X-ray Structure-Guided Discovery of a Potent, Orally Bioavailable, Dual Human Indoleamine/Tryptophan 2,3-Dioxygenase (hIDO/hTDO) Inhibitor That Shows Activity in a Mouse Model of Parkinson’s Disease

Article abstract of DOI:10.1021/acs.jmedchem.1c00303

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan 2,3-dioxygenase (hTDO) have been closely linked to the pathogenesis of Parkinson’s disease (PD); nevertheless, development of dual hIDO1 and hTDO inhibitors to evaluate their potential efficacy against PD is still lacking. Here, we report biochemical, biophysical, and computational analyses revealing that 1H-indazole-4-amines inhibit both hIDO1 and hTDO by a mechanism involving direct coordination with the heme ferrous and ferric states. Crystal structure-guided optimization led to23, which manifested IC₅₀values of 0.64 and 0.04 μM to hIDO1 and hTDO, respectively, and had good pharmacokinetic properties and brain penetration in mice.23showed efficacy against the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse motor coordination deficits, comparable to Madopar, an anti-PD medicine. Further studies revealed that different from Madopar,23likely has specific anti-PD mechanisms involving lowering IDO1 expression, alleviating dopaminergic neurodegeneration, reducing inflammatory cytokines and quinolinic acid in mouse brain, and increasing kynurenic acid in mouse blood.

Full text of DOI:10.1021/acs.jmedchem.1c00303

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Article
X‑ray Structure-Guided Discovery of a Potent, Orally Bioavailable,
Dual Human Indoleamine/Tryptophan 2,3-Dioxygenase (hIDO/
hTDO) Inhibitor That Shows Activity in a Mouse Model of
Parkinson’s Disease
Xiang-Li Ning,∥ Yu-Zhi Li,∥ Cui Huo, Ji Deng, Cheng Gao, Kai-Rong Zhu, Miao Wang, Yu-Xiang Wu,
Jun-Lin Yu, Ya-Li Ren, Zong-Yuan Luo, Gen Li, Yang Chen, Si-Yao Wang, Cheng Peng, Ling-Ling Yang,
Zhou-Yu Wang, Yong Wu, Shan Qian,* and Guo-Bo Li*
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ABSTRACT: Human indoleamine 2,3-dioxygenase 1 (hIDO1)
and tryptophan 2,3-dioxygenase (hTDO) have been closely linked
to the pathogenesis of Parkinson’s disease (PD); nevertheless,
development of dual hIDO1 and hTDO inhibitors to evaluate their
potential efficacy against PD is still lacking. Here, we report
biochemical, biophysical, and computational analyses revealing
that 1H-indazole-4-amines inhibit both hIDO1 and hTDO by a
mechanism involving direct coordination with the heme ferrous
and ferric states. Crystal structure-guided optimization led to 23,
which manifested IC50 values of 0.64 and 0.04 μM to hIDO1 and
hTDO, respectively, and had good pharmacokinetic properties and
brain penetration in mice. 23 showed efficacy against the 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse motor coordination deficits, comparable to Madopar, an anti-PD medicine.
Further studies revealed that different from Madopar, 23 likely has specific anti-PD mechanisms involving lowering IDO1
expression, alleviating dopaminergic neurodegeneration, reducing inflammatory cytokines and quinolinic acid in mouse brain, and
increasing kynurenic acid in mouse blood.
toxicity via multiple mechanisms, for example, activating NMDA
receptors, inhibiting glutamatergic neurotransmission, and
inducing oxidative stress.10,11 The high KYN/Trp ratio has
been observed in serum and the cerebral spinal fluid (CSF) of
PD patients.12,13 Abnormal levels of the free-radical generator 3-
HK and the excitotoxin QUIN have also been found in the
putamen, prefrontal cortex, and substantia nigra pars compacta
(SNpc) of PD patients.14 By contrast, reduced concentrations of
neuroprotective KYNA have been reported in PD patients’
CSF.15 These studies suggested that targeting KP probably
represents a potential direction to develop new treatments for
PD.
INTRODUCTION
■
Parkinson’s disease (PD) is a chronic neurodegenerative
disorder which is characterized by progressive and selective
loss of nigrostriatal dopaminergic neurons. Numerous studies
have revealed that PD is related with neuroinflammation,
mitochondrial dysfunction, oxidative stress, metabolic disturb-
ance, and/or dysfunctional protein degradation.1,2 In recent
years, with the discovery of alteration of neuroactive kynurenine
pathway (KP) metabolite levels and the activity of associated
enzymes in the central nervous system (CNS), KP has been
considered as one of the key factors contributing to the
development and progression of PD.3−7 KP is responsible for
consuming ∼95% of dietary tryptophan (Trp) in mammals and
produces an array of neuroactive metabolites, including
kynurenic acid (KYNA), picolinic acid, quinolinic acid
(QUIN), 3-hydroxykynurenine (3-HK), and 3-hydroxyanthra-
nilic acid.7,8 These KP metabolites have important but different
roles in neuromodulation and disease progression. For example,
KYNA possesses neuroprotective properties mainly through the
inhibition of N-methyl-D-aspartate (NMDA) receptors to
counteract the toxic effects of excess inflow of Ca2+ and also
has antioxidant properties;9 in contrast, QUIN exerts neuro-
The initial rate-limiting step of KP is mediated by three
evolutionarily related heme enzymes in mankind: indoleamine
Received: February 16, 2021
Published: June 10, 2021
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© 2021 American Chemical Society
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Figure 1. (A) Current mechanistic proposals for IDO/TDO-mediated oxidative cleavage of tryptophan, where the initial indole activation is either by
an electrophilic or radical addition.16 (B) Representative hIDO1/hTDO inhibitors: epacadostat (INCB024360),18 navoximod (NLG-919),19 I-1,23
linrodostat (BMS-986205),20,33 PF-06840003,21 LY3381916,22 NLG8189,44 I-2,24 I-3,25 LM10,34 I-4,35 I-5,38 I-6,39 I-7,40 I-8,41 and I-9,41 of which
the potency values are from the corresponding cited references.
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Figure 2. Biochemical assays reveal 1 and 2 as substrate-competitive, dual hIDO1/hTDO inhibitors. (A) IC50 curves of 1 and 2 against hIDO1 and
hTDO; (B,C) kinetic analyses using the Cornish-Bowden method46 revealed 1 and 2 are substrate-competitive inhibitors of hIDO1 and hTDO.
2,3-dioxygenase 1 (hIDO1), hIDO2, and tryptophan 2,3-
dioxygenase (hTDO). These dioxygenases catalyze oxidative
cleavage of the indole ring of Trp at the 2,3-position to generate
N-formyl kynurenine (NFK) using dioxygen (Figure 1A).16
hTDO is a tryptophan-specific enzyme, while hIDO1 has a
broader scope of substrates with the indole ring, for example,
serotonin, melatonin, and tryptamine.16,17 hIDO1 is a
monomeric heme enzyme that is widely expressed in various
cell types, including microglia, astrocytes, and neurons; notably,
hIDO1 can be up-regulated by neuroinflammatory cytokines
and has been closely linked to various diseases including PD.7,13
As an isozyme of hIDO1, hTDO is a tetrameric heme enzyme
that constitutively expresses at high levels in the liver.8 hTDO is
also expressed in the brain (e.g., in neurons, astrocytes, and
endothelial cells)8 and has been involved in regulation of
neurodegeneration and associated phenotypes in models of PD
and other CNS disorders.13 The biological roles of hIDO2 are
still relatively poorly understood.
inhibitors (e.g., I-1,23 I-2,24 and I-325) were continuously
identified by the DNA-encoded library technology, virtual
screening, or other strategies (Figure 1B), providing new
candidates for drug development.24−28 Mechanistic studies of
hIDO1 inhibitors have revealed distinct inhibition modes, for
example, via binding to chelate with heme iron,23,29−31 binding
to heme without iron chelation,21 binding to trigger heme
release,32,33 or only binding with apo-hIDO1,22 suggesting
different perspectives in inhibitor development. In contrast, a
few selective hTDO inhibitors (e.g., LM1034 and I-435) have
been reported; among them, LM10 (Figure 1B) shows
therapeutically relevant immunostimulatory effects in the mice
model with hTDO-mediated tumors.34,36 Some dual hIDO1/
hTDO inhibitors such as M4112,37 tryptanthrins I-5,38 I-6,39 I-
7,40 I-8,41 and I-941 (Figure 1B) were reported in recent years,
which are likely to achieve a more comprehensive regulation of
KP and related disease states. Notably, M4112, as a first-in-class,
potent, dual hIDO1/hTDO inhibitor, is being tested in the
phase I clinical trial for patients with advanced solid tumors.37
Although structurally different inhibitors for hIDO1 and hTDO
are available for drug development, none of them has been
approved for clinical use so far. Additionally, most inhibitors
have been or are currently being tested for anticancer activity,
while there are very few studies evaluating the potential of
particularly dual hIDO1/hTDO inhibitors for anti-PD activity.
Since hIDO1 inhibitors have great potential for cancer
immunotherapy other than CNS disorders, a number of highly
potent and selective hIDO1 inhibitors have been developed,
many of which have entered clinical development, such as
epacadostat (INCB024360),18 navoximod (NLG-919),19 linro-
dostat (BMS-986205),20 PF-06840003,21 and LY338191622
(Figure 1B). Very recently, several structurally novel hIDO1
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Figure 3. Optical absorption assays revealed 1 and 2 directly binding to the heme iron of hIDO1 and hTDO. (A) UV−vis absorption spectra of hIDO1
in complex with dimethylsulfoxide (DMSO), 1, or 2 under the assay buffer (100 mM potassium phosphate buffer, pH 6.5, 40 mM ascorbic acid, 200
μg/mL catalase, 7 μM methylene blue, 0.01% Triton X-100) over 2 h; the original spectra (above) were further processed by the second-order
derivative to obtain the corresponding characteristic peaks (below). The results revealed that the binding of 1 or 2 led to obvious red shift of the Soret
peak, indicating that both compounds are heme-iron coordination inhibitors of hIDO1. (B) UV−vis absorption spectra of hTDO in complex with
DMSO, 1, or 2 under the assay buffer, revealing that 1 and 2 bind to coordinate with the hTDO heme iron.
We previously identified 6-bromo-1H-indazol-4-amine de-
rivatives as hIDO1 inhibitors through structure-based rational
design.42,43 We herein report biochemical, biophysical, and
computational analyses revealing that these compounds inhibit
both hIDO1 and hTDO by coordination bonding with the heme
ferrous and ferric states. The subsequent X-ray structure-guided
optimization led to several new potent, dual hIDO1/hTDO
inhibitors, of which 23 manifested IC50 values of 0.64 and 0.04
μM to hIDO1 and hTDO, respectively. With good pharmaco-
kinetic (PK) properties and blood−brain barrier (BBB)
permeability, 23 showed efficacy to ameliorate motor
dysfunction behaviors in a 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine (MPTP)-induced PD mouse model with no
obvious toxicity. Furthermore, we performed mechanistic
studies to investigate effects of 23 on dopaminergic neuro-
degeneration, microglial activation, inflammatory cytokines, and
tryptophan metabolites, with the aim of providing evidence for
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Figure 4. Crystallographic and computational analyses reveal inhibition modes of 1 and 2 to hIDO1 and hTDO. (A) View from a crystal structure of
hIDO1/1 (PDB code 7E0O), with the mFo − DFc electron density (OMIT maps, blue mesh, contoured to 3σ) around 1, revealing that 1 binds to form
a coordination bond with heme iron and a hydrogen bond with the heme carboxyl group (for further details of interactions, please see Figure S6). (B)
View from a crystal structure of hIDO1/2 (PDB code 7E0P), with the mFo − DFc electron density (blue mesh, contoured at 3.0σ), revealing a similar
binding mode to 1. (C) Typical halogen bond (XB) between C6−Br and Cys129 observed in the hIDO1/2 structure. (D) Superimposing the docking
pose of 2 to hTDO with the crystal structure of hIDO1/2 using our MeCOM tool, revealing that 2 has a similar binding mode to hIDO1 and hTDO.
(E) Density functional theory (DFT) calculations for complexes of heme(FeIII/FeII) with 1 and its structural isomer (1-isomer) reveal similar
coordination bond energies of indazole 2-N with the heme FeIII and FeII states.
dual hIDO1/hTDO inhibitors as tools to exploit the potential
for PD treatment.
competitive inhibition patterns with Ki values of 2.98 and 0.39
μM to hIDO1 and hTDO, respectively (Figure 2C).
Since hIDO1 and hTDO have a characteristic optical
absorption peak (Soret peak) around 405 nm that could be
used as a reporter of the heme-iron electronic state, we next
investigated whether the binding of 1 or 2 impacts the heme-iron
electronic state by ultraviolet−visible (UV−vis) spectrum
analyses (for details, please see the Experimental Section).47,48
We observed that for hIDO1 in the reductive assay buffer (100
mM potassium phosphate buffer, pH 6.5, 40 mM ascorbic acid,
200 μg/mL catalase, 7 μM methylene blue, 0.01% Triton X-
100), the optical density at 405.5 nm (OD405.5) decreases, while
OD421.5 increases during incubation (Figure 3A, left), partly
reflecting that the hIDO1 enzymes in the heme ferric (FeIII)
state are gradually transformed to the heme ferrous (FeII) state
under these conditions.47 Incubation of hIDO1 with 1 under the
assay buffer resulted in a slight red shift of the Soret peak from
405.5 to 409 nm, indicating that the binding of 1 impacts the
electronic state of heme iron (Figure 3A, middle). By
comparison, hIDO1 in complex with 2 shows a Soret peak at
413.5 nm and another small shoulder peak at 422.5 nm (Figure
3A, right), which is similar to that of the hIDO1/NLG919
complex but different from that of the hIDO1/INCB024360
RESULTS AND DISCUSSION
■
Indazole Compounds 1 and 2 Inhibit hIDO1 and hTDO
via Coordination Bonding with Heme Ferric and Ferrous
States. Using a biochemical assay as described in the
Experimental Section, we tested 6-bromo-1H-indazol-4-amine
compounds 1 and 2 (Figure 2A) against hIDO1 and hTDO;
since hIDO2 has a sequence identity of 43% to hIDO1 but has a
low catalytic efficiency to L-Trp (hIDO2 Km = 6809 917 μM,
K
cat = 0.103 0.006 s−1),45 here, we only use hIDO1 for activity
testing. The results revealed that 1 inhibits both hIDO1 and
hTDO with IC50 values of 8.49 and 1.39 μM, particularly with
high ligand efficiencies (LEs) of 0.65 and 0.75, respectively
(Figure 2A). Compound 2 shows similar inhibitory potency to 1
to hIDO1 (IC50 = 5.64 μM) and hTDO (IC50 = 2.04 μM)
(Figure 2A). The followed substrate (L-Trp) titration of steady-
state velocity for hIDO1 and hTDO in the presence of 1 at
varying concentrations revealed that 1 displays a substrate-
competitive inhibition manner to hIDO1 and hTDO (Figure
2B), and the calculated Ki values of 1 to hIDO1 and hTDO are
4.48 and 0.28 μM, respectively. Similarly, 2 showed substrate-
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Table 1. Inhibitory Activity (IC50, μM) and LE of 6-Bromo-1H-indazol-4-amine Derivatives 1−21 with hIDO1 and hTDO
Enzymes
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Table 1. continued
a
b
The IC50 values were measured as described in the Experimental Section, and the IC50 curves are given in Figure S8. LE = 1.4 × pIC50/N, where
N is the number of non-hydrogen atoms.51,52 The clog P and clog D values were calculated using the ADMETlab program.53
c
complex (Figure S1), implying that 2 is likely to form a N−Fe
coordination bond (likely involving both the FeII and FeIII
states).29 Similar to hIDO1, hTDO enzymes were observed with
a decrease of OD407 and an increase of OD421.5 during
incubation, indicating the ever-growing number of active
hTDO enzymes (heme FeII state) in the assay buffer. The
binding of 1 or 2 to hTDO led to the red shift of the Soret peak
from 407 to 413.5 nm. More interestingly, an additional
characteristic peak at 422.5 nm was observed for hTDO in
complex with 2, illustrating that 2 likely coordinates to both the
hTDO heme FeIII and FeII states. As a comparison, we further
analyzed these complexes in the nonreductive phosphate buffer
(100 mM potassium phosphate buffer, pH 6.5). Only the Soret
peaks at 406 and 407 nm were observed for hIDO1 and hTDO,
respectively, representing the simplex heme FeIII state for both
enzymes (Figure S2). During incubation in 2 h, the hIDO1
OD406 and hTDO OD407 values slightly increase (Figure S2),
which indicate that hIDO1 and hTDO enzymes (in the heme
FeIII state) are very stable in the phosphate buffer. The hIDO1/1
and hIDO1/2 complexes exhibit characteristic peak maxima at
410/538.5/563.5 and 413/538.5/563.5 nm, respectively
(Figure S2A); the latter is similar to that of the hIDO1/
NLG919 complex in the phosphate buffer (Figure S3A).29
Similar optical absorption spectra were observed for hTDO/1,
hTDO/2, and the hTDO/NLG919 complex (Figures S2B and
S3C), indicating that 1 and 2 coordinate to heme FeIII of hIDO1
and hTDO in the phosphate buffer.
