Discovery of 2-arylbenzoxazoles as upregulators of utrophin production for the treatment of duchenne muscular dystrophy

Article abstract of DOI:10.1021/jm200135z

Figure Presented. A series of novel 2-arylbenzoxazoles that upregulate the production of utrophin in murine H2K cells, as assessed using a luciferase reporter linked assay, have been identified. This compound class appears to hold considerable promise as a potential treatment for Duchenne muscular dystrophy. Following the delineation of structure-activity relationships in the series, a number of potent upregulators were identified, and preliminary ADME evaluation is described. These studies have resulted in the identification of 1, a compound that has been progressed to clinical trials.

Full text of DOI:10.1021/jm200135z

ARTICLE
pubs.acs.org/jmc
Discovery of 2-Arylbenzoxazoles as Upregulators of Utrophin
Production for the Treatment of Duchenne Muscular Dystrophy
Daniel R. Chancellor,† Kay E. Davies,‡ Olivier De Moor,† Colin R. Dorgan,† Peter D. Johnson,†
Adam G. Lambert,† Daniel Lawrence,† Cristina Lecci,† Carole Maillol,† Penny J. Middleton,† Gary Nugent,†
Sꢀeverine D. Poignant,† Allyson C. Potter,‡ Paul D. Price,† Richard J. Pye,† Richard Storer,†
Jonathon M. Tinsley,† Renate van Well,† Richard Vickers,† Julia Vile,† Fraser J. Wilkes,† Francis X. Wilson,†
Stephen P. Wren,*,† and Graham M. Wynne*,†
†Summit plc, 91 Milton Park, Abingdon, Oxfordshire, OX14 4RY, United Kingdom
‡Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, South Parks Road, Oxford,
OX1 3QX, United Kingdom
S Supporting Information
b
ABSTRACT: A series of novel 2-arylbenzoxazoles that upregulate the production of utrophin in
murine H2K cells, as assessed using a luciferase reporter linked assay, have been identified. This
compound class appears to hold considerable promise as a potential treatment for Duchenne
muscular dystrophy. Following the delineation of structureꢀactivity relationships in the series, a
number of potent upregulators were identified, and preliminary ADME evaluation is described.
These studies have resulted in the identification of 1, a compound that has been progressed to
clinical trials.
’ INTRODUCTION
Ataluren treatment is associated with the production of functional
dystrophin and statistically significant reductions in the leakage of
muscle-derived creatine kinase into the blood. An alternative
nonsymptomatic pharmacological approach, which has only begun
to be investigated for DMD comparatively recently, is upregulation
therapy. This is based on increasing the expression of alternative
genes to replace those that are defective. Upregulation of utrophin,
an autosomal paralogue of dystrophin, has been proposed as a
potential treatment paradigm for DMD.6,7 It has been estimated that
a 2ꢀ3-fold increase in levels of systemic utrophin may be sufficient
to ablate the dystrophic phenotype and partially or completely
restore normal muscle function.8,9
Previously, the development of a cell-based assay was reported,
which was designed to allow assessment of utrophin upregulation in a
physiologically relevant environment and was also suitable for screen-
ing thousands of compounds.10 In this assay, murine H2K cells were
engineered to express the UTRN promoter linked to a luciferase
reporter gene. Using this construct, measurement of utrophin
upregulation could be made using a simple luminescence readout.
Following an in vitro screen of a focused set of compounds using the
aforementioned assay system, a number of small molecule upregu-
lators of utrophin were identified (Figure 1). Among these were
benzoxazole11 2 as well as the structurally related benzotriazole 3.
Although both screening hits showed encouraging levels of
potency in vitro, as well as some limited activity after dosing
in vivo,8 the compounds clearly contain several structural fea-
tures that give cause for concern, most notably the presence of
Duchenne muscular dystrophy (DMD) is one of the most
common of the muscular dystrophies, affecting approximately 1 in
3500 males. The disorder is caused by a mutation in the gene DMD,
located in humans on the X chromosome (Xp21). The DMD gene
codes for the protein dystrophin, which plays an essential role in
linking the internal cytoskeleton to the extracellular matrix as part of
the dystrophin-associated protein complex (DAPC). Loss of func-
tional dystrophin causes destabilization of the DAPC, and this
results in a breakdown of muscle fibers and a collapse of membrane
integrity.1 Death from DMD is currently inevitable and typically
occurs when the sufferer is in their twenties, usually due to cardiac or
respiratory failure. Accordingly, an effective therapy is essential to
treat this debilitating and ultimately fatal disease.2
Since the discovery of the gene responsible for causing DMD
about 20 years ago,3 a number of pharmacological approaches have
been investigated for the treatment of this and other muscular
dystrophies, and these have been recently reviewed.4 In the majority
of these studies, treatment is symptomatic only, being intended to
improve the phenotype by means such as decreasing inflammation,
improving calcium homeostasis, or increasing muscle progenitor
proliferation or commitment. A number of small molecule, non-
symptomatic approaches to treat DMD have also been described.
