Isolation, Structural Identification, Synthesis, and Pharmacological Profiling of 1,2- trans-Dihydro-1,2-diol Metabolites of the Utrophin Modulator Ezutromid

Article abstract of DOI:10.1021/acs.jmedchem.9b01547

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

Full text of DOI:10.1021/acs.jmedchem.9b01547

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Article
Isolation, structural identification, synthesis, and pharmacological profiling
of 1,2-trans-dihydro-1,2-diol metabolites of the utrophin modulator ezutromid
Maria Chatzopoulou, Tim D. W. Claridge, Kay E. Davies, Stephen G. Davies, David Elsey, Enrico
Emer, Ai M. Fletcher, Shawn Harriman, Neil Robinson, Jessica Rowley, Angela J. Russell, Jonathan
Tinsley, Richard Weaver, Isabel Wilkinson, Nicky Willis, Francis Wilson, and Graham M. Wynne
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01547 • Publication Date (Web): 10 Oct 2019
Downloaded from pubs.acs.org on October 12, 2019
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Isolation, structural identification, synthesis, and
pharmacological profiling of 1,2-trans-dihydro-1,2-
diol metabolites of the utrophin modulator ezutromid
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Maria Chatzopoulou, Tim D.W. Claridge, Kay E. Davies, Stephen G. Davies, David J. Elsey,
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Enrico Emer, Ai M. Fletcher, Shawn Harriman, Neil Robinson, Jessica A. Rowley, Angela
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J. Russell, * Jonathon M. Tinsley, Richard Weaver, Isabel V.L. Wilkinson, Nicky J. Willis,
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Francis X. Wilson, and Graham M. Wynne
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Department of Chemistry, University of Oxford, Chemistry Research Laboratory,
Mansfield Road, Oxford, OX1 3TA, UK
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Department of Physiology, Anatomy and Genetics, MDUK Oxford Neuromuscular
Centre, University of Oxford, Oxford, OX1 3PT, UK
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Summit Therapeutics plc, 136a Eastern Avenue, Milton Park, Abingdon, OX14 4SB, UK
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Summit Therapeutics plc, One Broadway, 14th Floor, Cambridge, MA 02142, US
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S.H.B. Enterprises Ltd, 55 Station Road, Beaconsfield, HP19 1QL
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Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3PQ,
UK
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XenoGesis Ltd, BioCity Nottingham, Pennyfoot Street, Nottingham, NG1 1GF, UK
KEYWORDS: Duchenne muscular dystrophy, ezutromid, 1,2-dihydrodiol, beta-
substituted naphthalene metabolism
ABSTRACT
5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first in class
utrophin modulator which has been evaluated in a phase 2 clinical study for the
treatment of Duchenne Muscular Dystrophy (DMD). Ezutromid was found to undergo
hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-
dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral
administration in humans and rodents. In many patients, plasma levels of the DHD
metabolites were found to exceed ezutromid. Herein, we describe the structural
elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical
assignments of DHD1 and DHD3, their de novo chemical synthesis and their production
in in vitro systems. We further elucidate the likely metabolic pathway and CYP isoforms
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responsible for DHD1 and DHD3 production and characterise their physicochemical,
ADME, pharmacological properties and preliminary toxicological profile.
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Introduction
Duchenne muscular dystrophy (DMD) is one of the most prevalent neuromuscular
disorders and is caused by mutations on the dystrophin gene that result in loss of the key
structural protein dystrophin. This is an essential component of the dystrophin-
associated protein complex that connects the actin cytoskeleton to the basal lamina. Loss
of dystrophin results in chronic inflammation and a vicious cycle of muscle necrosis and
regeneration that leads to the eventual replacement of muscle by adipose and connective
tissue. The gradual muscle degeneration causes loss of ambulation in adolescence but
ultimately respiratory and cardiac failure, significantly reducing the life expectancy of
DMD patients.1, 2
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Dystrophin is the largest gene known in humans and shows high susceptibility to
spontaneous mutations.3, 4 As a result, DMD patients can have different mutations and
this has created a challenge in the design of disease-modifying therapeutics as many are
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targeted to a specific mutation and thus a sub-population rather than all patients. Until
recently, patients could only be treated with symptomatic therapy like corticosteroids to
reduce inflammation; however this is now changing with the conditional approval of
ataluren (PTC Therapeutics) for nonsense mutations by the European Medicines Agency
(
EMA), although it is not yet approved by the FDA. An exon skipping drug, eteplirsen
(Sarepta Therapeutics) has also been granted conditional approval by the U.S Food and
Drug Administration (FDA). Other disease modifying approaches that are currently
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under clinical investigation include gene therapy and exon skipping candidates for
mutations other than exon 51. For many years we have pursued an alternative disease-
modifying approach in DMD: to replace defective dystrophin with utrophin, an approach
in principle applicable to all DMD patients irrespective of their mutation.
