S. S. Kher et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Me
Me
O
O
O
Me
O
H
N
H
N
H
N
H
N
Me
N
H
N
H
CO2H
O
O
O
HO
Me
O
NH2
18
(7 µM)
NH2*HCl
Figure 4.
a-Ketoamide 18 bearing a glutamine P1 side chain as an analogue of the
KITAQ substrate prime side sequence.
O
H
N
CbzHN
O
*
CO2t-Bu
~5%
O
Figure 3. Binding mode of compound 6 in PfSUB1 (PDB code: 4LVO) obtained by
docking calculations using the GOLD docking program. Hydrogen bonds between
the compound and the PfSUB1 structure are indicated by yellow dashed lines.
O
H
33a
NH2
CbzHN
O
N
*
a
CO2t-Bu
O
HN
CO2t-Bu
HO
NHcumyl
Table 2
*
CbzHN
O
*
Deletion of the P5 amino acid and importance of P2–P4 amino acids in peptidic
NH
32
a
-ketoamide PfSUB1 inhibitors
O
33b
~95%
P4
P2
Scheme 5. Synthesis of model
chain (a) TFA/DCM (1:1).
a-ketoamide 33 with a deprotected glutamine side
R4
O
O
R6
R3
O
O
H
N
H
N
H
N
OH
*
Me
N
H
N
H
R5
P3
O
Me
O
O
P1
studies, the side chain of P3 points away from the binding site
towards the solvent. The decreased inhibitory activity of 16 can
be explained by an increased solvatation penalty resulting from
replacing the polar threonine side chain with a methyl group. On
the other hand, the isoleucine in P4 is well accommodated by the
hydrophobic S4 pocket. Changing its side chain to a methyl group
likely results in loss of favourable Van der Waals interactions,
explaining the low potency of compound 17.
To investigate the importance of the P1 substituent, we initially
attempted preparation of compound 18 (Fig. 4) which bears a glu-
tamine side chain in the P1 position as in the preferred KITAQ/
DDEES substrate sequence. Unfortunately we discovered that the
glutamine side chain is not compatible with the electrophilic keto
Compd
R3, R4 (P2)
Me, H
R5 (P3)
R6 (P4)
IC50 (lM)
Me
Me
Me
Me
Me
Me
12
2.5 0.1
>50
HO
HO
HO
Me
Me
Me
Me
Me
Me
Me
Me
13
14
15
16
17
Me, Me
CH2CH2
H, H
>50
0.9 0.2
HO
Me, H
H, H
Me
30
1
group. The characteristic
a-proton signal of the a-ketoamide was
Me
>50
HO
Me
shifted up-field after protecting groups in a precursor were cleaved
off, indicating the disappearance of an adjacent carbonyl group The
structurally simplified model compound 33 was prepared from
precursor 32 (Scheme 5). Compound 33 was investigated by 2D
NMR using TOCSY and HMBC methods which revealed that the
equilibrium is shifted to a cyclic tautomer of 33b and only ꢁ5%
of 33a was present in the solution. The formation of cyclic tauto-
mer is expected also in the case of compound 18 which may
explain its unexpectedly low enzyme inhibitory activity.
of PfSUB1obtained by docking calculations using the X-ray struc-
ture of the enzyme (Fig. 3).25 MD simulation studies indicated that
the P5 substituent might not provide an important contribution to
the binding free energy.24 On this basis, compound 12 (Table 2)
lacking a P5 lysine residue was synthesised and found to display
about two times lower enzymatic activity compared to the original
inhibitor 6. However, the structure of 12 was considerably simpli-
fied, facilitating synthesis of analogues and further SAR studies.
Based on modeling of substrate binding24 and molecular dock-
ing of 6 (Fig. 3), the P2 position appears restricted to small amino
A limited series of pentapeptidic a-ketoamide analogues 19–27
was prepared to explore the importance of the P1 side chain
(Table 3). These studies showed that the S1 sub pocket can accom-
modate only relatively small substituents which is line with MD
simulations.24 Only the ethyl group in the P1 position in compound
25 produced a similar level of inhibitory activity as the methyl ana-
logue 15. Compound 23 bearing an N,N0-disubstituted glutamine
side chain and compound 24 with its glutamine side chain con-
strained in the lactam cycle were prepared as analogues not able
to form a cyclic tautomers; however these were inactive as PfSUB1
inhibitors. Given the preference for polar P1 side-chains in the sub-
strates of PfSUB1,8,11,24 further work is needed to develop the ana-
logues of P1 amino acids which could provide additional binding
acids such as alanine or glycine. Other small acids such as
a-Me-
alanine and 1-aminocyclopropylcarboxylic acid were incorporated
instead of alanine, but this resulted in inactive compounds (Table 2,
compounds 13 and 14). In contrast, incorporation of glycine
instead of alanine in P2 position was beneficial leading to com-
pound 15 with slightly increased inhibitory activity compared to
analogue 12 (Table 2). The importance of the P3 and P4 residues
was investigated by substitution with alanine (Table 2, compounds
16 and 17) According to substrate binding and inhibitor 6 docking