Abstract
Cytochrome P450 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.
Prescription pharmaceuticals are the most common treatment option for physicians. A recent study found 10% of individuals age 75 to 85 were taking drug combinations that were likely to have drug-drug interactions and that 37% of these patients were concurrently 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, occasionally 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 concomitantly 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 enzymes 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.
Associated toxicities due to CYP3A4 related drug-drug interactions have led to clinical failure and withdrawal from market of previously approved pharmaceuticals. Mibefradil (Posicor®), a potent inhibitor of CYP3A4, was withdrawn from the market after numerous reports of serious drug-drug interactions. Terfenadine (Seldane®) and Cisapride (Propulsid®) were withdrawn after patients taking the recommended dose along with CYP3A4 inhibitors such as ketoconazole developed heart arrhythmia leading to heart attack or death 2.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 recombinantly 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 five-fold preference for one of the two enzymes 4. This has been shown to have clinical importance because of the polymorphic expression of CYP3A5. CYP3A5*1 leads to the expression of active, full length CYP3A5, but the CYP3A5*3 (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 5. The frequency of having at least one functional CYP3A5*1 allele has varied with different reports, but averages approximately 10–30% in the Caucasian population and raise to 50–70% in African Americans 5. This has been shown to have clinical implications with the immunosuppressant tacrolimus. Plasma concentrations of tacrolimus are vitally important and are regularly 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 express CYP3A5 to maintain appropriate tacrolimus concentration6. Additionally, clinical drug interactions between the pan-CYP3A4/5 substrate midazolam and the mildly CYP3A4 selective (5-fold vs. 3A5) inhibitor fluconazole were larger for individuals with the *3/*3 CYP3A5 genotype.
Access to highly selective inhibitors of CYP3A4 and CYP3A5 will allow researchers to better assess the potential for deleterious pharmacokinetic drug-drug interactions and make higher quality predictions of pharmacokinetic properties including the effect of CYP3A5*1, or *3 genotype.
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 80 and 90% inhibition at 10 μM were selected for further evaluation using recombinant CYP3A4 and CYP3A5. After retesting at 10 μM, full IC50 were determined for compounds that appeared to have a level of CYP3A4 vs. CYP3A5 selectivity 7. This led us to identify two imidazopyridines as promising leads (Figure 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.
Figure 1.
Structure of imidazopyridines.
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 IC50’s for CYP3A4 inhibition but had reduced 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 protected regioisomers (only one shown for clarity), but both protected 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.
P450 inhibition of substituted phenyl urea analogs.
| ||||
|---|---|---|---|---|
| Cmpd | R | enzyme IC50 (μM)a,b
|
Fold-Selective | |
| CYP3A4 | CYP3A5 | |||
| 3 | 4-Cl | 0.31 | 34% | >190 |
| 4 | 4-Br | 0.21 | 60 | 290 |
| 8 | 2-Cl | 0.60 | 6.1 | 10 |
| 9 | 3-F | .71 | 11.4 | 16 |
| 10 | 3-Cl | 0.48 | 52 | 110 |
| 11 | 3-Br | 0.092 | 11.4 | 124 |
| 12 | H | 1.2 | 44 | 37 |
| 13 | 3-Me | 1.5 | 34% | >40 |
| 14 | 3-NO2 | 0.061 | 31.5 | 518 |
| 15 | 3-CO2Me | 0.082 | 8% | >700 |
| 16 | 4-OMe | 0.20 | 39% | >300 |
| 17 | 4-Ph | 0.026 | 27%c | >80c |
Values are mean of two or more experiments using midazolam hydroxylation as a measure or CYP3A4/5 activity. The error in these values is within ±30% of the mean.
Values with a % designation are percent inhibition at the highest concentration tested (60 μM). For selectivity purposes 60 μM is used and a greater than designation is given.
Solubility prevented evaluation of CYP3A5 inhibition above 2 μM.
Scheme 1.
(a) PMB-Cl, K2CO3, DMAC; (b) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, Pd(PPh3)4, K2CO3, THF; (c) TFA; (d) RCNO, toluene.
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 selectivity 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 analogs (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 compound was further analoged as shown in Scheme 2.
Scheme 2.
(a) LiOH, methanol, THF, rt, 2 h, 90%; (b) (i) N(H)nR, HATU, N-methylmorpholine, DMF, rt, 14 h, (n=0–2).
While esters 15 and 18 exhibited strong selectivity and potency for CYP3A4 vs CYP3A5, they were not pursued because of stability concerns. When tested in incubations containing 1 mg/ml human liver microsomes or human hepatic S9, the compounds had a half life of approximately 8 minutes with or without the addition of NADPH indicating probably hydrolysis by hepatic esterases making 15 and 18 poor in vitro probes. Hence, amides were investigated as more stable surrogates. The parent ester was easily saponified, and a variety of amines were coupled to form the amides (Table 2).