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Utrophin is a structural paralogue of dystrophin discovered and characterised over two
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decades ago. Initial proof of principle studies in mdx mice
and DMD dog were
encouraging and this data supported the initiation of drug discovery programs from a
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number of companies. The most advanced small molecule utrophin modulator to date
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is ezutromid (formerly SMT C1100, Summit Therapeutics plc), a first in class utrophin
modulator that progressed further, to a Phase 2 clinical trial in DMD boys (PhaseOut
DMD). Encouraging interim 24-week data, demonstrated reduced muscle fibre damage
and increased levels of utrophin following ezutromid treatment, providing the first
evidence of target engagement and proof of mechanism. However, ezutromid failed to
meet its primary or secondary endpoints after 48 weeks of treatment and further
development was discontinued.12
OH
OH
CYPs
DHD1
N
O
S
O
ezutromid
O
OH
OH
DHD3
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Figure 1. Chemical structure of ezutromid and isolated major metabolites DHD1 and
DHD3 (in each case one of two possible enantiomers is drawn as absolute configuration
was not determined)
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Ezutromid (Figure 1) bears a naphthalene moiety and is extensively metabolised after
oral administration.13-15 Naphthalene moieties commonly undergo oxidative metabolism:
glutathione adducts and naphthols were the first isolated metabolites, both postulated to
arise from a common reactive intermediate later identified as the 1,2-naphthalene
oxide.16, 17 Several CYP isoforms (CYP1A1, CYP1A2, CYP2E1, CYP2F1, CYP3A4,
CYP2A6) have been found to produce this intermediate.18-21 1-Naphthol can be produced
by rearrangement of 1,2-naphthalene oxide, while 1,2-dihydro-1,2-diol formation can
proceed either by enzymatic or non-enzymatic hydrolysis.20, 22 In an in vitro study in
pooled human liver microsomes (HLM), naphthalene was metabolised to predominantly
1
,2-dihydrodiol along with 1-naphthol and 2-naphthol, a transformation subsequently
20
found to be mediated predominantly by CYP1A2. Similar metabolic pathways have also
been observed for 1-naphthalene-containing drugs or investigational drugs such as
setipiprant, cinacalcet and terbinafine, but to the best of our knowledge there are no
similar reports of 2-naphthalene-containing drug candidates.23, 24,23-26
Clinical studies of ezutromid in healthy volunteers and pediatric DMD patients
demonstrated two major abundant metabolites in plasma, accounting for >90% of total
drug exposure on Day 1 and approximately 70% on Day 11.13-15 These were tentatively
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assigned as regioisomeric 1,2-dihydrodiols and named DHD1 and DHD3 (Figure 1)
based on the order of elution in the analytical procedure employed. In urine, species
assigned as hydroxylated metabolites of ezutromid and their respective O-glucuronides
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were found. While the plasma concentration of ezutromid varied significantly between
subjects, the relative ratios between ezutromid and metabolites were very similar.13
Furthermore, exposure to ezutromid was observed to decrease with repeated dosing,
while metabolite levels increased. It was therefore important to determine the major route
of metabolism of ezutromid and consequent potential CYP induction in order to better
understand the compounds PK/PD profile.
Herein, we detail ezutromid’s metabolic profile in in vitro and in vivo systems, together
with the structural elucidation and pharmacological characterisation of the major
dihydrodiol metabolites DHD1 and DHD3.
Results and Discussion
Ezutromid’s metabolism was investigated using a human in vitro model. A preliminary
study in human liver microsomes (HLM) showed that after incubation for 45 minutes an
+
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1:19 mixture of parent (A) and a single metabolite was observed (B, T 5.7 min, [M+H]
R
m/z 372, Figures S1, S3). Mass spectrometry analysis showed this metabolite
corresponded to an increase of 34 mass units which indicated either addition of H O , H
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and CH OH, or H S. MS-MS fragmentation analysis (m/z 354, 344, 326, 279 and 261)
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demonstrated a loss of m/z 18 (H O, m/z 354), suggesting a formal addition of H O . Key
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fragments of ezutromid (m/z 310 (-C H ) and 245 (-C H SO )) were visible as 344 (-C H )
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and 279 (-C H SO ) within the MS-MS fragmentation of the metabolite (Figures S2-4)
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suggesting that this metabolite may be a dihydroxylated benzoxazole or a
dihydronaphthalenediol (Figure 2).
H
H
N
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+2H
O
S
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m/z 245
O
m/z 279 and m/z 261 (279 - H O)
2
m/z 310
m/z 344 and 326 (344 - H O)
2
ezutromid (A) m/z 338
ezutromid's metabolite in HLM (B) m/z 372
Figure 2. Proposed fragmentation pathway of ezutromid (A) and main metabolite (B) in
human liver microsomes (HLM).
A more comprehensive investigation of ezutromid metabolism was accomplished using
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a single oral administration of a radiolabelled sample of [ C-1]ezutromid in mice.
Metabolites and their relative proportions were measured in plasma, urine, faeces and
liver over 3 days. A total of 16 metabolites were observed in plasma, up to 19 in excreta
and liver and the identities of 14 of them were discerned from a combination of accurate
n
mass measurements and diagnostic MS fragments (Figures S5-9, Tables S1-2). Consistent
with the observations in human samples, the most prominent metabolites in excreta were
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consistent with two isomeric dihydrodiol derivatives of ezutromid (M4 and M8) and their
corresponding glucuronide adducts (M2 and M5) (Figure S5, Tables S1-2).
Production of metabolites using in vitro systems
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A route to larger quantities of ezutromid’s metabolites was required in order to
elucidate their structure and investigate their pharmacological profiles. Ezutromid
stability was investigated in a range of in vitro systems in an attempt to identify an
appropriate system for production of the DHDs. Ezutromid’s metabolism was found to
vary significantly between different species in vitro (Table S3).
Using MicroCyp enzymes, a recombinant human cytochrome P450 and a microbial
biocatalyst panel, failed to produce the desired metabolites in sufficient quantities.
Several attempts at a biomimetic approach based on epoxidation followed by hydrolysis
also gave only trace quantities of the desired metabolites.
