Discovery and SAR of 2-arylbenzotriazoles and 2-arylindazoles as potential treatments for Duchenne muscular dystrophy

Article abstract of DOI:10.1016/j.bmcl.2011.06.047

Families of 2-arylbenzotriazoles and 2-arylindazoles that show positive effects in screens predictive of endogenous utrophin upregulation have been identified. Synthesis and structure-activity relationships are described leading to compounds with attractive in vitro profiles.

Full text of DOI:10.1016/j.bmcl.2011.06.047

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4828–4831
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of 2-arylbenzotriazoles and 2-arylindazoles as potential
treatments for Duchenne muscular dystrophy
Olivier De Moor, Colin R. Dorgan, Peter D. Johnson, Adam G. Lambert, Cristina Lecci, Carole Maillol,
Gary Nugent, Séverine D. Poignant, Paul D. Price, Richard J. Pye, Richard Storer, Jonathon M. Tinsley,
⇑
Richard Vickers, Renate van Well, Fraser J. Wilkes, Francis X. Wilson, Stephen P. Wren , Graham M. Wynne
Summit plc., 91 Milton Park, Abingdon, Oxfordshire OX14 4RY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Families of 2-arylbenzotriazoles and 2-arylindazoles that show positive effects in screens predictive of
endogenous utrophin upregulation have been identified. Synthesis and structure–activity relationships
are described leading to compounds with attractive in vitro profiles.
Received 8 April 2011
Revised 9 June 2011
Accepted 11 June 2011
Available online 17 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Duchenne
Muscular
Dystrophy
Benzotriazole
Indazole
Duchenne muscular dystrophy (DMD) is a common, genetic
neuromuscular disease associated with progressive deterioration
of muscle function. DMD has been characterised as an X-linked
recessive disorder that affects 1 in 3500 males caused by mutations
in the dystrophin gene. The vast majority of DMD patients lack the
dystrophin protein. Several pharmacological approaches1 have
been investigated for the treatment of DMD and other muscular
dystrophies. In the majority of these studies, treatment is only
symptomatic and a pressing need for an effective therapy still
remains. A number of genetic approaches to repair the genetic de-
fect in dystrophin are in development capable of treating small
subsets of patients. PTC-124™ (Ataluren) represented the most ad-
vanced small molecule, nonsymptomatic approach to the treat-
ment of DMD. Its mode of action allows read-through of the
nonsense mutations (premature stop codons) in about 10% of the
DMD population.2 However, it failed to achieve the primary clinical
endpoint in a recent Phase IIb trial in ambulant DMD boys. The ap-
proaches to force skipping of damaged dystrophin exons using
antisense approaches are gathering momentum with Phase III tri-
als started with GSK2402968 a single-stranded RNA-based anti-
sense oligonucleotide which corrects the reading frame in ꢀ13%
of all DMD patients. Our strategy involves the pharmacological
upregulation of utrophin transcription (utrophin3 is the autosomal
paralogue of dystrophin) as an alternative pharmacological ap-
proach for the treatment of all DMD.
We have previously reported a series of benzoxazoles as small
molecule upregulators of utrophin.4 Herein, we report the synthe-
sis and biological activity of a series of benzotriazoles and related
indazole compounds. The activity of compounds was measured
in a H2K luciferase reporter assay.5,6 Benzotriazole screening hit
17 (Fig. 1) was used as an internal positive control. We noted that
1 was highly bound to plasma proteins and rapidly turned over
when incubated with mouse liver microsomes. The presence of a
toxicophoric aniline motif and a metabolically labile methyl group
indicated that 1 was unsuitable as a starting point for an optimisa-
tion programme despite showing some limited in vivo activity.8
Consequently, we initiated a systematic evaluation of this series
in an effort to identify compounds with a more suitable profile.
These studies were designed to both delineate structure–activity
relationships and profile crossover compounds to key benzoxazole
analogues,4 (Fig. 1).
The synthetic route used to prepare analogues of 1 bearing
amide substituents is depicted in Scheme 1.9 Reaction of the
Me
N
O
N
N
NEt2
Ar
N
1
H2N
R
⇑
Corresponding author. Tel.: +44 (0) 1235443932; fax: +44 (0) 1235443999.
E-mail address: stephen.wren@summitplc.com (S.P. Wren).
Figure 1. Structures of 1 and generic benzoxazole class.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.047

