazolam hydroxylation
60
as a measure or CYP3A4/5 activity.
b
a
Values with a % designation are percent inhibition at the highest concentration
Values are means of two or more experiments.
b
tested (60
lM). For selectivity purposes 60
lM is used and a greater than desig-
Values with a % designation are percent inhibition at the highest concentration
M). For selectivity purposes 60 M is used and a greater than desig-
nation is given.
nation is given.
tested (60
l
l
c
Solubility prevented evaluation of CYP3A5 inhibition above 2
lM.

X. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1611–1614
1613
CYP3A5 inhibition leading to increased selectivity (Table 1). Given
the improved CYP3A4/CYP3A5 selectivity in the phenyl urea series,
structure–activity relationship studies (SAR) were initiated on this
scaffold. Compounds were easily synthesized as described in
Scheme 1. The chloro imidazopyridine (5) was first protected with
a p-methoxybenzyl group. This gave a separable mixture of pro-
tected regioisomers (only one shown for clarity), but both pro-
tected imidazopyridines underwent smooth Suzuki coupling to
yield intermediate 6. TFA deprotection and exposure to a variety
of phenyl isocyanates afforded final products as solids.
Table 1 summarizes the effects of substitution of the terminal
phenyl ring upon inhibition of CYP3A4 and CYP3A5 activity using
recombinant enzymes.6 Substitution at all positions of the phenyl
ring led to potent CYP3A4 inhibitors with varying degrees of selec-
tivity against CYP3A5. 2- and 3-substituted analogs (8–11) showed
reduced selectivity whereas larger 3-substituted analogs (14,15)
showed improvement in selectivity for CYP3A4. 4-Substituted ana-
logs (16,17) also showed good levels of selectivity, however for 17,
precipitation at higher concentrations was observed. Given the
good potency and nice selectivity profile of ester 15, this com-
pound was further analoged as shown in Scheme 2.
Esters 15 and 18 exhibited strong selectivity and potency for
CYP3A4 versus CYP3A5 but when tested in incubations containing
1 mg/ml human liver microsomes or human hepatic S9 the com-
pounds had short half lives of approximately 8 min with or with-
out the addition of NADPH indicating probable hydrolysis by
hepatic esterases making 15 and 18 poor in vitro probes. Hence,
amides were investigated as more stable surrogates. The parent es-
ter was easily saponified, and a variety of amines were coupled to
form the amides (Table 2).
Several of the analogs were highly selective for the inhibition of
CYP3A4 versus CYP3A5. A wide range of substituents were accept-
able; however, larger substituents particularly with the disubsti-
tuted amides exhibited increased CYP3A5 inhibition, thus
decreasing the fold-selectivity.
Table 3
Imidazopyridine derivatives of compound 22
O
H
N
HN
R
N
H
O
a,b
Compd
R
Enzyme IC50
CYP3A4
(
l
M)
Fold-selective
>600
CYP3A5
N
22
0.090
0.56
41%
N
H
N
N
N
33
34
36
37
34
25%
60
61
>21
3
N
N
H
2.9
N
N
N
N
N
22
N
H
N
N
N
In order to evaluate the necessity of the imidazopyridine group,
a variety of compounds were generated using compound 22 as a
template (Table 3). Significant loss of CYP3A4 inhibition was ob-
served for all of the tested derivatives.
Of the compounds detailed, compound 17 was the most potent
CYP3A4 inhibitor identified; however, compound 17 suffered from
4.6
8.1
2
N
H
Cl
38
14
44
3
N
H
Table 4
Analogs of compound 17 to retain potency and increase solubility
39
40
36%
26
21%
47%
None
>2
N
N
R
H
O
HN
N
H
N
N
O
N
H
N
N
N
41
42
43
28%
33%
0.13
7%
None
None
8
NH2
NH2
N
N
N
H
a
Compd
R
Enzyme IC50
(
l
M)
Fold-selective
8%
CYP3A4
CYP3A5
N
44
45
46
47
48
49
50
4-Cyano
0.011
0.030
0.20b
0.048
1.7
33
21
3000
700
>300
143
8
4-Hydroxy
4-Amino
3-Methoxy
Naphthylc
Pyridin-3-ylc
Pyridin-4-ylc
26%
6.8
0.98
13
N
0.22
0.36
30%
29%
>270
>160
a
Values are means of two or more experiments.
a
b
c
b
Values are means of two or more experiments.
