sub-micromolar range out of which thirty-three were picked based on their selectivity against human DHODH. The selected molecules were further tested against
3D7 strain of P. falciparum from which five molecules showed activity in
sub-micromolar range. The selected molecules were then tested against multidrugresistant strains of P. falciparum, i.e., HB3 and Dd2. 5-(2-methyl-1H-indol-1-yl)-Npropylthiophene-2-carboxamide exhibited the most promising IC 50 value (42 nM)
against PfDHODH (Fig. 16, XV).
Molecular docking results revealed that the inhibitor may bind to the same site as
teriflunomide in X-ray crystal structure with PDB 1TV5. Additional modifications
of the indole ring system with piperidine, piperazine, pyrazol, benzimidazole, and
2-methyl benzimidazole highlighted that multi-ring substitutions are important for
activity. This might be due to the better interactions shown by multiple ring substitutions in the hydrophobic region of the inhibitor binding site. Replacement of
indole moiety with benzimidazole moiety retained the PfDHODH inhibitory
activity in addition to which physiochemical properties were considerably improved
[65]. Modifications in the amide region lead to the conclusion that secondary amide
is essential for the activity, whereas primary and tertiary amides are inactive.
Cyclopropyl group was found to be the best optimized group at this position as it
occupies the hydrophobic pocket which was later found out to be different from that
occupied by triazolopyrimidine class of compounds [80b]. Second position of the
benzimidazole moiety was also optimized with various alkyl (ethyl, n-propyl) and
polar (hydroxyl, dimethyl amino) substituents out of which simple methyl substitution was found to be the only suitable one and comparable to the unsubstituted
derivative. Methyl substitution at this position also improves the in vitro hepatic
metabolic stability in human microsomes. Replacement of the thiophene ring with
various aromatic substituents (such as 2,5-substituted N-methyl pyrrole, pyrrole,
thiazole, furan, oxazole, and m-/p-substituted phenyl) resulted in considerable
decrease in the potency. Also, substitution at 2,5-position on the thiophene ring is
essential and there is a loss of activity with substitution at 3,4-position (Fig. 16).
Further modifications were focused on the benzimidazole moiety at 4th to 7th
position (Fig. 16, XVI). Simple methyl substitution at 4th, 5th, and 6th position
resulted in a twofold increase in activity (Pf IC 50 39–56 nM) compared to the
corresponding unsubstituted molecule (Pf IC 50 80 nM). Substitution at the 7th
position causes an eightfold reduction in activity (Pf IC 50 609 nM). This can be
interpreted to be due to the steric hindrance in the cavity of the enzyme caused by
substitution at the 7th position (Fig. 16). Hydrophobic electron-withdrawing group
(OCF 3 and CF 3 ) at the 5th position is more favorable, with 2–3-fold increase
in activity (Pf IC 50 22–28 nM, respectively) compared to the 6th position substitution (Pf IC 50 52–98 nM, respectively). The 5-OCF 3- substituted derivative
(N-cyclopropyl-5-(2-methyl-5-(trifluoromethoxy)-1H-benzimidazole-1-yl)thiophene2-carboxamide, Genz-667348; Fig. 16, XVI) was further studied in the acute
P. berghei (ANKA strain) efficacy model and was found to be curative when dosed
orally. The X-ray crystal structure of PfDHODH with Genz-667348 (PDB ID 3O8A,
entry 5, Table 1) revealed an alternate hydrophobic binding pocket different from
that of triazolopyrimidine analogues (Fig. 17) [65]. This may be due to more flexible
Structure-Based Design of PfDHODH Inhibitors …
205
3D7 strain of P. falciparum from which five molecules showed activity in
sub-micromolar range. The selected molecules were then tested against multidrugresistant strains of P. falciparum, i.e., HB3 and Dd2. 5-(2-methyl-1H-indol-1-yl)-Npropylthiophene-2-carboxamide exhibited the most promising IC 50 value (42 nM)
against PfDHODH (Fig. 16, XV).
Molecular docking results revealed that the inhibitor may bind to the same site as
teriflunomide in X-ray crystal structure with PDB 1TV5. Additional modifications
of the indole ring system with piperidine, piperazine, pyrazol, benzimidazole, and
2-methyl benzimidazole highlighted that multi-ring substitutions are important for
activity. This might be due to the better interactions shown by multiple ring substitutions in the hydrophobic region of the inhibitor binding site. Replacement of
indole moiety with benzimidazole moiety retained the PfDHODH inhibitory
activity in addition to which physiochemical properties were considerably improved
[65]. Modifications in the amide region lead to the conclusion that secondary amide
is essential for the activity, whereas primary and tertiary amides are inactive.
Cyclopropyl group was found to be the best optimized group at this position as it
occupies the hydrophobic pocket which was later found out to be different from that
occupied by triazolopyrimidine class of compounds [80b]. Second position of the
benzimidazole moiety was also optimized with various alkyl (ethyl, n-propyl) and
polar (hydroxyl, dimethyl amino) substituents out of which simple methyl substitution was found to be the only suitable one and comparable to the unsubstituted
derivative. Methyl substitution at this position also improves the in vitro hepatic
metabolic stability in human microsomes. Replacement of the thiophene ring with
various aromatic substituents (such as 2,5-substituted N-methyl pyrrole, pyrrole,
thiazole, furan, oxazole, and m-/p-substituted phenyl) resulted in considerable
decrease in the potency. Also, substitution at 2,5-position on the thiophene ring is
essential and there is a loss of activity with substitution at 3,4-position (Fig. 16).
Further modifications were focused on the benzimidazole moiety at 4th to 7th
position (Fig. 16, XVI). Simple methyl substitution at 4th, 5th, and 6th position
resulted in a twofold increase in activity (Pf IC 50 39–56 nM) compared to the
corresponding unsubstituted molecule (Pf IC 50 80 nM). Substitution at the 7th
position causes an eightfold reduction in activity (Pf IC 50 609 nM). This can be
interpreted to be due to the steric hindrance in the cavity of the enzyme caused by
substitution at the 7th position (Fig. 16). Hydrophobic electron-withdrawing group
(OCF 3 and CF 3 ) at the 5th position is more favorable, with 2–3-fold increase
in activity (Pf IC 50 22–28 nM, respectively) compared to the 6th position substitution (Pf IC 50 52–98 nM, respectively). The 5-OCF 3- substituted derivative
(N-cyclopropyl-5-(2-methyl-5-(trifluoromethoxy)-1H-benzimidazole-1-yl)thiophene2-carboxamide, Genz-667348; Fig. 16, XVI) was further studied in the acute
P. berghei (ANKA strain) efficacy model and was found to be curative when dosed
orally. The X-ray crystal structure of PfDHODH with Genz-667348 (PDB ID 3O8A,
entry 5, Table 1) revealed an alternate hydrophobic binding pocket different from
that of triazolopyrimidine analogues (Fig. 17) [65]. This may be due to more flexible
Structure-Based Design of PfDHODH Inhibitors …
205