These are based on either restoring the function of the mutated gene
or slowing/preventing degradation of the resulting protein and
muscle. The most advanced of these is PTC-124, the mode of
action of which is to allow read-through of the nonsense mutations
(premature stop codons), which are present in around 5ꢀ15% of
DMD sufferers, thereby restoring gene function and production of
dystrophin protein.5 Phase 2a clinical trial data have shown that
Received: November 25, 2010
Published: April 04, 2011
r
2011 American Chemical Society
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potential toxicophores (e.g., a primary aniline function in both 2
and 3, Figure 1) or of metabolically vulnerable groups (e.g.,
benzylic methyl and phenolic units present in 2).12 This latter
assertion was confirmed by the low systemic exposure seen when
2 was dosed orally to mice (Table 6). In this paper, we report the
synthesis and biological evaluation of a family of 2-substituted
benzoxazoles,10,13 culminating in the discovery of 1, a compound
that has been progressed to clinical trials. Further work on the
benzotriazole series will be reported elsewhere.14
16. In the latter case, Fmoc protection was employed (15) to
simplify isolation of the final product. Keto analogue 17 was
synthesized in three steps from commercially available propio-
phenone 18, and Lawesson's reagent17 was used to convert 8 to
its corresponding thioamide analogue, 19.
Alternative cores were obtained according to the routes shown
in Scheme 3. Benzimidazole 20 and benzothiazole 21 were
prepared in three and four steps, respectively, from diamine 22
and fluoride 23. A tandem Sonogashira/cyclization strategy18
was utilized to furnish benzofuran 24, a precursor to 25.
Sulfonyl derivatives were prepared as outlined in Scheme 4. A
family of 5-ethylsulfonyl- substituted benzoxazoles were accessed
using either a cyclocondensation approach between 26 and the
appropriate acyl derivative or via oxidative cyclization of an imine
intermediate (methods A, B, or C). Replacement of the ethyl group
was accomplished via demethylation of anisoles 27 (R = Me, Pr, iPr,
and NEt2), giving 28, followed by PPA-mediated cyclization with the
appropriate aryl carboxylic acid. In some cases, 28 (R = OH, NH2,
and NHMe) was condensed with acyl chlorides to afford either sul-
fonic acid or additional 5-sulfonylamido-benzoxazole derivatives, res-
pectively. Preparation of 6-ethylsulfonyl-benzoxazoles was achieved
using a three step sequence. Aminophenol 29 was formed in two
steps via the SNAr reaction between sodium ethanesulfinate and
fluoronitrophenol 30, followed by hydrogenolysis of the nitro group.
Treatment of 29 with the requisite acid chlorides in dioxane, under
microwave activation, then furnished the desired compounds.
’ CHEMISTRY
There are a number of methodologies that would allow access
to the 2-aryl benzoxazole core.15 The most generally applicable
synthetic route employs a cyclocondensation approach between
an aminophenol and either a carboxylic acid, using polypho-
sphoric acid (PPA), or an acid chloride. While thermal activation
of the reaction was often the heating method of choice, we
discovered that microwave irradiation16 was also an extremely
effective accelerant, particularly for reactions using an acid
chloride. Compounds of general structure 4 and 5 were either
purchased from commercial vendors or prepared using one of the
routes indicated in Scheme 1. 5-Aminobenzoxazoles (4, R =
5-NH2) were converted to amide derivatives, 6, under standard
reaction conditions. Alternatively, reduction of the nitro function
present in 5 was effected using either tin(II) chloride or iron
powder and subsequent amide formation delivered 6.
A range of 2-(4-chlorophenyl)benzoxazoles bearing various
substituents at the 5-position were prepared using routes shown
in Scheme 2. The N-methyl derivative 7 was prepared from 8
using sodium hydride and iodomethane. Heating 3-amino-
4-hydroxybenzoic acid (9) and p-chlorobenzoyl chloride in
1,4-dioxane, under microwave activation, afforded carboxylic
acid 10. Subsequent O-(7-azabenzotriazol-1yl)-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate (HATU)-mediated amide
formation gave reversed amide 11. Sulfonamide 12 was prepared
from aniline 13 using standard conditions. Reductive amination
of 13, using sodium triacetoxyborohydride, gave 14 in moderate
yield and 13 was also used to prepare isosteric alanine analogue
’ RESULTS AND DISCUSSION
All analogues described herein were screened using the H2K
cell-based assay with benzotriazole 313 as an internal control com-
pound. Initial efforts focused on attempts to replace the substituents
present in 2-arylbenzoxazole 2, while still retaining some level of
biological activity (Table 1). Comparable activity was noted with the
somewhat larger 5-alkyl substituent (31), which indicated that, while
more investigation was clearly required, structural changes to the
periphery of the molecule were possible. The 6-desmethyl 5-NH2
variant 32 showed a modest level of activity as compared to 2. This
latter observation suggested that structureꢀactivity crossover existed
between the benzoxazole and the benzotriazole classes (as exempli-
fied by 2 and 3, respectively).
