Discovery of clinical candidate (1 R,4 r)-4-((R)-2-((S)-6-Fluoro-5 H-imidazo[5,1-A[isoindol-5-yl)-1-hydroxyethyl)cyclohexan-1-ol (Navoximod), a potent and selective inhibitor of indoleamine 2,3-dioxygenase 1

Article abstract of DOI:10.1021/acs.jmedchem.9b00662

A novel class of 5-substituted 5H-imidazo[5,1-a]isoindoles are described as potent inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1). A structure-based drug design approach was used to elaborate the 5H-imidazo[5,1-a]isoindole core and to improve potency and pharmacological properties. Suitably placed hydrophobic and polar functional groups in the lead molecule allowed improvement of IDO1 inhibitory activity while minimizing off-target liabilities. Structure-activity relationship studies focused on optimizing IDO1 inhibition potency and a pharmacokinetic profile amenable to oral dosing while controlling CYP450 and hERG inhibitory properties.

Full text of DOI:10.1021/acs.jmedchem.9b00662

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Article
The Discovery of Clinical Candidate (1R,4r)-4-((R)-2-((S)-6-Fluoro-5H-
imidazo[5,1-a]isoindol-5-yl)-1-hydroxyethyl)cyclohexan-1-ol (Navoximod),
a Potent and Selective Inhibitor of Indoleamine 2,3-dioxygenase 1
Sanjeev Kumar, Jesse P. Waldo, Firoz A. Jaipuri, Agnieszka Marcinowicz, Clarissa Van Allen, James Adams,
Tanay Kesharwani, Xiaoxia Zhang, Richard A. Metz, Angela Oh, Seth F. Harris, and Mario R. Mautino
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00662 • Publication Date (Web): 18 Jun 2019
Downloaded from http://pubs.acs.org on June 18, 2019
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The Discovery of Clinical Candidate (1R,4r)-4-((R)-
1
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-((S)-6-Fluoro-5H-imidazo[5,1-a]isoindol-5-yl)-1-
hydroxyethyl)cyclohexan-1-ol (Navoximod), a
Potent and Selective Inhibitor of Indoleamine 2,3-
dioxygenase 1.
†
¤
‡¤
§¤
†
Sanjeev Kumar , Jesse P. Waldo Firoz A. Jaipuri , Agnieszka Marcinowicz , Clarissa Van
,
†
†
‖
†
ǂ
Allen , James Adams , Tanay Kesharwani , Xiaoxia Zhang, Richard Metz , Angela J. Oh , Seth F.
ǂ
†
Harris , Mario R. Mautino *
†
‡
NewLink Genetics, Ames, IA 50010, United States
Discovery Chemistry, Janssen Research and Development, LLC, Welsh and McKean Roads,
Spring House, PA 19477, United States
§
Medicinal Chemistry, Biocon Bristol-Myers Squibb R&D Center, Biocon Park, Bommasandra
Industrial Area, Bengaluru, KA 560099, India
‖
Department of Chemistry, University of West Florida, Pensacola, FL 32514
ǂ Structural Biology, Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080
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KEYWORDS. Indoleamine 2,3-dioxygenase 1 inhibitors, Navoximod, Imidazoisoindoles,
Phenylimidazoles, Structure-based drug discovery
ABSTRACT. A novel class of 5-substituted 5H-imidazo[5,1-a]isoindoles are described as potent
inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1). A structure-based drug design approach was
used to elaborate the 5H-imidazo[5,1-a]isoindole core and to improve potency and
pharmacological properties. Suitably placed hydrophobic and polar functional groups in the lead
molecule allowed improvement of IDO1 inhibitory activity while minimizing off-target liabilities.
The structure-activity relationship (SAR) studies focused on optimizing IDO1 inhibition potency
and a pharmacokinetic profile amenable to oral dosing while controlling CYP450 and hERG
inhibitory properties.
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Introduction
Indoleamine 2,3-dioxygenase 1 (IDO1) is a 45 kDa cytosolic monomeric heme-dioxygenase that
catalyzes the initial and rate limiting oxidative cleavage of the 2,3-indole position of L-tryptophan
(
L-Trp) and incorporates both atoms of molecular oxygen (O ), resulting in the production of N-
2
formyl kynurenine. The main function of IDO1 is the regulation of acquired local and peripheral
immune tolerance in normal and pathological scenarios. IDO1-expressing cells are found
constitutively in many normal tissues where IDO1 regulates local inflammation and moderates
responses against pathogens as well as to foreign or uncommon non-pathological antigens.
Constitutive IDO1 activity is also found at the maternal-fetal interface, where IDO1 plays a critical
role in the induction of maternal immune tolerance to the father-derived allogeneic antigens
1
expressed by the fetus. IDO1 expression can also be induced in antigen-presenting cells (APCs)
of the immune system in response to inflammation signals and is subject to complex regulation by
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an array of immunological signals. In response to inflammatory signals, IDO1 exerts its
immunosuppressive effects by both local depletion of tryptophan (Trp) and by the generation of
kynurenine (Kyn). Degradation of tryptophan deprives cells of an essential nutrient, triggering
changes in metabolism and protein synthesis that can result in changes of cellular function. Cells
have sensing mechanisms to detect deficiency in any amino acid, thus allowing them to transiently
stop non-critical protein synthesis and coordinate a survival response. One such amino-acid
deficiency sensing mechanism is the stress kinase GCN2, which contains a regulatory domain that
binds uncharged amino acid free-tRNA. When GCN2 binds uncharged tRNA this activates its
1
1
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2
kinase domain, which phosphorylates the ribosomal initiation factor eIF2α. In its phosphorylated
configuration, p-eIF2α prevents the translation of most mRNA species, except those involved in
compensation responses to the starvation stress.2-3 In addition to depleting tryptophan, IDO1
