Important Hydrogen Bond Networks in Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitor Design Revealed by Crystal Structures of Imidazoleisoindole Derivatives with IDO1

Article abstract of DOI:10.1021/acs.jmedchem.5b01390

Indoleamine 2,3-dioxygenase 1 (IDO1), promoting immune escape of tumors, is a therapeutic target for the cancer immunotherapy. A number of IDO1 inhibitors have been identified, but only limited structural biology studies of IDO1 inhibitors are available t

Full text of DOI:10.1021/acs.jmedchem.5b01390

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Article
Important Hydrogen Bond Networks in Indoleamine 2,3-
dioxygenase 1 (IDO1) Inhibitor Design revealed by Crystal
Structures of Imidazoleisoindole Derivatives with IDO1
Yi-Hui Peng, Shau-Hua Ueng, Chen-Tso Tseng, Ming-Shiu Hung, Jen-Shin Song, Jian-Sung Wu, Fang-
Yu Liao, Yu-Shiou Fan, Mine-Hsine Wu, Wen-Chi Hsiao, Ching-Cheng Hsueh, Shu-Yu Lin, Chia-Yi
Cheng, Chih-Hsiang Tu, Lung-Chun Lee, Ming-Fu Cheng, Kak-Shan Shia, Chuan Shih, and suying wu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01390 • Publication Date (Web): 07 Dec 2015
Downloaded from http://pubs.acs.org on December 8, 2015
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Important Hydrogen Bond Networks in
Indoleamine 2,3ꢀdioxygenase 1 (IDO1)
Inhibitor Design revealed by Crystal
Structures of Imidazoleisoindole Derivatives
with IDO1
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YiꢀHui Peng, ShauꢀHua Ueng, ChenꢀTso Tseng, MingꢀShiu Hung, JenꢀShin Song,
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JianꢀSung Wu, FangꢀYu Liao, YuꢀShiou Fan, MineꢀHsine Wu, WenꢀChi Hsiao,
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ChingꢀCheng Hsueh, ShuꢀYu Lin, ChiaꢀYi Cheng, ChihꢀHsiang Tu, LungꢀChun
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Lee, MingꢀFu Cheng, KakꢀShan Shia, Chuan Shih, Su Ying Wu.
†
Institute of Biotechnology and Pharmaceutical Research, National Health Research
Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC
KEYWORDS: Indoleamine 2,3ꢀdioxygenase 1 (IDO1), imidazoleisoindole, NLG919,
IDO1 inhibitors, hydrogen bond network, intramolecular hydrogen bond, structural
biology
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ABSTRACT
Indoleamine 2,3ꢀdioxygenase 1 (IDO1), promoting immune escape of tumors, is a
therapeutic target for the cancer immunotherapy. A number of IDO1 inhibitors have
been identified but only limited structural biology studies of IDO1 inhibitors are
available to provide insights on the binding mechanism of IDO1. In this study, we
present the structure of IDO1 in complex with 24, a NLG919 analogue with potent
activity. The complex structure revealed the imidazole nitrogen atom of 24 to
coordinate with the heme iron, and the imidazoleisoindole core situated in pocket A
with the 1ꢀcyclohexyl ethanol moiety extended to pocket B to interact with the
surrounding residues. Most interestingly, 24 formed an extensive hydrogen bond
network with IDO1, which is a distinct feature of IDO1/24 complex structure and is
not observed in the other IDO1 complex structures. Further structureꢀactivity
relationship, UVꢀspectra and structural biology studies of several analogues of 24
demonstrated that extensive hydrophobic interactions and the unique hydrogen
bonding network contributes to the great potency of imidazoleisoindole derivatives.
These results are expected to facilitate the structureꢀbased drug design of new IDO
inhibitors.
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Introduction
Indoleamine 2,3ꢀdioxygenase 1 (IDO1) is a hemeꢀcontaining enzyme that catalyzes
the initial and rate limiting step in the catabolism of tryptophan via the kynurenine
pathway, leading to the formation of a series of biologically active metabolites,
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1,2
including the final product nicotinamide adenine dinucleotide (NAD). It catalyzes
the oxidative ring cleavage of various substrates, including tryptophan,
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ꢀhydroxytryptophan, serotonin and tryptamine. Through the catabolism of
tryptophan, IDO1 inhibits the proliferation and differentiation of T cells, which are
sensitive to the degradation of tryptophan and accumulation of tryptophan
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catabolites. IDO1 is found to be overꢀexpressed in cancer cells, acting as a direct
guard against Tꢀcell attack, and is also expressed within antigen presenting cells to
enhance peripheral tolerance to tumor associated antigens (TAAs) in tumor draining
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lymph nodes. By these mechanisms, IDO1 facilitates the survival, growth, invasion,
and metastasis of malignant cells expressing TAAs by protecting them from
recognition and attack by the immune system. In vivo studies showed that IDO1
deficiency resulted in impaired tumor growth, metastases development delay, and
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prolonged survival in an IDO1 animal model, validating IDO1 as a therapeutic
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target for the cancer immunotherapy.
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Several IDO1 inhibitors have showed efficacy when used alone or in combination
with other therapeutics, and are currently in the clinical trials. One of the most
advanced, hydroxyamidine compound (INCB024360, also named epacadostat),
developed by Incyte, is an orally bioavailable small molecule inhibitor of the IDO1
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protein, with potent enzymatic and cellular activity. It was also shown to enhance T
lymphocyte, dendritic cell and natural killer cell responses in vitro. As in the in vivo
study, INCB024360 was efficacious as a single agent or in combination use with
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cytotoxic and immunotherapy agents in cancer mouse models. INCB024360
currently undergoes several clinical trials, either used as a monotherapy or in
combination with various antibody, in subjects with advanced or metastatic cancers.
