Discovery of Highly Potent Benzimidazole Derivatives as Indoleamine 2,3-Dioxygenase-1 (IDO1) Inhibitors: From Structure-Based Virtual Screening to in Vivo Pharmacodynamic Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b01809

In this study, a successful medicinal chemistry campaign that exploited virtual, biophysical, and biological investigations led to the identification of a novel class of IDO1 inhibitors based on a benzimidazole substructure. This family of compounds is endowed with an extensive bonding network in the protein active site, including the interaction with pocket C, a region not commonly exploited by previously reported IDO1 inhibitors. The tight packing of selected compounds within the enzyme contributes to the strong binding interaction with IDO1, to the inhibitory potency at the low nanomolar level in several tumoral settings, and to the selectivity toward IDO1 over TDO and CYPs. Notably, a significant reduction of L-Kyn levels in plasma, together with a potent effect on abrogating immunosuppressive properties of MDSC-like cells isolated from patients affected by pancreatic ductal adenocarcinoma, was observed, pointing to this class of molecules as a valuable template for boosting the antitumor immune system.

Full text of DOI:10.1021/acs.jmedchem.9b01809

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Article
Discovery of highly potent benzimidazole derivatives as
indoleamine 2,3-dioxygenase-1 (IDO1) inhibitors: from structure-
based virtual screening to in vivo pharmacodynamic activity
Marta Serafini, Enza Torre, Silvio Aprile, Erika Del Grosso, Alessandro Gesù, Alessia Griglio,
Giorgia Colombo, Cristina Travelli, Salvatore Paiella, Annalisa Adamo, Elena Orecchini, Alice
Coletti, Maria Teresa Pallotta, Stefano Ugel, Alberto Massarotti, Tracey Pirali, and Silvia Fallarini
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01809 • Publication Date (Web): 09 Mar 2020
Downloaded from pubs.acs.org on March 10, 2020
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Discovery of highly potent benzimidazole
derivatives as indoleamine 2,3-dioxygenase-1
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(
IDO1) inhibitors: from structure-based virtual
screening to in vivo pharmacodynamic activity
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‡
1‡
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Marta Serafini, Enza Torre, Silvio Aprile, Erika Del Grosso, Alessandro Gesù, Alessia
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Griglio, Giorgia Colombo, Cristina Travelli, Salvatore Paiella, Annalisa Adamo, Elena
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Orecchini, Alice Coletti, Maria Teresa Pallotta, Stefano Ugel, Alberto Massarotti, * Tracey
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Pirali * and Silvia Fallarini
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Department of Pharmaceutical Sciences; Università del Piemonte Orientale; Novara, 28100;
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Italy; Department of Pharmaceutical Sciences, Università degli Studi di Pavia, Pavia, 27100,
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Italy; General and Pancreatic Surgery, Pancreas Institute; University of Verona, Verona, Italy
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7134; University Hospital and Department of Medicine, Section of Immunology; University of
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Verona, Verona, Italy 37126; Department of Experimental Medicine; University of Perugia;
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Perugia, 06132; Italy; Department of Medicine; University of Perugia, Piazza Lucio Severi 1,
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6132 Perugia, Italy
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ABSTRACT
In this study, a successful medicinal chemistry campaign that exploited virtual, biophysical and
biological investigations led to the identification of a novel class of IDO1 inhibitors based on a
benzimidazole substructure. This family of compounds is endowed with an extensive bonding
network in the protein active site, including the interaction with pocket C, a region not commonly
exploited by previously reported IDO1 inhibitors. The tight packing of selected compounds within
the enzyme contributes to the strong binding interaction with IDO1, to the inhibitory potency at
the low nanomolar level in several tumoral settings and to the selectivity towards IDO1 over TDO
and CYPs. Notably, a significant reduction of L-Kyn levels in plasma, together with a potent effect
on abrogating immunosuppressive properties of MDSC-like cells isolated from patients affected
by pancreatic ductal adenocarcinoma, were observed, pointing to this class of molecules as a
valuable template for boosting the antitumor immune system.
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Introduction
Cancer immunotherapy is well-established as an effective clinical option for cancer treatment,
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alongside surgery, chemotherapy and radiation. Indeed, immune checkpoint inhibitors (targeting
CTLA-4 or PD-1/PD-Ll pathways) have become first-line therapies in advanced non-small cell
lung cancer and melanoma.2
Nevertheless, this approach has shown substantial benefit to only some of the patients, while the
rest do not respond, and even the patients that have initially a benefit might fail to respond in later
stages. Mechanisms underlying resistance to cancer immunotherapy are complex and still under
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debate, but most of them rely on the immunosuppressive tumor microenvironment. Therefore, the
search for novel strategies able to boost the antitumor immune system and make cancer
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immunotherapy more versatile, durable and effective is an urgent need. In this context, among the
different combinatorial approaches that reverse tumor immunosuppression, the inhibition of
indoleamine 2,3-dioxygenase-1 (IDO1) has taken center stage.