In order to investigate the precise inhibitory modes of 1 and 2,
we then sought to obtain X-ray crystal structures for complexes
of hIDO1 with them; details of crystallization and structure
determinations are described in the Experimental Section. Co-
crystallization experiments yielded structures of hIDO1/1 (PDB
code 7E0O) and the hIDO1/2 (PDB code 7E0P) complex
(Table S1), which are crystallized in the P212121 space group
with two molecules in the asymmetric unit (ASU) (Table S2). A
disulfide covalent bond was observed between the two hIDO1
molecules (chain A Cys308−chain B Cys308, Figure S4) in ASU,
similar to those previously reported for hIDO1.29 The observed
mFo − DFc density adjacent to the active site heme allowed
unambiguous modeling of 1 and 2 (Figure 4A,B). The hIDO1/1
crystal structure revealed that 1 mainly engages in the A-site
(Figure 4A), corresponding to the binding site for the indole
moiety of L-Trp (Figure S5). The indazole 2-N atom of 1 is
positioned to form a coordinate bond with the heme iron (2.1
Å), as reflected by the continuous electron density connecting
the heme iron with the 2-N atom (Figure 4A), consistent with
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Table 2. IC50 and LE Values of 1H-Indazole Analogues 22−42 with hIDO1 and hTDO
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Table 2. continued
a
The IC50 values were measured as described in the Experimental Section, and the IC50 curves are given in Figure S12.
the observed optical absorption results (Figure 3A). In addition,
the C4−NH of 1 forms hydrogen-bonding interactions with
Gly262 (2.8 Å), and the indazole ring of 1 makes face-to-edge
π−π stacking interactions with Phe163 (Figures 4A and S6).
More interestingly, in the high-quality structure of IDO1/2, the
indazole 1-NH is positioned to form water-bridging interactions
with helix B Ser167, and C4−NH makes hydrogen-bonding
interactions with Gly262 (3.2 Å) on the DE loop (Figure 4B). We
also observed that the C6−Br atom is positioned to form a
classical halogen bond49 with the sulfur atom of Cys129, where
the Br···S distance is 3.47 Å (less than the sum of the van der
Waals radii of bromine atom RvdW of 1.83 Å and sulfur atom RvdW
of 1.80 Å), the C−Br···S angle is 162.32°, and the Br···S−C angle
is 145.35° (Figure 4C). Comparing with 1, 2 occupies both the
A-site and B-site and has additional hydrogen-bonding
interactions with Ser235 on the DE loop and hydrophobic
interactions with Phe226 and Arg231 on helix D (Figures 4B and
S6). We also performed molecular docking analysis to
investigate the possible mode of 2 binding to hTDO. The
results showed that 2 binds to hTDO likely via a mode similar to
that observed in the hIDO1/2 structure, although hTDO has a
shorter DE loop and different residues (e.g., Tyr45, His76, and
Ala150) in the active sites (Figure 4D).
noncovalent interactions that may influence the Fe−N
coordination modes. The coordination bond energy was
calculated using the computed Gibbs free energy of each
complex minus the energy of sole heme and compound (please
see the Experimental Section). The computational results
revealed that the coordination bond energy of heme FeIII−N
(1) is comparable to (slightly larger) that of heme FeII−N (1),
suggesting that the indazole core is likely suitable to coordinate
with both the heme FeIII and FeII states, consistent with that
observed by the optical absorption analyses (Figure 3).
Interestingly, a relatively high coordination bond energy was
observed for heme FeIII−N (1-isomer), implying that the isomer
transformation in iron coordination is likely. Overall, the results
clearly reveal the sophisticated inhibition mechanisms of 1 and 2
to hIDO1 and hTDO and provide an important structural basis
for subsequent structural optimization to obtain more potent
dual hIDO1/hTDO inhibitors.
X-ray Structure-Guided Optimization Design and
Structure−Activity Relationship Analyses. Comparison
of hIDO1/1 and hIDO1/2 with reported complex structures
revealed that 6-bromo-1H-indazol-4-amine matches well with
the anchor pharmacophore features of hIDO1/hTDO (i.e.,
coordination bonding with heme iron and hydrogen bonding
with Gly262),50 while unlike the potent inhibitors (e.g., NLG-919
and I-1, Figure 1B), 1 and 2 do not fit the hydrophobic B-site
properly and are not able to make electrostatic/hydrogen-
bonding interactions with the heme carboxyl group (Figure S7).
In subsequent structural optimization, we first focused on
searching for appropriate substituents at C4−NH to occupy the
To compare coordination strengths of indazole N-2 with the
heme FeIII and FeII states, we performed DFT calculations for
complexes of heme(FeIII)/1, heme(FeII)/1, heme(FeIII)/1-
isomer, and heme(FeII)/1-isomer (Figure 4E). All computa-
tions were performed by using the fit-LANZ2DZ basic set. The
heme carboxyl group was removed to reduce the effect of
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Figure 5. Crystallographic analyses reveal the binding mode of compounds 22 and 23 to hIDO1. (A) View of a crystal structure of the hIDO1/22
complex (PDB code 7E0Q) reveals that 22 makes coordination bonding with heme iron, halogen bonding with Cys129, and hydrogen bonding with
Gly262 on the DE loop. (B) View of a crystal structure of the hIDO1/23 complex (PDB code 7E0S) reveals that 23 binds via a similar mode with that of
22. (C) Comparison of hIDO1/22 and hIDO1/23 reveals that 23 has an additional hydrogen bond with the heme carboxyl group and fits better with
the B-site, compared with 22. The mFo − DFc electron densities (OMIT maps, blue mesh, contoured at 3.0σ) around 22 and 23 are calculated from the
last refinement models.
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Figure 6. Crystallographic analyses reveal the 36 binding mode to hIDO1 and structural features for hIDO1 selectivity. (A) View of a crystal structure
of the hIDO1/36 complex (PDB code 7E0T) reveals that 36 is positioned to make coordination bonding with heme iron, hydrogen bonding with
Gly262 on the DE loop and the heme carboxyl group, and hydrophobic interactions with residues on the B-site (for details, please see Figure S14); the
calculated mFo − DFc electron density (OMIT maps, blue mesh) around 36 is contoured at 3.0σ. (B) Comparison of hIDO1/36 and hTDO (PDB
code 6VBN)39 reveals that 36 C5−Br may have some collisions with Ala150 of hTDO, partly explaining the selectivity of 36 for hIDO1 over hTDO.
hydrophobic B-site and/or to engage in hydrogen-bonding
interactions with the heme carboxyl group. 6-Bromo-1H-
indazol-4-amine derivatives 3−21 were designed and synthe-
sized (for details, please see the Chemistry section), and their
inhibitory activities against hIDO1 and hTDO enzymes are in
Table 1.
hIDO1 and hTDO than 20, suggesting that the hydroxyl group
of 20 is important for dual hIDO1/hTDO inhibition.
Since 20 has similar clog P and clog D values with an NLG-
919 analogue (Table 1), we next investigated the stereochemical
isomers of 20 in hIDO1/hTDO inhibition. The mixtures of
(1′R,2′R)/(1′S,2′S)-isomers and (1′R,2′S)/(1′S,2′R)-isomers
were first synthesized as described in the Chemistry section. The
enzymatic assays revealed that the mixtures of (1′R,2′S)/
(1′S,2′R)-isomers had more potent inhibitory activity to hIDO1
(IC50 = 3.42 μM) and hTDO (IC50 = 0.02 μM), compared with
the mixtures of (1′R,2′R)/(1′S,2′S)-isomers (Figure S9). Then,
the stereochemically pure (1′S,2′R)-isomer (22) and (1′R,2′S)-
isomer (23) were synthesized (for details, please see the
Chemistry section). Compound 22 exhibited IC50 values of 2.85
and 0.09 μM to hIDO1 and hTDO, respectively. Notably, 23
manifested potent inhibition to hIDO1 (IC50 = 0.64 μM) and
had more potent hTDO (IC50 = 0.04 μM) inhibition than the
NLG-919 analogue and INCB024360 (Tables 1 and 2). X-ray
crystallographic analyses for complexes of hIDO1 with 22 or 23
were performed to uncover their precise binding modes (Table
S1). Fortunately, we obtained high-quality hIDO1/22 (PDB
code 7E0Q) and hIDO1/23 (PDB code 7E0S) complex
structures (Table S2); 22 and 23 could be clearly modeled
with the Fo − Fc electronic density in the active site (Figure
5A,B). The crystal structures revealed that 22 and 23 have
similar binding modes to hIDO1 (Figures 5 and S10), for
example, with respect to form a coordination bond with the
heme iron (1.9 Å) and make hydrogen-bonding interactions
with Gly262 (3.3 Å) on the DE loop, water-bridging interactions
with Ser167 on helix B, and halogen-bonding interactions with
Cys129 (3.6 Å) (Figures 5A,B and S10). The (1′R,2′S)-
cyclohexanol moiety of 23 has an additional hydrogen bond
with the heme carboxyl group (2.9 Å) and fits better with the
hydrophobic B-site surrounded by Phe163, Phe226, and Arg231
residues in the hIDO1 active site (Figure 5C), explaining why 23
is more potent than 22. UV−vis spectrum analyses were further
performed for complexes of hIDO1/hTDO and 22/23. The
results revealed that 22 and 23 have similar effects when binding
to hIDO1/hTDO in the reductive assay buffer and nonreductive
phosphate buffer (Figure S11).
Compounds 3 and 4 bearing hydroxyl-containing substituents
at C4−NH, which are expected to provide a hydrogen-bond
donor to interact with the heme carboxyl group, showed slightly
lower inhibition to hIDO1 and comparable activity to hTDO,
compared with 1 and 2 (Table 1). Another compound 5 with an
equivalent hydroxyl group (like 3/4) had similar inhibitory
potency to hIDO1 and hTDO; replacing the hydroxyl group
with the carbonyl (6) or oxime (7) group led to a decrease of
hIDO1/hTDO inhibitory activity. By comparison, compounds
8 and 9, which also contain a hydroxyl group at C4−NH
substituents, unfortunately showed low inhibitory activity to
hIDO1 and hTDO. We then introduced the amine-containing
saturated rings at C4−NH (10−15), which may be positioned
to form hydrogen-bonding or ionic-bonding interactions with
the heme carboxyl group; these compounds showed sub-
micromolar inhibitory activity to hIDO1 and hTDO, com-
parable to that of 1/2 (Table 1). Interestingly, 16 had IC50
values of 9.02 and 1.36 μM to hIDO1 and hTDO, respectively,
which is more potent than 14 (Table 1), likely due to the
carboxylate group of 16 having interactions with hIDO1 Arg231
or hTDO Arg144. Surprisingly, introducing a pyridin-2-ylmethyl
substituent at C4−NH (17) retained single-digit micromolar
activity to hTDO but only had a moderate activity to hIDO1,
whereas replacing with a cyclohexylmethyl substituent (18)
yielded improved inhibitory activity and LE to both hIDO1
(IC50 = 1.09 μM and LE = 0.46) and hTDO (IC50 = 0.86 μM
and LE = 0.47), compared with 1 and 2 (Table 1). Further
introduction of hydroxylamine (19) or hydroxyl (20) on the
cyclohexylmethyl led to better hTDO inhibition and com-
parable hIDO1 inhibition in comparison with 18. In particular,
20 manifested an IC50 of 0.12 μM to hTDO, which is
comparable to the NLG-919 analogue (an hTDO IC50 of 0.15
μM), and also had an IC50 of 4.12 μM to hIDO1. In contrast, 21
without the hydroxyl group showed less potent activity to both
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Figure 7. Crystallographic analyses reveal the binding mode of 41 to hIDO1 and importance of the JK loop in substrate/inhibitor binding. (A) View of
a crystal structure of the hIDO1/41 complex (PDB code 7E0U) reveals that 41 binds to form coordination bonding with heme iron, halogen bonding
with Cry129, hydrogen bonding with Gly262 on the DE loop, and hydrophobic interactions with residues on the B-site and JK loop; the calculated mFo −
DFc electron density (OMIT maps, blue mesh) around 41 is contoured at 3.0σ. (B) Superimposing the structures of hIDO1/41 and hIDO1/L-Trp
(PDB code 5WMU)29 reveals evidence for JK loop flexibility, suggesting that the JK loop may play an important role in capturing L-Trp and delivering
it to heme iron for oxidative cleavage.
Subsequently, we investigated the structure−activity relation-
ship (SAR) for derivatives 24−35 with different linkers or C6-
substituents (Table 2). Shifting the position of the linker NH of
22/23 yielded compounds 24/25, resulting in loss of potency to
both hIDO1 and hTDO, and similarly, introducing the amide
linker (26) led to low hIDO1/hTDO inhibition, demonstrating
the importance of the linker NH of 22/23 to achieve potent
inhibitory activity, consistent with that observed in hIDO1/22
and hIDO1/23 structures (Figure 5). The addition of a methyl
group to N1 of the indazole core (27) led to a significant drop in
activity against hIDO1 and hTDO, which probably interferes
with the water-bridging interactions with Gly262 (hIDO1)/
Ala150 (hTDO) or coordination bonding interactions with the
heme iron. Changing the 23 4,6-substituted indazole core to 5,7-
substituted indazole (e.g., 28 and 29) unfortunately yielded
lower inhibitory potency to hIDO1 and hTDO (Table 2).
Replacing C6−bromine of 23 with chlorine (30), methyl (31),
and trifluoromethyl (32) also did not improve hIDO1/hTDO
inhibition (slightly decreased), and further replacement with
formate (33), carboxyl (34), and amine (35) resulted in a
significant decrease in potency to hIDO1 and hTDO (Table 2).
By analyses of ab initio electrostatic potential surfaces of 23 and
30−35, we observed that C6-substituents have no significant
impacts on electrostatic potentials of the indazole N2 atom
associated with heme-iron coordination bonding (Figure S13),
reflecting that the differences of these compounds in hIDO1/
hTDO inhibition are not due to variations in coordination
bonding energies but possibly because of the differences in size
and electronic features of C6-substituents (e.g., C6−Br with
halogen bonding to hIDO1 Cys129, Figure 5B).
hIDO1/36 structure revealed that 36 has a similar binding mode
to 23, for example, with respect to coordination bonding
interactions with heme iron (1.9 Å) and hydrogen-bonding
interactions with Gly262 (3.2 Å) and the heme carboxyl group
(3.3 Å) (Figure 6A). Different from 23, C5−Br of 36 does not
have features to form the typical halogen bond with Cys129
.
Superimposition of hIDO1/36 with an hTDO structure39 by
using our MeCOM tool revealed that C5−Br is likely positioned
to have some collisions with Ala150 on the shorter DE loop of
hTDO (Figure 6B), largely explaining why 36 has selective
inhibition for hIDO1 over hTDO. In addition, the hIDO1/36
structure also explains that the low hIDO1/hTDO inhibition of
37 likely results from C7−Br having collisions with residues on
α-helix B of hIDO1 and hTDO to hinder indazole binding,
suggesting that C7-substituents are not beneficial to interactions
with hIDO1 and hTDO. The observed differences in DE loops
will be the main features for developing selective inhibitors for
hIDO1 or hTDO.
We next moved to modify the hydroxyl group of the C4−NH
cyclohexyl substituent. When adding acetyl to the hydroxyl
group (38), we observed markedly decreased inhibitory
activities to both hIDO1 and hTDO. We then replaced the
hydroxyl group of 23 with a primary amine (39) that may
provide hydrogen-bonding/ionic-bonding interactions with the
heme carboxyl group. Unexpectedly, 39 had lower activity than
23 to both hIDO1 (IC50 = 0.70 μM) and hTDO (IC50 = 0.36
μM). Another isomer 40 also had much lower activity to hIDO1
and hTDO (Table 2). Nevertheless, replacement of the hydroxyl
group of 23 with a chlorine atom led to compound 41, which
showed slightly better hIDO1 inhibition (IC50 = 0.29 μM) but
lower hTDO inhibition (IC50 = 0.22 μM). Crystallographic
analyses of the hIDO1/41 complex (PDB code 7E0U) revealed
that similar to 23, 41 is positioned to make coordination
bonding interactions with heme iron (2.0 Å), halogen-bonding
interactions with Cys129 (3.4 Å), hydrogen-bonding interactions
with Gly262 (3.0 Å), and hydrophobic interactions with Phe226
and Arg231. More interestingly, the JK loop was positioned to
have interactions with the ((1′S,2′S)-2-chlorocyclohexyl)-
methyl moiety of 41 (Figure 7A). Superimposing the structures
of hIDO1/41 with hIDO1/L-Trp (PDB code 5WMU)29
revealed evidence for flexibility in the JK loop, suggesting that
Interestingly, 36 bearing a C5−Br atom had slightly lower
inhibition to hIDO1 (IC50 = 1.23 μM) than 23 but showed 10-
fold selectivity for hIDO1 over hTDO (IC50 = 11.24 μM); in
contrast, 37 with two bromine atoms at the C5- and C7-
positions showed little activity against hIDO1 and hTDO
(Table 2). In order to understand the selective hIDO1 inhibition
for 36, we attempted co-crystallization experiments for the
hIDO1/36 complex (Table S1). Fortunately, we obtained a
high-quality crystal structure of hIDO1/36 (PDB code 7E0T)
(Table S2), in which 36 could be clearly modeled with the mFo
− DFc density in the hIDO1 active site (Figure 6A). The
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Table 3. Physicochemical, PK, and BBB Permeability Properties for 23
physicochemical property
MW (g/mol)
TPSA (Å2)
clog P
324.22
56.65
3.29
clog D (pH 7.4)
HBD
2.988
3
N + O
4
volume (Å3)
molecular flexibility
225.69
3 rotatable bonds
a
in vitro permeability
MDCKII-MDR1
Caco-2-BCRP
Caco-2-BCRP + novobiocin
b
PappA−B (10−6 cm/s)
13.46 0.2
10.34 0.1
0.77
55.13 2.2
81.92 0.4
18.8 0.4
10.4 0.1
0.56
42.2 1.4
74.5 2.0
19.4 0.1
9.97 0.7
0.51
43.8 0.6
74.9 2.1
b
PappB−A (10−6 cm/s)
c
efflux ratio
solution recoveryA−B (%)
solution recoveryB−A (%)
d
d
protein binding properties
e
f
u,plasma (%)
0.73 0.04
f
mouse plasma protein binding rate (%)
99.27
0.25
g
f
u,brain (%)
mouse brain homogenate binding rate (%)
pharmacokinetic property
0
h
99.75
a
b
po (100 mg/kg) /iv (100 mg/kg) (mean SEM, n = 5)
T
T
C
1/2 (h)
max (h)
max (mg/L)
4.27 0.66/10.07 4.71
0.42 0.11
6.87 2.14
AUC(0−12) (mg/L·h)
AUC(0−∞) (mg/L·h)
22.20 4.07/30.32 2.81
26.56 3.66/43.82 5.25
25.45 2.30/28.05 11.31
4.18 0.77/2.43 0.03
73.2
V
ss (L/kg)(0−12)
CL ((L/h)/kg)(0−12)
oral bioavailability F (%)
i
BBB permeability parameters
j
Kp
0.81
0.28
k
Kp,uu
a
b
Novobiocin was a BCRP transporter inhibitor. Papp was the apparent permeability coefficient from the apical (A) to the basolateral (B) direction
c
d
(A−B) or from B to A. ER was calculated by taking the ratio of Papp from the B−A to A−B direction. Solution recovery was calculated by taking
the ratio of the final amount of the compound in donor and receiver chambers to the initial amount of the compound in the donor chamber.
e
f
g
f
u,plasma, fraction unbound of 23 in mouse plasma. Mouse plasma protein binding rate was calculated by subtracting fu,plasma from 100%. fu,brain,
h i
fraction unbound of 23 in the mouse brain. Mouse brain homogenate binding rate was calculated by subtracting fu,brain from 100%. Oral
bioavailability F was the dose-corrected area under the curve (AUC) oral divided by AUC intravenous. Kp, total brain to total plasma
concentration ratio. Kp,uu, unbound brain to unbound plasma concentration ratio. Values are expressed as the mean SEM for n ≥ 3 experiments.
j
k
the JK loop may contribute to the nature of hIDO1 catalysis by
capturing L-Trp and delivering it to the heme-iron-containing
active site for oxidative cleavage. By comparison, 42 with a
((1′R,2′R)-2-chlorocyclohexyl)methyl at C4−NH had low
inhibition to both hIDO1 and hTDO, further reflecting high
requirements of inhibitor binding in the hydrophobic B-site.
