Synthesis, Identification, and Structure-Activity Relationship Analysis of GATA4 and NKX2-5 Protein-Protein Interaction Modulators

Article abstract of DOI:10.1021/acs.jmedchem.9b01086

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.

Full text of DOI:10.1021/acs.jmedchem.9b01086

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Article
Synthesis, identification and structure-activity relationship analysis
of GATA4 and NKX2-5 protein-protein interaction modulators
Mikael Jumppanen, Sini M. Kinnunen, Mika J. Välimäki, Virpi Talman, Samuli Auno, Tanja Bruun, Gustav
Boije af Gennäs, Henri Guillaume Xhaard, Ingo B. Aumüller, Heikki J Ruskoaho, and Jari Yli-Kauhaluoma
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01086 • Publication Date (Web): 20 Aug 2019
Downloaded from pubs.acs.org on August 21, 2019
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Synthesis, identification and structure-activity
relationship analysis of GATA4 and NKX2-5
protein-protein interaction modulators
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Mikael Jumppanen , Sini M. Kinnunen , Mika J. Välimäki , Virpi Talman , Samuli Auno ,
Tanja Bruun1,2 Gustav Boije af Gennäs , Henri Xhaard , Ingo B. Aumüller , Heikki
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Ruskoaho , Jari Yli-Kauhaluoma
1Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty
of Pharmacy, Viikinkaari 5 E (P.O. Box 56), FI-00014 University of Helsinki, Finland
2Drug Research Program, Division of Pharmacology and Pharmacotherapy, Faculty of
Pharmacy, Viikinkaari 5 E (P.O. Box 56), FI-00014 University of Helsinki, Finland
3National Heart and Lung Institute, Imperial College London, Imperial Centre for
Translational and Experimental Medicine, Du Cane Road, London, W12 0NN, UK
KEYWORDS: GATA4, NKX2-5, isoxazole, drug discovery, structure-activity analysis
ABSTRACT: Transcription factors GATA4 and NKX2-5 directly interact and
synergistically activate several cardiac genes and stretch-induced cardiomyocyte
hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound,
which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space
around the molecular structure of 1 was explored by synthesizing and characterizing 220
derivatives and structurally related compounds. In addition to the synergistic
transcriptional activation, selected compounds were evaluated for their effects on
transcriptional activities of GATA4 and NKX2-5 individually as well as potential
cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic
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isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5
transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated
with the reduced cell viability. In summary, comprehensive SAR analysis accompanied
with data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-
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transcriptional synergy and revealed structural features important for it.
INTRODUCTION
Protein-protein interactions (PPIs) are critical regulatory events in physiology and
diseases, and they represent an attractive target for pharmacological interventions.1
However, targeting PPIs with small molecules is challenging owing to the large surface
area involved in protein-protein binding and the lack of clear binding pockets for small
molecules at many protein-protein interfaces. Nevertheless, the number of successful
examples of small molecule modulators of PPIs have been growing,1,2 and Lawson et al.
recently concluded that small molecules provide significant opportunities for allosteric
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modulation of PPIs. Transcription factors are the fundamental regulators of gene
expression and their PPIs are of pivotal importance in the regulation of biological
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systems. The GATA family of transcription factors consists of six proteins (GATA1-6)
which are involved in a variety of physiological and pathological processes.5–7 GATA1-3
are required for differentiation of mesoderm and ectoderm-derived tissues, whereas
GATA4-6 are implicated in development and differentiation of endoderm- and mesoderm-
derived tissues such as induction of differentiation of embryonic stem cells and
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cardiovascular embryogenesis. In the developing heart, GATA4 is one of the earliest-
8
9–12
expressed transcription factors and is essential for normal cardiac development.