Another IDO1 inhibitor, NLG919 (an imidazoleisoindole derivative; the chemical
structure is undisclosed), developed by NewLink Genetics and later licensed to
Genentech as GDCꢀ0919, is undergoing phase I clinical trials in the treatment of
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recurrent advanced solid tumors. NLG919 showed potent inhibition of IDO1 in both
enzymatic and cellular assays. The in vivo study revealed that oral administration of
NLG919 reduced the concentration of kynurenine in plasma and tissue by around
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0%. Treatment with NLG919 showed doseꢀdependent activation and proliferation of
effector T cells, and led to the dramatic regression of tumor size. The ability of
NLG919 to reduce tumor growth was highly correlated with its functional immune
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response, which is consistent with its proposed mechanism of action as an immune
modulator. The dose repeat toxicity (NOAEL) of rat and dog were both at 80
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mg/kg/day in 28 days, BID. In addition, when combined with Indoximod, NLG919
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showed synergistic T cell activation and tumor regression. No dose limiting toxicities
have been observed soꢀfar, and the safety and toxicity of NLG919 in patients is
currently being evaluated. In addition to INCB024360 and NLG919, various IDO
inhibitors have been discovered by several pharma companies, and are now in the
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different stages of drug development.
Although various IDO1 inhibitors with diverse chemical structures have been reported,
only few crystal structures of IDO1 bound to inhibitors are available in the public
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domain.
The first is the structure of IDO1 in complex with 4ꢀphenylimidazole
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(4ꢀPI), a fragmentꢀlike ligand with weak inhibition against IDO1 (IC50 = 48 ꢀM).
As revealed by ligandꢀbound structures, 4ꢀPI was found to be located in the binding
site (pocket A) with its phenyl ring inserting into a lipophilic cavity, and one nitrogen
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atom of the imidazole ring interacting to the heme iron. In addition to pocket A,
however, the binding site could extend to the adjacent hydrophobic pocket (pocket B),
consisting of Phe163, Phe226, Arg231, Leu234 and Ile354 residues, which larger
ligands might target. Whereas pocket A is the major binding site involving
hemeꢀbinding, pocket B could be targeted to improve the binding affinity. Recently,
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Tojo et al. reported two inhibitors coꢀcrystallized with IDO1 :
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2ꢀ(5ꢀpꢀtolylthiazolo[2,3ꢀc][1,2,4]triazolꢀ3ꢀylthio)ꢀNꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)aceta
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mide) (IC50 = 3.0 ꢁM); and the imidazothiazole derivative 27 (IC50 = 1.9 ꢁM).
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These two compounds induced a conformation change in pocket A, resulting from the
movement of main chain Ala260ꢀSer263 and the shift of Phe226 and Arg231, and
extended into the pocket B. The structural information suggests that the interactions
with Phe226 and Arg231 contribute to the increased potency of these imidazothiazole
derivatives to IDO1.
As could be seen from the foregoing discussion, all the published IDO structures
upꢀtoꢀdate are complexed with weak inhibitors with inhibition potency at micromolar
range. Therefore, the structures of IDO in complex with potent inhibitors are needed
to gain a better understanding of the binding mechanism of IDO1 and further provide
molecular insights to design novel and potent inhibitors. In this study, we present the
structure of IDO1 in complex with 24 at 2.2 Å. Compound 24, a NLG919 analogue,
contains an imidazoleisoindole core and exhibits potent inhibitory activity to IDO1
with the IC50 value of 38 nM. The crystal structure revealed 24 to occupy both pocket
A and B, coordinating with the heme iron atom through the imidazole nitrogen and
making hydrophobic interactions with the surrounding residues. Interestingly, 24
forms an extensive hydrogen bond network with IDO1; there are two intermolecular
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hydrogen bonds between 24, the 7ꢀ propionic acid group of the heme and the main
chain NH group of Ala264; and an intramolecular hydrogen bond within 24 between
the isoindole nitrogen and the hydroxyl group. This hydrogen bond network is a
distinct feature of the IDO1/24 complex structure and is not observed in the other
IDO1 complex structures. Moreover, the detailed structureꢀactivity relationship (SAR)
information of NLG919 analogues, i.e. imidazoleisoindole derivative, are not
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disclosed and UVꢀspectra studies of these analogues are unavailable. In this study,
structureꢀactivity relationship, UVꢀspectra, and several coꢀcrystal structures of
imidazoleisoindole derivatives are also presented to elucidate the inhibition
mechanisms of this series of compounds.
Results and Discussion
Biological
Activity
of
24.
Compound
24
(1ꢀcyclohexylꢀ2ꢀ(5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol), a NLG919 analogue, was
synthesized and evaluated by biological assays. Compound 24 contains two chiral
centers; one adjacent to the cyclohexyl group, and the other at the 5ꢀposition of the
imidazoleisoindole ring (Figure S1 in the supporting information). This results in four
stereoisomers,
designated
as
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(
(
(R)ꢀ1ꢀcyclohexylꢀ2ꢀ((S)ꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol),
(S)ꢀ1ꢀcyclohexylꢀ2ꢀ((S)ꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol),
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((S)ꢀ1ꢀcyclohexylꢀ2ꢀ((R)ꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol)
and
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((R)ꢀ1ꢀcyclohexylꢀ2ꢀ((R)ꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol)
(Figure
S1).
Compound 24, a mixture of four stereoisomers (28, 29, 30 and 31), inhibited IDO1
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with IC50 value of 38 nM.
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A UVꢀvisible spectra study was performed to evaluate the binding of compound
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4. IDO1 is a hemeꢀcontaining protein and the unique UV absorption properties of
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porphyrins are useful in studies of hemeꢀproteins.
The absorbance spectra of the
heme group is highly sensitive to the changes in the polarity of heme surroundings
upon the ligand/substrate binding, which changes the spectral properties of the
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heme. Therefore, the changes in the UVꢀvisible spectra causing by the IDO1ꢀligand
interaction could be utilized to evaluate the binding of compounds to IDO1. The
UVꢀvisible spectra of ferric IDO1 were measured in the presence and absence of
compound 24 (Figure 1). In the absence of compound 24, the absorption spectrum of
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ferric IDO1 exhibited a Soret peak at 403 nm, similar to the previous publications.