The optimal, most cost-effective and scalable conditions for the synthesis of the major
metabolites was found to be by metabolic conversion of ezutromid with induced rat S9
fractions ( β -naphthoflavone/phenobarbital) (Table S4). UV-Vis spectroscopy showed
conversion to a 18:77:5 mixture of ezutromid, dihydrodiol-1 (D1) and dihydrodiol-2 (D2)
after 4 h of incubation (Figure S10). Though this procedure was low yielding for diol D2
it was found to be amenable to scale-up: scaling up to 200 mL incubation volume gave
similar levels of conversion to D1 and D2 (Table S5, Figure S11). The isolated sample of
D1 was compared to the samples produced from the radiochemical study in mice and
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was found to have similar retention times to M4 (T 26.7 and 27.5 min, respectively) and
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identical fragmentation by MS/MS, suggesting these had the same composition (Figures
S12-13, Table S6).
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Moreover, the analytical data of dihydrodiol metabolites DHD1 and DHD3 from
human plasma samples, were compared to those from induced rat S9 D1 and D2 using
LC-MS/MS (Figure 3). D1 eluting at T = 8.2 min with m/z 372.0898 matched the sample
R
of DHD1 from human patients M372a, T = 8.1 min with m/z 372.0899. D2 eluting at T =
R
R
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.0 min with m/z 372.0899 matched the sample of DHD3 from human patients M372b, TR
= 8.9 min with m/z 372.0898. Further accurate mass measurement of both samples gave
identical fragmentation for the two samples supporting the same composition (Table S7,
Figures S14-17).
Figure 3. Cumulative accurate mass summed ion chromatograms for: A) an equimolar
mixture of ezutromid, D1, and D2 reference standards B) plasma following multiple oral
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doses of ezutromid (1.0 g, BID) in paediatric DMD subjects – 8 hours post-AM dose on
day 7
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9
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9
0
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6
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8
9
0
1
2
3
4
5
6
7
8
9
0
De novo synthesis of dihydrodiols DHD1 and DHD3
In order to assign the correct regio- and relative stereochemistries to the dihydrodiols
DHD1 and DHD3 we carried out a de novo chemical synthesis. Several previous studies
on 1-naphthyl-containing molecules report preferential formation of 7,8-dihydrodiol or
5
,6-dihydrodiol as the major (or only) metabolites.27,28,29-32 Based on the proposed 2-step
2
4
epoxidation-transdiaxial ring opening mechanism of 1,2-dihydrodiol formation, it was
envisaged that the relative stereochemistry in both dihydrodiols was likely to be trans.
Thus, authentic samples of trans-dihydrodiols 9 and 10, reasoned to be the major
metabolites from ezutromid, were synthesized from the corresponding naphthalenes
(Scheme 1).
O
N
OH
OH
c
Et
S
O
>
10 g
O
7a
79%
e
R1
O
O
O
O
N
OH
OH
c
R1
R2
d
a
Et
HO
O
N
R2
Et
86%
1 g
S
O
N
O
S
27%
6
7-84%
7
9
Et
O
O O
S
1
2
R1: H R2: OMe
R1: OMe R2: H
O
O
3
4
5
6
R1: H R2: OMe
R1: OMe R2: H
O
O
HO
OH
b
61-70%
R1: H R2: OH
R1: OH R2: H
O
O
N
c
d
Et
Et
N
23%
S
O
95%
S
O
8
O
10
O
Scheme 1. Synthetic route to trans-1,2-dihydrodiols 9 and 10 (one of two possible
enantiomers is depicted as absolute configuration was not unambiguously determined).
1
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1
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2
2
2
2
2
3
3
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3
3
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5
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5
5
5
5
6
o
Reagents and conditions: (a) (1) SOCl , DMF, 1,4-dioxane, 80 C, 1 h; (2) 2-amino-4-
2
o
o
(
ethylsulfonyl)phenol, MsOH, p-xylene, 140 C, 48 h. (b) BBr , DCM, 0-40 C, 24 h. (c) IBX,
3
DMF, rt, 90 min. (d) LiBH , O , rt, 24 h (e) o-chloranil, DMF, rt, 24 h.
4
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Regioisomeric 6-methoxy-2-naphthoic acid 1 and 7-methoxy-2-naphthoic acid 2 were
treated sequentially with thionyl chloride, 2-amino-4-(ethylsulfonyl)phenol and BBr to
3
afford naphthols 5 and 6 in good yields (67-84% over three steps). Oxidation of 5 and 6
with IBX gave 5,6-diketo and 7,8-diketo species 7 and 8 respectively. Initial attempts to
scale up the production of 9 resulted in either partial oxidation of 5 to naphthalene diol
7
a, or a mixture of 7 and 7a. However, treatment of 7a with o-chloranil gave complete
conversion to the diketo species 7. Reduction of the diketo-species to the final dihydrodiol
products 9 and 10 was attempted under a range of conditions including NaBH , KBH ,
4
4
Pd/C, LiAlH , LiAlH(OtBu ) , REDAL, DIBAL, in air, under oxygen or an inert
4
3 3
atmosphere. Optimal conditions were found to be treatment with LiBH under an oxygen
4
atmosphere. The final products 9 and 10 were isolated as racemic mixtures after
chromatography (SI). 9 and 10 were resolved into single enantiomers following chiral
chromatography.
