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

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.

and related compounds pp. S59-96  Synthesis of compounds in Tables 6-8 (compounds not included in Schemes 1-4 and Table 1) pp. S97-106 List of commercially obtained compounds (Table S1) pp. S107-112 Structures of the previously published compound (SI137) not presented in the manuscript pp. S113 Hierarchical clustering of the luciferase activity data pp. S113-114 General procedure II: Acyl chloride-mediated amide synthesis When the required acyl chloride was not commercially available, it was prepared according to the following procedure. Unless otherwise noted, one equivalent of carboxylic acid was dissolved in anhydrous dichloromethane under argon. Three equivalents of a 2 M solution of oxalyl choride in dichloromethane and a catalytic amount of anhydrous DMF (0.1 equiv) were added, and the reaction mixture was stirred for 2 h at room temperature. The solvent was removed with a rotary evaporator, and the residue was dissolved in anhydrous pyridine. The required amine was added, and the reaction mixture was stirred overnight at room temperature. Diethyl ether was added and the organic phase was washed three times with water. When phase separation was slow, 5-30% of brine was added to the aqueous phase to aid separation. The organic solvents were removed with a rotary evaporator. The crude product was either directly recrystallized from the solvent mixtures indicated or purified by automated preparative chromatography. Unless otherwise indicated, the chromatography on silica gel was performed with an increasing gradient of ethyl acetate in n-hexane, starting with 0% of ethyl acetate.
Unless otherwise noted, anhydrous sodium sulphate (3 equiv) was flame-dried in an evacuated Schlenk flask. Absolute THF, acetic acid (0.25-1 equiv) as well as an equimolar amount of aromatic aldehyde and aromatic amine were added to the flask containing Na2SO4. The reaction mixture was stirred at room temperature for the indicated time, and a 1.0 M solution of sodium cyanoborohydride in THF (3-5 equiv) was added. The solution was stirred at room temperature until TLC indicated consumption of the starting materials and intermediate Schiff base (3-5 d). During this period of time additional sodium cyanoborohydride may be added (1-2 equiv). The reaction mixture was quenched with a diluted solution of sodium hydroxide in water or a saturated solution of sodium hydrogencarbonate in water. After 20-60 min ethyl acetate was added, and the organic phase was washed three times with a mixture of water and brine (typically 2:1). The organic solvents were removed with a rotary evaporator. The crude product was purified by automated preparative chromatography. The chromatography on silica gel was performed with an increasing gradient of ethyl acetate containing triethylamine (1%) in n-hexane, starting with 0% of ethyl acetatetriethylamine mixture.

4-Methoxy-N-(5-methyl-3-phenylisoxazol-4-yl)benzamide (SI32)
5-Methyl-3-phenylisoxazol-4-amine, 28 (105 mg, 0.500 mmol) was dissolved in anhydrous pyridine (2 mL) under argon and cooled to 0 °C. Anisoyl chloride (102 mg, 0.600 mmol) was added, and the reaction mixture was stirred overnight while it was allowed to warm to rt. DMAP (61.1 mg, 0.500 mmol) was added, and the reaction mixture was stirred overnight. HBTU (189 mg, 0.500 mmol) and DIPEA (0.261 mL, 1.50 mmol) were added, and the reaction mixture was stirred overnight. Diethyl ether was added, and the organic phase was washed with water, twice with a 2 M solution of sodium hydroxide in water, twice with 2 M hydrochloric acid and several times with water until the aqueous phase remained neutral. The organic solvents were removed with a rotary evaporator, and the crude product was purified by automated preparative chromatography on silica gel with an increasing gradient of ethyl acetate in n-hexane, starting with 0% of ethyl acetate to yield compound SI32 ( 1, 159.3, 129.8, 129.5, 128.8, 128.3, 127.0, 125.4, 113.7 113.3, 55.4, 10.7