generates a series of bioactive downstream metabolites along the kynurenine pathway. Kynurenine
and other Trp metabolites are immunologically-active ligands for the aryl hydrocarbon receptor
4
(
AhR). The AhR is a ligand-activated transcription factor, which regulates transcription of genes
5
+
that include IDO1 and FoxP3, a key transcription factor that controls differentiation of Foxp3
4
regulatory T cells (Tregs) , which are a population of cells that play a key role in the regulation of
6
immune responses. The activation of both the GCN2 and AhR pathways leads to differentiation
and activation of Tregs, the induction of anergy of effector T cells and modulation of function and
phenotype in antigen presenting cells. Together, these IDO1-driven changes mediate local and
systemic immunosuppressive effects that block immune response.
Given the important role of IDO1 in control of acquired immune tolerance, it is not surprising that
the IDO1 pathway is induced in tumor cells and in host immune cells and contributes to acquired
7
immunologic tolerance towards those tumors. IDO1 expression and activity is observed in many
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types of tumors including melanoma, leukemia, pancreatic, ovarian, colorectal, endometrial and
8
prostate cancers, and its expression is associated with significantly worse clinical outcomes. IDO1
can also be expressed by cells of the host immune system that are associated with tumors, where
it contributes to acquired immunologic tolerance of tumors.
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Consequently, inhibition of IDO1 activity to restore immune response against tumors is an area of
active interest, triggering the development of numerous classes of IDO1 inhibitory compounds in
9
the last decade. Here, we describe our structure-activity relationship (SAR) strategy and efforts
that resulted in the selection of the clinical development candidate navoximod (NLG-919), starting
from the available crystal structure of IDO1 complexed with the inhibitory compound 4-phenyl
imidazole (1).10
Results and Discussions
Substitution of the phenyl ring of 1. Our present work in this area stems from the published X-
1
0
ray crystal data of IDO1 complexed with 1 and the preliminary studies by Kumar et al. on these
11
structures. In 5-(2-hydroxyphenyl)imidazole (3), the increase of 10-fold in potency with respect
to 1 (hIDO1 IC = 28 µM) was attributed to the hydrogen bonding interactions of hydroxyl with
5
0
Ser167 in the IDO1 active site. Docking studies with 3 showed that small substituents might be
accommodated at 3', 5' and 6'-positions of the phenyl ring. By diligently selecting a combination
of small hydrophilic and hydrophobic moieties, we were able to increase the potency about 5-fold
over 3 to generate 11 (Table 1). 4-6, 8 and 11 showed submicromolar activity, indicating that the
IDO1 active site has a small pocket that could accommodate a hydrophobic group para to the 2'-
OH. Switching the Cl and F positions in 11 resulted in substantial loss of activity (14).
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Table 1. Effects of Substitution on the Phenyl Ring of 1.
X
H
3'
N
4
'
N
R
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
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4
4
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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
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5
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0
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0
6' 5'
LEa
compound
X
H
3'
-
-
-
-
5'
-
-
6'
-
-
-
-
-
-
-
IC50 (µM)
28
1.7 (4.8)b
0.49
0.64
0.76
1.7
0.96
4.7
EC50 (µM)
1
3
4
5
6
7
8
9
-
0.58
0.67
0.68
0.67
0.66
0.62
0.60
0.57
0.65
0.48
0.43
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
7.2
0.94
2.4
1.7
-
-Cl
-Me
-Br
-F
-F
-
-Cl
-
-F
-
-
-F
-
-F
-Cl
-Cl
-
b
-OH
60
1.9
-
1
1
1
1
3
4
-
-
-
0.3
32
47
-
a
b
11
Biochemical LE; Value determined previously
Figure 1. Predicted binding mode for 11 (magenta) minimized in the IDO1 pocket using the crystal
IDO1 structure 2D0T (Hydrogen bonding and heme interaction with 11 shown with red dotted
line)
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1
1 was the most potent compound of this series and showed very good biochemical ligand
efficiency; however, it showed a significant 6-fold shift in cellular potency and had a very short
half-life in vivo (t1/2 < 1h). The short half-life was attributed to high clearance due to the phase 2
metabolization of phenol (data not reported). Several prodrugs of phenol were synthesized to
overcome the metabolic liability, but in vivo PK could not be improved (data not reported). We
decided to remove the phenolic hydroxyl group and attempted substitutions on the N-1 nitrogen to
achieve favorable interactions within the active site. In an attempt to gain hydrogen bond
interactions between hydrogen bond donors and the heme propionate, compounds with different
linker lengths such as a primary alcohol 15, amides 18 and 23-24 were prepared. However, these
attempts led to compounds with only marginal inhibitory activity (23) or inactive compounds (15,
0
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1
8 and 24). Hydrophobic side chains such as 2,2-dimethylbutyl 21 and cyclohexyl 22 were
explored in an attempt to gain favorable Van der Waals interactions with Phe226. However, these
approaches did not afford any improvement in the activity compared with parent compounds 3 and
4
(Table 2). Nonetheless, they indicated that substitutions on the N-1 imidazole could be tolerated,
confirming that N-1 of imidazole ring points towards the open region of the active site.11
Table 2. Effects of Substitution on the N-1 of 1.
R
N1
6' 5'
N
3
X
2'
3'
compound
