Probing Mechanisms of CYP3A Time-Dependent Inhibition Using a Truncated Model System

Article abstract of DOI:10.1021/acsmedchemlett.5b00191

Time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes may incur serious undesirable drug-drug interactions and in rare cases drug-induced idiosyncratic toxicity. The reactive metabolites are often generated through multiple sequential biotransformations and form adducts with CYP enzymes to inactivate their function. The complexity of these processes makes addressing TDI liability very challenging. Strategies to mitigate TDI are therefore highly valuable in discovering safe therapies to benefit patients. In this Letter, we disclose our simplified approach toward addressing CYP3A TDI liabilities, guided by metabolic mechanism hypotheses. By adding a methyl group onto the α carbon of a basic amine, TDI activities of both the truncated and full molecules (7a and 11) were completely eliminated. We propose that truncated molecules, albeit with caveats, may be used as surrogates for full molecules to investigate TDI.

Full text of DOI:10.1021/acsmedchemlett.5b00191

Letter
pubs.acs.org/acsmedchemlett
Probing Mechanisms of CYP3A Time-Dependent Inhibition Using a
Truncated Model System
Xiaojing Wang,* Minghua Sun, Connie New,† Spencer Nam,‡ Wesley P. Blackaby,§ Alastair J. Hodges,∥
David Nash,§ Mizio Matteucci,§ Joseph P. Lyssikatos,⊥ Peter W. Fan, Suzanne Tay, and Jae H. Chang
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Time-dependent inhibition (TDI) of cytochrome
P450 (CYP) enzymes may incur serious undesirable drug−drug
interactions and in rare cases drug-induced idiosyncratic toxicity.
The reactive metabolites are often generated through multiple
sequential biotransformations and form adducts with CYP
enzymes to inactivate their function. The complexity of these
processes makes addressing TDI liability very challenging.
Strategies to mitigate TDI are therefore highly valuable in
discovering safe therapies to benefit patients. In this Letter, we
disclose our simplified approach toward addressing CYP3A TDI
liabilities, guided by metabolic mechanism hypotheses. By adding a methyl group onto the α carbon of a basic amine, TDI
activities of both the truncated and full molecules (7a and 11) were completely eliminated. We propose that truncated molecules,
albeit with caveats, may be used as surrogates for full molecules to investigate TDI.
KEYWORDS: TDI, metabolic mechanism, CYP3A, Pim kinases
Aside from having potent PIM kinase inhibition,18
embers of the cytochrome P450 (CYP) 3A subfamily
Mare responsible for the metabolism of roughly half of all
compound 1 (Figure 1) was found to have potent CYP3A
marketed pharmaceutical drugs.1−3 Altered CYP activity
TDI activity. Two methods were used to assess the TDI
potency as described by Kenny et al.19 In an area under the
resulting from time-dependent inhibition (TDI) can lead to
clearance changes of drugs, thus contributing to drug−drug
interactions (DDI)4 that can cause efficacy and safety
problems.1,5,6 These concerns are becoming increasingly
relevant in the face of growing polypharmacy.4,5,7 CYP
inhibition is a collective term for a decrease in CYP activity
and can be classified into three groups: reversible, quasi-
irreversible, and irreversible.8,9 More specifically, the latter two
modes often fall under the heading of TDI because in these
cases CYP inactivation is caused by the formation of either an
adduct or a metabolic-inhibitory (or -intermediate) complex
(MIC) between the enzyme and reactive metabolites of the
drug.6,8 The inactivating species can result from sequential
biotransformations through multiple routes and the complexity
of these possibilities makes the accurate analysis of TDI
challenging.7,8,10,11 Previously, researchers have demonstrated
the possibility of dialing out TDI by (i) blocking metabolic sites
that lead to the generation of reactive metabolites, (ii) diverting
the site of bioactivation to other metabolic hotspots, and (iii)
reducing the binding affinity of the substrates to the CYP
enzymes through the modulation of their physicochemical
properties.12−15 Despite some successes, dialing out TDI may
still be complicated by multiple species responsible for
mechanism-based inhibition (MBI).16,17 Our present work
takes a simplified approach toward studying the TDI
mechanisms by removing potential sites of metabolism.
curve (AUC) shift dilution assay, 1 inhibited CYP3A with a
TDI IC50 of below 0.1 μM and an AUC shift of 57.3% when
Figure 1. (a) Compound 1 shown with likely sites of metabolism by
the CYP3A enzyme, as predicted by MetaSite without reactivity
correction. Predicted metabolic hotspots are represented by red circles.