This shifted to 413 nm in the presence of compound 24, demonstrating that compound
4 binds to IDO1 and coordinates with the iron of the heme group.
Structural Biology Studies of Compound 24 To further elucidate the binding mode
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.
and to gain insights into the detailed interactions of compound 24 with IDO1, human
IDO1 protein was purified and coꢀcrystallized with 24. The IDO1/24 coꢀcrystals
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formed in 1~2 weeks and were red in color. The structure of IDO1 in complex with
compound 24 was solved to a resolution of 2.2 Å. The space group was found to be
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P2 2 2 with two IDO1 monomers in the asymmetric unit. The electron density maps
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of IDO1/24 were clear, except for some disordered regions that included residues
60ꢀ380 in monomer A, and residues 361ꢀ375 in monomer B. The monomer of IDO1
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consists of two domains: one is the large domain and the other is the small domain.
The hemeꢀbinding pocket is located in the large domain and residue His346 was
coordinated to the heme iron atom at the fifth coordination position. The electron
density map clearly showed that 24 was located at the sixth coordination site of the
heme, and covalently bound to the heme iron (Figure 2 and Figure S1). Since IDO1
was crystallized with a mixture of all four stereoisomers of 24, all of these
stereoisomers were built into the density map to investigate which isomers majorly
bind to IDO1. The 2FoꢀFc and FoꢀFc map of all four stereoisomers were presented in
Figure S1. A difference map, showing differences between the model and the
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experimental data, could detect the misplacement of the ligand
and also the wrong
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conformation or stereochemistry of the ligand.
Positive difference density were
found near two chiral centers of 30 and 31. In contrast, no positive difference density
were observed around 28 and 29, suggesting 28 and 29 fit properly in the density map.
These two stereoisomers were therefore selected for further model building and
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refinement. The results showed that both stereoisomers, 28 (R,Sꢀstereoisomer) and 29
(S,Sꢀstereoisomer), superimposed well with each other and made almost identical
interactions with IDO1. Therefore, 28 (R,Sꢀstereoisomer) and 29 (S,Sꢀstereoisomer),
both contribute to the inhibition of IDO1, which is consistent with the later biological
analysis of 28 and 29 showing that they inhibited IDO1 with very similar IC50 values.
From the foregoing stereoisomer study, the R,S configuration of 24 is used for
the discussion in the IDO1/24 complex structure. 24 was found to coordinate with the
heme iron through the imidazole nitrogen atom (Figure 2A). This direct binding of 24
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with the heme iron is characterized as a type II interaction and is supported by the
UVꢀvisible spectra data: 24 was found to induce a Soret band red shift from 403 to
413 nm upon the binding to heme iron. The imidazoleisoindole core is situated deep
in pocket A and formed extensive hydrophobic interactions with Tyr126, Cys129,
Val130, Phe163, Phe164, Gly262, Ala264 and the porphyrin ring. The 1ꢀcyclohexyl
ethanol moiety extended to pocket B and formed hydrophobic interactions with
Phe226, Ile354 and Leu384, and also had close contacts with Arg231 in the entrance
region (Figure 2A). Most interestingly, 24 formed an extensive hydrogen bond
network with the heme group and the protein residues in the active site (Figure 2B).
This hydrogen bond network consisted of a hydrogen bond formed by the hydroxyl
group of 24 with the 7ꢀ propionic acid of the heme; a hydrogen bond formed by the
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propionic acid side chain with the main chain NH group of Ala264; and an
intramolecular hydrogen bond formed within 24 by the isoindole nitrogen and the
hydroxyl group.
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The IDO1/24 complex structure suggests that some functional groups of 24
would contribute to the binding of 24 to IDO1 and thus affect the inhibitory potency.
For example, the imidazoleisoindole group coordinated to the heme iron and formed
hydrophobic interactions with residues in pocket A; the hydroxyl group was involved
in the hydrogen bond network with IDO1; and the isoindole nitrogen participated in
the intramolecular hydrogen bonding to stabilize 24 and the binding to IDO1.
Preorganization of ligand by the intramolecular hydrogen bond would lead to
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decreased entropic cost of binding and consequently result in increased affinity.
To
further investigate the contributions of these pharmacophores in the binding to and
inhibition of IDO1, and advance structureꢀbased drug design, several analogues of 24
were synthesized to explore structureꢀactivity relationship (SAR), and structural
biology studies performed to elucidate the detailed interactions between these
analogues with IDO1 (Table 1).
Chemistry. Compound 22–26 were synthesized according to the procedures
30
established by the Newlink Genetics Co and the compound identification data of
those was listed in the supporting information. The synthesis of
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1ꢀcyclohexylꢀ2ꢀ(3ꢀmethylꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol
(8)
from
2
ꢀmethylimidazole 1 followed the synthetic paths established by the Newlink
30
Genetics Co. and described in scheme 1.
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Compound 15 was synthesized by the methods shown in scheme 2.
βꢀOxoꢀcyclohexanepropanal 10 was prepared by the condensation of
cyclohexylethanone (6) and ethyl formate (9) following the literature reported
31
procedure. 4ꢀ(2ꢀAminophenyl)imidazole 13 was synthesized by a Suzuki coupling
of iodoimidazole 11 and pinacol borate 12, and then compound 13 was reacted with
1
0 to give tricyclic compound 14. Reduction of the ketone group of compound 14
with sodium borohydride gave the resulted imidazo[1,5ꢀc]quinazolinyl ethanol 15.
Compounds 18, 20, and 21 were prepared using established methods with necessary
modifications
(scheme
3).