R
-CH
-CH
-CH
Cyclohexyl
2'
-
-
OH
OH
-
5'
-
-
IC50 (µM)
>1000
>1000
23
1.9
187
>1000
EC50 (µM)
1
1
2
2
2
2
5
8
1
2
3
4
2
2
CH
CH
2
OH
NHCOCH
-
-
-
6.1
-
-
2
3
2
CH
2
t-Bu
Cl
-
-
-CH
-CH
2
2
CONHMe
CH CONHMe
2
-
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Next, we turned our attention to the hydrophobic pocket formed by Phe163 and Phe226,
independently of the interactions between 2-OH and Ser167. In order to access the hydrophobic
pocket, ether analogues of 3 were synthesized (Table 3). The active site seems to be able to
accommodate a wide range of hydrophobic groups and linker lengths while showing improvement
in enzymatic inhibition with respect to the parent compound 1. A comparison between benzyl,
phenethyl and phenpropyl ethers showed that the ethylene linker was more favorable than
methylene and propylene linkers (26-28). t-Butyl 29 and i-propyl 30 were well tolerated with
acceptable biochemical ligand efficiencies. Cycloalkyl compounds with 3-, 5- and 6-membered
rings (31-33) joined to an O-ethylene linker were nearly equipotent in biochemical assays and also
had good biochemical ligand efficiencies. Notably, an O-ethylene linker terminating with a
cyclohexyl group (33) was nearly equipotent with phenol (3) in biochemical assay. Side chains
terminating with polar heterocycles such as pyrimidine 34 or pyrazole 35 resulted in the loss of
biochemical IDO1 inhibition and decreased ligand efficiencies.
1
1
1
1
1
1
1
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1
1
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2
2
2
2
2
2
2
2
2
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3
3
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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
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Table 3. Effect of Linker Length and Distal Hydrophobic Group on 2'-Phenylethers.
R
HN
N
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
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7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
compound
1
R
H
-OCH
-O(CH
-O(CH
-O(CH
-O(CH
IC50 (µM)
27
18
9.1
16
7.8
6.6
5.1
EC50 (µM)
LEa
0.58
0.35
0.35
0.32
0.39
0.43
0.44
2
2
2
2
3
6
7
8
9
0
2
Ph
-
65
110
-
-
-
2
2
2
2
)
2
)
3
)
2
)
2
Ph
Ph
t-Bu
i-Pr
31
O(CH2)2
O(CH2)2
3
2
7.2
-
0.38
3
3
3
4
3.8
26
-
0.38
0.26
O(CH2)2
N
N
154
O(CH2)2
3
5
N
44
-
0.32
O(CH2)2
N
aBiochemical LE. -: Not determined
With these data in hand, we investigated whether a hybrid molecule of 3 and 33 would provide
a synergistic effect on improving potency (Figure 2). A combination of 2'-OH and 6'-
ethylcyclohexyl ether side chain resulted in slight improvement in biochemical activity (36) as
compared to the parent 33, but was equipotent with 3. This data suggests that only one component,
2
'-hydroxyl or the 6'-ethylcyclohexyl ether, is contributing to the binding energy of 36, and that
the optimal conformation adopted within the active site for each group is not compatible with the
preferred binding mode provided by the combined substituents. The molecular conformation of 36
shows that due to increased torsional strain between the phenyl and imidazole rings, the phenyl
1
2
and imidazole rings are at a dihedral angle of 45 degrees. Compound 36 was not considered for
further optimization as it exhibited a large shift in cellular activity.
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HO
O
NH
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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6
0
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9
0
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9
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36
IC50 = 1.7 M
EC50 = 80 M
Figure 2. Effect of Combination on IDO1 Inhibition.
Effect of molecular rigidification on IDO1 activity. One established strategy to increase
membrane permeability and cellular potency of small molecule inhibitors is to reduce the number
1
3
of rotatable bonds. In order to reduce the number of rotatable bonds, we envisioned restricting
the rotation of the carbon-carbon single bond of phenyl imidazole by synthesizing fused rigid
derivatives (Table 4). A methylene linker fusing the N-1 and 2'-position of the phenyl ring (37)
improved biochemical IDO1 inhibition 4-fold over 1 with improved ligand efficiency. Two carbon
linker containing compounds oxazepine derivative 44 and imidazo[5,1-a]isoquinoline 45 resulted
in the loss of IDO1 inhibition. In order to exploit interactions of the imidazoisoindole ring with
Ser167, we explored whether the addition of a 9-OH group would substantially improve binding
affinity (41). However, 41 was found to be biochemically equipotent with 37, suggesting that the
phenolic OH group on 41 does not interact with Ser167 in a similar orientation as the 2'-OH group
of phenylimidazole series. Energy minimization of 37 in the IDO1 active site revealed that the
methylene carbon vector towards the opening of the pocket could potentially accommodate various
substituents (Figure 3). This presented an excellent opportunity for further modifications to explore
5
H-substituted imidazoisoindole analogues. Given the comparable biochemical potency of the
imidazoisoindoles 37 and 41, and the potential metabolic sensitivity of phenolic compounds to
1
4
glucuronidation, we chose to explore substituents on 37 instead of 41.
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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
Table 4. Effect of Rigidification on Phenylimidazole Derivatives.
LEa
compound
Structure
IC50 (µM)
5.7
EC50 (µM)
>25
3
4
4
7
1
4
0.61
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
N
N
HO
3.1
312
22
7.9
0.59
0.35
0.50
N
N
O
-
-
N
N
45
N
N
aBiochemical LE. -: Not determined
