Design and Synthesis of Small Molecules Based on a Substructural Analysis of the Histone Deacetylase Inhibitors TSA and SAHA

Inhibitors of histone deacetylases (HDACs) are patent inducers of differentiation and bear considerable potential as drugs for chemoprevention and treatment of cancer. In this paper, we have investigated three synthetic, inhibitors A1a,b, A2a. Analogue hybrid trichostatine A (TSA), suberoylanilide hydroxamic acid SAHA, in order to seek new histone deacetylases (HDACs) inhibitors.


H
Hi is st to on ne e d de ea ac ce et ty yl la as se es s ( (H HD DA AC Cs s) ) a an nd d h hi is st to on ne e a ac ce et ty yl l transferases (HATs) are known to play an important role in the regulation of gene expression 1,2 .HATs mediate hyperacetylation of positively charged lysine residues in the N-terminal tail of core histone and loosen the histone-DNA binding.As a result, activation of genes transcription can occur.In contrast HDACs catalyse deacetylation of the acetyl ε amino group residues and lead to the tight histone-DNA binding 3 .In this case, the access to transcription factors is restricted.These enzymes correlate with cell cycle progression, differentiation and apoptosis 4 and their deregulation is associated with tumorogenisis 5 .
HDACs inhibitors have demonstrated potential for the prevention and treatment of cancer in numerous cell culture 6,7 and animals models 8 .HDACs have emerged as an attractive target for new anticancer drugs and there is a great demand for new inhibitors 9,10 .

Figure 1. Structures of known HDAC inhibitors
These compounds consist of a hydrophobic scaffold with a spacer that is attached to a functional group which can interact with zinc ion present in the active site pocket [25][26] .Compounds containing hydroxamic acid as a functional group are reported as the most potent inhibitors for HDACs 16 .

Experimental
Nuclear magnetic resonance spectra ( 1 H NMR and 13 C NMR) were recorded on a Bruker 300 and 75 MHz.Flash column chromatography was performed using MACHEREY-NAGEL silica gel 60 (15-40 µm) as the stationary phase.All reactions were run under a positive pressure of nitrogen unless otherwise stated.

Results and Discussion
As part of our efforts to search for novel HDACs inhibitors, we investigated a series of new hydroxamate analogues (A) which were designed as hybrides of TSA/SAHA (Figure 2).Analogues hybrides TSA/SAHA The rationale for the structure relied on the following parameters.The presence of the amide function on the lateral chain instead of substituted alkene on the TSA, is one common feature shared by these analogues.We can then consider these analogues as TSA isosters.The presence of the carbonyl function on the lateral chain instead of the methyl one, introduces a new hydrophilic feature which can allow the formation of hydrogen binding in the tubular pocket of the enzyme.Such a modification could have an impact on the affinity toward HDACs.These analogues have an aryl recognition moiety which can be substituted in different ways.The orientation of the methyl in the chain is identical to that of TSA.The lateral chain comes from the acylation of an aromatic amine and leads to a pattern which is identical to that of SAHA.This chain will be saturated or unsaturated.In this paper we report the synthesis of these compounds.Scheme 1 shows a general synthetic route of the desired analogues A1-a,b and A2-a.The N-tert-butoxycarbonyl-L-alanine 5 was coupled with para substituted aniline 6a,b in several conditions.The use of N,N-bis(2-oxo-3-oxazolidinyl)-phosphinique chloride (BOP-Cl) as coupling reagent resulted in low yield 41%. Using DPPA to activate the carboxylic acid gave a 90% and 78% yield for respectively 7a and 7b.The N protecting group was removed after treatment with trifluoroacetic acid.The amine 8a,b reacted in DMF with succinic anhydride in the presence of TEA to give carboxylic acid 9a: 93%, and 9b: 61%.The resulting acid was converted to the O-benzyl protected hydroxamate.Subsequent catalytic hydrogenation of the protected group was used to generate the desired hydroxamates A1-a (qt), A1-b (73%).
The synthesis of the analogue A2-a started with the coupling of amine 8a with the monoethyl ester fumaric acid (Scheme 1).The ethyl ester 10 was cleaved under basic conditions in the presence of LiOH-MeOH and generated the corresponding acid with 57% yield.The acid was then converted to the O-THP (protected hydroxamate), using TBTU as coupling agent.The hydroxamic acid A2-a was isolated after purification of a small sample on the preparative plates with 66% yield.

Conclusion
We have synthesized three analogues hybrides of TSA/SAHA with good yields.From these analogues, some novel synthetic inhibitors can be developed.

Figure 2 .
Figure 2. Analogues hybrides TSA/SAHAThe rationale for the structure relied on the following parameters.The presence of the amide function on the lateral chain instead of substituted alkene on the TSA, is one common feature shared by these analogues.We can then consider these analogues as TSA isosters.The presence of the carbonyl function on the lateral chain instead of the methyl one, introduces a new hydrophilic feature which can allow the formation of hydrogen binding in the tubular pocket of the enzyme.Such a modification could have an impact on the affinity toward HDACs.These analogues have an aryl recognition moiety which can be substituted in different ways.The orientation of the methyl in the chain is identical to that of TSA.The lateral chain comes from the acylation of an aromatic amine and leads to a pattern which is identical to that of SAHA.This chain will be saturated or unsaturated.In this paper we report the synthesis of these compounds.Scheme 1 shows a general synthetic route of the desired analogues A1-a,b and A2-a.