Table 2.
Phenyl amide and ester derivatives.
| |||||
|---|---|---|---|---|---|
| Cmpd | X | R | enzyme IC50 (μM)a
|
Fold-Selective | |
| CYP3A4 | CYP3A5 | ||||
| 15 | O | Me | 0.082 | 8% | >700 |
| 18 | O | Et | 0.044 | 27% | >1300 |
| 19 | O | H | 39% | 33% | None |
| 20 | N | Me | 0.098 | 45% | >600 |
| 21 | N | Et | 0.055 | 20.3 | 368 |
| 22 | N | iPr | 0.090 | 41% | >600 |
| 23 | N | Di-Me | 0.11 | 60 | 544 |
| 24 | N | Di-Et | 0.047 | 8.6 | 183 |
| 25 | N | Di-iPr | 0.060 | 1.6 | 26 |
| 26 | N | phenyl | 0.053 | 18% | >1100 |
| 27 | N | cyclopentyl | 0.070 | 6.9 | 98 |
| 28 | N | piperidine | 0.079 | 5.5 | 70 |
| 29 | N | morpholine | 0.29 | 60 | 208 |
Values are means of two or more experiments. The error in these values is within ±30% of the mean.
Not determined.
Several of the analogs were highly selective for the inhibition of CYP3A4 vs CYP3A5. A wide range of substituents were acceptable; however, larger substituents particularly with the disubstituted amides exhibited increased CYP3A5 inhibition, thus decreasing the fold-selectivity.
In order to evaluate the necessity of the imidazopyridine group, a variety of compounds were generated using compound 22 as a template (Table 3).
Table 3.
Imidazopyridine derivatives of compound 22.
| ||||
|---|---|---|---|---|
| Cmpd | R | enzyme IC50 (μM)a
|
Fold-Selective | |
| CYP3A4 | CYP3A5 | |||
| 22 |
|
0.090 | 41% | >600 |
| 33 |
|
0.56 | 34 | 61 |
| 34 |
|
2.9 | 25% | >21 |
| 36 |
|
22 | 60 | 3 |
| 37 |
|
4.6 | 8.1 | 2 |
| 38 |
|
14 | 44 | 3 |
| 39 |
|
36% | 21% | None |
| 40 |
|
26 | 47% | >2 |
| 41 |
|
28% | 7% | None |
| 42 |
|
33% | 8% | None |
| 43 |
|
0.13 | 0.98 | 8 |
Values are means of two or more experiments. The error in these values is within ±30% of the mean.
The most potent CYP3A4 inhibitor identified within the series was compound 17; however, compound 17 suffered from poor solubility. A series of substituted 4-phenyl analogs were prepared using similar strategies as in Scheme 1.
In summary, we have synthesized a series of imidazopyridines that are highly potent inhibitors of CYP3A4 and show the highest degree of specificity between CYP3A4 and CYP3A5 ever presented in the literature. Since many drugs are metabolized by CYP3A4/5 it would be beneficial to have the appropriate in vitro tools to understand the relative roles of CYP3A4 and CYP3A5 to enable quantitative prediction of the impact on pharmacokinetics. Assessment of the inhibitors for in vitro metabolism studies is currently underway.
Table 4.
Analogs of Compound 17 to retain potency and increase solubility.
| ||||
|---|---|---|---|---|
| Cmpd | R | enzyme IC50 (μM)a
|
Fold-Selective | |
| CYP3A4 | CYP3A5 | |||
| 44 | 4-cyano | 0.011 | 33 | 3000 |
| 45 | 4-hydroxy | 0.030 | 21 | 700 |
| 46 | 4-amino | 0.20b | 26% | >300c |
| 47 | 3-methoxy | 0.048 | 6.8 | 143 |
| 48 | naphthylc | 1.7 | 13 | 8 |
| 49 | pyridin-3-ylc | 0.22 | 30% | >270 |
| 50 | pyridin-4-ylc | 0.36 | 29% | >160 |
Values are means of two or more experiments. The error in these values is within ±30% of the mean.
CYP3A4 inhibition curve was not sigmoidal
Phenyl was replaced with naphthylene and pyridine
Acknowledgments
We acknowledge grant R21DK091630 (MDC) for funding.
Contributor Information
Theodore M. Kamenecka, Email: kameneck@scripps.edu.
Michael D. Cameron, Email: cameron@scripps.edu.
References and notes
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