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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. 37 (magenta) minimized in the IDO1 (2D0T) pocket (receptor surface shown in grey,
heme and ligand interaction shown by red dashes).
Substitution at the C5-position of the imidazo[5,1-a]isoindole core and lead identification.
Aiming to engage favorable interactions within the IDO1 active site, our focus turned towards
synthesizing imidazo[5,1-a]isoindole analogues containing functional groups that might form
favorable interactions with the heme propionate side chain or residues Phe226, Ser263, Ser235 or
Arg231. We extended 37 by substituting the 5-methylene carbon of 5H-imidazo[5,1-a]isoindole.
Because phenylimidazole 33 containing an ethylcyclohexyl ether side chain had good biochemical
IDO1 inhibition as compared to 1, we synthesized fused analogue 46 by incorporating an
ethylcyclohexyl side chain onto the 5-position of the imidazoisoindole core. Racemic
imidazoisoindole 46 exhibited superior biochemical (IC = 0.135 µM) and cellular inhibition of
5
0
IDO1 (EC = 1.1 µM) as compared to 33 and to 37 representing a >20-fold improvement in
5
0
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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
cellular potency, suggesting that molecular rigidification improved the biochemical and cellular
potency. Docking studies suggested that the S-enantiomer of 46 would be best accommodated,
establishing hydrophobic interactions with Phe226, while the R-enantiomer of 46 would likely
make an unfavorable clash with Ser263 and Ala264 residues. (Figure 4).
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 4. S-Isomer of racemate 46 (magenta) minimized in IDO1 (2D0T), cyan dashes shows
distance (4.2Å) between heme propionate side chain and methylene carbon linker.
Targeting interactions with heme propionate. In the minimized structure of S-46, the linker
methylene adjacent to the cyclohexyl ring is within close proximity (4.2 Å) of the heme propionate
side chain (Figure 4). We envisioned that a suitably placed hydrogen bond donor or basic amine
could form a hydrogen bond or ionic interaction respectively with the heme propionate side chain,
while at the same time reduce its cLogP to improve solubility and cellular potency. Hydroxyl and
amino groups were introduced into 46 giving rise to 52 and 54. Amine analogue 54 was not
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9
tolerated in the side chain. Interestingly, diastereomeric mixture 52 bearing a hydroxyl group on
the side chain was 13-fold more potent (EC = 83 nM) in cellular IDO1 inhibition assay as
5
0
compared to parent imidazo[5,1-a]isoindole 46. An analogous non-cyclized derivative 48 was
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
found to be >100-fold less active than 52, highlighting the importance of restricting conformational
freedom and orienting the hydroxyl group vector toward the heme propionate to form a hydrogen
bond (Figure 5).
7
8
6
9
5
10
O
1
1
1
1
5
N
N 4
N
OH
2
1
NH
HO
14
H N
3
13
2
N
N
2
N
N
46
52
54
48
IC50 = 0.135 M
EC50 = 1.1 M
cLogP = 5.28
IC50 = 0.060 M
EC50 = 0.083 M
cLogP = 3.48
IC50 = 1.1 M
EC50 = 3.1 M
cLogP = 3.57
IC50 = 7.2 M
cLogP = 3.08
Figure 5. Effect of C11 substitution to target heme propionate interactions
Effect of various substituents at C5 of imidazo[5,1-a]isoindole core on IDO1 inhibition. We
evaluated different substituents at the C5 position that could afford favorable interactions with the
IDO1 enzyme (Table 5). Owing to the difficulty in separating and isolating individual
diastereomers for each compound, the initial efforts were focused in the evaluation of IDO1
inhibition for the mixture of diastereomers of each compound. Compounds lacking a cycloalkyl
group such as 57 and 58 were 100-fold less potent than 52, suggesting that a combination of Van
der Waals interactions arising from the cyclohexyl moiety and hydrogen bonding by the hydroxyl
group is required for potent IDO1 inhibition. Cyclopentyl derivative 61 resulted in a 2.5 fold
decrease in biochemical IDO1 inhibition compared to that of 52. Increasing the linker length by
one carbon between the hydroxyl and cyclohexyl of 52 resulted in significant loss in potency (63).
A ketone functional group (64) was not tolerated in the side chain. Presumably, electron pair
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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
repulsion between the carbonyl oxygen’s lone pair and the heme propionate side chain decreases
the binding efficiency of 64, resulting in a loss of potency. In case of pyridine analogues 67, 69
and 71, the 4-pyridyl derivative 71 was equipotent with the corresponding phenyl analogue 65,
indicating that binding to IDO1 was highly dependent upon the position of heteroatom in the
aromatic ring side chain, presumably the heteroatom in the 4-pyridyl analogue occupies the
solvent-exposed region. Polar functional groups were not tolerated near hydrophobic residue
Phe226 within the IDO1 pocket. Pyran analogue 73 resulted in a 10-fold decrease in IDO1 potency.
A similar trend was observed with 5-membered azoles such as thiazole 75, N-methyl-imidazole 77
and N-methyl-pyrazole 79 or with basic secondary amines such as piperidine 84 and azetidine 85.
However, the carbamate derivatives of azetidine or piperidine (81 and 83) were found to be >25-
fold more active as compared to the corresponding free amines. Six-membered ring carbamate 83
was preferred over the corresponding azetidine 81.
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 5. Cyclic Side Chain Modification of 5H-Imidazo[5,1-a]isoindoles
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R
compound
IC50 (µM)
EC50 (µM)
N
N
HO
5
5
5
2
7
8
0.060
6.7
0.083
5.9