In
the postnatal heart, GATA4 acts as a critical regulator of hormone response and
mechanical stress13–16 as well as cardiac repair and regeneration.17–20
GATA4 actions involve combinatorial interactions with a number of other nuclear proteins,
reinforcing their activity and tissue specificity.5–7 For example, functional cardiomyocytes
can be directly induced from fibroblasts by a combination of three cardiac transcription
factors, GATA4, MEF2C and TBX5, in vitro and in vivo.21,22 Most of the PPIs occur through
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the C-terminal zinc finger which also mediates DNA binding and is highly conserved
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throughout the GATA family. GATA4 regulates myocardial gene expression by
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interacting with different cardiac specific transcription factors, such as NKX2-5, NFAT and
MEF2.5–7 NKX2-5, a member of the evolutionary conserved NK family of homeobox
proteins, is a critical GATA4 cofactor and essential for heart development.23–25 GATA4
and NKX2-5 directly interact and synergistically activate several genes including those
encoding atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP).7,26
Mutational studies have shown that the tandem GATA consensus sites of the proximal
promoter in combination with an NKX2-5 binding element are important for the stretch-
activated BNP transcription.27 As the precise balance of GATA4-NKX2-5 interaction is
essential for mechanical stretch-induced cardiomyocyte hypertrophy, the functional
modulation of their interaction could present a novel approach for cardiac repair under
pathophysiological conditions.
We have previously shown that single point mutations can interfere with GATA4 and
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NKX2-5 interaction, implicating that this PPI is targetable by small molecules to achieve
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direct inhibition, activation or allosteric modulation. Recently, we reported the
identification of several compounds that affect the transcriptional synergy of GATA4 and
NKX2-5.29,30 In addition, we have shown that a small molecule inhibitor of GATA4-NKX2-5
transcriptional synergy reduces cardiomyocyte hypertrophic response in vitro,
ameliorates hypertrophic signaling in vivo, and improves cardiac function in vivo in
experimental models of myocardial infarction and hypertension.29,31,32 These studies led
to the identification of compounds 1-3 (Figure 1) which either increase (2) or inhibit (1 or
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3) transcriptional synergy of GATA4 and NKX2-5. Our aim in this study was to optimize
the original isoxazole hit compound 1 by means of synthesis and biological investigation
of 220 structurally related compounds with modified or alternative northern, central and
southern parts (Figure 2). Additionally, this structure-activity relationship (SAR) analysis
was augmented by 29 commercial and 8 previously published compounds33 that were
tested with a luciferase reporter assay to examine their effects on transcriptional synergy
of GATA4 and NKX2-5. The most potent compounds were also tested independently in
luciferase reporter assays for NKX2-5 and GATA4 activity. Furthermore, toxicity of the
selected
compounds
was
studied
with
3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays in the COS-1 cell
line.
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N
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Figure 1. Representative examples of the previously reported modulators of GATA4-
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NKX2-5 transcriptional synergy. Compounds 1 and 3 inhibit and compound 2 enhances
transcriptional synergy of GATA4 and NKX2-5.
RESULTS
CHEMISTRY
Synthesis of compounds
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To gain information on the chemical space of the hit compound 1 in regard to the ability
of such molecules to inhibit GATA4-NKX2-5 transcriptional synergy, we synthesized 220
structurally related compounds. For a simplified view, we divided the chemical structure
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into three sections, i.e. northern, central and southern part (Figure 2). The original
central amide linker in 1 was replaced with other linkers, such as alkyl, amino, inverse
amide, imino, ether and thio ether linkers. The alternative southern parts comprise
structural changes of the isoxazole substituents (Ph or Me), scaffold hopping as well as
exploration of other heterocycles as surrogates of the original isoxazole ring. In the
northern part, we mainly experimented with substituted phenyl moieties in the para
position. A few compounds comprised of heterocycles in the northern part and some
compounds devoid of a central or northern part were prepared.
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Synthesis of structurally
related compounds
Northern parts (Table 4)
Ethers (Table 3)
N
to improve efficacy
to reduce toxicity
Northern part
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Central part
Various central parts (Table 2)
NH
Southern part
N
O
Various isoxazole substituents (Table 5)
Scaffold hopping (Table 1)
Figure 2. Synthesis strategy for structural modifications of compound 1.