(b) CYP3A TDI parameters for 1, using testosterone (or midazolam)
as the probe substrate.
Received: May 7, 2015
Accepted: July 12, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00191
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ACS Medicinal Chemistry Letters
Letter
testosterone20 was used as a probe substrate for one binding
site of CYP3A. A similar result (IC50 of <0.1 μM and AUC shift
of 55.6%) was obtained with midazolam as the probe substrate
for the second binding site of CYP3A. Furthermore, a manual
kinetic assay allowed for measurement of both the maximal rate
of enzyme inactivation (kinact) and the inhibitor concentration
that supports half the maximal rate of inactivation (KI), thereby
confirming TDI activity. Compound 1 has a kinact of 0.06 min−1
and KI of 1.2 μM, resulting in a high ratio of kinact/KI of 52 mL/
min/μmol. The high degree of TDI exhibited by 1 is
comparable to potent known clinical time-dependent CYP3A
inhibitors such as mifepristone16 (kinact = 0.08 min−1, KI = 1.3
μM, and kinact/KI = 62 mL/min/μmol). Major metabolites of
compound 1 were identified through compound incubation
with human hepatocytes. Not surprisingly, the azepane ring was
the hotspot for phase I and II metabolism (Supporting
Information, Figure s1). Phase I metabolism such as oxidation
was predicted by MetaSite21 (Figure 1). In order to mitigate
TDI activity, truncated tool molecules were designed to probe
the molecular mechanism of metabolism by focusing on the
major metabolic hotspot.
Figure 2. Proposed mechanisms of action.
Given the preponderance of metabolism on the azepane ring,
fragments 2−4 (Table 1) were designed with a range of polarity
compound 1 and its enantiomer (CYP3A IC50 of <0.1 μM and
AUC shift of 54%) demonstrated potent TDI activity.
Therefore, racemic compounds 6 and 9 were used in further
studies. In striking contrast with 2, compounds 5a,b and 6
lacked difluoro substitution and showed no TDI liability. This
implies that fluorine groups played a critical role in the
formation of reactive intermediates. In this regard, the lack of
TDI activity for 5a,b seemed to negate the quasi-irreversible
pathway hypothesis (Figure 2). In addition, a dialysis
experiment (data not shown) confirmed 1 as an irreversible
CYP inhibitor and therefore invalidated the quasi-irreversible
pathway I.
As in pathway II, we hypothesized that a Michael
acceptor22−24 generated through an oxidative cleavage of the
amine and HF elimination could react with a CYP enzyme
covalently. In fact, glutathione trapping experiment of
compound 1 supported this hypothesis (Supporting Informa-
tion, Figure s2). Logically, 7a,b were designed to block the α
position of the basic amine from being oxidized and thus
prevent the formation of a Michael acceptor. Satisfyingly, 7a,b
were both devoid of TDI activity despite having significantly
higher lipophilicity (cLogP: 3.3) than compound 2 (cLogP:
2.3).
Compound 8, which has a tertiary amine in place of the
primary amine on 2, was designed for two purposes; first to
block the nitroso group formation and second to slow down
the α carbon oxidation by steric hindrance. Encouraged by the
lack of TDI activity of 8, we made the fully decorated analogue
S4 (Supporting Information, Table s2). Despite with slightly
decreased activity compared to 1, S4 still maintained strong
TDI activity surprisingly. Compound 9, which features a
secondary alcohol and gem-difluoro group, had weak TDI
activity with midazolam as the probe substrate. This is
consistent with the hypothesis that secondary alcohol being
oxidized to the ketone ultimately gives rise to the same Michael
acceptor intermediate. Further confirming this hypothesis, the
fully assembled analogue S5 (Supporting Information, Table
s2) had strong TDI potency. However, one cannot exclude the
possibility that the TDI activities of S4 and S5 are net results of
metabolic modifications of multiple sites of the molecules.