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

OH
OH

7.7
6.7
HO
61
0.16
0.906
0.78
0.23


6
3
OH
O
HO
HO
HO
6
6
6
4
5
7
5.2
0.33
8.8
15
0.82
-




N
N
69
2.9
-
HO
7
7
1
3
N
0.37
0.39


O
0.79
1.8
0.51
0.76
-
HO


S
7
7
5
7
N
HO
N
127
1.2
N
HO

N
N
79
4.2
-
HO
HO
8
4
NH
120


8
8
5
1
NH
24
-
HO

N Boc
0.81
0.14
0.49
0.24
HO
HO
83
N Boc

Optimization of piperidine analogues of 5H-imidazo[5,1-a]isoindoles. The nitrogen atom in
piperidine analogue 84 offered a convenient handle for derivatization to possibly improving
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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
potency and drug-like properties.15 A library of piperidine amide and urea analogues were
prepared and representative examples are shown in Table 6. Acyclic and cyclic aliphatic or
aromatic R-groups 87-89 resulted in a decrease in potency compared to 83. A methylene linker
between the phenyl ring and amide bond was required to maintain potent IDO1 inhibition as
evidenced by a 60-fold improvement in IDO1 inhibition from 89 to 90 and 91. The urea derivate
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
9
4 also exhibited a similar profile. Unfortunately, these compounds showed potent CYP3A4
inhibition. Lowering the cLogP by introducing heteroatoms in the phenyl ring (92-93) improved
the CYP3A4 profile at the expense of a decrease in IDO1 potency. In general, structural
modifications that were incorporated to mitigate CYP liabilities resulted in loss of IDO1 potency.
Due to their propensity to be potent inhibitors of multiple CYP isoforms, piperidine derivatives
were no longer considered for further development as potent IDO1 inhibitors.
Table 6. Analogues of N-Piperidine Amides of 5H-Imidazo[5,1-a]isoindoles
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R
N
CYP 450 IC50 (µM)
IC50
(µM)
EC50
(µM)
compound
cLogP
N
HO
O
3
A4
5
2D6
208
1A2
2B6
N
8
8
7
8
2.5
1.1
>100
>100
0.11
2.15
*
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
O
*
0.67
0.33
0.70
-
9.5
-
>100
-
61
-
O
*
8
9
2.4
-
2.15
F
O
90
0.031
0.040
0.23
0.7
0.078
0.13
1.3
0.13
0.06
3.8
19.5
4.8
40
>100
>100
28
39
50
2.38
2.53
0.88
-0.07
*
F
O
9
9
1
2
*
O
N
N
56
*
*
N
O
93
3.8
8.60
152
>100
>100
O
9
4
0.045
0.059
0.51
10
93
75
1.98
*
N
H
-: Not determined
Lead optimization of 52. Since optimization of the piperidine series based on 84 did not yield
good lead candidates, we focused the lead optimization efforts on cyclohexyl derivative 52. Due
to difficulty in developing methods to synthesize and separate pure stereoisomers for each
compound, the initial lead optimization efforts were carried out on mixtures of diastereomers.
Compound 52 exhibited high in vitro clearance (HLM Clint = 18.03 mL/min/Kg) and potent
CYP3A4 inhibition (0.17 µM). Therefore, we focused our efforts on mitigating CYP3A4
inhibition and improving the metabolic stability of 52 while maintaining potency. First, the effect
of small halogen substitutions on the phenyl ring of the imidazo[5,1-a]isoindole core was
investigated (Table 7). Substitutions with fluoro or chloro at the 7 or 8-position resulted in a loss
of potency. Specifically, substitution at the 8-position by fluoro or chloro (100 and 106) resulted
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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
in a >60-fold decrease in biochemical inhibition, suggesting tight fitting of the imidazo[5,1-
a]isoindole core in the IDO1 pocket ceiling and in a different position compared to what was
inferred for the mode of binding of phenylimidazole 11. However, a fluoro group placed at the 6-
position in 96 maintained potent IDO1 inhibition and improved the CYP3A4 profile about 10-fold.
Table 7. Effect of Substituents on the Phenyl Ring of the 5H-Imidazo[5,1-a]isoindole
Pharmacophore
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
7
8
X
6
9
CYP 450 IC50 (µM)
compound
IC50 (µM)
EC50 (µM)
N4
3
1
HO
N
2
X
3A4
0.17
2D6
2.2
1A2
2B6
5.9
5
9
9
2
6
8
-H
0.06
0.03
0.42
4
0.083
0.2
>100
6-F
7-F
2.0
2.0
-
>100
10
-
0.34
2.7
-
-
-
-
1
00
8-F
-
-
102
104
6-Cl
7-Cl
8-Cl
0.61
0.21
6.9
1.9
1.70
0.01
-
88
4.7
-
-
4.8
4.5
-
2.2
6.1
-
1
06
5.4
-: Not determined
Mitigating CYP450 and Human Liver Microsomal Clearance. We then focused our efforts on
improving the hepatic clearance of lead molecule 96 (18.91 mL/min/Kg). We hypothesized that
the high clearance of 96 could be due to poor metabolic stability of the cyclohexyl group. First
16
pass metabolism of cycloalkyl groups is a well-documented pathway. We presumed the C15 of
the cyclohexyl side chain to be the hot spot for metabolization, and several strategies were explored
to circumvent high clearance in human liver microsomes (Table 8). Blocking the C15-positon by
a geminal-difluoro group offered a modest improvement in clearance as observed in compounds
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1
08 and 110. Other C15-cyclohexyl modifications such as methylcarboxylate 112 and ketone 114
resulted in decreased IDO1 inhibition. Benzamides 116, 118 derived from 4-trans and 4-cis
cyclohexyl amines respectively exhibited promising activity against IDO1, but these compounds
exhibited poor aqueous solubility near neutral pH and suffered from moderate CYP3A4 and
CYP2D6 inhibition.
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
Modeling of the (C5 S, C11 R) stereoisomer of 52 within the IDO1 active site revealed that the
C15-position of the cyclohexyl ring is within close proximity to Ser235 and Arg231 (Figure 7).
