Discovery of Imidazoisoindole Derivatives as Highly Potent and Orally Active Indoleamine-2,3-dioxygenase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00114

A novel series of imidazoisoindoles were identified as potent indoleamine-2,3-dioxygenase (IDO) inhibitors. Lead optimization toward improving potency and eliminating CYP inhibition resulted in the discovery of lead compound 25, a highly potent IDO inhibitor with favorable pharmacokinetic properties. In the MC38 xenograft model in hPD-1 transgenic mice, 25 in combination with the anti-PD-1 monoclonal antibody (SHR-1210) achieved a synergistic antitumor effect superior to each single agent.

Full text of DOI:10.1021/acsmedchemlett.9b00114

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Letter
Discovery of Imidazoisoindole Derivatives as Highly Potent
and Orally Active Indoleamine-2,3-dioxygenase Inhibitors
Wangyang Tu, Fanglong Yang, Guoji Xu, Jiangtao Chi, Zhiwei Liu, Wei Peng, Bing Hu, Lei Zhang,
Hong Wan, Nan Yu, Fangfang Jin, Qiyue Hu, Lianshan Zhang, Feng He, and Weikang Tao
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00114 • Publication Date (Web): 03 Jun 2019
Downloaded from http://pubs.acs.org on June 4, 2019
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ACS Medicinal Chemistry Letters
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Discovery of Imidazoisoindole Derivatives as Highly Potent and
Orally Active Indoleamine-2,3-dioxygenase Inhibitors
Wangyang Tu,†,§ Fanglong Yang,† Guoji Xu,† Jiangtao Chi,† Zhiwei Liu,† Wei Peng, † Bing Hu,† Lei
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†
†
,†
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‡
†
,¶
Zhang, Hong Wan, Nan Yu, * Fangfang Jin, Qiyue Hu, Lianshan Zhang, Feng He, Weikang
Tao†,
¶
†Shanghai Hengrui Pharmaceutical Co., Ltd., 279 Wenjing Road, Shanghai 200245, China
‡Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, Jiangsu 222047, China
¶Chengdu Suncadia Medicine Co., Ltd., 88 South Keyuan Road, Chengdu, Sichuan 610000, China
KEYWORDS: IDO, immuno-oncology, SAR, lead optimization
ABSTRACT: A novel series of imidazoisoindoles were identified as potent IDO inhibitors. Lead optimization toward improving
potency and eliminating CYP inhibition resulted in the discovery of lead compound 25, a highly potent IDO inhibitor with
favorable pharmacokinetic properties. In the MC38 xenograft model in hPD1 transgenic mouse, 25 in combination with the anti-
PD1 monoclonal antibody (SHR-1210) achieved a synergistic antitumor effect, superior to each single agent.
The immune system plays a vital role in the regulation of
tumor growth, invasion, and metastasis. Immune checkpoint
inhibitors that restore the capability of the immune system to
recognize and eliminate malignant cells have produced
impressive clinical benefit, but many patients across a wide
range of malignancies still do not respond.1 These findings
imply there are additional immunoregulators that maintain
productive immunosurveillance in cancer. Indeed, tumor cells
escape immune elimination through evolving various tactics to
evade, subvert, and manipulate innate and adaptive immunity.
As a result, combinatory regimens with different targeted
therapeutic agents are necessary to produce sustained
therapeutic effect.2,3 One of these agents is a family of
tryptophan catabolizing enzymes including indoleamine-2,3-
dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO)
which convert tryptophan first to N-formylkynurenine and
further to kynurenine and additional metabolites. Both the
depletion of tryptophan and signals generated by its
which leads to accelerated differentiation of CD4+ T cell into
regulatory T cells, as well as suppression of CD8+ effector T
cell and impaired dendritic cell functions. On the other hand,
tumor cells evade immune-mediated eradication via PD-L1
expression, since the interaction of PD-L1 with PD1 inhibits
the secretion of cytotoxic mediators by CD8+ T cells. In
addition, IDO was further upregulated upon blockade of
PD1/PD-L1 interaction in mice due to compensatory
mechanism.15 Therefore, simultaneous blockade of both
pathways may represent an opportunity to accomplish greater
antitumor effects by complementary regulations of the
cytotoxic T cells.
NLG-919 is one of the IDO/TDO dual inhibitors that have
been evaluated in clinical trials alone or in combination with
anti-PD-L1 antibody for various solid tumors.16-18 Herein, we
report the synthesis and SAR study of a novel series of
imidazoisoindoles as potent IDO inhibitors and the
identification of lead compound that synergized with PD1
blockade in a murine tumor model.
metabolites are important contributors to immunosuppresion. 4-
6
F
Expression of IDO is widespread in human body, being
most abundant in antigen-presenting cells such as
macrophages and dendritic cells. IDO activity is increased in
several tumor types and is correlated with a poor prognosis.7,8
TDO is exclusively produced in the liver to maintain the
systemic tryptophan levels in response to food uptake.
Although the major role of IDO in immune regulation has
been validated, there are recent evidences that suggest TDO
might regulate immunosuppression similar to that of IDO.9
IDO selective and IDO/TDO dual inhibitors have been the
focus of research,10-13 whereas TDO selective inhibitors
remain elusive.9,14
OH
H
N
HO
NLG-919
N
Figure 1. Imidazoleisoindole derivative as IDO inhibitor in
clinical trial.
NLG-919 interacts with IDO via imidazoleisoindole core
coordinating to the iron center of heme. Hydroxyl group on the
side chain engages in an extensive hydrogen bond network
and contributes to the biological activity.19 However, three
consecutive chiral centers exert tremendous structural
complexities and synthetic challenges. We hypothesized that
Tumor cells hijack the immunosuppressive process by
upregulating IDO activity in the tumor microenvironment,
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modification of the side chain of NLG-919 with