NꢀAlkylation
of
norharmane
(16)
and
2
ꢀbromoꢀ1ꢀcyclohexylethanꢀ1ꢀone gave ketone 17, which was reduced using sodium
borohydride to give 2ꢀβꢀcarbolinꢀ9ꢀylꢀ1ꢀcyclohexylꢀethanol 18. The synthesis of
compounds 20 and 21 followed the same procedure as mentioned above with
αꢀcarboline 19 as the starting material.
SAR Studies. When a fluoro group was introduced in the imidazoleisoindole to
generate 25, it showed 2ꢀfold improvement in the inhibition to IDO1 compared to 24.
As shown in UVꢀvisible spectra, 25 induced the same red shift as 24, from 403 nm to
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413 nm, indicating 25 could also make the coordinated covalent bond with the heme
iron. The replacement of the hydroxyl group with carbonyl group (22) resulted in a
1
2ꢀ fold decrease in inhibition of IDO1 as compared with 24. Similarly, replacement
10
11
12
13
14
15
16
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18
19
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23
24
25
26
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of the hydroxyl group with carbonyl group (23) resulted in a 14ꢀfold decrease in IDO1
inhibitory activity, compared to its hydroxyl analogue 25. These results shows the
important role played by the hydroxyl group in the inhibition to IDO1. The effect of a
halogen group on the potency was also investigated: compound 23 with an additional
fluorine group relative to compound 22 showed 2ꢀfold higher potency than 22.
Further modifications of the imidazoleisoindole ring of 24 to give 8 and 26 led to a
complete loss of activity toward IDO1. In 8, the imidazole ring was replaced with the
2ꢀmethylimidazolyl ring and the activity decreased significantly. The reason for this
dramatic loss of activity could be that the additional methyl group at the C2ꢀposition
of the imidazole could result in a steric clash with the heme group in IDO1 and thus
prevent the formation of the coordination covalent bond with the heme iron. This
hypothesis is supported by the UVꢀvisible spectra of 8, which showed only a weak
Soret signal from 403 to 405 nm, suggesting that 8 was unable to coordinate with
heme iron. Compound 24, in contrast, induced an obvious Soret signal, from 403 to
4
13 nm. Compound 26, a triazolyl analogue formed by modification of the Cꢀ2
position of the imidazole ring of 24, showed no IDO1 inhibitory activity, consistent
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with a described study. Modifications of the isoindole ring of 24 were also found to
affect the binding to and inhibition of IDO1. Expansion of the 5ꢀmembered isoindole
ring of 24 to a sixꢀmembered quinazoline ring generated 15, resulting in a 29ꢀfold
decrease in IDO1 activity. A molecular docking study of 15 in the active site of IDO1
was performed and compared with the IDO1/24 complex structure (Figure S2) to
elucidate the difference in binding interactions between 15 and 24. It was found that
although the imidazole nitrogen formed the coordinate covalent bond with the heme
iron as evidenced by its induced Soret red shift from 403 to 413 nm, the ring
expansion of 15 rearranged the orientation of the 1ꢀcyclohexyl ethanol moiety such
that the hydroxyl group became located away from the quinazoline nitrogen (distance
= 4.3 Å) and was therefore unable to form an intramolecular hydrogen bond within
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1
5.
Another
structural
modification,
replacement
of
the
fiveꢀmembered
imidazoleisoindole ring of 24 with a sixꢀmembered, 9Hꢀpyrido[3,4ꢀb]indole ring to
generate 18, resulted in almost complete loss of activity (IC50＞10 ꢁM). The
UVꢀvisible spectra revealed that 18 showed a smaller spectral change (403 to 407 nm)
upon binding to IDO1 than that observed for 24, indicating the weaker interactions of
1
8 with the heme group. A coꢀcrystal structure of IDO1 with 18 revealed that the
hydroxyl group of 18 was unable to form the hydrogen bond with the heme (Figure 4)
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and the nitrogen atom in the pyridine ring was further away from the heme iron (see
below section of Structureꢀbased Drug Design of imidazoisoindole Compounds).
Finally, the modification of imidazoleisoindole ring of 24 to a 9Hꢀpyrido[2,3ꢀb]indole,
10
11
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14
15
16
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18
19
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23
24
25
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2
0 or 21, led to the complete loss of activity. 20 and 21 showed no inhibition against
IDO1 and the UVꢀvisible spectra of IDO1 in the presence of 21 (or 20) was the same
as that of the native IDO1 protein alone. These results suggest that 20 and 21 were
unable to form the coordinated covalent bond with the heme iron, which may explain
the complete loss of inhibitory activity.
StructureꢀBased Drug Design of imidazoisoindole Compounds. In order to
elucidate the binding mode of imidazoisoindole derivatives at the molecular level,
compounds 23, 25, and 18 were coꢀcrystallized with the IDO1 protein and the
structures were solved (Table S1). Superimposition of the structure of IDO1/25
complex (Figure 3A) and that of the IDO1/24 complex revealed (Figure 3C) these two
compounds adopted very similar conformations in the active site, and formed similar
coordinated covalent bond, hydrogen bond networks and hydrophobic interactions
with the surrounding residues. The difference between these two inhibitors is the
additional fluorine group to make an extra halogen bonding interaction with the
oxygen atom in the main chain of Gly 262 in pocket A (Figure 3A), which contributes
to the 2ꢀfold improved potency of 25 as compared with 24.
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As revealed in the above mentioned SAR studies, the replacement of the hydroxyl
group of 25 with a carbonyl group to give 23, decreased the inhibitory potency
1
4ꢀfold. The same trend was also observed for 22 and 24. To investigate the role
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played by this hydroxyl group in the binding to the IDO1 protein, the structure of the
IDO1/23 complex (Figure 3B) was solved and compared with that of IDO1/25
complex. The two structures superimposed well (Figure 3C) to each other and both
coordinated with heme iron, and also formed extensive hydrophobic interactions and a
halogen bond with the protein. However, in the IDO1/23 complex structure, the
carbonyl group was unable to serve as a hydrogen bond donor to allow the
intramolecular hydrogen bond to the isoindole nitrogen. The loss of this
intramolecular hydrogen bond could be the reason for the decrease in inhibitory
activity of 23. This comparison of IDO1/23 and IDO1/25 emphasizes the contribution
of the intramolecular hydrogen bond on the inhibition of IDO1.