Metabolite Structure Determination/Verification
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9
Unambiguous structure elucidation of D1/DHD1 and D2/DHD3 was achieved with full
NMR assignment and comparison with the authentic samples obtained from chemical
1
synthesis. Overlaying of the H-NMR spectra of 9 and D1 showed complete overlap,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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0
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0
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9
0
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5
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7
8
9
0
similarly overlaying of the spectra of 10 and DHD3 also showed complete overlap
(
Figures S18-19). Full assignments were based on COSY for J-coupled pairs of protons,
and edited HSQC and HMBC correlations for the assignments of aromatic, aliphatic
carbon-13 chemical shifts and their attached protons. The regiochemistry of both
dihydrodiols was assigned by NOESY. For D1, determined to be the 6-benzoxazol-2-yl
regioisomer, characteristic NOE correlations were observed between H and H , and
8
1
alkene proton H with naphthalene proton H (Figure 4a). In D2, determined to be the 7-
4
5
benzoxazol-2-yl regioisomer, characteristic NOE correlations were observed between H8
and H , and H with alkene proton H (Figure 4b). In both regioisomers a NOE
1
5
4
correlation was observed between H and H , and no strong NOE correlation was
2
3
observed between H and H , consistent with trans relative stereochemistry in both cases.
1
2
The observed H -H cross peak is dominated by scalar coupling effects, with evidence of
1
2
only a very weak NOE between the protons contributing to it. The assignment of trans
relative stereochemistry was further supported by analysis of vicinal coupling constants
1
3
= 10.5 Hz (3J(H2,H3) = 2.4 Hz). This is
in the H-NMR spectra: for both D1 and D2 J
(H1,H2)
3
3
consistent with data for other reported trans-dihydrodiols, while cis-dihydrodiols have
much lower recorded J values (i.e 4.8 Hz).34
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0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. NOESY spectra and NOE correlations for structure elucidation of a) D1 and b)
D2. One of two possible enantiomers is shown, absolute configuration was not
determined.
Determination of CYP-mediated metabolism of ezutromid
Naphthalene has been shown to be predominantly metabolised by human CYP1A2 into
the trans-1,2-dihydrodiol and to the 1-naphthol. CYP3A4 has also shown to induce
metabolism to the 2-naphthol.20 To identify the main CYP isoforms involved in
ezutromid’s metabolism two approaches were employed. Firstly, a panel of 8
recombinant CYP isoform expression systems (Bactosomes™) were incubated with
ezutromid (Table S8). Secondly, CYP isoform specific chemical inhibitors were used to
determine their effects on ezutromid metabolism in human liver microsomes (Table S9).
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9
The shortest half-life for ezutromid was observed in the presence of CYP1A1 (T1/2 2.16
min) and CYP1A2 (T1/2 2.62 min) bactosomes (Table S8). Turnover of ezutromid in
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 bactosomes was minimal
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(T1/2 ≥180 min). Formation of DHD1 was observed upon incubation of ezutromid with
CYP1A2, CYP2B6 and CYP2D6 although peak area ratios were small. Formation of DHD3
was not observed under any conditions.
In human liver microsomes, turnover of ezutromid was decreased in the presence of
inhibitors of CYP1A (CLint 5.06 μL/min/mg), CYP2B6 (CLint 21.37 μL/min/mg) and
CYP2D6 (CL 35.1 μL/min/mg) compared to control (CL 50.67 μL/min/mg) (Table S9).
int
int
Ticlopidine was used as an FDA recommended inhibitor for CYP2B6, however it has been
shown to inhibit other CYP isoforms, including CYP1A2, at the concentration used in this
study. It was therefore plausible, and consistent with our observations of CYP1A1/2-
mediated metabolism of ezutromid in bactosomes, that the decrease in turnover of
ezutromid in the presence of ticlopidine might result from CYP1A2 inhibition rather than
CYP2B6. This was further supported by the observation that decreased formation of both
DHD1 and DHD3 was observed only in the presence of inhibitors of CYP1A (6% and 11%
of control) and to a lesser extent CYP2D6 (65% and 66% of control). The CYP2D6 inhibitor
used, quinidine, has been reported to be a relatively selective inhibitor, inhibiting
3
5, 36
CYP3A4 only at higher concentrations (IC = 28 μM),
thus CYP2D6 appears to also
5
0
be involved in ezutromid’s metabolism, but to a lesser extent that CYP1A1/2.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In summary, the CYP1A1 and CYP1A2 enzymes appear to be the main P450 isoforms
mediating ezutromid’s metabolism and the formation of DHD1 and DHD3 in vitro. Based
on the methods used, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 do not appear to be
involved in the metabolism of this molecule, but CYP2D6 may also contribute to a lesser
extent.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ezutromid metabolite profiling upon repeated dosing
As there was a marked decrease in exposure after repeated dosing of ezutromid in
1
4
DMD patients, the metabolic profile of ezutromid was investigated in rat after 1 and 28
days (2000 mg/kg). Consistent with the results from DMD patients, it was found that
ezutromid’s clearance was higher after 28 days of dosing compared to day 1. After 24
hours, notable levels of ezutromid (38-89%) were found in plasma (Table S10). A large
variation in the extent of ezutromid metabolism was observed between animals. The
most abundant metabolite observed (T 4.57-4.61 min, 8-44%) was a dihydrodiol (I)
R
followed by an isomeric dihydrodiol (II) (T 4.98-5.00 min, 3-14%).