2,2'-[[4-[[(5-Methyl-3-phenylisoxazol-4-yl)amino]methyl]phenyl]azanediyl]bis(ethan-1-ol) (SI43)
SI42 (0.200 g, 0.547 mmol) was dissolved under argon in a mixture of absolute THF (3.5 mL) and absolute MeOH (2 mL). Sodium cyanoborohydride (124 mg, 3.28 mmol, 6 equiv) was added and the reaction mixture was stirred for 2 d at room temperature. A saturated solution of sodium hydrogen carbonate in H2O (8 mL) was added and the solution was stirred for 20 min at room temperature. EtOAc was added and the organic phase was washed three times water. The solvent was removed with a rotary evaporator, and the residue was subjected to automated preparative chromatography. The chromatography on silica gel was performed with an increasing gradient of MeOH in EtOAc, starting with 0% of MeOH. The solvents of the fractions still containing both the reaction product and starting material were removed with a rotary evaporator. Treating the residue with a small amount of MeOH led to crystallization of the starting material overnight (85.9 mg, 0.235 mmol). The solvent of the mother liquor was removed with a rotary evaporator and the residue was subjected to automated preparative chromatography. The chromatography on silica gel was performed with an increasing gradient of ethyl acetate in n-hexane, starting with 0% of ethyl acetate gave compound SI43 (18.9 mg, 0.0514 mmol, 9%) as a yellow oil. 1   159. 1, 157.4, 146.9, 129.4, 129.4, 128.9, 128.7, 127.1, 126.2, 123.2, 110.9, 58.1, 53.3, 51.4, 10.2 ppm. HRMS calc. for C21H26N3O3 [M+Na] + : 390.1794, found 390.1797.

5-Methyl-3-(3-nitrophenyl)isoxazole-4-carboxylic acid 41g and 5-methyl-3-(2nitrophenyl)isoxazole-4-carboxylic acid (41h)
Note: The reaction procedure has not been optimized. The compound 41g can likely be obtained in much higher yield. 41h is only a minor side product. The synthesis of 41g is known ( 5-Methyl-3-phenylisoxazole-4-carboxylic acid 6 (10.0 g, 49.2 mmol) was added in small portions during a period of 40 min to fuming nitric acid (60 mL), cooled to 0 °C in an ice bath. The reaction mixture was stirred at 0 °C for 4 h and quenched by addition of crushed ice (100 mL). Water (180 mL) was added slowly and the precipitated mixture of intermediates was obtained by filtration. The precipitate was washed with water and the wet solid was recrystallized from methanol to give an intermediate mixture of differently nitrated regioisomers (11.0 g, 44.2 mmol, 90%) as yellowish white crystals. This mixture of isomers was dissolved in DMSO (50 mL). Potassium carbonate (2.98 g, 53.0 mmol, 1.2 equiv) and iodomethane (5.50 mL, 88.4 mmol, 2 equiv) were added, and the reaction mixture was stirred for 5 d at room temperature. The reaction mixture was diluted with ethyl acetate S47 and washed three times with a mixture of water and brine (30%) and the organic solvent was removed with a rotary evaporator. The crude product was divided into five portions, which were separately purified by automated chromatography on silica gel, using a gradient of increasing acetone in nhexane starting with 100% n-hexane. Hereby, the smaller second fraction was partially separated from the major first fraction. The combined second fractions were recrystallized from methanol/water (10+1) to give the intermediate methyl 5-methyl-3-(2-nitrophenyl)isoxazole-4-carboxylate (314 mg, 1.20 mmol, which was hydrolyzed by lithium hydroxide hydrate (64.8 mg, 1.54 mmol, 1.5 equiv) at room temperature overnight in a mixture of THF (1 mL), methanol (1 mL) and water (0.5 mL). This reaction mixture was diluted with water, washed once with diethyl ether, which in turn was reextracted once with water. Ethyl acetate was added to the combined aqueous phases, which were acidified with 1 M hydrochloric acid. The phases were separated, and the organic phase was washed neutral with portions of water. The organic solvent was removed with a rotary evaporator, and the crude product was recrystallized from ethanol/water (10+1) to give compound 41h (0.250 g, 1.01 mmol, 2%).
The combined major first fractions of the chromatography were recrystallized from n-hexane/benzene (2+1) and twice from ethanol/water (10 + 1) to give a mixture of the corresponding 3-and 4-nitrosubstituted isoxazole methyl ester (3.48 g, 13.3 mmol) that contains methyl 5-methyl-3-(3nitrophenyl)isoxazole-4-carboxylate as the major component. This mixture of intermediates was hydrolyzed by lithium hydroxide hydrate (835 mg, 19.9 mmol, 1.5 equiv) at room temperature overnight in a mixture of THF (12 mL), methanol (12 mL) and water (6 mL). This reaction mixture was diluted with water and washed once with diethyl ether. Diethyl ether was added, and the aqueous phase was acidified with 1 M hydrochloric acid. The phases were separated and the organic phase was washed neutral with portions of a mixture of water and brine (20%). The organic solvent was removed with a rotary evaporator, and the crude product was recrystallized twice from ethanol to give compound 41g (741 mg, 2.99 mmol, 6%).   1, 162.4, 160.5, 147.4, 135.7, 129.9, 129.7, 124.5, 124.0, 108.7, 13.1 ppm.