SAR studies found that replacement of the imidazoisoindole core of (24) with the
9
Hꢀpyrido[3,4ꢀb]indole core to give 18 led to a dramatic loss of IDO1 inhibitory
activity and also resulted in a smaller UVꢀvis shift (403 to 407 nm). To understand
these changes, 18 was coꢀcrystallized with IDO1 and the structure was solved (Figure
4
A and Table S1). The hydroxyl group of 18 was found to be moved away from the
propionic acid with distance > 4.3Å, with loss of the intermolecular hydrogen bond
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with the heme (Figure 4). In comparison, the hydroxyl group of 24 was shifted toward
the propionic acid group where it formed a strong intermolecular hydrogen bond with
the heme. In addition, the 9Hꢀpyrido[3,4ꢀb]indole core was found to be shifted away
from the heme, a distance of 2.44Å separating the pyridine nitrogen and the heme iron.
In comparison, the imidazoisoindole core of 24 is closer to the heme iron. The
absence of the hydrogen bonding interactions with the heme group in IDO1 and the
weaker coordinate covalent interactions would provide the explanations for the
shorter UVꢀvis shift signal induced by 18. Also, the hydroxyl group of IDO1/24
formed an intramolecular hydrogen bond with the isoindole nitrogen, but in the
IDO1/18 complex structure this cannot take place due to an unfavorable spatial
arrangement between the hydroxyl group and the indole nitrogen that precludes the
formation of stable fiveꢀmembered hydrogen bond. In a fiveꢀmembered hydrogen
bonding system, a planar hydrogen bonded conformation with a dihedral angle
between the hydrogen bond donor and acceptor of less than 30° is preferred for steric
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33
and electrostatic reasons. The dihedral angle between the hydroxyl group (Hꢀbond
donor) and the indole nitrogen (Hꢀbond acceptor) of 18 deviates from planarity by
6
3.8° (Figure 4A) and it is therefore unlikely to form an intramolecular hydrogen
bond. In conclusion, the loss of the hydrogen bonding interactions with the heme
group, weaker coordinate covalent interactions and the absence of the intramolecular
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hydrogen bonding interactions result in the loss of inhibition against IDO1, and
shorter red shift in UVꢀvisible spectrum.
Comparison with the Structures of Other Inhibitors Bound to IDO1. The
xꢀray coꢀcrystal structure of the IDO1/24 complex was first compared with that of
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IDO1/4PI (Figure 5A and 5C). 4PI is a fragmentꢀlike compound that showed weak
inhibition against IDO1 with an IC50 value of 48 ꢀM. Superimposing the structures of
these two complexes revealed the imidazole ring of 4PI to be located at the sixth
coordination site of the heme, and the imidazole nitrogen atom to be covalently
bonded to the heme iron. Hydrophobic interactions with Ala264 and Phe163 were also
observed. The phenyl ring (ringꢀ2) of 4PI superimposed well with the
imidazoleisoindole (ringꢀ3) of 24 and extended to pocket A to form the hydrophobic
interactions with Val130, Phe163, Phe164 and Gly262 (Figure 5A). However, in the
IDO1/4PI complex structure, 4PI only occupied in the pocket A. In the IDO1/24
complex structure, however, 24 also directed toward and formed extensive
interactions with pocket B. The cyclohexyl group was located in pocket B and made
interactions with Phe226, Ile354, Leu384 and Arg231. Furthermore, in the IDO1/4PI
complex structure, the propionic acid side chain of the heme was away from the
coordinated site and had no interactions with 4PI, whereas in the IDO1/24 complex
structure, the 7ꢀ propionic acid of the heme moved toward the coordinated site and
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formed a strong hydrogen bond with the hydroxyl group of 24. Finally, an
intramolecular hydrogen bond formed within 24 by the isoindole nitrogen and the
hydroxyl group was absent in the IDO1/4PI structure. In conclusion, the additional
interactions with pocket B and the extensive hydrogen bonding network with IDO1
are thought to explain the better inhibition of IDO1 by 24 compared with 4PI.
10
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18
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23
24
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15
The structure of IDO1/24 and was also compared with that of IDO1/27 (Figure
5
B and 5C). By superimposing the structure complex of 27 onto 24, it was found that
the paraꢀtolyl imidazothiazole moiety (rings 1ꢀ3) of 27 occupied a similar position to
the imidazoleisoindole core of 24. Both compounds formed a coordinated covalent
bond with the heme iron through the nitrogen atom in the core structures, and made
extensive hydrophobic interactions with the surrounding residues in the pocket A. The
differences were the hydrogen bond network and the orientation of chloroꢀphenyl
moiety of 27. The chloroꢀphenyl moiety of 27 adopted a spatial position different
from that of the cyclohexyl moiety of 24. Compound 27 induced a conformation
1
5
change by shifting Phe226, and consequently disrupting the edge to face πꢀπ
interactions between Phe163 and Phe226. Compound 27 was located next to Phe226
and had no interactions with Arg231. On the other hand, in the structure of IDO1/24,
Phe226 adopted a conformation similar to that of IDO1/4PI. Compound 24 was
positioned between Phe226 and Arg231 and formed the interactions with these two
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residues. A mutagenesis study showed that Phe226 and Arg231 play an important role
14
in the catalytic activity of IDO1. Moreover, the hydrogen bond interactions between
the 24 and IDO1 are different from that of 27 and IDO1. As described above,
extensive hydrogen bond networks were formed by the hydroxyl group of 24, the 7ꢀ
propionic acid of the heme, and the main chain NH group of Ala264 and also included
an intramolecular hydrogen bond formed within 24. In the structure of IDO1/27
complex, the amide group of 27 occupied a similar space to the hydroxyl group of 24
and hence formed the hydrogen bond with the heme, but the 7ꢀ propionic acid of the
heme formed the hydrogen bond with the side chain of Ser263 instead of the main
chain NH group of Ala264. Also, no intramolecular hydrogen bond was observed
within 27. Taken together, these findings explain the decreased activity of 27
compared to 24.