Northern part. The synthesis of selected derivatives with alternative northern parts is
shown in Scheme 1. The amides 4a-k were obtained from various amines 5a-k and 5-
methyl-3-phenylisoxazole-4-carboxylic acid 6 in the presence of N,N,N′,N′-tetramethyl-O-
(
1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) or in some cases O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) as a
coupling agent and Hünig’s base (DIPEA). The quaternary ammonium salt 7 was
obtained by alkylating compound 4i with iodomethane in methanol under microwave
irradiation. The nitro derivative 8 was obtained from compound 5i with NOBF in
4
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acetonitrile. The reductive treatment of compound 8 with zinc in an equimixture of acetic
acid and methanol produced the corresponding amine 9. The esters 10a-d were then
prepared from the methyl benzoate 11 by a base-assisted ester hydrolysis followed by
alkylation of the resulting benzoic acid derivative 12 with alkyl halides in the presence of
K CO in N,N-dimethylformamide (DMF). Compound 11 was prepared via acid chloride
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of 6, generated with oxalyl chloride in the presence of catalytic amount of DMF. 4-(tert-
Butyl)thiazol-2-amine 5k was synthesized by letting thiourea and 1-bromopinacolone 14
34
to react in ethanol under microwave irradiation at 120 C. Compound 15 was prepared
from the corresponding ethynyl(trimethylsilyl) derivative 4j in the presence of tert-butyl
bromide and n-tetrabutylammonium fluoride (TBAF) in THF.
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Scheme 1. Synthesis of the compounds with different northern parts.a
R1 R1=
R1=
R1=
N
O
4a, 5a
4d, 5d
4g, 5g
O
O
OH
O
O
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R1=
R1=
R1=
R1
N
O
N
R1
i
N
H2N
N
H
O
4b, 5b
4e, 5e
4h, 5h
5a-j
4a-j
O
S
ii
R1=
R1=
H2N
N
Br
N
Cl
4c, 5c
14
5k
4f, 5f
i
NH2
R1=
N
N
iii
R1=
iv
R1=
N
N
O
N
O
4i, 5i
v
8
9
NO2
S
N
HN
vi
R1=
R1=
R1=
CN
4k
I
SiMe3
4
j, 5j
7
15
R2
O
OMe
CN
R2=
R2=
CN
O
O
O
O
vii
viii
OMe
ix
1
0a
10c
HN
N
O
OH
R2
N
NH2
13
O
R2=
R2=
11
12
10a-d
1
0b
10d
aReagents and conditions: (i) 5-methyl-3-phenylisoxazole-4-carboxylic acid (6), HBTU,
DIPEA, DMF, rt, 1-2 d; (ii) EtOH, mw (100 °C, 30 min); (iii) NOBF , MeCN, 0 C, 2 h; (iv)
4
Zn, AcOH/MeOH (1:1), rt, overnight; (v) MeI, MeOH, mw (100 °C, 8 h); (vi) t-BuBr, TBAF,
THF; (vii) 6, (COCl) , DMAP, py, rt, 3 d; (viii) LiOH∙H O, THF/MeOH/H O (1+1+1), rt, 2 d;
2
2
2
2
(
ix) 10a-c: R Br, K CO , DMF, rt, 1-4 d; 10d: HO(CH ) N(CH ) , HBTU, DIPEA, DMF, rt,
2
3
2 2
3 2
3 d.
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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
Central part. The investigation of the central part was important to evaluate the role of
changes in hydrogen bond accepting or donating properties of the cental linker, the
importance of its electrostatic effects and aspects of flexibility in this molecular region.
The central part of the lead structure 1 (an amide linker) has both hydrogen bond donating
and accepting properties. It can be considered as relatively polar and due to the
delocalized lone pair of nitrogen electrons as quite rigid. Selected compounds are
presented in the Schemes 2 and 3. The acetamide derivative 16 was prepared from N,N-
diethyl-p-phenylenediamine 17 and 5-methyl-3-phenylisoxazole-4-carbaldehyde 18 via the
intermediate imine 19. The imine 19 was subsequently reduced to the corresponding
amine 20 with sodium borohydride, followed by treatment with acetyl chloride in pyridine.