3-(4-Bromophenyl)-N-[4-(diethylamino)phenyl]-5-
methylisoxazole-4-carboxamide 39i (0.075 g, 0.175 mmol) was dissolved under argon in absolute toluene (2 mL). boronic acid (27.0 mg, 0.193 mmol, 1.1 equiv), ethanol (1 mL) and a 2.0 M solution of sodium carbonate in H2O (1 mL) were added, and the mixture was degassed. Tetrakis(triphenylphosphine)palladium(0) (10.1 mg, 0.00875 mmol, 5 mol%) was added under argon, and the reaction mixture was heated at 80 °C under reflux conditions for 5 h and and to 70 °C overnight. Ethyl acetate was added, and the organic phase was washed three times with a mixture of water and brine (3:1). The solvent was removed with a rotary evaporator, and the crude product was subjected to automated preparative chromatography. The chromatography on silica gel was performed with an increasing gradient of ethyl acetate in n-hexane, starting with 0% of ethyl acetate gave compound 39j (

ii. 3-(4-Bromothiophen-2-yl)-5-methylisoxazole-4-carboxylate (45l)
Diacetoxyiodobenzene (354 mg, 1.10 mmol, 1.1 equiv) was dissolved in a mixture of MeOH (4 mL) and water (400 µL) and stirred at 0 °C in an ice bath. Trifluoroacetic acid (15 µL) and (E/Z)-4bromothiophene-2-carbaldehyde oxime 43l (206 mg, 1.00 mmol) were added, and after 2 min ethyl but-2-ynoate (82.8 µL, 1.10 mmol, 1.1 equiv) was added. The reaction mixture was stirred for 30 min at 0 °C, after which the ice-bath was removed and stirring was continued at room temperature for 1.5 h. The solvent was removed with a rotary evaporator, and the crude product mixture was subjected to automated preparative chromatography. The chromatography on silica gel was performed with an increasing gradient of ethyl acetate in n-hexane, starting with 0% of ethyl acetate. The chromatography gave 81.1 mg of a product mixture containing ethyl 3-(4-bromothiophen-2-yl)-5methylisoxazole-4-carboxylate 45l, which was used without further purification in the next step. (41l) 3-(4-Bromothiophen-2-yl)-5-methylisoxazole-4-carboxylate 45l was dissolved in an equimixture of THF, MeOH and water (1.5 mL). Lithium hydroxide hydrate (16.1 mg, 0.385 mmol) was added and the reaction mixture was stirred overnight at room temperature. Ethyl acetate was added, and the organic phase was extracted twice with water. The combined aqueous phases were washed once with diethyl ether, acidified with a 1 M solution of HCl in H2O and extracted with ethyl acetate. The organic phase was washed twice with water and once with a mixture of water and brine (2:1) until the aqueous phase remained neutral. Evaporation of the organic phase with a rotary evaporator and drying in an oil pump vacuum gave crude 3-(4-bromothiophen-2-yl)-5-methylisoxazole-4-carboxylic acid 41l (19.3 mg), which was used without purification.

N,N-Diethyl-p-phenylenediamine hydrochloride (50)
N,N-Diethyl-p-phenylenediamine 17 (4.77 mL, 29.0 mmol) was dissolved in diethyl ether (13 mL). A 4 M solution of hydrochloric acid in 1,4-dioxane (7 mL) was added. The mixture was stirred for 2 h at room temperature. The solvent was removed with a rotary evaporator. The residue was treated with methanol and the solvent was removed with a rotary evaporator to give a solid material, which was recrystallized from 2-propanol, filtered and washed with ethyl acetate to give compound 50 (3.00 g, 14.9 mmol, 51%) as brownish white crystals, which was used without further purification or characterization.