R
After 28 days of dosing, more extensive metabolism of ezutromid was observed (Table
S11). Ezutromid was detected at low levels and the two main metabolites were found in
similar proportions to day 1 [T 4.57-4.61 (50-70%), T 4.98-5.00 min (10-12%), dihydrodiol
R
R
I and II respectively]. Two other metabolites were also present in significant amounts, a
glucuronide conjugate of a dihydrodiol, and the O-glucuronide conjugate of an
1
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aromatised diol. In urine, the composition of metabolites changed significantly with
dihydrodiol glucuronides being the most abundant (Table S12). The relative proportions
of the remaining metabolites varied significantly between male and female rats.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
6
0
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2
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7
8
9
0
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2
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8
9
0
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2
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9
0
CYP Induction
As exposure to ezutromid was observed to decrease over time with a concurrent
1
3
increase in the levels of metabolites in both humans and rodents, the propensity of
ezutromid and its two major metabolites DHD1 and DHD3 to induce CYP expression
was investigated. No significant induction of CYP1A1/2 was observed using in vitro
systems where only a single dose was administered: after 48 h incubation of ezutromid
(0.1-10 μM) with rat hepatocytes no significant difference in CYP1A1/2 mediated enzyme
activity was observed (data not shown). Similarly, in human hepatocytes, incubation
with ezutromid for 48 h did not result in significant induction of CYP1A2 (data not
shown). However, ezutromid was observed to inhibit CYP1A2 enzymic activity in
human liver microsomes with an IC of 5.4 μM.
5
0
Although no evidence in support of CYP induction in vitro was observed, a marked
elevation in 7-ethoxyresorufin O-deethylase (EROD) activity was observed after treatment
with ezutromid (2000 mg/kg/day, p.o) for 28 days in rats. EROD enzyme activity was
elevated 17-18 fold, 21-22 fold, and 13-18 fold when expressed per mg protein, per g liver
and per nmole P450, respectively (Table S13). No significant changes were observed in
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microsomal protein or total P450 concentration. EROD activity is used as a proxy for
37
induction of the CYP1A subfamily (CYP1A1, CYP1A2); thus these data suggest that
repeated oral administration of ezutromid in rats can result in induction of CYP1A, which
are the principal enzymes responsible for mediating metabolism of ezutromid.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
A similar trend was observed after administration of ezutromid (500 mg/kg/day) for 39
weeks in minipigs. No evidence of significant increase in microsomal protein
concentration and total cytochrome P450 content were observed (data not shown), but a
small increase in EROD and 7-pentoxyresorufin O-depentylase (PROD) activity (2.1 fold
and 2.0 fold, respectively) was observed in female minipigs (Table S14). The magnitude
of this effect was very low in comparison to that observed with other CYP1A and CYP2B
inducers. The marked gender difference between the basal levels of CYP1A activity in
minipigs (CYP1A2 and CYP2B activity is higher in female minipigs by 4- and 3.8-fold
3
8
respectively), could account for the observed lack of increased EROD or PROD activity
in male minipigs. No effect was observed on testosterone hydroxylase, lauric acid 11- and
1
2-hydroxylase activity, suggesting no induction of CYP3A, CYP2E1 and CYP4A
respectively.
Pharmacological characterisation and in vitro PK profiling of DHD1 and DHD3
As the two major metabolites DHD1 and DHD3 were produced in significant amounts
1
5
in vivo, and exposure exceeded that of ezutromid in some patients, a systematic analysis
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of the pharmacological activity, physicochemical properties, pharmacokinetic and
toxicological profile of DHD1 and DHD3 was undertaken.
Although both metabolites appeared to show comparable levels of activity to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
9
ezutromid in the H2K-mdx utrnA-luc assay, a luciferase reporter gene assay using a
human muscle specific utrophin A promoter in mouse mdx myoblasts, differences were
observed in the Homogenous Time-Resolved Fluorescence (HTRF) assay quantifying
human utrophin protein in cell lysates of iDMD cells (Table 1). In the latter assay, no
increase in utrophin protein was observed upon treatment with DHD1 up to the highest
concentration tested (10 M), while DHD3 was found to increase utrophin protein but
with an approximately 6-fold reduction in potency compared to ezutromid. The
molecular target of ezutromid and the mechanism through which it increases utrophin
protein levels is not yet known, and so it is unclear at this stage whether DHD3 and
ezutromid act through a common molecular target or distinct pathways leading to an
increase in utrophin protein. Studies are ongoing to resolve this question.
Table 1. Assay data to monitor for utrophin upregulation for DHD1 and DHD3
metabolites of ezutromid
a
b
c
d
Compound
DHD1
H2K EC ± SEM (μM) HTRF EC ± SEM (μM)
5
0
50
0.37 ± 0.08
>10 μM
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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5
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6
DHD3
0.12 ± 0.03
0.11 ± 0.01
8.35 ± 0.62
1.28 ± 0.24
ezutromid
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
firefly luciferase reporter gene assay in H2K-mdx utrnA-luc cells; b
EC values were calculated using non-linear regression (curve fit) from
5
0
c
2 biological replicates (3 technical replicates each); HTRF, homogenous
time-resolved fluorescence assay; d EC values were calculated using
5
0
non-linear regression (curve fit) from 2 biological replicates (3 technical
replicates each)
Physicochemical and in vitro PK properties were also determined for DHD1 and DHD3
(Tables 2-3). Both metabolites had a measured logD of around 2, with increased solubility
and permeability, and a higher unbound fraction to mouse and human plasma proteins
compared to ezutromid (Table 2). Metabolic stability was measured in vitro in mouse and
human S9 fractions and hepatocytes. DHD3 was found to be the more stable of the two
metabolites in both mouse and human hepatocytes (Table 3).