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Stereoisomers of 24. Compound 24 was synthesized as a mixture of four
stereoisomers and chiral chromatography was performed by the Daicel Chiral
Technologies Co., LTD to separate the mixture (Figure S3 in supporting information).
Four peaks were obtained and their inhibitory activities toward IDO1 were evaluated.
Peaks 3 and 4 showed potent inhibition of IDO1 with IC50 values of 19.5 nM and 28.9
nM, respectively. Peaks 1 and 2 showed weak inhibition to IDO1 with IC50 values of
2
437 nM and >10000 nM, respectively. These results are consistent with a described
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study. In this patent, compounds 1415, 1416, 1417 and 1418 are four stereoisomers
and present the same chemical structure as 31, 30, 29 and 28, respectively. The IC50
value of compound 1415 (31) and compound 1416 (30) reported in the patent are 1~
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1
0 ꢁM while the IC50 value of compound 1417 (29) and compound 1418 (28) are < 1
M. This data suggests that two stereoisomers, 28 and 29, significantly contribute to
ꢁ
the observed inhibition of IDO1 by 24 (the mixture), whereas the other two
stereoisomers, 31 and 30, do not.
Conclusions
In this study, the structure of IDO1 bound to a potent inhibitor, 24 (a NLG919
analogue), was solved at the resolution of 2.2Å. The density map clearly showed 24 to
be located at the sixth coordination site of the heme and covalently bound with the
heme iron through the imidazole nitrogen, consistent with the observation by
UVꢀvisible spectra that 24 induced a Soret peak shift from 403 to 413 nm. The
imidazoleisoindole core was found deep in pocket A, and where it formed extensive
hydrophobic interactions with Tyr126, Cys129, Val130, Phe163, Phe164, Gly262,
Ala264 and the porphyrin ring. The 1ꢀcyclohexyl ethanol moiety extended to pocket
B and formed hydrophobic interactions with Phe226, Ile354 and Leu384. It also had
close contacts with Arg231 in the entrance region. Most interestingly, 24 formed an
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extensive hydrogen bond network: two intermolecular hydrogen bonds between 24,
the 7ꢀ propionic acid of the heme and the main chain NH group of Ala264; and an
intramolecular hydrogen bond within 24 by the isoindole nitrogen and the hydroxyl
group. This hydrogen bond network is a distinct feature of IDO1/24 complex structure
and is not observed in the other IDO1 complex structures.
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Introduction of a fluoro group to the structure of 24 resulted in 25, which exhibited a
2
ꢀfold improvement (IC50 = 19 nM) in inhibition of IDO1. Comparison of the
IDO1/25 and IDO1/24 complex structures revealed that the additional fluorine group
of 25 formed an extra halogen bonding interaction with the oxygen atom in the main
chain of Gly 262 in pocket A, which could account for the improved potency of 25
compared to 24.
The role of the hydroxyl group in the binding of IDO1 protein was further
investigated by synthesizing compound 23. Comparison of the structure of IDO1/23
with that of IDO1/25 revealed the replacement of hydroxyl group with the carbonyl
group resulted in the loss of the intramolecular hydrogen bond, leading to the
decreased potency of 23. The intramolecular hydrogen bond plays a unique role in
29,34ꢀ36
drug discovery as it could alter the conformation of the molecules
and modulate
37ꢀ39
the physicochemical properties of the designed compounds.
Studies have shown
that intramolecular hydrogen bonds contribute to the improved potency, better cell
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permeability, enhanced oral absorption and improved pharmacokinetic properties of
3
3,37ꢀ41
compounds.
Finally, the slight modification of the imidazoleisoindole core of 24 to give 8, 15, 18,
0, 21 and 26 led to a dramatic loss of IDO1 inhibitory activity and also resulted in
10
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2
the shorter Soret band red shift. Further structural biology studies demonstrated that
the weaker coordinate covalent interactions and the loss of the hydrogen bonding
network completely eliminate the capacity of these analogues to inhibit IDO1.
Novel, small molecular inhibitors of IDO1 are becoming increasingly important due
to mounting evidence of the essential role played by IDO1 in cancer immunotherapy.
Although a number of IDO1 inhibitors have been identified and developed, only
limited structural biology studies of IDO1 inhibitors are available, all of which are
weak inhibitors with IC50 values in the µM range. The structural biology studies of 24
and its analogues revealed extensive hydrophobic interactions and a unique hydrogen
bonding network contributes to the great potency of imidazoleisoindole derivatives.
These insights are expected to facilitate the structureꢀbased drug design of new IDO
inhibitors.
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EXPERIMENTAL Section
General Remarks
Chemicals and solvents were purchased and used as received without further
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purification. All reactions were carried out under argon atmosphere. Flash column
chromatography was performed with Merck Kieselgel 60, No. 9385, 230ꢀ400 mesh
ASTM silica gel as the stationary phase. Automated flash chromatography was carried
out by Teledyne Isco CombiFlash® Automated Flash Chromatography systems.
1
Proton ( H) nuclear magnetic resonance spectra were measured on a Varian
Mercuryꢀ300 or Varian Mercuryꢀ400 spectrometer. Chemical shifts were recorded in
parts per million (ppm) on the delta (δ) scale relative to the resonance of the solvent
peak. Coupling description was shown by abbreviation: s = singlet; d = doublet; t =
triplet; q = quartet; quin = quintet; br = broad; m = multiplet. Highꢀresolution mass
spectra (HRMS) were measured by a VARIAN 901ꢀMS electrospray ionization (ESI)
or JEOL JMSꢀ700 fast atom bombardment (FAB) mass spectrometer. Purity of the
final compounds was determined with an Hitachi 2000 series HPLC system using a
C18 column (Agilent ZORBAX Eclipse XDBꢀC18 5 mm, 4.6 mm × 150 mm),
operating at 25 °C. Peaks were detected UV absorption at 254 nm.