Table 1. CYP3A TDI of Substituted Pyrazoles vs Calculated
Physicochemical Properties
a
b
CYP3A_T (M)
calculated
ID
R
IC50 (μM)
%AUC shift
tPSA
cLogP
1
2
3
4
c
<0.1 (<0.1)
4.3 (5.0)
2.5 (7.7)
no (no)
57.3 (55.6)
40.1 (28.7)
22.5 (−21.1)
11.8 (7.2)
115
76
3.5
2.3
2.7
1.7
Bz
Cbz
85
d
pic
89
a
CYP3A AUC shift dilution assay with testosterone (or midazolam) as
the probe substrate. IC50 values were calculated from the AUC shift
assay. “No” in IC50 column indicates there is no evidence of TDI.
b
tPSA, topological polar surface area; cLogP, calculated partition
c
d
coefficient. See Figure 1. Picolinoyl.
(tPSA: 76−89) and lipophilicity (cLogP: 1.7−2.7). Indeed, the
more lipophilic compounds (i.e., 2 and 3 compared to 4) were
found to have moderate TDI activity in AUC shift dilution
assays with either testosterone or midazolam as the probe
substrate. The overall reduced magnitude of TDI activity
relative to 1 (cLogP: 3.5) is probably related to the decreased
lipophilicity of 2 and 3. Fragment 4 represents a set of
picolinoyl analogues with various azepane substitutions
(Supporting Information, Table s1) that all had no CYP3A
TDI activity. Redirection of metabolism to the picolinoyl
group, reduction of metabolism due to lower lipophilicity, or
both factors together may be responsible for this observation.
Guided by the hypothetical metabolic pathways delineated in
Figure 2, we investigated TDI structure−activity relationships
(SARs) of benzoyl derivatives with various azepane sub-
stitutions (Table 2). To gauge the impact of stereochemistry on
TDI activity, two pairs of enantiomers (e.g., 5a/5b and 7a/7b)
were compared. There was no difference in TDI potency of the
enantiomeric pairs, which agreed well with the fact that both
B
DOI: 10.1021/acsmedchemlett.5b00191
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ACS Medicinal Chemistry Letters
Letter
Table 2. CYP3A TDI of Substituted Pyrazoles: Probing the Main Site of Metabolism
a
b
c
d
Same as that in Table 1. Same as that in Table 1. Calculated logarithm of acid dissociation constant for the most basic group. Compounds 6 and
9 are racemic, 10 is achiral, and the rest are all single enantiomers.
To probe the mechanism in a different way, we designed 10
by removing the basic amine or alcohol. In view of the
significantly increased lipophilicity, it was not surprising that 10
had nearly the same magnitude of TDI activity as 2. However,
from a mechanistic point of view, it prompted us to add
metabolic pathway III, involving oxidation at the α carbon (C2)
to the endocyclic nitrogen of the azepane ring. The fully
decorated analogue S6 (Supporting Information, Table s2)
exhibited even stronger TDI activity than 10. Collectively, these
data imply that fully decorated molecules with higher
lipophilicity are more prone than fragments to undergo
metabolic modifications leading to TDI activities. In addition,
TDI investigation is complicated by sequential metabolic
modifications and multiple-site metabolisms at play. Truncated
molecules with fewer sites for biotransformation may provide a
straightforward way of understanding the mechanism of action
of the main metabolic site.
Figure 3. CYP3A TDI of compound 11 with testosterone (or
midazolam) as the substrate probe.