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IDO1 is an intracellular enzyme catalyzing the first, rate-limiting step of tryptophan (L-Trp)
catabolism resulting in the production of L-kynurenine (L-Kyn). IDO1 is expressed by primary
and metastatic tumor cells, intra-tumoral endothelial cells, immune cells of peri-tumoral stroma
and tumor-draining lymph nodes. This enzyme is a key mediator in the establishment of tumor
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immune escape through different mechanisms. In particular, L-Trp degradation and L-Kyn
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production induce: a) inhibition of mTOR1 and activation of GCN2, resulting in dysfunction and
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apoptosis of T effector cells; b) suppression of NK cell proliferation and functions; c)
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differentiation and activation of regulatory T (Treg) cells, tolerogenic dendritic (DC) and
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myeloid-derived suppressor cells (MDSCs); d) tumor neovascularization. Moreover, IDO1
expression and/or activity have been observed in several cancer types and are well correlated with
poor prognosis and low survival of patients.13
Recently, a better understanding of the molecular pathways linking L-Trp catabolism and tumor
immune tolerance has provided novel potential targets. In particular, L-Trp dioxygenase (TDO),
initially thought to be expressed only in liver, can be found in some other tumor types (e.g.
glioblastoma, breast cancer). Furthermore, preliminary in vitro studies revealed its involvement in
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tumor induce immune tolerance, tumor cell resistance to anoikis, and metastatic prowess.
Besides TDO, IDO2, whose functions are currently under investigation, is overexpressed in tumor
microenvironment, functionally enabling IDO1-dependent Treg and tolerogenic DC generation
and contributing to B cell-mediated autoantibody production. This latter aspect is an important
issue in the development of those types of cancer that rely upon B-cell to induce inflammation.16
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Among the three L-Trp metabolizing enzymes, most attention has been paid to IDO1 and over the
last decades thousands of compounds have been reported as IDO1 selective inhibitors in cancer
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immunotherapy. Only few have reached clinical trials, and none of these has been approved so
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far, suggesting that the identification of potent and clinically useful IDO1 inhibitors is an open
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challenge. Epacadostat (1, INCB024360, Figure 1), indoximod (2, 1-methyl-D-tryptophan,
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Figure 1), navoximod (3, NLG-919, Figure 1), EOS-200271 (4, PF-06840003, Figure 1) and
BMS-986205 (5, Figure 1) have been/are currently being tested in human clinical trials.
Based on promising results in Phase 1/2 studies, epacadostat has proceeded to Phase 3 trial
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ECHO-301) in combination with pembroluzimab in metastatic melanoma.23 Recent results
coming from the pivotal Phase 3 of ECHO-301 have shown no indication that epacadostat provides
an increased benefit compared to pembrolizumab alone, questioning the effectiveness of IDO1
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inhibitors. This failure led to the interruption of other phase III trials and to reconsider whether
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some elements had been neglected in the landscape of IDO1 inhibitors. Among them, a nodal
point lies in the identification of biomarkers to stratify patients that respond better to this therapy.
In this regard, the detection of IDO1 expression and L-Kyn/L-Trp levels in tumor and metastatic
cells, as well as in peripheral blood mononuclear cells (PBMCs), would represent good prognostic
and response markers.26
On a different note, availability of novel IDO1 inhibitors with different profiles in terms of PD and
PK properties may be necessary to restore hope to IDO1 inhibition and achieve clinical benefit.
Prompted by the challenge of discovering novel and effective IDO1 inhibitors, we have performed
a medicinal chemistry campaign that has started with a structure-based virtual screening approach
and has led to the identification of potent benzimidazole derivatives.
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Figure 1. Chemical structures of IDO inhibitors that have reached clinical development.
Results
Structure-based virtual screening
Most IDO1 inhibitors are characterized by a binding mode that exploits the coordination with heme
iron and the interaction with pocket A, a large lipophilic pocket localized above the sixth
coordination site of the iron-heme, and pocket B, a lipophilic area located at the binding site
entrance (for a representation of IDO1 active site see Figure 2).18b In 2018 our research group
published a class of imidazothiazoles featuring a peculiar binding mode in the IDO1 active site,
with the side chain protruding into an additional pocket, named C, where a crucial hydrogen bond
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is formed with Lys238. We therefore integrated this novel information in a structure-based
virtual screening. To this aim, all the bioactivity data of already known tested molecules were
extracted and downloaded from scientific literature, following an approach already reported in our
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previous paper, in order to validate the protocol. The database chosen for our screening purpose
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was ZINC15, which contains readily purchasable and drug-like compounds. OMEGA2 program
was used to generate a maximum of 500 conformations for each molecule with a root mean square
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deviation (RMSD) of 0.8 Å between conformers. The processed database was docked into IDO1
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active site (PDB structure 2D0T) with HYBRID using default parameters, the results were sorted
using the predicted binding energy (Chemgauss4), and duplicates were removed. The top ranked
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00 molecules were analyzed by visual inspection and by evaluating their pose in the active site
of IDO1. Among them, the first 50 commercially available molecules were selected and purchased
from eMolecules®.31
Evaluation of cellular inhibitory activity and identification of compound 10
All the purchased molecules were tested for their IDO1 inhibition activity in a cell-based assay.