O
3
4
969
189
5344
NDb
1
2
3
4
5
6
7
8
imidazoleisoindole core kept intact could offer the best
opportunity to fine tune potency and physicochemical
properties. Compounds 1-8 were synthesized via the route
shown in Scheme 1.20 Regioselective lithiation of m-
bromofluorobenzene with LDA followed by nucleophilic
addition to a variety of substituted aldehydes gave rise to
corresponding alcohols. The resulting alcohols were mesylated
and subsequently substituted by imidazole. The final
intramolecular Pd-mediated cyclization furnished the tricyclic
imidazoleisoindole core decorated with various appendages.
NH
O

>10000
>10000
N
N


5
3563
1275
NDb
9
6
7
8
87
338
374
793
544
NDb
NDb
10
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25
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Scheme 1. Synthesis of 5-substituted imidazoleisoindoles a

2741
2734
F
OH
F
OMs
R
F
a
b
R

Br
Br
Br
aValues are expressed as the mean of three independent
determinations. NDb = not determined.
N
F
F
N
Compound 6 was selected as the start for the next round
c
d
SAR study.
A series of substituted piperidinyls were
R
R
synthesized and assessed (Table 2). In order to lower
lipophilicity of compound 6, replacement of the phenyl group
with heteroaryls was investigated. Heteroaryls such as
thiadiazole (9), indole (10) and benzothiazole (11)
demonstrated enhanced potency toward IDO enzymatic assay
but not cellular assay. Simple substituents such as fluorine (12)
and cyano (13) didn’t improve potency. The morpholine 14
showed 3-fold lower potency than 6. Replacement of
morpholine with pyridine (15) resulted in a 10-fold boost
toward IDO activity. Among the aromatic substitutions tested,
methylpyrazole 18 was the most potent in all three assays
(IDO IC50 = 26 nM, TDO IC50 = 132 nM, Hela IC50 =101 nM,
respectively). The m-substituted methylpyrazole 17 was 2-fold
less potent than 18 due to unfavorable orientation of the
aromatic substitution. Fluorine (19, 20) and methyl (21, 22)
were tolerated on the central phenyl ring. Fluorine 20 was 2-
fold more potent than 18. And fluorine 19 and methyl 21
showed comparable potency to 18. In compound 23, central
phenyl ring was replaced by pyrimidine and the potency
decreased by 4-fold compared to 18.
N
Br
N
aReagents and conditions: (a) (1) LDA, THF, –78°C, 1 h; (2)
RCHO, –78°C, 1 h; (b) NaH, THF, MsCl, reflux, 48 h; (c)
imidazole, NaH, DMF, 100°C, 12 h; (d) Pd(OAc)2, PPh3,
Cy2NMe, DMF, 100°C, 5 h.
The screening assays include enzymatic assays with
purified recombinant human IDO/TDO proteins and cellular
IDO inhibition assay in the Hela cell line. Cyclohexyl 1
exhibited comparable potency to NLG-919 as shown in Table
1. However, smaller cyclopentyl 2 was less potent in IDO
assay. Tetrahydropyranyl 3 was a much weaker inhibitor
compared to the all-carbon counterpart 1. Piperidinyl 4
completely lost potency in all the assays since hydrogen bond
donor at this position may not be tolerated. Blocking NH with
amide (5) didn’t improve potency. To our delight,
phenylpiperidinyl 6 restored the potency similar to NLG-919.
Replacement of cyclohexyl (1) with phenyl (7) led to a 20-fold
drop of potency in the enzymatic assays. And benzyl 8 showed
similar potency to phenyl 7.
Table 2. SAR of 5-piperidinyl substituted
imidazoleisoindoles
Table 1. SAR of 5-substituted imidazoleisoindoles
F
F
R
N
R
N
N
N
N
IDO
TDO
Hela
IDO
TDO
Hela
Compd
9
R
IC50
IC50
IC50
Compd
R
IC50
IC50
IC50
(nM)a
(nM)a
(nM)a
(nM)a
(nM)a
(nM)a
S
OH