N-[4-(Diethylamino)phenyl]-5-phenyloxazole-4-carboxamide (47a)
Synthesis according to the General procedure I. Deviating from the General procedure I, the reaction mixture was diluted with ethyl acetate for the work-up and extracted once with a 2 M solution of sodium hydroxide in water and twice with 4 M hydrochloric acid. The combined hydrochloric acid phases were made alkaline with a 2 M solution of sodium hydroxide in water and extracted twice with ethyl acetate. The combined organic phases were washed twice with a mixture of water and brine (10:1).  1, 150.1, 144.4, 129.8, 129.3, 128.4, 127.7, 126.9, 126.9, 122.2, 111.6, 43.7, 12.4

(E)-N-[4-(Diethylamino)phenyl]-5-[[(4-(diethylamino)phenyl]imino]methyl]-3phenylisoxazole-4-carboxamide (48)
5-Methyl-3-phenylisoxazole-4-carboxylic acid (2.03 g, 10.0 mmol) was dissolved in carbon tetrachloride (40 mL) under argon. N-Bromosuccinimide (1.78 g, 10.0 mmol) and benzoylperoxide (242 mg, 1.00 mmol) were added, and the reaction mixture was heated under reflux conditions for 8.5 h at 60 °C, stirred at room temperature overnight and heated for 6 h at 60 °C. The solution was filtered after cooling to rt, washed once with water and the aqueous phase was extracted once with DCM. The combined organic phases were extracted with a 2 M solution of sodium hydroxide in H2O. The aqueous phase was acidified with 1 M hydrochloric acid and extracted with ethyl acetate. The organic S72 phase was washed neutral with water (twice). The solvent was removed with a rotary evaporator and the crude product was subjected to automated chromatography. The chromatography on silica gel was performed with an increasing gradient of ethyl acetate in n-hexane, starting with 0% of ethyl acetate, but it was not able to separate the mixture of intermediates. The resulting crude product mixture (380 mg) was dissolved in absolute DMF (5 mL). N,N-Diethyl-p-phenylenediamine hydrochloride 50 (442 mg, 1.87 mmol), HBTU (1.42 g, 3.74 mmol) and DIPEA (1.63 mL, 9.35 mmol) were added to the solution, and the reaction mixture was stirred for 2 d at room temperature. The reaction mixture was diluted with ethyl acetate, and washed with a mixture of water and brine (50%). The phases were separated and the aqueous phase was extracted once with ethyl acetate. The combined organic phases were washed once with a small amount of water and the solvent was removed with a rotary evaporator. The crude product was subjected to automated chromatography.  1, 144.6, 138.0, 136.3, 130.3, 128.9, 128.9, 127.9, 127.1, 124.1, 121.8, 115.9, 111.7, 111.5, 43.9, 43.8, 12.5, 12.4

Methyl (E)-5-[[[4-(diethylamino)phenyl]imino]methyl]-3-phenylisoxazole-4-carboxylate (51)
i.  Methyl 5-methyl-3-phenylisoxazole-4-carboxylate (2.17 g, 10.0 mmol) was dissolved in carbon tetrachloride (40 mL) under argon. N-Bromosuccinimide (1.78 g, 10.0 mmol) and benzoylperoxide (242 mg, 1.00 mmol, 0.1 equiv) were added, and the reaction mixture was heated under reflux conditions for 6 h to 60 °C and stirred at room temperature for 4 d. The solution was dissolved to ethyl acetate and extracted three times with H2O. The solvent was removed with a rotary evaporator, and the crude product was subjected to automated chromatography. The chromatography on silica gel, with an increasing gradient of ethyl acetate in n-hexane, starting with 0% of ethyl acetate gave compound 53, which was used in a next step without further purification.

Hierarchical clustering
To identify compounds which inhibit the GATA4-NKX2-5 transcriptional synergy but have no effect on either NKX2-5 or GATA4 transcriptional activity, we performed hierarchical clustering for biological data to get two-dimensional presentation of the three-dimensional activity data. It has been shown previously b that cluster analysis can be used to identify compounds with a similar activity pattern, since they are close to each other in an activity space determined by activity measures. Hierarchical clustering of the activity patterns of selected compounds is presented in SFigure 1. We identified two potentially interesting groups of compounds with different activity patterns based on hierarchical clustering. In the first group, there are two novel compounds which inhibited transcriptional synergy of GATA4 and NKX2-5 without affecting GATA4 transcriptional activity similar to that of compound 3. In the second group, compounds 39k and 47l did not affect NKX2-5 transcriptional activity. In addition, compound 39k slightly increased GATA4 transcriptional activity at 10 µM concentration. A common structural feature for all selective (i.e. not affecting GATA4 transcriptional activity) inhibitors of GATA4-NKX2-5 transcriptional synergy is a hydrogen bond acceptor in the para position in the northern part benzene ring.