Following these results, we initiated a more extensive hit-to-lead
evaluation by investigating structural regions A and C (Figure 2) in
parallel. Although only modestly active, 32 bore functionality more
amenable to synthetic exploration. Thus, our attention centered on
introduction of an amine functionality at the 5-position, for facile
Figure 1. Screening hits (EC50).
Scheme 1a
a Conditions: (i) Arylcarboxylic acid, PPA, 120ꢀ190 °C or ArCOCl, 1,4-dioxane, microwave, 210 °C (8ꢀ97%). (ii) R1COCl, pyridine, room
i
temperature or R1COCl, Et3N or Pr2NEt, DCM, room temperature (37ꢀ60%). (iii) Arylcarboxylic acid, PPA, 180 °C or RCOCl, 1,4-dioxane,
microwave, 210 °C (49ꢀ73%). (iv) Pd/C, H2, EtOAc, AcOH or SnCl2, industrial methylated spirit (IMS) (or EtOH), 70 °C, room temperature or Fe,
NH4Cl, IMS (or tetrahydrofuran), H2O, 70ꢀ80 °C (16ꢀ80%). (v) Acid chloride, pyridine, or iPr2NEt/Et3N, CH2Cl2, (4-dimethylaminopyridine),
room temperature or carboxylic acid, HATU, pyridine, N,N-dimethylformamide (DMF), room temperature (8ꢀ93%).
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Scheme 2a
a Conditions: (i) NaH, MeI, DMF (32%). (ii) 4-Chlorobenzoyl chloride, 1,4-dioxane, microwave, 210 °C (79%). (iii) HATU, DIPEA, DMF, iPrNH2,
room temperature (18%). (iv) Isopropylsulfonyl chloride, pyridine, CH2Cl2 (11%). (v) Isobutyraldehyde, NaBH(OAc)3, DCE, room temperature
(48%). (vi) FmocAlaOH, HATU, pyridine, DMF (16%). (vii) Piperidine, DMF (34%). (viii) Fe(NO3)3 9H2O/IMS, 80 °C (25%). (ix) Fe, NH4Cl,
3
IMS, 80 °C (60%). (x) p-ClPhCO2H, PPA, 180 °C (10%). (xi) Lawesson's reagent, PhMe, reflux (26%).
Scheme 3a
a Conditions: (i) 4-Chlorobenzoyl chloride, PPA, 150 °C (36%). (ii) SnCl2, IMS, 70 °C (product used crude). (iii) Isobutyryl chloride, pyridine, room
temperature (11% over 2 steps for 20; 44% for 21). (iv) Na2S 9H2O, aqueous sodium bicarbonate, MW (product used crude). (v) 4-Chlorobenzoyl chloride,
3
Eaton's reagent, MW (product used crude). (vi) Fe, NH4Cl, IMS, reflux (13% over 3 steps from 23). (vii) HNO3, CH2Cl2 (43%). (viii) 1-Chloro-4-
ethynylbenzene, prolinol, Pd(C), CuI, PPh3, H2O (7%). (ix) Fe, NH4Cl, IMS, H2O, reflux; isobutyryl chloride, pyridine (42% overall).
derivatization and variation of the C2-substituent, thereby allowing
for the preparation of a wider range of compounds. Alkyl and aryl
amides with four different C2-aryl substituents were initially targeted
(Table 1).
Importantly, the activity of 2-(3,4-dichlorophenyl)-substituted
aniline 33 confirmed that the phenolic and aniline functions in
region C were not essential. Furthermore, the size and nature of the
amide substituent appeared to be critical. For analogues bearing a
C2-phenyl residue, the smaller acetyl group (35) was moderately
active, with the ethyl substituent (36) conferring increased activity
and n-propyl derivative 38 exhibiting lower activity. Branching of
the alkyl chain was tolerated, with the isopropyl unit (34 and 39)
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Scheme 4a
a Conditions: Method A: R-CO2H, PPA, 125-190 °C (7ꢀ63%). Method B: RCOCl, 1,4-dioxane, microwave, 210 °C (7ꢀ85%). Method C: OHC-R,
EtOH, Δ; PhI(OAc)2, MeOH, room temperature (7%). Method D: SOCl2, CHCl3, reflux (55%) following method A. Other conditions: (i) BBr3,
CH2Cl2 or 1,2-DCE, reflux (products used crude). (ii) Aryl-CO2H, PPA, 120ꢀ190 °C (5-76%). (iii) Aryl-COCl, 1,4-dioxane, microwave, 210 °C
(36ꢀ41%). (iv) EtSO2Na, DMSO, 100 °C. (v) H2, Pd(C), IMS (46% over 2 steps from 30).
Table 1. Initial StructureꢀActivity Relationships (Varying
C5 and C2 Substituents)
Figure 2. Compound 32 and structural regions for exploration.
