Synthesis and evaluation of compounds that induce readthrough of premature termination codons

Article abstract of DOI:10.1016/j.bmcl.2011.07.107

A structure-activity relationship (SAR) study was carried out to identify novel, small molecular weight compounds which induce readthrough of premature termination codons. In particular, analogs of RTC13, 1, were evaluated. In addition, hypothesizing that these compounds exhibit their activity by binding to the ribosome, we prepared the hybrid analogs 13 containing pyrimidine bases and these also showed good readthrough activity.

Full text of DOI:10.1016/j.bmcl.2011.07.107

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5842–5848
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of compounds that induce readthrough
of premature termination codons
Michael E. Jung a, , Jin-Mo Ku a, Liutao Du b, Hailiang Hu b, Richard A. Gatti b,c
⇑
a Department of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, CA 90095, United States
b Department of Pathology and Laboratory Medicine, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, CA 90095, United States
c Department of Human Genetics, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, CA 90095, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A structure–activity relationship (SAR) study was carried out to identify novel, small molecular weight
compounds which induce readthrough of premature termination codons. In particular, analogs of
RTC13, 1, were evaluated. In addition, hypothesizing that these compounds exhibit their activity by bind-
ing to the ribosome, we prepared the hybrid analogs 13 containing pyrimidine bases and these also
showed good readthrough activity.
Received 26 October 2010
Revised 26 July 2011
Accepted 27 July 2011
Available online 4 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Premature termination codons
Organic synthesis
Readthrough compounds
Thiazolidinones
A nonsense mutation is a point mutation in a sequence of DNA
that results in a premature termination codon (PTC) or a nonsense
codon in the transcribed mRNA.1 These cause the formation of
either no protein or of a truncated, unstable, nonfunctional protein.
Approximately 30% of common genetic diseases result from non-
sense mutations,2 such as in Ataxia-telangiectasia (A-T), Duchenne
muscular dystrophy (DMD), cystic fibrosis (CF), and spinal muscu-
lar dystrophy (SMA).3 A-T is a rare autosomal recessive neurode-
generative disease that affects primarily the cerebellum and
causes truncal ataxia.4 A-T patients also suffer from an increased
risk of cancer, immunodeficiency and hypersensitivity to ionizing
radiation and some chemotherapeutics, such as alkylating agents.
Mutations in the ATM (Ataxia-Telangiectasia Mutated) gene cause
A-T. ATM is a 370 kD ‘high molecular weight’ protein kinase that
is found in all cells, primarily in the nucleus. It phosphorylates over
700 downstream protein targets, which are involved primarily in
the recognition and repair of DNA double strand breaks but also
in responding to oxidative stress, cell cycle checkpoints, and non-
sense mediated decay. Our previous efforts3a,5 have been directed
towards the development of drugs for A-T with either antisense
morpholino oligonucleotides (AMO) or aminoglycoside read-
through compounds (RTCs). Delivery has been a major obstacle
in bringing AMOs to therapeutic fruition. RTCs like gentamycin,
an aminoglycoside,6 have undesirable off-target effects, such as
nephrotoxicity and hearing loss.7 Furthermore, because the cere-
bellum is the major site of pathology in A-T8 and aminoglycosides
are too large to cross the blood–brain barrier, we sought to develop
more specific and potent small molecules. We report herein a
structure–activity relationship study, which has identified novel
small molecular weight nonaminoglycoside readthrough
compounds.
For the purpose of finding novel readthrough compounds, we
recently9 reported a high-throughput screen (HTS) of 34,000 com-
pounds using a PTT-ELISA assay that resulted in the identification
of 12 low molecular weight compounds with PTC readthrough
activity. From these, two potent lead compounds, RTC13 1 and
RTC14 2, (Fig. 1) consistently induced functional ATM protein in
ATM-deficient cells containing disease-causing nonsense muta-
tions, as demonstrated by direct measurement of ATM protein, re-
stored ATM kinase activity, and colony survival assays for cellular
radiosensitivity. We exposed A-T lymphoblastoid cell lines (LCLs)
to each RTC compound for 4 days prior to harvesting the cells.9
Although 2 showed a slightly higher in vitro readthrough signal
than 1, 1 was less cytotoxic to LCLs.9 Therefore we concentrated
our SAR efforts on analogs of 1. Herein, we report the synthesis
and evaluation of several 2-imino and 2-thioxothiazolidin-4-one
derivatives with regard to their ability to translate through the pre-
mature termination codons directly caused by nonsense
mutations.
We examined four structural changes in 1 (Fig. 2), namely:
change in the heteroatom of the 2-carbonyl unit of the thiazoli-
din-4-one (A); variation of the aryl group on the furan ring (B);
introduction of an alkyl group on the ring nitrogen of the thiazoli-
⇑
Corresponding author.
E-mail address: jung@chem.ucla.edu (M.E. Jung).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.107