H
NLG-
919

297
27
586
376
571
203
79
247
434
N
HO
N

1
2
108
764
85
95
568
10

1239

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S
Lead compound 18 was synthesized by the route shown in
11

19
195
440
1
Scheme 2. Chiral separation of the racemate 18 gave rise to
the desired enantiomer 25, while another enantiomer 26 was
totally inactive in all IDO assays (IC50 > 10 μM).
Scheme 2. Synthesis of lead compound 25a
N
2
3
4
12
13
3-F-Ph
4-CN-Ph
72
81
371
394
167
353
5
6
7
8
N
O
F

14
15
358
33
1029
353
1712
280
F
a
b
N
NH
N
N
Br
N

N
N
24
4
9
N
10
11
12
13
14
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17
18
19
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24
25
26
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16
17
70
79
183
639
240
253
F

N
N
N
N
N
F
N
N
c
25

N
N
N
+
F
N
N
18
N
N
N
N
N
18
19
26
21
132
215
101
57

N
N
26
aReagents and conditions: (a) 1,4-dibromobenzene, Pd2(dba)3,
BINAP, tBuONa, toluene, 80°C, 12 h; (b) 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(dppf)Cl2,
Na2CO3, DME/H2O, microwave ,120°C, 40 min; (c) chiral
separation.
F
N
N


F
20
21
8
65
69
N
N
Compound 25 was fully profiled in vitro and in vivo as
shown in Table 4. It was a highly potent dual IDO/TDO
inhibitor in both enzymatic assays and cellular assay. It was
clean in in vitro toxicity studies including CYP and hERG. It
showed high plasma protein bonding in mouse, dog and
human and high liver microsome stability in rat and human.
Pharmacokinetic profiling of 25 in mouse, rat and dog
demonstrated good oral exposure and bioavailability (F =
59.6%, 60.3%, 27.3%, respectively) in all species.
N
N
14
114
121