Overall, X-ray structure-guided optimization identified new
potent, dual hIDO1/hTDO inhibitors, for example, 23,
providing new chemical tools for investigating their therapeutic
potentials for PD and other related diseases. Moreover, SAR and
crystallographic analyses demonstrated the importance of
proposed anchor pharmacophore features for hIDO1/hTDO
in lead discovery and optimization,50 which will be useful for
discovering other chemotypes targeting hIDO1/hTDO. Since
23 manifested potent inhibition to both hIDO1 and hTDO, we
then tested cellular activity of 23 in HeLa (for hIDO1),38 SW48
(for hTDO),39 and A172 (for hTDO)43 cell lines (for details,
please see the Experimental Section); the NLG-919 analogue
and epacadostat were used as positive controls. We observed
that 23 exhibited EC50 values of 5.50, 1.07, and 3.03 μM to
inhibit Trp hydrolysis in HeLa, SW48, and A172 cell lines,
respectively (Figure S15). The NLG-919 analogue showed EC50
values of 0.10, 1.51, and 3.0 μM, respectively, in HeLa, SW48,
and A172 cell lines, under our assay conditions, largely
consistent with previously reported data;39 epacadostat showed
activity in the HeLa cell line (EC50 = 0.08 μM) but less activity to
SW48 and A172 cell lines. We next investigated the PK
properties, BBB permeability, and therapeutic potentials of 23 in
a mouse model of PD in the following sections.
PK Properties and BBB Permeability of 23. We first
calculated physicochemical properties for 23; the results
indicated that it has the physicochemical features that favor
BBB permeability (Table 3).54,55 We then performed in vitro
analysis of compound permeability in Caco-2-BCRP and
MDCKII-MDR1 cell lines (Table 3); the former is the most
commonly used model for intestinal permeability, while the
latter is used to characterize BBB permeability.56 Compound
transport efficiency is largely related to two efflux drug
transporters, breast cancer resistance protein (BCRP) and P-
glycoprotein (MDR1), which are both highly expressed at the
BBB;57 BCRP is also expressed in small intestinal epithelial
cells.58 In the permeability assays, the transport of the
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compound across the cell layer was measured by the apical to the
basolateral (A−B, absorptive direction) and the basolateral to
the apical (B−A, secretory direction) at 37 °C. Compound 23
showed high permeability in both cell lines, with apparent
permeability coefficients (Papp) of 13.46 and 10.34 × 10−6 cm/s
in the A−B and B−A directions in MDCKII-MDR1 cells and
18.8 and 10.4 × 10−6 cm/s in the A−B and B−A directions in
Caco-2-BCRP cells, respectively (Table 3). To assess the
potential limitation of the efflux transporter in 23 transport, the
efflux ratio (ER) was calculated by taking the ratio of Papp from
the B−A to A−B directions. The ER values from the MDCKII-
MDR1 and Caco-2-BCRP assays were 0.77 and 0.56,
respectively (Table 3), suggesting minor contribution of
MDR1 and BCRP to 23 transport.59 We further used
novobiocin, a known inhibitor of BCRP,60 to examine whether
23 is a BCRP substrate. In Caco-2-BCRP cell assays, the Papp
values of 23 in the presence of novobiocin were 19.4 and 9.97 ×
10−6 cm/s in the A−B and B−A directions with an ER of 0.51
(Table 3). Very small differences between ERs/apparent
permeabilities with and without novobiocin were observed
(Table 3), indicating that 23 is likely not the BCRP substrate.
The solution recovery of 23 in the A−B direction of all assays
was lower than 75%, which may be due to cellular retention,
metabolism, adsorption, or other issues.61 After transport assays,
Lucifer yellow leakage assays were used to examine the integrity
of the cell monolayer; in all wells, the recovery of Lucifer yellow
in the basolateral side was less than 1%, indicating that cell
monolayers were not damaged.62
Figure 8. PK and brain penetration properties of 23. (A) Plasma
concentration−time curve of 23 in SD rats with an oral (red) and iv
(green) administration of 100 mg/kg. Blood was collected at the
indicated times, and the concentrations of 23 in plasma were
determined. Points, mean; bars, SEM; n = 5. (B) Concentrations of
23 in plasma and the brain in Kunming mice with an oral administration
of 100 mg/kg. Blood and brains samples were collected at 0.5, 1.0, 2.0,
4.0, 8.0, and 12.0 h, respectively. Points, mean; bars, SEM; n = 4.
We then measured unbound fractions of 23 in mouse blood
plasma and brain homogenate in vitro by equilibrium dialysis
(for details, please see the Experimental Section). The fractions
unbound of 23 in the brain (fu,brain) and plasma (fu,plasma) were
0.25 and 0.73% (Table 3), respectively, indicating that 23 has
high plasma protein and brain tissue binding.
We next conducted a preliminary assessment of PK properties
of 23 on Sprague-Dawley (SD) rats by intravenous (iv) and per
os (po) administration of a single 100 mg/kg dose of 23 (for
details, please see the Experimental Section). The plasma
concentrations of 23 versus time profile is shown in Figure 8A,
and the calculated PK parameters of 23 by a noncompartment
model are summarized in Table 3. With an oral administration of
100 mg/kg 23, the obtained area under the concentration−time
curve (AUC0−∞) was about 26.56 mg/L·h and the half-life
(T1/2) was about 4.27 h (Table 3). With a clearance (CL)(0−12)
permeability properties of 23, which will be an important basis
to understand its efficacy in the PD mouse model. By comparing
the crystal structures of hIDO1 and hTDO with the homology
modeling structures of mouse IDO1 (mIDO1) and mTDO,
respectively, we observed that these two enzymes are highly
conservative in the active site between human and mouse species
(Figure S17), suggesting that the mouse model is suitable to
evaluate the effects for 23 in vivo.
Compound 23 Shows Protective Effects in the MPTP-
Induced PD Mouse Model. Motor dysfunction is a common
symptom occurring in PD. To assess the potential protective
effects of 23 on motor dysfunction, we established a PD mouse
model by treating with the neurotoxin MPTP to induce motor
dysfunction (please see the Experimental Section) and
performed behavior tests on the MPTP-treated mice with or
without treatment of 23. Madopar was used as a positive control.
After seven daily injections of MPTP (30 mg/kg per day
intraperitoneally), the mice were administered with 100 mg/kg
23, 30 mg/kg 23, 10 mg/kg 23, 50 mg/kg Madopar, or the
vehicle solvent by oral gavage once daily for an additional 14
days. During this time, we tested the mice for motor
coordination on the rota-rod test. As observed in the three
independent tests on days 6, 9, and 12, MPTP injections caused
significant impairment with decreases in time spent on the
rotating rod (Figure 9 and Table S3). Treatments of 23 even in
30 and 10 mg/kg could attenuate or prevent the MPTP-induced
deficits (P < 0.001), comparable to that by treatment of 50 mg/
kg Madopar (Figure 9). In the grip test, compared with MPTP-
treated mice, 23-treated mice also have improved performance
of 4.18 (L/h)/kg and an apparent distribution volume (Vss(0-12)
)
of 25.45 L/kg (Table 3), compound 23 was rapidly absorbed
(Tmax = 0.42 h) with a Cmax of 6.87 mg/L and high oral
bioavailability (F = 73.2%) (Table 3).
We further performed brain penetration studies in Kunming
mice to determine the total brain/total plasma concentration
ratio (Kp).59 Compound concentrations were measured for
plasma and brain samples collected at 0.5, 1.0, 2.0, 4.0, 8.0, and
12.0 h after a single oral dose of 100 mg/kg. The observed
concentrations of 23 in plasma and brain samples are consistent
with the PK trends in SD rats (Figure S16), and the calculated Kp
was 0.81. To better establish exposure/pharmacokinetics−
efficacy/pharmacodynamics relationships, Kp was further trans-
formed into the unbound concentration ratio (Kp,uu) based on
the above fu,brain and fu,plasma data59 to characterize the
distribution equilibrium of free compound 23 available between
the brain and blood plasma (Table 3). The calculated Kp,uu was
0.28, indicating that 23 is likely the active efflux at the BBB.
These results clearly reveal the PK properties and BBB
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Figure 9. In vivo effects of 23 on MPTP-induced motor deficits in rota-rod tests on day 6, day 9, and day 12 from modeling. The motor coordination
was compared in the control group (NS), the MPTP-treated group (MPTP + NS), and the MPTP-induced PD mice treated with 23 100 mg/kg, 23 30
mg/kg, 23 10 mg/kg, or Madopar 50 mg/kg groups. The data are expressed as the mean SEM (n = 10). P-values were calculated by comparing with
the MPTP-treated group.
(Figure S18). Moreover, the weights of the mice body and
organs were not affected by treatment of MPTP, 23, and
Madopar (Figure S19 and Table S4). The results demonstrate
that 23 has comparable in vivo protective effects to Madopar on
PD mouse models and has no obvious toxicity under the tested
doses and conditions.
α, whereas 23 appears to inhibit MPTP-induced expression of
IL-1β and TNF-α (Figure 11). In addition, compared with 23,
Madopar displayed different effects on the level of inflammatory
cytokines. Similar MPTP- and 23-treated effects of inflamma-
tory cytokines were observed in striatum (Figure S20). The
results indicate that different from Madopar, 23 has specific
mechanisms to protect MPTP-induced neuronopathy by
changing IDO1 expression, dopaminergic neurodegeneration,
and inflammatory cytokines.
Furthermore, the blood and substantia nigra and striatum
samples from the vehicle-, MPTP-, 23- (30 mg/kg), and
Madopar-treated mice were submitted to perform absolute
quantitative analysis of tryptophan metabolites. Except for
KYNA, no significant differences of tryptophan metabolites were
observed in the blood of all the four groups (Figures 12A and
S21). The 23-treated mice had a high level of the neural
protective substance KYNA in blood, compared with other
groups (P < 0.05). In substantia nigra and striatum of MPTP-
treated mice, the amount of NFK production was increased,
whereas 23-treated mice displayed low NFK production (Figure
12B). Notably, an increase of neurotoxic QUIN was observed in
substantia nigra and striatum of MPTP-treated mice, while 23
could result in a decreased amount of QUIN (Figure 12B).
Other tryptophan metabolites were not observed with obvious
changes in bloods and substantia nigra and striatum (Figures
S21 and S22). In contrast, Madopar did not have similar impacts
on the tryptophan metabolites, further confirming that 23 has
specific mechanisms, for example, probably by blocking IDO-/
TDO-mediated NFK and QUIN production in substantia nigra
and striatum and increasing KYNA production in blood, to
protect MPTP-induced motor dysfunction.
Besides, we further used IFPTarget, a tool for virtual target
screening,64 to predict other possible targets for 23. By screening
against more than 1500 different types of targets using
IFPTarget, we found that 23 might bind with the heme-
containing enzyme CYP2C9 and some kinases (e.g., Flt3). The
activity test revealed that 23 did indeed inhibit CYP2C9 with an
IC50 value of 9.27 μM (Figure S23); sulfaphenazole, a positive
control, showed 83.4% inhibition to CYP2C9 at 3 μM. By using
the KINOMEscan service in Eurofins DiscoverX to perform
kinase profiling for 23 against a panel of kinases, we found that
23 manifested >90% inhibition at 10 μM for Flt3, DYRK1B, and
GSK3β (Table S5). Previously, Flt3 has been linked to dendritic
cell-mediated immune responses,65 which may be possibly
Effects of 23 on Dopaminergic Neurodegeneration,
Microglial Activation, Inflammatory Cytokines, and
Tryptophan Metabolites. We then investigated whether 23
has impacts on the generation of dopaminergic neurons and
neurites by comparing the SNpc and striatum of mice from the
vehicle-, MPTP-, 23- (30 mg/kg), and Madopar-treated groups.
Immunohistochemistry (IHC) was used to detect tyrosine
hydroxylase (TH), which is a rate-limiting enzyme in the
formation of dopamine in the cytoplasm63 and has been well
established to label dopaminergic neurons. Compared with
vehicle-treated mice, the MPTP-treated mice had a lower TH
level and an impaired structure of the dopaminergic neurons and
neuritis in SNpc (Figure 10A,B), indicating that MPTP resulted
in dopaminergic neuron loss and fiber bridge impairment. 23-
treated mice showed a slight increase of TH-positive neuron
cells in SNpc and approaching the normal cell morphology and
fiber innervation (Figure 10A,B), comparable to Madopar-
treated mice, further confirming the protective effect of 23 in
MPTP-induced dopaminergic neuronopathy. By using IHC, we
next detected the number of microglia cells in SNpc and
striatum by staining ionized calcium binding adaptor molecule 1
(Iba-1). We observed that in the MPTP-treated group, the
number of Iba-1-positive cells was increased in striatum, and 23-
treated mice had less Iba-1-positive cells in striatum (Figure
10C,D), indicating that 23 is likely to prevent the MPTP-
induced increase of at least Iba-1-positive microglia cells.
Subsequent western blotting (WB) experiments were carried
out to detect the expression of IDO1 and TDO in substantia
nigra and striatum of mice. We observed that MPTP likely
increased the expression of both IDO1 and TDO in substantia
nigra and striatum. In 23-treated mice, there were no significant
changes of TDO expression, while a substantially low level of
IDO1 expression was observed (Figure 10E). Different from 23,
Madopar treatment did not markedly change the expression of
IDO1 and TDO (Figure 10E). Further examination of the
expression of inflammation-related cytokines, including IFN-γ,
IL-1β, and TNF-α, indicated that in SNpc, MPTP is likely to
induce up-regulation of inflammatory cytokines IL-1β and TNF-
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Figure 10. Analyses of TH, Iba-1, IDO1, and TDO in mice brain. (A) IHC staining of TH in SNpc and striatum of mice shown in brown. (B) Number
of TH-positive cells in SNpc and striatum (n = 3). (C) IHC staining of Iba-1 in SNpc and striatum shown in brown. (D) Number of Iba-1-positive cells
in SNpc and striatum (n = 3). (E) WB analysis of IDO1 and TDO in substantia nigra and striatum of mice. The data are expressed as the mean SEM,
and the P-values are also shown in figures.
involved in regulation of inflammatory factors in the PD model.
Moreover, both DYRK1B and GSK3β have been identified as
potential targets for PD and other neurodegenerative
diseases.66,67 These off-targets may not only help to interpret
the efficacy of 23 in the MPTP-induced PD mouse models but
also suggest new possible directions (e.g., multiple-targeting
IDO/TDO and GSK3β) to develop agents for PD treatment.
amine (1 or A1) was synthesized starting from 2-methyl-1,3-
dinitrobenzene via four steps (Scheme S1), which were
described in our previous report.42,43 The noncommercially
available substituted aldehydes were synthesized from the
corresponding esters via LiAlH4 reduction and Dess−Martin
oxidation, as shown in Schemes S2 and S3.
As shown in Scheme 1, 6-bromo-1H-indazole-4-amine
derivatives 3−9 were synthesized from tert-butyl 4-amino-6-
bromo-1H-indazole-1-carboxylate (A6), which was prepared
starting from 6-bromo-4-nitro-1H-indazole (Scheme S1). The
substituted epoxides were synthesized from the corresponding
CHEMISTRY
■
The general procedures for the target compounds in Tables 1
and 2 are described in Schemes 1−4. 6-Bromo-1H-indazole-4-
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Figure 11. IHC analyses of inflammatory cytokines in SNpc. (A) IHC staining of IFN-γ, IL-1β, iNOS, and TNF-α in SNpc of mice shown in brown.