The sulfonamide 21 was obtained from a reaction between 5-methyl-3-phenylisoxazole-
4-sulfonyl chloride 22 and diamine 17 in pyridine. Thioamide 23 was obtained by reacting
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
1
with Lawesson’s reagent and could be derivatized by base treatment (Li CO and NaH)
2
3
and a reaction with alkyl halides to give S-alkylated derivatives 24a-b. The alkylated
amides 25a-c were obtained from 1 by deprotonation with sodium hydride and
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subsequent alkylation, and the thioamide analog 26, in turn, was obtained from the N-
methylated amide 25a and Lawesson’s reagent.
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
The inverse amide 27 was prepared from 5-methyl-3-phenylisoxazol-4-amine 28 and 4-
(diethylamino)benzoic acid 29. Due to the poor reactivity of the electron rich carboxyl
group of 29 (electron donating effects of its para substituent) a reaction sequence via the
acid chloride (generated with oxalyl chloride and DMF, cat.) was given preference over
the generally applied HBTU-mediated coupling. The N,N-disubstituted amines 30a-d
were prepared through the initial imine intermediate 31 of the reaction between 4-
(diethylamino)benzaldehyde 32 and isoxazolylamine 28. The carbon-nitrogen double
bond of the imine was reduced with NaBH to give the corresponding secondary amine
4
33, followed by a reaction with an appropriate reagent (alkyl halides, anhydride or
isocyanate) under basic conditions.
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2
3
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7
8
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
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
Scheme 2. Selected compounds with different central partsa
N
N
N
N
N
N
iv
i
ii
iii
O
NH
O
N
NH
O
S
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
N
O
NH2
N
O
N
O
N O
16
21
17
19
20
N
N
N
1
R
Z=O, R1=
F
NH2
O
N
viii
v
25b
Z
Z
NH
N
R1 Z=O, R1=
N
HN
N
O
O
28
CN
N
N
25c
N
O
O
O
N
H
1
ix
27
Z = O
1
Z = O, R =Me
1
25a
vi
vi
N
O
0d
Z = S 23
Z = S, R =Me 26
xii
3
R1
vii
N
3
R = Me
N
N
N
30a
R2
R3=
S
2
N
R = Me, (E/Z)~3:124a
x
xi
30b
R3
N
R2=
F
(E/Z)~5:1 24b
N
NH
O
R3=
F
N
R2
O
N
N
N
F
24a,b
O
O
O
F
31
33
30a-c
30c
R3
aReagents and conditions: (i) 5-methyl-3-phenylisoxazole-4-carbaldehyde (18), AcOH,
Na SO (anhydr.), THF, rt, 5 h; (ii) NaBH , THF, MeOH, rt, overnight; (iii) AcCl, py, rt, 4
2
4
4
d; (iv) 5-methyl-3-phenylisoxazole-4-sulfonyl chloride (22), py, 0 C  rt, overnight; (v)
1
1
1
.) NaH, DMF, 0 °C, 1-2 h, 2.) R Br or R I, 0 C  rt, 1-4 d; (vi) Lawesson’s reagent,
PhMe, 110 °C, 6-8 h; (vii) 24a: MeI, Li CO , MeCN/DMSO, 38 °C, overnight; 24b: 1.) NaH,
2
3
DMF, 0 C, 2 h, 2.) Br(CH ) F, rt, 3 d; (viii) 1.) 4-(diethylamino)benzoic acid (29), (COCl) ,
2
2
2
cat. DMF, DCM, rt, 2 h, 2.) 28, py, rt, overnight; (ix) 4-(diethylamino)benzaldehyde (32),
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AcOH, Na SO (anhydr.), PhMe, rt, overnight; (x) NaBH , MeOH/THF, rt, 2 d; (xi) 30a:
2
4
4
3
Me I, Cs CO , DMF, rt, 3 d; 30b: allyl iodide, DMF, rt, 3 d; 30c: or TFAA, DMAP, py, rt, 2
2
3
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
d; (xii) 4-isocyanatobenzonitrile, THF, rt, 4 d.