We hypothesized that a polar group at the C15-position could improve IDO1 activity by interacting
with Ser235 or Arg231. Furthermore, introduction of a polar group would lower cLogP, which
could improve CYP3A4 inhibition profile. Several case studies have established that compounds
with lower cLogP tend to decrease the risk of potent CYP3A4 inhibition and drug-drug
1
7
18
interactions, while also leading to improvement in metabolic stability. Substitution at the C15-
position by a hydroxyl group resulted in significant improvement in human microsomal clearance
(4.06 mL/min/Kg) and improved CYP profile (120) while maintaining IDO1 potency. By
decreasing the cLogP of lead candidate 96 (cLogP = 3.64), CYP inhibition and high clearance
were greatly improved. Overall, 120 (cLogP = 1.55) had the best balance of potency, metabolic
stability, off-target liabilities, and physiochemical properties (Table 8).
Table 8. Effect of Substituents on the Distal Cyclohexyl Ring
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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
R1
R2
CYP 450 (IC50 (µM))
EC50
HLM
hERG
EC50
N
IC50
(
fu
(%)
cmpd
HO
cLogP
µM) (µM)
Ph1
N
(µM)
3
A4
2D6
1A2
2B6
R1
R2
H
mL/min/Kg
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
96
F
H
F
0.03
0.2
2.0
1.2
2.6
-
2.0
5.7
20
-
>100
>100
10
41
23
18.91
14.60
15.27
-
6.1
3.64
2.87
3.02
2.34
1.15
F
1
1
1
1
08
10
12
14
*
0.046 0.079
13.2
>100
-
17.9
F
F
F
*
0.10
0.28
0.6
0.093
0.22
0.2
>100 >100
-
-
-
H
F
CO
2
Me
-
-
*=O
O
10.9
95
>100 >100
65
20.29
1
16
H
Ph
Ph
0.05
0.086 1.20
6.8
>100
>100
82
74
30
-
2.7
3.03
*
*
NH
O
1
1
18
20
H
F
0.072 0.079 1.10
0.2 0.25
1.40
16
34
8.23
4.06
2.9
3.03
1.55
NH
OH
3.30 >100 >100 >100
43.6
*Solubility data in µM: 120 (pH1=353; pH6.5=329; pH7.4=316); 118 (pH1=400; pH6.5=65;
pH7.4=56); 116 (pH1=363; pH6.5=22; pH7.4=25).
Stereochemistry and IDO1 activity relationship. Since 120 is a mixture of 8 stereoisomers, we
first determined the effect of C5 and C11 stereochemistry on isomers isolated from the active
analogue 52, which only contains 2 chiral centers. To this purpose, diastereomers of compound 52
were isolated by chiral SFC, giving rise to stereoisomers 121-124. The absolute stereochemistry
1
of each stereo isomer was determined by X-ray crystallography and H-NMR. These syn and anti
1
enantiomer pairs showed a typical splitting pattern by H-NMR for C5-H, with a triplet δ 5.39 (J
= 6.3 Hz, 1H) or a doublet of doublet δ 5.48 (J = 10.6, 3.2 Hz, 1H), respectively. The
stereochemistry at the C5 ring junction plays an important role in the binding of imidazo[5,1-
a]isoindole derivatives within the IDO1 active site. C5 S-substituted imidazoisoindole compounds
were drastically more potent than the corresponding C5 R-stereoisomers, just as it had been
predicted by in silico modeling. Stereoisomers 124 and 123 were 100-fold to 1000-fold more active
than their corresponding C5 R-stereoisomers 121 and 122 in biochemical IDO1 assay. C11 S-
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hydroxy stereoisomer 123 was over 5-fold more potent than C11 R-hydroxy stereoisomer 124 in
a IDO1 biochemical inhibition assay (Figure 6).
(
R)
(R)
(S)
(S)
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
C5
C11
R)
N
N
N
N
(
(S)
(S)
(R)
HO
HO
HO
HO
N
N
N
N
121
122
123
124
IC50 = >25 M
IC50 = 18 M
 5.39 (t, 1H)
IC50 = 0.021 M
 5.48 (dd, 1H)
IC50 = 0.11 M
d .39 (t, 1H)
C5-H:  5.48 (dd, 1H)
Figure 6. Evaluation of 52 isomers towards IDO1 inhibition
We hypothesized that the cyclohexyl moiety occupies the hydrophobic region near Phe226 and
the hydroxyl group forms a hydrogen bond interaction with the heme propionate side chain (Figure
7
). Our modeling hypothesis was later confirmed by recently published X-ray co-crystal structure
of various 5-H substituted imidazoisoindole analogues bound to IDO1.19
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7
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
Figure 7. 123 minimized in the IDO1 (2D0T) pocket; ligand-receptor interactions including
hydrogen bond shown by red dashes. The cyan dashes shows the distance between C15 of
cyclohexyl and Ser235 oxygen (4.4 Å) and Arg231 nitrogen (4.4 Å).
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
Effect of Stereochemistry in 120 and selection of NLG-919. Encouraged by the overall profile
of 120, we synthesized both cis (126) and trans-cyclohexanol (127) derivatives. Cis and trans-
cyclohexanol derivatives 126 and 127 respectively, were equipotent in biochemical assays as
mixtures of diastereomers (Table 9). Diastereomeric mixtures 126 and 127 were separated by
chiral SFC. As observed with the stereoisomers of compound 52, the predominant effect on IDO1
activity was determined by the stereochemistry of C5.
Trans-cyclohexanol C5-S isomers NLG-919 and 132 were 100-fold and 70-fold more potent
than their corresponding C5-R isomers 133 and 134, respectively. Similarly, cis-cyclohexanol
isomers 130 and 128 were 35-fold and 3-fold more potent than their C5 R-isomers 129 and 131,
respectively. The stereochemistry of the C11-OH group also played an important secondary role
in IDO1 inhibition, with the R-isomer being 2-fold to 5-fold more active than the corresponding
S-isomer. Similarly, the stereo configuration of the cyclohexanol group played an important role,
with the trans configuration being 3-fold to 10-fold more potent than the cis form. NLG-919
exhibited superior biochemical and cellular potency, an acceptable CYP450 profile, low hERG