Coupled with maintaining PIM target potency (reports to be
published separately), the potential strategies to mitigate TDI
were applied to the full molecules. In fact, inclusion of a methyl
group alpha to the primary amine in 11 (Figure 3) not only
maintained potent PIM inhibition but also obviated the TDI
activity similar to analogous fragments 7a,b. With this data, the
pathway II hypothesis of generating a Michael acceptor was
further supported. In summary, the TDI potency of appropriate
C
DOI: 10.1021/acsmedchemlett.5b00191
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ACS Medicinal Chemistry Letters
Letter
fragments trends well with the full molecules. The absolute
TDI potencies are reduced for fragments that are in all cases
less lipophilic than full molecules. We concluded that as a
general strategy in mitigating TDI, carefully selected fragments
may be used to investigate the underlying metabolic
mechanisms. The benefits of using fragments include simpler
syntheses, shorter turnaround time, better material economy,
and more focused study of the major metabolic soft spots than
full molecules.
⊥Biogen Idec, 12 Cambridge Center, Cambridge, Massachu-
setts 02142, United States.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Thanks to Genentech, a member of the Roche group, for the
research funds.
The syntheses of 1−11 are outlined in Schemes s1−s3
(Supporting Information). Gem-difluoro azepanes were not
readily available, for which novel routes were developed starting
from α,β-unsaturated ketones (Scheme s1). Michael addition of
trimethylsilyl azide to t-butylcarboxyl (Boc) protected azepine
(S16a,b) provided the desired adducts (S17a,b). Subsequent
ketone reduction, Boc removal, and SnAr reactions afforded
azepane substituted nitro pyrazoles (S20a,b). Staudinger
reduction of the azide followed by Boc protection transformed
azides S20a,b to Boc-protected amines S20d,e, respectively.
Hydroxyl azepane S19c was readily available commercially and
was converted to S20c by SnAr reaction with S13. Desired
products 1−4, 7, 10, and 11 were then obtained in a sequence
of hydroxyl oxidation to ketone using Dess−Martin period-
inane, difluorination using Deoxo-Fluor, catalytic hydrogena-
tion of the nitro group, amide coupling, and acidic removal of
the Boc group. Similarly, compound 8 was prepared by Boc
removal of S22a, methylation through reductive amination,
catalytic hydrogenation of the nitro group, and amide coupling
in sequence.
Notes
The views expressed are those of the authors and do not
necessarily reflects the opinions of the Department of Defense
or the Uniformed Services University of the Health Sciences.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to analytical, purification chemistry, and
compound management group for compound characterization,
purification, and handling. We are also thankful to chem-
informatics and computational chemistry group for all the
calculated properties. Special thanks to Mr. Huiyong Hu and
Ms. Chenghong Zhang for technical assistance.
ABBREVIATIONS
■
TDI, time-dependent inhibition; CYP, Cytochrome P450;
DDI, drug−drug interactions; SAR, structure−activity relation-
ship; SnAr, nucleophilic aromatic substitution
In summary, molecular fragments were used as surrogates to
probe the TDI SAR and mechanism. A hypothesis-driven
approach was instrumental in designing key molecules devoid
of TDI liabilities, e.g., 7a, 7b, and 11. In understanding TDI
SAR, it is important to closely track physiochemical properties,
particularly lipophilicity. Metabolic hotspot migration should be
anticipated when designing molecules to mitigate metabolism.
Our work adds to the SAR database of culprit pharmacophores
incurring TDI beyond the traditional motifs.6,17 In addition,
robust syntheses of decorated azepanes, broadly useful for other
chemical series, are described.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, compound characterization, assay
protocols, additional TDI data, and metabolite identification
data. The Supporting Information is available free of charge on
the ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00191.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: wang.xiaojing@gene.com.
Present Addresses
†Impossible Foods, Redwood City, California 94063, United
States.
‡Uniformed Services, University of the Health Sciences,
Bethesda, Maryland 20814, United States.
§BioFocus, a Charles River Company, Chesterford Research
Park, Saffron Walden, Essex, CB10 1XL, United Kingdom.
∥Dotmatics Limited, the Old Monastery, Windhill, Bishops
Stortford, Herts, CM23 2ND, United Kingdom.
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