Table 2. Physicochemical profile and plasma protein binding (100% plasma) of DHD1
and DHD3 metabolites of ezutromid
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PPB F (%)
b
Compoun logD
KinSol
PAMPApH
6cm/s)
(10-
7.4
d
(pH 7.4) (μM)
mouse
human
63.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
4
4
4
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4
4
4
4
4
4
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5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
2
3
4
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0
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2
3
4
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9
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Highly
DHD1
DHD3
2.04
1.95
ND
96
33.3
34.4
9.5
unbound
210
1-2
92.3
99.5
87.2
ezutromi
d
99.0
Abbreviations: F , fraction bound; KinSol, kinetic aqueous solubility; PAMPA, parallel
b
artificial membrane permeability assay; PPB, plasma protein binding
Table 3. In vitro metabolic stability of DHD1 and DHD3 metabolites of ezutromid in
mouse and human microsomes (S9 fraction) and hepatocytes (Hep)
Compou T1/2 ± SD (min)
CL ± SD (μL/min.mg)
int
nd
mouse
human
S9
mouse
S9
human
S9
Hep
Hep
Hep
S9
Hep
DHD1
39 ± 1.8 7 ± 0.7 >138
85 ± 8.2 35 ± 1.3 98 ± 9.9 <10
8 ± 0.8
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DHD3
112
6.2
± 143 ± >138
16.3
142
± 13 ± 3.1 5 ± 0.6 <10
5 ± 0.4
2
10.3
ezutromi 74 ± 7.8
d
101 ±
4.5
19 ± 2.0
14 ± 0.6
0
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0
Preliminary toxicology and safety assessment of metabolites
UGT turnover and UGT inhibition by ezutromid metabolites
No detectable turnover of DHD1 and DHD3 was observed in all uridine glucuronate
transferase (UGT) incubations (T1/2 >240 min). The CL for both DHD1 and DHD3 with
int
all UGT enzymes was determined to be <5.8 μL/min/pmol, for UGT1A4 it was
determined to be <2.9 μL/min/pmol. The propensities of DHD1 and DHD3 to act as UGT
inhibitors were also investigated. DHD1 was found to be an inhibitor of UGT1A9 and
UGT1A3 (12% and 62% residual activity respectively at 10M for DHD1); DHD3 was
found to be a weak inhibitor of UGT1A9, UGT1A3 and UGT1A4 (45, 79 and 84% residual
activity respectively at 10μM for DHD3, Table S15).
Interactions of ezutromid metabolites with liver transporter proteins
The potential of DHD1 and DHD3 to cause drug-induced liver injury was evaluated by
screening them against a panel of hepatic transporters in vitro (Table S16). Both
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metabolites were found to inhibit the breast cancer resistance protein (BCRP) efflux
transporter with an IC50 value in the low micromolar range, and the organic anion
transporter protein (OATP) uptake receptors OATP1B1 and OTAP1B3 with IC values
5
0
1
1
1
1
1
1
1
1
1
1
2
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0
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9
0
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9
0
in the mid micromolar range. DHD1 was also observed to inhibit the multidrug and toxic
compound extrusion (MATE) transporter 1 with an IC value of 10 M (Table S16). No
5
0
other interactions were observed. DHD1 and DHD3 were also assessed as substrates for
BCRP, MDR1, MDR4, OAT1B1 and OAT1B3. No evidence of transport or uptake was
observed for either metabolite (Table S16).
Secondary pharmacology of ezutromid metabolites
DHD1 and DHD3 were screened at 10 M against a panel of 87 receptors and enzymes
(CEREP). No significant interactions were noted with DHD3, but DHD1 was found to
inhibit monoamino oxidase B (MAO-B) and the adenosine transporter (Graph S1, Table
S17). In a follow up study, DHD1 was found to inhibit MAO-B and the adenosine
transporter with IC values of 4.0 μM, and 3.9 μM respectively.
5
0
Conclusions
The metabolic fate of ezutromid was evaluated using in vitro and in vivo systems.
Hepatic clearance was found to be the main route of ezutromid’s metabolism. Oxidative
metabolism, predominantly mediated by CYP1A led to the formation of 1,2-dihydrodiols
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DHD1 and DHD3, produced as the two major metabolites in mice, rats and minipigs,
consistent with observations in human plasma. Comparison of analytical and
spectroscopic data of DHD1 and DHD3, the dihydrodiol metabolites produced from mice
and authentic samples accessed through chemical synthesis allowed the regio- and
stereochemical assignment of the structures of both metabolites. While the metabolic fate
of 1-substituted naphthalene containing drugs have been profiled, this study provides an
insight into the metabolic fate of a 2-substituted naphthalene in mammals and humans,
which are less well exemplified in the literature.
0
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The pharmacological, pharmacokinetic and toxicological profile of the two metabolites
DHD1 and DHD3 were investigated. Both appear to be equipotent to ezutromid in the
luciferase reporter gene assay, however DHD3 was found to be 6-fold less potent
compared to ezutromid in an HTRF assay monitoring increases in utrophin protein
levels. DHD1 was inactive in the HTRF assay. The physicochemical and in vitro
pharmacokinetic properties of DHD1 and DHD3 were shown to be more favourable than
ezutromid; they showed improved solubility, permeability, metabolic stability, and
reduced plasma protein binding. DHD1 or DHD3 were found to have an acceptable
toxicological profile: no significant interactions with hepatic transporters or other
receptors, ion channels or enzymes were observed for either metabolite.