N
N
22
23
20
226
579
691
324


Table 4. Profiling of compound 25
N
N
N
113
N
F
N
N
N
aValues are expressed as the mean of three independent
determinations.
N
N
NLG-919 was reported as a potent inhibitor of CYP3A4
(IC50 = 2.8 uM).21 Consequently, CYP inhibition assay was
included at this stage of lead optimization. As shown in Table
3, substitutions on the central phenyl ring inevitably led to
inhibition of different CYP isoforms. The most potent
compound 20 significantly inhibited CYP3A4 (IC50 = 2.6 uM).
Compound 18 had a clean CYP profile with only moderate
inhibition of CYP3A4.
Assay
25
9.7
47
76
Enzymatic IDO IC50 (nM)a
Enzymatic TDO IC50 (nM)a
Cellular Hela IC50 (nM)a
Table 3. CYP inhibition of compounds 18-21
CYP inhibition (1A2, 2C9, 2C19,
2D6, 3A4)
> 30 uM
> 30 uM
Compd
1A2
2C9
2C19
2D6
3A4
IC50
IC50
IC50
IC50
IC50 (uM)a
m/tb
(uM)a
(uM)a
(uM)a
(uM)a
hERG
18
19
20
21
>10
8.95
>10
>10
>10
0.94
7.4
>10
1.15
6.81
1.57
>10
9.61
>10
4.51
8.4/6.08
0.35/0.95
4.57/2.6
0.21/0.44
99.6% / 99.2% /
99.7%
PPB (mouse/dog/human)
Liver microsome stability (rat/human)
T1/2 (min)
142 / 147
0.3
aValues are expressed as the mean of three independent
determinations. bMidazolam as substrate/testosterone as substrate.
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kynurenine reduction by 57% was observed at 2 h after dosing
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Mouse PK@10 mg/kg
Cmax(ng/mL)
1
2
3
4
5
6
7
8
(see Supporting Information Figure S1).
3605
29116
4.55
The hPD1 transgenic mice implanted with MC38 tumors
were treated with 25 to evaluate the antitumor efficacy. As
shown in Figure 3, oral treatment with 25 (bid × 14)
demonstrated dose-dependent tumor growth inhibition (20
mg/kg, TGI = 39%; 50 mg/kg, TGI = 78%). The anti-PD1
monoclonal antibody SHR-1210 was only modestly
efficacious in this tumor model (5 mg/kg, ip, qod×8, TGI =
65%).23 Compound 25 was dosed in combination with SHR-
1210 using the following schedule: (SHR-1210: 5 mg/kg, ip,
qod × 8; 25: 20 mg/kg or 50 mg/kg, po, bid × 14). The
combination regimen at both dose levels achieved
significantly enhanced antitumor activity (TGI > 90%)
compared to either treatment alone. It was noted that no body
weight loss was observed for all the treatment groups.
AUC (ng/mL*h)
t1/2 (h)
Bioavailability F%
59.6%
Rat PK@10mg/kg
Cmax(ng/mL)
1848
6133
1.22
9
AUC (ng/mL*h)
t1/2 (h)
10
11
12
13
14
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18
19
20
21
22
23
24
25
26
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Bioavailability F%
60.3%
Dog PK@2 mg/kg
Cmax(ng/mL)
166
1340
6.49
AUC (ng/mL*h)
t1/2 (h)
Bioavailability F%
27.3%
aValues are expressed as the mean of three independent
determinations.
In order to gain insight into the superior potency of 25,
computational study was performed. As shown in Figure 2, 25
coordinated to the iron center of heme in a similar manner to
NLG-919, while the side chain of 25 stuck deeper into pocket
B. The methylpyrazol ring engaged in a cation-π interaction
with Arg231 with a distance of 3.38Å. Cation-π interactions
are ubiquitously found in proteins, protein-ligand and protein-
DNA complexes, and impose influence on biological
structures, molecular recognition and catalysis.22 Due to the
additional cation-π interaction, 25 served as a more potent
inhibitor, despite the lack of the hydroxyl group which formed
a hydrogen bonding with Ser235 as in NLG-919.
Figure 3. Efficacy study of 25 alone and in combination with
SHR-1210 in the MC38 xenograft model in hPD1 transgenic
mouse.
In summary, a highly potent IDO inhibitor 25 with
favorable preclinical toxicity and pharmacokinetic properties
was discovered through several rounds of SAR studies with
the aim of improving potency and eliminating CYP inhibition
liabilities. Compound 25 proved to be orally efficacious in the
MC38 xenograft model and its combination with anti-PD1
monoclonal antibody showed a synergistic antitumor effect.
ASSOCIATED CONTENT