(B) IHC staining density of IFN-γ, IL-1β, iNOS, and TNF-α in SNpc of mice (n = 3). The data are expressed as the mean SEM, and the P-values are
also shown in figures.
Figure 12. Quantitative analyses of KYNA, NFK, and QUIN in mouse (A) blood serum and (B) substantia nigra and striatum, revealing that 23
treatment led to increased KYNA production in blood serum and lower NFK and QUIN production in substantia nigra and striatum compared with
other groups (P < 0.05). Blood serum and substantia nigra and striatum samples were taken from the NS, MPTP + NS, MPTP + 23 30 mg/kg, and
MPTP + Madopar 50 mg/kg groups. The data are expressed as the mean SEM (n = 3), and the P-values are also shown in figures.
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a
Scheme 1. Synthesis of the Target Compounds 3−9 via Epoxide-Opening Reaction and Nucleophilic Substitution
a
Reagents and conditions: (a) epoxide, NaOH, EtOH/H2O (1:1), 100 °C; (b) TFA, DCM, rt; (c) 2-bromo-1-(4-hydroxyphenyl)ethan-1-one,
Cs2CO3, DMF, 60 °C; (d) hydroxylamine hydrochloride, pyridine, 110 °C.
a
Scheme 2. Synthesis of the Target Compounds 10−23 and 40−42 via Hantzsch Ester-Mediated Reductive Amination
a
Reagents and conditions: (a) Hantzsch ester, TFA, DCM/MeOH (3:1), 40 °C; (b) TFA, DCM, rt; (c) NCS, PPh3, DCM, rt; (d) phthalimide,
PPh3, DIAD, THF, rt; hydrazine hydrate, EtOH, rt.
alkenes via MCPBA-mediated oxidation, as shown in Scheme
S4. Then, A6 was reacted with the substituted epoxides through
an epoxide-opening reaction, followed by trifluoroacetic acid
(TFA)-mediated deprotection, to obtain the target compounds
3−5 and 8−9. A6 was reacted with 2-bromo-1-(4-
hydroxyphenyl)ethan-1-one through an alkaline nucleophilic
substitution to afford compound 6, which was further reacted
with hydroxylamine hydrochloride to obtain compound 7
(Scheme 1).
Derivatives 10−23 and 40−42 were synthesized from A1 and
the substituted aldehydes via the Hantzsch ester-mediated
reductive amination (Scheme 2).42,43 Among these compounds,
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a
Scheme 3. Synthesis of the Target Compounds 24−26
a
Reagents and conditions: (a) 2-aminocyclohexan-1-ol, EDCI, HOBT, DIEA, DCM, rt; (b) LiAlH4, THF, 60 °C.
a
Scheme 4. Synthesis of the Target Compounds 27−37
a
Reagents and conditions: (a) Hantzsch ester, TFA, DCM/MeOH (3:1), 40 °C; (b) NaOH, EtOH/H2O (1:1), 80 °C.
amine-containing compounds 10−15 were subsequently
prepared via acidic hydrolysis in TFA from the corresponding
tert-butyl carbonates. The racemic compound 20 was
synthesized from B7 (Scheme S3), and four chiral isomers can
be clearly observed in the chiral high-performance liquid
chromatography (HPLC) spectra (Supporting Information).
The chiral compounds 22 and 23 were synthesized from
(1R,2R)- and (1S,2S)-2-hydroxycyclohexane-1-carbaldehyde
(B13 and B14), respectively. B13 and B14 were prepared via
aldol condensation of pimelic dialdehyde using L-proline and D-
proline as the catalyst, as shown in Scheme S5, according to the
previous report by List et al.68 The chiral purities of 22 and 23
were also analyzed through chiral chromatography, and the
chiral purities are above 98%. However, the identification of
chiral structures of 22 and 23 was proved to be difficult; we
attempted various crystallization conditions but failed to obtain
single crystals for 22 and 23. We thus moved to convert the key
immediate B13 to its derivative IV-2 through a reductive
amination with (2,4-dinitrophenyl)hydrazine and sulfonylation
with p-TsCl (Supporting Information). The single crystal of IV-
2 was obtained in the mixed solution of DCM and petroleum
ether. The chiral structure of B13 was finally assigned according
to the X-ray diffraction data of the IV-2 single crystal
(Supporting Information). Besides, the absolute configurations
of 22 and 23 were further confirmed by the crystal structures of
hIDO1/22 (PDB code 7E0Q) and hIDO1/23 (PDB code
7E0S) (Figure 5). Compound 23 was then converted to 40
through the Mitsunobu reaction with phthalimide and
subsequent hydrazinolysis; this synthetic procedure can result
in the configuration inversion of chiral carbon. Compounds 22
and 23 were respectively converted to halides 42 and 41 through
a neutral halogenation with N-chlorosuccinimide (NCS) and
PPh3, also along with the configuration inversions of chiral
carbon; the chirality of 41 was confirmed by crystallographic
analysis of the hIDO1/41 complex (PDB code 7E0U, Figure 7).
Derivatives 24−26 were synthesized from A13 (Schemes 3
and S1). A13 was reacted with (1R,2R)-2-aminocyclohexan-1-ol
through 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hy-
drochloride (EDCI)-mediated condensation reaction in the
presence of hydroxybenzotriazole (HOBT) and N,N-diisopro-
pylethylamine (DIEA) to obtain amide 26, which was reduced
with LiAlH4 to produce amine 25. Similarly, compound 24 was
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a
Scheme 5. Synthesis of the Target Compound 38
a
Reagents and conditions: (a) (Boc)2O, DMAP, Et3N, DCM, rt; (b) CH3COOH, DCC, DMAP, DCM, rt; (c) TFA, DCM, rt.
a
Scheme 6. Synthesis of the Target Compound 39
a
Reagents and conditions: (a) NaN3, DMF, rt; (b) H2, Pd/C, rt.
glassware under an atmosphere of nitrogen and under anhydrous
conditions (as required). All progress was monitored by thin layer
chromatography using GF254 silica gel precoated glass-backed plates,
and the compounds were detected under a UV lamp at 254 nm.
Purification was performed by flash column chromatography
separations using silica gel (200−300 mesh). 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance NEO 400 or Bruker DPX
300 spectrometer with tetramethylsilane as an internal standard in d6-
DMSO or CDCl3. Multiplicities are used with the following
abbreviations: s = singlet, br = broad singlet, d = doublet, t = triplet,
q = quartet, dd = doublet of doublets, and m = multiplet. HRMS was
performed using the micrOTOF-Q II 10203 mass spectrometer with an
AP-ESI ion source. All purities were determined by HPLC using a C18
column (Agilent Technologies, 1220 Infinity LC). The mobile phase
was a mixture of CH3CN and H2O containing 0.1% formic acid. Peaks
were detected at 254 nm. The chiral column is an AD-H column LC16
(Shimadzu, Japan). The temperature was maintained at 30 °C at a flow
rate of 1 mL/min. The mobile phase was a mixture of n-hexane and
isopropanol (70:30, v/v). The injection volume was 10 μL.
General Procedure I: Hantzsch Ester-Mediated Reductive
Amination. To a solution of amine (1.0 mmol) and aldehyde (1.2
mmol) in DCM/MeOH (3:1, 15 mL), Hantzsch ester (1.2 mmol) and
TFA (0.1 mmol) were added, and the resulting mixture was stirred at 40
°C for 12 h. The solution was adjusted to pH 7−8 by addition of
NaHCO3, and the crude residue was obtained by concentrating in
vacuo. Finally, the crude residue was purified by column chromatog-
raphy to give the intermediate or target compounds.
General Procedure II: TFA-Mediated Boc-Deprotection. To a
solution of carbamate (1.0 mmol) in DCM (10 mL), TFA (1 mL) was
added, and the resulting mixture was stirred at rt for 4 h. The solution
was adjusted to pH 7−8 by addition of NaHCO3. The mixture was
dissolved with DCM, washed with brine, and dried over anhydrous
Na2SO4. The solvent was evaporated, and the crude product was
charged on a silica gel column chromatograph with DCM and MeOH as
the eluent to afford the target compounds.
General Procedure III: Epoxide-Opening Reaction. The
solution of indazole A6 (311 mg, 1.0 mmol) and epoxide (1.5 mmol)
in EtOH/H2O (1:1, 20 mL) was stirred at 100 °C for 12 h. The solution
was adjusted to pH 10 by addition of NaOH during the synthetic
process. The mixture was cooled to rt and concentrated under reduced
pressure. The residue was dissolved with ethyl acetate (EA), washed
with brine, and dried over anhydrous Na2SO4. The solvent was
evaporated, and the crude product was charged on a silica gel column
chromatograph with DCM and MeOH as the eluent to afford the target
compounds.
synthesized from A13 and (1S,2S)-2-aminocyclohexan-1-ol.
Derivatives 27−37 were synthesized via Hantzsch ester-
mediated reductive amination from B13 and indazole
derivatives A1−A13 (Schemes 4 and S1). As shown in Scheme
5, compound 23 was protected with (Boc)2O in the presence of
4-dimethylaminopyridine (DMAP) and Et3N and then reacted
with CH3COOH through an N,N′-dicyclohexylcarbodiimide
(DCC)-mediated condensation to afford the ethyl ester
derivative, which was further reacted with TFA catalytic
hydrolysis to obtain the target compound 38. As shown in
Scheme 6, compound 41 was reacted with NaN3 and then
reduced through hydrogenolysis to afford the target compound
39. The chemical structures of the target compounds 1−42 were
1
confirmed via H NMR, 13C NMR, and high-resolution mass
spectrometry (HRMS) (Supporting Information).
CONCLUSIONS
■
This study presents X-ray structure-guided optimization of 6-
bromo-1H-indazol-4-amine derivatives, leading to identification
of new potent, dual hIDO1/hTDO inhibitors. The SAR and
crystallographic analyses demonstrated the proposed anchor
pharmacophore features for hIDO1/hTDO, which will be useful
for discovering other chemotypes targeting hIDO1/hTDO. The
observed structural differences between hIDO1 and hTDO
particularly in DE loops provided insights to develop selective
inhibitors for hIDO1 or hTDO. The crystal structures also
provided high-quality examples of Br···S halogen bonds to
investigate the halogen-bonding interactions. The resulting dual
hIDO1/hTDO inhibitor 23 displayed in vivo protective effects
in the MPTP-induced PD mouse model. Further mechanistic
studies revealed that 23 likely works through a specific
mechanism involving changes in IDO1 expression, dopaminer-
gic neurodegeneration and inflammatory cytokines, an increase
of the neural protective substance KYNA in mouse blood, and
inhibition of IDO1-/TDO-mediated NFK and QUIN produc-
tion to protect MPTP-induced motor dysfunction. Overall, this
work investigates therapeutic potentials of hIDO1/hTDO
inhibitor 23 in the PD mice model and suggests a new
possibility to exploit hIDO1/hTDO inhibitors for treating PD
and other neurodegenerative diseases.
Synthesis of 4-(((6-Bromo-1H-indazol-4-yl)amino)methyl)phenol
(2). The title compound was obtained as a yellow solid via general
procedure I from A1 and 4-hydroxybenzaldehyde in 59% yield, 98%
HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.80 (s, 1H), 9.29 (s, 1H),
8.21 (s, 1H), 7.18 (d, J = 8.5 Hz, 2H), 6.81 (s, 1H), 6.73 (d, J = 8.5 Hz,
EXPERIMENTAL SECTION
■
Chemistry. All chemicals and reagents were purchased from
commercial sources and used without further purification unless
otherwise specified. The reactions were performed using oven-dried
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2H), 6.06 (s, 1H), 4.29 (d, J = 5.8 Hz, 2H). 13C NMR (100 MHz, d6-
DMSO): δ 156.7, 143.2, 132.7, 129.7, 128.8, 121.7, 115.5, 112.5, 101.1,
100.0, 46.0. HRMS (AP-ESI): 318.0236 (M + H)+ (calcd for
C14H12BrN3O: 318.0237).
8.30 (s, 1H), 6.86 (s, 1H), 6.10 (d, J = 1.0 Hz, 1H), 4.36 (dd, J = 13.5,
3.1 Hz, 1H), 4.16 (dd, J = 13.5, 8.4 Hz, 1H), 3.66 (dd, J = 8.1, 3.4 Hz,
2H), 3.37 (dd, J = 10.8, 4.7 Hz, 1H), 3.27 (dd, J = 10.8, 6.5 Hz, 1H),
3.17 (dd, J = 11.1, 5.3 Hz, 1H), 1.86−1.45 (m, 10H). HRMS (AP-ESI):
338.0858 (M + H)+ (calcd for C15H20BrN3O: 338.0863). 9: 1H NMR
(400 MHz, CDCl3): δ 7.82 (s, 1H), 6.89 (s, 1H), 6.40 (d, J = 1.0 Hz,
1H), 4.27 (dd, J = 14.0, 2.3 Hz, 1H), 4.14−4.08 (m, 3H), 3.77 (t, J = 6.1
Hz, 1H), 3.49−3.36 (m, 1H), 3.06 (s, 1H), 1.78−1.51 (m, 9H). 13C
NMR (100 MHz, d6-DMSO): δ 143.7, 142.5, 131.8, 121.4, 113.1,
104.3, 99.9, 73.9, 52.9, 41.5, 29.5, 27.3, 26.6, 26.3, 26.2. HRMS (AP-
ESI): 338.0857 (M + H)+ (calcd for C15H20BrN3O: 338.0863).
Synthesis of (S)-6-Bromo-N-(pyrrolidin-2-ylmethyl)-1H-indazol-
4-amine (10). The title compound was obtained as a yellow solid via
general procedure I and general procedure II from A1 and aldehyde B3
Synthesis of 2-((6-Bromo-1H-indazol-4-yl)amino)-2-phenyle-
than-1-ol (3). The title compound was obtained as a yellow solid via
general procedure III and general procedure II from A6 and epoxide
1
B10 (Scheme S4) in 24% yield for two steps, 98% HPLC. H NMR
(400 MHz, d6-DMSO): δ 12.84 (s, 1H), 8.34 (s, 1H), 7.44 (d, J = 7.3
Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 6.89 (d, J =
6.7 Hz, 1H), 6.81 (s, 1H), 5.94 (s, 1H), 5.06 (t, J = 5.7 Hz, 1H), 4.57
(dd, J = 12.3, 6.7 Hz, 1H), 3.80−3.58 (m, 2H). 13C NMR (100 MHz,
d6-DMSO): δ 142.7, 142.0, 141.7, 132.7, 128.8, 127.5, 127.2, 121.5,
112.7, 101.9, 100.3, 66.2, 60.0. HRMS (AP-ESI): 332.0381 (M + H)+
(calcd for C15H14BrN3O: 332.0393).
1
(Scheme S2) in 76% yield for two steps, 94% HPLC. H NMR (400
Synthesis of 2-((6-Bromo-1H-indazol-4-yl)amino)-2-(3-
chlorophenyl)ethan-1-ol (4) and 2-((6-Bromo-1H-indazol-4-yl)-
amino)-1-(3-chlorophenyl)ethan-1-ol (5). The title compound 4
and 5 were obtained as a yellow solid via general procedure III and
general procedure II from A6 and epoxide B11 (Scheme S4) in 24 and
30% yields for two steps, respectively, 95 and 96% HPLC. 4: 1H NMR
(400 MHz, d6-DMSO): δ 12.86 (s, 1H), 8.33 (s, 1H), 7.50 (s, 1H), 7.43
(d, J = 7.6 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H),
6.89 (d, J = 7.1 Hz, 1H), 6.84 (s, 1H), 5.97 (s, 1H), 5.08 (t, J = 5.8 Hz,
1H), 4.63 (dd, J = 12.6, 6.7 Hz, 1H), 3.83−3.59 (m, 2H). 13C NMR
(100 MHz, d6-DMSO): δ 144.6, 142.3, 142.0, 133.4, 132.7, 130.6,
127.5, 127.0, 126.3, 121.5, 112.7, 101.9, 100.6, 65.8, 59.1. HRMS (AP-
ESI): 366.0008 (M + H)+ (calcd for C15H13BrClN3O: 366.0003). 5: 1H
NMR (400 MHz, d6-DMSO): δ 12.82 (s, 1H), 8.20 (s, 1H), 7.48 (s,
1H), 7.43−7.30 (m, 3H), 6.85 (s, 1H), 6.65 (t, J = 5.4 Hz, 1H), 6.22 (s,
1H), 5.73 (s, 1H), 4.85 (t, J = 5.5 Hz, 1H). 13C NMR (100 MHz, d6-
DMSO): δ 147.0, 143.2, 133.3, 130.4, 127.4, 126.4, 125.3, 121.9, 112.5,
100.9, 100.1, 70.3, 51.1. HRMS (AP-ESI): 366.0008 (M + H)+ (calcd
for C15H13BrClN3O: 366.0003).
MHz, d6-DMSO): δ 12.91 (s, 1H), 8.14 (s, 1H), 6.93 (s, 1H), 6.85 (t, J
= 5.8 Hz, 1H), 6.28 (d, J = 0.8 Hz, 1H), 3.79 (dd, J = 14.6, 7.3 Hz, 1H),
3.51−3.46 (m, 2H), 2.20−1.66 (m, 6H). 13C NMR (100 MHz, d6-
DMSO): δ 142.6, 132.4, 121.8, 112.7, 101.0, 58.4, 56.5, 45.2, 44.1, 28.1,
23.3, 19.0.
