ACS Medicinal Chemistry Letters
Letter
compounds were calculated; these are shown in Table 1.
The calculated pKa values showed some correlation with the
enzymatic activity. All the activities were similar to that of the
reference compound except compound 3 which might be due
to suboptimal binding reflected in the weaker docking score.
Based on the enzymatic and cellular data, as well as the
calculated pKa, compound 5 was selected as the starting point
for the next round SAR study. A series of cyclohexane
substitutions were designed and synthesized (Table 2,
improved enzymatic potency. As inspired by our previous
work,26 substituted piperidinyls were investigated (Table 2).
Weak electron-withdrawing substitutions such as benzene
(compound 11, Scheme S11) and benzaldehyde (compound
12, Scheme S12) showed similar potencies in IDO1 enzymatic
assays as compound 5, with reduced cellular potencies.
Interestingly, the phenylformamide substitution (compound
13, Scheme S13) showed 3-fold increase in the enzymatic
potency compared to compound 5. Further substitution at the
para-position with 1-methylpyrazol-4-yl (compound 14,
Scheme S14) resulted in a 10-fold and a 4-fold increase in
the enzymatic and cellular potencies, respectively. The
benzylformamide substitution (compound 15, Scheme S15)
and the sulfonyl substitution (compound 18, Scheme 1)
showed similar potency levels in both enzymatic and cellular
assays as compound 14.
To further analyze binding poses of the designed
compounds, we also performed molecular modeling studies
on compound 14 and 18. As shown in Figure 2, both
compounds shared a similar binding pose in the pocket A. As a
result, the deprotonated oxygen of the N-hydroxylamidine was
positioned to bind the heme iron similarly as epacadostat. In
the pocket B, compound 18 formed the aformentioned
hydrogen bond with the residue Arg231 (Figure 2B).
Interestingly, a significant cation−π interaction was formed
at a distance of 2.47 Å between the Arg231 and the phenyl
group in compound 14 (Figure 2A). Cation−π interactions
were quite common in proteins, protein−ligands and protein−
DNA complexes, and important for protein folding, molecular
recognition and catalysis.29 Thus, it was reasonable to expect
that the similar cation-π interactions in the pocket B could
contribute to the improvement of binding potencies in
compounds, 13−16.
To further evaluate the ADMET properties of compounds
13−15 and 18, we profiled them in CYP and hERG inhibition
assays. As shown in Table S2, all the compounds except 15 had
clean CYP and hERG profiles. In addition to compound 18, we
selected the most potent compound 14 for further in vivo
studies among the compounds forming cation−π interactions.
Two animal models (rat and dog) were used to evaluate the
pharmacokinetics of those two compounds. As listed in Table
3, compound 14 showed poor oral pharmacokinetics in dog,
while compound 18 had a better profile and good oral
exposure in both species.
The synthesis route and in vitro and in vivo Profile for
compound 18 are shown in Scheme 1 and Table 4. In vitro
data indicated that compound 18 was a highly potent and
selective IDO1 inhibitor with clean CYP and hERG profiles. Its
pharmacokinetic profiles in animal models (mouse, rat, and
dog) demonstrated an increased oral exposure and bioavail-
ability from mouse, rat, to dog (F = 44%, 58%, and 102.1%,
respectively). Meanwhile, compared with epacadostat, com-
pound 18 exhibited a superior pharmacokinetic profile in a
Figure 1. (A) Hydroxyamidine derivative as IDO1 inhibitor; (B)
INCB-24360 (navy) binding mode in the crystal structure (PDB
6E40).
has led to the identification of novel IDO1 inhibitors by
bioisosteric replacements of the furazan group, as well as the
alternative groups to the sulfamide side chain.
We designed a set of electron-withdrawing carbonyl groups
to replace the furazan ring (compounds 1−6; the synthesis
to keep the acidity of the hydroxylamidine group. Sub-
sequently, biological assays were employed to evaluate the
biological activities of compounds 1−6, including enzymatic
assays with purified recombinant human IDO1/TDO proteins
and cellular IDO1 inhibition assays using HeLa cell lines. As
shown in Table 1, compound 1 with thiazole substitution
exhibited the best enzymatic activity (IDO1 IC50 = 51 nM)
among those designs. It showed micromolar level activity in
the HeLa cell line, which could be related to the membrane
permeability of the compound. Compounds 2−4 with
aromatic or heterocyclic aromatic substitutions also showed
less potencies compared to epacadostat in enzymatic and
cellular assays. Compound 5 with saturated six-member ring
showed a modest enzymatic activity, and the best cellular
activity among the designed compounds. As shown by the
structure−activity relationship (SAR) data of compound 6, the
carbonyl group of compound 5 was quite important for its
enzymatic and cellular potencies.
A molecular modeling study was carried out to further
understand the SAR of these compounds. It was well-known
that the interaction between the deprotonated oxygen and the
heme iron is important for biological activity.18,19 So we
hypothesized that the oxygen of the N-hydroxyamidine was in
the deprotonated state and the heme iron was in its ferrous
state (Fe2+). Compounds 1−6 were docked into the binding
pocket using MOE.23,24 A commonly used docking score in
MOE, GBVI/WSA dG28 alone, could not be used to
distinguish actives from inactives (Table 1). Then, the pKa
values25 of the heme interaction oxygens from each
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ACS Med. Chem. Lett. 2021, 12, 195−201