Supporting Information
Biological
assays,
pharmacokinetic
assays,
in
vivo
pharmacodynamic study, in vivo efficacy study, computational
methods, experimental procedures and analytical data for key
compounds are included.
The Supporting Information is available free of charge on the
ACS Publications website.
Figure 2. Molecular docking of 25 (cyan) binding to the IDO
active site (PDB code: 2D0T). NLG-919 (salmon) and 25 are
superimposed for comparison. The key cation-π interaction is
indicated by brown dashed line.
AUTHOR INFORMATION
Corresponding Author
*E-mail: yunan@shhrp.com
Based on the promising in vitro activity and
pharmacokinetic data, 25 was further evaluated in the in vivo
pharmacodynamic study. A single dose of 100 mg/kg of 25
was administrated orally to C57 mice. The maximum
Present Address
§Janssen(China) R&D Center, Jinchuang Mansion, 4560 Jinke
Road, Shanghai 201210, China.
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ACS Medicinal Chemistry Letters
inhibitor for immuno-oncology. ACS Med. Chem. Lett. 2017, 8, 486–
491.
Author Contributions
1
2
3
4
5
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7
8
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the
manuscript.
(13) Crosignani, S.; Bingham, P.; Bottemanne, P.; Cannelle, H.;
Cauwenberghs, S.; Cordonnier, M.; Dalvie, D.; Deroose, F.; Feng, J.
L.; Gomes, B.; Greasley, S.; Kaiser, P. E.; Kraus, M.; Négrerie, M.;
Maegley, K.; Miller, N.; Murray, B. W.; Schneider, M.; Soloweij, J.;
Stewart, A. E.; Tumang, J.; Torti, V. R.; Van den Eynde, B.; Wythes,
M. Discovery of a novel and selective indoleamine 2,3-dioxygenase
(IDO-1) inhibitor 3 ‑ (5-fluoro ‑ 1H ‑ indol-3-yl)pyrrolidine-2,5-dione
(EOS200271/PF-06840003) and its characterization as a potential
clinical candidate. ACS Med. Chem. Lett. 2017, 60, 9617–9629.
(14) Pei, Z.; Mendonca, R.; Gazzard, L.; Pastor, R.; Goon, L.;
Gustafson, A.; VanderPorten, E.; Hatzivassiliou, G.; Dement, K.;
Cass, R.; Yuen, P.-w.; Zhang, Y.; Wu, G.; Lin X.; Liu, Y.; Sellers, B.
D. Aminoisoxazoles as potent inhibitors of tryptophan 2,3-
dioxygenase 2 (TDO2). ACS Med. Chem. Lett. 2018, 9, 417–421.
(15) Spranger, S.; Koblish, H. K.; Horton, B.; Scherle, P. A.; Newton,
R.; Gajewski, T. F. Mechanism of tumor rejection with doublets of
CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2
production and proliferation of CD8+ T cells directly within the tumor
microenvironment. J. Immunother. Cancer 2014, 2:3.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank analytical group, DMPK group of the company for their
contributions.
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ABBREVIATIONS
PD-1, programmed death 1; LDA, lithium diisopropylamide; THF,
tetrahydrofuran; DME, 1,2-dimethoxyethane; TEA: triethylamine;
MsCl: methanesulfonyl chloride; DMF, dimethyl formamide;
BINAP,
2,2'-bis(diphenylphosphino)-1,1'-binaphthyl;
dba:
dibenzylideneacetone; SAR, structure and activity relationship;
CYP, cytochrome p450 enzyme; hERG, human ether-a-go-go-
related gene; PPB, plasma protein bonding; PK, pharmacokinetic;
po, orally; ip, intraperitoneally; bid, twice daily; qod, every other
day; TGI, tumor growth inhibition.
(16) Nayak, A.; Hao, Z.; Sadek, R.; Dobbins, R.; Marshall, L.;
Vahanian, N. N.; Ramsey, W. J.; Kenedy, E.; Mautino, M. R.; Link,
C. J.; Lin, R. S.; Royer-Joo, S.; Liang, X.; Salphati, L.; Morrissey, K.
M.; Mahrus, S.; McCall, B.; Pirzkall, A.; Munn, D. H. Janik, J. E.;
Khleif, S. N. Phase Ia study of the indoleamine 2,3-dioxygenase 1
(IDO1) inhibitor navoximod (GDC-0919) in patients with recurrent
advanced solid tumors. J. Immunother. Cancer 2018, 6: 61.
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IDO IC50: 9.7 nM
TDO IC50: 47 nM
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Good bioavailability in preclinical species
Efficacious in MC38 xenograft model
Synergistic antitumor effect in combination with
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