M. E. Jung et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5842–5848
5843
tBu
H
O
O
NO2
NO2
S
NH
OH
NH
1 RTC13
2 RTC14
Figure 1. Structures of HTS hits RTC13 1 and RTC14 2.
H
H
O
O
O
O
R
NO2
S
NH
S
NH
X
NH
A
B
H
H
O
X
O
N
O
R
NO2
S
S
NH
R'
NH
NH
C
D
Figure 2. Four structural variations of lead compound RTC13 1.
Table 1
The evaluation of readthrough activity of compounds 7
H
H
O
O
HetAr
O
R
S
N
R'
S
N
R'
X
X
7
9
Entry
Compd
X
R
R0
FC-ATM (Delta-FI, 30 lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
RTC13
7a
7b
7c
7d
7e
NH
NH
S
2-NO2
H
H
H
H
3.27
Negative
Negative
Negative
Negative
Negative
Negative
Negative
1.90
2-NO2
2-NO2
2-CF3
2-OMe
2-Cl
2-F
3-MeO
3-Cl
NH
NH
NH
NH
NH
NH
NH
NH
S
S
S
S
S
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
2.27
3-F
Negative
Negative
Negative
3.55
2.86
2.66
2-CF3
2-MeO
2-Cl
2-F
3-OMe
3-Cl
S
S
2.86
3.44
3-F

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M. E. Jung et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5842–5848
Figure 3. Measurement of readthrough activity of compounds 7 using FC-ATMs1981 in A-T cells. After 4 days of compound treatment, A-T cells were irradiated for 10 Gy and
collected for assaying. Wild type cells (WT) were used as a positive assay control. The data from 3 sets of independent of experiments are summarized here. The increased
Delta-FI (difference of fluorescent intensity before and after IR) indicated the restoration of ATM autophosphorylation at Serine 1981. All RTC-treated cell samples showed
significant differences as compared to untreated cells (0), except 7h (⁄p <0.05, by t-tests comparing paired concentrations of treated to untreated cells).
Table 2
The evaluation of readthrough activity of compound 9
Entry
Compd
HetAr
R0
FC-ATM (Delta-FI, 30 lM)
1
2
3
4
5
6
7
8
9
9a
9b
9c
9d
9e
9f
9g
9h
9i
2-Benzofuranyl
2-Indolyl
3-Indolyl
5-(Pyridin-2-yl)-furan-2-yl
5-Phenylthiophen-2-yl
Furan-2-yl
2-Benzofuranyl
2-Indolyl
NH
NH
NH
NH
NH
NH
S
S
S
S
Negative
Negative
Negative
Negative
Negative
Negative
3.50
Negative
Negative
Negative
3-Indolyl
5-(Pyridin-2-yl)-furan-2-yl
10
9j
Pd(PPh3)4
K2CO3
O
Br
CHO
+
O
CHO
tol/EtOH
120 °C
70-90%
B(OH)2
R
R
3
4
5
O
NaOAc
H
AcOH
130 °C
50-95%
O
+
S
N
O
R'
R
X
S
N
6a X = NH R = H
R'
6b X = S R = H
6c X = NH R = Me
X
7
6c
H
O
N
O
1) NH4OH
85%
2) ClCH2CO2H
Δ/75%
HetAr
HetAr-CHO
8
S
S
N
R'
NaOAc
R'
AcOH
Me-N=C=S
X
X
130 °C
6a X = NH R = H
50-95%
9
6b X = S R = H
6c X = NH R = Me
Scheme 1. Synthesis of compounds 7 and 9.

