Discovery of 5-(N-hydroxycarbamimidoyl) benzofuran derivatives as novel indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2021.127963

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) are rate-limiting enzymes in the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism and are becoming key drug targets in the combination therapy of checkpoint inhibitors in immunoncology. To discover a selective and potent IDO1 inhibitor, a structure–activity relationship (SAR) study of N-hydroxybenzofuran-5-carboximidamide as a novel scaffold was investigated in a systematic manner. Among the synthesized compounds, the N-3-bromophenyl derivative 19 showed the most potent inhibition, with an IC₅₀ value of 0.44 μM for the enzyme and 1.1 μM in HeLa cells. The molecular modeling of 19 with the X-ray crystal structure of IDO1 indicated that dipole-ionic interactions with heme iron, halogen bonding with Cys129 and the two hydrophobic interactions were important for the high potency of 19.

Full text of DOI:10.1016/j.bmcl.2021.127963

Bioorg. Med. Chem. Lett. 40 (2021) 127963
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 5-(N-hydroxycarbamimidoyl) benzofuran derivatives as novel
indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors
a
,
c
b
b
b
b
b
Juyoung Jung , Hongchul Yoon , Te-ik Sohn , Kyusic Jang , Yeongran Yoo , Ilji Jeong ,
b
b
c
c,*
Jae Eui Shin , Jin Hee Lee , Jihyae Ann , Jeewoo Lee
a
iLeadBMS Co., Ltd., Hwaseong-si, Gyeonggi-do 18469, Republic of Korea
b
Research Laboratories, Ildong Pharmaceutical Co., Hwaseong-si, Gyeonggi-do 445-170, Republic of Korea
c
Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) are rate-limiting enzymes
in the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism and are becoming key drug targets in the
combination therapy of checkpoint inhibitors in immunoncology. To discover a selective and potent IDO1 in-
hibitor, a structure–activity relationship (SAR) study of N-hydroxybenzofuran-5-carboximidamide as a novel
scaffold was investigated in a systematic manner. Among the synthesized compounds, the N-3-bromophenyl
Indoleamine 2
3
-Dioxygenase 1
Kynurenine Pathway
Immunotherapy
derivative 19 showed the most potent inhibition, with an IC50 value of 0.44
μ
M for the enzyme and 1.1 M in
μ
HeLa cells. The molecular modeling of 19 with the X-ray crystal structure of IDO1 indicated that dipole-ionic
interactions with heme iron, halogen bonding with Cys129 and the two hydrophobic interactions were impor-
tant for the high potency of 19.
L-Tryptophan (L-Trp) metabolism follows three significant pathways:
the kynurenine pathway (KP), the serotonin production pathway, and
the direct transformation to indole derivatives by intestinal microor-
ganisms.1 In mammalian cells, most L-Trp molecules are metabolized
mainly via the KP, which is known to control immunoregulatory func-
tions, cell proliferation suppression, reproduction and the central ner-
vous system. In the KP, L-Trp is converted first into N-formyl-L-
kynurenine by the rate-limiting enzymes, indoleamine 2,3-dioxygenase
immunotherapy of IDO inhibition have been conducted as either a single
agent or combinatorial regimen with monoclonal antibodies (mAbs)
(anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb and
anti-programmed cell death protein 1(PD-1)/programmed death-ligand
1 (PD-L1) mAb) or radiation, but there has been no remarkable success
to date.
Among the IDO1-selective inhibitors, epacadostat (INCB-024360)
(1), navoximod (GDC-0919) (2), and linrodostat (BMS-986205) (3)
4
–8
1
and 2 (IDO1 and IDO2) or tryptophan 2,3-dioxygenase (TDO), which
showed high affinity for the IDO1 enzyme or its apo-form (Fig. 1).
are attractive targets in immunotherapeutics. Indeed, the over-
expression of these enzymes is commonly found in various types of
cancers, such as glioblastoma, melanoma, lung cancer, ovarian cancer
and colon cancer, in which the prognosis and survival rates are poor.2
The metabolic depletion of L-Trp and the production of kynurenine
metabolites by IDO1/IDO2 and TDO can cause immunosuppression by
suppressing effector T cells and NK cells, activating circulating T regu-
latory cells (Tregs), promoting the differentiation of CD4+ T cells to
Tregs and stimulating the immunosuppressive microenvironment
through the secretion of soluble mediators. Therefore, IDO1/IDO2 and
TDO inhibition is a promising new cancer immunotherapeutic strategy
that effectively enhances antitumor immunity.3 Clinical trials on the
Epacadostat has been the leading molecule in the clinical development
of IDO1 inhibitors but failed to demonstrate its efficacy in a phase 3
clinical trial in patients with unresectable or metastatic melanoma.