The melanoma derived A375 cell line was used as cellular model, since it expresses high levels of
IDO1 (RT-PCR) and L-Kyn (HPLC) when treated with recombinant human IFN-γ.27a For the first
screening, A375 cells were untreated/treated with 10 µM of each compound for 48 h and L-Kyn
levels were measured. A number of ten compounds out of 50 displayed an inhibitory activity >30%
at the tested concentration and in all cases cell viability was >85%, confirming that the observed
reduction in L-Kyn levels was not related to direct compound cytotoxicity. For the seven most
active compounds, the IC50 value was determined in the cell-based assay (see Supporting
Information for the IC50 values) and ranged from 4.4 µM to 0.0016 µM. The seven identified
compounds displayed no similarities in their structures and, among them, the most potent molecule
was 10 (Figure 2B), with an IC50 value of 16 nM. We therefore decided to focus our efforts on this
hit, prompted by the fact that it was a novel chemotype (while other emerged compounds had
already been reported in the literature), that it was far more potent than the other identified
molecules (with at least a ten-fold lower IC50 compared to the other emerged compounds), and that
no IDO1 inhibitor with similar structure had been previously described.
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To validate our discovery, the hit was synthesized in our laboratory according to Scheme 1. The
synthetic protocol afforded the desired amine 9 in two steps: substitution between 1H-
benzo[d]imidazole and 3-(bromomethyl)benzonitrile and subsequent reduction of the aromatic
nitrile 8. Coupling between intermediate 9 and 4-bromo-1H-pyrrole-2-carboxylic acid provided
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0 in good yield, highlighting the high synthetic feasibility displayed by our hit. The compound
was re-tested at the same conditions and its potency was in accordance with the compound
purchased from eMolecules®.
Scheme 1. Re-synthesis of the hit compound 10.
Reagents and conditions: (a) KOH, acetone, 60 °C, 4 h, 99%. (b) LiAlH , dry THF, 65 °C - rt, 18
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h, 70%. (c) TEA, HOBt, EDCI, 4-bromo-1H-pyrrole-2-carboxylic acid, dry CH
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Cl , rt, 18 h, 50%.
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Ligand binding experiments
In order to confirm target engagement of IDO1 by compound 10, binding experiments were carried
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out using MicroScale Thermophoresis (MST) assays on recombinant human IDO1. Epacadostat
(
1) was used as positive control in this assay. As a result, a low dissociation constant was found
for 10 (K = 0.55 ± 0.36 µM; see Supporting Information), whereas a higher dissociation constant
was determined for epacadostat (1, K
d
d
= 3.46 ± 0.86 M, see Supporting Information). The K
d
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value of compound 10 suggests that it does bind to IDO1, and therefore proves a direct target
engagement, accounting for the potent reduction of L-Kyn production in melanoma derived A375
cell line.
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Docking pose and SAR study of compound 10
The docking pose of 10 (Figure 2) reveals a peculiar putative binding mode: the benzimidazole
group is accommodated in pocket A (Tyr126, Cys129, Val130, Phe163 and Phe164) and its
nitrogen coordinates the iron of the heme group. In contrast with other reported inhibitors, pocket
B (Phe226, Arg231 and Ser235) is not fully exploited since the phenyl ring is located in the
proximity of this pocket. Here, the carbonyl group establishes a hydrogen bond with Ser235. Two
additional hydrogen bonds are formed between the carboxylic moiety of the heme group and both
the amide and the pyrrole NH moieties. The long side-chain protrudes from the solvent-exposed
region of the active site towards pocket C, with the bromine that forms a putative halogen bond
with the close carbonyl group of Lys238.33
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Figure 2. Structure and docking pose of the identified hit compound 10. (A) Location of the
binding region on IDO1 crystal structure (PDB id: 2D0T); (B) Schematic representation of
predicted interactions of compound 10 within IDO1 binding pockets; (C) Docking pose of hit
compound 10. Aminoacids of pocket A, pocket B and pocket C are depicted in red, green and blue
respectively. Heme group and compound 10 are depicted as cyan and pink sticks.
Starting from the scaffold of compound 10 and its putative binding mode, a SAR study was
undertaken, with the aim of validating the predicted interactions by chemical modification of 10
and biological evaluation of the obtained analogues. The synthesized analogues were tested as
IDO1 inhibitors in a cellular-based assay. The A375 cells unstimulated/stimulated with
recombinant human IFN-γ were untreated/treated with 1 µM of each compound for 48 h and L-
Kyn levels were measured by HPLC.27a For compounds showing an inhibitory activity >80% and
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cell viability >80% the concentration/response curves were determined and IC50 values calculated
(Table 1).