M. E. Jung et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5842–5848
5845
the synthesis of the heteroarylmethylene analogs 9. Most of the
heteroaryl aldehydes 8 were commercially available while those
that were not, for example, the benzofuran-2-, indole-2-, 5-phenyl-
thiophene-2-, and 5-(2-pyridyl)-furan-2-carboxaldehydes, were
prepared by straightforward routes (see Supplementary data).
These two series, 7 and 9, were tested for readthrough activity
using an assay in which the ATM protein kinase activity was mea-
sured using a flow cytometry-based ATMs1981 autophosphoryla-
tion assay (FC-ATM).11 ATM kinase activity is demonstrated by
the change in the fluorescence intensity before and after ionizing
radiation (Delta-FI) (Fig. 3). An increased Delta-FI indicates the res-
toration of ATM kinase activity by a compound. Table 1 shows the
data for the analogs 7 in which the groups X, R, and R0 were varied.
Several analogs showed reasonably good readthrough activity,
with the 2-chlorophenyl and the 3-fluorophenyl 2-thioxo analogs
7m and q being the best of this group. Among the heteroaryl ana-
logs 9, only the 2-benzofuranyl 2-thioxo analog, 9g, gave positive
results (Table 2).
O
O
N
NH
S
NH
X
Y
R
E
F R = H, F, Me
Y = O, NH
Figure 4. Change of thiazolidinone unit to pyrimidinone.
din-4-one (C); and introduction of different aryl groups as the cen-
tral ring unit (D).
The syntheses of the compounds listed in Tables 1 and 2 are
shown in Scheme 1.10 Coupling of the arylboronic acids 3 with
commercially available 5-bromofurfural 4 afforded the 5-arylfurf-
urals 5 in good yields. Condensation of these compounds with
any of the thiazolidinones 6a–c furnished the desired products 7
in 50–95% yield. The two thiazolidinones 6a and b were commer-
cially available and the N-methyl analog 6c was prepared in two
steps from methyl isothiocyanate as shown. Also substitution of
any of several heteroaryl aldehydes 8 for the 5-arylfurfural allowed
We hypothesized that the mechanism of action of these novel
tricyclic aromatic compounds might be similar to that of the ami-
noglycosides, which have been reported to exert their readthrough
effects through an interaction with the ribosome.12 In particular
we wondered whether the 2-imino and 2-thioxothiazolidin-4-
one units E might be binding to nucleobases in the ribosome via
1) NaBH4
O
O
MeOH
CHO
CH2X
90-95%
2) PBr3
R
+
R
5
10 X = OH
11 X = Br
O
O
HN
K2CO3
DMF
O
NH
N
NH
45 °C
24 h
NO2
O
O
57-88%
R'
R'
12a R' = H
13a R' = H
12b R' = F
13b R' = F
12c R' = Me
13c R' = Me
13d R' = CHO
12d R' = CHO
O
O
O
HN
N
NH
NH
NH
R
S
O
N
O
O
H
R'
13e R = H R' = H
13f R = H R' = Me
13k
13g R = 2-F R' = H
13h R = 2-F R' = Me
13i R = 3-Cl R' = H
6a
NaOAc
AcOH
O
13d
O
N
NH
NH
NO2
S
O
O
N
H
13j
Scheme 2. Synthesis of compounds 13.