O. De Moor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4828–4831
4829
R1
R1
H2N
N
H2N
R2
R1
N
R2
+
NH2
a, b
N
B
H2N
A
N
N
O
N
R2
c
R
N
H
2-6, 8 or 10
Scheme 1. Synthesis of benzotriazoles. (a) Aniline, NaNO2, aq HCl, NH4SO3NH2, diamine A; (b) CuSO4, 28% NH3 in H2O, pyridine, reflux (9–58% overall); (c) RCOCl, CH2Cl2,
i
Et3N, RT (10–75%, compounds 2–6) or Ac2O, pyridine (21%, compound 10) or PrCOCl, pyridine, DMAP (35%, compound 8).
Y
appropriate aniline with nitrous acid followed by stepwise addi-
H
N
Y
.HCl
H2N
tion of ammonium sulphamate and the required diamine (A), fol-
lowed by copper sulphate in aqueous ammonia, delivered
benzotriazole-amines, B. Subsequent coupling with acid chlorides
gave the requisite amides. However, this sequence was handi-
capped by low yields and the need for extensive purification.
The synthetic tractability of this series was enhanced by utilis-
ing an alternative reaction sequence, as exemplified by the conver-
sion of C (X = Cl) to 8 and C (X = F) to 11; Scheme 2.9,10
Cl
X
N
N
N
Cl
a
O2N
NO2
O2N
C
X = Cl, F
Y
N
N
O
N
Cl
b, c
N
H
Screening results5 (Table 1) indicated that deletion of the 6-
methyl group marginally improved activity (compare 1 and 7)
whereas replacement of the terminal 4-dimethylamino-group with
a 4-chloro residue was detrimental (7 and 9). 5-Amido-6-methyl
analogues 2–6 all exhibited less activity than aniline 1 whereas ali-
phatic amide analogues of 9 were active in some cases (8 and 10).
8
(Y=H), 11 (Y=Cl)
Scheme 2. Alternative benzotriazole syntheses. (a) NaOAc, EtOH, reflux (40% for 8)
or NaOAc, toluene, 120 °C (68% for 11); (b) Fe, NH4Cl, THF:water (3–4:1), 80 °C
(product used crude for 8; crude treated with Na2S2O4, THF:water and refluxed
overnight and used without purification (compound 11); (c) iPrCOCl, pyridine,
CH2Cl2 (4–33% over two steps).
Table 1
Early benzotriazole SAR
R1
R2
N
N
N
HN
R3
Compound
R1
R2
R3
H
EC50
1.8
/l
M or % vs 1a(concn)b
1
Me
4-NEt2
O
O
2
3
Me
Me
4-Cl
3.07
>30
N
O
4-NEt2
O
O
O
4
5
6
Me
Me
Me
4-NEt2
4-Cl
>30
>30
>30
Et
Pri
Pri
4-NEt2
7
8
H
H
4-NEt2
4-Cl
H
H
0.70
0.76
O
Pri
9
H
H
4-Cl
4-Cl
>30
O
O
10
1.80
Me
Pri
11
H
3,4-di-Cl
87% (3 lM)
a
No EC50 available (DR line shape, etc.).
Concentration at max value.
b