Epacadostat in combination with pembrolizumab and the anti-PD-1
mAb also did not improve progression-free survival or overall survival
compared with placebo plus pembrolizumab, leading to the early
termination of the ECHO-301/Keynote-252 trial.9
Although the development of IDO1 inhibitors has lost momentum
due to these failures, translational and clinical trials to confirm new
possibilities are still actively underway. Moreover, it was observed
increased expression of IDO and PD-L1 along with penetration of Treg in
10
tumor microenvironment, and it is known that PD-1 or CTLA4
*
Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
https://doi.org/10.1016/j.bmcl.2021.127963
Received 20 January 2021; Received in revised form 21 February 2021; Accepted 10 March 2021
Available online 17 March 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

J. Jung et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127963
MeOH to produce 4-hydroxy-3-iodobenzonitrile 9. Benzonitrile 9 was
reacted with propargyl alcohol in the presence of 10 mol % Pd(II)
catalyst and diisopropyl amine to undergo the sequential two-step re-
action in which the Sonogashira coupling of 9 with propargyl alcohol
formed an acetylene intermediate and then cyclized into 2-(hydrox-
ymethyl)benzofuran 10.11 Thereafter, the hydroxymethyl group of 10
was either protected by the tert-butyldimethylsilyl (TBS) group or
alkylated with a methyl or cyclopropylmethyl group to produce 11. The
nitrile group of 11 was reduced to the corresponding aldehyde 12,
which was converted to the final N-hydroxyamidine 13 in three steps by
following the same route described in Scheme 1. For the 2-(hydrox-
ymethyl)benzofuran derivatives, the TBS group of 13 was deprotected
using tetrabutylammonium fluoride to produce the final 14 (Scheme 2).
The inhibition of synthesized compounds for IDO1 was evaluated by
measuring the kynurenine concentration from the absorbance at 480 nm
after incubation with substrate L-Trp in enzyme or cellular assays con-
ducted in HeLa cells treated with IFN-γ. The half maximal inhibitory
concentration (IC50) values were calculated based on percent inhibition
compared to that of the vehicle control and are represented in
Fig. 1. IDO-1-selective inhibitors.
5
,12,13
blockades can induce upregulation of IDO1, which can cause an
immunosuppressive environment around tumor.2,10 Therefore, the
development of IDO inhibitors still has significant value in that immu-
notherapy combined with these inhibitors may produce synergistic ef-
fects with excellent anticancer activity and highly favorable safety
profiles.
Tables 1–4
′
First, the SAR of N -hydroxy-N-phenylbenzofuran-5-carbox-
imidamide derivatives was investigated (Table 1). The unsubstituted
phenyl derivative 15 showed weak activity with an IC50 value of 19 µM
in HeLa cells. The 4-chlorophenyl derivative 16 also did not exhibit any
activity in either enzyme or cell-based assays. Among the 3-halogenated
phenyl derivatives, the 3-fluorophenyl derivative 17 did not improve the
inhibitory activity compared to 15, but the 3-chlorophenyl 18 and 3-
bromophenyl 19 derivatives showed potent inhibition with IC50 values
In this study, N-hydroxybenzofuran-5-carboximidamide as a novel
scaffold designed from epacadostat was investigated as a novel IDO1
selective inhibitor. The structure–activity relationship (SAR) was stud-
′
ied by varying the N-substituent in N -hydroxyamidine and the 2-substit-
of 0.69 and 0.44
μ
M in enzymes and IC50 values of 1.5 and 1.1 M in
μ
uent in benzofuran to evaluate its inhibition in enzyme and cell-based
assays. Molecular modeling of selective inhibitors was performed to
identify the binding mode in the active site.
HeLa cells, respectively. On the other hand, the 3-trifluoromethylphenyl
derivative 20 also displayed good inhibition but had a lower inhibition
than that of 18 and 19.