inhibition (71 µM), a low protein binding (f 52%), low clearance in human liver microsomes (8.52
u
mL/min/Kg), and excellent solubility at wide pH range (>200 µM at pH 1-7.4).
Table 9. Effect of stereochemistry on C5 and C11
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9
hERG
EC50
(µM)
F
6
Human Liver
µsome
OH
CYP 450 (IC50 (µM))
f
u
IDO1
IC50
5
EC50
(µM)
compound
11
HO
N
(µM)
3
A4 2D6 1A2
2B6
Ph1
%
1
N
C5,C11
cis/trans
mL/min/Kg
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
Mix of
isomers
126
cis
cis
0.26
0.44
3.4
-
-
-
-
-
-
-
-
-
-
-
-
-
1
28
(S, S)
(R, R)
(S, R)
(R, S)
-
-
-
12.74
129
cis
-
-
-
-
-
-
-
1
1
1
1
30
31
27
32
cis
0.09
1.5
0.17
3.10
38
-
94
51
-
>100
18.51
46
-
cis
-
-
-
-
-
-
-
Mix of
isomers
trans
trans
trans
trans
trans
0.2
-
0.22
-
-
-
-
-
6.20
-
-
(S, S)
(R, S)
(R, R)
(S, R)
0.042
2.9
5.6
-
94
-
>100
43
-
-
35
-
133
34
NLG-919
-
-
-
1
3.7
-
-
-
-
-
-
-
0.028
0.075
5.7
86
>100 >100
71
8.52
52
-
: Not determined
The biochemical potency of NLG-919 (IC 28 nM) was close to the concentration of enzyme
50
being used in the enzymatic potency assay, indicating that NLG-919 is a tight binding inhibitor of
IDO1. Determination of the K was performed by measuring vi/v0 at IDO1 enzyme concentrations
i
of 63 to 1080 nM in the presence of varying concentrations of NLG-919 (0-8100 nM) followed
2
0
by non-linear fitting of the data to the Morrison equation. This analysis indicated that NLG-919
has a K of 5.8 nM. An analysis of the mode of inhibition by determination of the IC at different
i
50
concentrations of L-Trp (10-400 M) indicated that NLG-919 inhibits IDO1 activity in an
uncompetitive or non-competitive mode with respect to L-Trp substrate with α<1. The non-
competitive or uncompetitive mode of inhibition with respect to L-Trp was determined under
conditions of pseudo-first order assuming a non-limiting concentration of O , the second substrate
2
of the reaction, and is in agreement with the proposed mode of binding of NLG-919 directly
coordinated to the heme Fe+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
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 8. Crystal structure of NLG-919 bound to human IDO1 (6O3I). The protein is colored in
ivory. The heme molecule is displayed in light blue ball-and-stick, highlighting protein:heme
interactions at the propionate (yellow dashes) and the iron atom (grey dashes). Hydrogen bonds
from NLG-919 to the protein are indicated in red dashes, and select residues in the immediate
vicinity of the binding site are shown in stick and labeled.
The binding mode was corroborated by determination of the crystal structure of NLG-919 bound
to hIDO1. The core imidazoisoindole maintains its anticipated iron-coordinated position with the
upper tricyclic scaffold forming tight Van der Waals contacts in the hydrophobic cavity created by
IDO1 residues Val130, Phe163, Phe164, and Leu234. This positioning accounts for the dramatic
negative impact of halogens at C-7 and C-8 in compounds 98 and 100, while the design of the C-
6
fluoro-substituent is well-braced to the C- of Gly262 providing a positive contribution to
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potency. The stereochemistry of the linker positions its hydroxyl group to achieve the designed
hydrogen bond with the heme propionate, while the cyclohexanol moiety continues outward
toward the solvent accessible opening of the heme-proximal cavity, its saturated bulk affording
contacts in the widening channel. Additionally, the terminal hydroxyl is able to form a hydrogen
bond interaction with Ser235, giving a specific orientation to the distal end of the ligand.
Combined, the interplay of the stereochemistry and the observed interactions provides a consistent
accounting for the potency and selectivity of NLG-919 (Figure 8).
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
Pharmacological properties of NLG-919
NLG-919 showed favorable pharmacological properties that prompted us to select it as a clinical
development candidate. First, NLG-919 was not extensively metabolized by human or rat
microsomes, with >85% remaining after 90 min incubation in the presence of Phase I+II cofactors.
Second, NLG-919 showed no CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 (IC > 100
5
0
µM) inhibition; weak inhibitory effect for CYP2D6 (IC 86 µM) and moderate inhibitory effect
5
0
on CYP3A4 with IC values of 5.7 µM (midazolam) or 15 µM (testosterone). In Caco-2 assay,
5
0
-6
-6
NLG-919 was highly permeable (22.7 × 10 cm/s from A to B, 35.0 × 10 cm/s from B to A) and
demonstrated an efflux ratio <2 in the presence of verapamil at 100 µM indicating that NLG-919
is not a potential substrate for P-glycoprotein (P- gp). NLG-919 did not activate arylhydrocarbon
receptor (AhR) at 10 µM and it exhibited low transactivation of PXR at 300 µM, suggesting low
probability of CYP450 induction.
The pharmacokinetics of NLG-919 was studied in rats, mice and dogs. Following single
intravenous doses at 10-50 mg/kg in SD rats, NLG-919 exhibited a half-life (t ) of 1 h, and the
1
/2
clearance (CL) was about 23 – 39.1 mL/min/kg. The clearance decreased with increased dose level,
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1
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
indicating inverse dose-dependent clearance and saturation of the elimination pathways. In
C57B16 mice, the clearance was found to be ~26 mL/min/kg with a t of 3-4 h. Following a single
1
/2