First of all, in order to investigate the role of the nitrogen of the benzimidazole in the coordination
with the iron of the heme group, compound 14 was synthesized. Substitution between 1H-indole
and 3-(bromomethyl)benzonitrile and subsequent reduction of the nitrile group afforded the
intermediate 13, that, after coupling with 4-bromo-1H-pyrrole-2-carboxylic acid, yielded
compound 14 (Scheme 2). As expected, the nhibitory activity was completely abolished (0% of
inhibition, Table 1), confirming that the presence of the benzimidazole ring is crucial for the
coordination with the heme group in the active site.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of compound 14.
Reagents and conditions: (a) KOH, acetone, 60 °C, 4 h, 71%. (b) LiAlH
4
, dry THF, 65 °C - rt, 18
Cl , rt, 18 h, 54%.
h, 46%. (c) TEA, HOBt, EDCI, 4-bromo-1H-pyrrole-2-carboxylic acid, dry CH
2
2
Next, the role of the key hydrogen bond between the amide and the carboxylic moiety of the heme
group was confirmed by the synthesis and evaluation of compound 17, where the -NH- of the
secondary amide is substituted with the -O- of the ester. 17 was prepared according to Scheme 3,
starting from 1H-benzo[d]imidazole and (3-(bromomethyl)phenyl)methanol to afford intermediate
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6 that underwent esterification with 4-bromo-1H-pyrrole-2-carboxylic acid to provide the desired
compound. 17 is devoid of inhibitory activity at 1 µM, substantiating that the -NH- of the
secondary amide is involved in the formation of a pivotal hydrogen bond within the IDO1 active
site.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of compound 17.
Reagents and conditions: (a) KOH, acetone, 60 °C, 2 h, 34%. (b) TEA, DMAP, EDCI, 4-bromo-
1
H-pyrrole-2-carboxylic acid, dry CH
2
Cl , rt, 18 h, 40%.
2
Furthermore, the importance of the halogen bond between the bromine at 3 position on the pyrrole
ring and the close carbonyl group of Lys238 was investigated by the synthesis of compound 18
that was obtained by coupling the amine 9 with the appropriate carboxylic acid, according to
Scheme 4. As suggested by the docking pose, the presence of the bromine contributes to the
binding as its removal leads to a decrease in activity (18, 72% of inhibition).
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4. Synthesis of compounds 18-39.
Reagents and conditions: (a) TEA, HOBt, EDCI, dry CH
2
Cl , rt, 18 h, 13-86%.
2
Finally, compounds 19 and 20, displaying a furan and a thiophene ring, respectively, were prepared
as described in Scheme 4 and evaluated with the aim of confirming the role of the -NH- of the
pyrrole ring of 10 in estabilishing the hydrogen bond with the carboxylic moiety of the heme group.
As expected, a decrease in the inhibitory activity occurs in both cases (19 and 20, 25% and 62%
of inhibition, respectively).
Besides the above SAR study, in order to investigate the role of the 4-bromopirrole moiety placed
in pocket C and further improve the potency of compound 10, a series of analogues was in silico-
designed and synthesized (21-39, 43, 46, 47). To this aim, about 44,000 purchasable carboxylic
acids have been virtually combined with compound 9, and the resulted library of candidates has
been screened in the IDO1 active site, using the same virtual screening procedure described before.
Compounds have been ranked according to their binding energies in the protein, as well as their
drug-like profiles. The virtual candidates that displayed the highest score have been selected for
synthesis.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
By coupling amine 9 with different carboxylic acids (Scheme 4), 19 benzimidazoles have been
obtained in good to excellent yields (22-24, 26-39, 43), with only two exceptions (21, 25) showing
poor yield (Table 1). For the synthesis of compound 43 protection and deprotection of the
additional amine group were necessary, according to Scheme 5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5. Synthesis of compound 43.
Reagents and conditions: (a) 2 M aq. NaOH, Boc
2
O, THF, 16 h, 86%. (b) Compound 9, TEA,
Cl2, 0 °C, 30 min, 71%.
HOBt, EDCI, dry CH Cl , rt, 18 h, 57%. (c) CF COOH, dry CH
2
2
3
2
Benzofuran 21 shows an IC50 value of 413 nM, while benzothiophene (22) provides a lower
inhibitory activity (77% of inhibition). If pyrrole is fused with both a non-substituted (23) or a
substituted (24, 25) phenyl ring, the activity is retained (IC50 = 72, 636, 781 nM, respectively),
while when a 5-methoxy-1H-indole substituent is present (26) a significant cytotoxicity is
observed (55% of viability at 1 µM). Finally, the benzimidazole 27 is well tolerated (IC50 = 407
nM), while a benzodioxolane ring is not (28, 5% of inhibition).
The SAR study reveals that for the analogues displaying a phenyl ring at the R position (29-38),
1
when unsubstituted, the inhibitory activity is lost (29, 32% of inhibition). Electron-donating (30)
and electron-withdrawing groups such as trifluoromethyl (31), bromine (32), chlorine (33) and
fluorine (34) at position 4' reduce potency compared to 10. On the other hand, compound 35
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9
displaying a cyano group at position 4' retains a good potency (IC50 = 90 nM), while the activity
is lower when other substituents are added on the -CN substituted phenyl ring (36, 37, 38).