′
The final 5-(N-substituted-N -hydroxycarbamimidoyl) benzofuran
In the di-substituted derivatives, when a 4-fluoro atom was incor-
porated into 17–20, thereby producing 21–24, the activities did not
improve, but 23 and 24 still showed good inhibition. The 3-fluoro-4-
chlorophenyl 25 and 3,4-dimethylphenyl 26 derivatives were
confirmed to be inactive, as expected. Since the incorporation of a
polarized bulky group into the 3-position provided good inhibition,
other types of bulky groups were further explored. Unfortunately, most
of these bulky groups, including alkylcarbonyl (27), N-alkylamide
(28–30), alkyloxy (31), aryloxy (32), and arylalkyl (33, 34), were found
derivatives were generally synthesized by a three-step synthesis process,
which included aldoxime formation from the corresponding benzofuran
aldehyde, -chlorination of aldoxime and nucleophilic substitution of
α
amine to form N-hydroxyamidine (Scheme 1).
Commercially available benzofuran-5-carboxaldehyde 4 was con-
verted to the corresponding aldoxime 5 by treating hydroxylamine and
resulted in a very high yield. -Chlorination on aldoxime 5 was per-
α
formed by electrophilic addition of N-chlorosuccinimide (NCS) to pro-
vide oxyimidic chloride 6. It should be noted that chlorination was
′
to be inactive in both enzyme and cell-based assays. Only the 1,1 -
◦
conducted under temperature control by maintaining 50 C, otherwise
biphenyl derivative 35 displayed moderate inhibition.
this reaction resulted in a low yield. Initially, oxyimidic chloride was
used directly for the next step without further purification but provided
intractable side products. Therefore, to obtain the final compound with
high purity, oxyimidic chloride 6 was purified and then reacted with the
corresponding primary amines to afford the final N-hydroxyamidine 7.
For the syntheses of 2-alkoxymethyl benzofuran derivatives, 4-cya-
nophenol 8 was iodinated by treating with iodine and ammonia in
Scheme 2. Synthesis of the 2-alkoxymethyl 5-(N-hydroxycarbamimidoyl)
benzofuran derivatives Reagents and conditions: (a) I
2
, 20% NH
4
OH, MeOH, r.t.,
◦
2
1
h; (b) propargyl alcohol, Pd(dppf)Cl
2
, DIPA, CuI, toluene/THF (2:1), 100 C,
0 h; (c) i) TBSCl, imidazole, DMF, r.t., 30 min; ii) MeI, NaH, DMF, r.t., 1 h; iii)
Scheme 1. Synthesis of the 5-(N-hydroxycarbamimidoyl)benzofuran de-
(chloromethyl)cyclopropane, NaH, DMF, r.t., 12 h; (d) DIBAL-H, CH
2
Cl
2
, ꢀ 10
◦
◦
rivatives Reagents and conditions: (a) NEt
3
, NH
2
OH∙HCl, CH
2
Cl
2
, r.t., 16 h; (b)
C, 30 min; (e) NEt
3
, NH
2
OH∙HCl, CH
2
Cl
2
, r.t., 3 h; (f) NCS, DMF, 50 C, 3 h;
◦
3
2
NCS, DMF, 50 C, 3 h; (c) R (CH
2
)
n
NH
2
, THF, r.t., 16 h.
(g) R (CH
2
)
n
NH
2
, THF, r.t., 16 h; (h) TBAF, THF, r.t., 1 h.
2

J. Jung et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127963
Table 1
Table 3
Inhibitory activity of the substituents of the phenyl group.
Inhibitory activity of the benzofuran C(2)-substituents.
IDO1 (IC50,
Enzyme
μ
M)
1
Compd
R
R
IDO1 (IC50, M)
μ
HeLa cells
Compd
R
3
R
4
Enzyme
HeLa cells
4
2
F
1.7
8.0
2.4
5.9
1.2
6.1
2.2
2.5
8.0
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
H
H
Cl
H
H
H
H
F
>30
>30
32.7
0.69
0.44
1.2
19
43
44
45
46
47
48
Cl
Br
F
H
>30
20
0.71
7.1
F
Cl
Br
CF
F
1.5
Cl
Br
F
1.0
1.1
0.93
2.2
3
1.9
22.7
5.2
14.0
1.8
4
9
Cl
Br
F
1.5
0.4
11
17.6
3.2
Cl
Br
CF
F
F
F
1.6
2.6
50
3
F
1.2
2.2
5
5
1
2
10.6
Cl
Me
H
H
H
H
H
H
H
H
H
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
11.0
10.3
>30
>30
>30
>30
>30
>30
>30
>30
>30
11.9
Me
Cl
Br
0.9
0.9
10.8
8.6
COCH
3
CONHCH
CONHCH
2
(c-hexyl)
Ph
53
2
CO-(4-Me-piperidin-1-yl)
O(CH
2 3 3
) CH
OPh
Table 4
CH
CH
Ph
2
Ph
OPh
Inhibitory activity of the dibenzofuran scaffold.