4830
O. De Moor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4828–4831
Table 3
Indazole series SAR
.HCl HN Ar
H2N
F
N
N
N
+
Ar
Ar
b
a
MeO2S
EtO2S
MeO2S
NO2
N
Ar
OH
N
-
R
N
N
N
N
Compound
R
Ar
EC50
/l
M or % vs 1a(concn)b
Ar
c
N
N
MeO2S
D
E
17
SO2Me
>30
Cl
O
Scheme 3. Preparation of sulfonyl-benzotriazoles. (a) NaOAc, EtOH, reflux (56–
87%); (b) Fe, NH4Cl, THF:water (5:1), 80 °C (7–26%) (c) LiHMDS, THF, À78 °C, 1h
then MeI, RT (19–54%).
Cl
18
1.73
N
H
iPr
19
20
SO2iPr
54% (10
>30
lM)
Cl
Cl
Table 2
5-Sulfonyl-benzotriazoles
SO2Et
N
a
N
Ar
No EC50 available (DR line shape, etc.).
Concentration at max value.
b
N
R
Compound
R
Ar
EC50
/
l
M or % vs 1a(concn)b
Disappointingly, methyl sulphone 12 and ethyl sulphones 13
12
SO2Me
11.67
Cl
and 14 all exhibited lower activity compared to amide 8 (Table
2). In addition, naphthyl sulphones 15 and 16 were not deemed ac-
tive enough for further profiling and so this area of work was
discontinued.
We also explored the replacement of the benzotriazole nucleus
by a bioisosteric indazole ring to determine if activity could be en-
hanced. This was accomplished by conversion of commercially
available aldehydes (F) to G or 17; Scheme 4.9,11 Standard manip-
ulations were then used to prepare test compounds.
The assay data shown in Table 3 indicate that direct analogues
of 12 and 14 (17 and 20) were inactive. Isopropyl sulphone 19,
formed as a by-product in the synthesis of 20, exhibited only mod-
erate activity.
Cl
13
SO2Et
52% (10
>30
lM)
Cl
Cl
14
15
SO2Et
SO2Me
63% (3
73% (3
l
M)
16
SO2Et
l
M)
a
No EC50 available (DR line shape, etc.).
Concentration at max value.
b
However, isopropylamide 18 (the crossover to 8), exhibited
good levels of activity and results from further in vitro evaluation
of both these compounds are shown in Table 4. Incubation of 8 and
18 in human liver microsomes indicated encouraging in vitro sta-
bility with half-lives of 4.85 and 3.3 h, respectively. The series also
Isopropylamide 8, bearing a 4-chlorophenyl unit at the 2-position,
was favoured and a reasonable level of activity was retained after
incorporation of a second chloro group at the meta-position of the
2-aryl ring (11).
Next, we investigated whether the understanding of SAR derived
from previous work on related benzoxazoles4 would help to further
increase the activity of 8. Consequently, sulphone analogues were
prepared as outlined in Scheme 3. Formation of N-oxides was ob-
served after reaction of 1-fluoro-4-(methylsulfonyl)-2-nitroben-
zene and the requisite aryl hydrazines. Subsequent iron-mediated
reduction provided 5-methylsulfonyl derivatives (D) and standard
alkylation conditions were used, if required, to afford ethylsul-
phones, E.9
exhibited low-moderate turbidometric aqueous solubility (1
lM,
3–10 M). Complications arising from luciferase-based assays
l
due to stabilisation and inhibition of luciferase are well docu-
mented.12 The clear structure–activity relationships described
herein suggest that this was not a concern for either chemotype.
In addition, significant upregulation of utrophin levels was ob-
served compared to background (DMSO controls) when 18 was
evaluated in a real time quantitative PCR assay.5 This result pro-
vides comfort that in vitro activity is not attributed to any ‘off
target’ effects.
CHO
NHAr
H2N
Cl
+
a
b
R
NO2
R
R
NO2
F
Y
N
N
Cl
Cl
N
Cl
c
N
S
O
O
(R = SO2Me; 17)
Y = H (20) + Y = Me (19)
O
N
N
N
Cl
d, e
N
R
N
H
(R = NO2; G)
18
Scheme 4. Synthesis of indazoles. (a) ArNH2, EtOH, RT then 70 °C (used crude in next step); (b) P(OEt)3, 105 °C, 3 h (74%); (c) LiHMDS, THF, À78 °C, then MeI, RT (8% 19, 27%
i
20); (d) Fe, NH4Cl, THF:water (5:1), 80 °C (92%); (e) PrCOCl, pyridine, RT (10%).

