Su obra

Synthesis of N -Boc protected α-amino amides allows facile transformation to the desired final product, an α-amino amide salt through the removal of the Boc group.

Table 2.2: [α]D measurements of matching enantiomers.

NH_2.HCl N

O NH_2.HCl

N

O NH_2.HCl

N

O NH_2.HCl

N O

[α]D -30.4° 36 +33.0° 46 -7.8° 37 +7.2° 47

NH_2.HCl N

O

OH

NH_2.HCl N

O

OH

NH_2.HCl N

O

OH

NH_2.HCl N

O

OH

[α]D 34.9° 43 +32.4° 48 -9.0° 44 +12.1° 49

Removal of the Boc protecting group proceeded with ease requiring only a short treatment with ethereal hydrogen chloride (generally <2 h). The N -Boc-α-amino amides 32 and 50 were sucessfully deprotected following the procedure used in literature¹⁴⁷ to synthesise the lead compound 4. Similar treatment with ethereal hydrogen chloride afforded α-amino amide salts 36 and 46 in excellent yields (>90

%).

N -Boc protected α-amino amide 47 has been N -Boc-deprotected previously via a similar method using trifluoroacetic acid in DCM. Following a basic workup the amine of 47 was obtained as an oil²⁰³. Solid compounds are more stable and easier to handle, in this case it was therefore desirable to obtain the hydrochloride salt for this final product (and all future cases). The oil was dissolved in diethyl ether then treated with ethereal hydrochloric acid to precipitate the insoluble hydrochloride salt.

In contrast to the easily isolable salts 36 and 46, the hygroscopic nature of the analogues 37 and 47 (which have tetrahydroisoquinolyl STBGs) caused difficulties in isolation. Upon evaporation of the solvent from the ethereal suspension of 37 the white precipitate turned into a colourless oil with an apparent >100% yield. The

NMR spectrum of the salt revealed complete N -Boc deprotection but also contained a large water peak. It was therefore determined that these hydrochloride salts were hygroscopic and that the ethereal hydrochloric acid had a small amount of water which remained as the diethyl ether evaporated and was subsequently absorbed by the salt turning it into an oil. This was confirmed when trituration of the oil with dry diethyl ether to remove water did give a small amount of white hydrochloride salt.

N

O NH_2.HCl

N

O NH_2.HCl N

O NH_2.HCl N

O NH_2.HCl

36 46 37 47

Figure 2.2: The non-hygroscopic (36, 46) and the very hygroscopic (37, 47) N -Boc-α-amino amide salts.

The salt 37 was so hygroscopic that the initial solid obtained after vacuum filtra-tion became an oil within 5 seconds. The filtrafiltra-tion of the hydrochloride salts was further complicated by the latent enthalpy of evaporation of diethyl ether. As the diethyl ether evaporates it cools the surrounding glassware and atmospheric water condenses on the surfaces which is absorbed by the filtered product. This problem was overcome by the use of gravity filtration over diethyl ether heated to reflux.

The filter paper was then moved quickly to a warm drying cupboard to prevent the cooling of the surfaces and the resultant condensation and absorption of atmo-spheric water into the salt. Different levels of hygroscopicity were encountered for various other salts and so gravity filtration over boiling diethyl ether was utilised in the isolation of all subsequent salts. The solid salts were kept in a desiccator and were exposed as little as possible to open atmosphere. Yields for the deprotection of N -Boc α-amino amides can therefore only be taken as approximate due to the unknown level of hygroscopiscity for every individual hydrochloride salt.

A stronger N -Boc deprotection strategy was required for α-amino amides 40 and 51 (Figure 2.3) with phenol ARBGs. After a 16 h treatment with ethereal hydrochloric

N

O NH

O O

OH N

O NH

O O

OH

40 51

Figure 2.3: The α-amino amides 40 & 51 which require HCl bubbling through an ethe-real solution.

acid some starting material was present (as determined by TLC), and was still present after a further 2 h at reflux. A modified deprotection strategy attempted which conveniently also avoids the use of slightly wet ethereal hydrochloric acid.

Hydrogen chloride is bubbled through a dry ethereal solution of the N -Boc-α-amino amide deprotecting it and forming an insoluble hydrochloride salt which can be collected by filtration as described above.

2.3 1

Generation α-amino amide inhibition assay results

Inital efforts of this study were aimed at the investigation of the chemical variability of the novel inhibitor 4 through alteration of both the STBG and ARBG components whilst keeping the α-amino amide ZBG constant.

Initially exploration of the ARBG and STBG was undertaken separately to assess the possible routes to improve the potency and selectivity of the lead compound 4. Relative initial steady-state rates collected for inhibition of HDAC8 by lead compound 4 are shown in Figure 2.4. The lead compound 4 has a measured IC50

of 206 ±51 nM which corresponds to a ki of 204 ±50 nM using the Cheng-Prusoff equation (Equation 2.1)²⁰⁴, in this case because the KM of the MAL substrate (17 mM) is 85 times that of the substrate concentration ([S]=200 µM), _K^[S]_M is small

and Ki values are very close to IC50 values. The binding energy of 4 to HDAC8 is calculated using Gibbs free energy of binding equation (Equation 2.2).

Ki = IC50

1 + _K^[S]_M (2.1)

∆Gbinding = −RT lnKi (2.2)

The IC50 obtained for 4 differs slightly from the literature reported value of 90 nM¹⁴⁷. Values reported for other inhibitors against HDAC8 can vary greatly - for example Vorinostat (5) has reported IC50 values between 49 nM²⁰⁵ and 2185 nM²⁰⁶. Additionally the precise assay conditions and error values are not reported in the literature and so conclusions about this discrepancy cannot be made. Here assay conditions were kept consistent where possible and comparisons between inhibitors are therefore possible here.

RelativeActivity

[ ] / M

1 10- 1 10- 1 10- 1 10- 1

10-0 0.2 0.4 0.6 0.8 1.0

0.5

IC50= 210 nM

4

Figure 2.4: Experimental data points recorded for 20 HDAC8-4 inhibition assays. Dif-ferent symbols represent data from independent repeats. Fitted line is a calculated inhibiton curve for IC₅₀= 210 nM, the average calculated for 4