EC50 or %
compound
R1
R2
vs 3a(conc/μM)b
Table 2. StructureꢀActivity Relationships for “C” Region
2
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
5,6-di-Me
5-iPr
5-NH2
5-NH2
2-OH, 4-NH2
2-OH, 4-NH2
2-OH, 4-NH2
3,4-di-Cl
3,4-di-Cl
H
H
Cl
H
H
H
H
4-Cl
4-Cl
4-Cl
4-Cl
0.7
1
3.5
107% (3)
1.5
3
0.8
85% (3)
3
5-NHC(O)iPr
5-NHC(O)Me
5-NHC(O)Et
EC50 or %
vs 3a (conc/μM)b
5-NHC(O)Et
R2
4-Cl
5-NHC(O)Pr
5-NHC(O)iPr
5-NHC(O)Bu
5-NHC(O)Ph
5-NHC(O)Pr
5-NHC(O)Ph
5-NHC(O)CH2Ph
5-NHC(O)(CH2)2Ph
compound
0.8
8
34
46
47
48
49
50
51
52
53
54
55
0.4
69% (10)
>30
3.5
>30
5
3,4-di-Cl
4-Me
1.5
95% (10)
132% (10)
12% (10)
22% (10)
0.9
4-OMe
4-NEt2
4-CF3
4-F
>30
a Versus 3 (max value vs max value); no EC50 available. b Concentration
at max value.
3-Cl
4.5
appearing to be particularly favorable. Use of the larger n-butyl
substituent (40) or replacing the alkyl group with a carbocyclic aryl
group (41) gave a dramatic decrease in activity.
Introduction of the bulkier 4-chlorophenyl group at the
2-position provided a propylamide (42) with comparable activity
to the unsubstituted example 38. In this 2-(4-chlorophenyl) series,
ethyl amide 37 exhibited activity, and the trend of replacing the
amide alkyl group with a phenyl residue leading to reduced activity
was again observed (c.f. 42 and 43). Alkaryl analogues did not offer
any advantage, as exemplified by 44 and particularly 45.
Having established some preliminary structureꢀactivity relation-
ships, a more detailed investigation of region C was undertaken
(Figure 2). Screening data for a series of analogues bearing a 5-iso-
propylamido unit and substituents on the C2-phenyl ring designed
to confer a range of electronic and steric properties are summarized
2-Cl
1.1
2,3-di-Cl
2,4-di-Cl
2,5-di-Cl
10
53% (10)
>30
a Versus 3 (max value vs max value); no EC50 available. b Concentration
at max value.
in Table 2. Variation at the C2-position was found to have a signi-
ficant effect on activity. For example, the para-substituted chloro
(8), fluoro (50), methoxy (47), andmethyl(46) derivatives all gave
good levels of activity. Conversely, introduction of a basic diethy-
lamino group (48) or a strongly electron-withdrawing trifluoro-
methyl group (49) at the 4-position was less favored. The position
of the substituent was also important. para-Chloro substitution was
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favored over ortho and meta substitution (compare results for 8, 51,
and 52). Disubstitution was also investigated on the C2-phenyl ring.
The 3,4-disubstitution pattern (34) proved to be the most favorable
combination by comparison to the corresponding 2,3-dichloro, 2,
4-dichloro, and 2,5-dichloro analogues (53ꢀ55).
Table 3. Regiochemical and Other SAR
Next, we investigated the position of the amide substitution on
the core. Screening results (Table 3) indicated that transposition of
the isopropyl amide of 8 to the 6-position resulted in a notable drop
in potency (57) and the relative activities of 57, 59, and 60 indicated
that addition of a second halogen atom to the C2-phenyl group was
not advantageous. Interestingly, introduction of a second substituent
on the core of the compound (58) also did not improve activity as
compared to 8. Substitution at the 4-position was also disfavored, as
demonstrated by comparing 46 and 56.
substitution
EC50 or %
compound
site (NHC(O)iPr)
R2
4-Cl
4-Me
4-Me
4-Cl
4-Cl
2,3-di-Cl
3,4-di-Cl
R3
vs 3a (conc/μM)b
8
46
56
57
58
59
60
5
5
4
6
6
6
6
H
H
H
H
5-Cl
H
H
0.4
95% (10)
Further replacements to the 5-isopropylamido residue were
also examined by screening analogues of 8 (Table 4). N-Methylation
(7) and an isopropylsulfonamide (12) were not tolerated.
Introduction of either a thioamido (19), isobutylamino (14)
motif, or reversal of the amido function (11) similarly resulted
in loss of activity. Isosteric alanine analogue 16 also exhibited
reduced activity, whereas introduction of a 5-keto group (17)
resulted in good activity.
>30
3
>30
>30
4
a Versus 3 (max value vs max value); no EC50 available. b Concentration
at max value.