4
ꢀIodoꢀ2ꢀmethylꢀ1ꢀtritylꢀ1Hꢀimidazole
(3).
The
synthesis
of
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5ꢀiodoꢀ2ꢀmethylꢀ1Hꢀimidazole (2) and 4ꢀiodoꢀ2ꢀmethylꢀ1ꢀtritylꢀ1Hꢀimidazole (3)
42
followed
the
established
procedure
with
necessary
modification.
4
ꢀIodoꢀ2ꢀmethylꢀ1Hꢀimidazole (2) (3.12 g, 15.0 mmol) in THF (100 mL) was added
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triethylamine (4.18 mL, 30.0 mmol) and trityl chloride (5.02 g, 18.0 mmol), and then
the reaction mixture was reflux for 3 h. After cooling, the suspension was filtered. The
filtrate was evaporated in vacuo. The residue was dissolved in dichloromethane (100
mL) and washed with sodium thiosulfate (100 mL, 5% solution in water). The water
layer was extracted with dichloromethane (100 mL). The combined organic layer
4
were dried over MgSO and concentrated to afford the crude. The crude mixture was
2 2
purified by automated flash chromatography using 0–4% MeOH : CH Cl gradient to
1
6
give 3 as a paleꢀyellow solid (3.22 g, 47%). H NMR (DMSOꢀd , 400 MHz) δ 7.45–
7
2
2
.39 (m, 9 H), 7.07–7.04 (m, 6 H), 6.65 (s, 1 H), 1.49 (s, 3 H).
ꢀ(2ꢀMethylꢀ1ꢀtritylꢀ1Hꢀimidazolꢀ4ꢀyl)benzaldehyde (5). The synthesis of
ꢀ(2ꢀmethylꢀ1ꢀtritylꢀ1Hꢀimidazolꢀ4ꢀyl)benzaldehyde (5) followed the established
30
procedure of the NewLink Genetics Corporation, with necessary modifications.
A
mixture of 4ꢀiodoꢀ2ꢀmethylꢀ1ꢀtritylꢀ1Hꢀimidazole (3) (1.80 g, 4.00 mmol),
ꢀfoymylphenylboronic acid (4) (0.90 g, 6.00 mmol), and potassium phosphate
tribasic (2.55 g, 12.0 mmol) in DMF (20 mL) and water (4 mL) was purged with
2
nitrogen for 15 minutes. Pd(PPh
3
)
4
(0.460 g, 0.400 mmol) was added to the reaction
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mixture, and the mixture was purged with nitrogen for another 15 minutes. The
o
reaction mixture was stirred at 90 C for 16 h. After cooling, the solution was filtered
through a pad of celite. The mixture was diluted with water and extracted with ethyl
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acetate. The combined organic layer were washed with brine, dried over MgSO and
concentrated to afford the crude. The crude was purified by automated flash
1
2 2
chromatography using 0–6% MeOH : CH Cl gradient to give 5 (0.220 g, 13%). H
NMR (CDCl 400 MHz) δ 10.41 (s, 1 H), 7.92 (d, 1 H), 7.74–7.63 (m, 1 H), 7.57 (t, 1
3,
H), 7.41–7.28 (m, 10 H), 7.20–7.16 (m, 6 H), 6.95 (s, 1 H), 1.79 (s, 3 H).
1
ꢀCyclohexylꢀ2ꢀ(3ꢀmethylꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanone
(7).
The
synthesis of 1ꢀcyclohexylꢀ2ꢀ(3ꢀmethylꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanone (7)
followed the established procedure of the NewLink Genetics Corporation with
3
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necessary
ꢀ(2ꢀmethylꢀ1ꢀtritylꢀ1Hꢀimidazolꢀ4ꢀyl)benzaldehyde (5) (0.220 g, 0.500 mmol) and
cyclohexylmethyl ketone (6) (70.0 ꢁL, 0.500 mmol) in THF (3 mL) was added NaOEt
0.20 mL, 0.630 mmol, 21 wt % in ethanol) and the mixture was stirred at room
temperature for 3 h. The solvent was concentrated to give the residue, and the residue
modifications.
To
a
solution
of
2
(
4
was diluted with saturated NH Cl(aq) (10 mL) and extracted with dichloromethane (2 x
1
4
0 mL). The combined organic layer were dried over MgSO and concentrated to
afford the crude product. The crude was purified by automated flash chromatography
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2 2
using 0–10% MeOH : CH Cl gradient to give 7 as a white solid (75.0 mg, 51%). H
NMR (CDCl 400 MHz) δ 7.49 (d, 1 H), 7.35 (t, 1 H), 7.27–7.25 (m overlapped with
3,
s at 7.26, 1 H), 7.20 (t, 1 H), 7.07 (s, 1 H), 5.72 (dd, 1 H), 3.24 (dd, 1 H), 2.95 (dd, 1
H), 2.47 (s, 3 H), 2.38–2.32 (m, 1 H), 1.95–1.60 (m, 4 H), 1.41–1.18 (m, 6 H).
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1
ꢀCyclohexylꢀ2ꢀ(3ꢀmethylꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol
(8).
The
synthesis of 1ꢀcyclohexylꢀ2ꢀ(3ꢀmethylꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanol (8)
followed the established procedure of the NewLink Genetics Corporation with
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necessary
modifications.