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2
3
4
5
6
7
8
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
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
Central part: Ethers. The synthesis of the compounds 34a-e, 35a-e and 36a,b with an
ether linkage in the central part is presented in Scheme 3. First, the carboxylic acid 6 was
reduced to the corresponding primary alcohol 37 with borane-THF complex. Using the
Mitsunobu reaction with triphenylphosphine, diisopropyl azodicarboxylate (DIAD) and
suitable phenols, compound 37 was converted to the derivatives 34a, 34b and 34c. The
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
ketone 34a was reduced with NaBH in the presence of diethylamine to the corresponding
4
alcohol 34d, which in turn could be alkylated with iodomethane after deprotonation with
NaH to give the ether compound 34e. Furthermore, the ester-substituted northern part
3
4c was hydrolyzed with LiOH∙H O to give the corresponding benzoic acid 38, which was
2
coupled with five other alcohols and two amines to give the esters 35a-e and amides 36a-
b, respectively. Except for the synthesis of 35b, all these coupling reactions were
performed with HBTU, employing amines and alcohols. In contrast, compound 35b was
prepared by a nucleophilic substitution of 4-bromobutanenitrile under basic conditions
(K CO ).
2
3
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Scheme 3. Synthesis of ethersa
R1
O
OH
O
iii
iv
R1=
R1=
R1=
3
4a
34d
34e
i
ii
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
N
N
N
O
R1=
O
O
O
NO2
OH
OH
O
R1
3
4b
4c
6
37
34a-e
O
O
R1=
3
v
R2
R2=
R2=
R2=
R2=
N
vi
N
CN
5a
O
O
O
O
O
O
3
35c
35e
O
O
R2=
OH
R2
38
vii
35a-e
CN
O
35b
35d
R3
N
R3=
R3=
N
OH
O
O
36a
O
R3
N
36a, b
36b
a
1
Reagents and conditions: (i) BH -THF, THF, 0 C  rt, 7 d; (ii) R OH, Ph P, DIAD, THF,
3
3
0
C  rt, overnight; (iii) NaBH , Et NH, EtOH, 35-40 C, 3 d; (iv) 1.) NaH (60 w/w %,
4
2
mineral oil), DMF, 0 C, 45 min, 2.) MeI, 0 C  rt, overnight; (v) LiOH∙H O,
2
2
THF/MeOH/H O (1+1+1), rt, overnight; (vi) 35a and 35c-e: R OH, HBTU, DIPEA, DMF,
2
3
rt, 2-4 d; 35b: 4-bromobutanenitrile, K CO , DMF, rt, 4 d; (vii) R H (sec. amine), HBTU,
2
3
DIPEA, DMF, rt, overnight.
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2
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5
6
7
8
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
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
Southern part. The synthetic routes to isoxazoles with substituted phenyl moieties,
heteroaryl residues or ethyl substituent in 3 position (39a-o and 40) are described in
Scheme 4. The amide couplings of the building blocks 41a-o and the phenylenediamines
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
17 and 42, respectively, were performed with HBTU or HATU. The bromophenyl
derivative
39i
was
further
converted
to
the
biphenyl
39j
via
tetrakis(triphenylphosphine)palladium(0)-catalyzed Suzuki reaction. In addition, the
Scheme 4 presents syntheses for the isoxazolecarboxylic acid building blocks that were
not commercially available (41f and 41l-p). The carboxylic acids 41l-p were synthesized
via 1,3-dipolar cycloaddition reactions between nitrile oxides and ethyl 2-butynoate. The
nitrile oxide intermediates were generated from the corresponding oximes 43l-p that were,
in turn, prepared from the respective aromatic aldehydes 44l-p. The 1,3-dipolar additions
were mediated by (diacetoxyiodo)benzene (DIB)35 or alternatively by Oxone® . The
36
resulting isoxazole esters 45l-p were hydrolyzed with either LiOH∙H O or NaOH to
2
produce the intermediate carboxylic acids 41l-p. Carboxylic acid 41f was prepared from
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the 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic acid 41e via nitration of the
phenyl ring by HNO in the presence of H SO .