Table 4. Analogues of 8
In addition, benzimidazole, benzothiazole, and benzofuran
ring systems were investigated as replacements19 for the benzoxazole
core (region B, Figure 2). The activity observed for benzothiazole 21
was lower as compared to 8 (Table 5), and the corresponding
benzimidazole and benzofuran derivatives were considerably less
active (20 and 25). Hence, focus was maintained on benzoxazoles.
To establish its in vitro metabolic profile, lead compound 8
was incubated with both mouse and human liver microsomes (MLM
and HLM). As can be seen from these data (Table 6), while the
compound appeared to be relatively stable in HLM, it exhibited only
modest stability in MLM. Turbidometric aqueous solubility assess-
ment of 8was also undertaken (3 μM) to further profile this family of
compounds. Pleasingly, intraperitoneal dosing of 8in the mouse at 10
mg kgꢀ1 indicated its observed plasma exposure was far superior as
compared to the original hit compound 2 (Table 6), although still in
need of further optimization. Accordingly, our chemistry program
then centered on generating new leads with improved microsomal
stability and solubility while retaining high activity. Gratifyingly, sul-
fone 63 (Table 7) showed improved aqueous solubility (turbido-
metric solubility, 100 μM) and encouraging activity as compared to
its isopropylamide analogue, 39 (Table 2). These results prompted
the preparation of the 4-chloro-analogue 61, a compound exhi-
biting reasonable activity and microsomal stability (Tables 6 and 7).
The improved pharmacokinetic characteristics of 61 relative to 8
(Table 6), following dosing at 10 mg kgꢀ1 (i.p.) in the mouse,
coupled with the lack of any significant in vivo efficacy after dosing 8
EC50 or %
compound
R
vs 3a (conc/μM)b
7
8
NMeC(O)iPr
NHC(O)iPr
C(O)NHiPr
>30
0.4
11
12
14
16
17
19
>30
>30
>30
35% (3)
0.9
i
NHSO2 Pr
i
NHCH2 Pr
NHC(O)CcH(Me)NH2
C(O)Et
NHC(S)iPr
>30
a Versus 3 (max value vs max value); no EC50 available. b Concentration
at max value. c (S)-stereochemistry.
Table 5. Core SAR
compound
X
Y
EC50 or % vs 3a (conc/μM)b
8
20
21
25
O
N
N
N
CH
0.4
10
1.2
>30
NH
S
O
Table 6. In Vitro and in Vivo Profiles of Lead Compounds
compound
MLMa
HLMa
i.p. dose (mg/kg)
Tmax (min)
Cmax (ng/mL)
T1/2 (min)
AUCc
aqueous solubility (μM)d
1
2
8
61
62
81.5
22
19.3
30.6
101
105
NT
286
69
50b
10b
10
15
5
15
5
1486b
10.9
2807
5650
NT
N/Ab
40.9
136
131
NT
420
0.6
229
579
NT
3ꢀ20
1
3
10
10
105
NT
3ꢀ10
a T1/2 (min). b Study conducted by Cyprotex (address in Experimental Section). Time points (h): 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24. Male CD1 mice dosed
at 50 mg/kg using 95% PBS containing 0.1% Tween 20 and 5% DMSO as vehicle. Cmax = 1.23ꢀ1.75 μg/mL (∼4 μM), three animals; data are shown as a
mean value in ng/mL. AUC = 7 μg h/mLe. Studies were conducted by Wickham laboratories (address in Experimental Section). Male CD1 mice dosed
at 10 mg/kg using 95% PBS containing 0.1% Tween 20 and 5% DMSO as vehicle. Time points for compound 2 (min): 5, 15, 30, 60, 90, 120, 240, and
360; time points for compounds 8 and 61 (min): 5, 15, 30, 60, 90, 120, 240, and 480. The half-life was not calculated because the log concentration vs
time plot did not show a linear region of at least three points. μg.min/mL. d Turbidometric (final test compound concentration); NT, not tested.
c
Further details can be found here: http://www.cyprotex.com/cloescreen/physicochemical-properties/turbidimetric-solubility/.
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Table 7. Sulfone SAR
a Versus 3 (max value vs max value); no EC50 available. b Concentration at max value.
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at 10 mg kgꢀ1 (i.p.) in the mdx mouse8 provided additional drives to
focus our investigations on the sulfone class.
gave improved activity. Benzothiophene 78 and partially saturated
analogue 81 provided additional actives, compounds 82 and 83
were moderately active, whereas tetrahydronaphthalenes 79 and 80
were weakly active. A 2-naphthyl substituent (1) gave an additional
increase in activity, and pleasingly, both compound 1 and 62 showed
enhanced microsomal stability as compared to 8 (Table 6). Other
results suggested that 78 and 81 (HLM T1/2 = 59 and 20 min,
respectively) would be less stable than 1 in vivo.
Other types of C2 substituent were also examined. The
presence of a biphenyl unit (84), phenoxyphenyl groups (85
and 86), or a methylene spacer between the core and a naphthyl
residue (87) gave poorly active compounds. Results in Table 7
also show that stilbene 88 was moderately active, whereas the
absence of an aromatic C2 substituent appears to be detrimental
(2-propyl derivative 89 and cyclohexane 90).