Compounds 46 and 47 have been synthesized by using Ugi multicomponent reaction (MCR) and
exploiting ammonium chloride as a nitrogen source. The required isocyanide 45 has been
synthesized starting from amine 9 that was formylated and dehydrated. Then, two MCRs have
been set up using isocyanide 45, paraformaldehyde, ammonium chloride and acetic or benzoic
acid, yielding compounds 46 and 47, respectively (Scheme 6). The yields of both reactions were
very poor (30-32%), not surprisingly since it is well known that Ugi MCR does not work well
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
when ammonia is employed as nitrogen source. The introduction of a polar moiety (39, 43) at R
1
position or a peptidic substructure (46, 47) in the side chain are not tolerated, inducing a drop in
activity.
Scheme 6. Synthesis of compounds 46 and 47.
Reagents and conditions: (a) Ac
2
O, HCOOH, 3 h, 98%. (b) TEA, POCl
3
, dry CH
2
Cl , 0 °C, 2.5 h,
2
4
4%. (c) NH Cl, CH O, TEA, R
4
2
2
COOH, MeOH, H O, 65 °C, 8 h, 30-32%.
2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Since pocket A is not completely occupied by compound 10 and other reported IDO1 inhibitors
display halogen atoms to increase the affinity with this buried pocket, a bromine atom was
introduced at position 6 of the benzimidazole core with the aim of improving the affinity with
pocket A. Compound 53 was synthesized following the synthetic route outlined in Scheme 7. The
desired amine 52 was prepared via a three-step procedure: nucleophilic aromatic substitution,
cyclization and reduction of the nitrile group (Scheme 7).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 7. Synthesis of compound 53.
Reagents and conditions: (a) K
2
CO
3
, DMSO, 72 °C, 16 h, 13%. (b) formic acid, Fe, 85 °C, 16 h,
Cl , rt, 18 h, 39%.
6
9%. (c) LiAlH , dry THF, rt, 16 h, 40%. (d) TEA, HOBt, EDCI, dry CH
4
2
2
Compound 53 shows an IC50 value of 19 nM, but its chemical instability together with the lack of
improvement in ADME properties compared to the hit compound (data not shown) have prompted
us to discard the compound.
Taking into consideration all these data, the main findings are represented in Figure 3 and the
compounds 10, 23 and 35 that display no significant cytotoxicity and IC50 values of 16, 72 and 90
nM, respectively, have been selected for further evaluation.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Graphical representation of SAR study around compound 10.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1. SAR study around compound 10.
0
1
2
3
4
5
6
7
8
9
0
1
Cell
IDO cellular
assay
inhibition
(%) @ 1 µM
Cell
IDO cellular
assay
inhibition
(%) @ 1 µM
Cpd,
IC50
Cpd,
IC50
viability
(%) @ 1
viability
(%) @ 1
µM ± SD
X
Y
R
1
R
3
X
Y
R
1
R
3
Yield (%)
(nM)
Yield (%)
(nM)
µM ± SD
1
-
-
-
-
97 ± 2.5
92 ± 3.6
96 ± 0.2
7.7
16
29, 54%
30, 69%
NH
NH
N
N
H
H
98 ± 2
94 ± 3
32 ± 15
88 ± 2.1
-
2 10, 50%
3
4
5
NH
N
H
H
H
100 ± 0.5
327
6 14, 54%
NH
O
H
N
100 ± 0
100 ± 0
0 ± 4
0 ± 2
-
-
31, 82%
32, 77%
NH
NH
H
N
H
H
97 ± 3
96 ± 4
81 ± 4.9
98 ± 1.4
-
7
8
9
1
7, 40%
134
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1
1
8, 62%
9, 65%
NH
NH
N
N
H
H
94 ± 6
72 ± 2.1
-
-
33, 60%
34, 78%
NH
NH
N
N
H
H
96 ± 4
89 ± 7
99 ± 0
477
961
100 ± 0
25 ± 11.4
88 ± 7.1
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8
Cell
viability
%) @ 1
µM ± SD
IDO cellular
assay
inhibition
(%) @ 1 µM
Cell
IDO cellular
assay
inhibition
(%) @ 1 µM
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Cpd,
IC50
Cpd,
IC50
viability
(%) @ 1
µM ± SD
X
Y
R
1
R
3
X
Y
R
1
R
3
0
1
2
3
(
Yield (%)
(nM)
Yield (%)
(nM)
4 20, 78%
NH
NH
N
N
H
H
98 ± 1
62 ± 9.9
91 ± 4
-
35, 77%
36, 51%
NH
NH
N
N
H
H
100 ± 1
94 ± 1
88 ± 6.9
90
-
5
6
7
2
2
2
1, 37%
2, 52%
3, 54%
100 ± 0
88 ± 1
99 ± 4
99 ± 1
413
44 ± 16.9
8
9
0
1
2
3
4
5
6
7
NH
NH
NH
NH
N
N
N
N
H
H
H
H
77 ± 1.2
94 ± 3.5
90 ± 7.5
71 ± 15.5
-
37, 60%
38, 48%
39, 55%
43, 71%
NH
NH
NH
NH
N
N
N
N
H
H
H
H
99 ± 1
100 ± 0