O. De Moor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4828–4831
4831
Table 4
5. Assay details: H2K cells were plated (96 well) 24 h prior to dosing then cultured
with compound for a further 48 h before the luciferase signal was measured.
The level of luciferase was determined using the Steady-Glo™ Luciferase Assay
System (Promega) and the plates were read using a FLOUStar plate reader and
Selected in vitro data for compounds 8 and 18
In vitro parameter
8
18
Stacker unit (BMG Labtech). The primary screen was performed at 10 lM in
triplicate. A secondary screen was performed at six concentrations in triplicate
to generate EC50 values.
EC50
% UTRN
Aq Sol (
HLM Clint (ml/mg/ml)
HLM T1/2 (mins)
(
l
M)
0.76
–
1
4.77
291
1.73
137.6b
3–10
7.02
l
M)a
The target sequence for the real time quantitative PCR screen was an amplicon
that spans the exon 3–4 boundary, and an amplicon in the beta actin RNA
message was used as the endogenous control. Cells were seeded in 6 well
plates at 25,000 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 RNeasyÒ Plus
kit (Qiagen 79654) and QiaShredder (Qiagen 74134), using the manufacturer’s
instructions. For reverse transcription, 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
DDCT protocol described by Livaks and Schmittgen Livak, Methods, 2001, 25,
402). 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.
a
a
198
a
Turbidometric aqueous solubility and microsomal stability assessments were
conducted by Cyprotex Discovery Limited, 15 Beech Lane, Macclesfield, Cheshire,
SK10 2DR, United Kingdom. See http://www.cyprotex.com/cloescreen/physico-
chemical-properties for further details.
b
Background (DMSO control) = 100; standard deviation = 28; P value = 0.021.
In summary, families of 2-arylbenzotriazoles and related 2-aryl-
indazoles that show positive effects in screens predictive of endog-
enous utrophin upregulation have been discovered. Compounds 8
and 18 showed promising in vitro ADMET properties and were pro-
gressed for in vivo evaluation.
6. Wynne, G.M.; Wren, S.P.; Johnson, P.D.; Price, P.D.; De Moor, O.; Nugent, G.;
Tinsley, J.M.; Storer, R.; Mulvaney, A.; Pye, R.J.; Dorgan, C.R. WO 2007/091106.
7. Compound 1 was purchased from Chembridge (San Diego, California, USA). 95%
purity was determined by LCMS (RT = 7.55 min, MH+ 296.2;) and 1H NMR (D6-
DMSO): 7.89 (2H, d, J 8.6 Hz), 7.56 (1H, s), 6.73 (3H, m), 5.21 (2H, br), 3.34 (4H,
q, J 7.1 Hz), 2.19 (3H, s), 1.06 (6H, t, J 7.1 Hz). HPLC-UV-MS was performed on a
Gilson 321 HPLC with detection performed by a Gilson 170 DAD and a Finnigan
Acknowledgement
AQA mass spectrometer operating in electrospray ionisation mode.
A
Phenomenex Gemini C18 150 Â 4.6 mm column was used. 1H NMR spectra
were recorded on a Bruker instrument operating at 300 MHz.
8. For a description of mdx mouse model see: Tinsley, J.; Deconinck, N.; Fisher, R.;
Kahn, D.; Phelps, S.; Gillis, J.-M.; Davies, K. Nature Med. 1998, 4, 1441.
9. Wynne, G.M.; Wren, S.P.; Johnson, P.D.; Price, P.D.; De Moor, O.; Nugent, G.;
Dorgan, C.R.; Tinsley, J.M.; Storer, R.; Mulvaney, A.; Pye, R.J. WO 2007/091107;
Wynne, G.M.; Wren, S.P.; Johnson, P.D.; Poignant, S.; De Moor, O.; Lecci, C.;
Storer, R., van Well, R. WO 2009/021747.
10. Rogers, J.; Maike, F.; Dan, N. WO 2005/054212.
11. Armour, M. A.; Cadogan, J. I. G.; Grace, D. S. B. J. Chem. Soc., Perkin Trans. 2 1975,
1185.
12. Auld, D. S.; Lovell, S.; Thorne, N.; Lea, W. A.; Maloney, D. J.; Shen, M.; Rai, G.;
Battaile, K. P.; Thomas, C. J.; Simeonov, A.; Hanzlik, R. P.; Inglese, J. J. Med. Chem.
2008, 51, 2372; Auld, D. S.; Lovell, S.; Thorne, N.; Lea, W. A.; Maloney, D. J.;
Shen, M.; Rai, G.; Battaile, K. P.; Thomas, C. J.; Simeonov, A.; Hanzlik, R. P.;
Inglese, J. Proc. Natl. Acad. Sci. 2010, 107, 4878.
We thank Mr. Stuart Whibley for analytical support.
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