CYP3A4 inhibition curve was not sigmoidal.
Phenyl was replaced with naphthylene and pyridine.
Values with a % designation are percent inhibition at the highest concentration
M). For selectivity purposes 60 M is used and a greater than desig-
nation is given.
tested (60
l
l

1614
X. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1611–1614
poor solubility. A series of substituted 4-phenyl analogs were
prepared using similar strategies as in Scheme 1 to try to retain
potency but increase solubility (Table 4).
References and notes
1. Qato, D. M.; Alexander, G. C.; Conti, R. M.; Johnson, M.; Schumm, P.; Lindau, S. T.
JAMA 2008, 300, 2867.
The reported series of imidazopyridines are highly potent inhib-
itors of CYP3A4 and show the highest degree of specificity between
CYP3A4 and CYP3A5 ever presented in the literature. Of particular
interest are compounds 14, 22, 44 and 45 which had high selectiv-
ity and were not rapidly depleted in HLM incubations. Since many
drugs are metabolized by CYP3A4/5 these molecules represent the
first chemical tools to help understand the relative roles of CYP3A4
and CYP3A5 to enable quantitative prediction of the impact on
pharmacokinetics and drug–drug interactions. Assessment of the
inhibitors for in vitro metabolism studies is currently underway.
2. Zechnich, A. D.; Hedges, J. R.; Eiselt-Proteau, D.; Haxby, D. West J. Med. 1994, 160,
321.
3. Ekins, S.; Stresser, D. M.; Williams, J. A. Trends Pharmacol. Sci. 2003, 24, 161.
4. (a) Rendic, S. Drug Metab Rev 2002, 34, 83; (b) Williams, J. A.; Hyland, R.; Jones, B.
C.; Smith, D. A.; Hurst, S.; Goosen, T. C.; Peterkin, V.; Koup, J. R.; Ball, S. E. Drug
Metab. Dispos. 2004, 32, 1201.
5. (a) Stresser, D. M.; Broudy, M. I.; Ho, T.; Cargill, C. E.; Blanchard, A. P.; Sharma, R.;
Dandeneau, A. A.; Goodwin, J. J.; Turner, S. D.; Erve, J. C.; Patten, C. J.; Dehal, S. S.;
Crespi, C. L. Drug Metab. Dispos. 2004, 32, 105; (b) Pearson, J. T.; Wahlstrom, J. L.;
Dickmann, L. J.; Kumar, S.; Halpert, J. R.; Wienkers, L. C.; Foti, R. S.; Rock, D. A.
Chem. Res. Toxicol. 2007, 20, 1778; (c) Khan, K. K.; He, Y. Q.; Correia, M. A.;
Halpert, J. R. Drug Metab. Dispos. 2002, 30, 985.
6. Daly, A. K. Clin. Pharmacokinet. 2006, 45, 13.
7. Zhao, Y.; Song, M.; Guan, D.; Bi, S.; Meng, J.; Li, Q.; Wang, W. Transplant Proc.
2005, 37, 178.
8. CYP3A inhibition assays: Incubations contained 1 nM recombinant CYP3A4 or
Acknowledgments
CYP3A5 Supersomes,
5
l
M
midazolam, 1 mM NADPH, and variable
We thank the PDM group of Pfizer Inc. for a research fellowship
to X.L. and Drs. Ruth Hyland, Michael Zientek and Scott Obach for
productive scientific discussion.
concentrations of inhibitor in 100 mM phosphate buffer, pH 7.4. Incubations
were conducted at 37 °C and terminated by the addition of acetonitrile after
5 min. 10-Hydroxymidazolam formation was quantitated by LC–MS/MS.