100 ± 1
100 ± 0
37 ± 0
32 ± 14.5
0 ± 0
-
-
-
-
72
8 24, 49%
636
781
9
0
1
2
2
2
5, 13%
6, 56%
7, 86%
99 ± 1
55 ± 11
100 ± 0
0 ± 0
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
NH
NH
N
N
H
H
-
-
46, 30%
47, 32%
NH
NH
N
N
H
H
100 ± 0
99 ± 0
0 ± 0
-
-
83 ± 10.6
407
13 ± 0.7
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Cell
viability
%) @ 1
µM ± SD
IDO cellular
assay
inhibition
(%) @ 1 µM
Cell
IDO cellular
assay
inhibition
(%) @ 1 µM
Cpd,
IC50
Cpd,
IC50
viability
(%) @ 1
µM ± SD
X
Y
N
R
1
R
3
X
Y
N
R
1
R
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
(
Yield (%)
(nM)
Yield (%)
(nM)
2
8, 84%
NH
H
96 ± 3
5 ± 6.4
-
53, 39%
NH
Br
99 ± 1
98 ± 1.7
19 nM
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Profiling of compounds 10, 23 and 35
IDO1 and TDO selectivity
The three selected compounds were tested in a mastocytoma cell line stably expressing mouse
IDO1 (P1.IDO1) or TDO (P1.TDO) in order to evaluate their selectivity. To this aim, cells
P1.IDO1 and P1.TDO were treated with different dilutions of 10, 23 and 35 for 16 h and L-Kyn
secretion in cell culture supernatants was detected by HPLC analysis. In these settings, the
compounds behaved as selective inhibitors of IDO1 against TDO at 1 µM, yet when the
concentration was increased up to 10 µM, an inhibition of TDO was observed to some extent. We
measured the IC50 value of each compound in the two cell lines and we found that IC50 of
compound 10 is 12.7 nM in P1.IDO1 and 5.46 µM in P1.TDO. Moreover, for 23 IC50 is 9.3 nM in
P1.IDO1 and 13.2 µM in P1.TDO, whereas for 35 IC50 is 111.7 nM and 32.3 µM, respectively,
suggesting a high selectivity of the three compounds towards IDO1 over TDO .
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IDO1 inhibitory activity in a set of different tumoral cell lines
Compared to epacadostat (1, Figure 1), compound 10 showed a very similar activity in the A375
cell line, with IC50 values of 7.7 nM and 16 nM, respectively. The cellular potency of 10 in
inhibiting L-Kyn production was further investigated using several human cell lines, including
cervical cancer derived HeLa cells, lung cancer derived LXF-289 cells, breast cancer derived
MCF-7 cells, liver cancer derived HepG2 cells and pancreas cancer derived DAN-G cells. As
shown in Table 2, compound 10 is able to inhibit L-Kyn production in most cell lines with potency
comparable with that observed in A375 cell line, suggesting that it effectively inhibits IDO1
enzyme activity regardless of cell source. Compared to the other cell lines, in the pancreatic
derived DAN-G cells 10 showed an IC50 value of one order of magnitude greater (0.605 µM ±
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9
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
.0626) than those observed in the other cells. This result could be ascribed to the concomitant
expression of other Trp degrading enzymes such as TDO, as suggested by Western Blot analysis
(data not shown), or IDO2, and to the fact that, as reported above, compound 10 is a selective
inhibitor of IDO1.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Potency of compound 10 on different cell lines.
Cell line
A375
IC50 (μM)
0.016 ± 0.0004
0.064 ± 0.0018
0.014 ± 0.0002
0.021 ± 0.0006
0.043 ± 0.0016
0.605 ± 0.0626
HeLa
LXF-289
MCF-7
HepG2
DAN-G
On the same note, the activity of 23 and 35 was measured on human breast derived MCF-7 and
human pancreas derived DAN-G cell lines as in vitro models. As shown in Table 3, the IC50 values
obtained in MCF-7 cell line were slightly increased for both compounds (0.083 µM ± 0.005 and
0
.11 µM ± 0.23 for 23 and 35, respectively). As observed for compound 10, the IC50 values
measured in DAN-G cell line were higher than those observed in A375 cell line for both
compounds, in particular of one and two order of magnitude greater for compounds 23 and 35,
respectively.
Table 3. Potency of compounds 23 and 35 on different cell lines.