Experimental Section
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General procedures. All reactions involving moisture-sensitive reagents were carried
out under a nitrogen or argon atmosphere using standard vacuum line techniques and
glassware that was flame dried and cooled under nitrogen before use. Water was
purified by an Elix® UV-10 system. All other reagents were used as supplied (analytical
or HPLC grade) without prior purification. Thin layer chromatography was performed
on aluminium plates coated with 60 F254 silica. Flash column chromatography was
performed either on Kieselgel 60 silica on a glass column, or on a Biotage SP4 automated
flash column chromatography platform. Melting points were recorded on a EZ-Melt
Automated Melting Point Apparatus (EZ Melt) and are uncorrected. NMR spectra were
recorded on Bruker Avance III spectrometers (at 400 or 500 MHz) using the deuterated
solvent stated and at rt. The field was locked by external referencing to the relevant
deuteron resonance. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter
1
1
1
1
1
1
1
1
1
1
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0
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0
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9
0
1
2
3
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7
8
9
0
−
1
2
−1
with a water-jacketed 1 cm cell. Specific rotations are reported in 10 deg cm g and
concentrations in g/100 mL. Accurate mass measurements were run on either a Bruker
MicroTOF internally calibrated with polyalanine, or a Micromass GCT instrument fitted
with a Scientific Glass Instruments BPX5 column (15 m  0.25 mm) using amyl acetate as
a lock mass. Purity of the compounds was determined by high performance liquid
chromatography (HPLC) and was 95% or higher in all cases. The instrument used was a
Waters Acquity H-Class. Consisting of Acquity UPLC PDA Detector, Acquity
Quaternary Solvent Manager and Acquity QDa mass detector. The columns and
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conditions varied. For chemical purity, the column was an Acquity BEH C18 1.7 μm
particle size (50x2.1 mm); eluent A 0.1% TFA, eluent B 0.1% TFA in MeCN, gradient (see
SI), with a flow rate of 0.6 mL/min; column temperature 40 oC; injection volume 1.0 μL.
Enantiopurity was determined on the same instrument with a Lux iC5 particle size 5 μM
0
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(4.6mm×250mm) or a Lux iC5 particle size 5 μM (4.6mm×250mm) column for D1/9 and
D2/10, respectively; 50:50 EtOH(or MeOH):CO2 (0.2% v/v NH3) isocratic, with a flow rate
o
of 4 mL/min; column temperature 40 C; injection volume 1.0 μL. An EnVision™ 2103
Multilabel plate reader – Perkin Elmer: 620 and 665nm emission filters was used for the
HTRF assay. Experiments conducted in CROs used their standard equipment.
The 28 day oral (gavage) administration of ezutromid study in the rat and the 39 week
oral (gavage) administration of ezutromid study in minipig were designed to meet the
known requirements of European Directive 2001/83/EC and all subsequent amendments
together with any relevant International Conference on Harmonisation (ICH) guidelines.
Materials. The starting material for DHD1 (7-methoxy-2-naphthoic acid) was provided
14
by Synthesis Med Chem Pty Ltd; [ C]ezutromid was synthesised at Covance
Laboratories Ltd (synthetic scheme and analytical data reported in SI, Scheme S1). Liver
and intestine microsomes for intrinsic clearance of ezutromid were obtained from
Xenotech: human microsomes (pooled and mixed gender), monkey microsomes
(Cynomolgus, pooled male), dog microsomes (Beagle, pooled male), rat microsomes
(Sprague-Dawley, pooled male); pooled human liver microsomes (pooled male and
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female) for the in vitro metabolite identification study in human liver microsomes were
prepared at Cyprotex.
Microsomes were stored at -80°C prior to use; β-
naphthoflavone/phenobarbital induced rat S9 was obtained from Xenotech (Product No:
R1081.S9, Lot No: 710507 (day 1) and Lot No: 1510257 (days 2-5)); UltraPool™ human
liver microsomes 150 were used in the P450 time-dependent inhibition and UGT
phenotyping and inhibition studies.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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1
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Metabolite extractions from induced rat S9
In brief, ezutromid (100μg/ml), was incubated with β-naphthoflavone/phenobarbital
induced rat S9 [0.5mg/ml protein concentration] and NADPH (3 mM) for 7 h. The mixture
was then quenched with ice-cold methanol, centrifuged to remove excess protein and
solvents were removed. After chiral separation (Reach separations, complete method
described in SI), 120 mg D1 and 11 mg D2 were obtained from 188 mg of ezutromid.
D1
1
H NMR (400 MHz, MeOH-d ) δ 8.29 (dd, J = 1.8, 0.6 Hz, 1H), 8.18 (dd, J = 8.0, 1.8 Hz,
4
1
H), 8.01 (dd, J = 7.2, 1.8 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H), 7.94 (dd, J = 8.5, 0.7 Hz, 1H), 7.79
(dd, J = 7.9, 1.2 Hz, 1H), 6.59 (dd, J = 9.9, 2.3 Hz, 1H), 6.08 (dd, J = 9.8, 2.4 Hz, 1H), 4.77
(dd, J = 10.5, 1.2 Hz, 1H), 4.43 (dt, J = 10.5, 2.4 Hz, 1H), 3.29 (q, J = 7.4 Hz, 2H), 1.26 (t, J =
13
7
.4 Hz, 3H). C NMR (125.5 MHz, MeOH-d ) δ 166.8, 155.2, 143.7, 136.9, 135.1, 134.2,
4
2
5
128.2, 127.4, 126.8, 126.7, 126.3, 121.5, 112.9, 75.5, 73.7, 51.6, 7.7. [훼 ] ― 46.8 [푐 0.1, 푀푒푂퐻].