3
2
4
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
Scheme 4. Synthesis of compounds 39a-o and 40 with substituted aryl and heteroaryl
moieties attached to the isoxazole ringa
R
O2N
F
R=
R=
R=
R=
R=
R=
R=
N
R
O
O2N
39g, 41g, 40
39a, 41a
39d, 41d
Cl
i
N
H
N
O
39a-o
R
O
R=
R=
Et
OH
N
O
O2N
Cl
39e, 41e
NO2
Cl
N
O
39b, 41b
39h, 41h
39k, 41k
41a-o
i
N
H
N
O
Cl
F
Br
N
R=
R=
ii
40
O2N
39i, 41i
Cl
iii
39j
39c, 41c
39f, 41f
R
Br
O
O
O
R=
HO
R
OEt
OH
R=
R=
R=
R=
S
O
N
R
R
S
iv
v
vi
R
H
H
N
N
39l, 41l, 43l,
4l, 45l
39n, 41n, 43n,
44n, 45n
39p, 41p, 43p,
44p, 45p
O
O
41l-p
4
44l-p
43l-p
45l-p
S
O
N
3
4
9m, 41m, 43m,
4m, 45m
39o, 41o, 43o,
44o, 45o
aReagents and conditions: (i) N,N-diethyl-p-phenylenediamine (17) or N,N-dimethyl-p-
phenylenediamine (42), HBTU or HATU, DIPEA, DMF, rt, 1-2 d; (ii) HNO (65%), H SO
3
2
4
(
conc.), 0 ºC, 45 min, rt, 2 h; (iii) (4-fluorophenyl)boronic acid, Pd(Ph P) , Na CO ,
3
4
2
3
PhMe/EtOH/H O, EtOH, 80 C, 5 h, 70 C, overnight. (iv) H NOH∙HCl, py, EtOH,rt, 1−4
2
2
h; (v) 45l-n: ethyl but-2-ynoate, DIB, TFA, MeOH or MeOH/H O, 0 C  rt, 2-3 h; 45o:
2
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6
7
8
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
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
ethyl but-2-ynoate, PhI, Oxone®, H O, rt, 18 h; 45p: ethyl but-2-ynoate, KCl, Oxone®,
2
H O, rt, 5 h; (vi) 45l: LiOH∙H O, THF/MeOH/H O, rt, overnight; 41m-o: NaOH,
2
2
2
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
MeOH/H O, 60 C (or rt for 41o), 20-28 h.
2
Further, we embarked on identifying the surrogates of isoxazole ring in the southern part.
The carboxyl scaffolds 46a-ac were either commercially available or synthesized.
Synthesis of the triazole scaffolds is presented in Supporting Information. Carboxylic
acids 46a-ac were linked to the northern part 17 via a HBTU or HATU-mediated amide
coupling in the presence of DIPEA (Table 1) to give the amides 47a-ac for subsequent
biological studies.
Surprisingly, our attempt to extend the isoxazole core from the 5-position via substitution
with bromine followed by an amide coupling led to a spontaneous formation of a small
amount of imine 48 in the absence of an oxidizing reagent (Scheme 5). 5-Methyl-3-
phenylisoxazole-4-carboxylic acid 6 was brominated in the presence of N-
bromosuccinimide (NBS) and benzoylperoxide. The attempt to couple the formed crude
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product 49 with N,N-diethyl-p-phenylenediamine hydrochloride 50 via HBTU and DIPEA
gave a small amount (3% yield over 2 steps) of the imine 48. We confirmed this finding
by synthesizing the imine derivative 51 without coupling reagents under microwave
irradiation (120 C, 30 min). Imine 51 was synthesized from methyl 5-methyl-3-
phenylisoxazole-4-carboxylate 52 via bromide intermediate 53. A possible explanation for
the formation of an imine could be an autoxidation of the corresponding amine obtained
by a nucleophilic substitution of bromide. Autoxidations of amines of highly conjugated
compounds have been reported previously.37
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
Scheme 5. Autoxidation of imines via a secondary amine intermediatea
N
O
O
O
OH
OH
NH
i
i
ii
N
N
N
N
N
Br
O
O
O
O
6
49
48
O
O
O
OMe
OMe
OMe
N
iii
N
N
N
N
Br
O
O
52
53
51
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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
a
Reagents and conditions: (i) (products not isolated and characterized) NBS, (PhCO2)2,
CCl , reflux, 15 h (or for 53: reflux for 6 h and rt for 4 d); (ii) N,N-diethyl-p-
4
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
phenylenediamine hydrochloride (50), HBTU, DIPEA, DMF, rt, 2 d; (iii) N,N-diethyl-p-
phenylenediamine, MeOH, mw (120 C, 30 min).