Variation of the sulfonyl function present in 61 provided the
screening data shown in Table 8. Switching the ethyl group of 61
for methyl, n-propyl, or isopropyl groups lead to reduced activity
(91ꢀ93). Also, 5-isopropylsulfonyl derivative 94 displayed
marginally weaker activity than its 5-ethylsulfonyl counterpart,
62. Sulfonic acid 96 and sulfonamide analogues (95 and 97)
exhibited lower activity as compared to 61. Moving the ethyl-
sulfonyl substituent from the 5-position to the 6-position of the
core structure was also disfavored (cf. 61ꢀ62 and 98ꢀ99).
5-Methylsulfone 100 provided another example of an active
compound bearing a 2-naphthyl group (Table 8) but was not
progressed further due to its lower murine microsomal stability
(MLM T1/2 = 33.4 min) as compared to 1.
Although amido and keto analogues 17 and 50, respectively,
also exhibited encouraging activity profiles (Tables 2 and 4, vide
infra), their murine microsomal stability was significantly less as
compared to 61 (MLM T1/2 = 13 and 16.1 min, respectively);
therefore, these compounds were not progressed any further.
Lead optimization of 61 was achieved by systematic modification
of the 2-aryl and sulfonyl substituents. First, an extensive range of C2-
substituted analogues of 61 was screened (Table 7). Switching the
para-chloro substituent to the ortho and meta positions was disfavored
(64 and 65), in accordance with earlier observations on amide 8. In
addition, introduction of 2,3-dichlorophenyl, 2-fluoro-3-chlorophe-
nyl, and other monocyclic groups at the 2-position (66ꢀ70) gaverise
to low activity. Conversely, 3,4-disubstitution was favored (62 and 71
to a lesser extent), as discussed previously in the context of amido
derivatives. At this juncture, we extended this latter observation by
testing compounds bearing 3,4-bicyclic units. However, analogues
72ꢀ74, 76, and 77 exhibited weak to mode-
rate activity, whereas introduction of a 6-quinolinyl motif (75)
Table 8. Variation of Sulfone
EC50 or %
compound
R
R2
vs 3a (concn/μM)b
Auld et al. have published20,21 examples of complications
arising from luciferase-based assays due to stabilization and inhibi-
tion of luciferase. It was not believed that this was an issue for the
2-arylbenzoxazoles described herein because clear structureꢀactiv-
ity relationships were observed, for example, in the 5-alkylamido
series. In any event, we also evaluated selected compounds in a real-
time quantitative polymerase chain reaction (RT-PCR) assay, to
monitor utrophin expression, as an adjunct to the engineered H2K
cell-based assay results. Active compounds were identified by
significantly upregulated utrophin levels when compared to back-
ground [dimethylsulfoxide (DMSO) control] with 1 and 2 showing
the best results. Amides 34 and 60 and sulfone 62 provided
additional examples of active compounds in this assay (Table 9
and Figure 3). These observations provide support for the in vitro
1
61
62
91
92
93
94
95
96
97
98
99
100
5-SO2Et
5-SO2Et
5-SO2Et
5-SO2Pr
5-SO2Me
3,4-benzo fused
4-Cl
3,4-di-Cl
4-Cl
4-Cl
4-Cl
3,4-di-Cl
4-Cl
4-Cl
4-Cl
4-Cl
0.91
5
3
>30
53% (10)
10.2
45% (10)
60% (30)
>30
28% (10)
>30
10.6
i
5-SO2 Pr
i
5-SO2 Pr
5-SO2NH2
5-SO3H
5-SO2NEt2
6-SO2Et
6-SO2Et
5-SO2Me
3,4-di-Cl
3,4-benzo fused
95% (3)
a Versus 3 (max value vs max value); no EC50 available. b Concentration
at max value.
Figure 3. RT-PCR assay results. Compounds 1, 2, 60, and 62 were tested at 10 μM; compound 34 was tested at 3 μM.
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Table 9. RT-PCR Data
compound
R1
R2
EC50 or % vs 3a(conc/μM)b
% UTRNc
P value
1
2
5-SO2Et
3,4-benzofused
2-OH, 4-NH2
3,4-di-Cl
0.91
0.7
1.5
4
143.0
144.8
128.2
111.5
138.3
0.002
0.008
0.058
0.077
0.088
5,6-di-Me
34
60
62
5-NHC(O)iPr
6-NHC(O) iPr
5-SO2Et
3,4-di-Cl
3,4-di-Cl
3
a Versus 3 (max value vs max value); no EC50 available. b Concentration at max value. c Background (DMSO control) = 100.