Synthesis of (R)-6-Bromo-N-(pyrrolidin-2-ylmethyl)-1H-indazol-
4-amine (11). The title compound was obtained as a yellow solid via
general procedure I and general procedure II from A1 and aldehyde B2
1
(Scheme S2) in 40% yield for two steps, 97% HPLC. H NMR (400
MHz, d6-DMSO): δ 12.91 (s, 1H), 8.15 (s, 1H), 6.93 (s, 1H), 6.86 (t, J
= 5.7 Hz, 1H), 6.28 (s, 1H), 3.83−3.77 (m, 2H), 2.14 (dt, J = 12.5, 7.5
Hz, 2H), 1.98−1.87 (m, 4H), 1.77−1.68 (m, 2H). 13C NMR (100
MHz, d6-DMSO): δ 142.6, 121.8, 112.7, 101.0, 58.4, 56.5, 45.2, 44.0,
28.1, 23.3, 19.0. HRMS (AP-ESI): 295.0549 (M + H)+ (calcd for
C12H15BrN4: 295.0553).
Synthesis of (S)-6-Bromo-N-(piperidin-2-ylmethyl)-1H-indazol-4-
amine (12). The title compound was obtained as a yellow solid via
general procedure I and general procedure II from A1 and aldehyde B5
1
(Scheme S2) in 65% yield for two steps, 97% HPLC. H NMR (400
MHz, d6-DMSO): δ 12.90 (s, 1H), 8.16 (s, 1H), 6.91 (s, 1H), 6.83 (t, J
= 5.9 Hz, 1H), 6.28 (s, 1H), 3.46 (dd, J = 12.7, 6.0 Hz, 2H), 2.92 (td, J =
12.5, 2.7 Hz, 1H), 2.00−1.94 (m, 1H), 1.80−1.43 (m, 7H). 13C NMR
(100 MHz, d6-DMSO): δ 142.7, 132.5, 121.8, 116.2, 112.7, 100.8, 55.0,
45.3, 44.4, 44.3, 26.7, 22.4, 21.8. HRMS (AP-ESI): 309.0718 (M + H)+
(calcd for C13H17BrN4: 309.0709).
Synthesis of 2-((6-Bromo-1H-indazol-4-yl)amino)-1-(4-
hydroxyphenyl)ethan-1-one (6). To a solution of indazole A6 (311
mg, 1.0 mmol) and 2-bromo-1-(4-hydroxyphenyl)ethan-1-one (258
mg, 1.2 mmol) in DMF (5 mL), Cs2CO3 (977 mg, 3.0 mmol) was
added, and the mixture was stirred at 60 °C for 3 h. The mixture was
dissolved with EA, washed with brine, and dried over anhydrous
Na2SO4. The solvent was evaporated, and the crude product was
charged on a silica gel column chromatograph with petroleum ether and
EA as the eluent to afford the target compounds in 32% yield, 93%
HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.91 (s, 1H), 8.26 (s, 1H),
8.00 (d, J = 8.8 Hz, 2H), 6.99−6.83 (m, 3H), 6.78 (t, J = 5.5 Hz, 1H),
6.14 (d, J = 0.7 Hz, 1H), 4.74 (d, J = 5.6 Hz, 2H). 13C NMR (100 MHz,
d6-DMSO): δ 194.5, 163.0, 143.1, 131.9, 131.0, 126.9, 121.7, 115.8,
112.7, 112.6, 101.5, 100.6, 49.3, 21.2, 14.5. HRMS (AP-ESI): 346.0182
(M + H)+ (calcd for C15H12BrN3O2: 346.0186).
Synthesis of (Z)-2-((6-Bromo-1H-indazol-4-yl)amino)-1-(4-
hydroxyphenyl)ethan-1-one Oxime (7). The solution of 6 (345 mg,
1.0 mmol) and hydroxylamine hydrochloride (417 mg, 6.0 mmol) in
pyridine (10 mL) was stirred at 110 °C for 3 h. The mixture was
extracted with EA, and the organic layers were washed with brine and
dried over anhydrous Na2SO4. The solvent was evaporated, and the
crude product was charged on a silica gel column chromatograph with
petroleum ether and EA as the eluent to afford the target compound 7 in
40% yield, 92% HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.77 (d, J =
7.0 Hz, 1H), 11.45 (s, 1H), 9.59 (s, 1H), 8.09 (s, 1H), 7.52−7.47 (m,
2H), 6.91 (t, J = 6.5 Hz, 1H), 6.83−6.68 (m, 4H), 6.26 (s, 1H), 4.45 (d,
J = 5.6 Hz, 2H). 13C NMR (100 MHz, d6-DMSO): δ 194.5, 163.0,
143.1, 132.4, 131.9, 131.0, 126.9, 121.7, 116.5, 115.8, 115.5, 112.7,
112.6, 101.5, 100.6, 49.3, 21.2, 14.5. HRMS (AP-ESI): 361.0293 (M +
H)+ (calcd for C15H13BrN4O2: 361.0295).
Synthesis of (R)-6-Bromo-N-(piperidin-2-ylmethyl)-1H-indazol-4-
amine (13). The title compound was obtained as a yellow solid via
general procedure I and general procedure II from A1 and aldehyde B4
1
(Scheme S2) in 35% yield for two steps, 93% HPLC. H NMR (400
MHz, d6-DMSO): δ 12.87 (s, 1H), 8.51 (s, 1H), 8.18 (s, 1H), 6.90 (s,
1H), 6.80 (t, J = 5.8 Hz, 1H), 6.27 (d, J = 0.8 Hz, 1H), 3.47−3.37 (m,
2H), 2.88 (td, J = 12.3, 2.8 Hz, 1H), 1.99−1.41 (m, 8H). 13C NMR
(100 MHz, d6-DMSO): δ 142.8, 142.1, 132.5, 121.8, 116.2, 112.7,
100.8, 55.0, 45.6, 44.6, 27.1, 22.7, 22.0. HRMS (AP-ESI): 309.0714 (M
+ H)+ (calcd for C13H17BrN4: 309.0709).
Synthesis of 6-Bromo-N-(pyrrolidin-3-ylmethyl)-1H-indazol-4-
amine (14). The title compound was obtained as a yellow solid via
general procedure II from 16 in 92% yield, 98% HPLC. 1H NMR (400
MHz, d6-DMSO): δ 12.86 (s, 1H), 9.00 (s, 2H), 8.18 (s, 1H), 6.86 (s,
1H), 6.72 (s, 1H), 6.20 (s, 1H), 3.25 (s, 3H), 3.21−3.14 (m, 1H),
3.02−2.93 (m, 1H), 2.67 (dt, J = 14.5, 7.1 Hz, 1H), 2.11 (td, J = 12.7,
7.0 Hz, 1H), 1.72 (dq, J = 15.5, 7.8 Hz, 1H), 1.24 (s, 1H). 13C NMR
(100 MHz, d6-DMSO): δ 143.2, 142.1, 132.5, 121.8, 112.5, 100.7,
100.4, 48.5, 45.1, 44.9, 37.2, 28.4. HRMS (AP-ESI): 295.0554 (M +
H)+ (calcd for C12H15BrN4: 295.0553).
Synthesis of N-(Azetidin-3-ylmethyl)-6-bromo-1H-indazol-4-
amine (15). The title compound was obtained as a yellow solid via
general procedure I and general procedure II from A1 and aldehyde B6
1
(Scheme S2) in 45% yield for two steps, 92% HPLC. H NMR (400
MHz, d6-DMSO): δ 12.89 (s, 1H), 8.95 (s, 2H), 8.15 (s, 1H), 6.88 (s,
1H), 6.76 (s, 1H), 6.20 (s, 1H), 4.06 (t, J = 9.5 Hz, 2H), 3.87−3.72 (m,
2H), 3.51−3.42 (m, 3H), 3.22−3.05 (m, 1H). 13C NMR (100 MHz, d6-
DMSO): δ 143.2, 142.1, 132.5, 121.8, 119.2, 112.5, 100.7, 56.5, 49.4,
45.1, 31.2. HRMS (AP-ESI): 231.0411 (M + H)+ (calcd for
C11H13BrN4: 231.0396).
Synthesis of 2-((6-Bromo-1H-indazol-4-yl)amino)-2-cyclohexyle-
than-1-ol (8) and 2-((6-Bromo-1H-indazol-4-yl)amino)-1-cyclohex-
ylethan-1-ol (9). The title compound 8 and 9 were obtained as a yellow
solid via general procedure III and general procedure II from A6 and
epoxide B12 (Scheme S4) in 16 and 67% yields for two steps,
respectively, 93 and 92% HPLC. 8: 1H NMR (400 MHz, d6-DMSO): δ
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Synthesis of tert-Butyl 3-(((6-Bromo-1H-indazol-4-yl)amino)-
methyl)pyrrolidine-1-carboxylate (16). The title compound was
obtained as a yellow solid via general procedure I from A1 and
Synthesis of (1R,2S)-2-(((6-Bromo-1H-indazol-4-yl)amino)-
methyl)cyclohexan-1-ol (23). The title compound was obtained as a
yellow solid via general procedure I from A1 and B13 (Scheme S5) in
79.7% yield, 98% HPLC. 1H NMR (400 MHz, CDCl3): δ 7.83 (s, 1H),
6.83 (s, 1H), 6.21 (d, J = 1.0 Hz, 1H), 3.43 (td, J = 10.1, 4.3 Hz, 1H),
3.19 (qd, J = 12.3, 6.2 Hz, 2H), 1.93−1.90 (m, 1H), 1.85−1.42 (m,
5H), 1.38−1.09 (m, 4H). 13C NMR (100 MHz, CDCl3): δ 142.7,
141.7, 131.9, 123.1, 112.6, 102.9, 101.1, 49.3, 44.1, 36.1, 29.6, 25.2,
24.5, 1.0. HRMS (AP-ESI): 324.0714 (M + H)+ (calcd for
C14H18BrN3O: 324.0706).
1
aldehyde B1 (Scheme S2) in 42% yield, 92% HPLC. H NMR (400
MHz, CDCl3): δ 10.79 (s, 1H), 7.96 (s, 1H), 7.01 (s, 1H), 6.31 (s, 1H),
4.53 (d, J = 72.9 Hz, 1H), 3.62 (t, J = 9.72 Hz, 1H), 3.39−3.15 (m, 4H),
2.61 (s, 1H), 2.11 (tt, J = 11.0, 5.5 Hz, 1H), 1.80−1.68 (m, 1H), 1.50 (s,
9H). 13C NMR (101 MHz, CDCl3): δ 154.7, 142.0, 141.9, 131.6, 122.8,
112.4, 102.6, 101.6, 79.5, 49.4, 46.2, 45.1, 37.5, 28.5, 28.4. HRMS (AP-
ESI): 395.1085 (M + H)+ (calcd for C17H23BrN4O2: 395.1077).
Synthesis of 6-Bromo-N-(pyridin-2-ylmethyl)-1H-indazol-4-
amine (17). The title compound was obtained as a yellow solid via
general procedure I from A1 and picolinaldehyde in 43% yield, 98%
HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.86 (s, 1H), 8.63−8.48
(m, 1H), 8.23 (s, 1H), 7.76 (td, J = 7.7, 1.7 Hz, 1H), 7.39 (t, J = 6.9 Hz,
2H), 7.28 (dd, J = 6.9, 5.3 Hz, 1H), 6.85 (s, 1H), 6.04 (s, 1H), 4.53 (d, J
= 6.0 Hz, 2H). 13C NMR (100 MHz, d6-DMSO): δ 159.5, 149.5, 143.0,
142.1, 137.2, 132.6, 122.6, 121.6, 112.6, 101.2, 100.5, 48.6. HRMS (AP-
ESI): 303.0237 (M + H)+ (calcd for C13H11BrN4: 303.0240).
Synthesis of 6-Bromo-N-(cyclohexylmethyl)-1H-indazol-4-amine
(18). The title compound was obtained as a yellow solid via general
procedure I from A1 and cyclohexanecarbaldehyde in 60% yield, 95%
HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.78 (s, 1H), 8.22 (s, 1H),
6.81 (s, 1H), 6.57 (t, J = 5.5 Hz, 1H), 6.09 (d, J = 0.9 Hz, 1H), 3.00 (t, J
= 6.2 Hz, 2H), 1.83 (d, J = 12.7 Hz, 2H), 1.67 (ddd, J = 10.7, 9.8, 3.1 Hz,
4H), 1.32−1.08 (m, 4H), 1.05−0.85 (m, 2H). 13C NMR (100 MHz, d6-
DMSO): δ 143.7, 142.1, 132.7, 121.9, 112.4, 100.3, 99.6, 49.6, 37.0,
31.2, 26.6, 25.9. HRMS (AP-ESI): 308.0744 (M + H)+ (calcd for
C13H11BrN4: 308.0757).
Synthesis of (1S,2S)-2-(((6-Bromo-1H-indazol-4-yl)methyl)-
amino)cyclohexan-1-ol (24). According to the synthesis method of
25, compound 24 was synthesized from A13 and (1S,2S)-2-
1
aminocyclohexan-1-ol in 39% yield for two steps, 93% HPLC. H
NMR (400 MHz, d6-DMSO): δ 13.04 (s, 1H), 8.19 (s, 1H), 7.41 (d, J =
8.3 Hz, 1H), 7.28 (dd, J = 8.2, 7.0 Hz, 1H), 7.07 (d, J = 6.9 Hz, 1H), 4.68
(s, 1H), 4.14 (d, J = 13.7 Hz, 1H), 3.99 (d, J = 13.7 Hz, 1H), 2.31 (d, J =
2.7 Hz, 1H), 2.08−1.98 (m, 1H), 1.82 (d, J = 8.7 Hz, 1H), 1.61 (d, J =
7.7 Hz, 3H), 1.35−0.93 (m, 6H). 13C NMR (100 MHz, d6-DMSO): δ
140.5, 134.2, 132.8, 126.2, 122.6, 119.2, 109.0, 72.9, 63.2, 48.8, 34.4,
30.1, 24.5. HRMS (AP-ESI): 324.0701 (M + H)+ (calcd for
C14H18BrN3O: 324.0706).
Synthesis of (1R,2R)-2-(((6-Bromo-1H-indazol-4-yl)methyl)-
amino)cyclohexan-1-ol (25). The solution of 26 (337 mg, 1.0
mmol) in THF (10 mL) was slowly added LiAlH4 (152 mg, 4.0
mmol) at 0 °C. After stirring at 60 °C for 3 h, the reaction was quenched
by aq NaOH. The mixture was filtered, and the filtrate was dried. The
solvent was evaporated, and the crude product was charged on a silica
gel column chromatograph with DCM and MeOH as the eluent to
afford the target compound 26 in 80% yield, 97% HPLC. 1H NMR (400
MHz, d6-DMSO): δ 13.03 (s, 1H), 8.18 (s, 1H), 7.41 (d, J = 8.3 Hz,
1H), 7.28 (dd, J = 8.3, 7.0 Hz, 1H), 7.07 (d, J = 6.9 Hz, 1H), 4.66 (s,
1H), 4.13 (d, J = 13.7 Hz, 1H), 3.97 (d, J = 13.7 Hz, 1H), 3.19 (d, J = 3.4
Hz, 1H), 2.30 (td, J = 10.4, 3.9 Hz, 1H), 2.03 (d, J = 12.2 Hz, 1H),
1.87−1.76 (m, 1H), 1.65−1.56 (m, 2H), 1.28−0.96 (m, 6H). 13C NMR
(100 MHz, d6-DMSO): δ 140.5, 134.5, 132.8, 126.3, 122.6, 119.1,
109.0, 73.0, 63.2, 48.8, 34.3, 30.2, 24.6, 24.5. HRMS (AP-ESI):
324.0701 (M + H)+ (calcd for C14H18BrN3O: 324.0706).
Synthesis of 6-Bromo-N-((1R,2S)-2-hydroxycyclohexyl)-1H-inda-
zole-4-carboxamide (26). To a solution of acid A13 (241 mg, 1.0
mmol) in DCM (15 mL) was added (1R,2R)-2-aminocyclohexan-1-ol
(287 mg, 2.5 mmol), EDCI (287 mg, 1.5 mmol), HOBT (202 mg, 1.5
mmol), and DIEA (193 mg, 1.5 mmol) at 0 °C. The reaction mixture
was stirred at rt for 12 h. The mixture was washed with aq NaHCO3 and
brine and dried over anhydrous Na2SO4. The solvent was evaporated,
and the product was obtained in 88% yield, 95% HPLC. 1H NMR (400
MHz, d6-DMSO): δ 13.33 (s, 1H), 8.39 (s, 1H), 8.34 (d, J = 8.1 Hz,
1H), 7.92 (s, 1H), 7.77 (d, J = 1.4 Hz, 1H), 4.71 (d, J = 5.3 Hz, 1H),
3.68 (d, J = 8.4 Hz, 1H), 3.45 (dt, J = 9.5, 4.8 Hz, 1H), 2.04−1.82 (m,
2H), 1.67 (d, J = 10.1 Hz, 2H), 1.33−1.19 (m, 4H). 13C NMR (303
MHz, d6-DMSO): δ 165.1, 141.4, 134.8, 130.0, 122.9, 120.5, 119.0,
115.5, 71.5, 55.8, 35.0, 31.7, 24.9, 24.6. HRMS (AP-ESI): 338.0499 (M
+ H)+ (calcd for C14H16BrN3O2: 338.0499).