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Compound
Cell line
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IC50 (μM) 23
IC50 (μM) 35
0.072 ± 0.002
0.083 ± 0.005
0.14 ± 0.007
0.09 ± 0.006
0.11 ± 0.23
1.94 ± 0.23
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Molecular dynamics
The docking results of compounds 10 (Figure 2), 23 and 35 (see Supporting Information) were
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further analyzed with Molecular Dynamics (MD) simulations using Desmond package. The MD
of IDO1/compound complexes were simulated for 100ꢀnsꢀat 300ꢀK using a standard protocol. The
protein structure was stabilized, as shown in the RMSDs for both the IDO1 Cα and the ligand (see
Supporting Information). In the MD simulation the benzimidazole moiety of 10 is stabilized in
pocket A, where it is able to interact, for the majority of time, with the amino acidic residues of
Phe163 and Tyr126, as well as with the heme group. This behavior is consistent with the docking
results, as well as the contacts near the pocket B, such as with Arg231. On the other hand, the
hydrogen bond interaction with Ser235 occurs for only the 50% of the simulation time. The pyrrole
group of 10 makes interactions with the amino acids in pocket C, that are present for up to 10% of
the simulation time. The conformation of hIDO1 changes during the MD, and in this
relaxed/dynamic situation the side-chain of 10 that is more exposed to the solvent is shifted near
the pocket B. In this new conformation, the pyrrole group can give a cation-π interaction with
Arg231.
Compound 23 adopts two different poses during the MD, one within pocket C and the other near
pocket B, and, as for compound 10, a cation-π interaction with Arg231 is formed for the 80% of
the simulation time. Similarly, compound 35 can adopt two different poses: half of the time it
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maintains the interaction with pocket C, while in the other half the aromatic ring bearing a nitrile
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In vitro metabolic stability
The in vitro metabolic stability of 10, 23 and 35 was evaluated in mouse liver microsomes (MLM)
activated by NADPH by measuring the substrate residual after one hour of incubation. All three
compounds undergo oxidative metabolism, with a residual substrate of 32%, 65% and 59%,
respectively. Similar percentages have been observed in the incubation mixtures supplied with
glutathione (GSH). Next, we performed the metabolites structural characterization by high
resolution mass spectrometry (HRMS), processing the raw data with a workflow aimed for drug
metabolites discovery provided by Compound Discoverer 3.0 software (Thermo Scientific).
Overall, the three compounds undergo two main transformations (Figure 4): benzimidazole
oxidation at different positions and oxidative N-dealkylation at both the benzylic carbon atoms,
the latter leading to the formation of the corresponding carboxylic acid further reduced to aldehyde
and alcohol. Despite a higher metabolic stability compared to the other two analogues, the indole
heterocycle of 23 is susceptible to microsomal oxidation and at least three metabolites have been
detected. Interestingly, data analysis did not highlight the formation of GSH adducts, suggesting
that metabolism is not driven toward the formation of reactive species. Full data of metabolites
structures and mass spectral data, as well as the metabolic pathways, are given in the Supporting
Information.
Based on the observed liability, strategies aimed at reducing the susceptibility toward metabolism
might be worth of investigation. The blockage of the putative sites of hydroxylation by using
fluorine or deuterium atoms would presumably not impare the activity of the compounds on IDO1,
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while improving resistance toward metabolism (Figure 4). While the use of fluorine is well
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established in medicinal chemistry, deuterium incorporation has only recently exploded, after the
approval by FDA of the first deuterated drug. Within our class of molecules, the deuterium kinetic
isotope effect (DKIE) might be exploited to mitigate both the aldehyde oxidase (AO)-driven
oxidation of benzimidazole ring and the amide N-dealkylation.37
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Figure 4. Metabolic pathways of compounds 10, 23 and 35 in MLM and possible strategies to
improve metabolic stability. The labile soft spots are highlighted in blue.
CYP inhibition
Since cytochrome P450 (CYP) family shares with IDO1 the iron-containing heme group, all the
candidates have been evaluated for their inhibitory activity on these enzymes. Aminopyrine N-
demethylase assay was performed in order to evaluate the inhibition of CYPs by 10, 23 and 35 and
to predict the risk of pharmacokinetic drug interactions. 10 showed a mild inhibitory activity with
an IC50 value of 67.7 µM compared to incubations with ketoconazole (CYP3A inhibitor), which
gave an IC50 value of 6.4 µM in our assay. For 23 and 35, IC50s curves could not be fitted for the
absence of the bottom plateau phase which is related to their inability to inhibit CYP.
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Inhibition of immunosuppressive activity of human monocytes from PDAC patients
In order to further validate the activity of our compounds, the two hits, 10 and 23, were evaluated
for their ability to inhibit the immunosuppressive activity of monocytes from patients affected by
pancreatic ductal adenocarcinoma (PDAC) towards proliferation of T cells. PDAC represents the
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fourth leading cause of cancer death especially because of its high resistance to therapy. PDAC
cells release several pro-inflammatory molecules, thus favoring the expansion and accumulation
of monocytic MDSCs that are strongly involved in promoting immune escape mechanisms
exploited by cancer. We validated the immunosuppressive activity of monocytes isolated from
PDAC patients (n=16). As previously shown by Trovato et al.,39 on the basis of such
immunosuppressive activity, our cohort of PDAC patients could be divided into two sub-groups:
“
suppressive PDAC” (n=11, red), whose monocytes were able to reduce T cell proliferation, and
non-suppressive PDAC” (n=5, blue), whose monocytes did not inhibit T cell proliferation,
“
similarly to healthy donor (HD)-derived monocytes (n=7, black) (Figure 5A). In order to evaluate
the capability of IDO1 inhibitors to reduce the immunosuppressive activity of monocytes from
PDAC patients, we first selected the higher concentration that would not affect the viability of
both monocytes isolated from HD (30 µM) (Figure 5B) and PDAC patients (Figure 5C).
