Limits...
Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain.

Bonomi R, Mukhopadhyay U, Shavrin A, Yeh HH, Majhi A, Dewage SW, Najjar A, Lu X, Cisneros GA, Tong WP, Alauddin MM, Liu RS, Mangner TJ, Turkman N, Gelovani JG - PLoS ONE (2015)

Bottom Line: Histone deacetylases (HDAC's) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors.PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo.Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Wayne State University, Detroit, MI 48202, United States of America.

ABSTRACT
Histone deacetylases (HDAC's) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa-specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [18F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [18F]DFAHA and previously reported [18F]FAHA. PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.

No MeSH data available.


Synthesis of DFAHA and TFAHA.Reaction conditions are as follows: a) RT overnight; b) 2mL DCM, stirred overnight.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526562&req=5

pone.0133512.g001: Synthesis of DFAHA and TFAHA.Reaction conditions are as follows: a) RT overnight; b) 2mL DCM, stirred overnight.

Mentions: The syntheses of DFAHA (3a) and TFAHA (3b) are shown in Fig 1. Compound 1 was synthesized following a previously published method in 80% yield [35]. Compound 1 was the key intermediate for the synthesis of the precursor compounds for both DFAHA (3a) and TFAHA (3b). Reaction of 1 with bromofluoro acetyl chloride (freshly prepared by reacting bromofluoro acetic acid with thionyl chloride), and triethylamine in DCM at 0°C, produced compound 2a in 15% yield. Due to the low boiling point of bromofluoro acetyl chloride, it was not possible to evaporate excess thionyl chloride prior to adding the compound 1; for this reason, less than 1 equivalent of thionyl chloride was used in the reaction. Compound 2a was obtained as a diastereoisomeric mixture and characterized by 1H, 13C and 19F NMR spectroscopy and high-resolution mass spectroscopy. The 1H NMR spectrum of compound 2a showed 2 doublets corresponding to the fluorobromomethyl proton at 7.03 and 6.74 ppm with coupling constants of 49.45 Hz (typical for F-H coupling). Based on the relative integration of these two doublets the, diasteromeric mixture was obtained in an 80:20 ratio. The corresponding 19F-NMR spectrum showed 2 doublet peaks at -147.34 and -143.52 ppm with coupling constants 50.35 Hz and 48.83 Hz respectively, and the same diasteromeric ratio of 80:20. Resolving the diastereomeric mixture was unnecessary, because the subsequent reaction (radio-fluorination) led to the formation of difluroacetyl amide, which is optically inactive. The yield of this reaction is quite low, only 15%. As an alternative, we performed this reaction using the isobutyl chloroformate as intermediary step to aid the coupling. Also, we tested N,N'-dicyclohexylcarbodiimide (DCC) as an alternative coupling agent with 4-dimethylaminopyridine (DMAP) as a catalyst. Although not yet tested, other alternative coupling agents such as hydroxybenzotriazole (HOBT) or (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU) can be explored with N,N-diisopropylethylamine (DIPEA) as catalyst. One possible reason for the consistently low yield is the bromine atoms ability to couple with the amine of compound 1. The competition for coupling site between the bromine and the chlorine will inherently lower the correct product formation. Reaction of compound 1 with difluorobromo acetyl chloride in DCM in the presence of triethylamine [36] produced the precursor, compound 2b, in 50% yield. Consistent with the structure of 2b, a singlet at -60.07 ppm was observed in the 19F NMR spectrum.


Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain.

Bonomi R, Mukhopadhyay U, Shavrin A, Yeh HH, Majhi A, Dewage SW, Najjar A, Lu X, Cisneros GA, Tong WP, Alauddin MM, Liu RS, Mangner TJ, Turkman N, Gelovani JG - PLoS ONE (2015)

Synthesis of DFAHA and TFAHA.Reaction conditions are as follows: a) RT overnight; b) 2mL DCM, stirred overnight.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526562&req=5

pone.0133512.g001: Synthesis of DFAHA and TFAHA.Reaction conditions are as follows: a) RT overnight; b) 2mL DCM, stirred overnight.
Mentions: The syntheses of DFAHA (3a) and TFAHA (3b) are shown in Fig 1. Compound 1 was synthesized following a previously published method in 80% yield [35]. Compound 1 was the key intermediate for the synthesis of the precursor compounds for both DFAHA (3a) and TFAHA (3b). Reaction of 1 with bromofluoro acetyl chloride (freshly prepared by reacting bromofluoro acetic acid with thionyl chloride), and triethylamine in DCM at 0°C, produced compound 2a in 15% yield. Due to the low boiling point of bromofluoro acetyl chloride, it was not possible to evaporate excess thionyl chloride prior to adding the compound 1; for this reason, less than 1 equivalent of thionyl chloride was used in the reaction. Compound 2a was obtained as a diastereoisomeric mixture and characterized by 1H, 13C and 19F NMR spectroscopy and high-resolution mass spectroscopy. The 1H NMR spectrum of compound 2a showed 2 doublets corresponding to the fluorobromomethyl proton at 7.03 and 6.74 ppm with coupling constants of 49.45 Hz (typical for F-H coupling). Based on the relative integration of these two doublets the, diasteromeric mixture was obtained in an 80:20 ratio. The corresponding 19F-NMR spectrum showed 2 doublet peaks at -147.34 and -143.52 ppm with coupling constants 50.35 Hz and 48.83 Hz respectively, and the same diasteromeric ratio of 80:20. Resolving the diastereomeric mixture was unnecessary, because the subsequent reaction (radio-fluorination) led to the formation of difluroacetyl amide, which is optically inactive. The yield of this reaction is quite low, only 15%. As an alternative, we performed this reaction using the isobutyl chloroformate as intermediary step to aid the coupling. Also, we tested N,N'-dicyclohexylcarbodiimide (DCC) as an alternative coupling agent with 4-dimethylaminopyridine (DMAP) as a catalyst. Although not yet tested, other alternative coupling agents such as hydroxybenzotriazole (HOBT) or (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU) can be explored with N,N-diisopropylethylamine (DIPEA) as catalyst. One possible reason for the consistently low yield is the bromine atoms ability to couple with the amine of compound 1. The competition for coupling site between the bromine and the chlorine will inherently lower the correct product formation. Reaction of compound 1 with difluorobromo acetyl chloride in DCM in the presence of triethylamine [36] produced the precursor, compound 2b, in 50% yield. Consistent with the structure of 2b, a singlet at -60.07 ppm was observed in the 19F NMR spectrum.

Bottom Line: Histone deacetylases (HDAC's) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors.PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo.Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Wayne State University, Detroit, MI 48202, United States of America.

ABSTRACT
Histone deacetylases (HDAC's) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa-specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [18F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [18F]DFAHA and previously reported [18F]FAHA. PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.

No MeSH data available.