BIOLOGY
Luciferase reporter assays. To screen compounds for GATA4-NKX2-5 interaction we
have developed a luciferase reporter assay based on their property to activate gene
28
transcription synergistically through the NKX2-5 binding site on DNA. In this assay, we
transfected mammalian COS-1 cells with protein expression vectors for GATA4 and
NKX2-5 proteins together with artificial luciferase reporter vector containing three high
affinity binding sites for NKX2-5 (NKE). As shown in Figure 3A, NKX2-5 activates the
luciferase reporter vector and together with GATA4 the activation is synergistic, while
GATA4 alone has only a minor effect. All 257 compounds were tested in two
concentrations, 3 µM and 10 µM. Compounds either inhibited, enhanced or had no effect
on the GATA4-NKX2-5 transcriptional synergy. To investigate the selectivity of the active
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compounds, we tested their effects on NKX2-5 transcriptional activity by using the same
luciferase reporter vector as in transcriptional synergy assay with three NKE sites (Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
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0
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9
0
1
2
3
4
5
6
7
8
9
0
3B). Furthermore, we used GATA luciferase reporter assay to examine if compounds
have an effect on GATA4 transcriptional activity. In this assay, we co-transfected the cells
with a GATA4 protein expression vector and a reporter vector with tandem GATA4
binding sites (Figure 3C). Previously we have reported that compound 1 inhibited the
29
GATA4-NKX2-5 transcriptional synergy at micromolar concentrations (IC =3 µM).
50
However, at higher concentrations 1 also influenced NKX2-5 (Figure 3B) and GATA4
(Figure 3C) transcriptional activity and showed toxicity in human induced pluripotent stem
cells (iPSCs), mouse embryonic stem cells (mESCs) and mESC-derived progenitor
cells.33
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9
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Luciferase screening assays. (A) The effects of compounds 1, 2, and 3 on
GATA4 and NKX2-5 induced synergistic activation of the luciferase reporter gene
containing three NKX-binding sites. The assay can detect both compounds that inhibit
and compounds that increase synergistic activation. Results are average (±SD) of several
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independent experiments (1, n=87; 3, n=4) except for compound 2 one experiment with
three technical replicates (±SD) is presented. (B) The effects of compounds 1, 2, and 3
on NKX2-5-induced reporter gene activation. Results are average (±SD) of several
independent experiments (1, n=22; 2, n=2; 3, n=3). (C) The effects of 1, 2, and 3 on
GATA4-induced activation of a tandem GATA-site containing luciferase reporter
construct. Results are average (±SD) of several independent experiments (1, n=25; 2,
n=3; 3, n=5). N denotes NKX2-5; G, GATA4; NKE, NKX-binding site; GATA, GATA-
1
1
1
1
1
1
1
1
1
1
2
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4
5
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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
*
**
***
binding site. P < 0.05, P < 0.01, P < 0.001 (randomized block ANOVA followed by a
Dunnet’s post hoc test).
SAR ANALYSIS
Southern part: Scaffold hopping. To identify structural features affecting inhibition of
GATA4 and NKX2-5 transcriptional synergy, we first present an exploration of the
chemical space of the southern part of the original hit compound 1. The Table 1 presents
the biological activity of selected derivatives comprising different heterocyclic scaffolds in
the southern part, which in turn are differently substituted by aromatic and heteroaromatic
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1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
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5
5
5
6
residues as well as other structures. Herein, we focus on the heterocyclic core of the
southern part, i.e. isoxazole in 1. The compounds in Table 1 comprise other heterocycles
in this region, or include an isoxazole with an aromatic residue in its 5 position). While the
isoxazole in 1 is substituted by a phenyl group in 3 position, many of the derivatives in
Table 1 include heteroaryl residues instead of a phenyl substituent. The presumed effects
of switching the phenyl unit to a heteroaryl moiety are discussed below together with other
derivatives (Table 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
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
The enhancers of the GATA4-NKX2-5 transcriptional synergy (Table 6) seem to contain
a hydrogen bond acceptor in the same position, indicating a role in the binding affinity of
the compounds. In general, hydrogen bond acceptor capacity of the isoxazole structures
are strongly localized on the nitrogen atom. In line with the reference compound 1, the
methyl-substituted furan 54 and thiophene 56 also confirm this hypothesis. We found the
hydrogen bond acceptor with one or two atom distance to the position at which an
aromatic moiety is attached to interfere with GATA4-NKX2-5 synergy in most structures.
