(Fig. 14a) position shows optimal activity and introduction of heteroatom in the
naphthyl ring leads to decrease in enzymatic activity. A smaller aromatic group
other than naphthyl and anthracene moiety leads to decrease in activity. Even
though DSM1 showed good in vitro and whole-cell activity, it was not active
in vivo against P. berghei. It was further observed that the compound showed
reduced plasma concentration on repeated exposure [78, 87]. In 2009, Gujjar et al.
prepared a series of forty new compounds with different substituted phenyl moieties
at R 3 position [87]. Para substitution was found to give active compound compared
to the unsubstituted and o-/m-substituted analogues and large electron-withdrawing
hydrophobic substituents were found to be preferred in the order of
CF 3 > Br > OCF 3 > CH 3 > NO 2 > F > Cl. DSM74 was found to be the best
choice among the prepared series with PfDHODH IC 50 0.28 ± 0.02 µM (Pf3D7
cells EC 50 0.34 ± 0.04 µM). The compound DSM74 was equally potent in
P. falciparum and P. berghei and showed good plasma exposure in mice in vivo
studies. This hit was also more stable in human microsomes (in vitro). This study
established the confidence that this class of compounds can be active in in vivo
studies and there is a scope for further improvement of its metabolic profile.
However, DSM74 showed activity in mid-nanomolar range leaving a wide berth for
further improvement [87].
In 2009, Deng et al. reported the crystal structures of lead PfDHODH inhibitors
(DSM1, DSM2, DSM74) in the inhibitor binding site of PfDHODH (PDB ID 3I65,
3I68, 3I6R, entry 2–4, Table 1) [64]. The triazolopyrimidine ring in all three
inhibitors binds to the polar region at the end of the inhibitor binding tunnel similar
to the teriflunomide binding in crystal structure 1TV5. The amino acids involved
are His185 forming hydrogen bond with N 1 (Fig. 14a) and Arg265 forming
hydrogen bond with N 5 . In case of DSM2 and DSM74, the triazolopyrimidine ring
tilts slightly inside the polar region with the slight reorientation of amino acid
Leu176. Tyr528 show water-mediated hydrogen bond with N 3 of the inhibitor in all
three crystal structures. The orientation of the inhibitor in the cavity is such that C 5
position lies closest to the FMN (6 Å distance) and there is a small channel which
can be further exploited for structure-based drug design. The hydrophobic pocket in
these three crystal structures is different from that of 1TV5. The amino acid residues
of the hydrophobic pocket comprises of Ile237, Leu189, Leu197, Met536, Phe227,
and Phe188. The large aromatic ring of DSM2 is accommodated by small rotational
changes of amino acid residues Leu197 and Met536, which results in the expansion
of the hydrophobic cavity. The smaller phenyl group of DSM74 does not fill the
hydrophobic cavity completely and this might be the reason for its 10-fold less
activity than that of DSM1 and DSM2. There is an extended aromatic stacking
network from FMN to Tyr528 carried forward toward the hydrophobic pocket
through Phe227 which forms edge-to-face p-p interaction with the inhibitor
naphthyl (DSM1)/phenyl (DSM74) ring followed by p-p interaction with Phe188.
The mutagenesis studies of the inhibitor binding pocket suggested that His185 and
Arg265 mutation to Ala raises the IC 50 by 80–90-folds. Other mutations have less
effect on the activity with a raise of 5- to 30-fold in IC 50 value. Also, a very
interesting observation was noted during the crystal structure of the inhibitor. It was
202
S. Bhagat et al.
naphthyl ring leads to decrease in enzymatic activity. A smaller aromatic group
other than naphthyl and anthracene moiety leads to decrease in activity. Even
though DSM1 showed good in vitro and whole-cell activity, it was not active
in vivo against P. berghei. It was further observed that the compound showed
reduced plasma concentration on repeated exposure [78, 87]. In 2009, Gujjar et al.
prepared a series of forty new compounds with different substituted phenyl moieties
at R 3 position [87]. Para substitution was found to give active compound compared
to the unsubstituted and o-/m-substituted analogues and large electron-withdrawing
hydrophobic substituents were found to be preferred in the order of
CF 3 > Br > OCF 3 > CH 3 > NO 2 > F > Cl. DSM74 was found to be the best
choice among the prepared series with PfDHODH IC 50 0.28 ± 0.02 µM (Pf3D7
cells EC 50 0.34 ± 0.04 µM). The compound DSM74 was equally potent in
P. falciparum and P. berghei and showed good plasma exposure in mice in vivo
studies. This hit was also more stable in human microsomes (in vitro). This study
established the confidence that this class of compounds can be active in in vivo
studies and there is a scope for further improvement of its metabolic profile.
However, DSM74 showed activity in mid-nanomolar range leaving a wide berth for
further improvement [87].
In 2009, Deng et al. reported the crystal structures of lead PfDHODH inhibitors
(DSM1, DSM2, DSM74) in the inhibitor binding site of PfDHODH (PDB ID 3I65,
3I68, 3I6R, entry 2–4, Table 1) [64]. The triazolopyrimidine ring in all three
inhibitors binds to the polar region at the end of the inhibitor binding tunnel similar
to the teriflunomide binding in crystal structure 1TV5. The amino acids involved
are His185 forming hydrogen bond with N 1 (Fig. 14a) and Arg265 forming
hydrogen bond with N 5 . In case of DSM2 and DSM74, the triazolopyrimidine ring
tilts slightly inside the polar region with the slight reorientation of amino acid
Leu176. Tyr528 show water-mediated hydrogen bond with N 3 of the inhibitor in all
three crystal structures. The orientation of the inhibitor in the cavity is such that C 5
position lies closest to the FMN (6 Å distance) and there is a small channel which
can be further exploited for structure-based drug design. The hydrophobic pocket in
these three crystal structures is different from that of 1TV5. The amino acid residues
of the hydrophobic pocket comprises of Ile237, Leu189, Leu197, Met536, Phe227,
and Phe188. The large aromatic ring of DSM2 is accommodated by small rotational
changes of amino acid residues Leu197 and Met536, which results in the expansion
of the hydrophobic cavity. The smaller phenyl group of DSM74 does not fill the
hydrophobic cavity completely and this might be the reason for its 10-fold less
activity than that of DSM1 and DSM2. There is an extended aromatic stacking
network from FMN to Tyr528 carried forward toward the hydrophobic pocket
through Phe227 which forms edge-to-face p-p interaction with the inhibitor
naphthyl (DSM1)/phenyl (DSM74) ring followed by p-p interaction with Phe188.
The mutagenesis studies of the inhibitor binding pocket suggested that His185 and
Arg265 mutation to Ala raises the IC 50 by 80–90-folds. Other mutations have less
effect on the activity with a raise of 5- to 30-fold in IC 50 value. Also, a very
interesting observation was noted during the crystal structure of the inhibitor. It was
202
S. Bhagat et al.