Synthesis of (1R,2S)-2-(((6-Bromo-1-methyl-1H-indazol-4-yl)-
amino)methyl)cyclohexan-1-ol (27). The title compound was
obtained as a yellow solid via general procedure I from A5 (Scheme
S1) and B13 in 18.2% yield, 91% HPLC. 1H NMR (400 MHz, CDCl3):
δ 7.74 (s, 1H), 6.78 (s, 1H), 6.21 (d, J = 14.3 Hz, 1H), 4.14 (s, 1H), 3.87
(s, 3H), 3.42 (td, J = 9.8, 4.3 Hz, 1H), 3.26−3.13 (m, 3H), 1.95−1.85
(m, 1H), 1.83−1.54 (m, 6H), 1.29 (t, J = 22.0 Hz, 3H). 13C NMR (100
MHz, CDCl3): δ 142.8, 141.6, 129.8, 122.6, 113.2, 103.3, 102.4, 100.4,
62.2, 49.3, 44.1, 36.1, 35.6, 31.9, 29.7, 25.2, 24.5, 22.7, 14.1. HRMS
(AP-ESI): 338.0859 (M + H)+ (calcd for C15H20BrN3O: 338.0863).
Synthesis of (1R,2S)-2-(((5-Bromo-1H-indazol-7-yl)amino)-
methyl)cyclohexan-1-ol (28). The title compound was obtained as a
yellow solid via general procedure I from A7 (Scheme S1) and B13 in
40.7% yield, 93% HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.78 (s,
1H), 7.90 (s, 1H), 7.10 (s, 1H), 6.41 (s, 1H), 5.76 (s, 1H), 5.15−4.04
(m, 1H), 3.56 (dd, J = 12.5, 2.8 Hz, 1H), 3.23 (dd, J = 11.3, 7.4 Hz, 1H),
2.97 (dd, J = 12.5, 8.4 Hz, 1H), 1.91 (s, 1H), 1.67−1.50 (m, 4H), 1.30−
Synthesis of 4-(((6-Bromo-1H-indazol-4-yl)amino)methyl)-
cyclohexan-1-one Oxime (19). The title compound was obtained as
a yellow solid via general procedure I from A1 and aldehyde B9
1
(Scheme S3) in 54% yield, 92% HPLC. H NMR (400 MHz, d6-
DMSO): δ 12.78 (s, 1H), 10.17 (s, 1H), 8.21 (s, 1H), 6.81 (s, 1H), 6.63
(t, J = 5.3 Hz, 1H), 6.12 (s, 1H), 3.35 (s, 1H), 3.07 (t, J = 5.9 Hz, 2H),
2.30 (d, J = 13.9 Hz, 1H), 2.18−1.83 (m, 4H), 1.74 (td, J = 13.6, 4.9 Hz,
1H), 1.28−1.01 (m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 157.5,
143.5, 142.1, 132.7, 121.9, 112.4, 100.4, 99.7, 48.4, 36.2, 31.0, 29.7,
23.2. HRMS (AP-ESI): 335.0513 (M + H)+ (calcd for C14H18BrN3:
335.0513).
Synthesis of 2-(((6-Bromo-1H-indazol-4-yl)amino)methyl)-
cyclohexan-1-ol (20). The title compound was obtained as a yellow
solid via general procedure I from A1 and aldehyde B7 (Scheme S3) in
41% yield, 95% HPLC. 1H NMR (400 MHz, CDCl3): δ 11.21 (s, 1H),
7.92 (s, 1H), 6.90 (d, J = 7.2 Hz, 1H), 6.27 (d, J = 10.0 Hz, 1H), 5.41 (s,
1H), 5.01 (s, 1H), 4.14 (s, 1H), 3.51 (td, J = 10.0, 4.2 Hz, 1H), 3.43−
3.13 (m, 2H), 2.15−1.97 (m, 1H), 1.93−1.48 (m, 6H), 1.44−0.77 (m,
4H). 13C NMR (100 MHz, CDCl3): δ 142.7, 142.7, 141.8, 141.7, 131.8,
131.7, 123.2, 123.1, 112.6, 112.4, 102.8, 102.3, 101.2, 100.86 (s), 75.8,
68.2, 49.2, 45.9, 44.1, 40.1, 36.0, 33.0, 29.6, 24.9, 24.5, 20.5. HRMS
(AP-ESI): 324.0714 (M + H)+ (calcd for C14H18BrN3O: 324.0706).
Synthesis of N-((1,4-Dioxaspiro[4.5]decan-6-yl)methyl)-6-bromo-
1H-indazol-4-amine (21). The title compound was obtained as a
yellow solid via general procedure I from A1 and aldehyde B8 (Scheme
S3) in 77% yield, 96% HPLC. 1H NMR (400 MHz, CDCl3): δ 10.51 (s,
1H), 7.87 (s, 1H), 6.86 (s, 1H), 6.20 (s, 1H), 5.18 (s, 1H), 4.04−3.73
(m, 4H), 3.38−3.19 (m, 1H), 3.13−2.97 (m, 1H), 2.10−1.89 (m, 1H),
1.85−1.02 (m, 10H). 13C NMR (100 MHz, d6-DMSO): δ 143.5, 142.1,
132.5, 121.9, 112.4, 109.9, 100.3, 99.6, 64.7, 64.6, 43.4, 42.4, 34.5, 28.5,
24.2, 23.9. HRMS (AP-ESI): 366.0838 (M + H)+ (calcd for
C16H20BrN3O2: 366.0812).
Synthesis of (1S,2R)-2-(((6-Bromo-1H-indazol-4-yl)amino)-
methyl)cyclohexan-1-ol (22). The title compound was obtained as a
yellow solid via general procedure I from A1 and B14 (Scheme S5) in
61.7% yield, 96% HPLC. 1H NMR (400 MHz, CDCl3): δ 7.94 (s, 1H),
6.96 (s, 1H), 6.33 (s, 1H), 3.53 (dt, J = 14.3, 4.9 Hz, 1H), 3.30 (qd, J =
12.4, 6.2 Hz, 2H), 2.07−1.95 (m, 1H), 1.91−1.66 (m, 5H), 1.44−1.21
(m, 4H). 13C NMR (100 MHz, CDCl3): δ 142.7, 141.8, 131.8, 123.0,
112.6, 102.7, 101.1, 49.3, 44.0, 36.0, 30.9, 29.6, 25.2, 24.5. HRMS (AP-
ESI): 324.0714 (M + H)+ (calcd for C14H18BrN3O: 324.0706).
8324
https://doi.org/10.1021/acs.jmedchem.1c00303
J. Med. Chem. 2021, 64, 8303−8332

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1.11 (m, 4H). 13C NMR (100 MHz, d6-DMSO): δ 135.9, 124.3, 115.1,
109.2, 104.8, 71.4, 46.1, 44.7, 36.0, 29.4, 25.5, 24.9. HRMS (AP-ESI):
324.0715 (M + H)+ (calcd for C14H18BrN3O: 324.0706).
Synthesis of (1R,2S)-2-(((6-Amino-1H-indazol-4-yl)amino)-
methyl)cyclohexan-1-ol (35). Cbz-immediate was obtained as a
yellow solid via general procedure I from A11 (Scheme S1) and B13
in 46% yield. To a solution of Cbz-immediate (39 mg, 0.1 mmol) and
Pd/C (5%, 10 mg) in MeOH (5 mL), hydrogen gas was filled, and the
mixture was stirred at 10 MPa for 12 h. The mixture was filtrated to
remove Pd/C. The solvent was evaporated, and the crude product was
charged on a silica gel column chromatograph with DCM and MeOH as
the eluent to afford the target compound 36 in 36% yield, 92% HPLC.
1H NMR (400 MHz, d6-DMSO): δ 12.81 (s, 1H), 8.17 (s, 1H), 7.28 (s,
Synthesis of (1R,2S)-2-(((5-Chloro-1H-indazol-7-yl)amino)-
methyl)cyclohexan-1-ol (29). The title compound was obtained as a
yellow solid via general procedure I from A8 (Scheme S1) and B13 in
33% yield, 91% HPLC. 1H NMR (400 MHz, CDCl3): δ 7.95 (s, 1H),
7.03 (d, J = 8.1 Hz, 1H), 6.08 (d, J = 8.1 Hz, 1H), 5.92 (s, 1H), 3.43 (td,
J = 10.1, 4.2 Hz, 1H), 3.25 (dd, J = 12.3, 7.8 Hz, 1H), 3.15 (dd, J = 12.4,
4.3 Hz, 1H), 1.92 (dd, J = 9.5, 3.3 Hz, 1H), 1.69 (dd, J = 37.3, 14.3 Hz,
5H), 1.45 (dd, J = 12.4, 9.2 Hz, 1H), 1.35−1.22 (m, 2H). 13C NMR
(100 MHz, CDCl3): δ 140.5, 138.7, 132.5, 127.5, 115.1, 103.7, 101.0,
49.9, 43.9, 35.9, 29.7, 25.2, 24.5, 22.7. HRMS (AP-ESI): 280.1205 (M +
H)+ (calcd for C14H18ClN3O: 280.1211).
1H), 6.71 (s, 1H), 6.04 (s, 1H), 2.97−2.86 (m, 1H), 2.01−1.92 (m,
1H), 1.90−1.83 (m, 1H), 1.66−1.55 (m, 4H), 1.42−0.75 (m, 9H). 13
C
NMR (100 MHz, d6-DMSO): δ 142.4, 141.3, 132.2, 115.6, 99.6, 98.1,
72.1, 46.7, 44.6, 36.0, 29.5, 25.5, 24.9.
Synthesis of (1R,2S)-2-(((5-Bromo-1H-indazol-4-yl)amino)-
methyl)cyclohexan-1-ol (36). The title compound was obtained as a
yellow solid via general procedure I from A9 (Scheme S1) and B13 in
40% yield, 96% HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.91 (s,
1H), 8.39 (s, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.64 (d, J = 8.6 Hz, 1H),
5.82 (t, J = 5.4 Hz, 1H), 5.05 (s, 1H), 4.03−3.88 (m, 1H), 3.49−3.23
(m, 4H), 1.85 (d, J = 12.6 Hz, 3H), 1.75−1.44 (m, 4H), 1.32−0.90 (m,
6H). HRMS (AP-ESI): 324.0715 (M + H)+ (calcd for C14H18BrN3O:
324.0706).
Synthesis of (1R,2S)-2-(((5,7-Dibromo-1H-indazol-4-yl)amino)-
methyl)cyclohexan-1-ol (37). The title compound was obtained as a
yellow solid via general procedure I from A10 (Scheme S1) and B13 in
47% yield, 98% HPLC. 1H NMR (400 MHz, DMSO): δ 13.25 (s, 1H),
8.55 (s, 1H), 7.48 (s, 1H), 6.06 (t, J = 5.4 Hz, 1H), 5.10 (d, J = 4.4 Hz,
1H), 4.02−3.85 (m, 1H), 3.32−3.21 (m, 1H), 1.94−1.76 (m, 2H), 1.60
(dd, J = 24.0, 12.8 Hz, 3H), 1.22 (dd, J = 20.1, 11.1 Hz, 2H), 1.13−0.96
(m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 139.8, 131.9, 113.5, 95.4,
73.0, 49.8, 44.8, 36.1, 31.1, 29.2, 25.3, 24.8. HRMS (AP-ESI): 401.9823
(M + H)+ (calcd for C14H17Br2N3O: 401.9811).
Synthesis of (1R,2S)-2-(((6-Bromo-1H-indazol-4-yl)amino)-
methyl)cyclohexyl Acetate (38). To a solution of acetic acid (0.1
mL, 1.8 mmol) in DCM (20 mL) were added DCC (366 mg, 1.8
mmol) and DMAP (15 mg, 0.1 mmol) at 0 °C, and the reaction mixture
was stirred at 0 °C for 0.5 h. Then, the residue (300 mg, 0.7 mmol) was
added, and the reaction mixture was stirred at rt for 1 h. The mixture
was filtered, and the filtrate was washed with aq NaHCO3 and brine and
dried over anhydrous Na2SO4. The solvent was evaporated, and the
ethyl ester derivative was obtained in 76% yield. The title compound 38
was obtained as a yellow solid via general procedure II in 77% yield,
98% HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.78 (s, 1H), 8.18 (s,
1H), 6.82 (s, 1H), 6.59 (t, J = 5.7 Hz, 1H), 6.12 (s, 1H), 4.54 (td, J = 9.9,
4.4 Hz, 1H), 3.36−3.27 (m, 1H), 3.00−2.90 (m, 1H), 2.05 (s, 3H), 1.97
(s, 1H), 1.84−1.47 (m, 5H), 1.26 (dd, J = 19.8, 6.4 Hz, 3H). 13C NMR
(100 MHz, d6-DMSO): δ 170.5, 143.5, 142.2, 132.6, 122.0, 112.5,
100.4, 99.8, 74.9, 45.4, 41.4, 33.8, 31.7, 29.5, 25.8, 24.8, 24.3, 21.5.
HRMS (AP-ESI): 366.0809 (M + H)+ (calcd for C16H20BrN3O2:
366.0817).
Synthesis of N-(((1S,2R)-2-Aminocyclohexyl)methyl)-6-bromo-
1H-indazol-4-amine (39). The mixture of 41 (11 mg, 0.03 mmol)
and NaN3 (3 mg, 0.04 mmol) in DMF (2 mL) was stirred at rt for 12 h.
Then, to this solution was added Pd/C (10%); hydrogen gas was filled,
and the mixture was stirred for 2 h. The mixture was filtrated to remove
Pd/C. The filtrate was extracted with EA, and the organic layers were
washed with brine and dried over anhydrous Na2SO4. The solvent was
evaporated, and the crude product was purified by column
chromatography with DCM and MeOH as the eluent to obtain 39 as
a yellow-white solid in 74% yield for two steps, 92% HPLC. 1H NMR
(400 MHz, d6-DMSO): δ 12.83 (s, 1H), 8.20 (s, 1H), 7.76−7.40 (m,
2H), 6.83 (s, 2H), 6.61 (s, 2H), 6.11 (s, 2H), 4.71 (s, 2H), 3.23−2.90
(m, 3H), 2.74−2.57 (m, 1H), 2.11−1.93 (m, 5H), 1.86−1.78 (m, 2H),
1.75−1.57 (m, 8H), 1.53−1.30 (m, 9H). 13C NMR (100 MHz, d6-
DMSO): δ 143.4, 132.6, 129.8, 128.4, 121.9, 100.5, 99.8, 48.2, 40.0,
34.5, 27.0, 25.4, 20.9.
Synthesis of (1R,2S)-2-(((6-Chloro-1H-indazol-4-yl)amino)-
methyl)cyclohexan-1-ol (30). The title compound was obtained as a
yellow solid via general procedure I from A2 (Scheme S1) and B13 in
30% yield, 92% HPLC. 1H NMR (400 MHz, CDCl3): δ 7.86 (s, 1H),
6.63 (s, 1H), 6.03 (s, 1H), 3.44 (td, J = 10.0, 4.2 Hz, 1H), 3.17 (qd, J =
12.4, 6.2 Hz, 2H), 2.03−1.85 (m, 1H), 1.81−1.53 (m, 4H), 1.38−1.10
(m, 4H), 1.10−0.89 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 142.5,
141.3, 135.2, 131.5, 112.2, 100.3, 98.0, 49.3, 44.0, 36.0, 31.9, 29.7, 25.2,
24.5, 22.7. HRMS (AP-ESI): 280.1219 (M + H)+ (calcd for
C14H18ClN3O: 280.1211).
Synthesis of (1R,2S)-2-(((6-Methyl-1H-indazol-4-yl)amino)-
methyl)cyclohexan-1-ol (31). The title compound was obtained as a
yellow solid via general procedure I from A3 (Scheme S1) and B13 in
51% yield, 94% HPLC. 1H NMR (400 MHz, CDCl3): δ 7.83 (s, 1H),
6.49 (s, 1H), 5.99 (s, 1H), 3.40 (td, J = 10.0, 4.2 Hz, 1H), 3.26 (dd, J =
12.2, 7.8 Hz, 1H), 3.16 (dd, J = 12.3, 4.4 Hz, 1H), 2.28 (s, 3H), 1.93−
1.83 (m, 1H), 1.82−1.51 (m, 5H), 1.48−1.22 (m, 3H). 13C NMR (100
MHz, CDCl3): δ 142.0, 141.6, 139.0, 131.4, 112.5, 101.9, 98.7, 49.6,
44.3, 35.7, 29.6, 25.3, 24.5, 22.4. HRMS (AP-ESI): 260.1752 (M + H)+
(calcd for C15H21N3O: 260.1757).
Synthesis of (1R,2S)-2-(((6-(Trifluoromethyl)-1H-indazol-4-yl)-
amino)methyl)cyclohexan-1-ol (32). The title compound was
obtained as a yellow solid via general procedure I from A4 (Scheme
S1) and B13 in 57% yield, 93% HPLC. 1H NMR (400 MHz, CDCl3): δ
7.93 (s, 1H), 6.95 (s, 1H), 6.25 (s, 1H), 5.46 (s, 1H), 3.45 (d, J = 3.9 Hz,
1H), 3.24 (d, J = 6.8 Hz, 2H), 1.94−1.82 (m, 1H), 1.82−1.56 (m, 4H),
1.49 (s, J = 20.8, 10.1 Hz, 1H), 1.38−1.19 (m, 3H). 13C NMR (100
MHz, CDCl3): δ 142.7, 140.3, 131.6, 130.9, 130.6, 126.0, 123.3, 115.0,
96.1, 95.1, 60.5, 49.2, 44.1, 36.0, 29.6, 25.2, 24.5. HRMS (AP-ESI):
314.1476 (M + H)+ (calcd for C15H18F3N3O: 314.1475).