5846
M. E. Jung et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5842–5848
O
11
HN
O
Cs2CO3
DMF
O
N
N
25 °C
12 h
NO2
R'
NH2
NH2
R'
14a R' = H
14b R' = F
15a R' = H
15b R' = F
67%
69%
Scheme 3. Synthesis of compounds 15.
Table 3
The evaluation of readthrough activity of compounds 13 and 15
O
O
O
O
HN
N
NH
NH
NH
NH
R" =
R
S
S
O
O
N
H
X
NH
R'
13j
13/15
13k
Entry
Compd
R
R0
X
FC-ATM (Delta-FI, 30
l
M)
IRIF-ATM (%) (30 lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
13a
13b
13c
13d
13e
13f
13g
13h
13i
2-NO2
2-NO2
2-NO2
2-NO2
H
H
2-F
2-F
3-Cl
2-NO2
H
F
O
O
O
O
O
O
O
O
O
O
4.94
2.08
Negative
2.24
3.42
3.80
5.43
6.16
5.07
2.42
5.32
Negative
Negative
10.67
14.5
Negative
16.0
9.4
22.75
20
9.6
11.33
18
17
Negative
Negative
Me
CHO
H
Me
H
Me
H
R00
13j
13k
15a
15b
2-NO2
2-NO2
H
F
NH
NH
Figure 5. Measurement of readthrough activity of compounds 13 using FC-ATMs1981 in A-T cells. After 4 days of treatment with a compound, A-T cells were irradiated for
10 Gy and collected for assaying. Wild type cells (WT) were used as a positive assay control. The data from 3 sets of independent experiments are summarized here. The
increased Delta-FI (difference of fluorescent intensity before and after IR) indicated the restoration of ATM autophosphorylation of at Serine 1981. All RTC-treated cell samples
showed significant difference as compared to untreated cells (0), except 13b (⁄p <0.05, by t-tests comparing paired concentrations of treated to untreated cells).

M. E. Jung et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5842–5848
5847
Figure 6. Measurement of readthrough activity of compounds 13a using an ATMs1981 nuclear foci formation assay (IRIF-ATM) in A-T cells. After 4 days of treatment with a
compound, A-T cells were irradiated for 10 Gy and collected for assaying. Wild type cells (WT) were used as a positive assay control. The data from >3 sets of independent of
experiments are summarized here. All RTC-treated cell samples showed significant differences as compared to untreated cells (0), except 13g (⁄p <0.05, by t-tests comparing
paired concentrations of treated to untreated cells).
hydrogen bonding (Fig. 4). Consequently, we decided to substitute
these thiazolidinone units with various pyrimidine bases, F, for
example, uracil, 5-fluoro- and 5-methyluracil (thymine), and cyto-
sine units. The synthesis of these novel analogs was relatively easy
and involved hydride reduction of the 5-arylfurfurals 5 to give the
alcohols 10 in good yields (Scheme 2). Conversion of the alcohols
into the bromides 11 with PBr3 followed by reaction of the crude
bromides 11 with an excess (10 eq) of the desired pyrimidine bases
12a–d gave good yields (57–88%) of the 1-pyrimidinylmethyl fur-
ans 13a–d. An analogous series of reactions, beginning with other
aldehydes 5, permitted the formation of the pyrimidines 13e–i.
Condensation of the 2-imino thiazolidin-4-one 6a with the alde-
hyde 13d gave the analog 13j having both a pyrimidinone and
the thiazolidinone imine. Finally, condensation of the pyrimidine-
carboxaldehyde with 6a gave the novel bicyclic analog 13k having
just a pyrimidinone coupled to a thiazolidinone. The cytosine ana-
logs were prepared by reaction of cytosine and 5-fluorocytosine
14a and b with the nitro aldehyde 11 (R = 2-NO2) in the presence
of cesium carbonate in DMF to give 15a and b in 67% and 69% yield,
respectively (Scheme 3).
Moreover, we have designed and prepared novel compounds in
which a pyrimidinedione unit replaces the thiazolidinone ring 13
and these also show excellent readthrough activity. Further work
in this area is underway and will be reported in due course.
Acknowledgments
This work was partially supported by grants from the National
Institutes of Health (NS052528), the A-T Medical Research Founda-
tion, the AT Ease Foundation, and the Scott Richards Charitable
Trust. We thank Francesca Fike for technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.107.
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