’ EXPERIMENTAL SECTION
Assays. H2K cells13,23 were plated into 96-well plates 24 h prior to
dosing and then cultured with compound for a further 48 h before the
luciferase signal was measured. Then, the level of luciferase was deter-
mined using the Steady-Glo Luciferase Assay System (Promega), and
the plates were read using a FLOUStar plate reader and Stacker unit
(BMG Labtech). The primary screen was performed at 10 μM in
triplicate. A secondary screen was performed at six concentrations in
triplicate to generate EC50 values (Figure 5).
Figure 4. Structure of 1.
The target sequence for the real-time quantitative PCR screen was an
amplicon that spans the exon 3ꢀ4 boundary, and an amplicon in the β-actin
RNA message was used as the endogenous control. Cells were seeded in
6-well plates at 25000 cells per well in 3 mL of appropriate media and
incubated for 24 h prior to dosing. Compounds were dosed in a final
concentration of 1% DMSO for 72 h. RNA extractions were performed
using a QIAGEN RNeasy Plus kit (Qiagen 79654) and QiaShredder
(Qiagen 74134), using the manufacturer's instructions. For reverse tran-
scription, Applied Biosystems's (4368814) High-Capacity cDNA Reverse
Transcriptase Kit from was used according to manufacturer's instructions.
The quantitative PCR (qPCR) method used the ΔΔCT protocol described
by Livaks and Schmittgen.24 The 7300 Real-Time PCR System from
Applied Biosystems was used for this assay along with 7300 System SDS
software with the SDS Relative Quantification Study Plug in. 7300 System
SDS software with the SDS Relative Quantification Study Plug was used for
data analysis.
Figure 5. Typical doseꢀresponse curve. The green line represents the
test compound; the red line represents 3. Three replicates per data point.
Other in Vitro and in Vivo Studies. Turbidometric aqueous
solubility and microsomal stability assessments were conducted by Cypro-
tex Discovery Limited, 15 Beech Lane, Macclesfield, Cheshire, SK10 2DR,
United Kingdom. In vivo pharmacokinetic studies were either performed at
Cyprotex Discovery Limited (same address; compounds 2, 8, and 61) or
Wickham Laboratories Ltd., Winchester Road, Wickham, Fareham, Hamp-
shire, PO17 5 EU, United Kingdom (compound 1).
activity of this family being associated with utrophin upregulation
rather than any “off target” effects.
Compound 1 (Figure 4) was selected for further investigation
based on its overall in vitro and in vivo22 profile. Further
exploration of the 2-arylbenzoxazole class will be reported on
in due course.
Chemistry/Compound Characterization. HPLC-UV-MS was
performed on a Gilson 321 HPLC with detection performed by a Gilson
170 DAD and a Finnigan AQA mass spectrometer operating in electrospray
ionization mode. The HPLC column used is a Phenomenex Gemini C18
150 mm ꢁ 4.6 mm. Preparative HPLC was performed on a Gilson 321 with
detection performed by a Gilson 170 DAD. Fractions were collected using a
Gilson 215 fraction collector. The preparative HPLC column used was a
Phenomenex Gemini C18 150 mm ꢁ 10 mm, and the mobile phase was
acetonitrile/water.
’ SUMMARY
Following the appraisal of hits discovered from a focused
screening approach, a series of novel 2-arylbenzoxazoles have
been identified as potent upregulators of utrophin production
as assessed using an H2K cell-based assay with a luciferase
reporter readout. The most active examples have in vitro potency
below 1 μM and in vitro and in vivo ADME profiles supportive
of further investigation. Lead optimization studies have resulted
in the identification of 1 as a molecule for further progression.
The in vivo properties of compound 1 are discussed in more
detailed elsewhere.22
1H NMR spectra were recorded on a Bruker instrument operating at
300 MHz. NMR spectra were obtained as CDCl3 solutions (reported
in ppm), using chloroform as the reference standard (7.25 ppm) or
DMSO-d6 (2.50 ppm). When peak multiplicities are reported, the
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Journal of Medicinal Chemistry
ARTICLE
following abbreviations are used s (singlet), d (doublet), t (triplet), m
(multiplet), br (broadened), dd (doublet of doublets), dt (doublet of
triplets), and td (triplet of doublets). Coupling constants, when given,
are reported in Hertz (Hz).
Compounds were prepared, as assessed using the aforementioned
standard spectroscopic techniques, in g95% purity unless otherwise
stated. It is also stated herein if LC data were not available. Starting
materials were either procured from commercial sources or prepared as
described herein.
Column chromatography was performed either by flash chromatog-
raphy (40ꢀ65 μm silica gel) or using an automated purification system
(SP1 Purification System from Biotage). Microwave reactions were
performed in an Initiator 8 (Biotage) or an Explorer 48 system (CEM).