Synthesis of Methyl 4-((((1S,2R)-2-Hydroxycyclohexyl)methyl)-
amino)-1H-indazole-6-carboxylate (33). The title compound was
obtained as a yellow solid via general procedure I from A12 (Scheme
S1) and B13 in 58% yield, 92% HPLC. 1H NMR (400 MHz, CDCl3): δ
7.91 (s, 1H), 7.38 (s, 1H), 6.69 (s, 1H), 5.26 (s, 1H), 3.80 (s, 3H), 3.42
(td, J = 9.9, 4.0 Hz, 1H), 3.22 (ddd, J = 16.2, 12.2, 6.0 Hz, 2H), 1.91 (d, J
= 10.1 Hz, 1H), 1.67 (dd, J = 50.0, 14.1 Hz, 5H), 1.51−1.19 (m, 3H).
13C NMR (100 MHz, CDCl3): δ 168.3, 142.0, 140.7, 131.5, 130.0,
116.3, 101.3, 99.2, 75.7, 52.2, 49.3, 44.2, 35.9, 29.7, 25.2, 24.6. HRMS
(AP-ESI): 304.1647 (M + H)+ (calcd for C16H21N3O3: 304.1656).
Synthesis of 4-((((1S,2R)-2-Hydroxycyclohexyl)methyl)amino)-
1H-indazole-6-carboxylic Acid (34). To a solution of 34 (303 mg,
1.0 mmol) in EtOH/H2O (1:1) (40 mL), NaOH (2.0 mmol) was
added, and the mixture was stirred at 80 °C for 1 h. The solution was
adjusted to pH 6−7 by addition of HCl. The solvent was evaporated,
and the crude product was charged on a silica gel column
chromatograph with DCM and MeOH as the eluent to afford the
title compound 35 as a yellow solid in 41% yield, 91% HPLC. 1H NMR
(400 MHz, d6-DMSO): δ 12.97 (s, 1H), 8.21 (s, 1H), 7.32 (s, 1H), 6.71
(s, 1H), 6.13 (s, 1H), 3.62 (d, J = 9.5 Hz, 1H), 3.31−3.17 (m, 1H), 2.94
(t, J = 9.6 Hz, 1H), 2.03−1.79 (m, 2H), 1.76−1.48 (m, 3H), 1.31−0.87
(m, 5H). 13C NMR (100 MHz, d6-DMSO): δ 170.6, 142.1, 141.4,
134.8, 132.1, 115.3, 99.5, 98.5, 72.2, 56.5, 46.8, 44.7, 36.0, 29.6, 25.5,
24.9, 19.0. HRMS (AP-ESI): 290.1492 (M + H)+ (calcd for
C15H19N3O3: 290.1499).
Synthesis of N-(((1S,2S)-2-Aminocyclohexyl)methyl)-6-bromo-
1H-indazol-4-amine (40). 23 (100 mg, 0.3 mmol) was dissolved in
THF (10 mL), and phthalimide (91 mg, 0.6 mmol) and
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triphenylphosphine (162 mg, 0.6 mmol) were added at 0 °C. The
mixture was stirred for 10 min, and diisopropyl azodicarboxylate
(DIAD) (125 mg, 0.6 mmol) was then added. The mixture was stirred
at rt for 5 h. The mixture was extracted with EA, and the organic layers
were washed with brine and dried over anhydrous Na2SO4. The solvent
was evaporated to afford the crude immediate. The immediate (100 mg,
0.2 mmol) was dissolved in absolute ethanol (10 mL), and hydrazine
hydrate (66 mg, 1.3 mmol) was added at room temperature (rt). The
mixture was stirred for 2 h. The solvent was evaporated, and the crude
product was purified by column chromatography with DCM and
MeOH as the eluent to obtain 38 as a yellow-white solid in 30% yield for
The hIDO1 proteins were removed from the N-terminal his-tag with
TEV protease (1:25 w/w) at 4 °C overnight and captured on the Ni-
NTA column (Cube Biotech). The fraction was further purified by
loading onto a Superdex S75 column pre-equilibrated with 100 mM
potassium phosphate buffer (pH 6.5). Peak fractions were obtained for
crystallization and spectroscopic analysis. The protein concentration
was determined via measuring the A280 absorbance using a NanoDrop
2000c spectrophotometer (Thermo Fisher Scientific). The purity of the
collected proteins was >95% as determined by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (12.5%). The ratio of heme-
incorporated hIDO1 proteins was determined by the ratio of A404/A280
(a ratio of 2.2 was taken as a reference for 100% heme incorporation);69
the final IDO1 samples achieved was ∼90% heme incorporation. All the
proteins were stored at −80 °C.
Compound IC50 Determinations. The compound inhibitory
activities to hIDO1 and hTDO were tested in the assay buffer (100 mM
potassium phosphate buffer, pH 6.5, 40 mM ascorbic acid, 0.01%
Triton X-100, 7 μM methylene blue, 200 μg/mL catalase). All
compounds were dissolved in DMSO in 10−100 mM. The compounds
were preincubated with the enzymes for 10 min at rt, and the enzyme
reactions were initiated by adding L-Trp (400 μM) at 37 °C and
terminated after 30−60 min by adding 30 μL of 30% (w/v)
trichloroacetic acid (TCA) and further incubation of 30 min at 50 °C
to convert NFK into kynurenine. Then, 90 μL of acetic acids containing
2% (w/v) para-dimethylaminobenzaldehyde was added to react with
kynurenine for about 5 min at rt. The resulting products were measured
for the absorbance at 480 nm using a Thermo microplate reader. All
determinations were performed in triplicate. The IC50/pIC50/s.e. pIC50
values were calculated by using GraphPad Prism software.
Optical Absorption Spectroscopic Measurements. UV−vis
absorption spectra of hIDO1/hTDO in complex with compounds were
recorded with a UV-1900i spectrophotometer (Shimadzu Scientific
Instruments, Inc.) with a spectral slit width of 1 nm at rt. The samples
were prepared at rt with 6 μM ferric hIDO1/hTDO and 0.5 mM
compounds in the assay buffer (100 mM potassium phosphate buffer,
pH 6.5, 40 mM ascorbic acid, 200 μg/mL catalase, 7 μM methylene
blue, 0.01% Triton X-100) or potassium phosphate buffer (100 mM,
pH 6.5). The UV−vis scans (300−700 nm) were recorded over 2 h.
X-ray Crystallography. The crystal structures of IDO1/1 (PDB
code 7E0O), IDO1/2 (PDB code 7E0P), IDO1/22 (PDB code
7E0Q), IDO1/23 (PDB code 7E0S), IDO1/36 (PDB code 7E0T), and
IDO1/41 (PDB code 7E0U) were obtained by the hanging drop vapor
diffusion method. The purified IDO1 proteins (20 mg/mL) were mixed
with 100 mM inhibitors in the crystallization buffer (100 mM
potassium phosphate buffer pH 6.5) for a final molar ratio of
inhibitor/hIDO1 of 10:1. The mixtures were incubated on ice for 1 h
and then centrifuged at 12,000 rpm for 10 min to remove insoluble
materials. Co-crystals were grown in 15−23% (v/v) polyethylene glycol
8000 and 0.2 M ammonium acetate. Before co-crystals were flash-
frozen in liquid nitrogen for data collection, they were cryoprotected
using the mother liquor supplemented with 30% (v/v) glycerol. The X-
ray diffraction data were obtained at the Shanghai Synchrotron
Radiation Facility (SSRF) BL19U1 beamline and processed by using
AutoProcess. The molecular replacement and refinement were carried
out as previously described. Crystallization conditions are in Table S1,
and data collection and refinement statistic are in Tables S2.
Molecular Docking. All molecular docking studies were performed
using AutoDock Vina.70 The X-ray crystal structure of hTDO/GNE1
(PDB code 6VBN)39 was used as the docking template. All water
molecules and solvent ligands were removed. A grid box was set by
covering the whole heme-containing active site of hTDO. The ligand
structure was prepared and energy-minimized using the Discovery
Studio package. The best docking pose representing most of the
observed poses is designated as the most possible binding pose.
Quantum Mechanical Computations. All DFT computations
were performed by using the Qchem 5.3 program. Initial coordinates of
all complexes were prepared from hIDO1/1 (PDB code 7E0O) using
IQmol. All geometry optimizations were performed at the LANL2DZ
level of theory using the B3LYP method. The optimized conformations
of complexes were then selected to compute single-point free energies
1
two steps, 90% HPLC. H NMR (400 MHz, d6-DMSO): δ 12.78 (s,
1H), 8.20 (s, 1H), 6.81 (s, 1H), 6.66 (t, J = 5.5 Hz, 1H), 6.12 (s, 1H),
5.74 (m, 2H), 4.78 (m, 1H), 3.19−2.99 (m, 2H), 1.99 (d, J = 3.3 Hz,
3H), 1.82 (m, 1H), 1.78−1.67 (m, 1H), 1.65−1.43 (m, 2H), 1.42−1.27
(m, 2H). 13C NMR (100 MHz, d6-DMSO): δ 143.4, 132.6, 129.8,
128.4, 121.9, 100.5, 99.8, 48.2, 40.0, 34.5, 27.0, 25.4, 20.9.
Synthesis of 6-Bromo-N-(((1S,2S)-2-chlorocyclohexyl)methyl)-
1H-indazol-4-amine (41). NCS (133 mg, 1.0 mmol) and PPh3 (262
mg, 1.0 mmol) were added to the solution of 23 (323 mg, 1.0 mmol) in
DCM (20 mL) at 0 °C. The mixture was stirred at rt for 4 h. The
mixture was washed with aq NaHCO3 and brine and dried over
anhydrous Na2SO4. The solvent was evaporated, and the crude product
was charged on a silica gel column chromatograph with petroleum ether
and EA as the eluent to afford the title compound 41 as a yellow solid in
40% yield, 91% HPLC. 1H NMR (400 MHz, d6-DMSO): δ 12.79 (s,
1H), 8.20 (s, 1H), 6.83 (s, 1H), 6.59 (s, 1H), 6.12 (s, 1H), 4.71 (s, 1H),
3.26−3.15 (m, 1H), 3.11−3.01 (m, 1H), 2.15−2.05 (m, 1H), 1.76−
1.22 (m, 8H). 13C NMR (100 MHz, d6-DMSO): δ 143.4, 142.1, 132.6,
121.9, 112.5, 100.5, 100.1, 64.1, 46.5, 34.0, 24.9, 24.6, 20.4. HRMS (AP-
ESI): 342.0366 (M + H)+ (calcd for C14H17BrClN3: 342.0367).
Synthesis of 6-Bromo-N-(((1R,2R)-2-chlorocyclohexyl)methyl)-
1H-indazol-4-amine (42). According to the synthesis method of 41,
compound 42 was synthesized from 22 in 21% yield, 90% HPLC. 1H
NMR (400 MHz, CDCl3): δ 7.97 (s, 1H), 7.00 (s, 1H), 6.33 (s, 1H),
4.53 (s, 1H), 3.32 (qd, J = 13.7, 7.2 Hz, 2H), 1.84−1.77 (m, 5H), 1.64−
1.52 (m, 5H). HRMS (AP-ESI): 342.0366 (M + H)+ (calcd for
C14H17BrClN3: 342.0367).
1
The H NMR, 13C NMR, and HRMS spectra of all the target
compounds are given in the Supporting Information.
Constructs, Protein Expression, and Purification. The
truncated hIDO1 (12−403 aa) and hTDO (19−388 aa) with the N-
terminal hexa-histidine tag were cloned into pET28a vectors. The
proteins were expressed in Escherichia coli Transetta (DE3) using the
LB medium containing kanamycin sulfate (50 μg/mL) and
chloramphenicol (50 μg/mL) supplemented with 1 mM 5-amino-
levulinic acid and 30 μM hemin chloride at 37 °C. When the OD600
values reached 0.8−1.0, 0.5 mM isopropyl β-D-1-thiogalactopyranoside
was added to induce protein expression in a shaker at 150 rpm at 25 °C
for 6−8 h. The culture was harvested by centrifugation (4000 rpm, 20
min) and resuspended in lysis buffer A (50 mM potassium phosphate,
0.3 M sodium chloride, 5% glycerol, 25 mM imidazole, pH 7.1 for
hIDO1; 50 mM potassium phosphate, 0.3 M sodium chloride, 5%
glycerol, pH 7.8 for hTDO) and protease inhibitors, followed by cell
lysis using a ultrahigh-pressure homogenizer (JNBIO). After
centrifugation at 4 °C (13,000 rpm) for 30 min, the supernatants
through from an Ni-NTA column (Cube Biotech) were pre-
equilibrated with lysis buffer A and then washed extensively with
buffer B (50 mM potassium phosphate, 0.3 M sodium chloride, 5%
glycerol, 30 mM imidazole, pH 7.1 for hIDO1; 50 mM potassium
phosphate, 0.3 M sodium chloride, 5% glycerol, 30 mM imidazole, pH
7.8 for hTDO) to remove nonspecifically binding proteins. The
obtained proteins were eluted in buffer C (50 mM potassium
phosphate, 0.3 M sodium chloride, 5% glycerol, 350 mM imidazole,
pH 7.1 for hIDO1; 50 mM potassium phosphate, 0.3 M sodium
chloride, 5% glycerol, 250 mM imidazole, pH 7.8 for hTDO) and then
concentrated using an Amicon Ultra 10k (Millipore). The proteins
were dialyzed into 100 mM potassium phosphate buffer (pH 6.5) for
biochemical assays.
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using LANL2DZ through three different methods, including B3LYP,
PBE0, and m06. The coordination bond energy was finally obtained by
the computed Gibbs free energy of each complex minus the energy of
the corresponding heme and compound. All geometry optimizations
and free-energy computations were performed in a hidden solvent
environment using the polarizable continuum models. All optimized
coordinate figures were generated using IQmol 2.15.
carried out on the 6th, 9th, and 12th days after MPTP administration;
three replicates for each test were carried out.
Grip Tests. Grip tests were performed as that described by Moran et
al.71 after 18 days of modeling. Before the tests, the mice were trained
for the grip strength test. The apparatus was a string of a 50 cm length,
which was pulled taut between two vertical supports and raised 40 cm
above the ground. The mouse was placed in the middle of the string and
evaluated according to the following system: 0, falls off; 1, hangs onto
string for more than 60 s; 2, escapes in 40−60 s; 3, escapes in 20−40 s;
4, escapes in 10−20 s; and 5, escapes in 10 s. Each mouse was tested at
an interval of 20 min with three replicates.
Cellular IDO1/TDO Assays. The IDO1/TDO cellular activity
assays were carried out as previously reported.38,50 The HeLa cells
(with high IDO1 expression induced by IFN-γ) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM); the SW48 and A172
cells (with high TDO expression) were cultured in the L-15 medium
and DMEM medium, respectively. All the media were supplemented
with 10% fetal bovine serum and 1% penicillin−streptomycin solution.
The test compounds were diluted threefold in the corresponding
medium. The cells were counted to adjust the density of 1 × 104 for
HeLa cells, 2 × 104 for SW48 cells, and 1 × 104 for A172 cells per well in
96-well culture plates. On the next day, a total volume of 200 μL of the
medium containing the compound, L-tryptophan, and IFN-γ was added
to HeLa cells for a final concentration of 200 μM L-tryptophan and 50
ng/mL IFN-γ per well. A 200 μL medium containing compounds and L-
tryptophan were added to SW48 cells and A172 cells with a final
concentration of 200 μM for L-tryptophan per well. After incubation in
a 37 °C, 5% CO2 incubator for HeLa and A172 cells or in 100% air for
SW48 cells for 48 h, 150 μL of the supernatant was taken out and mixed
with 50 μL of 30% (w/v) TCA and further incubated with 30 min at 65
°C to convert NFK into kynurenine. The reaction mixture was
centrifuged at 13,000 rpm for 10 min. The supernatant (100 μL) from
each system was transferred into 96-well plates and mixed with 100 μL
of acetic acids containing 2% (w/v) para-dimethylaminobenzaldehyde.
The resulting products were measured for the absorbance at 480 nm.
All determinations were performed in triplicate. The EC50 values were
calculated by using GraphPad Prism software.
Animal Studies. Male C57BL/6J mice (10 weeks old, 22−26 g)
were purchased from Chengdu Dossy Experiment Animals Co., Ltd.
(Sichuan, China) and were housed five per cage under standard
conditions (a 12 h light/dark cycle; 22−25 °C; with free access to food
and water). All experimental procedures and protocols were reviewed
and approved by the National Institutional Animal Care and Ethical
Committee of Chengdu University of Traditional Chinese Medicine
and were in accordance with the Guide for the Care and Use of
Laboratory Animals. The mice were allowed to acclimate for 1 week
before the experiments.
Mice were randomly divided into six groups (10 mice in each group):
the vehicle group, MPTP group, MPTP + 30 100 mg/kg group, MPTP
+ 30 30 mg/kg group, MPTP + 30 10 mg/kg group, and MPTP +
Madopar 50 mg/kg group (as a positive control). Within the first 7 days
of the experiments, the mice in the vehicle group were treated with
saline, while the mice in other groups were given MPTP 30 mg/kg
intraperitoneally at a fixed time (9:00 a.m.) per day to induce
neurodegeneration. Subsequently, the mice were then administered
with various doses of 30 or Madopar (50 mg/kg) by oral gavage once
daily for an additional 14 days, and the mice in the vehicle and MPTP
groups were given normal saline by oral gavage. All experiments were
carried out blind. After behavioral tests, the mice were sacrificed and the
tissues were processed for further study.
Sample Collection and Tissue Preparations. After the
behavioral evaluations, all mice were anesthetized and their blood
was collected through abdominal aorta. The brains were taken to
perform the immunohistochemical staining or further dissected out the
striatum and substantia nigra to perform western blot analysis and
absolute quantitative study of tryptophan channel metabolites.