퐷
D2
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1
H NMR (500 MHz, MeOH-d ) δ 8.48 (d, J = 1.6 Hz, 1H), 8.33 – 8.27 (m, 1H), 8.17 (ddd,
4
J = 7.9, 1.9, 0.8 Hz, 1H), 7.98 (dd, J = 8.5, 1.8 Hz, 1H), 7.94 (dd, J = 8.5, 0.6 Hz, 1H), 7.34 (d,
J = 7.9 Hz, 1H), 6.56 (dd, J = 9.8, 2.3 Hz, 1H), 6.13 (dd, J = 9.8, 2.5 Hz, 1H), 4.78 (dd, J = 10.5,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
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8
9
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1
3
1
.3 Hz, 1H), 4.45 (dt, J = 10.5, 2.4 Hz, 1H), 3.29 (s, 2H), 1.26 (t, J = 7.4 Hz, 3H). C NMR
(
125.5 MHz, MeOH-d ) δ 166.9, 155.2, 143.8, 140.0, 138.3, 136.9, 135.6, 128.6, 128.0, 127.6,
4
2
5] ―177.5 [푐 0.1, 푀푒푂퐻]
126.7, 126.1, 121.5, 112.8, 75.2, 73.9, 51.6, 7.7;
[
훼
퐷
Chemistry
General method for the synthesis of naphthols
To a solution of 1 or 2 (3.0 g, 14.8 mmol) in 1,4-dioxane (20 mL), DMF (0.1 mL) and
SOCl (2.0 mL, 26.4 mmol) were added dropwise and refluxed for 1h. The solvents were
2
evaporated and the crude material was used in the next step.
6-Methoxy-2-naphthoyl chloride
1
H NMR (400 MHz, CDCl ) δ 8.67 (d, J = 2.0 Hz, 1H), 8.03 (dd, J = 8.7, 1.9 Hz, 1H), 7.90
3
(
d, J = 9.0 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.24 (s, 1H), 7.17 (d, J = 2.5 Hz, 1H), 3.97 (s, 3H).
7-Methoxy-2-naphthoyl chloride
1
H NMR (400 MHz, CDCl ) δ 8.64 (dt, J = 1.8, 0.7 Hz, 1H), 7.92 (dd, J = 8.6, 1.9 Hz, 1H),
3
7
.84 (d, J = 8.7 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.33 (dd, J = 8.9, 2.5 Hz, 1H), 7.28 (d, J = 2.5
Hz, 1H), 3.96 (s, 3H).
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A solution of the crude naphthoyl chloride (14.8 mmol) and 2-amino-4-
(
ethylsulfonyl)phenol (1.5 g, 14.8 mmol) was refluxed in p-xylene for 1 h. Then MsOH
(0.15 mL) was added and refluxed for 2 d. The reaction was cooled to room temperature
1
1
1
1
1
1
1
1
1
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2
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and partitioned between EtOAc (2 x 100 mL) and sat. aq. NaHCO (100 mL), washed with
3
brine (100 mL) and dried over MgSO . The crude material was used in the next step.
4
5-(Ethylsulfonyl)-2-(6-methoxynaphthalen-2-yl)benzo[d]oxazole (3)
1
H NMR (400 MHz, CDCl ) δ 8.73 – 8.69 (m, 1H), 8.33 (d, J = 1.6 Hz, 1H), 8.26 (dd, J =
3
8
.5, 1.7 Hz, 1H), 7.93 (dd, J = 8.5, 1.8 Hz, 1H), 7.88 (t, J = 9.0 Hz, 2H), 7.75 (d, J = 8.4 Hz,
1H), 7.24 (dd, J = 8.9, 2.5 Hz, 1H), 7.19 (d, J = 2.5 Hz, 1H), 3.97 (s, 3H), 3.19 (q, J = 7.4 Hz,
1
3
2
1
H), 1.31 (t, J = 7.4 Hz, 3H); C NMR (125.5 MHz, CDCl ) δ 166.0, 159.8, 154.0, 143.1, 137.0,
3
35.3, 130.8, 128.9, 128.4, 127.9, 125.3, 124.7, 121.2, 120.9, 120.3, 111.4, 106.1, 55.6, 51.2, 7.8.
5
-(Ethylsulfonyl)-2-(7-methoxynaphthalen-2-yl)benzo[d]oxazole (4)
1
H NMR (400 MHz, CDCl ) δ 8.67 (d, J = 1.7 Hz, 1H), 8.34 (dd, J = 1.8, 0.6 Hz, 1H), 8.13
3
(
dd, J = 8.5, 1.7 Hz, 1H), 7.93 (dd, J = 8.5, 1.8 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.81 – 7.76
m, 1H), 7.74 (dd, J = 8.4, 0.6 Hz, 1H), 7.29 – 7.20 (m, 2H), 3.94 (d, J = 8.3 Hz, 3H), 3.19 (q, J
(
= 7.4 Hz, 2H), 1.31 (t, J = 7.4 Hz, 3H).
BBr (1.0 M in CH Cl , 43.6 mL, 43.6 mmol) was added dropwise to 3 or 4 at 0 C and
o
3
2
2
o
o
the reaction was warmed to 40 C. After 24 h, the reaction was cooled at 0 C and
quenched with water, followed by extractions with CHCl /iPrOH (3:1, 3 x 50 mL), dried
3
over MgSO , and the solvents were evaporated in vacuo. The residue was triturated with
4
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