Interestingly, the treatment of suppressive monocytes with IDO1 inhibitors 1, 10 and 23
significantly reduced their capacity to restrict T cell proliferation (Figure 5D), suggesting the
impact of IDO1 in driving the inhibitory function of PDAC-isolated suppressive monocytes.
Notably, compound 10 showed a higher effect compared to epacadostat 1. Finally, as expected,
both 10 and 23 inhibitors resulted ineffective on non-suppressive PDAC-isolated monocytes
(Figure 5E).
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Figure 5. IDO-inhibitors abrogate immunosuppressive function of PDAC-derived monocytes. (A)
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Representative proliferation peaks of Cell Trace CD3 T lymphocytes following the co-culture
with HD (black), PDAC non-suppressive (blue), and PDAC suppressive monocytes (red) (left
+
panel) and relative proliferation perecentage of CD3 T cells after the co-culture with HD (n=7,
black), PDAC non-suppressive (n=5, blue), and PDAC suppressive monocytes (n=11, red) (right
panel). All values are normalized on activated T cells in the absence of myeloid cells. (B) HD
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monocytes (n=3) viability after a 24 h treatment with different concentrations of epacadostat 1
(black), compound 10 (light blue), compound 23 (purple) and DMSO (grey) as control. All values
are normalized on untreated cells. (C) PDAC monocytes (n=3) viability after a 24 h treatment with
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0 µM of epacadostat (black), compound 10 (light blue) and compound 23 (purple). (D, E)
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(D) and PDAC non-suppressive (E) monocytes pre-treated with 30 µM of epacadostat 1 (black),
compound 10 (light blue), compound 23 (purple) and DMSO (grey) as control. All values are
normalized on activated T cells in the absence of myeloid cells. Data are expressed as mean ±
SEM. Statistical analyses were performed using Mann-Whitney test. HD: healty donor PDAC:
pancreatic ductal adenocarcinoma.
In vivo PK/PD of compound 23
Considering the slightly higher in vitro metabolic stability shown by compound 23 compared to
1
0, we proceeded evaluating its pharmacokinetic parameters in mice. Briefly, mice were injected
with 23 (i.v. 10ꢀmg/kg, once) and serial blood sampling was performed. 23 showed a half-life of
.65ꢀh, with a clearance of 7.5ꢀmL/min/Kg, a volume of distribution of 6.6ꢀL/Kg and a Cmax of 2684
0
μg/L (see Supporting Information for the full set of PK parameters).
At selected time points, and in particular at 0, 2, 5, 15, 30 min and 8 h after the injection of the
compound, the L-Kyn plasmatic levels were measured. As expected, according to the short half-
life of 10, L-Kyn levels rapidly decreased up to 2 min and remained below the basal level until 30
min, while restoring the basal level in 8 h (see Supporting Information).
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These data were benchmarked against epacadostat 1, evaluating L-Kyn levels after oral
administration of compound 1: a tendency in decreasing L-Kyn levels was observed, in agreement
with literature data.40
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Discussion and Conclusions
In this study, structure-based virtual screening was instrumental to identify a potent benzimidazole
compound (10) that is endowed with attractive features. It is a novel chemotype, not sharing
structural similarities with previously reported IDO1 inhibitors, and displays high synthetic
feasibility, together with a peculiar predicted binding mode in the active site compared to the
previously reported IDO1 inhibitors. The benzimidazole group is accommodated in pocket A, the
nitrogen of the benzimidazole ring coordinates the heme group and the long side chain protrudes
towards pocket C. The tight packing within the enzyme allows for an extensive bonding network,
including one halogen and three hydrogen bonds that contribute to a strong binding interaction
(Kd = 0.36 µM) and a high inhibition potency (IC50 = 16 nM in A375). Interestingly, MD analysis
reveals that this pocket is not constantly occupied as 10 flips between two poses: one where it
interacts with pocket C and the other where it is shifted towards pocket B, in proximity to Arg231.
The in silico-aided SAR study around 10 afforded a derivative, 23, in which the bromopyrrole
moiety displayed by 10 is replaced by an indole group, that according to docking analysis is located
between pocket B and pocket C, and is able to form two hydrogen bonds with the carboxylic
moiety of the heme group. MD analysis highlights that 23 can flip between two different poses,
but simulation time spent near Arg231 is greater than compound 10. 23 is able to inhibit IDO1 at
the low nanomolar level in different tumour cell lines, is not cytotoxic up to 30 μM, is specific for
IDO1 over TDO (>1000-fold) and does not interfere with CYPs. Last, the compound can be
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