IV dose administration to dogs, NLG-919 exhibited variable t , ranging from 1.6 – 6.9 h. NLG-
1
/2
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
9
19 was rapidly eliminated in rats with a plasma clearance value ranging from 41.4 – 59.3
mL/min/kg. A similar trend of decreasing clearance with increased dose was observed in dogs,
though to a lesser extent than in rats. The systemic exposure increased proportionally as the dose
was increased from 1 to 25 mg/kg. Following single oral doses at 10, 25, and 50 mg/kg in SD rats,
NLG-919 was rapidly absorbed (Tmax = 0.5 h) and exhibited moderate oral bioavailability of 41-
6
0% with a t of about 1 h. In C57B16 mice, NLG-919 exhibited high oral bioavailability of 69%
1/2
and 87% for the dose levels of 10 and 100 mg/kg, respectively and a t of ~1 h for the lower doses
1
/2
and 3 h for the higher dose. In dogs, oral bioavailability increased from 14% at 5 mg/kg to 72% at
1
50 mg/kg, suggesting saturation of metabolic clearance at higher doses (Table 10).
Table 10. Single dose pharmacokinetic profile of NLG-919 in different species after iv and oral
dosing
Dose
mg/kg)
CL
AUC0-inf
(ngh/mL)
t
1/2
C
max
(ng/mL)
%F
(
(mL/min /kg)
(h)
Species
Rat
IV
44.6
32.0
23.2
27.5
25.0
-
IV
PO
1630
7975
15400
3777
-
IV
1.44
1.25
1.0
3.9
3.0
-
PO
1.05
1.15
1.06
1.2
-
PO
1
0
3930
13400
34950
6132
34633
-
1560
6420
10050
2257
-
41.5
60.4
44.1
69.0
-
25
5
1
0
0
Mice
Dog
50
100
1
60195
-
3.3
-
14165
-
87.0
-
59.3
282
2.2
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56.1
901
-
207
-
1.6
-
-
-
-
-
-
-
-
-
132
-
-
-
-
13.7
-
1
0
53.7
3127
4.8
-
15
-
-
1045
-
1400
-
22.3
-
2
5
5
0
41.4
10050
6.9
-
-
-
-
-
8645
57750
7855
40450
43.0
71.8
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
1
50
-
-: Not determined
NLG-919 was found to be moderately bound to plasma proteins (43% – 49% in SD rats, 48% –
2.0% in cynomolgus monkey and 52% – 55% in human plasma at 0.2, 2 and 20 μM
5
concentration). After a single oral dose of 50 mg/kg administered to C57Bl/6 mouse, NLG-919
was found to be present at higher concentration in plasma than in other organs, the maximum
exposure occurred at 1 h post-dose.
The pharmacodynamic effect of NLG-919 in vivo was measured in a mouse model where IDO1
2
1
activity is induced in the lungs by intranasal instillation of bacterial lipopolysaccharides. In this
mouse model, plasma Kyn was reduced by 50% and lung Kyn was reduced by 60%, 6 h after oral
dosing of NLG-919 at 50 mg/kg. Given that approximately 50% of systemic Kyn levels in plasma
is contributed by IDO1 and the rest is contributed by the liver enzyme tryptophan 2,3-dioxygenase
2
2
(TDO2), the results suggest that an oral dose of 50 mg/kg produces almost complete inhibition
of IDO1 activity in this model.
The antitumor activity of NLG-919 was determined in the Pan02 tumor model in C57Bl/6 mice,
where it demonstrated to reduce tumor growth and enhance survival as a single agent (p=0.0005;
Figure 9A) as well as to enhance the antitumor effect of cyclophosphamide in a combination
chemotherapy regimen (p=0.0083; Figure 9B).
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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
6
Figure 9. Antitumor activity of NLG-919 in Pan02 tumor model. A) 5x10 Pan02 cells were
implanted s.c. into the flank of C57Bl/6 mice (n=7/group). NLG-919 was dosed at 500 mg/kg/day,
from Days 7-32. B) Survival of C57Bl/6 mice bearing Pan02 tumors (n=12/group), where mice
were dosed with NLG-919 at 400 mg/kg/day from days 7 to 25 post-tumor inoculation, with or
without 3 doses of cyclophosphamide 100 mg/kg i.p. on Days 8, 13 and 15.
Toxicology studies were conducted in rats and dogs in 28-day repeat dose studies with 2-week
recovery. It was determined that the non-observed adverse event level (NOAEL) was 250
mg/kg/dose BID for rats (AUC0-inf = 22500 h*ng/mL) and 200 mg/kg/dose BID for dogs (AUC0-
of 22550 h*ng/mL).
1
2h
In summary, NLG-919 had a favorable drug-like profile and was selected as a clinical candidate
for further evaluation in Phase 1 clinical trials. Furthermore, this study describes for the first time,
the use of imidazo[5,1-a]isoindoles as a new class of heme-binding chemical pharmacophore,
which could become a structural scaffold for other heme-containing pharmacologic targets. In fact,
further SAR studies focusing on different C5 side chain substituents have afforded potent specific
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Page 29 of 108
Journal of Medicinal Chemistry
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inhibitors of the related enzyme tryptophan 2,3-dioxygenase (TDO2) and dual inhibitors of both
IDO1 and TDO2 enzymes.23
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Chemistry
The synthesis of 4-phenylimidazole derivatives 4-8 was achieved by the reaction of α-
24
bromoketone derivatives with formamide (Scheme 1). 3 and 9 were synthesized according to the
literature.11
a
Scheme 1 Synthesis of substituted 5-(phenyl)-1H-imidazole (1) derivatives
.
HO
HO
H
3'
3'
N
O
a
N
1'
1'
R
R
5'
5'
2a-f
3
5
7
, R = H; 4, R = 5'-Cl;
, R = 5'-CH ; 6, R = 5'-Br;
3
, R = 5'-F; 8, R = 3', 5'-F.
a
o
Reagents and conditions : (a) i) CuBr , CHCl , 60 C;
2
3
o
ii) H NCHO, 170-180 C, 33-61%.
2
1
1 was prepared by palladium-catalyzed Negishi cross-coupling25 of 4-iodo-1-trityl-1H-
2
6
imidazole with 4-chloro-2-fluoro-6-iodophenol followed by deprotection of the trityl group with
acetic acid in methanol (Scheme 2).
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