Method B. 5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole
(1). To 2-amino-4-(ethylsulfonyl)phenol (604 mg, 3.0 mmol) in dry 1,
4-dioxane (2.5 mL) was added 2-naphthoyl chloride (572 mg, 3.0 mmol) at
room temperature. The reaction vessel was heated, under microwave
activation, at 210 °C for 15 min. After it was cooled, the mixture was
slowly poured into water, and the resulting precipitate was filtered and
washed with aqueous sodium hydroxide (1 M NaOH) and then water to
give the crude product. Purification by flash chromatography, eluting with
4:1 petrol ether:ethyl acetate (EtOAc) gave 750 mg (75%) of the title
compound. LCMS RT = 6.94 min, MHþ 338.1. 1H NMR (DMSO-d6):
8.90 (1H, br), 8.34 (1H, d, J = 1.4 Hz), 8.30 (1H, dd, J = 8.6 and 1.7 Hz),
8.24ꢀ8.05 (4H, m), 7.99 (1H, dd, J = 8.5 and 1.8 Hz), 7.73ꢀ7.64 (2H, m),
3.41 (2H, q, J = 7.3 Hz), 1.15 (3H, t, J = 7.3 Hz).
layer was washed with saturated aqueous sodium carbonate (Na2CO3).
The combined organic layers were dried over anhydrous MgSO4 and
evaporated. The resulting solid was dissolved in methanol, passed through
an acidic scavenger column (silica-based quaternary amine SPE-AX, sup-
plied by Biotage), and then evaporated to afford 25 mg (41%) of the title
1
compound. LCMS RT = 5.16 min, MHþ 253.1. H NMR (DMSO-d6):
10.14 (1H, s), 8.21ꢀ8.14 (3H, m), 7.71 (1H, d, J = 8.8 Hz), 7.65ꢀ7.60
(3H, m), 7.51 (1H, dd, J = 9.0 and 2.1 Hz), 2.09 (3H, s)
Method K. Method K was the same as Method J, except triethyla-
mine was used as a base instead of diisopropylamine.
N-(2-(4-Chlorophenyl)benzo[d]oxazol-5-yl)isobutyramide (8). Yield,
46%. LCMS RT = 7.04 min, MHþ 315.1. 1H NMR (DMSO-d6): 10.03
(1H, s), 8.22ꢀ8.18 (3H, m), 7.74ꢀ7.67 (3H, m), 7.56 (1H, dd, J = 8.9 and
2.1 Hz), 2.67ꢀ2.59 (1H, m), 1.14 (6H, d, J = 6.8 Hz).
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis details and analytical
b
data for all other methods and compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ44(0)1235443932. Fax: þ44(0)1235443999. E-mail:
stephen.wren@summitplc.com.
Method A. 2-Phenylbenzo[d]oxazol-5-amine (Intermediate for
35). To PPA at 110 °C were added simultaneously 2,4-diaminophenol
dihydrochloride (7.88 g, 40.0 mmol) and benzoic acid (4.88 g, 40.0
mmol). The resulting mixture was then heated to 180 °C for 3ꢀ5 h. The
solution was then poured into water. The resulting precipitate was
collected by filtration and washed with saturated sodium bicarbonate
solution. The crude product was recrystallized from ethanol/water to
afford 8.15 g (97%) of the title compound. LCMS RT = 5.17 min, MHþ
211.1. 1H NMR (DMSO-d6): 8.15ꢀ8.12 (2H, m), 7.60ꢀ7.56 (3H, m),
7.42 (1H, d, J = 8.7 Hz), 6.89 (1H, d, J = 2.1 Hz), 6.68 (1H, dd, J = 8.6
and 2.2 Hz), 5.12 (2H, s).
All compounds below were prepared following the same general
method and purified either by trituration, recrystallization, or column
chromatography. In some cases, the crude precipitate was basified with
aqueous NaOH solution and then collected by filtration (prior to
purification). Reaction temperatures ranged from 125 to 190 °C, and
some reactions were left stirring overnight.
’ ACKNOWLEDGMENT
We thank Stuart Whibley for analytical support (LCMS and
HPLC).
’ ABBREVIATIONS USED
DAPC, dystrophin-associated protein complex; DMD, Duchenne
muscular dystrophy; DMF, N,N-dimethylformamide; DMSO, dime-
thylsulfoxide; EtOAc, ethyl acetate; HATU, O-(7-azabenzotriazol-
1yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate; HLM,
human liver microsomes; IMS, industrial methylated spirit; MLM,
mouse liver microsomes; NaOH, sodium hydroxide; Na2CO3, so-
dium carbonate; PPA, polyphosphoric acid
2-(4-Chlorophenyl)benzo[d]oxazol-5-amine (13). Yield, 95%.
LCMS RT = 6.01 min, MHþ 245.1 (93% purity). 1H NMR (CDCl3):
8.08 (2H, d, J = 9.0 Hz), 7.43 (2H, d, J = 9.0 Hz), 7.28 (1H, d, J = 9.0 Hz),
6.97 (1H, d, J = 2.2 Hz), 6.66 (1H, dd, J = 8.6 and 2.3 Hz), 3.67 (2H,
br s). Compound 13 was used to prepare 8 (see Method K).
2-(4-Chlorophenyl)-5-(ethylsulfonyl)benzo[d]oxazole (61). Yield,
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