IHC for TH, Iba-1, IFN-γ, IL-1β, TNF-α, and iNOS. The expression of
TH, Iba-1, IFN-γ, IL-1β, TNF-α, and iNOS in the SNpc and striatum
was examined by IHC, which were commissioned by Yibaidao
Technology Co., Ltd., Chengdu, China. Briefly, the brains of mice
were taken and fixed in 10% formaldehyde for one more day and then
dehydrated and embedded to prepare the paraffin section. 3 μm serial
sagittal brain sections were cut in a cryostat and exposed to repair with
ethylenediaminetetraacetic acid antigen retrieval solution (pH 9.0),
followed by incubation with 0.3% H2O2 and the blocking reagent (3%
bovine serum albumin). Then, the primary antibody rabbit IgG TH
(1:500 dilutions, 25859-1-AP, Proteintech, Chicago, USA) or the
rabbit polyclonal antibody against Iba-1 (1:400 dilutions, 10904-1-AP,
Proteintech, Chicago, USA) or TNF-α (1:200 dilutions, GB11188,
Servicebio) or IL-1β (1:500 dilutions, GB11113, Servicebio) or iNOS
(1:200 dilutions, 18985-1-AP, Proteintech, Chicago, USA) or IFN-γ
(1:100 dilutions, A12450, ABclonal) was added to incubate overnight
at 4 °C. The brain sections were washed with phosphate-buffered saline
buffer (pH 7.4) and incubated with the secondary goat antirabbit
antibody conjugated to horse radish peroxidase (HRP) (Dako,
Denmark, A/S, K5007) at rt for 50 min, visualized with HRP’s
chromogenic substrate DAB (Dako, Denmark, A/S, K5007), and then,
the cell nucleus was stained with hematoxylin. Color development was
monitored under the microscope, and then, the images were collected
and analyzed. The brain sections of three individual mice in each group
were used to analyze the number of TH-positive cells (or Iba-1-positive
cells) or the optical intensity of IFN-γ, IL-1β, TNF-α, and iNOS of the
SNpc and striatum at ×400 original magnification by Image-Pro Plus
6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The number of
cells and the optical density in the experimental group were normalized
to the value from the vehicle-treated group.
WB Analysis of the Expression of IDO1 and TDO in Substantia
Nigra and Striatum. WB analysis was carried out to detect the
expression of IDO1 and TDO in substantia nigra and striatum. Briefly,
the substantia nigra and striatum were separated, weighed, and
homogenized using ice-cold RIPA lysis buffer. After comminution by
ultrasonication, centrifugation, quantification, and boiling, the protein
samples were separated by 10% SDS polyacrylamide gel and transferred
onto polyvinylidene difluoride membranes. The membranes were
blocked and probed by anti-IDO1 and anti-TDO antibodies. The
expression of IDO1 and TDO in the vehicle group, MPTP group, 23-
treated (30 mg/kg) group, and Ma-treated (50 mg/kg) group was
analyzed to study the pathogenic and promoting effects of IDO1/TDO
on PD and the therapeutic effects of 23 on PD.
Absolute Quantitative Analysis of Tryptophan Metabolites in
Substantia Nigra and Striatum. After the behavioral evaluations, three
mice per group were sacrificed for detection of tryptophan metabolites
by MetWare (http://www.metware.cn/) based on the AB Sciex
QTRAP 6500 liquid chromatography with tandem mass spectrometry
(LC−MS/MS) platform.72−74 Briefly, 50 μL of peripheral blood
plasma was precipitated and extracted with 250 μL of methanol. The
substantia nigra and striatum were taken and weighed (0.05 g of the
sample), and 500 μL of methanol was added to homogenize with a
tissue homogenizer. The resulting samples were then vortexed and
General Behavioral Tests. The mice were allowed to familiarize
with the testing room, equipment, and experimenter before behavioral
testing, and the specific screening conditions and training methods are
described below. General indexes including body weight and acute
reaction within hours after administration were monitored at 1 day
before modeling and 1, 3, 5, 7, and 14 days after modeling.
Rota-Rod Tests. The global motor coordination and balance of mice
were evaluated and quantitatively analyzed using rota-rod equipment,
which was able to measure six mice parallelly. The animals were
preadapted to the experimental apparatus and exercise method. The
motor function was assessed by placing the animals on the rod which
rotated at 25 rpm, and the latency time for the first fall to the floor was
recorded; the maximum execution time was set to 180 s. The tests were
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centrifuged to obtain the supernatant for further LC−MS analysis. The
effects of compounds on the changes of tryptophan metabolites were
analyzed by referring to that reported by Breda et al.75
In Vivo Safety Evaluation. After the end of PD model efficacy
experiments, the body weights and main organ coefficients of the mice
in the different groups were determined to preliminarily evaluate in vivo
safety of treated compounds.
homogenate) to achieve a final concentration of 2 μM. An aliquot of
150 μL of the loading matrix was loaded to the donor side of each
dialysis well in triplicate, and 150 μL of the dialysis buffer was
transferred to the receiver side of the well. The dialysis device was
placed in an incubator at 37 °C, 5% CO2, on a shaking platform with
rotation at 100 rpm for 4 h. After reaching equilibrium, 50 μL samples
from the buffer side and the matrix side were taken and transferred into
a 96-well plate to prepare the test solutions. Each sample well was
supplemented with an equal volume of the opposite blank matrix
(buffer or matrix) to achieve a 1:1 (v/v) dialysis buffer/matrix system.
All samples were then added 500 μL of 100% acetonitrile containing an
internal standard, and the mixture obtained was vortexed and
centrifuged for 20 min at 4000 rpm. 100 μL of the supernatant of
samples was then analyzed by LC−MS/MS. For plasma, the unbound
fraction (fu,plasma) was determined as the ratio of concentrations in the
buffer and plasma via eq 4, and the unbound fraction in the undiluted
brain (fu,brain) was calculated via eq 5
Permeability Assays with MDR1-MDCKII Cells. The perme-
ability assay was commissioned by WuXi AppTec Co. Ltd., Shanghai,
China. Briefly, the human MDR1-transfected Madin−Darby canine
kidney II (MDR1-MDCKII) cell line (obtained from Piet Borst at the
Netherlands Cancer Institute) was cultured on polyethylene mem-
branes (PET) at a cell density of 2.5 × 105 cells/mL in 96-well insert
systems for 4−7 days for the formation of the confluent cell monolayer.
Compound 23 was diluted with Hanks’ balanced salt solution
containing 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid (transport buffer, pH 7.4) to a final concentration of 2 μM (DMSO
< 1%) and was added to the donor compartment, with the receiver side
containing the vertical buffer. The 96-well plate was incubated at 37 °C
with 5% CO2, and after 2.5 h, the test compound in both compartments
was analyzed by LC−MS/MS. The permeation assays from the A to B
direction or B to A direction were conducted in triplicate. After the
permeability experiments, the cell monolayer integrity was determined
by the Lucifer yellow rejection assay (100 μM Lucifer yellow in the
apical side and the blank transport buffer in the basolateral side). The
relative fluorescence unit of Lucifer yellow is measured at 425/528 nm
(excitation/emission) with an EnVision plate reader.
F
T
fu,plasma
=
× 100
(4)
where F is the free concentration of 23 on the dialysis buffer (receiver)
side of the membrane and T is the total concentration of 23 on the
matrix (donor) side of the membrane
1/D
− 1 + 1/D
fu,brain
=
× 100
1
(F / T)
(5)
The parameters including apparent permeability coefficient (Papp),
ER, and solution recovery were calculated using eqs 1−3, respectively,
where F is the free concentration of 23 on the dialysis buffer (receiver)
side of the membrane, T is the total concentration of 23 on the matrix
(donor) side of the membrane, and D is the dilution factor.76
The unbound brain to unbound plasma concentration ratio (Kp,uu) of
23 was calculated via eq 6
dCr
dt
P
=
× V ÷ (A × C0)
app
r
(1)
where dCr/dt is the cumulative concentration of 23 in the receiver side
as a function of time, Vr is the solution volume in the receiver side, A is
the surface area for the transport, and C0 is the initial concentration of
23 added to the donor chamber
fu,brain
Kp,uu = Kp ×
fu,plasma
(6)
where Kp is total brain to total plasma concentration of 23 ratio.59
PK Determination and Analysis. The oral and iv formulation was
freshly prepared on the day of the experiment. Compound 23 was
dissolved in 5% v/v DMSO, added with 20% v/v PEG 400 to form a
uniform solution, and finally 75% v/v normal saline (NS) to make up
the final volume. Male SD rats or male Kunming mice were
administered orally or intravenously at a single dose of 100 mg/kg.
The volume of the dose was 10 mL/kg for both the groups. Blood
samples were collected from the retro-orbital plexus of each SD rat at
0.083, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 12.0, and 24.0 h
after oral administration or at 0.083, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 5.0,
8.0, 12.0, and 24.0 h after iv administration and then centrifuged at 3500
rpm and 4 °C for 15 min to harvested plasma samples. Brain samples
from male Kunming mice were collected in addition to whole blood at
0.5, 1.0, 2.0, 4.0, 8.0, and 12.0 h. Brain samples were homogenized using
acetonitrile−water mixtures. All the samples were temporarily stored at
−20 °C until HPLC analysis using sulfamethoxazole as an internal
standard. The plasma concentration−time data were fitted to a
noncompartmental model using DAS (version 3.0) to obtain the PK
parameters. The percentage of oral bioavailability (F) was calculated
using eq 7
P (BA)
app
Efflux ratio =
P (AB)
app
(2)
where Papp(BA) is the apparent permeability coefficient from the
basolateral to apical direction and Papp(AB) is the apparent permeability
coefficient from the apical to basolateral direction
V × Cr + V × Cd
r
d
Solution recovery =
× 100
V × C0
(3)
d
where Vd is the volume in the donor chamber and Cr and Cd are the final
concentrations of the transport compound in the receiver and donor
compartments, respectively.
Permeability Assays with Caco-2-BCRP Cells. The human
BCRP-transfected human colorectal carcinoma (Caco-2-BCRP) cells
(purchased from ATCC) were seeded on PET at 1 × 105 cells/cm2 for
21−28 days for the formation of the confluent cell monolayer with a
change of the medium every 4−5 days. The steps of the subsequent
transport assay and monolayer integrity assessment were consistent
with the MDR1-MDCKII cell assay, and the only difference was that
the permeation of compound 23 in Caco-2-BCRP cells was tested in the
presence or absence of 30.0 μM novobiocin (a BCRP inhibitor)
bidirectionally.
AUCoral
AUCiv
doseiv
doseoral
F =
×
× 100
(7)
Mouse Plasma/Brain Protein Binding Assays. The binding
assays of 23 were commissioned by WuXi AppTec Co. Ltd., Shanghai,
China. In brief, the 96-well equilibrium dialysis device was used to
determine the fraction of unbound compound 23 in plasma and the
brain of Kunming mice. The molecular mass cutoff of dialysis
membrane strips was 12−14 kDa. Before the experiments, the
membranes were rinsed and socked in ultrapure water for 20 min.
The male Kunming mice plasma and 1/3 brain homogenate samples
were prepared for the assay. To prepare the loading matrix, an aliquot of
compound 23 was spiked in the blank matrix (plasma or brain
where AUCoral and AUCiv are the areas under the plasma concentration
time after oral or iv administration.
CYP2C9/Kinase Inhibition. The inhibitory activity of 23 to
CYP2C9 was commissioned by WuXi AppTec Co. Ltd., Shanghai,
China. Briefly, the CYP2C9 substrate 4′-hydroxy diclofenac solution
and 23 (or the positive control sulfaphenazole) were added with human
liver microsome in the incubation plate. After reduced nicotinamide
adenine dinucleotide phosphate cofactor solution was supplemented to
the above mixtures, the plate was incubated for 10 min at 37 °C in a
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Article
water bath. The reaction was terminated, and the mixture was
centrifuged to collect the supernatant for LC/MS/MS analysis. The
kinase profiling of 23 was performed by using the KINOMEscan service
in Eurofins DiscoverX.
Statistical Analysis. All data were expressed as the mean
standard error of the mean (SEM). The P-values among different
groups of mice were calculated by using the t-test tool in Excel. The PK
parameters were analyzed by DAS (version 3.0). P < 0.05 was regarded
as a significant difference.
Yu-Xiang Wu − Department of Pharmaceutical Engineering,
College of Food and Bioengineering, Xihua University,
Chengdu 610039, China
Jun-Lin Yu − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu 610041,
China
Ya-Li Ren − State Key Laboratory of Southwestern Chinese
Medicine Resources, School of Pharmacy, Chengdu University
of Traditional Chinese Medicine, Chengdu 611137, China
Zong-Yuan Luo − Department of Pharmaceutical Engineering,
College of Food and Bioengineering, Xihua University,
Chengdu 610039, China
Gen Li − Key Laboratory of Drug-Targeting and Drug Delivery
System of the Education Ministry and Sichuan Province,
Department of Medicinal Chemistry, West China School of
Pharmacy, Sichuan University, Chengdu 610041, China
Yang Chen − Department of Pharmaceutical Engineering,
College of Food and Bioengineering, Xihua University,
Chengdu 610039, China
Si-Yao Wang − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu 610041,
China
Cheng Peng − State Key Laboratory of Southwestern Chinese
Medicine Resources, School of Pharmacy, Chengdu University
of Traditional Chinese Medicine, Chengdu 611137, China;
orcid.org/0000-0003-3303-906X
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00303.
■
sı
Synthetic procedure of intermediates, data collection and
refinement statistics for X-ray crystallography, and NMR
and HRMS spectra of target compounds (PDF)
Molecular formula strings for 1−42 (CSV)
Accession Codes
PDB codes: 7E0O (hIDO1/1), 7E0P (hIDO1/2), 7E0Q
(hIDO1/22), 7E0S (hIDO1/23), 7E0T (hIDO1/36), and
7E0U (hIDO1/41).
AUTHOR INFORMATION
Corresponding Authors
■
Shan Qian − Department of Pharmaceutical Engineering,
College of Food and Bioengineering, Xihua University,
Chengdu 610039, China; orcid.org/0000-0002-9550-
6885; Email: qians33@163.com
Guo-Bo Li − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu 610041,
China; orcid.org/0000-0002-4915-6677;
Email: liguobo@scu.edu.cn
Ling-Ling Yang − Department of Pharmaceutical Engineering,
College of Food and Bioengineering, Xihua University,
Chengdu 610039, China
Zhou-Yu Wang − Department of Pharmaceutical Engineering,
College of Food and Bioengineering, Xihua University,
Chengdu 610039, China
Yong Wu − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu 610041,
China; orcid.org/0000-0003-0719-4963
Authors
Xiang-Li Ning − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu 610041,
China
Complete contact information is available at:
Yu-Zhi Li − State Key Laboratory of Southwestern Chinese
Medicine Resources, School of Pharmacy, Chengdu University
of Traditional Chinese Medicine, Chengdu 611137, China
Cui Huo − Department of Pharmaceutical Engineering, College
of Food and Bioengineering, Xihua University, Chengdu
610039, China
Ji Deng − Key Laboratory of Drug-Targeting and Drug Delivery
System of the Education Ministry and Sichuan Province,
Department of Medicinal Chemistry, West China School of
Pharmacy, Sichuan University, Chengdu 610041, China
Cheng Gao − Department of Pharmaceutical Engineering,
College of Food and Bioengineering, Xihua University,
Chengdu 610039, China
Kai-Rong Zhu − Key Laboratory of Drug-Targeting and Drug
Delivery System of the Education Ministry and Sichuan
Province, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu 610041,
China
Miao Wang − State Key Laboratory of Southwestern Chinese
Medicine Resources, School of Pharmacy, Chengdu University
of Traditional Chinese Medicine, Chengdu 611137, China
https://pubs.acs.org/10.1021/acs.jmedchem.1c00303
Author Contributions
∥X.-L.N. and Y.-Z.L. are co-first authors.
Author Contributions
X.-L.N. and Y.-Z.L. contributed equally to this work. G.-B.L. and
S.Q. designed the project. C.H., C.G., and Y.-X.W. carried out
chemical synthesis and structural identification, as supervised by
S.Q. Y.C., L.-L.Y., Z.-Y.L., and Y.W. performed the identification
of chiral structures and chiral chromatographic analysis. X.-L.N.,
J.D., and K.-R.Z. expressed and purified recombinant hIDO1
and hTDO enzymes. X.-L.N. carried out all other biochemical
studies, determined the absorption spectra, and crystallized all
complexes, as supervised by G.-B.L. J.-L.Y. and G.L. obtained
and analyzed the diffraction data of all complexes and performed
structural determination, as supervised by G.-B.L. Y.-Z.L., M.W.,
Y.-L.R., and C.P. performed in vivo pharmacodynamics and
mechanistic studies. X.-L.N., J.D., and S.-Y.W. determined PK
properties and tryptophan metabolites. J.-L.Y. carried out all
computation studies. G.-B.L., S.Q., and X.-L.N. wrote the initial
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and approved the final version for submission.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the funds from the National Natural
Science Foundation of China (81973189, 8187429, and
82073698), the Sichuan Outstanding Young Scientific and
Technological Talents Project (2020JDJQ0054), the Sichuan
Science and Technology Program (2018HH0100), the 111
project (B18035), the Chengdu Technology Innovation
Research Project (2018-YFYF-00195-SN), the Scientific Re-
search Foundation of Sichuan University (2082604401098 and
20826044A2035), the Sichuan Education Department
(18TD0023), the Outstanding Interdiscipline Project of West
China Hospital of Sichuan University (ZYJC18024), and the
Innovation Fund of Xihua Scholars, Undergraduate and
Postgraduate from Xihua University (202010650017). The
authors thank the staff of the BL19U1 beamline of the National
Center for Protein Science Shanghai at the Shanghai
Synchrotron Radiation Facility for assistance during data
collection.
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