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A few derivatives are formally characterized by lacking the methyl group in the 5 position
of the isoxazole ring in 1. The activities of 47d, 47e, 47n, 47p, 47t, 47z and 54 in luciferase
assay indicate that this methyl group is not necessary for inhibiting GATA4-NKX2-5
transcriptional synergy. Particularly noteworthy in this regard is the isothiazole 47s and
the thiophene 47u that are missing both substituents (Ph, Me). These compounds had
clear inhibitory effect on transcriptional synergy. A change of the position of the aryl
sustituent further away from the central part (compounds 47b and 47y) did not seem a
promising approach. The aromatic substituent with a one-atom spacer (compound 47q)
could be further explored, but a two-atom spacer (compound 47i) did not seem to enhance
inhibitory effect on GATA4-NKX2-5 interaction. Also, replacing the isoxazole with a
bicyclic unit, i.e. 47j, 47v, 47aa, and 47ab, was not a promising approach, although the
number of these derivatives is too small to allow drawing final conclusions. We also note
that some derivatives with a bicyclic unit, such as 47v and 47aa, indicate promising
features for the development of enhancers of GATA4-NKX2-5 transcriptional synergy.
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
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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
Table 1. Activities of derivatives with structurally diverse southern parts (% of control) at
1
0 µM concentrationa
O
R
HN
N
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
47a-ac
Cmpd
R
NKX2-5
(%
of
Synergy
GATA4
(% of control) control)
(% of control)
Mean ± SD
Mean ± SD
Mean ± SD
3b
O
N
7
8 ± 13
84 ± 17
ND
100 ± 13
ND
O
O
N
47a
N
110 ± 4.0
47b
N
N
N
100 ± 16
ND
21 ± 0.88
50 ± 0.012
ND
68 ± 9.1
62 ± 8.9
N
N
S
47c
47d
47e
2
4 ± 0.9
7 ± 9.8
O
N
4
O
N
N
N
62 ± 1.5
ND
ND
47f
O
N
N
N
9
9 ± 11
93 ± 3.9
ND
130 ± 13
ND
N
N
4
7g
7h
N
N
110 ± 15
N
NH
4
O
N
S
O
1
00 ± 6.1
110 ± 13
ND
140 ± 30
ND
47i
100 ± 0.24
O
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7j
N
87 ± 5.8
91 ± 10
ND
ND
ND
ND
N
47k
N
N
N
O
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
47l
O
O
N
N
S
85 ± 6.5
96 ± 3.8
97 ± 35
47m
N
110 ± 14
110 ± 4.1
49 ± 7.7
ND
110 ± 1.6
52 ± 0.65
ND
4
7n
7o
S
N
41 ± 2.2
N
N
4
N
N
84 ± 4.5
47p
N
N
N
78 ± 11
ND
ND
47q
O
CF3
N
N
68 ± 3.1
ND
ND
ND
ND
47r
140 ± 4.3
7 ± 12
O
N
47s
2
16 ± 2.7
48 ± 35
N
S
47t
S
3
3 ± 6.2
5 ± 6.3
39 ± 4.5
26 ± 3.4
58 ± 6.7
23 ± 2.7
47u
47v
S
2
NO2
S
S
N
1
30 ± 14
10 ± 5.6
20 ± 18
ND
ND
ND
ND
ND
ND
N
N
4
7w
7x
S
S
1
S
4
N
O
1
O
47y
CF3
O
85 ± 5.4
ND
ND
Br
F
47z
N
N
S
52 ± 2.7
65 ± 5.8
45 ± 2.8
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