2
Table 2
Inhibitory activity of the substituents of the benzyl group.
IDO1 (IC50,
μM)
Compd
R
n
Enzyme
HeLa cells
5
5
5
5
5
5
4
5
6
7
8
9
3-Cl
0
0
0
0
0
1
1.8
1.3
9.6
2.4
>30
3.6
7.6
3-Br
10.8
22.8
16.2
>30
29.6
3-Cl-4-F
3-Br-4-F
3,5-(OMe)
3-Cl
IDO1 (IC50, M)
μ
2
Compd
R
Enzyme
HeLa cells
28.2
3
6
7
>30
explored (Table 3). SAR analysis indicated that they showed a similar
pattern as that examined in Table 1, in which the activity increased as
the size of the halogen increased. The side chains at the 2-position of
benzofuran appeared to be tolerated for activity.
3
>30
5.8
3
8
22
17.6
Finally, the dibenzofuran surrogates (54–59) of potent inhibitors
3
4
4
9
0
1
>30
>30
>30
16.2
>30
>30
(
18, 19, 22, 23) were explored (Table 4). Their activities were similar to
those of the 2-phenylbenzofuran derivatives in Table 3 (52 vs 54, 53 vs
5), indicating that the dibenzofuran ring is the cyclized form of 2-phe-
5
nylbenzofuran. The SAR showed a similar pattern as that of the benzo-
furan derivatives.
To investigate the binding mode of the new scaffold for IDO1,
SAR analysis indicated that the 4-substitution is detrimental to ac-
docking studies of 19, the most potent inhibitor, with the IDO1 X-ray
tivity, but the incorporation of a bulky halogen at the 3-position is
critical for activity; their potencies seem to be correlated to their cor-
responding molar refractivity (MR) values (Br = 0.888 > C1 = 0.603 >
14,15
crystal structure were performed.
As shown in Fig. 2, the binding
16
mode of 19 was found to be similar to that of epacadostat. The hy-
droxyl group of N-hydroxyamidine forms a strong dipole-ionic interac-
tion with the heme iron. The 3-bromophenyl group binds deep into the
hydrophobic pocket (Pocket A), which is comprised of Tyr126, Val130,
CF
3
= 0.502 > F = 0.092), which account for the size and polarity of a
certain group.
Next, according to the results in Table 1, the one-carbon homologous
analogs (36–41) of phenyl, 3-chlorophenyl, 3-chloro-4-fluorophenyl,
heterocyclic and cyclopropyl rings were examined (Table 2). However,
most of them exhibited weak or little activity, indicating that they were
less active than the corresponding directly linked derivatives.
Next, the 2-hydroxymethyl (42–44), 2-methoxymethyl (45–47), 2-
Phe164, and Leu234 and forms a
π
- stacking interaction with Phe163.
π
In addition, the 3-Br group forms a strong halogen bonding interaction
with the sulfur of Cys129. On the other hand, the benzofuran ring
occupied the second hydrophobic pocket (Pocket B)
′
In summary, a series of N-hydroxy-N -substituted benzofuran-5-
carboximidamide compounds were investigated as IDO1 inhibitors in
enzyme and cell-based assays. SAR analysis indicated that in N-phenyl
substituted derivatives, the position of the substituents at the phenyl
(
(
cyclopropylmethoxy)methyl (48–50), and 2-phenyl benzofuran
51–53) analogs of potent 3-halophenyl derivatives (17–19) were
3

J. Jung et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127963
the work reported in this paper.
Acknowledgments
This research was supported by a grant of the Korea Health Tech-
nology R&D Project through the Korea Health Industry Development
Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic
of Korea (grant number: HR20C0021).
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
4