To
a
solution
of
1
ꢀcyclohexylꢀ2ꢀ(3ꢀmethylꢀ5Hꢀimidazo[5,1ꢀa]isoindolꢀ5ꢀyl)ethanone (7) (72.0 mg,
o
0.240 mmol) in methanol (2 mL) at 0 C was added sodium borohydride (27.8 mg,
0.730 mmol), and the mixture was stirred at room temperature for 2 h. The solvent
was removed under reduced pressure and 1N HCl (1 mL) was added to the crude. The
2
solution was stirred for 10 min and was neutralized by saturated. K CO3(aq) solution.
The aqueous layer was extracted with dichloromethane (2 x 5 mL). The combined
4
organic layer was dried over MgSO and concentrated to afford a crude product. the
crude was purified by automated flash chromatography using 1–10% MeOH : CH
1
2 2
Cl
gradient to give 8 as a white solid (54.0 mg, 75%). H NMR (CDCl 300 MHz) (a
3,
mixture of 4 stereoisomers) δ 7.51 (d, 1 H), 7.46 (d, 1 H), 7.36 (t, 1 H), 7.22 (t, 1 H),
7
.06 (s, 1 H), 5.40 (dd, 1 H), 3.59–3.51 ( m, 1 H), 2.53 (s, 3 H), 2.42–2.27 (m, 1 H),
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2.02–1.90 (m, 1 H), 1.72–1.52 (m, 5 H), 1.40–0.88 (m, 6 H). ESMS m/z 297.1 (M +
1
24 2
); HRMS (FAB) m/z calcd for C19H N O (M): 296.1889, found 296.1892; HPLC
purity of 4 stereoisomers of compound 8 > 99%,
ꢀ(1ꢀTritylꢀ1Hꢀimidazolꢀ4ꢀyl)aniline (13). Following the procedure of the synthesis
of 5, a mixture of 4ꢀiodoꢀ1ꢀtritylꢀ1Hꢀimidazole (11) (1.75 g, 4.00 mmol),
ꢀaminophenylboronic acid pinacol ester (12) (1.31 g, 6.00 mmol), and potassium
phosphate tribasic (2.55 g, 12.0 mmol) in DMF (20 mL) and water (4 mL) under
R
t = 21.15 min.
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nitrogen was added Pd(PPh ) (0.460 g, 0.400 mmol). After workꢀup, the crude
mixture was purified by automated flash chromatography using 0–10% MeOH :
1
2 2 3
CH Cl gradient to give the desired product 13 (0.860 g, 53%). H NMR (CDCl , 400
MHz) δ 7.56 (s, 1 H), 7.40–7.34 (m, 9 H), 7.28 (d, 1 H), 7.23–7.18 (m, 6 H), 7.08 (s, 1
H), 7.04 (t, 1 H), 6.73 (d, 1 H), 6.66 (t, 1 H).
1
ꢀCyclohexylꢀ2ꢀ(5,6ꢀdihydroimidazo[1,5ꢀc]quinazolinꢀ5ꢀyl)ethanone
(14).
Following the procedure of the synthesis of 7, to
a
solution of
2
ꢀ(1ꢀtritylꢀ1Hꢀimidazolꢀ4ꢀyl)aniline (13) (0.270
g, 0.670 mmol) and
3
ꢀcyclohexylꢀ3ꢀoxopropanal (10) (0.120 g, 0.800 mmol) in methanol (4 mL) was
added acetic acid (1 mL). After workꢀup, the crude mixture was purified by
2 2
automated flash chromatography using 3–8% MeOH : CH Cl gradient to give 14 as a
1
white solid (100 mg, 51 %). H NMR (CDCl
3
, 400 MHz) δ 7.76 (s, 1 H), 7.48 (d, 1
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H), 7.37 (s, 1 H), 7.13 (dd, 1 H), 6.91 (dd, 1 H), 6.76 (d, 1 H), 5.90 (dd, 1 H), 4.94 (br
s, 1 H), 3.16–3.05 (m, 2 H), 2.34–2.05 (m overlapped with br s at 2.22, 1 H), 1.90–
1
.60 (m, 4 H), 1.40–1.19 (m, 6 H).
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ꢀCyclohexylꢀ2ꢀ(5,6ꢀdihydroimidazo[1,5ꢀc]quinazolinꢀ5ꢀyl)ethanol (15). Following
the
procedure
of
the
synthesis
of
8,
1
ꢀcyclohexylꢀ2ꢀ(5,6ꢀdihydroimidazo[1,5ꢀc]quinazolinꢀ5ꢀyl)ethanone (14) (90.0 mg,
0
.300 mmol) was reacted with sodium borohydride (35.0 mg, 0.900 mmol) in
methanol (3 mL). After workꢀup, the crude mixture was purified by automated flash
2 2
chromatography using 1–8% MeOH : CH Cl gradient to give 15 as a white solid
1
(63.0 mg, 69%), which is a mixture of 4 stereoisomers H NMR (CDCl 400 MHz)
3,
(a mixture of 4 stereoisomers) δ 7.85 (s, 1 H), 7.46 (d, 1 H), 7.32 (s, 1 H), 7.11 (t, 1 H),
6
.90 (t, 1 H), 6.84–6.70 (m, 1 H), 5.78 (dd, 0.4 H), 5.65 (d, 0.6 H), 3.75–7.63 (m, 0.4
H), 3.46 (dd, 0.6 H), 2.20–1.83 (m, 2 H), 1.83–1.58 (m, 5 H), 1.42–0.83 (m, 6 H).
23 3
ESMS m/z 298.1 (M + 1); HRMS (FAB) m/z calcd for C18H N O (M): 297.1841,
R
found 297.1841. HPLC purity of 4 stereoisomers of compound 15 = 92.8%, t =
2
0.63 min.
ꢀβꢀCarbolinꢀ9ꢀylꢀ1ꢀcyclohexylꢀethanol (18). Powdered KOH (126.2 mg, 2.25 mmol)
2
was added to a solution of norharmane (16) (252 mg, 1.50 mmol) in anhydrous DMF
(1.5 mL) at 0 °C
.
After stirring at room temperature for 30 min until dissolved,
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